OXM3 preservatives, OXM3 formulations and preparation methods

Abstract

The present invention discloses an OXM3 preservative, an OXM3 formulation and a preparation method. The OXM3 preservative comprises 0.5 mg / mL to 5 mg / mL tromethamine, 0.1 mg / mL to 100 mg / mL stabilizer, 0.01 mg / mL to 5 mg / mL chelating agent, and a solvent, the stabilizer comprises one or more of mannitol, propylene glycol, arginine, arginine hydrochloride, histidine and histidine hydrochloride, the chelating agent comprises edetate disodium, and the solvent comprises water. The OXM3 formulation composed of the OXM3 preservative can ensure that the active ingredient OXM3 is stably stored for at least 6 months, preferably 12 months or more, more preferably 18 months to 24 months or more.

Description

[Technical field]

[0001] REFERENCE TO RELATED APPLICATIONS The present invention claims priority to a patent application filed in China on April 30, 2021, entitled "OXM3 preservative, OXM3 formulation and preparation method" with application number 202110485578.0, and to a patent application filed in China on April 20, 2022, entitled "OXM3 preservative, OXM3 formulation and preparation method" with application number 202210420561.1, the contents of which are incorporated herein by reference in their entirety.

[0002] The present invention belongs to the field of pharmacology, specifically, to an OXM3 preservative, an OXM3 formulation and a preparation method. [Background technology]

[0003] Drug stability is an important indicator to ensure the efficacy and safety of drugs. Obtaining a drug formulation that gives a drug good stability is an important condition for maintaining its efficacy and safety within the shelf life of the drug. Through forced, accelerated, shaking and light irradiation stability tests, the effects of different excipients and inner packaging materials on the quality of active ingredients are investigated and evaluated for each drug formulation. The detection items in the research process mainly include appearance, visible foreign matter, purity (SEC-HPLC method and RP-HPLC method), charge variants (AEX method), and biological activity (cell method).

[0004] OXM3 is a glucagon-like peptide-1 (GLP-1) and glucagon receptor dual agonist, which can bind to and activate the glucagon-like peptide-1 receptor (GLP-1R) and glucagon receptor (GCGR). It was first disclosed in CN201680036771.3, and its molecular formula is HXaa2QGTFTSDYSKYLDEKKAKEFVEWLLEGPSSG, where Xaa2 is Aib, K at position 20 is chemically modified by ([2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γGlu)1-CO-(CH2)18-CO2H binding to the ε-amino group of the K side chain, and the carboxyl group of the C-terminal glycine is amidated to the C-terminal primary amide, and the chemical formula of OXM3 is as shown in the figure below.

[0005] [ka]

[0006] In the prior art, there is no liquid preservative capable of preserving OXM3 well, and therefore there is a strong demand for a preservative capable of stably preserving OXM3 and an OXM3 formulation capable of stably preserving OXM3. Summary of the Invention

[0007] In view of the lack of a liquid preservative that can preserve OXM3 well and an OXM3 formulation that can stably preserve OXM3 in the prior art, the present invention provides an OXM3 preservative, an OXM3 formulation and a preparation method. The OXM3 formulation composed of the above OXM3 preservative can ensure that the active ingredient OXM3 can be stably preserved for at least 6 months, preferably 12 months or more, more preferably 18 to 24 months or more.

[0008] In order to solve the above technical problems, the technical solution 1 provided by the present invention is an OXM3 preservative comprising 0.5 mg / mL-5 mg / mL tromethamine, 0.1 mg / mL-100 mg / mL stabilizer, 0.01 mg / mL-5 mg / mL chelating agent, and a solvent, wherein the stabilizer comprises one or more of mannitol, propylene glycol, arginine, arginine hydrochloride, histidine, and histidine hydrochloride, preferably, the stabilizer comprises mannitol and propylene glycol, the chelating agent comprises edetate disodium, and the solvent comprises water.

[0009] Preferably, the preservative comprises 1 mg / mL to 3 mg / mL tromethamine, 10 mg / mL to 66 mg / mL stabilizing agent, and 0.03 mg / mL to 1 mg / mL chelating agent. Preferably, the preservative comprises 1.21 mg / mL tromethamine, 20 mg / mL to 46 mg / mL stabilizing agent, and 0.05 mg / mL to 0.5 mg / mL chelating agent.

[0010] In one preferred example of Technical Scheme 1, the preservative consists of 1.21 mg / mL tromethamine, 46 mg / mL mannitol, 0.5 mg / mL edetate disodium, and water as a solvent.

[0011] In one preferred example of Technical Scheme 1, the preservative consists of 1.21 mg / mL tromethamine, 20 mg / mL propylene glycol, 0.5 mg / mL edetate disodium, and water as a solvent.

[0012] In one preferred example of Technical Scheme 1, the preservative consists of 1.21 mg / mL tromethamine, 10 mg / mL propylene glycol, 23 mg / mL mannitol, 0.05 mg / mL edetate disodium, and water as a solvent.

[0013] Preferably, the preservative further comprises a surfactant, preferably Tween 80.

[0014] In order to solve the above technical problems, the technical solution 2 provided by the present invention is the preparation method of the OXM3 preservative as mentioned above.

