In-situ phase change gel sustained release preparation and preparation method and application thereof

Abstract

The invention provides an in-situ injection phase change gel sustained-release preparation as well as a preparation method and application thereof, and particularly relates to a stable injectable gel sustained-release preparation prepared from phospholipid, oil for injection and active pharmaceutical ingredients, and the stable injectable gel sustained-release preparation has the characteristics of good biocompatibility, remarkable sustained-release effect, wide application range, simplicity in preparation and the like, and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to an in situ injection phase change gel sustained-release preparation, in particular to a stable injectable gel sustained-release preparation prepared from phospholipids and an injection oil, belonging to the field of medical technology. BACKGROUND

[0002] Peptide drugs have good selectivity for human specific targets, and their large molecular size and diverse structures enhance their interaction with specific active sites, making peptide drugs have better biological activity and lower toxicity than small molecules. However, peptide drugs have low stability, are easily degraded, have short half-lives in vivo, and have low bioavailability, especially in the treatment of chronic diseases, which often require long-term frequent injections, resulting in low compliance, high economic cost, and other adverse reactions. Developing injectable long-acting sustained-release preparations for peptide drugs is an important means to improve bioavailability, facilitate medication, stabilize drug concentration, reduce toxicity and side effects, and improve patient compliance.

[0003] At present, various sustained-release systems for injection have been designed and manufactured to release drugs. Among them, microspheres are the most studied and successfully marketed injectable long-acting sustained-release preparations, and PLGA (poly-lactic-co-glycolic acid) microspheres are the most widely used. Exenatide microspheres for injection (trade name: Bydureon) is the first long-acting hypoglycemic drug in China, which is one of the most successful varieties. Exenatide is a glucagon-like peptide-1 (GLP-1) receptor agonist polypeptide used to improve blood glucose control in patients with type 2 diabetes, and its ordinary dosage form exenatide injection (trade name: Byetta) requires subcutaneous injection twice a day. Subsequently, Amylin successfully developed exenatide microspheres for injection using PLGA as a carrier, which only needs to be injected once a week, greatly reducing the frequency of administration and improving patient compliance. Although PLGA microspheres have good sustained-release effect, their preparation process is complex, the drug loading capacity is low, and the organic solvents used in the preparation process are left in the preparation. In addition, the lactic acid and glycolic acid produced during the degradation process can cause a decrease in the pH value of the injection site, which may cause an inflammatory response. If a biological macromolecule drug is encapsulated, the acidic environment caused by the decrease in pH value can cause the activity of the biological macromolecule drug to decrease and denature. The above shortcomings limit the application of PLGA microspheres.

[0004] Multivesicular liposome (MVL) is a sustained-release preparation first discovered and studied by Kim team of University of Washington in 1983. It is a spherical preparation composed of multiple non-concentric aqueous chambers, i.e. vesicles, with phospholipid, cholesterol and neutral lipid as the vesicular material. As the vesicles break one by one, the drug is released to achieve the purpose of sustained release. Compared with traditional liposomes, MVL has the advantages of high encapsulation efficiency and less drug leakage, and is suitable for encapsulating water-soluble small molecule drugs and bioactive macromolecules. One of the most successful and marketed MVLs is bupivacaine liposome injection suspension (trade name: Exparel) prepared by Pacira Company in the United States using DepoFoam multivesicular liposome technology, which can continuously release drugs for 72 hours. Patent CN102274183B reports a preparation method and application of a multivesicular liposome, which encapsulates exenatide and releases continuously for 160 hours in vitro. Patent CN110339166B reports a liraglutide multivesicular liposome, which has a hypoglycemic effect for 312 hours in vivo. Although MVL achieves good sustained-release effect, it still has many shortcomings: first, the preparation of MVL at least contains amphiphilic lipids (such as phospholipids), neutral lipids (such as triglycerides), emulsifiers and osmotic pressure regulators, etc. The prescription and preparation process are complex, and there is a problem of residual organic solvents; secondly, there is a difference in drug concentration between the inner and outer water phases of MVL, and the problem of drug penetration still exists, and it is easy to appear sedimentation and aggregation during storage, which affects the stability of the preparation and is not convenient for transportation and storage; in addition, the existing MVL shows that the drug release time is usually difficult to exceed 2 weeks in vivo and in vitro experiments, and the release time is short. The above shortcomings limit the development of MVL.

[0005] Vesicular phospholipid gels (VPG) are also being studied. VPG is a semi-solid phospholipid dispersion system, which is similar in shape to vesicles, but different from traditional liposome gels and ordinary liposome chamber structures. Due to the formation of a unique three-dimensional network structure, VPG can act as a drug reservoir to achieve sustained release of drugs. Patents CN102626381A, CN102697741A and CN102716095A respectively report injectable vesicular phospholipid gels of latamoxef sodium, oxaliplatin and paclitaxel, but the drug release time of these preparations does not exceed 24 hours, and the release time is short.

