WO2021093553A1 - 缺血性脑损伤的新型治疗药物 - Google Patents

缺血性脑损伤的新型治疗药物 Download PDF

Info

Publication number
WO2021093553A1
WO2021093553A1 PCT/CN2020/123215 CN2020123215W WO2021093553A1 WO 2021093553 A1 WO2021093553 A1 WO 2021093553A1 CN 2020123215 W CN2020123215 W CN 2020123215W WO 2021093553 A1 WO2021093553 A1 WO 2021093553A1
Authority
WO
WIPO (PCT)
Prior art keywords
glutamate
brain injury
glt
membrane
glutamate transporter
Prior art date
Application number
PCT/CN2020/123215
Other languages
English (en)
French (fr)
Inventor
王以政
王宇青
Original Assignee
中国科学院脑科学与智能技术卓越创新中心
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 中国科学院脑科学与智能技术卓越创新中心 filed Critical 中国科学院脑科学与智能技术卓越创新中心
Publication of WO2021093553A1 publication Critical patent/WO2021093553A1/zh

Links

Images

Classifications

    • 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
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • 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
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4433—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a six-membered ring with oxygen as a ring hetero atom
    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/444—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring heteroatom, e.g. amrinone
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2503/00—Use of cells in diagnostics
    • C12N2503/02—Drug screening

