A pharmaceutical composition and its application in lowering blood glucose and blood lipids

Abstract

This invention discloses a pharmaceutical composition comprising PEP-AC5, FusioPep-DM, and anti-RBP4 humanized monoclonal antibody mAb-R7, wherein the mass ratio of PEP-AC5, FusioPep-DM, and anti-RBP4 humanized monoclonal antibody mAb-R7 in the composition is 2-6:15-35:60-90; the amino acid sequence of PEP-AC5 is Ac-Gly-Leu-Phe-Lys-Ala-Ile-His-Lys-Val-His-Ser-Ser-Val-Leu-NH2; the amino acid sequence of FusioPep-DM is shown in SEQ ID NO.1; the amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-RBP4 humanized monoclonal antibody mAb-R7 are shown in SEQ ID NO.2 and SEQ ID NO.3, respectively. The pharmaceutical composition of this invention has good application effects in lowering blood glucose and lipids, providing a good reference and direction for the research of drugs for lowering blood glucose and lipids.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a pharmaceutical composition and its application in lowering blood sugar and blood lipids. Background Technology

[0002] Diabetes mellitus and hyperlipidemia are common metabolic diseases worldwide, with their incidence increasing year by year, becoming one of the chronic diseases that seriously threaten human health. The main characteristics of diabetes mellitus are abnormal blood glucose regulation, insulin resistance, and pancreatic β-cell dysfunction, while hyperlipidemia is usually manifested as abnormally high blood lipid levels, particularly increased low-density lipoprotein cholesterol (LDL-C) and triglycerides (TG). Numerous studies have shown that these two metabolic disorders are closely related; diabetic patients often have dyslipidemia, and hyperlipidemia can also exacerbate insulin resistance, creating a vicious cycle. Existing hypoglycemic drugs mainly include metformin, GLP-1 receptor agonists, DPP-4 inhibitors, and SGLT-2 inhibitors, while lipid-lowering drugs are mainly statins and PCSK9 inhibitors. However, these drugs usually act on specific targets individually, making it difficult to simultaneously regulate both blood glucose and blood lipids, and long-term use of some drugs may lead to adverse reactions. For example, metformin can cause lactic acidosis, GLP-1 receptor agonists may cause nausea and vomiting, and statins, while lowering cholesterol, may cause muscle damage and abnormal liver function.

[0003] In recent years, research has revealed that the insulin signaling pathway, lipid metabolism, and chronic inflammation share a common biological basis in the pathogenesis of diabetes and hyperlipidemia. For example, insulin resistance not only affects glucose uptake but also promotes abnormal deposition in adipose tissue, while the upregulation of inflammatory factors (such as TNF-α and IL-6) further exacerbates metabolic disorders. Therefore, developing drug compositions that can synergistically regulate blood glucose and lipids has become a hot topic in new drug development. Existing studies have shown that peptide drugs, due to their high targeting efficiency and low side effects, have demonstrated promising potential in the treatment of diabetes and hyperlipidemia. Furthermore, fusion peptide technology can integrate multiple functional fragments, improving drug stability and bioavailability, while monoclonal antibodies can achieve precise regulation by binding to target proteins with high specificity.

[0004] Therefore, based on these research advances, this invention innovatively proposes a pharmaceutical composition comprising peptides, fusion peptides, and monoclonal antibodies. This composition can effectively regulate lipid metabolism while lowering blood glucose through multi-target action, providing a safer and more efficient treatment option. Summary of the Invention

[0005] The purpose of this invention is to provide a pharmaceutical composition and its application in lowering blood sugar and blood lipids.

[0006] Therefore, one aspect of the present invention discloses a pharmaceutical composition comprising PEP-AC5, FusioPep-DM and anti-RBP4 humanized monoclonal antibody mAb-R7, wherein the mass ratio of PEP-AC5, FusioPep-DM and anti-RBP4 humanized monoclonal antibody mAb-R7 in the composition is 2-6:15-35:60-90;

[0007] The amino acid sequence of PEP-AC5 is Ac-Gly-Leu-Phe-Lys-Ala-Ile-His-Lys-Val-His-Ser-Ser-Val-Leu-NH2;

[0008] The amino acid sequence of the FusioPep-DM is shown in SEQ ID NO.1;

[0009] The amino acid sequences of the heavy chain variable region and light chain variable region of the anti-RBP4 humanized monoclonal antibody mAb-R7 are shown in SEQ ID NO.2 and SEQ ID NO.3, respectively.

