Polypeptides and their use as CCK receptor agonists / antagonists - Patents.com

Abstract

Polypeptide and its application as CCK receptor agonist / antagonist. This polypeptide has relatively high agonist / antagonist activity on CCK receptor. Compared with polypeptide CCK4, this polypeptide has a long half-life and long duration of therapeutic effect in vivo. Experiments have shown that this polypeptide has obvious improving effect on the spatial memory impairment of aged mice, aged memory impairment mice and Alzheimer's disease mice, so that said polypeptide has great application potential.

Description

[Technical field]

[0001] The present invention relates to the fields of chemical and pharmaceutical technology, in particular to polypeptides and their use as CCK receptor agonists / antagonists. [Background technology]

[0002] Cholecystokinin (CCK) is a humoral regulator synthesized and secreted by duodenal and jejunal cells, and is a polypeptide consisting of 33 amino acids. It is widely distributed in the brain, especially in the cortex, striatum, hippocampus, pretectal area, diaphragm, and hypothalamus, and is also secreted to the periphery through the small intestine, where it plays an important role as a neurotransmitter or neuromodulator. Its main physiological effects are contraction of the gallbladder, stimulation of secretion of pancreatic enzymes, increase in insulin release, increase in hepatic bile secretion, delay of gastric emptying, stimulation of secretion of mucous glands, enhancement of peristalsis of the small intestine, and inhibition of absorption of potassium, sodium, chloride, and liquid by the jejunum and ileum. In some cases, it can affect arterial pressure and affect the immune system. In some central nerve cells, CCK coexists with dopamine. It plays an important role in the mechanisms of acetylcholine, gamma-aminobutyric acid, serotonin, opiates, growth hormone inhibition, substance P, and ion channels. Ingestion of CCK can cause physiological changes, ptosis, hypothermia, hyperglycemia and rigidity, as well as some behavioral changes, lack of exercise, reduced aggression, lack of pain, effects on the learning of knowledge, changes in sexual behavior, and feelings of fullness when eating.

[0003] For CCK to play its biological role, it must first bind to CCK receptors present on the surface of target cells. CCK receptors belong to the G protein-coupled receptors and are widely distributed in vivo. There are two subtypes of CCK receptors based on their affinity for endogenous ligands: CCK-A receptors and CCK-B receptors. Two subtypes have been identified in both the peripheral and central nervous systems. Experiments have shown that CCK receptor agonists and antagonists can be used to treat diets, obesity, gallbladder cancer, pancreatic cancer, epilepsy, depression, and digestive disorders caused by excess stomach acid. However, no treatment of amnesia and dementia with CCK receptor agonists has been reported. Summary of the Invention [Problem to be solved by the invention]

[0004] At present, existing peptide CCK receptor agonists and antagonists have short half-lives and cannot exert long-term effects in vivo by oral administration or injection, so they are of little pharmaceutical value. Therefore, the development of new CCK receptor agonists is of great importance for application in the treatment of related diseases. [Means for solving the problem]

[0005] Summary of the invention In view of this, the present invention provides a polypeptide and its use as CCK receptor agonist / antagonist.The polypeptide has high agonist / antagonist activity on CCK receptor.Compared with CCK4, the polypeptide according to the present invention has a long half-life and effective period in vivo, and improves the spatial memory impairment of memory-impaired aged mice and Alzheimer's disease mice.

[0006] In order to achieve the above objectives of the invention, the present invention provides the following technical solutions:

[0007] The present invention relates to a compound of formula (I): [ka] Formula (I) [In the formula, X is an amide bond or a single bond; [ka] The amino acids at the corresponding positions may be D- or L-amino acids; R1, R2, R3 and R4 are independently: [ka] and R I , R II , R III and R IV is independently selected from the group consisting of halogen, nitro, C1-C4 alkyl, azido N3 and cyano; R5 and R6 are independently selected from the group consisting of H, and substituted or unsubstituted C1-C4 alkyl; R7 has the following structure: Biotin, AC, Fmoc, Cbz, PEG100, PEG200, PEG300, PEG400, PEG600, PEG800, PEG1000, PEG1500, PEG2000, [ka] selected from the group consisting of or a stereoisomer, prodrug, pharma- ceutically acceptable solvate or salt thereof.

[0008] Preferably, R1 has the following structure: [ka] selected from the group consisting of; R2 has the following structure: [ka] selected from the group consisting of; R3 has the following structure: [ka] selected from the group consisting of; R4 has the following structure: [ka] selected from the group consisting of; R I , R II , R III and R IV is independently selected from the group consisting of halogen, nitro, C1-C4 alkyl, azide and cyano.