[0015] In order to solve the above technical problems, the technical solution 3 provided by the present invention is an OXM3 formulation containing the above-mentioned OXM3 preservative and 1mg / mL-100mg / mL of OXM3, and the above OXM3 is, for example, 2mg / mL, 3mg / mL, 4mg / mL, 5mg / mL, 6mg / mL, 7mg / mL, 8mg / mL, 9mg / mL, 10mg / mL, 15mg / mL, 18mg / mL, 20mg / mL, 30mg / mL, 40mg / mL, 50mg / mL, 60mg / mL, 70mg / mL, 80mg / mL, 90mg / mL or 100mg / mL. Preferably, the above OXM3 is at a concentration of 3mg / mL-50mg / mL. More preferably, the OXM3 has a concentration of 6 mg / mL to 20 mg / mL, even more preferably, the OXM3 has a concentration of 15 mg / mL to 20 mg / mL, and even more preferably, the OXM3 has a concentration of 3 mg / mL, 4 mg / mL, 6 mg / mL, 8.37 mg / mL, 12 mg / mL, 15 mg / mL, 18 mg / mL or 20 mg / mL.

[0016] Preferably, the OXM3 formulation has a pH of 7 to 9. Preferably, the OXM3 formulation has a pH of 7.5 to 8.5. More preferably, the OXM3 formulation has a pH of 7.7.

[0017] In one preferred example of technical proposal 3, the OXM3 formulation is composed of 8.37 mg / mL OXM3, 1.21 mg / mL tromethamine, 46 mg / mL mannitol, 0.5 mg / mL edetate disodium, and water as a solvent, and the OXM3 formulation has a pH of 7.7.

[0018] In one preferred example of technical proposal 3, the OXM3 formulation is composed of 8.37 mg / mL OXM3, 1.21 mg / mL tromethamine, 20 mg / mL propylene glycol, 0.5 mg / mL edetate disodium, and water as a solvent, and the OXM3 formulation has a pH of 7.7.

[0019] In one preferred example of technical proposal 3, the OXM3 formulation is composed of 3 mg / mL OXM3, 1.21 mg / mL tromethamine, 10 mg / mL propylene glycol, 23 mg / mL mannitol, 0.05 mg / mL edetate disodium, and water as a solvent, and the OXM3 formulation has a pH of 7.7.

[0020] In one preferred example of technical proposal 3, the OXM3 formulation is composed of 4 mg / mL OXM3, 1.21 mg / mL tromethamine, 10 mg / mL propylene glycol, 23 mg / mL mannitol, 0.05 mg / mL edetate disodium, and water as a solvent, and the OXM3 formulation has a pH of 7.7.

[0021] In one preferred example of technical proposal 3, the OXM3 formulation is composed of 6 mg / mL OXM3, 1.21 mg / mL tromethamine, 10 mg / mL propylene glycol, 23 mg / mL mannitol, 0.05 mg / mL edetate disodium, and water as a solvent, and the OXM3 formulation has a pH of 7.7.

[0022] In one preferred example of technical proposal 3, the OXM3 formulation is composed of 12 mg / mL OXM3, 1.21 mg / mL tromethamine, 10 mg / mL propylene glycol, 23 mg / mL mannitol, 0.05 mg / mL edetate disodium, and water as a solvent, and the OXM3 formulation has a pH of 7.7.

[0023] In one preferred example of technical proposal 3, the OXM3 formulation is composed of 15 mg / mL OXM3, 1.21 mg / mL tromethamine, 10 mg / mL propylene glycol, 23 mg / mL mannitol, 0.05 mg / mL edetate disodium, and water as a solvent, and the OXM3 formulation has a pH of 7.7.

[0024] In a preferred embodiment of technical proposal 3, the OXM3 formulation is composed of 18 mg / mL OXM3, 1.21 mg / mL tromethamine, 10 mg / mL propylene glycol, 23 mg / mL mannitol, 0.05 mg / mL edetate disodium, and solvent water, and the OXM3 formulation has a pH of 7.7. In a preferred embodiment of technical proposal 3, the OXM3 formulation is composed of 20 mg / mL OXM3, 1.21 mg / mL tromethamine, 10 mg / mL propylene glycol, 23 mg / mL mannitol, 0.05 mg / mL edetate disodium, and solvent water, and the OXM3 formulation has a pH of 7.7.

[0025] In order to solve the above technical problems, the technical solution 4 provided by the present invention is a preparation method for the above-mentioned OXM3 preparation, the preparation method includes: (1) mixing tromethamine, a stabilizer and a chelating agent to obtain an OXM3 preservative; (2) mixing the OXM3 and the OXM3 preservative obtained in step (1) to obtain a mixed solution; and (3) adjusting the pH of the mixture and then filtering to obtain an OXM3 preparation. In order to solve the above technical problems, the fifth technical solution provided by the present invention is the use of the above-mentioned OXM3 preservative in preserving OXM3.

[0026] In order to solve the above technical problems, the technical solution 6 provided by the present invention is a liquid preparation prepared with the preservatives as mentioned above, and the liquid preparation is preferably an aqueous injection.

[0027] In order to solve the above technical problems, the present invention provides a technical solution 7, which is a delivery device comprising the above-mentioned OXM3 preservative, the above-mentioned OXM3 formulation, or the above-mentioned liquid formulation, The delivery device preferably includes a container, a sealing element, and a needle tip; Where: The container is preferably a vial, a syringe, or a vial; The sealing part is preferably a sealing plug or a sealing ring; The injection needle tip is preferably a water needle or a single needle-micro needle set.

[0028] In a preferred embodiment of technical solution 7, the delivery device is a pre-filled syringe.

[0029] In order to solve the above technical problems, the technical solution 8 provided by the present invention is a reagent kit including the above-mentioned OXM3 formulation, the above-mentioned liquid formulation, or the above-mentioned delivery device.

