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WO2021249376A1 - Tlr4通路抑制剂和/或拮抗剂在制备药物中的用途 - Google Patents

Tlr4通路抑制剂和/或拮抗剂在制备药物中的用途 Download PDF

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WO2021249376A1
WO2021249376A1 PCT/CN2021/098812 CN2021098812W WO2021249376A1 WO 2021249376 A1 WO2021249376 A1 WO 2021249376A1 CN 2021098812 W CN2021098812 W CN 2021098812W WO 2021249376 A1 WO2021249376 A1 WO 2021249376A1
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alkyl
tlr4
antagonist
reperfusion
ischemia
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French (fr)
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周宏伟
徐开宇
高徐璇
尹恝
何彦
陈慕璇
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Southern Medical University Zhujiang Hospital
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Southern Medical University Zhujiang Hospital
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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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7024—Esters of saccharides
    • 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
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • 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
    • A61K31/66—Phosphorus compounds
    • A61K31/683—Diesters of a phosphorus acid with two hydroxy compounds, e.g. phosphatidylinositols
    • A61K31/685—Diesters of a phosphorus acid with two hydroxy compounds, e.g. phosphatidylinositols one of the hydroxy compounds having nitrogen atoms, e.g. phosphatidylserine, lecithin
    • 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
    • A61K31/66—Phosphorus compounds
    • A61K31/683—Diesters of a phosphorus acid with two hydroxy compounds, e.g. phosphatidylinositols
    • A61K31/688—Diesters of a phosphorus acid with two hydroxy compounds, e.g. phosphatidylinositols both hydroxy compounds having nitrogen atoms, e.g. sphingomyelins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • This application relates to the field of biomedicine, and specifically relates to the use of TLR4 pathway inhibitors and/or antagonists 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
  • This application provides the use of TLR4 pathway inhibitors and/or antagonists in the preparation of medicaments for the prevention, alleviation and/or treatment of gastrointestinal ischemia-reperfusion related distal injury in subjects.
  • the TLR4 pathway inhibitor and/or antagonist has the structure shown in Formula I:
  • G 1 is a 4- to 12-membered heterocyclic group containing 1 to 3 heteroatoms independently selected from N, O and S, -NR 1 R 2 , -OH, -OC 1-4 alkyl, C 3 -8 cycloalkyl, or a 5- to 12-membered heteroaryl group containing 1 to 3 heteroatoms independently selected from N, O and S; wherein the cycloalkyl, the heterocyclic group, the hetero The aryl group is optionally substituted with 1 to 4 substituents independently selected from halogen, C 1-4 alkyl, C 1-4 haloalkyl, -OH, -OC 1- 4 alkyl and OXO;
  • R 1 and R 2 are each independently hydrogen or C 1-4 alkyl
  • G 2 is selected from (i) to (xiii):
  • X 1 is O or S
  • X 2 is O, S, NH or NC 1-4 alkyl
  • R 5 is Or C 1-4 alkyl
  • R 3 , R 7 , R 13 , R 17 , R 19 , R 21 , R 27 , R 29 and R 33 are each independently selected from hydrogen and C 1-4 alkyl;
  • R 11 and R 31 are each independently selected from hydrogen, C 1-4 alkyl, and optionally substituted with 1-3 substituents independently selected from halogen, C 1-4 alkyl, C 1- 4 haloalkyl, -OH, and -Phenyl substituted by a substituent of OC 1-4 alkyl;
  • R 4a , R 4b , R 4c , R 5a , R 6 , R 8 , R 9 , R 10 , R 12 , R 14 , R 15 , R 16 , R 18 , R 20 , R 22 , R 23 , R 24 , R 25 , R 26 , R 28 , R 30 and R 32 are each independently -OH, halogen, nitro, cyano, C 1-4 alkyl, C 1-4 haloalkyl, -OC 1 -4 alkyl, -OC 1-4 haloalkyl, -NH 2 , -NH (C 1-4 alkyl), -N (C 1-4 alkyl) (C 1-4 alkyl), -NHC( O) C 1-4 alkyl, -N (C 1-4 alkyl) C (O) C 1-4 alkyl, -NHC (O) OC 1-4 alkyl, -N (C 1- 4 alkoxy Base) C(O)OC 1-4 alky
  • R 22 , R 24 , R 25 , R 26 , R 28 , R 30 or R 32 form a condensed ring together with the atoms to which they are attached
  • n1 and n2 are independently 1, 0, 2, 3, 4, or 5;
  • n3 and n4 are independently 0, 1, 2 or 3
  • the TLR4 pathway inhibitor and/or antagonist includes OxPAPC, TAK-242 and/or LPS-RS.
  • the TLR4 pathway inhibitor OxPAPC blocks the binding of LPS to LBP and CD14; TAK-242 binds to the cysteine CYS747 of the intracellular domain of TLR4 to inhibit the TLR4 signaling pathway.
  • the TLR4 pathway antagonist LPS-RS directly competes between acylated lipid A and hexaacylated lipid A for binding to MD-2.
  • the medicament is formulated so that the TLR4 pathway inhibitor and/or antagonist is locally effective in the gastrointestinal tract.
  • the medicament is formulated so that the TLR4 pathway inhibitor and/or antagonist still prevents, alleviates and/or treats the gastrointestinal deficiency about 1 hour or later after administration.
  • the effective amount of the distal injury related to blood reperfusion exists locally in the gastrointestinal tract.
  • the drug is formulated such that at most 50% of the TLR4 pathway inhibitor and/or antagonist in the drug is absorbed by the subject about 24 hours or later after administration. Enter the blood circulatory system.
  • the concentration of the TLR4 pathway inhibitor and/or antagonist 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 suffering from a disease or condition associated with 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 related to gastrointestinal ischemia-reperfusion includes ischemic stroke.
  • the medicament is configured to be suitable for oral administration.
  • the TLR4 pathway inhibitor and/or antagonist is not substantially decomposed and/or inactivated by digestive juice.
  • the application also provides a pharmaceutical composition comprising the TLR4 pathway inhibitor and/or antagonist described in the application and optionally a pharmaceutically acceptable carrier.
  • the present application also provides a method for preventing, alleviating and/or treating distal injury related to gastrointestinal ischemia-reperfusion in a subject, the method comprising administering to the subject the TLR4 pathway inhibitor and/or antagonist.
  • the administration includes administration via the gastrointestinal tract.
  • the TLR4 pathway inhibitor and/or antagonist exerts its effect locally in the gastrointestinal tract.
  • the TLR4 pathway inhibitor and/or antagonist is still used to prevent, alleviate and/or treat the gastrointestinal ischemia-reperfusion-related distal injury after about 1 hour or after administration
  • the effective amount is present in the local gastrointestinal tract.
  • At most 50% of the TLR4 pathway inhibitor and/or antagonist is absorbed by the subject and enters the blood circulatory system about 24 hours or after administration.
  • the dosage of the TLR4 pathway inhibitor and/or antagonist is about 0.01 to 200 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.
  • the application also provides the use of TLR4 for screening drugs, wherein the drugs are used to prevent, alleviate and/or treat gastrointestinal ischemia-reperfusion-related distal injury in a subject.
  • the drug inhibits the activation and/or signal transduction of the TLR4/TRAF6/NF ⁇ B pathway.
  • 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-3B show the changes in the expression levels of TLR4 pathway-related genes in colon tissue after gastrointestinal ischemia-reperfusion caused by ischemic stroke in this application.
  • Figure 4 shows the results of the intervention of the TLR4 pathway inhibitor (OxPAPC) described in the present application on the relief of brain damage after ischemic stroke in mice.
  • OxPAPC TLR4 pathway inhibitor
  • FIG. 5 shows the results of the intervention of the TLR4 pathway inhibitor (TAK-242) described in the present application on the alleviation of brain damage after ischemic stroke in mice.
  • Figure 6 shows the results of the intervention of the TLR4 pathway antagonist (LPS-RS) described in this application on the relief of brain damage after ischemic stroke in mice before modeling.
  • LPS-RS TLR4 pathway antagonist
  • Figure 7 shows the results of the intervention of the TLR4 pathway antagonist (LPS-RS) described in this application on the relief of brain damage after ischemic stroke in mice after modeling.
  • LPS-RS TLR4 pathway antagonist
  • TLR4 is also referred to as Toll-like receptor 4, and generally refers to a class of transmembrane proteins belonging to the Toll-like receptor family.
  • TLR4 is usually a type I transmembrane protein, which can be divided into three parts: extracellular domain, cytoplasmic domain and transmembrane domain. Normally, through its extracellular leucine-rich repeat domain (LRR) or intracellular Toll The /IL-1 receptor (TIR) domain forms a complex with other molecules in response to signals.
  • LRR leucine-rich repeat domain
  • TIR Toll The /IL-1 receptor
  • TLR4 ligands can include viral proteins, polysaccharides, and endogenous proteins, such as low-density lipoproteins, ⁇ -defensins, and heat shock proteins; TIR has homophilic interactions. After recognizing signal molecules, TLR4 can occur The molecular conformation changes to recruit downstream signal molecules containing TIR to form a signal complex. TLR4 can play a basic role in pathogen recognition and innate immune activation. For example, TLR4 can cooperate with LY96 and CD14 to mediate the innate immune response to bacterial lipopolysaccharide (LPS) (Tatematsu M, etc., J Immunol.
  • LPS bacterial lipopolysaccharide
  • TLR4 can act through MYD88, TIRAP and TRAF6 to cause NF- ⁇ B activation, cytokine secretion and inflammation (Medzhitov R, etc., Nature. 1997 Jul 24; 388 (6640):394-7; Arbour NC, etc., Nat Genet. 2000 Jun; 25(2): 187-91; Tatematsu M, etc., J Immunol. 2016 May 1; 196(9): 3865-76 );
  • TLR4 capable of binding with TLR6, in response to oxidized LDL (of oxLDL) or amyloid ⁇ 42 promotes monocyte / macrophages of sterile inflammation; e.g., TLR4 capable of binding to negatively charged LDL (LDL -) And mediate LDL - induced cytokine release (Estruch M, etc., Atherosclerosis.
