WO2025091065A1 - Flavonoid composition - Google Patents

Abstract

A composition for delivering a flavonoid to a human or non-human animal subject, the composition comprising: a flavonoid such as pinocembrin in combination with one or more of: a fatty acid, a solubility enhancer configured to increase the aqueous solubility of the fatty acid, and a matrix.

Description

FLAVONOID COMPOSITION

FIELD OF THE INVENTION

[001]. The present invention relates generally to compositions for the delivery of a flavonoid to a human or other animal. More particularly, the compositions are used for application via routes that avoid first pass metabolism, such as via the buccal, sublingual, nasal, pharyngeal, routes.

BACKGROUND TO THE INVENTION

[002]. Flavonoids, a diverse class of polyphenolic compounds ubiquitously present in the plant kingdom, have garnered significant scientific interest due to their wide range of biological activities. Comprising over 6,000 identified structures, flavonoids can be further subdivided into several subclasses including flavonols, flavones, flavanones, flavan-3-ols, anthocyanidins, and isoflavones, based on variations in their central pyran ring.

[003]. Historically, the dietary significance of flavonoids was underscored by their ability to impart coloration to flowers, fruits, and leaves. However, a growing body of evidence from epidemiological, in vitro, and in vivo studies now suggests that these compounds play pivotal roles far beyond their pigmentary properties. Their ubiquitous presence in a variety of fruits, vegetables, teas, wines, and traditional medicinal herbs implicates them as integral components of human diet and ancient pharmacopeia.

[004]. Biochemically, flavonoids have been shown to modulate a multitude of signaling pathways, receptors, and enzymes. Their multifaceted interactions with the cellular machinery contribute to their antioxidative, anti-inflammatory, antiviral, and antitumoral properties. The inherent structure of flavonoids, characterized by hydroxylated phenolic structures, is pivotal to their radical scavenging potential, thereby conferring protection against oxidative stress — a major contributor to the pathophysiology of numerous chronic diseases. [005]. However, the therapeutic utilization of flavonoids is not without challenges. Despite their promising in vitro activities, the in vivo bioavailability of flavonoids is often limited due to their extensive first-pass metabolism by the liver, leading to rapid conjugation, methylation, and excretion. Thus, while dietary consumption of flavonoids is associated with several health benefits, direct therapeutic applications often necessitate modifications to improve their pharmacokinetic profiles. In many cases, such modifications have an adverse effect on efficacy, or are otherwise not possible or practicable.

[006]. Where an improvement in pharmacokinetic profile is not readily achievable, the only option is to increase flavonoid dosage (unit dosage amount and/or frequency of dosing) so as to maintain minimal effective serum concentrations. Flavonoids are typically obtained from low abundance natural sources, with the cost of isolating meaningful amounts of active often resulting in any therapeutic regime being prohibitively expensive.

[007]. It is an aspect of the present invention to provide an improvement to prior art laboratory-based analyte detection hardware and methods. It is a further aspect of the present invention to provide a useful alternative to prior art laboratory-based analyte detection hardware and methods.

[008]. The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

SUMMARY OF THE INVENTION

[009]. In a first aspect, but not necessarily the broadest aspect, the present invention provides a composition for delivering a flavonoid to a subject, the composition comprising: a flavonoid in combination with one or more of: a fatty acid, a solubility enhancer configured to increase the aqueous solubility of the fatty acid, and a matrix.

[010]. In one embodiment of the first aspect, the fatty acid has a chain length of: between about 13 to 23 carbon atoms, or about 14 to 22 carbon atoms, or about 15 to 21 carbon atoms, or about 16 to 20 carbon atoms, or about 17 to 19 carbon atoms, or 18 carbon atoms.

[Oi l]. In one embodiment of the first aspect, the fatty acid is a dietary fatty acid.

[012]. In one embodiment of the first aspect, the fatty acid is a monounsaturated fatty acid.

[013]. In one embodiment of the first aspect, the fatty acid is a cis fatty acid

[014]. In one embodiment of the first aspect, the double bond of the monounsaturated fatty acid is at or about midway along the carbon chain.

[015]. In one embodiment of the first aspect, the fatty acid is an omega-9 fatty acid.

[016]. In one embodiment of the first aspect, the omega-9 fatty acid is oleic acid.

[017]. In one embodiment of the first aspect, the solubility enhancer is an emulsifier capable of emulsifying the fatty acid.

[018]. In one embodiment of the first aspect, the solubility enhancer is a polymer.

[019]. In one embodiment of the first aspect, the polymer is a polymer of ethylene oxide.

[020]. In one embodiment of the first aspect, the solubility enhancer is polyethylene glycol

(PEG). [021]. In one embodiment of the first aspect, the PEG has a molecular weight or an average molecular weight of: between about 10 and 1000 Da, or between about 50 and 900 Da, or between about 100 and 800 Da, or between about 150 and 700 Da, between about 200 and 600 Da, or between about 300 and 500 Da, or about 400 Da.

[022]. In one embodiment of the first aspect, the fatty acid and the solubility enhancer are provided as separate molecules, or combined in a single molecule.

[023]. In one embodiment of the first aspect, the single molecule is formed by reaction of a fatty acid with a solubility enhancer at a ratio of about 1 : 1 , or another suitable ratio.

[024]. In one embodiment of the first aspect, the single molecule is a PEG oleate.

[025]. In one embodiment of the first aspect, the PEG oleate is PEG-7 oleate, PEG-8 oleate, PEG-9 oleate, PEG- 10 oleate, or PEG-11 oleate,

[026]. In one embodiment of the first aspect, the matrix is a polymer or a biodegradable polymer.

[027]. In one embodiment of the first aspect, the polymer or biodegradable polymer is an anionic or cationic polymer.

[028]. In one embodiment of the first aspect, the matrix is stable under a physiological condition for less than about 20 minutes, including or absent any enzyme capable of breaking down the matrix.

[029]. In one embodiment of the first aspect, the matrix is a gel or a hydrogel.

[030]. In one embodiment of the first aspect, the matrix is positively charged at a physiological pH, including a pH between about 5.0 and 8.5. [031]. In one embodiment of the first aspect, the matrix is charged so as to bind a mucosal surface of a living animal or a cadaver animal, including the buccal, sublingual, pharyngeal, nasopharyngeal, pulmonary or nasal mucosa of an animal.

