JP6288815B2 - Pharmaceutical composition for treating pneumonia, etc. - Google Patents

Description

  The present invention relates to a compound for preventing and / or treating pneumonia and / or pulmonary fibrosis, and a pharmaceutical composition containing the compound.

  Interstitial pneumonia (IP) is one of those classified by the form of lesions of pneumonia, an inflammatory disease of the lung. Interstitial pneumonia (IP) is an intractable disease that causes inflammation in the interstitial tissue of the lungs, and as it progresses, the inflamed tissue becomes fibrotic and causes pulmonary fibrosis. Interstitial pneumonia (IP) that does not have a clear cause, such as viral infection or drug addiction, is called idiopathic interstitial pneumonitis (IIP). Pneumonia (IIP) is classified into seven types including normal interstitial pneumonia (UIP) and the like. In normal interstitial pneumonia (UIP), areas where fibroblasts and myofibroblasts aggregate (Fibroblastic foci) are observed, and excessive collagen and extracellular matrix accumulate in the lung interstitium. It is a chronic disease that presents. Normal interstitial pneumonia (UIP) is characterized by the fact that fibrotic lesions exhibit spatial and temporal heterogeneity between normal lung tissues. Idiopathic Pulmonary Fibrosis (IPF) is characterized by idiopathic interstitial pneumonia (IIP), which is characterized by fibrosis and restrictive ventilatory impairment, and is usually pathologically normal. Unexplained lung disease characterized by type interstitial pneumonia (UIP). Idiopathic pulmonary fibrosis (IPF) is designated as a specific disease in Japan, and is said to have the highest incidence among idiopathic interstitial pneumonia (IIP).

  Treatment of idiopathic pulmonary fibrosis (IPF) includes administration of steroids, colchicine, cyclosporin A, endothelin receptor antagonist bosentan, and TNF receptor inhibitor etanercept to suppress inflammation in the stroma. Yes, but only in a few patients with IPF. In addition, IPF is a disease with a poor prognosis, and is often fatal within 3 to 5 years after diagnosis. In some cases, lung transplantation is indicated, and there is no effective treatment until now.

  The alveoli are mainly composed of type I alveolar epithelial cells, type II alveolar epithelial cells, vascular endothelial cells, and fibroblasts, and type I alveolar epithelial cells occupy most of the alveolar surface. Type II alveolar epithelial cells are interspersed between type I alveolar epithelial cells. Type II alveolar epithelial cells are considered as reserve cells. When an alveolar injury, ie type I alveolar epithelial cell injury occurs, type II alveolar epithelial cells proliferate and type I alveolar epithelial cells The shape is changed to repair. In pulmonary fibrosis, tissue repair is repeated, resulting in hyperplasia of type II alveolar epithelial cells. So far, many studies have been made on the damage of type I cell epithelial cells (Non-patent Document 1), but the expected results have not been achieved.

Idiopathic interstitial pneumonias: Primum movens: epithelial, endothelial or whatever.Calabrese F, Giacometti C, Rea F, Loy M, Valente M. Sarcoidosis Vasc Diffuse Lung Dis. 2005 Dec; 22 Suppl 1: S15-23. Review.

  Therefore, the object of the present invention is to prevent and / or treat pneumonia causing pulmonary fibrosis including idiopathic pulmonary fibrosis (IPF) that cannot be solved by conventional pharmaceutical compositions, and / or It is to provide a pharmaceutical composition for preventing and / or treating pulmonary fibrosis including idiopathic pulmonary fibrosis (IPF).

  Reported that IPF increases sialidase activity in the alveoli of IPF, which is thought to cause abnormal inflammatory reaction and fibrosis as a result of repeated tissue injury and tissue repair due to persistent stimulation to alveolar tissue (CR Lambre et al., Clin. Exp. Immunol. 73: 230-235, 1988) and one type II alveolar epithelial cell that constitutes the alveoli may be damaged by sialidase Although there are reports (Honda Takayuki, Shinshu Medical Journal, 47 (6): 481-488, 1999), there is no report on the relationship between sialidase and pulmonary fibrosis.

  Under such circumstances, the present inventors have continued intensive research to solve the above-mentioned problems, and paying attention to the possibility that sialidase is involved in the progression of pulmonary fibrosis, influenza virus known as an anti-influenza drug A compound having sialidase inhibitory activity was tested using interstitial pneumonia model rats. Surprisingly, it was found to have therapeutic and preventive effects on pneumonia and pulmonary fibrosis, and the present invention was completed. It was.

