WO2021233948A1 - Method to treat a pathogen lung infection - Google Patents

Abstract

The present invention relates to the treatment of pathogen lung infection. Using a self-resolving influenza infection model, the inventors show for the first time that influenza infection leads to the accumulation of senescent cells in the lungs and that this process contributes to the acute lung illness and to the altered lung functions that persists on the long term. Thus, using this model of respiratory virus infection, the inventors show that senolytic compounds could be useful to treat pathogen lung infection and particularly, viral lung infection or at least abolish the symptoms of lung infection induced by respiratory viruses like, among others, Influenza A and B viruses, rhinoviruses, Respiratory Syncytial Virus, and coronaviruses such as severe acute respiratory syndrome coronavirus (SARS-CoV)-2. This would be very beneficial notably for the ongoing epidemics coronavirus disease 19 (COVID-19). Thus, the present invention relate to a senotherapeutic compound for use in the treatment of a pathogen lung infection in a subject in need thereof.

Description

METHOD TO TREAT A PATHOGEN LUNG INFECTION

FIELD OF THE INVENTION:

The present invention relates to a senotherapeutic compound for use in the treatment of a pathogen lung infection in a subject in need thereof.

BACKGROUND OF THE INVENTION:

Respiratory viral infections remain a major economical and health issue worldwide as exemplified by the emerging COVID-19 pandemics. Despite the application of vaccination programs and antiviral drug treatments, influenza infection is one of the most important causes of respiratory tract diseases and is responsible for widespread morbidity and mortality every winter (Morens 2008, McCullers 2006, 2014). Influenza infections can also result in sporadic and often devastating pandemics; the 1918 pandemic led to the death of 50 million people (McCullers 2006, 2014). The outbreak of the 2009 influenza virus highlights the threat of recurrent pandemia and the urgent need to propose novel therapies for the acute phase of the infection, but also for preventing lung sequelae. Most studies on influenza pathogenesis focus on the acute phase of the disease including viral pneumonia, respiratory dysfunctions and secondary bacterial infection. Severe influenza infection can also result in chronic lung diseases involving both airway and alveolar sites (up to 6 months). This includes bronchiolitis, mucous cell metaplasia and associated airway hyperreactivity, fibrosis and emphysema (Keeler SP JI 2019). Moreover, excess of hyaluronic acid in alveoli appears to reduce respiratory functions (Bell TJ Matrix Biol 2019). As accumulation of senescent cells associates with numerous chronic lung diseases including COPD and lung fibrosis, we herein investigated the possibility that IAV infection might be associated with accumulation of senescent cells in the lungs and that senescent cell accumulation might promote early as well as long term effects of influenza.

Cell senescence consists in a stable proliferation arrest and acquisition of a specific senescence-associated secretory phenotype (SASP) characterized by the release of inflammatory cytokines, immune modulators, proteases, growth factors, pro-fibrotic factors, and various effectors that can among others promote senescent cells clearance, reinforce senescent phenotypes, and alter tissue microenvironments (Childs 2017). Cell senescence can be triggered either by progressive telomere shortening secondary to incomplete chromosomal replication or by various stress stimuli such as oxidative stress, DNA damaging agents, and inflammation. These processes produce a DNA damage response with p53-dependent upregulation of the cyclin-dependent kinase inhibitor p21 and/or expression of pl6INK4A, a classical marker of senescent cells (Childs 2017).

SUMMARY OF THE INVENTION:

Using a self-resolving influenza infection model, the inventors show for the first time that influenza infection leads to the accumulation of senescent cells in the lungs and that this process contributes to the acute lung illness and to the altered lung functions that persists on the long term. Thus, using this model of respiratory virus infection, the inventors show that senolytic compounds could be useful to treat pathogen lung infection and particularly, viral lung infection or at least abolish the symptoms of lung infection induced by respiratory viruses like, among others, Influenza A and B viruses, rhinoviruses, Respiratory Syncytial Virus, and coronaviruses such as severe acute respiratory syndrome coronavirus (SARS-CoV)-2. This would be very beneficial notably for the ongoing epidemics coronavirus disease 19 (COVID- 19).

Thus, the present invention relate to a senotherapeutic compound for use in the treatment of a pathogen lung infection in a subject in need thereof. Particularly, the invention is defined by its claims.

PET ATT /ED DESCRIPTION OF THE INVENTION:

A first aspect of the invention relates to a senotherapeutic compound for use in the treatment of a pathogen lung infection in a subject in need thereof.

Particularly, using the example of the Influenza A virus (IAV), the inventors showed that this virus induced severe lung damage and massive accumulation of senescent cells that persisted after complete clearance of the virus. Moreover they showed that lung-cell senescence, resulting from IAV infection directly increases IAV infection severity, delays airway epithelium recovery and, subsequently, leads to post-viral lung pathologies. Counteracting the cell- senescence process or eliminating senescent lung cells might limit infection duration and lung damage severity, lung inflammation, expedite epithelial repair, hasten viral clearance, and protect against secondary infections and post-viral lung sequelae.

Thus, in other words, the invention relates to a senotherapeutic compound for use in the inhibition / suppression of senescent lung cells in a treatment of a pathogen lung infection of the respiratory tract in a subject in need thereof.

In other words, the invention relates to a senotherapeutic compound for use in the treatment of a pathogen respiratory tract infection in a subject in need thereof. The invention also relates to a senotherapeutic compound for use in the improvement of lung cells and/or the functions of the lung after a pathogen lung infection in a subject in need thereof.

According to the invention, the lung inflammation can be induced by the viral infection which induced senescent lung cells which produced cytokines like interleukin (IL)-6 and IL-

1b·

As used herein, the term “a pathogen lung infection” denotes a lung infection induced by a biological pathogen or in other word an infectious agent.

According to the invention, the pathogen can be a virus, bacterium, protozoan, prion, viroid, or fungus.

According to the invention, the bacterium can be selected from the group consisting of: Streptococcus pneumoniae; Staphylococcus aureus; Haemophilus influenza, Myoplasma species, Moraxella catarrhalis, Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella enterica serovar, Typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis, Pseudomonas such as P. aeruginosa, Campylobacter, Mycobacterium tuberculosis, and Streptomyce.

According to the invention, the fungus can be selected from the group consisting of: aspergillus, Candida albicans and Cryptococcus neoformans.

More particularly, the pathogen lung infection is induced by a respiratory virus.

Particularly, the respiratory virus can be Influenza virus, such as the Influenza A virus (IAV) or the Influenza B virus (IAB), adenovirus, metapneumovirus, cytomegalovirus, parainfluenza virus (e.g., hPIV-1, hPIV-2, hPIV-3, hPIV-4), the human rhinovirus (HRV), the Human respiratory syncytial virus (HRSV) or a coronavirus.