[0030] In order to solve the above technical problems, the technical solution 9 provided by the present invention is the use of the above-mentioned OXM3 formulation, the above-mentioned liquid formulation, the above-mentioned delivery device, or the above-mentioned reagent kit in the preparation of a product for treating or preventing a disease in a subject, wherein the above-mentioned disease is preferably obesity, diabetes, and non-alcoholic steatohepatitis.

[0031] Based on common knowledge in the art, each of the above preferred conditions can be arbitrarily combined to obtain each preferred embodiment of the present invention.

[0032] All of the reagents and materials used in the present invention are commercially available.

[0033] The positive advances of the present invention are as follows: The present invention provides an OXM3 preservative, an OXM3 formulation and a preparation method, and the OXM3 formulation composed of the above OXM3 preservative meets the criteria of no change in quality after being detected in aspects such as appearance, visible foreign matter, content, purity, charge variants and biological activity in the consideration of forced, accelerated and long-term stability, and can be stably stored for at least 6 months and at most 12 months or more, which makes it extremely easy to store, transport and use OXM3. [Brief description of the drawings]

[0034] [Figure 1] This is a graph showing the change trend of monomer purity (SEC-HPLC method) (40°C ± 2°C). [Diagram 2] This is a graph showing the change trend of monomer purity (SEC-HPLC method) (25°C ± 2°C). [Diagram 3] FIG. 1 is a graph showing the change trend of main peak purity (RP-HPLC method) (40° C.±2° C.). [Figure 4] FIG. 1 is a graph showing the change trend of main peak purity (RP-HPLC method) (25° C.±2° C.). [Diagram 5] This is a trend diagram of charge variants-principal components (AEX method) (40°C±2°C). [Figure 6] This is a trend diagram of charge variants-acidic components (AEX method) (40°C±2°C). [Figure 7] This is a trend diagram of charge variants-principal components (AEX method) (25°C ± 2°C). [Figure 8] This is a trend diagram of charge variants-acidic components (AEX method) (25°C ± 2°C). [Figure 9] FIG. 1 is a graph showing the change trend of main peak purity (RP-HPLC method) (40° C.±2° C.). [Figure 10] FIG. 1 is a graph showing the change trend of main peak purity (RP-HPLC method) (25° C.±2° C.). [Figure 11] The trend of change in total impurities (RP-HPLC method) (40℃±2℃). [Figure 12] This shows the trend of change in total impurities (RP-HPLC method) (25℃±2℃). [Figure 13] This is a trend diagram of charge variants-principal components (AEX method) (40°C±2°C). [Figure 14] This is a trend diagram of charge variants-acidic components (AEX method) (40°C±2°C). [Figure 15] This is a trend diagram of charge variants-principal components (AEX method) (25°C ± 2°C). [Figure 16] This is a trend diagram of charge variants-acidic components (AEX method) (25°C ± 2°C). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] The present invention will be further described below with reference to examples, but the present invention is not limited to the scope of these examples. In the following examples, the experimental methods not specified in the specific conditions are selected according to conventional methods and conditions or product specifications.

[0036] 1. Experimental materials and equipment 1.1 Experimental samples OXM3: Content factor 0.837, purchased from Eli Lily, lot number BO1706P002, or OXM3: Purchased from Asymchem, lot number CPo125104-01-03-02-41-01.

[0037] 1.2 The experimental materials are shown in Table 1.

[0038] [Table 1]

[0039] 1.3 Equipment is shown in Table 2.

[0040] [Table 2]

[0041] 2. Criteria for determining whether quality has not changed To determine whether the sample has changed, a criterion was set to determine whether the quality has not changed by comparing the sample detection index value with the initial value based on the product recognition and the precision of the equipment and method, and the details are shown in Table 3.

[0042] [Table 3]

[0043] The specific detection methods for each of the items in Table 3 are as follows: Appearance: The appearance of this product was visually inspected. Content: RP-HPLC method: Using a reversed-phase chromatography column, mobile phase A is 0.1% (v / v) TFA aqueous solution, mobile phase B is 0.085% (v / v) TFA acetonitrile solution; diluent is 980 μL of ultrapure water, 1 mol / L Tris-HCl Add 20μL of pH8.0 buffer, mix evenly, and prepare at the time of use; For the control solution, take one IBI362 control and gently invert to mix thoroughly; For the sensitivity solution, take 50μL of IBI362 control into a 10mL measuring flask, add 20mmol / L Tris buffer to make the volume 10mL; Take the test sample and dilute it with ultrapure water to 1.0mg / mL to obtain the test solution; Chromatography conditions: detection wavelength 220nm, column temperature 60℃, column pressure 400bar, sample plate temperature 5℃, flow rate 1.2mL / min, injection volume 10μL, collection time 60min, elution gradient is as follows:

[0044] [Table 4]

[0045] 10 μL of each of the diluent, control solution, sensitivity solution and test solution was precisely measured, and the sample was injected to start detection.

[0046] Visible foreign bodies: According to the method described in the Pharmacopoeia Commission of the People's Republic of China (2015 edition, Four Parts General Principle 0904 "Testing Method for Visible Foreign Bodies", Beijing: China Pharmaceutical Science and Technology Press, 2015), samples were tested for visible foreign bodies using a transparency meter (manufactured by Tianjin Tianda Tianfa, model number YB-2) and an insoluble particle detector (manufactured by Tianjin Tianda Tianfa, model number GWJ-8).