[0009] Preferably, the polypeptide has the formula (II): [ka] Formula (II) It has a structure shown below.

[0010] Preferably, in the polypeptide of formula (II), R1 has the following structure: [ka] selected from the group consisting of; R2 has the following structure: [ka] selected from the group consisting of; R3 has the following structure: [ka] selected from the group consisting of; R4 has the following structure: [ka] selected from the group consisting of; R5 is selected from the group consisting of H and CH3.

[0011] Preferably, the polypeptide has formula (III) or (IV): [ka] Formula (III) [ka] Formula (IV) [In the formula, R I , R II , R III and R IV is independently selected from the group consisting of halogen, nitro, C1-C4 alkyl, azido N3 and cyano, and R V is a carbon or sulfur atom. It has a structure shown below.

[0012] Preferably, in the polypeptide of formula (III), R I , R II , R III and R IV is independently selected from the group consisting of H, F, Cl, Br, I, CN, N3, Me, or NO2.

[0013] In particular, the polypeptide provided by the present invention has any of the structures in Table 1.

[0014] Preferably, the polypeptide has the structure of compounds 1-4: (1) Ac-(D-Trp)-Met-Asp-Phe(3-Br)-NH2 [ka] 1 (2) Ac-Trp-Nle-Asp-Phe(3-Br)-NH2 [ka] 2 (3) Ac-(D-Trp)-Nle-Asp-Phe(3-Br)-NH2 [ka] 3 (4) Fmoc-(N-Me-Trp)-Nle-Asp-Phe(3-Br)-NH2 [ka] 4

[0015] The polypeptide of the present invention is produced by condensation reaction using amino acids and modified amino acids as raw materials. In the present invention, there is no particular limitation on the synthesis method, and solid phase synthesis or liquid phase synthesis can be used. In the present invention, there is no particular limitation on the specific steps of the condensation reaction and the reagents used, which are commonly used or well known in the art.

[0016] In another aspect, the present invention relates to a pharmaceutical composition comprising a polypeptide according to the present invention, a pharma- ceutically acceptable salt, stereoisomer or prodrug molecule thereof, and a pharma- ceutically acceptable excipient.

[0017] "Pharmaceutically acceptable excipients" may include pharma- ceutically acceptable carriers, diluents, preservatives, solubilizers, stabilizers, disintegrants, adhesives, lubricants, wetting agents, emulsifiers, sweeteners, colorants, flavors, salts, buffers, coating agents, and antioxidants. Those skilled in the art know suitable excipients and techniques for formulating pharmaceutical compositions.

[0018] In the present invention, in vitro CCK-B receptor agonism and antagonism experiments are carried out on the compounds.The experiments show that the above compounds have good agonist or antagonist effects on CCK-B receptor, and can be used to treat and prevent diseases such as obesity, depression, amnesia, or senile dementia, which require the stimulation of cholecystokinin receptor by full or partial agonism or antagonism.Therefore, the present invention also relates to the use of the polypeptide of any structure shown in formula (I), (II), (III) or (IV) as CCK receptor agonist or antagonist, or in the preparation of medicaments for treating or preventing CCK receptor-related diseases.

[0019] In the present invention, the CCK receptor-related disease includes at least one of amnesia, dementia, epilepsy, depression, obesity, gallbladder cancer, pancreatic cancer, and digestive system diseases.

[0020] Among these, amnesia includes anterograde amnesia and retrograde amnesia, and dementia includes Alzheimer's disease (senile dementia), frontotemporal dementia, Parkinson's disease, prion disease (Creutzfeldt-Jakob disease), Lewy body dementia, Huntington's disease, etc. "Treatment or prevention" of amnesia or dementia includes, but is not limited to, amelioration or partial amelioration of amnesia, improvement of learning process, improvement of memory ability, prevention or reversal of amnesic episodes, and amelioration or prevention of complications of amnesia or dementia such as anxiety, depression, irritability, loss of interest, and social withdrawal. In certain embodiments of the present invention, administration of a polypeptide having the structure of formula (I) to a subject significantly improves the subject's learning ability and improves the subject's memory impairment, resulting in long-term effects.

[0021] The term "effective amount" generally refers to an amount sufficient to produce a therapeutically desirable result, although the exact nature of the result will vary depending on the particular condition being treated. The polypeptide of the present invention having the structure of formula (I) may be included in a composition, particularly a pharmaceutical composition, in an effective amount, i.e., an amount suitable for treating or preventing amnesia or dementia in a subject, particularly a mammal.

