Compositions containing cannabinoids and olive oil phenols and medical uses thereof

Abstract

The present invention describes chemical compositions including cannabinoids and olive oil phenols such that the amount of cannabinoids in the chemical compositions is always less than the amount of olive oil phenol and medical-related uses thereof.

Description

[0001] COMPOSITIONS CONTAINING CANNABINOIDS AND OLIVE OIL PHENOLS AND MEDICAL USES THEREOF

[0002] DESCRIPTION

[0003] Technical field

[0004] The present disclosure relates to chemical compositions containing cannabinoids and olive oil phenols and medical-related uses thereof.

[0005] Cannabinoids, primarily derived from the Cannabis sativa plant, encompass a diverse range of compounds known for their interaction with the endocannabinoid system. These compounds exert their effects through interactions with the endocannabinoid system, a complex network comprising cannabinoid receptors (CB1 and CB2) and their endogenous ligands, which regulate a plethora of physiological processes including synaptic plasticity regulation, neurodevelopment, immune system modulation, pain sensation, inflammation, and mood and memory regulation. As such, cannabinoids have garnered considerable interest for their potential therapeutic applications.

[0006] Additionally, cannabinoids exert their effects through interactions with other receptor systems beyond the endocannabinoid system. Notably, cannabinoids act on G protein-coupled receptors (GPCRs) such as GPR3, GPR6, GPR5, and GPR12, which are involved in various cellular signaling pathways. Activation of these receptors by cannabinoids can modulate neurotransmitter release and intracellular signaling cascades, contributing to the diverse pharmacological effects of cannabinoids.

[0007] Moreover, cannabinoids also interact with serotonin receptors, particularly the 5HT1A receptor subtype, which plays a crucial role in mood regulation and anxiety modulation. By modulating serotonin receptor activity, cannabinoids may exert anxiolytic and antidepressant effects, further expanding their therapeutic potential.

[0008] In addition to these receptor systems, cannabinoids interact with ion channels, particularly transient receptor potential (TRP) channels, which are involved in many physiological and pathological functions. Among TRP channels, TRPA1 is of particular interest due to its role in nociception and its modulation by cannabinoids. TRPA1 is a redox-sensitive, non-selective cation channel that mediates extracellular Ca2+entry when intracellular Reactive Oxygen Species (ROS) is increased. Modulation of overexpressed TRPA1 channels, such in cancer pathologies or in hippocampal astrocytes and brain endothelium which correlates with key pathological features of Alzheimer’s Disease (AD), is also of particular interest. Thus, by influencing TRPA1 activity, cannabinoids can provide additional avenues for therapeutic intervention in pathologies such as cancer, AD, seizures, pain management and inflammation- related disorders.

[0009] A critical aspect in the pharmacokinetics of cannabinoids, especially when administered orally, is their low bioavailability. Bioavailability refers to the proportion of a drug that enters the systemic circulation and is available for active effect. Oral administration of cannabinoids is hindered by the phenomenon of first-pass hepatic metabolism, leading to extensive biotransformation in the liver and significantly reducing the active concentration reaching the bloodstream.

[0010] As a result of this low bioavailability, the efficacy of cannabinoids, for instance, in modulating TRPA1 channel activity may be compromised. To compensate for low bioavailability and achieve sufficient plasma concentrations of cannabinoids, higher doses may be required. However, this increases the risk of adverse effects and significantly increase costs. Further, low bioavailability may result in delayed onset of action, as it takes longer for cannabinoids to reach the required concentrations in target tissues.

[0011] Olive oil phenols are compounds found naturally in olive oil. Among these phenolic compounds, secoiridoids such as oleocanthal, oleacein, and oleuropein aglycone have drawn particular interest for their potential health benefits, including anti-cancer and neuroprotective properties. Recent studies have suggested that certain secoiridoids possess the ability to modulate TRPA1 channel activity but, despite their potential, secoiridoids remain relatively unexplored compared to cannabinoids.

[0012] Thus, there exists a need for achieving optimal bioavailability of cannabinoids, enhancing its efficacy in modulating TRPA1 channel activity, while minimizing potential adverse effects.

[0013] Summary of the invention

[0014] According to a first aspect of the present invention, there is provided a chemical composition comprising at least one cannabinoid and at least one olive oil phenol, wherein the amount of cannabinoid is always less than the amount of olive oil phenol. Advantageously, the inventors have surprisingly found that by combining cannabinoids with olive oil phenols, the amount of cannabinoids being less than the amount of olive oil phenols, not only a reduced amount of cannabinoids may be used but also the efficacy in modulating TRPA1 channel activity is greatly improved, compared to compositions not combining cannabinoids with olive oil phenols as well as compared to compositions combining cannabinoids with olive oil phenols wherein the amount of cannabinoids is higher than the amount of olive oil phenols. Preferably, the at least one olive oil phenol is a secoiridoid.

[0015] Optionally, the chemical composition consists essentially of at least one cannabinoid and at least one olive oil phenol, wherein the amount of cannabinoid is always less than the amount of olive oil phenol. Preferably, the at least one olive oil phenol is a secoiridoid. The term “consisting essentially of” shall mean that specific further components can be present, namely those not materially affecting the essential characteristics of the compound or composition. Optionally, said further components not materially affecting the essential characteristics of the composition may be one or more pharmaceutically acceptable excipients. Optionally, a pharmaceutically acceptable excipient may be an aqueous solution or carrier. Optionally, said aqueous solution is a buffer of normal pH or near normal, such as Phosphate Buffer Saline (PBS). Optionally, the pharmaceutically acceptable excipient may be an emulsifier, a buffering agent, a pH adjusting agent, a tonic modifier, a preservative, an antioxidant, a stabilizer, or a combination of the above.

[0016] Optionally, the at least one cannabinoid is selected from the group having general formula I-

[0017] V: and the at least one olive oil phenol is selected from the group having general formula VI-VI I: wherein R1 = H or COOX, X being either H or a straight or branched alkyl group, alkenyl group or alkynyl group having a carbon atom number between 1-5, R2 = H or OH, R3 = H or COOCH3

[0018] Optionally, the at least one cannabinoid is selected from the group of: cannabidiolic acid (CBDA), cannabinolic acid (CBNA), cannabigerolic acid (CBGA), cannabichromenic acid (CBCA), A9-tetrahydrocannabinolic acid (A9-THCA), cannabidiol (CBD), cannabinol (CBN), cannabigerol (CBG), cannabichromene (CBC), A9-THC, cannabidiolic acid methyl ester (CBDA-Me), cannabinolic acid methyl ester (CBNA-Me), cannabigerolic acid methyl ester (CBGA-Me), cannabichromenic acid methyl ester (CBCA-Me), A9-tetrahydrocannabinolic acid methyl ester (A9-THCA-Me). Preferably, the at least one cannabinoid is CBD, CBN, CBG. More preferably, the at least one cannabinoid is CBD.

[0019] Optionally, the at least one olive oil phenol is a secoiridoid selected from the group: oleocanthal (OLC), oleacein (OLA), oleomissinional (OM), oleuropein aglycone (OPA), ligstroside aglycone (LIGA). Preferably, the at least one secoiridoid is OLC, OLA.

[0020] These compound selections allow, for instance, for tailoring the pharmacological profile of the composition depending on the desired target.

[0021] Optionally, the molar ratio of the at least one cannabinoid and the at least one olive oil phenol is 1 :3-1 :40, preferably 1 :3-1 :8. The inventors have surprisingly found that by maintaining a molar ratio cannabinoid / olive oil phenol of about 1 :3, 1 :4, 1 :8 the synergistic effect observed by having a combination of cannabinoids and olive oil phenols wherein the amount of cannabinoids is always less than the amount of olive oil phenols is greatly optimized.

[0022] Optionally, the at least one cannabinoid is CBD and the at least one olive oil phenol is OLC. Optionally, the molar ratio CBD / OLC is 1 :3.

