WO2022008597A1

WO2022008597A1 - Methods and pharmaceutical composition for the treatment of infectious diseases

Info

Publication number: WO2022008597A1
Application number: PCT/EP2021/068845
Authority: WO
Inventors: Patrick AUBERGER; Paul CHAINTREUIL; Océane DUFIES; Johan COURJON; Laurent Boyer; Arnaud JACQUEL
Original assignee: INSERM (Institut National de la Santé et de la Recherche Médicale); Centre Hospitalier Universitaire De Nice; Universite Cote D'azur
Priority date: 2020-07-08
Filing date: 2021-07-07
Publication date: 2022-01-13

Abstract

The invention relates to a pan caspase inhibitor for use in the treatment of lung infection (such as Syndrome-Corona Virus (SARS-CoV) infection and SARS-COV related disorders) in a subject in need thereof. The current COVID-19 pandemic is predicted to last several additional months, further increasing mortality rates, especially in the more fragile populations. Inventors have surprisingly found that Emricasan is able to efficiently inhibit caspase-8 which is involved in the inflammation process. More particularly, inventors have shown that Emricasan blocks caspase-1 activation in monocytes from healthy donor or COVID19 patient. This new finding with either Emricasan alone or in combination with other therapeutics seem to be very promising in patients suffering from an infectious disease, more particularly an infection caused by Syndrome-Corona Virus.

Description

METHODS AND PHARMACEUTICAL COMPOSITION FOR THE TREATMENT OF INFECTIOUS DISEASES

FIELD OF THE INVENTION:

The present invention is in the field of infectiology. More particularly, the invention relates to methods and compositions for treatment of Severe Acute Respiratory Syndrome caused by Coronavirus infection (SARS-CoV-2) during COVID-19.

BACKGROUND OF THE INVENTION:

Coronaviruses cause severe diseases mainly of the respiratory and gastrointestinal tract. The infection of humans with coronaviruses have been known since the sixties to be associated with respiratory tract i.e. common cold-like diseases. Severe Acute Respiratory Syndrome- Corona Virus (SARS-CoV) is a highly aggressive in humans with often fatal outcome. In late December 2019, a new betacoronavirus SARS-CoV-2 has emerged in Wuhan China. The World Health Organization has named the severe pneumonia caused by this new coronavirus COVID-19 (for Corona Virus Disease 2019, WHO, 2020). Since its emergence, the COVID- 19 has spread to 159 countries across the five continents.

Thus, the COVID-19 pandemic constitutes a major problem for all health systems, with its ongoing extension and possible resurgences, and while considering current mortality, as well as potential morbidity due to fibrotic sequels. Fortunately, there is a mobilization on a scale never before attained worldwide for medical research and with an extensive number of clinical trials conducted on new treatments. Vaccine are being tested in healthy volunteers, but large- scale vaccination cannot be expected before 1 or 2 years.

Thus, there is still an urgent need to find a new treatments of infectious diseases infection, more particularly, lung infection caused by Severe Acute Respiratory Syndrome- Corona Virus (SARS-CoV).

SUMMARY OF THE INVENTION:

The invention relates to methods and compositions for treatment of an infectious disease, more particularly infections caused by Coronaviruses. In particular, the invention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION:

The current COVID-19 pandemic is predicted to last several additional months, further increasing mortality rates, especially in the more fragile populations. In addition, at a longer term, there are major concerns for possible resurgences and for the potential morbidity due to fibrotic sequels. Inventors have surprisingly found that Emricasan is able to efficiently inhibit caspase-8 which is involved in the inflammation process.

This new finding with either Emricasan alone or in combination with other therapeutics seem to be very promising in patients suffering from COVID-19, Syndrome-Corona Virus infection (SARS-CoV-2).

Method for treating infectious disease

Accordingly, in a first aspect, the invention relates to a method for treating an infectious disease in a subject in need thereof comprising a step of administering to said subject a therapeutically effective amount of a pan-caspase inhibitor.

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 patient at risk of contracting the disease or suspected to have contracted the disease as well as patients 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 patient 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 patient 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 patient 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 patient during treatment of an illness, e.g., to keep the patient 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 interval, 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., pain, disease manifestation, etc.]). As used herein, the term “infectious disease” refers to disorders caused by organisms such as bacteria, viruses, fungi or parasites. In a particular embodiment, such organism can cause an infection in different parts of body such as vessels, tissues and/or organs. In a particular embodiment, the infectious disease causes a lung infection. In another embodiment, the infectious disease causes blood infection.

As used herein, the term “lung infection” has its general meaning in the art and means the invasion of lung tissues of a patient by disease-causing microorganisms, their multiplication and the reaction of lung tissues to these microorganisms and the toxins that they produce. In some embodiments, the lung infections include but are not limited to pneumonia (including community -acquired pneumonia, nosocomial pneumonia (hospital-acquired pneumonia, HAP; health-care associated pneumonia, HCAP), ventilator-associated pneumonia (VAP)), ventilator-associated trachebronchitis (VAT), and bronchitis. There are two types of lung infection: chronic lung infection and acute lung infection.

As used herein, the term “chronic lung infection” refers to a long-term infection which may be an apparent, unapparent or latent infection.

As used herein, the term “acute lung infection” has its general meaning in the art and refers to a disease of the lungs characterized by inflammation and consolidation followed by resolution and caused by infection from viruses, fungi, or bacteria. The term is also known as “pneumonia”. Typically acute lung infection is associated with lung inflammation that is the rapid onset of progressive malfunction of the lungs, and is usually associated with the malfunction of other organs due to the inability to take up oxygen.

In some embodiments, the infectious disease is caused by a bacterial infection, such as bacterial pneumonia. In some embodiments, the bacterial infection is caused by a bacterium selected from the group consisting of Streptococcus pneumoniae (also referred to as pneumococcus), Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pyogenes, Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Moraxella catarrhalis, Chlamydophila pneumoniae, Mycoplasma pneumoniae, Legionella pneumophila, Serratia marcescens, Burkholderia cepacia, Burkholderia pseudomallei, Bacillus anthracis, Bacillus cereus, Bordatella pertussis, Stenotrophomonas maltophilia, a bacterium from the citrobacter family, a bacterium from the ecinetobacter family, and Mycobacterium tuberculosis.

In some embodiments, the bacterial infection of the invention is not caused by Haemophilus influenza or Haemophilus parainfluenzae. In some embodiments, the infectious disease is caused by a fungal infection. In some embodiments, the fungal infection is caused by a fungus selected from the group consisting of Histoplasma capsulatum, Cryptococcus neoformans, Pneumocystis jiroveci, Coccidioides immitis, Candida albicans, and Pneumocystis jirovecii (which causes pneumocystis pneumonia (PCP), also called pneumocystosis).

In some embodiments, the infectious disease is caused by a viral infection, such viral pneumonia. In some embodiments, the viral infection is caused by a virus selected from the group consisting of respiratory syncytial virus, adenovirus, metapneumovirus, cytomegalovirus, , rhinovirus, coxsackie virus, echo virus, herpes simplex virus, coronavirus (SARS-coronavirus such as SARS-Covl or SARS-Cov 2), and smallpox. In some embodiments, the viral lung infection may be due to a member of the Pneumoviridae, Paramyxoviridae and/or Coronaviridae families are in particular selected from the group consisting of upper and lower respiratory tract infections due to: human respiratory syncytial virus (hRSV), type A and type B, human metapneumovirus (hMPV) type A and type B; measles virus, endemic human coronaviruses (HCoV-229E, -NL63, -OC43, and -HKU1), severe acute respiratory syndrome (SARS), Severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle-East respiratory syndrome (MERS) coronaviruses. In particular, the method of the present invention is suitable for the treatment of Severe Acute Respiratory Syndrome (SARS). More particularly, the method of the present invention is suitable for the treatment of COVID- 19. In particular, the method of the present invention is suitable for the treatment of SARS- COV related disorder.

In some embodiments, the viral infection of the invention is not caused by influenza virus (e.g., Influenza virus A, Influenza virus B).

In some embodiments, the viral lung infection of the invention is not be due to a member of the parainfluenza virus type 3 (PIV-3).

In some embodiments, the viral lung infection of the invention is not caused by Zika virus (ZKV).

As used herein, “SARS-CoV infection” refers to the transmission of severe acute respiratory syndrome coronavirus from an animal and/or human to another animal and/or human primarily via respiratory droplets from coughs and sneezes within a range of about 2 meters. Indirect contact via contaminated surfaces is another possible cause of infection.

As used herein, the term "severe acute respiratory syndrome coronavirus” or “SARS- coronavirus” is a strain of virus that causes severe acute respiratory syndrome. It is an enveloped, positive-sense, single-stranded RNA virus which infects the epithelial cells within the lungs. The virus enters the host cell by binding to the ACE2 receptor. SARS-coronavirus comprises SARS-CoV 1 or SARS-CoV 2.

