CN116897040A

CN116897040A - Compounds and methods for treating coronaviruses

Info

Publication number: CN116897040A
Application number: CN202180055906.1A
Authority: CN
Inventors: T·贝根; B·德佩兹; S·贝卢扎德; P·布罗丁
Original assignee: Apteeus; Centre National de la Recherche Scientifique CNRS; Universite Lille 2 Droit et Sante; Institut National de la Sante et de la Recherche Medicale INSERM; Centre Hospitalier Regional Universitaire de Lille CHRU
Current assignee: Apteeus; Centre National de la Recherche Scientifique CNRS; Universite Lille 2 Droit et Sante; Institut National de la Sante et de la Recherche Medicale INSERM; Centre Hospitalier Universitaire de Lille
Priority date: 2020-06-10
Filing date: 2021-06-08
Publication date: 2023-10-17

Abstract

The present invention relates to the use of clofoglic phenol in the prevention or treatment of diseases caused by coronaviruses, in particular Covid-19.

Description

Compounds and methods for treating coronaviruses

Technical Field

The present invention relates to the use of clofoc phenol (cloftol) for the treatment of diseases caused by coronaviruses, in particular Covid-19.

Background

Coronaviruses belong to the family Coronaviridae (Coronaviridae) (order of nested viruses (Nidovirales)), including viruses having a single-stranded, positive-sense RNA genome, and are about 26 to 32 kilobases in size. The family coronaviridae includes alpha coronaviruses (alpha CoV), beta coronaviruses (beta CoV), delta coronaviruses (delta CoV), and gamma coronaviruses (gamma CoV). Bats and rodents are considered hosts of alpha CoV and beta CoV. At present, it is not clear which animals are hosts of delta CoV and gamma CoV. Coronaviruses are named according to their appearance under electron microscopy, and because of the presence of spike glycoproteins on their envelope, these viruses appear to cover sharp structures around them, like corons (corona) or crowns.

Since the beginning of the 21 st century, three coronaviruses have crossed species barriers, causing fatal pneumonia in humans: severe acute respiratory syndrome coronavirus (SARS-CoV), middle east respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2 (also known as 2019-nCoV). SARS-CoV-2 is an enveloped, positive-sense, single-stranded RNA virus belonging to the genus beta CoV, which also includes SARS-CoV and MERS-CoV. SARS-CoV-2 has 89% nucleotide identity with bats SARS-like CoV and 82% identity with human SARS-CoV. Other coronaviruses that may infect humans include human coronavirus NL63 (Hcov-NL 63), human coronavirus OC43 (Hcov-OC 43), human coronavirus HKU1 (Hcov-HKU 1) and human coronavirus 229E (Hcov-229E).

Coronavirus entry into host cells is mediated by transmembrane spike (S) glycoproteins that form homotrimers that protrude from the viral surface. In addition to direct membrane fusion, SARS-CoV has been shown to enter cells via an endocytic pathway, which leads to viral gene expression (Cell Research 2008; 18:290-301). Regarding SARS-CoV-2, ACE2 (angiotensin converting enzyme 2) has been proposed to mediate SARS-CoV-2S-mediated entry into cells, making it a functional receptor for this emerging coronavirus; SARS-CoV-2S binds to human ACE2 with an affinity comparable to that of SARS-CoV S (Cell 2020; 180:281-292). SARS-CoV-2 has also been demonstrated to use the transmembrane protease serine 2 (TMPRSS 2) for the initiation of the (prime) S protein (Cell 2020; 181:271-280).

Since the advent of SARS-CoV-2 and the outbreak of the related disease Covid-19, many drugs (whether in research or already marketed) have been tested for their potential therapeutic effect in treating/alleviating the effects of Covid-19. To the inventors' knowledge, when SARS-CoV-2 can enter (i.e., endocytose into and fuse into) cells in two ways, none of these drugs has been shown to block the viral cycle at the concentrations that can be reached (at tolerable doses) in the infected tissue.

The clofoc phenol (2- (2, 4-dichlorophenyl) -4- (1, 3-tetramethylbutyl) phenol, CAS number 37693-01-9,PubChem CID 2799) is a bacteriostatic antibiotic suitable for use in treating infections caused by gram-positive bacteria. The chlorofoci phenol is first described in french patent application 2101076. The compound was then demonstrated to activate the response pathway of unfolded proteins, making it a potential candidate for the treatment of prostate cancer (British Journal of Pharmacology2014;171: 4478-4489). Recently, clofogliol has been shown to activate EBV lytic gene expression (Journal of Virology 2019;93 (20): e 00998-19) and inhibit proliferation of glioma stem cells by activating KLF13 (J Clin invest.2019;129 (8): 3072-3085).

