Aconitic acid for treatment of pulmonary viral infections

Abstract

The present invention relates to aconitic acid or a pharmaceutically acceptable salt thereof for use in a method of treating a viral pulmonary infection and / or an adverse immune response to a viral pulmonary infection, the method comprising administering to a subject in need thereof an effective amount of aconitic acid or a pharmaceutically acceptable salt thereof, or to a pharmaceutical composition comprising aconitic acid or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier for use in a method as defined above.

Description

Background Art

[0001] Lung infections can be a persistent and widespread burden, such as influenza, or sudden and devastating, as with SARS-CoV-2. The recent pandemic has further highlighted the key role of viruses in respiratory infections.

[0002] Since the 1918 pandemic, influenza viruses have caused significant morbidity and mortality annually, and the pathogenesis of influenza and anti-influenza therapeutic strategies have been extensively studied. The pathophysiology of influenza lung infection is the result of two phenomena: (i) the intrinsic pathogenicity of the virus, which is related to its tropism for host airway cells, and (ii) the adverse immune response of the subject, which often includes a hyperinflammatory immune response. Indeed, a robust host immune response is required for viral clearance, but the recruitment of massive cells and the release of cytotoxic molecules lead to excessive lung inflammation and may be associated with lung damage, morbidity, and mortality.

[0003] Two classes of anti-influenza drugs have been discovered, but WHO currently recommends only one of them (neuraminidase inhibitors) [7]. Antiviral therapies for influenza, such as The introduction of oseltamivir has resulted in a reduction in the duration of flu symptoms in patients with influenza worldwide. However, there remains some degree of skepticism about the actual efficacy of NA inhibitors, particularly after a 2014 Cochrane clinical meta-analysis showed a slight reduction in flu symptoms in children and adults with uncomplicated influenza, but not in hospitalized patients [8]. In particular, current antiviral therapies address the intrinsic pathogenicity of the virus but have little or no effect on the host's hyperinflammatory immune response to viral infection.

[0004] Therefore, rapid development of new strategies to treat viral lung infections and / or adverse immune responses to viral lung infections remains crucial.

[0005] Summary of the Invention

[0006] The present invention is based on the inventor's surprising discovery that aconitic acid can modulate the immune response triggered by viral infection in the lungs. In particular, the inventor has demonstrated that aconitic acid has anti-inflammatory properties with potential sufficient to block the inflammatory cascade during influenza and SARS-CoV-2 infection. In particular, aconitic acid exhibits favorable immunomodulatory activity in the lungs of subjects with viral infections caused by respiratory viruses such as influenza or coronavirus, and more surprisingly, exhibits powerful antiviral activity against some respiratory viruses including influenza.

[0007] Thus, embodiment E1 of the present disclosure relates to aconitic acid or a pharmaceutically acceptable salt thereof for use in a method for treating viral lung infection and / or an adverse immune response to viral lung infection, the method comprising administering an effective amount of aconitic acid or a pharmaceutically acceptable salt thereof to a subject in need thereof.

[0008] Embodiment E2 of the present disclosure relates to aconitic acid for use in a method according to embodiment El, wherein the aconitic acid is cis-aconitic acid or trans-aconitic acid.

[0009] Embodiment E3 of the present disclosure relates to aconitic acid for use in a method according to any one of embodiments E1 or E2, wherein the aconitic acid has immunomodulatory and / or antiviral properties, wherein:

[0010] -The immunomodulatory properties of aconitic acid include reducing or suppressing the inflammatory response to viral lung infections;

[0011] -The antiviral properties of aconitic acid include inhibition of viral replication.

[0012] Embodiment E4 of the present disclosure relates to aconitic acid for use in a method according to any one of embodiments E1 to E3, wherein the viral lung infection is caused by a respiratory virus, preferably selected from influenza virus, coronavirus, respiratory syncytial virus, parainfluenza virus, metapneumovirus, rhinovirus, adenovirus, varicella-zoster virus, cytomegalovirus, paramyxovirus such as Nipah virus, and bocavirus.

[0013] Embodiment E5 of the present disclosure relates to aconitic acid for use in a method according to any one of embodiments E1 to E4, wherein the viral lung infection is caused by influenza virus or coronavirus.

[0014] Embodiment E6 of the present disclosure relates to aconitic acid according to any one of embodiments E1 to E5 for use in a method for treating a viral lung infection.

[0015] Embodiment E7 of the present disclosure relates to aconitic acid according to any one of embodiments 1-5 for use in a method for treating an adverse immune response to a viral lung infection in a subject having a lung viral infection.

[0016] Embodiment E8 of the present disclosure relates to aconitic acid according to any one of embodiments E1 to E5 for use in a method for preventing escalation of a pulmonary viral infection to an adverse immune response associated with a viral lung infection in a subject having the pulmonary viral infection.

[0017] Embodiment E9 of the present disclosure relates to aconitic acid for use in a method according to any one of embodiments E1 to E10, wherein the adverse immune response to a viral lung infection is an adverse immune response to a viral infection of the lungs that is an exaggerated inflammatory immune response, dyspnea, shortness of breath, pneumonia (particularly acute pneumonia), chronic respiratory disease (such as asthma or exacerbation of chronic obstructive pulmonary disease), sepsis, septic shock, cytokine storm, or acute respiratory distress syndrome (ARDS).

[0018] Embodiment E10 of the present disclosure relates to aconitic acid for use in a method according to embodiments E1-E12, wherein the aconitic acid is administered 4-14 days after infection.

[0019] Embodiment E11 of the present disclosure relates to aconitic acid for use in a method according to any one of embodiments E1 to E10, wherein the subject is an animal, preferably a human, a domestic bird, or a pig.

[0020] Embodiment E12 of the present disclosure relates to aconitic acid for use in a method according to any one of embodiments E1 to E11, wherein aconitic acid is used alone or in combination with one or more active substances selected from the group consisting of antiviral agents, antibiotics, and / or analgesics.

[0021] Embodiment E13 of the present disclosure relates to aconitic acid for use in a method according to any one of embodiments E1 to E12, wherein the treatment is for preventing the development of a pulmonary viral infection, the method comprising administering aconitic acid in a subject not infected by a respiratory virus.

[0022] Embodiment E14 of the present disclosure relates to aconitic acid for use in a method according to any one of embodiments E1-E13, wherein the composition is administered intrapulmonary, nasally, orally, enterally, intravenously, intramuscularly, and subcutaneously.

[0023] Another aspect of the present disclosure relates to a pharmaceutical composition comprising aconitic acid or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, for use in a method as defined in any one of embodiments E1 to E14. Detailed Description of the Invention

[0025] The inventors have now demonstrated that, surprisingly, aconitic acid exerts favorable immunomodulatory activity in lung cells infected with influenza virus and, even more surprisingly, also exerts a powerful antiviral activity. In fact, the inventors have demonstrated that aconitic acid has immunomodulatory (particularly downregulating) properties that are sufficiently effective to block the inflammatory cascade during influenza and coronavirus infection.

[0026] They also confirmed that aconitic acid strongly inhibited the influenza virus polymerase and prevented the expression of viral mRNA and protein synthesis, thereby preventing influenza virus replication. This antiviral effect was observed in multiple influenza virus types (e.g., type A, type B, ...) and subtypes (e.g., H3N2, H1N1, ...). The inventors therefore demonstrated that aconitic acid has a direct inhibitory effect on two components of the pathophysiology of influenza virus infection: the intrinsic pathogenicity of the virus and the hyperinflammatory immune response associated with influenza virus infection.

[0027] Notably, the inventors have demonstrated that aconitic acid treatment can reduce IL-6 production in a dose-dependent manner in bronchial epithelial cells stimulated with various inflammatory agonists. Without wishing to be bound by any theory, the inventors believe that these findings strongly suggest that aconitic acid has potent intrinsic downregulatory properties and has therapeutic effects on a variety of viral infections.

[0028] Importantly, the inventors have demonstrated the protective effects of aconitic acid not only in vitro but also in vivo in a murine model and in isolated human lungs.

[0029] Thus, one aspect of the present disclosure relates to aconitic acid, or a pharmaceutically acceptable salt thereof, for use in a method of treating a viral lung infection and / or an adverse immune response to a viral lung infection, the method comprising administering to a subject in need thereof an effective amount of aconitic acid, or a pharmaceutically acceptable salt thereof.

[0030] Aconitic acid or a pharmaceutically acceptable salt thereof

[0031] The aconitic acid may be cis-aconitic acid or trans-aconitic acid.

[0032] The aconitic acid is preferably cis-aconitic acid.

[0033] In the present disclosure, the term "pharmaceutically acceptable salt" refers to salts of aconitic acid that have little or no adverse toxicological effects. The counterion can be, for example, selected from the group consisting of aluminum, arginine, benzathine penicillin, calcium, chloroprocaine, choline, diethanolamine, ethanolamine, ethylenediamine, lysine, magnesium, histidine, lithium, meglumine, potassium, procaine, sodium, triethylamine, and zinc, preferably selected from the group consisting of sodium and potassium.

[0034] In some embodiments, the pharmaceutically acceptable salt of aconitic acid is the sodium salt of cis-aconitic acid or trans-aconitic acid.

[0035] In some embodiments, the pharmaceutically acceptable salt of aconitic acid is the potassium salt of cis-aconitic acid or trans-aconitic acid.

[0036] Hereinafter, the expressions "aconitic acid" and "aconitic acid or a pharmaceutically acceptable salt thereof" are used interchangeably.

[0037] Viral lung infections

[0038] As used in this disclosure, the term "pulmonary viral infection" refers to a condition characterized by: (i) the proliferation of one or more viruses in the lungs of a subject, and / or (ii) one or more adverse immune responses associated with or induced by the viral infection in the lungs of a subject. The lung is a specific organ within the respiratory system. Humans typically have two lungs. According to this disclosure, a pulmonary viral infection may affect other organs of the respiratory system selected from the group consisting of the pharynx, larynx, or trachea.

[0039] Adverse immune response

[0040] As used herein, the expression "adverse immune response" or "host adverse immune response" or "subject adverse immune response" refers to a syndrome of physiological, pathological, and / or biochemical abnormalities triggered by viral infection. The expression "adverse immune response" also refers to organ dysfunction caused by a dysregulated host response to infection, which can be defined as "sepsis."

[0041] Clinical manifestations of an adverse immune response to a viral infection of the lungs typically include one or more of the following: abnormal body temperature (typically characterized by a body temperature greater than 38°C or less than 36°C), dyspnea, tachypnea (typically characterized by a respiratory rate greater than 20 / min), bronchitis, pneumonia, sepsis-induced organ dysfunction, and acute respiratory distress syndrome (ARDS).

[0042] Biological manifestations usually include one or more of the following: abnormal white blood cell count (typically characterized by a WBC count greater than 12,000 / mm 3 or less than 4000 / mm 3 or immature granulocytes greater than 10%), excessive release of systemic cytokines such as tumor necrosis factor (TNF), interleukin-1 (IL-1), interleukin-6 (IL-6), and interleukin-8 (IL-8), an exaggerated inflammatory immune response (such as a cytokine storm or cytokine release syndrome), coagulopathy (consumption of clotting factors, fibrinogen, and platelets), and hypoxemia assessed by arterial blood gas analysis (typically characterized by a PaO2 / FiO2 ratio less than or equal to 300 mmHg). Imaging findings typically include chest radiographs and / or CT scans showing pulmonary infiltrates.

[0043] In some embodiments, the pulmonary viral infection induces or is capable of inducing an adverse immune response in the subject. In some embodiments, the adverse immune response to the pulmonary viral infection is an excessive inflammatory immune response, dyspnea, shortness of breath, pneumonia (particularly acute pneumonia), exacerbation of chronic respiratory diseases (such as asthma or chronic obstructive pulmonary disease), sepsis, septic shock, cytokine storm or acute respiratory distress syndrome (ARDS).

[0044] In some embodiments, the adverse immune response to a viral infection of the lung is pulmonary hypertension or hypotension.

[0045] In some embodiments, the adverse immune response to the viral infection in the lungs is multiple organ failure. Multiple organ failure can include heart failure, liver failure, lung failure, kidney failure, or gastrointestinal (GI) system failure.

