WO2022144048A2 - 多酚类化合物在防治脑水肿中的应用 - Google Patents

多酚类化合物在防治脑水肿中的应用 Download PDF

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WO2022144048A2
WO2022144048A2 PCT/CN2022/085834 CN2022085834W WO2022144048A2 WO 2022144048 A2 WO2022144048 A2 WO 2022144048A2 CN 2022085834 W CN2022085834 W CN 2022085834W WO 2022144048 A2 WO2022144048 A2 WO 2022144048A2
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procyanidin
cerebral edema
polyphenolic compound
pharmaceutically acceptable
expression
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PCT/CN2022/085834
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French (fr)
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WO2022144048A3 (zh
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昌军
刘新华
丁琛
王静欢
金琳
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复旦大学
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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
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin

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  • the invention belongs to the field of biomedicine, and particularly relates to the application of a class of polyphenolic compounds in preventing and treating cerebral edema, especially its application in preparing a medicine for preventing and treating cerebral edema caused by high altitude hypobaric hypoxia and cerebral edema caused by cerebral apoplexy.
  • Cerebral edema is a series of serious pathological changes, such as a series of serious pathological changes, such as increased brain volume, increased intracranial pressure, decreased cerebral perfusion pressure and cerebral blood flow, caused by various internal and external factors acting on the brain tissue, causing excessive fluid accumulation in brain cells or intercellular spaces. Brain herniation can even be life-threatening. Common causes of cerebral edema include cerebrovascular disease, craniocerebral trauma, cerebral stroke, brain tumor, and any diseases that can affect the blood supply and oxygen supply to the brain, such as cardiac and respiratory arrest, explosive liver failure, high altitude, carbon monoxide poisoning, etc. Brain edema is mainly divided into 4 types: vasogenic cerebral edema, cytotoxic cerebral edema, interstitial cerebral edema, and osmotic cerebral edema.
  • the drug treatment of cerebral edema includes: 1) dehydration treatment, which can quickly reduce intracranial pressure and prevent brain herniation.
  • Commonly used drugs mannitol, hypertonic saline, furosemide; 2) sedation treatment, rapid sedation to prevent accident
  • Drugs propofol, fentanyl
  • Hormone therapy to improve brain cell function and reduce cerebral edema commonly used drugs: dexamethasone.
  • dexamethasone There is no targeted drug for high altitude cerebral edema.
  • dexamethasone high glucose, acetazolamide, furosemide, etc. are also given. But so far there is no drug specifically for the treatment of cerebral edema.
  • the technical problem to be solved by the present invention is to provide the application of a polyphenolic compound for the lack of effective prevention and treatment of cerebral edema in the prior art, especially in the preparation and prevention of cerebral edema caused by hypobaric hypoxia and cerebral apoplexy caused by cerebral apoplexy.
  • procyanidins B 1 -B 4 in cerebral edema caused by hypobaric hypoxia and/or stroke.
  • the present invention unexpectedly found that proanthocyanidins B 1 -B 4 can inhibit the aquaporin AQP4.
  • stroke or trauma can cause severe edema in the normal brain, while the brain edema in the APQ4 aquaporin-deficient brain is very mild and recovers quickly.
  • the present invention discovers for the first time the pharmacological effects and uses of polyphenolic compounds, especially procyanidins B 1 -B 4 in cerebral edema, especially the application in preventing and treating high altitude cerebral edema-related diseases.
  • the present invention mainly solves the above technical problems through the following technical solutions.
  • One of the technical solutions of the present invention is: the application of a polyphenolic compound in the preparation of a medicine for preventing and treating cerebral edema; the structural formula of the polyphenolic compound is as follows:
  • R 1 to R 6 may be hydrogen, hydroxyl, methoxy or fluorine at the same time or not at the same time.
  • the polyphenolic compound in the present invention is procyanidin, a pharmaceutically acceptable salt thereof, a solvate thereof, a solvate of a pharmaceutically acceptable salt thereof, or a crystal form thereof.
  • the procyanidins are preferably selected from procyanidin B 1 , procyanidin B 2 , procyanidin B 3 and procyanidin B 4 , gallocatechin-(4 ⁇ 8)-catechin (CAS No. 78392-25-3) and One or more of theacin-(4 ⁇ 8)-gallocatechin (CAS No. 135095-45-3).
  • the polyphenolic compound is procyanidin B 1 , procyanidin B 2 , procyanidin B 3 or procyanidin B 4 .
  • procyanidin B 3 (CAS No. 23567-23-9) has the best effect in treating cerebral edema, especially for high-altitude cerebral edema.
  • the cerebral edema described in the present invention can be conventional in the art, such as cerebral edema caused by hypoxia and/or stroke.
  • the cerebral edema may be acute cerebral edema; for example, high-altitude cerebral edema.
  • Procyanidin B 1 , procyanidin B 2 , procyanidin B 3 and procyanidin B 4 are a class of compounds extracted from grape seeds, and their structural formulas and gallocatechin-(4 ⁇ 8)-catechin, or catechin-
  • the structural formula of (4 ⁇ 8)-gallocatechin is as follows:
  • the acute cerebral edema has one or both of the following manifestations:
  • the polyphenolic compound can be the only active ingredient of the medicine.
  • the second technical solution of the present invention is: the application of polyphenolic compounds in the preparation of medicines for recovering damage caused by hypoxia of astrocytes;
  • the structural formula of the polyphenolic compound is as follows:
  • R 1 to R 6 may be hydrogen, hydroxyl, methoxy or fluorine at the same time or not at the same time.
  • the polyphenolic compound is preferably procyanidin, a pharmaceutically acceptable salt thereof, a solvate thereof, a solvate of a pharmaceutically acceptable salt thereof, or a crystal form thereof.
  • the procyanidins in the present invention can be selected from procyanidin B 1 , procyanidin B 2 , procyanidin B 3 , procyanidin B 4 , gallocatechin-(4 ⁇ 8)-catechin and catechin-(4 ⁇ ) ⁇ 8) - One or more of gallocatechins; eg, procyanidin B 1 , procyanidin B 2 , procyanidin B 3 or procyanidin B 4 , preferably procyanidin B 3 .
  • the injury may have one or both of the following manifestations: 1) the expression of AQP4 and HIF-1 ⁇ is increased; 2) the expression of inflammatory proteins is increased; the inflammatory proteins are preferably vcam-1, cox-2, iNOS, One or more of IL-6 and IL-1 ⁇ .
  • the polyphenolic compound can be the only active ingredient of the medicine.
  • the third technical solution of the present invention is: the application of polyphenolic compounds in the preparation of AQP4 inhibitors; the structural formula of the polyphenolic compounds is as follows:
  • R 1 to R 6 may be hydrogen, hydroxyl, methoxy or fluorine at the same time or not at the same time.
  • pharmaceutically acceptable means that salts, solvents, excipients, etc. are generally non-toxic, safe, and suitable for use by a subject.
  • subject is preferably a mammal, more preferably a human.
