EP4117669A1 - Composition comprising diltiazem for treating a viral infection caused by sars-cov-2 viruses - Google Patents

Abstract

The present invention relates to a pharmaceutical composition comprising Diltiazem in a suitable pharmaceutical carrier for its therapeutic use in the prevention and/or treatment of a viral infection caused by the SARS-CoV-2 virus, referred to as COVID-19 disease.

Description

COMPOSITION INCLUDING DILTIAZEM FOR TREATING VIRAL INFECTION WITH SARS-COV-2 VIRUSES

FIELD OF THE INVENTION

The present invention relates to a novel therapeutic use of known therapeutic compounds, alone or in combination with other active agents, to treat viral infection with the severe acute respiratory syndrome coronavirus SARS-CoV-2, otherwise referred to as coronavirus disease 2019. (COVID-19).

STATE OF THE ART

Human acute respiratory infections (ARIs) represent one of the main causes of consultations, hospitalizations and deaths worldwide, being the leading cause of death in young children with nearly 2 million deaths per year. Among the etiological agents responsible for acute respiratory infections, viruses occupy a prominent place. They are indeed found in the majority of cases of childhood pneumonia and are a predisposing factor for bacterial pneumonia in adults.

Coronaviruses are enveloped viruses, possessing a capsid exhibiting helical symmetry. They have a single-stranded, positive-sense RNA genome and are capable of infecting cells of birds and mammals.

Coronavirus infections can cause respiratory pathologies associated with symptoms similar to the common cold (caused in particular by the hCoV and OC43 viruses), bronchiolitis (caused by the NL63 virus) or even more serious illnesses such as severe acute respiratory syndrome. caused by SARS-CoV (Severe Acute Respiratory Syndrome Coronavirus) which generated an epidemic in 2003, and Middle East respiratory syndrome due to MERS-CoV, which generated an epidemic in 2012.

The Severe Acute Respiratory Syndrome Coronavirus 2, or SARS-CoV-2, is the coronavirus that caused the 2019-2020 coronavirus outbreak, generating the form of pneumonia referred to as 2019 coronavirus disease (COVID-19). This epidemic was declared a "public health emergency of international concern" by the World Health Organization (WHO) on January 30, 2020. The first patient was reported for the first time in the city of Wuhan (Hubei province, People's Republic of China). As of March 9, 2020, 111,321 cases have been confirmed worldwide, including 3,892 deaths in 109 countries. Related to the SARS-CoV virus, the SARS-CoV-2 virus belongs to the species Severe acute respiratory syndrome-related coronavirus, in the genus Betacoronavirus and the family Coronaviridae (Gorbalenya et al., 2020). The morphology of the virions is typical of that of the coronaviruses, with in particular the halo of protein protuberances (“Spike”) which gave them their name of “crown virus”.

Its genome, consisting of a single-stranded RNA of 29,903 nucleotides, was sequenced for the first time on January 5, 2020 by a team from Fudan University in Shanghai (China). Its genome is 79.5% similar to that of SARS-CoV and 96% to that of bat coronavirus, which suggests that the origin of the SARS-CoV-2 virus is zoonotic and is found in bats (Zhou et al., 2020).

Symptoms of SARS-CoV-2 infection roughly resemble those of seasonal flu: they include fever, fatigue, dry cough, shortness of breath, difficulty breathing, pneumonia, kidney failure , and can go as far as the death of the patient in severe cases (Hui et al., 2020).

A case-fatality calculation based on figures provided by different countries to the WHO on confirmed infections and deaths estimates the death rate to be around 2% (data from February 2020). However, this case fatality rate remains uncertain, due to the difficulty in estimating in the field the number of confirmed cases and the number of deaths directly attributable to SARS-CoV-2. It should be noted that according to the WHO, most of the patients who died had a weakened immune system due to other health problems such as hypertension, diabetes or cardiovascular disease.

The incubation period of SARS-CoV-2 is estimated to be between 2 and 14 days, but the incubation period would be longer in some cases, up to 24 days.

No specific antiviral treatment or vaccine is currently available for the prevention and / or treatment of viral SARS-CoV-2 infection.

The majority of treatments currently received by patients infected with this virus are essentially aimed at alleviating the symptoms of fever, cough and dyspnea, in order to promote their spontaneous recovery.

Antiviral compounds, known for their therapeutic activities on other types of virus, are currently being tested in phase 2 and 3 clinical trials. They are in particular compounds that are inhibitors of cellular or viral proteases, and of nucleoside analogues. These various therapeutic compounds are in particular Remdesivir, Galidesivir, Lopinavir, Camostat mesilate and Chloroquine. Remdesivir

Remdesivir is a broad spectrum antiviral compound, acting as a nucleoside analogue, more specifically an adenosine analogue. Available as a lyophilisate, this compound is administered intravenously. It is metabolized to its active nucleotide analog form (GS-441524): its presence misleads viral polymerase and causes reduced viral RNA synthesis.

Remdesivir was developed by the company Gilead Science for the treatment of infections with the Ebola and Marburg viruses. It also exhibits antiviral activity against other single-stranded RNA viruses such as Nipah, Hendra, Lassa viruses, respiratory syncytial virus (RSV) and SARS and MERS coronaviruses, in preclinical models (Lo et al., 2017) .

Clinically, Remdesivir has been studied so far in healthy volunteers and in patients infected with the Ebola virus. Treatments prescribed for a few patients with SARS-CoV-2 infection have not provided information on the safety and / or effectiveness in the treatment of SARS-CoV-2 infection. In addition, based on limited clinical experience, the toxicity and therefore safety profile of this compound is not characterized at this stage. Remdesivir has not yet received marketing authorization.

In this context, Gilead supplied Remdesivir to China for evaluation in several clinical trials including patients infected with SARS-CoV-2 (with and without symptoms). Gilead announced on February 26, 2020 the implementation of two Phase 3 clinical trials to test this compound.

Galidesivir

Galidesivir (BCX4430) is a nucleoside analogue, in particular adenosine, developed by the company BioCryst Pharmaceuticals.

This antiviral compound was initially developed for use against the hepatitis C virus and subsequently against filoviruses such as Ebola virus and Marburg virus. Galidesivir protects against Ebola virus disease and Marburg virus disease in rodents and monkeys when administered up to 48 hours after exposure to the virus. Its development for use in humans has been accelerated in order to address the lack of available treatments to combat the Ebola virus disease epidemic in West Africa.

This compound has broad spectrum antiviral activity against a set of other families of RNA viruses, such as bunyaviruses, arenaviruses, paramyxoviruses, coronaviruses and flaviviruses. Molnupiravir

Molnupiravir (also referred to as EIDD-2801 and MK-4482) is an antiviral compound which has the advantage of being in a form suitable for oral administration.

This compound is a prodrug which is metabolized after ingestion into N4-hydroxycytidine, a nucleoside derivative which inhibits RNA viruses by introducing RNA replication errors by the viral RNA-dependent RNA polymerase.

This compound has been tested for its anti-influenza activity. It also has some activity against coronaviruses such as SARS-CoV, MERS-CoV and SARS-CoV-2, responsible respectively for so-called SARS, MERS and COVID-19 diseases. (Painter et al., 2021); (Sheahan et al., 2020).

Lopinavir

Lopinavir is a viral protease inhibitor, used as an antiviral against the human immunodeficiency virus (HIV). It is marketed in association with Ritonavir by Abbott laboratories, mainly under the name Kaletra.

Lopinavir works by inhibiting the production of functional proteins and enzymes by new virions, which blocks the spread of the virus.

In humans, Lopinavir is rapidly broken down in the body by the cytochrome P450 system. For this reason, it is only given in a fixed combination with Ritonavir. This second drug, which is also a protease inhibitor, has the function of inhibiting cytochrome P450 monooxygenases, and therefore of slowing down the degradation of Lopinavir by these enzymes. This makes it possible to significantly reduce the dose required, and therefore the number of tablets to be absorbed by the patient.