[0046] In some embodiments, the viral infection of the lungs and / or the adverse immune response to the viral infection of the lungs causes a viral respiratory illness in the subject, commonly known as influenza (also known as "flu"), coronavirus disease 2019 (COVID-19), severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), or the common cold.

[0047] In some embodiments, the viral infection of the lung and / or adverse immune response to the viral infection of the lung favors the development of bacterial lung co-infection or superinfection.

[0048] Immunomodulatory and antiviral properties

[0049] In some embodiments, aconitic acid for use according to the present disclosure has immunomodulatory and / or antiviral properties, preferably wherein:

[0050] -The immunomodulatory properties of aconitic acid include reducing or suppressing the inflammatory response to viral lung infections;

[0051] -The antiviral properties of aconitic acid include reducing or inhibiting viral replication.

[0052] Immunomodulatory properties

[0053] In some embodiments, aconitic acid has immunomodulatory properties.

[0054] In the present disclosure, the term "immunomodulatory drug" or "immunomodulation" refers to a substance that inhibits or reduces one or more adverse immune responses to viral lung disease in a subject, particularly an excessive inflammatory immune response to viral infection of the lungs. For example, the ability of a substance to reduce an inflammatory response can be confirmed by measuring a decrease in the expression of some components of the inflammatory cascade (such as IL-6, IL-8, or tumor necrosis factor-α (TNF-α)) in the presence of the substance.

[0055] In some embodiments, the immunomodulatory properties of aconitic acid include inhibiting or reducing viral-induced immune inflammatory responses in the lungs.

[0056] In some embodiments, aconitic acid for its use according to the present disclosure is used as an anti-inflammatory drug for treating viral infections of the lungs or pneumonia, in particular for treating or preventing adverse immune responses to viral infections of the lungs.

[0057] In some embodiments, aconitic acid for its use according to the present disclosure is used to treat an adverse immune response to a pulmonary viral infection, the adverse immune response being selected from an excessive inflammatory immune response selected from an acute exacerbation of a chronic respiratory disease (such as asthma or chronic obstructive pulmonary disease), pneumonia, sepsis, septic shock, cytokine storm and acute respiratory distress syndrome, particularly in moderate to advanced cases of the pulmonary infection, in particular, wherein the pulmonary infection is a respiratory viral infection caused by influenza virus or a coronavirus (such as SARS-CoV-2).

[0058] Antiviral properties

[0059] In some embodiments, aconitic acid has antiviral properties.

[0060] In the present disclosure, the term "antiviral drug" or "antiviral" refers to a substance that inhibits or reduces viral replication in a subject.

[0061] In some embodiments, the antiviral properties of aconitic acid include inhibition of viral replication.

[0062] In some embodiments, aconitic acid for its use according to the present disclosure acts as an antiviral drug to treat viral infections of the lungs, more particularly, by disrupting the life cycle of respiratory viruses.

[0063] In some preferred embodiments, aconitic acid for its use according to the present disclosure acts as an immunomodulator and as an antiviral agent to treat respiratory viral infections.

[0064] respiratory viruses

[0065] In this disclosure, the term "respiratory virus" refers to a virus that has a tropism for airway cells, particularly the lungs of a subject, and is capable of eliciting an adverse immune response as described herein.

[0066] In some embodiments, the respiratory virus is selected from influenza virus, coronavirus, respiratory syncytial virus, parainfluenza virus, metapneumovirus, rhinovirus, adenovirus, varicella zoster virus, cytomegalovirus, paramyxovirus (such as Nipah virus), and bocavirus.

[0067] influenza virus

[0068] In some embodiments, the respiratory virus is influenza virus.

[0069] In some embodiments, the influenza virus is influenza A virus, influenza B virus, influenza C virus, or influenza D virus.

[0070] In some embodiments, the influenza A virus is serotype H1N1, H1N2, H2N2, H2N3, H3N1, H3N2, H3N8, H5N1, H5N2, H5N3, H5N6, H5N8, H5N9, H6N1, H6N2, H7N1, H7N2, H7N3, H7N4, H7N7, H7N9, H9N2, or H10N7.

[0071] In some embodiments, the influenza virus is an influenza B virus.

[0072] In some embodiments, the influenza B virus is a Victoria or Yamagata serotype.

[0073] In some embodiments, the influenza virus is an influenza virus that commonly causes influenza in humans, such as an H1N1, H2N2, H3N2, H5N1, H7N7, H1N2, H9N2, H7N2, H7N3, or H10N7 influenza virus.

[0074] In some embodiments, the influenza virus is an influenza virus that commonly causes avian influenza, such as an H5N1 or H7N9 influenza virus.

[0075] In some embodiments, the influenza virus is an influenza virus that typically causes swine or pig influenza, such as an H1N1, H1N2, H2N1, H3N1, H3N2, H2N3, or influenza C virus.

[0076] In some embodiments, the influenza virus is an influenza virus that commonly causes equine influenza, such as an H7N7 or H3N8 influenza virus.

[0077] In some embodiments, the influenza virus is an influenza virus that is typically canine influenza, such as an H3N8 influenza virus.

[0078] In some embodiments, aconitic acid for its use according to the present disclosure reduces or inhibits influenza virus replication and immune inflammatory responses to influenza virus infection.

[0079] Coronavirus

[0080] In some embodiments, the respiratory virus is a coronavirus of the Orthocoronavirinae subfamily of coronaviruses.

[0081] In some embodiments, the coronavirus is selected from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), severe acute respiratory syndrome coronavirus (SARS-CoV) or Middle East respiratory syndrome coronavirus (MERS-CoV) or beta-coronavirus.

[0082] In some embodiments, the coronavirus is SARS-CoV-2.

[0083] In some embodiments, aconitic acid for its use according to the present disclosure at least reduces or inhibits the immune inflammatory response to coronavirus infection, and optionally also reduces the immune inflammatory response to coronavirus infection.

[0084] Treatment

[0085] As used herein, the term "treatment" or "therapy" refers to any action that can alleviate or suppress the symptoms associated with a pathological condition, including curative treatment and preventive treatment of a disease.

[0086] Curative treatment is defined as treatment that results in a cure or treatment that alleviates, improves and / or eliminates, reduces and / or stabilizes symptoms of a disease or the pain it causes. The term "curative treatment" may refer to one or more of the following: (1) inhibiting the disease; for example, inhibiting the disease, condition or disorder of an individual who is experiencing or exhibiting the pathology or symptoms of the disease, condition or disorder (i.e., preventing further development of the pathology and / or symptomology); and (2) ameliorating the disease; for example, ameliorating the disease, condition or disorder of an individual who is experiencing or exhibiting the pathology or symptoms of the disease, condition or disorder (i.e., reversing the pathology and / or symptoms), such as reducing the severity of the disease or reducing or alleviating one or more symptoms of the disease. In particular, with respect to the treatment of pulmonary viral infections, the term "curative treatment" may refer to inhibiting viral infection and / or adverse immune responses associated with the viral infection. Preventive treatment includes treatment for the prevention of disease, as well as treatment that reduces and / or delays the incidence of the disease or the risk of its occurrence. The terms "improve" and "reduce" include but do not require complete recovery or complete prevention. The term "prophylactic treatment" can refer to preventing one or more of the following: (i.e., preventing the disease, condition, or disorder in an individual at risk of experiencing or exhibiting the pathology or symptoms of the disease, condition, or disorder (i.e., preventing the development of the pathology and / or symptoms); and (2) reducing and / or delaying the incidence of the disease in an individual who is experiencing or exhibiting the pathology or symptoms of the disease, condition, or disorder (i.e., reversing the pathology and / or symptoms), such as reducing the severity of the disease or reducing or alleviating one or more symptoms of the disease.

[0087] In the context of the present disclosure, the term "treatment" may preferably refer to the curative or prophylactic treatment of a viral lung infection as described herein, or the curative or prophylactic treatment of a viral lung infection and / or an adverse immune response to a viral lung infection. In particular, with respect to the methods of the present disclosure, the term "prophylactic treatment" may refer to the prevention of a viral infection and / or an adverse immune response to said viral infection.

[0088] The present disclosure also provides use of aconitic acid or a pharmaceutically acceptable salt thereof as described herein, optionally in combination with a pharmaceutically acceptable carrier and / or one or more active substances as described herein, in the preparation of a medicament for treating a viral lung infection as described herein and / or an adverse immune response to the viral lung infection.

[0089] In another embodiment, the present disclosure provides a method for treating viral lung infection and / or adverse immune responses associated with viral lung infection, comprising administering to a subject in need thereof an effective amount of aconitic acid or a pharmaceutically acceptable salt thereof, optionally in combination with a pharmaceutically acceptable carrier and / or one or more active substances described herein.

[0090] dose

[0091] The aconitic acid used according to the present disclosure is administered to the patient with an effective dose. As used herein, the term "effective" dose or "therapeutically effective dose" refers to an amount observed to treat or prevent active lung infection, such as an amount observed to suppress or reduce viral infection and / or an adverse immune response to lung viral infection. The amount of aconitic acid to be administered and the duration of treatment are assessed by those skilled in the art according to standard criteria, such as the physiological condition of the experimenter to be treated, the lung infection to be treated or the nature of the lung inflammatory reaction and the route of administration used. The aconitic acid used according to the present disclosure can be administered in the form of a single dose or multiple doses.

[0092] In some embodiments, aconitic acid for use thereof according to the present disclosure is administered to the subject in a therapeutically effective dose, e.g., to a concentration dose of about 0.3 to about 10 mM at the treatment site (e.g., respiratory tract). However, this exemplary dose can vary within a wide range and is suitable for the individual circumstances of each individual case.

[0093] Patient selection

[0094] The subject or patient to be treated is an animal, preferably a mammal.

[0095] According to a preferred embodiment, the subject to be treated is a human, preferably an adult. In some embodiments, the subject is an elderly patient, particularly older than 50, 60, 70, 80, 90 years old, more particularly older than 65 years old. In some embodiments, the subject is a child, particularly a child younger than 2, 5, 7 or 10 years old.

[0096] In some embodiments, the subject to be treated is a subject susceptible to pulmonary viral infection. As used herein, "susceptible" refers to an individual who may have difficulty protecting themselves and is therefore at a higher risk of suffering from pulmonary viral infection and its complications. Subjects susceptible to pulmonary viral infection include, but are not limited to, elderly patients, particularly elderly patients 65 years of age or older, children, particularly children 2 years of age or younger, and pregnant women.

[0097] According to one embodiment, the subject to be treated is a non-human animal, preferably a domesticated animal selected from the group consisting of birds, dogs, cats, horses, cows, sheep, pigs and non-human primates.

[0098] The domesticated poultry is, for example, chicken, duck, goose or turkey.

[0099] In some embodiments, the subject is an animal, preferably a human or domesticated bird.

[0100] Subjects to be treated

[0101] Subjects to be treated

[0102] The methods of the present disclosure can be administered to a subject before or after a viral lung infection, before or after an adverse immune response to the viral infection occurs.

[0103] Thus, the methods of the present disclosure can be applied to infected or uninfected subjects, ie, subjects with varying degrees of severity of viral infection as described herein.

[0104] In some embodiments, the subject is at risk for developing an adverse immune response to a viral lung infection, such as developing pneumonia.

[0105] In some embodiments, the subject at risk for developing an adverse immune response to a viral lung infection is an elderly human patient, preferably a human patient older than 50, 60, 70, 80, 90 years of age.

[0106] Subject infection

[0107] In some embodiments, the methods of the present disclosure are used in infected subjects, ie, subjects with a viral lung infection.

[0108] In some embodiments, the methods of the present disclosure are used to treat viral lung infections.

[0109] In some embodiments, the methods of the present disclosure are used to prevent a subject from developing an adverse immune response to a viral lung infection.

[0110] In some embodiments, the methods of the present disclosure are used to prevent a subject from developing an excessive inflammatory immune response, dyspnea, shortness of breath, pneumonia (particularly acute pneumonia), exacerbation of chronic respiratory diseases (such as asthma or chronic obstructive pulmonary disease), sepsis, septic shock, cytokine storm, or acute respiratory distress syndrome (ARDS).

[0111] In some embodiments, the methods of the present disclosure are used to prevent escalation of a viral infection in the lungs into an adverse immune response.

[0112] The subject is infected and develops an adverse immune response to the infection

[0113] In some embodiments, the methods of the present disclosure are used to treat an adverse immune response to a viral lung infection.

