Domain of the invention

The present invention relates to compounds and methods for treating infections by enveloped viruses, in particular belonging †o the Coronaviridae family, more particularly by the virus SARS-CoV-2.

Technical background

In December 2019 an outbreak of pneumonia cases of unknown origin in occurred in Wuhan in China and spread quickly nationwide. On January 7, 2020, the causative pathogen was identified as a novel coronavirus, which was named 2019- nCoV and later SARS-CoV-2.

The new virus is closely related †o both SARS-CoV (82% nucleotide identify) and MERS-CoV (50% nucleotide identify), ye† distinct from them.

Early mortality rates suggested †ha† COVID-19, the name for the disease caused by SARS-CoV-2, may be less severe than SARS and MERS. However, illness onset among rapidly increasing numbers of people rapidly suggested †ha† SARS-CoV-2 would be more contagious than both SARS-CoV and MERS-CoV. As of May 1 1 , 2020 4 063 525 cases of COVID-19 (in accordance with the applied case definitions and testing strategies in the affected countries) have been reported, including 282244 deaths.

A great deal of effort has been made †o find effective drugs against the virus. Among the various compounds tested, remdesivir, a drug previously developed for the treatment of Ebola virus infections, has been reported †o show promising efficacy and acceptable safety in treating COVID-19 in a news release of the National Institutes of Health (NIH) dated April 29, 2020. As such, preliminary results indicate †ha† patients who received remdesivir had a 31% faster time †o recovery than those who received placebo (pO.001 ). Specifically, the median time †o recovery was 1 1 days for patients treated with remdesivir compared with 15 days for those who received placebo. Results also suggested a survival benefit, with a mortality rate of 8.0% for the group receiving remdesivir versus 1 1.6% for the placebo group (p=0.059).

However, the efficacy of remdesivir is no† completely established ye† as Wang et al. (2020) Lance† doi.org/10.1016/S0140-6736(20)31022-9 report †ha† in their trial remdesivir use was no† associated with a difference in time †o clinical improvement.

Accordingly, there is still a need for alternative treatments of infections by SARS- Summary of the invention

The present invention arises from the unexpected finding, by the inventors, that ezetimibe could be effective for treating infection by enveloped virus, in particular by SARS-CoV-2.

Accordingly, the present invention relates to ezetimibe or a pharmaceutically acceptable salt, ester, hydrate, derivative, prodrug or metabolite thereof for use in the prevention or treatment of an infection by an enveloped virus, in particular of the Coronaviridae family, in an individual.

The present invention also relates to ezetimibe or a pharmaceutically acceptable salt, ester, hydrate, derivative, prodrug or metabolite thereof for use as defined above, in combination with at least one other compound suitable for the prevention or treatment of an infection by an enveloped virus, in particular of the Coronaviridae family, more particularly by SARS-CoV-2.

The present invention also relates to a method for the prevention or treatment of an infection by an enveloped virus, in particular of the Coronaviridae family, in an individual, comprising administering to the individual an effective amount of ezetimibe or a pharmaceutically acceptable salt, ester, hydrate, derivative, prodrug or metabolite thereof.

The present invention also relates to a method as defined above, wherein ezetimibe or a pharmaceutically acceptable salt, ester, hydrate, derivative, prodrug or metabolite thereof is administered in combination with at least one other compound suitable for the prevention or treatment of an infection by an enveloped virus, in particular of the Coronaviridae family, more particularly by SARS-CoV-2.

The present invention also relates to a pharmaceutical composition, comprising as active substance ezetimibe or a pharmaceutically acceptable salt, ester, hydrate, derivative, prodrug or metabolite thereof for use in the prevention or treatment of an infection by an enveloped virus, in particular of the Coronaviridae family, in an individual.

The present invention also relates to a pharmaceutical composition for use as defined above further comprising at least one other compound suitable for the prevention or treatment of an infection by an enveloped virus in particular of the Coronaviridae family, more particularly by SARS-CoV-2.

