Augmentation of Anti-viral and Anti-cancer treatments by combinations of poly(i:c) and cannabidiol

Abstract

The present invention relates to pharmaceutical compositions of Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol or their combination and methods of administering such compositions for treating viral infections particularly caused by RNA type viruses. The present invention further provides pharmaceutical compositions of Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol or their combination which can be combined with pharmaceutical compositions of Poly(LC) to make one single composition or a kit having a separate composition of Cannabidiol and a separate composition of Poly(LC) so that they can be separately administered as needed wherein such combined use of Cannabidiol and Poly(LC) augments positive action / effects of Poly(LC) including but not limited to cancer.

Description

[0001] Augmentation of anti-viral and anti-cancer treatments by combinations of Poly(I:C) and Cannabidiol

[0002] Field Of the Invention

[0003] The invention relates to pharmaceutical compositions of Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol or their combination and methods of administering such compositions for treating viral infections particularly caused by RNA type viruses. The compositions can be prepared and administered with RNA vaccines to improve effects of vaccines, to reduce their side effects and to reduce frequency of administration of vaccines.

[0004] Invention provides pharmaceutical compositions of Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol or their combination which can be combined with pharmaceutical compositions of Poly(I:C) to make one single composition or a kit having a separate composition of Cannabidiol and a separate composition of Poly(I:C) so that they can be separately administered as needed wherein such combined use of Cannabidiol and Poly(I:C) augments positive action / effects of Poly(I:C) including but not limited to cancer. Alternatively, compositions of Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol or their combination and compositions of Poly(I:C) can be separately procured and administered.

[0005] Cancer includes one or more of breast cancer, Cervical cancer, ovarian cancer, bladder cancer, Metastatic Cancer, colorectal cancer, liver cancer, Gynecologic Cancers, Head and Neck Cancers, Kidney cancer, lung cancer, lymphoma, leukemia, mesothelioma, myeloma, prostate cancer, skin cancer, thyroid cancer, uterine cancer, brain cancer and cancers of the central and peripheral nervous system, cancers of the hematopoietic tissue, vaginal and vulvar cancers.

[0006] Invention provides pharmaceutical compositions of Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol or their combination which can be combined with pharmaceutical compositions of Poly(I:C) to make one single composition or a kit having a separate composition of Cannabidiol and a separate composition of Poly(I:C) so that they can be separately administered as needed wherein such combined use of Cannabidiol and Poly(I:C) mitigates the adverse effects / negative effects of Poly(I:C) such as excess general toxicity in therapeutic applications for use in cancer. Cancer includes one or more of breast cancer, Cervical cancer, ovarian cancer, bladder cancer, Metastatic Cancer, colorectal cancer, liver cancer, Gynecologic Cancers, Head and Neck Cancers, Kidney cancer, lung cancer, lymphoma, leukemia, mesothelioma, myeloma, prostate cancer, skin cancer, thyroid cancer, uterine cancer, brain cancer and cancers of the central and peripheral nervous system, cancers of the hematopoietic tissue, vaginal and vulvar cancers.

[0007] Background of The Invention

[0008] Recently, several studies involving poly(I:C) have been emerged in the area of immunotherapy as well as for identifying treatment of various infections.

[0009] Poly(I:C) Poly inosinic:poly cytidylic acid (usually abbreviated poly I:C or poly(I:C)) is an immunostimulant. It is used in the form of its sodium salt to simulate viral infections. It is a general model of all RNA-type viral infections and is a mixture of short units of artificial, mismatched double- stranded RNA. With uneven overhanging regions that are single-stranded.

[0010] Lei Fang et al have developed a light-controllable charge-reversal nanoparticle (I:CCN) with controlled release of polyinosinic -poly cytidylic acid [Poly(I:C)] to treat triple negative breast cancer (TNBC) by enhanced photodynamic immunotherapy .

[0011] Mustafa Yalcinkaya et al have examined the effects of the Sars-Cov-2 envelope (E) protein, a virulence factor in coronaviruses, on inflammasome activation and pulmonary inflammation. In cultured macrophages the E protein suppressed inflammasome priming and NLRP3 inflammasome activation. Similarly, in mice transfected with E protein and treated with poly(I:C) to simulate the effects of viral RNA, the E protein, in an NLRP3 -dependent fashion, reduced expression of pro- IL-ip, levels of IL-ip and IL- 18 in broncho-alveolar lavage fluid, and macrophage infiltration in the lung. To simulate the effects of more advanced infection, macrophages were treated with both LPS and poly(I:C).

[0012] Summary of the Invention

[0013] The invention relates to establishing role of Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol or their combination as an antiviral particularly for RNA type viruses. The invention provides pharmaceutical composition of Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol or their combination and methods of administering such compositions for treating viral infections or for prophylactic use in situations of viral infections or for priming an individual with Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol or their combination compositions where such individual is likely to endure such viral infections. In such situations, Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol or their combination acts by augmenting induction of interferons and interferon stimulating factors or genes and also by causing apoptosis of viral infected cells. All these actions of cannabidiol are immunogenic and induced by an action of an immuno stimulant such as virus or vaccine. Here Poly(I:C) is used as an immuno stimulant to test several actions of Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol or their combination. This has also unfolded that Cannabidiol enhances positive effects of Poly(I:C) and can be employed with Poly(I:C) in treatment such as in treatment of cancer, breast cancer, Cervical cancer, ovarian cancer, bladder cancer, Metastatic Cancer, colorectal cancer, liver cancer, Gynecologic Cancers, Head and Neck Cancers, Kidney cancer, lung cancer, lymphoma, leukemia, mesothelioma, myeloma, prostate cancer, skin cancer, thyroid cancer, uterine cancer, brain cancer and cancers of the central and peripheral nervous system, cancers of the hematopoietic tissue, vaginal and vulvar cancers.

[0014] In such treatments, Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol or their combination augments both early and late apoptosis of cancer cells transfected with Poly(I:C) which simulate cancer cells treated with or under treatment of Poly(I:C) and Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol is found to act as a synergist and an adjuvant by enhancing action of Poly(I:C) multi-fold, while potentially reducing negative effects / adverse effects due to multiple factors including but not limited to the reduction in effective dose, reduction in general or non-specific toxicity and augmentation and synergy caused by co-administration of CBD to Poly(I:C) as suggested in the invention.

[0015] Invention provides pharmaceutical compositions of Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol or their combination which can be combined with pharmaceutical compositions of Poly(I:C) to make one single composition or a kit encompassing a separate composition of Cannabidiol and a separate composition of Poly(I:C) so that they can be separately administered as needed wherein such combined use of Cannabidiol and Poly(I:C) augments positive action / effects of Poly(I:C) such as its use in cancer. Cancer includes one or more of breast cancer, Cervical cancer, ovarian cancer, bladder cancer, Metastatic Cancer, colorectal cancer, liver cancer, Gynecologic Cancers, Head and Neck Cancers, Kidney cancer, lung cancer, lymphoma, leukemia, mesothelioma, myeloma, prostate cancer, skin cancer, thyroid cancer, uterine cancer, brain cancer and cancers of the central and peripheral nervous system, cancers of the hematopoietic tissue, vaginal and vulvar cancers.

[0016] Alternatively, compositions of Cannabidiol and compositions of Poly(hC) can be separately procured and administered.

[0017] Brief Description of Figures

[0018] Figure 1A and IB provide Effect of untransfected cells and poly (I:C) transfection, with and without CBD, on the mRNA levels of IFN type I viz. alpha and beta interferons.

[0019] Figure 1C, ID and IE provide Effect of poly (I:C) transfection, with and without CBD, on the mRNA levels of IFN type II viz. gamma interferons and type III viz. lambda interferons. Figure 2 A, 2B, 2C and 2D provide Effect of poly (I:C) transfection, with and without CBD, on the mRNA levels of interferon induced genes OAS1, OAS2, OAS3 and OASL.

[0020] Figures 1A - IE and figures 2 A - 2D provide effects when cells are transfected with Iμg / ml poly (I:C) and treated with vehicle control (0.1% ethanol) or 2 pm CBD.

[0021] Figure 3A provides dose-dependent effects of CBD on HEK293 relative number 24 h after transfection with Poly (I:C) at the concentrations shown (n=4), with IC50 values and 95% confidence intervals (CI) for CBD denoted beside each Poly (I:C) treatment level (Figure 3 Al), and with IC50 values and 95% confidence intervals (CI) for Poly (I:C) denoted beside each CBD treatment level (Figure 3A2).

[0022] Figures 3B and 3C respectfully provide Early and late apoptotic index measures at 24 h in HEK293 cells transfected with 5 μg / ml Poly (I:C), and treated with CBD at increasing concentrations (n=4). *P<0.05, **P<0.01, ****P<0.0001.

[0023] Figures. 4A to 41 provide effect of poly (I:C) transfection, with and without CBD, on the mRNA levels of IFN types I, II and III, and OAS genes. Expression of

[0024] IFN a (Figure 4A), IFNp (Figure 4B), IFN γ (Figure 4C), IFN λI (Figure 4D), IFN λ2 / 3 (Figure 4E), OAS1 (Figure 4F), OAS2 (Figure 4G), OAS3 (Figure 4H) and OASL (Figure 41), in untransfected cells (transfection reagents only - marked “control”), or cells transfected with 5 μg / ml poly (I:C) (marked “Poly(I:C)”) and treated with vehicle alone (0.1% ethanol, black bars) or 2 pm CBD (white bars). Data are means + SEM, *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001.

[0025] Figures 4A - 41 provide effects when cells are transfected with 5μg / ml poly (I:C) and treated with vehicle control (0.1% ethanol) or 2 pM CBD.

[0026] Figure 5A provides the relative number of MCF-10A epithelial breast cells and MCF-7 estrogen-dependent breast cancer cells after transfection with Poly(I:C) at the levels indicated, or transfection reagents alone (Poly I:C 0 μg / ml), and treatment with CBD. Cells were transfected with Poly(I:C) and then treated with CBD as indicated for 24 hours, after which cells were counted using a Cytation 5 Cell Imaging Multi-Mode Reader (Biotek Instruments). Figure 5B provides area-under-the-curve (AUC) quantitation representing the concentration-dependent effects of CBD on the relative number of MCF-10A epithelial breast cells and MCF-7 estrogen-dependent breast cancer cells after transfection with Poly(I:C) at the levels indicated, or with transfection reagents alone (Poly I:C 0 μg / ml).

[0027] Figure 6A provides the early apoptosis index of MCF-10A epithelial breast cells and MCF-7 breast cancer cells after transfection with 5 μg / ml Poly(I:C) or transfection reagents alone (Poly I:C 0 μg / ml), and treatment with CBD at increasing concentrations. Data are means ± SEM. Asterisks on MCF-10A (5 μg / ml Poly(I:C)) line points denote differences versus MCF-10A (0 μg / ml Poly(I:C)) line points for comparisons between the same CBD concentrations, *P<0.05, **P<0.01. Asterisks on MCF-7 (5 μg / ml Poly(I:C)) line points denote differences versus MCF-7 (0 μg / ml Poly(I:C)) line points for comparisons between the same CBD concentrations, *P<0.05, ****P<0.0001.abcMeasures with different superscripts are significantly different for MCF-7 cells treated with 5 ug / ml Poly(I:C) between concentrations of CBD, showing a dose-dependent effect of CBD, P<0.0001.

[0028] Figure 6B provides area-under-the curve (AUC) analysis for each cell / Poly(I:C) treatment combination highlights that the Early Apoptosis Index response is augmented only by a small degree by 5 μg / ml Poly(I:C) in MCF-10A noncancerous cells, while this is augmented to a very large degree in MCF-7 cells by 5 μg / ml Poly(I:C), as a result of the significant dose-dependent effects of CBD on MCF-7 cells treated with 5 μg / ml Poly(I:C), but not MCF-10A cells treated with 5 μg / ml Poly(I:C). Data were analyzed by 1-way ANOVA with Tukey’s post-hoc analysis, and are means ± SEM. ***P<0.001, ****P<0.0001, n=5..

[0029] Figure 7A provides the late apoptosis index of MCF-10A epithelial breast cells and MCF-7 breast cancer cells after transfection with 5 μg / ml Poly(I:C) or transfection reagents alone (Poly I:C 0 μg / ml), and treatment with CBD at increasing concentrations. Cells were transfected with Poly (I:C) and then treated with CBD as indicated for 24 hours, after which a late apoptosis index was calculated based on incorporation of the late apoptosis indicator PI, normalized to cell number. Data were analyzed by 2-way ANOVA with Tukey’s post-hoc analysis. Data are means ± SEM. Asterisks on MCF-10A (5 μg / ml Poly(I:C)) line points denote differences versus MCF- 10A (0 μg / ml Poly(I:C)) line points for comparisons between the same CBD concentrations, *P<0.05, **P<0.01. Asterisks on MCF-7 (5 μg / ml Poly(I:C)) line points denote differences versus MCF-7 (0 μg / ml Poly(I:C)) line points for comparisons between the same CBD concentrations, *P<0.05, ****P<0.0001.abcMeasures with different superscripts are significantly different for MCF-7 cells treated with 5 ug / ml Poly(I:C) between concentrations of CBD, showing a dosedependent effect of CBD P< 0.0001.

[0030] Figure 7B provides area-under-the curve (AUC) analysis for each cell / Poly(I:C) treatment combination highlighting that the Fate Apoptosis Index response is augmented only by a small degree by 5 μg / ml Poly(I:C) in MCF-10A noncancerous cells, while this is augmented to a very large degree in MCF-7 cells by 5 μg / ml Poly(I:C), as a result of the significant dose-dependent effects of CBD on MCF-7 cells treated with 5 μg / ml Poly(I:C), but not on MCF-10A cells treated with 5 μg / ml Poly(I:C). Data were analyzed by 1-way ANOVA with Tukey’s post-hoc analysis, and are means ± SEM. **P<0.01, ***P<0.001, ****P<0.0001. n=5..

[0031] Figure 8 provides Effect of poly (I:C) transfection, with and without 1 pM CBG, on the mRNA levels of IFN types I, II and III. Expression of IFNa (Figure 8A), IFNb (Figure 8B), IFNg (Figure 8C), IFN11 (Figure 8D), and IFN12 / 3 (Figure 8E), in untransfected cells (transfection reagents only), or cells transfected with 5μg / ml poly (I:C) and treated with vehicle control (0.1% ethanol) or 1 pm CBG. Data are means ± SEM, **P<0.01, ***P<0.001, and ****P<0.0001.

[0032] Figure 9. Provides Effect of poly (I:C) transfection , with and without CBG, on the mRNA levels of OAS family members. Expression of OAS1 (Figure 9A), OAS2 (Figure 9B), OAS3 (Figure 9C) and OASL (Figure 9D), in untransfected cells (transfection reagents only), or cells transfected with 5 μg / ml poly (I:C) and treated with vehicle control (0.1% ethanol) or 1 pm CBG. Data are means ± SEM, **P<0.01, ***P<0.001, and ****P<0.0001. Figure 10. Effect of Poly (I:C) with and without CBG on relative cell number and early and late apoptosis indexes. Figure 10A1 provides Dose-dependent effects of CBG on HEK293 relative number 24 h after transfection with Poly (I:C) at the concentrations shown (n=4), with IC50 values for CBG denoted beside each Poly (I:C) treatment level.

[0033] Figure 10A2 shows the dose-dependent effects of Poly(I:C) on HEK293 relative cell number 24 hours after transfection with Poly (I:C) at the concentrations shown (n=4), with IC50 values denoted beside each CBG treatment level. When cells were not treated with CBG, 7.35 ug / ml Poly(I:C) was needed to cause a 50% reduction of cell number.

[0034] When cells were treated with 0.5 pM CBG, only 3.33 ug / ml Poly(I:C) was needed to cause the same reduction in cell number. This means that when 0.5 pM CBG is present, less than 1 / 2 of the amount of Poly(I:C) is needed to cause the same reduction in cell number. When 1 pM CBG is used, only about 1 / 3 of the amount of Poly(I:C) is needed to cause a 50% reduction in cell numbers. CBG alone does not reduce cell numbers.

[0035] However, CBG makes Poly(I:C) more effective at reducing cell numbers so that a lower dose of Poly(I:C) is needed to obtain the same effect when CBG is present.

[0036] Figure 10B and Figure 10C provide early and late apoptotic index measures at 24 h in HEK293 cells transfected with 5 μg / ml Poly (I:C), and treated with CBG at increasing concentrations (n=4). *P<0.05, **P<0.01, ****P<0.0001.

[0037] Figure 11 provides the relative number of MCF-10A epithelial breast cells and MDA-MB-231 triple-negative breast cancer cells after transfection with Poly(I:C) at the levels indicated, or transfection reagents alone (Poly I:C 0 ug / ml), and treatment with CBD at the concentrations indicated. Cells were transfected with Poly(I:C) and then treated with CBD as indicated for 24 hours, after which cells were counted using a Cytation 5 Cell Imaging Multi-Mode Reader (Biotek Instruments). Figure 12 provides Area-unde-the-curve (AUC) quantitation representing the concentration-dependent effects of CBD on the relative number of MCF-10A epithelial breast cells and MDA-MB-231 triple-negative breast cancer cells after transfection with Poly(I:C) at the levels indicated, or with transfection reagents alone (Poly I:C 0 ug / ml). Poly(I:C) treatment significantly decreased levels of both MCF-10A cells and MDA-MB-231 cells over increasing levels of CBD. However, when cells were treated with 2.5 ug / ml Poly(I:C), the AUC was significantly lower for MDA-MB-231 cells than for MCF-10A cells, indicating that CBD has a greater effect on the reduction in the number of cancerous cells than non-cancerous control cells when combined with this level of Poly(I:C) treatment. Data were analyzed by 1-way ANOVA with Tukey’s post-hoc analysis, and are means ± SEM. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. n=5.

[0038] Figure 13 provides the early apoptosis index of MCF-10A epithelial breast cells and MDA-MB-231 breast cancer cells after transfection with 5 ug / ml Poly(I:C) or transfection reagents alone (Poly I:C 0 ug / ml), and treatment with CBD at increasing concentrations. Cells were transfected with Poly(I:C) and then treated with CBD as indicated for 24 hours, after which an early apoptosis index was calculated based on incorporation of the early apoptosis indicator pSIVA, normalized to cell number counted using a Cytation 5 Imager.

