CN113797337A

CN113797337A - Application of small molecule inhibitor in preventing and treating respiratory viral pneumonia

Info

Publication number: CN113797337A
Application number: CN202010919429.6A
Authority: CN
Inventors: 黄波; 刘玉英; 佟伟民
Original assignee: Institute of Basic Medical Sciences of CAMS
Current assignee: Institute of Basic Medical Sciences of CAMS
Priority date: 2020-06-17
Filing date: 2020-09-04
Publication date: 2021-12-17
Anticipated expiration: 2040-09-04
Also published as: CN111803635A; CN111803635B; WO2021253647A1; CN113797337B

Abstract

The application provides application of a small molecule inhibitor in preventing and treating respiratory viral pneumonia. In particular, it relates to a novel use of AhR inhibitors in the treatment or amelioration of viral infections. The inhibitor for treating virus infection provided by the application can effectively inhibit the expression of angiotensin converting enzyme 2, so that the virus infection lung tissue is blocked, and the AhR inhibitor is expected to become a potential drug for preventing and treating lung diseases caused by virus infection.

Description

Application of small molecule inhibitor in preventing and treating respiratory viral pneumonia

This application claims priority from patent application 202010552732.7 (priority date 2020, 6/17).

Technical Field

The present application relates to the fields of biology, medicine, and clinics. In particular to a small molecule inhibitor and application thereof in preventing and treating viral pneumonia.

Background

The global coronavirus pandemic has caused a tremendous impact on countries around the world. The virus has spread to almost every country around the world in less than 6 months. Some of these countries have experienced a second wave burst, while others, russia, brazil, india and parts of africa, remain in the first wave burst.

This new pathogen was named Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) by the World Health Organization (WHO) at 11/2/2020. SARS-CoV-2 is the cause of the 2019 coronavirus (COVID-19), belonging to the same virus family as the severe acute respiratory syndrome coronavirus (SARS-CoV) and the middle east respiratory syndrome coronavirus (MERS-CoV), respectively, which were named in 2003 and 2012, respectively. However, unlike previous new coronavirus related diseases, the COVID-19 pandemic has led to higher morbidity and mortality in various countries.

The progress of the novel coronavirus pneumonia COVID-19 becomes very difficult to control once it enters the severe stage and can easily lead to death of the patient. At present, the cause of death of patients due to SARS-COV-2 infection is not clear. Viral entry is generally considered to be the first step in mediating pathogenic processes. Angiotensin converting enzyme 2(ACE2) is a membrane receptor expressed on the surface of airway epithelial cells. Scientists began intensive research into ACE2 at the beginning of the 21 st century. ACE2 has been shown to be a host cell receptor for both the SARS-COV and HCoV-NL63 human coronaviruses, and interacts with the spike protein of coronaviruses. This interaction is mediated by the RBD domain of the spike protein and is thought to be a key step in the membrane fusion of the virus with the cell. ACE2 is a newly discovered host cell receptor for the lethal new coronavirus SARS-CoV-2 (2019-nCoV). Blocking the interaction between the S protein and ACE2 is an effective method for treating coronavirus infections.

Therefore, how to provide effective intervention that can use the receptor ACE2 as a therapeutic target for virus infection, so as to effectively prevent new corona from infecting lung tissues, becomes a problem to be solved at present.

Disclosure of Invention

According to some embodiments of the present disclosure, there is provided an AhR (arene receptor) inhibitor which is useful as a viral pneumonia formulation. The viral pneumonia formulation comprises an AhR inhibitor.

In the present disclosure, the term "inhibitor" refers to a natural or synthetic compound that inhibits (or reduces or down-regulates) the expression of a gene and/or protein, and/or inhibits (or reduces or down-regulates) the activity of a gene and/or protein, and/or modulates the associated signaling pathway with the gene and/or protein. By way of non-limiting example, the inhibitor may act on any one or a combination of genes and/or proteins: such as, but not limited to, translation, post-translational processing, stability, degradation, nuclear localization, cytoplasmic localization, transcription, post-transcriptional processing, activation, inactivation, modification, signaling. The inhibitor is allowed to be competitive, noncompetitive, fully antagonistic, or partially antagonistic.

