Pharmaceutical formulations containing GLP-1 fusion protein and their uses

Abstract

This invention relates to a pharmaceutical formulation containing a GLP-1 fusion protein and its applications. The pharmaceutical formulation of this invention comprises a GLP-1 fusion protein and a buffer, and the pH value of the pharmaceutical formulation is in the range of about 6.0 to about 7.0. The pharmaceutical formulation of this invention is long-acting, stable, has few side effects, has a simple preparation process, a clear pathway, is safe and reliable, has few side effects, and is easy to use.

Description

[Technical Field]

[0001] This invention belongs to the field of pharmaceutical formulations, and more particularly to pharmaceutical formulations containing GLP-1 fusion proteins and their uses.

[0002] Glucagon-like peptide-1 (GLP-1), also known as incretin, is secreted from L cells in the small intestine. It exerts regulatory effects targeting multiple organs, including promoting insulin secretion, inhibiting glucagon release, delaying gastric emptying, suppressing appetite, and playing a crucial role in regulating nutrient intake and absorption. The biological effects of GLP-1 are primarily mediated through the activation of the GLP-1 receptor (GLP-1R). GLP-1R is a G protein-binding membrane protein mainly expressed in pancreatic beta cells, but is also expressed to varying degrees in other tissues and cells such as the lungs, heart, kidneys, gastrointestinal tract, and brain. When GLP-1 binds to its receptor, it activates adenylyl cyclase (AC), thereby stimulating the production of the second messenger cyclic adenosine monophosphate (cAMP), which then interacts with protein kinase A (PKA) and the Epac family's cAMP-regulated guanine nucleotide exchange factor (camp GEF) (Leech, CA, et al., Expression of cAMP-regulated guanine nutrote exchange factors in pancreatic beta-cells. Biochem Biophys Res Commun, 2000. 278(1): p.44-7).

[0003] The natural half-life of GLP-1 in the human body is only 1-2 minutes, mainly due to rapid enzymatic inactivation by dipeptidyl peptidase IV (DPP-IV) and renal clearance. Therefore, scientists have developed various long-acting GLP-1 analogs that are resistant to degradation. For example, human GLP-1 analogs have been modified through amino acid substitutions and N-terminal modifications (including fatty acid acylation and N-acetylation) to extend their circulating half-life. Albumin-bound GLP-1 (albiglutide) also has an extended half-life. In recent years, various GLP-1 receptor agonists and analogs have been widely used to treat metabolic disorders related to glucose and lipid metabolism, particularly type 2 diabetes mellitus (T2DM) and obesity. GLP-1 receptor agonists play a crucial role in diabetes treatment and also have preventive and therapeutic effects on cardiovascular and neurological diseases. Furthermore, GLP-1 binds to GLP-1 receptors in organs such as the kidneys and skin, influencing tissue metabolism and related diseases. The GLP-1 fusion protein disclosed in U.S. Patent US8658174 consists of a GLP-1 peptide fused with an IgG / Fc domain and can be used for the treatment of diabetes.

[0004] The use of genetic engineering recombinant protein technology to produce fusion proteins for therapeutic purposes aims to express the characteristics of natural polypeptides. This process involves cell engineering steps of transcription, translation, and post-translational modification. This process directly affects the physicochemical properties, conformation, in vivo half-life, biological activity, and production yield of the drug. Therefore, there is still a need for and it is of great importance for improved GLP-1 fusion proteins that are suitable for large-scale production, have high yield and activity, and have long half-lives for clinical treatment.

[0005] There is an urgent need for a fusion protein formulation that can be used and has guaranteed long-term storage. Conventional protein freeze-drying methods are very time-consuming and energy-consuming, and freeze-dried powders are cumbersome to use and unsuitable for patients' home use. Therefore, the research and development of a GLP-1 analog formulation that is long-acting, stable, has few side effects, has a simple preparation process, a clear pathway, is safe and reliable, has few side effects, and is easy to use is of great significance.

[0006] Invention Description The objective of the present invention is to provide a pharmaceutical preparation containing an improved GLP-1 receptor agonist fusion protein that offers many advantages, including long-term stability, fewer side effects, a safe and reliable preparation process, and ease of use.

[0007] In the pharmaceutical formulations comprising the GLP-1 fusion protein provided by the present invention, the GLP-1 fusion protein is an improved GLP-1 fusion protein characterized by increased yield and activity, and / or extended half-life. The fusion protein can be obtained by various methods or means, such as selecting amino acid substitution, hydroxylation, oxidation, etc., at specific protein positions, including hydroxylation of K34 of the GLP-1 polypeptide and / or reduction of oxidation of the GLP-1 polypeptide. Furthermore, amino acid substitution at specific positions of the GLP-1 fusion protein in the pharmaceutical formulations provided by the present invention significantly extends the half-life of the improved fusion protein and exhibits excellent preventive and therapeutic effects in human and animal disease models. In one embodiment, the present invention provides a pharmaceutical formulation comprising the following: (a) GLP-1 fusion protein, the GLP-1 fusion protein comprises a GLP-1 polypeptide and an immunoglobulin Fc domain, the GLP-1 polypeptide covalently bound to the immunoglobulin Fc domain, the GLP-1 polypeptide selected from human GLP-1(7-37), human GLP-1(7-36) amide and DPP-IV resistant human GLP-1, and the GLP-1 polypeptide contains one or more amino acid substitutions selected from the following groups relative to natural human GLP-1: A8G, G22E and R36G; The immunoglobulin Fc domain contains an IgG2-Fc domain or is an IgG2-Fc domain, and the IgG2-Fc domain contains one or more amino acid substitutions selected from the group C222S, A330S and P331S; and (b) Buffer solution; here, the pH value of the pharmaceutical preparation is in the range of approximately 6.0 to approximately 7.0.

[0008] In another embodiment, the present invention provides pharmaceutical formulations comprising: (a) GLP-1 fusion protein, the fusion protein contains a GLP-1 polypeptide and an immunoglobulin Fc domain, the GLP-1 polypeptide is covalently bound to the immunoglobulin Fc domain, The GLP-1 polypeptide is selected from human GLP-1(7-37), human GLP-1(7-36) amide, and DPP-IV resistant human GLP-1, and the GLP-1 polypeptide contains one or more amino acid substitutions selected from the following groups relative to natural human GLP-1: A8G, G22E, and R36G; The immunoglobulin Fc domain contains or is an IgG2-Fc domain, and the IgG2-Fc domain contains one or more amino acid substitutions selected from the groups C222S, A330S, and P331S. and (b) Buffer. The buffer is selected from the group consisting of phosphate buffer, citrate buffer, borate buffer, histidine buffer, and acetate buffer.

[0009] In some embodiments, the buffer is a phosphate buffer. In some embodiments, the phosphate buffer includes dihydrogen phosphate (e.g., disodium hydrogen phosphate, dipotassium hydrogen phosphate, etc.), dihydrogen phosphate (e.g., sodium dihydrogen phosphate, potassium dihydrogen phosphate, etc.), or a combination thereof. In some embodiments, the phosphate buffer is a disodium hydrogen phosphate / sodium dihydrogen phosphate buffer. In some embodiments, the phosphate buffer is prepared from disodium hydrogen phosphate hydrate and sodium dihydrogen phosphate hydrate. In some embodiments, the phosphate buffer is prepared from disodium hydrogen phosphate dodecahydrate and sodium dihydrogen phosphate monohydrate. In some embodiments, the concentration of the phosphate buffer in the pharmaceutical formulation is about 5 mM to about 15 mM (e.g., about 10 mM).

[0010] In some embodiments, the pharmaceutical formulations provided by the present invention further include a carbohydrate. In some embodiments, the carbohydrate is selected from the group consisting of mannitol, sorbitol, maltitol, erythritol, arabitol, xylitol, sucrose, lactose, trehalose, dextran, and combinations thereof. In some embodiments, the carbohydrate is selected from one or more of mannitol, sucrose, and sorbitol, for example, the carbohydrate is mannitol. In some embodiments, the concentration of the carbohydrate in the pharmaceutical formulation is 1 to 10% (w / v), for example, about 4.6% (w / v).

[0011] In some embodiments, the pharmaceutical formulations provided by the present invention further include a surfactant. In some embodiments, the surfactant is selected from the following group: polysorbate (e.g., polysorbate 80), polyoxyethylene castor oil derivatives, poloxamer (e.g., poloxamer 188), lecithin, polyethylene glycol 15-hydroxystearate, cyclodextrin, and combinations thereof. In some embodiments, the surfactant is polysorbate 80. In some embodiments, the concentration of the surfactant in the pharmaceutical formulation is 0.01% (w / v) to 0.04% (w / v), for example, about 0.02% (w / v).

[0012] In some embodiments, the pharmaceutical formulation comprises a GLP-1 fusion protein, a phosphate buffer, a carbohydrate, and a surfactant.

[0013] In certain embodiments, the pharmaceutical formulation comprises a GLP-1 fusion protein, a phosphate buffer (e.g., disodium hydrogen phosphate / sodium dihydrogen phosphate buffer), mannitol, and polysorbate.

[0014] In certain embodiments, the concentration of the GLP-1 fusion protein in the pharmaceutical formulation is 0.2 to 20 mg / mL (for example, 1 to 5 mg / mL).

[0015] In certain embodiments, the pharmaceutical formulation provided by the present invention comprises (a) a GLP-1 fusion protein (wherein the GLP-1 fusion protein has the amino acid sequence shown in SEQ ID NO: 7), (b) disodium hydrogen phosphate / sodium dihydrogen phosphate buffer, (c) mannitol, and (d) polysorbate 80.

[0016] In certain embodiments, the pharmaceutical formulation provided by the present invention comprises (a) 0.2 to 20 mg / mL of GLP-1 fusion protein (wherein the GLP-1 fusion protein has the amino acid sequence shown in SEQ ID NO: 7), (b) 5 to 15 mM of disodium hydrogen phosphate / sodium dihydrogen phosphate buffer, (c) 1 to 10% (w / v) of mannitol, and (d) 0.01% (w / v) to 0.04% (w / v) of polysorbate 80.

[0017] In certain embodiments, the pharmaceutical formulation provided by the present invention comprises (a) about 2 mg / mL, about 4 mg / mL, or about 6 mg / mL of GLP-1 fusion protein (where the amino acid sequence of the GLP-1 fusion protein is as shown in SEQ ID NO: 7), (b) about 10 mM disodium hydrogen phosphate / sodium dihydrogen phosphate buffer, (c) about 4.6% (w / v) mannitol, and (d) about 0.02% (w / v) polysorbate 80.

[0018] In certain embodiments, the pharmaceutical formulations provided by the present invention include (a) a GLP-1 fusion protein in an amount of about 5.3 mg / mL, about 6.67 mg / mL, about 10 mg / mL, about 12 mg / mL, about 13.3 mg / mL, or about 20 mg / mL (the amino acid sequence of the GLP-1 fusion protein is as shown in SEQ ID NO: 7); (b) about 10 mM disodium hydrogen phosphate / sodium dihydrogen phosphate buffer; (c) about 4.6% (w / v) mannitol; and (d) about 0.02% (w / v) polysorbate 80.

[0019] In certain embodiments, the unit dose of the pharmaceutical formulation provided by the present invention is approximately 0.5 mL and comprises (a) approximately 1 mg, approximately 2 mg, or approximately 3 mg of GLP-1 fusion protein (the amino acid sequence of the GLP-1 fusion protein is as shown in SEQ ID NO: 7), (b) approximately 0.58 mg of disodium hydrogen phosphate dodecahydrate, (c) approximately 0.465 mg of sodium dihydrogen phosphate monohydrate, (d) approximately 23.2 mg of mannitol, and (e) approximately 0.1 mg of polysorbate 80, with the remainder being water for injection.

[0020] In certain embodiments, the unit dose of the pharmaceutical formulation provided by the present invention is approximately 0.75 mL and contains (a) approximately 4 mg, approximately 5 mg, approximately 7.5 mg, approximately 9 mg, approximately 10 mg, or approximately 15 mg of GLP-1 fusion protein (the amino acid sequence of the GLP-1 fusion protein is as shown in SEQ ID NO: 7), (b) approximately 0.87 mg of disodium hydrogen phosphate dodecahydrate, (c) approximately 0.6975 mg of sodium dihydrogen phosphate monohydrate, (d) approximately 34.8 mg of mannitol, and (e) approximately 0.15 mg of polysorbate 80, with the remainder being water for injection.

[0021] In certain embodiments, the pharmaceutical formulation provided by the present invention has a pH value in the range of 6.4 to 7.0.

[0022] In certain embodiments, the pharmaceutical formulation provided by the present invention is isotonic. In certain embodiments, the pharmaceutical formulation provided by the present invention is a liquid formulation (e.g., an injectable formulation). In certain embodiments, the pharmaceutical formulation is administered by intravenous injection, subcutaneous injection, or intramuscular injection.

[0023] In certain embodiments, the pharmaceutical preparation provided by the present invention is stable at 25 ± 2°C for at least 3 months or at 40 ± 2°C for at least 2 weeks.

[0024] In certain embodiments, after the pharmaceutical preparation provided by the present invention is left standing for at least 1 month, the pH value changes by about 10% or less (for example, about 5% or less, about 4% or less, about 3% or less, about 2% or less, about 1% or less).

[0025] In certain embodiments, after the pharmaceutical preparation provided by the present invention is left standing for at least 1 month, the content of high molecular weight (HMW) derivatives or low molecular weight (LMW) derivatives does not exceed about 10% (for example, about 5% or less, about 4% or less, about 3% or less, about 2% or less, about 1% or less, etc.). In certain embodiments, the content of HMW derivatives and / or LMW derivatives is determined by size exclusion chromatography (SEC) or reverse phase liquid chromatography (RP-LC).

[0026] In certain embodiments, after the pharmaceutical preparation provided by the present invention is left standing for at least 1 month, the purity changes by only about 5% or less (for example, about 3% or less, about 2% or less, about 1% or less, etc.). In certain embodiments, the purity of the pharmaceutical preparation provided herein is determined by SDS capillary electrophoresis (for example, non-reducing SDS capillary electrophoresis).

[0027] In certain embodiments, the isoelectric point of the pharmaceutical preparation provided by the present invention does not change by more than about 5% (for example, about 4% or less, about 3% or less, about 2% or less, about 1% or less, etc.) after being left standing for at least 1 month. In certain embodiments, the isoelectric point of the pharmaceutical preparation is determined by capillary focusing isoelectric point electrophoresis (cIEF).

[0028] In certain embodiments, the GLP-1 polypeptide in the GLP-1 fusion protein has a certain level of hydroxylation at lysine 34 (K34) compared to natural human GLP-1. In certain embodiments, the hydroxylation level is 10% to 100%, for example, 10% or more, 15% or more, 20% or more, 26% or more, 27% or more, 28% or more, 29% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more.

[0029] In certain embodiments, the GLP-1 polypeptide in the GLP-1 fusion protein exhibits a lower degree of oxidation at tryptophan 31 (W31) compared to natural human GLP-1. In certain embodiments, the GLP-1 polypeptide in the GLP-1 fusion protein is substantially unoxidized at W31 compared to natural human GLP-1. In certain embodiments, the GLP-1 polypeptide in the GLP-1 fusion protein exhibits an oxidation level of less than 0.5% (e.g., less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1%) at W31 compared to natural human GLP-1, or is undetectable.

[0030] In certain embodiments, the GLP-1 polypeptide in the GLP-1 fusion protein has at least 90% sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, and contains one or more amino acid substitutions selected from the group consisting of A8G, G22E, and R36G compared to natural human GLP-1. In certain embodiments, the amino acid sequence of the GLP-1 polypeptide in the GLP-1 fusion protein is shown in SEQ ID NO: 3.

[0031] In certain embodiments, the IgG2-Fc domain in the GLP-1 fusion protein is an Fc domain derived from human IgG2. In certain embodiments, the IgG2-Fc domain has a reduced degree of oxidation at methionine 253 (M253) corresponding to SEQ ID NO: 7. In certain embodiments, the IgG2-Fc domain has a specific level of oxidation at M253 corresponding to SEQ ID NO: 7. In certain embodiments, the oxidation level of the IgG2-Fc domain at M253 corresponding to SEQ ID NO: 7 is approximately 15% or less, approximately 10% or less, approximately 9% or less, approximately 8% or less, approximately 7% or less, approximately 6% or less, approximately 5% or less, approximately 4% or less, approximately 3% or less, approximately 2% or less, approximately 1% or less, approximately 0.5% or less, or undetectable.

[0032] In certain embodiments, the IgG2-Fc domain in the GLP-1 fusion protein has at least 90% sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 5 or SEQ ID NO: 6 and includes one or more amino acid substitutions selected from the group consisting of C222S, A330S, and P331S. In certain embodiments, the amino acid sequence of the IgG2-Fc domain in the GLP-1 fusion protein is as shown in SEQ ID NO: 6.

[0033] In certain embodiments, the GLP-1 fusion protein in the pharmaceutical formulation provided by the present invention comprises the GLP-1 polypeptide shown in SEQ ID NO: 3 and comprises the immunoglobulin Fc domain shown in SEQ ID NO: 6.

[0034] In certain embodiments, in the GLP-1 fusion protein in the pharmaceutical formulation provided by the present invention, the GLP-1 polypeptide is covalently bound to the immunoglobulin Fc domain via a linker. In certain embodiments, in the GLP-1 fusion protein in the pharmaceutical formulation provided by the present invention, the amino acid sequence of the GLP-1 polypeptide is shown in SEQ ID NO: 3, the amino acid sequence of the immunoglobulin Fc domain is shown in SEQ ID NO: 6, and the amino acid sequence of the linker is shown in SEQ ID NO: 9.

[0035] In certain embodiments, the GLP-1 fusion protein in the pharmaceutical formulation provided by the present invention has the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 7.

[0036] In another aspect, the present invention also provides the use of pharmaceutical formulations comprising the GLP-1 fusion protein described herein in the manufacture of agents for the treatment or prevention of disease.

[0037] In another aspect, the present invention also provides the use of a pharmaceutical formulation comprising the GLP-1 fusion protein and additional therapeutic agents described herein in the manufacture of a drug for the treatment or prevention of a disease.

[0038] In another embodiment, the present invention also provides a pharmaceutical combination comprising a pharmaceutical formulation containing the GLP-1 fusion protein described herein and an additional therapeutic agent.

[0039] In certain embodiments, the disease is selected from the group consisting of metabolic diseases related to disorders of glucose metabolism and / or lipid metabolism, complications of metabolic diseases, and neurological diseases and other related diseases.

[0040] In certain embodiments, additional therapeutic agents are selected from the group consisting of insulin, metformin, sulfonylurea drugs (e.g., glimepiride, glibenclamide, gliclazide, glikidone), α-glucosidase inhibitors (e.g., acarbose), and γ-aminobutyric acid.

[0041] Other features and advantages of the present invention will become apparent from the following detailed description of embodiments. While the detailed description and specific examples are provided only as illustrations of preferred embodiments of the present invention, various changes and modifications within the spirit and scope of the invention will be apparent to those skilled in the art. [Brief explanation of the drawing]

[0042] [Figure 1] This is a schematic diagram of the pKN012-GLP1-IgG2 / Fc vector. [Figure 2] This shows the mass spectra of peptide segments 27-34:EFIAWLVK(+16Da) of the GLP-1 fusion protein YN-011 after Lys-C digestion. A: Major mass spectrum of EFIAWLVK, B: Major mass spectrum of EFIAWLVK(+16Da) modification, C: Secondary mass spectrum of EFIAWLVK, D: Secondary mass spectrum of EFIAWLVK(+16Da) modification. [Figure 3A] This shows UV detection data for modified peptides 27-34 (aa21-28) after Lys-C digestion: EFIAWLVK (+16Da) modified peptides 54-79 (aa48-73), unmodified peptides 224-240 (aa218-234). Figure 3B is an enlarged view of Figure 3A. [Figure 4] Figures 4A and 4B show the mean blood concentration-time profiles after single and multiple subcutaneous injections of 1 mg, 2 mg, 3 mg, and 4 mg of YN-011 in T2DM patients. Figures 4C and 4D show the mean blood concentration-time profiles after a single subcutaneous injection, while Figures 4C and 4D show the mean blood concentration-time profiles after multiple subcutaneous injections. [Figure 5] This diagram shows a schematic of a Phase IIa double-blind, placebo-controlled trial conducted in patients with T2DM to evaluate the efficacy and safety of YN-011 administered subcutaneously at dose levels of 1 mg, 2 mg, 3 mg, and 4 mg. Black triangles indicate administration of YN-011 at specified dose levels, black circles indicate oral glucose tolerance tests (OGTT) measurements, white triangles indicate indicated administration, and pentagrams indicate safety evaluations. [Figure 6] This shows the effect of multiple doses of YN-011 on fasting blood glucose levels in patients with T2DM. (At each time point, YN-011 showed a significant difference compared to placebo at dose levels of 3 mg and 4 mg, with a p-value of less than 0.05.) [Figure 7] This shows the effect of multiple doses of YN-011 on HbA1c levels in patients with T2DM. (At each time point, YN-011 showed a significant difference compared to placebo at dose levels of 1 mg, 3 mg, and 4 mg, with a p-value of <0.05.) [Figure 8]This shows the change in body weight (BW) of obese rhesus monkeys after repeated subcutaneous injections of YN-011. [Figure 9] The results after stimulating SH-SY5Y neurons with TNF-α at different concentrations for 48 hours are shown (*P<0.05 TNF-α (20 ng / ml) vs control; **P<0.01 TNF-α (40, 60, 80, 100 ng / ml) vs control). [Figure 10] YN-011 and GABA have been shown to inhibit the reduction in SH-SY5Y cell viability caused by TNF-α (**P<0.01 TNF-α vs control;#P<0.05 YN-011 (10nM or 100nM) or GABA (100μM) + TNF-α vs TNF-α;##P<0.01 YN-011 (500nM) + TNF-α vs TNF-α;n=6). [Figure 11] This study demonstrates that the combined use of YN-011 and GABA significantly increases SH-SY5Y cell viability (**P<0.01 TNF-α vs control, #P<0.05 TNF-α+GABA+YN-011 vs TNF-α+YN-011; n=6). [Figure 12] This shows that TNF-α apoptosis in SH-SY5Y neurons was reduced by YN-011 (**P<0.01 TNF-α vs control, #P<0.05 10nM YN-011+TNF-α vs TNF-α, ##P<0.01 100nM or 500nM YN-011+TNF-α vs TNF-α, n=3). [Figure 13] This study demonstrates that TNF-α-induced apoptosis in SH-SY5Y neurons is reduced by GABA (**P<0.01 TNF-α vs control, #P<0.05 10μM or 100μM GABA+TNF-α vs TNF-α, n=3). [Figure 14]This study demonstrates a reduction in TNF-α-induced apoptosis in SH-SY5Y neurons with the combined use of YN-011 and GABA (**P<0.01 TNF-α vs control, #P<0.05 GABA or YN-011+TNF-α vs GABA+YN-011+TNF-α, n=3). [Figure 15] These are live-cell staining images showing TNF-α-promoted damage in SH-SY5Y neurons (**: significant, ***: very significant). [Figure 16] These are live-cell staining images showing the protective effect of YN-011 against TNF-α-induced injury in nerve cells (**P<0.01 TNF-α vs control;#P<0.05 10nM YN-011+TNF-α vs TNF-α;##P<0.01 100nM or 500nM YN-011+TNF-α vs TNF-α;n=3). [Figure 17] Live cell staining images showing the protective effect of GABA against TNF-α-induced damage in neuronal cells (**P<0.01 TNF-α vs control; #P<0.05 10μM GABA+TNF-α vs TNF-α;##P<0.01 100μM GABA+TNF-α vs TNF-α; n=3). [Figure 18] These are live-cell staining images showing that the combined use of YN-011 and GABA provides protective effects against TNF-α-induced damage in nerve cells (**P<0.01 TNF-α vs control; #P<0.05 GABA or YN-011+TNF-α vs GABA+YN-011+TNF-α; n=3). [Figure 19] This shows a reduction in TNF-α-induced neuronal apoptosis by YN-011 (**P<0.01 TNF-α vs control; #P<0.05 100nM or 500nM YN-011+TNF-α vs TNF-α; n=3). [Figure 20]This shows a reduction in apoptosis induced by GABA in TNF-α-induced neurons (**P<0.01 TNF-α vs control; #P<0.05 100μM GABA+TNF-α vs TNF-α; n=3). [Figure 21] This shows a reduction in TNF-α-induced neuronal apoptosis by the combined use of YN-011 and GABA (**P<0.01 TNF-α vs control;#P<0.05 100nM YN-011+TNF-α vs 100μM GABA+100nM YN-011+TNF-α;##P<0.01 100μM GABA+TNF-α vs 100μM GABA+100nM YN-011+TNF-α;n=3). [Figure 22] This shows a reduction in TNF-α-induced neuronal apoptosis by YN-011 (**P<0.01 TNF-α vs control; #P<0.05 10nM, 100nM, or 500nM YN-011+TNF-α vs TNF-α; n=3). [Figure 23] This shows a reduction in TNF-α-induced neuronal apoptosis by GABA (**P<0.01 TNF-α vs control; #P<0.05 100μM GABA+TNF-α vs TNF-α; n=3). [Figure 24] This shows a reduction in TNF-α-induced neuronal apoptosis by the combined use of YN-011 and GABA (**P<0.01 TNF-α vs control; #P<0.05 TNF-α+100μM GABA+100nM YN-011 vs TNF-α+100μM GABA; ##P<0.05 TNF-α+100μM GABA+100nM YN-011 vs TNF-α+100nM YN-011; n=3). [Figure 25] This shows that inflammatory cytokine mRNA expression induced by Aβ1-42 oligomers in HMC3 microglia cells was reduced by GABA (*P<0.05, **P<0.01 Aβ1-42 oligomers vs control; #P<0.05 GABA+Aβ1-42 oligomers vs Aβ1-42 oligomers; n=3). [Figure 26] This shows that Aβ1-42 oligomer-induced inflammatory cytokine mRNA expression in HMC3 microglia cells was reduced by YN-011 (*P<0.05, **P<0.01 Aβ1-42 oligomer vs control; #P<0.05, ##P<0.01 YN-011+Aβ1-42 oligomer vs Aβ1-42 oligomer; n=3). [Figure 27] The combined use of YN-011 and GABA reduces the expression of Aβ1-42 oligomer-induced inflammatory cytokine mRNA in HMC3 microglia cells (*P<0.05, **P<0.01 Aβ1-42 oligomer vs control; #P<0.05, GABA+YN-011+Aβ1-42 oligomer vs GABA+Aβ1-42 oligomer or YN-011+Aβ1-42 oligomer; n=3). [Figure 28] This shows that inflammatory cytokine expression induced by Aβ1-42 oligomers in HMC3 microglial cells was reduced by GABA (**P<0.01 Aβ1-42 oligomers vs control; #P<0.05 GABA + Aβ1-42 oligomers vs Aβ1-42 oligomers; n=3). [Figure 29] This shows that Aβ1-42 oligomer-induced inflammatory cytokine expression in HMC3 microglia cells was reduced by YN-011 (**P<0.01 Aβ1-42 oligomer vs control; #P<0.05 YN-011 + Aβ1-42 oligomer vs Aβ1-42 oligomer; n=3). [Figure 30] This shows that Aβ1-42 oligomer-induced inflammatory cytokine expression in HMC3 microglia cells was reduced by the combination of YN-011 and GABA (**P<0.01 Aβ1-42 oligomer vs. control; #P<0.05 GABA+YN-011+Aβ1-42 oligomer vs. GABA+Aβ1-42 oligomer or YN-011+Aβ1-42 oligomer; n=3).

[0043] Detailed explanation of the disclosure: The following is a detailed description to be helpful to those skilled in the art in carrying out the invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art in which the invention pertains. The terms used herein are for the sole purpose of describing specific embodiments and are not intended to limit the invention. All publications, patent applications, patents, figures, and other references referenced herein are incorporated herein by reference in their entirety.

[0044] I. Definition Unless otherwise indicated, terms defined and used in this document should be understood as dictionary definitions, definitions in incorporated documents, and / or the generally known meanings of the defined terms.

[0045] All references, patents, and patent applications cited in this document are incorporated by reference in their entirety with respect to the respective subject matter they refer to, and in some cases may encompass the entire content of the referenced documents.

[0046] All features disclosed in this specification can be combined in any way. Each feature disclosed herein can be replaced by an alternative feature that serves the same, equivalent, or similar purpose. Thus, unless otherwise noted, each disclosed feature is merely an example of a set of equivalent or similar features.

[0047] As used herein, the terms “peptide,” “polypeptide,” and “protein” refer to an amino acid chain comprising two or more natural or non-natural amino acid residues, regardless of whether they are post-translationally modified (e.g., glycosylated or phosphorylated). Polypeptides disclosed herein may, for example, contain 3 to 3500 natural or non-natural amino acid residues. Proteins referred to may be a single peptide chain or a multi-subunit protein (e.g., composed of two or more polypeptides). The terms “peptide,” “polypeptide,” and “protein” as described herein are interchangeable and may include not only natural amino acids but also non-natural amino acids or amino acid analogs or mimics. Peptides, polypeptides, or proteins described herein may be obtained by any method known in the art, including but not limited to natural isolation, recombinant expression, and chemical synthesis.

[0048] The term "amino acid" as used here refers to an organic compound containing an amino group (-NH2), a carboxyl group (-COOH), and a side chain specific to each amino acid. In this application, amino acid names are also represented by standard one- or three-letter codes, which are summarized as follows:

[0049] [Table 1]

[0050] As used herein, the term "GLP-1 polypeptide" includes GLP-1 receptor agonist polypeptides having lysine at amino acid residue 34, or corresponding thereto, such as the polypeptide shown in SEQ ID NO: 1 or SEQ ID NO: 2. Specifically, this includes, but is not limited to, GLP-1(7-37), GLP-1(7-36)amide (also interchangeably referred to herein as GLP-1(7-36)amide, GLP-1(7-36amide), and GLP-1(7-36)), DPP-IV resistant GLP-1, and other GLP-1 analogs having lysine at amino acid residue 34, or corresponding thereto thereto. For example, GLP-1 polypeptides may include liraglutide (VICTOZA®, Novo Nordisk), semaglutide (OZEMPIC®, Novo Nordisk), albiglutide (SYNCRIA®, GlaxoSmithKline), tapoglutide (Roche), dulaglutide (TRULICITY®, Eli Lilly), or GLP-1 polypeptides derived from LY2428757 (Eli Lilly), and may also include GLP-1 polypeptides disclosed in WO2021163972A1, CN111217915A, WO2011056713A2, and WO2000034332A1, which are incorporated herein by reference, respectively. For example, the polypeptides may be GLP-1 analogs containing the "KG" amino acid motif sequence.

[0051] As used in this document, the terms “polynucleotide” or “oligonucleotide” refer to two or more covalently bonded nucleotides. Unless otherwise specified in the context, the term generally includes, but is not limited to, single-stranded (ss) or double-stranded (ds) deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). For example, the polynucleotide molecules or oligonucleotides of the present invention may consist of single-stranded and double-stranded DNA, DNA containing a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA. The mixture of single-stranded and double-stranded regions may include hybrid molecules containing both DNA and RNA, which may be either single-stranded or, more typically, a mixture of single-stranded and double-stranded regions. Furthermore, polynucleotide molecules may consist of triple-stranded regions containing RNA or DNA or both RNA and DNA. As used herein, the term “oligonucleotide” generally refers to a polynucleotide having a length of 200 base pairs or less and which may be single-stranded or double-stranded. The sequences provided herein may be DNA sequences or RNA sequences. However, unless otherwise specified in the context, it is important to understand that the provided sequences will include both DNA and RNA, as well as complementary RNA and DNA sequences. For example, the sequence 5'-GAATCC-3' should be understood to include 5'-GAAUCC-3', 5'-GGATTC-3', and 5'-GGAUUC-3'.

[0052] The terms “sequence identity” or “sequence similarity” used in this document refer to the percentage of sequence similarity between two peptide sequences or two nucleotide sequences. To determine the percentage of sequence identity between two amino acid sequences or two nucleotide sequences, the sequences are aligned for optimal comparison purposes (for example, by introducing a gap in the sequence of the first amino acid or nucleotide sequence to best align it with the second amino acid or nucleotide sequence), and the amino acid residues or nucleotides at corresponding positions are compared. In other words, the percentage (%) sequence identity of an amino acid sequence (or nucleic acid sequence) can be calculated by dividing the number of identical amino acid residues (or bases) in the same reference sequence being compared by the total number of amino acid residues (or bases) in the candidate or reference sequence (based on the shorter one). If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the residues are considered identical at that position. The percentage of similarity between two sequences is a function of the number of shared identical positions in the sequences (i.e., Percentage of Similarity = Number of Overlapping Identical Positions / Total Number of Positions × 100%). In one embodiment, the lengths of the two sequences are the same. A mathematical algorithm can also be used to determine the percentage of sequence similarity between the two sequences. One preferred non-restrictive example of a mathematical algorithm used to compare two sequences is the Karlin-Altschul algorithm, which was later modified as the Karlin-Altschul algorithm. This algorithm is incorporated into the NBLAST and XBLAST programs and can be used with the NBLAST nucleotide program parameter set (e.g., score=100, word length=12) to obtain nucleotide sequences homologous to a particular polynucleotide molecule. BLAST protein search can be performed using the XBLAST program parameter set (e.g., score=50, word length=3) to obtain amino acid sequences homologous to the protein molecules described in this document. Gapded BLAST can be used to obtain gapped alignments for comparison purposes.Alternatively, you can use PSI-BLAST to perform iterative searches and detect distant relationships between molecules (ibid.). When using the BLAST, Gapded BLAST, and PSI-Blast programs, you can use the default parameters for each program (e.g., XBLAST and NBLAST) (e.g., see the NCBI website). Another preferred non-restrictive example of a mathematical algorithm used for sequence comparison is the algorithm proposed by Myers and Miller, which is incorporated into the ALIGN program (version 2.0), part of the GCG sequence alignment software package. When comparing amino acid sequences using the ALIGN program, you can use the PAM120 weight residue table, gap length penalty 12, and gap penalty 4. The percentage of sequence identity between two sequences can be determined using similar techniques as above, with or without allowing gaps. When calculating the percentage of identity, typically only perfect matches are considered.

[0053] In the present invention, "conservative amino acid substitution" means substituting one amino acid residue with another amino acid residue without impairing the essential properties of the protein. Appropriate conservative amino acid substitutions can be made by substituting amino acids having similar hydrophobicity, polarity, and R-chain length. Examples of conservative substitutions include substituting one nonpolar (hydrophobic) residue with another nonpolar residue (e.g., alanine, isoleucine, valine, leucine, or methionine), substituting one polar (hydrophilic) residue with another polar residue (e.g., between arginine and lysine), substituting one basic residue with another basic residue (e.g., lysine, arginine, or histidine), or substituting one acidic residue with another acidic residue (e.g., aspartic acid or glutamic acid). The term "conservative substitution" also includes using chemically derived residues or non-natural amino acids in place of underived residues, as long as the peptide exhibits the required activity.

[0054] In this invention, the term "fusion protein" refers to a protein containing two or more peptides that form different functional domains. For example, the GLP-1 fusion protein described in this paper contains a GLP-1 peptide and an immunoglobulin Fc domain.

[0055] In this invention, the term "linker" refers to any chemical moiety that can be covalently bonded to another part. For example, a linker is a sequence of 1, 2, 3, 4, or 5 amino acid residues, or an artificial amino acid sequence having a length of 5 to 15, 20, 30, 50, or more amino acid residues, which is linked by a peptide bond and used to link one or more peptides. A linker may or may not have a secondary structure. Linker sequences are known in the art; see, for example, Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993); Poljak et al., Structure 2:1121-1123 (1994).

[0056] In this invention, the term "CH2" refers to the constant domain 2 of the immunoglobulin heavy chain. Similarly, the term "CH3" refers to the constant domain 3, which is another structural domain of the immunoglobulin heavy chain.