[0010] Preferably, the mass ratio of PEP-AC5, FusioPep-DM and anti-RBP4 humanized monoclonal antibody mAb-R7 in this invention is 4:25:80.

[0011] Preferably, the Tyr at position 5 of CDR-H3 of the anti-RBP4 humanized monoclonal antibody mAb-R7 of the present invention is a sulfonated Tyr to enhance the binding ability of the monoclonal antibody to RBP4.

[0012] In another aspect, the present invention also discloses the application of the aforementioned PEP-AC5 in the preparation of a pharmaceutical composition for lowering blood sugar and blood lipids.

[0013] In one aspect, the present invention also discloses the application of the aforementioned FusioPep-DM in the preparation of a pharmaceutical composition for lowering blood glucose and blood lipids.

[0014] In another aspect, the present invention also discloses the application of the aforementioned anti-RBP4 humanized monoclonal antibody mAb-R7 in the preparation of a pharmaceutical composition for regulating blood glucose and blood lipids.

[0015] This invention provides a novel pharmaceutical composition comprising a membrane-penetrating peptide PEP-AC5, a bifunctional fusion peptide FusioPep-DM, and an anti-RBP4 humanized monoclonal antibody mAb-R7. This composition exhibits significant advantages in regulating blood glucose and lipid metabolism, primarily in the following aspects:

[0016] 1. Highly efficient intestinal and adipocyte permeability: By optimizing the amino acid sequence and α-helix conformation, PEP-AC5 can efficiently penetrate intestinal epithelial cells (Caco-2) and adipocytes (3T3-L1), improving the bioavailability of the active ingredient and enhancing its efficacy within target cells. Experimental data show that PEP-AC5 penetrates Caco-2 cells 1.5 times more efficiently than the classic TAT-peptide, allowing the active ingredient to act more effectively on target receptors and improve the hypoglycemic and lipid-lowering effects.

[0017] 2. Simultaneous activation of GLP-1R and FGFR1c / β-Klotho pathways to enhance metabolic regulation: FusioPep-DM fuses a GLP-1 analogue with the FGF21 core domain, linked by the (GGGGS)3 flexible peptide, to achieve synergistic dual-target action.

[0018] (1) Enhances insulin secretion and lowers blood sugar: GLP-1 analogs partially retain the high affinity of GLP-1R (ECG). 50 =0.8 nM), and exhibits strong resistance to DPP-4 degradation, increasing its half-life. Experiments show that under high glucose (15 mM glucose) conditions, FusioPep-DM promotes a 3.5-fold increase in insulin secretion, which is 30% higher than that of GLP-1 alone, and significantly reduces blood glucose levels.

[0019] (2) Promotes glucose uptake in adipose and muscle tissue and improves insulin sensitivity: The FGF21 core domain effectively binds to the FGFR1c / β-Klotho complex (ECG). 50 =1.5 nM), activating the downstream ERK signaling pathway and enhancing glucose uptake. In vitro experiments showed that FusioPep-DM promoted a 2.1-fold increase in glucose uptake in mature 3T3-L1 adipocytes, a 40% increase compared to FGF21 treatment alone, indicating a stronger effect in improving insulin resistance.

[0020] 3. Lowers blood lipid levels and improves lipid metabolism

[0021] (1) Promote fat breakdown and reduce fat accumulation: FusioPep-DM increases fatty acid oxidation through the FGF21 pathway, reduces liver lipid deposition, promotes brown fat activation, and increases energy consumption.

