CN113768943A

CN113768943A - Application of TLR4 pathway inhibitor in preparation of medicine

Info

Publication number: CN113768943A
Application number: CN202010525515.9A
Authority: CN
Inventors: 周宏伟; 徐开宇; 高徐璇; 尹恝; 何彦; 陈慕璇
Original assignee: Southern Medical University Zhujiang Hospital
Current assignee: Southern Medical University Zhujiang Hospital
Priority date: 2020-06-10
Filing date: 2020-06-10
Publication date: 2021-12-10
Also published as: WO2021249376A1

Abstract

The present application relates to the use of a TLR4 pathway inhibitor for the manufacture of a medicament for preventing, ameliorating and/or treating ischemia-reperfusion-related distal injury of the gastrointestinal tract in a subject. The application also relates to the use of TLR4 for screening a medicament for preventing, ameliorating and/or treating ischemia-reperfusion-related distal injury of the gastrointestinal tract in a subject. The application also relates to pharmaceutical compositions comprising the TLR4 pathway inhibitor and methods of preventing, ameliorating and/or treating distal injury associated with ischemia-reperfusion of the gastrointestinal tract in a subject.

Description

Application of TLR4 pathway inhibitor in preparation of medicine

Technical Field

The application relates to the field of biomedicine, in particular to application of a TLR4 pathway inhibitor in preparation of a medicament.

Background

Stroke is one of the most widespread diseases in the world today, the incidence rate of stroke increases year by year, and the life quality of stroke patients is reduced, and death is caused in severe cases. Worldwide, the annual incidence and mortality of ischemic stroke increased by 37% and 21%, respectively, from 1990 to 2010.

Currently, the treatment focus of stroke patients is intravenous thrombolysis or intravascular treatment, such as intravenous injection of recombinant tissue plasminogen activator (r-tPA), which is still considered to be the most important treatment. In addition, depending on the disease state of each patient, there are antiplatelet therapies, anticoagulants, neuroprotective agents and other symptomatic therapies, including the treatment and prevention of hyperglycemia, hypertension and acute stroke complications. However, in view of the increasingly severe forms of stroke, new therapeutic modalities, targets and drugs are urgently needed for further development.

Disclosure of Invention

The present application provides the use of an inhibitor of the TLR4 pathway in the manufacture of a medicament for preventing, ameliorating and/or treating distal injury associated with ischemia-reperfusion of the gastrointestinal tract in a subject.

In certain embodiments, the TLR4 pathway inhibitor has a structure according to formula I:

wherein G is¹Is a 4-to 12-membered heterocyclyl containing 1 to 3 heteroatoms independently selected from N, O and S, -NR¹R²，-OH，-OC_1-4Alkyl radical, C_3-8Cycloalkyl, or 5 to 12 membered heteroaryl comprising 1 to 3 heteroatoms independently selected from N, O and S; wherein, theSaid cycloalkyl, said heterocyclyl, said heteroaryl optionally substituted with 1 to 4 substituents, said 1 to 4 substituents independently selected from halogen, C_1-4Alkyl radical, C_1-4Haloalkyl, -OH, -OC_1-4Alkyl and OXO;

R¹and R²Each independently is hydrogen or C_1-4An alkyl group;

L¹is-C_1-4alkylene-O-, -C_1-5alkylene-or-C (o) -CH ═ CH-, wherein said-C_1-5Alkylene group of the formula_1-4alkylene-O-optionally substituted with 1 to 2 halogens or-OH, or L¹Comprises the following steps:

wherein C is_1-4Alkylene and G¹Bonded and imidazole in a position opposite to G²The phenyl groups of (a) are fused meta and para;

G²selected from (i) to (xiii):

R⁴is composed of

X¹Is O or S;

X²o, S, NH or NC_1-4An alkyl group;

R⁵is composed of

Or C_1-4An alkyl group;

R³、R⁷、R¹³，R¹⁷、R¹⁹、R²¹、R²⁷、R²⁹and R³³Each independently selected from hydrogen and C_1-4An alkyl group;

R¹¹and R³¹Each independently selected from hydrogen and C_1-4Alkyl and optionally substituted by 1-3 substituents independently selected from halogen, C_1-4Alkyl radical, C_1-4Haloalkyl, -OH and-OC_1-4Phenyl substituted with a substituent of alkyl;

R^4a、R^4b、R^4c、R^5a、R⁶、R⁸、R⁹、R¹⁰、R¹²、R¹⁴、R¹⁵、R¹⁶、R¹⁸、R²⁰、R²²、R²³、R²⁴、R²⁵、R²⁶、R²⁸、R³⁰and R³²Each occurrence is independently-OH, halogen, nitro, cyano, C_1-4Alkyl radical, C_1-4Haloalkyl, -OC_1-4Alkyl, -OC_1-4Haloalkyl, -NH₂、–NH(C_1-4Alkyl), -N (C)_1-4Alkyl) (C_1-4Alkyl), -NHC (O) C_1-4Alkyl, -N (C)_1-4Alkyl group(s) C (O) C_1-4Alkyl, -NHC (O) OC_1-4Alkyl, -N (C)_1-4Alkyl) C (O) OC_1-4Alkyl, -C (O) OC_1-4Alkyl, -C (O) OH, -C (O) NH₂、-C(O)NH(C_1-4Alkyl) or-C (O) N (C)_1-4Alkyl) (C_1-4Alkyl), and optionally two R^4a、R^4b、R^5a、R⁶、R⁸、R⁹、R¹⁰、R¹²、R¹⁴、R¹⁵、R¹⁶、R¹⁸、R²⁰、R²¹、R²²、R²⁴、R²⁵、R²⁶、R²⁸、R³⁰Or R³²Form fused rings 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

In certain embodiments, the TLR4 pathway inhibitor comprises oxpc, Eritoran, and/or TAK-242.

In certain embodiments, the TLR4 pathway inhibitors OxPAPC, Eritoran block binding of LPS to LBP and CD 14; TAK-242 binds to cysteine CYS747 of the intracellular domain of TLR4 and inhibits the TLR4 signaling pathway.

In certain embodiments, the medicament is formulated such that the TLR4 pathway inhibitor exerts its effect locally in the gastrointestinal tract.

In certain embodiments, the medicament is formulated such that, about 1 hour or after administration, the TLR4 pathway inhibitor is still present in the gastrointestinal tract local area in an amount effective to prevent, ameliorate and/or treat the distal injury associated with ischemia-reperfusion of the gastrointestinal tract.

In certain embodiments, the medicament is formulated such that at most 50% of the TLR4 pathway inhibitor in the medicament is absorbed by the subject into the blood circulation system about 24 hours or later after administration.

In certain embodiments, the concentration of the TLR4 pathway inhibitor in the medicament is from about 0.0001% (w/w) to about 90% (w/w).

In certain embodiments, the subject has, is having, or is at risk of having a disease or condition associated with ischemia-reperfusion of the gastrointestinal tract.

In certain embodiments, the disease or condition associated with ischemia reperfusion of the gastrointestinal tract comprises stroke, trauma, shock, sepsis, acute pancreatitis, or inflammatory bowel disease.

In certain embodiments, the disease associated with ischemia reperfusion of the gastrointestinal tract comprises ischemic stroke.

In certain embodiments, the subject has been, is, or is at risk of experiencing the gastrointestinal ischemia-reperfusion.

In certain embodiments, the distal injury associated with ischemia-reperfusion of the gastrointestinal tract comprises ischemic stroke.

In certain embodiments, the medicament is configured for oral administration.

In certain embodiments, the TLR4 pathway inhibitor is not substantially degraded and/or inactivated by digestive fluids.

The present application also provides a pharmaceutical composition comprising a TLR4 pathway inhibitor described herein and optionally a pharmaceutically acceptable carrier.

The present application also provides a method of preventing, ameliorating and/or treating distal injury associated with gastrointestinal ischemia-reperfusion in a subject comprising administering to the subject an inhibitor of the TLR4 pathway described herein.

In certain embodiments, the administering comprises gastrointestinal administration.

In certain embodiments, the TLR4 pathway inhibitor exerts its potency locally in the gastrointestinal tract.

In certain embodiments, the TLR4 pathway inhibitor is still present locally in the gastrointestinal tract in an amount effective to prevent, ameliorate, and/or treat the ischemia-reperfusion-associated distal injury of the gastrointestinal tract at about 1 hour or after administration.

In certain embodiments, at most 50% of the TLR4 pathway inhibitor is absorbed by the subject into the blood circulation system about 24 hours or after administration.

In certain embodiments, the TLR4 pathway inhibitor is administered at a dose of about 0.01 to 200mg/kg body weight.

The present application also provides a method of preventing distal injury associated with ischemia-reperfusion of the gastrointestinal tract in a subject, the method comprising:

a) monitoring a gastrointestinal condition of the subject;

b) administering a TLR4 pathway inhibitor to the subject when the monitoring indicates that the subject is at risk of experiencing gastrointestinal ischemia-reperfusion, or while or after experiencing gastrointestinal ischemia-reperfusion.

The present application also provides for the use of TLR4 for screening a medicament for preventing, ameliorating and/or treating ischemia-reperfusion-related distal injury of the gastrointestinal tract in a subject.

In certain embodiments, the agent inhibits activation and/or signaling of the TLR4/TRAF 6/nfkb pathway.

Other aspects and advantages of the present application will be readily apparent to those skilled in the art from the following detailed description. Only exemplary embodiments of the present application have been shown and described in the following detailed description. As those skilled in the art will recognize, the disclosure of the present application enables those skilled in the art to make changes to the specific embodiments disclosed without departing from the spirit and scope of the invention as it is directed to the present application. Accordingly, the descriptions in the drawings and the specification of the present application are illustrative only and not limiting.

Drawings

The specific features of the invention to which this application relates are set forth in the appended claims. The features and advantages of the invention to which this application relates will be better understood by reference to the exemplary embodiments described in detail below and the accompanying drawings. The brief description of the drawings is as follows:

FIG. 1 shows the cecal blood flow changes observed by the laser speckle imaging system (Ravorder RFLSI Pro) in mice of the present application.

FIG. 2 shows the statistics of the mouse cecal blood flow ROI propagation observed by the laser speckle imaging System (Rewold RFLSI Pro) in the present application.

FIG. 3 shows the change in expression level of TLR4 pathway-associated genes in colon tissue after ischemia reperfusion of gastrointestinal tract caused by ischemic stroke in the present application.

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification.

