WO2024229503A1 - Agarwood oil nanoemulsions, methods pertaining to and uses thereof - Google Patents

Abstract

An oil-in-water nanoemulsion comprising a continuous aqueous phase, a surfactant, and a particulate oil phase, wherein the particulate oil phase is dispersed within the aqueous phase, wherein the particulate oil phase comprises particles comprising extracted agarwood oil having a diameter in the range of about and including 160 nm to about 200 nm.

Description

Title of Invention

[0001] Agarwood oil nanoemulsions, methods pertaining to and uses thereof

Technical Field

[0002] This invention relates to an oil-in-water nanoemulsion comprising an extracted agarwood oil and a surfactant, wherein the oil-in-water nanoemulsion comprises particles comprising an extracted agarwood oil having specific size ranges. This invention further relates to methods pertaining to and uses of the oil-in-water nanoemulsion comprising an extracted agarwood oil. Specifically, the ability to modulate several biological markers associated with diseases such as cancer, chronic inflammation and chronic obstructive pulmonary disease (COPD).

Background of Invention

[0003] Agarwood is an evergreen plant endemic to areas in south-east and subcontinental Asia. An oil can be extracted from the plant, for example by various distillation methods. The oil extracted from some agarwood pants has been previously described as biologically active but there are significant barriers to formulating this oil in a way suitable for administration to a patient and to be taken into biological tissues.

[0004] Inflammation refers to an evolutionarily conserved process that involves the activation of both immune and non-immune cells. It is characterized symptoms which can include redness, pain, swelling, heat, and loss of physiological function. The pathophysiological reasoning behind these signs and symptoms is modulated by complex biological processes that occur seconds to hours following exposure to causative factors that can be any external stimulus like pathogens, allergens, toxic materials, or foreign bodies, or it may be an internal stimulus due to some impairment in tissue functioning.

[0005] Ideally, such an inflammatory response can help defend the host from viruses, bacteria, toxins (such as inhalants used to deliver pharmaceutically active material), and infections via the elimination of pathogens, thereby promoting tissue repair and healing. As a result, the impending injury can be effectively minimized, thereby facilitating the restoration of tissue homeostasis, leading to the subsequent resolution of acute inflammatory processes. Nevertheless, if inflammation remains uncontrolled or unresolved, it may lead to chronic inflammatory responses that occur well beyond the presence of the causative stimuli and lead to development of conditions such as various cancers and chronic obstructive pulmonary inflammation disorder (COPD).

[0006] Cancers, such as lung cand breast cancers, are a diverse set of diseases with complex aetiologies. Patient prognosis is variable and remains poor for sub-sets of the population.

[0007] Diseases such as cancer and those associated with chronic inflammation affect significant proportions of the world's population causing significant personal and financial difficulty. Typically available current treatments for various cancers and inflammation diseases such as COPD rely heavily on involved processes requiring complex synthetic chemistry and/or biology in their production. Many of the currently available therapies are highly toxic in their therapeutic window, produce iatrogenic reactions, as well as adverse reactions that may affect the eventual therapeutic outcomes.

[0008] There is therefore an ongoing need for the development of additional potential therapies for diseases such as those set out hereinbefore. Particularly therapies that are available and derivable from sustainable sources including plant stock.

[0009] A reference herein to a patent document or other matter which is given as prior art is not to be taken as an admission that the document or matter was known or that the information it contains was part of the common general knowledge as at the priority date of any of the claims.

Summary of Invention

[0010] The inventors have now demonstrated that an oil-in-water nanoemulsion comprising an extracted agarwood oil and a surfactant where the oil-in-water nanoemulsion comprises particles comprising an extracted agarwood oil, where the particles have a specific ranges of diameter have significant activity for several biological markers associated with cancers and conditions associated with inflammation such as COPD. [0011] Without wishing to be bound by theory, the nanoemlustion of these specific parameters may improve cell permeation and uptake to enable delivery of the extracted agarwood oil. Without wishing to be bound by theory, this improvement may be due to improved water solubility, cellular uptake and possibly bioavailability and pharmacokinetic profile.

[0012] Additionally, the extracted agarwood oil can be obtained from plant stock in a manner than can be considered sustainable. The extraction is by a distillation process that does not involve the resources typically associated with complex synthetic biological or chemical processes.

[0013] In accordance with a first aspect the invention provides a n oil-in-water nanoemulsion comprising a continuous aqueous phase, a surfactant, and a particulate oil phase, wherein the particulate oil phase is dispersed within the aqueous phase, wherein the particulate oil phase comprises particles comprising extracted agarwood oil having a diameter in the range of about and including 160 nm to about 200 nm.

[0014] In some embodiments the particles have a diameter in the range of about and including 165 nm to about 195 nm. In some embodiments the particles have a diameter in the range of about and including 170 nm to about 190 nm. In some embodiments the particles have a diameter in the range of about and including 175 nm to about 185 nm. In some embodiments the particles have a diameter in the range of about and including 182 nm to about 178 nm. In some embodiments, the particles have a diameter of about 180 nm.

[0015] Without wishing to be bound by theory, these particle sizes range may aid in the improvements of water solubility and cellular uptake.

[0016] In some embodiments the particles have a substantially spherical morphology.

[0017] In some embodiments the oil-in-water nanoemulsion has a polydispersity index in the range of about and including 0.30 to about 0.40. In some embodiments the oil-in-water nanoemulsion has a polydispersity index in the range of about and including 0.32 to about 0.38. In some embodiments the oil-in-water nanoemulsion has a polydispersity index of about 0.36. [0018] In some embodiments the surfactant is a non-ionic surfactant.

[0019] In some embodiments the non-ionic surfactant is a Poloxomer.

[0020] In some embodiments the Poloxomer is Poloxomer 407.

[0021] In some embodiments the surfactant is present in a concentration of between and including about 1.5 to 3.5 mg/mL. In some embodiments the surfactant is present in a concentration of between and including about 2.0 mg/mL to 3.0 mg/mL. In some embodiments the surfactant is present in a concentration of between and including about 2.5 mg/mL.

[0022] In some embodiments the extracted agarwood oil is present in the nanoemulsion in a concentration of between and including about 5 mg/mL to about 15 mg/mL. In some embodiments the extracted agarwood oil is present in the nanoemulsion is present in the nanoemulsion in a concentration of about 7 mg/mL to about 13 mg/mL. In some embodiments the extracted agarwood oil is present in the nanoemulsion is present in the nanoemulsion in a concentration of about 10 mg/mL.

[0023] In some embodiments the an extracted agarwood oil comprises about 12.31±5 percentage weight valerianol, about 8.03±2 percentage weight gamma-eudesmol, about 3.71±1 percentage weight epi-cyclocolorenone, about 3.71±1 percentage weight nootkatone, about 3.69±1 percentage weight beta-eudesmol, about 3.02±l percentage weight methyl phenethyl ketone, about 2.90±l percentage weight 10-epi-gamma-eudesmol, about 1.74±0.5 percentage weight hinesol, about 1.6810.5 percentage weight dihydro-columellarin, about 0.8810.25 percentage weight alpha-curcumene, about 0.8510.25 percentage weight alpha- humulene, about 0.5610.1 percentage weight alpha-bulnesene, about 0.4510.1 percentage weight Selina-4, 11-diene, about 0.3810.1 percentage weight debromofiliformin, about 0.2610.05 percentage weight 4,5-di-epi-Aristolochene, about 0.2510.05 percentage weight elemol, about 0.1610.05 percentage weight alpha-guaiene and about 0.1110.05 percentage weight alpha-selinene.

[0024] In some embodiments the invention provides a method of inhibiting transcription of a pro-inflammatory cytokine in at least one cell exposed to an inflammation inducer comprising administering to the cells an effective amount of the oil-in-water nanoemulsion hereinbefore described.

[0025] In some embodiments the pro-inflammatory cytokine is IL-8.

[0026] In some embodiments the invention provides a method of inhibiting protein expression of pro-inflammatory mediators and/or cytokines in at least one cell exposed to an inflammation inducer comprising administering to the cells an effective amount of the oil-in- water nanoemulsion hereinbefore described.

