JP6800591B2 - Influenza remedy - Google Patents

Description

The present invention relates to a therapeutic agent for influenza. More specifically, it relates to inflammation associated with influenza virus infection, especially to the prevention and / or treatment of pneumonia. Furthermore, the present invention relates to a combination with other influenza therapeutic agents having different mechanisms of action.

About 90% or more of the causes of cold syndrome are infected with viruses, and it is said that there are more than 200 types of causative viruses. Influenza is a type of cold syndrome, and the causative influenza viruses (mainly A and B) are extremely infectious and cause various systemic symptoms characterized by high fever. Cold syndrome initially presents with symptoms of upper respiratory tract inflammation of the throat and nose. Influenza, on the other hand, has a rapid fever, followed by systemic symptoms such as fatigue, myalgia, and arthralgia, as well as symptoms of upper respiratory tract inflammation. Most people heal spontaneously in 1 to 2 weeks, but weak elderly people and infants are more likely to develop bronchitis and pneumonia, and when they become severe, they may develop encephalitis and heart failure.

Regarding the relationship between influenza and pneumonia, there are viral pneumonia caused by influenza virus and pneumonia (secondary infection) that develops after influenza. Pneumonia tends to become more severe, such as ARDS (acute respiratory distress syndrome) that develops afterwards and pneumonia caused by a secondary infection that develops when the influenza seems to have been cured, and it is said that most people who die from influenza are caused by pneumonia. There is.

Antiviral agents for influenza include oseltamivir phosphate (trade name: Tamiflu), zanamivir hydrate (trade name: Relenza), amantadine hydrochloride (trade name: Symmetrel, etc.), peramivir hydrate (commodity). Name: Rapiacta), laninamivir octanoic acid ester hydrate (trade name: Inavir), etc. The effective time of these drugs after infection is approximately 48 hours, and their use is limited. The emergence of antiviral resistant strains has also been reported. Furthermore, a stable supply system of vaccines has not been established for the highly pathogenic avian influenza epidemic that is feared. The aforementioned oseltamivir phosphate, zanamivir hydrate, peramivir hydrate, and laninamivir octanoate ester hydrate are all inhibitors of neuraminidase (NA) present on the surface of influenza virus, and are only in the early stages of infection. Is valid at. After 48 hours from the onset, almost no individual healing effect can be expected. Also, these drugs are not effective against resistant viruses. As a resistant virus, for example, the 274th amino acid of neuraminidase is replaced with a normal histidine to tyrosine.

It is desired to develop a drug having a mechanism of action different from that of an antiviral agent and capable of improving the symptoms of influenza without being affected by drug-resistant influenza virus and virus subtype.

HMGB1 (High Mobility Group Box 1: hereinafter referred to as "HMGB1") protein is a typical DAMP (danger-associated molecular patterns). HMGB1 is a member of the non-histone chromatin-related protein family involved in DNA organization and transcriptional regulation. Similar to IL-1α, it is released extracellularly from the cell nucleus by cell necrosis and induces an inflammatory reaction. It has been suggested that HMGB1 acts on dendritic cells and monocytes to induce maturation, infiltration, and release of cytokines and inflammatory mediators. In addition, recognition of TLR4 on neutrophils suggests that HMGB1 contributes to the inflammatory response in arthritis and Sjogren's syndrome in relation to the disease. In addition, HMGB1 is highly expressed in cancer, suggesting that it promotes cell survival and proliferation through activation of NF-κB. In recent years, HMGB1 is released extracellularly due to cell necrosis, and active secretion is observed extracellularly in response to vasculitic signal, and it is attracting attention as a new inflammatory marker. HMGB1 is a protein that has homology over 99% or more of the amino acid sequence from rodents to humans. This HMGB1 is also present in normal cells, but its blood concentration increases due to stimulation by LPS (lipopolysaccharide), which is an intracellular toxin released in sepsis (systemic inflammatory response syndrome), resulting in ultimate tissue damage. .. Cerebral vasospasm inhibitor (Patent Document 1) and cerebral infarction inhibitor (Patent Document 2) have already been patented for drugs containing the anti-HMGB1 monoclonal antibody against HMGB1 as an active ingredient, but they are associated with influenza or influenza virus infection. No disclosure is made regarding the prevention and / or treatment of pneumonia.