[0114] Subjects not infected

[0115] In some embodiments, the methods of the present disclosure are used in subjects who are not infected with a viral infection.

[0116] In some embodiments, the methods of the present disclosure are used to treat viral lung infections and / or adverse immune responses to viral lung infections.

[0117] Severity / stage of viral infection

[0118] The method can be applied to various stages of lung infection.

[0119] The inventors have shown that, surprisingly, aconitic acid is effective even when administered some time after infection (4 days), whereas oseltamivir is not.

[0120] This result strongly supports the use of aconitic acid in the treatment of advanced lung infections. As used herein, "advanced lung infection" refers to patients with lung infections who require oxygen therapy. This result is particularly beneficial because current antiviral treatments, such as It is ineffective when used alone for late-stage infections.

[0121] In some embodiments, treatments according to the present disclosure are used to treat a subject in an advanced stage of a viral infection in the lungs.

[0122] In these embodiments, the aconitic acid for use thereof according to the present disclosure is preferably administered to the subject 4-14 days after infection, preferably 5-11 days after infection, and more preferably 7-11 days after infection. In some embodiments, the aconitic acid for use thereof according to the present disclosure is preferably administered to the subject 4-11 days after infection, and more preferably 4-7 days after infection.

[0123] In some other embodiments, the treatment according to the present disclosure is used to treat a subject in the early stages of a lung infection.

[0124] In these embodiments, aconitic acid for its use according to the present disclosure is preferably administered to the subject immediately after infection, 1 hour, 6 hours, 12 hours, 1 day, 2 days, 3 days, 8-24 hours after infection, more preferably 10-20 hours after infection, even more preferably 12-16 hours after infection.

[0125] In some embodiments, the methods according to the present disclosure are used to prevent a pulmonary viral infection from escalating into an adverse immune response associated with a viral lung infection in a subject having the pulmonary viral infection.

[0126] Without wishing to be bound by any theory, the inventors believe that aconitic acid may be most effective when administered as soon as possible. However, as mentioned above, the inventors have found that, quite surprisingly, aconitic acid is still effective 4 days after infection, which from a practical and / or clinical perspective is significantly better than existing antiviral treatments such as (oseltamivir) has significant advantages.

[0127] The methods of the present disclosure are applicable to subjects at various stages of viral infection, ie, subjects exhibiting varying degrees of severity of viral lung infection and / or adverse immune responses to viral infection of the lungs.

[0128] The severity of the subject's condition can be assessed by scoring according to the following ordinal scale, where increasing numbers indicate increasing severity:

[0129]

[0130] In some embodiments, the subject's severity assessment is non-infected / ambulatory.

[0131] In some embodiments, the subject's severity assessment is hospitalization but not requiring ventilatory support.

[0132] In some embodiments, the subject's severity assessment is hospitalization with ventilatory support.

[0133] Combination therapy

[0134] The aconitic acid used according to the present disclosure can be used as the sole active ingredient or in combination with one or more active substances. The aconitic acid and the active substance can be administered simultaneously or sequentially.

[0135] In the present disclosure, the term "administering" means administering a therapeutic agent alone or in combination with another therapeutic agent.

[0136] According to one embodiment, the aconitic acid is administered in combination with one or more active substances selected from the group consisting of antiviral agents, antibiotics and / or analgesics.

[0137] In some embodiments, aconitic acid is used in combination with one or more antiviral agents, particularly antiviral agents that are standard of care for respiratory viral infections, such as neuraminidase inhibitors (e.g., oseltamivir). Zanamivir Peramivir Favipiravir, remdesivir, ribavirin, interferon α2a or 2b, monopiravir, sotovir, casprevir / idezumab, mabaloxavir ).

[0138] In some embodiments, aconitic acid is used in combination with one or more antibiotics used to treat bacterial co-infections, particularly antibiotics used in standard antibiotic therapy, such as penicillins, cephalosporins, fluoroquinolones, aminoglycosides, carbapenems, and macrolides.

[0139] In some embodiments, aconitic acid is used in combination with one or more analgesics, such as acetaminophen (paracetamol), nefopam, tramadol, and opioids.

[0140] Route of administration

[0141] Aconitic acid used in accordance with the present disclosure can be administered by any known route of administration, including intrapulmonary, systemic (parenteral, intravenous, etc.), oral, rectal, topical, or subcutaneous.

[0142] In some preferred embodiments, aconitic acid for use in accordance with the present disclosure is administered intrapulmonary, nasally, orally, enterally, intravenously, intramuscularly, and subcutaneously.

[0143] In some preferred embodiments, aconitic acid for use in accordance with the present disclosure is administered intrapulmonary.

[0144] As used herein, "intrapulmonary" refers to a route of administration that can deliver aconitic acid to the lungs and / or bronchi, where it is particularly concentrated in the alveoli and / or bronchial epithelium.

[0145] In some embodiments, aconitic acid for its use according to the present disclosure is preferably administered as an aerosol of a powder or an aqueous solution or suspension, in particular using a nebulizer or dry powder inhaler.

[0146] In some embodiments, aconitic acid for its use according to the present disclosure is administered as an aerosol of a powder or an aqueous solution or suspension in a subject who is breathing spontaneously or receiving supplemental oxygen (including hyperoxia equipment) or assisted by mechanical ventilation (non-invasive or invasive).

[0147] As used in this disclosure, the term "aerosol" refers to a dispersion of solid particles or liquid droplets in a gas suitable for targeting the lower respiratory tract, preferably the lungs. A nebulizer is defined as a device capable of aerosolizing a liquid material (solution or dispersion) in the form of inhalable droplets. A nebulizer can administer the composition via a mask or nozzle placed in the mouth and / or nose of a subject.

[0148] Administration regimen

[0149] In some embodiments, aconitic acid for use thereof according to the present disclosure and optionally one or more other active substances described herein are administered simultaneously, separately, or sequentially in a single dose or in a divided dose regimen.

[0150] In some embodiments, aconitic acid for use thereof according to the present disclosure and optionally one or more additional active substances as described herein are administered to a subject in a divided dose regimen.

[0151] In some embodiments, a split dose regimen as described herein comprises 2-10 split doses.

[0152] In a preferred embodiment, the split administration regimen described herein is administered once a day or once every two days.

[0153] In one embodiment, the divided doses described herein are administered with a time interval of 4 hours to 48 hours, preferably 4 hours to 12 hours, more preferably 4 hours to 10 hours, such as 6 hours between the two divided doses.

[0154] Pharmaceutical composition

[0155] When used as a medicament, the aconitic acid provided herein for its use can be administered in the form of a pharmaceutical composition.

[0156] Pharmaceutical compositions for use thereof according to the present disclosure typically comprise aconitic acid and a pharmaceutically acceptable carrier for use as described herein.

[0157] In the context of this disclosure, the term "pharmaceutically acceptable carrier" refers to substances conventionally used in combination with active ingredients for the preparation of medicaments, such as excipients, vehicles, adjuvants, buffers, etc. The choice of such carriers essentially depends on the envisaged route of administration. Pharmaceutically acceptable carriers include diluents (fillers, expanders, such as lactose, microcrystalline cellulose), disintegrants (e.g., sodium starch glycolate, croscarmellose sodium), binders (e.g., PVP, HPMC), lubricants (e.g., magnesium stearate), glidants (e.g., colloidal SiO2), solvents / co-solvents (e.g., aqueous vehicles, propylene glycol, glycerol), buffers (e.g., citrate, gluconate, lactate), preservatives (e.g., sodium benzoate, parabens (Me, Pr and Bu), BKC), antioxidants (e.g., BHT, BHA, ascorbic acid), wetting agents (e.g., polysorbates, sorbitan esters), thickeners (e.g., methylcellulose or hydroxyethylcellulose), sweeteners (e.g., sorbitol, saccharin, aspartame, acesulfame potassium), and humectants (e.g., propylene glycol, ethylene glycol, glycerol, sorbitol). Other suitable pharmaceutically acceptable carriers are described in particular in Remington's Pharmaceutical Sciences, 15 th Ed., Mack Publishing Co., New Jersey (1991) and Bauer et ak, Pharmazeutische Technologic, 5 thEd., Govi-Verlag Frankfurt (1997). Those skilled in the art can easily select a suitable pharmaceutically acceptable carrier depending on, for example, the formulation and administration route of the pharmaceutical composition.

[0158] In some embodiments, the aconitic acid can be in an encapsulated form, for example, by being introduced into a microsphere or microcapsule, which is a reservoir consisting of an active ingredient core surrounded by a coating material membrane. The polymer forming the coating material can be of natural origin (gelatin, chitosan, etc.), semi-synthetic origin (cellulose derivatives, etc.) or synthetic origin, such as commonly used lactic acid and glycolic acid copolymers. The compounds of the present disclosure can also be encapsulated in nanoparticles, which are based on biodegradable polymers or colloidal systems that can retain one or more active molecules by sealing and / or adsorption, and have a size of 10-1000 nm.

[0159] The pharmaceutical composition according to the present disclosure preferably comprises aconitic acid in an amount of 5 μg-1000 mg, preferably 1-500 mg, preferably 5-100 mg.

[0160] The weight ratio of the compound according to the present disclosure and the pharmaceutically acceptable carrier is 5 / 95-95 / 5, preferably 20 / 80-80 / 20.

[0161] Pharmaceutical compositions according to the present disclosure can, for example, be formulated as tablets, capsules, granules, powders, sachets, resolvable powders, dry powder inhalers, and / or chewable tablets. Such solid formulations can include appropriate amounts of excipients and other ingredients. Such solid formulations can contain, for example, cellulose, cellulose microcrystals, povidone, magnesium stearate, etc.

[0162] In some preferred embodiments, the pharmaceutical composition is for inhalation. In this case, the dosage can be reduced because the drug is directly administered to the site of action, i.e., the lungs.

[0163] The present disclosure also relates to a method for treating a lung infection, comprising administering to a subject an effective amount of aconitic acid and / or a pharmaceutical composition containing aconitic acid.

[0164] The presently disclosed subject matter also relates to the use of aconitic acid in the preparation of a pharmaceutical composition for treating lung infection.

[0165] Other aspects and advantages of the present disclosure will appear from a reading of the following examples, which are to be considered as non-limiting illustrations. BRIEF DESCRIPTION OF THE DRAWINGS

[0166] Figure 1. Evaluation of anti-influenza virus activity of metabolites derived from glycolysis or the TCA cycle.

[0167] ( Figure 1a ) Detection of metabolites derived from glycolysis or the TCA cycle. Bronchial epithelial (BEAS-2B) cells were infected with A / Scotland / 20 / 74 (H3N2) virus at an MOI of 1 (IAV) or without virus (PBS) for 4 hours and treated with 3.4 mM of the following TCA cycle or glycolysis metabolites (Metabolites) or without treatment (PBS) for 16 hours: cis-aconitic acid (CA), itaconic acid (Ita), oxaloacetic acid (Oxa), isocitrate (Isoc), citrate (Cit), fumaric acid (Fum), pyruvate (Pyr), or glucose (Glc). (Figure 1b) Viral particle production assessed by neuraminidase activity assay. (Figure 1c) hIL-6 production in cell supernatants was measured by ELISA. BEAS-2B cells were transfected with cis-aconitic acid decarboxylase (CAD) or control (scramble) siRNA. 48 hours after transfection, cells were infected with A / Scotland / 20 / 74 (H3N2) virus (IAV) or without virus at an MOI of 1 for 4 hours and treated with 3.4 mM cis-aconitic acid (CA) or without it for 16 hours. (Figure 1d) Gene knockdown was assessed by RT-qPCR. (Figure 1e, Figure 1f) The production of viral particles was assessed by plaque assay (Figure 1e) and neuraminidase activity assay (f). (Figure 1g) The production of hIL-6 in cell supernatants was measured by ELISA. All data are expressed as mean ± SEM and are the accumulation of 7 (Figure 1b) or 4 (Figure 1c and Figure 1e-g) independent experiments. Statistical analysis was performed using the Kruskal-Wallis test and Dunn's multiple comparison test ( Figure 1a -g). Trans-aconitic acid (TA) also exhibited similar antiviral and anti-inflammatory activities, indicating that both isomers of aconitic acid have anti-influenza properties ( Figure 1h-i ).

[0168] Figure 2: The anti-influenza virus properties of cis-aconitic acid are mediated by inhibition of viral polymerase activity.