[0027] In some embodiments the pro-inflammatory mediators and/or cytokines are selected from the list consisting of IL-la, I L-ip, IL-IRa and GDF-15.

[0028] In some embodiments the invention provides a method of stimulating protein expression of anti-inflammatory mediators in at least one cell exposed to an inflammation inducer comprising administering to the cells an effective amount of the oil-in-water nanoemulsion hereinbefore described.

[0029] In some embodiments the anti-inflammatory mediators are selected from the list consisting of IL-10, IL-18Bpa, GH, VDBP, relaxin-2, 1 FN-y, PDGF-BB and TFF3.

[0030] In some embodiments the invention provides a method of stimulating transcription of antioxidant genes in at least one cell exposed to an inflammation inducer comprising administering to the cells an effective amount of the oil-in-water nanoemulsion hereinbefore described.

[0031] In some embodiments the antioxidant gene is GCLA or GSTP1.

[0032] In some embodiments the invention provides a pro-survival gene in at least one cell exposed to an inflammation inducer comprising administering to the cells an effective amount of the oil-in-water nanoemulsion hereinbefore described.

[0033] In some embodiments the pro-survival gene is PI3K. [0034] In some embodiments the inflammation inducer is a respiratory drug deliverer. In some embodiments the respiratory drug deliverer is cigarette smoke, vaporizer liquid or cigarette smoke extract (CSE).

[0035] In some embodiments the invention provides a method of suppressing cell proliferation and/or colony formation comprising administering to the cell an effective amount of the oil-in-water nanoemulsion hereinbefore described.

[0036] In some embodiments the cell proliferation and/or colony formation is associated with lung cancer activity.

[0037] In some embodiments the cell proliferation associated with lung cancer activity is A549.

[0038] In some embodiments the invention provides a method of stimulating expression of a tumour suppressor gene in at least one cell comprising administering to the cells an effective amount of the oil-in-water nanoemulsion hereinbefore described.

[0039] In some embodiments the tumour suppressor gene is p53.

[0040] In some embodiments the invention provides a method of inhibiting expression of a protein involved in cancer cell migration in at least one cell comprising administering to the cells an effective amount of the oil-in-water nanoemulsion of hereinbefore described.

[0041] In some embodiments that the cancer cell is A549.

[0042] In embodiments the protein involved in cancer cell migration is selected from the list consisting of cathepsin B, cathepsin D, cathepsin S and cathepsin G.

[0043] In some embodiments the invention provides a method of inhibiting expression of a cancer associated gene in at least one cell comprising administering to the cells an effective amount of the oil-in-water nanoemulsion hereinbefore described.

[0044] In some embodiments the cancer associated gene is KRAS or EGFR. [0045] In some embodiments the invention provides a method of inhibiting expression of a cancer associated protein in at least one cell comprising administering to the cells an effective amount of the oil-in-water nanoemulsion hereinbefore described.

[0046] In some embodiments the cancer associated protein is selected from the list consisting of Dkk-1, vimentin, GM-CSF, survivin, amphiregulin and urokinase.

[0047] In some embodiments the methods hereinbefore described are performed in vivo or in vitro.

[0048] In some embodiments nanoemulsion hereinbefore described is administered to a human.

[0049] In some embodiments the invention provides a method for the treatment and/or prevention of a condition associated by chronic inflammation, comprising administering to a subject in need thereof an effective amount of the oil-in-water nanoemulsion hereinbefore described.

[0050] In some embodiments the condition is associated with a change in expression or transcription are selected from the following list consisting of IL-8, IL-la, I L-ip, IL-IRa, GDF- 15, IL-10, IL-18Bpa, GH, VDBP, relaxin-2, IFN-y, PDGF-BB, TFF3, GCLA, GSTP1 and PI3K.

[0051] In some embodiments the condition is COPD or a disease associated with chronic exposure to an inflammation inducer.

[0052] In some embodiments the invention provides a use of the oil-in-water nanoemulsion of hereinbefore described in the manufacture of a medicament for the treatment and/or prophylaxis for the treatment and/or prevention of a condition medicated by chronic inflammation.

[0053] In some embodiments the condition is associated with a change in expression or transcription are selected from the following list consisting of L-8 IL-la, I L-ip, IL-IRA GDF-15 IL-10, IL-18Bpa, GH, VDBP, relaxin-2, IFN-y, PDGF-BB, TFF3, GCLC, GSTP1 and PI3K. [0054] In some embodiments the condition is COPD or a disease associated with chronic exposure to an inflammation inducer.

[0055] In some embodiments the invention provides a method for the treatment and/or prevention of cancer comprising administering to a subject in need thereof an effective amount of the oil-in-water nanoemulsion hereinbefore described.

[0056] In some embodiments the cancer is associated with a change in expression of the following list consisting of p53, KRAS and EGFR.

[0057] In some embodiments treatment of the cancer is associated with inhibition of expression of the proteins selected from the list consisting of casthespins B, casthespins D, casthespins S, casthespins G, Dkk-1, vimentin, GM-CSF, survivin, amphiregulin and urokinase.

[0058] In some embodiments the cancer is lung cancer or breast cancer.

[0059] In some embodiments the invention provides a use of the oil-in-water nanoemulsion hereinbefore described in the manufacture of a medicament for the treatment and/or prophylaxis for the treatment and/or prevention cancer.

[0060] In some embodiments cancer is associated with a change in expression or transcription of the following selected from the list consisting of p53, KRAS and EGFR.

[0061] In some embodiments the cancer is lung cancer or breast cancer.

[0062] In some embodiments the invention provides methods or uses hereinbefore described which are conducted using the oil-in-water nanoemulsion corresponding to less than and including about 100 pg/mL. In some embodiments the method or use is conducted using the oil-in-water nanoemulsion corresponding to up to and including about 50 pg/mL. In some embodiments the method or use is conducted using the oil-in-water nanoemulsion corresponding to between and including about 25 pg/mL to about 50 pg/mL. In some embodiments methods or uses hereinbefore described are conducted using the oil- in-water nanoemulsion at a concentration of about 25 pg/mL or about 50 pg/mL. [0063] Where the terms "comprise", "comprises" and "comprising" are used in the specification (including the claims) they are to be interpreted as specifying the stated features, integers, steps or components, but not precluding the presence of one or more other features, integers, steps or components, or group thereof.

[0064] Further aspects of the invention appear below in the detailed description of the invention.

Brief Description of Drawings

[0065] Embodiments of the invention will herein be illustrated by way of example only with reference to the accompanying drawings in which:

[0066] Figure 1 is a chart showing the that the oil-in-water nanoemulsion comprising an extracted agarwood oil is well tolerated in 5% CSE-induced BCi-NSl.l cells. BCi-NSl.l cells were pre-incubated for 1 h in the presence of increasing concentrations of agarwood-NE (10, 50, 100, 500, or 1000 pg/mL), followed by exposure to 5% CSE for 24h. Upon treatment, MTT assay was used to measure cell viability. Cell viability was normalised as a percentage compared to untreated control. The results are mean ± SEM of 3 independent experiments (****; _p <0.0001).

[0067] Figure 2 is a chart showing Inhibition of transcription of pro-inflammatory cytokines by the oil-in-water nanoemulsion comprising an extracted agarwood oil in cells exposed to an inflammation inducer. The pro-inflammatory cytokine IL-8. BCi-NSl.l cells were pre-incubated for 1 h in the presence of 25 and 50 pg/mL the oil-in-water nanoemulsion comprising an extracted agarwood oil, followed by exposure to 5% CSE for 24h. The mRNA levels of IL-8 were determined via RT-qPCR. Values are expressed as mean ± SEM (n = 4, *: p < 0.05; ****: p < 0.0001).