Definitions

  • the present invention belongs to the field of biomedicine. More specifically, the present invention relates to a novel therapeutic drug for ischemic brain injury.
  • Stroke has become the number one killer of Chinese people’s health. So far, the only drug used to treat ischemic stroke is t-PA, but t-PA has the hidden danger of causing cerebral hemorrhage and only about 5% of patients benefited. In addition, although there are many drugs that have shown good protective effects in rodent cerebral ischemia models, they all ended in failure in clinical transformation. Therefore, finding new drugs for the treatment of ischemic stroke is an extremely urgent topic in the field of stroke.
  • Ischemic stroke refers to the blockage of blood vessels in the human body, especially the blockage of blood vessels in the brain. There are many blood vessels in the brain. However, clinical data shows that the probability of blockage of the middle cerebral artery is the greatest, so the commonly used animal model They all block the middle cerebral artery. Ischemic brain injury refers to a series of brain parenchymal damage caused by cerebrovascular blockage. Therefore, an ischemic stroke will result in ischemic brain damage.
  • the main reason for the early damage of ischemic stroke is that glutamate is released in large quantities and cannot be absorbed effectively. It accumulates in a large amount outside the cell, which causes the combination of glutamate and NMDARs to cause calcium ion overload on neurons and cause a series of infarctions Surrounding depolarization, immune, programmed cell death and other reactions produce excitotoxic effects.
  • experiments with NMDARs as drug targets can improve ischemic brain injury. Because of the low efficiency of clinical trials and the large side effects, they have not been successfully applied. Therefore, without affecting NMDARs, inhibiting excitotoxicity has great potential in the treatment of ischemic stroke.
  • the concentration of glutamate between cells is mainly balanced by release and reabsorption.
  • the reabsorption of glutamate is mainly eliminated by transporting glutamate into the cell through glutamate transporters.
  • glutamate transporters There are five phenotypes of glutamate transporters, EAAT1-5, including GLT-1 (EAAT2).
  • GLT-1 GLT-1
  • Ischemic stroke is divided into an acute phase and a recovery phase.
  • the acute phase is generally a few hours after ischemia occurs, and the recovery phase is more than ten hours after ischemia and several days later.
  • the acute phase of ischemia is a very dangerous period for patients. If effective treatment can be carried out at this stage, it is of great significance for saving patients' lives and improving their life after illness.
  • the purpose of the present invention is to provide a novel therapeutic drug for ischemic brain injury.
  • the use of the compound of the core structure represented by formula (I) or its isomers, derivatives, solvates or precursors, or their pharmaceutically acceptable salts is provided for : Preparation of a composition for relieving or treating ischemic brain injury; preparation of a composition for maintaining the glutamate transporter GLT-1 on the astrocyte membrane and reducing the concentration of extracellular glutamate during the process of ischemic brain injury;
  • the ischemic brain injury is brain injury in the acute phase of ischemia or brain injury in the early phase of ischemia.
  • the acute phase or early phase is: the early phase or acute phase of ischemic stroke (for example, it is called Acute Ischemic Stroke).
  • the acute phase or early stage is usually within 6 hours after the occurrence of ischemia, such as within 5, 4, 3, 2 or 1 hour.
  • the ischemic brain damage includes: ischemic cerebral stroke, brain damage caused by ischemic cerebral stroke.
  • the maintenance of the glutamate transporter GLT-1 on the astrocyte membrane includes: promoting the glutamate on the astrocyte membrane
  • the expression of the acid transporter GLT-1 increases the activity (function) of the glutamate transporter GLT-1 on the membrane of astrocytes.
  • the compound removes extracellular glutamate by maintaining the glutamate transporter GLT-1 on the membrane of astrocytes.
  • the compound inhibits the activation of the SHH signaling pathway and thereby inhibits the decrease in the activity of the glutamate transporter GLT-1 on the membrane, thereby effectively reducing the extracellular glutamate concentration.
  • the maintenance of the on-membrane glutamate transporter GLT-1 of astrocytes is maintenance during the acute phase of ischemia (for example, known as Acute Ischemic Stroke).
  • R there is at least one substituent R on the phenyl group of the compound of the core structure represented by formula (I), and R is independently selected from: hydrogen, C1-C4 alkyl, hydroxyl, C2-C4 chain Alkenyl, C2-C4 alkynyl, halogen.
  • R is independently selected from: hydrogen, hydroxyl, and C1-C2 alkyl.
  • the solvate is a hydrate.
  • the composition is a pharmaceutical composition.
  • a method for preparing the on-membrane glutamate transporter GLT-1 for maintaining astrocytes during ischemic brain injury, and reducing the extracellular glutamine of astrocytes includes: adding a compound of formula (I) or its isomers, derivatives, solvates or precursors thereof, or their
  • the pharmaceutically acceptable salt is mixed with a pharmaceutically or foodstuff acceptable carrier.
  • a method for maintaining the glutamate transporter GLT-1 on the membrane of astrocytes or reducing the extracellular glutamate of astrocytes during ischemic brain injury includes: treating astrocytes with a compound of the core structure represented by formula (I) or an isomer, derivative, solvate or precursor thereof, or a pharmaceutically acceptable salt thereof.
  • the method is an in vitro method.
  • the method is a method that is not for the purpose of treatment.
  • the method is a method of maintaining the glutamate transporter GLT-1 on the membrane of astrocytes in astrocyte culture.
  • a method for screening substances that alleviate or treat ischemic brain injury comprising: (1) providing astrocytes that express SHH signaling pathway member proteins and grains Glutamate transporter GLT-1; (2) cells treated with candidate substances (1); (3) regulation of SHH signaling pathway member proteins in the cells of (2) on the glutamate transporter GLT-1 on the cell membrane, If the candidate substance can maintain the expression or activity of the glutamate transporter GLT-1 on the cell membrane, it indicates that the candidate substance is a substance (or a potential substance) that alleviates or treats ischemic brain injury.
  • the "inhibition” is statistical inhibition (significant inhibition), such as inhibition of 10%, 20%, 40%, 50%, or 60% or more.
  • step (2) before the candidate substance is treated, it further includes treatment with a SMO (Smoothened) specific agonist to down-regulate the GLT-1 activity.
  • SMO Smoothened
  • the SHH signaling pathway member protein includes SMO protein.
  • step (2) includes: in the test group, treating with an SMO specific agonist, and adding the candidate substance to the cells of (1); and/or step (3) includes: detecting the test group The regulation of SHH signaling pathway member proteins on the cell membrane of the glutamate transporter GLT-1 in the cells, and compared with the control group, wherein the control group is treated with SMO-specific agonists without adding the candidate substance (1) cells; if the candidate substance can maintain the expression or activity of the glutamate transporter GLT-1 on the cell membrane and reduce the concentration of extracellular glutamate, it indicates that the candidate substance is for alleviating or treating ischemic brain injury Substance (or potential substance).
  • the regulation of SHH signaling pathway member proteins on the cell membrane of the glutamate transporter GLT-1 in the cells of (2) is determined.
  • the candidate substance includes (but is not limited to): small molecule compounds (eg, small molecule compounds from a compound library or gene library); genes or proteins, such as interference molecules, gene editing reagents, nucleic acids Inhibitors, small RNAs, etc.
  • small molecule compounds eg, small molecule compounds from a compound library or gene library
  • genes or proteins such as interference molecules, gene editing reagents, nucleic acids Inhibitors, small RNAs, etc.
  • the cells are cultured in vitro.
  • the cell is a cell culture.
  • FIG. 1 During ischemia in rodents, the expression of SHH on the ischemic side increases. In the figure, 0 is the time when the blood vessel is blocked, that is, the ischemia starts.
  • FIG. 2A-B During ischemia, the activation of SHH signaling pathway can promote the increase of extracellular glutamate concentration.
  • Figure 3A Using different drugs to treat astrocytes, record the current of GLT-1 protein by electrophysiological method; among them, when processing the cells, the final concentration of SAG (3 ⁇ M); the final concentration of CYC (10 ⁇ M); the final SAG+CYC Concentration (3 ⁇ M+10 ⁇ M).
  • FIG. 3B Experimental isotope demonstrates that SHH reduces the uptake of extracellular glutamate by astrocytes through SMO.
  • NVP-LDE225 can completely inhibit the effect of SAG.
  • Figure 4 The test procedure for evaluating the efficacy of NVP-LDE225 in the C57 mouse cerebral ischemia model.
  • Figure 6 The test procedure for evaluating the efficacy of NVP-LDE225 in a cerebral ischemia model in cynomolgus monkeys.
  • Figure 7 Evaluation results of the efficacy of NVP-LDE225 in the cynomolgus monkey cerebral ischemia model.
  • Picture A shows the MRI scan DWI sequence (48h after ischemia), and the highlighted signal represents the area of brain damage.
  • Figure B is a statistical graph. The horizontal axis is the time point of the MRI scan. Both T2 and DWI highlight signals can reflect the severity of brain injury.
  • Panel C counts the behavioral changes of monkeys in different treatment groups during the time course of 30 days after ischemia.
  • a SHH (Sonic hedgehog) signaling pathway inhibitor NVP-LDE225 can quickly regulate the activity of GLT-1 on the cell membrane through the SHH signaling pathway protein SMO, and quickly clear extracellular glutamine. Acid, and can be used to prepare medicines for relieving or treating ischemic brain damage (including ischemic stroke and brain damage caused by ischemic stroke).
  • the present invention first provides a compound having a core structure represented by structural formula (I):
  • the present invention also includes the isomers, solvates, precursors, or pharmaceutically acceptable salts of the above-mentioned compounds having the core structure represented by formula (I), as long as they also have the same properties as those represented by formula (I).
  • the compounds of the core structure have the same or substantially the same functions.
  • pharmaceutically acceptable salt refers to a salt formed by the reaction of a compound with inorganic acid, organic acid, alkali metal or alkaline earth metal.
  • salts include (but are not limited to): (1) salts formed with the following inorganic acids: such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid; (2) salts formed with the following organic acids, such as acetic acid, oxalic acid, succinic acid, and tartaric acid , Methanesulfonic acid, maleic acid, or arginine.
  • Other salts include those formed with alkali metals or alkaline earth metals (such as sodium, potassium, calcium, or magnesium) in the form of esters, carbamates, or other conventional "prodrugs". Compounds have one or more asymmetric centers. Therefore, these compounds can exist as racemic mixtures, individual enantiomers, individual diastereomers, mixtures of diastereomers, cis or trans isomers.
  • the "precursor of the compound” refers to a compound that undergoes metabolism or chemical reaction in the patient's body after being taken by an appropriate method to transform into a compound of the core structure shown in structural formula (I), or chemical A salt or solution composed of a compound of the core structure shown in structural formula (I).
  • the compound of the present invention can be obtained by a variety of methods well known in the art and using known raw materials, such as chemical synthesis or from biological (such as animal or plant) The method of extraction or modification on the basis of extraction in ), these methods are all included in the present invention.
  • the compound of the present invention can be synthesized by a well-known method; the synthesized compound can be further purified by column chromatography, high performance liquid chromatography and the like. In addition, the compound of the present invention can also be obtained commercially.
  • the present invention also provides a composition (for example, a pharmaceutical composition), which contains an effective amount of the compound described in the core structure represented by formula (I), or an isomer, solvate, or precursor thereof. That is, the present invention provides a composition containing NVP-LDE225 or an analog thereof.
  • the "contains", “having” or “including” includes “including”, “mainly composed of”, “essentially composed of”, and “consisted of”; Mainly composed of", “basically composed of” and “consisted of” belong to the subordinate concepts of "contains", “has” or “includes”.
  • the composition is a pharmaceutical composition, and the composition further contains a pharmaceutically acceptable salt and/or a pharmaceutically acceptable carrier or excipient.
  • pharmaceutically acceptable ingredients are substances that are suitable for humans and/or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), that is, substances with a reasonable benefit/risk ratio.
  • a “pharmaceutically acceptable carrier” is a pharmaceutically or food acceptable solvent, suspending agent, or excipient used to deliver the compound of the present invention to animals or humans.
  • the carrier can be liquid or solid.
  • the pharmaceutical composition contains 0.001-50% by weight of the compound represented by the core structure of formula (I) or a pharmaceutically acceptable salt thereof.
  • the pharmaceutical composition contains 0.05-30% by weight of the compound represented by the core structure of formula (I) or a pharmaceutically acceptable salt thereof; more preferably, the pharmaceutical The composition contains 0.01-20% by weight of the compound represented by the core structure of formula (I) or a pharmaceutically acceptable salt thereof.
  • the dosage form of the pharmaceutical composition of the present invention can be various, as long as it can make the active ingredient reach the mammalian body effectively.
  • it can be selected from: gels, aerosols, tablets, capsules, powders, granules, syrups, solutions, or suspensions.
  • the preferred pharmaceutical compositions are solid compositions, especially tablets and solid-filled or liquid-filled capsules.
