WO2021170073A1 - 小分子化合物在治疗肺上皮细胞损伤和/或血管内皮细胞损伤介导的疾病中的用途 - Google Patents

小分子化合物在治疗肺上皮细胞损伤和/或血管内皮细胞损伤介导的疾病中的用途 Download PDF

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WO2021170073A1
WO2021170073A1 PCT/CN2021/078061 CN2021078061W WO2021170073A1 WO 2021170073 A1 WO2021170073 A1 WO 2021170073A1 CN 2021078061 W CN2021078061 W CN 2021078061W WO 2021170073 A1 WO2021170073 A1 WO 2021170073A1
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lung
damage
injury
cells
acute
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PCT/CN2021/078061
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English (en)
French (fr)
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陆秉政
陈玉嫔
王亚娜
黄家瑜
黄春晖
陈婕思
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广州市赛普特医药科技股份有限公司
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Priority to US17/802,870 priority Critical patent/US20230089123A1/en
Priority to EP21760885.0A priority patent/EP4129299A4/en
Priority to JP2022551743A priority patent/JP7464737B2/ja
Publication of WO2021170073A1 publication Critical patent/WO2021170073A1/zh

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    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/565—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol
    • A61K31/568—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol substituted in positions 10 and 13 by a chain having at least one carbon atom, e.g. androstanes, e.g. testosterone
    • A—HUMAN NECESSITIES
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    • A—HUMAN NECESSITIES
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
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    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • 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
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives

Definitions

  • the present invention relates to the use of small molecule compounds in the treatment of diseases mediated by lung epithelial cell damage and/or vascular endothelial cell damage, and specifically relates to 5 ⁇ -androsta-3 ⁇ ,5,6 ⁇ -triol (herein sometimes referred to as "YC -6" or “YC6”) and its analogues, especially the application of these compounds in the treatment of lung injury and cerebral small vessel disease.
  • YC -6 5 ⁇ -androsta-3 ⁇ ,5,6 ⁇ -triol
  • Lung injury is common in lung diseases, especially in diseases such as acute lung injury/acute respiratory distress syndrome (ALI/ARDS), pulmonary hypertension, sepsis, etc., which seriously endangers health, and some have a high mortality rate.
  • ALI/ARDS acute lung injury/acute respiratory distress syndrome
  • pulmonary hypertension pulmonary hypertension
  • sepsis etc.
  • Acute lung injury refers to a variety of non-cardiogenic serious internal and external pathogenic factors (such as viral infection, bacterial infection, trauma, shock, ischemia reperfusion, acute pancreatitis, inhalation).
  • Acute hypoxic respiratory insufficiency or failure caused by sexual injury, diffuse alveolar injury, poisoning caused by other toxins, etc., its pathological features are pulmonary edema and alveoli caused by increased permeability of alveolar epithelium and pulmonary capillary endothelium Collapse and expansion, alveolar wall thickening and inflammatory cell infiltration, etc. (Butt et al., 2016).
  • ALI acute respiratory distress syndrome
  • ARDS acute respiratory distress syndrome
  • the mortality rate of ALI/ARDS can reach 40% to 60%, and there is still no specific drug therapy.
  • Alveoli is the basic unit for gas exchange in the lung, and its inner surface is covered with type I and type II alveolar epithelial cells.
  • Type I squamous cells make up 90% of the alveolar surface area and are easily injured.
  • Type II three-dimensional cells constitute the remaining 10% of the alveolar surface area and are not easily damaged. Their functions include surfactant production, ion transport, and proliferation and differentiation of type I cells after injury.
  • Alveolar epithelium not only forms a dense barrier to isolate foreign pathogens, but also maintains lung homeostasis and relative sterility through the interaction of its surface receptors and secreted products with immune cells. Among them, alveolar epithelial cells play a leading role in lung homeostasis, and are directly related to lung-related diseases such as acute lung injury, lung fibrosis, and tissue reconstruction diseases.
  • Pulmonary vascular endothelial cells form a monolayer lining the vasculature. Because their physiological locations are exposed to multiple injury factors, such as LPS, endotoxin, TNF- ⁇ and oxidative stress, pulmonary vascular endothelial cells are a variety of lung injury factors Attacked target cells play an important role in the pathogenesis of ALI/ARDS. Studies have shown that bronchoalveolar lavage fluid from patients at risk or with early and late ARDS is cytotoxic to human lung microvascular endothelial cells.
  • LPS bacterial endotoxin
  • cytokines cytokines
  • oxygen free radicals etc.
  • secretion and release are different
  • a variety of inflammatory mediators and cytokines make the pro-inflammatory and anti-inflammatory mediators imbalance, and the coagulation and anticoagulation systems are imbalanced, causing pulmonary microcirculation disorders and pulmonary hypertension, which can promote pulmonary interstitial edema, pulmonary hemorrhage and progressive breathing difficulties, leading to The patient developed progressive hypoxemia and respiratory distress (Du Jingxia et al., 2012).
  • Screening drugs that can alleviate pulmonary vascular endothelial cell damage caused by unfavorable factors is expected to be used in the prevention and treatment of various lung injury-related diseases, including various infectious pneumonia, acute lung injury, ARDS, pulmonary hypertension, etc.
  • Phosphofructokinase-2/fructose-2,6-bisphosphatase3 is a key regulator of glycolysis pathway in the sugar metabolism pathway.
  • PFKFB3 plays a key role in the process of lung injury.
  • Glycolytic enzyme PFKFB3 inhibitors can improve the survival rate, lung inflammation, increase in lactic acid, and lung cell apoptosis in ALI mice induced by cecal ligation and puncture (CLP) (Gong Y et al, 2017). Anaerobic glycolysis is also an important factor in sepsis-related ALI cell apoptosis.
  • PFKFB3 inhibitors can significantly reduce LPS-induced acute lung injury/acute respiratory distress syndrome (ALI/ARDS) lung injury in experimental animals (Wang L et al, 2019).
  • ALI/ARDS acute lung injury/acute respiratory distress syndrome
  • PFKFB3 is specifically knocked out in vascular endothelial cells, it significantly reduces the glycolysis level of endothelial cells, reduces the expression of growth factors, pro-inflammatory cytokines, and cell adhesion factors, thereby inhibiting the abnormal proliferation of pulmonary vascular smooth muscle cells and the lungs.
  • the infiltration of perivascular inflammatory cells inhibits the development of hypoxia-induced pulmonary hypertension (Cao Y, 2019).
  • the blood-brain barrier (Blood-Brain Barrier, BBB) is the cell interface between the central nervous system and the circulatory system, and is a kind of brain barrier.
  • the structure of the blood-brain barrier includes vascular endothelial cells, pericytes, astrocytes, foot processes, basement membranes, etc., and together with neurons constitute neurovascular units.
  • Vascular endothelial cells constitute the anatomical basis of the blood-brain barrier, allowing various selective transport systems to transport nutrients and other substances into and out of the brain, ensuring the low permeability of the intercellular space to hydrophilic solutes.
  • vascular endothelial cell damage and endothelial tissue dysfunction can increase the permeability of the blood-brain barrier, causing blood components to enter the potential perivascular space and brain substance, causing damage to nerve cells and glial cells. It has been reported that the increase in permeability of the blood-brain barrier precedes the appearance of nerve damage and clinical symptoms.
  • the inventors unexpectedly discovered that the compound 5 ⁇ -androsta-3 ⁇ ,5,6 ⁇ -triol can significantly inhibit the up-regulation of PFKFB3 expression, significantly inhibit the accumulation of lactic acid, reduce vascular endothelial cell damage, reduce alveolar epithelial cell damage, inhibit alveolar septal thickening, and reduce Alveolar injury and pneumoniae cell infiltration can be used to treat various diseases mediated by alveolar epithelial cell injury and/or vascular endothelial cell injury.
  • one aspect of the present invention provides the use of the compound of formula I, its deuterated product or pharmaceutically acceptable salt in the preparation of a medicament for the prevention or treatment of diseases mediated by damage to lung epithelial cells and/or vascular endothelial cells:
  • R 1 is selected from H, -CN, fluorine, chlorine, C 1-10 alkyl, fluorine or chlorine substituted C 1-10 alkyl, C 1-10 alkoxy, fluorine or chlorine substituted C 1-10 Alkoxy and C 3-10 cycloalkyl.
  • the R 1 is H, -CHCH 2 CH 3 , -CH(CH 3 ) 2 , -CH(CH 2 ) 3 CH 3, or -CH(CH 3 )(CH 2 ) 3 CH( CH 3 ) 2 .
  • said R 1 is H.
  • the present invention provides any of the above-mentioned compounds, their deuterated products or pharmaceutically acceptable salts for the prevention or treatment of diseases mediated by lung epithelial cell damage and/or vascular endothelial cell damage.
  • the present invention provides a method for preventing or treating diseases mediated by damage to lung epithelial cells and/or vascular endothelial cells, the method comprising administering to a subject in need thereof a prophylactically or therapeutically effective amount of Any of the above-mentioned compounds, deuterated products or pharmaceutically acceptable salts thereof.
  • the disease mediated by damage to lung epithelial cells and/or vascular endothelial cells is acute lung injury, acute respiratory distress syndrome, pulmonary hypertension, pulmonary edema , Pulmonary fibrosis, chronic lung disease of premature infants, chronic obstructive pulmonary disease, pneumocystis disease and pulmonary embolism.
  • the acute lung injury, acute respiratory distress syndrome, pulmonary hypertension, or pulmonary edema is caused by hyperoxia, viral infection, bacterial infection, trauma, shock, ischemia reperfusion, acute pancreatitis, inhalation injury , Diffuse alveolar injury and/or poisoning.
  • the acute lung injury, acute respiratory distress syndrome, pulmonary hypertension, or pulmonary edema is caused by hyperoxia, viral infection, bacterial infection, trauma, shock, ischemia reperfusion, acute pancreatitis, inhalation injury , Diffuse alveolar damage and/or poisoning, and not caused by hypoxia (such as hypoxia in high altitude environment).
  • the virus is a coronavirus (for example, a novel coronavirus SARS-CoV-2), influenza virus, respiratory syncytial virus, adenovirus, parainfluenza virus, measles virus, cytomegalovirus, or a combination thereof.
  • the virus is a coronavirus (for example, a novel coronavirus SARS-CoV-2).
  • the acute lung injury is lung injury caused by surgery, such as sublobectomy, lobectomy, or pneumonectomy, such as pneumonectomy, lung tumor resection, or lung transplantation.
  • the pulmonary fibrosis is idiopathic pulmonary fibrosis or pneumoconiosis.
  • the disease mediated by damage to vascular endothelial cells includes cerebral small vessel disease mediated by the destruction of the blood-brain barrier, but does not include cerebral microhemorrhage, cerebral microhemorrhage, and cerebral microhemorrhage. Stroke and cerebral edema.
  • the destruction of the blood-brain barrier is manifested as increased permeability of the blood-brain barrier. In some embodiments, the destruction of the blood-brain barrier is manifested as damage to vascular endothelial cells of the blood-brain barrier.
  • the clinical manifestations of cerebrovascular disease mediated by the destruction of the blood-brain barrier are cognitive impairment, gait disorder, mood disorder, urinary incontinence, and/or depression.
  • the imaging manifestations of cerebral small vessel disease mediated by the destruction of the blood-brain barrier include white matter lesions. In some embodiments, the imaging manifestations of cerebral small vessel disease mediated by the destruction of the blood-brain barrier are only white matter lesions.
  • the disease mediated by damage to lung epithelial cells and/or vascular endothelial cells is cardiovascular disease or diabetic vascular complications.
  • the cardiovascular disease is selected from one or more of acute myocardial infarction (AMI), angina pectoris, coronary heart disease, ischemic heart disease, heart failure, hypertension, and cardiovascular intervention thrombosis .
  • the diabetic vascular complication is one or more of diabetic retinopathy, diabetic nephropathy, and impaired diabetic wound healing.
  • FIG. 1 YC-6 significantly inhibited the increase in PFKFB3 expression caused by glutamate. Stimulate primary cultured neurons with 200 ⁇ M glutamate for 5mins and 15mins, 10 ⁇ M YC-6, corresponding solvent HP- ⁇ -CD and NMDA receptor blocker 10 ⁇ M MK-801 pre-incubation for 20mins as the experimental group, solvent control group and Positive control drug group. After collecting the protein, PFKFB3 immunoblotting analysis was performed.
  • YC-6 significantly inhibits the intracellular accumulation of lactic acid, the end product of glycolysis downstream of PFKFB3, caused by glutamate.
  • the primary cultured neurons were stimulated with 200 ⁇ M glutamate for 15mins, and 10 ⁇ M YC-6 and the corresponding solvent HP- ⁇ -CD were pre-incubated for 20mins as the experimental group, solvent control group and positive control drug group. After the cells were collected, the intracellular lactate content was determined.
  • YC-6 significantly reduces the damage of vascular endothelial cells caused by OGD-R.
  • Human umbilical vein endothelial cells (HUVEC) and rat vascular endothelial cells (RAOEC) were treated with oxygen and glucose deprivation for 4 hours, and then reoxygenated to normal culture conditions for 24 hours, then LDH detection and analysis were performed.
  • YC-6 prophylactically administered cells are pre-applied with solvents or drugs (final concentrations of 1 ⁇ M, 3 ⁇ M, and 10 ⁇ M) for 1 hour before oxygen-glucose deprivation treatment.
  • solvents are given at the same time as reoxygenation treatment.
  • YC-6 final concentration is 1 ⁇ M, 3 ⁇ M, 10 ⁇ M).
  • YC-6 significantly reduces the damage of human alveolar epithelial cells caused by hypoxia and LPS.
  • the final concentration of pre-incubation YC-6 was 1 ⁇ M, 5 ⁇ M, 10 ⁇ M. After 1 hour, 10 ⁇ M dexamethasone was used as the drug control, and the corresponding volume of 20% hydroxypropyl cyclodextrin was used as the solvent control. Then the cells were subjected to 20 ⁇ g/mL LPS and 1% O 2 Hypoxia treatment for 24 hours.
  • FIG. 1 Pathological effects of YC-6 in protecting LPS from acute lung injury.
  • Red arrows indicate alveolar and pulmonary interstitial edema
  • blue arrows indicate loose lesions around blood vessels
  • yellow arrows indicate inflammatory cell infiltration
  • green arrows indicate bronchial mucosal epithelial injury and shedding
  • orange arrows indicate alveolar congestion.
  • the black scale indicates 100 ⁇ m.
  • Figure 7 The process of making a model of cynomolgus monkey lung injury caused by acute hypobaric hypoxia. At 320 meters (A), 3000 meters (B), 4500 meters (C), 6000 meters (D), 7500 meters for 24 hours (E) and 7500 meters for 48 hours (F), after hypoxia and drug treatment, the final collection A sample of the lungs of a cynomolgus monkey. The syringe shows the time point of administration.
  • FIG. 8 YC-6 inhibits pulmonary vascular congestion and alveolar septal thickening caused by acute hypobaric hypoxia in cynomolgus monkeys.
  • Normal pressure normoxia group (Normobaric normoxia, NN); acute hypobaric hypoxia group (Hypobaric hypoxia, HH); YC-6 administration group (HH+YC-6).
  • the red arrow indicates the blood vessel section with congestion and swelling of the lung tissue.
  • the second row of pictures is an enlarged view of the blue frame area of the first row of pictures, and the second row of red frame is a further enlarged view of the black frame area.
  • the black scale indicates 500 ⁇ m, and the red scale indicates 100 ⁇ m.
  • Figure 9 Statistical analysis of YC-6 significantly inhibited the thickening of alveolar septum in cynomolgus monkeys caused by acute hypobaric hypoxia.
  • NN normobaric normoxia
  • HH Hapobaric hypoxia
  • YC-6 inhibits the formation of alveolar hyaline membrane and red blood cell leakage in cynomolgus monkeys caused by acute hypobaric hypoxia.
  • NN Normal pressure normoxia group
  • HH Hapobaric hypoxia
  • HH+YC-6 YC-6 administration group.
  • the red arrow indicates the transparent protein membrane in the alveolar cavity
  • the black arrow indicates the fibrous hyperplasia of the alveolar septum
  • the blue arrow indicates the red blood cell in the alveolar cavity.
  • the black scale indicates 100 ⁇ m.
  • YC-6 inhibits inflammatory infiltration of cynomolgus monkey lung tissue caused by acute hypobaric hypoxia.
  • NN Normal pressure normoxia group
  • HH Hapobaric hypoxia
  • HH+YC-6 YC-6 administration group.
  • the black arrow indicates the inflammatory cell infiltration in the interstitium of the alveolar membrane
  • the red arrow indicates the inflammatory cell infiltration in the alveolar cavity
  • the blue arrow indicates the exfoliated lung epithelial cells.
  • the black scale indicates 100 ⁇ m.
  • YC-6 up-regulates the expression of NR4A3 protein in vascular endothelial cells and reduces cell damage under oxygen and glucose deprivation-reoxygenation/hypoxia stimulation.
  • (b) is a representative picture of NR4A3 immunofluorescence staining of HUVEC and RAOEC cells under the same treatment in a picture. The scale bar indicates 50 ⁇ m.
  • (c) is the quantification of the average fluorescence intensity of NR4A3 processed in (b).
  • (d) NR4A3/CD31 fluorescent double-stained representative pictures of non-human primate cynomolgus monkey lung tissue under normal environment (normal pressure and normal oxygen), hypobaric hypoxia and hypobaric hypoxia + YC-6. The white ruler indicates 100 ⁇ m.
  • (e) is the quantification of the average fluorescence intensity of NR4A3 and the average fluorescence intensity of CD31 co-localized with the CD31 signal processed in the figure (d). The number of samples for normal, hypobaric hypoxia, and hypobaric hypoxia+YC-6 were 3, 5, and 5, respectively.
  • YC-6 inhibits the ubiquitination and degradation of vascular endothelial cells NR4A3 caused by oxygen glucose deprivation-reoxygenation.
  • (a) Real-time fluorescent quantitative PCR to detect the relative expression of NR4A3mRNA in HUVEC and RAOEC cells under normal culture, oxygen glucose deprivation, oxygen glucose deprivation-reoxygenation and oxygen glucose deprivation-reoxygenation+YC-6 treatment.
  • Solvent or 10 ⁇ M YC-6 is given to the cells at the same time as the reoxygenation treatment.
  • composition refers to a formulation suitable for administration to an intended animal subject for therapeutic purposes, which contains at least one pharmaceutically active ingredient, such as a compound.
  • the composition further contains at least one pharmaceutically acceptable carrier or excipient.
  • pharmaceutically acceptable means that the substance does not have the property that, taking into account the disease or condition to be treated and the respective route of administration, this property will enable rational and prudent medical practitioners to avoid administering the substance to patients substance. For example, for injectables, it is generally required that such materials be substantially sterile.
  • prophylactically effective amount and “therapeutically effective amount” mean that the substance and the amount of the substance are effective in preventing, reducing or ameliorating one or more symptoms of a disease or disorder, and/or prolonging the treatment of the subject. Survival is effective.
  • Treatment includes administering a compound of the application or a pharmaceutically acceptable salt thereof to reduce the symptoms or complications of the disease or disorder, or to eliminate the disease or disorder.
  • the term "alleviation” as used herein is used to describe the process of reducing the severity of the signs or symptoms of a disorder. Symptoms can be alleviated but not eliminated. In one embodiment, administration of the pharmaceutical composition of the application results in the elimination of signs or symptoms.
  • prevention includes administering the compound of the present application or a pharmaceutically acceptable salt thereof to prevent or tissue from producing a specific disease, symptom, or complication.
  • C 1-10 or “C 3-10 " or similar expressions as used herein refers to having 1 to 10 or 3 to 10 carbon atoms.
  • a C 1-10 alkyl group refers to an alkyl group having 1 to 10 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, decyl and the like.
  • diseases mediated by damage to lung epithelial cells and/or vascular endothelial cells includes diseases mediated by damage to lung epithelial cells, diseases mediated by damage to vascular endothelial cells, and diseases mediated by lung epithelial cells and vascular endothelial cells. Damage-mediated diseases.
  • R 1 is selected from H, -CN, fluorine, chlorine, C 1-10 alkyl, fluorine or chlorine substituted C 1-10 alkyl, C 1-10 alkoxy, fluorine or chlorine substituted C 1-10 Alkoxy and C 3-10 cycloalkyl.
  • the compound of formula I, its deuterated product, or a pharmaceutically acceptable salt thereof is also referred to herein as "the compound of the present invention” or "the compound”.
  • R 1 is H
  • the compound is 5 ⁇ -androsta-3 ⁇ ,5,6 ⁇ -triol (abbreviated as "YC-6" or "YC6")
  • its structural formula is as formula (II ) Shown.
  • R 1 is -CHCH 2 CH 3 and the compound is 17-propylene-androst-3 ⁇ ,5 ⁇ ,6 ⁇ -triol. In one embodiment, R 1 is -CH(CH 3 ) 2 and the compound is 17-isopropyl-androst-3 ⁇ ,5 ⁇ ,6 ⁇ -triol. In one embodiment, R 1 is -CH(CH 2 ) 3 CH 3 and the compound is 17-butyl-androst-3 ⁇ ,5 ⁇ ,6 ⁇ -triol. In one embodiment, R 1 is -CH(CH 3 )(CH 2 ) 3 CH(CH 3 ) 2 , and the compound is cholestane-3 ⁇ ,5 ⁇ ,6 ⁇ -triol.
  • the compounds of the present invention can be formulated in the form of pharmaceutically acceptable salts.
  • Expected pharmaceutically acceptable salt forms include, but are not limited to, mono-salt, double-salt, tri-salt, tetra-salt and the like.
  • the pharmaceutically acceptable salts are non-toxic at the amount and concentration at which they are administered. Without preventing it from exerting its physiological effects, by changing the physical properties of the compound, the preparation of such a salt can facilitate pharmacological applications. Useful changes in physical properties include lowering the melting point for transmucosal administration, and increasing solubility for administration of higher concentrations of drugs.
  • Pharmaceutically acceptable salts include acid addition salts, such as those containing sulfate, chloride, hydrochloride, fumarate, maleate, phosphate, sulfamate, acetate, citrate, milk Salts of acid salt, tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, cyclamate, and quinic acid salt.
  • acid addition salts such as those containing sulfate, chloride, hydrochloride, fumarate, maleate, phosphate, sulfamate, acetate, citrate, milk Salts of acid salt, tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, cyclamate, and quinic acid salt.
  • Pharmaceutically acceptable salts can be obtained from acids such as hydrochloric acid, maleic acid, sulfuric acid, phosphoric acid, sulfamic acid, acetic acid, citric acid, lactic acid, tartaric acid, malonic acid, methanesulfonic acid, ethanesulfonic acid, Benzenesulfonic acid, p-toluenesulfonic acid, cyclamic acid, fumaric acid and quinic acid.
  • acids such as hydrochloric acid, maleic acid, sulfuric acid, phosphoric acid, sulfamic acid, acetic acid, citric acid, lactic acid, tartaric acid, malonic acid, methanesulfonic acid, ethanesulfonic acid, Benzenesulfonic acid, p-toluenesulfonic acid, cyclamic acid, fumaric acid and quinic acid.
  • pharmaceutically acceptable salts also include base addition salts, such as those containing benzathine penicillin, chloroprocaine, choline, diethanolamine, ethanolamine, tert-butylamine, ethylenediamine , Meglumine, procaine, aluminum, calcium, lithium, magnesium, potassium, sodium, ammonium, alkylamine and zinc salts.
  • base addition salts such as those containing benzathine penicillin, chloroprocaine, choline, diethanolamine, ethanolamine, tert-butylamine, ethylenediamine , Meglumine, procaine, aluminum, calcium, lithium, magnesium, potassium, sodium, ammonium, alkylamine and zinc salts.
  • bases such as those containing benzathine penicillin, chloroprocaine, choline, diethanolamine, ethanolamine, tert-butylamine, ethylenediamine , Meglumine, procaine, aluminum, calcium, lithium, magnesium, potassium, sodium, ammonium, alky
  • salts can be prepared.
  • the free base form of the compound is dissolved in a suitable solvent, such as an aqueous solution or a water-alcohol solution containing a suitable acid, and then the solution is evaporated for separation.
  • a suitable solvent such as an aqueous solution or a water-alcohol solution containing a suitable acid
  • the salt is prepared by reacting the free base and acid in an organic solvent.
  • the desired pharmaceutically acceptable salt can be prepared by any suitable method available in the art, for example, treating the free base with an inorganic acid or an organic acid, such as hydrochloric acid, Hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and similar acids, such as acetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid , Pyranosidyl acid (such as glucuronic acid or galacturonic acid), ⁇ -hydroxy acid (such as citric acid or tartaric acid), amino acid (such as aspartic acid or glutamic acid), aromatic acid ( Such as benzoic acid or cinnamic acid), sulfonic acid (such as p-toluenesulfonic acid or ethanesulfonic acid) or the like.
  • an inorganic acid or an organic acid such as hydrochloric acid, Hydrobromic
  • the desired pharmaceutically acceptable salt can be prepared by any suitable method, for example, treating the free acid with an inorganic or organic base, such as an amine (primary amine). , Secondary or tertiary amine), alkali metal hydroxide or alkaline earth metal hydroxide or the like.
  • an inorganic or organic base such as an amine (primary amine). , Secondary or tertiary amine), alkali metal hydroxide or alkaline earth metal hydroxide or the like.
  • suitable salts include organic salts, which are derived from amino acids (such as L-glycine, L-lysine, and L-arginine), ammonia, primary, secondary, and tertiary amines, and cyclic amines (such as Hydroxyethylpyrrolidine, piperidine, morpholine and piperazine), and inorganic salts derived from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum and lithium.
  • amino acids such as L-glycine, L-lysine, and L-arginine
  • ammonia such as L-glycine, L-lysine, and L-arginine
  • primary, secondary, and tertiary amines such as Hydroxyethylpyrrolidine, piperidine, morpholine and piperazine
  • inorganic salts derived from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum and lithium.
