KR20200135043A - Composition for prevention or treatment of lung damage diseases comprising inhibitor of macrophage-1 antigen - Google Patents

Abstract

The present invention relates to: a composition for preventing or treating lung damage disease which contains an inhibitor of the expression or activity of a macrophage-1 antigen (Mac-1) in neutrophils in pulmonary capillaries, and which alleviates microcirculation disorders in the lungs; a screening method; a method for providing information for diagnosis of lung damage disorder; and a composition and a kit for diagnosing pulmonary microcirculation disorder. The composition according to an embodiment of the present invention inhibits the expression or activity of the Mac-1 in neutrophils in pulmonary capillaries, so that red blood cells can smoothly pass through pulmonary capillaries to increase gas exchange in individuals with pulmonary microcirculation disorders to alleviate pulmonary microcirculation disorder, thereby having an excellent effect as the composition for preventing or treating pulmonary microcirculation disorder.

Description

Composition for prevention or treatment of lung damage diseases comprising inhibitor of macrophage-1 antigen}

The present specification includes inhibitors of the expression or activity of macrophage-1 antigen (Mac-1) in neutrophils in pulmonary capillaries, and for preventing or treating lung damage diseases that improves microcirculation disorders in the lungs. Disclosed are a composition, a screening method, a method of providing information for diagnosing whether or not a lung injury disorder, a composition and a kit for diagnosing whether or not a pulmonary microcirculation disorder.

Sepsis is the largest share of deaths during hospitalization (Torio CM, Moore BJ. National Inpatient Hospital Costs: The Most Expensive Conditions by Payer, 2013: Statistical Brief #204. Healthcare Cost and Utilization Project (HCUP) ) Statistical Briefs, Rockville (MD) , 2016; Hall MJ, Levant S, DeFrances CJ Trends in inpatient hospital deaths:... National hospital Discharge Survey, 2000-2010 NCHS Data Brief 2013 (118): 1-8), the pathogen It is a syndrome characterized by the host's dysregulated response to invasion, and involves hemodynamic changes that lead to several life-threatening organ dysfunctions (Singer M, Deutschman CS, Seymour CW, Shankar- Hari M, Annane D, Bauer M, Bellomo R, Bernard GR, Chiche JD, Coopersmith CM, Hotchkiss RS, Levy MM, Marshall JC, Martin GS, Opal SM, Rubenfeld GD, van der Poll T, Vincent JL, Angus DC. . The Third International Consensus Definitions for Sepsis and septic Shock (Sepsis-3) JAMA 2016: 315 (8):. 801-810; Angus DC, van der Poll T. Severe sepsis and septic shock N Engl J Med 2013: 369 ( 9): 840-851). Of the organs damaged by sepsis, the lungs are the first and most often damaged, and whether acute respiratory distress syndrome (ARDS) or acute lung injury (ALI) predicts the likelihood of death in sepsis patients. It is the most important factor (Lagu T, Rothberg MB, Shieh MS, Pekow PS, Steingrub JS, Lindenauer PK. Hospitalizations, costs, and outcomes of severe sepsis in the United States 2003 to 2007. Crit Care Med 2012: 40(3): 754-761.). Despite intensive research efforts aimed at treating acute lung injury due to sepsis, there is no effective treatment aimed at microcirculation (Thompson BT, Chambers RC, Liu KD. Acute Respiratory Distress Syndrome. N Engl J Med. 2017: 377(6): 562-572). It is known that measuring dead space can provide significant clinical data for acute lung injury (Nuckton TJ, Alonso JA, Kallet RH, Daniel BM, Pittet JF, Eisner MD, Matthay MA.Pulmonary dead space). . -space fraction as a risk factor for death in the acute respiratory distress syndrome N Engl J Med 2002: 346 (17): 1281-1286), the supply of oxygen in the blood from the damaged side of the waste skeleton currently only occur (ventilate) It just stays on the hypothesis that doesn't perfuse. In particular, acute respiratory distress syndrome is a syndrome that has not been clearly identified for the association between lung injury and microcirculation (Ryan D, Frohlich S, McLoughlin P. Pulmonary vascular dysfunction in ARDS. Ann Intensive Care 2014: 4: 28). Recently, in one study, evidence of blood clots in pulmonary blood vessels, which were limited to ex vivo studies, was reported, but the in vivo process of neutrophil influx and the contributing factors that disturb the pulmonary microcirculation have not yet been studied ( .. Matthay MA, Ware LB, Zimmerman GA The acute respiratory distress syndrome J Clin Invest 2012: 122 (8): 2731-2740; Yuan Y, Alwis I, Wu MCL, Kaplan Z, Ashworth K, Bark D, Jr., Pham A, McFadyen J, Schoenwaelder SM , Josefsson EC, Kile BT, Jackson SP. neutrophil macroaggregates promote widespread pulmonary thrombosis after gut ischemia. Sci Transl Med 2017: 9(409)).

Unregulated recruitment and activation of neutrophils can induce tissue damage through the release of inflammatory mediators including cytokines and reactive oxygen species (ROS) (Grommes J, Soehnlein O. Contribution of neutrophils to acute lung injury. Mol Med 2011: 17(3-4): 293-307; Matute-Bello G, Downey G, Moore BB, Groshong SD, Matthay MA, Slutsky AS, Kuebler WM, Acute Lung Injury in Animals Study G. An official American Thoracic Society workshop report: features and measurements of experimental acute lung injury in animals. Am J Respir Cell Mol Biol 2011: 44(5): 725-738). However, the results of previous studies on specific dynamic factors of neutrophils themselves in pulmonary microcirculation are mostly limited to speculation obtained by observing systemic circulation (Phillipson M, Kubes P. The neutrophil in vascular inflammation. Nat Med 2011: 17(11) ): 1381-1390). Since the diameter of neutrophils is larger than the diameter of pulmonary capillaries, neutrophils must be deformed to pass through the capillaries, which is a relatively long process (Doerschuk CM. Mechanisms of leukocyte sequestration in inflamed lungs. Microcirculation 2001: 8 (2): 71-88). This process, called neutrophil sequestration, was originally described as involving cells rather than groups of neutrophils circulating freely in the lungs, and to some extent was observed using a visible radiation modeling imaging device (MacNee W, Selby C. New perspectives on basic mechanisms in lung disease. 2. Neutrophil traffic in the lungs: role of haemodynamics, cell adhesion, and deformability. Thorax 1993: 48(1): 79-88). Indeed, previous studies have demonstrated neutrophil sequestration in pulmonary capillaries, but the mechanism by which neutrophil sequestration leads to acute lung injury or acute respiratory distress syndrome is unknown (Kuebler WM, Borges J, Sckell A, Kuhnle GE. , Bergh K, Messmer K, Goetz AE.Role of L-selectin in leukocyte sequestration in lung capillaries in a rabbit model of endotoxemia.Am J Respir Crit Care Med 2000: 161(1): 36-43; Lien DC, Henson PM, Capen RL, Henson JE, Hanson WL, Wagner WW, Jr., Worthen GS. Neutrophil kinetics in the pulmonary microcirculation during acute inflammation. Lab Invest 1991: 65(2): 145-159).

Accordingly, the present inventors observe the neutrophil of a model with pulmonary microcirculation disorder using a custom-designed video-speed laser scanning confocal microscope to study a composition for preventing or treating lung injury disease, and By searching for a target for improving microcirculation disorder, the present invention was completed.

In one aspect, an object of the present invention is to inhibit the expression or activity of macrophage-1 antigen (Mac-1) in neutrophils in pulmonary capillaries so that red blood cells pass smoothly through pulmonary capillaries. To provide a composition for preventing or treating lung damage disease and a method for screening a material for preventing or treating lung damage disease, which can improve gas exchange in a pulmonary microcirculation disorder individual to improve gas exchange in the lung. .

In another aspect, an object of the present invention is to provide a method for providing diagnostic information on whether or not a pulmonary microcirculation disorder useful for diagnosing whether or not a pulmonary microcirculation disorder, a composition and a kit for diagnosing pulmonary microcirculation disorder.

In one aspect, the present invention comprises an inhibitor of the expression or activity of macrophage-1 antigen (Mac-1) in neutrophils in pulmonary capillaries, and is a composition for improving pulmonary microcirculation disorder, It provides a composition for preventing or treating lung injury disease.

In another aspect, the present invention, (a) preparing a lung injury model; (b) treating a test substance in the lung injury model; And (c) determining whether the test substance inhibits the expression or activity of macrophage-1 antigen (Mac-1) in neutrophils in the lung capillaries of the lung injury model. It provides a method of screening a substance for preventing or treating an injured disease.

In another aspect, the present invention comprises the step of measuring the expression or activity of macrophage-1 antigen (Mac-1) in neutrophils isolated from pulmonary capillaries of a test subject. Provides a method of providing information for diagnosing microcirculation disorders.

In another aspect, the present invention provides a composition for diagnosing pulmonary microcirculation disorders comprising a reagent for detecting mRNA or protein of macrophage-1 antigen (Mac-1) in neutrophils in pulmonary capillaries.

In another aspect, the present invention provides a kit for diagnosing pulmonary microcirculation disorders comprising a reagent for detecting mRNA or protein of macrophage-1 antigen (Mac-1) in neutrophils in pulmonary capillaries.

The composition according to an embodiment of the present invention suppresses the expression or activity of macrophage-1 antigen (Mac-1) in neutrophils in the pulmonary capillaries, so that red blood cells smooth the pulmonary capillaries. By allowing the passage to increase gas exchange in pulmonary microcirculation disorder individuals, the microcirculation disorder of the lung can be improved, and thus, there is an excellent effect as a composition for preventing or treating lung damage diseases. In addition, by measuring the expression or activity of macrophage-1 antigen in neutrophils isolated from pulmonary capillaries, it is possible to diagnose pulmonary microcirculation disorder more quickly, simply and accurately.

1 schematically shows a process of manufacturing a lung injury mouse model (N-Dep+LPS mouse model) depleted of neutrophils using an acute lung injury mouse model (ALI mouse model) according to an embodiment of the present invention Is also.
2 is a control mouse model (PBS), an ALI mouse model (LPS), and the neutrophil removal model of Example 2 (N-Dep mouse model and N-Dep+LPS mouse model) according to an embodiment of the present invention. A diagram showing the results of photographing the lung microcirculation by the imaging system according to an embodiment of the present invention and processing the image obtained therefrom according to the image processing process. In Figure 2, green (Capillary, TMR Dextran) represents anatomical capillaries, red (Functional, DiD-RBC) represents functional capillary, and magenta (magenta) (LysM, LysM ^{GFP /+} ) represents neutrophils. , A white asterisk (*) indicates a dead space, and a white arrowhead indicates a trapped or isolated neutron. In FIG. 2, Merge is a combination of anatomical capillary, functional capillary, and neutrophil imaging, and the magnified image is a combination of anatomical capillary and neutrophil imaging, and the scale in the magnified image of FIG. The bar is 20 μm and the scale bar in the remaining images is 100 μm.
3A is a control mouse model (PBS), an ALI mouse model (LPS), and a neutrophil removal model of Example 2 (N-Dep mouse model and N-Dep+LPS mouse model) according to an embodiment of the present invention. It is a graph showing the functional capillary ratio (FCR).
3B is a control mouse model (PBS), an ALI mouse model (LPS), and a neutrophil removal model of Example 2 (N-Dep mouse model and N-Dep+LPS mouse model) according to an embodiment of the present invention. This is a graph showing the number of neutrophils per unit area (512 X 512 μm).
4 is a diagram schematically showing a process of separating neutrophils from each of the left ventricle (LV) and lungs of the mouse model according to an embodiment of the present invention.
5 is a diagram showing the results of flow cytometric analysis on neutrophils separated from each of the left ventricle (LV, red) and lungs (Lung, blue) of a mouse model according to an embodiment of the present invention.
6A to 6D are CD11a, CD11b, and neutrophils separated from each of the left ventricle (LV) and lungs of the control (PBS) mouse model and the ALI mouse model (LPS) according to an embodiment of the present invention. It is a graph comparing the expression levels of each of CD18 and CD62L. In FIGS. 6A to 6D, MFI represents mean fluorescence intensity.
7A and 7B are isolated neutrophils of the control (PBS) mouse model and the ALI mouse model (LPS) according to an embodiment of the present invention, and the expression on the cell surface of each of CD11b and CD18 in the neutrophil in vivo It is a diagram showing the result of visualization. In Figures 7a and 7b, red (Ly6G+) represents neutrophils, green (CD11b or CD18) represents expression on the cell surface of each of CD11b and CD18 in neutrophils, and Merge represents the expression of neutrophils and CD11b, or between neutrophils and CD18. This is the sum of expressions. The scale bars in FIGS. 7A and 7B are 100 μm.
8A to 8D are graphs comparing the number of neutrophils expressing CD11b or CD18 between an ALI mouse model (LPS) and a control mouse model (PBS) according to an embodiment of the present invention. Figure 8a shows the number of neutrophils expressing CD11b per unit area (512 X 512 μm), Figure 8b shows the ratio of the number of neutrophils expressing CD11b to the total number of neutrophils, and Figure 8c shows the unit area (512 X 512 μm). The number of neutrophils expressing CD18 per μm), and FIG. 8D shows the ratio of the number of neutrophils expressing CD18 to the total number of neutrophils.
9 is a CLP mouse model (Fc), an anti-Mac-1 mouse model (Anti-CD11b) and apsiksimab mouse model model (Abc), and a normal group (Sham) mouse model according to an embodiment of the present invention ( Sham)'s lung microcirculation is photographed by the imaging system according to an embodiment of the present invention, and an image obtained therefrom is processed according to an image processing process. In FIG. 9, green (Capillary, TMR Dextran) represents anatomical capillaries, red (Functional, DiD-RBC) represents functional capillary, magenta (Ly6G) represents neutrophils, and white asterisks. (*) represents a dead space. In FIG. 9, Merge is the sum of anatomical capillaries, functional capillaries, and neutrophil imaging, and the scale bar is 100 μm.
10A is a CLP mouse model (Fc), an anti-Mac-1 mouse model (Anti-CD11b), and apsiksimab mouse model model (Abc), and a normal group (Sham) mouse model according to an embodiment of the present invention ( Sham)'s functional capillary fraction (FCR).
10B is a CLP mouse model (Fc), an anti-Mac-1 mouse model (Anti-CD11b), and apsiksimab mouse model model (Abc), and a normal group (Sham) mouse model according to an embodiment of the present invention ( Sham) is a graph comparing the number of neutrophils (Ly6G+ cells) according to an embodiment of the present invention.
11 is a pulmonary microcirculation of a CLP mouse model (pre-Abc) before administration of absiksimab and a CLP mouse model (post-Abc) after administration of absiksimab according to an embodiment of the present invention. It is a diagram showing a result of processing an image taken by the imaging system according to the following and an image obtained therefrom according to an image processing process. In Figure 11, green (Capillary, FITC Dextran) represents anatomical capillaries, red (Functional, DiD-RBC) represents functional capillary, and magenta (Magenta) (LysM, LysM ^GFP/+ ) represents neutrophils. And white arrowheads indicate recovery of red blood cell perfusion. In FIG. 11, Merge is the sum of anatomical capillaries, functional capillaries, and neutrophil imaging, and the scale bar is 100 μm.
12 is a graph comparing the functional capillary fraction (FCR) of a CLP mouse model (pre-Abc) before absiksimab administration and a CLP mouse model (post-Abc) after absiksimab administration according to an embodiment of the present invention to be.
13A and 13B are oxygen in arterial blood of a normal mouse model (Sham), a CLP mouse model (Fc) before administration of absiksimab, and a CLP mouse model (Abc) after administration of absiksimab according to an embodiment of the present invention This is a graph comparing the partial pressure and the partial pressure of carbon dioxide.

