WO2021115412A1 - 一氧化氮合酶通路抑制剂在制备药物中的用途 - Google Patents

一氧化氮合酶通路抑制剂在制备药物中的用途 Download PDF

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WO2021115412A1
WO2021115412A1 PCT/CN2020/135595 CN2020135595W WO2021115412A1 WO 2021115412 A1 WO2021115412 A1 WO 2021115412A1 CN 2020135595 W CN2020135595 W CN 2020135595W WO 2021115412 A1 WO2021115412 A1 WO 2021115412A1
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nitric oxide
oxide synthase
reperfusion
gastrointestinal
ischemia
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PCT/CN2020/135595
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English (en)
French (fr)
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周宏伟
徐开宇
高徐璇
尹恝
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南方医科大学珠江医院
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Priority to CN202410036265.0A priority Critical patent/CN117867100A/zh
Priority to EP20899407.9A priority patent/EP4074341A4/en
Priority to US17/784,608 priority patent/US20220401386A1/en
Priority to CN202080005818.6A priority patent/CN113164605A/zh
Publication of WO2021115412A1 publication Critical patent/WO2021115412A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/155—Amidines (), e.g. guanidine (H2N—C(=NH)—NH2), isourea (N=C(OH)—NH2), isothiourea (—N=C(SH)—NH2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/17—Amides, e.g. hydroxamic acids having the group >N—C(O)—N< or >N—C(S)—N<, e.g. urea, thiourea, carmustine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
    • A61K31/573—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone substituted in position 21, e.g. cortisone, dexamethasone, prednisone or aldosterone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • This application relates to the field of biomedicine, and specifically relates to the use of nitric oxide synthase pathway inhibitors in the preparation of medicines.
  • Stroke is one of the most widespread diseases in the world today, and its incidence is increasing year by year. The quality of life of stroke patients declines, and it can lead to death in severe cases. Worldwide, from 1990 to 2010, the annual morbidity and mortality of ischemic stroke increased by 37% and 21%, respectively.
  • r-tPA tissue-type plasminogen activator
  • the application provides the use of a nitric oxide synthase pathway inhibitor in the preparation of a medicament for the prevention, alleviation and/or treatment of gastrointestinal ischemia-reperfusion related distal injury in a subject.
  • the nitric oxide synthase pathway inhibitor inhibits the activity of nitric oxide synthase.
  • the nitric oxide synthase includes inducible nitric oxide synthase iNOS.
  • the nitric oxide synthase pathway inhibitor includes an amino acid inhibitor and/or a non-amino acid inhibitor.
  • the nitric oxide synthase pathway inhibitor includes an amino acid inhibitor, and the amino acid inhibitor includes aminoguanidine AG, 1400W, L-NIL and/or isothiourea.
  • the nitric oxide synthase pathway inhibitors include non-amino acid inhibitors, and the non-amino acid inhibitors include glucocorticoids, flavonoids, 2-amino-4-methylpyridine and/ Or amino piperidine.
  • the medicament is formulated so that the nitric oxide synthase pathway inhibitor is locally effective in the gastrointestinal tract.
  • the medicament is formulated such that the nitric oxide synthase pathway inhibitor is still effective in preventing, alleviating, and/or treating the gastrointestinal ischemia about 48 hours or after administration.
  • An effective amount of reperfusion-related distal injury is present locally in the gastrointestinal tract.
  • the drug is formulated such that at most 50% of the nitric oxide synthase pathway inhibitor in the drug is absorbed by the subject and enters about 1 hour or later after administration. blood circulation system.
  • the concentration of the nitric oxide synthase pathway inhibitor in the drug is about 0.0001% (w/w) to about 90% (w/w).
  • the subject has been, is currently or is at risk of having a surgery, disease or condition related to the ischemia-reperfusion of the gastrointestinal tract.
  • the disease or condition related to gastrointestinal ischemia-reperfusion includes stroke, trauma, shock, sepsis, acute pancreatitis, or inflammatory bowel disease.
  • the disease associated with gastrointestinal ischemia-reperfusion includes ischemic stroke.
  • the subject has experienced, is, or is at risk of experiencing ischemia-reperfusion of the gastrointestinal tract.
  • the distal injury associated with gastrointestinal ischemia-reperfusion includes ischemic stroke.
  • the medicament is configured to be suitable for oral administration.
  • the nitric oxide synthase pathway inhibitor is not substantially decomposed and/or inactivated by digestive juice.
  • the application also provides the use of nitric oxide synthase for screening drugs for the prevention, alleviation and/or treatment of gastrointestinal ischemia-reperfusion related distal injury in a subject.
  • the drug inhibits the expression and/or activity of the nitric oxide synthase.
  • the nitric oxide synthase includes inducible nitric oxide synthase iNOS.
  • the application also provides a pharmaceutical composition comprising the nitric oxide synthase pathway inhibitor described in the application and optionally a pharmaceutically acceptable carrier.
  • the application also provides the use of nitric oxide synthase pathway inhibitors in the preparation of health care products for preventing, alleviating and/or treating the remote gastrointestinal ischemia-reperfusion in subjects. damage.
  • the application also provides the use of nitric oxide synthase pathway inhibitors in the preparation of biological products for the prevention, alleviation and/or treatment of gastrointestinal ischemia-reperfusion related distal end damage.
  • the present application also provides a method for preventing, alleviating and/or treating a distal injury related to gastrointestinal ischemia-reperfusion in a subject, the method comprising administering the subject described in the present application to the subject Nitric oxide synthase pathway inhibitor.
  • the administration includes administration via the gastrointestinal tract.
  • the nitric oxide synthase pathway inhibitor exerts its effect locally in the gastrointestinal tract.
  • the nitric oxide synthase pathway inhibitor is still effective in preventing, alleviating and/or treating the gastrointestinal ischemia-reperfusion-related distal injury
  • the effective amount is present locally in the gastrointestinal tract.
  • At most 50% of the nitric oxide synthase pathway inhibitor is absorbed by the subject and enters the blood circulatory system about 1 hour or after administration.
  • the administration dose of the nitric oxide synthase pathway inhibitor is about 0.01 to 1000 mg/kg body weight.
  • the present application also provides a method for preventing distal injury related to gastrointestinal ischemia-reperfusion in a subject, the method comprising:
  • the administration includes administration via the gastrointestinal tract.
  • Figure 1 shows the changes in blood flow in the cecum of mice observed by the laser speckle imaging system (Reward RFLSI Pro) in this application.
  • Figure 2 shows the statistical results of the ROI proportion of the cecal blood flow in mice observed by the laser speckle imaging system (Reward RFLSI Pro) in this application.
  • Figures 3A-3C show the changes in the expression levels of Nos2, Nox1 and Duox2 genes in the intestinal (colon) tissue after gastrointestinal ischemia-reperfusion caused by ischemic stroke in the present application.
  • Figure 4 shows the change of nitrate concentration in the mucus layer of the cecum after gastrointestinal ischemia and reperfusion caused by ischemic stroke in the present application.
  • Figures 5A-5C show the effect of aminoguanidine (AG) intervention in this application on the expression levels of Nos2, Nox1 and Duox2 genes in colon tissue after gastrointestinal ischemia-reperfusion caused by ischemic stroke.
  • AG aminoguanidine
  • Figure 6 shows the effect of aminoguanidine (AG) intervention in this application on the nitrate concentration in the cecal mucus layer after gastrointestinal ischemia-reperfusion caused by ischemic stroke.
  • Figures 7A-7J show the effect of aminoguanidine (AG) intervention in this application on the expression levels of intestinal barrier-related factors and pro-inflammatory cytokines in the intestinal tissue after gastrointestinal ischemia-reperfusion caused by ischemic stroke .
  • AG aminoguanidine
  • Figure 8 shows the effect of aminoguanidine (AG) intervention in this application on brain injury after gastrointestinal ischemia-reperfusion in mice.
  • FIG 9 shows the effect of aminoguanidine (AG) intervention in the present application on the results of improved neurological deficit scores in mice.
  • FIGS 10A-10B show the effects of aminoguanidine (AG) intervention shown by Nissl staining in the present application on neuronal death in the brain of mice.
  • AG aminoguanidine
  • Figures 11A-11E show the effect of aminoguanidine (AG) intervention in this application on the serum midgut barrier markers and inflammatory factor levels after gastrointestinal ischemia-reperfusion caused by ischemic stroke.
  • AG aminoguanidine
  • Figures 12A-12B show the effect of aminoguanidine (AG) intervention at different times or doses in this application on ischemic stroke after intestinal ischemia reperfusion.
  • AG aminoguanidine
  • nitric oxide synthase generally refers to a group of enzymes that can synthesize nitric oxide from nitrogen atoms in arginine in an aerobic environment, and its English abbreviation is usually NOS.
  • the activity of nitric oxide synthase usually requires the participation of cofactors, which can include nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), Flavin Mononucleotide (FMN), Proheme and Tetrahydrobiopterin (BH4), etc.
  • cofactors can include nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), Flavin Mononucleotide (FMN), Proheme and Tetrahydrobiopterin (BH4), etc.
  • Nitric oxide synthase usually includes the following three types: I neural nitric oxide synthase (nNOS or NOS1), II inducible nitric oxide synthase (iNOS or NOS2), III endothelial nitric oxide synthase (eNOS) Or NOS3).
  • Type I and Type III enzymes are native enzymes (cNOS), also called constitutive NOS (cNOS), which are basically an enzyme that exists in many normal tissues.
  • inducible nitric oxide synthase iNOS generally refers to nitric oxide synthase that is generally induced to be expressed under certain pathological or physiological conditions. Its activity usually does not depend on calcium ions and calmodulin, and can synthesize large amounts of nitric oxide. Factors that induce iNOS can include heme, cytokines, aerobic stress, IFN- ⁇ , TNF- ⁇ , IL-1 ⁇ , and so on.
  • the inducible nitric oxide synthase iNOS may comprise human inducible nitric oxide synthase iNOS.
  • the human inducible nitric oxide synthase iNOS gene is located at position 17cen-q11.2 of the 17th pair of autosomes and is about 37 kb in length. Its transcription is controlled by cytokines, lipopolysaccharide (LPS) and other inflammatory mediators.
  • the human iNOS promoter is one of the largest and most complex known so far, and it interacts with transcriptional regulators to control the expression of iNOS genes.
  • the open reading frame of iNOS is encoded by 27 exons, including a translation start position (in exon 2) and a stop position (in exon 27).
  • the TATA box of human iNOS is located 30bp upstream of the transcription initiation site.
  • iNOS mRNA obtained by alternative splicing is more diverse and has different stability.
  • nitric oxide synthase pathway generally refers to intracellular and/or extracellular signaling pathways that regulate the expression or activity of nitric oxide synthase.
  • ischemia usually refers to a situation where the blood supply of a tissue or organ is insufficient, which leads to hypoxia and nutrient deficiency.
  • ischemia can be caused by vascular problems, such as vascular embolism, vascular compression; it can also be caused by vasoconstriction, thrombosis, or ischemia caused by embolism, or it can be caused by accidental trauma, surgical intervention, or other Diseases of organs or tissues, such as ischemic diseases of other organs or tissues, such as gastrointestinal ischemia caused by ischemic stroke in the present application.
  • ischemia-reperfusion generally refers to the process of returning the blood supply to the tissue after ischemia or hypoxia (hypoxia or hypoxia).
  • ischemia-reperfusion usually refers to the process of resuming blood flow after the ischemic inducement is eliminated/offset/compensated/slowed.
  • the reperfusion of ischemic tissue can usually be accompanied by any of the following: damage to the microvascular system at the reperfusion site, for example, due to increased permeability of capillaries and arterioles, resulting in fluid filtration and Increased diffusion; activated endothelial cells produce more reactive oxygen species or free radicals after reperfusion, leading to subsequent inflammation; newly returned blood transports white blood cells to the reperfusion area, and white blood cells respond to tissue damage to release inflammatory factors such as white blood cells Interleukins and free radicals, white blood cells may also bind to the endothelium of small capillaries, blocking them and causing new ischemia; on the other hand, the restored blood flow reintroduces oxygen into the tissues, which may be destroyed in this specific case
  • the cell’s protein, nucleic acid and plasma membrane structure, and the resulting reactive substances may indirectly act on redox signaling to initiate cell apoptosis, and cell membrane damage may further lead to the release of more free radicals.
  • the free radicals may include nitroxide radicals, such as nitric oxide or its derivatives.
  • Ischemia-reperfusion usually leads to reperfusion injury, such as cerebral infarction, acute myocardial infarction, no reflow phenomenon after cardiopulmonary resuscitation, stress ulcer, pancreatitis, burns, transplantation of isolated organs, intestinal ischemia, necrosis Enterocolitis, intermittent claudication, acute tubular necrosis, liver failure after shock, and multiple system organ failure.
  • reperfusion injury such as cerebral infarction, acute myocardial infarction, no reflow phenomenon after cardiopulmonary resuscitation, stress ulcer, pancreatitis, burns, transplantation of isolated organs, intestinal ischemia, necrosis Enterocolitis, intermittent claudication, acute tubular necrosis, liver failure after shock, and multiple system organ failure.
  • distal reperfusion injury When the location of the reperfusion injury is different from the tissue or organ that has been ischemia-reperf
  • the term "distal injury” generally refers to a pathophysiological process involving organs or tissues that are different from the local organs or tissues that have been ischemia-reperfused. This process is usually caused by the exposure of cell products produced by ischemia-reperfusion organs or tissues to other organs through the circulatory system.
  • the distal injury may include a disease, symptom, or medical intervention that causes the ischemia-reperfusion, for example, stroke, trauma, shock, sepsis, acute pancreatitis, inflammatory bowel disease, or brain Trauma surgery.
  • the ischemia-reperfusion may include gastrointestinal ischemia-reperfusion.
  • the distal injury may include ischemic stroke injury related to gastrointestinal ischemia-reperfusion. For example, gastrointestinal ischemia-reperfusion-related ischemic stroke damage caused by ischemic stroke.
  • distal injury related to gastrointestinal ischemia-reperfusion generally refers to the pathophysiological process of an organ or tissue that is different from the gastrointestinal tract where ischemia-reperfusion occurs.
  • This pathophysiological process is related to gastrointestinal ischemia-reperfusion.
  • it can be manifested as a pathophysiological process of organs or tissues different from the gastrointestinal tract that occurs at the same time, before or after gastrointestinal ischemia-reperfusion.
  • the pathophysiological process of the organ or tissue shows corresponding changes, for example, its symptoms are alleviated or alleviated.
  • nitric oxide synthase inhibitors are administered based on gastrointestinal ischemia-reperfusion to alleviate the symptoms of ischemic stroke.
  • the term "inhibitor” generally refers to a compound/substance or composition that can completely or partially prevent or reduce the physiological function of one or more specific proteins.
  • the reduction of the physiological function of one or more specific proteins may include a reduction in the activity of the protein itself or a reduction in the amount of the protein itself.
  • the inhibitor may exist as different crystals, amorphous substances, pharmaceutically acceptable salts, hydrates, and solvates.
  • the term "administration” generally refers to the introduction of the inhibitor into the body of the subject by any route of introduction or delivery. Any method known to those skilled in the art for contacting cells, organs or tissues with the inhibitor can be used. Including but not limited to intra-arterial, intranasal, intra-abdominal, intravenous, intramuscular, subcutaneous transdermal or oral administration.
  • the daily dose can be divided into one, two or more suitable form doses to be administered at one, two or more times during a certain period of time.
  • the term “effective amount” or “effective dose” generally refers to an amount sufficient to achieve or at least partially achieve the desired effect.
  • the "therapeutically effective dose” or “therapeutically effective dose” of a drug or therapeutic agent is usually used alone or in combination with another therapeutic agent to promote the regression of the disease (this is achieved by reducing the severity of disease symptoms and the frequency of the asymptomatic period of the disease). Increase in degree and duration, or prevention of damage or disability due to disease).
  • the “prophylactically effective dose” or “prophylactically effective dose” of a drug generally refers to the amount of the drug that inhibits the development or recurrence of the disease when administered to a subject at risk of disease development or disease recurrence, alone or in combination with another therapeutic agent .
  • a variety of methods known to those skilled in the art can be used to evaluate the ability of the therapeutic or preventive agent to promote the regression of the disease or inhibit the development or recurrence of the disease, such as in a human subject during a clinical trial, in an animal model system Predict the efficacy on humans or determine the activity of the agent in an in vitro assay.
  • stroke generally refers to an acute cerebrovascular disease, also known as “stroke” or “cerebral vascular accident (CVA)”.
  • Stroke can be a group of diseases that cause brain tissue damage due to sudden rupture of blood vessels in the brain or blood can not flow into the brain due to blood vessel blockage, including ischemic stroke and hemorrhagic stroke.
  • ischemic stroke generally refers to a group of diseases that cause the nervous tissue dysfunction in a specific area of the brain or a broad brain tissue area due to insufficient blood supply.
  • Insufficient blood supply to the brain can be caused by a variety of diseases or abnormalities, such as sickle cell anemia, vascular compression, ventricular tachycardia, arterial plaque accumulation, thrombosis, severe hypotension, and congenital heart defects.
  • vascular compression may cause cerebral ischemia by blocking the arteries that carry oxygen into the brain, and the causes of vascular compression such as tumors;
  • ventricular tachycardia may cause Complete cardiac arrest causes blood flow to stop, and arrhythmia may also lead to the formation of blood clots, leading to cerebral ischemia;
  • arterial blockage caused by the accumulation of arterial plaque may also cause cerebral ischemia, in the case of a small amount of plaque accumulation It can also cause the passage to narrow and tend to form thrombus, which can lead to cerebral ischemia;
  • abnormal blood coagulation function large blood clots can also cause cerebral ischemia by blocking blood flow;
  • heart attack may also cause cerebral ischemia, heart attack may cause Blood flow is slow, blood may start to clot and prevent blood from flowing to the brain; there is a correlation between heart attack and low blood pressure, improper use of drugs and reactions to drugs may also lead to low blood pressure, which usually means low blood pressure.