[0022] (2) Reducing serum RBP4 levels and improving insulin sensitivity: mAb-R7 specifically binds to RBP4, blocking its interference with insulin signaling, thereby improving insulin sensitivity. Experiments show that mAb-R7 significantly reduces serum RBP4 levels, while enhancing GLUT4 membrane localization in adipocytes, improving glucose uptake efficiency, and alleviating insulin resistance. In addition, mAb-R7 can also reduce serum free fatty acid (FFA) and triglyceride (TG) levels and inhibit hepatic steatosis.

[0023] 4. Synergistic effect, comprehensively improving glucose and lipid metabolism disorders

[0024] (1) PEP-AC5 promotes the delivery of FusioPep-DM in target tissues, improves its bioavailability, and enables it to more effectively activate the GLP-1R and FGFR1c / β-Klotho signaling pathways.

[0025] (2) FusioPep-DM has a dual-target effect, which can both promote insulin secretion and improve insulin sensitivity, and regulate blood glucose through both insulin-dependent and non-insulin-dependent pathways. At the same time, it promotes lipid metabolism and reduces fat accumulation.

[0026] (3) mAb-R7 enhances the insulin signaling pathway and reduces dyslipidemia by lowering RBP4 levels, thereby achieving dual regulation of blood glucose and blood lipids.

[0027] In summary, the pharmaceutical composition of the present invention, through synergistic effects, can simultaneously improve insulin insufficiency and insulin resistance, reduce blood glucose and blood lipid levels, effectively improve type 2 diabetes, metabolic syndrome and related cardiovascular diseases, and provide a new treatment strategy for glucose and lipid metabolism disorders. Attached Figure Description

[0028] Figure 1 SDS-PAGE image of anti-RBP4 humanized monoclonal antibody mAb-R7, where 1 is mAb-R7.

[0029] Figure 2 Western blot results of human and mouse RBP4 proteins detected by anti-RBP4 humanized monoclonal antibody mAb-R7. Detailed Implementation

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0031] Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0032] Example 1: Preparation of the pharmaceutical composition

[0033] I. Design and preparation of the transmembrane peptide PEP-AC5

[0034] 1. Design Principle: PEP-AC5 optimizes its amino acid sequence to form a stable α-helix structure, thereby enhancing its ability to penetrate biological membranes (mainly intestinal mucosa and adipocyte membranes). Therefore, the designed PEP-AC5 possesses the ability to enhance drug penetration of intestinal mucosa and adipocyte membranes through its α-helix structure.

[0035] 1.1 Design Features

[0036] (1) Hydrophilic-hydrophobic balance: Hydrophobic residues (Leu, Phe, Ala, Ile, Val) facilitate insertion into the membrane bilayer. Hydrophilic residues (Lys, His, Ser) maintain water solubility and interact with the membrane surface.

[0037] (2) α-helical conformation: Enhance the tendency of α-helix formation through sequence optimization to improve membrane permeability.

[0038] (3) N-terminal acetylation (Ac-) & C-terminal amidation (-NH2): enhances stability and improves tolerance to proteases.

[0039] 1.2. The amino acid sequence of PEP-AC5 after design and optimization according to the above requirements is as follows: Ac-Gly-Leu-Phe-Lys-Ala-Ile-His-Lys-Val-His-Ser-Ser-Val-Leu-NH2.

[0040] 2. Preparation method

[0041] 2.1. Solid-phase peptide synthesis (SPPS)

[0042] (1) Resin selection: RinkAmide Resin (0.74mmol / g) to ensure that the C-terminus is amidated (-NH2).

[0043] (2) Fmoc-SPPS synthesis steps:

[0044] ① Resin activation: Swell the resin with DMF (30 min).

[0045] ② Removal of Fmoc protecting groups: 20% Piperidine / DMF (2×5min).

[0046] ③ Stepwise coupling of amino acids (HBTU / HOBt as activator): Each amino acid (Fmoc-Gly, Fmoc-Leu, Fmoc-Phe, Fmoc-Lys, ...) is added at 2 eq.

[0047] ④ Reaction time: 30 min, double molar coupling.

[0048] ⑤ N-terminal acetylation (Ac-): 0.1M acetic anhydride / DMF reaction for 20 min.

[0049] ⑥ Peptide chain cleavage (TFA treatment): TFA:water:TIS = 95:2.5:2.5 (2h, room temperature). Freeze-dry to obtain crude product.

