Co-assembly of macrocyclic amphiphilic compound and insulin as well as preparation method and application of co-assembly

Abstract

The invention discloses a co-assembly of a macrocyclic amphiphilic compound and insulin as well as a preparation method and application of the co-assembly, and relates to the technical field of medicines. The co-assembly of the macrocyclic amphiphilic compound and the insulin is co-assembled by utilizing the insulin and the macrocyclic amphiphilic compound. According to the present invention, the treatment effect of the co-assembly of the macrocyclic amphiphilic compound and the insulin on the Alzheimer's disease can be enhanced by improving the metabolic injury, the limitation that only the A beta is eliminated in the prior art is improved, the adopted GCD-INS co-assembly treatment strategy breaks through the tradition, the plaque deposition and the insulin resistance can be reduced, and the treatment effect of the GCD-INS co-assembly on the Alzheimer's disease can be improved; and nerve cell apoptosis and synaptic injury can be reduced, and cognitive impairment can be improved, so that a more comprehensive treatment effect can be provided. The compound can be widely applied to preparation of drugs for treating or improving metabolic disorder of Alzheimer's disease.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, in particular to a co-assembly of a macrocyclic amphiphilic compound and insulin, and a preparation method and application thereof. Background Art

[0002] Treatment for Alzheimer's disease (AD) usually uses a single drug or treatment method, mainly focusing on eliminating amyloid beta (Aβ) and improving insulin resistance. The prevention and treatment of AD mainly focus on Aβ targeted therapy, including exosome-based drug carriers, nanoparticle-based targeted therapy, monoclonal antibody-based immunotherapy, etc. These traditional methods have certain limitations in improving the clinical symptoms of AD patients and fail to effectively solve other problems related to AD, such as metabolic abnormalities.

[0003] Treatments targeting Aβ deliver drugs to damaged nerve cells through specific carriers to promote the degradation and clearance of Aβ. These methods usually only target Aβ itself and fail to address other pathological processes associated with AD, such as insulin resistance. Treatments targeting insulin resistance usually provide insulin through direct administration to restore the sensitivity of nerve cells to insulin. However, this method has certain limitations in accuracy and effectiveness, which may lead to increased side effects and toxicity of the drug.

[0004] Although existing technologies have alleviated the symptoms of AD to a certain extent, there are still problems such as limited therapeutic effect and imprecise drug administration. Therefore, a new treatment method is needed to more effectively improve the metabolic abnormalities and pathological characteristics of AD.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The purpose of the present invention is to provide a co-assembly of a macrocyclic amphiphilic compound and insulin, and a preparation method and application thereof.

[0007] The present invention is achieved in that:

[0008] In a first aspect, the present invention provides a co-assembly of a macrocyclic amphiphilic compound and insulin, which comprises insulin and a macrocyclic amphiphilic compound, wherein the macrocyclic amphiphilic compound is a co-assembly of a guanidino-modified calixarene and a cyclodextrin.

[0009] In an optional embodiment, the molar ratio of the insulin to the macrocyclic amphiphilic compound is 0.8-1.2:1.

[0010] In a second aspect, the present invention provides a method for preparing a co-assembly of a macrocyclic amphiphilic compound and insulin as described in any one of the aforementioned embodiments, comprising mixing insulin and the macrocyclic amphiphilic compound.

[0011] In an optional embodiment, before mixing with the macrocyclic amphiphilic compound, the insulin is dissolved in a buffer solution to prepare a stock solution with a concentration of 80-120 mM, and then the stock solution is mixed with the insulin and the macrocyclic amphiphilic compound at a molar ratio of 0.8-1.2:1.

[0012] In an optional embodiment, the method for preparing the macrocyclic amphiphilic compound comprises:

[0013] The guanidino-modified calixarene is dissolved in methanol to prepare a calixarene methanol solution with a concentration of 1-2 mM, and cyclodextrin is dissolved in chloroform to prepare a cyclodextrin chloroform solution with a concentration of 1-2 mM;

[0014] The calixarene methanol solution and the cyclodextrin chloroform solution are mixed in a molar ratio of 1:1, dried to obtain a film on the container wall, a buffer solution is added to the container, and the solution is ultrasonically treated at 70-90° C. for 2-4 hours to obtain a co-assembly of guanidino-modified calixarene and cyclodextrin;

[0015] Preferably, the buffer is a HEPES buffer with a concentration of 8-12 mM and a pH of 7-8.

[0016] In a third aspect, the present invention provides use of a co-assembly of a macrocyclic amphiphilic compound and insulin as described in any one of the aforementioned embodiments in the preparation of a drug for treating or improving metabolic abnormalities in Alzheimer's disease.

