WO2013044871A1

WO2013044871A1 - The use of chloroquine, chlorpromazine, derivatives thereof, or mixtures thereof in preparing medications for treating and/or preventing pulmonary infection and injury

Info

Publication number: WO2013044871A1
Application number: PCT/CN2012/082480
Authority: WO
Inventors: 蒋澄宇; 金宁一; 孙阳; 邹镇; 李霄; 闫毅武
Original assignee: 中国医学科学院基础医学研究所; 中国人民解放军军事医学科学院军事兽医研究所
Priority date: 2011-09-29
Filing date: 2012-09-29
Publication date: 2013-04-04
Also published as: CN103027915A; CN103687599A

Abstract

The present invention relates to the use of chloroquine for the treatment and chlorpromazine for the prevention of pulmonary infection and injury. In particularly, the present invention relates to the use of chloroquine, chlorpromazine, derivatives thereof, mixtures thereof, or medicinal compositions containing the same for the preparation of medications for treating and/or preventing pulmonary infection and/or injury in mammals, including humans, caused by influenza viruses.

Description

Use of chloroquine treatment and chlorpromazine for prevention of lung infection and injury

The present invention relates to the use of chloroquine or chlorpromazine or a derivative thereof or a mixture thereof for the preparation of a medicament for the treatment and/or prevention of influenza virus-induced lung infection and/or lung injury in a mammal, including a human. Background technique

Influenza virus, referred to as influenza virus, is divided into three types: spherical or filamentous, with a diameter of 80~120nm. The three types of viruses have similar biochemical and biological characteristics, which is a kind of epidemic caused by humans and animals. A cold RNA virus. In taxonomy, the influenza virus belongs to the Orthomyxoviridae family, which causes acute upper respiratory tract infections, and is rapidly spread by air, and there are often periodic pandemics around the world. Influenza viruses can cause more serious symptoms, such as pneumonia or heart and lung failure, in elderly and children with weak immunity and some immune-impaired patients.

Influenza A virus is a common influenza virus and is most susceptible to mutation. It is divided into many subtypes according to H and N antigens. H can be divided into 15 subtypes (H1~H15) and N has 9 subtypes ( N1~N9). Among them, only H1N1, H2N2, and H3N2 mainly infect humans, and many other subtypes of natural hosts are a variety of birds and animals. The subtype of influenza A virus is known as Avian Influenza Virus (AIV), and the most harmful to birds is the H5, H7 and H9 subtypes.

The host range of avian influenza viruses is very wide. The majority of birds in the natural world are poisoned by poultry. Most wild birds are poisonous or latent infections. Poultry are most susceptible to turkeys, followed by chickens, and ducks and geese. Under normal circumstances, the avian flu virus does not infect animals other than birds and pigs. However, in 1997, Hong Kong reported for the first time 18 cases of H5N1 human avian influenza infection, including 6 deaths, which caused widespread concern worldwide. Since 1997, there have been several incidents of avian influenza virus infection in the world. The highly pathogenic H5N1, H7N7, H9N2, and other avian influenza viruses, once mutated and have human-to-human transmission ability, can lead to the epidemic of human avian influenza. The source of human avian influenza is mainly chickens, ducks, geese, especially chickens infected with the avian flu virus. The main route of transmission is contact or airborne.

So far, the number of cases of bird flu in the world is the highest, and the highest case fatality rate is H5N1. Avian influenza, with a mortality rate of up to 60%. The clinical features of human infection with H5N1 avian influenza virus are: Long incubation period, most patients with high fever, accompanied by flu-like symptoms and lower respiratory tract symptoms, sometimes with upper respiratory symptoms, rarely appear like H7 flu Conjunctivitis caused by the virus, some patients will have diarrhea, vomiting, abdominal pain, rib pain, bleeding gums, nosebleeds in the early stage. Compared with the common cold, watery diarrhea is more common. As the infection time prolongs, respiratory distress, breathing Burst sounds are common when rushing and inhaling. The amount of sputum is uncertain, sometimes it is bloody. Clinical pneumonia occurs in almost all patients; X-ray changes include diffuse, multiple or scattered exudation; exudation of interstitial space; partial fusion or lobular fusion with bronchial aeration. Studies have shown that radiological examination abnormalities usually occur at a median time of 7 days (3 to 17 days) after fever.