Disclosure of Invention

It has now been found that when SARS-CoV-2 can enter cells by two entry routes, endocytic entry and fusion, clofoci blocks the viral cycle. Thus, it has been found that clofoglic phenol can effectively inhibit the cytopathic effect caused by SARS-CoV-2, reducing the viral load in cells infected with the virus.

Accordingly, the present invention relates to the use of clofoglic phenol for the prevention or treatment of a disease caused by a coronavirus, such as SARS-CoV, MERS-CoV, SARS-CoV-2, HCoV-NL63, HCoV-OC43, HCoV-HKU1 or HCoV-229E. The invention also relates to a method of treating a disease caused by a coronavirus, such as SARS-CoV, MERS-CoV, SARS-CoV-2, HCoV-NL63, HCoV-OC43, HCoV-HKU1 or HCoV-229E, comprising administering to a subject in need thereof clofogliclade.

Drawings

FIGS. 1 and 3 show the effects of different concentrations of clofogliflozin on the cytopathic effects of SARS-CoV-2 infected Vero81 cells (either non-transduced TMPRSS2 gene (FIG. 1) or transduced TMPRSS2 gene (FIG. 3)).

FIGS. 2 and 4 show the effect of different concentrations of clofogliflozin on the growth enhancement of SARS-CoV-2 infected Vero81 cells (either non-transduced TMPRSS2 gene (FIG. 2) or transduced TMPRSS2 gene (FIG. 4)).

FIGS. 5 and 6 show the effect of clofogliflozin on SARS-CoV-2 genome replication in SARS-CoV-2 infected Vero81 cells and SARS-CoV-2 infected TMPRSS2 transduced Vero81 cells.

FIG. 7 shows the effect of clofogliclan on SARS-CoV-2 loading in SARS-CoV-2 infected Vero81 cells and SARS-CoV-2 infected TMPRSS2 gene transduced Vero81 cells.

FIG. 8 shows the effect of clofogliol on the genomic replication of SARS-CoV-2 in SARS-CoV-2 infected Calu-3 cells.

FIG. 9 shows the pharmacokinetics of clofogliflozin in C57BL/6J mice.

FIG. 10 shows the effect of clofogliflozin on SARS-CoV-2 infection in K18-hACE2 transgenic C57BL/6J mice.

FIG. 11 shows the effect of clofoglic on genome replication of SARS-CoV-2 in Vero81 cells infected with two different variants of SARS-CoV-2.

FIG. 12 shows the inhibition of HCoV-229E-Rluc virus growth in Huh-7 cells by clofogliol.

FIG. 13 shows the effect of clofoglic on viral secretion in infected Vero81 cells (lower curve) and TMPRSS2 transduced infected Vero81 cells (upper curve).

FIG. 14 shows the effect of clofoglic phenol on viral replication in infected Vero81 cells (upper curve) and TMPRSS2 transduced infected Vero81 cells (lower curve).

Figure 15 shows the effect of clofogliflozin on viral secretion in infected human bronchial epithelial cells.

FIG. 16 shows the effect of clofogliflozin on viral secretion in infected human bronchial epithelial cells.

Detailed Description

In the context of the present disclosure, various embodiments described below may be combined.

In a first aspect, the invention relates to the use of clofoglic phenol for the prevention or treatment of a disease caused by a coronavirus, such as SARS-CoV, MERS-CoV, SARS-CoV-2, HCoV-NL63, HCoV-OC43, HCoV-HKU1 or HCoV-229E. In particular, the clofogliclan is particularly useful for preventing or treating diseases caused by coronaviruses such as SARS-CoV, MERS-CoV, SARS-CoV-2.

In one embodiment, the coronavirus is SARS-CoV-2 and the corresponding disease is Covid-19. Those skilled in the art will appreciate that variants of SARS-CoV-2 are encompassed by the generic term "SARS-CoV-2". Examples of such variants include the b.1 variant with the D614G mutation, whose passage (descends) produces the b.1.1.7 variant; variant b.1.526; b.1.351 variety; b.1.1.248 variants; in particular variants B.1.427 and B.1.429; variant b.1.617.

In one embodiment, the clofoglic phenol is administered in combination with at least one other (small molecule or biological) active ingredient that is being or has been used in research to treat SARS-CoV, MERS-CoV or SARS-CoV-2. The administration of at least one other active ingredient may be simultaneous, sequential or over a period of time compared to the administration of clofogliclan.

