EP4739147A1

EP4739147A1 - Composition

Info

Publication number: EP4739147A1
Application number: EP24742647.1A
Authority: EP
Inventors: Charlotte CADDICK; Cai READ; Tommaso IANNITTI
Original assignee: Nicoventures Trading Ltd
Current assignee: Nicoventures Trading Ltd
Priority date: 2023-07-06
Filing date: 2024-07-04
Publication date: 2026-05-13
Also published as: WO2025008633A1

Abstract

The present invention relates to a synthetic composition comprising a first component and a second component, wherein the first component comprises at least one compound selected from caffeine, theophylline and paraxanthine, and the second component comprises at least one compound selected from L-theanine and a flavonoid.

Description

COMPOSITION

FIELD OF THE INVENTION

The present invention relates to compositions comprising a first component and a second component, to articles comprising the compositions, as well as to uses, methods and systems for administering the compositions to a user.

BACKGROUND

It is known that certain active compounds have a pharmacological effect when administered to a user. In some instances, the pharmacological effect may be utilized to influence a medical state of the user, i.e. the active compound is administered as a medicine. In other instances, the pharmacological effect may be utilized to influence a non-medical state of the user, e.g. the active compound is not administered as a medicine. An example of influencing a non-medical state might be the administration of caffeine to modulate alertness.

The pharmacological effect of a particular active compound may depend on its ability to interact with one or more biological targets within the body of the user. The extent of the interaction with the one or more targets will influence the extent to which a pharmacological effect is perceived.

In some instances, multiple active compounds can be administered to a user. The active compounds may each act on one or more biological targets to a different extent.

It would be advantageous to develop improved compositions comprising multiple actives.

SUMMARY OF THE INVENTION

In one aspect, the present invention relates to a synthetic composition comprising a first component and a second component, wherein the first component comprises at least one compound selected from caffeine, theophylline and paraxanthine, and the second component comprises at least one compound selected from L-theanine and a flavonoid. In a further aspect, the present invention relates to the use of a synthetic composition as defined herein to modulate the neural state of a user.

In a further aspect, the present invention relates to a method of modulating the neural state of a user, the method comprising administering to the user a synthetic composition as defined herein.

In a further aspect, the present invention relates to an article comprising a synthetic composition as defined herein.

These and other aspects of the present invention will now be further described.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1a provides a concentration-response curve for L-theanine used in combination 1 Figure 1 b provides a concentration-response curve for theophylline used in combination 1

Figure 2a provides a concentration-response curve for EGCG used in combination 2 Figure 2b provides a concentration-response curve for paraxanthine used in combination 2

Figure 3a provides a concentration-responsecurve for flavone used in combination 3 Figure 3b provides a concentration-response curve for caffeine used in combination 3 Figure 4a provides a concentration-response curve for hispidulin from used in combination 4

Figure 4b provides a concentration-response curve for caffeine used in combination 4

DETAILED DESCRIPTION

Synergy

The concept of “synergy” is generally understood as being present where the effect which results from a combination comprising multiple individual components is greater than the sum of the effect of each individual component in isolation. In the context of the present disclosure, the relevant effect is that which results from the application of the components (in isolation and in combination) to biological targets of interest. To quantify the interaction between the components, the observed combination effect is compared to the expected effect predicted by a reference model. When the combination effect is greater than expected, the combination is classified as synergistic. This is further explained in Yadav et al (2015, Computational and Structural Biotechnology Journal 13 (2015) 504-513).

Since the presence of synergy depends on the difference between the observed combination effect and the expected effect predicted by a reference model, the choice of the reference model can influence the conclusion as to whether synergy is determined to be present or not.

Reference models commonly used for the determination of synergy in the context of active components include: the Loewe model (Loewe S. The problem of synergism and antagonism of combined drugs. Arzneimittelforschung. 1953 Jun;3(6):285-90) the Bliss model (Bliss Cl. The toxicity of poisons applied jointly. Ann App Biol. 1939; 26: 585-615)', the zero interaction potency (ZIP) model (Yadav B, Wennerberg K, Aittokallio T, Tang J. Searching for Drug Synergy in Complex Dose-Response Landscapes Using an Interaction Potency Model. Comput Struct Biotechnol J. 2015 Sep 25;13:504-13) and the highest single agent (HSA) model (Foucquier J, Guedj M. Analysis of drug combinations: current methodological landscape. Pharmacol Res Perspect. 2015 Jun;3(3):e00149. doi: 10.1002/prp2. 149. Epub 2015 May 20. Erratum in: Pharmacol Res Perspect. 2019 Dec;7(6):e00549). These reference models will be referred to throughout the present disclosure as: the Loewe model; the Bliss model; the ZIP model and the HSA model.

According to the present invention, the determination of synergy for a particular combination of components involves the step of evaluating which one, or combination, of the above reference models is most applicable.

Such an approach has resulted in the identification of synergistic synthetic combinations comprising a first component and a second component as defined herein.

Synthetic Composition In one aspect, the present invention relates to a synthetic composition comprising a first component and a second component, wherein the first component comprises a compound selected from caffeine, theophylline and paraxanthine, and the second component comprises at least one compound selected from L-theanine and a flavonoid.

According to the present disclosure, the term “synthetic composition” refers to a composition which is produced by combining multiple individual and/or isolated compounds to form a composition, rather than via an extraction process whereby a starting composition containing multiple compounds is extracted and then purified or otherwise modified to reduce its constituent components.