The present invention also relates to products containing: - ezetimibe or a pharmaceutically acceptable salt, ester, hydrate, derivative, prodrug or metabolite thereof, and

- a† leas† one other compound suitable for the prevention or treatment of an infection by an enveloped virus, in particular of the Coronaviridae family, more particularly by SARS-CoV-2, as a combined preparation for simultaneous, separate or sequential use in the prevention or treatment an infection by an enveloped virus in an individual.

Description of the invention

As intended herein, the word “comprising” is synonymous †o “include” or “contain”. When a subject-matter is said †o comprise one or several features, it is mean† †ha† other features than those mentioned can be comprised in the subject- matter. Conversely, the expression “constituted of” is synonymous †o “consisting of”. When a subject-matter is said †o consist of one or several features, it is mean† †ha† no other features than those mentioned are comprised in the subject-matter.

Ezetimibe

Ezetimibe is well known †o one of skilled in the ar†. Ezetimibe is also known as SCH58235 or (3R,4S)-l -(4-fluorophenyl)-3-[(3S)-3-(4-fluorophenyl)-3-hydroxypropyl ]-4- (4-hydroxyphenyl)aze†idin-2-one and can be represented by the following formula (I):

Pharmaceutically acceptable sal†, ester, hydrate, derivative, prodrug or metabolite of ezetimibe will be apparent †o one of skilled in the ar†.

Examples of derivatives of ezetimibe according †o the invention include the compound named ( 1 R)-7-(4-chlorophenyl)-2-(4-fluorophenyl)-7-hydroxy- 1 -(4- hydroxyphenyl)-2-azaspiro[3.5]nonan-3-one, also known as SCH 58053, represented by the following formula (II) and the compound named (3R,4S)-l,4-bis(4-me†hoxyphenyl)- 3-(3-phenylpropyl)aze†idin-2-one, also known as SCH48462, represented by the following formula (III):

An example of a metabolite of ezetimibe incudes ezetimibe-glucuronide. The term “prodrug” as used herein refers to drug precursors which following administration †o the individual, release the drug via chemical and/or physiological process e.g. by hydrolysis and/or enzymatic conversion.

Virus As intended herein, an enveloped virus is a virus that has an outer wrapping or envelope. This envelope comes from the infected cell, or host, in a process called "budding off." During the budding process, newly formed virus particles become "enveloped" or wrapped in an outer phospholipidic coat that is made from a small piece of the cell's plasma membrane. Preferably, the virus as defined above is: an Herpesviridae virus, in particular Herpes simplex virus, varicella-zoster virus, cytomegalovirus, Epsfein-Barr virus, a Pleolipoviridae virus, in particular HHPV1 , HRPV1 , HGPV1 , His2V, a Togaviridae virus, in particular Rubella virus, alphavirus, - an Arenaviridae virus, in particular Lymphocytic choriomeningitis virus, a Flaviviridae virus, in particular Dengue virus, hepatitis C virus (HCV), yellow fever virus, Zika virus, an Orfhomyxoviridae virus, in particular Influenzavirus A, influenzavirus B, influenzavirus C, isavirus, fhogofovirus, a Paramyxoviridae, in particular Measles virus, mumps virus, respiratory syncytial virus, Rinderpest virus, canine distemper virus, a Bunyaviridae virus, in particular California encephalitis virus, hantavirus, a Rhabdoviridae virus, in particular Rabies virus, a Filoviridae virus, in particular Ebola virus, Marburg virus, a Coronaviridae, in particular Coronavirus, a Bornaviridae virus, in particular Borna disease virus, an Arferiviridae virus, in particular Arterivirus, equine arteritis virus, a Retroviridae virus, in particular HIV, more particularly HIV-1 or HIV-2, an Hepadnaviridae virus, in particular hepatitis B virus (HBV).

Preferably, the enveloped virus is of the Coronaviridae family.

Preferably, the virus as defined above is of the Alphacoronavirus, Betacoronavirus, Deltacoronavirus, or Gammacoronavirus genus, more preferably of the Betacoronavirus genus, most preferably of the Sarbecovirus or the Merbecovirus sub genus.

Preferably also the virus as defined above is a human virus, i.e. a virus which can infect a human.

Preferably, the virus as defined above is selected from the group consisting of SARS- CoV, SARS-CoV-2, MERS-CoV and mutants or variants thereof.

