WO2023065716A1 - 一种多肽及其作为cck受体激动剂/拮抗剂的应用 - Google Patents

一种多肽及其作为cck受体激动剂/拮抗剂的应用 Download PDF

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WO2023065716A1
WO2023065716A1 PCT/CN2022/102744 CN2022102744W WO2023065716A1 WO 2023065716 A1 WO2023065716 A1 WO 2023065716A1 CN 2022102744 W CN2022102744 W CN 2022102744W WO 2023065716 A1 WO2023065716 A1 WO 2023065716A1
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polypeptide
mice
following structures
group
compound
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French (fr)
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托特瑞拉米琦
贺菊方
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Centre For Regenerative Medicine And Health Hong Kong Institute Of Science & Innovation Chinese Academy Of Sciences Ltd
City University of Hong Kong CityU
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Centre For Regenerative Medicine And Health Hong Kong Institute Of Science & Innovation Chinese Academy Of Sciences Ltd
City University of Hong Kong CityU
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Priority to US18/702,790 priority Critical patent/US20240415918A1/en
Priority to JP2024547806A priority patent/JP7745858B2/ja
Priority to CA3224247A priority patent/CA3224247A1/en
Priority to AU2022370113A priority patent/AU2022370113B2/en
Priority to KR1020247016670A priority patent/KR102902210B1/ko
Priority to EP22882338.1A priority patent/EP4421082A4/en
Publication of WO2023065716A1 publication Critical patent/WO2023065716A1/zh
Priority to ZA2024/02706A priority patent/ZA202402706B/en
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Definitions

  • the invention relates to the technical field of chemistry and medicine, in particular to a polypeptide and its application as a CCK receptor agonist/antagonist.
  • Cholecystokinin is a humoral regulator synthesized and secreted by cells of the duodenum and jejunum, and is a polypeptide composed of 33 amino acids. It is widely distributed in the brain, especially in the cortex, striatum, hippocampus, cerebrum, septum, and hypothalamus, and is also secreted peripherally through the small intestine, where it plays an important role as a neurotransmitter or modulator.
  • CCK coexists with dopamine. It plays an important role in mechanisms including acetylcholine, gamma-aminobutyric acid, serotonin, opiates, growth hormone inhibition, hormone P and ion channels.
  • CCK can cause physiological changes, ptosis, hypothermia, hyperglycemia and stubbornness, and some behavioral changes, lack of exercise, decreased aggressiveness, lack of pain sensation, impact on learning knowledge, sexual behavior and satiety when eating Changes in abdominal sensation.
  • CCK receptors belong to G protein-coupled receptors and are widely distributed in the body.
  • CCK receptors include two subtypes, CCK-A receptors and CCK-B receptors, depending on their affinity for endogenous ligands. Both subtypes have been found in the peripheral and central nervous system.
  • CCK receptor agonists and antagonists can be used in the treatment of diet, obesity, gallbladder cancer, pancreatic cancer, epilepsy, depression and digestive system diseases caused by hyperacidity. But CCK receptor agonists have not been reported in the treatment of amnesia and dementia.
  • the present invention provides a polypeptide and its application as a CCK receptor agonist/antagonist.
  • the polypeptide has higher agonist/antagonist activity on CCK receptors.
  • the polypeptide of the present invention has a longer half-life in vivo and a longer-lasting curative effect, and has the effect of improving spatial memory defects in aged memory-deficient mice and Alzheimer's disease mice.
  • the present invention provides a polypeptide having the structure shown in formula I or its stereoisomer, prodrug, pharmaceutically acceptable solvate or salt:
  • X is an amide bond or a single bond
  • amino acid at the corresponding position is a D-type amino acid or an L-type amino acid
  • R 1 , R 2 , R 3 and R 4 are independently selected from any of the following structures:
  • R I , R II , R III , R VI are independently selected from halogen, nitro, C1-C4 alkyl, azido N 3 or cyano;
  • R 5 and R 6 are independently selected from H, substituted or unsubstituted C1-C4 alkyl groups
  • R 7 is selected from any of the following structures:
  • Biotin, AC, Fmoc, Cbz PEG100, PEG200, PEG300, PEG400, PEG600, PEG800, PEG1000, PEG1500, PEG2000,
  • R is selected from any of the following structures:
  • R is selected from any of the following structures:
  • R 3 is selected from any of the following structures:
  • R is selected from any of the following structures:
  • R I , R II, R III and R IV are independently selected from halogen, nitro, C1-C4 alkyl, azido or cyano.
  • polypeptide has the structure shown in formula II:
  • the R1 is selected from any of the following structures:
  • R is selected from any of the following structures:
  • R 3 is selected from any of the following structures:
  • R is selected from any of the following structures:
  • R 5 is selected from H or CH 3 .
  • polypeptide has the structure shown in formula III or formula IV:
  • R I , R II , R III , R IV are independently selected from halogen, nitro, C1-C4 alkyl, azido N 3 or cyano, and Rv is a carbon atom or a sulfur atom.
  • said R I , R II , R III , R IV are independently selected from H, F, Cl, Br, I, CN, N 3 , Me or NO 2 .
  • polypeptide provided by the present invention has any one of the structures in Table 1.
  • polypeptide structure compound 1-4 is shown as:
  • the polypeptide of the present invention is prepared by condensation reaction using amino acid and modified amino acid as raw material.
  • the present invention has no special limitation on the synthesis method, and either solid-phase synthesis or liquid-phase synthesis can be used.
  • the present invention relates to a pharmaceutical composition, comprising the polypeptide of the present invention, its pharmaceutically acceptable salt, stereoisomer or prodrug molecule, and a pharmaceutically acceptable excipient.
  • “Pharmaceutically acceptable excipients” may include pharmaceutically acceptable carriers, diluents, preservatives, solubilizers, stabilizers, disintegrants, binders, lubricants, wetting agents, emulsifiers, sweeteners, Flavoring agents, coloring agents, flavoring agents, salts, buffers, coating agents and antioxidants. Those skilled in the art know suitable excipients and techniques for formulating pharmaceutical compositions.
  • the present invention conducts in vitro CCK-B receptor agonistic and antagonistic experiments on the compound.
  • the above-mentioned compounds have better agonistic or antagonistic effects on CCK-B receptors, and can be used for diseases that require stimulation of cholecystokinin receptors through full or partial agonism or antagonism, such as obesity, depression, amnesia, or old age Treatment and prevention of diseases such as dementia. Therefore, the present invention also relates to the application of a polypeptide of any structure shown in formula I, II, III or IV as a CCK receptor agonist or antagonist, or in the preparation of medicines for treating or preventing CCK receptor-related diseases .
  • the CCK receptor-related diseases include at least one of amnesia, dementia, epilepsy, depression, obesity, gallbladder cancer, pancreatic cancer, and digestive system diseases.
  • amnesia includes anterograde amnesia and retrograde amnesia
  • dementia includes Alzheimer's disease (senile dementia), frontotemporal dementia, Parkinson's disease, Prion's disease (CJD), Lewy body dementia, Huntington's disease illness etc.
  • "Treatment or prevention" of amnesia or dementia specifically includes, but is not limited to, amelioration or partial amelioration of amnesia, improvement of learning processes, improvement of memory capacity, arrest or reversal of amnesia episodes, amelioration or arrest of amnesia or dementia complications such as anxiety, depression, irritability , loss of interest, social withdrawal, etc.