The present inventors have determined that such synthetic compositions display synergy or a synergistic behavior when applied in vitro to a biological target, in particular neural cells. The stimulation of neural cells by active components can be used to modulate a particular mood state of the subject. For example, certain active components, such as some cannabinoids, may elicit a feeling of relaxation. Other active components, such as caffeine, may elicit a feeling of stimulation.

In the context of the present invention, eliciting neuro-stimulation extends to one or more of increasing alertness, increasing awareness, and increasing responsiveness in a user. Moreover, the present invention is generally aimed at eliciting a response which is not curative in the medical sense. Accordingly, the composition and uses described herein are for non-medical uses. In one embodiment, the composition is not used to treat neurodegenerative disorders. An example of a neurological disorder is narcolepsy.

By determining the synergistic effect with respect to the stimulation of neural cells, it is possible to identify synergistic synthetic combinations that can be used to modulate the mood state of a user.

According to the present invention, the synthetic composition comprises a first component. According to the present invention, the first component comprises at least one compound selected from caffeine, theophylline and paraxanthine. Adenosine acts as modulator in various physiological and pathophysiological processes in central nervous system (CNS). Caffeine, theophylline and paraxanthine (and other derivatives of xanthine) are known to play a role in the blocking of adenosine receptors to limit signalling through adenosine-mediated mechanisms’ . In this way, caffeine, theophylline and paraxanthine are capable of acting as neuro-stimulants.

In one embodiment, the first component comprises caffeine.

In one embodiment, the first component comprises theophylline.

In one embodiment, the first component comprises paraxanthine.

The first component may be present in the synthetic composition in a range of from 0.25 to 150pM.

In one embodiment, the first component may be present in the synthetic composition in a range of from 0.25 to 125 pM, or from 0.25 to 100 pM, or from 0.25 to 75 pM, or from 0.25 to 50 pM, or from 0.25 to 30 pM, or from 0.25 to 25 pM, or from 0.25 to 20 pM, or from 0.25 to 15 pM, or from 0.25 to 10 pM.

In one embodiment, the first component may be present in the synthetic composition in a range of from 1 to 125 pM, or from 5 to 125 pM, or from 10 to 125 pM, or from 15 to 125 pM, or from 20 to 125 pM, or from 25 to 125 pM, or from 30 to 125 pM, or from 40 to 125 pM, or from 50 to 125 pM, or from 60 to 125 pM, or from 70 to 125 pM, or from 80 to 125 pM, or from 90 to 125 pM, or from 100 to 125 pM.

Preferably, the first component may be present in the synthetic composition in a range of from 0.25 to 5pM, or from 0.25 to 4.5pM, or from 0.25 to 4pM, or from 0.25 to 3.5pM, or from 0.25 to 3pM, or from 0.25 to 2.5pM, or from 0.25 to 2pM.

Preferably, the first component may be present in the synthetic composition in a range of from 0.5 to 5pM, or from 0.75 to 5pM, or from 1 to 5pM, or from 1 .25 to 5pM, or from 1 .5 to 5pM. Preferably, the first component may be present in the synthetic composition in a range of from 0.5 to 4.5pM, or from 0.75 to 4pM, or from 0.75 to 3.5pM, or from 0.75 to 3pM, or from 0.75 to 2.5pM, or from 0.75 to 2pM, or from 1 to 2pM, or from 1 to 1 ,75pM.

Preferably where the first component is caffeine, it may be present in the synthetic composition in a range of from 0.5 to 4.5pM, or from 0.75 to 4pM, or from 0.75 to 3.5pM, or from 0.75 to 3pM, or from 0.75 to 2.5pM, or from 0.75 to 2pM, or from 1 to 2pM, or from 1 to 1.75pM. In particular, where the first component is caffeine, it may be present in the synthetic composition in an amount of about 0.39 pM.

Preferably, the first component may be present in the synthetic composition in a range of from 1 to 50pM, or from 1 to 45pM, or from 1 to 45pM, or from 1 to 40pM, or from 1 to 35pM, or from 1 to 30pM, or from 1 to 25pM.

Preferably, the first component may be present in the synthetic composition in a range of from 5 to 50pM, or from 10 to 50pM, or from 15 to 50pM, or from 20 to 50pM, or from 25 to 50pM, or from 30 to 50pM, or from 35 to 50pM.

Preferably, the first component may be present in the synthetic composition in a range of from 1 to 10pM, or from 2 to 9pM, or from 3 to 8pM, or from 4 to 7pM, or from 5 to 7pM.

Preferably where the first component is theophylline, it may be present in the synthetic composition in a range of from 1 to 10pM, or from 2 to 9pM, or from 3 to 8pM, or from 4 to 7pM, or from 5 to 7pM. In particular, where the first component is theophylline, it may be present in the synthetic composition in an amount of about 6.25 pM.

Preferably, the first component may be present in the synthetic composition in a range of from 50 to 120pM, or from 50 to 110pM, or from 50 to 100pM, or from 50 to 90pM, or from 50 to 80pM, or from 50 to 70pM, or from 50 to 60pM.

Preferably, the first component may be present in the synthetic composition in a range of from 60 to 120pM, or from 70 to 110pM, or from 80 to 100pM, or from 90 to 100pM. Preferably, where the first component is paraxanthine, it may be present in the synthetic composition in a range of from 15 to 125pM, or from 20 to 120pM, or from 25 to 115pM, or from 30 to 110pM, or from 35 to 105pM, or from 40 to 10OpM, or from 45 to 95pM.

