WO2021043718A1

WO2021043718A1 - Antiviral pharmaceutical composition containing 6-aminonicotinamide

Info

Publication number: WO2021043718A1
Application number: PCT/EP2020/074231
Authority: WO
Inventors: Gilles Duverlie; Lynda HANDALA; Etienne BROCHOT
Original assignee: Universite De Picardie Jules Verne; Centre Hospitalier Universitaire D'amiens-Picardie
Priority date: 2019-09-03
Filing date: 2020-08-31
Publication date: 2021-03-11

Abstract

The invention relates to a pharmaceutical composition containing 6-aminonicotinamide as the pharmaceutically active compound, for use as an antiviral agent in the treatment of a viral infection, specifically a polyomavirus infection, in a subject requiring such treatment.

Description

ANTIVIRAL PHARMACEUTICAL COMPOSITION CONTAINING 6

AMINONICOTINAMIDE

The present invention relates to a pharmaceutical composition which contains as a pharmaceutically active compound 6-aminonicotinamide for its use as an antiviral agent in the treatment of viral infection, in particular polyomavirus infection. This composition can be used during the complications of polyomavirus infections requiring medical management of patients (nephropathies, hemorrhagic cystitis, etc.)

Most people are asymptomatically infected with polyomaviruses from childhood. Polyomaviruses remain latent or persistent in healthy people whose immune systems can control the infection. On the other hand, their reactivation in immunocompromised people (elderly, transplanted, infected with HIV, for example) can cause very serious illnesses. Thus, in the case of a kidney transplant, the BK virus can lead to rejection of the transplant, if it reactivates in the transplant patient or if it infects the transplant patient through the organ of the donor. The BK virus can also cause, especially in immunocompromised patients, nephritis, urethral stricture or hemorrhagic cystitis. In patients who have received a bone marrow transplant, it is not uncommon for the BK virus to cause hemorrhagic cystitis.

Other polyomaviruses cause cancer or neurological pathologies. Thus, the JC virus which remains latent in the urinary tract, can cause, upon reactivation in an immunocompromised subject, progressive multifocal leukoencephalitis (PML). The MCPyV virus is associated with cancer, Merkel cell carcinoma. The SV 40 virus, which is a simian virus, has been able to infect humans because of the first polio vaccination campaigns with the oral Sabin-type vaccine prepared from cell culture in monkey kidneys. In immunocompromised monkeys, it can cause kidney disease and sometimes demyelinating disease similar to PML.

The publication “Human polyomavirus reactivation: disease pathogenesis and treatment approaches” by De Gascun and Carr, published in 2013 in the journal “Clinical and Developmental Immunology” edited by Hindawi Publishing Corporation (ID 373579) reviews most of the known polyomaviruses, pathologies that they are likely to cause, at risk subjects and known antivirals. This publication indicates that no antiviral tested in clinical trials offers a sufficiently significant therapeutic benefit. The aforementioned publication describes the drug candidates used against polyomaviruses: certain nucleosides or other chemical compounds such as myrtazapine, chlorpromazine, mefloquine or leflunomide have been tested as antivirals. Fluoroquinolones can also be used as an antiviral in the case of the BK virus. In the case of kidney transplant rejection caused by the BK virus, inhibitors of the mTOR enzyme have also been tested; their antiviral effectiveness is not proven.

The publication entitled "45 years after the discovery of human polyomaviruses BK and JC: Time to speed up the understanding of associated diseases and treatment approaches" by H. Barth et al. and published in the journal Crit Rev Microbiol., in March 2017 volume 43 (2), pages 178-195, indicates that Cidofovir®, a nucleoside which inhibits the replication of the herpes virus makes it possible to reduce the replication of the BK virus in human kidney cells. This nucleoside is nevertheless nephrotoxic which decreases its interest. A lipid derivative of Cidofovir®, Brincidofovir® is found to be less nephrotoxic, but these compounds are not currently used because their clinical benefit remains insufficient.

In practice, in transplant patients under immunosuppressive treatment, direct virological monitoring (detection of the virus or a constituent of the virus) or indirect (detection of an antibody or a biomarker) is carried out. Beyond a certain virus threshold detected, the action to be taken is generally a reduction or an end to the treatments in progress for the initial pathology treated (transplant, multiple sclerosis, HIV infection, etc.). Only the reduction, modulation or suspension of immunosuppressive treatments can restore sufficient immunity to control polyomavirus infections. This takes at least a week and is often done at the expense of treating the initial pathology (loss of the graft, etc.). In the case of patients infected with HIV, reactivation of polyomaviruses can be a real problem if it is not possible to restore sufficient immunity to stop the viral spread.

An aim of the present invention is therefore to provide a pharmaceutical composition which makes it possible to limit or prevent viral replication / propagation, in particular of a polyomavirus, and in particular of the BK virus and / or of the SV 40 virus. Another aim of the present invention is to provide a pharmaceutical composition which makes it possible to limit or prevent the dissemination of a virus in the body, in particular a polyomavirus, in particular in the blood, in the kidneys or in the brain.