[0068] Figure 3 is a chart showing inhibition of protein expression of pro-inflammatory cytokines and mediators by the oil-in-water nanoemulsion comprising an extracted agarwood oil in cells exposed to an inflammation inducer. BCi-NSl.l cells were pre-incubated for 1 h in the presence of 25 and 50 pg/mL the oil-in-water nanoemulsion comprising an extracted agarwood oil, followed by exposure to 5% CSE for 24 h. The protein levels of IL-la (A), I L-ip (B), IL-IRa (C), and GDF-15 (D) were determined via human cytokine protein array.

[0069] Figure 4 is a chart showing stimulation of protein expression of anti-inflammatory mediators by the oil-in-water nanoemulsion comprising an extracted agarwood oil in cells exposed to an inflammation inducer. BCi-NSl.l cells were pre-incubated for 1 h in the presence of 25 and 50 pg/mL agarwood-NE, followed by exposure to 5% CSE for 24 h. The protein levels of IL-10 (A), IL-18Bpa (B), GH (C), VDBP (D), relaxin-2 (E), I FN-y (F), PDGF-BB (G), and TFF3 (H) were determined via human cytokine protein array.

[0070] Figure 5 is a chart showing stimulation of transcription of antioxidant genes by the oil-in-water nanoemulsion comprising an extracted agarwood oil in cells exposed to an inflammation inducer. BCi-NSl.l cells were pre-incubated for 1 h in the presence of 25 and 50 pg/mL the oil-in-water nanoemulsion comprising an extracted agarwood oil, followed by exposure to 5% CSE for 24 h. The mRNA levels of GCLC (A) and GSTP1 (B) were determined via RT-qPCR. Values are expressed as mean ± SEM (n = 3-4, *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001).

[0071] Figure 6 is a chart showing stimulation of transcription of a pro-survival gene by the oil-in-water nanoemulsion comprising an extracted agarwood oil in cells exposed to an inflammation inducer. BCi-NSl.l cells were pre-incubated for 1 h in the presence of 25 and 50 pg/mL the oil-in-water nanoemulsion comprising an extracted agarwood oil, followed by exposure to 5% CSE for 24 h. The mRNA levels of PI-3K were determined via RT-qPCR. Values are expressed as mean ± SEM (n = 4, ***: p < 0.001; ****: p < 0.0001).

[0072] Figure 7 is a photograph showing surpressed cell migration of A549 cells using the oil-in-water nanoemulsion comprising an extracted agarwood oil. A549 cells were seeded at low density before adding the nanoemaulsion and growing for 10-14 days at 25 and 50 pg/mL. The sample were then stained with crystal violet to enable visualisation versus a control.

[0073] Figure 8 is a chart showing the effect of the oil-in-water nanoemulsion comprising an extracted agarwood oil on human lung adenocarcinoma cells (A549) cell proliferation. A549 cells were seeded in a growth medium and the nanoemulsion was added at lpg/mL, 5, pg/mL 10 pg/mL, pg/mL 25 pg/mL and 50 pg/mL concentrations. The oil-in-water nanoemulsion comprising an extracted agarwood oil significantly decreased A549 cell proliferation in a dose dependent manner versus a control.

[0074] Figure 9 is a chart showing the effect of the oil-in-water nanoemulsion comprising an extracted agarwood oil on mRNA gene expression was measured in A549 cells. This chart shows an increase in the suppressor p53 gene at 25 and 50 pg/mL concentrations versus a control.

[0075] Figure 10 is a chart showing the effect of the oil-in-water nanoemulsion comprising an extracted agarwood oil on mRNA gene expression was measured in A549 cells. This chart shows and decrease in the expression of KRAS and EGFR proteins at 25 and 50 pg/mL concentrations versus a control.

[0076] Figure 11 is a chart showing the effect A549 cells incubated with the oil-in-water nanoemulsion comprising an extracted agarwood oil 25 pg/mL and 50 pg/mL concentrations versus a control. This chart shows a decrease in expression of casthespin B, casthespins D, casthespins S and casthespins G at 25 pg/mL and 50 pg/mL concentrations versus a control.

[0077] Figure 12 is a photograph showing that the nanoemulsion comprising an extracted agarwood oil was then added at 25 pg/mL and 50 pg/mL concentrations versus a control significantly decreases A549 cell migration over 24 to 48 hours.

[0078] Figure 13 is a chart showing inhibition of expression of the proteins Dkk-1, vimentin and CM-CSF using the nano emulsion comprising an extracted agarwood oil at 25 pg/mL and 50 pg/mL concentrations versus a control significantly decreases A549 cell migration over 24 to 48 hours.

[0079] Figure 14 is a chart showing a decrease in expression of Dkk-1, vimentin and GM- CSF in A459 cells at 25 pg/mL and 50 pg/mL concentrations versus a control.

[0080] Figure 15 is a chart showing a decrease in expression of survivin, amphiregulin and urokinase in A459 cells at 5 pg/mL and 50 pg/mL concentrations versus a control. Detailed Description

[0081] The present invention relates to an oil-in-water nanoemulsion comprising an extracted agarwood oil and a surfactant.

[0082] As used herein the term "nanoemulsion" relates to are relatively kinetically stable oil-in-water dispersions with droplet sizes on the order of 100s of nm. Specifically, the size range of about and including 160 nm to about 200 nm is to be understood to fall within the meaning of this term.

[0083] As used herein the term "an extracted agarwood oil" includes an oil extracted from plants of the Aquilaria genus, such as the species including Aquilaria Sinesis, Aquilaria Crassna, Aquilaria acuminata, Aquilaria apiculate, Aquilaria baillonii, Aquilaria banaensis, Aquilaria beccariana, Aquilaria brachyantha, Aquilaria cumingiana, Aquilaria filaria, Aquilaria grandiflora, Aquilaria hirta, Aquilaria khasiana, Aquilaria malaccensis, Aquilaria macrocarpa, Aquilaria rostrata and Aquilaria subintegra.

[0084] As used herein, the term "subject" shall be taken to mean any mammalian animal, including a human.

[0085] As used herein, "disease", "disorder", "condition" and the like, as they relate to a subject's health, are used interchangeably and have meanings ascribed to each and all such terms.

[0086] As used herein, the term "preventing", "prevent" or "prevention" includes providing prophylaxis with respect to occurrence or recurrence of a specified disease. An individual may be predisposed to or at risk of developing the disease or relapse but has not yet been diagnosed with the disease or the relapse.

[0087] An "effective amount" refers to at least an amount effective, at dosages and for periods of time necessary, to achieve the desired result. For example, the desired result may be a therapeutic or prophylactic result. An effective amount can be provided in one or more administrations. In some examples of the present disclosure, the term "effective amount" is meant an amount necessary to effect treatment or prevention of a disease as described herein. In some examples of the present disclosure, the term "effective amount" is meant an amount necessary to treat or prevent a given disease. The effective amount may vary according to the disease to be treated or factor to be altered and according to the weight, age, racial background, sex, health and/or physical condition and other factors relevant to the subject being treated. Typically, the effective amount will fall within a relatively broad range (e.g., a "dosage" range) that can be determined through routine trial and experimentation by a medical practitioner. Accordingly, this term is not to be construed to limit the disclosure to a specific quantity. The effective amount can be administered in a single dose or in a dose repeated once or several times over a treatment period.

[0088] As used herein, the terms "treating", "treat" or "treatment" include administering a therapeutic agent, for example, to thereby reduce or eliminate at least one symptom of a specified disease or to slow progression of the disease.

[0089] A protein or gene associated with cancer or chronic inflammation is one where a change in transcription or expression has been correlated to the aetiology or manifestation of symptoms or progression of the disease in question.

[0090] The invention relates to an oil-in-water nanoemulsion. A nanoemulsion of this type typically is comprised of a continuous aqueous phase and a particulate oil phase which is dispersed throughout the continuous phase. The dispersed particulate oil phase is comprised of discrete particles comprising the extracted agarwood oil. The particles are typically substantial spherical and have a diameter in the ranges of about and including 160 nm to about 200 nm.

[0091] A surfactant is often required to stabilise an emulsion where the continuous phase is aqueous and having dispersed within the nanoparticles comprising an oil.