In the present application, the term "TLR 4," also known as Toll-like receptor 4, generally refers to a class of transmembrane proteins belonging to the Toll-like receptor family. TLR4 is generally a type I transmembrane proteinThey can be divided into extracellular, cytoplasmic and transmembrane regions, and typically respond to signals by complexing their extracellular leucine-rich repeat domain (LRR) or intracellular Toll/IL-1 receptor (TIR) domains with other molecules. Ligands for TLR4 may include viral proteins, polysaccharides, and endogenous proteins such as low density lipoproteins, beta-defensins, and heat shock proteins; TIR has homophilic interaction (homotrophic interaction), and after recognition of a signaling molecule, TLR4 can undergo a molecular conformational change to recruit a downstream signaling molecule containing TIR to form a signaling complex. TLR4 has a fundamental role in pathogen recognition and innate immune activation, e.g., TLR4 is able to mediate, in concert with LY96 and CD14, the innate immune response to bacterial Lipopolysaccharide (LPS) (Tatematsu M, etc., J Immunol.2016May 1; 196(9): 3865-76); for example, TLR4 may act through MYD88, TIRAP and TRAF6 to cause NF-. kappa.B activation, cytokine secretion and inflammatory response (Medzhitov R, etc., Nature.1997Jul 24; 388(6640): 394-7; Arbour NC, etc., Nat Genet.2000Jun; 25(2): 187-91; Tatematsu M, etc., J Immunol.2016May 1; 196(9): 3865-76); for example, from free fatty acids (e.g. palmitate) or Ni²⁺Triggered LPS-independent inflammatory responses (Schmidt M, etc., Nat Immunol.2010Sep; 11(9): 814-9); for example, NF-. kappa.B expression mediated by stimulation by Mycobacterium tuberculosis HSP70(dnaK) and HSP65(groEL-2) (Bulut Y, etc., J Biol chem.2005Jun3; 280(22): 20961-7); for example, TLR4 can bind to TLR6 to promote aseptic inflammation of monocytes/macrophages in response to oxidized low density lipoprotein (oxLDL) or amyloid β 42; for example, TLR4 is capable of binding negatively charged LDL (LDL)^-) And mediates LDL^-Induced cytokine release (Estruch M, etc., Atherosclersis.2013Aug; 229(2): 356-62). In humans, the gene encoding TLR4 is located on chromosome 9q33.1, and comprises 4 exons; in mice, the gene encoding TLR4 is located on chromosome 4C 1; 34.66cM, comprising 3 exons.

In the present application, the term "TLR 4 pathway" generally refers to an intracellular and/or extracellular signaling pathway capable of modulating the expression or activity of TLR4, and/or, triggered by the activation of TLR 4.

In the present application, the term "alkyl" generally refers to straight or branched chain saturated hydrocarbons. 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-dimethylpentyl, 2, 3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, and n-decyl.

In the present application, the term "alkylene" generally refers to a divalent group derived from a straight or branched chain saturated hydrocarbon. Including but not limited to-CH₂-、-CH₂CH₂-、-CH₂CH₂CH₂-、-CH₂CH(CH₃)CH₂-or CH₂CH(CH₃)CH(CH₃)CH₂-。

In the present application, the term "haloalkyl" generally means an alkyl group, as defined herein, in which one, two, three, four, five, six or seven hydrogen atoms are replaced with a halogen. For example, 2-fluoroethyl, difluoromethyl, trifluoromethyl, 2,2, 2-trifluoroethyl, 2,2, 2-trifluoro-1 or 1-dimethylethyl and the like.

In the present application, the term "heteroaryl" generally refers to an aromatic heterocycle. For example an aromatic ring containing at least one heteroatom selected from O, N or S. Heteroaryl groups may 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. A 5 membered monocyclic heteroaryl ring may comprise two double bonds and one, two, three or four heteroatoms as ring atoms, for example furyl, imidazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, 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. Bicyclic heteroaryls are 8-to 12-membered ring systems having a monocyclic heteroaryl fused to an aromatic, saturated or partially saturated carbocyclic ring or fused to a second monocyclic heteroaryl ring. For example benzofuranyl, benzooxadiazolyl, 1, 3-benzothiazolyl, benzimidazolyl, benzothienyl, indolyl, indazolyl, isoquinolyl, naphthyridinyl, oxazolopyridine, quinolinyl. The heteroaryl group may be attached to the parent molecule through any substitutable carbon atom or any substitutable nitrogen atom contained in the group.

In the present application, the term "cycloalkyl" generally refers to a carbocyclic ring that does not contain heteroatoms as ring atoms, and does not contain double bonds. For example cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl. The cycloalkyl group may be attached to the parent molecule through any substitutable carbon atom.

In the present application, the term "heterocycle" or "heterocyclic" generally refers to a ring system comprising at least one heteroatom as a ring atom, wherein the heteroatom may be selected from O, N or S. The heterocycle may be a monocyclic heterocycle, a fused bicyclic heterocycle, or a spirocyclic heterocycle. Monocyclic heterocycles include, but are not limited to, azetidinyl, diazacyclopentyl, 1, 3-dioxolanyl, 1, 4-dioxolanyl, 1, 3-dioxolanyl, 4, 5-dihydroisoxazol-5-yl, 3, 4-dihydropyranyl, 1, 3-dithiothienyl, 1, 3-dithienyl, imidazolinyl, isothiazolinyl, isoxazolinyl, morpholinyl, oxadiazolinyl, oxazolinyl, piperazinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothienyl, thiadiazolinyl, thiazolinyl, thiazolidinyl, thiomorpholinyl, 1, 1-dioxothiomorpholinyl, thiopyranyl and trithioenyl.

The fused bicyclic heterocycle generally refers to a 7-12 membered ring system having a monocyclic heterocycle fused to a phenyl, saturated or partially saturated carbocycle, or to another monocyclic heterocycle or to a monocyclic heteroaryl ring. For example, 1, 3-benzodioxazol-4-yl, 1, 3-benzodithiol, 3-azabicyclo [3.1.0] hexyl, hexahydro-1H-furo [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-tetrahydroquinolyl.

In the present application, the term "OXO" generally refers to an oxygen atom bonded to a parent molecular moiety. The OXO can be linked to a carbon or sulfur atom by a double bond, or to a nitrogen atom by a single bond (e.g. an N-oxide).

In the present application, terms such as "alkyl," "cycloalkyl," "alkylene," and the like may be preceded by a designation to indicate the number of atoms present in a group at a particular instance, e.g., C, as would be known to one skilled in the art_1-4Alkyl radical, C_3-6Cycloalkyl radical, C_1-4Alkylene, etc., the subscript number following "C" indicates the number of carbon atoms present in the group. E.g. C₃Alkyl refers to alkyl having three carbon atoms (i.e., n-propyl, isopropyl); c_1-4The members of the group may have any number of carbon atoms falling within the range of 1-4.

In the present application, the term "ischemia" generally refers to a condition in which the tissue or organ is under-supplied with blood, resulting in hypoxia and nutrients. Ischemia can generally be caused by vascular problems, such as vascular embolism, vascular compression; it may also be caused by ischemia due to vasoconstriction, thrombosis or embolism, by accidental trauma, surgical intervention, or by disease of other organs or tissues, such as ischemic disease of other organs or tissues, such as the gastrointestinal ischemia resulting from ischemic stroke in this application.

In the present application, the term "ischemia reperfusion" generally refers to the process of blood supply back to tissue after ischemia or hypoxia (hypoxia or hypoxia). In other words, ischemia-reperfusion generally refers to the process by which the tissue or organ in which ischemia occurs resumes blood flow after the cause of ischemia is eliminated/counteracted/compensated/slowed. In certain embodiments, reperfusion of ischemic tissue may generally be accompanied by the occurrence of any of the following: reperfusion site microvascular system damage, for example due to increased permeability of capillaries and arterioles, resulting in increased fluid filtration and diffusion in the tissue; activated endothelial cells produce more reactive oxygen species or free radicals after reperfusion leading to a subsequent inflammatory response; the new return blood transports the white blood cells from the blood to the reperfusion area, where they respond to tissue damage to release inflammatory factors such as interleukins and free radicals, and may also bind to the endothelium of small capillaries, blocking them and leading to new ischemia; on the other hand, the restored blood flow reintroduces oxygen into the tissue, which in this particular case may destroy the proteins, nucleic acids and plasma membrane structures of the cells, and the reactive substances thus produced may act indirectly on redox signaling to initiate apoptosis, and damage to the cell membrane may further lead to the release of more free radicals. The free radical may comprise a nitroxide radical, such as nitric oxide or a derivative thereof. Ischemia reperfusion usually leads to reperfusion injury, such as cerebral infarction, acute myocardial infarction, brain no-reflow phenomenon after cardiopulmonary resuscitation, stress ulcer, pancreatitis, burn, transplantation of isolated organs, intestinal ischemia, necrotizing enterocolitis, intermittent claudication, acute tubular necrosis, liver failure after shock, and multi-system organ failure. When the site of the reperfusion injury occurs at a different site than the tissue or organ that has been ischemia-reperfused, it may be generally referred to as a distal reperfusion injury.

In the present application, the term "distal injury" generally refers to a pathophysiological process involving an organ or tissue different from a local organ or tissue that has been ischemia-reperfused. This process may often result from exposure of cellular products produced at the site of ischemia-reperfusion organ or tissue to other organs via the circulatory system. In certain embodiments, the distal injury may comprise a disease, condition, or medical intervention that causes the ischemia-reperfusion, e.g., stroke, trauma, shock, sepsis, acute pancreatitis, inflammatory bowel disease, or brain trauma surgery. In certain embodiments, the ischemia reperfusion may comprise gastrointestinal ischemia reperfusion. In certain embodiments, the distal injury may comprise an injury from ischemic stroke associated with ischemia-reperfusion of the gastrointestinal tract. For example, ischemic stroke damage associated with ischemia reperfusion of the gastrointestinal tract caused by ischemic stroke.

In the present application, the term "ischemia-reperfusion-related distal injury of the gastrointestinal tract" generally refers to a pathophysiological process of an organ or tissue different from the part of the gastrointestinal tract where ischemia-reperfusion occurs. Such pathophysiological processes are associated with ischemia-reperfusion of the gastrointestinal tract, and may for example be manifested as pathophysiological processes of organs or tissues different from the gastrointestinal tract site that occur simultaneously with, before or after the ischemia-reperfusion of the gastrointestinal tract. For example, when intervening on gastrointestinal ischemia-reperfusion or a gastrointestinal ischemia-reperfusion-related effect, the pathophysiological process of the organ or tissue exhibits a corresponding change, e.g., a reduction or alleviation of its symptoms. For example, administration of a TLR4 pathway inhibitor based on gastrointestinal ischemia reperfusion in the present application reduces the symptoms of ischemic stroke.

In the present application, the term "inhibitor" generally refers to a compound/substance or composition that is capable of preventing or reducing, in whole or in part, the physiological function of one or more specific proteins. The reduction in physiological function of one or more specific proteins may comprise a reduction in the activity of the protein itself or a reduction in the amount of the protein itself present, which would normally result in inhibition of the physiological function in which it is involved. For example, in the present application the inhibitor is capable of binding to TLR4 and thereby blocking TLR4 from binding to its activator to be activated, or to a downstream signaling pathway molecule and thereby unable to mediate signaling. In certain embodiments, the inhibitors may exist as distinct crystalline, amorphous materials, pharmaceutically acceptable salts, hydrates, and solvates.

In the present application, the term "administering" generally refers to introducing the inhibitor into the body of a subject by any route of introduction or delivery. Any method known to those skilled in the art for contacting a cell, organ or tissue with the inhibitor may be employed. Including but not limited to intra-arterial, intranasal, intra-abdominal, intravenous, intramuscular, subcutaneous, transdermal, or oral. A daily dose may be divided into one, two or more doses of suitable form to be administered at one, two or more times during a certain period of time.