Clinical and pathological examinations suggest that the lesions in critically ill patients are mainly in the respiratory system. Pathological examination showed that the lungs of severe patients showed consolidation, often accompanied by pathological changes such as hemorrhage, exudation and abscess. Palate fluid or fibrinous exudation can be seen in the alveolar space, with varying degrees of clear membrane formation, suggesting diffuse lung tissue damage. It is currently believed that the basic lesions of lung tissue damage in patients with severe H5N1 avian influenza are similar to those of other types of influenza, SARS and influenza A (H1N1) viruses, all of which are diffuse lung tissue lesions of varying severity.

Up to now, there is still no effective treatment for H5N1 avian influenza virus, and because of the high mutation rate and the increased drug resistance of influenza viruses, there is still a need for new types of influenza treatment drugs.

Chloroquine is mainly used to treat acute episodes of malaria and to control symptoms of malaria. It can't stop the recurrence, but because it lasts longer, it can delay the recurrence, and it can't prevent and block the spread of malaria. It has a curative effect on falciparum malaria, and can also be used to treat hepatic amebiasis, clonorchiasis, paragonimiasis, connective tissue disease and the like. It can also be used to treat photosensitivity disorders such as erythema. The pharmacological action of chloroquine is: chloroquine acts on the intra-erythrocytic schizonts, and after 48 to 72 hours, the schizont in the blood is killed. Through the action of chloroquine, the nuclear fragmentation of the malaria parasite, the cell paddle appears vacuolization, and the malaria pigment aggregates into a mass.

Chlorpromazine is mostly white phosphate or milky white crystalline powder; it has slight odor and tastes extremely bitter; it has hygroscopicity; it has a light gradient; the aqueous solution is acidic. Chlorpromazine pharmacological action is a blocker of central dopamine receptors, with sedative, antipsychotic, antiemetic, hypothermia and basal metabolism, alpha-adrenergic receptors and m-cholinergic receptors Body block, antihistamine, affecting endocrine, etc., clinically used to control schizophrenia or other mental disorders such as agitation, nervousness, hallucinations, delusions; treatment of vomiting caused by various causes; also used for low temperature anesthesia and artificial Hibernation; combined with analgesics to treat severe pain in patients with advanced cancer.

The use of chloroquine and chlorpromazine and its derivatives, respectively, in combination or in combination for the treatment and/or prevention of influenza has not been reported. Summary of the invention

Accordingly, the technical solution of the present invention is the use of chloroquine or chlorpromazine or a derivative thereof or a mixture thereof for the preparation of a medicament for treating and/or preventing influenza virus-induced lung infection and/or lung injury in a mammal, including a human.

The technical solution of the present invention further relates to a pharmaceutical composition comprising chloroquine or chlorpromazine or a derivative thereof or a mixture thereof for the preparation of a lung infection and/or lung injury in a mammal, including a human, caused by the treatment and/or prevention of influenza virus. Use in medicine.

In one embodiment of the present invention, the derivative of chloroquine is selected from the group consisting of hydroxychloroquine, hydroxychloroquine sulfate, and dichloroquine phosphate; and the derivative of the chlorpromazine is selected from the group consisting of acetyl pepazine, perphenazine, and triflurane. Oxazine.

In another embodiment of the present invention, the influenza virus is an influenza A virus, preferably an influenza A H1, H3, H5, H7 or H9 subtype strain, more preferably an influenza A H5 subtype strain. More preferably, it is an influenza A H5N1 virus, and most preferably an influenza A H5N1 influenza virus A/Jilin/9/2004 (H5N1) strain.

In another embodiment of the invention, the pharmaceutical composition comprises a therapeutically effective amount of chloroquine or chlorpromazine or a derivative thereof or a mixture thereof, and one or more pharmaceutically acceptable carriers, the carrier These include, but are not limited to, diluents, excipients, fillers, binders, wetting agents, disintegrants, surfactants, adsorbent carriers.

In another embodiment of the present invention, the pharmaceutical composition may further comprise one or more therapeutically effective doses of a pharmaceutically active ingredient in synergy with the chloroquine or chlorpromazine or a derivative thereof or a mixture thereof. .

In another embodiment of the invention, the pharmaceutical composition may be formulated into any pharmaceutically acceptable dosage form including, but not limited to, an injection, a spray, a nasal drop, an inhalant or an oral dose. . The invention also relates to a method of treating and/or preventing a lung infection and/or lung injury in a mammal, including a human, caused by an influenza virus, comprising administering to a patient infected with an influenza virus a therapeutically effective amount of chloroquine or chlorpromazine or a derivative thereof Or a mixture thereof.