  • ischemic stroke can also be achieved by surgically blocking the blood flow of the middle cerebral artery in mice.
  • the nerve tissue dysfunction may include neuronal death.
  • the main symptoms of nerve tissue dysfunction in different areas can include: air/taste/hearing or visual changes, swallowing/pupil response to light, physical movement disorders, aphasia, changes in breathing and heart rate, ischemia of other tissues or organs, Loss of consciousness, etc.
  • Digestive juice generally refers to the fluid secreted by the digestive system that digests food.
  • Digestive juice is mainly composed of organic matter, ions and water.
  • the main functions of digestive juice can include: diluting food to make it equal to the osmotic pressure of plasma to facilitate absorption; changing the pH in the digestive cavity to adapt to the needs of digestive enzyme activity; hydrolyzing complex food components to make it easier Absorption; protect the mucous membrane of the digestive tract and prevent physical and chemical damage by secreting mucus, antibodies and a large amount of fluid.
  • Digestive juice can include the following: saliva, gastric juice, pancreatic juice, bile, small intestinal juice, etc.
  • prevention generally refers to preventive administration of the combination to healthy subjects to prevent the occurrence of a certain disease or condition. It may also include prophylactic administration of the combination to patients in the pre-stage of the allergic disease to be treated. "Prevention” does not require 100% elimination of the possibility of occurrence of a disease or condition. In other words, “prevention” generally means that the probability or degree of occurrence of a disease or condition is reduced in the presence of the administration combination.
  • the term "alleviation” refers to the reduction, reduction or delay of a certain condition, disease, disorder or phenotype.
  • the pathology, disease, disorder or phenotype may include subjective perceptions such as pain, dizziness or other physiological disorders, or medically detectable indications, such as lesions detected by medical testing means.
  • treatment generally refers to clinical intervention for changing the natural course of the treated individual or cell in the clinical pathological process. This may include improving the state of the disease, eliminating the focus of the disease, or improving the prognosis.
  • the term "administered via the gastrointestinal tract” generally refers to allowing the specific release of the therapeutic agent at or near the gastrointestinal tract site.
  • the bioavailability of the drug can be increased in the gastrointestinal tract and/or decreased in the systemic circulation.
  • Lower systemic drug levels can lead to reduced toxicity and reduced immunogenicity (e.g. in In the case of biological preparations), on the one hand, it leads to improved overall safety and fewer adverse side effects, on the one hand, it increases the dose in the gastrointestinal tract to achieve a more efficient and targeted effect.
  • topical administration of therapeutic agents also provides new modes of action, such as combined administration of different local locations.
  • the term "works locally in the gastrointestinal tract” generally means that the administered therapeutic agent can maintain an effective dose in the gastrointestinal tract and can change the local gastrointestinal tract disease or phenotype. Exercising the effect locally in the gastrointestinal tract does not exclude the observation of the therapeutic agent in organs or tissues other than the gastrointestinal tract.
  • the therapeutic agent may have stability in the gastrointestinal tract and/or tissue penetration ability (the ability to penetrate into the gastrointestinal tissue), and may be substantially distributed in the gastrointestinal tissue.
  • the stability may include the stability before and/or after administration, such as the stability in the delivery device, the stability of the formulation and/or drug in the gastrointestinal environment after administration, including the disease state gastrointestinal tract Environment, such as temperature stability, pH stability, oxidation stability.
  • the part of the gastrointestinal tract may include part or sub-portion of one or more parts of the gastrointestinal tract of the subject.
  • subject generally refers to human or non-human animals, including but not limited to cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats, or monkeys.
  • the term "pharmaceutical composition” generally refers to a mixture comprising at least one active ingredient to be administered to a subject to treat a specific disease or condition affecting the individual. It allows the active ingredient to be in an effective form and does not contain additional components that have unacceptable toxicity to the subject to which the composition is to be administered.
  • This composition may be sterile, or it may contain a pharmaceutically acceptable carrier.
  • the term "pharmaceutically acceptable carrier” generally refers to a pharmaceutically acceptable substance, composition or vehicle involved in carrying or transporting a chemical agent.
  • a pharmaceutically acceptable substance for example, buffers, surfactants, stabilizers, preservatives, absorption enhancers for enhancing bioavailability, liquid or solid fillers, diluents, excipients, solvents, encapsulating materials and/or other conventional enhancements Solvent or dispersant.
  • the term "about” generally refers to a range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, above or below the specified value. Variation within the range of 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
  • the present application provides a use of a nitric oxide synthase pathway inhibitor in the preparation of a medicament for the prevention, alleviation and/or treatment of gastrointestinal ischemia-reperfusion related distal damage.
  • the application also provides a use of a nitric oxide synthase pathway inhibitor in the preparation of a medicament for the prevention, alleviation and/or treatment of neurological-related diseases or disorders in subjects.
  • the drug is formulated so that the nitric oxide synthase pathway inhibitor exerts its effect locally in the gastrointestinal tract.
  • the part of the gastrointestinal tract may include parts of the stomach, small intestine, and large intestine.
  • the stomach may include the gastric cardia, the fundus of the stomach, the body of the stomach, and the pylorus of the stomach.
  • the small intestine may include duodenum, jejunum, and ileum.
  • the large intestine may include cecum, colon, and rectum.
  • the local effect on the gastrointestinal tract may include the nitric oxide synthase pathway inhibitor inhibiting the activity of the local nitric oxide synthase in the gastrointestinal tract or reducing the local monoxide in the gastrointestinal tract. Expression of nitrogen synthase.
  • the local effect on the gastrointestinal tract may include a decrease in the expression level of the iNOS gene in the intestinal tissue.
  • the intestinal tissue may include colonic tissue.
  • the local effect on the gastrointestinal tract may include a decrease in the content of nitrate in the intestinal tissue.
  • the intestinal tissue may comprise cecal tissue.
  • the local effect on the gastrointestinal tract may include a decrease in the expression level of Nox1 and Duox2 genes in the intestinal tissue.
  • the intestinal tissue may include colonic tissue.
  • the local effect on the gastrointestinal tract may include changes in the expression level of intestinal barrier-related genes in the intestinal tissue.
  • genes related to the intestinal barrier may include Tjp1, Ocln, and Cldn2.
  • Tjp1 is tight junction protein 1
  • Ocln is Occludin
  • Cldn2 is Claudin-2.
  • the local effect on the gastrointestinal tract may include increased expression of Tjp1 and Ocln in the intestinal tissue.
  • the local effect on the gastrointestinal tract may include a decrease in Cldn2 expression in the intestinal tissue.
  • the local effect of the gastrointestinal tract may include reducing the expression of pro-inflammatory cytokine genes in the intestinal tissue
  • the pro-inflammatory cytokine genes are selected from any one of the following group: Tnf, Il17 , Ifng, Il1b, Il6, Cxcl2 and Kc; among them, Tnf is tumor necrosis factor, Il17 is interleukin 17, Ifng is interferon ⁇ , Il1b is interleukin 1 ⁇ , Il6 is interleukin 6, Kc and Cxcl2 are Chemokine members.
  • the intestinal tissue may comprise jejunum, ileum, cecum, or colon.
  • the drug is formulated so that the nitric oxide synthase pathway inhibitor is still used to prevent, alleviate, and/or treat the gastrointestinal ischemia-reperfusion-related
  • the effective amount of distal injury is present locally in the gastrointestinal tract.
  • the drug is formulated so that at most 50% of the nitric oxide synthase pathway inhibitor in the drug is absorbed by the subject and enters the blood circulation about 1 hour or later after administration system.
  • the concentration of the nitric oxide synthase pathway inhibitor in the drug is about 0.0001% (w/w) to about 90% (w/w).
  • the concentration of the nitric oxide synthase pathway inhibitor is about 0.0005% (w/w) to about 90% (w/w), about 0.001% (w/w) to about 85% (w/w) , About 0.0015% (w/w) to about 80% (w/w), about 0.002% (w/w) to about 75% (w/w), about 0.0025% (w/w) to about 70% ( w/w), about 0.003% (w/w) to about 65% (w/w), about 0.0035% (w/w) to about 60% (w/w), about 0.004% (w/w) to About 55% (w/w), about 0.0045% (w/w) to about 50% (w/w), about 0.005% (w/w) to about 45% (w/w), about 0.0055% (w /w) to about 40% (w/w), about 0.006% (w/w) to about 35% (w/w), about 0.0065% (w/w) to about 30% (w/w),
  • the subject has been, is currently or is at risk of suffering from a disease or disorder related to the gastrointestinal ischemia-reperfusion.
  • the diseases or conditions related to gastrointestinal ischemia-reperfusion may include natural events, trauma, or one or more surgical operations or other therapeutic interventions to reduce/stop blood flow in the gastrointestinal tract.
  • the natural event may include arterial infarction, venous obstruction, or systemic hypotension that destroys or reduces blood flow to internal organs, and the systemic hypotension may include hemorrhagic shock due to blood loss, myocardial infarction, or heart failure The resulting cardiogenic shock, neurogenic shock, renal shock or allergic reaction.
  • the diseases and/or conditions related to gastrointestinal ischemia-reperfusion may include stroke, trauma, shock, sepsis, acute pancreatitis, and/or inflammatory bowel disease.
  • the disease related to gastrointestinal ischemia-reperfusion may include stroke.
  • the stroke may include a group of diseases that cause brain tissue damage due to a sudden rupture of blood vessels in the brain or blood can not flow into the brain or blood flow into the brain is reduced due to blood vessel obstruction.
  • the disease may include ischemic stroke.
  • the ischemic stroke may include atherosclerotic occlusion of large arteries, cerebral embolism (embolic infarction), non-embolic infarction of small deep perforating arteries (lacunar infarction), and by distal arteries.
  • Watershed area ischemia (hemodynamic stroke) caused by stenosis and decreased cerebral blood flow.
  • the subject has ever suffered from the above-mentioned diseases or conditions related to the gastrointestinal ischemia-reperfusion.
  • the once may include that the subject has the aforementioned disease associated with the gastrointestinal ischemia-reperfusion before the administration of the drug to the subject.
  • the subject is suffering from the above-mentioned diseases related to the gastrointestinal ischemia-reperfusion.
  • the treatment may include that the subject suffers from the above-mentioned disease associated with the gastrointestinal ischemia-reperfusion when the drug is administered to the subject.
  • the subject is at risk of suffering from the above-mentioned diseases related to the gastrointestinal ischemia-reperfusion.
  • the risk may include that the patient may suffer from the above-mentioned diseases related to the gastrointestinal ischemia-reperfusion after administering the drug to the subject.
  • the subject has experienced, is or is at risk of experiencing the gastrointestinal ischemia-reperfusion.
  • the subject has experienced ischemia-reperfusion of the gastrointestinal tract.
  • the once may comprise that the subject has experienced the gastrointestinal ischemia-reperfusion prior to administering the drug to the subject.
  • the subject is experiencing ischemia-reperfusion of the gastrointestinal tract.
  • the being may include that the subject is experiencing ischemia-reperfusion of the gastrointestinal tract when the drug is administered to the subject.
  • the subject is at risk of experiencing the gastrointestinal ischemia-reperfusion.
  • the at-risk may include that the subject has undergone the gastrointestinal ischemia-reperfusion after administering the drug to the subject.
  • the distal injury related to gastrointestinal ischemia-reperfusion includes ischemic stroke.
  • the drug is configured to be suitable for oral administration.
  • oral administration for example, oral administration.
  • the oral dosage form may include capsules, tablets, pills, granules, or syrups.
  • the oral dose can be in the range from about 0.01 to 1000 mg/kg, from about 0.01 to 200 mg/kg, from about 0.01 to 180 mg/kg, from about 0.01 to 160 mg/kg, from about 0.01 to 140 mg/kg, from about 0.01 to 120 mg/kg, from about 0.01 to 100 mg/kg, from about 0.1 to 200 mg/kg, from about 0.1 to 150 mg/kg, from about 0.1 to 100 mg/kg, from about 0.1 to 80 mg/kg, from about 1 to 60mg/kg, from about 0.1 to 40mg/kg, from about 0.1 to 20mg/kg, 25-200mg/kg, about 50-200mg/kg, about 100-200mg/kg, about 25-50mg/kg, about 25- It is administered at a dose of 100 mg/kg, about 50-100 mg/kg, or about 50-200 mg/kg of body weight.
  • the oral mode may include taking one or more times a day, daily, every other day, every week, every two weeks, every month, or every two months.
  • the oral administration time is about 1-7 days, about 1-6 days, about 1-5 days, about 1-4 days, about 1-3 days, about 1-2 days, 1-24 days after stroke. Hours, about 3-12 hours, about 6-12 hours, about 1-3 hours, about 1-6 hours, about 3-6 hours, about 3-12 hours, about 6-12 hours.
  • the nitric oxide synthase pathway inhibitor is basically not decomposed and/or inactivated by the digestive juice.
  • the digestive juice may include saliva, gastric juice, small intestinal juice, pancreatic juice, and bile.
  • saliva has a pH of 6.6 to 7.1, and its main components include salivary amylase, lysozyme, and a small amount of inorganic substances (such as inorganic salts containing sodium, potassium, and calcium).
  • the nitric oxide synthase pathway inhibitor is basically It is not broken down and/or inactivated by the saliva.
  • gastric juice has a pH of 0.9 to 1.5, and its main components include pepsin, gastric acid (ie, hydrochloric acid), mucus, and inorganic substances such as sodium and potassium salts.
  • the nitric oxide synthase pathway inhibitor is basically not affected by the said nitric oxide synthase pathway inhibitor.
  • the gastric juice is broken down and/or inactivated.
  • pancreatic juice has a pH of 7.8 to 8.4, and its main components include sodium bicarbonate, pancreatic amylase, pancrelipase, trypsinogen, and chymotrypsinogen, etc.
  • the nitric oxide synthase pathway inhibitor is basically not affected.
  • the pancreatic juice is decomposed and/or inactivated.
  • bile has a pH of about 6.8 to 7.4, and its main components are bile salts and bile pigments, and the nitric oxide synthase pathway inhibitor is basically not decomposed and/or inactivated by the bile.
  • the small intestinal juice which has a pH of about 7.6, contains various digestive enzymes such as amylase, maltase, sucrase, lactase, peptidase, and lipase.
  • the nitric oxide synthase pathway inhibitor is basically not affected.
  • the small intestinal juice is decomposed and/or inactivated.
  • the nitric oxide synthase pathway inhibitor is not substantially decomposed and/or inactivated by the saliva, gastric juice, pancreatic juice, bile, and small intestinal juice.
  • the nitric oxide synthase pathway inhibitor is basically not decomposed and/or inactivated when the pH is 6.6-7.1, 0.9-1.5, 7.8-8.4, 6.8-7.4, or 7.6.
  • the nitric oxide synthase pathway inhibitor is not substantially decomposed and/or inactivated by the digestive juice. It may include that the nitric oxide synthase pathway is inhibited in the digestive juice or can be substantially maintained after being in contact with the digestive juice. Inhibit the performance of the nitric oxide synthase.
  • the application also provides the use of nitric oxide synthase for screening drugs, wherein the drugs are used to prevent, alleviate and/or treat gastrointestinal ischemia-reperfusion related distal injury in a subject .
  • the application also provides the use of nitric oxide synthase pathway inhibitors for screening drugs for the prevention, alleviation and/or treatment of neurological-related diseases or disorders in subjects.
  • the screening of drugs may include the evaluation process of biological activity, pharmacological effects and medicinal value of substances that may be used as drugs.
  • the screening drugs may include screening at the biochemical level and at the cellular level.
  • the drug selection may also include high-throughput screening and virtual drug screening.
  • the drug inhibits the expression and/or activity of the nitric oxide synthase.
  • the nitric oxide synthase is an inducible nitric oxide synthase iNOS.
  • the present application also provides a pharmaceutical composition, which may include the nitric oxide synthase pathway inhibitor described in the present application and optionally a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may include health care products.
  • the health product generally refers to a product that has the common characteristics of general foods, can regulate the functions of the human body, and is suitable for consumption by a specific population, but is not for the purpose of curing diseases, and is also called a dietary supplement. Including tea, wine, bee products, drinks, soups, fresh juices, medicated food, etc.
  • the pharmaceutical composition may include a biological product.
  • the biological product usually refers to the preparation of biological materials such as microorganisms, cells and various animal and human-derived tissues and liquids obtained by ordinary or genetic engineering, cell engineering, protein engineering, fermentation engineering and other biotechnologies. Products for the prevention, treatment and diagnosis of human diseases. Including vaccines, vaccines, toxins, toxoids, blood products, immunoglobulins, antigens, allergens, cytokines, hormones, enzymes, fermentation products, monoclonal antibodies, DNA recombinant products, etc.
  • the nitric oxide synthase pathway inhibitor may include an amino acid inhibitor and/or a non-amino acid inhibitor.
  • the amino acid inhibitor may include aminoguanidine AG, 1400W, L-NIL and/or isothiourea
  • the non-amino acid inhibitor may include glucocorticoids, flavonoids, 2-amino-4-methylpyridine and / Or aminopiperidine.
  • the pharmaceutically acceptable carrier may include pharmaceutically acceptable substances, compositions or vehicles involved in carrying or transporting chemical reagents, such as buffers, surfactants, stabilizers, preservatives, for enhancing bioavailability Absorption enhancers, liquid or solid fillers, diluents, excipients, solvents, encapsulating materials and/or other conventional solubilizers or dispersants.