[0169] ( Figure 2a ) Left: Human bronchial epithelial BEAS-2B cells were infected with influenza A / Scotland / 20 / 74 (H3N2) virus at an MOI of 1 or 5 (a) for 4 hours, then washed and treated with 3.4 mM or the indicated concentrations of cis-aconitic acid (CA) or not treated with it (PBS). Right: Representative images of 2 different transmission electron microscopy (TEM) analyses (top) or 2 scanning electron microscopy (SEM) experiments (bottom). Arrows indicate IAV particles budding from bronchial epithelial cells 20 h after infection (scale bar: 1 μm). ( Figure 2b-e) 8 hours after infection, viral protein expression and trafficking were analyzed by confocal microscopy and Western blotting to detect viral NP, NS1, and PA proteins using specific antibodies. Figure 2b ) Viral proteins are shown in grey (scale bar = 20 μM). Figure 2c ) The numerical raw integer density (RawIntDen, which is the sum of all pixel values in the ROI (region of interest)) was further measured and normalized to the mean of the IAV conditions for each experiment. Figure 2d 、 Figure 2e ) Viral protein expression was assessed by Western blotting. Relative protein quantification was obtained by normalizing to the mean of the "IAV condition" samples; (β-actin was used as a loading control) (Figure 2f). Six hours after infection, IAV transcription was assessed by RT-qPCR to quantify M1 viral mRNA. (Figure 2g) Minigenome analysis was performed on HEK-293T cells to determine the effect of cis-aconitic acid on viral polymerase activity. 293T cells were transfected with pRF483-PA-RT, pRF483-PB2-RT, pRF483-NP-RT, pRF483-PB1-RT and the reporter plasmid pPolI-WSN-NA-firefly luciferase. 20 hours after transfection, cells were treated with 0, 1.2, 2.3 or 3.4 mM cis-aconitic acid (CA). Luciferase activity was measured 48 hours after transfection. Results are expressed as 3 ( Figure 2a 、 2b , 2c), 4 ( Figure 2d 、 2e , 2f) or the mean ± SEM of 5 independent experiments (Fig. 2f). Kruskal-Wallis test with Dunn's multiple comparison test (Fig. 2g), Mann-Whitney test ( Figure 2c ) or Wilcoxon matched-pairs signed-rank test ( Figure 2e , 2f) for statistical analysis.

[0170] Figure 3. Cis-aconitic acid blocks the proliferation of different influenza virus types and subtypes.

[0171] Human bronchial epithelial BEAS-2B cells were infected with influenza A / pandemic / 2009 H1N1 (H1N1p) strain (Figure 3a), influenza A / Puerto Rico / 8 / 1934 H1N1 (PR8) virus (Figure 3b), or influenza B / Yamagata (B / Paris / 234 / 2013) virus (IBV) (Figure 3c). Four hours after infection, cells were washed and treated with or without 3.4 mM cis-aconitic acid (CA) for 16 hours. Plaque-forming unit assays were used to quantify the yield of infectious virus particles in cell supernatants. Data are presented as mean ± SEM of three (Figure 3b) or four (Figures 3a and 3c) independent experiments. Statistical analysis was performed using a paired t-test for proportions.

[0172] Figure 4. Cis-aconitic acid reduces proinflammatory responses and signaling in lung epithelial cells.

[0173] (Figure 4a-b) Human bronchial epithelial BEAS-2B cells were infected with influenza A / Scotland / 20 / 74 (H3N2) virus at an MOI of 1 for 4 hours or were not infected with the virus and subsequently treated with 3.4mM cis-aconitic acid (CA) for 20 hours or were left untreated. (Figure 4a) Phosphorylated forms (P) of ERK1 / 2, AKT, and p65 proteins were detected using Western blotting (β-actin was used as a loading control). (Figure 4b) Levels of 6 immune mediators in cell supernatants were determined using specific protein arrays. Green bars represent IAV-induced secretion of mediators (expressed as fold change relative to non-infected (NI) conditions). Blue bars represent secretion of mediators induced by IAV+CA (expressed as fold change relative to NI conditions). ( Figure 4c BEAS-2B cells were transfected with different plasmids expressing NF-κB, IL-8, ISG54, or ISG56 luciferase. Twenty-four hours after transfection, cells were stimulated or treated with 2 μg / mL Poly(I:C) (PIC) for 4 hours, or left unstimulated (gray) and treated with 3.4 mM cis-aconitic acid (CA) for 16 hours. Luciferase levels were measured 24 hours after stimulation. Figure 4d ) BEAS-2B cells were stimulated with IAV at an MOI of 1 (IAV), 2 μg / mL of Poly(I:C) (PIC), 50 nM phorbol 12-myristate 13-acetate (PMA), or 20 ng / mL tumor necrosis factor α (TNFα) for 4 hours, or left unstimulated (PBS) and treated with increasing doses of cis-aconitic acid (CA) for 16 hours. IL-6 levels in cell supernatants were determined by ELISA. Data are presented as mean ± SEM of at least 5 independent experiments. Statistical analysis was performed, and individual differences were calculated for each comparison using a standard two-way ANOVA Turkey multiple comparison test ( Figure 4c ), Kruskal-Wallis( Figure 4d :IAV, PIC and PMA) or Friedman ( Figure 4d TNFα) with Dunn's multiple comparison test. (Figures 4e-h) Cis-aconitic acid exhibits antiviral and anti-inflammatory properties in primary human bronchial epithelial cells (PBECs). (Figures 4e-f) PBECs in liquid culture were infected with influenza A / Scotland / 20 / 74 (H3N2) virus at an MOI of 1 for 4 hours and subsequently treated with various concentrations of cis-aconitic acid (CA) for 44 hours or left untreated. At 48 hours post-infection, neuraminidase activity (Figure 4e) and hIL-6 (Figure 4f) were measured in cell supernatants to assess viral particle production and proinflammatory cytokine release, respectively. (Figures 4g-h) PBECs in liquid culture were incubated with or without poly(I:C) or PMA for 4 hours and subsequently treated with various concentrations of cis-aconitic acid (CA) for 20 hours or left untreated. IL-6 levels were further measured by ELISA. (Fig. 4i-j) PBECs in liquid culture were infected with A / Scotland / 20 / 74 (H3N2) at an MOI of 1 and then treated with 3.4 mM cis-aconitic acid (CA) or left untreated. SYTOXTM labeling was monitored for 18 hours. At 18 hours of infection, cell death was assessed using representative images (Fig. 4a) and quantification of labeling (Fig. 4b). PBECs were obtained from four independent patients. (Fig. 4k-l) Human precision-cut lung sections (PCLS) were performed on lung tissue collected from patients undergoing thoracic surgery (first panel, Fig. 4k). Lung explants were cut into 400 μm slices (second panel, Fig. 4k). Individualized PCLS were placed at the air-liquid interface (third and fourth panels, Fig. 4k). 2.10 4 PCLS were infected with pfu of influenza A / Scotland / 20 / 74 (H3N2) virus (IAV) and treated with or without 3.4 mM cis-aconitic acid 2 h post-infection. hIL-6 was measured in PCLS supernatants 48 h post-infection ( Figure 41 ). Data are presented as mean ± SEM. Statistical analysis was performed using the Kruskal-Wallis and Dunn multiple comparison tests.

[0174] Figure 5. Cis-aconitic acid (CA) reduces viral load, lung inflammation, and tissue damage in influenza virus-infected mice.

[0175] (Figures 5a-5d) Seven-week-old female C57Bl / 6 mice were infected intranasally with 200 pfu of A / Scotland / 20 / 74 (H3N2) virus (IAV) and treated intranasally with or without 0.6 mg of cis-aconitic acid (CA) 2 days after infection. Mice were euthanized 4 or 8 days after infection. On day 4 after infection, lungs and BAL fluid were collected to determine: (Figure 5a) viral load by plaque-forming units (n=11); (Figure 5b) levels of 50 mediators using a specific protein array on BAL fluid. On day 8 after infection, the number and activation of immune and inflammatory cells in BAL fluid (Figure 5c) and lungs (Figure 5d) were determined by flow cytometry. All data are expressed as mean ± SEM and are the cumulative of 3 (Figure 5a) or 2 (Figures 5c, 5d) independent experiments.

[0176] ( Figures 5e-5h Nine NF-κB transgenic Balb / c mice were infected with 300 PFU of A / Scotland / 20 / 74 (H3N2) IAV and treated intranasally with 0.6 mg CA 2 days post-infection. On day 8 post-infection, mice were anesthetized and intranasally instilled with luciferin (0.75 mg kg-1). (Figures 5g, 5h) Bioluminescence was measured using an IVIS imaging system. (Figures 5e, f) Lung sections were stained with hematoxylin-eosin, and tissue damage was further assessed by microscopy and inflammation scoring. Scale bar: x6 / 20 μM. Statistical analysis was performed using the Mann-Whitney test.

[0177] Figure 6. Cis-aconitic acid protects mice from influenza infection more effectively than oseltamivir in late-stage treatment.

[0178] Seven-week-old female C57Bl / 6 mice were intranasally infected with 200 pfu of A / Scotland / 20 / 74 (H3N2) virus (IAV) and treated (CA) or left untreated (PBS) with 30 mg / kg cis-aconitic acid (CA) or 20 mg / kg oseltamivir (Osel) (intranasally) 20 minutes after infection (Fig. 6a, 6b) or 2 days after infection (pi) (Fig. 6c, 6d). Animal survival (Fig. 6a, 6c) and weight loss (Fig. 6b, 6d) were monitored daily. All data are expressed as mean ± SEM and are the cumulative total of 1 (Fig. 6a, 6b) or 2 (Fig. 6c, 6d) independent experiments. Statistical analysis was performed using the Log-rank (Mantel-Cox) test. Figure 7. Cis-aconitic acid protects mice from influenza infection within a timeframe consistent with patient care.

[0179] Patients hospitalized with community-acquired pneumonia (CAP) were studied, and the results are shown in Tables 1 and 2 (see page 27). Influenza was of type A and type B strain. Figure 7a shows the time between symptom onset and first hospital admission. Each dot is a patient; the line represents the median. Seven-week-old female C57Bl / 6 mice were infected intranasally with 200 pfu of influenza A / Scotland / 20 / 74 (H3N2) virus (IAV) and treated (blue) or left untreated (green) with 30 mg / kg cis-aconitic acid (CA) or 20 mg / kg oseltamivir (Osel) (via intranasal route) on days 4 and 5 post-infection (Figure 7b). Animal survival was monitored daily (Figure 7b). Data are expressed as mean ± SEM and are cumulative of three independent experiments. Statistical analysis was performed using the Log-rank (Mantel-Cox) test.

[0180] Figure 8. Tolerance of bronchial epithelial cells to cis-aconitic acid exposure.

[0181] ( Figure 8a ) Human bronchial epithelial BEAS-2B cells were treated with PBS or 3.4 mM cis-aconitic acid (CA), itaconic acid (Ita), oxaloacetic acid (Oxa), isocitrate (IsoC), citric acid (Cit), fumaric acid (Fum), pyruvic acid (Pyr) or glucose (Glc) for 16 hours, and cytotoxicity was assessed by MTS assay. BEAS-2B cells were treated with 3.4 mM CA (CA+) for 6 or 24 hours, or not treated with it (CA-) (Figures 8b, 8c, 8d), or treated with 0, 1.2, 2.3 or 3.4 mM CA for 20 hours (Figure 8e). Cell proliferation (Figure 8b), mitochondrial markers (Figure 8c), ROS production (Figure 8d) and cell viability (Figure 8e) were analyzed by Ki67, mitotracker, DHR123 and Live Dead staining, respectively. ( Figure 8f ) Human primary bronchial epithelial cells (PBEC) were treated with 0.6, 3.4 or 5.7 mM CA for 16 h, and cytotoxicity was assessed by MTS assay. Data are presented as 4 (Fig. 8b-f) or 5 ( Figure 8a ) are means + / - SEM of independent experiments. Kruskal-Wallis test and Dunn's multiple comparison test were used ( Figure 8a Statistical analysis was performed using the Friedman test and Dunn's multiple comparison test (Figures 8b-8d).

[0182] Figure 9. In vivo tolerability and safety of cis-aconitic acid.