[0039] Figure 14 provides Area-under-the-curve (AUC) quantitation representing the concentration-dependent effects of CBD on the Early Apoptosis Index of MCF-10A epithelial breast cells and MDA-MB-231 estrogen-independent breast cancer cells after transfection with 5 ug / ml Poly(I:C) or with transfection reagents alone (PolyI:C 0 ug / ml). This analysis highlights that CBD augments the anti- cancer response to a single dose of Poly(I:C) to a significantly greater extent in MDA-MB- 231 cancer cells compared to MCF10A cells.

[0040] Data were analyzed by 1-way ANOVA with Tukey’s post- hoc analysis, and are means ± SEM. **P<0.01, ***P<0.001, ****P<0.0001. n=4. Figure 15 provides a late apoptosis index of MCF-10A epithelial breast cells and MDA-MB-231 breast cancer cells after transfection with 5 ug / ml Poly(I:C) or transfection reagents alone (Poly I:C 0 ug / ml), and treatment with CBD at increasing concentrations. Cells were transfected with Poly(I:C) and then treated with CBD as indicated for 24 hours, after which a late apoptosis index was calculated based on incorporation of the late apoptosis indicator propidium iodide, normalized to cell number. Data were analyzed by 2-way ANOVA with Tukey’s post-hoc analysis. Data are means ± SEM.

[0041] Figure 16 provides Area-under-the-curve (AUC) quantitation representing the concentration-dependent effects of CBD on the Late Apoptosis Index of MCF-10A epithelial breast cells and MDA-MB-231 estrogen-independent breast cancer cells after transfection with 5 ug / ml Poly(I:C) or with transfection reagents alone (Poly I:C 0 ug / ml). The Late Apoptosis Index response to 5 ug / ml Poly(I:C) in cells treated with increasing concentrations of CBD is augmented to a smaller degree in MCF-10A noncancerous cells compared to MDA-MB-231 cells. Data were analyzed by 1-way ANOVA with Tukey’s post-hoc analysis, and are means ± SEM. **P<0.01, ****P<0.0001.n=4.

[0042] Detailed Description of the Invention

[0043] The present invention establishes anti-viral role of Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol or their combination. This role is established through an immunogenic action of Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol or their combination wherein it augments induction of interferons and interferon stimulating factors or genes when it is employed in presence of an immunostimulant. An immunostimulant can be any agent which induces immunogenic response such as a virus causing infection or it can be a vaccine. Since presence of immunostimulant is required for such action of Cannabinoid, either Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol or their combination is administered with immuno stimulant in cases such as where it is a vaccine or it is administered when immunostimulant is likely to be present in the body or likely to attack body of a mammal or a human.

[0044] The invention provides a pharmaceutical composition comprising therapeutically effective amount of a Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol or their combination. The composition can be used in treating virus infection in a patient caused by an RNA type virus wherein said Cannabinoid or composition produces an enhancement / augmentation of innate immunity of the patient. This is due to at least one of the following effects, i) augmenting induction of interferons; ii) augmenting induction of interferon stimulated factors or genes; iii) by causing apoptosis of virus infected cells.

[0045] The invention provides method of treating a virus infection in a patient caused by an RNA type virus by administering a pharmaceutical composition comprising therapeutically effective amount of a Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol or their combination. The said Cannabinoid or composition produces an enhancement / augmentation of innate immunity of the patient. This is produced due to at least one of the following effects, i) augmenting induction of interferons; ii) augmenting induction of interferon stimulated factors or genes; iii) by causing apoptosis of virus infected cells.

[0046] Induction of one or more interferons include induction of one or more of Type I (alpha and beta) or Type II (gamma) or Type III (lambda) Interferons or any combination thereof.

[0047] Induction of one or more interferon stimulated factors or genes include interferon stimulated factors or genes selected from one or more of OAS1, OAS2, OAS3, OASL.

[0048] Apoptosis of virus infected cells includes both early and late apoptosis and their combination.

[0049] The invention further provides a pharmaceutical composition comprising therapeutically effective amount of a Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol or their combination for use in treating cancer which is treated or can be treated or controlled or can be controlled by using poly(I:C). Such pharmaceutical compositions can be combined with poly(I:C) or composition of poly(I:C). When combined, such compositions are administered concomitantly or one after other.

[0050] The invention provides treatment involving administering a pharmaceutical composition comprising therapeutically effective amount of a Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol or their combination along with poly(I:C) or composition of poly(I:C).

[0051] Such administration effectively achieve one or more of the following: a. reduces dose of Poly(I:C); b. reduces toxicity / cytotoxicity of Poly(I:C) associated with higher dose; c. enhances efficacy of Poly (I:C); d. provides synergistic or adjunct therapy with Poly(I:C).

[0052] Further such administration and such compositions achieve above without increasing damage to non-cancerous cells. In the present case, an immuno stimulant Poly (I:C) is employed to simulate viral infection and as a model for both RNA virus and RNA vaccine.

[0053] The present study has also unfolded immunogenic response of Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol or their combination by augmenting both early and late apoptosis of cells transfected with Poly (I:C). Cells transfected with Poly (I:C) represent or simulate infected cells and hence Cannabidiol can exhibit immunogenic action by causing apoptosis of infected cells.

[0054] In this new role, Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol or their combination can be administered in a viral infection particularly in an infection caused by RNA type virus. Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol or their combination can also be administered as a prophylactic in a viral infection or to prime an individual who is likely to endure such viral attack. Alternatively, Cannabidiol can be administered with an immuno stimulant such as RNA vaccine to enhance and / or prolong its effect and to control / reduce frequency of administering vaccines or to reduce any side effects of such vaccines etc.

[0055] The invention relates to pharmaceutical compositions of Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol or their combination and methods of administering Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol or their combination to augment immunogenic responses of an immunostimulant.

[0056] The study has unfolded another role of Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol or their combination. Apart from using Poly(I:C) as a model, it is proposed in some treatments. Cannabidiol can enhance positive effects of Poly(I:C). One such role of Poly(I:C) is in treating cancers. Cancer includes one or more of breast cancer, Cervical cancer, ovarian cancer, bladder cancer, Metastatic Cancer, colorectal cancer, liver cancer, Gynecologic Cancers, Head and Neck Cancers, Kidney cancer, lung cancer, lymphoma, leukemia, mesothelioma, myeloma, prostate cancer, skin cancer, thyroid cancer, uterine cancer, brain cancer and cancers of the central and peripheral nervous system, cancers of the hematopoietic tissue, vaginal and vulvar cancers.

[0057] Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol or their combination has exhibited multi-fold augmentation in both early and late apoptosis of cancer cells transfected with Poly(I:C) without affecting non-cancerous cells, or affecting non-cancerous cells to a much smaller extent, when similarly treated. Thus, Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol or their combination acts as a synergist and as an adjuvant to increase the pro-apoptotic and anti-cancer capacity of Poly(I:C), without increasing damage or only minimally increasing damage to non-cancerous cells and therefore avoiding general or nonspecific toxicity in healthy cells, or by lowering the effective dose of Poly(I:C) needed to cause significant anti-cancer effects to a dose that does not cause damage or only minimally causes damage to non-cancerous cells.

[0058] Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol or their combination is found to enhance the cellular anti-viral response to RNA-type viruses (modelled generally by Poly(I:C)) resulting in better protection upon infection, and it would also enhance the inflammatory response to any RNA-type vaccines (modelled also by Poly(I:C)), resulting in a better immunogenic response and better or earlier immunity after vaccination.

[0059] Invention provides pharmaceutical compositions of Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol or their combination which can be combined with pharmaceutical compositions of Poly(I:C) to make one single composition or a kit having a separate composition of Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol or their combination and a separate composition of Poly(I:C) so that they can be separately administered as needed wherein such combined use of Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol or their combination and Poly(I:C) augments positive action / effects of Poly(I:C) such as its use in cancer. Cancer includes one or more of breast cancer, Cervical cancer, ovarian cancer, bladder cancer, Metastatic Cancer, colorectal cancer, liver cancer, Gynecologic Cancers, Head and Neck Cancers, Kidney cancer, lung cancer, lymphoma, leukemia, mesothelioma, myeloma, prostate cancer, skin cancer, thyroid cancer, uterine cancer, brain cancer and cancers of the central and peripheral nervous system, cancers of the hematopoietic tissue, vaginal and vulvar cancers.

[0060] Alternatively, compositions of Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol or their combination and compositions of Poly(I:C) can be separately procured and administered.

[0061] In recent past, Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol or their combination has shown promising role in priming, prophylaxis and treatment of Covid-19 infection (WO2021199078 and WO2022018754). Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol or their combination can produce an enhancement / augmentation of innate immunity of the patient due to at least one of the following effects, i) infected patient cells undergo apoptosis early after infection; ii) induction of interferon transcription in the patient; iii) induction of interferon-induced antiviral effectors in the patient.

[0062] In another study submitted in Indian Patent Application 202121045573, the applicant had proposed that the pathogens and parasites in elimination of which interferons and their downstream effectors play a key role can be eliminated by administration of one or more Cannabinoids.

[0063] Research on Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol or their combination is continued after exploring its multifaceted roles in Covid- 19 infection caused by SARS-COV-2 which is an RNA virus. The present study is one more step to evaluate and establish antiviral role of Cannabidiol for all RNA type viruses. This study was prioritized when recently (PCT / IB2022 / 051304), the applicant has established Cannabidiol’s role in augmenting effects of Covid- 19 vaccine by sustaining / enhancing response / level of IgG or IgM antibodies where Cannabidiol was administered with the said vaccine in mice. In that study, not only Cannabidiol augmented immune response in mice by exhibiting continuous higher levels of IgG or IgM antibodies but it helped in quickly attaining higher response / level of IgG or IgM antibodies in mice compared to vaccine alone.

[0064] The present study establishes antiviral role of Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol or their combination in all RNA viruses. The study also proposes augmentation of effect of a vaccine by administration of Cannabidiol with such vaccines.

[0065] To evaluate and establish such roles of Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol or their combination, present study involves transfecting Human Embryonic Kidney Cells (HEK293 cells) with Poly (I:C). Polyinosinic acid:polycytidylic acid (Poly (I:C)) acts as an immunostimulant and has been used to simulate viral infections. If Poly (I:C) is introduced into mouse lungs, it will cause pneumonia. An anti-viral response to double-stranded RNA is so strong that cells (and animals) that are treated with Poly(I:C) respond as though they have been infected by a virus - even though a virus isn't present.

[0066] Poly (I:C) is a general model of all RNA-type viral infections. Poly (I:C) is a mixture of short units of artificial, mismatched double-stranded RNA. with uneven overhanging regions that are single-stranded.

[0067] In the present study, Poly(I:C) is chosen as a broad model to study the effects of Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol or their combination on the cellular responses to RNA-type viruses.

[0068] A general mechanism when any RNA virus invades cells is as follows. When an RNA-based virus (like coronaviruses, influenza, and other RNA-type viruses) get into cells, they will copy their large genome, making a big strand of double stranded RNA. Cells typically recognize this as foreign, and the cellular endoribonucleases like Dicer will chop up this double-stranded RNA into very small double- stranded RNAs. These little bits of RNA do not look like the normal RNA transcripts that cells make, and cells ’sense’ these as an indication of viral infection. One way they do this is through OAS proteins. OAS proteins bind to these small, double stranded RNA, and this activates them to produce signaling molecules that activate RNAseL, and stimulate it to start chopping up viral RNA, but also normal cellular RNA, which can lead to apoptosis, shutting down virus reproduction in a cell.

[0069] In the present study, Human Embryonic Kidney Cells (HEK293 cells) are transfected with Poly (I:C) (Polyinosinic acid:polycytidylic acid) using jetPRIME® (Polyplus Transfection, New York, NY, U.S.A.). jetPRIME® is employed as a transfection reagent which is a powerful and versatile DNA and siRNA transfection reagent with superior cell viability and high transfection efficiency. As a control, Human Embryonic Kidney Cells (HEK293 cells) are treated with the transfection mixture alone (1.25 pL jetPRIME reagent and 50 pL buffer diluted in medium to provide untransfected cells. Both transfected and untransfected cells are treated with Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol or their combination. The detailed process of preparing transfected cells and untransfected control are provided under example 1.

[0070] It is interestingly found that Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol or their combination with poly (I:C) augments the anti-viral response to Poly(I:C) by inducing all types of interferons and interferon stimulated genes OAS1, OAS2, OAS3 and OASL suggesting that cells would have a better anti-viral response to any RNA-type virus, including influenza, coronaviruses, etc. when Cannabidiol is administered.

[0071] Following observations are made when HEK293 cells are employed, i) When untransfected cells are treated with Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol or their combination (with transfection reagent alone) induces all of the interferons (except alpha) and all of the OAS genes. Thus, significant enhancement in levels of interferons beta, gamma, lambda 1, lambda 2 / 3, and interferon induced genes OAS1, OAS2, OAS3 and OASL are observed. ii) Poly(I:C) transfection alone (without Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol or their combination) induces a response in all of the inflammatory / anti- viral genes tested, including IFNalpha and IFNbeta, demonstrating that it works as a model (cells sense it is an RNA-type virus, and respond appropriately). Thus, significant enhancement in levels of interferons alpha, beta, gamma, lambda 1, lambda 2 / 3, and interferon induced genes OAS1, OAS2, OAS3 and OASL are observed. iii) In cells that are transfected with Poly(I:C) and treated with Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol or their combination, the induction of all of the genes is significantly elevated relative to levels in cells treated with Poly(I:C) but not treated with Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol or their combination, indicating that adding Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol or their combination enhances the anti-viral response of cells to RNA-type viruses, alone. Thus, significant enhancement in levels of interferons gamma, lambda 1, lambda 2 / 3, and interferon induced genes OAS1, OAS2, OAS3 and OASL are observed. iv) In cells that are transfected with Poly(I:C) and treated with Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol or their combination, the induction of all of the genes is significantly elevated relative to levels in cells treated with Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol or their combination (but not exposed to Poly(I:C), indicating that Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol or their combination particular augments the response when evidence of a virus is present. Thus, significant enhancement in levels of interferons alpha, beta, gamma, lambda 1, lambda 2 / 3, and interferon induced genes OAS1, OAS2, OAS3 and OASL are observed. v) In cells that are transfected with Poly(I:C) and treated with Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol or their combination, it augments early and late apoptosis indicating that virus-infected cells can undergo greater levels of apoptosis when treated with Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol or their combination.

[0072] Following observations are made when cancer cells are employed, i) A differential susceptibility has been achieved wherein cancer cells are found more highly susceptible to combination of Poly(I:C) and Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol or their combination than non-cancer cells. ii) Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol or their combination significantly augments effects of Poly(I:C) including augmentation of both early and late apoptosis of cancer cells transfected with Poly (I:C) and treated with Cannabidiol or Cannabigerol indicating selective apoptosis of cancer cells. iii) Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol or their combination acts as a synergist and an adjuvant in treatments of cancer using Poly(I:C).

[0073] This suggests that Poly(I:C) could be used in lower doses when combined with Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol or their combination to achieve the same level of efficacy, thereby reducing toxicity and adverse effects of the treatment, since each compound augments the effectiveness of the other, reducing effective doses.

[0074] Combinations of Poly (I:C) and Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol or their combination for the treatment of cancer MDA-MB-231 cells are a well-established estrogen-independent human breast cancer cell line, first isolated from the pleural effusion of a 51 year-old Caucasian woman with metastatic breast cancer by the MD Anderson Cancer Centre. These cells are negative for estrogen, progesterone, and HER2 (human epidermal growth factor 2) receptors. This type of cancer is particularly difficult to treat, since it is not responsive to hormonal inhibitors like Tamoxifen, or treatments that target the HER2 receptor. There are therefore fewer treatment options for this type of cancer, and it is more deadly.

[0075] These cells will form mammary tumors in athymic nude mice if grafted intraductally. MCF-10A cells are a mammary epithelial cell line that was first isolated in 1984 by the Michigan Cancer Foundation. They were taken from a 36 year-old female with fibrocystic disease, and are considered noncancerous, since they will not form tumors in immunosuppressed (e.g. athymic nude or SCID mice). They are not ‘normal’ cells, but rather non-cancerous cells. MCF-10A cells are commonly used as a control cell line for studies on cancer cells. Cancer cells are typically more susceptible to apoptosis induction, since they often lack cell-cycle checkpoint regulators (or have a dysregulation of these regulators). This lack of regulation both drives atypical growth, resulting in tumor formation, and also prevents appropriate cell cycle exit to facilitate repair. This can contribute to the accumulation of DNA mutations, which can contribute to uncontrolled and inappropriate growth - but can also make cells more susceptible to death through processes like apoptosis, rather than repair, when damaged.

[0076] This differential susceptibility is exploited in cancer therapies. Most cancer therapies are cytotoxic, causing damage to rapidly dividing cells, and other types of cells. The greater susceptibility of cancer cells to death following exposure to cytotoxic agents is therefore used in therapies that seek to do greater damage to cancer cells than normal cells, with the hope that normal cells will undergo repair, while cancer cells will undergo cell death.

[0077] There are many types of anti-cancer therapeutics. Therapies such as radiation and some chemotherapeutic s are genotoxic. This is not ideal, since genotoxicity can increase susceptibility of an individual to the development of new neoplasms, even if treatment of the original cancer is successful. In some cases, use of these agents can be targeted to the site of a tumor, to shrink it. However, because tumors can metastasize, and metastatic tumors are the most dangerous to survival, and it is very difficult to locate metastases, treatments that can be given systemically to reach all parts of the body are best for treating metastatic disease. In this regard, treatments that show a high degree of toxicity towards tumor cells, but a low degree of toxicity towards normal cells, are ideal.