  • the pharmaceutically acceptable salt of the compound may exist as a complex.
  • complexes include 8-chlorotheophylline complexes (for example, dimenhydrinate: diphenhydramine 8-chlorotheophylline (1:1) complex; diphenhydramine) and various cyclodextrin-containing compounds The complex.
  • the present invention is also expected to include pharmaceutically acceptable deuterated compounds or other non-radioactive substituted compounds using the compound.
  • Deuteration is the replacement of one or more or all of the hydrogen in the active molecular group of a drug with the isotope deuterium. Because it is non-toxic and non-radioactive, it is about 6 to 9 times more stable than carbon-hydrogen bonds, and can block metabolic sites and extend the drug. The half-life of the drug is considered to be an excellent modification method, thereby reducing the therapeutic dose without affecting the pharmacological activity of the drug.
  • Another aspect of the present invention provides a pharmaceutical composition, which comprises an effective amount of a compound of formula I, a deuterated product or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
  • composition refers to a composition comprising a compound of formula I and a pharmaceutically acceptable carrier, wherein the compound and the pharmaceutically acceptable carrier are present in the composition in a mixed form.
  • the composition will generally be used in the treatment of human subjects. However, they can also be used to treat similar or identical conditions in other animal subjects.
  • subject refers to human and non-human vertebrates, such as mammals, such as non-human primates, sports animals, and commercial animals, such as horses, cows, pigs, sheep, Rodents, and pets (such as dogs and cats).
  • the appropriate dosage form depends in part on the use or route of administration, such as oral, transdermal, transmucosal, inhalation or by injection (parenteral). Such dosage forms should enable the compound to reach target cells. Other factors are well known in the art and include considerations such as toxicity and dosage forms that delay the compound or composition from exerting its effects.
  • Carriers or excipients can be used to produce the composition.
  • the carrier or excipient may be selected to facilitate the administration of the compound.
  • carriers include calcium carbonate, calcium phosphate, various sugars (such as lactose, glucose, or sucrose), or starch types, cellulose derivatives, gelatin, vegetable oils, polyethylene glycol, and physiologically compatible solvents.
  • physiologically compatible solvents include sterile water for injection (WFI) solutions, saline solutions, and dextrose.
  • compositions or components of the composition can be administered by different routes, including intravenous, intraperitoneal, subcutaneous, intramuscular, oral, transmucosal, rectal, transdermal, or inhalation. In some embodiments, injections or freeze-dried powder injections are preferred.
  • the compound can be formulated into conventional oral dosage forms such as capsules, tablets, and liquid preparations such as syrups, elixirs, and concentrated drops.
  • compositions for oral use can be obtained, for example, by combining the composition or its components with solid excipients, optionally grinding the resulting mixture, and processing the mixture of particles after adding suitable adjuvants (if necessary), thereby Obtain tablets or dragees.
  • suitable excipients are especially fillers, such as sugars, including lactose, sucrose, mannitol or sorbitol; cellulose preparations, such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methyl fiber Vitamins, hydroxypropyl methylcellulose, sodium carboxymethyl cellulose (CMC) and/or polyvinylpyrrolidone (PVP: povidone).
  • disintegrating agents may be added, such as cross-linked polyvinylpyrrolidone, agar, or alginic acid or their salts, such as sodium alginate.
  • injection parenteral administration
  • the composition of the present invention or its components is formulated as a sterile liquid solution, preferably in a physiologically compatible buffer or solution, such as saline solution, Hank's solution or Ringer's solution.
  • the composition or its components may be formulated in a solid form and re-dissolved or suspended immediately before use. It can also be produced in freeze-dried powder form.
  • Administration can also be via transmucosal, topical or transdermal means.
  • penetrants suitable for the barrier to be penetrated are used in the formulation.
  • penetrating agents include, for example, for transmucosal administration, bile salts and fusidic acid derivatives.
  • detergents can be used to promote penetration.
  • Transmucosal administration for example, can be by nasal spray or suppository (transrectally or vaginally).
  • Effective amounts of the various components can be determined by standard procedures to be administered, for example, the considerations compound IC 50, the biological half life of the compound, the age, size and weight of the object-related disorders. The importance of these and other factors is well known to those of ordinary skill in the art. Generally speaking, the dosage will be between approximately 0.01 mg/kg and 50 mg/kg of the subject being treated, preferably between 0.1 mg/kg and 20 mg/kg. Multiple doses can be used.
  • composition of the present invention or its components can also be used in combination with other therapeutic agents for the treatment of the same diseases. Such combined use includes administering these compounds and one or more other therapeutic agents at different times, or using this compound and one or more other therapeutic agents at the same time.
  • the dosage of one or more compounds of the present invention or other therapeutic agents used in combination can be modified, for example, by methods known to those skilled in the art to reduce the dosage relative to the compound or therapeutic agent used alone. dose.
  • the combined use or combination includes use with other therapies, drugs, medical procedures, etc., where the other therapies or procedures can be used at a time different from the composition of the present invention or its components (for example, in a short period of time). (Such as several hours, such as 1, 2, 3, 4-24 hours) or in a longer period of time (such as 1-2 days, 2-4 days, 4-7 days, 1-4 weeks)) (or in It is administered at the same time as the composition of the present invention or its components.
  • Combined use also includes use with a therapy or medical procedure (such as surgery) that is administered once or infrequently, and is accompanied by the composition of the present invention or its components. Administration in a short or longer period of time before or after the other therapy or procedure.
  • the present invention is used to deliver the composition of the present invention or its components and one or more other pharmaceutical therapeutic agents, They are delivered by the same or different routes of administration.
  • the combined administration of any route of administration includes the delivery of the composition of the present invention or its components and one or more other pharmaceutical therapeutic agents together in any preparation form through the same route of administration, including the two compounds being chemically linked and they are in A formulation that maintains the respective therapeutic activity at the time of administration.
  • this other drug therapy can be co-administered with the composition of the invention or its components.
  • the combined use by co-administration includes the administration of co-formulations or formulations of chemically linked compounds, or two administrations within a short period of time (for example, within 1 hour, within 2 hours, within 3 hours, or up to 24 hours).
  • the co-administration of separate formulations includes co-administration of delivery via one device, such as the same inhalation device, the same syringe, etc., or administration by different devices within a short period of time relative to each other.
  • the co-formulation of the compound of the present invention and one or more additional drug therapies delivered by the same route of administration includes preparing the materials together so that they can be administered through one device, including different compounds combined in one formulation, or compound They are modified so that they are chemically linked together but still maintain their respective biological activities.
  • Such chemically linked compounds may include a linker that separates the two active ingredients, which linker is basically maintained in the body or may be degraded in the body.
  • the present invention provides the use of any of the compounds, their deuterated substances or pharmaceutically acceptable salts in the preparation of drugs for the prevention or treatment of diseases mediated by damage to lung epithelial cells and/or vascular endothelial cells .
  • the disease mediated by damage to lung epithelial cells and/or vascular endothelial cells is selected from the group consisting of acute lung injury, acute respiratory distress syndrome, pulmonary hypertension, pulmonary edema, pulmonary fibrosis, chronic lung disease of premature infants One or more of chronic obstructive pulmonary disease, pneumocystis disease and pulmonary embolism.
  • the acute lung injury, acute respiratory distress syndrome, pulmonary hypertension, or pulmonary edema is caused by hyperoxia, viral infection, bacterial infection, trauma, shock, ischemia reperfusion, acute pancreatitis, inhalation injury , Diffuse alveolar injury and/or poisoning.
  • the acute lung injury, acute respiratory distress syndrome, pulmonary hypertension, or pulmonary edema is caused by hyperoxia, viral infection, bacterial infection, trauma, shock, ischemia reperfusion, acute pancreatitis, inhalation injury , Diffuse alveolar damage and/or poisoning, and not caused by hypoxia.
  • the virus is a coronavirus (for example, a novel coronavirus SARS-CoV-2), influenza virus, respiratory syncytial virus, adenovirus, parainfluenza virus, measles virus, cytomegalovirus, or a combination thereof.
  • the virus is a coronavirus (for example, a novel coronavirus SARS-CoV-2).
  • the acute lung injury is lung injury caused by surgery, such as sublobectomy, lobectomy, or pneumonectomy, such as pneumonectomy, lung tumor resection, or lung transplantation. .
  • the disease mediated by damage to lung epithelial cells and/or vascular endothelial cells is lung injury.
  • the disease mediated by damage to lung epithelial cells and/or vascular endothelial cells is acute lung injury.
  • the disease mediated by damage to lung epithelial cells and/or vascular endothelial cells is acute respiratory distress syndrome.
  • the disease mediated by damage to lung epithelial cells and/or vascular endothelial cells is pulmonary hypertension.
  • the disease mediated by damage to vascular endothelial cells includes cerebral small vessel disease mediated by the destruction (or damage) of the blood-brain barrier, but does not include cerebral microhemorrhage, stroke, and cerebral edema.
  • the destruction of the blood-brain barrier is manifested by increased permeability of the blood-brain barrier.
  • the breakdown of the blood-brain barrier is manifested as damage to vascular endothelial cells of the blood-brain barrier.
  • the clinical manifestations of cerebral small vessel disease mediated by the destruction of the blood-brain barrier are cognitive impairment, gait disorder, mood disorder, urinary incontinence, and/or depression.
  • the imaging manifestations of cerebral small vessel disease mediated by the destruction of the blood-brain barrier include white matter lesions. In some embodiments, the imaging manifestations of cerebral small vessel disease mediated by the destruction of the blood-brain barrier are only white matter lesions.
  • the disease mediated by damage to lung epithelial cells and/or vascular endothelial cells is cardiovascular disease or diabetic vascular complications.
  • the cardiovascular disease is selected from one or more of acute myocardial infarction (AMI), angina pectoris, coronary heart disease, ischemic heart disease, heart failure, hypertension, and cardiovascular intervention thrombosis .
  • the diabetic vascular complication is one or more of diabetic retinopathy, diabetic nephropathy, and impaired diabetic wound healing.
  • the present invention provides a method for preventing or treating diseases mediated by damage to lung epithelial cells and/or vascular endothelial cells, the method comprising administering to a subject in need thereof a prophylactically or therapeutically effective amount of this Any compound of the invention, its deuterated substance or pharmaceutically acceptable salt.
  • the disease mediated by damage to lung epithelial cells and/or vascular endothelial cells is selected from the group consisting of acute lung injury, acute respiratory distress syndrome, pulmonary hypertension, pulmonary edema, pulmonary fibrosis, chronic lung disease of premature infants One or more of chronic obstructive pulmonary disease, pneumocystis disease and pulmonary embolism.
  • the acute lung injury, acute respiratory distress syndrome, pulmonary hypertension, or pulmonary edema is caused by hyperoxia, viral infection, bacterial infection, trauma, shock, ischemia reperfusion, acute pancreatitis, inhalation injury , Diffuse alveolar injury and/or poisoning.
  • the acute lung injury, acute respiratory distress syndrome, pulmonary hypertension, or pulmonary edema is caused by hyperoxia, viral infection, bacterial infection, trauma, shock, ischemia reperfusion, acute pancreatitis, inhalation injury , Diffuse alveolar damage and/or poisoning, and not caused by hypoxia.
  • the virus is a coronavirus (for example, a novel coronavirus SARS-CoV-2), influenza virus, respiratory syncytial virus, adenovirus, parainfluenza virus, measles virus, cytomegalovirus, or a combination thereof.
  • the virus is a coronavirus (for example, a novel coronavirus SARS-CoV-2).
  • the acute lung injury is lung injury caused by surgery, such as sublobectomy, lobectomy, or pneumonectomy, such as pneumonectomy, lung tumor resection, or lung transplantation. .
  • the disease mediated by damage to lung epithelial cells and/or vascular endothelial cells is lung injury.
  • the disease mediated by damage to lung epithelial cells and/or vascular endothelial cells is acute lung injury.
  • the disease mediated by damage to lung epithelial cells and/or vascular endothelial cells is acute respiratory distress syndrome.
  • the disease mediated by damage to lung epithelial cells and/or vascular endothelial cells is pulmonary hypertension.
  • the disease mediated by damage to vascular endothelial cells includes cerebral small vessel disease mediated by the destruction (or damage) of the blood-brain barrier, but does not include cerebral microhemorrhage, stroke, and cerebral edema.
  • the destruction of the blood-brain barrier is manifested by increased permeability of the blood-brain barrier.
  • the destruction of the blood-brain barrier is manifested as damage to vascular endothelial cells of the blood-brain barrier.
  • the clinical manifestations of cerebral small vessel disease mediated by the destruction of the blood-brain barrier are cognitive impairment, gait disorder, mood disorder, urinary incontinence, and/or depression.
  • the imaging manifestations of cerebral small vessel disease mediated by the destruction of the blood-brain barrier include white matter lesions. In some embodiments, the imaging manifestations of cerebral small vessel disease mediated by the destruction of the blood-brain barrier are only white matter lesions.
  • the disease mediated by damage to lung epithelial cells and/or vascular endothelial cells is cardiovascular disease or diabetic vascular complications.
  • the cardiovascular disease is selected from one or more of acute myocardial infarction (AMI), angina pectoris, coronary heart disease, ischemic heart disease, heart failure, hypertension, and cardiovascular intervention thrombosis .
  • the diabetic vascular complication is one or more of diabetic retinopathy, diabetic nephropathy, and impaired diabetic wound healing.
  • Another aspect of the present invention provides any compound of the present invention, its deuterated product or pharmaceutically acceptable salt for use in the prevention or treatment of diseases mediated by damage to lung epithelial cells and/or vascular endothelial cells.
  • the disease mediated by damage to lung epithelial cells and/or vascular endothelial cells is selected from the group consisting of acute lung injury, acute respiratory distress syndrome, pulmonary hypertension, pulmonary edema, pulmonary fibrosis, chronic lung disease of premature infants One or more of chronic obstructive pulmonary disease, pneumocystis disease and pulmonary embolism.
  • the acute lung injury, acute respiratory distress syndrome, pulmonary hypertension, or pulmonary edema is caused by hyperoxia, viral infection, bacterial infection, trauma, shock, ischemia reperfusion, acute pancreatitis, inhalation injury , Diffuse alveolar injury and/or poisoning.
  • the acute lung injury, acute respiratory distress syndrome, pulmonary hypertension, or pulmonary edema is caused by hyperoxia, viral infection, bacterial infection, trauma, shock, ischemia reperfusion, acute pancreatitis, inhalation injury , Diffuse alveolar damage and/or poisoning, and not caused by hypoxia.
  • the virus is a coronavirus (for example, a novel coronavirus SARS-CoV-2), influenza virus, respiratory syncytial virus, adenovirus, parainfluenza virus, measles virus, cytomegalovirus, or a combination thereof.
  • the virus is a coronavirus (for example, a novel coronavirus SARS-CoV-2).
  • the acute lung injury is lung injury caused by surgery, such as sublobectomy, lobectomy, or pneumonectomy, such as pneumonectomy, lung tumor resection, or lung transplantation. .
  • the disease mediated by damage to lung epithelial cells and/or vascular endothelial cells is lung injury.
  • the disease mediated by damage to lung epithelial cells and/or vascular endothelial cells is acute lung injury.
  • the disease mediated by damage to lung epithelial cells and/or vascular endothelial cells is acute respiratory distress syndrome.
  • the disease mediated by damage to lung epithelial cells and/or vascular endothelial cells is pulmonary hypertension.
  • the disease mediated by damage to vascular endothelial cells includes cerebral small vessel disease mediated by the destruction (or damage) of the blood-brain barrier, but does not include cerebral microhemorrhage, stroke, and cerebral edema.
  • the destruction of the blood-brain barrier is manifested by increased permeability of the blood-brain barrier.
  • the destruction of the blood-brain barrier is manifested as damage to vascular endothelial cells of the blood-brain barrier.
  • the clinical manifestations of cerebral small vessel disease mediated by the destruction of the blood-brain barrier are cognitive impairment, gait disorder, mood disorder, urinary incontinence, and/or depression.
  • the imaging manifestations of cerebral small vessel disease mediated by the destruction of the blood-brain barrier include white matter lesions. In some embodiments, the imaging manifestations of cerebral small vessel disease mediated by the destruction of the blood-brain barrier are only white matter lesions.
  • the disease mediated by damage to lung epithelial cells and/or vascular endothelial cells is cardiovascular disease or diabetic vascular complications.
  • the cardiovascular disease is selected from one or more of acute myocardial infarction (AMI), angina pectoris, coronary heart disease, ischemic heart disease, heart failure, hypertension, and cardiovascular intervention thrombosis .
  • the diabetic vascular complication is one or more of diabetic retinopathy, diabetic nephropathy, and impaired diabetic wound healing.
  • ALI/ARDS Acute Lung Injury/Acute Respiratory Distress Syndrome
  • Acute lung injury is an acute and progressive hypoxic respiratory failure caused by lung inflammation and increased pulmonary microvascular permeability, and its final stage is acute respiratory distress syndrome (ARDS).
  • ARDS acute respiratory distress syndrome
  • a variety of factors can cause acute lung injury, including but not limited to hyperoxia, viral infection, bacterial infection, trauma, shock, ischemia reperfusion, acute pancreatitis, inhalation injury, diffuse alveolar injury, poisoning, etc.
  • acute lung injury/acute respiratory distress syndrome in or caused by infectious pneumonia (such as bacterial pneumonia or viral pneumonia).
  • the pathophysiological characteristics of the new coronavirus-infected pneumonia include: the new coronavirus mainly attacks the lungs of patients, causing diffuse alveolar damage, accompanied by cell fiber mucin-like exudates, and at the same time The shedding of lung cells and the formation of transparent lung membrane can be seen. This severely affects the ventilation/ventilation function of the lungs and develops into acute respiratory distress syndrome. Clinical studies have also found that some patients still have lung damage for a long time after the viral nucleic acid test turns negative, and it will even worsen.
  • PAH Pulmonary hypertension
  • ALI the changes of pulmonary vascular endothelial cells are particularly noticeable.
  • the damage of vascular endothelial cells has been regarded as the pathological basis of the occurrence and development of ALI.
  • Pulmonary hypertension (PAH) is one of the main early manifestations of ALI caused by various reasons. PAH can promote pulmonary interstitial edema, pulmonary hemorrhage and progressive dyspnea. Dysfunction and damage of pulmonary vascular endothelial cells may be the key factors in the formation of PAH during ALI.
  • vascular endothelial dysfunction appears before vascular changes and is reversible. Therefore, research and understanding of its occurrence and development and its regulatory mechanism are of great significance to the prevention and treatment of ALI.
  • alveolar epithelial cells are very obvious. Although the lung capillaries are also damaged to a certain extent, the epithelial cells are still mainly damaged. At the liquid-air interface of the alveoli, the alveolar epithelial cells maintain the appropriate water and solute content in the alveolar lining fluid, which is essential for gas exchange and the host's resistance to pathogens such as viruses and bacteria. If the endothelial permeability changes in the lung, the edema fluid in the lung interstitium and alveolar cavity will eventually be cleared, and pulmonary interstitial fibrosis will not develop.
  • alveolar epithelial cells will increase protein permeability, reduce alveolar fluid and alveolar protein clearance, and then cause the accumulation of large molecular weight proteins in the alveoli and disorderly repair, leading to deterioration of gas exchange.
  • the destruction of alveolar epithelium during the ALI process leads to the shedding of epithelial cells and the subsequent infiltration of protein-rich edema fluid into the alveolar cavity, accelerating the destruction of the alveolar barrier.
  • the forms of epithelial cell destruction include apoptosis and necrosis.
  • necrosis leads to the destruction of cell membranes and cytoplasm overflow, which further stimulates the inflammatory response.
  • Alveolar mechanical damage, high temperature, ischemia or bacterial product stimulation can directly lead to necrosis.
  • Apoptosis is related to surface death receptors, and only mild inflammation is caused by cell clearance. Extensive epithelial cell apoptosis and separation lead to the exposure of the cell basement membrane to the inflammatory products of the alveolar cavity, such as oxidants, proteases and inflammatory factors.
  • the destruction of epithelial cells causes the proliferation of fibroblasts and the formation of collagen, which may lead to pulmonary fibrosis.
  • repairing the integrity of alveolar epithelium is very important for restoring the normal function of epithelial cells and removing pulmonary edema.
  • a series of studies have confirmed that certain factors produced by ALI/ARDS patients themselves or whether they enter the alveolar cavity can promote epithelial repair. These soluble factors are derived from fibroblasts, macrophages, endothelial cells, epithelial cells, extracellular matrix, and plasma exudates in the patient's lungs, and constitute cytokines, chemokines, growth factors, prostaglandins and mechanism components.
  • ALI/ARDS Although there are many studies on ALI/ARDS, the mortality rate of patients remains high. Due to the poor effect of existing drug treatments, the treatment of ARDS is still based on supportive treatment.
  • One of the main factors that determine the severity and progression of ALI is alveolar epithelial injury. From the perspective of epithelial cell therapy, inhibiting the early damage of epithelial cells, accelerating the repair of epithelial cells, and eliminating pulmonary edema will all be effective measures to improve the survival rate of patients.
  • IPF Idiopathic Pulmonary Fibrosis
  • Idiopathic pulmonary fibrosis is a lung disease caused by alveolar epithelial cell damage and abnormal tissue proliferation. Studies have confirmed that the pathogenesis is the damage of alveolar epithelial cells for a variety of reasons, and finally leads to the proliferation of fibroblasts and the aggregation of myofibroblasts to form fibroblast foci. Epithelial cell apoptosis may be an important factor in the early development of IPF. Inflammation, intracellular tension, telomerase activity and other factors participate in the apoptosis of alveolar epithelial cells, and play an important role in the early stages of pulmonary fibrosis.
  • IPF The important pathological feature of IPF is the fibrotic foci formed by the active proliferation of fibroblasts. Fibroblast foci are the site of epithelial cell damage and repair. Various mediators secreted by epithelial cells promote the migration, proliferation, and differentiation of fibroblasts, which leads to extensive fibrosis in the alveoli and ultimately to progressive dyspnea.
  • the pathogenesis of IPF may be caused by abnormal re-epithelialization of alveolar epithelial cells and abnormal proliferation of fibroblasts after injury. Cell death in IPF is manifested as apoptosis after epithelial cell injury, which plays an important role in the occurrence of diseases.
  • Pneumoconiosis is a systemic disease mainly caused by irreversible fibrosis of lung tissue caused by long-term inhalation of productive dust. At present, there is no specific treatment for pneumoconiosis. In the pathological process of pulmonary fibrosis caused by re-dust, some alveolar epithelial cells are abnormally activated, but also damaged and apoptosis.
  • EMT epithelial-mesenchymal transition
  • Hyperoxia lung injury is a lung inflammatory disease based on diffuse lung cell injury that can quickly affect gas exchange.
  • Hyperoxia lung injury is a general term for acute and chronic non-injury, including pulmonary oxygen poisoning, caused by breathing high partial pressure oxygen with a volume fraction of 21% higher than normal pressure or hyperbaric oxygen higher than 1 atmosphere. Because newborns, especially premature babies, the antioxidant enzyme system and lung surfactants are immature, after high oxygen enters the lungs, oxygen free radicals are generated, causing cell damage, apoptosis, and necrosis, and at the same time, inflammatory mediators and inflammatory cells are produced. Infiltration, leading to lung inflammation, tissue damage and abnormal repair.
  • Hyperoxia causes oxidative damage to alveolar epithelial cells and inhibits the process of alveolarization. Extensive alveolar epithelial cell damage and the decrease in damage repair ability significantly increase the permeability of the air-blood barrier and cause pulmonary edema, which promotes the occurrence of ALI.
  • Chronic lung disease (CLD) of premature infants is the most common and most serious complication of premature infants after long-term inhalation of high concentrations of oxygen or mechanical ventilation treatment or infection.
  • CLD in preterm infants was first described by Northway in 1967 as a complication of the treatment of hyaline membrane disease in preterm infants, also known as bronchopulmonary dysplasia (BPD).
  • Oxygen therapy is the most commonly used treatment for premature infants with cardiopulmonary diseases. Although this measure can change the hypoxic state of children and save their lives, long-term inhalation of high concentrations of oxygen can cause lung damage to varying degrees, and severe cases can develop into CLD.
  • ELBW extremely low birth weight
  • ELBW extremely low birth weight
  • COPD chronic obstructive pulmonary disease
  • COPD chronic obstructive pulmonary disease
  • CS Cigarette smoke
  • alveolar epithelial cells are damaged by the oxidants in CS. The main part. Studies have shown that the apoptotic index of alveolar epithelial cells in COPD patients is significantly higher than that of pulmonary vascular endothelial cells, and the two are positively correlated.
  • Endothelial cell damage and apoptosis first destroy the integrity of alveolar structure, and then damage or apoptosis of alveolar epithelial cells may occur. After endothelial cell injury or apoptosis, it may be triggered by direct cell-cell interaction and some indirect mechanism to trigger the apoptosis of other cells, especially alveolar epithelial cells, thereby forming COPD and emphysema.
  • patients with COPD have vascular endothelial dysfunction, which is related to the severity of the disease. Abnormal function of vascular endothelial cells is closely related to the occurrence and development of COPD.
  • Inflammation vascular injury, hemodynamic changes, hypoxia, oxidative stress and apoptosis can promote vascular endothelial cell dysfunction.
  • Studies have shown that even in the early stages of COPD, even in healthy smokers with normal lung function, the function of vascular endothelial cells has changed. Abnormal changes in the function of vascular endothelial cells may play an important role in the occurrence and development of COPD and the high incidence of related cardiovascular events.
  • Pneumocystis disease is caused by Pneumocystis sp Rapid recovery occurs mostly in people with weakened immune function, and the incidence of AIDS patients is as high as 70-80%.
  • the virulence of Pneumocystis spp. is weak, and most healthy people have a recessive infection. Only when the host's immune function is low, the latent Pneumocystis spp. multiplies in large numbers, leading to the occurrence of PCP.