[0183] Seven-week-old female and male C57Bl / 6 mice were intranasally instilled with PBS or 0.6 mg of cis-aconitic acid (CA) every two days for 15 days. (Figure 9a) Weight loss was monitored (n=15). On day 15, mice were euthanized, and lungs, bronchoalveolar lavage (BAL) fluid, and serum were collected to determine: (Figure 9b) levels of 111 other mediators in BAL using a specific protein array; (Figure 9c) levels of ALAT activity in serum; (Figure 9d) alterations in the microbial community in mouse fecal pellets were assessed following genomic DNA extraction and 16S rRNA sequencing. Microbial community diversity was quantified using Aitchison distance values (a beta diversity metric). Each point represents the Aitchison distance between CA-treated and PBS-treated mice on days 0, 7, or 14. The overall fecal microbial composition did not differ over time between control and CA-treated mice (PERMANOVA; p<0.05). (Fig. 9e) The number and activation of immune cells in BAL were analyzed by flow cytometry, as well as complete blood counts (Fig. 9f). All data are expressed as mean ± SEM and are cumulative (Fig. 9a, 9c) or representative (Fig. 9b, 9e) of three independent experiments. Statistical analysis was performed using the Kruskal-Wallis test and Dunn's multiple comparison test.

[0184] Figure 10 .Cis-aconitic acid inhibits proinflammatory responses in SARS-CoV-2-infected epithelial cells.

[0185] ( Figure 10 a) Epithelial cells were infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at an MOI of 1. Four hours after infection, cells were washed and treated with or without 1 mg / mL cis-aconitic acid for 16 hours. IL-6 levels in cell supernatants were measured by ELISA. Figure 10 b: Vero epithelial cells were infected with the SARS-CoV-2 Wuhan strain. Figure 10 c: Human primary bronchial epithelial cells were infected with the Delta strain of SARS-CoV-2. Data are presented as mean ± SEM and are the cumulative total of three independent experiments. *For p < 0.05, ns: not significant.

[0186] Figure 11 .The effects of cis-aconitic acid on inflammation in the body.

[0187] 10 μg of lipopolysaccharide (LPS) was instilled into NF-κB transgenic Balb / c ( Figure 11 a, 11b) or C57Bl / 6( Figure 11c) Mice were treated intranasally with 0.6 mg of cis-aconitic acid (CA) 15 minutes after the stimulation. 24 hours after the instillation, the mice were anesthetized. In the NF-κB transgenic Balb / c animal group, luciferin (0.75 mg.kg -1 ), and bioluminescence was measured using the IVIS imaging system ( Figure 11 a, 11b). In the C57Bl / 6 mouse group, BAL fluid was collected to determine TNFα levels ( Figure 11 c). Bioluminescence was then measured using an IVIS system. All data are presented as mean ± SEM and represent one independent experiment. Statistical analysis was performed using the Mann-Whitney test.

[0188] Figure 12 .Confirmation of the protective effect of cis-aconitic acid in IAV-infected Balb / c mice.

[0189] To validate the findings observed in C57Bl / 6 mice within a different mouse species (see Figures 6a-d), we performed experiments on Balb / c mice. These (NF-κB transgenic) Balb / c animals were infected with 300 PFU of A / Scotland / 20 / 74 (H3N2) IAV and, 2 days after infection, were treated intranasally with 0.6 mg of cis-aconitic acid (CA). On day 8 post-infection, mice were anesthetized and luciferin (0.75 mg / kg) was instilled intranasally. -1 Animal survival was monitored daily ( Figure 12 a) and weight loss ( Figure 12 b). All data are expressed as mean ± SEM. Statistical analysis was performed using the Log-rank (Mantel-Cox) test.

[0190] Figure 13 The anti-influenza activity of cis-aconitic acid is independent of that of itaconic acid.

[0191] CAD-deficient and WTC57B1 / 6 mice were intranasally infected with 100 pfu of A / Scotland / 20 / 74 (H3N2) virus (IAV) and treated intranasally with 30 mg / kg cis-aconitic acid (CA) 20 minutes after infection or without treatment. Animal survival was monitored daily. All data are expressed as mean ± SEM. Statistical analysis was performed using the Log-rank (Mantel-Cox) test. DETAILED DESCRIPTION

[0192] Example 1: Mitochondrial-derived metabolite cis-aconitic acid prevents influenza through dual antiviral and anti-inflammatory activities die

[0193] 1.1 / Materials and Methods

[0194] VirusMouse-adapted influenza A virus / Scotland / 20 / 74 (H3N2) was generously provided by the group of Pr. Sylvie van der Werf (Pasteur Institute, Paris, France). Influenza A virus / PR / 8 / 34 (H1N1) strain was a kind gift from Dr. Georg Kochs (Freiburg University, Germany). The H1N1 pandemic IAV strain was kindly provided by Dr. Trottein (Center for Infection and Immunity of Lille) was generously provided. Influenza B virus / Paris / 234 / 2013 (Yamagata) lineage was obtained from the European Virus Archive Global (EVAg).

[0195] Cell culture . In vitro experiments were performed using human bronchial epithelial BEAS-2B cells, while plaque assays used Madin-Darby canine kidney (MDCK) cells, and minigenome assays used HEK-293T. These cells were cultured in F-12K medium (BEAS-2B), or in MEM supplemented with 10% FBS and 100U / mL penicillin, 100μg / mL streptomycin (HEK-293T and MDCK). All cells were mycoplasma-free. BEAS-2B cells were infected with IAV Scotland at an MOI=1 for 4 hours in medium without FBS (MOI=5 was used for TEM and SEM analysis). Cells were also stimulated in FBS medium containing 2μg / mL Poly(I:C), or in medium containing 2μg / mL PMA or 20ng / ml TNFα in a medium without FBS. Four hours after stimulation, the cells were washed with PBS and subsequently exposed to different concentrations of cis-aconitic acid or other test metabolites for 4 or 16 hours.

[0196] Neuraminidase (NA) assay This assay measures the release of the fluorescent product 4-methylumbelliferyl from the substrate 2'-(4-methylumbelliferyl)-α-DN-acetylneuraminic acid sodium salt hydrate (MU-NANA). 67 μL of cell supernatant was incubated with 33 μL of MU-NANA (50 μM) in a black 96-well microplate. Kinetics were immediately performed, and fluorescence was measured for 1 hour at Ex = 355 nm and Em = 460 nm.

[0197] Protein array and ELISACell supernatants or BAL were centrifuged at 500 g for 5 minutes and the supernatants were stored at -80° C. Protein arrays and DuoSet ELISA (human IL-6, and mouse MPO and ALT) were performed according to the manufacturer's instructions (R&D Systems or Clinisciences for ALT ELISA).

[0198] Minigenome assay . Minigenome studies were performed in 24-well plates. Briefly, 293T cells were transfected with 50ng pRF483-PA-RT, 50ng pRF483-PB2-RT, 100ng pRF483-NP-RT, 50ng pRF483-PB1-RT and 150ng reporter plasmid pPolI-WSN-NA-firefly luciferase, which contains the firefly luciferase ORF, flanked by the non-coding region of the NA fragment under the control of the human polymerase I promoter. As a negative control, 293T cells were transfected with the same plasmids, except for the PB1 expression plasmid. The method used Fugene HD transfection reagent (Promega) according to the manufacturer's instructions. 20 hours after transfection, the cells were treated with different concentrations of cis-aconitic acid. 48 hours after transfection, the cells were washed twice with phosphate-buffered saline (PBS) and lysed in 100 μl of lysis buffer provided by the Firefly Luciferase Assay System (Promega). Firefly luciferase activity was measured in 20 μl of cell extract using the firefly luciferase substrate provided by the above kit and a Centro luminometer (Berthold).

[0199] RNA isolation and RT-qPCR Lyse cells in 6-well plates with 350 μL of "RA1" buffer (included in the Macherey-Nagel RNA extraction kit) and 1 / 100 diluted β-mercaptoethanol. Total RNA was extracted from cells using an RNA kit, including a step for genomic DNA degradation with DNase. Nucleic acid samples were quantitatively analyzed using a Nanodrop 2000 UV-visible spectrophotometer. For each sample, single-stranded cDNA was synthesized from 500 ng of total RNA using a high-capacity cDNA reverse transcription kit (Applied Biosystem) using specific sense IAV M1 primers or random primers. Quantitative real-time PCR was performed using a LightCycler 480 instrument (Roche Diagnostics) to determine mRNA levels. PCR used 10 ng of reverse transcribed total RNA as a template, 10 μM (each) forward and reverse primers, and 10 μL Premix Ex Taq was performed in a final volume of 20 μL. Each reaction was performed in a white 96-well plate, with two replicates per well. The thermal reaction protocol consisted of an initial denaturation step at 95°C for 30 seconds, followed by 40 cycles of denaturation at 95°C for 5 seconds and primer annealing and extension at 60°C for 20 seconds (read at 83°C). For each amplified cDNA, a melting curve was plotted to check reaction specificity.

[0200] Western-blotting . Cells in 6-well plates were lysed with 150 μL RIPA buffer (150 mM sodium chloride, 50 mM Tris-HCl, 1 mM ethylenediaminetetraacetic acid, 1% Triton X100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate) and a protease inhibitor cocktail (1 / 200 dilution) or PhosphoSafe extraction reagent. The samples were centrifuged at 12,000 g for 10 minutes to remove debris. Protein concentration was measured using the Pierce™ Protein BCA assay kit. 10 μg of total protein was diluted with reducing Laemmli buffer, heated at 100°C for 5 minutes, and loaded onto a 12% SDS-PAGE. Proteins were then transferred to nitrocellulose membranes and probed with anti-NP (1 / 500), anti-NS1 (1 / 1000), anti-PA (1 / 1000), anti-PB2 (1 / 500), anti-M1 (1 / 1000), anti-(β)ERK1 / 2 (1 / 2000), anti-(β)AKT (1 / 1000), anti-(β)p65 (3000), or anti-β-actin (1 / 5000). Bound antibodies were revealed using anti-rabbit IgG for NS1, PB2, (β)AKT, (β)ERK1 / 2, and (β)p65, and anti-mouse IgG (HRP-linked) for the other proteins, and ECL detection reagents. Detection was performed using an automated imaging system (MF ChemiBis 3.2, DNR BioImaging Systems), followed by analysis and quantification using FUJI FILM MultiGauge software.

[0201] Confocal fluorescence microscopy. Cells were grown in 12-well plates with coverslips at the bottom of the wells. After different treatments, cells were fixed with 4% formaldehyde for 30 minutes at room temperature and then permeabilized in PBS 0.1% Triton X-100 for 30 minutes at room temperature. After saturation with PBS 1% bovine serum albumin, 0.1% Tween20 for 1 hour, cells were stained with anti-NP-FITC (1 / 30), anti-NS1 (1 / 200), anti-M1 (1 / 150), anti-PB2 (1 / 200) and anti-PA (1 / 50) antibodies for 2 hours at room temperature. Anti-rabbit AF488 was used as a secondary antibody for NS1 and PB2 for 2 hours at room temperature, and anti-mouse AF488 was used for other proteins. Then, actin was stained with ActinRen 555 reagent for 30 minutes and nuclei were stained with NucBlue reagent for 5 minutes. Samples were analyzed with a Leica SP8 confocal microscope and Leica LasX Life Science Software.

[0202] Transmission electron microscopy . The cells were washed with PBS, detached with trypsin and centrifuged. The cells were fixed by incubation in 4% paraformaldehyde, 1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) for 24 hours. The samples were further washed in PBS and fixed by incubation with 2% osmium tetroxide for 1 hour. Next, the samples were completely dehydrated in a series of gradient ethanol solutions and propylene oxide. The impregnation step was carried out with a mixture of (1:1) propylene oxide / Epon resin, and the samples were then placed in pure resin overnight. They were then embedded in Epon resin and polymerized at 60°C for 48 hours. Ultrathin sections (90 nm) of these blocks were obtained using a Leica EMUC7 ultramicrotome (Wetzlar, Germany). The sections were stained with 2% uranyl acetate and 5% lead citrate. Observation was performed with a transmission electron microscope (JEOL 1011) and analysis was performed using a Digital Micrograph.