[0078] We expect that many treatments will kill cancer cells, so studies on the efficacy of new treatments need to look for an effect in cancer cells that is greater, or much greater than the effect that is observed in normal cells. Ideally, a potential treatment will have no effect in normal cells, but a big effect in cancer cells. Detailed Description Of Figures

[0079] Figure 1 (figures 1A - IE) provides Effect of poly (I:C) transfection, with and without CBD, on the mRNA levels of IFN types I, II and III. Expression of IFNα (1A), IFNβ (IB), IFNγ (1C), IFNλ1 (ID), and IFNλ2 / 3 (IE), in untransfected cells (transfection reagents only), or cells transfected with 1 μg / ml poly (I:C) and treated with vehicle control (0.1% ethanol) or 2 pm CBD for 24 hours. Data are means ± SEM, *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001.

[0080] Figure 1A and IB provide Effect of untransfected cells and poly (I:C) transfection, with and without CBD, on the mRNA levels of IFN type I viz. alpha and beta interferons.

[0081] When untransfected cells are treated with Cannabidiol, CBD (with transfection reagent alone) does not induce interferons alpha and beta significantly.

[0082] Poly(I:C) transfection alone (without CBD) induces mRNA levels of IFN type I viz. alpha and beta interferons. This demonstrates that cells sense poly (I:C) as an RNA-type virus, and respond appropriately.

[0083] When cells that are transfected with Poly(I:C) and treated with CBD, the induction of both alpha and beta interferons is not significantly elevated relative to levels in cells transfected with Poly(I:C) but not treated with CBD.

[0084] When cells that are transfected with Poly(I:C) and treated with CBD, the induction of both alpha and beta interferons is significantly elevated relative to levels in cells treated with CBD (but not exposed to Poly(I:C), indicating that CBD particularly augments the response when evidence of a virus is present.

[0085] Figure 1C, ID and IE provide Effect of poly (I:C) transfection, with and without CBD, on the mRNA levels of IFN type II viz. gamma interferons and type III viz. lambda interferons.

[0086] When untransfected cells are treated with Cannabidiol, CBD (with transfection reagent alone) induces interferons gamma and lambda (lambda 1 and 2 / 3) significantly. Poly(I:C) transfection alone (without CBD) induces mRNA levels of IFN type II and III viz. gamma and lambda interferons. This demonstrates that cells sense poly (I:C) as an RNA-type virus, and respond appropriately.

[0087] When cells are transfected with Poly(I:C) and treated with CBD, CBD induces interferons gamma and lambda to a significantly greater extent relative to levels in cells treated with Poly(I:C) but not treated with CBD, indicating that adding CBD enhances the anti-viral response of cells to RNA-type viruses, alone.

[0088] In cells that are transfected with Poly(I:C) and treated with CBD, CBD induces interferons gamma and lambda to a significantly greater extent relative to levels in cells treated with CBD (but not exposed to Poly(I:C), indicating that CBD particularly augments the response when evidence of a virus is present.

[0089] Fig. 2. Effect of poly (I:C) transfection, with and without CBD, on the mRNA levels of OAS family members. Expression of 0AS1 (2A), OAS2 (2B), OAS3 (2C) and OASL (2D), in untransfected cells (transfection reagents only), or cells transfected with 1 μg / ml poly (I:C) and treated with vehicle control (0.1% ethanol) or 2 pm CBD for 24 hours. Data are means ± SEM, *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001.

[0090] Figure 2 A, 2B, 2C and 2D provide Effect of poly (I:C) transfection, with and without CBD, on the mRNA levels of interferon induced genes OAS1, OAS2, OAS3 and OASL.

[0091] When untransfected cells are treated with Cannabidiol, CBD (with transfection reagent alone) induces interferon induced genes OAS1, OAS2, OAS3 and OASL significantly.

[0092] Poly(I:C) transfection alone (without CBD) induces mRNA levels of interferon induced genes OAS1, OAS2, OAS3 and OASL. This demonstrates that cells sense poly (I:C) as an RNA-type virus, and respond appropriately. When cells are transfected with Poly(I:C) and treated with CBD, CBD induces interferon induced genes OAS1, OAS2, OAS3 and OASL to a significantly greater extent relative to levels in cells treated with Poly(I:C) but not treated with CBD, indicating that adding CBD enhances the anti-viral response of cells to RNA-type viruses, alone.

[0093] In cells that are transfected with Poly(I:C) and treated with CBD, CBD significantly induces interferon induced genes OAS1, OAS2, OAS3 and OASL to a significantly greater extent relative to levels in cells treated with CBD (but not exposed to Poly(I:C)), indicating that CBD particularly augments the response when evidence of a virus is present.

[0094] Figures 1A - IE and figures 2 A - 2D provide effects when cells are transfected with Iμg / ml poly (I:C) and treated with vehicle control (0.1% ethanol) or 2 pm CBD for 24 hours.

[0095] Another interesting study taken up includes Effect of Poly (I:C) with and without CBD on relative cell number and early and late apoptosis indexes. Figure 3A1 provides dose-dependent effects of CBD on HEK293 relative cell number 24 hours after transfection with Poly (I:C) at the concentrations shown (n=4), with IC50 values for CBD denoted beside each Poly (I:C) treatment level. These values are IC50 of CBD alone when no Poly(I:C) is used and IC50 of CBD when combinations of Poly(I:C) and CBD both are used. The CBD concentrations from 0 pM (no CBD), 0.5 pM, 1 pM, 1.5 pM and 2 pM are employed along with the control (no Poly(I:C)), 2.5 pg / ml, 5 pg / ml and 10 pg / ml of Poly (I:C). Relative cell number from 100 % (no CBD, No Poly(I:C)) drops to 50 % when a combination of Poly(I:C) at 5 pg / ml and CBD at 1.01 pM is employed and does not change much even when Poly(I:C) is employed at 10 pg / ml wherein required amount of CBD drops from 1.01 pM to 0. 96 pM to cause 50 % reduction in cell number.

[0096] The IC50 for CBD was well above a pharmacological range in cells treated only with transfection reagent (51.65 pM). However, the IC50 for CBD was 1.87 pM for cells exposed to 2.5 μg / ml Poly (I:C), 1.01 pM for cells exposed to 5 μg / ml Poly (I:C), and 0.96 pM for cells exposed to 10 μg / ml Poly (I:C). Based on this analysis, 5 μg / ml Poly (I:C) was used in subsequent apoptosis and gene expression analyses. Further, at IC50 of combination of Poly(I:C) at 5 pg / ml and CBD at 1.01 pM, such combination has much lower dose of CBD than when CBD is used alone at 51.65 pM to achieve IC50. The reduction is almost 50 times.

[0097] When Poly(I:C) is employed alone at a concentration of 5 pg / ml in absence of CBD, a reduction of around 25 % is noted in relative cell number which reduces to 50 % when 1.01 pM of CBD is used. Shown another way as provided in figure 3A2, when cells were not treated with CBD, almost 20 ug / ml Poly(I:C) was needed to cause a 50% reduction of cell number. When cells were treated with 0.5 pM CBD, only ~6 ug / ml Poly(I:C) was needed to cause the same reduction in cell number. This means that when 0.5 pM CBD is present, less than l / 3rdof the amount of Poly(I:C) is needed to cause the same reduction in cell number. When 2 pM CBD is used, less than 1 / 4th of the amount of Poly(I:C) is needed to cause a 50% reduction in cell numbers. CBD alone does not reduce cell numbers. However, CBD makes Poly(I:C) more effective at reducing cell numbers so that a lower dose of Poly(I:C) is needed to obtain the same effect when CBD is present.

[0098] Figures 3B and 3C provide Early and late apoptotic index respectively measures at 24 h in HEK293 cells transfected with 5 μg / ml Poly (I:C), and treated with CBD at increasing concentrations (n=4). *P<0.05, **P<0.01, ****P<0.0001.

[0099] The relative index of cells undergoing early apoptosis rises from around 20,000 (no Poly(I:C), no CBD) to 70,000 when cells are transfected with 5 μg / ml Poly (I:C), and treated with 1.5 - 2 pM of CBD.

[0100] The relative index of cells undergoing late apoptosis rises from around 300 - 350 (no Poly(I:C), no CBD) to 2600 - 2800 when cells are transfected with 5 μg / ml Poly (I:C), and treated with 1.5 - 2 pM of CBD.

[0101] In cells that were not transfected with Poly (I:C), there was no significant effect of CBD on early or late apoptotic indexes, while 5 μg / ml Poly (I:C) transfection in cells significantly increased apoptosis, regardless of the level of CBD treatment (Fig. 3B, 3C).

[0102] However, the addition of 1 to 2 pM CBD to cells transfected with 5 pg / ml Poly (I:C) significantly augmented the early apoptotic index of cells above those cells treated with 0 pM CBD (vehicle alone), or 0.5 pM CBD (Fig. 3B), and significantly augmented the late apoptotic index over those cells treated only with vehicle (Fig. 3C).

[0103] Figures 4A - 41 provide effects when cells are transfected with 5μg / ml poly (I:C) and treated with vehicle control (0.1% ethanol) or 2 pM CBD for 24 hours.

[0104] The control cells treated with 2 pM CBD did not show an increase in IFNa (Fig. 4A), although IFNβ gene expression was significantly elevated in this group (Fig. 4B). The control cells treated with 2 pM CBD had significantly elevated levels of other IFN genes and ISG, including IFNγ, IFNλ, 1 IFNλ2 / 3, OAS1, OAS2, OAS3, and OASL (Figs. 4C-I). Poly (I:C) significantly increased gene expression of all IFN and ISG tested, except for IFNa. Notably, 2 pM CBD without Poly (I:C) increased expression of IFNy, OAS2, OAS3, and OASL to a greater extent than Poly (I:C) without CBD (Figs. 4C, G, H and I), yet CBD alone did not increase apoptosis or reduce cell numbers (Figs. 3A-C) in the manner that Poly (I:C) did, indicating that CBD may offer an immunogenic priming effect without harming cells. When combined, 2 pM CBD plus 5 μg / ml Poly (I:C) increased the expression of all interferons and ISGs (Figs. 4A-I). In some cases, the magnitude of increase was remarkable. The increase for IFNλ 1 and OAS 1 was over 300-fold above levels in cells treated only with transfection reagents and no Poly(I:C) without CBD, the increase for OAS2 was over 800-fold, and the increase for OAS3 and OASL was over 100-fold (Figs. 4D, 4F-I).

[0105] Combination of Poly(I:C) and Cannabidiol led to synergistic effects in augmenting IFNγ, IFNλ1, IFNλ2 / 3, OAS1, OAS2, OAS3, and OASL to such a level never thought or achieved before.

[0106] Combination of Poly(I:C) and Cannabidiol led to synergistic effects in augmenting both early and late apoptosis to such a level never thought or achieved before. Poly(I:C) as a model of all RNA viruses along with Cannabidiol has exhibited immunogenic action enhancing induction of interferons and interferon stimulating factors or genes and early and late apoptosis. These actions establish anti-viral role of Cannabidiol. The cells which are transfected with Poly(I:C) and treated with CBD simulate / represent those cells which are infected with any RNA virus and treated with CBD. Early and late apoptosis augmentation of such cells establishes elimination / death of infected cells and control of spread of infection.

[0107] The present invention relates to establishing anti-viral role of Cannabidiol. This role is established through immunogenic action of Cannabidiol wherein it augments induction of interferons and interferon stimulating factors or genes when it is employed with an immuno stimulant and augments both early and late apoptosis of transfected cells (which are transfected by Poly(I:C) and represent virus infected cells.

[0108] With the present study, anti-viral role of Cannabidiol is established since CBD augments the anti-viral response to Poly(I:C), suggesting that cells would have a better anti-viral response to any RNA-type virus when Cannabidiol is administered. The present study in combination with one or more previous studies conclude that Cannabidiol if administered with any RNA-type vaccine can sustain or enhance effects of such vaccine. It has been seen in case of Covid- 19 vaccines (PCT / IB2022 / 051304) that Cannabidiol in suitable form when administered with Covid- 19 vaccines (such as Covishield and Covaxin) exhibited enhanced levels of IgG and IgM antibodies for 28 days compared to vaccine alone wherein the Cannabidiol administration was before or at the time or after vaccine administration or employed any combination thereof.

[0109] Poly(I:C) is an immuno stimulant and its role in simulating viral infection has been established. Administration of Cannabidiol with Poly(I:C) substantially enhancing all interferons and interferons stimulating genes and administration of Cannabidiol with Covid-19 vaccine enhancing levels of antibodies point out and clearly establishes that CBD augments the immunogenic response to vaccination with an RNA-type vaccine. This is particularly true as RNA-type vaccines also deliver RNAs coding for one or more viral proteins (typically the SPIKE protein) into the muscle of a mammal similar to effect exhibited by Poly (I:C). Alternatively, a combination of Poly (I:C) and Cannabidiol can act as a better adjuvant for vaccines by boosting the immunogenic response, making a dose of vaccine more effective, and / or potentially reducing the negative effects / adverse effects of the vaccines.

[0110] Once RNAs coding for one or more viral proteins (typically the SPIKE protein) is delivered into the muscle of a mammal, cells in the muscle must make these proteins and then display them so that helper T-cells can recognize the foreign protein as a foreign antigen, and can process that antigen, and display it to B-cells that can make antibodies to that foreign antigen. Those antibodies provide defense against an initial infection, helping both to prevent infection, and to fight off an infection. To prevent or fight off an infection, immunosurveillance by circulating T-cells is necessary, a therapy that can increase the innate immune response of muscle cells to the presence of viral RNA or viral DNA, such as by increasing the production of interferons by cells, can help to attract and recruit T-cells and other immunocytes to the area of inoculation. An enhanced T-cell presence at the site of inoculation would be expected to increase antigen uptake through phagocytosis, which is followed by T-cell presentation of antigen fragments to B-cells, resulting in an enhanced antibody generation by B-cells in response to the vaccine.

[0111] Previously applicant has also shown (Indian patent application - 202121045573) that when cells are transfected with adenoviral-type vectors similar to the one used in the COVISHIELD vaccine, cells induce interferons alpha, beta, gamma, lambda 1, and lambda 2 / 3.

[0112] The mechanism of action of Cannabidiol is interestingly unfolded through various studies conducted by the applicant including its administration with i) Covid-19 vaccine (PCT / IB2022 / 051304) and ii) Poly (I:C) (Present study) and iii) adenoviral type vectors (Indian patent application - 202121045573).

[0113] Cannabidiol is found to enhance the cellular anti-viral response to RNA-type viruses (modelled generally by Poly(I:C)) resulting in better protection upon infection, and it would also enhance the inflammatory response to any RNA-type vaccines (modelled also by Poly(I:C)), resulting in a better immunogenic response and better or earlier immunity after vaccination.

[0114] Cannabidiol augments immunogenic response of virus, vaccine, vector or Poly(I:C). This role contradicts with the established anti-inflammatory role of Cannabidiol reported in several literatures. Studies conducted by the applicant point out towards immunogenic role for inducing immune response. Thus, Cannabidiol can be administered with vaccine for RNA virus as well as with RNA vaccines.

[0115] This study also proposes without restriction or limitation, role of Cannabidiol with one or more vaccines such as viral or bacterial or parasite vaccines ; Cancer vaccines - Allogeneic and Autologous, Whole Tumor Cell Vaccine, Peptide and Protein Vaccines, Immune Cell Vaccine, Genetic Vaccine, Therapeutic Cancer Vaccines and combinations thereof with other therapeutics; Haemophilus b Conjugate Vaccines, Pertussis Vaccines, Cholera Vaccines, Malaria Vaccines, Helminth Vaccines, Influenza Vaccines, Respiratory Syncytial Virus vaccines, Hepatitis B Vaccines, Poliovirus Vaccines, Herpes Simplex Vaccines, Rotavirus Vaccines, Flavivirus Vaccine, Human Immunodeficiency Virus vaccine, Synthetic Peptides as Vaccines, Pneumococcal vaccine, Yellow fever vaccine, Smallpox vaccine and SARS COV-2 vaccines such as the Component Viral Vaccines and Whole Virus Vaccines.

[0116] The present study has demonstrated augmentation of apoptosis, augmentation of induction of interferons and interferons stimulating genes and helps in proposing that

[0117] 1) Cannabidiol can be used in treatment of infection by any RNA virus; and

[0118] 2) Cannabidiol can augment effects of Poly(I:C). Hence, if Poly(I:C) is employed in any treatment, Cannabidiol can certainly augment beneficial effects of Poly(I:C) in such treatment.

[0119] 3) Cannabidiol can reduce the adverse effects / negative effects and general or non-specific toxicity of Poly(I:C) in healthy or non-cancerous cells while augmenting the response and vice versa for conditions including but not limited to Cancer, autoimmune, neurodegenerative and other disorders where Poly(I:C) and / or CBD will be used as treatment. Cancer includes one or more of breast cancer, Cervical cancer, ovarian cancer, bladder cancer, Metastatic Cancer, colorectal cancer, liver cancer, Gynecologic Cancers, Head and Neck Cancers, Kidney cancer, lung cancer, lymphoma, leukemia, mesothelioma, myeloma, prostate cancer, skin cancer, thyroid cancer, uterine cancer, brain cancer and cancers of the central and peripheral nervous system, cancers of the hematopoietic tissue, vaginal and vulvar cancers.

[0120] Interestingly as Cannabidiol is found to enhance effects of Poly(I:C), the inventors further propose role of Cannabidiol with Poly (I:C) to enhance beneficial effects of Poly (I:C). One such beneficial effect which has been tested is elimination of cancerous cells.

[0121] The inventors have discovered that a combination of Poly(I:C) and Cannabidiol acts as a synergistic combination in enhancing anti-cancer effects of Poly(I:C) as well as Cannabidiol when used alone. Not only this but the combination exhibits much higher anticancer effects than a mere additive effects of the two.

[0122] Cancer cells are typically more susceptible to apoptosis induction, since they often lack cell-cycle checkpoint regulators (or have a dysregulation of these regulators). This lack of regulation both drives atypical growth, resulting in tumor formation, and also prevents appropriate cell cycle exit to facilitate repair. This can contribute to the accumulation of DNA mutations, which can contribute to uncontrolled and inappropriate growth - but can also make cells more susceptible to death through processes like apoptosis, rather than repair, when damaged.

