NOVEL USE OF FIBROBLAST GROWTH FACTOR 2

Abstract

The present invention provides a novel use of fibroblast growth factor 2 (FGF-2), i.e., a use of FGF-2 in preparation of medicine. The uses of the medicine are the following (a) and/or (b) and/or (c): (a) the prevention and/or treatment of lung injury; (b) the prevention and/or treatment of influenza; (c) the prevention and/or treatment of diseases caused by influenza viruses.

Claims

1-20. (canceled)

21. A method of protecting a mammal from a lung injury, an influenza or a disease induced by an influenza virus or treating a lung injury, an influenza or a disease induced by an influenza virus in a mammal, said mammal being in need of said protecting or treating, said method comprising administering FGF-2 to said mammal.

22. The method according to claim 21, wherein the FGF-2 is a human-derived FGF-2.

23. The method according to claim 22, wherein the FGF-2 is: (A) a protein as set forth by SEQ ID NO. 1 in the Sequence Listing; or (B) a protein derived from (A) with a substitution and/or a deletion and/or an addition of one or more amino acid residues and having the same activity as (A).

24. The method according to claim 21, wherein the mammal is in need of the protecting or treating for a lung injury.

25. The method according to claim 24, wherein the lung injury is a lung injury induced by a virus, a bacterium or a fungus.

26. The method according to claim 24, wherein the lung injury is a lung injury induced by septicemia.

27. The method according to claim 21, wherein the mammal is in need of the treating for influenza, or the protecting or the treating for a disease induced by an influenza virus.

28. The method according to claim 27, wherein the influenza virus is influenza A virus H1N1.

Description

DESCRIPTION OF DRAWINGS

[0053] FIG. 1 shows the results in Example 1.

[0054] FIG. 2 shows the slice staining results in Example 2.

[0055] FIG. 3 shows the wet/dry ratio results in Example 2.

[0056] FIG. 4 shows the statistical results of survival rate in Example 3.

[0057] FIG. 5 shows the statistical results of body weight changes in Example 3.

[0058] FIG. 6 shows the slice staining results in Example 3.

[0059] FIG. 7 shows the wet/dry ratio results in Example 3.

[0060] FIG. 8 shows the statistical results of survival rate in Example 4.

[0061] FIG. 9 shows the slice staining results in Example 4.

[0062] FIG. 10 shows the wet/dry ratio results in Example 4.

[0063] FIG. 11 shows the slice staining results in Example 5.

BEST MODES TO CARRY OUT THE INVENTION

[0064] Following Examples are intended to facilitate better understanding of the present invention, but not for limiting the present invention. Those experimental methods used in the following Examples are conventional methods, unless otherwise specified. Those experimental materials used in the following Examples are commercially available from a conventional biochemical reagent supplier, unless otherwise specified. Those quantitative tests in the following Examples each were conducted triplicate, with the results averaged. The data is analyzed and processed with software GraphPad Prism 5. In the statistics of survival rate, after being infected with viruses, those mice which died in 24 h are considered as non-specific death, and are excluded from the statistics of survival rate.

[0065] C57 BL/6 mice (4-week old): Laboratory Animal Center of The Academy of Military Medical Science. FGF-2 gene knocked-out mice (with a background of SPF grade C57 BL/6 mice): American Jackson Laboratory, Catalog No. 003256. LPS (LPS, from LPS of E. coli 0111: B4): Sigma, L2630. zymosan A (Zymosan A, from S. cerevisiae): Sigma, Z4250. FGF- 2 recombinant protein (Human recombinant FGF-2/basic FGF protein): protein sequence is shown as SEQ NO: 1 in the Sequence Listing, encoding gene thereof is shown as SEQ NO: 2 in the Sequence Listing; Millipore, Catalog No. 01-106, when used, it is diluted into desired concentration with PBS buffer.