  • Pulmonary embolism is a pathological process in which a pulmonary artery or its branches is blocked by thrombus or other substances. It is a common disease with high morbidity and mortality.
  • Vascular endothelial cell damage is the first element that causes pulmonary embolism. Endothelial cells act on platelets and neutrophils through biological molecules such as microparticles and tissue factor, and participate in thrombosis. Since the damage and repair of vascular endothelial cells play an important role in the process of pulmonary embolism, in recent years, research on the occurrence and development of pulmonary embolism has also focused more and more on the molecular, cytological, and pathological aspects of endothelial cell damage and repair. explore.
  • Vascular endothelial cell damage is related to the occurrence and development of many cardiovascular diseases. Thrombosis caused by vascular endothelial damage is the pathological basis of many cardiovascular diseases. Shedding vascular endothelial cells were first found in the peripheral blood of patients with subacute endocarditis. With the deepening of research, it has been found that many cardiovascular diseases have varying degrees of changes in circulating endothelial cells. Circulating endothelial cells in patients with acute myocardial infarction (AMI) and angina pectoris increase significantly and can last for several days.
  • AMI acute myocardial infarction
  • AMI acute myocardial infarction
  • angina pectoris increase significantly and can last for several days.
  • vascular endothelial dysfunction plays an important role in the occurrence and development of hypertension.
  • hypertension itself aggravates vascular endothelial disorders.
  • the endothelial cells of hypertensive rats are significantly increased in the blood of the damaged organs, and the circulating endothelial cells of hypertensive patients are significantly higher than that of ordinary people, and the increase is higher than that of patients with coronary heart disease.
  • Cardiovascular interventions mainly mechanically stimulate the vascular endothelium and cause damage. Different surgical procedures have different degrees of damage to the vascular endothelium.
  • vascular endothelial cells When vascular endothelial cells are damaged, it causes endothelial dysfunction and changes in hemodynamics, which causes a large number of traps and vasoactive substances to accumulate locally in the injured blood vessel, and platelet activation is the main pathophysiological mechanism of thrombosis in cardiovascular interventional procedures.
  • vascular endothelial dysfunction is an important cause of diabetic vascular complications (especially retinopathy, nephropathy and impaired wound healing).
  • the change of vascular endothelial function is also an important factor in other pathogenesis of diabetes, mainly reflected in insulin resistance, lipotoxicity and Impaired insulin secretion.
  • vascular endothelial function damage is the initiating factor and the main pathophysiological basis of diabetic vascular disease. Even diabetic patients who have not yet experienced chronic vascular complications have experienced a significant decrease in endothelial function.
  • Cerebral small blood vessels refer to the small perforating arteries and arterioles (40-200 ⁇ m in diameter), capillaries and venules of the brain. They constitute the basic unit of blood supply of brain tissue and play an important role in the maintenance of brain function.
  • the large and small blood vessels of the brain together constitute the vascular tree. They have structural continuity, are affected by hemodynamics, and are exposed to risk factors. Therefore, the lesions of the large and small blood vessels in the brain should theoretically have Parallel correlation of severity. However, in clinical work, the inconsistency between the two is often found. For example, patients with severe cerebral small vessel disease but not accompanied by cerebral arterial stenosis are often found, and vice versa.
  • Cerebral small vessel disease generally refers to the clinical, cognitive, imaging, and pathological syndromes caused by the various lesions of the above small blood vessels. It is customary to refer to small perforating arteries and small arteries. Clinical and imaging manifestations caused by the disease. CSVD is mainly characterized by stroke (deep small infarction, cerebral hemorrhage), cognitive and affective disorders, and overall functional decline as prominent clinical manifestations. In imaging, the prominent manifestations are lacunar infarction (LI) and lacune ), white matter lesions (WML), enlarged perivascular space (EPVS) and cerebral microbleeds (CMB), etc.
  • LI lacunar infarction
  • WML white matter lesions
  • EPVS enlarged perivascular space
  • CMB cerebral microbleeds
  • Cerebral small vessel disease can involve small arteries, capillaries, and venules, with perforating arteries being the most common.
  • Hypertension, vascular inflammation or genetic defects caused vascular endothelial cell damage, smooth muscle hyperplasia, and thickening of the basement membrane of small blood vessel walls can all cause chronic cerebral ischemia.
  • Loss and proliferation of vascular smooth muscle cells, thickening of blood vessel walls, stenosis of vascular lumen causing chronic and progressive local or even diffuse subclinical ischemia, demyelination of nerve cells, loss of oligodendrocytes, and axonal damage, resulting in Incomplete ischemia.
  • other studies suggest that increased vascular permeability after endothelial injury leads to extravasation of intravascular materials, causing damage to blood vessels and surrounding tissues, which may also play an important role in the progression of the disease at this stage.
  • the diseases mediated by damage to vascular endothelial cells include cerebral small vessel diseases mediated by the destruction or damage of the blood-brain barrier, but do not include cerebral microhemorrhage, stroke, and cerebral edema.
  • the cerebral small vessel disease mediated by the destruction of the blood-brain barrier includes white matter lesions.
  • the cerebral small vessel disease mediated by the destruction of the blood-brain barrier only manifests as white matter lesions.
  • Example 1 YC-6 significantly and quickly inhibits the up-regulation of PFKFB3 expression caused by glutamate
  • PFKFB3 phosphofructokinase-2/fructose-2,6-bisphosphatase 3
  • PFKFB3 phosphofructokinase-2/fructose-2,6-bisphosphatase 3
  • the cells were taken from SD P7-8 neonatal rats, the cerebellum was taken out, and the meninges and blood vessels were further removed using micro tweezers; the separated cerebellum tissue was transferred to another petri dish containing dissecting fluid.
  • Digestion Use a dropper to add the chopped tissue to 7mL 0.25% trypsin digestion solution, digest it at 37°C for 15mins; arrange the cell surface and clean the instruments. Turn it upside down several times every five minutes to make the tissues more fully contact the digestive juices. Termination of digestion: After digestion, it can be seen that the release of DNA due to broken cells will cause the tissues to stick together and be suspended in the liquid.
  • Inoculate cells centrifuge at 1000 rpm for 5 minutes, collect the cell pellet, and resuspend the cells in DMEM containing 10% FBS. Count with a handheld cell counter, and the seeding density is 4.0-5.0 ⁇ 10 5 cells/mL. The inoculation volume of 35mm petri dishes is 2mL, and the inoculation volume of 48-well plates is 300 ⁇ L.
  • Cell seeding and culture 24 hours after seeding, supplementation of cytarabine (final concentration of 10 ⁇ M) inhibits the growth of glial cells. Supplement glucose (5mM) to maintain nutrition on the seventh day, and use it on the eighth day. Glutamate-induced primary cerebellar granule neuron injury model and drug treatment.
  • the culture medium of the primary cerebellar granule neurons planted in the six-well plate was replaced with 2mL kreb's buffer, which served as a normal control group.
  • glutamic acid with a final concentration of 200 ⁇ M for 5mins and 15mins for the corresponding time and use this as the model group; pre-incubate with 10 ⁇ M YC-6 or NMDA receptor blocker MK-801 20mins in advance, and then add the above-mentioned concentration of glutamic acid
  • the corresponding time of treatment was used as the drug treatment group; the same amount of solvent HP- ⁇ -CD as the drug treatment group was added as the solvent control group, and the pre-incubation time was the same as that of the drug treatment group.
  • a 12% SDS-polyacrylamide gel was prepared with a volume fraction of 12%, and the sample was injected according to the total amount of protein loaded in each well of 20 ⁇ g, and the electrophoresis separation was carried out under the condition of 100V constant pressure; then it was transferred to the PVDF membrane by the wet transfer method.
  • YC-6 significantly and rapidly inhibits the up-regulation of PFKFB3 expression caused by glutamate
  • Example 2 The effect of YC-6 on lactic acid, a key metabolic molecule in lung epithelial injury
  • Lactic acid is a product of anaerobic glycolysis and downstream of the metabolic pathway regulated by PFKFB3, which directly damages lung epithelial cells.
  • Research by Gong Y et al. confirmed that in vitro LPS caused apoptosis of human alveolar epithelial A549 cells, inflammatory cytokine production, increased glycolytic flux, and increased reactive oxygen species (ROS), and these changes were reversed by the PFKFB3 inhibitor 3PO. More importantly, lactic acid is also a key metabolite that causes lung injury. The treatment of A549 cells with lactic acid will lead to apoptosis and enhancement of ROS.
  • Pretreatment The number of cells used in the determination of intracellular lactic acid content is 1-2*10 ⁇ 6.
  • the specific experimental steps of cell pretreatment include the following, washing the cells with pre-chilled Kreb's buffer three times. Add 220 ⁇ L of cell lysate to fully lyse. After the cells, the lysate was collected into a 1.5mL EP tube. Centrifuge at 16000g for 5 minutes at 4°C, collect 160 ⁇ L of the supernatant into a new EP tube, and quantify the remaining supernatant for BCA protein. Add 56 ⁇ L 4mM HClO 4 , mix upside down and mix thoroughly, and let it react on ice for five minutes to remove the existing LDH of the sample itself to prevent endogenous interference.
  • the determination method of intracellular lactic acid content was determined according to the instruction of L-Lactate Assay kit (abcam, ab65331). The specific steps are: take 50 ⁇ L of the dilution and add it to a 96-well black-bottomed cell culture plate, then add an equal volume of the substrate solution containing LDHA, shake and mix, and place it in a 37°C carbon dioxide-free incubator for incubation 30mins. Measure the fluorescence intensity with a microplate reader in the dark and the excitation light wavelength is 535nm. Bring the fluorescence intensity read from the sample to the standard curve of the fluorescence intensity corresponding to the lactic acid concentration of different standards, and convert the fluorescence intensity into the actual concentration of lactic acid in the sample solution.
  • YC-6 significantly inhibits the intracellular accumulation of lactic acid, the end product of PFKFB3 downstream glycolysis caused by glutamate
  • the primary cerebellar granule neuron cells were stimulated with 200uM glutamate for 15 minutes, and then the cell lysate was collected to study the effect of YC-6 treatment on the glycolysis product lactic acid (Figure 2).
  • the intracellular lactic acid of neurons was stimulated by glutamate for 15 minutes. The content increased significantly, and YC-6 treatment suppressed this increase.
  • the above experimental results show that YC-6 inhibits the key enzyme of glycolysis, PFKFB3, while significantly inhibiting the accumulation of lactic acid.
  • Example 3 YC-6 significantly reduces the damage of vascular endothelial cells
  • Pulmonary vascular endothelial cells form a monolayer lining the vasculature. Because their physiological positions are exposed to direct stimulation of bacterial endotoxin, LPS, inflammatory factors such as TNF- ⁇ , chemical poisons, and oxidative stress, endothelial cells are damaged. And apoptosis. The damage of pulmonary vascular endothelial cells causes increased capillary permeability, which leads to increased lung water content, resulting in pulmonary edema and difficulty breathing. At the same time, pulmonary vascular endothelial cells secrete and release various inflammatory mediators and cytokines, which make pro-inflammatory and anti-inflammatory.
  • Inflammatory media blood coagulation and anticoagulation systems are unbalanced, causing pulmonary microcirculation disorders and pulmonary hypertension, which can promote pulmonary interstitial edema, pulmonary hemorrhage and progressive dyspnea, and patients develop progressive hypoxemia and respiratory distress.
  • OGD-R oxygen-glucose deprivation and restoration
  • HUVEC cells are cultured in modified DMEM medium containing 10% fetal bovine serum. When the cells grow to about 80% confluency, the cells are digested with 0.25% trypsin, and then cultured in modified DMEM containing 10% peptide bovine serum Pipette the base uniformly, adjust the cell concentration to 1 ⁇ 10 5 cells/mL, inoculate 400uL/well in a 24-well plate, and perform the experiment when the cells grow to about 70% confluence.
  • RAOEC cells were cultured in high-sugar DMEM medium containing 10% fetal bovine serum, and the inoculation conditions were the same as HUVEC.
  • the oxygen concentration of the hypoxic workstation is set to 1% (1% O 2 , 5% CO 2 , 94% N 2 ), the temperature is 37° C., and the humidity is 85%.
  • Preventive administration Before the cells are treated with oxygen and glucose deprivation, they are pre-applied with solvents or drugs (final concentration of 1 ⁇ M, 3 ⁇ M, 10 ⁇ M) for 1 hour, and then during the oxygen and glucose deprivation treatment, add the corresponding sugar-free DMEM medium. When reoxygenation treatment, add the corresponding solvent or drug to the normal medium until the end of the experiment. The model control group was not treated with any drugs, and each treatment group was subjected to 3 repetitions.
  • OGD-R treatment caused an increase in the release of LDH from human umbilical vein endothelial cells (HUVEC) and rat vascular endothelial cells (RAOEC), indicating that OGD-R injury treatment caused the damage and death of HUVEC cells and RAOEC cells.
  • YC-6 dose-dependently significantly reduced the increase in LDH release caused by OGD-R and reduced cell damage.
  • YC-6 significantly reduced vascular endothelial cell damage in a dose-dependent manner, suggesting that YC-6 can play an effective protective role in the process of vascular endothelial cell-related lung injury caused by various pathological factors.
  • Example 4 YC-6 reduces the damage of alveolar epithelial cells caused by hypoxia and LPS
  • Alveolar epithelial cells and capillary endothelial cells are the direct targets of various damaging pathogenic factors such as inflammation, toxic inhalation, virus infection and sepsis. After injury, the permeability increases and apoptosis occurs, resulting in diffuse interpulmonary Quality and alveolar edema, leading to progressive hypoxemia and respiratory distress. During the onset of new coronary pneumonia, lung epithelial cells are the main targets of virus attack.
  • the new type of coronavirus (SARS-CoV-2, hereinafter referred to as CoV) is a single positive-stranded RNA virus with a length of about 27-32 kb.
  • the viral genome is mainly composed of two parts: the replicase coding region and the structural protein coding region.
  • Pulmonary epithelial cells include all two types of type I or type II alveolar epithelial cells.
  • this section uses bacterial lipopolysaccharide LPS and hypoxia injury models, through Detection of cellular LDH release evaluates the protective effect of YC-6 on lung epithelial cells, and provides experimental evidence for YC-6 to treat ARDS caused by different factors including the new coronavirus.
  • the experimental results showed that YC-6 significantly reduced the damage of alveolar epithelial cells caused by hypoxia and LPS in a dose-dependent manner.
  • HPAEpiC cells are cultured with alveolar epithelial cell culture medium. When the cells grow to about 80% confluence, the cells are digested with 0.25% trypsin, and then blow evenly with alveolar epithelial cell culture medium to adjust the cell concentration to 2 ⁇ 10 5 cells /mL, inoculate 400uL/well in a 24-well plate, and perform the experiment when the cells grow to about 70% confluence.
  • the oxygen concentration of the hypoxic workstation is set to 1% (1% O 2 , 5% CO 2 , 94% N 2 ), the temperature is 37° C., and the humidity is 85%. After the conditions of the hypoxic workstation were stabilized, 1mg/mL LPS was directly added to the cells to make the final concentration of LPS 20 ⁇ g/mL, and then the cells were placed in the hypoxic workstation for 24 hours.
  • the drug group Before cell modeling, the drug group is pre-incubated with solvent (20% hydroxypropyl cyclodextrin solution) or drug (final concentration of YC-6 is 1 ⁇ M, 5 ⁇ M, 10 ⁇ M) for 1 hour, and then LPS is added and placed in hypoxia The workstation is modeled until the end of the experiment.
  • the model control group was not treated with any drugs, and the normal group was placed in a cell incubator for routine culture. 10 ⁇ M dexamethasone was used as the drug control. Each treatment group performed 3 replicates.
  • Example 5 YC-6 significantly improved LPS-induced acute lung injury in rats
  • Animal grouping Select 60 rats that have passed the quarantine and uniform body weight to be included in the experiment. They are randomly divided into 6 groups according to their body weight, with 10 animals per group. The animal grouping and the treatment dose of each group are as follows:
  • test drug was administered via tail vein at 0.3mL/100g body weight, and hydrocortisone (25mg/kg) was administered intraperitoneally at 0.5mL/100g body weight; all test agents were administered 0.5h before LPS administration.
  • test drug solvent and YC-6 were injected intravenously every 6h for a total of 4 injections, and the hydrocortisone (HC) group was injected intraperitoneally every 12h for a total of 2 injections.
  • the rats were weighed, the rats were anesthetized by isoflurane, and the rats were fixed in the supine position, and the neck skin was prepared and disinfected with 75% ethanol.
  • the neck skin was cut in the middle, the subcutaneous tissue was bluntly separated, and the upper trachea was exposed.
  • the rat was erected, shaken, and rotated to make the LPS solution evenly distributed in the lungs.
  • the skin is sutured and disinfected with iodophor.
  • the animals were anesthetized by intraperitoneal injection of 20% uracil solution at a volume of 6 mL/kg body weight. After bleeding from the abdominal aorta, the chest cavity was exposed and lung tissue was taken. Put the left lung into 10% paraformaldehyde aqueous solution and fix it for 48 hours. Transect the left lung coronal plane into the upper and lower parts of equal width, and then cut the lower half of the left lung into two parts of equal width according to the coronal plane. The sagittal plane of the upper part of the left lung and two coronal breads of the lower part of the left lung were buried in a paraffin block, sectioned and stained with hematoxylin-eosin (HE).
  • HE hematoxylin-eosin
  • Tissue dehydration and paraffin embedding remove the tissue from the fixative solution and soak in 50% ethanol (30min)-70% ethanol (overnight)-80% ethanol (30min)-90% ethanol (30min)-95% ethanol (30min) )-Anhydrous ethanol (2 times, 30min each time)-Xylene (2 times, 5-10min each time, until the sample is completely transparent)-62°C paraffin wax (3 times, 1 hour each time), and then embed the tissue .
  • Tissue paraffin section sections the thickness is 3 ⁇ m, the sections are dried on a dryer and then placed in a 37°C oven to dry overnight, and then used for HE staining.
  • HE staining Take out the paraffin sections stored at room temperature and bake them in an oven at 65°C for 30 minutes, then immediately immerse the sections in xylene for dewaxing three times, respectively, for 5 minutes, 2 minutes, and 2 minutes; rehydration: the third soak After toluene, the slices are immersed in 100% ethanol-100% ethanol-95% ethanol-90% ethanol-80% ethanol-70% ethanol-50% ethanol-distilled water for rehydration, each time for 1min; take out the slices and dry slightly , Then place the section in a wet box, add hematoxylin dye solution dropwise to the tissue, make sure that the dye solution completely covers the tissue, incubate at room temperature for 5 minutes; gently rinse the section with distilled water to wash off the excess hematoxylin, and then place the section Re-humidify the box, drop the eosin staining solution on the tissue, incubate for 2 min at room temperature; rinse the sections gently with distilled water; soak
  • Observation of lung tissue pathology Nikon Eclipse Ti-U inverted fluorescence microscope was used to observe lung tissue pathology changes under light microscope. The analysis was performed according to the level of changes in inflammatory cell infiltration, alveolar hemorrhage, alveolar wall thickening, alveolar expansion, and bronchial epithelial shedding. According to the Smith scoring system, the lung injury scores were performed by two experimenters who did not know the experimental group and dosing information. The six indicators of inflammatory cell infiltration, alveolar hemorrhage, alveolar wall thickening, alveolar expansion, and bronchial epithelial shedding were performed respectively for lung injury. Severity score.
  • the total lung injury pathology score is the sum of the above scores.
  • Figure 5 The pathological observation results in Figure 5 show that the normal control group (Figure 5A) has clear lung tissue structure, the alveolar structure is polygonal or round thin-walled vacuoles, the alveolar space is clean, the boundaries are clear, the alveolar wall is not thickened, and the alveoli are not thickened. There is no inflammatory infiltration in the interstitium; the structure of the bronchial tube wall is clear, and no epithelial cells are shed; the blood vessels are normal.
  • the scores of the LPS model group were significantly higher than those of the normal control group (P ⁇ 0.001); the YC-6 treatment group and the LPS model group were given doses of 12, 40, and 120 mg/kg/d
  • the pathological score was significantly lower (P ⁇ 0.01 or P ⁇ 0.001); and the treatment of YC-6 in the low-dose group was significantly better than the protective effect of 50mg/kg/d hydrocortisone (P ⁇ 0.05).
  • LPS causes significant lung damage, manifested by a large number of inflammatory cell infiltration, alveolar wall thickening, alveolar congestion, expansion and collapse, and bronchial mucosal epithelial shedding.
  • YC-6 significantly reduces the permeability of the vascular endothelial barrier caused by LPS, reduces inflammatory cell infiltration, and reduces alveolar damage.
  • Example 6 YC-6 reduces acute lung injury in cynomolgus monkeys
  • Non-human primates are closer to humans in terms of phylogeny, anatomical structure, etc., which can reduce the bias in drug efficacy evaluation caused by species differences; at the same time, the pharmacological dose, toxicology and drug efficacy obtained in non-human primates
  • the research data can provide a more reliable basis for follow-up clinical trials.
  • the altitude rose to 3000 meters at a speed of 3 m/s; after the animals stayed at the simulated altitude of 3000 meters in the hypobaric cabin for 30 minutes, 10ml of blood from each experimental monkey was taken, and then the acute hypobaric hypoxia group was injected with glucose intravenously Normal saline 10ml; simulated elevation to 4500m at a speed of 3m/s; after the animals stayed at a simulated altitude of 4500m in the low-pressure cabin for 30 minutes, 10ml of blood from each experimental monkey was taken, and the acute hypobaric hypoxia group was intravenously injected with glucose.
  • the YC-6 injection was diluted to 10ml with glucose saline at a dose of 10mg/kg, and the YC-6 treatment group was given a one-time intravenous bolus; the acute hypobaric hypoxia group was only intravenously A bolus injection of 10ml glucose saline.
  • the YC-6 injection was diluted with glucose saline at a dose of 10 mg/kg to 10 ml, and the YC-6 treatment group was given an intravenous bolus; acute hypotension In the oxygen group, only 10ml glucose saline was injected intravenously; after the animals in the drug treatment group stayed in the hypobaric chamber at a simulated altitude of 4500 meters for 30 minutes, the YC-6 injection was diluted with glucose saline at a dose of 10mg/kg to 10ml, YC-6
  • the treatment group received an intravenous bolus injection; the acute hypobaric hypoxia group received an intravenous bolus injection of 10ml glucose saline; the animals in the drug treatment group stayed in the hypobaric chamber at a simulated altitude of 6000 meters for 30 minutes, and the YC-6 sustained-release agent was dosed at 30 mg/kg
  • the YC-6 injection was diluted with glucose saline at a dose of 10 mg/kg to 10 ml, and the YC-6 treatment group received an intravenous bolus injection.
  • the sustained-release agent is divided into 5 points for skeletal muscle intramuscular injection at a dose of 30 mg/kg. In the acute hypobaric hypoxia group, only 10ml glucose saline was injected intravenously.
  • the altitude was lowered to 6000 meters at a speed of 3 meters per second, and the experimental animals were anesthetized (0.06ml/kg ketamine hydrochloride injection, 0.02ml/kg xylazine hydrochloride injection), and carotid bloodletting was sacrificed. Animals, dissected, collected materials, and fixed.
  • Tissue paraffin embedding and fixation After the lung tissue is taken, the lung tissue is trimmed into a tissue block with a thickness of no more than 1 cm, and it is fixed in 10 times the volume of paraformaldehyde. When fixing, the floating lung tissue is pressed to the liquid surface with cotton Make the organization fully fixed. After 48 hours, change the fixative solution and continue fixation for 48 hours, and then it can be used for paraffin embedding.
  • Tissue paraffin section section the thickness is 3mm, the section is dried on a dryer and then placed in a 37° oven to dry overnight, and then used for HE staining.
  • Hematoxylin-Eosin (HE) staining Take out the paraffin sections stored at room temperature and bake them in an oven at 65°C for 30 minutes, then immediately immerse the sections in xylene for dewaxing three times, respectively, for 5 min, 2 min, and 2 min; rehydrate : After soaking in xylene for the third time, soak the slices in 100% ethanol-100% ethanol-95% ethanol-90% ethanol-80% ethanol-70% ethanol-50% ethanol-distilled water for rehydration, 1 min each time ; Take out the section to dry a little, then place the section in a humid box, add hematoxylin dye solution dropwise on the tissue, make sure that the dye solution completely covers the tissue, incubate at room temperature for 5 minutes; rinse the section gently with distilled water to wash away excess hematoxylin , Then put the section back into the wet box, add the eosin staining solution dropwise to the tissue, and incubate at room temperature for 2
  • cynomolgus monkeys show significant symptoms of acute altitude sickness, such as shortness of breath, vomiting, ataxia, confusion, etc., indicating that non-human primate cynomolgus monkeys have acute altitude sickness.
  • the replication of the disease model was successful.
  • the early pathological features of ALI/ARDS are the expansion of alveolar wall capillaries, widening of alveolar septum, and exudation of serum, neutrophils and macrophages in the alveolar cavity, which then develops into diffuse lung congestion, edema, and hyaline membrane in the alveoli Formation and focal lung collapse.
  • HE staining was used to observe whether YC-6 could reduce lung injury caused by acute hypobaric hypoxia.
  • the alveolar structure of the cynomolgus monkey lung tissue in the normal control group is polygonal or round thin-walled vacuoles with clear boundaries.
  • the alveolar epithelial cells are thin-walled alveolar septa, and capillary sections can be seen in the septum.
  • acute hypobaric hypoxia caused a significant thickening of the alveolar septum; however, it was significantly improved after YC-6 administration.
  • the thickness of the alveolar septa of each group was measured and statistically showed (Figure 9) that YC-6 significantly reduced the injury of alveolar septal thickening.
  • DAD diffuse alveolar damage
  • hyaline membrane is also one of the characteristic pathologies of ARDS, which was found in the pathological examination of patients with new coronary pneumonia [1] .