[0203] Scanning electron microscopy . The cells were washed with PBS, detached with trypsin and centrifuged. The cells were fixed by incubation in 4% paraformaldehyde, 1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) for 24 h. Then, the samples were washed in phosphate-buffered saline (PBS) and fixed by incubation with 2% osmium tetroxide for 1 h. The samples were then completely dehydrated in a series of gradient alcohol solutions and dried in hexamethyldisilazane (HMDS, Sigma, St-Louis, MO). Finally, the dried samples were spread on carbon disks and analyzed on a GATAN PECS 682 instrument (Pleasanton, CA). The platinum coating was observed under a Zeiss Ultra plus FEG-SEM scanning electron microscope (Oberkochen, Germany).

[0204] IAV titration by plaque-forming unit assay Titrations in cell supernatants and mouse lungs were performed as previously described

[38] .

[0205] Primary human bronchial epithelial cell culture Primary human bronchial epithelial cells (PBECs) were isolated from macroscopically normal bronchial tissue obtained from patients undergoing lobectomy at the University Hospital of Tours ("CHRU") in Tours, France. Cancer-free tissue sections were washed and incubated with 0.018% (w / v) protease type XIV (Sigma-Aldrich) in the absence of Ca2+ at 37°C. 2+ / Mg 2+The epithelial cells were gently scraped from the luminal surface, washed, and then cultured in serum-free keratinocyte medium (Gibco) supplemented with 2.4 ng / ml epidermal growth factor (Gibco), 25 μg / ml bovine pituitary extract (Gibco), 1 μM isoproterenol (Sigma-Aldrich), 100 U / mL penicillin and 100 mg / ml streptomycin (Lonza) on 6-well plates (37°C, 5% CO2 with 30 μg / ml PureCol (Advanced BioMatrix, San Diego, CA)). Diego, CA, USA), 10 μg / ml bovine serum albumin (Sigma-Aldrich) and 5 μg / ml fibronectin (isolated from human plasma and diluted in PBS) were coated for 24 h. During the first week of culture, 1 / 500 Primocin (Invivogen, France) was added to the culture medium. After the cells reached near confluence, they were trypsinized (0.03% [w / v] soft trypsin (Gifco, Detroit, USA), 0.01% (w / v) EDTA (VWR, France), 0.1% glucose (VWR, France) in PBS) and stored in liquid nitrogen. These PBECs were used to generate mucociliary differentiated cells by differentiation in liquid culture as described previously

[83] , except that we used Stemcell's PneumaCult EX instead of BEGM / DMEM medium. Before PBECs were stimulated with 2 μg / ml PIC or 50 mM PMA, PneumaCult The initial proliferation period was 3-4 days in EX medium. Subsequent stimulation was performed in a medium consisting of a 1:1 mixture of BEGM medium and complete DMEM / F12 medium. The medium was supplemented with 100 U / mL penicillin, 100 μg / mL streptomycin, 12.5 ml Gibco TM HEPES (1M) and 5ml Gibco TM GlutaMAX TM Supplements.

[0206] Human Precision Cut Lung Slices (PCLS) PCLS were obtained from lung resections of surgical patients from the CHRU of Tours. Lung explants were cut into 400 μm slices using a McIIwain Tissus choper. Individualized PCLS were placed at the air-liquid interface and thawed with 2.10 4PCLS were infected with pfu of influenza A / Scotland / 20 / 74 (H3N2) virus (IAV). Two hours later, PCLS were treated with 3.4 mM cis-aconitic acid and maintained at 37°C, 5% CO2. 48 hours after infection, PCLS supernatants were collected to measure cytokine release. PCLS were fixed in formalin for histological imaging.

[0207] Animal infection and fluid collection . Seven-week-old female or male C57Bl / 6 mice were infected intranasally with 200 pfu of influenza A / Scotland / 20 / 74 (H3N2) IAV, or were not infected with the virus and treated with 0.6 mg of cis-aconitic acid at different time points, or were left untreated. On the day of sacrifice, blood was collected and centrifuged at 10,000 g for 10 minutes for serum analysis, or it was heparinized and examined using a ProCyte Dx blood cell counter (Idexx, France). The airways were washed four times with 0.5 ml of PBS for BAL collection. After centrifugation, the BAL fluid was stored at -80°C for subsequent measurement of inflammatory mediators, and the precipitate was recovered in PBS 2% FBS. Red blood cells were discarded using red blood cell lysis buffer, and white blood cells were counted and analyzed by flow cytometry. After BAL, 10 mL of PBS was injected into the heart for lung perfusion. The left lung was placed in PBS containing 4% paraformaldehyde for histological analysis. Lung suspensions were obtained by enzymatic digestion using a gentleMACS dissociator (Miltenyi Biotech) according to the kit manufacturer's instructions. Groups of nine Balb / c NF-κB transgenic immunocompetent mice were also infected with 300 PFU of A / Scotland / 20 / 74 (H3N2) IAV. Eight days after infection, the mice were anesthetized and fluorescein (0.75 mg / kg) was instilled intranasally. -1 ) and quantified luciferase activity. Bioluminescence was measured using an IVIS imaging system. Weight loss was monitored daily and mice were sacrificed when they lost 25% of their initial body weight.

[0208] Groups of 20 wild-type (WT) or cis-aconitate decarboxylase (CAD)-deficient C57Bl / 6 mice were infected with 100 PFU of A / Scotland / 20 / 74 (H3N2) IAV and treated with 0.6 mg of cis-aconitate 20 minutes after infection or left untreated. Weight loss was monitored daily and mice were sacrificed when they lost 20% of their initial body weight.

[0209] Flow cytometric analysis BAL or human bronchial epithelial cells were aliquoted into round-bottom 96-well plates and centrifuged at 300 g for 5 min at 4 °C. Samples were further stained with specific antibodies and appropriate isotype controls. One well was inoculated for each antibody and used for fluorescence minus one control. Flow cytometry data were collected on the analyzer and analyzed using VenturiOne software.

[0210] Histopathology . Lung tissue was collected after BAL and airway lavage and placed in PBS containing 4% paraformaldehyde. Lung sections approximately 4 μm thick were cut and stained with hematoxylin-eosin. The study pathologist used a blinded experimental protocol and examined the tissue sections using light microscopy on a Leica Diaplan microscope. All histopathological results were graded semiquantitatively on a scale of 0-4 (0: none, 1: mild, 2: moderate, 3: severe, 4: very severe). All lung tissue preparation and analysis were performed at LAPV (Amboise, France).

[0211] Genomic DNA extraction Genomic DNA was extracted from mouse fecal pellets by transferring them to tubes containing 2.8 mm ceramic beads (VWR), 0.1 mm glass beads (VWR), 100 μl of GES, and 800 μl of 200 mM sodium phosphate buffer (pH 8). The samples were bead-beaten using a Powerlyzer 24 benchtop homogenizer at 3000 rpm for three minutes. After centrifugation at 15,000 rpm for 10 minutes, the supernatant was processed using a MagMAX Express 96 Deep Well Magnetic Particle Processor (Applied Biosystems) and a DNA Multi-Sample Kit (Life Technologies).

[0212] 16S rRNA sequencing and analysis. The v34 region of the 16S rRNA gene was amplified using PCR. The reaction contained 50 ng of template DNA, 5 pmol of 341F and 806R Illumina adapter primers, 1 U of Taq polymerase, 1× buffer, 1.5 mM MgCl2, 0.4 mg / mL bovine serum albumin, and 0.2 mM of each dNTP. The PCR program included an initial denaturation at 94°C for 5 minutes, followed by 5 cycles of 94°C for 30 seconds, 47°C for 30 seconds, and 72°C for 40 seconds, followed by 25 cycles of 94°C for 30 seconds, 50°C for 30 seconds, and 72°C for 40 seconds, and a final extension at 72°C for 10 minutes. The amplicons were normalized using the SequalPrep Normalization Kit (ThermoFisher) and then sequenced on the Illumina MiSeq platform at the McMaster Genomics Facility. Cutadapt (DOI: 10.14806 / ej.17.1.200) was used to trim raw reads, remove adapters, and delete reads less than 100 bp based on a minimum quality score of 30. The DADA2 pipeline (10.1038 / nmeth.3869) was used to determine amplicon sequence variants (ASVs) for each Illumina run. The sequence variant tables from each Illumina run were then merged and dimers were removed using DADA2. Each ASV was classified based on the SILVA database v1.3.2. Statistical analysis and visualization of fecal microbial data were performed using RStudio v4.1.2. ASVs not classified at the kingdom or phylum level, as well as ASVs classified as eukaryotic or mitochondrial organisms, were excluded. Aitchison distances were measured using the microbiome (http: / / microbiome.github.io) and phyloseq packages (10.1371 / journal.pone.0061217). Permutational multivariate analysis of variance (PERMANOVA) was performed using the adonis function in the vegan software package (10.1111 / j.1654-1103.2003.tb02228.x).

[0213] Cell proliferation and cytotoxicity assays For cell proliferation assays, cells in 96-well plates were washed twice with PBS and incubated with 100 μL of a 1 / 5 diluted MTS reagent at 37°C for 1 hour. Optical density was measured at 490 nm. For cytotoxicity assays, cells were stained with Live / Dead or anti-Ki67 mitotracker for 15 minutes at 4°C, followed by flow cytometric analysis.

[0214] siRNA transfectionOne day before siRNA transfection, Beas-2B cells (1.25×10 5 ) were seeded in 12-well plates. Negative control scramble (Sigma-Aldrich) siRNA was transfected. Each siRNA stock solution was diluted to 50 nM in 100 μL of OptiMEM (Gibco) containing 1.5 μL of RNAiMax reagent (Invitrogen). After incubation at room temperature for 5 minutes, 100 μL of each siRNA mixture was added to 900 μL of fresh culture medium per well. RNA interference (RNAi) was performed for 48 hours (medium was replaced after 24 hours), and gene knockdown was assessed by RT-qPCR.

[0215] Patient data collection . The study was conducted over 18 months in a 37-bed ICU of the University Hospital of Tours (“CHRU”) in Tours (France). Pneumonia was defined as pulmonary infiltrates on chest radiograph, accompanied by one or more of the following symptoms: dyspnea, cough (with or without sputum), fever (temperature ≥38.0°C) or hypothermia (temperature <35.0°C). Community-acquired infection was defined as infection occurring within 48 hours of admission, excluding nosocomial pneumonia. Cases of pneumonia due to Pneumocystis jiroveci or aspiration were not included. Cases with a PaO2 ≥60 mmHg in ambient air or requiring oxygen therapy ≤4 L / min or not requiring mechanical ventilation (invasive or non-invasive) were not included. Baseline patient information was collected at the time of case presentation through a face-to-face semi-structured interview with the patient or relative. Observations from the physical examination at the time of presentation, including vital signs and lung auscultation, were recorded. Microbiological studies including blood culture, Legionella and pneumococcal urinary antigen testing, bacterial culture of tracheal aspirate, multiplex PCR Respifinder SMART of respiratory fluids (sputum and / or nasal washes and / or endotracheal aspirates and / or bronchoalveolar lavage [BAL]) (Pathofinder B.V., Oxfordlaan, Netherlands).

[0216] Statistical analysis Statistical analyses were performed using Graphpad Prism. Data are presented as mean ± SEM. Statistical values, including the number of replicates (n) and the statistical tests used, can be found in the figure legends. *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.0001. For in vitro experiments, n = number of separate experiments. For in vivo experiments, n = number of individual animals.

[0217] Study Approval . All animal experiments were performed in accordance with national government guidelines and approved by local and national ethics committees (CEEA.19, #201604071220401-4885). Informed consent was obtained from each patient in accordance with the Declaration of Helsinki, and tissue and cell collection was declared to the French Ministry of Graduate study, Research and Innovation (DC-2008-308, MERI Ministère de l'Enseignement Supérieur, de la Recherche et de l'Innovation). Human data collection complied with the French law of observational studies, approved by the ethics committee of the French Intensive Care Society (CE SRLF 13-28) and approved by the Commission Nationale de l'Informatique et des Libertés (CNIL) for the treatment of personal health data. Written and oral information was provided to the patients or their next of kin. Oral informed consent was given by the patients or their next of kin according to the approval of the ethics committee.

[0218] 1.2 / Results

[0219] Cis-aconitic acid inhibits influenza A virus replication and inflammatory mediator production in infected lung epithelial cells.