Hereinafter, the present invention will be described in detail.

In one aspect, the present invention is a composition for preventing, improving or treating lung injury diseases, the composition as an active ingredient of macrophage-1 antigen (Mac-1) in neutrophils in pulmonary capillaries. It provides a composition comprising an inhibitor of expression or activity. In addition, the present invention provides a composition for improving pulmonary microcirculation disorders comprising an inhibitor of expression or activity of macrophage-1 antigen (Mac-1) in neutrophils in pulmonary capillaries as an active ingredient. do.

In one aspect of the present specification, microcirculation is blood circulation found in small blood vessels such as capillary arteries, capillary veins, capillaries, and capillary lymphatic vessels, and is also referred to as microcirculation or capillary circulation, and is the center of metabolism in tissues. It is a place where necessary materials are supplied and discharged. In one aspect of the present invention, the microcirculation may be microcirculation in the lungs, but is not limited thereto.

In one aspect of the present invention, an individual to be prevented or treated is not particularly limited as long as it is an individual for the purpose of preventing or treating a lung injury disease, and any individual can be applied. Specifically, the individual may be a non-human animal or human such as a monkey, dog, cat, rabbit, morpho, rat, mouse, cow, sheep, pig, goat, etc., but is not limited thereto. Further, the individual may be an individual having a microcirculation disorder, a microcirculation disorder, a capillary circulation disorder, or a peripheral circulation disorder, but is not limited thereto.

The neutrophil is a type of granular leukocyte (granulocyte) mainly made in the bone marrow, and is a cell of the same family as monocytes, and occupies 50-70% of leukocytes and about 90% of granulocytes in human blood, neutrophils, and neutral cells. Also called neutrophil leukocyte and neutrophil leukocyte. The neutrophils are leukocytes that first reach the damaged or infected site when tissue is damaged or infected with microorganisms, and various chemotaxis factors such as interleukin 8 (IL8) are made at the damaged or infected site, and neutrophils are attracted to the neutrophils. Is known to cause a strong acute inflammatory reaction. Among the functions of the neutrophils, the most remarkable is the predation and sterilization of bacteria. In one aspect of the present invention, the neutrophils may be neutrophils in pulmonary capillaries.

The macrophage-1 antigen (Mac-1) is an adhesive molecule belonging to the integrin family, and is a glycoprotein expressed in neutrophils, monocytes, macrophages, and parts of activated lymphocytes, and CD11b (αM chain, molecular weight of about 170,000 Da) and CD18 (β2 chain, molecular weight of about 95,000 Da) are non-covalently bonded heterodimer, which is also called CD11b/CD18. The macrophage-1 antigen is also stored in secreted granules within cells and is rapidly expressed on the cell surface upon activation. There are three divalent metal ion binding sites in the αM chain, and the adhesion of this molecule is dependent on divalent metal ions, and the ligands are ICAM-1, iC3b, fibrinogen, blood coagulation factor X, and LPS (lipopolysaccharide). . The molecular-mediated adhesion is involved in adhesion of leukocytes to vascular endothelial cells, infiltration into tissues, and phagocytosis, and the molecule associates with the cytoskeleton or protein kinase in the cytoplasm, and respiration explosion of leukocytes ( It is known to be involved in signaling in respiratory burst, oxidative burst, etc.

According to an embodiment of the present invention, neutrophil removal improves pulmonary microcirculation disorders, but the effect of treatment-induced neutrophil removal in sepsis is unclear because the effect of bacterial clearance and improvement of systemic inflammatory response are unclear. It is unclear. Accordingly, the present inventors explored and evaluated a subpopulation of neutrophils capable of alleviating lung damage, and as a result of flow cytometry, Mac-1 (CD11b/CD18) reacting with ICAM-1 in endothelial cells and various coagulation factors It was confirmed that the expression level was increased in the isolated neutrophils of the lung of the lung injury model.

In the present specification, "expression of a gene" is a concept in the broadest sense including transcription, translation, and post-translational modification.

The composition according to an aspect of the present invention may include an inhibitor of the expression or activity of macrophage-1 antigen in neutrophils in pulmonary capillaries as an active ingredient.

The macrophage-1 antigen expression or activity inhibitor may be a substance that inhibits translation of the mRNA encoding the macrophage-1 antigen, and specifically, may be an oligonucleotide that binds to at least a portion of the mRNA encoding the macrophage-1 antigen. , siRNA, shRNA and miRNA may be any one or more. The macrophage-1 antigen expression or activity inhibitor may be any one or more of siRNA, shRNA, and miRNA that induces RNA interference (RNAi), and to inhibit mRNA expression of a gene encoding the macrophage-1 antigen. Using the RNAi phenomenon that induces interference of the mRNA encoding the macrophage-1 antigen can be used to prevent or treat lung damage. miRNA is a type of endogenous small RNA (RNA) that exists in cells and is derived from DNA that does not synthesize proteins and is produced from a hairpin-shaped transcript. miRNA binds to the complementary sequence of 3'-UTR of target mRNA and induces translational inhibition or destabilization of the mRNA, and ultimately acts as a repressor to inhibit protein synthesis of the target mRNA. . One miRNA targets multiple mRNAs, and it is known that mRNA can also be regulated by multiple miRNAs. Other RNAs that induce RNAi phenomena include short interfering RNA (siRNA), which is a short RNA between 19 and 27 mer, and shRNA having a short hairpin structure.

The macrophage-1 antigen expression or activity inhibitor may include a peptide that specifically binds to macrophage-1 antigen in neutrophils, and specifically, an antibody that specifically binds to macrophage-1 antigen in neutrophils. It may include. The antibody may be an antibody that specifically binds to CD11b or CD18, and more specifically, may specifically bind to CD11b represented by the amino acid sequence of SEQ ID NO: 1 or 2, and more specifically, the following sequence 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% of the amino acid sequence of number 1 or 2 It may specifically bind to a peptide having homology above, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. And, more specifically, BC Bioscience's BD Pharmingen ^TM (Catalog Number: BD 553307) may be, but is not limited thereto.

[SEQ ID NO: 1] Amino acid sequence of CD11b (Integrin alpha-M isoform 1 precursor)

1 malrvlllta ltlchgfnld tenamtfqen argfgqsvvq lqgsrvvvga pqeivaanqr

61 gslyqcdyst gscepirlqv pveavnmslg lslaattspp qllacgptvh qtcsentyvk

121 glcflfgsnl rqqpqkfpea lrgcpqedsd iaflidgsgs iiphdfrrmk efvstvmeql

181 kksktlfslm qyseefrihf tfkefqnnpn prslvkpitq llgrthtatg irkvvrelfn

241 itngarknaf kilvvitdge kfgdplgyed vipeadregv iryvigvgda frseksrqel

301 ntiaskpprd hvfqvnnfea lktiqnqlre kifaiegtqt gssssfehem sqegfsaait

361 sngpllstvg sydwaggvfl ytskekstfi nmtrvdsdmn daylgyaaai ilrnrvqslv

421 lgapryqhig lvamfrqntg mwesnanvkg tqigayfgas lcsvdvdsng stdlvligap

481 hyyeqtrggq vsvcplprgq rarwqcdavl ygeqgqpwgr fgaaltvlgd vngdkltdva

541 igapgeednr gavylfhgts gsgispshsq riagsklspr lqyfgqslsg gqdltmdglv

601 dltvgaqghv lllrsqpvlr vkaimefnpr evarnvfecn dqvvkgkeag evrvclhvqk

661 strdrlregq iqsvvtydla ldsgrphsra vfnetknstr rqtqvlgltq tcetlklqlp

721 nciedpvspi vlrlnfslvg tplsafgnlr pvlaedaqrl ftalfpfekn cgndnicqdd

781 lsitfsfmsl dclvvggpre fnvtvtvrnd gedsyrtqvt fffpldlsyr kvstlqnqrs

841 qrswrlaces asstevsgal kstscsinhp ifpensevtf nitfdvdska slgnklllka

901 nvtsennmpr tnktefqlel pvkyavymvv tshgvstkyl nftasentsr vmqhqyqvsn

961 lgqrslpisl vflvpvrlnq tviwdrpqvt fsenlsstch tkerlpshsd flaelrkapv

1021 vncsiavcqr iqcdipffgi qeefnatlkg nlsfdwyikt shnhllivst aeilfndsvf

1081 tllpgqgafv rsqtetkvep fevpnplpli vgssvgglll lalitaalyk lgffkrqykd

1141 mmseggppga epq

[SEQ ID NO: 2] Amino acid sequence of CD11b (Integrin alpha-M isoform 2 precursor)

1 malrvlllta ltlchgfnld tenamtfqen argfgqsvvq lqgsrvvvga pqeivaanqr

61 gslyqcdyst gscepirlqv pveavnmslg lslaattspp qllacgptvh qtcsentyvk

121 glcflfgsnl rqqpqkfpea lrgcpqedsd iaflidgsgs iiphdfrrmk efvstvmeql

181 kksktlfslm qyseefrihf tfkefqnnpn prslvkpitq llgrthtatg irkvvrelfn

241 itngarknaf kilvvitdge kfgdplgyed vipeadregv iryvigvgda frseksrqel

301 ntiaskpprd hvfqvnnfea lktiqnqlre kifaiegtqt gssssfehem sqegfsaait

361 sngpllstvg sydwaggvfl ytskekstfi nmtrvdsdmn daylgyaaai ilrnrvqslv

421 lgapryqhig lvamfrqntg mwesnanvkg tqigayfgas lcsvdvdsng stdlvligap

481 hyyeqtrggq vsvcplprgr arwqcdavly geqgqpwgrf gaaltvlgdv ngdkltdvai

541 gapgeednrg avylfhgtsg sgispshsqr iagsklsprl qyfgqslsgg qdltmdglvd

601 ltvgaqghvl llrsqpvlrv kaimefnpre varnvfecnd qvvkgkeage vrvclhvqks

661 trdrlregqi qsvvtydlal dsgrphsrav fnetknstrr qtqvlgltqt cetlklqlpn

721 ciedpvspiv lrlnfslvgt plsafgnlrp vlaedaqrlf talfpfeknc gndnicqddl

781 sitfsfmsld clvvggpref nvtvtvrndg edsyrtqvtf ffpldlsyrk vstlqnqrsq

841 rswrlacesa sstevsgalk stscsinhpi fpensevtfn itfdvdskas lgnklllkan

901 vtsennmprt nktefqlelp vkyavymvvt shgvstkyln ftasentsrv mqhqyqvsnl

961 gqrslpislv flvpvrlnqt viwdrpqvtf senlsstcht kerlpshsdf laelrkapvv

1021 ncsiavcqri qcdipffgiq eefnatlkgn lsfdwyikts hnhllivsta eilfndsvft

1081 llpgqgafvr sqtetkvepf evpnplpliv gssvggllll alitaalykl gffkrqykdm

1141 mseggppgae pq

Alternatively, the antibody may be specifically Absiksimab (Abciximab), and the Absiksimab may be Absiksimab (Clotinab) manufactured by ISU abxis, but is not limited thereto.