[0023] Optionally, the at least one cannabinoid is CBD and the at least one olive oil phenol is OLA. Optionally, the molar ratio CBD / OLA is 1 :3.

[0024] Optionally, the at least one cannabinoid is CBN and the at least one olive oil phenol is OLC. Optionally, the molar ratio CBN / OLC is 1 :3.

[0025] Optionally, the at least one cannabinoid is CBN and the at least one olive oil phenol is OLA. Optionally, the molar ratio CBN / OLA is 1 :3.

[0026] Optionally, the at least one cannabinoid is CBG and the at least one olive oil phenol is OLC. Optionally, the molar ratio CBG / OLC is 1 :3. Optionally, the at least one cannabinoid is CBG and the at least one olive oil phenol is OLA.

[0027] Optionally, the molar ratio CBG / OLA is 1 :3.

[0028] Optionally, the composition is in the form of an oral solution. Optionally, the composition is in a solid form such as that of a capsule, a tablet, a suppository, or granules.

[0029] Optionally, the composition is in the form of a solution for injection, transdermal solution, inhalation or sublingual administration.

[0030] Optionally, the composition is a chemical composition for oral administration comprising at least one cannabinoid and at least one olive oil phenol, wherein the amount of the at least one cannabinoid is greater than the amount of the at least one olive oil phenol. In such embodiment, the molar ratio of the at least one cannabinoid to the at least one olive phenol is X:1 , X being 1 or greater than 1. Preferably, X is between 3-40, more preferably 3-8, and even more preferably 3.

[0031] Optionally, the composition is an oral dosage form comprising a first composition in which the amount comprising a first composition in which the amount of at least one cannabinoid is less than the amount of at least one olive oil phenol, and a second composition I which the amount of at least one cannabinoid is greater than the amount of at least one olive oil phenol, such that the effective molar ratio at the site of action is within the range of 1 :3 to 1 :40, preferably 1 :3 to 1 :8, more preferably 1 :3.

[0032] Although certain embodiments of the invention rely on compositions wherein the amount of cannabinoid is less than the amount of olive oil phenol, the inventors have also found that in specific administration formats — particularly oral dosage forms — it may be advantageous to formulate the composition such that the amount of cannabinoid is greater than the amount of olive oil phenol. This is due to the differences in physicochemical properties and metabolic fate of cannabinoids and olive oil phenols, which affect their relative absorption, distribution, metabolism, and bioavailability. Cannabinoids, especially in their neutral form, typically exhibit low water solubility and undergo extensive first-pass metabolism, resulting in reduced systemic availability. In contrast, olive oil phenols such as secoiridoids may exhibit better absorption profiles or differing pharmacokinetics, potentially leading to a disproportionate concentration at the site of action when administered in equivalent amounts. Therefore, by increasing the initial amount of cannabinoids in the administered composition, the effective molar ratio of cannabinoid to olive oil phenol at the pharmacological target site can be adjusted to achieve the desired range, such as approximately 1 :3, which has been shown to result in optimal synergistic effects. In some embodiments, this principle is implemented through a dualcomposition dosage form, wherein a first component contains a higher proportion of olive oil phenols, and a second component contains a higher proportion of cannabinoids. This allows fine-tuned control over the temporal and spatial release of the active agents, accommodating their divergent absorption and distribution kinetics while ultimately delivering a targeted and functionally optimized ratio at the site of action.

[0033] According to a second aspect of the present invention, there is provided a composition according to any of the embodiments described for the first aspect of the present invention for use as a medicament.

[0034] Optionally, when the composition is for use as a medicament in the form of an oral solution such as a capsule, a tablet, a suppository or granules, the amount of the at least one cannabinoid is greater than the amount of the at least one olive oil phenol, such that the effective molar ratio at the site of action is within the range of 1 :3 to 1 :40, preferably 1 :3 to 1 :8, more preferably 1 :3. This allows for the differential pharmacokinetics of the components. To ensure that range of 1 :3 to 1 :40, preferably 1 :3 to 1 :8, and more preferably 1 :3 (cannabinoid:olive oil phenol), the formulation may be designed with an initial cannabinoid excess, enabling compensation for post-administration pharmacokinetic disparities and ensuring that the desired functional ratio is achieved at the biological target.

[0035] Optionally, the composition according to the first aspect of the present invention is for use in the treatment of pain. Optionally, the composition according to the first aspect of the present invention is for use in the treatment of cancer, central nervous system disorders, neurodegenerative diseases, psoriasis, Alzheimer’s, or epilepsy.

[0036] Brief description of the drawings

[0037] FIG 1. is a table showing the synergistic effect of CBD, CBN, CBG, CBC, OLC and OLA and combinations of them in rTRPAI or hTRPAI (Wild Type or Y840A mutant) channel through a calcium mobilization assay.

[0038] FIG 2. is a compilation of tables (A, B, C, D) showing the synergistic effect of quadruple combinations of CBN, CBG, CBD and CBC or the quadruple combination of three cannabinoids with an olive oil secoiridoid on MDA-MB 231 breast cancer cells, after 48 hours of exposure.

[0039] FIG 3. is a compilation of tables (A, B, C, D) showing the synergistic effect of the double combination of CBG with CBD and the double combination of cannabinoids or their esters with an olive oil secoiridoid on SK-BR-3 breast cancer cells, after 48 hours of exposure. FIG 4. is a table showing the synergistic effect of the double combination of CBG with CBD and the double combination of CBG with OPA on MCF7 breast cancer cells, after 48 hours of exposure.

[0040] FIG 5. is a table showing the synergistic effect of the double combination of CBG and CBD on A2058 melanoma cancer cells, after 48 hours of exposure.

[0041] FIG 6. is a compilation of tables (A, B, C) showing the synergistic effect of triple combinations of CBN, CBG, CBD and CBC or the quadruple combination of three cannabinoids with an olive oil secoiridoid in SK-M EL-28 melanoma cancer cells, after 48 hours of exposure.

[0042] FIG 7. is a compilation of tables (A, B) showing the synergistic effect of the quadruple combination of three cannabinoids with an olive oil secoiridoid on Huh7 liver cancer cells, after 48 hours of exposure.

[0043] FIG 8. is a compilation of tables (A, B) showing a comparison of the activity of the double and triple combinations of the three cannabinoids between the breast cancer lines and the non- oncogenic epithelial cell line MCF 10A, after 48 hours of exposure.

[0044] FIG 9. is a compilation of tables (A, B, C, D) showing the synergistic effect of the perfusion of CBD and OLC, following high K+ aCSF perfusion in hippocampal slices, in the number of spontaneous events.

[0045] FIG 10. is a compilation of tables (A, B, C) showing the reduction of the synergistic effect of the perfusion of CBD and OLC, in the presence of a TRPA1 antagonist (HC-030031), following high K+ aCSF perfusion in hippocampal slices, in the number of spontaneous events.

[0046] FIG 11. is a compilation of tables (A, B, C) showing the potentiation of the field excitatory post- synaptic potential (fEPSP) following tetanic stimulation in the hippocampus, in CBD and OLC treated hippocampal slices.

[0047] FIG 12. is a graphic representation showing the antitumor effect of the combination of OLC and CBD on MDA-MB 231 xenograft mouse model

[0048] FIG 13. is a compilation of tables (A, B) showing the discrimination and exploration index of a NOR experiment on 5xFAD mice after the oral administration of OLC and CBD.

[0049] Detailed description

[0050] Within the present invention, the term cannabinoids mean substances having a general formula of any of the following I to V, wherein R1 = H or COOX, X being either H or a straight or branched alkyl group, alkenyl group or alkynyl group having a carbon atom number between 1-5:

[0051] Examples of cannabinoids according to the invention are: cannabidiolic acid (CBDA) (I, R1 = COOH), cannabinolic acid (CBNA) (II, R1 = COOH), cannabigerolic acid (CBGA) (III, R1 = COOH), cannabichromenic acid (CBCA) (IV, R1 = COOH) and A9-tetrahydrocannabinolic acid (A9-THCA) (V, R1 = COOH) are phytocannabinoids of the Cannabis sativa plant.

