FR3119540A1 - DHODH inhibitor therapeutic composition - Google Patents

Abstract

[Title: DHODH Inhibitory Therapeutic Composition A therapeutic composition comprising a mother tincture of a leaf extract of a plant belonging to the genus "Neurolaena" and species "lobata" for use as a dihydroorotate dehydrogenase inhibitor drug human (DHODH).]

Description

DHODH inhibitor therapeutic composition

RNA genome viruses are responsible for many human pathologies such as influenza, dengue fever, hepatitis C, measles, bronchiolitis in infants or, more recently, the so-called Covid-19 coronavirus.

The classic therapeutic arsenal for combating viruses with an RNA genome consists of targeting the activity of a viral protein essential to the cycle of the virus. Such essential proteins may in particular consist of RNA polymerase, integrase, a helicase or a protease. However, although these viral proteins may be conserved in several viruses with RNA genomes, the development of broad-spectrum therapeutic molecules, active against different viruses, remains relatively limited.

In addition, the plasticity of viral genomes, as well as the adaptability and evolutionary capacity of RNA-genome viruses, facilitate the rapid appearance of escape mutants. These mutants render ineffective treatment with broad-spectrum therapeutic molecules that target a viral protein essential to the virus cycle.

In order to circumvent these problems of mutation of therapeutic targets, a new therapeutic approach targeting the host cell, rather than the virus itself, has been developed in recent years. The principle of this new approach is to block the cellular mechanisms essential to viral replication, so as to prevent viral proliferation by stimulating the innate immune response in cells infected by a virus with an RNA genome.

One of the known cellular mechanisms essential for viral replication is the pyrimidine biosynthesis cycle. Indeed, pyrimidine is crucial for the survival of human cells, in particular when the latter are host cells of pathogens, in particular viruses with an RNA genome.

More particularly, in mammals and in human cells, the synthesis of pyrimidine takes place by two biosynthetic pathways: the "de novo pyrimidine pathway", and another "rescue pathway" which is carried out under specific physiological conditions. . Most human parasites do not possess the "rescue pathway" of pyrimidine synthesis. However, pyrimidines are necessary for the manufacture of pyrimidine nucleotides. These pyrimidine nucleotides are essential for cell survival and multiplication. Thus, blocking the “de novo pyrimidine pathway” is considered an effective therapeutic means to selectively target human parasites without affecting the human host and its normal cellular functioning.

In other words, all human pathogens, including RNA viruses, are deficient in the “rescue pathway” of pyrimidine biosynthesis. Therefore, the "de novo pyrimidine pathway", of the host cell, is a targeted pathway in therapy to eliminate these pathogens, in particular to prevent the replication of RNA viruses in host cells.

The de novo pyrimidine biosynthetic pathway is a synthetic pathway that takes place in several successive steps. This path and its different stages are illustrated on the drawings attached.

More specifically, the fourth step in the de novo pyrimidine biosynthetic pathway consists of dehydrogenation of the dihydroorotate called "DHO" which leads to the formation of orotate "ORO".

The enzyme, of the oxidoreductase type, which catalyzes the dehydrogenation reaction of dihydroorotate, is dihydroorotate dehydrogenase called "DHODH".

During the dehydrogenation of DHO to ORO, there is an electron transfer between two cofactors, one of which is an electron donor and the other is an electron acceptor. For example, the electron transfer, during this dihydroorotate dehydrogenation reaction, takes place thanks to the oxidation-reduction couple of flavin mononucleotides FMN/FMNH ₂ with the couple of ubiquinones QH ₂ /Q or with the couple nicotinamide adenine NAD+/ NADH. DHODH binds to its cofactor FMN in conjunction with ubiquinone to catalyze the oxidation of dihydroorotate to orotate.

The de novo biosynthetic pathway of pyrimidines ensures the synthesis of uridine 5-monophosphate called UMP. UMP serves as a precursor for other pyrimidine nucleotides. The latter are necessary and essential for cell division and the metabolic activity of the host cell which is infected by the RNA virus. Thus, it has been shown that inhibiting DHODH in host cells leads to a drop in the amount of pyrimidines in infected cells, which amplifies the innate antiviral immune response.

In a known manner, DHODH enzymes are separated into two groups, class 1 DHODH and class 2 DHODH. These two classes of DHODH are established according to their sequence similarity, their binding sites, their cellular localization and their preferred substrate.

Class 1 DHODHs are cytosolic enzymes present in protozoan pathogens.

Class 2 DHODHs are monomeric protein-like enzymes that bind to the inner membrane of eukaryotic mitochondria.

In other words, in humans, DHODH, belongs to class 2, it is a mitochondrial protein located on the outer surface of the inner mitochondrial membrane. Human DHODH exhibits two domains consisting of the alpha/beta-barrel domain containing the active site and the alpha-helical domain, the latter forms the opening of a tunnel leading to the active site.

To date, there are several known inhibitors of human DHODH, such as brequinar, teriflunomide or even leflunomide.

For example, leflunomide is used to treat rheumatoid arthritis or multiple sclerosis. The immunosuppressive effects of leflunomide have been attributed to depletion of pyrimidine supply to T cells or to more complex pathways mediated by interferon or interleukin. The inhibition of human DHODH by leflunomide is due to the binding of leflunomide to the alpha-helical domain which forms the opening of a tunnel leading to the active site of DHODH. Although marketed as an active drug ingredient, leflunomide causes side effects in 1% to 10% of patients, including diarrhoea, nausea, vomiting, mouth ulcers, abdominal pain, inflammation of the colon, headache, inflammation of the tendons, accentuation of natural hair loss, eczema, dry skin, increase in transaminases, or decrease in white blood cells.

Inhibition of human DHODH by brequinar occurs by the same mechanism as leflunomide, by occupying the opening of the tunnel leading to the active site. Brequinar was used as an anti-cancer drug in the late 1980s; however, as it is responsible for multiple undesirable side effects, it has not been accepted as a drug. In addition, brequinar is recognized as very toxic to human cells. As a result, the idea of using it in therapy to fight viral infections was quickly abandoned by the medical profession.

Thus, in the case of the development of a pathology linked to a viral infection by an RNA genome virus, the known inhibitors of DHODH seem inappropriate to treat the viral symptoms of a patient, in particular because of their side effects. and cytotoxic on cells, regardless of cell type.

This is why, in the case of a viral infection, in particular by an RNA virus, an alternative solution to known DHODH inhibitors, such as brequinar, teriflunomide or even leflunomide, should be found. This alternative solution, in addition to preventing the de novo pyrimidine pathway by inhibiting the enzymatic activity of DHODH, is desired to be non-cytotoxic for all human cells and with a minimum of side effects when taken in the form of medicine.

In addition, in the current context, most patients are attached to the origin and design methods of drugs. In general, patients want to minimize the share of synthetic components that make up the drug as much as possible. These synthetic components can be responsible for side effects just like the active ingredient. Accordingly, in addition to inhibiting human DHODH, the object of the present invention is also to find a DHODH inhibitor which is as much as possible of natural origin, of simple design with a low carbon footprint, in order to limit side effects while treating the RNA virus infection and associated symptoms.

The aim of the present invention is to overcome the drawbacks of the state of the art, by proposing a therapeutic composition comprising a mother tincture of an extract of leaves of a plant belonging to the genus " Neurolaena " and to the species " lobata for use as a human dihydroorotate dehydrogenase (DHODH) inhibitor drug.

This therapeutic composition has the advantage of having, as an active ingredient that inhibits the activity of DHODH, said mother tincture of Neurolaena lobata . This mother tincture is advantageously easy and quick to prepare, at lower cost on an industrial scale. In addition, said mother tincture of the therapeutic composition of the invention is advantageously of natural origin, that is to say non-synthetic, while being recognized in the literature as being non-cytotoxic in vivo .

Consequently, said therapeutic composition has few, if any, side effects related to its active ingredient of natural origin and non-cytotoxic. Following ingestion by a patient, said therapeutic composition of the invention inhibits the activity of DHODH without having any cytotoxic effect. Taking said therapeutic composition, including said mother tincture, generates an increase in the innate immune response in patients whose immune system is weakened by pathogens or any other disease.

Further, according to further features of the invention, said therapeutic composition is for use in a method for treating symptoms associated with viral infection of RNA genome viruses.

Said therapeutic composition, comprising a mother tincture of Neurolaena lobata having the property of inhibiting the activity of DHODH and preventing the de novo pathway of pyrimidine synthesis, is suitable in the case of viral infection by a RNA, especially positive RNA.