According to the present invention, the synthetic composition comprises a second component. According to the present invention, the second component comprises at least one compound selected from L-theanine and a flavonoid.

The flavonoid may be selected from (-)-Epigallocatechin 3-gallate (EGCG), baicalin, galangin, quercetin, flavone, genistein, kaempferol, hispidulin, and tetra-O-methyl scutellarein.

The second component may be present in the synthetic composition in a range of from 0.1 to 125 pM, or from 0.1 to 100 pM, or from 0.1 to 75 pM, or from 0.1 to 50 pM, or from 0.1 to 30 pM, or from 0.1 to 25 pM, or from 0.1 to 20 pM, or from 0.1 to 15 pM, or from 0.1 to 10 pM.

Preferably, the second component may be present in the synthetic composition in a range of from 0.1 to 29pM, or from 0.1 to 28pM, or from 0.1 to 27pM, or from 0.1 to 26pM, or from 0.1 to 25pM, or from 0.1 to 24pM, or from 0.1 to 23pM, or from 0.1 to

22pM, or from 0.1 to 21 pM, or from 0.1 to 20pM, or from 0.1 to 19pM, or from 0.1 to

18pM, or from 0.1 to 17pM, or from 0.1 to 16pM, or from 0.1 to 15pM, or from 0.1 to

14pM, or from 0.1 to 13pM, or from 0.1 to 12pM, or from 0.1 to 11 pM, or from 0.1 to

10pM, or from 0.1 to 9pM, or from 0.1 to 8pM, or from 0.1 to 7pM, or from 0.1 to 6pM, or from 0.1 to 5pM, or from 0.1 to 4pM, or from 0.1 to 3pM, or from 0.1 to 2pM, or from 0.1 to 1 pM.

Preferably, the second component may be present in the synthetic composition in a range of from 0.2 to 2pM, or from 0.3 to 2pM, or from 0.4 to 2pM, or from 0.5 to 2pM, or from 0.6 to 2pM, or from 0.7 to 2pM, or from 0.8 to 2pM, or from 0.9 to 2pM, or from 1 to 2pM.

In one embodiment, the compound of the first component is caffeine and the compound of the second component is L-theanine. In one embodiment, the compound of the first component is caffeine and the compound of the second component is a flavonoid. Preferably, the compound of the first component is caffeine and the compound of the second component is a flavonoid selected from (-)-Epigallocatechin 3-gallate, baicalin, galangin, quercetin, flavone, genistein, kaempferol, hispidulin, and tetra-O-methyl scutellarein. Particularly preferred is where the compound of the first component is caffeine and the compound of the second component is flavone. Particularly preferred is where the compound of the first component is caffeine and the compound of the second component is hispidulin.

In one embodiment, the compound of the first component is theophylline and the compound of the second component is a flavonoid. Preferably, the compound of the first component is theophylline and the compound of the second component is a flavonoid selected from (-)-Epigallocatechin 3-gallate, baicalin, galangin, quercetin, flavone, genistein, kaempferol, hispidulin, and tetra-O-methyl scutellarein. Most preferably, the compound of the first component is theophylline and the compound of the second component is L-theanine.

In one embodiment, the compound of the first component is paraxanthine and the compound of the second component is L-theanine. In one embodiment, the compound of the first component is paraxanthine and the compound of the second component is a flavonoid. Preferably, the compound of the first component is paraxanthine and the compound of the second component is a flavonoid selected from (-)-Epigallocatechin 3- gallate, baicalin, galangin, quercetin, flavone, genistein, kaempferol, hispidulin, and tetra-O-methyl scutellarein. Most preferably, the compound of the first component is caffeine and the compound of the second component is (-)-Epigallocatechin 3-gallate.

The first component and the second component may be present in the synthetic composition in a defined molar ratio.

Preferably, the molar ratio of first component to the second component may be in a range selected from:

4 (first component): 1 to 64 (second component);

64 to 256 (first component): 1 to 4 (second component); or

1 (first component):! to 4 (second component). Preferably, the molar ratio of first component to the second component may be in a range selected from:

4 (Theophylline): 1 to 64 (second component);

64 to 256 (Paraxanthine): 1 to 4 (second component); or

1 (Caffeine): 1 to 4 (second component).

4 (Theophylline): 1 to 64 (L-Theanine);

64 to 256 (Paraxanthine): 1 to 4 (Flavanoid); or

1 (Caffeine): 1 to 4 (Flavanoid).

64 to 256 (Paraxanthine): 1 to 4 ((-)-Epigallocatechin 3-gallate);

1 (Caffeine): 1 to 4 (Flavone); or

1 (Caffeine): 1 to 4 (hispidulin).

The synthetic composition of the present invention may be formulated as a delivery vehicle for delivery of the first and second components to a user. In this regard, the synthetic compositions defined herein may be formulated to deliver the first and second components to a user orally, nasally, respiratorily or transdermally.

In one embodiment, the synthetic composition is formulated to deliver the first and second components to a user respiratorily. In this regard, the synthetic composition may be formulated so as to be capable of forming an aerosol (either via condensation or atomization) for inhalation.

One example of such a formulation is as an aerosolisable liquid, gel or solid. In the context of the present disclosure, such a formulation is referred to as an aerosolgenerating material. Thus, in the context of this formulation of the synthetic composition, the terms “synthetic composition” and “aerosol-generating material” may be used interchangeably. In one embodiment, the aerosol-generating material may comprise one or more aerosol former materials. The one or more aerosol-former materials may be selected from one or more of glycerol, propylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, 1 ,3-butylene glycol, erythritol, meso-Erythritol, ethyl vanillate, ethyl laurate, a diethyl suberate, triethyl citrate, triacetin, a diacetin mixture, benzyl benzoate, benzyl phenyl acetate, tributyrin, lauryl acetate, lauric acid, myristic acid, and propylene carbonate.