The invention further discloses chloroquine or chlorpromazine or a derivative thereof or a mixture thereof for use in the treatment and/or prevention of lung infections and/or lung damage in mammals, including humans, caused by influenza viruses.

In the present invention, the chloroquine, chlorpromazine and derivatives thereof may be used alone or in combination for pulmonary infection and/or lung injury in mammals including humans caused by influenza viruses.

The present invention utilizes the H5N1 avian influenza virus to attack mice to demonstrate that chloroquine or chlorpromazine plays an important role in the pathological injury and death of acute lung tissue in mice infected with avian influenza virus A/Jilin/9/2005 (H5N1) strain. Interventions against chloroquine or chlorpromazine play an important role in preventing damage caused by infection with the avian influenza virus A/Jilin/9/2005 (H5N1) strain. Thus, the present invention demonstrates for the first time that chloroquine or chlorpromazine can prevent or slow the severe consequences of H5N1 avian influenza virus infection.

At the same time, it is well known to those skilled in the art that when it is confirmed that chloroquine or chlorpromazine plays an important preventive and/or therapeutic role in pathological damage, death, etc. caused by infection of avian influenza virus, related derivatives or mixtures thereof can also Play the same or similar role. As is well known to those skilled in the art, derivatives refer to compounds formed by the substitution of atoms or groups of atoms in the parent compound molecule by other atoms or groups of atoms.

Likewise, pharmaceutical compositions comprising chloroquine or chlorpromazine or derivatives thereof or mixtures thereof according to the invention can also be used for the prevention and/or treatment of H5N1 avian influenza viruses, in particular A/Jilin/9/2005 (H5N1) strain. The active ingredient of the pharmaceutical composition is chloroquine or chlorpromazine or a derivative thereof or a mixture thereof, and may also include other synergistic effects with the chloroquine or chlorpromazine or a derivative thereof or a mixture thereof. Active ingredient. DRAWINGS

Figure 1: Wild-type BALB/c mice infected with virus control or TCID ₅ . For the H6N1 avian influenza virus of 10 ⁶ , the solvent control or chloroquine was intraperitoneally injected 6 hours, 1 day, 2 days, 3 days, 4 days and 5 days after infection, respectively. Record the survival curve in 8 days and plot the mortality curve. 10 mice per group.

Figure 2: Wild-type BALB/c mice infected with virus control or TCID ₅ . For the H6N1 avian influenza virus of 10 ⁶ , two intraperitoneal injections of solvent pairs were given 2 hours and 0.5 hours before infection, respectively. Or chloroquine. Record the survival curve in 8 days and plot the mortality curve. 10 small images in each group: Wild-type BALB/c mice infected with virus control or H5N1 avian influenza virus with TCID _5Q of 10 ⁶ were injected intraperitoneally with solvent control or chloropropion 2 hours and 0.5 hours before infection, respectively. Oxazine. Record the survival curve in 8 days and plot the mortality curve. 10 mice per group.

Figure 4: Wild-type BALB/c mice infected with virus control or H5N1 avian influenza virus with TCID _5Q of 10 ⁶ were injected intraperitoneally 6 hours, 1 day, 2 days, 3 days, 4 days and 5 days after infection, respectively. Solvent control or chlorpromazine. Record the survival curve in 8 days and plot the mortality curve. 10 mice per group.

Figure 5: Wild-type BALB/c mice infected with virus control or H5N1 avian influenza virus with TCID _5Q of 10 ⁶ were injected intraperitoneally 6 hours, 1 day, 2 days, 3 days, 4 days and 5 days after infection, respectively. Solvent control or chloroquine. After 4 days of infection, the lung tissue of the mice was taken for lung tissue wet-dry ratio detection. 4 mice per group.

Figure 6: Wild type BALB/c mice infected with virus control or H5N1 avian influenza virus with TCID _5Q of 10 ⁶ were injected intraperitoneally with solvent control or chloroquine 2 hours and 0.5 hours before infection, respectively. After 4 days of infection, the lung tissue of the mice was taken for lung tissue wet-dry ratio detection. 4 mice per group.

FIG Ί wildtype BALB / c mice infected with a virus or TCID _5Q controls the H5N1 avian influenza virus ¹⁰⁶ respectively 2 hours and 0.5 hours prior to infection, by intraperitoneal injection twice a solvent control or chlorpromazine. After 4 days of infection, the lung tissue of the mice was taken for lung tissue wet-dry ratio detection. 4 mice per group.