  • the compound of the present invention or its pharmaceutical composition can also be stored in a sterile device suitable for injection or drip infusion.
  • the effective dosage of the core structure compound represented by formula (I) as an active ingredient may vary with the mode of administration and the severity of the disease to be treated.
  • NVP-LDE225 regulates glutamate by regulating the SHH signal pathway.
  • the SHH signal pathway was originally a signal pathway involved in the process of neural development.
  • the inventor’s research proves for the first time that SHH can regulate the upper and lower membranes of glutamate transporters on astrocytes, and can quickly achieve changes in the number of glutamate transporters on the membrane, thereby reducing extracellular glutamate. The density is adjusted quickly.
  • ischemic brain damage including ischemic stroke, brain damage caused by ischemic stroke
  • the expression of SHH is significantly increased.
  • the down-regulation of the transporter on the membrane caused by the activation of the SHH signal pathway reduces the reabsorption of glutamate and accumulates in a large amount outside the cell.
  • the compound of the present invention can change this situation, increase the expression or activity of the transporter on the membrane, and accelerate The elimination of extracellular glutamate has a significant improvement effect in ischemic stroke.
  • SHH signaling pathway can help the brain to repair a few days after ischemia, so it is considered a signaling pathway beneficial to neuroprotection.
  • the function of SHH signaling pathway has not been reported in the acute phase of ischemia, and the inventors found that it does not play a neuroprotective role in the acute phase of ischemia. Its activation actually leads to the glutamate transporter (GLT-1 ) Decreases in expression or activity on the cell membrane, and its inhibitor (preferably NVP-LDE225) can effectively reverse this situation.
  • GLT-1 glutamate transporter
  • NVP-LDE225 its inhibitor
  • the SHH signaling pathway inhibitor NVP-LDE225 has been used by those in the art to treat advanced basal cell tumors. It is currently on the market in the United States and Europe. Its main mechanism of action is to inhibit the protein Smoothened activity in the SHH signaling pathway.
  • the inventors unexpectedly discovered that NVP-LDE22 only needs to be injected once during the ischemic process to quickly reach the drug target in the early stage of ischemia (for example, within 6 hours or less), and realize the effective value of the drug. , It has a good improvement effect on the development of ischemic stroke. It has been proved to have a good neuroprotective effect in animal experiments in rodents and non-human primate animal models. Among them, the cynomolgus monkey is related to the structure of the human brain. Highly similar species. Currently, there are no drugs of cerebral ischemia neuroprotection on the market, and the present invention is likely to fill this gap.
  • SHH regulates extracellular glutamate in a model of ischemic stroke it is demonstrated for the first time that SHH regulates extracellular glutamate in a model of ischemic stroke.
  • the inventor’s previous research has proposed the concept of SHH regulating extracellular glutamate, but it is limited to cell (neuron) experiments based on physiological conditions, and the cells targeted are not astrocytes.
  • the amino acid transporter is not GLT-1.
  • SHH regulates the activity of the glutamate transporter GLT-1 specifically expressed on astrocytes which is the same as the previous SHH for the glutamate transporter EAAC1 specifically expressed on neurons.
  • the regulation is completely different. Therefore, although the present inventors have previously proposed the concept of SHH regulating extracellular glutamate, the mechanism in the present invention is different, and the present invention has confirmed the existence of such regulation in an in vivo ischemia model for the first time.
  • the present inventors transformed the clinical drug NVP-LDE225, a specific inhibitor of the SHH signaling pathway into drugs for the treatment of ischemic brain injury, and expanded the field’s understanding of the SHH signaling pathway during ischemia. Know the clinical indications of NVP-LDE225 or its analogues.
  • NVP-LDE225 or its analogues is provided for preparing a composition (medicine) for relieving or treating ischemic brain injury.
  • ischemic brain injury During ischemia, the amount of GLT-1 protein on the cell membrane of astrocytes will decrease, and the role of NVP-LDE225 is to maintain the amount of GLT-1 protein on the cell membrane during ischemic brain injury.
  • NVP-LDE225 or its analogues is provided for preparing the expression of the glutamate transporter GLT-1 on the membrane of astrocytes during ischemic brain injury.
  • active composition drug
  • NVP-LDE225 or its analogues is provided to prepare a composition (medicine) for reducing the amount of extracellular glutamate in astrocytes.
  • the SHH signal pathway protein includes SMO protein.
  • the present invention provides a method for screening potential substances for alleviating or treating ischemic brain injury, the method comprising: (1) providing astrocytes that express SHH signaling pathway proteins and glutamate transport (2) Treat the cells of (1) with the candidate substance; (3) Determine the regulation of the SHH signal pathway protein in the cell of (2) on the glutamate transporter on the cell membrane, if the candidate substance inhibits the SHH signal pathway protein Decrease or increase the expression or activity of the regulated glutamate transporter indicates that the candidate substance maintains or up-regulates the glutamate transporter on the astrocyte membrane and reduces the astrocyte’s cellularity. The amount of extraglutamate, so as to alleviate or treat the substance (or potential substance) of ischemic brain injury.
  • a control group in order to make it easier to observe changes in the expression or activity of the signal pathway, gene or protein during screening, a control group may also be set, and the control group may not be added.
  • the candidate substance system in order to make it easier to observe changes in the expression or activity of the signal pathway, gene or protein during screening, a control group may also be set, and the control group may not be added.
  • candidate substances can be a wide range of substances of interest.
  • small molecule compounds, genes, or proteins from compound libraries or gene libraries or small molecule compounds, agonists, upregulators, and molecules designed for SHH signaling pathway proteins, genes or proteins or their upstream or downstream molecules.
  • the method further includes: conducting further cell experiments and/or animal experiments on the obtained potential substances to further select and determine the treatment of ischemic brain injury (including ischemic stroke, Brain damage caused by ischemic stroke) a really useful substance.
  • ischemic brain injury including ischemic stroke, Brain damage caused by ischemic stroke
  • the present invention also provides potential substances for alleviating or treating ischemic brain injury obtained by using the screening method.
  • These preliminary screening substances can constitute a screening library so that people can finally screen out drugs that can be really useful for the treatment of ischemic brain injury (including ischemic stroke).
  • Example 1 During ischemia in rodents, the expression of SHH on the ischemic side increased
  • a mouse ischemia-reperfusion model was established and then tested.
  • the detection area is the mouse striatum.
  • the middle cerebral artery occlusion (MCAO) lasts for 120 minutes, after which the embolus is taken out to realize the recanalization of the blood vessel.
  • the ischemic side (ipsi) and the ischemic contralateral (contra) striatum were taken out and immersed in artificial cerebrospinal fluid to collect SHH protein secreted outside the cells.
  • the SHH protein content in the artificial cerebrospinal fluid enriched with SHH was measured by ELISA (enzyme-linked immunosorbent assay) experiment, and it was found that the SHH protein of the striatum on the ischemic side was released in the interval 30 to 60 minutes after ischemia. Significantly more than the contralateral ischemia.
  • mice 15 minutes before ischemia or 45 minutes after ischemia, mice were intraperitoneally injected with SHH signaling pathway specific inhibitor CYC (cyclopamine) or its solvent control HBC, and it was found that extracellular glutamine in the HBC group The acid concentration increases significantly after ischemia, and CYC can effectively reduce the extracellular glutamate concentration. Therefore, the activation of SHH signaling pathway during ischemia can indeed promote the increase of extracellular glutamate concentration.
  • SHH signaling pathway specific inhibitor CYC cyclopamine
  • Astrocytes are cultured in vitro, treated with SMO specific inhibitors and activators, and recorded the current of GLT-1 protein by electrophysiological methods.
  • the current of GLT-1 protein can directly reflect the activity of GLT-1 protein on the cell membrane.
  • the left picture of Fig. 3A is the recorded current graph, and the right picture of Fig. 3A is the statistical graph of the current changes of GLT-1 after treatment with different drugs.
  • the incubation time of all drugs is 30 minutes.
  • the specific agonist of SMO SAG can reduce the activity of GLT-1 by about 50%
  • the SMO specific inhibitor CYC which specifically acts on the protein SMO of the SHH signaling pathway and inhibits its activity
  • SHH+CYC SHH+CYC
  • FIG. 3B an isotope experiment is used to demonstrate that SHH reduces the uptake of extracellular glutamate by astrocytes through SMO.
  • Astrocytes were incubated with 3 H-labeled glutamate. After treatment with different drugs (as shown in the figure), the cells were collected and the amount of 3 H-labeled glutamate was measured. This reflects the ability of astrocytes to take up glutamate. The uptake of glutamate is completely dependent on the glutamate transporter on the cell membrane. SHH down-regulates the activity of GLT-1. Therefore, SHH-treated astrocytes have a significantly reduced ability to take up glutamate, and this phenomenon can be SMO antagonist CYC blocked.
  • the SHH signal pathway rapidly regulates the number of glutamate transporter GLT-1 on the membrane and regulates the concentration of extracellular glutamate. Inhibiting the activation of the SHH signaling pathway can inhibit the decrease in the activity of the glutamate transporter GLT-1 on the membrane, thereby effectively reducing the extracellular glutamate concentration.
  • NVP-LDE225 protects brain damage and improves behavior in a cerebral ischemia model
  • NVP-LDE225 is used in C57 mouse ischemia model
  • the results are shown in Figure 5.
  • the left image is the actual image. TTC brain slices are stained.
  • the red area is the active area of the brain tissue, and the white area is the brain injury.
  • the middle statistical graph is the calculation of the volume of the white area. It is found that NVP-LDE225 can significantly reduce the size of the white area, which in turn reflects the reduction of brain damage.
  • the picture on the right is an overall assessment of the neurobehavioral improvement of NVP-LDE225 in mice after ischemia. It can be seen that NVP-LDE225 can significantly improve neurobehavioral defects in mice after ischemia.
  • NVP-LDE225 is used in cynomolgus monkey ischemia model
  • An ischemia model was established with cynomolgus monkeys to analyze the mitigation or treatment effects of NVP-LDE225 obtained by the inventors on cerebral ischemia.
  • Clamping the middle cerebral artery in cynomolgus monkeys the specific process is: 2-3 days before the operation, first measure MRI, CBF and behavior to obtain basic values, and then clamp the middle cerebral artery on day 0, and clamp until The administration was started at 25 minutes, the total volume of the administration was 5ml, and the duration of administration was 5 minutes.
  • the monkeys in the first group were given 0.91mg/ml NVP-LDE225 137.5ul, and the monkeys in the second group were given 137.5ul of solvent; the blood gas sampling time point was: clip 15 minutes before closure, 5 minutes after clamping and 15 minutes after administration. Behavior is determined on the first day. On the second day, MRI, CBF and behavior were measured again.
  • Figure 6 shows the procedure of NVP-LDE225 drug efficacy evaluation test in the cynomolgus monkey cerebral ischemia model.
  • Figure A shows the MRI scan DWI sequence (48h after ischemia).
  • the highlight signal represents the area of brain damage. It can be seen from the figure that in the NVP-LDE225 group, the highlight signal is higher than that of the placebo. The group is significantly reduced.
  • Figure B is a statistical graph. The horizontal axis is the time point of the MRI scan. Both T2 and DWI highlight signals can reflect the severity of brain injury. It can be seen that whether it is 48h or 30 days after ischemia, the ischemic brain injury in the NVP-LDE225 treatment group is significantly reduced.
  • Figure C is a statistical analysis of the behavioral changes of monkeys in different treatment groups during the 30-day period after ischemia. Among them, the performance of consciousness, muscle coordination and motor system were better in the NVP-LDE225 group, combining the four items. The overall score has improved significantly.
  • NVP-LDE225 can effectively reduce cerebral infarction.
  • astrocytes which express the SHH signaling pathway and the glutamate transporter GLT-1. This cell is used as a cell model for screening drugs that inhibit the SHH signaling pathway.
  • Test group a culture of the above-mentioned cells treated with candidate substances
  • Control group a culture of the above-mentioned cells not treated with the candidate substance.
  • NVP-LDE225 was tested as a candidate substance, and the results showed that the down-regulation of glutamate transporter activity was significantly inhibited, so that NVP-LDE225 is a useful candidate substance for the treatment of ischemic brain injury.
  • the inventors also used the compounds in the compound library to process the cultures of the above-mentioned cells in order to screen other drugs.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Epidemiology (AREA)
  • Biomedical Technology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Immunology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Urology & Nephrology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Hematology (AREA)
  • Microbiology (AREA)
  • Neurosurgery (AREA)
  • Neurology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Cardiology (AREA)
  • Vascular Medicine (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Biophysics (AREA)
  • Food Science & Technology (AREA)
  • Cell Biology (AREA)

