A composition

Abstract

There is disclosed a pharmaceutical composition comprising: a bisabolol compound, valencene and a terpinene compound.

Description

[0001] A composition

[0002] The present invention relates to a composition that has antiviral, antibacterial, anti- carcinogenic and anti-tumour properties.

[0003] Background Bacterial, fungal and viral infections are common in both humans and animals throughout the world. In recent years antibiotic and antifungal resistance have become more common and are an increasing risk to global health. Many of the standard antibiotics and antifungal drugs are no longer effective against certain antibiotic resistant bacteria and antifungal resistant fungi. Thus, there is a growing need for new drugs which can treat bacterial and fungal infections. During 2020, a novel coronavirus (SARS-CoV-2) spread rapidly through the world causing a global pandemic. Treating viral infections can be particularly challenging and antiviral drugs often have a limited effect, particularly for emerging novel viruses such as SARS-CoV-2. Thus, there is a global need for new antiviral drugs to help fight viral infections of all kinds in both humans and other animals, and particularly highly contagious and pathogenic viruses such as SARS-CoV-2.

[0004] Cancer is a common disease which in 2015 caused 8.8 million deaths worldwide1. There is an ongoing need for new drugs which can both help to prevent cancer occurring in the first place and also treat cancer if it forms.

[0005] In a first aspect of the present invention there is provided a composition comprising a bisabolol compound, valencene and a terpinene compound.

[0006] Bisabolol compounds are sesquiterpenes that include alpha-bisabolol (formula 1) and beta- bisabolol (Formula 2) .

[0007] Valencene is a sesquiterpene that has the following formula:

[0008] Formula 3 Terpinene compounds are a group of isomeric monoterpene compounds that include alpha-terpinene (formula 4), beta-terpinene (formula 5), gamma-terpenine (formula 6) and delta-terpenine (formula 7).

[0009] Formula 4 Formula 5 Formula 6 Formula 7 The applicant has found that compositions which include a bisabolol compound, valencene and a terpinene compound have a range of properties and physiological effects, including anti-bacterial activity, anti-viral activity and anti-fungal activity. Additionally, the compositions of the invention have been found to have anti-carcinogenic properties and anti-tumour properties.

[0010] The amount of the bisabolol compound may be from about 10% to about 40% by weight, for example from about 15% to about 35% by weight, for example from about 20% to about 30% by weight, for example from about 22% to about 28% by weight, for example from about 23% to about 27% by weight, for example about 25% by weight of the total amount of the bisabolol compound, valencene and the terpinene compound; or for example from about 20% to about 35% by weight, for example from about 25% to about 35% by weight, for example from about 27% to about 33% by weight, for example from about 28% to about 32% by weight, for example from about 29% to about 31% by weight.

[0011] The amount of valencene may be from about 22% to about 53% by weight, for example from about 27% to about 48% by weight, for example from about 32% to about 43% by weight, for example from about 35% to about 41% by weight, for example from about 36% to about 39% by weight, for example about 37.5% by weight of the total amount of the bisabolol compound, valencene and the terpinene compound. Alternatively, the amount of valencene may be from about 20% to about 35% by weight, for example from about 25% to about 35% by weight, for example from about 27% to about 33% by weight, for example from about 28% to about 32% by weight, for example from about 29% to about 31% by weight of the total amount of the bisabolol compound, valencene and the terpinene compound.

[0012] The amount of the terpinene compound may be from about 22% to about 53% by weight, for example from about 27% to about 48% by weight, for example from about 32% to about 43% by weight, for example from about 35% to about 41% by weight, for example from about 36% to about 39% by weight, for example from about 36% to about 38% by weight, for example about 37.5% by weight of the total amount of the bisabolol compound, valencene and the terpinene compound.

[0013] The composition may comprise each of the bisabolol compound, valencene and the terpinene compound in any of the amounts set out above.

[0014] The bisabolol compound may be alpha-bisabolol and / or beta-bisabolol. Preferably, the bisabolol compound is alpha-bisabolol. In the case that the bisabolol compound is alpha-bisabolol, the alpha-bisabolol may be in the form of a-(-)-bisabolol as shown in formula 1a or the alpha-bisabolol may be in the form of the enantiomer a-(+)-bisabolol, or another enantiomeric form. The bisabolol may be a racemic mixture of both alpha-bisabolol enantiomers or other enantiomers thereof.

[0015] Formula 1a

[0016] In the case that the bisabolol compound is beta-bisabolol, the beta-bisabolol may be in the form of an enantiomer as shown in formula 2a, or an enantiomer thereof, or a racemic mixture of enantiomeric forms.

[0017] Formula 2a

[0018] The valencene may be in the form of the enantiomer shown in formula 3a or it may be in the form of other enantiomers thereof or a racemic mixture of enantiomeric forms. Formula 3a The terpinene compound may be one or more of alpha-terpinene, beta-terpinene, gamma- terpenine or delta-terpenine. Preferably, the terpinene compound is alpha-terpinine, beta- terpinene or gamma-terpinene, for example gamma-terpinene.

[0019] Preferably, the composition comprises: alpha-bisabolol in an amount from about 10% to about 40% by weight, for example from about 15% to about 35% by weight, for example from about 20% to 35% by weight, for example from about 25% to about 35% by weight, for example from about 27% to about 33% by weight, for example from about 28% to about 32% by weight, for example from about 29% to about 31% by weight of the total amount of the alpha-bisabolol, valencene and the terpinene compound; valencene in an amount from about 22% to about 53% by weight, for example about 20% to about 35% by weight, for example from about 25% to about 35% by weight, for example from about 27% to about 33% by weight, for example from about 28% to about 32% by weight, for example from about 29% to about 31% by weight of the total amount of the alpha-bisabolol, valencene and the terpinene compound; a terpinene compound in an amount from about 22% to about 53% by weight, for example from about 27% to about 48% by weight, for example from about 32% to about 43% by weight, for example from about 35% to about 41% by weight, for example from about 36% to about 39% by weight, for example from about 36% to about 38% by weight of the total amount of the alpha-bisabolol, valencene and the terpinene compound, wherein the terpinene compound is alpha terpinene, beta-terpinene or gamma terpinene, for example gamma-terpinene.