[0052] Cannabinoid acids are considered precursors of neutral cannabinoids (R1 = H). cannabidiol (CBD) (I, R1 = H), cannabinol (CBN) (II, R1 = H), cannabigerol (CBG) (III, R1 = H) and cannabichromene (CBC) (IV, R1 = H) and A9-THC (V, R1 = H) are the neutral phytocannabinoids of the Cannabis sativa plant.

[0053] Compounds with R1 = COOX, wherein X consists of a straight or branched alkyl group having carbon numbers from C1 to C5, are the esters of cannabinoid acids. For example, cannabidiolic acid methyl ester (CBDA-Me) (I, R1 = COOCH3), cannabinolic acid methyl ester (CBNA-Me) (I, R1 = COOCH3), cannabigerolic acid methyl ester (CBGA-Me) (III, R1 = COOCH3), cannabichromenic acid methyl ester (CBCA-Me) (I, R1 = COOCH3) and A9- tetrahydrocannabinolic acid methyl ester (A9-THCA) (V, R1 = COOCH3).

[0054] Within the present invention, the term olive oil phenols mean substances having a general formula according to VI or VII:

[0055] Examples of olive oil phenols according to the invention are: oleocanthal (OLC) (VI, R2 = R3 = H), oleacein (OLA) (VI, R2 = OH, R3 = H) and oleomissinional (OM) (VI, R2 = OH, R3 = COOCH3) oleuropein aglycone (OPA) (VII, R2 = OH) and ligstroside aglycone (LIGA) (VII, R2 = H)

[0056] An in vitro study evaluating the effect on TRPA1 channel activity of cannabinoids of general formula I or II or III or IV or V in combination with secoiridoids of general formula VI or VII was performed using a calcium mobilization assay. HEK293E cells were cultured at 37 °C in DMEM (Thermo Fisher Scientific), supplemented with 10% fetal bovine serum (FBS) and 1 % penicillin / streptomycin. The cells were seeded onto 96-well plates (Clear Bottom Black Polystyrene Poly-D-Lysine Coated Microplates, Greiner Bio-One) at a density of 75,000 (in 100 pl DMEM) and cultured at 37 °C overnight. On the second day following seeding, the medium was changed to OPTI-MEM supplemented with 5% FBS (filtered) before cotransfection using polyethylenimine (PEI) (1.6 pg / well) with plasmid DNAs encoding rTRPAI or WT-hTRPA1 or Y840-hTRPA1 (0.1 pg / well) and the GCaMP-6s calcium sensor (0.035 pg / well). After 4 h, the medium was changed to fresh OPTI-MEM supplemented with 5% FBS (filtered) and 0.1 % Plasmocin (InvivoGen). After an additional 44 h, the cells were washed with Dulbecco's phosphate-buffered saline (DPBS, no calcium, no magnesium, Thermo Fisher Scientific Inc. supplemented with 20 mM HEPES. The plates were then placed into a FlexStation 3 microplate reader (Molecular Devices) to monitor fluorescence changes (excitation, 488 nm; emission, 525 nm; cutoff, 515 nm) after the addition of 50 pl of test compounds (prepared at a 2x working concentration in Hank’s Buffered Salt Solution (HBSS) supplemented with 20 mM HEPES (HBSSH)). The stock solution of the tested compounds was prepared at a concentration of 200mM in dimethyl sulfoxide (DMSO). For each trace, a compound was added at 30 s following the start of the measurement. Readings were collected for an additional 150 s, and data were collected every 2 s. Calcium mobilization in response to test compounds was quantified as the percentage of change (peak fluorescence — baseline fluorescence level, denoted as AF) from its baseline fluorescence level (denoted as F). Data from single dose or dose response experiments were collected in sextuplicate. Allyl isothiocyanate (AITC) and GNE551 were used as positive control. Analysis of the results was performed in GraphPad. Results were expressed as mean ± SEM. Y840A point mutation was introduces using a standard Pfu-based mutagenesis technique according to the QuickChange protocol (Agilent). Y840A point mutation was verified by sequencing.

[0057] Example 1 : Effect of combining different cannabinoids and secoiridoids on TRPA1 channel activity.

[0058] Reference is made to FIG 1., which is a table showing the synergistic effect of CBD, CBN, CBG, CBC, OLC and OLA and combinations of these compounds in rTRPAI or hTRPAI channels through a calcium mobilization assay in a single-dose experiment. The effect of CBD, CBN, CBG, CBC, oleacein, oleocanthal and combinations thereof in rTRPAI channels using a calcium mobilization assay in a single-dose experiment is shown. As blank, HEK293E cells expressing only the calcium sensor (GCaMP6s) were used. As positive control, the natural agonist of TRPA1 , AITC was used. * = p < 0.05; ** = p < 0.01 ; *** = p < 0.001 ; **** = p < 0.0001 .

[0059] Electrophilic agonists, such as allyl isothiocyanate (AITC), activate TRPA1 through covalent modification of cysteine residues located within the N-terminal cytoplasmic domain of the receptor. However, neither secoiridoids nor cannabinoids seem to activate this channel in the same manner as the electrophilic TRPA1 agonist AITC. Oleocanthal seems to maintain its effect on TRPA1 channels after mutation of the TRPATs cysteine domain (Cys621 , Cys641 , Cys665) while cannabinoids, such as cannabidiol and cannabichromene, are proposed to bind to a hydrophobic region of TRPA1 channel. Additionally, secoiridoids, such as oleocanthal are highly unlikely to activate TRPA1 channel by entering the hydrophobic pocket, given their lower logP values (e.g., oleocanthal logP = 1.1541 vs. cannabidiol logP = 6.342.

[0060] This hypothesis is confirmed by the experiments on WT-hTRPA1 and Y840A-hTRPA1 variants.

[0061] The cryo-EM structure of hTRPAI in complex with GNE551 (PDB: 6X2J) provides structural insight into ligand binding. Y840 appears to be a critical residue for the binding of GNE551 and cannabinoids to the TRPA1 channel, as the agonistic effects of CBC, CBN, CBD, and CBG are abolished in the Y840A-hTRPA1 mutant. In contrast, oleocanthal (OLC) and oleacein (OLA) retain their activation effect on Y840A-hTRPA1 . Specifically, at a concentration of 50 pM, CBC, CBN, CBD, CBG, GNE551 , OLC, OLA, and allyl isothiocyanate (AITC) exhibited strong agonistic effects on WT-hTRPA1. However, the agonistic effects of CBC, CBN, CBD, CBG, and GNE551 were completely abolished in the Y840A-hTRPA1 mutant.

[0062] This hypothesis is also confirmed by the experiments on rTRPAI or hTRPAI channel activation. A synergistic effect on rTRPAI channel activation upon combination of cannabinoids with secoiridoids is observed. While each compound individually exhibits an agonistic effect comparable to the positive control, AITC, the combination notably surpasses this baseline. Specifically, a mixture of oleocanthal (OLC) or oleacein (OLA) with cannabidiol (CBD) in a 3:1 ratio (30 pM secoiridoid and 10 pM cannabinoid) achieves superior TRPA1 activation. This enhanced effect significantly outperforms equal concentrations of OLC / OLA and CBD (20 pM each) and other ratios like 1 :3 (10 pM secoiridoid and 30 pM cannabinoid), allowing for reduced amounts of cannabinoids to be used. Triple cannabinoid combinations (CBN / CBG / CBD) did not show a significant increase on the channel activation confirming the hypothesis that they share the same binding site. Cannabinoids and secoiridoids showed an enhanced effect in combination with AITC, further supporting the hypothesis that neither cannabinoids nor secoiridoids share the same binding site as AITC.