Abstract

本发明提供了一种缺血性脑损伤的新型治疗药物。首次揭示了一种SHH(Sonic hedgehog)信号通路抑制剂可以通过SHH信号通路的蛋白SMO(Smoothened)快速地调节细胞膜上GLT-1的数量，快速清除胞外的谷氨酸，并能够用于制备缓解或治疗缺血性脑损伤的药物。

Description

缺血性脑损伤的新型治疗药物 技术领域
本发明属于生物医药领域，更具体地，本发明涉及缺血性脑损伤的新型治疗药物。
背景技术
中风已经成为国人健康的头号杀手，目前为止，用于治疗缺血性脑中风的药物只有溶栓药物t-PA，但t-PA具有引发脑出血的隐患且受益病人只有约5％。另外，虽然有非常多的药物在啮齿类动物脑缺血模型中展示了很好的保护效果，但在临床转化中都以失败告终。因此，寻找新的治疗缺血性脑中风的药物是中风领域极为迫切的课题。
缺血性脑中风是指人体中的血管堵塞，尤其是大脑中的血管发生堵塞，大脑中的血管有很多，但是临床数据显示，大脑中动脉发生堵塞的概率是最大的，所以常用的动物模型都是堵塞大脑中动脉。缺血性脑损伤是指脑血管堵塞后引起的一系列脑实质的损伤。所以发生了缺血性脑中风随之就会产生缺血性脑损伤。
缺血性脑中风早期损伤的主要原因是谷氨酸大量释放，不能有效的吸收，在细胞外大量堆积，导致谷氨酸与NMDARs结合后引起神经元上的钙离子过载而引起一系列如梗死周围去极化，免疫，细胞程序性死亡等反应，产生兴奋性毒作用。目前以NMDARs为药物靶点的实验能够改善缺血性脑损伤，由于临床试验效率较低且具有较大的副作用而尚未成功应用。因此，在不影响NMDARs的情况下，抑制兴奋性毒性在缺血性脑中风的治疗中具有巨大的潜力。细胞间谷氨酸的浓度主要是由释放和重吸收来维持平衡。谷氨酸的重吸收主要通过谷氨酸转运体将谷氨酸转运到细胞内进行清除，谷氨酸转运体有五种表型，EAAT1-5，包括GLT-1(EAAT2)。尽管已有研究认为增加膜上谷氨酸转运体GLT-1表达量可以降低缺血性脑损伤，但由于此过程较缓慢，并不足以在脑缺血早期产生神经保护作用。已有研究中，通过上调GLT-1的基因转录表达来实现其蛋白水平的上调，进而在细胞膜上GLT-1的蛋白水平也有升高，但是从基因的转录到蛋白水平上调和细胞膜上蛋白水平增加之间需要几天时间，因此在 缺血性脑中风的急性期，这种调控并不具有实际意义。
缺血性脑中风分为急性期和恢复期，急性期一般在缺血发生后几个小时，恢复期是在缺血十几个小时及以后数天。缺血急性期是一个对于患者而言非常危险的时期，若是能在这一阶段进行有效的治疗，则对于挽救患者生命、改善患者病后生活状态均具有极为重要的意义。
因此，如何在缺血性脑损伤(如脑中风)早期快速降低胞外谷氨酸浓度是本领域亟待解决的一个问题。
发明内容
本发明的目的在于提供缺血性脑损伤的新型治疗药物。
在本发明的第一方面，提供式(I)所示母核结构的化合物或其异构体、衍生物、溶剂合物或前体，或它们的药学上可接受的盐的用途，用于：制备缓解或治疗缺血性脑损伤的组合物；制备在缺血性脑损伤过程中维持星形胶质细胞膜上谷氨酸转运体GLT-1并降低细胞外谷氨酸浓度的组合物；
Figure PCTCN2020123215-appb-000001
在一个优选例中，所述的缺血性脑损伤为缺血急性期的脑损伤或缺血早期的脑损伤。
在另一优选例中，所述的急性期或早期为：缺血性脑中风的早期或急性期(如称为Acute ischemic stroke)。
在另一优选例中，所述的急性期或早期通常为缺血发生后6小时内，例如5、4、3、2或1小时内。
在另一优选例中，所述的缺血性脑损伤包括：缺血性脑中风，由缺血性脑中风引起的脑损伤。
在另一优选例中，所述的维持星形胶质细胞膜上谷氨酸转运体GLT-1(进而降低缺血过程中细胞外谷氨酸浓度)包括：促进星形胶质细胞的膜上谷氨酸转运体GLT-1的表达，提高星形胶质细胞的膜上谷氨酸转运体GLT-1的活性(功能)。
在另一优选例中，所述化合物通过维持星形胶质细胞的膜上谷氨酸转运体GLT-1，清除胞外的谷氨酸。
在另一优选例中，所述化合物通过抑制SHH信号通路的激活进而抑制膜上谷氨酸转运体GLT-1的活性下降，从而有效降低细胞外谷氨酸浓度。
在另一优选例中，所述的维持星形胶质细胞的膜上谷氨酸转运体GLT-1为在缺血急性期(如称为Acute ischemic stroke)的维持。
在另一优选例中，式(I)所示母核结构的化合物的苯基上，存在至少一个取代基R，R独立地选自：氢、C1-C4烷基、羟基、C2-C4链烯基、C2-C4链炔基、卤素。
在另一优选例中，R独立地选自：氢、羟基、C1-C2烷基。
在另一优选例中，所述的溶剂合物为水合物。
在另一优选例中，所述的组合物为药物组合物。
在本发明的另一方面，提供一种制备用于在缺血性脑损伤过程中维持星形胶质细胞的膜上谷氨酸转运体GLT-1、减少星形胶质细胞的胞外谷氨酸量或缓解或治疗缺血性脑损伤的组合物的方法，包括：将式(I)所示母核结构的化合物或其异构体、衍生物、溶剂合物或前体，或它们的药学上可接受的盐与药学或食品学上可接受的载体混合。
在本发明的另一方面，提供一种在缺血性脑损伤过程中维持星形胶质细胞的膜上的谷氨酸转运体GLT-1或减少星形胶质细胞的胞外谷氨酸量的方法，包括：以式(I)所示母核结构的化合物或其异构体、衍生物、溶剂合物或前体，或它们的药学上可接受的盐处理星形胶质细胞。
在另一优选例中，所述的方法为体外方法。
在另一优选例中，所述的方法为不是以治疗为目的的方法。
在另一优选例中，所述的方法为维持星形胶质细胞培养物中星形胶质细胞的膜上谷氨酸转运体GLT-1的方法。
在本发明的另一方面，提供一种筛选缓解或治疗缺血性脑损伤的物质的方法，所述方法包括：(1)提供星形胶质细胞，该细胞表达SHH信号通路成员蛋白以及谷氨酸转运体GLT-1；(2)用候选物质处理(1)的细胞；(3)测定(2)的细胞中SHH信号通路成员蛋白对细胞膜上谷氨酸转运体GLT-1的调节情况，若 所述候选物质能够维持细胞膜上谷氨酸转运体GLT-1的表达或活性，则表明该候选物质是缓解或治疗缺血性脑损伤的物质(或潜在物质)。
在另一优选例中，所述的“抑制”为统计学上的抑制(显著性抑制)，如抑制10％、20％、40％、50％或60％以上。
在另一优选例中，步骤(2)中，候选物质处理之前，还包括以SMO(Smoothened)特异性激动剂处理，下调GLT-1活性。
在另一优选例中，SHH信号通路成员蛋白包括SMO蛋白。
在另一优选例中，步骤(2)包括：在测试组中，以SMO特异性激动剂处理，并将候选物质加入(1)的细胞中；和/或步骤(3)包括：检测测试组的细胞中SHH信号通路成员蛋白对细胞膜上谷氨酸转运体GLT-1的调节情况，并与对照组比较，其中所述的对照组是以SMO特异性激动剂处理但不添加所述候选物质的(1)的细胞；若所述候选物质能够维持细胞膜上谷氨酸转运体GLT-1的表达或活性并降低细胞外谷氨酸浓度，则表明该候选物质是缓解或治疗缺血性脑损伤的物质(或潜在物质)。
在另一优选例中，在候选物质处理后的1小时内、较佳地30分钟内，测定(2)的细胞中SHH信号通路成员蛋白对细胞膜上谷氨酸转运体GLT-1的调节情况。
在另一优选例中，所述的候选物质包括(但不限于)：小分子化合物(如，来自化合物库或基因库的小分子化合物)；基因或蛋白，如干扰分子，基因编辑试剂，核酸抑制物，小RNA等。
在另一优选例中，所述的细胞为离体培养的细胞。
在另一优选例中，所述的细胞为细胞培养物。
本发明的其它方面由于本文的公开内容，对本领域的技术人员而言是显而易见的。
附图说明
图1、啮齿类动物缺血过程中，缺血侧SHH表达升高。图中，0为血管堵塞、即缺血开始的时间。
图2A-B、在缺血过程中，SHH信号通路的激活可以促进细胞外谷氨酸浓 度的上升。
图3A、利用不同药物处理星形胶质细胞，通过电生理的方法记录GLT-1蛋白的电流；其中，处理细胞时，SAG终浓度(3μM)；CYC终浓度(10μM)；SAG+CYC终浓度(3μM+10μM)。
图3B、同位素的实验论证SHH通过SMO降低星形胶质细胞对于细胞外谷氨酸的摄取能力。
图3C、NVP-LDE225可以完全抑制SAG的作用。
图4、C57小鼠脑缺血模型中NVP-LDE225药效评估试验流程。
图5、C57小鼠脑缺血模型中NVP-LDE225药效评估试验结果。