[0017] In a fourth aspect, the present invention provides a co-assembly of a macrocyclic amphiphilic compound and insulin as described in any one of the aforementioned embodiments for use in the preparation of a method for reducing Aβ in the hippocampus. 1-42 The signal intensity and plaque number of the preparations were applied.

[0018] In a fifth aspect, the present invention provides a use of a co-assembly of a macrocyclic amphiphilic compound and insulin as described in any one of the aforementioned embodiments in the preparation of a preparation for reducing insulin resistance;

[0019] Preferably, the co-assembly of the macrocyclic amphiphilic compound and insulin is used in the preparation of a preparation for reducing abnormal phosphorylation of insulin receptor substrate-1 to improve insulin resistance;

[0020] Preferably, the co-assembly of the macrocyclic amphiphilic compound and insulin is used in the preparation of a preparation for increasing the expression of glucose transporter 3 to improve insulin resistance.

[0021] In a sixth aspect, the present invention provides use of a co-assembly of a macrocyclic amphiphilic compound and insulin as described in any one of the aforementioned embodiments in the preparation of a preparation for reducing neuronal apoptosis or reducing synaptic damage.

[0022] In a seventh aspect, the present invention provides use of a co-assembly of a macrocyclic amphiphilic compound and insulin as described in any one of the aforementioned embodiments for preparing a preparation for improving cognitive impairment in mice suffering from Alzheimer's disease.

[0023] The present invention has the following beneficial effects:

[0024] The co-assembly of macrocyclic amphiphilic compounds and insulin provided by the present invention utilizes insulin and macrocyclic amphiphilic compounds for co-assembly. The macrocyclic amphiphilic compounds have a certain cavity. When insulin is co-assembled with the macrocyclic amphiphilic compounds, it is contained in the cavity of the macrocyclic amphiphilic compounds. Among them, insulin can restore the sensitivity of nerve cells to insulin, and it can enhance the therapeutic effect of the co-assembly of macrocyclic amphiphilic compounds and insulin on Alzheimer's disease by improving metabolic damage, thereby improving the limitation of the prior art that only eliminates Aβ. The GCD-INS co-assembly treatment strategy adopted by the present invention breaks through the tradition, which can not only reduce plaque deposition and insulin resistance, but also reduce nerve cell apoptosis and synaptic damage, and improve cognitive impairment, thereby providing a more comprehensive therapeutic effect. It can be widely used in the preparation of drugs for treating or improving metabolic abnormalities in Alzheimer's disease, and the preparation of drugs for reducing Aβ in the hippocampus. 1-42 The invention is used in a preparation for measuring the signal intensity and plaque quantity of the peptide, a preparation for reducing insulin resistance, a preparation for reducing nerve cell apoptosis or reducing synaptic damage, or a preparation for improving cognitive impairment in mice with Alzheimer's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0026] Figure 1 A schematic diagram of the possible mechanism of GCD-INS co-assembly improving AD-like pathology provided by the present invention;

[0027] Figure 2 It is a graph showing the binding characteristics of GCD and insulin in Experimental Example 1 of the present invention, wherein a is a Zeta potential graph of GCD, GCD-INS and GCD-INS+Aβ, and b is a DLS data statistical graph of GCD and GCD-INS;

[0028] Figure 3Schematic diagram of the assembly and release of GCD-INS and ELISA detection in Experimental Example 1 of the present invention, wherein a is a schematic diagram of the principle of insulin loading and release, and b is a statistical diagram of the free insulin content per 20 μM GCD-INS or GCD-INS+Aβ;

[0029] Figure 4 This is a schematic diagram of the immunofluorescence intensity of the hippocampal DG region of 6-month-old 5×FAD mice after intranasal administration using anti-Aβ1-42 antibody (green) in Experimental Example 2 of the present invention;

[0030] Figure 5 This is a schematic diagram of the average intensity of Aβ labeling in Experimental Example 2 of the present invention;

[0031] Figure 6 This is a schematic diagram of Thioflavin T staining of the hippocampus of 5×FAD mice after intranasal administration in Experimental Example 2 of the present invention;

[0032] Figure 7 Schematic diagram of representative WB bands of IRS-1, p-IRS-1 and GLUT-3 in the hippocampus in Experimental Example 2 of the present invention;

[0033] Figure 8 Schematic diagram of quantitative analysis of the optical density ratio of IRS-1 / β-Tubulin (a), p-IRS-1 / β-Tubulin (b) and GLUT-3 / β-Tubulin (c) in Experimental Example 2 of the present invention;

[0034] Fig. 9 It is a schematic diagram of quantitative analysis of insulin displaced after incubation of GCD-INS co-assembly with different proteins in Experimental Example 3 of the present invention;