FIG. 8: Wild-type BALB / c mice infected with a virus or TCID _5Q controls the H5N1 avian influenza virus to ^106, respectively, 6 hours after the infection, 1 day, 2 days, 3 days, 4 days, 5 days, intraperitoneal injection Solvent control or chloroquine. Four days after infection, mouse lung tissue was taken for pathological examination of lung tissue. 4 mice per group. Wherein A: PBS + allantoic fluid control; B means: chloroquine (treatment) + allantoic fluid control; C means: PBS + H5N1 avian influenza virus; D means: chloroquine (treatment) + H5N1 avian influenza virus; E means: lung Statistical results of histopathological sections of inflammatory cell counts.

Figure 9: wild-type BALB / c mice infected with a virus or TCID _5Q controls the H5N1 avian influenza virus ¹⁰⁶ respectively 2 hours prior to infection and 0.5 hours, two intraperitoneal injection of solvent Or chloroquine. Four days after infection, mouse lung tissue was taken for pathological examination of lung tissue. 4 mice per group. Wherein A: PBS + allantoic fluid control; B means: chloroquine (treatment) + allantoic fluid control; C means: PBS + H5N1 avian influenza virus; D means: chloroquine (prevention) + H5N1 avian influenza virus; E means: lung Statistical results of histopathological sections of inflammatory cell counts.

Figure 10: Wild-type BALB/c mice infected with virus control or H5N1 avian influenza virus with TCID _5Q of 10 ⁶ were injected intraperitoneally with solvent control or chlorpromazine 2 hours and 0.5 hours before infection, respectively. Four days after infection, mouse lung tissue was taken for pathological examination of lung tissue. 4 mice per group. Wherein A: PBS + H5N1 avian influenza virus; B means: chlorpromazine (prevention) + H5N1 avian influenza virus.

Figure 11: wild-type BALB / c mice infected with a virus or TCID _5Q controls the H5N1 avian influenza virus to ^106, respectively, 6 hours after the infection, 1 day, 2 days, 3 days, 4 days, 5 days, intraperitoneal injection Solvent control or chlorpromazine. Four days after infection, mouse lung tissue was taken for pathological examination of lung tissue. 4 mice per group. Wherein A: PBS + H5N1 avian influenza virus; B means: chlorpromazine (treatment) + H5N1 avian influenza virus.

Figure 12: Wild-type BALB/c mice infected with virus control or H5N1 avian influenza virus with TCID _5Q of 10 ⁶ were injected intraperitoneally 6 hours, 1 day, 2 days, 3 days, 4 days and 5 days after infection, respectively. Solvent control or chloroquine. Mouse lung tissue was taken for detection of Western blot at 0 hours, 3 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 72 hours and 96 hours after infection. 4 mice per group.

Figure 13: wild-type BALB / c mice infected with a virus or TCID _5Q controls the H5N1 avian influenza virus to ^106, respectively, 6 hours after the infection, 1 day, 2 days, 3 days, 4 days, 5 days, intraperitoneal injection Solvent control or chloroquine. Mouse lung tissues were taken at 0 hours, 3 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 72 hours, and 96 hours after infection for RNA extraction and detection of M1 and M2 gene expression levels. 4 mice per group. Wherein A indicates: M1 gene expression level of H5N1 avian influenza virus in mouse lung tissue; B indicates: M2 gene expression level of H5N1 avian influenza virus in mouse lung tissue.

Figure 14: Wild-type BALB/c mice infected with virus control or H5N1 avian influenza virus with TCID _5Q of 10 ⁶ were injected intraperitoneally 6 hours, 1 day, 2 days, 3 days, 4 days and 5 days after infection, respectively. Solvent control or chloroquine. Mouse lung tissue was extracted at 0 hours, 3 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 72 hours and 96 hours after infection for RNA extraction and interleukin-6, interleukin-1β and tumor necrosis Factor-α gene expression level Detection. 4 mice per group. Wherein A indicates: the expression level of interleukin-1β gene in mouse lung tissue; Β indicates: the expression level of interleukin-6 gene in mouse lung tissue; C indicates: the expression level of tumor necrosis factor-α gene in mouse lung tissue. detailed description

The invention will be further clarified by the following non-limiting examples, and it is to be understood by those skilled in the art that many modifications may be made without departing from the spirit of the invention.