[0063] Similar results were observed for the combination of cannabinoids and secoiridoids in activating the hTRPAI channel. Notably, a molar ratio of 1 :3 (10 pM cannabinoid and 30 pM secoiridoid) exhibited a strong synergistic effect. Furthermore, the half-maximal effective concentration (ECS0) of oleocanthal (OLC) for hTRPAI activation was determined to be 2.3 pM, which decreased to 0.8 pM when co-administered with 9 pM cannabidiol (CBD), indicating a significant enhancement in potency.

[0064] This discovery highlights the unique efficacy of specific proportional combinations of these compounds in TRPA1 activation, through distinct binding site interactions, presenting a significant advancement in pharmacological applications.

[0065] In vitro cytotoxic study of the medicinal properties of cannabinoids of general formula I or II or III or IV or V in combination with secoiridoids of general formula VI or VII.

[0066] The anti-proliferative I cytotoxic activity of cannabinoids of general formula I or II or III or IV or V in combination with secoiridoids of general formula V or VII was studied by the in vitro MTT colorimetric procedure. This method is widely used to measure cellular metabolic activity as an indicator of cell viability, division, and cytotoxicity, and based on the reduction of a yellow salt of tetrazole ((3- (4,5-dimethylthiazol-2-yl) -2). 5-diphenyltetrazolium bromide (MTT) in violet formazan crystals from metabolically active cells.

[0067] The synergistic effect of cannabinoids with phenols was evaluated based on the coefficient of drug interaction (Coefficient of drug Interaction, GDI) which is calculated based on the following formula: GDI = 100 x AB% survival I survival x B% survival) where AB % is the % cell viability after combination drug action, A% cell viability after component A action and B% cell viability after component B action. When the GDI coefficient is greater than 1 the examined drugs show a competitive effect, when it is equal to 1 they show an additive effect while when the GDI coefficient is less than 1 we have a synergistic effect. In fact, when the coefficient is less than 0.7 then the synergy is considered strong.

[0068] Briefly for this cytotoxicity test, the MDA-MB 231 cancer cell line (human breast cancer cell line), the SK-BR-3 cancer cell line (human breast cancer cell line), the MCF-7 cancer cell line (human breast cancer cell line), the cancer cell line A2058 (melanoma line), the SK-MEL-28 line (melanoma line), the Huh7 cell line (human liver cancer cell line) and MCF 10A cell line (non-oncogenic breast epithelial cell line) were used. All cell lines were tested under O2 conditions of 20% v / v, in the presence of FBS 10% v / v after 48 h or 72 h of incubation with the selected substances listed in each example below.

[0069] The study of the cytotoxic activity of cannabinoids of general formula I or II or III or IV or V in combination with secoiridoids of general formula V or VII by the measurement procedure using MTT, showed that the substances at a concentration of less than 20 pM (for each ) can lead to a 50% reduction in the population of cancer cells MDA-MB-231 , SK-BR-3, MCF-7, A2058, SK-MEL-28 and Huh7 and therefore these substances and any pharmaceutical preparations resulting in these can be used for the treatment of breast, melanoma and liver cancer.

[0070] Example 2: Effect of a combination of cannabinoids and secoiridoids on breast cancer cell line MDA-MB 231.

[0071] Example 2a: Double and triple combinations of the cannabinoids CBG, CBN, CBD

[0072] In the MDA-MB 231 cell line, the combination of CBG with CBN showed better activity than CBN and CBG separately after 48 hours, at concentrations of 10 - 30 pM (Figure 1A). Specifically, the combination of CBG with CBN, at concentrations of 20 pM each, resulted in a 75.2% reduction in the cell population, while in the same cell line CBN has EC50 = 35.1 pM and CBG has EC50 = 33.7 pM. The CDI coefficient was calculated to be 0.3, a value indicating strong synergistic activity.

[0073] In the same cancer cell line, the combination of CBD with CBN showed a stronger effect than either cannabinoid alone after 48-hour incubations (Figure 1A). Specifically, the combination of CBN with CBD, at concentrations of 20 pM and 10 pM respectively, caused a 72% reduction in the cell population, while the EC50 values of CBN and CBD are 35.1 pM and 29.6 pM respectively. The CDI coefficient was estimated to be 0.4, a value indicating a high synergistic effect.

[0074] The triple combination of CBN, CBG and CBD showed better activity after 48 hours upon increasing the CBN concentration to 20 pM (Figure 1 B). Specifically, the triple combination of CBN, CBG and CBD at concentrations of 20, 10, 5 pM respectively, caused a reduction of the cell population by 59.6%, while their combination at lower concentrations of CBN had a much smaller effect.

[0075] Example 2b: Combinations of CBG, CBN, CBD and CBC with olive oil secoiridoids

[0076] A quadruple combination of CBG, CBN, CBD and OLC showed better performance compared to the triple combination of CBG, CBN, CBD or OLC alone after 48 hours of exposure (Figure 1C). Specifically, the combination of CBG, CBN, CBD, OLC at concentrations of 10 pM for each cannabinoid and 5 pM for OLC, resulted in a 33.2% reduction in the cell population, while the triple combination of CBG, CBN, CBD caused only a 3.2% reduction in cell population and OLC alone reduced it by 22.5%. The CDI coefficient was calculated to be 0.9, a value indicating a slight synergistic effect.

[0077] The combination of CBG, CBN, CBD and OLA showed increased activity compared to both the triple combination of CBG, CBN, CBD and OLA alone after 48 hours (Figure 1C). Specifically, the combination of CBG, CBN, CBD and OLA, at concentrations of 10 pM for each cannabinoid and 15 pM of OLA resulted in a 34% reduction in the cell population. The triple combination of CBG, CBN, CBD did not show any activity (97% survival) while OLA alone caused only 17.6% reduction. The CDI coefficient was calculated to be 0.8, indicating synergistic effect.

[0078] The combination of CBG, CBN, and CBD with LIGA showed better but relatively low activity compared to both the triple combination of CBG, CBN, CBD and LIGA alone afterr 48 hours (Figure 1C). Specifically, the quadruple combination of CBG, CBN, CBD and LIGA, at concentrations of 10 pM for each cannabinoid and 15 pM for LIGA, resulted in 83.9% cell survival compared with 97% survival using the triple combination alone and 87.5% survival using LIGA alone. The CDI coefficient was calculated to be 1 , indicating additive activity.

[0079] The combination of CBG, CBN and CBD with OPA showed better activity (Figure 1C). Specifically at concentrations of 10 pM for each cannabinoid and for OPA, the quadruple combination resulted in the survival of 69.7% of the cells. Incubation for 48 hours with the triple combination of CBG, CBN and CBD showed 97% cell survival and OPA 84.7%. The CDI coefficient was calculated to be 0.8, indicating a slight synergistic effect.

[0080] The combination of CBG, CBN and CBD with OM showed improved activity over both the triple combination of CBG, CBN and CBD and OM separately at 48 hours (Figure 1C). Specifically, the combination of CBG, CBN, CBD and OM, at concentrations of 10 pM for each cannabinoid and 35 pM for OM, resulted in a 40.2% reduction in the cell population, a statistically significantly greater reduction than that observed with the triple combination of CBG, CBN, CBD (only 3% reduction) or with OM alone (25.9% reduction). The GDI coefficient was calculated to be 0.8, indicating synergistic effect.

[0081] The combination of CBG, CBN, CBC and OLA showed increased activity compared to both the triple combination of CBG, CBN, CBC and OLA alone when used separately for 48-hour cell incubation (Figure 1 D). Specifically, the combination of CBG, CBN, CBC and OLA, in concentrations of 10 pM for each cannabinoid and 15 pM of OLA resulted in a 22% reduction in the cell population. The triple combination of CBG, CBN and CBC did not show any activity, while OLA alone caused only 17% reduction in the cell population after 48 hours of incubation. The CDI coefficient was calculated to be 0.9, indicating a slight synergistic effect.