In the context of being formulated to deliver the first and second components to a user respiratorily, the aerosol-generating material may be used in a non-combustible aerosol provision system that releases compounds from an aerosol-generating material without combusting the aerosol-generating material, such as electronic cigarettes, tobacco heating products, and hybrid systems to generate aerosol using a combination of aerosol-generating materials.

According to the present disclosure, a “non-combustible” aerosol provision system is one where the aerosol-generating material of the aerosol provision system (or component thereof) is not combusted or burned in order to facilitate delivery of at least one substance to a user.

In some embodiments, the delivery system is a non-combustible aerosol provision system, such as a powered non-combustible aerosol provision system.

In some embodiments, the non-combustible aerosol provision system is an electronic cigarette, also known as a vaping device or electronic nicotine delivery system (END), although it is noted that the presence of nicotine in the aerosol-generating material is not a requirement.

In some embodiments, the non-combustible aerosol provision system is an aerosolgenerating material heating system, also known as a heat-not-burn system. An example of such a system is a tobacco heating system (wherein the aerosol-generating material comprises tobacco or a component derived therefrom). In some embodiments, the non-combustible aerosol provision system is a hybrid system to generate aerosol using a combination of aerosol-generating materials, one or a plurality of which may be heated. In particular, each of the aerosol-generating materials may be, for example, in the form of a solid, liquid or gel and may or may not contain nicotine. In some embodiments, the hybrid system comprises a liquid or gel aerosolgenerating material and a solid aerosol-generating material. The solid aerosolgenerating material may comprise, for example, tobacco or a non-tobacco product. Typically, the non-combustible aerosol provision system may comprise a noncombustible aerosol provision device and a consumable for use with the non- combustible aerosol provision device.

In some embodiments, the disclosure relates to consumables comprising aerosolgenerating material and configured to be used with non-combustible aerosol provision devices. These consumables are sometimes referred to as articles throughout the disclosure.

In some embodiments, the non-combustible aerosol provision system, such as a non- combustible aerosol provision device thereof, may comprise a power source and a controller. The power source may, for example, be an electric power source or an exothermic power source. In some embodiments, the exothermic power source comprises a carbon substrate which may be energised so as to distribute power in the form of heat to an aerosol-generating material or to a heat transfer material in proximity to the exothermic power source.

In some embodiments, the non-combustible aerosol provision system may comprise an area for receiving the consumable, an aerosol generator, an aerosol generation area, a housing, a mouthpiece, a filter and/or an aerosol-modifying agent.

In some embodiments, the consumable for use with the non-combustible aerosol provision device may comprise aerosol-generating material, an aerosol-generating material storage area, an aerosol-generating material transfer component, an aerosol generator, an aerosol generation area, a housing, a wrapper, a filter, a mouthpiece, and/or an aerosol-modifying agent. In one embodiment, the synthetic composition is formulated to deliver the first and second components to a user orally. In this regard, the synthetic composition may be formulated so as to be capable of delivering the first and second components to the buccal cavity of a user and/or the gastrointestinal tract of a user. In one embodiment, the synthetic composition may take the form of a lozenge, tablet, gum, gel, powder, chew, melt, liquid, or semi-solid lozenges, or article comprising inhalable powders, and oral products such as oral tobacco which includes snus or moist snuff, wherein the synthetic composition may or may not comprise nicotine. Such synthetic compositions are configured to release the first and second components to the buccal cavity of a user and/or the gastrointestinal tract of a user. The synthetic composition may be present in a pouch or capsule for buccal or Gl release.

When formulated for oral delivery, the synthetic composition can comprise the first and second components, and one or more fillers. The one or more fillers may generally comprise a sugar alcohol or a combination of sugar alcohols.

In any of the above synthetic compositions which are formulated to deliver the first and second components orally or respiratorily, the synthetic composition may comprise one or more further constituents. In particular, one or more further constituents may be selected from one or more physiologically and/or olfactory active constituents, and/or one or more functional constituents.

In some embodiments, the active constituent is an olfactory active constituent and may be selected from a "flavour" and/or "flavourant" which, where local regulations permit, may be used to create a desired taste, aroma or sensation in a product for adult consumers. In some instances such constituents may be referred to as flavours, flavourants, cooling agents, heating agents, or sweetening agents, and may include one or more of extracts (e.g., licorice, hydrangea, Japanese white bark magnolia leaf, chamomile, fenugreek, clove, menthol, Japanese mint, aniseed, cinnamon, herb, Wintergreen, cherry, berry, peach, apple, Drambuie, bourbon, scotch, whiskey, spearmint, peppermint, lavender, cardamom, celery, cascarilla, nutmeg, sandalwood, bergamot, geranium, honey essence, rose oil, vanilla, lemon oil, orange oil, cassia, caraway, cognac, jasmine, ylang-ylang, sage, fennel, piment, ginger, anise, coriander, coffee, or a mint oil from any species of the genus Mentha), flavour enhancers, bitterness receptor site blockers, sensorial receptor site activators or stimulators, sugars and/or sugar substitutes (e.g., sucralose, acesulfame potassium, aspartame, saccharine, cyclamates, lactose, sucrose, glucose, fructose, sorbitol, or mannitol), and other additives such as charcoal, chlorophyll, minerals, botanicals, or breath freshening agents. They may be imitation, synthetic or natural ingredients or blends thereof. They may be in any suitable form, for example, oil, liquid, or powder.