[0066] Influenza A virus H1N1 A strain /Beijing/501/2009 (H1N1) (abbreviated as BJ501 strain): http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=info&id=6 488568&lvl=3&keep=1&srchmode=1&unlock&lin=s; Yang P, Deng J, Li C, Zhang P, Xing L, Li Z, Wang W, Zhao Y, Yan Y, Gu H, Liu X, Zhao Z, Zhang S, Wang X, Jiang C. Characterization of the 2009 pandemic A/Beijing/501/2009 H1N1 influenza strain in human airway epithelial cells and ferrets. PLoS One. 2012;7(9):e46184. doi: 10.1371/journal.pone.0046184. Epub 2012 September 26.

[0067] Influenza A virus H1N1 influenza strain in human airwa epithelial cells and ferrets. PLoS One.

[0068] Influenza A virus H1N1 A strain/Puerto Rico/8/1934 (H1N1) (abbreviated as PR8 strain):

[0069] http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode32 Info&id=21 1044&lvl=3&lin=f&keep=1&srchmode=1&unlock; Li C, Yang P, Sun Y, Li T, Wang C, Wang Z, Zou Z, Yan Y, Wang W, Wang C, Chen Z, Xing L, Tang C, Ju X, Guo F, Deng J, Zhao Y, Yang P, Tang J, Wang H, Zhao Z, Yin Z, Cao B, Wang X, Jiang C. IL-17 response mediates acute lung injury induced by the 2009 pandemic influenza A (H1N1) virus Cell Res. 2012 Mar; 22(3):528-38. doi: 10.1038/cr.2011.165. Epub 2011 October 25.

[0070] The PBS buffer used in the Examples is 0.01 mol/L PBS buffer, pH7.2, unless otherwise specified.

Example 1. Increased FGF-2 Level in Mouse Lung Lavage Fluid Induced by Influenza A virus H1N1

[0071] For the experimental group (5 of 4-6 weeks old C57 BL/6 mice): each of the mice was securely fixed, and intraperitoneally injected with 50-60 μL of 1 g/100 mL solution of pentobarbital sodium for anesthesia; the anesthetized mice were kept with their head backward and upward leaned to bring their nasal cavities into an upward position, and 10 μL virus solution of BJ501 strain (10.sup.5.5 TCID.sub.50/mouse) was added dropwise to their nasal cavity at each side through a pipette; the mice were kept in this posture for 15 seconds, to allow the virus to enter into their respiratory tracts; 24 hours after the infection with BJ501 strain, the mice were killed by means of intraperitoneal injection of excessive anesthetic; the killed mouse was fixed on a small animal operating table, and the skin and bone of its chest were removed, a small opening was cut on its trachea, and 800 μL PBS buffer was injected into the mouse through the opening; finally, lung lavage fluid (LLF) was aspirated by three repeated imbibitions, and detected for the concentration of FGF-2 therein by a Bio-Plex Mouse Cytokine 23-flex kit.

[0072] For the control group (5 of 4-6 weeks old C57 BL/6 mice): the mice were treated in the same way as for the experimental group, except that an equal volume of allantoic fluid of chick embryo was used instead of the virus solution of BJ501 strain.

[0073] The results are shown in FIG. 1. The mice infected with influenza A virus H1N1 have a FGF-2 concentration in LLF higher than that of the control group (*P<0.05), that is, the expression level of FGF-2 in LLF is significantly higher in the mice infected with influenza A H1N1 than that in the control group. The results indicate that FGF-2 plays an important role in influenza A H1N1 induced lung injury, and an intervention targeting FGF-2 can hold an important position in the treatment of injuries induced by infection with influenza A H1N1.