[0022] The subject can be human or animal, but is particularly mammalian, preferably human.Therefore, the subject is preferably a human suffering from memory loss or dementia.The effective amount of the polypeptide of the present invention having the structure of formula (I) depends on the species, weight, age and individual condition of the subject, and can be determined by standard methods such as experimental animals.

[0023] Those skilled in the art can determine whether a subject needs to be treated for amnesia or dementia according to the present invention based on predisposing factors. Predisposing factors for amnesia or dementia can include, but are not limited to, any kind of damage or trauma to the central nervous system (CNS), including degenerative diseases of the central nervous system, such as Alzheimer's disease, frontotemporal dementia, prion disease (Creutzfeldt-Jakob disease), Lewy body dementia, Parkinson's disease, Huntington's disease, and non-degenerative diseases of the central nervous system, such as vascular dementia, space-occupying lesions (tumors, chronic subdural hematomas, chronic brain abscesses), infectious diseases (meningoencephalitis, neurosyphilis, AIDS dementia, prion disease), traumatic brain dementia, normal intracranial pressure hydrocephalus, endocrine metabolic disorders, poisoning, hypoxia, and paraneoplastic syndromes.

[0024] The polypeptide according to the present invention having the structure of formula (I) can be administered to a subject orally, by injection, rectally, topically, parenterally, transdermally, or by inhalation.In an embodiment in which the subject is a mouse, the polypeptide having the structure of formula (I) is administered to the subject by injection.The term injection includes intraperitoneally, intravenously, intramuscularly, subcutaneously, and intradermally.

[0025] Compared with the prior art, the polypeptide of the present invention has a longer half-life in vivo and a longer effective period. Experiments have shown that the polypeptide can significantly improve the memory of aged mice and mice with memory impairment, and has great potential for application. [Brief description of the drawings]

[0026] [Figure 1]FIG. 1 is a graph showing the effect of CCK8 on CHO-CCK cells. [Diagram 2] Figure 2 shows the results of observing the fluorescent signals in the posterior visual cortex in different treatment groups. A shows the results of the blank control group not injected with compound HT-177, and B shows the results of the group intraperitoneally injected with compound HT-177. [Diagram 3] Figure 3 shows that intraperitoneal injection of CCK4 improves the behavioral performance of aged mice in the Morris water maze test. (A) Daily learning performance of treated and control mice in a positioning navigation experiment; (B) Tracking of treated and control mice in a spatial exploration experiment. [Figure 4] Figure 4 shows that intraperitoneal injection of CCK4 and compound HT-267 improves the behavioral performance of Alzheimer's disease mice in the Morris water maze test. (A) Daily learning performance of CCK4-treated and control mice in the positional navigation experiment; (B) Tracking heat maps of CCK4-treated and control mice in the spatial exploration experiment, where the dotted circle indicates the original platform location; (C) Percentage of target quadrant exploration time of CCK4-treated and control mice in the spatial exploration experiment; (D) Daily learning performance of HT-267-treated and control mice in the positional navigation experiment; (E) Tracking heat maps of HT-267-treated and control mice in the spatial exploration experiment, where the dotted circle indicates the original platform location; (F) Percentage of target quadrant exploration time of HT-267-treated and control mice in the spatial exploration experiment. [Diagram 5] Figure 5 shows that intraperitoneal injection of CCK4 and compounds HT-267, HT-177 and HT-178 improves the behavioral performance of memory-impaired mice in the Morris water maze test. (A) The percentage of time spent exploring the target quadrant in the CCK4-treated group and the control group in the spatial exploration experiment; the percentage of time spent exploring the target quadrant in the HT-267, HT-177 and HT-178-treated group and the control group in the spatial exploration experiment. [Figure 6]Figure 6 shows that intraperitoneal injection of compound HT-267 improves the behavioral performance of Alzheimer's disease mice in a novel object recognition experiment. (A) Example of tracking of mice during the habituation and testing periods, where blue squares represent old objects and yellow triangles represent new objects; (B) Novel object recognition index by treated and control mice. [Figure 7] The novel object recognition index by mice in the treatment and control groups in the novel object recognition experiment is shown in Figure 7. Intraperitoneal injection of compounds HT-267 (treatment group 1), HT-177 (treatment group 2), and HT-178 (treatment group 3) improved the behavioral performance of memory-impaired mice in the novel object recognition experiment. [Figure 8] 8 shows the long-term potentiation effect of the compound HT-267 (treated group) on the cortex of aged mice. The left side of time 0 is the basal field potential level before treatment, and the right side of time 0 is the field potential recording after treatment. [Figure 9] 9 shows the long-term potentiation effect of the compound HT-267 (administration group) on the cortex of Alzheimer's mice. The left side of time 0 is the basal field potential level before administration, and the right side of time 0 is the field potential recording after administration. [Figure 10] 10 shows the long-term potentiation effects of compound HT-267 (treatment group 1), compound HT-177 (treatment group 2), and compound HT-178 (treatment group 3) on the cortex of memory-impaired mice. 100% indicates the basal field potential level before treatment and the field potential level after treatment after normalization. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] Detailed Description The present invention provides a polypeptide and its use as a CCK receptor agonist / antagonist.Those skilled in the art can appropriately improve process parameters by referring to the disclosure of this application.In particular, all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention.Although the method and use of the present invention have been described by preferred embodiments, it is obvious that those skilled in the art can modify or appropriately change, combine, realize and apply the method and use of the present specification without departing from the disclosure, spirit and scope of the present invention.

