CN108685858B - Pramipexole sustained release preparation for injection and preparation method thereof - Google Patents

Pramipexole sustained release preparation for injection and preparation method thereof Download PDF

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CN108685858B
CN108685858B CN201710225964.XA CN201710225964A CN108685858B CN 108685858 B CN108685858 B CN 108685858B CN 201710225964 A CN201710225964 A CN 201710225964A CN 108685858 B CN108685858 B CN 108685858B
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pramipexole
plga
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CN108685858A (en
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王淑君
童超
叶田田
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Shenyang Pharmaceutical University
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Abstract

The invention belongs to the technical field of medicines, and relates to a pramipexole sustained-release preparation for injection and a preparation method thereof, wherein the preparation method comprises the following steps: dissolving the main drug and the internal water phase additive in water for injection to form a drug-containing internal water phase solution; dissolving PLGA and an organic phase additive in an organic solvent to form an organic phase solution; mixing the medicated inner water phase solution and the organic phase solution, and ultrasonically treating to form water-in-oil colostrum; adding the water-in-oil primary emulsion into the external water phase solution, and stirring to form water-in-oil-in-water double emulsion; solidifying the water-in-oil-in-water double emulsion to form microspheres, centrifugally washing and collecting the microspheres to obtain wet microspheres; adding a gel solution and a freeze-drying protective agent into the wet microspheres to obtain a microsphere gel solution; and (4) freeze-drying the microsphere gel solution to obtain the microsphere gel. The pramipexole sustained release preparation for injection has high drug loading, good temperature sensitivity and biocompatibility, and can release drug continuously and stably, effectively reduce the occurrence of motion complications, and improve the compliance of patients.

Description

Pramipexole sustained release preparation for injection and preparation method thereof
Technical Field
The invention relates to the technical field of medicines, in particular to a pramipexole sustained-release preparation for injection and a preparation method thereof.
Background
Parkinson's Disease (PD), also known as parkinsonism, belongs to extravertebral disorders and is one of the most common neurodegenerative diseases. The most important pathological change of the Parkinson's disease is the degenerative death of midbrain nigral Dopamine (DA) neurons, which causes the disease to be caused by the significant reduction of striatal dopamine content.
Traditional parkinson's disease treatment usually employs levodopamine, but long-term application is likely to result in motor complications. Currently, the new concept of parkinson's disease treatment is Continuous Dopaminergic Stimulation (CDS), which is mainly achieved by continuous administration of levodopa or dopamine receptor agonists, however, due to the "peak-valley" phenomenon in blood concentrations, treatment of parkinson's disease by continuous dopaminergic stimulation easily causes pulse-like stimulation, resulting in difficulty in controlling motor-related symptoms, while reducing patient compliance.
Therefore, there is a need to develop a pramipexole sustained release preparation for injection, which has good curative effect and is convenient to use and is used for treating the Parkinson's disease.
Disclosure of Invention
The invention aims to provide a pramipexole sustained release preparation for injection and a preparation method thereof, the pramipexole sustained release preparation for injection has high drug loading, good temperature sensitivity and biocompatibility, and can continuously and stably release drugs, effectively reduce the occurrence of motion complications and improve the compliance of patients.
In order to achieve the above object, the present invention provides a pramipexole sustained release preparation for injection, which comprises the following raw materials: 5mg to 5000mg of main drug, 1mg to 1000mg of internal water phase additive, 0.05g to 25g of PLGA, 0.05g to 2g of organic phase additive, 1ml to 100ml of gel solution and 10mg to 1000mg of freeze-drying protective agent.
Preferably, the raw materials consist of: 100 mg-500 mg of main drug, 10 mg-50 mg of internal water phase additive, 0.5 g-2.5 g of PLGA, 0.1 g-0.5 g of organic phase additive, 5 ml-20 ml of gel solution and 40 mg-200 mg of freeze-drying protective agent.
The main medicine is pramipexole hydrochloride and various pramipexole salts.
The inner water phase additive is one or more of poloxamer, chitosan, alginate, carbomer and span.
When the inner water phase additive is a mixture of poloxamer and chitosan, the mass ratio of the poloxamer to the chitosan is (0.5-20): 1. when the span is included in the internal water phase additive, the specific gravity of the span in the internal water phase additive is 0.05-10%.
Preferably, when the internal water phase additive is a mixture of poloxamer and chitosan, the mass ratio of the poloxamer to the chitosan is (2-6): 1. when the span is included in the internal water phase additive, the specific gravity of the span in the internal water phase additive is 0.2% -1%.
The organic phase additive is one or a mixture of poloxamer and chitosan.
The molar ratio of lactide to glycolide in the PLGA is 75/25 or 50/50, the end group of the PLGA is one or more of ester end capping, carboxyl end group and hydroxyl end group, the molecular weight of the PLGA is 15 kDa-60 kDa, and the intrinsic viscosity is 0.18 dl/g-0.55 dl/g.
The gel solution is one or more mixture water solutions of poloxamer and chitosan, and the concentration of the gel solution is 20 mg/ml-2000 mg/ml.
Preferably, the gel solution is one or more mixture aqueous solutions of poloxamer and chitosan, and the concentration of the gel solution is 100 mg/ml-500 mg/ml.
The poloxamer is one or more of poloxamer 188, poloxamer 237, poloxamer 338 and poloxamer 407.
The freeze-drying protective agent is one or a mixture of mannitol and sodium chloride.
The invention also provides a preparation method of the pramipexole sustained-release preparation for injection, which comprises the following steps:
step 1, dissolving a main drug and an internal water phase additive in water for injection to form a drug-containing internal water phase solution (internal water phase for short);
step 2, dissolving PLGA and an organic phase additive in an organic solvent to form an organic phase solution;
step 3, mixing the drug-containing internal water phase solution and the organic phase solution, and ultrasonically forming water-in-oil colostrum;
step 4, adding the water-in-oil primary emulsion into an external water phase solution, and stirring to form a water-in-oil-in-water multiple emulsion;
step 5, solidifying the water-in-oil-in-water double emulsion to form microspheres, and centrifugally washing and collecting the microspheres to obtain wet microspheres;
step 6, adding a gel solution and a freeze-drying protective agent into the wet microspheres to obtain a microsphere gel solution;
and 7, freeze-drying the microsphere gel solution to obtain the pramipexole sustained-release preparation for injection, namely the pramipexole sustained-release preparation for injection for treating the Parkinson's disease.