Indeed, RNA viruses are deficient in the “pyrimidine rescue pathway”. RNA viruses for their cell infection mechanism must use the "de novo pathway" of the host cell to synthesize pyrimidine which is necessary in the cell replication mechanism, in other words necessary for viral multiplication. Thus, inhibition of the de novo pathway of the host cell prevents cell replication, i.e. viral replication of RNA viruses. Therefore, the use of said therapeutic composition of the invention in a method for treating the symptoms associated with a viral infection of RNA genome viruses is appropriate.

According to a preferred embodiment, the therapeutic composition of the invention is used in a method making it possible to treat the symptoms associated with a viral infection of viruses with an RNA genome chosen from the families of viruses from the following list: the Coronaviridae, the Flaviviridae, the Orthomyxoviridae, or the Togaviridae.

Most preferably, the present invention also relates to a method for preparing a dry extract of a diluted mother tincture of dry leaves of Neurolaena lobata , characterized in that it comprises the following steps:

i) preparing a mixture based on dry leaves of Neurolaena lobata in sugar cane alcohol at 50°, said mixture having a mass concentration of between 15 and 20 g/L;

ii) said mixture is left to macerate with stirring for about 21 days;

iii) After maceration, the mixture is filtered through “cartridges” with a porosity of 50-75 μm and a liquid filtrate is obtained, corresponding to a mother tincture of dry leaves of Neurolaena lobata , and a solid retentate;

iv) the said liquid mother tincture is diluted to ¼ by adding an aqueous solution and a hydroalcoholic solution is then obtained;

v) the alcohol contained in this solution is evaporated by rotary evaporator, until an aqueous solution is obtained;

vi) the aqueous solution thus obtained is frozen, then it is freeze-dried to obtain a dry extract of the diluted mother tincture of dry leaves of Neurolaena lobata .

Advantageously, the mixture prepared in step i) has a mass concentration of between 16 and 17g of dry leaves of Neurolaena lobata per liter (L) of sugar cane alcohol at 50°, and preferably equal to 16g /I.

Most preferably, during step iv), said mother tincture is diluted by mixing a volume equal to 0.75L of filtrate and a volume equal to 2.25L of water.

The invention also relates to a method for preparing a liquid solution from a dry extract of diluted and freeze-dried mother tincture of dry leaves of Neurolaena lobata , said dry extract being obtained according to the method described above, said liquid solution being obtained by diluting said dry extract in water or in an aqueous solvent such as dimethyl sulfoxide (DMSO), said liquid solution having a concentration of between 6,500 and 20,000 ng of dry extract per mL of aqueous solvent, preferably between 6,667 and 20,000 ng/mL.

The present application also relates to a mother tincture extract of dry leaves of Neurolaena lobata diluted, in particular according to a ¼ dilution of the mother tincture, and freeze-dried, said extract being in liquid solution and having a final concentration of between 6,500 and 20,000 ng/mL (mass of freeze-dried dry extract of diluted mother tincture / volume of aqueous solution), for its use in the treatment of a viral infection due to the SARS-CoV-2 virus responsible for Covid 19.

Neurolaena lobata leaf mother tincture extract is particularly indicated for use in decreasing the production of cytokines, including IL-6 and IP-10, in the treatment of severe forms of viral infection due to the virus SARS-CoV-2 responsible for Covid 19.

Other characteristics and advantages of the invention will emerge from the detailed description which will follow of the non-limiting embodiments of the invention, with reference to the appended figures, in which:

: there schematically represents the stages of the de novo pyrimidine synthesis pathway in human cells, including the fourth stage which consists of the dehydrogenation reaction, i.e. the action of the enzyme dihydroorotate dehydrogenase "DHODH" to transform dihydroorotate “DHO” in orotate ORO;

: there schematically represents the inhibitory action of said composition of the invention on mitochondrial human DHODH, within a human host cell infected with a virus with an RNA genome;

: there shows the evolution curve, as a function of the contact time between DHODH and its substrate, of the optical density measured at 600 nm of a sample at a concentration of 0.01 µg/mL consisting either of brequinar, or of a mother tincture of an extract of leaves of Neurolaena lobata, named “H1”, or either by another mother tincture of an extract of leaves of Neurolaena lobata named “H2”;

: there shows the evolution curve, as a function of the contact time between DHODH and its substrate, of the optical density measured at 600 nm of a sample at a concentration of 0.1 µg/mL consisting either of brequinar, or of “H1”, or either by “H2”;

: there shows the evolution curve, as a function of the contact time between DHODH and its substrate, of the optical density measured at 600 nm of a sample at a concentration of 1 µg/mL consisting either of brequinar, or of “H1 “, or either by “H2”;

: there represents the evolution curve of the percentage inhibition of the activity of DHODH on its substrate, observed between 0 and 275 seconds of contact with the sample, as a function of the concentration of the sample H1 or H2;

: there represents the evolution curve of the percentage inhibition of the activity of DHODH on its substrate, observed between 0 and 275 seconds of contact with the sample, as a function of the concentration of the sample H1, H2 or of brequinar.

The present invention relates to a therapeutic composition comprising a mother tincture of an extract of the leaves of a plant belonging to the genus " Neurolaena " and to the species " lobata " for use as an inhibitor of human dihydroorotate dehydrogenase (DHODH ).

There schematically represents the de novo synthesis pathway of pyrimidines comprising several steps, in which step 4 involves the action of the human dihydroorotate dehydrogenase "DHODH".

In the de novo pyrimidine synthesis pathway, the precursors of the pyrimidine core are glutamine, aspartic acid and CO ₂ . As seen on the , in the first step, there is formation of carbamylphosphate by carbamylphosphate synthetase. In the second step, aspartate transcarbamylase catalyzes the formation of carbamylaspartate. The latter is transformed in the third step, by dihydroorotase, into dihydroorotate “DHO”. Then in the fourth step, the dihydroorotate dehydrogenase “DHODH” will catalyze the transformation of “DHO” into orotate “ORO” which will serve as a reaction precursor to obtain Uridine monophosphate “UMP”. UMP is useful in the mechanism of polymerization with RNA necessary for cell multiplication.

According to the invention, the mother tincture of an extract from the leaves of a plant belonging to the genus “ Neurolaena ” and to the species “ lobata ” is advantageously of natural origin. Indeed, Neurolaena lobata is a plant belonging to the Asteraceae family found in the West Indies and Central America, especially in Guadeloupe. In these territories, this plant is grown and harvested easily because the agroecological and pedoclimatic conditions are favorable there.

Thus, by virtue of its natural origin, the use of an extract of Neurolaena lobata advantageously makes it possible to reduce the risk of side effects during its use within a therapeutic composition for treating a disease.

In addition, to support this idea, several studies and scientific publications have demonstrated that an extract of Neurolaena lobata has no toxicity in vivo (Gracioso JS et al., J. Pharm. Pharmacol. 1998, 50: 1425 – 1429; Gracioso JS et al., Phytomedecine, 2000, Vol.7(4), pp.283 – 289). In particular, in mice, after oral ingestion of a dose of 5000 mg/kg of hydroalcoholic extract of the aerial parts of Neurolaena lobata , no physiological toxicity was observed after several days.

Consequently, in addition to its natural origin, the choice of an extract of Neurolaena lobata, as the active principle of a therapeutic composition aimed at inhibiting the activity of DHODH, advantageously respects the objective of the invention of be non-cytotoxic for all human cells, in particular those involved in the immune response, at the concentrations which have been chosen.

According to the invention, said mother tincture of an extract of Neurolaena lobata leaves consists of a hydroalcoholic solution.

According to a preferred embodiment, the mother tincture is produced from an extract solely of the leaves of a Neurolaena lobata plant.

According to the invention, said therapeutic composition is in a pharmaceutical form for oral ingestion. For example, said therapeutic composition is in a liquid form, for example in the form of a syrup, or else in a solid form, for example in the form of a tablet.

According to the invention, said therapeutic composition comprises, as active principle, said mother tincture of Neurolaena lobata having an inhibitory effect on the activity of DHODH, as well as other excipients allowing its galenic formulation. The interactions that may exist between these excipients and said mother tincture do not influence or impact the inhibitory effect of DHODH.

Preferably, said therapeutic composition of the invention comprises only excipients of natural origin, which cause little or no side effects when they are formulated with said mother tincture of Neurolaena lobata .

According to a preferred embodiment, said therapeutic composition is used in a method for treating symptoms associated with a viral infection.

More specifically, said therapeutic composition of the invention is used in a method making it possible to treat the symptoms associated with an infection by a virus with an RNA genome chosen from the families of viruses from the following list: the Coronaviridae, the Flaviviridae, or else Togaviridae:

- among the Coronaviridae, we can cite by way of example SARS-CoV-2, responsible for Covid 19;

- among the Flaviviridae, mention will be made of the Hepacivirus genus, the only representative of which is the virus responsible for hepatitis C, and, in the Flavivirus genus, the virus responsible for yellow fever or the Zika virus and,

- among the Togaviridae, we can cite the virus responsible for chikungunya.