  • the transparent membrane is a uniform red-stained membrane formed on the surface of the respiratory bronchioles, alveolar ducts, and alveoli. It is composed of exuded plasma protein, cellulose and disintegrated alveolar epithelial cell debris.
  • Hyaline membrane formation and alveolar interstitial fibrous hyperplasia increase the alveolar septum, resulting in decreased alveolar membrane permeability and dysfunction of blood gas exchange.
  • acute hypobaric hypoxia caused fibrous hyperplasia of the alveolar septum (black arrow), and at the same time caused the formation of a transparent protein membrane in the alveolar cavity (red arrow), indicating that acute hypobaric hypoxia caused diffuse alveolar injury; acute low pressure Hypoxia also caused leakage of red blood cells into the alveolar cavity (blue arrow), indicating that acute low-pressure hypoxia caused the destruction of the blood-air barrier; but no such obvious changes were observed in the YC-6 drug group, suggesting that YC-6 has an effect on the alveolar epithelium Cells and vascular endothelial cells have a protective effect.
  • the above results suggest that YC-6 may protect alveolar epithelial cells and vascular endothelial cells, reduce the permeability of the air-blood barrier caused by acute low-
  • inflammatory cell infiltration and alveolar epithelial injury are important pathological factors.
  • the alveolar capillary wall is diffusely damaged and permeability is enhanced, and pulmonary edema and cellulose exudation occur.
  • Type II alveolar epithelial cells are damaged, and alveolar surfactants are reduced or disappeared, leading to the formation of lung hyaline membrane and lung collapse.
  • the above changes can cause oxygen diffusion disorder in the alveoli, imbalance of the ventilation blood flow ratio, hypoxemia and respiratory distress. As shown in Figure 11, compared with the normal group, acute hypobaric hypoxia caused the infiltration of inflammatory cells in the lung tissue.
  • a large number of inflammatory cells can be observed in the alveolar mesangial interstitium (black arrow) and alveolar cavity (red arrow). In some alveolar cavities, damaged lung epithelial cells (blue arrows) were seen, while no obvious inflammatory infiltration and damaged lung epithelial cells were observed in the YC-6 treatment group.
  • the above results show that YC-6 reduces vascular permeability and can effectively reduce inflammatory infiltration of lung tissue and lung epithelial cell damage.
  • YC-6 significantly inhibited pulmonary vascular congestion and swelling and alveolar septum thickening, and reduced lung gas-blood barrier damage. YC-6 showed significant lung injury. Protective medicinal effect.
  • Example 7 YC-6 reduces vascular endothelial cell damage caused by hypoxia-related stimulation by promoting the protein expression of NR4A3
  • Nuclear receptor subfamily 4 group A, member 3 (Nuclear Receptor Subfamily 4 Group A Member 3, NR4A3), also known as Neuron-derived Orphan Receptor-1 (NOR1).
  • NR4A3 participates in the regulation of physiological and pathological processes such as metabolism, inflammation, cell proliferation, apoptosis and differentiation. Knockdown of NR4A3 significantly inhibits the capillary transformation ability of vascular endothelial cells, indicating that NR4A3 has important physiological functions in vascular endothelial cells. Previous studies have shown that the up-regulation of NR4A3 can promote cell survival under different pathological injuries.
  • NR4A3 in vascular endothelial cells can inhibit apoptosis by up-regulating cellular inhibitor of apoptosis 2, cIAP2, thereby improving the survival of vascular endothelial cells under hypoxic conditions; in neurons Increasing the expression of NR4A3 can reduce neuronal damage caused by oxidative stress and glutamate-induced excitotoxicity.
  • Human umbilical vein endothelial cells were purchased from Guangzhou Saiku Biotechnology Co., Ltd. and cultured in modified DMEM medium (CellCook, CM2007) containing 10% fetal bovine serum.
  • modified DMEM medium CellCook, CM2007
  • Rat vascular endothelial cells were purchased from Guangzhou Geneo Biotechnology Co., Ltd. and cultured in DMEM medium (Coring, 10-013-CV) containing 10% fetal bovine serum.
  • Paraffin section of cynomolgus monkey lung tissue including 3 normal control group (normobaric normoxia, NN), acute hypobaric hypoxia treatment group 5 (hypobaric hypoxia, HH), acute hypobaric hypoxia + YC-6 treatment group 5 (HH +YC-6).
  • High-sugar DMEM medium (corning, 10-03-CV)
  • Skimmed milk powder (Wako, 190-12865)
  • Anti-actin antibody (Arigo, arg62346)
  • HRP Goat anti-mouse IgG antibody
  • Donkey anti-Mouse IgG H+L
  • Highly Cross-Adsorbed Secondary Antibody Alexa Fluor 488 fluorescent secondary antibody (Invitrogen, A-21202)
  • Donkey anti-Rabbit IgG H+L Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 555 fluorescent secondary antibody (Invitrogen, A-31572)
  • Hoechst33342 (Sigma-Aldrich, 14533, the use concentration is 5 ⁇ g/mL)
  • Chemiluminescent fluid (Merck Millipore, WBKLS0500)
  • MIK Mayer hematoxylin staining solution
  • Ultra-clean workbench cell incubator, hypoxia workstation, microplate reader, chemiluminescence imager, laser confocal microscope, low-temperature high-speed centrifuge, real-time fluorescent quantitative PCR system
  • HUVEC cells are cultured in modified DMEM medium containing 10% fetal bovine serum. When the cells grow to about 80% confluency, the cells are digested with 0.25% trypsin, and then cultured in modified DMEM containing 10% fetal bovine serum Pipette the base uniformly, adjust the cell concentration to 1 ⁇ 10 5 cells/mL, inoculate 400uL/well in a 24-well plate, and perform the experiment when the cells grow to about 70% confluence.
  • RAOEC cells were cultured in high-sugar DMEM medium containing 10% fetal bovine serum, and the inoculation conditions were the same as HUVEC.
  • the oxygen concentration of the hypoxic workstation When performing OGD-R damage treatment, set the oxygen concentration of the hypoxic workstation to 1% (1% O 2 , 5% CO 2 , 94% N 2 ), the temperature is 37° C., and the humidity is 85%. Place the sugar-free DMEM medium in the hypoxia workstation for pre-hypoxia for 3 hours. Aspirate the medium in the culture plate, wash it twice with pre-warmed sugar-free DMEM medium, then add 100uL of sugar-free DMEM medium to each well, place the culture plate in the hypoxic workstation, and add 300uL to each well. Oxygen-free sugar-free DMEM medium, and then place the culture plate in the hypoxia workstation to continue hypoxia for 4 hours.
  • the cells in the drug treatment group were treated with oxygen and glucose deprivation, they were given the specified final concentration of drug (the final concentration of YC-6 was 1 ⁇ M, 3 ⁇ M or 10 ⁇ M while reoxygenation treatment, and the solvent control was given the corresponding 20% hydroxypropyl cyclodextrin, the final concentration of cycloheximide (CHX) is 100 ⁇ M, the final concentration of chloroquine (CQ) is 10 ⁇ M, 30 ⁇ M or 100 ⁇ M, and the final concentration of MG132 is 10nM, 30nM or 100nM) Until the end of the experiment.
  • the model control group was not treated with any drugs.
  • the cells were quickly washed twice with PBS, and then 4% paraformaldehyde was added and left at room temperature for 15 minutes. Then, it was incubated with 0.2% TritonX-100 in PBS for 15 minutes at room temperature to break the membrane. Wash the cells three times with PBS, then add the corresponding primary antibody diluted with DAKO antibody diluent, and incubate overnight at 4°C. Aspirate the primary antibody, wash the cells with PBS 3 times, then add the diluted fluorescent secondary antibody, and incubate for 1 hour at room temperature in the dark.
  • Aspirate the secondary antibody wash the cells 3 times with PBS, then add hoechst33342 to stain the nucleus, and incubate for 5 minutes at room temperature in the dark.
  • centrifuge at 12000g for 15 minutes at 4°C carefully discard the isopropanol, add 1 mL of pre-cooled 75% ethanol (dilute absolute ethanol with DEPC water to 75%) to the bottom of the tube, and invert the centrifuge tube to float the bottom of the tube. Then centrifuge at 12000g at 4°C for 15 minutes, discard 75% ethanol, and wash again with fresh pre-cooled 75% ethanol. Centrifuge at 12000g for 15 minutes at 4°C, discard 75% ethanol, then centrifuge briefly, and suck up the remaining ethanol with a pipette. Place the centrifuge tube on the ultra-clean workbench and leave the tube opening open for 5-10 minutes to allow the ethanol to evaporate cleanly.
  • pre-cooled 75% ethanol dilute absolute ethanol with DEPC water to 75%)
  • RNA quality is high.
  • the primer sequence includes: ⁇ -actin(human)forward: 5'-GATTCCTATGTGGGCGACGA-3'; reverse: 5'-AGGTCTCAAACATGATCTGGGT-3'; NR4A3(human)forward: 5'-AGCGGCGGCATCCTC-3'; reverse: 5'-CTAAGGGTCCAGGCTCAGG -3'; ⁇ -actin(rat)forward: 5'-CGCGAGTACAACCTTCTTGC-3'; reverse: 5'-CGTCATCCATGGCGAACTGG-3'; NR4A3(rat)forward: 5'-GGAAACGTGGCGACATCCT-3'; reverse: 5'-CAGTGGGCTTTGGGTTCTGTG -3'.
  • IP lysis buffer to adjust the protein concentration of each sample to 1mg/mL, take an equal volume of each sample to a new centrifuge tube (the remaining small amount of each sample is used as input), add the corresponding amount of antibody according to the antibody instruction Mix by inverting slowly at 4°C overnight. Then wash the IP magnetic beads twice with the IP lysis solution, add the same amount of IP magnetic beads to each sample, and continue to invert and mix slowly at 4°C for 2 hours. Then separate the magnetic beads, wash the IP magnetic beads 5 times with IP lysis solution, and mix by inversion at 4°C for 5 minutes each time.
  • each sample was added with an equal amount of 1 ⁇ protein loading buffer (IP lysate and 5 ⁇ protein loading buffer were mixed in a 4:1 ratio), centrifuged briefly, and placed in a boiling water bath Boil for 5 minutes, then centrifuge briefly to collect the sample to the bottom of the tube. Transfer the boiled sample to a new centrifuge tube. Discard the magnetic beads.
  • the sample can be placed on ice for later use or frozen at -80°C.
  • the protein samples were subjected to 10% SDS-PAGE electrophoresis, and the subsequent operation was the same as that of western blotting.
  • Reoxygenation injury and YC-6 administration can increase the protein expression of NR4A3 in a dose-dependent manner ( Figure 12a), while dose-dependently reduce the cell damage caused by oxygen glucose deprivation-reoxygenation ( Figure 3), indicating that YC-6 can be upregulated by
  • the expression of NR4A3 plays a role in protecting vascular endothelial cells.
  • the expression of NR4A3 was analyzed by the method of cellular immunofluorescence. It was consistent with the results of immunoblotting.
  • the reoxygenation injury and YC-6 administration at the same time can increase the protein expression of NR4A3 in a dose-dependent manner ( Figure 12b-c).
  • hyaline membrane is considered to be a typical case feature of diffuse alveolar injury, indicating that acute low pressure hypoxia caused diffuse alveolar injury and increased permeability of the lung endothelial barrier.
  • YC-6 After administration of YC-6, it can improve the pathological changes of lung tissue caused by acute hypobaric hypoxia and reduce the increase in permeability of the air-blood barrier.
  • Our results show the importance of NR4A3 in reducing vascular endothelial cell damage caused by hypoxia-related stimuli, and YC-6 can up-regulate the protein expression of NR4A3 and reduce the vascular endothelial cell damage and acute hypobaric hypoxia caused by oxygen glucose deprivation-reoxygenation Caused endothelial barrier damage.
  • YC-6 inhibits the ubiquitination and degradation of NR4A3 caused by oxygen glucose deprivation-reoxygenation