[0006] A kind of in-situ phase change gel sustained-release system (CN102526753A) with high concentration phospholipid as main matrix and ethanol as solvent is developed in our laboratory. It has good biocompatibility, significant sustained-release effect, good in-vivo degradation, etc. and has good sustained-release effect when applied to protein and peptide drugs. For example, octreotide acetate can be steadily released in rats, rabbits and dogs for about a month, with little drug burst, which is better than the commercially available octreotide acetate microspheres (M Wang, et al. Pharmacokinetic and pharmacodynamic study of a phospholipid-based phase separation gel for once a month administration of octreotide, Journal of Controlled Release 230 (2016) 45-56); Exenatide acetate phospholipid gel has little burst release and can be released in rats for up to a month, maintaining stable blood glucose-lowering effect for more than 20 days (M Hu, et al. Long-Acting Phospholipid Gel of Exenatide for Long-Term Therapy of Type II Diabetes, Pharmaceutical research 33 (2016): 1318-1326). In the prior art including CN102526753A, the common solvents used in phospholipid gel system are organic solvents such as ethanol and NMP. The reason for using such organic solvents is not only due to their good solubility for various drugs and excipients, but also due to their good biocompatibility and safety. However, we also found in our research that on the one hand, such phospholipid gel preparations using ethanol and NMP as solvents still inevitably cause irritation to some extent, leading to excessive inflammation at the injection site. On the other hand, it is known to those skilled in the art that ethanol and other organic solvents are common chemical factors that cause peptide degradation and inactivation. The presence of ethanol and other organic solvents tends to cause adsorption and aggregation of peptide drugs, thereby reducing stability and efficacy, and is also not conducive to the transportation and storage of the preparation.

[0007] Therefore, on the basis of the prior art, how to ensure the stability of peptide drugs in the preparation, improve the preparation efficiency of the injection drug sustained-release system, reduce local irritation, and achieve good sustained-release effect is a technical problem to be solved. SUMMARY

[0008] The purpose of the present application is to overcome the defects of the prior art and provide an in-situ injection phase change gel sustained-release preparation, a preparation method and application thereof.

[0009] It is accidentally found in the research that phospholipid mixed with ethyl oleate and / or ethyl linoleate, an injection oil, at a certain temperature condition can form a clear, transparent and uniform system, which remains clear when cooled to room temperature and has good fluidity, and can replace the organic solvent in the prior art as a drug solvent, and the viscosity of the formed gel system is suitable for injection and has good biocompatibility. Moreover, the inventors find through a large number of tests that for peptide drugs, ethyl oleate and / or ethyl linoleate as a solvent in the phospholipid gel system can achieve a technical effect far superior to other similar injection oils, can significantly improve the stability of peptide drugs, and can replace the organic solvents such as ethanol and NMP commonly used in the gel drugs in the prior art, and unexpected technical effects are achieved. When the system encounters a small amount of body fluid, the fluidity will decrease significantly and then solidify. When the drug is dissolved or dispersed in the system and injected into the body, the body fluid penetrates to reduce the viscosity of the preparation at the injection site, and a semi-solid gel is formed, thereby becoming a carrier and barrier for drug sustained release and effectively controlling the release of the drug.

[0010] According to the above-mentioned creative finding, we take octreotide acetate as a model drug to prepare a preparation of phospholipid and ethyl oleate in a ratio of 1:1 (w:w), and after subcutaneous injection of 0.8 mL to rats, the drug can be released for nearly one month. Under the same dosage, the release curve of octreotide acetate is more gentle than that of the phospholipid gel prepared according to the patent CN102526753A, the release time is longer, the irritability to the injection site is significantly reduced, and the stability is significantly increased.

[0011] According to the above-mentioned research, we find that without using organic solvents such as ethanol, phospholipid mixed with ethyl oleate and / or ethyl linoleate, an injection oil, at a suitable ratio still has good fluidity, and can prolong the release time of the drug after injection. Without using organic solvents, the risk of degradation of peptide biological macromolecular drugs is also greatly reduced. We respectively load thymalfasin, liraglutide and semaglutide, three kinds of peptide drugs, into the phospholipid and injection oil system of the present application and the phospholipid gel prepared according to the patent CN102526753A, and the results show that the system of the present application can significantly improve the stability of biological macromolecular drugs.

[0012] One of the purposes of the present application is to provide an in-situ injection phase change gel sustained release system prepared from phospholipid, injection oil and a drug active ingredient, which does not use organic solvents, has very low local irritability, greatly improves the stability of the drug, and the drug active ingredient is a peptide.

[0013] One of the purposes of the present application is to provide a phospholipid gel sustained release preparation containing a peptide active ingredient, which has a wide range of applications, rapidly undergoes phase change after injection, can achieve long-acting sustained release, and is easy to inject.

[0014] The phospholipid gel sustained release formulation according to the present application comprises phospholipid 10-60 parts by weight, injection oil 45-90 parts by weight, and active pharmaceutical ingredient 0.0025-20 parts by weight. The active pharmaceutical ingredient is a peptide.

[0015] In the phospholipid gel sustained release formulation system, the ratio of phospholipid to injection oil in the carrier is 2:8-6:4 (w:w), preferably, the ratio of phospholipid to injection oil is 3:7-5.5:4.5 (w:w), more preferably 3:7-5:5 (w:w).

[0016] In the phospholipid gel sustained release formulation system, the content of injection oil is 40%-90% by weight, preferably 45%-80%, preferably 50%-70%, more preferably 50%-60%.

