WO2022095057A1 - 一种医药组合物及其医药用途 - Google Patents

一种医药组合物及其医药用途 Download PDF

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WO2022095057A1
WO2022095057A1 PCT/CN2020/127619 CN2020127619W WO2022095057A1 WO 2022095057 A1 WO2022095057 A1 WO 2022095057A1 CN 2020127619 W CN2020127619 W CN 2020127619W WO 2022095057 A1 WO2022095057 A1 WO 2022095057A1
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lithium
pharmaceutical composition
blood
endothelial cells
brain barrier
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French (fr)
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畅君雷
冀雅彬
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Shenzhen Institute of Advanced Technology of CAS
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Shenzhen Institute of Advanced Technology of CAS
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/14—Alkali metal chlorides; Alkaline earth metal chlorides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/49—Urokinase; Tissue plasminogen activator
    • 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

Definitions

  • the present application relates to the technical field of medicine, in particular to a pharmaceutical composition and its medical use.
  • Ischemic stroke also known as cerebral infarction
  • the optimal treatment methods include alteplase (rtPA) and surgical thrombectomy.
  • rtPA alteplase
  • thrombectomy surgical thrombectomy
  • the time window of vascular recanalization therapy is mainly set to avoid or reduce the damage of blood-brain barrier (BBB) caused by reperfusion. If the BBB can be effectively protected, the time window of revascularization therapy may be extended, which will help more people receive revascularization therapy.
  • BBB blood-brain barrier
  • the existing main treatment methods are intravenous alteplase rtPA thrombolysis and intra-arterial thrombectomy, and they all have strict time window restrictions, the former being 4.5 hours, the latter is 6-8 hours (a few patients can be extended to 24 hours).
  • Clinical studies have shown that serious complications such as hemorrhagic transformation and cerebral edema will occur when vascular patency is performed beyond this time window. Therefore, a considerable proportion of patients with acute cerebral infarction do not seek medical treatment in time, the distance from the hospital is far away, and the traffic jams on the road are subjective and objective factors.
  • BBB protection drugs there are no targeted BBB protection drugs in clinical practice.
  • cerebral edema or hemorrhagic transformation if cerebral herniation has been induced, use 20% mannitol, hypertonic salt, diuretics and other dehydration treatments for mild cases, and "decompressive craniectomy" for severe cases; and if no cerebral herniation is induced , and generally give patients supportive treatment such as sedation, blood pressure reduction, correction of water, electricity and acid-base balance disorders.
  • the main technical problem to be solved by the present application is to provide a pharmaceutical composition and its medical use, which can play an effective pharmaceutical role in the treatment of ischemic stroke and the blood-brain barrier damage caused by it.
  • a technical solution adopted in the present application is to provide a pharmaceutical composition for the treatment of ischemic stroke, the pharmaceutical composition comprising: an effective dose of lithium for the treatment of ischemic stroke and a pharmaceutically acceptable compound.
  • the accepted carrier, wherein lithium is used to upregulate the Wnt/ ⁇ -catenin signaling pathway of cerebral vascular endothelial cells, and to protect the structure of tight junction proteins in endothelial cells of the blood-brain barrier.
  • the lithium agent is lithium chloride and/or lithium carbonate.
  • the pharmaceutical composition is in dosage unit form.
  • the dosage of the lithium agent in the pharmaceutical composition is one of 1.5-6.0 mmol/kg, wherein mmol/kg is the amount of the lithium agent relative to the mass of the patient.
  • the dosage of the lithium agent in the pharmaceutical composition is 3.0 mmol/kg.
  • the pharmaceutical composition also includes an effective dose of alteplase for treating ischemic stroke, and the dose of alteplase in the pharmaceutical composition is 0.9 mg/kg, wherein mg/kg is the amount ofreteplase Quality relative to the quality of the patient.
  • another technical solution adopted in the present application is to provide the use of a lithium agent in the preparation of a drug for the treatment of ischemic stroke, wherein the lithium agent is used to upregulate the Wnt/ ⁇ of cerebral vascular endothelial cells -catenin signaling pathway, and protects the structure of tight junction proteins in endothelial cells of the blood-brain barrier.
  • the lithium agent is lithium chloride and/or lithium carbonate.
  • another technical solution adopted in the present application is to provide a pharmaceutical composition for the treatment of blood-brain barrier damage, the pharmaceutical composition comprising: an effective dose of lithium for treating blood-brain barrier damage and a medicinally acceptable drug.
  • the accepted carrier, wherein lithium is used to upregulate the Wnt/ ⁇ -catenin signaling pathway of cerebral vascular endothelial cells, and to protect the structure of tight junction proteins in endothelial cells of the blood-brain barrier.
  • another technical solution adopted in the present application is to provide a use of a lithium agent in the preparation of a medicine for treating blood-brain barrier damage, wherein the lithium agent is used to upregulate the Wnt/ ⁇ of cerebral vascular endothelial cells -catenin signaling pathway, and protects the structure of tight junction proteins in endothelial cells of the blood-brain barrier.
  • another technical solution adopted in the present application is to provide a pharmaceutical composition for the treatment of blood-encephalopathy, the pharmaceutical composition comprising: an effective dose of lithium for the treatment of blood-encephalopathy and a pharmaceutically acceptable The carrier, wherein lithium is used to upregulate the Wnt/ ⁇ -catenin signaling pathway of cerebral vascular endothelial cells, and to protect the structure of tight junction proteins in endothelial cells of the blood-brain barrier.
  • another technical solution adopted in the present application is to provide the use of a lithium agent in the preparation of a medicine for the treatment of blood and brain diseases, wherein the lithium agent is used to upregulate the Wnt/ ⁇ - catenin signaling pathway, and protects the structure of tight junction proteins in endothelial cells of the blood-brain barrier.
  • the lithium agent in the pharmaceutical composition of the present application can up-regulate the Wnt/ ⁇ -catenin signaling pathway in cerebral vascular endothelial cells, as well as on the tight junction protein of endothelial cells of the blood-brain barrier.
  • the protection of the structure plays an important role, and then can protect the blood-brain barrier. Therefore, lithium can play an effective drug role in the treatment of ischemic stroke and its resulting blood-brain barrier damage.
  • Fig. 1 is a schematic diagram of experimental comparison after staining the coronal section of mouse brain with TTC;
  • Figure 2 is a schematic diagram of the relative proportion of mouse infarct volume in the cerebral hemisphere
  • Figure 3 is a schematic diagram of the relative proportion of Evans blue staining
  • Figure 4 is a schematic diagram of the relative percentage of Evans blue exudation to hemisphere weight
  • Figure 5 is a schematic diagram of the exudation of endogenous IgG from blood vessels observed by immunofluorescence double staining
  • Fig. 6 is the relative density columnar schematic diagram of the exudation of IgG from blood vessels
  • Fig. 7 is a columnar schematic diagram of neurobehavioral scoring
  • Figure 8 is a schematic diagram of the observation of neuronal apoptosis by immunofluorescence double staining
  • Figure 9 is a histogram of the relative density of the number of apoptotic neurons in the infarcted area of mice.
  • Figure 10 is a schematic diagram of the expression of active ⁇ -catenin on endothelial cells observed by immunofluorescence double staining
  • Figure 11 is a histogram of the relative density of active ⁇ -catenin expression on endothelial cells
  • Figure 12 is a schematic diagram of the active and total ⁇ -catenin protein levels in the brain tissue in the infarcted area of each group;
  • Figure 13 is a histogram of the relative density of immunoblotting in Figure 12;
  • Figure 14 is another histogram of the relative density of immunoblotting in Figure 12;
  • Figure 15 is a histogram of axin2 levels in cerebral infarction tissues in each group
  • Figure 16 is a histogram of apcdd1 mRNA levels in cerebral infarction tissues in each group;
  • Figure 17 is a schematic diagram of the effect of different concentrations of lithium chloride on bEnd.3 Axin2 mRNA levels
  • Figure 18 is a schematic diagram of the influence of different concentrations of lithium chloride on the bEnd.3 TCF/LEF TOPflash value
  • Figure 19 is a schematic diagram of the effect of lithium chloride corresponding to the construction of a Wnt/ ⁇ -catenin pathway-deficient bEnd.3 cell line by knocking out Fzd4;
  • Figure 20 is a schematic diagram of another effect of lithium chloride corresponding to the construction of a Wnt/ ⁇ -catenin pathway-deficient bEnd.3 cell line by knocking out Fzd4;
  • Figure 21 is a histogram of axin2 mRNA levels in each group.
  • the present application provides a pharmaceutical composition for treating ischemic stroke, the pharmaceutical composition comprising: an effective dose of lithium for treating ischemic stroke and a pharmaceutically acceptable carrier.
  • Ischemic stroke is a general term for brain tissue necrosis caused by stenosis or occlusion of the arteries supplying blood to the brain (carotid and vertebral arteries) and insufficient blood supply to the brain.