A study published in 2004 showed that the Lopinavir / Ritonavir combination represented "substantial clinical benefit" for patients with SARS (Chu et al., 2004).

Regarding the treatment of SARS-CoV-2, Jinyintan Hospital in Wuhan, where the first 41 confirmed patients of COVID-19 disease were treated, has started a randomized controlled trial with this combination of anti-HIV drugs. .

Camostat mesilate Camostat mesilate or Camostat mesilate (FOY-305) is a synthetic low molecular weight protease inhibitor. It is able to inhibit trypsin, prostasin, matriptase and plasma kallikrein. This compound is used in therapy to treat chronic inflammation of the pancreas. In addition, this compound attenuates the function of the epithelial sodium ducts of the respiratory tract, and improves mucociliary clearance.

Camostat mesilate tablets are approved in Japan and marketed under the brand name FOI PAN®. They are used for the treatment of remission of acute symptoms of chronic pancreatitis and postoperative reflux esophagitis.

Patent EP 2 435 064 proposes targeting the serine proteases HAT and TMPRSS2 for the treatment of viral infections, in particular by influenza viruses.

Recently, it was shown that Camostat mesilate inhibits the transmembrane serine protease 2 TMPRSS2 of SARS-CoV-2, an enzyme necessary for the multiplication of the virus (Hoffmann et al., 2020).

Chloroquine

Chloroquine, initially known for its antimalarial activity, has an antiviral action on SARS-CoV according to data obtained in vitro (Vincent et al., 2005).

Recently published studies have described an apparent efficacy of Chloroquine Phosphate in the treatment of the SARS-CoV-2 virus. (Wang et al., 2020; Gao et al., 2020)

Thus, a consensus of Chinese experts now recommends including Chloroquine phosphate in the treatment of patients, at a rate of 500 mg twice a day for 10 days for patients diagnosed as mild, moderate and severe cases of disease, and without contraindication to Chloroquine.

However, clinical data are still limited.

New antiviral compounds are actively sought for the treatment and prevention against this emerging SARS-CoV-2 virus.

Among the new antiviral compounds identified in recent years, we can mention in particular Diltiazem, which is an active compound which acts on the host cell of the virus rather than directly on the virus, thus making it possible to have a broad spectrum of action and power. treat different viral infections. International application WO 87/07508 describes the use of Diltiazem for the treatment of viral infections linked to cytomegalovirus or to herpes.

International application WO 2011/066657 describes the use of Diltiazem for the treatment or prevention of viral infections such as oral herpes, genital herpes and shingles.

International application WO 2016/146836 describes the use of Diltiazem to treat infections by influenza viruses.

The article by (Fujioka et al., 2018) teaches that Diltiazem, known for its role as an inhibitor of Ca2 + ion transport, also acts as an inhibitor of cellular infection by the Influenza A virus.

International application WO 2019/224489 details some of the biological effects of Diltiazem, which induces the expression of genes encoding type III interferons in cells of the respiratory epithelium and of the intestinal epithelium. Diltiazem has thus been proposed for various therapeutic applications, in particular for the treatment of viral and bacterial respiratory infections in respiratory and intestinal epithelia.

Like Diltiazem, Berberine is a known active compound which may exhibit antiviral activity.

Berberine is an isoquinoline alkaloid produced by certain plants, in particular by the Berberi species, which is widely used in the Asian pharmacopoeia.

Studies carried out in vitro and in mouse models have shown that Berberine acts on various cellular pathways, and has antibacterial, antifungal, antiviral, anti-inflammatory and metabolic properties, improving hyperglycemia, type 2 diabetes, as well as hyperlipidemia. . (Mohan et al., 2017)

Berberine is particularly active in the fight against pulmonary arterial hypertension (PAH), a diffuse disease of pulmonary microvascular remodeling accompanied by a malignant proliferation of smooth muscle cells in the pulmonary artery, which causes a persistent rise in pulmonary arterial pressure. leading to right ventricular hypertrophy (HVD).

Berberine also has excellent anti-inflammatory properties, reducing joint swelling, tissue infiltration and destruction by inflammatory cells. Berberine regulates the polarization of macrophages, decreases the phagocytic function of macrophages, reduces the levels of M1 cytokines and increases the levels of M2 cytokines (IL-10 and transforming growth factor-bΐor TGF-b1). Berberine has a broad antibacterial spectrum, especially against infection with methicillin-resistant Staphylococcus aureus (MRSA). When applied in vitro and in combination with methoxyhydnocarpine, Berberine inhibits the growth of Staphylococcus aureus.

Berberine exhibits antiviral activity on various viruses, including:

- on the herpes virus HSV (Song et al. 2014)

- on influenza viruses (Wu et al. 2011)

- on cytomegalovirus (HCMV): low (micromolar) concentrations of Berberine inhibit the replication of different strains of HCMV, including clinical isolates and strains resistant to approved DNA polymerase inhibitors, by interfering with transactivating activity Immediate Early-2 (IE2) viral protein (Luganini et al., 2019)

- on the hepatitis C virus (HCV): Berberine would inhibit the entry of HCV into cells (Ting-Chun Hung et al., 2019)

- on the Zika virus (ZIKV): Berberine and emodin have been shown to be particularly effective against the entry and replication of several viruses, in particular triggering a powerful virucidal effect on ZIKV (Batista et al. 2019).

Regarding coronaviruses:

- Berberine may exhibit antiviral action against SARS-CoV (WO 2013/185126), although no experimental data are presented; and

- Berberine has an antiviral action against MERS-CoV (WO 2018/073549).

In the context of the present invention, several compounds were selected and evaluated in a cellular test for viral infection as well as on a model of human respiratory epithelium cultured in vitro at the air / liquid interface. Some of these compounds, alone or in combination, exhibited an antiviral effect on the SARS-CoV-2 coronavirus, quite unexpectedly.

The selected compounds had been previously described for other therapeutic applications. Surprisingly, it has now been shown that these compounds, alone or in combination, have antiviral activity against SARS-CoV-2, and allow the treatment and / or prevention of infection with this coronavirus.

In particular, combination therapies based on the association of at least two antiviral compounds appear to be particularly effective for the treatment and / or prevention of infection by the SARS-CoV-2 coronavirus. DISCLOSURE OF THE INVENTION

The present invention relates to a compound selected from Diltiazem, Berberine and their combination, for its therapeutic use in the treatment of viral infection with the SARS-CoV-2 virus (known as COVID-19 disease).

In particular, the present invention relates to Diltiazem for its therapeutic use in the prevention and / or treatment of viral infection with the SARS-CoV-2 virus, known as the COVID-19 disease.

In addition, the present invention relates to a pharmaceutical composition comprising, in a suitable pharmaceutical vehicle, at least one compound chosen from Diltiazem and Berberine, for its therapeutic use in the treatment of viral infection by the SARS-CoV virus. -2 (known as COVID-19 disease).

The present invention also relates to a pharmaceutical composition comprising Diltiazem in a suitable pharmaceutical vehicle, for its therapeutic use in the prevention and / or treatment of viral infection by the SARS-CoV-2 virus.

The present invention also relates to a composition for its use as described above, comprising at least one other active agent chosen from:

- a nucleoside analogue;

- an inhibitor of viral protease (s);

- a transmembrane serine protease inhibitor;

- Chloroquine, and

- any mixture of the aforementioned compounds.

Among the nucleoside analogs, the preferred compounds are Remdesivir, Galidesivir, Molnupiravir, and combinations thereof.

Among the inhibitors of viral protease (s), the preferred compound is Lopinavir, and more preferably Lopinavir combined with Ritonavir.

Among transmembrane serine protease inhibitors, the preferred compound is Camostat mesilate.