  • pharmaceutically acceptable salts refers to salts of compounds of the present invention, and pharmaceuticals, pharmaceutical compositions containing them, prepared with relatively non-toxic, pharmaceutically acceptable acids or bases.
  • a relatively acidic functional group is contained in the compounds of the present invention and the drugs and pharmaceutical compositions containing them, the compounds of the present invention can be combined with such drugs by using a sufficient amount of a pharmaceutically acceptable base in a pure solution or a suitable inert solvent.
  • the base addition salt is obtained by contacting the neutral form.
  • Pharmaceutically acceptable base addition salts include, but are not limited to, lithium, sodium, potassium, calcium, aluminum, magnesium, zinc, bismuth, ammonium, diethanolamine.
  • acid additions can be obtained by contacting the neutral form of such drugs with a sufficient amount of a pharmaceutically acceptable acid in neat solution or in a suitable inert solvent.
  • a pharmaceutically acceptable acid include inorganic acids, including but not limited to: hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, carbonic acid, phosphoric acid, phosphorous acid, sulfuric acid, and the like.
  • Described pharmaceutically acceptable acid includes organic acid, described organic acid includes but is not limited to: acetic acid, propionic acid, oxalic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid , fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, salicylic acid, tartaric acid, methanesulfonic acid, isonicotinic acid, acid citric acid, oleic acid , tannic acid, pantothenic acid, hydrogen tartrate, ascorbic acid, gentisic acid, fumaric acid, gluconic acid, sugar acid, formic acid, ethanesulfonic acid, pamoic acid (i.e.
  • the “multiple” in the term “one or more” can mean 2, 3, 4, 5, 6, 7, 8, 9 or more.
  • the compounds of the present invention, medicines or pharmaceutical compositions containing them can be administered in unit dosage form, and the route of administration can be enteral or parenteral, such as oral administration, topical application, intravenous injection, intramuscular injection, subcutaneous injection, nasal cavity, oral mucosa , eyes, lungs and respiratory tract, skin, vagina, rectum, etc.
  • the dosage form for administration can be a liquid dosage form, a solid dosage form or a semi-solid dosage form.
  • Liquid dosage forms can be solutions (including true solutions and colloidal solutions), emulsions (including o/w, w/o and double emulsion), suspensions, injections (including water injection, powder injection and infusion), eye drops solid dosage forms can be tablets (including ordinary tablets, enteric-coated tablets, buccal tablets, dispersible tablets, chewable tablets, effervescent tablets, orally disintegrating tablets), capsules ( Including hard capsules, soft capsules, enteric-coated capsules), granules, powders, pellets, drop pills, suppositories, films, patches, gas (powder) aerosols, sprays, etc.; semi-solid dosage forms can be ointments, Gels, pastes, etc.
  • the medicament or pharmaceutical composition of the present invention can be made into ordinary preparations, as well as sustained-release preparations, controlled-release preparations, targeted preparations and various microparticle drug delivery systems.
  • “Pharmaceutical composition” refers to mixing one or more of the compounds of the present invention, or a pharmaceutically acceptable salt, solvate, hydrate or prodrug thereof, with another chemical ingredient, such as a pharmaceutically acceptable carrier, .
  • a pharmaceutical composition is to facilitate the process of administration to an animal.
  • “Pharmaceutically acceptable carrier” refers to an inactive ingredient in a pharmaceutical composition that does not cause significant irritation to the organism and does not interfere with the biological activity and properties of the administered compound, such as, but not limited to: calcium carbonate, calcium phosphate , various sugars (such as lactose, mannitol, etc.), starch, cyclodextrin, anhydrous stearate, cellulose, anhydrous carbonate, acrylic acid polymer or methacrylic acid polymer, gel, water, polyethylene glycol, Propylene glycol, ethylene glycol, EZ sesame oil or hydrogenated EZ sesame oil or polyethoxy hydrogenated EZ sesame oil, sesame oil, corn oil, peanut oil, etc.
  • compositions in addition to a pharmaceutically acceptable carrier, it may also include adjuvants commonly used in pharmacy (agents), such as: antibacterial agents, antifungal agents, antimicrobial agents, preservatives, conditioning agents. Colorants, solubilizers, thickeners, surfactants, complexing agents, proteins, amino acids, fats, carbohydrates, vitamins, minerals, trace elements, sweeteners, pigments, flavors or their combinations, etc.
  • treatment refers to therapeutic therapy.
  • treatment refers to: (1) ameliorating one or more biological manifestations of the disease or disorder, (2) interfering with (a) one or more points in the biological cascade leading to or causing the disorder or (b) ) one or more biological manifestations of the disorder, (3) amelioration of one or more symptoms, effects or side effects associated with the disorder, or one or more symptoms, effects or side effects associated with the disorder or its treatment, or (4) slowing the progression of the disorder or one or more biological manifestations of the disorder.
  • solvate refers to a substance formed by combining a compound of the present invention with a stoichiometric or non-stoichiometric amount of a solvent. Solvent molecules in solvates can exist in ordered or non-ordered arrangements.
  • the solvent includes, but is not limited to, water, ethanol, and the like.
  • “Pharmaceutically acceptable salt” and “solvate” in the term “solvate of a pharmaceutically acceptable salt”, as described above, refer to a compound of the present invention with a relatively non-toxic, pharmaceutically acceptable acid or bases, substances formed in combination with stoichiometric or non-stoichiometric amounts of solvents.
  • Said “solvates of pharmaceutically acceptable salts” include, but are not limited to, hydrochloric acid monohydrates of the compounds of the present invention.
  • crystal form means that the ions or molecules in it are arranged strictly periodically in three-dimensional space in a definite manner, and have the regularity of periodic repetition at a certain distance; crystal form, or polymorphism.
  • amorphous means that the ions or molecules are in a disordered distribution state, that is, there is no periodic arrangement between ions and molecules.
  • the "comprising, comprising or containing” may mean that there are other components in addition to the components listed below; it may also mean “consisting of”, that is, only the components listed below are included without Other ingredients are present.
  • the reagents and raw materials used in the present invention are all commercially available.
  • polyphenolic compounds such as procyanidin B3
  • procyanidin B3 can reduce leakage after intervening in brain edema, improve the overall lengthening of the hippocampus caused by brain edema, and narrow the DG area. It can improve the expression of AQP4 and HIF-1 ⁇ caused by cerebral edema, and the expression of ZO-1 and claudin-1 is decreased; it can make the expression of inflammatory proteins (vcam-1, cox-2, iNOS). expression decreased.
  • FIG. 1 shows the water content detection
  • the results show that procyanidin B 3 (MT-8) can significantly reduce the water content.
  • Figure 2 shows the damage to the barrier of acute brain edema detected by the EB method.
  • the results show that MT-8 can significantly reduce leakage.
  • Figure 3 shows HE staining to observe the effect of acute cerebral edema on the morphology of the hippocampus.