  • the gene encoding TLR4 is located on chromosome 9 9q33.1, containing 4 exons; in mice, the gene encoding TLR4 is located on chromosome 4 C1; 34.66cM, containing 3 exons.
  • TLR4 pathway generally refers to an intracellular and/or extracellular signaling pathway that can regulate the expression or activity of TLR4, and/or that is triggered by the activation of TLR4.
  • alkyl generally refers to a linear or branched saturated hydrocarbon. Including but not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3 -Methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl and n-decyl.
  • alkylene generally refers to a divalent group derived from a linear or branched saturated hydrocarbon. Including but not limited to -CH 2 -, -CH 2 CH 2 -, -CH 2 CH 2 CH 2 -, -CH 2 CH(CH 3 )CH 2 -or CH 2 CH(CH 3 )CH(CH 3 )CH 2 -.
  • haloalkyl generally means that one, two, three, four, five, six, or seven hydrogen atoms of an alkyl group as defined in this application are replaced by halogen.
  • heteroaryl generally refers to an aromatic heterocyclic ring.
  • Heteroaryl groups can contain 5 to 12 ring atoms.
  • the heteroaryl group may be a 5- to 6-membered monocyclic heteroaryl group or an 8- to 12-membered bicyclic heteroaryl group.
  • the 5-membered monocyclic heteroaryl ring can contain two double bonds and one, two, three, or four heteroatoms as ring atoms, for example, furyl, imidazolyl, isoxazolyl, isothiazolyl, oxadiene Azolyl, oxazolyl, pyrazolyl, pyrrolyl, tetrazolyl, thiadiazolyl, thiazolyl, thienyl and triazolyl.
  • the 6-membered heteroaryl ring may contain three double bonds and one, two, three, or four heteroatoms as ring atoms, for example, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, and triazinyl.
  • a bicyclic heteroaryl group is an 8- to 12-membered ring system having a monocyclic heteroaryl group fused to an aromatic, saturated or partially saturated carbocyclic ring or to a second monocyclic heteroaryl ring.
  • benzofuranyl benzoxadiazolyl, 1,3-benzothiazolyl, benzimidazolyl, benzothienyl, indolyl, indazolyl, isoquinolinyl, naphthyridyl, Oxazolopyridine, quinolinyl.
  • the heteroaryl group can be attached to the parent molecule through any substitutable carbon atom or any substitutable nitrogen atom contained in the group.
  • cycloalkyl generally refers to a carbocyclic ring that does not contain heteroatoms as ring atoms and does not contain double bonds.
  • cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl can be attached to the parent molecule through any substitutable carbon atom.
  • heterocyclic or “heterocyclic” generally refers to a ring system containing at least one heteroatom as a ring atom, where the heteroatom can be selected from O, N or S.
  • the heterocyclic ring may be a monocyclic heterocyclic ring, a fused bicyclic heterocyclic ring or a spirocyclic heterocyclic ring.
  • Monocyclic heterocycles include, but are not limited to, azetidinyl, azetidinyl, diazacyclopentane, 1,3-dioxolane, 1,4-dioxolane Pentyl, 1,3-dioxolane, 4,5-dihydroisoxazol-5-yl, 3,4-dihydropyranyl, 1,3-dithiothienyl, 1,3-Dithienyl, imidazolinyl, imidazolinyl, isothiazolinyl, isothiazolinyl, isothiazolinyl, isoxazolinyl, isoxazolinyl, morpholinyl, oxadiazolinyl, oxadiazolinyl Oxazolinyl, oxazolinyl, oxazolinyl, piperazinyl, piperazinyl, pyrrolidinyl,
  • the fused bicyclic heterocyclic ring generally refers to 7-12 having a monocyclic heterocyclic ring fused with a phenyl, saturated or partially saturated carbocyclic ring, or with another monocyclic heterocyclic ring or with a monocyclic heteroaryl ring Yuan ring system.
  • 1,3-benzodiaxazol-4-yl 1,3-benzodisulfanyl, 3-azabicyclo[3.1.0]hexyl, hexahydro-1H-furan[3,4-c ]Pyrrolyl, 2,3-dihydro-1,4-benzodioctyl, 2,3-dihydro-1-benzofuranyl, 2,3-dihydro-1-benzothienyl, 2 ,3-Dihydro-1H-indolyl, 5,6,7,8-tetrahydroimidazo[1,2-a]pyrazinyl and 1,2,3,4-tetrahydroquinolinyl.
  • OXO generally refers to an oxygen atom bonded to the parent molecular moiety. OXO can be connected to a carbon atom or a sulfur atom through a double bond, and can also be connected to a nitrogen atom through a single bond (for example, N-oxide).
  • alkyl cycloalkyl
  • alkylene alkylene
  • C 3 alkyl refers to an alkyl group having three carbon atoms (ie, n-propyl, isopropyl); in C 1-4 , the members of the group can have any number falling within the range of 1-4 Of carbon atoms.
  • 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 in the tissue 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 particular 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 site 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 via 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 part 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 relieved.
  • the TLR4 pathway inhibitor and/or antagonist is administered based on gastrointestinal ischemia-reperfusion to alleviate the symptoms of ischemic stroke.
  • the term "inhibitor” generally refers to a polymerization retarder, which is a substance used to retard or reduce the rate of a chemical reaction.
  • the inhibitor can inhibit or moderate the chemical reaction, for example, the inhibitor can slow the reaction.
  • 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 reduction in the activity of the protein itself or the reduction in the amount of the protein itself usually results in the inhibition of the physiological function in which it participates.
  • the inhibitor described in the present application can bind to TLR4 to prevent TLR4 from binding to its activator to be activated, or prevent it from binding to downstream signaling pathway molecules and thus cannot mediate signal transduction.
  • the inhibitor may exist as different crystals, amorphous substances, pharmaceutically acceptable salts, hydrates, and solvates.
  • the TLR4 inhibitor can block the signal pathway of TLR4.
  • the TLR4 inhibitor may include OxPAPC and/or TAK-242.
  • the term "antagonist” generally refers to the ability to partially or completely block, inhibit, reduce or neutralize the biological activity of a target (for example, one or more specific proteins).
  • the antagonist may not cause a biological effect by itself after binding to the receptor, but block the effect mediated by the receptor agonist.
  • the antagonist can specifically bind to and inhibit the activity of the target (e.g., one or more specific proteins).
  • the antagonist can specifically bind to and inhibit the activity of TLR4.
  • the TLR4 antagonist can compete with TLR4 ligand for binding.
  • the TLR4 antagonist may include LPS-RS.
  • the term "administration” generally refers to the introduction of the inhibitor and/or antagonist into the body of the subject through any route of introduction or delivery. Any method known to those skilled in the art for contacting cells, organs or tissues with the inhibitor and/or antagonist 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). The increase in degree and duration, or the 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 a 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 therapeutics or prophylactics to promote the regression of the disease or inhibit the development or recurrence of the disease, such as in human subjects during clinical trials, in animal model systems 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 the sudden rupture of blood vessels in the brain or blood can not flow into the brain due to blood vessel obstruction, 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 can 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 due to 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 clotting 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.
  • 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 regions can include: air/taste/auditory 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; protects the mucous membrane of the digestive tract by secreting mucus, antibodies and a large amount of fluid, preventing physical and chemical damage.
  • 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 the prophylactic administration of the combination to patients in the prophase 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 conditions, diseases, disorders or phenotypes 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 used to alter the natural course of the treated individual or cell in the clinical pathological process. This can 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 agents), 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 "work 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 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 TLR4 pathway inhibitor and/or antagonist in the preparation of a medicament for the prevention, alleviation and/or treatment of gastrointestinal ischemia-reperfusion related diseases in a subject End injury.
  • the application also provides the use of a TLR4 pathway inhibitor and/or antagonist in the preparation of a medicament for the prevention, alleviation and/or treatment of neurological-related diseases or disorders in a subject.
  • the TLR4 pathway may include the TLR4/TRAF6/NF ⁇ B pathway.
  • the drug is formulated so that the TLR4 pathway inhibitor and/or antagonist exerts local effects 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 drug is formulated such that the TLR4 pathway inhibitor and/or antagonist can still prevent, alleviate, and/or treat the gastrointestinal ischemia-reperfusion-related remoteness about 1 hour or later after administration.
  • the effective amount of end injury is present locally in the gastrointestinal tract.
  • the drug is formulated so that at most 70% of the TLR4 pathway inhibitor and/or antagonist in the drug is absorbed by the subject and enters the blood circulatory system about 24 hours or later after administration.
  • the locally effective gastrointestinal tract may include the TLR4 pathway inhibitor and/or antagonist locally inhibiting the activation of TLR4 or the binding of TLR4 to its downstream signal molecules in the gastrointestinal tract.
  • the local effect on the gastrointestinal tract may include a decrease in the expression level of TLR4 and/or its downstream signal molecules in the intestinal tissue.
  • the expression level may include mRNA level.
  • the downstream signal molecule may include adaptor protein (MYD88), tumor necrosis factor receptor related factor 6 (TRAF6), nuclear factor kappa B (NF ⁇ B).
  • MYD88 adaptor protein
  • TNF ⁇ B tumor necrosis factor receptor related factor 6
  • NF ⁇ B nuclear factor kappa B
  • the local effect on the gastrointestinal tract may include changes in the expression level of intestinal barrier-related genes in the intestinal tissue.
  • the intestinal barrier-related genes 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 exerting effect on 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 chemokines member.
  • the intestinal tissue may comprise jejunum, ileum, cecum, or colon.
  • the concentration of the TLR4 pathway inhibitor and/or antagonist in the drug is about 0.0001% (w/w) to about 90% (w/w).
  • the concentration of the TLR4 pathway inhibitor and/or antagonist 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), About 0.00 0.00
  • the subject has been, is currently or is at risk of suffering from a disease and/or disorder related to the ischemia-reperfusion of the gastrointestinal tract.
  • the diseases and/or conditions related to gastrointestinal ischemia-reperfusion may include natural events, trauma, or one or more surgical operations or other therapeutic interventions to reduce/block the blood flow of 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 the sudden rupture of blood vessels in the brain or the inability of blood to flow into the brain or the decrease of blood flow into the brain 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 the subject suffering from the above-mentioned disease related to the gastrointestinal ischemia-reperfusion before the administration of the drug to the subject.