The flavor may be added to the aerosolisable material as part of a so-called “flavour block”, where one or more flavours are blended together and then added to the aerosolisable material.

In some embodiments, the active constituent may be or derived from plants.

In one embodiment, the synthetic composition is formulated to deliver the first and second components to a user transdermally. In this regard, the synthetic composition may be formulated as an ointment, cream, gel, or rub.

In one embodiment, the synthetic composition is formulated to deliver the first and second components to a user nasally. In this regard, the synthetic composition may be formulated as a powder, liquid, or semi-solid.

As described generally herein, there is provided a synthetic composition comprising a synergistic combination of a first component and a second component. Whilst the first and second components have been described above as being present in the composition in certain concentrations and/or molar ratios, it will be appreciated to one skilled in the art that when an active component is present in a composition which is to be delivered to a user, certain factors may lead to less than 100% of the component present in the composition being available at the site of the biological target. Such factors include the “release” of the component from the composition (this may also include “transfer” of the component from one phase to another), as well as the bioavailability of the component once administered to a user.

One skilled in the art is able to perform routine testing to determine the “release” of the component from a particular formulation. For example, considering a liquid formulation which is to be delivered to the user via inhalation, one skilled in the art can determine the concentration of the relevant component in the formulation (since the formulation can be made and tested according to known chemical techniques) and then the amount of the relevant component present in an aerosol produced from the formulation can be tested by capturing the aerosol on, for example, a Cambridge filter pad and performing an analysis of the of the component captured (using standard analytical techniques). It is then possible to calculate the efficiency of the transfer from the liquid composition to the aerosol. An appropriate release scaling factor (F_reiease) can then be applied to the amounts or molar ratios of the components in the composition. The scaling factor represents the extent to which the amount or molar ratio of a particular component needs to be increased so as to approximate the composition amount or molar ratio in the formulated composition. Where there is 100% release, Freiease is 1. Where there is 50% release, Freiease is 2.

According to the present invention, Freiease for any given synthetic formulation may be 1 ,

1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0,

3.1 , 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0,

5.1 , 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1 , 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0,

7.1 , 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1 , 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0,

9.1 , 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 11 , 12, 13, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, or 500.

With respect to oral bioavailability of a released component, a similar approach can be taken. For example, if the first component has a bioavailability of 100% and the second component has a bioavailability of 50% and the calculated molar ratio in order to achieve synergy in vitro are 2 (first component): 1 (second component), then the synthetic composition formulated for delivery would have a molar ratio of 1 (first component):1 (second component), since twice as much of the second component would needed to achieve the desired ratio in vivo. According, when formulating the synthetic composition for a particular mode of administration, a bioavailability scaling factor (F availability) can also be applied to the amounts or molar ratios of the components in the composition. The skilled person is aware of suitable software which can be used to predict bioavailability of various actives. A particular example in this regard is ADMET Predictor® from SimulationsPlus (htps . //www. sim u I a tjpns-plu s . com/software/a metpredictor/)

In the context of the present disclosure, Favaiiab ty for following first and second components as determined ADMET Predictor® are as follows:

• Caffeine:

• theophylline:

• paraxanthine:

• L-theanine:

• flavone:

• Hispidulin:

• (-)-Epigallocatechin 3-gallate):

Accordingly, the concentrations and/or molar ratio of each component may need to be adjusted to account for said factors F_reiease and/or F_avaiiabiiity. The extent of the adjustment will depend on the formulation used for delivery. For example, when the synthetic composition is formulated for delivery via inhalation, account can be taken firstly of the transfer of the component from the composition to an aerosol (if not already present as an aerosol), and secondly the bioavailability of the component once administrated respiratorily. The skilled person is able to determine both the transfer and bioavailability of each of the first and second component and adjust the concentration and/or molar ratio accordingly.

Thus, in one embodiment, the synthetic composition is formulated to deliver the first and second components in a synergistic amount to a user.

Use and method for modulating the relaxation state of a user

In a further aspect, the present invention relations to the use of a synthetic composition as defined herein to modulate the neural- state, such as stimulation state, of a user. Accordingly, in respect of the use, the specific first and second components, and their amounts in the composition, may be as outlined for the synthetic composition above. In a further aspect, the present invention relations to a method of modulating the neural state, such as neural-stimulation state, of a user, the method comprising administering to the user a synthetic composition as defined herein. Accordingly, in respect of the method, the specific first and second components, and their amounts in the composition, may be as outlined for the synthetic composition above.

Said modulation for the use or the method may be to increase the neural-stimulation state of the user.

In one embodiment of the use or method to modulate the neural state of a user, the synthetic composition is administered one, twice, three, four, five or more times per day.

In one embodiment of the use or method to modulate the neuro state of a user, the synthetic composition is administered one or two hours after waking. In one embodiment of the use or method to modulate the neuro-stimulation state of a user, the synthetic composition is administered after a meal, for example, within 5, 10, 15, 20, 25, 30 minutes of finishing the meal.

Method for determining the extent of synergy between a first component and a second component

The present disclosure utilizes a method for determining the extent of synergy between a first component and a second component when applied to a target together via a synthetic composition.

