CN109939220B - Polypeptide microsphere with quick-release and slow-release effects and preparation method thereof - Google Patents
Polypeptide microsphere with quick-release and slow-release effects and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a preparation method of polypeptide microspheres with quick-release and slow-release effects, which comprises the following steps: co-lyophilizing water-soluble polypeptide and surfactant to obtain powdery co-lyophilized extract, and dispersing the co-lyophilized extract in organic solvent to obtain oil phase solution, wherein the organic solvent is dissolved with high molecular polymer containing hydrophobic chain segment; and uniformly mixing the oil phase solution and a poor solvent of the high molecular polymer to form polypeptide microspheres wrapped with the high molecular polymer, and then adding the mixture into a curing agent to completely cure the polypeptide microspheres to obtain the polypeptide microspheres with quick-release and slow-release effects. The invention adopts a phase separation method to prepare the polypeptide microspheres, avoids the use of polar solvents, effectively improves the safety, and simultaneously has quick release and slow release effects.
Description
Technical Field
The invention relates to the field of pharmaceutical preparations, in particular to a polypeptide microsphere with quick-release and slow-release effects and a preparation method thereof.
Background
Polypeptide protein drugs generally need to be frequently injected for a long time due to the stability, half-life and the like, so that the compliance of patients is poor. In recent years, sustained-release preparations are developed rapidly, and the development of sustained-release long-acting injection preparations of polypeptide protein medicines is also advanced to a certain extent, and the sustained-release long-acting injection preparations mainly comprise two main types, namely implants and microsphere injection preparations.
The preparation of the protein polypeptide medicament into the microspheres can achieve the effects of taste masking, improvement of the stability of the protein polypeptide medicament, targeting of the preparation, prolongation of the action time of the medicament and further improvement of the curative effect. The microsphere preparation method generally comprises an emulsion solvent volatilization method, a phase separation method, a hot-melt extrusion method and a spray drying method.
The hot-melt extrusion method is less used for preparing microspheres of unstable drugs such as protein polypeptide due to higher requirements on temperature, and in addition, the spray drying method is not suitable for protein polypeptide drugs with higher values due to low yield and higher loss. The multiple emulsion solvent volatilization method is a common method for preparing protein polypeptide drug microspheres, however, the problems of low drug loading rate and encapsulation efficiency and high burst release are the most common problems of the method, for example, the burst release effect of triptorelin acetate microspheres in Chinese invention patent CN104010629 is too large, which brings about many side effects. The application of the phase separation method can effectively improve the drug loading rate and the encapsulation efficiency, but generally, the dispersion condition of the water-soluble protein polypeptide drug can obviously influence the drug loading rate, the encapsulation efficiency and the release condition of the obtained microspheres.
Chinese patent CN201811434326 discloses a polypeptide microsphere and a preparation method thereof, wherein the polypeptide microsphere is prepared by a phase separation method, but polar solvents such as glacial acetic acid and dimethyl sulfoxide are adopted in the process to dissolve protein polypeptide drugs. According to the specification of the Chinese pharmacopoeia of the 2015 edition, glacial acetic acid (acetic acid) and dimethyl sulfoxide are all third solvents, and the safety of the glacial acetic acid (acetic acid) and the dimethyl sulfoxide is proved by detecting residual solvents, so that a great safety problem exists. In addition, the release profile shown in this patent is too low in the initial stage and slow in onset after injection.
Disclosure of Invention
In order to solve the technical problems, the invention aims to provide a polypeptide microsphere with quick release and slow release effects and a preparation method thereof.
In order to achieve the purpose, the invention provides the following technical scheme:
the first purpose of the invention is to provide a preparation method of polypeptide microspheres with quick release and slow release effects, which comprises the following steps:
(1) Co-lyophilizing water-soluble polypeptide and surfactant to obtain powdery co-lyophilized extract, and dispersing the co-lyophilized extract in organic solvent to obtain oil phase solution, wherein the organic solvent is dissolved with high molecular polymer containing hydrophobic chain segment;
(2) And uniformly mixing the oil phase solution and a poor solvent of the high molecular polymer to form polypeptide microspheres wrapped by the high molecular polymer, and then adding the mixture into a curing agent to completely cure the polypeptide microspheres to obtain the polypeptide microspheres with quick-release and slow-release effects.