图6、食蟹猴脑缺血模型中NVP-LDE225药效评估试验流程。
图7、食蟹猴脑缺血模型中NVP-LDE225药效评估结果。A图所示为MRI扫描DWI序列(缺血后48h)，高亮信号表征的是脑损伤的区域。B图是统计图，横轴为MRI扫描时间点，T2和DWI的高亮信号都可以反映脑损伤严重程度。C图统计了缺血后30天这一时程中，不同处理组猴子在行为学上的变化。
具体实施方式
本发明人经过深入的研究，首次揭示了一种SHH(Sonic hedgehog)信号通路抑制剂NVP-LDE225可以通过SHH信号通路蛋白SMO快速地调节细胞膜上GLT-1的活性，快速清除胞外的谷氨酸，并能够用于制备缓解或治疗缺血性脑损伤(包括缺血性脑中风，由缺血性脑中风导致的脑损伤)的药物。
化合物及组合物
本发明首先提供了一种具有结构式(I)所示的母核结构的化合物：
Figure PCTCN2020123215-appb-000002
本发明还包括上述具有式(I)所示母核结构的化合物的异构体、溶剂合物、前体，或它们的药学上可接受的盐，只要它们也具有与式(I)所示母核结构的化 合物具有相同或基本相同的功能。所述的“药学上可接受的盐”是指化合物与无机酸、有机酸、碱金属或碱土金属等反应生成的盐。这些盐包括(但不限于)：(1)与如下无机酸形成的盐：如盐酸、硫酸、硝酸、磷酸；(2)与如下有机酸形成的盐，如乙酸、草酸、丁二酸、酒石酸、甲磺酸、马来酸、或精氨酸。其它的盐包括与碱金属或碱土金属(如钠、钾、钙或镁)形成的盐，以酯、氨基甲酸酯，或其它常规的“前体药物”的形式。化合物具有一个或多个不对称中心。所以，这些化合物可以作为外消旋的混合物、单独的对映异构体、单独的非对映异构体、非对映异构体混合物、顺式或反式异构体存在。
所述的“化合物的前体”指当用适当的方法服用后，该化合物的前体在病人体内进行代谢或化学反应而转变成结构式(I)所示母核结构的一种化合物，或化学结构式(I)所示母核结构的一个化合物所组成的盐或溶液。
本领域人员应理解，在得知了本发明化合物的结构以后，可通过多种本领域熟知的方法、利用公知的原料，来获得本发明的化合物，比如化学合成或从生物(如动物或植物)中提取或在提取基础上进行改造的方法，这些方法均包含在本发明中。
可以利用公知的方法来合成本发明的化合物；合成的化合物可以进一步通过柱层析法、高效液相色谱法等方式进一步纯化。此外，也可以通过商购的方式获得本发明的化合物。
本发明还提供了一种组合物(如，药物组合物)，含有有效量的式(I)所示母核结构所述的化合物、或其异构体、溶剂合物、前体。也即，本发明提供了含有NVP-LDE225或其类似物的组合物。
本发明中，所述的“含有”，“具有”或“包括”包括了“包含”、“主要由……构成”、“基本上由……构成”、和“由……构成”；“主要由……构成”、“基本上由……构成”和“由……构成”属于“含有”、“具有”或“包括”的下位概念。
作为一种优选方式，所述的组合物为药物组合物，所述组合物中还含有药学上可接受的盐和/或药学上可接受的载体或赋形剂。
如本文所用，“药学上可接受的”成分是适用于人和/或动物而无过度不良副反应(如毒性、刺激和变态反应)的物质，即有合理的效益/风险比的物质。 “药学上可接受的载体”是用于将本发明的化合物传送给动物或人的药学上或食品上可接受的溶剂、悬浮剂或赋形剂。载体可以是液体或固体。
在本发明中，所述的药物组合物含有按照重量比例为0.001-50％的式(I)所示母核结构所示的化合物或其药学上可接受的盐。较佳的，所述的药物组合物含有按照重量比例为0.05-30％的式(I)所示母核结构所示的化合物或其药学上可接受的盐；更佳地，所述的药物组合物含有按照重量比例为0.01-20％的式(I)所示母核结构所示的化合物或其药学上可接受的盐。本领域人员应理解，根据临床实际需求或制药学中的药物设计方式，其它的重量比例也是可行的。
本发明所述的药物组合物的剂型可以是多种多样的，只要是能够使活性成分有效地到达哺乳动物机体的剂型都是可以的。比如可选自：凝胶剂、气雾剂、片剂、胶囊、粉末、颗粒、糖浆、溶液、或悬浮液。根据本发明的化合物所治疗的疾病类型，本领域人员可以选择方便应用的剂型。从易于制备和给药的立场看，优选的药物组合物是固态组合物，尤其是片剂和固体填充或液体填充的胶囊。本发明的化合物或其药物组合物也可储存在适宜于注射或滴注的消毒器具中。式(I)所示母核结构化合物作为活性成分的有效施用剂量可随给药的模式和待治疗的疾病的严重程度而变化。
应用
本发明人发现，NVP-LDE225通过调控SHH信号通路来发挥对于谷氨酸的调节作用。SHH信号通路原是一条参与神经发育过程的信号通路。本发明人的研究首次证明，SHH可以对星形胶质细胞上的谷氨酸转运体的上下膜进行调节，可以快速的实现膜上谷氨酸转运体数量变化，从而对胞外的谷氨酸浓度进行快速调节。并且，在缺血性脑损伤(包括缺血性脑中风，由缺血性脑中风导致的脑损伤)中，SHH的表达明显升高。SHH信号通路激活引起的膜上转运体下调导致的谷氨酸重吸收降低而在细胞外大量堆积，而本发明的化合物可以改变这一情况，提高膜上谷氨酸转运体的表达或活性，加速胞外谷氨酸的清除，在缺血性脑中风中具有明显的改善效果。
已有的科学研究中显示，SHH信号通路可以在缺血发生后几天帮助大脑进行修复，因此被认为是一种有益于神经保护的信号通路。但是SHH信号通路功 能在缺血急性期并未有报道，而本发明人发现其在缺血急性期发挥的并非是神经保护的作用，其激活实际上导致了谷氨酸转运体(GLT-1)的在细胞膜上表达量下降或活性下降，其抑制剂(优选地为NVP-LDE225)则可以有效地逆转这一情况。本发明中首次揭示，SHH信号通路在缺血急性期有激活并且可以快速调控细胞外谷氨酸浓度，谷氨酸是缺血急性期导致脑损伤的最强因素。
针对SHH信号通路抑制剂NVP-LDE225，已往被本领域人员用于治疗晚期基底细胞瘤，目前已在美国及欧洲上市，其主要作用机制为抑制SHH信号通路中的蛋白Smoothened活性。而本发明人意外地发现，NVP-LDE22只需要在缺血过程中注射一次，就能够在缺血早期(如在6小时内或更短的时间内)快速到达药物靶标，实现药物的有效价值，对于缺血性脑中风的发展具有良好的改善效果，在啮齿类和非人灵长类动物模型的动物实验中均证明其具有很好的神经保护作用，其中食蟹猴是与人类大脑结构高度相近的物种。目前市场上并没有脑缺血神经保护类型的药物，而本发明很有可能填补了这一空白。
本发明中，首次论证在缺血性脑中风的模型中存在SHH调控细胞外谷氨酸的情形。本发明人的在先研究中提出过SHH调控细胞外谷氨酸的概念，但仅限于基于生理状态下的细胞(神经元)实验，且所针对的细胞并非星形胶质细胞，针对的谷氨酸转运体并非为GLT-1。本发明中，首次确认了SHH对于特异表达于星形胶质细胞上的谷氨酸转运体GLT-1活性的调控，这与之前的SHH对于特异表达在神经元上的谷氨酸转运体EAAC1的调控截然不同。所以，尽管本发明人之前关于SHH调控细胞外谷氨酸的概念有所提出，但是本发明中的机制是不同的，并且本发明首次在在体缺血模型中证实了这种调控的存在。
在上述发现的基础上，本发明人将临床用药NVP-LDE225，SHH信号通路特异性抑制剂转化为治疗缺血性脑损伤的药物，拓展了本领域对于SHH信号通路在缺血过程中的认知及NVP-LDE225或其类似物的临床适应症。
因此，作为本发明的一种应用方式，提供了NVP-LDE225或其类似物的用途，用于制备缓解或治疗缺血性脑损伤的组合物(药物)。在缺血过程中，星形胶质细胞的细胞膜上GLT-1蛋白量会下降，而NVP-LDE225的作用在于在缺血性脑损伤过程中维持细胞膜上GLT-1的蛋白量。
作为本发明的一种应用方式，提供了NVP-LDE225或其类似物的用途，用 于制备在缺血性脑损伤过程中维持星形胶质细胞的膜上谷氨酸转运体GLT-1的表达或活性的组合物(药物)。
作为本发明的一种应用方式，提供了NVP-LDE225或其类似物的用途，用于制备减少星形胶质细胞的胞外谷氨酸量的组合物(药物)。
药物筛选
在得知了SHH信号通路蛋白与细胞膜上谷氨酸转运体以及胞外谷氨酸量的相互调控的分子机制之后，可以基于该特征来筛选通过在该分子机制中起作用，从而缓解或治疗缺血性脑损伤的物质。优选地，SHH信号通路蛋白包括SMO蛋白。