Indeed, for their viral replication within the host cell, RNA viruses need the de novo pathway of pyrimidine synthesis of the host cell to be functional. These viruses lack a pyrimidine synthesis pathway. However, if this cellular pathway is blocked by the use of the therapeutic composition of the invention inhibiting the activity of DHODH, the viral replication of RNA viruses within the host cell will no longer be possible, the latter being deficient both from the de novo pathway but also from the salvage pathway of pyrimidine synthesis.

There shows the mechanism of action of the composition of the invention on the inhibition of DHODH in a human cell infected with a virus with an RNA genome. Thanks to the mother tincture of an extract of Neurolaena lobata leaves of the composition of the invention, the dehydrogenation reaction carried out by DHODH in the mitochondria does not take place. The de novo pyrimidine synthesis pathway is blocked, and the salvage pathway cannot be used by the virus, which lacks it. UMP and pyrimidine bases of nucleotides cannot be used by RNA virus to start viral replication.

Consequently, no virion can be produced by the host cell, even after introduction of the RNA viral genome into said host cell. Inhibition of DHODH prevents viral multiplication of RNA-genome viruses and prevents production and egress of virions outside the host cell membrane. Thus, the use of the composition of the invention, with an inhibitory action on the activity of DHODH, prevents viral multiplication and the development of symptoms associated with the presence of the pathogen within the organism. The composition of the invention therefore constitutes an effective therapeutic means for treating viral infections, in particular by viruses with RNA genomes.

It follows that the use of the therapeutic composition of the invention in a method for treating the symptoms associated with an infection by an RNA genome virus consists of a solution for combating the infection and limiting viral multiplication.

The therapeutic composition of the invention is a good alternative to existing viral disease treatment solutions targeting DHODH inhibition. This specific use makes it possible to prevent viral replication within the host cell, by inhibiting the de novo pyrimidine synthesis pathway, while increasing the immune response by the defense cells and without cytotoxic effect.

The present therapeutic composition of the invention therefore consists of an alternative solution making it possible to inhibit DHODH, that is to say to block the de novo pathway for the synthesis of pyrimidines necessary for the replication of RNA viruses, this without being invasive and destructive of the cells involved in the immune response against the pathogen.

The therapeutic composition of the invention also has the advantage of being easy to manufacture while being as natural as possible in the eyes of consumers and patients who would use it.

The results of experiments below are intended to illustrate the effect of inhibition of DHODH by the therapeutic composition of the invention.

Other results of experiments obtained in vitro detailed below illustrate, in particular, the action of the therapeutic composition of the invention in particular on the severe forms caused by a viral infection with the SARS-CoV-2 virus responsible for the Covid-19.

Indeed, these results demonstrate a particularly interesting action of the therapeutic composition of the invention comprising a mother tincture of an extract from the leaves of a Neurolaena lobata plant on the reduction in the level of certain cytokines released by the cells after an infection. by the SARS-CoV-2 virus.

Cytokines are proteins naturally synthesized by immune cells to mediate the immune response following infection by a pathogen. They promote a natural inflammatory reaction allowing the infected organism to defend itself against the pathogen.

However, in certain cases of SARS-CoV-2 infections, the release of cytokines, in particular in the lung cells, is so important that it triggers a "cytokine storm". This runaway immune system leads to a hyper-inflammatory reaction that can destroy tissues, cause acute respiratory distress syndromes, can lead to physiological deterioration, or even become lethal for the person in whom this reaction is triggered.

The results of tests, illustrated below, carried out in vitro on a therapeutic composition in accordance with the invention comprising a mother tincture of an extract of the leaves of a plant of Neurolaena lobata demonstrate that the production of cytokines can be substantially reduced by the action of said composition.

To return now to the results of tests relating to the inhibition of the action of DHODH, tests were carried out on a therapeutic composition of the invention comprising a mother tincture of an extract of leaves of a plant of Neurolaena lobata.

This mother tincture constitutes the sample to be tested. The measurement of the inhibition of the action of DHODH on its substrate is carried out in a standard multi-well plate with a transparent wall. The wells contain the samples to be tested, the DHODH and its substrate.

To evaluate the inhibition of DHODH by the sample, the optical density parameter called “OD” is used. Indeed, we measure, in each well, the "DO" at a wavelength of 600 nm, at several time intervals, for a period of 5 min.

More specifically, each well includes the sample diluted or not, the DHODH enzyme and its colorimetric substrate diluted in a test buffer. Said colorimetric substrate comprises DHO which can be transformed into ORO by the action of DHODH.

Over time, the consumption of the colorimetric substrate DHO by the enzyme DHODH results in a decrease in OD. This decrease signifies the transformation of colored DHO into uncolored ORO by the activity of DHODH. In other words, DHO is consumed, reduced to ORO by the activity of DHODH, which causes a change in the measured DO.

In case of inhibition of DHODH activity by the sample, the DO remains stable over time. Indeed, in the case of an inhibition, the colorimetric DHO substrate will not be transformed by the DHODH into ORO, the DO will therefore remain that of the initial DHO.

• une solution d’enzyme de classe 1 nommée « rhDHODH » pour « recombinant humain DHODH »,
• un tampon test constitué de 50 mM Tris, 150 mM KCl et 0,1% Triton ® X-100, à pH 8,
• un mélange de substrat de la DHODH contenant : 2 mM de L-dihydroorotique dit « DHO », 0,2 mM de décylubiquinone dit « Q » et 0,12 mM de 2,6 Dichloroindophenol sodium salt hydrate dit « DPIP » dans le tampon test,
• un échantillon de teinture mère d’un extrait de feuilles d’une plante deNeurolaena lobata, dilué ou non, dans un tampon de dilution constitué de diméthylsulfoxyde dit « DMSO ».

To conduct the experiments, it was used:

• a class 1 enzyme solution called “rhDHODH” for “recombinant human DHODH”,
• a test buffer consisting of 50 mM Tris, 150 mM KCl and 0.1% Triton ® X-100, at pH 8,
• a mixture of DHODH substrate containing: 2 mM of L-dihydroorotic called “DHO”, 0.2 mM of decylubiquinone called “Q” and 0.12 mM of 2.6 Dichloroindophenol sodium salt hydrate called “DPIP” in the buffer test,
• a sample of mother tincture of an extract from the leaves of a Neurolaena lobata plant, diluted or not, in a dilution buffer made up of dimethyl sulfoxide called “DMSO”.

It should be noted that in the substrate mixture of DHODH, the L-dihydroorotic called “DHO” constitutes the colorimetric substrate used by DHODH during the dehydrogenation reaction.

In the substrate mixture, decylubiquinone called “Q” and Dichloroindophenol sodium salt hydrate called “DPIP” are the electron acceptors and donors. The transfer of these electrons allows the realization of the dehydrogenation reaction by the oxidoreductase DHODH.

Preparation of the mother tincture of an extract from the leaves of a plant of Neurolaena lobata constituting the sample H1:

• On récolte les feuilles deNeurolaena lobata,
• On sèche lesdites feuilles,
• On broie lesdites feuilles sèches jusqu’à obtention d’une poudre,
• On fait macérer la poudre dans une solution d’alcool de canne à sucre pendant 21 jours jusqu’à obtention d’un macérat, le ratio des quantités étant 1 g de poudre pour 62,5 mL de solution d’alcool de canne à sucre,
• On filtre ledit macérat jusqu’à obtention d’un filtrat, nommé H1.

The H1 mother tincture of an extract from the leaves of a Neurolaena lobata plant is obtained by carrying out the following process steps:

• We harvest the leaves of Neurolaena lobata ,
• These leaves are dried,
• Said dry leaves are ground until a powder is obtained,
• The powder is macerated in a sugar cane alcohol solution for 21 days until a macerate is obtained, the ratio of the quantities being 1 g of powder for 62.5 mL of sugar cane alcohol solution ,
• Said macerate is filtered until a filtrate, named H1, is obtained.

In the aforementioned method, according to a preferred embodiment, the sheets can be dried under a current of hot air, at a temperature preferably below 40° C., for about 120 hours, until they present a residual moisture content of the order of 6 to 9, preferably 6.5 to 9%.

The humidity level is determined by any appropriate methods known to those skilled in the art. For example, the humidity level can be determined by using a desiccator installed in a room with a temperature below 40°C, with a relative humidity level below 85% without direct exposure to sunlight, electricity air or vibration.

For example, the desiccator referenced XM60 marketed by PRECISA MOLEN France, with a standard precision of 1 mg in high resolution and temperature ranges ranging from 30°C to 230°C with an increment of 1°C can be used to measure the residual leaf moisture content.