[0028] All of the materials used in the present invention are commercially available and can be purchased on the market.

[0029] The present invention will be further described below in conjunction with examples: EXAMPLES

[0030] Relationship between the structure of the polypeptide compound of the present invention and the agonist activity of the CCK receptor Amino acids and modified amino acids were used as raw materials to produce polypeptide analogues through condensation reactions, and the activity of the polypeptides was measured according to the following process. The structures and activities are shown in Table 1.

[0031] (1) Reagents and raw materials: Cell line: CHO-CCK model cells Detection Reagent: Fluo-8 No Wash Calcium Assay Kit (AAT Bioquest, #36316) Positive control: CCK8 (2) Testing: Changes in intracellular calcium ion concentration are detected after a compound acts on model cells containing CCK-B receptors (CHO-CCK). (3) Manufacturing method of the compound: 1. Compounds were formulated in 10 mM stock solution using DMSO and diluted in a 3-fold gradient to a series of 9 concentrations to form a 1000× stock solution. 2.1 Agonists Compound stock solutions were diluted 200-fold with HHBS buffer to make 5-fold working solutions. 2.2 Antagonists First, CCK8 was added to HHBS buffer to a final concentration of 50 nM, and then the compound was diluted 200-fold with the resulting HHBS buffer to give a 5× working solution.

[0032] (4) Test Scheme: 1. CHO-CCK cells are seeded at 30,000 cells / well / 100 μL into 96-well plates in medium containing 1% FBS and incubated overnight at 37° C. 2. Prepare 1x Fluo-8 assay buffer: 9 mL of HHBS, 1 mL of 10x Pluronic F127 Plus, and 20 µL of Fluo-8 thoroughly mixed for later use. 3. Add 100 μL / well of Fluo-8 dye working solution to the cell plate. Incubate in a 37°C incubator for 30 minutes, then remove and incubate at room temperature in the dark for 30 minutes. 4. During the cell culture process, the compounds to be detected were prepared in HHBS. 5. Add the compounds to the 96-well CHO-CCK cell plate at 50 μL / well, one line at a time, and then immediately place the plate in a microplate reader to detect the dynamic fluorescence signal changes at Ex / Em=490 nm / 520 nm, one line at a time. (5) Test results and analysis methods: 1. The effect of CCK8 on CHO-CCK cells is shown in Figure 1.

[0033] The results showed that CCK8, as a positive control, was considered to have the strongest agonist activity, and its high concentration effect was in the saturation state of the cells. The concentration-effect curve of CCK8 was analyzed, and the concentration at which its agonist effect reached 90% saturation, i.e., EC 90was calculated to be approximately 10 nM, so the concentration of CCK8 in the antagonist screening method was set to 10 nM. When analyzing the results, the ratio of the signal from the cell group treated with both the compound and 10 nM CCK8 to the signal from the cell group treated with 10 nM CCK8 alone was calculated.

[0034] Because the agonist effect of CCK8 at 20 nM reached substantial saturation, the fluorescence intensity corresponding to this concentration was used as the 100% control in the analysis of the agonist screening results. When analyzing the results, the ratio of the signal of the compound alone cell group to the signal of the 20 nM CCK8 alone cell group was calculated.

[0035] Compound agonism / antagonism EC 50 is obtained by plotting the fitting curve with the percentage of agonism / antagonism on the Y-axis and the concentration of compound on the X-axis.