That is, the present invention relates to the following.
[1] A pharmaceutical composition for preventing and / or treating pneumonia and / or pulmonary fibrosis, comprising a compound having sialidase inhibitory activity.
[2] The pharmaceutical composition according to [1], wherein the compound having sialidase inhibitory activity has mammalian sialidase inhibitory activity.
[3] The pharmaceutical composition according to [2], wherein the compound having sialidase inhibitory activity has viral sialidase inhibitory activity and / or bacterial sialidase inhibitory activity.
[4] The pharmaceutical composition according to [2] or [3], wherein the mammalian sialidase inhibitory activity is human sialidase inhibitory activity.
[5] The pharmaceutical composition according to any one of [1] to [4], wherein the compound having sialidase inhibitory activity is at least one compound selected from the group consisting of peramivir, oseltamivir, zanamivir and laninamivir.
[6] The pharmaceutical composition according to any one of [1] to [5], wherein the pneumonia is interstitial pneumonia.
[7] The pharmaceutical composition according to any one of [1] to [6], wherein the pneumonia is pneumonia caused by injury of type II alveolar epithelial cells.
[8] A mammalian sialidase inhibitor comprising peramivir and / or laninamivir.

[9] A method for preventing and / or treating pneumonia and / or pulmonary fibrosis using a compound having sialidase inhibitory activity.
[10] The method according to [9], wherein the compound having sialidase inhibitory activity is a compound having mammalian sialidase inhibitory activity.
[11] The method according to [10], wherein the compound having sialidase inhibitory activity is a compound having viral sialidase inhibitory activity and / or bacterial sialidase inhibitory activity.
[12] The method according to [10] or [11], wherein the mammalian sialidase inhibitory activity is human sialidase inhibitory activity.

[13] The method according to any one of [9] to [12], wherein the compound having sialidase inhibitory activity is at least one compound selected from the group consisting of peramivir, oseltamivir, zanamivir and laninamivir.
[14] The method according to any one of [9] to [13], wherein the pneumonia is interstitial pneumonia.
[15] The method according to any one of [9] to [14], wherein the pneumonia is pneumonia caused by injury of type II alveolar epithelial cells.

According to the present invention, a compound useful for the treatment and / or prevention of pneumonia and / or the prevention and / or treatment of pulmonary fibrosis and a pharmaceutical preparation containing the same can be provided.
According to the present invention, it is preferable to provide a compound useful for preventing and / or treating idiopathic fibrosis, and a pharmaceutical preparation containing the same.
According to the present invention, it is preferable to provide a compound useful for protecting type II alveolar epithelial cells from injury, and a pharmaceutical preparation containing the same.
According to the present invention, it is possible to provide a compound useful for the treatment and / or prevention of a disease caused by an increase in sialidase, and a pharmaceutical preparation containing the same.
ADVANTAGE OF THE INVENTION According to this invention, the compound which shows viral sialidase inhibitory activity and has the therapeutic and preventive effect of pneumonia and pulmonary fibrosis, and a composition containing this can be provided.

The mechanism of action for preventing and / or treating pneumonia / pulmonary fibrosis is not always clear.
A relatively old paper, CR Lambre et al., Clin. Exp. Immunol. 73: 230-235, 1988, describes that sialidase activity is increased in the alveoli of idiopathic interstitial pneumonia. It can be said that there is a suggestion of the relationship between sialidase and sialidase, but here, no mention is made of prevention or treatment of IPF by a sialidase inhibitor, and nothing is made clear about its mechanism of action. In fact, the present inventors tried to measure the sialidase activity in the bronchioloalveolar lavage fluid by the same method in order to try the method described in the document, but the sialidase activity was too low to measure. However, according to the findings of the present inventors, rather than the damage of type I cell epithelial cells, which was the subject of conventional research, Takayuki Honda, Shinshu Medical Journal, 47 (6): 481-488, 1999 It is considered that the type II alveolar epithelial cell injury caused by sialidase is related to the mechanism of pulmonary fibrosis.

FIG. 1 is a schematic diagram showing the mechanism by which type II alveolar epithelial cells are damaged by sialidase.

FIG. 2 is a schematic diagram showing a part of a method for preparing a sample for flow cytometry measurement in Example 1.

FIG. 3 shows the results of flow cytometry using an anti-TF antibody in Example 1. “Count” on the vertical axis represents the number of cells, and the horizontal axis represents FITC fluorescence intensity. A) A549 cells treated with S-Sia: Fluorescence intensity increased at sialidase concentration of 1 U / L or more, and exposure of TF antigen was observed. B) CHO cells treated with S-Sia: Fluorescence intensity increased at sialidase concentration of 1 U / L or more, and exposure of TF antigen was observed. C) Lec2 cells treated with S-Sia: Fluorescence intensity increased regardless of sialidase concentration, and exposure of TF antigen was observed. D) A549 cells treated with A-Sia: Fluorescence intensity increased at sialidase concentration of 0.1 U / L or more, and exposure of TF antigen was observed. E) CHO cells treated with A-Sia: Fluorescence intensity increased at sialidase concentration of 0.1 U / L or more, and exposure of TF antigen was observed. F) Lec2 cells treated with A-Sia: Fluorescence intensity increased regardless of sialidase concentration, and exposure of TF antigen was observed.