The method comprises the steps of: i) performing an in vitro [Ca2+]i FLIPR Assay on the target using a first component at a plurality of concentrations of the first component to generate a raw data set in respect of the first component; ii) performing an in vitro [Ca2+]i FLIPR Assay on the target using a second component at a plurality of concentrations of the second component to generate a raw data set in respect of the second component; iii) performing an in vitro [Ca2+]i FLIPR Assay on the target using compositions comprising the first component and the second component at a plurality of concentrations to generate a raw data set in respect of the compositions; iv) evaluating the raw data in respect of the first and the second component to categorize each raw data set as being effect-based or concentration-based; v) applying one or more reference models of synergy to the raw data set in respect of the compositions based on the categorization of the raw data for the first and the second component; and vi) based on the application of the one or more reference models of synergy to raw data set in respect of the compositions, determining the extent of synergy between the first component and second component in the compositions.

The target can be a collection of cells, for example, neural cells.

The reference models can be selected from the Loewe model, the Bliss model, the ZIP model and/or the HSA model.

In one embodiment, step iv) includes the step of identifying and excluding positive outliers in the data. A suitable methodology for this may be Tukey’s test.

At least two of steps i), ii) or iii) can be performed using the same test cell source. This can result in improved accuracy of the assay.

Synergy can be determined based on the statistical significance of at least one reference model producing a synergistic result for at least concentration combination.

The raw data for each component can be categorized as being effect-based or concentration-based, dependent on its concentration-response curve profile.

Typically, when the concentration-response curve for the first component is not sigmoidal, the reference model applied for step v) can comprise the Bliss and/or the HSA model. When the concentration-response curve for the second component is not sigmoidal, the reference model applied for step v) can comprise the Bliss and/or the HSA model. When the concentration-response curve for both the first and the second component is not sigmoidal, the reference model applied for step v) can comprise the Bliss and/or the HSA model.

When the concentration-response curve for the first component is different to the concentration-response curve for the second, the reference model applied for step v) can comprise the Bliss and/or the HSA model.

When the normalized response for at least one compound is above 100%, the reference model can be selected from the Loewe model and/or the HSA model.

When the majority of the normalized responses are above 100%, the reference model can be selected from the Loewe model, the ZIP model and/or the HSA model.

When the first component and the second component demonstrate different concentration-response behaviors, the reference model can be selected from the Bliss model and/or the HSA model. An example of the first component and the second components demonstrating different concentration-response behaviors, would be when the two components possess discordant slopes (a positive concentration-response trend for one and a negative concentration-response trend for the other).

The first component for the method can be as described above with respect to the synthetic composition. The second component for the method can be as described above with respect to the synthetic composition. The compositions comprising the first and second components for the method are the synthetic compositions described herein.

EXAMPLES

Example 1 - Evaluation of component mixtures on rat cortical culture using intracellular calcium oscillation

1.1 - Introduction

Rat primary brain cortical culture was selected as a system to investigate the effect of a list of compounds on intracellular calcium and cell viability. 2 - Test items

2.1 - Test items 2.2 - Test mixtures

2.3 - Test Item Formulation

Test items were dissolved in DMSO, at a concentration of 50 mM, and then stored at - 20°C until use.

2.3.1. Test Item Assay Plate Preparation

Test compound stock solutions (50 mM in neat DMSO) were 4- fold serial diluted in

DMSO using a Biomek NX to generate 7-point concentration response curves (ORC) at a concentration which is 250-fold the final assay concentration. Of this compound plate, appropriate volume of each well were stamped into V-bottom drug plates and diluted prior to the experiment with assay buffer to obtain 0.4% DMSO final concentration and the following test item final concentrations in the assay plate: 100 pM, 25 pM, 6.25 pM, 1.56 pM, 0.39 pM, 0.0 pM. 3. EQUIPMENT

3.1. FLI PR Tetra

FLIPR Tetra (Molecular Devices, San Jose, CA, USA) was used to detect intracellular calcium levels. The instrument is provided with excitation led at 470-495 nm wavelength and emission filter at 515-575 nm wavelength. The instrument is equipped with an automated 384-tips head which allows simultaneous compound dispensing and fluorescence reading. Temperature controller was set at 37°C for the duration of the experiment.

4 - EXPERIMENTAL DESIGN

The experimental design aimed to assess the effect of test items on rat brain cortical culture using intracellular calcium oscillations.

The combination studies were performed in 384 wells plates. Each combination was performed in triplicate, in the same plate.

5 - METHODS AND PROCEDURES

Rat cortical cell culture derived using the methodology described below was used at 14 DIV for the assays.

5.1 - Rat Cortical Culture

Rat cortical neurons were obtained from rat (Sprague-Dawley) foetuses at embryonic day 18. After the pregnant rat sacrifice, foetuses were decapitated and cortices isolated. Cortices were enzymatically digested with Neuron Isolation Enzyme (ThermoFisher), following manufacturer’s instructions. Isolated cortical neurons were counted and suspended in complete neuronal medium containing Neurobasal Plus (ThermoFisher) supplemented with 2% B27 Plus (ThermoFisher) and 1 % Pen/Strep. Plating into 384- wells plates coated with 50 pg/mL poly-D-lysine (Sigma) was performed at a density of 7000 cells/well for calcium and viability assays. The cells were cultured in a 37 °C humidified incubator with 5% CO2. Half volume medium was removed and replenished with fresh medium once a week.

5.2 - [Ca2+]i FLIPR Assay

5.2.1 - FLIPR Solutions Assay buffer was prepared, containing 145 mM NaCI, 5 mM KCI, 2 mM CaCI2, 1 g/litre D-(+)-glucose, 20 mM HEPES (pH 7.4).

