CN115212204B - Application of compound in preparation of medicine for treating Alzheimer disease - Google Patents
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- CN115212204B CN115212204B CN202210884725.6A CN202210884725A CN115212204B CN 115212204 B CN115212204 B CN 115212204B CN 202210884725 A CN202210884725 A CN 202210884725A CN 115212204 B CN115212204 B CN 115212204B
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
Abstract
The invention relates to a compound, or pharmaceutically acceptable salts, deuterated compounds, solvates, stereoisomers, esterification products and amidation products thereof, which are used for preparing a medicine for treating Alzheimer's disease, wherein the compound is ZL001 and has a structure shown in the following formula (I). Compound ZL001 is a mature tool drug whose compound patent has expired. In the prior art, most GLP-1R agonists are polypeptide macromolecules, ZL001 is taken as a GLP-1R agonist of a small molecule, and the production cost and the stability have incomparable advantages in clinical compliance. The compound ZL001 can improve the cognitive dysfunction of a3 xTg-AD mouse and reverse the LTP inhibition of the 3 xTg-AD mouse by acting on a postsynaptic mechanism, so that the compound ZL001 is a novel medicine compound for treating/relieving Alzheimer's disease, which is expected to be clinically applied.
Description
Technical Field
The invention relates to the field of new application of biological medicines, in particular to application of a compound in preparation of a medicine for treating Alzheimer's disease.
Background
Alzheimer's Disease (AD), also known as primary senile dementia, is a neurodegenerative Disease, which is manifested clinically by memory decline, persistent cognitive decline and dyskinesia. With the development of modern medical technology and the improvement of life quality, the aging degree of population is continuously increased, and Alzheimer's disease brings heavy burden to society and families.
There is no fully clear theorem about the pathogenesis of Alzheimer's disease. There are also a number of hypotheses concerning the various factors involved in its pathogenesis. Mainly comprises the assumptions of central cholinergic injury, abeta cascade theory, aging hypothesis, metabolic disorder, free radical apoptosis, tau protein phosphorization, antioxidant stress, calcium imbalance, genetics and the like. There is no mechanism or hypothesis to fully and accurately elucidate the pathogenesis of AD.
Numerous studies have shown that GLP-1 and its receptor (GLP-1 receptor), GLP-1R, are present in structural regions of the central nervous system that regulate cognitive functions. GLP-1R agonist can activate neuron pathway at specific part, and can remarkably increase nerve excitability of Hippocampus, hypothalamic collateral chamber nucleus, ventral medial nucleus and parabrainstem, and has effects of appetite regulation, cognitive function regulation, etc. GLP-1 and its mimics pretreatment can reduce cell death induced by beta-amyloid plaque accumulation, protect synapses from adverse effects of beta-amyloid on hippocampal synaptic plasticity, protect Long-term potentiation (Long-term potentiation, LTP) of AD model animals, and improve learning and memory and other cognitive dysfunction of AD model animals.
The inventors' prior patent CN202110103556.3 reports a known compound (hereinafter referred to as ZL 001) acting as a GLP-1R agonist. Glucagon-like peptide-1 (glp-1) enhances beta cell insulin secretion, and also reduces glucagon secretion by acting on alpha cells, thereby reducing hepatic glucose output, and acts on the center and stomach, suppressing appetite and slowing gastric emptying, thereby reducing beta cell load. GLP-1R agonists are therefore an effective hypoglycemic agent. In addition, GLP-1R agonists have been reported to have therapeutic effects including weight loss, cardiovascular benefits, and improvement of Alzheimer's disease and fatty liver.
GLP-1R agonists known in the prior art for treating or preventing diabetes mellitus are generally macromolecular polypeptides, such as liranafide, liraglutide and exenatide, and have great difficulty in synthesis and preparation and high cost; the need for injection administration results in poor patient compliance; GLP-1R agonists that have been approved for use today rely on importation and are expensive; and the stability of the polypeptide substances is easily influenced by temperature and pH. Therefore, the development of a small-molecule GLP-1R agonist for treating Alzheimer's disease has important significance.
Previous patents by the inventors have investigated that ZL001 is effective in stimulating insulin secretion and exhibits GLP-1R correlation. Based on the fact that ZL001 may be a small molecule GLP-1R agonist, which has some incomparable advantages over the GLP-1R agonists of the polypeptide macromolecules, the inventors continued further research into other therapeutic effects of ZL001 compounds as GLP-1R agonists, such as alzheimer's disease. The ZL001 is found to be a novel drug for treating AD, and has important scientific significance and commercial value.