Japanese Patent No. 3882090 Japanese Patent No. 3876325

An object of the present invention is to provide a therapeutic agent for influenza. More specifically, regarding inflammation associated with influenza virus infection, it is an object of the present invention to provide an influenza therapeutic agent for the prevention and / or treatment of pneumonia. Another object of the present invention is to provide an influenza therapeutic agent in combination with another influenza therapeutic agent having a different mechanism of action.

The inventors of the present application administered an anti-HMGB1 monoclonal antibody to an influenza virus infection model in order to solve the above problems, and the administration of the anti-HMGB1 monoclonal antibody improved the survival rate. As a result of further diligent studies, it was found that the anti-HMGB1 monoclonal antibody improved the inflammatory symptoms caused by influenza virus infection, and the present invention was completed. When used in combination with other influenza therapeutic agents having different mechanisms of action, a more effective influenza therapeutic agent can be provided.

That is, the present invention comprises the following.
1. 1. An influenza therapeutic agent containing an anti-HMGB1 monoclonal antibody as an active ingredient.
2. 2. The influenza therapeutic agent according to item 1 above, wherein the influenza therapeutic agent is for inflammation associated with influenza virus infection.
3. 3. The influenza therapeutic agent according to item 1 above, wherein the influenza therapeutic agent is influenza treatment by preventing and / or treating pneumonia associated with influenza virus infection.
4. The influenza therapeutic agent according to any one of the above items 1 to 3, wherein the anti-HMGB1 monoclonal antibody is administered at 0.2 to 5 mg / kg at a time.
5. The influenza therapeutic agent according to any one of the preceding items 1 to 4, which is characterized in that an influenza therapeutic agent having a different mechanism of action is used in combination with the anti-HMGB1 monoclonal antibody.
6. The influenza therapeutic agent according to item 5 above, wherein the influenza therapeutic agent having a mechanism of action different from that of the anti-HMGB1 monoclonal antibody is a neuraminidase inhibitor.
7. The influenza therapeutic agent according to item 5 above, wherein the influenza therapeutic agent having a mechanism of action different from that of the anti-HMGB1 monoclonal antibody is peramivir.

According to the influenza therapeutic agent containing the anti-HMGB1 monoclonal antibody of the present invention as an active ingredient, the risk of death due to influenza virus infection can be reduced. The anti-HMGB1 monoclonal antibody as the active ingredient of the present invention does not act directly on influenza virus, but can be an effective preventive and / therapeutic agent for pneumonia associated with influenza virus infection. According to the therapeutic agent for influenza containing the anti-HMGB1 monoclonal antibody of the present invention as an active ingredient, it acts on HMGB1 in the living body increased by influenza virus infection and exerts its effect. Therefore, it is extremely useful because it can suppress inflammation associated with influenza virus infection without affecting the subtype of influenza therapeutic agent resistant influenza virus.