Another aim of the present invention is to provide a pharmaceutical composition which limits or prevents the spread of a virus, in particular a polyomavirus, in an immunosuppressed subject, in particular a patient who has undergone a kidney transplant.

Another object of the present invention is to provide a pharmaceutical composition in which the pharmaceutically active compound exhibits a good selectivity index as an antiviral.

Another aim of the present invention is to provide a combination product which makes it possible to prevent infection and / or reactivation of polyomaviruses, in particular the BK virus and / or the SV 40 virus, in immunocompromised patients.

The present invention relates to a pharmaceutical composition containing as a pharmaceutically active compound 6-aminonicotinamide for its use as an antiviral agent in the treatment of a viral infection, in particular a polyomavirus infection, in a subject having such. need.

The inventors have in fact shown that such a pharmaceutical composition is effective in reducing or suppressing the replication of a virus in the case of a primary infection and in the case of virus reactivation. The composition of the invention makes it possible either to avoid infection, when it is administered as a preventive measure, for example, before a kidney transplant, or to avoid or limit the reactivation of the virus, in particular of the polyomavirus, when 'it is administered, for example on a preemptive basis, before too great a drop in the control of this infection by the subject's immune system, this infection being able to be induced by an immunosuppressive or immunomodulatory treatment, or even curatively in order to limit the spread of the virus when administered after the primary infection or after reactivation of the virus.

Furthermore, the targets of 6-ANA are the same as those of nicotinamide. Thus, the viral inhibition of 6-ANA is competitive and the administration of nicotinamide can therefore serve as an antidote by restoring the metabolic inhibitions induced by 6-ANA. The publication entitled “JC T-antigen Regulatory Glucose Metabolic Pathways in Brain Tumor Cells” by Evan Noch et al., Published April 2, 2012 in volume 7 (issue 4) of the journal PLos ONE, indicates that 6-aminonicotinamide reduces the amount of T-antigen in BsB8 cells. BsB8 cells are a medulloblastoma cell line which expresses the T-antigen without being infected with any virus. The T-antigen is an oncogenic protein produced by the JC polyomavirus and which interacts with a wide variety of cellular proteins to promote tumor development. Therefore, it was not clear that 6-aminonicotinamide could have any effect on the polyomavirus itself and therefore the polyomavirus infection itself.

According to one embodiment of the present invention, said virus is a polyomavirus, in particular the BK virus or the SV 40 virus.

According to a particular embodiment which can be combined with any embodiment of the present invention, the pharmaceutical composition of the invention is contained in a formulation suitable for oral administration or in a formulation suitable for administration. parenterally, in particular intravenously or in a formulation suitable for administration by the bladder route. In the case of polyomaviruses which can remain latent in renal or urothelial cells, such as the BK virus, for example, local administration in the bladder, by a catheter, can make it possible to treat the viral focus located in the urinary tract. The pharmaceutical composition of the invention which is in the bladder moves up the ureters due to the outflow or reflux of urine and helps treat the viral focus in the kidneys. This local administration near the viral focus considerably reduces the side effects of 6-ANA, in particular its ototoxicity. 6-ANA is indeed likely to cause edema of the pericaryon at this level.

According to an embodiment which can be combined with any one of the embodiments of the invention, the pharmaceutical composition of the invention is in a form suitable for being administered at a pharmaceutical active dosage equal to or greater than 0, 1 g / kg / day, in particular equal to 0.2 g / kg / day, in particular for a period equal to or greater than 5 days or equal to or less than 10 days.

According to one variant, the pharmaceutical composition of the invention is in a form suitable for parenteral or bladder administration and suitable for to be administered at a daily pharmaceutical active dosage equal to or greater than 0.1 mg / kg, in particular equal to 0.2 mg / kg for 2, 3 or 4 weeks or at a dosage equal to or greater than 0.3 mg / kg and in particular equal to 0.4 mg / kg for 1 or 2 weeks.

The subject is not limited according to the invention. It may be an uninfected subject or an already infected subject in whom the virus, in particular the polyomavirus and in particular the BK virus, is reactivated or latent. According to an embodiment which can be combined with any one of the embodiments of the invention, the subject is an immunosuppressive subject and in particular a human subject immunosuppressed following an immunosuppressive or immunomodulatory treatment, in particular an immunosuppressive treatment. or immunomodulator which makes it possible to avoid the rejection of a transplant, in particular of a kidney transplant.

The composition of the invention can further comprise any excipient. A person skilled in the art is able to choose an excipient, in particular an excipient as defined in the 21 st edition of Remington, "the Science and practice of Pharmacy". By way of example, the pharmaceutical composition of the invention may contain, whatever its embodiment, at least one excipient chosen from water, lactose, corn or potato starch, dextrose , sorbitol, microcrystalline cellulose, dibasic calcium phosphate hydrate, acacia gum, gelatin, polyvinyl pyrrolidone, glucose, carboxy-methyl cellulose, povidone iodine, talc, stearic acid , calcium stearate, magnesium stearate, polyethylene glycol, surfactants, oils, hydrogenated oils, possibly hydrogenated vegetable oils, waxes, in particular vegetable, colloidal silica, clays, cellulose, starch modified, copolymers, silica gel, activated carbon, hydroxypropylmethyl cellulose, zein, polysaccharides, propylene glycol, aqueous solvents, in particular water, alcohols, acetic acid, acetone, ethyl acetates, ethanol, ci acid teric acid, DMSO, benzyl alcohol, butyl paraben, phenol, ascorbic acid, sodium bisulfate, tocopherols, soy lecithin, EDTA, tartaric acid, sodium alginates.