[0036] Table 1: Polypeptide structures and activities [Table 1] TIFF2024541119000021.tif209170 TIFF2024541119000022.tif212170 TIFF2024541119000023.tif211170 TIFF2024541119000024.tif212170 TIFF2024541119000025.tif211170 TIFF2024541119000026.tif210170 TIFF2024541119000027.tif212170 TIFF2024541119000028.tif211170 TIFF2024541119000029.tif210170 TIFF2024541119000030.tif212170 TIFF2024541119000031.tif209170 TIFF2024541119000032.tif212170 TIFF2024541119000033.tif211170 TIFF2024541119000034.tif211170 TIFF2024541119000035.tif211170 TIFF2024541119000036.tif211170 TIFF2024541119000037.tif212170 TIFF2024541119000038.tif211170 TIFF2024541119000039.tif211170 TIFF2024541119000040.tif211170 TIFF2024541119000041.tif211170 TIFF2024541119000042.tif212170 TIFF2024541119000043.tif212170 TIFF2024541119000044.tif211170 TIFF2024541119000045.tif212170 TIFF2024541119000046.tif211170 TIFF2024541119000047.tif211170 TIFF2024541119000048.tif210170 TIFF2024541119000049.tif213170 TIFF2024541119000050.tif210170 TIFF2024541119000051.tif211170 TIFF2024541119000052.tif209170 TIFF2024541119000053.tif212170 TIFF2024541119000054.tif213170 TIFF2024541119000055.tif214170 TIFF2024541119000056.tif213170 TIFF2024541119000057.tif213170 TIFF2024541119000058.tif212170 TIFF2024541119000059.tif212170 TIFF2024541119000060.tif211170 TIFF2024541119000061.tif212170 TIFF2024541119000062.tif211170 TIFF2024541119000063.tif230170 TIFF2024541119000064.tif229170 TIFF2024541119000065.tif211170 TIFF2024541119000066.tif212170 TIFF2024541119000067.tif210170 TIFF2024541119000068.tif210170 TIFF2024541119000069.tif211170 TIFF2024541119000070.tif211170 TIFF2024541119000071.tif212170 TIFF2024541119000072.tif212170 TIFF2024541119000073.tif212170 TIFF2024541119000074.tif211170 TIFF2024541119000075.tif211170 TIFF2024541119000076.tif212170 TIFF2024541119000077.tif222170 TIFF2024541119000078.tif171170Note: "NA" indicates "not detected."

[0037] From the activity data in Table 1, it can be seen that the first amino acid, tryptophan, can be in the D- or L-form, but the L-form is more active, and the amino acids in positions 2-4 are methionine / n-leucine, aspartic acid, and phenylalanine, respectively. The amino acids in positions 2-4 are L-form amino acids and are necessary for the activity of the polypeptide analog. When R2 is the side chain of n-leucine and R4 is the side chain of 3-bromophenylalanine, the compound is more active. When R7 is an acetyl group, compounds such as HT-177 and HT-267 are more active. EXAMPLES

[0038] In the examples, the pharmacokinetic characteristics of HT-267, HT-177 and CCK4 (HT-9) after intravenous administration in KM mice (male) were measured (n=4). Specifically, the above three polypeptides were dissolved in a solution of 5% DMSO and 95% secondary deionized water, respectively, and injected into the tail vein of KM mice at a dose of 1 mg / kg, respectively. After a single dose, whole blood (100 μL) was taken from the orbital vein at different time points, and the whole blood samples were collected in heparinized tubes. Plasma components were immediately separated by centrifugation, and the corresponding concentrations were analyzed by LC / MS / MS.

[0039] Table 2 [Table 2] Note: "~" indicates "not detected."

[0040] The results showed that the presence of CCK4 could not be detected in blood samples after tail vein injection, indicating that its concentration was lower than the detection limit (50 ng / ml), probably due to the rapid degradation of CCK4 after intravenous injection. Both compounds HT-267 and HT-177 showed good half-lives of 1.601 and 0.721 hours, respectively. At the same time, the Tmax times were 0.033 and 0.25 hours, respectively. They also showed good results in terms of maximum concentration (Cmax), especially HT-267, which reached 1929.095 μg / L. EXAMPLES

[0041] After anesthetizing with pentobarbital at a dose of 100 mg / kg, adult (8-week-old) wild-type mice (C57BL / 6) were gently fixed onto a digital brain stereotaxic apparatus (RWD Life Science, China) via the mouse head and injected with AAV9-hsyn-cck-2.0-GFP virus (3.2 × 10 12 vg / ml) was injected (DV=300 μm). For cortical imaging, a rectangular craniotomy of 3 mm × 3 mm was performed and a cover slip was embedded as an observation window, and the dura was not removed. Two weeks after the virus injection, CCK-B receptors with green fluorescence were expressed. When the ligand bound to this artificial receptor, a green fluorescent signal was observed, as shown in Figure 2, which was observed under a wide-field fluorescence microscope.