The concentration of the main drug in the inner water phase is 200-500 mg/ml, the concentration of the inner water phase additive in the inner water phase is 50-300 mg/ml, wherein the inner water phase comprises the main drug, the inner water phase additive and water for injection, and the pH value of the inner water phase is adjusted to 4-6 by adopting phosphoric acid.
The volume of the inner water phase is 0.1 ml-0.5 ml.
The concentration of the organic phase additive in the organic phase is 20 mg/ml-100 mg/ml, and the concentration of the PLGA in the organic phase is 50 mg/ml-400 mg/ml, wherein the organic phase consists of PLGA, the organic phase additive and an organic solvent.
The organic solvent is one or a mixture of dichloromethane, ethyl acetate and acetone, and the volume of the organic phase is 3ml to 20 ml.
The volume ratio of the inner water phase to the organic phase in the water-in-oil colostrum is 1/10-1/30.
In the step 3, the ultrasonic power is 100 w-400 w, the ultrasonic time is 1 min-5 min, and the temperature is 0-15 ℃.
The external water phase in the step 4 is PVA aqueous solution
The volume ratio of the organic phase to the external water phase in the water-in-oil colostrum in the step 4 is 1/3-1/30, the stirring speed is 3000 rpm-10000 rpm, the time is 1 min-5 min, and the temperature is 0-15 ℃.
The external water phase solution is obtained by adjusting the pH value of 0.2-3% PVA aqueous solution to 8-10 by adopting phosphate buffer solution, wherein the polymerization degree of PVA is 1500-2000, and the alcoholysis degree is 87-89%.
The volume of the external water phase is 3ml to 20 ml.
The curing temperature of the whole curing in the step 5 is 10-45 ℃ and the time is 4-10 h; the primary solidification adopts solvent diffusion, the next solidification adopts solvent evaporation,
in the step 5, the preliminary solidification is to slowly add a diluent to the water-in-oil-in-water composite emulsion under stirring to diffuse an organic solvent, perform preliminary solidification of the water-in-oil-in-water composite emulsion, and then perform next solidification of the preliminarily solidified water-in-oil-in-water composite emulsion.
In the step 5, solvent evaporation is adopted in the next step of solidification, and the solvent evaporation is performed for 4-10 h by using a constant-temperature water bath magnetic stirrer for magnetic stirring
The volume ratio of the external water phase to the diluent is 1/0.5-1/5.
The diluent is 0-5% ethanol water solution; the volume of the diluent is 10 ml-150 ml, and the adding speed is 2 ml/min-10 ml/min.
And the centrifugal washing in the step 5 adopts equal-volume injection water to centrifugally wash for 2-5 times in a low-speed centrifuge with the rotating speed of 1500-4000 rpm, and the centrifugal washing is carried out for 5-15 min each time.
The pre-freezing temperature of the freezing in the step 7 is-45 ℃ to-55 ℃, the pre-freezing and heat preservation time is 4h to 8h, then the temperature is increased to 30 ℃ in a gradient manner at 5 ℃ to 15 ℃ per hour under the vacuum degree of less than 10pa, the heat preservation is carried out for 12h to 24h at 30 ℃, and the total freezing and drying time is 24h to 48 h.
The average diameter of microspheres in the pramipexole sustained release preparation for injection is 10-200 mu m.
Preferably, the average diameter of the microspheres in the pramipexole sustained release preparation for injection is 40-80 μm.
The pramipexole sustained release preparation for injection has temperature sensitive property after freeze-drying and redissolving, and can be coagulated at 32-40 ℃.
The pramipexole sustained release preparation for injection consists of microspheres in the middle and temperature-sensitive gel wrapping the microspheres, wherein the drug-loading rate of the pramipexole sustained release preparation for injection is more than 10%, and the encapsulation rate is more than 60%.
The pramipexole sustained-release preparation for injection is used for treating mild, moderate and severe Parkinson's disease.
Compared with the prior art, the invention has the beneficial effects.
The pramipexole sustained release preparation for injection for treating Parkinson disease and the preparation method thereof provided by the invention take semisolid as a local administration dosage form, can better adapt to complex physiological environment in vivo, and improve biocompatibility; in addition, the pramipexole sustained release preparation for injection has high drug loading amount of microspheres and good temperature sensitivity of gel, realizes continuous and stable drug release, effectively reduces the occurrence of motion complications, improves the compliance of patients, and is suitable for treating the mild, moderate and severe Parkinson's disease.
Drawings
Fig. 1 is a flowchart of a method for preparing pramipexole dihydrochloride sustained release preparations for injection according to example 1 of the present invention.
Fig. 2 is an optical microscope photograph of microspheres in the pramipexole sustained release preparation for injection described in example 1 of the present invention.
Fig. 3 is a graph showing the in vitro release profile of pramipexole dihydrochloride sustained release preparations for injection prepared in test example 1 of the present invention in example 1.
Fig. 4 is a graph showing the in vitro release profile of pramipexole dihydrochloride sustained release preparations for injection prepared in test example 2 of example 1 of the present invention.
Fig. 5 is a graph showing the in vitro release profile of pramipexole dihydrochloride sustained release preparations for injection, prepared in test example 3 of example 1 of the present invention.
Fig. 6 is a graph showing the in vitro release profile of pramipexole dihydrochloride sustained release preparations for injection, prepared in test example 4 of example 1 of the present invention.
Fig. 7 is a graph showing the in vitro release profile of pramipexole dihydrochloride sustained release preparations for injection, prepared in test example 5 of example 1 of the present invention.
Fig. 8 is a graph showing the blood concentration in rats of the pramipexole dihydrochloride sustained-release preparation for injection prepared in test example 6 of example 1 of the present invention.
Detailed Description
In order to make the objects, technical solutions and effects of the present invention clearer and clearer, the present invention is further described in detail below with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1.
The embodiment provides a pramipexole sustained-release preparation for injection, which consists of the following raw materials: 150mg of pramipexole dihydrochloride, 25mg of an internal water phase additive, 0.75g of PLGA, 0.25g of an organic phase additive, 10ml of a gel solution formed by poloxamer 188 and 20mg/ml of chitosan, and 100mg of mannitol.