Compound buffer was prepared from assay buffer, by addition of 0.05% Pluronic F-127 (Sigma).

Loading solution was prepared from assay buffer, by addition of 5 pM Cal-520 (Abeam), a cytoplasmic calcium indicator in the acetoxy I methyl (AM) ester form and 2.5 mM probenecid (Sigma). Probenecid, an inhibitor of organic anion transport, was added to calcium sensitive dye efflux.

5.2.2 - FLIPR Protocol

Fluorescent dye loading: cell plate was taken from 37° C incubator. Cell medium was removed by plate inversion, and 40 pL/well of loading solution was added manually. Plate was incubated for 1 h at 37° C, 0% CO2.

Compound assay ready plate preparation: preparation was performed as described in paragraph 2.3. Plate was incubated for 10 min at 37°C.

FLIPR compound addition: both fluorescent dye loaded cell plate and compound assay ready plate were placed in 37°C pre heated FLIPR chamber, for automated compound addition and simultaneous fluorescence reading. Cell plate fluorescence was read for 2 minutes before addition (baseline), then 10 pl/well (out of 30 pl/well) of compound plate were dispensed by FLIPR in cell plate (0.4% final DMSO concentration), and fluorescence reading continued for additional 22 minutes.

Cell plate was then repositioned in 37° C incubator for 1 h at 37° C, 0% CO2.

6 - ANALYSIS

6.1 - FLIPR Data Analysis

FLIPR peak analysis: [Ca2+], peaks were measured during a 2 minute time-frame at 21- 22 min (20 min). [Ca2+], peak detection and count was performed by Peak Pro software (Molecular Devices).

6.2. SynergyFinder Analysis

Analysis was conducted to assess which mixtures were synergistic. The data from the 20 minutes time point were considered. An outlier analysis was performed on the response of positive and negative controls, applying the Tukey’s method. It calculated thresholds based on the quartiles of the data. An extreme outlier is defined as values more than 1.5 times the interquartile range from the quartiles.

Once the extreme observations were excluded, the response was normalized on the mean of positive and negative controls and expressed in percentage.

For each drug, the concentration-response curve was estimated fitting a four-parameter logistic function.

The test item interaction was evaluated performing the 4-reference models: Loewe additivity model, the Bliss model, the Zero Interaction Potency model (ZIP) and the Higher Single Agent model (HSA) (Yadav B et al, 2015; Foucquier J et al, 2015). The synergy score matrix was presented in the heat map and graphically represented with a surface plot. The heatmap reported the overall average of synergy scores and the single scores estimated, with 95% confidence interval, for each combination.

An additional T-Test analysis was performed on bootstrapped estimated synergy scores under the null hypothesis of no-interaction. P-values lower than 0.05 were considered statistically significant.

Data elaboration was performed with SAS V.9.4 and RStudio V.1.4.1717; drug interaction assessment was performed with the SynergyFinder package V.3.0.13.

7 - RESULTS

7.1 - [Ca2+]i FLIPR Assays on Compounds Mixtures Spontaneous calcium oscillations were present in 14 DIV cultures, before test item addition. Time matched vehicle controls data were used for statistical analysis. The results derived from the ZIP, HSA, Bliss and Loewe model were evaluated simultaneously to define the interaction of each drug in combination. The most appropriate model was considered to evaluate the interaction according to model assumptions. Effect-based models, such as Bliss and HSA, were preferred when the single agents concentration-response curves were not sigmoidal or had different concentration-response behaviour. The Bliss model was not suggested when most of normalized responses were above 100%. Otherwise, the dose-effect based Loewe model was preferred. Each combination was analysed by applying the models to the three replicates not averaged and the evaluation of the effects (Synergy I No effect I Antagonism) was performed after an accurate comparison with the concentration-response matrices. Table 1 summarizes the effects of the compounds mixtures on calcium oscillations, the chosen models and the concentration ranges where the effects were observed.

Table 1

Combination 1 Theophylline vs L-theanine: The compounds exhibited different concentration-response curves. Synergy was observed at 6.25 pM for theophylline and in a range of 1.56 - 100 pM for L-theanine. In addition, strong antagonism peaks were detected but not consistent in the context of the models matrices and in comparison with the concentration-response matrix. HSA model was applied for the estimation of the combinations effects with the results shown in Table 2. Concentration-response Curves for the individual compounds are given in Figure 1a and Figure 1b. Table 2

24

SUBSTITUTE SHEET (RULE 26) Combination 2 Paraxanthine vs ECGC: The compounds exhibited an opposite trend in the concentration-response curves. Synergy was observed in a range of 25 - 100 pM for paraxanthine and in a range of 0.39 - 1 .56 pM for ECGC. In addition, strong antagonism peaks were detected but not consistent in comparison with the concentration-response matrix. HSA model was applied for the estimation of the combinations effects with the results shown in Table 3. Concentration-response Curves for the individual compounds are given in Figure 2a and Figure 2b.