As used herein, the term “coronavirus” has its general meaning in the art and refers to any member of members of the Coronaviridae family. Coronavirus is a virus whose genome is plus-stranded RNA of about 27 kb to about 33 kb in length depending on the particular virus. The virion RNA has a cap at the 5’ end and a poly A tail at the 3’ end. The length of the RNA makes coronaviruses the largest of the RNA virus genomes. In particular, coronavirus RNAs encode: (1) an RNA-dependent RNA polymerase; (2) N-protein; (3) three envelope glycoproteins; plus (4) three non- structural proteins. In particular, the coronavirus particle comprises at least the four canonical structural proteins E (envelope protein), M (membrane protein), N (nucleocapsid protein), and S (spike protein). The S protein is cleaved into 3 chains: Spike protein SI, Spike protein S2 and Spike protein S2'. Production of the replicase proteins is initiated by the translation of ORFla and ORFlab via a -1 ribosomal frame-shifting mechanism. This mechanism produces two large viral polyproteins, ppla and pplab, that are further processed by two virally encoded cysteine proteases, the papain-like protease (PLpro) and a 3C-like protease (3CLpro), which is sometimes referred to as main protease (Mpro). Coronaviruses infect a variety of mammals and birds. They cause respiratory infections (common), enteric infections (mostly in infants >12 mo.), and possibly neurological syndromes. Coronaviruses are transmitted by aerosols of respiratory secretions. Coronaviruses are exemplified by, but not limited to, human enteric coV (ATCC accession # VR-1475), human coV 229E (ATCC accession # VR-740), human coV OC43 (ATCC accession # VR-920), Middle East respiratory syndrome-related coronavirus (MERS-Cov) and SARS-coronavirus (Center for Disease Control), in particular SARS-Covl and SARS-Cov2.

According to the invention, the coronavirus can be a MERS-CoV, SARS-CoV, SARS- CoV-2 or any new future family members.

Thus, particularly, the invention also relates to a senotherapeutic compound for use in the treatment of a viral lung infection induced by a SARS-CoV-2 in a subject in need thereof.

As used herein, the term “senescent cells” or “cellular senescence” has its general meaning in the art and denotes a phenomenon characterized by the cessation of cell division and acquisition of a senescence-associated secretory program (SASP). Cellular senescence can be initiated by a wide variety of stress inducing factors. These stress factors include both environmental and internal damaging events, abnormal cellular growth, oxidative stress, autophagy factors, among many other things. The physiological importance for cell senescence has been attributed to prevention of carcinogenesis, and more recently, aging, development, and tissue repair.

As used herein, the term “senescent lung cells” denotes the senescence of the pulmonary cells inducing damage of these cells and of the lung function.

As used herein, the term “senotherapeutic compound” denotes therapeutic agents and strategies which to specifically target cellular senescence. According to the invention, the term “senotherapeutic compound” regroup the senolytic compound and the senomorphic compound. As used herein, the term “senolytic compound” denotes molecules which can induce death of senescent cells. Senolytic compounds are well known in the art (see for example Myrianthopoulos, V. et al., Pharmacol. Ther. 2019; Myrianthopoulos, V. Future Med. Chem. 2018 and . Vasanti Suvarnaa, et al., European Journal of Pharmacology (2019)).

According to the invention, a senolytic compound can be selected in the group consisting in but to limited to FOX04-related peptides, BCL-2 inhibitor such as navitoclax (ABT-263) or ABT-737, dasatinib, quercetin or combination of dasatinib and quercetin, tocotrienol, Fisetin, piperlongumine, azithromycin or roxithromycin.

Thus, the invention also relates to a senolytic compound for use in the treatment of a viral lung infection in a subject in need thereof.

As used herein, the term “FOX04-related peptides” refers to a FOX04-derived peptide that is capable of inhibiting FOX04. In particular, the FOX04-related peptides pertubs the FOX04 interaction with p53.

As used herein, the term “FOX04” has its general meaning in the art and refers to a member of the forkhead family transcription factors O subclass, which is characterized by a winged helix domain used for DNA binding.

As used herein, the term "BCL-2 inhibitor" refers to an agent that is capable of inhibiting one or more proteins in the BCL-2 family of anti-apoptotic proteins, e.g., BCL-2, BCL-xL, and BCL-w. In certain embodiments, a BCL-2 inhibitor of the disclosure inhibits one protein of the BCL-2 family selectively, e.g., a BCL-2 inhibitor may selectively inhibit BCL-2 and not BCL- xl or BCL-w.

The BCL-2 inhibitor described herein may inhibit one or more of BCL-2, BCL-xL, and BCL-w. In certain embodiments, the inhibitor of BCL-2 anti-apoptotic family of proteins inhibits BCL-2. In certain embodiments, the inhibitor of BCL-2 anti-apoptotic family of proteins inhibits BCL-2 and does not inhibit other members of the BCL-2 family of proteins, e.g., does not inhibit BCL-xL or BCL-w. In certain embodiments, the BCL-2 inhibitor is aBH3- mimetic.

In certain embodiments, the BCL-2 inhibitor of the disclosure inhibits BCL-xL function. In addition to inhibition of BCL-xL, the inhibitor may also interact with and/or inhibit one or more functions of BCL-2, e.g., BCL-xL/BCL-2 inhibitors. In certain embodiments, a BCL-2 inhibitor of the disclosure inhibits each of BCL-xL and BCL-w. In certain embodiments, a BCL- 2 inhibitor of the disclosure inhibits BCL-xL, BCL-2, and BCL-w. In certain embodiments, a BCL-2 inhibitor interferes with the interaction between the BCL-2 anti-apoptotic protein family member and one or more ligands or receptors to which the BCL-2 anti-apoptotic protein family member would bind in the absence of the inhibitor. In other embodiments, an inhibitor of one or more BCL-2 anti-apoptotic protein family members, wherein the inhibitor inhibits at least one BCL-2 protein specifically, binds only to one or more of BCL-xL, BCL-2, BCL-w and not to other Bcl-2 anti-apoptotic Bcl-2 family members, such as Mcl-1 and BCL2A1.