  • Solvent treatment after reoxygenation had no significant effect on the ubiquitination of NR4A3, but The administration of YC-6 can inhibit the ubiquitination of NR4A3 caused by reoxygenation, and the administration of MG132 and YC-6 after reoxygenation did not significantly increase the ubiquitination of NR4A3, indicating that YC-6 inhibited the ubiquitination of NR4A3.
  • YC-6 can reduce the damage of vascular endothelial cells caused by oxygen glucose deprivation-reoxygenation.
  • One of its mechanisms is that YC-6 inhibits the ubiquitination and degradation of NR4A3, thereby promoting the survival of vascular endothelial cells under pathological stimulation.
  • YC-6 promotes the protein expression of NR4A3 in vascular endothelial cells of lung tissue under hypoxia stimulation, reduces the permeability of the lung endothelial barrier and related pathological changes caused by hypoxia, and plays a role in lung protection.
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  • PFKFB3 mediates CD163+ tumor-associated macrophage infiltration to regulate oral cancer angiogenesis[C]// Proceedings of the Ninth General Stomatology Conference of Chinese Stomatological Association. 2018.

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Abstract

5α-雄甾-3β,5,6β-三醇及其类似物、其氘代物或其药学上可接受的盐在制备治疗患者的肺部疾病的药物中的应用。这些化合物可以显著抑制PFKFB3表达上调、显著抑制乳酸堆积、显著减轻血管内皮细胞损伤、显著减轻肺泡上皮细胞损伤、显著抑制肺泡间隔增厚、显著减轻肺泡损伤，从而能够用于治疗肺部等肺上皮细胞损伤和/或血管内皮细胞损伤介导的疾病。

Description

小分子化合物在治疗肺上皮细胞损伤和/或血管内皮细胞损伤介导的疾病中的用途 技术领域
本发明涉及小分子化合物在治疗肺上皮细胞损伤和/或血管内皮细胞损伤介导的疾病中的用途，具体涉及5α-雄甾-3β,5,6β-三醇(在本文中有时简称“YC-6”或“YC6”)及其类似物的上述用途，特别是这些化合物在肺损伤和脑小血管病的治疗中的应用。
背景技术
肺损伤常见于肺部疾病，特别是如急性肺损伤/急性呼吸窘迫综合征(ALI/ARDS)、肺动脉高压、败血症等疾病中，严重危害身体健康，有些还具备很高死亡率。
急性肺损伤(Acute lung injury,ALI)是指由非心源性的各种严重肺内、外致病因素(如病毒感染、细菌感染、创伤、休克、缺血再灌注、急性胰腺炎、吸入性损伤、弥漫性肺泡损伤、其他毒素导致的中毒等)导致的急性低氧性呼吸功能不全或衰竭，其病理特征表现为肺泡上皮和肺毛细血管内皮通透性增加所致的肺水肿、肺泡塌陷和扩张、肺泡壁增厚和炎性细胞浸润等(Butt et al.,2016)。ALI发展至严重阶段被称为急性呼吸窘迫综合征(Acute respiratory distress syndrome,ARDS)，表现为顽固性低氧血症和呼吸窘迫，可出现呼吸衰竭、多器官功能障碍甚至死亡。ALI/ARDS死亡率能达到40％～60％，当前仍无特效药物疗法。
新型冠状病毒(SARS-CoV-2)(Wu F et al,2020)引起的2019冠状病毒疾病(Corona Virus Disease 2019,COVID-19)中的29％并发急性呼吸窘迫综合征(Acute respiratory distress syndrome,ARDS)(Huang C et al,2020)。新型冠状病毒病引发ARDS进一步得到病理学证据的证实(Xu Z et al,2020)，在两例死亡患者的组织学检查中发现双侧弥漫性肺泡损伤，伴随细胞纤维粘液样渗出物，存在肺水肿、肺细胞脱落和肺透明膜形成，从而严重影响肺的通气/换气功能，发展为ARDS。如何防止患者转为重型和对数以万计的重型/危重型患者进行救治是紧迫的医学与社会需求。
肺泡是肺脏进行气体交换的基本单位，其内表面覆盖着I型和II型肺泡上皮细胞。I型扁平细胞构成90％的肺泡表面积并且容易受伤。II型立体细胞构成剩余10％的肺泡表面积且不易受损，它们的功能包括产生表面活性剂、转运离子以及对I型细胞损伤后的增殖和分化。肺泡上皮除了形成致密的屏障以隔绝外源性致病原外，也通过其表面受体和分泌产物与免疫细胞相互作用来维持肺部的稳态和相对无菌性。其中，肺泡上皮细胞在肺部稳态中占主导作用，和肺部相关疾病如急性肺损伤、肺纤维化以及组织重建疾病都直接相关。
肺血管内皮细胞形成脉管系统内衬的单层，由于其生理位置暴露于多种损伤因素，例如LPS、内毒素、TNF-α和氧化应激，因此肺血管内皮细胞是各种肺损伤因素攻击的靶细胞，在ALI/ARDS的发病机制中担当着重要角色。研究表明处于危险期或具有早期和晚期ARDS患者的支气管肺泡灌洗液对人肺微血管内皮细胞具有细胞毒性。肺血管内皮细胞在细菌内毒素(lippopolysaccharide,LPS)、细胞因子、氧自由基等的作用下，导致毛细血管通透性增加和肺含水量增加，从而出现肺水肿，呼吸困难；分泌和释放各种炎性介质和细胞因子，使得促炎和抗炎介质失衡，同时凝血与抗凝系统失衡，引起肺微循环障碍和肺动脉高压，可以促进肺间质水肿、肺出血和进行性呼吸困难，导致患者出现进行性低氧血症和呼吸窘迫(杜景霞等，2012)。筛选可减轻不利因素导致的肺血管内皮细胞损伤的药物，有望应用于各种肺损伤相关的疾病的防治，包括各种感染性肺炎、急性肺损伤、ARDS、肺动脉高压等。
磷酸果糖激酶-2/果糖-2,6-二磷酸酶3(phosphofructokinase-2/fructose-2,6-bisphosphatase3,PFKFB3)是糖代谢通路中糖酵解途径的关键调节蛋白。PFKFB3在肺损伤过程中扮演关键角色。糖酵解酶PFKFB3抑制剂可以改善盲肠结扎和穿刺(CLP)诱导的ALI小鼠的存活率、肺部炎症、乳酸增加和肺细胞凋亡损伤(Gong Y et al,2017)。厌氧糖酵解也是脓毒症相关ALI细胞凋亡的重要因素，PFKFB3抑制剂可显著减轻LPS诱导的急性肺损伤/急性呼吸窘迫综合征(ALI/ARDS)实验动物的肺损伤(Wang L et al,2019)。在血管内皮细胞特异性敲除PFKFB3后，显著降低了内皮细胞的糖酵解水平，使生长因子、促炎细胞因子和细胞黏附因子的表达降低，进而抑制肺血管平滑肌细胞的异常增殖，以及肺血管周炎症细胞的浸润，抑制了缺氧诱导的肺动脉高压的发展(Cao Y,2019)。在平滑肌细胞特异性敲除Pfkfb3后，糖酵解的代谢产物乳酸含量减少，导致依赖ERK1/2的calpain-2的磷酸化激活被减少，引起肺血管平滑肌细胞中骨胶原合成减少，平滑肌细胞的异常增殖也减弱，进而抑制了肺动脉高压发展过程中的肺血管重构(Kovacs et al.,2019)。
血脑屏障(Blood-Brain Barrier,BBB)是中枢神经系统与循环系统之间的细胞界面，是脑屏障的一种。血脑屏障的结构包括血管内皮细胞、周细胞、星形胶质细胞足突、基膜等，并与神经元一起构成神经血管单元。血管内皮细胞构成血脑屏障的解剖基础，允许各种选择性转运系统转运营养物质及其他物质进出脑，保证细胞间隙对亲水性溶质的低通透性。越来越多的来自临床研究、神经病理、流行病学及动物模型等的证据表明，血管通透性增加引起的血脑屏障破坏是脑小血管病的始动因素之一。血管内皮细胞损伤以及内皮组织功能紊乱会引发血脑屏障的通透性增加，导致血液中的成分进入潜在的血管周围间隙及脑实 质，造成神经细胞及胶质细胞的损害。有报道指出血脑屏障的通透性增加早于神经损害及临床症状的出现。
多项临床研究显示，脑小血管病患者存在脑血流量减少和血管自动调节功能存在障碍，PET和MRI检查提示白质高信号患者呈现低灌注状态并存在血管通透性增加以及血脑屏障损害，而灰质无明显变化，提示血脑屏障受损区域以白质为主。研究显示，白质病变患者存在血脑屏障通透性的长期改变，同时临近皮质的白质高信号进展与血脑屏障的受损程度有关，血脑屏障通透性变化造成血浆外渗和周围组织损害是导致白质病变持续恶化的重要原因。利用DCE-MRI进行的研究显示，脑小血管病患者较多脑组织存在潜在的血脑屏障渗漏，进一步支持血脑屏障完整性受损是脑小血管病的主要发病机制。
目前临床上仍然缺乏有效的药物来治疗各种肺部疾病的肺损伤或血管内皮细胞受损介导的疾病，因此提供一种能够有效治疗这些疾病的药物具有重要的临床意义。
发明内容
发明人意外发现，化合物5α-雄甾-3β,5,6β-三醇可以显著抑制PFKFB3表达上调、显著抑制乳酸堆积、减轻血管内皮细胞损伤、减轻肺泡上皮细胞损伤、抑制肺泡间隔增厚、减轻肺泡损伤和肺炎性细胞浸润，从而能够用于治疗由肺泡上皮细胞损伤和/或血管内皮细胞损伤介导的各种疾病。
因此，本发明一方面提供式I的化合物、其氘代物或药学上可接受的盐在制备用于预防或治疗肺上皮细胞和/或血管内皮细胞受损介导的疾病的药物中的应用：
Figure PCTCN2021078061-appb-000001
其中R 1选自H、-CN、氟、氯、C 1-10烷基、氟或氯取代的C 1-10烷基、C 1-10烷氧基、氟或氯取代的C 1-10烷氧基和C 3-10环烷基。在一些实施方式中，所述R 1是H、-CHCH 2CH 3、-CH(CH 3) 2、-CH(CH 2) 3CH 3或-CH(CH 3)(CH 2) 3CH(CH 3) 2。在优选的实施方式中，所述R 1是H。
在另一个方面，本发明提供上述任一化合物、其氘代物或药学上可接受的盐用于预防或治疗肺上皮细胞受损和/或血管内皮细胞受损介导的疾病。
在又一个方面，本发明提供一种预防或治疗肺上皮细胞和/或血管内皮细胞受损介导的疾病的方法，所述方法包括向有此需要的对象施用预防上或治疗上有效量的上述任一化合物、其氘代物或药学上可接受的盐。
在本公开中，对于上述任一个方面：在一些实施方式中，所述肺上皮细胞和/或血管内皮细胞受损介导的疾病是急性肺损伤、急性呼吸窘迫综合症、肺动脉高压、肺水肿、肺纤维化、早产儿慢性肺疾病、慢性阻塞性肺病、肺孢子菌病和肺栓塞中的一种或多种。在一些实施方式中，所述急性肺损伤、急性呼吸窘迫综合症、肺动脉高压或肺水肿是由高氧、病毒感染、细菌感染、创伤、休克、缺血再灌注、急性胰腺炎、吸入性损伤、弥漫性肺泡损伤和/或中毒引起的。在一些实施方式中，所述急性肺损伤、急性呼吸窘迫综合症、肺动脉高压或肺水肿是由高氧、病毒感染、细菌感染、创伤、休克、缺血再灌注、急性胰腺炎、吸入性损伤、弥漫性肺泡损伤和/或中毒引起的，并且不是由低氧(例如高原环境低氧)引起的。在一些实施方式中，所述病毒是冠状病毒(例如是新型冠状病毒SARS-CoV-2)、流感病毒、呼吸道合胞病毒、腺病毒、副流感病毒、麻疹病毒、巨细胞病毒或其组合。在一些实施方式中，所述病毒是冠状病毒(例如是新型冠状病毒SARS-CoV-2)。在一些实施方式中，所述急性肺损伤是手术导致的肺损伤，所述手术例如是亚肺叶切除术、肺叶切除术或全肺切除术等肺切除术、肺部肿瘤切除术或肺移植。在一些实施方式中，所述肺纤维化是特发性肺纤维化或尘肺病。
在本公开中，对于上述任一个方面：在一些实施方式中，所述血管内皮细胞受损介导的疾病包括由血脑屏障破坏介导的脑小血管病，但不包括脑微出血、脑卒中以及脑水肿。在一些实施方式中，所述血脑屏障破坏表现为血脑屏障的通透性增加。在一些实施方式中，所述血脑屏障破坏表现为血脑屏障的血管内皮细胞的损伤。在一些实施方式中，由血脑屏障破坏介导的脑小血管病的临床表现为认知损害、步态障碍、情绪障碍、尿失禁和/或抑郁。在一些实施方式中，由血脑屏障破坏介导的脑小血管病的影像学表现包括脑白质病变。在一些实施方式中，由血脑屏障破坏介导的脑小血管病的影像学表现仅为脑白质病变。
在本公开中，对于上述任一个方面：在一些实施方式中，所述肺上皮细胞和/或血管内皮细胞受损介导的疾病是心血管疾病或糖尿病血管并发症。在一些实施方式中，所述心血管疾病选自急性心肌梗死(AMI)、心绞痛、冠心病、缺血性心脏病、心力衰竭、高血压、心血管介入术血栓形成中的一种或多种。在一些实施方式中，所述糖尿病血管并发症是糖尿病性视网膜病变、糖尿病性肾病、糖尿病性伤口愈合受损中的一种或多种。
附图说明
图1.YC-6显著抑制谷氨酸引起的PFKFB3的表达增加。用200μM谷氨酸刺激原代培养的神经元5mins和15mins，10μM YC-6、相应溶剂HP-β-CD以及NMDA受体阻断剂10μM MK-801预孵20mins作为实验组、溶剂对照组以及阳性对照药物组。收集蛋白后进行PFKFB3免疫印迹分析。
图2.YC-6显著抑制谷氨酸引起的PFKFB3下游糖酵解终产物乳酸的胞内堆积。用200μM谷氨酸刺激原代培养的神经元15mins，10μM YC-6、相应溶剂HP-β-CD预孵20mins作为实验组和溶剂对照组以及阳性对照药物组。收集细胞后进行胞内乳酸含量测定。
图3.YC-6显著减轻OGD-R引起的血管内皮细胞损伤。人脐静脉内皮细胞(HUVEC)和大鼠血管内皮细胞(RAOEC)给予氧糖剥夺损伤4小时后，再复氧至正常培养条件24小时后，进行LDH检测分析。YC-6预防性给药细胞在进行氧糖剥夺处理前，先预敷溶剂或药物(终浓度为1μM、3μM、10μM)1小时，而治疗性给药是在复氧处理的同时给与溶剂或YC-6(终浓度为1μM、3μM、10μM)。
图4.YC-6显著减轻缺氧和LPS引起的人肺泡上皮细胞的损伤。预孵YC-6的终浓度为1μM、5μM、10μM 1小时后，10μM地塞米松为药物对照，相应体积20％羟丙环糊精为溶剂对照，然后细胞进行20μg/mL LPS和1％O 2缺氧处理24小时。
图5.YC-6保护LPS引起急性肺损伤的病理学效应。红色箭头指示肺泡和肺间质水肿，蓝色箭头指示血管外周疏松的病变组织，黄色箭头指示炎性细胞浸润，绿色箭头指示支气管粘膜上皮损伤脱落，橙色箭头指示肺泡充血。黑色标尺表示100μm。
图6.YC-6显著降低LPS致大鼠急性肺损伤的病理评分。n＝9-10；*：p<0.05；**：p<0.01；***：p<0.001。
图7.急性低压缺氧导致食蟹猴肺损伤模型制作流程。在320米(A)、3000米(B)、4500米(C)、6000米(D)、7500米24小时(E)以及7500米48小时(F)缺氧并给予药物处理后，最后采集食蟹猴的肺样本。注射器显示给药时间点。
图8.YC-6抑制急性低压缺氧引起的食蟹猴肺血管充血和肺泡间隔增厚。常压常氧组(Normobaric normoxia,NN)；急性低压缺氧组(Hypobaric hypoxia，HH)；YC-6给药组(HH+YC-6)。红色箭头指示肺组织充血肿胀的血管断面。第二行图片为第一行图片蓝框区域的放大图，第二行红框是黑框区域进一步的放大图。黑色标尺表示500μm，红色标尺 表示100μm。
图9.YC-6显著抑制急性低压缺氧引起的食蟹猴肺泡间隔增厚统计分析。NN(Normobaric normoxia):常压常氧组，n＝3；HH(Hypobaric hypoxia):急性低压缺氧组，n＝5；HH+YC-6:YC-6给药组，n＝5；**：与NN比较，p<0.01；#：与HH组比较，p<0.05。
图10.YC-6抑制急性低压缺氧引起的食蟹猴肺泡腔蛋白透明膜形成和红细胞渗漏。NN(Normobaric normoxia):常压常氧组；HH(Hypobaric hypoxia):急性低压缺氧组；HH+YC-6:YC-6给药组。红色箭头指示肺泡腔内蛋白透明膜，黑色箭头指示肺泡间隔纤维性增生，蓝色箭头指示肺泡腔内的红细胞。黑色标尺表示100μm。
图11.YC-6抑制急性低压缺氧引起的食蟹猴肺组织炎性浸润。NN(Normobaric normoxia):常压常氧组；HH(Hypobaric hypoxia):急性低压缺氧组；HH+YC-6:YC-6给药组。黑色箭头指示肺泡膜间质的炎性细胞浸润，红色箭头指示肺泡腔内的炎性细胞浸润，蓝色箭头指示脱落肺上皮细胞。黑色标尺表示100μm。
图12.氧糖剥夺-复氧/缺氧刺激下YC-6上调血管内皮细胞NR4A3蛋白表达和减轻细胞损伤。(a)Western blot检测HUVEC和RAOEC细胞在正常培养、氧糖剥夺和氧糖剥夺-复氧状态下NR4A3蛋白表达水平。溶剂或YC-6在复氧处理的同时给与。(b)是a图同样处理下HUVEC和RAOEC细胞NR4A3免疫荧光染色的代表性图片。标尺表示50μm。(c)是对(b)图处理的NR4A3平均荧光强度的定量。(d)在正常环境(常压常氧)、低压缺氧和低压缺氧+YC-6条件下非人灵长类食蟹猴肺组织的NR4A3/CD31荧光双染代表性图片。白色标尺指示100μm。(e)是对(d)图处理的与CD31信号共定位的NR4A3的平均荧光强度和CD31的平均荧光强度的定量。正常、低压缺氧和低压缺氧+YC-6的样本个数分别是3、5和5。(c)和(e)图的统计方法采用单因素方差分析进行统计分析，并使用Tukey法进行多重比较；n.s.表示没有统计学差异；**表示P小于0.01；***表示P小于0.001。
图13.YC-6抑制氧糖剥夺-复氧引起的血管内皮细胞NR4A3的泛素化降解。(a)实时荧光定量PCR检测正常培养、氧糖剥夺、氧糖剥夺-复氧和氧糖剥夺-复氧+YC-6处理下HUVEC和RAOEC细胞NR4A3mRNA的相对表达量。溶剂或10μM的YC-6在复氧处理的同时给与细胞。(b)Western blot检测正常培养、氧糖剥夺、氧糖剥夺-复氧和氧糖剥夺-复氧+不同药物处理下HUVEC和RAOEC细胞NR4A3蛋白表达量。CHX、MG132和CQ在复氧处理的时候给与。(c)抗泛素抗体的免疫沉淀实验检测氧糖剥夺-复氧和氧糖剥夺- 复氧+药物处理后NR4A3泛素化修饰的改变。YC-6的使用浓度为10μM，MG132的使用浓度为100nM。(a)图中NR4A3mRNA的相对表达量的统计方法采用单因素方差分析进行统计分析，并使用Tukey法进行多重比较，n.s.指示没有统计学差异；**表示P小于0.01；***表示P小于0.001。CHX表示放线菌酮，CQ表示氯喹。Input表示用于免疫沉淀的初始样本，IB表示免疫印迹检测。
具体实施方式
如本文所用，术语“组合物”指适于给预期动物对象施用以达到治疗目的的制剂，其含有至少一种药物活性组分，例如化合物。任选地，所述组合物还含有至少一种药物学上可接受的载体或赋形剂。
术语“药学上可接受的”表示所述物质不具有这样的特性，即考虑到将被治疗的疾病或病症以及各自的施用途径，该特性将会使理性谨慎的医学从业者避免给患者服用该物质。例如，对于可注射物来说，通常要求这样的物质是基本无菌的。
在本文中，术语“预防有效量”和“治疗有效量”表示所述物质和物质的量对于预防、减轻或改善疾病或病症的一种或多种症状，和/或延长接受治疗的对象的存活是有效的。
本文使用的“治疗”包括给予本申请的化合物或其药学上可接受的盐，以减轻疾病或病症的症状或并发症，或消除疾病或病症。本文使用的术语“减轻”用于描述病症的迹象或症状的严重性降低的过程。症状可减轻而没有消除。在一种实施方案中，给予本申请的药物组合物导致消除迹象或症状。
本文使用的“预防”包括给予本申请的化合物或其药学上可接受的盐，以防止或组织产生特定疾病、症状或并发症。
本文使用的术语“C 1-10”或“C 3-10”或类似表达是指具有1至10个或3至10个碳原子。例如C 1-10烷基是指具有1至10个碳原子的烷基，如甲基、乙基、丙基、异丙基、丁基、异丁基、戊基、癸基等。
本文使用的术语“肺上皮细胞和/或血管内皮细胞受损介导的疾病”包括肺上皮细胞受损介导的疾病、血管内皮细胞受损介导的疾病以及肺上皮细胞和血管内皮细胞受损介导的疾病。
式I的化合物、其氘代物及药学上可接受的盐
可用于本发明的方法或应用的化合物包括式I的化合物、其氘代物或其药学上可接受的盐，
Figure PCTCN2021078061-appb-000002
其中R 1选自H、-CN、氟、氯、C 1-10烷基、氟或氯取代的C 1-10烷基、C 1-10烷氧基、氟或氯取代的C 1-10烷氧基和C 3-10环烷基。式I的化合物、其氘代物或其药学上可接受的盐在本文也称为“本发明的化合物”或“所述化合物”。
在一种实施方式中，其中R 1为H，即所述化合物为5α-雄甾-3β,5,6β-三醇(简称“YC-6”或“YC6”)，其结构式如式(II)所示。
Figure PCTCN2021078061-appb-000003
在一个实施方式中，R 1为-CHCH 2CH 3，所述化合物是17-亚丙基-雄甾-3β,5α,6β-三醇。在一个实施方式中，R 1为-CH(CH 3) 2，所述化合物是17-异丙基-雄甾-3β,5α,6β-三醇。在一个实施方式中，R 1为-CH(CH 2) 3CH 3，所述化合物是17-丁基-雄甾-3β,5α,6β-三醇。在一个实施方式中，R 1为-CH(CH 3)(CH 2) 3CH(CH 3) 2，所述化合物是胆甾烷-3β，5α，6β-三醇。
本发明的化合物可以被配制为药学上可接受盐的形式。预期的药学上可接受的盐形式包括，但不限于，单盐、双盐、三盐、四盐等。药学上可接受盐在它们被施用的量和浓度下是无毒的。在不阻止其发挥生理效应的情况下，通过改变化合物的物理特性，这样的盐的制备可以便于药理学应用。在物理性质上有用的改变包括降低熔点以便经粘膜给药，以及增加溶解度以便施用更高浓度的药物。
药学上可接受的盐包括酸加成盐，例如那些含硫酸盐、氯化物、氢氯化物、延胡索酸盐、马来酸盐、磷酸盐、氨基磺酸盐、乙酸盐、柠檬酸盐、乳酸盐、酒石酸盐、甲磺酸盐、乙磺酸盐、苯磺酸盐、对甲苯磺酸盐、环己氨基磺酸盐和奎尼酸盐的盐。药学上可接受的盐可以从酸获得，所述酸例如盐酸、马来酸、硫酸、磷酸、氨基磺酸、乙酸、柠檬酸、乳 酸、酒石酸、丙二酸、甲磺酸、乙磺酸、苯磺酸、对甲苯磺酸、环己氨基磺酸、延胡索酸和奎尼酸。
当酸性官能团例如羧酸或酚存在时，药学上可接受的盐也包括碱加成盐，例如那些含有苄星青霉素、氯普鲁卡因、胆碱、二乙醇胺、乙醇胺、叔丁胺、乙二胺、葡甲胺、普鲁卡因、铝、钙、锂、镁、钾、钠、铵、烷基胺和锌的盐。使用合适的相应的碱可以制备此类盐。
通过标准技术，可以制备药学上可接受的盐。例如，将游离碱形式的化合物溶解在合适的溶剂中，例如含有适宜酸的水性溶液或水-醇溶液中，然后蒸发溶液进行分离。在另一个实例中，通过使游离碱和酸在有机溶剂中反应来制备盐。
例如，如果特定化合物是碱，则可以通过本领域可得到的任何合适方法制备所需的药学上可接受的盐，例如，用无机酸或有机酸处理游离碱，所述无机酸例如是盐酸、氢溴酸、硫酸、硝酸、磷酸和类似酸，所述有机酸例如是乙酸、马来酸、琥珀酸、扁桃酸、富马酸、丙二酸、丙酮酸、草酸、乙醇酸、水杨酸、吡喃糖苷酸(pyranosidyl acid)(如葡糖醛酸或半乳糖醛酸)、α-羟基酸(如柠檬酸或酒石酸)、氨基酸(如天冬氨酸或谷氨酸)、芳香酸(如苯甲酸或肉桂酸)、磺酸(如对甲苯磺酸或乙磺酸)或类似物。
同样，如果特定化合物是酸，则可以通过任何合适方法制备所需的药学上可接受的盐，例如，用无机碱或有机碱处理游离酸，所述无机碱或有机碱例如是胺(伯胺、仲胺或叔胺)、碱金属氢氧化物或碱土金属氢氧化物或类似物。合适的盐的示范性例子包括有机盐，其衍生自氨基酸(如L-甘氨酸、L-赖氨酸和L-精氨酸)、氨、伯胺、仲胺和叔胺，以及环胺(如羟乙基吡咯烷、哌啶、吗啉和哌嗪)，以及无机盐，其衍生自钠、钙、钾、镁、锰、铁、铜、锌、铝和锂。
化合物的药学上可接受的盐可以作为络合物存在。络合物的例子包括8-氯茶碱络合物(例如，茶苯海明：苯海拉明8-氯茶碱(1:1)络合物；晕海宁)和各种包含环糊精的络合物。
本发明还预期包括使用该化合物的药学上可接受的氘代化合物或其他非放射性取代化合物。氘代是将药物活性分子基团中的一个或多个或全部氢替换成同位素氘，因其无毒无放射性，又比碳氢键稳定约6～9倍，可以封闭代谢位点而延长药物的半衰期，从而降低治疗剂量，同时又不影响药物的药理活性，而被认为是一种优良的修饰方法。
药物组合物
本发明另一方面提供一种药物组合物，其包含有效量的式I的化合物、其氘代物或药学上可接受的盐，以及药学上可接受的载体。
在本发明中，“药物组合物”是指包含式I化合物和药学上可接受的载体的组合物，其中化合物和药学上可接受的载体以混合形式存在于组合物中。所述组合物一般将被用于人类对象的治疗。然而，它们也可以被用于治疗在其它动物对象中的相似的或相同的病症。在本文中，术语“对象”、“动物对象”和类似术语指人和非人类脊椎动物，例如哺乳动物，如非人类灵长类，竞技动物和商业动物，例如马、牛、猪、绵羊、啮齿类动物，和宠物(如狗和猫)。
合适的剂型，部分地取决于用途或给药的途径，例如经口、经皮、经粘膜、吸入或通过注射(肠胃外)。此类剂型应当使该化合物能够到达靶细胞。其它因素在本领域中是熟知的，包括需要考虑的事项，诸如毒性和延迟化合物或组合物发挥其效应的剂型。
载体或赋形剂可以被用于生产组合物。所述载体或赋形剂可以被选择为促进化合物的给药。载体的例子包括碳酸钙、磷酸钙、各种糖(例如乳糖、葡萄糖或蔗糖)、或淀粉类型、纤维素衍生物、明胶、植物油、聚乙二醇和生理相容性溶剂。生理上相容性溶剂的例子包括注射用水(WFI)无菌溶液、盐溶液和葡萄糖。
可以通过不同的路径施用组合物或组合物的组分，包括静脉内、腹膜内、皮下、肌内、经口、经粘膜、直肠、经皮或吸入。在一些实施方式中，优选注射剂或冻干粉针剂。对口服而言，例如，化合物可以被配制为常规口服剂型，例如胶囊、片剂，以及液体制剂，例如糖浆、酏剂和浓缩滴剂。
可以获得口服用途的药物制剂，例如通过将组合物或其组分与固体赋形剂组合，任选研磨所形成的混合物，以及在加入合适的辅剂之后(如需要)加工颗粒的混合物，从而获得片剂或糖衣丸。合适的赋形剂特别是填料，如糖，包括乳糖、蔗糖、甘露糖醇或山梨醇；纤维素制剂，例如玉米淀粉、小麦淀粉、大米淀粉、马铃薯淀粉、明胶、黄蓍树胶、甲基纤维素、羟丙基甲基纤维素、羧甲基纤维素钠(CMC)和/或聚乙烯吡咯烷酮(PVP：聚维酮(povidone))。如果需要，可以加入崩解剂，例如交联的聚乙烯吡咯烷酮、琼脂或藻酸或它们的盐，例如藻酸钠。
作为选择，可以使用注射(肠胃外给药)，例如肌内的、静脉内的、腹膜内的和/或皮 下的。对于注射而言，本发明的组合物或其组分被配制为无菌液体溶液，优选在生理相容的缓冲液或溶液中，例如盐水溶液、Hank溶液或Ringer溶液。另外，组合物或其组分可以被配制为固体形式，并在使用之前一刻被再溶解或悬浮。也可以生产冻干粉形式。
给药也可以通过经粘膜、局部或经皮方式。对于经粘膜、局部或经皮给药，在配方中使用适合待穿透的屏障的穿透剂。这样的穿透剂在本领域中是普遍已知的，包括，例如，对于经粘膜给药，胆汁盐和梭链孢酸衍生物。另外，去垢剂可以用于促进穿透。经粘膜给药，例如，可以通过鼻喷雾或栓剂(经直肠或阴道)。