It is a figure which shows the amino acid sequence which identifies HMGB1 of human, rat and bovine. The anti-HMGB1 monoclonal antibody of the present invention is a monoclonal antibody having the C-terminal region specified by SEQ ID NO: 5 as an epitope among these amino acid sequences. It is a figure which shows the method and result of the epitope mapping about the anti-HMGB1 monoclonal antibody of this invention based on the amino acid sequence which identifies HMGB1. (Example 1) It is a figure which shows the experimental method for confirming the effect of an anti-HMGB1 monoclonal antibody against influenza. (Example 2) It is a result figure which confirmed the survival rate when the anti-HMGB1 monoclonal antibody was administered to the mouse infected with influenza virus. (Example 2) It is a figure which shows the experimental method for confirming the effect of an anti-HMGB1 monoclonal antibody against influenza. (Example 3) It is a photograph figure which confirmed the pulmonary pathological findings when the anti-HMGB1 monoclonal antibody was administered to the mouse infected with influenza virus. (Example 3) It is a photograph figure which confirmed the histological findings (neutrophil infiltration) when the anti-HMGB1 monoclonal antibody was administered to the influenza virus infected mouse. (Example 3) It is a figure which measured the histological findings and the neutrophil count in the bronchoalveolar lavage fluid (BALF) when the anti-HMGB1 monoclonal antibody was administered to the influenza virus infected mouse. (Example 3) It is a figure which shows the result of having measured the amount of infected virus when the anti-HMGB1 monoclonal antibody was administered to the influenza virus infected mouse. (Example 3) It is a figure which shows the result of having measured the amount of HMGB1 about serum and BALF when the anti-HMGB1 monoclonal antibody was administered to the mouse infected with influenza virus. (Example 3) It is a figure which shows the result of having measured the amount of oxidative stress when the anti-HMGB1 monoclonal antibody was administered to the mouse infected with influenza virus. (Example 3) It is a result figure which confirmed the survival rate when the anti-HMGB1 monoclonal antibody and other influenza therapeutic agents having a different mechanism of action were administered to influenza virus-infected mice. (Example 4) It is a result figure which confirmed the effect on the weight loss when the anti-HMGB1 monoclonal antibody and other influenza therapeutic agents having different mechanisms of action were administered to influenza virus-infected mice. (Example 4)

The therapeutic agent for influenza of the present invention contains an anti-HMGB1 monoclonal antibody as an active ingredient. In principle, the anti-HMGB1 monoclonal antibody binds only to HMGB1 and detoxifies it, and does not act on other compounds. Therefore, it is considered that side effects are unlikely or extremely rare. By administering an influenza therapeutic agent containing an anti-HMGB1 monoclonal antibody as an active ingredient, pneumonia associated with influenza virus infection can be prevented and / or treated, and as a result, influenza can be treated.

The preparation of anti-HMGB1 monoclonal antibodies herein can follow methods known per se. For example, a commercially available HMGB1 can be used to immunize a mouse, rat or the like, and the antibody-producing cells or splenocytes thereof can be fused with myeloma cells to obtain a hybridoma. This hybridoma can be cloned and screened for clones producing antibodies that specifically react with HMGB1. This clone can be cultivated to purify the secreted monoclonal antibody. For example, it can be obtained by the method shown in Patent Document 1 or 2. For reference, the amino acid sequences of human, rat and bovine HMGB1 proteins are shown below. In the C-terminal part, the underlined site is the species-specific sequence. As used herein, a suitable anti-HMGB1 monoclonal antibody is a monoclonal antibody having an epitope at the C-terminal portion of HMGB1, for example, any of SEQ ID NOs: 4 to 6. In particular, the anti-HMGB1 monoclonal antibody using the amino acid sequence shown in SEQ ID NO: 5 as an epitope shows a strong binding ability to HMGB1.

Human HMGB1 (NCBI Reference Sequence: NP_002119.1) (SEQ ID NO: 1)
mgkgdpkkpr gkmssyaffv qtcreehkkk hpdasvnfse fskkcserwk tmsakekgkf edmakadkar yeremktyip pkgetkkkfk dpnapkrpps afflfcseyr pkikgehpgl sigdvakklg emwnntaadd kqpyekkaak lkekyekdia ayrakgkpda akkgvvkaek skkkkeeeed eedeedeeee e d eede d eee dddde

Rat HMGB1 (NCBI Reference Sequence: NP_037095.1) (SEQ ID NO: 2)
mgkgdpkkpr gkmssyaffv qtcreehkkk hpdasvnfse fskkcserwk tmsakekgkf edmakadkar yeremktyip pkgetkkkfk dpnapkrpps afflfcseyr pkikgehpgl sigdvakklg emwnntaadd kqpyekkaak lkekyekdia ayrakgkpda akkgvvkaek skkkkeeedd eedeedeeee e e eededeee dddde

Bovine HMGB1 (SEQ ID NO: 3)
mgkgdpkkpr gkmssyaffv qtcreehkkk hpdasvnfse fskkcserwk tmsakekgkf edmakadkar yeremktyip pkgetkkkfk dpnapkrpps afflfcseyr pkikgehpgl sigdvakklg emwnntaadd kqpyekkaak lkekyekdia ayrakgkpda akkgvvkaek skkkkeeeed eedeedeeee edeede e eee dddde