[0057] In this invention, the term "hinge region" refers to a flexible region between an antigen-binding fragment (Fab) and a crystallizable fragment (Fc) in the context of immunoglobulins such as IgG.

[0058] As used in this document, the term “vector” refers to a medium that can be used to operationally insert a gene element, express the gene element, produce a protein, RNA, or DNA encoded by the gene element, or replicate the gene element. Vectors can be used to transform, transduce, or transfect host cells, thereby allowing the delivered gene element to be expressed within the host cell. Examples of vectors include plasmids, phages, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phages or M13 phages, and animal viruses. Vectors may contain various regulatory elements for expression control, such as promoter sequences, transcription start sequences, enhancer sequences, selection elements, and reporter genes. In addition, vectors may contain origin sites for replication. Vectors may also contain components that facilitate entry into cells. These include, but are not limited to, viral particles, liposomes, or protein coats. Vectors can be expression vectors or cloning vectors.

[0059] The terms "DPPIV" and "DPP-IV" refer to dipeptidyl peptidase-IV, an enzyme that can inactivate natural GLP-1.

[0060] The term "hydroxylation level" refers to the percentage of residues modified by hydroxylation at specific amino acid positions within a peptide sample. For example, a hydroxylation level of 20% means that 20% (mole fraction) of the peptide molecule is hydroxylated at a particular amino acid position. Modulators can be used to increase or decrease the hydroxylation level of an expressed protein. For example, when present in an expression system, minoxidil and Zn2+ (e.g., from ZnSO4) can inhibit hydroxylation and decrease the hydroxylation level. The hydroxylation level can be measured using the method described in this embodiment or by mass spectrometry as described by Hou et al. (Hou, Y., et al., Nutrient Optimization Reduces Phosphorylation and Hydroxylation Level on an Fc-Fusion Protein in a CHO Fed-Batch Process. Biotechnol J, 2019. 14(3):p.e1700706.), or as further described in this document.

[0061] The term "oxidation level" refers to the percentage of amino acid residues in a peptide sample that are modified by oxidation. For example, an oxidation level of 2% means that 2% (molar fraction) of the peptide molecule is oxidized at a particular amino acid position. The oxidation level can be measured by the method described in this embodiment, or by the mass spectrometry method described in WO2002046227A2, or by the protein oxidation assay method by Bettinger et al. (Bettinger, JQ, et al., Quantitative Analysis of in Vivo Methionine Oxidation of the Human Proteome. J Proteome Res, 2020. 19(2): p.624-633.), or as further described in this document.

[0062] In this invention, the term "pharmaceutical grade" refers to the chemical purity or proportion of a drug, biological molecule, or reagent that meets the requirements for pharmaceutical manufacturing.

[0063] In the present invention, the term “treatment” means administering an effective amount of a compound, composition, or formulation to a subject, which may consist of a single dose or optionally include a series of steps. As is known in the art, “treatment” means a method used to achieve beneficial or desirable outcomes, including clinical outcomes. Beneficial or desirable clinical outcomes include, but are not limited to, relief or improvement of one or more symptoms or conditions, reduction of disease severity, stabilization of the disease state (i.e., no worsening), prevention of disease progression, recovery from disease, improvement or relief of disease, and relief of symptoms associated with the disease (partial, complete, or temporary). Beneficial or desirable clinical outcomes include improvement of fasting blood glucose levels and / or HbA1c levels, weight loss, improvement of liver lipid content, and improvement of cognitive function, motor coordination, etc. In the present invention, the term “subject” as used herein is also called “patient,” and includes all animals, including mammals, and preferably refers to humans. Furthermore, the term "subject" also includes livestock such as cattle, pigs, sheep, chickens, and horses, as well as rodents such as rats and mice, primates such as apes, monkeys, chimpanzees, gorillas, orangutans, and baboons, and domesticated animals such as dogs and cats.

[0064] In this invention, the term “pharmaceutically acceptable carrier” refers to any carrier, excipient, or formulation that is acceptable in the biological or other aspects of a pharmaceutical product. The use of a carrier, excipient, or formulation in a therapeutic formulation is considered acceptable as long as it is not incompatible with the active ingredient. The use of such pharmaceutically acceptable carriers is well known in the art, and various components that can be included in pharmaceutical formulations are described in the reference (Allen, LV, Jr., Remington: The Science and Practice of Pharmacy: from the past into the future. Int J Pharm Compd, 2012. 16(5): p.358-62).

[0065] In this invention, the term "therapeutic dose" refers to any amount that produces a desired effect on a subject, such as symptom relief, delay of disease progression, or prevention of disease onset. This amount can be administered multiple times over a period of time to achieve the desired effect. In the context of this document, this term may refer to an amount that lowers the subject's blood glucose level.

[0066] In this invention, the term "simultaneous administration" refers to the administration of two or more substances (compounds, compositions, etc.) to a target, and these substances have biological activity. The specific administration regimen depends on the pharmacokinetics of the two or more substances in their presence.

[0067] For understanding the scope of this invention, the term “contains” and its derivatives as used in this document are open-ended terms that specify the presence of the features, elements, components, parts, integers, and / or steps described, and do not exclude the presence of other features, elements, components, groups, integers, and / or steps not described. The same applies to similar terms with similar meanings, such as the terms “contains,” “has,” and their derivatives.

[0068] The terms “~composed of” and “~consisting of” used in this document, and their derivatives, are closed-end terms that identify the presence of the features, elements, components, groups, integers, and / or steps described, and further exclude the presence of any other features, elements, components, groups, integers, and / or steps that are not described.

[0069] The numerical ranges listed through the endpoint list in this document include all numbers and fractions within that range (e.g., 1-5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, 5). It should also be understood that all numbers and fractions are considered to be approximate.

[0070] Furthermore, the degree terms used in this document, such as "substantially," "approximately," and "approximately," indicate a reasonable deviation from the modifying phrase and do not significantly alter the final result. If such deviations do not negate the meaning of the modifying phrase, these degree terms should be interpreted as including a deviation of at least ±5% from the modifying phrase. More specifically, the term "approximately" refers to a deviation of approximately ±0.1–25%, ±1–20%, ±1–15%, ±1–10%, for example, a maximum of 10% or a maximum of 5% from the baseline value.

[0071] As used herein and in the appended claims, the singular forms “a,” “an,” and “one” include a reference to the plural unless otherwise specified. Therefore, for example, a composition containing “a compound” includes a mixture of two or more compounds. Also, unless otherwise specified, the term “or” is used in a general sense, meaning “and / or.”

[0072] Furthermore, the definitions and embodiments described in certain sections are intended to be applicable to other embodiments described in this document, as will be understood by those skilled in the art. For example, the following paragraphs define various aspects of the present invention in more detail. Each of the aspects thus defined may be combined with other aspects or more of aspects unless otherwise explicitly stated. Specifically, any feature indicated as preferred or advantageous may be combined with other features indicated as preferred or advantageous.

[0073] Methods and materials similar to or equivalent to those described in this document may be used in carrying out or testing the present invention, although specific methods and materials are also described in this embodiment.

[0074] II. Pharmaceutical Preparations The pharmaceutical formulation provided by the present invention comprises a GLP-1 fusion protein and a buffer. In one embodiment, the present invention provides a pharmaceutical formulation comprising: (a) GLP-1 fusion protein, where the GLP-1 fusion protein comprises a GLP-1 polypeptide and an immunoglobulin Fc domain, where the GLP-1 polypeptide is covalently bound to the immunoglobulin Fc domain, The GLP-1 polypeptide is selected from human GLP-1(7-37), human GLP-1(7-36) amide, and DPP-IV resistant human GLP-1, and the GLP-1 polypeptide contains one or more amino acid substitutions selected from the following groups relative to natural human GLP-1: A8G, G22E, and R36G; The immunoglobulin Fc domain contains an IgG2-Fc domain or is an IgG2-Fc domain, and the IgG2-Fc domain contains one or more amino acid substitutions selected from the group C222S, A330S, and P331S. and (b) Buffer solution.

[0075] Here, the pH value of the pharmaceutical preparation is in the range of approximately 6.0 to 7.0.

[0076] In the present invention, the buffer may be any suitable buffer. In certain embodiments, the buffer is a pH adjuster. In certain embodiments, the buffer is selected from the group consisting of phosphate buffer, citrate buffer, borate buffer, histidine buffer, and acetate buffer.

[0077] In certain embodiments, the pH value of the pharmaceutical formulation provided by the present invention is approximately 6.0, approximately 6.1, approximately 6.2, approximately 6.3, approximately 6.4, approximately 6.5, approximately 6.6, approximately 6.7, approximately 6.8, approximately 6.9, approximately 7.0, or any value within the range of any two of the above values. In certain embodiments, the pH value of the pharmaceutical formulation provided by the present invention is in the range of approximately 6.4 to approximately 7.0. In certain embodiments, the pH value of the pharmaceutical formulation provided by the present invention is in the range of approximately 6.5 to approximately 7.0. In certain embodiments, the pH value of the pharmaceutical formulation provided by the present invention is in the range of 6.4 to 7.0. In certain embodiments, the pH value of the pharmaceutical formulation provided by the present invention is in the range of 6.5 to 7.0. In certain embodiments, the pharmaceutical formulation provided by the present invention has a pH value in the range of 6.4 to 7.0. In certain embodiments, the pharmaceutical formulation provided by the present invention has a pH value in the range of 6.5 to 7.0. In certain embodiments, the pharmaceutical formulation provided by the present invention has a pH value in the range of approximately 6.4. In certain embodiments, the pharmaceutical formulation provided by the present invention has a pH value in the range of about 6.5. In certain embodiments, the pH value of the pharmaceutical formulation provided by the present invention is about 6.6. In certain embodiments, the pH value of the pharmaceutical formulation provided by the present invention is about 6.7. In certain embodiments, the pH value of the pharmaceutical formulation provided by the present invention is 6.4. In certain embodiments, the pH value of the pharmaceutical formulation provided by the present invention is 6.5. In certain embodiments, the pH value of the pharmaceutical formulation provided by the present invention is 6.6. In certain embodiments, the pH value of the pharmaceutical formulation provided by the present invention is 6.7.

[0078] In another embodiment, the present invention also provides pharmaceutical formulations including: (a) GLP-1 fusion protein, the fusion protein contains a GLP-1 polypeptide and an immunoglobulin Fc domain, where the GLP-1 polypeptide is covalently bound to the immunoglobulin Fc domain, The GLP-1 polypeptide is selected from human GLP-1(7-37), human GLP-1(7-36) amide, and DPP-IV resistant human GLP-1, and the GLP-1 polypeptide contains one or more amino acid substitutions selected from the following groups relative to natural human GLP-1: A8G, G22E, and R36G; The immunoglobulin Fc domain contains an IgG2-Fc domain or is an IgG2-Fc domain, and the IgG2-Fc domain contains one or more amino acid substitutions selected from the group C222S, A330S and P331S; and (b) Buffer. The buffer is selected from the group consisting of phosphate buffer, citrate buffer, borate buffer, histidine buffer, and acetate buffer.

[0079] In some embodiments, the buffer is a phosphate buffer. Various phosphate buffers are known in the art. In some embodiments, the phosphate buffer comprises a dihydrogen phosphate, dihydrogen phosphate, or a combination thereof. In some embodiments, the phosphate buffer comprises a dihydrogen phosphate or its hydrate. In some embodiments, the phosphate buffer comprises dihydrogen phosphate or its hydrate.

[0080] Examples of exemplary hydrogen phosphate disaliencies include disodium hydrogen phosphate, dipotassium hydrogen phosphate, diammonium hydrogen phosphate, and dicalcium hydrogen phosphate.

[0081] In some embodiments, the phosphate buffer is a disodium hydrogen phosphate / sodium dihydrogen phosphate buffer. In some embodiments, the phosphate buffer is prepared from disodium hydrogen phosphate and sodium dihydrogen phosphate. In some embodiments, the phosphate buffer is prepared from disodium hydrogen phosphate hydrate and sodium dihydrogen phosphate hydrate.

[0082] In the prior art, many types of disodium hydrogen phosphate hydrates are known, including disodium hydrogen phosphate monohydrate, disodium hydrogen phosphate dihydrate, disodium hydrogen phosphate heptahydrate, and disodium hydrogen phosphate dodecahydrate. Also, in the prior art, many types of sodium dihydrogen phosphate hydrates are known, including sodium dihydrogen phosphate monohydrate and sodium dihydrogen phosphate dihydrate. In some embodiments, phosphate buffers are prepared from disodium hydrogen phosphate dodecahydrate and sodium dihydrogen phosphate monohydrate. In some embodiments, the disodium hydrogen phosphate dodecahydrate and sodium dihydrogen phosphate monohydrate used in the preparation of phosphate buffers meet the criteria of the Chinese Pharmacopoeia (e.g., 2020 edition, Part IV).

[0083] In certain embodiments, the concentration of phosphate buffer in the pharmaceutical formulation is approximately 5 mM to approximately 15 mM (for example, approximately 5 mM, approximately 6 mM, approximately 7 mM, approximately 8 mM, approximately 9 mM, approximately 10 mM, approximately 11 mM, approximately 12 mM, approximately 13 mM, approximately 14 mM, approximately 15 mM, or any value between any two of the above numerical ranges). In certain embodiments, the concentration of phosphate buffer in the pharmaceutical formulation is approximately 10 mM. In certain embodiments, the concentration of phosphate buffer in the pharmaceutical formulation is 10 mM.

[0084] "mM" represents molar concentration, which is a commonly used unit of concentration in this field, indicating the amount of moles of a substance per unit volume in "mmol / L". For example, "The concentration of phosphate buffer in the pharmaceutical formulation is 10 mM" means that there are 10 mmol of phosphate per liter of the pharmaceutical formulation, or in other words, the concentration of phosphate in the pharmaceutical formulation is 10 mmol / L.

[0085] In certain embodiments, the buffer may be a disodium hydrogen phosphate / sodium dihydrogen phosphate buffer having a concentration of about 5 mM to about 15 mM (for example, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, or any value between any two of the above ranges). In certain embodiments, the buffer may be a disodium hydrogen phosphate / sodium dihydrogen phosphate buffer with a concentration of about 10 mM. In certain embodiments, the buffer may be a disodium hydrogen phosphate / sodium dihydrogen phosphate buffer with a concentration of 10 mM. In certain embodiments, the buffer may be a disodium hydrogen phosphate / sodium dihydrogen phosphate buffer with a concentration of about 6.65 mM. In certain embodiments, the buffer may be a disodium hydrogen phosphate / sodium dihydrogen phosphate buffer with a concentration of 6.65 mM.

[0086] In certain embodiments, the unit dose of the pharmaceutical formulation provided by the present invention is approximately 0.5 mL and contains approximately 0.6 mg of disodium hydrogen phosphate dodecahydrate and approximately 0.47 mg of sodium dihydrogen phosphate monohydrate. In certain embodiments, the unit dose of the pharmaceutical formulation provided by the present invention is approximately 0.5 mL / tube and contains approximately 0.6 mg of disodium hydrogen phosphate dodecahydrate and approximately 0.47 mg of sodium dihydrogen phosphate monohydrate. In certain embodiments, the unit dose of the pharmaceutical formulation provided by the present invention is approximately 0.5 mL and contains approximately 0.58 mg of disodium hydrogen phosphate dodecahydrate and approximately 0.465 mg of sodium dihydrogen phosphate monohydrate. In certain embodiments, the unit dose of the pharmaceutical formulation provided by the present invention is approximately 0.5 mL / tube and contains approximately 0.58 mg of disodium hydrogen phosphate dodecahydrate and approximately 0.465 mg of sodium dihydrogen phosphate monohydrate.

[0087] In certain embodiments, the unit dose of the pharmaceutical formulation provided by the present invention is approximately 0.75 mL and contains approximately 0.9 mg of disodium hydrogen phosphate dodecahydrate and approximately 0.7 mg of sodium dihydrogen phosphate monohydrate. In certain embodiments, the unit dose of the pharmaceutical formulation provided by the present invention is approximately 0.75 mL / tube and contains approximately 0.9 mg of disodium hydrogen phosphate dodecahydrate and approximately 0.7 mg of sodium dihydrogen phosphate monohydrate. In certain embodiments, the unit dose of the pharmaceutical formulation provided by the present invention is approximately 0.75 mL and contains approximately 0.87 mg of disodium hydrogen phosphate dodecahydrate and approximately 0.6975 mg of sodium dihydrogen phosphate monohydrate. In certain embodiments, the unit dose of the pharmaceutical formulation provided by the present invention is approximately 0.75 mL / tube and contains approximately 0.87 mg of disodium hydrogen phosphate dodecahydrate and approximately 0.6975 mg of sodium dihydrogen phosphate monohydrate.

[0088] In certain embodiments, the pharmaceutical formulation further comprises an osmotic regulator (e.g., a carbohydrate). In certain embodiments, the pharmaceutical formulation further comprises a carbohydrate. In some embodiments, the carbohydrate is selected from the group consisting of mannitol, sorbitol, maltitol, erythritol, arabitol, xylitol, sucrose, lactose, trehalose, dextran, and combinations thereof. In some embodiments, the carbohydrate is selected from one or more of mannitol, sucrose, and sorbitol. In some embodiments, the carbohydrate is mannitol. In some embodiments, the carbohydrate is sucrose. In some embodiments, the carbohydrate is sorbitol.

[0089] In some embodiments, the carbohydrate concentration in the pharmaceutical formulation is 1-10% (w / v), for example, about 1% (w / v), about 2% (w / v), about 3% (w / v), about 4% (w / v), about 5% (w / v), about 6% (w / v), about 7% (w / v), about 8% (w / v), about 9% (w / v), about 10% (w / v), or any value between any two of the above numerical ranges. In some embodiments, the carbohydrate concentration in the pharmaceutical formulation is about 4.6% (w / v) (for example, 4.64% (w / v)). In some embodiments, the carbohydrate concentration in the pharmaceutical formulation is 4.64% (w / v). In some embodiments, the carbohydrate concentration in the pharmaceutical formulation is 4.6% (w / v). In some embodiments, the carbohydrate concentration in the pharmaceutical formulation is about 3.1% (w / v) (for example, 3.09% (w / v)). In some embodiments, the concentration of carbohydrates in the pharmaceutical formulation is 3.1% (w / v).

[0090] "w / v" represents mass concentration, which is a commonly used unit of concentration in this field. It indicates the mass of a substance per unit volume, and its units are "kg / L", "kg / m3", or "g / mL". For example, "The concentration of carbohydrates in the pharmaceutical preparation is 4.6% (w / v)" means that there are 0.046 g of carbohydrates per milliliter of the pharmaceutical preparation, or in other words, the concentration of carbohydrates in the pharmaceutical preparation is 0.046 g / mL.

[0091] In certain embodiments, the pharmaceutical formulations provided herein contain mannitol in a concentration of 1 to 10% (w / v) (e.g., about 1% (w / v), about 2% (w / v), about 3% (w / v), about 4% (w / v), about 5% (w / v), about 6% (w / v), about 7% (w / v), about 8% (w / v), about 9% (w / v), about 10% (w / v), or any number between any two of the above numerical ranges). In certain embodiments, the pharmaceutical formulations provided herein contain mannitol in a concentration of about 4.6% (w / v) (e.g., 4.64% (w / v)). In certain embodiments, the pharmaceutical formulations provided herein contain mannitol in a concentration of 4.6% (w / v). In certain embodiments, the pharmaceutical formulations provided herein contain mannitol at a concentration of about 3.1% (w / v) (e.g., 3.09% (w / v)).

[0092] In certain embodiments, the unit dose of the pharmaceutical formulation provided herein is approximately 0.5 mL and contains approximately 23 mg of mannitol. In certain embodiments, the unit dose of the pharmaceutical formulation provided herein is approximately 0.5 mL / tube and contains approximately 23 mg of mannitol. In certain embodiments, the unit dose of the pharmaceutical formulation provided herein is approximately 0.5 mL / tube and contains approximately 23.2 mg of mannitol. In certain embodiments, the unit dose of the pharmaceutical formulation provided herein is approximately 0.5 mL / tube and contains approximately 23.2 mg of mannitol.

[0093] In certain embodiments, the unit dose of the pharmaceutical formulation provided herein is approximately 0.75 mL and contains approximately 35 mg of mannitol. In certain embodiments, the unit dose of the pharmaceutical formulation provided herein is approximately 0.75 mL / tube and contains approximately 35 mg of mannitol. In certain embodiments, the unit dose of the pharmaceutical formulation provided by the present invention is approximately 0.75 mL and contains approximately 34.8 mg of mannitol. In certain embodiments, the unit dose of the pharmaceutical formulation provided by the present invention is approximately 0.75 mL / tube and contains approximately 34.8 mg of mannitol.

[0094] In certain embodiments, the pharmaceutical formulation further comprises a protein stabilizer (e.g., a surfactant). In certain embodiments, the pharmaceutical formulation further comprises a surfactant such as an ionic surfactant or a nonionic surfactant. In certain embodiments, the surfactant is selected from the group consisting of polysorbate (e.g., polysorbate 80), polyoxyethylene castor oil derivatives, poloxamer (e.g., poloxamer 188), lecithin, polyethylene glycol 15-hydroxystearate, cyclodextrin, and combinations thereof. In certain embodiments, the surfactant is polysorbate 80. In this application, “polysorbate 80” is also referred to as “Tween 80” or “Tween-80”.

[0095] In certain embodiments, the concentration of the surfactant in the pharmaceutical formulation is 0.01%(w / v) to 0.04%(w / v), for example, about 0.01%(w / v), about 0.02%(w / v), about 0.03%(w / v), about 0.04%(w / v), or any value between any two of the above numerical ranges. In certain embodiments, the concentration of the surfactant in the pharmaceutical formulation is about 0.02%(w / v). In certain embodiments, the concentration of the surfactant in the pharmaceutical formulation is 0.02%(w / v). In certain embodiments, the concentration of the surfactant in the pharmaceutical formulation is about 0.01%(w / v) (for example, 0.013%(w / v)). In certain embodiments, the concentration of the surfactant in the pharmaceutical formulation is 0.01%(w / v).

[0096] In certain embodiments, the pharmaceutical formulations provided herein contain polysorbate 80 at a concentration of 0.01%(w / v) to 0.04%(w / v) (e.g., about 0.01%(w / v), about 0.02%(w / v), about 0.03%(w / v), about 0.04%(w / v), or any value between the above two numerical ranges). In certain embodiments, the pharmaceutical formulations provided herein contain polysorbate 80 at a concentration of about 0.02%(w / v). In certain embodiments, the pharmaceutical formulations provided herein contain polysorbate 80 at a concentration of about 0.02%(w / v). In certain embodiments, the pharmaceutical formulations provided herein contain polysorbate 80 at a concentration of about 0.01%(w / v) (e.g., 0.013%(w / v)). In certain embodiments, the pharmaceutical formulations provided herein contain polysorbate 80 at a concentration of 0.01% (w / v).

[0097] In certain embodiments, the unit dose of the pharmaceutical formulation provided by the present invention is approximately 0.5 mL and contains approximately 0.1 mg of polysorbate 80.

[0098] In certain embodiments, the unit dose of the pharmaceutical formulation provided by the present invention is approximately 0.75 mL and contains approximately 0.15 mg of polysorbate 80.

[0099] In certain embodiments, the pharmaceutical formulation provided by the present invention comprises a GLP-1 fusion protein, a phosphate buffer, a carbohydrate, and a surfactant. In certain embodiments, the pharmaceutical formulation provided by the present invention comprises a GLP-1 fusion protein, a phosphate buffer (e.g., disodium hydrogen phosphate / sodium dihydrogen phosphate buffer), mannitol, and a polysorbate (e.g., polysorbate 80). In certain embodiments, the pharmaceutical formulation provided by the present invention comprises a GLP-1 fusion protein, a disodium hydrogen phosphate / sodium dihydrogen phosphate buffer, mannitol, and polysorbate 80.

[0100] In certain embodiments, the concentration of GLP-1 fusion protein in the pharmaceutical formulation is 0.2 to 25 mg / mL, for example, about 0.5 mg / mL, about 1 mg / mL, about 1.5 mg / mL, about 2 mg / mL, about 2.5 mg / mL, about 3 mg / mL, about 3.5 mg / mL, about 4 mg / mL, about 4.5 mg / mL, about 5 mg / mL, about 5.5 mg / mL, about 6 mg / mL, about 6.5 mg / mL, about 7 mg / mL, about 7.5 mg / mL, about 8 mg / mL, about 8.5 mg / mL, about 9 mg / mL, about 9.5 mg / mL, about 10 mg / mL, about 10.5 mg / mL, about 11 mg / mL, about 11.5 mg / mL, about 12 mg / mL, about 12.5 mg / mL, approximately 13 mg / mL, approximately 13.5 mg / mL, approximately 14 mg / mL, approximately 14.5 mg / mL, approximately 15 mg / mL, approximately 15.5 mg / mL, approximately 16 mg / mL, approximately 16.5 mg / mL, approximately 17 mg / mL, approximately 17.5 mg / mL, approximately 18 mg / mL, approximately 18.5 mg / mL, approximately 19 mg / mL, approximately 19.5 mg / mL, approximately 20 mg / mL, approximately 20.5 mg / mL, approximately 21 mg / mL, approximately 21.5 mg / mL, approximately 22 mg / mL, approximately 22.5 mg / mL, approximately 23 mg / mL, approximately 23.5 mg / mL, approximately 24 mg / mL, approximately 24.5 mg / mL, approximately 25 mg / mL, or any value between any two of the above numerical ranges. In certain embodiments, the concentration of GLP-1 fusion protein in the pharmaceutical formulation is 0.2 to 20 mg / mL. In certain embodiments, the concentration of the GLP-1 fusion protein in the pharmaceutical formulation is 1 to 5 mg / mL.

[0101] In certain embodiments, the concentration of GLP-1 fusion protein in the pharmaceutical formulation is approximately 2 mg / mL. In certain embodiments, the concentration of GLP-1 fusion protein in the pharmaceutical formulation is 2 mg / mL. In certain embodiments, the concentration of GLP-1 fusion protein in the pharmaceutical formulation is approximately 3 mg / mL. In certain embodiments, the concentration of GLP-1 fusion protein in the pharmaceutical formulation is 3 mg / mL. In certain embodiments, the concentration of GLP-1 fusion protein in the pharmaceutical formulation is approximately 4 mg / mL. In certain embodiments, the concentration of GLP-1 fusion protein in the pharmaceutical formulation is 4 mg / mL. In certain embodiments, the concentration of GLP-1 fusion protein in the pharmaceutical formulation is approximately 6 mg / mL. In certain embodiments, the concentration of GLP-1 fusion protein in the pharmaceutical formulation is 6 mg / mL.

[0102] In certain embodiments, the concentration of GLP-1 fusion protein in the pharmaceutical formulation is approximately 5.3 mg / mL. In certain embodiments, the concentration of GLP-1 fusion protein in the pharmaceutical formulation is 5.3 mg / mL. In certain embodiments, the concentration of GLP-1 fusion protein in the pharmaceutical formulation is approximately 6.67 mg / mL. In certain embodiments, the concentration of GLP-1 fusion protein in the pharmaceutical formulation is 6.67 mg / mL. In certain embodiments, the concentration of GLP-1 fusion protein in the pharmaceutical formulation is approximately 10 mg / mL. In certain embodiments, the concentration of GLP-1 fusion protein in the pharmaceutical formulation is 10 mg / mL. In certain embodiments, the concentration of GLP-1 fusion protein in the pharmaceutical formulation is approximately 12 mg / mL. In certain embodiments, the concentration of GLP-1 fusion protein in the pharmaceutical formulation is 12 mg / mL. In certain embodiments, the concentration of GLP-1 fusion protein in the pharmaceutical formulation is approximately 13.3 mg / mL. In certain embodiments, the concentration of GLP-1 fusion protein in the pharmaceutical formulation is 13.3 mg / mL. In certain embodiments, the concentration of GLP-1 fusion protein in the pharmaceutical formulation is approximately 20 mg / mL.

[0103] In certain embodiments, the pharmaceutical formulations provided herein contain 0.2 to 20 mg / mL of GLP-1 fusion protein, and the GLP-1 fusion protein has the amino acid sequence shown in SEQ ID NO: 7. In certain embodiments, the pharmaceutical formulations provided herein contain about 2 mg / mL, about 4 mg / mL, or about 6 mg / mL of GLP-1 fusion protein, and the amino acid sequence of the GLP-1 fusion protein is shown in SEQ ID NO: 7. In certain embodiments, the pharmaceutical formulations provided herein contain 2 mg / mL, 4 mg / mL, or 6 mg / mL of GLP-1 fusion protein, and the amino acid sequence of the GLP-1 fusion protein is shown in SEQ ID NO: 7.

[0104] In certain embodiments, the pharmaceutical formulations provided by the present invention contain approximately 5.3 mg / mL, approximately 6.67 mg / mL, approximately 10 mg / mL, approximately 12 mg / mL, approximately 20 mg / mL of GLP-1 fusion protein, and the amino acid sequence of the GLP-1 fusion protein is as shown in SEQ ID NO: 7.

[0105] In certain embodiments, the unit dose of the pharmaceutical formulation provided by the present invention is approximately 0.5 mL and contains approximately 1 mg, approximately 2 mg, or approximately 3 mg of GLP-1 fusion protein, the amino acid sequence of the GLP-1 fusion protein being as shown in SEQ ID NO: 7.

[0106] In certain embodiments, the unit dose of the pharmaceutical formulation provided by the present invention is approximately 0.75 mL and contains approximately 4 mg, approximately 5 mg, approximately 7.5 mg, approximately 9 mg, approximately 10 mg, or approximately 15 mg of GLP-1 fusion protein, the amino acid sequence of the GLP-1 fusion protein is shown in SEQ ID NO: 7.

[0107] In certain embodiments, the pharmaceutical formulation provided by the present invention includes: (a) GLP-1 fusion protein (wherein the GLP-1 fusion protein has the amino acid sequence shown in SEQ ID NO: 7), (b) Disodium hydrogen phosphate / sodium dihydrogen phosphate buffer, (c) Mannitol, (d) Polysorbate 80.

[0108] In certain embodiments, the pharmaceutical formulation provided by the present invention includes: (a) 0.2-20 mg / mL GLP-1 fusion protein (where the GLP-1 fusion protein has the amino acid sequence shown in SEQ ID NO: 7), (b) 5-15 mM disodium hydrogen phosphate / sodium dihydrogen phosphate buffer, (c) 1-10% (w / v) mannitol, (d) 0.01% (w / v) - 0.04% (w / v) polysorbate 80.

[0109] In certain embodiments, the pharmaceutical formulation provided by the present invention includes: (a) Approximately 2 mg / mL GLP-1 fusion protein (the amino acid sequence of the GLP-1 fusion protein is as shown in SEQ ID NO: 7); (b) Approximately 10 mM disodium hydrogen phosphate / sodium dihydrogen phosphate buffer; (c) Approximately 4.6% (w / v) mannitol; and (d) Approximately 0.02% (w / v) polysorbate 80.

[0110] In certain embodiments, the pharmaceutical formulation provided by the present invention includes: (a) Approximately 4 mg / mL GLP-1 fusion protein (the amino acid sequence of the GLP-1 fusion protein is as shown in SEQ ID NO: 7); (b) Approximately 10 mM disodium hydrogen phosphate / sodium dihydrogen phosphate buffer, (c) Approximately 4.6% (w / v) mannitol, and (d) Approximately 0.02% (w / v) polysorbate 80.

[0111] In certain embodiments, the pharmaceutical formulation provided by the present invention includes: (a) Approximately 6 mg / mL GLP-1 fusion protein (the amino acid sequence of the GLP-1 fusion protein is as shown in SEQ ID NO: 7), (b) Approximately 10 mM disodium hydrogen phosphate / sodium dihydrogen phosphate buffer, (c) Approximately 4.6% (w / v) mannitol, and (d) Approximately 0.02% (w / v) polysorbate 80.

[0112] In certain embodiments, the pharmaceutical formulation provided by the present invention includes: (a) Approximately 5.3 mg / mL GLP-1 fusion protein (the amino acid sequence of the GLP-1 fusion protein is as shown in SEQ ID NO: 7); (b) Approximately 10 mM disodium hydrogen phosphate / sodium dihydrogen phosphate buffer; (c) Approximately 4.6% (w / v) mannitol; and (d) Approximately 0.02% (w / v) polysorbate 80.

[0113] In certain embodiments, the pharmaceutical formulation provided by the present invention includes: (a) Approximately 6.67 mg / mL GLP-1 fusion protein (the amino acid sequence of the GLP-1 fusion protein is as shown in SEQ ID NO: 7); (b) Approximately 10 mM disodium hydrogen phosphate / sodium dihydrogen phosphate buffer; (c) Approximately 4.6% (w / v) mannitol, and (d) Approximately 0.02% (w / v) polysorbate 80.

[0114] In certain embodiments, the pharmaceutical formulation provided by the present invention includes: (a) Approximately 10 mg / mL GLP-1 fusion protein (the amino acid sequence of the GLP-1 fusion protein is shown in SEQ ID NO: 7) (b) Approximately 10 mM disodium hydrogen phosphate / sodium dihydrogen phosphate buffer (c) Approximately 4.6% (w / v) mannitol, and (d) Approximately 0.02% (w / v) polysorbate 80.

[0115] In certain embodiments, the pharmaceutical formulation provided by the present invention includes: (a) GLP-1 fusion protein at approximately 12 mg / mL (the amino acid sequence of the GLP-1 fusion protein is as shown in SEQ ID NO: 7) (b) Approximately 10 mM disodium hydrogen phosphate / sodium dihydrogen phosphate buffer (c) Approximately 4.6% (w / v) mannitol and (d) Approximately 0.02% (w / v) of polysorbate 80 In certain embodiments, the pharmaceutical formulation provided by the present invention includes: (a) Approximately 13.3 mg / mL of GLP-1 fusion protein (the amino acid sequence of the GLP-1 fusion protein is as shown in SEQ ID NO: 7) (b) Approximately 10 mM disodium hydrogen phosphate / sodium dihydrogen phosphate buffer (c) Approximately 4.6% (w / v) mannitol and (d) Approximately 0.02% (w / v) polysorbate 80.

[0116] In certain embodiments, the pharmaceutical formulation provided by the present invention includes: (a) Approximately 20 mg / mL GLP-1 fusion protein (the amino acid sequence of the GLP-1 fusion protein is shown in SEQ ID NO: 7). (b) Approximately 10 mM disodium hydrogen phosphate / sodium dihydrogen phosphate buffer. (c) Approximately 4.6% (w / v) mannitol. (d) Approximately 0.02% (w / v) polysorbate 80.

[0117] The pharmaceutical formulations provided by the present invention can be manufactured, packaged, and / or sold in batches as unit doses and / or multiple unit doses. In certain embodiments, the unit dose of the pharmaceutical formulation provided by the present invention is approximately 0.5 mL and includes: (a) Approximately 1 mg of GLP-1 fusion protein (the amino acid sequence of the GLP-1 fusion protein is shown in SEQ ID NO: 7); (b) Approximately 0.58 mg of disodium hydrogen phosphate dodecahydrate; (c) Sodium dihydrogen phosphate monohydrate approximately 0.465 mg; (d) Approximately 23.2 mg of mannitol; and (e) Approximately 0.1 mg of polysorbate 80, the remainder being water for injection.

[0118] In certain embodiments, the unit dose of the pharmaceutical formulation provided by the present invention is approximately 0.5 mL and includes the following: (a) Approximately 2 mg of GLP-1 fusion protein (the amino acid sequence of the GLP-1 fusion protein is as shown in SEQ ID NO: 7); (b) Approximately 0.58 mg of disodium hydrogen phosphate dodecahydrate; (c) Approximately 0.465 mg of sodium dihydrogen phosphate monohydrate; (d) Approximately 23.2 mg of mannitol; and (e) Contains approximately 0.1 mg of polysorbate 80, with the remainder being water for injection.