  • the results show that the acute cerebral edema caused the overall length of the hippocampus and the narrowing of the DG area, and the staining found that the cells were arranged in disorder, some diffuse, and the CA3 area expanded outwards , and accompanied by diffusion; improved after MT-8 intervention.
  • Figure 4 shows the expression of AQP4, HIF-1 ⁇ and barrier proteins in the hippocampus after acute brain edema injury by Western blot.
  • Figure 5 shows the Western blot method to detect the expression of inflammatory proteins in the hippocampus after acute brain edema injury.
  • Control control group (given the corresponding volume of normal saline, 40mg/kg/day, ip.); HACE: high altitude cerebral edema model group (given the corresponding volume of normal saline, 40mg/kg/day, ip.); MT-8 : 40mg/kg/day, ip. *** p ⁇ 0.001.
  • Figure 6A shows the immunofluorescence method to detect the expression of AQP4 in astrocytes damaged by OGD
  • Figure 6B and Figure 6C show the Western blot method to detect the expression of AQP4, HIF-1 ⁇ and inflammatory proteins in astrocytes damaged by OGD.
  • Control control group; Hypoxia: OGD injury 6h model group; MT-8: 10 ⁇ M; *** p ⁇ 0.001.
  • Figure 7 shows the effects of procyanidin B 1 , procyanidin B 2 , procyanidin B 3 and procyanidin B 4 on the protein expressions of AQP4, TRPV4, HIF-1 ⁇ , HIF-2 ⁇ and SNX13 after OGD-induced astrocyte injury by Western blot. .
  • Figure 8 is the magnetic resonance image of the cynomolgus monkey administration group G2-1 on the 8th day.
  • Fig. 9 is the magnetic resonance image of the cynomolgus monkey administration group G2-2 on the 8th day.
  • Procyanidin B 3 is abbreviated as MT-8 in the following.
  • Example 1 The preventive effect of MT-8 on high altitude cerebral edema
  • mice in the plain control group were routinely reared in cages without exercise and decompression and hypoxia treatment, with free food and water, and natural day and night lighting.
  • the mice in the high-altitude hypoxia group were trained on the animal experimental treadmill for 2 days. Specific training method: Start at 8:30 every morning, set the treadmill slope to 0°, the speed to 12m/min, the continuous exercise time to be 15min, and to continue this training after a 20min rest, for a total of 6 times.
  • the daily exercise time is 90 minutes, and the rest of the time is free to eat and drink, and natural day and night lighting.
  • the animal acute hypoxia exposure experiment was carried out in a high altitude environment simulation chamber.
  • the specific preparation process of the disease model in a plateau environment simulation cabin with a simulated altitude of 5000m, the full-time experimenter in the cabin put the mice into the animal experimental treadmill for 30 minutes, and then immediately allowed the mice to exercise on the treadmill.
  • Animal treadmill setting parameters the slope is 10°, the speed is 12m/min, and the mice are kept in motion by electrical stimulation. After the mice continued to exercise for 4 hours, the experimenter in the cabin took the mice out of the track and let them rest for 15-20 minutes, during which the mice could eat and drink freely.
  • mice After the rest, continue to perform 11 times of exhaustive exercise in this way, with a total exercise time of 2640min.
  • the mice were placed in the experimental cabin, and the connecting cabin door was closed.
  • the extravehicular technicians quickly raised the altitude of the experimental cabin from 5000m to 6000m.
  • the altitude of the experimental cabin was increased from 6000m to 8000m, and the mice were allowed to continue to be exposed to decompression and hypoxia for 3 days without exercise at the plateau.
  • the temperature in the cabin is about 18°C
  • the light-dark cycle is 24h, and there is free food and water.
  • mice After the mice were anesthetized, the brains were quickly decapitated, and the wet weight of the brain tissue was weighed, then placed in an oven at 60°C for 3 d, and then the dry weight of the brain tissue was weighed.
  • the water content of each group of mice was calculated according to (wet weight-dry weight)/wet weight*100%.
  • mice 1h before sampling, 2% EB solution (4 mL/kg) was injected into the tail vein of each mouse. After 1h, the mice were anesthetized and perfused with 0.9% normal saline until the color of the heart outflow solution was transparent. The head was decapitated, and a general photograph was taken. Then freeze at -20°C for 30min, section 6 coronal slices, and photograph EB leakage.
  • mice brain tissue was homogenized to detect the expressions of AQP4, HIF-1 ⁇ , inflammatory proteins (vcam-1, cox-2, iNOS) and barrier proteins (ZO-1, claudin-1).
  • Acute cerebral edema caused a significant increase in water content, which was significantly reduced after MT-8 intervention (see Table 1 and Figure 1).
  • Acute cerebral edema caused barrier disruption and marked EB leakage; MT-8 intervention reduced leakage (Figure 2).
  • Acute cerebral edema caused the overall length of the hippocampus, the DG area became narrow, and the staining found that the cells were arranged disorderly and diffused.
  • Acute cerebral edema caused the expression of AQP4 and HIF-1 ⁇ to increase, and the expression of ZO-1 and claudin-1 to decrease; both were improved after MT-8 treatment (see Table 2 and Figure 4).
  • Acute cerebral edema caused an increase in the expression of inflammatory proteins (vcam-1, cox-2, iNOS), which was significantly decreased after MT-8 treatment (see Table 3 and Figure 5).
  • the SD rats within 24 hours of the newborn were taken, and the skin was wiped with alcohol, and then the rats were killed by dismembering their necks and disinfected in 75% alcohol for several minutes.
  • the cortex was then isolated, the cortex was chopped, and the chopped cortex was collected and centrifuged at 1000 rpm for 8 min. Discard the supernatant, add 0.25% trypsin to digest for 10 minutes, shake once every 5 minutes, add DMEM/F12 medium containing 10% FBS to stop digestion for 10 minutes, shake once every 5 minutes, and pass through a 200-mesh sieve.
  • the cells When the cells are more than 80% confluent, they are subcultured, digested with 0.25% trypsin (including EDTA) for a few minutes to terminate the digestion, adjust the cell density, and inoculate to the coated cells.
  • trypsin including EDTA
  • the third generation can be used for experiments.
  • the serum-free DMEM medium was replaced, and it was placed in a three-gas incubator with 1% O 2 , 5% CO 2 and 94% N 2 to prepare the hypoxia model. After 6 hours of hypoxia, the subsequent index detection was performed.
  • astrocytes were labeled with GFAP, and it was found that the expression of AQP4 was significantly increased, while the expression of AQP4 and HIF-1 ⁇ was significantly down-regulated after MT-8 treatment, and the inflammatory response was alleviated (vcam-1, cox -2, iNOS, IL-6, IL-1 ⁇ ) (see Table 4 and Figures 6A-6C).
  • the method of MT-8 treatment is that after the primary astrocytes have been cultured, they are passaged and seeded. When the cells grow to about 80%, they are pretreated with MT-8 (10 ⁇ M) for 4 hours, and then replaced with DMEM sugar-free culture. base, placed in a (1% O 2 , 5% CO 2 , 94% N 2 ) three-gas incubator to continue culturing for 6 hours and then collect the samples for relevant detection.