Pramipexole dihydrochloride is a dopamine receptor agonist, has high selectivity and specificity in combination with the D2 subfamily of dopamine receptors, has preferential affinity for D2 receptors, has complete intrinsic activity, and relieves dyskinesia of Parkinson patients by exciting striatal dopamine receptors. The half-life period of the pramipexole dihydrochloride is short and is 8-12 hours.
The Poloxamer (Poloxamer) is a polyoxyethylene-polyoxypropylene block copolymer, the trade name is Pluronic (Pluronic), the Poloxamer has a series of varieties with different relative molecular masses and the contents of polyoxypropylene and polyoxyethylene according to the proportion of ethylene oxide and propylene oxide in polymerization, and the Poloxamer is a nonionic surfactant and can be used as an emulsifier of intravenous injection fat emulsion. The poloxamer solution is a temperature sensitive in-situ gel.
The PLGA is poly (lactic-co-glycolic acid) copolymer, which is formed by random polymerization of two monomers, namely lactic acid and glycolic acid, and is a degradable functional polymer organic compound.
The molar ratio of lactide to glycolide in the PLGA is 75/25, the end group of the PLGA is a terminal carboxyl group, the molecular weight of the PLGA is 30kDa, and the intrinsic viscosity is 0.3 dl/g.
The gel solution is a mixture aqueous solution formed by the poloxamer 188 and the chitosan, and the concentration of the gel solution is 220 mg/ml.
The chitosan is also called chitosan, because the chitosan is a cationic polyamino sugar and has alkaline amino groups, when the pH value is less than 6.5, the chitosan is dissolved in water to form a gel solution; when the pH value is more than 7, the chitosan solution becomes gel. The chitosan aqueous solution is a pH sensitive in-situ gel.
The mannitol is used as a freeze-drying protective agent.
The embodiment also provides a preparation method of the pramipexole sustained-release preparation for injection, which comprises the following steps:
step 1, dissolving 150mg of pramipexole dihydrochloride and the internal water phase additive in 200ul of water for injection to form a pramipexole dihydrochloride water solution with the pramipexole concentration of 0.75g/ml, and adjusting the pH value to 5 by using phosphoric acid.
And 2, dissolving 0.75g of PLGA and 0.25g of organic phase additive in 5ml of dichloromethane to form a PLGA dichloromethane solution with the PLGA concentration of 150 mg/ml.
And 3, mixing the pramipexole dihydrochloride injection aqueous solution and the PLGA dichloromethane solution, and ultrasonically forming water-in-oil colostrum (hereinafter referred to as w/o colostrum) by adopting an ultrasonic cell crusher under the conditions of ultrasonic power of 300w, ultrasonic time of 3min and temperature of 0 ℃.
And 4, adding the w/o colostrum into 30ml of external water phase solution, and stirring by adopting a high-speed shearing machine under the conditions of stirring speed of 4000rpm, time of 3min and temperature of 5 ℃ to form water-in-oil-in-water multiple emulsion (hereinafter referred to as w/o/w multiple emulsion).
And 5, solidifying the w/o/w multiple emulsion to form microspheres, centrifugally washing and collecting the microspheres, and removing the organic solvent, the PVA aqueous solution and the diluent to obtain wet microspheres.
And 6, adding 10ml of the gel solution and 100mg of mannitol powder into wet microspheres, and uniformly stirring to obtain a microsphere gel solution.
And 7, freeze-drying the microsphere gel solution in a freeze dryer to obtain the pramipexole sustained-release preparation for injection.
The concentration of the pramipexole dihydrochloride in the inner water phase is 750mg/ml, the concentration of an inner water phase additive (namely a mixture formed by 20mg of poloxamer and 5mg of chitosan) in the inner water phase is 125mg/ml, wherein the inner water phase consists of 150mg of pramipexole dihydrochloride, the inner water phase additive formed by 20mg of poloxamer and 5mg of chitosan and 200ul of water for injection, and the pH value of the inner water phase is adjusted to 5 by adopting phosphoric acid.
The volume of the inner aqueous phase was 200 ul.
The concentration of the poloxamer 407 in the organic phase is 100mg/ml, and the concentration of the PLGA in the organic phase is 150mg/ml, wherein the organic phase consists of PLGA, an organic phase additive and an organic solvent.
The external water phase solution is obtained by adjusting the pH value of a 1% PVA aqueous solution (pre-saturated dichloromethane) to 9 by adopting a phosphate buffer solution, wherein the polymerization degree of PVA is 1700, and the alcoholysis degree is 88%.
The volume ratio of the inner aqueous phase and the organic phase of the w/o colostrum was 1/10.
The volume ratio of the organic phase and the external aqueous phase of the w/o colostrum in the step 4 is 1/6.
The curing temperature of the whole curing in the step 5 is 30 ℃, and the time is 6h and 6 min; and the primary solidification adopts solvent diffusion, the solvent diffusion adopts 30ml of 0.5% ethanol water solution with the pH value of 9, the 0.5% ethanol water solution is added into the w/o/w composite emulsion under the stirring state at the adding speed of 5ml/min, the water-in-oil-in-water composite emulsion is primarily solidified, and then the primarily solidified water-in-oil-in-water composite emulsion is further solidified. And the solvent evaporation is adopted in the next step of solidification, and the solvent evaporation is performed for 6 hours by adopting a constant-temperature water bath magnetic stirrer for magnetic stirring to remove the organic solvent dichloromethane.
The volume ratio of the external aqueous phase to the diluent was 1/1.
And 5, carrying out centrifugal washing for 10min each time by adopting equal volume of injection water and carrying out centrifugal washing for 3 times in a low-speed centrifuge with the rotating speed of 2000 rpm.
The pre-freezing temperature of the freezing in the step 7 is-50 ℃, the pre-freezing and heat preservation time is 6h, the temperature is increased to 30 ℃ in a gradient manner at 5 ℃ per hour under the vacuum degree of less than 10pa, the heat preservation is carried out for 12h at 30 ℃, and the total freeze drying time is 24 h.
The freeze drying in step 7 removes water, also including water for injection.
It will be appreciated that the water for injection, the organic solvent, the external aqueous phase solution and the diluent are all process additives, which mainly function to aid in the formation of the formulation and are therefore removed during the preparation of the pramipexole sustained release formulation for injection.