[0020] The total amount of the bisabolol compound, valencene and the terpinene compound may be from about 0.5% to about 6% by weight, for example 0.5% to about 5% by weight, for example from 0.5% by weight to 2% by weight, for example 1 % by weight of the composition.

[0021] The composition may further comprise a carrier. The carrier may function to aid the administration of the composition and may also function to stabilise or preserve the active ingredients. Preferably, the carrier comprises a carrier oil, for example one or more of gardenia oil, cherry oil or pine oil. Preferably, the carrier oil comprises gardenia oil, cherry oil and pine oil; or olive oil.

[0022] The carrier oil may further comprise olive oil. The composition may further comprise vitamin E. Herein vitamin E means one or more of the following compounds: alpha-tocopherol, beta-tocopherol, gamma-tocopherol, delta- tocopherol, alpha-tocotrienol, beta-tocotrienol, gamma-tocotrienol or delta-tocotrienol.

[0023] The amount of olive oil may be from about 50% to about 90% by weight, for example from about 60% to about 80% by weight, for example about 70% by weight of the total amount of olive oil, gardenia oil, cherry oil and pine oil. The amount of gardenia oil, cherry oil and pine oil may each be from about 5% to about 15%, for example from about 7% to about 13% by weight, for example about 10% by weight of the total amount of olive oil, gardenia oil, cherry oil and pine oil.

[0024] The total amount of olive oil, gardenia oil, cherry oil and pine oil may be from about 94% to about 98.5% by weight, for example from about 97% to about 98.5% by weight, for example about 98% by weight of the composition.

[0025] The amount of vitamin E may be about from 0.5% to about 5% by weight, for example from about 1% to about 5% by weight, for example from about 0.5% to about 2% by weight, for example about 1% by weight of the composition.

[0026] The composition may comprise alpha-bisabolol, valencene, alpha-terpinene, olive oil, gardenia oil, cherry oil, pine oil and vitamin E; or the composition may comprise alpha- bisabolol, valencene, beta-terpinene, olive oil, gardenia oil, cherry oil, pine oil and vitamin E; or the composition may comprise alpha-bisabolol, valencene, gamma-terpinene, olive oil, gardenia oil, cherry oil, pine oil and vitamin E; or the composition may comprise alpha- bisabolol, valencene, gamma-terpinene and olive oil. The amounts of alpha-bisabolol valencene, terpinene compound, olive oil, gardenia oil, cherry oil, pine oil and vitamin E in each of these compositions may be as set out above.

[0027] In a further aspect of the present invention there is provided a method for making the compositions as set out above, the method comprising mixing together each component in a suitable vessel.

[0028] The method may comprise a first step of mixing the bisabolol compound, valencene and the terpinene compound; a second step of forming the carrier; and a third step of mixing the mixture formed in the first step with the carrier.

[0029] The carrier may be formed by mixing the components making up the carrier in a vessel. For example, one or more of gardenia oil, cherry oil or pine oil may be mixed in a vessel to form a carrier oil. Olive oil may also be included and mixed with the other oils. Vitamin E may also be added to the carrier oil followed by mixing.

[0030] Preferably, the composition is a pharmaceutical composition, for example a veterinary pharmaceutical composition or a human pharmaceutical composition.

[0031] Preferably, the composition (e.g. pharmaceutical composition) is in the form of an oil.

[0032] The pharmaceutical composition may be in a form suitable for one or more of oral, topical, rectal, parenteral, transdermal, intravenous, intra-arterial, intraosseous infusion, intracerebral, intracerebroventricular, intrathecal, intramuscular, subcutaneous, intravaginal, intraperitoneal, epidural, intracerebral, intraosseous infusion, intravitreal, transmucosal, buccal, or nasal administration. The pharmaceutical composition may be in a form suitable for oral, topical or intravenous administration. Preferably, the pharmaceutical composition is in a form suitable for oral administration.

[0033] The pharmaceutical composition may further comprise one or more of a non-aqueous pharmaceutical carrier, an aqueous pharmaceutical carrier, a diluent, solvent, vehicle or a non-toxic excipient, including salts and preservatives, buffers and the like.

[0034] Examples of suitable aqueous and non-aqueous pharmaceutical carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils, and injectable organic esters such as ethyl oleate.

[0035] Examples of pharmaceutically acceptable excipients include antiadherents, colourings, flavourings, glidants, lubricants, preservatives, sorbents and sweeteners.

[0036] The pharmaceutical compositions of the present invention may also contain additives such as but not limited to preservatives, wetting agents, emulsifying agents, surfactants and dispersing agents. Antibacterial and antifungal agents can be included to prevent growth of microbes and includes, for example, m-cresol, benzyl alcohol, paraben, chlorobutanol, phenol, sorbic acid, and the like. If a preservative is included, the preservative may be benzyl alcohol, phenol and / or m-cresol. Furthermore, it may be desirable to include isotonic agents such as sugars, sodium chloride, and the like.

[0037] A pharmaceutical composition suitable for oral administration may be in the form of, for example, a capsule, a liquid, a gel, an emulsion, a syrup, and the like. In a further aspect of the present invention, there is provided a composition according to any statement set out above for use as a medicament.

[0038] In a further aspect of the present invention, there is provided a composition according to any statement set out above for use in treating viral infections. The viral infection may be caused by a coronavirus or infectious bronchitis virus. The coronavirus may be a severe acute respiratory syndrome-related coronavirus, for example SARS-CoV-2.

[0039] In a further aspect of the present invention there is provided the composition according to any statement set out above for use in treating a bacterial infection. The bacteria responsible for the bacterial infection may be selected from one or more of Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Salmonella enteric serovar Typhimurium, Pseudomonas aeruginosa, or Micrococcus luteus.