  • chemical reagents such as buffers, surfactants, stabilizers, preservatives, for enhancing bioavailability Absorption enhancers, liquid or solid fillers, diluents, excipients, solvents, encapsulating materials and/or other conventional solubilizers or dispersants.
  • the pharmaceutical composition may contain any one or any combination of the following components: gum arabic, alginate, alginic acid, aluminum acetate, benzyl alcohol, butyl paraben, butyl Hydroxytoluene, antioxidant, citric acid, calcium carbonate, candelilla wax, croscarmellose sodium, candy sugar, colloidal silicon dioxide, cellulose, carnauba wax, corn starch, carboxymethyl cellulose Calcium, calcium stearate, calcium disodium EDTA, copovidone, hydrogenated castor oil, calcium hydrogen phosphate dehydrated, cetylpyridinium chloride, cysteine HC1, crospovidone, calcium hydrogen phosphate, phosphoric acid Disodium hydrogen, dimethicone, sodium erythrosine, ethyl cellulose, gelatin, glyceryl monooleate, glycerin, glycine, glyceryl monostearate, glyceryl behenate, hydroxypropyl Cellu
  • the pharmaceutical composition may be in solid form, for example, capsules, tablets, pills, granules, sachets or lozenges; or may be in liquid form, such as solutions, suspensions, emulsions or syrups.
  • the present application also provides a method for preventing, alleviating and/or treating gastrointestinal ischemia-reperfusion-related distal injury in a subject, the method comprising administering the present application to the subject The nitric oxide synthase pathway inhibitor.
  • the application also provides a prevention, alleviation and/or treatment of a neurological-related disease or disorder in a subject, the method comprising administering the nitric oxide synthase pathway inhibitor described in the application to the subject.
  • the administration includes administration via the gastrointestinal tract.
  • the administration via the gastrointestinal tract includes the specific release of the nitric oxide synthase pathway inhibitor at or near a region of the gastrointestinal tract.
  • the nitric oxide synthase pathway inhibitor is delivered to the gastrointestinal tract by a mechanical device.
  • the nitric oxide synthase pathway inhibitor is delivered to the gastrointestinal tract through an endoscope or a spray catheter.
  • oral sustained-release agents and disintegrants are used to release the nitric oxide synthase pathway inhibitor in the gastrointestinal tract.
  • sustained-release agent and disintegrant can initiate release or disintegration depending on the pH of the gastrointestinal tract.
  • the nitric oxide synthase pathway inhibitor can be released in the gastrointestinal tract through enteric coated capsules and liposome microcapsules.
  • the nitric oxide synthase pathway inhibitor is delivered to the gastrointestinal tract by gavage.
  • the time of administration via the gastrointestinal tract is about 1-7 days after stroke, about 1-6 days, about 1-5 days, about 1-4 days, about 1-3 days, about 1-2 days, 1 -24 hours, about 3-12 hours, about 6-12 hours, about 1-3 hours, about 1-6 hours, about 3-6 hours, about 3-12 hours, about 6-12 hours.
  • the nitric oxide synthase pathway inhibitor works locally in the gastrointestinal tract.
  • the nitric oxide synthase pathway inhibitor is still present in the stomach in an effective amount to prevent, alleviate, and/or treat the distal injury caused by the gastrointestinal ischemia-reperfusion. Local intestines.
  • nitric oxide synthase pathway inhibitor For example, about 1 hour or later after administration, up to 50% of the nitric oxide synthase pathway inhibitor is absorbed by the subject and enters the blood circulatory system.
  • the administration dose of the nitric oxide synthase pathway inhibitor is from about 0.01 to 1000 mg/kg, from about 0.01 to 200 mg/kg, from about 0.01 to 180 mg/kg, from about 0.01 to 160 mg/kg, from About 0.01 to 140 mg/kg, from about 0.01 to 120 mg/kg, from about 0.01 to 100 mg/kg, from about 0.1 to 200 mg/kg, from about 0.1 to 150 mg/kg, from about 0.1 to 100 mg/kg, from about 0.1 To 80mg/kg, from about 1 to 60mg/kg, from about 0.1 to 40mg/kg, from about 0.1 to 20mg/kg, 25-200mg/kg, about 50-200mg/kg, about 100-200mg/kg, about 25-50mg/kg, about 25-100mg/kg, about 50-100mg/kg, about 50-200mg/kg body weight.
  • this application also provides a method for preventing gastrointestinal ischemia-reperfusion-related distal injury in a subject, the method comprising: (1) monitoring the gastrointestinal condition of the subject (2) When or after the monitoring shows that the subject has undergone gastrointestinal ischemia-reperfusion, administer a nitric oxide synthase pathway inhibitor to the subject.
  • the method further includes monitoring diseases or conditions related to gastrointestinal ischemia-reperfusion before step (1).
  • the detection of the gastrointestinal condition of the subject includes clinical examination, clinical examination and/or clinical examination to assess the gastrointestinal condition of the subject.
  • the clinical examination may include observing the patient’s appetite, dysphagia, abdominal pain, nausea, vomiting, hematemesis, hematochezia, stool characteristics, whether there are symptoms including abdominal pain and bloating during defecation, whether the bowel habits are changed, and the abdomen Physical examination.
  • clinical testing includes three major routines and so on.
  • the clinical test may include blood routine, urine routine, and stool routine.
  • clinical examination can include abdominal X-ray, ultrasound, CT, endoscopy, ERCP, and PTC.
  • monitoring the condition of the gastrointestinal tract of the subject may include observing changes in blood flow in the gastrointestinal area.
  • the administration includes administration via the gastrointestinal tract.
  • sustained-release agent and disintegrant can initiate release or disintegration depending on the pH of the gastrointestinal tract.
  • the nitric oxide synthase pathway inhibitor can be released in the gastrointestinal tract through enteric coated capsules and liposome microcapsules.
  • the nitric oxide synthase pathway inhibitor is delivered to the gastrointestinal tract by gavage.
  • the nitric oxide synthase can include any one of the following types: I neural nitric oxide synthase (nNOS or NOS1), II inducible nitric oxide synthase (iNOS or NOS2) , III Endothelial nitric oxide synthase (eNOS or NOS3).
  • I neural nitric oxide synthase nNOS or NOS1
  • II inducible nitric oxide synthase iNOS or NOS2
  • III Endothelial nitric oxide synthase eNOS or NOS3
  • the nitric oxide synthase may include an inducible nitric oxide synthase (iNOS or NOS2).
  • iNOS inducible nitric oxide synthase
  • the nitric oxide synthase pathway inhibitor inhibits the expression and/or activity of nitric oxide synthase.
  • the nitric oxide synthase pathway inhibitor inhibits the expression/or activity of inducible nitric oxide synthase iNOS.
  • the nitric oxide synthase pathway inhibitor inhibits the expression/or activity of inducible nitric oxide synthase iNOS in the gastrointestinal tract.
  • the nitric oxide synthase pathway inhibitor reduces the expression of inducible nitric oxide synthase iNOS in the gastrointestinal tract.
  • the nitric oxide synthase pathway inhibitor reduces the concentration of nitrate in the gastrointestinal tract.
  • the nitric oxide synthase pathway inhibitor may include an amino acid inhibitor.
  • the nitric oxide synthase pathway inhibitor may include a non-amino acid inhibitor.
  • the amino acid inhibitor may include aminoguanidine AG, 1400W, L-NIL and/or isothiourea and the like.
  • the structural formula of 1400W is as follows:
  • L-NIL The structural formula of L-NIL is as follows:
  • non-amino acid inhibitors may include glucocorticoids, flavonoids, 2-amino-4-methylpyridine and/or aminopiperidine and the like.
  • the distal injury related to ischemia-reperfusion of the gastrointestinal tract may include pathological or physiological processes of any organ or tissue different from the gastrointestinal tract.
  • the pathological or physiological process of any organ or tissue different from the gastrointestinal tract may occur simultaneously with the gastrointestinal ischemia-reperfusion.
  • the simultaneous occurrence may be that the pathological or physiological process of any organ or tissue different from the gastrointestinal tract and the occurrence time of gastrointestinal ischemia-reperfusion are within 10 minutes, such as within 5 minutes, such as 3 minutes. Within, for example, within 1 minute.
  • the pathological or physiological process of any organ or tissue different from the gastrointestinal tract may occur before the gastrointestinal ischemia-reperfusion.
  • the pathological or physiological process of any organ or tissue different from the gastrointestinal tract may occur within 24 hours before the gastrointestinal ischemia-reperfusion. For example, within 22 hours, such as within 20 hours, such as within 18 hours, such as within 16 hours, such as within 14 hours, such as within 12 hours, such as within 10 hours, such as within 8 hours, such as within 6 hours, such as within 5 hours
  • the pathological or physiological process of any organ or tissue different from the gastrointestinal tract may occur after the gastrointestinal tract ischemia-reperfusion.
  • the pathological or physiological process of any organ or tissue different from the gastrointestinal tract may occur within 30 days after the gastrointestinal ischemia-reperfusion. For example, within 28 days, such as 26 days, such as 24 days, such as 22 days, such as 20 days, such as 18 days, such as 16 days, such as 14 days, such as 12 days, such as 10 days, such as 8 days, such as 1 day, such as 6 days , Such as within 5 days, such as within 4 days, such as within 3 days, such as within 2 days, such as within 1 day, such as within 12 hours, such as within 9 hours, such as within 6 hours, such as within 3 hours, such as within 1 hour, such as within 0.5 hour .
  • the pathological or physiological process of any organ or tissue different from the gastrointestinal tract can be expressed as when the gastrointestinal ischemia-reperfusion or gastrointestinal ischemia-reperfusion related effects or characteristics are intervened, the organ Or the pathophysiological process of the tissue is alleviated or reduced.
  • the effects or characteristics related to intestinal ischemia-reperfusion may include gastrointestinal tissue Nos2, Nox1, Duox2 expression changes, gastrointestinal tissue nitrate content changes, intestinal barrier changes, gastrointestinal tissue pro-inflammatory factors; wherein, Nos2 Encoding inducible nitric oxide synthase, Nox1 encoding NADPH oxidase 1, Duox2 encoding dioxygenase 2, NADPH oxidase 1 and dioxygenase 2 are related to the production of reactive oxygen species.
  • the change in expression of iNOS, Nox1, Duox2 in gastrointestinal tissue may include increased expression of iNOS, Nox1, and Duox2 in gastrointestinal tissue
  • the change in nitrate content in gastrointestinal tissue may include an increase in nitrate content in gastrointestinal tissue.
  • Changes in the intestinal barrier may include damage to the intestinal barrier, changes in gastrointestinal tissue pro-inflammatory factors, and elevated gastrointestinal tissue pro-inflammatory factors.
  • the intestinal barrier damage may include decreased expression levels of Tjp1, Ocln genes, and increased expression levels of Cldn2 genes.
  • the inflammatory factors may include Tnf, Il17, Ifng, Il1b, Cxcl2, Kc, and/or Il6; wherein, Ocln, Cldn2 is an intestinal barrier related gene, Ocln is Occludin, Cldn2 is Claudin-2; Tnf is tumor necrosis factor, Il17 is interleukin 17, Ifng is interferon ⁇ , Il1b is interleukin 1 ⁇ , Il6 is interleukin 6, Kc and Cxcl2 chemokines.
  • the changes in the expression of iNOS, Nox1, Duox2 in gastrointestinal tissues may include an increase in the expression of iNOS, Nox1, and Duox2 in gastrointestinal tissues and then a decrease, and the changes in nitrate content in gastrointestinal tissues may include an increase in nitrate content in gastrointestinal tissues. Then drop again.
  • the pathological or physiological process of any organ or tissue of the stomach that is different from the gastrointestinal tract may include a systemic or local distal reaction caused by the reperfusion of ischemic tissue of the gastrointestinal tract, and the reaction may be Including extensive microvascular dysfunction, changes in tissue barrier function, and inflammation.
  • the pathological or physiological process of any organ or tissue different from the gastrointestinal tract may involve the lung.
  • the pathological or physiological process of any organ or tissue different from the gastrointestinal tract may include pulmonary edema, intrapulmonary thrombosis process, pulmonary embolism, and/or inflammation of lung tissue.
  • the pathological or physiological process of any organ or tissue different from the gastrointestinal tract may involve the kidney.
  • the pathological or physiological process of any organ or tissue different from the gastrointestinal tract may include renal failure, edema formation, thrombosis, thromboembolism, and/or inflammation of the kidney tissue.
  • the pathological or physiological process of any organ or tissue different from the gastrointestinal tract may involve the central nervous system.
  • the pathological or physiological process of any organ or tissue different from the gastrointestinal tract may include blood-brain barrier destruction, silent cerebral ischemia, stroke, cerebral edema, increase in intracranial pressure, inflammation of neuronal tissue, Nerve cell death, brain damage and/or nervous system dysfunction.
  • the pathological or physiological process of any organ or tissue different from the gastrointestinal tract may include ischemic stroke and related disorders.
  • the ischemic stroke and related disorders may include the ischemic stroke area that is not stained (off-white) in the TTC staining in the mouse MCAO model, and the ischemia in the Nissl staining of the frozen section of the tissue The purple Nissl body disappeared in the area of acute stroke, or the modified neurological deficit score of the mouse increased.
  • the pathological or physiological process of any organ or tissue different from the gastrointestinal tract may include inflammation.
  • the pathological or physiological process of any organ or tissue different from the gastrointestinal tract may include systemic inflammation.
  • the inflammation can affect the lungs, gastrointestinal system, cardiovascular system, other limbs, and/or central nervous system.
  • the pathological or physiological process of any organ or tissue different from the gastrointestinal tract may include systemic inflammatory response syndrome (severe inflammatory response syndrome) and/or multiple organ dysfunction syndrome (MODS).
  • systemic inflammatory response syndrome severe inflammatory response syndrome
  • MODS multiple organ dysfunction syndrome
  • the pathological or physiological process of any organ or tissue different from the gastrointestinal tract may include diseases, disorders or medical interventions that cause ischemia and reperfusion of the gastrointestinal tract, such as stroke, trauma, shock, sepsis, Surgery for acute pancreatitis, inflammatory bowel disease or brain injury.
  • diseases, disorders or medical interventions that cause ischemia and reperfusion of the gastrointestinal tract such as stroke, trauma, shock, sepsis, Surgery for acute pancreatitis, inflammatory bowel disease or brain injury.
  • the trauma includes damage to human tissues or organs caused by external forces, such as traffic injuries, fall injuries, mechanical injuries, sharp weapon injuries, fall injuries, and firearm injuries.
  • the shock usually refers to a clinical syndrome in which the effective circulating blood volume drops sharply after the body is attacked by a strong pathogenic factor, the body loses compensation, tissue ischemia and hypoxia, and neuro-humoral factor imbalance. Its main characteristics can include insufficient microcirculation perfusion in important organs and tissues, metabolic disorders, and dysfunctions of various systems throughout the body.
  • hypovolemic shock vasodilatory shock, cardiogenic shock.
  • the hypovolemic shock may include hemorrhagic shock, burn shock, and traumatic shock; the vasodilatory shock may include septic shock, anaphylactic shock, and neurogenic shock.
  • the nervous system-related diseases may include blood-brain barrier destruction, silent cerebral ischemia, stroke, cerebral edema, increased intracranial pressure, inflammation of neuronal tissue, neuronal cell death, brain damage, and/ Or nervous system dysfunction.
  • the nervous system dysfunction may include motor dysfunction and internal environment regulation disorder.
  • the internal environment regulation disorder may include an activity disorder related to innervation of internal organs, body fluids, or blood circulatory system.
  • the neurological related disorders may include cerebral ischemic diseases and related disorders.
  • the cerebral ischemic disease may include a group of diseases in which blood cannot flow into the brain or the blood flow into the brain is reduced due to vascular obstruction, disease, trauma, etc., causing brain tissue damage.
  • the cerebral ischemic disease may include ischemic stroke and related conditions.
  • the ischemic stroke-related disorder may include cerebral infarction.
  • the ischemic stroke may include atherosclerotic occlusion of large arteries, cerebral embolism (embolic infarction), non-embolic infarction of small deep perforating arteries (lacunar infarction), and remote Watershed area ischemia (hemodynamic stroke) caused by end artery stenosis and decreased cerebral blood flow.
  • embolism embolic infarction
  • non-embolic infarction of small deep perforating arteries lacunar infarction
  • remote Watershed area ischemia hemodynamic stroke
  • the ischemic stroke-related disorders may include brain nerve cell death, brain function damage, changes in breath/taste/hearing or vision, impaired swallowing/pupil response to light, physical movement disorders, aphasia, changes in breathing and heart rate, Other tissues or organs have neuroregulation disorders, loss of consciousness, etc.
  • the ischemic stroke-related disorder may include any index in NIHSS.
  • the NIHSS is the National Institute of Health Stroke Scale (National Institute of Health stroke scale), and the scoring method of the NIHSS can be referred to Williams LS, Yilmaz EY, Lopez-Yunez AM. Retrospective Assessment of Initial Stroke Severity with The NIH Stroke Scale. Stroke. 2000; 31:858–862).
  • the ischemic stroke and related conditions in the mouse MCAO model can be expressed as the ischemic stroke area is not stained (off-white) in TTC staining, and the tissue frozen section Nissl staining is ischemic Nissl bodies disappeared in the stroke area, or the modified neurological deficit score of mice increased.
  • the following examples are only to illustrate the use of the nitric oxide synthase pathway inhibitor of the present application in the preparation of drugs, the use of nitric oxide synthase in screening drugs, and the use of Pharmaceutical compositions of nitrogen synthase pathway inhibitors, etc., are not used to limit the scope of the present invention.