As can be seen from fig. 2, after the prepared pramipexole sustained release preparation for injection is redissolved, microspheres in the pramipexole sustained release preparation for injection are observed under an optical microscope, are spherical, are not damaged, have narrow particle size distribution, have an average particle size of 60 μm, are suitable for in vivo injection, and do not generate irritation in a rat experiment.
In this example 1, in order to further verify the advantageous effects of the present invention, the following test examples are provided.
1. The influence of the internal water phase additive on the particle size and the encapsulation efficiency of the microspheres in the pramipexole sustained-release preparation for injection is examined.
The additive of the internal water phase is a mixture of poloxamer and chitosan, wherein the mass ratio of poloxamer to chitosan is 2mg/0.5mg, 20mg/0.5mg, 2mg/5mg, 20mg/5mg and 40mg/10mg respectively. The results are shown in Table 1 and FIG. 3.
Table 1: the particle size and encapsulation efficiency of the microspheres under the condition of different mass ratios of the internal water phase additives.
Poloxamer/chitosan Particle size (. mu.m) Encapsulation efficiency (%)
2mg/0.5mg 43 55
20mg/0.5mg 51 69
2mg/5mg 49 68
20mg/5mg 58 78
40mg/10mg 66 80
As can be seen from table 1, the inner water phase additive has a significant effect on the particle size and the encapsulation efficiency of the microsphere, poloxamer and chitosan can increase the viscosity of the inner water phase and reduce the loss of the main drug from the inner water phase to the outer water phase, but poloxamer and chitosan increase the particle size of the microsphere when the dosage is increased. As can be seen from FIG. 3, when the mass ratio of poloxamer to chitosan was 2mg/0.5mg, the release was relatively rapid; when the mass ratio of the poloxamer to the chitosan is 40mg/10mg, the release is slow; when the mass ratios of poloxamer and chitosan are 20mg/0.5mg, 2mg/5mg and 20mg/5mg respectively, the release is in the middle, the release speeds are appropriate and are mutually overlapped, which shows that the controlled release effects of poloxamer and chitosan in the internal water phase additive can not be superposed.
Therefore, in this example 1, the mass ratio of poloxamer and chitosan in the internal water phase additive is determined to be 40mg/10mg, and preferably, the amount of poloxamer in the internal water phase additive is 20mg and the amount of chitosan in the internal water phase additive is 5 mg.
2. The influence of the organic phase additive on the particle size and the encapsulation rate of the microspheres in the pramipexole sustained-release preparation for injection is examined.
The organic phase additive is a mixture of poloxamer 407 and chitosan, wherein the mass ratio of poloxamer 407 to chitosan is 0g/0g, 0.5g/0g, 0g/0.1g, 0.5g/0.1g and 1g/0.2 g. The results are shown in Table 2 and FIG. 4.
Table 2: the particle size and encapsulation efficiency of the microspheres with different mass ratios of the organic phase additives.
Poloxamer 407/chitosan Particle size (. mu.m) Encapsulation efficiency (%)
0g/0g 37 50
0.2g/0g 57 60
0g/0.05g 55 57
0.2g/0.05g 59 80
0.4g/0.1g 70 79
As can be seen from table 2, the organic phase additive has significant influence on the particle size and encapsulation efficiency of the microsphere, and poloxamer 407 and chitosan can increase the organic viscosity and reduce the dissolution of the internal aqueous phase additive in the organic phase, thereby reducing the loss of the main drug from the internal aqueous phase to the external aqueous phase, but poloxamer 407 and chitosan greatly increase the particle size of the microsphere when the dosage is increased. As can be seen from fig. 4, when the mass ratio of poloxamer 407 to chitosan was 0.4g/0.1g, the burst release was small, and the release within 15 days was in a plateau phase, followed by an increase in release, and overall a slow release; when the mass ratio of poloxamer 407 to chitosan is 0.2g/0g, 0g/0.05g and 0.2g/0.05g respectively, the release degree is uniform, and the release is finished in 30 days; when the mass ratio of the poloxamer 407 to the chitosan is 0g/0g, burst release is large, early-stage release is rapid, and later-stage release is similar to that of the poloxamer 407 to the chitosan when the mass ratio of the poloxamer 407 to the chitosan is 0.2g/0g, 0g/0.05g and 0.2g/0.05g respectively, which shows that the addition of the organic phase additive poloxamer 407 and the chitosan can influence the burst release effect, but when the addition exceeds 0.4g/0.1g, complete release cannot be realized within 30 days.
Therefore, in this example 1, the mass ratio of poloxamer 407 to chitosan in the organic phase additive is determined to be 0.5mg/0.1mg, and preferably poloxamer 407 is used in an amount of 0.2g and chitosan is used in an amount of 0.05 g.
3. The influence of the PLGA concentration on the particle size and encapsulation efficiency of the microspheres in the pramipexole sustained-release preparation for injection is examined.
Here, the amounts of PLGA used were 0.25g, 0.5g, 0.75g, 1g and 2g, respectively. Wherein the molar ratio of lactide to glycolide in the PLGA is 75/25, and the molecular weight of the PLGA is 30 kDa.
Specifically, 0.25g, 0.5g, 0.75g, 1g, 2g of PLGA and the organic phase additive (i.e., poloxamer 407 in the organic phase additive is 0.5g and chitosan is 0.1g) were dissolved in 5ml of dichloromethane to form PLGA dichloromethane solutions with PLGA concentrations of 50mg/ml, 100mg/ml, 150mg/ml, 200mg/ml, 400 mg/ml. The results are shown in Table 3 and FIG. 5.
Table 3: microsphere particle size and encapsulation efficiency at different PLGA concentrations.
PLGA concentration Particle size (. mu.m) Encapsulation efficiency (%)
50mg/ml 49 45
100mg/ml 57 69
150mg/ml 60 77
200mg/ml 62 80
400mg/ml 80 81
As can be seen from Table 3, the concentration of PLGA has a significant effect on the particle size and encapsulation efficiency of the microspheres, and PLGA is the main carrier material of the microspheres, the amount of the PLGA is reduced, the particle size of the microspheres is reduced, the bulk density is reduced, and the porosity is increased, therefore, the encapsulation efficiency of the microspheres formed by PLGA with the concentration of 50mg/ml is very low. When the PLGA concentration was 400mg/ml, the microspheres increased in particle size without increasing encapsulation efficiency, but the microspheres decreased in moldability. As can be seen from fig. 5, the burst release rate and the release rate decrease with increasing PLGA dosage (or concentration), wherein PLGA concentrations of 100mg/ml, 150mg/ml and 200mg/ml respectively have similar PLGA dosage but different release curves, which indicates that the PLGA dosage greatly affects the release of the pramipexole sustained release injection.