通过标准程序可以确定待施用的各种组分的有效量，考虑的因素例如所述化合物IC 50、所述化合物的生物半衰期、对象的年龄、大小和体重以及与对象有关的病症。这些因素和其它因素的重要性对本领域普通技术人员而言是熟知的。一般而言，剂量将在被治疗的对象的大约0.01mg/kg至50mg/kg之间，优选在0.l mg/kg至20mg/kg之间。可以使用多次剂量。
本发明的组合物或其组分还可以与治疗相同疾病的其他治疗剂结合使用。这种结合使用包括在不同时间施用这些化合物以及一种或多种其他治疗剂，或同时使用这种化合物和一种或多种其他治疗剂。在一些实施方式中，可对本发明的一种或多种化合物或结合使用的其他治疗剂的剂量进行修改，例如，通过本领域技术人员已知的方法降低相对于单独使用的化合物或治疗剂的剂量。
要理解的是，结合使用或联用包括与其他疗法、药物、医学程序等一起使用，其中该其他疗法或程序可在不同于本发明的组合物或其组分的时间(例如，在短期内(如几个小时，如1、2、3、4-24小时)或在较长时间内(如1-2天、2-4天、4-7天、1-4周))(或在与本发明的组合物或其组分相同的时间被施用。结合使用还包括与一次或不频繁施用的疗法或医学程序(如手术)一起使用，并伴随本发明的组合物或其组分在该其他疗法或程序之前或之后的短期或较长时间段内的施用。在一些实施方式中，本发明用于递送本发明的组合物或其组分和一种或多种其他药物治疗剂，它们通过相同或不同给药途径递送。
任何给药途径的结合施用包括通过相同给药途径将本发明的组合物或其组分和一种或多种其他药物治疗剂以任何制剂形式一起递送，包括两种化合物化学地相连且它们在施用时保持各自治疗活性的制剂。在一个方面，该其他药物疗法可与本发明的组合物或其组分共同施用。通过共同施用的结合使用包括施用共制剂(co-formulation)或化学上连接的化合物的制剂，或在短期内(例如，1小时内、2小时内、3小时内、直至24小时内)施 用两种或多种独立制剂形式的化合物，它们以相同或不同的途径给药。
独立制剂的共同施用包括经由一个装置的递送的共同施用，例如相同吸入装置、相同注射器等，或相对彼此短期内由不同装置施用。通过相同给药途径递送的本发明的化合物和一种或多种额外的药物疗法的共制剂包括将材料一起制备从而它们可通过一个装置被施用，包括不同化合物组合在一种制剂中，或化合物被修饰从而使得它们在化学上连接在一起但仍保持各自的生物学活性。这种化学上连接的化合物可包括将两个活性成分分开的连接体，该连接体在体内基本维持，或在体内可能降解。
医药用途及治疗方法
在一个方面，本发明提供所述任一化合物、其氘代物或药学上可接受的盐在制备用于预防或治疗肺上皮细胞和/或血管内皮细胞受损介导的疾病的药物中的应用。在一些实施方式中，所述肺上皮细胞和/或血管内皮细胞受损介导的疾病选自急性肺损伤、急性呼吸窘迫综合症、肺动脉高压、肺水肿、肺纤维化、早产儿慢性肺疾病、慢性阻塞性肺病、肺孢子菌病和肺栓塞中的一种或多种。在一些实施方式中，所述急性肺损伤、急性呼吸窘迫综合症、肺动脉高压或肺水肿是由高氧、病毒感染、细菌感染、创伤、休克、缺血再灌注、急性胰腺炎、吸入性损伤、弥漫性肺泡损伤和/或中毒引起的。在一些实施方式中，所述急性肺损伤、急性呼吸窘迫综合症、肺动脉高压或肺水肿是由高氧、病毒感染、细菌感染、创伤、休克、缺血再灌注、急性胰腺炎、吸入性损伤、弥漫性肺泡损伤和/或中毒引起的，并且不是由低氧引起的。在一些实施方式中，所述病毒是冠状病毒(例如是新型冠状病毒SARS-CoV-2)、流感病毒、呼吸道合胞病毒、腺病毒、副流感病毒、麻疹病毒、巨细胞病毒或其组合。在一些实施方式中，所述病毒是冠状病毒(例如是新型冠状病毒SARS-CoV-2)。在一些实施方式中，所述急性肺损伤是手术导致的肺损伤，所述手术例如是亚肺叶切除术、肺叶切除术或全肺切除术等肺切除术、肺部肿瘤切除术或肺移植术。在一些实施方式中，其中所述肺纤维化是特发性肺纤维化或尘肺病。优选的实施方式中，所述肺上皮细胞和/或血管内皮细胞受损介导的疾病是肺损伤。在优选的实施方式中，所述肺上皮细胞和/或血管内皮细胞受损介导的疾病是急性肺损伤。优选的实施方式中，所述肺上皮细胞和/或血管内皮细胞受损介导的疾病是急性呼吸窘迫综合症。优选的实施方式中，所述肺上皮细胞和/或血管内皮细胞受损介导的疾病是肺动脉高压。
在一些实施方式中，所述血管内皮细胞受损介导的疾病包括由血脑屏障破坏(或受损)介导的脑小血管病，但不包括脑微出血、脑卒中以及脑水肿。在一些实施方式中，其中所述血脑屏障破坏表现为血脑屏障的通透性增加。在一些实施方式中，其中所述血脑屏障破 坏表现为血脑屏障的血管内皮细胞的损伤。在一些实施方式中，其中由血脑屏障破坏介导的脑小血管病的临床表现为认知损害、步态障碍、情绪障碍、尿失禁和/或抑郁。在一些实施方式中，其中由血脑屏障破坏介导的脑小血管病的影像学表现包括脑白质病变。在一些实施方式中，其中由血脑屏障破坏介导的脑小血管病的影像学表现仅为脑白质病变。
在一些实施方式中，所述肺上皮细胞和/或血管内皮细胞受损介导的疾病是心血管疾病或糖尿病血管并发症。在一些实施方式中，所述心血管疾病选自急性心肌梗死(AMI)、心绞痛、冠心病、缺血性心脏病、心力衰竭、高血压、心血管介入术血栓形成中的一种或多种。在一些实施方式中，所述糖尿病血管并发症是糖尿病性视网膜病变、糖尿病性肾病、糖尿病性伤口愈合受损中的一种或多种。
在一个方面，本发明提供一种预防或治疗肺上皮细胞和/或血管内皮细胞受损介导的疾病的方法，所述方法包括向有此需要的对象施用预防上或治疗上有效量的本发明的任一化合物、其氘代物或药学上可接受的盐。在一些实施方式中，所述肺上皮细胞和/或血管内皮细胞受损介导的疾病选自急性肺损伤、急性呼吸窘迫综合症、肺动脉高压、肺水肿、肺纤维化、早产儿慢性肺疾病、慢性阻塞性肺病、肺孢子菌病和肺栓塞中的一种或多种。在一些实施方式中，所述急性肺损伤、急性呼吸窘迫综合症、肺动脉高压或肺水肿是由高氧、病毒感染、细菌感染、创伤、休克、缺血再灌注、急性胰腺炎、吸入性损伤、弥漫性肺泡损伤和/或中毒引起的。在一些实施方式中，所述急性肺损伤、急性呼吸窘迫综合症、肺动脉高压或肺水肿是由高氧、病毒感染、细菌感染、创伤、休克、缺血再灌注、急性胰腺炎、吸入性损伤、弥漫性肺泡损伤和/或中毒引起的，并且不是由低氧引起的。在一些实施方式中，所述病毒是冠状病毒(例如是新型冠状病毒SARS-CoV-2)、流感病毒、呼吸道合胞病毒、腺病毒、副流感病毒、麻疹病毒、巨细胞病毒或其组合。在一些实施方式中，所述病毒是冠状病毒(例如是新型冠状病毒SARS-CoV-2)。在一些实施方式中，所述急性肺损伤是手术导致的肺损伤，所述手术例如是亚肺叶切除术、肺叶切除术或全肺切除术等肺切除术、肺部肿瘤切除术或肺移植术。在一些实施方式中，其中所述肺纤维化是特发性肺纤维化或尘肺病。优选的实施方式中，所述肺上皮细胞和/或血管内皮细胞受损介导的疾病是肺损伤。在优选的实施方式中，所述肺上皮细胞和/或血管内皮细胞受损介导的疾病是急性肺损伤。优选的实施方式中，所述肺上皮细胞和/或血管内皮细胞受损介导的疾病是急性呼吸窘迫综合症。优选的实施方式中，所述肺上皮细胞和/或血管内皮细胞受损介导的疾病是肺动脉高压。
在一些实施方式中，所述血管内皮细胞受损介导的疾病包括由血脑屏障破坏(或受损)介导的脑小血管病，但不包括脑微出血、脑卒中以及脑水肿。在一些实施方式中，其中所述血脑屏障破坏表现为血脑屏障的通透性增加。在一些实施方式中，其中所述血脑屏障破坏表现为血脑屏障的血管内皮细胞的损伤。在一些实施方式中，其中由血脑屏障破坏介导的脑小血管病的临床表现为认知损害、步态障碍、情绪障碍、尿失禁和/或抑郁。在一些实施方式中，其中由血脑屏障破坏介导的脑小血管病的影像学表现包括脑白质病变。在一些实施方式中，其中由血脑屏障破坏介导的脑小血管病的影像学表现仅为脑白质病变。
在一些实施方式中，所述肺上皮细胞和/或血管内皮细胞受损介导的疾病是心血管疾病或糖尿病血管并发症。在一些实施方式中，所述心血管疾病选自急性心肌梗死(AMI)、心绞痛、冠心病、缺血性心脏病、心力衰竭、高血压、心血管介入术血栓形成中的一种或多种。在一些实施方式中，所述糖尿病血管并发症是糖尿病性视网膜病变、糖尿病性肾病、糖尿病性伤口愈合受损中的一种或多种。
本发明另一方面提供本发明的任一化合物、其氘代物或药学上可接受的盐用于预防或治疗肺上皮细胞和/或血管内皮细胞受损介导的疾病。在一些实施方式中，所述肺上皮细胞和/或血管内皮细胞受损介导的疾病选自急性肺损伤、急性呼吸窘迫综合症、肺动脉高压、肺水肿、肺纤维化、早产儿慢性肺疾病、慢性阻塞性肺病、肺孢子菌病和肺栓塞中的一种或多种。在一些实施方式中，所述急性肺损伤、急性呼吸窘迫综合症、肺动脉高压或肺水肿是由高氧、病毒感染、细菌感染、创伤、休克、缺血再灌注、急性胰腺炎、吸入性损伤、弥漫性肺泡损伤和/或中毒引起的。在一些实施方式中，所述急性肺损伤、急性呼吸窘迫综合症、肺动脉高压或肺水肿是由高氧、病毒感染、细菌感染、创伤、休克、缺血再灌注、急性胰腺炎、吸入性损伤、弥漫性肺泡损伤和/或中毒引起的，并且不是由低氧引起的。在一些实施方式中，所述病毒是冠状病毒(例如是新型冠状病毒SARS-CoV-2)、流感病毒、呼吸道合胞病毒、腺病毒、副流感病毒、麻疹病毒、巨细胞病毒或其组合。在一些实施方式中，所述病毒是冠状病毒(例如是新型冠状病毒SARS-CoV-2)。在一些实施方式中，所述急性肺损伤是手术导致的肺损伤，所述手术例如是亚肺叶切除术、肺叶切除术或全肺切除术等肺切除术、肺部肿瘤切除术或肺移植术。在一些实施方式中，其中所述肺纤维化是特发性肺纤维化或尘肺病。优选的实施方式中，所述肺上皮细胞和/或血管内皮细胞受损介导的疾病是肺损伤。在优选的实施方式中，所述肺上皮细胞和/或血管内皮细胞受损介导的疾病是急性肺损伤。优选的实施方式中，所述肺上皮细胞和/或血管内皮细胞受损介导的疾 病是急性呼吸窘迫综合症。优选的实施方式中，所述肺上皮细胞和/或血管内皮细胞受损介导的疾病是肺动脉高压。
在一些实施方式中，所述血管内皮细胞受损介导的疾病包括由血脑屏障破坏(或受损)介导的脑小血管病，但不包括脑微出血、脑卒中以及脑水肿。在一些实施方式中，其中所述血脑屏障破坏表现为血脑屏障的通透性增加。在一些实施方式中，其中所述血脑屏障破坏表现为血脑屏障的血管内皮细胞的损伤。在一些实施方式中，其中由血脑屏障破坏介导的脑小血管病的临床表现为认知损害、步态障碍、情绪障碍、尿失禁和/或抑郁。在一些实施方式中，其中由血脑屏障破坏介导的脑小血管病的影像学表现包括脑白质病变。在一些实施方式中，其中由血脑屏障破坏介导的脑小血管病的影像学表现仅为脑白质病变。
在一些实施方式中，所述肺上皮细胞和/或血管内皮细胞受损介导的疾病是心血管疾病或糖尿病血管并发症。在一些实施方式中，所述心血管疾病选自急性心肌梗死(AMI)、心绞痛、冠心病、缺血性心脏病、心力衰竭、高血压、心血管介入术血栓形成中的一种或多种。在一些实施方式中，所述糖尿病血管并发症是糖尿病性视网膜病变、糖尿病性肾病、糖尿病性伤口愈合受损中的一种或多种。
疾病或病症
急性肺损伤/急性呼吸窘迫综合症(ALI/ARDS)
急性肺损伤(ALI)是肺脏炎症和肺微血管通透性增加的急性、进行性缺氧性呼吸衰竭，其最终阶段即急性呼吸窘迫综合症(ARDS)。多种因素可引起急性肺损伤，包括但不限于，高氧、病毒感染、细菌感染、创伤、休克、缺血再灌注、急性胰腺炎、吸入性损伤、弥漫性肺泡损伤、中毒等。例如，在感染性肺炎(如细菌性肺炎或病毒性肺炎)中的或由其引起的急性肺损伤/急性呼吸窘迫综合症。例如，新型冠状病毒感染的肺炎(Corona Virus Disease 2019,COVID-19)的病理生理学特征包括：新型冠状病毒主要攻击患者肺部，导致患者弥漫性肺泡损伤，伴随细胞纤维黏液样渗出物，同时可见肺细胞脱落和肺透明膜形成。从而严重影响肺的通气/换气功能，发展为急性呼吸窘迫综合征。临床研究还发现，有些患者在病毒核酸检测转阴之后，肺部损伤仍然长时间存在，甚至还会进一步恶化。
在ALI中，肺血管内皮细胞的改变尤为引人注目，血管内皮细胞受损已被视为ALI发生、发展的病理基础。肺动脉高压(PAH)是各种原因引起ALI早期的主要表现之一。PAH可以促进肺间质水肿，肺出血和进行性呼吸困难，肺血管内皮细胞的功能障碍和损伤可能 是ALI时PAH形成的关键因素。血管内皮功能紊乱出现于血管改变之前，是可逆的，因此研究和认识其发生和发展规律及其调控机制，对防治ALI具有重要意义。
研究证实，ALI/ARDS死亡的患者中肺泡上皮细胞的破坏非常明显，尽管肺毛细血管也有一定程度的损伤，但是仍以上皮细胞的损伤为主。在肺泡的液-气界面，肺泡上皮细胞使肺泡内衬液维持适当的水和溶质含量，这对气体交换以及宿主抵抗病毒及细菌等病原体至关重要。若肺内存在内皮通透性改变，肺间质及肺泡腔内的水肿液最终将被清除，也不会发展为肺间质纤维化。相反，肺泡上皮细胞的大量损伤会增加蛋白通透性、降低肺泡液体和肺泡蛋白清除率，进而造成肺泡内聚集大分子量的蛋白及无序修复，导致气体交换恶化。在ALI过程中肺泡上皮的破坏导致上皮细胞的脱落及随之富含蛋白的水肿液渗入肺泡腔，加速肺泡屏障的破坏。上皮细胞破坏的形式包括凋亡及坏死。在ARDS死亡患者及高氧环境、LPS刺激、盲肠结扎穿孔、缺血再灌注、呼吸机相关肺炎及呼吸机相关肺损伤动物模型均可发现肺泡壁存在这两种死亡方式。坏死导致细胞膜破坏，胞质溢出，进一步激发炎症反应。肺泡收到机械损伤、高温、局部缺血或细菌产物刺激均可直接导致坏死。凋亡与表面死亡受体相关，通过细胞清除仅引起轻微炎症。广泛上皮细胞凋亡及分离导致细胞基底膜暴露于肺泡腔的炎症产物，如氧化剂、蛋白酶及炎症因子。上皮细胞破坏引起成纤维细胞的增殖及胶原形成可能导致肺纤维化。在损伤之后，修复肺泡上皮完整性对于恢复上皮细胞的正常功能及清除肺水肿十分重要。一系列研究证实ALI/ARDS患者自身产生或是否入肺泡腔内的某些因子会促进上皮修复。这些可溶性因子来自于患者肺内的成纤维细胞、巨噬细胞、内皮细胞、上皮细胞、细胞外基质、血浆渗出物，组成细胞因子、趋化因子、生长因子、前列腺素及机制成分。尽管对ALI/ARDS研究很多，但是患者病死率居高不下。由于现有药物治疗效果不佳，所以ARDS的治疗仍以支持治疗为主。决定ALI严重性和进展的主要因素之一是肺泡上皮损伤成都。从上皮细胞的治疗角度出发，抑制上皮细胞的早起损伤，加速上皮细胞修复，消除肺水肿，都将是提高患者生存率的有效措施。
特发性肺纤维化(IPF)
特发性肺纤维化(IPF)是肺泡上皮细胞损伤、组织异常增生导致的肺部疾病。研究证实，发病机制为多种原因导致肺泡上皮细胞的损伤，最后导致成纤维细胞增殖和肌成纤维细胞聚集，形成成纤维细胞灶。上皮细胞凋亡可能是IPF早期发生发展的重要因素。炎症反应、细胞内张力、端粒酶活性等因素参与了肺泡上皮细胞凋亡，并在肺纤维化发病的早期阶段起到重要作用。IPF的重要病理特征表现为成纤维细胞活跃增殖形成的纤维化灶。成纤维细胞灶是上皮细胞损伤和修复的部位，上皮细胞损伤后能分泌的多种介质促使成纤 维细胞的迁移、增殖、分化，从而引起肺泡内广泛的纤维化，最终导致进行性呼吸困难。IPF的发病机制可能是肺泡上皮细胞损伤后的异常再上皮化和成纤维细胞异常增殖所致。IPF中的细胞死亡表现为上皮细胞损伤后的细胞凋亡，在疾病发生中起重要作用。研究证实，诱导肺上皮细胞凋亡能导致肺组织发生纤维化。肺泡上皮细胞损伤作为肺纤维化发生的最初因素是目前最为学者们接受的理论假设，靶向降低肺泡上皮细胞凋亡的药物可能为防止IPF提供新的治疗方案。研究表明，肺组织中的有氧糖酵解与肺纤维化密切相关。Hu等(2020)发现PFKFB3抑制剂3PO可以逆转LPS诱导的肺纤维化的小鼠模型中PFKFB3表达的显著上调和有氧糖酵解增强。
尘肺病
尘肺病是长期吸入生产性粉尘引起的以肺组织不可逆性纤维化为主的全身性疾病。目前尘肺病尚缺乏特效治疗方法，再粉尘导致肺纤维化的病理过程中，部分肺泡上皮细胞异常激活，也有损伤和凋亡，由于上皮细胞修复紊乱、促纤维化因子释放以及上皮间质转化(EMT)等过程，促进成纤维细胞灶和肌成纤维细胞灶形成以及大量细胞外基质产生，最终引起肺泡结构破坏和肺纤维化发生。
高氧肺损伤
高氧肺损伤是一种以弥漫性肺细胞损伤为基础，可迅速影响气体交换的肺部炎症性疾病。高氧肺损伤是呼吸高于常压下体积分数为21％的高分压氧气或高于1个绝对大气压的高压氧而发生的包括肺型氧中毒在内的急慢性非损害的统称。由于新生儿，特别是早产儿，抗氧化酶系统和肺表面活性物质发育不成熟，高氧进入肺部后，产生氧自由基，引起细胞损伤、凋亡、坏死，同时产生炎症介质，炎症细胞浸润，从而导致肺部炎症反应、组织损伤和异常修复的发生。高氧引起肺泡上皮细胞氧化损伤，抑制了肺泡化的进程，广泛肺泡上皮细胞的损伤及损伤修复能力的下降使气血屏障通透性明显增加出现肺水肿的表现，促进了ALI的发生。
早产儿慢性肺疾病
早产儿慢性肺疾病(chronic lung disease,CLD)是早产儿长时间吸入高浓度氧或机械通气治疗或感染后最常见的和最严重的并发症。早产儿CLD作为早产儿肺透明膜病治疗的并发症在1967年首次被Northway描述，也称为支气管肺发育不良(bronchopulmonary dysplasia,BPD)。氧疗法是患有心肺疾病的早产儿最常应用的一种治疗手段。这一措施虽可改变患儿的低血氧状态、挽救患儿的生命，但长期吸入高浓度的氧气，可引起不同程度的 肺损伤，严重者可发展为CLD。随着机械通气的广泛开展、早产儿管理技术的日益提高及肺表面活性物质的普遍应用，使早产儿，特别是超低出生体重儿(extremely low birth weight,ELBW)(出生体重1000g存活率有了明显改善,但随之而来的是早产儿慢性肺疾病发生率的提高,国外已达30％-40％,国内也有上升趋势。由于尚缺乏有效的监控和防治手段,其中10％-15％CLD患儿因严重肺功能障碍而死于呼吸衰竭,存活者也需长期依赖氧气或机械通气治疗。初期肺泡上皮细胞(alveolar epithelial cell，AEC)损伤和晚期肺纤维化是CLD主要病理特征。目前多试图通过减轻AEC损伤程度或减少肺组织胶原沉积而达到有效防止CLD的目的。
慢性阻塞性肺病
慢性阻塞性肺病(chronic obstructive pulmonary disease，COPD)是全球范围内呼吸系统疾病中发病率和死亡率居于前位的疾病，并且其发病还呈蔓延之势。已知吸烟是导致COPD的主要病因，香烟烟雾(cigarette smoke，CS)是一种富含氧化剂成份的复杂的混合物，在构成肺脏的多种细胞类型中，肺泡上皮细胞是遭受CS中氧化剂损伤的主要部位。研究显示，COPD患者肺泡上皮细胞凋亡指数明显高于肺血管内皮细胞凋亡指数，且二者呈正相关。内皮细胞的损伤与凋亡首先破坏肺泡结构的完整性，而后可能出现肺泡上皮细胞的损伤或凋亡。内皮细胞损伤或凋亡后，可能通过直接的细胞间相互作用和间接的某种机制，启动触发了其他细胞尤其是肺泡上皮细胞凋亡，从而形成COPD及肺气肿。此外，COPD患者存在血管内皮功能紊乱，且与疾病的严重程度具有相关性。血管内皮细胞功能异常与COPD的发生、发展有着紧密的关联。炎症、血管损伤、血液动力学改变、低氧、氧化应激和凋亡等可促进血管内皮细胞功能紊乱。研究显示，即使是COPD早期，甚至在肺功能正常的健康吸烟者中，血管内皮细胞功能已发生改变。血管内皮细胞功能的异常变化可能在COPD的发生、发展以及相关的心血管事件高发中具有重要作用。
肺孢子菌病
肺孢子菌病是由肺孢子菌引起的间质性肺炎(肺孢子菌肺炎，PCP)，临床主要表现为发热、干咳、进行性呼吸困难等，单纯吸氧不能缓解，经对因治疗后可迅速恢复，多发生于免疫功能低下者，艾滋病患者的发病率高达70-80％。肺孢子菌毒力弱，健康人多呈隐性感染，只有在宿主免疫功能低下时，潜伏的肺孢子菌才大量繁殖，导致PCP发生。吸入下呼吸道的肺孢子菌直接导致I型肺泡上皮细胞损伤并坏死，肺泡毛细血管通透性增加，肺泡内充满肺孢子菌和泡沫状嗜酸性物质，使肺泡的表面活性物质减少，影响气体交换，出现低氧血症(PCP患者最主要的特点)。肺泡II型上皮细胞代偿性肥大，肺泡间隙上皮细 胞增生、肥厚、部分脱落，同时间质内巨噬细胞和浆细胞增生，间质纤维化，造成肺功能严重障碍。
肺栓塞
肺栓塞是脱落的血栓或其他物质阻塞肺动脉或其分支的病理过程，是一种具有较高发病率和死亡率的常见疾病。血管内皮细胞损伤是引发肺栓塞的第一要素。内皮细胞通过微粒、组织因子等生物分子作用于血小板和中性粒细胞，参与血栓形成。由于血管内皮细胞损伤和修复在肺栓塞过程中具有重要地位，近年来关于肺栓塞的发生、发展机制的研究也越来越着重于对内皮细胞损伤和修复的分子学、细胞学、病理学的探索。
心血管疾病
血管内皮细胞受损与许多心血管疾病的发生和发展有关，血管内皮损伤引发血栓形成是许多心血管疾病的病理基础。最早在亚急性心内膜炎病人的外周血中发现有脱落的血管内皮细胞，随着研究的深入，人们发现许多心血管疾病的循环内皮细胞均有不同程度的变化。急性心肌梗死(AMI)和心绞痛患者的循环内皮细胞明显增加并可持续数天。冠心病急性发作和家中是由于内皮细胞损伤后发生功能变化，血管内皮细胞损伤程度和病情严重程度一致。国内研究发现，缺血性心脏病中AMI 46小时内循环内皮细胞较正常人明显增加，可持续24小时。再灌注时造成的损伤亦可致循环内皮细胞增多。冠心病合并其他危险因素如心力衰竭(CHF)时，可使循环内皮细胞升高明显。研究发现，CHF越重，血管内皮受损越明显，血管内皮受损反过来加重病情。此外，很多研究表明，内皮细胞损伤导致的功能障碍与高血压密切相关，一方面血管内皮功能障碍在高血压的发生、发展过程中起重要作用，另一方面高血压本身又加重血管内皮障碍，形成恶性循环。高血压大鼠的内皮细胞在损伤的脏器血液中明显提高，高血压患者的循环内皮细胞明显高于常人，其增加数量较冠心病患者更高。心血管介入术主要是机械刺激血管内皮，导致损伤。不同术式对血管内皮的损伤程度不同。血管内皮细胞受损时引起内皮功能失调，血液动力学发生改变，由此引发大量诱聚物和血管活性物质在损伤血管局部堆积，血小板活化，是心血管介入术血栓形成的主要病理生理机制。
糖尿病血管并发症
研究证实，无论是长期高血糖还是急性高血糖都能对人和动物内皮功能造成损伤。血管内皮功能障碍是糖尿病血管并发症(尤其是视网膜病变、肾病和伤口愈合受损)发生的重要原因，血管内皮功能的改变也是糖尿病其他发病机理的重要因素，主要体现在胰岛素 抵抗、脂毒性和胰岛素分泌受损。目前认为，血管内皮功能损伤是糖尿病血管病变发生的始动因素和主要病理生理学基础，甚至尚未出现慢性血管并发症的糖尿病患者已出现内皮功能明显降低。
脑小血管病
脑小血管指脑的小的穿支动脉和小动脉(直径40～200μm)、毛细血管及小静脉,它们构成了脑组织血供的基本单位，对脑功能的维持起着重要作用。脑大、小血管共同构成了脑血管树(vascular tree)，它们在结构上有连续性，共同受到血流动力学影响，共同暴露于危险因素，因此脑大、小血管病变从理论上应具有严重程度的平行相关性。但在临床工作中常常会发现二者的不一致性，例如常常发现有严重脑小血管病变但并不合并脑大动脉狭窄的患者，反之亦然。
脑小血管病(cerebral small vessel disease,CSVD)泛指上述小血管的各种病变所导致的临床、认知、影像学及病理表现的综合征，习惯上多指小的穿支动脉和小动脉病变所导致的临床和影像学表现。CSVD主要以卒中(深部小梗死、脑出血)、认知和情感障碍及总体功能下降为突出的临床表现，影像学上则突出表现为腔隙性梗死(lacunar infarction，LI)、腔隙(lacune)、脑白质病变(white matter lesions,WML)、血管周围间隙扩大(enlarged perivascular space,EPVS)及脑微出血(cerebral microbleeds,CMB)等。
脑小血管病可以累及小动脉、毛细血管以及小静脉，以穿通动脉受累最为常见。高血压、血管炎症或遗传缺陷引起的血管内皮细胞损伤、平滑肌增生、小血管壁的基底膜增厚都可以引起慢性脑组织缺血。血管平滑肌细胞丢失和增生、血管壁增厚、血管官腔狭窄，引起慢性、进行性的局部甚至是弥散性亚临床缺血，神经细胞脱髓鞘、少突胶质细胞丢失、轴索损伤，造成不完全性缺血。此阶段没有临床症状，核磁共振检查显示为脑白质病变。此外，另一些研究结果认为内皮损伤后血管通透性增加导致血管内物质外渗，引起血管及血管周围组织损伤，对这一阶段疾病的进展可能也发挥着重要作用。
在本公开中，所述血管内皮细胞受损介导的疾病包括由血脑屏障破坏或受损介导的脑小血管病，但不包括脑微出血、脑卒中以及脑水肿。在本发明的特别优选的实施方式中，由血脑屏障破坏介导的脑小血管病包括脑白质病变。在本发明的特别优选的实施方式中，由血脑屏障破坏介导的脑小血管病仅表现为脑白质病变。
实施例
实施例1.YC-6显著快速抑制谷氨酸引起的PFKFB3的表达上调
研究表明，磷酸果糖激酶-2/果糖-2,6-二磷酸酶3(phosphofructokinase-2/fructose-2,6-bisphosphatase 3,PFKFB3)表达增加及其下游产物乳酸的堆积在急性肺损伤、肺动脉高压等多种肺部疾病中扮演病理损伤性角色。在急性肺损伤、肺动脉高压等疾病发生发展中有关键性病理损伤作用。我们分析了YC-6对肺损伤关键分子PFKFB3蛋白水平的影响。结果出人意料地显示，YC-6快速抑制PFKFB3蛋白表达上调和乳酸堆积，提示了这可能是YC-6肺保护的独特机制。
原代小脑颗粒神经元培养
细胞取材自SD P7-8新生乳鼠，取出小脑，使用显微镊进一步除去脑膜和血管；将分离出的小脑组织转移至另一装有解剖液的培养皿。利用组织剪尽可能将组织剪碎。消化：用滴管将剪碎的组织加入7mL 0.25％胰蛋白酶消化液中，在37℃消化15mins；整理细胞超净台台面并清洗器械。每五分钟上下颠倒几次使得组织与消化液更充分接触。终止消化：消化结束后，可以看到由于细胞破碎DNA释放会使得组织粘连在一起悬浮在液体中。加入3mL含DNA酶的10％FBS DMEM培养基终止消化，上下颠倒几次可同时观察到由于DNA的水解使得剩余的组织分散开来。1000rpm离心5mins。小心尽可能去除上清。收集单细胞悬液：用滴管吸取7mL含DNA酶的10％FBS DMEM培养基，轻柔吹打组织沉淀。快速离心使转速达到1500rpm即停止，收集单细胞悬液至一新的15mL离心管中。接种细胞：1000rpm离心5分钟，收集细胞沉淀，用含10％FBS的DMEM重悬细胞。手持细胞计数仪进行计数，接种密度为4.0-5.0×10 5个/mL。35mm培养皿接种体积为2mL，48孔板接种体积为300μL。细胞接种与培养：接种24小时后,补充阿糖胞苷(终浓度为10μM)抑制胶质细胞的生长。第七天时补充葡萄糖(5mM)维持营养,第八天使用。谷氨酸诱导的原代小脑颗粒神经元损伤模型及药物处理。
细胞处理与给药
将种植在六孔板的原代小脑颗粒神经元的培养基换成2mL kreb’s buffer,以此作为正常对照组。加入终浓度为200μM的谷氨酸处理5mins以及15mins相应的时间并将此作为模型组；提前20mins预孵10μM YC-6或者NMDA受体阻断剂MK-801,然后加入上述浓度的谷氨酸进行处理相应时间作为药物处理组；加入和药物处理组等量的溶剂HP-β-CD作为溶剂对照组，并且其预孵时间和药物处理组相同。
蛋白免疫印迹
配制体积分数为12％SDS-聚丙烯酰胺凝胶，按照每孔上样蛋白总量为20μg进行样 品加注，100V恒压条件下电泳分离；之后用湿转法转移到PVDF膜。5％脱脂奶粉室温封闭1h；加入稀释后的一抗(抗体稀释液为3％BSA，抗体稀释比例为1：1000)，4℃孵育过夜；TBST洗涤3次,每次5mins,加入相应二抗(抗体稀释液为3％BSA，抗体稀释比例为1：5000),室温孵育1h，TBST洗涤3次，每次5mins。化学发光法显色曝光拍照。
YC-6显著快速抑制谷氨酸引起的PFKFB3的表达上调
如图1所示，与正常对照组相比，谷氨酸刺激导致胞内PFKFB3表达快速上调，而YC-6处理显著抑制这种急速上升。
实施例2.YC-6对肺上皮损伤关键代谢分子乳酸的作用