Further, in the step (1), the water-soluble polypeptide is selected from one or more of triptorelin or a salt thereof, goserelin or a salt thereof, leuprorelin or a salt thereof, buserelin or a salt thereof, octreotide or a salt thereof, exenatide or a salt thereof, lanreotide or a salt thereof, liraglutide or a salt thereof, thymopentin and growth hormone.
Preferably, the water-soluble polypeptide is selected from triptorelin or a salt thereof. More preferably, the water-soluble polypeptide is triptorelin acetate.
Further, in the step (1), the surfactant is one or more selected from the group consisting of a polyol-type copolymer, a polyoxyethylene-type copolymer, and a polyoxyethylene-polyoxypropylene copolymer.
Preferably, the surfactant is selected from one or more of polysorbate, maize, brij and poloxamer.
More preferably, the surfactant is selected from polysorbates, such as tween 80 or tween 20.
Further, in the step (1), in the freeze-drying process, the cold trap temperature of a freeze-drying machine is-50 ℃ to-60 ℃, and the freeze-drying pressure is 0.01 mbar to 0.52mbar. The water-soluble polypeptide and the surfactant are co-lyophilized, so that the specific surface area of the polypeptide can be increased, and the obtained microspheres have good shapes.
Further, in the step (1), the mass ratio of the water-soluble polypeptide, the surfactant and the high molecular polymer is 1.
Preferably, the mass ratio of the water-soluble polypeptide to the surfactant is 1. When the amount of the surfactant is too high, the microspheres are relatively more viscous in the preparation process, the obtained microspheres are more adhered, and the particle size is measured to be increased due to agglomeration, so that the microspheres are poor in shape.
Further, in the step (1), the organic solvent is one or more selected from dichloromethane, ethyl acetate, acetone and methyl ethyl ketone.
Preferably, the organic solvent is selected from dichloromethane or ethyl acetate.
Further, in the step (1), the high molecular polymer is selected from one or more of lactide-glycolide copolymer, polylactic acid (PLA), polylactic-polyethylene glycol (PLGA) and polycaprolactone.
Preferably, the high molecular polymer is selected from lactide-glycolide copolymer or polylactic acid. The monomer proportion of the lactide-glycolide copolymer is 50-85, and the molecular weight of the lactide-glycolide copolymer is 10000-60000 g/mol.
Further, in the step (2), the poor solvent is selected from dimethylsilicone oil and/or liquid paraffin.
Further, in the step (2), the oil phase solution and the poor solvent of the high molecular polymer are mixed uniformly at a rotation speed of 400 to 1200 rpm.
Further, in step (2), the ratio of the oil phase solution to the poor solvent is 2.
Further, in the step (2), the curing agent is selected from one or more of n-heptane, n-octane and n-hexane.
Further, in the step (2), the mixture is added into a curing agent at a rotation speed of 200-600 rpm for curing.
The second purpose of the invention is to provide a polypeptide microsphere with quick release and slow release effects, which is prepared by the preparation method and is characterized in that: the polypeptide microsphere comprises water-soluble polypeptide and a high polymer coated on the surface of the water-soluble polypeptide, wherein the high polymer contains a hydrophobic chain segment, the median diameter of the particle diameter of the polypeptide microsphere is 40-100 mu m, and the release rate of the water-soluble polypeptide in the polypeptide microsphere is quick release at the early stage and slow release at the later stage.