  • administering the polypeptide having the structure of formula I to the subject can significantly improve the subject's learning ability and improve the subject's memory deficit, and has a long-lasting effect.
  • the expression "effective amount” generally means an amount sufficient to produce a therapeutically desired result, wherein the exact nature of the result varies with the particular condition being treated.
  • the polypeptide having the structure of formula I of the present invention can be contained in a composition, especially a pharmaceutical composition, in an effective amount, that is, an amount suitable for treating or preventing amnesia or dementia in a subject, especially a mammal.
  • the subject may be a human or an animal, in particular the subject is a mammal, preferably a human. Therefore, the subject is preferably a human suffering from memory loss or dementia.
  • the effective amount of the polypeptide having the structure of Sequence I of the present invention may depend on the species, body weight, age and individual condition of the subject, and can be determined by standard methods such as experimental animals.
  • Predisposing factors for amnesia or dementia can include, but are not limited to, any type of injury or trauma to the central nervous system (CNS), including CNS degenerative and nondegenerative diseases, the former such as Alzheimer's disease, frontotemporal dementia , Prion's disease (Creut-Jakob disease), Lewy body dementia, Parkinson's disease, Huntington's disease, etc. Encephalitis, neurosyphilis, AIDS dementia, Ruan disease), traumatic brain dementia, normal intracranial pressure hydrocephalus, endocrine and metabolic disorders, poisoning, hypoxia and paraneoplastic syndromes, etc.
  • CNS central nervous system
  • the polypeptide having the structure of formula I according to the present invention can be administered to a subject by oral, injection, rectal, topical, parenteral, transdermal or inhalation routes.
  • the polypeptide having the structure of Formula I is administered to the subject by injection.
  • injection includes intraperitoneal, intravenous, intramuscular, subcutaneous and intradermal administration.
  • the polypeptide of the present invention has a longer half-life in vivo and a longer duration of curative effect.
  • the peptide has a significant improvement effect on the memory of aged and memory-deficiency mouse models, so it has great application potential.
  • Fig. 1 shows the curve diagram of CCK8 to CHO-CCK cell
  • Fig. 2 shows the fluorescence signal observation results of the posterior visual cortex of different treatment groups, A is the result of the blank control group without injection of compound HT-177, and B is the result of intraperitoneal injection of compound HT-177;
  • FIG 3 shows that intraperitoneal injection of CCK4 improves the behavioral performance of aged mice in the Morris water maze test; (A) the learning performance of the mice in the administration group and the control group in the positioning navigation experiment every day; (B) the administration group and the control group Trajectories of group mice in the space exploration experiment;
  • FIG. 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 the daily learning of mice in the CCK4 administration group and the control group in the positioning and navigation experiment Performance;
  • B Trajectory heat map of the mice in the CCK4-administered group and the control group in the space exploration experiment, and the dotted circle indicates the location of the original platform;
  • C The mice in the CCK4-administered group and the control group were in the space exploration experiment Proportion of exploration time in the target quadrant;
  • D The daily learning performance of mice in the HT-267 administration group and control group in the positioning navigation experiment;
  • E The space exploration experiment in the HT-267 administration group and control group mice Trajectory heat map, the dotted circle indicates the location of the original platform;
  • F HT-267 administration group and control group mice in the space exploration experiment in the proportion of the exploration time in the target quadrant;
  • Figure 5 shows that intraperitoneal injection of CCK4 and compounds HT-267, HT-177 and HT-178 improved the behavioral performance of memory-deficient mice in the Morris water maze test; Proportion of exploration time in the target quadrant in the exploration experiment; (B) Proportion of exploration time in the target quadrant of the mice in the compound HT-267, HT-177 and HT-178 administration groups and the control group in the space exploration experiment;
  • Figure 6 shows that intraperitoneal injection of compounds in compound HT-267 improves the behavioral performance of Alzheimer's mice in the novel object recognition test;
  • A The trajectory example of the mice in the familiarization period and the test period, the blue square represents the old objects, and yellow triangles represent new objects.
  • B Recognition index of new objects in administration group and control group mice;
  • Fig. 7 shows the recognition index of administration group and control group mice to new object in novel object recognition experiment;
  • Administration group 3 the compound improved the behavioral performance of memory-deficiency mice in the new object recognition experiment;
  • Figure 8 shows the long-term potentiation effect of compound HT-267 (administration group) on the cortex of aged mice; the left side of time point 0 is the basal field potential level before administration, and the right side of time point 0 is the field potential record after administration ;
  • Figure 9 shows the long-term potentiation effect of compound HT-267 (administration group) on the cortex of Alzheimer's disease mice; the left side of time point 0 is the basal field potential level before administration, and the right side of time point 0 is administration After the field potential recording;
  • Figure 10 shows the compound HT-267 (administration group 1), compound HT-177 (administration group 2), compound HT-178 (administration group 3) in the long-term enhancement of the compound on the cortex of memory-deficient mice
  • the effect, 100% is normalized to the basal field potential level before administration, and the graph shows the field potential level after administration.
  • the invention provides a polypeptide and its application as CCK receptor agonist/antagonist.
  • CCK receptor agonist/antagonist Those skilled in the art can refer to the content of this article to appropriately improve the process parameters to achieve.
  • all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention.
  • the method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously make changes or appropriate changes and combinations to the method and application herein without departing from the content, spirit and scope of the present invention to realize and apply the present invention Invent technology.
  • test materials used in the present invention are all common commercially available products, which can be purchased in the market.
  • Embodiment 1 The relationship between the structure of the polypeptide compound of the present invention and the agonistic activity of CCK receptors
  • Cell line CHO-CCK model cells
  • Detection reagent Fluo-8 No Wash Calcium Assay Kit (AAT Bioquest, #36316)
  • the compounds to be detected were prepared with HHBS.
  • the agonistic effect of CCK8 at 20nM has basically reached saturation, so the fluorescence intensity corresponding to this concentration is used as the 100% control in the analysis of agonist screening results. percentage.
  • the agonist/antagonist EC 50 of the compound can be obtained by drawing a fitting curve with the agonist/antagonist percentage as the ordinate and the compound action concentration as the abscissa.
  • NA means not detected.
  • the first amino acid tryptophan can be either D-type or L-type, but the L-type is more active, and the 2nd to 4th amino acids are respectively methionine/norleucine acid, aspartic acid and phenylalanine.
  • the 2nd to 4th amino acid is an L-type amino acid, which is necessary for the activity of polypeptide analogs; R2 is a norleucine side chain, and R4 is a 3-Br-substituted phenylalanine side chain, and the compound activity is better; R7 is acetyl Base time compounds are more active, such as compounds HT-177 and HT-267.
  • the above three polypeptides were respectively dissolved in a solution of 5% DMSO and 95% secondary deionized water. And injected into the tail vein of KM mice according to the dose of 1mg/kg respectively.
  • whole blood 100 ⁇ L was collected from the orbital vein at different time points, and whole blood samples were collected in heparinized tubes, and the plasma components were immediately separated by centrifugation, and the corresponding concentrations were analyzed by LC/MS/MS .
  • mice Male wild-type mice (C57BL/6) were housed under standard conditions with free access to food and water.