The present invention also relates to a combination product comprising at least one compound chosen from Diltiazem and Berberine, and at least one other active agent chosen from:

- a nucleoside analogue;

- an inhibitor of viral protease (s);

- a transmembrane serine protease inhibitor; - Chloroquine, and

- any mixture of the aforementioned compounds, for its simultaneous, separate or sequential use in the prevention and / or treatment of a viral infection by the SARS-CoV-2 virus.

The present invention also relates to a combination product comprising Diltiazem and at least one other active agent chosen from:

- a nucleoside analogue;

- an inhibitor of viral protease (s); a transmembrane serine protease inhibitor;

- Chloroquine, and

- any mixture of the aforementioned compounds, for its simultaneous, separate or sequential use in the prevention and / or treatment of a viral infection with the SARS-CoV-2 virus.

Finally, the invention also relates to a pharmaceutical composition comprising, in a suitable pharmaceutical vehicle, a combination of Diltiazem with Remdesivir; or a combination of Diltiazem with Molnupiravir.

DESCRIPTION OF FIGURES

FIG. 1 represents the antiviral effects of Berberine and Remdesivir as monotherapy on cultured Vero E6 cells infected with the SARS-CoV-2 virus.

Figure 1A: chronogram of the experiment.

Figure 1 B: Dose-effect curves of the molecules Berberine and Remdesivir in monotherapy and determination of the IC50 associated with 48 and 72 hours post-infection (hpi). Remdesivir and Berberine exhibit an antiviral effect at 48 hpi, with an IC50 determined at 0.98 and 17.47 mM, respectively. This antiviral effect is even more pronounced at 72 hpi with IC50s of less than 0.72 and 5.60 mM for Remdesivir and Berberine, respectively.

FIG. 2 represents the antiviral effects of the Diltiazem / Remdesivir, Berberine / Remdesivir combinations on cultured Vero E6 cells infected with the SARS-CoV-2 virus.

Figure 2A: chronogram of the experiment.

Figure 2B: Dose-effect curves of the Diltiazem / Remdesivir and Berberine / Remdesivir combinations, and determination of the IC50 associated with 48 and / or 72 hours post infection (hpi). The combination of treatment with Remdesivir in the presence of a fixed concentration of Diltiazem (11.5 pM), makes it possible to obtain antiviral efficacy with a IC50 of 0.32 mM, at 48hpi. In an equivalent manner, the combination of a treatment with Diltiazem in the presence of a fixed concentration of Remdesivir (2.5 mM), makes it possible to have antiviral efficacy with an IC50 of 0.55 mM, at 48hpi. Moreover, the combination of a treatment with Remdesivir in the presence of a fixed concentration of berberine (12.5 pM) makes it possible to have antiviral efficacy with an IC50 of 0.65 pM, at 48 hpi. At a more advanced stage of the infection (72hpi), the combination of treatment with Remdesivir in the presence of a fixed concentration of Diltiazem (11 .5 mM), makes it possible to obtain antiviral efficacy with an IC50 of 0.35 pM.

Figure 3 illustrates some of the results presented in Figures 1B and 2B visually.

Figure 3A: chronogram of the experiment.

FIG. 3B: Observation by light microscopy of the antiviral effect of Remdesivir and Diltiazem in monotherapy and in combination on a layer of Vero E6 cells in culture, and infected with SARS-CoV-2. Under the same experimental conditions as before (Fig. 2), the combination of Remdesivir (0.625 pM) and Diltiazem (11.5 pM) makes it possible to very visibly reduce the cytopathic effects of the infection (rounded cells and detached from the mat) in comparison untreated control, and monotherapy treatments.

Figure 4: Chronogram of the experiments of Example 3.

Figure 5. Chronogram of the experiment of example 4. The A549-ACE2 cells are seeded and then infected with a viral strain of SARS-CoV-2, before being treated 1 h post-infection with Diltiazem or Remdesivir. .

Figure 6. (A) A549-ACE2 cells infected at MOI of 10 ^L ; (B) A549-ACE2 cells infected at an MOI of 10 ²

Cells were untreated (black lines) or treated with 5 pM Remdesivir (gray squares) or 45 pM Diltiazem (gray triangles). The results are expressed as a percentage of viral titer measured in the supernatant, relative to the viral titer measured in the wells of the untreated cells, as a function of the time post-viral infection.

Figure 7. (A). Measurement of the IC50 of Diltiazem on A549-ACE2 cells infected with a viral strain of SARS-CoV-2. The viral titer is expressed as a percentage of the viral titer measured in the supernatant, relative to the viral titer measured in the wells of the untreated cells, as a function of the concentration of Diltiazem used. (B). Measurement of CC50 of Diltiazem on A549-ACE2 cells after 72 hours of treatment. The viability of the cells is expressed as a percentage of viability measured for the untreated cells. The results are presented according to the concentration of Diltiazem used. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to Diltiazem for its therapeutic use in the prevention and / or treatment of viral infection with the SARS-CoV-2 virus, known as the COVID-19 disease.

The present invention also relates to a pharmaceutical composition comprising, in a suitable pharmaceutical vehicle, at least one compound chosen from Diltiazem and Berberine, for its therapeutic use in the treatment of viral infection by the SARS-CoV-2 virus (COVID -19).

The present invention also relates to a pharmaceutical composition comprising Diltiazem in a suitable pharmaceutical vehicle, for its therapeutic use in the prevention and / or treatment of viral infection by the SARS-CoV-2 virus.

By "viral infection by the SARS-CoV-2 virus" is meant the fact that an organism, human or animal, has cells that have been infected with the SARS-CoV-2 coronavirus, also referred to by the name COVID-19 .

For the purposes of the invention, the expression “SARS-CoV-2 virus” designates on the one hand the coronavirus identified for the first time in Wuhan in China, and sequenced in early 2020 by a team from Fudan University in Shanghai ( Zhou et al., 2020); and on the other hand, includes all variants related to this first identified viral strain, which appeared subsequently, and in particular the following SARS-CoV-2 variant strains: i. the “Wuhan-like” strain used by the inventors, described in the examples; ii. the hCoV-19 / France / ARA-104350/2020 (GISAID ID: EPIJSL_683350) strain of the B.1 lineage (this strain has at least the D614G mutation in its spike protein; it is considered today as the circulating wild-type strain in Europe, by comparison with the variants cited below); iii. a viral strain called “English variant” hCoV-19 / France / ARA-SC2118 / 2020 (GISAID ID: EPI_ISL_900512) of lineage B.1.1.7; iv. a South African strain (501Y.V2.HV001) of lineage B.1.3.5.1 and v. a Brazilian variant strain of lineage B.1 .1 .28.

A viral infection is usually diagnosed by a healthcare professional, based on observing the symptoms of the infected patient. Additional laboratory tests may be necessary to confirm the diagnosis: blood and / or sputum and / or bronchoalveolar fluid tests. The infection can in particular be established by carrying out a detection by molecular biology and / or a viral titration from respiratory samples, or by assaying the specific antibodies of SARS-CoV-2 circulating in the blood.

The detection in infected individuals of this specific virus is carried out by conventional diagnostic methods, in particular molecular biology (PCR) which make it possible to establish that it is indeed this SARS-CoV-2 virus, well known to the person skilled in the art.

The term "treatment" refers to combating infection with the SARS-CoV-2 coronavirus in a human or animal body. Thanks to the administration of at least one composition according to the invention, the level of viral infection (infectious titer) in the organism will decrease, and preferably the virus will disappear completely from the organism within a period of less than that expected for a cure without treatment.

The term "treatment" also refers to alleviating the symptoms associated with the viral infection (respiratory syndrome, kidney failure, fever, etc.).

Certain compositions according to the invention are also intended for use in the prevention of infection with SARS-CoV-2.