Binding affinity of a BCL-2 inhibitor for BCL-2 family proteins may be measured. By way of example, binding affinity of a BCL-xL inhibitor may be determined using a competition fluorescence polarization assay in which a fluorescent BAR BI 13 domain peptide is incubated with BCL-xL protein (or other BCL-2 family protein) in the presence or absence of increasing concentrations of the BCL-XL inhibitor as previously described (see, e.g., U.S. Patent Publication 20140005190; Park et al. Cancer Res. 73 :5485-96 (2013); Wang et al., Proc. Natl. Acad Sci USA 97:7124-9 (2000); Zhang et al, Anal. Biochem. 307:70-5 (2002); Bruncko et al, J. Med. Chem. 50:641 -62 (2007)). Percent inhibition may be determined by the equation: 1 - [(mP value of well - negative control)/range)] x 100%. Inhibitor}' constant 0¾) value is determined by the formula: Kj = [I]5o/([h]5o/Ki+[P]o/Ki÷ V) as described in Bruncko et al ., J. Med. Chem. 50:641-62 (2007) (see, also, Wang, FEBS Lett. 360: 111-1 14 (1995)).

Examples of BCL-2 inhibitors include ABT-263 (4-[4-[[2-(4-chlorophenyl)-5,5- dimethylcyclohexen-l-yl]methyl]piperazin-l-yl]-N-[4-[[(2R)-4-mo holin-4-yl-l- phenylsulfanylbutan-2-yl]amino]-3-(trifluoromethylsulfonyl)phenyl]sulfonylbenzamide or IUPAC, (R)-4-(4-((4'-chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[l,r-biphenyl]-2- yl)methyl)piperazin-l-yl)-N-((4-((4-morpholino-l-(phenylthio)butan-2-yl)amino)-3- ((trifluoromethyl)sulfonyl)phenyl)sulfonyl)benzamide) (see, e.g., Park et al., 2008, J. Med. Chem. 51 :6902; Tse et al., Cancer Res., 2008, 68:3421; Int'l Patent Appl. Pub. No. WO2009/155386; U.S. Patent Nos. 7390799, 7709467, 7906505, 8624027) and ABT-737 (4- [4-[(4'- Chloro[l,r-biphenyl]-2-yl)methyl]-l-piperazinyl]-N-[[4-[[(lR)-3-(dimethylamino)-l- [(phenylthio)methyl]propyl]amino]-3-nitrophenyl]sulfonyl]benzamide, Benzamide, 4-[4-[(4'- chloro[l,l'-biphenyl]-2-yl)methyl]-l-piperazinyl]-N-[[4-[[(lR)-3-(dimethylamino)-l- [(phenylthio)methyl]propyl]amino]-3-nitrophenyl]sulfonyl]- or 4-[4-[[2-(4- chlorophenyl)phenyl]methyl]piperazin-l-yl]-N-[4-[[(2R)-4-(dimethylamino)-l- phenylsulfanylbutan-2-yl]amino]-3-nitrophenyl]sulfonylbenzamide) (see, e.g., Oltersdorf et al, Nature, 2005, 435:677; U.S. Pat. No. 7973161; U.S. Pat. No. 7642260). In other embodiments, the BCL-2 inhibitor is a quinazoline sulfonamide compound {see, e.g., Sleebs et al., 2011, J. Med. Chem. 54: 1914). In still another embodiment, the BCL- inhibitor is a small molecule compound as described in Zhou et al, J Med. Chem., 2012, 55:4664 {see, e.g., Compound 21 (R)-4-(4-chlorophenyl)-3-(3-(4-(4-(4-((4-(dimethylamino)-l- (phenylthio)butan-2-yl)amino)-3-nitrophenylsulfonamido)phenyl)piperazin-l-yl)phenyl)-5- ethyl-1 -methyl- lH-pyrrole-2-carboxylic acid) and Zhou et al., J Med. Chem., 2012, 55:6149 {see, e.g., Compound 14 (R)-5-(4-Chlorophenyl)-4-(3-(4-(4-(4-((4-(dimethylamino)-l-

(phenylthio)butan-2-yl)amino)-3 -nitrophenylsulfonamido)phenyl)piperazin- 1 -yl)phenyl)- 1 - ethyl-2-methyl-lH-pyrrole-3-carboxylic acid; Compound 15 (R)-5-(4-Chlorophenyl)-4-(3-(4- (4- (4-((4-(dimethylamino)-l-(phenylthio)butan-2-yl)amino)-3- nitrophenylsulfonamido)phenyl)piperazin-l-yl)phenyl)-l-isopropyl-2-methyl-lH-pyrrole-3- carboxylic acid). In other embodiments, the BCL- inhibitor is a BCL-2/BCL-xL inhibitor such as BM-1074 {see, e.g., Aguilar et al., 2013, J. Med. Chem. 56:3048); BM-957 {see, e.g., Chen et al., 2012, J. Med. Chem. 55:8502); BM-1197 {see, e.g., Bai et al., PLoS One 2014 Jun 5;9(6):e99404. Doi: 10.1371/journal.pone. 009904); U.S. Patent Appl. No. 2014/0199234; N- acylsufonamide compounds (see, e.g., Int'l Patent Appl. Pub. No. WO 2002/024636, Int'l Patent Appl. Pub. No. WO 2005/049593, Int'l Patent Appl. Pub. No. WO 2005/049594, U.S. Pat. No. 7767684, U.S. Pat. No. 7906505). In still another embodiment, the BCL-2 inhibitor is a small molecule macrocyclic compound (see, e.g., Int'l Patent Appl. Pub. No. WO 2006/127364, U.S. Pat. No. 7777076). In yet another embodiment, the BCL-2 inhibitor is an isoxazolidine compound (see, e.g., Int'l Patent Appl. Pub. No. WO 2008/060569, U.S. Pat. No. 7851637, U.S. Pat. No. 7842815). In yet another embodiment, the BCL-2 inhibitor is S44563 (see, e.g., Loriot et. al., Cell Death and Disease, 2014, 5, el423). In one embodiment, the BCL-2 inhibitor is (R)- 3-((4'-chloro-[l,r-biphenyl]-2-yl)methyl)-N-((4-(((R)-4-(dimethylamino)-l-(phenylthio)butan- 2-yl)amino)-3-nitrophenyl)sulfonyl)-2,3,4,4a,5,6-hexahydro-lH-pyrazino[l,2-a]quinoline-8- carboxamide. In another embodiment, the BCL-2 inhibitor is a small molecule heterocyclic compounds (see, e.g.,XJ.S. Pat. No. 9018381).

According to the invention, “senomorphic compound” can be selected in the group consisting in but to limited to SASP inhibitors (e.i. compounds interfering with pro- inflammatory Senescence Associated Secretory Phenotype (SASP)) production, including the glucocorticoids, the statins such as simvastatin, that can reduce the expression of pro- inflammatory cytokines (IL-6, IL-8, and MCP-1), the JAK1/2 inhibitors such as ruxolitinib, the NF-KB and p38 inhibitors, the IL-la blockers and the inhibitors of mTOR like rapamycin. Thus, the invention also relates to a senomorphic compound for use in the treatment of a viral lung infection in a subject in need thereof.