[0040] In a further aspect of the present invention, there is provided the composition according to any statement set out above for use in treating a fungal infection. The fungi responsible for the fungal infection may be a yeast, for example Candida albicans.

[0041] In a further aspect of the present invention there is provided the composition according to any statement set out above for use in preventing or treating cancer. The composition for use may be for preventing the formation of tumours, for example the composition for use may act as an anti-carcinogen. The composition for use may be for treating cancer, for example by reducing the mass of a tumour.

[0042] The composition for use as set out according to any statement above may be administered orally, topically or intravenously. Preferably, the composition for use may be administered orally.

[0043] The composition may be administered in a dose of 1 to 2 ml, 2 to 3 times per day.

[0044] In a further aspect of the present invention, there is provided a method of treatment of a viral infection, a bacterial infection, a fungal infection and / or a method of preventing or treating cancer, comprising a step of administering to a subject a composition as set out above.

[0045] The viral infection may be caused by a coronavirus or infectious bronchitis virus. The coronavirus may be a severe acute respiratory syndrome-related coronavirus, for example SARS-CoV-2. The bacteria responsible for the bacterial infection may be selected from one or more of Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Salmonella enteric serovar Typhimurium, Pseudomonas aeruginosa, or Micrococcus luteus. The fungi responsible for the fungal infection may be a yeast, for example Candida albicans. The method may be for preventing the formation of tumours, for example the composition may act as an anti-carcinogen. The method may be for treating cancer, for example by reducing the mass of a tumour.

[0046] The composition may be administered orally, topically or intravenously. Preferably, the composition may be administered orally.

[0047] The composition may be administered in a dose of 1 to 2 ml, 2 to 3 times per day.

[0048] In a further aspect of the present invention, there is provided the use of a composition as set out above in the manufacture of a medicament for treating a viral infection, a bacterial infection, a fungal infection and or preventing or treating cancer.

[0049] The viral infection may be caused by a coronavirus or infectious bronchitis virus. The coronavirus may be a severe acute respiratory syndrome-related coronavirus, for example SARS-CoV-2. The bacteria responsible for the bacterial infection may be selected from one or more of Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Salmonella enteric serovar Typhimurium, Pseudomonas aeruginosa, or Micrococcus luteus. The fungi responsible for the fungal infection may be a yeast, for example Candida albicans. The method may be for preventing the formation of tumours, for example the composition may act as an anti-carcinogen. The method may be for treating cancer, for example by reducing the mass of a tumour.

[0050] The composition may be administered orally, topically or intravenously. Preferably, the composition may be administered orally.

[0051] The composition may be administered in a dose of 1 to 2 ml, 2 to 3 times per day.

[0052] Herein the expression one or more of a, b or c includes the possibility of a, b or c alone; a and b; a and c; b and c; or a, b and c.

[0053] The present invention will now be described with reference to the following examples and the attached drawings, figures 1 to 6, in which

[0054] Figure 1 shows the chicken embryos for groups 1 to 5 following harvest as described in example 1; Figure 2 shows the chicken embryos of the positive and negative control groups following harvest, as described in example 1 ;

[0055] Figure 3 shows the results of testing a composition according to the invention in a nanoluciferase SARS-CoV-2 reporter assay as described in Example 5;

[0056] Figure 4 shows the results of a SARS-CoV-2 Plaque Reduction Assay, and particularly cytotoxicity of a composition according to the invention after 28 hours of incubation with Vero E6 cells, as described in Example 6;

[0057] Figure 5 shows the results of a SARS-CoV-2 Plaque Reduction Assay, and particularly the antiviral effect of a composition of the invention 28 hours after infection, as described in Example 6;

[0058] Figure 6 shows the results of a SARS-CoV-2 Plaque Reduction Assay, and specifically the effect of a composition of the invention at reducing viral plaques, as described in Example 6.

[0059] Examples

[0060] Composition 1 alpha-bisabolol, valencene, gamma-terpinene, olive oil, gardenia oil, cherry oil, pine oil, vitamin E

[0061] The amount of alpha-bisabolol was 25% by weight of the total amount of alpha bisabolol, valencene and gamma-terpinene. The amount of valencene was 37.5% by weight of the total amount of alpha bisabolol, valencene and gamma-terpinene. The amount of gamma- terpinene was 37.5% by weight of the total amount of alpha bisabolol, valencene and gamma-terpinene. The total amount of alpha-bisabolol, valencene and gamma-terpinene was 1% by weight of the total composition. The amount of vitamin E was 1 % by weight of the total composition, the amount of olive oil was 68.6% by weight of the total composition, the amount of gardenia oil was 9.8% by weight of the total composition, the amount of cherry oil was 9.8% by weight of the total composition and the amount of pine oil was 9.8% by weight of the total composition.

[0062] Composition 2 alpha-bisabolol, valencene, alpha-terpinene, olive oil, gardenia oil, cherry oil, pine oil, vitamin E

[0063] The amount of alpha-bisabolol was 25% by weight of the total amount of alpha bisabolol, valencene and alpha-terpinene. The amount of valencene was 37.5% by weight of the total amount of alpha bisabolol, valencene and alpha-terpinene. The amount of alpha-terpinene was 37.5% by weight of the total amount of alpha bisabolol, valencene and alpha- terpinene.

[0064] The total amount of alpha-bisabolol, valencene and alpha-terpinene was 1% by weight of the total composition. The amount of vitamin E was 1% by weight of the total composition, the amount of olive oil was 68.6% by weight of the total composition, the amount of gardenia oil was 9.8% by weight of the total composition, the amount of cherry oil was 9.8% by weight of the total composition and the amount of pine oil was 9.8% by weight of the total composition.