乳酸是厌氧糖酵解产物，是PFKFB3调控的代谢通路下游，对肺上皮细胞直接产生损伤作用。Gong Y等研究证实，在体外LPS引起人肺泡上皮A549细胞的凋亡、炎性细胞因子产生、糖酵解通量增强和活性氧(ROS)增加，并且这些变化被PFKFB3抑制剂3PO逆转。更为重要的是，乳酸也是导致肺损伤的关键代谢物，乳酸处理A549细胞会导致细胞凋亡和ROS增强。这些结果表明厌氧糖酵解可能是脓毒症相关ALI肺上皮细胞凋亡损伤的重要因素。在YC-6抑制PFKFB3表达基础上，我们进一步探讨YC-6对其下游具备肺上皮细胞损伤作用的乳酸水平的影响。结果显示，YC-6抑制糖酵解关键酶PFKFB3的同时，显著抑制了乳酸堆积。乳酸堆积的减轻可能是YC-6减轻肺上皮细胞损伤的机制之一。
细胞胞内乳酸测定
预处理：胞内乳酸含量测定使用的细胞数量为1-2*10^6。细胞前处理具体实验步骤包括以下，用预冷的Kreb’s buffer清洗细胞三次。加入220μL的细胞裂解液充分裂解。细胞后收集裂解液至1.5mL EP管内。4℃16000g条件下离心5mins，收集上清160μL至新的EP管内，其余上清做BCA蛋白定量。加入56μL 4mM HClO 4,上下颠倒充分混匀，至冰上反应五分钟除去样品本身已有的LDH，防止内源性干扰，此步骤应观察到管底有白色蛋白沉淀。4℃16000g离心5mins。收集上清160μL，将其置于新的EP管内。加入68μL 2mM KOH充分上下混匀，至冰上反应5mins。4℃16000g条件下离心15mins,收集160μL上清至新的EP管内，如果上清有白色絮状物，则再次离心15mins。将上清按照600-800稀释倍数用细胞裂解液稀释，并将此稀释液作为待测样品液备用。
胞内乳酸含量测定方法根据L-Lactate Assay试剂盒(abcam,ab65331)说明书来测定。具体步骤为：取50μL的稀释液加入到96孔黑底的细胞培养板中，随即加入等体积的含有 LDHA的底物溶液，摇晃混匀后将其放置在37℃无二氧化碳的孵箱中孵育30mins。用酶标仪在避光，激发光波长为535nm的条件下测定荧光强度。将样品中读出的荧光强度带入到不同标准品乳酸浓度对应的荧光强度的标准曲线中，将荧光强度转化为样品液中乳酸的实际浓度。
YC-6显著抑制谷氨酸引起的PFKFB3下游糖酵解终产物乳酸的胞内堆积
将原代小脑颗粒神经元细胞置于200uM谷氨酸刺激15mins后收集细胞裂解液研究YC-6处理对糖酵解产物乳酸的影响(图2)，谷氨酸刺激15mins后神经元胞内乳酸含量显著增加，而YC-6处理抑制了这种增加。以上实验结果表明，YC-6抑制糖酵解关键酶PFKFB3的同时，显著抑制了乳酸堆积。
实施例3.YC-6显著减轻血管内皮细胞损伤
肺血管内皮细胞形成脉管系统内衬的单层，由于其生理位置暴露于细菌内毒素、LPS、炎症因子如TNF-α、化学毒物和氧化应激等直接刺激之下，导致内皮细胞的损伤与凋亡。肺血管内皮细胞受损伤后引起毛细血管通透性增加导致肺含水量增加，从而出现肺水肿，呼吸困难；同时肺血管内皮细胞分泌和释放各种炎性介质和细胞因子，使得促炎和抗炎介质、凝血与抗凝系统失衡，引起肺微循环障碍和肺动脉高压，可以促进肺间质水肿、肺出血和进行性呼吸困难，患者出现进行性低氧血症和呼吸窘迫。为评价YC-6是否对血管内皮细胞损伤具有保护作用，应用氧糖剥夺/再复氧(oxygen-glucose deprivation and restoration,OGD-R)损伤模型，通过检测细胞LDH释放评价预防以及治疗性给药后细胞损伤情况。结果显示YC-6剂量依赖性地显著减轻血管内皮细胞损伤，提示在各种病理因素引起的血管内皮细胞相关的肺损伤过程中YC-6可发挥有效保护作用。
细胞培养
HUVEC细胞用含10％胎牛血清的改良型DMEM培养基培养，待细胞长至约80％融合度的时候，用0.25％胰蛋白酶消化细胞，然后用含10％肽牛血清的改良型DMEM培养基吹打均匀，把细胞浓度调整至1×10 5个/mL，按400uL/孔接种于24孔板，待细胞长至约70％融合度的时候进行实验。RAOEC细胞用含10％胎牛血清的高糖DMEM培养基培养，接种条件与HUVEC一致。
构建氧糖剥夺-复氧(OGD-R)损伤模型
设置缺氧工作站的氧气浓度为1％(1％O 2，5％CO 2，94％N 2)，温度为37℃，湿度为 85％。把无糖DMEM培养基置于缺氧工作站预缺氧3小时。吸走培养板中的培养基，用预热的无糖DMEM培养基洗2遍，然后每孔加入100uL的无糖DMEM培养基，将培养板置于缺氧工作站，每孔再加入300uL预缺氧的无糖DMEM培养基，然后将培养板静置于缺氧工作站继续缺氧4小时。然后进行复氧处理，把培养板取出，把无糖DMEM培养基更换为400uL含10％肽牛血清的正常培养基，然后置于37℃5％CO 2细胞培养箱继续培养24小时。正常组的细胞仅把培养基更换为400uL新的含10％胎牛血清的正常培养基。
给药方法
预防性给药：细胞在进行氧糖剥夺处理前，先预敷溶剂或药物(终浓度为1μM、3μM、10μM)1小时，然后在氧糖剥夺处理时，在无糖DMEM培养基中加入对应的溶剂或药物，复氧处理时，在正常培养基中加入对应的溶剂或药物，直至实验终点。模型对照组不加任何药物处理，每个处理组进行3个复孔重复。
治疗性给药：对细胞进行氧糖剥夺处理后，在复氧处理的同时，给与溶剂或药物(终浓度为1μM、3μM、10μM)，直至实验终点。模型对照组不加任何药物处理，每个处理组进行3个复孔重复。
细胞损伤检测(LDH检测)
实验终点时，每孔各取50uL培养基到96孔板，然后按照LDH检测试剂盒的操作指引进行细胞毒性检测，结果计算如下：
Figure PCTCN2021078061-appb-000004
实验结果用One-way Anova单因素方差分析法进行统计分析，并使用Tukey法进行多重比较，P<0.05认为具有显著性统计学差异。
实验结果
如图3结果显示，OGD-R处理引起人脐静脉内皮细胞(HUVEC)和大鼠血管内皮细胞(RAOEC)LDH释放的增加，说明OGD-R损伤处理引起了HUVEC细胞和RAOEC细胞的损伤和死亡；而无论给予损伤处理之前一小时预防性给药还是在复氧处理时治疗性给药，YC-6剂量依赖性显著减轻OGD-R引起的LDH释放增加，减轻细胞损伤。YC-6剂量依赖性地显著减轻血管内皮细胞损伤，提示在各种病理因素引起的血管内皮细胞相关的肺损伤过程中YC-6可发挥有效保护作用。
实施例4.YC-6减轻缺氧和LPS引起的肺泡上皮细胞损伤
肺泡上皮细胞及毛细血管内皮细胞是各种炎症、有毒物吸入、病毒感染以及脓毒症等损伤性致病因素打击的直接目标，损伤后通透性增加、发生凋亡，造成弥漫性肺间质及肺泡水肿，导致进行性低氧血症和呼吸窘迫。在新冠肺炎发病过程中，肺上皮细胞就是病毒攻击的主要目标。新型冠状病毒(SARS-CoV-2，以下简称CoV)是一种单正链RNA病毒，长度约为27-32kb，病毒基因组主要由复制酶编码区和结构蛋白编码区两部分构成。来自NIH团队等报道确定了2019-nCoV进入人体细胞的受体为ACE2。CoV特异识别和入侵血管紧张素转化酶2受体ACE2表达丰富的人体细胞，尤其是呼吸道上皮细胞和肺泡细胞，因此极易在下呼吸道感染和扩散，引发肺炎。但在临床上可以观察到在病毒转阴后，肺部炎症仍然可以存在甚至恶化，单纯的抗病毒治疗并不能挽救许多重症和危重症患者的生命。肺上皮细胞(HPAEpiC)包括全部两种Ⅰ或Ⅱ型肺泡上皮细胞，为评价YC-6是否直接对肺上皮细胞(HPAEpiC)具有保护作用，本部分应用细菌脂多糖LPS和缺氧损伤模型，通过检测细胞LDH释放评价YC-6对肺上皮细胞的保护作用，为YC-6治疗包括新冠病毒在内等不同因素引起的ARDS提供实验依据。实验结果显示YC-6剂量依赖性显著减轻缺氧和LPS引起的肺泡上皮细胞损伤。
细胞培养
HPAEpiC细胞用肺泡上皮细胞培养基培养，待细胞长至约80％融合度的时候，用0.25％胰蛋白酶消化细胞，然后用肺泡上皮细胞培养基吹打均匀，把细胞浓度调整至2×10 5个/mL，按400uL/孔接种于24孔板，待细胞长至约70％融合度的时候进行实验。
LPS+缺氧模型的制作
设置缺氧工作站的氧气浓度为1％(1％O 2，5％CO 2，94％N 2)，温度为37℃，湿度为85％。待缺氧工作站条件稳定后，将1mg/mL的LPS直接加入细胞中，使LPS终浓度为20μg/mL，然后把细胞放到缺氧工作站培养24小时。
给药方法
细胞在进行造模前，药物组先预孵溶剂(20％羟丙环糊精溶液)或药物(YC-6的终浓度为1μM、5μM、10μM)1小时，然后加入LPS并置于缺氧工作站进行造模，直至实验终点。模型对照组不加任何药物处理，正常组置于细胞培养箱进行常规培养。10μM地塞米松为药物对照。每个处理组进行3个复孔重复。
实验结果
实验结果如图4显示，缺氧和LPS刺激引起人肺泡上皮细胞LDH的释放增加，说明缺氧和LPS处理引起人肺泡上皮细胞的损伤，而YC-6可以剂量依赖性地减少缺氧和LPS刺激引起的肺泡上皮细胞的损伤。10μM的地塞米松也显著减轻这种损伤，10μM的YC-6的保护效应显著优于地塞米松。以上结果表明，YC-6可以剂量依赖性地显著减轻缺氧和LPS刺激引起的肺泡上皮细胞的损伤。
实施例5.YC-6显著改善LPS引起的大鼠急性肺损伤
动物分组和给药
动物分组：选取60只大鼠检疫合格且体重均一的大鼠纳入实验，按体重随机分为6组，10只/组，动物分组与每组药物处理剂量如下：
Figure PCTCN2021078061-appb-000005
给药方式：受试药物按0.3mL/100g体重尾静脉给药，氢化可的松(25mg/kg)按0.5mL/100g体重腹腔给药；所有受试试剂在LPS给予造模前0.5h进行第一次给予药物，受试药物溶剂与YC-6每6h静脉注射一次，共注射4次，氢化可的松(HC)组每12h腹腔注射一次，共注射2次。
造模方法
大鼠称重后，异氟烷诱导麻醉大鼠后，仰卧位固定，颈部皮肤备毛，75％乙醇消毒，正中切开颈部皮肤，钝性分离皮下组织，暴露上段气管。分别用注射器经气管注入8mg/mL的LPS溶液，分别0.2mL/只，每只大鼠按照250mg体重计算，剂量为6.4mg/kg。注射后立即将大鼠立直并摇晃、旋转，使LPS溶液均匀分布于肺内。缝合皮肤，碘伏消毒。
肺组织处理
LPS注射造模24h后，动物按6mL/kg体重的体积腹腔注射20％乌来糖溶液麻醉，腹主动脉放血死后，暴露胸腔，取肺组织。将左肺投入10％多聚甲醛水溶液固定48h后，将左肺冠状面横切为等宽的上下两部分，再将左肺下半部分按冠状面横切为等宽的两部分，然后将左肺上半部分的矢状面和两截左肺下半部分冠状面包埋在一个石蜡块中，切片和进行苏木素-伊红(HE)染色。
组织化学HE染色
组织脱水和石蜡包埋：将组织从固定液中取出，依次浸泡50％乙醇(30min)-70％乙醇(过夜)-80％乙醇(30min)-90％乙醇(30min)-95％乙醇(30min)-无水乙醇(2次，每次30min)-二甲苯(2次，每次5～10min，直至样品完全透明)-62℃石蜡(3次，每次1小时)，然后进行组织包埋。组织石蜡切片切片：厚度为3μm，切片在烘片机上烘干水分后放37℃烘箱烘烤干燥过夜，然后用于HE染色。HE染色：取出室温保存的石蜡切片，置于65℃烘箱中烘烤30min，然后立刻将切片浸于二甲苯中进行脱蜡三次，分别为5min、2min、2min；复水：第三次浸泡二甲苯后，将切片依次浸泡于100％乙醇-100％乙醇-95％乙醇-90％乙醇-80％乙醇-70％乙醇-50％乙醇-蒸馏水进行复水，每次1min；取出切片稍稍晾干，然后将切片置于湿盒，将苏木素染液滴加在组织上，确保染液将组织完全覆盖，室温孵育5min；用蒸馏水轻轻冲洗切片，将多余的苏木素洗去，然后将把切片放回湿盒，将伊红染色液滴加在组织上，室温孵育2min；用蒸馏水轻轻冲洗切片；将切片依次浸泡90％乙醇(1min)-95％乙醇(1min)-100％乙醇(1min)-100％乙醇(1min)-二甲苯(5min)-二甲苯(5min)进行脱水透明，然后用中性树脂(用适量二甲苯稀释，约50％二甲苯)封片。
肺组织病理观察与评分
肺组织病理形态学观察：使用Nikon Eclipse Ti-U倒置荧光显微镜光镜下观察肺组织病理学变化。按炎性细胞浸润、肺泡出血、肺泡壁增厚、肺泡扩张和支气管上皮脱落的改变等级进行分析。肺损伤评分按照Smith评分体系，由两位不知实验分组和给药信息的实验员，对炎性细胞浸润、肺泡出血、肺泡壁增厚、肺泡扩张和支气管上皮脱落这6项指标分别进行肺损伤严重程度评分。0分：正常；1分：轻度病变，病变范围小于整个视野面积的25％；2分：中度病变，病变范围为整个视野面积的25％-50％；3分：重度病变，病变范 围为整个视野面积的50％-75％；4分：极重度病变，病变范围大于整个视野面积的75％。总的肺损伤病理评分为上述各项评分的总和。
实验结果
如图5病理镜下观察结果显示，正常对照组(图5A)大鼠肺组织结构清晰，肺泡结构为多边形或圆形薄壁空泡，肺泡空间干净，边界清楚，肺泡壁无增厚，肺泡间质没有炎性浸润；支气管管壁结构清晰，未见上皮细胞脱落；血管正常。LPS模型组(图5B)大鼠肺组织可见大量炎性细胞浸润(黄色箭头)，肺泡腔和肺间质水肿，具有严重的纤维性渗出(红色箭头)，肺泡壁增厚，肺泡扩张和萎陷；支气管粘膜上皮脱落(绿色箭头)；肺泡充血，肺泡内可见大量红细胞(橙色箭头)；肺血管周边组织疏松，出现明显的渗出(蓝色箭头)，表明LPS引起肺血管通透性增加。给予不同剂量的YC-6组(图5C-E)与LPS模型组比较，减少炎性细胞浸润，减轻肺泡损伤，减轻血管屏障通透性增加；病理结果表明YC-6可以显著改善LPS引起的急性肺损伤。
如图6肺损伤病理评分统计结果所示，LPS模型组评分显著高于正常对照组(P<0.001)；而给予12、40、120mg/kg/d剂量的YC-6处理组与LPS模型组比病理评分显著降低(P<0.01或P<0.001)；并且低剂量组YC-6的处理显著优于50mg/kg/d氢化可的松的保护效果(P<0.05)。
以上结果表明，LPS导致显著的肺损伤，表现为大量炎性细胞浸润、肺泡壁增厚、肺泡充血、扩张和萎陷以及支气管粘膜上皮脱落。YC-6显著地减轻LPS引起的血管内皮屏障通透性增加，减少炎性细胞浸润、减轻肺泡损伤。
实施例6.YC-6减轻急性食蟹猴肺损伤
非人灵长类动物在种系发生、解剖结构等方面更接近人类，可减少种属差异带来的药效评价偏差；同时在非人灵长类得到的药理学剂量、毒理和药物疗效的研究数据可为后续临床试验提供更可靠的依据。我们使用非人灵长类动物食蟹猴和高原减压舱建立了急性低压缺氧对食蟹猴的肺损伤模型。应用HE染色等病理生化检测，评价YC-6是否减轻急性低压缺氧导致的肺损伤。
实验动物分组
健康雄性食蟹猴(Macaca fascicularis)24只，6至6.5岁，体重6.5-7.5Kg，购于广东省高要市康达实验动物中心。17只雄性食蟹猴分为3组，如下表：
Figure PCTCN2021078061-appb-000006
低压舱模拟7500米致食蟹猴急性低压缺氧肺损伤模型的制作
将饲养于动物房的食蟹猴每次随机选取3只，每只采血10ml后静脉推注葡萄糖生理盐水10ml，并做好标识。然后将动物放入低压舱内饲养1天以适应实验环境。急性低压缺氧组以3米/秒速度海拔模拟上升至3000米；动物在低压舱内模拟高度3000米停留30分钟后，采取每只实验猴血10ml，然后急性低压缺氧组静脉推注葡萄糖生理盐水10ml；以3米/秒速度海拔模拟上升至4500米；动物在低压舱内模拟高度4500米停留30分钟后，采取每只实验猴血10ml后，急性低压缺氧组静脉推注葡萄糖生理盐水10ml；以3米/秒速度海拔模拟上升至6000米；动物在低压舱内模拟高度6000米停留30分钟后，并采取每只实验猴血10ml并静脉推注葡萄糖生理盐水10ml；以2米/秒速度海拔模拟上升至7500米；动物在低压舱内模拟高度7500米停留24小时后，以3米/秒速度将海拔下降到6000米，采取每只实验猴血10ml并静脉推注葡萄糖生理盐水10ml；以2米/秒速度海拔模拟上升至7500米；在7500米停留48小时后，以3米/秒速度将海拔下降到6000米，麻醉(0.06ml/kg盐酸氯胺酮注射液，0.02ml/kg盐酸赛拉嗪注射液)实验动物，颈动脉放血处死动物，并进行解剖、取材、固定。常压常氧对照组食蟹猴在平原(海拔352米)麻醉(0.06ml/kg盐酸氯胺酮注射液，0.02ml/kg盐酸赛拉嗪注射液)后，颈动脉放血处死动物，并进行解剖、取材、固定。
给药时间、剂量及方式
药物处理组动物在未开始模拟升高前，YC-6注射液按10mg/kg的剂量用葡萄糖生理盐水稀释至10ml，YC-6处理组进行一次性静脉推注；急性低压缺氧组只静脉推注葡萄糖生理盐水10ml。药物处理组动物在低压舱内模拟高度3000米停留30分钟后，YC-6注射液按10mg/kg的剂量用葡萄糖生理盐水稀释至10ml，YC-6处理组进行一次静脉推注；急性低压缺氧组只静脉推注葡萄糖生理盐水10ml；药物处理组动物在低压舱内模拟高度 4500米停留30分钟后，YC-6注射液按10mg/kg的剂量用葡萄糖生理盐水稀释至10ml，YC-6处理组进行一次静脉推注；急性低压缺氧组只静脉推注葡萄糖生理盐水10ml；药物处理组动物在低压舱内模拟高度6000米停留30分钟后，YC-6缓释剂按30mg/kg的剂量分成5点进行骨骼肌肌肉注射；急性低压缺氧组只静脉推注葡萄糖生理盐水10ml。药物处理组动物在低压舱内模拟高度7500米停留24小时后，YC-6注射液按10mg/kg的剂量用葡萄糖生理盐水稀释至10ml，YC-6处理组进行一次静脉推注，并且YC-6缓释剂按30mg/kg的剂量分成5点进行骨骼肌肌肉注射。急性低压缺氧组只静脉推注葡萄糖生理盐水10ml。在7500米停留48小时后，以3米/秒速度将海拔下降到6000米，麻醉(0.06ml/kg盐酸氯胺酮注射液，0.02ml/kg盐酸赛拉嗪注射液)实验动物，颈动脉放血处死动物，并进行解剖、取材、固定。常压常氧组食蟹猴在平原(海拔320米)麻醉(0.06ml/kg盐酸氯胺酮注射液，0.02ml/kg盐酸赛拉嗪注射液)后，颈动脉放血处死动物，并进行解剖、取材、固定。
组织石蜡包埋固定、切片和苏木素-伊红(HE)染色
组织石蜡包埋固定：进行肺部组织取材后将肺组织修整为厚度不超过1cm的组织块，放进10倍体积的多聚甲醛中固定，固定时用棉花将漂浮的肺组织压到液面下使组织充分固定。48小时后更换一次固定液继续固定48小时，然后可以用于石蜡包埋。将组织从固定液中取出，依次浸泡50％乙醇(30min)-70％乙醇(过夜)-80％乙醇(30min)-90％乙醇(30min)-95％乙醇(30min)-无水乙醇(2次，每次30min)-二甲苯(2次，每次5～10min，直至样品完全透明)-62℃石蜡(3次，每次1小时)，然后进行组织包埋。组织石蜡切片切片：厚度为3mm，切片在烘片机上烘干水分后放37度烘箱烘烤干燥过夜，然后用于HE染色。苏木素-伊红(HE)染色：取出室温保存的石蜡切片，置于65℃烘箱中烘烤30min，然后立刻将切片浸于二甲苯中进行脱蜡三次，分别为5min、2min、2min；复水：第三次浸泡二甲苯后，将切片依次浸泡于100％乙醇-100％乙醇-95％乙醇-90％乙醇-80％乙醇-70％乙醇-50％乙醇-蒸馏水进行复水，每次1min；取出切片稍稍晾干，然后将切片置于湿盒，将苏木素染液滴加在组织上，确保染液将组织完全覆盖，室温孵育5min；用蒸馏水轻轻冲洗切片，将多余的苏木素洗去，然后将把切片放回湿盒，将伊红染色液滴加在组织上，室温孵育2min；用蒸馏水轻轻冲洗切片，将多余的伊红染色液洗去，然后用试剂盒配套的增色液轻轻冲洗两次，再用蒸馏水稍稍冲洗；将切片依次浸泡90％乙醇(1min)-95％乙醇(1min)-100％乙醇(1min)-100％乙醇(1min)-二甲苯(5min)-二甲苯(5min)进行脱水透明，然后用中性树脂(用适量二甲苯稀释，约50％ 二甲苯)封片。
YC-6对肺血管充血肿胀和肺泡间隔增厚的影响
如图7所建立的模型，随着海拔上升，食蟹猴表现出显著的急性高原病症状，例如呼吸急促、呕吐、共济失调、意识模糊等，表明非人灵长类食蟹猴急性高原病模型的复制成功。ALI/ARDS早期病理特征在于肺泡壁毛细血管扩张、肺泡间隔增宽，存在肺泡腔内浆液、嗜中性粒细胞和巨噬细胞渗出，进而发展为肺弥漫性充血、水肿，肺泡内透明膜形成和局灶性肺萎陷。本研究应用HE染色观察YC-6是否减轻急性低压缺氧导致的肺损伤。
如图8所示，正常对照组的食蟹猴肺组织肺泡结构为多边形或圆形薄壁空泡，边界清楚，肺泡上皮细胞间为薄壁肺泡隔，隔内可见毛细血管断面。如图8结果所示，急性低压缺氧引起肺泡间隔显著增厚；而YC-6给药处理后得到显著改善。对各组肺泡间隔厚度进行测量并统计显示(图9)，YC-6显著减轻肺泡间隔增厚性损伤。以上结果显示YC-6可以有效减轻急性低压缺氧引起的肺泡间隔增厚，提示YC-6维系肺血管、肺泡正常组织结构与功能，减轻气血屏障通透性损伤，有望对新冠肺炎等肺损伤疾病有治疗作用。
YC-6对肺泡腔蛋白透明膜形成和红细胞渗漏的影响
已有报道指出，人类急性肺损伤标志性的病理改变之一是弥漫性肺泡损伤(diffuse alveolar damage,DAD)，弥漫性肺泡损伤其中一个重要特征性表现是肺泡通透性改变导致血液蛋白进入肺泡、蛋白沉积形成的透明膜 [8]。同时，透明膜的形成也是ARDS的特征性病理之一，在新冠肺炎患者病理检查中被发现 [1]。透明膜是在呼吸细支气管、肺泡管、肺泡表面形成一层均匀红染的膜状物，由渗出的血浆蛋白、纤维素及崩解的肺泡上皮细胞碎屑构成。透明膜形成、肺泡间质纤维性增生使肺泡间隔增，导致肺泡膜通透性降低，引起血气交换功能障碍。如图10所示，急性低压缺氧引起肺泡间隔纤维性增生(黑色箭头)，同时引起部分肺泡腔内蛋白透明膜的形成(红色箭头)，说明急性低压缺氧引起弥漫性肺泡损伤；急性低压缺氧还引起红细胞渗漏进入肺泡腔(蓝色箭头)，说明急性低压缺氧引起了气血屏障的破坏；而YC-6药物组未观察到这种明显变化，提示YC-6对肺泡上皮细胞和血管内皮细胞具有保护作用。以上结果提示，YC-6可能通过保护肺泡上皮细胞和血管内皮细胞，减轻急性低压缺氧引起的气血屏障通透性增加，维持了正常的结构和功能。
YC-6对肺上皮细胞损伤、炎细胞浸润的影响
ALI/ARDS除与原发疾病有关外，炎性细胞浸润和肺泡上皮损伤是重要病理因素。肺泡毛细血管壁弥漫性损伤和通透性增强，发生肺水肿和纤维素渗出。Ⅱ型肺泡上皮细胞损 伤，肺泡表面活性物质减少或消失，导致肺透明膜形成和肺萎陷。上述改变均可引起肺泡内氧弥散障碍，通气血流比值失调，发生低氧血症和呼吸窘迫。如图11所示，和正常组比较，急性低压缺氧引起炎性细胞的肺组织的浸润，在肺泡膜间质(黑色箭头)和肺泡腔(红色箭头)中可观察到大量炎性细胞，部分肺泡腔可见脱落受损的肺上皮细胞(蓝色箭头)，而YC-6处理组没有观察到明显的炎性浸润和受损的肺上皮细胞脱落。以上结果显示YC-6减轻血管通透性，可以有效减轻肺组织的炎性浸润以及肺上皮细胞损伤。
实验结论
应用体内急性低压缺氧导致的食蟹猴肺损伤模型，通过病理学检测发现，YC-6显著抑制肺血管充血肿胀和肺泡间隔增厚、减轻肺气血屏障损伤，YC-6表现出显著肺保护的药效。
实施例7.YC-6通过促进NR4A3的蛋白表达减少缺氧相关刺激引起的血管内皮细胞损伤
核受体亚家族4，A组，成员3(Nuclear Receptor Subfamily 4 Group A Member 3，NR4A3)，也被称为神经元衍生孤儿素受体1(Neuron-derived Orphan Receptor-1，NOR1)。NR4A3作为一个转录因子，参与代谢、炎症、细胞增殖、凋亡和分化等生理和病理过程的调节。敲低NR4A3显著抑制血管内皮细胞的毛细血管转化能力，表明了NR4A3在血管内皮细胞中具有重要的生理功能。以往的研究表明，NR4A3的上调可以促进细胞在不同病理损伤下的存活。例如，在血管内皮细胞中增加NR4A3表达可以通过上调细胞凋亡抑制因子2(cellular inhibitor of apoptosis 2，cIAP2)来抑制细胞凋亡，从而提高缺氧条件下血管内皮细胞的存活；在神经元中增加NR4A3的表达可以减少氧化应激和谷氨酸诱导的兴奋性毒性造成的神经元损伤。
本实验探究YC-6是否对缺氧相关刺激引起的血管内皮细胞损伤具有保护作用，以及相关保护作用是否与调节NR4A3的表达相关。
实验材料
细胞株
人脐静脉内皮细胞(HUVEC)购于广州赛库生物技术有限公司，培养于含10％胎牛血清的改良型DMEM培养基(CellCook，CM2007)。大鼠血管内皮细胞(RAOEC)购于广州吉妮欧生物科技有限公司，培养于含10％胎牛血清的DMEM培养基(Coring，10-013-CV)。
组织切片
食蟹猴肺组织石蜡切片，包括正常对照组3个(normobaric normoxia，NN)，急性低压缺氧处理组5个(hypobaric hypoxia，HH)，急性低压缺氧+YC-6处理组5个(HH+YC-6)。
主要试剂和药物
高糖DMEM培养基(corning，10-03-CV)
改良型DMEM培养基(CellCook，CM2007)
澳洲胎牛血清(Gibco，10099141)
0.25％胰蛋白酶(Gibco，25200072)
无糖DMEM培养基(Gibco，A1443001)
LDH检测试剂盒(Promega，G1780)
20％羟丙环糊精溶液(Hydroxypropyl-β-Cyclodextrin，HP-β-CD)(广州市赛普特医药科技股份有限公司，安瓿瓶装，批号为180502，规格5mL：1g，浓度为0.2g/mL)
YC-6注射液(广州市赛普特医药科技股份有限公司，安瓿瓶装，批号20010201，规格5mL：50mg，浓度为10mg/mL，溶于20％羟丙环糊精溶液)
放线菌酮cycloheximide(MCE，HY-12320)
氯喹chloroquine(MCE，HY-17589)
MG132(Selleck，S2619)
NAC(Selleck，S1623)
PBS(Gibco，C10010500BT)
蛋白酶抑制剂(Targetmol，C0001)
磷酸酶抑制剂(Targetmol，0004)
细胞裂解液(Thermofisher Scientific，78501)
BCA蛋白定量试剂盒(Thermofisher Scientific，23225)
5×蛋白上样缓冲液(Beyotime，P0015L)
SDS-PAGE分离胶缓冲液(Bio-rad，161-0798)
SDS-PAGE浓缩胶缓冲液(Bio-rad，161-0799)
10％SDS溶液(Bio-rad，161-0416)
30％Acr-Bis丙烯酰胺(MIK，DB240)
过硫酸铵(MPbio，193858)
TEMED(Macklin，T6023-100mL)
PVDF膜(Roche，3010040001)
脱脂奶粉(Wako，190-12865)
TBS(Boster，AR0031)
Anti-NR4A3antibody(Santa cruz，sc-393902)
Anti-actin antibody(Arigo，arg62346)
Anti-CD31antibody(Abcam，ab28364)
Goat anti-mouse IgG antibody(HRP)二抗(Arigo，arg65350)
Donkey anti-Mouse IgG(H+L)Highly Cross-Adsorbed Secondary Antibody,Alexa Fluor 488荧光二抗(Invitrogen，A-21202)
Donkey anti-Rabbit IgG(H+L)Highly Cross-Adsorbed Secondary Antibody,Alexa Fluor 555荧光二抗(Invitrogen，A-31572)
Hoechst33342(Sigma-Aldrich，14533，使用浓度为5μg/mL)
水溶性封片剂(Boster，AR1018)
化学发光液(Merck Millipore，WBKLS0500)
10×EDTA抗原修复液(Boster，AR0023)
DAKO抗体稀释液(Dako，S-3022)
二甲苯(广州化学试剂厂，33535)
无水乙醇(广州化学试剂厂，32061)
Mayer苏木素染色液(MIK，BL003)
伊红Y(威佳科技，17372-87-1).