Thus, AhR inhibitors refer to compounds having the following effects: inhibiting (or reducing or down-regulating) expression of an AhR-encoding gene, and/or expression of AhR, and/or activity of AhR, and/or modulating an AhR-associated signaling pathway (e.g., without limitation, upstream of an AhR-associated signaling pathway). According to some embodiments of the present disclosure, there is provided a use of an AhR inhibitor in the manufacture of a medicament, wherein the medicament is for any one or combination selected from: preventing the occurrence or recurrence of viral pneumonia, and treating viral pneumonia or its symptoms.

In some embodiments, the virus is selected from one or a combination of: coronavirus, influenza a virus, influenza b virus, influenza c virus, measles virus, mumps virus, respiratory syncytial virus, parainfluenza virus, human metapneumovirus, hendra virus, nipah virus, rubella virus, rhinovirus, adenovirus, reovirus, coxsackie virus, ECHO virus, and variants thereof.

In some embodiments, the medicament is prepared in a dosage form selected from the group consisting of: injection, spray, aerosol, nasal drop, oral preparation, and dosage form suitable for mucosal administration.

AhR inhibitors suitable for use in the present disclosure such as, but not limited to, compounds disclosed in the prior art: WO2019036657, WO2018195397, CN106860471A, WO2013034685, WO 20120152015914.

In some exemplary embodiments, the AhR inhibitor is selected from one or a combination of: AhR antagonist 1, alpha-NF, CB7993113, CMLD-2166, CH223191, DMF, GNF351, PDM2, StemRegenin 1, SR1, IDO inhibitor.

In some embodiments, an IDO (indoleamine 2,3-dioxygenase) inhibitor is provided that is useful as a viral pneumonia formulation. The viral pneumonia formulation comprises an IDO inhibitor.

IDO (e.g., IDO1) is an active molecule located upstream of the AhR signaling pathway. Therefore, inhibitors acting on IDO can indirectly modulate AhR activity.

IDO inhibitors refer to compounds that have the following effects: inhibiting (or reducing or down-regulating) expression of an IDO-encoding gene, and/or expression of IDO, and/or activity of IDO.

In the art, IDO inhibitors can be generally classified by structure into several types:

-a tryptophan analogue: exemplary compounds are the N-methyl derivative of tryptophan L1 MT;

-quinone compounds: mainly obtained from natural products;

imidazole and triazole compounds: 4-benzimidazole and its derivatives can coordinate with iron atom of heme, and have strong IDO inhibitory activity. Exemplary compounds are NLG919 from NewLink Genetics;

-N-hydroxyamidines: the N-hydroxy amidine compound can be combined with the iron atom of the heme and simultaneously form a hydrogen bond with the nitrogen atom on the side amide group. An exemplary compound is INCB 024360.

IDO inhibitors suitable for use in the present disclosure are for example, but not limited to, compounds disclosed in the prior art: CN106866648B, CN106883224B, CN107501272B, CN109438513B, CN109748838B, CN105567690B, CN107260743B, WO 2015173764.

In some exemplary embodiments, the IDO inhibitor is selected from one or a combination of: tryptophan analogues, quinone and derivatives thereof, imidazole and derivatives thereof, triazole and derivatives thereof, and N-hydroxyamidine and derivatives thereof.

In some embodiments, the IDO inhibitor is selected from one or a combination of: 1-MT, Epacadostat, DO-IN-2, NLG919, PF-06840003, INCB024360, Exiguamine A and benzimidazole.

In some embodiments, the amount of the AhR inhibitor in a unit formulation is 10mg to 10 g. Mention may be made of 10mg, 20mg, 30mg, 40mg, 50mg, 60mg, 70mg, 80mg, 90mg, 100mg, 200mg, 300mg, 400mg, 500mg, 600mg, 700mg, 800mg, 900mg, 1g, 1.5g, 2g, 2.5g, 3g, 3.5g, 4g, 4.5g, 5g, 5.5g, 6g, 6.5g, 7g, 7.5g, 8g, 8.5g, 9g, 9.5g, 10g or a range between any two of the foregoing values.

In some embodiments, the amount of IDO inhibitor in a unit formulation is 1g to 10 g.

In other embodiments, the amount of the AhR inhibitor in a unit formulation is 10mg to 80 mg.

In some embodiments, the coronavirus is selected from the group consisting of: SARS, SARS-COV2, and variants thereof.

In some embodiments, the treatment is embodied as one or a combination selected from:

improve the blood oxygen saturation and improve PaO₂/FiO₂Relieving respiratory distress, reducing resistance of RRS respiratory system, improving ERS, improving PV-k level, improving Eta level, improving DLCO level, regulating expression level of ACE2, delaying or preventing disease progression to severe state, improving survival rate, and prolonging life time.