In particular, the composition of the invention can comprise DMSO as excipient and be provided in liquid form. The solubility of the major part of the compounds in this excipient is such that the required doses allow a dissolves at low, non-toxic concentrations of DMSO (<1 ° L ·). The composition of the invention can also be suitable for oral administration and not comprise any excipient.

According to another embodiment, the pharmaceutical composition of the invention is in the form of a capsule or a tablet with a delayed effect and in particular in that it contains as excipient at least one hydrogenated oil and polyethylene glycol. It may also, in this embodiment, contain DMSO in a mixture of excipients or DMSO alone as an excipient.

According to an embodiment which can be combined with any one of the embodiments of the invention, the pharmaceutical composition of the invention contains a therapeutically active quantity of 6-aminonicotinamide and in particular a therapeutic quantity substantially equal to or greater than 2 g, in particular substantially equal to 3 g or 4 g and substantially equal to or less than 6 grams.

According to a particular embodiment which can be combined with any one of the aforementioned embodiments, the pharmaceutical composition of the invention is suitable for being administered as a preventive measure before the primary infection or before the reactivation of said polyomavirus. This, in both cases, in particular, in the case of a patient who is going to undergo a kidney transplant.

According to a particular embodiment which can be combined with any one of the aforementioned embodiments, the pharmaceutical composition of the invention is capable of being administered preemptively during the reactivation of said polyomavirus before the appearance of clinical signs. .

According to another embodiment, capable of being combined with any one of the aforementioned embodiments, the composition of the invention is administered curatively after the primary infection or after the reactivation of said polyomavirus.

The present invention also relates to a combination product comprising a pharmaceutical composition according to any one of the aforementioned embodiments and at least one immunosuppressive agent chosen from steroidal anti-inflammatory drugs, calcineurin inhibitors, in particular cyclosporine or tacrolimus, cell multiplication inhibitors, in particular azathioprine, mycophenolate mofetil, inhibitors of the mTOR protein, such as vererolimus® in particular and agents blocking the second signal, in particular LEA29Y®, for their simultaneous delayed or sequenced use in time. According to the invention, the The composition of the invention can be separated from the immunosuppressive agent (s) or can be mixed with the latter (s).

The present invention also relates to a method of therapeutic treatment according to which a patient infected with a polyomavirus, in particular the BK virus and / or the SV40 virus, is administered a quantity / therapeutic dose of a pharmaceutical composition containing as active agent of 6-aminonicotinamide.

The composition can be administered via the bladder, parenterally or orally.

The patient can be a patient as defined above.

The aforementioned information relating to the composition of the invention or its mode of administration, applies to the aforementioned therapeutic method. The method can be curative or preventive.

Definitions

The term "polyomavirus" denotes a polyomavirus capable of infecting a mammal. It can be chosen from polyomaviruses that are human or likely to be pathogenic in humans, such as, for example, the BK virus, the SV40 virus or the JC virus (JCPyV).

The term “immunocompromised subject” designates a subject exhibiting at least one of the criteria defined below:

- insufficient immunoglobulins, especially immunoglobulins G, A, D, M and E;

- insufficient T lymphocytes and / or B lymphocytes;

an insufficiency of the cells involved in the elimination of the foreign antigenic particles, in particular an insufficient quantity of macrophages and / or polynuclear and / or neutrophils and / or dendritic cells;

- a deficit of phagocytosis; and

- a deficiency of the complement system.

The subject can be a mammal, especially a human. It may be a patient who is immunocompromised due to the taking of an immunosuppressive or immunomodulatory drug (for example, due to the taking of an anti-rejection drug, in particular in the case of transplants, in particular of kidney). The term “treatment” encompasses both prophylactic (or preventive) treatment which aims to avoid a primary infection or reactivation and preemptive or therapeutic treatment which aims to limit the spread of the virus during a primary infection or reactivation of the virus. virus in an already infected person.

The purpose of "pre-emptive treatment" is to treat early any patient suspected of developing a symptomatic clinical infection from biological markers or additional examinations (imaging, etc.) in a population particularly at risk of developing or reactivating a disease. initially asymptomatic infection which will progress beyond a certain threshold to a serious symptomatic clinical pathology. One example may be an elevation of a marker of an infectious agent in an asymptomatic patient beyond a threshold which indicates a high probability of developing a clinical pathology and its complications. In this case, it is an increase in the urinary or blood level of genomic copies of BK virus beyond the threshold where the infection becomes symptomatic such as nephropathies or hemorrhagic cystitis.