FIG. 4 shows the results of peramivir inhibition assay using 4MU-NANA in Example 2. “RFU” on the vertical axis represents the fluorescence intensity, and the horizontal axis represents the elapsed time (minutes) after the addition of 4MU-NANA. As the peramivir concentration increased, the fluorescence intensity decreased, and S-Sia activity was inhibited by peramivir.

FIG. 5 shows the results of a flow cytometry inhibition assay using peramivir in Example 2. “Count” on the vertical axis represents the number of cells, and the horizontal axis represents FITC fluorescence intensity. A) A549 cells: Fluorescence intensity decreased with increasing peramivir concentration, and S-Sia activity was suppressed by peramivir. B) CHO cells: The fluorescence intensity decreased with increasing peramivir concentration, and S-Sia activity was inhibited by peramivir. C) Lec2 cells: Fluorescence intensity is constant regardless of sialidase digestion and peramivir concentration.

FIG. 6 is a view showing complement cell injury nonspecific for anti-TF antibody in Example 3. The vertical axis represents absorbance at 490 nm, the horizontal axis represents sialidase concentration, “Iso” represents an isotype control antibody, and “TF” represents an anti-TF antibody. A) A549 cells: Absorbance is decreased by high-concentration sialidase digestion (1 U / L or more) regardless of antibody, and cytotoxicity is observed. B) CHO cells: Absorbance is decreased by high-concentration sialidase digestion (1 U / L or more) regardless of antibody, and cytotoxicity is observed. C) Lec2 cells: Absorbance is reduced by high-concentration sialidase digestion (1 U / L or more) regardless of antibody, and cytotoxicity is observed.

FIG. 7 is a view showing complement cell damage caused by sialidase digestion in Example 3. The vertical axis represents absorbance (490 nm), and the horizontal axis represents sialidase concentration. A) No complement added-A549 cells: Absorbance is constant regardless of sialidase concentration, and no cytotoxicity is observed. B) Complement addition-A549 cells: Absorbance decreases at sialidase concentration of 1 (U / L) or more, and cytotoxicity is observed. C) Complement-free-CHO cells: Absorbance is constant regardless of sialidase concentration, and no cytotoxicity is observed. D) Complement addition-CHO cells: Absorbance decreases at sialidase concentration of 1 (U / L) or more, and cytotoxicity is observed.

FIG. 8 is a schematic diagram showing a drug administration regimen for rats in Example 4.

FIG. 9 is a table showing changes in rat body weight after administration of peramivir in Example 4 and PaO2 / FlO2 (ratio of oxygen partial pressure in arterial blood to inhaled oxygen concentration) after 14 days.

FIG. 10 is a graph comparing PaO2 / FlO2 in the peramivir administration group and the control group in Example 4.

FIG. 11 is a graph comparing the body weights of the peramivir administration group and the control group in Example 4.

FIG. 12 is a pathological finding of pulmonary fibrosis in the control group in Example 4.

FIG. 13 shows the pathological findings of pulmonary fibrosis in the peramivir administration group in Example 4.

Hereinafter, the present invention will be described in detail.
Unless defined otherwise herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. In general, the terms used in connection with the compounds, cells, biochemistry, and techniques described herein are well known and commonly used in the art.

  One aspect of the present invention is to prevent and / or prevent pneumonia comprising a compound having viral sialidase inhibitory activity and mammalian sialidase inhibitory activity, bacterial sialidase inhibitory activity and mammalian sialidase inhibitory activity, or mammalian sialidase inhibitory activity. The present invention relates to a pharmaceutical composition for treating or preventing and / or treating pulmonary fibrosis.

  Sialidase, also known as neuraminidase, is a sugar hydrolase that cleaves sialic acid residues from oligosaccharide components of glycoproteins and glycolipids, including sialo-oligosaccharides, gangliosides, or sialoglycoproteins. This is what separates this. Sialidases are found in various organisms including bacteria, viruses, protozoa, and vertebrates, and are roughly divided into three types: viral sialidases, bacterial sialidases, and mammalian sialidases.

  In the present specification, the sialidase inhibitor is not particularly limited as long as it is a composition containing a compound having viral sialidase inhibitory activity, bacterial sialidase inhibitory activity, and / or mammalian sialidase inhibitory activity. The composition further comprises a compound having activity and further having viral sialidase inhibitory activity or bacterial sialidase inhibitory activity.

  Viral sialidase inhibitors include, for example, peramivir (trade name: Rapiacta (registered trademark)), oseltamivir (trade name: Tamiflu (registered trademark)), zanamivir (known as those that inhibit influenza virus sialidase) Trade name: Relenza (registered trademark)) and laninamivir (trade name: Inavir (registered trademark)). They inhibit sialidase, which is required when influenza virus is released by budding from infected host cells, thereby maintaining the binding between hemagglutinin on the surface of influenza virus and sialic acid on the surface of the host cell. Inhibits the release of virus from the host cell. In addition, it is known that these administrations are effective only at the early stage of influenza infection and have almost no individual healing effect after 48 hours from the onset of influenza.