In some embodiments, the viral pneumonia is selected from: light, common, heavy and dangerous.

According to some embodiments, there is provided a pharmaceutical composition comprising an AhR inhibitor, and optionally a pharmaceutically acceptable carrier.

In some embodiments, the pharmaceutical composition is for use in any one or combination selected from the group consisting of: preventing the occurrence or recurrence of viral pneumonia, and treating viral pneumonia or its symptoms.

In some embodiments, the pharmaceutical composition is a dosage form selected from the group consisting of: injection, spray, aerosol, nasal drop, oral preparation, and dosage form suitable for mucosal administration.

According to some embodiments, there is provided a method of preventing or treating viral pneumonia, comprising administering to a subject a prophylactically effective amount or a therapeutically effective amount of an AhR inhibitor. In some embodiments, routes of administration that may be mentioned include, but are not limited to: intramuscular, intravenous, subcutaneous, intradermal, oral, intranasal, respiratory, transmucosal, sublingual, parenteral.

In some embodiments of the methods according to the present application, an "effective amount" or "effective dose" refers to the amount of a drug, compound, pharmaceutical composition necessary to achieve any one or more beneficial or desired therapeutic results. Beneficial or desired results include: improving clinical outcome (e.g., reducing morbidity, mortality, ameliorating one or more symptoms), reducing severity, delaying onset of a disorder (including the disorder or its complications, intermediate pathological phenotypes presented during the development of the disorder, biochemical, histological, and/or behavioral symptoms).

In some embodiments, the unit formulation is a formulation of the active ingredient (AhR inhibitor) required for one administration, such as a unit (needle) injection, or the like.

The amount of drug required for a single administration to a patient can conveniently be calculated by multiplying the weight of the patient by the unit weight dose required for a single administration to the patient. For example, in the manufacture of a medicament, which is generally considered to be 50-70kg in weight of an adult human, the amount may be initially determined by equivalent dose conversion between the unit weight doses of the experimental animal and the human. For example, the determination can be made by referring to the guidelines proposed by the drug administration such as FDA and SFDA (Huang-Ji Han, et al, "equivalent dose conversion between animals and humans in pharmacological tests", Chinese clinical pharmacology and therapeutics, 2004 Sep; 9 (9): 1069-.

In embodiments of the present disclosure, a dose conversion between human and mouse may be used according to a human and mouse body surface area conversion factor of 0.0026.

In the presently disclosed regimen, for example, an IDO inhibitor is administered to a mouse in an amount of 1000mg/kg for a mouse weighing 20g (e.g., for a 20g mouse, the IDO inhibitor is formulated with water as a 5mg/mL solution that the mouse is drenched with 4mL per day).

According to some embodiments, there is also provided a method for preventing a patient with respiratory viral pneumonia from turning into an acute respiratory viral pneumonia, comprising a process of administering the anti-respiratory viral pneumonia formulation to a patient with respiratory viral pneumonia or an individual having a tendency to turn into an acute respiratory viral pneumonia. The specific process is intravenous or oral AhR inhibitor.

According to the scheme disclosed by the invention, the amount of the AhR inhibitor in the preparation for resisting respiratory viral pneumonia can be controlled as required, and the administration to patients with viral pneumonia at different stages is convenient.

Without being bound by any theory, interferon is the first alarm of viral invasion, and its signal activates a series of antiviral genes to exert a direct antiviral effect. However, delayed interferon responses may cause immunopathological changes in the body through recruitment and activation of innate immune cells that produce high levels of inflammatory factors. In the research of the applicant, IFN-beta and IFN-gamma are found to up-regulate the expression of angiotensin converting enzyme 2 in BAES-2B cells, thereby causing virus infection of lung tissues. The experimental research unexpectedly discovers that the AhR inhibitor can effectively block angiotensin converting enzyme 2 generated by IFN-beta and IFN-gamma induction, and can realize the clinical value of preventing patients with severe respiratory viral pneumonia virus pneumonia.

In some embodiments of the methods according to the present application, the patient refers to a viral carrier, in particular a patient who has already developed or may develop symptoms due to the presence of a virus. In particular embodiments, the patient is particularly a patient at risk of developing severe or critically ill illness.