HMGB1 Peptide No. 41: e d eede d eee dddde (SEQ ID NO: 4)
HMGB1 Peptide No. 41 Part: eee dddde (SEQ ID NO: 5)
HMGB1 peptide No.40: deeeee d eede d eee (SEQ ID NO: 6)

The dosage form and administration form of the influenza therapeutic agent containing the anti-HMGB1 monoclonal antibody of the present invention as an active ingredient are not particularly limited. Together with the anti-HMGB1 monoclonal antibody as the active ingredient, it can be formulated in solutions, microemulsions, dispersions, liposomes, or other suitable shapes suitable for high drug concentrations. It can be prepared by incorporating the required amount of anti-HMGB1 monoclonal antibody, optionally with one or a combination of the components listed above, into a suitable solvent and then filtering and sterilizing.

The therapeutic agent for influenza of the present invention can be administered by various methods known in the art. The form of administration is not particularly limited, but in consideration of influenza treatment, intravenous administration as an injection is preferable. In that case, as the solvent, an isotonic solution of plasma such as a physiological saline solution having an adjusted pH or an aqueous glucose solution can be used. When the antibody is freeze-dried together with salts and the like, pure water, distilled water, sterilized water and the like can also be used. The concentration may be that of a normal antibody preparation, and can be 1 to 5 mg / mL. However, the osmotic pressure of the injection should be the same as that of plasma.

The administration time of the therapeutic agent for influenza of the present invention is not particularly limited, but it is preferable that the earliest is after influenza infection, but specifically, within 7 days, preferably within 48 hours, and more preferably within 24 hours after influenza virus infection. Is.

As shown in the examples described later, when 2 mg / kg of anti-HMGB1 monoclonal antibody was administered to mice at a time, a remarkable improvement effect was observed in the mortality rate associated with influenza virus infection (see FIG. 4). ). In addition, administration of anti-HMGB1 monoclonal antibody improved neutrophil infiltration and inflammatory symptoms in lung tissue associated with influenza virus infection (see FIGS. 6, 7, and 8). On the other hand, the copy number of infected influenza virus was not reduced by administration of anti-HMGB1 monoclonal antibody. From this, the anti-HMGB1 monoclonal antibody of the present invention does not act directly on the influenza virus itself, but is effective for the prevention and / or treatment of inflammation associated with influenza virus infection, particularly pneumonia associated with influenza virus infection. It is thought that it acts on.

Considering these results, the dose to humans can be 0.2 to 5 mg / kg of anti-HMGB1 monoclonal antibody, and more preferably 1 to 2 mg / kg per dose. However, the dose of these drugs can be appropriately changed depending on the age and sex of the patient, the severity of the disease, and the like. In addition, the therapeutic agent for influenza of the present invention can be administered multiple times or continuously.

Furthermore, other influenza therapeutic agents can be used in combination with the influenza therapeutic agent of the present invention. The anti-HMGB1 monoclonal antibody, which is the active ingredient of the therapeutic agent for influenza of the present invention, may be formulated with one or more other therapeutic agents for influenza and / or administered in combination. The other therapeutic agent for influenza that can be used in combination is not particularly limited, and may be either a therapeutic agent already on the market or a therapeutic agent to be developed in the future. As the other influenza therapeutic agent, an influenza therapeutic agent having a mechanism of action different from that of the anti-HMGB1 monoclonal antibody is preferable. Examples thereof include neuraminidase inhibitors, M2 protein inhibitors and the like. Examples of the neuraminidase therapeutic agent include peramivir (trade name: Rapiacta), oseltamivir (trade name: Tamiflu), zanamivir (trade name: Relenza), and the like, and peramivir is particularly preferably used. Peramivir, oseltamivir, zanamivir and the like as active ingredients also contain pharmaceutically acceptable salts and hydrates. As the dose of the therapeutic agent already on the market, the existing dose can be applied, or the anti-HMGB1 monoclonal antibody of the present invention is expected to have a combined effect, and the dose is lower than the existing dose. You can also do it.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited by the following examples as well as the present invention, and appropriate modifications are made to the extent that it can be adapted to the gist described in the specification. It is also possible to carry out, all of which are within the technical scope of the invention.

