JP6944701B2 - Composition for the treatment of fulminant acute pneumonia containing a CD11b antagonist - Google Patents

Description

The present invention relates to a drug or pharmaceutical composition comprising a CD11b antagonist and used to prevent and / or treat fulminant acute pneumonia. The present invention also relates to an intraalveolar neutrophil aggregation inhibitor containing a CD11b antagonist. Furthermore, the present invention relates to a lung neutrophil infiltration inhibitor containing a CD11b antagonist. In addition, the present invention relates to prophylactic and / or therapeutic agents for fulminant acute pneumonia, including CD11b antagonists. The present invention also relates to a method for preventing and / or treating fulminant acute pneumonia, which comprises administering a CD11b antagonist.

Fulminant acute pneumonia refers to respiratory distress syndrome that presents with more serious symptoms than acute respiratory distress syndrome (hereinafter, may be abbreviated as ARDS). Acute pneumonia generally develops symptoms rapidly and then worsens rapidly and heals rapidly after a short period of improvement, whereas fulminant acute pneumonia develops symptoms rapidly. , Pneumonia progresses rapidly and the prognosis is poor, that is, it tends to become severe and the risk of death is high.

ARDS is a non-cardiogenic pulmonary edema caused by nonspecific inflammation of the alveolar region triggered by some invasion of the body, characterized by severe hypoxemia and decreased lung compliance. It is also characterized by a pathological image that presents with severe neutrophil infiltration and extensive alveolar damage (diffuse alveolar damage; DAD). The time from invasion to onset is usually within a few days, with post-onset mortality rates exceeding 40%. It is a disease that has become a major problem in clinical practice, such as occurring in about 20% of patients receiving artificial respiration in an intensive care unit or the like. With the onset of ARDS, endothelial cells and alveolar epithelial cells of the capillaries of the lung are damaged, edema and fibrosis of the lung are induced, and dyspnea leads to death. Currently, in addition to mechanical ventilation, drug therapies such as steroids and neutrophil esterase inhibitors have been tried, but they have not been able to be effective treatments. In addition, previous studies have reported that the onset of ARDS involves lipid mediators produced by infiltrated activated neutrophils and cytokines such as interferon-γ (IFNγ). The onset mechanism has not been clarified.

Recently, there have been signs of human infection of bird flu (H7N9, H5N1, etc.) and a worldwide epidemic, with a high infection lethality rate of up to 30-60%, but the main cause of death is virus proliferation. It has been found to be fulminant ARDS (Fulminant ARDS; hereinafter abbreviated as FARDS), that is, fulminant acute pneumonia, which occurs as a result of an excessive immune response on the host side rather than a direct effect. Therefore, establishing a treatment method for a future pandemic is an urgent issue.

The distinction between ARDS and FARDS is made based on the ratio of the partial pressure of oxygen in the arterial blood to the inhaled oxygen concentration (P / F ratio). The P / F ratio of a healthy person is about 400 to 500. When the P / F ratio is 300 or less, it is judged as acute lung injury (ALI), when it is 200 or less, it is judged as ARDS, and when it is 100 or less, it is judged as FARDS. ALI is an acute / progressive respiratory disease caused by causes such as sepsis, pneumonia, trauma, and aspiration, and when it becomes severe, it leads to ARDS and FARDS.

The CD11b molecule (hereinafter, may be simply referred to as CD11b) is a type I transmembrane glucose-binding protein belonging to the c-type lectin family, and is also called an integrin αM chain. The CD11b molecule forms a complex with the CD18 molecule to form a Mac-1 antigen and is expressed on the cell surface. In addition, the CD11b molecule is strongly expressed in monocytes / macrophages, neutrophils, NK cells and the like. As a function, it is known to be involved in binding to complement and fibrinogen and adhesion to vascular endothelial cells, but there are many unclear points about its specific role in inducing inflammation.

The present inventors have previously produced CD69 knockout mice and analyzed them to determine that the CD69 molecule on neutrophils is important for the development of arthritis (Non-Patent Document 1), and allergic asthma in CD69 knockout mice. It has been clarified that asthma does not occur (Non-Patent Documents 2 and 3). Then, allergic asthma therapeutic agents (Patent Documents 1 and 2), enteritis therapeutic agents (Patent Documents 3 and 4), and hepatitis therapeutic agents (Patent Documents 3 and 5-7) targeting the CD69 molecule have been disclosed. It has also been reported that in CD69 knockout mice, lung inflammation and fibrosis induced by intratracheal administration of bleomycin were alleviated as compared with wild mice (Non-Patent Document 4). However, the role of CD11b in the onset and exacerbation of fulminant acute pneumonia is unknown.

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An object of the present invention is to provide an effective means for the prevention and / or treatment of fulminant acute pneumonia.

In order to achieve the above object, the present inventors have analyzed the onset and fulminant mechanism of FARDS, and have diligently searched for a therapeutic target molecule for the purpose of developing a new therapeutic method. Then, the symptoms of FARDS in the mouse FARDS model, such as high mortality rate and marked infiltration of neutrophils into the lung, are alleviated by administering an antibody that specifically recognizes CD11b (hereinafter referred to as anti-CD11b antibody). It was also found that the lung infiltration of neutrophils derived from CD11b knockout mice was small in the mouse FARDS model. Then, it was clarified that the CD11b molecule could be a target molecule for a novel therapeutic method for FARDS, and the present invention was completed.

That is, the present invention relates to a pharmaceutical composition for preventing and / or treating fulminant acute pneumonia containing a CD11b antagonist.

The present invention also relates to the above-mentioned pharmaceutical composition in which the CD11b antagonist is an anti-CD11b antibody.

In addition, the present invention relates to prophylactic and / or therapeutic agents for fulminant acute pneumonia, including CD11b antagonists.

The present invention also relates to a prophylactic and / or therapeutic agent for fulminant acute pneumonia, which comprises an antibody that specifically recognizes CD11b.

In addition, the present invention presents a CD11b antagonist in an amount effective to prevent and / or treat pneumonia in a subject diagnosed in need of prevention and / or treatment of fulminant acute pneumonia. It relates to a method for preventing and / or treating fulminant acute pneumonia, including administration.

In addition, the present invention relates to the above method, wherein the CD11b antagonist is an anti-CD11b antibody.

INDUSTRIAL APPLICABILITY The present invention provides a pharmaceutical composition for preventing and / or treating fulminant acute pneumonia, which comprises a CD11b antagonist, for example, an anti-CD11b antibody, and a prophylactic and / or therapeutic agent for fulminant acute pneumonia, which comprises a CD11b antagonist. can do.

The present invention can also provide a method for preventing and / or treating fulminant acute pneumonia, which comprises administering a CD11b antagonist, such as an anti-CD11b antibody.

Fulminant acute pneumonia, which develops when a patient is receiving artificial respiration in an intensive care unit or is infected with highly pathogenic avian influenza, is characterized by a rapid onset and subsequent rapid progression of the condition, and has a poor prognosis. The risk of death is also extremely high. In the treatment of fulminant acute pneumonia for which no effective treatment method has been developed at present, the present invention may provide a novel and breakthrough treatment.

Since the pharmaceutical composition according to the present invention has shown an effective therapeutic effect in both presymptomatic administration and post-onset administration of fulminant acute pneumonia, it is used for both prevention and treatment of fulminant acute pneumonia. It can be used and contributes greatly to the pharmaceutical field as a treatment method for future pandemics such as bird flu.