  • the subject is suffering from the above-mentioned diseases or conditions 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 or conditions 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 undergone ischemia-reperfusion of the gastrointestinal tract before administering the drug to the subject.
  • the subject is experiencing ischemia-reperfusion of the gastrointestinal tract.
  • the being can 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 ischemia-reperfusion of the gastrointestinal tract.
  • the at-risk may include that the subject has experienced 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 a range from about 0.01 to 200 mg/kg body weight, 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 60 mg/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- It is administered at a dose of 100 mg/kg, about 50-200 mg/kg.
  • the oral route 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 TLR4 pathway inhibitor and/or antagonist 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), etc., the TLR4 pathway inhibitor and/or antagonist It is not substantially decomposed 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.
  • gastric acid ie, hydrochloric acid
  • mucus ie, mucus
  • inorganic substances such as sodium and potassium salts.
  • the TLR4 pathway inhibitors and/or antagonists are basically not affected.
  • the gastric juice is decomposed 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 TLR4 pathway inhibitors and/or antagonists are basically not The pancreatic juice is broken down and/or inactivated.
  • bile has a pH of about 6.8 to 7.4, and its main components are bile salts and bile pigments.
  • the TLR4 pathway inhibitors and/or antagonists are 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 TLR4 pathway inhibitors and/or antagonists are basically not The small intestinal juice is decomposed and/or inactivated.
  • the TLR4 pathway inhibitor and/or antagonist is not substantially decomposed and/or inactivated by the saliva, gastric juice, pancreatic juice, bile, and small intestinal juice.
  • the TLR4 pathway inhibitor and/or antagonist 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 TLR4 pathway inhibitor and/or antagonist is not substantially decomposed and/or inactivated by the digestive juice.
  • the function of suppressing the TLR4 can be basically maintained. For example, the function combined with TLR4.
  • the application also provides a pharmaceutical composition, which may include the TLR4 pathway inhibitor and/or antagonist described in the 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, 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 biotechnology such as genetic engineering, cell engineering, protein engineering, fermentation engineering, etc., for Products for the prevention, treatment and diagnosis of human diseases.
  • biological materials such as microorganisms, cells and various animal and human-derived tissues and liquids obtained by ordinary or biotechnology such as genetic engineering, cell engineering, protein engineering, fermentation engineering, etc.
  • 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 pharmaceutically acceptable carrier may include pharmaceutically acceptable substances, compositions or vehicles involved in carrying or transporting chemical agents, 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 agents 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, capsule, tablet, pill, granule, sachet or lozenge; or may be in liquid form, for example, solution, suspension, emulsion or syrup.
  • 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 TLR4 pathway inhibitor and/or antagonist.
  • the present application also provides a prevention, alleviation and/or treatment of a nervous system-related disease or disorder in a subject, the method comprising administering the TLR4 pathway inhibitor and/or antagonist described in the present application to the subject .
  • the administration includes administration via the gastrointestinal tract.
  • the administration via the gastrointestinal tract includes the specific release of the TLR4 pathway inhibitor and/or antagonist at or near the gastrointestinal tract.
  • the TLR4 pathway inhibitor and/or antagonist is delivered to the gastrointestinal tract through a mechanical device.
  • the TLR4 pathway inhibitor and/or antagonist can be delivered to the gastrointestinal tract through an endoscope or a spray catheter.
  • oral sustained-release agents and disintegrants allow the TLR4 pathway inhibitor and/or antagonist to be released in the gastrointestinal tract.
  • sustained-release agent and disintegrant can initiate release or disintegration depending on the pH of the gastrointestinal tract.
  • the TLR4 pathway inhibitor and/or antagonist can be released in the gastrointestinal tract through enteric coated capsules and liposome microcapsules.
  • the TLR4 pathway inhibitor and/or antagonist is delivered to the gastrointestinal tract by gavage.
  • the time of administration via the gastrointestinal tract 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 after stroke. -24 hours, 1-12 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 TLR4 pathway inhibitor and/or antagonist exerts efficacy locally in the gastrointestinal tract.
  • the TLR4 pathway inhibitor and/or antagonist is still present in an effective amount to prevent, alleviate and/or treat the distal injury caused by the gastrointestinal ischemia-reperfusion Local gastrointestinal tract.
  • TLR4 pathway inhibitor and/or antagonist For example, about 24 hours or later after administration, up to 50% of the TLR4 pathway inhibitor and/or antagonist is absorbed by the subject and enters the blood circulatory system.
  • the locally effective gastrointestinal tract may include the TLR4 pathway inhibitor and/or antagonist locally inhibiting the activation of TLR4 or the binding of TLR4 to its downstream signal molecules in the gastrointestinal tract.
  • the local effect on the gastrointestinal tract may include a decrease in the expression level of TLR4 and/or its downstream signal molecules in the intestinal tissue.
  • the expression level may include mRNA level.
  • the downstream signal molecule may include adaptor protein (MYD88), tumor necrosis factor receptor related factor 6 (TRAF6), nuclear factor kappa B (NF ⁇ B).
  • MYD88 adaptor protein
  • TNF ⁇ B tumor necrosis factor receptor related factor 6
  • NF ⁇ B nuclear factor kappa B
  • the local effect on the gastrointestinal tract may include changes in the expression level of intestinal barrier-related genes in the intestinal tissue.
  • the intestinal barrier-related genes 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 exerting effect on 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 chemokines member.
  • the intestinal tissue may comprise jejunum, ileum, cecum, or colon.
  • the dosage of the TLR4 pathway inhibitor and/or antagonist is from about 0.01 to 200 mg/kg body weight, from about 0.01 to 180 mg/kg, from about 0.01 to 160 mg/kg, from about 0.01 to 140 mg/kg.
  • 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 TLR4 pathway inhibitor and/or antagonist to the subject.
  • this application also provides a method for preventing neurological-related diseases or disorders in a subject, the method comprising: (1) monitoring the gastrointestinal condition of the subject; (2) When or after the monitoring shows that the subject undergoes ischemia-reperfusion of the gastrointestinal tract, the TLR4 pathway inhibitor and/or antagonist is administered to the subject.
  • the method for preventing distal injury caused by gastrointestinal ischemia-reperfusion in a subject further includes monitoring diseases or conditions related to gastrointestinal ischemia-reperfusion before step (1).
  • the method for preventing a disease or disorder related to the nervous system in a subject further includes monitoring a disease or disorder 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 evaluate the gastrointestinal condition of the subject.
  • the clinical examination may include observing the patient’s appetite, dysphagia, abdominal pain, nausea, vomiting, hematemesis, blood in the stool, stool characteristics, whether there are symptoms such as 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.
  • 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.
  • enteric-coated capsules and liposome microcapsules allow the TLR4 pathway inhibitor and/or antagonist to be released in the gastrointestinal tract.
  • the TLR4 pathway inhibitor and/or antagonist is delivered to the gastrointestinal tract by gavage.
  • this application also provides the use of TLR4 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 TLR4 pathway inhibitors and/or antagonists 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 activation of TLR4 or the binding of TLR4 to its downstream signal molecules.
  • the drug inhibits the activation and/or signal transduction of the TLR4/TRAF6/NF ⁇ B pathway.
  • the drug inhibits the binding of lipopolysaccharide (LPS) to lipopolysaccharide binding protein (LBP) and/or cluster of differentiation 14 protein (CD14).
  • LPS lipopolysaccharide
  • LBP lipopolysaccharide binding protein
  • CD14 cluster of differentiation 14 protein
  • the drug can bind to the cysteine CYS747 of the intracellular domain of TLR4.
  • the TLR4 pathway inhibitor and/or antagonist can inhibit the activation of TLR4 or the binding of TLR4 to its downstream signal molecules.
  • the TLR4 pathway inhibitor and/or antagonist includes inhibiting the activation of TLR4 or the binding of TLR4 to its downstream signal molecules by binding to TLR4.
  • the TLR4 pathway inhibitor and/or antagonist can decrease the expression level of TLR4 and/or its downstream signal molecules.
  • the expression level may include mRNA level.
  • the downstream signal molecule may include adaptor protein (MYD88), tumor necrosis factor receptor related factor 6 (TRAF6), nuclear factor kappa B (NF ⁇ B).
  • MYD88 adaptor protein
  • TNF ⁇ B tumor necrosis factor receptor related factor 6
  • NF ⁇ B nuclear factor kappa B
  • the TLR4 pathway inhibitor and/or antagonist can inhibit the activation and/or signal transduction of the TLR4/TRAF6/NF ⁇ B pathway.
  • the TLR4 pathway inhibitor and/or antagonist can inhibit the binding of lipopolysaccharide (LPS) to lipopolysaccharide binding protein (LBP) and/or cluster of differentiation 14 protein (CD14).
  • LPS lipopolysaccharide
  • LBP lipopolysaccharide binding protein
  • CD14 cluster of differentiation 14 protein
  • the TLR4 pathway inhibitor and/or antagonist can bind to the cysteine CYS747 of the intracellular domain of TLR4.