Therefore, in order to comprehensively analyze the influence on the particle size and encapsulation efficiency and the release rate, the PLGA concentration of example 1 was determined to be 100mg/ml to 200mg/ml, i.e., the amount of PLGA was 0.5g to 1 g. Preferably, the amount of PLGA is 0.75 g.
4. The influence of the ethanol concentration of the diluent on the particle size and the encapsulation efficiency of the microspheres in the pramipexole sustained-release preparation for injection is examined.
Here, the diluent is an aqueous ethanol solution having a pH of 9, and the ethanol concentrations of the aqueous ethanol solution are 0%, 0.25%, 0.5%, and 1%, respectively. The results are shown in Table 4 and FIG. 6.
Table 4: the particle size and encapsulation efficiency of the microspheres under different ethanol concentrations of the diluent.
Ethanol concentration of the dilution Particle size (. mu.m) Encapsulation efficiency (%)
0％ 49 45
0.25％ 57 75
0.5％ 60 77
1％ 62 60
As can be seen from Table 4, the ethanol concentration of the diluent has significant influence on the particle size and encapsulation efficiency of the microspheres, the diluent is mainly used for pre-curing the w/o/w multiple emulsion microspheres, the pre-curing speed is a key factor influencing the leakage of the main drug, but the faster pre-curing speed influences the microsphere form, and the 1% ethanol diluent reduces the microsphere balling rate and deteriorates the form. As can be seen from fig. 6, when the ethanol concentration of the diluent is 1%, the 24-hour burst release and the release increase in the first 5 days are probably due to the large degree of pre-curing, which makes the surface of the microspheres large and numerous in pores; when the ethanol concentration of the diluent is 0%, the release speed after 10 days is increased relative to other three groups, and the precuring degree is low, so that the main drug is approximately diffused to the edge of the microsphere, the microsphere PLGA is degraded in the later release period, and the release is increased.
Therefore, in this example 1, the ethanol concentration of the dilution was determined to be 0.25% to 0.5%, in view of the effects of the particle size, the encapsulation efficiency and the release rate. Preferably, the diluent is 0.5% ethanol aqueous solution with pH value of 9.
5. The influence of the 'pH value gradient of the internal water phase and the diluent' on the particle size and the encapsulation rate of microspheres in the pramipexole sustained-release preparation for injection is examined.
Here, the pH ratios of the internal aqueous phase and the diluent are 4/10, 5/9, 6/8, 7/7, respectively. The results are shown in Table 5 and FIG. 7.
Table 5: the particle size and encapsulation efficiency of the microspheres under different pH value gradients.
Ratio of pH values Particle size (. mu.m) Encapsulation efficiency (%)
4/10 56 70
5/9 58 81
6/8 60 75
7/7 62 66
As can be seen from table 5, the pH gradient (ratio of pH values) of the internal aqueous phase and the diluent has a significant effect on the particle size and the encapsulation efficiency of the microspheres, the pH gradient is mainly used to prevent the leakage of the main drug phase of the internal aqueous phase from the external aqueous phase, but the higher pH gradient causes the water of the internal aqueous phase to drive the main drug to enter the external aqueous phase, resulting in a decrease in the encapsulation efficiency. As can be seen from fig. 7, when the pH ratio of the internal aqueous phase to the diluent is 4/10, the release of the main drug is very slow, probably because the hydroxide ions of the diluent diffuse into the internal aqueous phase during the solidification of the additive of the internal aqueous phase, which causes the hydroxide ions to become solid quickly and wrap the main drug, and during the in vitro release, the release of the main drug becomes very slow; when the pH ratio of the internal water phase to the diluent is 5/9, 6/8 and 7/7, the release of the main drug is finished in 30 days.
Therefore, in this example 1, the pH gradient of the internal aqueous phase and the diluent was determined to be 5/9 to 6/8 by comprehensively analyzing the influence on the particle size, the encapsulation efficiency and the release rate. Preferably, the ratio of the pH of the internal aqueous phase and the diluent is 5/9.
6. Subcutaneous administration test in rats.
The pramipexole sustained release preparations for injection containing 5mg, 10mg and 20mg of pramipexole dihydrochloride are respectively subcutaneously administered to rats and divided into S1 groups, S2 groups and S3 groups; carrying out subcutaneous administration on rats by using a pramipexole dihydrochloride microsphere injection containing 10mg of pramipexole dihydrochloride, and dividing the rats into S4 groups; pramipexole dihydrochloride injection containing 0.3mg of pramipexole dihydrochloride was subcutaneously administered to rats, and divided into S5 groups. The plasma concentration profile in rats is shown in FIG. 8.
As can be seen from fig. 8, the pramipexole dihydrochloride injection in the S5 group is rapidly eliminated, the pramipexole dihydrochloride microspheres in the S4 group have a severe burst release behavior, the pramipexole sustained release preparation for injection in the S1, S2 and S3 groups has a small burst release effect, the release of the main drug in vivo is smoother than that in the S4 group, which indicates that the burst release of the main drug can be reduced by wrapping the microspheres in gel, and the area under the blood concentration-time curve (AUC) in vivo is linearly related to the dose under different doses of administration in the S1, S2 and S3 groups, and the pramipexole dihydrochloride is a linear pharmacokinetic process in vivo.
Example 2.
The embodiment provides a pramipexole sustained-release preparation for injection, which consists of the following raw materials: 150mg of pramipexole hydrochloride, 25mg of internal water phase additive, 1.0g of PLGA, 0.25g of organic phase additive, 10ml of gel solution and 100mg of mannitol.
The inner water phase additive is a mixture of poloxamer and chitosan, wherein the mass ratio of poloxamer chitosan to chitosan is 20mg/5 mg.
The molar ratio of lactide to glycolide in the PLGA was 75/25, and the molecular weight of the PLGA was 50 kDa.