It is a figure explaining the method of making a mouse FARDS model. In the mouse FARDS model, α-galactosylceramide (hereinafter abbreviated as αGalCel) was nasally administered to wild-type mice (hereinafter abbreviated as WT) (Day -1), and 24 hours later, lipopolysaccharide (hereinafter abbreviated as LPS) was administered. It was prepared by nasal administration (Day 0). (Example 1) It is a figure which shows the result of having examined the survival rate after LPS administration of a mouse FARDS model (see FIG. 1A) with time. The vertical axis of the figure shows the survival rate (Survival (%)), and the horizontal axis shows the time after LPS administration (Time after LPS challenge (h)). (Example 1) In the mouse FARDS model, a diagram explaining that significant neutrophil infiltration into the lung was observed as compared with mice that did not develop FARDS administered only with phosphate buffered saline (hereinafter abbreviated as PBS). be. In the figure, No treatment indicates a mouse that does not develop FARDS administered only with PBS, and treatment indicates a mouse FARDS model. (Example 1) It is a figure explaining that the P / F ratio (P / Fratio) was significantly lower in the mouse FARDS model as compared with the mouse which did not develop FARDS to administer PBS alone. In the figure, No treatment indicates a mouse that does not develop FARDS administered only with PBS, and treatment indicates a mouse FARDS model. (Example 1) It is a figure explaining the imaging technique used to examine the neutrophil infiltration into the lung in a mouse FARDS model. Using transgenic mice incorporating the green fluorescent protein gene (hereinafter referred to as GFP Tg) as donors, neutrophils and CD4 T cells (CD4 T cells) were collected. Then, wild-type mice were used as recipients, and the neutrophils and CD4 T cells were transferred via veins (iv). Forty-eight hours later, αGalCel was administered, and 24 hours later, LPS was administered. Neutrophil infiltration into the lungs was observed 6 hours, 12 hours, 24 hours, and 48 hours after LPS administration. (Example 2) It is a figure which shows the result of having measured the neutrophil infiltration into the lung 48 hours after LPS administration in a mouse FARDS model. ^{Mice treated with αGalCel and LPS had marked GFP-positive neutrophils (GFP +} neurofils) into the lungs compared to mice treated with PBS alone, mice treated with αGalCel and PBS, and mice treated with PBS and LPS. ) Infiltration was observed. (Example 2) It is a figure which shows the result of having measured the neutrophil infiltration into the lung with time in the mouse FARDS model. About 6 hours after LPS administration, ^{significant infiltration of GFP-positive neutrophils (GFP +} neurofils) into the lung was observed. (Example 2) It is a figure which shows the result of having measured the CD4 T cell infiltration into a lung with time in a mouse FARDS model. About 24 hours after LPS administration, infiltration of GFP-positive CD4 T cells (GFP ⁺ CD4 T cells) into the lung was observed. (Example 2) It is a figure which shows the result of having measured the type and the number of the immunoinflammatory cells in the alveolar lavage fluid in the mouse FARDS model. In the mouse FARDS model (black bar), increased infiltration of immunoinflammatory cells into the lung was observed as compared with mice that did not develop FARDS with PBS alone (white bar). The vertical axis of the figure shows the number ^{(Cell number (× 10 7)} / lung) of each cell per lung. In the figure, T lymphocyte. Are T lymphocytes, Blympho. Is a B lymphocyte, Mac. Is a macrophage, Neutor. Means neutrophils and total means all cells. (Example 3) ^{FIG. 5 showing that the significant infiltration of GFP-positive neutrophils (GFP +} neurofils) into the lung observed after FARDS induction in a mouse FARDS model was suppressed by pre-administration of an anti-CD11b antibody (Anti-CD11b mAb). Is. In the figure, PBS / PBS indicates a mouse in which FARDS was not induced, αGalCer / LPS indicates a mouse in which FARDS was induced, and Anti-CD11b mAb indicates a mouse in which FARDS was induced after administration of an anti-CD11b antibody. (Example 4) It is a figure explaining the drug administration procedure for examining the effect of anti-CD11b antibody pre-administration in a mouse FARDS model. Wild-type mice were administered anti-CD11b antibody (Day-2), nasally αGalCel 24 hours later (Day-1), and 24 hours later LPS nasally (Day 0). In the figure, Ab means an antibody. (Example 5) It is a figure which shows the result of having examined the effect of the anti-CD11b antibody pre-administration on the survival rate after LPS administration of a mouse FARDS model over time. As a result of administration of the anti-CD11b antibody (Anti-CD11b Ab) before the administration of αGalCer, the survival rate was significantly increased as compared with the administration of the control antibody (Control Ab). The vertical axis of the figure shows the survival rate (Survival (%)), and the horizontal axis shows the time after LPS administration (Time after LPS challenge (h)). (Example 5) It is a figure which shows that the remarkable neutrophil infiltration to the lung observed in a mouse FARDS model was reduced by administration of an anti-CD11b antibody (Anti-CD11b mAb) before administration of αGalCer. In the figure, No treatment shows a lung tissue image of a mouse that does not develop FARDS administered only with PBS, and Anti-CD11b mAb shows a lung tissue image when an anti-CD11b antibody is administered to a mouse FARDS model before administration of αGalCer. In addition, Control mAb shows a lung tissue image when a control antibody is administered after administration of LPS to a mouse FARDS model. (Example 5) It is a figure which shows that the low P / F ratio (P / Fratio) observed in a mouse FARDS model was significantly improved by administration of an anti-CD11b antibody (Anti-CD11b mAb) before administration of αGalCer. .. In the figure, (-) indicates a wild-type mouse to which only PBS was administered, αGC + LPS indicates a mouse that developed FARDS by administering α-GalCel and LPS, and Control mAb indicates a mouse to which a control antibody was administered. (Example 5) It is a figure explaining the drug administration procedure for examining the effect of the anti-CD11b antibody administration after FARDS induction in a mouse FARDS model. Wild-type mice were nasally administered αGalCel (Day -1), 24 hours later, LPS was nasally administered (Day 0) to induce FARDS, and 24 hours after LPS administration, anti-CD11b antibody was administered (Day -1). 1). In the figure, Ab means an antibody. (Example 6) It is a figure which shows the result of having examined the effect of the anti-CD11b antibody administration after administration of LPS on the survival rate of a mouse FARDS model over time. As a result of administration of the anti-CD11b antibody (Anti-CD11b Ab) after the administration of LPS, the survival rate was significantly increased as compared with the administration of the control antibody (Control Ab). The vertical axis of the figure shows the survival rate (Survival (%)), and the horizontal axis shows the time after LPS administration (Time after LPS challenge (h)). (Example 6) It is a figure which shows that the remarkable neutrophil infiltration to the lung observed in a mouse FARDS model was reduced by administration of an anti-CD11b antibody (Anti-CD11b mAb) after administration of LPS. In the figure, No treatment shows a lung tissue image of a mouse that does not develop FARDS administered only with PBS, and Anti-CD11b mAb shows a lung tissue image when an anti-CD11b antibody is administered to a mouse FARDS model after administration of LPS. In addition, Control mAb shows a lung tissue image when a control antibody is administered after administration of LPS to a mouse FARDS model. (Example 6) It is a figure which shows the result of having examined the effect of the anti-CD11b antibody pre-administration on the survival rate after LPS administration of a mouse FARDS model over time. As a result of administration of the anti-CD11b antibody (Anti-CD11b mAb) before the administration of αGalCer, the survival rate was significantly increased as compared with the administration of the control antibody (Control mAb). The vertical axis of the figure shows the survival rate (Survival (%)), and the horizontal axis shows the time after LPS administration (Time after LPS challenge (h)). (Example 7) It is a figure which shows that the remarkable neutrophil infiltration to the lung observed in a mouse FARDS model was reduced by administration of an anti-CD11b antibody (Anti-CD11b mAb) before administration of αGalCer. In the figure, No treatment shows a lung tissue image of a mouse that does not develop FARDS administered only with PBS, and Anti-CD11b mAb shows a lung tissue image when an anti-CD11b antibody is administered to a mouse FARDS model before administration of αGalCer. In addition, Control mAb shows a lung tissue image when a control antibody is administered after administration of LPS to a mouse FARDS model. (Example 7) It is a figure which shows that the low P / F ratio (P / Fratio) observed in a mouse FARDS model was significantly improved by administration of an anti-CD11b antibody (Anti-CD11b mAb) after administration of LPS. In the figure, (-) indicates a wild-type mouse to which only PBS was administered, αGC + LPS indicates a mouse that developed FARDS by administering α-GalCel and LPS, and Control mAb indicates a mouse to which a control antibody was administered. (Example 8)