Disclosure of Invention
The invention provides an application of a compound ZL001 serving as an oral small molecule GLP-1R agonist in treating Alzheimer's disease. ZL001 is a known compound having a molecular weight of < 300 and patents relating to ZL001 compounds have expired. ZL001 is taken as a known tool medicine of p38 MAPK and JNK signal paths, and participates in various cell activities such as cell proliferation, differentiation, apoptosis and the like through the p38 and JNK signal paths, so that the ZL001 has potential anti-tumor activity, can be used for developing anti-tumor medicines, and has been reported in a plurality of documents. However ZL001 has the effect of agonizing GLP-1R and is the first discovery of the inventors. Based on GLP-1R agonist action of ZL001, the invention provides a new application of ZL001 in preparing a medicament for treating Alzheimer's disease.
The invention solves the technical problems by the following technical proposal:
use of a compound ZL001, or a pharmaceutically acceptable salt, a deuterated compound, a solvate, a stereoisomer, an esterification product and an amidation product thereof, in the preparation of a medicament for treating alzheimer's disease, wherein the compound ZL001 has a structure shown in the following formula (I):
CAS number of compound ZL 001: 22862-76-6, methyl (2R, 3S, 4S) -2- [ (4-methoxyphenyl) methyl ] -3, 4-pyrrolidinediol 3-acetate, a pyrrolidine antibiotic isolated from Streptomyces griseus, is a known tool for the p38 MAPK and JNKs signaling pathway.
Further, the solvate is a hydrate, the esterification product is the product of the reaction esterification of hydroxyl and carboxylic acid on the compound ZL001, and the amidation product is the product of the amidation of secondary amino and carboxyl on the ZL 001. Still further, the carboxylic acid is at least one selected from formic acid, acetic acid, benzoic acid, citric acid, malic acid, gallic acid.
Furthermore, the invention also provides a pharmaceutical composition which takes the compound shown in the formula (I) or pharmaceutically acceptable salts, solvates, stereoisomers, esterification products and amidation products thereof as active ingredients and also comprises auxiliary materials.
Preferably, the compound ZL001 represented by formula (I) accounts for 1-95wt%, preferably 2-50wt%, more preferably 3-20wt% in the composition.
The auxiliary materials are selected conventionally in the art, and are specifically selected from at least one of excipient, stabilizer, pharmaceutically acceptable carrier, correctant, solvent, solubilizer, emulsifier, binder, disintegrating agent, filler, lubricant, osmotic pressure regulator, antiseptic, suspending agent, pH regulator, buffer, thickener, humectant and diluent.
Preferably, the compositions of the present invention are administered orally, by injection, nasally, orally, etc., preferably orally. ZL001 is used as a GLP-1R agonist of a small molecule, has better stability and compliance to patients compared with the GLP-1R agonist of a polypeptide macromolecule in the prior art, and is very suitable for oral administration. Specifically, the composition comprises oral solution, tablet, capsule, spray inhalant, granule, pill, gel, effervescent tablet, syrup, granule, etc.
The invention also provides application of the compound ZL001 or pharmaceutically acceptable salts, solvates, stereoisomers, esterification products and amidation products thereof or application of the pharmaceutical composition in preparing medicines for treating Alzheimer's disease. Experiments prove that the composition can relieve the symptoms of the AD model mice and improve the spatial learning and memory behaviors of the AD mice in the water maze.
Preferably, the pharmaceutical composition is administered at a dose of 0.5-30mg/kg/d, preferably 1-10mg/kg/d, more preferably 1-3mg/kg/d, based on the compound according to formula (I).
Preferably, the pharmaceutical composition is administered 1-3 times a day, such as once daily (QD), twice daily (BID), three times daily (TID).
Compared with the prior art, the invention has the following beneficial improvements:
1. the compound ZL001 is a mature tool medicine, the patent of the compound is expired, and the report of the compound as GLP-1R agonist is the first time. Compared with GLP-1R agonists which are polypeptide macromolecules in the prior art, ZL001 is taken as a GLP-1R agonist of a small molecule, and has incomparable advantages in clinical compliance, cost and stability.
2. The compound ZL001 provided by the invention has the advantages that the spatial learning and memory behaviors of the 3 xTg-AD mice are obviously improved, and the compound ZL001 effectively improves the synaptic plasticity injury of the 3 xTg-AD mice, which proves that the LTP inhibition of the 3 xTg-AD mice is reversed by acting on a postsynaptic mechanism. The compound ZL001 is a novel drug compound which is expected to be clinically applied for treating/relieving Alzheimer's disease.
Drawings
FIG. 1ForteBio Octet molecular interactions to detect the affinity of ZL001 with GLP-1R;
FIG. 2 effect of ZL001 on rat insulin secretion with or without GLP-1R blocker Exendin (9-39) intervention;
FIG. 3 effect of different doses ZL001 on glucose tolerance (OGTT) in C57BL/6 mice;
FIG. 4GLP-1R knockout mice were perfused with physiological saline, ZL001 or subcutaneously injected with Liraglutide, respectively, for blood glucose level detection;
FIG. 5 shows the results of a positioning navigation experiment for 5 consecutive days in the Morris water maze of each group of mice;
FIG. 6 is a representative swim trace plot of groups of mice in a Morris water maze space exploration experiment;
FIG. 7 the percent of total time in target quadrant swim time for each group of mice;
figure 8 escape latency in visual plateau experiments for each group of mice;
FIG. 9 is a graph showing the trend of the slope of fEPSP in the CA1 region of the hippocampus of each group of mice with time, and FIG. 9 is a statistical histogram of the slope of fEPSP in the CA1 region of the hippocampus of mice before and after high-frequency stimulation for 0min, 30min, and 60 min;
figure 10 statistical histograms of paired pulse facilitated comparisons for groups of mice.