Table 3

SUBSTITUTE SHEET (RULE 26) Combination 3 Caffeine vs flavone: The compounds exhibited an opposite trend in the concentration-response curves. Synergy was observed at 0.39 pM for caffeine and in a range of 0.39 - 1 .56 pM for flavone. The 100 pM concentration of flavone was not considered in the antagonism scores because of the strong effect of the compound. HSA model was applied for the estimation of the combination effects with the results shown in Table 4. Concentration-response Curves for the individual compounds are given in Figure 3a and Figure 3b. 1 Table 4

SUBSTITUTE SHEET (RULE 26) Combination 4 Caffeine vs hispidulin: The compounds exhibited different concentrationresponse curves. Synergy was observed at 0.39 pM for caffeine and in a range of 0.39 - 1 .56 pM for hispiduline. In addition, strong antagonism peaks were detected but not consistent in comparison with the dose-reponse matrix. HSA model was applied for the estimation of the combinations effects with the results shown in Table 5. Concentrationresponse Curves for the individual compounds are given in Figure 4a and Figure 4b.

Table 5

SUBSTITUTE SHEET (RULE 26) In conclusion, in order to address various issues and advance the art, this disclosure shows by way of illustration various embodiments in which the claimed invention(s) may be practiced. The advantages and features of the disclosure are of a representative sample of embodiments only, and are not exhaustive and/or exclusive. They are presented only to assist in understanding and to teach the claimed invention(s). It is to be understood that advantages, embodiments, examples, functions, features, structures, and/or other aspects of the disclosure are not to be considered limitations on the disclosure as defined by the claims or limitations on equivalents to the claims, and that other embodiments may be utilised and modifications may be made without departing from the scope of the claims. Various embodiments may suitably comprise, consist of, or consist essentially of, various combinations of the disclosed elements, components, features, parts, steps, means, etc. other than those specifically described herein. The disclosure may include other inventions not presently claimed, but which may be claimed in future.

Claims

1 . A synthetic composition comprising a first component and a second component, wherein the first component comprises at least one compound selected from caffeine, theophylline and paraxanthine, and the second component comprises at least one compound selected from L-theanine and a flavonoid.

2. The synthetic composition according to claim 1 , wherein the compound of the first component is caffeine.

3. The synthetic composition according to claim 1 or claim 2, wherein the compound of the second component is a flavonoid, such as one selected from (-)-Epigallocatechin 3-gallate (EGCG), baicalin, galangin, quercetin, flavone, genistein, kaempferol, hispidulin, and tetra-O-methylscutellarein.

4. The synthetic composition according to claim 1 , wherein the compound of the first component is theophylline.

5. The synthetic composition according to claim 4, wherein the compound of the second component is L-theanine.

6. The synthetic composition according to claim 1 , wherein the compound of the first component is paraxanthine.

7. The synthetic composition according to claim 6, wherein the compound of the second component is L-theanine.

8. The synthetic composition according to any one of preceding claims, wherein the composition is formulated to deliver the first and second components to a user orally, nasally, respiratorily or transdermally.

9. The synthetic composition according to claim 8, wherein the composition is formulated to deliver the first and second components to a user respiratorily and is an aerosol generating material.

10. The synthetic composition according to claim 9, wherein the aerosol generating material comprises one or more aerosol former materials.

11 . The synthetic composition according to claim 10, wherein the one or more aerosol-former materials may comprise one or more of glycerol, propylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, 1 ,3-butylene glycol, erythritol, meso-Erythritol, ethyl vanillate, ethyl laurate, a diethyl suberate, triethyl citrate, triacetin, a diacetin mixture, benzyl benzoate, benzyl phenyl acetate, tributyrin, lauryl acetate, lauric acid, myristic acid, and propylene carbonate.

12. The synthetic composition according to claim 8, wherein the composition is formulated to deliver the composition to a user orally, nasally, or transdermally.

13. The synthetic composition according to claim 12, wherein the composition is formulated to deliver the composition orally and takes the form of a lozenge, tablet, gum, gel, powder, chew, melt, liquid, or semi-solid lozenges, or article comprising inhalable powders, and oral products such as oral tobacco which includes snus or moist snuff, wherein the synthetic composition may or may not comprise nicotine.

14. The synthetic composition according to claim 12, wherein the composition is configured to deliver the composition transdermally and takes the form of an ointment, cream or rub.

15. The synthetic composition according to claim 12, wherein the composition is configured to deliver the composition nasally and takes the form of a powder, liquid, or semi-solid.

16. The synthetic composition according to any of the preceding claims, wherein the molar ratio of the first component to the second component may be in a range selected from:

4 (first component): 1 to 64 (second component);

64 to 256 (first component): 1 to 4 (second component); or

1 (first component):! to 4 (second component).

17. The synthetic composition according to any of the preceding claims, wherein the molar ratio of the first component to the second component may be in a range selected from:

4 (Theophylline): 1 to 64 (second component);

64 to 256 (Paraxanthine): 1 to 4 (second component); or

1 (Caffeine): 1 to 4 (second component).

18. The synthetic composition according to any of the preceding claims, wherein the molar ratio of the first component to the second component may be in a range selected from:

4 (Theophylline): 1 to 64 (L-Theanine);

64 to 256 (Paraxanthine): 1 to 4 (Flavanoid); or

1 (Caffeine): 1 to 4 (Flavanoid).

19. The use of a synthetic composition as defined in any one of claims 1 to 18 to modulate the neural- state of a user, preferably the neural-stimulation state.

20. The use according to claim 19, wherein the neural-stimulation state of the user is increased following administration to a user.

21 . A method of modulating the neuro-stimulation state of a user, the method comprising administering to the user a synthetic composition as defined in any one of claims 1 to 18.

22. An article comprising a synthetic composition as defined in any one of claims 1 to 18.

23. The article according to claim 22, wherein the article contains the synthetic composition, and wherein the article comprises a container, a pouch, a patch, or a capsule.

24. The article according to claim 23, wherein the container is for use with an aerosol provision system.