A long-acting sustained-release liposome of semaglutide and a preparation method and application thereof

Abstract

The application relates to the technical field of pharmaceutical preparations, in particular to a long-acting and slow-release liposome of semaglutide and a preparation method and application thereof. The liposome comprises semaglutide, phospholipid, cholesterol, charge-regulating lipid and rigidity-regulating lipid; the charge-regulating lipid is selected from at least one of distearoyl phosphatidylglycerol and dipalmitoyl phosphatidylglycerol; the molar ratio of the charge-regulating lipid and the phospholipid is 1:(5-20); the rigidity-regulating lipid is selected from at least one of distearoyl phosphatidylcholine and hydrogenated soybean phosphatidylcholine; the molar ratio of the rigidity-regulating lipid and the phospholipid is 1:(3-10); the average particle size of the liposome is 80-180 nm, and the polydispersity coefficient is less than or equal to 0.20. The charge-regulating lipid and the rigidity-regulating lipid are introduced into the bilayer of the liposome, the synergistic effect of the two is utilized to construct a hierarchical buffer release structure, and long-acting and slow-release, high encapsulation rate and good stability of the semaglutide are realized.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical formulation technology, and in particular to a long-acting sustained-release liposome of semaglutide, its preparation method, and its application. Background Technology

[0002] Smegglutide is a glucagon-like peptide-1 (GLP-1) receptor agonist used to treat type 2 diabetes and obesity. Smegglutide promotes insulin secretion in a glucose concentration-dependent manner by activating the GLP-1 receptor, while simultaneously inhibiting glucagon secretion, delaying gastric emptying, and achieving weight loss through central appetite suppression.

[0003] Currently, commercially available semaglutide formulations mainly include subcutaneous injections and oral tablets. While subcutaneous injections have achieved once-weekly dosing, there is still room for further reduction in injection frequency, and patients have a clinical need for even lower dosing regimens. Simultaneously, existing semaglutide injection formulations exhibit some fluctuations in blood drug concentration after administration. Further stabilizing the drug release curve would help improve efficacy and reduce the safety risks associated with excessively high blood drug concentrations. Oral tablets have extremely low bioavailability. Although absorption enhancers can improve oral absorption to some extent, this regimen still falls short of fully meeting clinical needs, with most of the administered dose remaining unabsorbed and wasting drug resources.

[0004] To address the aforementioned issues, those skilled in the art have attempted to load semaglutide into liposomes to improve its in vivo behavior. However, existing liposome formulations generally suffer from insufficient sustained-release performance: once inside the body, the bilayer structure of liposomes has limited ability to encapsulate semaglutide, resulting in rapid drug release and making it difficult to achieve truly long-acting sustained release. Furthermore, existing semaglutide liposome formulations are prone to aggregation, particle size increase, and drug leakage during storage, affecting the shelf life and reliability of clinical use.

[0005] Therefore, there is an urgent need in the field for a novel formulation that can achieve long-acting sustained release of semaglutide while possessing high encapsulation efficiency and good stability, in order to solve the aforementioned problems existing in the prior art. Summary of the Invention

[0006] The purpose of this invention is to address the problems existing in the prior art by providing a long-acting sustained-release liposome of semaglutide, its preparation method, and its application. By introducing charge-regulated lipids and rigid-regulated lipids into the liposome bilayer, a hierarchical buffer release structure is constructed using their synergistic effect, thereby achieving long-acting sustained release, high encapsulation efficiency, and good stability of semaglutide.

[0007] To achieve the above objectives, the present invention provides a long-acting sustained-release liposome of semaglutide, comprising semaglutide, phospholipids, cholesterol, charge-regulated lipids, and rigid-regulated lipids; The charge-regulated lipid is selected from at least one of distearylphosphatidylglycerol and dipalmitoylphosphatidylglycerol; the molar ratio of the charge-regulated lipid to the phospholipid is 1:(5-20). The rigid regulatory lipid is selected from at least one of distearylphosphatidylcholine and hydrogenated soybean phosphatidylcholine; the molar ratio of the rigid regulatory lipid to the phospholipid is 1:(3-10). The liposomes have an average particle size of 80-180 nm and a polydispersity index ≤0.20.

