WO2020253179A1 - Application d'un composé à petites molécules dans la préparation d'un médicament destiné à inhiber l'expression de la protéine tau - Google Patents

Application d'un composé à petites molécules dans la préparation d'un médicament destiné à inhiber l'expression de la protéine tau Download PDF

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WO2020253179A1
WO2020253179A1 PCT/CN2019/126698 CN2019126698W WO2020253179A1 WO 2020253179 A1 WO2020253179 A1 WO 2020253179A1 CN 2019126698 W CN2019126698 W CN 2019126698W WO 2020253179 A1 WO2020253179 A1 WO 2020253179A1
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tau protein
adda
disease
drug
medicine
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曾长江
赵�权
刘明
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成都山权江生物科技有限公司
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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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • 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
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • 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

Definitions

  • the present invention relates to the field of medicine. Specifically, the present invention relates to the application of a small molecule compound in the preparation of a medicine that inhibits the expression of Tau protein.
  • Alzheimer's disease also called senile dementia
  • AD Alzheimer's disease
  • senile dementia is a degenerative disease of the central nervous system with an insidious onset and a chronic progressive course. It is the most common type of senile dementia. [Burns A et al, BMJ., 338:467-471, 2009; WHO, “Dementia Fact sheet N°362", 2015] Mainly manifested as progressive memory impairment, cognitive impairment, personality change and language impairment Mental symptoms seriously affect social, professional and life functions [National Institute on Aging, "About Alzheimer's Disease: Symptoms", 2012].
  • AD Alzheimer's disease
  • AD Alzheimer's disease
  • Age gender (females are higher than males), education level, brain trauma, AD is also related to genetics, hypothyroidism, exposure to heavy metals, toxic chemicals and organic solvents, and others such as cerebrovascular disease, diabetes And the first depression in old age is also a risk factor for AD [Querfurth HW, LaFerla FM, NEJM, 362(4):329–44, 2010)].
  • APOE4 carriers have a significantly higher risk of early-onset and late-onset Alzheimer's disease. Carrying one copy of the gene will increase the risk by 4 times, while carrying two gene copies will increase the risk by 12 times.
  • APOE4 will affect the normal intake of blood sugar in the brain, leaving the brain in a state of energy deficiency. Compared with APOE2 and APOE3 carriers, APOE4 carriers have a lower blood glucose metabolism rate. Due to the long-term energy shortage of the brain, the function of neurons will be damaged, which will lead to the formation of plaques and tangles, and finally lead to neuronal apoptosis.
  • Tau protein Tau protein associated unit
  • the hyperphosphorylation of Tau protein will lead to the formation of tangles in neurons, causing microtubules to fall off and affect the transport of neurotransmitters and other substances in neurons, and gradually cause synapses to degenerate, axons disappear, and only left The remnant cell body of a neuron.
  • the uniqueness of the Tau protein theory is that it does not describe the cause of the disease.
  • amyloid or APOE4 or other factors that cause the disease, they will make neurons move toward a common fate: abnormal phosphorylation of Tau protein, synaptic degeneration and neuronal death.
  • Tau protein hypothesis can also provide a target for the development of new drugs, a major flaw of the Tau protein hypothesis is that there is currently little genetic evidence supporting the hypothesis. ⁇ Mudher M, Lovestone S, Trends in Neurosciences, 25:22-6, 2002. ⁇
  • the Tau protein hypothesis has not received the same attention as the amyloid hypothesis for a long time. But when the amyloid hypothesis encountered numerous obstacles, some proponents of the amyloid hypothesis began to turn to the Tau hypothesis.
  • Therapies supported by the Tau protein hypothesis so far mainly have the following directions: targeting tau protein-related kinase/phosphatase, or active and passive immunity, and reducing Tau protein phosphorylation through strategies such as anti-Tau protein aggregation inhibitors. Or inhibit Tau protein aggregation.
  • TauRx Therapeutics a company focused on the study of Tau protein aggregation inhibition, was established, and in 2004, the first drug, methylene blue, was advanced to clinical trials.
  • TauRx announced the results of phase II clinical trials at a conference, but the results caused a lot of controversy: the data showed that the two low-dose regimens of the drug are effective for moderate patients (non-early patients), and the clinical design The plan was also seriously questioned, and the company did not publish complete data on clinical trials after the meeting.