Drawings

Figure 1 shows that treatment of BAES-2B and mouse type II alveolar epithelial cells with Kyn found that Kyn significantly induced ACE2 expression at the protein level and that Kyn-induced ACE2 expression was abolished by addition of AhR inhibitor CH 223191.

FIG. 2 shows that treatment of BAES-2B cells and mouse type II alveolar epithelial cells with IFN- β or IFN- γ also resulted in upregulation of ACE2 expression, but could be disrupted by either the IDO inhibitor 1-MT or the AhR inhibitor CH 223191.

FIG. 3 shows that treatment of BAES-2B and mouse type II alveolar epithelial cells with FICZ results in upregulation of ACE2 expression in BAES-2B and primary murine alveolar epithelial cells.

FIG. 4 shows that both Kyn and interferon are effective in stimulating the transfer of AhR from the cytoplasm of BAES-2B cells or mouse type II alveolar epithelial cells to the nucleus.

FIG. 5 shows that lung injury caused by SARS-COV-2 can be effectively inhibited by an AHR inhibitor.

FIG. 6 shows that the viral load of SARS-COV-2 can be effectively reduced with an AHR inhibitor.

Detailed Description

Various cell lines, drugs and experimental animals were used in the following examples: the BEAS-2B human lung epithelial cell line is purchased from China typical Collection center (CCTCC); SARS-COV-2 is derived from institute of animal testing and medicine of Chinese academy of medical sciences; 6-8 week-old female Balb/c mice were purchased from the Experimental animals center of the cooperative medical college of Chinese medical sciences; rhesus monkeys of 3-4 years old were from the institute of laboratory animals of the cooperative medical college of science of china; IDO inhibitor (1-MT) was purchased from SIGMA corporation, USA; AhR inhibitor (CH223191) was purchased from MCE, usa.

Example 1 significant induction of ACE2 expression by Kyn through AHR pathway

Firstly, an experiment step:

1. preparing a reagent: ca-free²⁺/Mg²⁺HBSS + 5% FBS +2mM EDTA + collagenase + hyaluronidase, pre-heated to 37 ℃; the mice were sacrificed and the lungs were harvested; removing bronchi and lymph nodes; with no Ca²⁺/Mg²⁺Washing lung contents with PBS; cutting the lung into small pieces as small as possible, putting the small pieces into a 50mL centrifuge tube, and adding 20mL of the reagent prepared in the first step; shaking with a horizontal shaker at 37 deg.C and 250rpm for 20 min; filtering the supernatant with a 200-mesh filter screen into a new 50mL centrifuge tube, and adding 20mL of the reagent prepared in the first step into the residual intestinal tissue; shaking with a horizontal shaker at 37 deg.C and 250rpm for 20 min; filtering the supernatant with a 200-mesh filter screen to a previous 50mL centrifuge tube; the filtrate was centrifuged at 400g for 5min and the resulting cell pellet was lung epithelial cells, washed three times with PBS and used for subsequent experiments.

2. The method comprises the following steps: control (PBS treatment) and Kyn (0.4mM Kyn for 48 hours), Kyn plus AHR inhibitor (0.4mM Kyn and 4. mu.M CH223191 for 48 hours).

Western blot detection of protein expression: adding protease inhibitor into different treated samples; cracking on ice for 30 min; centrifuging at 12000g and 4 ℃ for 20min, and sucking the supernatant into a new 1.5mL EP tube, namely the total cell protein; the protein can be directly quantified or temporarily stored at-80 ℃.

BCA protein quantification: taking a small amount of each protein sample, diluting the protein sample by 5 times and 10 times by using RIPA lysate, sucking 25 mu L of the diluted protein sample, and adding the diluted protein sample into a 96-well plate; the protein standard stock solution (4 mug/mu L) is diluted by RIPA lysate in a multiple ratio, 25 mu L of the protein standard stock solution is absorbed by each concentration and added into a 96-well plate, and 25 mu L of RIPA lysate is added into the last well, wherein the concentrations are as follows: 1.6. mu.g/. mu.L, 0.8. mu.g/. mu.L, 0.4. mu.g/. mu.L, 0.2. mu.g/. mu.L, 0.1. mu.g/. mu.L, 0.05. mu.g/. mu.L, 0.025. mu.g/. mu.L, 0. mu.g/. mu.L; mixing the protein color development solution with the diluent at a ratio of 1:50, and adding 200 mu L of the protein color development solution into each hole of a 96-hole plate; reacting at 37 ℃ for 30 min; and reading the absorbance value of each hole at 562nm by using a microplate reader, drawing a standard curve according to the concentration and the absorbance of the standard substance, and calculating the protein concentration according to the absorbance value of the protein sample.