  • cerebral ischemia transient ischemic attack (TIA); reversible neurological impairment (RIND); progressive stroke (SIE); complete stroke (CS).
  • TIA transient ischemic attack
  • RIND reversible neurological impairment
  • SIE progressive stroke
  • CS complete stroke
  • Cerebral ischemia can be divided into localized cerebral ischemia and diffuse cerebral ischemia from the scope of ischemia.
  • the causes of localized cerebral ischemia are: middle cerebral artery embolism; extracranial internal carotid artery or vertebral artery stenosis, occlusion or thrombosis; cerebral artery spasm.
  • the causes of diffuse cerebral ischemia include cardiac arrest, hypotension, anemia, and hypoglycemia.
  • ischemic stroke aka cerebral infarction
  • revascularization therapy including alteplase (rtPA) and surgical thrombectomy, all of which have strict Due to the time window limitation, a considerable proportion of patients cannot receive such treatment.
  • the time window setting of vascular recanalization therapy is mainly to avoid or reduce the damage of the blood-brain barrier caused by reperfusion, and alteplase rtPA itself has damage to the blood-brain barrier, even if it is used within the time window, some patients will still have it.
  • BBB-damaging phenotypes such as hemorrhagic transformation, cerebral edema.
  • a BBB protectant and rtPA can be used in combination to reduce its damage to the BBB, it will improve the effectiveness of revascularization therapy, and the time window of revascularization therapy may be extended, which is conducive to making more Many people receive revascularization therapy.
  • lithium in current clinical or basic research is to prevent and treat bipolar disorder with alternating episodes of mania and depression.
  • the inventors of the present application have found in experimental studies that when lithium is used for ischemic stroke in mice, it can upregulate the Wnt/ ⁇ -catenin pathway of cerebral vascular endothelial cells, and it has a positive effect on BBB in the acute phase of cerebral infarction in mice. Destruction also has a clear protective effect, potentially increasing the indications for lithium.
  • Lithium can improve the functional integrity of cerebral vascular endothelial cells by up-regulating the Wnt/ ⁇ -catenin pathway of cerebral vascular endothelial cells, and can protect the structure of tight junction proteins of endothelial cells of the blood-brain barrier, thereby realizing the protection of BBB. effect.
  • the lithium agent is lithium chloride (LiCl) and/or lithium carbonate (Li2CO3).
  • the lithium agent may also be any reasonable mixture of one or more of lithium compounds such as lithium hydride, lithium nitride, and lithium hydroxide, which is not limited in this application.
  • the pharmaceutical composition is in dosage unit form.
  • the dose of the lithium agent in the pharmaceutical composition is one of 1.5-6.0 mmol/kg, and the mmol/kg is the amount of the lithium agent relative to the mass of the patient.
  • the dosage of the lithium agent in the pharmaceutical composition can also comprehensively consider changes in factors such as the toxic and side effects of the drug or blood drug concentration, and use other arbitrary A reasonable dose, which is not limited in this application.
  • the dosage of the lithium agent in the pharmaceutical composition is 3.0 mmol/kg.
  • the pharmaceutical composition also includes an effective dose of alteplase for treating ischemic stroke, and the dose of alteplase in the pharmaceutical composition is 0.9 mg/kg, and the mg/kg is the mass ofreteplase relative to patient quality.
  • the application also provides the use of a lithium agent in the preparation of a drug for the treatment of ischemic stroke, wherein the lithium agent is used to upregulate the Wnt/ ⁇ -catenin signaling pathway of cerebral vascular endothelial cells, and to regulate the endothelial cells of the blood-brain barrier.
  • the lithium agent is used to upregulate the Wnt/ ⁇ -catenin signaling pathway of cerebral vascular endothelial cells, and to regulate the endothelial cells of the blood-brain barrier.
  • the structure of the tight junction protein is protected.
  • the lithium agent is lithium chloride and/or lithium carbonate.
  • the application also provides a pharmaceutical composition for treating blood-brain barrier damage, the pharmaceutical composition comprising: an effective dose of lithium for treating blood-brain barrier damage and a pharmaceutically acceptable carrier, wherein the lithium is used to upregulate brain
  • a pharmaceutical composition for treating blood-brain barrier damage comprising: an effective dose of lithium for treating blood-brain barrier damage and a pharmaceutically acceptable carrier, wherein the lithium is used to upregulate brain
  • the Wnt/ ⁇ -catenin signaling pathway in vascular endothelial cells protects the structure of tight junction proteins in endothelial cells of the blood-brain barrier.
  • the lithium agent is lithium chloride and/or lithium carbonate.
  • the pharmaceutical composition is in dosage unit form.
  • the dosage of the lithium agent in the pharmaceutical composition is one of 1.5-6.0 mmol/kg, and the mmol/kg is the mass of the lithium agent relative to the patient.
  • the dosage of the lithium agent in the pharmaceutical composition is 3.0 mmol/kg.
  • the application also provides the use of a lithium agent in the preparation of a medicine for treating blood-brain barrier damage, wherein the lithium agent is used to upregulate the Wnt/ ⁇ -catenin signaling pathway of cerebral vascular endothelial cells, and to regulate the endothelial cells of the blood-brain barrier.
  • the lithium agent is used to upregulate the Wnt/ ⁇ -catenin signaling pathway of cerebral vascular endothelial cells, and to regulate the endothelial cells of the blood-brain barrier.
  • the structure of the tight junction protein is protected.
  • the lithium agent is lithium chloride and/or lithium carbonate.
  • the present application also provides a pharmaceutical composition for treating blood-brain disease, the pharmaceutical composition comprising: an effective dose of lithium for treating blood-brain disease and a pharmaceutically acceptable carrier, wherein the lithium is used to upregulate cerebral vascular endothelial cells Wnt/ ⁇ -catenin signaling pathway, and protects the structure of tight junction proteins in endothelial cells of the blood-brain barrier.
  • the lithium agent is lithium chloride and/or lithium carbonate.
  • the pharmaceutical composition is in dosage unit form.
  • the dosage of the lithium agent in the pharmaceutical composition is one of 1.5-6.0 mmol/kg, and the mmol/kg is the mass of the lithium agent relative to the patient.
  • the dosage of the lithium agent in the pharmaceutical composition is 3.0 mmol/kg.
  • the present application also provides the use of a lithium agent in the preparation of a medicine for treating blood-brain diseases, wherein the lithium agent is used to upregulate the Wnt/ ⁇ -catenin signaling pathway of cerebral vascular endothelial cells, and to regulate the endothelial cells of the blood-brain barrier.
  • the structure of the tight junction protein is protected.
  • the lithium agent is lithium chloride and/or lithium carbonate.
  • the lithium agents described herein may constitute the active ingredient of a pharmaceutical composition, and may generally be administered in the form of oral tablets, intravenous injections, or capsules in admixture with an appropriately selected suitable excipient or carrier.
  • Dosage compositions such as tablets, capsules, pills, suppositories, and powders depend upon the intended mode of administration, which may be by any acceptable route. These routes of administration include oral, intravenous (intravenous), intramuscular (intramuscular), subcutaneous (subcutaneous). One or more of these approaches can be used in a patient.
  • the compounds of the present application are administered as intravenous injection dosage forms and may be combined with non-toxic pharmaceutically acceptable inactive carriers such as water, glycerol, ethanol, physiological saline, and the like.
  • inactive carriers such as water, glycerol, ethanol, physiological saline, and the like.
  • Inert excipients which are commonly used as binders, disintegrating agents and coloring agents can also be added to the oral mixture.
  • the administered pharmaceutical compositions may also contain minor amounts of non-toxic substances such as pH buffering agents, emulsifiers, sodium acetate, and the like.
  • the dosage regimen for use of the compound will depend on the patient's species, sex, weight, age, medical condition, route of administration, and the severity of the condition being treated. A skilled physician can easily determine and prescribe an effective dose of a drug to treat the disease.
  • treatment includes one or more of curative treatment, palliative treatment, and prophylactic treatment, depending on the disease and condition of the patient.
  • the precise dose of each active compound administered will vary depending on a number of factors including, but not limited to, the type of patient and disease state being treated, the age of the patient, and the route of administration.
  • the dose administered will, of course, vary with the mode of administration, the treatment desired, and the disease indicated.
  • the total daily dose can be administered in a single dose or in divided doses.
  • Sustained release compositions are also encompassed by this application.
  • the pharmaceutical composition can be in a form suitable for oral administration, such as one of tablets, capsules, pills, powders, sustained-release formulations and solutions; or, the pharmaceutical composition can also be in a form suitable for intravenous injection , such as infusion bottles or infusion bags.
  • Pharmaceutical compositions can be presented in unit dosage forms suitable for single administration of precise dosages.
  • Pharmaceutical compositions will include conventional pharmaceutical carriers and active compounds. In addition, it may include other pharmaceutical or pharmaceutical agents, carriers, adjuvants, and the like.
  • Suitable pharmaceutical carriers include inert diluents or fillers, water. If desired, the pharmaceutical composition may contain additional ingredients such as flavoring agents, binders, and the like.