[0017] The phospholipid suitable for the phospholipid gel sustained release formulation according to the present application includes, but is not limited to, one or more combinations of natural phospholipid, semi-synthetic phospholipid, and synthetic phospholipid.

[0018] The natural phospholipid is selected from egg yolk lecithin, soybean phospholipid, or a combination thereof; the semi-synthetic phospholipid is selected from hydrogenated egg yolk lecithin, hydrogenated soybean phospholipid, or a combination thereof; and the synthetic phospholipid is selected from one or more combinations of dipalmitoyl phosphatidyl ethanolamine, dipalmitoyl phosphatidyl choline, distearoyl phosphatidyl choline, dimyristoyl phosphatidyl choline, dioleoyl phosphatidyl ethanolamine, dipalmitoyl phosphatidyl glycerol, and dipalmitoyl phosphatidyl.

[0019] In a specific embodiment, the present application preferably uses soybean phospholipid S100 and / or egg yolk phospholipid E80.

[0020] The injection oil suitable for the phospholipid gel sustained release formulation according to the present application includes one or a combination of ethyl oleate and ethyl linoleate. Preferably, the injection oil is ethyl oleate.

[0021] Peptides suitable for use in the preparation of the phospholipid gel sustained release formulations of the present application include, but are not limited to, hirudin, histrelin, ghrelin-2, ghrelin-6, cetrorelix, bivalirudin, arginine vasopressin, lysine vasopressin, desmopressin, pasireotide, goserelin, leuprolide, deslorelin, buserelin, triptorelin, goserelin, alarelin, semorelin, nafarlin, luteinizing hormone releasing hormone, fentanyl, octreotide, lanreotide, exenatide, liraglutide, albiglutide, dulaglutide, lixisenatide, benralizumab, semaglutide, tirzepatide, lomacanatide, human glucagon-like peptide-1, growth hormone, somatostatin, ziconotide, sincalide, thymopentin, abarelix, alprostadil, leucine enkephalin, methionine enkephalin, tetroxinal, gonadotropin-releasing hormone, thyrotropin-releasing hormone, growth hormone-releasing hormone (GHRH), growth hormone inhibiting hormone (GHIH), melanocyte-inhibiting hormone (MRIH), melanocyte-releasing hormone (MRH), corticotropin-releasing hormone (CRH), secretin, substance P, neurotensin, human brain natriuretic peptide, atrial natriuretic peptide, angiotensin I, angiotensin II, angiotensin III, sleep peptide, memory enhancing peptide, dynorphin, endorphin, angiotensin, enfuvirtide, CJC-1295, elcatonin, thymosin beta 4, vasopressin, vasopressin, vasopressin, ornithine vasopressin, melanolitan II, spleen penta-peptide, pramlintide, vapreotide, corticotropin, silk corticotropin 18 peptide, corticotropin 18 peptide, zinc corticotropin 24 peptide, corticotropin 24 peptide, corticotropin 25 peptide, corticotropin 28 peptide, vasoactive intestinal peptide, brain natriuretic peptide, bradykinin, orphanin, rezafirenin, trofinetide, motisafin, tildiparatide, or one or more combinations of pharmaceutically acceptable salts of the above.

[0022] Preferably, the peptide is selected from one or more of semaglutide, tirzepatide, leuprolide, goserelin, triptorelin, octreotide, lanreotide, pasireotide.

[0023] Further, the above-mentioned peptide drugs can be in the form of pharmaceutically acceptable salts thereof, including but not limited to hydrochloride, sulfate, acetate, salicylate, sulfonate, citrate and other pharmaceutically acceptable salt forms.

[0024] Further, the in-situ phase transition gel of the present application can further comprise pharmaceutically acceptable excipients.

[0025] Further, the pharmaceutically acceptable excipients are selected from one or more of preservatives, surfactants, antioxidants, metal ion chelating agents.

[0026] Further, the in-situ injection phase transition gel sustained release system of the present application does not contain organic solvents.

[0027] Further, the organic solvent is selected from one or more of methanol, ethanol, propylene glycol, tert-butyl alcohol, dimethyl sulfoxide and N,N-dimethylpyrrolidone.

[0028] Further, the in-situ injection phase change gel sustained release system of the present application is preferably used for preparing drugs for treating diabetes, tumors, cardiovascular and cerebrovascular diseases.

[0029] The in-situ injection phase change gel sustained release system of the present application is a novel dosage form, in which the pharmaceutically active ingredient is dissolved or dispersed in a composition of phospholipid and injection oil to form an injectable preparation with good fluidity. After injection into the body, body fluid infiltrates into the preparation, and a semi-solid gel encapsulating the pharmaceutically active ingredient is immediately formed at the injection site, thus achieving good sustained release effect.

[0030] The pharmaceutically active ingredient and the blank phospholipid injection oil base can be separately stored, and then dissolved or dispersed uniformly before administration.