  • the abnormal phosphorylation state caused by the imbalance between the phosphorylation and dephosphorylation of Tau protein may play a very important role in the formation of neurofibrillary tangles, which can cause the Tau protein to detach from the microtubules and then aggregate with each other.
  • the abnormal phosphorylation state may be caused by increased kinase activity or decreased phosphatase activity.
  • GSK-3 plays a key role in the phosphorylation of Tau protein under physiological and pathological conditions. However, the development of GSK-3 inhibitors is extremely difficult, and it is difficult to obtain compounds with high selectivity. Due to too many GSK-3 substrates, it is difficult to control toxic and side effects.
  • microtubule stabilizers are commonly used drugs in tumor chemotherapy. Similar to GSK-3 inhibitors, the side effects of these drugs are also difficult to control.
  • BMS Bristol-Myers Squibb
  • Tau protein immunotherapy is also divided into active immunity and passive immunity. Active immunization uses antigens to activate human immune cells to produce antibodies against a certain type of pathogenic Tau protein, while passive immunization uses monoclonal antibodies directly. So far, active immunotherapy has entered the clinic with two vaccines: Axon Neuroscience SE's AADvac-1, and Janssen's ACI-35. ACI-35 is undergoing a phase Ib clinical trial, and AADvac-1 will complete the clinical trial for effectiveness verification in 2019.
  • AbbVie's monoclonal antibody ABBV-8E12 in passive immunotherapy is undergoing phase II clinical trials. After the failure of Verubecestat, Merck purchased the rights to a Tau monoclonal antibody from Teijin. Biogen purchased BMS-986168 from BMS, which is also in phase II clinical trials.
  • Parkinson's disease is another chronic central nervous system degenerative disease that mainly affects the motor nervous system.
  • the disease is named after the British doctor James Parkinson, who published the book "An Essay on the Shaking Palsy” in 1817, which first detailed the symptoms of Parkinson's disease. .
  • the symptoms of Parkinson’s disease usually appear slowly over time. The most obvious early symptoms are tremor, limb stiffness, hypokinesia, and abnormal gait. There may also be cognitive and behavioral problems [Parkinson's Disease Information Page.NINDS.2016-06 -30]. Dementia is quite common in severely ill patients, and major depressive disorder and anxiety disorders also occur in more than one-third of cases. Other possible symptoms include perception, sleep, and emotional problems [Shulman JM et al, Annual Review of Pathology, 6:193–222, 2011].
  • Parkinson's disease usually occurs in people over 60 years old, and about 1% of the elderly suffer from the disease; men are more likely to get Parkinson's disease than women. If the patient is younger than 50 years of age, it is called early-onset Parkinson's disease. The expected life expectancy after Parkinson’s disease is confirmed is about 7-14 years [Sveinbjornsdottir S, Journal of Neurochemistry, 139:318-324, 2016].
  • Parkinson's disease The cause of Parkinson's disease is still unclear, but it is generally believed to be related to genetic and environmental factors. People in the family with Parkinson's disease are more likely to get this disease, and those who have been exposed to certain pesticides and have had head trauma are also at higher risk.
  • the main motor symptoms caused by Parkinson's disease are collectively called Parkinson's syndrome.
  • the main motor symptom of Parkinson's disease is caused by the death of substantia nigra cells of the midbrain, resulting in insufficient dopamine in the relevant brain areas of the patient.
  • the cause of cell death is currently poorly understood, but it is known to be related to the process of neuronal protein forming Lewy bodies [Kalia LV; Lang AE, Lancet, 386(9996):896–912, 2015].
  • Parkinson’s disease is regarded as a synuclein disease, which is the same as the nerve formed by the accumulation of Tau protein in Alzheimer’s disease. Fiber entanglement is different.
  • synucleinopathy and Tau proteinopathy have clinical overlaps. Patients with severe Parkinson's disease often have typical symptoms of Alzheimer's disease (dementia), and they often find it in their brains. Nerve fiber entanglement [Galpern WR, Lang AE, Annals of Neurology, 59(3):449-458, 2006].
  • Tau is an important participant in neurodegenerative diseases and is related to Parkinson's disease.
  • the concentration of total Tau (t-Tau) in cerebrospinal fluid (CSF) and the ratio of cerebrospinal fluid/serum albumin gradually increase during the H and Y stages of Parkinson's disease.