[0008] In some alternative embodiments, the phospholipid is selected from at least one of soybean phosphatidylcholine, egg yolk phosphatidylcholine, and myristoyl phosphatidylcholine.

[0009] In some optional embodiments, the charge-regulated lipid is distearate phosphatidylglycerol; the molar ratio of the charge-regulated lipid to the phospholipid is 1:(8-12).

[0010] In some optional embodiments, the rigid regulatory lipid is distearate phosphatidylcholine; the molar ratio of the rigid regulatory lipid to the phospholipid is 1:(4-8).

[0011] In some alternative embodiments, the molar ratio of cholesterol to phospholipid is 1:(1-3).

[0012] This invention also provides a method for preparing long-acting sustained-release liposomes of semaglutide, comprising the following steps: S1. Phospholipids, cholesterol, charge-regulated lipids, and rigidity-regulated lipids are dissolved in an organic solvent to obtain a lipid solution; S2. Mix the smegglutinin solution and the lipid solution, and homogenize them to obtain a liposome suspension; S3. The liposome suspension is subjected to solid-liquid separation to obtain liposomes; S4. Mix the liposomes and the lyophilization protectant, and freeze-dry to obtain smegglutinin long-acting sustained-release liposomes.

[0013] In some optional embodiments, in S1, the organic solvent is selected from at least one of chloroform, dichloromethane, and ethanol; the mass ratio of the phospholipid to the volume ratio of the organic solvent is (100-300) mg: (5-15) mL.

[0014] In some optional embodiments, in S2, the solvent of the semaglutide solution is selected from phosphate buffer (pH 7.4); the concentration of semaglutide in the semaglutide solution is 1-10 mg / mL; the mass ratio of the phospholipid in S1 to the semaglutide in S2 is (100-300):(10-30).

[0015] In some optional embodiments, in S2, the mixing includes: slowly adding the smegglutinin solution to the lipid solution while stirring at a speed of 300-700 r / min, and continuing to stir for 20-40 min.

[0016] In some alternative implementations, in S2, the homogenization process is carried out at a pressure of 500-1500 bar and for 3-10 cycles.

[0017] In some optional embodiments, in S3, the solid-liquid separation includes: passing the liposome suspension sequentially through 0.4-0.5 μm and 0.2-0.25 μm polycarbonate filter membranes, each membrane being squeezed 3-7 times.

[0018] In some optional embodiments, in S4, the freeze-drying protectant is selected from at least two of trehalose, sucrose, and mannitol; the mass ratio of the liposomes to the freeze-drying protectant is 100:(20-60); the freeze-drying includes the following procedure: pre-freezing at -40°C to -50°C for 2-4 hours, performing a first drying at -10°C to -30°C for 12-24 hours, and performing a second drying at 20-30°C for 4-8 hours.

[0019] The present invention also provides the use of the semaglutide long-acting sustained-release liposome or the semaglutide long-acting sustained-release liposome prepared by the above preparation method in the preparation of a medicament for the treatment of obesity and / or type 2 diabetes.

[0020] The present invention also provides a pharmaceutical formulation comprising the semaglutide long-acting sustained-release liposome or the semaglutide long-acting sustained-release liposome prepared by the aforementioned preparation method, and pharmaceutically acceptable excipients.

[0021] In some alternative embodiments, the pharmaceutical preparation is an injectable preparation, an oral preparation, or a transdermal preparation.

[0022] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention constructs a liposome with a hierarchical buffer release structure by simultaneously introducing charge-regulated lipids and rigid-regulated lipids into the liposome bilayer. Smegglutinin has an isoelectric point of approximately pH 5.4 and carries a net negative charge under neutral pH conditions (such as in vivo physiological environments and conventional release media). The electrostatic repulsion between this charge-regulated lipid and the surface negative charge generated by the anionic charge-regulated lipids causes smegglutinin to be primarily retained in the aqueous phase of the liposome, thus slowing its transmembrane release. Simultaneously, the rigid-regulated lipids have a higher phase transition temperature, which increases the order and density of the lipid bilayer, reduces membrane fluidity, and further hinders transmembrane diffusion of the drug. The charge-regulated lipids and rigid-regulated lipids synergistically achieve drug retention and release inhibition, respectively, resulting in no significant burst release in the initial release phase of the liposomes and maintaining a stable release curve within 72 hours, thus realizing the long-acting sustained release of smegglutinin.