Detailed Description
Compound ZL001 was purchased from shanghai Tao Su biochemical technology limited.
Example 1
The inventors have previously disclosed in patent CN202110103556.3 that ZL001 compounds act through GLP-1R agonists in stimulating insulin secretion in rats. Specifically, rat islets were incubated with 10. Mu.M ZL001 and 100nM Exendin (9-39) at a concentration of 11.1mmol/L glucose solution (11.1G). Exendin (9-39) is a specific competitive GLP-1R antagonist capable of blocking GLP-1R agonism by GLP-1R agonists. Exendin (9-39) blocked ZL001 insulinotropic effect at 11.1mmol/L glucose solution (11.1G), indicating that ZL001 insulinotropic effect is associated with agonism of GLP-1R.
In addition, the inventors have previously examined the affinity of ZL001 to GLP-1R by forteBio Octet molecular interactions in patent CN202110103556.3, demonstrating that there is a clear interaction and stronger affinity for ZL001 and GLP-1R.
To further verify that compound ZL001 is a small molecule GLP-1R agonist, the following experiments were also performed:
example 1 Compounds ZL001 and GCorrelation verification of LP-1receptor
1. ForteBio Octet molecular interaction detection of ZL001 affinity with GLP-1R
Construction of a rat GLP-1R vector: mRNA sequence of GLP-1R CDS region is obtained from NCBI, and total gene synthesis method is used together with PCR technology to obtain sequence fragment of rat GLP-1R. Agarose gel electrophoresis and gel cutting to recover GLP-1R rate gene fragments. Cloning the target gene GLP-1R into a vector pEX-3, carrying out enzyme digestion and electrophoresis on the PEX-3 by using EcoRI and BamHI enzymes, recovering the vector pEX-3 by using a DNA gel recovery kit, and cloning amplified fragments onto a linearized pEX-3 vector. Competent cells were obtained by calcium chloride method. The recombinant ligation product was transformed into competent cells, cloned colonies were picked, plasmids were minidrawn and positive clones were picked for identification. Sequencing and verifying recombinant plasmid, extracting in large quantity, sequencing 200ul of positive clone bacterial liquid, preserving the residual bacterial liquid by glycerol, comparing the sequencing result with the target gene sequence, inoculating the residual bacterial liquid to LB culture medium after the sequencing result and the target gene sequence are completely correct, and extracting in large quantity to enough recombinant plasmid. Cell-free GLP-1R protein expression: amplifying a target gene by adopting Hangzhou and Australian biotechnology Z1 high-fidelity DNA polymerase G-POL-001 or Z2 super-fidelity DNA polymerase G-POL-002, performing work over-expression on the target protein by using a TOB cell-free eukaryotic expression system, and then observing an expression result by using SDS-PAGE. And verifying an expression result by Western blot.
GLP-1R protein purification and concentration: 1) Construction of a rat GLP-1R vector containing his-tag and Tev cleavage sites at the N-terminus: a novel rat GLP-1R vector is constructed by adopting a QR molecular cloning and mutagenesis kit of Hangzhou and Australian biotechnology company, adding 6 his labels and Tev restriction sites at the N end of a target gene fragment of the constructed GLP-1R plasmid, and subcloning the target gene fragment into a PET vector. 2) Expression and purification of GLP-1R protein containing His tag and Tev restriction enzyme cleavage site at N end: successfully expressing GLP-1R protein by adopting an E.coli dialysis cell-free protein expression system of Hangzhou, happy Australian biotechnology limited company; the protein of interest was purified using a Histrap column. After obtaining the purified sample, 3. Mu.l was taken and loaded onto SDS-PAGE to observe the purification.
Detection was performed using a Fortebio Octet RE E detector available from Hangzhou double-day biotechnology Co. 1) Biotinylation of purified GLP-1R protein according to 1:1 to biotinylate the target protein. Proper amount of biotinylation reagent is added into the mixture to be in a water bath at 30 ℃ for 2 hours. After the reaction, excess biotin was removed by a gravity desalting column, and the SSA chip was solidified by eluting with PBS. 2) The compound ZL001 was diluted, ZL001 was dissolved in DMSO until completely dissolved, diluted 100-fold with PBST after dissolution, and diluted 100 μm with 1% dmso+pbst for subsequent detection. 3) The SSA chip solidified GLP-1R protein to detect ZL001, 21.2. Mu.M, 42.4. Mu.M, 84.8. Mu.M, 169.5. Mu.M, 339. Mu.M ZL001 solution and related reagents were added to the sample plate in the order of the order, and the instrument program was set and run. 4) Experimental data were processed using Fortebio data analysis 10.0.10.0 software.