In the present application, the term "effective amount" or "effective dose" generally refers to an amount sufficient to achieve, or at least partially achieve, a desired effect. A "therapeutically effective amount" or "therapeutically effective dose" of a drug or therapeutic agent is generally any amount of drug that promotes disease regression, as evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of the asymptomatic phase of the disease, or the prevention of damage or disability due to the development of the disease, when used alone or in combination with another therapeutic agent. A "prophylactically effective amount" or "prophylactically effective dose" of a drug generally refers to an amount of the drug that inhibits the development or recurrence of a disease when administered, alone or in combination with another therapeutic agent, to a subject at risk of disease development or disease recurrence. The ability of a therapeutic or prophylactic agent to promote disease regression or inhibit disease progression or recurrence can be assessed using a variety of methods known to those skilled in the art, such as predicting efficacy in humans in a human subject during clinical trials, in animal model systems, or by assaying the activity of the agent in an in vitro assay.

In the present application, the term "stroke" generally refers to an acute cerebrovascular disorder, also known as "stroke", "cerebrovascular accident (CVA)". Stroke can be a group of diseases that cause damage to brain tissue due to sudden rupture of a cerebral vessel or the inability of blood to flow into the brain due to vessel occlusion, including ischemic stroke and hemorrhagic stroke.

In the present application, the term "ischemic stroke" generally refers to a group of diseases that result in dysfunction of neural tissue in a specific region of the brain or a wide range of brain tissue regions due to insufficient blood supply to the region. 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. Wherein sickle blood cells clot more readily than normal blood cells, impeding blood flow to the brain; vascular compression may lead to cerebral ischemia by blocking the arteries that carry oxygen into the brain, the cause of vascular compression such as tumors; ventricular tachycardia can cause the heart to stop fully, resulting in blood flow cessation, and arrhythmia can also cause the formation of blood clots, resulting in cerebral ischemia; cerebral ischemia may also result from arterial occlusion due to arterial plaque build-up, which in the case of small plaque build-up can also result in narrowing of the channel prone to thrombus formation, leading to cerebral ischemia; blood coagulation dysfunction, large blood clots can also cause cerebral ischemia by preventing blood flow; heart attacks may also cause cerebral ischemia, heart attacks may slow blood flow, blood may begin to clot and stop blood flow to the brain; there is a correlation between heart attack and hypotension, and improper use and response to drugs can also lead to too low a blood pressure, which often represents inadequate oxygenation of the tissue. In the present application, ischemic stroke can also be achieved by surgically blocking arterial blood flow in the middle cerebral artery of a mouse. The neural tissue dysfunction may include neuronal death. The main symptoms due to dysfunction of nerve tissue in different areas may include: changes in smell/taste/hearing or vision, disorders of swallowing/pupil light response, physical movement disorders, aphasia, changes in breathing and heart rate, ischemia of other tissues or organs, loss of consciousness, and the like.

In this application, the term "digestive fluid" generally refers to a fluid secreted by the digestive system that acts to digest food. The digestive juice mainly comprises organic matters, ions and water. The main functions of the digestive juices may include: diluting the food to make it equal to the osmotic pressure of the plasma for absorption; changing the pH value in the digestive cavity to adapt to the requirement of digestive enzyme activity; hydrolyzes complex food ingredients to facilitate absorption; by secreting mucus, antibodies and large amounts of fluid, the digestive tract mucosa is protected against physical and chemical damage. The digestive juices may include the following: saliva, gastric juice, pancreatic juice, bile, intestinal juice, etc.

In the present application, the term "prevention" generally refers to prophylactic administration of a combination to a healthy subject to prevent the occurrence of a certain disease or disorder. It may also comprise prophylactic administration of the combination to a patient at a pre-stage of the allergic disease to be treated. "preventing" does not require 100% elimination of the likelihood of the occurrence of the disease or disorder, in other words "preventing" generally means that the likelihood or extent of occurrence of the disease or disorder is reduced in the presence of the administered combination.

In this application, the term "alleviating" refers to reducing, diminishing or delaying a condition, disease, disorder or phenotype. The condition, disease, disorder or phenotype may include subjective perception by the subject, such as pain, dizziness or other physiological disorder, or medically detectable indication, such as a disease condition detected by medical testing means.

In the present application, the term "treatment" generally refers to clinical intervention to alter the natural course of the treated individual or cell in the course of clinical pathology. May include improving the disease state, eliminating the lesion, or improving prognosis.

In the present application, the term "gastrointestinal administration" generally refers to allowing for the specific release of a therapeutic agent at or near a site in the gastrointestinal tract. By releasing the therapeutic agent locally rather than systemically, the bioavailability of the drug may be increased at the site of the gastrointestinal tract and/or decreased in the systemic circulation, and lower systemic drug levels may lead to reduced toxicity and reduced immunogenicity (e.g. in the case of biologics), leading on the one hand to improved overall safety and fewer adverse side effects, and on the other hand to increased dosages at the site of the gastrointestinal tract, leading to more efficient, more targeted effects. In some cases, topical administration of therapeutic agents also provides a novel mode of action, e.g., co-administration of different local sites.

In the present application, the term "exert its effect locally in the gastrointestinal tract" generally means that the therapeutic agent administered is capable of maintaining an effective dose locally in the gastrointestinal tract and of altering the condition or phenotype locally in the gastrointestinal tract. Local efficacy in the gastrointestinal tract does not preclude the observation of the therapeutic agent in organs or tissues other than local to the gastrointestinal tract. Generally, the therapeutic agent may have stability at the site of the gastrointestinal tract and/or tissue penetration (ability to penetrate into the tissue of the gastrointestinal tract) at the site of the gastrointestinal tract, and may be substantially distributed in the tissue of the gastrointestinal tract. Such stability may include stability before and/or after administration, e.g., stability within the delivery device, stability of the formulation and/or drug in the gastrointestinal environment after administration, including the gastrointestinal environment of the disease state, e.g., temperature stability, pH stability, oxidative stability. The portion of the gastrointestinal tract may comprise a portion or sub-portion of one or more sites of the gastrointestinal tract of the subject.

In this application, the term "subject" generally refers to a human or non-human animal, including but not limited to a cat, dog, horse, pig, cow, sheep, rabbit, mouse, rat, or monkey.

In the present application, the term "pharmaceutical composition" generally refers to a mixture comprising at least one active ingredient to be administered to a subject for the treatment of a specific disease or condition affecting the individual. Which allows the active ingredient to be in an effective form and which does not contain additional components having unacceptable toxicity to the subject to which the composition is to be administered. Such compositions may be sterile and may contain a pharmaceutically acceptable carrier.

In the present application, the term "pharmaceutically acceptable carrier" generally refers to a pharmaceutically acceptable substance, composition or vehicle involved in carrying or transporting a chemical agent. Such as buffers, surfactants, stabilizers, preservatives, absorption enhancers to enhance bioavailability, liquid or solid fillers, diluents, excipients, solvents, encapsulating materials, and/or other conventional solubilizing or dispersing agents.

In the present application, the term "comprising" is generally intended to include the explicitly specified features, but not to exclude other elements.

In the present application, the term "about" generally means varying from 0.5% to 10% above or below the stated value, for example, varying from 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%, or 10% above or below the stated value.

In one aspect, the present application provides the use of an inhibitor of the TLR4 pathway in the manufacture of a medicament for preventing, ameliorating and/or treating ischemia-reperfusion-related distal injury of the gastrointestinal tract in a subject.

The present application also provides for the use of an inhibitor of the TLR4 pathway in the manufacture of a medicament for preventing, ameliorating and/or treating a neurological-related disease or disorder in a subject.

For example, the TLR4 pathway may include the TLR4/TRAF 6/nfkb pathway.

In the present application, the medicament is formulated such that the TLR4 pathway inhibitor exerts its effect locally in the gastrointestinal tract.

For example, the gastrointestinal tract may locally comprise the stomach, small intestine, large intestine sites. The stomach may include the cardia portion, fundus, body, pylorus portion of the stomach. The small intestine may comprise the duodenum, jejunum, ileum. The large intestine may comprise the cecum, colon, rectum.

For example, the medicament is formulated such that, about 1 hour or after administration, the TLR4 pathway inhibitor is still present in the gastrointestinal tract as a local part thereof in an amount effective to prevent, ameliorate and/or treat the ischemia-reperfusion-related distal injury of the gastrointestinal tract.

For example, the medicament is formulated such that at most 70% of the TLR4 pathway inhibitor in the medicament is absorbed by the subject into the blood circulation system about 24 hours or later after administration.

For example, the local effect in the gastrointestinal tract may comprise that the TLR4 pathway inhibitor inhibits TLR4 activation or TLR4 binding to its downstream signaling molecule locally in the gastrointestinal tract.

For example, the local effect in the gastrointestinal tract may comprise a decrease in the expression level of TLR4 and/or a signaling molecule downstream thereof in intestinal tissue.

For example, the expression level may comprise an mRNA level.

For example, the downstream signaling molecule may include adaptor protein (MYD88), tumor necrosis factor receptor-related factor 6(TRAF6), nuclear factor kb (nfkb).

For example, the local effect in the gastrointestinal tract may comprise a change in the expression level of an intestinal barrier-associated gene in intestinal tissue.

For example, the intestinal barrier-associated genes may include Tjp1, Ocln, Cldn 2. Wherein Tjp1 is Claudin 1, Ocln is Ocplus, Cldn2 is Claudin-2.

For example, said local effect in the gastrointestinal tract may comprise an increase in Tjp1, Ocln expression in intestinal tissue.

For example, the local effect in the gastrointestinal tract may comprise a decrease in Cldn2 expression in intestinal tissue.

For example, the local effect in the gastrointestinal tract may comprise causing a decrease in the expression level of a proinflammatory cytokine gene in intestinal tissue, the proinflammatory cytokine gene being selected from any one of the group consisting of: tnf, Il17, Ifng, Il1b, Il6, Cxcl2 and Kc; wherein, Tnf is tumor necrosis factor, Il17 is interleukin 17, Ifng is interferon gamma, Il1b is interleukin 1 beta, Il6 is interleukin 6, and Kc and Cxcl2 are chemokine members.

For example, the intestinal tissue may comprise the jejunum, ileum, caecum or colon.

In the present application, the concentration of the TLR4 pathway inhibitor in the medicament is from about 0.0001% (w/w) to about 90% (w/w).