[0035] Fig.10 Schematic diagram of the experimental timetable (a) and experimental scheme (b) of the MWM test experiment in Experimental Example 4 of the present invention;

[0036] Fig.11 It is a schematic diagram of the results of the escape latency of the MWM test experiment in Experimental Example 4 of the present invention;

[0037] Fig.12 Schematic diagram of the target quadrant residence time (a) and the number of platform crossings (b) in the MWM test experiment in Experimental Example 4 of the present invention;

[0038] Fig.13 Schematic diagram of fluorescent Nissl immunostaining (a) and average fluorescence intensity of Nissl in the hippocampus (b) in 5×FAD mice after intranasal administration in Experimental Example 5 of the present invention;

[0039] Fig.14Schematic diagram of representative WB bands of NR2A, PSD95 and SYP in the hippocampus in Experimental Example 5 of the present invention;

[0040] Fig.15 Schematic diagram of quantitative analysis of the optical density ratios of NR2A / β-Tubulin (a), PSD95 / β-Tubulin (b) and SYP / β-Tubulin (c) in Experimental Example 5 of the present invention. DETAILED DESCRIPTION

[0041] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0042] See also Figure 1 The present invention provides a co-assembly of a macrocyclic amphiphilic compound and insulin (GCD-INS), which comprises insulin (INS) and a macrocyclic amphiphilic compound (GCD), wherein the macrocyclic amphiphilic compound is a co-assembly of a guanidino-modified calixarene and cyclodextrin. The molar ratio of insulin to the macrocyclic amphiphilic compound is 0.8-1.2:1.

[0043] In the present invention, insulin and macrocyclic amphiphilic compounds are co-assembled, and the macrocyclic amphiphilic compounds have a certain cavity. When insulin is co-assembled with the macrocyclic amphiphilic compounds, it is contained in the cavity of the cyclic amphiphilic compounds. Among them, insulin can restore the sensitivity of nerve cells to insulin, and it can enhance the therapeutic effect of the co-assembly of macrocyclic amphiphilic compounds and insulin on Alzheimer's disease by improving metabolic damage, improving the limitation of the existing technology that only eliminates Aβ. The GCD-INS co-assembly treatment strategy adopted by the present invention breaks through the tradition, which can not only reduce plaque deposition and insulin resistance, but also reduce nerve cell apoptosis and synaptic damage, and improve cognitive impairment, thereby providing a more comprehensive therapeutic effect.

[0044] The invention provides a method for preparing a co-assembly of a macrocyclic amphiphilic compound and insulin, which comprises mixing insulin and the macrocyclic amphiphilic compound. The preparation method is simple and easy to operate.

[0045] Specifically, the preparation method of the macrocyclic amphiphilic compound includes: dissolving guanidine-modified calixarene in methanol to prepare a calixarene methanol solution with a concentration of 1-2mM, and dissolving cyclodextrin in chloroform to prepare a cyclodextrin chloroform solution with a concentration of 1-2mM; mixing the calixarene methanol solution and the cyclodextrin chloroform solution in a molar ratio of 1:1, drying, and obtaining a thin film on the wall of the container, adding a buffer solution to the container, and ultrasonically treating the solution at 70-90°C for 2-4h to obtain a co-assembly of guanidine-modified calixarene and cyclodextrin; preferably, the buffer solution is a HEPES buffer solution with a concentration of 8-12mM and a pH of 7-8.

[0046] Before mixing insulin with the macrocyclic amphiphilic compound, insulin is dissolved in a buffer solution to prepare a stock solution with a concentration of 80-120 mM, and then the stock solution is mixed with insulin and the macrocyclic amphiphilic compound at a molar ratio of 0.8-1.2:1.

[0047] The use of a buffer solution to achieve the dissolution and dispersion of insulin facilitates subsequent uniform mixing with macrocyclic amphiphilic compounds, resulting in a better co-assembly effect.

[0048] The preparation method provided by the present invention is simple and easy to operate. The prepared GCD-INS co-assembly treatment strategy breaks through the tradition and can not only reduce plaque deposition and insulin resistance, but also reduce neuronal apoptosis and synaptic damage, and improve cognitive impairment, thereby providing a more comprehensive therapeutic effect.

[0049] Correspondingly, the present invention provides the use of the co-assembly of the above-mentioned macrocyclic amphiphilic compound and insulin in the preparation of a drug for treating or improving metabolic abnormalities in Alzheimer's disease.

[0050] The present invention provides a co-assembly of the above-mentioned macrocyclic amphiphilic compound and insulin for preparing a method for reducing Aβ in the hippocampus region. 1-42 The signal intensity and plaque number of the preparations were applied.