Experimental results: for small molecules (molecular weight<2 kDa), the affinity is generally 10 -4 -10 -7 M. KD values represent objectively existing affinity levels. For accurate calculation of the affinity of the compound to GLP-1R, 5 concentration points (21.2. Mu.M, 42.4. Mu.M, 84.8. Mu.M, 169.5. Mu.M, 339. Mu.M) were chosen for the experiment for fitting. FIG. 1ForteBio Octet molecular interaction detection of the affinity of ZL001 to GLP-1R, the combination of ZL001 and GLP-1R is a reversible process, the combination time of the two is 60s, the dissociation time of the two is 60s, and the characteristics of rapid combination and rapid dissociation are presented. Affinity constant KD of ZL001 to GLP-1R is 2.41×10 -4 。
2. Insulin secretion test
1. Preparing a solution:
1) Preparation of Collagenase P: 50mg of Collagenase P powder and 500mg of BSA powder are weighed into a 50mL sterile centrifuge tube, 50mL of RPMI 1640 culture medium is added into a super clean bench for dissolution, and 1mg/mL of Collagenase P is prepared after uniform mixing. Filtering with 0.22 μm sterile filter, packaging 10mL of the filtrate, sealing, and placing in a refrigerator at-20deg.C.
2) Preparation of DispaseII solution: weighing 50mg of DispaseII powder in a 50mL centrifuge tube, adding 10mL of PBS (containing EDTA and no Ca) under aseptic condition 2+ 、Mg 2+ ) After being evenly mixed, the Dis with the concentration of 5mg/mL is preparedpaseII solution. Filtering, packaging 200 μl of each tube, sealing, and placing in a refrigerator at-20deg.C for use.
3) Preparing EDTA solution: 0.05583g EDTA powder was weighed into a 50mL centrifuge tube, and 50mL PBS (containing EDTA and no Ca) was added under aseptic conditions 2+ 、Mg 2+ ) And mixing uniformly to prepare 30mmol/L EDTA solution. Filtering, packaging 1mL of the mixture in each tube, sealing, and placing the mixture in a refrigerator at the temperature of minus 20 ℃ for standby.
4) Preparation of KRBH (Krebs-Ringer Bicarbonate HEPES) buffer solution: 128.8mmol/L NaCl,10HEPES,4.8KCl,2.5mmol/L CaCl 2 ，1.2mmol/LKH 2 PO 4 ，1.2mmol/L MgSO 4 ，5mmol/L NaHCO 3 The pH of the solution was adjusted to 7.4 and 2% BSA was added as required by the experiment.
5) Preparation of 1mol/L glucose: 9g of glucose powder is weighed into a 50mL centrifuge tube, 50mL of triple distilled water is added under the aseptic condition, and the mixture is uniformly mixed. Filtering, packaging 1mL of the mixture in each tube, sealing, and placing the mixture in a refrigerator at the temperature of minus 20 ℃ for standby.
6) Preparation of 1640 medium containing 10% fetal bovine serum: taking 500mL of RPMI 1640 culture based on an ultra-clean workbench, taking out 55mL of the culture medium, adding 50mL of fetal bovine serum and 5mL of 100 Xgreen streptomycin mixed solution, fully shaking the culture medium uniformly, sealing the culture medium, and placing the culture medium in a refrigerator at 4 ℃ for standby.
2. Isolation and culture of rat islet tissue
1) Preparation: a) The constant temperature oscillator was turned on to raise the temperature and kept at 37℃and the oscillation frequency was adjusted to 80r/min. b) Taking out the Collagenase P in a refrigerator at-20deg.C, thawing at normal temperature, pumping into a 20mL syringe, connecting PE pipe with the syringe needle, removing air, and placing on ice blocks. c) A sterile workbench is prepared, and surgical instruments required for experiments are placed.
2) Male wistar rats were sacrificed, and after the abdomen was sterilized with 75% ethanol, the abdomen was dissected with surgical scissors until the heart was exposed. Transferring the rat to a stereoscopic microscope to find the common bile duct, and clamping the junction of the common bile duct and the duodenum by using a hemostatic forceps. The common bile duct is dissociated under a body microscope, and a V-shaped small opening is sheared by the ophthalmology to prepare the intubation tube.
3) Carefully insert the PE catheter-attached syringe into the common bile duct, inject Collagenase P into the common bile duct at a constant speed, and ligate the cannula.