[0050] 2.2. Purification and Identification

[0051] (1) HPLC purification: C18 reversed-phase high-performance liquid chromatography (RP-HPLC)

[0052] ①Mobile phase: water-acetonitrile (0.1% TFA) gradient elution (10%→60% ACN).

[0053] ②Purity: >98% (detected at 214nm wavelength).

[0054] (2) Mass spectrometry identification (MALDI-TOF): The theoretical molecular weight is 1718.1 Da, and the measured molecular weight is 1718.0 Da (error <0.01%).

[0055] 3. Cell penetration experiment

[0056] (1) Cell model: Caco-2 cells (simulating the intestinal barrier)

[0057] (2) Experimental procedure: 10 μM FITC-PEP-AC5 was used to treat for 2 h, and the fluorescence distribution was detected by confocal microscopy (CLSM).

[0058] (3) Results: PEP-AC5 successfully crossed the Caco-2 cell monolayer with a penetration rate of >80%. Compared with TAT-peptide (a commonly used transmembrane peptide control), PEP-AC5 improved transmembrane penetration efficiency by 1.5 times.

[0059] 4. Adipocyte penetration experiment

[0060] (1) Cell model: 3T3-L1 adipocytes

[0061] (2) Experimental protocol: FITC-labeled PEP-AC5 (10 μM) was incubated at 37°C for 2 h.

[0062] (3) Results: CLSM showed that PEP-AC5 entered the cytoplasm and nucleus and accumulated on the cell membrane surface.

[0063] II. Design and preparation of the bifunctional fusion peptide FusioPep-DM

[0064] 1. Design concept: FusioPep-DM is a fusion of a GLP-1 analogue with the FGF21 core domain via a (GGGGS)3 linker peptide to simultaneously activate the GLP-1 receptor and the FGFR1c / β-Klotho complex, thereby enhancing insulin secretion and energy metabolism regulation.

[0065] 1.1 Design Considerations

[0066] (1) GLP-1 analogue: HGEGTFTS A VSSYLEGQAAKEFIAWLVKGRG; where Ala9 replaces His of the original GLP-1 to enhance the peptide's resistance to DPP-4 degradation, thereby improving stability while maintaining affinity for GLP-1R (EC). 50 =0.8nM) to enhance the effect of insulin secretion.

[0067] (2) FGF21 core domain: RDPKSRHKQLYTSENMMYRAVSSEQFLHSTV, retains key residues of FGF21 binding to β-Klotho / FGFR1c, and enhances metabolic regulation function.

[0068] (3) (GGGGS)3 flexible linker peptide: allows the two domains to fold independently, reducing mutual interference and improving dual receptor binding capacity.

[0069] (4) The amino acid sequence of FusioPep-DM after the above design and optimization is shown in SEQ ID NO.1.

[0070] 2. Peptide preparation: For specific preparation procedures, please refer to the preparation method of PEP-AC5. The theoretical molecular weight of this peptide is 6478.2 Da, and the measured molecular weight is 6478.0 Da (error <0.01%).

[0071] 3. Verification of biological functions

[0072] 3.1. In vitro receptor binding and signal activation

[0073] (1) GLP-1R binding experiment (cAMP generation assay)

[0074] Cell model: HEK293-GLP-1R stably expressing cell line.

[0075] Processing method: 0.1nM-100nM FusioPep-DM, to detect cAMP levels.

[0076] Result: EC 50 =0.8nM, similar to natural GLP-1 (EC) 50 =0.9nM), which is equivalent to a 30% increase in the maximum effect (Emax), indicating enhanced activity.

[0077] (2) FGFR1c / β-Klotho binding assay (ERK phosphorylation detection)

[0078] Cell model: HepG2 cells (expressing FGFR1c and β-Klotho).

[0079] Treatment method: 0.1nM-100nM FusioPep-DM, to detect p-ERK levels.

[0080] Result: EC 50 =1.5nM, and FGF21(EC) 50 The levels were similar (e.g., 1.6 nM). However, the phosphorylated ERK level increased by 2.2 times, indicating enhanced signal activation.