Trizol(Invitrogen，15596)
逆转录酶(Thermofisher Scientific，EP0442)
dNTPs(Sigma-Aldrich，D7295)
SuperReal PreMix SYBR Green qPCR试剂盒(Tiangen,FP205)
IP裂解液(Beyotime，P0013)
IP磁珠(Bimake，B23202)
主要实验设备
超净工作台、细胞培养箱、缺氧工作站、酶标仪、化学发光成像仪、激光共聚焦显微镜、低温高速离心机、实时荧光定量PCR系统
实验方法
细胞培养、氧糖剥夺-复氧(oxygen-glucose deprivation and restoration,OGD-R)损伤模型和 药物处理
HUVEC细胞用含10％胎牛血清的改良型DMEM培养基培养，待细胞长至约80％融合度的时候，用0.25％胰蛋白酶消化细胞，然后用含10％胎牛血清的改良型DMEM培养基吹打均匀，把细胞浓度调整至1×10 5个/mL，按400uL/孔接种于24孔板，待细胞长至约70％融合度的时候进行实验。RAOEC细胞用含10％胎牛血清的高糖DMEM培养基培养，接种条件与HUVEC一致。进行OGD-R损伤处理时，设置缺氧工作站的氧气浓度为1％(1％O 2，5％CO 2，94％N 2)，温度为37℃，湿度为85％。把无糖DMEM培养基置于缺氧工作站预缺氧3小时。吸走培养板中的培养基，用预热的无糖DMEM培养基洗2遍，然后每孔加入100uL的无糖DMEM培养基，将培养板置于缺氧工作站，每孔再加入300uL预缺氧的无糖DMEM培养基，然后将培养板静置于缺氧工作站继续缺氧4小时。然后进行复氧处理，把培养板取出，把无糖DMEM培养基更换为400uL含10％胎牛血清的正常培养基，然后置于37℃、5％CO 2细胞培养箱继续培养24小时或指定的时间。正常组的细胞仅把培养基更换为400uL新的含10％胎牛血清的正常培养基。药物处理组的细胞，在对细胞进行氧糖剥夺处理后，在复氧处理的同时，给与指定终浓度的药物(YC-6的终浓度为 1μM、3μM或10μM，溶剂对照给与相应的20％羟丙环糊精，放线菌酮(cycloheximide，CHX)的终浓度为100μM，氯喹(chloroquine，CQ)的终浓度为10μM、30μM或100μM，MG132的终浓度为10nM、30nM或100nM)，直至实验终点。模型对照组不加任何药物处理。
免疫印迹(Western blot)
细胞用预冷的PBS洗2次，然后用细胞刮把细胞刮下来并转移至1.5mL的离心管，于4℃、1000g离心5分钟，弃去上清，向细胞沉淀加入适量的含有蛋白酶抑制剂和磷酸酶抑制剂的细胞裂解液，充分重悬细胞并于冰上放置5分钟使细胞充分裂解。然后4℃、12000g离心10分钟，分离上清，上清即为提取的细胞蛋白。然后用BCA蛋白定量试剂盒对各蛋白样品进行定量。蛋白定量后，用蛋白裂解液把各样品的蛋白浓度调整至与最低浓度的蛋白样品一致，然后分别把各蛋白样品与5×蛋白上样缓冲液按4:1的比例充分混匀，在沸水浴中煮5分钟，然后涡旋混匀，短暂离心收集样品至管底，置于冰上备用或冻存于-80℃。对蛋白样品进行10％SDS-PAGE电泳，然后把分离的蛋白转移至PVDF膜。用5％脱脂牛奶在室温对PVDF膜进行封闭2小时。封闭结束后，用TBST(即含1‰吐温20的TBS)洗膜3次，每次5分钟。然后用5％BSA稀释好对应的抗体，把PVDF膜置于相应的稀释好的抗体中，4℃过夜孵育。孵育结束后，用TBST洗膜3次，每次5分钟。用5％BSA稀释好对应的二抗，把PVDF膜置于相应的稀释好的二抗中，室温孵育1小时。孵育结束后，用TBST洗膜3次，每次5分钟，把PVDF膜置于化学发光液中，用化学发光成像仪进行成像。
组织免疫荧光
把石蜡切片置于65℃烘箱30分钟，然后迅速转移至100％二甲苯中进行脱蜡，按照常规流程(100％二甲苯，5min——100％二甲苯，2min——100％二甲苯，2min——100％乙醇，1min——100％乙醇，1min——95％乙醇，1min——90％乙醇，1min——80％乙醇，1min——70％乙醇，1min——50％乙醇，1min——蒸馏水，1min×3)进行复水，然后用EDTA抗原修复液(微波加热)进行抗原修复。修复结束后自然冷却至室温，然后用PBS洗3次，每次5分钟。甩干组织切片上残余的PBS，用免疫组化笔在组织外周画一圈，然后把对应的稀释于DAKO抗体稀释液的一抗滴加在组织上，使其完全覆盖组织。把组织切片置于4℃湿盒中过夜孵育。然后用PBS洗3次，每次5分钟。然后在组织上滴加对应的稀释好的荧光二抗，于湿盒中避光室温孵育1小时。然后用PBS洗3次，每次5分钟。用Hoechst33342 染核，湿盒中避光室温孵育5分钟。然后用PBS洗3次，每次5分钟，用水溶性封片剂封片，然后用激光共聚焦显微镜进行图像拍摄。
苏木素-伊红(hematoxylin-eosin，HE)染色
按之前所述对石蜡切片进行复水。然后将切片浸泡于苏木素染色液中，室温染色10分钟。用流水轻轻冲洗切片，将多余的苏木素洗去，然后用1％盐酸乙醇分化10秒。用流水轻轻冲洗切片5分钟，然后用1％氨水返蓝10秒或流水冲洗30分钟。用流水轻轻冲洗切片5分钟，然后浸泡于1％伊红中，室温染色5分钟。用流水轻轻冲洗切片，将多余的伊红染色液洗去。将切片依次浸泡70％乙醇(1min)-80％乙醇(1min)-90％乙醇(1min)-95％乙醇(1min)-100％乙醇(1min)-100％乙醇(1min)-二甲苯(5min)-二甲苯(5min)进行脱水透明，然后用中性树脂(用适量二甲苯稀释，约50％二甲苯)封片。把切片置于通风厨中，待封片剂干透后，使用Nikon Eclipse Ti-U倒置荧光显微镜进行明场拍照，每个个体进行随机视野拍照，供后续病理分析。
细胞免疫荧光染色
实验终点时，用PBS迅速洗细胞2次，然后加入4％多聚甲醛，室温放置15分钟。然后用含0.2％TritonX-100的PBS室温孵育15分钟进行破膜。用PBS洗细胞三次，然后加入相应的用DAKO抗体稀释液稀释好的一抗，4℃孵育过夜。吸走一抗，用PBS洗细胞3次，然后加入稀释好的荧光二抗，室温避光孵育1小时。吸走二抗，用PBS洗细胞3次，然后加入hoechst33342对细胞核进行染色，室温避光孵育5分钟。吸走hoechst33342，用PBS洗3次，加入200uL PBS，然后用激光共聚焦显微镜进行图像拍摄。
实时荧光定量PCR
实验终点时，弃去细胞培养基，然后直接加入适量的Trizol试剂，冰上充分裂解细胞后，把Trizol裂解物转移至无RNA酶的1.5mL离心管，然后加入Trizol用量的20％的氯仿，剧烈摇晃15秒，冰上放置10分钟，于4℃12000g离心15分钟，然后把上层液体转移至新的1.5mL离心管(不要触碰到中间层和下层)，加入0.8倍上层液体体积的异丙醇，颠倒数次混匀，冰上放置10分钟。然后4℃12000g离心15分钟，小心弃去异丙醇，向管底沉淀加入1mL预冷的75％乙醇(用DEPC水稀释无水乙醇至75％)，颠倒离心管使管底沉淀漂浮。然后于4℃12000g离心15分钟，弃去75％乙醇，用新的预冷的75％乙醇再洗一次。4℃12000g离心15分钟，弃去75％乙醇，然后再短暂离心，用移液枪吸走剩余的乙醇。把离心管置于超净工作台，敞开管口放置5～10分钟，让乙醇挥发干净。加入适量的 DEPC水溶解RNA，使用Nanodrop超微量紫外分光光度计测OD值定量，OD260/280在1.8-2.0范围内表明RNA质量较高。按以下方法对RNA进行逆转录和实时荧光定量PCR：
1)预变性：Total RNA 2μg、Oligo dT 1μL、DEPC·H 2O to 13μL混匀，65℃，5min，结束后立即置于冰上
2)逆转录：5×Reaction Buffer 4μL、dNTP mix 2μL、Reverse Transcriptase 1μL混匀，42℃60min，70℃10min，4℃forever
3)Real-time PCR(ABI 7500fast real-time PCR system)
应用引物母液：100nmol/μL(引物冻干粉+nmoles*10μL RNase-free ddH 2O)、引物工作液2nmol/μL(4μL FP+4μL RP+192μL RNase-free ddH 2O)以及SYBR Green Mix/ROX＝1.25mL/50μL，混匀以下溶液SYBR Green/ROX 5μL、cDNA 1μL、Primer 2μL、DEPC·H 2O 2μL混匀；进行PCR反应，循环参数Holding stage：95℃,15min；Cycling stage(40cycles)：95℃,10s→56℃,20s→72℃,30s；Melt Curve stage(continuous)：95℃,15s→65℃,60s→95℃,15s→65℃,15s；相对表达量的计算采用ComparativeΔΔCt法(RQ＝2 -ΔΔCt)进行数据分析。引物序列包括：β-actin(human)forward:5’-GATTCCTATGTGGGCGACGA-3’；reverse:5’-AGGTCTCAAACATGATCTGGGT-3’；NR4A3(human)forward:5’-AGCGGCGGCATCCTC-3’；reverse:5’-CTAAGGGTCCAGGCTCAGG-3’；β-actin(rat)forward:5’-CGCGAGTACAACCTTCTTGC-3’；reverse:5’-CGTCATCCATGGCGAACTGG-3’；NR4A3(rat)forward:5’-GGAAACGTGGCGACATCCT-3’；reverse:5’-CAGTGGGCTTTGGGTTCTGTG-3’。
免疫沉淀
细胞用预冷的PBS洗2次，然后用细胞刮把细胞刮下来并转移至1.5mL的离心管，于4℃、1000g离心5分钟，弃去上清，向细胞沉淀加入适量的含有蛋白酶抑制剂和磷酸酶抑制剂的IP裂解液，充分重悬细胞并于冰上放置5分钟使细胞充分裂解。然后4℃、12000g离心10分钟，分离上清，上清即为提取的细胞蛋白。然后用BCA蛋白定量试剂盒对各蛋白样品进行定量。蛋白定量后，用IP裂解液把各样品的蛋白浓度调整至1mg/mL，各样品取等体积至一个新离心管(每个样品剩余少量作为input)，按照抗体说明书加入相应量的抗体，于4℃缓慢颠倒混匀过夜。然后用IP裂解液洗IP磁珠两次，向各样品分别加入等量的IP磁珠，继续于4℃缓慢颠倒混匀2小时。然后分离磁珠，用IP裂解液洗IP磁珠5次，每次于4℃颠倒混匀5分钟。最后一次分离磁珠后，各样品加入等量的1×蛋白 上样缓冲液(IP裂解液与5×蛋白上样缓冲液按4:1的比例混匀)，短暂离心后，在沸水浴中煮5分钟，然后短暂离心收集样品至管底，转移煮沸后的样品至一个新离心管，弃去磁珠，样品可置于冰上备用或冻存于-80℃。对蛋白样品进行10％SDS-PAGE电泳，后续操作与免疫印迹一致。
数据统计
统计结果以均值±标准差呈现。运用单因素方差分析法(ONE-WAY ANOVA)进行统计分析，并使用Tukey法进行多重比较，P<0.05认为具有显著性统计学差异。
实验结果
氧糖剥夺-复氧/缺氧刺激下YC-6提高血管内皮细胞NR4A3蛋白表达和减少细胞损伤
已有报道指出，血管内皮细胞在缺氧刺激下通过上调NR4A3促进下游cIAP2表达从而减少细胞凋亡，说明缺氧相关刺激下NR4A3的表达对促进血管内皮细胞的存活具有重要作用。我们的结果显示，氧糖剥夺4小时引起HUVEC和RAOEC的NR4A3的蛋白表达增加，但复氧24小时引起NR4A3蛋白水平的下调(图12a)，同时引起细胞LDH的释放增加(图3)，提示了氧糖剥夺-复氧可能通过下调NR4A3的表达而促进细胞损伤。复氧损伤同时给与YC-6可以剂量依赖地提高NR4A3的蛋白表达(图12a)，同时剂量依赖地减少氧糖剥夺-复氧引起的细胞损伤(图3)，说明YC-6可以通过上调NR4A3的表达发挥保护血管内皮细胞的作用。通过细胞免疫荧光的方法对NR4A3的表达进行分析，与免疫印迹的结果一致，复氧损伤同时给与YC-6可以剂量依赖地提高NR4A3的蛋白表达(图12b-c)。
我们在急性低压缺氧致食蟹猴肺组织损伤的病理模型上进一步考察YC-6对NR4A3的表达影响。结果显示，在急性低压缺氧刺激下YC-6显著提高食蟹猴肺组织内皮细胞NR4A3的蛋白表达，以及抑制急性低压缺氧引起的血管内皮细胞标志物CD31的表达下调(图12d-e)。而之前组织病理学结果显示(图8-图11)，正常对照组的食蟹猴肺组织肺泡结构为多边形或圆形薄壁空泡，边界清楚，肺泡隔为薄壁结构，隔内可见毛细血管断面。急性低压缺氧引起严重的组织疏松、血管充血、肺泡壁增厚、肺泡腔内红细胞的渗出和透明膜的形成，以及炎性细胞对肺泡的浸润。透明膜的形成被认为是弥漫性肺泡损伤的典型病例特征，说明急性低压缺氧引起了弥漫性肺泡损伤以及肺内皮屏障的通透性增加。给与YC-6后能改善急性低压缺氧引起的肺组织病理改变和减轻气血屏障通透性增加。我们的结果显示NR4A3在减少缺氧相关刺激引起的血管内皮细胞损伤的重要性，而YC-6可以上调NR4A3 的蛋白表达和减轻氧糖剥夺-复氧引起的血管内皮细胞损伤和急性低压缺氧引起的内皮屏障损伤。
YC-6抑制氧糖剥夺-复氧引起的NR4A3的泛素化降解
以往的研究表明NR4A3的蛋白表达主要受转录水平的调控，HIF-1α是缺氧刺激下调控NR4A3表达的上游转录因子。因此，我们考察YC-6是否通过促进NR4A3的转录水平来促进其蛋白表达。结果显示(图13)，氧糖剥夺引起NR4A3转录水平的显著上升，表明氧糖剥夺通过激动HIF-1α的转录而促进NR4A3的表达，可能是血管内皮细胞对抗缺氧相关损伤的一种自我保护机制。复氧引起NR4A3转录水平的下降，给与YC-6以后没有显著上调NR4A3的转录水平，说明YC-6并不是通过促进NR4A3的转录而增加其蛋白表达。因此，我们进一步考察YC-6抑制NR4A3降解的可能性。对HUVEC和RAOEC进行氧糖剥夺处理，复氧的同时给与放线菌酮(CHX)去抑制蛋白质的合成，结果显示抑制蛋白合成导致NR4A3蛋白几乎完全耗竭，说明NR4A3蛋白在复氧损伤中发生快速降解。为了进一步考察NR4A3的降解途径，在复氧损伤给与CHX的同时，分别给与不同浓度的泛素蛋白酶体抑制剂MG132和溶酶体降解抑制剂氯喹CQ，来阻断不同方式的蛋白质降解。结果显示，复氧损伤下，同时给与CHX和MG132，可以抑制NR4A3的降解，但给与CHX和CQ没有明显抑制NR4A3的降解，表明复氧损伤过程中NR4A3的降解主要是通过泛素蛋白酶体系统。
为了进一步探索YC-6是否在复氧条件下抑制了NR4A3蛋白的泛素化降解，我们对复氧处理后的细胞进行蛋白提取和用抗泛素抗体进行免疫沉淀实验。结果显示，复氧损伤引起NR4A3的泛素化修饰，复氧损伤同时给与MG132能导致泛素化的NR4A3的增加，复氧后给与溶剂处理对NR4A3的泛素化修饰没有明显影响，但给与YC-6可以抑制复氧引起的NR4A3泛素化修饰，复氧后给与MG132和YC-6没有明显增加泛素化的NR4A3，表明YC-6抑制了NR4A3的泛素化修饰。
我们的结果表明，YC-6在复氧损伤情况下通过抑制NR4A3的泛素化修饰而抑制其降解，从而促进血管内皮细胞在损伤刺激下的存活。
综上，YC-6可以减少氧糖剥夺-复氧引起的血管内皮细胞的损伤，其机制之一是YC-6抑制NR4A3的泛素化降解，从而促进病理刺激下血管内皮细胞的存活。在整体动物上，YC-6在缺氧刺激下促进肺组织血管内皮细胞NR4A3的蛋白表达，减轻缺氧引起的肺内皮屏障通透性增加及相关病理改变，发挥肺保护的作用。
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Claims (23)

  1. 式I的化合物、其氘代物或药学上可接受的盐在制备用于预防或治疗对象的肺上皮细胞和/或血管内皮细胞受损介导的疾病的药物中的应用：
    Figure PCTCN2021078061-appb-100001
    其中R 1选自H、-CN、氟、氯、C 1-10烷基、氟或氯取代的C 1-10烷基、C 1-10烷氧基、氟或氯取代的C 1-10烷氧基和C 3-10环烷基。
  2. 根据权利要求1所述的应用，其中所述R 1是H、-CHCH 2CH 3、-CH(CH 3) 2、-CH(CH 2) 3CH 3或-CH(CH 3)(CH 2) 3CH(CH 3) 2。
  3. 根据权利要求1所述的应用，其中所述R 1是H。
  4. 根据权利要求1-3任一项所述的应用，其中所述肺上皮细胞和/或血管内皮细胞受损介导的疾病选自急性肺损伤、急性呼吸窘迫综合症、肺动脉高压、肺水肿、肺纤维化、早产儿慢性肺疾病、慢性阻塞性肺病、肺孢子菌病和肺栓塞中的一种或多种。
  5. 根据权利要求4所述的应用，其中所述急性肺损伤、急性呼吸窘迫综合症、肺动脉高压或肺水肿是由高氧、病毒感染、细菌感染、创伤、休克、缺血再灌注、急性胰腺炎、吸入性损伤、弥漫性肺泡损伤或中毒引起的。
  6. 根据权利要求4所述的应用，其中所述急性肺损伤、急性呼吸窘迫综合症、肺动脉高压或肺水肿是由高氧、病毒感染、细菌感染、创伤、休克、缺血再灌注、急性胰腺炎、吸入性损伤、弥漫性肺泡损伤和/或中毒引起的，并且不是由低氧引起的。
  7. 根据权利要求5或6所述的应用，其中所述病毒是冠状病毒、流感病毒、呼吸道合胞病毒、腺病毒、副流感病毒、麻疹病毒、巨细胞病毒或其组合。
  8. 根据权利要求7所述的应用，其中所述病毒是冠状病毒。
  9. 根据权利要求8所述的应用，其中所述冠状病毒是新型冠状病毒SARS-CoV-2。
  10. 根据权利要求4所述的应用，其中所述急性肺损伤是手术导致的肺损伤。
  11. 根据权利要求10所述的应用，所述手术是肺切除术、肺部肿瘤切除术或肺移植术。
  12. 根据权利要求11所述的应用，所述肺切除术是亚肺叶切除术、肺叶切除术或全肺切除术。
  13. 根据权利要求4所述的应用，其中所述肺纤维化是特发性肺纤维化或尘肺病。
  14. 根据权利要求1-3任一项所述的应用，其中所述血管内皮细胞受损介导的疾病包括由血脑屏障破坏介导的脑小血管病，但不包括脑微出血、脑卒中以及脑水肿。
  15. 根据权利要求14所述的应用，其中所述血脑屏障破坏表现为血脑屏障的通透性增加。
  16. 根据权利要求14或15所述的应用，其中所述血脑屏障破坏表现为血脑屏障的血管内皮细胞的损伤。
  17. 根据权利要求14所述的应用，其中由血脑屏障破坏介导的脑小血管病的临床表现为认知损害、步态障碍、情绪障碍、尿失禁和/或抑郁。
  18. 根据权利要求14所述的应用，其中由血脑屏障破坏介导的脑小血管病的影像学表现包括脑白质病变。
  19. 根据权利要求14所述的应用，其中由血脑屏障破坏介导的脑小血管病的影像学表现仅为脑白质病变。
  20. 根据权利要求1-3任一项所述的应用，其中所述血管内皮细胞受损介导的疾病包括心血管疾病或糖尿病血管并发症。
  21. 根据权利要求20所述的应用，其中所述心血管疾病选自急性心肌梗死(AMI)、心绞痛、冠心病、缺血性心脏病、心力衰竭、高血压、心血管介入术血栓形成中的一种或多种。
  22. 根据权利要求20所述的应用，其中所述糖尿病血管并发症是糖尿病性视网膜病变、糖尿病性肾病、糖尿病性伤口愈合受损中的一种或多种。
  23. 根据权利要求1-3任一项所述的应用，其中肺上皮细胞和/或血管内皮细胞受损介导的疾病表现为PFKFB3蛋白的过表达。
PCT/CN2021/078061 2020-02-28 2021-02-26 小分子化合物在治疗肺上皮细胞损伤和/或血管内皮细胞损伤介导的疾病中的用途 WO2021170073A1 (zh)

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