  • the treatment process of glucose and oxygen deprivation is as follows: after the primary astrocytes are cultured, they are passaged and seeded. When the cells grow to about 80%, they are replaced with DMEM sugar-free medium and placed in (1% O 2 , 5 %CO 2 , 94% N 2 ) in a three-gas incubator for 6 hours, and then collect samples for relevant testing.
  • proanthocyanidin treatment is as follows: after the primary astrocytes are cultured, passaged and seeded, when the cells grow to about 80%, they are pretreated with procyanidin B 1 -B 4 (10 ⁇ M) for 4 hours, and then replaced with DMEM sugar-free culture. base, placed in a (1% O 2 , 5% CO 2 , 94% N 2 ) three-gas incubator to continue culturing for 6 hours and then collect the samples for relevant detection.
  • the surgical window was closed with layer-by-layer sutures, the wound was disinfected, and ceftriaxone sodium (0.1 g/kg) and painridin (2 times a day, 4 mg/kg, i.m.) were continuously used for 7 days for veterinary care such as anti-infection and analgesia.
  • cynomolgus monkeys were randomly divided into 2 groups according to body weight, with 2 animals in each group, namely: 1) Model group: G1-1 and G1-2 (given the corresponding volume of normal saline, iv. daily for 7 consecutive days) 2) Administration group: G2-1 and G2-2 (administered with MT-8, 12 mg/kg/day, iv. administered every day for 7 consecutive days). The time point of the first administration was 1 hour after the start of stroke surgery.
  • the cynomolgus monkeys were fasted for 10-16 hours before MRI scan, and the animals were anesthetized by intramuscular injection of atropine (0.04 mg/kg) 15 minutes before anesthesia.
  • MRI scans were performed 3 days before surgery, Day 2, Day 4, and Day 8.
  • the scanning machine model was a Siemens SKyra 3.0T superconducting magnetic resonance imager.
  • the scanning sequence included: T1WI, T2WI, T2FLAIR, and DWI.
  • the scanning parameters are as follows. Comparing the image information of T1 and T2 images (Fig. 8 and Fig.
  • the edema brain tissue volume in the left brain in the T2 image was manually circled and compared with the left brain volume in each slice thickness by the Sante MRI Viewer software, and the edema volume percentage was obtained. ( ⁇ left brain edema volume in each layer/ ⁇ left brain volume in each layer*100%). Open the high B value and ADC images of DWI in the ITK-SNAP software, and mark the high signal area in the DWI high B value image and the low signal area in the ADC image as the core infarct area on the ADC image to obtain the core infarction area. volume of the area.

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Abstract

本发明公开了一种多酚类化合物的应用，特别是其在制备防治脑水肿的药物中的应用。本发明所述的多酚类化合物尤其涉及原花青素，例如原花青素B 3。本发明在体外糖氧剥夺诱导的星胶细胞损伤模型中，发现原花青素B 1、原花青素B 2、原花青素B 3和原花青素B 4给药后可显著下调水通道蛋白AQP4表达。体内低压低氧诱导的小鼠急性脑水肿模型中，原花青素B 3给药后可以明显降低小鼠脑含水量，下调水通道蛋白AQP4表达。体内食蟹猴脑卒中模型中，脑部磁共振成像扫描发现原花青素B 3给药组与模型组相比明显降低脑水肿体积。

Description

多酚类化合物在防治脑水肿中的应用 技术领域
本发明属于生物医药领域，具体涉及一类多酚类化合物在防治脑水肿中的应用，特别是其在制备防治高原低压低氧导致脑水肿和脑卒中导致脑水肿药物中的应用。
背景技术