5. Preparing a rubber plate: assembling a glue-making plate, preparing separation glue, adding TEMED, blowing uniformly, and immediately pouring glue (3.2 mL separation glue is poured into a 0.75mm glue groove, and 5mL separation glue is poured into a 1mm glue groove) to avoid generating bubbles; adding a layer of isopropanol on the separation gel to ensure that the edge of the separation gel is smooth, and observing a folding line at a two-phase interface after the separation gel is solidified for about 40 min; pouring off isopropanol, drying with filter paper, standing for 10min until isopropanol volatilizes; preparing concentrated glue, adding TEMED, blowing uniformly, immediately filling the glue, immediately inserting a comb to avoid bubbles in the glue, and solidifying the concentrated glue for about 30 min; the prepared rubber plate can be immediately spotted for electrophoresis, and can also be wrapped by filter paper wetted by water and stored at 4 ℃.

SDS-PAGE electrophoresis: calculating the volume of each sample according to the total amount of the protein to be loaded and the protein concentration of each sample, sucking the corresponding volume to a new 1.5mL EP tube, adding a loading buffer solution, and boiling for 10min at 100 ℃ in a metal bath; assembling an electrophoresis assembly, pouring 1 xSDS-PAGE electrophoresis buffer solution, removing upper-layer foam, and vertically pulling out a comb upwards in the electrophoresis solution; spotting the cooked protein sample and protein marker, and filling each sample volume with 1 × loading buffer; putting the electrophoresis assembly into an electrophoresis tank, and pouring 1 xSDS-PAGE electrophoresis buffer solution to a scale mark; and (3) starting electrophoresis, wherein the sample is subjected to constant pressure of 80V when the gel is concentrated, is subjected to constant pressure of 120V after entering the separation gel, and stops electrophoresis when bromophenol blue reaches the bottom of the gel.

7. Film transfer: soaking the filter paper and the sheared NC membrane in a pre-cooled membrane-transferring buffer solution at 4 ℃ half an hour before electrophoresis; taking out the gel after electrophoresis is finished, and cutting out the region where the target protein is located according to the molecular weight standard; assembling a membrane conversion component in a membrane conversion buffer solution, and sequentially from a cathode to an anode: filter paper, glue, NC membrane, filter paper; putting the membrane conversion assembly into an electrophoresis tank, and pouring 1L of membrane conversion buffer solution; and (3) switching on a power supply, carrying out film transfer in ice at a constant current of 200mA, wherein the film transfer time is determined according to the molecular weight of the protein.

8. Antibody incubation: after the membrane is transferred, taking out the NC membrane, putting the NC membrane into 5% BSA blocking solution (BSA is dissolved by TBST), and slowly shaking the horizontal shaking table at room temperature to block for 2 hours; placing the membrane in a primary antibody diluted by 5% BSA, slowly shaking at room temperature, incubating for 1h, and then incubating overnight at 4 ℃; after the night, taking out the membrane (the primary sodium azide is recycled and stored at 4 ℃), putting the membrane into a dish containing TBST, and quickly shaking the membrane at room temperature for washing 4 times for 10min each time; the membrane is placed in a secondary antibody diluted by 5% BSA and incubated for 2h at room temperature with slow shaking; the membrane was taken out and placed in a dish containing TBST, and the membrane was washed with shaking at room temperature for 10min each time for 4 times.

9. And (3) chemiluminescence development: after the membrane is washed, soaking the membrane in TBST and taking the membrane into a dark room; mixing ECL developer A, B solution in equal volume, 5cm²The total amount of the film color developing agent is 1 mL; laying a layer of PE gloves on a darkroom table, taking a film by a pair of tweezers, putting the film on the PE gloves after dipping the film on absorbent paper, and uniformly dropwise adding a developer mixed solution on the film; turning off the lamp, gradually lightening the strips on the visible film, clamping the film, dipping the film on the absorbent paper with dry color development liquid, putting the film into an X-ray box, and covering a layer of transparent plastic paper; taking out an X-ray film, putting the X-ray film into a box, covering a box cover, and adjusting exposure time according to different brightness; and after the time is up, taking out the X-ray film and putting the X-ray film into a developing machine, and if the strip is not satisfactory, taking out the X-ray film and putting the X-ray film into a box to adjust the exposure time.