Stabilisation by the surfactant reduces the likelihood of separation of the nanoemlusion into a discrete aqueous phase and a hydrophobic phase.

[0092] Examples of surfactants that can stabilise such emulsions include, ionic, nonionic, hydrophilic and hydrophobic surfactants. [0093] In some embodiments the surfactant can be a non-ionic surfactant. Examples of non-ionic surfactants include polyethylene glycol monocetyl Ether, poloxamers, pan 80, glycerol tristearate, sorbitan monopalmitate, triolien, span 20, hydroxypropyl methyl cellulose, tween 60, polysorbate 20, polyoxyethylene stearate, glyceryl monooleate, span 60, docosanamide, sorbitan trioleate, polyoxyethylene lauryl ether, polysorbate 40, emulsifier FM, propyleneglycol alginate, glycerol monohyroxystearate, fatty acids, lanolin, isopropyl esters, emulsifier LAE-9, polyethylene glycol) distearate, myristyl myristate, sucrose disterate, sorbitan sesquiolate, sorbitan tristate, glycerine monostearate, fatty alcohol polyoxyethylene ether N=3, castor oil polyoxyethylene (90) ether, monomystrin, alkyl polyglucoside, monocabrylin, alkyl phenyl polyoxyethylene ether, hydroxyethyl cellulose, dilaurin, decyl oleate, tween 80, tween 85, emulsifier EL-40, trimethylolpropane t, sucrose cocate, cetyl lactate, polyethylene glycol octadecyl ether, sucrose stearate, isooctyl palmitate, p entaerythrityl tetrastearate, Isopropyl myristate, monluarin, , octyl phenyl polyoxyethylene (30) ether, dibenzyl biphenyl polyoxyethylene etheri sooctadecanoic acid, ester with 1,2,3-propanetriol, 2-[bis(2-hydroxyethyl)amino]ethyl stearate, alkyl polyglucoside, oleyloleate, coconut oil alcohol acylamide, castor oil poloxyethylene (30) ether, ethylene glycol monostearate, glycerine monolaurate, glycerides, lard mono-, acetates and dodecyl polyglucoside.

[0094] In some embodiments the non-ionic surfactant is a poloxamer. Poloxamers are non-ionic surfactants of copolymers. The structure of this copolymer includes a central hydrophobic chain with hydrophilic chains extending therefrom. The general formula can be represented as follows:

[0095]

[0096] wherein the above general formula has: a = 2-130 and b = 15-67.

[0097] In some embodiments the poloxamer is poloxamer 407 which has a polyoxypropylene molecular mass of 4000 g/mol and a 70% polyoxyethylene content. [0098] Preferably the surfacing is present in an amount to stabilise the emulsion, such as in the amounts described herein.

[0099] COPD is a slow-developing, irreversible disease and causing approximately 3 million deaths per year. The principal features of COPD are chronic airway inflammation leading to irreversible damage of the lung parenchyma. This results in mucus retention and severe airflow limitation that leads to symptoms such as progressive and irreversible hyperresponsiveness of the airways. COPD is characterized by acute worsening of the disease's symptoms.

[0100] Chronic exposure to an inflammation inducer can lead to the development of conditions such as COPD. The term "inflammation inducer" used herein includes a respiratory drug deliverer, endemic pollution, industrial and work-place pollutants for example.

[0101] Respiratory drug deliverer can be for example, vaporiser liquid, cigarette smoke extract (CSE), cigarette smoke (CS), smoke associated with other tobacco-based products such as cigars, cigarillos, pipe tobacco and shisha. Additionally, this can be the smoke and/or vapours inhaled when administering other medical products, such as medical cannabis where legal.

[0102] Exposure to an inflammation inducer as described, such as CS, can include the promotion of the release of pro-inflammatory cytokines and mediators such as the interleukins (IL) IL-la, IL-ip, IL-8, IL-18, and growth/differentiation factor-15 (GDF15).

[0103] Additionally, exposure to an inflammation inducer as described, such as CS, can include inhibition of the release of anti-inflammatory cytokines such as IL-10.

[0104] GDF-15 has also been previously described to be a biomarker for COPD, and circulating GDF-15 to be increased in COPD patients when compared to healthy subjects.

[0105] Additional anti-inflammatory mediators are impacted by exposure to an inflammation induce, such as CS, to an inflammation inducer can include IL-18 binding protein (IL-18BP), growth hormone (GH), and vitamin D binding protein (VDBP). IL-18BP is a protein which can act as an IL-18 decoy, blocking the IL-18-mediated inflammatory response. Expression of this is reduced in the alveolar macrophages of rats exposed to second-hand smoke. As well as activity as a stimulator of tissue growth, cell reproduction, and cell regeneration, GH is also to reprogram macrophages towards an anti-inflammatory, reparative phenotype, and chronic exposure to exposure to an inflammation inducer has been described as reducing circulating GH levels.

[0106] VDBP is has cytokine-like activity and is an important mediator of inflammatory tissue injury. VDBP levels are downregulated in the plasma of people exposed to an inflammation induce, such as smokers compared to non-smokers.

[0107] Platelet-derived growth factor (PDGF) is a family of proteins regulating inflammation in the airways. In particular, PDGF-BB has a complex immunomodulatory role in many conditions including asthma, where it was shown to orchestrate lung tissue remodelling. It has been described to inhibit inflammatory responses during sepsis through the inhibition of pro-inflammatory cytokines including tumour necrosis factor-a (TNF-a), IL-6, IL-13, and IL-8.

[0108] Another anti-inflammatory protein with a relevant role in lung health is relaxin-2, which was recently shown, in a guinea pig model of exposure to an inflammation induce, such as CS exposure, to counteract CS-induced inflammation, remodelling, and tissue damage when administered exogenously.

[0109] The neuropeptide trefoil factor 3 (TFF3) is expressed by many cells of the respiratory tract and modulates the cytokine-induced secretion of inflammatory mediators. It affects airway mucus secretion and is involved in maintaining epithelial integrity and healing after mucosal injury. Expression of TFF3 has been described as reducing in a rat model of COPD obtained through exposure to an inflammation induce, such as CS. This could potentially contribute to tissue damage caused by CS exposure.

[0110] Further contribution to tissue damage by exposure to an inflammation induce, such as CS is caused by the direct induction of airway epithelial cell death, which is mediated by many mechanisms including inhibition of the protein arginine methyltransferase 6 (PRMT6)-phosphatidylinositol 3-kinase (PI3K)-Akt cell survival signalling pathway. Moreover, exposure to an inflammation induce, such as CS impairs the antiviral response of airway epithelial cells by inhibiting the production of interferon gamma (IFN-y) and IFN-y-dependent signalling resulting in further increased susceptibility to infection-associated tissue damage which can, in turn, fuel COPD progression.

[0111] Another fundamental driving factor of COPD is oxidative stress, which is caused by an imbalance between the production and elimination, through antioxidant detoxification mechanisms, of reactive oxygen species (ROS). A mediator of cellular detoxification is glutathione. This molecule is produced by a biosynthetic pathway whose initial and ratelimiting step is catalysed by the enzyme glutamate-cysteine ligase (GCLC). It has antioxidant activity as it acts as ROS scavenger. Carcinogenic products of tobacco smoke are detoxified upon conjugation with glutathione, and this reaction is catalysed by the enzyme glutathione S-transferase P (GSTP1). GSTP1 expression is reduced in lung and sputum specimens of patients with severe COPD.

[0112] The inventors have demonstrated that the nanoemulsion herein described is active for many of these biological markers associated with inflammation, including those brought about by an inflammation inducer, such as CS, as well as associated with COPD.

[0113] Additionally, the inventors have demonstrated that the nanoemulsion herein described is effective in preventing colony formation and cell proliferation of human lung adenocarcinoma cells (A549). The cell proliferation assay was displayed dose dependent behaviour. Additionally, migration in a wound healing assay has been demonstrated to be reduced for A549 cells.

[0114] The nanoemulsion herein described has been shown to promote expression of the p53 tumour suppressor gene. This gene has been implicated in supressing cancers that develop as tumours.

