Combined preparation of parathyroid hormone analogue drug-loaded core-shell microspheres and method for preparing parathyroid hormone analogue drug-loaded core-shell microspheres

Abstract

The invention provides a combined preparation of parathyroid hormone analogue drug loaded core-shell microspheres and a method for preparing the parathyroid hormone analogue drug loaded core-shell microspheres. The parathyroid hormone analogue drug-loaded core-shell microsphere combined preparation comprises a first core-shell microsphere, a second core-shell microsphere and a third core-shell microsphere. The ratio of the first core-shell type microsphere to the second core-shell type microsphere to the third core-shell type microsphere is 1: 1: 1, and the drug release time of the first core-shell type microsphere, the drug release time of the second core-shell type microsphere and the drug release time of the third core-shell type microsphere are different. The first core-shell type microsphere is prepared from the following components in percentage by weight: 4.76 to 16.67 percent of parathyroid hormone analogue medicine and 83.33 to 95.24 percent of high-molecular polymer material. And the second core-shell type microsphere comprises the following components in percentage by weight: 4.76%-16.67% of parathyroid hormone analogue medicine and 83.33%-95.24% of high-molecular polymer material, wherein the parathyroid hormone analogue medicine accounts for 4.76%-16.67%, and the high-molecular polymer material accounts for 83.33%-95.24%. And the third core-shell type microsphere comprises the following components in percentage by weight: 4.76%-16.67% of parathyroid hormone analogue medicine and 83.33%-95.24% of high-molecular polymer material, wherein the parathyroid hormone analogue medicine accounts for 4.76%-16.67%, and the high-molecular polymer material accounts for 83.33%-95.24%.

Description

Technical Field

[0001] The present invention relates to the technical field of drug controlled release materials, and particularly relates to a combined preparation of a core-shell microsphere loaded with a parathyroid hormone analogue drug and a method for preparing the core-shell microsphere loaded with the parathyroid hormone analogue drug. Background Art

[0002] Osteoporosis is a bone metabolism disease caused by the imbalance between bone resorption and formation after the body functions decline with the increase of age, resulting in increased bone fragility. Osteoporosis is divided into postmenopausal female osteoporosis and senile osteoporosis. Fractures in different parts caused by osteoporosis will seriously affect the survival rate of the elderly. Given the huge population base and the increasing aging trend in China, developing drugs for the prevention and treatment of osteoporosis has great market demand and practical significance.

[0003] Parathyroid hormone (PTH), as a key peptide hormone regulating calcium and phosphorus metabolism and bone turnover, can regulate the synthesis and catabolism of bone, and plays an important role in the differentiation, maturation and apoptosis of osteoblasts and osteoclasts.

[0004] Parathyroid hormone analogue (PTHa) is a class of synthetic polypeptide drugs based on the structure of parathyroid hormone. The commonly used parathyroid hormone analogue at present is teriparatide. Compared with parathyroid hormone, teriparatide is the first 34 amino acid fragments (rhPTH 1-34) with biological activity at the amino terminus of parathyroid hormone.

[0005] Based on the action characteristics of parathyroid hormone analogue, it is mainly used to treat osteoporosis-related diseases clinically. However, parathyroid hormone analogue has a very unique pharmacokinetic mechanism. Only intermittent use of a small dose of parathyroid hormone analogue can stimulate the activity of osteoblasts, promote bone formation, increase bone density and improve bone quality. Specifically, only when the blood drug concentration of teriparatide is lower than the endogenous parathyroid hormone concentration for less than 4 hours after a single administration, can it play a role in promoting bone formation and then treat osteoporosis. On the contrary, when the blood drug concentration of teriparatide is higher than the endogenous parathyroid hormone concentration for more than 4 hours after a single administration, it will instead show certain side effects, promote the activity of osteoclasts, and then exacerbate osteoporosis.

[0006] Currently, the only teriparatide product on the market is an injection, which requires daily subcutaneous injection to ensure the therapeutic effect. However, frequent injections cause great pain to elderly patients and reduce patient compliance. Although the scientific research field has conducted research on long-acting parathyroid hormone analog drug preparations, such as controlling drug release by preparing sustained-release microspheres, these preparations can effectively control burst release, but it is difficult to avoid side effects such as osteoclast activation and bone loss caused by sustained stable blood drug concentrations.

[0007] On the one hand, the unique pharmacokinetic mechanism means that the commercially available injection preparations need to be administered daily to ensure the therapeutic effect, which is very unfavorable to improve the patient's compliance with treatment. On the other hand, teriparatide is a single-chain polypeptide compound with strong hydrophilicity, which is difficult to penetrate the gastrointestinal mucosa and is extremely unstable under the action of pepsin, resulting in extremely low oral bioavailability and difficulty in oral administration.

[0008] In view of this, it is indeed necessary to propose a combined preparation of core-shell microspheres loaded with parathyroid hormone analog drugs and a method for preparing core-shell microspheres loaded with parathyroid hormone analog drugs to solve the above problems. Summary of the invention

[0009] The object of the present invention is to provide a combined preparation of parathyroid hormone analog drug-loaded core-shell microspheres with long-term controllable release capability and a method for preparing the parathyroid hormone analog drug-loaded core-shell microspheres.

[0010] To achieve the above object, the technical solution of the present invention provides a combined preparation of core-shell microspheres loaded with parathyroid hormone analogs, comprising:

[0011] The first core-shell microspheres have the following components and weight percentages: the parathyroid hormone analog drug accounts for 4.76%-16.67%, and the high molecular polymer material accounts for 83.33%-95.24%;

[0012] The second core-shell microspheres, the components and weight percentages are: the parathyroid hormone analog drug accounts for 4.76%-16.67%, and the high molecular polymer material accounts for 83.33%-95.24%;

[0013] The third core-shell microspheres, the components and weight percentages are: the parathyroid hormone analog drug accounts for 4.76%-16.67%, and the high molecular polymer material accounts for 83.33%-95.24%;

[0014] Among them, the ratio of the first core-shell microspheres, the second core-shell microspheres and the third core-shell microspheres is 1:1:1, and the drug release times of the first core-shell microspheres, the second core-shell microspheres and the third core-shell microspheres are different.

[0015] Optionally, the polymer material includes polylactic-co-glycolic acid and polylactic acid. The polylactic acid includes racemic polylactic acid and / or L-polylactic acid. The molecular weight of the polylactic-co-glycolic acid is 10,000 - 40,000 Da. The molecular weight of the polylactic acid unit in the polylactic acid is 3,000 - 800,000 Da, and the molecular weight of the polylactic acid unit in the third core-shell microspheres is greater than that in the second core-shell microspheres, and the molecular weight of the polylactic acid unit in the second core-shell microspheres is greater than that in the first core-shell microspheres.

[0016] Optionally, the drug release times of the first core-shell microspheres, the second core-shell microspheres, and the third core-shell microspheres are in a multiple relationship.

[0017] Optionally, the first core-shell microspheres, the second core-shell microspheres, and the third core-shell microspheres all include a core structure and a shell structure, and the parathyroid hormone analogue drug is present in the core structure.

[0018] Optionally, the average particle size of the first core-shell microspheres, and / or the second core-shell microspheres, and / or the third core-shell microspheres is 5 - 200 μm; the particle size distribution coefficient Span of the first core-shell microspheres, and / or the second core-shell microspheres, and / or the third core-shell microspheres ≤ 1.5.

[0019] Optionally, the entrapment efficiency of the first core-shell microspheres, and / or the second core-shell microspheres, and / or the third core-shell microspheres ≥ 70%, and the drug burst release in the first 2 h of the first core-shell microspheres, and / or the second core-shell microspheres, and / or the third core-shell microspheres does not exceed 10% of the overall drug loading.