  • CSF cerebrospinal fluid
  • BBB blood-brain barrier
  • Parkinson's disease There is currently no cure for Parkinson's disease.
  • the initial symptoms are usually treated with L-dopa.
  • dopamine agonists are used together.
  • neurons will continue to be lost, so the dose of the drug must be increased accordingly, but when the dose is just increased, side effects of dyskinesia such as involuntary tics will occur.
  • deep brain stimulation surgery in neurosurgery can be considered, which uses microelectrode discharge to reduce motor symptoms.
  • Parkinson's disease As for non-exercise-related symptoms of Parkinson's disease (such as patients with sleep disturbance or emotional problems), the treatment effect is usually poor [The National Collaborating Centre for Chronic Conditions, Symptomatic pharmacological therapy in Parkinson's disease. Parkinson's Disease.London: Royal College of Physicians. 2006: 59-100.; Kalia LV, Lang AE, Lancet, 386(9996):896-912, 2015.].
  • Histone post-translational modification regulates the organization and function of vertebrate genomes, and is a key participant in epigenetic regulation of gene activity [Di Lorenzo A, Bedford MT, FEBS Letters, 585(13): 2024-2031, 2011 ]. So far, most of the modified enzymes of PTM have been studied through isolation, in vitro modification or chemical inhibition or silencing/overexpression methods.
  • PRMT protein arginine methyltransferase
  • PRMT uses S-adenosyl-L-methionine (AdoMet) to catalyze the methylation of arginine to form monomethylarginine (MMA) and asymmetric (u-NG, u-NG-dimethyl- arginine or ADMA (Type I) or symmetric (u-NG, u-N0G-dimethylarginine or SDMA) (Type II) [Bedford MT, Clarke SG, Molecular Cell, 33:1-13, 2009].
  • PRMT participates in the regulation of chromatin structure and function through transcriptional activation, inhibition and its interaction with chromatin barrier elements [Aravind L, et al., Progress in Molecular Biology and Translational Science, 101: 105-76, 2011].
  • PRMT is also involved in pre-mRNA splicing, nuclear/cytoplasmic shuttling, cell cycle and DNA repair [Bezzi M, et al., Genes & Development, 27:1903-1916, 2013].
  • PRMT8 is a type I PRMT, which mainly exists in the neuronal area of the brain. It can asymmetrically dimethyl histone H4R3 to form H4R3me2a [Scaramuzzino C, PLoS One, 8: e61576, 2013].
  • the subcellular location of the enzyme is located in the plasma membrane because it can interact with membrane lipids using its N-terminal myristoylation motif.
  • the N-terminal region also contains two proline-rich motifs, allowing it to interact with several SH3 domains and PRMT2.
  • the enzyme activity of this enzyme exists in its N-terminal conformation, and its enzyme activity increases with the loss of this domain [Sayegh J, et al., Journal of Biological Chemistry, 282:36444-36453, 2007].
  • ADDA adenosine-2', 3'-dialdehyde
  • 2', 3'-dialdehyde adenosine its C 10 H 11 N 5 O 4 molecular weight is 265.23, and the structural formula is:
  • ADDA Alzheimer's disease and Parkinson's disease.
  • the purpose of the present invention is to provide ADDA for preparing inhibitors of Tau protein, and new uses in preparing drugs for treating Alzheimer's disease and Parkinson's disease.
  • the technical scheme of the present invention includes:
  • the Tau protein inhibitor is a drug that inhibits the expression of Tau protein.
  • the Tau protein inhibitor is a drug that inhibits the asymmetric dimethylation of the third arginine residue of histone H4.
  • the Tau protein inhibitor is a drug for inhibiting the formation of A ⁇ senile plaques in the brain.
  • the drug is a drug for treating diseases of the central nervous system.
  • the drug is a drug for treating chronic diseases of the central nervous system.
  • the drug is a drug for treating Alzheimer's disease and Parkinson's disease.
  • a drug for the treatment of central nervous system diseases which is a compound represented by formula (I) or its metabolites, tautomers, stereoisomers, pharmaceutically acceptable salts, hydrates and/or solvents
  • the compound is the active ingredient, plus pharmaceutically acceptable excipients or auxiliary ingredients to prepare a preparation; formula (I):
  • the auxiliary materials include conventional diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants, and synergists in the pharmaceutical field. Agent.