  • Stool sample genomic DNA extraction kit (MinkaGene Stool DNA Kit) is a product of Minka Gene; primers are synthesized by Thermo Fisher; TaqMan reverse transcription reagents and SYBR Green are products of Takara Bio; ViiA 7 real-time PCR system is a product of Applied Biosystems; nitric acid
  • the salt/nitrite test kit is a product of Sigma; TTC powder is a product of Sigma; D-Lac, LBP, and LPS serum ELISA kits are products of Genemei; other materials, reagents, etc., are available unless otherwise specified Obtained from commercial sources.
  • R analysis software is used for data analysis of non-microbial informatics data. Normally distributed data are expressed as mean ⁇ standard deviation, and non-normally distributed data are displayed in the form of median (interquartile range).
  • Non-parametric test uses Kruskal-Wallis rank sum test or Mann-Whitney U test, parametric test Use unpaired Student's test or ONE-WAY ANOVA for analysis. Categorical variables are expressed in proportions.
  • Use the Shapiro-Wilk test to check the normality of the data.
  • the Adonis test implemented in QIIME 1.9.1 is used. P ⁇ 0.05 (two-tailed) was considered a significant difference.
  • mice SPF-grade 8-week-old male C57BL/6 wild-type mice were selected as experimental subjects, and they were randomly grouped as follows: SHAM group, sham operation treatment; MCAO group, MCAO modeling treatment; Post-AG group (AG group ), after the mice were modeled by MCAO, they were given AG intervention (100mg/kg, gavage, 200 ⁇ l/1 time); in the Pre-AG group, mice were given AG intervention before being modeled by MCAO ( 1mg/ml, drinking 3-5 ⁇ l daily for 5 days).
  • mice were weighed and anesthetized with 0.2ml/10g tribromoethanol, a 1cm median incision in the neck was cut, and the tissue was carefully peeled off to expose the right carotid artery triangle.
  • external carotid artery external carotid artery needs to be peeled up as far as possible until it enters the anterior bifurcation of the skull
  • internal carotid artery ligate the proximal end of the common carotid artery (slip knot), use an electrocoagulator
  • the tip coagulates the small branches of the external carotid artery, and ligates the proximal end (slip knot) and the distal end (dead knot) of the external carotid artery.
  • mice Observe the blood flow of the blood vessels in the cecum of the mice in the SHAM group and the MCAO group.
  • the time points of the mice in the MCAO group were before the modeling operation, after the embolization line, after the embolization line, and 1 hour after the embolization line.
  • Group mice underwent sham operation and observed blood flow at the response time point. The specific steps are as follows:
  • mice Before the operation, the mice should be anesthetized, the abdomen should be disinfected, and the skin and muscle layer should be cut along the midline of the abdomen to expose the cecum;
  • the analysis adopts the blood flow changes in the Region of Interest (ROI), the region of interest is the six branches of the cecum, and the value of the region of interest is extracted as the blood flow value after recording.
  • ROI Region of Interest
  • the relative regional blood flow value at different time points in the detection process is the average blood flow value in the region of interest at the corresponding time divided by the blood flow value at the initial time point.
  • mice The core body temperature of mice was controlled at 37+0.5°C during the whole operation.
  • RNA extraction with Trizol reagent method (Invitrogen): Add 1ml TRIZOL to every 50-100mg homogeneous tissue sample for homogenization. Then add 0.2ml of chloroform, shake vigorously for 15 seconds after covering, place it at 15-30°C for 2-3 minutes, and then centrifuge at 12,000g for 15 minutes at 2-8°C. Transfer the upper aqueous phase, add 0.5ml isopropanol, leave it for 10 minutes at 15-30°C, and then centrifuge at 12,000g for 10 minutes at 2-8°C. Add 1ml 75% ethanol to wash the RNA pellet for 5 minutes. Then dissolve RNA and determine the concentration;
  • the nitrate concentration of cecal mucus extract was measured at 3 hours, 6 hours, and 24 hours after stroke. The specific steps are as follows:
  • step (3) Take the sample obtained in step (2) and operate according to the instructions of the nitrate/nitrite detection kit (colorimetric method) kit (Sigma) to prepare a standard curve, and calculate the nitrate concentration according to the standard curve.
  • Example 1 According to the description in Example 1, the following three groups of mice were prepared: SHAM group, sham operation; MCAO group, MCAO modeling; Post-AG group, MCAO modeling, 1 hour after stroke (ie One hour after the tether was removed) AG intervention (100mg/kg, 0.2ml, gavage, once) was carried out, and the MCAO model was carried out according to the method described in Example 2; 24 hours after the stroke, it was carried out according to The method described in Example 4 sampled three groups of mice and determined the expression level of Nos2 gene in intestinal tissues.
  • aminoguanidine significantly reduced the expression level of Nos2 gene in the intestinal tissues of mice after stroke.
  • Example 7 The effect of aminoguanidine (AG) intervention on the expression levels of Nox1 and Duox2 genes in intestinal tissues
  • Example 1 According to the description in Example 1, the following three groups of mice were prepared: SHAM group, sham operation; MCAO group, MCAO modeling; Post-AG group, MCAO modeling, 1 hour after stroke (ie One hour after the tether was removed) AG intervention (100mg/kg, 0.2ml, gavage, once) was carried out, and the MCAO model was carried out according to the method described in Example 2; 24 hours after the stroke, it was carried out according to the implementation
  • the method described in Example 4 sampled three groups of mice and determined the expression levels of Nox1 and Duox2 genes in intestinal tissues.
  • Aminoguanidine reduced the expression levels of Nox1 and Duox2 genes in the intestinal tissues of mice after stroke.
  • Example 8 The effect of aminoguanidine (AG) intervention on the nitrate concentration of cecal mucus extract
  • Example 1 According to the description in Example 1, the following three groups of mice were prepared: SHAM group, MCAO group, MCAO modeling treatment; Post-AG group, MCAO modeling mice, 1 hour after stroke (that is, after removing the tether 1 hour) AG intervention (100mg/kg, gavage, once), MCAO model was performed according to the method described in Example 2; 24 hours after the stroke, according to the method described in Example 5 Three groups of mice were sampled and the concentration of nitrate in mucus extract from the cecum of the mice was determined.
  • Example 9 The effect of aminoguanidine (AG) intervention on the expression of intestinal barrier factors and proinflammatory cytokine in intestinal tissue
  • Tjp1, Ocln, Cldn2, Tnf, Il17, Ifng, Il1b, Cxcl2, Kc, and Il6 were detected.
  • Tjp1, Ocln, Cldn2 are intestinal barrier related genes
  • Tjp1 is tight junction protein 1
  • Ocln is Occludin
  • Cldn2 is Claudin-2
  • Tnf is tumor necrosis factor
  • Il17 is interleukin 17
  • Il1b are Interleukin 1 ⁇
  • Il6 interleukin 6
  • Kc and Cxcl2 are chemokines. Sampling and testing were performed 24 hours after the stroke.
  • the primer sequences of Tjp1, Ocln, Cldn2, Tnf, Il17, Ifng, Il1b, Cxcl2, Kc, and Il6 are as shown in SEQ ID NO: 3-22.
  • Example 10 The effect of aminoguanidine (AG) intervention on brain damage in ischemic stroke
  • Example 1 Prepare the following three groups of mice according to the description in Example 1: MCAO group, MCAO modeling treatment; Post-AG group, MCAO modeling, 1 hour after stroke (that is, after removing the tether 1 hour) AG intervention (100mg/kg, gavage, 1 time); Pre-AG group, before MCAO modeling of mice, AG intervention (1mg/ml, drinking water daily 3-5 ⁇ l , 5 days), MCAO modeling was performed after 5 days of AG intervention. MCAO model building was performed in the manner described in Example 2;
  • brain injury area direct injury volume-(ipsilateral hemisphere of the body-contralateral hemisphere of the body), and finally the proportion of brain injury volume in the whole cerebral hemisphere is obtained.
  • the scoring system can comprehensively evaluate neurological functions, including movement, sensation, balance and reflex: 1 Exercise test: lift the tail of the mouse to evaluate the degree of bending and torsion of the limbs (0-3 points); posture when walking on a plane Evaluation (0-3 points). 2Balance test: The mouse is placed on a beam.
  • the assessment of neurological impairment is mainly based on whether the mouse can balance on the crossbeam, the limbs fall from the crossbeam, and the ability to pass the crossbeam smoothly (0-6 points).
  • 3 Sensory and reflex test test auricle reflex and corneal reflex (0-2 points) respectively.
  • mice in each group SHAM group, MCAO group, Post-AG group
  • mice in each group SHAM group, MCAO group, Post-AG group
  • Sectioning Adjust the temperature of the cryostat to -20°C. After the temperature is reached, the specimen is taken out, and the brain tissue is taken from the range of 2.0 mm to 4.0 mm behind the bregma (including the entire thalamus tissue), and continuously sliced into coronal slices with a thickness of 5 ⁇ m. Use a fine brush to flatten the section, place the section on a glass slide, and store it in a refrigerator at -20°C;
  • Nissl staining the brain slices were immersed in Nissl staining solution for 5 minutes and washed twice with distilled water. 95% ethanol for about 5 seconds. Dehydrate with 95% ethanol for 2 minutes. Immerse in fresh xylene for 5 minutes and cover with neutral gum. After the film was dried, it was observed under a microscope and pictures were collected for analysis. The cell membrane of the normally surviving neuron cell is intact and blue-purple, with deep staining of the nucleus.
  • Example 13 The effect of aminoguanidine (AG) intervention on serum midgut barrier markers and inflammatory factors
  • mice After the mice were assessed for mNSS, blood was collected. Blood collection methods include orbital blood collection and posterior vena cava blood collection.
  • the mouse serum samples were stored at -80°C before testing.
  • Mouse serum intestinal barrier markers such as D-Lac, LBP, and LPS are operated in accordance with the instructions of the ELISA kit (Elisa Lab).
  • the content of inflammatory factors such as IL-6 and TNF- ⁇ is strictly in accordance with the ELISA kit (Gene U.S., Elisa Lab) operate according to the steps in the manual, make a standard curve, and calculate each expression amount according to the standard curve.
  • Example 14 The effect of aminoguanidine (AG) intervention at different times or doses on brain damage in ischemic stroke
  • the dose effect of AG In the first hour after the mouse stroke, the mice were treated with different doses of AG intervention, the concentration range: 25mg/kg, 50mg/kg, 100mg/kg, 200mg/kg (intragastric 200 ⁇ l, 1 time) . And 24 hours after stroke, the degree of brain damage in mice was detected.
  • AG intervention 25mg/kg, 200 ⁇ l intragastric gavage, once was performed on the mice at 1, 3, 6, and 12 hours after stroke. And the degree of brain damage in mice was detected 24 hours after stroke.
  • Example 15 The effect of 1400W, L-NIL or 2-amino-4-methylpyridine (AMP) intervention on the expression level of Nos2 gene in intestinal tissue
  • mice Prepare the following five groups of mice as described in Example 1: SHAM group, sham operation treatment; MCAO group, MCAO modeling treatment; Post-1400W group/Post-L-NIL group/Post-AMP group, mice Perform MCAO modeling, 1 hour after stroke (that is, 1 hour after removing the thrombus), respectively, 1400W, L-NIL or AMP intervention (0.01-1000mg/kg, 0.2ml, gavage, 1 time), MCAO modeling It was carried out in the manner described in Example 2; 24 hours after the stroke, five groups of mice were sampled according to the method described in Example 4 and the Nos2 gene expression level in intestinal tissues was determined.
  • Example 16 The effect of 1400W, L-NIL or 2-amino-4-methylpyridine (AMP) intervention on the nitrate concentration of cecal mucus extract
  • mice Prepare the following five groups of mice as described in Example 1: SHAM group, sham operation treatment; MCAO group, MCAO modeling treatment; Post-1400W group/Post-L-NIL group/Post-AMP group, mice Perform MCAO modeling, 1 hour after stroke (that is, 1 hour after removing the thrombus), respectively, 1400W, L-NIL or AMP intervention (0.01-1000mg/kg, 0.2ml, gavage, 1 time), MCAO modeling It was carried out in the manner described in Example 2; 24 hours after the stroke, the three groups of mice were sampled according to the method described in Example 5 and the nitrate concentration in the mucus extract of the mouse cecum was measured.
  • Example 17 The effect of 1400W, L-NIL or 2-amino-4-methylpyridine (AMP) intervention on brain damage in ischemic stroke
  • Example 2 Prepare the following 4 groups of mice according to the description in Example 1: MCAO group, MCAO modeling treatment; Post-1400W group/Post-L-NIL group/Post-AMP group, MCAO modeling mice, after stroke 1400W, L-NIL or AMP intervention (0.01-1000mg/kg, gavage, 1 time) were performed for 1 hour (that is, 1 hour after the tether was removed). MCAO modeling was performed in the manner described in Example 2. According to the method of Example 10, the effect of 1400W, L-NIL or 2-amino-4-methylpyridine (AMP) intervention on the brain damage of mice with ischemic stroke was detected.