[0020] To achieve the above object, the technical solution of the present invention also provides a method for preparing core-shell microspheres loaded with parathyroid hormone analogue drugs for preparing the aforementioned first core-shell microspheres / second core-shell microspheres / third core-shell microspheres, including the following steps:

[0021] S1) Dissolve the parathyroid hormone analogue drug in deionized water to form an inner aqueous phase W 1 ;

[0022] S2) Secondarily mix a variety of polymer materials and an organic solvent to form an oil phase solution, and add the inner aqueous phase W obtained in step S1 1 to the oil phase solution, and adopt a low-temperature homogenization method to form a W 1 / O primary emulsion;

[0023] S3) Add the W 1 / O primary emulsion to the outer aqueous phase, and emulsify to form a W 1 / O / W 2 pre-compound emulsion, and use the premixed membrane emulsification technology to form a W1 / O / W 2 The pre - multiple emulsion forms uniform W 1 / O / W 2 droplets, and solidify the W 1 / O / W 2 droplets to form core - shell microspheres; the outer aqueous phase includes an emulsifier, an osmotic pressure regulator and deionized water;

[0024] S4) Centrifuge, wash and lyophilize the core - shell microspheres in sequence to form core - shell microspheres loaded with parathyroid hormone analogue drugs.

[0025] Optionally, the concentration of the parathyroid hormone analogue drug is 10 - 200 mg / mL, and the concentration of the multiple polymer materials is 1 - 500 mg / mL.

[0026] Optionally, the polymer materials include poly (lactic - co - glycolic acid) and polylactic acid. The polylactic acid includes racemic polylactic acid and / or L - polylactic acid; the molar ratio of lactide to glycolide in the poly (lactic - co - glycolic acid) is 90:10 - 10:90; the molecular weight of the poly (lactic - co - glycolic acid) is 10000 - 40000 Da; the molecular weight of the polylactic acid unit in the polylactic acid is 3000 - 800000 Da.

[0027] Optionally, the osmotic pressure regulator includes sodium chloride, and / or potassium chloride, and the mass concentration of the osmotic pressure regulator in the outer aqueous phase is 0.5 - 5.0 wt%.

[0028] Compared with the prior art, the technical solutions of the embodiments of the present invention have the following beneficial effects: The combined preparation of the core - shell microspheres loaded with parathyroid hormone analogue drugs of the present invention obtains an approximate pulse - like combined release curve by mixing and injecting core - shell microspheres with different drug release times, so as to achieve a release behavior similar to pulsed drug release, thereby overcoming the side effects such as the continuous and stable blood drug concentration of parathyroid hormone analogue drugs caused by the sustained - release microspheres using a single polymer material, which in turn leads to the activation of osteoclasts. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is a schematic diagram of the preparation process of the core - shell microspheres loaded with parathyroid hormone analogue drugs prepared by the present invention;

[0030] Figure 2 It is an electron micrograph of the microspheres prepared in Example 1 of the present invention;

[0031] Figure 3 It is a cross - section electron micrograph of the microspheres prepared in Example 1 of the present invention;

[0032] Figure 4 It is a confocal laser scanning microscopy image of the microspheres prepared in Example 1 of the present invention;

[0033] Figure 5 It is the particle size distribution diagram of the microspheres prepared in Example 1 of the present invention;

[0034] Figure 6 It is the electron micrograph of the microspheres prepared in Example 7 of the present invention;

[0035] Figure 7 It is the conventional release curve diagram of the microspheres prepared in Example 1 of the present invention;

[0036] Figure 8 It is the conventional release curve diagram of the microspheres prepared in Comparative Example 1 of the present invention;

[0037] Figure 9 It is the release curve diagram finally achieved by the compounding of various microspheres in the present invention. Detailed implementation manners

[0038] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0039] Here, it should be noted that in order to avoid obscuring the present invention due to unnecessary details, only the structures and / or processing steps closely related to the solution of the present invention are shown in the drawings, while other details less related to the present invention are omitted.

[0040] In addition, it should also be noted that the term "comprises", "comprising" or any other variation thereof is intended to cover a non-exclusive inclusion, such that a process, method, article or device comprising a series of elements includes not only those elements, but also other elements not expressly listed, or elements inherent to such process, method, article or device.

[0041] Please refer to Figures 1 to 9 As shown, the embodiments of the present invention provide a method for preparing a core-shell microsphere loaded with a parathyroid hormone analogue drug. The preparation of the core-shell microsphere includes the following steps:

[0042] S1, dissolving the parathyroid hormone analogue drug in deionized water to form an inner aqueous phase W 1 .

[0043] S2, secondarily mixing a variety of polymer materials and an organic solvent to form an oil-phase solution, and adding the inner aqueous phase W prepared in S1 1 to the oil-phase solution, and obtaining a W 1 / O primary emulsion after low-temperature homogenization.

[0044] S3, adding the W 1 / O primary emulsion to the outer aqueous phase and emulsifying to form a W 1 / O / W2 Pre-emulsion. The W is formed by using the pre-mixed membrane emulsification technique 1 / O / W 2 The pre-emulsion (in emulsion form) forms uniform W 1 / O / W 2 droplets through the hydrophilic microporous membrane, and the W 1 / O / W 2 droplets are conventionally solidified by mechanical stirring to remove the organic solvent to form core-shell microspheres.

[0045] S4, the core-shell microspheres are centrifuged, washed and freeze-dried in sequence to form core-shell microspheres loaded with parathyroid hormone analogue drugs.

[0046] Specifically, during the conventional solidification of the W 1 / O / W 2 droplets, as the organic solvent continuously volatilizes, different polymers will spontaneously arrange inside the droplets to form a core-shell structure according to their hydrophilic and hydrophobic properties. This process is called emulsion evolution. After the organic solvent completely volatilizes, the polymers dissolved in the organic solvent will remain in place to form core-shell microspheres. After the emulsion evolution ends, the core-shell microspheres are centrifuged, washed and freeze-dried in sequence to obtain core-shell microspheres loaded with parathyroid hormone analogue drugs.

[0047] Specifically, in step S1, by adjusting the concentration of the parathyroid hormone analogue drug in the inner aqueous phase, the osmotic pressure between the inner and outer aqueous phases during the preparation of the core-shell microspheres can be improved, and the encapsulation rate can be enhanced. Because if the osmotic pressure difference between the inner and outer aqueous phases is too large, it will cause the drug to escape severely during the solidification process and the encapsulation rate will be low.

[0048] In some embodiments, the concentration of the parathyroid hormone analogue drug is 10 - 500 mg / mL.

[0049] In some preferred embodiments, the concentration of the parathyroid hormone analogue drug is 50 - 200 mg / mL.

[0050] Specifically, in step S2, using multiple polymer materials to prepare core-shell sustained-release microspheres can regulate the distribution of the inner aqueous phase according to the hydrophilic and hydrophobic properties of the polymer materials during the preparation process. Specifically, the inner aqueous phase will be more likely to be enriched in the polymer with weaker hydrophobicity, and the hydrophilic and hydrophobic properties of the polymer can be regulated by adjusting the monomer composition. The inner aqueous phase W carrying the drug 1It will be mainly enriched in polymers with weaker hydrophobicity. During the preparation process, by changing the volume of the oil phase, the polymer concentration, and controlling the emulsion evolution time, etc., the distribution of such polymers in the core of the core-shell microspheres can be controlled. With such a setting, during the release process of the microspheres, the drug-free shell (outer shell) of the core-shell microspheres degrades first, and then the drug loaded in the core is released. Further, by adjusting the degradation time of the shell, core-shell microspheres loaded with parathyroid hormone analog drugs with different drug release times can be designed.

[0051] In some embodiments, the concentration of various polymer materials is 1-500 mg / mL.

[0052] In some embodiments, the various polymer materials include polylactic-co-glycolic acid copolymer and polylactic acid. Further, the molar ratio of lactide to glycolide in the polylactic-co-glycolic acid copolymer is 90:10-10:90.