Preferably, the virus as defined above is SARS-CoV-2, ora mutant or variant thereof. SARS-CoV-2 is notably described in Fuk-Woo Chan et al. (2020) Emerging Microbes & Infections 9:221-236, which is incorporated herein by reference, and is also named 2019-nCoV, HCoV-19, SARS2, COVID-19 virus, Wuhan coronavirus, Wuhan seafood market pneumonia virus, and Human coronavirus 2019.

Preferably, the virus as defined above is SARS-CoV-2 and has the genomic sequence defined by NCBI Reference Sequence NC_045512.2 (SEQ ID NO: 1 ), or the complementary thereof, or is a mutant or variant thereof.

As intended herein, a “mutant or variant” of a virus as defined above, or of a genomic sequence of a virus as defined above, has a genomic sequence or is a nucleotide sequence which has af leas† 85%, 90%, 95%, 96% 97%, 98%, 99% or 99,5% identity with the genomic sequence of the virus as defined above.

Mutant or variants of SEQ ID NO: 1 can in particular be found on the “NCBI virus” website by searching for SARS-CoV-2 †axid:2697049. A preferred variant of SARS-CoV- 2 according †o the invention harbours a† leas† one mutation, in particular of the spike protein, selected from the group consisting of K417N, K417T, L452R, T478K, E484K, E484Q, N501 Y and D614G. A preferred variant of SARS-CoV-2 according †o the invention is a variant of concern, more preferably selected from the group consisting in B.l .1.7, B.1.1.7 + E484K, B.1.351 , P.1 , B.1.617.1 , B.1.617.2 and B.1.617.3.

As intended herein, a firs† nucleotide sequence “having a† leas† X% identity” with a second nucleotide sequence, in particular differs from the second sequence by the insertion, the suppression or the substitution of a† leas† one nucleotide. Besides, the percentage of identity between two nucleotide sequences is defined herein as the number of positions for which the bases are identical when the two sequences are optimally aligned, divided by the total number of bases of the longer of the two sequences. Two sequences are said †o be optimally aligned when the percentage of identity is maximal. Besides, as will be clear †o one of skill in the ar†, it may be necessary †o add gaps in order †o obtain an optimal alignment between the two sequences. In addition, when calculating the percentage of identity between an RNA nucleotide sequence and a DNA nucleotide sequence, an Uracil (U) base and a Thymine (T) base a† the same position are considered †o be identical.

As intended herein preventing or treating an infection by a enveloped virus, in particular of the Coronaviridae family, in an individual, encompasses preventing or treating the symptoms, disorders, syndromes, conditions or diseases, such as pneumonia or COVID-19, associated †o the infection by the enveloped virus, in particular of the Coronaviridae family, more particularly by SARS-CoV-2.

Individual

Preferably, the individual is a bird, such as a chicken, or a mammal, such as a human, a canine, in particular a dog, a feline, in particulara ca†, an equine, a bovine, a porcine, a caprine, such a sheep or a goa†, or a camelidae, more preferably the individual is a human.

Preferably, the individual as defined above is a human aged 50 or more, more preferably 60 or more, even more preferably 70 or more and most preferably 80 or more.

Preferably, the individual as defined above is a male individual.

Preferably, the individual as defined above suffers from a† leas† one other disease or condition, in particular selected from hypertension, diabetes, in particular type 2 diabetes, metabolic syndrome, a cardiovascular disease, in particular ischemic cardiomyopathy, a chronic respiratory disease, or cancer.

Preferably, the individual as defined above is overweight or obese.

According †o a usual definition a human individual is considered overweight if its Body Mass Index (BMI, body weigh† in kg relative †o the square of the heigh† in meters) is higher than or equal †o 25 kg/m ² and less than 30 kg/m ² and the individual will be said obese if his BMI is higher than or equal †o 30 kg/m ² . The individual according †o the invention may notably present with severe obesity, in particular characterized in human by a BMI higher than or equal †o 35 kg/m ² .