  • AMP 2-amino-4-methylpyridine

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Abstract

本申请涉及一氧化氮合酶通路抑制剂在制备药物中的用途，所述药物用于预防、缓解和/或治疗受试者中胃肠道缺血再灌注相关的远端损伤。本申请还涉及一氧化氮合酶用于筛选药物的用途，其中所述药物用于预防、缓解和/或治疗受试者中胃肠道缺血再灌注相关的远端损伤。本申请还涉及包含所述一氧化氮合酶通路抑制剂的药物组合物以及预防、缓解和/或治疗受试者中胃肠道缺血再灌注相关的远端损伤的方法。

Description

一氧化氮合酶通路抑制剂在制备药物中的用途 技术领域
本申请涉及生物医药领域，具体的涉及一氧化氮合酶通路抑制剂在制备药物中的用途。
背景技术
脑卒中是当今世界上最为广泛的疾病之一，其发病率逐年增加，脑卒中患者生活质量下降，严重时导致死亡。在世界范围内，从1990年到2010年缺血性卒中年发病率和死亡率分别增加了37％和21％。
目前，脑卒中患者的治疗重点是静脉溶栓或血管内治疗，例如静脉注射重组组织型纤溶酶原激活剂(r-tPA)，该方法仍被认为是最重要的治疗方法。此外，根据每位患者的疾病具体状况，还会有抗血小板治疗，抗凝血剂，神经保护剂以及其他的对症治疗，包括治疗和预防高血糖，高血压和急性中风并发症。然而鉴于中风发病日趋严重的形式，新的治疗方式、靶点及药物亟需进一步地开发。
发明内容
本申请提供了一氧化氮合酶通路抑制剂在制备药物中的用途，所述药物用于预防、缓解和/或治疗受试者中与胃肠道缺血再灌注相关的远端损伤。
在某些实施方式中，所述一氧化氮合酶通路抑制剂抑制一氧化氮合酶的活性。
在某些实施方式中，所述一氧化氮合酶包括诱导型一氧化氮合酶iNOS。
在某些实施方式中，所述一氧化氮合酶通路抑制剂包括氨基酸类抑制剂和/或非氨基酸类抑制剂。
在某些实施方式中，所述一氧化氮合酶通路抑制剂包括氨基酸类抑制剂，所述氨基酸类抑制剂包括氨基胍AG、1400W、L-NIL和/或异硫脲。
在某些实施方式中，所述一氧化氮合酶通路抑制剂包括非氨基酸类抑制剂，所述非氨基酸类抑制剂包括糖皮质激素、类黄酮、2-氨基-4-甲基吡啶和/或氨基哌啶。
在某些实施方式中，所述药物被配制为使得所述一氧化氮合酶通路抑制剂在胃肠道局部发挥效力。
在某些实施方式中，所述药物被配制为使得在施用后约48小时或之后，所述一氧化氮合酶通路抑制剂仍然以预防、缓解和/或治疗所述与胃肠道缺血再灌注相关的远端损伤的有效量存在于胃肠道局部。
在某些实施方式中，所述药物被配制为使得在施用后约1小时或之后，所述药物中至多50％的所述一氧化氮合酶通路抑制剂被所述受试者吸收而进入血液循环系统。
在某些实施方式中，所述药物中所述一氧化氮合酶通路抑制剂的浓度为约0.0001％(w/w)至约90％(w/w)。
在某些实施方式中，所述受试者曾经、正在或有风险患有与所述胃肠道缺血再灌注相关的手术、疾病或病症。
在某些实施方式中，所述与胃肠道缺血再灌注相关的疾病或病症包括脑卒中、创伤、休克、败血症、急性胰腺炎或炎症性肠病。
在某些实施方式中，所述与胃肠道缺血再灌注相关的疾病包括缺血性脑卒中。
在某些实施方式中，所述受试者曾经、正在或有风险经受所述胃肠道缺血再灌注。
在某些实施方式中，所述与胃肠道缺血再灌注相关的远端损伤包括缺血性脑卒中。
在某些实施方式中，所述药物被配置为适于经口施用。
在某些实施方式中，所述一氧化氮合酶通路抑制剂基本上不被消化液分解和/或灭活。
本申请还提供了一氧化氮合酶用于筛选药物的用途，所述药物用于预防、缓解和/或治疗受试者中与胃肠道缺血再灌注相关的远端损伤。
在某些实施方式中，所述药物抑制所述一氧化氮合酶的表达和/或活性。
在某些实施方式中，所述一氧化氮合酶包括诱导型一氧化氮合酶iNOS。
本申请还提供了一种药物组合物，其包含本申请所述的一氧化氮合酶通路抑制剂和任选地药学上可接受的载体。
本申请还提供了一氧化氮合酶通路抑制剂在制备保健制品中的用途，所述保健制品用于预防、缓解和/或治疗受试者中与胃肠道缺血再灌注相关的远端损伤。
本申请还提供了一氧化氮合酶通路抑制剂在制备生物制品中的用途，所述生物制品用于预防、缓解和/或治疗受试者中与胃肠道缺血再灌注相关的远端损伤。
本申请还提供了一种预防、缓解和/或治疗受试者中与胃肠道缺血再灌注相关的远端损伤的方法，所述方法包括向所述受试者施用本申请所述的一氧化氮合酶通路抑制剂。
在某些实施方式中，所述施用包括经胃肠道施用。
在某些实施方式中，所述一氧化氮合酶通路抑制剂在胃肠道局部发挥效力。
在某些实施方式中，在施用后约48小时或之后，所述一氧化氮合酶通路抑制剂仍然以预防、缓解和/或治疗所述胃肠道缺血再灌注相关的远端损伤的有效量存在于胃肠道局部。
在某些实施方式中，在施用后约1小时或之后，至多50％的所述一氧化氮合酶通路抑制剂被所述受试者吸收而进入血液循环系统。
在某些实施方式中，所述一氧化氮合酶通路抑制剂的给药剂量为约0.01至1000mg/kg体重。
本申请还提供了一种预防受试者中与胃肠道缺血再灌注相关的远端损伤的方法，所述方法包括：
a)监测所述受试者的胃肠道状况；
b)当所述监测显示所述受试者有风险经受胃肠道缺血再灌注，或在经历胃肠道缺血再灌注之时或之后，向所述受试者施用一氧化氮合酶通路抑制剂。
在某些实施方式中，所述施用包括经胃肠道施用。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明书如下：
图1显示的是本申请中通过激光散斑成像系统(瑞沃德RFLSI Pro)观察的小鼠盲肠血流变化情况。
图2显示的是本申请中通过激光散斑成像系统(瑞沃德RFLSI Pro)观察的小鼠盲肠血流ROI proportion的统计结果。
图3A-3C显示的是本申请中由缺血性脑卒中引起的胃肠道缺血再灌注后肠道(结肠)组织Nos2、Nox1与Duox2基因表达水平变化情况。
图4显示的是本申请中由缺血性脑卒中引起的胃肠道缺血再灌注后盲肠粘液层硝酸盐浓度变化情况。
图5A-5C显示的是本申请中氨基胍(AG)干预对于由缺血性脑卒中引起的胃肠道缺血再灌注后结肠组织Nos2、Nox1与Duox2基因表达水平的影响。
图6显示的是本申请中氨基胍(AG)干预对于由缺血性脑卒中引起的胃肠道缺血再灌注后盲肠粘液层硝酸盐浓度的影响。
图7A-7J显示的是本申请中氨基胍(AG)干预对于由缺血性脑卒中引起的胃肠道缺血再 灌注后肠组织中肠屏障相关因子及促炎性细胞因子表达水平的影响。
图8显示的是本申请中氨基胍(AG)干预对于小鼠胃肠道缺血再灌注后脑损伤的影响。
图9显示的是本申请中氨基胍(AG)干预对于小鼠改良神经缺损评分结果的影响。
图10A-10B显示的是本申请中尼氏(Nissl)染色示出的氨基胍(AG)干预对于小鼠脑部神经元死亡的影响。
图11A-11E显示的是本申请中氨基胍(AG)干预对于由缺血性脑卒中引起的胃肠道缺血再灌注后血清中肠屏障标记物和炎症因子含量的影响。
图12A-12B显示的是本申请中不同时间或剂量的氨基胍(AG)干预对于肠道缺血再灌注后缺血性脑卒中的影响。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
在本申请中，术语“一氧化氮合酶”通常指一组酶的统称，其通常能够将精氨酸中的氮原子在有氧环境下合成一氧化氮，其英文缩写形式通常为NOS。一般情况下，一氧化氮合酶的活性通常还需要辅助因子的参与，所述辅助因子可以包括烟酰胺腺嘌呤二核苷酸磷酸(NADPH)、黄素腺嘌呤二核苷酸(FAD)、黄素单核苷酸(FMN)、原血红素及四氢生物蝶呤(BH4)等。一氧化氮合酶通常可以包含以下三种：Ⅰ神经型一氧化氮合酶(nNOS或NOS1)，Ⅱ诱导型一氧化氮合酶(iNOS或NOS2)，Ⅲ内皮型一氧化氮合酶(eNOS或NOS3)。Ⅰ、Ⅲ型酶属于原生酶(cNOS)，也称作构成型酶(constitutiveNOS,cNOS)，是许多正常组织中基本存在的一种酶。
在本申请中，术语“诱导型一氧化氮合酶iNOS”通常指一般在某些病理或生理情况下被诱导才会表达的一氧化氮合酶。其活性通常不依赖钙离子和钙调蛋白，能够合成大量的一氧化氮。诱导iNOS的因素可以包括血红素、细胞因子、需氧应激、IFN-γ、TNF-α、IL-1α等。诱导型一氧化氮合酶iNOS可以包含人诱导型一氧化氮合酶iNOS。人诱导型一氧化氮合酶iNOS基因位于第17对常染色体的17cen-q11.2位上，长约37kb。其转录受细胞因子、脂多糖(LPS)和其他炎症介质控制。人的iNOS启动子是迄今己知的最大、最复杂的之一，其与转录调节因子相互作用控制iNOS基因的表达。iNOS的开放阅读框(open reading frame)由27个外显子所编码，包括翻译始动位(于外显子2)和终止位(于外显子27)。人iNOS的TATA盒位于转录始动部位上游30bp处，巨噬细胞和上皮细胞内的iNOS转录有6％左右在多个起始部位发动，部分甚至从TATA指导的主起始部位伸延开几百个硷基对的上游区。这样， 通过替代拼接(alternative splicing)获得的iNOS mRNA更多样化且稳定性不同。
在本申请中，术语“一氧化氮合酶通路”通常指调控一氧化氮合酶表达或活性的细胞内和/或细胞外信号通路。
在本申请中，术语“缺血”通常是指组织或器官供血量不足，进而导致缺氧及养分的情形。缺血一般情况下可以由血管问题所导致，例如血管栓塞，血管压迫；也可能因血管收缩、血栓形成或栓塞导致的局部贫血所导致，也可能由意外创伤、手术介入导致，也可能由其他器官或组织的疾病所导致，例如其他器官或组织的缺血性疾病，例如本申请中由缺血性脑卒中导致的胃肠道缺血。
在本申请中，术语“缺血再灌注”通常指在缺血或缺氧(缺氧或低氧)后血液供应返回组织的过程。换句话说，缺血再灌注通常指发生缺血的组织或器官在缺血诱因被消除/抵消/代偿/减缓后重新恢复血流的过程。在某些实施方式中，缺血组织的再灌注通常可以伴有如下任意情况的发生：再灌注部位微血管系统损伤，例如由于毛细血管和小动脉的渗透性增加，从而导致组织中的流体过滤及扩散增加；活化的内皮细胞在再灌注后产生更多的活性氧物质或者自由基导致随后的炎症反应；新返回血液将白血细胞由运送到再灌注区域，白细胞响应组织损伤从而释放炎症因子例如白细胞介素以及自由基，白细胞还可能与小毛细血管的内皮结合，阻塞它们从而导致新的缺血；另一方面，恢复的血流在组织内重新引入氧气，氧气在此特定情况下可能会破坏细胞的蛋白质，核酸和质膜结构，由此产生的反应性物质可能间接作用于氧化还原信号传导以开启细胞凋亡，细胞膜的损伤也可能进一步导致导致更多的自由基释放。所述的自由基可以包含氮氧自由基，例如一氧化氮或其衍生物。缺血再灌注通常会导致再灌注损伤，例如脑梗塞、急性心肌梗死、心肺复苏术后脑无再流现象、应激性溃疡、胰腺炎、烧伤、离体器官的移植、肠缺血、坏死性小肠结肠炎、间歇性跛行、急性肾小管坏死、休克后肝功能衰竭及多系统器官功能衰竭等。当所述再灌注损伤发生的部位与已经缺血再灌注的组织或器官不同时，通常可以称作远端再灌注损伤。
在本申请中，术语“远端损伤”通常指累及与已经缺血再灌注的局部器官或组织不同的器官或组织的病理生理过程。这一过程通常可能由局部缺血再灌注器官或组织部位生成的细胞产物经由循环系统暴露于其他器官所致。在某些实施方式中，所述远端损伤可以包括引起所述缺血再灌注的疾病、病征或医学介入，例如，脑卒中、创伤、休克、败血症、急性胰腺炎、炎症性肠病或脑外伤手术。在某些实施方式中，所述缺血再灌注可以包括胃肠道缺血再灌注。在某些实施方式中，所述远端损伤可以包括胃肠道缺血再灌注相关的缺血性脑卒中的损伤。例如，由缺血性脑卒中引起的胃肠道缺血再灌注相关的缺血性脑卒中的损伤。
在本申请中，术语“胃肠道缺血再灌注相关的远端损伤”通常是指与发生缺血再灌注的 胃肠道部位不同的器官或组织的病理生理过程。这种病理生理过程与胃肠道缺血再灌注有关，例如可以表现为与胃肠道缺血再灌注同时、在其之前或之后发生的与胃肠道部位不同的器官或组织的病理生理过程。例如，当对胃肠道缺血再灌注或者胃肠道缺血再灌注相关效应或表征进行干预时，所述器官或组织的病理生理过程表现出相应变化，例如其症状减轻或缓解。例如在本申请中基于胃肠道缺血再灌注施用一氧化氮合酶抑制剂后减轻了缺血性脑卒中的症状。
在本申请中，术语“抑制剂”通常指能够完全或部分地预防或降低一种或多种特定蛋白质的生理功能的化合物/物质或组合物。所述降低一种或多种特定蛋白质的生理功能可以包含蛋白质本身活性的降低或者本身存在量的降低。在某些实施方式中，所述抑制剂可以作为不同的晶体、无定形物质、药学上可接受的盐、水合物和溶剂化物而存在。
在本申请中，术语“施用”通常指通过任意引入或递送途径将所述抑制剂引入受试者的身体中。可以采用本领域技术人员已知的用于使细胞、器官或组织与所述抑制剂接触的任何方法。包括而不限于动脉内、鼻内、腹内、静脉内、肌内、皮下透皮或口服。每日剂量可以划分成一个、两个或更多个合适形式的剂量以在某个时间段期间的一个、两个或更多个时间施用。
在本申请中，术语“有效量”或“有效剂量”通常指足以实现或至少部分实现所需效果的量。药物或治疗剂的“治疗有效量”或“治疗有效剂量”通常是当单独使用或与另一种治疗剂组合使用时促进疾病消退(这通过疾病症状严重程度的降低、疾病无症状期的频度和持续时间的增加、或者由于罹患疾病而引起的损害或残疾的预防来证明)的任何药物量。药物的“预防有效量”或“预防有效剂量”通常是指当单独或与另一种治疗剂组合给有疾病发展或疾病复发的风险的受试者施用时抑制疾病的发展或复发的药物量。可以使用本领域技术人员已知的多种方法对治疗剂或预防剂促进疾病消退或抑制疾病发展或复发的能力进行评估，比如在处于临床试验期间的人类受试者中、在动物模型系统中预测对人类的功效、或者通过在体外测定中测定药剂的活性。
在本申请中，术语“脑卒中”通常指一种急性脑血管疾病，也称作“中风”、“脑血管意外(cerebralvascular accident，CVA)”。脑卒中可以是由于脑部血管突然破裂或因血管阻塞导致血液不能流入脑而引起脑组织损伤的一组疾病，包括缺血性脑卒中和出血性脑卒中。
在本申请中，术语“缺血性脑卒中”通常指由于脑的特定区域或广泛的脑组织区域供血量不足，导致所述区域的神经组织功能障碍的一组疾病。脑供血不足可以由多种疾病或异常引起，例如可以是镰状细胞性贫血、血管压迫、室性心动过速、动脉斑块积聚、血栓、严重低血压以及先天性心脏缺陷等。其中，镰状血细胞比正常血细胞更容易凝结，阻碍血液流向 脑部；血管压迫通过阻断携带氧气进入大脑的动脉，可能导致脑缺血，血管压迫的原因例如肿瘤；室性心动过速可能导致心脏完全停跳导致血液流动停止，而心率失常也可能导致血凝块的形成，导致脑缺血；由于动脉斑块积聚导致的动脉阻塞也可能导致脑缺血，在少量斑块积聚的情况下也会导致通道变窄倾向于形成血栓，从而导致脑缺血；凝血功能异常，大血块也可以通过阻止血流引起脑缺血；心脏病发作也可能引起脑缺血，心脏病发作可能会使血流缓慢，血液可能开始凝结而阻止血液流向脑部；心脏病发作和低血压之间存在相关性，药物的不当使用和对药物的反应也可能导致过低的血压，过低的血压通常代表组织的氧合不足。在本申请中，缺血性脑卒中还可以是通过手术阻塞小鼠大脑中动脉血流实现。所述神经组织功能障碍可以包括神经元死亡。由于不同区域的神经组织功能障碍其主要症状可以包括：气/味/听或视觉改变、吞咽/瞳孔对光反应障碍、身体运动障碍、失语、呼吸和心率改变、其他组织或器官的缺血、意识丧失等。
在本申请中，术语“消化液”通常是指消化系统分泌的对食物起消化作用的液体。消化液主要由有机物、离子和水组成。消化液的主要功能可以包括：稀释食物，使之与血浆的渗透压相等，以利于吸收；改变消化腔内的pH，使之适应于消化酶活性的需要；水解复杂的食物成分，使之便于吸收；通过分泌粘液、抗体和大量液体，保护消化道粘膜，防止物理性和化学性的损伤。消化液可以包括如下几种：唾液、胃液、胰液、胆汁、小肠液等。
在本申请中，术语“预防”通常是指对健康受试者预防性地施用组合，以预防某种疾病或病症的发生。其也可以包含对处于待治疗变应性疾病前期的患者预防性地施用组合。“预防”不需要100％消除疾病或病症发生的可能性，换句话说，“预防”通常指在所述施用组合的存在下疾病或病症发生的可能性或发生程度降低了。
在本申请中，术语“缓解”指减少、缩减或迟滞某种病状、疾病、病症或表型。所述病状、疾病、病症或表型可以包括包括受试者主观感知例如疼痛、晕眩或其他生理性障碍，或者医学上可以检测的指征，例如通过医学检验手段检测到的病灶情况。
在本申请中，术语“治疗”通常指用于改变所处理的个体或细胞在临床病理过程中的自然过程的临床干预。可以包括改善病状态、消除病灶或改善的预后。