[0053] In some preferred embodiments, the molar ratio of lactide to glycolide in the polylactic-co-glycolic acid copolymer is 75:25-25:75. For example, the molar ratio of lactide to glycolide in the polylactic-co-glycolic acid copolymer can be 90:10, 80:20, 75:25, 60:40, 50:50, 45:55, 35:65, 25:75, etc.

[0054] Further, by adjusting the molecular weight of the polymer used, the release time and release degree of the prepared core-shell microspheres are adjusted, so that the core-shell microspheres loaded with parathyroid hormone analog drugs have an adjustable release curve.

[0055] In some embodiments, the molecular weight of the polylactic-co-glycolic acid copolymer is 10000-40000 Da.

[0056] In some preferred embodiments, the molecular weight of the polylactic-co-glycolic acid copolymer is 14000-26000 Da. For example, the molecular weight of the polylactic-co-glycolic acid copolymer can be 10000 Da, 12000 Da, 14000 Da, 16000 Da, 18000 Da, 20000 Da, 22000 Da, 24000 Da, 26000 Da, 30000 Da, 40000 Da, etc.

[0057] In some embodiments, the molecular weight of the polylactic acid unit in the polylactic acid is 3000-800000 Da.

[0058] In some preferred embodiments, the molecular weight of the polylactic acid units in the polylactic acid is 50,000 to 800,000 Da. For example, the molecular weight of the polylactic acid units can be 50,000 Da, 100,000 Da, 200,000 Da, 300,000 Da, 500,000 Da, 600,000 Da, 700,000 Da, 800,000 Da, etc.

[0059] In some embodiments, the mass ratio of the poly(lactic-co-glycolic acid) copolymer to the polylactic acid is 10:1 to 1:1.

[0060] In some preferred embodiments, the mass ratio of the poly(lactic-co-glycolic acid) copolymer to the polylactic acid is 5:1 to 1:1. For example, the mass ratio of the poly(lactic-co-glycolic acid) copolymer to the polylactic acid can be 4:1, 3:1, 2:1, etc.

[0061] In some embodiments, the concentration of the poly(lactic-co-glycolic acid) copolymer and / or the polylactic acid is 1 to 500 mg / mL.

[0062] In some preferred embodiments, the concentration of the poly(lactic-co-glycolic acid) copolymer and / or the polylactic acid is 3 to 300 mg / mL.

[0063] In some preferred embodiments, the concentration of the poly(lactic-co-glycolic acid) copolymer and / or the polylactic acid is 5 to 50 mg / mL. For example, the concentration of the poly(lactic-co-glycolic acid) copolymer and / or the polylactic acid can be 1 mg / mL, 5 mg / mL, 10 mg / mL, 50 mg / mL, etc.

[0064] In some embodiments, the composition of the polylactic acid is racemic polylactic acid, L-polylactic acid, etc.

[0065] In this embodiment, by adjusting the optical activity of the polylactic acid used, L-polylactic acid with stronger hydrophobicity is used instead of racemic polylactic acid to promote the phase separation degree between various polymers and promote the formation of a core-shell structure.

[0066] In step S2, the organic solvent includes at least one of dichloromethane, ethyl acetate, chloroform, acetone, toluene, ethyl propionate, or ethanol.

[0067] In step S2, the low-temperature homogenization treatment method can avoid the destruction of the teriparatide structure by heat. If the high-speed homogenization treatment method is adopted, the heat generated during this process will destroy the teriparatide structure. The temperature of the low-temperature homogenization is controlled in the range of 4 to 14 °C. For example, the temperature of the low-temperature homogenization can be 4 °C, 6 °C, 8 °C, 10 °C, 12 °C, 14 °C, etc.

[0068] Further, the time for low-temperature homogenization is 40 to 120 s. For example, the time for low-temperature homogenization can be 40 s, 60 s, 80 s, 100 s, 120 s, etc.

[0069] Specifically, in step S3, the outer aqueous phase includes an emulsifier, an osmotic pressure regulator, and deionized water.

[0070] In this embodiment, by adding an emulsifier to the outer aqueous phase, the oil-water interface of the water-in-oil in the outer aqueous phase is stabilized, the coalescence between oil-phase droplets is inhibited, and the formation of microspheres with uniform size is promoted.

[0071] Specifically, the emulsifier includes at least one of polyvinyl alcohol, polyglycerol ester, polyoxyethylene sorbitan monooleate, and polyoxyethylene sorbitan monolaurate.

[0072] In some embodiments, the mass concentration of the emulsifier in the outer aqueous phase is 0.5 to 5.0 wt%. In some preferred embodiments, the mass concentration of the emulsifier in the outer aqueous phase is 1.0 to 2.5 wt%. For example, the mass concentration of the emulsifier in the outer aqueous phase can be 0.5 wt%, 1.0 wt%, 1.5 wt%, 2.0 wt%, 2.5 wt%, 4.0 wt%, 5.0 wt%, etc.

[0073] In this embodiment, by adding an osmotic pressure regulator to the outer aqueous phase, the inner aqueous phase is stabilized, and it is inhibited that the inner aqueous phase dissolved with the parathyroid hormone analogue drug escapes to the outer aqueous phase due to the osmotic pressure difference between the inner and outer aqueous phases during the preparation process, resulting in a decrease in the drug loading.

[0074] Specifically, in some embodiments, the osmotic pressure regulator includes sodium chloride or potassium chloride. In some other embodiments, the osmotic pressure regulator includes sodium chloride and potassium chloride.

[0075] In some embodiments, the mass concentration of the osmotic pressure regulator in the outer aqueous phase is 0.5 to 5.0 wt%. In some preferred embodiments, the mass concentration of the osmotic pressure regulator in the outer aqueous phase is 1.0 to 2.5 wt%. For example, the mass concentration of the osmotic pressure regulator in the outer aqueous phase can be 0.5 wt%, 1.0 wt%, 1.5 wt%, 2.0 wt%, 2.5 wt%, 4.0 wt%, 5.0 wt%, etc.

[0076] In some embodiments, the volume ratio of the outer aqueous phase in step S3 to the oil-phase solution in step S2 is 100:1 to 5:1. In some preferred embodiments, the volume ratio of the outer aqueous phase in step S3 to the oil-phase solution in step S2 is 80:1 to 20:1.

[0077] In some other preferred embodiments, the volume ratio of the external aqueous phase in step S3 to the oil phase solution in step S2 is 50:1 to 30:1. For example, the volume ratio of the external aqueous phase in step S3 to the oil phase solution in step S2 can be 50:1, 45:1, 40:1, 35:1, 30:1, etc.

[0078] In step S3, the pore size of the hydrophilic microporous membrane is 20 - 100 μm. In some preferred embodiments, the pore size of the hydrophilic microporous membrane is 40 - 60 μm. For example, the pore size of the hydrophilic microporous membrane can be 20 μm, 30 μm, 40 μm, 45 μm, 50 μm, 55 μm, 60 μm, 80 μm, 100 μm, etc.

[0079] Furthermore, in step S3, by controlling the transmembrane pressure and the number of transmembrane passes of the hydrophilic microporous membrane, it is possible to ensure good uniformity and high yield of the particle size of the core - shell microspheres loaded with parathyroid hormone - like drug. In addition, by adjusting the pore size of the microporous membrane and the transmembrane pressure, directional quality control of the particle size of the core - shell microspheres loaded with parathyroid hormone - like drug is beneficial to large - scale production.

[0080] Specifically, the number of times passing through the hydrophilic microporous membrane is at least 1 time. In some preferred embodiments, the number of times passing through the hydrophilic microporous membrane is 3 times.

[0081] In step S3, the transmembrane pressure of the hydrophilic microporous membrane is 1 - 1000 kPa. In some preferred embodiments, the transmembrane pressure of the hydrophilic microporous membrane is 1 - 500 kPa. In some other preferred embodiments, the transmembrane pressure of the hydrophilic microporous membrane is 1 - 50 kPa. For example, the transmembrane pressure of the hydrophilic microporous membrane can be 1 kPa, 5 kPa, 10 kPa, 20 kPa, 30 kPa, 40 kPa, 50 kPa, etc.