[0123] Thus, while normal cells are likely to adopt repair mechanism, cancer cells are more likely to adopt apoptosis. This differential susceptibility is exploited in cancer therapies. Most cancer therapies are cytotoxic, causing damage to rapidly dividing cells, and other types of cells. The greater susceptibility of cancer cells to death following exposure to cytotoxic agents is therefore used in therapies that seek to do greater damage to cancer cells than normal cells, with the hope that normal cells will undergo repair, while cancer cells will undergo cell death. There are many types of anti-cancer therapeutics. Therapies such as radiation and some chemotherapeutic s are genotoxic. This is not ideal, since genotoxicity can increase susceptibility of an individual to the development of new neoplasms, even if treatment of the original cancer is successful. Cancer includes one or more of breast cancer, Cervical cancer, ovarian cancer, bladder cancer, Metastatic Cancer, colorectal cancer, liver cancer, Gynecologic Cancers, Head and Neck Cancers, Kidney cancer, lung cancer, lymphoma, leukemia, mesothelioma, myeloma, prostate cancer, skin cancer, thyroid cancer, uterine cancer, brain cancer and cancers of the central and peripheral nervous system, cancers of the hematopoietic tissue, vaginal and vulvar cancers.

[0124] In some cases, use of these agents can be targeted to the site of a tumor, to shrink it. However, because tumors can metastasize, and metastatic tumors are the most dangerous to survival, and it is very difficult to locate metastases, treatments that can be given systemically to reach all parts of the body are best for treating metastatic disease. In this regard, treatments that show a high degree of toxicity towards tumor cells, but a low degree of toxicity towards normal cells, are ideal.

[0125] Therefore, the studies on the efficacy of new treatments for cancer need to look for an effect in cancer cells that is greater, or much greater than the effect that is observed in normal cells. Ideally, a potential treatment will have no effect in normal or noncancerous cells, or only a small effect in normal or noncancerous cells, but a big effect in cancer cells.

[0126] Individually, Poly(I:C) and Cannabidiol are mentioned in literature as cancer therapeutics but never ever a combination of the two received any mention wherein one augments treatment of another. This is because role of Cannabidiol which was exploited in treatment of cancer was based on its anti-inflammatory action which is a response to immunogenic action and not an immunogenic action in itself whereas the present data suggests immunogenic action of Cannabidiol when it is used in combination with Poly(I:C).

[0127] Petrosino S et al in a paper titled, “Anti-inflammatory Properties of Cannabidiol, a Nonpsychotropic Cannabinoid, in Experimental Allergic Contact Dermatitis” mentions that CBD "dose-dependently inhibits poly-(I:C)-induced release of MCP- 2, interleukin-6 (IL-6), IL-8, and tumor necrosis factor-a". This is in contrast to what is unfolded in the present study.

[0128] Salles EL et al in a paper titled “Cannabidiol (CBD) modulation of apelin in acute respiratory distress syndrome” shows that CBD has an anti-inflammatory effect on acute respiratory distress syndrome (ARDS) which is also opposite to findings of the present study.

[0129] Khodadadi H et al in an article titled, “Cannabidiol Modulates Cytokine Storm in Acute Respiratory Distress Syndrome Induced by Simulated Viral Infection Using Synthetic RNA” shows that CBD has an anti-inflammatory effect on ARDS.

[0130] To invoke immunogenic action and augmentation of immunogenicity, Poly(I:C) and Cannabidiol are present together in the cell environment. This can be achieved by a number of ways. These immunogenic agents can be administered together, simultaneously or sequentially or within certain time period etc. It is also possible to prime cells with CBD before transfection with Poly(I:C). Since Poly(I:C) shall be taken up by cells, the present study involves transfecting cells with Poly(I:C) first and treating with CBD after two hrs. after transfection. This sequence however does not exclude other methods where CBD is administered before transfection or along with Poly(I:C) and also a method which may have longer duration between transfection with Poly(I:C) and thereafter treatment with CBD.

[0131] MCF-7 cells are chosen as representative of cancer cell line. MCF-7 cells are a well- established estrogen-dependent human breast cancer cell line, first isolated by the Michigan Cancer Foundation in 1970 from a tumor taken from a 69 year-old Caucasian woman.

[0132] MCF-10A cells are a mammary epithelial cell line that was first isolated in 1984 by the Michigan Cancer Foundation. They were taken from a 36-year old female with fibrocystic disease, and are considered non-cancerous, since they will not form tumors in immunosuppressed animals (e.g. athymic nude or severe combined immunodeficient (SCID) mice). MCF-10A cells are commonly used as a control cell line for cancer cells.

[0133] Both MCF-7 and MCF-10A cells are transfected with Poly(I:C) at various levels or treated with transfection reagents alone (also termed as control / untransfected i.e. Poly (I:C) is 0 pg / ml) and further treated with CBD at various dose levels including 0 pM i.e. no CBD.

[0134] The figure 5A provides relative number of MCF-10A epithelial breast cells and MCF-7 estrogen-dependent breast cancer cells after transfection with Poly(I:C) at the levels indicated such as 2.5 pg / ml and 5 pg / ml or transfection reagents alone (Poly I:C 0 μg / ml), and treatment with CBD. Cells were transfected with Poly(I:C) and then treated with CBD as indicated for 24 hours, after which cells were counted using a Cytation 5 Cell Imaging Multi-Mode Reader (Biotek Instruments).

[0135] There is a greater effect of Poly(I:C) on MCF-7 cell numbers than MCF-10A cell numbers. This effect is more pronounced at 2.5 μg / ml concentration of Poly(I:C). A further lower concentration of Poly(I:C) is likely to produce less effects on MCF- 10A cell numbers and can be chosen.

[0136] Figure 5B provides area-under-the-curve (AUC) quantitation representing the concentration-dependent effects of CBD on the relative number of MCF-10A epithelial breast cells and MCF-7 estrogen-dependent breast cancer cells after transfection with Poly(I:C) at the levels indicated, or with transfection reagents alone (Poly I:C 0 μg / ml). Poly(I:C) treatment significantly decreased levels of both MCF-10A cells and MCF-7 cells. However, when cells were treated with 2.5 μg / ml Poly(I:C), the AUC was significantly lower for MCF-7 cells than for MCF-10A cells, indicating that CBD has a greater effect on the reduction in the number of cancerous cells than non-cancerous control cells when combined with this level of Poly(I:C) treatment. Data were analyzed by 1-way ANOVA with Tukey’s post-hoc analysis, and are means ± SEM. *P<0.05, **P<0.01, ****P<0.0001. n=5.

[0137] Figure 6A provides early apoptosis index of MCF-10A epithelial breast cells and MCF-7 breast cancer cells after transfection with 5 μg / ml Poly(I:C) or transfection reagents alone (Poly I:C 0 μg / ml), and treatment with CBD at increasing concentrations. Cells were transfected with Poly (I: C) and then treated with CBD as indicated for 24 hours, after which an early apoptosis index was calculated based on incorporation of the early apoptosis indicator pSIVA, normalized to cell number. Data were analyzed by 2-way ANOVA with Tukey’s post-hoc analysis. Data are means ± SEM.

[0138] There were no significant differences in the early apoptosis index of MCF-10A and MCF-7 cells treated only with transfection reagent (i.e., Poly I:C 0 μg / ml) at any level of CBD treatment.

[0139] MCF-10A cells treated with 5 pg / ml had a slightly higher (~2 to 3-fold) apoptotic index relative to control transfected MCF-10A or MCF-7 cells, and this was statistically significant at CBD treatment levels of 0 pM, 0.5 pM, 1 pM, and 1.5 pM, but not 2 pM,*P<0.05, **P<0.01 versus untransfected cell lines. CBD, however, did not increase the toxicity of 5 μg / ml Poly (I:C) in MCF-10A cells.

[0140] In contrast, MCF-7 cells transfected with Poly(I:C) at 5 μg / ml had a sizeable increase in the early apoptosis index that was dose — dependent on CBD. Asterisks denote significant differences between MCF-7 cells treated with 5 μg / ml Poly(I:C) and all other cell-treatment groups within each concentration level of CBD, *P<0.05, ****P<0.0001. While CBD treatment did not cause a dose-dependent increase in the early apoptosis index of MCF-10A cells treated with 5 μg / ml Poly(I:C), or in either control-transfected cell line, CBD did cause a dose-dependent increase in the early apoptosis index of MCF-7 cancer cells transfected with 5 μg / ml Poly(I:C), that was maximal at 1 pM.abcMeasures with different superscripts are significantly different for MCF-7 cells treated with 5 μg / ml Poly(I:C) between concentrations of CBD, showing a dose-dependent effect of CBD P<0.0001.

[0141] Taken together, these data show that Poly (I:C) only minimally induces early apoptosis in MCF-10A non-cancerous cells, but significantly induces early apoptosis in MCF-7 breast cancer cells, and this anti-cancer effect in MCF-7 cells is augmented in a dose-dependent manner by up to 12-fold by CBD. This differential susceptibility to an early apoptosis between non-cancerous MCF-10A cells and cancerous MCF-7 cells due to effects of a combination of Poly (I:C) and CBD and is the main reason of efficacy of such treatment. Thus, at pharmacologically relevant levels, CBD can increase the anti-cancer effect of a specific dose of Poly (I:C), while causing much lower toxicity to noncancerous cells, which may indicate a potential use for CBD as a synergist and an adjuvant to increase the pro-apoptotic and anti-cancer capacity of Poly(I:C), without increasing damage to non-cancerous cells.

[0142] This will also help in maintaining / controlling dose of Poly(I:C) to its lower dose levels in treating cancers. Cancer includes one or more of breast cancer, Cervical cancer, ovarian cancer, bladder cancer, Metastatic Cancer, colorectal cancer, liver cancer, Gynecologic Cancers, Head and Neck Cancers, Kidney cancer, lung cancer, lymphoma, leukemia, mesothelioma, myeloma, prostate cancer, skin cancer, thyroid cancer, uterine cancer, brain cancer and cancers of the central and peripheral nervous system, cancers of the hematopoietic tissue, vaginal and vulvar cancers. Trying lower levels of Poly(I:C) also seems to be feasible with synergist or adjuvant CBD. This potentially suggests that Poly(I:C) could be used in lower doses when combined with CBD, thereby reducing non-specific or general toxicity to the organism and to healthy cells, reducing adverse effects of the treatment while still resulting in augmented targeted efficacy of each compound by the other compound. Figure 6B provides an area-under-the curve (AUC) analysis for each cell (MCF-7 and MCF-10A) / Poly(I:C) treatment combination and highlights that the Early Apoptosis Index response is augmented only by a small degree by 5 pg / ml Poly(I:C) in MCF-10A noncancerous cells, while this is augmented to a very large degree in MCF-7 cells by 5 μg / ml Poly(I:C), as a result of the significant dose-dependent effects of CBD on MCF-7 cells treated with 5 μg / ml Poly(I:C), but not on MCF- 10A non-cancerous cell-treatment combinations.

[0143] Figure 7A provides the late apoptosis index of MCF-10A epithelial breast cells and MCF-7 breast cancer cells after transfection with 5 μg / ml Poly(I:C) or transfection reagents alone (Poly I:C 0 μg / ml), and treatment with CBD at increasing concentrations. Cells were transfected with Poly (I: C) and then treated with CBD as indicated for 24 hours, after which a late apoptosis index was calculated based on incorporation of the late apoptosis indicator PI, normalized to cell number. Data were analyzed by 2-way ANOVA with Tukey’s post-hoc analysis. Data are means + SEM.

[0144] There were no significant differences between the late apoptosis index of MCF- 10A and MCF-7 cells treated only with transfection reagent (i.e. Poly I:C 0 μg / ml) at any level of CBD treatment. MCF-10A cells treated with 5 μg / ml Poly(I:C) had a slightly higher late apoptotic index relative to control transfected MCF-7 cells at 1 pM, 1.5 pM, and 2 pM CBD, and control-transfected MCF-10A cells at 1 pM only, *P<0.05 versus control transfected cell lines. CBD did not increase the toxicity of 5 μg / ml Poly (I:C) in MCF-10A cells at any dose.

[0145] However, MCF-7 cells transfected with Poly(I:C) at 5 μg / ml had a sizeable increase in the late apoptosis index that was dose — dependent on CBD. Asterisks on the blue dashed line with open blue squares denote significant differences between MCF-7 cells treated with 5 μg / ml Poly(I:C) and all other groups within each concentration of CBD, *P<0.05, ****P<0.0001. While CBD treatment did not cause a dosedependent increase in the late apoptosis index of MCF-10A cells treated with 5 μg / ml Poly(I:C), or in either control-transfected cell line, CBD did cause a dosedependent increase in the late apoptosis index of MCF-7 cancer cells transfected with 5 μg / ml Poly(I:C), that was maximal at 1.5 pM CBD.abcMeasures with different superscripts are significantly different for MCF-7 cells treated with 5 μg / ml Poly(I:C) between concentrations of CBD, showing a dose-dependent effect of CBD, P<0.0001.

[0146] Taken together, these data show that 5 μg / ml Poly (I:C) only minimally induces late apoptosis in MCF-10A non-cancerous cells, but significantly induces late apoptosis in MCF-7 breast cancer cells, and this anti-cancer effect in MCF-7 cells is augmented in a dose-dependent manner by CBD. Thus, at pharmacologically relevant levels, CBD can increase the anti-cancer effect of a specific dose of Poly (I:C), while causing much lower toxicity to noncancerous cells, which may indicate a potential use for CBD as an adjuvant to increase the pro-apoptotic and anti-cancer capacity of Poly(I:C), without increasing damage to non-cancerous cells, thereby decreasing the non-specific or general toxicity to the organism and to healthy cells of any specific dose of Poly(I:C), while augmenting the anti-cancer effects of each compound compared to their individual, uncombined effects.

[0147] Figure 7B provides an area-under-the curve (AUC) analysis for each cell(MCF-7 and MCF-10A) / Poly(I:C) treatment combination and highlights that the Late Apoptosis Index response is augmented only by a small degree by 5 μg / ml Poly(I:C) in MCF-10A noncancerous cells, while this is augmented to a very large degree in MCF-7 cells by 5 μg / ml Poly(I:C), as a result of the significant dose-dependent effects of CBD on MCF-7 cells treated with 5 μg / ml Poly(I:C), but not on other cell-treatment combinations. The increase in AUC is less than 3-fold between MCF-10A cells treated with increasing concentrations of CBD and transfected with 5 μg / ml Poly(I:C) versus control transfection only, compared to over 20-fold between MCF-7 cells treated with increasing concentrations of CBD and transfected with 5 μg / ml Poly(I:C) versus control transfection only, highlighting the greater effect of Poly(I:C) with CBD in cancerous compared to non-cancerous cells.

[0148] Data on apoptosis as emerged is really interesting.

[0149] CBD alone at tested levels did not increase apoptosis in MCF-10A or MCF-7 cells, which implies essentially non-toxic nature of CBD.

[0150] Transfecting noncancerous MCF-10A cells with 5 μg / ml Poly(I:C) increased early and late apoptosis. However, CBD treatment didn’t augment the toxicity of this effect (the same level of increase was seen whether cells were exposed to no CBD or 2 pM CBD). This is important, because it means that the predicted toxicity of this combination of Poly(I:C) plus CBD is likely to be minor, and also well- tolerated by normal, healthy cells in an organism.

[0151] Transfecting cancerous MCF-7 cells with 5 μg / ml Poly(I:C) and then treating with up to IpM CBD significantly increased early apoptosis by over 12-fold, compared to MCF-7 cells that were mock transfected with 0 μg / ml Poly(I:C) and then treated with 1 pM CBD. Transfecting cancerous MCF-7 cells with 5 μg / ml Poly(I:C) and then treating with 1.5 μiM CBD significantly increased late apoptosis by almost 32-fold, compared to MCF-7 cells that were mock transfected with 0 μg / ml Poly(I:C) and then treated with 1.5 pM CBD.

[0152] Notably, significant and sizeable effects of only 0.5 pM CBD were also observed on MCF-7 cell apoptosis indexes, when combined with 5 μg / ml Poly(I:C) treatment (~7-fold increased early apoptosis index, and ~ 14.5-fold increased late apoptosis index).

[0153] After successful results are available with CBD, another Cannabinoid Cannabigerol is also tested to check a) if anti-cancer, pro-apoptotic effect of Poly(I:C) in MDA-MB-231 cells is augmented in a dose-dependent manner by Cannabigerol; b) whether detrimental effects on non-cancerous control cells were less c) whether at pharmacologically relevant levels, Cannabigerol can increase the anti-cancer effect of a specific dose of Poly (I:C), while causing much lower toxicity to noncancerous cells.

[0154] If such effects of Cannabigerol can be confirmed, it may indicate a potential use for Cannabigerol as an adjuvant to increase the pro-apoptotic and anti-cancer capacity of Poly(I:C), without increasing damage to non-cancerous cells (i.e., while having lower toxicity in normal or healthy cells).

[0155] CBG (with transfection reagent alone) induces all of the IFN (figures 8 A - 8E) and all of the OAS genes (figures 9A - 9D), Poly(i:c) transfection alone (without CBG) induces a response in all of the inflammatory / anti-viral genes tested, except for IFNgamma, demonstrating that it works (cells sense it is an RNA-type virus, and respond appropriately).

[0156] IFN gene and ISG expression in cells treated with Poly(I:C) and CBG Control cells treated with 1 pM CBG had significantly elevated levels of IFN and ISG, including IFNa, IFNb, IFNg, IFN11, IFN12 / 3, OAS1, OAS2, OAS3, and OASL (Figs. 8A-8E and 9A - 9D). Poly (I:C) alone significantly increased gene expression of all IFN and ISG tested, except for IFNg. Notably, 1 pM CBG without Poly(I:C) increased expression of IFN 2 / 3, OAS3, and OASL to a greater extent than Poly (I:C) without CBG (Figs. 8E and 9 C and 9D), yet CBG alone does not increase apoptosis or reduce cell numbers (Fig. 3) in the manner that Poly (I:C) did, indicating it is both safe, and useful on its own as an anti-viral agent. When combined, 1 pM CBG plus 5 μg / ml Poly (I:C) increases the expression of all interferons and ISGs (Figs.8A-8E and 9A - 9D). In some cases, the magnitude of increase was remarkable. The increase for OAS1 was over 500 fold (Fig.9A).

