FR2937538A1 - ANTIGRIPPAL PRODUCT - Google Patents

Abstract

The present invention relates to a plant composition having antibacterial and antiviral effects. This effective composition against the influenza virus is intended for the prevention and treatment of viral infections induced by viruses of the family Orthomyxoviridae.

Description

Influenza product and methods of preparation Field of the invention: Faced with the problem of the risk of avian flu pandemic, the existing therapeutic solutions are solely of synthetic and costly origins.

Indeed, like all type A viruses, the H5N1 subtype has a great ability to mutate over time, as well as to exchange its genes with influenza viruses belonging to other types of infecting other species. A subtype of virus may stop circulating for several years in the human population but remain present in an animal population. If the animal population is in direct contact with a human being, they can transmit the virus again. A virus subtype may also be newly created by a genetic reassortment that occurs when a host is co-infected with two different viruses. If this new virus has H5 and NI segments specific to the avian virus, it will completely escape the recognition of the human immune system. If he also possesses genes that allow him to multiply effectively in mammals, he will then be able to transmit himself from man to man as effectively as classical flu. The appearance of an influenza virus belonging to a viral subtype totally unknown to the human population, such as H5N1, renders the immune memory of humans totally ineffective. This makes plausible a rapid and worldwide spread of the virus. 25 Influenza experts today recognize that a new pandemic seems inevitable. It will probably be caused by an H5N1 virus derived from the avian influenza strain currently circulating in Asia and reaching Europe's doorstep. The idea of a vaccine is not a satisfactory solution in view of the possible evolution of the virus during the passage to humans.

What is known is that the production of a vaccine will take at least six months once the circulating strain is isolated and that, given the global production capacity of vaccine manufacturers, these six months will not allow to produce more than one billion monovalent doses. With two doses needed to gain protection, fewer than 500 million people, or 14 percent of the world's population, will be vaccinated. This places a special emphasis on antiviral treatments, which will therefore be at the forefront of fighting a possible pandemic in the first place. SUMMARY OF THE INVENTION The invention relates to a composition used for the preparation of a medicament for the prevention and treatment of viral infections induced by viruses of the family Orthomyxoviridae, it comprises: Olive tree Grapefruit seed extract Rosemary extract Green tea extract Curcumin This therapeutic composition is characterized by levels of olive leaf extract ranging from 0.01 to 100 mg per Kg.

This therapeutic composition is also characterized by grapefruit seed extract contents of between 0.01 and 100 mg per kg.

This therapeutic composition may be in various forms, in the form of compacted powders or not, in liquid form, solution or dispersion, in semi-solid or gelled or emulsified or dispersed form, or in aerosol form. This therapeutic composition may be administered orally, by intravenous injection, by inhalation, or by means of a spray, a diffuser, or by prior impregnation in a tissue, or by any other appropriate means known per se.

The present invention relates to the antiviral capabilities of various formulations from antioxidants, and more particularly from plant extracts having antioxidant properties. These antioxidants, by their antibacterial activity constitute a barrier helping in the prevention of new infections favored by the attack of the viruses. The present invention resides in the combination of said substances which makes it possible to potentiate their effectiveness.

The present invention relates to the viruses of the family Orthomyxoviridae including the influenza virus and more particularly that of the bird flu. The present invention also relates to a formulation using pharmaceutical or chemical compositions for the improvement, treatment and prevention of diseases associated with influenza viruses.

The involvement of the body with the H5N1 avian influenza virus is characterized by the triggering of an exaggerated inflammatory reaction, which results in a secretion of mucositis, such as pneumonia, which causes respiratory distress in the affected subject. . It is this symptom that is responsible for the exceptional severity of the disease. The action of the invention will be to reduce this inflammatory response, this is what the scientific literature tends to prove in the various publications that serve as references. These publications describe that the various components of the invention interfere with the general mechanisms of the body's inflammatory response to the avian influenza virus. Thus the action of the invention will reduce the phenomenon of respiratory distress which often has a lethal effect in the affected subject, allowing him to pass the critical stage of the disease. The diversity of modes of action on the virus, its multiplication and its effects make it possible to envisage many applications of the invention, in particular thanks to the various possible galenic forms which represent an essential advantage of combating the flu pandemic. bird. The invention represents a prophylactic barrier for contamination between humans, in the case of a pandemic, for example by impregnating the protective masks which assures them a safer and more durable efficacy. In another example, it may serve as an air freshener of public places by its aerosolization in the air ducts, which would reduce the financial consequences of a pandemic for society. DESCRIPTION