As used herein, the term “severe acute respiratory syndrome coronavirus 1” or SARS- CoV-1) is the coronavirus responsible for the SARS epidemic from 2002 to 2004. It is a strain of the coronavirus species SARSr-CoV. This infectious agent is said to have appeared in November 2002 in Guangdong province, China. Between November 1, 2002 and August 31, 2003, the virus would have infected 8,096 people in thirty countries, causing 774 deaths, mainly in China, Hong Kong, Taiwan, and Southeast Asia. It is a single-stranded RNA virus of positive polarity belonging to the genus betacoronavirus

As used herein, the term “severe acute respiratory syndrome coronavirus 2” or SARS- CoV-2 is a positive-sense single-stranded RNA virus. It causes coronavirus disease 2019 (COVID-19), a respiratory illness. In a particular embodiment, the SARS-CoV is COVID-19. SARS-CoV -2 is a member of the subgenus Sarbecovirus (beta-CoV lineage B). Its RNA sequence is approximately 30,000 bases in length. SARS-CoV -2 is unique among known betacoronaviruses in its incorporation of a polybasic cleavage site, a characteristic known to increase pathogenicity and transmissibility in other viruses.

As used herein, the term “SARS-COV related disorder” refers to all diseases and/or complications linked to SAR-CoV infection. In a particular embodiment, the SARS-CoV related disorder is selected from the group consisting of but not limited to: Covid-19; cytokine storm syndrome (CSS); Respiratory distress syndrome (RDS); gastroenteritis and respiratory infections; pneumonia, and/or lung fibrosis.

As used herein, the term “fibrosis” refers to the common scarring reaction associated with chronic injury that results from prolonged parenchymal cell injury and/or inflammation that may be induced by a wide variety of agents, e.g., drugs, toxins, radiation, any process disturbing tissue or cellular homeostasis, toxic injury, altered blood flow, infections (viral, bacterial, spirochetal, and parasitic), storage disorders, and disorders resulting in the accumulation of toxic metabolites. Fibrosis is most common in the heart, lung, peritoneum, and kidney.

In a particular embodiment, the SARS-COV related disorder is lung fibrosis.

As used herein, the term “lung fibrosis” also called pulmonary fibrosis refers to a condition in which the lungs become scarred over time. Symptoms are selected from the following symptoms consisting of but not limited to: shortness of breath, a dry cough, feeling tired, weight loss, and nail clubbing; complications may include pulmonary hypertension, respiratory failure, pneumothorax, and lung cancer. As used herein, the term “subject” refers to any mammals, such as a rodent, a feline, a canine, and a primate. 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 embodiments, the subject is a premature human infant. The subject is confirmed to have an infectious disease or who may be classified as having a probable or suspected case of an infectious disease based on epidemiological factors. Subjects include those who are diagnosed with an infectious disease, those who test positive for infection by an infectious agent (pathogen) associated with an infectious disease (e.g., SARS-CoV), those who are suspected of having an infectious disease based on epidemiological factors, or those who are at an imminent risk of contracting an infectious disease (e.g., one who has been exposed or will likely be exposed to an infectious disease in the near future).

In some embodiments, the subject is a human afflicted with or susceptible to be afflicted with an infectious disease.

In some embodiments, the subject is a human afflicted with or susceptible to be afflicted with lung infection.

In another embodiment, the subject is a human afflicted with or susceptible to be afflicted with an acute lung infection.

In another embodiment, the subject is a human afflicted with or susceptible to be afflicted with a chronic lung infection.

In some embodiments, the subject is a human afflicted with or susceptible to be afflicted wherein said lung infection is a bacterial infection.

In some embodiments, the subject is a human afflicted with or susceptible to be afflicted wherein said lung infection is a viral infection.

In a particular embodiment, the subject is a human afflicted with or susceptible to be afflicted with a severe acute respiratory syndrome-corona virus (SARS-COV) infection.

In another embodiment, the subject is a human afflicted with or susceptible to be afflicted with SARS-CoV 1 infection or SARS-CoV 2 infection.

In another embodiment, the subject is a human afflicted with or susceptible to be afflicted with a SARS-CoV 19 infection.

In another embodiment, the subject is a human afflicted with or susceptible to be afflicted with lung fibrosis.

In some embodiments, the subject suffers from COVID-19. In some embodiment, the subject is a human afflicted with or susceptible to be afflicted with acute Covid-19.

As used herein, the term “acute COVID-19” refers to severe form of COVID-19 with difficulty breathing or shortness of breath, feeling of tightness or pain in the chest, loss of speech or motor skills.

In some embodiment, the subject is a human afflicted with or susceptible to be afflicted with post-acute COVID-19. In some embodiment, the subject is a human afflicted with or susceptible to be afflicted with long COVID-19.

As used herein, the term “long COVID-19” or“post-acute COVID-19” refers to persistent symptoms and/or delayed or long-term complications of SARS-CoV-2 infection beyond 4 weeks from the onset of symptoms and for several weeks to several months after apparent recovery from infection. Based on recent literature, it is further divided into two categories: subacute or ongoing symptomatic COVID-19, which includes symptoms and abnormalities present from 4-12 weeks beyond acute COVID-19; and chronic or post-COVID- 19 syndrome, which includes symptoms and abnormalities persisting or present beyond 12 weeks of the onset of acute COVID-19 and not attributable to alternative diagnoses. Symptoms of long Covid-19 or post-acute included but are not limited to severe fatigue, cardiopulmonary symptoms (breathing difficulties, chest pain and / or tightness, cough, tachycardia), neurological disorders (headaches, sensory disorders, cognitive disorders) or even muscle and joint pain, digestive and skin symptoms.

As used herein, the term “pan-caspase inhibitor” refers to a natural or synthetic compound that has a biological effect to inhibit the activity or the expression of one or more of the known caspases. Caspases are a family of intracellular cysteine proteases critical to several cellular functions, including apoptosis and inflammation. Mammalian caspases are numbered 1 - 13 and are classified either as initiators or effectors of downstream functions.

In a particular embodiment, the pan-caspase inhibitor is a caspase 1 inhibitor.

In a particular embodiment, the pan-caspase inhibitor is a caspase 2 inhibitor.

In a particular embodiment, the pan-caspase inhibitor is a caspase 3 inhibitor.

In a particular embodiment, the pan-caspase inhibitor is a caspase 4 inhibitor.

In a particular embodiment, the pan-caspase inhibitor is a caspase 5 inhibitor.

In a particular embodiment, the pan-caspase inhibitor is a caspase 6 inhibitor.

In a particular embodiment, the pan-caspase inhibitor is a caspase 7 inhibitor.

In a particular embodiment, the pan-caspase inhibitor is a caspase 8 inhibitor.

In a particular embodiment, the pan-caspase inhibitor is a caspase 9 inhibitor. In a particular embodiment, the pan-caspase inhibitor is a caspase 10 inhibitor.

In a particular embodiment, the pan-caspase inhibitor is a caspase 11 inhibitor.

In a particular embodiment, the pan-caspase inhibitor is a caspase 12 inhibitor.

In a particular embodiment, the pan-caspase inhibitor is a caspase 13 inhibitor.

As used herein, the term “caspase 1” also known as interleukin- 1 converting enzyme (ICE) refers to an enzyme that proteolytically cleaves the precursors of the inflammatory cytokines. Typically, caspase 1 cleaves pro IL-Ib and pro IL-18 into their active forms, IL-Ib and IL-18. The active cytokines lead to a downstream inflammatory response. The naturally occurring human caspase 1 gene has nucleotide sequences as shown in Genbank Accession numbers: NM_001223, NM_001257118, NM_001257119, NM_033292 andNM_033293. The naturally occurring human caspase 1 gene has amino acid sequences as shown in Genbank Accession numbers: NP_001214, NP_001244047, NP_001244048, NP_150634 and NP_150635. The murine nucleotide and amino acid sequences have also been described (Genbank Accession numbers: NM_009807 and NP_033937.

As used herein, the term “caspase 2” also known as CASP2 belongs to a family of cysteine proteases called caspases that cleave proteins mainly at an amino acid following an aspartic acid residue. Within this family, caspase 2 is part of the Ich-1 subfamily. The naturally occurring human caspase 2 gene has nucleotide sequences as shown in Genbank Accession numbers: NM_032983, NM_001224 and NM_032982. The naturally occurring human caspase

2 gene has amino acid sequences as shown in Genbank Accession numbers: NP_001215, NP_116764 and NP_116765.

As used herein, the term “caspase 3” also known as CPP32, Yama or apopain is formed from a 32 kDa zymogen that is cleaved into 17 kDa and 12 kDa subunits. The naturally occurring human caspase 3 gene has nucleotide sequences as shown in Genbank Accession numbers :NM_004346, NM_032991 and NP_004337. The naturally occurring human caspase

3 gene has amino acid sequences as shown in Genbank Accession numbers: NP_116786, NP_001341706, NP_001341708 and NP_001341709.