The present invention relates to a pharmaceutical composition for the prevention and / or treatment of fulminant acute pneumonia containing a CD11b antagonist. The present invention also relates to an intraalveolar neutrophil aggregation inhibitor containing a CD11b antagonist. Furthermore, the present invention relates to an inhibitor of neutrophil infiltration in the lung. In addition, the present invention relates to prophylactic and / or therapeutic agents for fulminant acute pneumonia, including CD11b antagonists. The present invention also provides fulminant acute pneumonia to subjects diagnosed with CD11b antagonist as having the potential to develop fulminant acute pneumonia and subjects diagnosed with fulminant acute pneumonia. With respect to methods of preventing and / or treating fulminant acute pneumonia, including administration in an amount effective to prevent and / or treat.

As used herein, the term "fulminant acute pneumonia" is used interchangeably with the term "fulminant acute respiratory distress syndrome (FARDS)".

"Fulminant acute respiratory distress syndrome (FARDS)" refers to respiratory distress syndrome that presents with more serious symptoms than acute respiratory distress syndrome (ARDS). ARDS refers to a condition characterized by acute respiratory failure due to impaired uptake of oxygen into the body by breathing. Breathing refers to the work of taking in oxygen in the air into the body and discharging the carbon dioxide produced in the body into inhalation. Incorporation of oxygen into the body is a step of delivering air from the mouth and nasal cavity through the airway to the alveoli (step 1), a step of gas exchange in the lungs, that is, oxygen is alveolar epithelium from the air in the alveolar. A step of being taken up by cells and then binding to hemoglobin contained in red alveoli in the blood through tissue stroma and then vascular endothelial cells (step 2), and a step of being transported from the heart to the whole body using blood circulation. This is performed according to (step 3). Respiratory failure is a condition in which one of these processes is impaired for some reason and oxygen cannot be sufficiently delivered to the whole body. ARDS refers to a condition characterized by acute respiratory failure that occurs within 48 hours mainly due to a disorder in the gas exchange process (step 2 above) in the lungs during the process of oxygen uptake into the body.

In a joint study group (American-European Consensus Conference: AECC) with the American Thoracic Disease Society and the European Society of Intensive Care Medicine, ARDS said, "Acute onset hypoxemia with a preceding underlying disease, chest X-ray image. Above, bilateral pulmonary infiltrates are observed, and cardiogenic pulmonary edema can be ruled out. " That is, ARDS does not refer to a specific disease, but is characterized by sudden onset, overt hypoxemia, the presence of abnormal shadows throughout the chest X-ray image, and noncardiogenic pulmonary edema. Refers to the syndrome shown. The pathophysiology of ARDS is the accumulation of activated neutrophils in the lung, the production of active oxygen and proteolytic enzymes by cells such as neutrophils, and the capillaries and lung epithelial cells in the alveolar wall due to the produced substances. Injury, leakage of liquid components from capillaries and the resulting edema, and microscopic findings of extensive alveolar region damage. The mortality rate after the onset of ARDS exceeds 43%. Examples of the preceding underlying disease include direct disorders such as severe pneumonia and aspiration pneumonia, and indirect disorders such as sepsis. About 80% of ARDS and ALI are recognized to be associated with sepsis.

According to the definition given by AECC in 1994 (AECC definition), when the P / F ratio is 300 or less, it is determined to be acute lung injury (ALI), and when it is 200 or less, it is determined to be ARDS. However, according to the new definition presented in 2012 (Berlin definition), mild ARDS (mild ARDS) when the P / F ratio is 300 or less, ARDS when it is 200 or less, and severe ARDS when the P / F ratio is 100 or less (Berlin definition). It is determined to be seven ARDS). The terms "mild ARDS" and the term "ALI" are used interchangeably herein. Also, the terms "severe ARDS" and "FARDS" are used interchangeably herein. The P / F ratio of a healthy person is about 400 to 500 (arterial oxygen partial pressure (PaO ₂ ): 80 to 100 Torr (mmHg)).

In FARDS, symptoms occur more rapidly than in ARDS, the progression of pneumonia is rapid, the prognosis is worse, that is, it is more likely to become severe and the risk of death is high. In recent years, it has been reported that FARDS is caused by infection with influenza viruses such as avian influenza (H7N9, H5N1, etc.) and shows a high infection lethality rate of 30 to 60%. It has been found that the fulminant and high mortality of the condition in FARDS occurs as a result of an excessive immune response to the host-side virus rather than the direct effect of virus growth. In inflammation such as pneumonia, a series of global or partial defense responses to various injurious factors acting on the body, such as changes in the number of immune system cells, changes in the migration rate of the cells, and activity of the cells. Changes in the condition cause histological disorders or pathological conditions. Examples of immune system cells involved in the biological defense reaction include T cells, B cells, monospheres or macrophages, antigen-presenting cells (APCs), dendritic cells, globules, NK cells, NKT cells, neutrophils, and favourites. Examples include acid globules, obese cells, or any other cell specifically associated with immunity, such as cytokine-producing endothelial cells or cytokine-producing epithelial cells. In FARDS due to influenza virus infection, specifically, infection-induced overproduction of cytokines further induces infiltration of neutrophils and alveolar macrophages into the lungs, resulting in infiltrated alveolar epithelial cells. It is believed that the molecules produced by the drug cause tissue damage.

The pharmaceutical composition and method according to the present invention are applicable to "fulminant acute pneumonia". As a preferred embodiment of the present invention, fulminant acute pneumonia due to influenza virus infection, more preferably FARDS due to influenza virus infection can be exemplified. In this embodiment, examples of the influenza virus that causes fulminant acute pneumonia include so-called seasonal influenza such as A Hong Kong type, A Soviet Union type, and B type, and highly pathogenic avian influenza virus. As the avian influenza virus, H5N1 type and H1N1 type can be preferably exemplified. In cases infected with the highly pathogenic avian influenza virus and those that become severe after infection with other types of virus, acute respiratory distress may be induced and become fulminant in a short period of time, sometimes leading to multiple organ failure. It has been reported that it can be migrated and even fatal.

"Preventing fulminant acute pneumonia" means not causing fulminant acute pneumonia, or at least reducing post-occurrence symptoms, by taking some measures before the onset of fulminant acute pneumonia. To say.

"Treatment of fulminant acute pneumonia" means to eliminate, alleviate, or stop the progression of fulminant acute pneumonia by some treatment.

The "CD11b antagonist" refers to a substance that suppresses the function of the intracellular signal transduction system mediated by the CD11b molecule, which is generated by the binding of the ligand of CD11b to the CD11b molecule, and hinders the expression of cell activity by the ligand. "Antagonists" are also referred to as antagonists, antagonists, blockers.