[0082] Example 3: Effect of a combination of cannabinoids and secoiridoids on the breast cancer cell line SK-BR-3.

[0083] Example 3a: Double combination of the cannabinoids CBG and CBD

[0084] In the SK-BR-3 cancer cell line, the combination of CBG with CBD showed increased activity compared to CBD and CBG alone at 48 hours. Specifically, the combination of CBG with CBD at concentrations of 30 pM and 10 pM, respectively, resulted in the survival of 46.2% of the cell population (Figure 2A). CBD in this cell line has EC50 = 29.2 pM and CBG has EC50 = 38.6 pM in 48 hours, while the CDI coefficient for the combination was calculated to be 0.7, a value that indicates high synergistic activity.

[0085] Example 3b: Double combinations of cannabinoids and their esters with olive oil secoiridoids

[0086] In the same cancer line, the combination of CBG with OPA showed better activity than CBG and OPA alone after 48hours (Figure 2B). Specifically, the combination of CBG with OPA at a concentration of 20 pM each, caused a 62% reduction in cell population, a statistically significant decrease compared to CBG alone (10% reduction, EC50 = 38.6 pM) or OPA alone (38.4% reduction). The CDI coefficient for the combination was calculated to be 0.7, a value indicating a high synergistic effect.

[0087] The combination of CBG with LIGA showed better activity compared to CBG and LIGA separately at 48 h (Figure 2C). Specifically, the combination of CBG with LIGA, at concentrations of 30 pM and 10 pM respectively, resulted in a 54% reduction in the cell population, whereas CBG alone (EC50 = 38.6 pM) and LIGA alone induced reduction of the cell population by approximately 32%. The GDI coefficient for the combination was calculated to be 1 (additive effect).

[0088] In addition, the combination of CBDA-Me with OPA showed better activity compared to CBDA- Me and OPA separately at 48 hours (Figure 2D). Specifically, the combination of CBDA-Me with OPA at concentrations of 20 and 10 pM, respectively, resulted in a 36.6% reduction in the cell population, a statistically significant decrease compared to either CBDA-Me alone (93.4 % cell survival, EC50 = 37.8 pM) or OPA alone (80.7% cell survival). The GDI coefficient for the combination was calculated to be 0.8, a value indicating a synergistic effect.

[0089] In the same cancer cell line, the combination of CBGA-Me with OPA at concentrations of 20 and 10 pM respectively showed better activity compared to CBGA-Me and OPA separately after 48 h (Figure 2D). Specifically, the combination of CBGA-Me with OPA at the above concentrations resulted in the survival of 57.9% of the cells, CBGA-Me alone (EC50 = 27.5 pM) and OPA alone showed low activity (80 % cell survival). The GDI coefficient for the combination was calculated to be 0.9 (synergistic effect).

[0090] Finally, the combination of CBGA-Me with LIGA at concentrations of 20 and 10 pM, respectively, showed better activity compared to CBGA-Me and LIGA separately after 48 hours (Figure 2D). Specifically, the above combination resulted in the survival of 52.8% of cells, a statistically significant difference compared to CBGA-Me alone (EC50 = 27.5 pM, 82.1% survival) and LIGA alone (95.4% survival).

[0091] Example 4: Effect of a combination of cannabinoids and secoiridoids on the MCF7 breast cancer cell line. Example 4a: Double combination of the cannabinoids CBG and CBD

[0092] In the MCF-7 cell line, the combination of CBG with CBD showed better activity than each cannabinoid individually after 48 hours (Figure 3). Specifically, the combination of CBG with CBD, at concentrations of 10 pM each, resulted in a reduction of the cell population by 36.7%. In fact, increasing the concentrations to 20 pM and 10 pM for CBG and CBD, respectively, caused a 41.5% reduction, a statistically significant decrease compared to either cannabinoid alone. CBD has EC50 = 24.1 pM and CBG has EC50 = 29.6 pM in this cell line, and the GDI coefficient was estimated to be less than 0.7, a value indicating a strong synergistic effect.

[0093] Example 4b: Double combination of CBG with the secoiridoid OPA In the same cell line, the combination of CBG with OPA showed better activity than CBG and OPA individually after 48 hours (Figure 3). Specifically, the combination of CBG with OPA at a concentration of 20 pM each, caused a 23% reduction in cell population, while incubation with either CBG alone (14% reduction, EC50 = 38.6 pM) or OPA alone showed little or no activity. The CDI coefficient for the combination was calculated to be 0.8, a value indicating a synergistic effect.

[0094] Example 5: Effect of cannabinoid combination on the A2058 melanoma cell lineln the A2058 melanoma cell line, the combination of CBG with CBN showed better activity than CBN and CBG alone after 48 hours (Figure 4). Specifically, the combination of CBG with CBN, at concentrations of 20 pM each, resulted in the survival of 50.6% of cells, a statistically significant difference compared to CBN alone (EC50 = 35.5 pM) and CBG alone (EC50 = 40.8 pM), which did not show any effect at the concentrations used. The CDI coefficient was calculated to be 0.5, a value indicating a strong synergistic effect.

[0095] In the same cell line, the combination of CBN with CBD showed a stronger effect than CBN and CBD alone after 48 hours (Figure 4). Specifically, the combination of CBN with CBD, at concentrations of 20 pM and 10 pM respectively, caused a 40% reduction in cell population, a statistically significant decrease compared to CBN alone (EC50 = 35.5 pM) or CBD alone (EC50 = 24.3 pM)s. The CDI coefficient was estimated to be 0.6, a value indicating a strong synergistic effect.

[0096] The combination of CBG with CBD showed better activity than CBG and CBD separately at 48 (Figure 4). Specifically, the combination of CBG with CBD, at concentrations of 25 pM and 10 pM respectively, resulted in a 67.1 % reduction in the cell population, a statistically significant decrease compared to CBG alone (EC50 = 40.8 pM) or CBD (EC50 = 24.3 pM). The CDI coefficient was calculated to be 0.4, a value indicating strong synergistic activity.

[0097] Example 6: Effect of a combination of cannabinoids and secoiridoids on the SK-MEL- 28 melanoma cell line.

[0098] Example 6a: Double combinations of cannabinoids CBG, CBN and CBD

[0099] In the SK-MEL-28 melanoma cell line, the combination of CBN with CBG showed stronger activity than CBN and CBG alone at 48 hours (Figure 5A). Specifically, the combination of CBG with CBN at concentrations of 20 pM each , resulted in the survival of 52.6% of cells. The cannabinoids CBN (EC50 = 39.9 pM) and CBG (EC50 = 41.5 pM) did not show any effect at these concentrations individually. The GDI coefficient was calculated as 0.6, a value indicating a strong synergistic effect.

[0100] In the same cell line, the combination of CBN with CBD showed a stronger effect than CBN and CBD alone after 48 hours (Figure 5A). Specifically, the combination of CBN with CBD at concentrations of 20 pM and 10 pM respectively, caused a 47.3% decrease in cell population, a statistically significant decrease compared to CBN alone (EC50 = 39.9 pM) and CBD alone (EC50 = 23.7 pM). The CDI coefficient was calculated as 0.6, a value indicating a strong synergistic effect.

[0101] The combination of CBG with CBD showed stronger activity than CBG and CBD separately after 48 hours (Figure 5A). Specifically, the combination of CBG with CBD at concentrations of 20 pM and 10 pM respectively, results in the survival of 59.4% of cells. The cannabinoids CBG (EC50 = 39.9 pM) and CBD (EC50 = 23.7 pM) did not show any effect at these concentrations individually. The CDI coefficient was calculated as 0.7, a value indicating a strong synergistic effect.