[0065] Composition 3

[0066] Alpha-bisabolol, valencene, beta-terpinene, olive oil, gardenia oil, cherry oil, pine oil, vitamin E

[0067] The amount of alpha-bisabolol was 25% by weight of the total amount of alpha bisabolol, valencene and beta-terpinene. The amount of valencene was 37.5% by weight of the total amount of alpha bisabolol, valencene and beta-terpinene. The amount of beta-terpinene was 37.5% by weight of the total amount of alpha-bisabolol, valencene and beta-terpinene.

[0068] The total amount of alpha-bisabolol, valencene and alpha-terpinene was 1% by weight of the total composition. The amount of vitamin E was 1% by weight of the total composition, the amount of olive oil was 68.6% by weight of the total composition, the amount of gardenia oil was 9.8% by weight of the total composition, the amount of cherry oil was 9.8% by weight of the total composition and the amount of pine oil was 9.8% by weight of the total composition.

[0069] Composition 4

[0070] Alpha bisabolol Valencene Gamma terpinene Olive oil

[0071] The amount of alpha bisabolol was 1.6% by weight of the total composition; the amount of valencene was 1.6% by weight of the total composition; the amount of gamma terpinene was 1.9% by weight of the total composition; and the amount of olive oil was 94.9% by weight of the total composition.

[0072] The ratio of alpha bisabolol to valencene to gamma terpinene in composition 4 is 1:1:1.1875.

[0073] Composition 5

[0074] Alpha bisabolol Valencene Gamma terpinene Composition 5 is the same as composition 4, but does not include olive oil. Accordingly, the amounts of alpha bisabolol, valencene and gamma terpinene relative to each other are the same as for composition 4. The ratio of alpha bisabolol to valencene to gamma terpinene in composition 4 is 1:1:1.1875.

[0075] The components that make up each of compositions 1 to 5 were obtained from a commercial supplier and the terpene components were mixed in a flask to obtain a homogeneous terpene mixture. In a separate flask the oil components and vitamin E were mixed to form the carrier. Finally, the terpene mixture was mixed with the carrier to form the compositions. Compositions 1 to 5 were tested in a variety of studies which are detailed in the following examples.

[0076] Example 1 Antiviral effect against poultry corona virus (infectious bronchitis virus)

[0077] Reagent and materials

[0078] Composition 1 Saline solution 0.85%

[0079] Allantoic fluid of IB H120 Virus

[0080] Methods

[0081] The trial was conducted on 5 groups of embryonated eggs.

[0082] Table 1a

[0083] Table 1b

[0084] Inactivation

[0085] The diluted Composition 1 solution was mixed using a vortex for ± 30 seconds. The oil solution was transferred to Erlenmeyer flask containing the antigen. The specimen were incubated in a 37°C shaker incubator for 16-18 hours.

[0086] Inoculation

[0087] The 5 treatment groups, the positive control group and the negative control group each included 10 day embryonated eggs. The 5 treatment groups were inoculated with 0.2 ml of the mixture prepared as set out above in table 1a. The negative control group was not inoculated. The positive control group was inoculated with 0.2 ml IBV H120 virus. The eggs were incubated for 6 days at 36.5°C incubator under daily observation. Eggs with dead embryos within 24 hours post-inoculation were discarded as non-specific. At the end of the observation period, all eggs were chilled in a refrigerator and harvested.

[0088] Results

[0089] Candling observation using a bright light to observe the growth and development of the embryos indicated that all embryos in the five treatment groups were alive until day 6 of observation. All embryos in the positive control group were dead, and all embryos in the negative control group were alive.

[0090] The eggs were harvested by removing the portion of the shell above the air cell using sterile forceps. The embryo was collected and placed into a petri dish for observation. The results are shown in figures 1 and 2.

[0091] The positive control group embryos were dead and stunted. The negative control group embryos had a normal appearance. The embryos in the five treatment groups resembled those of the negative control group, so indicating that Composition 1 prevented the infectious bronchitis virus infection.

[0092] Genetic analysis of the genome sequence of infectious bronchitis virus indicates that this virus shares high genetic similarity with the human corona virus SARS-CoV-2, which was responsible for the global Covid-19 pandemic in 2019 and 2020. Accordingly, the results from example 1 indicate that the compositions of the present invention could be used in the treatment of infection caused by SARS-CoV-2.

[0093] Example 2

[0094] Antimicrobial activity

[0095] The following microorganisms were purchased from the American Type Culture Collection and were used as test strains: Staphylococcus aureus ATCC-6538, Staphylococcus epidermidis ATCC-12228, Escherichia coli ATCC-8739, Salmonella enteric serovar Typhimurium ATCC-14028, Pseudomonas aeruginosa ATCC-9027, Micrococcus luteus ATCC-10240, yeasts Candida albicans ATCC-10231, and Candida albicans NCTC-885- 653.

[0096] Screening of Compositions 1, 2 and 3 for antibacterial and antifungal activity was carried out by the disk diffusion method. Mueller-Hinton agar plates and Cetrimide agar plates (for Pseudomonas aeruginosa ATCC-9027) were evenly seeded throughout the plate with each of overnight cultures strains at a concentration 1x108cfu / ml and 1x106cfu / ml for Candida albicans ATCC-10231 and Candida albicans NCTC-885-653 respectively. Sterilized standard paper discs (6 mm diameter) were dipped into the test solutions including 10mI of each undiluted and 1:10 and 1:100 warm dH20 diluted compositions, and placed on the already inoculated culture plates. A standard disc containing 30 pg tetracycline and 40 pg fluconazole for bacterial cultures and Candida albicans respectively were used as reference controls. Tween-80-35%, tween-80-35% 1:10, and dH20 were used as negative controls.

[0097] Antibacterial / antifungal activities of the Compositions 1, 2 and 3 against the test strains were determined after incubation of the test plates for 24 h at 35°C by measuring the diameter of the zone of growth inhibition (IZ). Antimicrobial activity screening was conducted in triplicates and results were presented as an average mean ± SD.