For example, the concentration of the TLR4 pathway inhibitor is about 0.0005% (w/w) to about 90% (w/w), about 0.001% (w/w) to about 85% (w/w), about 0.0015% (w/w) to about 80% (w/w), about 0.002% (w/w) to about 75% (w/w), about 0.0025% (w/w) to about 70% (w/w), about 0.003% (w/w) to about 65% (w/w), about 0.0035% (w/w) to about 60% (w/w), about 0.004% (w/w) to about 55% (w/w), about 0.0045% (w/w) to about 50% (w/w), about 0.005% (w/w) to about 45% (w/w), about 0.0055% (w/w) to about 40% (w/w), about 0.006% (w/w) to about 35% (w/w), about 0.0065% (w/w) to about 30% (w/w), about 0.007% (w/w) to about 25% (w/w), about 0.0075% (w/w) to about 20% (w/w), about 0.01% (w/w) to about 80% (w/w), about 0.1% (w/w) to about 70% (w/w), about 0.5% (w/w) to about 60% (w/w), about 1% (w/w) to about 50% (w/w), about 5% (w/w) to about 90% (w/w), about 10% (w/w) to about 80% (w/w), about 20% (w/w) to about 70% (w/w), about 10% (w/w) to about 50% (w/w), about 20% (w/w) to about 50% (w/w), about 20% (w/w) to about 40% (w/w). About 30% (w/w) to about 50% (w/w), about 10% (w/w) to about 20% (w/w).

In the present application, the subject has, is suffering from, or is at risk of suffering from a disease and/or condition associated with ischemia-reperfusion of the gastrointestinal tract.

In the present application, the diseases and/or conditions associated with ischemia-reperfusion of the gastrointestinal tract may comprise natural events, trauma, or one or more surgical or other therapeutic interventions that reduce/prevent blood flow to the gastrointestinal tract. Wherein the natural event may comprise an arterial infarction, a venous occlusion, or systemic hypotension that disrupts or reduces blood flow to internal organs, which systemic hypotension may include hemorrhagic shock due to blood loss, cardiogenic shock due to myocardial infarction or heart failure, neurogenic shock, nephrogenic shock, or anaphylaxis.

For example, the disease and/or condition associated with ischemia reperfusion of the gastrointestinal tract may include stroke, trauma, shock, sepsis, acute pancreatitis, and/or inflammatory bowel disease.

For example, the disease associated with ischemia reperfusion of the gastrointestinal tract may comprise stroke. The stroke may comprise a group of diseases that cause damage to brain tissue due to sudden rupture of cerebral vessels or due to blood flow loss into the brain or decreased blood flow into the brain due to vessel occlusion.

For example, the disease may comprise ischemic stroke. The ischemic stroke may comprise atherosclerotic occlusion of the aorta, cerebral embolism (embolic infarction), non-embolic infarction of small deep perforator arteries (lacunar infarction), and watershed area ischemia resulting from distal arterial stenosis and decreased cerebral blood flow (hemodynamic stroke).

In the present application, the subject has been suffering from the above-mentioned diseases or disorders associated with ischemia-reperfusion of the gastrointestinal tract. Said administering may comprise administering said drug to said subject prior to said subject suffering from said disease associated with ischemia-reperfusion of the gastrointestinal tract.

In the present application, the subject is suffering from the above-mentioned diseases or disorders associated with ischemia-reperfusion of the gastrointestinal tract. The subject is suffering from the above-mentioned disease associated with ischemia-reperfusion of the gastrointestinal tract while the administration of the drug to the subject is being comprised.

In the present application, the subject is at risk for the above-mentioned diseases or disorders associated with ischemia-reperfusion of the gastrointestinal tract. The risk may comprise that the patient may suffer from the above-mentioned diseases associated with ischemia-reperfusion of the gastrointestinal tract after administration of the drug to the subject.

In the present application, the subject has been, is, or is at risk of experiencing the gastrointestinal ischemia-reperfusion.

For example, the subject has experienced the gastrointestinal ischemia reperfusion. Said administering may comprise subjecting said subject to said gastrointestinal ischemia reperfusion prior to administering said drug to said subject.

For example, the subject is undergoing the gastrointestinal ischemia-reperfusion. The subject is undergoing the gastrointestinal ischemia reperfusion while the administering the drug to the subject may be comprised.

For example, the subject is at risk of experiencing the gastrointestinal ischemia-reperfusion. The risk may comprise the subject experiencing the gastrointestinal ischemia reperfusion after administration of the drug to the subject.

For example, the distal injury associated with ischemia reperfusion of the gastrointestinal tract comprises ischemic stroke.

In the present application, the medicament is configured to be suitable for oral administration. For example orally.

For example, oral dosage forms may include capsules, tablets, pills, granules, or syrups.

For example, an oral dose may be administered in a dosage range of from about 0.01 to 200mg/kg body weight, from about 0.01 to 180mg/kg, from about 0.01 to 160mg/kg, from about 0.01 to 140mg/kg, from about 0.01 to 120mg/kg, from about 0.01 to 100mg/kg, from about 0.1 to 200mg/kg, from about 0.1 to 150mg/kg, from about 0.1 to 100mg/kg, from about 0.1 to 80mg/kg, from about 1 to 60mg/kg, from about 0.1 to 40mg/kg, from about 0.1 to 20mg/kg, 25-200mg/kg, about 50-200mg/kg, about 100mg/kg, about 25-50mg/kg, about 25-100mg/kg, about 50-200 mg/kg.

For example, the oral regimen may include taking 1 or more times daily, every 1 day, weekly, biweekly, monthly, or bimonthly.

In the present application, the TLR4 pathway inhibitor is not substantially degraded and/or inactivated by digestive fluids. The digestive fluid may comprise saliva, gastric juice, intestinal juice, pancreatic juice, bile.

For example, saliva, the pH value of which is 6.6-7.1, mainly comprises saliva amylase, lysozyme, a small amount of inorganic substances (such as inorganic salts containing sodium, potassium and calcium) and the like, and the TLR4 pathway inhibitor is not substantially decomposed and/or inactivated by the saliva.

For example, gastric juice has a pH of 0.9 to 1.5, and contains pepsin, gastric acid (i.e., hydrochloric acid), mucus, and inorganic substances such as sodium salt and potassium salt as main components, and the TLR4 pathway inhibitor is not substantially decomposed and/or inactivated by the gastric juice.

For example, pancreatic juice, which has a pH of 7.8 to 8.4, contains sodium bicarbonate, pancreatic amylase, pancreatic lipase, trypsinogen, chymotrypsinogen, and the like as main components, and the TLR4 pathway inhibitor is not substantially decomposed and/or inactivated by the pancreatic juice.

For example, bile, having a pH of about 6.8 to 7.4, and having as major components bile salts and bile pigments, the TLR4 pathway inhibitor is not substantially degraded and/or inactivated by the bile.

For example, a small intestinal fluid, having a pH of about 7.6, comprising a plurality of digestive enzymes such as amylase, maltase, sucrase, lactase, peptidase, lipase, and the like, the TLR4 pathway inhibitor is not substantially degraded and/or inactivated by the small intestinal fluid.

For example, the TLR4 pathway inhibitor is not substantially degraded and/or inactivated by the saliva, gastric fluid, pancreatic juice, bile, intestinal fluid.

For example, the TLR4 pathway inhibitor is not substantially degraded and/or inactivated at phs of 6.6-7.1, 0.9-1.5, 7.8-8.4, 6.8-7.4, 7.6.

The TLR4 pathway inhibitor is not substantially broken down and/or inactivated by digestive fluids may include the TLR4 pathway inhibitor being capable of substantially maintaining the function of inhibiting TLR4 in or after contact with the digestive fluids. E.g., functions that bind to TLR 4.

In another aspect, the present application also provides a pharmaceutical composition that may comprise a TLR4 pathway inhibitor as described herein and optionally a pharmaceutically acceptable carrier.

For example, the pharmaceutical composition may comprise a health care product.

The health care product is a product which has the common characteristics of common food, can regulate the functions of a human body, is suitable for specific people to eat, and does not aim at treating diseases, and is also called as a dietary supplement. Comprises tea, wine, bee products, drinks, soup, fresh juice, medicated diet and the like.

For example, the pharmaceutical composition may include a biologic.

The biological product is prepared by applying common or biological materials such as microorganisms and cells obtained by biotechnology such as genetic engineering, cell engineering, protein engineering, fermentation engineering and the like, tissues and liquid of various animal and human sources and the like, and is used for preventing, treating and diagnosing human diseases. Including bacterins, vaccines, toxins, toxoids, blood products, immunoglobulins, antigens, allergens, cytokines, hormones, enzymes, fermentation products, monoclonal antibodies, recombinant DNA products, and the like.

The pharmaceutically acceptable carrier may comprise a pharmaceutically acceptable substance, composition or vehicle involved in carrying or transporting the chemical agent, such as buffers, surfactants, stabilizers, preservatives, absorption enhancers for enhancing bioavailability, liquid or solid fillers, diluents, excipients, solvents, encapsulating materials, and/or other conventional solubilizing or dispersing agents.

In the present application, the pharmaceutical composition may comprise any one or any combination of the following components: acacia, alginate, alginic acid, aluminium acetate, benzyl alcohol, butyl p-hydroxybenzoate, butylated hydroxytoluene, antioxidants, citric acid, calcium carbonate, candelilla wax, croscarmellose sodium, fructose, colloidal silicon dioxide, cellulose, carnauba wax, corn starch, carboxymethylcellulose calcium, calcium stearate, calcium disodium EDTA, copovidone, hydrogenated castor oil, dibasic calcium phosphate dehydrate, cetylpyridinium chloride, cysteine HC1, crospovidone, dibasic calcium phosphate, dibasic sodium phosphate, polydimethylsiloxane, sodium erythorbate, ethylcellulose, gelatin, glyceryl monooleate, glycerol, glycine, glyceryl monostearate, glyceryl dibutyrate, hydroxypropylcellulose, hydroxypropylmethylcellulose, HPMC phthalate, iron oxide, yellow iron oxide, lactose (aqueous or anhydrous or monohydrate or spray-dried), magnesium stearate, microcrystalline cellulose, mannitol, methylcellulose, magnesium carbonate, mineral oil, methacrylic acid copolymers, magnesium oxide, methyl paraben, PEG, polysorbate 80, propylene glycol, polyethylene oxide, propyl paraben, poloxamer 407 or 188, potassium bicarbonate, potassium sorbate, starch, phosphoric acid, polyoxyethylene 40 stearate, sodium starch glycolate, pregelatinized starch, croscarmellose sodium, sodium lauryl sulfate, silicon dioxide, sodium benzoate, stearic acid, syrups for pharmaceutical candies, granulating agents, sorbic acid, sodium carbonate, sodium saccharin, sodium alginate, silica gel, sorbitol monooleate, sodium stearyl fumarate, sodium chloride, sodium metabisulfite, sodium citrate dehydrate, carboxymethylcellulose sodium, succinic acid, sodium propionate, titanium dioxide, talc, triacetin, triethyl citrate.

In the present application, the pharmaceutical composition may be in solid form, for example, capsules, tablets, pills, granules, sachets or lozenges; or may be in liquid form, such as a solution, suspension, emulsion or syrup.

In another aspect, the present application also provides a method of preventing, ameliorating and/or treating a distal injury associated with ischemia-reperfusion of the gastrointestinal tract in a subject, the method comprising administering to the subject an inhibitor of the TLR4 pathway described herein.

The present application also provides a method of preventing, ameliorating and/or treating a nervous system related disease or disorder in a subject comprising administering to the subject a TLR4 pathway inhibitor as described herein.