4) Loosening hemostatic forceps, peeling the complete pancreas into a culture dish, removing impurities, pouring into a 50mL centrifuge tube, and digesting for 11min in a constant temperature shaker.
5) Digestion was stopped by adding 10mL of 1640 medium containing 10% fetal bovine serum to the super clean bench, and 10mL of Hanks Buffer (HBSS) was added and the tube suspension was forced to shake to sediment. The 350 μm filter was filtered into a new sterile 50mL centrifuge tube and centrifuged at 1200r for 3 min.
6) The supernatant was discarded, 10mL of Hitopaque-1077 isolate was added, and the pellet was blown down with a pipette. Then 10mL of RPMI 1640 culture medium is sucked by a 10mL syringe, and the culture medium is injected at a constant speed along the wall of the centrifugal tube so as to cause obvious delamination. Centrifuge for 23min,3200 r.
7) The supernatant was aspirated by a pipette into a petri dish, islets were picked under a stereoscopic microscope with a 10. Mu.L pipette into another petri dish containing 10% fetal bovine serum 1640 medium, and placed at 37℃with 5% CO 2 Culturing in an incubator.
3. Experimental procedure
1) Preparation before experiment: (1) experiments were divided into 4 groups, ep tube numbers (7 per group); (2) preparing a KRBH solution, placing the solution in an incubator for incubation for 30min, and regulating the pH to 7.4 by NaOH; (3) preparing a sample to be tested: 11.1mmol/L glucose solution (11.1G), 11.1G+100nM Exendin (9-39), 11.1G+10. Mu.M ZL001, 11.1G+10. Mu.M ZL001+100nM Exendin (9-39).
2) Formal experiments: (1) adding 500 mu L of 2.8mmol/L glucose solution (2.8G) into each Ep tube, picking 5 islets (uniform in size and smooth in edge) into the Ep tubes under a split microscope, and placing into an incubator for incubation for 30min; (2) the supernatant was aspirated off with a pipette (note that islets were not aspirated), then 500. Mu.L of 11.1G, 11.1G+100nM Exendin (9-39), 11.1G+10. Mu.M ZL001, 11.1G+100nM Exendin (9-39) +10. Mu.M ZL001 were added to each group in order, and incubated in an incubator for 30min; (3) the supernatant was aspirated into an Ep tube labeled in advance with a pipette, mixed well, sealed, and subjected to Insulin (INS) radioimmunoassay.
4. Experimental results
The results of the experiment are shown in FIG. 2, in which Exendin (9-39) partially blocks ZL001 insulinotropic action at 11.1G concentration, indicating that ZL001 insulinotropic action is associated with GLP-1R agonism.
3. Effect of ZL001 on blood glucose in C57BL/6 mice
1) Male C57BL/6 mice of 6 weeks old, weight 18-22 g, purchased from Shanxi province people hospital laboratory animal center, adapted for one week, daily normal diet drinking water;
2) Mice were fixed with ear nails prior to the experiment, and were grouped and numbered. The randomization was divided into the following 3 groups: control group: lavage physiological saline; 15mg/kg ZL001 group: gavage 15mg/kg ZL001 solution; 30mg/kg ZL001 group: the stomach was irrigated with 30mg/kg ZL001 solution. Each group n=6.
3) The mice are fasted for 12 hours the day before the experiment, can drink water freely, and the wood chip padding in the animal cage is removed;
4) On the day of the experiment, a 20% dextrose solution was prepared: weighing 1g of anhydrous glucose, dissolving in 5ml of sterile triple distilled water, uniformly mixing, filtering with a 0.22 mu m filter, sterilizing, and preparing at present;
5) Preparation of ZL001 solution (5 mg/ml): ZL001 is weighed, physiological saline is added, hydrochloric acid is added, vortex ultrasonic treatment is carried out until the solution is uniform, and then NaOH is used for adjusting the PH to 6.8-7, so that the medicine is prepared.
6) Weighing the empty weight of the mice;
7) According to experimental design, the normal saline or ZL001 is infused into the stomach, the operation is gentle, and repeated stimulation to the mice is avoided. Each group of mice is respectively administrated with glucose solution (2 g/Kg) for gastric lavage, and the operation is gentle, so that repeated stimulation to the mice is avoided. Tail venous blood was collected for blood glucose level detection at 0min (baseline level), 15 min, 30min, 60min, 90 min,120 min, respectively, after gastric lavage. Blood glucose levels at different time points were measured with a blood glucose meter, respectively, and statistically processed.
As shown in FIG. 3, the ZL 001-administered group showed a different increase in the tolerance to sugar load after oral administration of glucose, compared with the control group, in which 30mg/kg ZL001 had a more remarkable effect of reducing sugar than 15mg/kg ZL 001.