The terms “limiting the propagation of a virus” mean that at least in vitro the replication of the virus is slowed down or reduced for a given period of time, or even stopped. In the patient, this can (but not necessarily) result in the fact that the concentration of the virus concerned in the urine of the patient after treatment with the composition of the invention remains below the threshold viruria for which symptoms due to appear. directly or indirectly to the infection, in particular in an immunocompromised patient, and in particular in the context of the treatment of the initial pathology, which causes immunosuppression, and / or that the viremia (concentration of viral particles in the blood) is less than threshold viraemia for which symptoms appear, in particular in an immunocompromised patient, in particular in the case of the treatment of the initial pathology, which causes immunosuppression. The cut-off values may already be defined by clinical consensus which has made it possible to define decisional algorithms for the treatment of patients. By way of example, for the BK virus, clinical problems during renal transplantation only arise when the viruria exceeds 10 ⁷ / mL or the viremia 10 ⁴ / mL expressed as the number of genomic copies per milliliter. The term CC50 (50% Cytotoxic Concentration) designates the so-called cytotoxic concentration of the tested pharmaceutical active compound for which 50% inhibition of cell viability is obtained at the end of a given period. The term IC50 (50% Inhibitory Concentration) designates the concentration of the tested pharmaceutical active compound for which a reduction in viral infection of 50% is obtained under given experimental conditions.

The ISso selectivity index denotes the CC50 / IC50 ratio; the higher the selectivity index, the greater the therapeutic margin, that is, the therapeutically effective concentrations are lower the higher the cytotoxic concentrations.

The term "MOI" = multiplicity of infection, defines the ratio of the number of infectious viral particles to the total number of cells in the same volume used for the test.

The infectious titer defines the number of infectious viruses in a given volume. It is measured here in FFU / mL (Focus Forming Unit / mL), ie in Focus Forming Units therefore initially corresponding to one infectious viral particle per milliliter.

The terms “pharmaceutically active” and “therapeutic amount” indicate that the compound induces a measurable improvement, immediate or delayed, transient or definitive, in the state of health or the well-being of a subject, in particular when it is found. administered in an appropriate therapeutic amount.

The abbreviation 6-ANA "denotes in the present application 6-aminonicotinamide, of formula C6H7N3O and also called 6-aminopyridine-3-carboxamide.

The term "tacrolimus" denotes 3S- [3R ^* [E (1 S ^* , 3S ^* , 4S ^* )], 4S ^* , 5R ^* , 8S ^* , 9E, 12R ^* , 14R ^* , 15S ^* , 16R ^* , 18S ^* , 19S ^* , 26aR ^* ]] -5,6,8, 11, 12,13,14,15,16, 17,18,19,24,25,26,26 a-hexadecahydro-5, 19- dihydroxy-3- [2- (4-hydroxy-3- methoxycyclohexyl) -1 -methylethenyl] -14,16-dimethoxy-4,10,12,18-tetramethyl-8- (2-propenyl) -15,19- epoxy-3H-pyrido [2,1 -c] [1, 4] oxaazacyclotricosine-

1, 7,20,21 (4H, 23H) -tetrone, monohydrate.

The term "azathioprine" refers to 6 - [(1 -methyl-4-nitro-4,5-dihydro-1 H-imidazol-5-yl) sulfanyl] -7H-purine.

The term “mycophenolate mofetil” designates the molecule of the crude formula C23H31 N07 marketed under the name Cellcept®.

The term “Everolimus®” denotes 2-0- (2-hydroxyethyl) rapamycin; the term “LEA29Y” corresponds to the molecule marketed under the name Belatacept®. The term “progeny” corresponds to the quantity of infectious virus released into the supernatant after 72 hours of culture.

FIGURES

Fig. 1A represents the measurement of intracellular ATR (normalized value (in%) according to the control) of human primary renal epithelial cells (RPTEC) after 72 h of contact with solutions of different concentration of 6ANA (logarithmic scale) and Fig. . 1 B represents the measurement of the reduction of resazurin as a function of the 6-ANA concentration of the solution tested;

Fig. 2A represents the measurement of intracellular ATR (value normalized as a function of the control (in%)) of Vero cells after 72 hours of contact with solutions of 6-ANA of different 6-ANA concentration (logarithmic scale) and FIG. 2 B represents the measurement of the reduction of resazurin as a function of the 6-ANA concentration of the solution tested.