[0157] In cells that are transfected with Poly(i:c) and treated with CBG, the induction of all of the genes is significantly elevated above levels seen in cells treated with Poly(i:c) but not treated with CBG, indicating that adding CBG enhances the antiviral response of cells to RNA-type viruses, alone.

[0158] In cells that are transfected with Poly(i:c) and treated with CBG, the induction of all of the genes is significantly elevated relative to levels in cells treated with CBG (but not exposed to poly(i:c), indicating that CBG in particular augments the response when evidence of a virus is present.

[0159] Thus, CBG augments the anti-viral response to poly(i:c), suggesting that cells would have a better anti-viral response to any RNA-type virus, including influenza, coronaviruses, etc.

[0160] Following observations are made when HEK293 cells are employed, i) When untransfected cells are treated with Cannabigerol it induces all of the interferons and all of the OAS genes. Thus, significant enhancement in levels of interferons alpha, beta, gamma, lambda 1, lambda 2 / 3, and interferon induced genes OAS1, OAS2, OAS3 and OASL are observed. ii) Poly(I:C) transfection alone induces a response in all of the inflammatory / anti-viral genes tested (except for IFNgamma), including IFNalpha and IFNbeta, demonstrating that it works as a model (cells sense it is an RNA-type virus, and respond appropriately). Thus, significant enhancement in levels of interferons alpha, beta, lambda 1, lambda 2 / 3, and interferon induced genes OAS1, OAS2, OAS3 and OASL are observed. iii) In cells that are transfected with Poly(I:C) and treated with Cannabigerol, the induction of all of the genes is significantly elevated relative to levels in cells treated with Poly(I:C) but not treated with Cannabigerol indicating that adding Cannabigerol enhances the anti-viral response of cells to RNA-type viruses, alone. Thus, significant enhancement in levels of interferons alpha, beta, gamma, lambda 1, lambda 2 / 3, and interferon induced genes OAS1, OAS2, OAS3 and OASL are observed. iv) In cells that are transfected with Poly(I:C) and treated with Cannabigerol, the induction of all of the genes is significantly elevated relative to levels in cells treated with Cannabigerol (but not exposed to Poly(I:C), indicating that Cannabigerol augments the response when evidence of a virus is present. Thus, significant enhancement in levels of interferons alpha, beta, gamma, lambda 1, lambda 2 / 3, and interferon induced genes OAS1, OAS2, OAS3 and OASL are observed. v) It is interestingly found that Cannabigerol with poly (I:C) augments the anti-viral response to Poly(I:C) by inducing all types of interferons and interferon stimulated genes OAS1, OAS2, OAS3 and OASL suggesting that cells would have a better anti-viral response to any RNA-type virus, including influenza, coronaviruses, etc. when Cannabigerol is administered.

[0161] CBG also augments the pro-apoptotic response of cells to Poly(I:C), although it is very safe when used alone, and does not cause any cell death, loss of cell number, or apoptosis. It is only when CBG is combined with Poly I:C, as a model of an RNA virus, that CBG increases the loss of cells and apoptosis.

[0162] In cells that are transfected with Poly(I:C) and treated with Cannabigerol, it augments early and late apoptosis indicating that virus infected cells can undergo greater levels of apoptosis when treated with Cannabigerol. The slopes of the line generated from concentration-responses to CBG in cells transfected with Poly(I:C) were significantly non-zero, while the slope of the line for control cells treated with increasing concentrations of CBG, but transfected only with reagent, was essentially zero (Fig. 10A1). IC50 values for CBG concentrations were affected by the concentration of Poly (I:C) transfected. The predicted IC50 for CBG was so high above a pharmacological range in cells treated only with transfection reagent as to be irrelevant (i.e. 10e32 pM), meaning that CBG displayed no toxicity in the current assay up to 2 pM, and was not predicted to show toxicity at higher levels. However, the IC50 for CBG was 1.08 pM for cells exposed to 2.5 μg / ml Poly (I:C), 0.59 pM for cells exposed to 5 μg / ml Poly (I:C), and 0.24 pM for cells exposed to 10 μg / ml Poly (I:C), which is pharmacologically relevant. Based on this analysis, 5 μg / ml Poly (I:C) was used in apoptosis and gene expression analyses.

[0163] When cells were not treated with CBG, 7.35 ug / ml Poly(I:C) was needed to cause a 50% reduction of cell number (Figure 10A2). When cells were treated with 0.5 pM CBG, only 3.33 ug / ml Poly(I:C) was needed to cause the same reduction in cell number. This means that when 0.5 pM CBG is present, less than 1 / 2 of the amount of Poly(I:C) is needed to cause the same reduction in cell number. When 1 pM CBG is used, only about 1 / 3rdof the amount of Poly(I:C) is needed to cause a 50% reduction in cell numbers. CBG alone does not reduce cell numbers. However, CBG makes Poly(I:C) more effective at reducing cell numbers so that a lower dose of Poly(I:C) is needed to obtain the same effect when CBG is present.

[0164] In cells that were not transfected with Poly (I:C), there was no significant effect of CBG on early or late apoptotic indexes, while 5 μg / ml Poly (I:C) transfection in cells significantly increased apoptosis, regardless of the level of CBG treatment (Figs. 10B and 10C). However, the addition of 1 pM CBG (or more) significantly augmented the early and late apoptotic indexes of cells above those cells treated with 0 pM CBG (vehicle alone) or 0.5 pM CBG (Fig. 10B and 10C). Following observations are made, i) A differential susceptibility can be achieved for cancer cells wherein cancer cells are expected to be more susceptible to combination of Poly(I:C) and Cannabigerol than non-cancer cells. This is because there is a greater effect on apoptosis and induction of interferons with CBG +poly I:C, which would be beneficial if CBG is used along with poly(I:C) for cancer therapy. ii) Cannabigerol significantly augments effects of Poly(I:C) including augmentation of both early and late apoptosis of cells transfected with Poly (I:C) and treated with Cannabigerol indicating enhanced apoptosis of cells. iii) Cannabigerol will act as a synergist and an adjuvant in treatments of cancer using Poly(I:C).

[0165] This suggests that Poly(I:C) could be used in lower doses when combined with Cannabigerol to achieve the same level of efficacy, thereby reducing toxicity and adverse effects of the treatment, since each compound augments the effectiveness of the other, reducing effective doses.

[0166] Further studies were conducted using MDA-MB-231 cells which are a well- established estrogen-independent human breast cancer cell line. Similar studies are also conducted on MCF-10A cells which are commonly used as a control cell line for cancer cells. In these studies, MCF-10A cells and MDA-MB-231 cells were treated with Poly(I:C) at various concentrations, in combination with cannabidiol at various concentrations. Following results are obtained,

[0167] Cell Number:

[0168] As provided in Figure 11, the relative number of MCF-10A epithelial breast cells and MDA-MB-231 triple-negative breast cancer cells after transfection with Poly(I:C) at the levels indicated, or transfection reagents alone (Poly I:C 0 μg / ml), and treatment with CBD at the concentrations indicated.

[0169] There was a greater effect of 2.5 μg / ml Poly(I:C) on MDA-MB-231 cell numbers, indicating a greater cytotoxicity of this compound (at the 2.5 μg / ml treatment level when CBD is present) against cancer cells compared to non-cancerous cells (MCF- 10A). Promising therapeutics for cancer treatment should show greater toxicity with cancer cells than non-cancer cells.

[0170] Figure 12 provides Area-under-the-curve (AUC) quantitation representing the concentration-dependent effects of CBD on the relative number of MCF-10A epithelial breast cells and MDA-MB-231 triple-negative breast cancer cells after transfection with Poly(I:C) at the levels indicated, or with transfection reagents alone (Poly I:C 0 μg / ml).

[0171] Poly(I:C) treatment significantly decreased levels of both MCF-10A cells and MDA-MB-231 cells over increasing levels of CBD. However, when cells were treated with 2.5 μg / ml Poly(I:C), the AUC was significantly lower for MDA-MB- 231 cells than for MCF-10A cells, indicating that CBD caused a greater reduction in the number of cancerous cells than non-cancerous control cells when combined with this level of Poly(I:C) treatment.

[0172] Apoptosis:

[0173] CBD alone (without Poly (I:C)) at levels up to 2 pM did not increase apoptosis in MCF-10A or MDA-MB-231 cells, so it is considered ‘non-toxic’. There was no general or non-specific toxicity observed.

[0174] Transfecting cancerous MDA-MB-231 cells with 5 μg / ml Poly(I:C) and then treating with 2 pM CBD significantly increased early apoptosis by over 4-fold, compared to MDA-MB-231 cells that were mock transfected with 0 ug / ml Poly(I:C) and then treated with 2 pM CBD.

[0175] Transfecting cancerous MDA-MB-231 cells with 5 μg / ml Poly(I:C) and then treating with 2 pM CBD significantly increased late apoptosis by approximately 17-fold, compared to MDA-MB-231 cells that were mock transfected with 0 μg / ml Poly(I:C) and then treated with 2 pM CBD.

[0176] Early Apoptosis

[0177] Figure 13 provides the early apoptosis index of MCF-10A epithelial breast cells and MDA-MB-231 breast cancer cells after transfection with 5 μg / ml Poly(I:C) or transfection reagents alone (Poly I:C 0 μg / ml ), and treatment with CBD at increasing concentrations.

[0178] To examine the overall effect of increasing CBD concentrations when Poly(I:C) is present, or not, the area-under-the-curve was calculated for each interaction between treatment conditions.

[0179] Figure 14 provides Area-under-the-curve (AUC) quantitation representing the concentration-dependent effects of CBD on the Early Apoptosis Index of MCF-10A epithelial breast cells and MDA-MB-231 estrogen-independent breast cancer cells after transfection with 5 ug / ml Poly(I:C) or with transfection reagents alone (PolyI:C 0 ug / ml).

[0180] Taken together, these data show that the anti-cancer, pro-apoptotic effect of Poly(I:C) in MDA-MB-231 cells is augmented in a dose-dependent manner by CBD. Furthermore, detrimental effects on non-cancerous control cells were less. Thus, at pharmacologically relevant levels, CBD can increase the anti-cancer effect of a specific dose of Poly (I:C), while causing much lower toxicity to noncancerous cells, which may indicate a potential use for CBD as an adjuvant to increase the pro-apoptotic and anti-cancer capacity of Poly(I:C), without increasing damage to non-cancerous cells (i.e., while having lower toxicity in normal or healthy cells).

[0181] Following are the observations in Early Apoptosis studies:

[0182] 1. In MDA-MB-231 cells treated with 5 μg / ml Poly(I:C), the Early Apoptotic Index area-under-the-curve (AUC) response to CBD was augmented >4-fold compared to when cells were not treated with Poly(I:C), indicating that the combination of these compounds is superior to use of either compound alone.

[0183] 2. The Early Apoptotic Index AUC response of MDA-MB-231 estrogen independent breast cancer cells treated with 5 μg / ml Poly(I:C) plus CBD is significantly higher (by ~ 1 / 3rd) than the AUC response of MCF- 10A non-cancerous cells treated under the same conditions, indicating that this combined treatment is safer (and less toxic) for non-cancerous cells, and is more toxic for cancerous cells, which is important for cancer therapeutics. Late Apoptosis

[0184] Figure 15 provides the late apoptosis index of MCF-10A epithelial breast cells and MDA-MB-231 breast cancer cells after transfection with 5 μg / ml Poly(I:C) or transfection reagents alone (Poly I:C 0 ug / ml), and treatment with CBD at increasing concentrations.

[0185] Figure 16 provides Area-under-the-curve (AUC) quantitation representing the concentration-dependent effects of CBD on the Late Apoptosis Index of MCF-10A epithelial breast cells and MDA-MB-231 estrogen-independent breast cancer cells after transfection with 5 ug / ml Poly(I:C) or with transfection reagents alone (Poly I:C 0 ug / ml).

[0186] From above studies, following are observed:

[0187] 1. In MDA-MB-231 cells treated with 5 ug / ml Poly(I:C), the Late Apoptotic Index area-under-the-curve (AUC) response to CBD was augmented >5 -fold compared to when cells were not treated with Poly(I:C), indicating that the combination of these compounds is superior to use of either compound alone.

[0188] 2. The Late Apoptotic Index AUC response of MDA-MB-231 estrogen independent breast cancer cells treated with 5 ug / ml Poly(I:C) plus CBD is significantly higher than the AUC response of MCF-10A non-cancerous cells treated under the same conditions, indicating that this combined treatment is safer (and less toxic) for non- cancerous cells, and is more toxic for cancerous cells, which is important for cancer therapeutics.

[0189] Following examples illustrate invention and methods without limiting the scope of the invention in any way.

[0190] Examples and Methods

[0191] Polyinosinic acid: poly cytidylic acid (Poly (I:C)) transfection

[0192] For the poly (I:C) transfection, HEK293 cells were seeded on a 24-well plate at a density of 0.05 x 106cells per well and transfected with Poly (I:C) 24 hours later using JetPRIME (Polyplus Transfection, New York, NY, U.S.A.), according to the manufacturer’s instructions. Briefly, poly (I:C) (Sigma- Aldrich, St. Louis, MO, USA), jetPRIME reagent and buffer are mixed, and incubated this mixture at room temperature for 10 min. The incubated solution was diluted in culture medium and the mixture replaced the culture medium of the cells, so that the concentrations used were as indicated. Control (untransfected) cells were treated with the transfection mixture alone, without Poly(I:C). Transfected and untransfected cells were treated with CBD at the concentrations indicated, or vehicle (0.1% ethanol) approximately 2h after incubation with the respective transfection mixture.

[0193] Apoptosis assay

[0194] Early and late apoptotic cells were detected using a Kinetic Apoptosis Kit (#abl29817, Abeam, Toronto, Ontario, Canada), according to the manufacturer's instructions. Briefly, cells were seeded (1 x 104cells) in 96-well plates and allowed to adhere for 24 hours, then transfected and treated with either CBD or vehicle for 24 hours, labelled with Polarity Sensitive Indicator of Viability & Apoptosis (pSIVA™), which detects early / ongoing apoptosis, and with Propidium Iodide (PI), which detects cells that are in late apoptosis. Live cells were maintained at 37 °C while fluorescence was recorded at 469 / 525 nm for the detection of pSIVA and at 531 / 647 nm for the detection of PI. Results are expressed as an index, with the early apoptosis index calculated as pSIVA absorbance at 525 nm / relative cell number per well, and the late apoptosis index calculated as PI absorbance at 647 nm / relative cell number per well.

[0195] IFN and ISG mRNA expression qPCR analysis was conducted as previously described. Cells were grown in 24 well plates and transfected with Poly(I:C) as described above, and then treated with either 2 pM CBD or vehicle overnight for 24 hours. Total RNA was isolated using TRIzol® Reagent (1 ml per well) as described by the manufacturer (Invitrogen, Waltham, MA).

[0196] Quantification of RNA samples was performed using a Nanodrop 2000 Spectrophotometer (Thermo Fisher, Waltham, MA) that was also used to check for A260 / 280 ratio as an indicator of quality, and 2 pg of RNA was used to synthesize cDNA via oligo(dT) priming using a High-Capacity cDNA Reverse Transcription kit from Applied Biosystems (Waltham, MS, USA). For the real-time PCR assays, cDNA was diluted 1:4 and 1 pl was added to a master mix with 9 pl of PerfeCTa SYBR® Green supermix (Quanta Bio, Beverly, MA), 0.5 pl forward and reverse primers (25 pM each) for the targeted gene (please see Table 1 for primer sequences), and 3 pl of ddH20. The cycling conditions for all genes were as follows:

[0197] 1 cycle of 95°C for 2 min, followed by 49 cycles of 95°C for 10 s, then 60°C for 20 s. Relative expression of the targeted gene was calculated using the delta-delta-Ct method with the Ct values normalized to glyceraldehyde 3 -phosphate dehydrogenase (GAPDH). Table 1: Primer sequences

[0198] Primer sequences (all are DNA sequences, used for amplifying cDNA

[0199] (complementary DNA), made from reverse transcribed messenger RNA (mRNA))

[0200] Example 1A

[0201] Polyinosinic acid:polycytidylic acid (Poly (I:C)) transfection for gene expression experiments for Cannabidiol

[0202] For gene expression experiments, HEK293 cells were seeded on a 24-well plate at a density of 0.05 x 106cells per well and transfected with Poly (I:C) 24 hours later using JetPRIME (Polyplus Transfection, New York, NY, U.S.A.), according to the manufacturer’s instructions.

[0203] Briefly, a mass of poly (I:C) (Sigma-Aldrich, St. Louis, MO, USA) as needed to produce the final concentration of Poly(I:C) required (such as 1 μg / ml, 2.5 μg / ml, 5 μg / ml and 10 μg / ml) was mixed with 1.25 pL JetPRIME reagent and 50 pL buffer, and the mixture was incubated at room temperature for 10 min. The incubated solution was diluted in culture medium to a volume of 500 pL and the mixture replaced the culture medium of the cells. Control (untransfected) cells were treated with the transfection mixture alone (1.25 pL jetPRIME reagent and 50 pL buffer diluted in medium). Media in transfected and control cells was replaced after 2 hours with fresh media containing CBD at various concentrations or vehicle (0.1% ethanol).

[0204] Example IB

[0205] Polyinosinic acid:polycytidylic acid (Poly (I:C)) transfection for gene expression experiments for Cannabigerol

[0206] For the Polyinosinic acid:polycytidylic acid (Poly (I:C)) transfection, HEK293 cells were seeded on a 24-well plate at a density of 0.05 x 106cells per well and transfected with 5 pg / ml Poly (I:C) 24 hours later using JetPRIME (Polyplus Transfection, New York, NY, U.S.A.), according to the manufacturer’s instructions. Briefly, poly (I:C) (Sigma-Aldrich, St. Louis, MO, USA), jetPRIME reagent and buffer are mixed, and incubated this mixture at room temperature for 10 min. The incubated solution was diluted in culture medium and the mixture replaced the culture medium of the cells, so that the concentration used was 5 μg / ml. Control (untransfected) cells were treated with the transfection mixture alone, without Poly(I:C). Transfected and untransfected cells were treated with either 1 pM CBG or vehicle (0.1% ethanol) ~2h after incubation with the respective transfection mixture, for an additional 24 hours prior to harvesting of cells in TRIzol® Reagent (1 ml per well) as described by the manufacturer (Invitrogen, Waltham, MA).