In one embodiment, the at least one other active ingredient is selected from:

abemaciailb, amitraz dimesylate (almitrine bismesylate), amodiaquine (amodiaquine), anabaziram (anakinra), angiotensin 1-7, anidulafungin (anidulafungin), apixaban (apixaban), aspirin, avastin (avathrombag), azithromycin, bani Wei Shankang (bamlanivimab), baratinib (baricitinib), bazedoxifene (bazedoxifene) berberine (berbamine), epipiprazole (brexpiprazole), bromhexine (bromohexine), catastatin (carbamostat), canavanab), carboximab (casivizumab), casivizumab (casivimab), cepharanthine (phasprandine), ceritinib (ceriib), acetylpyridine, chloroquine, chlorquine ciclesonide (ciclesonide), clomiphene citrate (clomifene citrate), cobicistat (cobicistat), cloconazole (croconazole), cyclosporine, danoprevir (danoprevir), darunavir (darunavir), dexamethasone, digitoxin, digoxin, dihydroartemisinin (dihydroarteminine), dihydrogambogic acid (dihydrogambogic acid), diltiazem, dronedarone (dronedarone), drotaverine (drotaverine), ebastine (ebastine), eltrapa (eltrombopag), anti-Ma Pashan (emaprasugrel), etallima Wei Shankang (anevaleviab), etaverine (favirverine), favirapivir (fingolimod), finnimodine (flunarizine), glisten (glisten), glisten (giverine), ebastine (geeridine) Harringtonine (harringtonine), hematoporphyrin, hexachlorophene, hydrocortisone, hydroxychloroquine, hydroxyprogesterone caproate (hydroxyprogesterone caproate), yin Dewei mab (immovimab), IMU-838, interferon beta-1A, interferon beta-1B, isoosajin, isoflavones (isopomoterin), isotretinoin, ivakator (ivacator), ivermectin (ivermectin), JS016, ketoconazole, lidofloxazine (lidofloxazine), loperamide (loperamide), lopinavir (lopinavir), loratadine, loteprednol (loteprednol etabonate), lu Shuqu-wave pa (luruptromidag), LY-CoV555, mefloquine, methylprednisolone, mo Nupi, naproxat (namostat), niclosamide (niclosamide), fluquine) nitazoxanide, homoharringtonine (omacetaxine mepesuccinate), oxalajin, oseltamivir (oseltamivir), octyitinib (osiertinib), octyimiab (otimizole), ouabain (ouabain), oxiconazole, hydroxychloro Liu Benan (oxazanide), ozamate (ozanimod), papaverine, pegylated interferon lambda, piperacillin maleate (perhexiline maleate), pexidanib (pexidantinib), PF-07321332, phenazopyridine (phenazopyrdine), polidocanol (docanol), posaconazole (posaconazole), prednisone, recombinant human interferon alpha 1 beta, recombinant interferon alpha-2 b, regorafenib (regorafenib), renavir (renatuwei), ribavirin, ritonavir, ruxotinib, sitagliptin, sofosbuvir, soradyl gei, sorafenib, tamoxifen, thalidomide, thimerosal, thiohydramine, thiopyridazine, thymosin alpha 1, telulone, thioguanine, tolizumab, toremifene, trapezinol, tyrphostin, and Wu Mi novei.

In one embodiment, the clofogliclan is administered in the form of a pharmaceutical composition comprising at least one pharmaceutically acceptable excipient (other than clofogliclan). Excipients, pharmaceutical compositions and methods of making them are well known in the art (see, e.g., handbook of pharmaceutical Excipients, rowe et al, seventh edition, month 6 2012; rules and Guidance For Pharma Manufacturers and distributors 205,Medecines and Healthcare products Regulatory Agency, london, UK).

In another aspect, the invention relates to a method of preventing or treating a disease caused by a coronavirus, such as SARS-CoV, MERS-CoV, SARS-CoV-2, HCoV-NL63, HCoV-OC43, HCoV-HKU1 or HCoV-229E, comprising administering to a subject in need thereof clofogliol. In some embodiments, the coronavirus is SARS-CoV, MERS-CoV, or SARS-CoV-2. In some embodiments, the coronavirus is SARS-CoV-2 and the corresponding disease is Covid-19.

The current route of administration of clofogliclan is the rectal route. Suppositories containing 100mg, 200mg or 750mg of clofogliclan may be prepared with suitable pharmaceutically acceptable excipients.