[0119] In certain embodiments, the unit dose of the pharmaceutical formulation provided by the present invention is approximately 0.5 mL and includes the following: (a) Approximately 3 mg of GLP-1 fusion protein (the amino acid sequence of the GLP-1 fusion protein is as shown in SEQ ID NO: 7); (b) Approximately 0.58 mg of disodium hydrogen phosphate dodecahydrate; (c) Approximately 0.465 mg of sodium dihydrogen phosphate monohydrate; (d) Approximately 23.2 mg of mannitol; and (e) Contains approximately 0.1 mg of polysorbate 80, with the remainder being water for injection.

[0120] In certain embodiments, the unit dose of the pharmaceutical formulation provided by the present invention is approximately 0.75 mL and includes the following: (a) Approximately 4 mg of GLP-1 fusion protein (the amino acid sequence of the GLP-1 fusion protein is as shown in SEQ ID NO: 7); (b) Approximately 0.87 mg of disodium hydrogen phosphate dodecahydrate; (c) Approximately 0.6975 mg of sodium dihydrogen phosphate monohydrate; (d) Approximately 34.8 mg of mannitol; (e) Contains approximately 0.15 mg of polysorbate 80, with the remainder being water for injection.

[0121] In certain embodiments, the unit dose of the pharmaceutical formulation provided by the present invention is approximately 0.75 mL and includes the following: (a) Approximately 5 mg of GLP-1 fusion protein (the amino acid sequence of the GLP-1 fusion protein is shown in SEQ ID NO: 7); (b) Approximately 0.87 mg of disodium hydrogen phosphate dodecahydrate; (c) Sodium dihydrogen phosphate monohydrate approximately 0.6975 mg; (d) Approximately 34.8 mg of mannitol; and (e) Approximately 0.15 mg of polysorbate 80, the remainder being water for injection.

[0122] In certain embodiments, the unit dose of the pharmaceutical formulation provided by the present invention is approximately 0.75 mL and includes the following: (a) Approximately 7.5 mg of GLP-1 fusion protein (the amino acid sequence of the GLP-1 fusion protein is as shown in SEQ ID NO: 7); (b) Approximately 0.87 mg of disodium hydrogen phosphate dodecahydrate. (c) Sodium dihydrogen phosphate monohydrate approximately 0.6975 mg, (d) Approximately 34.8 mg of mannitol, and (e) Approximately 0.15 mg of polysorbate 80, the remainder being sterile water for injection.

[0123] In certain embodiments, the unit dose of the pharmaceutical formulation provided by the present invention is approximately 0.75 mL and includes the following: (a) Approximately 9 mg of GLP-1 fusion protein (the amino acid sequence of the GLP-1 fusion protein is shown in SEQ ID NO: 7); (b) Approximately 0.87 mg of disodium hydrogen phosphate dodecahydrate; (c) Sodium dihydrogen phosphate monohydrate approximately 0.6975 mg; (d) Approximately 34.8 mg of mannitol; and (e) Approximately 0.15 mg of polysorbate 80, the remainder being water for injection.

[0124] In certain embodiments, the unit dose of the pharmaceutical formulation provided by the present invention is approximately 0.75 mL and includes the following: (a) Approximately 10 mg of GLP-1 fusion protein (the amino acid sequence of the GLP-1 fusion protein is as shown in SEQ ID NO: 7); (b) Approximately 0.87 mg of disodium hydrogen phosphate dodecahydrate; (c) Approximately 0.6975 mg of sodium dihydrogen phosphate monohydrate; (d) Approximately 34.8 mg of mannitol; and (e) Contains approximately 0.15 mg of polysorbate 80, with the remainder being water for injection.

[0125] In certain embodiments, the unit dose of the pharmaceutical formulation provided by the present invention is approximately 0.75 mL and includes the following: (a) Approximately 15 mg of GLP-1 fusion protein (the amino acid sequence of the GLP-1 fusion protein is as shown in SEQ ID NO: 7); (b) Approximately 0.87 mg of disodium hydrogen phosphate dodecahydrate; (c) Approximately 0.6975 mg of sodium dihydrogen phosphate monohydrate; (d) Approximately 34.8 mg of mannitol; and (e) Contains approximately 0.15 mg of polysorbate 80, with the remainder being water for injection.

[0126] The pharmaceutical formulations provided by the present invention have superior stability. In certain embodiments, the pharmaceutical formulations provided herein are stable at 2°C to 8°C for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 25 months, 26 months, 27 months, 28 months, 29 months, 30 months, 31 months, 32 months, 33 months, 34 months, 35 months, or 36 months, etc. In certain embodiments, the pharmaceutical formulations provided by the present invention are stable at 2°C to 8°C for at least 24 months. In certain embodiments, the pharmaceutical formulations provided by the present invention are stable at 2°C to 8°C for at least 36 months.

[0127] In certain embodiments, the pharmaceutical formulations provided by the present invention are stable at 25±2℃ for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months. In certain embodiments, the pharmaceutical formulations provided by the present invention are stable at 25±2℃ for at least 6 months. In certain embodiments, the pharmaceutical formulations provided by the present invention are stable at 25±2℃ for at least 3 months.

[0128] In certain embodiments, the pharmaceutical formulations provided by the present invention are stable at 40±2°C for at least one week, two weeks, three weeks, four weeks, or one month. In certain embodiments, the pharmaceutical formulations provided by the present invention are stable at 40±2°C for at least two weeks.

[0129] The stability of the pharmaceutical formulations provided by the present invention can be evaluated using various indicators, such as changes in pH value, changes in GLP-1 fusion protein concentration, the content of high molecular weight (HMW) derivatives and / or low molecular weight (LMW) derivatives, changes in purity, and changes in isoelectric point.

[0130] In certain embodiments, the pharmaceutical formulation provided by the present invention exhibits no change in pH value exceeding approximately 10% (for example, not exceeding approximately 9%, not exceeding approximately 8%, not exceeding approximately 7%, not exceeding approximately 6%, not exceeding approximately 5%, not exceeding approximately 4%, not exceeding approximately 3%, not exceeding approximately 2%, not exceeding approximately 1%) after at least one month (e.g., 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 25 months, 26 months, 27 months, 28 months, 29 months, 30 months, 31 months, 32 months, 33 months, 34 months, 35 months, or 36 months). For example, the pH value of the pharmaceutical formulation provided by the present invention remained within the range of 6.4 to 6.7 even after at least one month (e.g., 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 25 months, 26 months, 27 months, 28 months, 29 months, 30 months, 31 months, 32 months, 33 months, 34 months, 35 months, or 36 months). In some embodiments, the pH value of the pharmaceutical formulation provided by the present invention does not change by more than about 3% after being left for at least one month.

[0131] In certain embodiments, the pharmaceutical formulation provided by the present invention shows no change in GLP-1 fusion protein concentration exceeding approximately 10% (e.g., not exceeding approximately 9%, not exceeding approximately 8%, not exceeding approximately 7%, not exceeding approximately 6%, not exceeding approximately 5%, not exceeding approximately 4%, not exceeding approximately 3%, not exceeding approximately 2%, not exceeding approximately 1%) after at least one month (e.g., 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 25 months, 26 months, 27 months, 28 months, 29 months, 30 months, 31 months, 32 months, 33 months, 34 months, 35 months, or 36 months). For example, after leaving the pharmaceutical formulation provided by the present invention for at least one month, the change in GLP-1 fusion protein concentration was within the range of ±0.2 mg / mL. In certain embodiments, after leaving the pharmaceutical formulation provided by the present invention for at least one month, the change in GLP-1 fusion protein concentration does not exceed approximately 3%. In some embodiments, the pharmaceutical formulation provided by the present invention is left for at least one month such that the change in GLP-1 fusion protein concentration does not exceed 3%. Those skilled in the art can measure the GLP-1 fusion protein concentration in the pharmaceutical formulation, for example, by the method described in Example 12.1.3 or Example 13.2.9, by a method commonly used by those skilled in the art.

[0132] In certain embodiments, the pharmaceutical formulations provided by the present invention have a content of high molecular weight (HMW) derivatives or low molecular weight (LMW) derivatives that does not exceed about 10% (for example, not exceeding about 9%, not exceeding about 8%, not exceeding about 7%, not exceeding about 6%, not exceeding about 5%, not exceeding about 4%, not exceeding about 3%, not exceeding about 2%, not exceeding about 1%) after at least one month (e.g., 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 25 months, 26 months, 27 months, 28 months, 29 months, 30 months, 31 months, 32 months, 33 months, 34 months, 35 months, or 36 months).

[0133] In certain embodiments, the pharmaceutical formulations provided by the present invention have a content of high molecular weight (HMW) derivatives and low molecular weight (LMW) derivatives that does not exceed approximately 10% (for example, not exceeding approximately 9%, not exceeding approximately 8%, not exceeding approximately 7%, not exceeding approximately 6%, not exceeding approximately 5%, not exceeding approximately 4%, not exceeding approximately 3%, not exceeding approximately 2%, not exceeding approximately 1%) after at least one month (e.g., 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 25 months, 26 months, 27 months, 28 months, 29 months, 30 months, 31 months, 32 months, 33 months, 34 months, 35 months, or 36 months).

[0134] In certain embodiments, the pharmaceutical formulation provided by the present invention contains no more than about 5% of HMW derivatives after at least one month (for example, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 25 months, 26 months, 27 months, 28 months, 29 months, 30 months, 31 months, 32 months, 33 months, 34 months, or 1 month, 35 months, or 36 months). In certain embodiments, the pharmaceutical formulation provided by the present invention contains no more than approximately 5% LMW derivative after at least one month (for example, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 25 months, 26 months, 27 months, 28 months, 29 months, 30 months, 31 months, 32 months, 33 months, 34 months, 35 months, or 36 months). In certain embodiments, the pharmaceutical formulation provided by the present invention contains no more than approximately 5% HMW derivative and no more than approximately 5% LMW derivative after at least one month (for example, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty

[0135] In certain embodiments, the pharmaceutical formulation provided by the present invention will not exceed approximately 4% after at least one month (for example, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 25 months, 26 months, 27 months, 28 months, 29 months, 30 months, 31 months, 32 months, 33 months, 34 months, or 1 month, 35 months, or 36 months). In certain embodiments, the pharmaceutical formulation provided by the present invention will have been left for at least one month so that the HMW derivative content does not exceed 4%. In certain embodiments, the pharmaceutical formulation provided by the present invention contains no more than approximately 1% LMW derivative after at least one month (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 months). In certain embodiments, the pharmaceutical formulation provided by the present invention has been left for at least one month so that the LMW derivative content does not exceed 1%.

[0136] Those skilled in the art can measure the content of HMW derivatives and / or LMW derivatives in a pharmaceutical formulation by methods commonly used by those skilled in the art. In some embodiments, the content of HMW derivatives and / or LMW derivatives is determined by volume exclusion chromatography (SEC) or reverse liquid chromatography (RP-LC). Those skilled in the art can adjust the measurement conditions by experience or as needed. In some embodiments, the content of HMW derivatives and / or LMW derivatives is determined by the methods described in Examples 12.1.5, 12.1.6, 13.2.6, or 13.2.7 of this application.

[0137] In certain embodiments, the pharmaceutical formulations provided by the present invention are left standing for at least one month (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty, twenty, twenty, twenty, twenty, twenty, twenty, twenty, twenty, twenty, twenty, twenty, twenty, thirty

[0138] The "purity" of the pharmaceutical formulation provided in this application refers to the relative content of the active ingredient in the formulation, i.e., the GLP-1 fusion protein (e.g., YN-011). For example, when measuring purity using the SEC-HPLC method, the peak percentage of the SEC monomer calculated based on the peak area normalization method represents the protein purity. For example, when measuring purity using the SEC-HPLC method, "purity change not exceeding approximately 2%" means that after a certain period of time, the change in the percentage of the individual SEC peak does not exceed approximately 2%.

[0139] Those skilled in the art can measure the purity of a pharmaceutical formulation by methods commonly used by those skilled in the art, such as SDS-capillary electrophoresis (e.g., non-reducing SDS-capillary electrophoresis). In some embodiments, the purity of a pharmaceutical formulation is determined by the methods described in Examples 12.1.5, 12.1.7, 13.2.7, or 13.2.8 of this application.

[0140] In certain embodiments, the pharmaceutical formulation provided by the present invention is configured such that, at least one month later (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty, twenty, twenty, twenty, twenty, twenty, twenty, twenty, twenty, twenty, twenty, twenty, thirty

[0141] In some embodiments, the isoelectric point of the pharmaceutical formulation provided by the present invention is determined by capillary focused isoelectric electrophoresis (cIEF). In some embodiments, the isoelectric point of the pharmaceutical formulation provided by the present invention is determined by the method described in Example 12.1.8 or 13.2.5 of the present invention.

[0142] In some embodiments, the pharmaceutical formulations provided by the present invention may also include a suitable diluent or carrier.

[0143] In another preferred embodiment, the diluent is sterile. Suitable diluents for GLP-1 fusion proteins and / or cells include, but are not limited to, aqueous saline solutions, pH buffers, and the diluents described herein, as well as glycerin solutions or other solutions suitable for frozen polypeptides and / or cells. Suitable diluents for nucleic acids and / or carriers include, but are not limited to, aqueous saline solutions, pH buffers, the diluents described herein, and water.

[0144] In another preferred embodiment, the carrier is a pharmaceutically acceptable carrier.

[0145] Examples of pharmaceutically acceptable carriers used in pharmaceutical formulations provided by the present invention include, for example, pharmaceutically acceptable liquids, gels, solid carriers, aqueous media, non-aqueous media, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspensions / dispersants, chelating agents, diluents, adjuvants, excipients or non-toxic auxiliary substances, other components known in the art, or various combinations thereof.

[0146] Examples of pharmaceutically acceptable carriers for pharmaceutical formulations provided by the present invention include sodium chloride injection, Ringer's injection, isotonic dextrorotonic acid injection, sterile water injection, or dextrorotonic acid and lactated Ringer's injection, non-aqueous media such as plant-derived non-volatile oils, cottonseed oil, corn oil, sesame oil, and peanut oil, antimicrobial agents that inhibit bacteria or fungal concentrations, penetrating agents such as sodium chloride and dextrose, buffering agents such as phosphate or citrate buffers, antioxidants such as sodium bisulfate, local anesthetics such as procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethylcellulose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone, emulsifiers such as polysorbate 80 (TWEEN-80), chelating agents such as ethylenediaminetetraacetic acid (EDTA) or ethylene glycoltetraacetic acid (EGTA), ethanol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid. Antimicrobial agents used as carriers can be added to multi-dose container compositions containing phenol or cresol, mercury compounds, benzyl alcohol, chlorobutanol, methylparaben and propylparaben, thimerosal, benzalkonium chloride, and benzethonium chloride. Suitable excipients include, for example, water, physiological saline, dextrose, glycerin, or ethanol. Suitable non-toxic auxiliary substances include, for example, wetting or emulsifying agents, pH buffers, stabilizers, solubility enhancers, or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrin.

[0147] The pharmaceutical formulations provided by the present invention can be prepared in a variety of dosage forms. Suitable dosage forms include, but are not limited to, solutions, suspensions, pills, tablets, and lyophilized preparations. In certain embodiments, the pharmaceutical formulations provided by the present invention are prepared as injectable compositions. Injectable compositions can be prepared in any conventional form, such as liquid solutions, suspensions, emulsions, or solid forms suitable for producing liquid solutions, suspensions, or emulsions. Injectable formulations include preparing sterile and / or non-thermally active raw material solutions for injection, lyophilized powders including subcutaneous tablets, sterile suspensions for injection, sterile-dried insoluble products combined with a medium immediately before use, and sterile and / or non-thermally active raw material emulsions. Solutions may be aqueous or non-aqueous.

[0148] In some embodiments, the pharmaceutical formulation provided by the present invention is a liquid formulation. In some embodiments, the pharmaceutical formulation provided by the present invention is an aqueous solution. The aqueous solution may contain, for example, at least 70% w / w, at least 75% w / w, at least 80% w / w, at least 85% w / w, at least 90% w / w, or at least 95% w / w of water. In some embodiments, the pharmaceutical formulation provided by the present invention is an injectable formulation. In some embodiments, the pharmaceutical formulation provided by the present invention is administered by intravenous injection, subcutaneous injection, or intramuscular injection. In some embodiments, the pharmaceutical formulation provided by the present invention is an isotonic liquid formulation, i.e., a liquid formulation in which the osmotic pressure is equal to the plasma osmotic pressure.

[0149] In some embodiments, a sterile lyophilized powder is prepared by dissolving a GLP-1 fusion protein, such as those disclosed herein, in a suitable solvent. The solvent may contain excipients or other pharmacological components that improve the stability of the powder or the reconstituted solution produced from the powder. Suitable excipients include, but are not limited to, water, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, or other suitable agents. The solvent may also contain buffers such as citrate, sodium phosphate, or potassium phosphate, or other such buffers known to those skilled in the art, in which case the buffer exhibits a substantially neutral pH value. The solution is then sterile filtered and subsequently lyophilized under standard conditions known to those skilled in the art to obtain the desired preparation. In some embodiments, the obtained solution is partitioned into vials and lyophilized. Each vial may contain a single or multiple amount of the fusion polypeptide, polypeptide complex, or complex or composition provided herein. Overfilling of vials is acceptable if the amount exceeds a single dose or is less than the amount required for one set of doses (e.g., about 10%), in order to facilitate accurate extraction and administration of the sample. The lyophilized powder can be stored under suitable conditions such as about 4°C to room temperature.

[0150] Reconstitution of lyophilized powder with injectable water provides a formulation for parenteral administration. In some embodiments, sterile and / or heat-free raw water or other suitable liquid carrier is added to the lyophilized powder for reconstitution. The exact amount depends on the given selected treatment and can be determined empirically.

[0151] In some embodiments, parenteral formulations per unit dose are packaged in ampoules, vials, or syringes with needles. All formulations for parenteral administration must be sterile and free of thermogenic substances, as is known and practiced in the art.

[0152] The compositions described herein may be administered by any method known to be effective by a general technician. One embodiment is peripheral parenteral administration by a sterile syringe or other mechanical device such as an infusion pump. In some embodiments, the peripheral parenteral route is an intravenous, intramuscular, subcutaneous, or intraperitoneal route of administration.

[0153] In some embodiments, the GLP-1 fusion proteins described herein are prepared in a solid preparation, such as by lyophilization or spray drying, and then reconstituted in a suitable diluent solution before application.

[0154] This is explained in "Remington: The Science and Practice of Pharmacy" (edited by DB Troy, 21st edition, Lippincott, Williams & Wilkins, 2006).

[0155] The active ingredient in the pharmaceutical formulation provided by the present invention, namely the GLP-1 fusion protein, will be described in detail below.

[0156] 1. GLP-1 fusion protein The pharmaceutical formulation provided by the present invention contains a fusion protein of stable GLP-1 and IgG / Fc domain formation.

[0157] In some embodiments, the GLP-1 fusion protein in the pharmaceutical formulation provided by the present invention comprises a GLP-1 polypeptide comprising one or more amino acid substitutions from the following group to natural human GLP-1 (A8G, G22E, R36G), and an immunoglobulin Fc domain, wherein the immunoglobulin Fc domain comprises or includes an IgG2-Fc domain comprising one or more amino acid substitutions from the following group, C222S, A330S, and P331S.

[0158] GLP-1 polypeptide Surprisingly, the yield and activity of the GLP-1 fusion protein in the pharmaceutical formulations provided by the present invention are improved by increasing the hydroxylation level at lysine at position 34 (K34) relative to natural human GLP-1, or by decreasing the oxidation at serine at position 31 (W31) relative to natural human GLP-1, and / or by site mutations (e.g., one or more mutations in A8G, G22E, R36G) by introducing one or more GLP-1 polypeptides, or by site mutations in the IgG2-Fc domain (e.g., one or more mutations in C222S, A330S, P331S).

[0159] The unprocessed natural human GLP-1 polypeptide has 37 amino acids, and its amino acid sequence is shown as SEQ ID NO:43, and is commonly referred to as "GLP-1(1-37)". The unprocessed natural human GLP-1 polypeptide is processed in the pancreas and small intestine to form GLP-1(7-37) or GLP-1(7-36) amide. In addition to the specific explanation, the amino acid sites of the GLP-1 polypeptide described in this application are the amino acid sites corresponding to SEQ ID NO:43. For example, K 34 of the GLP-1 polypeptide described in this application corresponds to the 34th site of SEQ ID NO:43. Also, for example, GLP-1(7-36) amide refers to a GLP-1 polypeptide fragment formed from amino acids between positions 7 and 36 of SEQ ID NO:43, and GLP-1(7-37) refers to a GLP-1 polypeptide fragment formed from amino acids between positions 7 and 37 of SEQ ID NO:43. In certain embodiments, the amino acid sequence of GLP-1(7-36) amide is shown in SEQ ID NO:1. In some embodiments, the amino acid sequence of GLP-1(7-37) is shown in SEQ ID NO:2.

[0160] Unless otherwise specified, the nomenclature for amino acid mutations referred to in this application is: name of the amino acid before the mutation - amino acid site where the mutation occurred - name of the amino acid after the mutation. For example, the A8G mutation in the GLP-1 polypeptide refers to a mutation in which alanine (A) at position 8 of SEQ ID NO:43 is changed to glycine (G).

[0161] The hydroxylation level of the GLP-1 fusion protein described in this application can be measured by various methods known in the prior art. For example, it was measured by the mass spectrometry method described by Hou et al. (Hou, Y., et al., Nutrient Optimization Reduces Phosphorylation and Hydroxylation Level on an Fc-Fusion Protein a CHO Fed-Batch Process. Biotechnol J, 2019. 14(3):pe 170070706.). Unless otherwise specified, the hydroxylation levels described in this application are based on the percentage of molecules. For example, if 10 g of the GLP-1 fusion protein according to the present invention is hydroxylated to K34 of the GLP-1 polypeptide and the remaining 90 g of the GLP-1 fusion protein is not hydroxylated to K34 of the GLP-1 polypeptide, the hydroxylation level of the GLP-1 fusion protein is considered to be 10%.

[0162] In some embodiments, the GLP-1 fusion protein of the present invention improves the in vivo half-life and / or yield during recombinant production. Therefore, the GLP-1 fusion protein and reagents for producing the GLP-1 fusion protein can be used in the manufacture of drugs.

[0163] In one embodiment, the GLP-1 polypeptide has a certain level of hydroxylation at lysine 34 (K34) relative to natural human GLP-1. K34 refers to the lysine residue at position 34 of the natural human GLP-1 polypeptide. Those skilled in the art know that the same lysine can correspond to different positions in other GLP-1 polypeptide sequences.

[0164] The GLP-1 fusion protein can refer to multiple molecules that may or may not be hydroxylated at K34, and the hydroxylation levels of the molecules can vary.

[0165] The present invention also provides components comprising a GLP-1 fusion protein. In some embodiments, the hydroxylation level at K34 of the GLP-1 fusion protein is about 10% to 100%, for example, at least about 20%, at least about 26%, at least about 30%, at least about 40%, or at least about 50%. Any percentage or range from 10% to 100% is considered.

[0166] In some embodiments, the hydroxylation level is 10% or more, for example, 10% or more, 15% or more, or 20% or more, or 26% or more, 27% or more, 28% or more, 29% or more, or 30% or more, or 35% or more, or 40% or more, or 45% or more, or 50% or more, or 55% or more, or 60% or more, or 65% or more, or 70% or more, or 75% or more, or 80% or more, or 85% or more, or 90% or more, or 95% or more.

[0167] In some embodiments, in the pharmaceutical formulations provided by the present invention, the GLP-1 fusion protein hydroxylates 10% or more, 15% or more, at least 20% or more, 26% or more, or 30% or more, or 40% or more, or 50% or more, or 60% or more, or 70% or more, or 80% or more, or 90% or more of the GLP-1 polypeptide in the fusion protein on K34 relative to natural human GLP-1.

[0168] As shown in the embodiments of this application, the yield of hydroxylated GLP-1 fusion protein is approximately 100 to 500 times higher than that of GLP-1 fusion protein in which hydroxylation is not detected at K34 of GLP-1.

[0169] The present invention has also found that the GLP-1 polypeptide in the GLP-1 fusion protein has a low oxidation level relative to the leucine at position 31 of natural human GLP-1 (W31), and that a low oxidation level may be advantageous for the stability and / or activity of the GLP-1 fusion protein. In some embodiments, the GLP-1 polypeptide in the GLP-1 fusion protein is substantially unoxidized at the leucine at position 31 (W31) relative to natural human GLP-1. In certain embodiments, the oxidation level of the GLP-1 polypeptide in the GLP-1 fusion protein on W31 relative to natural human GLP-1 is less than 0.5% (e.g., less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1%) or undetectable. W31 refers to the leucine residue at position 31 of the natural human GLP-1 polypeptide. Those skilled in the art will know that the same leucine may correspond to different positions in other GLP-1 polypeptides.

[0170] Detection can be performed by methods known in embodiments of this application or in the prior art (e.g., the mass spectrometry method described in WO 2002046227 A2 or by Bettinger et al. (Bettinger, JQ, et al., Quantitative Analysis of in Vivo Methionine Oxidation of the Human Proteome. J Proteome Res, 2020.19(2):p.624-633). Unless otherwise specified, the oxidation levels described herein are calculated based on the percentage of molecules. For example, if 10 g of GLP-1 fusion protein in 100 g of the present invention is oxidized by GLP-1 W31 and the remaining 90 g of GLP-1 fusion protein is not oxidized by GLP-1 W31, the oxidation level of the GLP-1 fusion protein is considered to be 10%.

[0171] The GLP-1 polypeptide of the fusion protein disclosed herein may be human-derived GLP-1. In some embodiments, the GLP-1 polypeptide is selected from human GLP-1(7-37), human GLP-1(7-36) amide, and DPP-IV resistant human GLP-1, and includes A8G and G22E substitutions relative to natural human GLP-1.

[0172] In one embodiment, the GLP-1 polypeptide is selected from human GLP-1(7-37), human GLP-1(7-36) amide, and DPP-IV resistant human GLP-1, and includes A8G, G22E, and R36G substitutions relative to natural human GLP-1.

[0173] In some embodiments, the GLP-1 polypeptide is GLP-1(7-37). In some embodiments, the GLP-1 polypeptide is GLP-1(7-36) amide. In some embodiments, the GLP-1 polypeptide is DPP-IV resistant GLP-1. In some embodiments, the GLP-1 polypeptide may contain amino acid substitutions such as one, two, or three mutations among A8G, G22E, and R36G.

[0174] In some embodiments, the GLP-1 polypeptide in the GLP-1 fusion protein described herein has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity of the amino acid sequence shown in, for example, SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, and contains one or more amino acid substitutions selected from the following group to natural human GLP-1: A8G, G22E, and R36G. In some embodiments, the GLP-1 polypeptide in the GLP-1 fusion protein described herein has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity of the amino acid sequence shown in, for example, SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, and stimulates insulin secretion by β cells in a glucose-dependent manner. In one embodiment, the GLP-1 polypeptide in the GLP-1 fusion protein described herein has, for example, the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity, and comprises one or more amino acid substitutions selected from the following group to native human GLP-1: A8G, G22E, and R36G, which stimulate insulin secretion from β-cells in a glucose-dependent manner.

[0175] [Table 2]

[0176] In one embodiment, the GLP-1 polypeptide is human GLP-1(7-37), and includes A8G, G22E, and R36G substitutions relative to natural human GLP-1.

[0177] In one embodiment, the amino acid sequence of the GLP-1 polypeptide includes the amino acid sequence shown in SEQ ID NO:3.

[0178] In one embodiment, the GLP-1 polypeptide is DPP-IV resistant human GLP-1.

[0179] Natural GLP-1 has a short circulating half-life (t1 / 2 < 2 minutes), which is mainly due to rapid enzyme inactivation and / or renal clearance, including dipeptide peptidase IV (DPP-IV). Therefore, when using natural GLP-1, subcutaneous infusion by pump must be continued to maintain the action of GLP-1 in vivo. Accordingly, pharmaceutically effective anti-degradable GLP-1 analog peptides have been developed. For example, Dulaglutide is a DPP-IV protected GLP-1 analog fused with an IgG4 / Fc fragment, with a half-life of 4.7–5.5 days (Geiser, JS, et al., Clinical Pharmacokinetics of Dulaglutide in Patients with Type 2 Diabetes: Analysis of Data from Clinical Trials. Clin Pharmacokinetics, 2016. 55(5): p.625-34).

[0180] FC Domain In one embodiment, the IgG2-Fc domain in the GLP-1 fusion protein described in this application is an Fc domain derived from human IgG2.

[0181] In this application, "IgG2-Fc" and "IgG2 / Fc" are interchangeable terms that refer to the Fc domain of immunoglobulin IgG2.

[0182] In some embodiments, the IgG2-Fc domain described herein has a certain level of oxidation on the methionine at position 253 (M253) corresponding to SEQ ID NO:7. In some embodiments, the IgG2-Fc domain described herein has a reduced level of oxidation on the methionine at position 253 (M253) corresponding to SEQ ID NO:7. In some embodiments, the IgG2-Fc domain described herein is not oxidized on the methionine at position 253 (M253) corresponding to SEQ ID NO:7. In this application, M253 refers to the methionine residue in IgG2 at position 253 corresponding to SEQ ID NO:7. Those skilled in the art will recognize that the same methionine may correspond to different positions in other IgG polypeptides.

[0183] In some embodiments, the oxidation level of the IgG2-Fc domain on M253 corresponding to SEQ ID NO:7 is approximately 15% or less, approximately 10% or less, approximately 9% or less, approximately 8% or less, approximately 7% or less, approximately 6% or less, approximately 5% or less, approximately 4% or less, approximately 3% or less, approximately 2% or less, approximately 1% or less, approximately 0.5% or less, or undetectable.

[0184] As mentioned above, a GLP-1 fusion protein can refer to multiple molecules that are oxidized or not oxidized at one or two positions of the IgG2-Fc domain M253 and / or W31 of the GLP-1 polypeptide, and the oxidation levels of the molecules can vary. Oxidation occurs within the rectangular frame of the following amino acid structure:

[0185] [Table 3]

[0186] In one embodiment, the GLP-1 fusion protein according to the present invention shows no oxidation on W31 of the GLP-1 polypeptide relative to natural human GLP-1, and has an oxidation level of approximately 2% at M253 of the IgG2-Fc domain. The oxidized water levels of other GLP-1 analog fusion proteins on W31 (for the GLP-1 polypeptide) and / or M253 (for the IgG2-Fc domain) average 2–8%.

[0187] The GLP-1 polypeptide in the fusion protein covalently binds to the Fc portion of the immunoglobulin (either directly or via a binding peptide). In some embodiments, the immunoglobulin is IgG. The IgG-Fc of the fusion protein disclosed herein may be IgG2-Fc.

[0188] In some embodiments, the IgG2-Fc domain includes a C222S substitution. The C222S substitution of IgG2-Fc can increase the flexibility of the N-terminal hinge region by removing the disulfide bond between the two monomers of the same dimer. The increased flexibility of the N-terminal hinge region can reduce the binding affinity to the Fcγ receptor, thereby reducing antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).

[0189] In another embodiment, the IgG2-Fc domain includes A330S and P331S substitutions. The A330S and P331S substitutions reduce affinity for the Fcγ receptor and C1q complement protein.

[0190] In some embodiments, the IgG2-Fc domain includes C222S, A330S, and P331S substitutions.

[0191] In addition to the amino acid position M253 being the amino acid position corresponding to SEQ ID NO:7, other amino acid residue positions in the IgG2-Fc domain described in this application include, for example, A330, P331, and C222, corresponding to positions in human IgG2 as shown in Genbank registration number QRG 33935.1. The IgG2-Fc portion may also be, for example, the 228 amino acids shown in SEQ ID NO:5, which correspond to amino acids 219-445 of human IgG2 as shown in Genbank registration number QRG 33935.1. Those skilled in the art will readily identify amino acid residue positions, such as the residue positions of A330, P331, and C222 in the reference sequence SEQ ID NO:5 or SEQ ID NO:6, or in the short or long Fc fragment.

[0192] In some embodiments, the IgG2-Fc domain described herein has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity compared to the amino acid sequence shown in SEQ ID NO:5 or SEQ ID NO:6, and includes one or more amino acid substitutions selected from the following groups: C222S, A330S, and P331S. Here, the fusion protein has an improved half-life compared to GLP-1 polypeptides without IgG / Fc domain fusion or with IgG4 / Fc domain fusion.

[0193] In some embodiments, the IgG2-Fc domain has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 5 or SEQ ID NO: 6, and includes one or more amino acid substitutions selected from the following groups: C222S, A330S, and P331S.

[0194] In one embodiment, the IgG2-Fc domain has at least 90% sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 5 or SEQ ID NO: 6 and includes A330S and P331S substitutions. Preferably, the IgG2-Fc domain further includes a C222S substitution. More preferably, the amino acid sequence of the IgG2-Fc domain is shown in SEQ ID NO: 6.

[0195] In one embodiment, in the GLP-1 fusion protein described in this application, the amino acid sequence of the GLP-1 polypeptide is shown in SEQ ID NO:3, and the amino acid sequence of the immunoglobulin Fc domain is shown in SEQ ID NO:6.

[0196] [Table 4]

[0197] The table below shows the correspondence between the positions in SEQ ID NO:3 and SEQ ID NO:7 of the GLP-1 polypeptide as described in this application, due to A8G, G22E, R36G substitution and K34 hydroxylation. The table below also shows the correspondence between the positions in QRG 33935.1 and SEQ ID NO:7 of the sites generated by C222S, A330S, P331S substitution and M253 oxidation on the IgG2-Fc domain as described in this application.

[0198] [Table 5]

[0199] In one embodiment, the GLP-1 peptide is located at the N-terminus of the immunoglobulin Fc domain. In another embodiment, the GLP-1 peptide is located at the C-terminus of the immunoglobulin Fc domain.

[0200] Linker In one embodiment, the GLP-1 peptide is directly covalently bound to the immunoglobulin Fc domain.

[0201] In one embodiment, the GLP-1 peptide is covalently bound to the immunoglobulin Fc domain via a linker.

[0202] In one embodiment, the linker is selected from a group of severable linkers, inseverable linkers, flexible linkers, rigid linkers, helical linkers, and non-helical linkers.

[0203] In one embodiment, the linker contains a connecting peptide.

[0204] The GLP-1 peptide and IgG-Fc domain (such as the IgG2-Fc domain) of the fusion proteins disclosed in this article can be linked to each other via a linking peptide. As used herein, the term “linking peptide” refers to any portion that links different functional domains of a peptide. The linking peptide can have any suitable length and structure.

[0205] In this invention, the term "linked peptide" refers to a preferred segment of amino acids that links different functional domains of a peptide. Various linked peptides have been investigated, and linked peptides can have any suitable length and structure.

[0206] In this invention, the term "cleavable linker" refers to a linker that is sensitive to intracellular proteases, pH, or chemical factors and is readily cleaved in the presence of such factors.

[0207] In this invention, the term "uncleavable linker" refers to a linker that is stable and resistant to intracellular proteases, pH, or chemical factors and is not easily cleaved.

[0208] In this invention, the term "flexible linker" refers to a linker that enhances spatial flexibility when linking different protein components, enabling the protein components to fold and form a three-dimensional structure without significant interference from one another.

[0209] In this invention, the term "rigid linker" refers to a linker that maintains a constant distance between different protein components when linking them together.

[0210] In the present invention, the term "helical linker" refers to a linker in which rigid units can form helical structures (such as α-helices) internally or between identical adjacent sequences, thereby providing a linker that gives the resulting fusion protein a relatively stable structure.

[0211] In this invention, the term "non-helical linker" refers to a linker that cannot form a helical structure.