A methodical combination of different active ingredients each of which is described in the scientific literature as acting on several pathogenic bacteria and viruses. The concept of the invention is based on the synergy of these various described actives. Indeed, the multiplication of the influenza virus is a complex mechanism described in the form of several stages. The combination of these different active ingredients makes it possible to act on most of these steps instead of focusing on a particular step; it is this approach that makes it possible to gain in efficiency. On the other hand, the invention represents a derivative of non-toxic natural products that can be used in several forms and that it can therefore be used more easily in terms of the various propagation factors of the virus without toxicity to the environment and its inhabitants. Prototypes were manufactured according to the invention, these prototypes were tested at several levels. It was first checked the activity of the active principles contained in the invention. They have been successfully tested against various microbial strains such as Escherichia coli; Staphylococcus aureus, Salmonella spp. Or Streptococcus pneumoniae. The in vitro efficacy against Haemophilus influenzae was then carried out in dose-response. These preliminary results, which are reported in Example 1 below, demonstrate an activity. Example 1: Date Jo J + 1 day of analysis Results: CFU / ml Concentration Extract 2 Extract 2 Extract 2 Extract 2 (ppm) without PA with PA without PA with PA 250 18 16 0 0 500 18 1 0 0 1000 11 0 0 0 2000 16 0 0 0 Table 1: Comparative effect of component of ND235 and its solvent on haemophilus influenzae by enumeration after initial contamination of about 100 CFU / ml. Simplified Interpretation: From day 0 the bacillus is blocked with the active ingredient (PA) is at 12% with 250 10 ppm PA or 94.4% with 500 ppm PA or 100% with higher amounts of AP. Example 2: We then developed and realized a pharmaceutical formulation allowing easier and more efficient use of ND235 facilitating in vitro acute toxicity studies of ND235. The results of this study presented in Table 2 below show the very low toxicity of ND235. Concentration (g / 1) ND235 Indicator Cells Average Deviation Type Average Deviation Type CELLULAR VIABILITY INDEX 7,50E + 00 0,553 0,18275393 0,126 0,05201653 2,50E + 00 0,84733333 0,26023515 0,173 0,05201653 8,33E -01 1.073 0.20751626 0.273 0.05201653 2.78E-01 1.70266667 0.07965132 0.282 0.05201653 9.26E-02 1.44666667 0.168122 0.266 0.05201653 3.09E-02 0.70166667 0, 04101626 0.302 0.05201653 1.03E-02 0.361 0.09534674 0.265 0.05201653 3.43E-03 0.24666667 0.09856132 0.235 0.05201653 1.14E-03 0.219 0.08758425 0.283 0.05201653 3.81E 0.119466667 0.0903567 0.269 0.05201653 1.27E-04 0.206 0.10742905 0.298 0.05201653 4.23E-05 0.203 0.05973274 0.259 0.05201653 Table 2: Dose-response effect of ND235 on the viability of Human lymphocytes in culture. Simulated interpretation:

1) Regardless of the concentration of ND235, the values of the viability indices (from 0.2 to 1.7) are greater than the values of the control cells without product (from 0.2 to 0.3). This shows that ND235 is not toxic even at the highest concentrations of 7g / 1.