Oral glucose tolerance test in 4GLP-1R knockout mice
1)GLP-1R -/- Mice purchased from the biotechnology company of the racing industry were fed adaptively for one week with daily normal diet;
2) Mice were fixed with ear nails prior to the experiment, and were grouped and numbered. GLP-1R -/- +saline group: administration of GLP-1R -/- Gastric lavage physiological saline of mice; GLP-1R -/- +zl001 group: administration of GLP-1R -/- Mice were lavaged with 30mg/kg ZL001 solution; GLP-1R -/- +liraglutide group: administration of GLP-1R -/- Mice were subcutaneously injected with 0.2mg/kg Liraglutide solution. Each group n=4.
3) The mice are fasted for 12 hours the day before the experiment, can drink water freely, and the wood chip padding in the animal cage is removed;
4) On the day of the experiment, a 20% dextrose solution was prepared: weighing 1g of anhydrous glucose, dissolving in 5ml of sterile triple distilled water, uniformly mixing, filtering with a 0.22 mu m filter, sterilizing, and preparing at present;
5) Preparation of ZL001 solution (5 mg/ml): weighing ZL001, adding normal saline, adding hydrochloric acid, performing vortex ultrasonic treatment to obtain uniform solution, and then adjusting the pH to 6.8-7 with NaOH; liraglutide solution (0.04 mg/ml) was prepared: liraglutide is weighed, added with triple distilled water and fully dissolved by ultrasonic treatment. ZL001 and Liraglutide solutions are all used in the prior art
6) Weighing the empty weight of the mice;
7) According to experimental design, respectively lavage normal saline, ZL001 or subcutaneous injection of Liraglutide, the operation is gentle, and repeated stimulation of mice is avoided. Each group of mice is respectively administrated with glucose solution (2 g/Kg) for gastric lavage, and the operation is gentle, so that repeated stimulation to the mice is avoided. After completion of the lavage, tail venous blood was collected at 0min (baseline level), 15 min, 30min, 60min, 90 min,120 min, respectively, for blood glucose level detection. Blood glucose levels at different time points were measured with a blood glucose meter, respectively, and statistically processed.
As shown in fig. 4, when the GLP-1R of the mice is knocked out, the effect of ZL001 and Liraglutide on the blood sugar of the mice is not significantly different from that of the physiological saline group, which indicates that the effect of the compound ZL001 and the known GLP-1R agonist Liraglutide is basically the same, and indicates that the compound ZL001 is a GLP-1R agonist.
Example 2 mouse Water maze test
2.1 test apparatus
2.2 test animals
Female APPSwe/PS1M146V/tauP301L 3 xTg-AD mice with a weight of 40.29 + -1.99 g and wild-type 31.52 + -1.52 g control mice were used for the experiment. Of these, 3×Tg-AD mice were purchased from Jakson Lab, U.S., and control mice were purchased from Peking Violet laboratory animal technologies Co. All animals are raised in SPF-class animal houses, the temperature of the animal houses is 20-25 ℃, and the animals are irradiated for 12 hours for dark circulation, so that adequate feeding and drinking water are ensured. All the operation flows accord with the administration and use guidelines of laboratory animals at Shanxi medical university.
At the time of rearing to 12 months of age, two mice were randomly divided into three groups: WT mouse control group (wt+ns); 3 XTg-AD mouse control group (3 XTg+NS); 3 XTg-AD mice ZL 001-dosed group (3 XTg+ZL001). Wherein, 25mg/kg ZL001 is administrated by intragastric administration in the administration group, equivalent physiological saline (NS) is administrated by intragastric administration in the control group, 1 time a day for 4 weeks, and then the behavioural experiment and the in vivo hippocampal field potential recording experiment (LTP) experiment are started, and the administration is still continuous during the experiment period.
2.3 Water maze test
The classical water maze test is mainly used for testing the long-term spatial reference learning and memory capacity of mice. The water maze consists of a circular uterine body with the diameter of 120cm and the height of 50cm, and different graphic marks are arranged on and around the uterine body wall. Before the experiment, tap water is injected into the uterus and titanium dioxide is added for uniform stirring. The water temperature is kept at (22+/-2) DEG C. With the aid of image acquisition and analysis software (Noldus Information Technology, the Netherlands), the uterus is divided into four quadrants, and an escape platform with the diameter of 12cm is placed in the center of a specific quadrant. The first stage of the classical water maze experiment is a positioning navigation experiment, and the surface of the platform is 1cm below the water surface for 5 days. The mice are put into water from any quadrant from the palace wall every day, the underwater platform searching study is carried out, and the escape latency period of the mice, namely the time required for the mice to enter water until the hidden target platform is found and climbs up, is recorded. If within 1min, the mouse is free to swim in the water and climb up the hidden target platform, it is allowed to rest on the platform for 15s to become familiar with the platform and markers around the platform. If the mouse did not find a hidden target platform in the water within 1min, it was manually guided to climb up the platform and stayed on the platform for 15s to familiarize with the platform and markers around the platform, with an escape latency of 60s. The second stage of the experiment is a space exploration experiment. The underwater platform is withdrawn the next day after the positioning navigation experiment is completed (namely, the sixth day of the experiment), two quadrants (except the target quadrant) are randomly selected, the mice are put into water twice, the mice are free to explore for 1min, and the swimming time and the track of the mice in the target quadrant are recorded. The third stage of the experiment is a visual platform experiment. To exclude interference of the mice with the experiment from differences in vision and swimming ability. The platform was raised 1cm above the water surface, two quadrants were randomly selected, mice were placed in sequence, and the average time and swimming speed of the mice to reach the platform were recorded. All data are expressed as mean±sem, data from day 1 to day 5 of the water maze and slope change of fEPSP are analyzed by repeated measures of ANOVA with SPSS18.0, and the remaining data are analyzed by one-way ANOVA with SPSS 18.0. Differences were considered statistically significant at P < 0.05.