  • TLR4 pathway inhibitor and/or antagonist may have the structure shown in Formula I:
  • G 1 is a 4- to 12-membered heterocyclic group containing 1 to 3 heteroatoms independently selected from N, O and S, -NR 1 R 2 , -OH, -OC 1-4 alkyl, C 3 -8 cycloalkyl, or a 5- to 12-membered heteroaryl group containing 1 to 3 heteroatoms independently selected from N, O and S; wherein the cycloalkyl, the heterocyclic group, the hetero The aryl group is optionally substituted with 1 to 4 substituents independently selected from halogen, C 1-4 alkyl, C 1-4 haloalkyl, -OH, -OC 1- 4 alkyl and OXO;
  • R 1 and R 2 are each independently hydrogen or C 1-4 alkyl
  • G 2 is selected from (i) to (xiii):
  • X 1 is O or S
  • X 2 is O, S, NH or NC 1-4 alkyl
  • R 5 is Or C 1-4 alkyl
  • R 3 , R 7 , R 13 , R 17 , R 19 , R 21 , R 27 , R 29 and R 33 are each independently selected from hydrogen and C 1-4 alkyl;
  • R 11 and R 31 are each independently selected from hydrogen, C 1-4 alkyl, and optionally substituted with 1-3 substituents independently selected from halogen, C 1-4 alkyl, C 1- 4 haloalkyl, -OH, and -Phenyl substituted by a substituent of OC 1-4 alkyl;
  • R 4a , R 4b , R 4c , R 5a , R 6 , R 8 , R 9 , R 10 , R 12 , R 14 , R 15 , R 16 , R 18 , R 20 , R 22 , R 23 , R 24 , R 25 , R 26 , R 28 , R 30 and R 32 are each independently -OH, halogen, nitro, cyano, C 1-4 alkyl, C 1-4 haloalkyl, -OC 1 -4 alkyl, -OC 1-4 haloalkyl, -NH 2 , -NH (C 1-4 alkyl), -N (C 1-4 alkyl) (C 1-4 alkyl), -NHC( O) C 1-4 alkyl, -N (C 1-4 alkyl) C (O) C 1-4 alkyl, -NHC (O) OC 1-4 alkyl, -N (C 1- 4 alkoxy Base) C(O)OC 1-4 alky
  • R 22 , R 24 , R 25 , R 26 , R 28 , R 30 or R 32 form a condensed ring together with the atoms to which they are attached
  • n1 and n2 are independently 1, 0, 2, 3, 4, or 5;
  • n3 and n4 are independently 0, 1, 2, or 3.
  • the structure shown in formula I also includes all isomeric forms of the structure, for example, enantiomers, diastereomers and/or geometric (or conformational) forms, for example, each Center of symmetry, R and S configurations of (Z) and (E) double bond isomers and (Z) and (E) conformational isomers.
  • the TLR4 pathway inhibitor may also include OxPAPC and/or TAK-242.
  • the OxPAPC may have the structural formula shown below:
  • the TAK-242 may have the structural formula shown below:
  • the TLR4 pathway inhibitor OxPAPC can block the binding of LPS to LBP and CD14; the TAK-242 can bind to the cysteine CYS747 of the intracellular domain of TLR4 to inhibit the TLR4 signaling pathway.
  • the TLR4 pathway antagonist may also include LPS-RS.
  • the LPS-RS may have a structural formula as shown below:
  • the TLR4 pathway antagonist LPS-RS can directly compete for the binding of MD-2 between acylated lipid A and hexaacylated lipid A.
  • the distal injury related to gastrointestinal ischemia-reperfusion 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.
  • 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
  • within 4.5 hours such as within 4 hours, such as within 3.5 hours, such as within 3 hours, such as within 2.5 hours, such as within 2 hours, such as within 1.5 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 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 are intervened, the organ or tissue The pathophysiological process is alleviated or reduced.
  • the effects related to intestinal ischemia-reperfusion may include activation of the TLR4 signaling pathway in gastrointestinal tissues, changes in the intestinal barrier, and changes in inflammatory factors in gastrointestinal tissues.
  • the activation of the TLR4 signaling pathway in gastrointestinal tissues may include increased expression of Tlr4, Myd88, Traf6 and/or Nfkb in gastrointestinal tissues; wherein Tlr4 encodes Toll-like receptor 4 (TLR4) and Myd88 encodes an adaptor protein (MYD88), Traf6 encodes tumor necrosis factor receptor-related factor 6 (TRAF6), and Nfkb encodes nuclear factor kappa B protein (NF ⁇ B).
  • TLR4 Toll-like receptor 4
  • Myd88 encodes an adaptor protein
  • Traf6 encodes tumor necrosis factor receptor-related factor 6 (TRAF6)
  • NF ⁇ B nuclear factor kappa B protein
  • the intestinal barrier changes may include intestinal barrier damage,
  • the gastrointestinal tissue inflammatory factor changes and the gastrointestinal tissue pro-inflammatory factor expression increases.
  • 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 reperfusion of ischemic tissue of the gastrointestinal tract, and the reaction may be Including extensive microvascular dysfunction and 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, increased 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 TTC staining in the mouse MCAO model, and ischemia in Nissl staining of tissue frozen sections.
  • 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 a disease, disorder or medical intervention that causes ischemia and reperfusion of the gastrointestinal tract, such as stroke, trauma, shock, sepsis, Surgery for acute pancreatitis, inflammatory bowel disease or traumatic brain injury.
  • a disease, disorder or medical intervention that causes ischemia and reperfusion of the gastrointestinal tract, such as stroke, trauma, shock, sepsis, Surgery for acute pancreatitis, inflammatory bowel disease or traumatic 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 diseases 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 disorders.
  • 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 disorder 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, Nervous regulation disorder of other tissues or organs, 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 disorders 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 TLR4 pathway inhibitors and/or antagonists of the present application in the preparation of drugs, the use of TLR4 for screening drugs, and the inclusion of TLR4 pathway inhibitors and / Or pharmaceutical compositions of antagonists, etc., are not used to limit the scope of the invention of the present application.
  • 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; TTC The powder is a product of Sigma; other materials, reagents, etc., unless otherwise specified, can be obtained from commercial sources.
  • R analysis software is used for data analysis of non-microbial informatics data. Normally distributed data is expressed as mean ⁇ standard deviation, and non-normally distributed data is displayed in the form of median (interquartile range).
  • the non-parametric test adopts 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.
  • 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 divided into the following groups: SHAM group, underwent sham operation; MCAO group, underwent MCAO modeling; Post-OxPAPC group, in small After MCAO modeling, mice were treated with OxPAPC; Pre-OxPAPC group, mice were treated with OxPAPC before MCAO modeling; Post-TAK group, mice were treated with MCAO after MCAO modeling TAK-242 intervention; Pre-TAK group, TAK-242 intervention before MCAO modeling in mice; Post-LPS-RS group, LPS-RS intervention after MCAO modeling in mice ; In the Pre-LPS-RS group, the mice were subjected to LPS-RS intervention before MCAO modelling.
  • mice were weighed and anesthetized with 0.2ml/10g of tribromoethanol, a 1cm median incision was cut in the neck, 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 for the mice in the MCAO group were before the modeling operation, after the suture plug, after the suture plug, and 1 hour after the suture plug.
  • 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 incised 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 branch of the six blood vessels in 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.
  • mice colon tissue was quickly collected and stored frozen at -80°C for the determination of TLR4 pathway related genes, including Tlr4, Traf6, For the relative expression levels of Tram, Myd88, and Nfkb genes, their primer sequences are shown in SEQ ID NO. 1-10. Specific steps are as follows:
  • 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 of 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;
  • Example 5 The effect of OxPAPC intervention on the expression level of genes related to the TLR4 pathway in intestinal tissues
  • mice Prepare the following three groups of mice according to the description in Example 1: SHAM group, undergoing sham operation; MCAO group, undergoing MCAO modelling; Post-OxPAPC group, mice undergoing MCAO modelling, 1 hour after stroke (ie One hour after the thread plug was removed) OxPAPC intervention (1mg/kg, gavage, 200ul/time) was performed on it, and the MCAO model was performed according to the method described in Example 2; 24 hours after the stroke, according to Example 4 The method described in the three groups of mice were sampled and the intestinal tissue Tlr4, Traf6, Tram, Myd88, Nfkb gene expression levels were measured.
  • OxPAPC reduced the expression levels of Tlr4, Traf6, Tram, Myd88, and Nfkb genes in the intestinal tissues of mice after stroke.
  • Example 6 The effect of OxPAPC intervention on brain damage in ischemic stroke
  • Example 1 (1) Prepare the following three groups of mice according to the description in Example 1: MCAO group, MCAO modeling treatment; Post-OxPAPC group, MCAO modeling mice, 1 hour after stroke (that is, after removing the thread plug 1 hour) OxPAPC intervention (1mg/kg, intragastric, 200 ⁇ L/time); Pre-OxPAPC group, before the MCAO modeling of mice, OxPAPC intervention (1mg/kg, intragastric, 200 ⁇ L/ Times), MCAO model was performed after 7 days of OxPAPC 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 value of the proportion of brain injury volume in the whole cerebral hemisphere was obtained.
  • the scoring system can comprehensively evaluate nerve function, 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 beam, the limbs hang down from the beam, and can pass the beam smoothly (0-6 points). 3 Sensory and reflex test: test auricle reflex and corneal reflex (0-2 points).
  • 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 9 The effect of TAK-242 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-TAK group, MCAO modeling, 1 hour after stroke (that is, after removing the thread plug 1 hour) TAK-242 intervention (9mg/kg, intragastric, 200 ⁇ L/time); Pre-TAK group, before the MCAO modeling of mice, TAK-242 intervention (3mg/kg, perfusion) Stomach, 200 ⁇ L/time), MCAO model was performed after 7 days of TAK-242 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 value of the proportion of brain injury volume in the whole cerebral hemisphere was obtained.
  • Example 10 The effect of LPS-RS intervention on brain damage in ischemic stroke
  • mice Prepare the following three groups of mice according to the description in Example 1: MCAO group, MCAO modeling treatment; Post-LPS-RS group, MCAO modeling mice, 1 hour after stroke (ie, remove the line One hour after embolization) they were treated with LPS-RS intervention (2.5 mg/kg, gavage, 200 ⁇ L/time); in the Pre-LPS-RS group, mice were treated with LPS-RS before MCAO modeling ( 2.5mg/kg, intragastrically every other day, 200 ⁇ L/time), MCAO modeling was performed after 4 LPS-RS interventions.
  • 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 value of the proportion of brain injury volume in the whole cerebral hemisphere was obtained.
  • TLR4 inhibitors and/or antagonists exert their effectiveness locally in the gastrointestinal tract
  • mice Before the mouse intervention (0h), and at different time points (1h, 3h, 6h, 12h, 24h) after the intervention, samples of mouse plasma and intestinal contents were collected.