The macrophage-1 antigen expression or activity inhibitor may be contained 0.2 to 20 mg/mL based on the total volume of the composition, and specifically 0.2 mg/mL or more, 0.3 mg/mL or more, 0.4 mg/mL or more, 0.5 mg/mL or more, 0.6 mg/mL or more, 0.7 mg/mL or more, 0.8 mg/mL or more, 0.9 mg/mL or more, 1 mg/mL or more, 1.1 mg/mL or more, 1.2 mg/mL or more, 1.3 mg/ mL or more, 1.4 mg/mL or more, 1.5 mg/mL or more, 1.6 mg/mL or more, 1.7 mg/mL or more, 1.8 mg/mL or more, 1.9 mg/mL or more, 2 mg/mL or more, 3 mg/mL or more , 4 mg/mL or more, 5 mg/mL or more, 6 mg/mL or more, 7 mg/mL or more, 8 mg/mL or more, 9 mg/mL or more, 10 mg/mL or more, 15 mg/mL or more, or 20 It may contain more than mg/mL, less than 20 mg/mL, less than 15 mg/mL, less than 10 mg/mL, less than 9 mg/mL, less than 8 mg/mL, less than 7 mg/mL, less than 6 mg/mL, 5 mg/mL or less, 4 mg/mL or less, 3.9 mg/mL or less, 3.8 mg/mL or less, 3.7 mg/mL or less, 3.6 mg/mL or less, 3.5 mg/mL or less, 3.4 mg/mL or less, 3.3 mg /mL or less, 3.2 mg/mL or less, 3.1 mg/mL or less, 3 mg/mL or less, 2.9 mg/mL or less, 2.8 mg/mL or less, 2.7 mg/mL or less, 2.6 mg/mL or less, 2.5 mg/mL Or less, 2.4 mg/mL or less, 2.3 mg/mL or less, 2.2 mg/mL or less, 2.1 mg/mL or less, 2 mg/mL or less, 1.5 mg/mL or less, 1 mg/mL or less, 0.5 mg/mL or less, or 0.2 mg/mL or less may be included, but is not limited thereto.

The microcirculation disorder refers to a case in which microcirculation is not normal, and means that microcirculation is not performed normally because white blood cells, red blood cells, platelets, and lymphocytes do not pass smoothly through capillaries. Specifically, the microcirculation disorder has a functional capillary ratio (FCR) according to Equation 1 below of 70% or less, 65% or less, 60% or less, 55% of the functional capillary fraction of the normal group without microcirculation disorder. Or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less, or the microcirculation disorder Is a functional capillary fraction of 0.4 or less, 0.38 or less, 0.36 or less, 0.34 or less, 0.32 or less, 0.3 or less, 0.28 or less, 0.26 or less, 0.24 or less, 0.22 or less, 0.2 or less, 0.18 or less, 0.16 or less, 0.14 or less, 0.12 or less , 0.1 or less, 0.08 or less, 0.06 or less, 0.04 or less, or 0.02 or less, but the range of the functional capillary fraction for determining whether the microcirculatory disorder is It may vary depending on the type, and is not limited to the above range.

[Equation 1]

Functional capillary fraction = functional capillary area / total capillary area

The improvement of the microcirculation disorder may be to increase the ratio of functional capillaries through which red blood cells pass among the total capillaries of the lung, and specifically, to increase the functional capillary ratio (FCR) according to the following equation 1. I can.

[Equation 1]

Functional capillary fraction = functional capillary area / total capillary area

The functional capillary refers to a capillary in which a function of capillaries among capillaries, for example, a function of exchanging oxygen, carbon dioxide, nutrients and other substances between blood and tissues by diffusion, occurs smoothly. The functional capillaries may be capillaries through which target elements in the bloodstream such as leukocytes, red blood cells, platelets, and lymphocytes move or pass. The more functional capillaries among the total capillaries, the smoother microcirculation of the individual or no microcirculation disorder.

The functional capillary area measurement may be to determine the same target element from a plurality of moving images in which the target element moves in the bloodstream to measure the area of the functional capillary, and the position difference according to the time difference of the target element in the blood flow It may be calculated by measuring the moving distance from the plurality of moving images. Specifically, the movement path of the target element in the bloodstream passing through the capillaries of the individual is measured over time from each of the plurality of moving images, and the movement of the target element in the plurality of blood flows It may be to measure the area of functional capillaries from the pathway. More specifically, by comparing a plurality of images with each other based on an image photographed with a time difference (t), target elements in the same blood flow are easily identified and tracked to measure the movement path of the target element in a single blood flow, The area of functional capillaries can be measured from the movement paths of target elements in a plurality of blood streams obtained by the same method.

The lung injury disease may be a disease caused by microcirculation disorders in the lungs, specifically pulmonary vasoconstriction, asthma, respiratory retardation, respiratory distress syndrome (RDS), acute respiratory distress syndrome (ARDS). ), cystic fibrosis (CF), allergic rhinitis (AR), pulmonary hypertension, emphysema, chronic obstructive pulmonary disease (COPD), lung transplant rejection, lung infection, bronchitis, and It may be one or more diseases selected from the group consisting of cancer, but is not limited thereto if it is a disease caused by microcirculation disorders in the lungs.

The expression or activity inhibitor of the macrophage-1 antigen according to an aspect of the present invention may be to increase the functional capillary fraction (FCR, %) in the pulmonary capillaries of an individual compared to a normal control or a control group before administration of the inhibitor. In particular, 1.1 times or more, 1.2 times or more, 1.3 times or more, 1.4 times or more, 1.5 times or more, 1.6 times or more, 1.7 times or more, 1.8 times or more, 1.9 times or more, 2 times or more, 2.1 times or more, 2.2 times It may increase more than, 2.3 times or more, 2.4 times or more, 2.5 times or more, 2.6 times or more, 2.7 times or more, 2.8 times or more, 2.9 times or more, or 3 times or more, but the pulmonary microcirculation disorder of the individual may be improved. The degree of increase in the functional capillary fraction is not limited to the above range. According to an embodiment of the present invention, in the CLP mouse model (CLP mouse model or pre-Abc mouse model) with pulmonary microcirculation disorder, FCR decreased by 50% or more compared to the normal group (Sham), and then Mac-1 expression or When the activity is inhibited (anti-Mac-1 mouse model, absiksimab mouse model, or post-Abc mouse model), FCR is increased by about 2 times or more to improve pulmonary microcirculation disorders, according to an embodiment of the present invention. It was found that the composition has an effect of preventing or treating lung injury diseases (Experimental Example 4-2 and FIG. 10A, and Experimental Example 4-3 and FIG. 12).

The macrophage-1 antigen expression or activity inhibitor according to an aspect of the present invention is the number of isolated neutrophils per unit area (512 X 512 μm) in the lung capillaries of an individual compared to the normal control or the control group before administration of the inhibitor. It may be to reduce, and specifically, the degree of reduction is 10% or more based on the number of isolated neutrophils per unit area (512 X 512 μm) in the pulmonary capillaries of the control group or the control group before administration of the inhibitor, It may be 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, or 55% or more, but the pulmonary microcirculation disorder of the individual is improved As long as the number of isolated neutrophils per unit area (512 X 512 μm) in capillaries is reduced, the range is not limited.

In the above aspect, the composition may be a pharmaceutical composition or a food composition.

The pharmaceutical composition may be formulated as an oral dosage form in a solid, semi-solid or liquid form by adding a commercially available inorganic or organic carrier using the composition as an active ingredient.

The preparations for oral administration include tablets, pills, granules, soft capsules, powders, fine granules, powders, emulsions, syrups, pellets, and the like. In order to formulate the active ingredient of the present invention, it can be easily formulated if carried out according to conventional methods, and surfactants, excipients, coloring agents, spices, preservatives, stabilizers, buffers, suspensions, and other commonly used auxiliary agents can be appropriately used.

The pharmaceutical composition according to the present invention may contain an increase in the number of neutrophils isolated in the pulmonary capillaries due to lung damage diseases, specifically lung microcirculation disorders, more specifically pulmonary microcirculation disorders, or dead space ), or a decrease in the number of red blood cells passing through capillaries can be usefully used for preventing or treating lung injury diseases. The lung damage diseases include pulmonary vasoconstriction, asthma, respiratory retardation, respiratory distress syndrome (RDS), acute respiratory distress syndrome (ARDS), cystic fibrosis (CF), allergies. Allergic rhintis (AR), pulmonary hypertension, emphysema, chronic obstructive pulmonary disease (COPD), lung graft rejection, lung infection, bronchitis, cancer, etc., but are not limited thereto.

The pharmaceutical composition may be administered orally, rectal, topical, transdermal, intravenous, intramuscular, intraperitoneal, subcutaneous, or the like.

In addition, the dosage of the composition or the active ingredient in the composition will vary depending on the age, sex, and weight of the subject to be treated, the specific disease or pathological condition to be treated, the severity of the disease or pathological condition, the route of administration, and the judgment of the prescriber. Dosage determination based on these factors is within the level of one of skill in the art. The dosage may be 0.001 mg/kg/day to 2000 mg/kg/day, specifically 0.5 mg/kg/day to 1500 mg/kg/day, more specifically 0.001 mg/kg/day or more, 0.01 mg/kg/day or more, 0.1 mg/kg/day or more, 0.5 mg/kg/day or more, 1 mg/kg/day or more, 10 mg/kg/day or more, 50 mg/kg/day or more, 100 mg/ kg/day or more, 150 mg/kg/day or more, 200 mg/kg/day or more, 250 mg/kg/day or more, 300 mg/kg/day or more, 350 mg/kg/day or more, 400 mg/kg/ Day or more, 450 mg/kg/day or more, 500 mg/kg/day or more, 550 mg/kg/day or more, 600 mg/kg/day or more, 650 mg/kg/day or more, 700 mg/kg/day or more , 750 mg/kg/day or more, 800 mg/kg/day or more, 850 mg/kg/day or more, 900 mg/kg/day or more, 950 mg/kg/day or more, 1000 mg/kg/day or more, 1050 mg/kg/day or more, 1100 mg/kg/day or more, 1150 mg/kg/day or more, 1200 mg/kg/day or more, 1250 mg/kg/day or more, 1300 mg/kg/day or more, 1350 mg/ kg/day or more, 1400 mg/kg/day or more, 1450 mg/kg/day or more, 1500 mg/kg/day or more, 1550 mg/kg/day or more, 1600 mg/kg/day or more, 1650 mg/kg/ Days or more, 1700 mg/kg/day or more, 1750 mg/kg/day or more, 1800 mg/kg/day or more, 1850 mg/kg/day or more, 1900 mg/kg/day or more, or 1950 mg/kg/day or more Can be, 2000 mg/kg/day or less, 1950 mg/kg/day or less, 1900 mg/kg/day or less, 1850 mg/kg/day or less, 1800 mg/kg/day or less, 1750 mg/kg/day or less , 1700 mg/kg/day or less, 1650 mg/kg/day or less, 1600 mg/kg/day or less, 1550 mg/k g/day or less, 1500 mg/kg/day or less, 1450 mg/kg/day or less, 1400 mg/kg/day or less, 1350 mg/kg/day or less, 1300 mg/kg/day or less, 1250 mg/kg/ 1 day or less, 1200 mg/kg/day or less, 1150 mg/kg/day or less, 1100 mg/kg/day or less, 1050 mg/kg/day or less, 1000 mg/kg/day or less, 950 mg/kg/day or less , 900 mg/kg/day or less, 850 mg/kg/day or less, 800 mg/kg/day or less, 750 mg/kg/day or less, 700 mg/kg/day or less, 650 mg/kg/day or less, 600 mg/kg/day or less, 550 mg/kg/day or less, 500 mg/kg/day or less, 450 mg/kg/day or less, 400 mg/kg/day or less, 350 mg/kg/day or less, 300 mg/ kg/day or less, 250 mg/kg/day or less, 200 mg/kg/day or less, 150 mg/kg/day or less, 100 mg/kg/day or less, 50 mg/kg/day or less, 10 mg/kg/ It may be less than or equal to 1 mg/kg/day, less than 0.5 mg/kg/day, less than 0.1 mg/kg/day, or less than 0.01 mg/kg/day.