The CD11b antagonist binds to CD11b, but unlike the ligand, which is a biological substance that binds to CD11b, does not cause a biological reaction, and the binding inhibits the binding between the ligand and CD11b, so that the ligand binds to the ligand and intracellularly via CD11b. It can be a substance that suppresses the function of the signal transduction system of the above and prevents the expression of cell activity by the ligand. Examples of such substances include anti-CD11b antibodies, CD11b aptamers, low molecular weight pharmaceuticals and the like.

CD11b antagonists can also be substances that can cause a decrease in CD11b expression. Such a substance may be any substance as long as it inhibits transcription and / or translation of the CD11b gene and suppresses the expression of the gene. Examples of such substances include CD11b mRNA antagonists, CD11b aptamers, low molecular weight double-stranded RNA, low molecular weight pharmaceuticals and the like.

As the CD11b antagonist, an anti-CD11b antibody can be preferably exemplified. Such an anti-CD11b antibody is preferably an antibody that specifically recognizes and binds to CD11b. Specific recognition and binding of CD11b means that it recognizes and binds to CD11b, but does not recognize or weakly recognizes proteins other than CD11b. Unlike a ligand, which is a biological substance that binds to CD11b, an anti-CD11b antibody that acts as a CD11b antagonist does not cause a biological reaction, and the binding inhibits the binding between the ligand and CD11b, so that the ligand binds to the ligand in the cell via CD11b. It is an antibody having an action of suppressing the function of a signal transduction system and preventing the expression of cell activity by the ligand.

The anti-CD11b antibody may be either a monoclonal antibody or a polyclonal antibody, and may be a chimeric antibody and / or a humanized antibody. Non-human antibodies can be humanized using methods known per se in the art of antibody production. For example, humanized antibodies can be made using transgenic animals whose immune system is partially or fully humanized. Any antibody or fragment thereof according to the present invention can be partially or completely humanized. The chimeric antibody can be produced by a method known per se in the technical field relating to the production of the antibody.

The anti-CD11b antibody can be produced by using a CD11b polypeptide or a fragment peptide containing an epitope thereof as an antigen and a method known per se in the technical field for producing the antibody. In the preparation of the anti-CD11b antibody, examples of the amino acid sequence of the CD11b polypeptide include a sequence having accession numbers NP_001139280, NP_000623, XP_016878705, XP_011544153, XP_011544521, XP_006721108, a part thereof or a combination thereof. be able to.

The CD11b polypeptide or fragment peptide containing an epitope thereof, which is an antigen used in the production of the anti-CD11b antibody, is preferably one depending on the species to which the anti-CD11b antibody is administered. When the subject to which the anti-CD11b antibody is administered is a human, the anti-CD11b antibody is preferably an antibody against a human CD11b polypeptide or a fragment polypeptide containing an antigen-binding fragment thereof. As such an antibody, various commercially available antibodies {NB110-89474 of Novus Biologicals, Anti-CD11b antibody of Abcam, APC anti-human CD11b (activated) Antibody of BioLegend, etc. can be used}.

The production of the antibody, specifically, in the case of the production of polyclonal antibodies, in animals, is frequent subcutaneous (sc) or intraperitoneal (ip) of the antigen in combination with an adjuvant such as Freund's adjuvant (complete or incomplete). .) It can be carried out by producing an antibody by injection. To improve immunogenicity, a protein that is immunogenic in the species that first immunizes the polypeptide or fragment containing the target amino acid sequence, such as keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin or soybean. Trypsin inhibitors and bifunctional agents or derivatizers such as maleimide benzoyl sulfosuccinimide ester (composite via cysteine residue), N-hydroxysuccinimide (via lysine residue), glutaaldehyde, succinic anhydride, SOCl _It is useful to use 2 etc. to form a complex. Alternatively, the immunogenic complex can be made as a fusion protein by recombinant techniques.

The production of a monoclonal antibody can be carried out by using a method known per se in the technical field related to the production of the antibody, for example, the hybridoma method described in the previous report (Non-Patent Document 5). In hybridoma methods, mice, hamsters or other suitable host animals are typically immunized with an immune agent to produce or be capable of producing antibodies that specifically bind to the immune agent. Guide the sphere. Alternatively, lymphocytes can be immunized in vitro. Monoclonal antibodies can be prepared by recovering spleen cells from immunized animals and by immunizing the cells in a conventional manner, eg, by fusion with myeloma cells. The prepared clones are then screened for those expressing the desired antibody. The monoclonal antibody is preferably one that does not cross-react with proteins other than CD11b. After identifying the desired hybridoma cells, the clones can be subcloned by limiting dilution procedures and grown by standard methods. Monoclonal antibodies secreted by subclones are isolated or purified from culture medium or ascites by conventional immunoglobulin purification procedures such as protein A-sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography. can do.

Further, in the present invention, an antibody fragment which is an anti-CD11b antibody fragment (hereinafter referred to as an antibody fragment) and contains an antigen-binding fragment of the anti-CD11b antibody can be used in the same manner as the anti-CD11b antibody. The structure of the antibody fragment is not particularly limited as long as it has an action similar to that of the anti-CD11b antibody. The antibody fragment contains a part of an intact antibody, for example, an antigen-binding region or a variable region of an intact antibody. Examples of antibody fragments include Fab fragments, Fab1 fragments, F (ab') 2 fragments and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Be done.

The antibody fragment can be produced by a method known per se in the technical field related to antibody production. For example, the Fab fragment is obtained by papain treatment of the antibody and the F (ab') 2 fragment is obtained by pepsin treatment. Also, the single-stranded Fv or sFv antibody fragment comprises the VH and VL domains of the antibody, in which case these domains are present in a single polypeptide chain. The Fv polypeptide can further contain a polypeptide linker between the VH and VL domains that allows sFv to form the desired structure for antigen binding. A diabody is a small antibody fragment with two antigen binding sites, which contains a heavy chain variable domain (VH) linked to a light chain variable domain (VL) with the same polypeptide chain (VH-VL). Short linkers can be used to allow pairing between two domains on the same strand, allowing the domain to pair with the complementary domain of another strand, resulting in two antigen binding sites. ..

Antibodies can be formulated as immunoliposomes. Liposomes containing an antibody can be prepared by a method known per se (Non-Patent Documents 6 and 7). Particularly useful liposomes can be produced by reverse phase evaporation with a lipid composition containing phosphatidylcholine, cholesterol and polyethylene glycol derivatized phosphatidylethanolamine (PEG-PE). The Fab fragment of the antibody according to the present invention can be complexed with liposomes via a disulfide exchange reaction (Non-Patent Document 8). Any therapeutic agent can be further contained in liposomes (Non-Patent Document 9).

As a CD11b antagonist, an aptamer that specifically recognizes and binds to CD11b and inhibits the binding between CD11b and its ligand can be exemplified. The aptamer may be either a nucleic acid aptamer or a peptide aptamer, and specifically may contain one or more of RNA, DNA and amino acids. Aptamers can be obtained using known methods (Non-Patent Documents 10-12). For example, an oligonucleotide library with variable regions ranging in length from 18 nucleotides to 50 nucleotides is used as a template for a run-off transcription reaction that produces a random pool of RNA aptamers. The aptamer pool is then exposed to an unconjugated matrix to remove species of non-specific interactions. The remaining pool is then incubated with the immobilized target. Most of the aptamer species in this pool have low affinity for the target, and washing can leave a pool bound to a small amount of more specific substrate. The pool is then eluted, precipitated, reverse transcribed and used as a template for run-off transfer. After performing such selection 5 times, a fixed amount is taken out, cloned and sequenced. Selection can continue until similar sequences are reproducibly recovered. Alternatively, aptamer production can be performed using a bead-based selection system. This process produces a library of beads in which each bead is coated with an aptamer population having the same sequence composed of natural and modified nucleotides. The beads library which may contain 1 × 10 ⁸ pieces unique sequence exceeds, complexed with tags such as fluorescent dyes, CD11b or a portion thereof, for example, incubated with peptides corresponding to the extracellular domain. After washing, the beads with the highest binding affinity are isolated and the aptamer sequence is determined for subsequent synthesis to give the aptamer with the desired function.