Thus, the present invention relates to: a pharmaceutical composition comprising Diltiazem in a suitable pharmaceutical vehicle, for its therapeutic use in the treatment of viral infection with the SARS-CoV-2 virus; a pharmaceutical composition comprising Berberine, in a suitable pharmaceutical vehicle, for its therapeutic use in the treatment of viral infection with the SARS-CoV-2 virus; and a pharmaceutical composition comprising Diltiazem in a suitable pharmaceutical vehicle, for its use in the prevention of viral infection with the SARS-CoV-2 virus.

For the purposes of the invention, the term "prevention" refers to preventing, or at least reducing the probability of occurrence, of an infection in a human or animal body by SARS-CoV-2. Thanks to the administration of at least one composition according to the invention, the human or animal cells of said organism become less permissive to infection, and are thus more likely not to be infected with said coronavirus, or to develop diseases. less severe symptoms during infection with said coronavirus.

The compositions according to the invention may be of pharmaceutical type, intended to be administered to a human being, or of veterinary type, intended to be administered to patients. non-human animals. With regard to animals, it is understood that the veterinary compositions for their use in the prevention and / or treatment of an infection by the SARS-CoV-2 coronavirus are intended to be administered to animals infected with this coronavirus.

According to the invention, the term "suitable pharmaceutical vehicle" denotes pharmaceutical vehicles or excipients, which are compounds having no specific action on the infection considered here. These vehicles or excipients are pharmaceutically acceptable, which means that they can be administered to an individual or an animal without generating significant deleterious effects.

The expression “at least one compound selected from Diltiazem and Berberine” means that the pharmaceutical composition comprises either Diltiazem or Berberine, or a combination of the two.

According to a first aspect, the pharmaceutical composition for its use according to the invention comprises at least an effective amount of Diltiazem. This pharmaceutical composition is intended for therapeutic and / or preventive use against infection by the SARS-CoV-2 virus.

According to a second aspect, the pharmaceutical composition for its use according to the invention comprises at least one effective amount of berberine.

According to a third aspect, the pharmaceutical composition for its use according to the invention comprises at least an effective amount of Diltiazem and an effective amount of Berberine.

By "effective amount" is meant within the meaning of the invention an amount of active compound sufficient to inhibit the proliferation and / or replication of the coronavirus, and / or the development of viral infection within the body. This inhibition can be quantified, for example by measuring the viral titer, as presented in the examples of the present application.

Thus, according to a particular aspect of the invention, the pharmaceutical composition for its use as described above comprises a combination of Diltiazem and Berberine.

For the purposes of the invention, the term "combination" means a composition comprising at least two distinct active compounds, the two compounds having antiviral action.

This combination includes either the same amount by weight of each antiviral compound, i.e. a combination of 50% Diltiazem and 50% Berberine by weight, or unequal doses of each compound, such as 90% Diltiazem. Diltiazem and 10% Berberine, 80% Diltiazem and 20% Berberine, 70% Diltiazem and 30% Berberine, 60% Diltiazem and 40% of Berberine, 40% of Diltiazem and 60% of Berberine, 30% of Diltiazem and 70% of Berberine, 20% of Diltiazem and 80% of Berberine, or even 10% of Diltiazem and 90% of Berberine, the percentages being expressed by weight of the compound relative to the total weight of the combination. Diltiazem

Diltiazem is a member of the benzothiazepine family, referenced under the CAS number 42399-41-7.

For the purposes of the present invention, the term “Diltiazem” denotes the molecule in the form of one of its L-cis or D-cis enantiomers, or of a racemic mixture of the two, or alternatively a Diltiazem salt as by example Diltiazem hydrochloride, the structural chemical formula of which is shown below, in formula (I):

Formula I

Diltiazem has been known for over 30 years and is approved by drug regulatory authorities in Europe and the United States. It can be given as Diltiazem hydrochloride. Cardizem®, Cartia®, Taztia® and Dilacor® are its most common trade names.

Many formulations are available, particularly sustained release formulations. Diltiazem is available in various dosage forms, such as a cream for topical application, as tablets or capsules for oral administration, as a powder for preparation of a solution for injection or as pharmaceutical preparations for inhalation (WO 02 / 094238, US 4,605,552).

The classic dosage in humans is 180 to 360 mg / day, administered in capsules or tablets, for its therapeutic use as a calcium channel blocker. The first identified physiological property of this compound is the inhibition of calcium channels, and therefore the inhibition of intracellular calcium fluxes. In particular, Diltiazem slows down the entry of transmembrane calcium into the myocardial muscle fiber and the smooth muscle fiber of the vessels. This makes it possible to decrease the intracellular calcium concentration reaching the contractile proteins.

In humans, the administration of Diltiazem is indicated for its vasodilator action, with the aim of reducing the work of the heart. It is thus used in the management of cardiac and circulatory disorders such as angina pectoris, high blood pressure, myocardial ischemias and tachycardia. Diltiazem also works by reversing the effects of angiotensin II, both renally and peripherally. Topically, Diltiazem may be indicated for chronic anal fissures.

Patent EP 1 117408 describes the use of Diltiazem, as a calcium channel inhibitor compound, to treat pathologies linked to the degeneration of photoreceptors of the retina.

Regarding the use of Diltiazem for the treatment of viral infections, as previously mentioned, this has already been described in several patent applications. In addition, a clinical trial is currently underway (FLUNEXT PHRC # 15-0442 ClinicalTrials.gov Identifier: NCT03212716), with the aim of obtaining marketing authorization for this new therapeutic antiviral indication.

Berberine

Berberine is a natural alkaloid found in a large number of plants, particularly in the Berber species. Its CAS number is 633 66 9

For the purposes of the present invention, the molecule in all its forms is designated by “Berberine”. Its developed chemical structure is shown schematically below: This molecule is widely used in the Asian pharmacopoeia, for its antifungal, antibacterial and anti-inflammatory properties.

In cells, Berberine is localized in particular in the mitochondria where it inhibits complex I of the respiratory chain, thus reducing the production of ATP and the subsequent activation of AMPK (Adenosine Monophosphate activated Protein Kinase). This ubiquitous enzyme plays a role in cellular energy homeostasis. The main effect of AMPK activation is to (i) stimulate hepatic fatty acid oxidation and ketogenesis, (ii) inhibit cholesterol synthesis, lipogenesis and triglyceride synthesis, (iii) stimulate oxidation of fatty acids in skeletal muscles and uptake of glucose by muscles; and (iv) modulate insulin secretion by beta cells in the pancreas.

The bioavailability of Berberine is low, but this does not present any difficulties because the systemic action of Berberine passes to a significant extent via either the modification of the intestinal microbiota and its metabolites, or after metabolization of Berberine by this same microbiota. .

The half-life of Berberine is also low, around 4 hours. This implies that the daily doses should ideally be divided into 3 doses. For an adult the daily dose is generally 500 mg to 1500 mg.

Combination with another active agent

It is understood that the composition for its use according to the invention comprises at least one compound chosen from Diltiazem and Berberine, and that it can therefore also include other active compounds, in addition to the suitable pharmaceutical vehicle.

In fact, Diltiazem and Berberine or their mixture can be used in therapy alone, or in combination with at least one other active agent.

They may be compounds which make it possible to improve the antiviral activity of Diltiazem and / or Berberine, or conversely, Diltiazem and Berberine may act as potentiating agents for these other active compounds.

Thus, the present invention relates to Diltiazem or Berberine or their combination, for their use in the potentiation of the antiviral effects of other therapeutic compounds used to treat and / or prevent viral infection by SARS-CoV-2, in particular those cited in the present application. These additional active compounds may be chosen from the pharmaceutical classes of agents mentioned in application WO 2015/157223, namely from antibacterial agents, antiparasitic agents, neurotransmission inhibitors, estrogen receptor inhibitors, inhibitors of DNA synthesis and replication, protein maturation inhibitors, kinase pathway inhibitors, cytoskeletal inhibitors, lipid metabolism inhibitors, anti-inflammatory agents, ion channel inhibitors, inhibitors of apoptosis and cathepsin inhibitors.