[0207] Quantification of RNA samples was performed using a Nanodrop 2000 Spectrophotometer (Thermo Fisher, Waltham, MA) that was also used to check for A260 / 280 ratio as an indicator of quality, and 2 pg of total RNA was used to synthesize cDNA via oligo(dT) priming using a High-Capacity cDNA Reverse Transcription kit from Applied Biosystems (Waltham, MS, USA). For real-time PCR assays, cDNA was diluted 1:4 and 1 pl was added to a master mix with 9 pl of PerfeCTa SYBR Green Supermix (Quanta Bio, Beverly, MA), 0.5 pl forward and reverse primers (25 pM each) for the targeted gene (please see Table 1 for primer sequences), and 3 pl of ddH20. The cycling conditions for all genes were as follows: 1 cycle of 95°C for 2 min, followed by 49 cycles of 95°C for 10 s, then 60°C for 20 s. Relative expression of the targeted gene was calculated using the deltadeltaCt method with the Ct values normalized to glyceraldehyde 3 -phosphate dehydrogenase (GAPDH).

[0208] Example 2: Polyinosinic acid: poly cytidylic acid (Poly (I:C)) transfection for cell number assessment and measures of early and late apoptosis

[0209] HEK293 cells were seeded on a 96-well plate at a density of 104cells per well and transfected with Poly (I:C) 24 hours later using JetPRIME (Polyplus Transfection, New York, NY, U.S.A.), according to the manufacturer’s instructions.

[0210] Briefly, a mass of poly (I:C) (Sigma-Aldrich, St. Louis, MO, USA) as needed to produce the final concentration of Poly(I:C) required was mixed with JetPRIME reagent (0.25 pL per well) and jetPRIME buffer (5 pL buffer per well), and the mixture was incubated at room temperature for 10 min. The incubated solution was diluted in culture medium (100 pL), and the mixture replaced the culture medium of the cells. Control (untransfected) cells were treated with the transfection mixture alone (i.e. the same amounts of jetPRIME reagent and buffer diluted in medium). Media in transfected and control cells was replaced after 2 hours with fresh media containing CBD at various concentrations or vehicle (0.1% ethanol).

[0211] Example 3: Polyinosinic add: poly cytidylic acid (Poly (I:C)) transfection for cell number assessment and measures of early and late apoptosis for cancer studies for CBD

[0212] MCF-10A or MCF-7 cells were seeded on a 96-well plate at a density of 104cells per well and transfected with Poly (I:C) 24 hours later using JetPRIME (Polyplus Transfection, New York, NY, U.S.A.), according to the manufacturer’s instructions.

[0213] Briefly, a mass of poly (I:C) (Sigma-Aldrich, St. Louis, MO, USA) as needed to produce the final concentration of Poly(I:C) required was mixed with JetPRIME reagent (0.25 pL per well) and jetPRIME buffer (5 pL buffer per well), and the mixture was incubated at room temperature for 10 min. The incubated solution was diluted in culture medium (100 pL), and the mixture replaced the culture medium of the cells. Control (untransfected) cells were treated with the transfection mixture alone (i.e., the same amounts of jetPRIME reagent and buffer diluted in medium). Media in transfected and control cells was replaced after 2 hours with fresh media containing CBD at various concentrations or vehicle (0.1% ethanol).

[0214] Example 4: Polyinosinic acid: poly cytidylic acid (Poly (I:C)) transfection for cell number assessment and measures of early and late apoptosis for studies for Cannabigerol

[0215] HEK293 cells were seeded on a 96-well plate at a density of 1 x 104cells per well and transfected with Poly (I:C) 24 hours later using JetPRIME (Polyplus Transfection, New York, NY, U.S.A.), according to the manufacturer’s instructions. Briefly, poly (I:C) (SigmaAldrich, St. Louis, MO, USA), jetPRIME reagent and buffer, are mixed and incubated this mixture at room temperature for 10 min. The incubated solution was diluted in culture medium and the mixture replaced the culture medium of the cells, so that the concentrations used were 5 μg / ml. Control (untransfected) cells were treated with the transfection mixture alone, without Poly(I:C). Transfected and untransfected cells were treated with either 1 pM CBG or vehicle (0.1% ethanol) approximately 2h after incubation with the respective transfection 4 mixture for 24 h.

[0216] Early and late apoptotic cells were detected using a Kinetic Apoptosis Kit (#abl29817, Abeam, Toronto, Ontario, Canada), according to the manufacturer's instructions. Briefly, cells were labelled with Polarity Sensitive Indicator of Viability & Apoptosis (pSIVA™), which detects early / ongoing apoptosis, and with Propidium Iodide (PI), which detects cells that are in late apoptosis. Live cells were maintained at 37 C while fluorescence was recorded at 469 / 525 nm for the detection of pSIVA and at 531 / 647 nm for the detection of PI. Results are expressed as an index, with the early apoptosis index calculated as pSIVA absorbance at 525 nm / relative cell number per well, and the late apoptosis index calculated as PI absorbance at 647 nm / relative cell number per well. Total cell numbers were calculated by counting cells per well using a Cytation 5 cell imager.

[0217] Example 5 - Pharmaceutical Compositions of Cannabidiol and Poly(I:C)

[0218] Separate or combined pharmaceutical compositions of Cannabinoids such as Cannabidiol and / or Cannabigerol and Poly(I:C) are prepared in accordance with the present invention as follows.

[0219] The active moieties embodied in the scope of this invention are Cannabidiol (CBD) and Poly inosine-poly cytidylic acid (Poly (I:C)). The latter is a double stranded RNA molecule with a MW distribution up to, for instance 3.600.000 Daltons. Poly (I:C) is a Toll Like Receptor 3 (TLR3) ligand that mimics viral double- stranded RNA and is a known stimulant of the innate immune response.

[0220] The Dose

[0221] Dose of Cannabinoids, preferably Cannabidiol and Cannabigerol (CBG)

[0222] Dose of Cannabinoids, particularly Cannabidiol and / or Cannabigerol (CBG) preparations is crucial since it is observed that it has concentration dependent effects and it is desired that the compositions are produced in multiple strengths to address the treatment of various levels of pathologies. A suitable dose of 0.1 mg / kg of body weight to 4000 mg / kg of body weight. The suitable dose can also be 0.1 to 1000 mg / kg of body weight or 0.1 to 500 mg / kg of body weight. The preferred dose can be 0.1 to 100 mg / kg of body weight or from 0.1 to 20 mg / kg of body weight.

[0223] Dose of Poly (I:C)

[0224] The dose will depend on the nature and status of health of an individual as well as the presence or absence of co-morbidities. It will also depend on age. Further, dose will depend on type of composition for example, whether oral or parenteral or topical or nasal or otic or ocular and also on the type of drug delivery system deployed for example liposomal, micellar, particulate - nano or micro which may enhance the availability of the active and thereby facilitate dose reduction. Typical dose ranges from a few nanograms to a few milligrams ranging but not limited to 0.001 nanogram to 100 mg / kg body weight, preferably 0.01 nanogram to 80 mg / kg body weight, more preferably 0.1 nanogram to 50 mg / kg body weight and most preferred from 0.1 microgram to 20 microgram / kg body weight

[0225] The Cannabinoids such as CBD and or Cannabigerol (CBG) and Poly (I:C) will be referred to as the active / s hereon in this document encompassing the formulation aspects.

[0226] The Dosing

[0227] The dosing encircles the following regimen / s but is not restricted to:

[0228] 1] Pre-priming with Poly (I:C) followed by a Cannabinoid such as CBD and / or Cannabigerol (CBG) 2] Pre-priming with a Cannabinoid such as CBD and / or Cannabigerol (CBG) followed by Poly (I:C)

[0229] 3] Concomitant administration of Poly (I:C) and a Cannabinoid such as CBD and / or Cannabigerol (CBG)

[0230] The above regimen / s may be used either alone or in a combination or sequentially as per the diagnosis and severity of the disease in an affected individual, enveloping all living beings especially mammals.

[0231] The scheduling

[0232] The scheduling of the administration of the above-mentioned first two regimens may be spaced apart by a few minutes to several minutes or hours and may accommodate a time difference of 1 minutes to 24 hours, but preferably from 2 minutes to 16 hours and even more preferably from 30 minutes to 12 hours and most preferred from 90 minutes to 6 hours. The same scheduling ranges apply for the various concentrations of Poly (I:C), CBD and or Cannabigerol (CBG)

[0233] The dosage forms

[0234] The dosage form can be preferably oral but can be administered parenterally when an urgent treatment is expected or when the patient is not capable of receiving an oral treatment. Formulations can be administered via any suitable administration route. For example, the formulations (and / or compositions) can be administered to the subject in need thereof orally, intravenously, intramuscularly, intravaginally, intraperitoneally, rectally, parenterally, intraocularly, topically, intranasally, or subcutaneously.

[0235] Suitable oral dosage forms include tablets - sublingual, buccal, effervescent, chewable; troches, lozenges, dispersible powders or granules and dragees; capsules, solutions, suspensions, syrups, lozenges, medicated gums (graphene and / or graphene oxide nano-particles loaded with the active / s further suitably incorporated into chewing gums), buccal gels or patches. Tablets can be made using compression or molding techniques well known in the art. The other dosage forms can also be prepared by 3-Dimensional (3D) or 4D printing and also by Carbon graphene loaded nano-particles and micro-particles. Gelatin or non-gelatin capsules can be formulated as hard or soft capsule shells, which can encapsulate liquid, solid, and semisolid fill materials, using techniques well known in the art.

[0236] Oral compositions

[0237] The compositions are designed to modify, alter and particularly improve solubility of Cannabidiol and or Poly(I:C) and or Cannabigerol (CBG). Cannabidiol has good lipid solubility but its aqueous solubility is poor. Poly(I:C) also has a low solubility in water. Hence, compositions of Cannabidiol and or Poly (I:C) may contain soluble or disintegrating excipients or binders and particularly those excipients which enhance solubility of Cannabidiol and or Poly (I:C) and or Cannabigerol (CBG) in water or in a solvent or co-solvent used in case of liquid preparations. The compositions may additionally contain stabilizers, anti-oxidants, sweeteners, flavours and colourants.

[0238] Suitable diluents include, but are not limited to, dicalcium phosphate dihydrate, calcium sulfate, lactose, sucrose, mannitol, sorbitol, cellulose, microcrystalline cellulose, kaolin, sodium chloride, dry starch, hydrolyzed starches, pregelatinized starch, silicone dioxide, titanium oxide, magnesium aluminum silicate and powdered sugar. The usual diluents include inert powdered substances such as starches, powdered cellulose, especially crystalline and microcrystalline cellulose, sugars such as fructose, mannitol and sucrose, grain flours and similar edible powders. Typical diluents include, for example, various types of starch, lactose, mannitol, kaolin, calcium phosphate or sulfate, inorganic salts such as sodium chloride and powdered sugar. Powdered cellulose derivatives are also useful. Binders can impart cohesive qualities to a solid dosage formulation, and thus can ensure that a tablet or bead or granule remains intact after the formation of the dosage forms. Suitable binder materials include, but are not limited to, starch, pregelatinized starch, gelatin, sugars (including sucrose, glucose, dextrose, lactose and sorbitol), polyethylene glycol, waxes, natural and synthetic gums such as acacia, tragacanth, sodium alginate, cellulose, including hydroxypropylmethylcellulose, hydroxypropylcellulose, ethylcellulose, and magnesium aluminum silicate (Veegum®), and synthetic polymers such as acrylic acid and methacrylic acid copolymers, methacrylic acid copolymers, methyl methacrylate copolymers, aminoalkyl methacrylate copolymers, poly aery lie acid / polymethacrylic acid and polyvinylpyrrolidone .

[0239] Typical tablet binders include substances such as starch, gelatin and sugars such as lactose, fructose, and glucose. Natural and synthetic gums, including acacia, alginates, methylcellulose, and polyvinylpyrrolidone can also be used. Polyethylene glycol, hydrophilic polymers, ethylcellulose and waxes can also serve as binders. Lubricants can be included to facilitate tablet manufacture. Suitable lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, glycerol behenate, polyethylene glycol, talc, and mineral oil.

[0240] A lubricant can be included in a tablet formulation to prevent the tablet and punches from sticking in the die. The lubricant can be chosen from such slippery solids as talc, magnesium and calcium stearate, stearic acid and hydrogenated vegetable oils. Disintegrants can be used to facilitate dosage form disintegration or "breakup" after administration, and generally include, but are not limited to, starch, sodium starch glycolate, sodium carboxymethyl starch, sodium carboxymethylcellulose, hydroxypropyl cellulose pregelatinized starch, clays, cellulose, alginine, gums or cross-linked polymers, such as cross linked PVP (Polyplasdone® XL from GAF Chemical Corp). Stabilizers can be used to inhibit or retard drug decomposition reactions which include, by way of example, oxidative reactions. Suitable stabilizers include, but are not limited to, antioxidants, butylated hydroxytoluene (BHT); ascorbic acid, its salts and esters; Vitamin E, tocopherol and its salts; sulfites such as sodium metabisulphite; cysteine and its derivatives; citric acid; propyl gallate, and butylated hydroxy anisole (BHA).

[0241] Solubilizers may contain surfactants. Suitable surfactants can be anionic, cationic, amphoteric or non-ionic surface-active agents. Suitable anionic surfactants include, but are not limited to, those containing carboxylate, sulfonate and sulfate ions. Delayed release / Sustained release / extended-release compositions Delayed release dosage compositions as described herein can be prepared as described in standard references such as "Pharmaceutical dosage form tablets", eds. Liberman et. al. (New York, Marcel Dekker, Inc., 1989), "Remington - The science and practice of pharmacy", 20th ed., Lippincott Williams & Wilkins, Baltimore, MD, 2000, and "Pharmaceutical dosage forms and drug delivery systems", 6th Edition, Ansel et al., (Media, PA: Williams and Wilkins, 1995). These references provide information on excipients, materials, equipment and process for preparing tablets and capsules and delayed release dosage forms of tablets, capsules, and granules. These references provide information on carriers, materials, equipment and process for preparing tablets and capsules and delayed release dosage forms of tablets, capsules, and granules. Alternative to a delayed release delivery, the compositions can also be prepared in a sustained release, or an extended release, or a combined sustained release and extended-release fraction dosage form, or in an immediate release dosage form, or a combined sustained release fraction and immediate release fraction dosage form, or a combination thereof. The compositions where release is modified can be formulated as matrix preparations, coated preparation, multilayer or tablet in tablet preparations, osmotic preparations etc. The compositions described herein can be coated with a suitable coating material, for example, to delay release once the particles have passed through the acidic environment of the stomach. Suitable coating materials include, but are not limited to, cellulose polymers such as cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate and hydroxypropyl methylcellulose acetate succinate; polyvinyl acetate phthalate, acrylic acid polymers and copolymers, and methacrylic resins that are commercially available under the trade name EUDRAGIT® (Roth Pharma, Westerstadt, Germany), zein, shellac, and polysaccharides. Coatings can be formed with a different ratio of water - soluble polymer, water insoluble polymers and / or pH dependent polymers, with or without water insoluble / water soluble non polymeric excipient, to produce the desired release profile. The coating can be performed on a dosage form (matrix or simple) which includes, but is not limited to, tablets (compressed with or without coated beads), capsules (with or without coated beads), beads, particle compositions, "ingredient as is" formulated as, but not limited to, suspension form or as a sprinkle dosage form Additionally, the coating material can contain conventional carriers such as plasticizers, pigments, colorants, glidants, stabilization agents, pore formers and surfactants. Optional pharmaceutically acceptable excipients include, but are not limited to, diluents, binders, lubricants, disintegrants, colorants, stabilizers, and surfactants. Diluents, also referred to as "fillers," can be used to increase the bulk of a solid dosage form so that a practical size is provided for compression of tablets or formation of beads and granules.

[0242] Suitable diluents include, but are not limited to, dicalcium phosphate dihydrate, calcium sulfate, lactose, sucrose, mannitol, sorbitol, cellulose, microcrystalline cellulose, kaolin, sodium chloride, dry starch, hydrolyzed starches, pregelatinized starch, silicone dioxide, titanium oxide, magnesium aluminum silicate and powdered sugar. The usual diluents include inert powdered substances such as starches, powdered cellulose, especially crystalline and microcrystalline cellulose, sugars such as fructose, mannitol and sucrose, grain flours and similar edible powders. Typical diluents include, for example, various types of starch, lactose, mannitol, kaolin, calcium phosphate or sulfate, inorganic salts such as sodium chloride and powdered sugar. Powdered cellulose derivatives are also useful. Binders can impart cohesive qualities to a solid dosage formulation, and thus can ensure that a tablet or bead or granule remains intact after the formation of the dosage forms. Suitable binder materials include, but are not limited to, starch, pregelatinized starch, gelatin, sugars (including sucrose, glucose, dextrose, lactose and sorbitol), polyethylene glycol, waxes, natural and synthetic gums such as acacia, tragacanth, sodium alginate, cellulose, including hydroxypropylmethylcellulose, hydroxypropylcellulose, ethylcellulose, and magnesium aluminum silicate (Veegum®), and synthetic polymers such as acrylic acid and methacrylic acid copolymers, methacrylic acid copolymers, methyl methacrylate copolymers, aminoalkyl methacrylate copolymers, polyacrylic acid / polymethacrylic acid and polyvinylpyrrolidone. Typical tablet binders include substances such as starch, gelatin and sugars such as lactose, fructose, and glucose. Natural and synthetic gums, including acacia, alginates, methylcellulose, and polyvinylpyrrolidone can also be used. Polyethylene glycol, hydrophilic polymers, ethylcellulose and waxes can also serve as binders. Lubricants can be included to facilitate tablet manufacture.