[0212] In one embodiment, the linker comprises a sequence containing a glycine residue and a serine residue. Preferably, the linker comprising the glycine residue and serine residue contains 1, 2, 3, 4, or more repeats, as shown in SEQ ID NO: 39 (GGGS), SEQ ID NO: 40 (GGGGS), SEQ ID NO: 41 (GGGGGS), or SEQ ID NO: 42 (GGGGGGGS).

[0213] In one embodiment, the linker includes an amino acid sequence selected from the following group: SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19.

[0214] [Table 6]

[0215] In certain embodiments, the linker peptide linking the GLP-1 polypeptide to the IgG2 / Fc structural domain may have at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity with respect to the amino acid sequence shown in SEQ ID NO:9. In certain embodiments, the linker peptide comprises the amino acid sequence shown in SEQ ID NO:9. In certain embodiments, the linker peptide has an amino acid sequence as shown in SEQ ID NO:9.

[0216] In a particular embodiment, in the GLP-1 fusion protein described herein, the GLP-1 polypeptide has the amino acid sequence shown in SEQ ID NO:3, the immunoglobulin Fc structural domain has the amino acid sequence shown in SEQ ID NO:6, and the linker has the amino acid sequence shown in SEQ ID NO:9.

[0217] Fusion protein In certain embodiments, the GLP-1 fusion proteins described herein have at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity with respect to the amino acid sequence shown in SEQ ID NO:7. In certain embodiments, the GLP-1 fusion proteins described herein have at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity with respect to the amino acid sequence shown in SEQ ID NO:7, and the GLP-1 polypeptide includes A8G, G22E, and R36G substitutions, and IgG2-Fc structural domain C222S, A330S, and P331S substitutions. In certain embodiments, the GLP-1 fusion protein described herein has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity with respect to the amino acid sequence shown in SEQ ID NO:7, and stimulates glucose-dependent insulin secretion from β-cells. In certain embodiments, the GLP-1 fusion protein described herein has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity with respect to the amino acid sequence shown in SEQ ID NO:7, and the GLP-1 polypeptide consists of A8G, G22E, R36G substitutions, IgG2-Fc structural domain C222S, A330S, and P331S substitutions, and stimulates glucose-dependent insulin secretion from β-cells.

[0218] In certain embodiments, the GLP-1 fusion protein described herein has an amino acid sequence shown in SEQ ID NO:7, or an amino acid sequence having at least 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) sequence identity with SEQ ID NO:7.

[0219] In certain embodiments, the GLP-1 fusion protein described herein has the amino acid sequence shown in SEQ ID NO:7, or an amino acid sequence having at least about 90% sequence identity with SEQ ID NO:7.

[0220] In certain embodiments, the GLP-1 fusion protein described herein has the amino acid sequence shown in SEQ ID NO:7.

[0221] [Table 7]

[0222] As shown in the examples, by modifying the sequence, the GLP-1 fusion proteins disclosed herein have a longer half-life, for example, the GLP-1 fusion protein YN-011 has a half-life of approximately 8.6 days (207 hours).

[0223] In the preparation of the GLP-1 fusion protein described herein, a signal peptide may also be included in the GLP-1 fusion protein.

[0224] In this specification, the term “signal peptide” refers to a polypeptide that enables a fusion protein to be secreted into an extracellular medium. Such peptides are sometimes also called “lead peptides,” “peptide precursors,” or “prepeptides.” The use of signal peptides to induce protein secretion is well known in the art (e.g., U.S. Patent US8658174, the contents of which are incorporated herein by reference in their entirety). Examples of signal peptides include, but are not limited to, human CD33 signal peptide, human growth hormone-releasing hormone (GHRH) signal peptide, human α-1-microglobulin / bikunin precursor (AMBP) signal peptide, Gauss luciferase signal peptide, mouse immunoglobulin heavy chain signal peptide, and mouse immunoglobulin κ light chain signal peptide. Signal peptides are cleaved during secretion. In some embodiments, cleavage of the signal peptide generates an active histidine residue at the N-terminus of the GLP-1 polypeptide.

[0225] In some embodiments, the signal peptide is a human CD33 signal peptide.

[0226] In certain embodiments, the signal peptide has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity to the amino acid sequence shown in SEQ ID NO:4 (MPLLLLPLWAGALA), enabling the secretion of the GLP-1 fusion protein. In certain embodiments, the signal peptide has the amino acid sequence shown in SEQ ID NO:4, or an amino acid sequence having at least 90% sequence identity to SEQ ID NO:4. In certain embodiments, the signal peptide has the amino acid sequence shown in SEQ ID NO:4.

[0227] In certain embodiments, the GLP-1 fusion proteins described herein have at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity to the amino acid sequence shown in SEQ ID NO:8. In certain embodiments, the GLP-1 fusion proteins described herein have at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity to the amino acid sequence shown in SEQ ID NO:8, and the GLP-1 polypeptide comprises A8G, G22E, and R36G substitutions, and IgG2-Fc structural domain C222S, A330S, and P331S substitutions. In certain embodiments, the GLP-1 fusion proteins described herein have at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity to the amino acid sequence shown in SEQ ID NO:8, and stimulate glucose-dependent insulin secretion from β cells. In certain embodiments, the GLP-1 fusion proteins described herein have at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity to the amino acid sequence shown in SEQ ID NO:8, the GLP-1 polypeptide consists of A8G, G22E, R36G substitutions, IgG2-Fc structural domain C222S, A330S, and P331S substitutions, and stimulates glucose-dependent insulin secretion from β cells.

[0228] In certain embodiments, the GLP-1 fusion proteins described herein have the amino acid sequence shown in SEQ ID NO:8, or an amino acid sequence having at least 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) sequence identity to SEQ ID NO:8.

[0229] In certain embodiments, the GLP-1 fusion protein described herein has an amino acid sequence set forth in SEQ ID NO:8 or an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:8.

[0230] In certain embodiments, the GLP-1 fusion protein described herein has the amino acid sequence set forth in SEQ ID NO:8. In certain embodiments, the GLP-1 fusion protein described in the present application has the amino acid sequence set forth in SEQ ID NO:8.

[0231]

Table 8

[0232] In certain embodiments, the half-life of the GLP-1 fusion protein described in the present application in a subject (e.g., a human subject) is at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days.

[0233] In another preferred embodiment, the GLP-1 fusion protein described in the present application is of pharmaceutical grade.

[0234] 2. Nucleic Acids, Recombinant Vectors and Cells The GLP-1 fusion protein in the pharmaceutical preparation provided by the present invention can be produced by various methods. For example, it is prepared using a recombinant cell containing a polynucleotide encoding the GLP-1 fusion protein, such as a Chinese hamster ovary cell that recombinantly expresses the GLP-1 fusion protein.

[0235] As used herein, the terms “nucleic acid” or “nucleotide” refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in single-stranded or double-stranded forms, and their polymers. Unless otherwise specified, a particular nucleotide sequence also includes its conserved modified variants (e.g., degenerate codon substitutions), alleles, orthogonal homology, SNPs and complementary sequences, and sequences explicitly noted. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more (or all) selected codons is substituted with a mixed base and / or deoxyinosine residue (see Batzer et al., Nucleic Acid Res. 19:5081 (1991), Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985), and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0236] In some embodiments, the polynucleotides described herein are codon-optimized, for example, for humans. Nucleic acid molecules can be used in the methods described herein.

[0237] In some embodiments, the polynucleotide encoding the GLP-1 fusion protein described herein includes a polynucleotide sequence shown in SEQ ID NO:26 or SEQ ID NO:27, or a polynucleotide sequence having at least 70% sequence identity with SEQ ID NO:26 or SEQ ID NO:27.

[0238] In certain embodiments, the polynucleotide encoding the GLP-1 fusion protein described herein comprises a polynucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the polynucleotide sequence shown in SEQ ID NO:26. In another embodiment, the polynucleotide encoding the GLP-1 fusion protein described herein is the polynucleotide sequence shown in SEQ ID NO:26.

[0239] In certain embodiments, the polynucleotide encoding the GLP-1 fusion protein described herein comprises a polynucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the polynucleotide sequence shown in SEQ ID NO:27. In another embodiment, the polynucleotide encoding the GLP-1 fusion protein described herein is the polynucleotide sequence shown in SEQ ID NO:27.

[0240] In one embodiment, the polynucleotide encoding the GLP-1 polypeptide described in the present application includes a polynucleotide sequence shown in any of SEQ ID NO:20-22, or a polynucleotide sequence having at least 70% (e.g., at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%) sequence identity with the polynucleotide sequences shown in SEQ ID NO:20-22.

[0241] In certain embodiments, the polynucleotide encoding the IgG2-Fc domain in the GLP-1 fusion protein described herein comprises a polynucleotide sequence shown in SEQ ID NO:24 or SEQ ID NO:25, or a polynucleotide sequence having less than 70% sequence identity (e.g., at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%) with the polynucleotide sequence shown in SEQ ID NO:24 or SEQ ID NO:25.

[0242] In one embodiment, the polynucleotide encoding the signal peptide described herein includes the polynucleotide sequence shown in SEQ ID NO:23, or a polynucleotide sequence having at least 70% (e.g., at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%) sequence identity with the polynucleotide sequence shown in SEQ ID NO:23.

[0243] In one embodiment, the polynucleotide encoding the linker described herein includes a polynucleotide sequence shown in any of SEQ ID NO:28-38, or a polynucleotide sequence having at least 70% (e.g., at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%) sequence identity with a sequence shown in any of SEQ ID NO:28-38.

[0244] [Table 9-1] [Table 9-2]

Table 9-3

Table 9-4

Table 9-5

[0245] A polypeptide and a fusion protein (Bodanszky, M., Principles of peptide synthesis. 2nd rev. ed. Springer laboratory. 1993, Berlin, New York: Springer-Verlag. xii, 329 p.) can be synthesized using standard protein chemistry techniques. Furthermore, automated peptide synthesizers are commercially available (e.g., Advanced ChemTech Mode 1396, Milligen / Biosearch 9600). Alternatively, the peptides, polypeptides, or fragments or variants thereof described herein can be recombinantly produced using a variety of expression systems well known in the art.

[0246] Any of the expected carriers can be used. For example, several vectors can be introduced into an expression system such as a mammalian, insect or bacterial expression system for the expression and purification of the expressed protein. Some vectors can be used to produce viruses. These different carriers are known in the art. Suitable carriers include, but are not limited to, pMPGCR 5 and pAV 0243.

[0247] In some embodiments, the vector is used with an in vitro expression system to produce a fusion protein. The expression and purification of the fusion protein can be performed by any suitable method known in the art.

[0248] Possible expression vectors include, but are not limited to, plasmids or modified viruses (e.g., lentiviral vectors, adenoviruses, and replication-defective reverse transcription viruses including adenoviruses). In one embodiment, the expression vector that can be used with the GLP-1 fusion protein of the present invention is pKN 012, which can be obtained by commercial means (Beijing Kohnoor Science & Technology Co., Ltd).

[0249] The carrier may include appropriate regulatory sequences and components. Appropriate regulatory sequences can be selected from a variety of sources, including bacterial, fungal, viral, mammalian, or insect genes. Examples of such regulatory sequences include transcription promoters and enhancers or RNA polymerase binding sequences, ribosome binding sequences, and translation initiation signals. Furthermore, depending on the cells to be transfected / infected / transmigrated and the vector used, other sequences such as replication origins, additional DNA restriction sites, enhancers, and sequences conferring transcriptional induction ability may be incorporated into the expression vector. In one embodiment, the modulation of sequences induces or increases expression in nerve tissue and / or cells. In one embodiment, the vector is a viral vector. The recombinant expression vector may also include marker genes to facilitate vector conversion for expressing the antibodies described herein, infection, or selection of host cells for transfection. The recombinant expression vector may also include other expression cassettes encoding fusion regions (e.g., for generating antibody "fusion proteins") that can be easily detected, including, for example, labels and markers as described herein.

[0250] In one embodiment, the carrier optionally includes one or more components as shown in Figure 1. For example, in one embodiment, the vector containing a polynucleotide encoding a GLP-1 fusion protein is pKN 012-GLP 1-IgG2.

[0251] Various methods for transforming cells can be used, including viral vectors, "naked" DNA, DNA in lipids or other nanoparticles, adjuvant-assisted DNA, and gene guns. For example, reverse transcription viral vectors, such as slow viral vectors, can also be used in cells within a transconductor. Other vector systems useful for carrying out the present invention include adenoviruses and adenovirus-based vectors.

[0252] Another aspect of the present invention provides recombinant cells comprising a polynucleotide encoding a GLP-1 fusion protein, or comprising the vector described in this application.

[0253] Stable recombinant cells can be prepared, for example, by transforming, transfecting, or transfecting recombinant cells with a vector containing polynucleotides, preferably any polynucleotide as described herein.

[0254] In one embodiment, the cells of the present invention also recombinantly or naturally express lysine hydroxyanase.

[0255] In one embodiment, the lysinase level or activity of the cells expressed in the present invention is higher than the lysinase level or activity of COS-7 cells. In other words, the cells have increased levels of lysinohydroxyidase activity compared to COS-7 cells. For example, the cells may be modified cells to increase the expression of lysine hydroxytransferase (e.g., by creating cells that recombinantly express lysine hydroxytransferase), or they may be cells that have naturally increased levels of lysine hydroxytransferase compared to, for example, COS-7 cells. For example, the lysine hydroxytransferase level or activity of the cells expressed in the present specification is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% higher than the lysine hydroxytransferase level or activity of COS-7 cells.

[0256] In one embodiment, the cells described herein are eukaryotic cells. In another embodiment, the eukaryotic cells are mammalian cells. In another embodiment, the mammalian cells are human-derived cells. For example, the mammalian cells are human embryonic kidney cells 293 (HEK 293 cells), e.g., HEK 293 T cells, HEK 293 S cells, or HEK 293 F cells. In another embodiment, the mammalian cells are Chinese hamster ovary (CHO) cells, e.g., CHO-K 1 cells, CHO-S cells, or CHO-GG 44 cells.

[0257] In one embodiment, recombinant cells of the present invention are obtained by acclimatizing CHOK 1 cells, for example, with Chinese hamster ovary cells.

[0258] Accordingly, the present invention also provides a method for producing a GLP-1 fusion protein, comprising culturing recombinant cells expressing the GLP-1 fusion protein, wherein the culturing comprises one or more process steps or materials described in the Examples. For example, the method may include one or more process steps described in Table 2 and / or one or more materials described in Table 3 or 4.

[0259] III. Methods and Uses of Treatment and Prevention of Diseases As described in this text, the drug formulations provided by the present invention are long-acting, stable, have few side effects, are easy to formulate, have a clear route of development, are safe and reliable, have few side effects, are convenient to use, and can be used for appropriate drug therapy or preventive purposes.

[0260] In another aspect of the present invention, a pharmaceutical formulation comprising the GLP-1 fusion protein disclosed herein may be used in a subject to treat, prevent, or delay the progression of an attack of a disease or disorder.

[0261] Another aspect of the present invention provides the use of a pharmaceutical formulation containing a GLP-1 fusion protein as a GLP-1 receptor agonist.

[0262] In another aspect of the present invention, a pharmaceutical formulation comprising a GLP-1 fusion protein for treating, preventing, or mitigating the progression of a disease or disorder is provided.

[0263] In another aspect of the present invention, a pharmaceutical formulation comprising a GLP-1 fusion protein for treating, preventing, or mitigating the progression of a disease or disorder is provided.

[0264] Another aspect of the present invention provides a method for treating, preventing, or mitigating the progression of a disease or disorder by administering a therapeutically effective dose to a subject who requires a pharmaceutical formulation containing a GLP-1 fusion protein.

[0265] Another aspect of the present invention provides for the use of a pharmaceutical formulation comprising the GLP-1 fusion protein described herein in the manufacture of a pharmaceutical for the treatment or prevention of a disease.

[0266] Another aspect of the present invention provides the use of a pharmaceutical formulation comprising the GLP-1 fusion protein described herein for the treatment or prevention of a disease.

[0267] Another aspect of the present invention provides a method for treating or preventing a disease, comprising administering a pharmaceutical formulation containing the GLP-1 fusion protein described herein to a target.

[0268] In certain embodiments, the pharmaceutical formulation containing the GLP-1 fusion protein is administered in amounts of approximately 0.2 mg to approximately 20 mg (per person). In some embodiments, the amount of fusion protein is approximately 1 mg to approximately 10 mg. In some embodiments, the amount of fusion protein is approximately 1 mg to approximately 5 mg. In some embodiments, the amount of fusion protein is approximately 0.2 mg, 0.25 mg, approximately 0.3 mg, approximately 0.35 mg, approximately 0.4 mg, approximately 0.45 mg, approximately 0.5 mg, approximately 0.55 mg, approximately 0.6 mg, approximately 0.65 mg, approximately 0.7 mg, approximately 0.75 mg, approximately 0.8 mg, approximately 0.85 mg, approximately 0.9 mg, approximately 0.95 mg, approximately 1 mg, approximately 1.5 mg, approximately 2 mg mg, approximately 2.5 mg, approximately 3 mg, approximately 3.5 mg, approximately 4 mg, approximately 4.5 mg, approximately 5.5 mg, approximately 6 mg, approximately 6.5 mg, approximately 7 mg, approximately 7.5 mg, approximately 8 mg, approximately 8.5 mg, approximately 9 mg, approximately 9.5 mg, approximately 10 mg, approximately 11 mg, approximately 12 mg, approximately 13 mg, approximately 14 mg, approximately 15 mg, approximately 16 mg, approximately 17 mg, approximately 18 mg, approximately 19 mg, or approximately 20 mg.

[0269] In certain embodiments, the pharmaceutical formulation containing the GLP-1 fusion protein was administered in therapeutically effective doses ranging from 0.01 mg / kg to approximately 100 mg / kg (e.g., approximately 0.01 mg / kg, approximately 0.5 mg / kg, approximately 1 mg / kg, approximately 2 mg / kg, approximately 5 mg / kg, approximately 10 mg / kg, approximately 15 mg / kg, approximately 20 mg / kg, approximately 25 mg / kg, approximately 30 mg / kg, approximately 35 mg / kg, approximately 40 mg / kg, approximately 45 mg / kg, approximately 50 mg / kg, approximately 55 mg / kg, approximately 60 mg / kg, approximately 65 mg / kg, approximately 70 mg / kg, approximately 75 mg / kg, 80 mg / kg, approximately 85 mg / kg, approximately 90 mg / kg, approximately 95 mg / kg, or approximately 100 mg / kg). In some of these embodiments, the pharmaceutical formulations containing the GLP-1 fusion protein provided herein are administered at doses of approximately 50 mg / kg or less of the fusion protein, and in some of these embodiments, the doses are 10 mg / kg or less, 5 mg / kg or less, 1 mg / kg or less, 0.5 mg / kg or less, or 0.1 mg / kg or less. In some embodiments, the dose may be varied during treatment. For example, in some embodiments, the initial dose may be higher than the subsequent dose. In some embodiments, the dose may be varied during treatment in response to the subject's response. In some embodiments, the pharmaceutical formulations provided herein are administered to a subject (e.g., a human) in a dosing scheme not exceeding once daily, once every three days, or once a week, once every two weeks, once every three weeks, or once a month. In some embodiments, the pharmaceutical formulations provided herein may be administered to a subject (e.g., a human) at dosing intervals of twice a week, once a week, once every two weeks, once every three weeks, once a month, or once every two months. The therapeutic effect of low-dose administration may improve patient compliance and lead to successful long-term treatment. The currently available therapeutic somallu peptide is administered once a week.

[0270] In some embodiments, the present invention provides pharmaceutical formulations containing a GLP-1 fusion protein for administration, for example, extra-gastrointestinal, intravenous, subcutaneous, or intramuscular administration. In some embodiments, the pharmaceutical formulations containing a GLP-1 fusion protein provided by the present invention can be administered extra-gastrointestinally or formulated for extra-enteral administration.

[0271] In certain embodiments, the pharmaceutical formulations containing the GLP-1 fusion protein provided by the present invention can be formulated as subcutaneous administration or as a formulation for subcutaneous administration.

[0272] In some embodiments, pharmaceutical formulations containing the GLP-1 fusion protein provided by the present invention can be formulated for intravenous administration or for intravenous administration.

[0273] In some embodiments, the pharmaceutical formulations containing the GLP-1 fusion protein provided by the present invention may be administered intramuscularly for intramuscular administration, or may be formulated for intramuscular administration.

[0274] In some embodiments, the treatment scheme may include multiple doses.

[0275] In some embodiments, the present invention provides a frequency of administration of a pharmaceutical formulation containing a GLP-1 fusion protein, either once every three days, once a week, or once every two weeks.

[0276] In one embodiment, the administration scheme for the pharmaceutical formulation containing the GLP-1 fusion protein provided by the present invention involves a drug-free period of one week, followed by administration once a week thereafter. In another preferred example, the drug-free period is two weeks.

[0277] In some embodiments, the pharmaceutical formulations containing the GLP-1 fusion protein provided by the present invention can be administered as a single dose followed by a 2-week rest period, and then four consecutive times per week. In another preferred embodiment, the treatment further includes administering an initial dose of 1 mg one week before the start of the administration scheme.

[0278] In some embodiments, the disease is selected from the group of metabolic diseases associated with glucose and / or lipid metabolism disorders, complications of metabolic diseases and neurological diseases, and other related diseases.

[0279] In one embodiment, the disease or disorder is a metabolic disorder related to impaired glucose and / or lipid metabolism.

[0280] In certain embodiments, metabolic disorders associated with impaired glucose and / or lipid metabolism are selected from the group consisting of diabetes mellitus, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), obesity, and metabolic syndromes.

[0281] In some embodiments, the metabolic disorder associated with impaired glucose and / or lipid metabolism is diabetes mellitus or includes diabetes mellitus. In certain embodiments, the metabolic disorder associated with impaired glucose and / or lipid metabolism is type 2 diabetes mellitus. In some embodiments, the metabolic disorder associated with impaired glucose and / or lipid metabolism is type 2 diabetes mellitus with inadequate glycemic control after diet and exercise interventions.

[0282] In another embodiment, the metabolic disorder associated with impaired glucose and / or lipid metabolism is obesity or includes obesity. In another embodiment, the metabolic disorder associated with impaired glucose and / or lipid metabolism is non-alcoholic fatty liver disease (NAFLD) or includes non-alcoholic steatohepatitis (NAFLD). In another embodiment, the metabolic disorder associated with impaired glucose and / or lipid metabolism is non-alcoholic liver fibrosis (NASH).

[0283] In some embodiments, subjects were newly diagnosed or previously diagnosed with diabetes (e.g., type 2 diabetes). Diabetes can be diagnosed in various ways, including fasting blood glucose (FPG). According to the American Diabetes Association, diabetes is diagnosed when fasting blood glucose levels are 126 mg / dl or higher.

[0284] In some embodiments, the likelihood of a subject developing diabetes (e.g., type 2 diabetes) increases. For example, a subject may be more susceptible to diabetes if they are obese or have a family history of diabetes due to genetic susceptibility.

[0285] In one embodiment, the subject is obese. Obesity can be defined using body weight index (BMI) as a reference. For example, the World Health Organization (WHO) defines obesity as a BMI of 30 or higher. In another embodiment, the subject is at least about 20 kg / m². 2 The subject has a BMI of [value missing]. In another embodiment, the subject may be an average person of the same age with blood glucose levels significantly higher than their body weight, but insufficient to diagnose diabetes. In another embodiment, the subject of the embodiment may be an individual with a family history of diabetes.

[0286] In some embodiments, the subject is a newly diagnosed or previously diagnosed patient with NAFLD or NASH. In other embodiments, the subject is at increased risk of developing NAFLD or NASH. For example, the subject may be genetically susceptible to NAFLD or NASH.

[0287] In some embodiments, complications of metabolic diseases include metabolic diseases (e.g., coronary artery disease, sudden cardiac death, heart failure, etc.), renal complications (e.g., acute kidney injury, diabetic nephropathy), or hepatic complications.

[0288] In some embodiments, the disease or disorder is a neurological disorder. In some embodiments, the neurological disorder is a neurodegenerative disorder. In some embodiments, the neurodegenerative disorder is selected from the following group: Alzheimer's disease (AD), motor neuron disease, Huntington's disease, and Parkinson's disease (PD).

[0289] In one embodiment, the neurodegenerative disease is Alzheimer's disease. In another embodiment, the neurodegenerative disease is Parkinson's disease. In yet another embodiment, the neurodegenerative disease is motor neuron disease. In yet another embodiment, the neurodegenerative disease is Huntington's disease.

[0290] In one embodiment, the object is a new diagnosis or has been previously diagnosed with Alzheimer's disease. In another embodiment, the subject has an increased likelihood of developing Alzheimer's disease. For example, the subject may be genetically predisposed to Alzheimer's disease, such as having a family history of Alzheimer's disease or having Tau or APP mutations associated with Alzheimer's disease.

[0291] In some embodiments, the subject is newly diagnosed with Parkinson's disease or has been previously diagnosed. In other embodiments, the subject's likelihood of developing Parkinson's disease is increased. For example, the subject may be genetically predisposed to Parkinson's disease, such as having a family history of Parkinson's disease.

[0292] In some embodiments, pharmaceutical formulations comprising the GLP-1 fusion protein provided by the present invention can be used in combination with any other known pharmaceutical or therapy for the treatment or prevention of a disease.

[0293] In one embodiment, the present invention provides for the use of pharmaceutical formulations and additional therapeutic agents comprising the GLP-1 fusion protein according to the present invention in the manufacture of pharmaceuticals for the treatment or prevention of disease.

[0294] In another aspect, the present invention provides the use of pharmaceutical formulations and additional therapeutic agents comprising the GLP-1 fusion protein according to the present invention for the treatment or prevention of diseases.

[0295] In another embodiment, the present invention provides a method for treating or preventing a disease, comprising administering to a subject a therapeutically effective amount of a pharmaceutical formulation containing the GLP-1 fusion protein described herein and an additional therapeutic agent.

[0296] In some embodiments, the additional therapeutic agent is selected from the group consisting of insulin, metformin, sulfonylurea (e.g., glimethyleneurea, glibenurea, glidide, glikidone), α-glucosidase inhibitors (e.g., acabonose), and γ-aminobutyric acid. In some embodiments, the additional therapeutic agent is metformin. In some embodiments, the additional therapeutic agent is γ-aminobutyric acid.

[0297] In some embodiments, the disease is selected from the group of metabolic diseases associated with glucose and / or lipid metabolism disorders, complications of metabolic diseases and neurological diseases, and other related diseases.

[0298] In certain embodiments, metabolic disorders associated with impaired glucose and / or lipid metabolism are selected from the group of diabetes mellitus, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), obesity, and metabolic syndromes. In some embodiments, metabolic disorders associated with impaired glucose and / or lipid metabolism are diabetes mellitus (e.g., type 2 diabetes mellitus, type 2 diabetes mellitus with inadequate glycemic control after diet and exercise interventions).

[0299] In some embodiments, complications of metabolic diseases include metabolic diseases (e.g., coronary artery disease, sudden cardiac death, heart failure, etc.), renal complications (e.g., acute kidney injury, diabetic nephropathy), or hepatic complications.

[0300] In some embodiments, the disease or disorder is a neurological disorder. In some embodiments, the neurological disorder is a neurodegenerative disorder. In some embodiments, the neurodegenerative disorder is selected from the following group: Alzheimer's disease (AD), motor neuron disease, Huntington's disease, and Parkinson's disease (PD).

[0301] In some embodiments, pharmaceutical formulations comprising the GLP-1 fusion protein disclosed herein can be administered in combination with pharmaceuticals for the treatment of diabetes, including currently marketed pharmaceuticals such as insulin and metformin, sulfonylureas mainly including glimeurea, glibenurea, glidite, and glikidone, and other pharmaceuticals marketed and under development for the treatment of diabetes, such as alpha-glucosidase inhibitors, such as acabose. In some embodiments, the diabetes drug is metformin or insulin. In some embodiments, the diabetes drug is metformin or insulin.

[0302] Accordingly, in one embodiment, the present invention provides a pharmaceutical formulation containing the GLP-1 fusion protein according to the present invention and the use of metformin in the manufacture of a pharmaceutical for the treatment of diabetes (e.g., type 2 diabetes). In one embodiment, the amino acid sequence of the GLP-1 fusion protein in the pharmaceutical formulation is shown in SEQ ID NO:7.

[0303] In another embodiment, the present invention provides a pharmaceutical formulation containing a GLP-1 fusion protein and the use of metformin for the treatment of diabetes (e.g., type 2 diabetes) according to the present invention. In one embodiment, the amino acid sequence of the GLP-1 fusion protein in the pharmaceutical formulation is shown in SEQ ID NO:7.

[0304] In another embodiment, the present invention provides a method for treating diabetes (e.g., type 2 diabetes), the method comprising administering to a subject a therapeutically effective amount of a pharmaceutical formulation containing the GLP-1 fusion protein described herein and metformin. In one embodiment, the amino acid sequence of the GLP-1 fusion protein in the pharmaceutical formulation is shown in SEQ ID NO:7.

[0305] In some embodiments, pharmaceutical formulations containing the GLP-1 fusion protein disclosed herein can be administered in combination with Alzheimer's disease drugs and / or non-pharmacological interventions, such as cognitive therapy. In certain embodiments, pharmaceutical formulations containing the GLP-1 fusion protein disclosed herein are administered in combination with gamma-aminobutyric acid for the treatment of neurodegenerative diseases. In some embodiments, pharmaceutical formulations containing the GLP-1 fusion protein disclosed herein are administered in combination with gamma-aminobutyric acid for the treatment of Alzheimer's disease.

[0306] As used herein, “combined administration” includes partial simultaneous administration as the same pharmaceutical composition, simultaneous administration as independent compositions, or administration at different times at different times as independent compositions. Where the phrase “combination” is used herein, a composition administered before or after another drug is considered to be administered “in combination” with the drug, even if the composition and the second drug are administered via different routes. Where possible, additional therapeutic agents administered in combination with fusion polypeptides, polypeptide complexes or conjugates provided herein are administered based on the time indicated on the product information form of the additional therapeutic agent, or in accordance with the “Physicians’ Desk Reference” (“Physicians’ Desk Reference,” 70th edition (2016)) or methods well known in the art.

[0307] In some embodiments, pharmaceutical formulations containing the GLP-1 fusion protein disclosed herein, when used in combination with γ-aminobutyric acid, have the effect of suppressing the reduction of SH-SY5 Y cell activity by TNF-α.

[0308] In some embodiments, pharmaceutical formulations comprising the GLP-1 fusion protein disclosed herein, when used in combination with γ-aminobutyric acid, can reduce apoptosis of TNF-α-induced neuronal cells SH-SY5Y.

[0309] In certain embodiments, a pharmaceutical formulation comprising the GLP-1 fusion protein disclosed herein can provide protection to TNF-α-damaged neurons when used in combination with γ-aminobutyric acid.

[0310] In some embodiments, pharmaceutical formulations comprising the GLP-1 fusion protein disclosed herein, when used in combination with γ-aminobutyric acid, can reduce apoptosis in TNF-α-induced neuronal cells.

[0311] In certain embodiments, a pharmaceutical formulation comprising the GLP-1 fusion protein disclosed herein is used in combination with γ-aminobutyric acid to reduce the expression of Aβ1-42 oligomer-induced inflammatory factors (e.g., TNF-α, IL-6) mRNA in HMC 3 small colloidal cells.

[0312] Accordingly, in another embodiment, the present invention provides a pharmaceutical formulation comprising the GLP-1 fusion protein according to the present invention and the use of γ-aminobutyric acid in the manufacture of pharmaceuticals for the treatment of neurodegenerative diseases (e.g., Alzheimer's disease, motor neuron disease, Huntington's disease, Parkinson's disease). In one embodiment, the amino acid sequence of the GLP-1 fusion protein in the pharmaceutical formulation is shown in SEQ ID NO:7.

[0313] In another embodiment, the present invention provides a pharmaceutical formulation comprising the GLP-1 fusion protein according to the present invention and the use of gamma-aminobutyric acid for the treatment of neurodegenerative diseases (e.g., Alzheimer's disease, motor neuron disease, Huntington's disease, Parkinson's disease). In one embodiment, the amino acid sequence of the GLP-1 fusion protein in the pharmaceutical formulation is shown in SEQ ID NO:7.

[0314] In another embodiment, the present invention provides a method for treating neurodegenerative diseases (e.g., Alzheimer's disease, motor neuron disease, Huntington's disease, Parkinson's disease), the method comprising administering a pharmaceutical formulation containing the GLP-1 fusion protein described herein and gamma-aminobutyric acid to a subject. In one embodiment, the amino acid sequence of the GLP-1 fusion protein in the pharmaceutical formulation is shown in SEQ ID NO:7.

[0315] In another embodiment, the present invention also provides a combination of pharmaceuticals comprising a pharmaceutical formulation containing a GLP-1 fusion protein described according to the present invention and an additional therapeutic agent. In some embodiments, the additional therapeutic agent is a drug for treating diabetes or a drug for treating neurodegenerative diseases. In some embodiments, the additional therapeutic agent is selected from the group consisting of insulin, metformin, sulfonylurea (e.g., glimethyleneurea, glibenurea, glidide, glikidone), α-glucosidase inhibitors (e.g., acabonose), and γ-aminobutyric acid. In some embodiments, the additional therapeutic agent is metformin. In some embodiments, the additional therapeutic agent is γ-aminobutyric acid.

[0316] IV. Kit The present invention also provides a kit for carrying out the method of the present invention. Such a kit may include the pharmaceutical formulation described herein, which can be provided in a sterile container. Optionally, it may also include instructions on how to treat or prevent a disease using the provided pharmaceutical formulation, or such instructions may be made available to the patient or healthcare provider.

[0317] In one embodiment, the kit comprises (a) a pharmaceutical formulation comprising a GLP-1 fusion protein according to the present invention, and (b) one or more containers for the pharmaceutical formulation. Such a kit may also include instructions for its use, which can be customized depending on the specific disease to be treated or prevented. The instructions may describe the uses and properties of the materials provided in the kit. In some embodiments, the kit includes instructions for administration to a patient to treat or prevent a disease, the disease being selected from a group of metabolic diseases related to glucose and / or lipid metabolism disorders, complications of metabolic diseases and neurological diseases, and other related diseases.

[0318] The instructions can be printed on a substrate such as paper or plastic, and may exist as a package page within the kit, such as on the kit container or on the labels of its components (i.e., associated with the packaging). In other embodiments, the specification may exist as an electronic storage data file located on a suitable computer-readable storage medium (e.g., CD-ROM, floppy disk, etc.). In yet another embodiment, the actual specification may not be present in the kit, but a means for obtaining the specification from a remote source via the internet is provided. An example of this embodiment is a kit that includes a URL from which the instructions can be viewed and / or downloaded. Typically, it is desirable to package some or all of the components of the kit in a suitable package to maintain sterility. The kit assembly may be packaged in a kit containment element (e.g., a case or similar structure) which may be an airtight container to further maintain the sterility of some or all of the kit assembly, or it may not be, to produce a single, easy-to-handle unit.

[0319] The present invention also provides the following embodiments.

[0320] Embodiment 1. (a) A pharmaceutical formulation comprising a GLP-1 polypeptide and a GLP-1 fusion protein comprising an immunoglobulin Fc domain, wherein the GLP-1 polypeptide is selected from human GLP-1(7-37), human GLP-1(7-36)amide and DPPIV-resistant human GLP-1, the GLP-1 polypeptide is covalently bound to an immunoglobulin Fc domain, the GLP-1 polypeptide comprises an A8G and / or G22E and / or R36G substitution, the immunoglobulin Fc domain comprises an IgG-containing G2 / Fc domain comprising a C222S substitution and / or an IgG2 / Fc domain comprising one or two selected from A330S substitution and P331S substitution, and (b) a buffer, in which case the pH value of the pharmaceutical formulation is in the range of 6.0 to 7.0, preferably in the range of 6.5 to 7.0, more preferably 6.7.