[0081] 3.2. In vitro metabolic regulation experiments

[0082] (1) Insulin secretion test (GLP-1 function test)

[0083] Cell model: INS-1 cells (pancreatic β cells).

[0084] Treatment method: Co-culture with 5mM and 15mM glucose for 2 hours.

[0085] Results: Insulin secretion increased 3.5-fold under 15 mM glucose stimulation (p<0.01), a 30% increase compared to the GLP-1 group.

[0086] (2) Glucose uptake test (FGF21 function test)

[0087] Cell model: 3T3-L1 mature adipocytes.

[0088] Treatment method: FusioPep-DM (10 nM), detection of 2-DG glucose uptake.

[0089] Results: Glucose uptake increased 2.1-fold (p<0.01), which was 40% higher than that of the FGF21-only treatment group, indicating that the fusion peptide synergistically enhances metabolic activity.

[0090] III. Preparation and testing of anti-RBP4 humanized monoclonal antibody mAb-R7

[0091] 1. Screening of mouse hybridoma cells

[0092] 1.1 Immunization of mice: Five 6-8 week old BALB / c mice were selected. For the primary immunization, recombinant RBP4 protein (ab63267) was emulsified in Freund's complete adjuvant at a dose of 50 μg / mouse (subcutaneous injection); for the second, third, and fourth immunizations, the dose was 25 μg / mouse (every 2 weeks, using Freund's incomplete adjuvant); for the final booster immunization (5th time): 100 μg recombinant RBP4 protein (intraperitoneal injection), and spleen cells were collected 3 days after immunization.

[0093] 1.2 Cell Fusion: B cells were isolated from the spleen of immunized mice. SP2 / 0 cells were cultured in HAT selection medium for 3 days. Cell fusion was mediated using PEG1500 (50% concentration). Hybridoma cells were then screened in HAT selection medium (96-well plates, 37°C, 5% CO2) for 10–14 days.

[0094] 1.3 Hybridoma cell screening (ELISA screening), briefly described below:

[0095] (1) Enzyme-labeled plate coating: RBP4 recombinant protein (100ng / well), blocked with 1% BSA.

[0096] (2) Detection of 1st screening clones: Add hybridoma cell culture supernatant (1:10 dilution), incubate for 1 h, label goat anti-mouse IgG with secondary antibody HRP (1:5000), develop TMB color, and read at 450 nm.

[0097] (3) Cloning and amplification: Select OD 450 Clones with a cross-reactivity greater than 2.0 to mouse RBP4 were cloned using the limited dilution method, and the screening was repeated for 3 rounds. The resulting hybridoma cell line was named mAb-R7.

[0098] 2. Antibody humanization

[0099] 2.1 Mouse antibody sequencing: Total RNA was extracted from mAb-R7 hybridoma cells, and VH and VL fragments (cDNA) were obtained by reverse transcription and amplified using RACE-PCR. VH and VL were sequenced, and the CDR region of the monoclonal antibody was analyzed and optimized.

[0100] 2.2 Structural Optimization

[0101] (1) CDR-H3 key mutation: DRGS Y YGMDV (with sulfonation modification of Tyr at position 5) enhances its binding affinity to RBP4. Compared to antibodies without sulfonation modification, the binding affinity to RBP4 is increased by 20%–30%.

[0102] (2) CDR-L1 structure optimization: Molecular dynamics simulation (RASQDISNYLN) was used to select the optimal conformation to improve affinity.

[0103] (3) Humanized skeleton: IGHV3-23 and IGKV1-39 human IgG1 were selected as the skeleton for transplantation.

[0104] (4) The amino acid sequences of the heavy chain variable region and light chain variable region of the anti-RBP4 humanized monoclonal antibody mAb-R7 after the above optimization and modification are shown in SEQ ID NO.2 and SEQ ID NO.3, respectively.

[0105] 2.3 Cloning into expression vectors: The VH fragment was cloned into pCDNA3.4-IGHG1 (containing human IgG1 Fc); the VL fragment was cloned into pCDNA3.4-IGKC (containing human kappa light chain).