[0023] (2) The introduction of charge-regulated lipids gives the liposome bilayer an appropriate amount of negative charge. During liposome formation, smegglutinin molecules are more effectively encapsulated in the aqueous phase within the liposome due to electrostatic interactions, reducing drug leakage during preparation and thus achieving a higher encapsulation efficiency. The encapsulation efficiency of the liposomes of this invention can reach over 75%, effectively reducing the proportion of free drug and improving the dosing efficiency per unit dose.

[0024] (3) The liposomes of the present invention impart a negative charge to the surface of the liposomes through charge regulation, thereby inhibiting the aggregation of liposomes during storage by utilizing electrostatic repulsion. At the same time, a suitable lyophilization protectant is used to form a glassy protective layer during the freeze-drying process, which effectively protects the integrity of the liposome bilayer structure and prevents drug leakage. The combined effect of these two factors enables the liposomes of the present invention to maintain good particle size uniformity and encapsulation stability under storage conditions of 2-8℃, thus extending the shelf life of the formulation.

[0025] (4) The preparation method of the present invention does not involve complex equipment or harsh process conditions, the parameters are controllable, it is easy to scale up production, and it has good prospects for industrial application. Attached Figure Description

[0026] Figure 1 This is a comparison chart of the cumulative release rate of liposomes in Examples 1-3 and Comparative Examples 1-3 of the present invention. Detailed Implementation

[0027] The following embodiments are provided to better understand the present invention and are not limited to the described embodiments. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0028] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0029] Example 1 This embodiment provides a method for preparing long-acting sustained-release liposomes of semaglutide, including the following steps: Weigh 200 mg of soybean phosphatidylcholine (SPC), cholesterol, distearylphosphatidylglycerol (DSPG), and distearylphosphatidylcholine (DSPC) into a round-bottom flask, wherein the molar ratio of DSPG to SPC is 1:10, the molar ratio of DSPC to SPC is 1:5, and the molar ratio of cholesterol to SPC is 1:1; add 10 mL of chloroform to the round-bottom flask and stir magnetically at room temperature until completely dissolved to obtain a lipid solution.

[0030] Weigh 20 mg of smegglutinin and dissolve it in phosphate buffer at pH 7.4 to obtain a smegglutinin solution with a concentration of 4 mg / mL.

[0031] Under a stirring speed of 500 rpm, the smegglutinin solution was slowly added dropwise to the lipid solution. After the addition was complete, stirring was continued for 30 min to form a colostrum. The obtained colostrum was transferred to a high-pressure homogenizer and homogenized 6 times at a pressure of 1000 bar to obtain a liposome suspension.

[0032] The liposome suspension was passed sequentially through 0.45 μm and 0.22 μm polycarbonate membranes, with each membrane being squeezed 5 times to obtain liposomes.

[0033] A lyophilization protectant (trehalose and sucrose, in a 1:1 mass ratio) was added to the liposomes, resulting in a liposome to lyophilization protectant mass ratio of 100:40. After thorough mixing, the mixture was dispensed into vials. The vials were placed in a freeze dryer and pre-frozen at -45°C for 3 hours, then heated to -20°C for a first drying process of 18 hours, and finally heated to 25°C for a second drying process of 6 hours to obtain smegglutinin long-acting sustained-release liposomes.

[0034] Example 2 This embodiment provides a method for preparing long-acting sustained-release liposomes of smegglutinin, which differs from Example 1 only in that the molar ratio of DSPG and SPC is adjusted to 1:15 and the molar ratio of DSPC and SPC is 1:7.5.

[0035] The remaining steps and conditions are exactly the same as in Example 1.