[032]. In one embodiment of the first aspect, the matrix is configured to retain the flavonoid and the fatty acid therein and/or thereabout, and controllably release the flavonoid to a mucosa of an animal that is proximal to or in contact with the composition.

[033]. In one embodiment of the first aspect, the matrix comprises an organic polymer.

[034] . In one embodiment of the first aspect, the organic polymer is a polysaccharide.

[035]. In one embodiment of the first aspect, the polysaccharide is a linear polysaccharide

[036]. In one embodiment of the first aspect, the matrix is selected from a chitosan, an aminated cellulose, a starch, an alginate, a collagen, a gelatin, a chitin, a polylysine, a pollyallylamine, a polyethylenimine, a polyhydroxy alkanoate, a cellulose nanofiber, and a cellulose nanocrystal.

[037] . In one embodiment of the first aspect, the flavonoid is a flavanone.

[038]. In one embodiment of the first aspect, the flavanone has a chemical structure according to formula 1

[039], wherein R2’, R3, R3’, R4’, R5, R6, R7 are each independently: H,

OH,

O-,

O-CH3, a glucoside (including a rhamnosidoglucoside), or any other organic functional group.

[040]. In one embodiment of the first aspect, R2’, R3, R3’, R4’, R5, R6, R7 are as follows:

R3 R5 R6 R7 R2' R3' R4' Name

H H H H H Flavanone

H OCH₃ H H H 5-Methoxyflavanone

H H OH H H 6-Hydroxyflavanone

H H OCH₃ H H 6-Methoxyflavanone

H H H OH H 7-Hydroxyflavanone

H H H H OH 2'-Hydroxyflavanone

H H H H H 4'-Hydroxyflavanone

H H H H H 4'-Methoxyflavanone

H OH H OH H Pinocembrin

H OH H OCH3 H Pinocembrin-7-methylether

H OH H OH H Naringenin

H OH H OH H Isosakuranetin

H OH H OCH3 H Sakuranetin

H OH H Gia H Naringenin-7-glucoside

H OH H Rh-Glb H Naringin

H OH H OH H Eriodictyol

H OH H OH H Homoeriodictyol

H OH H OH H Hesperetin

OH OH H OH H

Taxifolin a; Gl=Glucoside. b; Rh-Gl=Rhamnosidoglucoside.

[041]. In one embodiment of the first aspect, the flavanone is dihydroxyflavanone and/or a (2S)-flavan-4-one, or a functional derivative thereof. [042]. In one embodiment of the first aspect, the flavanone is (2S)-5,7-dihydroxy-2- phenyl-2,3-dihydrochromen-4-one, or a functional derivative thereof.

[043]. In one embodiment of the first aspect, the flavonoid is of the type naturally synthesized in a plant cell, although is not necessarily obtained from a plant cell.

[044]. In one embodiment of the first aspect, the composition is formulated as a gel, a lozenge, a troche, a dragee, a viscous composition, a bioadhesive, a medication stick, a tablet, a capsule, a spray, or a slurry

[045]. In one embodiment of the first aspect, the composition comprises a taste-masking agent, and/or a taste modifying agent, and/or a sweetener.

[046]. In a second aspect, the present invention provides a method of administering a flavonoid to a subject in need thereof, or desirous thereof, the method comprising contacting the composition of any embodiment of the first aspect to a target issue of the body of the subject.

[047]. In one embodiment of the second aspect, the target tissue is an external surface or an internal surface.

[048]. In one embodiment of the second aspect, the external surface is a skin surface, and the internal surface is a mucosal surface.

[049]. In one embodiment of the second aspect, the mucosal surface is a buccal, sublingual, pharyngeal, nasopharyngeal, pulmonary or nasal mucosal surface.

[050]. In one embodiment of the second aspect, the step of contacting is for a period of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 minutes, or until the matrix of the composition has mostly or fully degraded. [051]. In one embodiment of the second aspect, the step of contacting comprises actuation of a dispenser about the surface, the dispenser comprising an outlet configured to direct the composition toward or onto the surface.

[052]. In one embodiment of the second aspect, the method comprises causing the dispenser to release the composition via the outlet.

[053]. In one embodiment of the second aspect, release of the composition is caused by decreasing the internal volume of a container holding the composition, compressing a flexible container holding the composition, or moving a plunger through a container holding the composition.

BRIEF DESCRIPTION OF THE FIGURES

[054]. FIG. 1 is a calibration curve of pinocembrin in KBR (50 - 5000 ng/mL), R2 = 0.9956. Data are presented as the mean of two duplicate injections of each calibration standard.

[055]. FIG. 2 shows chromatograms of (A) blank KBR, (B) pinocembrin spiked into KBR at 50 ng/mL (retention time: 3.8 min), and (C) a representative receptor chamber sample at 240.

[056]. FIG. 3 shows a graph for quantification (in percentage terms) of applied pinocembrin dose permeating into the receptor chamber after application of pinocembrin in chitosan gel (3% w/v) with or without oleic acid (5% w/w) and/or PEG400 (5% w/w). Data are presented as mean ± SD (n=5-6). **p<0.01 and ****p<0.0001 indicates a significant difference in % permeating from the chitosan/oleic acid/PEG400 gel relative to the chitosan only gel using a two-way analysis of variance with a Dunnett’s posthoc test [057]. FIG. 4 is a calibration curve of pinocembrin in mouse plasma (50 - 5000 ng/mL), R2 = 0.9986. Data are presented as the mean of two duplicate injections of each calibration standard.

[058]. FIG. 5 shows chromatograms of (A) blank plasma, (B) pinocembrin spiked into mouse plasma at 50 ng/mL (retention time: 3.8 min), and (C) a representative mouse plasma sample at 240 min post dose.