Fig. 3A represents the measurement of intracellular ATR of CV1 cells (normalized value (in%) according to the control) brought into contact for 72 h with solutions having different 6-ANA concentrations (logarithmic scale) and FIG. 3 B represents the measurement of the reduction of resazurin as a function of the 6-ANA concentration of the solution tested (logarithmic scale);

Fig. 4A represents the inhibitory effect of 6-aminonicotinamide (in percentage) after 72 h of infection of RPTEC cells on the expression of the messenger RNA of the T antigen of the BK virus measured by qRT-PCR (measurement standardized against the expression of a cellular gene (GAPDH)) as a function of the 6-ANA concentration of the solution tested (logarithmic scale); Fig. 4 B represents the inhibitory effect of 6-ANA (in%) on the amount of extracellular DNA of BK virus measured by qPCR as a function of the 6-ANA concentration of the solution tested (logarithmic scale) and Fig. 4 C represents the inhibitory effect of 6-ANA (in%) on the percentage of RPTEC cells infected with the BK virus, which percentage is measured by immunofluorescence of the T viral antigen (T Ag) after 72 h of contact with the 6-ANA solution tested as a function of the 6-ANA concentration of said solution (logarithmic scale);

Fig. 5 represents the inhibitory effect of 6-ANA (in%) on the percentage of Vero cells infected with the BK virus, said percentage being measured by immunofluorescence of the viral T antigen (T Ag) after 72 hours of contact with a 6-ANA solution as a function of the 6-ANA concentration of said solution (logarithmic scale).

Fig. 6 represents the inhibitory effect of 6-ANA (in%) on the percentage of CV1 cells infected with the SV40 virus, said percentage being measured by immunofluorescence of the viral T antigen (T Ag) after 72 h of contact with a solution of 6-ANA as a function of the 6-ANA concentration of said solution tested (logarithmic scale);

Fig. 7 represents the antiviral effect of 6-ANA (in%) on the extracellular progeny of BK virus after 72 h of infection of RPTEC cells. (measurement standardized as a percentage relative to the control) as a function of the 6-ANA concentration of the solution tested (logarithmic scale);

Fig. 8 represents the antiviral effect of 6-aminonicotinamide (in%) on the extracellular progeny of the BK virus after 72 h of infection of RPTEC cells (measurement standardized as a percentage relative to the control) as a function of the 6-ANA concentration of the solution tested (logarithmic scale); and

Fig. 9 represents the antiviral effect of 6-aminonicotinamide (in%) on the extracellular progeny of the SV40 virus after 72 h of infection of CV1 cells (measurement standardized as a percentage relative to the control) as a function of the 6-ANA concentration of the solution tested (logarithmic scale).

EXAMPLES

1.1. Cell cultures

Cells: Vero and CV1 lines and primary cells (RPTEC)

The primary cells and cell lines used were cultured according to the recommendations of the suppliers and correspond to those used in the scientific literature.

Vero cells and CV1 cells are simian epithelial renal cells cultured according to the ATCC recommendations in DMEM medium (Gibco, Thermofisher Scientific, France) supplemented with 10% fetal calf serum.

Primary human renal epithelial proximal tubular cells (RPTECs Sciencell, Clinisciences, France) were cultured in growth medium for renal epithelial cells (REGM Bulletkit at Lonza, France) supplemented with 0.1% recombinant EGF (epithelial growth factor), 0.1% hydrocortisone, 0.1% epinephrine, 0.1% insulin, 0.1% triodothyronine, 0.1% transferrin, 0.1% GA-100 (amphotericin B and gentamycin) and 0.5% fetal calf serum. The cells are cultured at 37 ° C. in a 5% CO2 incubator according to the supplier's recommendations.

Two polyomaviruses were used, the human BK virus which causes kidney disease in humans and the simian virus SV40 as a prototype of the polyomaviruses.

The Dunlop strain of the BK virus was produced from the plasmid pUC19pBKv (34-2) under the reference 45025 at the ATCC. The plasmid is amplified after transformation of competent Escherichia Coli HB101 bacteria (Invitrogen) cultured at 37 ° C. in LB medium with 50 μg / ml of ampicillin. The plasmids produced are purified and linearized by enzymatic digestion with the restriction enzyme BamHI before being used to transfect the BK virus permissive Vero cells. The infected Vero cells are stored and passed until a cytopathic effect appears. The cells are then frozen and thawed 3 times to obtain cell lysis and release the virus which can be purified by ultracentrifugation.

The viral stock is titrated by serial dilution of a ratio of 10 (10 ^-1 to 10 ⁸ ) on the Vero cells distributed 24 h before in 96-well plates and then incubated at 37 ° C. The infected cells are revealed by immunofluorescence of the T antigen of the virus after three days of culture. The titer is expressed in focus units or FFU / mL.

SV40 virus strain 777 was cultured on CV1 cells. The stocks were produced after the onset of the cytopathic effect and the titrations carried out on the same cells in the same way as the BK virus as described above.

For testing, infections were made at a multiplicity of infection of 1 (MOI = 1).

Each experiment was carried out at least in triplicate to calculate the mean and the standard deviations which are reported in the curves carried out using the GraphPad Prism 8.0 software (San Diego, California).

The 6-aminonicotinamide used (marketed by Sigma-Aldrich, France; Ref. A68203) is described by the manufacturer as an inhibitor antimetabolite of vitamin B3 derivatives.

The 6-ANA was solubilized at 100 mM in dimethylsulfoxide (DMSO, Ref D2650 Sigma-Aldrich). The stock solution can be stored at 20 ° C. It is diluted for make concentration ranges in the culture media used respectively for each cell type. The ranges are prepared extemporaneously before each experiment.