[0047] Monomer purity: SEC-HPLC method: Separated using a size exclusion chromatography column, the mobile phase was 80% 20 mmol / L Tris buffer (pH 8.0) + 20% acetonitrile, and the diluent was 980 μL of ultrapure water and 1 mol / L Tris-HCl. Add 20μL of pH8.0 buffer, mix evenly, and prepare at the time of use; For the control solution, take one IBI362 control, gently invert and mix thoroughly; For the sensitivity solution, take 50μL of IBI362 control into a 10mL measuring flask, add 20mmol / L Tris buffer to make the volume 10mL; Take the test sample and dilute it with ultrapure water to 1.0mg / mL to prepare the test solution; Chromatography conditions: injection volume 10μL, flow rate 0.5mL / min, collection time 35min, column temperature 25℃, column pressure 1000psi / 70bar, sample plate temperature 6℃, detection wavelength 214nm. Elution method is isocratic elution. Accurately measure 10μL of diluent, control solution, sensitivity solution and test solution, respectively, and inject the sample to start detection.

[0048] Main peak purity: Same as RP-HPLC method and content measurement method.

[0049] Charge variants: Anion exchange chromatography (AEX-HPLC method combined with UV detection). Separation was performed using YMC-Biopro QA-F100×4.6mmS-5μm, mobile phase A was 100mM sodium acetate, pH 4.6, 50% acetonitrile (8.20g sodium acetate was added to 400mL water in a 1000mL graduated cylinder. The pH was adjusted to 4.6 with acetic acid. The total volume was made up to 500mL with water and then made up to 1000mL with acetonitrile), mobile phase B was 50% acetonitrile / water (500mL acetonitrile was added to a 1000mL graduated cylinder, then made up to 1000mL with water). A 1mg / mL solution was prepared in 20mM TRIS buffer, pH 8.0 (2.42g TRIS was dissolved in 1000mL water and the pH was adjusted to 8.0±0.1 with 1.0N HCl). 20 mM TRIS buffer was taken as the blank solution. The reference standard was used as the detection standard (concentration: 1.0 mg / mL). 10 μL of each of the blank solution, detection standard solution and test solution were taken and injected into the liquid chromatograph, with the mobile phase flow rate of 1.0 mL / min, the collection time of 45 min, the column temperature of 25°C, the detection wavelength of 280 nm, and the autosampler temperature of 2°C-8°C. The sample was injected and analyzed, and the contents of the main components, acidic components and basic components were calculated.

[0050] Biological activity: (including detection of GLP-1 activity and glucagon activity) 1) GLP-1 activity: Cultured HEK293-GLP1R cells were taken, the old medium was aspirated, washed once with 5 mL of PBS, digested with 1 mL of Accutase solution until the cells were detached, and digestion was terminated with Assay Buffer (10% FBS (56 mL), 90% DMEM (1x) + GlutaMAXTM-1 medium (500 mL)). The cell suspension was collected and centrifuged at 1000 r / min for 5 min, the supernatant was discarded, and the cells were resuspended in Assay Buffer and counted. The density of the cell suspension was adjusted to 0.2 x 10 6The concentration of each well was adjusted to 100 cells / mL. The control and test products were diluted 3-fold with Assay Buffer in 10 gradient dilutions starting at 534ng / mL, resulting in a total of 10 gradient samples. According to the experimental layout, the treated samples and cell suspensions were added to a 96-well white cell culture plate at 50μL / well, the negative control group was added with 50μL / well of Assay Buffer, and the surrounding wells were added with 100μL / well of Assay Buffer and sealed. The plates were then incubated at 37℃±2℃, 5%±1% CO 2 The plate was incubated in an incubator for 6 hours. The previously prepared color development solution (100 mL of Bio-Glo™ Luciferase Assay buffer was dissolved in Bio-Glo™ Luciferase Assay Substrate) was equilibrated to room temperature. The cell culture plate was removed and left to stand for 10 minutes until equilibrated to room temperature. 100 μL of the color development solution was added to each well and left to stand at room temperature for 5 minutes. The chemiluminescence values ​​at all wavelengths were read using a microplate reader, and a four-parameter curve fitting was performed with the protein concentration as the abscissa and the induction effect fold as the ordinate to obtain the EC 50 , R 2 The following data was obtained: Biological activity % = Control product EC 50 / Sample EC 50 ×100%, 2) Glucagon activity: Cultured CHO-K1 / GCGR / Ga15 cells were taken, the old medium was aspirated, and the cells were washed once with 1x PBS. 1 mL of Accutase solution was added, and the cells were incubated at 37°C ± 2°C, 5% ± 1% CO 2 Incubate in an incubator for 4-5 min, terminate digestion with F-12 (1x), centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend in an appropriate amount of analysis medium, and count. The density of the cell suspension was 1.8 × 10 6The starting concentration of the control and test products was 267 ng / mL, and 10 consecutive gradient dilutions were performed by 3-fold dilution using sample diluent (20% Stimulation Buffer, 80% sterile water (prepared at the time of use according to the dose)) to obtain a total of 10 gradient samples. According to the predetermined layout, the treated samples and cell suspensions were added to a 96-well plate at 5 μL / well, centrifuged immediately, the plate was sealed with a plate sealing membrane, and incubated at 37°C ± 2°C, 5% ± 1% CO 2 The plates were incubated in an incubator for 30 min. The 96-well plate was removed, the plate sealing membrane was peeled off, and centrifuged immediately. The prepared cAMP-d2 stock solution and Anti-cAMP-Cryptate stock solution were thawed in advance and equilibrated to room temperature to prepare working solutions. 5 μL of cAMP-d2 working solution (20% cAMP-d2 stock solution was taken and 80% Lysis & Detection Buffer was added (prepared at the time of use according to the dose)) and 5 μL of Anti-cAMP-Cryptate working solution (20% Anti-cAMP-stock solution was taken and 80% Lysis & Detection Buffer was added (prepared at the time of use according to the dose)) were added sequentially to each well, centrifuged immediately, and allowed to develop at room temperature for 60 min in the dark. The chemiluminescence values ​​at all wavelengths were read using a microplate reader, and a four-parameter curve fitting was performed with the protein concentration as the abscissa and the ratio value as the ordinate, and the EC 50 , R 2 The data obtained was biological activity % = control product EC 50 / Sample EC 50 ×100%. EXAMPLES