脑水肿就是各种内外因素作用于脑组织，使脑细胞内或细胞间隙空间过多积液，引起脑容积增大，颅内压增高、脑灌注压和脑血流量下降等一系列严重病理改变甚至产生脑疝而危及生命。脑水肿常见病因包括脑血管病变、颅脑创伤、脑中风、脑肿瘤、以及凡是能影响到大脑供血供氧的疾病，如心跳呼吸骤停、爆发性肝功衰竭、高海拔、一氧化碳中毒等。脑水肿主要分为4种类型：血管源性脑水肿、细胞毒性脑水肿、间质性脑水肿、和渗透性脑水肿。
脑水肿的药物治疗包括：1)脱水治疗，快速降低颅内压，防止脑疝，常用药物：甘露醇、高渗盐水、呋塞米；2)镇静治疗，快速镇静，防止患者出现意外，常用药物：丙泊酚、芬太尼；3)激素治疗，改善脑部细胞功能，减轻脑水肿，常用药物：地塞米松。对于高原脑水肿也没有针对性的药物，除加大吸氧量外，也是给予地塞米松，高葡萄糖，乙酰唑胺、呋塞米等。但迄今为止尚未有专门针对于脑水肿治疗的药物上市。
发明内容
本发明所要解决的技术问题是针对现有技术中缺乏有效防治脑水肿的缺陷，提供一种多酚类化合物的应用，尤其是其在制备防治低压低氧导致的脑水肿和脑卒中导致的脑水肿的药物中的应用，特别是高原脑水肿的药物。
目前，在低压低氧和/或脑卒中导致的脑水肿中，多酚类化合物特别是原花青素B 1-B 4的作用还没有任何相关报道。本发明意外发现原花青素B 1-B 4可以抑制水通道蛋白AQP4，小鼠模型中，卒中或外伤会让正常大脑发生严重水肿，而APQ4水通道蛋白缺失的大脑则水肿很轻微并很快恢复。本发明首次发现多酚类化合物，特别是原花青素B 1-B 4在脑水肿方面的药理作用和用途，尤其是在防治高原脑水肿相关疾病的应用。
本发明主要通过以下技术方案解决上述技术问题。
本发明的技术方案之一为：多酚类化合物在制备用于防治脑水肿的药物中的应用；所述多酚类化合物的结构式如下：
Figure PCTCN2022085834-appb-000001
其中，R 1～R 6可以同时或不同时为氢、羟基、甲氧基或者氟。
较佳地，本发明中所述多酚类化合物为原花青素、其药学上可接受的盐、其溶剂合物、其药学上可接受的盐的溶剂合物、或其晶型。
所述原花青素较佳地选自原花青素B 1、原花青素B 2、原花青素B 3和原花青素B 4、没食子儿茶素-(4α→8)-儿茶素(CAS号为78392-25-3)以及儿茶素-(4α→8)-没食子儿茶素(CAS号为135095-45-3)中的一种或多种。
在本发明一较佳实施方案中，所述多酚类化合物为原花青素B 1、原花青素B 2、原花青素B 3或者原花青素B 4。
特别地，本发明人意外发现原花青素B 3(CAS号为23567-23-9)治疗脑水肿效果最好，特别是对于高原脑水肿。
本发明中所述的脑水肿可为本领域常规，例如低氧低压和/或脑卒中导致的脑水肿。
本发明中，所述脑水肿可为急性脑水肿；例如高原脑水肿。
原花青素B 1、原花青素B 2、原花青素B 3以及原花青素B 4，是从葡萄籽中提取的一类化合物，其结构式以及没食子儿茶素-(4α→8)-儿茶素，或者儿茶素-(4α→8)-没食子儿茶素的结构式如下：
Figure PCTCN2022085834-appb-000002
在本发明一较佳实施方案中，所述的急性脑水肿具有如下一种或两种表现：
(1)脑部水肿，水通道蛋白AQP4、HIF-1α表达升高；
(2)脑屏障被破坏，ZO-1、claudin-1表达下降。
本发明中所述多酚类化合物可为所述药物的唯一活性成分。
本发明的技术方案之二为：多酚类化合物在制备恢复星形胶质细胞缺氧导致的损伤的药物中的应用；
所述多酚类化合物的结构式如下：
Figure PCTCN2022085834-appb-000003
其中，R 1～R 6可以同时或不同时为氢、羟基、甲氧基或者氟。
如技术方案之一所描述，所述多酚类化合物较佳地为原花青素、其药学上可接受的盐、其溶剂合物、其药学上可接受的盐的溶剂合物、或其晶型。
如上所述，本发明中所述原花青素可选自原花青素B 1、原花青素B 2、原花青素B 3、原花青素B 4、没食子儿茶素-(4α→8)-儿茶素以及儿茶素-(4α→8)-没食子儿茶素中的一种或多种；例如原花青素B 1、原花青素B 2、原花青素B 3或者原花青素B 4，优选原花青素B 3。
本发明中，所述损伤可具有如下一种或两种表现：1)AQP4和HIF-1α表达上升；2)炎症蛋白表达升高；所述炎症蛋白优选vcam-1、cox-2、iNOS、IL-6和IL-1β中的一种或多种。
如技术方案之一所述，所述多酚类化合物可为所述药物的唯一活性成分。
本发明的技术方案之三为：多酚类化合物在制备AQP4抑制剂中的应用；所述多酚类化合物的结构式如下：
Figure PCTCN2022085834-appb-000004
其中，R 1～R 6可以同时或不同时为氢、羟基、甲氧基或者氟。
对于所述多酚类化合物的优选限定，详见技术方案之一和技术方案之二。
术语解释
术语“药学上可接受的”是指盐、溶剂、辅料等一般无毒、安全，并且适合于受试者使用。所述的“受试者”优选哺乳动物，更优选为人类。
术语“药学上可接受的盐”是指本发明的化合物及包含其的药物、药物组合物与相对无毒的、药学上可接受的酸或碱制备得到的盐。当本发明的化合物及包含其的药物、药物组合物中含有相对酸性的功能团时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的药学上可接受的碱与这类药物的中性形式接触的方式获得碱加成盐。药学上可接受的碱加成盐包括但不限于：锂盐、钠盐、钾盐、钙盐、铝盐、镁盐、锌盐、铋盐、铵盐、二乙醇胺盐。当本发明的药物中含有相对碱性的官能团时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的药学上可接受的酸与这类药物的中性形式接触的方式获得酸加成盐。所述的药学上可接受的酸包括无机酸，所述无机酸包括但不限于：盐酸、氢溴酸、氢碘酸、硝酸、碳酸、磷酸、亚磷酸、硫酸等。所述的药学上可接受的酸包括有机酸，所述有机酸包括但不限于：乙酸、丙酸、草酸、异丁酸、马来酸、丙二酸、苯甲酸、琥珀酸、辛二酸、反丁烯二酸、乳酸、扁桃酸、邻苯二甲酸、苯磺酸、对甲苯磺酸、柠檬酸、水杨酸、酒石酸、甲磺酸、异烟酸、酸式柠檬酸、油酸、单宁酸、泛酸、酒石酸氢、抗坏 血酸、龙胆酸、富马酸、葡糖酸、糖酸、甲酸、乙磺酸、双羟萘酸(即4,4’-亚甲基-双(3-羟基-2-萘甲酸))、氨基酸(例如谷氨酸、精氨酸)等。当本发明的药物中含有相对酸性和相对碱性的官能团时，可以被转换成碱加成盐或酸加成盐。具体可参见Berge et al.,"Pharmaceutical Salts",Journal of Pharmaceutical Science 66:1-19(1977)、或、Handbook of Pharmaceutical Salts:Properties,Selection,and Use(P.Heinrich Stahl and Camille G.Wermuth,ed.,Wiley-VCH,2002)。
术语“一种或多种”中的“多种”可以是指2种、3种、4种、5种、6种、7种、8种、9种或者更多种。