By the scheme, the invention at least has the following advantages:
the invention co-freezes the water-soluble polypeptide and the surface active agent, the co-freezed matter is directly dispersed in the oil phase solution containing the high molecular polymer, and the preparation of the polypeptide microsphere is carried out by adopting a phase separation method. Firstly, a polar solvent is not adopted to dissolve the water-soluble polypeptide, so that the safety of the preparation is improved; secondly, the reasonable surfactant is adopted to be co-lyophilized with the water-soluble polypeptide in a proper mass ratio, so that the microspheres have good shapes, are not adhered and are not agglomerated. The invention adopts a method of co-freeze-drying water-soluble polypeptide and a surfactant and combines a phase separation method to prepare the microspheres, thereby well solving the problem of curative effect caused by over-high or over-low burst release in the release of the microspheres.
The polypeptide microsphere prepared by the method can effectively improve the release of the polypeptide, and the early stage of the release of the polypeptide microsphere is a quick release stage to achieve the drug effect, and the later stage is a slow release stage to maintain the blood concentration.
The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following detailed description is given with reference to the preferred embodiments of the present invention and the accompanying drawings.
Drawings
FIG. 1 is a scanning electron micrograph (800X) of triptorelin acetate sustained release microspheres prepared in example 1;
FIG. 2 is an in vitro release profile of triptorelin acetate sustained release microspheres prepared in example 1;
FIG. 3 is the in vitro release curve of triptorelin acetate sustained release microspheres prepared in example 2;
FIG. 4 is an in vitro release profile of triptorelin acetate sustained release microspheres prepared in example 4.
Detailed Description
The following detailed description of the present invention is provided in connection with the accompanying drawings and examples. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Example 1
Weighing 15mg triptorelin acetate and 2mg tween 80, and freeze-drying at-50 deg.C and 0.52mbar. Weighing 500mg of PLGA, dissolving the PLGA in 20g of dichloromethane, directly dispersing the obtained freeze-dried substance in a dichloromethane solution of the PLGA, and stirring and adding 12.5g of simethicone at the rotating speed of 1000rpm after ultrasonic dispersion so that the PLGA is coated on the surface of the triptorelin acetate. The resulting mixture was then added to a 750mL n-heptane bath and cured at 400rpm for 30min. Standing, removing the upper layer of clarified n-heptane after the suspended matter settles to the bottom, washing with petroleum ether for three times, drying, and collecting the polypeptide microspheres.
Example 2
Weighing 15mg triptorelin acetate and 4mg tween 80, and lyophilizing at-50 deg.C and 0.52mbar. 500mg of PLGA was weighed and dissolved in 20g of methylene chloride, and the lyophilizate was directly dispersed in a methylene chloride solution of PLGA, after ultrasonic dispersion, stirred at 1000rpm and 12.5g of dimethicone was added, and the resulting mixture was added to a 750mL n-heptane bath, solidified at 400rpm for 30min. Standing, removing the upper layer of clarified n-heptane after the suspended matter settles to the bottom, washing with petroleum ether for three times, drying, and collecting the polypeptide microspheres.
Example 3
Weighing 40mg of exenatide and 2mg of Tween 20, and lyophilizing at-60 deg.C and 0.37mbar. 400mg of PLA was weighed and dissolved in 20g of ethyl acetate, and the lyophilizate was directly dispersed in an ethyl acetate solution of PLA, after ultrasonic dispersion, the mixture was stirred at 1000rpm and 12.5g of dimethylsilicone oil was added, and then the mixture was added to 750mL of an n-heptane bath, solidified at 400rpm and solidified for 30min. Standing, removing the upper layer of clarified n-heptane after the suspended matter settles to the bottom, washing with petroleum ether for three times, drying, and collecting the polypeptide microspheres.
Example 4
This example is given as a comparative example: weighing 500mg of PLGA to dissolve in 20g of dichloromethane, weighing 15mg of triptorelin acetate to directly disperse in a dichloromethane solution of PLGA, stirring at the rotation speed of 1000rpm and adding 12.5g of dimethyl silicone oil after ultrasonic dispersion, then adding the obtained mixture into 750mL of n-heptane bath, solidifying at the rotation speed of 400rpm and solidifying for 30min. Standing, removing the upper layer of clarified n-heptane after the suspended matters settle, washing with petroleum ether for three times, drying, and collecting the polypeptide microspheres.