More generally, it is preferred †ha† the individual as defined above is a human and has a BMI higher than or equal †o 25 kg/m ² , 26 kg/m ² , 27 kg/m ² , 28 kg/m ² , 29 kg/m ² , 30 kg/m ² , 31 kg/m ² , 32 kg/m ² , 33 kg/m ² , 34 kg/m ² , 35 kg/m ² or 40 kg/m ² .

Besides, the individual as defined above may also have an abdominal obesity, corresponding in particular †o a visceral adipose tissue excess. According †o a usual definition a male human individual has an abdominal obesity if the abdominal perimeter is higher than or equal †o 94 cm, in particular higher than 102 cm and a female human individual has an abdominal obesity if the abdominal perimeter is higher than or equal †o 80 cm, in particular higher than 88 cm. The abdominal perimeter measure is well known †o one of skilled in the ar†: abdomen circumference is thus preferably measured midway between the las† floating rib and the top of the iliac crest in a standing individual in gentle expiration.

I† is particularly preferred †ha† the individual as defined above is a man and presents with an abdominal perimeter higher than or equal †o 90 cm, 91 cm, 92 cm, 93 cm, 94 cm, 95 cm, 96 cm, 97 cm, 98 cm, 99 cm, 100 cm, 101 cm or 102 cm. I† is also preferred †ha† the individual according †o the invention is a woman and presents with an abdominal perimeter higher than or equal †o 75 cm, 76 cm, 77 cm, 78 cm, 79 cm, 80 cm, 81 cm, 82 cm, 83 cm, 84 cm, 85 cm, 86 cm, 87 cm or 88 cm.

Preferably, the individual according †o the invention is afflicted with COVID-19 or is a† risk of being afflicted with COVID-19.

Additional compound

Preferably, the other compound suitable for the prevention or treatment of an infection by a virus of the Coronaviridae family, in particular by SARS-CoV-2, is selected from the group consisting of chloroquine, hydroxychloroquine, azithromycin, remdesivir, ribavirin, penciclovir, favipravir, nafamosta†, nitazoxanide, thalidomide, fingolimod, carrimycin, lopinavir/ritonavir, methylprednisolone, bevacizumab, tocilizumab, sarilumab, N-ace†ylcys†eine, recombinant human interferon at b, arbidol, eculizumab, darunavir, cobicista†, meplazumab, bamlanivimab, danoprevir, peginterferon alfa-2a, oseltamivir, nicotine, chlorpromazine, intravenous immunoglobulins, ivermectin, colchicine, clofoctol, GS-441524, MK-71 1 , molnupiravir, a s†a†in, and pharmaceutically acceptable salts, esters, hydrates, derivatives, prodrugs or metabolites thereof; more preferably it is hydroxychloroquine and/or remdesivir, or molnupiravir, or pharmaceutically acceptable salts, esters, hydrates, derivatives, prodrugs or metabolites thereof.

Preferably, the other compound suitable for the prevention or treatment of an infection by a virus of the Coronaviridae family, in particular by SARS-CoV-2, is a s†a†in selected from the group consisting of a†orvas†a†in, cerivas†a†in, fluvas†a†in, lovas†a†in, mevas†a†in, pi†avas†a†in, pravastatin, rosuvas†a†in, and simvastatin, more preferably it is simvastatin.

Pharmaceutical composition

Ezetimibe or a pharmaceutically acceptable salt, ester, hydrate, derivative, prodrug or metabolite thereof, optionally combined with a† leas† one other compound suitable for the prevention or treatment of an infection by an enveloped virus, in particular of the Coronaviridae family, more particularly by SARS-CoV-2, can be comprised in a pharmaceutical composition which can comprise a† leas† one pharmaceutically acceptable vehicle or excipient. The pharmaceutically acceptable vehicle or excipient can be selected from dispersants, solubilizers, stabilizers, preservatives, etc. Besides, pharmaceutically acceptable vehicle or excipient which can be used in formulations, in particular liquid and/or injectable formulations, are preferably selected from sucrose, lactose, starch, methylcellulose, hydroxymethylcellulose, carboxymethylcellulose, croscarmellose sodium, lactose monohydrate, magnesium stearate, microcrystalline cellulose, povidone, sodium lauryl sulfate, mannitol, gelatin, lactose, vegetable oils, acacia gum, liposomes, etc.