[0051] Example 1: Composition of OXM3 formulations F1 to F4 A buffer solution for each formulation was prepared according to Table 4, and OXM3 was added to each formulation solution in the prescribed amount. The pH was adjusted to 7.7. After filtering, 1 mL was dispensed into a 2R vial, which was then stoppered and capped.

[0052] [Table 5]

[0053] Example 2 Stability experiment of formulations F1 to F4 The above formulation samples were tested for forced, accelerated and long-term stability. The specific methods are shown in Table 5.

[0054] [Table 6]

[0055] Result analysis: (1) Appearance and visible foreign matter Samples F1 to F4 were left at 40°C ± 2°C for one month, at 25°C ± 2°C for three months, and at 5°C ± 3°C for three months, and all of their appearance and visible foreign matter passed the test.

[0056] (2)Content The results of the drug content are shown in Table 6. The results show that after 1 month at 40°C ± 2°C, the drug content of F1 and F2 samples was significantly reduced, and compared with 0 days, the drug content of F1 and F2 samples was reduced by 1.1 mg / mL and 1.0 mg, respectively, and the drug content of F3 and F4 samples was not significantly changed and was reduced by 0.2 mg / mL and 0.4 mg / mL, respectively. Similarly, after 3 months at 25°C ± 2°C, the drug content of F1 and F2 samples was significantly reduced, and compared with 0 days, the drug content of F1 and F2 samples was reduced by 1.1 mg / mL and 0.9 mg, respectively, and the drug content of F3 and F4 samples was not significantly changed and was reduced by 0.2 mg / mL and 0.3 mg / mL, respectively. After 3 months at 5°C ± 3°C, the drug content of each sample was not significantly changed. Thus, OXM3 is relatively stable in F3 and F4.

[0057] [Table 7]

[0058] (3) Purity The purity results are shown in Table 7. The change trend of the monomer purity (SEC-HPLC method) is shown in Figures 1 and 2. The results show that after being left at 40°C ± 2°C for one month, the monomer purity of samples F1 to F4 decreased significantly, and compared with 0 days, the monomer purity decreased by 3.0%, 1.7%, 1.1%, and 1.0%, respectively, and the decrease in F3 and F4 was obviously smaller than that of F1 and F2. After being left at 25°C ± 2°C for three months, the monomer purity (SEC-HPLC method) of F1 and F2 decreased by 4.2% and 3.5%, exceeding the judgment criterion, whereas the monomer purity of F3 and F4 did not change significantly, both decreasing by 0.3%. After being left at 5°C ± 3°C for three months, the monomer purity of all samples F1 to F4 did not change significantly.

[0059] The change trend of the main peak purity (RP-HPLC method) is shown in Figures 3 and 4. After being left at 40°C ± 2°C for one month, the main peak purity of samples F1 to F4 decreased significantly, and compared with 0 days, the main peak purity of samples F1 to F4 decreased by 5.9%, 4.3%, 2.2% and 2.5%, respectively, and the decrease in F3 and F4 was obviously smaller than that of F1 and F2. After being left at 25°C ± 2°C for three months, the main peak purity of samples F1 and F2 decreased by 6.9% and 5.5%, respectively, and the main peak purity of samples F3 and F4 decreased by 2.6% and 2.8%, respectively, and the decrease in F3 and F4 was obviously smaller than that of F1 and F2. After being left at 5°C ± 3°C for three months, the monomer purity of samples F1 to F4 did not change significantly.

[0060] For these reasons, F3 and F4 are superior to F1 and F2.

[0061] [Table 8]

[0062] (4) Charge variants The results for the charge mutants are shown in Table 8. The change trends are shown in Figures 5 to 8.

[0063] The results showed that after 1 month at 40℃±2℃, the main components and acidic components of the charge variants of each formulation sample changed obviously. Compared with 0 days, the main components of F1-F4 samples decreased by 14.4%, 11.3%, 9.0% and 9.0%, respectively, and the acidic components increased by 13.9%, 11.1%, 8.7% and 8.5%, respectively, and the change range of F3 and F4 was obviously smaller than that of F1 and F2. After 3 months at 25℃±2℃, the main components and acidic components of the charge variants of each formulation sample changed obviously. Compared with 0 days, the main components of F1-F4 samples decreased by 9.2%, 8.3%, 7.8% and 7.5%, respectively, and the acidic components increased by 8.4%, 7.4%, 6.7% and 6.6%, respectively, and the change range of F3 and F4 was smaller than that of F1 and F2. After 3 months at 5℃±3℃, the main components and acidic components of the charge mutants of each formulation sample showed no obvious changes. Therefore, F3 and F4 are superior to formulations F1 and F2.