[059]. FIG. 6 is a graph quantifying plasma concentrations of pinocembrin following buccal application in a chitosan gel with and without oleic acid (5% w/w) and PEG400 (5% w/w) to C57BL/6 mice, normalised to a nominal dose of 40 mg/kg. Data are presented as mean ± SD (n=6). A significant difference in area under the pinocembrin plasma concentration versus time curve (z<-5.27, p<0.05) exists following application of pinocembrin in chitosan gel and pinocembrin in chitosan gel with oleic acid and PEG400.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS THEREOF

[060]. After considering this description it will be apparent to one skilled in the art how the invention is implemented in various alternative embodiments and alternative applications. However, although various embodiments of the present invention will be described herein, it is understood that these embodiments are presented by way of example only, and not limitation. As such, this description of various alternative embodiments should not be construed to limit the scope or breadth of the present invention. Furthermore, statements of advantages or other aspects apply to specific exemplary embodiments, and not necessarily to all embodiments, or indeed any embodiment covered by the claims.

[061]. Throughout the description and the claims of this specification the word "comprise" and variations of the word, such as "comprising" and "comprises" is not intended to exclude other additives, components, integers or steps. [062]. Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment, but may.

[063]. The term "subject" is used to refer to a member of an animal species of mammalian origin, including but not limited to, a mouse, a rat, a cat, a goat, sheep, horse, hamster, ferret, platypus, pig, a dog, a guinea pig, a rabbit and a primate, such as, for example, a monkey, ape, and human.

[064]. The terms "treat", "treating", “prevent”, and “preventing”, includes abrogating, substantially inhibiting, slowing or reversing the progression of a disease, condition or disorder, substantially ameliorating clinical or symptoms of a condition, substantially preventing the appearance of clinical or esthetical symptoms of a disease, condition, or disorder, and protecting from harmful or annoying symptoms. Treating further refers to accomplishing one or more of the following: (a) reducing the severity of the disorder; (b) limiting development of symptoms characteristic of the disorder(s) being treated; (c) limiting worsening of symptoms characteristic of the disorder(s) being treated; (d) limiting recurrence of the disorder(s) in patients that have previously had the disorder(s); and (e) limiting recurrence of symptoms in patients that were previously asymptomatic for the disorder(s).

[065]. In a first aspect, the present invention provides a composition for delivering a flavonoid to a subject, the composition comprising: a flavonoid in combination with one or more of: a fatty acid, a solubility enhancer configured to increase the aqueous solubility of the fatty acid, and a matrix. The present invention is predicated at least in part on the inventors’ discovery that such a composition is useful for the delivery of a flavonoid across mucosal surfaces having a venous return that does not drain into the portal venous system. According to the present invention, the flavonoid may cross the mucosal surface and enter the general circulation without passing through the liver, thereby avoiding first pass metabolism. In some circumstances, avoidance of first pass metabolism allows for the use of lower amounts of flavonoid in a dosage form, or for less frequent dosing. The need for less flavonoid to achieve useful plasma concentrations is advantageous where the cost of the active is a negative factor in a treatment decision.

[066]. The flavonoid of the present composition may be selected for any clinical, health, or lifestyle-related reason. In some cases, the aim of administration is to prevent or treat a condition, such as a disease or a disorder, in the subject. In other cases, the flavonoid is administered to improve function in an otherwise healthy subject. Some subjects may seek administration of a flavonoid for reasons unrelated to health, and for some other perceived benefit.

[067]. In the context of the present invention, the term "flavonoid" is intended to include flavanols, flavones, and flavanones. In some embodiments of the composition the flavonoid is a flavanone, and in some embodiments a chiral flavanone existing as optical isomers, and in which case the flavanone may be either the D- isomeror the S-isomer. In some embodiments an uneuqal mixture of the D-isomer and S-isomer is used, or a racemic mixture is used. In some embodiments of the compositions the S-isomer is used in the present compositions.

[068]. In some embodiments, the flavonoid is the flavanone pinocembrin, or a modified form of pinocembrin. Advantageously, pinocembrin is a naturally occurring compounding having a known safety profile. Moreover, the compound is not controlled to the extent that a medical practitioner must prescribe the compound.

[069]. The composition may comprise an effective amount of the flavonoid, having regard to the use of concern and the characteristics of the subject. According to some embodiments, the effective amount of the flavonoid compound of the composition is of an amount from about 0.000001 mg/kg body weight of the subject to about 100 mg/kg body weight. According to another embodiment, the effective amount of the flavonoid compound of the composition is of an amount from about 0.00001 mg/kg body weight to about 100 mg/kg body weight. According to another embodiment, the effective amount of the flavonoid compound of the composition is of an amount from about 0.0001 mg/kg body weight to about 100 mg/kg body weight. According to another embodiment, the effective amount of the flavonoid compound of the composition is of an amount from about 0.001 mg/kg body weight to about 10 mg/kg body weight. According to another embodiment, the effective amount of the flavonoid compound is of an amount from about 0.01 mg/kg body weight to about 10 mg/kg body weight. According to another embodiment, the effective amount of the flavonoid compound of the composition is of an amount from about 0.1 mg/kg (or 100 pg/kg) body weight to about 10 mg/kg body weight. According to another embodiment, the effective amount of the flavonoid compound of the composition is of an amount from about 1 mg/kg body weight to about 10 mg/kg body weight. According to another embodiment, the effective amount of the flavonoid compound of the composition is of an amount from about 10 mg/kg body weight to about 100 mg/kg body weight. According to another embodiment, the effective amount of the flavonoid compound of the composition is of an amount from about 2 mg/kg body weight to about 10 mg/kg body weight. According to another embodiment, the effective amount of the flavonoid compound of the composition is of an amount from about 3 mg/kg body weight to about 10 mg/kg body weight. According to another embodiment, the effective amount of the flavonoid compound of the composition is of an amount from about 4 mg/kg body weight to about 10 mg/kg body weight. According to another embodiment, the effective amount of the flavonoid compound of the composition is of an amount from about 5 mg/kg body weight to about 10 mg/kg body weight. According to another embodiment, the effective amount of the flavonoid compound of the composition is of an amount from about 20, 30, 40, 50, or 60 mg/kg body weight to about 100 mg/kg body weight, including . According to another embodiment, the effective amount of the flavonoid compound of the composition is of an amount from about 70 mg/kg body weight to about 100 mg/kg body weight. According to another embodiment, the effective amount of the flavonoid compound of the composition is of an amount from about 80 mg/kg body weight to about 100 mg/kg body weight. According to another embodiment, the effective amount of the flavonoid compound of the composition is of an amount from about 90 mg/kg body weight to about 100 mg/kg body weight. According to another embodiment, the effective amount of the flavonoid compound of the composition is of an amount from about 0.000001 mg/kg body weight to about 90 mg/kg body weight. According to another embodiment, the effective amount of the flavonoid compound of the composition is of an amount from about 0.000001 mg/kg body weight to about 80 mg/kg body weight. According to another embodiment, the effective amount of the flavonoid compound of the composition is of an amount from about 0.000001 mg/kg body weight to about 70 mg/kg body weight. According to another embodiment, the effective amount of the flavonoid compound of the composition is of an amount from about 0.000001 mg/kg body weight to about 60 mg/kg body weight. According to another embodiment, the effective amount of the flavonoid compound of the composition is of an amount from about 0.000001 mg/kg body weight to about 50 mg/kg body weight. According to another embodiment, the effective amount of the flavonoid compound of the composition is of an amount from about 0.000001 mg/kg body weight to about 40 mg/kg body weight. According to another embodiment, the effective amount of the flavonoid compound is of an amount from about 0.000001 mg/kg body weight to about 30 mg/kg body weight. According to another embodiment, the effective amount of the flavonoid compound of the composition is of an amount from about 0.000001 mg/kg body weight to about 20 mg/kg body weight. According to another embodiment, the effective amount of the flavonoid compound of the composition is of an amount from about 0.000001 mg/kg body weight to about 10 mg/kg body weight. According to another embodiment, the effective amount of the flavonoid compound of the composition is of an amount from about 0.000001 mg/kg body weight to about 1 mg/kg body weight. According to another embodiment, the effective amount of the flavonoid compound of the composition is of an amount from about 0.000001 mg/kg body weight to about 0.1 mg/kg body weight. According to another embodiment, the effective amount of the flavonoid compound of the composition is of an amount from about 0.000001 mg/kg body weight to about 0.1 mg/kg body weight. According to another embodiment, the effective amount of the flavonoid compound of the composition is of an amount from about 0.000001 mg/kg body weight to about 0.01 mg/kg body weight. According to another embodiment, the effective amount of the flavonoid compound of the composition is of an amount from about 0.000001 mg/kg body weight to about 0.001 mg/kg body weight. According to another embodiment, the effective amount of the flavonoid compound of the composition is of an amount from about 0.000001 mg/kg body weight to about 0.0001 mg/kg body weight. According to another embodiment, the effective amount of the flavonoid compound of the composition is of an amount from about 0.000001 mg/kg body weight to about 0.00001 mg/kg body weight.