  • tablets containing various excipients such as citric acid can be combined with various disintegrants such as starch, alginic acid and certain complex silicates and binders such as sucrose, gelatin and gum arabic) together.
  • disintegrants such as starch, alginic acid and certain complex silicates and binders such as sucrose, gelatin and gum arabic
  • lubricants such as magnesium stearate, sodium lauryl sulfate, and talc are frequently used in the preparation of tablets.
  • Solid compositions of a similar type can also be used in soft and hard filled gelatin capsules. Useful components of these compositions include lactose or milk sugar and high molecular weight polyethylene glycols.
  • the active compounds therein may be mixed with various sweetening or flavoring agents, coloring or dyeing agents and, if desired, emulsifying or suspending agents and diluents such as water, ethanol , propylene glycol, glycerol) or a combination thereof.
  • various sweetening or flavoring agents such as water, ethanol , propylene glycol, glycerol
  • emulsifying or suspending agents and diluents such as water, ethanol , propylene glycol, glycerol
  • intravenous administration it can be used with distilled water or normal saline.
  • doses can be adjusted based on pharmacokinetic or pharmacodynamic parameters, which can include clinical effects such as toxic effects and/or laboratory values. Accordingly, this application encompasses intra-patient dose escalation as determined by one of skill in the art. Determining appropriate dosages and regimens for administering chemotherapeutic agents is well known in the relevant art and, once provided with the teachings disclosed herein, should be understood to be encompassed by those skilled in the art.
  • compositions of the present application can be prepared, packaged, or sold in bulk, in a single unit dose, or in multiple unit doses.
  • a "unit dose" is an individual quantity of a pharmaceutical composition containing a predetermined quantity of an active compound.
  • the amount of active compound is generally equal to the dose of active compound to be administered to a subject, or a convenient fraction of such a dose, such as, for example, one-half or one-third of such a dose.
  • compositions of the present application will vary depending on the identity, size and condition of the subject being treated and further depending on the route of administration of the composition.
  • the composition may contain between 0.1% and 100% (w/w) active ingredient.
  • compositions of the present application may further comprise one or more additional therapeutically effective compounds as discussed above.
  • lithium agent is lithium chloride as an example.
  • the model used in this application is a cerebral ischemia-reperfusion model of mouse middle cerebral artery occlusion (MCAO) for 1 hour (h) and recanalization for 48 hours. Specific steps are as follows:
  • mice 8 to 10 weeks old and weighing 20 to 23 g were first obtained. These mice were housed in a pathogen-free animal facility on a 12-hour light and dark cycle. Mice were randomly selected for sham surgery, ischemia models, or post-ischemia treatment studies. During surgery, anesthesia was first induced with 4% isoflurane in an induction chamber and maintained with 2% isoflurane delivered through a mask. The core temperature of each mouse was maintained at 37 ⁇ 0.5°C using a heating pad throughout the procedure, and the mice were treated with induced, transient middle cerebral artery occlusion (MCAO) using a modified intraluminal fiber model. After 60 min of MCAO, reperfusion was established by retracting the fibers.
  • MCAO transient middle cerebral artery occlusion
  • mice had free access to food and water throughout the reperfusion period. After 48 hours of MCAO, the mice were evaluated for neurological function by the observer, eg, on a deficit score (20), a grip test score (21), and a horizontal ladder test score (22). Mice without neurological deficits after surgery were excluded according to a pre-established exclusion plan.
  • mice were injected intraperitoneally (ip) with lithium chloride (LiCl) at a concentration of 2%, starting with intraperitoneal (ip) reperfusion, and then re-injected at the same dose 24 hours later.
  • LiCl lithium chloride
  • mice were deeply anesthetized with isoflurane.
  • the whole brain of the mouse was removed and coronal sectioning (thickness 2mm) was performed in a special groove.
  • coronal sectioning Immediately immerse the sections in 1 ml of 1% 2,3,5-triphenyltetrazolium chloride (TTC) and incubate at 37°C for 10 minutes. Then, the TTC solution was replaced with 4% paraformaldehyde for 1 h at room temperature.
  • Sections were photographed with a digital camera, and infarct size was measured by ImageJ software (a java-based public image processing software). To eliminate the effect of post-ischemic edema on lesion volume, infarct size was corrected as previously described (24).
  • the formula for calculating the infarct area (%) is [((volume of left hemisphere ⁇ non-infarct volume of right hemisphere)/volume of left hemisphere)100%.
  • the brains were then divided into the ipsilateral ischemic hemisphere and the contralateral non-ischemic hemisphere, homogenized in 1 ml of 50% trichloroacetic acid and centrifuged (10,000 rpm, 20 minutes), and the above solution was diluted four-fold with ethanol and used Evans blue concentrations were measured by a fluorescence reader (620 nm excitation; 680 nm emission) and expressed as ⁇ g/g brain tissue.
  • TUNEL terminal deoxynucleotidyl transferase-mediated dUTP (deoxyuridine triphosphate)-digoxigenin terminal labeling
  • DAPI 4,6-diamidino-2-phenylindole
  • Slides were mounted with DAPI (4',6-diamidino-2-phenylindole) in a fade-resistant mounting medium and imaged with a microscope to obtain 10, 20 or 40 images.
  • Immunofluorescence signal area or density was quantified by ImageJ (a public image processing software based on java) and normalized by vessel area (CD31 signal area) in 5 to 8 random ischemic areas per mouse.
  • Proteins were extracted from the ischemic side (at approximately 3.30 mm) of 2 mm thick coronal sections into the cerebral forebrain and analyzed by Western blotting. Brain tissue was collected 48 h after occlusion. Brain tissue proteins were isolated and collected by RIPA (Soluble Protein Extracted from Animal Tissue and Animal Cells) lysis buffer supplemented with protease and phosphatase inhibitors and quantified by BCA (BCA Protein Concentration Assay) assay according to standard protocols. Equal amounts of protein lysates from each sample were separated on 10% SDS-PAGE (polyacrylamide gel electrophoresis) gels.
  • RIPA Soluble Protein Extracted from Animal Tissue and Animal Cells
  • BCA BCA Protein Concentration Assay
  • the protein was transferred to a polyvinylidene fluoride membrane, which was then incubated with primary antibodies, including rabbit anti-claudin-5, rabbit anti-ZO-1, rabbit anti-occludin, rabbit anti-MMP-9, Rabbit anti- ⁇ -catenin, rabbit anti- ⁇ -catenin, rabbit anti-phospho-JNK Thr183/Tyr185 (rabbit anti-phospho-c-Jun N-terminal kinase-threonine 286/threonine 185), rabbit anti-JNK (c -Jun amino-terminal kinase), rabbit anti-phospho-Camk II Thr286 (calmodulin kinase II-threonine 286), rabbit anti-CamkII (calmodulin-dependent protein kinase II) at a dilution ratio of 1:1,000. ⁇ -Actin was used as a normalized loading control. Membranes were incubated with the corresponding secondary antibodies and blots were developed using the GelView 6000
  • RNA was reverse transcribed using RT-qPCR (real-time quantitative PCR) using Reverse Transcription Supermix, an object-based macrolanguage.
  • RT-qPCR was performed on a real-time PCR system using the Power SYBRi Script Green (powerful master mix with green excitation wavelength dye) method.
  • RNA expression was calculated using the comparative Ct (electron computed tomography) method normalized to actin. Data are expressed relative to a calibrator using the 2- ⁇ Ct method.
  • Mouse brain-derived endothelial cells 3 were grown in Dulbecco's (Dulbecco's phosphate buffered saline) modified Eagle's medium (DMEM, a medium containing various amino acids and glucose) containing 10% fetal bovine serum and 1% penicillin. ) were cultivated. All cultures were maintained in a CO2 incubator with 5% humidity at 37°C and routinely passaged at 80-90% confluence. After 15 hours of LiCl or vehicle treatment, cells were exposed to OGD/R (ex vivo model of cerebral ischemia from oxygen and glucose deprivation).
  • DMEM Dulbecco's phosphate buffered saline modified Eagle's medium
  • the culture medium was replaced with Dulbecco's modified medium with LiCl or vehicle control, and the cultured cells were placed in a modular incubator containing 0.5–1% O2 and 99% N2 (nitrogen) with O2 (Oxygen) analyzer to monitor. After 6 hours of OGD (oxygen glucose deprivation), cells were returned to normal culture conditions with LiCl to restore oxyglucose for 3 hours.
  • OGD oxygen glucose deprivation
  • CCK-8 Cell viability was measured with CCK-8 and cells were seeded in 96-well plates at a concentration of 5,000 cells/well. Ten mL of CCK-8 solution was added to each well containing 100 mL of medium. Cells were incubated at 37°C for 2 hours. OD values (optical density values) were measured at 450 nm with a MULTISKAN GO (multi-scanner).