Example 2. Induction of More Serious ALI by Influenza A Virus H1N1 in FGF-2 Deficient Mice

[0074] Each of 6 of 4-6 weeks old C57BL/6 mice (or 6 of 4-6 weeks old FGF-2 gene knocked-out mice) was securely fixed, and intraperitoneally injected with 50-60 μL of 1 g/100 mL solution of pentobarbital sodium by a 1 mL sterile syringe for anesthesia; the anesthetized mice were kept with their head backward and upward leaned to bring their nasal cavities into an upward position, a virus solution of BJ501 strain (10.sup.5.5 TCID.sub.50/mouse) was added dropwise to their nasal cavity at each side through a pipette; the mice were kept in this posture for 15 seconds, to allow the virus to enter into their respiratory tracts; 5 days after infection with the influenza A virus, the mice were killed by means of intraperitoneal injection of excessive anesthetic; three of the killed mice were fixed on a small animal operating table, and skin and bone of their chest were removed to expose thoracic cavity, from which lung together with heart were removed, and washed with sterile PBS buffer solution to wash off the blood on the surface, and then placed into a paraformaldehyde fixing solution for fixation at room temperature for 48 h, followed by a series of treatments such as embedding, slicing, HE staining; the other three of the killed mice were fixed on a small animal operating table, and skin and bone of their chest were removed to expose thoracic cavity, from which the entire lungs were taken off, subjected to removal of surface blood and excessive connective tissues, and weighed, and the wet weight of the lung was recorded; then, the lungs were placed in a tissue drier at a high temperature of 55° C. and dried; after 24 h, the lungs were taken out and weighed for dry weight when cooled to room temperature, so as to obtain a wet/dry ratio=wet lung weight/dry lung weight.

[0075] The results of slice staining are shown in FIG. 2 (×200 folds), wherein A represents the lung tissue of a C57BL/6 mouse, and B represents the lung tissue of a FGF-2 gene knocked-out mouse. In the C57BL/6 mouse infected with influenza A virus H1N1, serious pathological injuries occurred in lung tissue, normal lung tissue structure was destroyed, and the lung markings were disordered, accompanied with pathological injuries such as bleeding, inflammatory effusion, as well as massive red cells, inflammatory cell infiltration, and the like. The FGF-2 gene knocked-out mouse infected with the same titer of virus had more significant pathological injuries in lung tissue, and more significant pathological changes such as bleeding, effusion, inflammatory cell infiltration, or the like, unclear lung marking, and non-intact structure.

[0076] The results of “wet/dry ratio” are shown in FIG. 3. The “wet/dry ratio” of lung can reflect the extent of acute pulmonary edema in a mouse. In a FGF-2 gene knocked-out mouse infected with influenza A virus H1N1, the wet/dry ratio of lung was significantly increased than that of a C57BL/6 mouse (*P<0.05), which indicates that knockout of FGF-2 can deteriorate pulmonary edema in a mouse infected with influenza A virus H1N1. The results indicate that more serious ALI can be induced by influenza A virus H1N1 in a FGF-2 deficient mouse.

Example 3. Induction of More Serious ALI by Influenza A Virus H1N1 in FGF-2 deficient Mice

[0077] Each of 20 of 4-6 weeks old C57BL/6 mice (or 20 of 4-6 weeks old FGF-2 gene knocked-out mice) was securely fixed, and intraperitoneally injected with 1 mL sterile syringe 50-60 μL of 1 g/100 mL solution of pentobarbital sodium for anesthesia; the anesthetized mice were kept with their head backward and upward leaned to bring their nasal cavities into an upward position, 10 μL virus solution of PR8 strain (10.sup.5.5 TCID.sub.50/mouse) was added dropwise to their nasal cavity at each side through a pipette; the mice were kept in this posture for 15 seconds, to allow the virus to enter into their respiratory tracts; 14 of the mice were subjected to survival statistics (the day before the infection with influenza A virus was recorded as Day 0; from the point of the infection with influenza A virus, 24 hours later was recorded Day 1, and so on) and weight monitoring; the remaining 6 mice were killed by means of intraperitoneal injection of excessive anesthetic 5 days after infection with influenza A virus; three of the killed mice were fixed on a small animal operating table, and skin and bone of their chest were removed to expose thoracic cavity, from which lung together with heart were removed, and washed with a sterile PBS buffer solution to wash off the blood on the surface, and then placed into a paraformaldehyde fixing solution for fixation at room temperature for 48 h, followed by a series of treatments such as embedding, slicing, HE staining; the other three of the killed mice were fixed on a small animal operating table, and skin and bone of their chest were removed to expose thoracic cavity, from which their entire lungs were taken off, and subjected to removal of surface blood and excessive connective tissues, weighed, and the wet weight of the lung was recorded; then, the lungs were placed in a tissue drier at a high temperature of 55° C. and dried; after 24 h, the lungs were taken out and weighed for dry weight when cooled to room temperature, so as to obtain a wet/dry ratio=wet lung weight/dry lung weight.