  • the Morris water maze is an experimental method designed by the British psychologist Morris in 1981 and applied to the study of brain learning and memory mechanisms. It is widely used in learning and memory, senile dementia, intelligence and aging, new drug development/screening/evaluation, etc. in the scientific research of the subject.
  • the Morris water maze experiment we used to test the spatial memory ability of mice mainly includes two parts: the positioning navigation experiment and the space exploration experiment. Among them, the positioning navigation experiment lasted 10 days. The mice were put into the water four times from 4 water entry points facing the pool wall every day, and the time it took to find the platform hidden under the water surface (escape latency period) was recorded.
  • the space exploration experiment is to remove the platform after 24 hours of the positioning and navigation experiment, then put the mouse into the pool, record its swimming track within one minute, examine the mouse's memory of the original platform, and calculate the mouse's time on the original platform.
  • the Morris water maze experiment we used to test Alzheimer's disease mice mainly includes two parts: positioning navigation experiment and space exploration experiment. Wherein the positioning navigation experiment lasted 8-10 days, the mice were put into the water from 4 water entry points facing the pool wall for four times every day, and the time it took to find the platform hidden under the water surface (escape latency period) was recorded.
  • the space exploration experiment is to remove the platform after the positioning navigation experiment, then put the mouse into the pool, record its swimming track within one minute, examine the mouse's memory of the original platform, and calculate the quadrant where the mouse is located on the original platform ( Proportion of exploration time in the target quadrant).
  • Alzheimer's mice have been shown to have memory deficits in the Morris water maze test, not only showing longer escape latencies for locating hidden platforms in the locating navigation test, but also showing poorer memory recall in the space exploration test ability.
  • mice with Alzheimer's disease were divided into four groups, the first group was given intraperitoneal injection of CCK4 solution, the second group was given intraperitoneal injection of the solvent of CCK4 solution as a control; the third group was given intraperitoneal injection of compound HT-267 solution, the Four groups were given intraperitoneal injection of the solvent of HT-267 solution as a control.
  • intraperitoneal injection of drugs was given before each training, and the treatment was performed four times a day, and the training was performed four times, for a total of 10 days of training.
  • FIG 4A As shown in Figure 4A, after 10 days of training, the escape latency of the CCK4 administration group was shorter than that of the control group under the same operation, indicating that the administration group showed better learning ability than the control group.
  • Figure 4B is a heat map of the trajectories of the two groups of mice in the space exploration experiment. In the figure below, the CCK4-administered group showed more space exploration trajectories at the original position of the platform, indicating that the mice have better memory recall ability for the platform position.
  • Figure 4C compares the proportion of time spent exploring in the target quadrant between the two groups of mice, and the CCK4-administered group was significantly higher than the control group.
  • Figure 4F compares the proportion of time spent exploring in the target quadrant between the two groups of mice, and the HT-267-administered group was significantly higher than the control group. Comparing Figure 4F with Figure 4C, it can be seen that the performance of the HT-267-administered group is slightly better than that of the CCK4-administered group. Considering that the number of treatment times and treatment days of HT-267 is less than that of CCK4, we believe that HT-267 is comparable to that of CCK4. It has better therapeutic effect than CCK4.
  • mice were housed under standard conditions with free access to food and water.
  • the Morris water maze experiment we used to test memory-deficient mice mainly includes two parts: positioning navigation experiment and space exploration experiment.
  • the experimental method is the same as in Example 5.
  • mice We randomly divided the memory-deficient mice into six groups, the first group was given intraperitoneal injection of CCK4 solution, the second group was given intraperitoneal injection of the solvent of CCK4 solution as a control; the third group was given intraperitoneal injection of compound HT-267 solution, the fourth group The compound HT-177 solution was given intraperitoneal injection, the fifth group was given intraperitoneal injection of compound HT-178 solution, and the sixth group was given intraperitoneal injection of compound solution solvent as a control.
  • the treatment and training methods of the first group and the second group are the same as those of the first group and the second group in Example 5, that is, four times of treatment every day, four times of training, and a total of 10 days of training;
  • the treatment and training methods of the third group to the sixth group are the same as those of the third group and the fourth group in Example 5, that is, treatment once a day, training four times, a total of 8 days of training.
  • each group tested the spatial memory ability of the mice in the same way.
  • Figure 5 compares the proportion of the time spent exploring the target quadrant in the space exploration experiment of each group of mice. It can be seen from Figure 5A that the proportion of the explored time in the CCK4 treatment group is significantly higher than that in the control group, indicating that CCK4 has a significant effect on memory deficit mice.
  • the spatial memory deficits of HT-267, HT-177 and HT-178 can be improved; as shown in Figure 5B, the proportion of exploration time in the HT-267, HT-177 and HT-178 treatment groups was significantly higher than that in the control group, indicating that the compounds HT-267, HT-177 and HT- 178 has the effect of improving spatial memory deficits in memory-deficient mice, and the HT-267 treatment group has the best effect; considering that the therapeutic effects of compounds HT-267, HT-177 and HT-178 are comparable to CCK4, but the number of treatments And the number of treatment days is less than CCK4, we think that the compounds HT-267, HT-177 and HT-178 have greater therapeutic potential for CCK4.
  • the new object recognition experiment is a highly verified method of cognitive memory. It was designed by Ennaceur & Delacour (1988) based on the exploration characteristics of rodents to new environments. The length of object exploration time is used to evaluate the memory function of the tested animals. If the tested animals do not forget the familiar objects they have seen in the environment, they will spend more time exploring the new objects they have not seen; Exploration times for familiar objects seen should be roughly the same.
  • the experiment is usually divided into three stages: 1. Adaptation period: Let the animals get familiar with the experimental environment without placing any objects in the experimental box; 2. Familiarization period: Place two identical objects in the experimental box and let the mice explore freely for 10 minutes ;3.
  • Test period put a new object in the experimental box to replace one of the old objects, and the position of the object remains unchanged. Record the mice's exploration with video, and analyze the time for mice to explore new and old objects, and calculate the ratio of the time that mice spend exploring new objects in the test period to the total time for exploring objects, that is, the recognition index for new objects.
  • mice Male triple transgenic Alzheimer's mice (3xTg-AD) were housed under standard conditions with free access to food and water. Alzheimer's mice have been shown to have memory deficits in new object recognition experiments, and their ability to recognize new objects is reduced.
  • the recognition index of the new object in the administration group was significantly higher than that in the control group, indicating that the administration improved the recognition memory of the mice.
  • mice were housed under standard conditions with free access to food and water.
  • the novel object recognition experiment we used to test memory-deficient mice mainly includes an adaptation period, a familiarization period, and a test period.
  • the experimental method is the same as in Example 12.
  • LTP Long-term potentiation
  • In vitro electrophysiological electrical signal recording method was used to investigate whether the compound HT-267 could induce long-term potentiation in the neocortex of Alzheimer's disease mice.
  • the MED64 low-noise multi-electrode recording system was used to record electrical signals from brain slices cultured in vitro. After stable recording of excitatory postsynaptic field potentials for at least 15 minutes, brain slices were infused with the compound HT-267 for five minutes, and the basal field potential was enhanced, and the enhancement lasted for more than one hour, as shown in Figure 9. It shows that the compound HT-267 can induce long-term potentiation in the neocortex of Alzheimer's disease mice.