[0031] In a specific embodiment, the preparation method of the present application comprises the following steps:

[0032] (1) mixing phospholipid and injection oil, and dissolving the phospholipid completely at 0-120°C, wherein the mixing method is selected from one or more of stirring, vortexing and ultrasonication, and the mixing temperature is preferably 30-60°C, to obtain a clear and transparent phospholipid gel;

[0033] (2) preparing drug microparticles by a common crystallization or crushing method in pharmacy;

[0034] (3) mixing the phospholipid gel of step (1) and the drug microparticles of step (2) uniformly, removing air bubbles in the preparation by standing for a while, dispensing, and sealing to obtain the product.

[0035] The in-situ injection phase change gel sustained release preparation of the present application can be administered by subcutaneous injection, intramuscular injection, intracavitary injection through tissue, or intracavitary injection into an open body cavity without penetrating tissue.

[0036] Through creative research, the present application uses phospholipid and injection oil as main raw materials, and uses peptide drugs such as octreotide acetate, thymalfasin, liraglutide and semaglutide as model drugs, to obtain an in-situ injection phase change gel sustained release preparation with good fluidity and easy injection administration through simple mixing. Animal in vivo experiments prove that the preparation has good sustained release effect. The irritation to the injection site is investigated, and the results show that the preparation has good biocompatibility and does not irritate the administration site. The drug stability in the preparation is investigated, and the results show that the preparation has good stability at 4°C and 25°C.

[0037] The present application provides an in-situ gel preparation which can universally realize the sustained release of various drugs, and can well solve the problems of local irritation and decreased drug stability caused by the existing in-situ gel preparation of drugs, and has a good application prospect.

[0038] The in-situ phase change gel sustained release preparation provided by the present application has the following remarkable advantages:

[0039] (1) Good fluidity, easy to inject and administer, after injection into the body, body fluid infiltrates to make the preparation at the injection site solidify rapidly and phase change to form a semi-solid gel, thereby becoming a drug release depot for slow release of drugs.

[0040] (2) The raw and auxiliary materials used all have good biocompatibility and biodegradability, do not contain organic solvents, and almost do not cause irritation at the administration site.

[0041] (3) Wide application range, can significantly improve the stability of active drugs, and different drugs are not easily degraded.

[0042] (4) Simple preparation method, easy to realize industrialized production. BRIEF DESCRIPTION OF DRAWINGS

[0043] Figure 1 It is the appearance chart of the in-situ phase change gel sustained release preparation before and after phase change.

[0044] Figure 2 It is the photo of the in-situ phase change gel injected into water through a 26G injection needle.

[0045] Figure 3 It is the elastic modulus and viscous modulus of Example 1 at different time points after being placed in PBS.

[0046] Figure 4 It is the elastic modulus and viscous modulus of Example 1 and Example 1 after subcutaneous injection for 15 minutes.

[0047] Figure 5 It is the HE staining chart (×5) of the skin at the injection site after subcutaneous injection of the in-situ phase change gel sustained release preparation of the present application and the phospholipid gel of patent CN102526753A for 7 days and 14 days.

[0048] Figure 6 It is the HE staining chart (×200) of the skin tissue at the injection site after subcutaneous injection of the in-situ phase change gel sustained release preparation of the present application and the phospholipid gel of patent CN102526753A for 7 days and 14 days.

[0049] Figure 7 It is the HE staining chart (×200) of the subcutaneous tissue at the injection site after subcutaneous injection of the in-situ phase change gel sustained release preparation of the present application and the phospholipid gel of patent CN102526753A for 7 days and 14 days.

[0050] Figure 8 Stability study results of the comparative formulation of Example 2-4 and thymalfasin, liraglutide, semaglutide.

[0051] Figure 9 Pharmacokinetic profile after subcutaneous injection of Example 1 and octreotide acetate comparative formulation.

[0052] Figure 10 Pharmacokinetic profile after subcutaneous injection of Example 5, semaglutide solution and semaglutide comparative formulation. DETAILED DESCRIPTION

[0053] The following examples are further illustrations of the application but are not intended to limit the scope of the application. The application is further described in detail by reference to the following examples. However, this application should not be construed as limited to these examples and the preparation methods used therein. Moreover, equivalent alternatives, modifications, combinations, and improvements to the application will be apparent to those skilled in the art from the description contained herein. The examples are intended to be exemplary only and should not be construed as limiting the scope of the application.

[0054] Example 1

[0055] Take 5 g of soybean phospholipid S100 and 5 g of ethyl oleate, stir at 50°C to completely dissolve the phospholipid, and cool to room temperature (25°C) to obtain a clear transparent blank phospholipid gel with good flowability. Take 50 mg of thymalfasin fine powder, add it to the blank phospholipid gel, stir thoroughly, and stand until the bubbles completely disappear. Seal to obtain a thymalfasin phospholipid gel sustained-release preparation. Figure 2 Take 5 g of soybean phospholipid S100 and 5 g of ethyl oleate, stir at 50°C to completely dissolve the phospholipid, and cool to room temperature (25°C) to obtain a clear transparent blank phospholipid gel with good flowability. Take 50 mg of thymalfasin fine powder, add it to the blank phospholipid gel, stir thoroughly, and stand until the bubbles completely disappear. Seal to obtain a thymalfasin phospholipid gel sustained-release preparation.