The organic phase additive is a mixture of poloxamer 407 and chitosan, wherein the mass ratio of poloxamer 407 to chitosan is 0.2g/0.05 g.
The gel solution was formed from 150mg/ml poloxamer 188 and 75mg/ml chitosan.
The embodiment also provides a preparation method of the pramipexole sustained-release preparation for injection, which comprises the following steps:
step 1, dissolving 150mg of pramipexole dihydrochloride and 25mg of internal water phase additive in 200ul of water for injection to form a pramipexole dihydrochloride water solution with the pramipexole concentration of 0.75g/ml, and adjusting the pH value to 5 by using phosphoric acid.
And 2, dissolving 1.0g of PLGA and 0.25g of organic phase additive in 5ml of dichloromethane to form a PLGA dichloromethane solution with the PLGA concentration of 200 mg/ml.
And 3, mixing the pramipexole dihydrochloride injection aqueous solution and the PLGA dichloromethane solution, and ultrasonically forming w/o colostrum by adopting an ultrasonic cell crusher under the conditions of ultrasonic power of 300w, ultrasonic time of 3min and temperature of 0 ℃.
And 4, adding the w/o colostrum into 30ml of external water phase solution, and stirring by adopting a high-speed shearing machine under the conditions of stirring speed of 6000rpm, time of 2min and temperature of 5 ℃ to form w/o/w multiple emulsion.
And 5, solidifying the w/o/w multiple emulsion to form microspheres, centrifugally washing and collecting the microspheres, and removing the organic solvent, the PVA aqueous solution and the diluent to obtain wet microspheres.
And 6, adding 10ml of the gel solution and 100mg of mannitol powder into wet microspheres, and uniformly stirring to obtain a microsphere gel solution.
And 7, freeze-drying the microsphere gel solution to obtain the pramipexole sustained-release preparation for injection.
The external water phase solution is obtained by adjusting the pH value of a 2% PVA aqueous solution to 9 by adopting a phosphate buffer solution, wherein the polymerization degree of PVA is 1700, and the alcoholysis degree is 88%.
The curing temperature of the whole curing in the step 5 is 30 ℃, and the time is 8h6 min; the primary curing adopts solvent diffusion. Wherein, solvent diffusion is carried out by adding 30ml of 0.25 percent ethanol water solution with the pH value of 9 into the w/o/w composite emulsion under the stirring state at the adding speed of 5ml/min, carrying out primary solidification on the water-in-oil-in-water composite emulsion, and then carrying out next solidification on the primarily solidified water-in-oil-in-water composite emulsion. The next step of solidification adopts solvent evaporation, and the solvent evaporation adopts a constant-temperature water bath magnetic stirrer to carry out magnetic stirring for 8 hours
And 5, carrying out centrifugal washing for 10min each time by adopting equal volume of injection water and carrying out centrifugal washing for 3 times in a low-speed centrifuge with the rotating speed of 2000 rpm.
The pre-freezing temperature of the freezing in the step 7 is-55 ℃, the pre-freezing and heat preservation time is 6h, the temperature is increased to 30 ℃ in a gradient manner at 5 ℃ per hour under the vacuum degree of less than 10pa, the heat preservation is carried out for 12h at 30 ℃, and the total freeze drying time is 24 h.
The freeze drying in step 7 removes water, also including water for injection.
Example 3.
The embodiment provides a pramipexole sustained-release preparation for injection, which consists of the following raw materials: 150mg of pramipexole hydrochloride, 25mg of internal water phase additive, 1.0g of PLGA, 0.25g of organic phase additive, 10ml of gel solution and 100mg of mannitol.
The inner water phase additive is a mixture of poloxamer and chitosan, wherein the mass ratio of the poloxamer to the chitosan is 20mg/5 mg.
The molar ratio of lactide to glycolide in the PLGA was 50/50, and the molecular weight of the PLGA was 60 kDa.
The organic phase additive is a mixture of poloxamer 407 and chitosan, wherein the mass ratio of poloxamer 407 to chitosan is 0.2g/0.05 g.
The gel solution was formed from 50mg/ml chitosan and 200mg/ml poloxamer 188.
The embodiment also provides a preparation method of the pramipexole sustained-release preparation for treating injection, which comprises the following steps:
step 1, dissolving 150mg of pramipexole dihydrochloride and 25mg of internal water phase additive in 200ul of water for injection to form a pramipexole dihydrochloride water solution with the pramipexole concentration of 0.75g/ml, and adjusting the pH value to 5 by using phosphoric acid.
And 2, dissolving 1.0g of PLGA and 0.25g of organic phase additive in 5ml of dichloromethane to form a PLGA dichloromethane solution with the PLGA concentration of 200 mg/ml.
And 3, mixing the pramipexole dihydrochloride injection aqueous solution and the PLGA dichloromethane solution, and ultrasonically forming w/o colostrum by adopting an ultrasonic cell crusher under the conditions of ultrasonic power of 300w, ultrasonic time of 4min and temperature of 0 ℃.
And 4, adding the w/o colostrum into 30ml of external water phase solution, and stirring by adopting a high-speed shearing machine under the conditions of a stirring speed of 5000rpm, a time of 3min and a temperature of 5 ℃ to form w/o/w multiple emulsion.
And 5, solidifying the w/o/w multiple emulsion to form microspheres, centrifugally washing and collecting the microspheres, and removing the organic solvent, the PVA aqueous solution and the diluent to obtain wet microspheres.
And 6, adding 10ml of the gel solution and 100mg of mannitol powder into wet microspheres, and uniformly stirring to obtain a microsphere gel solution.
And 7, freeze-drying the microsphere gel solution to obtain the pramipexole sustained-release preparation for injection.
The external water phase solution is obtained by adjusting the pH value of a 1% PVA aqueous solution to 9 by adopting a phosphate buffer solution, wherein the polymerization degree of PVA is 1700, and the alcoholysis degree is 88%.