For example, the administration includes gastrointestinal administration. The gastrointestinal administration comprises specific release of the TLR4 pathway inhibitor at or near a site in the gastrointestinal tract.

For example, the TLR4 pathway inhibitor is delivered to the gastrointestinal tract by a mechanical device.

For example, the TLR4 pathway inhibitor is delivered to the gastrointestinal tract via an endoscope, a spray catheter.

For example, the TLR4 pathway inhibitor is released in the gastrointestinal tract by oral sustained release, disintegrating agents.

For example, the sustained release agent, disintegrant, can initiate release or disintegrate depending on the pH of the gastrointestinal tract.

For example, the TLR4 pathway inhibitor is released in the gastrointestinal tract by enteric-coated capsules, liposomal microcapsules.

For example, the TLR4 pathway inhibitor is delivered to the gastrointestinal tract by gavage.

For example, the time period for gastrointestinal administration is about 1-7 days, about 1-6 days, about 1-5 days, about 1-4 days, about 1-3 days, about 1-2 days, 1-24 hours, 1-12 hours, about 3-12 hours, about 6-12 hours, about 1-3 hours, about 1-6 hours, about 3-12 hours, about 6-12 hours after stroke.

For example, the TLR4 pathway inhibitor exerts its effect locally in the gastrointestinal tract.

For example, the TLR4 pathway inhibitor is still present in the gastrointestinal tract as an effective amount to prevent, ameliorate and/or treat the distal injury caused by ischemia-reperfusion of the gastrointestinal tract at about 1 hour or after administration.

For example, about 24 hours or later after administration, up to 50% of the TLR4 pathway inhibitor is absorbed by the subject into the blood circulation system.

For example, the expression level may comprise an mRNA level.

For example, the TLR4 pathway inhibitor is administered at a dose of from about 0.01 to 200mg/kg body weight, from about 0.01 to 180mg/kg, from about 0.01 to 160mg/kg, from about 0.01 to 140mg/kg, from about 0.01 to 120mg/kg, from about 0.01 to 100mg/kg, from about 0.1 to 200mg/kg, from about 0.1 to 150mg/kg, from about 0.1 to 100mg/kg, from about 0.1 to 80mg/kg, from about 1 to 60mg/kg, from about 0.1 to 40mg/kg, from about 0.1 to 20mg/kg, 25-200mg/kg, about 50-200mg/kg, about 100mg/kg, about 25-50mg/kg, about 25-100mg/kg, about 50-200 mg/kg.

In another aspect, the present application also provides a method of preventing ischemia reperfusion-related distal injury of the gastrointestinal tract in a subject, the method comprising: (1) monitoring a gastrointestinal condition of the subject; (2) administering a TLR4 pathway inhibitor to the subject when or after the monitoring shows that the subject is experiencing gastrointestinal ischemia reperfusion.

In another aspect, the present application also provides a method of preventing a nervous system-related disease or disorder in a subject, the method comprising: (1) monitoring a gastrointestinal condition of the subject; (2) administering a TLR4 pathway inhibitor to the subject when or after the monitoring shows that the subject is experiencing gastrointestinal ischemia reperfusion.

For example, the method of preventing distal injury caused by ischemia-reperfusion of the gastrointestinal tract in a subject further comprises monitoring a disease or condition associated with ischemia-reperfusion of the gastrointestinal tract prior to step (1).

For example, the method of preventing a neurological-related disease or disorder in a subject further comprises monitoring the disease or disorder associated with ischemia-reperfusion of the gastrointestinal tract prior to step (1).

For example, the detecting the gastrointestinal condition of the subject comprises clinical screening, clinical examination and/or clinical examination, and assessing the gastrointestinal condition of the subject.

For example, upon monitoring that a patient has, is, or is at risk of experiencing a disease or condition associated with ischemia-reperfusion of the gastrointestinal tract, the patient is clinically challenged, examined and examined to assess the gastrointestinal condition of the subject.

For example, the clinical physical examination may include observing the patient's appetite, the presence or absence of dysphagia, the presence or absence of abdominal pain, nausea, vomiting, hematemesis, hematochezia, stool characteristics, the presence or absence of symptoms including abdominal pain, abdominal distension during defecation, and changes in defecation habits, and abdominal physical examination.

For example, clinical trials include three major routines, and the like.

For example, clinical examinations may include abdominal X-ray, ultrasound, CT, endoscopy, ERCP, PTC, and the like.

For example, monitoring the gastrointestinal condition of the subject may comprise observing a change in blood flow at a gastrointestinal site.

For example, the administration includes gastrointestinal administration.

For example, the TLR4 pathway inhibitor agent is released in the gastrointestinal tract by enteric-coated capsules, liposomal microcapsules.

In another aspect, the present application also provides the use of TLR4 for screening for a medicament for preventing, ameliorating and/or treating ischemia-reperfusion-related distal injury of the gastrointestinal tract in a subject.

The present application also provides the use of a TLR4 pathway inhibitor for screening for a medicament for preventing, ameliorating and/or treating a neurological-related disease or disorder in a subject.

In the present application, the screening of a drug may comprise a process of evaluating the biological activity, pharmacological effect and medicinal value of a substance that may be used as a drug. The screening for drugs may comprise screening at a biochemical level and a cellular level. The drug selection may also include high throughput screening and virtual drug screening.

For example, the drug inhibits the activation of TLR4 or the binding of TLR4 to its downstream signaling molecule.

For example, the drug inhibits activation and/or signaling of the TLR4/TRAF 6/nfkb pathway.

For example, the drug suppresses the binding of Lipopolysaccharide (LPS) to Lipopolysaccharide Binding Protein (LBP) and/or cluster of differentiation 14 protein (CD 14).

For example, the agent is capable of binding to cysteine CYS747 of the intracellular domain of TLR 4.

In the present application, the TLR4 pathway inhibitor is capable of inhibiting the activation of TLR4 or the binding of TLR4 to its downstream signaling molecule.

In the present application, the TLR4 pathway inhibitor includes inhibiting the activation of TLR4 or the binding of TLR4 to its downstream signaling molecule by binding to TLR 4.

For example, the TLR4 pathway inhibitor is capable of causing a decrease in the expression level of TLR4 and/or a signaling molecule downstream thereof.

For example, the expression level may comprise an mRNA level.

For example, the TLR4 pathway inhibitor is capable of inhibiting the activation and/or signaling of the TLR4/TRAF 6/nfkb pathway.

For example, the TLR4 pathway inhibitor is capable of blocking the binding of Lipopolysaccharide (LPS) to Lipopolysaccharide Binding Protein (LBP) and/or cluster of differentiation 14 protein (CD 14).

For example, the TLR4 pathway inhibitor is capable of binding to cysteine CYS747 of the intracellular domain of TLR 4.

In the present application, the TLR4 pathway inhibitor may have a structure represented by formula I:

wherein G is¹Is a 4-to 12-membered heterocyclyl containing 1 to 3 heteroatoms independently selected from N, O and S, -NR¹R²，-OH，-OC_1-4Alkyl radical, C_3-8Cycloalkyl, or 5 to 12 membered heteroaryl comprising 1 to 3 heteroatoms independently selected from N, O and S; wherein said cycloalkyl, heterocyclyl, heteroaryl are optionally substituted with 1 to 4 substituents independently selected from halogen, C_1-4Alkyl radical, C_1-4Haloalkyl, -OH, -OC_1-4Alkyl and OXO;

R¹and R²Each independently is hydrogen or C_1-4An alkyl group;

G²selected from (i) to (xiii):

R⁴is composed of

X¹Is O or S;

X²o, S, NH or NC_1-4An alkyl group;

R⁵is composed of

Or C_1-4An alkyl group;

R^4a、R^4b、R^4c、R^5a、R⁶、R⁸、R⁹、R¹⁰、R¹²、R¹⁴、R¹⁵、R¹⁶、R¹⁸、R²⁰、R²²、R²³、R²⁴、R²⁵、R²⁶、R²⁸、R³⁰and R³²Each occurrence is independently-OH, halogen, nitro, cyano, C_1-4Alkyl radical, C_1-4Haloalkyl, -OC_1-4Alkyl, -OC_1-4Haloalkyl, -NH₂、–NH(C_1-4Alkyl), -N (C)_1-4Alkyl) (C_1-4Alkyl), -NHC (O) C_1-4Alkyl, -N (C)_1-4Alkyl group(s) C (O) C_1-4Alkyl, -NHC (O) OC_1-4Alkyl, -N (C)_1-4Alkyl) C (O) OC_1-4Alkyl, -C (O) OC_1-4Alkyl, -C (O) OH, -C (O) NH₂、-C(O)NH(C_1-4Alkyl) or-C (O) N (C)_1-4Alkyl) (C_1-4Alkyl), and optionally two R^4a、R^4b、R^5a、R⁶、R⁸、R⁹、R¹⁰、R¹²、R¹⁴、R¹⁵、R¹⁶、R¹⁸、R²⁰、R²¹、

R²²、R²⁴、R²⁵、R²⁶、R²⁸、R³⁰Or R³²Form fused rings 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.

In the present application, the structure of formula I also includes all isomeric forms of the structure, e.g., enantiomeric, diastereomeric and/or geometric (or conformational) forms, e.g., the R and S configurations of each of the asymmetric centers, (Z) and (E) double bond isomers and (Z) and (E) conformational isomers.

In the present application, the entire contents of the structures described in WO2019136147a1 with respect to the formula I are incorporated by reference.

In the present application, the TLR4 pathway inhibitor may also include oxpcpc, Eritoran, and/or TAK-242.

The oxapc may have a structural formula as shown below:

the Eritoran may have the structural formula shown below:

the TAK-242 may have the structural formula shown below:

for example, the TLR4 pathway inhibitors oxpsac, Eritoran can block binding of LPS to LBP and CD 14; the TAK-242 may bind to cysteine CYS747 of the intracellular domain of TLR4 to inhibit the TLR4 signaling pathway.

In the present application, the distal injury associated with ischemia-reperfusion of the gastrointestinal tract may comprise a pathological or physiological process of any organ or tissue other than the gastrointestinal tract.

For example, the pathological or physiological process of any organ or tissue other than the gastrointestinal tract may be concurrent with ischemia-reperfusion of the gastrointestinal tract.

For example, the coincidence may be that the pathological or physiological process of any organ or tissue other than the gastrointestinal tract and the occurrence of ischemia-reperfusion of the gastrointestinal tract are within 10 minutes, such as within 5 minutes, such as within 3 minutes, such as within 1 minute.

For example, the pathological or physiological process of any organ or tissue other than the gastrointestinal tract may occur prior to ischemia-reperfusion of the gastrointestinal tract.

For example, the pathological or physiological process of any organ or tissue other than the gastrointestinal tract may occur within 24 hours prior to ischemia-reperfusion of the gastrointestinal tract. For example within 22 hours, for example within 20 hours, for example within 18 hours, for example within 16 hours, for example within 14 hours, for example within 12 hours, for example within 10 hours, for example within 8 hours, for example within 6 hours, for example within 5 hours, for example within 4.5 hours, for example within 4 hours, for example within 3.5 hours, for example within 3 hours, for example within 2.5 hours, for example within 2 hours, for example within 1.5 hours, for example within 1 hour, for example within 0.5 hours.