[0051] The present invention provides the use of the co-assembly of the above-mentioned macrocyclic amphiphilic compound and insulin in the preparation of a preparation for reducing insulin resistance;

[0052] Preferably, the co-assembly of the macrocyclic amphiphilic compound and insulin is used in the preparation of a preparation for reducing abnormal phosphorylation of insulin receptor substrate-1 to improve insulin resistance;

[0053] Preferably, the co-assembly of the macrocyclic amphiphilic compound and insulin is used in the preparation of a preparation for increasing the expression of glucose transporter 3 to improve insulin resistance.

[0054] The present invention provides the use of the co-assembly of the above-mentioned macrocyclic amphiphilic compound and insulin in the preparation of a preparation for reducing nerve cell apoptosis or reducing synaptic damage.

[0055] The present invention provides the use of the co-assembly of the above-mentioned macrocyclic amphiphilic compound and insulin in the preparation of a preparation for improving cognitive impairment in mice suffering from Alzheimer's disease.

[0056] It can be seen that the co-assembly of the macrocyclic amphiphilic compound and insulin provided by the present invention can simultaneously intervene in multiple pathological processes such as plaque deposition, insulin resistance, neuronal apoptosis and synaptic damage, thereby achieving a multi-effect treatment effect. Compared with a single drug or treatment method, this comprehensive treatment strategy has more advantages and can comprehensively consider multiple pathological factors to improve the treatment effect.

[0057] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0058] Example 1

[0059] This embodiment provides a co-assembly of a macrocyclic amphiphilic compound and insulin, and the preparation method thereof comprises:

[0060] (1) Preparation of guanidine-modified calixarene and cyclodextrin co-assembly (GCD):

[0061] Guanidino-modified calixarene was dissolved in methanol to prepare a 1 mM calixarene methanol solution, and cyclodextrin was dissolved in chloroform to prepare a 1 mM cyclodextrin chloroform solution; the calixarene methanol solution and the cyclodextrin chloroform solution were mixed in a ratio of 1:1 and dried under high vacuum for 4 hours to produce a thin film in a glass bottle. HEPES {4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid, N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid)} buffer (10 mM, pH 7.4) was added to the glass bottle, and the solution was ultrasonically treated at 80°C for 2 hours to co-assemble the guanidino-modified calixarene and cyclodextrin to obtain a GCD co-assembly.

[0062] (2) Preparation of GCD and insulin co-assembly (GCD-INS):

[0063] Insulin was dissolved in HEPES buffer to prepare a 100 mM high concentration stock solution, and the insulin stock solution was added to the GCD co-assembly at a ratio of 1:1 to obtain a GCD-INS co-assembly.

[0064] Comparative Example 1

[0065] This comparative example provides a GCD co-assembly, the preparation method of which only includes step (1) of Example 1.

[0066] Comparative Example 2

[0067] This comparative example provides an insulin.

[0068] Comparative Example 3

[0069] This comparative example provides a HEPES buffer.

[0070] Experimental Example 1

[0071] In this experimental example, the single GCD co-assembly provided in Comparative Example 1 was used as the control group (GCD), and the GCD-INS provided in Example 1 was used as the experimental group (GCD-INS).

[0072] (1) This experimental example measured the zeta potential of Example 1 and Comparative Example 1, and also detected the change in the zeta potential of GCD-INS after adding Aβ in Example 1.

[0073] The detection method includes: taking about 1 mg of the samples of Example 1 and Comparative Example 1, placing them in 5 mL centrifuge tubes respectively, adding 2 mL of water, and ultrasonically dispersing for 5 minutes. Taking 1 mL of the upper solution, slowly adding it to the sample pool (container for holding the sample) to avoid bubbles in the sample pool. The Malvern laser particle size analyzer ZS90 was used for testing, and the instrument was preheated for 30 minutes; the test software was started, the test zeta program was selected, water was selected as the solvent, and the test was performed after preheating at 25°C for 30 seconds. Each sample was measured in parallel 3 times. For the test results, please refer to Figure 2 .

[0074] from Figure 2 As can be seen from a, the zeta potential of GCD provided in Comparative Example 1 is 23 mV, and the zeta potential of GCD-INS provided in Example 1 is 12 mV, indicating that after adding insulin to GCD, the significant negative shift of the zeta potential indicates that insulin is bound to GCD. After adding Aβ to GCD-INS, the zeta potential changes from 12 mV to 10 mV, indicating that Aβ interacts with GCD and Aβ can displace insulin out of the cavity.