[0042] The results showed that after intraperitoneal injection of compound HT-177 solution into the mice injected with the above virus, a green fluorescent signal was observed (Figure 2B), which indicated that the HT-177 drug crossed the blood-brain barrier and bound to the CCK-B receptor expressed by the virus. EXAMPLES

[0043] Adult wild-type mice (C57BL / 6) were raised under standard conditions with free access to food and water. The Morris water maze is an experimental technique devised by British psychologist Morris in 1981 and applied to the study of brain learning and memory mechanisms, and has been widely used in scientific research in learning and memory, senile dementia, intelligence and aging, new drug development / screening / evaluation and other fields. The Morris water maze experiment to test the spatial memory ability of mice mainly consisted of two parts: a positioning navigation experiment and a spatial exploration experiment. The positioning navigation experiment lasted for 10 days, and the mice were placed in the water four times a day from four entry points facing the pool wall, and the time they spent finding the hidden platform under the water surface (escape latency) was recorded. For the spatial exploration experiment, the platform was removed 24 hours after the positioning navigation experiment, and then the mice were placed in the pool and the swimming trajectory within 1 minute was recorded, the memory of the mice on the original platform was investigated, and the proportion of the mice's exploration time in the quadrant where the original platform was (target quadrant) was calculated.

[0044] Morris water maze experiments were used to test the spatial memory ability of aged wild-type mice and CCK4 (HT-9) treatment. Aged mice were randomly divided into two groups, one of which was intraperitoneally injected with CCK4 solution, and the other was intraperitoneally injected with the drug's solvent as a control. As shown in Figure 3A, the positioning navigation experiment showed that after several days of training, the escape latency of the treatment group was shorter than that of the control group, indicating that the treatment group showed better learning ability than the control group. Figure 3B is a trajectory diagram of the two groups of mice in the spatial exploration experiment, in which the treatment group showed more spatial exploration trajectories at the original position of the platform, indicating that the mice had better memory retrospective ability to the platform position. This experiment showed that CCK4 improved the spatial memory impairment of aged mice. EXAMPLES

[0045] Adult male 3xTg-AD transgenic mice were raised under standard conditions with free access to food and water. The Morris water maze experiment for testing Alzheimer's disease mice mainly consisted of two parts: a positional navigation experiment and a spatial exploration experiment. The positional navigation experiment lasted for 8 to 10 days, during which the mice were placed in the water four times a day from four entry points facing the walls of the pool, and the time they spent finding the hidden platform under the water surface (escape latency) was recorded. In the spatial exploration experiment, the platform was removed after the positional navigation experiment, and then the mice were placed in the pool, the trajectory they swam within one minute was recorded, the memory of the mice on the original platform was investigated, and the proportion of the exploration time of the mice in the quadrant where the original platform was located (target quadrant) was calculated. Alzheimer's disease mice were confirmed to have memory impairment in the Morris water maze experiment, which indicates not only a longer escape latency when locating the hidden platform in the positional navigation experiment, but also a reduced ability to retroactively recall memory in the spatial exploration experiment.

[0046] Alzheimer's disease mice were randomly divided into four groups, the first group was intraperitoneally injected with CCK4 solution, the second group was intraperitoneally injected with the solvent of CCK4 solution as a control, the third group was intraperitoneally injected with compound HT-267 solution, and the fourth group was intraperitoneally injected with the solvent of HT-267 solution as a control. In the positioning navigation training of the CCK4 treatment experiment, because the half-life of CCK4 is too short (less than 5 minutes), the drug was intraperitoneally injected before each training, four treatments were performed per day, and training was performed four times per day for a total of 10 days. As shown in Figure 4A, after 10 days of training, the escape latency of the CCK4-treated group was shorter than that of the control group in the same task, indicating that the treatment group showed better learning ability than the control group. Figure 4B shows that in the lower map, the CCK4-treated group showed more spatial exploration trajectories at the original position of the platform, indicating that the mice had better memory tracing ability to the platform position. FIG. 4C compares the percentage of exploration time in the target quadrant between the two mouse groups, which was significantly higher in the CCK4-treated group than in the control group.