  • the preparation is an injection preparation or an oral preparation.
  • the injection preparation is a powder injection.
  • pharmaceutically acceptable salt refers to a salt formed by the compound of the present invention and an acid or a base suitable for use as a medicine.
  • Pharmaceutically acceptable salts include inorganic salts and organic salts.
  • the pharmaceutically acceptable excipient has certain physiological activity, but the addition of the component will not change the dominant position of the above-mentioned pharmaceutical composition in the course of disease treatment, but only exerts auxiliary functions. These auxiliary functions are only for the component The utilization of known activity is a commonly used adjuvant therapy in the medical field. If the aforementioned auxiliary components are used in combination with the pharmaceutical composition of the present invention, they should still fall within the protection scope of the present invention.
  • the ADDA of the present invention can effectively inhibit the expression of Tau protein, and thus can theoretically be used to treat various central nervous system diseases caused by Tau protein, especially neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease.
  • the ADDA of the present invention can inhibit the asymmetric double methylation of the third arginine residue of histone H4.
  • the ADDA of the present invention has the ability to penetrate the blood-brain barrier and is suitable for various administration methods.
  • ADDA of the present invention can inhibit the formation of A ⁇ senile plaques in the brain, so that it can be used to treat AD in theory; at the same time, the experiment of the present invention also proves that ADDA can be used to treat AD.
  • Figure 1 A statistical diagram of the effects of different concentrations of ADDA on the transcription of Tau genes in human astrocytes.
  • FIG. 1 Western blot detection of Tau protein in human astrocytes treated with different concentrations of ADDA.
  • Figure 3 The effect of PRMT8 expression interference on the level of histone modification H4R3me2a.
  • Figure 4 Detection diagram of PRMT8 and Tau gene expression.
  • Figure 5 HPLC detection chart of ADDA content in orbital blood (serum sample 0-4h) and brain tissue (brain tissue 0-4h).
  • Figure 6 A statistical diagram of the effect of ADDA on the expression of Tau mRNA in primary neurons of the mouse cerebral cortex.
  • Figure 7 Immunoblotting image of Tau protein in primary neurons of mouse cerebral cortex under ADDA treatment.
  • Figure 8 Immunoblot of H4R3me2a in primary neurons of mouse cerebral cortex under ADDA treatment.
  • Figure 9 The escape latency and escape route diagram of AD model mice in the water maze experiment.
  • Figure 10 Detection of senile plaque formation in the brain region of AD model mice.
  • ADDA adenosine-2', 3'-dialdehyde, 2', 3'-dialdehyde adenosine
  • ADDA adenosine-2’, 3’-dialdehyde, 2’, 3’-dialdehyde adenosine
  • add pharmaceutically acceptable excipients and make a medicine.
  • Test Example 1 Real-time PCR detection of the effect of ADDA on Tau mRNA expression
  • human astrocyte SVG p12 As the research object, the cells were treated with different ADDA concentrations to explore the effect of different ADDA on the expression of Tau mRNA in SVG p12. As shown in Figure 1, in human astrocyte SVG p12, compared with the control group, ADDA can significantly inhibit Tau mRNA expression; and, the higher the concentration of ADDA, the more obvious the inhibitory effect on Tau mRNA.
  • the EP tubes and Tip tips used must be treated with 0.1% DEPC water and used after damp heat sterilization), and let stand at room temperature for 5 minutes; add 200 mL of chloroform (chloroform), shake vigorously for 15 seconds, and stand at room temperature for 2 to 3 minutes. Centrifuge for 15 minutes at 12000'g, 4°C. Transfer the upper aqueous phase to a new enzyme-free EP tube, add 500 mL of isopropanol, and let it stand at room temperature for 10 minutes. Centrifuge at 12000g, 4°C for 10min. Remove the supernatant, add 1ml of 75% pre-cooled ethanol (prepared with 0.1% DEPC water), and vortex for a while.
  • chloroform chloroform
  • RNA reverse transcription procedures 7500'g, 4°C, centrifuge for 5min. Remove the supernatant, dry, and dissolve it in 20 mL of 0.1% DEPC water. Place it at 55°C for 10 minutes to promote dissolution. Use a spectrophotometer to determine the concentration and purity, and perform the following RNA reverse transcription procedures:
  • the product can be used in PCR reaction immediately or stored at -30°C for later use.