The following experimental methods are conventional methods unless otherwise specified, and the experimental materials used can be easily obtained from commercial companies unless otherwise specified. Example

Example 1 : Chloroquine and chlorpromazine reduce the mortality rate of mice infected with H5N1 avian influenza virus

Experimental Materials:

1) Main experimental instruments: Class III biosafety laboratory, tertiary biological safety cabinet, animal breeding cabinet, mouse breeding cage, small animal surgical instruments, sterile syringes, pipettes, pipettes, etc.

2) Main experimental reagents: chloroquine (C6628, purchased from Sigma-Aldrich), chlorpromazine (C8138, purchased from Sigma-Aldrich), solvent control PBS, 1% (w/v) sodium pentobarbital solution , virus dilution, disinfectant (2.5% iodine and 75% alcohol).

3) Virus: H5N1 avian influenza virus A/Jilin/9/2004 (H5Nl)

4) Experimental animals:

SPF grade wild type (WT) BALB/c mice (4 weeks old): purchased from the China National Institute for the Control of Pharmaceutical and Biological Products.

experimental method:

1) Grouping: virus control + solvent control group, virus control + drug group, virus + solvent control group, virus + drug group;

2) The mice were intraperitoneally injected with solvent control or drugs. Each mouse was intraperitoneally injected with chloroquine (50 mg/kg) and chlorpromazine (15 mg/kg) for 2 times, respectively, 2 h and 0.5 h before infection; The treatment was given a total of 6 injections, 6 hours after infection, 1 day, 2 days, 3 days, 4 Days and 5 days;

3) Safely immobilize mice, anesthetized with 1% (w/v) sodium pentobarbital solution by intraperitoneal injection with a lmL sterile syringe;

4) Keep the head of the anesthetized mouse tilted up and down to make the nasal cavity upward. The neck was disinfected with alcohol, the neck skin was cut with scissors, the trachea was separated, and a titer of 10 ⁶ TCID ₅₀ H5N1 avian influenza virus was injected with a syringe, and each group was infected with 10 mice;

5) Keep the mouse in this position for 5 minutes to distribute the virus evenly in the lung tissue. The mice were placed in a squirrel cage, and after they were awake, water and food were given;

6) After infection, the mice that died within 24 hours were non-specific deaths and were not counted when performing mortality statistics;

7) Continuous observation for 8 days, record the death of each group of mice every day;

8) Statistics on mouse mortality using GraphPad Prism 5 software.

Experimental results:

1) Wild-type BALB/c mice were intraperitoneally injected with solvent control or chloroquine and chlorpromazine, and the virus titer was 10 ⁶ TCID ₅ . The mortality rate after the H5N1 avian influenza virus is shown in Figure 1, Figure 2, Figure 3, and Figure 4.

2) This result indicates that chloroquine plays an important role in the treatment and chlorpromazine in preventing the death of mice caused by H5N1 avian influenza virus infection, caused by chloroquine in the treatment and prevention of H5N1 avian influenza virus infection. It plays an important role in injury; chloroquine has no clear effect in preventing and chlorpromazine in the treatment of mice with H5N1 avian influenza virus infection. Example 2 Chloroquine and chlorpromazine alleviate pulmonary edema in mice after infection with H5N1 avian influenza virus

Experimental Materials:

2) Main experimental reagents: chloroquine (C6628, purchased from Sigma-Aldrich), chlorpromazine (C8138, purchased from Sigma-Aldrich), solvent control PBS, 1% (w/v) sodium pentobarbital solution , virus dilution, disinfectant (2.5% iodine and 75% alcohol). 3) Virus: H5N1 avian influenza virus A/Jilin/9/2004 (H5Nl)

4) Experimental animals: SPF wild type (WT) BALB/C mice (4 weeks old): purchased from the China National Institute for the Control of Pharmaceutical and Biological Products.

experimental method:

2) The mice were intraperitoneally injected with solvent control or drugs. Each mouse was intraperitoneally injected with chloroquine (50 mg/kg) and chlorpromazine (15 mg/kg) for 2 times, respectively, 2 h and 0.5 h before infection; The treatment was injected a total of 6 times, 6 hours, 1 day, 2 days, 3 days, 4 days and 5 days after infection;

4) Keep the head of the anesthetized mouse tilted up and down to make the nasal cavity upward. Disinfect the neck with alcohol, cut the neck skin with scissors, separate the trachea, and inject a titer of 10 ⁶ TCID ₅ with a syringe. H5N1 avian influenza virus, each group infected 4 mice;