The food composition may be a health food composition, the macrophage-1 antigen expression or activity inhibitor of the present invention may be added as it is or may be used with other foods or food ingredients, and may be appropriately used according to a conventional method.

There is no particular limitation on the kind of the health food. Examples of foods to which the green tea extract can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, and drinks. , Alcoholic beverages and vitamin complexes, and includes all health foods in the usual sense.

The health beverage composition of the present invention may contain various flavoring agents or natural carbohydrates as an additional component, like a conventional beverage. The natural carbohydrates described above are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. As the sweetener, natural sweeteners such as taumatin and stevia extract, and synthetic sweeteners such as saccharin and aspartame can be used. The ratio of the natural carbohydrate may be 0.01 to 0.04% by weight, specifically 0.02 to 0.03% by weight, based on the total weight of the composition of the present invention.

In addition to the above, the health food of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, colorants, pectic acids and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols. , Carbonated beverages used in carbonated beverages, and the like. In addition, it may contain flesh for the manufacture of natural fruit juice, fruit juice beverage and vegetable beverage. These ingredients may be used independently or in combination. The proportion of these additives may be 0.01 to 0.1% by weight based on the total weight of the composition of the present invention.

In another aspect, the present invention comprises the steps of: (a) preparing a lung injury model; (b) treating a test substance in the lung injury model; And (c) measuring a change in the expression or activity of macrophage-1 antigen (Mac-1) in neutrophils in pulmonary capillaries of the lung injury model by the test substance; It provides a method of preventing, improving, or screening a therapeutic substance. The description of the microcirculation, microcirculation disorder, neutrophil, macrophage-1 antigen, and lung injury disease are as described above.

The lung injury model may be a non-human animal such as monkey, dog, cat, rabbit, mormote, rat, mouse, cow, sheep, pig, goat, etc., and specifically, may be a non-human animal with lung damage due to sepsis, More specifically, it may be a CLP (cecal ligation and puncture) model, which is a non-human animal that caused sepsis by LPS administration or a non-human animal that made a puncture and ligated it in the cecum, but is limited thereto. It is not.

In one embodiment of the present invention, the "relative expression level" is macrophage in neutrophils in pulmonary capillaries after treatment with a test substance for expression or activity of macrophage-1 antigen in neutrophils in pulmonary capillaries before treatment of the test substance. 1 When the expression or activity of the antigen is compared, the expression or activity may be suppressed. Alternatively, the "relative expression level" is compared with the expression or activity of the macrophage-1 antigen in neutrophils in pulmonary capillaries treated with the test substance and the expression or activity of the macrophage-1 antigen in neutrophils in the pulmonary capillaries treated with the test substance. It may be the degree to which the expression is suppressed. The relative expression level may include, for example, a relative expression level of an mRNA or a relative expression level of a protein.

The step (c) may include comparing the expression or activity of the macrophage-1 antigen in neutrophils in the pulmonary capillaries before and after treatment of the test substance in the lung injury model. Alternatively, the step (c) may include comparing the expression or activity of the macrophage-1 antigen in neutrophils in the lung capillaries of the lung injury model treated with the test substance and the lung injury model not treated with the test substance. .

In addition, the screening method is a result of measuring the expression or activity of step (c), when the expression or activity of the macrophage-1 antigen decreases compared to before treatment of the test substance, the test substance is used to prevent, improve or treat lung injury diseases. It may further include the step of determining the substance.

The step of determining as a substance for preventing, improving, or treating lung damage disease is when the expression or activity (relative expression amount) of the macrophage-1 antigen decreases by about 10% or more as a result of measuring the expression or activity in step (c). It may include determining as a substance for preventing, improving, or treating damage diseases. That is, after treatment of the test substance for the expression or activity of the macrophage-1 antigen in neutrophils in the lung capillaries of the lung injury model before treatment of the test substance, the neutrophils in the pulmonary capillaries of the lung injury model were treated with the macrophage-1 antigen. When the expression or activity is reduced by about 10% or more when the degree of expression or activity is compared, it can be determined as a substance for preventing, improving, or treating lung injury diseases. Alternatively, the expression or activity of the macrophage-1 antigen in neutrophils in the pulmonary capillaries of the lung injury model treated with the test substance was determined by the expression of the macrophage-1 antigen in neutrophils in the pulmonary capillaries of the lung injury model without the test substance, or When the expression increases by about 10% or more compared to the level of activity, it can be determined as a substance for preventing, improving or treating lung injury diseases. For example, the expression or activity (relative expression level) of the macrophage-1 antigen is 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 15% or more, 16% than before treatment with the test substance. More than, 17% or more, 18% or more, 19% or more, 20% or more, 21% or more, 22% or more, 23% or more, 24% or more, 25% or more, 26% or more, 27% or more, 28% or more, 29% or more, 30% or more, 31% or more, 32% or more, 33% or more, 34% or more, 35% or more, 36% or more, 37% or more, 38% or more, 39% or more, 40% or more, 41% More than, 42% or more, 43% or more, 44% or more, 45% or more, 46% or more, 47% or more, 48% or more, 49% or more, 50% or more, 51% or more, 52% or more, 53% or more, 54% or more, 55% or more, 56% or more, 57% or more, 58% or more, 59% or more, 60% or more, 61% or more, 62% or more, 63% or more, 64% or more, 65% or more, 66% More than, 67% or more, 68% or more, 69% or more, 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, A decrease of 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, or 90% or more, It may be determined as a substance for preventing, improving, or treating lung damage disease, but is not limited thereto. The level of expression or activity is a result measured while ensuring statistical significance. The concept of statistical significance is a case in which a significant difference is shown through biological statistical analysis, and in a quantitative case, the p value is less than 0.05.

In addition, as an example, the degree of expression or activity of the macrophage-1 antigen is determined by known techniques, such as reverse transcription polymerase chain reaction (RT-PCR), ELISA, Western Blot, or Immune. It can be confirmed using a blot (Immune Blot), but is not limited thereto.

In another aspect, the present invention comprises the steps of: (a) preparing a lung injury model; (b) treating a test substance in the lung injury model; And (c) measuring a change in the expression or activity of macrophage-1 antigen (Mac-1) in neutrophils in pulmonary capillaries of the lung injury model by the test substance; It provides a method for screening substances for improving microcirculation disorders. The lung injury model, microcirculation, microcirculation disorder, neutrophil, macrophage-1 antigen, and the relative expression levels are as described above.

The step (c) may include comparing the expression or activity of the macrophage-1 antigen in neutrophils in the pulmonary capillaries before and after treatment of the test substance in the lung injury model. Alternatively, the step (c) may include comparing the expression or activity of the macrophage-1 antigen in neutrophils in the lung capillaries of the lung injury model treated with the test substance and the lung injury model not treated with the test substance. .

In addition, the screening method is a result of measuring the expression or activity in step (c), when the expression or activity of the macrophage-1 antigen decreases compared to before treatment of the test substance, the test substance is used as a substance for improving the microcirculation disorder of the lung. It may further include determining.

The step of determining as a substance for improving the microcirculation disorder in the lungs is when the expression or activity (relative expression amount) of the macrophage-1 antigen decreases by about 10% or more as a result of measuring the expression or activity in step (c), It may include determining as a circulatory disorder improving substance. In other words, after treatment of the test substance for the expression or activity of the macrophage-1 antigen in neutrophils in the lung capillaries of the lung injury model before treatment with the test substance, the neutrophils in the lung capillaries of the lung injury model When the expression or activity level is compared and the expression or activity decreases by about 10% or more, it can be determined as a substance for improving microcirculation disorders in the lungs. Alternatively, the expression or activity of the macrophage-1 antigen in neutrophils in the pulmonary capillaries of the lung injury model treated with the test substance was determined by the expression of the macrophage-1 antigen in neutrophils in the pulmonary capillaries of the lung injury model not treated with the test substance, or When the expression decreases by about 10% or more compared to the level of activity, it can be determined as a substance for improving microcirculation disorders in the lungs. For example, the expression or activity (relative expression level) of the macrophage-1 antigen is 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 15% or more, 16% than before treatment with the test substance. More than, 17% or more, 18% or more, 19% or more, 20% or more, 21% or more, 22% or more, 23% or more, 24% or more, 25% or more, 26% or more, 27% or more, 28% or more, 29% or more, 30% or more, 31% or more, 32% or more, 33% or more, 34% or more, 35% or more, 36% or more, 37% or more, 38% or more, 39% or more, 40% or more, 41% More than, 42% or more, 43% or more, 44% or more, 45% or more, 46% or more, 47% or more, 48% or more, 49% or more, 50% or more, 51% or more, 52% or more, 53% or more, 54% or more, 55% or more, 56% or more, 57% or more, 58% or more, 59% or more, 60% or more, 61% or more, 62% or more, 63% or more, 64% or more, 65% or more, 66% More than, 67% or more, 68% or more, 69% or more, 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, A decrease of 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, or 90% or more, It may be determined as a substance for improving the microcirculation disorder of the lungs, but is not limited thereto. The level of expression or activity is a result measured while ensuring statistical significance. The concept of statistical significance is a case in which a significant difference is shown through biological statistical analysis, and in a quantitative case, the p value is less than 0.05.

In addition, as an example, the degree of expression or activity of the macrophage-1 antigen is determined by known techniques, such as reverse transcription polymerase chain reaction (RT-PCR), ELISA, Western Blot, or Immune. It can be confirmed using a blot (Immune Blot), but is not limited thereto.

Since one embodiment of the present invention can provide a macrophage-1 antigen in neutrophils in the pulmonary capillaries as a biomarker capable of diagnosing pulmonary microcirculation disorders, another embodiment of the present invention uses this A composition or kit for diagnosing microcirculatory disorders may be provided. In addition, another embodiment of the present invention may provide a method of providing information for diagnosing pulmonary microcirculation disorder using the same.

Specifically, the present invention comprises the step of measuring the expression or activity of macrophage-1 antigen (Mac-1) in neutrophils isolated from pulmonary capillaries of a test subject, pulmonary microcirculation disorder A method of providing information for diagnosis may be provided.

In one embodiment, the method is to determine the level of expression or activity of macrophage-1 antigen in neutrophils isolated from pulmonary capillaries of the test subject and the level of expression or activity of macrophage-1 antigen in neutrophils isolated from pulmonary capillaries of a normal control group. It may further include comparing with.

In addition, as an embodiment, the method includes the expression or activity of macrophage-1 antigen in neutrophils isolated from pulmonary capillaries of the test subject in which the expression or activity of macrophage-1 antigen in neutrophils isolated from pulmonary capillaries of the test subject is If it is higher than the degree, the step of providing information that there is a pulmonary microcirculation disorder may be further included. For example, the level of macrophage-1 antigen expression or activity in neutrophils isolated from pulmonary capillaries of the test subject is 10% or more than the expression or activity of macrophage-1 antigen in neutrophils isolated from pulmonary capillaries of the normal control , 11% or more, 12% or more, 13% or more, 14% or more, 15% or more, 16% or more, 17% or more, 18% or more, 19% or more, 20% or more, 21% or more, 22% or more, 23 % Or more, 24% or more, 25% or more, 26% or more, 27% or more, 28% or more, 29% or more, 30% or more, 31% or more, 32% or more, 33% or more, 34% or more, 35% or more , 36% or more, 37% or more, 38% or more, 39% or more, 40% or more, 41% or more, 42% or more, 43% or more, 44% or more, 45% or more, 46% or more, 47% or more, 48 % Or more, 49% or more, 50% or more, 51% or more, 52% or more, 53% or more, 54% or more, 55% or more, 56% or more, 57% or more, 58% or more, 59% or more, 60% or more , 61% or more, 62% or more, 63% or more, 64% or more, 65% or more, 66% or more, 67% or more, 68% or more, 69% or more, 70% or more, 71% or more, 72% or more, 73 % Or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more , 86% or more, 87% or more, 88% or more, 89% or more, or 90% or more, it can be determined that there is a pulmonary microcirculation disorder. The level of expression or activity is a result measured while ensuring statistical significance. The concept of statistical significance is a case in which a significant difference is shown through biological statistical analysis, and in a quantitative case, the p value is less than 0.05.

In another aspect, the present invention can provide a composition for diagnosing pulmonary microcirculation disorders, including a reagent for detecting mRNA or protein of macrophage-1 antigen (Mac-1) in neutrophils in pulmonary capillaries.

In another aspect, the present invention can provide a kit for diagnosing pulmonary microcirculation disorders comprising a reagent for detecting mRNA or protein of macrophage-1 antigen (Mac-1) in neutrophils in pulmonary capillaries, The kit may further include an instruction sheet describing a method of providing information for diagnosing microcirculation disorders as described above.