[0321] Embodiment 2. A pharmaceutical formulation containing the GLP-1 fusion protein described in Embodiment 1, wherein the buffer is one or more selected from phosphate buffer, citrate buffer and borate buffer, preferably phosphate buffer, more preferably disodium hydrogen phosphate / sodium dihydrogen phosphate buffer, even more preferably 5-15 mM disodium hydrogen phosphate / sodium dihydrogen phosphate buffer, and more preferably 10 mM disodium hydrogen phosphate / sodium dihydrogen phosphate buffer.

[0322] Embodiment 3. The pharmaceutical preparation further contains as an excipient a sugar selected from the group consisting of mannitol, sorbitol, maltitol, erythritol, arabitol, xylitol, sucrose, lactose, trehalose, dextran, or a mixture thereof, preferably one or more selected from the group consisting of mannitol, sucrose, and sorbitol, more preferably one or two selected from the group consisting of mannitol and sucrose, and even more preferably the sugar is mannitol, and the mannitol concentration is preferably 1 to 10% (w / v), and more preferably 4.6 (w / v).

[0323] Embodiment 4. The pharmaceutical formulation further comprises one or more surfactants selected from Eon 80 (polysorbate 80), polyoxyethylene castor oil derivatives, poloxam, lecithin, polyethylene glycol 15-hydroxystearate ester, and cyclodextrins, more preferably Eon 80, with a concentration of Eon 80, preferably 0.01% (w / v) to 0.04% (w / v), more preferably 0.02% (w / v), as described in any one of Embodiments 1 to 3.

[0324] Embodiment 5. GLP-1 fusion protein, disodium hydrogen phosphate / sodium dihydrogen phosphate buffer, mannitol, pH 6.5-7.0.

[0325] Embodiment 6. A pharmaceutical formulation comprising the GLP-1 fusion protein according to any one of Embodiments 1 to 4, further comprising emetic temperature 80.

[0326] Embodiment 7. GLP-1 fusion protein, 10 mM disodium hydrogen phosphate / sodium dihydrogen phosphate buffer, 4.6% (w / v) mannitol, 0.02% (w / v) emetic temperature 80, pH 6.5-7.0.

[0327] Embodiment 8. A pharmaceutical formulation comprising the GLP-1 fusion protein according to any one of Embodiments 1 to 7, wherein the concentration of the GLP-1 fusion protein is 0.2 to 20 mg / ml, preferably 1 to 5 mg / ml, and more preferably 3 mg / ml.

[0328] Embodiment 9. Use in the manufacture of a pharmaceutical formulation comprising a GLP-1 fusion protein as described in any one of Embodiments 1 to 7, which is a pharmaceutical for treating or preventing a glycolipid metabolic disorder or a neurodegenerative disorder, preferably a GLP-1 receptor agonist, or preferably, the glycolipid metabolic disorder is selected from diabetes mellitus, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), obesity, metabolic syndrome, preferably, the neurodegenerative disorder is selected from Alzheimer's disease, motor neuron disease, Huntington's disease, or Parkinson's disease, more preferably, the glycolipid metabolic disorder is diabetes mellitus, more preferably type 2 diabetes mellitus.

[0329] The above disclosure provides a general overview of the application. A more complete understanding can be obtained by referring to the following specific examples. These examples are provided for illustrative purposes only and are not intended to limit the scope of the application. Where circumstances may be proposed or convenient, modifications of form and substitution of equivalents may be considered. Certain terms are used here, but these terms are for illustrative purposes only and not for limiting purposes. The following non-limiting embodiments are used to illustrate the content of the invention.

[0330] Examples Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0331] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercially available suppliers.

[0332] Example 1: Fusion protein expression and activity assay 1.1 Plasmid Construction We constructed a vector encoding a GLP-1 fusion protein. The fusion protein consists of human GLP-1 (7-37) and human IgG2 / Fc (including the hinge region, CH2, and CH3 of the human IgG2 heavy chain). We fused the signal peptide sequence of human CD33 (hCD33) with the GLP-1 sequence to induce secretion of the peptide into culture medium. We chemically synthesized a cDNA fragment encoding the fusion protein hCD33-GLP-1-IgG2 / Fc (hinge-ch2-ch3) (as shown in SEQ ID NO:27) and inserted it into the NcoI and HindIII sites of the pKN012 vector to generate pKN012-GLP-1-IgG2 / Fc.

[0333] The pKN012-GLP-1-IgG2 / Fc stable expression vector was used to transform E. coli DH5α-competent cells. Plasmid DNA was extracted from bacterial strains containing pKN012-GLP-1-IgG2 / Fc and digested with PvuI. After electrophoresis, target bands with the correct molecular weight were observed. Linearized plasmids were quantified after ethanol precipitation and used for stable transfection.

[0334] To establish CHO-K1 cells (lot number 58995535 / ATCC) that stably express GLP-1-IgG2 / Fc, linearized pKN012-GLP-1-IgG2 / Fc (2 μg) was transfected into CHO-K1 cells grown in 6-well plates (2.5 x 10⁵ cells / well) using electroporation (electroporation-mediated transfection). 24 hours after transfection, the cells were dispersed and cultured in CD-CHO medium containing MSX (methionine sulfoximine, 100 μM / L), and cells with stably incorporated recombinant plasmids into their genomes were selected. The culture medium was changed every 3 days until colonies formed. Individual colonies were isolated and expanded into stable cell lines. The GLP-1 fusion protein in the culture supernatant of cell lines grown in 24-well plates was tested using the rat GLP-1 RIA kit (YN-011). We selected and further identified cells capable of secreting the fusion protein. The amino acid sequence of the prepared GLP-1 fusion protein is shown in SEQ ID NO:7 (referred to as "YN-011" in this application).

[0335] 1.2 Evaluation of YN-011 activity by induction of cAMP levels Natural GLP-1 stimulates insulin secretion from beta cells in a glucose-dependent manner. To assess whether purified YN-011 possesses the function of natural GLP-1, its effect on insulin secretion from clonal insulin-secreting INS-1 cells was measured. After serum starvation and glucose starvation, INS-1 cells were treated with different amounts of purified YN-011 in the presence of 0, 5, or 20 mM glucose, as shown. In the absence of glucose, YN-011 did not stimulate insulin secretion from beta cells. However, in the presence of 5 mM or 20 mM glucose, YN-011 dose-dependently stimulated insulin secretion from beta cells. The data indicate that the GLP-1-IgG2 / Fc fusion protein YN-011 possesses biological activity and can stimulate glucose-dependent insulin secretion in INS-1 cells.

[0336] In the absence of glucose, INS-1 cells treated with YN-011 (120 nM) maintained baseline levels of cAMP. However, in the presence of 5 mM glucose, cAMP levels in YN-011-treated cells increased significantly, reaching levels comparable to those induced by exendin-4.

[0337] Example 2: Increased GLP-1 K34 hydroxylation levels improve protein yield and activity. 2.1 Determination of modification levels by LC-MS / MS peptide mapping Mass spectrometry was performed using Lys-C digestion for identification. The YN-011 sample was denatured, reduced, and alkylated, followed by Lys-C enzymatic hydrolysis to produce the peptide. Online LC-MS / MS analysis was performed using a Thermo LTQ Velos Orbitrap instrument, and the data were processed and analyzed using Mascot software. Peptide segments 21-28:EFIAWLVK were observed to have a molecular weight increase of 16 daltons (Da) due to modification. The primary and secondary mass spectra of the YN-011 sample with and without the +16Da modification are shown in Figure 2. The mass measurement error of the primary spectrum was within 10 ppm. By analyzing the mass-to-charge ratio of the secondary fragment ions, the +16Da modification site was determined to be K28. K28 is also called K34 because active GLP-1 (e.g., amino acids 7-37 of GLP-1) lacks the first six residues. Peptides 21-28 (also known as peptides 27-34) are also found in naturally occurring human GLP-1.

[0338] The modification level of the K34 site in YN-011 was determined using the UV280 method and EIC (extraction ion chromatography), as shown in Table 1 and Figures 3A and 3B. The UV280 calculation method is as follows:

[0339]

number

[0340] [Table 10] *Note: UV280 and EIC methods were used to detect the K34 modification level of YN-011.

[0341] 2.2 K34 Hydroxide Level of YN-011 After performing mass spectrometry and confirming that a +16Da modification occurs at the 34th position of lysine (Lys), and considering that +16Da corresponds to the molecular weight of the oxygen atom, the possibility of an oxidation reaction is suggested. In fact, there are two enzymes in cells that catalyze modification of the lysine side chain. One is called lysyloxidase (see https: / / en.wikipedia.org / wiki / Lysyl_oxidase) and the other is lysylhydroxylase (see https: / / en.wikipedia.org / wiki / Lysyl_hydroxylase). Lysyloxidase can oxidize the amino group at the 6th carbon of the lysine side chain to form an aldehyde group. However, this modification results in only a 1Da difference compared to the unmodified form, which does not match the observed +16Da modification. Lysylhydroxylase, on the other hand, adds a hydroxyl group to the carbon (γ position) of the lysine side chain to form stable hydroxylated lysine (hydroxylysine). Hydroxylation of lysine adds a hydroxyl group and reduces the number of hydrogen atoms in the side chain, resulting in a 16 Da increase in molecular weight. Therefore, the observed +16 Da modification is consistent with lysine hydroxylation. The hydroxylation site at position K34 is shown below.

[0342] [ka]

[0343] In conclusion, the +16Da modification observed in K34 of YN-011 is very likely due to lysine hydroxylation. Therefore, by adding a hydroxylation inhibitor during cell culture, it is possible to verify that the +16Da modification is lysine hydroxylation. Furthermore, by collecting proteins with different percentages of +16Da modification, it will be possible to investigate the effect of the degree of +16Da modification on the biological activity of the protein.

[0344] Table 2 shows the cell culture process for stable CHOK1 cells capable of secreting the fusion protein YN-011, as described in Example 1. The relevant materials and cell culture media required for cell culture are shown in Tables 3 and 4, respectively. As previously mentioned, mass spectrometry is used to detect the modification level of the fusion protein. The inventors found that the fusion protein was indeed hydroxylated with K34, with hydroxylation levels ranging from 15% to 40%.

[0345] To investigate the biological effects of K34 hydroxylation, eight shaking flasks (SF1-SF8) were set up to culture stable CHOK1 cells expressing the fusion protein YN-011. Various conditions were evaluated using SF1-SF8. Hydroxylation inhibitors were added during the cell culture process to collect proteins with different hydroxylation modification ratios and to investigate the effect of the degree of hydroxylation on the biological activity of the protein. Minoxidil is an inhibitor of lysyl hydroxylase, which suppresses lysine hydroxylation, while Zn2+ ions competitively inhibit the activity of the hydroxylation enzyme by competing with Fe2+ ions during the reaction. SF1 was used as a control without the addition of an inhibitor. The minoxidil used in this experiment was dissolved in 0.1N HCl solution. HCl was added to SF2 as an HCl control. SF3-SF5 were added to different concentrations of minoxidil: 0.1mM, 0.5mM, and 1.0mM, respectively. SF6 and SF7 were experimental groups to which Zn2+ was added. The Zn2+ reagent used was ZnSO4, with concentrations of 200 μM and 400 μM, respectively. SF8 contained both minoxidil and Zn2+ (provided by ZnSO4) as inhibitors, with total concentrations of 0.5 mM and 200 μM, respectively.

[0346] [Table 11]

[0347] [Table 12]

[0348] [Table 13]

[0349] 2.3 Increasing the hydroxylation level of K34 in GLP-1 can enhance protein yield and activity. Table 5 shows the results for protein yield and activity. SF1 was a blank control, and SF2 was treated with HCl only, resulting in hydroxylation levels of 15.7%–16.0%. From SF3 to SF5, the hydroxylation rate gradually decreased as the concentration of minoxidil (one of the hydroxylation inhibitors) increased. When the minoxidil dose reached 0.5 mM, the hydroxylation rate was 8.7%. When the minoxidil concentration increased to 1 mM, the rate was 9.4%. Cell proliferation and protein expression were significantly inhibited, with peak cell density decreasing by approximately 17% and protein yield decreasing by 57.0%–67.9%. Furthermore, when 400 μM of Zn2+ (another hydroxylation inhibitor) was added to the cell culture, the hydroxylation level also decreased to 10.7%. In the experiment where a combination of inhibitors (0.5 mM minoxidil, 200 μM Zn2+) was added to SF8, the hydroxylation level decreased to 9.6%.

[0350] In the presence of hydroxylation inhibitors such as minoxidil, the rate of hydroxylation gradually decreases as the concentration increases. Cell growth and protein expression are significantly inhibited, peak cell density decreases by approximately 17%, and yield gradually decreases in the range of 57.0% to 67.9%. Similarly, in the presence of the hydroxylation inhibitor Zn2+, as the Zn2+ concentration increases, the hydroxylation level also decreases, and protein yield decreases by 22.9% to 34.2%. When the hydroxylation level reaches 15.7% to 16.0%, the yield increases, doubling compared to lower hydroxylation levels. Therefore, the higher the hydroxylation level, the higher the yield.

[0351] SUPA-1 is a GLP-1-IgG2 / Fc fusion protein disclosed in U.S. Patent US8658174. No linking peptide is used between the GLP-1 peptide and IgG2 / Fc. Aside from the A8G substitution of the GLP-1 peptide, there are no other site mutations in the GLP-1 peptide or IgG2 / Fc. SUPA-1 was detected as unmodified (not hydroxylated or otherwise modified), and under the same conditions, the protein yield was 22 mg / L. In contrast, when the K34 hydroxylation of YN-011 was 8.7% to 9.6%, the yield reached 0.77 g / L to 1.03 g / L, more than 100 times that of SUPA-1. When the K34 hydroxylation exceeded 15%, the yield of YN-011 was more than 500 times that of SUPA-1.

[0352] Furthermore, the biological activity of YN-011 was tested using the method described in Example 1 for a blank control (SF1) and two samples with the lowest hydroxylation levels (SF4 and SF5). The results showed that the activity of SF1 was 88%, while the activities of SF4 and SF5 were 83% and 68%, respectively. Therefore, there is a tendency for biological activity to increase with increasing hydroxylation levels.

[0353] In summary, it was confirmed that K34 in the fusion protein is indeed hydroxylated, and this modification significantly improves the yield of YN-011 and tends to increase its biological activity.

[0354] Furthermore, by scaling up the cell culture to a 15L reactor and three 200L reactors, the YN-011 obtained from these batches showed 20%–30% hydroxylation at position 34, with a yield of approximately 2.7 g / L.

[0355] [Table 14]

[0356] Example 3 Measurement of oxidation level by LC-MS / MS The oxidation levels were measured according to the protein oxidation measurement method described in Example 2, or by referring to Bettinger et al. (Bettinger, JQ, et al., Quantitative Analysis of in Vivo Methionine Oxidation of the Human Proteome. J Proteome Res, 2020. 19(2): p.624-633.). It was observed that the GLP-1 fusion protein YN-011 of the present invention was not oxidized at the W31 position, while the oxidation level of degludec at the same position exceeded 5%. Oxidation occurred at the position indicated by the square in the W31 structure.

[0357] [ka]

[0358] Furthermore, while the GLP-1 fusion protein YN-011 of the present invention showed an oxidation level of approximately 2% to 4% at position M253, degludec showed an oxidation level of over 5% at the same position.

[0359] Example 4: Preventive and therapeutic effects of YN-011 in db / db mice (type 2 diabetes model) The effect of multiple subcutaneous injections of YN-011 on lowering blood glucose levels was evaluated in db / db mice (Jackson Laboratories, 000642). The mice were housed under normal lighting conditions (12 hours light / 12 hours dark) and at room temperature, and had free access to food (normal rodent diet) and water. Db / db mice lack leptin receptors and spontaneously develop obesity, hyperglycemia, and pancreatic β-cell atrophy by 4 weeks of age.

[0360] Sixty affected db / db mice (body weight 33-45 grams) were divided into six groups of 10 mice each (5 males, 5 females). Each group was administered YN-011 at doses of 0 (PBS buffer control), 0.15, 0.3, 0.6, or 1.2 mg / kg, or degludec at 0.3 mg / kg via subcutaneous injection. Another group of wild-type mice (body weight 17-22 grams) with the same genetic background were used as a normal control. Administration was performed once every three days (Q3D), for a total of nine doses over 26 days.

[0361] Throughout the experiment, random plasma glucose (RPG) levels in the db / db model control group were consistently high and significantly higher than those in normal control mice (P<0.001). After the initial administration, YN-011 significantly reduced RPG concentrations in db / db mice, and a therapeutic effect was observed 3 hours after administration at a low dose of 0.15 mg / kg. The duration of the hypoglycemic effect was 34 to 72 hours in the dose range of 0.15 to 1.2 mg / kg. YN-011 also significantly promoted insulin secretion, and an increase in serum insulin levels was observed 3 hours after administration.

[0362] After the ninth dose of YN-011, the RPG levels of mice treated with YN-011 were significantly lower than those of the control group at all dose groups and measurement time points, except for the 0.15 mg / kg group after the fourth dose (day 10). In this group, the difference was not statistically significant (P=0.07). To gain a more intuitive understanding of the reduction in blood glucose levels in each mouse group, the mean percentage reduction in random blood glucose levels at each measurement time point during the experiment was calculated. The results showed that the mean percentage reduction in random blood glucose levels at 72 hours after the seventh dose was 31.8%, 46.2%, and 50.8% for the 0.15, 0.3, and 0.6 mg / kg YN-011 groups, respectively, and reached 54.3% for the 1.2 mg / kg YN-011 group. The hypoglycemic effect of YN-011 lasted for 72 hours after repeated administration.

[0363] Long-term administration of YN-011 significantly reduced fasting blood glucose levels in db / db mice. Throughout the experiment, fasting blood glucose levels in db / db mice were consistently high and significantly higher than those of normal control mice (P<0.001). After subcutaneous injection of different doses of YN-011 every three days, fasting blood glucose levels in all YN-011 groups were significantly lower compared to the model control group (P<0.001). The fasting blood glucose reduction rates at 54 hours (day 21) after the seventh dose were 46.8%, 55.6%, 59.5%, and 64.7% for the 0.15, 0.3, 0.6, and 1.2 mg / kg YN-011 groups, respectively. This suggests that the hypoglycemic effect of YN-011 at doses of 0.15, 0.3, 0.6, and 1.2 mg / kg administered every three days can be maintained for at least 54 hours (3 days after administration). The positive control group, administered 0.3 mg / kg of degludec, also showed a significant decrease in fasting blood glucose levels at all measurement points.

[0364] To provide a more intuitive understanding of the reduction in blood glucose levels in each mouse group, we calculated the mean percentage reduction in fasting blood glucose levels at each measurement point throughout the experiment. The mean percentage reductions in fasting blood glucose levels for the YN-011 groups receiving 0.15, 0.3, 0.6, and 1.2 mg / kg were 55.0%, 61.8%, 63.7%, and 66.1%, respectively. The mean percentage reduction in fasting blood glucose levels in the positive control group administered 0.3 mg / kg of degludec was 63.6%.

[0365] Therefore, subcutaneous injection of YN-011 multiple times every three days significantly reduced fasting blood glucose levels in type 2 diabetic db / db mice. This effect was evident at a dose of 0.15 mg / kg, and the hypoglycemic effect of YN-011 at doses of 0.15, 0.3, 0.6, and 1.2 mg / kg was maintained for at least 54 hours (3 days after administration).

[0366] Subcutaneous injection of YN-011 at doses of 0.15–0.6 mg / kg into db / db mice tended to decrease serum fructosamine levels, but serum fructosamine levels decreased significantly in the group administered 1.2 mg / kg of YN-011.

[0367] In the model control group, random body weight and fasting body weight of db / db mice continued to increase throughout the experiment, whereas in the YN-011 treatment groups at doses of 0.3, 0.6, and 1.2 mg / kg, random body weight and fasting body weight were significantly reduced (P<0.05, P<0.01, P<0.001). In contrast, the SUPA-1 fusion protein disclosed in U.S. Patent US8658174 had no significant effect on the body weight of db / db mice. This indicates that the GLP-1 fusion protein of this application is superior.

[0368] Compared to the solvent control group, the YN-011 treatment group showed a significant reduction in epididymal fat content and its ratio to body weight. Scapular fat content was significantly reduced in the 0.15, 0.3, and 0.6 mg / kg YN-011 groups. Subcutaneous fat volume, inguinal fat volume, and their ratios to body weight were significantly reduced in the 0.3, 0.6, and 1.2 mg / kg YN-011 groups. Perirenal fat volume was significantly reduced in the 1.2 mg / kg YN-011 group.

[0369] After the final dose (9th dose, day 26), YN-011 dose-dependently increased fasting serum insulin levels in db / db mice, accompanied by a significant increase in pancreatic β-cell volume.

[0370] Compared to the solvent control group, multiple subcutaneous injections of YN-011 significantly reduced serum triglyceride levels, and 0.6 mg / kg YN-011 significantly reduced serum free fatty acid levels in db / db mice.

[0371] In summary, multiple subcutaneous injections of YN-011 showed a significant therapeutic effect in db / db mice. This not only improved glucose metabolism but also had a significant therapeutic effect on abnormal lipid metabolism.

[0372] Example 5: Phase IIa clinical trial of YN-011 5.1 Test Design This Phase IIa clinical trial is a double-blind, placebo-controlled study to evaluate the efficacy and safety of subcutaneous administration of YN-011 at doses of 1 mg, 2 mg, 3 mg, and 4 mg in subjects with type 2 diabetes mellitus (T2DM). The design of this trial is shown in Figure 5.

[0373] Main eligibility criteria for Phase IIa trials: • Patients with T2DM (WHO 1999) who have not taken metformin for more than one week and have not taken other oral antidiabetic drugs for more than two weeks. • HbA1c value at screening: 7.0% ≤ HbA1c ≤ 10.0%. • Age at screening is between 18 and 65 years old. • BMI ≥ 20 kg / m² 2 Furthermore, ≤40 kg / m 2 .

[0374] Main exclusion criteria for Phase IIa trials: · Type 1 diabetes. • Fasting C-peptide level <0.81 ng / mL. • Laboratory values ​​meet one of the following criteria: alanine aminotransferase (ALT) level ≥ 2.5 x ULN, and / or aspartate aminotransferase (AST) level ≥ 2.5 x ULN, fasting triglycerides > 5.6 mmol / L, and estimated glomerular filtration rate (eGFR) calculated using the CKD-EPI (EPI-(Scr)) formula < 45 mL / min / 1.73 m². 2 , • A history of type 2 multiple endocrine neoplasms or medullary thyroid carcinoma, either familial (first-degree relative) or personal. • Poorly controlled hypertension. • History of pancreatitis, pancreatic cancer, serum amylase >1.2 x ULN, or high-risk factors for pancreatitis at the time of screening. • Patients with uncontrolled hypothyroidism. • Suspected active infection. • Positive for hepatitis B surface antigen (HBsAg), hepatitis C antibody (HCV-Ab), human immunodeficiency virus antibody (HIV-Ab), or treponema pallidum antibody (TPAb). • Patients received treatment with a GLP-1 receptor agonist, DPP-4 inhibitor, or insulin during the three months prior to randomization. • History of grade 3-4 allergies to protein-based drugs based on CTCAE. • The individual had donated blood or lost more than 450 mL of blood in the three months prior to screening. • There is a significant endocrine disorder, immune disorder, coagulation disorder, genitourinary disorder, or blood disorder. Clinically significant gastric emptying disorders (e.g., gastric outlet obstruction), severe chronic gastrointestinal diseases (e.g., active ulcers within the last 6 months), long-term use of medications that directly affect gastrointestinal motility, or previous gastrointestinal surgery. • Any other condition that the principal investigator or attending physician deems inappropriate for participation in this study.

[0375] Participants were randomly assigned in a 4:1 ratio to receive either YN-011 or placebo. YN-011 was administered in doses of 1 mg, 2 mg, and 3 mg, with a single dose followed by a 2-week rest period, and then weekly for 4 consecutive weeks. Participants in the 4 mg group followed the same dosing regimen, except they received an initial dose of 1 mg one week prior to administration. All participants received a total of five random doses.

[0376] The primary endpoint was the safety and tolerability of YN-011 in patients with type 2 diabetes. Secondary endpoints included weekly changes from baseline in fasting blood glucose, changes from baseline in HbA1c at weeks 4 and 7, changes from baseline in glycated albumin at weeks 4 and 7, changes in glucose tolerance, and changes in pancreatic beta-cell function as assessed by oral glucose tolerance tests. Blood samples were collected from all subjects for pharmacokinetic studies. Safety assessments included adverse events, laboratory tests, vital signs, 12-lead electrocardiogram, physical examination, and assessment of anti-drug antibodies (ADAs).

[0377] 5.2 Pharmacokinetics of YN-011 (single or multiple doses) In the dose range of 1.0 mg to 4.0 mg, YN-011 showed an increasing trend. After a single dose, the half-life (T1 / 2) of YN-011 was approximately 207 hours (8.6 days), and the median Tmax was 60-84 hours (Figure 4). After the initial dose of YN-011 following randomization, administration was performed once a week, with the fourth dose being consecutive. Plasma concentrations of YN-011 reached a steady state after the fourth dose (Figure 4). The fusion protein in this application showed a significantly extended half-life compared to SUPA-1 after amino acid substitution. Furthermore, compared to commercially available drugs such as dulaglutide (elimination half-life 4.7-5.5 days (Geiser, JS, et al., Clinical Pharmacokinetics of Dulaglutide in Patients with Type 2 Diabetes: Analysis of Data from Clinical Trials. Clin Pharmacokinetics, 2016. 55(5): p. 625-34)) and semaglutide (elimination half-life 5.7-6.7 days (Pratley, RE, et al., Semaglutide versus dulaglutide once weekly in patients with type 2 diabetes (SUSTAIN 7): a randomised, open-label, phase 3b trial. Lancet Diabetes Endocrinol, 2018. 6(4): p. 275-286)), YN-011 showed a significantly longer half-life than dulaglutide and semaglutide.

[0378] 5.3 Efficacy of YN-011 in type 2 diabetes YN-011 was administered subcutaneously at formal doses of 1 mg, 2 mg, 3 mg, and 4 mg for the treatment of type 2 diabetes (according to the administration plan shown in Figure 5).

[0379] A total of 40 subjects received at least one dose of YN-011 or placebo and were included in the safety analysis. The mean age of the subjects was 51.7 ± 10.32 years, and the mean BMI was 25.80 ± 2.875 kg / m². 2 The average weight was 71.91 ± 12.360 kg. 40% of the subjects were female. Of the 40 subjects, 38 (95%) had complications. Fatty liver was confirmed by ultrasound in 32 (80%), hyperlipidemia in 25 (62.5%), hypertension in 19 (47.5%), arteriosclerosis in 10 (22.5%), and pulmonary lesions in 10 (22.5%). Throughout the study, none of the 40 subjects were taking any concomitant medications. The baseline characteristics of the subjects are summarized in the following table (Table 6).

[0380] [Table 15]

[0381] The effects of different doses of YN-011 on fasting blood glucose levels are shown in Figure 6. Multiple subcutaneous administrations of 3 mg or 4 mg of YN-011 resulted in sustained and significantly improved fasting blood glucose levels compared to placebo, and these improvements were clinically and statistically significant.

[0382] The effects of different doses of YN-011 on HbA1c are shown in Figure 7. Multiple subcutaneous administrations of 1 mg, 3 mg, or 4 mg of YN-011 resulted in sustained improvement in HbA1c levels, which were clinically and statistically significant compared to placebo. No serious side effects or safety risks were observed.

[0383] Example 6: Preventive and therapeutic effects of YN-011 on obesity 6.1 Effects of YN-011 on high-fat diet-induced obesity in mice The effects of multiple doses of YN-011 on glucose tolerance, insulin sensitivity, metabolism, and weight loss in high-fat diet (HFD) induced obese mice were evaluated. A diet-induced obesity (DIO) mouse model was established by feeding 5-month-old male C57BL / 6 mice (obtained from commercial suppliers such as Shanghai Slake Laboratory Animals Co., Ltd., under standard rearing conditions) an HFD (60% fat, 20% carbohydrates) for 6 months. Subsequently, DIO mice (body weight > 50g) were divided into two groups (5 mice per group). The experimental group received 0.3 mg / kg of YN-011 subcutaneously twice weekly (BIW), while the control group received phosphate-buffered saline (PBS) injections. Treatment was administered for 4 weeks.

[0384] Experimental results showed that 4 weeks of BIW injection with YN-011 resulted in a significant reduction in the body weight of DIO mice. Compared to the PBS control group, mice treated with YN-011 showed a substantial reduction in visceral fat, particularly epididymal fat. YN-011 did not affect the weight of other body-related tissues, such as brown adipose tissue (BAT), groin white adipose tissue (WAT), pancreas, and calf muscles.

[0385] Compared to the control group, 4 weeks of BIW injections with YN-011 significantly reduced ectopic lipid accumulation, liver triglycerides, serum ALT levels (YN-011 vs. Ctrl = 34.2±7.7 vs. 153.4±18.7, P<0.01), and AST levels (YN-011 vs. Ctrl = 72.20±19.29 vs. 145.6±16.8, P<0.05). After 4 weeks of BIW treatment, YN-011 also significantly improved the lipid profile, with a 30% reduction in total cholesterol (TC) (P<0.001), a 68% reduction in triglycerides (TG) (P<0.001), and a 57% reduction in non-esterified fatty acids (NEFA) (P<0.001). Compared to the control group, DIO mice administered multiple times with YN-011 showed a significantly reduced food intake. YN-011 administration tended to increase metabolic rate (VO2 and VCO2) and energy expenditure (EE), but this was not statistically significant. When normalized to body weight, YN-011-administered mice showed significantly increased VO2, VCO2, and EE at night. YN-011 did not affect UcP1 expression in BAT and epididymal WAT, but significantly upregulated UcP1 expression in inguinal WAT. This indicates that YN-011 did not enhance BAT thermogenesis but promoted browning of inguinal white adipose tissue. Therefore, DIO mice administered with YN-011 exhibited higher trunk body temperature at room temperature, maintained higher rectal temperature even when exposed to cold environments, and showed increased thermogenesis compared to the control group. These results suggest that YN-011 enhances the adaptation of obese mice to cold environments by generating more calories. Blood glucose fluctuations, intraperitoneal glucose tolerance tests, and insulin resistance experiments showed that YN-011 significantly reduced blood glucose levels (P<0.01) and improved insulin sensitivity (P<0.01) in DIO mice after 4 weeks of administration.

[0386] In conclusion, YN-011 effectively reduced body weight in obese mice and improved obesity-related metabolic disorders such as hyperglycemia, hyperlipidemia, and fatty liver. The beneficial effects of YN-011 on metabolism were associated with suppression of food intake, browning of WAT, and remodeling.

[0387] 6.2 Therapeutic effects of YN-011 on obese rhesus monkeys The obese rhesus monkeys used in this study were procured from Sichuan Primate Biotechnology Co., Ltd. Fifteen obese male rhesus monkeys were selected, aged 8–21 years (equivalent to 30–60 years in humans), weighing 9.25–15.70 kg, with fasting blood glucose (FPG) levels of 5.50–8.58 mmol / L and HbA1c levels of 4.5–5.0%. These monkeys had been diagnosed within the past year and had not received any medication. Liver and kidney function was normal. The animals were stratified based on FPG levels and randomly assigned to different groups. Selected animals underwent an intravenous glucose tolerance test (IVGTT) to measure blood glucose and insulin levels before administration of the test compound. The experimental groups included a placebo group and two YN-011 treatment groups (YN-011 50 μg / kg and YN-011 25 μg / kg), with five animals in each group. The treatment was administered once a week via subcutaneous injection (SC) for four consecutive weeks, on days 0 (D0), 7 (D7), 14 (D14), and 21 (D21).

[0388] The effects of YN-011 on body weight (BW) in obese rhesus monkeys are shown in Table 7 and Figure 8. In the placebo group, no significant changes in body weight were observed throughout the study period, demonstrating the stability of the model. In the YN-011 25 μg / kg group, body weight consistently decreased from D7 to D28 compared to baseline (2.60%–6.71%, P<0.05 or P<0.01), with a 6.17% decrease at D28. Compared to the placebo group, BW in the YN-011 25 μg / kg group was significantly reduced at D14 and D21 (P<0.05). In the YN-011 50 μg / kg group, body weight consistently decreased from D7 to D28 compared to baseline (4.06%–7.32%, P<0.05), with a significant decrease of 7.32% at D28 (P<0.05). Compared to the placebo group, BW in the YN-01 150 μg / kg group decreased significantly or very significantly from D7 to D28 (P<0.05 or P<0.01).

[0389] [Table 16] Note: "a" represents the pre-administration measurement. # indicates P<0.05 compared to baseline, ## indicates P<0.01 compared to baseline. SC refers to subcutaneous injection.

[0390] Example 7: Preventive and therapeutic effects of YN-011 in a non-alcoholic steatohepatitis (NASH) model in rhesus monkeys. 7.1 Experimental Method 7.1.1 GE Ultrasound-Guided Liver Biopsy Number of animals: 15 Number of specimens collected: 2 needles or less, with each needle specimen measuring 1.5 cm or less in length, and the total specimen length measuring 3 cm or less.

[0391] Approximately 2 cm of the collected tissue was immediately placed in 4% buffered formalin at room temperature and fixed for at least 24 hours. After appropriate modifications, dehydration, embedding, sectioning, HE staining, and Masson staining were performed.

[0392] Equipment: For ultrasound, we used the GE Vivid S5 ultrasound system; for sectioning, the LEICA RM2135 microtome; for slide examination, the OLYMPUS BX43 microscope; and for microscopic imaging, the OLYMPUS DP22-CU camera.

[0393] Tissue sections were stained with H&E and Masson stain. Pathology slides were evaluated by pathologists according to the diagnostic and treatment guidelines for non-alcoholic fatty liver disease (NAFLD) jointly developed by the American Association for Liver Disease (AASLD), the American College of Gastroenterology (ACG), and the American Gastroenterological Association (AGA). Diagnostic criteria are listed in Tables 2 and 3.

[0394] 7.1.2 Tissue preparation, embedding, and sectioning After properly fixing the liver biopsy specimen, it was dehydrated, impregnated with paraffin, embedded, and direct sections (5 μm) were prepared.

[0395] 7.1.3 H&E staining The sections were subjected to standard deparaffinization. The sections were immersed in 100% ethanol I, 100% ethanol II, 95%, 85%, and 75% ethanol for 3 minutes each. Next, they were rinsed with tap water for 3 minutes, stained with hematoxylin-eosin (HE) stain for 12 minutes, rinsed with tap water for 5 minutes, and stained with eosin Y solution (aqueous) for 3 minutes. Dehydration was performed quickly with 95% ethanol, followed by two dehydrations with anhydrous ethanol for 2-5 minutes each. Next, the sections were washed twice with xylene for 10 minutes each. Neutral mounting medium was applied, and microscopic examination was performed.

[0396] 7.1.4 Masson staining The sections were subjected to the usual deparaffinization process. The sections were stained with Weigert's iron hematoxylin for 5-7 minutes, and a weak acid working solution was prepared by mixing distilled water and a weak acid solution in a 2:1 ratio. The sections were washed with the weak acid working solution for 1 minute. After washing with phosphomolybdic acid solution for 1-2 minutes, the sections were immersed directly in aniline blue staining solution for 1-2 minutes, and then washed with the prepared weak acid working solution for 1 minute. The sections were quickly dehydrated with 95% ethanol, followed by two dehydrations with anhydrous ethanol for 2-5 minutes each. Next, the sections were washed with xylene for 10 minutes each. A neutral mounting medium was applied, and microscopic examination was performed.

[0397] 7.1.5 Histopathological examination of tissues Sections were observed under a microscope (using a digital imaging system), and pathological diagnoses and photographs were taken. Based on the histopathological diagnosis, HE and Masson results were scored according to the scoring criteria for the Non-Alcoholic Fatty Liver Disease Activity Score (NAS, Table 8) and liver fibrosis stage (Table 9).