According to the invention, the term “subject” denotes a mammal, such as a rodent, a feline, a canine, and a primate. In some embodiments, the subject is a human. In some embodiments, the subject is a human infant. In some embodiments, the subject is a human child. In some embodiments, the subject is a human adult. In some embodiments, the subject is an elderly human. In some embodiment, the subject is an elderly human over 60, 65, 70, 75, 80 or 85 year old. In some embodiments, the subject is a premature human infant. Particularly, the subject denotes a human with a pathogen lung infection. Particularly, the subject denotes a human with a viral lung infection. In a particular embodiment the subject is a human with co morbidities and in the elderly (see for example Guan et al., 2020). In particular embodiment, the subject suffers from viral infection. In particular embodiment, the subject suffers from viral lung infection, and more particular from viral lung infection induced by a SARS-CoV-2 infection. In particular embodiment, the subject suffers from viral lung infection and is over 60, 65, 70, 75, 80 or 85 year old.

In some embodiments, the subject can be symptomatic or asymptomatic. As used herein, the term "asymptomatic" refers to a subject who experiences no detectable symptoms for the coronavirus infection. As used herein, the term "symptomatic" refers to a subject who experiences detectable symptoms of a pathogen lung infection and particularly a coronavirus infection. Symptoms of coronavirus infection include: fatigue, anosmia, headache, cough, fever, difficulty to breathe.

In certain embodiments, the subject may suffer from respiratory failure corresponding to the severe form of infection by the pathogen and requiring hospitalization in an intensive care unit and assisted ventilation.

In a particular embodiment, the senotherapeutic compound of the invention can be administrated orally, intra-nasally, parenterally, intraocularly, intravenously, intramuscularly, intrathecally, or subcutaneously to subject in need thereof. The senotherapeutic compound of the invention may also be administrated by inhalation.

In particular embodiment, the senotherapeutic compound of the invention is administrated by systemic administration. As used herein, the term “systemic administration” has its general meaning in the art and refers to a route of administration of medication into the circulatory system so that the entire body is affected.

Ideally, the senotherapeutic compound is administrated to the subject in prevention, before the apparition of the symptoms of the pathogen lung infection, during the acute phase of the infection, or after complete clearance of the virus, at the time of persisting symptomatic or non- symptomatic lung dysfunction.

Particularly, the senotherapeutic compound is administrated to the subject after 5, 6, 7 days or more after the infection.

In specific embodiments, it is contemplated that the compounds of the present invention may be modified in order to improve their therapeutic efficacy and/or their specificity. Such modification of therapeutic compounds may be used to decrease toxicity, increase circulatory time, or modify biodistribution. For example, the toxicity of potentially important therapeutic compounds can be decreased significantly by combination with a variety of drug carrier vehicles that modify biodistribution.

A strategy for improving drug viability is the utilization of water-soluble polymers. Various water-soluble polymers have been shown to modify biodistribution, improve the mode of cellular uptake, change the permeability through physiological barriers; and modify the rate of clearance from the body. To achieve either a targeting or sustained-release effect, water- soluble polymers have been synthesized that contain drug moieties as terminal groups, as part of the backbone, or as pendent groups on the polymer chain.

Polyethylene glycol (PEG) has been widely used as a drug carrier, given its high degree of biocompatibility and ease of modification. Attachment to various drugs, proteins, and liposomes has been shown to improve residence time and decrease toxicity. PEG can be coupled to active agents through the hydroxyl groups at the ends of the chain and via other chemical methods; however, PEG itself is limited to at most two active agents per molecule. In a different approach, copolymers of PEG and amino acids were explored as novel biomaterials which would retain the biocompatibility properties of PEG, but which would have the added advantage of numerous attachment points per molecule (providing greater drug loading), and which could be synthetically designed to suit a variety of applications.

Those of skill in the art are aware of PEGylation techniques for the effective modification of drugs. For example, drug delivery polymers that consist of alternating polymers of PEG and tri-functional monomers such as lysine have been used by VectraMed (Plainsboro, N. J.). The PEG chains (typically 2000 daltons or less) are linked to the a- and e-amino groups of lysine through stable urethane linkages. Such copolymers retain the desirable properties of PEG, while providing reactive pendent groups (the carboxylic acid groups of lysine) at strictly controlled and predetermined intervals along the polymer chain. The reactive pendent groups can be used for derivatization, cross-linking, or conjugation with other molecules. These polymers are useful in producing stable, long-circulating pro-drugs by varying the molecular weight of the polymer, the molecular weight of the PEG segments, and the cleavable linkage between the drug and the polymer. The molecular weight of the PEG segments affects the spacing of the drug/linking group complex and the amount of drug per molecular weight of conjugate (smaller PEG segments provides greater drug loading). In general, increasing the overall molecular weight of the block co-polymer conjugate will increase the circulatory half- life of the conjugate. Nevertheless, the conjugate must either be readily degradable or have a molecular weight below the threshold-limiting glomular filtration (e.g., less than 45 kDa).

The invention also relates to a method for treating a pathogen lung infection in a subject in need thereof comprising administering to said subject in need thereof a therapeutically effective amount of a senotherapeutic compound.

As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subjects at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).

Therapeutic composition

A second object of the invention relates to a therapeutic composition comprising a senotherapeutic compound for use in the treatment of a pathogen lung infection in a subject in need thereof.

Any therapeutic agent of the invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.

"Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

The form of the pharmaceutical compositions, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the patient, etc.

The pharmaceutical compositions of the invention can be formulated for a topical, oral, intranasal, intrathecal, parenteral, intraocular, intravenous, intramuscular, hippocampal stereotactic or subcutaneous administration and the like.

In particular embodiment, the pharmaceutical compositions of the invention is formulated for systemic administration.

Preferably, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.

The doses used for the administration can be adapted as a function of various parameters, and in particular as a function of the mode of administration used, of the relevant pathology, or alternatively of the desired duration of treatment.

In addition, other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; time release capsules; and any other form currently can be used.