7) On the 4th or 5th day after infection, the mice were killed by intraperitoneal injection of excess anesthetic;

8) Fix the mouse on the small animal operating table, remove the chest skin and bones, expose the chest cavity, remove the whole lung of the mouse, remove the surface blood and excess connective tissue, weigh and record the wet weight of the lung;

9) The lungs were placed in a 55 ° C high temperature tissue desiccator for dry roasting, and taken out after 24 hours. When the temperature was lowered to room temperature, the dry weight of the lungs was weighed and recorded;

10) Calculate the mouse wet weight/dry ratio (Wet/Dry Ratio) for statistical analysis. Experimental results:

1) The results of wet-to-dry ratio of H5N1 avian influenza virus infected with virus titer of 10 ⁶ TCID50 after intraperitoneal injection of solvent control or chloroquine or chlorpromazine in wild-type BALB/c mice, as shown in Figure 5. Figure 6 and Figure 7. 2) This result indicates that chloroquine plays an important role in the treatment and chlorpromazine in preventing pulmonary edema in mice caused by H5N1 avian influenza virus infection.

Pathological damage

The experimental material and the experimental method in this embodiment are basically the same as in the second embodiment. The difference between the experimental methods is that the lung tissue damage effect is determined by the present example. Specifically, the experimental method is different in that the operation of the experimental mice after excessive anesthesia is performed to fix the mice. On the small animal operating table, remove the chest skin and bones, expose the chest cavity, remove the mouse lungs together with the heart, wash the surface blood with sterile PBS, and fix it in formaldehyde fixative for 48h at room temperature; The laboratory was subjected to embedding, sectioning, HE staining, etc.; the pathological sections were observed under a microscope and recorded.

Experimental results:

1) In the wild-type BALB/c mice, after intraperitoneal injection of solvent control or chloroquine, chlorpromazine, lung pathological changes and pulmonary inflammation after infection with the virus titer of 10 ⁶ TCID50 of H5N1 avian influenza virus The results of cell infiltration statistics are shown in Figures 8, 9, 10 and 11.

2) Figure 8 (X200 times, HE staining) Pathological photographs showed that severe pathological damage occurred in the lung tissue of 4 weeks old wild-type C57 BL/6 mice injected with PBS control after intraperitoneal injection of H5N1 avian influenza virus. The normal structure of the lung tissue is destroyed, the texture of the lung tissue is disordered, accompanied by hemorrhage, inflammatory exudation, and pathological damage such as infiltration of a large number of red blood cells and inflammatory cells. There was no significant pathological damage in the lung tissue of mice infected with the same titer virus by intraperitoneal injection of chloroquine (6 times of treatment, 6 hours, 1 day, 2 days, 3 days, 4 days and 5 days after infection). Significant hemorrhage, exudation or inflammatory cell infiltration and other pathological changes, lung tissue texture is clear, structural integrity; Figure 9 results show that intraperitoneal injection of chloroquine for prevention (preventive co-injection 2 times, respectively 2h and 0.5h before infection) There was no significant difference in the pathological damage of rat lung tissue compared with the PBS solvent group. The results showed that chloroquine treatment significantly improved the degree of pathological damage in acute lung tissue of mice infected with H5N1 influenza virus, while chloroquine prevention had no significant effect.

3) Figure 10 (X200 times, HE staining) Pathological photo shows: Infected H5N1 type After avian influenza virus, severe pathological damage occurred in the lung tissue of 4 weeks old wild-type C57 BL/6 mice injected intraperitoneally with PBS. The normal structure of the lung tissue is destroyed, the texture of the lung tissue is disordered, accompanied by hemorrhage, inflammatory exudation and pathological damage such as a large number of red blood cells and inflammatory cell infiltration. Infection with the same titer virus intraperitoneal injection of chlorpromazine for prevention (preventive co-injection 2 times, 2 h and 0.5 h before infection) showed no significant pathological damage in the lung tissue of the mice, no significant bleeding, exudation or inflammatory cell infiltration Such pathological changes, lung tissue texture is clear, structural integrity; Figure 11 results suggest intraperitoneal injection of chlorpromazine (treatment a total of 6 injections, respectively, 6 hours after infection, 1 day, 2 days, 3 days, 4 days and 5 There was no significant difference in the pathological damage of mouse lung tissue compared with the PBS solvent group. The results showed that chlorpromazine prevention can significantly improve the degree of acute lung tissue pathological damage in mice infected with H5N1 influenza virus, while chloropropaquine treatment has no significant effect. Example 4 Treatment with chloroquine reduced autophagy in lung tissue of mice infected with H5N1 avian influenza virus

Experimental Materials:

2) Main experimental reagents: chloroquine (C6628, purchased from Sigma-Aldrich), β-actin and LC-3 protein antibodies (both purchased from Sigma-Aldrich), solvent control PBS, 1% (W/V) Sodium pentobarbital solution, virus dilution, disinfectant (2.5% iodine and 75% alcohol).