  In the present invention, “a compound having a viral sialidase inhibitory activity”, “a compound having a bacterial sialidase inhibitory activity”, or “a compound having a mammalian sialidase inhibitory activity” refers to a sialidase derived from a virus, a bacterium or a mammal, respectively. The compound is not particularly limited as long as it is a compound that inhibits the ability to cleave an acid from a sugar chain. Such compounds include not only a single compound or a plurality of compound groups but also those having the respective sialidase inhibitory activities for the first time as a whole compound group.

Among those having the sialidase inhibitory activity of the aforementioned influenza virus, zanamivir is known to have a human sialidase inhibitory activity (Japanese Patent Laid-Open No. 2011-136964).
The inventors have discovered that some of the compounds known as influenza virus sialidase inhibitory activity, such as peramivir, have human sialidase inhibitor activity.

  The present inventors have further discovered that some of the compounds known as influenza virus sialidase inhibitory activity, such as peramivir, have therapeutic and prophylactic effects for pneumonia and pulmonary fibrosis.

  The dosage of the compound having sialidase inhibitory activity when the pharmaceutical composition of the present invention is used for the prevention or treatment of actual diseases can be appropriately selected depending on the sex, age, weight, degree of disease, and administration method of the patient. For example, when it is administered to an adult patient, it is not particularly limited as long as it is an effective amount for the treatment and prevention of pneumonia and pulmonary fibrosis. In the case of parenteral administration three times, generally 1 mg to 100 mg can be selected within a range of 1 to 3 times per day. Furthermore, it can be appropriately increased or decreased according to symptoms and side effects. In the case of children, the effective dose for adults can be referred to and appropriately selected according to age, weight, and degree of disease.

  The pharmaceutical composition of the present invention comprises a compound having sialidase inhibitory activity and, if necessary, a pharmaceutically acceptable carrier (for example, excipient, binder, disintegrant, lubricant, stabilizer, preservative). , PH adjusting agents, flavoring agents, diluents, solvents for injections, etc.) can be appropriately prepared by those skilled in the art. For example, dosage forms such as tablets, powders, and aqueous solutions can be appropriately selected by those skilled in the art according to the administration route described below.

  According to the present invention, interstitial pneumonia and pulmonary fibrosis, preferably acute or chronic lung such as idiopathic interstitial pneumonia (IPF), collagen disease lung, drug-induced interstitial pneumonia, radiation pneumonia, hypersensitivity pneumonia, etc. Diseases, pneumoconiosis, more preferably pneumonia resulting from injury of type II alveolar epithelial cells, can be treated and / or prevented.

  In the present invention, “pneumonia” refers to general pneumonia without particular limitation, but preferably refers to chronic lung disease such as interstitial pneumonia and pneumonia resulting from injury of type II alveolar epithelial cells.

  In the present invention, “pulmonary fibrosis” is not particularly limited, but preferably refers to chronic lung disease such as idiopathic pulmonary fibrosis (IPF). Pulmonary fibrosis treatable according to the present invention refers to a reversible one that has a small degree of fibrosis and is treatable, and is irreversible where fibrosis has progressed and treatment is no longer possible Is not included.

  In the present invention, “treating pneumonia” refers to alleviating or suppressing inflammation by administering the pharmaceutical composition of the present invention to a subject who already suffers from pneumonia. In particular, pneumonia caused by injury of type II alveolar epithelial cells due to high sialidase activity (FIG. 1) can be treated by inhibiting the activity of sialidase.

  In the present invention, “treating pulmonary fibrosis” means a reversible pulmonary fiber that has a small degree of fibrosis and can be treated by administering the pharmaceutical composition of the present invention to a subject already suffering from pneumonia. Symptoms include making the degree of fibrosis even smaller.

  In addition, a “subject” may be healthy or suffer from some kind of disease, but when treatment for various pneumonia is intended, an individual living with pneumonia or an experiment Infected organisms such as rodents such as mice, rats, gerbils and guinea pigs, felines such as cats, pumas and tigers, deer animals such as deer and elk, rabbits, dogs and minks Sheep, goat, cow, horse, monkey, human and the like.

In the present invention, “preventing pneumonia and / or pulmonary fibrosis” means inflammation and / or fibrosis by administering the pharmaceutical composition of the present invention to a subject not suffering from pneumonia and / or pulmonary fibrosis. It means not to cause. In particular, pneumonia and / or pulmonary fibrosis is prevented by administering a compound according to the present invention and / or a pharmaceutical composition containing the same to a subject in which sialidase having relatively high activity is detected in the lung stroma. can do.
“Administration” in the present invention can be carried out by routes such as oral, nasal, intravenous, and transdermal routes. For example, peramivir is preferably transvenous (intravenous injection), oseltamivir is oral, and zanamivir and laninamivir are nasal (inhalation), but are not particularly limited.

  The present inventors speculated that pneumonia and the resulting pulmonary fibrosis were caused by injury of type II alveolar epithelial cells by sialidase (FIG. 1). According to the present speculation, the compound of the present invention capable of inhibiting the activity of such sialidase and the pharmaceutical composition comprising the same prevent and / or treat pneumonia or prevent and / or treat pulmonary fibrosis. I can expect that.