  • In vitro electrophysiological signal recording method was used to study whether compound HT-267, compound HT-177, and compound HT-178 could induce long-term potentiation in the neocortex of memory-deficient mice.
  • the MED64 low-noise multi-electrode recording system was used to record electrical signals from brain slices cultured in vitro. After stable recording of the excitatory postsynaptic field potential for at least 15 minutes, compound HT-267 (administration group 1), compound HT-177 (administration group 2), and compound HT were injected into the brain slices of each group for five minutes.

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Abstract

一种多肽及其作为CCK受体激动剂/拮抗剂的应用，所述多肽对CCK受体具有较高的激动活性/拮抗活性。相比多肽CCK4，所述多肽在体内具有更长的半衰期及疗效持续时间。实验证明所述多肽对老年鼠、老年记忆缺陷型小鼠、阿尔兹海默症小鼠的空间记忆缺陷有明显改善作用，具有很大的应用潜力。

Description

一种多肽及其作为CCK受体激动剂/拮抗剂的应用
本申请要求于2021年10月20日提交中国专利局、申请号为202111222161.1、发明名称为“一种多肽及其作为CCK受体激动剂/拮抗剂的应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及化学医药技术领域，尤其涉及一种多肽及其作为CCK受体激动剂/拮抗剂的应用。
背景技术
胆囊收缩素(CCK)是由十二指肠和空肠细胞合成和分泌的一种体液调节因子,是由33个氨基酸组成的多肤。它广泛分布于脑，尤其是在皮质，纹状体，海马，大脑前盖，隔膜和下丘脑，也在外周通过小肠分泌，作为神经递质或调质发挥重要作用。它的主要生理作用是使胆囊收缩,刺激胰酶分泌,增加胰岛素的释放,增加肝胆汁的分泌,延缓胃排空,刺激粘液腺分泌,增强小肠蠕动以及抑制空回肠对钾、钠、氯和液体的吸收。在一些情况下，它可对动脉压有作用且可影响免疫系统。在一些中枢神经元中，CCK是与多巴胺共存的。其在包括乙酰胆碱，γ-氨基丁酸，5-羟色胺，鸦片制剂，生长激素抑制，素物质P和离子通道的机理中起重要作用。服用CCK可引起生理变化，上睑下垂，低温，高血糖和倔强症及一些行为上的变化，不爱运动，进取心下降，缺少痛觉，对学习知识的影响，性行为和对饮食时的饱腹感的变化。
CCK要发挥其生物学作用，首先要和存在于其靶细胞表面的CCK受体结合。CCK受体属于G蛋白偶联受体，在体内分布十分广泛。CCK受体根据其对内源性配基亲和力的不同包括两种亚型，即CCK-A受体和CCK-B受体。目前在外周和中枢神经系统中均已发现两种亚型。实验表明，CCK受体激动剂和拮抗剂可用作治疗饮食、肥胖、胆囊癌、胰腺癌、癫痫、抑郁以及由胃酸过多引起的消化系统疾病。但CCK受体激动剂对失忆和痴呆的治疗尚未见报道。
目前已有的肽类CCK受体激动剂和拮抗剂，半衰期很短，无法通过口服或注射在体内长效作用，因此制药意义不大。所以，开发新型的CCK受体激动剂，对相关疾病的治疗将有极大的应用意义。
发明内容
有鉴于此，本发明提供了一种多肽及其作为CCK受体激动剂/拮抗剂的应用。该多肽对CCK受体具有较高的激动活性/拮抗活性。相比CCK4，本发明多肽在体内半衰期更长，疗效更持久，对老年记忆缺陷型小鼠、阿尔兹海默症小鼠的空间记忆缺陷有改善作用。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供一种多肽，具有式Ⅰ所示结构或其立体异构体、前药、药学上可接受的溶剂化物或盐：
Figure PCTCN2022102744-appb-000001
其中：X为酰胺键或单键；
Figure PCTCN2022102744-appb-000002
为
Figure PCTCN2022102744-appb-000003
以使对应位置的氨基酸为D-型氨基酸或L-型氨基酸；
R 1、R 2、R 3和R 4独立地选自以下任一结构：
Figure PCTCN2022102744-appb-000004
Figure PCTCN2022102744-appb-000005
Figure PCTCN2022102744-appb-000006
R I、R II、R III、R VI独立地选自卤素、硝基、C1～C4的烷基、叠氮基N 3或氰基；
R 5、R 6独立地选自H、取代或未取代的C1～C4的烷基；
R 7选自以下任一结构：
Biotin、AC、Fmoc、Cbz、PEG100、PEG200、PEG300、PEG400、PEG600、PEG800、PEG1000、PEG1500、PEG2000，
Figure PCTCN2022102744-appb-000007
优选地，R 1选自以下任一结构：
Figure PCTCN2022102744-appb-000008
R 2选自以下任一结构：
Figure PCTCN2022102744-appb-000009
R 3选自以下任一结构：
Figure PCTCN2022102744-appb-000010
R 4选自以下任一结构：
Figure PCTCN2022102744-appb-000011
R I、R II、R III、R IV独立地选自卤素、硝基、C1～C4的烷基、叠氮基或氰基。
优选地，所述多肽具有式II所示的结构：
Figure PCTCN2022102744-appb-000012
优选地，式Ⅱ所示的多肽中，所述R 1选自以下任一结构：
Figure PCTCN2022102744-appb-000013
R 2选自以下任一结构：
Figure PCTCN2022102744-appb-000014
R 3选自以下任一结构：
Figure PCTCN2022102744-appb-000015
R 4选自以下任一结构：
Figure PCTCN2022102744-appb-000016
R 5选自H或CH 3。
优选地，所述多肽具有式III或式IV所示的结构：
Figure PCTCN2022102744-appb-000017
其中，R I，R II，R III，R IV独立地选自独立地选自卤素、硝基、C1～C4的烷基、叠氮基N 3或氰基，Rv为碳原子或硫原子。
优选地，式III所示的多肽中，所述R I，R II，R III，R IV独立地选自独立地选自H、F、Cl、Br、I、CN、N 3、Me或NO 2。
具体地，本发明提供的多肽具有表1结构中的任一种。
优选地，所述多肽结构化合物1～4所示：
(1)Ac-(D-Trp)-Met-Asp-Phe(3-Br)-NH 2
Figure PCTCN2022102744-appb-000018