Preferably, in the aforementioned protocol, the powder is macerated in a solution of sugar cane alcohol at a temperature of 25° C. to 30° C., preferably 30° C., for about 21 days, with stirring. slow every day for 12 hours.

Preparation of the mother tincture of an extract from the leaves of a plant of Neurolaena lobata constituting the sample H2

• On récolte les feuilles deNeurolaena lobata,
• On sèche lesdites feuilles,
• On fait macérer 100 g de feuilles séchées dans 1L d’une solution d’éthanol pur pendant 7 jours jusqu’à obtention d’un macérat,
• On filtre ledit macérat sur célite jusqu’à obtention d’un filtrat,
• On concentre par évaporateur rotatif ledit filtrat.

The H2 mother tincture of an extract from the leaves of a Neurolaena lobata plant is obtained by carrying out the following process steps:

• We harvest the leaves of Neurolaena lobata ,
• These leaves are dried,
• 100 g of dried leaves are macerated in 1L of a pure ethanol solution for 7 days until a macerate is obtained,
• Said macerate is filtered through celite until a filtrate is obtained,
• Said filtrate is concentrated by rotary evaporator.

Said concentrated filtrate is dried under pressure, in particular using a vacuum ramp, until sample H2 is obtained.

Protocol for analyzing the inhibition of DHODH by the composition of the invention .

In order to verify the inhibition of DHODH by the therapeutic composition of the invention, two stock solutions, named H1 and H2 respectively, were prepared.

The H1 stock solution is obtained by carrying out the aforementioned method after harvesting the leaves of a Neurolaena lobata plant.

The stock solution H2 is obtained by implementing the aforementioned method.

In order to test the impact of the sample on the inhibition of DHODH and to know the "dose-response effect", each H1 and H2 sample was diluted in a DMSO buffer.

The dilutions of samples H1 and H2 made it possible to obtain the following concentrations: in µg of sample/total mL of solution in the well: 0.01 µg/mL; 0.1 µg/mL; 1µg/mL; 10 µg/mL; 100 µg/mL; 1000 µg/mL indicated in Table 1 and Table 2 respectively below.

To start the protocol, each dilution of the H1 or H2 sample was placed in the presence of the rh DHODH enzyme and the substrate mixture in a well. In order to obtain an average of OD measured for a sample concentration H1 or H2, triplicates were carried out for each of the dilution concentrations of H1 and H2.

• On ajoute, dans chaque puit comportant les dilutions des échantillons H1 ou H2, l’enzyme rh DHODH préparée le jour même dans une solution tampon test,
• On laisse en présence l’enzyme et l’échantillon pendant 6 min à 37°C,
• On ajoute ensuite 50 µL du mélange de substrat coloré susmentionné, ceci consiste en le temps 0,
• On mesure dans chaque puits par lecture immédiate la DO à 600 nm, à intervalle régulier, toutes les 55 secondes, pendant 275 secondes.

More specifically, to obtain the results below, the following steps of the protocol were carried out:

• The rh DHODH enzyme prepared the same day in a test buffer solution is added to each well comprising the dilutions of the H1 or H2 samples,
• The enzyme and the sample are left in the presence for 6 min at 37°C,
• Then add 50 µL of the aforementioned colored substrate mixture, this consists of time 0,
• The OD at 600 nm is measured in each well by immediate reading, at regular intervals, every 55 seconds, for 275 seconds.

The amount of enzyme added to each well is the same. In the protocol, the enzyme is added to the well so as to have a concentration of 0.06 µg of rh DHODH enzyme / mL of total solution in the well.

Table 1 below shows the results obtained for sample H1:
Table 1: Inhibition of DHODH activity by sample H1

In Table 1, the first column gives the OD measurement time intervals at 600 nm. In other words, this corresponds to the contact time of the H1 sample with the rh DHODH in the presence of the substrate mixture.

In Table 1, the second line indicates the concentration of sample H1 in the well. This concentration is expressed in µg of sample H1/mL of total solution in the well.

Each column of Table 1 indicates the average OD value measured at 600 nm of the triplicates for the same concentration of sample H1, and for a defined time of contact with rh DHODH and its substrate.

For example, as seen in Table 1, after 110 seconds of contacting sample H1 at a concentration of 0.1 µg/mL with rh DHODH, and the colored substrate mixture, the average value of the DO measured on the three wells, of identical capacity, is 0.315.

The line with the Δ sign in Table 1 indicates the difference between the mean value of DO measured at 0 seconds of contact and the mean value of DO measured at 275 seconds of contact between sample H1, rh DHODH and its substrate.

The Δ sign gives the decrease in DO between 0 and 275 seconds of exposure, i.e. the capacity for transformation of DHO into ORO by the effective activity of DHODH.

In Table 1, the last two lines give, for each sample concentration of H1, the percentage of activity of transformation of DHO stained into ORO by the activity of rh DHODH, as well as the percentage of inhibition of rhDHODH activity by sample H1.

The % activity is calculated as follows, for a given concentration of sample H1 (hereafter H2):
% activity = (ΔH1X100) / Δ of the negative control.
For example, for sample H1 at 0.01µg/mL: % activity = (0.092 X 100) / 0.208 = 44.231.

The % inhibition is calculated by the following formula: 100- the value of the % activity.

To validate the effective activity of rh DHODH in transforming its DHO substrate during the experiment, a negative control was carried out in duplicate. This negative control is essential to validate the activity of DHODH on its substrate and to determine the % activity and the % inhibition of the samples.

The negative control column shows the mean values of DO measured, at 600 nm, at the different measurement intervals in seconds.

The negative control contains: rh DHODH denoted "E" in Table 1 at a concentration of 0.06 µg/mL of total solution in the well, with 50 µL of the colored substrate mixture denoted "S" and, replacing the sample H1, only a DMSO buffer solution denoted “T”.

The results show that between 0 and 275 seconds, the mean OD value measured at 600 nm decreases.

Consequently, the colored substrate DHO is well transformed into ORO, by the reduction activity by the enzyme rh DHODH. The rh DHODH enzyme is well functional with respect to the substrate mixture. In addition, neither the DMSO buffer solution nor the test buffer solution in which the DHO substrate is diluted affects the dehydrogenation activity by the rh DHODH enzyme.

As seen in Table 1, sample H1 of the invention inhibits the activity of DHODH for its substrate. For all concentrations of H1 tested, the percentage inhibition is between 51% and 56%. It is also found that for the concentrations tested, an increase in the concentration of sample H1 is not synonymous with an increase in the value of the percentage inhibition against the activity of DHODH.

The same protocol and the same DO measurements made it possible to quantify the percentage of inhibition of the H2 sample.

Table 2 below shows the results obtained for the different tested concentrations of sample H2
Table 2 : Inhibition of DHODH activity by sample H2

As seen in Table 2, the H2 sample of the invention inhibits the activity of DHODH.

For all concentrations of H2 tested, the percentage inhibition is between 48% and 67%.

Unlike the H1 sample, there seems to be a dose-response effect. Indeed, for a concentration of 1000 µg/mL, the percentage inhibition seems significantly higher than for a concentration of 0.01 µg/mL.

In other words, when the concentration of sample H2 is increased in the presence of the enzyme, the percentage inhibition increases. The H2 sample differs from the H1 sample in the protocol for the preparation of the Neurolaena lobata leaf extract. It would therefore seem that the elements responsible for the inhibitory activity of rh DHODH are concentrated in the leaves and that, depending on the mode of preparation of the mother tincture of leaves, the inhibitory activity is different.

Figures 3 to 5 illustrate the evolution of the optical density measured at 600 nm over time, for samples of H1, H2 or brequinar at a given concentration.

The curve of the presents the measurement of OD values at 600 nm, over time, for samples at a concentration of 0.01 µg/ml total in the well.

Similarly, the curve of the is for samples at a concentration of 0.1 µg/mL and the curve of the is for samples at a concentration of 1 µg/mL.

It should be noted that, in all cases, regardless of the concentration of the sample, for brequinar, a known inhibitor of DHODH, the OD at 600 nm remains almost stable over time. Brequinar is therefore a DHODH inhibitor.

For samples H1 and H2, a decrease in OD measured at 600 nm is observed. This decrease is synonymous with the activity of rh DHODH. Nevertheless, it is found that for each of the samples, the decrease in the DO is clear from 110 seconds of contacting.

Between 0 and 110 seconds, the OD measured at 600 nm is rather stable. This observation seems to mean that the DHODH enzyme is not immediately active. In the first 110 seconds, the DHODH does not carry out the dehydrogenation reaction, or only slightly, when it is in the presence of the H1 or H2 samples. The H1 and H2 samples therefore seem to slow down the onset of the activity of DHODH vis-à-vis its substrate.