As used herein, the terms “caspase 4” and “caspase 5” (homologous to murine caspase 11) refer to an enzyme that proteolytically cleaves other proteins at an aspartic acid residue (LEVD-).

As used herein, the term “caspase 6” also known as CASP6 is an enzyme which is involved in the early immune response via de-repression. It reduces the expression of the immunosuppressant cytokine interleukin- 10 and cleaves the macrophage suppressing IRAK- M. The naturally occurring human caspase 6 gene has nucleotide sequences as shown in Genbank Accession numbers: NM_001226 and NM_032992. The naturally occurring human caspase 6 gene has amino acid sequences as shown in Genbank Accession numbers: NP_001217 and NP_116787. The murine nucleotide and amino acid sequences have also been described (Genbank Accession numbers: NM_009811 and NP_033941).

As used herein, the term “caspase 7” refers to apoptosis-related cysteine peptidase, also known as CASP7. The naturally occurring human caspase 7 gene has nucleotide sequences as shown in Genbank Accession numbers: NM_001227, NM_001267056, NM_001267057, NM_001267058 and NM_033338. The naturally occurring human caspase 7gene has amino acid sequences as shown in Genbank Accession numbers: NP_001218, NP_001253985, NP_001253986, NP_001253987 and NP_001307840. The murine nucleotide and amino acid sequences have also been described (Genbank Accession numbers: NM_007611 and NP_031637).

As used herein, the term “caspase 8” refers to cysteine-dependent aspartate-directed proteases. Caspases are a family of cytosolic aspartate-specific cysteine proteases involved in the initiation and execution of apoptosis. Caspase-8 is a cysteine protease known for its roles in Fas-induced apoptosis and lymphocyte activation. Activation of caspase-8 is an initiator for several other members of the caspase family and can lead to downstream mitochondrial damage. The naturally occurring human caspase 8 gene has nucleotide sequences as shown in Genbank Accession numbers: NM_001080124, NM_001080125, NM_001228, NM_033355, NM_033356 and the naturally occurring human caspase 8 protein has aminoacid sequences as shown in Genbank Accession numbers: NP_001073593, NP_001073594, NP_001219, NP_203519, NP_203520. The murine nucleotide and amino acid sequences have also been described (Genbank Accession numbers NM_001080126, NM_001277926, NM_009812 and NP_001073595, NP_001264855, NP_033942).

As used herein, the term “caspase 9” refers to an enzyme which is an initiator caspase, critical to the apoptotic pathway found in many tissues. The naturally occurring human caspase 9 gene has nucleotide sequences as shown in Genbank Accession numbers: NM_001229, NM_001278054 and NM_032996. The naturally occurring human caspase 9 gene has amino acid sequences as shown in Genbank Accession numbers: NP_001220, NP_001264983 and NP_127463. The murine nucleotide and amino acid sequences have also been described (Genbank Accession numbers NM_001277932, NM_015733, NM_001355176 and NP_001264861, NP_056548, and NP_001342105).

As used herein, the term “caspase 10” also known as CASP10 refers to a member of the cysteine-aspartic acid protease. The naturally occurring human caspase 10 gene has amino acid sequences as shown in Genbank Accession numbers: NM_001206524, NM_001206542, NM_001230, NM_001306083 and NM_032974. The naturally occurring human caspase 10 gene has amino acid sequences as shown in Genbank Accession numbers: NP_001193453, NP_001193471, NP_001221, NP_001293012 and NP_116756.

As used herein, the term “caspase 12” refers to a protein that in humans is encoded by the CASP12 gene. The naturally occurring human caspase 12 gene has amino acid sequences as shown in Genbank Accession number NM_001191016 and the naturally occurring human caspase 12 gene has amino acid sequences as shown in Genbank Accession number NP_001177945.

In a particular embodiment, the pan-caspase inhibitor is a caspase 1 inhibitor or a caspase 8 inhibitor.

As used herein, the terms “caspase 1 inhibitor” and “caspase 8 inhibitor” refer to a natural or synthetic compound that has a biological effect to inhibit the activity or the expression of caspases 1 or 8. More particularly, such compound is capable of inhibiting the protease activity of caspases 1 or 8. In the context of the invention, such compound is able to reduce a pro-inflammatory environment. The method consists in the use of a caspases 1 or 8 inhibitor able to reduce and/or inhibit the secretion of pro-inflammatory cytokines secretion.

In a particular embodiment, the pan-caspase inhibitor is a caspase 1 inhibitor or caspase 8 inhibitor.

In a particular embodiment, the pan-caspase inhibitor is a peptide, petptidomimetic, small organic molecule, antibody, aptamers, siRNA or antisense oligonucleotide.

The term “peptidomimetic” refers to a small protein-like chain designed to mimic a peptide.

In a particular embodiment, the pan-caspase inhibitor is Z-VAD-FMK.

In another embodiment, the pan-caspase inhibitor is caspase 1 inhibitor, wherein said inhibitor is Ac-YVAD-CHO.

In another embodiment, the pan-caspase inhibitor is caspase 1 inhibitor, wherein said inhibitor is Z-IETD-FMK.

In another embodiment, the pan-caspase inhibitor is Z-DEVD-FMK.

In another embodiment, the pan-caspase inhibitor is Z-LEHD-FMK.

In a particular embodiment, the pan- caspase inhibitor is an aptamer. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. In a particular embodiment, the pan-caspase inhibitor is a small organic molecule. The term “small organic molecule” refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e.g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da.

In a particular embodiment, the pan-caspase inhibitor is a small molecule which is an selective inhibitor of caspase 8 selected among the following compounds: Emricasan, Nivocasan, Q-VD-OPh (1135695-98-5), PKR Inhibitor (CAS number: 608512-97-6), Q-VD- OPH (CAS 1135695-98-5), Gly-Phe b-naphthylamide (CAS number: 21438-66-4), BI-9B12 (CAS 848782-29-6).

In a particular embodiment, the pan-caspase inhibitor is Emricasan and its derivatives. As used herein, the term “Emricasan” also known as IDN-6556, 254750-02-2, PF-03491390, UNII-P0GMS9N47Q (S)-3-((S)-2-(2-(2-TERT-BUTYLPHENYLAMINO)-2-

OXO ACETAMIDO)PROPANAMIDO)-4-OXO-5-(2, 3,5,6- TETRAFLUOROPHEN OXY)PENT AN OIC ACID, PF 03491390, P0GMS9N47Q, (S)-3-((S)-

2-(2-((2-(tert-Butyl)phenyl)amino)-2-oxoacetamido)propanamido)-4-oxo-5-(2, 3,5,6- tetrafluorophenoxy)pentanoic acid refers to the first caspase inhibitor tested in human which has received orphan drug status by FDA. It is developed by Pfizer and made in such a way that it protects liver cells from excessive apoptosis. This molecule has the following formula, structure and the CAS number254750-02-2 in the art: C26H27F4N3O7:

In another embodiment, the pan-caspase inhibitor is Nivocasan and its derivatives. As used herein, the term “Nivocasan” also known as GS 9450 developed by Gilead Sciences, Inc (Ratziu V et al.2012; Arends JE et al.2011). Nivocasan has the following formula, structure and the CAS number 908253-63-4 in the art: C21H22FN3O5:

In some embodiments, the pan-caspase inhibitor is an antibody.

As used herein, the term “antibody” is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity. The term includes antibody fragments that comprise an antigen binding domain such as Fab', Fab, F(ab')2, single domain antibodies (DABs), TandAbs dimer, Fv, scFv (single chain Fv), dsFv, ds-scFv, Fd, linear antibodies, minibodies, diabodies, bispecific antibody fragments, bibody, tribody (scFv-Fab fusions, bispecific or trispecific, respectively); sc-diabody; kappa(lamda) bodies (scFv-CL fusions); BiTE (Bispecific T-cell Engager, scFv-scFv tandems to attract T cells); DVD-Ig (dual variable domain antibody, bispecific format); SIP (small immunoprotein, a kind of minibody); SMIP ("small modular immunopharmaceutical" scFv-Fc dimer; DART (ds -stabilized diabody "Dual Affinity ReTargeting"); small antibody mimetics comprising one or more CDRs and the like. The techniques for preparing and using various antibody-based constructs and fragments are well known in the art (see Rabat et ak, 1991, specifically incorporated herein by reference). Diabodies, in particular, are further described in EP 404, 097 and WO 93/1 1 161; whereas linear antibodies are further described in Zapata et al. (1995). Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab' and F(ab')2, scFv, Fv, dsFv, Fd, dAbs, TandAbs, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques or can be chemically synthesized. Techniques for producing antibody fragments are well known and described in the art. For example, each of Beckman et ak, 2006; Holliger & Hudson, 2005; Le Gall et ak, 2004; Reff & Heard, 2001 ; Reiter et ak, 1996; and Young et ak, 1995 further describe and enable the production of effective antibody fragments. In some embodiments, the antibody is a “chimeric” antibody as described in U.S. Pat. No. 4,816,567. In some embodiments, the antibody is a humanized antibody, such as described U.S. Pat. Nos. 6,982,321 and 7,087,409. In some embodiments, the antibody is a human antibody. A “human antibody” such as described in US 6,075,181 and 6,150,584. In some embodiments, the antibody is a single domain antibody such as described in EP 0 368 684, WO 06/030220 and WO 06/003388. In a particular embodiment, the inhibitor is a monoclonal antibody. Monoclonal antibodies can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique, the human B-cell hybridoma technique and the EBV-hybridoma technique.