For example, the pathological or physiological process of any organ or tissue other than the gastrointestinal tract may occur after ischemia-reperfusion of the gastrointestinal tract.

For example, the pathological or physiological process of any organ or tissue other than the gastrointestinal tract may occur within 30 days after ischemia-reperfusion of the gastrointestinal tract. For example within 28 days, for example within 26 days, for example within 24 days, for example within 22 days, for example within 20 days, for example within 18 days, for example within 16 days, for example within 14 days, for example within 12 days, for example within 10 days, for example within 8 days, for example within 1 day, for example within 6 days, for example within 5 days, for example within 4 days, for example within 3 days, for example within 2 days, for example within 1 day, for example within 12 hours, for example within 9 hours, for example within 6 hours, for example within 3 hours, for example within 1 hour, for example within 0.5 hours.

For example, the pathophysiological process of any organ or tissue other than the gastrointestinal tract may be manifested as a reduction or alleviation of the pathophysiological process of the organ or tissue when intervening on gastrointestinal ischemia-reperfusion or gastrointestinal ischemia-reperfusion-related effects.

For example, the intestinal ischemia reperfusion related effects may include activation of a TLR4 signaling pathway in gastrointestinal tissue, a change in intestinal barrier, a change in inflammatory factors in gastrointestinal tissue.

For example, activation of the TLR4 signaling pathway in gastrointestinal tissue may include increased expression of gastrointestinal tissue TLR4, Myd88, Traf6, and/or Nfkb; wherein Tlr4 encodes Toll-like receptor 4(TLR4), Myd88 encodes adaptor protein (MYD88), Traf6 encodes tumor necrosis factor receptor-related factor 6(TRAF6), and Nfkb encodes nuclear factor kappa B protein (NF kappa B).

For example, the intestinal barrier change may include an intestinal barrier injury,

for example, the gastrointestinal tissue inflammatory factor changes expression of a gastrointestinal tissue pro-inflammatory factor.

In the present application, the pathological or physiological processes of any organ or tissue of the stomach other than the gastrointestinal tract may include systemic or local distal reactions resulting from reperfusion of ischemic tissue of the gastrointestinal tract, which may include extensive microvascular dysfunction and altered tissue barrier function as well as inflammatory reactions.

In the present application, the pathological or physiological processes of any organ or tissue other than the gastrointestinal tract may involve the lungs.

For example, the pathological or physiological process of any organ or tissue other than the gastrointestinal tract may comprise pulmonary edema, a process of intrapulmonary thrombosis, pulmonary embolism, and/or inflammation of pulmonary tissue.

In the present application, the pathological or physiological processes of any organ or tissue other than the gastrointestinal tract may involve the kidney.

For example, the pathological or physiological process of any organ or tissue other than the gastrointestinal tract may comprise renal failure, edema formation, thrombosis, thromboembolism, and/or inflammation of renal tissue.

In the present application, the pathological or physiological processes of any organ or tissue other than the gastrointestinal tract may involve the central nervous system.

For example, the pathological or physiological process of any organ or tissue other than the gastrointestinal tract may include blood brain barrier disruption, silent cerebral ischemia, stroke, cerebral edema, raised intracranial pressure, inflammation of neuronal tissue, neuronal cell death, brain injury, and/or neurological dysfunction.

For example, the pathological or physiological processes of any organ or tissue other than the gastrointestinal tract may include ischemic stroke and related conditions.

For example, the ischemic stroke and related conditions can include non-staining (off-white) areas of ischemic stroke as evidenced by TTC staining in the mouse MCAO model, disappearance of purple Nissl bodies in areas of ischemic stroke as evidenced by cryo-microtomy (Nissl) staining, or an increase in the mouse modified neurological deficit score.

For example, the pathological or physiological process of any organ or tissue other than the gastrointestinal tract may comprise inflammation.

For example, the pathological or physiological process of any organ or tissue other than the gastrointestinal tract may comprise systemic inflammation. The inflammation may affect the lungs, gastrointestinal system, cardiovascular system, other extremities and/or central nervous system.

For example, the pathological or physiological process of any organ or tissue other than the gastrointestinal tract may comprise systemic inflammatory response syndrome (severe inflammatory response syndrome) and/or Multiple Organ Dysfunction Syndrome (MODS).

For example, the pathological or physiological process of any organ or tissue distinct from the gastrointestinal tract may include a disease, disorder, or medical intervention that causes ischemia-reperfusion of the gastrointestinal tract, e.g., stroke, trauma, shock, sepsis, acute pancreatitis, inflammatory bowel disease, or brain trauma surgery.

For example, the trauma includes a disruption of a tissue or organ of the human body caused by an external force factor, such as, for example, a traffic injury, a fall injury, a mechanical injury, a sharps injury, a fall injury, a firearm injury.

For example, shock is usually a clinical syndrome in which the body suffers from a strong pathogenic factor, the amount of effective circulating blood is sharply reduced, the body is decompensated, ischemia and hypoxia of tissues, and neuro-humoral factor imbalance. Its main features include insufficient perfusion of microcirculation in vital organs and tissues, metabolic disturbance and dysfunction of all systems of the whole body. For example hypovolemic shock, vasodilatory shock, cardiogenic shock. The hypovolemic shock may include hemorrhagic shock, burn shock, traumatic shock; the vasodilative shock may include septic shock, anaphylactic shock, neurogenic shock.

In the present application, the neurological-related disorder may include blood brain barrier disruption, silent cerebral ischemia, stroke, cerebral edema, raised intracranial pressure, inflammation of neuronal tissue, neuronal cell death, brain injury, and/or neurological dysfunction.

For example, the neurological dysfunction may include motor dysfunction, dysregulation of the internal environment. The disorders of homeostasis can include disorders of functioning of the internal organs, body fluids or blood circulation system in connection with innervation.

In the present application, the nervous system-related disorder may include cerebral ischemic diseases and related disorders thereof.

For example, the cerebral ischemic disease may include a group of diseases in which cerebral tissue is damaged due to blood failure to flow into the brain or decreased blood flow into the brain caused by vascular occlusion, lesions, trauma, etc.

For example, the cerebral ischemic disease may include ischemic stroke and its related conditions.

For example, the ischemic stroke-related disorder may include cerebral infarction.

For example, the ischemic stroke may comprise atherosclerotic occlusion of the aorta, cerebral embolism (embolic infarction), non-embolic infarction of small deep perforator arteries (lacunar infarction), and watershed area ischemia resulting from distal arterial stenosis and decreased cerebral blood flow (hemodynamic stroke).

For example, the ischemic stroke-related disorder may include brain nerve cell death, brain function impairment, changes in smell/taste/hearing or vision, swallowing/pupil photoreaction disorders, physical movement disorders, aphasia, changes in breathing and heart rate, neuromodulation disorders of other tissues or organs, loss of consciousness, and the like.

For example, the ischemic stroke-related disorder may include any of the indicators in NIHSS. Wherein The NIHSS is National Institute of Health and Health Scale (National Institute of Health Structure scale), and The scoring method of The NIHSS can be found in Williams LS, Yilmaz EY, Lopez-Yunez AM.retrodirective Association of Initial Stroke Severity with The NIH Stroke Scale.2000; 31:858-862).

For example, the ischemic stroke and related conditions may be manifested in a mouse MCAO model as non-staining (off-white) areas of ischemic stroke in TTC staining, disappearance of Nissl bodies in areas of ischemic stroke in cryo-microtomy (Nissl) staining, or an increase in the mouse modified neurological deficit score. Without wishing to be bound by any theory, the examples below are merely intended to illustrate the use of the TLR4 pathway inhibitors of the present application in the manufacture of a medicament, the use of TLR4 for screening for a medicament, a pharmaceutical composition comprising a TLR4 pathway inhibitor, etc., and are not intended to limit the scope of the invention of the present application.

Embodiments of the present application:

use of an inhibitor of the TLR4 pathway in the manufacture of a medicament for preventing, ameliorating and/or treating distal injury associated with ischemia-reperfusion of the gastrointestinal tract in a subject.

2. The use according to embodiment 1, wherein the TLR4 pathway inhibitor has a structure according to formula I:

R¹and R²Each independently is hydrogen or C_1-4An alkyl group;

G²selected from (i) to (xiii):

R⁴is composed of

X¹Is O or S;

X²o, S, NH or NC_1-4An alkyl group;

R⁵is composed of

Or C_1-4An alkyl group;

n1 and n2 are independently 1, 0, 2,3, 4 or 5;

n3 and n4 are independently 0, 1, 2 or 3.

3. The use according to embodiment 1, wherein the TLR4 pathway inhibitor comprises OxPAPC, Eritoran and/or TAK-242.

4. The use according to any one of embodiments 1-3 wherein the medicament is formulated such that the TLR4 pathway inhibitor is effective locally in the gastrointestinal tract.

5. The use according to any one of embodiments 1-4, wherein the TLR4 pathway inhibitor is still present in the gastrointestinal tract as long as about 1 hour or later after administration in an amount effective to prevent, ameliorate and/or treat the gastrointestinal ischemia-reperfusion-related distal injury.

6. The use according to any one of embodiments 1-5, wherein at most 50% of the TLR4 pathway inhibitor is absorbed by the subject into the blood circulation system about 24 hours or after administration.

7. The use according to any one of embodiments 1 to 6, wherein the concentration of the TLR4 pathway inhibitor in the medicament is from about 0.0001% (w/w) to about 90% (w/w).

8. The use according to any one of embodiments 1-7, wherein the subject has, is having, or is at risk of having a disease and/or condition associated with ischemia-reperfusion of the gastrointestinal tract.

9. The use according to embodiment 8, wherein the disease and/or condition associated with ischemia-reperfusion of the gastrointestinal tract comprises stroke, trauma, shock, sepsis and/or acute pancreatitis.

10. The use according to any one of embodiments 8-9, wherein the disease associated with ischemia reperfusion of the gastrointestinal tract comprises ischemic stroke.

11. The use according to any one of embodiments 1-10, wherein the subject has been, is, or is at risk of experiencing the gastrointestinal ischemia-reperfusion.

12. The use according to any one of embodiments 1-11, wherein the distal injury associated with ischemia-reperfusion of the gastrointestinal tract comprises ischemic stroke.

13. The use according to any one of embodiments 1-12, wherein the medicament is configured for oral administration.

14. The use according to any one of embodiments 1-13, wherein the TLR4 pathway inhibitor is not substantially degraded and/or inactivated by digestive fluids.

15. A pharmaceutical composition comprising the TLR4 pathway inhibitor of any one of embodiments 1-14 and optionally a pharmaceutically acceptable carrier.

16. A method of preventing, ameliorating and/or treating distal injury associated with ischemia-reperfusion of the gastrointestinal tract in a subject, the method comprising administering to the subject an inhibitor of the TLR4 pathway of any one of embodiments 1-14.