[0064] [Table 9]

[0065] (5) Biological activity (cell method) The results are shown in Table 9. The results showed that the biological activity (cell method) of samples F1 to F4 was acceptable within the range of 60% to 140% when placed under conditions of 40°C ± 2°C for 1 month, 25°C ± 2°C for 3 months, and 5°C ± 3°C for 3 months.

[0066] [Table 10]

[0067] In summary, F3 and F4 were superior to formulations F1 and F2, indicating that the addition of the stabilizer disodium edetate had an obvious effect, whereas F3 and F4 showed no significant difference, and mannitol and propylene glycol showed no significant effect on the stability of the samples.

[0068] Example 3: Construction of optimized formulation of OXM3 formulation F5 Based on the results of the above examples, combined with the development experience of pharmaceutical formulations, the formulations were optimized as follows: 3.0mg / mL, 4.0mg / mL, 6.0mg / mL, 12.0mg / mL, 15.0mg / mL, 18.0mg / mL, 20.0mg / mL OXM3, 1.21mg / mL tromethamine (i.e., trihydroxymethyl, Tris), 23.0mg / mL mannitol, 10.0mg / mL propylene glycol, 0.05mg / mL disodium edetate at pH 7.7, and filled into 1mL elongated slim prefilled syringes or 2R vials at 0.5mL to obtain formulations F5-1, F5-2, F5-3, F5-4, F5-5, F5-6 and F5-7. Prescription information is shown in Table 10 below.

[0069] [Table 11]

[0070] Example 4 Stability study of F5 formulation 1. The F5-1 and F5-2 samples obtained in Example 3 were tested for forced, accelerated and long-term stability. The specific methods are shown in Table 11.

[0071] [Table 12]

[0072] Results analysis The data in Tables 12 and 13 show that when the F5-1 and F5-2 samples were stored at 40°C ± 2°C for 4 weeks, the test items such as appearance, activity, and visible foreign matter did not change significantly, and the test items such as content, purity, and related substances changed, but were still within the quality standards.

[0073] [Table 13]

[0074] [Table 14]

[0075] The data in Tables 14 and 15 show that when the F5-1 and F5-2 samples were stored at 25°C ± 2°C for 3 months, the test items such as appearance, activity, and visible foreign matter did not change significantly, and the test items such as content, purity, and related substances changed, but were still within the quality standards.

[0076] [Table 15]

[0077] [Table 16]

[0078] The data in Tables 16 and 17 show that all test items of the F5-1 and F5-2 samples met the quality standards when stored at 5°C ± 3°C for 6 months.

[0079] [Table 17]

[0080] [Table 18]

[0081] Second, the F5-3 sample obtained in Example 3 was tested for its stability under forced, accelerated, shaking and light irradiation. The specific method is shown in Table 18.

[0082] [Table 19]

[0083] Results analysis 1. Appearance and visible foreign matter The F5-3 samples passed all appearance and visible foreign matter tests when placed at 40°C ± 2°C, 25°C ± 2°C, and 5°C ± 3°C for 1 month, 3 months, and 6 months, respectively. Throughout the 0-day and 1-month forced samples, the appearance was described as a clear to slightly opalescent, colorless or nearly colorless liquid.

[0084] 2, Content The results of the drug content are shown in Table 19. The results showed that the drug content of each sample did not change significantly when stored for 1 month, 3 months, and 6 months at 40°C ± 2°C, 25°C ± 2°C, and 5°C ± 3°C, respectively.

[0085] [Table 20]

[0086] 3, purity The purity results are shown in Table 20. The change trends are shown in Figures 9 to 12.

[0087] The results of monomer purity showed that the purity of the F5-3 sample (SEC-HPLC method) did not change significantly when stored at 40°C ± 2°C for 1 month, at 25°C ± 2°C for 3 months, and at 5°C ± 3°C for 6 months.

[0088] [Table 21]

[0089] The results of main peak purity showed that the main peak purity of F5-3 sample had different fluctuation ranges when stored at 40℃±2℃, 25℃±2℃, and 5℃±3℃ for 1 month, 3 months, and 6 months, respectively. When stored at 40℃±2℃ for 1 month, the main peak purity of F5-3 sample was significantly decreased but still within the quality standard. Compared with 0 days, the main peak purity of F5-3 sample decreased by 2.9%, total impurities increased by 2.9%, and the maximum single impurity increased by only 0.4% with no obvious change. When stored at 25℃±2℃ for 3 months, the main peak purity of F5-3 sample decreased by 1.9%, total impurities increased by 1.9%, and the maximum single impurity increased by only 0.1% with no obvious change. When stored at 5°C ± 3°C for 6 months, the main peak purity, total impurities, and maximum single impurity of the F5-3 sample did not change significantly.

[0090] The above purity test results indicate that the F5 formulation is stable.

[0091] 4. Charge variants The results for the charge mutants are shown in Table 21. The change trends are shown in Figures 13 to 16.

[0092] The results showed that after 1 month at 40°C ± 2°C, the main components and acidic components of the charge variants of F5-3 sample had no significant changes. Compared with 0 days, the main components of F5-3 sample decreased by 8.1%, and the acidic components increased by 6.6%. After 3 months at 25°C ± 2°C, the main components and acidic components of F5-3 sample had no significant changes, but were still within the quality standards. Compared with 0 days, the main components of F5-3 sample had decreased by 5.0%, and the acidic components increased by 4.1%. The results showed that after 6 months at 5°C ± 3°C, the main components and acid-base components of the charge variants of F5-3 sample had no significant changes.