(Example 1) Preparation of anti-HMGB1 monoclonal antibody An anti-HMGB1 monoclonal antibody was prepared by the same method as described in Patent Document 1. Specifically, it is as follows.

1. 1. Rat immunity Take 1 mg / mL of a mixture of commercially available bovine thymus-derived HMGB1 and HMGB2 (manufactured by Wako Pure Chemical Industries, Ltd., code number: 080-070741) in a 2 mL glass injection tube, and take another 2 mL glass injection tube. Emulsion was obtained by gradually mixing with the same volume of Freund's complete adjuvant through the connecting tube. 0.05 mL of the obtained emulsion was injected into the hind limb footpads of rats anesthetized with sevoflurane, for a total of 0.1 mL. Two weeks later, a test blood sample was taken from the jugular vein, and an increase in antibody titer was confirmed. The swollen iliac lymph nodes were then aseptically removed 3 weeks after the injection. From the two lymph nodes obtained, about 6 × 10 ⁷ cells could be recovered.

2. 2. Cell fusion and cloning The above iliac lymph node cells and mouse myeloma SP2 / O-Ag14 (SP2) cells were fused using polyethylene glycol, and the obtained fused cells were sown on a 96-well microplate. One week later, the first Elisa screening was performed and the well samples containing positive cells were secondarily screened by Western blotting. Cells in positive wells are transferred to a 24-well microplate, the cells are grown to a nearly confluent state (approximately 2 × 10 ⁵ ), and then 0.5 mL of frozen medium (10 v / v% fetal bovine serum in GIT medium). And dimethyl sulfoxide (10 v / v% added) were cryopreserved in liquid nitrogen. The cryopreserved cells were thawed and then cloned on a 96-well microplate.

3. 3. Purification of antibody The positive cells were mass-cultured for 2 weeks in a rotary culture device (manufactured by SARSTEDT) to obtain an antibody solution having a concentration of 2 to 3 mg / mL. This antibody solution was mixed with an affinity gel (MEP-HyperCel manufactured by Invitrogen) under a neutral pH, and the anti-HMGB1 antibody was specifically bound to the gel. The antibody specifically bound to the gel was eluted with sodium acetate buffer (pH 4). The eluate was concentrated by an ultrafiltration device and then further purified by a Sepharose CL6B gel filtration column (2 cm in diameter x 97 cm in length) to obtain an anti-HMGB1 monoclonal antibody.

4. Confirmation of Epitope Epitope mapping was performed on the anti-HMGB1 monoclonal antibody (clone # 10-22 antibody) obtained by the above purification (Fig. 2). As a result, the clone # 10-22 antibody showed a strong binding constant (Kd) using the peptide shown in SEQ ID NO: 4, particularly the peptide shown in SEQ ID NO: 5 as an epitope (Tables 1 and 2). In the following examples, clone # 10-22 antibody was used as the anti-HMGB1 monoclonal antibody.
HMGB1 Peptide No. 41: e d eede d eee dddde (SEQ ID NO: 4)
HMGB1 Peptide No. 41 Part: eee dddde (SEQ ID NO: 5)

(Example 2) Effect on influenza virus infection model mouse In this example, the effect on survival rate was confirmed as the effect of the anti-HMGB1 monoclonal antibody on the influenza virus infection model mouse.

1. 1. Influenza infection and antibody administration schedule 8-9 week old male mice (C57 / BL6J) were used. Influenza virus (A / Puerto Rico / 8/34 (H1N1)) was nasally infected with 100 PFU / mouse and used as the influenza virus infection model of this example. Antibodies were administered at 2 mg / kg tail vein 1 hour, 24 hours and 48 hours after influenza virus infection. The antibody administration was divided into 15 animals in the anti-HMGB1 monoclonal antibody (# 10-22) administration group prepared in Example 1 and 15 animals in the control antibody (anti-Keyhole Limpet hemocyanin (KLH) antibody: homemade) administration group (Fig.). 3).