[0243] Suitable lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, glycerol behenate, polyethylene glycol, talc, and mineral oil. A lubricant can be included in a tablet formulation to prevent the tablet and punches from sticking in the die. The lubricant can be chosen from such slippery solids as talc, magnesium and calcium stearate, stearic acid and hydrogenated vegetable oils. Disintegrants can be used to facilitate dosage form disintegration or "breakup" after administration, and generally include, but are not limited to, starch, sodium starch glycolate, sodium carboxymethyl starch, sodium carboxymethylcellulose, hydroxypropyl cellulose pregelatinized starch, clays, cellulose, alginine, gums or cross-linked polymers, such as cross linked PVP (Polyplasdone® XL from GAF Chemical Corp). Stabilizers can be used to inhibit or retard drug decomposition reactions which include, by way of example, oxidative reactions.

[0244] Suitable stabilizers include, but are not limited to, antioxidants, butylated hydroxytoluene (BHT); ascorbic acid, its salts and esters; Vitamin E, tocopherol and its salts; sulfites such as sodium metabisulphite; cysteine and its derivatives; citric acid; propyl gallate, and butylated hydroxyanisole (BHA). Parenteral compositions The active / s embodied can be formulated for parenteral delivery, such as injection or infusion, in the form of a solution or suspension. The formulation can be administered via any route, such as, the blood stream or directly to the organ or tissue to be treated. Parenteral formulations can be prepared as aqueous compositions using techniques known in the art. Typically, such compositions can be prepared as injectable formulations, for example, solutions or suspensions; solid forms suitable for using to prepare solutions or suspensions upon the addition of a reconstitution medium prior to injection; emulsions, such as water-in-oil (w / o) emulsions, oil-in-water (o / w) emulsions, and microemulsions thereof, liposomes, or emulsomes such as Self Micro-emulsifying Drug Delivery Systems (SMEDDS) and or Micellar. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, one or more polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), oils, such as vegetable oils (e.g., peanut oil, com oil, sesame oil, etc.), and combinations thereof. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and / or by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Solutions and dispersions as described herein can be prepared in water or another solvent or dispersing medium suitably mixed with one or more pharmaceutically acceptable excipients including, but not limited to, surfactants, dispersants, emulsifiers, pH modifying agents, and combination thereof. Suitable surfactants can be anionic, cationic, amphoteric or non-ionic surface-active agents. Suitable anionic surfactants include, but are not limited to, those containing carboxylate, sulfonate and sulfate ions. Suitable anionic surfactants include sodium, potassium, ammonium of long chain alkyl sulfonates and alkyl aryl sulfonates such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium bis-(2-ethylthioxyl)-sulfosuccinate; and alkyl sulfates such as sodium lauryl sulfate. Suitable cationic surfactants include, but are not limited to, quaternary ammonium compounds such as benzalkonium chloride, benzethonium chloride, cetrimonium bromide, stearyl dimethylbenzyl ammonium chloride, polyoxyethylene and coconut amine. Suitable nonionic surfactants include ethylene glycol monostearate, propylene glycol myristate, glyceryl monostearate, glyceryl stearate, polyglyceryl4-oleate, sorbitan acylate, sucrose acylate, PEG- 150 laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbates, polyoxyethylene octylphenylether, PEG- 1000 cetyl ether, polyoxyethylene tridecyl ether, polypropylene glycol butyl ether, Poloxamer® 401, stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallow amide. Examples of amphoteric surfactants include sodium N-dodecyl-p-alanine, sodium N lauryl-p-iminodipropionate, myristoamphoacetate, lauryl betaine and lauryl sulfobetaine.

[0245] The formulation can contain a preservative to prevent the growth of microorganisms. Suitable preservatives include, but are not limited to, parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. The formulation can also contain an antioxidant to prevent degradation of the embodied active / s. The formulation can be buffered to a pH of 3-8 for parenteral administration upon reconstitution. Suitable buffers include, but are not limited to, phosphate buffers, acetate buffers, and citrate buffers. Water-soluble polymers can be used in the compositions for parenteral administration. Suitable water-soluble polymers include, but are not limited to, polyvinylpyrrolidone, dextran, carboxymethylcellulose, and polyethylene glycol. Sterile injectable solutions can be prepared by incorporating the active / s in the required amount in the appropriate solvent or dispersion medium with one or more of the excipients listed above, as required, followed by filtered sterilization.

[0246] Dispersions can be prepared by incorporating the sterilized active / s into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those listed above. Sterile powders for the preparation of sterile injectable solutions can be prepared by vacuum-drying and freeze-drying techniques, which yields a powder of the active / s plus any additional desired ingredient from a previously sterile-filtered solution thereof. The powders can be prepared in such a manner that the particles are porous in nature, which can increase dissolution of the particles. Methods for making porous particles are well known in the art of pharmaceutical formulations for parenteral administration can be in the form of a sterile aqueous solution or suspension of particles formed from the active / s. Acceptable solvents include, for example, water, Ringer's solution, phosphate buffered saline (PBS), and isotonic sodium chloride solution.

[0247] The formulation can also be a sterile solution, suspension, or emulsion in a nontoxic, parenterally acceptable diluent or solvent such as 1,3 -butanediol. In some instances, the formulation can be distributed or packaged in a liquid form. In other embodiments, formulations for parenteral administration can be packed as a solid, obtained, for example by lyophilization of a suitable liquid formulation. The solid can be reconstituted with an appropriate carrier or diluent prior to administration. Solutions, suspensions, or emulsions for parenteral administration can be buffered with an effective amount of buffer necessary to maintain a pH suitable for ocular administration. Suitable buffers include, but are not limited to, acetate, borate, carbonate, citrate, and phosphate buffers. Solutions, suspensions, or emulsions for parenteral administration can also contain one or more tonicity agents to adjust the isotonic range of the formulation. Suitable tonicity agents include, but are not limited to, glycerin, mannitol, sorbitol, sodium chloride, and other electrolytes. Solutions, suspensions, or emulsions for parenteral administration can also contain one or more preservatives to prevent bacterial contamination of the ophthalmic preparations. Suitable preservatives include, but are not limited to, poly hexamethylenebiguanidine (PHMB), benzalkonium chloride (BAK), stabilized oxychloro complexes (otherwise known as Purite®), phenylmercuric acetate, chlorobutanol, sorbic acid, chlorhexidine, benzyl alcohol, parabens, thimerosal, and mixtures thereof. Solutions, suspensions, or emulsions, use of nanotechnology including nano-formulations for parenteral administration can also contain one or more excipients, such as dispersing agents, wetting agents, and suspending agents.

[0248] The Injection formulation contains the active / s either alone or in combination at concentrations embodied and solubilizers such as Ethyl alcohol 20% / ml and Propylene glycol 40% / ml and Water for injection -40% / ml. The solution should be isotonic and tonicity adjusting salts such as sodium chloride can be used. The pH range of 5-9 can be adjusted with suitable bufferants should be 6 - 8 preferably 6.5-7.5. It is a sterile, nonpyrogenic solution. The said Injection formulation can be in the form of a solution or micronized or nanosized dispersion. The said formulation can also be administered via inhalation with or without the aid of a medical device, metered or unmetered, and / or via nebulization for nasal administration for drug delivery to the lungs - viz. Pulmonary. The said formulation can also be administered via the buccal route as buccal drops or as buccal spray using appropriate medical device. The said formulation can be administered via the sublingual route as sublingual drops or as sublingual spray using appropriate medical device. Another variant of the sterile injectable formulation can also be a lyophilized injection. This injection may also contain sodium citrate dihydrate and citric acid anhydrous; and finally, be as a white to yellow lyophilized powder or plug. The solution should be prepared only with 1 to 2 mL of preservative-free Sterile Sodium Chloride Injection, 0.9 percent or preservative-free Sterile Water for Injection. The reconstituted solution is clear, slightly yellow and essentially free from visible particles. The specific gravity of the formulation can be between 1.01 to 1.7 g / ml. The particle size of the liquid droplets can range from 5 micron to 500 micron.

[0249] The Oral Spray formulation encompasses the active / s either alone or in combination; each at concentration of as described before and have excipients such as diluents viz. Mannitol ranging from 10 - 15 mg / ml; Sweeteners such as sucralose from 5- 10 mg / ml, Flavours as Raspberry, Strawberry from 5- 10 mg / ml and tonicity and taste modulators such as sodium chloride and propylene glycol from 0.1-0.5 mg / ml with purified water as the base solvent or carrier. The specific gravity of the formulation can be between 1.01 to 1.5 g / ml. The allied oral sprays can be categorized as buccal or sublingual sprays based on the area of application.

[0250] Additionally, the said Oral Spray may encompass surfactant- solubilizers and gelling agents such as Pluronic Fl 27 or Poloxamer 407 in the concentration ranging from 1 - 200 mg / ml. This formulation is liquid at temperatures less than 10 degree Celsius and starts gelling at temperature range above 30 degrees Celsius. It is a sterile, nonpyrogenic solution. The pH range if reconstituted should be 5-9 preferably 6.5-7.5. It can be administered using appropriate spray containers with specialised spray nozzle to facilitate spray below the tongue viz. sublingually or into the buccal or also the nasal cavity. The Spray can also be in the form of a micronized or nanosized suspension.

[0251] The Nasal Spray formulation would be devoid of the sweeteners and flavours. It gels at body temperature thereby facilitating longer dwell time possibly enhancing the drug penetration through the mucosal lining. This drug delivery mode bypasses the harsh acidic conditions of the stomach and also the hepatic breakdown thereby possibly increasing bio-availability. The specific gravity of the formulation can be between 1.01 to 1.7 g / ml. The said formulation can be administered via the nasal route as nasal drops or as nasal spray using appropriate medical device. The said formulation can be administered via inhalation with or without the aid of a medical device, metered or unmetered, and / or via nebulization.

[0252] The Inhalation or Pulmonary Capsule has the active / s either alone or in combination; each at concentration mentioned above and has excipients such as Magnesium stearate [Inhalation grade] or Lactose [Inhalation grade]. The core weight of the formulation can range from 25 - 500 mg / capsule. The Aerosol or Pulmonary delivery system has the active / s either alone or in combination; each at concentration mentioned above per actuation and has excipients such as propellent gases, propylene glycol, water, surfactants, anti-foam emulsion and anti-freeze excipients. The particle size of the liquid droplets can range from 5 micron to 500 micron.

[0253] Sublingual Tablets have the active / s either alone or in combination; each at concentration mentioned and have excipients such as diluents viz. Lactose monohydrate or Mannitol ranging from 10 - 30 mg / tablet; Disintegrants such as Starch or Crospovidone from 10- 15 mg / tablet; fillers such as Microcrystalline cellulose from 5-10 mg / tablet and lubricants such as Magnesium stearate from 0.5 - 1 mg / tablet. It may additionally contain or 5-10 mg / tablet of taste modulating or masking agents such as sodium chloride or buffers such as potassium dihydrogen phosphate. The core weight of the formulation can range from 50 - 80 mg / tablet.

[0254] The Orally Dispersible Tablets (ODT) have the active / s either alone or in combination; each at concentration mentioned above per tablet and have excipients such as diluents viz. Lactose monohydrate or Mannitol ranging from 10 - 15 mg / tablet; Disintegrants such as Starch or Crospovidone from 10- 15 mg / tablet; fillers such as Microcrystalline cellulose from 5-10 mg / tablet and lubricants such as Magnesium stearate from 0.5 - 1 mg / tablet. The core weight of the formulation can range from 50 - 80 mg / tablet.

[0255] The Buccal Tablets have the active / s either alone or in combination; each at a dose mentioned above tablet and have excipients such as polymers viz. polymers of acrylic acid and C10-C30 alkyl acrylate crosslinked with allyl pentaerythritol exemplified by Carbopol 934 ranging from 10 - 15 mg / tablet and or Hydroxy Propyl Methyl Cellulose (HPMC) K4M from 35-40 mg / tablet; Fillers such as Mannitol (directly compressible) from 10- 15 mg / tablet; and lubricants such as Magnesium stearate from 0.5 - 1 mg / tablet. The core weight of the formulation can range from 50 - 80 mg / tablet

[0256] The Delayed Release Tablets have the active / s either alone or in combination; each at concentration of 0.00001 mg to 200 mg / tablet and have excipients such as Mannitol, Microcrystalline cellulose (MCC PH 102), trisodium phosphate, Hydroxy Propyl Methyl Cellulose (HPMC 5cps), Hydroxy Propyl Methyl Cellulose (HPMC 15cps) and Crospovidone, Colloidal silicon dioxide, Magnesium stearate as the tablet core coated with a Seal Coating composition encompassing Ethyl cellulose using an appropriate solvent system viz. aqueous, non-aqueous; preferably non-aqueous ( Iso-propyl alcohol and Dichloromethane) to a 4-5% weight gain on the tablet cores finally coated with an aqueous gastro-resistant coating composition viz. Eudragit L100-55, Triethyl citrate, opacifier and colorant to a total weight gain of 26-30% of the tablet cores. The core weight of the formulation can range from 50 - 1200 mg / tablet. The Extended-Release Tablets have the active / s either alone or in combination; each at concentration mentioned above and have excipients such as fillers viz. Microcrystalline cellulose (MCC PH 101); polymers viz Hydroxy Propyl Methyl Cellulose (HPMC K100M) and Hydroxy Propyl Methyl Cellulose (HPMC K15M); binders viz. Povidone (PVP K29 / 32) and Lubricants viz. Magnesium stearate as the tablet core coated with a Film Coating composition using an appropriate solvent system viz. aqueous or non-aqueous; preferably non-aqueous (Iso-propyl alcohol and Dichloromethane) to a 2-3% weight gain on the tablet core. The core weight of the formulation can range from 50 - 1200 mg / tablet.

[0257] The Effervescent tablets have the active / s either alone or in combination; each at concentration mentioned above and have excipients such as citric acid, sodium bicarbonate, potassium citrate, mannitol, aspartame, strawberry flavour, bufferants, sodium benzoate and polyethylene glycol 6000. The core weight of the formulation can range from 50 - 2000 mg / tablet.

[0258] The Osmotic-controlled Release Oral delivery System (OROS) Tablets have the active / s either alone or in combination; each at concentration mentioned above and have excipients such as sorbitan monolaurate and Sodium chloride, microcrystalline cellulose (MCC PH 102), polymers viz Hydroxy Propyl Methyl Cellulose (HPMC K100M) and Hydroxy Propyl Methyl Cellulose (HPMC K15M), Colloidal silicon dioxide and Magnesium stearate as the tablet core; a Film coat to the tablet cores to a weight gain of 2.5 to 3.0 % w / w to the tablet core using a nonaqueous medium and a Functional Coat the tablet with Cellulose acetate nonaqueous dispersion in Iso-propyl alcohol to a weight gain of 25-30% w / w of the tablet core finally Laser drilled the tablets with an orifice of 150 - 250 micron. The core weight of the formulation can range from 50 - 1000 mg / tablet.

[0259] The Capsules have the active / s either alone or in combination; each at concentration mentioned above and have excipients such a microcrystalline cellulose (MCC PH 105), Colloidal silicon dioxide and Magnesium stearate as the core; encompassed in a hard gelatin capsule. The core weight of the formulation can range from 30 - 2055 mg / capsule. The Compressed lozenges or Chews or Lollipop have the active / s either alone or in combination; each at concentration mentioned above and have excipients such as ethoxylated hydrogenated castor oil Polyoxyl 35 Castor oil ( Cremophore EL / Kolliphor EL), Dextrate, Polyethylene glycol 6000, microcrystalline cellulose (MCC 102), povidone (PVP K29 / 32) and FD&C Yellow No. 6 and Magnesium stearate as the core. The core weight of the formulation can range from 100 - 3000 mg / unit

[0260] The Soft Gel Capsules have the active / s either alone or in combination; each at concentration mentioned above and have excipients such as propylene glycol, Poly Ethylene Glycol-400, Polyvinyl pyrrolidone K29 / 32, Butylated hydroxy toluene and ethanol-water blend as the core material filled into opaque soft gelatin capsules. The core weight of the formulation can range from 100 - 800 mg / capsule.

[0261] The Quick dissolving films - Oral and or Sublingual, have the active / s either alone or in combination; each at concentration as mentioned above and have excipients such as pullulan, sorbitol, polysorbate 80, sucralose, Monoammonium glycyrrhizinate and peppermint flavour. The core weight of the formulation can range from 50 - 800 mg / unit

[0262] The Oro-Buccal muco-adhesive films - Oral or Sublingual, have the active / s either alone or in combination; each at concentration mentioned above and have excipients such as Hydroxypropyl cellulose, Hydroxyethyl cellulose and Sodium carboxymethyl cellulose, Polyoxyl 35 Castor Oil (Cremophore EL / Kolliphor EL), Sodium benzoate, Parahydroxybenzoate methyl, Parahydroxybenzoate propyl, Sodium citrate and Sodium saccharine. The core weight of the formulation can range from 50 - 80 mg / unit.

[0263] The Oral Emulsions have the active / s either alone or in combination; each at concentration mentioned above and have excipients such as Polyoxyl 35 Castor Oil (Cremophore EL / Kolliphor EL), Saccharin Sodium, caramel, colorant, peppermint oil, com oil, sucrose and water. The specific gravity of the formulation can be between 0.5 - 1.5 g / ml The Eye drop formulations have the active / s either alone or in combination; each at concentration mentioned above and have excipients such as Polysorbate 20 / 80, Benzalkonium chloride, disodium EDTATE, Sodium Carboxymethyl Cellulose (Na CMC), Citric acid monohydrate, sodium hydroxide, hydrochloric acid and water. The final solution is sterile. The specific gravity of the formulation can be between 1.01 - 1.8 g / ml.

[0264] The Vaginal gels have the active / s either alone or in combination; each at concentration mentioned above and has excipients such as Polyoxyl 35 Castor Oil (Cremophore EL / Kolliphor EL), ascorbic acid, Glycerin or Propylene glycol, Hydroxypropyl Methylcellulose (HPMC E50), Trisodium Citrate dihydrate and water. The specific gravity of the formulation can be between 1.01 - 1.8 g / ml.