Pharmaceutical compositions of the present invention may comprise a further therapeutic active agent useful to treat the pathogen lung infection or the symptoms induced by the pathogen lung infection. For example, further agent may be selected in the group consisting bronchodilators like b2 agonists and anticholinergics, corticosteroids, beta2-adrenoceptor agonists like salbutamol, anticholinergic like ipratropium bromide or adrenergic agonists like epinephrine. Further agent may be also an antiviral compound like amantadine, rimantadine or pleconaril.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1. Weight loss and viral load during IAV infection. Mice were intranasally (i.n.) infected with 50 mΐ of PBS containing (or not, in a mock sample) 100 p.f.u. of H1N1 A/Califomia/04/2009 (pdm09) (Barth elemy 2018, Sencio 2020). This dose corresponds to a sub-lethal dose, which is necessary to investigate long term effect of infection. Body weight loss was monitored daily after IAV infection and mice were euthanized when they lost in excess of 20% of their initial body weight. Infected mice were killed at different time points p.L (A), Kinetic measurement of weight change (in % initial body weight, means ± SD) of IAV-infected mice. (B) IAV Ml mRNA levels were measured in the whole lungs by quantitative RT-PCR. Data are expressed as delta Ct values. The dashed line represents the detection threshold (n = 8, a representative experiment out of four is shown).

Figure 2. Induction of lung cell senescence following IAV infection in mice. (A) Changes in thoracic bioluminescence in pl6-luciferase knock-in mice following IAV infection. Mice expressing luciferase under the control of p!6 (p!6-luc mice) were used to monitor the appearance of lung senescent cells following IAV infection. Bioluminescence was then measured using the IVIS system. The scale indicates the average radiance : the sum of the photons per second from each pixel inside the ROI/number of pixels (photons/sec/cm2/sr). Thoracic bioluminescence increased as early as day 4 following IAV infection and continued to rise until day 28 (n=4). (B) Changes in the number of pl6 stained cells expressed as the number of stained nuclei over the total number of nuclei counted in at least four lung sections per mouse, (C) changes in the lung mRNA levels of the senescence markers pl6 and p21, and (D) changes in the lung mRNA levels of members of the senescent secretory phenotype osteopontin, IL-6 and IL-Ib at different times in lungs from mice subjected to IAV infection (n= 5-8).

Figure 3. Graph showing mean linear intercept of alveolar septa and representative lung sections showing emphysema lesions in mice infected by IAV (day 90 post-infection) in comparison with control mice (n=5-7). Results are expressed as the mean ± SD. Significant differences were determined using the Mann- Whitney U test. (*p < 0.05).

Figure 4. Effect of eliminating senescent pl6 expressing cells in pl6-ATTAC mice subjected to IAV infection and treated with the activator of the killer gene (AP20187). Mice were treated with AP20187 (0.5pg/g i.p. two times a week) or vehicle from day 1 to day 28. (A) Reduction of the lung protein levels of the senescence markers pl6 and p21 and of the DNA damage marker gH2AC (day 28 post-infection) (B) Reduction of emphysema lesions in AP20187-treated (0.5 pg/g 2x/week) mice compared to control (Ve/Veh) mice illustrated by a decrease in mean linear intercept of alveolar septa and. A and B, n= 5-7 (day 28 post-infection).

Figure 5. Effect of eliminating senescent cells by treating wild-type mice subjected to IAV infection with the senolytic molecule navitoclax. Mice were treated with navitoclax 5 times a week (50mg/kg/day by, gavage) or with vehicle from day 1 to day 28. (A) Reduction of the lung protein levels of the senescence markers pl6 and p21 and of the DNA damage marker gH2AC; (B) lung mRNA levels of the senescence markers p21and pl6 and of the SASP members IL6, IL8, MCP-1 and osteopontin in IAV-infected mice treated with Navitoclax in comparison with mice treated with vehicle (n=4). Results are expressed as the mean ± SD. Significant differences were determined using the Mann-Whitney U test. (**p < 0.01).

Figure 6. Effects of senescent cell’s depletion on lung emphysema and lung fibrosis development 1 month and 3 months after IAV infection, pi 6- ATT AC mice were infected with IAV (H1N1, 500 pfu, intranasal route) and senescent cells were depleted by activating the killer gene construct ATT AC with the drug AP20187. Lung emphysema as assessed by measurement of the mean linear intercept (MLI) (A) and lung fibrosis as assessed by the modified Ashcroft score (B). *P<0.05, **P<0.01 for comparison between group means as indicated.

Figure 7. Single cell RNAseq from COVID-19 patients revealed increased expression of senescence markers in epithelial cells. (A) In BALF obtained within 10 days after symptom onset from the GSE145926 dataset (Liao M et al, 2020), mRNA of the senescence marker CDKN2A, encoding pi 6, was mainly detected in epithelial cells, macrophages, and T cells, with higher levels in epithelial cells from patients with severe/critical COVID-19 infection compared to controls. (B-D) Expression of several senescence markers (i.e., (B) CDKN2A, CDKN1A, (C) uPAR, CXCL8, (D) IGFBP3, and GDF15) is significantly increased in ciliated and club cells in BALFs from patients with severe COVID-19 lung illness compared to patients with a moderate form and to healthy controls. Statistical tests were performed using the MAST package (Finak G et al. Genome Biology 2015) and reported values correspond to adjusted P values.

EXAMPLE:

Material & Methods

Mice and ethics statement

Specific pathogen-free C57BL/6J mice (8 week-old, male) were purchased from Janvier (Le Genest-St-Isle, France). Mice were maintained in a biosafety level 2 facility in the Animal Resource Center at the Lille Pasteur Institute for at least two weeks prior to usage to allow appropriate acclimatation. All experiments complied with current national and institutional regulations and ethical guidelines (Institut Pasteur de Lille/B59-350009 and CEEA 75. Nord Pas-de-Calais). All experiments were approved by the Ministere de FEducation Nationale, de FEnseignement Superieur et de la Recherche, France (00357.03 and APAFIS 13743- 2018022211144403).

Mice that express luciferase under the control of pl6 (pl6-luc mice) were used to monitor the appearance of lung senescent cells following IAV infection. P16-luc mice were obtained fromN-E Sharpless (Chapel Hill, NC, USA) by S Adnof s team and are currently being bred at the CNRS Orleans animal facility.

To genetically eliminate senescent cells in mice, we generated mice expressing a killer gene construct driven by the pl6 promoter (pi 6- ATT AC mice). Cells expressing the FKBP- Caspase8 construct are killed when the mice are given a rapalog (AP20187) that leads to caspase 8 activation. We have verified that AP20187 treatment did not induce side effects in our mouse models. The pl6-ATTAC mice were generated in France (M Do Cruzeiro, Plate- forme de Recombinaison Homologue, Inserm 1016-CNRS, Paris) and have been used and validated by S Adnot's team. The FKBP-Caspase8 construct was provided by PE Sherer (Dallas, USA) and has been inserted into the Rosa gene locus under the control of the pl6 INK promoter.