In a particular, the pan-caspase inhibitor is an intrabody having specificity for caspase 8. As used herein, the term "intrabody" generally refer to an intracellular antibody or antibody fragment. Antibodies, in particular single chain variable antibody fragments (scFv), can be modified for intracellular localization. Such modification may entail for example, the fusion to a stable intracellular protein, such as, e.g., maltose binding protein, or the addition of intracellular trafficking/localization peptide sequences, such as, e.g., the endoplasmic reticulum retention. In some embodiments, the intrabody is a single domain antibody. In some embodiments, the antibody according to the invention is a single domain antibody. The term “single domain antibody” (sdAb) or "VHH" refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such VHH are also called “nanobody®”. According to the invention, sdAb can particularly be llama sdAb.

In some embodiments, the inhibitor of pan-caspase expression (caspase 1 or caspase 8) is a short hairpin RNA (shRNA), a small interfering RNA (siRNA) or an antisense oligonucleotide which inhibits the expression of caspase 1 or caspase 8. In a particular embodiment, the inhibitor of JMY expression is siRNA. A short hairpin RNA (shRNA) is a sequence of RNA that makes a tight hairpin turn that can be used to silence gene expression via RNA interference. shRNA is generally expressed using a vector introduced into cells, wherein the vector utilizes the U6 promoter to ensure that the shRNA is always expressed. This vector is usually passed on to daughter cells, allowing the gene silencing to be inherited. The shRNA hairpin structure is cleaved by the cellular machinery into siRNA, which is then bound to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNAs that match the siRNA to which it is bound. Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, are a class of 20-25 nucleotide-long double- stranded RNA molecules that play a variety of roles in biology. Most notably, siRNA is involved in the RNA interference (RNAi) pathway whereby the siRNA interferes with the expression of a specific gene. Anti-sense oligonucleotides include anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of the targeted mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of the targeted protein, and thus activity, in a cell. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence can be synthesized, e.g., by conventional phosphodiester techniques. Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732). Antisense oligonucleotides, siRNAs, shRNAs of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid to the cells and typically mast cells. Typically, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art.

In some embodiments, the inhibitor of pan-caspase (caspase 1 or caspase 8) expression is an endonuclease. In the last few years, staggering advances in sequencing technologies have provided an unprecedentedly detailed overview of the multiple genetic aberrations in cancer. By considerably expanding the list of new potential oncogenes and tumor suppressor genes, these new data strongly emphasize the need of fast and reliable strategies to characterize the normal and pathological function of these genes and assess their role, in particular as driving factors during oncogenesis. As an alternative to more conventional approaches, such as cDNA overexpression or downregulation by RNA interference, the new technologies provide the means to recreate the actual mutations observed in cancer through direct manipulation of the genome. Indeed, natural and engineered nuclease enzymes have attracted considerable attention in the recent years. The mechanism behind endonuclease-based genome inactivating generally requires a first step of DNA single or double strand break, which can then trigger two distinct cellular mechanisms for DNA repair, which can be exploited for DNA inactivating: the errorprone nonhomologous end-joining (NHEJ) and the high-fidelity homology-directed repair (HDR).

In a particular embodiment, the endonuclease is CRISPR-cas. As used herein, the term “CRISPR-cas” has its general meaning in the art and refers to clustered regularly interspaced short palindromic repeats associated which are the segments of prokaryotic DNA containing short repetitions of base sequences.

In some embodiment, the endonuclease is CRISPR-cas9 which is from Streptococcus pyogenes. The CRISPR/Cas9 system has been described in US 8697359 B1 and US 2014/0068797. Originally an adaptive immune system in prokaryotes (Barrangou and Marraffmi, 2014), CRISPR has been recently engineered into a new powerful tool for genome editing. It has already been successfully used to target important genes in many cell lines and organisms, including human (Mali et ak, 2013, Science, Vol. 339 : 823-826), bacteria (Fabre et ak, 2014, PLoS Negk Trop. Dis., Vol. 8:e267T), zebrafish (Hwang et ak, 2013, PLoS One, Vol. 8:e68708.), C. elegans (Hai etak, 2014 Cell Res. doi: 10.1038/cr.2014.11.), bacteria(Fabre et ak, 2014, PLoS Negk Trop. Dis., Vol. 8:e267T), plants (Mali et ak, 2013, Science, Vol. 339 : 823-826), Xenopus tropicalis (Guo et ak, 2014, Development, Vol. 141 : 707-714.), yeast (DiCarlo et ak, 2013, Nucleic Acids Res., Vol. 41 : 4336-4343.), Drosophila (Gratz et ak, 2014 Genetics, doi:10.1534/genetics.113.160713), monkeys (Niu et ak, 2014, Cell, Vol. 156 : 836- 843.), rabbits (Yang et ak, 2014, J. Mol. Cell Biol., Vol. 6 : 97-99.), pigs (Hai et ak, 2014, Cell Res. doi: 10.1038/cr.2014.1T), rats (Ma et ak, 2014, Cell Res., Vol. 24 : 122-125.) and mice (Mashiko et ak, 2014, Dev. Growth Differ. Vol. 56 : 122-129.). Several groups have now taken advantage of this method to introduce single point mutations (deletions or insertions) in a particular target gene, via a single gRNA. Using a pair of gRNA-directed Cas9 nucleases instead, it is also possible to induce large deletions or genomic rearrangements, such as inversions or translocations. A recent exciting development is the use of the dCas9 version of the CRISPR/Cas9 system to target protein domains for transcriptional regulation, epigenetic modification, and microscopic visualization of specific genome loci. In some embodiment, the endonuclease is CRISPR-Cpfl which is the more recently characterized CRISPR from Provotella and Francisella 1 (Cpfl) in Zetsche et al. (“Cpfl is a Single RNA-guided Endonuclease of a Class 2 CRISPR-Cas System (2015); Cell; 163, 1-13).

As used herein the terms "administering" or "administration" refer to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g., pan-caspase inhibitor) into the subject, such as by oral, mucosal, intradermal, intravenous, subcutaneous, intramuscular delivery and/or any other method of physical delivery described herein or known in the art. When a disease, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof. When a disease or symptoms thereof, are being prevented, administration of the substance typically occurs before the onset of the disease or symptoms thereof. More particularly, the pan-caspase inhibitor is formulated for an oral administration to the subject. In a further embodiment, intravenous administration is performed to the subject.