[0075] Subsequently, this experimental example also conducted a dynamic light scattering (DLS) experiment on GCD and GCD-INS. The experimental method includes taking about 1 mg of the samples of Example 1 and Comparative Example 1, placing them in 5 mL centrifuge tubes respectively, adding 2 mL of water, and ultrasonically dispersing for 5 minutes. Take 1 mL of the upper solution and slowly add it to the sample pool to avoid bubbles in the sample pool. Use Malvern Laser Particle Sizer ZS90 for testing, and preheat the instrument for 30 minutes; start the test software, select the test particle size program, select water as the solvent, preheat at 25°C for 30 seconds and then measure. Each sample is measured in parallel 3 times. For the test results, please refer to Figure 2 Middle b.

[0076] from Figure 2As can be seen in b, the dynamic light scattering data of GCD and GCD-INS basically overlap, indicating that when GCD and INS are co-assembled, INS enters the cavity of GCD, and GCD-INS does not change the size of the GCD co-assembly.

[0077] (2) In order to detect the release of insulin after GCD-INS binds to Aβ, the concentration of free insulin released from GCD-INS+Aβ and GCD-INS solutions was measured by enzyme-linked immunosorbent assay (ELISA). In this experiment, free insulin in GCD-INS+Aβ and GCD-INS solutions of equal concentration (20 μM) was separated using ultrafiltration centrifuge tubes.

[0078] For the working mechanism, please refer to Figure 3 For test results, please refer to Figure 3 In middle b, it can be seen that after the addition of Aβ, the insulin level increased significantly, which fully proved that GCD-INS released insulin after binding with Aβ, and the released insulin was free in the solution.

[0079] Experimental Example 2

[0080] In this experimental example, the HEPES buffer provided in Comparative Example 3 was used as a blank group (HEPES), the separate GCD co-assembly and insulin provided in Comparative Examples 1 and 2 were used as control groups (GCD and INS), respectively, and the GCD-INS provided in Example 1 was used as an experimental group (GCD-INS).

[0081] (1) The effects of HEPES, INS, GCD and GCD-INS on the aggregation and level of Aβ in the brain of 5×FAD mice were studied.

[0082] In this experiment, Aβ 1-42 Immunostaining was performed using specific antibodies, including: 1-42 Antibodies (green) were used to stain the hippocampal DG region of 6-month-old 5×FAD mice after intranasal injection of 25 μL (5 μL per drop, 5 drops in total) of GCD-INS co-assembly solution (containing GCD 20 μM and insulin 50 μM), GCD co-assembly solution (20 μM), insulin (50 μM), or HEPES once a day for 1 month, and the immunofluorescence intensity and the average intensity of Aβ labeling were detected.

[0083] Please refer to the test results Figure 4 and Figure 5, it can be seen that the immunofluorescence intensity and the average intensity of Aβ labeling in the DG region of the hippocampus decreased in the 5×FAD+GCD group and the 5×FAD+INS group, and the 5×FAD+GCD-INS group was significantly lower than those in the 5×FAD+GCD group and the 5×FAD+INS group.

[0084] (2) To study the improvement effects of the above-mentioned HEPES, INS, GCD and GCD-INS on plaques in the hippocampus of 5×FAD mice.

[0085] In this experiment, thioflavin T was used to stain amyloid plaques. For the test results, please refer to Figure 6 , it can be seen that the number of plaques in the hippocampus of the 5×FAD+GCD-INS group was significantly reduced.

[0086] (3) The effects of the above HEPES, INS, GCD and GCD-INS on the representative WB bands of IRS-1, p-IRS-1 and GLUT-3 in the hippocampus of 5×FAD mice were studied. The specific experimental methods included: the hippocampus was isolated and homogenized with RIPA buffer containing 1% PMSF. The lysate was rotated at 4°C and 12,000 rpm for 20 minutes, and the supernatant was collected. After analyzing the protein concentration with a BCA protein detection kit, the supernatant was mixed with the loading buffer and boiled at 100°C for 15 minutes. The samples (40 μg per lane) were separated by 10-13% SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with 5% fat-free milk at room temperature for 1 hour, and then incubated with the primary antibody (1:1000) at 4°C overnight. Then, the membrane was incubated with the secondary antibody (1:2000) at room temperature for 1 hour. Finally, the protein band intensity was detected using a chemiluminescence imaging system.

[0087] Since serine phosphorylation of insulin receptor substrate-1 (IRS-1) affects the activity of IRS-1 and the stability of the protein, thus mediating feedback inhibition of insulin signaling, abnormal phosphorylation is a major mechanism of insulin resistance. Glucose transporter 3 (GLUT-3) is located on neurons and is a major factor affecting brain glucose transport and metabolism.