Thus, according to a particular implementation of the invention, the pharmaceutical composition for its use as described above comprises at least one other active agent, in particular an antiviral agent.

For the purposes of the present invention, the terms “antiviral agent” or “antiviral compound” are understood to mean active agents which act on the viral load (also called infectious titer), by inhibiting either directly or indirectly replication and / or the dissemination of a virus and in particular, in the present case, of the SARS-CoV-2 coronavirus, within an infected organism.

According to a particular aspect of the invention, the pharmaceutical composition for its use in the prevention and / or treatment of an infection by the SARS-CoV-2 coronavirus, comprises, in addition to Diltiazem and / or Berberine, at least another antiviral agent.

It is understood that this other antiviral agent will be used in the doses necessary to exhibit an antiviral action, this dose being designated as being "effective", this dosage being able to be easily determined by a person skilled in the art.

This combination comprises either the same amount by weight of each antiviral compound, i.e. a combination of 50% Diltiazem and / or Berberine and 50% of another antiviral agent by weight, or unequal doses of each compound, such as 90% Diltiazem and / or Berberine - 10% of the other antiviral agent, 80% - 20%, 70% - 30%, 60% - 40%, 40%

- 60%, 30% - 70%, 20% - 80%, or even 10% of Diltiazem and / or Berberine and 90% of another antiviral agent, the percentages being expressed by weight of the compound relative to the total weight of the combination.

By "antiviral activity" or "antiviral action" is meant either:

- a direct action on the virus, in particular the action of inhibiting the virus's replication cycle or its ability to infect and reproduce in host cells,

an indirect action on the target cells of said virus, by modulating the expression of certain genes of the target cells. The term 'target cells' is understood to mean cells infected with the coronavirus and / or likely to be infected in the near future, due to their immediate proximity to infected cells.

Antiviral agents are classified into different categories based on their mode of action. We can cite in particular:

- nucleotide or ribonucleoside analogues, which interfere with or stop the synthesis of DNA or RNA;

- inhibitors of enzymes involved in DNA or RNA synthesis (helicase, replicase);

- viral protease inhibitors;

- compounds which inhibit the stages of maturation of the virus during its replication cycle;

- compounds which interfere with the binding to the cell membrane, or the entry of viruses into host cells (fusion or entry inhibitors) such as transmembrane serine protease inhibitors, in particular those of type 2;

- agents which prevent the virus from expressing itself within the host cell after its entry, by blocking its disassembly within the cell;

- agents that restrict the spread of viruses to other cells.

Among these antiviral agents well known to those skilled in the art, the following are in particular used to fight against RNA viruses: nucleoside analogs, inhibitors of viral protease (s), helicase inhibitors, and inhibitors of cellular entry of the virus into target cells such as transmembrane serine protease inhibitors.

For the purposes of the invention, a "nucleoside analogue" refers to a compound used to prevent viral replication in infected cells, such as, for example, aciciovir. These compounds have structures similar enough to nucleosides to be integrated into replicating viral DNA strands, but they act as end-chain agents and stop the action of viral DNA polymerase.

Such a compound will be chosen from a nucleoside analog of guanosine (for example Ribavirin), adenosine (for example Remdesivir or Galidesivir), cytidine (Molnupiravir) or thymidine, or their deoxy versions. -.

For the purposes of the invention, a “viral protease inhibitor (s)” denotes an antiviral compound acting by inhibiting the action of at least one viral protease, a protein which allows the cleavage and assembly of viral proteins. , a process essential for obtaining new infectious virions. The virions obtained are then incapable of infecting new cells. This therapeutic strategy is used in particular to treat viral infections by HIV (Human Immunodeficiency Virus). For the purposes of the invention, a “transmembrane serine protease inhibitor” denotes an antiviral compound acting by inhibiting the entry of the virus into the cell, in particular by its action at the level of the transmembrane serine protease 2 designated by the abbreviation TMPRSS2.

According to a particular implementation of the invention, the composition for its use as described above comprises at least one other active agent chosen from:

- a nucleoside analogue;

- an inhibitor of viral protease (s);

- a transmembrane serine protease inhibitor;

- Chloroquine, and

- any mixture of the aforementioned compounds.

According to a first implementation of the invention, the combination comprises Diltiazem and at least one nucleoside analogue.

According to a second implementation of the invention, the combination comprises Diltiazem and at least one inhibitor of viral protease (s).

According to a third implementation of the invention, the combination comprises Diltiazem and at least one transmembrane protein serine inhibitor, in particular that of type 2.

According to a fourth embodiment of the invention, the combination comprises Diltiazem and Chloroquine.

According to a fifth implementation of the invention, the combination comprises Diltiazem and a mixture of at least two compounds chosen from:

- a nucleoside analogue;

- an inhibitor of viral protease (s);

- a transmembrane protein serine inhibitor, and

- Chloroquine.

According to a sixth implementation of the invention, the combination comprises Berberine and at least one nucleoside analogue.

According to a seventh implementation of the invention, the combination comprises berberine and at least one inhibitor of viral protease (s).

According to an eighth implementation of the invention, the combination comprises berberine and at least one transmembrane protein serine inhibitor, in particular that of type 2. According to a ninth implementation of the invention, the combination comprises Berberine and Chloroquine.

According to a tenth implementation of the invention, the combination comprises Berberine and a mixture of at least two compounds chosen from:

- a nucleoside analogue;

- an inhibitor of viral protease (s);

- a transmembrane protein serine inhibitor, and

- Chloroquine.

The at least one nucleoside analog may in particular be chosen from the group consisting of: Remdesivir, Galidesivir, Molnupiravir and their combinations.

The at least one viral protease inhibitor (s) may in particular be Lopinavir, and preferably Lopinavir combined with Rinotavir.

The at least one transmembrane protein serine inhibitor, in particular that of type 2, may in particular be Camostat mesilate.

According to other implementations, the pharmaceutical composition for its use according to the invention comprises or consists of:

- Diltiazem and Remdesivir;

- Diltiazem and Galidesvir;

- Diltiazem and Molnupiravir;

- Diltiazem and Lopinavir, preferably combined with Rinotavir;

- Diltiazem and Camostat mesilate;

- Diltiazem and Chloroquine, or even

- Diltiazem and all possible combinations of Remdesivir, Galidesvir, Molnupiravir, Lopinavir, Camostat mesilate and Chloroquine.

According to other implementations, the pharmaceutical composition for its use according to the invention comprises or consists of:

- Berberine and Remdesivir;

- Berbérine and Galidesvir;

- Berberine and Molnupiravir;

- Berberine and Lopinavir, preferably combined with Rinotavir;

- Berberine and Camostat mesilate;

- Berberine and Chloroquine, or even

- Berberine and all possible combinations of Remdesivir, Galidesvir, Molnupiravir, Lopinavir, Camostat mesilate and Chloroquine. According to other implementations, the pharmaceutical composition for its use according to the invention comprises or consists of:

- a combination of Diltiazem and Berberine, and Remdesivir;

- a combination of Diltiazem and Berberine, and Galidesvir;

- a combination of Diltiazem and Molnupiravir;

- a combination of Diltiazem and Berberine, and Lopinavir, preferably combined with Rinotavir;

- a combination of Diltiazem and Berberine, and Camostat mesilate;

- a combination of Diltiazem and Berberine, and Chloroquine, or even

- a combination of Diltiazem and Berberine, and all possible combinations of Remdesivir, Galidesvir, Molnupiravir, Lopinavir, Camostat mesilate and Chloroquine.

These combinations exhibit synergistic antiviral effects, as shown in the examples.