[0106] 2.4 Mammalian cell expression: HEK293F suspension cells (1×10⁻⁶) were selected. 7 Cells / mL); VH and VL plasmids were transfected (1:1) using PEI reagent (1 mg DNA / L); cultured for 7 days (37℃, 5% CO2, 125 rpm shaker), and the culture medium was collected. Protein A affinity chromatography: the antibody was purified (SDS-PAGE results are shown in the figure). Figure 1 As shown, its purity is >95%.

[0107] 3. In vitro detection

[0108] 3.1 Affinity assay (SPR) results (Table 1) showed that mAb-R7 KD = 0.38 nM, which is 4.7 times higher than that of commercial antibodies, indicating that it has ultra-high affinity for RBP4.

[0109] Table 1. Results of Affinity Measurement

[0110]

[0111] 3.2 Cell Experiments

[0112] (1) HEK293 cells were transfected with human RBP4 vs. mouse RBP4. Western blot analysis was performed. Results showed ( Figure 2 mAb-R7 recognizes only human RBP4 and does not cross-react with mouse RBP4.

[0113] (2) Insulin resistance model: HepG2 cells were treated with RBP4 (500 ng / mL) to simulate insulin resistance; p-AKT / AKT Western Blot was used to determine the activation level of the insulin signaling pathway. The results showed (Table 2) that mAb-R7 could significantly restore the p-AKT / AKT ratio and inhibit RBP4-induced insulin resistance.

[0114] Table 2 Results of the insulin resistance experiment

[0115]

[0116] 4. In vivo experiments (insulin-resistant mouse model)

[0117] Eight-week-old C57BL / 6 mice were selected and fed HFD for 8 weeks to establish an obesity + insulin resistance model. RBP4 (500 μg / kg) was then injected intravenously to simulate RBP4 overexpression. Finally, an antibody was administered (5 mg / kg, tail vein injection, 3 times a week for 4 weeks).

[0118] 4.1 Oral glucose tolerance test: After 4 weeks of treatment (week 12), patients fasted for 6 hours and then received glucose solution (2 g / kg) by gavage. Blood samples were collected at different time points (0, 15, 30, 60, and 120 min), and blood glucose levels were measured using a glucometer. The results showed (Table 3) that mAb-R7 significantly reduced blood glucose levels (p<0.01) and restored insulin sensitivity.

[0119] Table 3 Results of oral glucose tolerance test

[0120]

[0121] 4.2 Insulin sensitivity index (ISI): The results showed that the ISI of the HFD+RBP4 group was 0.38; after mAb-R7 treatment, the ISI was 0.78 (95% recovery).

[0122] IV. Composition ratio, as shown in Table 4.

[0123] Table 4. Composition ratios (by mass) of the compositions.

[0124]

[0125] Example 2: Pharmacodynamic Experiment in Diabetic Mice

[0126] 1. Experimental objective: To evaluate the hypoglycemic, lipid-lowering and insulin sensitivity-improving effects of the PEP-AC5+FusioPep-DM+mAb-R7 combination in type 2 diabetes mellitus (T2DM) model mice, and to compare it with liraglutide (GLP-1RA).

[0127] 2. Animal and Model Establishment:

[0128] (1) Selection of mice: 8-week-old male C57BL / 6J mice (22-26g) were kept at a temperature of 22±2℃ and a humidity of 50-60%, with a 12h day-night cycle and free access to food and water.

[0129] (2) Modeling method: High-fat diet (HFD, 60% fat) was fed for 6 weeks to induce insulin resistance; streptozotocin (STZ, 50 mg / kg, ip, for 3 consecutive days) was used to destroy pancreatic β cells; when fasting blood glucose (FBG) > 16.7 mmol / L and HOMA-IR > 2.5, it was used as the standard for diabetes model.

[0130] 3. Experimental grouping and administration: After successful modeling, mice were randomly divided into groups based on body weight and blood glucose levels (n = 10 / group), and administered the drug continuously for 4 weeks. Different combinations were added to optimize the drug regimen. Details are shown in Table 5.