3) Virus: H5N1 avian influenza virus A/Jilin/9/2004 (H5Nl)

experimental method:

1) Grouping: virus + solvent control group, virus + drug group;

2) Mice were injected intraperitoneally with solvent control or drug. Each mouse was intraperitoneally injected with chloroquine (50 mg/kg) for 6 times, 6 hours, 1 day, 2 days, 3 days, 4 days after infection. Days and 5 days;

3) Safely immobilize mice, intraperitoneal injection of 1% (W/V) pentabar with a lmL sterile syringe Anesthesia with sodium natto solution;

7) After infection with virus, mouse lung tissue was taken for detection of Western blot at 0 hours, 3 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 72 hours and 96 hours after infection, and β was detected separately. - The transition of actin and LC-3 I protein to LC-3 II protein. 4 mice per group.

Experimental result

The results in Fig. 12 show that the degree of conversion of the LC-3I protein to the LC-3 II protein in the lungs of the chloroquine-treated group was significantly lower than that of the vehicle control group after infection with the H5N1 avian influenza virus. It is well known to those skilled in the art that since the degree of conversion of LC-3 I protein to LC-3 II protein is a recognized standard reflecting autophagy, the results of this experiment can demonstrate that chloroquine treatment can significantly reduce the small cause caused by H5N1 avian influenza virus. The degree of autophagy in the lung tissue of rats.

The results suggest that the mechanism of action of chloroquine in relieving lung injury induced by H5N1 avian influenza virus may be achieved by reducing the degree of autophagy in lung tissue. Example 5 Treatment with chloroquine does not affect the replication of H5N1 avian influenza virus in mouse lung tissue

Experimental Materials:

2) Main experimental reagents: chloroquine (C6628, purchased from Sigma-Aldrich), H5N1 avian influenza virus M1 and M2 gene primers (Invitrogene), Trizol, 1% (W/V) sodium pentobarbital solution, virus Diluent, sterile PBS, chloroform, isopropanol, reverse transcription PCR Kit (Invitrogen), real-time quantitative PCR kit (Roche), solvent control for PBS, 1% (w/v) sodium pentobarbital solution, virus dilution, disinfectant (2.5% iodine and 75% alcohol) ) Wait.

3) Virus: H5N1 avian influenza virus A/Jilin/9/2004 (H5Nl)

experimental method:

1) Grouping: virus + solvent control group, virus + drug group;

2) Mice were injected intraperitoneally with solvent control or drug. Each mouse was intraperitoneally injected with chloroquine (50 mg/kg) for 6 times, 6 hours after infection, 1 day, 2 days, 3 days, 4 Days and 5 days;

3) The mice were fixed safely, and anesthetized with 1% (w/v) sodium pentobarbital solution by intraperitoneal injection with a lmL sterile syringe;

6) After infection, mice that died within 24 hours were non-specific deaths and were not counted in mortality statistics;

7) After infection with the virus, the mouse lung tissue was fixed at 0 hours, 3 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 72 hours and 96 hours after infection to fix the mouse to the small animal operating table. Remove the skin and bones of the chest, expose the chest cavity, remove the whole lung of the mouse, and immediately store it in liquid nitrogen for storage;

8) Remove the cryopreserved lung tissue, add 1.5 ml of Trizol, and grind for 20 seconds at the maximum speed of the homogenizer;

9) Extracting RNA from lung tissue, and measuring the expression of M1 and M2 genes in H5N1 avian influenza virus in lung tissue by reverse transcription PCR and real-time quantitative PCR;

10) Use Graphpad Prism5 to plot data for analysis.

Experimental result

The results of Figures 13A and B show that the mice in the chloroquine-treated group were compared to the solvent control group. There was no significant difference in the expression levels of M1 and M2 genes in the lung H5N1 avian influenza virus after infection with H5N1 avian influenza virus. The results indicate that treatment with chloroquine does not affect the replication of H5N1 avian influenza virus in mouse lung tissue.