Infections with IAV

Mice were anesthetized by intramuscular injection of 1.25 mg of ketamine plus 0.25 mg of xylazine in 100 mΐ of phosphate buffered saline (PBS), and then intranasally (i.n.) infected with 50 mΐ of PBS containing (or not, in a mock sample) 100 p.f.u. of H1N1 A/Califomia/04/2009 (pdm09) (Barth elemy 2018, Sencio 2020). This dose corresponds to a sub-lethal dose, which is necessary to investigate long term effect of infection. Body weight loss (in % initial body weight) was monitored daily after IAV infection and mice were euthanized when they lost in excess of 20% of their initial body weight. Infected mice were killed at different time points p.i..

Quantification of viral loads and assessment of gene expression by quantitative RT-PCR

Total RNA from lung tissues were extracted with the NucleoSpin® RNA kit (Macherey- Nagel, Hoerdt, Germany). RNA was reverse-transcribed with the High-Capacity cDNA Archive Kit (Life Technologies, USA). The resulting cDNA was amplified using SYBR Green- based real-time PCR and the QuantStudio™ 12K Flex Real-Time PCR Systems (Applied Biosystems™ , USA) following manufacturers protocol. Relative quantification was performed using the gene coding glyceraldehyde 3-phosphate dehydrogenase (Gapdh). Specific primers were designed using Primer Express software (Applied Biosystems, Villebon sur Yvette, France). Relative mRNA levels (2-D DCt) were determined by comparing (a) the PCR cycle thresholds (Ct) for the gene of interest and the house keeping gene Gadph (DCt) and (b) DCt values for treated and control groups (□ DCt). Data are expressed as a fold-increase over the mean gene expression level in mock-treated mice. Quantification of viral RNA was performed as described in (Paget 2011). Viral load is expressed as viral RNA normalized to gapdh expression level. Data were normalized against expression of the gapdh gene and were expressed as Ct.

Animal preparation lung emphysema assessments and lung tissue analysis Mice were anesthetized with an intraperitoneal injection of ketamine (60 mg/kg) and xylazine (10 mg/kg). The right lung was quickly removed and divided into two parts, which were immediately snap-frozen in liquid nitrogen then stored at -80°C until total RNA and protein extraction for real-time polymerase chain reaction, Western blot, and ELISA, as well as immunostaining. The left lungs were fixed by intratracheal infusion of 4% paraformaldehyde aqueous solution (Sigma Aldrich) at a transpleural pressure of 30 cmH O. For morphometry studies, 5-pm thick sagittal sections along the greatest axis of the left lung were cut in a systematic manner and stained with hematoxylin and eosin. Alveolar, bronchial and vascular morphometry studies were performed by an observer blinded to treatment.

Lung emphysema was assessed by measuring the mean-linear-intercept (MLI) method described by Knudsen L et al (6). Light microscope fields were quantitated at an overall magnification of 400, using a 42-point and 21-line grid. We examined 20 fields/animal (10 per lung), using a systematic sampling method from a random starting point. To correct area values for shrinkage associated with fixation and paraffin processing, we used the factor of 1.22 calculated during a previous study.

Immunofluorescence

Paraffin-embedded sections were deparaffmized using xylene and a graded series of ethanol dilutions then incubated in citrate buffer (0.01 M, pH 6) at 90°C for 20 minutes. Tissues were permeabilized with 0.1% Triton X-100 in phosphate-buffered saline (PBS) for 10 minutes.

For immunofluorescence, paraffin-embedded sections were prepared as described above; cells were fixed for 10 minutes in 1% formalin in PBS then permeabilized with 0.05% Triton X-100 in PBS for 10 minutes. Saturation was achieved using Dako antibody diluents with 10% goat serum. For double staining, first and second primary antibodies were diluted in Dako antibody diluents with 3% goat serum then incubated for 1 hour at 37°C in a humidified chamber. After PBS washes, the sections were covered with secondary antibody (mixed with mouse, rabbit, rat, or chicken Alexa Fluor^® 480, Alexa Fluor^® 555, or Alexa Fluor^® 660 [Abeam, Cambridge, UK]) for 40 min at 37°C in a humidified chamber. After 5 minutes of staining with DAPI, the sections were protected with coverslips secured with fluorescent mounting medium. The percentage of pl6 stained cells was determined by dividing the number of pl6 stained nuclei over the total number of DAPI stained nuclei counted in at least four lung sections per mouse.

Protein extraction and immunoblotting

Total proteins were extracted using RIPA lysis buffer (10 mM sodium phosphate pH 8, 150 mM NaCl, 1% sodium deoxycholate, 1% NP40, 0.5% SDS, 1 mM PMSF, 10 mM NaF, 1 mM sodium orthovanadate, and protease inhibitor cocktail [Roche, Meylan, France]). Immunoblots were carried out using the indicated antibodies and detected using an enhanced chemiluminescence detection system (GE Healthcare, Little Chalfont, UK). Densitometric quantification was normalized for the b-actin or GAPDH level using GeneTools software (Ozyme, Montigny le Bretonneux, France).

Materials Senolytic drugs. The senolytic molecule navitoclax, which induces senescent-cell apoptosis by counteracting the antiapoptotic function of Bcl-2 and Bcl-xL and used in these studies, was purchased from Med ChemTronica (Sweden) source AP20187??

The following primary antibodies were used: anti-beta-Actin (b-Act, A5316, Sigma); anti-alpha smooth muscle actin (a-SMA, ab5694, Abeam); GAPDH (sc-25778); anti- CDK2A/p 16INK4a (pl6, ab54210, Abeam; pl6, 92803, Cell signaling; pl6, PA520379, Thermoscientific); anti-p21 Wafl/Cipl (p21, 2946, Cell Signaling Technology [Danvers, MA]); anti phospho-gamma-H2AX (ab 9718, Cell Signaling Technology).

Statistical analysis

Results are expressed as the mean ± standard deviation (SD) unless otherwise stated. All statistical analysis was performed using GraphPad Prism 7 software following the guidelines in GraphPad Prism. A Mann- Whitney U test was used to compare two groups unless otherwise stated. P values less than 0.05 were considered significant.

Results

1) IAV infection associates with lung cell senescence

As a first approach, publicly available datasets from human bronchial epithelial cells (GSE71766) and mouse epithelial alveolar cells sorted from infected mice (GSE57008), three days after IAV infection in both cases were extracted. Gene Set Enrichment Analysis (GSEA) was performed using the GSEA v2.0.13 software with default parameters (www.broadinstitute.org/gsea/). Strikingly, IAV infection resulted in enrichment in cell senescence-related genes (Fridman senescence up signature, normalized enrichment score (NES) = 1.948, FDR q-val = 0.0054 for human cells and NES = 1.730, FDR q-val = 0.0011 for mouse cells) and aging-related genes (Demagalhaes aging up signature, NES = 1.949, FDR q- val = 0.0054 for human cells and NES = 1.954, FDR q-val = 0.0000 for mouse cells).