[0082] In step S3, the time for room - temperature curing is 3 - 10 h. In some preferred embodiments, the time for room - temperature curing is 5 - 8 h. For example, the time for room - temperature curing can be 5 h, 6 h, 7 h, 8 h, etc.

[0083] During the curing process, various polymer materials will form aggregates inside the emulsion droplets as the oil - phase solvent is gradually extracted. With the further extraction of the oil - phase solvent, various polymer materials will migrate to their respective layers under the action of thermodynamic and kinetic factors inside the emulsion droplets, and the curing ends when the aggregates of various polymer materials form metastable intermediates, forming core - shell microspheres.

[0084] After the microspheres are solidified, the internal aqueous phase loaded with the parathyroid hormone analogue drug is mainly distributed at the core position of the core-shell microspheres, and the outer shell part of the core-shell microspheres basically does not contain the drug. In terms of the release behavior of the microspheres, compared with the sustained and stable release curve of the sustained-release microspheres prepared using a single polymer material, the core-shell microspheres can hardly release the drug before the specified release time, and release the internal drug load almost completely after the expected release time arrives, achieving the effect of controllable drug release.

[0085] Furthermore, based on the special release behavior of the core-shell microspheres, the core-shell microspheres with different drug release times are mixed and injected to obtain a combined release curve similar to a pulse, so as to overcome the side effects such as the sustained and stable blood drug concentration of the parathyroid hormone analogue drug caused by the sustained-release microspheres using a single polymer material, which in turn leads to the activation of osteoclasts.

[0086] The core-shell microspheres loaded with the parathyroid hormone analogue drug prepared according to the above method can effectively improve the embedding rate. The embedding rate of the core-shell microspheres loaded with the parathyroid hormone analogue drug is ≥75%, and the drug loading amount can reach 4% - 10%. Moreover, the burst release of the core-shell microspheres loaded with the parathyroid hormone analogue drug in the first 2 hours is less than 15%, and the long-term controllable release of the parathyroid hormone analogue drug is also achieved.

[0087] In this embodiment, the calculation standard of the above embedding rate is as follows:

[0088] Embedding rate = (actual drug loading amount of the core-shell microspheres loaded with the parathyroid hormone analogue drug / theoretical drug loading amount of the core-shell microspheres loaded with the parathyroid hormone analogue drug) × 100%.

[0089] The average particle size of the core-shell microspheres loaded with the parathyroid hormone analogue drug is 5 - 200 μm, preferably 10 - 80 μm. For example, the average particle size of the core-shell microspheres loaded with the parathyroid hormone analogue drug can be 10 μm, 20 μm, 40 μm, 60 μm, 80 μm, 100 μm, 120 μm, 140 μm, 160 μm, 199 μm, etc.

[0090] Furthermore, the particle size distribution coefficient Span of the core-shell microspheres loaded with the parathyroid hormone analogue drug is < 1.5. Preferably, the particle size distribution coefficient Span of the core-shell microspheres loaded with the parathyroid hormone analogue drug is < 1.0.

[0091] The particle size distribution coefficient Span of the microspheres is a key parameter for the particle size distribution of the microspheres, and the specific definition is as follows:

[0092] Span = (D90 - D10) / D50. Among them, D90 represents the particle size corresponding to when the cumulative particle size distribution number of a sample reaches 90%, that is, the proportion of microsphere particles with a particle size smaller than D90 is 90%. D50 represents the particle size corresponding to when the cumulative particle size distribution number of a sample reaches 50%, that is, the proportion of microsphere particles with a particle size smaller than D50 is 50%. Generally, D50 can be used to represent the average particle size of the microsphere sample. D10 has a similar physical meaning to D50 and D90, which will not be elaborated here.

[0093] To further elaborate the technical solution of the present invention, the following examples are provided to explore the influence of different formulations on the structure of the core-shell microspheres.

[0094] Example 1: Preparation of core-shell microspheres with a 7-day delayed release after administration

[0095] Example 1 provides a method for preparing core-shell microspheres loaded with parathyroid hormone analog drugs. The core-shell microspheres prepared by this method can be released with a 7-day delay after administration. As Figure 1 shown, the method includes the following steps:

[0096] (1) Weigh 11.32 mg of teriparatide acetate (i.e., parathyroid hormone analog drug) and dissolve it in 0.1 mL of deionized water. Use a vortex mixer to fully dissolve teriparatide acetate in water to form the internal aqueous phase W 1 ;

[0097] (2) Weigh 100 mg of poly(lactic-co-glycolic acid) (the molar ratio of lactide to glycolide is 50:50, 20000 Da) and 200 mg of poly(L-lactic acid) (200000 Da) and dissolve them in 7.5 mL of dichloromethane organic reagent to form the oil phase. Add the internal aqueous phase W 1 to the oil phase and homogenize it in an ice-water bath to form W 1 / O primary emulsion. The homogenization rate is 24000 rpm, and the homogenization time is 90 s;

[0098] (3) Pour the W 1 / O primary emulsion into 75 mL of the external aqueous phase containing 1 wt% PVA and 1 wt% NaCl, and stir it evenly at a stirring rate of 300 rpm for 1 min to obtain W 1 / O / W 2 preliminary multiple emulsion. Subsequently, pass the W 1 / O / W 2 preliminary multiple emulsion through a hydrophilic microporous membrane with a pore size of 50 μm, the transmembrane pressure is 9 kPa, and pass through the membrane 3 times to obtain a uniformly sized W 1 / O / W 2The milk drops. Then, the milk drops were solidified at normal temperature and pressure for 6 h, and then, after being washed with water and centrifuged multiple times, long-acting controlled-release microspheres with uniform size and encapsulating teriparatide acetate were formed. The microspheres were vacuum freeze-dried for 72 h to obtain core-shell microspheres loaded with parathyroid hormone analog drugs.

[0099] Figure 3 It is shown that the core-shell microspheres loaded with parathyroid hormone analog drugs prepared in Example 1 have a core-shell structure. Figure 4 Among them, the green is the parathyroid hormone analog drug labeled with FITC, and the red is the polymer labeled with Nile red. It can be seen that the parathyroid hormone analog drug in the core-shell microspheres loaded with parathyroid hormone analog drugs prepared in Example 1 is mainly distributed at the central position of the core-shell microspheres. Figure 2 、 Figure 5 It is shown that the core-shell microspheres loaded with parathyroid hormone analog drugs prepared in Example 1 have a good spherical shape and uniform particle size. Specifically, the average particle size of the microspheres prepared in Example 1 is 18.96 μm, and the Span value of the particle size distribution coefficient is 0.78.

[0100] Example 2: Delaying the phase separation time of multiple polymers to optimize the core-shell microsphere structure

[0101] Example 2 provides a method for preparing core-shell microspheres loaded with parathyroid hormone analog drugs, and the method includes the following steps:

[0102] (1) Weigh 10.67 mg of teriparatide acetate (i.e., parathyroid hormone analog drug) and dissolve it in 0.1 mL of deionized water. Use a vortex mixer to fully dissolve teriparatide acetate in water to form an internal aqueous phase W 1 ;

[0103] (2) Weigh 100 mg of poly (lactic-co-glycolic acid) (the molar ratio of lactide to glycolide is 50:50, 20000 Da) and 200 mg of poly (L-lactic acid) (200000 Da) and dissolve them in 10 mL of dichloromethane organic reagent to form an oil phase. Add the internal aqueous phase W 1 to the oil phase and homogenize it in an ice-water bath to form a W 1 / O primary emulsion, with a homogenization rate of 24000 rpm and a homogenization time of 90 s;

[0104] (3) Pour the W 1 / O primary emulsion into 100 mL of an external aqueous phase containing 1 wt% PVA and 1 wt% NaCl, and stir it evenly at a speed of 300 rpm for 1 min to form a W 1 / O / W 2 pre-compound emulsion. Then, the W 1 / O / W 2The pre-emulsion passes through a hydrophilic microporous membrane with a pore size of 50 μm under a transmembrane pressure of 9 kPa three times to obtain W with a uniform particle size. 1 / O / W 2 droplets, which are solidified, washed, and freeze-dried to obtain the core-shell microspheres loaded with the parathyroid hormone analogue drug.