For example, the results presented in Table 2 make it possible to demonstrate the fact that, at 48 hours post-infection, the effects of Remdesivir are greater (+ 68%) in the presence of Diltiazem, as well as in the presence of Berberine. (+33%).

The results presented in Table 3 show that, on models of nasal epithelium, 48 hours post-infection, the effects of Remdesivir are potentiated by the presence of Diltiazem (+ 1.3 log reduction in viral production) or of Berberine. (+ 0.89 log reduction in viral production).

According to another implementation of the invention, the composition for its therapeutic use as described above further comprises at least one antibiotic.

Such an antibiotic will be particularly useful in preventing bacterial superinfection with the current viral infection.

The antibiotic is chosen from antibiotics well known to those skilled in the art, in particular those used in viral infections to prevent bacterial superinfection, and in particular those of the macrolide family.

The pharmaceutical compositions according to the present invention are suitable for nasal, oral, sublingual, inhalation, subcutaneous, intramuscular, intravenous, transdermal, ocular or rectal administration.

According to a preferred implementation, the composition for its use as described above is characterized in that it is in a galenic form suitable for nasal administration, in particular intranasal, in particular by inhalation. The intranasal route is a route of administration characterized in that the pharmaceutical composition is introduced directly into the nasal cavity of the patient, by various methods, for example: drops, spray, or inhaler. The use of a specific device, such as an intranasal mucosal spray device, is recommended.

The intranasal route offers the possibility of rapid, painless and non-invasive drug administration with an effectiveness often comparable to that of the intravenous route. It is particularly suitable in pediatrics or for the elderly, or in emergency medicine situations.

According to one implementation, the pharmaceutical composition for its use as described above is administered intranasally.

According to another preferred embodiment, the pharmaceutical composition for its use as described above is administered by inhalation.

Inhalation refers to absorption through the respiratory tract. It is in particular a method of absorbing compounds for therapeutic purposes, certain substances in the form of gas, micro-droplets or suspended powder.

The administration of pharmaceutical or veterinary compositions by inhalation, that is to say by the nasal and / or oral passages, is well known to those skilled in the art.

There are two types of administration by inhalation:

- administration by insufflation when the compositions are in the form of powders, and

- administration by nebulization when the compositions are in the form of aerosols (suspensions) or in the form of solutions, for example aqueous solutions, placed under pressure. The use of a nebulizer or sprayer will then be recommended to administer the pharmaceutical or veterinary composition.

The dosage form considered here is therefore chosen from: a powder, an aqueous suspension of droplets or a solution under pressure.

The present invention also relates to a combination product comprising at least one compound chosen from Diltiazem and Berberine, and at least one other active agent chosen from:

- a nucleoside analogue;

- an inhibitor of viral protease (s);

- a transmembrane serine protease inhibitor;

- Chloroquine, and

- any mixture of the aforementioned compounds, for simultaneous, separate or sequential use in the prevention and / or treatment of viral infection with the SARS-CoV-2 virus (COVID-19).

This combination product will in particular be composed of:

- Diltiazem and Remdesivir;

- Diltiazem and Galidesvir;

- Diltiazem and Molnupiravir;

- Diltiazem and Lopinavir;

- Diltiazem and Camostat mesilate;

- Diltiazem and Chloroquine, or even

- Diltiazem and all possible combinations of Remdesivir, Galidesvir, Molnupiravir, Lopinavir, Camostat mesilate and Chloroquine.

According to other implementations, this combination product will in particular be composed of:

- Berberine and Remdesivir;

- Berbérine and Galidesvir;

- Berberine and Molnupiravir;

- Berberine and Lopinavir;

- Berberine and Camostat mesilate;

- Berberine and Chloroquine, or even

- Berberine and all possible combinations of Remdesivir, Galidesvir, Monulpriravir, Lopinavir and Chloroquine.

According to other implementations, this combination product will in particular be composed of:

- a combination of Diltiazem and Berberine, and Remdesivir;

- a combination of Diltiazem and Berberine, and Galidesvir;

- a combination of Diltiazem and Berberine, and Molnupiravir;

- a combination of Diltiazem and Berberine, and Lopinavir;

- a combination of Diltiazem and Berberine, and Camostat mesilate;

- a combination of Diltiazem and Berberine, and Chloroquine, or even

- a combination of Diltiazem and Berberine, and all possible combinations of Remdesivir, Galidesvir, Molnupiravir, Lopinavir, Camostat mesilate and Chloroquine.

According to an implementation according to the invention, one of these combination products as described above is used simultaneously, separately or sequentially, for the prevention of a viral infection by the SARS-CoV-2 virus. (COVID-19).

According to another implementation according to the invention, one of these combination products as described above is used simultaneously, separately or sequentially, for the treatment of a viral infection by the SARS-CoV virus. 2 (COVID-19). This combination product may comprise other active compounds, and in particular at least one antibiotic.

The present invention also relates to a method for treating a patient infected with a SARS-CoV-2 virus (suffering from the disease known as COVID-19) comprising the administration to said patient of a pharmaceutical composition comprising, in a suitable pharmaceutical vehicle, the least one compound selected from Diltiazem and Berberine.

The present invention also relates to a method for preventing the appearance of a viral infection by a SARS-CoV-2 virus (called COVID-19 disease), in an individual likely to be infected with said virus, comprising the administration to said individual of a pharmaceutical composition comprising, in a suitable pharmaceutical vehicle, Diltiazem.

The present invention also relates to a method for treating a patient infected with a SARS-CoV-2 virus (suffering from the disease known as COVID-19), comprising the administration to said patient of a pharmaceutical composition comprising, in a suitable pharmaceutical vehicle, Diltiazem.

In particular, this method may also include the administration to said patient of another active compound, in particular chosen from the following compounds:

- a nucleoside analogue;

- an inhibitor of viral protease (s);

- a transmembrane serine protease inhibitor;

- Chloroquine, and

- any mixture of the aforementioned compounds.

The present invention also relates to a pharmaceutical composition comprising, in a suitable pharmaceutical vehicle, a combination of Diltiazem and / or Berberine, with Remdesivir.

The present invention also relates to a pharmaceutical composition comprising, in a suitable pharmaceutical vehicle, a combination of Diltiazem with Remdesivir.

The present invention also relates to a pharmaceutical composition comprising, in a suitable pharmaceutical vehicle, a combination of Diltiazem with Molnupiravir.

More precisely, said pharmaceutical composition comprises, in a suitable pharmaceutical vehicle, a combination of Diltiazem, Berberine, and Remdesivir. The present invention also relates to pharmaceutical compositions comprising, in a suitable pharmaceutical vehicle, the following combinations:

- a combination of Diltiazem and Berberine, and Galidesvir;

- a combination of Diltiazem and Berberine, and Lopinavir;

- a combination of Diltiazem and Berberine, and Molnupiravir;

- a combination of Diltiazem and Berberine, and Camostat mesilate;

- a combination of Diltiazem and Berberine, and Chloroquine, or even

- a combination of Diltiazem and Berberine, and all possible combinations of Remdesivir, Galidesvir, Molnupiravir, Lopinavir, Camostat mesilate and Chloroquine.

These combinations can be formulated according to all the possible ratios of each compound. In particular, they will comprise either the same amount by weight of each antiviral compound (33% of each), or unequal doses of each compound, the percentages being expressed by weight of the compound relative to the total weight of the combination.

All the therapeutic uses of these combinations are also objects of the invention.

EXAMPLES

Examples 1 to 3 demonstrate on the one hand the effectiveness of treatment with Diltiazem or Berberine as monotherapy, and on the other hand the advantage of using these molecules in combination with Remdesivir for the treatment of infections due to SARS-CoV-2.

Other data, not presented here, were obtained using apigenin, another compound showing antiviral action on certain viral strains, in particular against MERS-CoV (WO 2018/073549). However, this molecule did not show significant antiviral action against the SARS-CoV-2 coronavirus.