  • a stable bEnd.3 (mouse brain microvascular endothelial cell) cell line with Wnt/ ⁇ -catenin signaling TOP-flash firefly luciferase [with TCF/LEF (Constructs for binding sites of T-cell factor, lymphoid potentiator) and Renilla control reporters were seeded on 96-well plates. After 48 hours, cells were stimulated with 100 ng/ml Wnt3a for 24 hours. Using Dual Stop (double stop ) and Glo (globulin) systems to measure firefly and Renilla luciferase activity. Reporter activity was calculated as firefly/renilla activity in each well. In each graph, all data are normalized to The first bar represents the data point.
  • TEER was measured using a Millicell-ERS-2 (Millipore, Germany). bEnd.3 cells were seeded on Transwell permeable membranes (24-well cell culture inserts) with a pore size of 0.4 ⁇ m and allowed to grow for 3 days. Baseline TEER was measured before LiCl and OGD/R treatments. All measurements were normalized by subtracting TEER values measured in blank Transwell (invasion assay) filters. After OGD/R, the effect of LiCl on endothelial monolayer permeability of fluorescein isothiocyanate-conjugated 70 kDa FITC-dextran was evaluated.
  • FITC-dextran Ten microliters of FITC-dextran (5ug/ml) was added to the chamber and the mixture was incubated for 30 minutes. Permeability was assessed by measuring fluorescence at 520 nm from a 100 ⁇ l aliquot of medium taken from the chamber using a fluorescence plate reader MULTISKAN GO.
  • FIG. 1 is a schematic diagram of the relative proportion of mouse infarct volume in the cerebral hemisphere
  • Figure 3 is a schematic diagram of the relative proportion of Evans blue staining
  • Figure 4 is a schematic diagram of the relative proportion of Evans blue staining
  • Fig. 5 is a schematic diagram of the exudation of endogenous IgG from blood vessels observed by immunofluorescence double staining
  • Fig. 1 is a schematic diagram of the relative proportion of mouse infarct volume in the cerebral hemisphere
  • Figure 4 is a schematic diagram of the relative proportion of Evans blue staining
  • Fig. 5 is a schematic diagram of the exudation of endogenous IgG from blood vessels observed by immunofluorescence double staining
  • FIG. 6 is a schematic diagram of the relative density of IgG exuded from blood vessels; Fig. 7 is the histogram of the neurobehavioral score; Figure 8 is the schematic diagram of the neuron apoptosis observed by immunofluorescence double staining; Figure 9 is the histogram of the relative density of the number of apoptotic neurons in the infarcted area of mice; Figure 10 is the immunofluorescence double staining to observe the active ⁇ - Schematic diagram of the expression of catenin on endothelial cells; Figure 11 is a histogram of the relative density of active ⁇ -catenin expression on endothelial cells; Figure 12 is a schematic diagram of active and total ⁇ -catenin protein levels in brain tissue in the infarcted area of each group; Figure 13 is Figure 12 Figure 14 is another histogram of the relative density of immunoblotting in Figure 12; Figure 15 is a histogram of axin2 levels in cerebral infarction tissue in each group; Figure 16 is apcdd1
  • Figure 1 shows the ischemic area after staining the coronal section of the mouse brain with TTC, and the white area is the ischemic area;
  • Figure 3 shows the staining area of Evans blue (the blue area is the leakage area of Evans blue in the brain tissue);
  • Figure 5 shows the exudation of endogenous IgG (green) from blood vessels (CD31, red) observed by immunofluorescence double staining, and the corresponding scale is 100um;
  • Figure 8 shows the double immunofluorescence staining to observe the apoptosis of neurons (red) (TUNEL staining, green), the scale is 100um;
  • Figure 10 shows the expression of active ⁇ -catenin (green) on endothelial cells (red) observed by immunofluorescence double staining, the bar is 100um;
  • Figure 12 shows the active and total ⁇ -catenin protein levels in the brain tissue in the infarcted area of each group;