  • Real-time PCR uses the Rotor-gene 6000 quantitative PCR instrument from Gene's Corbett Research, the reagent is Roche FastStart Universal SYBR Green Master Mix, and the reaction system is as follows:
  • Tau primers The list of Tau primers is as follows:
  • the cells were treated with different ADDA concentrations to explore the effect of different ADDA on the expression of Tau protein in SVG p12.
  • RIPA cell lysate effective lysis component is 1% Triton X-100
  • the concentrated gel and separating gel of the appropriate concentration are prepared. Take 40-60 ⁇ g protein sample and mix it with 4'SDS loading buffer 1:4. Then heat at 100°C for 5 min, and centrifuge at 12000 rpm for 5 min. Add the protein sample to the loading channel, and add 5 ⁇ L of protein Marker to the left channel of the sample. After loading the sample, turn on the power and adjust the constant voltage for 90V electrophoresis. After the protein sample enters the separation gel, adjust the voltage to 120V to continue electrophoresis. When the bromophenol blue front reaches the bottom of the separation gel, stop the electrophoresis and take out the gel. . Remove the gel sandwich and take out the gel.
  • the gel corresponding to the target band.
  • Cut the PVDF membrane corresponding to the size of the gel, soak it in methanol for activation, and soak the transferred membrane filter paper in a semi-dry transfer buffer for 10 minutes.
  • the PVDF membrane was placed in a PBST solution containing 5% skimmed milk powder.
  • RNA PRMT8 interference small RNA
  • the interference sequence is GACAGUACAAGGACUUCAA
  • the cells were trypsinized, collected by centrifugation, and washed twice with 1′PBS. The cells were divided into two parts, one part was used to extract whole cell proteins, and the other part was used to extract nuclear histones.
  • RIPA cell lysate effective lysis component is 1% Triton X-100
  • lyse on ice for 30 minutes and centrifuge at 14000 rpm for 10 minutes, collect the protein supernatant, and determine the protein concentration by BSA method, -30°C Save it for later use.
  • Triton extraction solution containing 0.5% Triton X-100, 2mmol/L PMSF and 0.02% NaN 3 ) to resuspend the cells, lyse on ice for 10 minutes, centrifuge at 6500′g for 10 minutes, and discard The supernatant, washed the cells with half of the Triton extract in the previous step, added 0.2M HCl suspension cell mass, 4°C overnight, centrifuged at 6500'g for 10 minutes to collect the supernatant, measured the concentration and stored at -30°C for use.
  • Triton extraction solution containing 0.5% Triton X-100, 2mmol/L PMSF and 0.02% NaN 3
  • the blocking solution was discarded, the corresponding primary antibody and internal reference antibody (diluted according to the antibody instruction) were added, and incubated overnight at 4°C.
  • the PVDF membrane was taken out and washed 4 times with 1'PBST for 10 minutes each time.
  • RNA In human astrocytes SVG p12, specific interfering small RNA (siRNA) is used to silence the expression of protein arginine methyltransferase PRMT8 gene, and the expression changes of Tau mRNA are detected.
  • siRNA specific interfering small RNA
  • the results in Fig. 4 show that the expression level of Tau mRNA that interferes with PRMT8 expression is significantly reduced compared with the control.
  • RNA interfering small RNA
  • the interference sequence is GACAGUACAAGGACUUCAA
  • transfection can be performed.
  • the product can be used in PCR reaction immediately or stored at -30°C for later use.
  • Real-time PCR uses the Rotor-gene 6000 quantitative PCR instrument from Gene's Corbett Research, the reagent is Roche FastStart Universal SYBR Green Master Mix, and the reaction system is as follows:
  • tail vein injection to inject ADDA into mice to observe and analyze whether ADDA exists in the blood and brain tissues of mice to verify whether ADDA can penetrate the blood-brain barrier of mice.
  • tail vein injection of ADDA Later, we observed that ADDA was present in the blood and brain tissues of mice, and the ADDA content reached its peak 2 to 3 hours after injection.
  • mice were purchased from the Model Animal Research Institute of Nanjing University, females, 6-8 weeks old.
  • concentration of ADDA mother solution was 0.25mg/100 ⁇ L
  • the solvent was 0.04M HCl
  • 100 ⁇ L of ADDA solution was injected into mice by tail vein injection.