[0078] The statistics results of survival rate of mice infected with influenza A virus are shown in FIG. 4. Infected with the same titers of influenza A virus H1N1, the mortality of C57BL/6 mice was significantly lower than the FGF-2 gene knocked-out mice (*P<0.05). The statistical results of body weight change (i.e., a ratio of the body weight at a certain day to that at Day 0) of mice infected with influenza A virus are shown in FIG. 5. Infected with the same titers of influenza A virus H1N1, C57BL/6 mice had a body weight change significantly less than that of the FGF-2 gene knocked-out mice (*P<0.05). The results indicate that FGF-2 plays an essential role in the protection of mice infected with influenza A virus H1N1 from death, and an intervention targeting FGF-2 molecules may play an important role in the protection of treating the one infected with influenza A virus H1N1.

[0079] The results of slice staining are shown in FIG. 6 (×200 folds), wherein A represents the lung tissue of a C57BL/6 mouse, and B represents the lung tissue of a FGF-2 gene knocked-out mouse. In the C57BL/6 mouse infected with influenza A virus H1N1, serious pathological injuries occurred in lung tissue, normal lung tissue structure was destroyed, and lung markings were disordered, accompanied with pathological injuries such as bleeding, inflammatory effusion, as well as massive red cells, inflammatory cell infiltration, and the like. The FGF-2 gene knocked-out mouse infected with the same titer of virus had more significant pathological injuries in lung tissue, and more significant pathological changes such as bleeding, effusion, inflammatory cell infiltration, or the like, unclear lung markings, and non-intact structure.

[0080] The results of “wet/dry ratio” are shown in FIG. 7. In a FGF-2 gene knocked-out mouse infected with influenza A virus H1N1, the wet/dry ratio of lung was significantly increased than that of a C57BL/6 mouse (*P<0.05), which indicates that knockout of FGF-2 can deteriorate pulmonary edema in a mouse infected with influenza A virus H1N1. These results indicate that more serious ALI can be induced by influenza A virus H1N1 in a FGF-2 deficient mouse.

Example 4. Ability of FGF-2 Recombinant Protein to Mitigate Acute Lung Injury Induced by Infection with Influenza A Virus

[0081] I. Experiment I

[0082] For a experimental group (10 of 4-6 weeks old C57BL/6 mice): at Day 1, each of the mice was intravenously injected with 100 μl of a solution of FGF-2 recombinant protein (at a protein concentration of 0.5 mg/ml); at Day 2, each of the mice was securely fixed, and intraperitoneally injected with 50-60 μL of 1 g/100 mL solution of pentobarbital sodium by a 1 mL sterile syringe for anesthesia; the anesthetized mice were kept with their head backward and upward leaned to bring their nasal cavities into an upward position, and 10 μL virus solution of PR8 strain (10.sup.5.5 TCID.sub.50/mouse) was added dropwise to their nasal cavity at each side through a pipette; the mice were kept in this posture for 15 seconds to allow the virus to enter into their respiratory tract; at Day 3, each of the mice was intravenously injected with 100 μl of the FGF-2 recombinant protein solution (at a protein concentration of 0.5 mg/ml); at Day 5 , each of the mice was intravenously injected with 100 μl of the FGF-2 recombinant protein solution (at a protein concentration of 0.5 mg/ml); the survival of the mice was determined daily.