[0036] Example 3 This embodiment provides a method for preparing long-acting sustained-release liposomes of smegglutinin, which differs from Example 1 only in that: the freeze-drying protectant is adjusted to trehalose and mannitol in a mass ratio of 1:1, and the mass ratio of liposomes to freeze-drying protectant is 100:50.

[0037] The remaining steps and conditions are exactly the same as in Example 1.

[0038] Comparative Example 1 This comparative example provides a method for preparing smegglutinin liposomes, which differs from Example 1 only in that the addition of DSPG is omitted.

[0039] The remaining steps and conditions are exactly the same as in Example 1.

[0040] Comparative Example 2 This comparative example provides a method for preparing smegglutinin liposomes, which differs from Example 1 only in that the addition of DSPC is omitted.

[0041] The remaining steps and conditions are exactly the same as in Example 1.

[0042] Comparative Example 3 This comparative example provides a method for preparing smegglutinin liposomes, which differs from Example 1 only in that the addition of DSPG and DSPC is omitted.

[0043] The remaining steps and conditions are exactly the same as in Example 1.

[0044] Experimental Example 1 The liposomes prepared in Examples 1-3 and Comparative Examples 1-3 were reconstituted with water for injection to make the concentration of smegglutinin 2 mg / mL. The mixture was then gently shaken until completely dispersed to obtain the suspension of each liposome to be tested, which was then set aside for later use.

[0045] (1) Particle size and polydispersity index The average particle size and polydispersity index (PDI) of each liposome were determined by dynamic light scattering. The measurement temperature was 25℃, the equilibration time was 120s, and each sample was measured in triplicate, with the average value taken.

[0046] (2) Encapsulation rate Gel column preparation: Take Sephadex G-50 dextran gel, swell it, pack it into a column (1.5cm×30cm), and equilibrate it with pH 7.4 phosphate buffer.

[0047] Sample loading and elution: Load 0.5 mL of the liposome suspension to be tested onto the column, elute with pH 7.4 phosphate buffer at a flow rate of 1 mL / min, and collect the liposome eluent.

[0048] Membrane rupture treatment: Take the collected liposome eluent, add an equal volume of methanol / acetonitrile (1:1, v / v) mixed solvent, vortex mix, and then sonicate for 5 min to completely rupture the liposomes.

[0049] Content determination: The content of free drug and encapsulated drug in liposomes was determined by high performance liquid chromatography (HPLC). HPLC detection conditions were as follows: C18 reversed-phase column (4.6 mm × 150 mm, 5 μm); mobile phase A was 0.1% trifluoroacetic acid-water solution, mobile phase B was 0.1% trifluoroacetic acid-acetonitrile solution, gradient elution program was: 0-5 min, 5-25% B; 5-15 min, 25-45% B; 15-20 min, 45-70% B; 20-22 min, 70% B; 22-25 min, 5% B; detection wavelength was 214 nm; flow rate was 1 mL / min; column temperature was 40 ℃; injection volume was 20 μL.

[0050] Encapsulation efficiency calculation: Encapsulation efficiency (%) = (Amount of drug encapsulated in liposomes / Total amount of drug) × 100%.

[0051] The test results are shown in Table 1.

[0052] Table 1 Test Results

[0053] Table 1 shows that the liposomes of Examples 1-3 had an average particle size of 135.6-142.3 nm, a polydispersity index of 0.12-0.14, and an encapsulation efficiency of 82.5-86.7%, all exhibiting good particle size uniformity and high encapsulation efficiency. The encapsulation efficiency of Comparative Example 1 (DSPG omitted) was 65.3%, that of Comparative Example 2 (DSPC omitted) was 72.1%, and that of Comparative Example 3 (both DSPG and DSPC omitted) was only 52.6%, all significantly lower than those of the Examples. These results indicate that the introduction of charge-regulated lipids and rigid-regulated lipids can effectively improve the encapsulation efficiency of liposomes for smegglutinin, and that both have a synergistic effect.