本发明的化合物、含其的药物或药物组合物可以单位剂量形式给药，给药途径可为肠道或非肠道，例如口服、外敷、静脉注射、肌肉注射、皮下注射、鼻腔、口腔粘膜、眼、肺和呼吸道、皮肤、阴道、直肠等。
给药剂型可以是液体剂型、固体剂型或半固体剂型。液体剂型可以是溶液剂(包括真溶液和胶体溶液)、乳剂(包括o/w型、w/o型和复乳)、混悬剂、注射剂(包括水针剂、粉针剂和输液)、滴眼剂、滴鼻剂、洗剂和搽剂等；固体剂型可以是片剂(包括普通片、肠溶片、含片、分散片、咀嚼片、泡腾片、口腔崩解片)、胶囊剂(包括硬胶囊、软胶囊、肠溶胶囊)、颗粒剂、散剂、微丸、滴丸、栓剂、膜剂、贴片、气(粉)雾剂、喷雾剂等；半固体剂型可以是软膏剂、凝胶剂、糊剂等。
本发明的药物或药物组合物可以制成普通制剂、也可以制成缓释制剂、控释制剂、靶向制剂及各种微粒给药系统。
“药物组合物”指将本发明中的化合物中的一个或多个或其药学上可接受的盐、溶剂化物、水合物或前药与别的化学成分，例如药学上可接受的载体，混合。药物组合物的目的是促进给药给动物的过程。
“药学上可接受的载体”指的是对有机体不引起明显的刺激性和不干扰所给予化合物的生物活性和性质的药物组合物中的非活性成分，例如但不 限于：碳酸钙、磷酸钙、各种糖(例如乳糖、甘露醇等)、淀粉、环糊精、硬脂酸镇、纤维素、碳酸镇、丙烯酸聚合物或甲基丙烯酸聚合物、凝胶、水、聚乙二醇、丙二醇、乙二醇、EZ麻油或氢化EZ麻油或多乙氧基氢化EZ麻油、芝麻油、玉米油、花生油等。
前述的药物组合物中，除了包括药学上可接受的载体外，还可以包括在药(剂)学上常用的辅剂，例如：抗细菌剂、抗真菌剂、抗微生物剂、保质剂、调色剂、增溶剂、增稠剂、表面活性剂、络合剂、蛋白质、氨基酸、脂肪、糖类、维生素、矿物质、微量元素、甜味剂、色素、香精或它们的结合等。
术语“治疗”指治疗性疗法。涉及具体病症时，治疗指：(1)缓解疾病或者病症的一种或多种生物学表现，(2)干扰(a)导致或引起病症的生物级联中的一个或多个点或(b)病症的一种或多种生物学表现，(3)改善与病症相关的一种或多种症状、影响或副作用，或者与病症或其治疗相关的一种或多种症状、影响或副作用，或(4)减缓病症或者病症的一种或多种生物学表现发展。
术语“溶剂合物”是指本发明化合物与化学计量或非化学计量的溶剂结合形成的物质。溶剂合物中的溶剂分子可以有序或非有序排列的形式存在。所述的溶剂包括但不限于：水、乙醇等。
术语“药学上可接受的盐的溶剂合物”中的“药学上可接受的盐”和“溶剂合物”如上所述，是指本发明化合物与相对无毒的、药学上可接受的酸或碱、与化学计量或非化学计量的溶剂结合形成的物质。所述的“药学上可接受的盐的溶剂合物”包括但不限于本发明化合物的盐酸一水合物。
术语“化合物”、“药学上可接受的盐”、“溶剂合物”和“药学上可接受的盐的溶剂合物”可以以晶型或无定型的形式存在。术语“晶型”是指其中的离子或分子是按照一种确定的方式在三维空间作严格周期性排列，并具有间隔一定距离周期重复出现规律；因上述周期性排列的不同，可存在多种晶型，也即多晶型现象。术语“无定型”是指其中的离子或分子呈现杂乱无章的分布状态，即离子、分子间不具有周期性排列规律。
本发明中，所述“包括、包含或含有”可以是指除了包括后面所列举的成分，还存在其他成分；也可以是指“由……组成”，即只包括后面所列举的成分而不存在其他成分。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：
本发明中使用多酚类化合物，例如原花青素B 3干预脑水肿后能够减少渗漏，改善脑水肿引起的海马区整体变长，DG区变得狭窄，染色发现细胞排列紊乱、弥散，CA3区向外扩大，且伴随弥散等症状；并能够改善脑水肿引起的AQP4、HIF-1α表达升高，ZO-1、claudin-1表达下降；使炎症蛋白(vcam-1、cox-2、iNOS)的表达下降。
附图说明
图1为含水量检测，结果显示原花青素B 3(MT-8)可显著降低含水量。
图2为EB方法检测急性脑水肿对屏障的损伤情况，结果显示MT-8可显著减少渗漏。
图3为HE染色，观察急性脑水肿对海马区形态的影响，结果显示急性脑水肿引起海马区整体变长，DG区变得狭窄，且染色发现细胞排列紊乱，有些弥散，CA3区向外扩大，且伴随弥散；MT-8干预后有所好转。
图4为Western blot方法检测急性脑水肿损伤后海马区AQP4、HIF-1α及屏障蛋白表达。
图5为Western blot方法检测急性脑水肿损伤后海马区炎症蛋白表达。
Control：对照组(给予对应体积的生理盐水，40mg/kg/day,ip.)；HACE：高原脑水肿模型组(给予对应体积的生理盐水，40mg/kg/day,ip.)；MT-8：40mg/kg/day,ip.。 ***p<0.001。
图6A为Immunofluorescence方法检测星胶细胞受OGD损伤后AQP4表达情况；图6B和图6C显示Western blot方法检测星胶细胞受OGD损伤后AQP4、HIF-1α及炎症蛋白表达。
Control：对照组；Hypoxia：OGD损伤6h模型组；MT-8：10μM； ***p<0.001。
图7为Western blot方法检测原花青素B 1，原花青素B 2，原花青素B 3，原花青素B 4药物对OGD诱导的星胶细胞损伤后AQP4、TRPV4、HIF-1α、HIF-2α、SNX13蛋白表达情况的影响。
图8为食蟹猴给药组G2-1第8天磁共振影像。
图9为食蟹猴给药组G2-2第8天磁共振影像。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
原花青素B 3在以下内容中简称为MT-8。
实施例1 MT-8对高原脑水肿的防治作用
分组：将体重22-25g雄性C57小鼠(小鼠购自于上海B&K公司，饲养于复旦大学药学院动物实验中心，SPF级)随机分成3组，即：1)Control组(给予对应体积的生理盐水，40mg/kg/day,ip.n＝10)；2)模型组(给予对应体积的生理盐水，40mg/kg/day,ip.n＝10)；3)模型+MT-8组(40mg/kg/day,ip.n＝10)。
1)模型建立
在平原地区常氧环境下，平原对照组小鼠笼内常规饲养，不做运动和减压缺氧处理，自由进食饮水，自然昼夜照明。高原缺氧组小鼠在正式低氧暴露前，先利用动物实验跑台进行适应性跑台训练2d。具体训练方法：每日晨8:30开始，设置跑台坡度0°，速度12m/min，持续运动时间15min，休 息20min后继续以此训练，共6次。每日运动时间共计90min，其余时间自由进食饮水，自然昼夜照明。
参考高原肺水肿动物模型建立方法加以修改，利用高原环境模拟舱进行动物急性低氧暴露实验。疾病模型具体制备过程：在模拟海拔高度5000m的高原环境模拟舱内，由舱内专职实验员将小鼠放入动物实验跑台内稳定30min后，立即让小鼠进行跑台运动。动物跑台设置参数：坡度10°，速度12m/min，采用电刺激方式维持小鼠处于运动状态。小鼠持续运动4h后，舱内实验员将小鼠从跑道中取出，任其休息15～20min，期间小鼠可自由进食饮水。休息结束后继续以此方式循环往复进行力竭运动11次，运动时间共计2640min。高原缺氧组小鼠力竭运动结束后，将小鼠置入实验舱内，关闭连接舱门。舱外技术员迅速将实验舱海拔高度由5000m升高到6000m。力竭训练2d后，将实验舱海拔高度由6000m升高到8000m，让小鼠在高原无运动情况下继续减压缺氧暴露3d。舱内温度约18℃，光暗周期24h，自由进食饮水。