[0083] For a control group (10 of 4-6 weeks old C57 BL/6 mice): the mice were treated in the same way except that an equal volume of PBS buffer was used instead of the FGF-2 recombinant protein solution.

[0084] The statistic results of the survival rate from Day 1 to Day 14 are shown in FIG. 8. After infected with the same titer of influenza A virus H1N1, the control group had a mortality significantly higher than that of the mice of the experimental group (*P<0.05). The results indicate an important therapeutic effect of FGF-2 in death of the mice infected with influenza A virus H1N1, and an intervention targeting FGF-2 molecules may take an essential part in the recovery of treating the one infected with influenza A virus H1N1.

[0085] II. Experiment II

[0086] For a experimental group (6 of 4-6 weeks old C57BL/6 mice): at Day 1, each of the mice was intravenously injected with 100 μl of a solution of FGF-2 recombinant protein (at a protein concentration of 0.5 mg/ml); at Day 2, each of the mice was securely fixed, and intraperitoneally injected with 50-60 μL of 1 g/100 mL solution of pentobarbital sodium by a 1 mL sterile syringe for anesthesia; the anesthetized mice were kept with their head backward and upward leaned to bring their nasal cavities into an upward position, and 10 μL virus solution of PR8 strain (10.sup.5.5 TCID.sub.50/mouse) was added dropwise to their nasal cavity at each side through a pipette; the mice were kept in this posture for 15 seconds to allow the virus to enter into their respiratory tract; at Day 3, each of the mice was intravenously injected with 100 μl of the FGF-2 recombinant protein solution (at a protein concentration of 0.5 mg/ml), at Day 5 , each of the mice was intravenously injected with 100 μl of the FGF-2 recombinant protein solution (at a protein concentration of 0.5 mg/ml), at Day 6, the mice were killed by means of intraperitoneal injection of excessive anesthetic; three of the killed mice were fixed on a small animal operating table, and skin and bone of their chest were removed to expose thoracic cavity, from which lung together with heart were removed, and washed with a sterile PBS buffer solution to wash off the blood on the surface, and then placed into a paraformaldehyde fixing solution for fixation at room temperature for 48 h, followed by a series of treatments such as embedding, slicing, HE staining; the other three of the killed mice were fixed on a small animal operating table, and skin and bone of their chest were removed to expose thoracic cavity, from which their entire lungs were taken off, and subjected to removal of surface blood and excessive connective tissues, weighed and the wet weight of the lung was recorded; then, the lungs were placed in a tissue drier at a high temperature of 55° C. and dried; after 24 h, the lungs were taken out and weighed for dry weight when cooled to the room temperature, so as to obtain a wet/dry ratio=wet lung weight/dry lung weight.

[0087] For the control group (6 of 4-6 weeks old C57 BL/6 mice): the mice were similarly treated except that an equal volume of PBS buffer was used instead of the FGF-2 recombinant protein solution.

[0088] The results of slice staining are shown in FIG. 9 (×200 folds), wherein A represents the lung tissue of a mouse of the control group, and B represents the lung tissue of a mouse of the experimental group. In the mouse of the control group infected with influenza A virus H1N1, serious pathological injuries occurred in lung tissue, normal lung tissue structure was destroyed, and lung markings were disordered, accompanied with pathological injuries such as bleeding, inflammatory effusion, as well as massive red cells, inflammatory cell infiltration, and the like. However, in the mouse of the experimental group infected with the same titer of virus, neither significant pathological injury of lung tissue, nor significant pathological changes such as bleeding, effusion, inflammatory cell infiltration, or the like, was observed, and their lungs were observed having clear markings and perfect structure. The results indicate that FGF-2 plays an important role in the protection of a mouse from acute pathological injury of lung tissue induced by infection with influenza A virus H1N1.