  • lithium treatment enhanced monolayer cerebral vascular endothelial cell integrity and BBB function compared with control cells without lithium treatment.
  • Lithium has dual effects of BBB and nerve cell protection.
  • the BBB protection of lithium is achieved by up-regulating the Wnt/ ⁇ -catenin pathway in cerebral vascular endothelial cells and the structure of tight junction protein between endothelial cells that protects the BBB.
  • experiments confirmed that the protective effect of lithium on the BBB is mainly due to the protection of tight junctions between cerebral vascular endothelial cells.
  • lithium is a commonly used drug in clinic, its pharmacokinetics and side effects are clear, and its cost is low, so it is expected to be directly applied to clinical cerebral infarction patients.
  • lithium not only has BBB protection effect, but also has neuroprotective effect. Compared with other drugs targeting Wnt/ ⁇ -catenin pathway, lithium has better safety.
  • lithium can also be applied to other diseases that can cause BBB damage, including cerebral hemorrhage, toxic encephalopathy, metabolic encephalopathy, and cerebral hemorrhage secondary to central nervous system leukemia.
  • lithium in the pharmaceutical composition of the present application can up-regulate the Wnt/ ⁇ -catenin signaling pathway of cerebral vascular endothelial cells, and play an important role in protecting the structure of tight junction proteins in endothelial cells of the blood-brain barrier, This protects the blood-brain barrier. Therefore, lithium can play an effective drug role in the treatment of ischemic stroke and its resulting blood-brain barrier damage.

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Abstract

一种医药组合物及其医药用途。该医药组合物包括有效剂量的锂剂和医药上可接受的载体，在治疗缺血性脑卒中、血脑屏障损伤及血脑疾病方面起到有效的药物作用。

Description

一种医药组合物及其医药用途 【技术领域】
本申请涉及医药技术领域，特别是涉及一种医药组合物及其医药用途。
【背景技术】
缺血性脑卒中(又名脑梗死)是人类疾病谱当中致死率和致残率最高的疾病之一，对其最佳的治疗方法是包括阿替普酶(rtPA)和手术取栓在内的血管再通疗法，而他们均有严格的时间窗限制，导致相当比例的病人不能接受此类治疗。其中，血管再通疗法的时间窗的设定主要是为了避免或减少再灌注导致的血脑屏障(blood-brain barrier，BBB)损害。而如果能有效保护BBB，那么血管再通疗法的时间窗就有可能被延长，从而有利于使更多的人接受血管再通治疗。但目前临床尚无有效的BBB保护药物。
其中，急性缺血性脑卒中发病后的超早期，现有的主要治疗方法是阿替普酶rtPA静脉溶栓和动脉内手术取栓术，而它们均有严格的时间窗限制，前者为4.5小时，后者为6-8小时(少数病人可以延长到24小时)。临床研究显示，超过此时间窗进行血管开通治疗会出现诸如出血转化、脑水肿等严重并发症，因此，相当比例急性脑梗死病人由于没有及时就医、距离医院路程远、路上塞车等主、客观因素导致到达医院时已超过血管开通治疗的时间窗，而不能接受血管开通治疗。同时，研究表明，rtPA本身对于BBB有损害，即使在时间窗内使用，仍有相当比例病人会出现诸如出血转化、脑水肿等BBB损害表型。
目前，临床尚无针对性的BBB保护药物。有脑水肿或出血转化时，如已诱发脑疝，轻者使用20％甘露醇、高渗盐、利尿剂等脱水治疗，重者进行“去骨瓣减压术”；而如果未诱发脑疝，一般给予病人镇静、降压、纠正水电酸碱平衡紊乱等支持治疗。
【发明内容】
本申请主要解决的技术问题是提供一种医药组合物及其医药用途，能够在治疗缺血性脑卒中及其所导致的血脑屏障损伤起到有效的药物作用。
为解决上述技术问题，本申请采用的一个技术方案是：提供一种治疗缺血 性脑卒中的医药组合物，该医药组合物包括：治疗缺血性脑卒中有效剂量的锂剂和医药上可接受的载体，其中，锂剂用于上调脑血管内皮细胞Wnt/β-catenin信号通路，并对血脑屏障的内皮细胞的紧密连接蛋白的结构进行保护。
其中，锂剂为氯化锂和/或碳酸锂。
其中，医药组合物呈剂量单位形式。
其中，医药组合物中的锂剂的剂量为1.5-6.0毫摩尔/千克中的一种，其中，毫摩尔/千克为锂剂的物质的量相对于患者的质量。
其中，医药组合物中的锂剂的剂量为3.0毫摩尔/千克。
其中，医药组合物还包括治疗缺血性脑卒中有效剂量的阿替普酶，且医药组合物中的阿替普酶的剂量为0.9毫克/千克，其中，毫克/千克为阿替普酶的质量相对于患者的质量。
为解决上述技术问题，本申请采用的另一个技术方案是：提供一种锂剂在治疗缺血性脑卒中药物的制备中的用途，其中，该锂剂用于上调脑血管内皮细胞Wnt/β-catenin信号通路，并对血脑屏障的内皮细胞的紧密连接蛋白的结构进行保护。
其中，锂剂为氯化锂和/或碳酸锂。
为解决上述技术问题，本申请采用的又一个技术方案是：提供一种治疗血脑屏障损伤的医药组合物，该医药组合物包括：治疗血脑屏障损伤的有效剂量的锂剂和医药上可接受的载体，其中，锂剂用于上调脑血管内皮细胞Wnt/β-catenin信号通路，并对血脑屏障的内皮细胞的紧密连接蛋白的结构进行保护。
为解决上述技术问题，本申请采用的又一个技术方案是：提供一种锂剂在治疗血脑屏障损伤的药物的制备中的用途，其中，该锂剂用于上调脑血管内皮细胞Wnt/β-catenin信号通路，并对血脑屏障的内皮细胞的紧密连接蛋白的结构进行保护。
为解决上述技术问题，本申请采用的又一个技术方案是：提供一种治疗血脑疾病的医药组合物，该医药组合物包括：治疗血脑疾病的有效剂量的锂剂和医药上可接受的载体，其中，锂剂用于上调脑血管内皮细胞Wnt/β-catenin信号通路，并对血脑屏障的内皮细胞的紧密连接蛋白的结构进行保护。
为解决上述技术问题，本申请采用的又一个技术方案是：提供一种锂剂在治疗血脑疾病的药物的制备中的用途，其中，该锂剂用于上调脑血管内皮细胞 Wnt/β-catenin信号通路，并对血脑屏障的内皮细胞的紧密连接蛋白的结构进行保护。
本申请的有益效果是：区别于现有技术的情况，本申请医药组合物中的锂剂对上调脑血管内皮细胞Wnt/β-catenin信号通路，以及对血脑屏障的内皮细胞的紧密连接蛋白的结构的保护有着重要的作用，进而能够对血脑屏障进行保护。因此，锂剂能够在治疗缺血性脑卒中及其所导致的血脑屏障损伤起到有效的药物作用。
【附图说明】
图1是采用TTC对小鼠大脑的冠状切片进行染色后的试验对比示意图；
图2是小鼠梗死体积在脑半球内的相对占比示意图；
图3是伊文斯兰染色的相对占比的示意图；
图4是伊文斯兰渗出量占半球重量的相对百分比的示意图；
图5是免疫荧光双重染色观察内源性IgG从血管中的渗出情况的示意图；
图6是IgG从血管内渗出的相对密度柱状示意图；
图7是神经行为学评分柱状示意图；
图8是免疫荧光双重染色观察神经元凋亡情况示意图；
图9是小鼠梗死区神经元凋亡数量相对密度柱状图；
图10是免疫荧光双重染色观察activeβ-catenin在内皮细胞上的表达情况示意图；
图11是activeβ-catenin在内皮细胞上表达的相对密度柱状图；
图12是各组梗死区脑组织active和totalβ-catenin蛋白水平示意图；
图13是图12中免疫印迹法相对密度柱状图；