As described above, it has been shown that in interstitial pneumonia, sialidase activity is increased and type II alveolar epithelial cell injury may occur.
The present inventors thought that type II alveolar epithelial cells were damaged by the mechanism as shown in FIG. According to such a mechanism, injury is caused by an increase in the activity of an enzyme called sialidase in the alveoli. The present inventors have demonstrated that injury occurs when A549 cells, which are actually cultured cells of type II alveolar epithelial cells, are treated with sialidase.

  Thomsen-Friedenreich (TF) antigen is one of tissue blood group sugar chain antigens. Since humans have IgM-type natural antibodies against TF, there are sialylated TFs to which sialic acid that is not affected by natural antibodies is added in many tissues except testis and part of the brain where natural antibodies do not reach. In the living body, when there is an exposed TF having no sialic acid at the terminal, complement-mediated cytotoxicity works. In Pneumococcal Hemolytic Uremic Syndrome (P-HUS), TF of the erythrocytes, platelets, and glomerular endothelial cells are exposed by pneumococcal sialidase, causing natural antibody-induced cell damage. In healthy individuals, sialylated TF is observed on the surface of type II alveolar epithelial cells, but in patients with IPF, TF is exposed on the surface of some type II alveolar epithelial cells.

  In IPF, exposure of TF on the surface of type II alveolar epithelial cells may cause injury of type II alveolar epithelial cells in IPF as well as the onset mechanism of P-HUS. Applicants believe that type II alveolar epithelial cell injury is involved in the origin of IPF, and that cell line A549 (derived from type II alveolar epithelial cells) is treated with sialidase digestion, TF antibody and complement cells. Whether injuries occur was examined in the examples. The following experimental examples explain the present invention more specifically, and do not limit the scope of the present invention. Those skilled in the art having ordinary knowledge and techniques can make various modifications to the embodiments shown in the following experimental examples without departing from the spirit of the present invention. include.

[Example 1] Flow cytometry using anti-TF antibody <Cell culture>
A549 cells (human type II alveolar epithelial cancer cells) and CHO cells (Chinese hamster ovary cells) are 10% fetal calf serum (FCS) (Thermo Fisher Scientific, USA), 100 U / mL penicillin, 100 μg / mL streptomycin Culture was performed using an added DMEM liquid medium (DMEM) (nacalai tesque). Lec2 cells (CHO cell-derived sialyltransferase deficient cell line) were cultured using 10% FCS, 100 U / mL penicillin, 100 μg / mL streptomycin-added α-MEM liquid medium (α-MEM) (nacalai tesque). Each cell was cultured in a 5% CO ₂ incubator maintained at 37 ° C. and passaged twice a week.

<Flow cytometry>
(1) Cell preparation The culture solution of cultured A549 cells and CHO cells was removed, and the mixture was washed once by adding phosphate buffered saline (PBS) pH 7.0. PBS was removed, 1 mL of trypsin EDTA (SIGMA-ALDRICH, USA) was added, and the mixture was reacted for 5 minutes in a 37 ° C. incubator. After the reaction, cell suspension was confirmed, 10 mL of DMEM was added, and the entire amount was transferred to a 15 mL tube. Centrifugation was performed at 1000 rpm at room temperature for 5 minutes. The supernatant was removed, the cell suspension was adjusted to 1.0 × 10 ⁵ cells / mL with DMEM, and dispensed into 5 mL round tubes. In Lec2 cells, DMEM was replaced with α-MEM and the same operation was performed. Each tube into which the cells were dispensed was centrifuged at 1000 rpm for 5 minutes at room temperature. The supernatant was removed, and 500 μL of PBS was added to the tube and washed once.

(2) Digestion with sialidase After centrifugation, the supernatant was removed, and 200 μL of PBS was added to each tube. Next, Streptococcus 6646K-derived sialidase (S-Sia) (biochemical biobusiness) or Arthrobacter ureafaciens-derived sialidase (A-Sia) (nacalai tesque) is added to be 0, 0.01, 0.1, 1, 5 U / L, The reaction was allowed to proceed for 30 minutes at 37 ° C.

(3) After the blocking reaction, the mixture was centrifuged and the supernatant was removed. Then, PBS containing 0.1% bovine serum albumin (BSA) (SIGMA-ALDRICH) was added to the tube in a volume of 500 μL and washed. Washing with PBS supplemented with 0.1% BSA was performed three times. After washing, 500 μL of 3% BSA-added PBS was added to each tube, and a blocking reaction was performed at room temperature for 20 minutes.

(4) Primary antibody reaction After the reaction, the mixture was centrifuged, and the supernatant was removed. Then, 100% of PBS containing 0.1% BSA was added to the tube. Thereafter, 5 μl of mouse IgM anti-TF antibody (Glycotope, Germany) was added to the primary antibody and allowed to react on ice for 20 minutes. As an isotype control, 5 μl of mouse IgM anti-Lipopolysaccharide antibody (Beckman coulter, USA) was added and allowed to react on ice for 20 minutes.