]

[070]. According to some other embodiments, the composition comprises flavonoid in an amount sufficient to achieve (in a single dosage form, or across multiple dosage forms) from 1 pg/kg/day to 25 pg/kg/day. According to some other embodiments, the dose of the flavonoid compound of the composition ranges from 1 pg/kg/day to 2 pg/kg/day. According to some other embodiments, the dose of the flavonoid compound of the composition ranges from 2 pg/kg/day to 3 pg/kg/day. According to some other embodiments, the dose of the flavonoid compound of the composition ranges from 3 pg/kg/day to 4 pg/kg/day. According to some other embodiments, the dose of the flavonoid compound of the pharmaceutical ranges from 4 pg/kg/day to 5 pg/kg/day. According to some other embodiments, the dose of the flavonoid compound of the composition ranges from 5 pg/kg/day to 6 pg/kg/day. According to some other embodiments, the dose of the flavonoid compound of the composition ranges from 6 pg/kg/day to 7 pg/kg/day. According to some other embodiments, the dose of the flavonoid compound of the composition ranges from 7 pg/kg/day to 8 pg/kg/day. According to some other embodiments, the dose of the flavonoid compound of the composition ranges from 8 pg/kg/day to 9 pg/kg/day. According to some other embodiments, the dose of the flavonoid compound of the composition ranges from 9 pg/kg/day to 10 pg/kg/day. According to some other embodiments, the dose of the flavonoid compound of the composition ranges from 1 pg/kg/day to 5 pg/kg/day. According to some other embodiments, the dose of the flavonoid compound of the composition ranges from 5 pg/kg/day to 10 pg/kg/day. According to some other embodiments, the dose of the flavonoid compound of the composition ranges from 10 pg/kg/day to 15 pg/kg/day. According to some other embodiments, the dose of the flavonoid compound of the composition ranges from 15 pg/kg/day to 20 pg/kg/day. According to some other embodiments, the dose of the flavonoid compound of the composition ranges from 25 pg/kg/day to 30 pg/kg/day. According to some other embodiments, the dose of the flavonoid compound of the composition ranges from 30 pg/kg/day to 35 pg/kg/day. According to some other embodiments, the dose of the flavonoid compound of the composition ranges from 35 pg/kg/day to 40 pg/kg/day. According to some other embodiments, the dose of the flavonoid compound of the composition ranges from 40 pg/kg/day to 45 pg/kg/day. According to some other embodiments, the dose of the flavonoid compound of the composition ranges from 45 pg/kg/day to 50 pg/kg/day. According to some other embodiments, the dose of the flavonoid compound of the composition ranges from 50 pg/kg/day to 55 pg/kg/day. According to some other embodiments, the dose of the flavonoid compound of the composition ranges from 55 pg/kg/day to 60 pg/kg/day. According to some other embodiments, the dose of the flavonoid compound of the composition ranges from 60 pg/kg/day to 65 pg/kg/day. According to some other embodiments, the dose of the flavonoid compound of the composition ranges from 65 pg/kg/day to 70 pg/kg/day. According to some other embodiments, the dose of the flavonoid compound of the composition ranges from 70 pg/kg/day to 75 pg/kg/day. According to some other embodiments, the dose of the flavonoid compound of the composition ranges from 80 pg/kg/day to 85 pg/kg/day. According to some other embodiments, the dose of the flavonoid compound of the composition ranges from 85 pg/kg/day to 90 pg/kg/day. According to some other embodiments, the dose of the flavonoid compound of the composition ranges from 90 pg/kg/day to 95 pg/kg/day. According to some other embodiments, the dose of the flavonoid compound of the composition ranges from 95 pg/kg/day to 100 pg/kg/day.