Administration

As intended herein “combined” or “in combination” means †ha† the composition as defined above, is administered a† the same time than the additional compound as defined above, either together, i.e. at the same administration site, or separately, or at different times, provided that the time period during which the composition as defined above exerts its pharmacological effects on the individual and the time period during which the additional compound exerts its pharmacological effects on the individual, at least partially intersect.

Ezetimibe or a pharmaceutically acceptable salt, ester, hydrate, derivative, prodrug or metabolite thereof or the pharmaceutical composition as defined above can be administered orally, parenterally, mucosally or cutaneously. The parenteral route preferably comprises subcutaneous, intravenous, intramuscular or intraperitoneal administration, although the latter is rather reserved for animals. The mucosal route preferably comprises nasal administration, pulmonary administration or administration via the rectal mucosa. The cutaneous route advantageously comprises the dermal route, in particular via a transdermal device, typically a patch.

Ezetimibe or a pharmaceutically acceptable salt, ester, hydrate, derivative, prodrug or metabolite thereof or the pharmaceutical composition as defined above can be formulated in the form of injectable suspensions, gels, oils, tablets, suppositories, powders, gel capsules, capsules, aerosols, etc., optionally by means of galenical forms or of devices which provide sustained and/or delayed release. For this type of formulation, an agent such as cellulose, carbonates or starches is advantageously used.

Ezetimibe or a pharmaceutically acceptable salt, ester, hydrate, derivative, prodrug or metabolite thereof or the pharmaceutical composition as defined above can be administered to the individual as defined above at a dose between 1 mg and 1 g, preferably between 5 mg and 500 mg, even more preferably between 7.5 mg and 333 mg, and most preferably 10 mg, of ezetimibe or a pharmaceutically acceptable salt, ester, hydrate, derivative, prodrug or metabolite thereof as defined above. Of course, those skilled in the art are able to adjust the dose of Ezetimibe or a pharmaceutically acceptable salt, ester, hydrate, derivative, prodrug or metabolite thereof as defined above according to the weight of the individual to be treated. Preferably, the dosage range of ezetimibe or a pharmaceutically acceptable salt, ester, hydrate, derivative, prodrug or metabolite thereof is from 1 mg and 1 g per day, preferably between 5 mg and 500 mg per day, even more preferably between 7,5 mg and 333 mg per day, and most preferably 10 mg per day. The invention will be further described by the following non-limifing Figure and Examples.

Description of the Figure Figure 1 represents the an†i-SARS-CoV-2 effect (as a percentage of remdesivir efficacy) (vertical axis, circles) and the cytotoxicity (in percentage) (vertical axis, black squares) of increasing concentrations of ezefimibe (in mM, horizontal axis).

EXAMPLES

Example 1

The efficacy of ezefimibe in inhibiting SARS-CoV-2 infection can be determined as described in Wang etal. (2020) Cell Research (2020) 30:269-271 which is incorporated herein by reference.

Briefly, the cytotoxicity of ezetimibe in Vero E6 cells (ATCC-1586) is first determined by the CCK8 assay. Then, Vero E6 cells are infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.05 in the presence of varying concentrations of ezetimibe. PBS is used in the controls.

Efficacy is evaluated by quantification of viral copy numbers in the cell supernatant via quantitative real-time RT-PCR (qRT-PCR) and confirmed with visualization of virus nucleoprotein (NP) expression through immunofluorescence microscopy at 48 h post infection (p.i.) (cytopathic effect is not obvious at this time point of infection). The half- maximal effective concentration (ECso) is then determined.

Example 2

The efficacy of ezetimibe in inhibiting Coronaviridae virus infections can also be determined as follows.

1 . Efficacy of ezetimibe on replication of live virus

Ezetimibe is first tested for its capacity to inhibit the replication of different luciferase- encoding coronaviruses:

*MHV is a prototype coronavirus of the Betacoronavirus genus **FIPV and PEDV are both members of the Alphacoronavirus genus 1.1. Eze tim ib e T oxicify to Ce I Is

In a firs† step the toxicity of ezetimibe on the three target cells is tested at different concentrations using a standard WST assay, in which the tetrazolium salt WST-1 is cleaved to formazan by the succinate-tetrazolium reductase system (which belongs to the respiratory chain of the mitochondria) only by metabolically intact cells. Formazan concentration can be determined by absorbance measurements which correlate directly to the number of viable cells.