1 .2. 6-ANA Cytotoxicity Tests

The cytotoxicity tests were carried out in triplicate (triplicate) on 96-well cell culture microplates incubated for 3 days in an incubator at 5% CC> 2 under the same conditions as the tests used to evaluate the antiviral effects (see below )

Two classic test kits from the company Promega (France) were used according to the recommendations of the distributor / manufacturer:

The first test (CelITiter-GIo 2.0 Assay Ref. G9241) measures the amount of ATP by bioluminescence to test metabolically active cells. Briefly, the CelITiter Glo reagent is added to each well tested keeping the medium and the plate shaken for 2 minutes on an orbital shaker to induce cell lysis. The plate is then incubated at room temperature for 10 minutes to stabilize the signal which is finally read on a 96-well microplate luminometer (Berthold).

The results are visible in Fig. 1 A, FIG. 2A and FIG. 3A.

With reference to FIG. 1 A, it is observed that the measurement of intracellular ATR increases as a function of the logarithm of the 6-ANA concentration. In Fig. 1 a, the measurement of cellular ATR is 5% for a concentration of 1250 nM in 6-ANA; it increases to 50% for a concentration of 20,000 nM in 6-ANA.

In Fig. 2A, it is observed that the measurement of intracellular ATR of Vero cells is 5% for a 6-ANA concentration equal to 8 nM and 50% for a 6-ANA concentration of 80,000 nM. The measurement of intracellular GATR also increases with logarithm as the concentration of 6 ANA.

In Fig. 3A, it is observed that for the CV1 cells, the measurement of intracellular ATR is 5% for a 6-ANA concentration of 8 nM and 50% for a 6-ANA concentration of 80,000 nM.

It is therefore noted that 6-ANA has no negative influence on the proliferation of RPTEC, Vero and CV1 cells after 72 hours. The second test (CelITiter-Blue cell Viability Assay) uses a redox dye, ie the staining of a compound induced by the reducing capacity of cells. This is resazurin which is therefore reduced to resofurin and which can be measured by fluorimetry (Promega Fluorimeter).

Briefly, CelITiter-Blue reagent is added to each well. The microplate is shaken for 10 seconds on an orbital shaker and then incubated for 3 hours in a 5% CO2 incubator at 37 ° C. The reduction of the dye is then measured on a microplate fluorimeter with an excitation wavelength at 560 nm and emission at 590 nm.

The results are visible in Figs. 1 B, 2B and 3B

With reference to FIG. 1 B, the intracellular ATP measurement is 12% for a 6-ANA concentration equal to 1250 nM and 50% for a 6ANA concentration equal to 20,000 nM. The measurement of intracellular ATR increases exponentially with the logarithm of the 6-ANA concentration.

With reference to FIG. 2B, the measurement of intracellular ATR is 2% for a concentration of 8 nM; it increases to 50% for a 6-ANA concentration equal to 80,000 nM. It also increases with the concentration of 6-ANA.

With reference to FIG. 3B, the measurement of intracellular ATR is 2% for a 6-ANA concentration equal to 8 nM. The ATR measurement also increases exponentially with the concentration of 6-ANA.

It is therefore deduced from this that 6-ANA is not very cytotoxic on RPTEC, Vero and CV1 cells.

2. Testing the Antiviral Effects of 6-ANA

The cells are distributed ("plated") in 96-well cell culture microplates at densities between 10,000 and 20,000 cells per well, making it possible to obtain confluence on the fifth day of culture at 37 ° C. in a 5% incubator. of CO2. After 24 h of plating, the culture supernatants are removed, and the viral infections are carried out for 2 h at an MOI = 1. After these 2 hours, the inocula are removed by pipetting, and the cells are cultured with their respective media with or without drugs for 3 days.

Control wells of cells cultured respectively without 6-ANA, without virus, and without 6-ANA or virus are carried out for each experiment. The level of infection and its inhibition are evaluated by specific markers of the virus concerned: T antigen, viral DNA and viral messenger RNA, viral progeny.

The most used markers were the number of infected cells evaluated by immunofluorescence of the T antigen of the virus and the viral progeny (amount of infectious (infecting) virus released in the supernatant after 72 h of culture) in the supernatant after 3 days. corresponding to a complete cell cycle of the virus. For the BK virus, we also evaluated the extracellular viral DNA in the supernatants, the messenger RNA of the T antigen. The evaluation of these markers is described below.

2.1. Measurement of indirect immunofluorescence of T Antigen of polyomaviruses

The cells are washed three times with isotonic phosphate buffer at pH 7.4 (PBS) in the wells of the microplates after three days of culture. They are then fixed with 3.7% paraformaldehyde in PBS and permeabilized at room temperature (RT) for 15 minutes with 0.5% Triton X100 in filtered CSK buffer (cytoskeletal buffer; 10 mM PIPES pH 6, 8, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl, 1 mM EDTA) in equal parts with water (H2O 1: 1).