[0398] [Table 17] Note: NAS score = Fatty degeneration score + Interlobular inflammation score + Balloon-like degeneration score

[0399] [Table 18]

[0400] 7.1.6 Quantitative Analysis of Fatty Liver by GE 3.0T MRI In this study, a GE 3.0T MRI scanner (750W3T MRI, GE Healthcare) was used for liver imaging. The IDEAL-IQ sequence was used, combining the IDEAL (Iterative Water and Fat Separation by Echo Asymmetry and Least Squares Estimation) technique with rapid 3D multi-echo imaging. This technique includes multi-echo water-fat separation, region expansion algorithms, and various imaging methods for tissue fat quantification and R2* relaxation rates. Six contrast images, including in-phase, out-of-phase, water-only, fat-only, fat percentage map, and R2* map, were acquired in a single breath-hold scan by collecting signals from six different echo time (TE) values. The images were reconstructed using a computer algorithm to obtain the aforementioned contrast images. The IDEAL-IQ hybrid water-fat separation algorithm consists of two steps. The first step reconstructs complex domains to obtain water and fat images and a T2* map. The second step generates an additional set of estimated water and fat images. Next, these two sets of images are combined using a hybrid algorithm to produce the final image of water and fat.

[0401] Before conducting the experiment, a phantom test was performed. Five standard fat solutions with known fat content were prepared by modifying the body phantom preparation method used by Clare P. et al. (Elbassuoni, EA and RFAhmed, Mechanism of the neuroprotective effect of GLP-1 in a rat model of Parkinson's with pre-existing diabetes. Neurochem Int, 2019. 131: p.104583). Pure water was used as 0% fat. Sodium dodecyl sulfate (60 mmol) was dissolved in 1 L of deionized water, heated to 50°C, and then 40 g of gelatin was added and mixed well. Soybean oil (96, 108, 114, 117, and 120 ml, obtained from China National Pharmaceutical Group Chemical Reagents Co., Ltd.) was mixed with 0, 24, 12, 6, and 3 ml of the above solutions, respectively, to prepare solutions with fat content of 0%, 2.5%, 5%, 10%, 20%, and 100%. These solutions were filled into six 100 ml EP tubes for the subsequent scanning procedure.

[0402] Phantom validation experiments were conducted before each scan. All animals underwent a total of two scans: a baseline scan and an additional scan after drug administration was completed. Scans were performed in a 3.0T MRI room, and scan parameters are shown in Table 10. The anesthesia procedure involved intramuscular injection of 10 mg / kg ketamine hydrochloride, endotracheal intubation, and anesthesia maintenance with isoflurane and oxygen using a ventilator. Veterinary monitoring, including electrocardiogram, blood oxygenation, and respiratory rate, was performed throughout the scanning process and continued until the animals fully regained consciousness and resumed spontaneous breathing.

[0403] [Table 19]

[0404] Image Post-Processing and Analysis: Collected and stored rhesus macaque fatty liver MRI images were analyzed using a 750W 3T MRI workstation. Liver MRI images were selected to avoid large vessels, bile ducts, and gallbladder to prevent volumetric effects, and the maximum exposed area of ​​the right lobe region of the liver (layers 1-3) was chosen. Regions of interest (ROIs) were selected, with each ROI having an area of ​​90-110 mm². 2 Fatty liver was defined as MRI-PDFF% (magnetic resonance imaging proton density fat percentage) > 6% (according to clinical criteria).

[0405] 7.2 Experimental Results In vivo pharmacological studies of YN-011 were conducted in NASH rhesus monkeys. Clinical parameters evaluated included liver lipid content as assessed by MRI-PDFF%, NAFLD activity score (NAS), liver fibrosis stage as assessed by liver histology, body weight, body mass index (BMI), blood lipid profile, fructose metabolism, other biochemical parameters, and food intake.

[0406] The selected rhesus monkeys were 11–23 years old, which is equivalent to 30–70 years old in humans. All rhesus monkeys were male and weighed 13.63–22.85 kg. They had lipid metabolism abnormalities for more than two years, MRI-PDFF% was 7.8%–11.9%, NAS score was ≥3, and fibrosis score within 6 months was 0–1c. These animals clinically met the definition of NASH.

[0407] Fifteen rhesus monkeys were divided into three groups, with five monkeys in each group. These monkeys received subcutaneous injections of YN-011 once a week for 13 weeks (QW) at doses of 0 (solvent control, placebo control group), 0.050, or 0.150 mg / kg. The 0.150 mg / kg YN-011 group received the indicated dosage (0.1 mg / kg for the first dose, followed by 0.150 mg / kg for the next 12 doses).

[0408] During the study, all rhesus monkeys received the prescribed treatment, and no monkeys missed scheduled biopsies or discontinued treatment. No serious side effects were observed in any treatment group during the study.

[0409] As shown in Table 11, liver lipid content decreased by approximately 40% after 13 weeks of weekly YN-011 treatment compared to the placebo-controlled group.

[0410] To investigate whether YN-011 treatment resulted in histological improvement of the liver, liver biopsies were taken from all rhesus monkeys before the first injection and 89 days after the first injection. Liver sections were stained with HE stain and Masson stain and histologically analyzed. The degree of fatty degeneration, inflammation, hepatocyte swelling, and fibrosis was scored according to the criteria for NAFLD activity and NASH progression. Liver biopsy results showed that both NAS and liver fibrosis scores decreased in rhesus monkeys treated with YN-011, and no significant progression of NASH was observed (Table 12).

[0411] Liver fat detected by MRI-PDFF decreased from 9.0%±0.9% to 5.0%±0.2% in the 50 μg / kg YN-011 group and from 9.4%±1.5% to 5.6%±1.5% in the 150 μg / kg YN-011 group. Compared to baseline, the reduction rates at the end of treatment were 43.8% and 39.7%, respectively. There was no significant difference in the change in MRI-PDFF between the 50 μg / kg and 150 μg / kg YN-011 groups.

[0412] Histological analysis showed that in the 50 μg / kg YN-011 group, the mean NAS decreased from baseline 3.6 ± 0.5 to 1.6 ± 0.5 (P = 0.003). In the 150 μg / kg YN-011 group, the mean NAS decreased from baseline 3.6 ± 0.5 to 1.4 ± 0.5 (P < 0.001). There was no significant difference in NAS between the 50 μg / kg and 150 μg / kg YN-011 groups.

[0413] Regarding metabolic measurements such as liver damage biomarkers, lipid profiles, and body weight, YN-011 treatment showed excellent improvements in both the 50 μg / kg and 150 μg / kg groups.

[0414] [Table 20] Note: ROI: Area of ​​Interest; **Comparison with baseline, P<0.01; ##Comparison with placebo group, P<0.01; Rate of change = (Current value - Baseline value) / Baseline value × 100%.

[0415] [Table 21]

[0416] Furthermore, compared to baseline, body weight and BMI were significantly reduced in the YN-011 treatment group (days 7–84) of NASH rhesus monkeys. YN-011 treatment also significantly reduced serum low-density lipoprotein cholesterol (LDL-c), total cholesterol (TC), and total triglycerides (TG) at various measurement points throughout the treatment period, with 0.15 mg / kg YN-011 showing a more consistent lipid-lowering effect. High-density lipoprotein cholesterol (HDL-c) levels in the YN-011 treatment group showed a decreasing trend compared to baseline. These changes in serum lipid profiles were attributed to reduced food intake in animals administered YN-011. Throughout the study, plasma fasting blood glucose (FPG) levels remained within the normal range, and no significant differences were detected between the YN-011 and placebo groups after YN-011 and placebo administration. Plasma fructosamine (FRA), an effective biomarker reflecting mean glycemic control over the past 2-3 weeks, showed a decreasing trend in the YN-011 group one week after the first injection and a significant decrease of 4.3% after 12 weeks of treatment with 150 μg / kg YN-011. These results indicate that YN-011 is effective in controlling glycemic activity in NASH rhesus monkeys. The dose-dependent reduction in food intake observed in animals treated with YN-011 is a typical pharmacological effect of YN-011. In conclusion, these data indicate that multiple subcutaneous injections of YN-011 provide a significant therapeutic effect in NASH rhesus monkeys.

[0417] Example 8: In vitro experiments on neurodegenerative diseases 8.1 TNF-α concentration-dependent decline in the survival rate of SH-SY5Y neurons SH-SY5Y neurons were digested with 0.25% trypsin to prepare single-cell suspensions. The single-cell suspensions were seeded into 96-well plates with 100 μl of medium at a density of 10,000 cells per well. The plates were placed in a CO2 incubator (37°C, 5% CO2) overnight to allow the cells to adhere. The medium was replaced with a medium containing 10% FBS or 5% FBS, and the cells were stimulated with 20, 40, 60, 80, or 100 ng / ml of TNF-α for 48 hours. Each drug treatment was performed in six replication wells. 48 hours after drug treatment, 10 μl of CCK-8 solution was added to each well, and the plates were gently shaken to mix the reagents. The plates were then incubated in a CO2 incubator for 1–4 hours. Absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated using the following formula. Cell viability (%) = [A (treated) - A (blank)] / [A (control) - A (blank)] × 100 A (Processed): OD values ​​of wells containing cells, CCK-8 solution, and drug solution. A (Control): OD values ​​of wells containing CCK-8 solution without cells or drug solution. A (Blank): OD value of a well without cells

[0418] The experimental results (shown in Figure 9) indicate that an increase in TNF-α concentration significantly reduces the survival rate of SH-SY5Y neurons.

[0419] 8.2 Inhibitory effects of YN-011 and γ-aminobutyric acid (GABA) on TNF-α-induced SH-SY5Y cell viability decline Refer to Section 8.1 for experimental procedures. However, the drug treatment in this experiment differs from that in Section 8.1. Specifically, the drug treatment in this experiment was as follows: Using a medium containing 5% FBS, cells were treated for 48 hours with either 60 ng / ml TNF-α alone, in combination with 10, 100, or 500 nM YN-011, or in combination with 1, 10, or 100 μM GABA. Each drug treatment was performed in six repeat wells.

[0420] According to the experimental results (see Figure 10), the viability of SH-SY5Y cells decreased significantly in the presence of 60 ng / ml TNF-α, but cell viability improved significantly when treated with 10, 100, or 500 nM YN-011 or 100 μM GABA.

[0421] 8.3 Significant improvement in SH-SY5Y cell viability through the combined use of YN-011 and GABA. Refer to Section 8.1 for experimental procedures. However, the drug treatment in this experiment differs from that in Section 8.1. Specifically, the drug treatment in this experiment was as follows: Using a medium containing 5% FBS, cells were treated for 48 hours with either 60 ng / ml TNF-α alone or in combination with 100 nM YN-011 and / or 100 μM GABA. Each drug treatment was performed in six repeat wells.

[0422] The experimental results (see Figure 11) showed that the survival rate of SH-SY5Y neurons decreased significantly in the presence of 60 ng / ml TNF-α, while the combined treatment of 100 nM YN-011 and 100 μM GABA significantly improved cell viability. The combined use of YN-011 and GABA showed a stronger effect in improving cell viability than using either one alone.

[0423] 8.4 YN-011 reduces TNF-α-induced apoptosis in SH-SY5Y neurons. The experimental procedure is as follows: 1) Digest SH-SY5Y neurons in a 10cm culture dish using 0.25% trypsin and seed them into a 12-well plate. 2) Place the culture plate in a CO2 incubator and pre-culture overnight (37°C, 5% CO2) to promote cell adhesion. 3) Using a medium containing 5% FBS, treat the cells in each well with 60 ng / ml TNF-α alone or in combination with 10, 100, or 500 nM YN-011. Incubate the cells at 37°C for 48 hours. Each drug treatment should be performed on three replication wells. 4) After 48 hours, discard the supernatant, gently wash the cells once with PBS, and place the cell culture plate on ice. Add 120 μl of RIPA cell lysis buffer (Beyotime P0013B) containing protease and phosphatase inhibitors to each well and lyse the cells for 15 minutes. 5) Transfer the cell lysate to a 1.5 ml EP tube and centrifuge at 14,000 rpm for 30 minutes. 6) Carefully aspirate the supernatant into a new EP tube, add 5x loading buffer containing β-mercaptoethanol, and boil the sample at 100°C for 10 minutes to denature the protein. 7) Load 10 μg of protein per lane and perform SDS-PAGE gel electrophoresis. Position the gels perpendicular to the power rack in the electrophoresis tank, ensuring the concave side of the gels faces the power rack. Two gels can share one power rack. Secure the gels and power rack to the electrophoresis tank if necessary. Add electrophoresis buffer to create separate chambers between the two gels and the buffer in the electrophoresis tank. Gently remove the combs from the gels. 8) Electrophoresis: Connect the electrophoresis tank to the power supply using two electrodes, ensuring that the red and black electrode plugs match the corresponding ports. During electrophoresis, use low-voltage constant-voltage electrophoresis for the upper gel. Turn on the power and adjust the voltage to 80V (this usually takes about 15 minutes). Once the bromophenol blue dye has entered the lower gel, switch to high-voltage constant-voltage electrophoresis and adjust the voltage to 120V until the bromophenol blue dye reaches the bottom of the gel. 9) Perform a wet transfer. The sandwich configuration for the wet transfer is as follows: sponge / filter paper / gel / membrane / filter paper / sponge, firmly in place. There should be no air bubbles between the gel and the membrane, the sandwich orientation should be correct, with the negative electrode facing the protein side of the gel and moving towards the positive electrode (membrane). Once SDS-PAGE is complete, use a razor blade to gently separate the two glass plates of gel and place the gel on one of the glass plates. Using the blade, cut the gel at the boundary between the separated gel and the stacking gel, and cut off a small corner in the upper left corner of the separated gel to mark the order of the samples. Next, carefully transfer the gel to the transfer buffer. Cut a PVDF membrane the same size as the gel and immerse it in methanol for 5 seconds. Cut six pieces of filter paper the same size and equilibrate them in the transfer buffer for 15 minutes at the same time as the PVDF membrane and gel. Place the sponge pad, filter paper, gel, membrane, filter paper, and sponge pad (from bottom to top) on the transfer device, making sure that no air bubbles get trapped between the membrane and filter paper, gel and membrane, or filter paper and gel. Set the transfer current to a constant current of 200mA, and the transfer time is approximately 90 minutes. 10) Remove the transferred PVDF membrane, rinse it lightly with TBST, discard the TBST, and add 5% milk blocking solution to cover the PVDF membrane. Incubate at room temperature in a rocking shaker for 1 hour (30 rpm). 11) Incubate with primary antibody. Remove blocking solution and wash the membrane three times with TBST for 5 minutes each time, cleaving the membrane according to molecular weight. Add primary antibody at appropriate dilutions (Bcl-2, CST #3498S, 1:1000, Cleaved-caspase3, CST #9661S, 1:1000, Caspase3, CST #9662S, 1:1000, HSP90, Proteintech #13171-1-AP, 1:10000) and incubate overnight at 4°C in a rocking shaker. 12) Wash the membrane. Remove the PVDF membrane that has been incubated overnight and store the antibody at -20°C. Place the PVDF membrane in TBST solution and rinse quickly three times for 15 minutes each. 13) Incubate with secondary antibody. Add the corresponding species-specific secondary antibody (Jackson Lab, 1:10000) prepared in blocking solution to the membrane and incubate at room temperature for 1 hour with gentle shaking. 14) Wash the membrane. Remove the secondary antibody and place the PVDF membrane in TBST solution. Rinse quickly three times for 15 minutes each. 15) Perform ECL chemiluminescence. Under dark conditions, prepare a new developer (Solution A:Solution B = 1:1) according to the instructions on the ECL chemiluminescence kit (Millipore #WBKLS0500). Add the developer evenly to the film and place it in the imaging equipment for image acquisition. 16) Protein expression analysis. Using ImageJ software, the grayscale values ​​of the Western blot bands are analyzed and normalized to the grayscale values ​​of the reference protein HSP90 to calculate the relative expression level of the target protein.

[0424] The experimental results (shown in Figure 12) demonstrate that YN-011 significantly reduces TNF-α-induced apoptosis in SH-SY5Y neurons.

[0425] 8.5 GABA reduces TNF-α-induced apoptosis in SH-SY5Y neurons. Refer to Section 8.4 for the experimental procedure. However, the drug treatment in step 3) of this experiment differs from that in Section 8.4. In this experiment, the drug treatment in step 3) is as follows: Using a medium containing 5% FBS, treat the cells in each well with 60 ng / ml TNF-α alone, or in combination with 1, 10, or 100 μM GABA, at 37°C for 48 hours. Each drug treatment is performed in three replication wells.

[0426] The experimental results (shown in Figure 13) indicate that GABA reduces TNF-α-induced apoptosis in SH-SY5Y neurons.

[0427] 8.6 Reduction of TNF-α-induced apoptosis in SH-SY5Y neurons by combined use of YN-011 and GABA Refer to Section 8.4 for the experimental procedure. However, the drug treatment in step 3) of this experiment differs from that in Section 8.4. In this experiment, the drug treatment in step 3) is as follows: Using a medium containing 5% FBS, treat the cells in each well with 60 ng / ml TNF-α alone, or in combination with 100 μM GABA and / or 100 nM YN-011, at 37°C for 48 hours. Each drug treatment is performed in three replication wells.

[0428] The experimental results (shown in Figure 14) indicate that the combined use of YN-011 and GABA significantly reduces TNF-α-induced apoptosis in SH-SY5Y neurons.

[0429] 8.7 TNF-α damages SH-SY5Y neurons. Digeste SH-SY5Y neurons in a 10 cm culture dish with 0.25% trypsin to prepare a single-cell suspension. Seed the single-cell suspension into a 24-well plate. Place the culture plate in a CO2 incubator and pre-culture overnight at 37°C and 5% CO2 to allow cells to adhere. Treat the cells in each well with 20, 40, 60, or 80 ng / ml of TNF-α in a medium containing 5% FBS at 37°C for 48 hours. Each drug treatment is performed in three replication wells. Add the fluorescent dye Hoechst 33342 (Beyotime) at a dilution of 1:1000 to the wells and incubate the plate at 37°C for 10 minutes. Remove the culture medium, lightly wash twice with PBS, and immediately bring the plate to a fluorescence microscope for imaging (20x magnification). Take 10 fields of view of each cell well and count the number of positive cells stained blue in each field of view.

[0430] The experimental results (shown in Figure 15) indicate that TNF-α promotes damage to SH-SY5Y neurons.

[0431] 8.8 Protective effect of YN-011 against TNF-α-induced injury in nerve cells Refer to Section 8.7 for the experimental procedure. However, the drug treatment in this experiment differs from that in Section 8.7. The drug treatment in this experiment is as follows: Using a medium containing 5% FBS, treat the cells in each well with 60 ng / ml TNF-α alone, or in combination with 10, 100, or 500 nM YN-011, at 37°C for 48 hours. Each drug treatment is performed in three replication wells.

[0432] The experimental results (shown in Figure 16) indicate that YN-011 has a protective effect against TNF-α-induced injury in nerve cells.

[0433] 8.9 Protective effect of GABA against TNF-α-induced injury in nerve cells Refer to Section 8.7 for the experimental procedure. However, the drug treatment in this experiment differs from that in Section 8.7. In this experiment, the drug treatment is as follows: Using a medium containing 5% FBS, treat the cells in each well with 60 ng / ml TNF-α alone, or in combination with 1, 10, or 100 μM GABA, at 37°C for 48 hours. Each drug treatment is performed in three replication wells.

[0434] The experimental results (shown in Figure 17) indicate that GABA has a protective effect against TNF-α-induced damage in nerve cells.

[0435] 8.10 Protective effect of YN-011 and GABA on TNF-α-induced injury in nerve cells Refer to Section 8.7 for the experimental procedure. However, the drug treatment in this experiment differs from that in Section 8.7. In this experiment, the drug treatment is as follows: Using a medium containing 5% FBS, treat the cells in each well with 60 ng / ml TNF-α alone, or in combination with 100 μM GABA and / or 100 nMYN-011, at 37°C for 48 hours. Each drug treatment is performed in three replication wells.

[0436] The experimental results (shown in Figure 18) indicate that the combined use of YN-011 and GABA has a protective effect against TNF-α-induced damage in nerve cells.

[0437] 8.11 YN-011 reduces apoptosis in TNF-α-induced neurons. The experimental procedure is as follows: 1) Digest SH-SY5Y neurons in a 10 cm culture dish with 0.25% trypsin to prepare a single-cell suspension. Seed the single-cell suspension into a 24-well plate and place a sterile coverslip in each well. 2) Place the culture plate in a CO2 incubator and pre-culture it overnight at 37°C with 5% CO2 to allow the cells to adhere. 3) Using a medium containing 5% FBS, treat the cells in each well with 60 ng / ml TNF-α alone, or in combination with 10, 100, or 500 nM YN-011, at 37°C for 48 hours. Each treatment is performed in three repeating wells. 4) After 48 hours, remove the culture medium, add 400 μl of 4% paraformaldehyde (PFA) to each well, and fix at room temperature for 15 minutes. 5) Wash twice with PBS for 5 minutes each time. 6) To increase membrane permeability, add 0.1% Triton X-100 and wash at room temperature for 10 minutes. 7) Wash twice with PBS for 5 minutes each time. 8) Add 10% goat serum (GS) and incubate at room temperature for 30 minutes to block. 9) Remove the 10% GS and add the primary antibody Cleaved-caspase 3 (CST 9661S), diluted at a dilution ratio of 1:400 with 1% GS, in 30 μl of antibody solution per coverslip. 10) Leave it in the refrigerator at 4°C overnight. 11) The next day, remove the primary antibody and wash with PBS three times for 5 minutes each. 12) Add the fluorescent secondary antibody (Alexa Fluor® 488 Conjugate) and incubate in the dark at room temperature for 1 hour. 13) Wash with PBS for 5 minutes, three times. 14) Stain with DAPI for 5 minutes, then wash three times with PBS for 5 minutes each. 15) Apply mounting medium to a clean glass slide, then gently place the coverslip containing the cells upside down on top of the mounting medium and let it dry overnight at room temperature in a cool, well-ventilated place. 16) Images are taken using a fluorescence microscope (40x magnification). 17) Take 10 fields of view for each treatment group and count the number of positive cells stained green in each field of view.

[0438] The experimental results (shown in Figure 19) demonstrate that YN-011 significantly reduces apoptosis in TNF-α-induced neurons.

[0439] 8.12 GABA reduces apoptosis in TNF-α-induced neurons. Refer to Section 8.11 for experimental procedures. However, the drug treatment in this experiment differs from that in Section 8.11. In this experiment, the drug treatment in step 3) is as follows: Using a medium containing 5% FBS, treat the cells in each well with 60 ng / ml TNF-α alone, or in combination with 1, 10, or 100 μM GABA, at 37°C for 48 hours. Each drug treatment is performed in three replication wells.

[0440] The experimental results (shown in Figure 20) indicate that GABA reduces apoptosis in TNF-α-induced neurons.

[0441] 8.13 The combined use of YN-011 and GABA reduces apoptosis in TNF-α-induced neurons. Refer to Section 8.11 for experimental procedures. However, the drug treatment in this experiment differs from that in Section 8.11. In this experiment, the drug treatment in step 3) is as follows: Using a medium containing 5% FBS, treat the cells in each well with 60 ng / ml TNF-α alone, or in combination with 100 μM GABA and / or 100 nM YN-011, at 37°C for 48 hours. Each drug treatment is performed in three replication wells.

[0442] The experimental results (shown in Figure 21) demonstrate that the combined use of YN-011 and GABA significantly reduces apoptosis in TNF-α-induced neurons.

[0443] 8.14 YN-011 reduces apoptosis in TNF-α-induced neurons. Prepare a single-cell suspension by digesting SH-SY5Y nerve cells in a 10 cm culture dish with 0.25% trypsin. Seed the single-cell suspension into a 12-well plate. Place the culture plate in a CO2 incubator and pre-culture overnight at 37°C and 5% CO2 to allow cells to adhere. Treat the cells in each well with 60 ng / ml TNF-α alone, or in combination with 10, 100, or 500 nM YN-011, at 37°C for 48 hours using a medium containing 5% FBS. Each drug treatment is performed in three replication wells. Collect the cells and digest them with 0.25% trypsin without EDTA for 1 minute. Transfer the cell suspension to a 1.5 ml EP tube and centrifuge at 1000 rpm for 5 minutes, allowing the cells to settle at the bottom of the tube. Resuspend 105 cells in 100 μl of binding buffer, add 5 μl of PI and 5 μl of Annexin V-FITC solution, and incubate in the dark at room temperature for 15 minutes. Add 400 μl of binding buffer to each tube and detect using a flow cytometer (Annexin V-FITC excitation wavelength: 488 nm, emission wavelength: 520 nm, PI excitation wavelength: 535 nm, emission wavelength: 617 nm). Count the number of Annexin V-FITC-positive and PI-negative cells in each sample.

[0444] The experimental results (shown in Figure 22) demonstrate that YN-011 significantly reduces apoptosis in TNF-α-induced neurons.

[0445] 8.15 GABA reduces apoptosis in TNF-α-induced neurons. Refer to Section 8.14 for the experimental procedure. However, the drug treatment in this experiment differs from that in Section 8.14. In this experiment, the drug treatment is as follows: Using a medium containing 5% FBS, treat the cells in each well with 60 ng / ml TNF-α alone, or in combination with 1, 10, or 100 μM GABA, at 37°C for 48 hours. Each drug treatment is performed in three replication wells.

[0446] The experimental results (shown in Figure 23) indicate that GABA reduces apoptosis in TNF-α-induced neurons.

[0447] 8.16 The combined use of YN-011 and GABA reduces apoptosis in TNF-α-induced neurons. Refer to Section 8.14 for experimental procedures. However, the drug treatment in this experiment differs from that in Section 8.14. The drug treatment in this experiment is as follows: Using a medium containing 5% FBS, treat cells in each well with 60 ng / ml TNF-α alone, or in combination with 100 μM GABA and / or 100 nM YN-011, at 37°C for 48 hours. Each drug treatment is performed in three replication wells.

[0448] The experimental results (shown in Figure 24) indicate that the combined use of YN-011 and GABA significantly reduces apoptosis in TNF-α-induced neurons.

[0449] 8.17 GABA reduces mRNA expression of inflammatory factors in HMC3 microglia cells induced by Aβ1-42 oligomers. Under sterile conditions, the synthesized Aβ1-42 peptide (AnaSpec#AS-20276) is dissolved in hexafluoroisopropanol (Mecklin#H811026) to obtain a 1 mM solution. The solution is divided into 1.5 ml sterile centrifuge tubes, and the hexafluoroisopropanol is evaporated using a vacuum evaporator. The dried peptide forms a membrane in the tube and is stored at -20°C for later use. The Aβ1-42 oligomer is then freshly prepared from the dried peptide membrane as follows: The dried peptide membrane is dissolved in anhydrous DMSO to obtain a 5 mM solution, which is then diluted in cold cell culture basal medium to obtain a 100 μM stock solution. After incubation at 4°C for 24 hours, the stock solution is used for cell treatment.

[0450] HMC3 human microglia cells are seeded in a 24-well plate. After incubation overnight at 37°C and 5% CO2, cells in each well are treated for 48 hours with 10 μM Aβ1-42 oligomer alone, or in combination with 1, 10, or 100 μM GABA. Each drug treatment is performed in three replication wells. After 48 hours of treatment, total RNA is isolated from the cells in each well using 1 ml of Trizol (Invitrogen #15596026) according to the manufacturer's instructions. The RNA pellet is dissolved in 20 μl of DEPC-treated water. RNA concentration and purity are measured using NanoDrop 2000. RNA is converted to cDNA using a reverse transcription kit (Yeasen #11141ES60). Prepare the PCR reaction as follows: 5 μl PCR mix (Yeasen #10108ES03), 0.2 μl forward primer, 0.2 μl reverse primer, 1 μl cDNA, and 3.6 μl water. Perform qPCR on an Applied Biosystems 7500 real-time PCR system using the following thermal program: initial denaturation at 95°C for 3 minutes, followed by 40 cycles of denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 1 minute. Compare the relative mRNA expression of TNF-α and IL-6 between treated and untreated cells using the ΔΔCt method and normalize to the expression of the housekeeping gene RPLP0. The experimental results (shown in Figure 25) show that in the presence of 10 μM Aβ1-42 oligomer, TNF-α and IL-6 mRNA expression in HMC3 cells is significantly increased, while 100 μM GABA significantly decreases TNF-α mRNA expression, and both 10 μM and 100 μM GABA significantly decrease IL-6 mRNA expression.

[0451] 8.18 YN-011 reduces mRNA expression of inflammatory factors in HMC3 microglia cells induced by Aβ1-42 oligomers. Refer to Section 8.17 for the experimental procedure. However, the drug treatment in this experiment differs from that in Section 8.17. In this experiment, the drug treatment is as follows: Cells in each well are treated either with 10 μM Aβ1-42 oligomer alone or in combination with 10, 100, or 500 nM YN-011 for 48 hours. Each drug treatment is performed in three replication wells.

[0452] The experimental results (see Figure 26) show that the mRNA expression of TNF-α and IL-6 in HMC3 cells is significantly increased in the presence of 10 μM Aβ1-42 oligomers, while the mRNA expression of TNF-α and IL-6 is significantly decreased in the presence of 100 nM and 500 nM YN-011.

[0453] 8.19 The combined use of YN-011 and GABA reduces mRNA expression of inflammatory factors in HMC3 microglia cells induced by Aβ1-42 oligomers. Refer to Section 8.17 for the experimental procedure. However, the drug treatment in this experiment differs from that in Section 8.17. In this experiment, the drug treatment is as follows: Cells in each well are treated either with 10 μM Aβ1-42 oligomer alone or in combination with 100 μM GABA and / or 100 nM YN-011 for 48 hours. Each drug treatment is performed in three replication wells.

[0454] The experimental results (shown in Figure 27) indicate that the combined use of 100 μM GABA and 100 nM YN-011 shows a significantly stronger inhibitory effect on TNF-α mRNA expression induced by 10 μM Aβ1-42 oligomers compared to individual treatment with GABA or YN-011.

[0455] 8.20 GABA reduces the expression of inflammatory factors in HMC3 microglia cells induced by Aβ1-42 oligomers. HMC3 human microglia cells are seeded in a 12-well plate. After incubation overnight at 37°C and 5% CO2, cells in each well are treated for 48 hours with 10 μM Aβ1-42 oligomer alone or in combination with 1, 10, or 100 μM GABA. Each drug treatment is performed in three replication wells. After 48 hours of treatment, the cells are washed with PBS and lysed on ice for 15 minutes in RIPA buffer supplemented with protease inhibitors and phosphatase inhibitors. The cell lysates are centrifuged at 14,000 rpm for 30 minutes. The supernatant is collected and mixed with 5x loading buffer supplemented with β-mercaptoethanol and boiled at 100°C for 10 minutes. Denatured proteins are separated by SDS-PAGE. Briefly, 10 μg of protein is loaded onto a precast mini polyacrylamide gel (SurePAGE, GenScript#M00657) and run at a voltage of 120V. Next, the gel is transferred to a PVDF membrane (pore size 0.22 μm) at 200 mA for 90 minutes. After unblocking, the membrane is incubated overnight at room temperature at 4°C with primary antibodies (TNF-α, Proteintech #60291-Ig, IL-6, Proteintech #21865-1-AP, HSP90, Proteintech #13171-1-AP) diluted in 5% milk solution. After washing, the membrane is incubated at room temperature for 1 hour with the corresponding secondary antibody (Jackson Lab, 1:10000). After washing, the antibody signal on the membrane is visualized using an ECL kit (Millipore #WBKLS0500) according to the manufacturer's instructions. Relative protein levels normalized to the endogenous protein HSP90 are calculated using ImageJ.

[0456] The results (shown in Figure 28) show that in the presence of 10 μM Aβ1-42 oligomer, the expression of TNF-α and IL-6 in HMC3 cells was significantly increased, while 100 μM GABA significantly decreased IL-6 expression, and both 10 μM and 100 μM GABA significantly decreased TNF-α expression.

[0457] 8.21 YN-011 reduces the expression of inflammatory factors in HMC3 microglia cells induced by Aβ1-42 oligomers. Refer to Section 8.20 for the experimental procedure. However, the drug treatment in this experiment differs from that in Section 8.20. The drug treatment in this experiment is as follows: Treat the cells in each well with 10 μM Aβ1-42 oligomer alone, or in combination with 10, 100, or 500 nM YN-011 for 48 hours. Each drug treatment is performed in three replication wells.

[0458] The experimental results (see Figure 29) show that the expression of TNF-α and IL-6 in HMC3 cells is significantly increased in the presence of 10 μM Aβ1-42 oligomer, while the expression of TNF-α and IL-6 is significantly decreased in the presence of 100 nM and 500 nM YN-011.

[0459] 8.22 The combined use of YN-011 and GABA reduces the expression of inflammatory factors in HMC3 microglia cells induced by Aβ1-42 oligomers. Refer to Section 8.20 for the experimental procedure. However, the drug treatment in this experiment differs from that in Section 8.20. In this experiment, the drug treatment is as follows: Cells in each well are treated either with 10 μM Aβ1-42 oligomer alone or in combination with 100 μM GABA and / or 100 nM YN-011 for 48 hours. Each drug treatment is performed in three repeating wells.

[0460] The experimental results (shown in Figure 30) show that the combined use of 100 μM GABA and 100 nM YN-011 exhibits a significantly stronger inhibitory effect on the expression of TNF-α and IL-6 induced by 10 μM Aβ1-42 oligomers compared to individual treatment with GABA or YN-011.

[0461] Example 9: In vivo neurodegenerative diseases Neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD) are central nervous system disorders characterized by progressive loss of function of central nervous cells or their myelin sheaths. As a result, the structure and function of nerve cells are gradually lost, leading to symptoms such as dementia and motor impairment.

[0462] Among these, Alzheimer's disease (AD), also known as senile dementia, is characterized by progressive cognitive impairment and memory loss, and there is currently no effective treatment. Research shows that the brains of AD patients accumulate neurotoxic plaques formed by the abnormal aggregation of beta-amyloid (Aβ). Aβ can cause the formation of senile plaques in the brain and apoptosis of nerve cells, and is a key factor in the development of AD. Aβ is a peptide consisting of 39 to 43 amino acids, and the most common subtypes of Aβ in the human body are Aβ40 and Aβ42, which are prone to aggregation, leading to the formation of Aβ deposits and subsequent neurotoxic effects.

[0463] Studies have shown that GLP-1 has neuroprotective biological effects and can improve AD symptoms (Zheng, J., et al., GLP-1 improves the supportive ability of astrocytes to neurons by promoting aerobic glycolysis in Alzheimer's disease. Mol Metab, 2021. 47: p. 101180; Park, JS, et al., Blocking microglial activation of reactive astrocytes is neuroprotective in models of Alzheimer's disease. Acta Neuropathol Commun, 2021. 9(1): p. 78). In animal models, GLP-1 has demonstrated a neuroprotective role in AD by improving cognitive function in AD mice, reducing Aβ deposition, mitigating Aβ-induced glial cell overactivation, and alleviating oxidative stress and inflammation in the brain. This invention discloses a long-acting GLP-1 receptor agonist (GLP-1RA) that exerts biological effects by protecting neurons and improving central nervous system function, thereby helping to alleviate AD symptoms.

[0464] The experiment uses Alzheimer's disease model mice and wild-type control adult mice with the same genetic background. The animals are housed individually in cages, maintaining a 12 / 12 hour light-dark cycle (lights on at 08:00, off at 20:00), and the temperature is kept at 21.5°C. Food and water are freely available. Prior to the behavioral experiment, the mice are intraperitoneally (ip) injected with either YN-011 or a placebo for 16 weeks.