在本申请中，术语“经胃肠道施用”通常指允许在胃肠道部位处或附近区域特异性释放治疗剂。通过局部而不是全身释放治疗剂，药物的生物利用度可以在胃肠道部位增加和/或在体循环中减少，更低的全身药物水平可带来降低的毒性和降低的免疫原性(例如在生物制剂的情况下)，一方面导致改善的总体安全性以及较少的不良副作用，一方面提高在胃肠道部位的剂量，实现更高效、更加具有靶向的的效果。在一些情况下，局部施用治疗剂还提供了新的作用模式，例如不同局部位置的联合施用。
在本申请中，术语“在胃肠道局部发挥效力”通常是指所施用的治疗剂能够在胃肠道局部保持有效剂量并能够改变胃肠道局部的病症或表型。在胃肠道局部发挥效力不排除在胃肠道局部之外的器官或组织中观察到所述治疗剂。通常所述治疗剂可以具有在胃肠道部位的稳定性和/或胃肠道部位的组织穿透能力(渗透到胃肠道组织中的能力)，可以在胃肠道组织中实质分布。所述稳定性可以包括给药之前和/或之后的稳定性，例如在输送装置内的稳定性，给药后制剂和/或药物在胃肠道环境中的稳定性，包括疾病状态胃肠道环境，例如温度稳定性、pH稳定性、氧化稳定性。所述胃肠道局部可以包括受试者胃肠道一个或多个部位的部分或子部分。
在本申请中，术语“受试者”通常指人类或非人类动物，包括但不限于猫、狗、马、猪、奶牛、羊、兔、小鼠、大鼠或猴。
在本申请中，术语“药物组合物”通常指指一种混合物，其包含至少一种待对受试者施用以治疗影响该个体的具体疾病或病症的活性成分。其允许所述活性成分处于有效的形式并且不含有对该组合物将要给予的受试者具有不可接受的毒性的另外的组分。这种组合物可以是无菌的，也可以包含药学上可接受的载体。
在本申请中，术语“药学上可接受的载体”通常指药学上可接受的涉及携带或转运化学试剂的物质、组合物或媒介物。例如缓冲液、表面活性剂、稳定剂、防腐剂、用于增强生物利用度的吸收促进剂、液体或固体填充剂、稀释剂、赋形剂、溶剂、包囊材料和/或其他常规的增溶剂或分散剂。
在本申请中，术语“包含”通常是指包括明确指定的特征，但不排除其他要素。
在本申请中，术语“约”通常是指在指定数值以上或以下0.5％-10％的范围内变动，例如在指定数值以上或以下0.5％、1％、1.5％、2％、2.5％、3％、3.5％、4％、4.5％、5％、5.5％、6％、6.5％、7％、7.5％、8％、8.5％、9％、9.5％、或10％的范围内变动。
一方面，本申请提供一种一氧化氮合酶通路抑制剂在制备药物中的用途，所述药物用于预防、缓解和/或治疗受试者中胃肠道缺血再灌注相关的远端损伤。本申请还提供了一种一氧化氮合酶通路抑制剂在制备药物中的用途，所述药物用于预防、缓解和/或治疗受试者中神经系统相关疾病或病症。在本申请中，所述药物被配制为使得所述一氧化氮合酶通路抑制剂在胃肠道局部发挥效力。
在本申请中，所述胃肠道局部可以包含胃、小肠、大肠部位。所述胃可以包含胃贲门部、胃底、胃体、胃幽门部。所述小肠可以包含十二指肠、空肠、回肠。所述大肠可以包含盲肠、结肠、直肠。
在本申请中，所述在胃肠道局部发挥效力可以包含所述一氧化氮合酶通路抑制剂抑制所 述胃肠道局部一氧化氮合酶的活性或者降低所述胃肠道局部一氧化氮合酶的的表达。
例如，所述在胃肠道局部发挥效力可以包含肠组织中iNOS基因表达水平下降。所述肠组织可以包含结肠组织。
在本申请中，所述在胃肠道局部发挥效力可以包含肠组织中硝酸盐含量下降。所述肠组织可以包含盲肠组织。
在本申请中，所述在胃肠道局部发挥效力可以包含肠组织中Nox1、Duox2基因表达水平下降。所述肠组织可以包含结肠组织。
在本申请中，所述在胃肠道局部发挥效力可以包含肠组织中肠屏障相关基因表达水平的变化。
例如，肠屏障相关基因可以包括Tjp1、Ocln、Cldn2。其中，Tjp1为紧密连接蛋白1、Ocln为Occludin、Cldn2为Claudin-2。
例如，所述在胃肠道局部发挥效力可以包含肠组织中Tjp1、Ocln表达升高。
例如，所述在胃肠道局部发挥效力可以包含肠组织中Cldn2表达降低。
在本申请中，所述在胃肠道局部发挥效力可以包含使得肠组织中促炎性细胞因子基因表达量下降，所述促炎性细胞因子基因选自下组的任意一种：Tnf、Il17、Ifng、Il1b、Il6、Cxcl2和Kc；其中，Tnf为肿瘤坏死因子，Il17为白细胞介素17，Ifng为干扰素γ，Il1b为白细胞介素1β，Il6为白细胞介素6，Kc和Cxcl2是趋化因子成员。
例如，所述肠组织可以包含空肠、回肠、盲肠或结肠。
在本申请中，所述药物被配制为使得在施用后约48小时或之后，所述一氧化氮合酶通路抑制剂仍然以预防、缓解和/或治疗所述胃肠道缺血再灌注相关的远端损伤的有效量存在于胃肠道局部。
在本申请中，所述药物被配制为使得在施用后约1小时或之后，所述药物中至多50％的所述一氧化氮合酶通路抑制剂被所述受试者吸收而进入血液循环系统。
在本申请中，所述药物中所述一氧化氮合酶通路抑制剂的浓度为约0.0001％(w/w)至约90％(w/w)。
例如，所述一氧化氮合酶通路抑制剂的浓度为约0.0005％(w/w)至约90％(w/w)，约0.001％(w/w)至约85％(w/w)，约0.0015％(w/w)至约80％(w/w)，约0.002％(w/w)至约75％(w/w)，约0.0025％(w/w)至约70％(w/w)，约0.003％(w/w)至约65％(w/w)，约0.0035％(w/w)至约60％(w/w)，约0.004％(w/w)至约55％(w/w)，约0.0045％(w/w)至约50％(w/w)，约0.005％(w/w)至约45％(w/w)，约0.0055％(w/w)至约40％(w/w)，约0.006％(w/w)至约35％(w/w)，约0.0065％(w/w)至约30％(w/w)，约0.007％(w/w) 至约25％(w/w)，约0.0075％(w/w)至约20％(w/w)，约0.01％(w/w)至约80％(w/w)，约0.1％(w/w)至约70％(w/w)，约0.5％(w/w)至约60％(w/w)，约1％(w/w)至约50％(w/w)，约5％(w/w)至约90％(w/w)，约10％(w/w)至约80％(w/w)，约20％(w/w)至约70％(w/w)，约10％(w/w)至约50％(w/w)，约20％(w/w)至约50％(w/w)，约20％(w/w)至约40％(w/w)。约30％(w/w)至约50％(w/w)，约10％(w/w)至约20％(w/w)。
在本申请中，所述受试者曾经、正在或有风险患有与所述胃肠道缺血再灌注相关的疾病或病症。
在本申请中，所述与胃肠道缺血再灌注相关的疾病或病症可以包含天然事件、创伤或者减少/阻止胃肠道血流量的一种或多种外科手术或其他治疗性干预。其中，所述天然事件可以包含动脉梗死、静脉阻塞或者破坏或降低血液流向内脏器官的全身性低血压，所述全身性低血压可以包括因失血所致的出血性休克、因心肌梗死或心力衰竭所致的心源性休克、神经源性休克、肾源性休克或过敏反应。
例如，所述与胃肠道缺血再灌注相关的疾病和/或病症可以包括脑卒中、创伤、休克、败血症、急性胰腺炎和/或炎症性肠病。
例如，所述与胃肠道缺血再灌注相关的疾病可以包含脑卒中。所述脑卒中可以包含由于脑部血管突然破裂或因血管阻塞导致血液不能流入脑或入脑血流量减少而引起脑组织损伤的一组疾病。
例如，所述疾病可以包含缺血性脑卒中。所述缺血性脑卒中可以包含大动脉的动脉粥样硬化性闭塞、脑栓塞(栓塞性梗死)、小的深部穿支动脉的非栓塞性梗塞(腔隙性脑梗)、和由远端动脉狭窄及脑血流下降导致的分水岭区域缺血(血流动力学卒中)。
在本申请中，所述受试者曾经患有与所述胃肠道缺血再灌注相关的上述疾病或病症。所述曾经可以包含向所述受试者施用所述药物前所述受试者患有与所述胃肠道缺血再灌注相关的上述疾病。
在本申请中，所述受试者正在患有与所述胃肠道缺血再灌注相关的上述疾病。所述正在可以包含向所述受试者施用所述药物时所述受试者患有与所述胃肠道缺血再灌注相关的上述疾病。
在本申请中，所述受试者有风险患有与所述胃肠道缺血再灌注相关的上述疾病。所述有风险可以包含向所述受试者施用所述药物后所述患者可能患有与所述胃肠道缺血再灌注相关的上述疾病。
在本申请中，所述受试者曾经、正在或有风险经受所述胃肠道缺血再灌注。
例如，所述受试者曾经经受所述胃肠道缺血再灌注。所述曾经可以包含向所述受试者施用所述药物前所述受试者经受了所述胃肠道缺血再灌注。
例如，所述受试者正在经受所述胃肠道缺血再灌注。所述正在可以包含向所述受试者施用所述药物时所述受试者正在经受所述胃肠道缺血再灌注。
例如，所述受试者有风险经受所述胃肠道缺血再灌注。所述有风险可以包含向所述受试者施用所述药物后所述受试者经受了所述胃肠道缺血再灌注。
例如，所述与胃肠道缺血再灌注相关的远端损伤包括缺血性脑卒中。
在本申请中，所述药物被配置为适于经口施用。例如口服。
例如，口服的剂型可以包括胶囊、片剂、丸剂、粒剂或糖浆。
例如，口服剂量可以按照按范围从约0.01至1000mg/kg、从约0.01至200mg/kg、从约0.01至180mg/kg、从约0.01至160mg/kg、从约0.01至140mg/kg、从约0.01至120mg/kg、从约0.01至100mg/kg、从约0.1至200mg/kg、从约0.1至150mg/kg、从约0.1至100mg/kg、从约0.1至80mg/kg、从约1至60mg/kg、从约0.1至40mg/kg、从约0.1至20mg/kg、25-200mg/kg、约50-200mg/kg、约100-200mg/kg、约25-50mg/kg、约25-100mg/kg、约50-100mg/kg、约50-200mg/kg体重的剂量施用。
例如，所述口服方式可以包括每日1或多次、每日、每隔1日、每周、每两周、每月或每两月服用。
例如，所述口服的时间为脑卒中后约1-7天，约1-6天，约1-5天，约1-4天，约1-3天，约1-2天，1-24小时，约3-12小时，约6-12小时，约1-3小时，约1-6小时，约3-6小时，约3-12小时，约6-12小时。
在本申请中，所述一氧化氮合酶通路抑制剂基本上不被消化液分解和/或灭活。所述消化液可以包含唾液、胃液、小肠液、胰腺液、胆汁。
例如，唾液，其pH为6.6～7.1，主要成分包括唾液淀粉酶、溶菌酶和少量的无机物(如含钠、钾、钙的无机盐)等，所述一氧化氮合酶通路抑制剂基本上不被所述唾液分解和/或灭活。
例如，胃液，其pH为0.9～1.5，主要成分包括胃蛋白酶、胃酸(即盐酸)、黏液以及钠盐、钾盐等无机物，所述一氧化氮合酶通路抑制剂基本上不被所述胃液分解和/或灭活。
例如，胰液，其pH为7.8～8.4，主要成分包括碳酸氢钠、胰淀粉酶、胰脂肪酶、胰蛋白酶原和糜蛋白酶原等，所述一氧化氮合酶通路抑制剂基本上不被所述胰液分解和/或灭活。
例如，胆汁，其pH约为6.8～7.4，主要成分是胆盐和胆色素，所述一氧化氮合酶通路抑制剂基本上不被所述胆汁分解和/或灭活。
例如，小肠液，其pH约为7.6，包含淀粉酶、麦芽糖酶、蔗糖酶、乳糖酶、肽酶、脂肪 酶等多种消化酶，所述一氧化氮合酶通路抑制剂基本上不被所述小肠液分解和/或灭活。
例如，所述一氧化氮合酶通路抑制剂基本上不被所述唾液、胃液、胰液、胆汁、小肠液分解和/或灭活。
例如，所述一氧化氮合酶通路抑制剂在ph为6.6～7.1、0.9～1.5、7.8～8.4、6.8～7.4、7.6时基本上不被分解和/或灭活。
所述一氧化氮合酶通路抑制剂基本上不被消化液分解和/或灭活可以包括所述一氧化氮合酶通路抑制在所述消化液中或与所述消化液接触后能够基本保持抑制所述一氧化氮合酶的性能。
另一方面，本申请还提供了一氧化氮合酶用于筛选药物的用途，其中所述药物用于预防、缓解和/或治疗受试者中胃肠道缺血再灌注相关的远端损伤。
本申请还提供了一氧化氮合酶通路抑制剂用于筛选药物的用途，所述药物用于预防、缓解和/或治疗受试者中神经系统相关疾病或病症。
在本申请中，所述筛选药物可以包含对可能作为药物使用的物质进行生物活性、药理作用及药用价值的评估过程。所述筛选药物可以包含生化水平和细胞水平的筛选。所述选药物还可以包含高通量筛选和虚拟药物筛选。
在某些实施方式中，所述药物抑制所述一氧化氮合酶的表达和/或活性。
在某些实施方式中，所述一氧化氮合酶为诱导型一氧化氮合酶iNOS。
另一方面，本申请还提供了一种药物组合物，所述药物组合物可以包含本申请所述的一氧化氮合酶通路抑制剂和任选地药学上可接受的载体。
例如，所述药物组合物可以包括保健制品。
所述保健品通常是指具有一般食品的共性，能调节人体的机能，适用于特定人群食用，但不以治疗疾病为目的一类制品，也称作膳食补充剂。包括茶、酒、蜂制品、饮品、汤品、鲜汁、药膳等。
例如，所述药物组合物可以包括生物制品。
所述生物制品通常是指应用普通的或以基因工程、细胞工程、蛋白质工程、发酵工程等生物技术获得的微生物、细胞及各种动物和人源的组织和液体等生物材料制备的，用于人类疾病预防、治疗和诊断的制品。包括菌苗、疫苗、毒素、类毒素、血液制品、免疫球蛋白、抗原、变态反应原、细胞因子、激素、酶、发酵产品、单克隆抗体、DNA重组产品等。
所述一氧化氮合酶通路抑制剂可以包含氨基酸类抑制剂和/或非氨基酸类抑制剂。所述氨基酸类抑制剂可以包括氨基胍AG、1400W、L-NIL和/或异硫脲，所述非氨基酸类抑制剂可以包括糖皮质激素、类黄酮、2-氨基-4-甲基吡啶和/或氨基哌啶。所述药学上可接受的载体可以 包含药学上可接受的涉及携带或转运化学试剂的物质、组合物或媒介物，例如缓冲液、表面活性剂、稳定剂、防腐剂、用于增强生物利用度的吸收促进剂、液体或固体填充剂、稀释剂、赋形剂、溶剂、包囊材料和/或其他常规的增溶剂或分散剂。
在本申请中，所述药物组合物可以包含以下组分中的任何一种或其任何组合：阿拉伯胶，藻酸盐，海藻酸，乙酸铝，苯甲醇，对羟基苯甲酸丁酯，丁基化羟基甲苯，抗氧化剂，柠檬酸，碳酸钙，小烛树蜡，交联羧甲基纤维素钠，糖果糖，胶体二氧化硅，纤维素，巴西棕榈蜡，玉米淀粉，羧甲基纤维素钙，硬脂酸钙，EDTA二钠钙，共聚维酮，氢化蓖麻油，磷酸氢钙脱水，氯化十六烷基吡啶，半胱氨酸HC1，交联聚维酮，磷酸氢钙，磷酸氢二钠，聚二甲基硅氧烷，赤藓红钠，乙基纤维素，明胶，单油酸甘油酯，甘油，甘氨酸，单硬脂酸甘油酯，山丁酸甘油酯，羟丙基纤维素，羟丙基甲基纤维素，羟丙甲纤维素，HPMC邻苯二甲酸盐，氧化铁，氧化铁黄，乳糖(含水或无水或一水合物或喷雾干燥)，硬脂酸镁，微晶纤维素，甘露醇，甲基纤维素，碳酸镁，矿物油，甲基丙烯酸共聚物，氧化镁，对羟基苯甲酸甲酯，PEG，聚山梨醇酯80，丙二醇，聚环氧乙烷，对羟基苯甲酸丙酯，泊洛沙姆407或188，碳酸氢钾，山梨酸钾，淀粉，磷酸，聚氧乙烯40硬脂酸酯，羟基乙酸淀粉钠，预胶化淀粉，交联羧甲基纤维素钠，十二烷基硫酸钠，二氧化硅，苯甲酸钠，硬脂酸，用于药用糖果的糖浆，造粒剂，山梨酸，碳酸钠，糖精钠，海藻酸钠，硅胶，山梨糖醇单油酸酯，硬脂酰富马酸钠，氯化钠，偏亚硫酸氢钠，脱水柠檬酸钠，羧甲基纤维素钠，琥珀酸，丙酸钠，二氧化钛，滑石粉，甘油三乙酸酯，柠檬酸三乙酯。
在本申请中，所述药物组合物可以是固体形式，例如，胶囊，片剂，丸剂，粒剂，小药囊或锭剂；或者可以是液体形式，例如溶液，悬浮液，乳液或糖浆。
另一方面，本申请还提供了一种预防、缓解和/或治疗受试者中胃肠道缺血再灌注相关的远端损伤的方法，所述方法包括向所述受试者施用本申请所述的一氧化氮合酶通路抑制剂。
本申请还提供了一种预防、缓解和/或治疗受试者中神经系统相关疾病或病症，所述方法包括向所述受试者施用本申请所述的一氧化氮合酶通路抑制剂。例如，所述施用包括经胃肠道施用。所述经胃肠道施用包括在胃肠道部位处或附近区域特异性释放所述一氧化氮合酶通路抑制剂。
例如，通过机械装置将所述一氧化氮合酶通路抑制剂递送至胃肠道。
例如，通过内窥镜、喷雾导管将所述一氧化氮合酶通路抑制剂递送至胃肠道。
例如，通过口服缓释剂、崩解剂使得所述一氧化氮合酶通路抑制剂在胃肠道释放。
例如，所述缓释剂、崩解剂能够依赖胃肠道pH启动释放或崩解。
例如，通过肠溶衣胶囊、脂质体微胶囊使得所述一氧化氮合酶通路抑制剂在胃肠道释放。
例如，通过灌胃将所述一氧化氮合酶通路抑制剂递送至胃肠道。
例如，经胃肠道施用的时间为脑卒中后约1-7天，约1-6天，约1-5天，约1-4天，约1-3天，约1-2天，1-24小时，约3-12小时，约6-12小时，约1-3小时，约1-6小时，约3-6小时，约3-12小时，约6-12小时。
例如，所述一氧化氮合酶通路抑制剂在胃肠道局部发挥效力。
例如，在施用后约48小时或之后，所述一氧化氮合酶通路抑制剂仍然以预防、缓解和/或治疗所述胃肠道缺血再灌注引起的远端损伤的有效量存在于胃肠道局部。