因此，本发明提供一种筛选缓解或治疗缺血性脑损伤的潜在物质的方法，所述的方法包括：(1)提供星形胶质细胞，该细胞表达SHH信号通路蛋白以及谷氨酸转运体；(2)用候选物质处理(1)的细胞；(3)测定(2)的细胞中SHH信号通路蛋白对细胞膜上谷氨酸转运体的调节情况，若所述候选物质抑制SHH信号通路蛋白调节的谷氨酸转运体的表达或活性下降、或提高其表达或活性，则表明该候选物质是维持或上调星形胶质细胞的膜上谷氨酸转运体、减少星形胶质细胞的胞外谷氨酸量，从而缓解或治疗缺血性脑损伤的物质(或潜在物质)。
在本发明的优选方式中，在进行筛选时，为了更易于观察到所述信号通路、基因或蛋白的表达或活性的变化的改变，还可设置对照组，所述的对照组可以是不添加所述候选物质的体系。
对于候选物质没有特别的限制，可以是感兴趣的、广泛的物质。例如，但不限于：来自化合物库或基因库的小分子化合物、基因或蛋白；或针对SHH信号通路蛋白、基因或蛋白或它们的上游或下游分子设计的小分子化合物、激动剂、上调剂、促进剂、基因编辑试剂、干扰分子、核酸抑制物、结合分子(如抗体或配体)。在得知了本发明所设计的信号通路信息后，本领域技术人员可以作出多种多样的设计，这些均包含在本发明中。
作为本发明的优选方式，所述的方法还包括：对获得的潜在物质进行进一步的细胞实验和/或动物试验，以进一步选择和确定对于治疗缺血性脑损伤(包括缺血性脑中风，由缺血性脑中风导致的脑损伤)真正有用的物质。
另一方面，本发明还提供了采用所述筛选方法获得的对于缓解或治疗缺血性脑损伤的潜在物质。这些初步筛选出的物质可构成一个筛选库，以便于人们最终可以从中筛选出能够对于治疗缺血性脑损伤(包括缺血性脑中风)真正有用的药物。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。
实施例1、啮齿类动物缺血过程中，缺血侧SHH表达升高
建立小鼠缺血再灌注模型，之后进行检测。检测区域为小鼠纹状体，如图1所示，大脑中动脉堵塞(MCAO)时长为120分钟，之后将栓塞取出，实现血管再通。在如图所示的时间点取出缺血侧(ipsi)和缺血对侧(contra)纹状体，将其浸泡于人工脑脊液中，以收集分泌到细胞外的SHH蛋白。
将富集了SHH的人工脑脊液用ELISA(酶联免疫吸附测定)实验测定其中SHH蛋白含量，发现在缺血后30分钟到60分钟的区间内，缺血一侧的纹状体SHH蛋白的释放显著多于缺血对侧。
实施例2、调节SHH信号通路以调节GLT-1活性和细胞外谷氨酸浓度
1、SHH信号通路的激活促进细胞外谷氨酸浓度
啮齿类动物缺血过程中，缺血侧SHH信号通路的激活促进细胞外谷氨酸浓度升高。采用微透析的方法，从模型小鼠的缺血侧海马区域收集细胞外谷氨酸，然后用高效液相色谱的方法测量收集到的谷氨酸浓度。
结果如图2A-B所示，在缺血前15分钟或缺血后45分钟，小鼠腹腔注射SHH信号通路特异抑制剂CYC(cyclopamine)或其溶剂对照HBC，发现HBC组中细胞外谷氨酸浓度在缺血后有显著上升，而CYC可以有效降低细胞外谷氨酸浓度。因此，在缺血过程中SHH信号通路激活的确可以促进细胞外谷氨酸浓度上升。
2、SHH信号通路快速抑制GLT-1活性
体外培养星形胶质细胞，利用SMO特异性抑制剂和激活剂进行处理，通过电生理的方法记录GLT-1蛋白的电流，GLT-1蛋白电流可以直观反映细胞膜上GLT-1蛋白活性。图3A的左图是记录的电流图，图3A右图是对于不同药物处理后GLT-1电流变化的统计图，所有药物孵育时间均为30分钟，如图所示，SMO的特异性激动剂SAG可以使GLT-1活性下降约50％，而SMO特异性抑制剂CYC(其特异作用于SHH信号通路的蛋白SMO，抑制其活性)可以阻止SHH的作用(SHH+CYC)。这一实验结果进一步说明，在SHH信号通路中，SMO蛋白激活抑制了GLT-1活性。
在图3B中，用同位素的实验论证SHH通过SMO降低星形胶质细胞对于细胞外谷氨酸的摄取能力。星形胶质细胞孵育用 3H标记的谷氨酸，在不同药物(如图所示)的处理后，收集细胞，测量其中的 3H标记的谷氨酸的量。以此反映星形胶质细胞对于谷氨酸的摄取能力。谷氨酸的摄取完全依赖细胞膜上谷氨酸转运体，SHH下调了GLT-1活性，因此SHH处理的星形胶质细胞其对谷氨酸的摄取能力也有了显著降低，并且这一现象可以被SMO的拮抗剂CYC所阻断。
在图3C中，用NVP-LDE225替换CYC，SMO的特异性激动剂SAG可以使GLT-1活性下降约50％，而NVP-LDE225几乎可以完全抑制SAG的作用。这一结果为后续在小鼠及猴子的实验开展提供理论依据。
上述结果说明，SHH信号通路快速调节膜上谷氨酸转运体GLT-1的数量，调节胞外的谷氨酸的浓度。抑制SHH信号通路的激活可以抑制膜上谷氨酸转运体GLT-1的活性下降，从而有效降低细胞外谷氨酸浓度。
实施例3、NVP-LDE225在脑缺血模型中保护脑损伤及改善行为
1、NVP-LDE225用于C57小鼠缺血模型
以啮齿类C57小鼠建立缺血模型，用于分析本发明人筛选获得的NVP-LDE225对于脑缺血的缓解或治疗作用。
体重约25g的C57小鼠，在第1天对其腹腔注射NVP-LDE225，注射量为40mg/kg；同时设置溶剂对照(PEG400/5％Dextrose和水75:25(v/v))。20分钟后 进行缺血处理，缺血处理时间为2小时(h)，之后恢复供血。第二天，对小鼠进行行为学评分及TTC染色，确定脑损伤的程度。C57小鼠脑缺血模型中NVP-LDE225药效评估试验流程如图4。
结果如图5，左图为实物图，TTC脑片染色，红色区域为脑组织具有活性的区域，白色区域为脑损伤的部位。中间的统计图为对白色区域体积的计算，发现NVP-LDE225可以显著降低白色区域的大小，进而反映脑损伤的减少。右图为整体评估NVP-LDE225对于小鼠缺血后神经行为学的改善。可以看到NVP-LDE225可以显著改善缺血后小鼠神经行为缺陷。
2、NVP-LDE225用于食蟹猴缺血模型
以食蟹猴建立缺血模型，用于分析本发明人筛选获得的NVP-LDE225对于脑缺血的缓解或治疗作用。
对食蟹猴进行大脑中动脉夹闭，具体过程为：在术前2-3天，先测定MRI、CBF和行为学，获得基础值，然后第0天进行大脑中动脉夹闭，夹闭至25min时开始给药，给药总体积5ml，给药时长5分钟，第1组猴子给予0.91mg/ml NVP-LDE225 137.5ul，第2组猴子给予137.5ul的溶剂；血气采样时间点为：夹闭前15分钟，夹闭后5分钟和给药后15分钟。第1天测定行为学。第2天，再次测定MRI、CBF和行为学。之后分别在第5、7、14天测定行为学。最后，在第30天测定MRI、CBF和行为学，以及留脑切片。食蟹猴脑缺血模型中NVP-LDE225药效评估试验流程如图6。
结果如图7所示，A图所示为MRI扫描DWI序列(缺血后48h)，高亮信号表征的是脑损伤的区域，由图可见，NVP-LDE225组中，高亮信号比安慰剂组显著降低。B图是统计图，横轴为MRI扫描时间点，T2和DWI的高亮信号都可以反映脑损伤严重程度。可见，无论是在缺血后48h或者30天，NVP-LDE225处理组缺血性脑损伤都有显著减少。C图是统计了缺血后30天这一时程中，不同处理组猴子在行为学上的变化，其中，意识，肌肉协调及运动系统的表现均在NVP-LDE225组表现更好，综合四项的总体评分则有更加显著的改善。
因此，NVP-LDE225能够有效地减少脑梗死。
实施例4、细胞水平的药物筛选
取星形胶质细胞，该细胞表达SHH信号通路以及谷氨酸转运体GLT-1。将该种细胞作为用于筛选抑制SHH信号通路的药物的细胞模型。
测试组：用候选物质处理的上述细胞的培养物；
对照组：不用候选物质处理的上述细胞的培养物。
在处理后适当时间，采用常规方法测定SHH信号通路对细胞膜上谷氨酸转运体的调节情况，若所述候选物质抑制SHH信号通路调节的谷氨酸转运体的活性下调，则表明该候选物质是缓解或治疗缺血性脑损伤的物质。
采用NVP-LDE225作为候选物质进行了测试，结果显示谷氨酸转运体的活性下调被显著地抑制，从而NVP-LDE225是治疗缺血性脑损伤有用的候选物质。
同时，本发明人还运用化合物库中的化合物处理上述细胞的培养物，以期筛选其它药物。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (14)