图14是图12中免疫印迹法相对密度又一柱状图；
图15是各组脑梗死组织中axin2水平柱状图；
图16是各组脑梗死组织中apcdd1 mRNA水平柱状图；
图17是不同浓度氯化锂对于bEnd.3 Axin2 mRNA水平的影响示意图；
图18是不同浓度氯化锂对于bEnd.3 TCF/LEF TOPflash值的影响示意图；
图19是通过敲除Fzd4构建了Wnt/β-catenin通路缺陷的bEnd.3细胞系对应的氯化锂的效果示意图；
图20是通过敲除Fzd4构建了Wnt/β-catenin通路缺陷的bEnd.3细胞系对应的氯化锂的又一效果示意图；
图21是各组axin2 mRNA水平柱状图。
【具体实施方式】
下面结合附图和实施方式对本申请进行详细说明。
本申请提供一种治疗缺血性脑卒中的医药组合物，该医药组合物包括：治疗缺血性脑卒中有效剂量的锂剂和医药上可接受的载体。
缺血性脑卒中是指由于脑的供血动脉(颈动脉和椎动脉)狭窄或闭塞、脑供血不足导致的脑组织坏死的总称。有四种类型的脑缺血：短暂性脑缺血发作(TIA)；可逆性神经功能障碍(RIND)；进展性卒中(SIE)；完全性卒中(CS)。TIA无脑梗死存在，而RIND、SIE和CS有不同程度的脑梗死存在。
从缺血的影响范围可将脑缺血分为局限性脑缺血和弥漫性脑缺血。局限性脑缺血的病因有：大脑中动脉栓塞；颅外颈内动脉或椎动脉狭窄、闭塞或血栓形成；脑动脉痉挛。弥漫性脑缺血的病因有：心搏骤停、低血压、贫血、低血糖等。
其中，现有的对于缺血性脑卒中(又名脑梗死)的最佳治疗方法是采用包括阿替普酶(rtPA)和手术取栓在内的血管再通疗法，而他们均有严格的时间窗限制，导致相当比例病人不能接受此类治疗。血管再通疗法的时间窗设定主要是为了避免或减少再灌注导致的血脑屏障损害，而阿替普酶rtPA本身对于血脑屏障有损害，即使在时间窗内使用仍有部分病人会出现诸如出血转化、脑水肿等BBB损害表型。但如果能有一种BBB保护剂和rtPA联用，以减轻其对于BBB的损害，那将提高血管再通治疗的有效性，血管再通疗法的时间窗就有可能被延长，并有利于使更多的人接受血管再通治疗。
锂剂在目前的临床或基础研究中的主要用途是对治疗躁狂和抑郁交替发作的双相情感性精神障碍有预防和治疗作用。本申请的发明人在实验研究中发现，在将锂剂用于小鼠的缺血性脑卒中时，能够上调其脑血管内皮细胞Wnt/β-catenin通路，且对于小鼠脑梗死急性期BBB破坏也存在有明显的保护作用，从而潜在地增加了锂剂的适应症。
此外，虽然目前已有靶向性的Wnt/β-catenin通路激动剂，但尚处于实验阶 段。鉴于锂剂是一种临床常用的精神科药物，有望直接进行临床试验和应用。
而本申请的发明人在实验研究中发现，锂剂具有BBB功能和神经细胞的双重保护作用。且锂剂通过上调脑血管内皮细胞Wnt/β-catenin通路，能够改善脑血管内皮细胞的功能完整，并能够对血脑屏障的内皮细胞的紧密连接蛋白的结构进行保护，从而实现对BBB的保护作用。
其中，该锂剂为氯化锂(LiCl)和/或碳酸锂(Li2CO3)。而在其他实施例中，该锂剂还可以是氢化锂、氮化锂及氢氧化锂等锂的化合物中任一合理的一种或多种混合，本申请对此不做限定。
其中，医药组合物呈剂量单位形式。
其中，医药组合物中的锂剂的剂量为1.5-6.0毫摩尔/千克中的一种，该毫摩尔/千克为锂剂的物质的量相对于患者的质量。而在其他实施例中，该医药组合物在具体的人体上使用时，该医药组合物中的锂剂的剂量还可以综合考虑药物的毒副作用或血药浓度等因素的变化，而采用其他任一合理的剂量，本申请对比不做限定。
其中，医药组合物中的锂剂的剂量为3.0毫摩尔/千克。
其中，医药组合物还包括治疗缺血性脑卒中有效剂量的阿替普酶，且医药组合物中的阿替普酶的剂量为0.9毫克/千克，该毫克/千克为阿替普酶的质量相对于患者的质量。
本申请还提供一种锂剂在治疗缺血性脑卒中药物的制备中的用途，其中，该锂剂用于上调脑血管内皮细胞Wnt/β-catenin信号通路，并对血脑屏障的内皮细胞的紧密连接蛋白的结构进行保护。
其中，该锂剂为氯化锂和/或碳酸锂。
本申请还提供一种治疗血脑屏障损伤的医药组合物，该医药组合物包括：治疗血脑屏障损伤的有效剂量的锂剂和医药上可接受的载体，其中，该锂剂用于上调脑血管内皮细胞Wnt/β-catenin信号通路，并对血脑屏障的内皮细胞的紧密连接蛋白的结构进行保护。
其中，该锂剂为氯化锂和/或碳酸锂。
其中，医药组合物呈剂量单位形式。
其中，医药组合物中的锂剂的剂量为1.5-6.0毫摩尔/千克中的一种，该毫摩尔/千克为锂剂相对于患者的质量。
其中，医药组合物中的锂剂的剂量为3.0毫摩尔/千克。
本申请还提供一种锂剂在治疗血脑屏障损伤的药物的制备中的用途，其中，该锂剂用于上调脑血管内皮细胞Wnt/β-catenin信号通路，并对血脑屏障的内皮细胞的紧密连接蛋白的结构进行保护。
其中，该锂剂为氯化锂和/或碳酸锂。
本申请还提供一种治疗血脑疾病的医药组合物，该医药组合物包括：治疗血脑疾病的有效剂量的锂剂和医药上可接受的载体，其中，锂剂用于上调脑血管内皮细胞Wnt/β-catenin信号通路，并对血脑屏障的内皮细胞的紧密连接蛋白的结构进行保护。
其中，该锂剂为氯化锂和/或碳酸锂。
其中，医药组合物呈剂量单位形式。
其中，医药组合物中的锂剂的剂量为1.5-6.0毫摩尔/千克中的一种，该毫摩尔/千克为锂剂相对于患者的质量。
其中，医药组合物中的锂剂的剂量为3.0毫摩尔/千克。
本申请还提供一种锂剂在治疗血脑疾病的药物的制备中的用途，其中，该锂剂用于上调脑血管内皮细胞Wnt/β-catenin信号通路，并对血脑屏障的内皮细胞的紧密连接蛋白的结构进行保护。
其中，该锂剂为氯化锂和/或碳酸锂。
本文所述锂剂可构成医药组合物的活性成分，且通常可与适当选择的合适赋形剂或载体混合以口服片剂、静脉注射剂或胶囊的形式给药。例如片剂、胶囊、丸剂、栓剂和散剂等剂量组合物取决于预定的给药方式，其可通过任何可接受的途径。这些给药途径包括经口服、静脉注射(静注)、肌肉注射(肌注)、皮下注射(皮下)。在一名患者中可以使用这些途径中的一或多种。在一实施方式中，本申请的化合物用作静脉注射剂量形式给药且可与无毒的医药上可接受的非活性载体组合，例如水、甘油、乙醇、生理盐水等等。也可以在口服混合物中加入通常用作粘合剂、崩解剂和着色剂的惰性赋形剂。
需要时，所给予的医药组合物也可包含少量的无毒物质，例如pH缓冲剂、乳化剂、乙酸钠等。使用化合物的剂量方案将取决于患者的物种、性别、体重、年龄、医学状况、给药途径以及所治疗病症的严重程度。熟练的医师可以很容易地确定和规定治疗疾病的药物有效剂量。
视患者的疾病和状况而定，本文所用术语“治疗”包括治愈治疗、姑息治疗和预防性治疗中的一种或多种。各活性化合物所施用的精确剂量将根据许多 因素而变化，这些因素包括但不限于患者的类型和所治疗疾病状态的类型、患者的年龄、以及给药途径。
对于上述治疗用途来说，所施用的剂量当然会随所施用方式、所需治疗以及所指示的疾病而变化。总日剂量可以单次剂量或分次剂量施用。本申请也涵盖缓释组合物。
举例而言，医药组合物可以呈适合口服投药的形式，如片剂、胶囊、丸剂、散剂、缓释调配物及溶液中的一种；或，该医药组合物也可以呈适合静脉注射的形式，如输液瓶或输液袋。医药组合物可以呈适合于精确剂量的单次施用的单位剂量形式。医药组合物将包括常规的医药载体和活性化合物。另外，它可包括其他药用或医药剂、载体、佐剂等。
合适的医药载体包括惰性稀释剂或填充剂、水。若需要，医药组合物可包含另外的成分，例如调味剂、粘合剂和类似成分。因此对于口服给药，含例如柠檬酸等各种赋形剂的片剂可以与各种崩解剂(例如淀粉、海藻酸和某些复合硅酸盐)以及粘合剂(例如蔗糖、明胶和阿拉伯胶)一起采用。另外，例如硬脂酸镁、月桂基硫酸钠和滑石等润滑剂经常用于制备片剂。类似类型的固体组合物也可用于软和硬填充明胶胶囊中。这些组合物的有用组分包括乳糖或奶糖和高分子量聚乙二醇。当口服给药需要使用水混悬剂或酏剂时，其中的活性化合物可以与各种增甜剂或调味剂、色素或染料以及视需要乳化剂或助悬剂以及稀释剂(例如水、乙醇、丙二醇、甘油)或其组合相结合。而对于静脉注射给药，可以与蒸馏水或生理盐水一起使用。
所述领域的技术人员熟知或将明了制备具有特定量的活性化合物的各种医药组合物的方法。
本文所列剂量范围仅为例示性的，并不打算限制所主张组合物的范围或实践。举例而言，剂量可以根据药物代谢动力学或药效学参数调整，药物代谢动力学或药效学参数可包括临床效应如毒效应和/或实验室值。因此，本申请涵盖本领域技术人员所确定的患者内剂量递增。确定施用化疗剂的合适剂量和方案在相关领域中为人熟知，且一旦提供本文所公开的教义，应被理解为被所述领域的技术人员所涵盖。
本申请的医药组合物可以散装形式、以单个单位剂量形式、或以多个单位剂量形式制备、包装或出售。本文所用“单位剂量”是包含预定量的活性化合物的医药组合物的个别量。活性化合物的量通常等于将施用至受试者的活性化 合物的剂量、或此一剂量的一个方便的分数，例如，举例而言，此一剂量的一半或三分之一。
本申请医药组合物中活性化合物、医药上可接受的载体、及任何其他成分的相对量将根据所治疗受试者的身份、大小和状况且进一步根据组合物的投药途径而变化。举例来说，组合物可包含0.1％与100％(w/w)之间的活性成分。
除活性化合物外，本申请的医药组合物可进一步包含一种或多种另外的如上所讨论的治疗有效化合物。
下面以具体的数据来说明锂剂在治疗缺血性脑卒中方面提供有效的药物作用。其中，锂剂以氯化锂为例。