[0115] Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) is a gene that leads to expression of proteins involved in cell signalling pathways which have been described as controlling cell growth, cell maturation, and apoptosis. It has been linked with lung cancers such as non-small cell lung cancer. Epidermal growth factor receptor (EGFR) is another gene linked with cancers such as lung and breast cancer and is often over-expressed in such tissues and promotes cell proliferation. Both KRAS and EGFR have been demonstrated to be reduced in expression of A549 cells after treatment with the nanoemulsions herein described.

[0116] The protein Dickkopf-related protein 1 (Dkk-1) has been described as promoting migration and/or invasion of non-small cell lung cancer via the p-catenin signalling pathway. Vimentin is required for lung adenocarcinoma metathesis via heterotypic cell-cancer- associated fibroblast interactions during collective invasion.

[0117] Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been implicated in the development of lung cancer tumour development. The cathespins are a group of proteases that have been implicated in the degradation of the extracellular matrix as well as causing cancer metathesis.

[0118] Survivin expression inhibition aids in slowing tumor growth of non-small-cell lung cancer in cell both in vivo and in vitro. Amphiregulin promotes resilience to cancer therapeutics such as gefitinib in cancers such as non-small-cell lung cancers. Urokinase has been implicated in cancer invasion and metathesis mechanisms for lung cancers such as non- small-cell lung cancers.

[0119] The inventors have demonstrated that the nanoemulsion hereinbefore described is active in inhibiting expression of these cancer associated proteins.

Examples

[0120] Preparation of an extracted agarwood oil from Aquilaria Sinesis

[0121] The plant material was chopped and ground into power and left to air dry for 14 days to reduce moist contents. The essential oil was extracted from the dry agarwood powder through supercritical fluid carbon dioxide extraction at 0.005-0.006% per kg of raw agarwood powder. The extraction was performed at a pressure of 22 MPa and a temperature of 47 °C for 2 h, with a carbon dioxide fluid flow rate of 2 L/h. The separation was performed at 8 MPa and 40 °C. The essential oil obtained appeared as a transparent, slightly viscous liquid, with a brown colour and a deep woody aroma. The essential oil was soluble in alcohol and fixed oils and had the following typical composition (Table 1)

[0122] Table 1 - A typical composition of an extracted agarwood oil

[0123] The skilled person will appreciate that there will be natural variation in the exact components of an extracted agarwood oil due to for example seasonal variations and natural differences between particular plants. Thus, the above is provided so as to be representative of but not limiting to the exact chemical constituents of the extracted agarwood oil. Additionally, the applicant is not providing this example as an admission that any specific compound or combination of compounds may be responsible for the observed biological properties.

[0124] Preparation of an embodiment of an oil-in-water nanoemulsion comprising an extracted agarwood oil [0125] 200 mg of accurately weighed amount of an extracted agarwood oil extracted from Aquilaria Sinesis was taken in a 50 mL Falcon conical tube. In another tube, 50 mg of a surfactant, specifically a non-ionic surfactant Poloxamer 407 was dissolved with a required amount of purified distilled water (about 10 mL), and vortexed to ensure complete solubilization of the surfactant. The prepared solution was gradually added to the extracted agarwood oil at ambient temperature and vortexed for 1 minute. The coarse emulsion formed was subjected to probe sonication for 15 min at 80% amplitude in a 1 Hz on/off cycle to minimize heating. This resulted in the formation of a homogenised oil-in-water nanoemulsion, which was made up to a final volume of 20 mL by adding purified water. The obtained oil-in-water nanoemulsion was characterized for size and polydispersity index (dynamic light scattering), and morphology (transmission electron microscopy). The nanoemulsion was composed of droplets with substantially spherical morphology, of 180 ± 4.7 nm diameter and 0.36 ± 0.03 polydispersity index.

[0126] As will be understood by the skilled person, the polydispersity index measures the relative homogeneity of the particle sizes present in a given analyte.

[0127] Biological assessment of the oil-in-water nanoemulsion comprising an extracted agarwood oil

[0128] Cell Culture Treatment

[0129] Minimally immortalized human airway epithelium-derived basal cells ( BCi-NSl.1) were purchased from R. G. Crystal (Weill Cornell Medical College, New York, NY, USA). These cells were grown in broncho-epithelial basal media (BEBM) (Lonza, New York, NY, USA) supplemented with various growth factors and other supplements, including bovine pituitary extract, insulin, GA-1000 (Gentamicin sulfate-Amphotericin), retinoic acid, transferrin, triiodothyronine, epinephrine, human epidermal growth factor (BEGM Single Quots, Lonza), at 37 °C under humidified condition in the presence of 5% CO2. For experiments, the cells were seeded onto a 96-well plate (Corning, New York, NY, USA) or a 6-well plate (Corning) at a density of 1 x 104/well and 2 x 105/well, respectively. After 80% confluency, the cells were pre-treated for 1 h with agarwood-NE at the concentrations indicated, followed by the treatment of with or without 5% cigarette-smoke extract (CSE) for 24 h.

[0130] Cell Viability [0131] The cell viability of BCiNSl.l cells was assessed using 3-(4,5-Dimethylthiazol-2-yl)- 2,5- diphenyltetrazolium bromide (MTT, Merck, Rahway, NJ, USA). The cells were treated with different concentrations of the oil-in-water nanoemulsion comprising an extracted agarwood oil (10-1000 pg/mL) for 24 h in a 96-well plate. Then, MTT solution (250 pg/mL) was added into each well and incubated for 4 h. After incubation, the media was removed and the coloured formazan crystals formed in the reaction were dissolved with 100 pL dimethyl sulfoxide (DMSO, Merck, Rahway, NJ, USA). The absorbance at a wavelength of 540 nm was read using a POLARstar Omega microplate reader (BMG Labtech, Ortenberg, Germany).

[0132] Real Time-qPCR

[0133] The effects of the oil-in-water nanoemulsion comprising an extracted agarwood oil on mRNA expression levels of inflammation-related and oxidative stress-related genes in CSE-induced BCiNSl.l cells were determined by real time-qPCR. The cells were pre-treated with agarwood-NE at 25 and 50 pg/mL for 1 h, and then treated with or without 5% CSE for 24 h. The cells were then lysed with 500 pL TRI reagent (Merck). A total of 250 pL of chloroform was added and the mixture was centrifuged at 12,000x g, 4 °C, for 15 min. The aqueous phase was pipetted out into new Eppendorf tubes and 500 pL of isopropyl alcohol was added to precipitate the RNA. The tubes were then centrifuged at 12,000x g, room temperature, for 10 min. After centrifugation, the supernatant was removed, and the RNA pellets were washed 2x with 1 mL 75% ethanol. The tubes were centrifuged again at 8000x g, 4 °C, for 5 min. After the second round of centrifugation, the ethanol was removed, and the dry RNA pellets were dissolved in nuclease-free water. Nanodrop (Thermo Fisher Scientific, Waltham, MA, USA) was used to determine the concentration and purity of the RNA.

[0134] After subjecting to DNase I (Merck) treatment, 1 pg total RNA was reverse- transcribed to cDNA using the reaction mixture of M-MLV buffer (Thermo Fisher Scientific), random primers (0.5 pg/pL), dNTPs (10 mM), and DTT (100 mM). A thermal cycler (Eppendorf, Hamburg, Germany) was used in the subsequent steps involving denaturation (65 °C, 10 min), annealing (25 °C, 10 min), reverse transcription (37 °C, 50 min), and enzyme inactivation (70 °C, 15 min). Equal amounts (25 ng) of cDNA were then subjected to real-time qPCR with iTaq Universal SYBR green (BioRad, Hercules, CA, USA) and primers (forward and reverse, 0.5 pM each) using a CFX96 PCR system (BioRad). The real-time qPCR involved thermal cycles of 95 °C for 30 s (1 cycle), 95 °C for 15 s (40 cycles), and 60 °C for 30 s (1 cycle).