[0054] Experiment Example 2 The liposomes prepared in Examples 1-3 and Comparative Examples 1-3 were reconstituted with water for injection. A liposome suspension containing 2 mg of semaglutide was transferred to a dialysis bag (molecular weight cutoff 100 kDa). The dialysis bag was sealed and placed in 100 mL of release medium, which was a phosphate buffer solution at pH 7.4 (containing 0.02% sodium azide as an antibacterial agent). The release system was placed in a 37°C constant-temperature shaker and shaken at 100 rpm for release.

[0055] At predetermined time points (2h, 12h, 24h, 48h, and 72h), 1 mL of release medium was collected, and an equal amount of fresh release medium was immediately added. The concentration of smegglutinin in the release medium at each time point was determined using the HPLC method described in Example 1, and the cumulative release rate was calculated using the following formula: Cumulative release rate (%) = (Cumulative amount of drug in the release medium / Total amount of drug in the liposomes) × 100%. Each sample was measured in triplicate, and the average value was taken.

[0056] The cumulative release rate test results are shown in Table 2. Meanwhile, to more clearly and intuitively demonstrate the differences in release among the groups, a comparison chart of the cumulative release rates of liposomes in Examples 1-3 and Comparative Examples 1-3 was drawn, as shown below. Figure 1 As shown.

[0057] Table 2. Cumulative Release Rate Test Results

[0058] The results above show that the cumulative release rates of Examples 1-3 at each time point were significantly lower than those of the comparative examples. Example 1 showed a cumulative release rate of only 12.5% ​​at 2 hours, indicating no significant burst release; the cumulative release rate at 72 hours was 81.7%, with a stable release curve. Comparative Example 1 (omitting DSPG) achieved a cumulative release rate of 28.6% at 2 hours, Comparative Example 2 (omitting DSPC) achieved a cumulative release rate of 22.8% at 2 hours, and Comparative Example 3 (omitting both DSPG and DSPC) achieved a cumulative release rate as high as 35.2% at 2 hours, all significantly higher than the examples. These results indicate that the synergistic effect of charge-regulated lipids and rigidly regulated lipids can effectively delay the release rate of smegglutinin, achieving a long-lasting sustained-release effect.

[0059] Experimental Example 3 The liposomes prepared in Examples 1-3 and Comparative Examples 1-3 were placed in vials, sealed by stoppering and capping, and stored in the dark at 2-8°C for 6 months. Samples were taken before storage (0 months) and after 6 months of storage, reconstituted with water for injection, and the particle size and encapsulation efficiency were determined according to the method described in Experimental Example 1. The particle size change rate and encapsulation efficiency retention rate were calculated as follows: Particle size change rate (%) = (particle size at 6 months - particle size at 0 months) / particle size at 0 months × 100%, Encapsulation efficiency retention rate (%) = encapsulation efficiency at 6 months / encapsulation efficiency at 0 months × 100%.

[0060] The results of the storage stability test are shown in Table 3.

[0061] Table 3 Storage stability test results

[0062] Table 3 shows that after 6 months of storage at 2-8℃, Examples 1-3 exhibited excellent long-term storage stability with a particle size change rate of only 5.1-6.7% and an encapsulation retention rate of 94.7-96.0%. Comparative Examples 1-3 showed significantly increased particle size and a substantial decrease in encapsulation efficiency. These results indicate that the introduction of charge-regulated lipids and rigidity-regulated lipids can significantly improve the storage stability of liposomes.

[0063] Experiment Example 4 The liposomes prepared in Example 1 were reconstituted with water for injection to a concentration of smegglutinin of 2 mg / mL. The mixture was gently shaken until completely dispersed and then placed at 25°C. Samples were taken at 0 h, 4 h, 8 h, 12 h, and 24 h after reconstitution, and the particle size and polydispersity index (PDI) were determined according to the method described in Example 1. Each time point was measured in triplicate, and the average value was taken. The results of the reconstitution stability test are shown in Table 4.

[0064] Table 4 Results of Reconstitution Stability Test

[0065] As shown in Table 4, within 24 hours after the liposomes of Example 1 were reconstituted, the average particle size only increased slightly from 135.6 nm to 143.1 nm, and the polydispersity index remained between 0.12 and 0.15, with no significant changes. This indicates that the liposomes of the present invention have good stability after reconstitution, which facilitates preparation and operation before clinical use.