As a CD11b antagonist, a CD11b mRNA antagonist can also be exemplified. Examples of mRNA antagonists include at least one small interfering RNA (siRNA) or at least one ribozyme. According to the present invention, the CD11b antagonist is a therapeutic nucleic acid such as siRNA and can target the CD11b nucleotide sequence, its complements, or any combination thereof. Any suitable CD11b sequence can be utilized. Is the CD11b target sequence of the synthetic siRNA designed for a human CD11b nucleotide sequence having accession numbers AA436312, AK057656, AK127637, AY187247, AY187248, BC096346, BC096347, BC096348, BC099660, or any portion or combination thereof? , Or a subset of all or part of the transcript variants can be recognized.

The CD11b antagonist can be a nucleic acid of at least 10 nucleotides in length that specifically binds to and complements the target nucleic acid encoding CD11b or its complement. In this case, administration of the CD11b antagonist comprises introducing nucleic acid into cells of interest. RNA interference (RNAi) can be utilized and the CD11b antagonist may be siRNA. Administration of the CD11b antagonist involves introducing into the cells of interest, where the cells can transiently express CD11b as an effective amount of siRNA nucleic acid under conditions sufficient to interfere with the expression of CD11b. It is possible. The siRNA nucleic acid may contain protrusions. That is, not all nucleotides require binding to the target sequence. The siRNA nucleic acid may contain RNA. The siRNA nucleic acid may also contain DNA, a deoxyribonucleic acid nucleotide. Any type of suitable small interfering RNA can be utilized. Endogenous microRNAs (miRNAs) may be utilized. Other RNA interfering agents that can be used in accordance with the present invention include small interfering RNAs (SHRNA), trans-operated siRNAs (tasiRNAs), repeat sequence-related siRNAs ((repeat-associated siRNAs)), small interfering RNAs. Included are small interfering RNAs (small interfering RNAs) and Piwi interaction (pi) RNAs (piRNAs). The RNA interfering nucleic acids utilized are at least 10 nucleotides, at least 15 nucleotides, 16 nucleotides, 17 nucleotides, and 18 nucleotides. , 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, 30 nucleotides, 31 nucleotides, 32 nucleotides, 33 nucleotides, 34 nucleotides, at least. It can be 35 and / or 40 to 50 nucleotides in length. The RNAi agent can also include one or more deoxyribonucleotides. The RNAi agent, such as siRNA or shRNA, is a larger nucleic acid construct such as a suitable vector system. Examples of such vector systems include lentivirus vector systems and adenovirus vector systems. Examples of suitable systems are described in the report of Agaard et al. (Non-Patent Document 13). When present as part of a larger nucleic acid construct, the resulting nucleic acid can be longer than the RNAi nucleic acid contained, eg, greater than 50 nucleotides. The RNAi agent utilized can be cleaved by the target mRNA or You do not have to cut it.

Other nucleic acid antagonists can be utilized in addition to or as an alternative to RNA interference. The CD11b antagonist can be a ribozyme that specifically cleaves an RNA molecule transcribed from a gene encoding CD11b, where the ribozyme is a target substrate binding site, a catalytic sequence in the substrate binding site (the substrate binding site is CD11b). Complementary to some of the RNA molecules transcribed from the gene). The CD11b antagonist can be an antisense nucleic acid containing a nucleotide sequence that is complementary to at least 8 nucleotides of the nucleic acid encoding CD11b or its complement. The antisense nucleic acid can be complementary to the CD11b sequence of sufficient length and sequence content so that the antisense nucleic acid does not cross-react with the non-CD11b nucleotide sequence. Cross-reactivity may not cause substantial adverse side effects.

The CD11b antagonist can be a small molecule drug. For example, CD11b disabling small peptide mimetics can be utilized. Such mimetics can be configured to resemble the secondary structural features of the target protein CD11b.

As the CD11b antagonist, one type can be used alone, or any combination of a plurality of types can be used. CD11b antagonists can also be used in combination with other agents that prevent and / or treat fulminant acute pneumonia. Two or more therapeutic agents, including one or more CD11b antagonists, can be administered simultaneously, sequentially or in combination. Therefore, when administering two or more therapeutic agents, it is not necessary to administer them simultaneously, in the same way, or at the same dose. When administered simultaneously, the two or more therapeutic agents can be administered in the same composition or in different compositions. Two or more therapeutic agents can be administered using the same route of administration or different routes of administration. When administered at different times, the therapeutic agents may be administered before or after each other. The order of administration of the two or more therapeutic agents can be alternative. Each dose of one or more therapeutic agents may vary over time. The type of one or more therapeutic agents may vary over time. When administered at different times, the interval between two or more doses may be any period. When administered multiple times, the length of the period can be varied. The interval between administration of two or more therapeutic agents is 0 seconds, 1 second, 5 seconds, 10 seconds, 30 seconds, 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour. , 1.5 hours, 2 hours, 2.5 hours, 3 hours, 4 hours, 5 hours, 7.5 hours, 10 hours, 12 hours, 15 hours, 18 hours, 21 hours, 24 hours, 1.5 days It can be 2, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, or more.

The use of a pharmaceutically acceptable carrier for prescribing a CD11b antagonist in dosages suitable for systemic and topical administration with respect to the practice of the present invention is within the scope of the present invention. Compositions related to the present invention, especially those formulated as solutions, can be administered parenterally, eg, by intravenous injection, to suit the proper selection of carriers and the actual production. The compounds can be readily formulated in dosages suitable for oral administration using pharmaceutically acceptable carriers known in the art.

Examples of the pharmaceutical carrier include fillers, bulking agents, binders, wetting agents, disintegrants, lubricants, diluents and excipients, which are usually used depending on the usage form of the preparation. These are appropriately selected and used according to the administration form of the obtained preparation. More specifically, water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, xanthan gum, gum arabic, casein, Examples thereof include gelatin, agar, glycerin, propylene glycol, polyethylene glycol, vaseline, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, and lactose. These are used in combination of one type and two or more types as appropriate according to the dosage form of the target drug. In addition, stabilizers, bactericides, buffers, tonicity agents, chelating agents, surfactants, pH adjusters and the like can be appropriately used. Examples of the stabilizer include human serum albumin, ordinary L-amino acids, sugars, and cellulose derivatives. The L-amino acid is not particularly limited, and may be, for example, glycine, cysteine, glutamic acid, or the like. The saccharides are also not particularly limited, for example, monosaccharides such as glucose, mannose, galactose and fructose, sugar alcohols such as mannitol, inositol and xylitol, disaccharides such as sucrose, maltose and lactose, dextran, hydroxypropyl starch, chondroitin sulfate, etc. Any of polysaccharides such as hyaluronic acid and derivatives thereof may be used. The cellulose derivative is not particularly limited, and may be any of methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose and the like. The surfactant is not particularly limited, and either an ionic surfactant or a nonionic surfactant can be used. Surfactants include, for example, polyoxyethylene glycol sorbitan alkyl ester type, polyoxyethylene alkyl ether type, sorbitan monoacyl ester type, fatty acid glyceride type and the like. Buffering agents include boric acid, phosphoric acid, acetic acid, citric acid, ε-aminocaproic acid, glutamate and / and their corresponding salts (eg, alkali metal salts such as their sodium salts, potassium salts, calcium salts, magnesium salts and the like). Alkaline earth metal salt) can be exemplified. Examples of the tonicity agent include sodium chloride, potassium chloride, saccharides, and glycerin. Examples of the chelating agent include sodium edetate and citric acid.