To assess the extent of IAV-induced lung-cell senescence and lung damage, we investigated mice exposed to a sublethal dose of HlNlp2009 at different time points. IAV infection in C57BL/6 mice led to a body weight loss which peaked on day 8, followed by a rapid recovery on day 14 (Fig. 1 A). Lung IAV Ml mRNA levels as expressed as delta Ct values, became detectable on day 3, were maximum on day 7 and become undetectable again on day 14 (Fig. IB). Mice expressing luciferase under the control of the pl6 promoter were then infected with IAV to monitor thorax bioluminescence. As depicted in Figure 2A, an elevation of lung luminescence was evidenced in pl6-luciferase mice as early as day 4 post-infection. We then investigated the number of pi 6-positive cells in lung sections by immunofluorescence studies and expressed the number of pl6 stained cells as the number of stained nuclei over the total number of nuclei counted in at least four lung sections per mouse (Figure 2B). Interestingly, IAV infection in C57BL/6 mice led to the accumulation of pi 6-positive cells detected in the lungs on day 4 and increasing until day 28 post-infection to reach 40% of total lung cells (Figure 2B).. Collectively, these data show that IAV infection associates with accumulation of senescent cells in the lung compartment. Of note, lung senescent cells persisted after virus clearance was complete.

Together with this progressive accumulation of lung senescent cells, we measured an early rise of lung p21 (as early as day 3) and pi 6 (as early as day 7) mRNA levels which coincided with the peak of viral load but remained elevated until day 28 (Fig. 2C). A similar elevation in mRNA levels of some of the SASP components was measured, notably osteopontin, IL-6 and ILl-b (Fig.2D). It is noticeable that these transcripts remained more elevated at day 90 post-infection.

Histological examination of the day-28 lungs revealed severe remodeling with structural bronchial and alveolar damage, inflammatory infiltrates, airway epithelium abrasion, emphysema and fibrotic lesions (data not shown). Interestingly, the lung areas with the greatest lung damage were also the richest in pl6-stained cells. An important feature was the complete disappearance of the airway epithelium on day 7 post-infection, which persisted on day 28 and coincided with the presence of numerous senescent cells identified as pi 6-positive cells in the surrounding airway walls (data not shown). Moreover, emphysema lesions developed as early as day 28 (Fig. 3) and were also accompanied with numerous pl6-stained cells at the alveolar epithelial level, indicating a temporal and topographic relation between the pulmonary lesions and senescent cell accumulation.

2) Genetic or pharmacological elimination of senescent lung cells reduced lung damage and inflammation in IAV infected mice

To assess the effects of eliminating senescent cells, we first investigated pl6-INK- ATTAC mice that express the inducible suicide gene (caspase 8) that can be activated with the rapalog AP20187 (provider: clinisciences, HY-13992). In pl6-INK-ATTAC mice, treatment with AP20187, started at the time of IAV infectioneffectively eliminated pl6 expressing cells as shown by complete disappearance of pl6 stained cells in lungs from IAV infected mice (data not shown). This associatedwith a reduction in lung P16 and p21 protein levels and a reduction in the DNA damage marker gamma-H2AX protein (Fig 4 A). Moreover, senescent cell elimination allowed complete airway epithelium repair at this post-infection time-point (not shown), with a reduction in lung emphysema lesions as shown by normalization of the mean linear intercept (used as an index of lung emphysema) in pl6ATTAC mice treated with AP20187 (Fig. 4B).

Then the Effects of senescent cell’s depletion on lung emphysema and lung fibrosis development 1 month and 3 months after IAV infection was assessed.

The results show that lung emphysema and lung fibrosis are maximal at 1 month post infection but still persisted at 3 months post-infection (Figure 6 A and 6B). Mice treated with AP20187 were partially protected from lung emphysema and lung fibrosis at 1 month and were free of lung emphysema or of lung fibrosis at 3 months (Figure 6A and 6B).

To test whether pharmacological elimination of senescent cells in wild-type mice exhibited similar impacts as genetic interventions in pl6-ATTAC mice, we used the senolytic drug ABT263 (navitoclax), which induces senescent-cell apoptosis by counteracting the antiapoptotic function of Bcl-xL. Navitoclax given at the dose of 50mg/kg/day per gavage in wild-type mice infected by IAV and studied on day 28 effectively suppressed the accumulation of lung pl6-stained cells (immunofluorescence images not shown), together with a decrease in lung pi 6, p21 and gH2AX protein levels (Fig. 5 A). Navitoclax treatment also lead to complete airway epithelium recovery at this post-infection time-point (not shown), with a reduction in lung emphysema lesions (Data not shown) and together with a reduction in lung mRNA levels of pl6 and p21 as well as in mRNA levels of the SASP members IL6, IL8, ILl-b and osteopontin (Fig 5B).

Thus, eliminating senescent cells via suicide gene or senolytic strategies limited the severity of IAV infection, improved recovery, and protected against short- and long-term lung structural lung alterations. Such strategy could be useful to treat respiratory viral infection in general, including SARS-CoV-2), the etiologic agent of the new lung disease COVID-19.

EXAMPLE 2:

Material &Methods

First, we extracted data from publicly available datasets of scRNA-seq in BALF cells from patients with moderate (n=3) or severe/critical infection (n=6) versus three healthy controls (6) and analysed senescence-related genes. To further assess SARS-CoV-2-induced lung-cell senescence, we investigated lung samples from 3 year-old female cynomolgus macaques infected with 106 pfu of a SARS-CoV-2 clinical isolate (BetaCoV/France/IDF/0372/2020) or vehicle, by the combined intranasal and intratracheal route. All animal experiments were approved by the Animal Care and Use Committee of the French National Institute of Health and Medical Research. We have described the viral load characteristics and clinical and radiological parameters in this model (7). Two macaques were sacrificed at 4 days post-infection (dpi.) and two others at 30 dpi. Lung immunohistochemical analyses were performed using N-Histofme Simple Stain MAX PO (H1410I, Nichirei Bioscience Inc.) with anti-dsRNA-J2 used as primary anti-SARS-Cov-2 antibodiy (1:1000, RNT-SCI-10010200, Jena Bioscience). Primary antibodies for immunofluorescence were anti- pl6 (ab54210, Abeam); anti-p21 (ABIN6939038, antibodies-online GmbH); anti-mucinl (MUC1 abl09185, Abeam); anti-Von Willebrand Factor (vWF) (abl 17132, Abeam); anti- 53BP1 (NB 100-304, Novusbio); and anti-y-H2AX (MA5-33062, RRID AB-2810155, Thermo Fisher Scientific); secondary antibodies were anti-rabbit Alexa Fluor® 555 and anti-mouse Alexa Fluor® 660 (Invitrogen).