例如，在施用后约1小时或之后，至多50％的所述一氧化氮合酶通路抑制剂被所述受试者吸收而进入血液循环系统。
例如，所述一氧化氮合酶通路抑制剂的给药剂量为从约0.01至1000mg/kg、从约0.01至200mg/kg、从约0.01至180mg/kg、从约0.01至160mg/kg、从约0.01至140mg/kg、从约0.01至120mg/kg、从约0.01至100mg/kg、从约0.1至200mg/kg、从约0.1至150mg/kg、从约0.1至100mg/kg、从约0.1至80mg/kg、从约1至60mg/kg、从约0.1至40mg/kg、从约0.1至20mg/kg、25-200mg/kg、约50-200mg/kg、约100-200mg/kg、约25-50mg/kg、约25-100mg/kg、约50-100mg/kg、约50-200mg/kg体重。
另一方面，本申请还提供了一种预防受试者中胃肠道缺血再灌注相关的远端损伤的方法，所述方法包括：(1)监测所述受试者的胃肠道状况；(2)当所述监测显示所述受试者经受胃肠道缺血再灌注之时或之后，向所述受试者施用一氧化氮合酶通路抑制剂。
例如，所述方法还包括在步骤(1)前监测与胃肠道缺血再灌注相关的疾病或病症。
例如，所述检测所述受试者的胃肠道状况包括临床查体，临床检验和/或临床检查，评估受试者的胃肠道状况。
例如，在监测到患者曾经、正在或有风险经受与胃肠道缺血再灌注相关的疾病或病症时，对其进行临床查体，检验与检查，评估受试者的胃肠道状况。
例如，所述临床查体可以包括观察患者食欲、有无吞咽困难、有无腹痛、恶心、呕吐、呕血、便血、大便性状、排便时有无包含腹痛腹胀、排便习惯是否改变等症状，以及腹部查体。
例如，临床检验包括三大常规等。例如，所述临床检验可以包括血常规、尿常规和粪便常规。
例如，临床检查可以包括腹部X线、超声、CT、内镜、ERCP、与PTC等。
例如，监测所述受试者的胃肠道状况可以包含观察胃肠部位血流变化。
例如，所述施用包括经胃肠道施用。
例如，所述缓释剂、崩解剂能够依赖胃肠道pH启动释放或崩解。
例如，通过肠溶衣胶囊、脂质体微胶囊使得所述一氧化氮合酶通路抑制剂剂在胃肠道释放。
例如，通过灌胃将所述一氧化氮合酶通路抑制剂递送至胃肠道。
在本申请中，所述一氧化氮合酶可以包含如下几种中的任意一种：Ⅰ神经型一氧化氮合酶(nNOS或NOS1)，Ⅱ诱导型一氧化氮合酶(iNOS或NOS2)，Ⅲ内皮型一氧化氮合酶(eNOS或NOS3)。
例如，所述一氧化氮合酶可以包含诱导型一氧化氮合酶(iNOS或NOS2)。
在本申请中，所述一氧化氮合酶通路抑制剂抑制一氧化氮合酶的表达和/或活性。
例如，所述一氧化氮合酶通路抑制剂抑制诱导性一氧化氮合酶iNOS的表达/或活性。
例如，所述一氧化氮合酶通路抑制剂抑制诱导性一氧化氮合酶iNOS在胃肠道部的表达/或活性。
例如，所述一氧化氮合酶通路抑制剂使得诱导性一氧化氮合酶iNOS在胃肠道部的表达下降。
例如，所述一氧化氮合酶通路抑制剂使得胃肠道部的硝酸盐浓度下降。
例如，所述一氧化氮合酶通路抑制剂可以包括氨基酸类抑制剂。
例如，所述一氧化氮合酶通路抑制剂可以包括非氨基酸类抑制剂。
例如，所述氨基酸类抑制剂可以包括氨基胍AG、1400W、L-NIL和/或异硫脲等。其中，1400W的结构式如下所示:
Figure PCTCN2020135595-appb-000001
L-NIL的结构式如下所示：
Figure PCTCN2020135595-appb-000002
例如，所述非氨基酸类抑制剂可以包括糖皮质激素、类黄酮、2-氨基-4-甲基吡啶和/或氨基哌啶等。
在本申请中，所述与胃肠道缺血再灌注相关的远端损伤可以包括与胃肠道不同的任何器 官或组织的病理或生理过程。
例如，所述与胃肠道不同的任何器官或组织的病理或生理过程可以是与胃肠道缺血再灌注同时发生的。
例如，所述同时发生可以是所述与胃肠道不同的任何器官或组织的病理或生理过程与胃肠道缺血再灌注的发生时间相差在10分钟内，例如5分钟内，例如3分钟内，例如1分钟内。
例如，所述与胃肠道不同的任何器官或组织的病理或生理过程可以是在胃肠道缺血再灌注之前发生的。
例如，所述与胃肠道不同的任何器官或组织的病理或生理过程可以是在胃肠道缺血再灌注之前24小时内发生的。例如22小时内，例如20小时内，例如18小时内，例如16小时内，例如14小时内，例如12小时内，例如10小时内，例如8小时内，例如，6小时内，例如5小时内，例如4.5小时内，例如4小时内，例如3.5小时内，例如3小时内，例如2.5小时内，例如2小时内，例如1.5小时内，例如1小时内，例如，0.5小时内。例如，所述与胃肠道不同的任何器官或组织的病理或生理过程可以是在胃肠道缺血再灌注之后发生的。
例如，所述与胃肠道不同的任何器官或组织的病理或生理过程可以是在胃肠道缺血再灌注之后30天内发生的。例如28天内，例如26天内，例如24天内，例如22天内，例如20天内，例如18天内，例如16天内，例如14天内，例如12天内，例如10天内，例如8天内，例如1天内例如6天内，例如5天内，例如4天内，例如3天内，例如2天内，例如1天内，例如12小时内，例如9小时内，例如6小时内，例如3小时内，例如1小时内，例如0.5小时内。
例如，所述与胃肠道不同的任何器官或组织的病理或生理过程可以表现为当对胃肠道缺血再灌注或者胃肠道缺血再灌注相关效应或表征进行干预时，所述器官或组织的病理生理过程得到缓解或减轻。
例如，所述肠道缺血再灌注相关效应或表征可以包括胃肠组织Nos2、Nox1、Duox2表达变化、胃肠组织硝酸盐含量变化、肠屏障变化、胃肠组织促炎因子变化；其中，Nos2编码诱导型一氧化氮合酶，Nox1编码NADPH氧化酶1，Duox2编码双氧化酶2，NADPH氧化酶1和双氧化酶2与活性氧的生成有关。
例如，所述胃肠组织iNOS、Nox1、Duox2表达变化可以包括胃肠组织iNOS、Nox1、Duox2表达升高，所述胃肠组织硝酸盐含量变化可以包括胃肠组织硝酸盐含量升高，所述肠屏障变化可以包括肠屏障损伤，所述胃肠组织促炎因子变化胃肠组织促炎因子升高。
例如，所述肠屏障损伤可以包括Tjp1、Ocln基因表达水平降低，Cldn2基因表达水平升 高，所述炎症因子可以包括Tnf、Il17、Ifng、Il1b、Cxcl2、Kc和/或Il6；其中，Ocln、Cldn2为肠屏障相关基因，Ocln为Occludin、Cldn2为Claudin-2；Tnf为肿瘤坏死因子、Il17为白细胞介素17、Ifng为干扰素γ、Il1b为白细胞介素1β、Il6为白细胞介素6、Kc和Cxcl2趋化因子。
例如，所述胃肠组织iNOS、Nox1、Duox2表达变化可以包括胃肠组织iNOS、Nox1、Duox2表达升高之后再下降，所述胃肠组织硝酸盐含量变化可以包括胃肠组织硝酸盐含量升高之后再下降。
在本申请中，所述胃所述与胃肠道不同的任何器官或组织的病理或生理过程可以包括胃肠道缺血组织的再灌注导致的全身性或局部远端反应，所述反应可以包括广泛微血管功能障碍和组织屏障功能改变以及炎症反应。
在本申请中，所述与胃肠道不同的任何器官或组织的病理或生理过程可以累及肺。
例如，所述与胃肠道不同的任何器官或组织的病理或生理过程可以包含肺水肿、肺内血栓形成过程、肺栓塞和/或肺组织的炎症。
在本申请中，所述与胃肠道不同的任何器官或组织的病理或生理过程可以累及肾。
例如，所述与胃肠道不同的任何器官或组织的病理或生理过程可以包含肾衰竭、水肿形成、血栓形成、血栓栓塞和/或肾组织的炎症。
在本申请中，所述与胃肠道不同的任何器官或组织的病理或生理过程可以累及中枢神经系统。
例如，所述与胃肠道不同的任何器官或组织的病理或生理过程可以包括血脑屏障破坏、沉默型脑缺血、脑卒中、脑水肿、颅内压升高、神经元组织的炎症、神经细胞死亡、脑损伤和/或神经系统功能障碍。
例如，所述与胃肠道不同的任何器官或组织的病理或生理过程可以包含缺血性脑卒中及相关病症。
例如，所述缺血性脑卒中及相关病症可以包括在小鼠MCAO模型中表现为TTC染色中缺血性脑卒中区域为不着色(灰白色)，组织冰冻切片尼氏(Nissl)染色中缺血性脑卒中区域紫色尼氏小体消失，或者小鼠改良神经缺损评分升高。
例如，所述与胃肠道不同的任何器官或组织的病理或生理过程可以包含炎症。
例如，所述与胃肠道不同的任何器官或组织的病理或生理过程可以包含全身性炎症。所述炎症可以累及肺、胃肠系统、心血管系统、其他四肢和/或中枢神经系统。
例如，所述与胃肠道不同的任何器官或组织的病理或生理过程可以包含全身炎症反应综合征(重症炎性反应综合征)和/或多器官功能障碍综合征(MODS)。
例如，所述与胃肠道不同的任何器官或组织的病理或生理过程可以包括引起所述胃肠道缺血再灌注的疾病、病症或医学介入，例如，脑卒中、创伤、休克、败血症、急性胰腺炎、炎症性肠病或脑外伤手术。
例如，所述创伤包括由外力因素引起人体组织或器官的破坏，例如，交通伤、坠落伤、机械伤、锐器伤、跌伤、火器伤。
例如，所述休克通常是指机体遭受强烈的致病因素侵袭后，有效循环血量锐减，机体失去代偿，组织缺血缺氧，神经-体液因子失调的一种临床症候群。其主要特点可以包括重要脏器组织中的微循环灌流不足、代谢紊乱、全身各系统的机能障碍。例如，低血容量性休克、血管扩张性休克、心源性休克。所述低血容量性休克可以包括失血性休克、烧伤性休克、创伤性休克；所述血管扩张性休克可以包括感染性休克、过敏性休克、神经源性休克。
在本申请中，所述神经系统相关疾病可以包括血脑屏障破坏、沉默型脑缺血、脑卒中、脑水肿、颅内压升高、神经元组织的炎症、神经细胞死亡、脑损伤和/或神经系统功能障碍。
例如，所述神经系统功能障碍可以包括运动功能障碍、内环境调节紊乱。所述内环境调节紊乱可以包括内脏器官、体液或血液循环系统与神经支配相关的活动紊乱。
在本申请中，所述神经系统相关病症可以包含脑部缺血性疾病及其相关病症。
例如，所述脑部缺血性疾病可以包括由于血管阻塞、病变、创伤等导致血液不能流入脑或入脑血流量减少而引起脑组织损伤的一组疾病。
例如，所述脑部缺血性疾病可以包括缺血性脑卒中及其相关病症。
例如，所述缺血性脑卒中相关病症可以包括脑梗塞。
例如，所述缺血性脑卒中可以包含大动脉的动脉粥样硬化性闭塞、脑栓塞(栓塞性梗死)、小的深部穿支动脉的非栓塞性梗塞(腔隙性脑梗)、和由远端动脉狭窄及脑血流下降导致的分水岭区域缺血(血流动力学卒中)。
例如，所述缺血性脑卒中相关病症可以包括脑神经细胞死亡、脑功能损伤、气/味/听或视觉改变、吞咽/瞳孔对光反应障碍、身体运动障碍、失语、呼吸和心率改变、其他组织或器官的神经调节紊乱、意识丧失等。
例如，所述缺血性脑卒中相关病症可以包括NIHSS中的任意指标。其中，所述NIHSS为美国国立卫生研究院卒中量表(National Institute of Health stroke scale)，所述NIHSS的评分方法可以参见Williams LS,Yilmaz EY,Lopez-Yunez AM.Retrospective Assessment of Initial Stroke Severity with The NIH Stroke Scale.Stroke.2000；31:858–862)。
例如，所述缺血性脑卒中及相关病症在小鼠MCAO模型中可以表现为TTC染色中缺血性脑卒中区域为不着色(灰白色)，组织冰冻切片尼氏(Nissl)染色中缺血性脑卒中区域尼氏小 体消失，或者小鼠改良神经缺损评分升高。不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的一氧化氮合酶通路抑制剂在制备药物中的用途、一氧化氮合酶用于筛选药物的用途、包含一氧化氮合酶通路抑制剂的药物组合物等，而不用于限制本申请发明的范围。
实施例
材料和试剂
8-10周龄SPF级雄性C57BL/6小鼠，体重为22-24g，由广东省医学实验动物中心提供，具有实验动物质量合格证明。粪便样品基因组DNA提取试剂盒(MinkaGene Stool DNA Kit)为Minka Gene产品；引物由Thermo Fisher公司合成；TaqMan逆转录试剂、SYBR Green为Takara Bio公司产品；ViiA 7实时PCR系统为Applied Biosystems公司产品；硝酸盐/亚硝酸盐检测试剂盒为Sigma公司产品；TTC粉剂为Sigma公司产品；D-Lac、LBP、LPS血清ELISA试剂盒为基因美公司产品；其他材料、试剂等，如无特殊说明，均可从商业途径得到。
统计学分析
非微生物信息学数据数据分析采用R分析软件。正态分布数据以均数±标准差表示，非正态分布数据以中位数(四分位数间距)形式展示，非参数检验采用Kruskal-Wallis秩和检验或Mann-Whitney U检验，参数检验采用非成对Student’s test或ONE-WAY ANOVA进行分析。分类变量以比例表示。使用Shapiro-Wilk测试检查数据的正常性。对于微生物群分析，使用在QIIME 1.9.1中实施的Adonis测试。P<0.05(双尾)被认为具有显著差异。
实施例1.实验小鼠分组
选取SPF级8周龄雄性C57BL/6野生型小鼠为实验对象，并对其进行如下随机分组：SHAM组，进行假手术处理；MCAO组，进行MCAO造模处理；Post-AG组(AG组)，在小鼠进行MCAO造模后，对其进行AG干预(100mg/kg，灌胃，200μl/1次)；Pre-AG组，在小鼠进行MCAO造模前，对其进行AG干预(1mg/ml，饮水每日3-5μl，5天)。
实施例2.小鼠大脑中动脉梗死模型(MCAO)建立
小鼠称重，以0.2ml/10g的三溴乙醇进行麻醉，剪开颈部约1cm的正中切口，小心剥离组织，暴露右侧颈动脉三角区。小心分离出右侧颈总动脉、颈外动脉(颈外动脉需要尽量往上剥离，直到入颅前分叉处)和颈内动脉；结扎颈总动脉近心端(活结)，用电凝器尖端凝断颈外动脉小分支，结扎颈外动脉的近心端(活结)和远心端(死结)。剪开颈外动脉，将栓线(根据小鼠体重选择适当型号)往颈内动脉方向插入，略微感到阻力时停止插入(大约当栓线标记(离头端1cm)在颈总动脉分叉处)，固定栓线，随后缝合皮肤。插入栓线后1小时拔 栓，此时作为卒中后计时起点(即卒中后0点)。术中采用恒温毯保持小鼠核心体温在37+0.5℃，持续到小鼠麻醉效应过后苏醒，此操作是为了防止因低温脑保护作用影响小鼠脑缺血模型的建立。手术过程中因失血过多或造模时间超过15分钟的小鼠将舍弃，不纳入后期实验分析研究中。
实施例3.小鼠盲肠血流观察
对SHAM组以及MCAO组的小鼠盲肠部位血管进行血流观察，以MCAO组的小鼠造模操作前、插栓线后、拔栓线后、以及拔栓线后1小时为时间点，SHAM组小鼠进行假手术操作，并在响应时间点进行血流观察。具体操作步骤如下：
(1)操作前需对小鼠进行麻醉，腹部进行消毒，沿腹正中线逐层切开皮肤，肌层，暴露盲肠；
(2)使用激光散斑成像系统(瑞沃德RFLSI Pro)观察小鼠盲肠血流，首先观察小鼠盲肠全景成像，拍照后采用激光散斑成像系统(瑞沃德RFLSI Pro)软件记录盲肠血流动态图像，记录时间为10min，每帧间隔为1s；
(3)分析采用兴趣区域(Region of Interest，ROI)的血流变化，兴趣区域为盲肠六条血管分支，记录完毕后提取兴趣区域的数值作为血流值。
(4)检测过程中不同时间点的相对区域血流值为相应时刻的兴趣区内平均血流值除以最初时间点的血流值。
在整个手术过程中控制小鼠核心体温在37+0.5℃。
观察结果如图1和图2所示，在MCAO组小鼠移除栓线以及在此之后的1小时的时间点，与SHAM组小鼠相比，在MCAO组小鼠的盲肠血流减少，表明在MCAO小鼠脑卒中期间小鼠肠中发生缺血再灌注现象。
实施例4.肠道组织Nos2、Nox1、Duox2基因表达水平定量分析
在脑卒中后的不同时间点(3小时、6小时、12小时、24小时、3天、7天，其中起始计时时间为移除栓线的时间)，快速采取小鼠结肠组织并冷冻储存在-80℃用于测定Nos2基因相对表达水平。具体步骤如下：
(1)使用Minibeadbeater(Biospec Products，Bartlesville)均匀化结肠组织；
(2)Trizol试剂法(Invitrogen)提取RNA：每50-100mg均质组织样本中加入1ml TRIZOL进行匀浆。之后加入0.2ml氯仿，覆盖后剧烈摇动15秒，在15-30℃下放置2-3分钟，然后离心12,000g，15分钟，2-8℃。将上层水相转移，加入0.5ml异丙醇，放置10分钟，15-30℃，然后离心12,000g，10分钟，2-8℃。加入1ml 75％乙醇洗涤RNA沉淀5分钟。之后溶解RNA，并测定浓度；