Test example 1 microsphere particle size test
An appropriate amount of the microspheres prepared in examples 1 to 4 was divided into three portions, each portion was suspended in an appropriate amount of 0.1% tween 80 aqueous solution, each portion was subjected to ultrasonic treatment for 30 seconds, and the particle size of the microspheres was measured by a laser particle size distribution analyzer, and the results are shown in table 1, where the particle size (in particular, median size) of the microspheres was in the range of 40 to 100 μm, and the microspheres were suitable for injection.
TABLE 1 particle size of microspheres prepared in different examples
| Examples | Mean median diameter. + -. |
| 1 | 92.17±0.61 |
| 2 | 92.25±0.61 |
| 3 | 70.18±5.07 |
| 4 | 69.48±0.64 |
Test example 2 microsphere morphology testing
An appropriate amount of the microspheres prepared in example 1 were taken and the morphology of the microspheres was determined by scanning electron microscopy, the results of which are shown in FIG. 1. The results show that when the polypeptide and tween 80 were co-lyophilized in the mass ratio of example 1, the prepared microspheres were round.
Test example 3 microsphere drug loading and encapsulation efficiency test
30mg of triptorelin acetate microspheres prepared in examples 1-4 are weighed respectively, 3mL of ultrapure water is added, shaking is carried out for 1min, centrifugation (6000rpm, 10min) is carried out, supernate is collected, the steps are repeated for 3 times, the supernate is combined and centrifuged, the supernate is taken, HPLC analysis is carried out, and the amount of free medicine can be obtained.
Adding 1mL of dichloromethane into the residue, adding 4mL of ultrapure water, shaking for 1min, centrifuging, taking the water layer (upper layer), repeating the operation for 4 times, combining the water layers, centrifuging, taking the supernatant, and analyzing by HPLC to obtain the drug-loading amount. The results are shown in Table 2.
Wherein, the calculation formula of the drug loading rate and the encapsulation efficiency is as follows:
TABLE 2 drug loading and encapsulation efficiency of microspheres prepared in different examples
| Examples | Drug loading (%) | Encapsulation efficiency (%) |
| 1 | 2.19 | 74.87 |
| 2 | 1.64 | 56.21 |
| 3 | 5.30 | 57.89 |
| 4 | 0.78 | 26.48 |
Test example 4 in vitro release Curve testing of microspheres
Weighing 79mL of 0.1mol/L NaOH solution according to the specification of pharmacopoeia, adding 1.36g of KH 2 PO 4 Adding water to dilute to 200mL to obtain phosphate neutral buffer solution with pH 7.4, and adding 40 μ L of Tween 80 to obtain PBS-Tween 80 solution with Tween 80 concentration of 0.2%.
Weighing about 30mg of triptorelin acetate sustained-release microspheres prepared in examples 1, 2 and 4, respectively suspending the triptorelin acetate sustained-release microspheres with 3mL of PBS-Tween 80 solution, sampling in a constant-temperature 37 ℃ shaking table (100 rpm) for 4h, 1d, 3d, 7d, 14d, 21d, 28d, 35d, 42d, 49d and 56d respectively, using the supernatant for HPLC analysis, resuspending the remaining microspheres with a fresh medium and continuing to put out samples, calculating the cumulative release percentage, and drawing a release curve graph of the cumulative release percentage-time, wherein the results are shown in FIGS. 2, 3 and 4, and the results can be seen from the graph that the co-freeze-drying is carried out by adopting water-soluble polypeptide and surfactant, so that a release curve with both quick release and sustained release can be obtained, wherein the early stage is quick release and the later stage is sustained release; whereas the microspheres prepared without prior lyophilization (example 4) had only an immediate release phase. As can be seen from a comparison of FIGS. 3 and 2, when the ratio of the surfactant co-lyophilized with the water-soluble polypeptide was increased, there was a significant release-promoting effect during the release stage of PLGA erosion.