(2)Ac-Trp-Nle-Asp-Phe(3-Br)-NH 2
Figure PCTCN2022102744-appb-000019
(3)Ac-(D-Trp)-Nle-Asp-Phe(3-Br)-NH 2
Figure PCTCN2022102744-appb-000020
(4)Fmoc-(N-Me-Trp)-Nle-Asp-Phe(3-Br)-NH 2
Figure PCTCN2022102744-appb-000021
本发明多肽以氨基酸和修饰氨基酸为原料通过缩合反应制得。本发明对合成方法没有特殊限定，可以使固相合成也可以液相合成。本发明对缩合反应的具体过程以及采用的试剂没有特殊限定，为本领域常用或熟知的即可。
另一方面，本发明涉及一种药物组合物，包括本发明所述的多肽、其药学上可接受的盐、立体异构体或前药分子，以及药学上可接受的赋形剂。
“药学上可接受的赋形剂”可以包括药学上可接受的载体、稀释剂、防腐剂、增溶剂、稳定剂、崩解剂、粘合剂、润滑剂、润湿剂、乳化剂、甜味剂、着色剂、添味剂、盐、缓冲剂、包衣剂和抗氧化剂。本领域技术人员知道用于配制药物组合物的合适的赋形剂和技术。
本发明对所述化合物进行了体外CCK-B受体激动和拮抗实验。实验表明，上述化合物具有较好的对CCK-B受体的激动或拮抗作用，可用于需要通过全部或部分激动或拮抗作用刺激胆囊收缩素受体的疾病，例如肥胖，抑郁，失忆，或老年痴呆等疾病的治疗和预防。因此，本发明还涉及式I、II、III或IV所示的任一结构的多肽作为CCK受体激动剂或拮抗剂的应用，或在制备治疗或预防CCK受体相关疾病的药物中的应用。
本发明中，所述CCK受体相关疾病包括失忆、痴呆、癫痫、抑郁、肥胖症、胆囊癌、胰腺癌、消化系统疾病中的至少一种。
其中，失忆包括顺行性失忆症和逆行性失忆症，痴呆包括阿尔兹海默症(老年痴呆)、额颞叶痴呆、帕金森症、Prion病(克-雅病)、路易体痴呆、亨廷顿病等。“治疗或预防”失忆或痴呆特别包括但不限于改善或部分改善失忆症、改善学习过程、提高记忆能力、阻止或逆转失忆发作、改 善或阻止失忆或痴呆并发症，如焦虑、抑郁、易怒、兴趣缺失、社会性退缩等等。在本发明的具体实施方式中，向受试对象施用具有式I结构的多肽可显著提高受试对象的学习能力及改善受试对象的记忆缺陷，并具有长效作用。
表述“有效量”通常表示足以产生治疗上期望的结果的量，其中结果的确切性质根据所治疗的特定病症而变化。本发明的具有式I结构的多肽可以以有效量，即适于治疗或预防受试对象，特别是哺乳动物失忆或痴呆的量包含在组合物，特别是药物组合物中。
受试对象可以是人或动物，特别地受试对象是哺乳动物，优选人。因此，受试对象优选是罹患失忆或痴呆的人。本发明具有序列式I结构的多肽的有效量可取决于受试对象的种类、体重、年龄和个体状况，并可通过标准方法如实验动物测定。
技术人员可以基于诱病因素确定受试对象是否需要根据本发明进行对失忆或痴呆的治疗。失忆或痴呆的诱病因素可以包括但不限于中枢神经系统(CNS)的任何类型的损伤或创伤，包括中枢神经系统变性疾病和非变性病，前者如阿尔茨海默病、额-颞叶痴呆、Prion病(克-雅病)、路易体痴呆、帕金森病、亨廷顿病等，后者如血管性痴呆、占位性病变(肿瘤、慢性硬膜下血肿、慢性脑脓肿)、感染(脑膜脑炎、神经梅毒、艾滋病痴呆、阮蛋白病)、脑外伤性痴呆、正常颅压性脑积水、内分泌代谢障碍、中毒、缺氧及副肿瘤综合征等。
根据本发明的具有式I结构的多肽可通过口服、注射、直肠、局部、肠胃外、经皮或吸入途径施用至受试对象。在受试对象是小鼠的实施方式中，具有式I结构的多肽通过注射施用至受试对象。术语注射包括腹膜内、静脉内、肌内、皮下和皮内给药。
与现有技术相比，本发明具多肽具有更长的体内半衰期，疗效持续时间更长。实验表明，该多肽发现对老年和记忆缺陷型小鼠模型的记忆有显著的改善作用，因此具有很大的应用潜力。
附图说明
图1示CCK8对CHO-CCK细胞作用曲线图；
图2示不同处理组后视皮层的荧光信号观察结果，A为未注射化合物HT-177的空白对照组结果，B腹腔注射化合物HT-177后的结果；
图3示腹腔注射CCK4提高了老年小鼠在Morris水迷宫测试中的行为表现；(A)给药组及对照组小鼠在定位航行实验中每天的学习表现；(B)给药组及对照组小鼠在空间探索实验中的轨迹图；
图4示腹腔注射CCK4和化合物HT-267提高了阿尔兹海默症小鼠在Morris水迷宫测试中的行为表现；(A)CCK4给药组及对照组小鼠在定位航行实验中每天的学习表现；(B)CCK4给药组及对照组小鼠在空间探索实验中的轨迹热图，虚线圆圈表示原平台所在位置；(C)CCK4给药组及对照组小鼠在空间探索实验中在目标象限探索时长占比；(D)HT-267给药组及对照组小鼠在定位航行实验中每天的学习表现；(E)HT-267给药组及对照组小鼠在空间探索实验中的轨迹热图，虚线圆圈表示原平台所在位置；(F)HT-267给药组及对照组小鼠在空间探索实验中在目标象限探索时长占比；
图5示腹腔注射CCK4和化合物HT-267、HT-177和HT-178提高了记忆缺陷型小鼠在Morris水迷宫测试中的行为表现；(A)CCK4给药组及对照组小鼠在空间探索实验中在目标象限探索时长占比；(B)化合物HT-267、HT-177和HT-178给药组及对照组小鼠在空间探索实验中在目标象限探索时长占比；
图6示腹腔注射化合物HT-267中化合物提高了阿尔兹海默症小鼠在新物体识别实验中的行为表现；(A)小鼠在熟悉期和测试期的轨迹示例，蓝色方块代表旧物体，黄色三角形代表新物体。(B)给药组及对照组小鼠对新物体的识别指数；
图7示给药组及对照组小鼠在新物体识别实验中对新物体的识别指数；腹腔注射化合物HT-267(给药组1)、HT-177(给药组2)、HT-178(给药组3)中化合物提高了记忆缺陷型小鼠在新物体识别实验中的行为表现；
图8示化合物HT-267(给药组)在老年鼠皮层上的长时程增强效应；时间点0左边为给药前的基础场电位水平，时间点0右边为给药后的场电位记录；
图9示化合物HT-267(给药组)在阿尔兹海默症小鼠皮层上的长时程增强效应；时间点0左边为给药前的基础场电位水平，时间点0右边为给药后的场电位记录；
图10示化合物HT-267(给药组1)、化合物HT-177(给药组2)、化合物HT-178(给药组3)中化合物在记忆缺陷型小鼠皮层上的长时程增强效应，100％为归一化后给药前的基础场电位水平，图示为给药后的场电位水平。
具体实施方式
本发明提供了一种多肽及其作为CCK受体激动剂/拮抗剂的应用。本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
本发明采用的试材皆为普通市售品，皆可于市场购得。
下面结合实施例，进一步阐述本发明：
实施例1本发明多肽化合物的结构对CCK受体的激动活性的关系
以氨基酸和修饰氨基酸为原料，通过缩合反应，生成多肽类似物化合物，按照以下方法测定多肽的活性，结构和活性如表1所示。
(一)试剂、材料:
细胞系：CHO-CCK模型细胞
检测试剂：Fluo-8 No Wash Calcium Assay Kit(AAT Bioquest,#36316)
阳性对照药：CCK8
(二)测试内容：
检测化合物作用于含CCK-B受体的模型细胞(CHO-CCK)后胞内钙离子浓度的变化。
(三)化合物配制方法：
1、将化合物用DMSO配制成10mM母液，再3倍梯度稀释成9个浓度梯度，成1000×储存液
2、(1)激动剂
用HHBS缓冲液将化合物储存液200倍稀释，成5×工作液
(2)拮抗剂
先在HHBS缓冲液中加入CCK8至终浓度50nM，再用该HHBS缓冲液200倍稀释化合物成5×工作液