(3)使用TaqMan逆转录试剂(Takara Bio)分别从各RNA样品中获取cDNA；
(4)以20μl体积中加入2μl cDNA为模板(浓度为50-100ng/μl)，以250nM为最终浓度加入如SEQ ID NO：1-2和23-28所示的上游引物和下游引物，进行SYBR Green(Takara Bio)实时PCR，反应体系如表1所示：
表1
反应物 体积
上游引物F 0.4μl
下游引物R 0.4μl
ROX Reference Dye II 0.4μl
SYBR Green 10μl
cDNA 2μl
PCR水 6.8μl
(5)用ViiA 7实时PCR系统(Applied Biosystems)提取数据，并使用比较Ct方法进行数据分析。以各自Gapdh表达水平作为内参。
结果如图3A-3C所示，Nos2、Nox1、Duox2基因的表达在脑卒中后3-6小时内开始增加，在12-24小时时达到顶峰。
实施例5.盲肠粘液提取物硝酸盐浓度测定
在脑卒中后3小时、6小时、24小时三个时间点测定盲肠粘液提取物硝酸盐浓度，具体步骤如下：
(1)去除小鼠盲肠的肠腔内容物，收集盲肠粘液层并称重；
(2)每只小鼠的盲肠粘液用0.2ml超纯水(Gibco)萃取，通过4℃20,000g离心2分钟除去较大的颗粒，将过滤灭菌的上清液以-80℃储存待用；
(3)取步骤(2)所得样品按照硝酸盐/亚硝酸盐检测试剂盒(比色法)试剂盒(Sigma)说明书的步骤进行操作，制作标准曲线，按照标准曲线计算硝酸盐浓度。
结果如图4所示，脑卒中后3小时至6小时再至24小时小鼠盲肠粘液层中硝酸盐浓度显著增高。
实施例6.氨基胍(AG)干预对于肠道组织Nos2基因表达水平的影响
按照实施例1中的描述准备如下三组小鼠：SHAM组，进行假手术处理；MCAO组，进行MCAO造模处理；Post-AG组，小鼠进行MCAO造模，脑卒中后1小时(即移除栓线后1 小时)对其进行AG干预(100mg/kg，0.2ml，灌胃，1次)，MCAO造模按照实施例2所述的方式进行；在脑卒中后的24小时按照实施例4中描述的方法分别对三组小鼠进行取样并测定肠道组织Nos2基因表达水平。
结果如图5A所示，氨基胍(AG)显著降低了小鼠脑卒中后肠道组织Nos2基因的表达水平。
实施例7.氨基胍(AG)干预对于肠道组织Nox1与Duox2基因表达水平的影响
按照实施例1中的描述准备如下三组小鼠：SHAM组，进行假手术处理；MCAO组，进行MCAO造模处理；Post-AG组，小鼠进行MCAO造模，脑卒中后1小时(即移除栓线后1小时)对其进行AG干预(100mg/kg，0.2ml，灌胃，1次)，MCAO造模按照实施例2所述的方式进行；在脑卒中后的24小时按照实施例4中描述的方法分别对三组小鼠进行取样并测定肠道组织Nox1与Duox2基因表达水平。
结果如图5B-5C所示，氨基胍(AG)降低了小鼠脑卒中后肠道组织Nox1与Duox2基因的表达水平。
实施例8.氨基胍(AG)干预对于盲肠粘液提取物硝酸盐浓度的影响
按照实施例1中的描述准备如下三组小鼠：SHAM组，MCAO组，进行MCAO造模处理；Post-AG组，小鼠进行MCAO造模，脑卒中后1小时(即移除栓线后1小时)对其进行AG干预(100mg/kg，灌胃，1次)，MCAO造模按照实施例2所述的方式进行；在脑卒中后的24小时按照实施例5中描述的方法分别对三组小鼠进行取样并测定小鼠盲肠粘液提取物中硝酸盐浓度。
结果如图6所示，氨基胍(AG)显著降低了小鼠脑卒中盲肠粘液提取物中硝酸盐浓度。
实施例9.氨基胍(AG)干预对于肠组织中肠屏障因子和促炎性细胞因子(proinflammatory cytokine)表达量的影响
按照实施例4的方法检测Tjp1、Ocln、Cldn2、Tnf、Il17、Ifng、Il1b、Cxcl2、Kc、和Il6的表达水平。其中，Tjp1、Ocln、Cldn2为肠屏障相关基因，Tjp1为紧密连接蛋白1、Ocln为Occludin、Cldn2为Claudin-2、Tnf为肿瘤坏死因子、Il17为白细胞介素17、Ifng干扰素γ、Il1b为白细胞介素1β、Il6为白细胞介素6、Kc和Cxcl2为趋化因子。脑卒中后24小时进行取样检测。Tjp1、Ocln、Cldn2、Tnf、Il17、Ifng、Il1b、Cxcl2、Kc、和Il6的引物序列如如SEQ ID NO：3-22所示。
结果如图7A-7J所示，造模前氨基胍(AG)干预或者造模后氨基胍(AG)干预使Tjp1和Ocln基因表达水平显著升高，同时Cldn2基因表达水平显著降低，表明其改善了肠屏障损 伤；同时显著降低了Tnf、Il17、Ifng、Il1b、Cxcl2、Kc、和Il6的表达水平，表明其有效抑制了结肠组织的炎症反应。
实施例10.氨基胍(AG)干预对于缺血性脑卒中脑损伤的影响
(1)按照实施例1中的描述准备如下三组小鼠：MCAO组，进行MCAO造模处理；Post-AG组，小鼠进行MCAO造模，脑卒中后1小时(即移除栓线后1小时)对其进行AG干预(100mg/kg，灌胃，1次)；Pre-AG组，在小鼠进行MCAO造模前，对其进行AG干预(1mg/ml，饮水每日3-5μl，5天)，AG干预5天结束后进行MCAO造模。MCAO造模按照实施例2所述的方式进行；
(2)使用PBS配制TTC粉剂，TTC染色液浓度为2％，避光放置；
(3)小鼠麻醉(0.2ml/10g的三溴乙醇)后打开胸腔，剪开右心耳，心脏灌注冰冻PBS约2min；
(4)断头取脑，脑组织放置在脑槽中，随后将脑槽放置在-80℃冰箱7-8分钟后取出，行厚1.5mm的冠状切片；
(5)将切好的脑组织放置在TTC染色液中，避光染色10min。正常脑组织为深红色，缺血性脑卒中损伤区不着色(灰白色)。将染色后的切片加入甲醛固定后，取出拍照；
(6)采用ImagePlus Soft图像软件进行脑损伤体积分析。分析结果时排除脑水肿因素，通过水肿修正梗死体积的公式进行计算：脑损伤区域＝直接损伤体积-(躯体同侧半球-躯体对侧半球)，最终得到脑损伤体积占全大脑半球比例值。
结果如图8所示，无论是造模前氨基胍(AG)干预还是造模后氨基胍(AG)干预，小鼠脑缺血性脑卒中的损伤程度都显著缓解，相比于未干预的MCAO组。这说明通过抑制肠道一氧化氮合酶(iNOS)的表达，可以改善小鼠缺血性脑卒中后脑损伤。
实施例11.小鼠改良神经缺损评分(modified neurological severity score,mNSS)
脑卒中后24小时对MCAO组、Post-AG组、Pre-AG组进行此项操作。神经行为学检测由两位对实验分组不知情的测试者进行。mNSS评分有0-14分，0分代表正常，14分代表神经缺损最严重。该评分系统能综合性地评估神经功能，包括运动、感觉、平衡和反射：①运动试验：提起小鼠的尾部评估四肢的弯曲和扭转程度(0-3分)；在平面上走动时的姿势评估(0-3分)。②平衡试验：小鼠放置在一根横梁上。神经功能受损评估主要是根据小鼠是否能够在横梁上平衡，四肢从横梁上垂落以及能够平稳通过横梁进行评估(0-6分)。③感觉及反射试验：各自检测耳廓反射和角膜反射(0-2分)。
结果如图9所示，无论是造模前氨基胍(AG)干预还是造模后氨基胍(AG)干预，都显 著改善了小鼠神经缺损的情况。
实施例12.脑组织冰冻切片尼氏(Nissl)染色
(1)取脑：脑卒中后24小时，评估mNSS后，处死各组小鼠(SHAM组，MCAO组，Post-AG组)，取脑；
(2)固定：麻醉小鼠后打开胸腔充分暴露心脏，剪开右心耳，使用冰冻生理盐水进行心脏灌注10min，然后冰冻4％多聚甲醛(Paraformaldehyde，PFA)灌注10min。随后将组织固定于PFA 24h，30％PFA蔗糖48小时。充分脱水后脑组织完全沉底，取出脑组织；
(3)包埋：切取脑组织置于冰冻切片包埋模具中，常规OCT包埋，液氮冷冻，置于-80℃冰箱冻存；
(4)切片：将冰冻切片机温度调至-20℃。温度达到后取出标本，脑组织取前囟后2.0mm至4.0mm的范围(包含整个的丘脑组织)，连续切成厚度5μm的冠状面切片。用细毛笔展平切片，将切片置于载玻片上，置于-20℃冰箱中保存；
(5)尼氏染色：脑切片浸入尼氏染液5分钟，蒸馏水洗涤2次。95％乙醇约5秒。95％乙醇脱水2分钟。浸入新鲜二甲苯透明5分钟，中性树胶封片。片子晾干后置于显微镜下观察并采集图片分析。正常存活的神经元细胞胞膜完整呈蓝紫色，可见胞核深染。
结果如图10A-10B所示，AG干预显著减少了小鼠海马区域神经元的丢失。
实施例13.氨基胍(AG)干预对于血清中肠屏障标记物与炎症因子含量的影响
小鼠评估mNSS后进行采血。采血方式包括眼眶取血和后腔静脉取血。小鼠血清标本进行检测前储存于-80℃。小鼠血清肠屏障标记物如D-Lac、LBP、LPS按照ELISA试剂盒(基因美，Elisa Lab)说明书的步骤进行操作，炎症因子如IL-6、TNF-α含量严格按照ELISA试剂盒(基因美，Elisa Lab)说明书的步骤进行操作，制作标准曲线，按照标准曲线算出各表达量。
结果如图11A-11E所示，造模前氨基胍(AG)干预或者造模后氨基胍(AG)干预都显著降低了小鼠血清中D-Lac、LBP、LPS的含量，表明其改善了肠屏障损伤；同时造模后氨基胍(AG)干预显著降低了小鼠血清中IL-6、TNF-α的含量，表明其有效抑制了炎症反应。
实施例14.不同时间或剂量的氨基胍(AG)干预对于缺血性脑卒中脑损伤的影响
按照实施例8的实验方法检测不同时间、剂量的氨基胍(AG)干预对于肠道缺血再灌注后缺血性脑卒中的影响。
AG的剂量效应：在小鼠脑卒中后第1小时，对小鼠进行不同剂量AG干预，浓度范围：25mg/kg，50mg/kg，100mg/kg，200mg/kg(灌胃200μl，1次)。并在脑卒中后24小时检测 小鼠脑损伤程度。
AG的时间效应：分别在小鼠脑卒中后第1、3、6、12小时，对小鼠进行AG干预(25mg/kg，灌胃200μl，1次)。并在脑卒中后24小时检测小鼠脑损伤程度。
结果如图12A-12B所示，不同剂量的AG(范围：25-200mg/kg)均可显著减轻小鼠脑损伤。此外，在脑卒中后不同时间使用AG(脑卒中后1-12h)同样可以显著减轻小鼠脑损伤程度。
实施例15. 1400W、L-NIL或2-氨基-4-甲基吡啶(AMP)干预对于肠道组织Nos2基因表达水平的影响
按照实施例1中的描述准备如下五组小鼠：SHAM组，进行假手术处理；MCAO组，进行MCAO造模处理；Post-1400W组/Post-L-NIL组/Post-AMP组，小鼠进行MCAO造模，脑卒中后1小时(即移除栓线后1小时)分别进行1400W、L-NIL或AMP干预(0.01-1000mg/kg，0.2ml，灌胃，1次)，MCAO造模按照实施例2所述的方式进行；在脑卒中后的24小时按照实施例4中描述的方法分别对五组小鼠进行取样并测定肠道组织Nos2基因表达水平。
结果显示，相对于MCAO组，1400W、L-NIL或2-氨基-4-甲基吡啶(AMP)干预均降低了小鼠脑卒中后肠道组织Nos2基因的表达水平。
实施例16. 1400W、L-NIL或2-氨基-4-甲基吡啶(AMP)干预对于盲肠粘液提取物硝酸盐浓度的影响
按照实施例1中的描述准备如下五组小鼠：SHAM组，进行假手术处理；MCAO组，进行MCAO造模处理；Post-1400W组/Post-L-NIL组/Post-AMP组，小鼠进行MCAO造模，脑卒中后1小时(即移除栓线后1小时)分别进行1400W、L-NIL或AMP干预(0.01-1000mg/kg，0.2ml，灌胃，1次)，MCAO造模按照实施例2所述的方式进行；在脑卒中后的24小时按照实施例5中描述的方法分别对三组小鼠进行取样并测定小鼠盲肠粘液提取物中硝酸盐浓度。
结果显示，相对于MCAO组，1400W、L-NIL或2-氨基-4-甲基吡啶(AMP)干预降低了小鼠脑卒中盲肠粘液提取物中硝酸盐浓度。
实施例17. 1400W、L-NIL或2-氨基-4-甲基吡啶(AMP)干预对于缺血性脑卒中脑损伤的影响
按照实施例1中的描述准备如下4组小鼠：MCAO组，进行MCAO造模处理；Post-1400W组/Post-L-NIL组/Post-AMP组，小鼠进行MCAO造模，脑卒中后1小时(即移除栓线后1小时)分别进行1400W、L-NIL或AMP干预(0.01-1000mg/kg，灌胃，1次)。MCAO造模按照实施例2所述的方式进行。按照实施例10的方法检测1400W、L-NIL或2-氨基-4- 甲基吡啶(AMP)干预对小鼠缺血性脑卒中脑损伤的影响。
结果显示造模前或是造模后进行1400W、L-NIL或2-氨基-4-甲基吡啶(AMP)干预，相比于未干预的MCAO组，小鼠脑缺血性脑卒中的损伤程度都得到了缓解。
前述详细说明是以解释和举例的方式提供的，并非要限制所附权利要求的范围。目前本申请所列举的实施方式的多种变化对本领域普通技术人员来说是显而易见的，且保留在所附的权利要求和其等同方案的范围内。

Claims (24)

  1. 一氧化氮合酶通路抑制剂在制备药物中的用途，所述药物用于预防、缓解和/或治疗受试者中与胃肠道缺血再灌注相关的远端损伤。
  2. 根据权利要求1所述的用途，其中所述一氧化氮合酶通路抑制剂抑制一氧化氮合酶的活性。
  3. 根据权利要求1-2中任一项所述的用途，其中所述一氧化氮合酶包括诱导型一氧化氮合酶iNOS。
  4. 根据权利要求1-3中任一项所述的用途，其中所述一氧化氮合酶通路抑制剂包括氨基酸类抑制剂和/或非氨基酸类抑制剂。
  5. 根据权利要求1-4中任一项所述的用途，其中所述一氧化氮合酶通路抑制剂包括氨基酸类抑制剂，所述氨基酸类抑制剂包括氨基胍AG、1400W、L-NIL和/或异硫脲。
  6. 根据权利要求1-4中任一项所述的用途，其中所述一氧化氮合酶通路抑制剂包括非氨基酸类抑制剂，所述非氨基酸类抑制剂包括糖皮质激素、类黄酮、2-氨基-4-甲基吡啶和/或氨基哌啶。
  7. 根据权利要求1-5中任一项所述的用途，其中所述药物被配制为使得所述一氧化氮合酶通路抑制剂在胃肠道局部发挥效力。
  8. 根据权利要求1-7中任一项所述的用途，其中所述药物中所述一氧化氮合酶通路抑制剂的浓度为约0.0001％(w/w)至约90％(w/w)。
  9. 根据权利要求1-8中任一项所述的用途，其中所述受试者曾经、正在或有风险经受与所述胃肠道缺血再灌注相关的手术、疾病或病症。
  10. 根据权利要求9所述的用途，其中所述与胃肠道缺血再灌注相关的疾病或病症包括脑卒中、创伤、休克、败血症、急性胰腺炎或炎症性肠病。
  11. 根据权利要求9-10中任一项所述的用途，其中所述与胃肠道缺血再灌注相关的疾病包括缺血性脑卒中。
  12. 根据权利要求1-11中任一项所述的用途，其中所述受试者曾经、正在或有风险经受所述胃肠道缺血再灌注。
  13. 根据权利要求1-12中任一项所述的用途，其中所述胃肠道缺血再灌注相关的远端损 伤包括缺血性脑卒中。
  14. 根据权利要求1-13中任一项所述的用途，其中所述药物被配置为适于经口施用。
  15. 根据权利要求1-14中任一项所述的用途，其中所述一氧化氮合酶通路抑制剂基本上不被消化液分解和/或灭活。
  16. 一氧化氮合酶用于筛选药物的用途，其中所述药物用于预防、缓解和/或治疗受试者中与胃肠道缺血再灌注相关的远端损伤。
  17. 根据权利要求16所述的用途，所述药物抑制所述一氧化氮合酶的表达和/或活性。
  18. 根据权利要求16-17中任一项所述的用途，所述一氧化氮合酶包括诱导型一氧化氮合酶iNOS。
  19. 药物组合物，其包含权利要求1-15中任一项所述的一氧化氮合酶通路抑制剂和任选地药学上可接受的载体。
  20. 一种预防、缓解和/或治疗受试者中胃肠道缺血再灌注相关远端损伤的方法，所述方法包括向所述受试者施用权利要求1-15中任一项所述的一氧化氮合酶通路抑制剂。
  21. 根据权利要求20所述的方法，其中所述施用包括经胃肠道施用。
  22. 根据权利要求20-21中任一项所述的方法，其中述一氧化氮合酶通路抑制剂在胃肠道局部发挥效力。
  23. 一种预防受试者中胃肠道缺血再灌注相关的远端损伤的方法，所述方法包括：
    (b)监测所述受试者的胃肠道状况；
    (c)当所述监测显示所述受试者有风险经受胃肠道缺血再灌注，或在经历胃肠道缺血再灌注之时或之后，向所述受试者施用一氧化氮合酶通路抑制剂。
  24. 根据权利要求23所述的方法，其中所述施用包括经胃肠道施用。
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