  • Sample pretreatment Weigh approximately 10 mg of each stool sample, put them into a 1.5 mL tube, add 25 ⁇ L of water, and homogenize with zirconia beads for 3 minutes. Then, 185 ⁇ L of acetonitrile: methanol (8:2) was added to extract the metabolites, centrifuged at high speed (18000 g, 20 min), and the supernatant was transferred to a 96-well plate. Add 20 ⁇ L of freshly prepared derivatization reagent to each well, seal the plate, and place it at 30°C for about 60 minutes of derivatization.
  • UPLC-MS/MS (ACQUITY UPLC-Xevo TQS, Waters Corp., Milford, MA, USA) was used for drug concentration detection.
  • Use QuanMET software (v2.0, Metabo-Profile, Shanghai, China) to process the raw data files generated by UPLC-MS/MS, and perform peak integration, calibration and quantification for each metabolite.
  • OxPAPC, TAK-242, and LPS-RS were still present in the gastrointestinal tract in an effective amount to prevent, alleviate and/or treat the distal injury caused by ischemia-reperfusion of the gastrointestinal tract about 1 hour or later after administration. Part of the tract; about 24 hours or later after administration, up to 50% of OxPAPC, TAK-242, LPS-RS are absorbed by the subject and enter the blood circulatory system.

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Abstract

本申请涉及TLR4通路抑制剂和/或拮抗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TLR4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TLR4通路抑制剂和/或拮抗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

TLR4通路抑制剂和/或拮抗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TLR4通路抑制剂和/或拮抗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TLR4通路抑制剂和/或拮抗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TLR4通路抑制剂和/或拮抗剂具有式I所示的结构：
Figure PCTCN2021098812-appb-000001
其中，G 1为包含独立地选自N、O和S的1至3个杂原子的4至12元杂环基，-NR 1R 2，-OH，-OC 1-4烷基，C 3-8环烷基，或者包含独立地选自N、O和S的1至3个杂原子的5至12元杂芳基；其中，所述环烷基、所述杂环基、所述杂芳基任选地被1至4个取代基所取代，所述1至4个取代基独立地选自卤素、C 1-4烷基、C 1-4卤代烷基、-OH，-OC 1-4烷基和OXO；
R 1和R 2各自独立地为氢或C 1-4烷基；
L 1为-C 1-4亚烷基-O-、-C 1-5亚烷基-或-C(O)-CH＝CH-，其中所述-C 1-5亚烷基、所述-C 1-4亚烷基-O-可选地被1至2个卤素或-OH取代，或者L 1为：
Figure PCTCN2021098812-appb-000002
其中C 1-4亚烷基与G 1键合并且咪唑在相对于G 2的苯基间位和对位稠合；
G 2选自(i)至(xiii)：
Figure PCTCN2021098812-appb-000003
Figure PCTCN2021098812-appb-000004
R 4为
Figure PCTCN2021098812-appb-000005
X 1为O或S；
X 2为O、S、NH或NC 1-4烷基；
R 5为
Figure PCTCN2021098812-appb-000006
或C 1-4烷基；
R 3、R 7、R 13，R 17、R 19、R 21、R 27、R 29和R 33各自独立地选自氢和C 1-4烷基；
R 11和R 31各自独立地选自氢、C 1-4烷基和任选地被1-3个独立地选自卤素、C 1-4烷基、C 1- 4卤代烷基、-OH和-OC 1-4烷基的取代基取代的苯基；
R 4a、R 4b、R 4c、R 5a、R 6、R 8、R 9、R 10、R 12、R 14、R 15、R 16、R 18、R 20、R 22、R 23、R 24、R 25、R 26、R 28、R 30和R 32每次出现分别独立地为-OH、卤素、硝基、氰基、C 1-4烷基、C 1-4卤代烷基、-OC 1-4烷基、–OC 1-4卤代烷基、-NH 2、–NH(C 1-4烷基)、-N(C 1-4烷基)(C 1-4烷基)、-NHC(O)C 1-4烷基、-N(C 1-4烷基)C(O)C 1-4烷基、-NHC(O)OC 1-4烷基、-N(C 1- 4烷基)C(O)OC 1-4烷基、-C(O)OC 1-4烷基、-C(O)OH、-C(O)NH 2、-C(O)NH(C 1- 4烷基)或-C(O)N(C 1-4烷基)(C 1-4烷基)，并且可选地两个R 4a、R 4b、R 5a、R 6、R 8、R 9、 R 10、R 12、R 14、R 15、R 16、R 18、R 20、R 21、
R 22、R 24、R 25、R 26、R 28、R 30或R 32与它们所连接的原子一起形成稠环
Figure PCTCN2021098812-appb-000007
n1和n2独立地为1、0、2、3、4或5；
n3和n4独立地为0、1、2或3
在某些实施方式中，所述TLR4通路抑制剂和/或拮抗剂包括OxPAPC、TAK-242和/或LPS-RS。
在某些实施方式中，所述TLR4通路抑制剂OxPAPC阻断LPS与LBP和CD14的结合；TAK-242结合TLR4胞内域的半胱氨酸CYS747抑制TLR4信号通路。在某些实施方式中，TLR4通路拮抗剂LPS-RS在酰化脂质A和六酰化脂质A之间直接竞争与MD-2的结合。
在某些实施方式中，所述药物被配制为使得所述TLR4通路抑制剂和/或拮抗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1小时或之后，所述TLR4通路抑制剂和/或拮抗剂仍然以预防、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24小时或之后，所述药物中至多50％的所述TLR4通路抑制剂和/或拮抗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TLR4通路抑制剂和/或拮抗剂的浓度为约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TLR4通路抑制剂和/或拮抗剂基本上不被消化液分解和/或灭活。
本申请还提供了一种药物组合物，其包含本申请所述的TLR4通路抑制剂和/或拮抗剂和 任选地药学上可接受的载体。
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TLR4通路抑制剂和/或拮抗剂。
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TLR4通路抑制剂和/或拮抗剂在胃肠道局部发挥效力。
在某些实施方式中，在施用后约1小时或之后，所述TLR4通路抑制剂和/或拮抗剂仍然以预防、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24小时或之后，至多50％的所述TLR4通路抑制剂和/或拮抗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TLR4通路抑制剂和/或拮抗剂的给药剂量为约0.01至200mg/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TLR4通路抑制剂和/或拮抗剂。
在某些实施方式中，所述施用包括经胃肠道施用。
本申请还提供了TLR4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。
在某些实施方式中，所述药物抑制TLR4/TRAF6/NFκB通路的激活和/或信号传导。
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-3B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结肠组织TLR4途径相关基因表达水平的变化。
图4显示的是本申请所述的TLR4通路抑制剂(OxPAPC)干预对小鼠缺血性脑卒中后脑损伤的缓解的结果。
图5显示的是本申请所述的TLR4通路抑制剂(TAK-242)干预对小鼠缺血性脑卒中后脑损伤的缓解的结果。
图6显示的是造模前本申请所述的TLR4通路拮抗剂(LPS-RS)干预对小鼠缺血性脑卒中后脑损伤的缓解的结果。
图7显示的是造模后本申请所述的TLR4通路拮抗剂(LPS-RS)干预对小鼠缺血性脑卒中后脑损伤的缓解的结果。
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“TLR4”也称作Toll样受体4，通常是指一类属于Toll样受体家族的跨膜蛋白。TLR4通常都是Ⅰ型跨膜蛋白，可分为胞膜外区，胞浆区和跨膜区三部分，通常情况下，通过其细胞外富亮氨酸重复结构域(LRR)或细胞内Toll/IL-1受体(TIR)结构域与其他分子形成复合物来响应信号。TLR4的配体可以包括病毒蛋白、多糖以及内源蛋白，例如低密度脂蛋白，β-防御素和热休克蛋白；TIR具有嗜同性相互作用(homophilic interaction)，在识别信号分子后，TLR4可以发生分子构象变化从而募集下游含有TIR的信号分子组成信号复合体。TLR4能够在病原体识别和先天免疫激活中起着基本作用，例如，TLR4能够与LY96和CD14协同介导对细菌脂多糖(LPS)的先天免疫应答(Tatematsu M,etc.,J Immunol.2016 May 1；196(9):3865-76)；例如，TLR4可以通过MYD88、TIRAP和TRAF6起作用而导致NF-κB活化、细胞因子分泌以及炎症反应(Medzhitov R,etc.,Nature.1997 Jul 24；388(6640):394-7；Arbour NC,etc.,Nat Genet.2000 Jun；25(2):187-91；Tatematsu M,etc.,J Immunol.2016 May 1；196(9):3865-76)；例如，参与由游离脂肪酸(例如棕榈酸酯)或Ni 2+触发的非LPS依赖的炎症反应(Schmidt M,etc.,Nat Immunol.2010 Sep；11(9):814-9)；例如，介导结核分枝杆菌HSP70(dnaK)和HSP65(groEL-2)刺激的NF-κB表达(Bulut Y,etc.,J Biol Chem.2005 Jun  3；280(22):20961-7)；例如，TLR4能够与与TLR6结合，以响应氧化的低密度脂蛋白(oxLDL)或淀粉样β42而促进单核细胞/巨噬细胞的无菌炎症；例如，TLR4能够结合带负电的LDL(LDL -)并介导LDL -诱导的细胞因子释放(Estruch M,etc.,Atherosclerosis.2013 Aug；229(2):356-62)。在人类中，编码TLR4的基因位于9号染色体9q33.1，包含4个外显子；在小鼠中，编码TLR4的基因位于4号染色体4 C1；34.66cM，包含3个外显子。