本申请采用的模型是小鼠大脑中动脉栓塞(middle cerebral artery occlusion，MCAO)1小时(h)，血管再通48h的脑缺血再灌注模型，观察锂剂脑梗死超早期使用的干预效果。具体步骤如下：
1、动物规程和锂剂治疗
首先获取到8至10周大，且体重为20至23g的C57BL/6雄性小鼠。将这些小鼠在无病原体的动物设施中进行12小时的光照和黑暗循环饲养。随机选取小鼠用于假手术、缺血模型或缺血后的治疗研究。在对其进行外科手术时，首先在诱导室中用4％的异氟烷对其进行诱导麻醉，并通过面罩输送2％的异氟烷维持麻醉。在整个手术过程中，使用加热垫将每只小鼠的核心温度维持在37±0.5℃，并通过改良的腔内纤维模型对该小鼠进行诱导、短暂性大脑中动脉闭塞(MCAO)处理。在MCAO持续60分钟后，通过缩回纤维建立再灌注。
在整个再灌注期间，小鼠可以自由获取食物和水。在MCAO持续48小时后，由观察者对该小鼠的神经功能进行评估，比如，进行缺损评分(20)，抓握测试评分(21)和水平阶梯测试评分(22)。根据预先制定的排除计划，将手术后不存在神经功能缺损的小鼠排除。
在MCAO再灌注后，立即在小鼠的腹膜内(ip)注射浓度为2％的氯化锂(LiCl)，腹膜内(ip)再灌注开始，然后以相同剂量在间隔24小时后再次注射。
2、梗死面积分析
再灌注48小时后，用异氟烷深度麻醉小鼠。取出小鼠的整个大脑，并在一个特殊的凹槽中进行冠状切片(厚度2mm)。将切片立即浸入1ml 1％的2,3,5-三苯基四唑氯化物(TTC)中，并在37℃下孵育10分钟。然后，在室温下将TTC溶液替换为4％多聚甲醛1小时。用数码相机对切片进行拍照，并通过ImageJ 软件(基于java的公共的图像处理软件)测量梗死面积。为了消除缺血后水肿对损伤体积的影响，如先前所述纠正了梗死面积(24)。其中，梗塞面积(％)的计算公式为[((左半球的体积–右半球的非梗塞体积)/左半球的体积)100％。
3、伊文斯兰泄漏测量
使用伊文斯兰(Evans blue，一种生物染色剂，化学式为6,6'-[[3,3'-二甲基(1,1'-二苯基)-4,4'-二基]双(偶氮基)]双(4-氨基-5-羟基-1,3-萘二磺酸)四钠盐)进行灌注，通过眼眶后静脉内(i.v.)注射施用100μl2％伊文斯兰18-20h后，用异氟烷深度麻醉小鼠，然后经左心室经20ml冷盐水经心脏向搏动的心脏灌注，以去除血管内染料。取出小鼠大脑，并用数码相机拍照。然后将脑分为同侧缺血半球和对侧非缺血半球，并在1ml 50％三氯乙酸中匀浆并离心(10,000rpm，20分钟)，对上述溶液用乙醇稀释四倍后，用荧光读取器(620nm激发；680nm发射)测量伊文斯兰的浓度，并表示为μg/g脑组织。
4、免疫荧光和TUNEL检测染色
将冷冻的7μm厚切片在室温下在粘附显微镜载玻片上干燥，然后在PBS(磷酸缓冲盐溶液)中复水。在室温下，将切片在PBS+0.2％Triton X-100中的10％正常山羊血清中封闭1小时。将样品与以下一级抗体在PBS+5％山羊血清+0.2％Triton X-100中于4℃下孵育：仓鼠抗小鼠CD31(血小板-内皮细胞粘附分子)，驴抗小鼠IgG(免疫球蛋白G)，兔抗claudin-5(供紧密连接蛋白-5)，兔抗ZO-1(闭锁连接蛋白1)，兔抗胶原蛋白IV(静脉内)，兔抗laminin(层粘连蛋白)，兔抗结蛋白兔抗活性β-连环蛋白。通过在PBS中冲洗5分钟(3次)来去除多余的抗体。然后将样品与以下二级荧光标记的抗体在室温下孵育1小时：Cy3(受激发后肉眼可见的红光)山羊抗仓鼠IgG和Alexa Fluor(荧光染料)488驴抗兔IgG以PBS+5稀释1：500％山羊血清+0.2％Triton X-100在室温下放置1小时。
其中，对末端脱氧核苷酸转移酶介导的dUTP(脱氧尿嘧啶核苷三磷酸)-地高辛配基的末端标记(TUNEL)进行的染色，是使用TUNEL FITC(细胞凋亡检测试剂盒)进行的。而对于双免疫荧光染色，将TUNEL切片在PBS中洗涤，并在4℃下与兔抗NeuN(神经核抗原)一起孵育一整晚，然后在室温下与Alexa Fluor 647驴一起孵育1h抗兔IgG。通过在PBS中冲洗5分钟(3次)来去除多余的抗体。将载玻片用DAPI(4',6-二脒基-2-苯基吲哚)固定在防褪色固定介质中，并用显微镜成像，以获得10、20或40个图像。通过ImageJ(基于 java的公共的图像处理软件)定量免疫荧光信号面积或密度，并通过每只小鼠5至8个随机缺血区域中的血管面积(CD31信号面积)进行归一化。
5、蛋白质印迹分析
从2毫米厚的冠状切片的缺血侧(约3.30毫米处)提取蛋白质至大脑前脑，并通过Western(蛋白质印迹法)印迹进行分析。闭塞48h后收集脑组织。通过补充蛋白酶和磷酸酶抑制剂的RIPA(从动物组织和动物细胞中抽取的可溶性蛋白)裂解缓冲液分离并收集脑组织蛋白质，并根据标准方案通过BCA(BCA蛋白浓度检测)测定进行定量。在10％SDS-PAGE(聚丙烯酰胺凝胶电泳)凝胶上分离等量的每个样品的蛋白质裂解物。将蛋白质转移到聚偏二氟乙烯膜上，然后将聚偏二氟乙烯膜与一抗一起孵育，包括兔抗claudin-5，兔抗ZO-1，兔抗闭合蛋白，兔抗MMP-9，兔抗β-连环素，兔抗β-连环素，兔抗磷酸-JNK Thr183/Tyr185(兔抗磷酸-c-Jun氨基末端激酶-苏氨酸286/苏氨酸185)，兔抗JNK(c-Jun氨基末端激酶)，兔抗磷酸-Camk II Thr286(钙调蛋白激酶II-苏氨酸286)，兔抗CamkII(钙调蛋白依赖性蛋白激酶Ⅱ)，稀释比例为1：1,000。β-肌动蛋白用作标准化的加载对照。将膜与相应的二抗一起孵育，并使用GelView 6000M系统(核酸染料-6000M)显影印迹。
6、实时定量PCR(聚合酶链式反应)
使用Direct-RNA MiniPrep(直接-RNA-质粒提取试剂盒)试剂盒提取总RNA。使用逆转录Supermix(以对象为基础的宏语言)进行RT-qPCR(实时荧光定量PCR)逆转录RNA。RT-qPCR使用Power SYBRi Script Green(具有绿色激发波长染料的强效预混液)方法在实时荧光定量PCR仪的系统上进行。使用相对于肌动蛋白标准化的比较Ct(电子计算机断层扫描)方法计算RNA表达。相对于校准器，使用2-ΔΔCt方法表示数据。
7、细胞培养，氧葡萄糖剥夺和恢复(OGD/R)以及锂处理
将小鼠脑源性内皮细胞3在含有10％胎牛血清和1％青霉素的Dulbecco(杜尔贝科磷酸盐缓冲液)改良鹰培养基(DMEM，一种含各种氨基酸和葡萄糖的培养基)中进行培养。所有培养物均保持在湿度为5％的CO2培养箱中，温度为37℃，并在80-90％汇合时常规传代。LiCl或载体处理15小时后，将细胞暴露于OGD/R(糖氧剥夺离体脑缺血模型)。然后，将培养液替换为带有LiCl或媒介物对照的Dulbecco改良版培养基，并将培养细胞放入含0.5–1％O2的模块化培养箱中和99％的N2(氮气)，用O2(氧气)分析仪进行监测。在OGD (氧糖剥夺)6小时后，将细胞恢复为含LiCl的正常培养条件，以恢复3小时的氧葡萄糖。
8、细胞计数试剂盒8(CCK-8)测试
用CCK-8测量细胞活力，将细胞以5,000个细胞/孔的浓度接种在96孔板中。将十毫升的CCK-8溶液加入到每个含有100毫升培养基的孔中。将细胞在37℃下孵育2小时。用MULTISKAN GO(多扫描仪)在450nm下测量OD值(光密度值)。
9、萤光素酶测定
对于典型的萤光素酶测定，将稳定的bEnd.3(小鼠脑微血管内皮细胞)细胞系(其带有Wnt/β-catenin信号传导的TOP-flash萤火虫萤光素酶【具有TCF/LEF(T细胞因子、淋巴增强因子)结合位点的构建体】和海肾对照报道子接种在96孔板上。48小时后，将细胞用100ng/ml Wnt3a刺激24小时。使用Dual Stop(双重终止)和Glo(球蛋白)系统测量萤火虫和海肾荧光素酶的活性。将报告者的活性计算为每个孔中的萤火虫/海肾活性。在每个图中，所有数据均归一化为第一个条表示的数据点。
10、跨内皮电阻(TEER)和磁导率的测量
使用Millicell-ERS-2(德国密理博)测量TEER。将bEnd.3细胞播种在孔径为0.4μm的Transwell渗透膜(24孔细胞培养插入物)上，并使其生长3天。在LiCl和OGD/R处理之前测量基线TEER。通过减去空白Transwell(侵袭实验)滤波器中测得的TEER值对所有测量值进行归一化。在OGD/R之后，评估了LiCl对异硫氰酸荧光素共轭的70kDa FITC-葡聚糖的内皮单层渗透性的影响。将十微升的FITC-葡聚糖(5ug/ml)加入到腔室中，并将混合物温育30分钟。通过使用荧光板读数仪MULTISKAN GO测量从取自房室的100μl培养基等分试样发出的520nm处的荧光来评估通透性。
11、统计分析
使用Windows软件包SPSS 16.0(美国SPSS)进行统计分析。数据表示为平均值±标准误差(SE)。学生的t检验用于两组比较。使用单向方差分析(ANOVA)确定来自不同组的所有观察指标的统计差异。如果方差是异质的，则使用最低有效差(LSD)t检验或Dunnett T3(新复极差法检验)检验来进一步分析差异。统计学显着性定义为P<0.05。