[071]. The amount flavonoid that can be included in the present composition generally ranges from generally about 0.001 mg/kg body weight to about 10 g/kg body weight. However, dosage levels are based on a variety of factors, including the type of condition or disorder or other usage, the age, weight, sex, medical condition of the subject, the severity of the condition, the route and frequency of administration, and the particular flavonoid employed. Thus the dosage regimen may vary widely, but can be determined routinely by a physician or other health practitioner using standard methods, and having the benefit of the present specification. [072]. Without wishing to be limited by theory in any way, the fatty acid of the present composition is thought to function as a carrier, and possibly a permeation enhancer, which assists passage of the flavonoid through the relevant surface of the subject (such as the mucosa or the skin), and into the circulation. The flavonoid may be simply mixed with the fatty acid to form a suspension and/or a solution. Typically, this form of composition will be useful in delivery of flavonoid across the mucosa.

[073]. Alternatively, the flavonoid may be entrapped in fatty acid vesicles. For example, oleic acid vesicles may prepared using a film hydration method, whereby oleic acid and the flavonoid are dissolved or suspended in suitable solvent (such as methanol) followed by evaporation of solvent under vacuum using a rotary evaporator. The dried film is then hydrated at ambient temperature for several hours with phosphate buffer (pH 5.5). The prepared vesicular dispersion is sonicated to form the uniform size vesicular dispersion. Unentrapped flavonoid may be separated from the vesicle dispersion using gel chromatography (such as Sephadex™ G-50). Vesicular compositions may be more applicable to transdermal applications.

[074]. Advantage may be gained where the fatty acid is a dietary fatty acid. As used herein, the term “dietary” is used to mean that fatty acid is found in a food product such as fruits, vegetable oils, seeds, nuts, animal fats and fish oils. In the context of a composition, dietary fatty acids may be subject to lower or no regulatory requirements for supply to the public.

[075]. Omega-9 fatty acids, such as oleic acid, are generally preferred given their natural abundance in plant sources such as olives. Consumers have a general familiarity with olive oil and similar products and may therefore more readily accept a flavonoid provided in that context.

[076]. The present composition may comprise a solubility enhancer to increase the aqueous solubility of the fatty acid. As will be appreciated, the fatty acid (which carries the flavonoid) will be exposed to an aqueous environment of the subject. For example, if applied to the mucosa the composition is exposed the fluid bathing the mucosal cells, such fluid typically being a substantially aqueous solution of electrolytes, proteins, and carbohydrates. In a particular use application, the composition is for buccal or sublingual administration and in which case compatibility with the saliva is preferred. The fatty acid (with flavonoid carried therein) may be rendered generally miscible with or dispersible within the saliva by way of the solubility enhancer.

[077]. In some embodiments, the solubility enhancer has hydrophilic and hydrophobic regions and therefore able to interface with the fatty acid and an aqueous environment. The solubility enhancer may be an emulsifier such as a polysorbate, carboxymethyl cellulose, sodium lauryl sulfate, glyceryl ester, and the like. Preferably, the solubility enhancer is “tunable” in so far as various forms are available so as to permit the selection of a species that is appropriate to the flavonoid and/or aqueous environment at hand. Polymers having emulsification properties are useful in that regard given that shorter or longer forms of the polymer may be selected given the flavonoid and/or aqueous environment. Polyethylene glycols are one genus of polymers with many available lengths that are useful in the present compositions.

[078]. The present compositions may further comprise a matrix. For transdermal applications a matrix may not be required given that the fatty acid (carrying the flavonoid) may rapidly absorb into at least the stratum comeum layer of the skin, and be retained therein. In other applications (such as buccal and sublingual) the fluid overlaying the mucosal cells may undesirably wash away the composition thereby inhibiting contact of the flavonoid with the surface epithelium.

[079]. The matrix may mechanically retain the fatty acid and flavonoid, for example within voids or other structures. Alternatively, some type of physical force such as electrostatic attraction may be exploited to retain the fatty acid and flavonoid on or about the matrix. The matrix will typically not function so as to sequester the flavonoid for an indefinite period given the need for the active to be released into the target tissue. In some cases, the fatty acid may be retained within the matrix, with the flavonoid being transported out of the fatty acid and into the target tissue along a concentration gradient.

[080]. Where a matrix is used, it may be poorly water soluble so as to avoid rapid degradation in the saliva or other aqueous fluid with which the composition contacts. In some embodiments, the matrix is water soluble to a limited extent so as to degrade over a predetermined time period. As will be appreciated, some degradation of the matrix may be desirable for the purpose of slowly releasing the fatty acid (with flavonoid) to provide a depot-effect. The matrix may be configured to alter a pharmacokinetic parameter such as Tmax (time to peak plasma concentration), or AUC (area under the curve).

[081]. Degradation may also be desirable given that the composition should not remain in or on the body of the subject indefinitely. For example, for a buccal or a sublingual application, the composition should dissolve much like a lozenge when the flavonoid has been effectively delivered to the mucosa. Absent any degradation, the subject would be forced to remove the spent matrix from the mouth, or to swallow it.

[082]. Degradation may be achieved by means other than dissolution. As one example, the matrix may be configured to be susceptible to breakdown by resident enzymes. The oral cavity comprises amylases, and in that context a starch-based matrix may be suitably susceptible to enzyme-based degradation.

[083]. Having regard to the considerations above, an additional consideration is that the matrix may be generally inert in so far as no material leaches from it. The matrix may also be generally biocompatible so as to not trigger any undesirable biological process such as an immune or an inflammatory response. With that in mind, materials of a natural origin (i.e. obtained from a plant or an animal or the earth) may be preferable.

[084]. As discussed supra, the present composition may be washed away by saliva or some other fluid when contacted with the target tissue. To avoid that outcome the matrix may be configured to maintain contact with the target tissue. The matrix may be positively charged under physiological conditions, for example, so as to be electrostatically attracted to a negatively charged buccal or sublingual epithelium. The pH of buccal, nasal, pulmonary and pharyngeal cavities differ (varying between about 5.0 and 8.5) and accordingly, the matrix may be selected for an ability to be appropriately charged at the pH of the relevant cavity.