  • the mice were sacrificed by cervical dislocation, blood was collected from the orbit and brain tissue was extracted.
  • serum samples add 1 times the amount of normal saline to the blood, centrifuge at 10,000 rpm for 10 minutes, aspirate the supernatant, and filter with 0.22 ⁇ m for use.
  • the flushing speed is not too high. Adjust the flow rate. When using a new mobile phase for the first time, try the pressure first. The greater the flow rate, the greater the pressure, generally not more than 2000psi. Choose an appropriate flow rate. Set the detector analysis parameters for different samples. When the baseline becomes stable, the sample can be injected and analyzed. The pre-processing of the sample is very important when analyzing the sample. Before the sample is subjected to high-performance liquid chromatography (HPLC), analyze the ADDA standard, explore the column and mobile phase of ADDA detection and analysis, determine the detection wavelength of the standard, and determine the time and location of the peak of the standard. Then, compare the analysis results of the samples with those of the standard products.
  • HPLC high-performance liquid chromatography
  • Test Example 6 Real-time PCR detection of the effect of ADDA on the expression of Tau mRNA in primary neurons of mouse cerebral cortex
  • mice cerebral cortex primary neuron cells as the research object, we explored the effects of different ADDA treatment concentrations on the expression of Tau mRNA in mouse neuronal cells. It was found that as the concentration of ADDA increased, the expression of Tau mRNA decreased.
  • the primary neurons of the mouse cortex were extracted.
  • the pregnant mouse C57BL/6J
  • the abdominal cavity of the pregnant mouse was opened with dissecting scissors
  • the fetal mouse (E12.5) was taken out, and the brain of the fetal mouse was immersed in pre-cooled PBS.
  • the skin was removed, the skull was removed, and the fetal rat cortex was separated and placed in pre-cooled HBSS (Hanks balanced salt solution).
  • HBSS Hors balanced salt solution
  • the EP tube and Tip must be treated with 0.1% DEPC water and used after damp heat sterilization), stand at room temperature for 5 minutes; add 200 ⁇ L of chloroform (chloroform), shake vigorously for 15 seconds, and stand at room temperature for 2 to 3 minutes. Centrifuge for 15 minutes at 12000'g, 4°C. Transfer the upper aqueous phase to a new enzyme-free EP tube, add 500 ⁇ L of isopropanol, and let it stand at room temperature for 10 minutes. Centrifuge at 12000g, 4°C for 10min. Remove the supernatant, add 1ml of 75% pre-cooled ethanol (prepared with 0.1% DEPC water), and vortex for a while. 7500'g, 4°C, centrifuge for 5min. Remove the supernatant, dry, and dissolve it in 20 ⁇ L of 0.1% DEPC water. Place it at 55°C for 10 minutes to promote dissolution. Use a spectrophotometer to determine the concentration and purity, and perform the following RNA reverse transcription procedures:
  • the product can be used in PCR reaction immediately or stored at -30°C for later use.
  • Real-time PCR uses the Rotor-gene 6000 quantitative PCR instrument from Gene's Corbett Research, the reagent is Roche FastStart Universal SYBR Green Master Mix, and the reaction system is as follows:
  • Test Example 7 Western blot detection of the effect of ADDA on the expression of Tau protein in primary neurons of mouse cerebral cortex
  • the primary neuron cells of the mouse cerebral cortex were extracted according to the method described in Test Example 6, and the cells were treated with different ADDA concentrations to explore the effect of different ADDA on the expression of Tau protein in neuronal cells.
  • the concentrated gel and separating gel of the appropriate concentration are prepared. Take 40-60 ⁇ g protein sample and mix it with 4'SDS loading buffer 1:4. Then heat at 100°C for 5 min, and centrifuge at 12000 rpm for 5 min. Add the protein sample to the loading channel, and add 5 ⁇ L of protein Marker to the left channel of the sample. After loading the sample, turn on the power and adjust the constant voltage for 90V electrophoresis. After the protein sample enters the separation gel, adjust the voltage to 120V to continue electrophoresis. When the bromophenol blue front reaches the bottom of the separation gel, stop the electrophoresis and take out the gel. . Remove the gel sandwich and take out the gel.
  • the gel corresponding to the target band.
  • Cut the PVDF membrane corresponding to the size of the gel, soak it in methanol for activation, and soak the transferred membrane filter paper in a semi-dry transfer buffer for 10 minutes.