[0093] [Table 22]

[0094] 5, biological activity The biological activity results are shown in Table 22. The results showed that the biological activity (cell method) of the F5-3 sample was acceptable within the range of 60% to 140% when placed at 40°C ± 2°C for 1 month, at 25°C ± 2°C for 3 months, and at 5°C ± 3°C for 6 months.

[0095] [Table 23]

[0096] 6. Insoluble particulates The insoluble particulate results are shown in Table 23. The results showed that the F5-3 insoluble particulate was acceptable when stored at 40° C.±2° C. for two weeks and at 25° C.±2° C. for one month.

[0097] [Table 24]

[0098] (4) Results of shaking experiment The results of the shaking experiment are shown in Table 24. The results show that when shaken at room temperature, away from light, and at 650 r / min for 5 days, the appearance and visible foreign matter of F5-3 are all acceptable, and there is no obvious change in the purity, charge variants, and biological activity.

[0099] [Table 25]

[0100] (5) Light irradiation experiment results The light irradiation experiment results are shown in Table 25. The results show that after 10 days under the conditions of 25℃±2℃, 60%RH±5%RH, and 500Lux±50Lux, the appearance and visible foreign matter of the F5-3 sample all passed the test, and there was no obvious change in the purity, charge variants, and biological activity, and there was no obvious difference between the F5-3 samples.

[0101] [Table 26]

[0102] Third, the F5-4 and F5-5 samples obtained in Example 3 were tested for forced and accelerated stability. The specific methods are shown in Table 26.

[0103] [Table 27]

[0104] Results analysis The data in Tables 27 to 28 show that the test items such as activity and insoluble particulate matter of F5-4 and F5-5 samples did not change significantly when stored at 40°C ± 2°C for one month and at 25°C ± 2°C for three months, while the test items such as content, purity, and related substances changed but were still within the quality standards.

[0105] [Table 28]

[0106] [Table 29]

[0107] Fourth, the F5-7 sample obtained in Example 3 was tested for accelerated and forced stability. The specific method is shown in Table 29.

[0108] [Table 30]

[0109] Results analysis The data in Table 30 show that the test items such as activity of the F5-7 sample did not change significantly when it was stored at 40°C ± 2°C for one month and at 25°C ± 2°C for three months, while the test items such as content, purity, related substances, etc. showed changes, but were still within the quality standards.

[0110] [Table 31]

[0111] Based on the above experimental results and the experience of the formulation development platform, F5 was finally selected as the OXM3 formulation, with the composition of 3.0 mg / mL, 4.0 mg / mL, 6.0 mg / mL, 12 mg / mL, 15 mg / mL, 18 mg / mL or 20 mg / mL OXM3, 1.21 mg / mL tromethamine (i.e., trihydroxymethyl, Tris), 23.0 mg / mL mannitol, 10.0 mg / mL propylene glycol, and 0.05 mg / mL edetate disodium at pH 7.7.

Claims

1. OXM3 formulation comprising OXM3, comprising 0.5 mg / mL to 5 mg / mL tromethamine, 0.1 mg / mL to 100 mg / mL of a stabilizing agent, 0.01 mg / mL to 5 mg / mL of a chelating agent, and a solvent, wherein the stabilizing agent comprises one or more of mannitol, propylene glycol, arginine, arginine hydrochloride, histidine, and histidine hydrochloride, where the chemical formula of OXM3 is 【Chemistry 1】 OXM3 formulation.

2. 2. The OXM3 formulation of claim 1, wherein the formulation comprises 1 mg / mL to 3 mg / mL tromethamine, 10 mg / mL to 66 mg / mL of a stabilizing agent, 0.03 mg / mL to 1 mg / mL of a chelating agent, and a solvent.

3. The formulation comprises 1.21 mg / mL tromethamine, 46 mg / mL mannitol, 0.5 mg / mL edetate disodium, and water as a solvent; or The formulation comprises 1.21 mg / mL tromethamine, 20 mg / mL propylene glycol, 0.5 mg / mL edetate disodium, and water as a solvent; or 3. The OXM3 formulation of claim 2, wherein the formulation comprises 1.21 mg / mL tromethamine, 10 mg / mL propylene glycol, 23 mg / mL mannitol, 0.05 mg / mL edetate disodium, and water as a solvent.

4. The OXM3 formulation of claim 1, further comprising a surfactant.

5. 2. The OXM3 formulation of claim 1, wherein the OXM3 is at a concentration of 1 mg / mL to 100 mg / mL.