2) In concentrations greater than 10-3 g / l (lanes 8-1), ND235 stimulates cell growth relative to the cell control with a peak activity at the concentration of 0.3 g / l ( line 4)

It is noted that ND235 is not toxic because, on the contrary, it stimulates cell growth when its concentrations are greater than or equal to 2 × 10 -3 g / I. After verifying the non-toxicity of ND235 on human blood cells in culture, we investigated the possible toxicity to other tissue types that are more susceptible to infection with the influenza virus, kidney MDCK cells Example 3: Toxicity was evaluated as soon as the product was added (acute toxicity) and 24h after the addition of the product (possible cytostatic effect) Concentration Control Association 1 Association 2 Association 3 Association 4 ND235 (~) Without product CELL SIZE INDEX 1,00E-4-01 0,370 0,039 1,773 0,022 1, 8961-0.061 1.764 0.022 1.726 0.013 1.839 0.020 3.33E + 00 0.353 0.045 1.820 0.166 1.682 0.389 1.634 0.099 1.749 0.061 1.817 0.053 1.11 + 0.350 0.065 1.746 0.122 1.617 0.182 1.476 0.029 1.686 0.125 1.531 0.100 3.70E 01 0.364 0.067 1.545 0.089 1.3051-0.051 1.4431-0.127 1.421 0.036 1.1981-0.048 1.23E-01 0.391 0.081 0.772 0.088 0.6921-0.073 0.7201-0.082 0.7751-0.072 0.692 0.042 4, 12E-02 0.398 0.038 0.489 0.039 0.484 0.058 0.484 0.071 0.4791-0.075 0.515 0.055 1.37E-02 0.389 0.050 0.431 0.029 0.446 0.035 0.440 0.037 0.449 0.042 0.444 0.037 4.57E-03 0.336 0.061 0.412 0.053 0.411 0.075 0.393 0.059 0, 3761 - 0.068 0.365 0.078 Table 3: Direct Dose - Response Effect of ND235 and Various Associations of Its Active Ingredients on Viability of Culture MDCKs. 10 Simplified interpretation:

1) Whatever the concentration of ND235 or the different combinations of its active ingredients, the values of the viability indices (from 0.3 to 1.9) are greater than the values of the control cells without product (from 0.3 to 0). , 4, column 2). This shows that ND235 is not toxic even at the highest concentrations of 10 g / l.

2) In concentrations greater than 1.7 10-2 g / l (lines 7 to 1), ND235 and the different combinations of its active ingredients stimulate cell growth compared to cellular controls. 20 Concentration Control Association Association Association Association ND235 None 1 2 3 4 Product CELL SIZE INDEX 1,00E + 01 0.429 0.010 1.856 0.061 1.829 0.048 1.733 0.118 1.770 0.076 1.819 0.078 3.33E + 00 0.385 0.004 1.638t 0.044 1.750 0.046 1.657 0.044 1.715 0.080 1.839 0.079 1.11 + 00 0.429 0.011 0.962t 0.068 0.760 0.019 0.866 0.043 0.897 0.036 0.689 0.018 3.70E-01 0.448 0.018 0.610 0.036 0.469 0.027 0.610 0.066 0.601 0.030 0.477 0.027 1.23E-01 0.476 0.001 0.464 0.006 0.440 0.005 0.455 0.006 0.448 0.006 0.443 0.007 4.12E-02 0.443 0.018 0.427 0.023 0.436 0.028 0.437 0.035 0.437 0.012 0.440 0.026 1.37E-02 0.448 0.024 0.431 0.028 0.397 0.021 0.391 0.020 0.403 0.004 0.401 0.030 4.57E-03 0.454 0.047 0.462 0.053 0.422 0.021 0.413 0.009 0.406 0.034 0.389 0.009 1.52E-03 0.442 0.032 0.437 0.014 0.434 0.001 0.397 0.051 0.392 0.075 0.444 0.004 Table 4: Effect at 24 hours in dose - response of ND235 and various combinations of its active ingredients to the viability of cultured MDCKs. Simplified interpretation:

1) Regardless of the concentration of ND235 or the different combinations of its active ingredients, the values of the viability indices (from 0.38 to 1.84) are greater than the values of the control cells without product (from 0.38 to 0.45). This shows that ND235 is not toxic even at the highest concentrations of 10g / l (first line).

2) In concentrations greater than 3 10-1 g / l (lines 4 to 1), ND235 and various combinations of its active ingredients stimulate cell growth over cell controls.