[0088] from Figure 7 It can be seen that its expression is reduced in AD patients. Western blotting (WB) results showed that nasal injection of INS, GCD and GCD-INS into 5×FAD mice could reduce the level of p-IRS-1 (Ser612) in the hippocampus and increase the level of GLUT-3. Among them, the effect of GCD-INS injection was significantly better than that of INS alone and GCD alone.

[0089] Among them, β-Tubulin was used as the internal reference of WB experiment, and the above HEPES, INS, GCD and GCD-INS had no obvious effect on β-Tubulin in the brain of 5×FAD mice. Based on this, the optical density ratios of IRS-1 / β-Tubulin, p-IRS-1 / β-Tubulin and GLUT-3 / β-Tubulin were quantitatively analyzed (n=5). The analysis results can be found in Figure 8 .

[0090] from Figure 8 As can be seen in a, nasal injection of INS, GCD and GCD-INS into 5×FAD mice can increase the level of insulin receptor substrate-1 (IRS-1), among which the effect of GCD-INS injection is significantly better than that of INS alone and GCD alone.

[0091] from Figure 8 As can be seen in middle b, nasal injection of INS, GCD and GCD-INS into 5×FAD mice can reduce the level of p-IRS-1 (Ser612) in the hippocampus. Among them, the effect of GCD-INS injection is significantly better than that of INS alone and GCD alone.

[0092] from Figure 8 As can be seen in middle c, nasal injection of INS, GCD and GCD-INS into 5×FAD mice can increase the level of GLUT-3, among which the effect of GCD-INS injection is significantly better than that of INS alone and GCD alone.

[0093] Experimental Example 3

[0094] In this experiment, the GCD-INS co-assembly was incubated with different proteins (Aβ40, Aβ42, BSA, trypsin and β-galactosidase). The incubation conditions included: adding the co-assembly and proteins to the well plate, sealing the well plate with a film, and placing it on a horizontal microplate shaker for 2 hours at room temperature and 600 rpm. The displaced insulin was then quantitatively analyzed. The analysis results are shown in Fig. 9 .

[0095] from Fig. 9It can be seen that when the GCD-INS co-assembly is incubated on its own, the amount of insulin released is extremely low, while after incubation with Aβ40 or Aβ42, the amount of insulin displaced increases significantly, and after incubation with BSA, trypsin or β-galactosidase, the amount of insulin displaced is slightly higher than the effect of incubation with the GCD-INS co-assembly on its own, but significantly lower than the effect of incubation with Aβ40 or Aβ42, which proves that insulin cannot be effectively displaced by BSA, trypsin, and β-galactosidase, which are rich in tyrosine and lysine, but can be effectively displaced by Aβ40 and Aβ42, proving that the GCD-INS co-assembly can specifically bind to Aβ40 and Aβ42 and specifically displace insulin, while BSA, trypsin, and β-galactosidase, which are also rich in tyrosine and lysine, do not have a targeting effect and cannot effectively bind to GCD to displace insulin.

[0096] Experimental Example 4

[0097] In this experimental example, the HEPES buffer provided in Comparative Example 3 was used as a blank group (HEPES), the separate GCD co-assembly and insulin provided in Comparative Examples 1 and 2 were used as control groups (GCD and INS), respectively, and the GCD-INS provided in Example 1 was used as an experimental group (GCD-INS).

[0098] This experimental example investigated cognitive impairment. In order to evaluate the spatial learning and memory ability of mice, the mice were subjected to the Morris water maze (MWM) test to evaluate their spatial memory ability.

[0099] The Morris water maze test was conducted in a circular pool (90 cm in diameter and 60 cm in height) filled with opaque water maintained at 20-22°C. The movement of mice was tracked using an overhead camera connected to a computer and tracking software. The pool was divided into four quadrants of equal area. In the initial training phase, a platform with a diameter of 10 cm was located in the center of the northwest quadrant, submerged 1 cm below the water surface. The training phase lasted for 5 days, starting randomly from 4 points (northeast, east, south, and southwest) each day. The time required for mice to find the platform (escape latency) was recorded. In the spatial exploration test phase (day 6), the platform was removed and the mice were allowed to explore the pool starting from the southeast quadrant. The test phase examined the time the mice stayed in the target quadrant and the number of times they crossed the platform.

[0100] See also Fig.10In a and b, mice were first intranasally administered for 30 days (injection of 25 μL (5 μL per drop, 5 drops in total) of GCD-INS co-assembly solution (containing GCD 20 μM and insulin 50 μM), GCD co-assembly solution (20 μM), insulin (50 μM), or HEPES, once a day for 1 month to establish the model), and then trained according to the above test method for 31-35 days, and the platform was removed on the 36th day for testing.