[0056] Example 2

[0057] Take 5 g of soybean phospholipid S100 and 5 g of ethyl oleate, stir at 50°C to completely dissolve the phospholipid, and cool to room temperature (25°C) to obtain a clear transparent blank phospholipid gel with good flowability. Take 50 mg of thymalfasin fine powder, add it to the blank phospholipid gel, stir thoroughly, and stand until the bubbles completely disappear. Seal to obtain a thymalfasin phospholipid gel sustained-release preparation.

[0058] Example 3

[0059] Take 5 g of soybean phospholipid S100 and 5 g of ethyl oleate, stir at 50°C to completely dissolve the phospholipid, and cool to room temperature (25°C) to obtain a clear transparent blank phospholipid gel with good flowability. Take 50 mg of thymalfasin fine powder, add it to the blank phospholipid gel, stir thoroughly, and stand until the bubbles completely disappear. Seal to obtain a thymalfasin phospholipid gel sustained-release preparation.

[0060] Example 4

[0061] Take 5g soybean phospholipid S100 and 5g ethyl oleate, stir at 50°C to make the phospholipid completely dissolved, after cooling to room temperature (25°C), a clear transparent blank phospholipid gel is obtained. Take 50mg semaglutide fine powder, add it to the blank phospholipid gel, stir well, stand until the bubbles completely disappear, seal, and obtain the semaglutide phospholipid gel sustained-release preparation.

[0062] Example 5

[0063] Take 5g soybean phospholipid S100 and 5g ethyl oleate, stir at 50°C to make the phospholipid completely dissolved, after cooling to room temperature (25°C), a clear transparent blank phospholipid gel is obtained. Take 50mg semaglutide fine powder, add it to the blank phospholipid gel, stir well, stand until the bubbles completely disappear, seal, and obtain the semaglutide phospholipid gel sustained-release preparation.

[0064] Example 6

[0065] Take 5g soybean phospholipid S100 and 5g ethyl oleate, stir at 50°C to make the phospholipid completely dissolved, after cooling to room temperature (25°C), a clear transparent blank phospholipid gel is obtained. Take 50mg semaglutide fine powder, add it to the blank phospholipid gel, stir well, stand until the bubbles completely disappear, seal, and obtain the semaglutide phospholipid gel sustained-release preparation.

[0066] Example 7

[0067] Take 5g soybean phospholipid S100 and 5g ethyl oleate, stir at 50°C to make the phospholipid completely dissolved, after cooling to room temperature (25°C), a clear transparent blank phospholipid gel is obtained. Take 50mg semaglutide fine powder, add it to the blank phospholipid gel, stir well, stand until the bubbles completely disappear, seal, and obtain the semaglutide phospholipid gel sustained-release preparation.

[0068] Example 8

[0069] Take 5g soybean phospholipid S100 and 5g ethyl oleate, stir at 50°C to make the phospholipid completely dissolved, after cooling to room temperature (25°C), a clear transparent blank phospholipid gel is obtained. Take 50mg semaglutide fine powder, add it to the blank phospholipid gel, stir well, stand until the bubbles completely disappear, seal, and obtain the semaglutide phospholipid gel sustained-release preparation.

[0070] Example 9

[0071] Take 5g soybean phospholipid S100 and 5g ethyl oleate, stir at 50°C to make the phospholipid completely dissolved, after cooling to room temperature (25°C), a clear transparent blank phospholipid gel is obtained. Take 50mg semaglutide fine powder, add it to the blank phospholipid gel, stir well, stand until the bubbles completely disappear, seal, and obtain the semaglutide phospholipid gel sustained-release preparation.

[0072] Example 10

[0073] Take 5.5 g of soybean phospholipid S100, 4.5 g of ethyl oleate, and stir at 40°C until the phospholipid is completely dissolved. After cooling to room temperature (25°C), a clear and transparent blank phospholipid gel is obtained. Take 200 mg of tirofiban fine powder, add it to the blank phospholipid gel, stir thoroughly, and let it stand until the bubbles completely disappear. Seal it to obtain a tirofiban phospholipid gel sustained-release preparation.

[0074] Example 11

[0075] Take 5 g of hydrogenated egg yolk lecithin, 3 g of ethyl oleate, 2 g of medium-chain fatty acid glyceride, and 1 mg of butylated hydroxyanisole, and stir at 45°C until the phospholipid is completely dissolved. After cooling to room temperature (25°C), a clear and transparent blank phospholipid gel is obtained. Take 30 mg of growth hormone fine powder, add it to the blank phospholipid gel, stir thoroughly, and let it stand until the bubbles completely disappear. Seal it to obtain a growth hormone phospholipid gel sustained-release preparation.

[0076] Example 12

[0077] Take 5 g of hydrogenated soybean lecithin, 2 g of ethyl oleate, 3 g of medium-chain fatty acid glyceride, and 5 mg of EDTA-CaNa2, and stir at 37°C until the phospholipid is completely dissolved. After cooling to room temperature (25°C), a blank phospholipid gel is obtained. Take 50 mg of trofin peptide fine powder, add it to the blank phospholipid gel, stir thoroughly, and let it stand until the bubbles completely disappear. Seal it to obtain a trofin peptide phospholipid gel sustained-release preparation.