By a "therapeutically effective amount" is meant a sufficient amount of a pan-caspase inhibitor for use in a method for the treatment of infectious disease (such as SARS-CoV infection and/or SARS-CoV related disease) at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well known within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic 20 adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, typically from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day. In some embodiment, the inventors show that Emricasan treatment decreased a large panel of genes linked to COVID19. In some embodiment, the inventors show that Emricasan treatment decreased the following genes : RPS16 (Human Uniprot number: P62249), PIK3R1 (Human Uniprot number: P27986), RPS27A (Human Uniprot number: P62979), TLR8 (Human Uniprot number: Q9NR97), UBA52 (Human Uniprot number: P62987), CFD (Human Uniprot number: P00746), RPLP2 (Human Uniprot number: P05387), F13A1 (Human Uniprot number: P00488), RPL13 (Human Uniprot number: P26373), C2 (Human Uniprot number: P06681), RPL36 (Human Uniprot number: Q9Y3U8), IRAK4 (Human Uniprot number: Q9NWZ3), RPL1 (Human Uniprot number: 021235), TNFRSF1A (Human Uniprot number: PI 9438), RPL36AL (Human Uniprot number: Q969Q0), TYK2 (Human Uniprot number: P29597), RPL6 (Human Uniprot number: Q02878), RPS4Y1 (Human Uniprot number: P22090), RPL7 (Human Uniprot number: P18124), RPL37 (Human Uniprot number: P61927), RPS6 (Human Uniprot number: P62753), SYK (Human Uniprot number: P43405), RPS3 (Human Uniprot number: P23396), RPL22 (Human Uniprot number: P35268), RPSIO (Human Uniprot number: P46783), RPL30 (Human Uniprot number: P62888), RPS4X (Human Uniprot number: P62701), RPL26 (Human Uniprot number: P61254), RPS20 (Human Uniprot number: P60866), RPS27 (Human Uniprot number: P42677), RPS24 (Human Uniprot number: P62847), RPL23 (Human Uniprot number: P62829), RPL7A (Human Uniprot number: P62424), RPL15 (Human Uniprot number: P61313), RPL29 (Human Uniprot number: P47914), RPS23 (Human Uniprot number: P62266), RPS29 (Human Uniprot number: P62273), RPS12 (Human Uniprot number: P25398), RPS17 (Human Uniprot number: P08708), RPL31 (Human Uniprot number: P62899), RPL37A (Human Uniprot number: P61513), RPL5 (Human Uniprot number: P46777), RPL36AL (Human Uniprot number: Q969Q0), RPS15A (Human Uniprot number: P62244), RPLP0 (Human Uniprot number: P05388), RPS3A (Human Uniprot number: P61247), RPS5 (Human Uniprot number: P46782), RPLPl (Human Uniprot number: P05386), RPL10A (Human Uniprot number: P62906), RPS25 (Human Uniprot number: P62851), RPL3 (Human Uniprot number: P39023), RPL28 (Human Uniprot number: P46779), RPL18A (Human Uniprot number: Q02543), RPS2 (Human Uniprot number: P15880), RPS15 (Human Uniprot number: P62841), RPL12 (Human Uniprot number: P30050), RPL18A (Human Uniprot number: Q02543), RPL21 (Human Uniprot number: P46778), RPS19 (Human Uniprot number: P39019), RPL39 (Human Uniprot number: P62891), RPL35A (Human Uniprot number: PI 8077), RPL11 (Human Uniprot number: P62913), RPS21 (Human Uniprot number: P63220), RPS13 (Human Uniprot number: P62277), RPL24 (Human Uniprot number: P83731), RPL27A (Human Uniprot number: P46776), RPS7 (Human Uniprot number: P62081), RPS11 (Human Uniprot number: P62280), ADAR (Human Uniprot number: P55265), RPL19 (Human Uniprot number: P84098), STAT1 (Human Uniprot number: P42224), FAU (Human Uniprot number: P62861), STAT2 (Human Uniprot number: P52630), RPL38 (Human Uniprot number: P63173), IL6ST (Human Uniprot number: P40189), RPS8 (Human Uniprot number: P62241), MAVS (Human Uniprot number: Q7Z434), RPS28 (Human Uniprot number: P62857), OAS1 (Human Uniprot number: P00973), RPL41 (Human Uniprot number: P62945), IRF9 (Human Uniprot number: Q00978), RPL13A (Human Uniprot number: P40429), C1QC (Human Uniprot number: P02747), RPL34 (Human Uniprot number: P49207), C1QA (Human Uniprot number: P02745), RPL17 (Human Uniprot number: P18621), TLR4 (Human Uniprot number: 000206), RPS14 (Human Uniprot number: P62263), RPL32 (Human Uniprot number: P62910. In some embodiment, the genes listed above have their expression reduced when macrophages are treated with Emricasan.

Combined preparation

The pan-caspase inhibitor as described above is also combined with a classical treatment.

As used herein, the term “classical treatment” refers to antiviral treatment, antibiotics, anti-parasitic treatment, immunosuppressive corticosteroids or non-steroidal therapies; immunotherapy: recombinant human I L- 1 B receptor antagonist; neutralizing monoclonal anti- IL-Ib antibody; PAK-l/PAK-2 inhibitors; or NLRP3 inhibitor.

Accordingly, in a second aspect, the invention relates to i) a pan-caspase inhibitor and ii) a classical treatment used as a combined preparation for treating an infectious disease in a subject in need thereof.

In a further embodiment, the combined preparation according to the invention, wherein the infectious disease causes a lung infection.

In a particular embodiment, the combined preparation according to the invention, wherein the lung infection is SARS such as COVID-19.

In a particular embodiment, the invention relates to i) a pan-caspase inhibitor and ii) antiviral treatment used as a combined preparation for treating infectious disease in a subject in need thereof.

As used herein, the term “antiviral treatment” to an inhibitor which inhibits the viral entry, viral internalization, viral replication and/or viral release. Such inhibitor prevents or reduces the occurrence of viral resistance, and the like. Typically, the antiviral treatment includes but not limited to interferons (e.g., interferon-alpha, pegylated interferon-alpha), ribavirin, anti-HCV, (monoclonal or polyclonal) antibodies, RNA polymerase inhibitors, protease inhibitors, IRES inhibitors, helicase inhibitors, antisense compounds, ribozymes, neuraminidase inhibitor, nucleoside analogues of guanine, nucleoside analogue of thymidine, nucleoside reverse transcriptase inhibitors (NRTI), nucleotide reverse transcriptase inhibitor (NtRTI), protease inhibitors and any combination thereof.

Typically, the antiviral treatment is selected from the group consisting of but not limited to Abacavir, Acyclovir (Aciclovir), Adefovir, Amantadine, Ampligen, Amprenavir (Agenerase), Arbidol, Atazanavir, Atripla, Balavir, Baloxavir marboxil (Xofluza), Biktarvy, Boceprevir (Victrelis), Cidofovir, Cobicistat (Tybost), Combivir, Daclatasvir (Daklinza), Darunavir, Delavirdine, Descovy, Didanosine, Docosanol Dolutegravir, Doravirine (Pifeltro), Ecoliever, Edoxudine, Efavirenz, Elvitegravir, Emtricitabine, Enfuvirtide, Entecavir, Etravirine (Intelence), Famciclovir, Fomivirsen, Fosamprenavir, Foscamet, Fosfonet, Fusion inhibitor, Ganciclovir (Cytovene), Ibacitabine, Ibalizumab (Trogarzo), Idoxuridine, Imiquimod, Imunovir, Indinavir, Inosine, Integrase inhibitor, Interferon type I, Interferon type II, Interferon type III, Interferon, Lamivudine, Letermovir (Prevymis), Lopinavir, Loviride, Maraviroc, Methisazone, Moroxydine, Nelfmavir, Nevirapine, Nexavir, Nitazoxanide, Norvir, Nucleoside analogues, Oseltamivir (Tamiflu), Peginterferon alfa-2a, Peginterferon alfa-2b, Penciclovir, Peramivir (Rapivab), Pleconaril, Podophyllotoxin, Protease inhibitor, Pyramidine, Raltegravir, Remdesivir, Reverse transcriptase inhibitor Ribavirin, Rilpivirine (Edurant), Rimantadine, Ritonavir, Saquinavir, Simeprevir (Olysio), Sofosbuvir, Stavudine, Synergistic enhancer, Telaprevir, Telbivudine (Tyzeka), Tenofovir alafenamide, Tenofovir disoproxil, Tenofovir, Tipranavir, Trifluridine, Trizivir, Tromantadine, Truvada, Valaciclovir (Valtrex), Valganciclovir, Vicriviroc, Vidarabine, Viramidine, Zalcitabine, Zanamivir (Relenza) and/or Zidovudine.

In a particular embodiment, the invention relates to i) a pan-caspase inhibitor and ii) an antibiotic used as a combined preparation for treating an infectious disease in a subject in need thereof.

As used herein, the term “antibiotic” refers to an antimicrobial substance active against bacteria and is the most important type of antibacterial agent for fighting bacterial infections. Each antibiotic belongs to an antibiotic class which is a grouping of different drugs that have similar chemical and pharmacologic properties. Their chemical structures may look comparable, and drugs within the same class may kill the same or related bacteria. Typically, the antibiotic class includes but not limited to Penicillins, Tetracyclines, Cephalosporins, Quinolones, Lincomycins, Macrolides, Sulfonamides, Glycopeptides, Aminoglycosides, and Carbapenems.

Typically, the antibiotic is selected from the group consisting of but not limited to amoxicillin, amoxicillin and clavulanate, ampicillin, dicloxacillin, oxacillin, penicillin V potassium, demeclocycline, doxycycline, eravacycline, minocycline, omadacycline, tetracycline, cefaclor, cefdinir, cefotaxime, ceftazidime, ceftriaxone, cefuroxime, ciprofloxacin, levofloxacin, moxifloxacin, clindamycin, lincomycin, azithromycin, clarithromycin, erythromycin, sulfamethoxazole and trimethoprim, sulfasalazine, dalbavancin, oritavancin, telavancin, vancomycin, gentamicin, tobramycin, amikacin, imipenem and cilastatin, meropenem, doripenem and ertapenem.

In a particular embodiment, the invention relates to i) a pan-caspase inhibitor and ii) an anti-parasitic treatment used as a combined preparation for treating an infectious disease in a subject in need thereof.

As used herein, the term “anti-parasitic treatment” refers to a treatment of parasitic diseases, such as those caused by helminths, amoeba, ectoparasites, parasitic fungi, and protozoa. Antiparasitics target the parasitic agents of the infections by destroying them or inhibiting their growth.