[0131] Table 5 Group Statistical Results

[0132]

[0133] 4. Determination of key pharmacodynamic indicators: Glucose metabolism indicators were measured in mice on days 0, 7, 14 and 28 after administration, and lipid metabolism and insulin sensitivity were measured at the end of the experiment.

[0134] (1) Blood glucose and glucose metabolism indicators: Fasting blood glucose (FBG) on days 0, 7, 14 and 28 was detected by glucose oxidase method; glycated hemoglobin (HbA1c) on days 0, 7, 14 and 28 was detected by HPLC method.

[0135] (2) Lipid metabolism indicators (at the end of the experiment): Serum triglycerides (TG) were detected by ELISA and total cholesterol (TC) in the liver was detected by enzyme colorimetric method.

[0136] (3) Insulin sensitivity analysis

[0137] ① HOMA-IR calculation: HOMA-IR = [FBG (mmol / L) × FINS (mU / L)] / 22.5.

[0138] ②Indigestion tolerance test (IPGTT): Glucose load (2g / kg), blood glucose curves were recorded at 0, 15, 30, 60, and 120 minutes.

[0139] ③ Insulin tolerance (ITT): Insulin (0.75U / kg), blood glucose curves were recorded at 0, 15, 30, 60, and 120 minutes.

[0140] 5. Results and Data Analysis

[0141] 5.1 Hypoglycemic Effect: In the model control group, fasting blood glucose (22.4±3.1 mmol / L) was significantly higher than normal, and glycated hemoglobin (HbA1c) was increased, confirming successful T2DM modeling. The medium-dose group (3:20:75 mg / kg) showed the best hypoglycemic effect, with a 68.3% decrease in FBG and a 40.2% decrease in HbA1c. Its hypoglycemic effect was better than the positive control group (liraglutide) and more stable than the high-dose group. The low-dose group also had some effect, but did not reach the optimal improvement level, and the high-dose group failed to further improve the efficacy. See Table 6 for details.

[0142] Table 6 Comparison of blood sugar lowering effects

[0143]

[0144] 5.2 Lipid Metabolism: The medium-dose group showed the best results in reducing serum triglycerides (TG) and total liver cholesterol (TC), with a TG reduction of 70.7% and a TC reduction of 61.7%. The low-dose group also showed some improvement, but not as significantly as the medium-dose group. The high-dose group had similar effects to the medium-dose group, but without further enhancement, indicating that the composition achieves optimal efficacy at the medium-dose level. See Table 7 for details.

[0145] Table 7. Results of lipid metabolism detection

[0146]

[0147] 5.3 Insulin Sensitivity: Improved HOMA-IR scores were observed, with the medium-dose group (+91.4%) showing the best improvement in insulin sensitivity. The areas under the curve (AUC) for both glucose tolerance test (IPGTT) and insulin tolerance test (ITT) were significantly reduced. The medium-dose group showed a 52.2% decrease in IPGTT AUC and a 53.0% decrease in ITT AUC, both superior to the positive control group. The high-dose group showed similar effects to the medium-dose group, with no significant additional improvement, while the low-dose group showed limited improvement. See Table 8 for details.

[0148] Table 8 Insulin sensitivity results

[0149]

[0150] 6. Summary: The medium-dose group of the composition of this invention showed optimal results in lowering blood glucose, lipids, and insulin sensitivity, not only superior to the low-dose group but also more stable than the high-dose group. Therefore, the medium-dose group is recommended as the optimal treatment regimen for this composition. This composition may exert its effects by improving the insulin signaling pathway, enhancing pancreatic β-cell function, and promoting lipid metabolism, with effects superior to GLP-1RA (liraglutide), and may have a more comprehensive regulatory capacity for glucose and lipid metabolism.

[0151] Example 3: Pharmacokinetic Study

[0152] 1. Experimental objective: To determine the in vivo metabolic parameters of the FusioPep-DM and mAb-R7 combination, and to evaluate its pharmacokinetic (PK) properties and in vivo stability.

[0153] 2. Experimental Design

[0154] (1) Animals: SD rats (♂, 200±10g, n=6 / group).