[0465] Morris Water Maze Setup The maze is made of white opaque plastic, 120cm in diameter and 40cm high, and is filled with 25°C water to prevent excessive cooling of body temperature. A small escape platform (10 x 6.5 x 21.5cm) is placed in a fixed position within the quadrant, 25cm away from the surrounding walls and hidden 1cm below the water surface. There are several fixed visual cues on the walls of the room.

[0466] spatial memory Four points (north, south, east, and west) equidistant along the circumference of the pool serve as starting positions. The starting positions are randomly assigned in four trials per day. During training, mice are trained to find a fixed safety platform submerged in the pool. 24 hours after the end of the training period, the safety platform is removed from the pool, and the length and time of the mice's swimming paths are recorded, starting from a random starting position (n=12 per group). The collected data are analyzed using one-way and two-way ANOVA to assess spatial memory, represented by the time and path length required to reach the original location of the safety platform, and spatial awareness, represented by the time spent searching the area where the safety platform was located.

[0467] Detection of Aβ40 and Aβ42 levels Aβ40 and Aβ42 levels are measured using an Aβ detection kit. Briefly, brain hemispheres of AD model mice and control mice treated with YN-011 are homogenized with Tris-buffered saline (25 mM Tris HCl, pH 7.4, 150 mM NaCl) containing a proteinase inhibitor (Sigma, 250 ml / 5 ml buffer). The brain homogenates are then centrifuged at 100,000 g at 4°C for 1 hour. The supernatant is then diluted 1:10 and an ELISA is performed. In the ELISA, only soluble beta-amyloid oligomer levels are measured, not monomers. Protein quantification is performed using the Bradford protein assay. The final Aβ values ​​are determined after normalization to total protein levels (n=6 / group).

[0468] Example 10: Phase IIb and III clinical trials of YN-011 (monotherapy) 10.1 Test Design Phase IIb and Phase III clinical trials are multicenter, randomized, double-blind, placebo-controlled studies conducted to evaluate the efficacy and safety of YN-011 in patients with type 2 diabetes mellitus (T2DM) that is not adequately controlled despite diet and exercise therapy. Phase IIb is an exploratory clinical trial to investigate dose-response and safety evaluations (divided into four groups: YN-011 1 mg, YN-011 2 mg, YN-011 3 mg, and a placebo control group) with the aim of obtaining the Phase III recommended dose (RP3D) of YN-011 for Phase III. Phase III is a confirmation clinical trial for efficacy.

[0469] Key eligibility criteria for Phase IIb and III studies: • Age at screening is between 18 and 75 years old • Diagnosed with type 2 diabetes (according to WHO 2000 criteria) and not receiving antihyperglycemic agent treatment in the 8 weeks prior to screening. • Glycated hemoglobin HbA1c at screening: 7.5% ≤ HbA1c ≤ 11% · Glycated hemoglobin HbA1c before randomization: 7.5% ≤ HbA1c ≤ 10.5% · Fasting plasma glucose (FPG) at screening and before randomization < 13.9 mmol / L · BMI ≥ 18.5 kg / m 2 and ≤ 40 kg / m 2

[0470] Main exclusion criteria for Phase IIb and III studies: · Type 1 diabetes · Taking DPP-4 inhibitors and / or GLP-1 analogs during the 3 months prior to screening for the use of any drug · Continuing insulin treatment for 14 days or more during the 1 year prior to screening (excluding gestational diabetes requiring insulin treatment) · Fasting C-peptide < 0.3 nmol / mL · Suffering from diabetic ketoacidosis, diabetic lactic acidosis, or hyperosmolar nonketotic diabetic coma during the 6 months prior to screening · Suffering from proliferative retinopathy or macular disease, severe diabetic neuropathy, intermittent claudication, or diabetic foot disease during the 6 months prior to screening or requiring treatment for them · Severe hypoglycemic events (grade 3 hypoglycemia) without an obvious cause during the 6 months prior to screening, or more than 3 hypoglycemic events (blood glucose < 3.9 mmol / L) during the 1 month prior to screening, or recurrent symptoms related to hypoglycemia · Having a serious trauma, severe infectious disease, or surgery during the 1 month prior to screening and having a disease that may affect blood glucose control prior to screening · Donating blood, experiencing major blood loss (> 400 mL), or receiving a blood transfusion within the past 3 months · Uncontrolled hypertension · Patients with a history of acute or chronic pancreatitis, symptomatic gallbladder disease, pancreatic injury, or other high-risk factors for pancreatitis, or patients with amylase and / or lipase more than 1.5 times the upper limit of normal (ULN) at screening • History of medullary thyroid carcinoma, multiple endocrine neoplasia (MEN) 2A or 2B syndrome, or a related family history or a history of other malignancies. • Clinically significant gastric emptying abnormalities, severe chronic gastrointestinal disease, long-term use of medications that directly affect gastrointestinal motility, or gastrointestinal surgery within 6 months prior to screening (as determined by the investigator). • Any condition that causes blood disorders, hemolysis, or red blood cell instability • Uncontrolled hyperthyroidism or hypothyroidism • Positive for hepatitis B surface antigen (HBsAg) and hepatitis B viral load (HBV-DNA) exceeding the local laboratory detection limit; positive for hepatitis C antibody (HCV-Ab), human immunodeficiency virus antibody (HIV-Ab), treponema pallidum antibody (TP-Ab), or positive for COVID-19 nucleic acid test. • Acute or chronic hepatitis, or clinical laboratory findings meeting any of the following criteria: alanine aminotransferase (ALT) level ≥ 2.5 x ULN and / or aspartate aminotransferase (AST) ≥ 2.5 x ULN, fasting triglycerides > 5.7 mmol / L, estimated glomerular filtration rate (eGFR) calculated using the CKD-EPI (EPI-(Scr)) formula < 60 mL / min / 1.73 m² 2 • Any other condition that the principal investigator or attending physician deems inappropriate for participation in the clinical trial.

[0471] The Phase IIb dose-finding treatment regimen for enrolled subjects is as follows: Subjects enrolled in the Phase IIb efficacy confirmation stage:

[0472] [Table 22]

[0473] [Table 23]

[0474] The primary efficacy endpoint was a comparison of YN-011 with placebo in patients with type 2 diabetes regarding the change from baseline in HbA1c levels at 12 weeks (Phase IIb) or 24 weeks (Phase III) of double-blind administration. Secondary efficacy endpoints included primarily changes from baseline in FPG, fasting insulin, fasting C-peptide, fasting glucagon, fasting lipid profile, and fasting body weight (12 weeks in Phase IIb and 24 weeks in Phase III). Other secondary endpoints included HbA1c target achievement rate (percentage of subjects with HbA1c <7.0% and <6.5%), area under the curve of glucose during mixed meal tolerance tests (MMTT), and area under the curve of insulin or C-peptide during MMTT. Safety evaluations included adverse events, laboratory tests, vital signs, and 12-lead electrocardiogram assessments.

[0475] 10.2 Therapeutic effects of YN-011 in type 2 diabetes Clinical trial results showed that HbA1c levels decreased by 1.73% after 24 weeks of administration of 1 mg of YN-011. It has also been reported that HbA1c levels decreased by 1.55% after 30 weeks of administration of 1 mg of semaglutide (Sorli et al., Lancet Diabetes Endocrinol 2017;5:251-60), and HbA1c levels decreased by 1.46% after 26 weeks of administration of 1.5 mg of dulaglutide (Shi et al., J Diabetes Investig 2020;11:142-150).

[0476] Furthermore, clinical trial results showed that the incidence of hypoglycemia (<3.9 mmol / L) after 24 weeks of administration of YN-011 1 mg and 3 mg was 0.8% and 1.7%, respectively. The incidence of hypoglycemia after 26 weeks of administration of dulaglutide 0.75 mg and 1.5 mg was reported to be 4.1% and 6.3%, respectively (Shi et al., J Diabetes Investig 2020;11:142-150).

[0477] Furthermore, the incidence of nausea after 24 weeks of administration of YN-011 1 mg and 3 mg was 3.4% and 6.0%, respectively. The incidence of nausea after 30 weeks of treatment with semaglutide 0.5 mg and 1 mg doses was reported to be 20% and 24%, respectively (Sorli et al., Lancet Diabetes Endocrinol 2017;5:251-60), and the incidence of nausea after 26 weeks of treatment with dulaglutide 1.5 mg dose was 9.5% (Shi et al., J Diabetes Investig 2020;11:142-150). In the case of liraglutide, the incidence of nausea after 24 weeks of treatment with 1 mg and 3 mg doses was 5.6% and 10%, respectively (Shuai et al., Diabetes Obes Metab. 2021;23(1):116-124).

[0478] Example 11: Phase IIb and Phase III clinical trials of YN-011 in combination with metformin. 11.1 Test Design The Phase IIb and Phase III clinical trials were multicenter, randomized, double-blind, placebo-controlled studies evaluating the efficacy and safety of YN-011 in patients with T2DM whose glycemic control was inadequate with metformin therapy. The Phase IIb trial aimed to investigate dose-response, evaluate safety (divided into three groups: YN-011 1 mg + metformin, YN-011 3 mg + metformin, and placebo + metformin), and determine the recommended Phase III dose (RP3D) for the Phase III trial to confirm efficacy.

[0479] The inclusion criteria for subjects in the Phase IIb and Phase III trials of YN-011 in combination with metformin were essentially the same as those for Implementation Example 10, except for the following: • You have been diagnosed with type 2 diabetes for at least 8 weeks (WHO 2000) and meet one of the following conditions: a) Receiving metformin monotherapy for at least 8 weeks at a dose of 1500 mg or more per day or the maximum tolerated dose (less than 1500 mg per day, or 1000 mg or more per day) (Eligible subjects were able to enter a direct induction period). b) Metformin monotherapy was administered for less than 8 weeks at a dose of 1500 mg / day or greater, or at the maximum tolerated dose (less than 1500 mg / day but greater than 1000 mg / day) (eligible subjects were required to enter a metformin dose stabilization period). c) The patient received metformin monotherapy at a dose of less than 1500 mg / day and had not reached the maximum tolerated dose (eligible subjects were required to enter a metformin dose escalation and dose stabilization period).

[0480] For the main exclusion criteria for subjects in the Phase IIb and Phase III trials of YN-011 in combination with metformin, please refer to the exclusion criteria in Implementation Example 10.

[0481] The treatment regimens for subjects enrolled in the Phase IIb dose confirmation stage are as follows: Subjects enrolled in the Phase IIb dose confirmation stage:

[0482] [Table 24]

[0483] [Table 25]

[0484] The primary efficacy endpoint included a double-blind comparison of YN-011 in combination with metformin versus placebo in combination with metformin, regarding the change in HbA1c levels from baseline after 12 weeks (Phase IIb) or 24 weeks (Phase III) of administration in patients with type 2 diabetes whose blood glucose control was inadequately controlled with metformin therapy. For secondary efficacy endpoints and safety evaluations, please refer to Implementation Example 10.

[0485] 11.2 Therapeutic effects of YN-011 in combination with metformin in type 2 diabetes Clinical trial results showed that after 24 weeks of treatment with 3 mg of YN-011 in combination with metformin, HbA1c levels decreased by 1.8% and FPG levels decreased by 2.42%. It has been reported that administration of 1.5 mg of dulaglutide in combination with metformin resulted in a 1.42% decrease in HbA1c levels and a 1.93% decrease in FPG levels after 40 weeks of treatment (Dungan et al., Lancet 2014;384:1349-57). Furthermore, the clinical trial results showed that the incidence of hypoglycemia (<3.9 mmol / L) after 24 weeks of administration of 3 mg of YN-011 in combination with metformin was 1.8%, which was comparable to the incidence of hypoglycemia in the placebo group in combination with metformin (1.7%). The incidence of hypoglycemia after 40 weeks of administration of dulaglutide 1.5 mg in combination with metformin was reported to be 9% (Dungan et al., Lancet 2014;384:1349-57). Furthermore, the incidence of nausea after 24 weeks of administration of YN-011 3 mg in combination with metformin was 7.0%. The incidence of nausea after 40 weeks of administration of dulaglutide 1.5 mg in combination with metformin was reported to be 20% (Dungan et al., Lancet 2014;384:1349-57), and the incidence of nausea after 30 weeks of administration of semaglutide 1 mg in combination with metformin was reported to be 13.4% (Ji et al., Diabetes Obes Metab. 2021;23:404-414).

[0486] Example 12: Pharmaceutical formulation screening of GLP-1 fusion proteins In this example, the inventors purified the tissue culture supernatant containing the GLP-1 fusion protein (YN-011) obtained in Example 1, prepared different types and concentrations of excipients and different formulations, and screened for a preferred YN-011 formulation by detecting each formulation.

[0487] 12.1 Detection Method 12.1.1 Appearance Using a visual method, the sample bottles were wiped clean, and the color, transparency, and visible particles of the samples were observed by placing them in front of a black and white background on a transparency meter under a light irradiation intensity of 1500 lx.

[0488] 12.1.2 pH value Using the electropotential method, the pH meter was calibrated with a standard solution, and then 50-100 μl of sample was taken and the pH value was measured.

[0489] 12.1.3 Protein content Using the ultraviolet method, 2.5 μl of the sample was pipetted, and then the sample was absorbed at a wavelength of 280 nm using a Nano Drop 2000 to calculate the sample concentration. The measurement was repeated twice, and the average value was used as the final result.

[0490] 12.1.4 Turbidity (A350) Since proteins do not have an absorption peak at 350 nm, the absorption value of a sample in this wavelength range can reflect the turbidity of the sample itself. Therefore, the detection result of A350 is used as the basis for turbidity comparison. 100 μl of the sample was added to a 96-well plate using a pipette gun, and then the absorption value of the sample at 350 nm was measured using a SpectraMax M5e enzyme scale.

[0491] 12.1.5 Molecular exclusion chromatography purity (SEC-HPLC) In molecular sieve gel chromatography during the formulation development experimental stage, this measurement method was performed using an Agilent system, a TSK G3000SWXL gel column (5 μm, 7.8 × 300 mm, 25°C), with an injection temperature of 5°C, a column temperature of 25°C, and a detection wavelength of 280 nm. The fluid phase group consisted of 50 mM sodium phosphate, 300 mM sodium chloride, pH 7.0 ± 0.2, with isoelution and a flow rate of 1.0 mL / min. The sample concentration was 3 mg / mL, and the standard was diluted to 3 mg / mL in the fluid phase, maintaining a concentration consistent with the sample. The injection volume was 20 μL.

[0492] 12.1.6 Inverted Liquid Chromatography (RP-LC) Reverse high-efficiency liquid chromatography separates protein molecules after reduction denaturation based on the strength of their interaction with a C8 reversed-phase chromatography column. The solvent on the stationary phase surface flows at a constant rate, and highly polar molecules produce peaks first, while less polar molecules interact more strongly with the C8 column and produce peaks later. After separation, various substances were detected by a UV detector.

[0493] 12.1.7 Non-reducing tip gel electrophoresis (SDS_CALIPER_NR) A denatured solution (for current formulation) was prepared using the sample buffer mother liquor, 10% SDS, and 100 mM N-Ethylmaleimide from the reagent kit (purchased from SCIEX) in a volume ratio of 20:1:0.7. Simultaneously, the reference sample and the sample were diluted to 1 mg / mL with water. 2 μL of the sample dilution and 7 μL of the denatured solution were taken into new 1.5 mL EP tubes, mixed, and heated at 70°C for 10 mins (ladder was heated directly at 70°C without adding the denatured solution). After instantaneous centrifugation, 35 μL of water (120 μL with 12 μL ladder added) was added, and the mixture was mixed using an oscillator to complete the sample preparation. 42 μL of the prepared sample was taken and transferred to a 96-well plate, and centrifuged at 4000 rpm for 20 mins. Subsequently, the 96-well plate was placed in the instrument plate chamber (Note: there are two empty spaces in the plate chamber: a wash buffer and a ladder). The sample then completed steps such as sample aspiration, staining, separation, decolorization, and inspection in a chip equipped with destain-gel, gel-dye, and maker. The raw data was analyzed using LabChip GX Reviewer.

[0494] 12.1.8 Capillary Focused Isoelectrophoresis (cIEF) In electrophoresis systems such as the ProteinSimple iCE 3 all-column imaging capillary, a fluorine-coated capillary (ProteinSimple, FC coated cartridge, id100μm) is used to focus and separate charge heteromers in each sample according to their isoelectric point differences. The sample concentration in the sample solution was 0.3 mg / mL, the automatic injector temperature control was 15°C, and the injection duration was 90 seconds. The voltage and time for the first focus stage were 1500V, 1 minute, the voltage and time for the second focus stage were 3000V, 8 minutes, and the detection wavelength was 280 nm. Two types of labels with known isoelectric points (pI maker) can be added to the sample and focused simultaneously to determine the isoelectric point of the sample being measured. Data analysis was performed using Empower (Empower@3) or Chrom Perfect Software. The main peak content, peak group A (acidic peak) content, and peak group B (basic peak) content can be obtained using the peak area normalization method.

[0495] 12.2 Filtering of Buffer Systems 12.2.1 Filtering Strategy for Buffer Systems Four buffer systems were selected: 10 mM citrate, histidine, phosphate, and acetate. Ten different buffer systems were designed for consideration, ranging from pH 4.5 to 7.0 (see Table 13 for details). The YN-011 stock solution was replaced with buffer systems of 10 mM citrate at pH 6.2, 10 mM histidine at pH 6.0, 10 mM phosphate at pH 6.5, and 10 mM acetate at pH 5.0. The pH was adjusted with an acid or base (e.g., HCl / NaOH) to obtain the remaining 6 pH formulations, after which the protein concentration was adjusted to 3 mg / mL. 2 mL samples were filled into 5 mL neutral boron-silicon glass injectable bottles, sealed with rolled caps, and examined. The samples were left for 4 weeks under two different examination conditions, 25°C and 40°C. Samples were taken at different time points and each test was performed to evaluate the protein stability in the 10 buffer systems. The buffering system schemes are shown in Table 13.

[0496] [Table 26]

[0497] 12.2.2 Appearance and pH Testing The appearance of YN-011 was observed according to the buffer system schemes in Table 13. Under conditions of 25°C, all YN-011 samples exhibited colorless micro-emulsification. After 2 weeks, a small amount of fine particles were observed in the citrate / sodium citrate buffer system (pH 6.2), and a large amount of fine particles were observed after 4 weeks. Samples in the histidine / histidine hydrochloride buffer system at three pH values ​​(pH 5.5, 6.0, 6.5) all showed different particle conditions under 25°C conditions. After 2 weeks, a small amount of fine particles was observed in the pH 5.5 sample, but large particle proteins appeared in the pH 6.0 and 6.5 samples, and the number of particles increased over time. Samples in the disodium hydrogen phosphate / sodium dihydrogen phosphate buffer system (pH 6.0, 6.5, 7.0) all showed small amounts of particles or flocs under 25°C conditions. Samples of an acetic acid / sodium acetate buffer system at three different pH values ​​(pH 4.5, 5.0, and 5.5) showed very small amounts of particles appearing after being left for 4 weeks under 25°C conditions, with a slight increase in particle count after 4 weeks.

[0498] Under 40°C conditions, all YN-011 samples exhibited colorless slight emulsion. Samples in a citrate / sodium citrate buffer system (pH 6.2) showed small amounts of flocculated particles after 1 and 2 weeks at 40°C, while a large amount of fine particles were observed in the sample after 4 weeks. Samples in a histidine / histidine hydrochloride buffer system at three different pH values ​​(pH 5.5, 6.0, and 6.5) showed varying particle counts at different sampling points under 40°C conditions. Small amounts of fine particles were observed in the pH 5.5 samples after 1 and 2 weeks at 40°C, and in the pH 6.0 and pH 5.5 samples after 1 week at 40°C, with the particle count increasing over time. Samples in the disodium hydrogen phosphate / sodium dihydrogen phosphate buffer system at pH 6.0 and pH 6.5 showed small amounts of particles or flocs at different sampling points under 40°C conditions. Samples in the pH 7.0 system of the same system showed no particles after 1 and 2 weeks at 40°C, but small amounts of flocs were observed after 4 weeks. Samples in the acetic acid / sodium acetate buffer system at three pH values ​​(pH 4.5, 5.0, and 5.5) showed no particles after 1 week under 40°C conditions. After 2 weeks, very small amounts of particles appeared in the pH 5.0 and pH 5.5 samples, and after 4 weeks, small amounts of cotton-like material appeared in all three pH samples.

[0499] The initial pH and pH stability at different time points were measured for the YN-011 formulation system according to the buffering system schemes in Table 13. As shown in Table 14, the three pH values ​​(pH 6.0, 6.5, 7.0) of the disodium hydrogen phosphate / sodium dihydrogen phosphate buffer system and the three pH values ​​(pH 4.5, 5.0, 5.5) of the acetate / sodium acetate buffer system showed basically consistent measurement results at different sampling points, indicating pH stability. The samples in the citrate / sodium citrate buffer system (pH 6.2) and the three pH values ​​(pH 5.5, 6.0, 6.5) of the histidine / histidine hydrochloride buffer system showed pH increases to different degrees over time under conditions of 25°C and 40°C, indicating pH instability.

[0500] [Table 27] Note: W = period, T0 = start of analysis

[0501] 12.2.3 Measurement of protein content in different buffer systems As shown in Table 15, the samples in the citrate / sodium citrate buffer system (pH 6.2), the samples in the disodium hydrogen phosphate / sodium dihydrogen phosphate buffer system at three pH values ​​(pH 6.0, 6.5, 7.0), and the samples in the acetate / sodium acetate buffer system at three pH values ​​(pH 4.5, 5.0, 5.5) showed nearly identical measurement results at different sampling points, indicating the stability of their protein content. The samples in the histidine / histidine hydrochloride buffer system at pH 5.5 and 6.0 changed over time under 25°C conditions, and the detection results were basically identical. After leaving the pH 6.5 sample at 25°C for 4 weeks, the detected protein content increased. After leaving the three pH values ​​(pH 5.5, 6.0, 6.5) of this buffer system at 40°C for 4 weeks, an increase in protein content was detected in all of them. This change may be due to the increase in UV 280 absorption value due to the large number of particles observed in the visual results mentioned above.

[0502] [Table 28] Note: W = period, T0 = start of analysis

[0503] 12.2.4 Turbidity (A350) Detection As shown in Table 16, the buffer system for each study showed little change in detection results across different study conditions and different sampling points.

[0504] [Table 29] Note: W = period, T0 = start of analysis

[0505] 12.2.5 Detection of proteolysis levels by SEC-HPLC As shown in Table 17, under 25°C conditions, the SEC results showed no change in the purity of the main peak in the disodium hydrogen phosphate / sodium dihydrogen phosphate buffer systems at pH 6.5 and pH 7.0 after 4 weeks of analysis. The main peak in the disodium hydrogen phosphate / sodium dihydrogen phosphate buffer system at pH 6.0 decreased by 38.0%. In the citrate / sodium citrate buffer system and histidine / histidine hydrochloride buffer system, the main peaks of the samples were almost undetectable after 4 weeks, but the fragment peaks after proteolysis continued to increase. In the acetic acid / sodium acetate buffer systems at pH 4.5 and pH 5.0, the main peak at T0 point was already below 40%, and was almost undetectable after 4 weeks. In the acetic acid / sodium acetate buffer system at pH 5.5, the main peak decreased by 70.5% after 4 weeks, and the fragment peaks after proteolysis also increased.

[0506] Under 40°C conditions, the SEC results showed that after 4 weeks, the purity of the main peak in the disodium hydrogen phosphate / sodium dihydrogen phosphate buffer systems at pH 6.5 and pH 7.0 remained unchanged, and the fragment peaks after proteolysis remained at low levels. The main peak of the sample in the citrate / sodium citrate buffer system decreased by 53.2% after 4 weeks. The main peaks of the sample in the histidine / histidine hydrochloride buffer system, the disodium hydrogen phosphate / sodium dihydrogen phosphate buffer system at pH 6.0, and the acetate / sodium acetate buffer system were almost undetectable after 4 weeks, but the fragment peaks after proteolysis increased.

[0507] The SEC results demonstrate good stability of YN-011 in disodium hydrogen phosphate / sodium dihydrogen phosphate buffer systems at pH 6.5 and pH 7.0.

[0508] [Table 30] Note: W = period, T0 = start of analysis, "ND" indicates no detection.

[0509] 12.2.6 Stability of proteins tested by RP-HPLC As shown in Table 18, under 25°C conditions, the reverse-phase HPLC results after 4 weeks showed the least decrease in main peak purity in the disodium hydrogen phosphate / sodium dihydrogen phosphate buffer systems at pH 6.5 and pH 7.0, at 3.5% and 1.9%, respectively, while the decrease in the main peak in the disodium hydrogen phosphate / sodium dihydrogen phosphate buffer system at pH 6.0 was 9.7%. The main peaks of the samples in the citrate / sodium citrate buffer system and the histidine / histidine hydrochloride buffer system were almost undetectable after 4 weeks. The main peak of the samples in the acetic acid / sodium acetate buffer system at pH 4.5 was undetectable after 4 weeks, while the main peaks of the samples in the acetic acid / sodium acetate buffer systems at pH 5.0 and 5.5 decreased by 51.6% and 25.3%, respectively, after 4 weeks.

[0510] Under 40°C conditions, the results of inversion HPLC showed that after 4 weeks, the decrease in main peak purity was smallest in the disodium hydrogen phosphate / sodium dihydrogen phosphate buffer systems at pH 6.5 and pH 7.0, decreasing by 10.3% and 7.8%, respectively. The main peak in the disodium hydrogen phosphate / sodium dihydrogen phosphate buffer system at pH 6.0 decreased by 50.6%. The main peak in the citrate / sodium citrate buffer system samples decreased by 27.4% after 4 weeks. The main peaks in the histidine / histidine hydrochloride buffer system samples at pH 5.5 and pH 6.0 were almost undetectable after 4 weeks, and the main peak in the histidine / histidine hydrochloride buffer system sample at pH 6.5 decreased by 27.4% after 4 weeks. The main peaks in the acetic acid / sodium acetate buffer system samples were almost undetectable after 4 weeks.

[0511] Reverse-phase HPLC results show good stability of YN-011 in disodium hydrogen phosphate / sodium dihydrogen phosphate buffer systems at pH 6.5 and pH 7.0.

[0512] [Table 31] Note: W = period, T0 = start of analysis, "ND" indicates no detection.

[0513] 12.2.7 Stability of cIEF test proteins As shown in Table 19, the cIEF results at 25°C showed no significant changes in the main peak, peak group A, and peak group B of the disodium hydrogen phosphate / sodium dihydrogen phosphate buffer systems at pH 6.5 and pH 7.0 after 4 weeks of observation. The main peak of the disodium hydrogen phosphate / sodium dihydrogen phosphate buffer system at pH 6.0 decreased by 10.6%, peak group A decreased by 13.3%, and peak group B increased by 24.0%. In the citrate / sodium citrate buffer system, histidine / histidine hydrochloride buffer system, and acetate / sodium acetate buffer systems at pH 4.5 and 5.0, the sample main peak and peak group A decreased to 0, while peak group B reached 100%. In the acetate / sodium acetate buffer system at pH 5.5, the sample main peak decreased by 20.8%, peak group A decreased by 29.5%, and peak group B increased by 50.4%.

[0514] At 40°C, cIEF results showed that after 4 weeks of observation, the main peak, peak group A, and peak group B of the sodium hydrogen phosphate / sodium dihydrogen phosphate buffer systems at pH 6.5 and pH 7.0 showed the least change. After 4 weeks, the main peak and peak group A of the pH 6.0 disodium hydrogen phosphate / sodium dihydrogen phosphate buffer system approached 0, while peak group B reached 96.1%. The main peak of the pH 6.5 disodium hydrogen phosphate / sodium dihydrogen phosphate buffer system decreased by 14.5%, peak group A decreased by 2.5%, and peak group B increased by 17.0%. The main peak of the pH 7.0 disodium hydrogen phosphate / sodium dihydrogen phosphate buffer system decreased by 15.3%, peak group A increased by 7.6%, and peak group B increased by 7.7%. In the citrate / sodium citrate buffer system, the main peak of the sample decreased by 17.6%, peak group A decreased by 6.2%, and peak group B increased by 23.6%. In the histidine / histidine hydrochloride buffer systems at pH 5.5 and pH 6.0, the main peak of the sample decreased to 0. In the histidine / histidine hydrochloride buffer system (pH 6.5), the main peak of the sample decreased by 28.9% after 4 weeks, peak group A decreased by 14.4%, and peak group B increased by 43.2%. After 4 weeks, the main peak and peak group A of the sample in the acetate / sodium acetate buffer system decreased to 0, and peak group B reached 100%.

[0515] [Table 32] Note: W = period, T0 = start of analysis, "ND" indicates no detection.

[0516] 12.2.8 Summary Considering the results of all the tests above, YN-011 exhibits good stability in disodium hydrogen phosphate / sodium dihydrogen phosphate buffer systems at pH 6.5 and pH 7.0. Reverse-phase HPLC results show that protein stability in the phosphate buffer system at pH 7.0 is slightly better than at pH 6.5, but the difference between the two is not clear. Therefore, the effect of the disodium hydrogen phosphate / sodium dihydrogen phosphate buffer system at pH 6.5-7.0 is relatively good.

[0517] 12.3 Excipient Screening 12.3.1 Excipient Screening Plan A 10 mM disodium hydrogen phosphate / sodium dihydrogen phosphate buffer system with a pH of 6.7 was selected and combined with five common medicinal excipients (mannitol, sucrose, sorbitol, sodium chloride, and glycine). Additionally, a 10 mM citrate / sodium citrate buffer system with a pH of 6.5 was selected and combined with mannitol. Eight different formulations were created to investigate the thermal stability and freeze-thaw stability of YN-011. See Table 20 for details. The YN-011 stock solution was replaced with a 10 mM disodium hydrogen phosphate / sodium dihydrogen phosphate buffer system with a pH of 6.7 and a 10 mM citrate / sodium citrate buffer system with a pH of 6.5. Appropriate excipients were added, the protein concentration was adjusted to 3 mg / mL, and 2 mL of the sample was filled into a 5 mL neutral boron silicon glass injection bottle with a plug-rolled cap. The samples were left at 25°C for 2 weeks and 4 weeks, and at 40°C for 1 week, 2 weeks, and 4 weeks. The samples underwent two and five freeze-thaw cycles from -40°C to room temperature, and samples were taken at different time points to perform each test, screening and evaluation of candidate excipients.

[0518] [Table 33]

[0519] 12.3.2 Appearance In visual observation for thermal stability, all YN-011 samples were colorless. In the citrate buffer system, the emulsion phenomenon was amplified in samples left at 25°C, and small amounts of particulate matter were observed in samples left at 40°C for 4 weeks, along with the generation of particulate matter. In the phosphate buffer system, all samples observed with each excipient and sampling point showed micro-emulsification. Formulations with added sodium chloride increased particulate matter, consistent with the control without excipients, but no particle generation was observed in phosphate formulations containing several other excipients.

[0520] In visual observation of the freeze-thaw experiment, all YN-011 samples were colorless and slightly opaque. In the citrate buffer system with added mannitol, samples after two freeze-thaw cycles produced a small amount of fine particles, and the number of particles increased after five freeze-thaw cycles. In the phosphate buffer system, formulations with added mannitol and glycine showed similar particle behavior. In the phosphate formulations containing sucrose, sorbitol, and sodium chloride, no particles were generated in samples after two freeze-thaw cycles, while samples after five freeze-thaw cycles produced a small amount of fine particles.

[0521] 12.3.3 Measurement results of pH value and protein content As shown in Tables 21 and 22, no significant changes in protein content or pH value were observed over time in the six formulations including excipients.

[0522] [Table 34] Note: W = period, T0 = start of analysis

[0523] [Table 35] Note: W = period, T0 = start of analysis

[0524] 12.3.4 Detection of proteolysis levels by SEC-HPLC As shown in Table 23, after 4 weeks of observation at 25°C, the main SEC peak in phosphate buffer systems containing different excipients did not decrease significantly. The sucrose-containing formulation showed the smallest decrease at 0.4%, while the glycine-containing formulation showed the largest decrease at 1.2%. The main SEC peak in the citrate buffer system without excipients decreased by 0.2%, while the main SEC peak in the citrate buffer system containing mannitol decreased by 2.2%.

[0525] After four weeks of observation under 40°C conditions, a clear decrease in the SEC main peak was observed in phosphate buffer systems containing sorbitol and glycine, at 2.0% and 3.1%, respectively. The decrease in the SEC main peak in the phosphate buffer system containing mannitol was the smallest, at 0.9%. The SEC main peak in the citrate buffer system without excipients decreased by 0.5%, while the SEC main peak in the citrate buffer system containing mannitol decreased by 1.0%.

[0526] After five freeze-thaw cycles during the freeze-thaw experiment, the decrease in the SEC main peak was most pronounced in the phosphate buffer system containing sodium chloride, decreasing by 2.6%. The smallest decrease in the SEC main peak was observed in the glycine-containing phosphate formulation, which decreased by 0.5%. In the phosphate formulations containing sucrose, mannitol, and sorbitol, the main peak decreased by 0.8% after five freeze-thaw cycles. The SEC main peak in the citrate buffer system without excipients decreased by 2.0% after five freeze-thaw cycles, while the SEC main peak in the citrate buffer system containing mannitol decreased by 1.2%.

[0527] [Table 36] Note: W = period, T0 = start of analysis, "ND" indicates no detection.

[0528] 12.3.5 Stability of RP-HPLC-tested proteins As shown in Table 24, after 4 weeks of observation at 25°C, the phosphate-buffered system showed the most significant decrease in the RP main peak for formulations containing glycine, decreasing by 3.9%. Formulations without excipients showed the smallest decrease at 1.3%, while formulations with other excipients decreased by 1.5% for mannitol, 1.8% for sucrose, 1.8% for sorbitol, and 1.5% for sodium chloride. Compared to the phosphate-buffered system, the citrate-buffered system showed a significant decrease in the RP main peak, decreasing by 72.6% for formulations without excipients and by 9.4% for the citrate-buffered system containing mannitol.

[0529] After 4 weeks of observation at 40°C, the phosphate-buffered system showed the most significant decrease in the RP main peak for formulations containing glycine, at 20.3%. Formulations without excipients showed the lowest decrease in the main peak, at 6.1%. For formulations containing other excipients, the RP main peak decreased by 7.6% for mannitol, 8.2% for sucrose, 9.3% for sorbitol, and 6.7% for sodium chloride. In the citrate-buffered system, the RP main peak of formulations without excipients decreased by 6.1%, while the RP main peak of the citrate-buffered system containing mannitol decreased by 7.3%.

[0530] In the freeze-thaw experiment, no significant changes were observed in the RP peak of each formulation.

[0531] [Table 37] Note: W = period, T0 = start of analysis

[0532] 12.3.6 SDS_CALIPER_NR purity (%) measurement results As shown in Table 25, in non-reducing SDS-Caliper detection, no significant change in the purity of each formulation was observed after 4 weeks under 25°C conditions. After 4 weeks under 40°C conditions, the decrease in purity was most significant in the phosphate formulations containing glycine and sodium chloride, decreasing by 2.6% and 1.9%, respectively, while no significant change in the purity of the remaining formulations was observed. No significant change in the purity of each formulation was observed in the freeze-thaw experiment.

[0533] [Table 38] Note: W = period, T0 = start of analysis

[0534] 12.3.7 cIEF Detection Stability As shown in Table 26, after 4 weeks of observation under 25°C conditions, there was no change in the isoelectric point of each formulation. There was a significant decrease in the two main peaks of the citrate buffer system formulations, while there was no apparent change in the main peaks of the remaining formulations. After 4 weeks of observation under 40°C conditions, there was no change in the isoelectric point of each formulation, but all main peaks clearly decreased. Among these, the decrease in the main peak of the glycine-containing phosphate formulation was the most significant, decreasing by 18.0%, while the decrease in the main peak of the mannitol-containing citrate buffer system was the smallest, decreasing by 5.3%. In the freeze-thaw experiment, there was no apparent change in the main peaks of each formulation.