Analysis of test results: fig. 5 shows the results of the positioning navigation experiments for 5 consecutive days in the Morris water maze for each group of mice. It can be seen that the escape latency was reduced to varying degrees for each group of mice with increasing training days. Wherein, on day 5, the wt+ns group has significantly reduced escape latency (P < 0.05) compared to the 3 xtg+ns group; the escape latency was significantly reduced in the 3 xtg+zl001 group compared to the 3 xtg+ns group (P < 0.05). This suggests that 3×Tg-AD mice have significant spatial learning impairment, and administration of ZL001 significantly ameliorates spatial learning impairment. Fig. 6 is a representative swim trace plot of each group of mice in a Morris water maze space exploration experiment. Fig. 7 is a graph of the percentage of time that mice swim in the target quadrant, and it can be seen that the percentage of time that the 3 xtg+ns group swim in the target quadrant is significantly reduced (P < 0.01) compared to the wt+ns group; the 3 xtg+zl001 group showed a significant increase in the percentage of swimming time in the target quadrant compared to the 3 xtg+ns group (P < 0.05). This indicates that 3×Tg-AD mice have severely impaired spatial memory function, and ZL001 administration can significantly improve spatial memory function. Fig. 8 is the escape latency of mice in a visual plateau experiment, and it can be seen that there is no statistical difference in the time to plateau for each group of mice. It was shown that the prolongation of escape latency and the reduction of target quadrant swimming time ratio in 3 xTg-AD mice are not due to differences in vision and motor ability, but are due to learning memory cognitive dysfunction. The ZL001 compound provided by the invention can obviously reduce the escape latency of a3 xTg-AD model mouse in a positioning navigation stage and can increase the swimming time percentage of the 3 xTg-AD model mouse in a target quadrant in a space exploration stage under the dosage of 25 mg/kg/day, namely the ZL001 compound can obviously improve the space learning and memory capacity of the 3 xTg-AD model mouse.
Example 3In vivo hippocampal field potential recording experiment of mice
After anesthesia, 3×Tg-AD mice and wild-type control mice were fixed in a brain stereotactic apparatus (Ruiwod Co., china) and stimulation electrodes and recording electrodes were positioned on the hippocampal Schaffer collateral and CA1 region pyramidal cell radiation layer, respectively, according to brain map coordinates. The hippocampal Schaffer collateral was stimulated with concentric stimulation electrodes, the postsynaptic potential of extracellular field excitability in the CA1 region was recorded by recording electrodes (Field excitatory postsynaptic potential, fEPSP), and the Long-term potentiation of fEPSP (Long-term potentiation, LTP) after administration of High Frequency Stimulation (HFS) and Paired pulse facilitation by dipulse stimulation (Paired-pulse facilitation, PPF) were observed.
Mice were intraperitoneally injected with 5% chloral hydrate (7 ml/kg dose) and fixed on a brain stereotactic apparatus after anesthesia. The skull is exposed by cutting the skin, and a small hole with the diameter of about 2mm is drilled at the position of 2mm behind the bregma and 1.5mm aside. After parallel binding, the stimulating electrode and the recording electrode are positioned vertically above the small hole and inserted into the brain. The extracellular fEPSP changes were recorded by continuous electrical stimulation with the stimulator to ensure that the stimulating electrode was located on the Schaffer collateral of the CA3 region of the hippocampus and the recording electrode was located on the CA1 pyramidal cell layer of the hippocampus. After the stable basic fEPSP is recorded, the current intensity is increased from 0 to 0.002mA to 0.2mA each time, the variation amplitude of the fEPSP is recorded, an I-O curve is made, and 50% of the current stimulation intensity corresponding to the maximum amplitude of the fEPSP is selected as the test stimulation intensity. Test stimulus was given once every 30s, stimulus was repeated 60 times, and fEPSP was recorded for 30 min. 3 bursts of paired pulse stimulation at 50ms intervals were then administered, each burst at 30s intervals, to guide PPF. PPF refers to the enhancement of postsynaptic responses by the 2 nd stimulus administered immediately after the completion of the 1 st stimulus, reflecting the change in presynaptic transmitter release probability, used to determine whether changes in synaptic plasticity are related to presynaptic mechanisms. Followed by 3 strings of high frequency stimulation at intervals of 30s, each string having 20 stimulation at 200Hz to induce the production of hippocampal LTP. After high frequency stimulation, the regulatory stimulator was returned to single stimulation at 30s intervals, and fEPSP was recorded over 60 min. After the recording was completed, the percentage change in slope of fEPSP before and after induction of high frequency stimulation between the groups was compared.