The curing temperature of the whole curing in the step 5 is 30 ℃, and the time is 6h15 min; the primary curing adopts solvent diffusion. Wherein, solvent diffusion is adopted, 30ml of 0.25% ethanol water solution with the pH value of 9 is added into the w/o/w composite emulsion under the stirring state at the adding speed of 2ml/min, methylene dichloride is diffused, the water-in-oil-in-water composite emulsion is primarily solidified, and then the primarily solidified water-in-oil-in-water composite emulsion is further solidified. The next step of solidification adopts solvent evaporation, the solvent evaporation adopts a rotary evaporator, the conditions are that the rotary evaporation is carried out for 6 hours under the conditions that the temperature is 30 ℃ and the vacuum degree is 30Kpa, and dichloromethane is removed
And 5, carrying out centrifugal washing for 3 times by adopting equal volume of injection water in a low-speed centrifuge with the rotating speed of 2000rpm, wherein each time of centrifugal washing is 10min, and removing the PVA aqueous solution and the diluent.
The pre-freezing temperature of the freezing in the step 7 is-50 ℃, the pre-freezing and heat preservation time is 6h, the temperature is increased to 30 ℃ in a gradient manner at 5 ℃ per hour under the vacuum degree of less than 10pa, the heat preservation is carried out for 12h at 30 ℃, and the total freeze drying time is 24 h.
The freeze drying in step 7 removes water, also including water for injection.
Example 4.
The embodiment provides a pramipexole sustained-release preparation for injection for treating Parkinson's disease, which consists of the following raw materials: 180mg of pramipexole dihydrochloride, 40mg of an internal water phase additive, 1.0g of PLGA, 0.25g of an organic phase additive, 10ml of a gel solution and 100mg of mannitol.
The inner water phase additive is a mixture of carbomer and span 80, wherein the span 80 is 1mg, and the span is used as a surfactant.
The molar ratio of lactide to glycolide in the PLGA was 75/25, and the molecular weight of the PLGA was 50 kDa.
The organic phase additive is a mixture of poloxamer 407 and chitosan, wherein the mass ratio of poloxamer 407 to chitosan is 0.2g/0.05 g.
The gel solution was formed from 75mg/ml chitosan and 150mg/ml poloxamer 188.
The embodiment also provides a preparation method of the pramipexole sustained-release preparation for injection, which comprises the following steps:
step 1, dissolving 180mg of pramipexole dihydrochloride and 40mg of internal water phase additive in 300ul of water for injection to form a pramipexole dihydrochloride water solution with the pramipexole concentration of 0.6g/ml, and adjusting the pH value to 5 by using phosphoric acid.
And 2, dissolving 1.0g of PLGA and 0.25g of organic phase additive in 5ml of dichloromethane to form a PLGA dichloromethane solution with the PLGA concentration of 200 mg/ml.
And 3, mixing the pramipexole dihydrochloride injection aqueous solution and the PLGA dichloromethane solution, and ultrasonically forming w/o colostrum by adopting an ultrasonic cell crusher under the conditions of ultrasonic power of 300w, ultrasonic time of 3min and temperature of 0 ℃.
And 4, adding the w/o colostrum into 30ml of external water phase solution, and stirring by adopting a high-speed shearing machine under the conditions of a stirring speed of 5000rpm, a time of 3min and a temperature of 5 ℃ to form w/o/w multiple emulsion.
And 5, solidifying the w/o/w multiple emulsion to form microspheres, centrifugally washing and collecting the microspheres, and removing the organic solvent, the PVA aqueous solution and the diluent to obtain wet microspheres.
And 6, adding 10ml of the gel solution and 100mg of mannitol powder into wet microspheres, and uniformly stirring to obtain a microsphere gel solution.
And 7, freeze-drying the microsphere gel solution to obtain the pramipexole sustained-release preparation for injection.
The external water phase solution is obtained by adjusting the pH value of a 2% PVA aqueous solution to 9 by adopting a phosphate buffer solution, wherein the polymerization degree of PVA is 1700, and the alcoholysis degree is 88%.
The curing temperature of the whole curing in the step 5 is 15 ℃, and the time is 6h6 min. And the primary curing adopts solvent diffusion, wherein the solvent diffusion adopts 0.25% ethanol water solution with the pH value of 9 and 30ml, and the ethanol water solution is added into the w/o/w composite emulsion under the stirring state at the adding speed of 5ml/min, so that dichloromethane is diffused, the w/o/w composite emulsion primary curing is carried out, and then the water-in-oil-in-water composite emulsion of the primary curing is carried out the next curing. The next step of solidification adopts solvent evaporation, the solvent evaporation adopts a rotary evaporator, the rotary evaporation is carried out for 6 hours under the conditions that the temperature is 15 ℃ and the vacuum degree is 30Kpa, and dichloromethane is removed.
And 5, carrying out centrifugal washing for 3 times by adopting equal volume of injection water in a low-speed centrifuge with the rotating speed of 2000rpm, wherein each time of centrifugal washing is 10min, and removing the PVA aqueous solution and the diluent.
The pre-freezing temperature of the freezing in the step 7 is-55 ℃, the pre-freezing and heat preservation time is 6h, the temperature is increased to 30 ℃ in a gradient manner at 5 ℃ per hour under the vacuum degree of less than 10pa, the heat preservation is carried out for 12h at 30 ℃, and the total freeze drying time is 24 h.
The freeze drying in step 7 removes water, also including water for injection.
Example 5.
The embodiment provides a pramipexole sustained-release preparation for injection, which consists of the following raw materials: 180mg of pramipexole dihydrochloride, 25mg of an internal water phase additive, 1.0g of PLGA, 0.25g of an organic phase additive, 10ml of a gel solution and 100mg of mannitol.
The inner water phase additive is a mixture of poloxamer and chitosan, wherein the mass ratio of poloxamer chitosan to chitosan is 20mg/5 mg.
The molar ratio of lactide to glycolide in the PLGA was 75/25, and the molecular weight of the PLGA was 50 kDa.
The organic phase additive is a mixture of poloxamer 407 and chitosan, wherein the mass ratio of poloxamer 407 to chitosan is 0.2g/0.05 g.
The gel solution was formed from 75mg/ml chitosan and 150mg/ml poloxamer 188.
The embodiment also provides a preparation method of the pramipexole sustained-release preparation for injection, which comprises the following steps:
step 1, dissolving 180mg of pramipexole dihydrochloride and 25mg of internal water phase additive in 300ul of water for injection to form a pramipexole dihydrochloride water solution with the pramipexole concentration of 0.6g/ml, and adjusting the pH value to 5 by using phosphoric acid.
And 2, dissolving 1.0g of PLGA and 0.25g of organic phase additive in 5ml of organic solvent to form a PLGA ethyl acetate and acetone mixture solution with the PLGA concentration of 200mg/ml, wherein the organic solvent is a mixture of ethyl acetate and acetone in a volume ratio of 1: 1.