(5) Secondary antibody reaction After the reaction, it was washed 3 times with 500 μL of PBS containing 0.1% BSA. Thereafter, the cells were suspended in 500 μL of PBS containing 0.1% BSA, and 0.5 μL of FITC-labeled goat anti-mouse IgM antibody (Invitorogen, USA) was added as a secondary antibody and allowed to react on ice for 20 minutes (FIG. 2).

(6) Measurement After the reaction, the plate was washed 3 times with 500 μL of PBS containing 0.1% BSA. After washing, the suspension was suspended in 500 μL of FACS Flow (BD Biosciences, USA) and measured with a BD FACSCalibur (BD Biosciences). The analysis used CELLQUEST Software (BD Biosciences).

<Result> (Binding of anti-TF antibody by sialidase digestion)
In A549 cells and CHO cells, fluorescence intensity was enhanced when anti-TF antibody was added after S-Sia or A-Sia digestion. The fluorescence intensity changed depending on the sialidase concentration. Both cells showed the maximum fluorescence intensity at an S-Sia concentration of 5 U / L, and no fluorescence could be confirmed at 0.1 U / L or less (FIGS. 3-A and B). Similarly, A-Sia digestion of 5 U / L and 1 U / L showed the highest fluorescence intensity. The fluorescence intensity was confirmed up to 0.5 U / L (FIGS. 3-D and E).
In Lec2 cells, fluorescence intensity did not change even when anti-TF antibody was added after digestion with S-Sia or A-Sia (FIGS. 3-C, F).
When an isotype control antibody was added in place of the anti-TF antibody, there was no increase in fluorescence intensity in A549 cells, CHO cells, and Lec2 cells (1 in FIG. 3).

[Example 2]
<Flow cytometry inhibition assay with peramivir>
(1) Example 1 (1) Cells were prepared in the same manner as cell preparation.
(2) Sialidase digestion
S-Sia was adjusted to 2.5 U / L with physiological saline. The anti-influenza virus agent peramivir (BIOCRYST, USA), which inhibits sialidase with sialidase-added saline, was adjusted to 0.01, 0.1, 1, 10 mg / mL, added to each tube at 200 μL, and reacted at 37 ° C for 30 minutes. I let you.
Subsequent blocking, primary antibody reaction, secondary antibody reaction, and measurement were performed in the same manner as in (3) to (6) of Example 1.

<Sialidase inhibition assay using 4MU-NANA>
It adjusted with the physiological saline so that peramivir might be 0.01, 0.1, 1, and 10 mg / mL in a 1.5 mL tube. To each solution, add S-Sia to 2.5 U / L and 4-methylumbelliferyl-N-acetylneuraminic acid (4MU-NANA, Toronto Research Chemicals) to 0.154 mg / mL, and mix well. . Thereafter, 100 μL of each solution was dispensed into a 96-well plate, and fluorescence intensity was measured using Cytoflour Series 4000 (Applied Biosystems, USA). Gain 50 was measured every 5 minutes for 60 minutes at 37 ° C. 4-methylumbelliferone (4MU) was detected at an excitation wavelength of 365 nm and a fluorescence wavelength of 450 nm.

<Result>
(Suppression in inhibition assay using 4MU-NANA)
When peramivir was added, the fluorescence intensity was suppressed in a concentration-dependent manner. With 0.01 mg / mL peramivir, fluorescence intensity suppression could not be confirmed, but at 0.1 mg / mL or more fluorescence intensity suppression could be confirmed. At 10 mg / mL peramivir, fluorescence intensity suppression was maximized (FIG. 4).
When peramivir was not added, fluorescence intensity was increased over time.
(Suppression in flow cytometry inhibition assay)
The fluorescence intensity of A549 cells and CHO cells was suppressed depending on the peramivir concentration. In A549 cells, suppression started from 0.1 mg / mL of peramivir, and the fluorescence intensity was completely suppressed at 1 mg / mL (FIG. 5-A). In CHO cells, suppression was observed from 0.1 mg / mL of peramivir, and complete fluorescence intensity suppression was obtained at 1 mg / mL as in A549 cells (FIG. 5-B).
On the other hand, in Lec2 cells, no change in the fluorescence intensity could be confirmed regardless of the peramivir concentration (FIG. 5-C).

[Example 3]
<Complement cytotoxicity experiment using anti-TF antibody>
Cell preparation, sialidase digestion, blocking and antibody reaction were performed in the same manner as in (1) to (4) of Example 1.

(5) Preparation of complement reaction solution and complement reaction Rabbit complement (Cedarlane laboratories, Canada) dissolved in 1 mL of distilled water was diluted 1.5 times with serum-free DMEM to prepare a complement reaction solution. After the primary antibody reaction, the plate was washed 3 times with PBS containing 0.1% BSA, a complement reaction solution was added, and the mixture was reacted at 37 ° C for 1 hour.