In one embodiment, the subject has one or more of the following conditions or diseases: overweight, obesity, diabetes, hyperlipidemia, arterial hypertension, cardiovascular disease, chronic kidney disease, immunosuppression.

In one embodiment, the method further comprises administering at least one additional active ingredient to the subject. The administration of at least one other active ingredient may be simultaneous, sequential or over a period of time relative to the administration of clofogliclan.

In one embodiment, at least one other active ingredient is as defined above.

Chlorofol has been shown to inhibit the cytopathic effects of hSARS-CoV2 in Vero81 cells and TMPRSS2 transduced Vero81 cells, as well as in human Calu-3 cells. Vero81 cells allow entry of the virus in an endocytosis-based entry manner. When transduced with TMPRSS2, vero81 cells became permissive for viral entry in two ways (endocytosis and fusion). It has also been demonstrated that clofoglic acid inhibits the genomic replication of the virus in the same cells and in human Calu-3 cells, which also provide two modes of entry for the virus. It was also demonstrated that clofoglic phenol reduced viral load in cultures of cells infected with the virus (Vero 81 cells and Vero81 cells transduced with TMPRSS 2). It has further been demonstrated that clofogliflozin reduces viral load in the lung of SARS-CoV-2 infected mice (K18-hACE 2). It has further been demonstrated that clofogliflozin inhibits viral genome replication of 2 different varieties of SARS-CoV-2. It has further been demonstrated that clofoglic phenol inhibits replication of HCoV-229E (alpha coronavirus).

The following examples are included for illustrative purposes and should not be construed as limiting in any way.

Examples

Virus isolation and amplification

Human coronavirus SARS-CoV2 (hSARS-CoV 2) was isolated from patient samples and annotated as βCoV/French/IDF 0372/2020. It was used in vitro and expanded in TMPRSS2 expressing VERO-81 cells, cultured in plates and incubated until the cell monolayer was completely destroyed. The lineage B1.1.7 of SARS-CoV-2 (GISAID accession number EPI_ISL_ 1653931) is the second variant of SARS-CoV-2 to be used in vitro. After harvesting, the supernatant was diluted by successive 10-fold dilutions (10 ^-1 To 10 ^-8 ) Viral titration was performed on VERO-81 cells (incubated on DMEM supplemented with 2% FBS (fetal bovine serum)). The plates were incubated at 37℃with 5% CO ₂ Incubate under conditions for 5 days. Afterwards, the plates were examined using an inverted microscope (ZEISS Primovert) to assess the extent of the virus-induced cytopathic effect in the cell culture. The estimated virus concentration was calculated by the Spearman and Karber method and expressed as log10 TCID ₅₀ /mL (dose of tissue culture infection, i.e., the amount of virus that produces cytopathic effects in 50% of the infected cells). The detection limit of the method is 1.5TCID ₅₀ /ml。

The hCoV-19_IPL_French strain of SARS-CoV-2 (NCBI MW 575140) was also used for in vitro and in vivo experiments. Viruses were inoculated at MOI 0.01 and allowed to proliferate in Vero-E6 cells expressing TMPRSS 2. After infection, the cell supernatant medium was harvested at 72 hours and stored frozen in small aliquots at-80 ℃.

TMPRSS2 transduction

Vero-81 cells were transduced with lentiviruses expressing TMPRSS 2. TMPRSS2 was cloned into pTRIP vector by using pTRIP-TMPRSS2, phCMV-VSVG and HIV gag-pol and Turbolect according to the manufacturer's instructions ^TM (ThermoFischer) transfected HEK293T cells, generate lentiviral vectors. Supernatant containing lentiviral vectorIn transduced cells. Cells were then used for the experiment 72 hours after transduction.

Measurement of cytopathic effects

Cell culture:

at 37℃at 5% CO ₂ Vero cells are exposed to a moist atmosphereCCL-81 ^TM ECACC, sigma-Aldrich, france, hereinafter referred to as "Vero81" cells]In the presence of 10% of Fetal Bovine Serum (FBS) and Glutamax ^TM Is cultured in Dulbecco's Modified Eagle's Medium (DMEM).

At 37℃at 5% CO ₂ In Huh-7 cells were cultured in Dulbecco's modified eagle's medium (DMEM, gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS, eurobio).

Calu-3 cells (Clinisciences, EP-CL-0054) were grown in the most limiting essential medium (Gibco, MEM) supplemented with glutamax (Gibco) and 10% heat-inactivated FBS.

At 37℃at 5% CO ₂ In Vero-E6 cells (ATCC, CRL-1586) were cultured in Dulbecco's modified eagle's medium (DMEM, gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS, eurobio).