In order to validate, effectively, the activity of the enzyme rh DHODH of reduction of its substrate, one also carried out a positive control in duplicate. The positive control wells contain, in place of the diluted H1 or H2 sample, brequinar, which is a known inhibitor of DHODH activity.

In parallel with the measurement of OD of the samples, the OD of a solution of brequinar diluted in a DMSO buffer in the presence of rh DHODH and its substrate was measured.

In the same way as for the samples, the action of different concentrations of the brequinar solution on the inhibition of rh DHODH was measured.

In order to validate the activity of rh DHODH on its substrate, a positive control is carried out in triplicate, in parallel with the protocol for measuring the inhibition of DHODH by brequinar. The positive control wells include the rh DHODH called "E", and its substrate mixture called "T" as well as, replacing the brequinar, the DMSO buffer called "T".

Table 3 below shows the results obtained for the different tested concentrations of the brequinar sample
Painting 3 : Inhibition of DHODH activity by brequinar

In Table 3, the activity of the rh DHODH of transformation of its substrate is validated by the positive control. Indeed, over time, a decrease in the OD measured at 600 nm is effectively observed. This decrease indeed results from the transformation of DHO into ORO, by the dehydrogenation activity of rh DHODH. Neither the DMSO buffer nor the test buffer impact the activity of the rh DHODH enzyme. The rh DHODH enzyme is therefore functional in this protocol for measuring the inhibition of DHODH by brequinar.

Table 3 shows that the different concentrations of brequinar tested exhibit an rh DHODH inhibition activity of between 92% and 100%.

To highlight the inhibitory activity of the H1 and H2 samples, the shows the curve of the percentage inhibition of samples H1 and H2 with respect to each other, as a function of their concentration, after 275 seconds of contacting with DHODH and its substrate.

On the , it is found that, for concentrations ranging from 0.01 to 1000 μg/mL, H1 exhibits a relatively stable percentage inhibition of rh DHODH.

On the contrary, H2 shows a significant increase in its percentage inhibition for a concentration exceeding 100µg/mL. In particular, for a concentration of 1000 µg of H2/mL of total solution in the well, the percentage of inhibition of rh DHODH advantageously reaches 66.3%.

In the same way, the shows the evolution of the percentage of inhibition of samples H1, H2 and of brequinar according to their concentration after 275 seconds of bringing into contact with DHODH and its substrate. It is found that samples H1 and H2, like brequinar, are inhibitors of DHODH activity on substrate. Indeed, although the inhibition of DHODH by the H1 and H2 samples is not 100% and as efficient as the brequinar, it does exist.

Consequently, with regard to the results obtained, the samples of mother tinctures of Neurolaena lobata H1 and H2 of natural origin effectively exhibit, and in a significant manner, an inhibitory activity of the DHODH enzyme.

Thus, the therapeutic composition of the invention comprising a mother tincture of an extract from the leaves of a plant belonging to the genus “ Neurolaena ” and to the species “ lobata ” has the effect of inhibiting the activity of DHODH.

Therefore, a therapeutic composition comprising either of these samples, i.e. a leaf-based mother tincture of Neurolaena lobata is a promising natural, non-toxic product for treating diseases whose therapeutic target is the inactivation of DHODH.

In particular, the therapeutic composition of the invention is a possible avenue for treating diseases resulting from infection by a viral pathogen, in particular viruses with an RNA genome.

In vitro tests were also carried out to demonstrate the antiviral and virucidal efficacy, as well as to determine the inhibitory effect on the release of certain cytokines, of the therapeutic composition based on the mother tincture of Neurolaena lobata .

Preparation of samples to be tested:

Several samples were prepared from leaves of the plant Neurolaena lobata for the conduct of these tests.

The samples are denoted “TOTUM”.

“TOTUM 3” corresponds to a mother tincture obtained from the leaves of Neurolaena lobata ; it was prepared as follows:

i) A mass of 3.6 kg of dry leaves of Neurolaena lobata was mixed with a volume of 225L of sugar cane alcohol at 50°, which corresponds to a mass concentration of 16g of dry leaves of Neurolaena lobata per liter of cane alcohol; more generally, a mixture is prepared having a mass concentration of between 15 and 20 g/L, preferably between 16 and 17 g/L and, more preferably, equal to 16 g of dry leaves of Neurolaena lobata per liter of alcohol sugar cane at 50°;
Preferably, just as for the preparation of sample H1 mentioned above, the leaves can be dried under a stream of hot air, at a temperature preferably below 40° C., for approximately 120 hours, until that they have a residual moisture content of the order of 6 to 7%. The humidity level can be determined in the same way as for H1 as well.

ii) The mixture is left to macerate with stirring for about 21 days, at a temperature of around 30°C, with slow stirring every day for 12 hours;

iii) After maceration, the mixture is filtered through “cartridges” or filter bags with a porosity of 50-75 µm and a filtrate and a retentate are obtained;

iv) sampling of a volume of 3L of filtrate

v) evaporation of the alcohol contained in the latter by rotary evaporator, in three stages, until an aqueous solution with a volume approximately equal to 1L is obtained.

vi) Freezing of the aqueous solution thus obtained, then freeze-drying.

A lyophilisate having a mass equal to 12.3 g is finally obtained.

The "TOTUM 4" is a sample that is diluted from the mother tincture.

More specifically, to obtain this sample, in step iv) of the protocol for obtaining TOTUM 3 below, instead of taking 3L of filtrate, 0.75L of filtrate is taken and diluted in a volume equal to 2.25L of water. A hydroalcoholic solution having a total volume equal to 3L is then obtained. In general, a ¼ dilution of the liquid filtrate, or mother tincture, obtained in step iii) above is carried out.

Then, the following steps for obtaining TOTUM 4 are similar to those which are implemented for obtaining TOTUM 3 and which have been described above, namely:

v) evaporation of the alcohol contained in the hydroalcoholic solution, preferably having a volume equal to 3L, by rotary evaporator, for example in three times, until an aqueous solution is obtained, which may have a volume approximately equal to 1L;

vi) Freezing of the aqueous solution thus obtained, then freeze-drying.

A lyophilisate having a mass equal to 5.6 g and corresponding to a dry extract of a diluted mother tincture (one-quarter) of dry leaves of Neurolaena lobata is finally obtained.

As a negative control, a sample called “TOTUM 2” was prepared from the pulp of dried Musa sapentium bananas.

More specifically, to obtain this sample, 150g of dried banana pulp was diluted in a volume of 5L of sugar cane alcohol at 50°.

The mixture is left to macerate for about 5 days, with stirring for 2 to 3 hours a day, before being filtered on paper with a porosity between 10 and 20 µm, to obtain 4L of hydroalcoholic filtrate.

The filtrate is then evaporated by rotary evaporator, until a dry extract is obtained, with a mass equal to 6g.

Evaluation of the inhibition of the expansion of the SARS-CoV2 virus responsible for Covid 19 during the treatment of human lung epithelial cells (Calu-3) and kidney cells (VeroE6-TMPRSS2) by TOTUM 2, 3 and 4

The SARS-CoV2 virus strain that was used in conducting these tests is the European strain (a mutation of the original Wuhan strain to D614G), which corresponds to the Slovakia/SK reference SARS-CoV-2 strain. -BMC5/2020.

The virus strain was provided by the European Virus Archive goes Global (Evag) platform (https://www.european-virus-archive.com/).

The SARS-Cov2 viral strain was amplified and titrated on the VeroE6 TMPRSS2 cell line by Oncodesign.

Two cell lines were used in these evaluation tests: they are the following lines:

- Calu-3, human lung adenocarcinoma (origin ATCC – American Type Culture Collection);

- Vero E6-TMPRSS2, non-human primate kidney epithelial cells (origin NIBSC – National Institute for Biological Standards and Controls, UK).

The Calu-3 cellular model is already well described in the literature for Sars-CoV (see C.-T. K. Tseng, J. Tseng, L. Perrone, M. Worthy, V. Popov, and C. J. Peters, “Apical entry and release of severe acute respiratory syndrome-associated coronavirus in polarized Calu-3 lung epithelial cells,” J Virol, vol.79, no.15, pp. 9470–9479, Aug. 2005, doi: 10.1128/JVI.79.15.9470- 9479.2005).

Calu-3 cells were cultured in a monolayer at 37°C in a humidified atmosphere (5% CO2, 95% air) in the corresponding cell culture medium (MEM + 1% Pyruvate + 1% glutamine + 10% Fetal Bovine Serum ).