[0102] Example 6b: Triple combinations of cannabinoids CBG, CBN, CBD and CBC with the secoiridoid OLA

[0103] The combination of CBN, CBG and CBD with OLA showed better activity than both the triple combination of cannabinoids and OLA alone at 48 hours (Figure 5B). Specifically, the combination of CBG, CBN and CBD and OLA, at concentrations of 10 pM for each cannabinoid and 15 pM for OLA, resulted in a 46.9% reduction. Incubation with the triple combination of CBN, CBG and CBD showed 82.8% cell survival and with OLA alone 86.3%. The CDI coefficient was estimated to be 0.7, a value indicating strong synergistic activity.

[0104] The combination of CBN, CBG and CBC with OLA showed better activity than both the triple combination of cannabinoids and OLA alone after 48 hours (Figure 5C). Specifically, the combination of CBG, CBN, CBC and OLA, in concentrations of 10 pM for each cannabinoid and 15 pM for OLA, resulted in a 48% reduction. Incubation with the triple combination of CBN, CBG and CBC showed 82% cell survival and with OLA alone, 76%. The CDI coefficient was estimated to be 0.9, a value indicating synergistic activity.

[0105] Example 7: Effect of a combination of cannabinoids and secoiridoids on the Huh7 liver cancer cell line.

[0106] In the Huh7 liver cancer cell line, the combination of CBN, CBG, CBD with OLA showed better efficacy than the triple combination of cannabinoids alone and OLA separately after 48 (Figure 6A). Specifically, the combination of CBG, CBN, CBD and OLA, at concentrations of 10 pM for each cannabinoid and 25 pM for OLA, resulted in the survival of 66.2% of the cells. Incubation with the triple combination of CBN, CBG, CBD showed 93.5% survival, and with OLA alone, 80.8% survival. The CDI coefficient was calculated to be 0.9, a value indicating a synergistic effect.

[0107] The combination of CBN, CBG and CBC with OLA showed better activity in Huh7 cells than the triple combination of cannabinoids alone and OLA separately after 48 hours (Figure 6B). Specifically, the combination of CBG, CBN, CBC and OLA, at concentrations of 10 pM for each cannabinoid and 25 pM for OLA, resulted in a 69% reduction. Incubation with the triple combination of CBN, CBG and CBC showed 44% cell survival, and with OLA alone, 76%. The CDI coefficient was estimated to be 0.9, a value indicating synergistic activity.

[0108] Example 8: Effect of the combination of cannabinoids and secoiridoids on the non- oncogenic epithelial cell line MCF 10A.

[0109] In the MCF7 breast cancer cell line, the combination of CBG with CBD showed a stronger effect than in the non-oncogenic MCF10A cell line (Figure 7A).

[0110] In the breast cancer cell line MDA-MB 231 , the combination of CBN with CBD and CBN showed stronger activity compared to the same combination in the non-oncogenic MCF10A cell line (Figure 7B).

[0111] In the MDA-MB 231 breast cancer cell line, the combination of CBN, CBG and CBD showed stronger activity than when compared to the same combination in the non-oncogenic MCF10A cell line (Figure 7B).

[0112] In vivo study of the antitumor effect of combinations of cannabinoids of general formula I, II, III, IV, or V in combination with secoiridoids of general formula V or VII in an MDA- MB-231 xenograft model of cancer

[0113] Female nude mice (CB17-SCID), aged 6-8 weeks and weighing 15-20 g, were obtained from Charles River Laboratories and housed under controlled conditions. MDA-MB-231 cells were cultured in four T-175 flasks until they reached 90% confluency, harvested using trypsin, and resuspended in PBS solution (1.2 mL per flask). Each cell suspension was used to inoculate four mice, with cell suspensions (~1.5x10A6 cells / 100 pL) injected subcutaneously on both sides in the breast area to create orthotopic breast tumors. Treatments began 5 days postinjection. Mice were divided into two groups (n=10 per group): control (vehicle, corn oil) and a combination of oleocanthal (10 mg / kg) and CBD (50 mg / kg). Compounds were administered via oral gavage 3 times per week for 40 days. After 40 days, the animals were sacrificed. The tumors in each mouse were surgically removed and weighed using a precision balance. Data were analyzed using GraphPad, with tumor volumes and body weights expressed as mean ± SEM, and statistical significance determined by one-way ANOVA followed by Tukey's post- hoc test (p<0.05). All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) and complied with ARRIVE guidelines.

[0114] Example 9: Antitumor effect of the combination of cannabidiol and oleocanthal

[0115] A combination of oleocanthal (10 mg / kg) and CBD (50 mg / kg) was administered via oral gavage daily for 40 days. The results (Figure 12) demonstrated a significant reduction in tumor size and weight in the combination treatment group. Tumor weight in the control group averaged 0.26 g, whereas the combination treatment group showed a marked decrease to 0.13 g. Statistical analysis using one-way ANOVA followed by Tukey's HSD test confirmed the significance of these results (p<0.05). These findings indicate that the combination of CBD and OLC exhibits a significant effect in reducing tumor growth, providing a potential therapeutic composition for the treatment of breast cancer. The combination therapy offers enhanced efficacy over individual treatments, representing a significant advancement in cancer therapeutics.

[0116] Ex vivo study of the medicinal properties of cannabinoids of general formula I or II or III or IV or V in combination with secoiridoids of general formula V or VII.

[0117] Brain Slice Preparation

[0118] Mice were euthanized under halothane anesthesia. The brain was removed and placed in ice cold oxygenated artificial cerebrospinal fluid (aCSF) (95%O2 / 5% CO2) containing (in mM): 125 NaCI, 3.5 KCI, 26 NaHCO3, 1 MgCI2 and 10 glucose (pH =7.4, 315 mOsm / l). The brain was blocked and the part containing the hippocampus was glued onto the stage of a vibratome (Leica, VT1000S, Leica Biosystems GmbH, Wetzlar, Germany). The brain slices (400pm) containing the HPC were placed in a submerged chamber containing oxygenated (95% 02 / 5% CO2) aCSF (in mM): 125 NaCI, 3.5 KCI, 26 NaHCO3, 2CaCI2, 1 MgCI2 and 10 glucose (pH = 7.4, 315 mOsm / l) at 36,6 oC. The brain slices were allowed to equilibrate for at least 1 h in this chamber before recordings began. The slices were placed in a temperature-controlled slice chamber at 36,6 oC with 95% 02-5% CO2 under a stereoscope continuously perfused initially with control aCSF containing (in mM): 125 NaCI, 3.5 KCI, 26 NaHCO3, 2CaCI2, 1 MgCI2 and 10 glucose (pH = 7.4, 315 mOsm / l), followed by high K+ aCSF (aCSF containing, in mM: 125 NaCI, 7.5 KCI, 26 NaHCO3, 1 MgCI2, 2 CaCI2 and 10 glucose (pH = 7.4, 315 mOsm / l). To evaluate the effect of CBD and / or oleocanthal, different concentrations of each compound were diluted in high K+ aCSF.

[0119] Dose Response Analysis in electrophysiological recordings.

[0120] It is well established that spontaneous activity recordings from brain slices resemble the activity recorded in vivo. Moreover, an increase in the concentration of extracellular potassium ions [K+] in the perfusing solutions induces depolarization, increased firing rates and burst firing. According to previous studies, perfusion with high [K+] aCSF is often used to model seizures in brain slices from mice because high [K+]-induced activity closely resembles that observed in human epilepsy.

[0121] For the spontaneous local field potentials (LFPs) the recording electrode was filled with 3 M NaCI and was positioned in the CA1 region of distinct hippocampal brain slices. The recordings were amplified using the EXT-02F amplifier (National Instruments), digitized with ITC-18 (Instrutech, Inc.) and recorded on a computer running WindowslO with WinWCP software (Stratchclyde electrophysiology software). To quantify the number of spontaneous activity events the selectable high pass filter was adjusted at 3 Hz, for offset removal and elimination of line frequency noise, while the low-pass filter was adjusted at 300Hz.