2. 2. Survival rate The survival rate associated with influenza virus infection in the anti-HMGB1 monoclonal antibody-administered group and the control antibody-administered group of the present invention was confirmed by the Kaplan-Meier method. The Cox-Mantel test was performed for the difference in survival time. As a result, a clear improvement in survival rate was observed in the anti-HMGB1 monoclonal antibody-administered group (Fig. 4).

(Example 3) Effect on influenza virus infection model mouse In this example, pathological examination was conducted as the effect of the anti-HMGB1 monoclonal antibody on the influenza virus infection model mouse. Specifically, inflammation at the site of infection (pulmonary pathological examination (HE / Gr1)), neutrophil count in bronchoalveolar lavage fluid (BALF), viral load, HMGB1, and oxidative stress were confirmed.

1. 1. Influenza infection and antibody administration schedule An influenza virus infection model was prepared in the same manner as in Example 2, and 2 mg / kg of antibody was administered to the tail vein 1 hour, 24 hours, and 48 hours after the influenza virus infection. The antibody administration was divided into an anti-HMGB1 monoclonal antibody (# 10-22) administration group and a control antibody (anti-KLH antibody) administration group prepared in Example 1. Lungs, BALF and serum were collected 3 days, 5 days, 7 days and 10 days after influenza virus infection (Fig. 5).

2. 2. Pulmonary pathological examination (HE / Gr1)
Mice on the 10th day of infection were laparotomized and pulmonary pathological symptoms were confirmed. As a result, it was confirmed that the mouse to which the control antibody was administered showed an inflammatory state in and around the lung, whereas the mouse to which the anti-HMGB1 monoclonal antibody was administered suppressed the inflammation (Fig. 6).

3. 3. Histological findings (neutrophil infiltration)
Lung tissue was collected from mice 3 days, 5 days, 7 days and 10 days after infection and histological findings were performed. As a result, inflammatory cell infiltration was observed in the mice to which the control antibody was administered (Fig. 7). Furthermore, as a result of confirming the neutrophil count in BALF, it was confirmed that the neutrophil count was suppressed when the anti-HMGB1 monoclonal antibody was administered at any time of infection as compared with the control (Fig. 8). ).

4. Amount of Infected Virus The amount of influenza virus was measured for serum and BALF collected from mice on the 3rd, 5th, 7th and 10th days after infection. The amount of influenza virus was measured by the RT-PCR method. As a result, when the anti-HMGB1 monoclonal antibody was administered at any time of infection, no particular difference in the amount of influenza virus was observed as compared with the control (Fig. 9). From this, it was considered that the anti-HMGB1 monoclonal antibody did not act directly on the influenza virus.

5. Amount of HMGB1 in serum and BALF Amount of HMGB1 was measured in serum and BALF collected from mice 3, 5, 7 and 10 days after infection. The amount of HMGB1 was measured by the ELISA method. As a result, it was found that the HMGB1 level was suppressed in the serum when the anti-HMGB1 monoclonal antibody was administered at any time of infection as compared with the control (Fig. 10). In BALF, it was found that when the anti-HMGB1 monoclonal antibody was administered 7 days and 10 days after infection, the HMGB1 level was suppressed as compared with the control (Fig. 10).

6. Amount of oxidative stress (antioxidant effect)
Biosample analysis Vol.32, No.4 (2009) of d-ROMs test (Diacron Reactive Oxygen Metabolitie: active oxygen metabolitie test) for BALF collected from mice on the 3rd, 5th, 7th and 10th days after infection. ) Was used. The degree of oxidative stress was confirmed by the obtained U. CARR (unit curl) value. As a result, it was found that the U. CARR value was suppressed when the anti-HMGB1 monoclonal antibody was administered at any time of infection as compared with the control (Fig. 11). From this, it was considered that the anti-HMGB1 monoclonal antibody could reduce the oxidative stress caused by influenza virus infection.