[0135] The sequences of human primers used were as follows in Table 2:

[0136] Human Cytokine Protein Array

[0137] The effects of of the oil-in-water nanoemulsion comprising an extracted agarwood oil on cytokine expression levels in CSE-induced BCiNSl.l cells were assessed using a human cytokine protein array kit (R&D Systems, Minneapolis, MN, USA). The cells were seeded in 6-well plates as indicated previously and were pre-treated with of the oil-in-water nanoemulsion comprising an extracted agarwood oil at 25 and 50 pg/mL for 1 h, then treated with 5% CSE for 24 h. The cells were lysed using RIPA buffer (ThermoFisher Scientific, Sydney, NSW, Australia) that contained protease and phosphatase inhibitors (Roche Diagnostics GmbH, Mannheim, Germany). Equal amounts (300 pg) of protein were loaded onto human cytokine arrays and incubated overnight at 4 °C. Further incubation with antibodies and reagents were conducted in accordance with the manufacturer's instructions. The protein spots in the array were photographed using the ChemiDoc MP (Bio-Rad, Hercules, CA, USA) and analysed using Image J. (version 1.53c, Bethesda, MD, USA).

[0138] Statistical Analysis

[0139] Figures 1, 2, 5 and 6, the data were expressed as mean ± SEM and statistically analysed using 1-way ANOVA, followed by Dunnett multiple comparison test. A p-value of <0.05 we considered significant. In Figures 3 and 4, the individual measurements are indicated together with the mean value of each group. [0140] Identification of a tolerated concentration of the oil-in-water nanoemulsion comprising an extracted agarwood oil in cells exposed to an inflammation inducer

[0141] To find a safe concentration for cell treatment, a toxicity study was performed, using the MTT assay to measure cell viability upon exposure of 5% CSE-induced BCi-NSl.l cells to various concentrations of the oil-in-water nanoemulsion comprising an extracted agarwood oil. The findings are shown in Figure 1. Treatment with an extracted agarwood oil amounts corresponding to up to 50 pg/mL agarwood oil extract did not result in significant reduction of cell viability (Figure 1). Concentrations of 100, 500, and 1000 pg/mL oil extracted from an extracted agarwood oil significantly decreased cell viability. (Figure 1, ps < 0.0001 against untreated control). In the subsequent experiments, cells were exposed to the well tolerated concentrations of 25 and 50 pg/mL the oil-in-water nanoemulsion comprising an extracted agarwood oil.

[0142] Inhibition of transcription of pro-inflammatory cytokines by the oil-in-water nanoemulsion comprising an extracted agarwood oil in cells exposed to an inflammation inducer

[0143] The anti-inflammatory activity of the oil-in-water nanoemulsion comprising an extracted agarwood oil was studied on 5% CSE-induced BCi-NSl.l cells by measuring the mRNA levels of the pro-inflammatory cytokine IL-8. CSE induced a 6.2-fold increase of the transcription of the IL-8 mRNA compared to control (Figure 2, p < 0.0001). Treatment with agarwood-NE at 25 and 50 pg/mL concentration resulted in the concentration-dependent reduction of IL-8 mRNA levels by 16.1% and 54.9%, respectively, compared to CSE-treated cells (Figure 2, p < 0.05 and p < 0.0001, respectively).

[0144] Inhibition of protein expression of pro-inflammatory cytokines and mediators by the oil-in-water nanoemulsion comprising an extracted agarwood oil in cells exposed to an inflammation inducer

[0145] The protein levels of the pro-inflammatory cytokines and mediators IL-la, IL-ip, IL-IRa, and GDF-15 are shown in Figure 3. Exposure of BCi-NSl.l cells to 5% CSE induced a significant increase in the levels of IL-la (1.6-fold, Figure 3A), IL-ip (1.7-fold, Figure 3B), IL- lRa (1.1-fold, Figure 3C), and GDF-15 (1.1-fold, Figure 3D) compared to untreated control. The levels of these proteins were significantly reduced to similar extents upon treatment with the two concentrations of agarwood-NE tested (25 and 50 pg/mL). Upon treatment with 50 pg/mL of the oil-in-water nanoemulsion comprising an extracted agarwood oil, the levels of IL-la were reduced by 54.5% (Figure 3A), while the levels of IL-1 were reduced by 35.4% (p < 0.0001, Figure 3B). Furthermore, treatment with 50 pg/mL of the oil-in-water nanoemulsion comprising an extracted agarwood oil resulted in a 15.4% reduction of the levels of IL-IRa (Figure 3C) and in a 45.3% reduction of the levels of GDF-15 (Figure 3D). Although, treatment with 25 pg/mL the oil-in-water nanoemulsion comprising an extracted agarwood oil resulted in a slightly lower extent of reduction of the amount of these four cytokines, no statistically significant difference was detected between the two concentrations tested in all cases.

[0146] Stimulation of protein expression of anti-inflammatory mediators by the oil-in- water nanoemulsion comprising an extracted agarwood oil in cells exposed to an inflammation inducer

[0147] The protein levels of the investigated anti-inflammatory cytokines and mediators are shown in Figure 4. Treatment of BCi-NSl.l cells with 5% CSE caused a significant reduction of the protein levels of the following cytokines compared to untreated control: IL- 10 (13.3%, Figure 4A), IL-18 Bpa (18.9%, Figure 4B), growth hormone (GH, 14.5%, Figure 4C), vitamin D binding protein (VDBP, 7.3%, Figure 4D), relaxin-2 (14.0%, Figure 4E), interferon-y (IFN-y, 15.9%, Figure 4F), platelet-derived growth factor (PDGF-BB, 13.3%, Figure 4G), and trefoil factor 3 (TFF3, 17.5%, Figure 4H). Exposure to 50 pg/mL of agarwood-NE counteracted the effect of CSE treatment, significantly increasing the levels of all these proteins compared to cells treated with 5% CSE only. In particular, the 50 pg/mL easing the levels of all these proteins compared to cells treated with 5% CSE only. In particular, the 50 pg/mL concentration of the oil-in-water nanoemulsion comprising an extracted agarwood oil increased the levels of IL-10 by 13.1% (Figure 4A) and the levels of IL-18 Bpa by 11.7% (Figure 4B). The levels of GH were increased by 7.0% (Figure 4C) and the levels of VDBP were increased by 7.0% (Figure 4D). Furthermore, upon treatment with 50 pg/mL the oil-in-water nanoemulsion comprising an extracted agarwood oil, relaxin-2 levels resulted in an increase by 8.0% (Figure 4E), and the levels of IFN-y increased by 11.8% (Figure 4F). Finally, 50 pg/mL the oil-in-water nanoemulsion comprising an extracted agarwood oil treatment increased the levels of PDGF-BB by 10.6% (Figure 4G) and the levels of TFF3 by 12.7% (Figure 4H). Treatment with 25 pg/mL the oil-in-water nanoemulsion comprising an extracted agarwood oil significantly increased the levels of IL-18Bpa (6.8%, Figure 4B), relaxin-2 (8.8%, Figure 4E), and IFN-y (6.7%, Figure 4F).