[0098] A micro dilution susceptibility assay was performed to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The standard 96 well micro-titer plate method was used. The plates were prepared by dispensing 100 pi of TSB (Tryptic Soy Broth) and 20 pi of the inoculums into each well. Each well had a final volume of 120 pi. The plates were incubated at 37 ± 1 °C, for 24 hours for bacteria and at 34 ± 1 °C, 48 hours for yeasts. The inhibition of growth of bacteria was revealed by the addition of resazurin sterile solution (20 pi, 0.02 %, w / v) and re-incubation for 3 hours. MIC was determined by the permanence of blue coloration in the wells. A change of colour from blue to red indicated the bacterial growth. The MIC was calculated as the highest dilution showing complete inhibition of the tested strains. The wells that showed no apparent growth were selected to evaluate the MBC, which was determined by the absence of microbial growth on plates containing TSB. The tests were carried out in duplicates.

[0099] Degree of inhibition

[0100] Table 2: Antibacterial activity of Compositions 1, 2 and 3 against bacterial and yeast strains expressed by inhibition diameter (mm ± SD).

[0101] The degree of inhibition was determined by the values of inhibition zone (IZ) diameter of Compositions 1 , 2 and 3 (Table 2). Compositions 1 , 2 and 3 showed a good (+) inhibitory activity against the tested bacterial and fungal strains. MIC determination by microdilution in broth

[0102] The antibacterial potency of Composition 1 against a panel of pathogenic microorganisms was evaluated by the measurement of minimum inhibitory concentration (MIC). The results are presented in Table 3. Composition 1 elicited an antibacterial and antifungal activity. Particularly, Candida albicans strains exhibited a higher sensitivity to the tested composition.

[0103] Table 3: Activity of Composition 1 against bacterial and yeast strains expressed by MIC. Example 3

[0104] Anti-carcinogenesis activity Materials and Methods

[0105] Testing conditions: Route of administration: oral.

[0106] Solvent: Sodium chloride, solution for injection 9 mg / ml.

[0107] Carcinogen: 7,12-Dimethylbenz(a)anthracene DMBA (SIGMA 254-1G 055K1360).

[0108] Tumour models: Subcutaneous fibrosarcoma that occurs after a single subcutaneous injection of DMBA. Animals: Wistar rats.

[0109] Justification of the choice of animals: rats were selected as test models, taking into account the requirements of chemotherapeutic studies.

[0110] Number of animals: 50 rats.

[0111] 5 Body weight of animals: males -140-155g;

[0112] The animals were split into five groups and information on each group is set out in table 4. 0

[0113] To study the effect of Composition 1 on carcinogenesis, induction of subcutaneous tumours was carried out using 7,12-Dimethylbenz(a)anthracene (DMBA). DMBA dissolved in distilled olive oil was injected subcutaneously into the iliac region of the rats at a dose of 15 mg / kg in a volume of 0.2-0.3 ml. The dose of carcinogen provides almost 95-100% yield of tumours 5 at the injection site.

[0114] The investigated preparation, without dilution, was administered orally to animals at a dose of 6 ml / kg. The choice of the tested dose of Composition 1 was carried out on the basis of its acute toxicity data, and also took into account the experience of world practice, confirming 0 the “dose-effect” relationship under the action of a carcinogen / anticarcinogen. Individual doses for each animal were calculated based on their weight, followed by recalculation of doses per kilogram of body weight. For haematological examinations, blood was collected from rats from the tail vein. Each experimental group consisted of 10 healthy sexually mature rats. The control group, with the above quantities of intact animals, received an equivalent 5 volume of solvent. The studies were carried out in accordance with generally accepted regulatory documents: [Guidelines for conducting preclinical studies of drugs, A. Mironov, N. D. Bunatyan. et al 2012; Nonclinical Evaluation for Anticancer Pharmaceuticals, ICH Harmonized Tripartite Guideline S9, Current Step 4 version dated 29 October 2009.]. 0 Clinical observation during the adaptation period.

[0115] During the adaptation period and prior to the introduction of Composition 1 , the animals were under close observation. The observation included monitoring and record of changes in the animals’ skin and hair side, eyes and mucous tunics, mobility and general behaviour. As a 5 criterion of acceptance of randomization, an absence of external signs of diseases and homogeneity of groups by body weight (± 10%) were considered. Determination of weighing points

[0116] The determination of body mass, internal organs of animals and tumours was carried out on scales such as “Centaurus Scale TM 500g / 0.01g” and “BT-500”. Quantitative determination of blood corpuscles was carried out on Picoscale blood corpuscle counter, manufactured by MEDICOR Budapest.

[0117] Administration of the preparation

[0118] Oral administration of the preparation was carried out using syringes with appropriate atraumatic metal gastric probe (tubes).

[0119] Observations of animals

[0120] During the course of the experiment, the general condition of the animals, the features of their behaviour, the intensity and nature of the motor activity, the condition of the hair, skin and mucous membranes, the intake of food and water, changes in body weight, etc., were regularly recorded. After completion of the treatment period, the animals were sacrificed by deep anaesthesia with stabilized ether and subjected to macroscopic examination.

[0121] Statistical methods

[0122] The obtained results were statistically processed according to the Student-Fisher’s method (using a specialized program for biomedical purposes GraphPad Prism for Windows v. 5).

[0123] Two schemes of chemoprophylactic effects were chosen: on the initiation phase and postinitiation of carcinogenesis. In the first scheme, Composition 1 was administered to rats for 17 days - 10 days before and 7 days after a single injection of DMBA. In the second scheme, Composition 1 was administered to rats starting from day 7 after the introduction of the carcinogen, and then throughout the experiment (150 days) until the rats were sacrificed.