The dose range of the pharmaceutical composition according to the present invention is not particularly limited, and the administration form, administration route, type of disease, subject properties (body weight, age, medical condition and presence / absence of use of other drugs, etc.), and the doctor in charge It is appropriately selected according to the judgment and the like. Appropriate doses can be determined using common conventional experiments for optimization well known in the art, but generally, for example, about 0.01 μg to 1000 mg per kg body weight of the subject. It is preferably in the range of about 0.1 μg to about 100 mg. The above dose can be administered once to several times a day.

The preferred dose of the CD11b antagonist contained in the pharmaceutical composition or drug according to the present invention varies depending on the type of the CD11b antagonist, but it is easy to carry out a simple repeated experiment using a fulminant acute pneumonia model animal or the like. Can be decided. For example, the preferred dose of the anti-CD11b antibody is based on the fact that the effect in the mouse fulminant acute pneumonia model is observed at 200 μg / day (see Example 4-6 described later), and the neutralizing antibody titer of the antibody is used. In consideration of the above, 0.5 mg / kg to 100 mg / kg, preferably 0.5 mg / kg to 50 mg / kg, more preferably 0.5 mg / kg to 20 mg / kg, still more preferably 0.5 mg / kg per day. It is kg to 10 mg / kg, even more preferably 0.5 mg / kg to 10 mg / kg, still more preferably 0.5 mg / kg to 2.5 mg / kg.

The amount of the active ingredient contained in the pharmaceutical composition or drug according to the present invention is appropriately selected from a wide range. It is usually in the range of about 0.00001-70% by weight, preferably about 0.0001-5% by weight.

The route of administration can be either systemic or topical. In this case, an appropriate administration route is selected according to the disease, symptoms and the like. The agent according to the present invention can be administered by either an oral route or a parenteral route. Examples of the parenteral route include normal intravenous administration, intraarterial administration, and subcutaneous, intradermal, and intramuscular administration.

The dosage form is not particularly limited and may be various dosage forms. For example, in addition to being able to be used as a solution preparation, it is freeze-dried to a state where it can be stored, and then dissolved in a buffer solution containing water or physiological saline to prepare it to an appropriate concentration before use. You can also do it. It may also be a sustained release dosage form or a sustained release dosage form.

Specifically, for oral administration, tablets, capsules, powders, granules, pills, liquids, emulsions, suspensions, solutions, liquor, syrups, extracts, and elixirs should be used. Can be done. Parenteral agents include, for example, subcutaneous injections, intravenous injections, intramuscular injections, injections such as intraperitoneal injections, transdermal and patches, ointments and lotions, and sublingual agents for oral administration. , Oral patches, and aerosols and suppositories for nasal administration, but are not limited thereto. These preparations can be produced by a known method usually used in the preparation process.

When preparing an oral solid preparation, add an excipient, if necessary, a binder, a disintegrant, a lubricant, a colorant, a flavoring agent, a odorant, etc. to the above active ingredient, and then tablet by a conventional method. , Coated tablets, granules, powders, capsules and the like can be produced. Such additives may be those commonly used in the art, for example, excipients include lactose, sucrose, sodium chloride, glucose, starch, calcium carbonate, kaolin, microcrystalline cellulose, silicic acid. Etc., as the binder, water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl starch, methyl cellulose, ethyl cellulose, shelac, calcium phosphate, polyvinylpyrrolidone and the like. Disintegrants include dried starch, sodium alginate, canten powder, sodium hydrogencarbonate, calcium carbonate, sodium lauryl sulfate, monoglyceride stearate, lactose, etc., and lubricants include purified talc, stearate, borosand, polyethylene glycol, etc. As the flavoring agent, sucrose, orange peel, starch, starch acid and the like can be exemplified.

When preparing an oral liquid preparation, an oral liquid preparation, a syrup agent, an elixir agent and the like can be produced by adding a flavoring agent, a buffering agent, a stabilizer, a odorant and the like to the above compound by a conventional method. In this case, the flavoring agent may be those listed above, the buffering agent may be sodium citrate or the like, and the stabilizer may be tragant, gum arabic, gelatin or the like.

When preparing an injection, a pH regulator, buffer, stabilizer, isotonic agent, local anesthetic, etc. are added to the above compound to produce subcutaneous, intramuscular and intravenous injections by a conventional method. can do. Examples of the pH adjuster and buffer in this case include sodium citrate, sodium acetate, sodium phosphate and the like. Examples of the stabilizer include sodium pyrosulfite, ethylenediaminetetraacetic acid (EDTA), thioglycolic acid, thiolactic acid and the like. Examples of the local anesthetic include procaine hydrochloride, lidocaine hydrochloride and the like. Examples of the tonicity agent include sodium chloride, glucose and the like.

The CD11b antagonist can be provided as an intra-alveolar neutrophil aggregation inhibitor or a pulmonary neutrophil infiltration inhibitor.

"Inhibition of neutrophil aggregation in the alveoli" means to reduce the aggregation and accumulation of neutrophils occurring in the alveoli, for example, the aggregation and accumulation of neutrophils in the alveoli observed in diseases such as FARDS. Say that.

"Suppression of neutrophil infiltration in the lung" refers to inhibiting the invasion of neutrophils into the lung, for example, the invasion of neutrophils into the lung, which is observed in diseases such as FARDS.

The CD11b antagonist can also be provided as a prophylactic and / or therapeutic agent for fulminant acute pneumonia containing the CD11b antagonist.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the technical scope of the present invention is not limited thereto.

Fulminant acute pneumonia was induced in wild-type mice (mouse FARDS model) and mortality, lung findings, and P / F ratios were examined. As the mouse, a wild-type C57BL / 6 mouse was used. All animals were bred according to the guidelines of Yamaguchi University.

The mouse FARDS model is a solution prepared by dissolving αGalCel (manufactured by KIRIN), which is an activator of NKT cells, in pyrogen-free phosphate buffered saline (PBS) at a dose of 1 μg / 50 μl PBS / mouse. A wild mouse (C57BL / 6) was sensitized by nasal administration (Day -1), and 24 hours later, 50 μg / 50 μl of a solution prepared by dissolving lipopolysaccharide (LPS) in pyrogen-free PBS was prepared. Produced by nasal administration (Day 0) at a PBS / mouse dose to induce fulminant acute pneumonia (Fig. 1A). It has already been reported that fulminant acute pneumonia is induced by administration of LPS to wild-type mice following administration of αGalCel (Non-Patent Document 14).

After LPS administration, the life and death of the mice was confirmed every 12 hours, and the survival rate was calculated. Arterial blood was collected from individual mice 48 hours after LPS administration, and the partial pressure of arterial blood oxygen was measured using an automatic analyzer (ABS555, RADIOMETER COPENHAGEN, Denmark) to calculate the P / F ratio. For histological analysis, lungs obtained from individual mice 72 hours after LPS administration were fixed in 4% paraformaldehyde, paraffin-embedded, sectioned and stained with hematoxylin and eosin (H & E) for histological examination. bottom. Specimens were examined under a light microscope.

1. 1. Mortality rate of mouse FARDS model When examined using 8 mouse FARDS models, more than 80% died 2-3 days after LPS administration (Fig. 1B).