As an embodiment, the reagent for detecting mRNA or protein of macrophage-1 antigen included in the composition or kit for diagnosing whether or not pulmonary microcirculation disorder may include one or more of primers and probes that specifically bind to macrophage-1 antigen. . As an embodiment, the macrophage-1 antigen protein detection reagent may include at least one of an antibody and a probe that specifically binds to the macrophage-1 antigen.

In the present specification, "probe" refers to a polynucleotide having a base of a sequence capable of complementarily binding to a target site of a gene, a variant thereof, or a polynucleotide and a labeling material bound thereto.

In the present specification, "primer" refers to a polynucleotide having a base of a sequence capable of complementarily binding to the end of a specific region of a gene used to amplify a specific region corresponding to a target site of a gene using PCR, or Means that variant. The primer is not required to be completely complementary to the end of a specific region, and may be used as long as it is complementary enough to hybridize to the end to form a double chain structure.

In the present specification, "hybridization" means that two single-stranded nucleic acids form a duplex structure by pairing of complementary base sequences.

Hybridization can occur not only when the complementarity between single-stranded nucleic acid sequences is perfect, but also in the presence of some mismatch bases.

In the present specification, "polynucleotide" refers to a polymer of a plurality of nucleotides, and refers to a polynucleotide in a broad sense including an oligonucleotide, which is a polymer of dozens of nucleotides in a conventional sense.

Hereinafter, the configuration and effects of the present invention will be described in more detail with reference to examples and experimental examples. However, the following examples and experimental examples are provided for illustrative purposes only to aid understanding of the present invention, and the scope and scope of the present invention are not limited thereto.

On the other hand, all animal experiments in the following Examples and Experimental Examples were performed according to standard guidelines for the management and use of laboratory animals, and the Institutional Animal Ethics Committee of KAIST (protocol No. KA2014-30 and KA2016-55). Care and Use Committee, IACUC).

In addition, all data in the following experimental examples are expressed as an appropriate mean±SD or median±interquartile range in order to represent the values of each group. Statistical differences between means or median values were determined by the unpaired 2-tailed Student's t-test, Mann-Whitney test, one-way ANOVA using post hoc Holm-Sidak's multiple comparisons, or Kruskal-Wallis test using post hoc Dunn's multiple comparisons. Was appropriately determined by Statistical significance was set to P <0.05, and analysis was performed with Prism 6.0 (GraphPad).

[ Example 1] Sepsis-induced acute lung injury mouse model preparation

In order to study a composition for preventing or treating lung injury disease, a mouse model of acute lung injury induced by sepsis was prepared by the following method.

All mice used in this example were ventilated under a light:dark cycle of 12 hours: 12 hours (12:12h), and temperature (22.5 °C) and humidity (52.5%) were controlled in cages. They were housed individually and provided with a standard diet and ad libitum . Male mice (20 to 30 g) aged 8 to 20 weeks were used as the experimental group. LysM ^{GFP /+} mice were provided by Professor Minsoo Kim of the University of Rochester, USA (hereinafter referred to as LysM ^GFP/+ mouse model).

The LysM ^{GFP / +} mouse model administered with a high dose of LPS or a CLP (cecal ligation and puncture) mouse model as a sepsis-induced acute lung injury (ALI) mouse model. The experiment was carried out.

In the case of the high-dose LPS administration model, 3 to 6 hours before capillary angiography, LPS (10 mg/kg, E.coli serotype 055:B5, L2880, Sigma-Aldrich) was applied peritoneum to the LysM ^{GFP /+} mouse. peritoneum) intraperitoneally (hereinafter referred to as an ALI mouse model, a mouse model 3 hours after LPS administration among ALI mouse models is referred to as an LPS 3h mouse model, and a mouse model 6 hours after LPS administration is referred to as an LPS 6h mouse model). As a control group, mice injected with the same amount of PBS into the peritoneum (hereinafter referred to as a control group or a PBS mouse model) were prepared.

The CLP model was performed by a single experienced experimenter according to the method described above. Specifically, 75% of the appendix (membranous intestine, cecum) of the LysM ^{GFP /+} mouse is firmly housed with 6-0 black silk, and a single puncture with a double hole pierced at the end of the cecum with a 21-gauge needle I made it. Then, the cecum was gently squeezed to check whether a puncture for pushing the feces was open. The cecum was replaced with the abdominal cavity, and the abdominal incision was sutured with 4-0 black silk. The shame group went through the same surgical procedure except for cecal ligation and puncture.

[Example 2] Neutrophil removal model (N-Dep model and N-Dep+LPS model) preparation

In order to confirm the effect of neutrophils on lung damage caused by pulmonary microcirculation disorder, a mouse model depleted of neutrophils (hereinafter, neutrophil removal model) was prepared. Specifically, by intraperitoneally injecting 200 μg of an anti-Ly6G+ monoclonal antibody (Clone 1A8, 551459, BD Biosciences) to the LysM ^{GFP /+} mouse of Example 1 24 hours before in vivo lung imaging, neutrophils A mouse model (hereinafter, N-Dep model) without depleted lung damage was prepared. In the same way, 24 hours before the acute lung injury mouse model of Example 1 was prepared in the acute lung injury mouse model of Example 1, anti-Ly6G+ monoclonal antibody (Clone 1A8, 551459, BD Biosciences) 200 μg was injected intraperitoneally to prepare a lung injury mouse model (hereinafter, N-Dep+LPS model) depleted of neutrophils.

[Example 3] Neutrophil labeling and in vivo lung imaging

(1) neutrophil labeling

To visualize the molecular expression of sequenced neutrophils in the lungs in vivo , 2 hours before imaging, 25 μg of Alexa Fluor 555 (A-20005, ThermoFisher Scientific) conjugated to the fluorophore. CD11b (Clone M1/70, 553307, BD Biosciences) and 25 μg of CD18 (Clone GAME-46, 555280, BD Biosciences) were each injected through the tail of the mouse model of Example 1 to label neutrophils. .

In addition, erythrocytes and vasculature of the mouse model of Example 1 were fluorescently stained. Specifically, red blood cells were obtained through cardiac puncture, and then labeled with Vybrant DiD (V22887, ThermoFisher Scientific) according to the method described in the product information sheet. Then, just before photographing, the 50 million count of DiD-labeled red blood cells were injected through the vascular catheter of the tail vein of the mouse model of Example 1 above. In addition, in order to visualize the vasculature, FITC (molecular weight 2M Da, Sigma-Aldrich) or tetramethylrhodamine (TMR) bound with dextran dye was added to the mouse model of Example 1 as described above. It was injected through the same vascular catheter.

The procedure of injecting CD11b and CD18 with luminescent fluorophores bound to the mouse model, DiD-labeled red blood cells, and FITC or TMR bound with dextran dye was described in the following in vivo lung imaging.

(2) In vivo lung imaging

In vivo lung imaging was performed as follows.

Specifically, LysM of Example 1 ^{GFP /+} mouse model, ALI mouse model, PBS mouse model and Shame group mouse model, N-Dep mouse model and N-Dep+LPS mouse model of Example 2, Mac-1 of Example 3 Inhibitory mouse model was anesthetized with ketamine (80 mg/kg) and xylazine (12 mg/kg), and then intubated using a 20 gauge vascular catheter with a lighting guidewire and artificial It was connected to a respiratory (MouseVent, Kent Scientific). The respiration was performed by setting an inspiratory pressure of 24 to 30 mmHg, a respiratory rate of 120 to 130 per minute, and a positive-end expiratory pressure (PEEP) of ₂ cmH ₂ O. 2% isoflurane was administered to maintain anesthesia, and pulse oximetry was applied to monitor oxygen supply and survival. A heat needle from a constant temperature system (RightTemp, Kent Scientific) was injected into the rectum, and the body temperature was maintained at 37.0°C using a feedback-regulated heating pad. The tail vein was cannulated with a 30-gauge needle attached to a PE-10 tube for intravenous injection of the dye, red blood cells and neutrophils of (1) above. Then, the mouse model was placed on the right side, and left thoracotomy was performed. The skin and muscle were incised until the ribs were exposed, and the pleura was exposed by making an incision between the 3rd and 4th ribs. After thoracotomy, the imaging window of the following experimental example was applied to the pleural surface, and a pump (DOA-P704-AA, GAST) and a regulator (NVC 2300a, EYELA) through a tube connected to the lung imaging window were used to provide a negative suction pressure. ) Was added.

[Experimental Example 1] Pulmonary microcirculation photographing in a neutrophil removal model

To visualize the pulmonary microcirculation in vivo through the pulmonary imaging window, a custom-built video-rate laser-scanning confocal microscopy system was implemented.

Imaging system

Specifically, three types of continuous laser modules (488 nm (MLD488, Kobold), 561 nm (Jive, Kobold), and 640 nm (MLD640, Kobold) were used as excitation lightweight light sources for multicolor fluorescence imaging. light source). The laser beam was integrated collinearly by a dichroic beam splitter (DBS1; FF593-Di03, DSB2; FF520-Di02, Semrock), and a multi-edge dichroic It was sent to the laser-beam scanner by a beam splitting device (DBS3; Di01-R405/488/561/635, Semrock). The laser scanning unit includes X-axis scanning using a rotating polygonal mirror with 36 sides (MC-5, aluminum coating, Lincoln Laser) and Y-axis scanning using a galvanometer scanning mirror (6230H, Cambridge Technology). It consists of four axes. Control mouse model of Example 1 (PBS), ALI mouse model ( LPS), the neutrophil removal model of Example 2 (N-Dep mouse model and N-Dep+LPS mouse model) was transferred to each lung with a two-dimensional raster scanning laser beam. The fluorescence signal emitted from the lungs of the mouse model was detected in advance in the XYZ conversion 3D step (3DMS, Sutter Instrument) by the objective lens. The de-scanned three-color fluorescent signal is divided into a spectrum with a dichroic beam splitter (DBS4; FF560-Di01, DBS5; FF649-Di01, Semrock), and then a band pass filter (BPF1). ; FF02-525/50, BPF2; FF01-600/37, BPF3; FF01-685/40, Semrock) through a photomultiplier (PMT; R9110, Hamamatsu). The voltage output of each PMT was digitized by a 3-channel frame grabber (Solios, Matrox) with 8-bit resolution at a sampling rate of 10 MHz. Using customized imaging software based on Matrox Imaging Library (MIL9, Matrox) and Visual C#, movies with image speed were displayed and recorded in real time with a frame rate of 30 Hz and a frame size of 512 X 512 pixels. .

Image processing

Images captured using the imaging system were displayed and stored at an acquisition speed of 30 frames per second of 512 x 512 pixels per frame. Real-time image frames were averaged over 30 frames using MATLAB (Mathworks) code to improve contrast and signal-to-noise ratio. To minimize motion artifacts, each frame was processed with an image registration algorithm before averaging. Image rendering using 3D reconstruction, track analysis of neutrophils, and track displacement display were performed with IMARIS 8.2 (Bitplane).

The imaging of the lung microcirculation of each of the control mouse model (PBS) of Example 1, the ALI mouse model (LPS), and the neutrophil removal model of Example 2 (N-Dep mouse model and N-Dep+LPS mouse model) The result of processing the image photographed by the system and obtained therefrom according to the image processing procedure is shown in FIG. 2. At this time, the N-Dep+LPS mouse model among the neutrophil removal models of Example 2 was photographed by the image system by the above method 6 hours after LPS injection. On the other hand, in the sepsis-induced acute lung injury mouse model, the functional capillary ratio (FCR; calculated as the functional capillary area to the total capillary area) decreases at the beginning of the acute lung injury, as shown in FIG. 2. As shown, unlike the control group (PBS), in the ALI mouse model (LPS), a dead space in which red blood cells cannot pass through capillaries is formed, and isolated neutrophils are increased. On the other hand, the neutrophil removal model (N-Dep mouse model and N-Dep+LPS mouse model) has more functional capillaries where red blood cells pass through capillaries to facilitate microcirculation, resulting in a dead space and isolated It was confirmed that FCR increased and pulmonary microcirculation disorder was improved due to a decrease in neutrophils.

[ Experimental example 2] Neutral Confirmation of Functional Capillary Ratio (FCR) of removal model

Functional capillary fraction (FCR) comparison

It was confirmed from Experimental Example 1 that the pulmonary microcirculation disorder was improved in the neutrophil removal model, and in order to confirm this as quantified data, DiD-labeling moving in the capillaries obtained through the imaging system and image processing of Experimental Example 1 The functional capillary image was analyzed using real-time images of red blood cells. After dividing the color of the image, the sequential image of the channel for detecting DiD was processed with a median filter having a radius of 2 pixels to increase the signal-to-noise ratio. A maximal intensity projection of 600-900 frames (20-30 seconds) was created to show the functional capillaries perfused by red blood cells. Functional capillary ratio (FCR) was calculated by Equation 1 below.

[Equation 1]

Functional capillary fraction = area of functional capillaries / area of total capillaries.