The viral load was quantified in RT-qPCR and / or in TCID50 / ml in samples of infection supernatants from Vero E6 cells but also in apical washes and cell lysates of reconstituted human epithelium (HAE MucilAir, Epithelix), treated with Diltiazem, Berberine, or Remdesivir as monotherapy or in combinations of two of these molecules (Diltiazem + Remdesivir, or Berberine + Remdesivir).

The viral production relative to each of the concentrations of molecules was determined and is represented in relation to the viral production from infected cells or HAE in the same conditions but not treated. Median inhibitory concentrations (IC50) were determined for each treatment condition.

Example 1. Materials and methods

Virus used

The virus used was isolated from a sample of a patient infected with SARS-CoV-2.

The strain of SARS-CoV-2 used in this study was isolated from a 47-year-old female patient recruited from a French clinical cohort evaluating patients with COVID-19 (NCT04262921). This study was carried out in accordance with the Declaration of Helsinki and received the approval of the local ethics committee. The viral strain was sequenced with Illumina MiSeq and deposited in the GISAID EpiCoVTM database under the reference BetaCoV / France / IDF0571 / 2020 (EPI identification number _ISL_411218). For reference see (Pizzorno et al., 2020).

Isolation of the virus was performed by inoculation of Vero E6 cells (ATCC CRL-1586) and monitoring the appearance of cytopathic effects. After the appearance of the first virus-induced effects, the infection supernatant was harvested and the viral RNAs were extracted therefrom using the QIAmp Viral RNA kit (QIAGEN). The extracted RNAs were then subjected to Illumina MiSeq sequencing (Zymo-Seq RiboFree), with 500x coverage, and the sequences were assembled using the hisat2 and consensus alignment programs.

The sequence was then deposited on the GISAID EpiCoV platform (Accession ID EPI _ ISL _ 411218) under the name BetaCoV / France / IDF0571 / 2020.

This viral strain is phylogenetically very close to the strains of SARS-CoV-2 which circulated at the start of the epidemic in the Wuhan region in China in January / February 2020. This strain is therefore representative of the strains of SARS-CoV -2 at the origin of infectious diseases known as "COVID-19" currently observed today in the world.

Vero E6 cell infection protocol

Vero E6 cells (ATCC CRL-1586) were cultured in DMEM medium 4.5 g / l of Glucose, supplemented with L-Glutamine and Penicillin / streptomycin and 10% inactivated fetal calf serum, at 37 ° C 5% CO 2.

To carry out the infections, the cells were rinsed twice with serum-free medium and were covered with a minimum volume containing a dilution of virus, this dilution being determined from the infectious titer (see chapter determination of the infectious titer), in order to obtain an adequate multiplicity of infection (MOI). After an incubation of one hour in the presence of a minimum volume, the medium was replaced by DMEM medium 4.5 g / l of Glucose, supplemented with L-Glutamine and Penicillin / streptomycin and 2% of inactivated fetal calf serum, and the cells were incubated again at 37 ° C 5% CO 2.

Epithelial infection protocol

To carry out infections, we also used a model of reconstituted human epithelium (HAE MucilAir, Epithelix), obtained from human primary cells obtained by nasal biopsies, cultured at the air-liquid interface with a specific culture medium in Costar Transwell inserts (Corning, NY, USA. For infection experiments, the apical poles were gently washed twice with OptiMEM medium (Gibco, ThermoFisher Scientific), then infected with 150 µl of virus dilution in OptiMEM medium. , at a multiplicity of infection (MOI) of 0.1 After one hour of incubation at 37 ° C 5% CO 2, the viral suspension was removed.

Determination of the infectious titer in Vero E6 cells

The determination of the infectious titer was carried out by a limit dilution technique, on these Vero E6 cells in a 96-well plate. A volume of 50 ml of serial dilutions is deposited in wells in quadruplicates. The cells are then incubated at 37 ° C. 5% CO 2 and the presence of cytopathic effects is then monitored after 3 days of infection. The 50% tissue culture infectious dose (TCID50 / ml), that is, the viral titre required to cause infection in 50% of the inoculated cells, was calculated using the technique of Reed and Muench.

Quantification of the viral genome by quantitative PCR

The probes and primers used were described by the School of Public Health / University of Hong Kong (Table 1).

A quantitative “one-step” PCR was carried out using the StepOnePlus Real Time PCR System kit (Applied Biosystems), with the EXPRESS One-Step Superscript qRT-PCR reagent (Invitrogen), in a reaction volume of 20 mI containing 10 mI of supermix Express qPCR (2x), 1 mI of each primer (10 mM), 3.1 mI of water, 0.4 mI of Rox dye (25 mM) and 2 mI of viral RNA.

The following program was used: 15min at 50 ° C, followed by 40 cycles (15s 95 ° C; 1 min 60 ° C).

Table 1

FAM and TAMRA denote fluorescent markers.

Example 2. Comparison of IC50s of Diltiazem, Berberine, Remdesivir, and Diltiazem + Remdesivir and Berberine + Remdesivir Combinations on SARS-CoV-2 Virus in Vero E6 Cell Model.

Vero E6 cells infected with SARS-CoV-2 (MOI 0, 1) were treated one hour after infection with increasing concentrations:

- Diltiazem (2.8 to 45 mM),

- Berberine (1.6 to 25 pM) and - Remdesivir (0.6 to 10 pM) alone (Fig1) and in combination (Fig 2).

For each of the combined treatments tested, a dose of one of the molecules was set and associated with increasing concentrations of the other molecule (same concentration ranges as those used in monotherapy, respectively) and vice versa. The viral infectious titers measured in the culture supernatants of the infected cells (taken 48 and 72 hours post-infection) reflect the level of viral replication under the various treatment conditions.

Table 2 below summarizes the IC50 data for different monotherapy and / or combination treatments obtained in Vero E6 cells at different times of SARS-CoV-2 infection.

Table 2

This table clearly illustrates the gain of combinations of treatments in terms of reducing the IC50 of certain molecules compared to monotherapy treatments of these molecules. Thus, Remdesivir combined with a fixed concentration of Diltiazem (11.5 pM) allows to obtain a reduction of approximately 68% of its IC50 (0.32 versus 0.98 pM) at 48hpi, and of 52% of its IC50 (0.35 versus 0.72 pM). ) at 72hpi.

In parallel, Diltiazem combined with a fixed concentration of Remdesivir makes it possible to obtain an IC50 of 0.55 pM, while its IC50 is greater than 45 pM at 48hpi. In addition, Remdesivir combined with a fixed concentration of Berberine (12.5 pM) achieves a reduction of approximately 33% of its IC50 (0.65 versus 0.98 pM) at 48hpi.

Example 3. Comparison of the Antiviral Activities of Diltiazem, Berberine, Remdesivir, and Diltiazem + Remdesivir, Berberine + Remdesivir Combinations on SARS-CoV-2 Virus in a Reconstituted Human Respiratory Epithelium Infection Model.

Reconstituted human respiratory epithelia of nasal origin and cultured at an air / liquid interface (MucilAir® HAE, Epithelix) were infected with SARS-CoV-2 (MOI 0.1). The basal medium was treated once a day with the following molecules, as a single treatment or in combination: - Diltiazem (45 or 90 mM),

- Remdesivir (20 or 40 pM) and

- Berberine (4 pM) for 48 or 72 hours post-infection.

Figure 4 shows the chronogram of the experiments. At different times, the epithelia were harvested and lysed. Total RNA was extracted and viral genomes were quantified by RT-PCR by normalizing data using quantification of cellular gene product (GAPDH).

These data make it possible to assess the antiviral effect of monotherapy and combination treatments by relative measurement of viral replication, expressed as a percentage of viral replication in the control (untreated) or as -Iog10 of relative viral production.