[0148] Stimulation transcription of antioxidant genes by the oil-in-water nanoemulsion comprising an extracted agarwood oil in cells exposed to an inflammation inducer

[0149] The antioxidant activity of the oil-in-water nanoemulsion comprising an extracted agarwood oil was investigated on 5% CSE-induced BCiNSl.l cells by measuring the mRNA levels of the genes GCLC and GSTP1 (Figure 5). Compared to the untreated control, CSE induced a 32.7% reduction of the transcription of the GCLC mRNA (Figure 5A, p < 0.0001) and a 65.4% reduction of the transcription of the GSTP1 mRNA (Figure 5B, p < 0.01). Treatment with the oil-in-water nanoemulsion comprising an extracted agarwood oil at 50 pg/mL concentration resulted in a significant 63.6% increase of the mRNA levels of GCLC (Figure 5A, p < 0.0001) and in a significant 344.0% increase of the mRNA levels of GSTP1 (Figure 5B, p < 0.001), compared to the 5% CSE-treated group. Furthermore, treatment with the oil-in-water nanoemulsion comprising an extracted agarwood oil at 25 pg/mL concentration significantly increased the GSTP1 mRNA levels by 138.5% compared to the 5% CSE-treated group (Figure 5B, p < 0.05). Treatment with the oil-in-water nanoemulsion comprising an extracted agarwood oil at 50 pg/mL concentration resulted in 63.6% increase of the mRNA of GCLC mRNA (Figure 5A, p < 0.0001) and a 65.4% reduction of the transcription of the GSTP1 mRNA (Figure 5B, p < 0.01). Treatment with the oil-in-water nanoemulsion comprising an extracted agarwood oil at 50 pg/mL concentration resulted in a significant 63.6% increase of the mRNA levels of GCLC (Figure 5A, p < 0.0001) and in a significant 344.0% increase of the mRNA levels of GSTP1 (Figure 5B, p < 0,001), compared to the 5% CSE-treated group. Furthermore, treatment with the oil-in-water nanoemulsion comprising an extracted agarwood oil at 25 pg/mL concentration significantly increased the GSTP1 mRNA levels by 138.5% compared to the 5% CSE-treated group (Figure 5B, p < 0.05). [0150] Stimulating transcription of a pro-survival gene by the oil-in-water nanoemulsion comprising an extracted agarwood oil in cells exposed to an inflammation inducer

[0151] The effect of the oil-in-water nanoemulsion comprising an extracted agarwood oil on pro-survival pathways was investigated on 5% CSE-induced BCi-NSl.l cells by measuring the mRNA levels of the PI3K gene (Figure 6). Exposure of cells to CSE resulted in a significant 31.2% reduction of the PI3K mRNA levels compared to the untreated control group (p < 0.0001, Figure 6). Treatment with the oil-in-water nanoemulsion comprising an extracted agarwood oil at 25 pg/mL and 50 pg/mL concentration resulted in a significant, concentration dependent increase of PI3K mRNA levels by 26.5% and 54.8% (p < 0.001 and p < 0.0001, respectively), compared to the 5% CSE-treated group (Figure 6).

[0152] Suppressing colony formation by the oil-in-water nanoemulsion comprising an extracted agarwood oil

[0153] The effect of the oil-in-water nanoemulsion comprising an extracted agarwood oil on human lung adenocarcinoma cells (A549) cells were conducted using a colony forming assay as described in the art. A549 cells were seeded at low density in a growth medium and the nanoemulsion was added at 25 pg/mL to 50 pg/mL concentration. The cells were then grown for 10 - 14 days and images taken of the cells to assess the cell migration. After the growing period, the samples were stained with crystal violet to enable visualisation. The oil- in-water nanoemulsion comprising an extracted agarwood oil significantly decreased A549 cell migration at 25 pg/mL and 50 pg/mL concentrations as can be seen in Figure 7.

[0154] Suppressing cell proliferation formation by the oil-in-water nanoemulsion comprising an extracted agarwood oil

[0155] The effect of the oil-in-water nanoemulsion comprising an extracted agarwood oil on human lung adenocarcinoma cells (A549) cells were conducted using a cell proliferation assay as described in the art. A549 cells were seeded in a growth medium and the nanoemulsion was added at lpg/mL, 5, pg/mL 10 pg/mL, pg/mL 25 pg/mL and 50 pg/mL concentrations. After a period of growth, MTT was added and the absorbance was measured at 540 nm. The oil-in-water nanoemulsion comprising an extracted agarwood oil significantly decreased A549 cell proliferation in a dose dependent manner versus a control as shown in Figure 8. Statistical analysis was performed using 2-way ANOVA and Tukey multiple comparison.

[0156] Stimulating expression of a tumour suppressor gene p53 by the oil-in-water nanoemulsion comprising an extracted agarwood oil and inhibiting expression of a cancer mediating genes KRAS and EGFR by the oil-in-water nanoemulsion comprising an extracted agarwood oil

[0157] mRNA gene expression was measured using qPCT in a manner similar to that already described. A549 cells were seeded in 6 well plates with growth media. The cells were incubated for 24 hours with the oil-in-water nanoemulsion comprising an extracted agarwood oil and at 25 pg/mL and 50 pg/mL concentration. This was followed by RNA extraction, cDNA preparation and RT-CPR. Figure 9 shows an increase in the p53 tumour suppression gene. Figure 10 shows a decreased in both the KRAS and EGFR genes.

[0158] Inhibiting expression of cancer mediating proteins by the oil-in-water nanoemulsion comprising an extracted agarwood oil

[0159] A549 cells were seeded in well plates and incubated with the oil-in-water nanoemulsion comprising an extracted agarwood oil 25 pg/mL and 50 pg/mL concentrations versus a control. After incubation for 24 hours, the proteins were extracted and quantified using an overnight incubation protein array blot. Chemidoc imaging of the blog was used to visualise the results.

[0160] Figure 11 shows a decrease in expression of casthespins B, casthespins D, casthespins S and casthespins G at 25 pg/mL and 50 pg/mL concentrations versus a control in A549 cells.

[0161] Figure 13 shows a decrease in expression of Dkk-1, vimentin and GM-CSF 25 pg/mL and 50 pg/mL concentrations versus a control in A549 cells. [0162] Figure 14 shows a decrease in expression of survivin, amphiregulin and urokinase 25 pg/mL and 50 pg/mL concentrations versus a control in A549 cells.

[0163] Decreasing A549 cell migration in a wound hearing / cell migration assay by the oil-in-water nanoemulsion comprising an extracted agarwood oil [0164] A549 cells were seeded in well plants in a growth media. A scraping tool was used to make wounds. The nanoemulsion comprising an extracted agarwood oil was then added at 25 pg/mL and 50 pg/mL concentrations versus a control. The cells were then allowed to migrate for 24 to 48 hours. Inspection was then performed using visual microscopy. [0165] Figure 12 is a photograph showing that the nanoemulsion comprising an extracted agarwood oil was then added at 25 pg/mL and 50 pg/mL concentrations versus a control significantly decreases A549 cell migration over 24 to 48 hours.

[0166] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is understood that the invention includes all such variations and modifications which fall within the spirit and scope of the present invention.

Claims

Claims

1. An oil-in-water nanoemulsion comprising a continuous aqueous phase, a surfactant, and a particulate oil phase, wherein the particulate oil phase is dispersed within the aqueous phase, wherein the particulate oil phase comprises particles comprising extracted agarwood oil having a diameter in the range of about and including 160 nm to about 200 nm.

2. The oil-in-water nanoemulsion of claim 1, wherein the particles have a diameter in the range of about and including 170 nm to about 190 nm.

3. The oil-in-water nanoemulsion of claim 1 or claim 2, wherein the particles have a diameter of about 180 nm.

4. The oil-in-water nanoemulsion of claim any one of claims 1 to 3, wherein the particles have a substantially spherical morphology.

5. The oil-in-water nanoemulsion of any one of claims 1 to 4, having a polydispersity index in the range of about and including 0.30 to about 0.40.

6. The oil-in-water nanoemulsion of any one of claims 1 to 5, having a polydispersity index in the range of about 0.36.

7. oil-in-water nanoemulsion of any one of claims 1 to 6, wherein the surfactant is a nonionic surfactant.

8. The oil-in-water nanoemulsion of claim 7, wherein the non-ionic surfactant is a Poloxomer.

9. The oil-in-water nanoemulsion of claim 8, wherein the Poloxomer is Poloxomer 407.

10. The oil-in-water nanoemulsion of any one of claims 1 to 9, wherein the surfactant is present in a concentration of between and including about 1.5 to 3.5 mg/mL.

11. The oil-in-water nanoemulsion of any one of claims 1 to 10, wherein the surfactant is present in a concentration of between and including about 2.5 mg/mL

12. The oil-in-water nanoemulsion of any one of claims 1 to 11, wherein the extracted agarwood oil is present in a concentration of between and including about 5 mg/mL to about 15 mg/mL

13. The oil-in-water nanoemulsion of any one of claims 1 to 12, wherein the extracted agarwood oil is present in a concentration of about 10 mg/mL.