2. Pulmonary findings of mouse FARDS model In the mouse FARDS model, 72 hours after LPS administration, very severe neutrophil infiltration in lung tissue was observed, similar to the histopathological image of ARDS / FARDS in humans (Fig. 1C).

3. 3. P / F ratio of mouse FARDS model The P / F ratio of mouse FARDS model was 91.8 ± 34.6. On the other hand, the P / F ratio of untreated wild-type mice (hereinafter referred to as steady-state wild-type mice) was 507.1 ± 31.9. From this result, it was clarified that the P / F ratio of the wild-type mouse in the steady state is about the same as or slightly higher than that of the human, while the P / F ratio of the mouse FARDS model is a value corresponding to the human FARDS. (Fig. 1D).

The infiltration of neutrophils and CD4 T cells into the lungs of the mouse FARDS model was quantitatively measured by visualizing immune cells using imaging techniques.

The mouse FARDS model was prepared as shown below (Fig. 2A). A transgenic mouse (hereinafter referred to as GFP Tg) incorporating a green fluorescent protein gene was used as a donor, and neutrophils and CD4 T were used from the femoral bone marrow using an AutoMACS sorter (Miltenyi Biotec). The cells were purified to a purity of 98% each. GFP Tg (C57BL / 6-Tg (CAG-EGFP) C14-Y01-FM131Osb meeting) was purchased from RIKEN BioResource Center. Two million isolated neutrophils or CD4 T cells were administered intravenously (iv) into wild-type C57BL / 6 mice (Day-3). Then, C57BL / 6 mice, which are wild-type mice of the same strain, were used as recipients, and the neutrophils or CD4 T cells were transferred via veins. 48 hours after cell transfer (Day -1), αGalCel was nasally administered to wild-type mice at a dose of 1 μg / 50 μl PBS / mouse, and 24 hours later (Day 0), LPS was administered at a dose of 50 μg / 50 μl PBS / mouse. Nasal administration was performed to induce fulminant acute pneumonia.

Lungs of mice were removed 6 hours, 12 hours, 24 hours, and 48 hours after LPS administration, and GFP-positive neutrophils were observed using a fluorescence microscope (M250FA, manufactured by Nikon Corporation).

1. 1. Neutrophil Accumulation in the Lung of Mouse FARDS Model In mice treated with αGalCel and LPS, very severe GFP-positive neutrophil infiltration in lung tissue was observed 48 hours after LPS administration (Fig. 2B). Infiltration of neutrophils into the lungs was also observed in mice administered with LPS alone, but the number of infiltrated neutrophils was smaller than that in mice administered with αGalCel and LPS. The results revealed that LPS-induced lung neutrophil infiltration was dramatically increased by premedication with αGalCel. In addition, the infiltrated neutrophils were found to be agglomerated to the extent that the neutrophils were in contact with each other. In pneumonia other than fulminant acute pneumonia, neutrophils are contained only to the extent that they are scattered in the alveoli (Non-Patent Documents 15-18). The formation of aggregates by neutrophils infiltrating the lungs is one of the characteristics of fulminant acute pneumonia. In addition, in the mice to which αGalCel and LPS were administered, in addition to the transferred GFP-positive neutrophils, many GFP-unlabeled neutrophils originally present in the recipient were infiltrated, and agglomerates of neutrophils were present. Is forming. This result suggests that administration of αGalCel and LPS dramatically increases neutrophil infiltration into the lungs and forms aggregates, resulting in fulminant pneumonia.

2. Time-dependent changes in lung neutrophil accumulation after LPS administration In mice administered with αGalCel and LPS, significant infiltration of GFP-positive neutrophils into the lung was observed about 6 hours after LPS administration (Fig. 2C). ..

3. 3. Time-dependent changes in CD4 T cell accumulation in the lung after LPS administration In mice administered with αGalCel and LPS, accumulation of GFP-positive CD4 T cells in the lung was observed from about 24 hours after LPS administration (Fig. 2D). It was found that the accumulation of CD4 T cells in the lung was observed later than the accumulation of neutrophils in the lung.

The infiltration of immunoinflammatory cells into the lungs of a mouse FARDS model was examined. A mouse FARDS model prepared by the same method as in Example 1 was used.

Three days after LPS administration, alveolar lavage (BAL) was performed according to the previously reported (Non-Patent Document 19). All alveolar lavage fluids were collected and cells in 150 μl aliquots were counted. The number of each cell type in the lung of one mouse was calculated from the total number of cells and the percentage of each cell type by flow cytometry.

Infiltration of immunoinflammatory cells into the lungs of the mouse FARDS model increased dramatically after LPS administration, and in particular, neutrophil accumulation was characteristically observed (Fig. 3).

From the examination results of Example 3, the infiltration of immunoinflammatory cells into the lung of the mouse FARDS model increased dramatically after LPS administration, and in particular, the accumulation of neutrophils was characteristically observed. Therefore, the effect of anti-CD11b antibody administration on lung neutrophil infiltration in a mouse FARDS model was examined.

A mouse FARDS model prepared by the same method as in Example 2 was used. Twenty-four hours after cell transfer (Day-2), anti-CD11b mAb (BioLegend rat IgGM1 / 70) was injected intraperitoneally (ip) at a dose of 200 μg / mouse 24 hours later. ΑGalCel was nasally administered to (Day -1), and LPS was nasally administered 24 hours later (Day 0) to induce fulminant acute pneumonia. Twenty-four hours after LP administration, the lungs of the mice were taken out, and GFP-positive neutrophils were observed using a fluorescence microscope (M205FAA, manufactured by Nikon Corporation).

Significant lung infiltration of GFP-positive neutrophils was observed in mice treated with αGalCel and LPS, but presymptomatic administration of anti-CD11b antibody suppressed lung neutrophil infiltration (Fig. 4). In addition, the formation of aggregates by neutrophils infiltrating the lung was suppressed by presymptomatic administration of anti-CD11b antibody.

From the above results, the anti-CD11b antibody can be used for suppressing neutrophil infiltration into the lung and aggregation of infiltrated neutrophils.

The suppressive effect of fulminant acute pneumonia by administration of anti-CD11b antibody before the onset of FARDS was examined. A mouse FARDS model prepared by the same method as in Example 1 was used. Twenty-four hours before αGalCel administration (Day-2), anti-CD11b mAb (rat IgGM1 / 70 manufactured by BioLegend) was intraperitoneally (ip) administered at a dose of 200 μg (Fig. 5A). The same treatment was carried out using a control antibody (rat IgG2b, κ isotype control antibody manufactured by BioLegend) as a negative subject.

After LPS administration, the life and death of the mice was confirmed every 12 hours, and the survival rate was calculated. Arterial blood was collected from individual mice 48 hours after LPS administration, and the partial pressure of arterial blood oxygen was measured using an automatic analyzer (ABS555, RADIOMETER COPENHAGEN, Denmark) to calculate the P / F ratio. For histological analysis, lungs obtained from individual mice 72 hours after LPS administration were fixed in 4% paraformaldehyde, paraffin-embedded, sectioned and H & E stained for histological examination. Specimens were examined under a light microscope.

1. 1. Mortality rate of mouse FARDS model When examined using 8 mouse FARDS models, a significant reduction in mortality rate was observed by administration of anti-CD11b antibody before the onset of FARDS (Fig. 5B). Specifically, more than 80% of the mice survived with avoidance of lethality.

2. Pulmonary findings of mouse FARDS model In the histopathological image 72 hours after LPS administration, severe inflammatory cell infiltration was observed in the lung tissue of the mouse to which the control antibody was administered, but the mouse to which the anti-CD11b antibody was administered before the onset of FARDS. In the lung tissue, infiltration of inflammatory cells was suppressed and the degree of inflammation was reduced (Fig. 5C).