Table 3

Table 3 shows the effects of Diltiazem, Berberine and Remdesivir monotherapy treatments and Diltiazem + Remdesivir, Berberine + Remdesivir combined treatments on SARS-CoV-2 virus replication in a human respiratory epithelium model (MucilAir® HAE, Epithelix) of nasal origin.

Table 3 summarizes the data for antiviral activities of Diltiazem, Berberine, Remdesivir, and Diltiazem / Remdesivir, Berberine / Remdesivir combinations on SARS-CoV-2 virus in a reconstituted human respiratory epithelium infection model. nasal or bronchial origin. Remdesivir, as a single treatment, exhibits significant efficacy at 48hpi in epithelium of nasal origin (more than 7.75 Iog10 of reduction in viral replication).

The combination of Remdesivir with Diltiazem or with Berberine (under the same concentration conditions as in monotherapy) significantly increases the antiviral effect (8.6 and 8.29 Iog10 of reduction in viral replication for combination with Diltiazem and Berberine, respectively).

Monotherapy treatments with Diltiazem or Berberine also show significant antiviral efficacy at 48hpi (0.35 and 0.86 Iog10 reductions in viral replication with Diltiazem and Berberine, respectively).

The efficacy of Remdesivir at 72hpi, under our experimental conditions, although remaining very high, is comparatively less important than at 48hpi in epithelium of nasal origin (2.42 and 2.05 Iog10 of reduction at 20 mM and 2 , 30 and 2.24 log 10 of reduction at 40 mM).

At 72hpi, the Remdesivir / Diltiazem combinations exhibit efficacy in epithelium of nasal origin, but without significant difference with simple treatment with Remdesivir. This could be explained by the limited antiviral efficacy of Diltiazem under these experimental conditions (0.16 / 0.15 Iog10 reduction in nasal epithelium.

Example 4. Comparison of the antiviral activities of Diltiazem and Remdesivir, on the SARS-CoV-2 virus in cell model A549-ACE2

Cells of the A549 cell line are human alveolar basal epithelial cells derived from adenocarcinoma. This cell line is used as a model for the study of lung cancer, but also as target cells for infectious viruses targeting the respiratory tract. These cells were subsequently modified to express the AC E2 receptor, through which the SARS-CoV-2 virus enters host cells. This A549-ACE2 cell line was obtained from Creative Biogene (USA).

Unlike Vero cells, these A549-ACE2 cells have a complete and functioning interferon signaling pathway. They are therefore more suitable for studying the effects of Diltiazem, which acts on these signaling pathways.

The experimental protocol is as follows:

Seeding of A549-ACE2 cells,

- 24 hours later, infection of the A549-ACE2 cells with a Wuhan-like strain of

SARS-CoV-2 (MOI 10 ¹ and 10 ² ),

1 hour post infection (pi), cells are treated with 45 µM Diltiazem, The supernatants are taken at 24 hours, 48 hours, 72 hours and 96 hours after infection for viral quantification by RT-PCR.

The incubation media used are as follows:

• Inoculation medium: DMEM 1 g / L glucose (GIc), 200mM L-Glutamine (L-Glu), 104 units Penicillin / Streptomycin (P / S), 10% Fetal Calf Serum (FCS)

• Infection medium: DMEM 1 g / L GIc, 200mM L-Glu, 104 U P / S, 0% FCS

• Treatment medium: DMEM 1 g / L GIc, 200mM L-Glu, 104 U P / S, 2% FCS

FIG. 5 shows the experimental protocol: a single treatment is carried out at 1 h pi, then the supernatant is taken at 24, 48, 72 and 96 hpi.

Figure 6 shows the results obtained after treatment of infected cells at two different infection multiplicity rates (A: MOI = 10 ¹ ; B: MOI = 10 ² ) with Diltiazem (45 mM) or Remdesivir (5 pM) .

The two compounds Remdesivir and Diltiazem make it possible to obtain, with a single treatment, the same level of inhibition of the viral titer (measured by RT-PCR) over time, regardless of the rate of multiplicity of infection.

Example 5. Determination of IC50 on SARS-CoV-2 and CC50 of Diltiazem, in line A549 ACE2

The experimental protocol is as follows:

Seeding of A549-ACE2 cells,

- 24 hours later, infection of the A549-ACE2 cells with a Wuhan-like strain of

SARS-CoV-2 (ME 10 ¹ ),

1 hour post infection (pi), the cells are treated with Diltiazem at different concentrations,

The supernatants are taken 72 hours after infection for viral quantification by RT-PCR.

The IC50 is the median inhibitory concentration, that is, the amount of Diltiazem required to achieve 50% inhibition of viral replication of the strain SARS-CoV-2 tested.

The results obtained are presented in Figure 7A: the IC50 of Diltiazem is 19.7 pM, which is indicative of good inhibitory efficacy in vitro.

In parallel, the cytotoxicity of Diltiazem on these A549-ACE2 cells was verified under the same experimental conditions. For this, the CC50 (Cytotoxic concentration 50%), which corresponds to the dose of Diltiazem necessary to reduce the viability of the cells by half, has was measured by following the viability of A549-ACE2 cells in the presence of different concentrations of Diltiazem. Viability was determined by an MTS test performed 72 hours after the start of treatment.

The MTS test is a colorimetric method: the method is based on the reduction of the tetrazolium compound MTS by viable cells to generate a colored formazan product, allowing the counting of viable (stained) cells and dead cells.

The results obtained are presented in FIG. 7B: the viability of the cells is reduced by half with a concentration of Diltiazem equal to 374 mM, a dose much greater than the IC50 measured previously.

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Claims

1. Diltiazem for its therapeutic use in the prevention and / or treatment of viral infection with the SARS-CoV-2 virus, known as the COVID-19 disease.

2. Pharmaceutical composition comprising Diltiazem in a suitable pharmaceutical vehicle, for its therapeutic use in the prevention and / or treatment of viral infection with the SARS-CoV-2 virus, known as the COVID-19 disease.

3. Composition for its use according to claim 2, comprising at least one other active agent chosen from:

- a nucleoside analogue;

- an inhibitor of viral protease (s);

- a transmembrane serine protease inhibitor;

- Chloroquine, and

- any mixture of the aforementioned compounds.

4. A composition for its use according to claim 3, comprising a nucleoside analog selected from Remdesivir, Galidesivir, Molnupiravir and combinations thereof.

5. A composition for its use according to claim 4 comprising a combination of Diltiazem and Remdesivir.

6. A composition for its use according to claim 4 comprising a combination of Diltiazem and Galidesivir.

7. A composition for its use according to claim 4 comprising a combination of Diltiazem and Molnupiravir.

8. Composition for its use according to claim 3, comprising an inhibitor of viral protease (s), in particular Lopinavir, preferably Lopinavir combined with Rinotavir.

9. A composition for its use according to claim 3, comprising a transmembrane serine protease inhibitor, in particular Camostat mesilate.

10. A composition for its use according to one of claims 1 to 9, further comprising at least one antibiotic.

11. Composition for its use according to one of claims 1 to 10, characterized in that said composition is in a dosage form suitable for intranasal administration, in particular by inhalation.

12. Combination product comprising Diltiazem and at least one other active agent chosen from: - a nucleoside analogue;

- an inhibitor of viral protease (s);

- a transmembrane serine protease inhibitor;

- Chloroquine, and

- any mixture of the aforementioned compounds, for its simultaneous, separate or sequential use in the prevention and / or treatment of a viral infection with the SARS-CoV-2 virus.

13. A pharmaceutical composition comprising, in a suitable pharmaceutical vehicle, a combination of Diltiazem and Remdesivir.

14. A pharmaceutical composition comprising, in a suitable pharmaceutical vehicle, a combination of Diltiazem and Molnupiravir.