[0265] The Suppository formulations have the active / s either alone or in combination; each at concentration mentioned above and have excipients such as hard fat, surfactants, and the following inactive ingredients: butylated hydroxy anisole, butylated hydroxytoluene, edetic acid, glycerin, polyethylene glycol 3350, polyethylene glycol 8000, purified water and sodium chloride. The core weight of the formulation can range from 200- 3000 mg / unit

[0266] Topical and transdermal compositions

[0267] The active / s as described herein can be formulated for topical administration. The active / s can have a formula according to the ones mentioned herein. Suitable dosage forms for topical administration include creams, ointments, salves, sprays, gels, lotions, emulsions, liquids, and transdermal patches. The formulation can be formulated for transmucosal, transepithelial, transendothelial, or transdermal administration. The topical formulations can contain one or more chemical penetration enhancers, membrane permeability agents, membrane transport agents, emollients, surfactants, stabilizers, and combination thereof.

[0268] In some embodiments, the active / s can be administered as a liquid formulation, such as a solution or suspension, a semi-solid formulation, such as a lotion or ointment, or a solid formulation. In some embodiments, the active / s can be formulated as liquids, including solutions and suspensions, such as eye drops or as a semi-solid formulation, such as ointment or lotion for topical application to the skin, to the mucosa, such as the eye, to the vagina, or to the rectum. The formulation can contain one or more excipients, such as emollients, surfactants, emulsifiers, penetration enhancers, and the like.

[0269] Suitable emollients include, without limitation, almond oil, castor oil, cetostearoyl alcohol, cetyl alcohol, cetyl esters wax, cholesterol, cottonseed oil, cyclomethicone, ethylene glycol palmitostearate, glycerin, glycerin monostearate, glyceryl monooleate, isopropyl myristate, isopropyl palmitate, lanolin, lecithin, light mineral oil, medium-chain triglycerides, mineral oil and lanolin alcohols, petrolatum, petrolatum and lanolin alcohols, soybean oil, starch, stearyl alcohol, sunflower oil, xylitol and combinations thereof. In some embodiments, the emollients can be ethylhexylstearate and ethylhexyl palmitate. Suitable surfactants include, but are not limited to, emulsifying wax, glyceryl monooleate, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polysorbate, sorbitan esters, benzyl alcohol, benzyl benzoate, cyclodextrins, glycerin monostearate, poloxamer, povidone and combinations thereof. In some embodiments, the surfactant can be stearyl alcohol.

[0270] Suitable emulsifiers include, but are not limited to, acacia, metallic soaps, certain animal and vegetable oils, and various polar compounds, anionic emulsifying wax, calcium stearate, carbomers, cetostearyl alcohol, cetyl alcohol, cholesterol, diethanolamine, ethylene glycol palmitostearate, glycerin monostearate, glyceryl monooleate, hydroxpropyl cellulose, hypromellose, lanolin, hydrous, lanolin alcohols, lecithin, medium-chain triglycerides, methylcellulose, mineral oil and lanolin alcohols, monobasic sodium phosphate, monoethanolamine, nonionic emulsifying wax, oleic acid, poloxamer, poloxamers, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, propylene glycol alginate, self-emulsifying glyceryl monostearate, sodium citrate dehydrate, sodium lauryl sulfate, sorbitan esters, stearic acid, sunflower oil, tragacanth, triethanolamine, xanthan gum and combinations thereof. In some embodiments, the emulsifier can be glycerol stearate. Suitable classes of penetration enhancers include, but are not limited to, fatty alcohols, fatty acid esters, fatty acids, fatty alcohol ethers, amino acids, phospholipids, lecithins, cholate salts, enzymes, amines and amides, complexing agents (liposomes, cyclodextrins, modified celluloses, and diimides), macrocyclics, such as macrocylic lactones, ketones, and anhydrides and cyclic ureas, surfactants, N-methyl pyrrolidones and derivatives thereof, DMSO and related compounds, ionic compounds, azone and related compounds, and solvents, such as alcohols, ketones, amides, polyols (e.g., glycols).

[0271] Suitable emulsions include, but are not limited to, oil-in-water, water-in-oil emulsions or multiple emulsions. Either or both phases of the emulsions can include a surfactant, an emulsifying agent, and / or a liquid non-volatile non-aqueous material. In some embodiments, the surfactant can be a non-ionic surfactant. In other embodiments, the emulsifying agent is an emulsifying wax. In further embodiments, the liquid non-volatile non-aqueous material is a glycol. In some embodiments, the glycol is propylene glycol. The oil phase can contain other suitable oily pharmaceutically acceptable excipients. Suitable oily pharmaceutically acceptable excipients include, but are not limited to, hydroxylated castor oil or sesame oil can be used in the oil phase as surfactants or emulsifiers. Lotions containing the active / s as described herein are also provided. In some embodiments, the lotion can be in the form of an emulsion having a viscosity of between 100 and 1000 centistokes. The fluidity of lotions can permit rapid and uniform application over a wide surface area. Lotions can be formulated to dry on the skin leaving a thin coat of their medicinal components on the skin's surface.

[0272] Creams containing the active / s as described herein are also provided. The cream can contain emulsifying agents and / or other stabilizing agents. In some embodiments, the cream is in the form of a cream having a viscosity of greater than 1000 centistokes, typically in the range of 20,000-50,000 centistokes. Creams, as compared to ointments, can be easier to spread and easier to remove. One difference between a cream and a lotion is the viscosity, which is dependent on the amount / use of various oils and the percentage of water used to prepare the formulations. Creams can be thicker than lotions, can have various uses, and can have more varied oils / butters, depending upon the desired effect upon the skin. In some embodiments of a cream formulation, the water-base percentage can be about 60% to about 75% and the oil base can be about 20% to about 30% of the total, with the other percentages being the emulsifier agent, preservatives and additives for a total of 100%

[0273] Ointments containing the active / s as described herein and a suitable ointment base are also provided. Suitable ointment bases include hydrocarbon bases (e.g., petrolatum, white petrolatum, yellow ointment, and mineral oil); absorption bases (hydrophilic petrolatum, anhydrous lanolin, lanolin, and cold cream); waterremovable bases (e.g., hydrophilic ointment), and water-soluble bases (e.g., polyethylene glycol ointments). Pastes typically differ from ointments in that they contain a larger percentage of solids. Pastes are typically more absorptive and less greasy that ointments prepared with the same components.

[0274] Also described herein are gels containing the active / s as described herein, a gelling agent, and a liquid vehicle. Suitable gelling agents include, but are not limited to, modified celluloses, such as hydroxypropyl cellulose and hydroxyethyl cellulose; Carbopol® homopolymers and copolymers; thermos -reversible gels and combinations thereof.

[0275] Suitable solvents in the liquid vehicle include, but are not limited to, diglycol monoethyl ether; alklene glycols, such as propylene glycol; dimethyl isosorbide; alcohols, such as isopropyl alcohol and ethanol. The solvents can be selected for their ability to dissolve the drug. Other additives, which can improve the skin feel and / or emolliency of the formulation, can also be incorporated. Such additives include, but are not limited, isopropyl myristate, ethyl acetate, C12- C15 alkyl benzoates, mineral oil, squalane, cyclomethicone, capric / caprylic triglycerides, and combinations thereof.

[0276] Also described herein are foams that can include the active / s as described herein. Foams can be an emulsion in combination with a gaseous propellant. The gaseous propellant can include hydrofluoroalkanes (HFAs). Suitable propellants include HFAs such as 1,1 ,1,2-tetrafluoroethane (HFA 134a) and 1,1, 1,2, 3, 3, 3 heptafluoropropane (HFA 227), but mixtures and admixtures of these and other HFAs that are currently approved or can become approved for medical use are suitable. The propellants can be devoid of hydrocarbon propellant gases, which can produce flammable or explosive vapors during spraying. Furthermore, the foams can contain no volatile alcohols, which can produce flammable or explosive vapors during use. Buffers can be used to control pH of a composition. The buffers can buffer the composition from a pH of about 4 to a pH of about 7.5, from a pH of about 4 to a pH of about 7, or from a pH of about 5 to a pH of about 7. In some embodiments, the buffer can be triethanolamine.

[0277] Preservatives can be included to prevent the growth of fungi and microorganisms. Suitable preservatives include, but are not limited to, benzoic acid, butylparaben, ethyl paraben, methyl paraben, propylparaben, sodium benzoate, sodium propionate, benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, and thimerosal. In certain embodiments, the formulations can be provided via continuous delivery of one or more formulations to a patient in need thereof. For topical applications, repeated application can be done or a patch can be used to provide continuous administration of the active / s over an extended period of time.

[0278] Additional Active Agents

[0279] In some embodiments, an amount of one or more additional active agents are included in the pharmaceutical compositions containing the active / s or pharmaceutical salt thereof. Suitable additional active agents include, but are not limited to, DNA, RNA, amino acids, peptides, polypeptides, antibodies, aptamers, ribozymes, guide sequences for ribozymes that inhibit translation or transcription of essential tumor proteins and genes, hormones, immunomodulators, antipyretics, anxiolytics, antipsychotics, analgesics, antispasmodics, anti-inflammatories, antihistamines, anti-infectives, and chemo therapeutics. Other suitable additional active agents include, but are not limited to, statins, cholesterol lowering drugs, glucose lowering drugs. The active / s can be used as a monotherapy or in combination with other active agents for treatment of metabolic disorder (diabetes, high cholesterol, hyperlipidemia, high-triglycerides) .

[0280] Apart from the active - Cannabidiol (CBD), and Cannabigerol (CBG), other cannabinoids such asCannabidiolic acid (CBA), Cannabinol (CBN) and tetrahydrocannabivarin (THCV) in therapeutically effective concentrations can be incorporated either alone or in combinations to address the diseased state in Man and mammals.

[0281] Also, as the inventors progress with their research on Poly (I:C); the inventors further propose the role of Poly (ICI:C) as a promising candidate with Cannabidiol (CBD) and or Cannabigerol (CBG) and or with other minor cannabinoids including but not limited to cannabinol, cannabidiolic acid, delta8-tetrahydrocannabivarin, or delta9-tetrahydrocannabinol, or other isomers stereochemically related compounds, and their combinations thereof to effectively treat viral infections and cancer in mammals.

[0282] References:

[0283] Petrosino S, Verde R, Vaia M, Allara M, luvone T, Di Marzo V. Anti-inflammatory Properties of Cannabidiol, a Nonpsychotropic Cannabinoid, in Experimental Allergic Contact Dermatitis. J Pharmacol Exp Ther. 2018 Jun;365(3):652-663. doi: 10.1124 / jpet.117.244368. Epub 2018 Apr 9. PMID: 29632236.

[0284] Salles EL, Khodadadi H, Jarrahi A, Ahluwalia M, Paffaro VA Jr, Costigliola V, Yu JC, Hess DC, Dhandapani KM, Baban B. Cannabidiol (CBD) modulation of apelin in acute respiratory distress syndrome. J Cell Mol Med. 2020 Nov;24(21): 12869- 12872. doi: 10.11 H / jcmm.l5883. Epub 2020 Oct 15. PMID: 33058425; PMCID: PMC7686987.

[0285] Khodadadi H, Salles EL, Jarrahi A, Chibane F, Costigliola V, Yu JC, Vaibhav K, Hess DC, Dhandapani KM, Baban B. Cannabidiol Modulates Cytokine Storm in Acute Respiratory Distress Syndrome Induced by Simulated Viral Infection Using Synthetic RNA. Cannabis Cannabinoid Res. 2020 Sep 2;5(3): 197-201. doi: 10.1089 / can.2020.0043. PMID: 32923657; PMCID: PMC7480719.

[0286] Fang L, Zhao Z, Wang J, et al. Light-controllable charge-reversal nanoparticles with polyinosinic -poly cytidylic acid for enhancing immunotherapy of triple negative breast cancer. Acta Pharm Sin B. 2022;12(l):353-363. doi:10.1016 / j.apsb.2021.06.006

[0287] Yalcinkaya M, Liu W, Islam MN, et al. Modulation of the NLRP3 inflammasome by Sars-CoV-2 Envelope protein. Sci Rep. 2021;l l(l):24432. Published 2021 Dec 24. doi:10.1038 / s41598-021-04133-7

Claims

ClaimsWe claim:

1. A pharmaceutical composition comprising therapeutically effective amount of a Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol or their combination for use in treating virus infection in a patient caused by an RNA type virus wherein said Cannabinoid or composition thereof produces an enhancement / augmentation of innate immunity of the patient due to at least one of the following effects, i) augmenting induction of interferons; ii) augmenting induction of interferon stimulated factors or genes; iii) by causing apoptosis of virus infected cells.

2. The pharmaceutical composition according to the claim 1 wherein an enhancement / augmentation of innate immunity of the patient is due to apoptosis of virus infected cells.

3. The pharmaceutical composition according to the claim 1 wherein an enhancement / augmentation of innate immunity of the patient is due to induction of interferons4. The pharmaceutical composition according to the claim 1 wherein an enhancement / augmentation of innate immunity of the patient is due to induction of interferon stimulated factors or genes.

5. The pharmaceutical composition according to the claim 2 wherein an enhancement / augmentation of innate immunity of the patient is due to induction of early apoptosis or late apoptosis or both early and late apoptosis of virus infected cells.

6. The pharmaceutical composition according to the claim 3 wherein an enhancement / augmentation of innate immunity of the patient is due to inductionof one or more of Type I (alpha and beta) or Type II (gamma) or Type III (lambda) Interferons or any combination thereof.

7. The pharmaceutical composition according to the claim 4 wherein an enhancement / augmentation of innate immunity of the patient is due to induction of interferon stimulated factors or genes selected from one or more of OAS 1, OAS2, OAS3, OASL.

8. A pharmaceutical composition comprising therapeutically effective amount of a Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol or their combination for use in treating cancer which is treated or can be treated or controlled or can be controlled by using poly(I:C).

9. The pharmaceutical composition for use of claim 8 comprising therapeutically effective amount of a Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol or their combination and Poly(I:C).

10. The pharmaceutical composition for use of claim 8 comprising therapeutically effective amount of a Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol for combining with a composition of Poly(I:C).

11. The pharmaceutical composition for use of claim 10 wherein said two compositions are administered concomitantly.

12. The pharmaceutical composition for use of claim 10 wherein said two compositions are administered one after the other.

13. A kit comprising the pharmaceutical composition of claim 8 and a composition of Poly (I:C).

14. The pharmaceutical composition for use of claim 8 wherein cancer is selected from one or more of breast cancer, Cervical cancer, ovarian cancer, bladder cancer, Metastatic Cancer, colorectal cancer, liver cancer, Gynecologic Cancers, Head and Neck Cancers, Kidney cancer, lung cancer, lymphoma, leukemia, mesothelioma, myeloma, prostate cancer, skin cancer, thyroid cancer, uterine cancer, brain cancer and cancers of the central and peripheral nervous system, cancers of the hematopoietic tissue, vaginal and vulvar cancers.

15. The pharmaceutical composition for use of claim 14 wherein cancer is selected from one or more of breast cancer, Cervical cancer, ovarian cancer, bladder cancer, Metastatic Cancer, Gynecologic Cancers, uterine cancer, vaginal and vulvar cancers.

16. The pharmaceutical composition for use of claim 10 for achieving one or more of the following, a. for reducing dose of Poly(I:C); b. for reducing toxicity / cytotoxicity of Poly(I:C) associated with higher dose; c. for enhancing efficacy of Poly (I:C); d. for synergistic or adjunct therapy with Poly(I:C).

17. The pharmaceutical composition for use of claim 16 without increasing damage to non-cancerous cells.

18. A method of treating a virus infection in a patient caused by an RNA type virus by administering a pharmaceutical composition comprising therapeutically effective amount of a Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol or their combination wherein said Cannabinoid or composition thereof produces an enhancement / augmentation of innate immunity of the patient due to at least one of the following effects, i) augmenting induction of interferons; ii) augmenting induction of interferon stimulated factors or genes; iii) by causing apoptosis of virus infected cells.

19. The method according to the claim 18 wherein an enhancement / augmentation of innate immunity of the patient is due to apoptosis of virus infected cells.

20. The method according to the claim 18 wherein an enhancement / augmentation of innate immunity of the patient is due to induction of interferons.

21. The method according to the claim 18 wherein an enhancement / augmentation of innate immunity of the patient is due to induction of interferon stimulated factors or genes.

22. The method according to the claim 19 wherein an enhancement / augmentation of innate immunity of the patient is due to induction of early apoptosis or late apoptosis or both early and late apoptosis of virus infected cells.

23. The method according to the claim 20 wherein an enhancement / augmentation of innate immunity of the patient is due to induction of one or more of Type I (alpha and beta) or Type II (gamma) or Type III (lambda) Interferons or any combination thereof.

24. The method according to the claim 21 wherein an enhancement / augmentation of innate immunity of the patient is due to induction of interferon stimulated factors or genes selected from one or more of OAS1, OAS2, OAS3, OASL.

25. A method of treating cancer by administering a pharmaceutical composition comprising therapeutically effective amount of a Cannabinoid, preferably, selected from Cannabidiol and Cannabigerol or their combination wherein the cancer is treated or can be treated or controlled or can be controlled by using poly(I:C).

26. The method of claim 25 wherein the pharmaceutical composition is administered with Poly(I:C).

27. The method of claim 25 wherein the pharmaceutical composition is combined with a composition of Poly(I:C).

28. The method of claim 27 wherein said two compositions are administered concomitantly.

29. The method of claim 27 wherein said two compositions are administered one after the other.

30. The method of claim 25 wherein the cancer is selected from one or more of breast cancer, Cervical cancer, ovarian cancer, bladder cancer, Metastatic Cancer, colorectal cancer, liver cancer, Gynecologic Cancers, Head and Neck Cancers, Kidney cancer, lung cancer, lymphoma, leukemia, mesothelioma, myeloma, prostate cancer, skin cancer, thyroid cancer, uterine cancer, brain cancer andcancers of the central and peripheral nervous system, cancers of the hematopoietic tissue, vaginal and vulvar cancers.

31. The method of claim 25 wherein the cancer is selected from one or more of breast cancer, Cervical cancer, ovarian cancer, bladder cancer, Metastatic Cancer, Gynecologic Cancers, uterine cancer, vaginal and vulvar cancers.

32. The method of claim 27 for achieving one or more of the following, e. for reducing dose of Poly(I:C); f. for reducing toxicity / cytotoxicity of Poly(I:C) associated with higher dose; g. for enhancing efficacy of Poly (I:C); h. for synergistic or adjunct therapy with Poly(I:C).

33. The method of claim 32 without increasing damage to non-cancerous cells.

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