[0105] Example 2 provides a method for preparing core-shell microspheres loaded with a parathyroid hormone analogue drug, which optimizes the process prescription of Example 1. By reducing the polymer oil phase concentration, that is, increasing the oil phase volume, the time required for the complete volatilization of the organic solvent during the solidification of the microspheres is increased, thereby delaying the phase separation time between various polymers, giving different polymers more evolution time to optimize the core-shell structure of the microspheres, and thus obtaining a better controlled release effect.

[0106] Example 3: Further slowing down the polymer phase separation rate to optimize the core-shell structure

[0107] In order to explore whether giving more evolution time will enhance the degree of phase separation between various polymers, Example 3 is hereby set up. That is, Example 3 further increases the volume of the oil phase on the basis of Example 2. Specifically, Example 3 provides a method for preparing core-shell microspheres loaded with a parathyroid hormone analogue drug, and the method includes the following steps:

[0108] (1) Weigh 10.03 mg of teriparatide acetate (i.e., the parathyroid hormone analogue drug) and dissolve it in 0.1 mL of deionized water. Use a vortex mixer to fully dissolve teriparatide acetate in water to form the internal aqueous phase W 1 ;

[0109] (2) Weigh 100 mg of poly(lactic-co-glycolic acid) (the molar ratio of lactide to glycolide is 50:50, 20000 Da) and 200 mg of poly(L-lactic acid) (200000 Da) and dissolve them in 15 mL of dichloromethane organic reagent to form the oil phase. Add the internal aqueous phase W 1 to the oil phase and homogenize it in an ice-water bath to form the W 1 / O primary emulsion at a homogenization rate of 24000 rpm for 90 s;

[0110] (3) Pour the W 1 / O primary emulsion into 150 mL of the external aqueous phase containing 1 wt% PVA and 1 wt% NaCl, and stir it evenly at a speed of 300 rpm for 1 min to form the W 1 / O / W 2 pre-emulsion. Subsequently, pass the W 1 / O / W 2 pre-emulsion through a hydrophilic microporous membrane with a pore size of 50 μm under a transmembrane pressure of 9 kPa three times to obtain W with a uniform particle size.1 / O / W 2 The milk droplets are solidified, washed and freeze-dried to obtain the core-shell microspheres loaded with the parathyroid hormone analogue drug.

[0111] Example 4: Replace the core polymer material of the core-shell microspheres and attempt to optimize the core-shell structure

[0112] Example 4 provides a method for preparing core-shell microspheres loaded with a parathyroid hormone analogue drug, and the method includes the following steps:

[0113] (1) Weigh 10.04 mg of teriparatide acetate (i.e., the parathyroid hormone analogue drug) and dissolve it in 0.1 mL of deionized water. Use a vortex mixer to fully dissolve teriparatide acetate in water to form the internal aqueous phase W 1 ;

[0114] (2) Weigh 100 mg of poly(lactic-co-glycolic acid) (the molar ratio of lactide to glycolide is 75:25, 20000 Da) and 200 mg of poly(L-lactic acid) (200000 Da) and dissolve them in 7.5 mL of dichloromethane organic reagent to form the oil phase. Add the internal aqueous phase W 1 to the oil phase and homogenize it in an ice-water bath to form the W 1 / O primary emulsion, with a homogenization rate of 24000 rpm and a homogenization time of 90 s;

[0115] (3) Pour the W 1 / O primary emulsion into 75 mL of the external aqueous phase containing 1 wt% PVA and 1 wt% NaCl, and stir it evenly at a speed of 300 rpm for 1 min to form the W 1 / O / W 2 pre-compound emulsion. Subsequently, pass the W 1 / O / W 2 pre-compound emulsion through a hydrophilic microporous membrane with a pore size of 50 μm, with a transmembrane pressure of 9 kPa, and pass it through 3 times to obtain the W 1 / O / W 2 milk droplets, which are solidified, washed and freeze-dried to obtain the core-shell microspheres loaded with the parathyroid hormone analogue drug.

[0116] It can be understood that the special configuration of the core-shell microspheres is mainly achieved through the hydrophilic and hydrophobic properties of various polymers. During the emulsion evolution stage, as the organic solvent continuously volatilizes, the various polymers originally uniformly dissolved in the milk droplets gradually stratify according to their hydrophilic and hydrophobic properties, forming microspheres with a shell and core configuration. The polymer used in Example 4 is PLGA7525. Compared with PLGA5050 used in Example 1, PLGA7525 has stronger hydrophobic properties. By replacing this material, the influence of the hydrophobic properties of the polymer on the configuration of the core-shell microspheres can be explored.

[0117] Example 5: Controlling the thickness of the shell layer of core-shell microspheres by changing the polymer ratio in the oil phase

[0118] Example 5 provides a method for preparing core-shell microspheres loaded with parathyroid hormone analog drugs, and the method includes the following steps:

[0119] (1) Weigh 11.05 mg of teriparatide acetate (i.e., parathyroid hormone analog drug) and dissolve it in 0.1 mL of deionized water. Use a vortex mixer to fully dissolve teriparatide acetate in water to form an inner aqueous phase W 1 ;

[0120] (2) Weigh 200 mg of poly (lactic-co-glycolic acid) (the molar ratio of lactide to glycolide is 50:50, 20000 Da) and 100 mg of poly (L-lactic acid) (200000 Da) and dissolve them in 7.5 mL of dichloromethane organic reagent to form an oil phase. Add the inner aqueous phase W 1 to the oil phase and homogenize it in an ice-water bath to form a W 1 / O primary emulsion. The homogenization rate is 24000 rpm and the homogenization time is 90 s;

[0121] (3) Pour the W 1 / O primary emulsion into 75 mL of an outer aqueous phase containing 1 wt% PVA and 1 wt% NaCl, and stir it evenly at a speed of 300 rpm for 1 min to form a W 1 / O / W 2 pre-emulsion. Subsequently, pass the W 1 / O / W 2 pre-emulsion through a hydrophilic microporous membrane with a pore size of 50 μm. The pressure during membrane passing is 9 kPa, and pass through the membrane 3 times to obtain W 1 / O / W 2 droplets with uniform particle size. After solidification, washing and freeze-drying, core-shell microspheres loaded with parathyroid hormone analog drugs are obtained.

[0122] The shell layer of the core-shell microspheres prepared in Example 5 is thinner than that of the core-shell microspheres prepared in Example 1. Specifically, the shell layer thickness of the core-shell microspheres prepared in Example 1 is about 4 microns, and the shell layer thickness of the core-shell microspheres prepared in Example 5 is about 2 microns. Core-shell microspheres with a thinner shell layer will degrade faster and release drugs earlier.

[0123] Example 6: Extending the phase separation time on the basis of Example 5 to optimize the core-shell structure

[0124] On the basis of Example 5, in this Example 6, a larger volume of dichloromethane is used to extend the time required for emulsion evolution and attempt to optimize the core-shell structure. Specifically, Example 6 provides a method for preparing core-shell microspheres loaded with parathyroid hormone analog drugs, and the method includes the following steps:

[0125] (1) Weigh 10.92 mg of teriparatide acetate (i.e., the parathyroid hormone analogue drug) and dissolve it in 0.1 mL of deionized water. Use a vortex mixer to fully dissolve teriparatide acetate in water to form an inner aqueous phase W 1 ;

[0126] (2) Weigh 200 mg of poly (lactic - co - glycolic acid) (the molar ratio of lactide to glycolide is 50:50, 20000 Da) and 100 mg of poly - L - lactic acid (200000 Da) and dissolve them in 10 mL of dichloromethane organic reagent to form an oil phase. Add the inner aqueous phase W 1 to the oil phase and homogenize it in an ice - water bath to form a W 1 / O primary emulsion. The homogenization rate is 24000 rpm and the homogenization time is 90 s;

[0127] (3) Pour the W 1 / O primary emulsion into 100 mL of an outer aqueous phase containing 1 wt% PVA and 1 wt% NaCl, and stir it evenly at a speed of 300 rpm for 1 min to form a W 1 / O / W 2 pre - multiple emulsion. Subsequently, pass the W 1 / O / W 2 pre - multiple emulsion through a hydrophilic microporous membrane with a pore size of 50 μm. The pressure across the membrane is 9 kPa, and pass through the membrane 3 times to obtain W 1 / O / W 2 droplets with a uniform particle size. After solidification, washing, and freeze - drying, core - shell microspheres loaded with the parathyroid hormone analogue drug are obtained.