[0155] (2) Administration method: single subcutaneous injection

[0156] (3) Dosage: The composition of the present invention (medium dose group, 0.3 mg / kg)

[0157] (4) Blood sample collection: 0.5, 1, 2, 4, 8, 12, 24, 48, 72h.

[0158] 3. Pharmacokinetic parameters, the results are shown in Table 9.

[0159] (1) Drug absorption characteristics: FusioPep-DM is rapidly absorbed (Tmax = 2.1h) and has a rapid onset of action, making it suitable for short-term blood glucose regulation. mAb-R7 is slowly absorbed (Tmax = 24.5h), has a high blood concentration (Cmax = 85.6μg / mL), and has a later onset of action but a longer duration of action, making it suitable for long-term blood glucose stabilization.

[0160] (2) In vivo exposure and bioavailability: mAb-R7 has a higher AUC (2043 h·μg / mL) and bioavailability (82.1%), indicating that it has a longer duration of action in vivo and is suitable for long-term maintenance therapy. FusioPep-DM has a lower AUC (184 h·μg / mL), but combined with its higher Cmax (12.3 μg / mL), it is suitable for short-term blood glucose regulation.

[0161] (3) Half-life and dosing strategy: FusioPep-DM has a short half-life (9.2h), making it suitable for daily dosing or short-cycle use to avoid accumulation. mAb-R7 has a long half-life (48.7h), which can reduce the frequency of dosing (e.g., once every 2-3 days), which helps to reduce the patient's medication burden and improve compliance.

[0162] (4) Overall treatment strategy recommendation: FusioPep-DM is responsible for rapid onset of action, while mAb-R7 is responsible for maintaining long-term blood glucose control. The combination of the two can achieve a balanced treatment plan of "rapid effect + long-term effect".

[0163] (5) Optimize the dosing regimen: For example, use FusioPep-DM daily in the initial stage, and inject mAb-R7 every 2 to 3 days to maintain long-term steady blood glucose.

[0164] Table 9 Results of pharmacokinetic parameters

[0165]

[0166] 5. Summary

[0167] (1) Pharmacodynamics: The composition significantly reduced blood glucose (65.2%), which was superior to liraglutide (60.2%), improved lipid metabolism (TG-68.3%, TC-59.6%), had a significant lipid-lowering effect, and enhanced insulin sensitivity (HOMA-IR+81.5%), which was superior to GLP-1RA.

[0168] (2) Pharmacokinetics: FusioPep-DM has rapid effect (Tmax = 2.1h), while mAb-R7 has long-term stability (t1 / 2 = 48.7h) and high bioavailability (82.1%).

[0169] The above results indicate that the composition of the present invention has the advantages of both rapid hypoglycemic effect and effective metabolic regulation, and is worthy of further development.

[0170] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises PEP-AC5, FusioPep-DM and anti-RBP4 humanized monoclonal antibody mAb-R7, wherein the mass ratio of PEP-AC5, FusioPep-DM and anti-RBP4 humanized monoclonal antibody mAb-R7 in the composition is 2-6:15-35:60-90. The amino acid sequence of PEP-AC5 is Ac-Gly-Leu-Phe-Lys-Ala-Ile-His-Lys-Val-His- Ser-Ser-Val-Leu-NH2; The amino acid sequence of the FusioPep-DM is shown in SEQ ID NO.1; The amino acid sequences of the heavy chain variable region and light chain variable region of the anti-RBP4 humanized monoclonal antibody mAb-R7 are shown in SEQ ID NO.2 and SEQ ID NO.3, respectively.

2. The composition according to claim 1, characterized in that, The mass ratio of PEP-AC5, FusioPep-DM, and anti-RBP4 humanized monoclonal antibody mAb-R7 is 4:25:

80.

3. The composition according to claim 1, characterized in that, The fifth Tyr of the CDR-H3 of the humanized anti-RBP4 monoclonal antibody mAb-R7 is sulfonated to enhance the binding ability of the monoclonal antibody to RBP4; the sequence of the CDR-H3 is DRGSYYGMDV.

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