最后在平原地区取材进行检测。
2)脑组织含水量检测
麻醉小鼠后，迅速断头取脑，称量脑组织湿重，然后放于60℃烘箱烘烤3d，再称量脑组织干重。根据(湿重-干重)/湿重*100％计算各组小鼠的含水量。
3)EB渗漏检测通透性
取材前1h，每只小鼠尾静脉注射2％EB溶液(4mL/kg)，1h后，麻醉小鼠，用0.9％生理盐水灌注小鼠，至心脏流出溶液颜色透明为止。断头取脑，进行大体拍照。随后-20℃冷冻30min，冠状切片6片，拍摄EB渗漏情况。
4)HE染色
取材后送切片，染色HE，观察海马及皮质区形态变化。
5)Western Blot
取小鼠部分脑组织匀浆，检测AQP4、HIF-1α及炎症蛋白(vcam-1、cox-2、iNOS)、屏障蛋白(ZO-1、claudin-1)表达情况。
收集细胞裂解液，检测AQP4、HIF-1α表达情况。
6)实验结果：
急性脑水肿引起含水量显著升高，MT-8干预后可显著降低脑含水量(见表1和图1)。
表1含水量检测
Figure PCTCN2022085834-appb-000005
急性脑水肿引起屏障遭到破坏，EB渗漏明显；MT-8干预后可减少渗漏(图2)。急性脑水肿引起海马区整体变长，DG区变得狭窄，且染色发现细胞排列紊乱，有些弥散，CA3区向外扩大，且伴随弥散；MT-8干预后有所好转(图3)。急性脑水肿引起AQP4、HIF-1α表达升高，ZO-1、claudin-1表达下降；MT-8处理后均有所改善(见表2和图4)。
表2蛋白印迹检测
Figure PCTCN2022085834-appb-000006
急性脑水肿引起炎症蛋白(vcam-1、cox-2、iNOS)表达升高，MT-8处理后可显著下降其表达(见表3和图5)。
表3蛋白印迹检测
Figure PCTCN2022085834-appb-000007
实施例2 MT-8以及原花青素类似物对缺氧模型星形胶质细胞的保护作用
1)原代皮质星形胶质细胞培养与纯化
取新生24h内的SD大鼠，用酒精擦拭皮肤后断颈处死置于75％酒精内消毒数分钟，断头取脑，将脑组织置于冷却的PBS中，去除小脑、脑膜、及血管，随后分离皮质，将皮质剪碎，收集剪碎的皮质，离心，1000rpm，8min。弃上清，加入0.25％胰蛋白酶消化10分钟，每5min振摇一次，加入含10％FBS的DMEM/F12培养基终止消化10min，每5min振摇一次，过200目筛网。取滤液，离心，1000rpm，5min，弃上清，加含10％FBS的DMEM/F12培养基，吹打均匀，计数，调整细胞密度，接种在提前包被过L-多聚赖氨酸的塑料瓶中。置于37℃，5％CO 2的培养箱中培养30～60min进行差速分离，随后每隔两天更换培养液。培养至7-9d，更换新培养基，盖紧瓶盖，用无菌灭菌带包起，密封，将培养瓶置于摇床上，保持培养瓶水平，确保培养液覆盖细胞，然后以300rpm,在37℃培养箱中培养6h，以去除少胶质细胞，培养瓶中培养基换为新鲜的培养基，再以210rpm继续摇18h，将培养液转移至一新的用L-多聚赖氨酸包被的培养瓶中，置于培养箱中培养，待细胞融合80％以上时进行传代，以0.25％胰蛋白酶(含EDTA)消化数分钟，终止消化，调整细胞密度，接种到包被过L-多聚赖斯的培养瓶中，第三代可用于实验。
2)星形胶质细胞缺氧模型制备
更换无血清DMEM培养基，放置于1％O 2、5％CO 2、94％N 2的三气培养箱中进行缺氧模型制备，缺氧6h后进行后续指标检测。
3)实验结果：
星形胶质细胞缺氧后，用GFAP标记星形胶质细胞，发现AQP4表达明显增加，而MT-8处理后可显著下调AQP4和HIF-1α表达，并减轻炎症反应 (vcam-1、cox-2、iNOS、IL-6、IL-1β)(见表4以及图6A～图6C)。
其中，MT-8处理的方法为原代星胶细胞培养好后，传代、种板，待细胞长至80％左右时，给予MT-8(10μM)预处理4h后，更换为DMEM无糖培养基，放置于(1％O 2、5％CO 2、94％N 2)三气培养箱中继续培养6h后收样进行相关检测。
表4蛋白印迹检测
Figure PCTCN2022085834-appb-000008
Western blot方法检测原花青素B 1、原花青素B 2、原花青素B 3和原花青素B 4药物对OGD(糖氧剥夺，oxygen and glucose deprivation)诱导的星胶细胞损伤后AQP4、TRPV4、HIF-1α、HIF-2α、SNX13蛋白表达的影响情况(图7)，发现原花青素B 1、原花青素B 2、原花青素B 3和原花青素B 4处理后可显著下调AQP4、TRPV4、HIF-1α、HIF-2α表达，显示有治疗高原脑水肿的作用。
其中，糖氧剥夺的处理过程为：原代星胶细胞培养好后，传代、种板，待细胞长至80％左右时，更换为DMEM无糖培养基，放置于(1％O 2、5％CO 2、94％N 2)三气培养箱中继续培养6h后收样进行相关检测。
原花青素处理的过程为：原代星胶细胞培养好后，传代、种板，待细胞长至80％左右时，给予原花青素B 1-B 4(10μM)预处理4h后，更换为DMEM无糖培养基，放置于(1％O 2、5％CO 2、94％N 2)三气培养箱中继续培养6h后收样进行相关检测。
实施例3 MT-8对食蟹猴脑卒中模型中脑水肿的药效
1)动物：4只食蟹猴(Macaca Fascicularis)，雄性,8岁左右，(养殖方： 肇庆创药生物科技有限公司)；本实验方案中设计应用于动物的实验操作程序在澎立生物IACUC(实验动物管理和使用委员会)进行审批通过后执行。
2)环境：实验猴被安置于澎立生物动物房的不锈钢猴猴笼(适应期：1600mm×1400mm×1640mm；实验期800mm×700mm×820mm)中，适应期：每笼两只；实验期：每笼一只(配备镜子、塑料玩具球等动物福利器具)。整个实验期间安置动物的房间号记录在实验记录里。安置实验猴的房间区域过滤通风换气次数为每小时10-20次。温度保持在19-19℃(66-84°F)之间，相对湿度为35-75％。每天上下午观测并记录温度和湿度。照明条件为每天12小时(08:00-20:00)日光灯照射和12小时无照明。动物的福利措施按照澎立的标准操作程序执行，以减少并帮助安抚实验带来的压力。
3)食物和饮水：试验猴每日喂食两次。澎立生物接收的每一批动物饲料均会留样，如有需要可进行检测分析，所得分析证书由澎立生物归档保存。并向猴子提供额外的蔬菜和水果以保证其食欲。整个试验验期间，试验用猴可无限量获取经澎立生物内部处理的饮用水。当地市政供水经过滤系统净化后达到世界卫生组织(WHO)人类饮用水标准。