(6) Examination of the number of viable cells After the reaction, the cells were washed 3 times with PBS containing 0.1% BSA. After washing, 300 μL to 500 μL of serum-free DMEM was added to float the cells, and 100 μL each was dispensed into a 96 well plate. 20 μL of CellTiter 96 (registered trademark) AQueous One Solution Cell Proliferation Assay (Promega, USA) was added to each well and mixed well. The reaction was carried out at 37 ° C for 2 hours.

(7) Measurement After the reaction, absorbance was measured using SPECTRAmax PLUS ³⁸⁴ (Molecular Devices, USA). Absorbance was measured at 490 nm.

<Complement cytotoxicity experiment without antibody>
A549 cells and CHO cells were treated and measured in the same manner as described above except that the step of (4) antibody reaction in <Complement cell damage experiment using anti-TF antibody> was not performed.

<Result>
When A549 cells and CHO cells were digested with high-concentration sialidase, addition of anti-TF antibody or isotype control antibody caused cytotoxicity (FIGS. 6-A and B). There was no difference in cytotoxicity between the two antibodies.
In A549 cells and CHO cells, cytotoxicity occurred after digestion of high concentration S-Sia and only addition of complement without addition of antibody (FIG. 7). If complement was not added, cytotoxicity of both cells did not occur (FIGS. 7-A, C).
Cytotoxicity was observed at S-Sia 1 U / L or higher, and CHO cells were observed more strongly than A549 cells (FIGS. 7-B, D).
In Lec2 cells, there was no cytotoxicity with or without anti-TF antibody and complement (FIG. 6-C).

(Consideration of Examples 1 to 3)
A549 cells (derived from type II alveolar epithelial cells) and CHO cells have a sugar chain structure called sialylated TF on the surface, and cytotoxicity was caused by adding complement after sialidase digestion (FIG. 7). . Whether or not anti-TF antibody was added after sialidase digestion did not affect cytotoxicity, and the presence of anti-TF antibody was not involved in cytotoxicity (FIGS. 6 and 7). This result suggests that type II alveolar epithelial cell injury occurs if sialidase activity is increased and complement is involved in the alveoli of the living body. There is a report that sialidase activity is increased in bronchoalveolar lavage fluid of IPF patients, and mild type II alveolar epithelial cell injury may be a cause of the onset of IPF.

  The present inventors have increased sialidase activity in the alveolar space triggered by viral infection, etc., and anti-TFIgM, which is a natural antibody, binds to the TF antigen that appears on the surface of type II alveolar epithelial cells. This study was conducted under the hypothesis that induced type II alveolar epithelial cell injury occurs. This hypothesis is known as the onset mechanism of P-HUS.

  Sialyl TF, sialyl TF, and TF sugar chain structures are present on the surface of A549 cells, CHO cells, and Lec2 cells. Using flow cytometry, TF antigen was expressed on the surface of A549 cells and CHO cells after sialidase digestion, and TF antigen was expressed on the Lec2 cell surface regardless of sialidase digestion (FIG. 3). In the inhibition assay of sialidase inhibitor (peramivir) (FIGS. 4 and 5), sialidase was suppressed and TF antigen did not appear, so sialic acid was digested by sialidase and TF antigen appeared.

  High concentration S-Sia and A-Sia digestion expressed the TF antigen on the surface of type II alveolar epithelial cells in a concentration-dependent manner. On the other hand, when the S-Sia activity was 0.1 U / L or less, sialic acid was not digested and TF antigen was not expressed (FIGS. 3-A and B). This indicates that sialic acid may or may not be digested due to sialidase concentration differences. The difference in sialidase concentration in each alveoli can explain the spatial and temporal heterogeneity of fibrotic lesions, which is the greatest feature of IPF. The continuation of very mild inflammatory lesions in IPF may also be explained by sialidase concentrations.

  Complement was added to the anti-TF antibody to examine the cytotoxicity. However, in all cells, the cytotoxicity was caused by the complement regardless of the presence or absence of the anti-TF antibody, which was different from our hypothesis (FIG. 6). A, B). Three types of complement system activation pathways are known. The classical pathway produced by IgM or IgG antibodies bound to antigen, the alternative pathway produced without antibodies, and the lectin pathway. The type II alveolar epithelial cell injury of this study is unlikely to be caused by the classical pathway via IgM antibody like P-HUS, and since lectin is not used, the injury caused by activation of the lectin pathway is negative. is there. However, the presence of cytotoxicity due to complement suggests the possibility of cytotoxicity due to activation of alternative pathways. The alternative pathway is activated when the complement protein C3 metabolite C3b binds to the surface of pathogens and autologous cells and further forms a complex (C3 convertase) with C3b to which a protein called factor B is bound. The In autologous cells, alternative pathways are controlled to prevent injury by inhibiting inhibitory proteins called β1H (Factor H) and C3b inactivator (C3bINA) from competing with factor B. Kazatchkine et al. Report that the removal of sialic acid from sheep erythrocyte membranes attenuates the alternative pathway inhibitor protein β1H binding to C3b. In addition, Fearon et al. Have reported that the binding of β1H is suppressed, so that factor B binding occurs preferentially and activation of the alternative pathway causes sheep erythrocyte damage. β1H has been shown to have the ability to bind anions, and sialic acid is an important factor that controls the activation of alternative pathways. Regarding the relationship between IPF and complement, the relationship between the polymorphism of erythrocyte Complement receptor 1 and the incidence of IPF and the increase of each complement factor in IPF have been reported. To clarify the relationship between IPF and the alternative pathway, it is necessary to analyze β1H and sialic acid in IPF.