[0078] Experimental Example 1 Selection of injection oil

[0079] Weigh 4 g of egg yolk phospholipid E80 per portion, and add 6 g of each of the following six kinds of injectable oils in Table 9, stir, observe the appearance, and use a modular intelligent advanced rotational rheometer (model: MCR302, manufacturer: Anton Paar GmbH, Austria) to measure the viscosity (measurement parameters: temperature 25°C, shear rate 25 s -1 ). The results are shown in the following table:

[0080] Table 1: Solubility properties of injectable oils and egg yolk phospholipid E80

[0081]

[0082] As can be seen from the above table, ethyl oleate and ethyl linoleate have good solubility for E80.

[0083] S100, 4 g per portion, was added to 6 g of each of the following 9 kinds of injectable oils in Table 9, stirred, the appearance was observed, and the viscosity was measured using a modular intelligent advanced rotational rheometer (model: MCR302, manufacturer: Anton Paar GmbH, Austria) (measurement parameters: temperature 25°C, shear rate 25 s -1 ). The results are shown in the following table.

[0084] Table 2: Solubility properties of injectable oils and soybean phospholipid S100

[0085]

[0086]

[0087] As seen from the above table, ethyl oleate and ethyl linoleate have good solubility for S100.

[0088] Selection of the ratio of injectable oil in Experimental Example 2

[0089] Different ratios of phospholipid and injectable oil, 10 g in total, were weighed according to the following table, stirred, the appearance was observed, and the viscosity was measured using a modular intelligent advanced rotational rheometer (model: MCR302, manufacturer: Anton Paar GmbH, Austria) (temperature 25°C, shear rate 25 s -1 ). The results are shown in the following table.

[0090] Table 3: Screening of the ratio of phospholipid and injectable oil

[0091]

[0092]

[0093] As seen from the above table, when the ratio of phospholipid and injectable oil reaches 6:4 (w:w), the viscosity of the system increases significantly and cannot be smoothly injected through a syringe, therefore the ratio of phospholipid and injectable oil is preferably 2:8 to 5.5:4.5 (w:w), and preferably the ratio of phospholipid and injectable oil is 2:8 to 5:5 (w:w).

[0094] Investigation of rheological properties in Experimental Example 3

[0095] As a representative of Example 1, Example 1 was taken 0.5 mL / portion and put into a dialysis bag (molecular weight cut-off 8000-14000 Da, width 27 mm), both ends were closed, then put into a PBS (pH = 7.2) medium, 25°C, 100 rpm, at different time points, the elastic modulus (storage modulus) G' and the viscous modulus (loss modulus) G" were measured using a modular intelligent advanced rotational rheometer. The measurement parameters were: strain 1%, angular frequency 1 rad / s, temperature 25°C. The results are shown in the following table.Figure 3 As shown, initially, both the elastic modulus G' and the viscous modulus G" are very small, and G' is smaller than G", indicating that the preparation is in a liquid state. With the infiltration of water, G' and G" increase continuously, and at 4 h, G' is greater than G", indicating that the preparation has turned into a solid.

[0096] The product of Example 1 was injected subcutaneously into SD rats, and 15 minutes after injection, the elastic modulus (storage modulus) G' and the viscous modulus (loss modulus) G" were determined using a modular intelligent advanced rotational rheometer. The determination parameters were: strain 1%, angular frequency 0.1-100 rad / s, and temperature 25°C. The results are shown in Figure 4 As shown, it can be seen that 15 minutes after injection into the subcutaneous tissue of rats, G' is greater than G", and the gel has completely solidified.

[0097] Experimental Example 4: Investigation of Local Irritation

[0098] Preparation of the in-situ phase change gel sustained-release preparation of the present application: 5 g of soybean phospholipid S100 and 5 g of ethyl oleate were taken, and the phospholipid was completely dissolved by stirring at 50°C. After cooling to room temperature (25°C), the product was obtained.

[0099] Preparation of the phospholipid gel of patent CN102526753A: 7 g of soybean phospholipid S100, 1.5 g of medium-chain fatty acid glyceride, and 1.5 g of anhydrous ethanol were taken, and the phospholipid was completely dissolved by stirring. The product was obtained.

[0100] The in-situ phase change gel sustained-release preparation of the present application and the phospholipid gel of patent CN102526753A were injected subcutaneously into SD rats, and the occurrence of adverse reactions such as ulceration, edema, and erythema of the skin at the injection site was observed, and the skin tissue at the injection site was taken out on the 7th day and the 14th day for histopathological examination. The pathological results are as follows:

[0101] Table 4: Adverse reaction conditions

[0102]

[0103] After injection administration, all of the experimental animals behaved normally in terms of diet, and there was no obvious erythema, ulceration, or edema of the skin at the injection site. As shown in Figures 5 to 7 As shown in the figure, on the 7th day, the in-situ phase change gel sustained-release preparation of the present application and the phospholipid gel of patent CN102526753A both had different degrees of inflammatory reaction in the subcutaneous tissue after injection, the skin tissue of the former was normal, and the latter was accompanied by mild local necrosis of the skin tissue. The above pathological changes were recovered on the 14th day, the inflammatory reaction of the in-situ phase change gel sustained-release preparation of the present application changed from moderate to slight, and the change of the skin on the 14th day was almost within the normal range; the inflammatory reaction of the phospholipid gel of patent CN102526753A changed from slight to moderate.