The fixed cells are then washed with PBS and then incubated with the primary monoclonal antibody for 1 h at RT, washed again, then incubated with the secondary antibody for 30 minutes in the dark. Both antibodies were diluted in 0.1% PBS Tween 20. The primary antibody is a mouse monoclonal anti-SV40 TAg diluted ^1/500 [PAb416] (Abcam, France). This antibody reveals both the T antigen of the SV40 virus and the T antigen of the BK virus. The secondary antibody is a polyclonal goat anti-mouse IgG antibody conjugated with Alexa Fluor Plus 488 diluted to 1 / 1000th (ThermoFisher Scientific, France).

To facilitate the counting of the cells, the nuclei are stained with 1/1000 ^th DAPI for five minutes in the wells which are then washed three times with PBS. The number of infected cells and the number of total cells are counted by immunofluorescence on an inverted microscope at low magnification x10 (Axio Vert.AI Zeiss with a Colibri 7 LED source, France).

2.2. Quantification of extracellular DNA of BK virus

The supernatants of the infected cells are recovered after the three days of culture and clarified by centrifugation for 15 minutes at 1500 rpm, then stored at 4 ° C before performing the quantitative real-time PCR (qPCR Taqman, Applied Biosystems, France). Primers and a probe targeting a conserved region of the VP1 and VP2 genes in the BK virus genome were used. The reactions are carried out in a total volume of 25 μl containing the Applied Universal Master Mix 2x, 10 mM of each primer and of the probe labeled with 6-carboxyfluorescein (FAM) and finally 5 μL of cell supernatant.

Amplification is carried out in an ABI prism 7900 HT device (Applied Biosystems) according to the program: 2 minutes at 50 ° C followed by a cycle at 95 ° C for 10 minutes, and 45 cycles at two times of 15 seconds at 95 ° C and one minute at 60 ° C.

Quantification is carried out using a 4-point calibration range containing known concentrations of plasmid pUC19pBKv (34-2) containing the genome of the BK virus (9,000,000 to 9,000 copies / mL in dilutions of ratio 10 ). The quantifications are carried out in triplicate and a negative control is added to each series.

2.3. Quantification by qRT-PCR of the intracellular messenger RNA of the T Antigen of BK virus

Total intracellular RNAs were extracted using the RNeasy® Mini Kit (Qiagen ™) on the QIAcube extractor (Qiagen ™) from the manufacturer's protocol in the presence of DNAse. The concentration of the extracted RNAs was evaluated on the Nanodrop ND 1000 spectrophotometer. The cDNAs were synthesized from the RNAs extracted by reverse transcription with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems ™) according to the manufacturer's recommendations.

The qPCR of the cDNA corresponding to the T Antigen was carried out in a total volume of 25 μl containing the Applied Universal Master Mix 2x, 10 mM of each primer and of the probe labeled with 6-carboxyfluorescein (FAM) and finally 2 μL of cDNA. PCR amplification was performed on ABI PRISM 7900 HT Sequence Detection System (Applied Biosystems ™) according to the manufacturer's recommendations. The quantification was carried out by the AACt method II is a relative quantification by comparison of the Ct (Cycle threshold) obtained with respect to the expression of the cellular gene for household b-Actin. This formula helps normalize gene expression from series to series.

3. Evaluation of the extracellular infectious progeny of BK and SV40 viruses The progeny here corresponds to the quantity of infectious virus released into the supernatant after 72 h of culture with or without 6-ANA. The supernatants are clarified by centrifugation for 15 minutes at 1500 rpm and then stored at 4 ° C. A volume of 100 microliters is taken to infect Vero (for the BK virus) or CV1 (for the SV40) cell culture wells. The supernatant is removed after 2 h of contact at 37 ° C. in a 5% CO 2 incubator with the cells and replaced with culture medium. After 3 days under these conditions, the infectious foci are counted in each well by indirect immunofluorescence of the viral T antigen according to the technique described above.

4. Statistical calculations, 50% concentrations and selectivity index.

The means of the results and their standard deviations are calculated using the GraphPad Prism 8 software according to the analysis recommendations given for the in vitro pharmacological study of the inhibitory effect of drugs on cell growth or the titer of markers. viral.

The concentrations are converted to a base 10 logarithm and the test results are normalized to 100% for the control wells without 6-ANA (= 100% control). The results of the wells with 6-ANA are therefore expressed as a percentage relative to the 100% control.

The program makes it possible to produce the curve (curve fitting) and to calculate the 50% inhibitory effects, ie either the cytotoxic effects on the cells (CC50), or the inhibitory concentrations of the viruses (IC50).

The SI selectivity index is then estimated by the CC50 / IC50 ratio. It increases (»1) when the test compound has a 50% antiviral effect at a dose that is all the smaller as the 50% cytotoxic effect is manifested at a dose that is all the stronger. This selectivity index is therefore an indicator of the therapeutic margin of the compound that one would expect if it were used for a clinical trial.

The numerical values extracted from the aforementioned figures are grouped together in Table 1 below according to each cell type studied.