FIG. 9 is a graph showing the trend of the slope of fEPSP in the hippocampal CA1 region of each group of mice over time, and FIG. 9 is a statistical histogram of the slope of fEPSP in the hippocampal CA1 region of mice before high-frequency stimulation and 0min, 30min, and 60min after high-frequency stimulation. It can be seen that there was no statistical difference in basal fEPSP slope for each group of mice prior to administration of high frequency stimulation, indicating that there was no dysfunction and the same synaptic transmission in each group of mice. In high frequency stimulation induced LTP experiments, both wt+ns and 3 xtg+zl001 groups induced LTP, and the 3 xtg+ns group failed to induce LTP; within 0-60 min after induction of high frequency stimulation, the fEPSP slope of the 3 xTg+NS group is significantly reduced compared to the WT+NS group (P <0.05; P < 0.01); the slope of fEPSP was significantly increased in the 3 xTg+ZL001 group (P < 0.05) compared to the 3 xTg+NS group. The above results indicate that ZL001 significantly reversed LTP inhibition in 3 x Tg-AD mice. Fig. 10 is a statistical histogram of PPF comparisons for each group of mice. It can be seen that there was no statistical difference in the percentage of PPF in each group of mice, indicating that the presynaptic transmitter release was not affected in the 3×tg-AD mice, nor was compound ZL001 affecting presynaptic transmitter release.
LTP results showed that the 3 xtg-AD model mice were not successfully induced to LTP, and that compound ZL001 was successfully induced to LTP and maintained, indicating that compound ZL001 effectively improved synaptic plasticity impairment in 3 xtg-AD mice, ZL001 reversed LTP inhibition in 3 xtg-AD mice by acting on postsynaptic mechanisms.
Claims (9)
1. Use of a pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof, and an adjuvant in the manufacture of a medicament for the treatment of alzheimer's disease;
(I)
the mass ratio of the compound shown in the formula (I) is 1-95wt%.
2. Use according to claim 1, characterized in that the mass ratio of the compound of formula (I) is 2-50wt%.
3. Use according to claim 1, characterized in that the mass ratio of the compound of formula (I) is 3-20wt%.
4. The use according to claim 1, wherein the adjuvant is selected from at least one of excipients, stabilizers, flavors, solvents, solubilizers, emulsifiers, binders, disintegrants, fillers, lubricants, osmotic pressure regulators, preservatives, suspending agents, pH regulators, buffers, thickeners, humectants, diluents.
5. The use according to claim 1, wherein the composition is administered orally, by injection, nasally, by oral inhalation.
6. The use according to claim 1, wherein the pharmaceutical composition is administered in an amount of 0.5-30mg/kg/d based on the compound of formula (I).
7. The use according to claim 1, wherein the pharmaceutical composition is administered in an amount of 1-10mg/kg/d based on the compound of formula (I).
8. The use according to claim 1, wherein the pharmaceutical composition is administered in an amount of 1-3mg/kg/d based on the compound of formula (I).
9. The use according to claim 1, wherein the pharmaceutical composition is administered 1-3 times a day.
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| CN105377284A (en) * | 2013-05-30 | 2016-03-02 | 莫尔研究应用有限公司 | Compositions and methods for treatment of retinal degenerative diseases |
| CN106420712A (en) * | 2015-08-10 | 2017-02-22 | 上海市中医老年医学研究所 | Application of anisomycin to preparation of medicine for preventing/treating ovarian cancer |
| CN111548311A (en) * | 2020-06-04 | 2020-08-18 | 山西医科大学 | Small molecule GLP-1R agonist and application thereof |
| CN114790160A (en) * | 2021-01-26 | 2022-07-26 | 山西医科大学 | Anisomycin derivative and application of anisomycin and derivative thereof as GLP-1R agonist |
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| CN104127408A (en) * | 2014-07-23 | 2014-11-05 | 暨南大学 | Application of bacteria-originated pyrrolidine compound in preparation of immunosuppressor |
| CN106420712A (en) * | 2015-08-10 | 2017-02-22 | 上海市中医老年医学研究所 | Application of anisomycin to preparation of medicine for preventing/treating ovarian cancer |
| CN111548311A (en) * | 2020-06-04 | 2020-08-18 | 山西医科大学 | Small molecule GLP-1R agonist and application thereof |
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