[0104] Experimental Example 5 Stability Investigation

[0105] Preparation of thymalfasin comparative formulation: 7 g of soybean phospholipid S100, 1.5 g of medium-chain fatty acid glyceride, 1.2 g of anhydrous ethanol and 0.3 g of water for injection were stirred to completely dissolve the phospholipid, and then 50 mg of thymalfasin was added, followed by thorough stirring, standing until the bubbles completely disappeared, filling with nitrogen, and then adding a plug and rolling to seal, thereby obtaining the thymalfasin comparative formulation.

[0106] Preparation of liraglutide comparative formulation: 7 g of soybean phospholipid S100, 1.5 g of medium-chain fatty acid glyceride, 1.2 g of anhydrous ethanol and 0.3 g of water for injection were stirred to completely dissolve the phospholipid, and then 50 mg of liraglutide was added, followed by thorough stirring, standing until the bubbles completely disappeared, filling with nitrogen, and then adding a plug and rolling to seal, thereby obtaining the liraglutide comparative formulation.

[0107] Preparation of semaglutide comparative formulation: 7 g of soybean phospholipid S100, 1.5 g of medium-chain fatty acid glyceride, 1.2 g of anhydrous ethanol and 0.3 g of water for injection were stirred to completely dissolve the phospholipid, and then 50 mg of semaglutide was added, followed by thorough stirring, standing until the bubbles completely disappeared, filling with nitrogen, and then adding a plug and rolling to seal, thereby obtaining the semaglutide comparative formulation.

[0108] The above comparative formulation and Examples 2, 3 and 4 were respectively placed in constant temperature conditions of 4°C and 25°C, and samples were taken at regular time intervals (0w, 1w, 2w, 4w, 8w, n = 3), and the contents of thymalfasin, liraglutide and semaglutide were determined by HPLC. The content at 0w was taken as the initial content and set as 100%, and the percentages of the polypeptides at each time point were calculated. The results are shown in the following table and the accompanying Figure 8 and the following table:

[0109] Table 5: Stability results

[0110]

[0111]

[0112] From the results in the above table and the accompanying Figure 8 It can be seen from the residual amount curves of each drug at different temperatures that at 25°C, thymalfasin in the comparative formulation degrades rapidly, followed by liraglutide and semaglutide; at 4°C, the drugs in the comparative formulation still have a certain amount of degradation. However, the drugs in Examples 2-4 have almost no degradation at 4°C and 25°C, indicating that the in-situ phase change gel sustained release formulation of the present application can significantly improve the stability of the drug, and can be stored at room temperature, thereby reducing transportation and storage costs.

[0113] Experimental Example 6 In vivo pharmacokinetic study

[0114] Preparation of octreotide acetate comparative preparation: take 6 g of soybean phospholipid S100, 3 g of medium-chain fatty acid glyceride and 1 g of anhydrous ethanol, stir to completely dissolve the phospholipid, then add 50 mg of octreotide acetate, stir thoroughly, stand until the bubbles completely disappear, and seal to obtain the octreotide acetate comparative preparation.

[0115] Twelve SD male rats (body weight 180±20 g) were divided into two groups, with 6 rats in each group. Each group was subcutaneously injected with 0.8 mL of Example 1 and octreotide acetate comparative preparation, respectively, and the dosage was 4 mg. After administration, 400 μL of blood was taken from the eye socket, centrifuged (6000 r, 5 min) to take 100 μL of the upper plasma, 400 μL of methanol was added as a precipitant, and then 0.22 μm filter was used for filtration. The sample was injected into the LC-MS / MS for determination of the blood drug concentration. The results are shown in the following table:

[0116] Table 6: Pharmacokinetic results of octreotide acetate preparation

[0117]

[0118] From the pharmacokinetic data results and the drug-time curve in the above table, Figure 9 It can be seen that, compared with the octreotide acetate comparative preparation, Example 1 prolongs the half-life and average residence time of octreotide acetate, and has good sustained-release performance.

[0119] Semaglutide solution: commercially available semaglutide injection was diluted with water for injection to obtain a semaglutide solution of 0.05 mg / mL.

[0120] Semaglutide comparative preparation: take 7 g of soybean phospholipid S100, 1.5 g of medium-chain fatty acid glyceride, 1.2 g of anhydrous ethanol and 0.3 g of water for injection, stir to completely dissolve the phospholipid, then add 15 mg of semaglutide, stir thoroughly, stand until the bubbles completely disappear, and seal to obtain the semaglutide comparative preparation.

[0121] Thirty-six SD male rats (body weight 180±20 g) were divided into three groups, with 6 rats in each group. Each group was subcutaneously injected with 0.8 mL of Example 5, semaglutide solution and semaglutide comparative preparation, respectively. After administration, blood samples were collected from each group at 0.25, 0.5, 1, 2, 4, 8, 12 h and 1, 2, 3, 5, 9, 13, 17, 21, 25, 30, 35 d. The blood drug concentration was determined by LC-MS / MS. The results are shown in the following table:

[0122] Table 7: Pharmacokinetic results of semaglutide preparation

[0123]

[0124] From the pharmacokinetic data results of the above table and the drug-time curve attached Figure 10 Example 5 significantly prolongs the half-life and mean residence time of semaglutide peptide, also with good sustained-release performance.