[0047] In the positioning navigation training of the compound HT-267 treatment experiment, the half-life of HT-267 is up to 1.601 hours, so only one intraperitoneal injection per day is required and four training sessions can work, so the treatment was performed once a day and the training was performed four times a day for a total of eight days. As shown in Figure 4D, after eight days of training, the escape latency of the HT-267-treated group was shorter than that of the control group in the same task, indicating that the treatment group showed better learning ability than the control group. Figure 4E is a tracking heat map of the two groups of mice in the spatial exploration experiment, and in the lower map, the HT-267-treated group showed more spatial exploration trajectories at the original position of the platform, indicating that the mice had better memory tracing ability to the platform position. Figure 4F compares the proportion of exploration time in the target quadrant between the two mouse groups, and the HT-267-treated group was significantly higher than the control group. Comparing Figure 4F with Figure 4C, it can be seen that the HT-267-treated group showed slightly better results than the CCK4-treated group. Also, considering that HT-267 had fewer treatment times and fewer treatment days than CCK4, it was considered that HT-267 had a better effect than CCK4.

[0048] The experiment showed that both CCK4 and the compound HT-267 improved spatial memory impairment in Alzheimer's disease mice, with HT-267 being more effective. EXAMPLES

[0049] Adult memory-impaired mice were raised under standard conditions with free access to food and water. The Morris water maze experiment for testing memory-impaired mice mainly consisted of two parts: positioning navigation experiment and spatial exploration experiment. The experimental method was the same as that in Example 5.

[0050] The memory-impaired mice were randomly divided into six groups, the first group was intraperitoneally injected with CCK4 solution, the second group was intraperitoneally injected with the solvent of the CCK4 solution as a control, the third group was intraperitoneally injected with compound HT-267 solution, the fourth group was intraperitoneally injected with compound HT-177 solution, the fifth group was intraperitoneally injected with compound HT-178 solution, and the sixth group was intraperitoneally injected with the solvent of the compound solution as a control. In the positioning navigation experiment, the treatment and training methods of the first and second groups were the same as those of the first and second groups in Example 5, i.e., four treatments per day, four trainings per day, and a total of 10 days of training. The treatment and training methods of the third to sixth groups were the same as those of the third and fourth groups in Example 5, i.e., four treatments per day, four trainings per day, and a total of 10 days of training. In the spatial exploration experiment, the spatial memory ability of the mice was similarly tested for each group.

[0051] Figure 5A compares the proportion of exploration time of each group of mice in the target quadrant of the spatial exploration experiment. From Figure 5A, it can be seen that the proportion of exploration time of the CCK4 treatment group was significantly higher than that of the control group, which indicates that this group CCK4 improved the spatial memory impairment of memory-impaired mice. As can be seen from Figure 5B, the proportion of exploration time of the HT-267, HT-177, and HT-178 treatment groups was significantly higher than that of the control group, which indicates that compounds HT-267, HT-177, and HT-178 improved the spatial memory impairment of memory-impaired mice, with the HT-267 treatment group having the best effect. Compounds HT-267, HT-177, and HT-178 have the same therapeutic effect as CCK4, but considering that the number of treatments and the number of treatment days are fewer than CCK4, it is believed that compounds HT-267, HT-177, and HT-178 have better therapeutic potential than CCK4.

[0052] The experiment showed that CCK4 and compounds HT-267, HT-177 and HT-178 improved spatial memory impairment in memory-impaired mice, and compounds HT-267, HT-177 and HT-178 have better therapeutic potential than CCK4. EXAMPLES

[0053] The novel object recognition experiment is a highly validated method of recognition memory designed by Ennaceur & Delacour (1988) based on the exploratory characteristics of rodents into novel environments, this model evaluates the memory function of the tested animals based on the amount of time the animals spend exploring familiar objects they have seen in the environment and novel objects they have not seen. If the tested animal has not forgotten the familiar object in the environment, it is expected to spend more time exploring the novel object they have not seen; if the familiar object they have seen is forgotten, the animal should spend essentially the same amount of time exploring the novel object in the environment and the familiar object they have seen. The experiment is usually divided into three stages: 1. Adaptation period: the animal is allowed to get used to the experimental environment, and no objects are placed in the experimental box; 2. Habituation period: two identical objects are placed in the experimental box and the mouse is allowed to explore freely for 10 minutes; 3. Test period: a novel object is placed in the experimental box to replace one of the old objects, and the object's position is not changed. The mouse's exploration was video-recorded, and the time the mouse spent exploring the old and new objects was analyzed. The proportion of the time the mouse spent exploring the novel object to the total time exploring the objects during the test period was calculated, i.e., the novel object recognition index.