In Equation 1, the total capillary area is the area of the blood vessel detected by the dextran signal, and the functional capillary area is the area to which the DiD-labeled red blood cells move. All image processing for calculating the functional capillary fraction was performed by ImageJ (https://imagej.nih.gov/ij/), and the results are as shown in FIG. 3A (n (number of fields) = 30, 10 FOV (Field of View) per mouse, 3 per group, P <0.05, one-way ANOVA using post hoc Holm-Sidak's multiple comparison test, data are mean ± sd).

As shown in Figure 3a, compared to the control (PBS) mouse model, compared to the control (PBS) mouse model, the FCR (%) of the ALI mouse model (LPS) decreased by 50% or more, confirming that pulmonary microcirculation disorders occurred due to lung damage, and the ALI mouse model Compared with (LPS), the neutrophil removal model (N-Dep mouse model and N-Dep+LPS mouse model) increased FCR (%) by about 3 times or more, confirming that the pulmonary microcirculation disorder was improved.

Histological analysis

The unit area of each of the control mouse model (PBS) of Example 1, the ALI mouse model (LPS), and the neutrophil removal model of Example 2 (N-Dep mouse model and N-Dep+LPS mouse model) (512 X 512 Histological analysis was performed to compare the number of neutrophils per μm).

Specifically, after in vivo imaging of the mouse model, lung tissue was collected, perfusion and fixation were performed with 4% paraformaldehyde, and then additionally fixed overnight in 4% paraformaldehyde. . For H&E (hematoxylin and eosin) staining, the fixed tissue was treated using a standard procedure, placed in paraffin, and cut into 4 μm sections, and H&E staining was performed using a conventional method. As shown in Figure 3b (n (number of fields) = 30, 10 FOV (Field of View) per mouse, 3 per each group, P <0.05, one-way ANOVA using a post hoc Holm-Sidak multiple comparison test , Data are mean ± sd). In FIG. 3B, cells/field is the number of neutrophils per unit area (512 X 512 μm), and the filed means unit area (512 X 512 μm).

In addition, as shown in FIG. 3B, the number of neutrophils (LysM+ cells) decreased to a certain level in the preparation of the neutrophil removal model of Example 2, but considering the magnified image of FIG. 2, in the extravascular space. It was confirmed that neutrophils were not completely depleted due to the debris of macrophages (LysM+ cells) presumed to be lung macrophages.

3A and 3B, it was found that when the number of LysM+ cells, which are neutrophils in most blood vessels, decreases, the functional capillary fraction (FCR) in the lung microcirculation is improved. In addition, it was found that neutrophils function as a major component of aggregates in the lung microcirculation and primary blockers of blood flow when systemic inflammation occurs.

[ Experimental example 3] In the capillaries for preventing or treating lung damage diseases Neutral target quest

From Experimental Example 2, when the number of neutrophils in the capillaries forming microcirculation is reduced, it can be seen that microcirculation disorders can be improved to prevent or treat lung damage diseases, but this neutrophil reduction strategy is difficult to perform clinically. There is this. Accordingly, the following experiment was performed to search for targets in isolated neutrophils for preventing or treating lung injury diseases. At this time, the present inventors hypothesized that the integrin expression pattern of neutrophils derived from the left ventricle that has already passed through the pulmonary capillaries and the pattern of neutrophils derived from the lungs are different.

Flow cytometry

First, neutrophils were separated from each of the left ventricle (LV) and lungs of the control (PBS) mouse model and the ALI mouse model (LPS) of Example 1, and the neutrophils derived from the left ventricle and the neutrophils derived from the lungs were To investigate integrin expression, flow cytometry was performed on the two isolated neutrophils.

First, in order to separate the isolated neutrophils of the lungs, the lungs were harvested without perfusion and were digested. Then, the lungs were put in a PBS solution, finely chopped, filtered through a 40 μm filter, and stained at 4° C. for 30 minutes. Meanwhile, in order to separate neutrophils from the left ventricle, 100 μl of blood was extracted from the left ventricle of a mouse model using injection, and then neutrophils 1.0 X 10 ⁶ cells were hemolyzed using flow cytometry (FACS) (BD, LSRFortessa ^TM ). Was separated. Clonal antibodies used in this experimental example were Ly6G-FITC (1A8, 551460, BD Biosciences), CD11a-BV510 (M17/4, 563669, BD Biosciences) CD11b-PE-Cy7 (M1/70, 552850, BD Biosciences), CD18-APC (C71/16, 562828, BD Biosciences), CD62L (MEL-14, 560514, BD Biosciences), Viability Dye eFluor 506 (65-0866-14, ThermoFisher Scientific), and stained cells are LSR Fortessa flow cytometer (BD Biosciences). Then, flow cytometry was performed on the two isolated neutrophils gated on Ly6G+ using Flowjo (FlowJo, LLC), and the results are as shown in FIGS. 5 and 6A to 6D (each group ( Group, n per group) (number of mice) = 5, * P <0.05, Mann-Whitney test, MFI represents mean fluorescence intensity, data is mean ± sd).

5 and 6A to 6D, the expression levels of CD11b and CD18 are higher in the neutrophils of the ALI mouse model (LPS) compared to the neutrophils of the control (PBS), and in the same mouse model, the neutrophils derived from the lungs were compared to the neutrophils derived from the left ventricle. It was confirmed that the expression level of CD18 was higher (n = 5 per each group, * P <0.05, Mann-Whitney test). MFI, mean fluorescence intensity. Data are means ± s.d.).

In vivo imaging

Integrins in the isolated neutrophils of the control (PBS) mouse model and the ALI mouse model (LPS) of Example 1 were photographed and imaged according to the method of Experimental Example 1. 7A and 7B are results of in vivo visualization of the expression on the cell surface of each of CD11b and CD18 in neutrophils and neutrophils.

As shown in FIGS. 7A and 7B, a very small number of neutrophils were identified in the control group (PBS), and CD11b and CD18 were hardly expressed on the surface of the neutrophils. In contrast, in the ALI mouse model (LPS), a very large number of neutrophils were identified, and it was confirmed that the expression levels of CD11b and CD18 were very high on the surface of the neutrophils.

Comparison of the number of neutrophils expressing CD11b or CD18

The number of neutrophils expressing CD11b or CD18 was compared between the ALI mouse model (LPS) of Example 1 and the control mouse model (PBS), and the results are the same as in FIGS. 8A to 8D. (n (number of fields) = 9, 3 FOV (Field of View) per mouse, 3 per each group, * P <0.05, Mann-Whitney test, data is mean ± sd) The filed of FIGS. 8A and 8C is a unit area (512 X 512 μm).

As shown in FIGS. 8A to 8D, the number of neutrophils expressing CD11b on the surface of neutrophils per unit area (CD11b+ Ly6G+ cells per field) is almost no control (PBS), whereas ALI mouse model (LPS) is about 300 ( 8a), the number of neutrophils expressing CD18 on the surface of neutrophils per unit area (CD18+ Ly6G+ cells per field) is also about 40 in the control group (PBS), but about 330 in the ALI mouse model (LPS), which is about 8 times as large. It was confirmed that the difference remained (FIG. 8C). In addition, the ratio of neutrophils expressing CD11b on the surface of neutrophils to all neutrophils (CD11b+ Ly6G+ / total Ly6G+) is about 0.05 in the control (PBS), but in the ALI mouse model (LPS), it is 0.8, which is about 16 times (Fig. 8b), the ratio of neutrophils expressing CD18 on the surface of neutrophils to all neutrophils (CD18b+ Ly6G+ / total Ly6G+) was about 0.4 in the control group (PBS), and 0.9, which is about twice or more of the ALI mouse model (LPS) ( Fig. 8d). Through this, it was found that the expression levels of CD11b and CD18 on the surface of neutrophils in the capillaries forming microcirculation were very increased due to lung damage.

From the above results, it was confirmed that the expression level of Mac-1 (CD11b/CD18) integrin was increased in neutrophils isolated from the lungs due to lung damage compared to neutrophils circulating in the microcirculation.

[Experimental Example 4] Checking the effect of improving lung damage according to Mac-1 inhibition

It was confirmed that the neutrophils isolated from the lungs due to lung damage from Experimental Example 3 increased the expression level of Mac-1 (CD11b/CD18) integrin. In the Mac-1 inhibitory mouse model prepared in Example 3, Mac- 1 It was confirmed whether the expression or activity inhibitor improves lung damage and further microcirculation disorder. In the following experimental examples, a mouse model with lung damage due to sepsis was used, but in order to expand this to a sepsis model caused by various bacteria, the CLP model of Example 1 was used as a sepsis model.

[Experimental Example 4-1] Mac-1 inhibition mouse model preparation

In order to confirm the effect of inhibiting Mac-1 activity in lung injury disease, a mouse model (hereinafter, Mac-1 inhibition model) in which the activity of Mac-1 was inhibited was prepared. Specifically, 1 hour after the preparation of the CLP mouse model of Example 1, 5 hours before in vivo imaging, an anti-CD11b antibody (5 mg/kg, Clone M1/70, 553307, BD Biosciences) was intraperitoneally injected to Mac-1 An anti-Mac-1 model, which is a mouse model with suppressed activity, was prepared. In addition, as another Mac-1 inhibition model, absiksimab (10 mg/kg, Clotinab, ISU Abxis) was injected into the CLP mouse model of Example 1 in the same manner as the anti-Mac-1 model. An absiksimab model was prepared.

[ Experimental example 4-2] Confirmation of the effect of improving pulmonary microcirculation disorder by inhibiting Mac-1

In the same manner as in Experimental Example 1, the CLP mouse model of Example 1 (Fc in FIGS. 9 and 10), the anti-Mac-1 mouse model of Experimental Example 4-1 (Anti-CD11b in FIGS. 9 and 10) And the absiksimab model (Abc in FIGS. 9 and 10), and the lung microcirculation of the normal group (Sham) mouse model (Sham in FIGS. 9 and 10) were photographed, and functional in the same manner as in Experimental Example 2 based on this Microcirculation was quantified by measuring the capillary fraction (FCR) and performing histological analysis, and the results are as shown in FIGS. 9 and 10 (n (number of fields) = 14-25, 3 mice per each group, * P <0.05, two-tailed t-test, data are mean ± sd).

9, unlike the normal group (Sham), in the CLP mouse model (Fc), a dead space in which red blood cells cannot pass through capillaries was formed, and then Mac-1 activity was suppressed (anti-Mac -1 mouse model and absiksimab mouse model), it was confirmed that the pulmonary microcirculation disorder was improved as red blood cells passed through the capillaries and the functional capillaries in which microcirculation smoothly occurred increased more and the dead space decreased.

In addition, as shown in Fig. 10a, compared to the normal group (Sham), the FCR (%) of the CLP mouse model (Fc) decreased by 50% or more, and it was confirmed that pulmonary microcirculation disorders occurred due to lung damage, and the CLP mouse Compared with the model (Fc), the Mac-1 inhibition model (anti-Mac-1 mouse model and absiksimab mouse model) increased FCR (%) by about 2 times or more, confirming that the pulmonary microcirculation disorder was improved.

Similarly, as shown in Figs. 9 and 10B, the number of neutrophils (Ly6G+ cells) isolated from the CLP mouse model (Fc) increased compared to the normal group (Sham), and then Mac By -1 inhibition (anti-Mac-1 mouse model and absiksimab mouse model), it was confirmed that the number of isolated neutrophils recovered to a level corresponding to that of the normal group.

[ Experimental example 4-3] Confirmation of the effect of improving pulmonary microcirculation disorder through comparison before and after Mac-1 inhibition

From Experimental Example 4-2, it was confirmed that microcirculation disorders can be improved through inhibition of Mac-1 expression or activity, and the effect was once again confirmed through a comparison before and after absiksimab administration.

Specifically, 6 hours after preparing the CLP mouse model of Example 1 (hereinafter, referred to as pre-Abc mouse model), the lung microcirculation was photographed in the same manner as in Experimental Example 1, and the same as in Experimental Example 2 The functional capillary fraction (FCR) was measured by the method. Then, absiksimab was administered to the CLP mouse model by the method of Experimental Example 4-1 (hereinafter, referred to as the post-Abc mouse model), and after 30 minutes, pulmonary microcirculation and functional capillary were photographed in the same manner as above. Blood vessel fraction (FCR) measurement was performed, and the results are as shown in FIGS. 11 and 12 (n (number of fields) = 20 and 24, 6-8 FOV (Field of View) per mouse, 3 per each group) , * P <0.05, two-tailed t-test, data are mean ± sd).

As shown in FIGS. 11 and 12, the lung injury mouse model (CLP mouse model) is a capillary in which red blood cells cannot pass because the specific gravity (FCR) occupied by functional capillaries among the total capillaries is less than 20% before the administration of absiksimab. Although there are many blood vessels, it was confirmed that the number of red blood cells passing through the capillaries increased rapidly, such as an increase in the functional capillary fraction (FCR) about 2 times or more when the expression or activity of Mac-1 was suppressed by administration of absiksimab.