In a particular embodiment, the anti-parasitic drug is selected from the group consisting of but not limited to: chloroquine, amodiaquine, mefloquine, halofantrine, artemether, artesunate, arteminol, sulfadoxine, pyrimethamine, proguanil, atovaquone, quinine, abamectine, albendazole, diethylcarbamazine, mebendazole, niclosamide, ivermectin, suramine, thiabendazole, levamisole, praziquantel, triclabendazole, flubendazole, metronidazole, tinidazole, secnidazole, tenonitrozole, pyrimethamine, amphotericin B, pentamidine, miltefosine, nifurtimox, benznidazole, amphotericin B, ketoconazole, econazole, griseofulvin, miconazole orfluconazole.

In a particular embodiment, the anti-parasitic drug is an anti-malaria drug. Typically, the anti-parasitic drug is chloroquine.

In a particular embodiment, emricasan and ii) hydroxychloroquine used as a combined preparation for treating an infectious disease in a subject in need thereof.

Accordingly, in a particular embodiment, the invention relates to i)a pan-caspase inhibitor and ii) an anti-parasitic treatment used as a combined preparation for treating an infectious disease in a subject in need thereof, wherein the pan-caspase inhibitor is emricasan and the anti-parasitic drug is hydroxychloroquine. As used herein, the term "hydroxychloroquine" or “HCQ” has its general meaning in the art and refers to 2-[[4-[(7-Chloro-4-quinolyl) amino] pentyl] ethylamino] ethanol sulfate (1:1). Methods of synthesis for hydroxychloroquine are disclosed in U.S. Pat. No. 2,546,658, herein incorporated by reference. The HCQ has the following structure:

HCQ has similar pharmacokinetics to chloroquine, with rapid gastrointestinal absorption and elimination by the kidneys. Cytochrome P450 enzymes (CYP2D6, 2C8, 3A4 and 3A5). HCQ is metabolized into three major metabolites, desethyl-chloroquine (DCQ), bisdesethylchloroquine (BDCQ), and N-desethylhydroxy chloroquine (DHCQ).

In some embodiments, the metabolites of HCQ are desethyl-chloroquine (DCQ), bisdesethylchloroquine (BDCQ), and N-desethylhydroxy chloroquine (DHCQ).

In a particular embodiment, the anti-parasitic drug is N-desethylhydroxychloroquine” (DHCQ). As used herein, the term "N-desethylhydroxychloroquine” or “DHCQ” has its general meaning in the art and has the following structure:

In a particular embodiment, the anti-parasitic drug is bisdesethylchloroquine (BDCQ). As used herein, the term "bisdesethylchloroquine” or “BDCQ” has its general meaning in the art, refers to 4-N-(7-chloroquinolin-4-yl)pentane- 1,4-diamine and has the following structure: In a particular embodiment, the anti-parasitic drug is desethyl-chloroquine (DCQ). As used herein, the term "desethyl-chloroquine” or “DCQ” has its general meaning in the art, refers 4-N-(7-chloroquinolin-4-yl)-l-N-ethylpentane-l, 4-diamine and has the following structure:

In a particular embodiment, the invention relates to i) a pan-caspase inhibitor and ii) immunosuppressive corticosteroids used as a combined preparation for treating an infectious disease infection in a subject in need thereof.

As used herein, the term “corticosteroid” is well known in the art and refers to class of steroid hormones that are produced in the adrenal cortex as well as the synthetic analogues of these hormones. Two types of classes of corticosteroid exist in the art: glucocorticoids and mineralocorticoids. The corticosteroid for use in the invention is selected from the group consisting of: Flugestone (flurogestone); Fluorometholone; Medrysone; Prebediolone acetate; chlormadinone acetate, cyproterone acetate, medrogestone, medroxyprogesterone acetate, megestrol acetate, and segesterone acetate; Chloroprednisone; Cloprednol; Difluprednate; Fludrocortisone; Fluocinolone; Fluperolone; Fluprednisolone; Loteprednol; Methylprednisolone; Prednicarbate; Prednisolone; Prednisone; Tixocortol; Triamcinolone; Alclometasone; Beclometasone; Betamethasone; Clobetasol; Clobetasone; Clocortolone; Desoximetasone; Dexamethasone; Diflorasone; Difluocortolone; Fluclorolone; Flumetasone; Fluocortin; Fluocortolone; Fluprednidene; Fluticasone; Fluticasone furoate; Halometasone; Meprednisone; Mometasone; Mometasone furoate; Paramethasone; Prednylidene; Rimexolone; Ulobetasol (halobetasol); Amcinonide; Budesonide; Ciclesonide; Deflazacort; Desonide; Formocortal (fluoroformylone); Fluclorolone acetonide (flucloronide); Fludroxycortide (flurandrenolone, flurandrenolide); Flunisolide; Fluocinolone acetonide; Fluocinonide; Halcinonide; Triamcinolone acetonide; Cortivazol; RU-28362.

In a particular embodiment, the invention relates to i) a pan-caspase inhibitor and ii) non-steroidal drug used as a combined preparation for treating an infectious disease in a subject in need thereof.

As used herein, the term “nonsteroidal drug” refers to a class of drugs which decrease inflammation. The nonsteroidal drug for use in the invention is selected from the group consisting of: Aspirin (acetylsalicylic acid); Diflunisal (Dolobid); Salicylic acid and other salicylates Salsalate (Disalcid); Ibuprofen; Dexibuprofen ; Naproxen ; Fenoprofen ; Ketoprofen ; Dexketoprofen ; Flurbiprofen ; Oxaprozin; Loxoprofen; Indomethacin; Tolmetin; Sulindac; Etodolac; Ketorolac; Diclofenac; Aceclofenac; Nabumetone; Piroxicam; Meloxicam; Tenoxicam; Droxicam; Lomoxicam; Phenylbutazone; Mefenamic acid; Meclofenamic acid; Flufenamic acid; Tolfenamic acid; Celecoxib; Clonixin.

In a particular embodiment, the invention relates to i) a pan-caspase inhibitor and ii) an immunotherapy treatment used as a combined preparation for treating an infectious disease in a subject in need thereof.

As used herein, the term “immunotherapy” has its general meaning in the art and refers to the treatment that consists in administering an immunogenic agent i.e. an agent capable of inducing, enhancing, suppressing or otherwise modifying an immune response.

In a further embodiment, the invention relates to i) a pan-caspase inhibitor and ii) a neutralizing monoclonal anti-IL-Ib antibody used as a combined preparation for treating an infectious disease in a subject in need thereof.

As used herein, the term “a neutralizing monoclonal anti-IL-Ib antibody” refers to an antibody that blocks or reduces at least one activity of a polypeptide comprising the epitope to which the antibody specifically binds. The neutralizing antibody reduces IL-Ib biological activity in in cellulo and/or in vivo tests. In the context of the invention, the neutralizing monoclonal anti-IL-Ib antibody is canakinumab (trade name Ilaris, developed by Novartis). In a particular embodiment, the invention relates i) a pan-caspase inhibitor and ii) a recombinant human IL-IB receptor antagonist used as a combined preparation for treating an infectious disease in a subject in need thereof.

As used herein, the term “a recombinant human IL-IB receptor antagonist” refers to an inhibitor which inhibits the activity of IL- la and IL-Ib by competitively blocking their binding to type I and type II receptors. IL-1RA is produced by comeal epithelial cells, monocytes, neutrophils, macrophages, and fibroblasts. In the context of the invention, the recombinant human IL-1B receptor antagonist is Anakinra (marketed as Kineret® by Swedish Orphan Biovitru).

In a particular embodiment, the invention relates i) a pan-caspase inhibitor and ii) a NLRP3 inhibitor used as a combined preparation for treating an infectious disease in a subject in need thereof.

As used herein, the term “NLRP3” refers to Nucleotide-binding oligomerization domain-like receptor including a pyrin domain 3. Nucleotide-binding oligomerization domain- like receptors ("NLRs") include a family of intracellular receptors that detects pathogen- associated molecular patterns ("PAMPs") and endogenous signal danger molecules. NLRPs represent a subfamily of NLRs that include a Pyrin domain and are constituted by proteins such as NLRPl, NLRP3, NLRP4, NLRP6, NLRP7, and NLRP12. NLRPs are involved in the formation of multiprotein complexes termed inflammasomes. The NLRP3 inflammasome forms a molecular platform inside macrophages and microglial cells, catalyzing the activation of the protease Caspase-1. Caspase-1 is responsible for converting the potent pro-inflammatory cytokine interleukin- 1 beta (IL-Ib) from an inactive to an active secreted form.

In a particular embodiment, the inhibitor of NLRP3 is MCC950. MCC950 blocks the release of IL-lbeta induced by NLRP3 activators, such as ATP, MSU and Nigericin, by preventing oligomerization of the inflammasome adaptor protein ASC (apoptosis-associated speck-like protein containing CARD). MCC950 is well known in the art and has the cas number 210826-40-7 and chemical formula: C20H24N2O5S.