[0128] Example 7: Increasing the proportion of the shell material of the core - shell microspheres to optimize the structure of the core - shell microspheres

[0129] Example 7 provides a method for preparing core - shell microspheres loaded with a parathyroid hormone analogue drug, and the method includes the following steps:

[0130] (1) Weigh 10.32 mg of teriparatide acetate (i.e., the parathyroid hormone analogue drug) and dissolve it in 0.1 mL of deionized water. Use a vortex mixer to fully dissolve teriparatide acetate in water to form an inner aqueous phase W 1 ;

[0131] (2) Weigh 100 mg of poly (lactic - co - glycolic acid) (the molar ratio of lactide to glycolide is 50:50, 20000 Da) and 300 mg of poly - L - lactic acid (200000 Da) and dissolve them in 10 mL of dichloromethane organic reagent to form an oil phase. Add the inner aqueous phase W 1 to the oil phase and homogenize it in an ice - water bath to form a W 1 / O primary emulsion. The homogenization rate is 24000 rpm and the homogenization time is 90 s;

[0132] (3) Pour the W 1 / O primary emulsion into the external aqueous phase containing 1 wt% PVA and 1 wt% NaCl in 100 mL, and stir uniformly at a speed of 300 rpm for 1 min to form the W 1 / O / W 2 pre - multiple emulsion. Subsequently, the W 1 / O / W 2 pre - multiple emulsion passes through a hydrophilic microporous membrane with a pore size of 50 μm, the pressure during membrane passing is 9 kPa, and it passes through the membrane 3 times to obtain W 1 / O / W 2 droplets of uniform particle size. After solidification, washing, and freeze - drying, core - shell microspheres loaded with parathyroid hormone - like drug are obtained.

[0133] In this example, the shell material of the core - shell microspheres is PLLA, and the core material is PLGA. In the conventional configuration, the mass ratio of the shell material PLLA to the core material PLGA is 2:1. This example is used to explore whether changing the mass ratio of the shell material PLLA to the core material PLGA will affect the structure of the core - shell microspheres. According to the results of this example, after changing the mass ratio of the shell material PLLA to the core material PLGA, the separation of the two polymer materials in the core - shell microspheres becomes unclear, and it is difficult to form a core - shell structure.

[0134] Comparative Example 1: Increasing the molecular weight of the shell material of core - shell microspheres to prepare core - shell microspheres with delayed drug release

[0135] Comparative Example 1 provides a method for preparing core - shell microspheres loaded with parathyroid hormone - like drug, including the following steps:

[0136] (1) Weigh 9.86 mg of teriparatide acetate (i.e., parathyroid hormone - like drug) and dissolve it in 0.1 mL of deionized water. Use a vortex mixer to fully dissolve teriparatide acetate in water to form the internal aqueous phase W 1 ;

[0137] (2) Weigh 100 mg of poly (lactic - co - glycolic acid) (the molar ratio of lactide to glycolide is 50:50, 20000 Da) and 200 mg of poly (L - lactic acid) (700000 Da) and dissolve them in 7.5 mL of dichloromethane organic reagent to form the oil phase. Add the internal aqueous phase W 1 to the oil phase and homogenize it in an ice - water bath to form the W 1 / O primary emulsion, with a homogenization rate of 24000 rpm and a homogenization time of 90 s;

[0138] (3) Pour the W 1 / O primary emulsion into 75 mL of the external aqueous phase containing 1 wt% PVA and 1 wt% NaCl, and stir uniformly at a speed of 300 rpm for 1 min to form the W1 / O / W 2 Pre-emulsion. Subsequently, W 1 / O / W 2 The pre-emulsion passed through a hydrophilic microporous membrane with a pore size of 50 μm under a transmembrane pressure of 9 kPa for 3 times to obtain W with a uniform particle size 1 / O / W 2 milk droplets, which were solidified, washed and freeze-dried to obtain core-shell microspheres loaded with parathyroid hormone analog drugs.

[0139] Compared with Example 1, in Comparative Example 1, the molecular weight of poly(L-lactic acid), the material used as the shell layer, was increased. A polymer with a larger molecular weight means a slower degradation rate, and the predetermined drug release time is also postponed accordingly.

[0140] Comparative Example 2: Continuing to increase the molecular weight of the shell layer material on the basis of Comparative Example 1 to delay the drug release time

[0141] Comparative Example 2 provides a method for preparing core-shell microspheres loaded with parathyroid hormone analog drugs, including the following steps:

[0142] (1) Weigh 10.76 mg of teriparatide acetate (i.e., parathyroid hormone analog drug) and dissolve it in 0.1 mL of deionized water. Use a vortex mixer to fully dissolve teriparatide acetate in water to form an internal aqueous phase W 1 ;

[0143] (2) Weigh 100 mg of poly(lactic-co-glycolic acid) (the molar ratio of lactide to glycolide is 50:50, 20000 Da) and 200 mg of poly(L-lactic acid) (800000 Da) and dissolve them in 7.5 mL of dichloromethane organic reagent to form an oil phase. Add the internal aqueous phase W 1 to the oil phase and homogenize it in an ice-water bath to form W 1 / O primary emulsion, with a homogenization rate of 24000 rpm and a homogenization time of 90 s;

[0144] (3) Pour the W 1 / O primary emulsion into 75 mL of an external aqueous phase containing 1 wt% PVA and 1 wt% NaCl, and stir it evenly at a speed of 300 rpm for 1 min to form W 1 / O / W 2 pre-emulsion. Subsequently, the W 1 / O / W 2 pre-emulsion passed through a hydrophilic microporous membrane with a pore size of 50 μm under a transmembrane pressure of 9 kPa for 3 times to obtain W 1 / O / W 2 milk droplets, which were solidified, washed and freeze-dried to obtain core-shell microspheres loaded with parathyroid hormone analog drugs.

[0145] Based on Comparative Example 1, in this Comparative Example 2, the molecular weight of the polymer - poly(L - lactic acid) as the shell layer was increased. A higher polymer molecular weight means a longer degradation time and a slower drug release rate.

[0146] Comparative Example 3: Decreasing the molecular weight of the core material of the core - shell microspheres to attempt to accelerate the drug release process

[0147] Comparative Example 3 provides a method for preparing core - shell microspheres loaded with a parathyroid hormone analogue drug, comprising the following steps:

[0148] (1) Weigh 10.76 mg of teriparatide acetate (i.e., the parathyroid hormone analogue drug) and dissolve it in 0.1 mL of deionized water. Use a vortex mixer to fully dissolve teriparatide acetate in water to form the internal aqueous phase W. 1 ;

[0149] (2) Weigh 100 mg of poly(lactic - co - glycolic acid) (the molar ratio of lactide to glycolide is 50:50, 5000 Da) and 200 mg of poly(L - lactic acid) (200000 Da) and dissolve them in 7.5 mL of dichloromethane organic reagent to form the oil phase. Add the internal aqueous phase W 1 to the oil phase and homogenize it in an ice - water bath to form the W 1 / O primary emulsion, with a homogenization rate of 24000 rpm and a homogenization time of 90 s.