Compounds tested: the solution of clofoci was prepared in DMSO at the desired concentration to achieve the desired concentration for the experiment. The solution was dispensed onto living cells. The maximum concentration of DMSO was 0.15%.

Cell infection: cells were infected after addition of different concentrations of clofoglic phenol or control solvents. The multiplicity of infection was 0.01.

Reagent:hurst dye 33342, tri-hydrochloride, tri-hydrate (reference H3570, thermoscher) and propidium iodide (reference P1304MP, thermoFisher) were used to monitor the viral cytopathic effect (CPE) of clofop-nol at concentrations of 10 μg/ml and 1 μg/ml, respectively. After an incubation time of 72 hours, these reagents were added.

Results were obtained by analyzing the percentage of PI positive nuclei, the toxicity exerted by the replacement virus on the cells, and the number of nuclei corresponding to the inhibition of cell growth exerted by the virus.

Measurement of viral genome replication

Cell culture:at 37℃at 5% CO ₂ Vero81 cells and TMPRSS2 transduced Vero-81 cells were supplemented with 10% Fetal Bovine Serum (FBS) and Glutamax in a humid atmosphere ^TM Is cultured in Dulbeck's Modified Eagle Medium (DMEM).

Cell infection: cells were infected after addition of different concentrations of clofoglic phenol or control solvents. The multiplicity of infection was 0.25.

By substitution of viral RNA (viral load) associated with total cellular RNAMetering (gating)Results were obtained.

RNA metering (gating): after 6 hours of incubation, total cellular RNA was extracted using a Nucleospin RNA kit (Macherey-Nagel) according to the manufacturer's instructions and the SARS-CoV-2 genome was measured by quantitative RT-PCR. RNA was reverse transcribed using a high capacity cDNA reverse transcription kit (Applied Biosystems). Real-time PCR was then performed using the TaqMan universal PCR reaction mixture (Applied Biosystems) in Quantum studio 3. Primers and probes for the open reading frame of the E gene were used (WHO protocol). A standard curve was created by using in vitro transcribed RNA for the amplicon.

Determination of in vitro viral load

Vero-81 and Vero-81-TMPRSS2 cells were infected at an MOI of 0.25 for 1 hour, then the cells were rinsed 3 times with PBS and further incubated in the presence of increasing concentrations of clofock phenol for 16 hours. For each condition, performed in duplicate. Cell supernatants were collected by TCID ₅₀ The method measures viral titers.

Determination of viral load in mouse lung

To determine the viral load of the lung, half of the right lobes were homogenized in a lying Matrix D tube (mpbio) containing 1mL of PBS using a Mixer Mill MM 400 (Retsch) (15 min-15 Hz). After centrifugation at 11,000rpm for 5 minutes, the clarified supernatant was collected for virus titration. The supernatant was diluted in DMEM containing 1% penicillin/streptomycin and the dilutions transferred to Vero-E6 cells in 96 well plates for TCID ₅₀ And (5) measuring.

Assessment of Gene expression by RTqPCR

Half of the right lung lobes were homogenized using 1mL RA1 buffer (containing 20mM TCEP) in a Nucleospin RNA kit. Total RNA in the tissue homogenate was extracted using Nucleospin RNA from Macherey Nagel. RNA was eluted with 60. Mu.L of water.

RNA was reverse transcribed using the high-capacity cDNA Archive kit (Life Technologies, USA). Real-time PCR and Quantum studio Using SYBR Green-based ^TM 12K Flex real-time PCR System (Applied Biosystems) ^TM U.S.) the resulting cDNA was amplified according to the manufacturer's protocol. The gene encoding glyceraldehyde 3-phosphate dehydrogenase (Gapdh) was used for relative quantification. Specific primers were designed using Primer Express software (Applied Biosystems, villebon-sur-Yvette, france) and ordered from Eurofins Scientifics (Ebersberg, germany). Determining the relative mRNA level by comparing (a) the PCR cycle threshold (Ct) of the gene of interest and the housekeeping gene (ΔCt), and (b) the ΔCt value (ΔΔCt) of the treatment and control groups (2 ^ΔΔCt ). Data were normalized to gapdh gene expression and expressed as fold increase relative to the average gene expression level in mice treated with the blank control. Viral load is expressed as viral RNA normalized to Gapdh expression level (Δct).