  1. 式(I)所示母核结构的化合物或其异构体、衍生物、溶剂合物或前体，或它们的药学上可接受的盐的用途，用于：
    制备缓解或治疗缺血性脑损伤的组合物；或
    制备在缺血性脑损伤过程中维持星形胶质细胞膜上谷氨酸转运体GLT-1并降低细胞外谷氨酸浓度的组合物；
    Figure PCTCN2020123215-appb-100001
  2. 如权利要求1或6所述的用途，其特征在于，所述的缺血性脑损伤为缺血急性期的脑损伤或缺血早期的脑损伤。
  3. 如权利要求1或2所述的用途，其特征在于，所述的缺血性脑损伤包括：缺血性脑中风，由缺血性脑中风引起的脑损伤。
  4. 如权利要求1所述的用途，其特征在于，所述的维持星形胶质细胞膜上谷氨酸转运体GLT-1包括：促进星形胶质细胞的膜上谷氨酸转运体GLT-1的表达或提高星形胶质细胞的膜上谷氨酸转运体GLT-1的活性。
  5. 如权利要求1或4所述的用途，其特征在于，所述化合物通过维持星形胶质细胞的膜上谷氨酸转运体GLT-1，清除胞外的谷氨酸。
  6. 如权利要求1或4所述的用途，其特征在于，所述的维持星形胶质细胞的膜上谷氨酸转运体GLT-1为在缺血急性期的维持。
  7. 一种制备用于在缺血性脑损伤过程中维持星形胶质细胞的膜上谷氨酸 转运体GLT-1并降低细胞外谷氨酸浓度或缓解或治疗缺血性脑损伤的组合物的方法，包括：将式(I)所示母核结构的化合物或其异构体、衍生物、溶剂合物或前体，或它们的药学上可接受的盐与药学或食品学上可接受的载体混合；
    Figure PCTCN2020123215-appb-100002
  8. 一种在缺血性脑损伤过程中维持星形胶质细胞的膜上谷氨酸转运体GLT-1并降低细胞外谷氨酸浓度的方法，其特征在于，所述方法包括：以式(I)所示母核结构的化合物或其异构体、衍生物、溶剂合物或前体，或它们的药学上可接受的盐处理星形胶质细胞；
    Figure PCTCN2020123215-appb-100003
  9. 一种筛选缓解或治疗缺血性脑损伤的物质的方法，其特征在于，所述方法包括：
    (1)提供星形胶质细胞，该细胞表达SHH信号通路成员蛋白以及谷氨酸转运体GLT-1；
    (2)用候选物质处理(1)的细胞；
    (3)测定(2)的细胞中SHH信号通路成员蛋白对细胞膜上谷氨酸转运体GLT-1的调节情况，若所述候选物质抑制SHH信号通路成员蛋白调节的谷氨酸转运体GLT-1的活性、维持星形胶质细胞膜上谷氨酸转运体GLT-1并降低细胞外谷氨酸浓度，则表明该候选物质是缓解或治疗缺血性脑损伤的物质。
  10. 如权利要求9所述的方法，其特征在于，步骤(2)中，候选物质处理时，还包括以SMO特异性激动剂处理，下调GLT-1。
  11. 如权利要求9所述的方法，其特征在于，SHH信号通路成员蛋白包括SMO蛋白。
  12. 如权利要求9～11任一所述的方法，其特征在于，步骤(2)包括：在测试组中，以SMO特异性激动剂处理，并将候选物质加入(1)的细胞中；和/或
    步骤(3)包括：检测测试组的细胞中SHH信号通路成员蛋白对细胞膜上谷氨酸转运体GLT-1的调节情况，并与对照组比较，其中所述的对照组是以SMO特异性激动剂处理但不添加所述候选物质的(1)的细胞；若所述候选物质抑制SHH信号通路成员蛋白调节的谷氨酸转运体GLT-1的活性、维持细胞膜上谷氨酸转运体GLT-1的表达或活性并降低细胞外谷氨酸浓度，则表明该候选物质是缓解或治疗缺血性脑损伤的物质。
  13. 如权利要求9所述的方法，其特征在于，所述的缺血性脑损伤为缺血急性期的脑损伤，缺血早期的脑损伤。
  14. 如权利要求9所述的方法，其特征在于，在候选物质处理后的1小时内、较佳地30分钟内，测定(2)的细胞中SHH信号通路成员蛋白对细胞膜上谷氨酸转运体GLT-1的调节情况。
PCT/CN2020/123215 2019-11-11 2020-10-23 缺血性脑损伤的新型治疗药物 WO2021093553A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201911095830.6 2019-11-11
CN201911095830.6A CN112773805B (zh) 2019-11-11 2019-11-11 缺血性脑损伤的新型治疗药物