[0122] For analysis of spontaneous events, 30 spontaneous voltage traces of 32 s duration were acquired under the experimental conditions described above. In off-line analysis, the 32- second voltage signals were first decimated (down-sampled) by a factor of 10. As a spontaneous event, any voltage response larger than 3 ■ ob was identified.

[0123] Before assessing the effect of CBD and oleocanthal diluted in the same high K+ aCSF solution, an initial dose response analysis of each compound separately was performed. Specifically, the following concentrations of CBD diluted in high K+ aCSF were tested: i) 1 pM, ii) 2,5pM, iii) 5pM, iv) 10pM, v) 20 pM and vi) 40 pM. For oleocanthal, the tested concentration were: i)1 pM, ii)10 pM, iii)20 pM, iv)50 pM and v)100 pM.

[0124] For the dose response analysis of the perfusion of CBD and oleocanthal inhigh K+ aCSF, the following concentrations were tested:

[0125] 1. i) CBD 1 pM, ii) CBD 1 pM plus oleocanthal 1 pM, iii) CBD 1 pM plus oleocanthal 10pM, iv) CBD 1 pM plus oleocanthal 20pM, v) CBD 1 pM plus oleocanthal 50pM and vi) CBD 1 pM plus oleocanthal 100pM. 2. i) CBD 2,5pM, ii) CBD 2,5pM plus oleocanthal 1 M, iii) CBD 2,5pM plus oleocanthal 10pM, iv) CBD 2,5pM plus oleocanthal 20pM, v) CBD 2,5pM plus oleocanthal 50pM and vi) CBD 2,5pM plus oleocanthal 100pM.

[0126] 3. i) CBD 5pM, ii) CBD 5pM plus oleocanthal 1 M, iii) CBD 5pM plus oleocanthal 10pM, iv) CBD 5pM plus oleocanthal 20pM, v) CBD 5pM plus oleocanthal 50pM and vi) CBD 5pM plus oleocanthal 100pM.

[0127] A combination of CBD, OLC and HC-030031 , a TRPA1 antagonist, was also evaluated by perfusion with 10 pM HC-030031 , before and during perfusion with CBD 2.5 pM and OLC 20 pM. Before applying the different compounds and doses, the brain slices were initially perfused with high K+ aCSF to induce seizures. The spontaneous activity after the CBD and / or oleocanthal and / or HC-030031 was normalized to the activity recorded after perfusing with high K+ aCSF. Carbamazepine (CBZ) was used as a positive control.

[0128] Example 10:

[0129] Effect of the combination of CBD and OLC on the reduction of high [K+]-induced activity

[0130] Independent applications of 2.5 pM CBD or 20 pM OLC following high K+ aCSF perfusion did not yield a notable decrease in the number of spontaneous events. In contrast, a 1 :8 ratio of CBD to OLC, with respective concentrations of 2.5 pM and 20 pM, resulted in a remarkable 74% reduction in spontaneous events, outperforming perfusion with 100 pM carbamazepine (CBZ), which achieved a 49% reduction. This indicates a synergistic interaction between CBD and OLC for reducing high [K+]-induced activity.

[0131] Effect of the combination of CBD and OLC on the reduction of high [K+]-induced activity in the presence of a TRPA1 antagonist.

[0132] To determine if part of the effect is through TRPA1 activation, 10 pM HC-030031 , a selective TPRA1 antagonist, was applied before and during perfusion with 2.5 pM CBD and 20 pM OLC. This combination resulted in 27.2% reduction in spontaneous events in comparison with the 74% reduction achieved by 2.5 pM CBD and 20 pM OLC alone. This indicates that part of the reduction in high [K+]-induced activity is mediated through TPRA1 activation.

[0133] Long-term Potentiation and memory Long Term Potentiation (LTP) is the most widely implicated mechanism of memory storage in the hippocampus. It is a process involving persistent strengthening of synapses resulting in a long-lasting increase in signal transmission among neurons. The synaptic enhancement is achieved after brief, high-frequency electrical stimulation in the hippocampus; the theta-burst stimulation. LTP recording is widely recognized as a cellular model for the study of learning and memory which is linked with activity-dependent, sustained increases in synaptic plasticity.

[0134] The role of CBD in memory enhancement has been thoroughly examined in previous reports by employing LTP experiments. Interestingly, the potentiation varies in a dose-dependent manner. Initially, a dose response analysis for CBD was conducted with the following concentrations: i) 1 pM, ii) 2,5 pM and iii) 5 pM. Next, the field excitatory post-synaptic potentials (fEPSPs) were acquired i) during perfusion with normal aCSF, ii) during perfusion with 2,5 pM CBD diluted in normal aCSF or 100pM oleocanthal diluted in normal aCSF and iii) during perfusion with 2,5 pM CBD combined withlOOpM oleocanthal diluted in normal aCSF.

[0135] For evoked recordings, the fEPSP value were measured from the minimum value of the synaptic response (4-5 ms following stimulation) compared to the baseline value prior to stimulation. Both parameters were monitored in real-time in every experiment. A stimulusresponse curve was then determined using stimulation intensities between 0.1 and 0.3 mA, in 0.1 mA steps. For each intensity level, two traces were acquired and averaged. Baseline stimulation parameters were selected to evoke a response of 1 mV. For the LTP experiments, synaptic responses were normalized to the average 10 min pre-theta burst fEPSPs.

[0136] Example 11 : Effect of CBD and OLC on Long-term Potentiation and memory in hippocampal slicesThe application of CBD and OLC individually resulted in a moderate increase in the percentage of baseline excitatory postsynaptic potentials (EPSPs), suggesting an enhancement of LTP. Remarkably, the combined application of CBD at 2.5 pM and OLC at 100 pM produced a significantly greater increase in EPSPs, far exceeding the effects observed with the individual compounds. This potentiation was sustained over a 50-minute period, as indicated by electrophysiological recordings. Statistical analysis using the Kruskal-Wallis test confirmed the significance of these observations (p < 0.0001), underscoring a clear synergistic interaction between the secoiridoids and cannabinoids in enhancing synaptic plasticity. These findings indicate potential treatment of neurological conditions where enhancement of synaptic function is desirable using the combination of a cannabinoid and a secoiridoid. Behavioral experiments on learning and memory

[0137] Adult male 5xFAD transgenic mice harboring the five familial Alzheimer’s disease-linked mutations (APP KM670 / 671 NL (Swedish), APP 1716V (Florida), APP V717I (London), PSEN1 M146L (A > C), PSEN1 L286V) were used in this study (Oakley et al., 2006). Original breeders were obtained from Jackson Laboratories and bred in-house on a C57BI / 6J background. For this study, cohorts of young (3-month-old) 5xFAD mice and age-matched wild-type C57BI / 6J littermates were used. The animals were group-housed (3-5 animals / cage) in climate- controlled conditions (30-50% humidity, 21 ± 2°C, 12:12 h light / dark cycle) with ad libitum access to food and water. Animals were habituated to housing conditions for 1 week prior to the experimental procedures. All procedures were performed under approval of the Veterinary Directorate of the Prefecture of Heraklion (Crete), complied with Greek Government guidelines and the guidelines of the FORTH ethics committee, and were performed in accordance with approved protocols from the Federation of European Laboratory Animal Science Associations (FELASA) and Use of Laboratory Animals (License number: EL91-BIOexp-02; Approval Code: 360667, Approval Date: 29 / 11 / 2021 , active for 3 years).