(四)测试方案：
1、用含1％FBS的培养基铺CHO-CCK细胞96孔板，30000个细胞/孔/100μL，37℃培养过夜。
2、配制1×Fluo-8分析缓冲液：将9mL HHBS,1mL 10×Pluronic F127 Plus和20μL Fluo-8充分混匀备用。
3、将Fluo-8染料工作溶液加入细胞板，100μL/孔，先置于37℃培养箱中孵育30min，再取出置于室温避光孵育30min。
4、孵育细胞的过程中用HHBS配制待检测化合物。
5、逐行将化合物加入96孔CHO-CCK细胞板中，50μL/孔，然后立即放入酶标仪逐行检测Ex/Em＝490nm/520nm动态荧光信号变化。
(五)测试结果分析方法：
1、CCK8对CHO-CCK细胞作用曲线图见图1。
结果显示，CCK8作为阳性对照，认为其激动活性为最强，其高浓度作用效果为细胞的饱和状态。分析CCK8的浓度作用曲线，计算其激动效果达到饱和的90％即其EC 90≈10nM，所以将拮抗剂筛选方法中的CCK8浓度设为10nM，分析结果时，计算化合物与10nM CCK8同时作用细胞组信号占10nM CCK8单独作用细胞组信号的百分率。
CCK8 20nM时激动效果基本已达到饱和，所以将该浓度对应的荧光强度作为激动剂筛选结果分析中的100％对照，分析结果时，计算化合物 单独作用细胞组信号占20nM CCK8单独作用细胞组信号的百分率。
以激动/拮抗百分比为纵坐标，化合物作用浓度为横坐标绘制拟合曲线，即可得到该化合物的激动/拮抗EC 50。
表1多肽结构和活性
Figure PCTCN2022102744-appb-000022
Figure PCTCN2022102744-appb-000023
Figure PCTCN2022102744-appb-000024
Figure PCTCN2022102744-appb-000025
Figure PCTCN2022102744-appb-000026
Figure PCTCN2022102744-appb-000027
Figure PCTCN2022102744-appb-000028
Figure PCTCN2022102744-appb-000029
Figure PCTCN2022102744-appb-000030
Figure PCTCN2022102744-appb-000031
Figure PCTCN2022102744-appb-000032
Figure PCTCN2022102744-appb-000033
Figure PCTCN2022102744-appb-000034
Figure PCTCN2022102744-appb-000035
Figure PCTCN2022102744-appb-000036
Figure PCTCN2022102744-appb-000037
Figure PCTCN2022102744-appb-000038
Figure PCTCN2022102744-appb-000039
Figure PCTCN2022102744-appb-000040
Figure PCTCN2022102744-appb-000041
Figure PCTCN2022102744-appb-000042
Figure PCTCN2022102744-appb-000043
Figure PCTCN2022102744-appb-000044
Figure PCTCN2022102744-appb-000045
Figure PCTCN2022102744-appb-000046
Figure PCTCN2022102744-appb-000047
Figure PCTCN2022102744-appb-000048
Figure PCTCN2022102744-appb-000049
Figure PCTCN2022102744-appb-000050
Figure PCTCN2022102744-appb-000051
Figure PCTCN2022102744-appb-000052
Figure PCTCN2022102744-appb-000053
Figure PCTCN2022102744-appb-000054
Figure PCTCN2022102744-appb-000055
Figure PCTCN2022102744-appb-000056
Figure PCTCN2022102744-appb-000057
Figure PCTCN2022102744-appb-000058
Figure PCTCN2022102744-appb-000059
Figure PCTCN2022102744-appb-000060
Figure PCTCN2022102744-appb-000061
Figure PCTCN2022102744-appb-000062
Figure PCTCN2022102744-appb-000063
Figure PCTCN2022102744-appb-000064
Figure PCTCN2022102744-appb-000065
Figure PCTCN2022102744-appb-000066
Figure PCTCN2022102744-appb-000067
Figure PCTCN2022102744-appb-000068
Figure PCTCN2022102744-appb-000069
Figure PCTCN2022102744-appb-000070
Figure PCTCN2022102744-appb-000071
Figure PCTCN2022102744-appb-000072
Figure PCTCN2022102744-appb-000073
Figure PCTCN2022102744-appb-000074
Figure PCTCN2022102744-appb-000075
Figure PCTCN2022102744-appb-000076
Figure PCTCN2022102744-appb-000077
Figure PCTCN2022102744-appb-000078
Figure PCTCN2022102744-appb-000079
Figure PCTCN2022102744-appb-000080
注：NA表示未检测。
由表1中活性数据可以看出，第一个氨基酸色氨酸为D-型和L-型都可，但L-型活性更强，第2～4位的氨基酸分别为蛋氨酸/正亮氨酸、天冬 氨酸和苯丙氨酸。第2～4位氨基酸为L-型氨基酸是多肽类似物活性必需；R2为正亮氨酸侧链，R 4为3-Br代苯丙氨酸侧链时化合物活性较好；R 7为乙酰基时化合物活性较高，如化合物HT-177和HT-267。
实施例2
本实施例中，通过静脉给药后测定HT-267、HT-177和CCK4(HT-9)在KM小鼠(雄性)的药代动力学特性(n＝4)。具体的，将以上三种多肽分别溶于5％DMSO和95％二次去离子水的溶液中。并分别按照1mg/kg剂量注入KM小鼠尾静脉。单次给药后，分别于不同时间点从眶静脉取全血(100μL)，并在肝素化管中收集全血样本，血浆组分立即通过离心分离，并通过LC/MS/MS分析相应浓度。
表2
Figure PCTCN2022102744-appb-000081
注：～表示未检测到。