在本申请中，术语“TLR4通路”通常是指能够调节TLR4的表达或活性的，和/或，由TLR4的激活所触发的细胞内和/或细胞外信号通路。
在本申请中，术语“烷基”通常是指直链或支链饱和烃。包括但不限于甲基、乙基、正丙基、异丙基、正丁基、仲丁基、异丁基、叔丁基、正戊基、异戊基、新戊基、正己基、3-甲基己基、2,2-二甲基戊基、2,3-二甲基戊基、正庚基、正辛基、正壬基和正癸基。
在本申请中，术语“亚烷基”通常是指衍生自直链或支链饱和烃的二价基团。包括但不限于-CH 2-、-CH 2CH 2-、-CH 2CH 2CH 2-、-CH 2CH(CH 3)CH 2-或CH 2CH(CH 3)CH(CH 3)CH 2-。
在本申请中，术语“卤代烷基”通常是指如本申请中所定义的烷基的中一个、两个、三个、四个、五个、六个或七个氢原子被卤素取代。例如，2-氟乙基、二氟甲基、三氟甲基、2,2,2-三氟乙基、2,2,2-三氟-1或1-二甲基乙基等。
在本申请中，术语“杂芳基”通常是指芳族杂环。例如包含至少一个选自O、N或S的杂原子的芳环。杂芳基可以包含5至12个环原子。杂芳基可以是5至6元单环杂芳基或8至12元双环杂芳基。5元单环杂芳基环可以包含两个双键以及一个、两个、三个或四个杂原子作为环原子，例如，呋喃基，咪唑基，异恶唑基，异噻唑基，恶二唑基，恶唑基，吡唑基，吡咯基，四唑基，噻二唑基，噻唑基，噻吩基和三唑基。6元杂芳基环可以包含三个双键以及一个、两个、三个或四个杂原子作为环原子，例如，吡啶基，哒嗪基，嘧啶基，吡嗪基和三嗪基。双环杂芳基是具有与芳族、饱和或部分饱和的碳环稠合或与第二单环杂芳基环稠合的单环杂芳基的8至12元环系统。例如，苯并呋喃基，苯并恶二唑基，1，3-苯并噻唑基，苯并咪唑基，苯并噻吩基，吲哚基，吲唑基，异喹啉基，萘吡啶基，恶唑并吡啶，喹啉基。杂芳基基团可以通过基团中包含的任何可取代的碳原子或任何可取代的氮原子连接至母体分子。
在本申请中，术语“环烷基”通常是指不含有杂原子作为环原子、不含有双键的碳环。例如，环丙基，环丁基，环戊基，环己基，环庚基或环辛基。环烷基可通过任何可取代的碳原子连接至母体分子。
在本申请中，术语“杂环”或“杂环的”通常是指包含至少一个杂原子作为环原子的环系统，其中杂原子可以选自O，N或S。杂环可以是单环杂环，稠合双环杂环或螺环杂环。单环杂环包括但不限于氮杂环丁烷基，氮杂环戊烷基，二氮杂环戊烷基，1，3-二氧杂环戊烷基，1，4- 二氧杂环戊烷基，1，3-二氧杂环戊烷基，4，5-二氢异恶唑-5-基，3，4-二氢吡喃基，1，3-二硫代噻吩基，1，3-二噻吩基，咪唑啉基，咪唑啉基，异噻唑啉基，异噻唑烷基，异恶唑啉基，异恶唑啉基，吗啉基，恶二唑啉基，恶二唑啉基，恶唑啉基，恶唑啉基，哌嗪基，哌嗪基，吡咯烷基，四氢呋喃基，四氢吡喃基，四氢噻吩基，噻二唑啉基，噻二唑烷基，噻唑啉基，噻唑烷基，硫代吗啉基，1，1-二氧代硫代吗啉基，硫代吡喃基和三噻吩基。
所述稠合双环杂环通常是指具有与苯基、饱和或部分饱和的碳环，或与另一个单环杂环或与单环杂芳基环稠合的单环杂环的7-12元环系统。例如，1，3-苯并二恶唑-4-基，1，3-苯并二硫基，3-氮杂双环[3.1.0]己基，六氢-1H-呋喃[3，4-c]吡咯基，2，3-二氢-1，4-苯并二辛基，2，3-二氢-1-苯并呋喃基，2，3-二氢-1-苯并噻吩基，2，3-二氢-1H-吲哚基，5，6，7，8-四氢咪唑并[1，2-a]吡嗪基和1，2，3，4-四氢喹啉基。
在本申请中，术语“OXO”通常是指键合至母体分子部分的氧原子。OXO可以通过双键连接至碳原子或硫原子，也可通过单键(例如N-氧化物)连接到氮原子上。
在本申请中，如本领域技术人员可知的，“烷基”、“环烷基”、“亚烷基”等之类的术语可以在名称前加一个标识表示在特定情况下基团中存在的原子数，例如，C 1-4烷基，C 3-6环烷基，C 1-4亚烷基等，“C”后所跟下标数字表示在基团中存在的碳原子数。例如，C 3烷基是指具有三个碳原子的烷基(即，正丙基，异丙基)；C 1-4中，基团的成员可具有落入1-4范围内的任何数目的碳原子。
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TLR4通路抑制剂和/或拮抗剂后减轻了缺血性脑卒中的症状。
在本申请中，术语“抑制剂”通常指缓聚剂，是一种用来阻滞或降低化学反应速度的物质。所述抑制剂可以抑制或缓和化学反应，例如，所述抑制剂可以减缓反应。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，所述蛋白质本身活性的降低或者本身存在量的降低通常会导致其参与的生理功能受到抑制。例如，在本申请中所述抑制剂能够与TLR4结合从而阻碍TLR4与其激活因子结合而被激活，或者阻碍其与下游信号通路分子结合从而不能够介导信号传导。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。例如，所述TLR4抑制剂可以阻断TLR4的信号通路。与例如，所述TLR4抑制剂可以包括OxPAPC和/或TAK-242。
在本申请中，术语“拮抗剂”通常指能够部分或全部阻断、抑制、降低或中和靶标(例如一种或多种特定蛋白质)的生物学活性。例如，所述拮抗剂可以与受体结合后本身不引起生物 学效应，但阻断该受体激动剂介导的作用。例如，所述拮抗剂可以特异性结合并抑制所述靶标(例如一种或多种特定蛋白质)的活性。在本申请所述，所述拮抗剂可以特异性结合并抑制TLR4的活性。例如，所述TLR4拮抗剂可以与TLR4的配体进行竞争结合。例如，所述TLR4拮抗剂可以包括LPS-RS。
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和/或拮抗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和/或拮抗剂接触的任何方法。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TLR4通路抑制剂和/或拮抗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TLR4通路抑制剂和/或拮抗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。
例如，所述TLR4通路可以包括TLR4/TRAF6/NFκB通路。
在本申请中，所述药物被配制为使得所述TLR4通路抑制剂和/或拮抗剂在胃肠道局部发挥效力。
例如，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。
例如，所述药物被配制为使得在施用后约1小时或之后，所述TLR4通路抑制剂和/或拮抗剂仍然以预防、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。
例如，所述药物被配制为使得在施用后约24小时或之后，所述药物中至多70％的所述TLR4通路抑制剂和/或拮抗剂被所述受试者吸收而进入血液循环系统。
例如，所述在胃肠道局部发挥效力可以包含所述TLR4通路抑制剂和/或拮抗剂在所述胃肠道局部抑制TLR4的激活或者TLR4与其下游信号分子的结合。
例如，所述在胃肠道局部发挥效力可以包含肠组织中TLR4和/或其下游信号分子表达水平下降。
例如，所述表达水平可以包括mRNA水平。
例如，所述下游信号分子可以包括衔接蛋白(MYD88)、肿瘤坏死因子受体相关因子6(TRAF6)、核因子κB(NFκB)。
例如，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，所述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。
例如，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，所述药物中所述TLR4通路抑制剂和/或拮抗剂的浓度为约0.0001％(w/w)至约90％(w/w)。
例如，所述TLR4通路抑制剂和/或拮抗剂的浓度为约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和/或病症。
在本申请中，所述与胃肠道缺血再灌注相关的疾病和/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或病症。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或病症。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200mg/kg体重、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。
在本申请中，所述TLR4通路抑制剂和/或拮抗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TLR4通路抑制剂和/或拮抗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TLR4通路抑制剂和/或拮抗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TLR4通路抑制剂和/或拮抗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TLR4通路抑制剂和/或拮抗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TLR4通路抑制剂和/或拮抗剂基本上不被所述小肠液分解和/或灭活。
例如，所述TLR4通路抑制剂和/或拮抗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TLR4通路抑制剂和/或拮抗剂在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TLR4通路抑制剂和/或拮抗剂基本上不被消化液分解和/或灭活可以包括所述TLR4通路抑制剂和/或拮抗剂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TLR4 的功能。例如，与TLR4结合的功能。
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TLR4通路抑制剂和/或拮抗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TLR4通路抑制剂和/或拮抗剂。
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TLR4通路抑制剂和/或拮抗剂。
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TLR4通路抑制剂和/或拮抗剂。
例如，通过机械装置将所述TLR4通路抑制剂和/或拮抗剂递送至胃肠道。
例如，通过内窥镜、喷雾导管将所述TLR4通路抑制剂和/或拮抗剂递送至胃肠道。
例如，通过口服缓释剂、崩解剂使得所述TLR4通路抑制剂和/或拮抗剂在胃肠道释放。
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TLR4通路抑制剂和/或拮抗剂在胃肠道释放。
例如，通过灌胃将所述TLR4通路抑制剂和/或拮抗剂递送至胃肠道。
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，1-12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TLR4通路抑制剂和/或拮抗剂在胃肠道局部发挥效力。
例如，在施用后约1小时或之后，所述TLR4通路抑制剂和/或拮抗剂仍然以预防、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24小时或之后，至多50％的所述TLR4通路抑制剂和/或拮抗剂被所述受试者吸收而进入血液循环系统。
例如，所述在胃肠道局部发挥效力可以包含所述TLR4通路抑制剂和/或拮抗剂在所述胃肠道局部抑制TLR4的激活或者TLR4与其下游信号分子的结合。
例如，所述在胃肠道局部发挥效力可以包含肠组织中TLR4和/或其下游信号分子表达水平下降。