In a particular embodiment, the inhibitor of NLRP3 is described in the following patent applications: WO2017/129897; WO2013/007763; WO2016/12322; W02017/031161;

WO2017/017469; WO2017/184746; WO2019/025467; WO2019/034693.

In a particular embodiment, the invention relates i) a pan-caspase inhibitor and ii) a PAK-1 and/or PAK-2 inhibitor used as a combined preparation for treating an infectious disease in a subject in need thereof. As used herein, the term "PAK-1" has its general meaning in the art and refers to P21- Activated Kinase 1, also known as Serine/threonine-protein kinase PAK-1, or P21 protein (Cdc42/Rac)-activated kinase 1. PAK-1 is a member of p21 -activated kinases family (PAKs) involved in the ERK activation, MAPK pathway activation and that are critical effectors that link the Rho GTPases to cytoskeleton reorganization and nuclear signaling and have been implicated in a wide range of biological activities.

As used herein, the term "PAK-1 inhibitor" refers to any compound that is able to inhibit the activity or expression of PAK-1. In particular the PAK-1 inhibitor inhibits the kinase activity of PAK-1. Typically, the PAK-1 inhibitor blocks PAK-1 interaction with proteins involved in ERK pathway and MAPK pathway such as RAF-1 (CRAF), inhibits its phosphorylation, or blocks MAPK cascade. The term "PAK-1 antagonist" refers to a compound that selectively blocks or inactivates PAK-1. As used herein, the term "selectively blocks or inactivates" refers to a compound that preferentially binds to and blocks or inactivates PAK-1 with a greater affinity and potency, respectively, than its interaction with the other sub-types or isoforms of the PAKs family.

Example of PAK-1 inhibitors include the compounds described in W02004007504, W02006072831, W02007023382, W02007072153, W02009086204, W02010071846, WO2011044264, WO2011044535, WO2011156640, WO2011156646, WO2011156775, WO2011156780, WO2011156786, and WO 2013026914.

Additional examples of PAK-1 inhibitors include, but are not limited to, staurosporine, 3-hydroxystaurosporine, K252a, CEP-1347, OSU-03012, DW12, FL172 (disclosed in Yi et ak, Biochemical Pharmacology, 2010, 80:683-689, the disclosure of which with respect to PAK-1 inhibitor compounds is hereby incorporated herein by reference), IP A3 (commercially available from Tocris), PF-3758309, PAK10 (available from Calbiochem), EKB569, TKI258, FRAX- 597 (available from Tocris) and SU-14813. In some embodiments, the PAK-1 inhibitor is a macrocyclic lactone. As used herein, the term "macrocyclic lactones" has its general meaning in the art and refers to macrocyclic lactones and macrocyclic lactones derivatives described in Lespine A. Lipid-like properties and pharmacology of the anthelmintic macrocyclic lactones. Expert Opin Drug Metab Toxicol. 2013 Dec; 9(12): 1581-95. Macrocyclic lactones, like ivermectin, are capable of inhibiting PAK-1 activity (e.g. HASMIMOTO ET AL: "Ivermectin inactivates the kinase PAK-1 and blocks the PAK-1 dependent growth of human ovarian cancer and NF2 tumor cell lines", DRUG DISCOV. THERAPEUTICS, vol. 3, no. 6, 2009, - 2009, pages 243-246). Examples of macrocyclic lactones include those described in WO 2012078605, WO 2012150543, WO2011075592, W0199316189, and WO2012028556. In some embodiments, examples of macrocyclic lactones include but are not limited to Ivermectin (Stromectol), Doramectin, Selamectin, Moxidectin, Milbemycin, Abamectin, Nemadectin and Eprinomectin. In a particular embodiment, the inhibitor of PAK-1 is AZ13711265. AZ13711265 is well known in the art, its CAS number is 2016806-55-4 and has the following chemical formula and structure in the art C28H35FN603S:

As used herein, the terms “combined treatment”, “combined therapy” or “therapy combination” refer to a treatment that uses more than one medication. The combined therapy may be dual therapy or bi-therapy.

In a particular embodiment, i) a pan-caspase inhibitor and ii) a classical treatment as a combined preparation according to the invention for simultaneous, separate or sequential use in the treatment of an infectious disease in a subject in need thereof.

In a particular embodiment, the invention relates to i) a pan-caspase inhibitor and ii) antiviral treatment used as a combined preparation according to the invention for simultaneous, separate or sequential use in the treatment of an infectious disease in a subject in need thereof.

In a particular embodiment, the invention relates to i) a pan-caspase inhibitor and ii) an antibiotic used as a combined preparation according to the invention for simultaneous, separate or sequential use in the treatment of an infectious disease in a subject in need thereof.

In a particular embodiment, the invention relates to i) a pan-caspase inhibitor and ii) an anti-parasitic treatment used as a combined preparation according to the invention for simultaneous, separate or sequential use in the treatment of an infectious disease in a subject in need thereof.

In a particular embodiment, i) a pan-caspase inhibitor and ii) a neutralizing monoclonal anti-IL-Ib antibody as a combined preparation according to the invention for simultaneous, separate or sequential use in the treatment of an infectious disease in a subject in need thereof.

In a particular embodiment, i) a pan-caspase inhibitor and ii) recombinant human IL- 1B receptor antagonist as a combined preparation according to the invention for simultaneous, separate or sequential use in the treatment of an infectious disease in a subject in need thereof. In a particular embodiment, i) a pan-caspase inhibitor and ii) a PAK-1 and/or PAK-2 inhibitor as a combined preparation according to the invention for simultaneous, separate or sequential use in the treatment of an infectious disease in a subject in need thereof.

As used herein, the term “administration simultaneously” refers to administration of 2 active ingredients by the same route and at the same time or at substantially the same time. The term “administration separately” refers to an administration of 2 active ingredients at the same time or at substantially the same time by different routes. The term “administration sequentially” refers to an administration of 2 active ingredients at different times, the administration route being identical or different.

Pharmaceutical composition

The pan-caspase inhibitor for use according to the invention alone and/or combined with a classical treatment as described above may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions.

Accordingly, in a third aspect, the invention relates to a pharmaceutical composition comprising a pan-caspase inhibitor for treating an infectious disease in a subject in need thereof.

In a particular embodiment, the pharmaceutical composition according to the invention, wherein the pan-caspase inhibitor is Emricasan.

In a particular embodiment, the pharmaceutical composition according to the invention comprising i) a pan-caspase inhibitor and ii) a classical treatment.

In a particular embodiment, the pharmaceutical composition according to the invention comprising i) a pan-caspase inhibitor and ii) an antiviral treatment.

In a particular embodiment, the pharmaceutical composition according the invention comprising i) a pan-caspase inhibitor and ii) an antibiotic.

In a particular embodiment, the pharmaceutical composition according the invention comprising i) a pan-caspase inhibitor and ii) an anti-parasitic drug.

In a particular embodiment, the pharmaceutical composition according the invention comprising i) a pan-caspase inhibitor and ii) immunosuppressive corticosteroids.

In a particular embodiment, the pharmaceutical composition according the invention comprising i) a pan-caspase inhibitor and ii) non-steroidal drug.

In a particular embodiment, the pharmaceutical composition according the invention comprising i) a pan-caspase inhibitor and ii) a neutralizing monoclonal anti-IL-Ib antibody.

In a particular embodiment, the pharmaceutical composition according the invention comprising i) a pan-caspase inhibitor and ii) a recombinant human IL-1B receptor antagonist. In a particular embodiment, the pharmaceutical composition according the invention comprising i) a pan-caspase inhibitor and ii) PAK-1 and/or PAK-2 inhibitor.

In a particular embodiment, the pharmaceutical composition according the invention comprising i) a pan-caspase inhibitor and ii) a NLRP3inhibitor.

As used herein, the terms "pharmaceutically" or "pharmaceutically acceptable" refer 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 anon-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms. Typically, 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 pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The polypeptide (or nucleic acid encoding thereol) can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or inj ected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

Method for screening

In a forth aspect, the invention relates to a method of screening a drug suitable for the treating an infectious disease in a subject in need thereof comprising i) providing a test compound and ii) determining the ability of said test compound to inhibit the expression or activity of pan-caspase.

Any biological assay well known in the art could be suitable for determining the ability of the test compound to inhibit the activity or expression of caspase such as caspase 1 or caspase 8. In some embodiments, the assay first comprises determining the ability of the test compound to bind to caspase such as caspase 1 or caspase 8.