For the cytotoxicity tests, it can be seen from the results grouped together in Table 1 below that the cell lines are more robust than the primary cells (RPTEC). The results differ but are comparable with the two tests used. The results for the BK virus on the primary lines show that the antiviral effects increase as they progress through the viral cycle of cell infection (messenger RNA, genomic DNA, number of infected cells and viral progeny). 6-ANA therefore has an inhibitory effect at all stages of the virus cycle, including more advanced stages of its cycle where 6-ANA is potentially even more effective. 6-ANA can therefore be used preventively, preemptively and curatively.

The most significant results in relation to the pathophysiology of BK virus infection concern those obtained for viral progeny on primary lines (RPTEC).

The selectivity indices of 6-aminonicotinamide are greater than 20. The indices obtained are even higher with the lines. These results are encouraging for setting up clinical trials aimed at limiting viruria and viremia due to BK virus respectively below the consensus thresholds of 10 ⁷ / mL and 10 ⁴ / mL to avoid clinical pathologies.

The results obtained with the SV40 in Table 1 show that 6-ANA is also effective against the SV40 virus. They therefore show that 6-aminonicotinamide is effective on several polyomaviruses and very probably on all of their representatives, including the JC virus, the Merkel cell polyomavirus, etc. 6-ANA can be used as a local treatment or systemic.

Table 1

In the table above cc ATP and cc resazurin refer to the cytotoxicity of 6-ANA on the cell type indicated in the table to the left. The Applicant is not bound by the following explanation of the mechanism of action of 6-aminonicotinamide. However, the Applicant considers that the antiviral action of 6- aminonicotinamide can be explained by inhibition of NAD + and NADP + enzymes in the cell. The main mechanism probably involves the inhibition of the pentose-phosphate pathway used by many viruses for the rapid synthesis of their nucleic acids.

Claims

1. A pharmaceutical composition containing as a pharmaceutically active compound 6-aminonicotinamide for use as an antiviral agent in the treatment of viral infection, especially polyomavirus infection, in a subject in such need.

2. Pharmaceutical composition according to claim 1, for its use according to claim 1, characterized in that said virus is a polyomavirus selected from BK virus, SV 40 virus and JC virus.

3. Pharmaceutical composition according to any one of claims 1 and 2, for its use according to claim 1, characterized in that it is contained in a formulation suitable for oral administration or in a formulation suitable for administration by parenteral route, in particular intravenously or in a formulation suitable for administration by the bladder route

4. Pharmaceutical composition according to any one of the preceding claims for its use according to claim 1, characterized in that it is in a form suitable for administration at a pharmaceutical active dosage of said pharmaceutical active compound equal to or greater than 0.1. g / kg / day, in particular equal to 0.2 g / kg / day, in particular for a period equal to or greater than 5 days or equal to or less than 10 days.

5. Pharmaceutical composition according to any one of the preceding claims, for its use according to claim 1, characterized in that it further comprises at least one excipient selected from water, lactose, corn starch. or potato, dextrose, sorbitol, microcrystalline cellulose, dibasic calcium phosphate hydrate, acacia gum, gelatin, polyvinyl pyrrolidone, glucose, carboxy-methyl cellulose, povidone iodine , talc, stearic acid, calcium stearate, magnesium stearate, polyethylene glycol, surfactants, oils, hydrogenated oils, vegetable oils, hydrogenated vegetable oils, waxes, in particular vegetable waxes, silica colloidal, clays, cellulose, modified starch, copolymers, silica gel, activated carbon, hydroxypropylmethyl cellulose, zein, polysaccharides, propylene glycol, aqueous solvents, especially water, alcohols, ac acetic ide, DMSO, acetone, ethyl acetates, ethanol, citric acid, benzyl alcohol, butyl paraben, phenol, ascorbic acid, sodium bisulfate, tocopherols, soy lecithin, EDTA, l tartaric acid, sodium alginates.

6. Pharmaceutical composition according to claim 5, for its use according to claim 1, characterized in that it is in the form of a capsule or a tablet with delayed effect and in particular in that it contains as that excipient is hydrogenated oil and polyethylene glycol.

7. Pharmaceutical composition according to claim 6, for its use according to claim 1, characterized in that it contains a therapeutically active amount of 6-aminonicotinamide equal to or greater than 2 g, in particular substantially equal to 3 g or 4 g and substantially equal to or less than 6 grams.

8. Pharmaceutical composition according to any one of the preceding claims, for its use according to claim 1, characterized in that it is administered as a preventive measure before the primary infection or before the reactivation of said polyomavirus.

9. Pharmaceutical composition according to any one of the preceding claims, for its use according to claim 1, characterized in that it is administered curatively after primary infection or after reactivation of said polyomavirus.

10. Combination product comprising a composition according to any one of the preceding claims and at least one immunosuppressive agent chosen from steroidal anti-inflammatory drugs, calcineurin inhibitors, in particular cyclosporine or tacrolimus®, cell multiplication inhibitors. , in particular azathioprine, mycophenolate mofetil, inhibitors of the mTOR protein, in particular Vererolimus® and agents blocking the second signal, in particular LEA29Y®.