In addition, in order to measure the oxygen partial pressure and carbon dioxide partial pressure in arterial blood of the pre-Abc mouse model and the post-Abc mouse model, arterial blood gas analysis was performed. Specifically, a 1 mL syringe containing a 22 gauge needle was coated with heparin and the normal group mouse model (Sham) (n = 8), pre-Abc mouse model (Fc) (n = 10) and post-Abc mouse model (Abc)(n = 6) was introduced into the left ventricle of each heart. Then about 200 μl of blood was sampled and analyzed with an i-STAT handheld blood analyzer (G3 cartridge, Abbott Point of Care Inc.), and the mouse models were put into a CO ₂ chamber immediately after blood sampling. Was euthanized. The arterial blood gas analysis results are the same as those of FIGS. 13A and 13B (* P <0.05, Kruskal-Wallis test using post hoc Dunn's multiple comparison test, data are mean ± sd).

13A and 13B, the partial pressure of oxygen in the arteries of the CLP mouse model (corresponding to the Fc, pre-Abc mouse model) decreased compared to the normal group (Sham) (FIG. 13A ), and the partial pressure of carbon dioxide increased ( 13B), it was confirmed that the reduction in the functional capillary fraction in the lung injury mouse model (CLP mouse model) was a result of hypoxemia and hypercapnia. Pulmonary microcirculation disorders due to hypoxia and hypercapnia were improved by suppression of the expression or activity of Mac-1 following administration of absikimab, which is the normal group in which the partial pressure of oxygen and carbon dioxide in the arteries of the post-Abc mouse model (Abc) were normal. It can be confirmed by changing the degree corresponding to (Figs. 13a and 13b). Through this, it was found that the pulmonary microcirculation disorder can be improved by increasing gas exchange in individuals with pulmonary microcirculation disorders through inhibition of Mac-1 expression or activity.

Accordingly, the composition comprising the inhibitor of Mac-1 expression or activity in neutrophils according to an aspect of the present invention has an excellent effect of preventing or treating lung damage diseases by improving microcirculation disorders in the lungs.

<110> SEOUL NATIONAL UNIVERSITY HOSPITAL Korea Advanced Institute of Science and Technology <120> Composition for prevention or treatment of lung damage diseases comprising inhibitor of macrophage-1 antigen <130> 19p027ind <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 1153 <212> PRT <213> Artificial Sequence <220> <223> Integrin alpha-M isoform 1 precursor <400> 1 Met Ala Leu Arg Val Leu Leu Leu Thr Ala Leu Thr Leu Cys His Gly 1 5 10 15 Phe Asn Leu Asp Thr Glu Asn Ala Met Thr Phe Gln Glu Asn Ala Arg 20 25 30 Gly Phe Gly Gln Ser Val Val Gln Leu Gln Gly Ser Arg Val Val Val 35 40 45 Gly Ala Pro Gln Glu Ile Val Ala Ala Asn Gln Arg Gly Ser Leu Tyr 50 55 60 Gln Cys Asp Tyr Ser Thr Gly Ser Cys Glu Pro Ile Arg Leu Gln Val 65 70 75 80 Pro Val Glu Ala Val Asn Met Ser Leu Gly Leu Ser Leu Ala Ala Thr 85 90 95 Thr Ser Pro Pro Gln Leu Leu Ala Cys Gly Pro Thr Val His Gln Thr 100 105 110 Cys Ser Glu Asn Thr Tyr Val Lys Gly Leu Cys Phe Leu Phe Gly Ser 115 120 125 Asn Leu Arg Gln Gln Pro Gln Lys Phe Pro Glu Ala Leu Arg Gly Cys 130 135 140 Pro Gln Glu Asp Ser Asp Ile Ala Phe Leu Ile Asp Gly Ser Gly Ser 145 150 155 160 Ile Ile Pro His Asp Phe Arg Arg Met Lys Glu Phe Val Ser Thr Val 165 170 175 Met Glu Gln Leu Lys Lys Ser Lys Thr Leu Phe Ser Leu Met Gln Tyr 180 185 190 Ser Glu Glu Phe Arg Ile His Phe Thr Phe Lys Glu Phe Gln Asn Asn 195 200 205 Pro Asn Pro Arg Ser Leu Val Lys Pro Ile Thr Gln Leu Leu Gly Arg 210 215 220 Thr His Thr Ala Thr Gly Ile Arg Lys Val Val Arg Glu Leu Phe Asn 225 230 235 240 Ile Thr Asn Gly Ala Arg Lys Asn Ala Phe Lys Ile Leu Val Val Ile 245 250 255 Thr Asp Gly Glu Lys Phe Gly Asp Pro Leu Gly Tyr Glu Asp Val Ile 260 265 270 Pro Glu Ala Asp Arg Glu Gly Val Ile Arg Tyr Val Ile Gly Val Gly 275 280 285 Asp Ala Phe Arg Ser Glu Lys Ser Arg Gln Glu Leu Asn Thr Ile Ala 290 295 300 Ser Lys Pro Pro Arg Asp His Val Phe Gln Val Asn Asn Phe Glu Ala 305 310 315 320 Leu Lys Thr Ile Gln Asn Gln Leu Arg Glu Lys Ile Phe Ala Ile Glu 325 330 335 Gly Thr Gln Thr Gly Ser Ser Ser Ser Phe Glu His Glu Met Ser Gln 340 345 350 Glu Gly Phe Ser Ala Ala Ile Thr Ser Asn Gly Pro Leu Leu Ser Thr 355 360 365 Val Gly Ser Tyr Asp Trp Ala Gly Gly Val Phe Leu Tyr Thr Ser Lys 370 375 380 Glu Lys Ser Thr Phe Ile Asn Met Thr Arg Val Asp Ser Asp Met Asn 385 390 395 400 Asp Ala Tyr Leu Gly Tyr Ala Ala Ala Ile Ile Leu Arg Asn Arg Val 405 410 415 Gln Ser Leu Val Leu Gly Ala Pro Arg Tyr Gln His Ile Gly Leu Val 420 425 430 Ala Met Phe Arg Gln Asn Thr Gly Met Trp Glu Ser Asn Ala Asn Val 435 440 445 Lys Gly Thr Gln Ile Gly Ala Tyr Phe Gly Ala Ser Leu Cys Ser Val 450 455 460 Asp Val Asp Ser Asn Gly Ser Thr Asp Leu Val Leu Ile Gly Ala Pro 465 470 475 480 His Tyr Tyr Glu Gln Thr Arg Gly Gly Gln Val Ser Val Cys Pro Leu 485 490 495 Pro Arg Gly Gln Arg Ala Arg Trp Gln Cys Asp Ala Val Leu Tyr Gly 500 505 510 Glu Gln Gly Gln Pro Trp Gly Arg Phe Gly Ala Ala Leu Thr Val Leu 515 520 525 Gly Asp Val Asn Gly Asp Lys Leu Thr Asp Val Ala Ile Gly Ala Pro 530 535 540 Gly Glu Glu Asp Asn Arg Gly Ala Val Tyr Leu Phe His Gly Thr Ser 545 550 555 560 Gly Ser Gly Ile Ser Pro Ser His Ser Gln Arg Ile Ala Gly Ser Lys 565 570 575 Leu Ser Pro Arg Leu Gln Tyr Phe Gly Gln Ser Leu Ser Gly Gly Gln 580 585 590 Asp Leu Thr Met Asp Gly Leu Val Asp Leu Thr Val Gly Ala Gln Gly 595 600 605 His Val Leu Leu Leu Arg Ser Gln Pro Val Leu Arg Val Lys Ala Ile 610 615 620 Met Glu Phe Asn Pro Arg Glu Val Ala Arg Asn Val Phe Glu Cys Asn 625 630 635 640 Asp Gln Val Val Lys Gly Lys Glu Ala Gly Glu Val Arg Val Cys Leu 645 650 655 His Val Gln Lys Ser Thr Arg Asp Arg Leu Arg Glu Gly Gln Ile Gln 660 665 670 Ser Val Val Thr Tyr Asp Leu Ala Leu Asp Ser Gly Arg Pro His Ser 675 680 685 Arg Ala Val Phe Asn Glu Thr Lys Asn Ser Thr Arg Arg Gln Thr Gln 690 695 700 Val Leu Gly Leu Thr Gln Thr Cys Glu Thr Leu Lys Leu Gln Leu Pro 705 710 715 720 Asn Cys Ile Glu Asp Pro Val Ser Pro Ile Val Leu Arg Leu Asn Phe 725 730 735 Ser Leu Val Gly Thr Pro Leu Ser Ala Phe Gly Asn Leu Arg Pro Val 740 745 750 Leu Ala Glu Asp Ala Gln Arg Leu Phe Thr Ala Leu Phe Pro Phe Glu 755 760 765 Lys Asn Cys Gly Asn Asp Asn Ile Cys Gln Asp Asp Leu Ser Ile Thr 770 775 780 Phe Ser Phe Met Ser Leu Asp Cys Leu Val Val Gly Gly Pro Arg Glu 785 790 795 800 Phe Asn Val Thr Val Thr Val Arg Asn Asp Gly Glu Asp Ser Tyr Arg 805 810 815 Thr Gln Val Thr Phe Phe Phe Pro Leu Asp Leu Ser Tyr Arg Lys Val 820 825 830 Ser Thr Leu Gln Asn Gln Arg Ser Gln Arg Ser Trp Arg Leu Ala Cys 835 840 845 Glu Ser Ala Ser Ser Thr Glu Val Ser Gly Ala Leu Lys Ser Thr Ser 850 855 860 Cys Ser Ile Asn His Pro Ile Phe Pro Glu Asn Ser Glu Val Thr Phe 865 870 875 880 Asn Ile Thr Phe Asp Val Asp Ser Lys Ala Ser Leu Gly Asn Lys Leu 885 890 895 Leu Leu Lys Ala Asn Val Thr Ser Glu Asn Asn Met Pro Arg Thr Asn 900 905 910 Lys Thr Glu Phe Gln Leu Glu Leu Pro Val Lys Tyr Ala Val Tyr Met 915 920 925 Val Val Thr Ser His Gly Val Ser Thr Lys Tyr Leu Asn Phe Thr Ala 930 935 940 Ser Glu Asn Thr Ser Arg Val Met Gln His Gln Tyr Gln Val Ser Asn 945 950 955 960 Leu Gly Gln Arg Ser Leu Pro Ile Ser Leu Val Phe Leu Val Pro Val 965 970 975 Arg Leu Asn Gln Thr Val Ile Trp Asp Arg Pro Gln Val Thr Phe Ser 980 985 990 Glu Asn Leu Ser Ser Thr Cys His Thr Lys Glu Arg Leu Pro Ser His 995 1000 1005 Ser Asp Phe Leu Ala Glu Leu Arg Lys Ala Pro Val Val Asn Cys Ser 1010 1015 1020 Ile Ala Val Cys Gln Arg Ile Gln Cys Asp Ile Pro Phe Phe Gly Ile 1025 1030 1035 1040 Gln Glu Glu Phe Asn Ala Thr Leu Lys Gly Asn Leu Ser Phe Asp Trp 1045 1050 1055 Tyr Ile Lys Thr Ser His Asn His Leu Leu Ile Val Ser Thr Ala Glu 1060 1065 1070 Ile Leu Phe Asn Asp Ser Val Phe Thr Leu Leu Pro Gly Gln Gly Ala 1075 1080 1085 Phe Val Arg Ser Gln Thr Glu Thr Lys Val Glu Pro Phe Glu Val Pro 1090 1095 1100 Asn Pro Leu Pro Leu Ile Val Gly Ser Ser Val Gly Gly Leu Leu Leu 1105 1110 1115 1120 Leu Ala Leu Ile Thr Ala Ala Leu Tyr Lys Leu Gly Phe Phe Lys Arg 1125 1130 1135 Gln Tyr Lys Asp Met Met Ser Glu Gly Gly Pro Pro Gly Ala Glu Pro 1140 1145 1150 Gln <210> 2 <211> 1152 <212> PRT <213> Artificial Sequence <220> <223> Integrin alpha-M isoform 2 precursor <400> 2 Met Ala Leu Arg Val Leu Leu Leu Thr Ala Leu Thr Leu Cys His Gly 1 5 10 15 Phe Asn Leu Asp Thr Glu Asn Ala Met Thr Phe Gln Glu Asn Ala Arg 20 25 30 Gly Phe Gly Gln Ser Val Val Gln Leu Gln Gly Ser Arg Val Val Val 35 40 45 Gly Ala Pro Gln Glu Ile Val Ala Ala Asn Gln Arg Gly Ser Leu Tyr 50 55 60 Gln Cys Asp Tyr Ser Thr Gly Ser Cys Glu Pro Ile Arg Leu Gln Val 65 70 75 80 Pro Val Glu Ala Val Asn Met Ser Leu Gly Leu Ser Leu Ala Ala Thr 85 90 95 Thr Ser Pro Pro Gln Leu Leu Ala Cys Gly Pro Thr Val His Gln Thr 100 105 110 Cys Ser Glu Asn Thr Tyr Val Lys Gly Leu Cys Phe Leu Phe Gly Ser 115 120 125 Asn Leu Arg Gln Gln Pro Gln Lys Phe Pro Glu Ala Leu Arg Gly Cys 130 135 140 Pro Gln Glu Asp Ser Asp Ile Ala Phe Leu Ile Asp Gly Ser Gly Ser 145 150 155 160 Ile Ile Pro His Asp Phe Arg Arg Met Lys Glu Phe Val Ser Thr Val 165 170 175 Met Glu Gln Leu Lys Lys Ser Lys Thr Leu Phe Ser Leu Met Gln Tyr 180 185 190 Ser Glu Glu Phe Arg Ile His Phe Thr Phe Lys Glu Phe Gln Asn Asn 195 200 205 Pro Asn Pro Arg Ser Leu Val Lys Pro Ile Thr Gln Leu Leu Gly Arg 210 215 220 Thr His Thr Ala Thr Gly Ile Arg Lys Val Val Arg Glu Leu Phe Asn 225 230 235 240 Ile Thr Asn Gly Ala Arg Lys Asn Ala Phe Lys Ile Leu Val Val Ile 245 250 255 Thr Asp Gly Glu Lys Phe Gly Asp Pro Leu Gly Tyr Glu Asp Val Ile 260 265 270 Pro Glu Ala Asp Arg Glu Gly Val Ile Arg Tyr Val Ile Gly Val Gly 275 280 285 Asp Ala Phe Arg Ser Glu Lys Ser Arg Gln Glu Leu Asn Thr Ile Ala 290 295 300 Ser Lys Pro Pro Arg Asp His Val Phe Gln Val Asn Asn Phe Glu Ala 305 310 315 320 Leu Lys Thr Ile Gln Asn Gln Leu Arg Glu Lys Ile Phe Ala Ile Glu 325 330 335 Gly Thr Gln Thr Gly Ser Ser Ser Ser Phe Glu His Glu Met Ser Gln 340 345 350 Glu Gly Phe Ser Ala Ala Ile Thr Ser Asn Gly Pro Leu Leu Ser Thr 355 360 365 Val Gly Ser Tyr Asp Trp Ala Gly Gly Val Phe Leu Tyr Thr Ser Lys 370 375 380 Glu Lys Ser Thr Phe Ile Asn Met Thr Arg Val Asp Ser Asp Met Asn 385 390 395 400 Asp Ala Tyr Leu Gly Tyr Ala Ala Ala Ile Ile Leu Arg Asn Arg Val 405 410 415 Gln Ser Leu Val Leu Gly Ala Pro Arg Tyr Gln His Ile Gly Leu Val 420 425 430 Ala Met Phe Arg Gln Asn Thr Gly Met Trp Glu Ser Asn Ala Asn Val 435 440 445 Lys Gly Thr Gln Ile Gly Ala Tyr Phe Gly Ala Ser Leu Cys Ser Val 450 455 460 Asp Val Asp Ser Asn Gly Ser Thr Asp Leu Val Leu Ile Gly Ala Pro 465 470 475 480 His Tyr Tyr Glu Gln Thr Arg Gly Gly Gln Val Ser Val Cys Pro Leu 485 490 495 Pro Arg Gly Arg Ala Arg Trp Gln Cys Asp Ala Val Leu Tyr Gly Glu 500 505 510 Gln Gly Gln Pro Trp Gly Arg Phe Gly Ala Ala Leu Thr Val Leu Gly 515 520 525 Asp Val Asn Gly Asp Lys Leu Thr Asp Val Ala Ile Gly Ala Pro Gly 530 535 540 Glu Glu Asp Asn Arg Gly Ala Val Tyr Leu Phe His Gly Thr Ser Gly 545 550 555 560 Ser Gly Ile Ser Pro Ser His Ser Gln Arg Ile Ala Gly Ser Lys Leu 565 570 575 Ser Pro Arg Leu Gln Tyr Phe Gly Gln Ser Leu Ser Gly Gly Gln Asp 580 585 590 Leu Thr Met Asp Gly Leu Val Asp Leu Thr Val Gly Ala Gln Gly His 595 600 605 Val Leu Leu Leu Arg Ser Gln Pro Val Leu Arg Val Lys Ala Ile Met 610 615 620 Glu Phe Asn Pro Arg Glu Val Ala Arg Asn Val Phe Glu Cys Asn Asp 625 630 635 640 Gln Val Val Lys Gly Lys Glu Ala Gly Glu Val Arg Val Cys Leu His 645 650 655 Val Gln Lys Ser Thr Arg Asp Arg Leu Arg Glu Gly Gln Ile Gln Ser 660 665 670 Val Val Thr Tyr Asp Leu Ala Leu Asp Ser Gly Arg Pro His Ser Arg 675 680 685 Ala Val Phe Asn Glu Thr Lys Asn Ser Thr Arg Arg Gln Thr Gln Val 690 695 700 Leu Gly Leu Thr Gln Thr Cys Glu Thr Leu Lys Leu Gln Leu Pro Asn 705 710 715 720 Cys Ile Glu Asp Pro Val Ser Pro Ile Val Leu Arg Leu Asn Phe Ser 725 730 735 Leu Val Gly Thr Pro Leu Ser Ala Phe Gly Asn Leu Arg Pro Val Leu 740 745 750 Ala Glu Asp Ala Gln Arg Leu Phe Thr Ala Leu Phe Pro Phe Glu Lys 755 760 765 Asn Cys Gly Asn Asp Asn Ile Cys Gln Asp Asp Leu Ser Ile Thr Phe 770 775 780 Ser Phe Met Ser Leu Asp Cys Leu Val Val Gly Gly Pro Arg Glu Phe 785 790 795 800 Asn Val Thr Val Thr Val Arg Asn Asp Gly Glu Asp Ser Tyr Arg Thr 805 810 815 Gln Val Thr Phe Phe Phe Pro Leu Asp Leu Ser Tyr Arg Lys Val Ser 820 825 830 Thr Leu Gln Asn Gln Arg Ser Gln Arg Ser Trp Arg Leu Ala Cys Glu 835 840 845 Ser Ala Ser Ser Thr Glu Val Ser Gly Ala Leu Lys Ser Thr Ser Cys 850 855 860 Ser Ile Asn His Pro Ile Phe Pro Glu Asn Ser Glu Val Thr Phe Asn 865 870 875 880 Ile Thr Phe Asp Val Asp Ser Lys Ala Ser Leu Gly Asn Lys Leu Leu 885 890 895 Leu Lys Ala Asn Val Thr Ser Glu Asn Asn Met Pro Arg Thr Asn Lys 900 905 910 Thr Glu Phe Gln Leu Glu Leu Pro Val Lys Tyr Ala Val Tyr Met Val 915 920 925 Val Thr Ser His Gly Val Ser Thr Lys Tyr Leu Asn Phe Thr Ala Ser 930 935 940 Glu Asn Thr Ser Arg Val Met Gln His Gln Tyr Gln Val Ser Asn Leu 945 950 955 960 Gly Gln Arg Ser Leu Pro Ile Ser Leu Val Phe Leu Val Pro Val Arg 965 970 975 Leu Asn Gln Thr Val Ile Trp Asp Arg Pro Gln Val Thr Phe Ser Glu 980 985 990 Asn Leu Ser Ser Thr Cys His Thr Lys Glu Arg Leu Pro Ser His Ser 995 1000 1005 Asp Phe Leu Ala Glu Leu Arg Lys Ala Pro Val Val Asn Cys Ser Ile 1010 1015 1020 Ala Val Cys Gln Arg Ile Gln Cys Asp Ile Pro Phe Phe Gly Ile Gln 1025 1030 1035 1040 Glu Glu Phe Asn Ala Thr Leu Lys Gly Asn Leu Ser Phe Asp Trp Tyr 1045 1050 1055 Ile Lys Thr Ser His Asn His Leu Leu Ile Val Ser Thr Ala Glu Ile 1060 1065 1070 Leu Phe Asn Asp Ser Val Phe Thr Leu Leu Pro Gly Gln Gly Ala Phe 1075 1080 1085 Val Arg Ser Gln Thr Glu Thr Lys Val Glu Pro Phe Glu Val Pro Asn 1090 1095 1100 Pro Leu Pro Leu Ile Val Gly Ser Ser Val Gly Gly Leu Leu Leu Leu 1105 1110 1115 1120 Ala Leu Ile Thr Ala Ala Leu Tyr Lys Leu Gly Phe Phe Lys Arg Gln 1125 1130 1135 Tyr Lys Asp Met Met Ser Glu Gly Gly Pro Pro Gly Ala Glu Pro Gln 1140 1145 1150