(Example 4) Effect on influenza virus infection model mouse In this example, the combined effect of an anti-HMGB1 monoclonal antibody and an existing anti-influenza virus agent having a different mechanism of action was confirmed on an influenza virus infection model mouse. As an effect, the effect by survival rate and body weight was confirmed.

1. 1. Influenza infection and antibody administration schedule 8-9 week old male mice (C57 / BL6J) were used. A nasal infection of 400 PFU / mouse with influenza virus (A / Puerto Rico / 8/34 (H1N1)) was used as the influenza virus infection model of this example. The anti-HMGB1 monoclonal antibody (# 10-22) prepared in Example 1 was used as the anti-HMGB1 monoclonal antibody. Peramivir (trade name: Rapiacta) was used as an existing anti-influenza virus agent to be used in combination.

Peramivir was intramuscularly administered at 10 mg / kg / day 2-4 days after influenza virus infection. The anti-HMGB1 monoclonal antibody was administered to the tail vein at 5 mg / kg / day 2-4 days after influenza virus infection (see FIG. 12). The effects of the anti-HMGB1 monoclonal antibody and peramivir in combination (n = 9) and peramivir alone (n = 14) on survival and weight loss were confirmed.

2. 2. Survival rate The survival rate associated with influenza virus infection in each of the above-mentioned administration groups was confirmed by the Kaplan-Meier method. The Cox-Mantel test was performed for the difference in survival time. As a result, an improvement in survival rate was observed in the combined administration group of anti-HMGB1 monoclonal antibody and peramivir as compared with the single administration group of peramivir (Fig. 12).

3. 3. Body Weight For each of the above mice, the body weight immediately before influenza virus infection was set to 100% for each drug-administered group after influenza virus infection, and the rate of change in body weight thereafter was confirmed. As a result, a tendency of weight loss was observed after influenza virus infection in all groups, and the weight loss was most severe around 9 to 11 days after infection. However, it was confirmed that the rate of weight loss was suppressed in the combined administration group of anti-HMGB1 monoclonal antibody and peramivir (Fig. 13).

As described in detail above, the influenza therapeutic agent containing the anti-HMGB1 monoclonal antibody of the present invention as an active ingredient can reduce the risk of death from influenza. The anti-HMGB1 monoclonal antibody as the active ingredient of the present invention does not act directly on influenza virus, but exerts its effect by suppressing the symptoms associated with influenza virus infection. In particular, it can be an effective prophylactic and / or therapeutic agent for pneumonia. According to the influenza therapeutic agent of the present invention, since it acts on HMGB1 in the living body increased by influenza virus infection and exerts an anti-pneumonia effect, it does not affect the subtype of influenza therapeutic agent resistant influenza virus and causes influenza virus infection. It is extremely useful because it can suppress the accompanying pneumonia.

Further, since the influenza therapeutic agent of the present invention has a different mechanism of action from the conventional influenza therapeutic agent, it can be used in combination with a conventionally known influenza therapeutic agent, and a more reliable influenza therapeutic effect can be expected.

Claims (6)

An influenza therapeutic agent containing an anti-HMGB1 monoclonal antibody that does not act directly on influenza virus as an active ingredient, and is characterized by being used in combination with a neuraminidase inhibitor selected from peramivir, oseltamivir, or zanamivir . The influenza therapeutic agent according to claim 1, wherein the influenza therapeutic agent is for inflammation associated with influenza virus infection. The influenza therapeutic agent according to claim 1, wherein the influenza therapeutic agent is influenza treatment by preventing and / or treating pneumonia associated with influenza virus infection. The influenza therapeutic agent according to any one of claims 1 to 3, wherein the anti-HMGB1 monoclonal antibody is administered at a dose of 0.2 to 5 mg / kg. The influenza therapeutic agent according to any one of claims 1 to 4, wherein the neuraminidase inhibitor is peramivir. The influenza therapeutic agent according to any one of claims 1 to 5, wherein the influenza therapeutic agent containing an anti-HMGB1 monoclonal antibody as an active ingredient is an influenza therapeutic agent for treating influenza for which a neuraminidase inhibitor alone does not have a therapeutic effect. ..