例如，所述表达水平可以包括mRNA水平。
例如，所述下游信号分子可以包括衔接蛋白(MYD88)、肿瘤坏死因子受体相关因子6(TRAF6)、核因子κB(NFκB)。
例如，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，所述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。
例如，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。
例如，所述TLR4通路抑制剂和/或拮抗剂的给药剂量为从约0.01至200mg/kg体重、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。
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TLR4通路抑制剂和/或拮抗剂。
另一方面，本申请还提供了一种预防受试者中神经系统相关疾病或病症的方法，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TLR4通路抑制剂和/或拮抗剂。
例如，所述预防受试者中胃肠道缺血再灌注引起的远端损伤的方法还包括在步骤(1)前监测与胃肠道缺血再灌注相关的疾病或病症。
例如，所述预防受试者中神经系统相关疾病或病症的方法还包括在步骤(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，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TLR4通路抑制剂和/或拮抗剂剂在胃肠道释放。
例如，通过灌胃将所述TLR4通路抑制剂和/或拮抗剂递送至胃肠道。
另一方面，本申请还提供了TLR4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TLR4通路抑制剂和/或拮抗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。
例如，所述药物抑制TLR4的激活或者TLR4与其下游信号分子的结合。
例如，所述药物抑制TLR4/TRAF6/NFκB通路的激活和/或信号传导。
例如，所述药物阻抑脂多糖(LPS)与脂多糖结合蛋白(LBP)和/或分化簇14蛋白(CD14)的结合。
例如，所述药物能够结合TLR4胞内域的半胱氨酸CYS747。
在本申请中，所述TLR4通路抑制剂和/或拮抗剂能够抑制TLR4的激活或者TLR4与其下游信号分子的结合。
在本申请中，所述TLR4通路抑制剂和/或拮抗剂包括通过与TLR4的结合而抑制TLR4的激活或者TLR4与其下游信号分子的结合。
例如，所述TLR4通路抑制剂和/或拮抗剂能够使TLR4和/或其下游信号分子表达水平下降。
例如，所述表达水平可以包括mRNA水平。
例如，所述下游信号分子可以包括衔接蛋白(MYD88)、肿瘤坏死因子受体相关因子6(TRAF6)、核因子κB(NFκB)。
例如，所述TLR4通路抑制剂和/或拮抗剂能够抑制TLR4/TRAF6/NFκB通路的激活和/或信号传导。
例如，所述TLR4通路抑制剂和/或拮抗剂能够阻抑脂多糖(LPS)与脂多糖结合蛋白(LBP)和/或分化簇14蛋白(CD14)的结合。
例如，所述TLR4通路抑制剂和/或拮抗剂能够结合TLR4胞内域的半胱氨酸CYS747。
在本申请中，所述TLR4通路抑制剂和/或拮抗剂可以具有式I所示的结构：
Figure PCTCN2021098812-appb-000008
其中，G 1为包含独立地选自N、O和S的1至3个杂原子的4至12元杂环基，-NR 1R 2，-OH，-OC 1-4烷基，C 3-8环烷基，或者包含独立地选自N、O和S的1至3个杂原子的5至12元杂芳基；其中，所述环烷基、所述杂环基、所述杂芳基任选地被1至4个取代基所取代，所述1至4个取代基独立地选自卤素、C 1-4烷基、C 1-4卤代烷基、-OH，-OC 1-4烷基和OXO；
R 1和R 2各自独立地为氢或C 1-4烷基；
L 1为-C 1-4亚烷基-O-、-C 1-5亚烷基-或-C(O)-CH＝CH-，其中所述-C 1-5亚烷基、所述-C 1-4亚烷基-O-可选地被1至2个卤素或-OH取代，或者L 1为：
Figure PCTCN2021098812-appb-000009
其中C 1-4亚烷基与G 1键合并且咪唑在相对于G 2的苯基间位和对位稠合；
G 2选自(i)至(xiii)：
Figure PCTCN2021098812-appb-000010
Figure PCTCN2021098812-appb-000011
R 4为
Figure PCTCN2021098812-appb-000012
X 1为O或S；
X 2为O、S、NH或NC 1-4烷基；
R 5为
Figure PCTCN2021098812-appb-000013
或C 1-4烷基；
R 3、R 7、R 13，R 17、R 19、R 21、R 27、R 29和R 33各自独立地选自氢和C 1-4烷基；
R 11和R 31各自独立地选自氢、C 1-4烷基和任选地被1-3个独立地选自卤素、C 1-4烷基、C 1- 4卤代烷基、-OH和-OC 1-4烷基的取代基取代的苯基；
R 4a、R 4b、R 4c、R 5a、R 6、R 8、R 9、R 10、R 12、R 14、R 15、R 16、R 18、R 20、R 22、R 23、R 24、R 25、R 26、R 28、R 30和R 32每次出现分别独立地为-OH、卤素、硝基、氰基、C 1-4烷基、C 1-4卤代烷基、-OC 1-4烷基、–OC 1-4卤代烷基、-NH 2、–NH(C 1-4烷基)、-N(C 1-4烷基)(C 1-4烷基)、-NHC(O)C 1-4烷基、-N(C 1-4烷基)C(O)C 1-4烷基、-NHC(O)OC 1-4烷基、-N(C 1- 4烷基)C(O)OC 1-4烷基、-C(O)OC 1-4烷基、-C(O)OH、-C(O)NH 2、-C(O)NH(C 1- 4烷基)或-C(O)N(C 1-4烷基)(C 1-4烷基)，并且可选地两个R 4a、R 4b、R 5a、R 6、R 8、R 9、R 10、R 12、R 14、R 15、R 16、R 18、R 20、R 21、
R 22、R 24、R 25、R 26、R 28、R 30或R 32与它们所连接的原子一起形成稠环
Figure PCTCN2021098812-appb-000014
n1和n2独立地为1、0、2、3、4或5；
n3和n4独立地为0、1、2或3。
在本申请中，所述式I所示的结构还包括该结构的所有异构形式，例如，对映异构，非对映异构和/或几何(或构象)形式，例如，每个不对称中心，(Z)和(E)双键异构体以及(Z)和(E)构象异构体的R和S构型。
在本申请中，WO2019136147A1中记载的关于所述式I所示的结构的全部内容以引用的方式并入本申请。
在本申请中，所述TLR4通路抑制剂还可以包括OxPAPC和/或TAK-242。
所述OxPAPC可以具有如下所示的结构式：
Figure PCTCN2021098812-appb-000015
所述TAK-242可以具有如下所示的结构式：
Figure PCTCN2021098812-appb-000016
例如，所述TLR4通路抑制剂OxPAPC可以阻断LPS与LBP和CD14的结合；所述TAK-242可以结合TLR4胞内域的半胱氨酸CYS747抑制TLR4信号通路。
在本申请中，所述TLR4通路拮抗剂还可以包括LPS-RS。
所述LPS-RS可以具有如下所示的结构式：
Figure PCTCN2021098812-appb-000017
TLR4通路拮抗剂LPS-RS可在酰化脂质A和六酰化脂质A之间直接竞争与MD-2的结合。
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可以包括胃肠组织TLR4信号通路的激活、肠屏障变化、胃肠组织炎症因子变化。
例如，所述胃肠组织TLR4信号通路的激活可以包括胃肠组织Tlr4、Myd88、Traf6和/或Nfkb表达升高；其中Tlr4编码Toll样受体4(TLR4)，Myd88编码衔接蛋白(MYD88)、Traf6编码肿瘤坏死因子受体相关因子6(TRAF6)、Nfkb编码核因子κB蛋白(NFκB)。
例如，所述肠屏障变化可以包括肠屏障损伤，
例如，所述胃肠组织炎症因子变化胃肠组织促炎因子表达升高。
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TLR4通路抑制剂和/或拮抗剂在制备药物中的用途、TLR4用于筛选药物的用途、包含TLR4通路抑制剂和/或拮抗剂的药物组合物等，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；TTC粉剂为Sigma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-OxPAPC组，在小鼠进行MCAO造模后，对其进行OxPAPC干预；Pre-OxPAPC组，在小鼠进行MCAO造模前，对其进行OxPAPC干预；Post-TAK组，在小鼠进行MCAO造模后，对其进行TAK-242干预；Pre-TAK组，在小鼠进行MCAO造模前，对其进行TAK-242干预；Post-LPS-RS组，在小鼠 进行MCAO造模后，对其进行LPS-RS干预；Pre-LPS-RS组，在小鼠进行MCAO造模前，对其进行LPS-RS干预。
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，将线栓(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(大约当线栓标记(离头端1cm)在颈总动脉分叉处)，固定线栓，随后缝合皮肤。插入线栓后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、插线栓后、拔线栓后、以及拔线栓后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组小鼠移除线栓以及在此之后的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现象。
实施例4.肠道组织TLR4通路相关基因表达水平定量分析
在脑卒中后的24小时时间点(其中起始计时时间为移除线栓的时间)，快速采取小鼠结肠组织并冷冻储存在-80℃用于测定TLR4通路相关基因，包括Tlr4、Traf6、Tram、Myd88、Nfkb基因的相对表达水平，其引物序列如SEQ ID NO.1-10所示。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/ul)，以250nM为最终浓度加入如SEQ ID NO：1-10所示的上游引物和下游引物，进行SYBR Green(Takara Bio)实时PCR，反应体系如下：
表1
反应物 体积
上游引物F 0.4ul
下游引物R 0.4ul
ROX Reference Dye II 0.4ul
SYBR Green 10ul
cDNA 2ul
PCR水 6.8ul
(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，其引物序列如SEQ ID NO.11-12所示。
观察结果如图3所示，结果显示相比于Sham组，MCAO组Traf6、Myd88、Nfkb基因的相对表达水平升高。
实施例5.OxPAPC干预对于肠道组织TLR4通路相关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-OxPAPC组，小鼠进行MCAO造模，脑卒中后1小时(即移除线栓后1小时)对其进行OxPAPC干预(1mg/kg，灌胃，200ul/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Tlr4、Traf6、Tram、Myd88、Nfkb基因表达水平。
结果显示OxPAPC降低了小鼠脑卒中后肠道组织Tlr4、Traf6、Tram、Myd88、Nfkb基因的表达水平。
实施例6.OxPAPC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-OxPAPC组，小鼠进行MCAO造模，脑卒中后1小时(即移除线栓后1小时)对其进行OxPAPC干预(1mg/kg，灌胃，200μL/次)；Pre-OxPAPC组，在小鼠进行MCAO造模前，对其进行OxPAPC干预(1mg/kg，灌胃，200μL/次)，OxPAPC干预7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。