  • the PVDF membrane was placed in a PBST solution containing 5% skimmed milk powder.
  • the blocking solution was discarded, the corresponding primary antibody and internal reference antibody (diluted according to the antibody instruction) were added, and incubated overnight at 4°C.
  • the PVDF membrane was taken out and washed 4 times with 1'PBST for 10 minutes each time.
  • Test Example 8 Western blot detection of the effect of ADDA on the H4R3me2a level of primary neurons in the cerebral cortex of mice
  • mouse cerebral cortex primary neuron cells as the research object, to explore the effect of ADDA treatment on the level of mouse neuronal cells H4R3me2a.
  • the primary neuron cells of the mouse cerebral cortex were extracted according to the method described in Example 6, and the mouse cerebral cortex neuron cells were treated with ADDA to explore the effect of ADDA on the level of H4R3me2a in neuronal cells.
  • ADDA Alzheimer's disease
  • the cells were collected by centrifugation at 1000 rpm and washed twice with 1 ⁇ PBS.
  • the cells were resuspended in Triton extract (containing 0.5% Triton X-100, 2mmol/L PMSF and 0.02% NaN3), and lysed on ice for 10 minutes, 6500 ⁇ Centrifuge for 10 minutes at g, discard the supernatant, and add 0.2M HCl suspension cell pellet, overnight at 4°C, and centrifuge at 6500 ⁇ g for 10 minutes to collect the supernatant. After determining the concentration, store at -30°C for later use.
  • Triton extract containing 0.5% Triton X-100, 2mmol/L PMSF and 0.02% NaN3
  • the PVDF membrane was taken out and washed 4 times with 1 ⁇ PBST for 10 minutes each time.
  • Test Example 9 The effect of ADDA on spatial learning and memory in AD mice
  • APP/PS1 double transgenic Alzheimer's disease model mice (C57BL/6J) as the research object, after intraperitoneal injection, observe the effect of ADDA on the spatial learning and memory ability of APP/PS1 mice.
  • the water maze experiment shows that as the number of training days increases, the time for the mice in the control group and the administration group to find an underwater platform is significantly shortened. And, starting from the fourth day, compared with the APP/PS1 mice in the control group (0.04M HCl treatment), the time for the ADDA-administered mice to find an underwater platform was significantly shorter. It shows that ADDA treatment can improve the spatial learning and memory ability of APP/PS1 mice.
  • the experimental APP/PS1 double transgenic Alzheimer's disease model mice (C57BL/6J) were purchased from the Institute of Model Animals of Nanjing University, males, and started to be administered at 6 months of age.
  • Use 0.04M HCl as a solvent to dissolve ADDA the dosage is 12.5mg/kg
  • control mice (Control) are injected with the same volume of 0.04M HCl solvent
  • 100 ⁇ L of the prepared ADDA solution and control solvent are injected by intraperitoneal injection Mice were injected every two days for a duration of 2.5 months.
  • the water maze experiment is a classic method to test the spatial learning and memory abilities of mice. It mainly includes a round game pool with a diameter of 1.5m, a detachable platform, and a video tracking system.
  • the swimming pool is divided into four quadrants.
  • the whole experiment should be kept in a quiet environment, and the water temperature should be maintained at 23 ⁇ 1°C.
  • the mice adapt to the familiar environment, put all the mice in the swimming pool in turn, let them swim freely for 1 minute, and then guide the mice to stay on the platform for 20 seconds to help them become familiar with the surrounding environment.
  • the mice were placed in different quadrants for training in sequence, the mice were faced to the pool wall, gently put into the water, and the swimming time (escape latency) of the mice in the water was recorded. The time is 1 min. If the mouse can find the hidden platform within 1 min and stay on the platform for 5 seconds, it is deemed to have successfully found the platform.
  • the mouse fails to find the platform within 1 min, it will be artificially guided to the platform to stay for 20 seconds, and the swimming time (escape latency) is 1 min by default.
  • the swimming time is 1 min by default.
  • the training interval is no less than 30 minutes, and the training lasts for a total of 5 days.
  • the training is performed at the same time every day.
  • the video analysis system is used to record and analyze the mouse activity track. During the water maze experiment, the mice still kept the drug once every two days.