[0066] In summary, this invention constructs a semaglutide liposome with a hierarchical buffer-release structure by introducing charge-regulated lipids and rigidity-regulated lipids into the liposome bilayer. The electrostatic repulsion provided by the charge-regulated lipids and the enhancement of bilayer rigidity by the rigidity-regulated lipids are utilized. These liposomes exhibit a stable release curve and no significant burst release in in vitro release experiments, achieving long-acting sustained release of semaglutide. They also demonstrate high encapsulation efficiency and good storage and reconstitution stability. Furthermore, the preparation method of this invention is simple, with controllable parameters, suitable for industrial production, providing a new formulation option for the clinical application of semaglutide.

[0067] Finally, it should be noted that the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A long-acting sustained-release liposome of semaglutide, characterized in that, Including smegglutinin, phospholipids, cholesterol, charge-regulated lipids, and rigidly regulated lipids; The charge-regulated lipid is selected from at least one of distearylphosphatidylglycerol and dipalmitoylphosphatidylglycerol; the molar ratio of the charge-regulated lipid to the phospholipid is 1:(5-20). The rigid regulatory lipid is selected from at least one of distearylphosphatidylcholine and hydrogenated soybean phosphatidylcholine; the molar ratio of the rigid regulatory lipid to the phospholipid is 1:(3-10). The liposomes have an average particle size of 80-180 nm and a polydispersity index ≤0.

20.

2. The semaglutide long-acting sustained-release liposome according to claim 1, characterized in that, The phospholipid is selected from at least one of soybean phosphatidylcholine, egg yolk phosphatidylcholine, and myristoyl phosphatidylcholine.

3. The semaglutide long-acting sustained-release liposome according to claim 1, characterized in that, The charge-regulated lipid is distearate phosphatidylglycerol; the molar ratio of the charge-regulated lipid to phospholipid is 1:(8-12).

4. The semaglutide long-acting sustained-release liposome according to claim 1, characterized in that, The rigid regulatory lipid is distearate phosphatidylcholine; the molar ratio of the rigid regulatory lipid to phospholipid is 1:(4-8).

5. The semaglutide long-acting sustained-release liposome according to claim 1, characterized in that, The molar ratio of cholesterol to phospholipids is 1:(1-3).

6. A method for preparing semaglutide long-acting sustained-release liposomes as described in any one of claims 1-5, characterized in that, Includes the following steps: S1. Phospholipids, cholesterol, charge-regulated lipids, and rigidity-regulated lipids are dissolved in an organic solvent to obtain a lipid solution; S2. Mix the smegglutinin solution and the lipid solution, and homogenize them to obtain a liposome suspension; S3. The liposome suspension is subjected to solid-liquid separation to obtain liposomes; S4. Mix the liposomes and the lyophilization protectant, and freeze-dry to obtain smegglutinin long-acting sustained-release liposomes.

7. The preparation method according to claim 6, characterized in that, In S2, the homogenization process is carried out at a pressure of 500-1500 bar and the number of cycles is 3-10.

8. The preparation method according to claim 6, characterized in that, In S4, the freeze-drying protectant is selected from at least two of trehalose, sucrose, and mannitol; the freeze-drying includes the following procedure: pre-freezing at -40°C to -50°C for 2-4 hours, performing a first drying at -10°C to -30°C for 12-24 hours, and performing a second drying at 20-30°C for 4-8 hours.

9. The use of the semaglutide long-acting sustained-release liposome according to any one of claims 1-5 or the semaglutide long-acting sustained-release liposome prepared by the preparation method according to any one of claims 6-8 in the preparation of a medicament for treating obesity and / or type 2 diabetes.

10. A pharmaceutical preparation, characterized in that, The product comprises semaglutide long-acting sustained-release liposomes according to any one of claims 1-5 or semaglutide long-acting sustained-release liposomes prepared by the preparation method according to any one of claims 6-8, and pharmaceutically acceptable excipients.

Images