[085]. The matrix may consist of or comprise a bioadhesive configured to adhere to the target tissue, and in which case mucoadhesion may be employed. Mucoadhesion arises from contact between mucins of the mucosa and the mucoadhesive (typically comprising polymers). Hydration (typically by the saliva) is important in consolidating the mucoadhesive joint. Formation of the joint involves the penetration of the polymer chains of the adhesive into the mucus layer and generation of polymer-mucin bonding arising from weak bonding, van der Waals forces, hydrogen bonding and electrostatic interactions. The adhesive polymers typically possess a high number of functional groups, as hydroxyl groups or unionized carboxylate groups, capable of participating in weak bonding. The molecular weight of the polymers, and the polymer chain flexibility may be manipulated to facilitate penetration of polymer chains into the mucus, to provide a suitable level of hydration and entanglement with mucins.

[086]. Chitosan is a natural polysaccharide derived from the shell of crustaceans that has been found useful as a matrix where the target tissue is the buccal or sublingual mucosa. The fatty acid and flavonoid are retained by the chitosan, with the overall complex having a net positive charge. The amino group in chitosan has a pKb value of ~6.5, resulting significant protonation in solution, increasing according to a decrease in pH. This affords chitosan a bioadhesive property, allowing it to bind to negatively charged mucosal membranes. The free amine groups on chitosan chains can form crosslinked polymeric networks with dicarboxylic acids to afford some mechanical properties which may give the composition some three-dimensional form as well as retaining any fatty acid and flavonoid.

[087]. In some embodiments, the function of the matrix is fulfilled by pharmaceutical excipients such as binders, fillers, disintegrants and the like. Such compositions may be deposited in the buccal cavity, for example, with saliva causing the composition to decompose so as to expose and release the flavonoid into the cavity which in turn contacts the buccal mucosa.

[088]. In that regard, the present compositions may take the form of a pharmaceutical composition or a single unit dosage form comprising a flavonoid. Individual dosage forms of the invention may be suitable for mucosal (including sublingual, buccal, rectal, pulmonary, nasal, or vaginal), transdermal, or topical administration. Pharmaceutical compositions and dosage forms of the invention typically also comprise one or more pharmaceutically acceptable excipients. Sterile dosage forms are also contemplated.

[089]. Examples of dosage forms include, but are not limited to: tablets; caplets; capsules, such as soft elastic gelatin capsules; cachets; troches; lozenges; dispersions; suppositories; ointments; cataplasms (poultices); pastes; powders; dressings; creams; plasters; solutions; patches; aerosols (e.g., nasal sprays, nebulizers or inhalers); gels; liquid dosage forms suitable for oral or mucosal administration to a patient, including suspensions (e.g., aqueous or non-aqueous liquid suspensions, oil-in-water emulsions, or a water-in-oil liquid emulsions), solutions, and elixirs; and solids that can be reconstituted to provide liquid dosage forms suitable for parenteral administration to a subject.

[090]. The composition may comprise one or more compounds that control the rate by which the dosage form will decompose. Such compounds, which are referred to herein as "stabilizers," include, but are not limited to, antioxidants such as ascorbic acid, pH buffers, or salt buffers.

[091]. Pharmaceutical compositions of the invention that are suitable for oral administration (and be retained in the buccal cavity or sublingually) can be presented as discrete dosage forms, such as, but are not limited to, tablets (e.g., chewable tablets), caplets, capsules, and liquids (e.g., flavored syrups). Such dosage forms contain predetermined amounts of flavonoid. [092]. Typical oral dosage forms of the invention are prepared by combining the flavonoid in an intimate admixture with at least one excipient according to conventional pharmaceutical compounding techniques. Excipients can take a wide variety of forms depending on the form of preparation desired for administration. For example, excipients suitable for use in oral liquid or aerosol dosage forms include, but are not limited to, water, glycols, oils, alcohols, flavoring agents, preservatives, and coloring agents. Examples of excipients suitable for use in solid oral dosage forms (e.g., powders, tablets, capsules, and caplets) include, but are not limited to, starches, sugars, micro-crystalline cellulose, diluents, granulating agents, lubricants, binders, and disintegrating agents.

[093]. Because of their ease of administration, tablets and capsules represent the most advantageous buccal and sublingual dosage unit forms, in which case solid excipients are employed. If desired, tablets can be coated by standard aqueous or nonaqueous techniques. Such dosage forms can be prepared by any of the methods of pharmacy. In general, pharmaceutical compositions and dosage forms are prepared by uniformly and intimately admixing the active ingredients with liquid carriers, finely divided solid carriers, or both, and then shaping the product into the desired presentation if necessary.

[094]. For example, a tablet can be prepared by compression or molding. Compressed tablets can be prepared by compressing in a suitable machine the active ingredients in a free-flowing form such as powder or granules, optionally mixed with an excipient. Molded tablets can be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

[095]. Examples of excipients that can be used in oral dosage forms of the invention include, but are not limited to, binders, fillers, disintegrants, and lubricants. Binders suitable for use in pharmaceutical compositions and dosage forms include, but are not limited to, corn starch, potato starch, or other starches, gelatin, natural and synthetic gums such as acacia, sodium alginate, alginic acid, other alginates, powdered tragacanth, guar gum, cellulose and its derivatives (e.g., ethyl cellulose, cellulose acetate, carboxymethyl cellulose calcium, sodium carboxymethyl cellulose), polyvinyl pyrrolidone, methyl cellulose, pre -gelatinized starch, hydroxypropyl methyl cellulose, (e.g., Nos. 2208, 2906, 2910), macrocrystalline cellulose, and mixtures thereof.

[096]. Examples of fillers suitable for use in the pharmaceutical compositions and dosage forms disclosed herein include, but are not limited to, talc, calcium carbonate (e.g., granules or powder), microcrystalline cellulose, powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol, starch, pre -gelatinized starch, and mixtures thereof. The binder or filler in pharmaceutical compositions of the invention is typically present in from about 50 to about 99 weight percent of the pharmaceutical composition or dosage form.

[097]. Suitable forms of microcrystalline cellulose include, but are not limited to, the materials sold as AV1CEL-PH-101, AVTCEL-PH-103 AVICEL RC-581, AVICEL-PH- 105 (available from FMC Corporation, American Viscose Division, Avicel Sales, Marcus Hook, PA), and mixtures thereof. A specific binder is a mixture of microcrystalline cellulose and sodium carboxymethyl cellulose sold as AVICEL RC-581. Suitable anhydrous or low moisture excipients or additives include AVICEL-PH-103™ and Starch 1500 LM.