[0220] To examine the potential anti-influenza effects of metabolites from glycolysis or the TCA cycle, we infected human bronchial epithelial cells (BEAS-2B cells) with influenza A virus (IAV) (A / Scotland / 20 / 74, H3N2) and treated them with 3.4 mM metabolites at 4 h post-infection (hpi). This concentration corresponds to the highest dose at which no cytotoxic effects were observed for all tested metabolites ( Figure 8a As a surrogate marker for new virion release, we measured neuraminidase (NA) activity in cell supernatants at 20 hpi. We observed a 60-fold decrease in NA activity in infected cells treated with cis-aconitic acid (p = 0.0003) (Figure 1b). A decrease in NA activity was also observed with itaconic acid (4.8-fold, p = 0.028). None of the other tested metabolites had any effect on NA activity.

[0221] Consistently, cis-aconitic acid reduced interleukin 6 (IL-6) production, an inflammatory mediator, by IAV-infected epithelial cells ( FIG. 1 c ).

[0222] Cis-aconitate is a TCA intermediate that is essential for producing energy in the form of ATP. In addition, cis-aconitate decarboxylase (CAD, also known as ACOD1 or Irg1) converts cis-aconitate to itaconic acid. To ensure that the anti-influenza effect of cis-aconitate should not be attributed to itaconic acid, we used CAD siRNA to prevent BEAS-2B cells from metabolizing cis-aconitate to itaconic acid. Compared with the control scramble siRNA, CAD siRNA effectively blocked CAD expression (Figure 1d), but the antiviral and anti-inflammatory properties of cis-aconitate were retained (Figure 1e-g). This strongly suggests that regardless of whether itaconic acid is involved, cis-aconitate protects epithelial cells from IAV infection, revealing its independent protective mechanism.

[0223] Supplementary experiments also demonstrated that trans-aconitic acid exhibited similar antiviral and anti-inflammatory activities, indicating that both isomers of aconitic acid have anti-influenza properties ( Figure 1h-i ).

[0224] Cis-aconitic acid inhibits influenza polymerase.

[0225] To better understand how cis-aconitic acid inhibits viral particle production, we investigated its effects on different stages of the IAV replication cycle. IAV is an enveloped virus whose genome consists of negative-sense, single-stranded, segmented RNA. It has eight segments encoding 12 viral genes. The influenza virus life cycle can be divided into the following stages: (i) entry into the host cell; (ii) entry of viral ribonucleoproteins (vRNPs) into the cell nucleus; (iii) transcription and replication of the viral genome; (iv) translation of messenger viral RNA into viral proteins; (v) export of vRNPs from the cell nucleus; and (vi) assembly and budding at the host cell plasma membrane. We retraced all of these steps.

[0226] Therefore, we first examined IAV-infected cells by transmission and scanning electron microscopy (TEM, SEM, respectively) and observed a significant reduction in viral budding after cis-aconitic acid treatment (IAV+CA, Figure 2a Whether the reduction in viral budding is due to an impairment of the budding process itself or to a reduced production of viral material (viral RNA and / or protein) remains unclear. We quantified the expression of viral proteins and mRNAs in infected epithelial cells treated with or without cis-aconitic acid. Using Western-blot and confocal microscopy analysis, we observed a sharp decrease in viral protein expression of NP, NS1, and PA at 8 h post-infection under treated conditions ( Figure 2b-e). Using qRT-PCR, we found that at 6 h post-infection, the relative expression of viral matrix 1 (M1) mRNA was reduced 10-fold by cis-aconitic acid treatment (p < 0.01, Figure 2f). Delivery of vRNPs from the cell surface to the nucleus lasts for approximately 1 h, with cell entry and fusion processes being quite rapid (approximately 10 min) [33,34]. Since cis-aconitic acid was administered 4 h post-infection (and after a cell washing step), we speculated that cis-aconitic acid was unlikely to affect the early steps of viral entry, from cell fusion to nuclear trafficking. Overall, these results suggest that cis-aconitic acid inhibits transcription of the viral genome, and that the decrease in viral protein expression and ultimately the production of new virions are downstream effects. To test the effect of cis-aconitic acid on viral polymerase activity, we performed a minigenome assay in 293T cells transfected with the reporter plasmid pPolI-WSN-NA-firefly luciferase. After 24 hours of treatment, we observed a dose-dependent decrease in luciferase activity, which was reduced by approximately 75% using 3.4 mM cis-aconitic acid (p < 0.03, Figure 2g). In conclusion, we demonstrate that cis-aconitic acid disrupts the influenza virus life cycle by inhibiting viral polymerase activity.

[0227] Cis-aconitic acid exhibits antiviral properties against various types and subtypes of influenza virus

[0228] All previous data were from IAV (A / Scotland / 20 / 74(H3N2)) infections. However, influenza viruses show great diversity and a large number of subtypes. In 2018, the Global Influenza Surveillance, Epidemiology and Control Center reported that influenza A was the main type, with influenza A(H1N1)pdm09 (corresponding to the 2009 H1N1 pandemic strain) and influenza A(H3N2) being the majority

[35] . Influenza B viruses are also an important component of influenza infections. Thus, in 2013, 35% of respiratory specimens that tested positive for influenza were positive for influenza B viruses, of which 80% belonged to the B / Yamagata lineage and 20% to the B / Victoria lineage. Therefore, in addition to the previously tested influenza A / Scotland / 20 / 74H3N2 strain, we also evaluated the effects of cis-aconitic acid on influenza A / Pandemic / 2009H1N1 (Figure 3a), influenza A / Puerto Rico / 8 / 1934H1N1 (Figure 3b), and influenza B Yamagata strain B / Paris / 234 / 2013 (Figure 3c). Plaque-forming unit assays showed that cis-aconitic acid produced at least a 1-log reduction in viral particles, regardless of the type or subtype of influenza virus used (Figures 3a-c). These data demonstrate the broad-spectrum antiviral properties of cis-aconitic acid against influenza viruses.

[0229] Cis-aconitic acid is an anti-inflammatory mediator.

[0230] We wondered whether the reduction in inflammatory mediator production (IL-6) by cis-aconitic acid was a downstream effect of its antiviral effects or a consequence of its inherent immunomodulatory properties. This question is crucial for influenza pathophysiology, which is a consequence of both cytopathic viral effects and an excessive inflammatory response (immunopathology occurs when the immune balance is disturbed) [36-38].

[0231] Deleterious immune responses are characterized by dysregulated activation of intracellular signaling pathways and excessive production of inflammatory cytokines [39–41]. Compared with uninfected conditions, IAV-infected cells showed upregulation of Ras-dependent phosphorylation of extracellular signal-regulated kinases (ERK) 1 / 2, protein kinase B (PKB) (also known as Akt), and the p65 subunit of nuclear factor-κB (NF-κB) 20 hours after infection (Figure 4a). Treatment with cis-aconitic acid inhibited the accumulation of these phosphorylated forms in infected cells. IAV infection consistently induced a 4- to 7-fold increase in the expression of CCL2 / MCP-1, CCL5 / RANTES, CXCL1 / GROα, CXCL10 / IP-10, IL-6, and CXCL8 / IL-8 (Figure 4b, blue bars) compared with controls inhibited by cis-aconitic acid (Figure 4b, green bars).

[0232] Interestingly, treatment of uninfected lung epithelial cells with cis-aconitic acid also resulted in a reduction in key signaling pathways (Figure 4a, cis-aconitic acid inhibits ERK, AKT, and NF-κB signaling pathways). Notably, the dose of cis-aconitic acid used did not affect cell proliferation, mitochondrial accumulation, ROS production, or cell viability ( Figures 8b-8e ). To gain further insights into the intrinsic immunomodulatory properties of cis-aconitic acid, we stimulated bronchial epithelial cells with different inflammatory agonists: (i) Poly(I:C) (PIC), a synthetic double-stranded RNA poly(I:C) and a potent agonist of TLR3 signaling, which mimics IAV-triggered immune responses

[42] ; (ii) phorbol 12-myristate 13-acetate (PMA), a diester of phorbol, which activates the signal transduction enzyme protein kinase C (PKC); (iii) TNFα, an inflammatory cytokine responsible for multiple signaling events within cells leading to necrosis or apoptosis. All of these agonists induced the activation of signaling pathways, which led to the release of inflammatory cytokines. We observed a trend for cis-aconitic acid treatment to induce IL-6 production in a dose-dependent manner ( Figure 4c , TNFα) or significantly decreased ( Figure 4c , IAV, PIC, PMA).

[0233] Together, these findings demonstrate that cis-aconitic acid exhibits anti-inflammatory properties that can effectively disrupt the inflammatory cascade involved in influenza infection.

[0234] Preclinical models confirm anti-infective and anti-inflammatory properties of cis-aconitic acid

[0235] To gain insight into the generalization and translation of previous observations, we explored whether the antiviral and anti-inflammatory properties of cis -aconitic acid were confirmed in experimental models with complementary approaches: ex vivo models of infection in human bronchial epithelial cells and human lung slices, and an in vivo murine model of influenza pneumonia.

[0236] Effects of cis-aconitic acid in human PEBC and lung tissue .

[0237] Primary human bronchial epithelial cells (PBECs) were isolated from bronchial tissue obtained from patients undergoing lobectomy at the CHRU of the Tour Center and cultured ex vivo to regenerate a pseudo-stratified airway epithelium with basal cells, mucus-secreting cells, and ciliated cells [43, 44]. This model has always been highly relevant for the study of viral infection [45-48]. We observed that after influenza infection, cis-aconitic acid treatment significantly reduced NA activity (Figure 4e). IAV induced a large amount of hIL-6 production (~1300 pg / mL), which was significantly and dose-dependently inhibited by cis-aconitic acid (reduction of up to 80%, Figure 4f). In addition, the expression of proinflammatory cytokines induced by the immunostimulants PIC or PMA showed a dose-dependent reduction with cis-aconitic acid treatment (Figure 4f). Figure 4g, 4h Given the key role of cell death in the pathogenesis of influenza A virus (IAV) (

[62] ), we used the latest The impact of cis-aconitic acid on this critical process was carefully evaluated. TM The expression of cis-aconitic acid (a marker indicating cell death) was significantly reduced (about 70%), indicating that cis-aconitic acid has a significant protective effect against IAV-triggered cell death ( Figures 4i, 4j ). Finally, we investigated the anti-inflammatory effects of cis-aconitic acid on freshly collected ex vivo human lung tissue infected with IAV. As expected, IAV infection resulted in the secretion of proinflammatory cytokines from human precision-cut lung sections (PCLS; Figure 4k). Notably, cis-aconitic acid treatment resulted in a significant two-fold reduction in IL-6 production (Figure 4I). Together, these findings confirm that cis-aconitic acid possesses dual antiviral and anti-inflammatory properties in an ex vivo human model, highlighting its high translational relevance.

[0238] Anti-inflammatory effects of cis-aconitic acid in vivo

[0239] We then further investigated the anti-inflammatory effects of intranasally administered cis-aconitic acid in a valuable mouse model of acute lung injury induced by lipopolysaccharide, a cell wall component characteristic of Gram-negative bacteria (

[63] ).

[0240] However, prior to these experiments, the safety profile of cis-aconitic acid had been thoroughly evaluated in mice by chronic instillation for 15 days. Notably, no adverse effects or changes in microbial composition were observed under these conditions ( Figures 9a-9d Subsequently, we evaluated the activity of NF-κB, a key transcription factor in inflammatory signaling, induced by LPS instillation in NF-κB-luciferase transgenic mice, with or without cis-aconitic acid treatment (Fig. 6a-b). 24 hours after stimulation, LPS-induced inflammation ( Figure 11 a, middle panel) In animals treated with cis-aconitic acid ( Figure 11 a, right and Figure 11 Consistent with this observation, TNFα secretion in BAL fluid of mice stimulated with LPS was significantly reduced in mice treated with cis-aconitic acid ( Figure 11 c). Therefore, our in vivo experiments confirmed the inhibitory effect of cis-aconitic acid on inflammatory pathways, thereby reducing the secretion of downstream pro-inflammatory cytokines.