[0138] On p90, each mouse was habituated for 15 min in an open field (45 x 45 x 45 cm), once a day for 3 days. On p100, two identical objects were placed into the open field, and the mouse was allowed to explore them for 5 min. After 25 min, the mouse was returned to the open field chamber for 5 min to explore one familiar object from the previous trial and one new object. The time of object exploration was measured using JWatcher. Object exploration was defined as time spent exploring objects by physical proximity (i.e., touching, sniffing). The object exploration index was calculated as: [(novel object exploration time + familiar object exploration time) / total time in chamber]. The discrimination index of the test phase was calculated as: [(time exploring novel object - time exploring familiar object) / (time exploring novel object + time exploring familiar object)].

[0139] Example 12: Effect of OLC and CBD on Novel Object Recognition (NOR) in an Alzheimer’s disease mouse model (5xFAD)

[0140] The impact of oral administration of CBD and OLC on memory performance was evaluated using the NOR test in an Alzheimer’s disease mouse model (5xFAD). CBD (50 mg) or OLC (10 mg) administered individually did not produce significant improvements in discrimination or exploration indexes. However, co-administration of CBD (50 mg) and OLC (10 mg) significantly increased the discrimination index (p = 0.05, t-test), indicating a synergistic effect enhancing memory performance in 5xFAD mice. These results (Figure 13) suggest a significant synergistic improvement in memory performance with combined CBD and OLC administration in this model.

[0141] Example 13

[0142] Example 13a: Preparation of oral solution with cannabinoids of general formula I or II or III or IV in combination with secoiridoids of general formula V or VII.

[0143] Cannabinoids and secoiridoids are dissolved in PEG400 and the solution is placed in a dropper vial for use as a drop solution.

[0144] Example 13b: Preparation of soft capsules with cannabinoids of general formula I or II or III or IV in combination with secoiridoids of general formula V or VII.

[0145] Cannabinoids and secoiridoids are mixed with oily carrier in a ratio of 1 :10 by weight and incorporated into a soft capsule.

[0146] Example 13c: Preparation of hard capsules with cannabinoids of general formula I or II or III or IV in combination with secoiridoids of general formula V or VII.

[0147] Cannabinoids and secoiridoids are mixed with microcrystalline cellulose in a ratio of 1 :20 by weight and can be used to make hard capsules.

[0148] Unless defined otherwise all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the presently described subject matter pertains.

[0149] Where a range of values is provided, for example, concentration ranges, percentage range or ratio ranges, it is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the described subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and such embodiments are also encompassed within the described subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the described subject matter. It should be understood that the terms “a” and “an” as used above and elsewhere herein refer to “one or more” of the enumerated components. It will be clear to one of ordinary skill in the art that the use of the singular includes the plural unless specifically stated otherwise. Therefore, the terms “a”, “an” and “at least one” are used interchangeably in this application. Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as size, weight, reaction conditions and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about”. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present subject matter. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

Claims

CLAIMS1. A chemical composition comprising: at least one cannabinoid; at least one olive oil phenol; wherein the amount of cannabinoid is always less than the amount of olive oil phenol.

2. Composition according to claim 1 wherein the at least one cannabinoid is selected from the group having general formula l-V:and the at least one olive oil phenol is selected from the group having general formulaVI-VII:wherein:R1 = H or COOX, X being either H or a straight or branched alkyl group, alkenyl group or alkynyl group having a carbon atom number between 1-5.R2 = H or OHR3 = H or COOCH33. Composition according to any preceding claim, wherein the at least one cannabinoid is selected from the group of: cannabidiolic acid (CBDA), cannabinolic acid (CBNA), cannabigerolic acid (CBGA), cannabichromenic acid (CBCA), A9- tetrahydrocannabinolic acid (A9-THCA), cannabidiol (CBD), cannabinol (CBN), cannabigerol (CBG), cannabichromene (CBC), A9-THC, cannabidiolic acid methyl ester (CBDA-Me), cannabinolic acid methyl ester (CBNA-Me), cannabigerolic acidmethyl ester (CBGA-Me), cannabichromenic acid methyl ester (CBCA-Me), A9- tetrahydrocannabinolic acid methyl ester (A9-THCA-Me).

4. Composition according to any preceding, wherein the at least one olive oil phenol is a secoiridoid.

5. Composition according to claim 4 wherein the secoiridoid is selected from the group: oleocanthal (OLC), oleacein (OLA), oleomissinional (OM), oleuropein aglycone (OPA), ligstroside aglycone (LIGA).

6. Composition according to any preceding claim, wherein the at least one cannabinoid is CBD and the at least one olive oil phenol is OLC or OLA.

7. Composition according to any preceding claim, wherein the molar ratio of the at least one cannabinoid and the at least one olive oil phenol is 1:3.

8. Composition according to any preceding claim, wherein the composition is in the form of an oral solution.

9. Composition according to claim 8, wherein the composition is in the form of a capsule, a tablet, a suppository, or granules.

10. Composition according to any of claims 1-7, wherein the composition is in the form of a solution for injection or a transdermal solution.

11. Composition according to any of claims 1-10 for use as a medicament.

12. Composition according to claim 11 when dependent on claim 8 or 9, wherein the amount of the at least one cannabinoid is greater than the amount of the at least one olive oil phenol, such that the effective molar ratio at the site of action is within the range of 1 :3 to 1:40, preferably 1:3 to 1 :8, more preferably 1 :3.

13. Composition according to any of claims 1-12 for use in the treatment of cancer, central nervous system disorders, neurodegenerative diseases, psoriasis, Alzheimer’s, epilepsy.

14. Composition according to any of claims 1-12 for use in the treatment of pain.

15. An oral dosage form comprising a first composition according to any of claims 1-7 and a second composition comprising at least one cannabinoid and at least one oliveoil phenol, wherein the molar ratio of the at least one cannabinoid to the at least one olive oil phenol in the second composition is X:1 , X being greater than 1.

16. A chemical composition for oral administration comprising: at least one cannabinoid; at least one olive oil phenol; wherein the amount of the at least one cannabinoid is always greater than the amount of the at least one olive oil phenol.

17. Composition according to claim 16 wherein the at least one cannabinoid is selected from the group having general formula l-V:and the at least one olive oil phenol is selected from the group having general formulaVI-VII:wherein:R1 = H or COOX, X being either H or a straight or branched alkyl group, alkenyl group or alkynyl group having a carbon atom number between 1-5.R2 = H or OHR3 = H or COOCH318. Composition according to any preceding claim, wherein the at least one cannabinoid is selected from the group of: cannabidiolic acid (CBDA), cannabinolic acid (CBNA), cannabigerolic acid (CBGA), cannabichromenic acid (CBCA), A9- tetrahydrocannabinolic acid (A9-THCA), cannabidiol (CBD), cannabinol (CBN), cannabigerol (CBG), cannabichromene (CBC), A9-THC, cannabidiolic acid methylester (CBDA-Me), cannabinolic acid methyl ester (CBNA-Me), cannabigerolic acid methyl ester (CBGA-Me), cannabichromenic acid methyl ester (CBCA-Me), A9- tetrahydrocannabinolic acid methyl ester (A9-THCA-Me).

19. Composition according to any preceding, wherein the at least one olive oil phenol is a secoiridoid.

20. Composition according to claim 18 wherein the secoiridoid is selected from the group: oleocanthal (OLC), oleacein (OLA), oleomissinional (OM), oleuropein aglycone (OPA), ligstroside aglycone (LIGA).

21. Composition according to any preceding claim, wherein the at least one cannabinoid is CBD and the at least one olive oil phenol is OLC or OLA.

22. Composition according to any preceding claim, wherein the molar ratio of the at least one cannabinoid and the at least one olive oil phenol is X: 1 , X being greater than 1 .

23. Composition according to claim 22, wherein X is between 3-40, preferably 3-8, more preferably 3.

24. Composition according to any of claims 16-23, wherein the composition is in the form of a capsule, a tablet, a suppository, or granules.

25. Composition according to any of claims 16-24 for use as a medicament, preferably in the treatment of cancer, central nervous system disorders, neurodegenerative diseases, psoriasis, Alzheimer’s, epilepsy.

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