[0124] Starting from the 20th day after exposure to the carcinogen, daily palpations of rats were performed in the area of DMBA injection, in order to determine the latent period of occurrence of tumours. On the 15th day of the experiment and every 30 days, the animals were examined, and their body weights were determined. At the end of the experiment, on the 150th day after introduction of the carcinogen, the rats were sacrificed with overdosed ether narcosis. The animals were dissected for macroscopic examination. Subcutaneous tumours were separated and weighed. The mean latent period of tumour formation (MLP), mean tumour weight (MTW), as well as the index of tumour suppression (ITS) were taken as the main effect assessment criteria in the experiment.

[0125] The ITS was calculated according to the following formula:

[0126] MTW (control) - MTW (test)

[0127] MTW (control) A positive value of this index indicates a reduction of tumour growth, while a negative value attests to the stimulation of tumour growth.

[0128] Results

[0129] It was found that 100% of animals survived to the end of the experiment in all experimental and control groups. The physiological states and behavioural reactions in rats of the control and experimental groups did not differ significantly from each other during the whole experiment. In the three experimental groups (I, II, III) on the 15th and 30th day after the administration of DMBA, some lag in the weight gain of the rats was recorded. These changes are characteristic of the toxic effects of DMBA. During the following months, a uniform increase in the weight indices of animals in experimental and control groups were observed. In intact animals (group V) and in rats receiving only Composition 1 (group IV), a dynamic (physiological) increase in body weight was observed during the whole experiment. By the end of the experiment, the average body weight of animals of group I increased by 113 g compared to the initial weight, group 11 - 116 g, group III - 113 g, group IV - 117 g, the intact group - 119 g. Sexual differences between body mass indices of animals (Table 5) were not identified.

[0130] Almost all animals treated with DMBA (group I) developed tumours. Tumours appeared at the site of the injection of carcinogen and had the appearance of subcutaneous formations of dense consistency, irregular shape, and various sizes. Histologically, all developed neoplasms were classified as fibro-sarcomas. The first tumour in group I was detected in females after 29 days, and in males after 31 days from the administration of DMBA. The last tumours were detected on the 51st and 47th day of the experiment, in females and males, respectively. The average latent period of tumour formation in females and males was 39.4 and 39.6 days respectively. In general, in this group of rats, the average latent period of tumour development was 39.5 days. At the end of the experiment, the average mass of tumours in the group as a whole was 8.7 g. The average mass of tumours for females was 8.4 g, and the average mass of tumours for males was 9.1 g.

[0131] Animals of group II who received the test preparation before the introduction of DMBA for 10 days and 7 days after the carcinogen (the first scheme of the experiment), the first tumour was detected at 33 days. The last tumour was detected at 58 days. The mean latent period of development of neoplasms in the specified group for females and males corresponded to 41.3 and 44.1 days respectively (average 42.6 days). The increase in the latent period of tumour development was 8.1%. The tumour mass in this group averaged 7.3 g. At the same time, a pronounced sexual sensitivity of animals was established. In females, tumours were found in 10 out of 10 rats with an average tumour mass of 8.7 g. Unlike females, in males, tumours were detected in 8 out of 10 rats, with a tumour mass of 5.7 g. The tumour growth inhibition index in this subgroup was equal to 39.6% (P <0.05). This indicates a possible antipromoter effect of Composition 1 in rats, that is, the ability of the preparation to inhibit carcinogenesis.

[0132] In-group III of rats treated with Composition 1 after administration of DMBA (second experimental scheme), neoplasms developed in all animals. The first tumour was detected on day 31 , the last tumour was detected on day 61 of the experiment (Table 6). The MLP in this group was 43.2 days. At the same time, for females and males, the MLP was 40.6 and 46.1 days, respectively. The increase in the latent period of tumour development was 9.4%. Along with this, in group III, there was some inhibition of tumour growth. The average mass of tumours in females and males was 6.5 and 6.3 g, respectively, which confirms the antitumor property of Composition 1 (26.4%).

[0133] An increase in the time of formation and a decrease in the average mass of tumours in animals of the two experimental groups (II and III) as compared to the control (DMBA) indicates that Composition 1 has a protective and antitumor effect. Thus, Composition 1 has an inhibitory effect on carcinogenesis induced by DMBA.

[0134] Table 5. The average body weight of animals with a separate and combined use of DMBA and Composition 1.

[0135] Table 6. Influence of Composition 1 on the DMBA-induced carcinogenesis in rats. * p < 0,05

[0136] Composition 1 demonstrated significant inhibition of the growth of tumours in rats. Data on the effects on the stage of post-initiation of carcinogenesis indicate that Composition 1 influences the time of growth of tumours, somewhat increasing the main latent period of their development. In the rats of this group, there was also a slight decrease in the mass of induced tumours. Generally, Composition 1 exhibits an anti-carcinogenic effect, normalizes some physiological parameters of rats and reduces the growth of DMBA-induced neoplasms.

[0137] The results of the studies suggest that Composition 1, with repeated use in rats, increases the main latent period of tumours and causes a decrease in the average mass of DMBA- induced neoplasms, showing protective and antitumor effects. The inhibitory effect is particularly evident in the first scheme of the experiment when studying the effect of the preparation on the stage of carcinogenesis initiation.

[0138] Example 4

[0139] The antitumor activity of Composition 1 was investigated in vivo, on models of transplanted tumours of mice used for the initial selection of anti-tumour drugs. Studies of anti-tumour activity were carried out in accordance with generally accepted regulatory documents [Guidelines for preclinical drug research, Mironov A.N., Bunatyan N.D. and others 2012; Nonclinical Evaluation for Anticancer Pharmaceuticals, ICH Harmonized Tripartite Guideline, Having reached Step 4 of the ICH Process at the ICH Steering Committee meeting on October 29, 2009.].

[0140] Materials and Methods

[0141] Testing conditions

[0142] Tumour models: Sarcoma 180 (Crocker's sarcoma), which is a low-differentiated experimental sarcoma of mice, which emerged spontaneously as a carcinoma.

[0143] Animals: White breedless mice.

[0144] Justification of the choice of animals: mice were selected as test models, taking into account the requirements of chemotherapeutic studies.