具体地，实验结果请参见图1至图21(其中，图1、图3、图4、图8、图 10、图19以及图20的原图为彩图)，图1是采用TTC对小鼠大脑的冠状切片进行染色后的试验对比示意图；图2是小鼠梗死体积在脑半球内的相对占比示意图；图3是伊文斯兰染色的相对占比的示意图；图4是伊文斯兰渗出量占半球重量的相对百分比的示意图；图5是免疫荧光双重染色观察内源性IgG从血管中的渗出情况的示意图；图6是IgG从血管内渗出的相对密度柱状示意图；图7是神经行为学评分柱状示意图；图8免疫荧光双重染色观察神经元凋亡情况示意图；图9是小鼠梗死区神经元凋亡数量相对密度柱状图；图10是免疫荧光双重染色观察activeβ-catenin在内皮细胞上的表达情况示意图；图11是activeβ-catenin在内皮细胞上表达的相对密度柱状图；图12是各组梗死区脑组织active和totalβ-catenin蛋白水平示意图；图13是图12中免疫印迹法相对密度柱状图；图14是图12中免疫印迹法相对密度又一柱状图；图15是各组脑梗死组织中axin2水平柱状图；图16是各组脑梗死组织中apcdd1 mRNA水平柱状图；图17是不同浓度氯化锂对于bEnd.3 Axin2 mRNA水平的影响示意图；图18是不同浓度氯化锂对于bEnd.3 TCF/LEF TOPflash值的影响示意图；图19是通过敲除Fzd4构建了Wnt/β-catenin通路缺陷的bEnd.3细胞系对应的氯化锂的效果示意图；图20是通过敲除Fzd4构建了Wnt/β-catenin通路缺陷的bEnd.3细胞系对应的氯化锂的又一效果示意图；图21是各组axin2 mRNA水平柱状图。
其中，图1显示出了采用TTC对小鼠大脑的冠状切片进行染色后的缺血区，该白色区域即为该缺血区；图2显示出了梗死体积在脑半球内的相对占比(％)，n＝8/组；图3显示出了伊文斯兰的染色区(蓝色区域为伊文斯兰在脑组织内的渗漏区域)；图4对应为伊文斯兰渗出量占半球重量的相对百分比(％)，n＝6/组；图5显示了出免疫荧光双重染色观察内源性IgG(绿色)从血管(CD31，红色)中的渗出情况，对应标尺为100um；图6显示出了IgG从血管内渗出的相对密度柱状图，n＝3/组；图7显示出了神经行为学评分柱状图，n＝8/组。Data are mean(平均数据)±s.e.m.,*，P<0.05，**，P<0.01。综合图1-图7能够看出，氯化锂的使用能够减少小鼠脑梗死体积和BBB渗漏，且能够改善神经功能缺损症状。
其中，图8显示出了免疫荧光双重染色观察神经元(红色)凋亡情况(TUNEL染色，绿色)，标尺100um；图9显示出了梗死区神经元凋亡数量相对密度柱状图，n＝3/组。Data are mean±s.e.m.，*，P<0.05。综合图8-图9能够看出，氯化锂的使用减少了脑梗死组织的神经细胞凋亡。
其中，图10显示出了免疫荧光双重染色观察activeβ-catenin(绿色)在内皮细胞(红色)上的表达情况，标尺100um；图11显示出了activeβ-catenin在内皮细胞上表达的相对密度柱状图，n＝3/组；图12显示出了各组梗死区脑组织active和totalβ-catenin蛋白水平；图13和图14显示出了“C”图中免疫印迹法相对密度柱状图，n＝3/组；图15和图16显示出了各组脑梗死组织中axin2和apcdd1 mRNA水平柱状图，n＝3/组。缺血/再灌注：I/R，ischemia/reperfusion.n＝8/组。Data are mean±s.e.m.，*，P<0.05,**，P<0.01。综合图10-图16能够看出，氯化锂的使用能够减少小鼠脑梗死体积和BBB渗漏，且能够改善神经功能缺损症状。
其中，图17显示出了不同浓度氯化锂对于bEnd.3 Axin2 mRNA水平的影响，n＝4/组；图18显示出了不同浓度氯化锂对于bEnd.3 TCF/LEF TOPflash值的影响，n＝5/组；图19和图20显示出了通过敲除Fzd4构建了Wnt/β-catenin通路缺陷的bEnd.3细胞系用来观察氯化锂的效果，发现氯化锂能够显著提高WT和Fzd4-/-bEnd.3细胞系OGD/R后降低的细胞电阻值，并降低FITC-dextran渗漏率，n＝3/组；图21显示出了各组axin2 mRNA水平柱状图，n＝3/组。Data are mean±s.e.m.,*P<0.05，**P<0.01.糖氧剥夺/恢复：OGD/R,oxygen-glucose deprivation and recovery；正常对照：NC，normal control；野生型：WT，wild type；内皮细胞穿膜电阻：TEER,trans-endothelial electrical resistance。综合图17-图21能够看出，锂剂的使用能够上调小鼠脑微血管内皮细胞(bEnd.3)糖氧剥夺/恢复(模拟缺血再灌注损伤)后Wnt/β-catenin通路，并减轻内皮细胞损害。
实验发现：1)在使用的氯化锂的给药量为1.5、3.0和6.0mmol/kg(毫摩尔/千克)三个剂量，以皮下注射，血管开通后即刻给药，1次/日时，均观察到了小鼠脑梗死体积减少，同时也观察到了梗死区神经细胞凋亡的减少；2)在观察使用3.0mmol/kg浓度氯化锂对于BBB功能的保护作用时，发现梗死半球BBB功能示踪剂伊文斯兰(Evans blue)和小鼠血清IgG蛋白渗漏明显减少；3)通过行为学测定，发现锂剂能够改善小鼠脑梗死早期的神经行为学缺损。
通过小鼠脑组织的分子生物学实验发现，Wnt/β-catenin通路的标志物active-β-catenin在脑血管内皮细胞上表达增加，同时其在脑组织中的蛋白水平在锂剂干预后也显著增高。此外，梗死脑组织内Wnt/β-catenin通路的标志性基因apcdd1在锂剂干预之后也显著增高。在脑血管内皮细胞的体外实验中发现，锂剂能够显著上调Wnt/β-catenin通路。同时，在体外糖氧剥夺/恢复模型中(模拟 体内的缺血再灌注损伤)，与无锂剂处理的对照细胞相比，锂剂处理可增强单层脑血管内皮细胞完整性和BBB功能。
且锂剂具有BBB和神经细胞保护双重作用；锂剂的BBB保护作用是通过上调脑血管内皮细胞Wnt/β-catenin通路和保护BBB的内皮细胞间紧密连接蛋白的结构实现的。同时，实验证实锂剂对于BBB的保护作用主要源于对于脑血管内皮细胞之间的紧密连接的保护。
此外，由于锂剂是临床常用药物，其药代动力学和毒副作用清晰，成本低廉，因此有望直接应用于临床脑梗死病人。
其中，锂剂不仅有BBB保护作用，同时也有神经保护作用，与其他靶向干预Wnt/β-catenin通路药物相比，锂剂具有更好的安全性。
其中，锂剂同样可应用与其他可导致BBB损害的疾病，包括脑出血、中毒性脑病、代谢性脑病、中枢神经系统白血病继发脑出血等。
综上所述，本申请中的医药组合物采用锂剂能够上调脑血管内皮细胞Wnt/β-catenin信号通路，并对血脑屏障的内皮细胞的紧密连接蛋白的结构的保护有着重要的作用，从而能够对血脑屏障进行保护。因此，锂剂能够在治疗缺血性脑卒中及其所导致的血脑屏障损伤起到有效的药物作用。
以上所述仅为本申请的实施方式，并非因此限制本申请的专利范围，凡是利用本申请说明书及附图内容所作的等效结构或等效流程变换，或直接或间接运用在其他相关的技术领域，均同理包括在本申请的专利保护范围内。

Claims (12)

  1. 一种治疗缺血性脑卒中的医药组合物，其特征在于，所述医药组合物包括：治疗缺血性脑卒中有效剂量的锂剂和医药上可接受的载体，其中，所述锂剂用于上调脑血管内皮细胞Wnt/β-catenin信号通路，并对血脑屏障的内皮细胞的紧密连接蛋白的结构进行保护。
  2. 根据权利要求1所述的医药组合物，其特征在于，所述锂剂为氯化锂和/或碳酸锂。
  3. 根据权利要求1或2所述的医药组合物，其特征在于，所述医药组合物呈剂量单位形式。
  4. 根据权利要求3所述的医药组合物，其特征在于，所述医药组合物中的所述锂剂的剂量为1.5-6.0毫摩尔/千克中的一种，其中，毫摩尔/千克为所述锂剂的物质的量相对于患者的质量。
  5. 根据权利要求4所述的医药组合物，其特征在于，所述医药组合物中的所述锂剂的剂量为3.0毫摩尔/千克。
  6. 根据权利要求1所述的医药组合物，其特征在于，所述医药组合物还包括治疗缺血性脑卒中有效剂量的阿替普酶，且所述医药组合物中的所述阿替普酶的剂量为0.9毫克/千克，其中，毫克/千克为所述阿替普酶的质量相对于患者的质量。
  7. 一种锂剂在治疗缺血性脑卒中药物的制备中的用途，其特征在于，所述锂剂用于上调脑血管内皮细胞Wnt/β-catenin信号通路，并对血脑屏障的内皮细胞的紧密连接蛋白的结构进行保护。
  8. 根据权利要求7所述的用途，其特征在于，所述锂剂为氯化锂和/或碳酸锂。
  9. 一种治疗血脑屏障损伤的医药组合物，其特征在于，所述医药组合物包括：治疗血脑屏障损伤的有效剂量的锂剂和医药上可接受的载体，其中，所述锂剂用于上调脑血管内皮细胞Wnt/β-catenin信号通路，并对血脑屏障的内皮细胞的紧密连接蛋白的结构进行保护。
  10. 一种锂剂在治疗血脑屏障损伤的药物的制备中的用途，其特征在于，所述锂剂用于上调脑血管内皮细胞Wnt/β-catenin信号通路，并对血脑屏障的内皮细胞的紧密连接蛋白的结构进行保护。
  11. 一种治疗血脑疾病的医药组合物，其特征在于，所述医药组合物包括：治疗血脑疾病的有效剂量的锂剂和医药上可接受的载体，其中，所述锂剂用于上调脑血管内皮细胞Wnt/β-catenin信号通路，并对血脑屏障的内皮细胞的紧密连接蛋白的结构进行保护。
  12. 一种锂剂在治疗血脑疾病的药物的制备中的用途，其特征在于，所述锂剂用于上调脑血管内皮细胞Wnt/β-catenin信号通路，并对血脑屏障的内皮细胞的紧密连接蛋白的结构进行保护。
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