It is noted that ND235 is not toxic since, on the contrary, it stimulates the cell growth of MDCK when its concentrations are greater than or equal to 0.3 g / 1 .5 Modes of action of ND235 at the level of the cell infected (Figure 1) Action of ND235 on Stage 1 Mooring of the influenza virus to the target cell is enabled by the interaction between a viral hemagglutinin and sialic acid receptors located on the cell surface (Lamb , 1989). This operation can be compared to a key and lock system. Part of the subunit HA1 (the key), facing outwards, has a very particular form and recognizes a specific molecule: sialic acid (the lock), which is present on the surface of certain cells. Both forms are perfectly recognizable. This recognition between the HA and the sialic acid causes the attachment of the viral particle to the target cell (the key is inserted into the lock). The blocking of this step of attaching the virus to the host cell by one of the active principles of ND235 has been clearly demonstrated by work published in good scientific journals (Impact Factor> 3). ND235 blocks the multiplication of the virus and therefore decreases the cellular interactions due to infection, this concept has a direct destructive effect of the virus on the affected cell, which reduces the excess of immune response which leads in the case bird flu respiratory distress often responsible for the death of the infected subject. - ~ ND235 action on step 12

Release of new viruses: The new virus particles remain attached to the membrane of the cell that produced them, because of the binding between the haemagglutinin (of the virus) and the sialic acid (of the cell). NA proteins break this link, allowing new viruses to break off and infect new target cells. One of the actions of the active ingredient contained in ND235 will prevent the action of neuraminidase (NA) which is responsible for the detachment of the host cell virus and the release of new infectious viruses. Blocking this release of infected particles in the cell will thereby decrease the number of viruses circulating. ND235 will moderate the inflammatory response of different cells affected by viral attack. 35 ù- Conclusion on cellular action modes of ND235

Several active ingredients contained in ND235 have a recognized action against the bird flu virus. This action has been the subject of publications 40 recognized in various scientific journals of good level. The antiviral actions described demonstrate their effectiveness by blocking the production of the virus in the cell, particularly in steps 6 and 7 for regulating the production of viral proteins and for synthesizing effective viral proteins. Thus the number of viruses produced will be decreased and ND235 will act on the cell identical to the previous mechanisms by acting on the infection and its consequences. A second study was conducted to verify the antiviral effect of ND235 against the virus. Influenza A Influenza A. Study Principle: MDCK cells are the reference cells for the study of the multiplication of the influenza A virus. For our antiviral effect study, we chose to use a reference strain of the HIN1 influenza virus strain A / PRI8 / 34. This viral strain was obtained from a Puerto Rican patient and this reference virus is accessible at the American Tissue and Collection Bank (American Type Culture Collection).

Viral infection is followed by its cytopathic effect (CPE) on cells in culture. This effect can be observed by the appearance of lysis plaques on cell monolayers (Plaque Forming Unit, PFU). If the viral concentration is increased, cell lysis can be extended to all or part of the cell monolayer. The experiments are carried out in 96-well culture plates which are stained with crystal violet. Staining will result from the presence of cells. Thus, the disappearance of the color will attest to the viro-induced destruction of the cells. In addition, we will verify in parallel wells containing different concentrations of our product without virus, it does not cause cell lysis confirming its lack of toxicity in our system. The antiviral activity of ND235 is established by comparing the effects of different dilutions of ND235 in the presence of decreasing amounts of virus. The amounts of virus used for infection are given in TCID50. TCID50 is the viral concentration that will give a CPE effect on 50% of cells in culture.

The experiments are performed in triplicate.

Cells: MDCK cells are derived from cells isolated by S.H. Madin and N.B. Darby in 1958 from a kidney of an adult female cocker spaniel breed. These cells are accessible at the American bank of ATCC tissues and viruses. MDCK cells are cultured at 37 ° C in a humid atmosphere containing 5% CO 2 in DMEM medium with 10% fetal calf serum in the presence of antibiotics Penicillin Streptomycin.

Virus: The viral influenza A strain used is A1PR / 8/34 strain HIN1 (ATCC VR-1249). During infection, the MDCK cells are cultured at 33 ° C. in a humid atmosphere containing 5% CO2 and in MEM medium with 0.125% BSA and in the presence of 1 g / ml trypsin treated with TPCK ( Perbio SA, Be). Protocol: 96-well culture plates are inoculated at 5 × 10 4 MDCK cells per well. These cells are cultured at 37 ° C. in a humid atmosphere containing 5% CO 2 in DMEM medium with 10% fetal calf serum in the presence of Penicillin Streptomycin antibiotics. When they form a confluent monolayer, they are infected with decreasing amounts of virus resulting from one-third dilutions of a stock solution of virus. The title of the starting virus stock solution established in 96-well culture plates is 81 TCID50.