Vero E6-TMPRSS2 cells were cultured in a monolayer at 37°C in a humidified atmosphere (5% CO2, 95% air) in the corresponding cell culture medium (DMEM + 1% pyruvate + 1% cocktail antibiotics (penicillin, streptomycin and geneticin) + 2% fetal bovine serum).

The cells of these two cell lines are adherent to the plastic vials. For cell passage procedures, cells were detached from the culture flask by a 20-minute (for Calu lineage cells) and 5-minute (for Vero lineage cells) treatment with trypsin- versene and neutralized by adding complete culture medium. For the study, the cells were deposited on 96-well plates.

Cells were counted and their viability was assessed using the Vi-cell cell counter.

In a first series of tests, denoted "CAS1", the cells of the two aforementioned cell lines are brought into contact with the compounds to be tested (TOTUM 2, 3 and 4 in particular), for a period of 24 hours, before exposure to the viral strain of SARS-CoV-2. This first “CAS 1” series makes it possible to study the antiviral effect, in other words the cells are treated with the compound before being infected.

In a second series of tests, denoted "CAS 2", the viral strain of SARS-CoV-2 is brought into contact with the various compounds to be tested, including TOTUMs 2, 3 and 4, for a period of 30 min at temperature ambient, before bringing the cells into contact with the virus. This second “CAS 2” series makes it possible to study the virucidal effect, in other words the virus is brought into contact with the compound before being brought into contact with the cells.

Testing protocol for CAS 1 assays with Calu-3 and Vero E6 TMPRSS2 cell lines

Cells were counted and their viability assessed using the Vi-CELL Cell Analyzer.

The cells were seeded to reach confluence:

- Vero E6 TMPRSS2 – 30,000 cells/well;

- Calu-3 – 90,000 cells/well.

From the lyophilisates and dry extracts of TOTUMs 2, 3 and 4, stock solutions are prepared in DMSO at 10mg/mL. From these stock solutions, seven concentrations of compounds to be tested were prepared in a complete growth medium and added to the cells: 10000, 3333, 1111, 370, 123, 41, 14 ng/mL.

The first biological replicate (N=1) was carried out with these concentrations.

The second biological replicate (N=2) was carried out with different concentrations. Indeed, the concentrations tested were adjusted after the analysis of the results of the first biological replicate.

Thus for the N=2 replicate, the following concentrations were used: 100000, 33333, 11111, 3704, 1235, 412 and 137 ng/mL for TOTUM 3 and 20000, 6667, 2222, 741, 247, 82 and 27 ng /mL for TOTUMs 2 and 4.

As a reference control compound, or positive control, the active metabolite of remdesivir was used. Seven concentrations of remdesivir (20000, 6667, 2222, 741, 247, 82, 27 nM), were prepared and added to the cells.

The active remdesivir metabolite was supplied by Oncodesign, as a 20mM stock solution in DMSO.

The plates were incubated for 24 h at 37°C.

Then, a volume of 10 μL of viral preparation equivalent to an MOI (Multiplicity Of Infection)=0.01 was added and incubated at 37° C. for 48 h for the VeroE6 TMPRSS2 cells and 72 h for the Calu-3 cells.

A fraction (50 μL) of the supernatants was collected and then stored at a temperature equal to -20° C. to determine the viral load.

A fraction (~200 μL in three aliquots: 2×50 μL + the remaining volume) of the supernatants was collected and then stored at a temperature equal to -20° C. for the cytokine assay.

Testing protocol for CAS 2 assays with Calu-3 and Vero E6 TMPRSS2 cell lines

Cells were counted and their viability was assessed using the Vi-CELL Cell Analyzer.

The cells were seeded to reach confluence:

- Vero E6 TMPRSS2 – 35,000 cells/well;

- Calu-3 – 80,000 cells/well.

From the lyophilisates and dry extracts of TOTUMs 2, 3 and 4, seven concentrations of compounds to be tested were prepared in a fresh growth medium: 10000, 3333, 1111, 370, 123, 41, 14 ng/mL.

The first biological replicate (N=1) was carried out with these concentrations.

As for CAS 1, the second biological replicate (N=2) was carried out with different concentrations.

Thus for the N=2 replicate, the following concentrations were used: 100000, 33333, 11111, 3704, 1235, 412 and 137 ng/mL for TOTUM 3 and 20000, 6667, 2222, 741, 247, 82 and 27 ng /mL for TOTUMs 2 and 4.

Similarly, seven concentrations of the reference control, or positive control, the active metabolite of remdesivir (20000, 6667, 2222, 741, 247, 82, 27 nM), were prepared.

A volume of 10 μL of viral preparation equivalent to an MOI=0.01 was mixed with the compounds to be tested and incubated at room temperature for 30 min.

The compound/virus mixture was then added to the cells.

Cells were incubated at 37°C for 48 h for VeroE6-TMPRSS2 cells and 72 h for Calu-3 cells.

A fraction (50 μL) of the supernatants was collected and then stored at -20° C. to determine the viral load.

A fraction (~200 μL in three aliquots: 2×50 μL + the remaining volume) of the supernatants was collected then stored at −20° C. for cytokine assay.

Note: a virus-free plate was produced to assess the cytotoxicity of the compounds tested on the two cell types. Cell viability was assessed by the CellTiter Glo assay for all conditions according to the manufacturer's recommendations (Promega, G7570).

For CAS 1 and CAS 2, viral load quantification by RTqPCR, targeting the viral ORF1ab gene, was performed at the end of the experiment.

The extraction of the viral RNA was carried out by the Macherey Nagel Viral RNA kit and the RNA was frozen at -80°C until completion of the RT-qPCR.

Full RT-qPCR was performed using the SuperScript ^TM One-Step qRT-PCR System kit, with qRT-PCR primers and conditions targeting the ORF1ab gene. The amplifications were carried out with a Bio-Rad CFX384 ^TM apparatus and corresponding software.

Still for CAS 1 and CAS 2, the CellTiter-Glo® luminescent cytotoxicity test or cell viability test was performed on both a control plate (without virus) and on the treated and infected plates to evaluate the cytotoxicity of the samples. tested.

The CellTiter-Glo® luminescent cell viability assay is a homogeneous method for determining the number of viable cells in culture based on the quantification of ATP present, an indicator of metabolically active cells.

The method was used for VeroE6-TMPRSS2 and Calu-3 cells in the absence of virus to establish the cytotoxicity of each of the compounds that were tested.

The method was also used for VeroE6-TMPRSS2 cells in the presence of virus 48 hours after infection in the event of cytopathogenic effects (presence of active virus); the presence of virus in the Vero cellular model results in cytopathogenic effects after use of the cellular machinery, whereas the virus is produced continuously in the Calu model.

The test was performed according to the supplier's protocol.

After removing all supernatant for PCR reactions and cytokine assays, add 100µL of fresh cell medium to 100µL of reagent and incubate until luminescence is recorded, at least 15 min after mixing.

For cases 1 and 2, the dosage of cytokines, more particularly of IL 6, MCP1 and IP10 were carried out by ELISA using commercial kits on cell culture supernatants collected 48h and 72h post -infection, respectively for the Vero and Calu-3 cell lines.

Results: evaluation of the cytotoxicity of TOTUMs (without virus)

The toxicity of the samples to be tested “TOTUMs” and of the active metabolite of remdesivir on Calu-3 cells not exposed to the virus was evaluated by measuring cell viability after exposure to the compounds for 96 hours.

The results are set out in the tables below, the cell viability being expressed in % relative to the untreated cells, after exposure to the compounds of the Calu-3 cell line, AND corresponding to the standard deviation:

After treatment with the active metabolite of remdesivir, the results are similar between the biological replicates with an average cell viability ranging from 91% (27 nM, N=1) to 116% (247nM and 20000 nM, N=1).

During the biological replicate N=1, cell viability after treatment with TOTUMS 2, 3 and 4, for the seven concentrations ranging from 14 ng/mL to 10,000 ng/mL, was similar to that obtained after treatment with the active metabolite of remedy.

Similar results were also obtained for TOTUMs 2 and 4 during the N=2 biological replicate (see tables below), for the concentrations tested ranging from 137 ng/mL to 100,000 ng/mL.

However, it should be noted that a decrease in cell viability was observed for TOTUM 3, for a concentration of 100,000 ng/mL.

The toxicity of TOTUMs 2, 3 and 4 and of the active metabolite of remdesivir on VeroE6-TMPRSS2 cells not exposed to the virus was also evaluated by measuring cell viability after exposure to the compounds for 72 hours.

The results are set out in the tables below, the cell viability being expressed in % relative to the untreated cells, after exposure to the compounds of the VeroE6 cell line, AND corresponding to the standard deviation:

After treatment with the active remdesivir metabolite, results are similar between biological replicates with average cell viability ranging from 92% (27 nM, N=1, left table above) to 109% (20,000 nM, N =2, right panel).