[0150] (3) Pour the W 1 / O primary emulsion into 75 mL of the external aqueous phase containing 1 wt% PVA and 1 wt% NaCl, and stir it evenly at a speed of 300 rpm for 1 min to form the W 1 / O / W 2 pre - multiple emulsion. Then, pass the W 1 / O / W 2 pre - multiple emulsion through a hydrophilic microporous membrane with a pore size of 50 μm, with a transmembrane pressure of 9 kPa, and pass through the membrane 3 times to obtain uniform - sized W 1 / O / W 2 droplets. After solidification, washing, and freeze - drying, core - shell microspheres loaded with the parathyroid hormone analogue drug are obtained.

[0151] Compared with Comparative Example 1, in this Comparative Example 3, the molecular weight of the poly(lactic - co - glycolic acid) (PLGA) as the core was decreased. The decrease in the molecular weight of the core material means that after the degradation of the shell - layer polymer, the arrangement at the core is looser, and the drug is released more completely.

[0152] Test conditions

[0153] The core - shell microspheres loaded with the parathyroid hormone analogue drug provided in Examples 1 to 7 and Comparative Examples 1 to 3 were tested. The test method is as follows:

[0154] (1) Morphological characterization

[0155] The prepared long-acting controlled-release microspheres encapsulating teriparatide acetate were redispersed in the aqueous phase, and the surface morphology of the microspheres was observed using a cold field emission electron scanning microscope.

[0156] (2) Average particle size and particle size distribution

[0157] The freeze-dried long-acting controlled-release microspheres encapsulating teriparatide acetate were redispersed in the aqueous phase, and the redissolved solution was dropped into a laser particle size analyzer to measure the particle size of the microspheres.

[0158] (3) Drug loading determination

[0159] ① Microsphere destruction method: Accurately weigh 20.63 mg of the freeze-dried finished microspheres and place them in a 50 mL centrifuge tube. Subsequently, add 20 mL of acetonitrile solution, and ultrasonicate in a water bath for 30 min to fully destroy the microsphere skeleton material. Then, precisely add 30 mL of deionized water for drug extraction. After standing for 20 min, centrifuge at 8000 rpm for 10 min, take the supernatant, filter it through a 0.45 μm microporous membrane, and take the filtrate as the test solution. The drug loading is determined using high performance liquid chromatography.

[0160] ② High performance liquid chromatography conditions: Octadecylsilyl silica gel is used as the filler (4.6×250 mm, 5 μm). Mobile phase A is an aqueous solution of 0.1% TFA, and mobile phase B is an acetonitrile solution of 0.1% TFA. Gradient elution is used, and the gradient elution program is that the proportion of mobile phase A changes with time as shown in Table 1; the injection volume is 20 μL; the flow rate is 1.0 mL per minute; the detection wavelength is 280 nm.

[0161] Table 1: Gradient elution program

[0162] Time Mobile phase A (%) 0 70 15 65 20 70

[0163] (4) In vitro release experiment

[0164] Experimental purpose: By simulating the in vivo release method, test the release pattern of the prepared controlled-release microspheres loaded with parathyroid hormone analog drugs within the test time, so as to verify the influence of different preparation process parameters on the drug release ability of the core-shell microspheres.

[0165] Experimental method: Accurately weigh 10.34 mg of the controlled-release microspheres loaded with parathyroid hormone analog drugs to be tested into a 50 mL centrifuge tube. Add 10 mL of commercially available 1× phosphate buffer solution to the centrifuge tube. Place the centrifuge tube on a vertical suspension instrument and perform vertical suspension at a rotation speed of 60 rpm under the condition of 37.5 °C. At 0.5 h, 2 h, 4 h, 8 h, 24 h, 28 h, 32 h, and 48 h before placing the sample; after 48 h, carefully take out 2 mL of the solution from the centrifuge tube every 1 day and accurately supplement 2 mL of the solution. Continuously sample for 28 days, detect the concentration of teriparatide acetate contained therein, and calculate the release curve of the controlled-release microspheres loaded with parathyroid hormone analog drugs.

[0166] The test results are shown in Table 2:

[0167] Table 2: Particle size distribution and encapsulation efficiency of the prepared microspheres

[0168]

[0169]

[0170] Figure 1 Shows the specific preparation process, mainly controlling the structure of the prepared core-shell microspheres through phase separation regulation during the evaporation of organic solvents. Figure 3 Shows the cross-sectional structure of the core-shell microspheres observed using cryosectioning combined with scanning electron microscopy, confirming that the prepared microspheres meet the theoretical expectations and have a core-shell structure. Figure 4 Shows the core-shell microspheres prepared using FITC-labeled parathyroid hormone drugs and Nile red-labeled polymers, confirming that the parathyroid hormone drugs are mainly distributed in the core of the core-shell microspheres, while the shell layer basically does not contain drugs. After the shell layer of the core-shell microspheres degrades, a large amount of drugs can be released in a short time to maximize the therapeutic effect of drug loading and inhibit the generation of side effects.

[0171] The in vitro release behavior of the core-shell microspheres is as Figure 7 shown. The core-shell microspheres prepared according to the scheme of Example 1 showed a controlled release behavior, with a low initial burst release and drug release after a predetermined release time. Figure 8 Shows the in vitro release behavior of Comparative Example 1. From Figure 7 and Figure 8 , it can be found that with the change of the molecular weight of the shell polymer, the core-shell microspheres showed different drug release behaviors. The controlled-release microspheres loaded with parathyroid hormone analog drugs prepared in Example 1 and Comparative Example 1 showed release behaviors in the first week and the second week after administration, respectively.

[0172] The core-shell microspheres prepared from the foregoing different embodiments will exhibit different drug release behaviors. Therefore, core-shell microspheres with different drug release times can be mixed to prepare a combined preparation of core-shell microspheres loaded with parathyroid hormone analog drugs with long-term controllable drug release ability, so as to obtain the effect of multiple drug releases. Such a setting can avoid certain side effects that occur when the blood drug concentration of teriparatide is higher than the endogenous parathyroid hormone concentration for more than 4 hours after a single administration, such as promoting osteoclast activity and further exacerbating osteoporosis.

[0173] Specifically, the preparation method of the combined preparation of core-shell microspheres loaded with parathyroid hormone analog drugs is as follows: accurately weigh a variety of core-shell microspheres with different predetermined drug release times, mix them according to a certain mass ratio, and use a vortex mixer to fully vortex after adding the release medium to make the microspheres form a uniform suspension to ensure uniform mixing.

[0174] The embodiment of the present invention also provides a combined preparation of core-shell microspheres loaded with parathyroid hormone analog drugs. In this embodiment, the preparation method of the combined preparation of core-shell microspheres loaded with parathyroid hormone analog drugs is as follows: accurately weigh three kinds of core-shell microspheres with different predetermined drug release times (for example, the core-shell microspheres prepared in Example 1, Comparative Example 1, and Comparative Example 2), mix them according to a mass ratio of 1:1:1, and use a vortex mixer to fully vortex after adding the release medium to make the core-shell microspheres form a uniform suspension to ensure uniform mixing.

[0175] That is to say, the combined preparation of core-shell microspheres loaded with parathyroid hormone analog drugs prepared by the above method includes a first core-shell microsphere (the core-shell microsphere prepared in Example 1), a second core-shell microsphere (the core-shell microsphere prepared in Comparative Example 1), and a third core-shell microsphere (the core-shell microsphere prepared in Comparative Example 2) with a ratio of 1:1:1, and the drug release times of the first core-shell microsphere, the second core-shell microsphere, and the third core-shell microsphere are different. Among them, the components and weight percentages of the first core-shell microsphere are: the proportion of parathyroid hormone analog drug is 4.76%-16.67%, and the proportion of high molecular polymer material is 83.33%-95.24%; the components and weight percentages of the second core-shell microsphere are: the proportion of parathyroid hormone analog drug is 4.76%-16.67%, and the proportion of high molecular polymer material is 83.33%-95.24%; the components and weight percentages of the third core-shell microsphere are: the proportion of parathyroid hormone analog drug is 4.76%-16.67%, and the proportion of high molecular polymer material is 83.33%-95.24%.