[0101] See also Fig.11 ,In the initial training stage, the escape latency of the 5×FAD+INS group and the 5×FAD+GCD group was shorter than that of the 5×FAD+HEPES group, and the escape latency of the 5×FAD+GCD-INS group was shorter than both, especially at 3-5 days, when it was close to the escape latency of the WT+HEPES group and the WT+GCD-INS group.

[0102] from Fig.12 As can be seen from a and b, the target quadrant residence time and platform crossing times of the 5×FAD+INS group and the 5×FAD+GCD group were significantly better than those of the 5×FAD+HEPES group, while the target quadrant residence time of the 5×FAD+GCD-INS group was significantly better than those of the 5×FAD+INS group and the 5×FAD+GCD group.

[0103] This suggests that GCD-INS co-assembly can significantly improve the learning ability of 5×FAD mice.

[0104] Experimental Example 5

[0105] In this experimental example, the HEPES buffer provided in Comparative Example 3 was used as a blank group (HEPES), the separate GCD co-assembly and insulin provided in Comparative Examples 1 and 2 were used as control groups (GCD and INS), respectively, and the GCD-INS provided in Example 1 was used as an experimental group (GCD-INS).

[0106] This experimental example examined the expression of synaptic-related proteins that are closely related to synaptic function and cognitive ability.

[0107] (1) In this experimental example, fluorescent Nissl was used for immunostaining. The specific method includes: using fluorescent Nissl to stain the hippocampal DG region of 6-month-old 5×FAD mice after intranasal administration (scale bar, 200 μm), and detecting the immunofluorescence intensity and the average intensity of Nissl labeling.

[0108] Please refer to the test results Fig.13In a and b, it can be seen that intranasal injection of INS, GCD and GCD-INS into 5×FAD mice can increase the fluorescence intensity in the hippocampus, and the fluorescence intensity of the 5×FAD+GCD-INS group is higher than that of the 5×FAD+HEPES group and higher than that of INS alone and GCD alone.

[0109] (2) The expression of N-methyl-D-aspartate receptor subunit 2A (NR2A), presynaptic membrane protein SYP, and postsynaptic density protein 95 (PSD95, a postsynaptic scaffold protein) in the hippocampus was detected by western blot. Fig.14 .

[0110] from Fig.14 It can be seen that intranasal injection of INS, GCD and GCD-INS into 5×FAD mice can increase the levels of NR2A, SYP and PSD95 in the hippocampus, while the levels of NR2A, SYP and PSD95 in the 5×FAD+GCD-INS group were higher than those in the 5×FAD+HEPES group and higher than those in INS alone and GCD alone.

[0111] Among them, β-Tubulin was used as the internal reference of WB experiment, and the above HEPES, INS, GCD and GCD-INS had no obvious effect on β-Tubulin in the brain of 5×FAD mice. Based on this, the optical density ratios of NR2A / β-Tubulin, PSD95 / β-Tubulin and SYP / β-Tubulin were quantitatively analyzed (n=5). The analysis results can be found in Fig.15 .

[0112] from Fig.15 As can be seen in a, nasal injection of INS, GCD, and GCD-INS into 5×FAD mice increased NR2A levels, among which the effect of GCD-INS injection was significantly better than that of INS alone and GCD alone. NR2A activation supports cell survival and synaptic plasticity (such as LTP), which is related to learning and memory. When NR2A levels are reduced, synaptic function is impaired and cell apoptosis increases.

[0113] from Fig.15 As shown in middle b, nasal injection of INS, GCD and GCD-INS into 5×FAD mice can increase the level of PSD95, among which the effect of GCD-INS injection is significantly better than that of INS alone and GCD alone. PSD95 is an important protein in the postsynaptic density region, and its decreased level can lead to impaired synaptic function and increased cell apoptosis. PSD95 reduction destroys synaptic plasticity and leads to impaired learning and memory ability; PSD95 reduction affects calcium signal regulation, which can cause mitochondrial dysfunction and cell apoptosis.

[0114] from Fig.15 As shown in middle c, nasal injection of INS, GCD and GCD-INS into 5×FAD mice can increase the level of SYP, among which the effect of GCD-INS injection is significantly better than that of INS alone and GCD alone. Reduced SYP levels will lead to decreased synaptic transmission efficiency, impaired synaptic function, and learning and memory disorders. Reduced SYP expression is associated with oxidative stress and mitochondrial dysfunction, and these changes will activate cell apoptosis signals and lead to neuronal death.

[0115] The results showed that GCD-INS co-assembly exhibited excellent recovery ability in the event of cell apoptosis and impaired synaptic function.