Results

In BALF obtained within 10 days after symptom onset, mRNA of the senescence marker CDKN2A, encoding pi 6, was mainly detected in epithelial cells, macrophages, and T cells, with higher levels in epithelial cells from patients with severe/critical COVID-19 infection compared to controls (Figure 7A). The expression of several senescence markers, i.e., CDKN2A, CDKN1A (encoding p21), Urokinase Plasminogen Activator Surface Receptor (uPAR), CXCL8, IGFBP3, and GDF15 was significantly increased in epithelial ciliated and club cells in BALF from patients with severe COVID-19 compared to those with moderate disease and to healthy controls, suggesting that lung-cell senescence induction was contemporary of viral detection (Fig 7B-D). To further assess the extent of SARS-CoV-2- induced lung-cell senescence and the fate of senescent lung cells over time, we investigated macaques 4 and 30 dpi, corresponding respectively to the viral load peak and to the absence of detectable viral RNA in BALF. Immunohistochemical studies of lung sections at 4 dpi revealed SARS-CoV-2 antigen-stained cells, including lung endothelial cells (ECs) and parenchymal cells, as well as numerous pi 6- and p21 -immunofluorescence-stained cells predominating at sites of alveolar damage. SARS-CoV-2 antigen-stained cells were rarer at 30 dpi, contrasting with massive accumulation of pi 6- and p21 -positive cells throughout the lung at this time, indicating that senescent lung cells persisted after virus clearance (data not shown).

Interestingly, the lungs at 30 dpi no longer contained the consolidated parenchymal areas seen at 4 dpi but showed extensive lung parenchyma remodelling, with thickening of the alveolar and pulmonary vessel walls and abundant extracellular matrix deposits as assessed by collagen staining (not shown). These advanced lesions were accompanied with massive accumulation of pi 6- and p21 -positive cells, mostly alveolar type II cells and ECs, as shown by double-immunofluorescence staining for pl6 and mucinl (MUC 1) and for von vWF, respectively (data not shown). Of note, pl6 staining of most ECs was seen in many pulmonary vessels, notably those occluded by thrombosis and showing intraluminal vWF staining. Cells stained for pl6 were also stained for the DNA damage markers g-H2AC protein and p53- binding protein 1. Collectively, our data constitute the first evidence of temporal and topographic relations between senescent-cell accumulation and pulmonary lesions induced by SARS-CoV-2 infection.

Cell senescence is usually viewed as a process occurring in response to chronic stressors that critically impedes healthy aging and promotes age-related noncommunicable diseases (Baker et al, 2016). Here, BALF cells from patients experiencing severe COVID-19 expressed high levels of senescent-cell markers. This original observation was confirmed in a macaque model of COVID-19: massive accumulation of senescent lung cells occurred early in areas of severe lung damage due to SARS-CoV-2 infection. Moreover, senescent cells persisted in the lungs over time, and new senescent cells appeared concomitantly with the development of sustained lung alterations including remodeling of the alveolar septa and pulmonary vessels. Given the deleterious effect of cell senescence on tissue repair and inflammation, these results suggest that senescent-cell accumulation may contribute to the early lung alterations caused by SARS-CoV-2 infection and, potentially, to the post-viral lung pathology that develops in a substantial number of individuals (Guler et al, 2021). A causal relationship between EC senescence and vascular thrombosis may also be suspected, as most ECs were senescent in thrombosed vessels. We suggest that counteracting the cell- senescence process or eliminating senescent lung cells might lessen lung damage severity. This may be of therapeutic importance since strategies are now proposed to control senescence in various lung diseases (Chang et al, 2016). This new concept may apply not only to chronic age-related diseases such as lung fibrosis or emphysema, but also to infections with respiratory viruses such as the coronavirus SARS-CoV-2.

REFERENCES:

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure. Ballinger, M.N., and Standiford, T.J. (2010). Postinfluenza bacterial pneumonia: host defenses gone awry. J. Interferon Cytokine Res. Off. J. Int. Soc. Interferon Cytokine Res. 30, 643-652.

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Claims

CLAIMS:

1. A senotherapeutic compound for use in the treatment of a pathogen lung infection in a subject in need thereof.

2. A senotherapeutic compound for use in the inhibition / suppression of senescent lung cells in a treatment of a pathogen lung infection of the respiratory tract in a subject in need thereof.

3. A senotherapeutic compound for use according to claims 1 or 2 wherein the pathogen lung infection is induced by a pathogen selected in the group consisting in virus, bacterium, protozoan, prion, viroid, or fungus.

4. A senotherapeutic compound for use according claim 3 wherein the virus can be the Influenza A virus (IAV), the human rhinovirus (HRV), the Human respiratory syncytial virus (HRSV) or a coronavirus.

5. A senotherapeutic compound for use according claim 4 wherein the coronavirus is the MERS-CoV, the SARS-CoV or the SARS-CoV-2.

6. A senotherapeutic compound for use according claims 1 to 5 wherein the senotherapeutic compound is a senolytic compound.

7. A senotherapeutic compound for use according claim 6 wherein the senolytic compound is selected in the group consisting in F0X04-related peptides, BCL-2 inhibitor such as navitoclax (ABT-263) or ABT-737, dasatinib, quercetin or combination of dasatinib and quercetin, tocotrienol, Fisetin, piperlongumine, azithromycin or roxithromycin.

8. A senotherapeutic compound for use according claims 1 to 5 wherein the senotherapeutic compound is a senomorphic compound.

9. A senotherapeutic compound for use according claim 8 wheren the senomorphic compound is in the group consisting in SASP inhibitors production, including the glucocorticoids, the statins such as simvastatin, the JAK1/2 inhibitors such as ruxolitinib, the NF-KB and p38 inhibitors, the IL-la blockers and the inhibitors of mTOR like rapamycin.

10. A therapeutic composition comprising a senotherapeutic compound for use in the treatment of a pathogen lung infection in a subject in need thereof.

11. A method for treating a pathogen lung infection in a subject in need thereof comprising administering to said subject in need thereof a therapeutically effective amount of a senotherapeutic compound.