Claims (19)

As a composition for preventing, improving or treating lung injury diseases,
The composition comprises an inhibitor of expression or activity of macrophage-1 antigen (Mac-1) in neutrophils in pulmonary capillaries as an active ingredient. The method of claim 1,
The prevention, improvement or treatment of lung injury disease,
By improving the microcirculation disorder of the lungs, the composition. A composition for improving pulmonary microcirculation disorders, comprising an inhibitor of expression or activity of macrophage-1 antigen (Mac-1) in neutrophils in pulmonary capillaries as an active ingredient. The method according to any one of claims 1 to 3,
The composition of the macrophage-1 antigen expression or activity inhibitor is an antibody that specifically binds to the macrophage-1 antigen in neutrophils. The method of claim 4,
The antibody is abciximab, the composition. The method according to claim 2 or 3,
The improvement of the microcirculation disorder is to increase the ratio of functional capillaries through which red blood cells pass among the total capillaries of the lung. The method of claim 6,
The improvement of the microcirculation disorder is to increase the functional capillary fraction (Functional Capillary Ratio, FCR) according to the following formula 1, composition:
[Equation 1]
Functional capillary fraction = area of functional capillaries / area of total capillaries. The method of claim 1,
The lung damage disease is a disease caused by microcirculation disorders of the lung, composition. The method of claim 1,
The lung injury diseases include pulmonary vasoconstriction, asthma, respiratory retardation, respiratory distress syndrome (RDS), acute respiratory distress syndrome (ARDS), cystic fibrosis (CF), allergic Rhinitis (allergic rhinitis, AR), pulmonary hypertension, emphysema, chronic obstructive pulmonary disease (COPD), lung transplant rejection, lung infection, bronchitis, and at least one disease selected from the group consisting of cancer, the composition. (a) preparing a lung injury model;
(b) treating a test substance in the lung injury model; And
(c) measuring a change in the expression or activity of the macrophage-1 antigen (Mac-1) in neutrophils in the pulmonary capillaries of the lung injury model by the test substance; Prevention, improvement or screening of therapeutic substances. The method of claim 10,
The lung injury model of step (a) is a sepsis model, a screening method. The method of claim 10,
The step (c) comprises comparing the expression or activity of the macrophage-1 antigen in neutrophils in pulmonary capillaries before and after treatment of the test substance in a lung injury model. The method of claim 10,
When the expression or activity of the macrophage-1 antigen is decreased as a result of measuring the expression or activity of the step (c) compared to before treatment of the test substance, the step of determining the test substance as a substance for preventing, improving or treating lung damage disease That, how to screen. (a) preparing a lung injury model;
(b) treating a test substance in the lung injury model; And
(c) measuring the change in the expression or activity of the macrophage-1 antigen (Mac-1) in neutrophils in the lung capillaries of the lung injury model by the test substance; Circulatory disorder improvement substance screening method. Providing information for diagnosis of pulmonary microcirculation disorder, including measuring the expression or activity of macrophage-1 antigen (Mac-1) in neutrophils isolated from pulmonary capillaries of the test subject Way. The method of claim 15,
Comprising the step of comparing the level of expression or activity of the macrophage-1 antigen in neutrophils isolated from the pulmonary capillaries of the test subject with the level of expression or activity of the macrophage-1 antigen in neutrophils isolated from the pulmonary capillaries of the normal control. , A method of providing information for diagnosis of pulmonary microcirculation disorder. The method of claim 15,
If the level of macrophage-1 antigen expression or activity in neutrophils isolated from the pulmonary capillaries of the test subject is higher than the level of macrophage-1 antigen expression or activity in neutrophils isolated from the pulmonary capillaries of the normal control, there is a pulmonary microcirculation disorder. A method of providing information for diagnosing whether or not a pulmonary microcirculation disorder further comprises providing information to be. A composition for diagnosing pulmonary microcirculation disorders comprising a reagent for detecting mRNA or protein of macrophage-1 antigen (Mac-1) in neutrophils in pulmonary capillaries. A kit for diagnosing pulmonary microcirculation disorders containing a reagent for detecting mRNA or protein of macrophage-1 antigen (Mac-1) in neutrophils in lung capillaries.

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