6. The OXM3 formulation of claim 1, wherein the OXM3 formulation has a pH of 7 to 9.

7. 7. The OXM3 formulation of claim 6, comprising 8.37 mg / mL OXM3, 1.21 mg / mL tromethamine, 46 mg / mL mannitol, 0.5 mg / mL edetate disodium, and water as a solvent, the OXM3 formulation having a pH of 7.7; or the OXM3 formulation comprises 8.37 mg / mL OXM3, 1.21 mg / mL tromethamine, 20 mg / mL propylene glycol, 0.5 mg / mL edetate disodium, and a solvent, water, the OXM3 formulation having a pH of 7.7; or the OXM3 formulation comprises 3 mg / mL OXM3, 1.21 mg / mL tromethamine, 10 mg / mL propylene glycol, 23 mg / mL mannitol, 0.05 mg / mL edetate disodium, and a solvent, water, the OXM3 formulation having a pH of 7.7; or the OXM3 formulation comprises 4 mg / mL OXM3, 1.21 mg / mL tromethamine, 10 mg / mL propylene glycol, 23 mg / mL mannitol, 0.05 mg / mL edetate disodium, and a solvent, water, the OXM3 formulation having a pH of 7.7; or the OXM3 formulation comprises 6 mg / mL OXM3, 1.21 mg / mL tromethamine, 10 mg / mL propylene glycol, 23 mg / mL mannitol, 0.05 mg / mL edetate disodium, and a solvent, water, the OXM3 formulation having a pH of 7.7; or the OXM3 formulation comprises 8 mg / mL OXM3, 1.21 mg / mL tromethamine, 10 mg / mL propylene glycol, 23 mg / mL mannitol, 0.05 mg / mL edetate disodium, and a solvent, water, the OXM3 formulation having a pH of 7.7; or the OXM3 formulation comprises 12 mg / mL OXM3, 1.21 mg / mL tromethamine, 10 mg / mL propylene glycol, 23 mg / mL mannitol, 0.05 mg / mL edetate disodium, and a solvent, water, the OXM3 formulation having a pH of 7.7; or the OXM3 formulation comprises 15 mg / mL OXM3, 1.21 mg / mL tromethamine, 10 mg / mL propylene glycol, 23 mg / mL mannitol, 0.05 mg / mL edetate disodium, and a solvent, water, the OXM3 formulation having a pH of 7.7; or the OXM3 formulation comprises 18 mg / mL OXM3, 1.21 mg / mL tromethamine, 10 mg / mL propylene glycol, 23 mg / mL mannitol, 0.05 mg / mL edetate disodium, and a solvent, water, the OXM3 formulation having a pH of 7.7; or OXM3 formulation comprising 20 mg / mL OXM3, 1.21 mg / mL tromethamine, 10 mg / mL propylene glycol, 23 mg / mL mannitol, 0.05 mg / mL edetate disodium, and a solvent, water, wherein the OXM3 formulation has a pH of 7.

7.

8. A method for preparing an OXM3 formulation, comprising: (1) mixing tromethamine, a stabilizer and a chelating agent to obtain an OXM3 preservative; (2) mixing OXM3 and the OXM3 preservative obtained in step (1) to obtain a mixed solution; and (3) adjusting the pH of the mixture and then filtering to obtain an OXM3 formulation, wherein the OXM3 formulation comprises OXM3, 0.5 mg / mL to 5 mg / mL tromethamine, 0.1 mg / mL to 100 mg / mL of a stabilizer, 0.01 mg / mL to 5 mg / mL of a chelating agent, and a solvent, wherein the stabilizer comprises one or more of mannitol, propylene glycol, arginine, arginine hydrochloride, histidine, and histidine hydrochloride, wherein the chemical formula of OXM3 is 【Chemistry 2】 A preparation method comprising the steps of:

9. 13. A liquid formulation prepared from the formulation of claim 1, wherein the liquid formulation is an aqueous injectable solution.

10. A delivery device comprising the OXM3 formulation of claim 1, The delivery device includes a container, a sealing element, and a needle tip; Where: the container is a vial, a syringe, or a vial; The sealing element is a sealing plug or a sealing ring; and The injection needle tip is a water needle or a single needle-micro needle set; or The delivery device is a pre-filled syringe.

11. A reagent kit comprising the OXM3 formulation of claim 1 and a delivery device, wherein the delivery device is a pre-filled syringe.

12. The OXM3 formulation of claim 1, wherein the stabilizing agent comprises mannitol and propylene glycol, the chelating agent comprises disodium edetate, and the solvent comprises water.

13. The OXM3 formulation of claim 5, wherein the OXM3 is at a concentration of 3 mg / mL to 20 mg / mL.

14. The OXM3 formulation of claim 13, wherein the OXM3 is at a concentration of 4 mg / mL, 8 mg / mL, or 12 mg / mL.

15. The OXM3 formulation of claim 2, comprising 1.21 mg / mL tromethamine, 20 mg / mL to 46 mg / mL of a stabilizer, 0.05 mg / mL to 0.5 mg / mL of a chelating agent, and a solvent.

16. The OXM3 formulation of claim 3, comprising 1.21 mg / mL tromethamine, 10 mg / mL propylene glycol, 23 mg / mL mannitol, 0.05 mg / mL edetate disodium, and water as a solvent.

17. The OXM3 formulation of claim 4, wherein the surfactant is Tween 80.

18. The OXM3 formulation of claim 6, wherein the OXM3 formulation has a pH of 7.5 to 8.

5.

19. The OXM3 formulation of claim 7, comprising 4 mg / mL OXM3, 1.21 mg / mL tromethamine, 10 mg / mL propylene glycol, 23 mg / mL mannitol, 0.05 mg / mL edetate disodium, and water as a solvent, the OXM3 formulation having a pH of 7.7; or the OXM3 formulation comprises 8 mg / mL OXM3, 1.21 mg / mL tromethamine, 10 mg / mL propylene glycol, 23 mg / mL mannitol, 0.05 mg / mL edetate disodium, and a solvent, water, the OXM3 formulation having a pH of 7.7; or OXM3 formulation comprising 12 mg / mL OXM3, 1.21 mg / mL tromethamine, 10 mg / mL propylene glycol, 23 mg / mL mannitol, 0.05 mg / mL edetate disodium, and a solvent, water, wherein the OXM3 formulation has a pH of 7.

7.

20. An OXM3 formulation as described in claims 1 to 7 and 12 to 19 for treating or preventing a disease in a subject, wherein the disease is preferably obesity, diabetes, and non-alcoholic steatohepatitis.

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