Second, experimental results

WESTERN imprinting showed that treatment of BAES-2B and mouse type II alveolar epithelial cells with Kyn found that Kyn significantly induced ACE2 expression at the protein level, and that Kyn-induced ACE2 expression was abolished by the addition of AhR inhibitor CH 223191.

Example 2 Induction of ACE2 expression by Interferon beta through the IDO-AHR pathway

Firstly, experimental steps

1. Preparing a reagent: ca²⁺/Mg²⁺HBSS + 5% FBS +2mM EDTA + collagenase + hyaluronidase, pre-heated to 37 ℃; the mice were sacrificed and the lungs were harvested; removing bronchi and lymph nodes; with no Ca²⁺/Mg²⁺Washing lung contents with PBS; cutting the lung into small pieces as small as possible, and placing into a 50mL centrifuge tubeAdding 20mL of the reagent prepared in the first step; shaking with a horizontal shaker at 37 deg.C and 250rpm for 20 min; filtering the supernatant with a 200-mesh filter screen into a new 50mL centrifuge tube, and adding 20mL of the reagent prepared in the first step into the residual intestinal tissue; shaking with a horizontal shaker at 37 deg.C and 250rpm for 20 min; filtering the supernatant with a 200-mesh filter screen to a previous 50mL centrifuge tube; the filtrate was centrifuged at 400g for 5min and the resulting cell pellet was lung epithelial cells, washed three times with PBS and used for subsequent experiments.

2. The method comprises the following steps: control (PBS treatment), interferon beta (1ng/ml INF beta 48 hours treatment), interferon beta plus IDO inhibitor (1ng/ml INF beta and 0.2mM 1-MT 48 hours treatment) and interferon beta plus AHR inhibitor (1ng/ml INF beta and 4. mu.M CH223191 48 hours treatment).

Western blot detection, BCA protein quantification, gel plate preparation, SDS-PAGE electrophoresis, membrane transfer, antibody incubation, and chemiluminescence visualization were performed as described in example 1 above.

Second, experimental results

WESTERN imprinting showed that treatment of BAES-2B and mouse type II alveolar epithelial cells with interferon beta resulted in significant induction of ACE2 expression at the protein level, and that interferon beta-induced ACE2 expression was abolished by addition of IDO1 inhibitor 1-MT or AhR inhibitor CH 223191.

Example 3 Induction of ACE2 expression by Interferon Gamma via the IDO-AHR pathway

Firstly, experimental steps

1. Mouse alveolar cell isolation was performed as described in example 1 above.

2. The method comprises the following steps: control (PBS treatment), interferon gamma (10ng/ml INF gamma for 48 hours), interferon gamma plus IDO inhibitor (10ng/ml INF gamma and 0.2mM 1-MT for 48 hours) and interferon gamma plus AHR inhibitor (10ng/ml INF gamma and 4. mu.M CH223191 for 48 hours).

Second, experimental results

The WESTERN blot showed that treatment of BAES-2B and mouse type II alveolar epithelial cells with interferon gamma resulted in significant induction of ACE2 expression at the protein level, and that interferon gamma-induced ACE2 expression was abolished by addition of IDO1 inhibitor 1-MT or AhR inhibitor CH 223191.

Example 4 FICZ expression in ACE2 Induction by AHR

Firstly, experimental steps

1. Mouse alveolar cell isolation was performed as described in example 1 above.

2. The method comprises the following steps: control (PBS treatment), FICZ (1. mu.M FICZ treatment for 48 hours).

Second, experimental results

The WESTERN blot showed that when BAES-2B and mouse type II alveolar epithelial cells were treated with FICZ, FICZ was found to significantly induce ACE2 expression at the protein level.

Example 5 Interferon stimulated Nuclear localization of AHR

Firstly, experimental steps

1. Mouse alveolar cell isolation was performed as described in example 1 above.

2. The method comprises the following steps: control (PBS treatment), interferon beta (1ng/ml interferon beta 48 hours), interferon gamma (10ng/ml interferon gamma 48 hours), Kyn (0.4mM Kyn 48 hours).

Second, experimental results

With interferon-treated BAES-2B and mouse type II alveolar epithelial cells, the inventors found that the results of immunofluorescent staining showed that AHR of the interferon-treated group and Kyn-treated group was significantly up-regulated in expression and appeared to be nuclear-entry localized, compared to the control group mice (PBS group).