Example 1

By adding a range of concentrations of clofoc phenol, followed by 1:3 and then using hsar-CoV 2 to infect at a MOI 0.01 (multiplicity of infection (MOI) 0.01 means that the amount of virus used is 0.01 virus per 1 cell) for 72 hours to assess the effect of clofop on Vero81 cells infected with hsar-CoV 2. Biological replicates were performed with two technical replicates.

Results were obtained by analysis of the percentage of PI positive nuclei, expressed as percentage CPE relative to the defined measure (scale) for positive (0% infected cells) and negative (100% infected cells) controls. As can be seen from FIG. 1, the clofogliflozin reduced CPE by 95% (IC) ₉₅ ) The concentration was 9.2. Mu.M.

Example 2

By adding a range of concentrations of clofoc phenol, followed by 1:3 and then infection with hSARS-CoV2 at MOI 0.01 for 72 hours to evaluate the effect of clofogliflozin on hSARS-CoV2 infected Vero81 cells. Biological experiments were repeated in duplicate or in triplicate techniques.

The results were obtained by analyzing the number of living cells expressed as a percentage of improvement in cell growth relative to the measure defined by normal cell growth (uninfected control) and no cell growth (infected control). As can be seen from fig. 2, 95% of the maximum reaction of clofogliflozin (EC ₉₅ ) The concentration of clofoglic phenol was estimated to be 21 μm.

Example 3

By adding a range of concentrations of clofoc phenol, followed by 1:3 and then infection with hsar-CoV 2 at MOI 0.01 for 72 hours to assess the effect of clofoc phenol on hsar-CoV 2 infected TMPRSS2 transduced Vero81 cells. Biological experiments were performed in triplicate.

Results were obtained by analysis of the percentage of PI positive nuclei expressed as a percentage of CPE relative to the measure defined for positive (0% infected cells) and negative (100% infected cells) controls. As can be seen from FIG. 3, the IC of the clofogliflozin ₉₅ 8.7. Mu.M.

Example 4

By adding a range of concentrations of clofoc phenol, followed by 1:3 and then infection with hsar-CoV 2 at MOI 0.01 for 72 hours to assess the effect of clofock phenol on TMPRSS2 transduced Vero81 cells infected with hsar-CoV 2. Biological experiments were performed in triplicate.

By passing throughThe results were obtained by analyzing the number of living cells expressed as a percentage of improvement in cell growth relative to the measure defined by normal cell growth (uninfected control) and no cell growth (infected control). As can be seen from fig. 4, estimated EC of clofogliflozin ₉₅ 25. Mu.M.

Example 5

By adding a range of concentrations of clofoc phenol, followed by 1:2,5 was diluted and then infected with hsar-CoV 2 at MOI 0.25 for 6 hours to assess the effect of clofoglic phenol on Vero81 and TMPRSS2 transduced Vero81 cells infected with hsar-CoV 2. Biological experiments were performed in duplicate.

The results were obtained by metering (rosin) the viral load expressed as the ratio of viral RNA to total sample RNA (RNAt). As can be seen from FIG. 5, the IC of the clofogliflozin ₉₅ 11. Mu.M.

The results in examples 1 to 5 show that the IC of the clofogliflozin ₉₅ Between about 9. Mu.M and about 11. Mu.M, i.e., about 25-fold lower than the Cmax (about 250. Mu.M) of clofazite reported in human lung tissue of a patient undergoing a lung resection procedure or a total lung resection (Pneumonectomy) in which clofazite is administered prior to or during the procedure (Journal of Antimicrobial Chemotherapy 1987; 19:679-683). Similarly, the EC of clofogliflozin ₉₅ From about 21 μm to about 25 μm, i.e., this value is about 10-fold lower than the Cmax reported in human lung tissue (see above).

Example 6

Vero-81 and Vero-81-TMPRSS2 cells were infected at an MOI of 0.25 in the presence of an increased concentration of clofock phenol for 6 hours. Then, total RNA was extracted and viral RNA was quantified by RT-qPCR. Results were normalized by the amount of total RNA and expressed as the average of three independent experiments performed in duplicate. The results are shown in FIG. 6, from which it can be seen that clofogliflozin inhibits genome replication of SARS-CoV-2.

Example 7

Vero-81 and Vero-81-TMPRSS2 cells were infected with SARS-CoV-2 at an MOI of 0.25. After 1 hour, the inoculum was removed, the cells were washed with PBS, and then with clofogliflozinAnd (5) processing. The cells were then further incubated for 16 hours. Thereafter, the supernatant is collected and the amount of secreted infectious virus is quantified. The dotted line indicates the detection limit (1.5 TCID ₅₀ /mL). These data represent the average of three independent experiments (n=3). Experiments were performed in duplicate for each condition. The results are shown in FIG. 7, from which it can be seen that the SARS-CoV-2 loading decreases as the concentration of chlorofluorophenol increases.