3. 3. P / F ratio of mouse FARDS model The P / F ratio of mouse FARDS model is 98.4 ± 36.2, and the P / F of wild-type mice to which only PBS is administered (hereinafter referred to as steady-state wild-type mice). The ratio was 575.2 ± 32.1. In the mice to which the anti-CD11b antibody was administered before the onset of FARDS, the P / F ratio was improved to 480.8 ± 49.5. On the other hand, in the mice to which the control antibody was administered, the P / F ratio was 109.9 ± 37.2, and no improvement was observed. From this result, it was clarified that the decrease in P / F ratio observed in the mouse FARDS model was significantly improved by administering the anti-CD11b antibody before the onset of FARDS (Fig. 5D).

It was revealed that the anti-CD11b antibody, when administered before the induction of FARDS, increases the survival rate of mice after the onset of FARDS, suppresses the infiltration of inflammatory cells into the lung, and significantly improves the P / F ratio. rice field.

The inhibitory effect of anti-CD11b antibody administration after the onset of FARDS on fulminant acute pneumonia was investigated. A mouse FARDS model prepared by the same method as in Example 1 was used. Twenty-four hours after LPS administration, anti-CD11b mAb (bioLegend rat IgGM1 / 70) was administered intraperitoneally (ip) at a dose of 200 μg (FIG. 6A). The same treatment was performed using a control antibody as a negative subject.

After LPS administration, the life and death of the mice was confirmed every 12 hours, and the survival rate was calculated. For histological analysis, lungs obtained from individual mice 72 hours after LPS administration were fixed in 4% paraformaldehyde, paraffin-embedded, sectioned and H & E stained for histological examination. Specimens were examined under a light microscope.

1. 1. Mortality rate of mouse FARDS model When examined using 10 mouse FARDS models, a significant decrease in mortality rate was observed by administration of anti-CD11b antibody after the onset of FARDS (Fig. 6B). Specifically, more than 80% of the mice survived with avoidance of lethality.

2. Pulmonary findings of mouse FARDS model In the histopathological image 72 hours after LPS administration, severe inflammatory cell infiltration was observed in the lung tissue of mice to which the control antibody was administered, but in mice to which anti-CD11b antibody was administered after the onset of FARDS. Infiltration of inflammatory cells was suppressed in lung tissue (Fig. 6C). Compared with the lung tissue of mice to which the anti-CD11b antibody was administered before the onset of FARDS, the degree of suppression of cell infiltration was small, but clearly different from the control antibody-administered group, a space for gas exchange remained in the lung tissue. .. Therefore, the survival rate increased.

As described above, it was clarified that the anti-CD11b antibody gave the same survival rate as the administration before the induction even after the administration after the induction of FARDS, and suppressed the infiltration of inflammatory cells into the lung.

Using an anti-CD11b antibody cloned differently from Example 5 above, the suppressive effect of fulminant acute pneumonia by administration of the anti-CD11b antibody before the onset of FARDS was examined.
A mouse FARDS model prepared by the same method as in Example 1 was used. Anti-CD11b mAb (ratIgG 3A33 manufactured by Southern Biotech) was intraperitoneally (ip) administered at a dose of 200 μg 24 hours before αGalCel administration (Day-2) in the same administration procedure as in Example 5. The same treatment was carried out using a control antibody (rat IgG2b, κ isotype control antibody manufactured by BioLegend) as a negative subject.
The anti-CD11b antibody 3A33 used in this example and the anti-CD11b antibody M1 / 70 used in Example 5 were both fluorescently labeled and the live mouse cells were directly stained, and the expression of CD11b was detected by a flow cytometer. I'm sure I can do it. Since the antibody molecule cannot enter the cell by direct staining of living cells, the fact that CD11b can be detected by the flow cytometer means that the two types of anti-CD11b antibodies used both recognize and bind to the extracellular region of the CD11b molecule. It means that.

After LPS administration, the life and death of the mice was confirmed every 12 hours, and the survival rate was calculated. For histological analysis, lungs obtained from individual mice 72 hours after LPS administration were fixed in 4% paraformaldehyde, paraffin-embedded, sectioned and H & E stained for histological examination. Specimens were examined under a light microscope.

1. 1. Mortality rate of mouse FARDS model When examined using 5 mouse FARDS models, a significant reduction in mortality rate was observed by administration of anti-CD11b antibody before the onset of FARDS (Fig. 7A). Specifically, more than 80% of the mice survived with avoidance of lethality.

2. Pulmonary findings of mouse FARDS model In the histopathological image 72 hours after LPS administration, severe inflammatory cell infiltration was observed in the lung tissue of the mouse to which the control antibody was administered, but the mouse to which the anti-CD11b antibody was administered before the onset of FARDS. In the lung tissue, the infiltration of inflammatory cells was suppressed and the degree of inflammation was reduced (Fig. 7B).

By administering the anti-CD11b antibody to an antibody cloned differently from Example 5 before the induction of FARDS, it is possible to increase the survival rate of mice after the onset of FARDS and suppress the infiltration of inflammatory cells into the lung. confirmed.

The inhibitory effect of anti-CD11b antibody administration after the onset of FARDS on fulminant acute pneumonia was investigated. A mouse FARDS model prepared by the same method as in Example 1 was used. Similar to Example 6, anti-CD11b mAb (rat IgGM1 / 70, manufactured by BioLegend) was intraperitoneally (ip) administered at a dose of 200 μg 24 hours after LPS administration. The same treatment was performed using a control antibody as a negative subject.

Arterial blood was collected from individual mice 48 hours after LPS administration, and the P / F ratio was calculated in the same manner as in Example 5.

The P / F ratio of the mouse FARDS model was 94.9 ± 31.2, and the P / F ratio of the steady-state wild-type mouse was 616.6 ± 22.2. In the mice to which the anti-CD11b antibody was administered after the onset of FARDS, the P / F ratio was improved to 269.5 ± 44.1. On the other hand, in the mice to which the control antibody was administered, the P / F ratio was 105.6 ± 36.4, and no improvement was observed. From this result, it was clarified that the decrease in P / F ratio observed in the mouse FARDS model was significantly improved even when the anti-CD11b antibody was administered after the onset of FARDS (Fig. 8).

(General)
The results of the above examples suggest that the CD11b molecule can be a good therapeutic target molecule for FARDS, a refractory inflammatory disease. By suppressing the expression and function of CD11b in immunoinflammatory cells, such as neutrophils, FARDS can be treated.

The present invention provides a pharmaceutical composition for preventing and / or treating fulminant acute pneumonia, which comprises a CD11b antagonist, for example, an anti-CD11b antibody, and a method for preventing and / or treating fulminant acute pneumonia. It can be used in the pharmaceutical field.

Claims (4)

A pharmaceutical composition comprising a CD11b antagonist for the prevention and / or treatment of fulminant acute pneumonia , wherein the CD11b antagonist is an antibody that specifically recognizes CD11b .
A neutrophil aggregation inhibitor containing a CD11b antagonist, which is an antibody that specifically recognizes CD11b by the CD11b antagonist, which is an inhibitor of neutrophil aggregation in the alveoli.
A neutrophil infiltration inhibitor containing a CD11b antagonist, which is an antibody that specifically recognizes CD11b by the CD11b antagonist .
A prophylactic and / or therapeutic agent for fulminant acute pneumonia containing a CD11b antagonist, which is an antibody in which the CD11b antagonist specifically recognizes CD11b .

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