Example 6 AHR inhibitors reverse the phenomenon of lung tissue damage caused by SARS-COV-2 virus and significantly reduce viral load

Firstly, experimental steps

Rhesus monkeys were injected non-exposed intratracheal. Normal group is injected with normal saline with the same amount as the treatment group at one time through the trachea, and the treatment group is irrigated with the lung 10 at one time through the trachea⁶Unit of SARS-CoV-2 virus particles. Immediately after injection, the animals were rotated upright enough to distribute the drug evenly in the lungs, and the treatment was waited for 1 week. Collection of lung tissue: the rhesus monkey is anesthetized, euthanized, the lung tissue is taken out after thoracotomy, the surface of the lung tissue is washed away with prepared normal saline, and the tissue is stored at-80 ℃ after the operation is carried out as quickly as possible. A portion of lung tissue was immediately stored in 4% paraformaldehyde for preparation of HE staining and RNA in situ hybridization.

Second, experimental results

Lung tissue of rhesus monkey infected with SARS-CoV-2, the inventors found that HE staining showed significant lung tissue damage in mice compared to control mice (MOCK group, no virus infection), whereas using AhR inhibitor significantly reversed the lung tissue damage in rhesus monkey caused by SARS-CoV-2 (fig. 5). Furthermore, the inventors found that the AhR inhibitor group showed a significant reduction in viral load in lung tissue of rhesus monkeys as compared to rhesus monkeys of the infected group as a result of RNA in situ hybridization, whereas the viral load of SARS-CoV-2 was significantly reduced using the AhR inhibitor (fig. 6).

In summary, the scheme of the present application has the following effects:

1. the anti-respiratory viral pneumonia preparation AhR inhibitor can effectively inhibit ACE2 expressed by virus-induced organism respiratory system, and is expected to become a potential medicine for treating lung diseases infected by coronavirus.

2. After viral infection, the body will produce a large amount of IFN-beta and IFN-gamma, which are two cytokines that are intended to exert antiviral effects, but produce other negative effects (i.e. promote the lung to produce more ACE2, and induce the virus to enter human lung cells more again). AhR inhibitors (IDO acting through the AhR pathway) reverse this negative effect.

3. The anti-coronavirus pneumonia preparation can also kill viruses by enhancing the systemic immune response, and has the advantages of high safety and no toxic or side effect.

Claims

Use of an AhR inhibitor in the manufacture of a medicament, wherein:

the medicament is for any one or combination selected from: preventing the occurrence or recurrence of viral pneumonia, treating viral pneumonia or its symptoms;

the virus is selected from one or a combination of the following: coronavirus, influenza a virus, influenza b virus, influenza c virus, measles virus, mumps virus, respiratory syncytial virus, parainfluenza virus, human metapneumovirus, hendra virus, nipah virus, rubella virus, rhinovirus, adenovirus, reovirus, coxsackie virus, ECHO virus, and variants thereof;

preferably, the virus is SARS or a variant thereof, SARS-COV2 or a variant thereof;

preferably, the medicament is prepared in a dosage form selected from the group consisting of: injections, sprays, aerosols, nasal drops, oral preparations, dosage forms suitable for mucosal administration;

preferably, the AhR inhibitor is selected from one or a combination of: AHR antagonist 1, alpha-NF, CB7993113, CMLD-2166, CH223191, DMF, GNF351, PDM2, StemRegenin 1, SR1, IDO inhibitors;

preferably, the IDO inhibitor is selected from one or a combination of the following: tryptophan analogues, quinone and derivatives thereof, imidazole and derivatives thereof, triazole and derivatives thereof, and N-hydroxyamidine and derivatives thereof;

more preferably, the IDO inhibitor is selected from one or a combination of the following: 1-MT, Epacadostat, DO-IN-2, NLG919, PF-06840003, INCB024360, Exiguamine A, benzimidazole; most preferably, the IDO is IDO 1.
2. The use according to claim 1, wherein the amount of AhR inhibitor in a unit formulation is from 10mg to 10 g;

preferably, the amount of said IDO inhibitor in a unit formulation is from 1g to 10 g;

preferably, the amount of AhR inhibitor other than IDO inhibitor in a unit formulation is 10mg to 80 mg.
3. Use according to claim 1 or 2, wherein:

the treatment is embodied as one or a combination selected from: improve the blood oxygen saturation and improve PaO₂/FiO₂Relieving respiratory distress, reducing RRS respiratory system resistance, improving ERS, improving PV-k level, improving Eta level, improving DLCO level, regulating angiotensin converting enzyme 2 expression level, improving survival rate, and prolonging life;

the prevention means delaying or preventing the progression of the disease to severe or critically ill state, and reducing the risk of progression to severe or critically ill state.
4. The use of claim 1, wherein the viral pneumonia is selected from the group consisting of: light, common, heavy and dangerous.
5. A pharmaceutical composition comprising:

AhR inhibitors, and

optionally, a pharmaceutically acceptable carrier:

the pharmaceutical composition is for any one or combination selected from the group consisting of: preventing the occurrence or recurrence of viral pneumonia, treating viral pneumonia or its symptoms;

the virus is selected from one or a combination of the following: coronavirus, influenza a virus, influenza b virus, influenza c virus, measles virus, mumps virus, respiratory syncytial virus, parainfluenza virus, human metapneumovirus, hendra virus, nipah virus, rubella virus, rhinovirus, adenovirus, reovirus, coxsackie virus, ECHO virus, and variants thereof;

preferably, the virus is SARS or a variant thereof, SARS-COV2 or a variant thereof;

the pharmaceutical composition is in a dosage form selected from the group consisting of: injections, sprays, aerosols, nasal drops, oral preparations, dosage forms suitable for mucosal administration;

preferably, the AhR inhibitor is selected from one or a combination of: AHR antagonist 1, alpha-NF, CB7993113, CMLD-2166, CH223191, DMF, GNF351, PDM2, StemRegenin 1, SR1, IDO inhibitors;

preferably, the IDO inhibitor is selected from one or a combination of the following: tryptophan analogues, quinone and derivatives thereof, imidazole and derivatives thereof, triazole and derivatives thereof, and N-hydroxyamidine and derivatives thereof;

more preferably, the IDO inhibitor is selected from one or a combination of the following: 1-MT, Epacadostat, DO-IN-2, NLG919, PF-06840003, INCB024360, Exiguamine A, benzimidazole;

the amount of the AhR inhibitor in a unit formulation is 10mg to 10 g;

preferably, the amount of said IDO inhibitor in a unit formulation is from 1g to 10 g;

preferably, the amount of AhR inhibitor other than IDO inhibitor in a unit formulation is 10mg to 80 mg;

preferably, the IDO is IDO 1.
6.A method of preventing or treating viral pneumonia, comprising:

administering to the subject a prophylactically or therapeutically effective amount of an AhR inhibitor;

the virus is selected from one or a combination of the following: coronavirus, influenza a virus, influenza b virus, influenza c virus, measles virus, mumps virus, respiratory syncytial virus, parainfluenza virus, human metapneumovirus, hendra virus, nipah virus, rubella virus, rhinovirus, adenovirus, reovirus, coxsackie virus, ECHO virus, and variants thereof;

preferably, the virus is SARS or a variant thereof, SARS-COV2 or a variant thereof;

the administration is selected from: intramuscular, intravenous, subcutaneous, intradermal, oral, intranasal, respiratory, transmucosal, sublingual, parenteral;

the AhR inhibitor is selected from one or a combination of: AHR antagonist 1, alpha-NF, CB7993113, CMLD-2166, CH223191, DMF, GNF351, PDM2, StemRegenin 1, SR1, IDO inhibitors;

preferably, the IDO inhibitor is selected from one or a combination of the following: 1-MT, Epacadostat, DO-IN-2, NLG919, PF-06840003, INCB024360, Exiguamine A, benzimidazole;

the amount of the AhR inhibitor in a unit formulation is 10mg to 10 g;

preferably, the amount of said IDO inhibitor in a unit formulation is from 1g to 10 g;

preferably, the amount of AhR inhibitor other than IDO inhibitor in a unit formulation is 10mg to 80 mg;

preferably, the IDO is IDO 1;

the prevention means delaying or preventing the progression of the disease to severe or critically ill, reducing the risk of progression to severe or critically ill;

the treatment is embodied as one or a combination selected from: improve the blood oxygen saturation and improve PaO₂/FiO₂Relieving respiratory distress, reducing RRS respiratory system resistance, improving ERS, improving PV-k level, improving Eta level, improving DLCO level, regulating angiotensin converting enzyme 2 expression level, improving survival rate, and prolonging life.