At the N=1, cell viability after treatment with TOTUMs 2, 3 and 4, for the seven concentrations ranging from 14 ng/mL to 10,000 ng/mL were similar to those obtained after treatment with the active metabolite of remdesivir.

Similar results were obtained for TOTUMs 2 and 4 during N=2 for the concentrations tested ranging from 137 ng/mL to 100,000 ng/mL.

Furthermore, for TOTUM 3, at concentrations of 33,333 ng/mL and 100,000 ng/mL, cytotoxicity is present.

Results: CASE 1 – Antiviral effect of compounds

These tests aim to assess the antiviral effect of compounds that must be analyzed, in other words, the cells are treated with the compound before being infected.

First, on the Calu-3 cell line, viral load was assessed by quantification of viral RNA targeting the ORF1ab gene. The infected cell control is the 100% reference.

After treatment with the active metabolite of Remdesivir, the results are similar between the biological replicates. The viral load decreases when the compound concentration increases, with:

- In N=1, a percentage compared to infected and untreated cells ranging from 84% for 27 nM of remdesivir to 0% (undetectable viral load) for 2,222 nM, 6,667 nM and 20,000 nM,

- In N=2, a percentage relative to infected and untreated cells ranging from 92% for 55 nM of remdesivir to 0% (undetectable viral load) for 4,444 nM, 13,333 nM and 40,000 nM.

For the compounds tested, at N=1, the viral load ranged from 67% (TOTUM 3 at 370 nM) to 280% (TOTUM 2 at 10,000).

At N=2, results similar to N=1 were observed for TOTUM 2, for all seven concentrations. For TOTUM 4 at 6,667 ng/mL and 20,000 ng/mL a viral load of 59% and 49% was observed.

Furthermore, for TOTUM 3 at 100,000 ng/mL an undetectable viral load (0%) was observed.

An assay of three cytokines (IL6, IP10 and MCP1) was performed on cell culture supernatants (in ng/mL) on the Calu 3 lung cell line inoculated with SARS-CoV-2. The cells were treated with the products tested for 24 hours then inoculated with the viral strain for 72 hours. The compound concentrations are indicated in nM for the active metabolite of remdesivir and in ng/mL for the TOTUMs tested.

IL6 is a pro-inflammatory cytokine, expressed at a basal level of 300pg/mL. In the event of infection, its level is greatly increased by around 2000 pg/mL. The chemokine IP10 is involved in inflammatory processes, undetectable in basal rate. In case of infection, its rate is greatly increased by around 400pg/ml

The MCP1 cytokine could not be detected in the studies performed. Cytokines have transient expression; consequently, at the time when the assay is carried out, the cytokine may already have been expressed or will be expressed later, given that a single point of reading is carried out, respectively at 48h and 72h post infection for the Vero and Calu models, and not a kinetic.

For IL6 (table below on the left for the first replicate N=1 and on the right for the second replicate N=2), a dose-response effect was observed for the active metabolite of remdesivir as expected, with cytokine concentrations decreasing as compound concentrations increased.

For the chemokine IP10 (table below on the left for replicate N=1 and on the right for replicate N=2) a dose-response effect is also observed:

It is particularly interesting to note that similar results, i.e. a dose-response effect, were observed for TOTUMs 3 and 4 for N=1 when assaying IL6.

The same is true for N=2.

A dose-response effect is also remarkable during tests with TOTUMs 3 and 4 for the N=1 for the IP10 assay, for the N=1 replicate:

The same is true for N=2:

No dose-response effect is observed for TOTUM 2 either in the context of the IL6 assay or in the context of the IP10 assay (results not shown).

On the VeroE6-TMPRSS2 cell line, viral load was assessed by quantification of viral RNA targeting the ORF1ab gene.

After treatment with the active metabolite of remdesivir, the results are similar between biological replicates. The viral load decreases when the compound concentration increases, with:

- In N=1, a percentage relative to infected and untreated cells ranging from 101% for 27 nM of remdesivir to 0% (undetectable viral load) for 6,667 nM and 20,000 nM,

- In N=2, a percentage relative to infected and untreated cells ranging from 70% for 27 nM of remdesivir to 0% (undetectable viral load) for 6,667 nM and 20,000 nM:

At N=1, the viral load compared to infected and untreated cells after treatment with TOTUMs 2, 3 and 4, for the seven concentrations ranging from 14 ng/mL to 10,000 ng/mL was similar to or greater than that obtained after treatment with the lowest dose of the active metabolite of remdesivir; antiviral activity does not appear to be diminished.

These results are not repeated here.

During N=2, results similar to N=1 were observed for TOTUM 2 for the seven concentrations ranging from 137 ng/mL to 100,000 ng/mL (no induction of reduction in viral load, therefore no antiviral activity of TOTUM 2).

Moreover :

- After treatment with TOTUM 3 at concentrations of 33,333 ng/mL and 100,000 ng/mL viral loads of 9% and 0%, respectively, were observed, in other words undetectable:

- After treatment with TOTUM 4 at 6667 ng/mL, a viral load of 78% compared to untreated infected cells is observed and, for treatment with a concentration of 20,000 ng/mL, a viral load reduced to 40% has been observed:

TOTUM 3 induces a significant decrease in the viral load, even undetectable, at concentrations of 33,333 ng/mL and 100,000 ng/mL.

TOTUM 4 induces a substantial decrease in viral load at a concentration of 20,000 ng/mL.

Results: CASE 2 – Virucidal effect of compounds

These tests aim to assess the virucidal effect of compounds to be analyzed, in other words the virus was incubated with the compounds for 30 minutes before the cells were cultured with the pretreated inoculums.

First, on the Calu-3 cell line, viral load was assessed by quantification of viral RNA targeting the ORF1ab gene.

After treatment with the active metabolite of remdesivir, the results are similar between biological replicates. The viral load decreases when the compound concentration increases, with:

- At N=1, a percentage relative to infected and untreated cells ranging from 113% for 27 nM of remdesivir to 0% for 6,667 nM and 20,000 nM:

- At N=2, a percentage relative to infected and untreated cells ranging from 109% for 27 nM of remdesivir to 0% for 2222 nM, 6667 nM and 20,000 nM:

At N=1, the viral load compared to infected and untreated cells after treatment with TOTUMs 2, 3 and 4, for the seven concentrations ranging from 14 ng/mL to 10,000 ng/mL were similar or higher than that obtained after treatment with the lowest dose of the active metabolite of remdesivir (results not shown).

At N=2, results similar to N=1 were observed for TOTUM 2 for the seven concentrations ranging from 27 ng/mL to 20,000 ng/mL and from 14 ng/mL to 10,000 ng/mL.

Moreover :
- After treatment with TOTUM 3 at a concentration of 100,000 ng/mL, a viral load of 0% (undetectable viral load) was observed;

After treatment with TOTUM 4 at concentrations of 2,222 ng/mL and 6,667 ng/mL, viral loads of 46% and 43%, respectively, were observed:

A three-cytokine assay was performed for IL6 and IP10 on cell culture supernatant samples.

For IL6, a dose-response effect was observed for the active metabolite of remdesivir with decreasing cytokine concentrations when the compound concentrations increased, for the replicate N=1 (table below on the left) and for the replicate N= 2 (table below right).

In the context of the IL6 assay, similar results were observed for TOTUMs 3 and 4 for N=1:

The same is true for the N=2 replicate:

For IP10 a dose-response effect is also to be noted when the virus is incubated with remdesivir for the N=1 replicate (table below on the left) and for the N=2 replicate (table below on the right) .

Similar results, i.e. a dose-response effect, were observed for TOTUMs 3 and 4 for N=1 during the IP10 assay:

The same is true for the N=2 replicate:

On the VeroE6-TMPRSS2 cell line at present, the viral a viral load was assessed by quantification of the viral RNA by targeting the ORF1ab gene.

After treatment with the active metabolite of remdesivir, the results are similar between biological replicates. The viral load decreases when the compound concentration increases, with:

- In N=1, a percentage compared to uninfected and untreated cells ranging from 97% for 27 nM of remdesivir to 0% and 1% (undetectable viral load) for 6,667 nM and 20,000 nM, respectively:

- In N=2, a percentage compared to uninfected and untreated cells ranging from 100% for 27 nM of remdesivir to 0% (undetectable viral load) for 2,222 nM, 6,667 nM and 20,000 nM:

At N=1, the viral load compared to infected and untreated cells after treatment with TOTUMs 2, 3 and 4, for the seven concentrations ranging from 14 ng/mL to 10,000 ng/mL was similar to or greater than that obtained after treatment with the lowest dose of active metabolite of remdesivir (results not shown, a priori no virucidal effect).