[0241] Therapeutic effect of cis-aconitic acid on mice infected with influenza virus

[0242] Next, we examined the therapeutic potential of cis-aconitic acid in a mouse model of acute IAV pneumonia. Mice were infected intranasally with a lethal dose of influenza A / Scotland / 20 / 74 (H3N2) and treated with 0.6 mg of cis-aconitic acid 2 days after infection. In the initial phase, mice were sacrificed 4 days after infection to analyze early events in influenza pathophysiology. Notably, mice treated with cis-aconitic acid showed a significant reduction in viral load in lung tissue compared to control animals (1-log reduction, p < 0.0001; Figure 5a). Further evaluation included examining the relative expression of 50 mediators in the bronchoalveolar lavage (BAL) compartment, including proinflammatory cytokines, interferons, stimulatory factors or growth factors, and proteases; Figure 5b). All of these mediators increased after IAV infection but were significantly reduced in animals treated with cis-aconitic acid (Figure 5b).

[0243] Extending the sacrifice time to 8 days after infection, our model exhibited characteristics of severe IAV-induced lung injury [49-53], including increased leukocyte numbers in BAL fluid, depletion of alveolar macrophages, neutrophil recruitment, and activation of T lymphocytes, NKT cells, and DCs (Figure 5c (BAL fluid), 5d (lung tissue)). Treatment with cis-aconitic acid consistently alleviated these responses: the number of alveolar macrophages was comparable to that in uninfected mice, while neutrophil recruitment was reduced by two-thirds (p < 0.02, Figure 5d), and the activation of NKT, DC, alveolar macrophages, and T cells was significantly lower than that observed in infected untreated mice (Figures 5c and 7d).

[0244] Considering the potential damage caused by these events in severe IAV infection, the observed trends of cis-aconitic acid in normalizing cellular infiltration and immune cell activation may have beneficial effects on lung tissue [54-59]. Consistently, histopathological analysis showed that IAV-induced alveolar wall thickening, hyaline membrane formation, and epithelial necrosis were significantly reduced in mice treated with cis-aconitic acid compared with untreated animals (Figure 5e, f). Treated mice showed reduced lung congestion and decreased inflammation scores compared with untreated mice (Figure 5e right panel and 5f).

[0245] To further evaluate IAV-induced inflammation more broadly, we measured NF-κB activity in NF-κB-luciferase transgenic mice infected with IAV and treated with or without cis-aconitic acid (Figures 5g and h). Eight days after infection, influenza infection-induced inflammation was systemic (i.e., inflammation in the digestive tract; Figure 5g, middle panel). This inflammation was significantly reduced in mice treated with cis-aconitic acid (Figures 5g, right panels and h).

[0246] Thus, cis-aconitic acid effectively prevents IAV-induced lung injury by reducing all components of influenza pathogenesis, including viral replication, secretion of inflammatory mediators, and recruitment / activation of inflammatory cells.

[0247] Cis-aconitic acid alleviates mortality in IAV-infected mice within a prolonged therapeutic window

[0248] To determine the anti-influenza potential of cis-aconitic acid, we evaluated its benefit on mortality in an infected mouse model compared to the reference anti-influenza drug oseltamivir (neuraminidase inhibitor)

[60] . IAV-infected but untreated mice showed zero survival, while mice treated with cis-aconitic acid or oseltamivir achieved 80% and 90% survival, respectively, 20 minutes after infection (Fig. 6a). After 15 days, the surviving mice had returned to their original weight. Delaying the administration of oseltamivir until 2 days after infection abrogated the beneficial effect of this treatment on the survival of infected mice (Fig. 6c). Notably, the protective effect of cis-aconitic acid persisted in C57Bl / 6 and Balb / c mice even when treatment was delayed until 2 days after infection (Fig. 6c, d).

[0249] Exploratory research should ultimately improve patient care. To this end, experimental designs must consider real-world concerns. The timing of appropriate anti-infective treatment is crucial because it is a predictor of efficacy. During influenza infection, there is a delay between the onset of symptoms and actual medical consultation or hospitalization. Therefore, experimental settings testing anti-influenza therapies should account for this treatment time; otherwise, it may bias efficacy. To help translate our findings to humans, we conducted an adjunct study to a prospective clinical trial

[61] to determine the time from symptom onset to hospitalization in patients with community-acquired pneumonia (CAP) due to influenza infection. We studied 153 patients with CAP: 37% had viral pneumonia, 24% had bacterial pneumonia, 20% had coinfections, and 19% had no respiratory pathogen identified. The most common viruses identified were influenza A, rhinovirus / enterovirus, and influenza B, accounting for 33%, 11%, and 10% of the viruses identified, respectively. The most common bacteria identified were Streptococcus pneumoniae, Staphylococcus aureus, and Legionella pneumophila, accounting for 50%, 16%, and 12% of the bacteria identified, respectively. The characteristics of CAP patients (with a particular focus on influenza) are shown in Figure 7 and Table 1 below:

[0250]

[0251] Table 1. Patient characteristics. Quantitative data are median and interquartile range (IQR), and qualitative data are n (%). 1 Defined as solid cancer, hematologic disease, organ transplant, bone marrow transplant, HIV infection, and splenectomy

[0252] The three main clinical symptoms of CAP reported due to influenza (A or B) infection are fever, cough, and dyspnea, as shown in Figure 7 and Table 2 below:

[0253]

[0254] Table 2. Symptoms and clinical signs. Quantitative data are median and interquartile range (IQR), qualitative data are n (%).

[0255] The time from onset of CAP to hospitalization is generally 3 (2-7) days, while that of CAP due to influenza (A or B) infection is 4 (3-6) days. Therefore, evaluating the efficacy of anti-influenza drugs by testing them 4 days after infection seems to be the most appropriate approach.

[0256] Therefore, we examined the effects of administering cis-aconitic acid to mice on day 4 after IAV infection. By this stage, IAV infection had progressed to pneumonia, characterized by the massive production of inflammatory mediators (Figure 5b) and manifested as a severe condition with ~20% body weight loss (Figure 6d). To better simulate potential clinical translation, we administered a second dose of cis-aconitic acid on day 5 after infection. In contrast to oseltamivir, cis-aconitic acid demonstrated significant efficacy in treating IAV-infected mice, significantly increasing survival from 0% to 50% (Figure 6b).

[0257] In conclusion, we demonstrated that mice with severe IAV pneumonia who received cis-aconitic acid in a curative manner were more effectively treated than mice treated with currently recommended antiviral drugs. Importantly, the protective benefit of cis-aconitic acid extended to situations where treatment was administered within a clinically relevant timeframe, further highlighting its potential as a promising therapeutic option for influenza infection.

[0258] Supplementary Data Confirmation of the protective effect of cis-aconitic acid in IAV-infected Balb / c mice.

[0259] To validate the findings observed in C57Bl / 6 mice (see Figures 6a-d) within a different mouse species, we performed experiments in Balb / c mice. These (NF-κB transgenic) Balb / c mice were infected with IAV (300 PFU) and treated intranasally with 0.6 mg of cis-aconitic acid (CA) for 2 days. Animal survival was monitored daily ( Figure 12 a) and weight loss ( Figure 12 b) In stark contrast, untreated Balb / c mice completely failed to survive IAV infection, whereas mice treated with cis-aconitic acid experienced a remarkable 90% survival rate. The significant protective effect of cis-aconitic acid was further demonstrated by its significant effect on alleviating weight loss in IAV-infected animals. These findings highlight the significant therapeutic potential of cis-aconitic acid in improving survival and weight loss across various mouse strains.

[0260] The anti-influenza activity of cis-aconitic acid is independent of that of itaconic acid.

[0261] Cis-aconitate decarboxylase (CAD), also known as ACOD1 or Irg1, facilitates the conversion of cis-aconitate to itaconate, a key player in linking innate immune responses and cellular metabolism, particularly in the context of IAV infection [18,18,26]. To determine whether the anti-influenza effects of cis-aconitate observed in our mouse model of IAV pneumonia were solely attributable to itaconate, we performed experiments using wild-type (WT) and CAD-deficient (CAD- / -) mice. These mice were infected intranasally with IAV and treated with cis-aconitate or PBS (as a vehicle control), and the animals were monitored daily for survival. Figure 13 As shown, the survival pattern in CAD- / - mice was very similar to that in wild-type mice. Therefore, these findings strongly suggest that the anti-influenza properties of cis-aconitic acid operate independently of itaconic acid.

[0262] Example 2: Cis-aconitic acid has anti-inflammatory properties that are sufficient to block the inflammatory cascade during SARS-CoV-2 infection reaction .

[0263] 2.1 / Method .

[0264] Viruses. SARS-CoV-2 strains (Wuhan and Delta) were isolated from patients at the Academic Hospital of Tours.

[0265] Cell Culture. In vitro experiments were performed using either the Vero cell line or primary human bronchial epithelial cells. The method for generating primary human bronchial epithelial cell cultures is described in Example 1.

[0266] Results .

[0267] To gain further insights into the anti-inflammatory properties of cis-aconitic acid during COVID-19, we infected various epithelial cell models (Wuhan and Delta, Figure 10 a). These infections induce the release of numerous inflammatory cytokines, exemplified in this article by IL-6. Notably, treatment with cis-aconitic acid significantly reduced IL-6 production, regardless of the epithelial cell model or SARS-CoV-2 strain used ( Figure 10 b, 10c).

[0268] These results suggest that cis-aconitic acid has anti-inflammatory properties and is able to disrupt the inflammatory cascade triggered during SARS-CoV-2 infection.

[0269] References

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Claims

1. Aconitic acid or a pharmaceutically acceptable salt thereof for use in a method for treating viral lung infection and / or adverse immune response to viral lung infection, the method comprising administering an effective amount of aconitic acid or a pharmaceutically acceptable salt thereof to a subject in need thereof.

2. Aconitic acid for use in a method according to claim 1, wherein the aconitic acid is cis-aconitic acid or trans-aconitic acid.

3. Aconitic acid for use in a method according to any one of claims 1 or 2, wherein the aconitic acid has immunomodulatory and / or antiviral properties, wherein: - the immunomodulatory properties of the aconitic acid include reducing or inhibiting the inflammatory response to the viral lung infection; - The antiviral properties of aconitic acid include inhibition of viral replication.

4. Aconitic acid for use in a method according to any one of claims 1 to 3, wherein the viral lung infection is caused by a respiratory virus, preferably selected from influenza virus, coronavirus, respiratory syncytial virus, parainfluenza virus, metapneumovirus, rhinovirus, adenovirus, varicella zoster virus, cytomegalovirus, paramyxovirus such as Nipah virus, and Bocavirus.

5. Aconitic acid for use in a method according to any one of claims 1 to 4, wherein the viral lung infection is caused by influenza virus or coronavirus.

6. Aconitic acid for use in a method according to any one of claims 1 to 5, wherein the method is for treating a viral lung infection.

7. Aconitic acid for use in a method according to any one of claims 1 to 5, wherein the method is for treating an adverse immune response to a viral lung infection in a subject suffering from a lung viral infection.

8. Aconitic acid for use in a method according to any one of claims 1 to 5, wherein the method is for preventing a pulmonary viral infection from escalating into an adverse immune response in a subject suffering from the pulmonary viral infection.

9. Aconitic acid for use in a method according to any one of claims 1 to 10, wherein the adverse immune response to a viral lung infection is an adverse immune response to a viral infection of the lungs which is an exaggerated inflammatory immune response, dyspnea, tachypnea, pneumonia, in particular acute pneumonia, exacerbation of a chronic respiratory disease such as asthma or chronic obstructive pulmonary disease, sepsis, septic shock, cytokine storm or acute respiratory distress syndrome (ARDS).

10. Aconitic acid for use in a method according to claims 1-12, wherein the aconitic acid is administered 4-14 days after infection.

11. Aconitic acid for use in a method according to any one of claims 1 to 10, wherein the subject is an animal, preferably a human, a domestic bird or a pig.

12. Aconitic acid for use in a method according to any one of claims 1 to 11, wherein aconitic acid is used alone or in combination with one or more active substances selected from the group consisting of antiviral agents, antibiotics and / or analgesics.

13. Aconitic acid for use in a method according to any one of claims 1 to 12, wherein the treatment is for preventing the development of a pulmonary viral infection, the method comprising administering aconitic acid in a subject not infected by a respiratory virus.

14. Aconitic acid for use in a method according to any one of claims 1 to 13, wherein the composition is administered intrapulmonary, nasally, orally, enterally, intravenously, intramuscularly and subcutaneously.

15. A pharmaceutical composition comprising aconitic acid or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier for use in a method according to any one of the preceding claims.

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