  • Test Example 10 Thioflavin-S (Thioflavin-S) and immunohistochemical staining methods were used to detect the effect of ADDA on the formation of senile plaques in AD mice
  • a ⁇ ( ⁇ -amyloid) deposition is the main cause of senile plaques in the brain, and the corresponding senile plaques are called A ⁇ senile plaques. According to the amyloid cascade hypothesis, A ⁇ senile plaque formation is the main pathological manifestation of Alzheimer's disease (AD).
  • AD Alzheimer's disease
  • the senile plaque referred to in this test example is A ⁇ senile plaque.
  • APP/PS1 double transgenic Alzheimer's disease model mice (C57BL/6J) will have senile plaques in the cerebral cortex and hippocampus at the age of 4 months, and the number and size of senile plaques will increase with the increase of months.
  • ADDA Alzheimer's disease model mice
  • Thioflavin-S Thioflavin-S
  • immunohistochemical staining to detect senile plaques in the brain cortex and hippocampus of control mice and ADDA-treated mice Formation.
  • mice 9 months old mice were sacrificed for senile plaque and IHC analysis.
  • the ADDA treatment group had significantly less senile plaque formation in the brain cortex and hippocampus. It shows that ADDA can inhibit the formation of senile plaques in the brain regions of AD mice.
  • the mouse's chest cavity was quickly opened, and 4% paraformaldehyde was infused from the left ventricle with a disposable syringe to remove blood from the brain.
  • the mouse skull was peeled off with dissecting scissors, the brain was exposed, and the brain was separated and placed in formalin fixation solution to prepare paraffin sections (thickness of 4 ⁇ m) and frozen sections (thickness of 30 ⁇ m).
  • paraffin sections were stained with immunohistochemistry, and frozen sections were stained with Thioflavin-S.
  • the sections are bleached in 0.25% permanganic acid solution for 30 minutes, rinsed in deionized water for 5 minutes, and then rinsed in 0.25% acetic acid solution for 5 seconds; after rinsing in deionized water for 5 minutes Put it in blocking solution (BSA) and block for 30 minutes; rinse with deionized water for 5 minutes and drip with Thioflavin-S dye solution (prepared with 50% ethanol) for 5-8 minutes; rinse with 50% ethanol and deionized water for 2 times and use glycerin Mount the gel and take pictures under a fluorescence microscope.
  • BSA blocking solution
  • Thioflavin-S dye solution prepared with 50% ethanol
  • tissue sections were baked in a 60°C incubator for 2 hours before deparaffinization. Soak and dewax in xylene and gradient ethanol respectively, incubate with 0.5% Triton X-100 at room temperature for 15 minutes, rinse with PBS for 5 minutes; immerse the slices in 0.01M citrate buffer (pH 6.0), heat to boiling for 10-8 minutes, and take out Then it was naturally cooled to room temperature, washed with PBS and incubated with 3% H 2 O 2 at room temperature for 10 min to remove endogenous peroxidase.
  • the ADDA of the present invention can be used to inhibit the expression of middle-aged dementia and Parkinson's disease related genes/protein Tau, easily penetrate the blood-brain barrier, and play a role in treating middle-aged dementia and Parkinson's disease.
  • ADDA can also inhibit histone H4 methylation (H4R3me2a), which may be achieved by inhibiting PRMT8.
  • ADDA is colorless, tasteless, easy to dissolve, low concentration, stable, and safe. It has broad application prospects in the treatment of brain degenerative diseases.

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Abstract

L'invention concerne une application d'un composé à petites molécules dans la préparation d'un médicament destiné à inhiber l'expression de la protéine Tau. L'invention concerne également une application de 2',3'-adénosine dialdéhyde (ADDA) ou d'un métabolite in vivo, un tautomère, un stéréoisomère, un sel pharmaceutiquement acceptable, un hydrate et/ou un solvate de celui-ci dans la préparation d'un inhibiteur de protéine Tau. L'invention concerne un médicament pour le traitement d'une maladie du système nerveux central. L'invention clarifie pour la première fois la fonction du composé ADDA dans l'inhibition de l'expression de la protéine Tau humaine, et fournit un médicament candidat pour le traitement de la maladie d'Alzheimer (AD) et de la maladie de Parkinson.
PCT/CN2019/126698 2018-06-22 2019-12-19 Application d'un composé à petites molécules dans la préparation d'un médicament destiné à inhiber l'expression de la protéine tau WO2020253179A1 (fr)

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