In the invention, when the polypeptide microspheres with quick release and slow release effects are prepared, other polypeptides can be selected from the water-soluble polypeptides, such as one or more of goserelin or salts thereof, leuprorelin or salts thereof, buserelin or salts thereof, octreotide or salts thereof, exenatide or salts thereof, lanreotide or salts thereof, liraglutide or salts thereof, thymopentin and growth hormone. The surfactant can also be one or more of maize, beneze and poloxamer.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and it should be noted that, for those skilled in the art, many modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.
Claims (1)
1. A preparation method of polypeptide microspheres with quick release and slow release effects is characterized by comprising the following steps:
(1) Co-lyophilizing water-soluble polypeptide and polysorbate to obtain powdery co-lyophilized matter, and then dispersing the co-lyophilized matter in an organic solvent to obtain an oil phase solution, wherein a high molecular polymer containing a hydrophobic chain segment is dissolved in the organic solvent; the mass ratio of the water-soluble polypeptide to the polysorbate to the high polymer is 1;
(2) Uniformly mixing the oil phase solution and a poor solvent of a high molecular polymer to form polypeptide microspheres wrapped by the high molecular polymer, and then adding the mixture into a curing agent to completely cure the polypeptide microspheres to obtain the polypeptide microspheres with quick-release and slow-release effects;
in the step (1), the water-soluble polypeptide is selected from one or more of triptorelin or a salt thereof, goserelin or a salt thereof, leuprorelin or a salt thereof, buserelin or a salt thereof, octreotide or a salt thereof, exenatide or a salt thereof, lanreotide or a salt thereof, liraglutide or a salt thereof, thymopentin and growth hormone; the organic solvent is selected from one or more of dichloromethane, ethyl acetate, acetone and methyl ethyl ketone; the high molecular polymer is selected from one or more of lactide-glycolide copolymer, polylactic acid-polyethylene glycol and polycaprolactone; in the freeze-drying process, the temperature of a cold trap of an adopted freeze-drying machine is between 50 ℃ below zero and 60 ℃ below zero, and the freeze-drying pressure is 0.01 to 0.52 mbar;
in the step (2), the poor solvent is selected from dimethyl silicone oil and/or liquid paraffin; the curing agent is selected from one or more of n-heptane, n-octane and n-hexane.
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| CN1575792A (en) * | 2003-07-25 | 2005-02-09 | 复旦大学 | Method for stablizing protein medicine and its application in microball preparation |
| CN102688198A (en) * | 2012-06-19 | 2012-09-26 | 广州帝奇医药技术有限公司 | Polypeptide drug sustained-release microsphere preparation and preparation method thereof |
| CN103585114A (en) * | 2013-11-25 | 2014-02-19 | 深圳翰宇药业股份有限公司 | Improved method for preparing exenatide sustained release microspheres |
| CN109432397A (en) * | 2018-11-28 | 2019-03-08 | 苏州天马医药集团天吉生物制药有限公司 | Polypeptide microballoon and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1575792A (en) * | 2003-07-25 | 2005-02-09 | 复旦大学 | Method for stablizing protein medicine and its application in microball preparation |
| CN102688198A (en) * | 2012-06-19 | 2012-09-26 | 广州帝奇医药技术有限公司 | Polypeptide drug sustained-release microsphere preparation and preparation method thereof |
| CN103585114A (en) * | 2013-11-25 | 2014-02-19 | 深圳翰宇药业股份有限公司 | Improved method for preparing exenatide sustained release microspheres |
| CN109432397A (en) * | 2018-11-28 | 2019-03-08 | 苏州天马医药集团天吉生物制药有限公司 | Polypeptide microballoon and preparation method thereof |
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