4)脑卒中模型建立：食蟹猴术前禁食10-16小时，麻醉前15分钟肌注阿托品(0.04mg/kg)，肌注3mg/kg的舒泰镇静，吸入1-3％异氟烷保持麻醉状态后进行气管插管，监护仪检测血氧饱和度、心率、体温。气管插管连接呼吸机，根据心率和血氧饱和度调整持续异氟烷吸入麻醉浓度(0.5％-3％)。头部备皮，将左侧颧弓上缘水平线与眶外缘垂直线连线交点，和颅骨矢状线与双外耳道连线(颅骨映射线)交点，两交点连线即为左侧大脑中动脉(middle cerebral artery,MCA)大致走行方向。颅骨开窗：切开皮肤及肌肉，暴露颅骨，以左侧颧弓上缘上侧与眶外缘后侧为边界做一约20mm×15mm颅骨开窗。
永久性结扎大脑中动脉：十字剪开硬脑膜，沿大脑外侧裂用脑棉保护向 内游离暴露MCA M1，顺着此血管向颅底深入，寻找MCA从willis环的发出端，此部分即为MCA M1近端。随后将MCA M1近端结扎，制作永久性脑缺血模型。逐层缝合关闭手术窗口，消毒创口，连续使用7天头孢曲松钠(0.1g/kg)和痛立定(一天2次，4mg/kg,i.m.)进行抗感染和镇痛等兽医护理。
5)分组与给药
4只食蟹猴按体重随机分为2组，每组2只，即：1)模型组：G1-1和G1-2(给予对应体积的生理盐水，iv.每天给药，连续7天)；2)给药组：G2-1和G2-2(给予MT-8，12mg/kg/day,iv.每天给药，连续7天)。首次给药时间点为开始脑卒中手术后1小时进行给药。
6)脑部磁共振成像扫描
食蟹猴核磁扫描前禁食10-16小时，麻醉前15分钟肌注阿托品(0.04mg/kg)，肌注3mg/kg的舒泰使动物麻醉。于手术前3天、Day2、Day4、Day8进行MRI扫描，扫描机器型号为西门子公司SKyra 3.0T超导磁共振成像仪，扫描序列包括：T1WI、T2WI、T2FLAIR、DWI，扫描参数如下表。对照T1、T2图片影像信息(图8和图9)，通过Sante MRI Viewer软件分别手动圈选T2图像中左脑中水肿脑组织体积与每个层厚中左脑体积相比，获得水肿体积百分率(∑每层中左脑水肿体积/∑每层中左脑体积*100％)。在ITK-SNAP软件中打开DWI的高B值和ADC图像，将DWI高B值图像中的呈现高信号且在ADC图像中呈现的低信号区域在ADC图像上标注成核心梗死区，获得核心梗死区的体积。
表5磁共振成像扫描参数
Figure PCTCN2022085834-appb-000009
Figure PCTCN2022085834-appb-000010
7)实验结果
表6磁共振成像扫描脑水肿结果
Figure PCTCN2022085834-appb-000011
表7磁共振成像扫描脑梗死体积结果
  day2体积(mm 3) day4体积(mm 3) day8体积(mm 3)
G1-1 3609.37 4117.83 5965.5
G1-2 6627.93 4977.21 5589.51
G2-1 9624.99 9238.29 5335.29
G2-2 1815.43 1195.96 812.83
从结果显示，脑卒中模型的猴脑水肿在第4天达到峰值，模型组第8天脑水肿体积均明显高于第2天(1.23倍和2.22倍)；而给药组G2-2猴子的脑水肿明显改善，第8天的脑水肿体积为第2天的66％，第8天脑梗死体积为第2天的45％；给药组G2-1猴子的第8天的脑水肿体积为第2天的92％，第8天脑梗死体积为第2天的55％。表明化合物MT-8有治疗脑卒中导致脑水肿的作用。

Claims (10)

  1. 多酚类化合物在制备用于防治脑水肿的药物中的应用；所述多酚类化合物的结构式如下：
    Figure PCTCN2022085834-appb-100001
    其中，R 1～R 6同时或不同时为氢、羟基、甲氧基或者氟。
  2. 如权利要求1所述的应用，其特征在于，所述多酚类化合物为原花青素、其药学上可接受的盐、其溶剂合物、其药学上可接受的盐的溶剂合物、或其晶型。
  3. 如权利要求2所述的应用，其特征在于，所述原花青素选自原花青素B 1、原花青素B 2、原花青素B 3、原花青素B 4、没食子儿茶素-(4α→8)-儿茶素以及儿茶素-(4α→8)-没食子儿茶素中的一种或多种；例如原花青素B 1、原花青素B 2、原花青素B 3或者原花青素B 4；优选原花青素B 3。
  4. 如权利要求1所述的应用，其特征在于，所述脑水肿为低氧低压和/或脑卒中导致的脑水肿。
  5. 如权利要求1所述的应用，其特征在于，所述脑水肿为急性脑水肿。
  6. 如权利要求5所述的应用，其特征在于，所述急性脑水肿具有如下一种或两种表现：
    (1)脑部水肿，AQP4、HIF-1α表达升高；
    (2)脑屏障被破坏，ZO-1、claudin-1表达下降。
  7. 如权利要求1～6任一项所述的应用，其特征在于，所述多酚类化合物 为所述药物的唯一活性成分。
  8. 多酚类化合物在制备恢复星形胶质细胞缺氧导致的损伤的药物中的应用；
    所述多酚类化合物的结构式如下：
    Figure PCTCN2022085834-appb-100002
    其中，R 1～R 6同时或不同时为氢、羟基、甲氧基或者氟。
  9. 如权利要求8所述的应用，其特征在于，所述多酚类化合物为原花青素、其药学上可接受的盐、其溶剂合物、其药学上可接受的盐的溶剂合物、或其晶型；较佳地：
    所述原花青素选自原花青素B 1、原花青素B 2、原花青素B 3、原花青素B 4、没食子儿茶素-(4α→8)-儿茶素以及儿茶素-(4α→8)-没食子儿茶素中的一种或多种；例如原花青素B 1、原花青素B 2、原花青素B 3或者原花青素B 4；优选原花青素B 3；
    和/或，所述损伤具有如下一种或两种表现：
    1)AQP4和HIF-1α表达上升；
    2)炎症蛋白表达升高；所述炎症蛋白优选vcam-1、cox-2、iNOS、IL-6和IL-1β中的一种或多种；
    和/或，所述多酚类化合物为所述药物的唯一活性成分。
  10. 多酚类化合物在制备AQP4抑制剂中的应用；所述多酚类化合物的结构式如下：
    Figure PCTCN2022085834-appb-100003
    其中，R 1～R 6同时或不同时为氢、羟基、甲氧基或者氟；
    较佳地，所述多酚类化合物为原花青素、其药学上可接受的盐、其溶剂合物、其药学上可接受的盐的溶剂合物、或其晶型；
    所述原花青素优选选自原花青素B 1、原花青素B 2、原花青素B 3、原花青素B 4、没食子儿茶素-(4α→8)-儿茶素以及儿茶素-(4α→8)-没食子儿茶素中的一种或多种；例如原花青素B 1、原花青素B 2、原花青素B 3或者原花青素B 4；更优选原花青素B 3。
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* Cited by examiner, † Cited by third party
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CN115998874A (zh) * 2022-12-07 2023-04-25 西南交通大学 水通道蛋白3和/或水通道蛋白5作为抑郁症药物靶点的用途

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