结果显示，CCK4在进行尾静脉注射后，在血液样本中不能被检测到CCK4的存在，说明其浓度低于检测线(50ng/ml)，可能的原因是CCK4在静脉注射后快速分解导致。检测化合物HT-267及HT-177都表现出较好的半衰期，分别为1.601小时和0.721小时。同时，Tmax的时间分别为0.033小时和0.25小时。此外，最大浓度(Cmax)也表现出较好的结果，尤其是HT-267达到了1929.095μg/L。
实施例3
成年(八周)野生型小鼠(C57BL/6)用100mg/kg剂量的戊巴比妥麻醉后，将小鼠头部轻轻固定在数字脑立体定位仪(RWD Life Science，China)上，注射AAV9-hsyn-cck-2.0-GFP(3.2×10 12vg/ml)病毒到皮层表面区域(DV＝300μm)的多个位点(250nl/每个位点)。对于皮质成像，进行3mm×3mm矩形开颅手术并植入盖玻片作为观察窗，硬脑膜没有被去除。病毒注射两周后，表达携带绿色荧光的CCK-B受体。当配体与这种人工受体结合时，会观察到绿色荧光信号，以宽场荧光显微镜来监测信号，如图2所示。
结果显示，打过上述病毒的小鼠在腹腔注射化合物HT-177溶液后，观察到绿色荧光信号(图2B)，说明HT-177药物穿过血脑屏障，与病毒表达的CCK-B受体结合。
实施例4
成年野生型小鼠(C57BL/6)在标准条件下饲养，可自由获得食物和水。Morris水迷宫是英国心理学家Morris于1981年设计并应用于脑学习记忆机制研究的一种实验手段，被广泛应用于学习记忆、老年痴呆、智力与衰老、新药开发/筛选/评价等多个学科的科学研究中。我们用于测试小鼠空间记忆能力的Morris水迷宫实验主要包括定位航行实验和空间探索实验两个部分。其中定位航行实验历时10天，每天将小鼠面向池壁分别从4个入水点放入水中四次，记录其寻找到隐藏在水面下平台的时间(逃避潜伏期)。空间探索实验是在定位航行实验24小时后，去除平台，然后将小鼠放入水池中，记录其在一分钟时间内的游泳轨迹，考察小鼠对原平台的记忆，并计算小鼠在原平台所在象限(目标象限)中探索时长占比。
使用Morris水迷宫实验测试老年野生型小鼠的空间记忆能力，并给予CCK4(HT-9)治疗。我们将老年小鼠随机分为两组，一组给予腹腔注射CCK4溶液，另一组给予腹腔注射药物溶剂作为对照。如图3A所示，在定位航行实验中，经过几天的训练，给药组的逃避潜伏期比对照组短，表示 给药组比对照组表现出更好的学习能力。图3B是两组小鼠在空间探索实验中的轨迹图，给药组在平台原位置表现出更多的空间探索轨迹，表示小鼠对平台位置有更好的记忆回溯能力。此实验表明CCK4对老年小鼠的空间记忆缺陷有改善作用。
实施例5
成年雄性三转基因阿尔兹海默症小鼠(3xTg-AD)在标准条件下饲养，可自由获得食物和水。我们用于测试阿尔兹海默症小鼠的Morris水迷宫实验主要包括定位航行实验和空间探索实验两个部分。其中定位航行实验历时8-10天，每天将小鼠面向池壁分别从4个入水点放入水中四次，记录其寻找到隐藏在水面下平台的时间(逃避潜伏期)。空间探索实验是在定位航行实验后去除平台，然后将小鼠放入水池中，记录其在一分钟时间内的游泳轨迹，考察小鼠对原平台的记忆，并计算小鼠在原平台所在象限(目标象限)中探索时长占比。阿尔兹海默症小鼠被证实在Morris水迷宫实验中有记忆缺陷，不仅在定位航行实验中表现出更长的定位隐藏平台的逃避潜伏期，在空间探索实验中也表现出更差的记忆回溯能力。
我们将阿尔兹海默症小鼠随机分为四组，第一组给予腹腔注射CCK4溶液，第二组给予腹腔注射CCK4溶液的溶剂作为对照；第三组给予腹腔注射化合物HT-267溶液，第四组给予腹腔注射HT-267溶液的溶剂作为对照。在CCK4治疗实验的定位航行训练中，因为CCK4半衰期太短(少于5分钟)，在每次训练前都给予腹腔注射药物，每天治疗四次，训练四次，一共训练10天。如图4A所示，经过10天的训练，CCK4给药组的逃避潜伏期比同样操作下的对照组短，表示给药组比对照组表现出更好的学习能力。图4B是两组小鼠在空间探索实验中的轨迹热图，下图CCK4给药组在平台原位置表现出更多的空间探索轨迹，表示小鼠对平台位置有更好的记忆回溯能力。图4C比较了两组小鼠在目标象限中探索的时长占比，CCK4给药组明显高于对照组。
在化合物HT-267治疗实验的定位航行训练中，因HT-267半衰期长达1.601小时，每天只需腹腔注射给药一次，即可作用于四次训练，因此每 天治疗一次，训练四次，一共训练8天。如图4D所示，经过8天的训练，HT-267给药组的逃避潜伏期比同样操作下的对照组短，表示给药组比对照组表现出更好的学习能力。图4E是两组小鼠在空间探索实验中的轨迹热图，下图HT-267给药组在平台原位置表现出更多的空间探索轨迹，表示小鼠对平台位置有更好的记忆回溯能力。图4F比较了两组小鼠在目标象限中探索的时长占比，HT-267给药组明显高于对照组。将图4F与图4C相比，可以看出，HT-267给药组的表现略优于CCK4给药组，考虑到HT-267的治疗次数和治疗天数少于CCK4，我们认为HT-267相比CCK4有更好的治疗效果。
此实验表明CCK4和化合物HT-267对阿尔兹海默症小鼠的空间记忆缺陷均有改善作用；且HT-267的治疗效果更好。
实施例6
成年记忆缺陷型小鼠在标准条件下饲养，可自由获得食物和水。我们用于测试记忆缺陷型小鼠的Morris水迷宫实验主要包括定位航行实验和空间探索实验两个部分。实验方法与实施例5相同。
我们将记忆缺陷型小鼠随机分为六组，第一组给予腹腔注射CCK4溶液，第二组给予腹腔注射CCK4溶液的溶剂作为对照；第三组给予腹腔注射化合物HT-267溶液，第四组给予腹腔注射化合物HT-177溶液，第五组给予腹腔注射化合物HT-178溶液，第六组给予腹腔注射化合物溶液的溶剂作为对照。其中，在定位航行实验中，第一组与第二组的治疗与训练方法与实施例5中第一组与第二组相同，即每天治疗四次，训练四次，一共训练10天；第三组到第六组的治疗与训练方法与实施例5中第三组与第四组相同，即每天治疗一次，训练四次，一共训练8天。空间探索实验中，各组对小鼠的空间记忆能力进行测试的操作相同。
图5比较了各组小鼠在空间探索实验中对目标象限进行探索的时长占比，由图5A可知，CCK4治疗组的探索时长占比明显高于对照组，说明CCK4对记忆缺陷型小鼠的空间记忆缺陷有改善作用；由图5B可知，HT-267、HT-177与HT-178治疗组的探索时长占比均明显高于对照组，说 明化合物HT-267、HT-177与HT-178对记忆缺陷型小鼠的空间记忆缺陷有改善作用，其中HT-267治疗组的作用最好；考虑到化合物HT-267、HT-177与HT-178的治疗效果与CCK4相当，但治疗次数和治疗天数少于CCK4，我们认为化合物HT-267、HT-177与HT-178相比CCK4有更大的治疗潜力。
此实验表明CCK4和化合物HT-267、HT-177和HT-178对记忆缺陷型小鼠的空间记忆缺陷均有改善作用，且化合物HT-267、HT-177与HT-178相比CCK4有更大的治疗潜力。
实施例7
新物体识别实验是认知记忆的一种高度验证方法，是Ennaceur&Delacour(1988)根据啮齿类对新环境的探索特性设计，该模型依据动物对环境中原来见过的熟悉物体和没有见过的新物体的探索时间的长短来评价被测试动物的记忆功能。倘若被测试动物没有遗忘环境中见过的熟悉物体，便会用更多的时间探索没有见过的新物体；若遗忘了见过的熟悉物体，则动物对环境中没有见过的新物体和见过的熟悉物体的探索时间应基本相同。实验通常分为三个阶段：1、适应期：让动物熟悉实验环境，实验箱内不放置任何物体；2、熟悉期：在实验箱内放置两个相同的物体，让小鼠自由探索10分钟；3、测试期：在实验箱中放入新物体以替换其中一个旧物体，物体位置保持不变。用视频记录小鼠探索情况，并分析小鼠探索新旧物体的时间，计算小鼠在测试期探索新物体的时间占探索物体总时间的比例，即对新物体的识别指数。