[0535] [Table 39] Note: W = period, T0 = start of analysis, "ND" indicates no detection.

[0536] 12.3.8 Summary YN-011 stabilizes other systems in a phosphate buffer system containing sucrose and mannitol.

[0537] 12.4 Screening of Surfactants Screening revealed that surfactants commonly used in injectable drugs, such as Twain 80 (polysorbate 80), polyoxyethylene castor oil derivatives, poloxamer, lecithin, polyethylene glycol 15-hydroxystearate ester, and cyclopaste extracts, can all be applied to this drug. As a surfactant, Twain 80 (polysorbate 80) is a stabilizer commonly used in injectable drugs. In this example, the concentration of Twain 80 was optimized.

[0538] 12.4.1 Optimized design for exhalation temperature of 80°C Based on the results of buffer system screening, a 10 mM disodium hydrogen phosphate / sodium dihydrogen phosphate buffer system at pH 6.7 was selected and combined with four different Emmodium 80 concentrations (0, 0.01%, 0.02%, 0.04%) to determine the optimal Emmodium 80 concentration for YN-011. See Table 27 for specific procedures. The YN-011 stock solution was replaced with a 10 mM disodium hydrogen phosphate / sodium dihydrogen phosphate buffer system at pH 6.7, Emmodium 80 mother liquor was added, and the protein concentration was adjusted to 3 mg / mL by adding the appropriate phosphate buffer. 2 mL of the sample was filled into a 5 mL neutral boron silicon glass injection bottle and sealed with a rolled cap. The samples were left at 40°C for 1, 2, and 4 weeks, and the samples were also shaken for 1 and 2 days in a rocker set to 25°C and 300 rpm. Samples were taken at different time points and each test was performed to screen and evaluate the four Emmodium 80 concentrations.

[0539] [Table 40]

[0540] 12.4.2 Appearance Visual observation revealed that all YN-011 samples were colorless and slightly opaque. In formulations without Twain 80, a very small amount of flocculated protein particles were observed under shaking conditions, and a very small amount of protein particles were observed after being left at 40°C for 4 weeks. In formulations containing 0.01% Twain 80, a very small amount of protein particulate matter was observed after being left at 40°C for 4 weeks, and no particulate matter was observed under vibration conditions. Formulations containing 0.02% and 0.04% Twain 80 showed no change in appearance.

[0541] 12.4.3 pH Value and Protein Content In the four formulations containing different concentrations of Twain 80, there were no significant changes in pH value or protein content under 40°C and vibration conditions.

[0542] 12.4.4.4 Detection of proteolysis levels by SEC-HPLC As shown in Table 28, after 2 days under vibration conditions, the main peak purity of the formulation without emetic heat 80 decreased by 1.8%, and polymer and fragment increased by 0.9%, respectively. However, the SEC purity of the three formulations containing emetic heat 80 remained unchanged. Under 40°C observation conditions, the SEC purity of all formulations decreased. After 4 weeks, the SEC purity of the formulations without emetic heat 80 decreased by 1.0% and 0.9%, respectively, while the SEC purity of the formulations containing 0.02% and 0.04% emetic heat 80 decreased by 0.8%.

[0543] [Table 41] Note: D = Day, W = Week, T0 = Start of analysis, "ND" indicates no detection.

[0544] 12.4.5 SDS_CALIPER_NR purity measurement results As shown in Table 29, no significant changes were observed in the purity of the unreduced SDS-Caliper for each formulation under vibrational conditions. After 4 weeks of observation under 40°C conditions, the purity of formulations containing 0.01%, 0.02%, and 0.04% Twain 80 decreased by 1.2%, 1.2%, and 1.3%, respectively.

[0545] [Table 42] Note: D = Day, W = Week, T0 = Start of analysis

[0546] 12.4.6 Stability of RP-HPLC-tested proteins As shown in Table 30, under vibrational conditions, the main RP peak of the formulation without emetic 80 decreased by 1.2%, but the main RP peak of the three formulations containing emetic 80 remained unchanged. Under 40°C observation conditions, the main RP peak of each formulation decreased. After 4 weeks, the RP purity of the formulation without emetic 80 decreased by 6.1%, while the main RP peaks of the formulations containing 0.01%, 0.02%, and 0.04% emetic 80 decreased by 8.1%, 8.5%, and 8.6%, respectively.

[0547] [Table 43] Note: D = Day, W = Week, T0 = Start of analysis

[0548] 12.4.7 cIEF Detection Stability As shown in Table 31, no significant changes were observed in any of the components of cIEF under vibrational conditions. Under the 40°C observation conditions, the main peak and peak group B of each formulation decreased, while peak group A increased, and there were no clear differences between the various points.

[0549] [Table 44] Note: D = Day, W = Week, T0 = Start of analysis, "ND" indicates no detection.

[0550] 12.4.8 Summary of Optimization of Emission Temperature 80 Concentration Experiments optimizing the concentration of Embryon 80 revealed that adding Embryon 80 to the formula is necessary to enhance protein stability under oscillating conditions. There was no significant difference in the effect of Embryon 80 concentrations of 0.01%, 0.02%, and 0.04% on protein stability. Considering potential errors that may occur in future production processes, an Embryon 80 concentration of 0.02% was selected.

[0551] Example 13: Stability test of pharmaceutical formulations 13.1 Method of manufacturing the formulation Based on the excipient compositions shown in Table 32, 0.5 mL / branched YN-011 formulations were prepared with YN-011 protein content of 1 mg, 2 mg, and 3 mg, respectively (shown in Table 34). Based on the excipient compositions shown in Table 33, 0.75 mL / branched YN-011 formulations were prepared with YN-011 protein content of 4 mg, 5 mg, 7.5 mg, and 9 mg, respectively (shown in Table 34). Long-term stability tests were then performed on each of the prepared formulations according to the testing method described in Example 13.2.

[0552] [Table 45]

[0553] [Table 46]

[0554] [Table 47]

[0555] The manufacturing methods for each formulation are briefly explained below: (1) Preparation of buffer solution: Based on the content shown in Tables 32 and 33, weigh out disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate monohydrate, mannitol, and polysorbate 80, dissolve each weighed additive in water for injection, and filter to remove sterilization. (2) Thawing of undiluted solution: Thaw the undiluted solution in a water bath at 18°C ​​to 26°C, avoiding light. (3) Dilution and mixing of stock solution: Mix the stock solution after thawing in step (2) and the buffer solution arranged in step (1) in a constant proportion by stirring. (4) Sterilization filtration: Sterilize and filter the chemical solution prepared in step (3) into a sterile container. (5) Dispensing: Dispense the drug solution prepared in step (4) in the amount corresponding to the target specifications.

[0556] 13.2 Detection Method 13.2.1 Exterior Approximately 1 ml of the sample was placed in a clean colorimetric tube, and approximately 1 ml of ultrapure water was placed in a clean colorimetric tube as a control. Under normal laboratory lighting, the sample and ultrapure water were placed against a white background and their colors were compared horizontally. Under a transparency meter, the light intensity was adjusted to 1000 lx, and the transparency of the sample and ultrapure water was observed by horizontal contrast under a black background. Visible particles were detected using visual methods.

[0557] 13.2.2 Photoresist Method Using an insoluble particulate detector, the insoluble particulate matter of the sample was measured according to the instrument's operating regulations, with at least four measurements taken, each time sampling of 5 ml or more. The data from the first measurement was discarded, and the average of the subsequent three measurements was used as the measurement result. The number of particulate matter contained in each container was calculated from the sampling volume and the display volume for each container.

[0558] 13.2.3 pH value Using the electropotential method, the pH meter was calibrated with a standard solution. A 0.2 ml sample was then taken, measured three times in parallel, and the measured pH values ​​were recorded. The test results were reported according to the average value.

[0559] 13.2.4 Osmotic pressure After calibrating the instrument using a freezing point osmometer, the osmotic molar concentration of the sample was measured according to the operating procedure. Before measuring the sample, the sample was mixed, the integrity of the sample tube was checked, and 50 μl of the sample was collected using a pipette into a clean, dried sample tube, and its osmotic molar concentration was measured. Each sample was tested three times in parallel, and the test results were reported based on the average value.

[0560] 13.2.5 icIEF method The Protein Simple Maurice C capillary electrophoresis system separates charge heteromers in each sample according to their isoelectric point differences. The instrument parameters are set as follows: prefocus: 1500V, 1 minute; focus: 3000V, 8 minutes; injection time: 90 seconds; UV detection wavelength: 280nm; sample plate temperature: 15°C. Two types of labels with known isoelectric points (pI makers) are added to the sample and focused simultaneously to determine the isoelectric point of the sample being measured. The spectrum is derived, the isoelectric point of the main peak of the sample is reported, and the peak area percentages of the main peak, Group A, and Group B are reported. The main peak content, Group A content, and Group B content can be obtained using the peak area normalization method.

[0561] 13.2.6 SEC-HPLC method The purity of YN-011 protein was measured using molecular exclusion chromatography with Thermo UltiMate 3000 high-efficiency liquid chromatography. TSKgelG 3000 column (5 μm, 7.8 × 300 mm); flow rate: 0.5 ml / min, injection volume: 25 μL, column temperature: 25 ± 3 °C, injection plate temperature: 5 ± 3 °C, detection wavelength: 280 nm, sampling time: 35 min, elution method: isoelution. Mobile phase A: 50 mmol / L phosphate buffer, 150 mmol / L NaCl, 500 mmol / L-arginine hydrochloride, 10% IPA, pH 6.3 ± 0.1.

[0562] 13.2.7 Non-reducing CE-SDS method (non-reducing sodium dodecyl sulfate capillary electrophoresis) The purity of YN-011 protein was measured using non-reducing sodium dodecyl sulfate capillary electrophoresis with a Beckman PA 800 Plus capillary electrophoresis system, SDS-MW Gel Buffer purchased from SCIEX, and an uncoated capillary. The system was set up according to the instrument's standard operating procedures. The output spectrum was integrated, and protein purity was calculated based on peak area normalization. For non-reducing samples, protein purity was expressed as the main peak area percentage, reporting the LMW and HMW content respectively. The LMW content is the sum of the corrected peak area percentages to the left of the main peak, and the HMW content is the sum of all corrected peak area percentages to the right of the main peak.

[0563] 13.2.8 Reduced CE-SDS method (Reduced sodium dodecyl sulfate capillary electrophoresis) The purity of YN-011 protein was measured using reduced sodium dodecyl sulfate capillary electrophoresis with a Beckman PA 800 Plus capillary electrophoresis system, SDS-MW Gel Buffer purchased from SCIEX, and an uncoated capillary. The system was set up according to the instrument's standard operating procedures. The output spectrum was integrated, and protein purity was calculated based on peak area normalization. For reduced samples, protein purity was expressed as the principal peak area percentage, and the protein purity and the content of "Others" were reported, where "Others" is the sum of the corrected peak area percentages of the remaining peaks excluding the principal peak.

[0564] 13.2.9 UV method (ultraviolet spectrophotometry) Using ultraviolet spectrophotometers (UV-1900, UV-1900i) purchased from Shimadzu, the YN-011 protein concentration was measured using ultraviolet spectrophotometric methods. Both photometric and spectral measurements were performed, and the wavelength at 280 nm was measured. The reported protein content value is the average of two parallel preparation tests of the sample, and the reporting unit is mg / mL.

[0565] 13.2.10 Biological activity testing The biological activity of the sample is determined by detecting cAMP activity. A cAMP reservoir was prepared, and the amount of cAMP produced in CHO K1 cells was measured using the cAMP-Gs Dynamic kit. The procedure is summarized below. • Sample preparation: Add the diluted sample to a 96-well cell culture plate at a rate of 20 μl / well according to the experimental setup. • Cell processing: Using a splatter gun, adjust the CHO K 1 cell density to 7.5 x 10⁵ cells / ml. Add the cell solution to rows 1-11 of a 96-well cell culture plate, 20 μl per well. • Cell and sample incubation: Centrifuge the reaction plate at 300 rpm for 30 seconds, then incubate at 25±2°C for 30±8 minutes. • Chromogenicity: Add 40 μl / well of a mixture of cAMP antibody, cAMP-d 2, and cAMP lysis buffer to a 96-well cell culture plate for 60 ± 9 minutes. • Reading board, • Data processing:

[0566]

number

[0567] 13.3 Test Results 13.3.1 Physicochemical Identification The isoelectric point was measured using the icIEF method, and in long-term stability studies under 2-8°C conditions, all formulations maintained a main peak isoelectric point within 6.2 ± 0.2 within 24 months of the start of the test, matching the working reference sample and meeting the specifications.

[0568] Visual inspection revealed that all formulation samples were colorless, transparent liquids. Visible foreign matter was detected using a visible foreign matter detection method, and all formulations met the specifications. Insoluble particulate matter was detected using a photoresistance method, and all formulations met the specifications.

[0569] During the sampling period, the pH values ​​of all samples remained stable within the range of 6.7 ± 0.3, indicating relatively good pH stability of the formulation.

[0570] The osmotic molar concentration of the samples was measured using an osmotic molar concentration measurement method. Within the sampling time, the osmotic molar concentration of all samples stabilized within the range of 290 ± 50 mOsmol / kg, indicating that the osmotic pressure of the formulations is relatively stable.

[0571] 13.3.2 Measurement of purity and charge variants Tables 35, 36, and 37 show the proteolysis levels of the formulations detected by SEC-HPLC, non-reducing CE-SDS, and reducing CE-SDS within the sampling time, respectively. All samples showed a main peak purity of over 95%.

[0572] [Table 48]

[0573] [Table 49]

[0574] [Table 50]

[0575] The proportion of the main peak measured for all formulations measured using the MICIEF method was greater than 46%, and as shown in Table 38, there was little change within the sampling interval.

[0576] [Table 51]

[0577] 13.3.3 Biological Value As shown in Table 39, relative biological activity was determined by detecting cAMP activity, and all test samples showed relative biological activity of 85% to 121%, meeting the relevant quality standards.

[0578] [Table 52]

[0579] 13.3.4 Protein Concentration As shown in Table 40, the protein concentration was determined by UV spectroscopy, and the concentrations of all test samples were within the standard range, indicating relatively good stability.

[0580] [Table 53]

[0581] The above examples enumerate what is currently considered to be ...

Claims

1. Pharmaceutical preparations containing the following ingredients: (a) GLP-1 fusion protein, the GLP-1 fusion protein comprises a GLP-1 polypeptide and an immunoglobulin Fc domain, the GLP-1 polypeptide is covalently bonded to the immunoglobulin Fc domain, the GLP-1 polypeptide is selected from human GLP-1(7-37), human GLP-1(7-36) amide, and DPP-IV resistant human GLP-1, the GLP-1 polypeptide contains one or more amino acid substitutions selected from the group consisting of A8G, G22E, and R36G compared to natural human GLP-1, the immunoglobulin Fc domain contains an IgG2-Fc domain or is an IgG2-Fc domain, the IgG2-Fc domain contains one or more amino acid substitutions selected from the group consisting of C222S, A330S, and P331S, (b) buffer solution, where the pH value of the pharmaceutical formulation is in the range of approximately 6.0 to approximately 7.

0.

2. Pharmaceutical preparations including the following: (a) GLP-1 fusion protein, the fusion protein contains a GLP-1 polypeptide and an immunoglobulin Fc domain, the GLP-1 polypeptide is covalently bound to the immunoglobulin Fc domain, The GLP-1 polypeptide is selected from human GLP-1(7-37), human GLP-1(7-36) amide, and DPP-IV resistant human GLP-1, and the GLP-1 polypeptide contains one or more amino acid substitutions selected from the following groups relative to natural human GLP-1: A8G, G22E, and R36G; The immunoglobulin Fc domain contains an IgG2-Fc domain or is an IgG2-Fc domain, and the IgG2-Fc domain contains one or more amino acid substitutions selected from the group C222S, A330S, and P331S; and (b) Buffering agent. The buffering agent is selected from the group consisting of phosphate buffering agents, citrate buffering agents, borate buffering agents, histidine buffering agents, and acetate buffering agents.

3. A pharmaceutical formulation according to claim 1 or 2, wherein the buffer is a phosphate buffer.

4. In the drug formulation according to claim 2 or 3, the phosphate buffer comprises a dihydrogen phosphate (e.g., disodium hydrogen phosphate, dipotassium hydrogen phosphate, etc.), dihydrogen phosphate (e.g., sodium dihydrogen phosphate, potassium dihydrogen phosphate, etc.), or a combination thereof.

5. A pharmaceutical preparation according to any one of claims 2 to 4, wherein the phosphate buffer is a disodium hydrogen phosphate / sodium dihydrogen phosphate buffer.

6. A pharmaceutical preparation according to any one of claims 2 to 5, wherein the phosphate buffer is prepared from disodium hydrogen phosphate hydrate and sodium dihydrogen phosphate hydrate.

7. A pharmaceutical preparation according to any one of claims 2 to 6, wherein the phosphate buffer is prepared from disodium hydrogen phosphate dodecahydrate and sodium dihydrogen phosphate monohydrate.

8. A pharmaceutical preparation according to any one of claims 2 to 7, wherein the concentration of phosphate buffer in the pharmaceutical preparation is about 5 mM to about 15 mM (for example, about 10 mM).

9. A pharmaceutical preparation according to any one of claims 1 to 4, wherein the pharmaceutical preparation further comprises a carbohydrate.

10. A pharmaceutical formulation according to claim 9, wherein the carbohydrate is selected from the group consisting of mannitol, sorbitol, maltitol, erythritol, arabitol, xylitol, sucrose, lactose, trehalose, dextran, and combinations thereof.

11. A pharmaceutical preparation according to any one of claims 9 to 10, wherein the carbohydrate is one or more selected from mannitol, sucrose, and sorbitol, for example, the pharmaceutical preparation in which the carbohydrate is mannitol.

12. A pharmaceutical preparation according to any one of claims 9 to 11, wherein the concentration of carbohydrates in the pharmaceutical preparation is 1 to 10% (w / v), for example, about 4.6% (w / v).

13. A pharmaceutical preparation according to any one of claims 1 to 11, further comprising a surfactant.

14. A pharmaceutical formulation according to claim 13, wherein the surfactant is selected from the group consisting of polysorbate (e.g., polysorbate 80), polyoxyethylene castor oil derivatives, poloxamer (e.g., poloxamer 188), lecithin, polyethylene glycol 15-hydroxystearate, cyclodextrin, and combinations thereof.

15. A pharmaceutical preparation according to any one of claims 13 to 14, wherein the surfactant is polysorbate 80.

16. A pharmaceutical preparation according to any one of claims 13 to 15, wherein the concentration of the surfactant in the pharmaceutical preparation is 0.01% (w / v) to 0.04% (w / v), for example, about 0.02% (w / v).

17. A pharmaceutical formulation according to any one of claims 1 to 15, wherein the pharmaceutical formulation comprises a GLP-1 fusion protein, a phosphate buffer, a carbohydrate, and a surfactant.

18. A pharmaceutical formulation according to any one of claims 1 to 15, wherein the pharmaceutical formulation comprises a GLP-1 fusion protein, a phosphate buffer (e.g., disodium hydrogen phosphate / sodium dihydrogen phosphate buffer), mannitol, and polysorbate.

19. A pharmaceutical formulation according to any one of claims 1 to 19, wherein the GLP-1 polypeptide in the GLP-1 fusion protein has a specific level of hydroxylation at lysine 34 (K34) compared to natural human GLP-1.

20. A pharmaceutical preparation according to claim 19, wherein the hydroxylation level is 10% to 100%, for example, 10% or more, 15% or more, 20% or more, 26% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more.

21. A pharmaceutical formulation according to any of the claims, wherein the GLP-1 polypeptide in the GLP-1 fusion protein is substantially less oxidized at the tryptophan position 31 (W31) compared to natural human GLP-1.

22. A pharmaceutical formulation according to any one of the above claims, wherein the oxidation level of the GLP-1 polypeptide in the GLP-1 fusion protein in W31 is less than 0.5% or undetectable compared to natural human GLP-1.

23. A pharmaceutical formulation according to any one of the above claims, wherein the GLP-1 polypeptide in the GLP-1 fusion protein has at least 90% sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, and comprises one or more amino acid substitutions selected from the group consisting of A8G, G22E, and R36G compared to natural human GLP-1.

24. A pharmaceutical formulation according to any one of the above claims, wherein the GLP-1 polypeptide in the GLP-1 fusion protein is selected from human GLP-1(7-37), human GLP-1(7-36) amide, and DPP-IV resistant human GLP-1, and comprises A8G and G22E substitutions relative to natural human GLP-1.

25. A pharmaceutical formulation according to any one of the above claims, wherein the GLP-1 polypeptide in the GLP-1 fusion protein is selected from human GLP-1(7-37), human GLP-1(7-36) amide, and DPP-IV resistant human GLP-1, and comprises A8G, G22E, and R36G substitutions relative to natural human GLP-1.

26. A pharmaceutical preparation according to any one of the above claims, wherein the GLP-1 polypeptide in the GLP-1 fusion protein is human GLP-1 (7-37), and comprises A8G, G22E and R36G substitutions compared to natural human GLP-1.

27. A pharmaceutical preparation according to any one of the above claims, wherein the amino acid sequence of the GLP-1 polypeptide in the GLP-1 fusion protein is as shown in SEQ ID NO:

3.

28. A pharmaceutical preparation according to any one of the above claims, wherein the IgG2-Fc domain in the GLP-1 fusion protein is an Fc domain derived from human IgG2.

29. A pharmaceutical preparation according to any one of the above claims, wherein the IgG2-Fc domain in the GLP-1 fusion protein has at least 90% sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 5 or SEQ ID NO: 6, and includes one or more amino acid substitutions selected from the group consisting of C222S, A330S, and P331S.

30. A pharmaceutical preparation according to any one of the above claims, wherein the IgG2-Fc domain in the GLP-1 fusion protein has at least 90% sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 5 or SEQ ID NO: 6, and includes A330S and P331S substitutions.

31. A pharmaceutical formulation according to any one of the above claims, wherein the IgG2-Fc domain in the GLP-1 fusion protein further comprises a C222S substitution.

32. A pharmaceutical preparation according to any of the above claims, wherein the amino acid sequence of the IgG2-Fc domain in the GLP-1 fusion protein is as shown in Sequence ID No.

6.

33. A pharmaceutical preparation according to any of the above claims, wherein the amino acid sequence of the GLP-1 polypeptide in the GLP-1 fusion protein is as shown in SEQ ID NO: 3, and the amino acid sequence of the immunoglobulin Fc domain is as shown in SEQ ID NO:

6.

34. A pharmaceutical formulation according to any of the above claims, wherein in the GLP-1 fusion protein, the GLP-1 polypeptide is located at the N-terminus or C-terminus of the immunoglobulin Fc domain.

35. A pharmaceutical preparation according to any one of claims 1 to 35, wherein in the GLP-1 fusion protein, the GLP-1 polypeptide is directly covalently bound to the immunoglobulin Fc domain.

36. A pharmaceutical formulation according to any one of claims 1 to 35, wherein in the GLP-1 fusion protein, the GLP-1 polypeptide is covalently bound to the immunoglobulin Fc domain via a linker.

37. A pharmaceutical preparation according to claim 36, wherein the linker is selected from the group consisting of a severable linker, an inseverable linker, a flexible linker, a rigid linker, a helical linker, and a non-helical linker.

38. A pharmaceutical preparation according to any one of claims 36 to 37, wherein the linker comprises a linker peptide that links a GLP-1 polypeptide and an IgG2-Fc domain.

39. A pharmaceutical preparation according to claim 38, wherein the linker peptide comprises a linker containing glycine and serine.

40. A pharmaceutical formulation according to claim 39, wherein the linker comprising glycine and serine comprises 1, 2, 3, 4 or more repetitions as shown in SEQ ID NO: 39 (GGGS), SEQ ID NO: 40 (GGGGGS), SEQ ID NO: 41 (GGGGGGGS), or SEQ ID NO: 42 (GGGGGGGGGS).

41. A pharmaceutical preparation according to any one of claims 36 to 40, wherein the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 9, SEQ ID NOs: 10, SEQ ID NOs: 11, SEQ ID NOs: 12, SEQ ID NOs: 13, SEQ ID NOs: 14, SEQ ID NOs: 15, SEQ ID NOs: 16, SEQ ID NOs: 17, SEQ ID NOs: 18, and SEQ ID NOs:

19.

42. A pharmaceutical preparation according to claim 41, wherein the linker comprises the amino acid sequence shown in SEQ ID NO:

9.

43. A pharmaceutical preparation according to any one of claims 36 to 42, wherein the amino acid sequence of the GLP-1 polypeptide is as shown in SEQ ID NO: 3, the amino acid sequence of the immunoglobulin Fc domain is as shown in SEQ ID NO: 6, and the amino acid sequence is as shown in SEQ ID NO:

7. The amino acid sequence of the linker is shown in SEQ ID NO:

9.

44. A pharmaceutical preparation according to any one of the above claims, wherein the GLP-1 fusion protein has the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence having at least 80% sequence identity with SEQ ID NO:

7.

45. A pharmaceutical preparation according to any one of the above claims, wherein the oxidation level of the IgG2-Fc domain in M253 corresponding to SEQ ID NO: 7 is 5% or less.

46. A pharmaceutical formulation according to any of the above claims, wherein the half-life of the GLP-1 fusion protein in a human subject is at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, or at least 14 days.

47. A pharmaceutical formulation according to any of the above claims, wherein in the GLP-1 fusion protein, 10% or more, or 15% or more, or at least 20% or more, or 26% or more, or 30% or more, or 40% or more, or 50% or more, or 60% or more, or 70% or more, or 80% or more, or 90% or more of the GLP-1 polypeptide in the fusion protein is hydroxylated with K34 compared to natural human GLP-1.

48. A pharmaceutical preparation according to any one of the claims, wherein the concentration of GLP-1 fusion protein in the pharmaceutical preparation is 0.2 to 20 mg / mL (for example, 1 to 5 mg / mL).

49. A pharmaceutical preparation according to any one of the claims, wherein the pharmaceutical preparation comprises: (a) GLP-1 fusion protein, where the GLP-1 fusion protein has the amino acid sequence shown in SEQ ID NO:

7. (b) Disodium hydrogen phosphate / sodium dihydrogen phosphate buffer, (c) Mannitol, and (d) Polysorbate 80.

50. A pharmaceutical preparation according to any one of the claims, wherein the pharmaceutical preparation comprises: (a) 0.2 to 20 mg / mL of GLP-1 fusion protein, where the GLP-1 fusion protein has the amino acid sequence shown in SEQ ID NO:

7. (b) 5-15 mM disodium hydrogen phosphate / sodium dihydrogen phosphate buffer, (c) 1-10% (w / v) mannitol, and (d) 0.01% (w / v) to 0.04% (w / v) polysorbate 80.

51. A pharmaceutical preparation according to any one of the above claims, wherein the pharmaceutical preparation comprises: (a) GLP-1 fusion protein in an amount of approximately 2 mg / mL, approximately 4 mg / mL, or approximately 6 mg / mL (the amino acid sequence of the GLP-1 fusion protein is as shown in SEQ ID NO: 7), (b) Approximately 10 mM disodium hydrogen phosphate / sodium dihydrogen phosphate buffer, (c) Approximately 4.6% (w / v) mannitol, and (d) Approximately 0.02% (w / v) polysorbate 80.

52. A pharmaceutical preparation according to any one of the claims, wherein the pharmaceutical preparation comprises: (a) GLP-1 fusion protein in concentrations of approximately 5.3 mg / mL, 6.67 mg / mL, 10 mg / mL, 12 mg / mL, 13.3 mg / mL, or 20 mg / mL (the amino acid sequence of the GLP-1 fusion protein is as shown in SEQ ID NO: 7); (b) Approximately 10 mM disodium hydrogen phosphate / sodium dihydrogen phosphate buffer; (c) Approximately 4.6% (w / v) mannitol; and (d) Approximately 0.02% (w / v) polysorbate 80.

53. A pharmaceutical preparation according to any one of the claims, wherein the unit dose of the pharmaceutical preparation is about 0.5 mL, and comprises the following: (a) Approximately 1 mg, 2 mg, or 3 mg of GLP-1 fusion protein (the amino acid sequence of the GLP-1 fusion protein is as shown in SEQ ID NO: 7); (b) Approximately 0.58 mg of disodium hydrogen phosphate dodecahydrate; (c) Approximately 0.465 mg of sodium dihydrogen phosphate monohydrate; (d) Approximately 23.2 mg of mannitol; and (e) Approximately 0.1 mg of polysorbate 80 (the remainder being sterile water for injection).

54. A pharmaceutical preparation according to any one of the above claims, wherein the unit dose of the pharmaceutical preparation is about 0.75 mL, and comprises the following components: (a) Approximately 4 mg, 5 mg, 7.5 mg, 9 mg, 10 mg, or 15 mg of GLP-1 fusion protein (the amino acid sequence of the GLP-1 fusion protein is as shown in SEQ ID NO: 7) (b) Approximately 0.87 mg of disodium hydrogen phosphate dodecahydrate (c) Approximately 0.6975 mg of sodium dihydrogen phosphate monohydrate (d) Approximately 34.8 mg of mannitol (e) Approximately 0.15 mg of polysorbate 80 The rest is water for injection.

55. A pharmaceutical preparation according to any one of the claims, wherein the pH value is in the range of 6.4 to 7.

0.

56. A pharmaceutical preparation according to any one of the claims, which is isotonic.

57. A pharmaceutical preparation according to any one of the claims, which is a liquid preparation.

58. A pharmaceutical preparation according to any one of the claims, which is administered by intravenous, subcutaneous, or intramuscular injection.

59. A pharmaceutical preparation according to any one of the claims, which is stable at 25±2°C for at least 3 months, or at 40±2°C for at least 2 weeks, or at 2-8°C for at least 24 months.

60. A pharmaceutical preparation according to any of the above claims, wherein the pH value of the preparation does not change to about 10% or less (for example, about 5% or less, about 4% or less, about 3% or less, about 2% or less, about 1% or less) after being left standing for at least one month.

61. A pharmaceutical formulation according to any of the above claims, wherein, after the formulation is left standing for at least one month, the concentration of the GLP-1 fusion protein does not change by approximately 10% or less (for example, approximately 5% or less, approximately 4% or less, approximately 3% or less, approximately 2% or less, approximately 1% or less).

62. A pharmaceutical preparation according to any of the above claims, wherein, after leaving the preparation for at least one month, the content of a high molecular weight (HMW) derivative or a low molecular weight (LMW) derivative does not exceed about 10% (for example, about 5% or less, about 4% or less, about 3% or less, about 2% or less, about 1% or less, etc.).

63. A pharmaceutical formulation according to any of the above claims, wherein the amount of the HMW derivative and / or LMW derivative is determined by size exclusion chromatography (SEC) or reversed-phase liquid chromatography (RP-LC).

64. A pharmaceutical preparation according to any of the above claims, wherein the purity of the preparation does not change by more than about 5% after being left for at least one month.

65. A pharmaceutical preparation according to any one of the claims, wherein the purity of the preparation is determined by SDS capillary electrophoresis (for example, non-reducing SDS capillary electrophoresis).

66. A pharmaceutical preparation according to any one of the claims, wherein, after being left for at least one month, the isoelectric point of the preparation does not change by more than about 5% (for example, about 4% or less, about 3% or less, about 2% or less, about 1% or less, etc.).

67. A pharmaceutical preparation according to any one of the claims, wherein the isoelectric point of the preparation is determined by capillary focusing isoelectric electrophoresis (cIEF).

68. Use of a pharmaceutical preparation according to any one of the claims in the manufacture of a medicinal product for the treatment or prevention of a disease.

69. The use according to claim 68, wherein the disease is selected from the group consisting of metabolic diseases related to disorders of glucose metabolism and / or lipid metabolism, complications of metabolic diseases, and neurological diseases and other related diseases.

70. The use according to claim 69, wherein the metabolic disorder related to impaired glucose metabolism and / or lipid metabolism is selected from the group consisting of diabetes mellitus, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), obesity, and metabolic syndrome.

71. Use according to claim 69 or 70, wherein the metabolic disorder associated with impaired glucose and / or lipid metabolism is diabetes mellitus (e.g., type 2 diabetes mellitus, type 2 diabetes mellitus with poor glycemic control after dietary and exercise therapy interventions).

72. The use according to claim 69, wherein the complications of metabolic disease include cardiovascular complications, renal complications, or hepatic complications caused by the metabolic disease.

73. The use according to claim 69, wherein the nervous system disease is a neurodegenerative disease.

74. The use according to claim 73, wherein the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, motor neuron disease, Huntington's disease, and Parkinson's disease.

75. The pharmaceutical preparation according to any one of claims 1 to 67, and the use of additional therapeutic agents in the preparation of a pharmaceutical for treating or preventing a disease.

76. The use according to claim 75, wherein the disease is selected from the group consisting of metabolic diseases related to disorders of glucose metabolism and / or lipid metabolism, complications of metabolic diseases, and neurological diseases and other related diseases.

77. The use according to claim 76, wherein the metabolic disorder associated with impaired glucose metabolism and / or lipid metabolism is selected from the group consisting of diabetes mellitus, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), obesity, and metabolic syndrome.

78. Use according to claim 76 or 77, wherein the metabolic disorder associated with impaired glucose and / or lipid metabolism is diabetes mellitus (e.g., type 2 diabetes mellitus, type 2 diabetes mellitus with poor glycemic control after dietary and exercise therapy interventions).

79. The use according to claim 76, wherein the complication of the metabolic disease includes a cardiovascular complication, a renal complication, or a hepatic complication caused by the metabolic disease.

80. The use according to claim 76, wherein the nervous system disease is a neurodegenerative disease.

81. The use according to claim 80, wherein the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, motor neuron disease, Huntington's disease, and Parkinson's disease.

82. A use according to any one of claims 75 to 81, wherein the additional therapeutic agent is selected from the group consisting of insulin, metformin, sulfonylurea (e.g., glimepiride, glibenclamide, gliclazide, glikidone), α-glucosidase inhibitors (e.g., acarbose), and γ-aminobutyric acid.

83. The use according to claim 82, wherein the additional therapeutic agent is metformin.

84. The use according to claim 82, wherein the additional therapeutic agent is γ-aminobutyric acid.

85. The pharmaceutical preparation described in any one of claims 49 to 54 and metformin are used in the manufacture of a drug for the treatment of diabetes.

86. In the use described in claim 85, diabetes is type 2 diabetes.

87. In the use described in claim 85 or 86, the amino acid sequence of the GLP-1 fusion protein in the pharmaceutical formulation is as shown in SEQ ID NO:

7.

88. The pharmaceutical preparation described in any one of claims 49 to 54 and γ-aminobutyric acid are used in the manufacture of a drug for the treatment of neurodegenerative diseases.

89. In the use described in claim 88, the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, motor neuron disease, Huntington's disease, and Parkinson's disease.

90. The use according to claim 88 or 89, wherein the amino acid sequence of the GLP-1 fusion protein in the pharmaceutical formulation is as shown in SEQ ID NO:

7.

91. A combination of drugs comprising a pharmaceutical preparation according to any one of claims 1 to 67 and an additional therapeutic agent.

92. A combination of drugs according to claim 91, wherein the additional therapeutic agent is a drug for treating diabetes or a drug for treating neurodegenerative diseases.

93. A combination of agents according to claim 91 or 92, wherein the additional therapeutic agent is selected from the group consisting of insulin, metformin, sulfonylurea (e.g., glimepiride, glibenclamide, gliclazide, glikidone), α-glucosidase inhibitors (e.g., acarbose), and γ-aminobutyric acid.

94. A combination of drugs according to claim 93, wherein the additional therapeutic agent is metformin.

95. A combination of drugs according to claim 93, wherein the additional therapeutic agent is γ-aminobutyric acid.

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