Example 8

Calu-3 cells were infected at an MOI of 0.25 in the presence of an increased concentration of clofosol for 24 hours. Then, RNA from the total cells was extracted and viral RNA was quantified by RT-qPCR. The results are shown in FIG. 8, from which it can be seen that the clofoglic phenol inhibits the replication of SARS-CoV-2 in Calu-3 cells.

The results in examples 6 to 8 show that clofogliclan shows antiviral activity against Sars-CoV2.

Example 9

Dilution of clofogliol in sodium chloride solution (1.75% final concentration)RH40 (07076, sigma) and 1.4% ethanol at final concentration) for intraperitoneal injection. Female C57BL/6J mice of 8 to 10 weeks of age were i.p. treated with a single dose of clofogliflozin (62.5 mg/kg) and thereafter sacrificed at different time points (fig. 9, left panel). In a separate experiment, mice were sacrificed by i.p. treatment with a dose of clofoc phenol (62.5 mg/kg) twice daily for two days 1 hour after the last injection (fig. 9, right panel). The concentrations of clofogliclan in plasma and lung of treated mice were measured by: plasma samples and lung tissue were collected and treated with absolute ethanol at a ratio of 1 to 10 and 1 to 50, respectively. Lung Tissue was homogenized with a mechanical lysis system (Tissue Lyzer II). The supernatant was obtained by centrifugation prior to injection into LC-MS/MS. Samples were analyzed using a UPLC system acquisition I Class (Waters) in combination with a triple quadrupole mass spectrometer Xex TQD (Waters). The column placed at 40 ℃ was an Acquity BEH C8 x 2.1mm,1.7 μm column (Waters) using the following mobile phases: 5mM ammonium formate in water (pH 3.75) as solventA) And 5mM ammonium formate in acetonitrile (pH 3.75) as solvent (B). The results shown in fig. 9 represent the average of three independent experiments performed in triplicate (lung) or in duplicate (plasma). It can be seen that the clofogliclan is distributed in the lungs at very significant levels.

Example 10

Eight week old female K18-human ACE2 expressing C57BL/6 mice (B6. Cg-Tg (K18-hACE 2) 2 Prlmn/J) were purchased from Jackson Laboratory. For infection, mice were anesthetized by intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg), followed by intranasal infection with 50 μl of DMEM containing 5×10 ² TCID ₅₀ hCoV-19_ipl_french strain of SARS-CoV-2 (NCBI MW 575140) (or not included in control samples). All experiments met the current national and institutional regulations and ethical guidelines. These protocols were approved by the institutional ethics committee "limit' Ethique en Experimentation Animale" (CEEA) 75, north garland straits (france).

After i.n. inoculation with SARS-CoV-2 (5X 10 per mouse ² TCID ₅₀ ) Mice were i.p. treated with clofogliflozin (62.5 mg/kg) or vehicle (fig. 10, left panel) 1 and 8 hours after infection, and 1 day after infection (twice a day). Animals were sacrificed on day 2 and day 4 post infection. Viral load was determined by titration of Vero-81 cells (fig. 10, middle panel) and by RT-qPCR (fig. 10, right panel) (day 2 post infection).

It can be seen that the treatment with clofoglic significantly reduced SARS-CoV-2 infection in mice.

Example 11

Vero-81 cells were infected with either SARS-CoV-2 lineage B1 (SARS-CoV-2/human/FRA/ville_vero-TMPRSS 2/2020) or SARS-CoV-2 lineage B1.1.7 (GISAID accession number epi_isl_ 1653931) containing the D614G mutation. Viral genomes were quantified by RT-qPCR. Results were normalized by the amount of total RNA and expressed as the average of three independent experiments performed in duplicate. As can be seen from FIG. 11, clofogliflozin inhibits the genome replication of SARS-CoV-2 variant.

Example 12

Huh-7 cells were infected with HCoV-229E-Rluc in the presence of different concentrations of clofosol. At 7 hours post infection, cells were lysed and luciferase activity was quantified. The results, expressed as a percentage of control, are shown in fig. 12, representing the average of three independent experiments performed in triplicate. Error bars represent Standard Error (SEM) of the mean. As can be seen from FIG. 12, clofogliflozin has activity against coronavirus HCoV-229E, which is different from Sars-CoV2.

The data in examples 1 to 12 above demonstrate the value of clofoglic phenol in the prevention or treatment of diseases caused by coronaviruses such as Covid-19.