Claims

1. An in-situ injectable phase change gel sustained-release formulation, characterized in that, The product contains, by weight, 10-60 parts phospholipids, 45-90 parts injectable oil, and 0.0025-20 parts active pharmaceutical ingredient; the active pharmaceutical ingredient is a peptide; the injectable oil is selected from one or a combination of ethyl oleate and ethyl linoleate.

2. The in-situ injection phase change gel sustained-release formulation according to claim 1, characterized in that, The phospholipids are selected from one or more combinations of natural phospholipids, semi-synthetic phospholipids, and synthetic phospholipids; the natural phospholipids are selected from egg yolk lecithin, soybean lecithin, or combinations thereof; the semi-synthetic phospholipids are selected from hydrogenated egg yolk lecithin, hydrogenated soybean lecithin, or combinations thereof; the synthetic phospholipids are selected from one or more combinations of dipalmitoyl phosphatidylethanolamine, dipalmitoyl phosphatidylcholine, distearyl phosphatidylcholine, dimyristoyl phosphatidylcholine, dioleoyl phosphatidylethanolamine, dipalmitoyl phosphatidylglycerol, and dipalmitoyl phosphate.

3. The in-situ injection phase change gel sustained-release formulation according to claim 1, characterized in that... The phospholipid is selected from one or a combination of soybean phospholipid S100 and egg yolk phospholipid E80.

4. The in-situ injection phase change gel sustained-release formulation according to claim 1, characterized in that, The peptides are selected from hirudin, histamine vasopressin, growth hormone-releasing peptide-2, growth hormone-releasing peptide-6, cetrorexate, bivalirudin, arginine vasopressin, lysine vasopressin, desmopressin, parretoxin, gonadorelin, leuprorelin, deselenorelin, buserelin, triptorelin, goselenorelin, alarrelin, sermorelin, nafarelin, progesterone, futorelin, hexarelin, octreotide, lanreotide, exenatide, liraglutide, abiglutide. Dulaglutide, Lixina peptide, Benalutide, Smegglutide, Telboride, Loxenatide, Human glucagon-like peptide-1, Growth hormone, Somatostatin, Ziconopeptide, Thymofasin, Sincalitone, Thymopentin, Abaricillin, Atedol, Leucine enkephalin, Methionine enkephalin, Ticoctin, Gonadotropin-releasing hormone, Thyrotropin-releasing hormone, Growth hormone-releasing hormone (GHRH), Somatostatin (GHIH), Melanocyte-stimulating hormone (MSH) Inhibitory pigmentocyte hormone (MRIH), melanocyte-releasing hormone (MRH), corticotropin-releasing hormone (CRH), secretin, substance P, neurotensin, human brain natriuretic peptide, atrial natriuretic peptide, angiotensin I, angiotensin II, angiotensin III, sleep peptide, memory-enhancing peptide, dynorphin, endorphin, angiotensin, enfuviride, CJC-1295, calcitonin, thymosin β4, vasopressin, tannins Vasopressin, phenylvasopressin, ornithine vasopressin, melanotan II, spleen pentapeptide, pramlintide, valproate, corticotropin, serine corticotropin-18, glargine corticotropin-18, zinc corticotropin-24, corticotropin-24, corticotropin-25, corticotropin-28, vasoactive intestinal peptide, brain natriuretic peptide, bradykinin, orphanin, rezafenidone, trifenidone, motixafotide, tedulglutide, or one or more pharmaceutically acceptable salts of the above drugs.

5. The in-situ injection phase change gel sustained-release formulation according to claim 1, characterized in that, The peptide is selected from one or more of the following: smegglutide, telpolide, leuprolide, goserelin, triptorelin, octreotide, lanreotide, and parretide.

6. The in-situ injection phase change gel sustained-release formulation according to claim 1, characterized in that, The ratio of phospholipids to injectable oil in the carrier is 2:8 to 6:4 (w:w), preferably 3:7 to 5.5:4.5 (w:w).

7. The in-situ injection phase change gel sustained-release formulation according to claim 1, characterized in that, The formulation also contains pharmaceutically acceptable excipients.

8. A method for preparing the in-situ injectable phase change gel sustained-release formulation according to any one of claims 1-7, characterized in that... Includes the following steps: (1) Mix phospholipids with injection oil and mix at 0-120°C to completely dissolve the phospholipids and obtain a clear and transparent phospholipid gel. (2) Prepare drug microparticles by crystallization or pulverization of the active pharmaceutical ingredient; (3) Mix the phospholipid gel from step (1) and the drug particles from step (2) evenly, let stand to remove air bubbles in the preparation, dispense, seal, and obtain the final product.

9. Use of the in-situ injection phase change gel sustained-release formulation according to any one of claims 1-7 in the preparation of drugs for treating diabetes, tumors, and cardiovascular and cerebrovascular diseases.

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