And 3, mixing the pramipexole dihydrochloride injection aqueous solution and the PLGA ethyl acetate and acetone mixture solution, and ultrasonically forming w/o colostrum by adopting an ultrasonic cell crusher under the conditions of ultrasonic power of 300w, ultrasonic time of 3min and temperature of 0 ℃.
And 4, adding the w/o colostrum into 30ml of external water phase solution, and stirring by adopting a high-speed shearing machine under the conditions of stirring speed of 4000rpm, time of 3min and temperature of 5 ℃ to form w/o/w multiple emulsion.
And 5, solidifying the w/o/w multiple emulsion to form microspheres, centrifugally washing and collecting the microspheres, and removing the organic solvent, the PVA aqueous solution and the diluent to obtain wet microspheres.
And 6, adding 10ml of the gel solution and 100mg of mannitol powder into wet microspheres, and uniformly stirring to obtain a microsphere gel solution.
And 7, freeze-drying the microsphere gel solution to obtain the pramipexole sustained-release preparation for injection.
The external water phase solution is obtained by adjusting the pH value of a 2% PVA aqueous solution to 9 by adopting a phosphate buffer solution, wherein the polymerization degree of PVA is 1700, and the alcoholysis degree is 88%.
The curing temperature of the whole curing in the step 5 is 15 ℃, and the time is 8h6 min. And the primary curing adopts solvent diffusion, wherein the solvent diffusion adopts 0.25% ethanol water solution with the pH value of 9 and 30ml, and the ethanol water solution is added into the w/o/w composite emulsion under the stirring state at the adding speed of 5ml/min, so that dichloromethane is diffused, the w/o/w composite emulsion primary curing is carried out, and then the water-in-oil-in-water composite emulsion of the primary curing is carried out the next curing. And the next step of solidification adopts solvent evaporation, the solvent evaporation adopts a rotary evaporator, the rotary evaporation is carried out for 8 hours under the conditions that the temperature is 15 ℃ and the vacuum degree is 20Kpa, and ethyl acetate and acetone are removed.
And 5, carrying out centrifugal washing for 3 times by adopting equal volume of injection water in a low-speed centrifuge with the rotating speed of 2000rpm, wherein each time of centrifugal washing is 10min, and removing the PVA aqueous solution and the diluent.
The pre-freezing temperature of the freezing in the step 7 is-55 ℃, the pre-freezing and heat preservation time is 6h, the temperature is increased to 30 ℃ in a gradient manner at 5 ℃ per hour under the vacuum degree of less than 10pa, the heat preservation is carried out for 12h at 30 ℃, and the total freeze drying time is 24 h.
The freeze drying in step 7 removes water, also including water for injection.
It should be understood that equivalents and modifications of the technical solution and inventive concept thereof may occur to those skilled in the art, and all such modifications and alterations should fall within the scope of the appended claims.

Claims (4)

1. The pramipexole sustained-release preparation for injection is characterized by comprising the following raw materials: 100 mg-500 mg of main drug, 10 mg-50 mg of internal water phase additive, 0.5 g-2.5 g of PLGA, 0.1 g-0.5 g of organic phase additive, 5 ml-20 ml of gel solution and 40 mg-200 mg of freeze-drying protective agent; the inner water phase additive is a mixture of poloxamer and chitosan, and the mass ratio of the poloxamer to the chitosan is (2-6): 1; the organic phase additive is a mixture of poloxamer 407 and chitosan, and the mass ratio of the poloxamer 407 to the chitosan is 4: 1;
the main drug is pramipexole dihydrochloride;
the molar ratio of lactide to glycolide in the PLGA is 75/25, and the concentration of the PLGA is 100-200 mg/ml;
the gel solution is formed by 200mg/ml poloxamer 188 and 20mg/ml chitosan, and the pramipexole sustained-release preparation for injection is prepared by the following method:
step 1, dissolving a main drug and an internal water phase additive in water for injection to form a drug-containing internal water phase solution;
step 2, dissolving PLGA and an organic phase additive in an organic solvent to form an organic phase solution;
step 3, mixing the drug-containing internal water phase solution and the organic phase solution, and ultrasonically forming water-in-oil colostrum;
step 4, adding the water-in-oil primary emulsion into an external water phase solution, and stirring to form a water-in-oil-in-water multiple emulsion;
step 5, solidifying the water-in-oil-in-water double emulsion to form microspheres, and centrifugally washing and collecting the microspheres to obtain wet microspheres;
step 6, adding a gel solution and a freeze-drying protective agent into the wet microspheres to obtain a microsphere gel solution;
step 7, freeze-drying the microsphere gel solution to obtain a pramipexole sustained-release preparation for injection;
in the step 3, the volume ratio of the internal aqueous phase to the organic phase is 1/10-1/30;
in the step 4, the external water phase solution is obtained by adjusting the pH value of a 1-2% PVA aqueous solution to 9 by adopting a phosphate buffer solution;
in the step 5, the solidification is to preliminarily solidify the water-in-oil-in-water composite emulsion, then carry out the next step of solidification on the preliminarily solidified water-in-oil-in-water composite emulsion, wherein the preliminary solidification adopts organic solvent diffusion, the organic solvent diffusion adopts 0.5-5 times of ethanol aqueous solution with the pH value of 9, the concentration of the ethanol aqueous solution is 0.25-0.5%, the next step of solidification adopts solvent evaporation, and the solvent evaporation adopts a constant temperature water bath magnetic stirrer for magnetic stirring;
in the step 6, the freeze-drying protective agent is mannitol.
2. The pramipexole sustained-release preparation for injection according to claim 1, wherein the PLGA has a molecular weight of 15kDa to 60kDa and an intrinsic viscosity of 0.18dl/g to 0.55 dl/g.
3. The pramipexole dihydrochloride sustained-release preparation for injection according to claim 1, wherein the pramipexole dihydrochloride sustained-release preparation for injection has a drug loading rate of more than 10%, an entrapment rate of more than 60% and a phase transition temperature of 32-40 ℃.
4. The pramipexole sustained-release preparation for injection according to claim 1, wherein the organic solvent is one or more of dichloromethane, ethyl acetate and acetone.
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