[098]. Disintegrants are used in the compositions of the invention to provide tablets that disintegrate when exposed to an aqueous environment. Tablets that contain an excess of disintegrant may disintegrate in storage, while those that contain too little may not disintegrate at a desired rate or under the desired conditions. Thus, a sufficient amount of disintegrant that is neither too much nor too little to detrimentally alter the release of the active ingredients should be used to form solid oral dosage forms of the invention. The amount of disintegrant used varies based upon the type of formulation, and is readily discernible to those of ordinary skill in the art. Typical pharmaceutical compositions comprise from about 0.5 to about 15 weight percent of disintegrant, specifically from about 1 to about 5 weight percent of disintegrant.

[099]. Disintegrants that can be used in pharmaceutical compositions and dosage forms of the invention include, but are not limited to, agar-agar, alginic acid, calcium carbonate, microcrystalline cellulose, croscarmellose sodium, crospovidone, polacrilin potassium, sodium starch glycolate, potato or tapioca starch, pre -gelatinized starch, other starches, clays, other algins, other celluloses, gums, and mixtures thereof.

[100]. Lubricants that can be used in pharmaceutical compositions and dosage forms of the invention include, but are not limited to, calcium stearate, magnesium stearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oil (e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, co oil, and soybean oil), zinc stearate, ethyl oleate, ethyl laureate, agar, and mixtures thereof. Additional lubricants include, for example, a syloid silica gel (AEROSIL 200, manufactured by W.R. Grace Co. of Baltimore, MD), a coagulated aerosol of synthetic silica (marketed by Degussa Co. of Piano, TX), CAB-O- SIL (a pyrogenic silicon dioxide product sold by Cabot Co. of Boston, MA), and mixtures thereof. If used at all, lubricants are typically used in an amount of less than about 1 weight percent of the pharmaceutical compositions or dosage forms into which they are incorporated.

[101]. The flavonoid used in the methods of the invention can be administered by controlled release means or by delivery devices that are well known to those of ordinary skill in the art. Examples include, but are not limited to, those described in U.S. Patent Nos.: 3,845,770; 3,916,899; 3,536,809; 3,598,123; and 4,008,719, 5,674,533, 5,059,595, 5,591,767, 5,120,548, 5,073,543, 5,639,476, 5,354,556, and 5,733,566. Such dosage forms can be used to provide slow or controlled-release of one or more active ingredients using, for example, hydropropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or a combination thereof to provide the desired release profile in varying proportions. Suitable controlled-release formulations known to those of ordinary skill in the art, including those described herein, can be readily selected for use with the active ingredients of the invention. The invention thus encompasses single unit dosage forms suitable for oral administration such as, but not limited to, tablets, capsules, gelcaps, and caplets that are adapted for controlled-release. [102]. All controlled-release pharmaceutical products have a common goal of improving drug therapy over that achieved by their non-controlled counterparts. Ideally, the use of an optimally designed controlled-release preparation in medical treatment is characterized by a minimum of drug substance being employed to cure or control the condition in a minimum amount of time. Advantages of controlled-release formulations include extended activity of the drug, reduced dosage frequency, and increased patient compliance. In addition, controlled-release formulations can be used to affect the time of onset of action or other characteristics, such as blood levels of the drug, and can thus affect the occurrence of side (e.g„ adverse) effects.

[103]. Most controlled-release formulations are designed to initially release an amount of drug (active ingredient) that promptly produces the desired therapeutic effect, and gradually and continually release of other amounts of drug to maintain this level of therapeutic or prophylactic effect over an extended period of time. In order to maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replace the amount of drug being metabolized and excreted from the body. Controlled-release of an active ingredient can be stimulated by various conditions including, but not limited to, pH, temperature, enzymes, water, or other physiological conditions or compounds.

[104]. Transdermal dosage forms may be used. Such forms include "reservoir type" or

"matrix type" patches, which can be applied to the skin and worn for a specific period of time to permit the penetration of a desired amount of active ingredients.

[105]. Suitable excipients (e.g., carriers and diluents) and other materials that can be used to provide transdermal and topical dosage forms encompassed by this invention are well known to those skilled in the pharmaceutical arts, and depend oa the particular tissue to which a given pharmaceutical composition or dosage form will be applied. With that fact in mind, typical excipients include, but are not limited to, water, acetone, ethanol, ethylene glycol, propylene glycol, butane- 1,3-diol, isopropyl myristate, isopropyl pal itate, mineral oil, and mixtures thereof.

[106]. Depending on the specific tissue to be treated, additional components may be used prior to, in conjunction with, or subsequent to treatment with active ingredients of the invention. For example, penetration enhancers can be used to assist in delivering the active ingredients to the tissue. Suitable penetration enhancers include, but are not limited to: acetone; various alcohols such as ethanol, oleyl, and tetrahydrofuryl; alkyl sulfoxides such as dimethyl sulfoxide; dimethyl acetamide; dimethyl formamide; polyethylene glycol; pyrrolidones such as polyvinylpyrrolidone; ollidon grades (Povidone, Polyvidone); urea; and various water-soluble or insoluble sugar esters such as Tween 80 (polysoibate 80) and Span 60 (sorbitan monostearate).

[107]. Topical dosage forms may be used. Such forms include, but are not limited to, creams, lotions, ointments, gels, solutions, emulsions, suspensions, or other forms known to one of skill in the art.

[108]. Mucosal dosage forms may be used which include, but are not limited to, ophthalmic solutions, sprays and aerosols. Dosage forms suitable for treating mucosal tissues within the oral cavity can be formulated as mouthwashes or as oral gels.

[109]. In some embodiments the flavonoid and/or the formulation and/or the dosage form is/are selected so as to limit or obviate first-pass metabolism of the flavonoid in the liver and/or facilitate passage (active or otherwise) across the blood-brain barrier.