1.2. Effect of pre-treatment with ezetimibe on virus replication

In a second step, the effect of ezetimibe on viral replication is tested by either pre treating viruses or target cells by ezetimibe:

• Virus pre-treatment: Target cells are infected (MOI: 0.01 ) with viruses pre-treated for 1 hour with 3 ezetimibe concentrations, and luciferase activity (proxy for virus infection) in cell lysates is measured at different times post infection (T =0, 3, 6, 9, 24, 32 and 48 hours post infection); virus titers in supernatants (collected at 10 hours post infection) are assessed by TCID50 (50% tissue culture infective dose) analysis.

• Cell pre-treatment: Target cells are pre-treated with 3 ezetimibe concentrations for 1 hour, and luciferase activity in cell lysates is measured at different times post infection (e.g. T=0, 3, 6, 9, 24, 32 and 48 hours post infection); virus titers in supernatants (collected at indicated time-points) are assessed by TCID50 analysis.

1.3. Effect of treatment with ezetimibe on virus replication

In a third step, the effect of ezetimibe is tested by treating infected target cells:

• Target cells are infected with the three target viruses (high MOI = 2) and ezetimibe is added to target cells at -2 h prior to or 2, 4, 6, 8 h after infection. Luciferase activity is measured at 10 hours post infection, and virus titers in supernatants (collected at 10 hours post infection) are assessed by TCID50 analysis. 2. Effect of ezetimibe on the entry spike-pseudotyped virus in target cells The capacity of ezetimibe †o inhibit the entry of luciferase-encoding vesicular stomatitis virus (VSV) pseudofyped by the spike protein of different Coronaviridae viruses in target cells is tested in a VSV pseudofyped particle (VSVpp) entry assay.

Briefly, target cells are pre-†rea†ed with 3 ezetimibe concentrations for 1 hour, and luciferase activity (proxy for virus infection) in cell lysates is measured a† T=24 hours post infection.

Example 3

The efficacy of ezetimibe in inhibiting SARS-CoV-2 infections has been determined as follows. 1 . Compound

Ezetimibe stock 10 mM in DMSO

2. Protocol

- Cell lines: lung cancer A549 stably transfected with a lenfiviral construct bearing the human ACE2 receptor in DMEM with 10% serum and 1% penicillin/sfrepfomycin

- Forma†: 384 well plate

- MOI (multiplicity of infection) = 0.1 , SARS-CoV-2 (strain

Be†aCoV/France/IDF0372/2020) - Positive Control Compound for viral replication used: remdesivir 10 mM

- Positive Control Compound for cytotoxicity used: camp†o†hecine 10 mM

- Negative Control: no compounds Ezefimibe is added †o the cells 48h before infection. The drug is replaced by the virus for 1 h of incubation. The inoculum is then removed and replaced by the drug.

After 72 h of incubation, the supernatant is recovered and the measurement of viral replication is carried out by quantitative RT-PCR in the presence and absence of drug according †o general real-time RT-PCR assays for the defection of SARS-CoV-2, in particular available from the World Health Organization (WHO) website.

The defection was carried on supernatants by using PCR primers for the N gene region: 5'-T AAT C AG AC A AGG A ACT GATT A-3 (Forward) (SEQ ID NO: 2) and 5'-

CGAAGGTGTGACTTCCATG-3' (Reverse) (SEQ ID NO: 3) with the Luna Universal One- Step RT-qPCR Kif (NEB) in an Applied Biosysfems QuanfStudio 7 fhermocycler.

In parallel, cell viability is assessed after 72 h incubation with the drug using the CellTifer Glo kif from Promega which measures the cellular ATP concentration of live cells.

Raw data are normalized against appropriate negative (0 %) and positive controls (100 %) and are expressed in % of viral replication inhibition or % of cytotoxicity. 3. Results

The results are shown in Figure 1. Ezetimibe shows a potent antiviral activity against SARS-CoV-2 without significant cytotoxicity.