17. The method of embodiment 16, wherein said administering comprises gastrointestinal administration.

18. The method according to any one of embodiments 16-17, wherein the TLR4 pathway inhibitor is effective locally in the gastrointestinal tract.

19. The method according to any one of embodiments 16-18, wherein the TLR4 pathway inhibitor is still present in the gastrointestinal tract as long as about 1 hour or later after administration in an amount effective to prevent, ameliorate and/or treat the gastrointestinal ischemia-reperfusion-related distal injury.

20. The method according to any one of embodiments 16-19, wherein at most 50% of the TLR4 pathway inhibitor is absorbed by the subject into the blood circulation system about 24 hours or after administration.

21. A method of preventing distal injury associated with ischemia-reperfusion of the gastrointestinal tract in a subject, the method comprising:

a) monitoring a gastrointestinal condition of the subject;

b) administering to the subject the TLR4 pathway inhibitor of any one of embodiments 1-14 when or after the monitoring shows that the subject is experiencing gastrointestinal ischemia reperfusion.

22. The method of embodiment 21, wherein said administering comprises gastrointestinal administration.

Use of TLR4 for screening a medicament, wherein the medicament is for preventing, ameliorating and/or treating distal injury associated with ischemia-reperfusion of the gastrointestinal tract in a subject.

24. The use according to embodiment 23, wherein the medicament inhibits activation and/or signalling of the TLR4/TRAF 6/nfkb pathway.

Examples

Materials and reagents

The SPF male C57BL/6 mice 8-10 weeks old have the body weight of 22-24g, are provided by Guangdong province medical experimental animal center, and have the quality qualification certification of experimental animals. The fecal sample genome DNA extraction Kit (MinkaGene pool DNA Kit) is a Minka Gene product; primers were synthesized by Thermo Fisher corporation; TaqMan reverse transcription reagent and SYBR Green are products of Takara Bio company; the ViiA 7 real-time PCR system is a product of Applied Biosystems; the TTC powder is a product of Sigma company; the ELISA kit of D-Lac, LBP and LPS serum is a product of GeneMei company; other materials, reagents, etc., are commercially available without specific reference.

Statistical analysis

The data analysis of the non-microbiological informatics data adopts R analysis software. Normal distribution data are expressed as mean. + -. standard deviation, non-normal distribution data are presented as median (interquartile range), non-parametric test using Kruskal-Wallis rank sum test or Mann-Whitney U test, parametric test using unpaired Student's test or ONE-WAY ANOVA for analysis. The classification variables are expressed in scale. The data were examined for normality using the Shapiro-Wilk test. For microbiota analysis, Adonis tests performed in QIIME 1.9.1 were used. P <0.05 (two-tailed) is considered to have significant differences.

Example 1 Experimental mice grouping

SPF class 8 week old male C57BL/6 wild type mice were selected as subjects and randomly grouped as follows: a SHAM group for performing a pseudo-surgical treatment; MCAO group, carry on MCAO molding treatment; the Post-OXPA group is used for carrying out OXPA intervention on the mice after MCAO molding; the Pre-OXPA group, on which OXPA intervention was performed before MCAO modeling of mice.

Example 2 establishment of mouse middle cerebral artery infarction Model (MCAO)

The mice were weighed, anesthetized with 0.2ml/10g of tribromoethanol, a median incision of about 1cm of the neck was cut, the tissue carefully dissected and the right carotid trigone exposed. Carefully separating the right common carotid artery, the external carotid artery (the external carotid artery needs to be stripped upwards as much as possible until the anterior cranial bifurcation) and the internal carotid artery; ligating the proximal end (slipknot) of common carotid artery, coagulating off the small branch of external carotid artery with the tip of electric coagulator, and ligating the proximal end (slipknot) and distal end (dead knot) of external carotid artery. The external carotid artery was cut open, a plug of thread (the appropriate size was chosen based on the mouse weight) was inserted into the internal carotid artery, the insertion was stopped when slight resistance was felt (approximately when the plug of thread marks (1 cm from the tip) at the common carotid bifurcation), the plug was secured, and the skin was then sutured. The plug was removed 1 hour after insertion of the plug, which served as the post-stroke timing start (i.e., post-stroke 0). The core body temperature of the mouse is kept at 37+0.5 ℃ by adopting a constant temperature blanket in the operation, and the constant temperature blanket is continuously recovered until the mouse is anaesthetized and revived, so that the operation is to prevent the establishment of a mouse cerebral ischemia model from being influenced by the low-temperature cerebral protection effect. Mice with excessive blood loss or modeling time exceeding 15 minutes in the operation process are discarded and are not included in the later experimental analysis research.

Example 3 Observation of mouse cecal blood flow

Blood flow observation is carried out on blood vessels at the cecum part of mice in the SHAM group and the MCAO group, and the mice in the SHAM group are subjected to pseudo-operation and blood flow observation at the response time point by taking the time points of before model making operation, after inserting a wire plug, after pulling the wire plug and 1 hour after pulling the wire plug of the MCAO group. The specific operation steps are as follows:

(1) before operation, a mouse needs to be anesthetized, the abdomen is disinfected, the skin and the muscular layer are cut layer by layer along the median line of the abdomen, and the cecum is exposed;

(2) observing the cecal blood flow of a mouse by using a laser speckle imaging system (Rewode RFLSI Pro), firstly observing panoramic imaging of the cecal of the mouse, recording a cecal blood flow dynamic image by adopting laser speckle imaging system (Rewode RFLSI Pro) software after photographing, wherein the recording time is 10min, and the interval between frames is 1 s;

(3) analyzing and adopting blood flow change of an Interest Region (ROI), wherein the Interest Region is six blood vessel branches of the caecum, and extracting the numerical value of the Interest Region as a blood flow value after recording.

(4) The relative regional blood flow values at different time points in the detection process are the average blood flow value in the interest region at the corresponding time divided by the blood flow value at the initial time point.

The core body temperature of the mice was controlled at 37+0.5 ℃ throughout the surgery.

As shown in fig. 1 and 2, at the time point of removal of the wire plugs from the MCAO group mice and 1 hour thereafter, the cecal blood flow was reduced in the MCAO group mice as compared with the SHAM group mice, indicating that ischemia occurred in the intestines of the mice during stroke of the MCAO mice.

Example 4 quantitative analysis of expression level of TLR4 pathway-associated genes in intestinal tissue

At the 24 hour time point after stroke (where the initial timing time is the time to remove the wire plug), mouse colon tissue was rapidly harvested and stored frozen at-80 ℃ for determination of relative expression levels of TLR4 pathway-associated genes, including TLR4, Traf6, Tram, Myd88, Nfkb genes, whose primer sequences are shown in SEQ ID nos. 1-10. The method comprises the following specific steps:

(1) colon tissue was homogenized using minibedbeater (Biospec Products, Bartlesville);

(2) RNA extraction by Trizol reagent method (Invitrogen): 1ml TRIZOL was added to each 50-100mg of the homogenized tissue sample for homogenization. Then 0.2ml chloroform was added, followed by covering, vigorous shaking for 15 seconds, standing at 15-30 ℃ for 2-3 minutes, and then centrifugation for 12,000g, 15 minutes, 2-8 ℃. The upper aqueous phase was transferred, 0.5ml isopropanol was added, left to stand for 10 minutes at 15-30 ℃ and then centrifuged at 12,000g for 10 minutes at 2-8 ℃. The RNA pellet was washed for 5 minutes by adding 1ml of 75% ethanol. Then dissolving RNA and determining the concentration;

(3) obtaining cDNA from each RNA sample using TaqMan reverse transcription reagent (Takara Bio), respectively;

(4) mu.l cDNA was added as template (concentration 50-100ng/ul) in a 20. mu.l volume and the final concentration 250nM was added as shown in SEQ ID NO: 1-10, SYBR Green (Takara Bio) real-time PCR was performed using the following reaction systems:

TABLE 1

(5) Data were extracted using the ViiA 7 real-time PCR system (Applied Biosystems) and analyzed using the comparative Ct method. The respective Gapdh expression levels are used as internal references, and the primer sequences are shown in SEQ ID NO. 11-12.

The observation results are shown in FIG. 3, and the results show that the relative expression levels of the genes of Traf6, Myd88 and Nfkb in the MCAO group are increased as compared with those in the Sham group.

Example 5 Effect of OxPAPC intervention on expression levels of genes associated with the TLR4 pathway in intestinal tissue

The following three groups of mice were prepared as described in example 1: a SHAM group for performing a pseudo-surgical treatment; MCAO group, carry on MCAO molding treatment; post-oxpo group, mice were modeled with MCAO, which was subjected to oxpo intervention (100mg/kg, 0.2ml, gavage, 1 time) 1 hour after stroke (i.e. 1 hour after removal of the wire plug), and MCAO modeling was performed as described in example 2; three groups of mice were each sampled 24 hours after stroke as described in example 4 and the intestinal tissue Tlr4, Traf6, Tram, Myd88, Nfkb gene expression levels were determined.

The results show that the oxPACs reduce the expression level of Tlr4, Traf6, Tram, Myd88 and Nfkb genes of intestinal tissues after the mice are subjected to stroke.

Example 6 Effect of OxPAPC intervention on brain injury in ischemic stroke

(1) The following three groups of mice were prepared as described in example 1: MCAO group, carry on MCAO molding treatment; in the Post-OXPAC group, mice are subjected to MCAO molding, and are subjected to OXPAC intervention (100mg/kg, intragastric administration and 1 time) 1 hour after cerebral apoplexy (namely 1 hour after removing a thread plug); the Pre-OXPAAPC group carries out OXPAC intervention (1mg/ml, drinking water, 5 days) before MCAO molding on mice, and MCAO molding is carried out after 5 days of OXPAC intervention. MCAO modelling was performed as described in example 2;

(2) preparing TTC powder by using PBS (phosphate buffer solution), wherein the concentration of TTC staining solution is 2%, and placing in a dark place;

(3) after anesthetizing the mice (0.2ml/10g tribromoethanol), the thoracic cavity was opened, the right auricle was cut open, and the heart was perfused with frozen PBS for about 2 min;

(4) cutting head, collecting brain, placing brain tissue in brain groove, placing brain groove in refrigerator at-80 deg.C for 7-8 min, taking out, and making into coronal slice with thickness of 1.5 mm;

(5) the cut brain tissue is placed in TTC staining solution and stained for 10min in the dark. Normal brain tissue is dark red, and the ischemic stroke injury area is not colored (grey white). Adding formaldehyde into the dyed slices for fixation, and taking out for photographing;

(6) brain injury volume analysis was performed using ImagePlus Soft image software. And (3) when analyzing the result, excluding the cerebral edema factor, and calculating by using an edema infarction volume correction formula: and (3) obtaining the proportion value of the brain injury volume in the whole cerebral hemisphere finally by directly damaging the volume (ipsilateral hemisphere-contralateral hemisphere).

The result shows that the injury degree of cerebral ischemic stroke of mice in the MCAO group is obviously relieved compared with that of the mice in the non-stem MCAO group whether the oxPAC intervention is performed before modeling or after modeling, which shows that the cerebral ischemic stroke injury of the mice can be improved by inhibiting the effect of TLR 4.