Publications (1)

Publication Number Publication Date
WO2021093553A1 true WO2021093553A1 (zh) 2021-05-20

Family

ID=75749771

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/123215 WO2021093553A1 (zh) 2019-11-11 2020-10-23 缺血性脑损伤的新型治疗药物

Country Status (2)

Country Link
CN (1) CN112773805B (zh)
WO (1) WO2021093553A1 (zh)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103893184A (zh) * 2009-07-21 2014-07-02 诺华股份有限公司 包含活性剂的局部药物组合物
CN101820950B (zh) * 2007-06-07 2015-01-21 Irm责任有限公司 作为刺猬蛋白信号通路调节剂的联苯基甲酰胺类化合物

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
NZ737399A (en) * 2015-05-21 2023-01-27 Chemocentryx Inc Ccr2 modulators
EP3332257A1 (en) * 2015-08-07 2018-06-13 Thomas Helledays Stiftelse För Medicinsk Forskning Method for diagnosisng cancer or cancer-associated thrombosis by measuring levels of h3cit in plasma
AR110995A1 (es) * 2017-02-24 2019-05-22 Bayer Ag Combinación de inhibidores de quinasa atr con sal de radio-223
WO2019100003A1 (en) * 2017-11-17 2019-05-23 Yoon Jaeyoung Combination therapy targeting cancer associated with the hedgehog pathway
CN109010333A (zh) * 2018-11-06 2018-12-18 杭州医学院 脱氢弯孢霉素在抑制Hedgehog通路中的应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101820950B (zh) * 2007-06-07 2015-01-21 Irm责任有限公司 作为刺猬蛋白信号通路调节剂的联苯基甲酰胺类化合物
CN103893184A (zh) * 2009-07-21 2014-07-02 诺华股份有限公司 包含活性剂的局部药物组合物
CN103893184B (zh) * 2009-07-21 2016-03-02 诺华股份有限公司 包含活性剂的局部药物组合物

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
WANG LEI: "The Roles of Astrocyte in Nervous System Diseases", BIOTECHNOLOGY BULLETIN, 26 June 2010 (2010-06-26), pages 33 - 36+51, XP055812516, DOI: 10.13560/j.cnki.biotech.bull.1985.2010.06.044 *

Also Published As

Publication number Publication date
CN112773805B (zh) 2024-05-10
CN112773805A (zh) 2021-05-11

Similar Documents

Publication Publication Date Title
Fink Ethyl pyruvate: a novel anti‐inflammatory agent
RU2731383C2 (ru) Композиции селеноорганических соединений и способы их применения
US20220117967A1 (en) Compositions and Methods for the Treatment of Prader-Willi Syndrome
EP1845970B1 (en) Tumor necrosis factor inhibitors
US20180251489A1 (en) Malt1 inhibitors and uses thereof
JP2019531320A (ja) Ｓｓａｏ／ｖａｐ−１阻害剤とｓｇｌｔ２阻害剤とを含む組合せ、その使用
US20120040956A1 (en) Inhibitors of hif-1 protein accumulation
JP2022504944A (ja) Ｎａｄ＋またはｎａｄ＋前駆体と組み合わせたｓａｒｍ１の阻害
EP2675275B1 (en) Compositions and methods for the treatment of obesity and related disorders
CN113813387A (zh) Ppar激动剂在制备治疗急性髓细胞白血病的药物中的应用
CN112839712A (zh) 用于减少或治疗发炎的组合物和方法
WO2021093553A1 (zh) 缺血性脑损伤的新型治疗药物
KR20190034609A (ko) 환상 아민 유도체의 결정 및 그 의약 용도
AU2004317897B2 (en) Activation of hypoxia-inducible gene expression
CN101658522B (zh) 他克林短链二聚体类化合物在制备治疗神经退行性疾病药物中的应用
WO2017058828A1 (en) Methods for treating diseases mediated by erbb4-positive pro-inflammatory macrophages
Koike et al. Pretreatment with olprinone hydrochloride, a phosphodiesterase III inhibitor, attenuates lipopolysaccharide-induced lung injury via an anti-inflammatory effect
JP5101306B2 (ja) 糖尿病治療剤
US11643428B2 (en) Therapeutic drug for neurodegenerative disease and application thereof
KR20140134170A (ko) 신경염증 질환의 예방 또는 치료용 약학 조성물
CN108601772A (zh) 用于治疗tdp-43蛋白质病的他克莫司
TWI641594B (zh) 用於治療中風的環烯醚萜苷類化合物，其醫藥組合物及其用途
CN116421605B (zh) Isx-9在治疗衰老相关的昼夜节律幅度下降和睡眠障碍方面的应用
EP4053129A1 (en) Use of ginkgolide a in the treatment of autism
WO2018236747A1 (en) COMPOUNDS AND COMPOSITIONS FOR EXTENDING THE DURATION OF A SUBJECT

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20887808

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20887808

Country of ref document: EP

Kind code of ref document: A1