These infections are carried out in the absence or presence of ND235 at different concentrations. The cells are then kept in culture at 33 ° C. in a humid atmosphere containing 5% CO 2 for four days and then stained with Crystal Violet. The cytopathogenic effects are then recorded in order to establish the percentage inhibition of the viral infection obtained by the different concentrations of ND235.

The remaining cell quantities are estimated by measuring the monolayer uptake at 595 nm performed in a microplate reader. The absorption values at 595 nm are corrected by those of the non-specific absorptions measured at 405 nm. The results of the mean values and the standard deviations are presented in Table 3 and in the form of a diagram. 10 Results: Viral InputConcentration in ND 235 0% 0% 0% 4% 4% 4% 0.4% 0.4% 0.4% 0.04% 0.04% 0.04% -0.007 0.005 0.002 -0.004 -0.006 0.024 -0.008 -0.007 -0.01 -.006 -.006 -.005 -0.012 0.018 -0.01 -0.009 0.03 0.053 0.073 -0.003 -0.005 0.034 0.02 0.004 -0.005 0.003 0.093 0.352 0.317 0.273 0.235 -0.004 0.049 -0.006 0.009 0.025 0.025 0.024 0.115 0.527 0.201 0.653 0.508 0.453 0.244 0.493 0.325 0.06 0.038 0.177 0.513 0.686 0.737 0.796 0.596 0.447 0.462 0.755 0.643 0.49 0.471 0.494 0.483 0.457 0.734 0.815 0.537 0.478 0.52 0.592 0.533 0.467 0.331 0.477 0.481 0.745 0.828 0.753 0.621 0.414 0.465 0.582 0.646 0.351 0.535 0.512 0.436 0.731 0.728 0.01 0.407 0.374 0.591 0.644 0.59 0.353 Table 1: Raw absorbance data as a function of ND23 concentration and quantities viral. Viral Input: amounts of virus in TCID50 used for infection at time O. 0.3 0.1 Ctrl30 Title CONTROL ND235 4% ND235 0.4% ND235 0.04% Viral 81 + + + + + + + + + + + + 27 + + + + + + + + + + + + 9 + + + + - + - + - + - + + + + + 3 + + + - + - _ _ + - - + - + 1 + + - - - - - - - - - - 0.3 - - - - - - - - - .. - - 0,1 - - - - - - - - - - - - Ctrl - - - - - - - Table 2: Inhibition of infection with Influenza A virus by different concentrations of ND235 viral titre: in TCID50.

Effect: +: destruction> 60% of the cells; + -: destruction of 30% to 60% of the cells; -: presence of 70% to 100% of the cells.

Interpretation: The different ND235 product concentrations are effective against the virus. Percent viral inhibition can be calculated by comparing wells infected with the lowest effective viral dilutions in the presence and absence of the product. Thus, it follows that: ND235 4%: 80% viral inhibition ND235 0.4%: 84% viral inhibition ND235 0.04%: 73% viral inhibition In the control wells containing the different concentrations of ND235 in absence of virus (Ctrl line) the staining is not diminished in the presence of ND235. The decrease in the intensity of the staining would have attested to a toxicity. The results of our previous toxicity studies confirm that ND235, even at the highest concentrations, is not toxic. Viral Input ND235 4% ND235 0.4% ND235 0.04% Control 0.1 0.7753 0.0458 0.5000 0.1078 0.5263 t 0.1552 0.4297 0.0855 0.3 0.6687 0.1877 0.5117 0.0304 0.5107 0.0945 0.4827 0.0115 1 0.7210 0.0303 0.5017 0.0820 0.6293 0.1330 0.2427 0.2442 3 0.4603 0, 2333 0.4017 0.193 0.2927 0.2183 0.0547 0.0523 9 0.3140 0.0396 ~ 0.0933 0.155 0.0093 0.0155 0.0303 0.0544 27 0.0247 0.0313 0.0217 0.0445 0.0193 0.0150 -0.0020 0.0173 81 0.0047 0.0168 -0.0083 0.0015 -0.0057 0.0006 0.0000 0.0060 - Table 3: Mean values and standard deviation of absorbances as a function of ND235 concentration and viral input. Viral input: in TCID50.