Example 13

Vero81 cells and TMPRSS2 transduced Vero-81 cells were infected with hSARS-CoV-2 at a MOI of 0.25 in DMEM medium containing 10% fetal bovine serum. After 1 hour, the inoculum was removed, the cells were washed with PBS and then treated with different concentrations of clofogliol. The cells were then exposed to 5% CO ₂ Incubated at 37℃for 16 hours in a humid atmosphere. Thereafter, the supernatant was collected by TCID ₅₀ The amount of virus secreted therein was evaluated, expressed as log10 TCID ₅₀ /mL. The data reported in fig. 13 represent the average of two different experiences (experiences) (n=2). For each condition, performed in duplicate. The broken line in the figure shows that the detection limit of the method is 1.5TCID ₅₀ /ml。

As can be seen from FIG. 13, clofogliflozin inhibits secretion of hSARS-CoV-2 by infected cells.

Example 14

Vero81 cells and TMPRSS2 transduced Vero-81 cells were infected with hSARS-CoV-2 at a MOI of 0.25 in the presence of different concentrations of clofoci in DMEM medium containing 10% fetal bovine serum. After 6 hours, the inoculum was removed and the cells lysed to allow total RNA to be extracted using the Nucleospin RNA kit (Macherey-Nagel) according to the manufacturer's instructions. SARS-CoV-2 genome was measured by quantitative RT-PCR using primers and probes for the open reading frame of the E gene (WHO protocol). The results were normalized by the total amount of RNA per sample, expressed as a percentage of untreated control, as shown in fig. 14. The experiment was performed twice (n=2). In each experiment, each condition was performed in duplicate.

As can be seen from FIG. 14, clofogliflozin inhibits replication of hSARS-CoV2 in infected cells.

Example 15

Human bronchial epithelial cells (Mucilair) ^TM Available from epichelix corporation) is treated by top-pole application of clofoglib phenol. Firstly using compositions containing different concentrations of clofogliclanThe epithelial cells were washed with culture medium and then infected with hSARS-CoV-2 at MOI 0.2 for 1 hour (chlorofosol still present). Then, the inoculum was removed and the epithelial cells were washed with PBS, after which 25. Mu.l of I.about.M. containing different concentrations of clodrol was added to the top electrode>Culture medium. After incubation at 37℃for 72 hours, 200. Mu.l of Mucilair was added to the top of the epithelial cells ^TM Culture medium, harvested after 5 minutes incubation. These supernatants are used to quantify the amount of secreted virus, e.g. TCID ₅₀ And (5) evaluating. The results are shown in fig. 15, wherein the dashed line represents the limit of detection of adefovir (remdis vir). In addition, total RNA was extracted using a Nucleospin RNA kit (Macherey-Nagel) prior to genome quantification by RT-qPCR. The results were not normalized due to the small total amount of RNA available, as shown in fig. 16. The data in fig. 15 and 16 represent the mean and standard deviation (displacement) of triplicate technical replicates (n=3).

As can be seen from fig. 15 and 16, 30 μm of clofoglic phenol reduced viral titers measured in mucus of human bronchial epithelial cells by more than 100-fold in vitro. This is confirmed by the quantitative decrease in viral RNA.

Claims

1. Use of clofoc phenol for the prevention or treatment of diseases caused by coronaviruses.

2. The clofoglic phenol for use according to claim 1, wherein the coronavirus is SARS-CoV, MERS-CoV or SARS-CoV2.

3. The clofoglic phenol for use according to claim 1, wherein the disease is Covid-19.

4. The clofoc phenol for use according to any one of the preceding claims, wherein the clofoc phenol is administered in combination with at least one other active ingredient.

5. The clofoc phenol for use according to claim 4, wherein the at least one other active ingredient is administered simultaneously, sequentially or over a period of time.

6. The clofoc phenol for use according to any one of the preceding claims, wherein the clofoc phenol is administered in the form of a pharmaceutical composition comprising at least one pharmaceutically acceptable excipient.

7. A method for preventing or treating a disease caused by a coronavirus comprising administering to a subject in need thereof clofogliclan.

8. The method of claim 7, wherein the coronavirus is SARS-CoV, MERS-CoV, or SARS-CoV2.

9. The method of claim 7, wherein the disease is Covid-19.

10. The method of any one of the preceding claims, further comprising administering at least one additional active ingredient to the subject.

11. The method of claim 4, wherein the at least one additional active ingredient is administered simultaneously, sequentially or over a period of time.