成年雄性三转基因阿尔兹海默症小鼠(3xTg-AD)在标准条件下饲养，可自由获得食物和水。阿尔兹海默症小鼠被证实在新物体识别实验中有记忆缺陷，对新物体的识别能力下降。我们将阿尔兹海默症小鼠随机分为两组，一组给予腹腔注射化合物HT-267溶液，另一组给予腹腔注射药物溶剂作为对照。如图6B所示，给药组对新物体的识别指数明显高于对照组，表示给药提高了小鼠的识别记忆。
实施例8
成年记忆缺陷型小鼠在标准条件下饲养，可自由获得食物和水。我们用于测试记忆缺陷型小鼠的新物体识别实验主要包括适应期、熟悉期、测试期。实验方法与实施例12相同。
我们将记忆缺陷型小鼠随机分为四组，一组给予腹腔注射药物溶剂作为对照组，给药组1给予腹腔注射化合物HT-267溶液，给药组2给予腹腔注射化合物HT-177溶液，给药组3给予腹腔注射化合物HT-178溶液。经过适应期和熟悉期训练，我们在测试期实验中对几组小鼠的识别记忆进行测试。图7比较了四组小鼠在测试期对新物体探索的识别指数，由图7可知，三个给药组对新物体的探索识别指数明显高于对照组。
此实验表明化合物HT-267、HT-177和HT-178对记忆缺陷型小鼠的识别记忆缺陷有改善作用。
实施例9
长时程增强(long-term potentiation，LTP)被研究者认为是记忆机制之一。我们使用体外电生理电信号记录方法，来研究化合物HT-267是否可以诱导老年鼠新皮层中的长时程增强。
我们使用MED64低噪音多电极记录系统来记录体外培养的脑片的电信号。在稳定记录至少15分钟的兴奋性突触后场电位之后，我们往脑片上注入五分钟的化合物HT-267，我们发现基础场电位得到增强，增强作用持续一小时以上，如图8所示。这个实验说明化合物HT-267能诱导老年鼠皮层中的长时程增强。
实施例10
使用体外电生理电信号记录方法，来研究化合物HT-267是否可以诱导阿尔兹海默症小鼠新皮层中的长时程增强。使用MED64低噪音多电极记录系统来记录体外培养的脑片的电信号。在稳定记录至少15分钟的兴奋性突触后场电位之后，往脑片上注入五分钟的化合物HT-267，结果发现基础场电位得到增强，增强作用持续一小时以上，如图9所示。说明，化合物HT-267能诱导阿尔兹海默症小鼠新皮层中的长时程增强。
实施例11
使用体外电生理电信号记录方法，来研究化合物HT-267、化合物HT-177、化合物HT-178是否可以诱导记忆缺陷型小鼠新皮层中的长时程增强。使用MED64低噪音多电极记录系统来记录体外培养的脑片的电信号。在稳定记录至少15分钟的兴奋性突触后场电位之后，分别往各组脑片上注入五分钟的化合物HT-267(给药组1)、化合物HT-177(给药组2)、化合物HT-178(给药组3)中化合物，发现各组脑片的基础场电位都得到增强，增强作用持续一小时以上，如图10所示，与给药前的基础场电位归一化计算之后，三组给药组的场电位都显著增强到120％-140％。这个实验说明化合物HT-267、化合物HT-177、化合物HT-178能诱导记忆缺陷型小鼠新皮层中的长时程增强。
以上仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (10)

  1. 一种多肽，其特征在于，具有式Ⅰ所示结构或其立体异构体、前药、药学上可接受的溶剂化物或盐：
    Figure PCTCN2022102744-appb-100001
    其中：X为酰胺键或单键；
    Figure PCTCN2022102744-appb-100002
    为
    Figure PCTCN2022102744-appb-100003
    以使对应位置的氨基酸为D-型氨基酸或L-型氨基酸；
    R 1、R 2、R 3和R 4独立地选自以下任一结构：
    Figure PCTCN2022102744-appb-100004
    Figure PCTCN2022102744-appb-100005
    R I、R II、R III、R IV独立地选自卤素、硝基、C1～C4的烷 基、叠氮基或氰基；
    R 5、R 6独立地选自H、取代或未取代的C1～C4的烷基；
    R 7选自以下任一结构：
    Biotin、AC、Fmoc、Cbz、PEG100、PEG200、PEG300、PEG400、PEG600、PEG800、PEG1000、PEG1500、PEG2000，
    Figure PCTCN2022102744-appb-100006
  2. 根据权利要求1所述的多肽，其特征在于，R 1选自以下任一结构：
    Figure PCTCN2022102744-appb-100007
    R 2选自以下任一结构：
    Figure PCTCN2022102744-appb-100008
    R 3选自以下任一结构：
    Figure PCTCN2022102744-appb-100009
    R 4选自以下任一结构：
    Figure PCTCN2022102744-appb-100010
    R I、R II、R III、R IV独立地选自卤素、硝基、C1～C4的烷基、叠氮基或氰基。
  3. 根据权利要求1所述的多肽，其特征在于，所述多肽具有式II所示的结构：
    Figure PCTCN2022102744-appb-100011
  4. 根据权利要求3所示的多肽，其特征在于，
    所述R 1选自以下任一结构：
    Figure PCTCN2022102744-appb-100012
    R 2选自以下任一结构：
    Figure PCTCN2022102744-appb-100013
    R 3选自以下任一结构：
    Figure PCTCN2022102744-appb-100014
    R 4选自以下任一结构：
    Figure PCTCN2022102744-appb-100015
    R 5选自H或CH 3。
  5. 根据权利要求1所述的多肽，其特征在于，所述多肽具有式III或式IV所示的结构：
    Figure PCTCN2022102744-appb-100016
    其中，R I，R II，R III，R IV独立地选自独立地选自卤素、硝基、C1～C4的烷基、叠氮基N3或氰基，Rv为碳原子或硫原子。
  6. 根据权利要求5所述的多肽，其特征在于，所述R I，R II，R III，R IV独立地选自独立地选自H、F、Cl、Br、I、CN、N 3、Me或NO 2。
  7. 根据权利要求1所述的多肽，其特征在于，为表1中HT-1～HT292化合物中的任一种。
  8. 一种药物组合物，其特征在于，包括权利要求1～7任一项所述的多肽、其药学上可接受的盐、立体异构体或前药分子，以及药学上可接受的辅料。
  9. 权利要求1～7任一项所述的多肽或权利8所述的药物组合物作为CCK受体激动剂或拮抗剂的应用，或在制备治疗或预防CCK受体相关疾病的药物中的应用。
  10. 根据权利要求所述的应用，其特征在于，所述CCK受体相关 疾病包括失忆、痴呆、癫痫、抑郁、肥胖症、胆囊癌、胰腺癌、消化系统疾病中的至少一种。
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