During N=2, results similar to N=1 were observed for TOTUM 2 for the seven concentrations ranging from 27 ng/mL to 20,000 ng/mL (results not shown, no virucidal effect of TOTUM 2) .

Moreover :

- After treatment with TOTUM 3 at a concentration of 33333 ng/mL, a viral load of 16% is observed; at a concentration of 100,000 ng/mL, an undetectable viral load (0%) was observed:

After treatment with TOTUM 4 at a concentration of 20,000 ng/mL, a viral load of 47% was observed:

Findings

The results obtained demonstrate the following:

On the one hand, the compound remdesivir, which was tested in vitro as a reference active metabolite against the SARS-CoV-2 virus, clearly shows antiviral and virucidal activity against said virus, without apparent toxicity on cells. The viral load decreases when the concentration of remdesivir increases for CAS 1 and CAS 2. In addition, for the cytokines IL6 and IP10, a dose-response effect is observed for the active metabolite of remdesivir, with decreasing concentrations of cytokines when the concentrations of said metabolite increase, as expected. These results validate the test protocols used.

However, remdesivir, although having an interesting activity in vitro on the SARS-CoV-2 virus, has in parallel notorious nephrotoxic effects which can prove harmful for a patient affected by Covid.

The sample referenced TOTUM 2 was obtained from dried banana pulp. The results obtained during the tests carried out demonstrate that such an extract has no antiviral or virucidal effect. Furthermore, no dose-response effect on cytokine release could be observed for this sample.

Here again, these test results, expected to be negative, confirm the protocol that has been implemented.

As regards the sample named TOTUM 3, this is obtained from a concentrated Neurolaena lobata mother tincture, while the sample referenced under the name TOTUM 4 corresponds to a dilution of the mother tincture which has also made it possible to obtain said TOTUM 3, as emerges from the detailed description of the protocol for obtaining these TOTUMs 3 and 4 described above.

The results obtained with the TOTUM 3 and detailed above demonstrate, on the one hand, that for compound concentrations ranging from 14 to 10,000 ng/mL, no toxicity is to be noted on Calu-3 and VeroE6 cells.

On the other hand, cytotoxicity is to be noted for a compound concentration of 100,000 ng/mL on the cells of the Calu-3 line. The same is true for the compound concentrations of 33,333 ng/mL and 100,000 ng/mL on the cells of the VeroE6 line, where cytotoxicity is present.

In parallel, for TOTUM 3, at a concentration of 100,000 ng/mL, an undetectable viral load is observed for the Calu-3 cell line. However, this concentration has been shown to exhibit cytotoxic effects. For the VeroE6 cell line, undetectable viral loads were observed for compound concentrations of 33,333 ng/mL and 100,000 ng/mL. However, here again cytotoxicity is present.

It seems that the concentration of active compound in this sample is too high and leads to cytotoxicity on the cells.

The results obtained with TOTUM 4, consisting of a quarter dilution of the mother tincture, compared to the TOTUM 3 mentioned above, are particularly interesting.

For the record, in the sample preparation process, following the step of filtration of the mother tincture, in step iv) of the protocol for obtaining TOTUM 3, 0.75L of filtrate is taken and diluted in a volume equal to 2.25L of water. A hydroalcoholic solution is then obtained having a total volume equal to 3L and corresponding to a ¼ dilution of the mother tincture of dry leaves of Neurolaena lobata .

The results obtained with this TOTUM 4 demonstrate, on the one hand, that it does not present any cytotoxicity on the model cell lines, whatever the concentration tested, even for the highest of them.

In parallel with this absence of cytotoxicity, an antiviral activity of TOTUM 4 is demonstrated for the concentrations 6,667 ng/mL and 20,000 ng/mL, for which the viral loads observed are respectively 59 and 49% for the Calu- 3. Antiviral activity was also detected for the VeroE6 line, after treatment with TOTUM 4 at 6667, at 10,000 ng/mL and at 20,000 ng/mL, for which viral loads of 78%, 66% and 40% were respectively observed.

It should also be noted that, in a particularly interesting way, during the assay of the cytokines IL6 and IP10 which were carried out for CAS 1 and for CAS 2, a dose-response effect is observed for TOTUM 4, with decreasing cytokine concentrations when compound concentrations increase.

As a result of the above results, and in particular those relating to the assay of the cytokines IL6 and IP10 in the lung cells, it is possible to affirm that a diluted mother tincture extract, in particular according to a ¼ dilution of the mother tincture, and freeze-dried leaves of Neurolaena lobata , in liquid solution having a concentration of between 6,500 and 20,000 ng/mL (mass of freeze-dried extract of diluted mother tincture/volume of aqueous solution), preferably between 6,667 and 20,000 ng/ mL, has antiviral activity and virucidal activity against the SARS-CoV-2 virus responsible for Covid 19, and is effective in the fight against severe forms of this disease.

Indeed, the results demonstrating a dose-dependent effect of TOTUM 4 on the release of the cytokines IL-6 and IP10, such an extract of the mother tincture of Neurolaena lobata is particularly indicated with a view to avoiding the cytokine storm likely to occur. produce, in particular, in the lungs of patients suffering from a severe form of Covid, in reaction to infection by the virus.

Here we mean by “patient with a severe form of Covid-19”, a patient hospitalized to fight against Covid-19 and placed on oxygen therapy.

Claims (8)

Therapeutic composition comprising a mother tincture of an extract of the leaves of a plant belonging to the genus " Neurolaena " and the species " lobata " for use as a medicament inhibiting human dihydroorotate dehydrogenase (DHODH) in the treatment of a viral infection of a virus with an RNA genome, characterized in that the said virus with an RNA genome is chosen from the viruses of the following list:
- viruses of the Coronaviridae family,
- the virus responsible for hepatitis C, the virus responsible for yellow fever or the Zika virus in the Flaviviridae family,
- the virus responsible for chikungunya in the Togaviridae family. Therapeutic composition according to Claim 1, characterized in that the said virus with an RNA genome is the SARS-CoV-2 virus responsible for Covid-19. Process for preparing a dry extract of a diluted mother tincture of dry leaves of Neurolaena lobata , characterized in that it comprises the following steps:
i) preparing a mixture based on dry leaves of Neurolaena lobata in sugar cane alcohol at 50°, said mixture having a mass concentration of between 15 and 20 g/L;
ii) said mixture is left to macerate with stirring for approximately 21 days;
iii) After maceration, the mixture is filtered through “cartridges” with a porosity of 50-75 μm and a liquid filtrate is obtained, corresponding to a mother tincture of dry leaves of Neurolaena lobata , and a solid retentate;
iv) said liquid mother tincture is diluted to ¼ by adding an aqueous solution and a hydroalcoholic solution is then obtained;
v) the alcohol contained in this solution is evaporated by rotary evaporator, until an aqueous solution is obtained;
vi) the aqueous solution thus obtained is frozen, then it is freeze-dried to obtain a dry extract of the diluted mother tincture of dry leaves of Neurolaena lobata . Process for the preparation of a dry extract from a diluted mother tincture of dry leaves of Neurolaena lobata according to the preceding claim, characterized in that the mixture prepared in step i) has a mass concentration of between 16 and 17 g of dry leaves of Neurolaena lobata per liter (L) of sugar cane alcohol at 50°, and preferably equal to 16 g/L. Process for the preparation of a dry extract from a diluted mother tincture of dry leaves of Neurolaena lobata according to Claim 3 or Claim 4, characterized in that, during stage iv), the said mother tincture is diluted in mixing a volume equal to 0.75L of filtrate and a volume equal to 2.25L of water. Process for the preparation of a liquid solution of a dry extract of diluted and freeze-dried mother tincture of dry leaves of Neurolaena lobata , obtained according to the process of any one of Claims 3 to 5, characterized in that the said liquid solution is obtained by diluting said dry extract in water or in an aqueous solvent such as dimethyl sulfoxide (DMSO), said liquid solution having a concentration of between 6,500 and 20,000 ng of dry extract per mL of aqueous solvent, preferably between 6 667 and 20,000 ng/mL. Extract of mother tincture of dry leaves of Neurolaena lobata diluted, in particular according to a ¼ dilution of the mother tincture, and freeze-dried, said extract being in liquid solution and having a final concentration of between 6,500 and 20,000 ng/mL (mass of 'freeze-dried dry extract of diluted mother tincture / volume of aqueous solution), for its use in the treatment of a viral infection due to the SARS-CoV-2 virus responsible for Covid 19. Extract of mother tincture of leaves of Neurolaena lobata according to the preceding claim for its use in reducing the production of cytokines, in particular IL-6 and IP-10, in the treatment of severe forms of a viral infection due to the SARS- CoV-2 responsible for Covid 19.