The absorbance values at 595 nm were corrected by absorption values at 405 nm. Figure 2 reports the effect of ND235 on MDCK cell infection by Influenza A Virus. Simplified interpretation of Figure 2:

The uptake values in the virus-free control wells (first series, TCID50) are of the order of 0.75 and 0.45 in the presence of 4% ND235 and 0 cYo (control) respectively of ND235. Thus, the ND235 at the highest concentrations (ND235 4%) will promote the development of cells compared to the control cells not having ND235 (Control). For the lower and lower concentrations of ND235, this cell growth stimulating effect is less pronounced but still appears to be present. These results are consistent with those obtained in the in vitro toxicity study of our product indicating a beneficial effect on cell growth.

The different concentrations presented of ND235 (series 1) effectively block doses of virus destroying 50% of the cells. For viral infections made with three and nine times more virus (series 3 and 9) there is still inhibition of the virus by ND235 but in a dose-dependent manner, the highest concentrations of ND235 conferring the best protection. Conclusion: ND235 is an effective antiviral against in vitro infection with influenza A virus. We have demonstrated that its efficacy is dose - dependent. This study shows that ND235 at a concentration of 0.4% has an antiviral efficacy of 84%. It is important to note that at a dose ten times lower, the efficacy of ND235 remains remarkable. Indeed it is of the order of 75%.

On the other hand, it is worth remembering that this product is completely natural and that the effectiveness of its constituents both at the antiviral level and at the level of the decrease of the immune response has been described in the scientific literature. Finally, the interest of this 40 product also lies in the fact that it will block the influenza virus by different mechanisms which, unlike a single product such as oseltamivir or amantadine, goes very well. greatly decrease the likelihood of resistant mutants. 45 LEGEND OF FIGURES:

Figure 1 shows the pattern of influenza A virus multiplication and the mode of action of ND235. In this figure, the interference of the ND235 is symbolized by the four arrows with thick lines starting from the Roman numerals I and II. In the figure, the numbers correspond to the different sequential stages of the infection cycle of the cell by the influenza A virus. These phases are as follows: 1) Attachment of the virus to the cell 2) Entry into the cell by endocytosis 3) Decrease of the pH 4) Fusion, release of the virus content into the cell 5) Entry of viral RNA into the cell nucleus 15 6) Production of new strands of viral RNA 7) Production of viral proteins 8) Output of viral RNA from the nucleus 9) Migration of elements 10) Assembly 2 0 11) Budding 12) Release of new viruses 25 Figure 2 represents the effect of ND235 on MDCK cell infection by Influenza A Virus. On the abscissa are given increasing concentrations of virus and on the ordinate the absorption values proportional to the number of cells. The measurements are made in the absence (Control) and in the presence of increasing amounts of ND235 (0.04%, 0.4% and 4%). 13

Claims (5)

REVENDICATIONS1. Composition used for the preparation of a medicament for the prevention and treatment of viral infections induced by the viruses of the family Orthomyxoviridae characterized in that it comprises: Olive leaf extracts Grapefruit seed extracts Extracts of rosemary Extracts of green tea 10 Curcumin 2. Therapeutic composition according to claim 1 characterized in that it comprises 0.01 to 100 mg per Kg of olive leaf extracts 15 3. Therapeutic composition according to claim 1 characterized in that it comprises 0.01 to 100 mg per Kg of grapefruit seed extract. 4. Therapeutic composition according to any one of claims 1 to 3, characterized in that it is in the form of compacted powders or not, in liquid form, solution or dispersion, in semi-solid or gelled or emulsified form or dispersed, or in aerosol form 5. Therapeutic composition according to any one of claims 1 to 3 characterized in that it is administered orally, by intravenous injection, by inhalation, or with the aid of a spray, a diffuser, or by prior impregnation in a fabric. 14