Example 7 modified neurological maintenance score (mNSS) in mice

This was done 24 hours after stroke on the MCAO, Post-OXPACs, Pre-OXPACs groups. Neuro-behavioral testing was performed by two testers blinded to the experimental group. The mNSS score was 0-14 points, with 0 points representing normal and 14 points representing the most severe neurological deficit. The scoring system can comprehensively assess neurological functions including motor, sensory, balance and reflexes: firstly, a motion test: lifting the tail of the mouse to evaluate the bending and twisting degree of the limbs (0-3 points); pose assessment while walking on a flat surface (score 0-3). ② balance test: the mouse was placed on a beam. The evaluation of impaired nerve function was based mainly on whether the mice were able to balance on the crossbeams, the limbs dropped from the crossbeams and were able to pass smoothly through the crossbeams (score 0-6). Sensory and reflex test: auricle reflex and corneal reflex (0-2 points) were each detected.

The results show that OxPAPC intervention before or after modeling improved the neurological deficit in mice compared to the non-primed MCAO group.

Example 8 Nissl staining of frozen sections of brain tissue

(1) Taking out the brain: 24 hours after stroke, after assessment of mNSS, each group of mice (SHAM group, MCAO group, Post-OXPA group) was sacrificed, and brains were taken;

(2) fixing: after anesthetizing the mice, the chest was opened to fully expose the heart, the right auricle was cut open, and cardiac perfusion was performed with frozen normal saline for 10min, followed by frozen 4% Paraformaldehyde (PFA) perfusion for 10 min. The tissue was then fixed to PFA for 24h, 30% PFA sucrose for 48 h. After full dehydration, the brain tissue is completely sunk and taken out;

(3) embedding: cutting brain tissue, placing in a frozen section embedding mold, embedding with conventional OCT, freezing with liquid nitrogen, and freezing at-80 deg.C in a refrigerator;

(4) slicing: the temperature of the ice-cold microtome was adjusted to-20 ℃. After the temperature reached, the specimen was removed, and the brain tissue (including the whole thalamus tissue) was cut into coronal sections of 5 μm in thickness in a range of 2.0mm to 4.0mm after bregma. Flattening the slices with a fine hairbrush, placing the slices on a glass slide, and storing in a refrigerator at-20 ℃;

(5) and (3) Nie dyeing: brain sections were immersed in Nise stain for 5 minutes and washed 2 times with distilled water. 95% ethanol for about 5 seconds. Dehydration with 95% ethanol for 2 min. Immersed in fresh xylene for 5 minutes, clear, and blocked with neutral gum. The film is dried and then placed under a microscope for observation and picture analysis. The cell membrane of the normally surviving neuron is complete and bluish purple, and the nucleus can be seen to be deeply stained.

The results show that OXPAPC intervention significantly reduced neuronal loss in mice.

Example 9 Effect of other TLR4 inhibitors on cerebral injury during ischemic stroke

The effect of Eritoran and TAK-242 on brain injury during cerebral arterial thrombosis in mice was examined as in example 7.

The results show that the groups subjected to the intervention of the inhibitors before or after the molding are all relieved in the degree of cerebral ischemic stroke damage of mice compared with the non-intervention MCAO group.

Example 10 local Effect of TLR4 inhibitors in the gastrointestinal tract

Samples of mouse plasma, intestinal contents were collected before (0h) and at various time points after (1h, 3h, 6h, 12h, 24h) the mice were intervened.

Sample pretreatment: about 10mg of each stool sample was weighed, and each stool sample was packed in a 1.5mL tube, 25. mu.L of water was added, and homogenized with zirconia beads for 3 min. Then, 185 μ L of acetonitrile: the metabolites were extracted with methanol (8:2), centrifuged at high speed (18000g,20min) and the supernatant transferred to 96-well plates. Add 20. mu.L of freshly prepared derivatizing reagent to each well, seal the plate, and place at 30 ℃ for about 60min for derivatization. Then, the sample was diluted by adding 350. mu.L of ice-bath 50% methanol solution, the plate was placed at-20 ℃ for 20min, followed by centrifugation at 4 ℃ (4000g,30min), 135. mu.L of supernatant was aspirated and transferred to a new 96-well plate, and 15. mu.L of internal standard was added to each well. A gradient dilution of the derivatized standard stock was added to the left well and the plate was finally sealed for LC-MS analysis.

In each serum sample 25. mu.L were removed and transferred to a 96-well plate. Add 100. mu.L of ice-cold methanol containing internal standard to each well and shake vigorously for 5 min. The 96-well plate was centrifuged at 4000g for 30min and replaced in the pipetting station. Transfer 30 μ L of supernatant to a new 96-well plate, add 20 μ L of freshly prepared derivatizing agent, seal the plate, and place at 30 ℃ for about 60min for derivatization. Then, the sample was diluted by adding 350. mu.L of ice-bath 50% methanol solution, the plate was placed at-20 ℃ for 20min, followed by centrifugation at 4 ℃ (4000g,30min), 135. mu.L of supernatant was aspirated and transferred to a new 96-well plate, and 15. mu.L of internal standard was added to each well. A gradient dilution of the derivatized standard stock was added to the left well and the plate was finally sealed for LC-MS analysis.

Drug concentration measurements were performed using an ultra performance liquid chromatography-tandem mass spectrometry instrument (UPLC-MS/MS) (ACQUITY UPLC-Xevo TQS, Waters Corp., Milford, MA, USA). The raw data file generated by UPLC-MS/MS was processed using quinmet software (v2.0, Metabo-Profile, Shanghai, China) to perform peak integration, calibration and quantification of each metabolite.

The results show that OxPAPC, Eritoran, TAK-242 are still present in the gastrointestinal tract locally at an amount effective to prevent, ameliorate and/or treat distal damage caused by ischemia-reperfusion of the gastrointestinal tract about 1 hour or more after administration; about 24 hours or later after administration, up to 50% of the OxPAPC, erithoran, TAK-242 is absorbed by the subject into the blood circulation system.

The foregoing detailed description is provided by way of illustration and example, and is not intended to limit the scope of the appended claims. Various modifications of the presently recited embodiments will be apparent to those of ordinary skill in the art and are intended to be within the scope of the appended claims and their equivalents.

Sequence listing

<110> Zhujiang Hospital of southern medical university

Application of <120> TLR4 pathway inhibitor in preparation of medicine

<130> 0133-PA-004

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<170> PatentIn version 3.5

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Claims

Use of an inhibitor of the TLR4 pathway in the manufacture of a medicament for preventing, ameliorating and/or treating distal injury associated with ischemia-reperfusion of the gastrointestinal tract in a subject.
2. The use of claim 1, wherein the TLR4 pathway inhibitor has a structure according to formula I:

wherein G is¹Is a 4-to 12-membered heterocyclyl containing 1 to 3 heteroatoms independently selected from N, O and S, -NR¹R²，-OH，-OC_1-4Alkyl radical, C_3-8Cycloalkyl, or 5 to 12 membered heteroaryl comprising 1 to 3 heteroatoms independently selected from N, O and S; wherein said cycloalkyl, heterocyclyl, heteroaryl are optionally substituted with 1 to 4 substituents independently selected from halogen, C_1-4Alkyl radical, C_1-4Haloalkyl, -OH, -OC_1-4Alkyl and OXO;

R¹and R²Each independently is hydrogen or C_1-4An alkyl group;

L¹is-C_1-4alkylene-O-, -C_1-5alkylene-or-C (o) -CH ═ CH-, wherein said-C_1-5Alkylene group of the formula_1-4alkylene-O-optionally substituted with 1 to 2 halogens or-OH, or L¹Comprises the following steps:

wherein C is_1-4Alkylene and G¹Bonded and imidazole in a position opposite to G²The phenyl groups of (a) are fused meta and para;

G²selected from (i) to (xiii):

R⁴is composed of

X¹Is O or S;

X²o, S, NH or NC_1-4An alkyl group;

R⁵is composed of
Or C_1-4An alkyl group;

R³、R⁷、R¹³，R¹⁷、R¹⁹、R²¹、R²⁷、R²⁹and R³³Each independently selected from hydrogen and C_1-4An alkyl group;

R¹¹and R³¹Each independently selected from hydrogen and C_1-4Alkyl and optionally substituted by 1-3 substituents independently selected from halogen, C_1-4Alkyl radical, C_1-4Haloalkyl, -OH and-OC_1-4Phenyl substituted with a substituent of alkyl;

R^4a、R^4b、R^4c、R^5a、R⁶、R⁸、R⁹、R¹⁰、R¹²、R¹⁴、R¹⁵、R¹⁶、R¹⁸、R²⁰、R²²、R²³、R²⁴、R²⁵、R²⁶、R²⁸、R³⁰and R³²Each occurrence is independently-OH, halogen, nitro, cyano, C_1-4Alkyl radical, C_1-4Haloalkyl, -OC_1-4Alkyl, -OC_1-4Haloalkyl, -NH₂、–NH(C_1-4Alkyl), -N (C)_1-4Alkyl) (C_1-4Alkyl), -NHC (O) C_1-4Alkyl, -N (C)_1-4Alkyl group(s) C (O) C_1-4Alkyl, -NHC (O) OC_1-4Alkyl, -N (C)_1-4Alkyl) C (O) OC_1-4Alkyl, -C (O) OC_1-4Alkyl, -C (O) OH, -C (O) NH₂、-C(O)NH(C_1-4Alkyl) or-C (O) N (C)_1-4Alkyl) (C_1-4Alkyl), and optionally two R^4a、R^4b、R^5a、R⁶、R⁸、R⁹、R¹⁰、R¹²、R¹⁴、R¹⁵、R¹⁶、R¹⁸、R²⁰、R²¹、R²²、R²⁴、R²⁵、R²⁶、R²⁸、R³⁰Or R³²Form fused rings 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.
3. The use of claim 1, wherein the TLR4 pathway inhibitor comprises OxPAPC, Eritoran, and/or TAK-242.
4. A pharmaceutical composition comprising the TLR4 pathway inhibitor of any one of claims 1-3 and optionally a pharmaceutically acceptable carrier.
5. A method of preventing, ameliorating and/or treating distal injury associated with ischemia-reperfusion of the gastrointestinal tract in a subject, the method comprising administering to the subject the TLR4 pathway inhibitor of any one of claims 1-3.
6. The method of claim 5, wherein the administering comprises gastrointestinal administration.
7. A method of preventing distal injury associated with ischemia-reperfusion of the gastrointestinal tract in a subject, the method comprising:

a) monitoring a gastrointestinal condition of the subject;

b) administering the TLR4 pathway inhibitor of any of claims 1-3 to the subject when or after the monitoring shows that the subject is experiencing gastrointestinal ischemia reperfusion.
8. The method of claim 7, wherein the administering comprises gastrointestinal administration.
Use of TLR4 for screening a medicament, wherein the medicament is for preventing, ameliorating and/or treating distal injury associated with ischemia-reperfusion of the gastrointestinal tract in a subject.
10. The use of claim 9, wherein the medicament inhibits activation and/or signalling of the TLR4/TRAF 6/nfkb pathway.