[0054] Adult male tritransgenic Alzheimer's disease mice (3xTg-AD) were housed under standard conditions with free access to food and water. Novel object recognition experiments showed that Alzheimer's disease mice had memory impairment and a reduced ability to recognize novel objects. The Alzheimer's disease mice were randomly divided into two groups, one of which was intraperitoneally injected with compound HT-267 solution, and the other was intraperitoneally injected with the drug's solvent as a control. As shown in Figure 6B, the novel object recognition index of the treatment group was significantly higher than that of the control group, indicating that the treatment improved the recognition memory of the mice. EXAMPLES

[0055] Adult memory-impaired mice are reared under standard conditions, with free access to food and water.The new object recognition experiment for testing memory-impaired mice mainly includes an adaptation period, a familiarization period and a test period.The experimental method is the same as that of Example 12.

[0056] The memory-impaired mice were randomly divided into four groups, one of which was intraperitoneally injected with the drug's solvent as a control group, group 1 was intraperitoneally injected with compound HT-267 solution, group 2 was intraperitoneally injected with compound HT-177 solution, and group 3 was intraperitoneally injected with compound HT-178 solution. After the adaptation and habituation period training, the recognition memory of several groups of mice was tested in the test period experiment. Figure 7 compares the novel object recognition index of the four mouse groups during the test period, and it can be seen from Figure 7 that the novel object recognition index of the three treatment groups was significantly higher than that of the control group.

[0057] This experiment showed that compounds HT-267, HT-177 and HT-178 improved recognition memory impairment in memory-impaired mice. EXAMPLES

[0058] Long-term potentiation (LTP) is considered by researchers to be one of the memory mechanisms. We used in vitro electrophysiological electrical signal recording methods to investigate whether the compound HT-267 can induce long-term potentiation in the neocortex of aged mice.

[0059] A MED64 low-noise multi-electrode recording system was used to record electrical signals from in vitro cultured brain slices. After stable recording of excitatory postsynaptic field potentials for at least 15 minutes, the compound HT-267 was injected into the brain slices for 5 minutes, and it was found that the basal field potentials were enhanced and the enhancement lasted for more than an hour, as shown in Figure 8. This experiment demonstrated that the compound HT-267 could induce long-term potentiation in the cortex of aged mice. EXAMPLES

[0060] Using an in vitro electrophysiological electrical signal recording method, we investigated whether the compound HT-267 can induce long-term potentiation in the neocortex of Alzheimer's disease mice. A MED64 low-noise multi-electrode recording system was used to record electrical signals from in vitro cultured brain slices. After stable recording of excitatory postsynaptic field potentials for at least 15 minutes, the compound HT-267 was injected into the brain slices for 5 minutes, and it was found that the basal field potentials were enhanced and the enhancement lasted for more than 1 hour, as shown in Figure 9. It was shown that the compound HT-267 can induce long-term potentiation in the neocortex of Alzheimer's disease mice. EXAMPLES

[0061] Using an in vitro electrophysiological electrical signal recording method, we investigated whether compound HT-267, compound HT-177, and compound HT-178 could induce long-term potentiation in the neocortex of memory-impaired mice. A MED64 low-noise multi-electrode recording system was used to record electrical signals in brain slices cultured in vitro. After stable recording of excitatory postsynaptic field potentials for at least 15 minutes, compound HT-267 (administration group 1), compound HT-177 (administration group 2), and compound HT-178 (administration group 3) were each injected into the brain slices for 5 minutes, and as shown in Figure 10, it was found that the basal field potentials of each group of brain slices were enhanced and the enhancement lasted for more than 1 hour. After normalization to the basal field potentials before administration, the field potentials of the three administration groups were significantly enhanced to 120-140%. This experiment showed that compound HT-267, compound HT-177, and compound HT-178 could induce long-term potentiation in the neocortex of memory-impaired mice.

[0062] It should be noted that the above is only a preferred embodiment of the present invention, and those skilled in the art can make many improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the protection scope of the present invention.

Claims

1. the below described: A polypeptide which is any one of the compounds HT-177, HT-178, HT-193, HT-199, HT-202, HT-230, HT-249, HT-251, HT-255, HT-256, HT-257, HT-258, HT-267, and HT-268 listed in the above.

2. A pharmaceutical composition comprising the polypeptide of claim 1 or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable excipient.

3. 13. Use of the polypeptide of claim 1 or the pharmaceutical composition of claim 2 in the preparation of a medicament for treating or preventing a CCK receptor-associated disease, wherein the CCK receptor-associated disease is selected from amnesia, dementia, epilepsy, depression, obesity, gallbladder cancer, and pancreatic cancer.

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