The results suggest that the mechanism of action of chloroquine in relieving H5N1 avian influenza virus-induced lung injury in mice is not achieved by affecting viral replication. Example 6 Chloroquine treatment reduced the expression levels of interleukin-1 and interleukin-6 genes in mouse lung tissue induced by H5N1 avian influenza virus, but did not affect the expression of tumor necrosis factor-α gene.

The experimental materials and experimental methods of this example are identical to those of Example 5 except that the primers in the experimental materials are mouse interleukin 1-β, interleukin-6 and tumor necrosis factor-α gene primers.

(Invitrogene)

Experimental result

The results in Fig. 14A show that the expression level of interleukin-1-beta gene in the chloroquine-treated group was significantly decreased at 12 hours, 24 hours, and 36 hours after infection with the H5N1 avian influenza virus compared with the vehicle control group; The results showed that the expression of interleukin-6 gene in the lung decreased significantly 24 hours after infection with the H5N1 avian influenza virus; Figure 14C shows that there was no significant difference in the expression of tumor necrosis factor-α gene in the lung. * Ρ<0.05, * *Ρ<0.01.

The results showed that chloroquine treatment significantly reduced the expression of mouse pro-inflammatory factors interleukin-1 and interleukin-6 in the early stage of infection, which was consistent with the results of lung histopathology (Fig. 11), indicating that chloroquine treatment has The effect of significantly reducing the degree of inflammatory lesions in the lung tissue of mice plays an important role in the treatment of lung injury in mice caused by H5N1 avian influenza virus.

Claims

Claims:

A use of chloroquine or chlorpromazine or a derivative thereof or a mixture thereof for the manufacture of a medicament for the treatment and/or prevention of influenza virus-induced lung infection and/or lung injury in a mammal, including a human.

The use according to claim 1, wherein the derivative of chloroquine is selected from the group consisting of hydroxychloroquine, hydroxychloroquine sulfate, and dichloroquine phosphate; and the derivative of chlorpromazine is selected from the group consisting of acetyl pepazine, perphenazine, Trifluoperazine.

3. The use according to claim 1, wherein the influenza virus is an influenza A virus, preferably a subtype H1, H3, H5, H7 or H9 strain, more preferably a type A H5 subtype The strain is more preferably an influenza A H5N1 virus, most preferably an influenza A H5N1 influenza virus A/Jilin/9/2004 H5N1 strain.

4. Use of a pharmaceutical composition comprising chloroquine or chlorpromazine or a derivative thereof or a mixture thereof for the manufacture of a medicament for the treatment and/or prevention of influenza virus-induced lung infection and/or lung injury in a mammal, including a human .

The use according to claim 4, wherein the derivative of chloroquine is selected from the group consisting of hydroxychloroquine, hydroxychloroquine sulfate, and dichloroquine phosphate; and the derivative of chlorpromazine is selected from the group consisting of acetyl pepazine, perphenazine, Trifluoperazine.

6. The use according to claim 4, wherein the influenza virus is an influenza A virus, preferably an influenza A H1, H3, H5, H7 or H9 subtype strain, more preferably an alpha H5 subtype The strain is more preferably an influenza A H5N1 virus, most preferably an influenza A H5N1 influenza virus A/Jilin/9/2004 strain.

7. The use according to any one of claims 4-6, wherein the pharmaceutical composition comprises a therapeutically effective amount of chloroquine or chlorpromazine or a derivative thereof or a mixture thereof, and one or more pharmaceutically acceptable Accepted carrier.

8. Use according to claim 7, characterized in that the pharmaceutical composition may further comprise one or more therapeutically effective doses which act synergistically with the chloroquine or chlorpromazine or a derivative thereof or a mixture thereof. The active ingredient of the drug.

9. Use according to any one of claims 4-8, wherein the pharmaceutical composition can be formulated into any of the pharmaceutically acceptable dosage forms.

The use according to claim 9, wherein the pharmaceutically acceptable dosage form is an injection, a spray, a nasal drop, an inhalant or an oral agent.

11. A method of treating and/or preventing influenza virus-induced lung infection and/or lung injury in a mammal, including a human, comprising administering to a patient infected with an influenza virus a therapeutically effective amount of chloroquine or chlorpromazine or a derivative thereof or a mixture of them.

12. Chloroquine or chlorpromazine or a derivative thereof or a mixture thereof for use in the treatment and/or prevention of a lung infection and/or lung injury in a mammal, including a human, caused by an influenza virus.