[0176] Such as Figure 9As shown, the in vitro drug release behavior of three core-shell microspheres with different predetermined drug release times, Example 1 (predetermined drug release time of 7 days), Comparative Example 1 (predetermined drug release time of 14 days) and Comparative Example 2 (predetermined drug release time of 21 days), after being fully mixed, is shown. Figure 9 The in vitro release curves in the figure show that the combination preparation of core-shell microspheres can release drugs three times within 21 days, achieving a release behavior similar to pulse release. Similarly, according to demand, core-shell microspheres with other different drug release times can be selected for mixing to prepare different combination preparations, thereby expanding the scope of use of combination preparations, meeting the needs of more different disease treatments, and providing strong support for personalized medicine.

[0177] Further, the weight percentage of each component in the first core-shell microsphere, the second core-shell microsphere, and the third core-shell microsphere can be dynamically adjusted according to their loading efficiency and release behavior. That is, the weight percentage of each component in the first core-shell microsphere, the second core-shell microsphere, and the third core-shell microsphere can be the same or different.

[0178] In summary, the present invention provides a combination preparation of core-shell microspheres loaded with parathyroid hormone analogs, and a combined release curve similar to a pulse is obtained by mixing and injecting core-shell microspheres with different drug release times, thereby achieving a release behavior similar to pulse release, so as to overcome the side effects such as osteoclast activation caused by the sustained-release microspheres of a single polymer material, which leads to a continuous and stable blood concentration of parathyroid hormone analogs. For example, three types of core-shell microspheres loaded with parathyroid hormone analogs (i.e., Example 1, Comparative Example 1, and Comparative Example 2) with uniform size, high drug loading, and long-term controlled release with drug release times of 7 days, 14 days, and 21 days are mixed, so that the drug is released every 7 days after injection to simulate a once-weekly dosing mode.

[0179] The present invention also provides a method for preparing long-acting core-shell microspheres loaded with parathyroid hormone analogs, and the particle size of the core-shell microspheres loaded with parathyroid hormone analogs can be directional controlled by adjusting the pore size of the microporous membrane and the transmembrane pressure to facilitate large-scale production.

[0180] Compared with the prior art, the present invention achieves the preparation of core-shell microspheres with controlled drug release capability by regulating the phase separation between multiple polymers during the preparation process of core-shell microspheres. The drug is controlled to be concentrated in the center of the core-shell microspheres, and after the shell of the core-shell microspheres is degraded, a large amount of drug is released in a short time to maximize the therapeutic effect of the drug and inhibit the occurrence of side effects. At the same time, the premixed membrane emulsification technology is combined in the preparation process. 1 / O / W 2After the formation of the pre-emulsion, the emulsion is pushed through a microporous membrane under nitrogen pressure to obtain emulsion droplets with a uniform particle size, and thus core-shell microspheres with a uniform particle size and a narrow particle size distribution are obtained.

[0181] The above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A combined preparation of core-shell microspheres loaded with parathyroid hormone analogs, characterized in that: include: The first core-shell microspheres, the components and weight percentages are: the parathyroid hormone analog drug accounts for 4.76%-16.67%, and the high molecular polymer material accounts for 83.33%-95.24%; The second core-shell microspheres, the components and weight percentages are: the parathyroid hormone analog drug accounts for 4.76%-16.67%, and the high molecular polymer material accounts for 83.33%-95.24%; The third core-shell microspheres, the components and weight percentages are: the parathyroid hormone analog drug accounts for 4.76%-16.67%, and the high molecular polymer material accounts for 83.33%-95.24%; Among them, the ratio of the first core-shell microsphere, the second core-shell microsphere and the third core-shell microsphere is 1:1:1, and the first core-shell microsphere, the second core-shell microsphere and the third core-shell microsphere have different drug release times.

2. The combined preparation according to claim 1, characterized in that The high molecular polymer material includes polylactic acid-hydroxyacetic acid copolymer and polylactic acid, and the polylactic acid includes racemic polylactic acid and / or left-handed polylactic acid; the molecular weight of the polylactic acid-hydroxyacetic acid copolymer is 10000-40000Da; the molecular weight of the polylactic acid unit in the polylactic acid is 3000-800000Da, and the molecular weight of the polylactic acid unit in the third core-shell microsphere is greater than the molecular weight of the polylactic acid unit in the second core-shell microsphere, and the molecular weight of the polylactic acid unit in the second core-shell microsphere is greater than the molecular weight of the polylactic acid unit in the first core-shell microsphere.

3. The combined preparation according to claim 1, characterized in that The drug release times of the first core-shell microsphere, the second core-shell microsphere and the third core-shell microsphere are in multiple relationship.

4. The combined preparation according to claim 1, characterized in that The first core-shell microsphere, the second core-shell microsphere and the third core-shell microsphere all include a core structure and a shell structure, and the parathyroid hormone analog drug exists in the core structure.

5. The combined preparation according to claim 1, characterized in that The average particle size of the first core-shell microsphere, and / or the second core-shell microsphere, and / or the third core-shell microsphere is 5 to 200 μm; the particle size distribution coefficient Span of the first core-shell microsphere, and / or the second core-shell microsphere, and / or the third core-shell microsphere is ≤1.

5.

6. The combined preparation according to claim 1, characterized in that The embedding rate of the first core-shell microsphere, and / or the second core-shell microsphere, and / or the third core-shell microsphere is ≥70%, and the drug burst released in the first 2 hours of the first core-shell microsphere, and / or the second core-shell microsphere, and / or the third core-shell microsphere does not exceed 10% of the total drug load.

7. A method for preparing core-shell microspheres loaded with parathyroid hormone analogs, for preparing the first core-shell microspheres / second core-shell microspheres / third core-shell microspheres as described in any one of claims 1 to 6, characterized in that: The following steps are involved: S1) dissolving the parathyroid hormone analog drug in deionized water to form an inner water phase W1; S2) a plurality of polymer materials and an organic solvent are mixed for a second time to form an oil phase solution, the inner water phase W1 obtained in step S1 is added to the oil phase solution, and a low temperature homogenization method is adopted to form W1 / O colostrum; S3) adding the W1 / O colostrum to the external aqueous phase, emulsifying to form a W1 / O / W2 pre-emulsion, passing the W1 / O / W2 pre-emulsion through a hydrophilic microporous membrane using a premixed membrane emulsification technique to form uniform W1 / O / W2 emulsion droplets, and solidifying the W1 / O / W2 emulsion droplets to form core-shell microspheres; the external aqueous phase comprises an emulsifier, an osmotic pressure regulator and deionized water; S4) sequentially centrifuging, washing and freeze-drying the core-shell microspheres to form parathyroid hormone analog drug-loaded core-shell microspheres.

8. The method according to claim 7, characterized in that The concentration of the parathyroid hormone analog drug is 10-200 mg / mL, and the concentration of the various high molecular polymer materials is 1-500 mg / mL.

9. The method according to claim 7, characterized in that: The high molecular polymer material includes polylactic acid-glycolic acid copolymer and polylactic acid, wherein the polylactic acid includes racemic polylactic acid and / or L-polylactic acid; the molar ratio of lactide to glycolide in the polylactic acid-glycolic acid copolymer is 90:10 to 10:90; the molecular weight of the polylactic acid-glycolic acid copolymer is 10000 to 40000 Da; and the molecular weight of the polylactic acid unit in the polylactic acid is 3000 to 800000 Da.

10. The method according to claim 7, characterized in that The osmotic pressure regulator includes sodium chloride and / or potassium chloride, and the mass concentration of the osmotic pressure regulator in the external water phase is 0.5-5.0 wt %.

Images