[0116] In summary, the co-assembly of macrocyclic amphiphilic compounds and insulin provided by the present invention utilizes insulin and macrocyclic amphiphilic compounds for co-assembly. The macrocyclic amphiphilic compounds have a certain cavity. When insulin is co-assembled with the macrocyclic amphiphilic compounds, it is contained in the cavity of the macrocyclic amphiphilic compounds. Among them, insulin can restore the sensitivity of nerve cells to insulin, and it can enhance the therapeutic effect of the co-assembly of macrocyclic amphiphilic compounds and insulin on Alzheimer's disease by improving metabolic damage, thereby improving the limitation of the prior art that only eliminates Aβ. The GCD-INS co-assembly treatment strategy adopted by the present invention breaks through the tradition, which can not only reduce plaque deposition and insulin resistance, but also reduce nerve cell apoptosis and synaptic damage, and improve cognitive impairment, thereby providing a more comprehensive therapeutic effect. It can be widely used in the preparation of drugs for treating or improving metabolic abnormalities in Alzheimer's disease, and the preparation of drugs for reducing Aβ in the hippocampus. 1-42 The invention is used in a preparation for measuring the signal intensity and plaque quantity of the peptide, a preparation for reducing insulin resistance, a preparation for reducing nerve cell apoptosis or reducing synaptic damage, or a preparation for improving cognitive impairment in mice with Alzheimer's disease.

[0117] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A co-assembly of a macrocyclic amphiphilic compound and insulin, characterized in that: The invention comprises insulin and a macrocyclic amphiphilic compound, wherein the macrocyclic amphiphilic compound is a co-assembly of calixarene modified with guanidino group and cyclodextrin.

2. The co-assembly of the macrocyclic amphiphilic compound and insulin according to claim 1, characterized in that: The molar ratio of the insulin to the macrocyclic amphiphilic compound is 0.8-1.2:

1.

3. A method for preparing a co-assembly of a macrocyclic amphiphilic compound and insulin as claimed in any one of claims 1 to 2, characterized in that: It comprises mixing said insulin and said macrocyclic amphiphile.

4. The method for preparing the co-assembly of the macrocyclic amphiphilic compound and insulin according to claim 3, characterized in that: Before the insulin is mixed with the macrocyclic amphiphilic compound, the insulin is first dissolved in a buffer solution to prepare a stock solution with a concentration of 80-120 mM, and then the stock solution is mixed with the macrocyclic amphiphilic compound at a molar ratio of 0.8-1.2:

1.

5. The method for preparing the co-assembly of the macrocyclic amphiphilic compound and insulin according to claim 3, characterized in that: The preparation method of the macrocyclic amphiphilic compound comprises: The guanidino-modified calixarene is dissolved in methanol to prepare a calixarene methanol solution with a concentration of 1-2 mM, and cyclodextrin is dissolved in chloroform to prepare a cyclodextrin chloroform solution with a concentration of 1-2 mM; The calixarene methanol solution and the cyclodextrin chloroform solution are mixed in a molar ratio of 1:1, dried to obtain a film on the container wall, a buffer solution is added to the container, and the solution is ultrasonically treated at 70-90° C. for 2-4 hours to obtain a co-assembly of guanidino-modified calixarene and cyclodextrin; Preferably, the buffer is a HEPES buffer with a concentration of 8-12 mM and a pH of 7-8.

6. Use of the co-assembly of the macrocyclic amphiphilic compound and insulin as claimed in any one of claims 1 to 2 in the preparation of a drug for treating or improving metabolic abnormalities in Alzheimer's disease.

7. The co-assembly of the macrocyclic amphiphilic compound according to any one of claims 1 to 2 and insulin in the preparation of a drug for reducing Aβ in the hippocampus 1-42 The signal intensity and plaque number of the preparations were applied.

8. Use of the co-assembly of the macrocyclic amphiphilic compound and insulin as claimed in any one of claims 1 to 2 in the preparation of a preparation for reducing insulin resistance; Preferably, the co-assembly of the macrocyclic amphiphilic compound and insulin is used in the preparation of a preparation for reducing abnormal phosphorylation of insulin receptor substrate-1 to improve insulin resistance; Preferably, the co-assembly of the macrocyclic amphiphilic compound and insulin is used in the preparation of a preparation for increasing the expression of glucose transporter 3 to improve insulin resistance.

9. Use of the co-assembly of the macrocyclic amphiphilic compound according to any one of claims 1 to 2 and insulin in the preparation of a preparation for reducing neuronal apoptosis or reducing synaptic damage.

10. Use of the co-assembly of the macrocyclic amphiphilic compound and insulin as claimed in any one of claims 1 to 2 in the preparation of a preparation for improving cognitive impairment in mice suffering from Alzheimer's disease.

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