  The ongoing stimulation of alveolar tissue, which is thought to be the mechanism of IPF development, has not been elucidated. In this study, it was found that type II alveolar epithelial cell A549, which has been desialylated by sialidase digestion, is complementarily damaged (FIG. 7-B). Therefore, if alveolar constituent cells are damaged in the IPF patient alveoli in relation to complement, cell damage caused by sialidase digestion may stimulate the alveolar tissue in the mechanism of IPF development. is there. If chronic stimulation in IPF is associated with alternative pathway activation by sialidase, sialidase activity needs to persist even at low levels. Therefore, a mechanism for maintaining sialidase activity continuously rather than a temporary high sialidase activity state such as viral infection is required, which will be a clue for IPF pathogenesis and treatment.

[Example 4] Test using interstitial pneumonia model rat <Material and method>
Rats were exposed to the trachea after pentobarbital anesthesia, and bleomycin (0.01 U / kg), known as an anticancer agent, was intratracheally administered using a 26G needle syringe. The surgical site was sutured and returned to the normal breeding room after anesthesia awakening. After follow-up for 2 weeks, the animals were euthanized again by pentobarbital anesthesia, and alveolar lavage and dissection were performed. For therapeutic intervention, a sialidase inhibitor (peramivir 10 mg / kg) was administered intraperitoneally. The administration was continued for 7 to 13 days after bleomycin administration (FIG. 8). For intraperitoneal administration, inhalation anesthesia with diethyl ether was used in consideration of animal pain, although only one injection was required each time. The number of animals used was 4 in each of the control and intervention groups, for a total of 8 animals.

<Result>
Changes in body weight and PaO2 / FIO2 are shown in FIGS. There was no significant change in body weight before administration between the control group (326.8 ± 6.7 g) and the intervention group (336.5 ± 8.3 g). After 14 days, the control group had a significant weight loss of 234.5 ± 25.3 g and the intervention group 362.2 ± 23.9 g. In addition, PaO2 / FIO2 was significantly worsened in the control group after 14 days in the control group 149.1 ± 23.9 and the intervention group 284.9 ± 23.9. Further, in the histopathological search of the lung after dissection, the lung fibrosis was clearly suppressed in the intervention group compared to the control group (FIGS. 12 and 13).

  Since the compound according to the present invention and the pharmaceutical composition containing the compound can treat and / or prevent pneumonia and pulmonary fibrosis caused by injury of type II alveolar epithelial cells, It was able to treat refractory idiopathic pulmonary fibrosis that was not found and fulfills the needs of the people.

1 ... No sialidase digestion (isotype control antibody)
2. No sialidase digestion (anti-TF antibody)
3 ... sialidase concentration 0.01 U / L (anti-TF antibody)
4 ... Sialidase concentration 0.1 U / L (anti-TF antibody)
5 ... Sialidase concentration 1 U / L (anti-TF antibody)
6 ... Sialidase concentration 5 U / L (anti-TF antibody)

11 ... Negative control without 4MU-NANA 12 ... Positive control without addition of peramivir 13 ... Peramivir concentration 0.01 mg / mL
14 ... Peramivir concentration 0.1 mg / mL
15 ... Peramivir concentration 1 mg / mL
16 ... peramivir concentration 10 mg / mL

21 ... No sialidase digestion (isotype control antibody) without peramivir 22 ... No sialidase digestion (anti-TF antibody) No peramivir addition 23 ... With sialidase digestion (anti-TF antibody) No peramivir 24 ... sialidase Digested (anti-TF antibody) peramivir concentration 0.01 mg / mL
25 ... with sialidase digestion (anti-TF antibody) peramivir concentration 0.1 mg / mL
26 ・ ・ ・ Sialidase digested (anti-TF antibody) Peramivir concentration 1 mg / mL
27 ... With sialidase digestion (anti-TF antibody) Peramivir concentration 10 mg / mL

Claims (1)

A pharmaceutical composition for preventing and / or treating pneumonia resulting from injury of type II alveolar epithelial cells by sialidase and / or pulmonary fibrosis resulting therefrom comprising a compound having sialidase inhibitory activity ,
The pharmaceutical composition, wherein the compound having sialidase inhibitory activity is peramivir .