In some embodiments, a population of cells then contacted and activated so as to determine the ability of the test compound to inhibit the activity or expression of caspase such as caspase 1 or caspase 8. In particular, the effect triggered by the test compound is determined relative to that of a population of immune cells incubated in parallel in the absence of the test compound or in the presence of a control agent either of which is analogous to a negative control condition. The term "control substance", "control agent", or "control compound" as used herein refers a molecule that is inert or has no activity relating to an ability to inhibit a biological activity or expression. It is to be understood that test compounds capable of inhibiting the activity or expression of caspase such as caspase 1 or caspase 8, as determined using in vitro methods described herein, are likely to exhibit similar modulatory capacity in applications in vivo. Typically, the test compound is selected from the group consisting of peptides, petptidomimetics, small organic molecules, antibodies (e.g. intraantibodies), aptamers or nucleic acids. For example the test compound according to the invention may be selected from a library of compounds previously synthesised, or a library of compounds for which the structure is determined in a database, or from a library of compounds that have been synthesised de novo.

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

FIGURES: Figure 1: Emricasan is an effective inhibitor of caspase-1. The ability of Emricasan to inhibit caspase-1 activity was assessed using active recombinant of caspase-1. YVAD-CHO is used as positive control in the in vitro assay. The results are expressed as A.U./min and represent the mean of 3 independent experiments realized in duplicate.

Figure 2: Emricasan blocks caspase-1 activation in monocytes from healthy donor or COVID19 patient. Whole peripheral blood cells of a healthy donor or a COVID-19 patient with critical symptoms were stained for active Caspase-1 (detected using the FAM-FLICA probe) and for CD45, CD 14, CD 16 markers. Cells were immunophenotyped by flow cytometry and monocytes were subdivided into 3 populations: non-classical monocytes (CD45+CD14dimCD16+), intermediate monocytes (CD45+CD14highCD16+) and classical monocytes (CD45+CD14highCD16-). A) Peripheral blood of a healthy donor was treated with 5 mM Emricasan for 3h or left untreated (upper panel). Nigericin (a specific NLRP3 activator) was added during 30 min to increase the basal activation of caspase-1 (lower panel). Active caspase-1 was detected using the FAM-FLICA probe and cells were immunophenotyped by flow cytometry as previously described. B) Peripheral blood of a COVID-19 patient was treated with 5 pM Emricasan for 3h or left untreated. Active Caspase-1 was detected using the FAM- FLICA probe in non-classical monocytes, intermediate monocytes and classical monocytes.

Figure 3. Main pathways regulated by Emricasan in human macrophages. The effects of Emricasan on macrophages were further explored by performing RNAseq analysis comparing cells treated with CSF-1 with or without Emricasan (3 pM). Genes clustering was partitioned into 5 clusters. Genes from the cluster 1 where extracted and over-representation analysis was performed on gene sets from the KEGG database (using clusterProfiler package enrichKEGG function, with adjusted p and q values cutoff of 0.05). The 5 top gene sets enriched in the cluster 1 are represented (draw on R with ggplot). Interestingly, Emricasan downregulates an important panel of coronavirus-COVID19 related genes.

EXAMPLE:

Materials and Methods:

Caspase activity measurement assay: Each assay (in triplicate) was performed with 0.25 units of active recombinant caspase-1. Briefly, active recombinant caspase-1 was incubated in a 96-well plate with 0.2 mM of YVAD-AMC as substrate for various times at 37°C. Caspase activity was measured following emission at 460 nm (excitation at 390 nm) in the presence or the absence of various concentrations of Emricasan or YVAD-CHO. Enzyme activities were expressed in arbitrary units (A.U.) per min. Ex vivo stimulation of whole blood and flow cytometry: Whole blood samples were collected into sodium citrate collection tubes and analyzed 24 h after collection. The peripheral blood was diluted 1 : 1 with RPMI medium and treated or not with 5 mM Emricasan (Sigma) for 3h and next with Nigericin (Invivogen) or vehicle for 30 min at 37 °C, under agitation (500 rpm on Eppendorf ThermoMixer). Caspase-1 activation was detected using the FAM-FLICA® Caspase-1 Assay kit (Immunochemistry) according to the manufacturer’s instructions. Briefly, cells were incubated with the FAM-FLICA probe for 30 min at 37 °C before to be washed: 1 mL of RPMI was added to dilute the non-bounded probe. Cell surface markers were stained for 10 min in the dark at room temperature using the following recombinant antibodies (1/100) (Miltenyi Biotec): CD45-VIoGreen (clone REA747), CD14-APC-VIo770 (clone REA599), CD16-PE (clone REA423). Red blood cells were lysed using the BD Pharm Lyse buffer (BD Biosciences) according to the manufacturer’s instructions. Cells were fixed with 4% PFA for 10 min. Cells were analyzed using MACSQuant 10 flow cytometer from Miltenyi. Data were analyzed with the FlowJo and GraphPad Prism softwares. After single cells were gated and debris excluded, peripheral blood mononuclear cells (PBMCs) were gated on CD45 positive cells. Monocytes were then gated as CD14 positive cells.

Results:

The ability of Emricasan to inhibit caspase-1 activity was first assessed using active recombinant of caspase-1 (Figure 1). Emricasan dose-dependently inhibits caspase-1 activity (maximal inhibition at 100 nM and IC50 at 6 nM). Strikingly, Emricasan was more efficient than YVAD-CHO, a potent inhibitor of caspase-1 to inhibit the recombinant enzyme (IC50 of 12 nM for YVAD-CHO). The ability of Emricasan to inhibit caspase-1 activity was also verified using whole blood from a healthy donor or a COVID-19 patient (Figure 2A and 2B). Thus, Emricasan blocks the basal activity of caspase-1 in classical, intermediate and non- classical monocytes detected using the FAM-FLICA probe (Figure 2A, upper panel) and dampens the Nigericin-mediated caspase-1 activation in classical and intermediate monocytes (Figure 2A, lower panel). Interestingly, we highlighted that Emricasan can also decrease the caspase-1 activity observed in all monocytes subsets from a COVID-19 patient. Altogether, these results indicate that Emricasan, a clinical available pan-caspase inhibitor, could be a new strategy to overcome cytokine storm by inhibiting caspase-1 activation.

As the previous severe acute respiratory syndrome coronavirus (SARS-CoV), Covid-19 uses the same cellular entry receptor, the angiotensin converting enzyme 2 (ACE2), which is present in high quantity in the lower respiratory tract of humans, especially on type II alveolar cells (AT2). COVIDS affects firstly the respiratory mucosa and it might further invade other organs, triggering a series of immune responses and possibly leading to a “cytokine storm production”, with an immune response out of control, and ultimately leading to critical conditions in certain COVID patients.

Inventor found the main pathways regulated by Emricasan in human macrophages. The effects of Emricasan on macrophages were further explored (Figure 3). Interestingly, Emricasan downregulates an important panel of coronavirus-COVID19 related genes.

Inventors have surprisingly found that Emricasan is able to efficiently inhibit caspase-8 which is involved in the inflammation process.

This new finding with either Emricasan alone or in combination with other therapeutics seem to be very promising in patients suffering from Syndrome-Corona Virus (SARS-CoV) infection.

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.

Claims

CLAIMS:

1. A method for treating an infectious disease in a subject in need thereof comprising a step of administering to said subject a therapeutically effective amount of a pan-caspase inhibitor.

2. The method according to claim 1, wherein the infectious disease is an disorder caused by organisms such as bacteria, viruses, fungi or parasites

3. The method according to claim 2, wherein the infectious disease causes a lung infection.

4. The method according to claim 2, wherein the lung infection is caused by a virus selected from the group consisting of respiratory syncytial virus, metapneumovirus, cytomegalovirus, rhinovirus, adenovirus, coxsackie virus, echo virus, herpes simplex virus, coronavirus and smallpox.

5. The method of claim 4, wherein the coronavirus is Severe Acute Respiratory Syndrome- Corona Virus (SARS-CoV).

6. The method of claim 5, wherein the SARS-CoV is Covid-19.

7. The method according to claim 1, wherein the pan-caspase inhibitor is Emricasan.

8. i) A pan-caspase inhibitor and ii) a classical treatment used as a combined preparation for treating infectious disease in a subject in need thereof.

9. i) A pan-caspase inhibitor and ii) an anti-parasitic treatment used as a combined preparation for treating infectious disease in a subject in need thereof.

10. The combined preparation according to claims 8 and 9, wherein the pan-caspase inhibitor is Emricasan and the anti-parasitic treatment is hydroxychloroquine.

11. i) A pan-caspase inhibitor and ii) a neutralizing monoclonal anti-IL-Ib antibody used as a combined preparation for treating infectious disease in a subject in need thereof.

12. i) A pan-caspase inhibitor and ii) a recombinant human IL-1B receptor antagonist used as a combined preparation for treating infectious disease in a subject in need thereof.

13. i) A pan-caspase inhibitor and ii) aNLRP3 inhibitor used as a combined preparation for treating infectious disease in a subject in need thereof.

14. A pharmaceutical composition comprising a pan-caspase inhibitor for treating an infectious disease in a subject in need thereof.

15. The pharmaceutical composition according to claim 14, wherein the pan-caspase inhibitor is Emricasan.