[0145] Number of animals: 52 mice.

[0146] Animal body weight: males-22-24g; females-20-22g. Principle of the method

[0147] The antitumor activity of Composition 1 was studied in mice with transplantable sarcoma 180 The studies were carried out in accordance with the methodological recommendations for the experimental study of new potential antitumor substances.

[0148] A transplant was formed from pieces of tumour tissue free of necrosis (the peripheral part of the tumour node) that were ground with scissors until a homogeneous mass was formed. A sterile saline solution was added to the tumour in a ratio of 1: 3 to 1 : 4, and the resulting slurry was injected under the animal's skin with a syringe. The transplantation was carried out under sterile conditions., A 12-14 day tumour was used as the donor.

[0149] 0.2-0.3 ml of 20% tumour suspensions in physiological solution was administered subcutaneously to the armpit of each mouse. 48 hours after tumour transplantation, the animals were divided into experimental and control groups, having 8 and 10 mice respectively. Animals of the experimental groups were injected with the test preparation once a day for 6 days. One day after the last injection, the animals were sacrificed with an overdose of ether anesthesia and weighed. The weight of the tumours was separately determined. The therapeutic effect was assessed by the percent inhibition of tumour growth (ITG) with respect to the control.

[0150] Clinical observation during the adaptation period.

[0151] During the adaptation period and prior to the introduction of Composition 1 , the animals were under close observation. The observation included monitoring and record of changes in the animals’ skin and hair side, eyes and mucous tunics, mobility and general behaviour. As a criterion of acceptance of randomization, an absence of external signs of diseases and homogeneity of groups by body weight (± 10%) was considered.

[0152] Determination of animals’ body weight.

[0153] The determination of body weight of animals and tumours was carried out on scales of the "Centaurus Scale TM 500g / 0.01 g" type.

[0154] Administration of the preparation.

[0155] Oral administration of Composition 1 was carried out using syringes with appropriate atraumatic metal gastric probe (tubes).

[0156] Observations of animals.

[0157] During the course of the experiment, the general condition of the animals, the features of their behaviour, the intensity and nature of the motor activity, the condition of the hair, skin and mucous membranes, the intake of food and water, changes in body weight, etc., were regularly recorded during the experiment. After completion of the treatment period, the animals were sacrificed by deep anaesthesia with stabilized ether and subjected to macroscopic examination. Statistical methods.

[0158] The obtained results were statistically processed according to the Student-Fisher’s method (used a specialized program for biomedical purposes GraphPad Prism).

[0159] Results

[0160] According to acute toxicity data, the maximum tolerated dose of the Composition 1 when administered orally to mice exceeded 32ml / kg. In chemotherapeutic experiments, the drug was used in the maximum allowable (0.6 ml) and slightly smaller (0.3 ml) for mice, which corresponded to 30 and 15 ml / kg. The individual doses for each animal were calculated taking into account their weight, with the subsequent recounting of doses per kg of body weight. Each experimental group consisted of 8-10 healthy mature females and males which had been quarantined for at least 10 days. Control animals, with the indicated ratio of females and males, received a physiological solution in the maximum permissible volume.

[0161] The results of the chemotherapeutic experiments summarized in Table 7 below show that Composition 1 at a dose of 30 ml / kg exhibits an antitumor effect on sarcoma 180, inhibiting tumour growth by 51.5% (p <0.05). At a dose of 15ml / kg, inhibition of sarcoma 180 growth was 23.5% (p<0.05). With this, the GF index was -1.2%. This value indicates a slight decrease in the body weight of animals during the course of the experiment. At a dose of 15ml / kg, GF is 0.7%, which indirectly indicates the absence of toxic effects of the drug on the animal body after repeated use.

[0162] Table 7. Antitumor activity of Composition 1 on the model of sarcoma 180

[0163] Thus, Composition 1 in a dose of 30 ml / kg shows a moderate anti-tumour effect against sarcoma 180.

[0164] The results of the study of antitumor activity indicate that Composition 1 , when administered orally at a dose of 30 ml / kg, exhibits a moderate antitumor effect against sarcoma 180, inhibiting tumour growth by 51.5%.

[0165] Example 5 - effect against Sars-CoV-2 virus

[0166] Composition 5, as described above, was tested using a nanoluciferase SARS-CoV-2 assay as described in Xie et al.; bioRxiv . 2020 Jun 23;2020.06.22.165712. A549-hACE2 cells (12,000 cells per well in phenol-red free medium containing 2% fetal bovine serum (FBS)) were plated into a white opaque 96-well plate (Corning). 3-fold serial dilutions from 10mM (final assay concentration) of the test composition were prepared in water in a 96 well format and further diluted 100-fold in the phenol-red free culture medium containing 2% FBS. Cell culture medium was removed and incubated with 50 pl_ of the diluted composition solutions and 50 pl_ of SARS-CoV2-Nluc viruses (at MOI of 0.025). At 48 h post-infection, 50 mI_ Nano luciferase substrates (Promega) were added to each well and Luciferase signals measured using a Synergy™ Neo2 microplate reader. Relative luciferase signals were calculated by normalizing the luciferase signals of compound-treated groups to that of the water-treated groups (set as 100%). The EC50 (compound concentration for reducing 50% of luciferase signal) data were calculated from technical repeats using a nonlinear regression model.

[0167] The results generated by the nanoluciferase SARS-CoV-2 assay are illustrated in Figure 3. Figure 3 shows that composition 5 inhibits the SARS-CoV-2 reporter gene in a dose dependent manner.

[0168] Example 6 - SARS-CoV-2 Plaque Reduction Assay

[0169] The plaque assay was performed in Vero E6 cells using standard methods (50 mI supernatants for the 1st dilution of the plaque assay). Plaque assays were performed in batches according to the following general protocol.