观察结果如图4所示，结果显示无论是造模前OxPAPC干预还是造模后OxPAPC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显著缓解，这说明通过抑制TLR4的作用可以改善小鼠缺血性脑卒中后脑损伤。
实施例7.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-OXPAPC组、Pre-OXPAPC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)。
结果显示，无论是造模前OxPAPC干预还是造模后OxPAPC干预，相比于未干预的MCAO组都改善了小鼠神经缺损的情况。
实施例8.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-OxPAPC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。
结果显示，OxPAPC干预显著减少了小鼠神经元的丢失。
实施例9.TAK-242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-TAK组，小鼠进行MCAO造模，脑卒中后1小时(即移除线栓后1小时)对其进行TAK-242干预(9mg/kg，灌胃，200μL/次)；Pre-TAK组，在小鼠进行MCAO造模前，对其进行TAK-242干预(3mg/kg，灌胃，200μL/次)，TAK-242干预7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。
观察结果如图5所示，结果显示无论是造模前TAK-242干预还是造模后TAK-242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显著缓解，这说明通过抑制TLR4的作用可以改善小鼠缺血性脑卒中后脑损伤。
实施例10.LPS-RS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-LPS-RS组，小鼠进行MCAO造模，脑卒中后1小时(即移除线栓后1小时)对其进行LPS-RS干预(2.5mg/kg，灌胃，200μL/次)；Pre-LPS-RS组，在小鼠进行MCAO造模前，对其进行LPS-RS干预(2.5mg/kg，隔天灌胃，200μL/次)，LPS-RS干预4次结束后进行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。
观察结果如图6-7所示，结果显示无论是造模前LPS-RS干预还是造模后LPS-RS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显著缓解，这说明通过抑制TLR4的作用可以改善小鼠缺血性脑卒中后脑损伤。
实施例11.TLR4抑制剂和/或拮抗剂在胃肠道局部发挥效力
在小鼠干预前(0h)，及干预后不同时间点(1h，3h，6h，12h，24h)，收集小鼠血浆、肠道内容物样本。
样本预处理：每个粪便样品称取约10mg，分别装进1.5mL管中，加入25μL的水，用 氧化锆珠匀浆3min。然后，加入185μL的乙腈：甲醇(8:2)提取代谢物，高速离心(18000g,20min)，将上清液转移至96孔板中。每个孔中加入20μL新鲜配制的衍生化试剂，将板密封，置于30℃进行约60min的衍生化。然后，加入350μL冰浴的50％甲醇溶液稀释样品，将板置于-20℃下20min，接着在4℃下离心(4000g,30min)，吸取135μL上清液，转移至新的96孔板，每个孔中加入15μL的内标。在左边孔中加入衍生化的标准品原液的梯度稀释液，最后将板密封，用于LC-MS分析。
在每个血清样品中移取25μL转移至96孔板中。每个孔中加入100μL含内标的冰冷甲醇，剧烈振荡5min。将96孔板在4000g下离心30min，再重新放置于移液工作站中。转移30μL上清于新的96孔板中，加入20μL新鲜配制的衍生化试剂，将板密封，置于30℃进行约60min的衍生化。然后，加入350μL冰浴的50％甲醇溶液稀释样品，将板置于-20℃下20min，接着在4℃下离心(4000g,30min)，吸取135μL上清液，转移至新的96孔板，每个孔中加入15μL的内标。在左边孔中加入衍生化的标准品原液的梯度稀释液，最后将板密封，用于LC-MS分析。
利用超高效液相色谱-串联质谱联用仪(UPLC-MS/MS)(ACQUITY UPLC-Xevo TQS,Waters Corp.,Milford,MA,USA)进行药物浓度检测。使用QuanMET软件(v2.0,Metabo-Profile,Shanghai,China)对UPLC-MS/MS生成的原始数据文件进行处理，对每一个代谢物进行峰的集成、校准和定量。
结果显示，施用后约1小时或之后，OxPAPC、TAK-242、LPS-RS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；在施用后约24小时或之后，至多50％的OxPAPC、TAK-242、LPS-RS被所述受试者吸收而进入血液循环系统。
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. TLR4通路抑制剂和/或拮抗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TLR4通路抑制剂和/或拮抗剂具有式I所示的结构：
    Figure PCTCN2021098812-appb-100001
    其中，G 1为包含独立地选自N、O和S的1至3个杂原子的4至12元杂环基，-NR 1R 2，-OH，-OC 1-4烷基，C 3-8环烷基，或者包含独立地选自N、O和S的1至3个杂原子的5至12元杂芳基；其中，所述环烷基、所述杂环基、所述杂芳基任选地被1至4个取代基所取代，所述1至4个取代基独立地选自卤素、C 1-4烷基、C 1-4卤代烷基、-OH，-OC 1-4烷基和OXO；
    R 1和R 2各自独立地为氢或C 1-4烷基；
    L 1为-C 1-4亚烷基-O-、-C 1-5亚烷基-或-C(O)-CH＝CH-，其中所述-C 1-5亚烷基、所述-C 1-4亚烷基-O-可选地被1至2个卤素或-OH取代，或者L 1为：
    Figure PCTCN2021098812-appb-100002
    其中C 1-4亚烷基与G 1键合并且咪唑在相对于G 2的苯基间位和对位稠合；
    G 2选自(i)至(xiii)：
    Figure PCTCN2021098812-appb-100003
    Figure PCTCN2021098812-appb-100004
    R 4为
    Figure PCTCN2021098812-appb-100005
    X 1为O或S；
    X 2为O、S、NH或NC 1-4烷基；
    R 5为
    Figure PCTCN2021098812-appb-100006
    或C 1-4烷基；
    R 3、R 7、R 13，R 17、R 19、R 21、R 27、R 29和R 33各自独立地选自氢和C 1-4烷基；
    R 11和R 31各自独立地选自氢、C 1-4烷基和任选地被1-3个独立地选自卤素、C 1-4烷基、C 1- 4卤代烷基、-OH和-OC 1-4烷基的取代基取代的苯基；
    R 4a、R 4b、R 4c、R 5a、R 6、R 8、R 9、R 10、R 12、R 14、R 15、R 16、R 18、R 20、R 22、R 23、R 24、R 25、R 26、R 28、R 30和R 32每次出现分别独立地为-OH、卤素、硝基、氰基、C 1-4烷基、C 1-4卤代烷基、-OC 1-4烷基、–OC 1-4卤代烷基、-NH 2、–NH(C 1-4烷基)、-N(C 1-4烷基)(C 1-4烷基)、-NHC(O)C 1-4烷基、-N(C 1-4烷基)C(O)C 1-4烷基、-NHC(O)OC 1-4烷基、-N(C 1- 4烷基)C(O)OC 1-4烷基、-C(O)OC 1-4烷基、-C(O)OH、-C(O)NH 2、-C(O)NH(C 1- 4烷基)或-C(O)N(C 1-4烷基)(C 1-4烷基)，并且可选地两个R 4a、R 4b、R 5a、R 6、R 8、R 9、R 10、R 12、R 14、R 15、R 16、R 18、R 20、R 21、R 22、R 24、R 25、R 26、R 28、R 30或R 32与它们所连接的原子一起形成稠环
    Figure PCTCN2021098812-appb-100007
    n1和n2独立地为1、0、2、3、4或5；
    n3和n4独立地为0、1、2或3。
  3. 根据权利要求1所述的用途，其中所述TLR4通路抑制剂和/或拮抗剂包括OxPAPC、TAK-242和/或LPS-RS。
  4. 根据权利要求1-3中任一项所述的用途，其中所述药物被配制为使得所述TLR4通路抑制剂和/或拮抗剂在胃肠道局部发挥效力。
  5. 根据权利要求1-4中任一项所述的用途，其中在施用后约1小时或之后，所述TLR4通路抑制剂和/或拮抗剂仍然以预防、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。
  6. 根据权利要求1-5中任一项所述的用途，其中在施用后约24小时或之后，至多50％的所述TLR4通路抑制剂和/或拮抗剂被所述受试者吸收而进入血液循环系统。
  7. 根据权利要求1-6中任一项所述的用途，其中所述药物中所述TLR4通路抑制剂和/或拮抗剂的浓度为约0.0001％(w/w)至约90％(w/w)。
  8. 根据权利要求1-7中任一项所述的用途，其中所述受试者曾经、正在或有风险患有与所述胃肠道缺血再灌注相关的疾病和/或病症。
  9. 根据权利要求8所述的用途，其中所述与胃肠道缺血再灌注相关的疾病和/或病症包括脑卒中、创伤、休克、败血症和/或急性胰腺炎。
  10. 根据权利要求8-9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。
  11. 根据权利要求1-10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。
  12. 根据权利要求1-11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远端损伤包括缺血性脑卒中。
  13. 根据权利要求1-12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。
  14. 根据权利要求1-13中任一项所述的用途，其中所述TLR4通路抑制剂和/或拮抗剂基本上不被消化液分解和/或灭活。
  15. 药物组合物，其包含权利要求1-14中任一项所述的TLR4通路抑制剂和/或拮抗剂和任选地药学上可接受的载体。
  16. 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权利要求1-14中任一项所述的TLR4通路抑制剂和/或拮抗剂。
  17. 根据权利要求16所述的方法，其中所述施用包括经胃肠道施用。
  18. 根据权利要求16-17中任一项所述的方法，其中所述TLR4通路抑制剂和/或拮抗剂在胃肠道局部发挥效力。
  19. 根据权利要求16-18中任一项所述的方法，其中在施用后约1小时或之后，所述TLR4通路抑制剂和/或拮抗剂仍然以预防、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。
  20. 根据权利要求16-19中任一项所述的方法，其中在施用后约24小时或之后，至多50％的所述TLR4通路抑制剂和/或拮抗剂被所述受试者吸收而进入血液循环系统。
  21. 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经受胃肠道缺血再灌注之时或之后，向所述受试者施用TLR4通路抑制剂和/或拮抗剂。
  22. 根据权利要求21所述的方法，其中所述施用包括经胃肠道施用。
  23. TLR4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。
  24. 根据权利要求23所述的用途，其中所述药物抑制TLR4/TRAF6/NFκB通路的激活和/或信号传导。
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