[0089] The results of wet/dry ratio are shown in FIG. 10. The mice of the experimental group, after being infected with influenza A virus H1N1, had a wet/dry ratio of lung significant lower than that of mice of the control group (*P<0.05), which indicates that FGF-2 can substantially mitigate pulmonary edema in a mouse induced by infection with influenza A virus H1N1. The results suggest that FGF-2 plays an important role in the protection from acute pathological injury of lung tissue induced by infection with influenza virus A.

Example 5. Ability of FGF-2 Recombinant Protein to Mitigate Pathological Injury of Mouse Lung Tissue After Co-infection with LPS and Zymosan A

[0090] For Group 1 (4 of 4-6 weeks old C57BL/6 mice): each of the mice were intravenously injected with 100 μl of a FGF-2 recombinant protein solution (at a protein content of 50 μg) 12 hours prior to LPS infection, 1 hours prior to LPS infection, and 8 hours after LPS infection, respectively.

[0091] Infection: each of the mice was intraperitoneally injected with 50-60 μL of a 1 g/100 mL solution of pentobarbital sodium by a 1 mL sterile syringe for anesthesia; the anesthetized mice were kept with their head backward and upward leaned to bring their nasal cavities into an upward position, and intranasally and dropwise administered with 50 μL of a LPS solution (100 μg LPS in a solvent of PBS buffer); the mice were kept in this posture for 5 minutes to allow the LPS to enter into their respiratory tracts; 1 hour after the infection with LPS, each of the mice was intraperitoneally injected with 50-60 μL of the 1 g/100 mL pentobarbital sodium solution by a 1 mL sterile syringe for anesthesia; the anesthetized mice were kept with their head backward and upward leaned to bring their nasal cavities into an upward position, and intranasally and dropwise administered with 50 μL of a zymosan A solution (60 μg LPS in a solvent of PBS buffer); the mice were kept in this posture for 5 minutes, to allow the LPS to enter into their respiratory tracts.

[0092] At 24 hours after the LPS infection, the mice were killed by means of intraperitoneal injection of excessive anesthetic, and fixed on a small animal operating table, and skin and bone of their chest were removed to expose thoracic cavity, from which lung together with heart were removed, and washed with a sterile PBS buffer solution to wash off the blood on the surface, and then placed into a paraformaldehyde fixing solution for fixation at room temperature for 48 h, followed by a series of treatments such as embedding, slicing, HE staining, etc.

[0093] Group 2: the mice were treated in the same way as Group 1 except for no injection of FGF-2 recombinant protein solution 12 hours prior to LPS infection, 1 hours prior to LPS infection, and 8 hours after LPS infection.

[0094] Group 3: the mice were treated in the same way as Group 1 except that an equal volume of PBS buffer solution was used instead of the LPS solution, and an equal volume of PBS buffer solution was used instead of the zymosan A solution.

[0095] The results are shown in FIG. 11, wherein A represents Group 1, B represents Group 2, and C represents Group 3. No significant pathological injury and pathological changes such as bleeding, effusion, inflammatory cell infiltration, or the like, but clear lung markings and perfect structure were observed in the lung tissue of the mice of Group 3. Significant pathological injury and pathological changes such as bleeding, effusion, inflammatory cell infiltration, or the like, unclear lung markings, and non-intact structure were observed in the lung tissue of the mice of Group 2. No significant pathological injury and pathological changes such as bleeding, effusion, inflammatory cell infiltration, or the like, but clear lung markings and perfect structure were observed in the lung tissue of the mice of Group 1. The results indicate that FGF-2 plays an important protective role in acute pathological injuries of lung tissue in mouse induced by co-infection with LPS and zymosan A.

INDUSTRIAL APPLICATION

[0096] The present invention discloses uses of FGF-2 in the manufacture of a drug for treating and/or preventing lung injury, for preventing and/or treating influenza, and for preventing and/or treating a disease induced by an influenza virus. The present invention may be valuable for the treatment and prevention of above diseases.