[0170] Vero E6 cells were seeded in 6-well plates overnight at 37 °C and infected with SARS-CoV- 2 virus in DMEM cell media. The diluted virus was added to each well and after viral adsorption, overlay media / oxoid agar mixture was added to ensure homogenous consistency. Incubation occurred at 37 °C in a cell culture incubator for 72h. Following incubation, the 6 wells were separated into two groups: a test group including three wells and a control group including three wells. Composition 5 was added to the wells in the test group. Composition 5 was not added to the wells in the control group. Afterwards, the plates were fixed with 5% formaldehyde (w / methanol) overnight to ensure virus inactivation. The samples were stained with crystal violet for 1 h in order to determine the viral titres (PFU / ml).

[0171] Cytotoxicity after 28 hours of incubation with composition 5 was measured and is shown in Figure 4. Cytotoxicity is presented as a percentage of control across varying dilutions of composition 5. As shown in Figure 4, high cell viability is demonstrated from serial dilution 3 to 6.

[0172] Figure 5 illustrates the observed antiviral (anti- SARS-CoV-2) effect 28 hours after infection. Figure 5 shows plaque forming unit (PFU) values for different concentrations of composition 5 and illustrates that a 1:2048 dilution of composition 5 led to a reduction in plaque forming units.

[0173] Figure 6 shows a photograph of the plate wells from the experiment detailed above in Example 6. The control plate wells (labelled “control”) were prepared as described above, but these plate wells were not treated with composition 5. The plate wells labelled “Composition 5 (2048 dilution)” were treated with composition 5 at a dilution of 1 :2048. Each plate well in each row shown in Figure 6 includes a successively higher viral load moving from the left plate to the right plate.

[0174] As illustrated in figure 6, the control plate wells demonstrated significant viral plaque growth. In contrast, the plate wells treated with composition 5 demonstrated a significant reduction in observable viral plaques.

Claims

Claims1. A pharmaceutical composition comprising: a bisabolol compound, valencene and a terpinene compound.

2. A pharmaceutical composition according to claim 1 , wherein the bisabolol compound is in an amount from about 10% to about 40% by weight of the total amount of the bisabolol compound, valencene and the terpinene compound.

3. A pharmaceutical composition according to claim 1 or 2, wherein the valencene is in an amount from about 22% to about 53% by weight of the total amount of the bisabolol compound, valencene and the terpinene compound.

4. A pharmaceutical composition according to any preceding claim, wherein the terpinene compound is in an amount from about 22% to about 53% by weight of the total amount of the bisabolol compound, valencene and the terpinene compound.

5. A pharmaceutical composition according to any preceding claim, wherein the bisabolol compound is alpha-bisabolol and / or beta-Bisabolol.

6. A pharmaceutical composition according to claim 5, wherein the bisabolol compound is alpha-bisabolol.

7. A pharmaceutical composition according to any preceding claim, wherein the terpinene compound is one or more of alpha-terpinene, beta-terpinene, gamma-terpenine or delta- terpenine,8. A pharmaceutical composition according to claim 7, wherein the terpinene compound is gamma-terpinene.

9. A pharmaceutical composition according to any preceding claim, comprising alpha- bisabolene in an amount from about 10% to about 40% by weight, valencene in an amount from about 22% to about 54% by weight, and a terpinene compound in an amount from about 22% to about 54% by weight, wherein the terpinene compound is alpha terpinene, beta-terpinene or gamma terpinene.

10. A pharmaceutical composition according to any preceding claim, further comprising a carrier.

11. A pharmaceutical composition according to any preceding claim, wherein the carrier is a carrier oil.

12. A pharmaceutical composition according to any preceding claim, wherein the carrier oil comprises one or more of gardenia oil, cherry oil or pine oil.

13. A pharmaceutical composition according to claim 12 wherein the carrier oil further comprises olive oil.

14. A pharmaceutical composition according to any preceding claim, further comprising vitamin E.

15. A pharmaceutical composition according to any one of claims 1 to 4, wherein the composition comprises alpha-bisabolol, valencene, alpha-terpinene, olive oil, gardenia oil, cherry oil, pine oil and vitamin E.

16. A pharmaceutical composition according to any one of claims 1 to 4, wherein the composition comprises alpha-bisabolene, valencene, beta-terpinene, olive oil, gardenia oil, cherry oil, pine oil and vitamin E.

17. A pharmaceutical composition according to any one of claims 1 to 4, wherein the composition comprises alpha-bisabolene, valencene, gamma-terpinene, olive oil, gardenia oil, cherry oil, pine oil and vitamin E.

18. A pharmaceutical composition according to any one of claims 1 to 4 wherein the composition comprises alpha-bisabolene, valencene, gamma-terpinene and olive oil.

19. A pharmaceutical composition according to any preceding claim for use as a medicament.

20. A pharmaceutical composition according to any preceding claim for use in treating viral infections.

21. A pharmaceutical composition for use according to claim 20, wherein the viral infection is caused by a coronavirus or infectious bronchitis virus.

22. A pharmaceutical composition for use according to claim 21, wherein the coronavirus is SARS-CoV-2.

23. A pharmaceutical composition according to any one of claims 1 to 18 for use in the treatment of bacterial infections.

24. A pharmaceutical composition for use according to claim 23, wherein the bacteria responsible for the bacterial infection is selected from one or more of Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Salmonella enteric serovar Typhimurium, Pseudomonas aeruginosa, or Micrococcus luteus.

25. A pharmaceutical composition according to any one of claims 1 to 18 for use in treating a fungal infection, for example a fungal infection caused by Candida albicans.

26. A pharmaceutical composition according to any one of claims 1 to 18 for use in preventing or treating cancer.

27. A method of treatment of a viral infection, a bacterial infection, a fungal infection and / or a method of preventing or treating cancer, comprising a step of administering to a subject a composition according to any one of claims 1 to 18.

28. Use of a pharmaceutical composition according to any one of claims 1 to 18 in the manufacture of a medicament for treating a viral infection, a bacterial infection, a fungal infection and / or for preventing or treating cancer.

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