14. The oil-in-water nanoemulsion of any one of claims 1 to 13, wherein the an extracted agarwood oil comprises about 12.31±5 percentage weight valerianol, about 8.03±2 percentage weight gamma-eudesmol, about 3.71±1 percentage weight epi-cyclocolorenone, about 3.71±1 percentage weight nootkatone, about 3.69±1 percentage weight beta-eudesmol, about 3.02±l percentage weight methyl phenethyl ketone, about 2.90+1 percentage weight 10-epi-gamma- eudesmol, about 1.74+0.5 percentage weight hinesol, about 1.68+0.5 percentage weight dihydro-columellarin, about 0.88±0.25 percentage weight alpha-curcumene, about 0.85±0.25 percentage weight alpha-humulene, about 0.56±0.1 percentage weight alpha-bulnesene, about 0.45±0.1 percentage weight Selina-4, 11-diene, about 0.38±0.1 percentage weight debromofiliformin, about 0.26±0.05 percentage weight 4,5-di-epi-Aristolochene, about 0.25±0.05 percentage weight elemol, about 0.16±0.05 percentage weight alpha-guaiene and about 0.11±0.05 percentage weight alpha-selinene.

15. A method of inhibiting transcription of a pro-inflammatory cytokine in at least one cell exposed to an inflammation inducer comprising administering to the cells an effective amount of the oil-in-water nanoemulsion of any one of claims 1 to 14.

16. The method of claim 15, wherein the pro-inflammatory cytokine is IL-8.

17. A method of inhibiting protein expression of pro-inflammatory mediators and/or cytokines in at least one cell exposed to an inflammation inducer comprising administering to the cells an effective amount of the oil-in-water nanoemulsion of any one of claims 1 to 14.

18. The method of claim 17, wherein the pro-inflammatory mediators and/or cytokines are selected from the list consisting of IL-la, I L-ip, IL-IRa and GDF-15.

19. A method of stimulating protein expression of anti-inflammatory mediators in at least one cell exposed to an inflammation inducer comprising administering to the cells an effective amount of the oil-in-water nanoemulsion of any one of claims 1 to 14.

20. The method of claim 19, wherein the anti-inflammatory mediators are selected from the list consisting of IL-10, IL-18Bpa, GH, VDBP, relaxin-2, IFN-y, PDGF-BB and TFF3.

21. A method of stimulating transcription of antioxidant genes in at least one cell exposed to an inflammation inducer comprising administering to the cells an effective amount of the oil-in-water nanoemulsion of any one of claims 1 to 14.

22. The method of claim 21, wherein the antioxidant gene is GCLA or GSTP1.

23. A method of stimulating transcription of a pro-survival gene in at least one cell exposed to an inflammation inducer comprising administering to the cells an effective amount of the oil-in-water nanoemulsion of any one of claims 1 to 14.

24. The method of claim 23, wherein the gene is PI3K.

25. The method of any one of claims 15 to 24, wherein the inflammation inducer is a respiratory drug deliverer.

26. The method of claim 25, wherein the respiratory drug deliverer is cigarette smoke, vaporizer liquid or cigarette smoke extract (CSE).

27. A method of suppressing cell proliferation and/or colony formation comprising administering to the cell an effective amount of the oil-in-water nanoemulsion of any one of claims 1 to 14.

28. The method of claim 27, wherein the cell proliferation and/or colony formation is associated with lung cancer activity.

29. The method of claim 28, wherein the cell is associated with lung cancer activity is A549.

30. A method of stimulating expression of a tumour suppressor gene in at least one cell comprising administering to the cells an effective amount of the oil-in-water nanoemulsion of any one of claims 1 to 14.

31. The method of claims 30, wherein the tumour suppressor gene is p53.

32. A method of inhibiting expression of a cancer associated gene in at least one cell comprising administering to the cells an effective amount of the oil-in-water nanoemulsion of any one of claims 1 to 14.

33. The method of claim 32, wherein the cancer associated gene is KRAS or EGFR.

34. A method of inhibiting expression of a cancer associated protein in at least one cell comprising administering to the cells an effective amount of the oil-in-water nanoemulsion of any one of claims 1 to 14.

35. The method according to claim 34, wherein the associated protein is selected from the list consisting of Dkk-1, vimentin, GM-CSF, survivin, amphiregulin and urokinase.

36. A method of inhibiting expression of a protein involved in cancer cell migration in at least one cell comprising administering to the cells an effective amount of the oil-in-water nanoemulsion of any one of claims 1 to 14.

37. The method according to claim 36, wherein the cancer cell is A549.

38. The method according to claim 36 or 37, wherein the protein involved in cancer cell migration is selected from the list consisting of cathepsin B, cathepsin D, cathepsin S and cathepsin G.

39. The method of any one of claims 15 to 38, wherein the method is performed in vivo or in vitro.

40. The method of any one of claims 15 to 38, wherein the oil-in-water nanoemulsion is administered to a human.

41. A method for the treatment and/or prevention of a condition associated with chronic inflammation, comprising administering to a subject in need thereof an effective amount of the oil-in-water nanoemulsion of any one of claims 1 to 14.

42. The method of claim 41, wherein the condition is associated with a change in expression or transcription are selected from the following list consisting of IL-8, IL-la, IL-ip, IL-IRa, GDF-15, IL-10, IL-18Bpa, GH, VDBP, relaxin-2, IFN-y, PDGF-BB, TFF3, GCLA, GSTP1 and PI3K.

43. The method of claim 42, wherein the condition is COPD or a disease associated with chronic exposure to inflammation inducer.

44. A use of the oil-in-water nanoemulsion of any one of claims 1 to 14 in the manufacture of a medicament for the treatment and/or prophylaxis for the treatment and/or prevention of a condition associated with by chronic inflammation.

45. The use of claim 44, wherein the condition is associated with a change in expression or transcription are selected from the following list consisting of L-8 IL-la, IL-ip, IL-IRA GDF-15 IL-10, IL-18Bpa, GH, VDBP, relaxin-2, IFN-y, PDGF-BB, TFF3, GCLC, GSTP1 and PI3K.

46. The use of claim 45, wherein the condition is COPD or a disease associated with chronic exposure to an inflammation inducer.

47. A method for the treatment and/or prevention of cancer comprising administering to a subject in need thereof an effective amount of the oil-in-water nanoemulsion of any one of claims 1 to 14.

48. The method of claim 42, wherein treatment of the cancer is associated with a change in expression of the following genes selected from the list consisting of p53, KRAS and EGFR.

49. The method of claim 43, wherein the cancer is lung cancer or breast cancer.

50. The method according to claim 47, wherein treatment of the cancer is associated with inhibition of expression of the proteins selected from the list consisting of casthespins B, casthespins D, casthespins S, casthespins G, Dkk-1, vimentin, GM-CSF, survivin, amphiregulin and urokinase.

51. A use of the oil-in-water nanoemulsion of any one of claims 1 to 14 in the manufacture of a medicament for the treatment and/or prophylaxis for the treatment and/or prevention cancer.

52. The use of claim 52, wherein the treatment of the cancer is associated with a change in expression of the genes selected from the list consisting of p53, KRAS and EGFR.

53. The use of claim 53, wherein the cancer is lung cancer or breast cancer.

54. The use according to claim 51, wherein treatment of the cancer is associated with inhibition of expression of the proteins selected from the list consisting of casthespins B, casthespins D, casthespins S, casthespins G, Dkk-1, vimentin, GM-CSF, survivin, amphiregulin and urokinase.

55. The method or use of any one of claims 15 to 54, wherein the method or use is conducted using the oil-in-water nanoemulsion corresponding to less than and including about 100 pg/mL.

56. The method or use of any one of claims 15 to 54, wherein the method or use is conducted using the oil-in-water nanoemulsion corresponding to up to and including about 50 pg/mL.

57. The method or use of any one of claims 15 to 54, wherein the method or use is conducted using the oil-in-water nanoemulsion corresponding to between and including about

25 to about 50 pg/mL.