[110]. In some embodiments of the composition there is no fatty acid, with the matrix nevertheless functioning as a means for retaining the flavonoid (with or without a carrier of some type) about the target tissue (whether mucosa, skin or otherwise). In such embodiments, the matrix nevertheless functions so as to maintain the flavonoid proximal to the target tissue so as to improve delivery of the active thereto. [111]. In terms of administration, the composition may be contacted to the target tissue in any appropriate manner. Where the composition is solid or semi-solid, the composition may be manually deposited into a relevant cavity (such as the mouth or the rectum) having the target tissue. Where the composition is flowable, the composition may be provided in a tube or a syringe-like contrivance and dispensed onto the target tissue. Where the composition is applied to the skin, the composition may be manually spread over the skin surface to facilitate absorption.

[112]. The present compositions may be administered to a subject for any reason, including in a method for treating or preventing a disease or a disorder, or for generally improving health.

[113]. For example, a flavonoid (and particularly pinocembrin) can be used to treat or prevent a fibrotic condition, such as pulmonary fibrosis (including idiopathic pulmonary fibrosis, infection-induced pulmonary fibrosis, radiation-induced pulmonary fibrosis, progressive massive fibrosis, cystic fibrosis), pancreatic fibrosis (including cystic fibrosis), retropertinoneal fibrosis, hepatic fibrosis (including bridging fibrosis and cirrhosis), arterial fibrosis (including arterial stiffness), intestinal fibrosis (including Crohn’s disease), joint fibrosis (including athrofibrosis of the knee, shoulder and other joints, adhesive capsulitis), manual/digital fibrosis (including Dupuytren’s contracture), dermal fibrosis (including keloid, nephrogenic systemic fibrosis, scleroderma), penis (including Peyronie’s disease), lymph node fibrosis (including mediastinal fibrosis) and myocardial fibrosis (including interstitial fibrosis and replacement fibrosis).

[114]. Pinocembrin is also effective in treating or preventing inflammatory conditions such as pulmonary inflammation (including COPD, asthma, rhinitis, bronchitis), dermal inflammation (including acne and scleroderma), gastrointestinal inflammation (including celiac disase, Crohn’s disease, colitis, diverticulitis), autoimmune diseases (such as SLE), urinary system diseases (including glomerulonephritis, cystitis, protastitis), sarcoidosis, transplant rejection, vasculitis, atherosclerosis, pelvic inflammatory disease, rheumatic fever, and otitis. [115]. Flavonoids have known efficacy in many other conditions including cancer, cardiovascular conditions (such as ischemia), neurological conditions (such as Alzheimer’s disease, Parkinson’s disease, and motor neurone disease) and infectious diseases.

[116]. Flavonoids have also non-therapeutic applications, for example to improve cognition, memory, mood, general wellbeing, and the like.

[117]. The present invention will now be more fully described by reference to the following non-limiting examples.

EXAMPLE 1: Materials and methods used in experimental work

[118]. Buccal tissue from pigs (Australian Food Group & Diamond Valley Pork,

Melbourne, Australia) was collected by careful removal of the epithelium from the inside of the cheek post-mortem. Tissue was mounted into modified Ussing chambers (with chamber volume of 3 mL) and pre-weighed gel administered to the tissue before the addition of Krebs bicarbonate ringer (KBR, pH 7.4) buffer (37°C) into both the donor and receptor chambers. Both donor and receptor chambers were maintained at 37°C and supplied with carbogen. Samples were collected from the receptor chamber (50 pL) at 15, 30, 45, 60, 120, 180 and 240 min, with 50 pL of fresh KBR added then added to the receptor chamber to maintain volume consistency. Pinocembrin concentration in the receptor chamber samples was determined by HPLC-UV.

[119]. Gel formulations were prepared using a chitosan base (3% w/v) with or without 5%

(w/w) oleic acid and 5% (w/w) PEG400. To prepare 3% (w/v) chitosan gel, 3 g of low MW chitosan was dissolved into 100 mL of 50 mM 2-(/V-morpholino)ethanesulfonic acid (MES) buffer, pH adjusted to 5.8, stirred for 2 h at room temperature on a magnetic stirrer and then heated in an oven overnight at 37°C. Permeation enhancers (oleic acid, PEG400 or both) were added at 5% (w/w) to the chitosan gel and mixed vigorously with a spatula to create a homogenous gel. Pinocembrin (S-isomer) obtained from a Eucalyptus species) was then added to gel formulations to give a final mass of 1 mg of pinocembrin in 33.3 mg of gel for use in vitro and 1 mg of pinocembrin in 8 mg of gel for use in vivo.

EXAMPLE 2: In vivo absorption studies

[120]. All animal experiments were approved by the Monash Institute of Pharmaceutical

Sciences Animal Ethics Committee and performed in accordance with the National Health and Medical Research Council Guidelines for the care and use of animals for scientific purposes. In vivo studies were conducted using female C57BL/6 mice between 6-10 weeks of age, sourced from the Monash Animal Research Platform, Monash University. Mice were anaesthetised with inhalational isoflurane and an approximate 8 mg mass of pinocembrin gel (with or without oleic acid and PEG400) applied to the buccal mucosa, to equate to an approximate dose of 40 mg/kg of pinocembrin. Mice remained under anaesthesia with isoflurane for the entire sampling period. Blood samples were taken at 15, 30, 60, 120, 240 and 480 min after buccal application and immediately centrifuged at 14,000 xg for 5 min, followed by plasma collection. Plasma samples were stored at -20°C and pinocembrin concentration quantified by HPLC-UV.

EXAMPLE 3: Preparation of calibration standards and samples and HPLC assay validation procedure

[121]. Stock solutions of pinocembrin were prepared at a concentration of 1 mg/mL in acetonitrile and stored in aliquots at -20°C. Calibration standards were prepared on the day of each analysis. Serial dilutions of spiking solutions were prepared in acetonitrile across a 500-50000 ng/mL range. 10 pL of spiking solution was added to 90 L of KBR buffer or blank mouse plasma to create standard solutions ranging from 50 - 5000 ng/mL. Receptor chamber and mouse plasma samples from relevant studies were prepared in the same manner except that 10 pL of acetonitrile was added in place of the spiking solution. Standard solutions and samples were then vortexed and proteins precipitated by the addition of 100 pL of acetonitrile and then vortexed a second time before centrifugation at 14,000 xg for 5 min. The supernatant was then removed and an aliquot of 50 pL injected onto the HPLC column. [122]. In order to assess for intra-assay accuracy and precision, six independently prepared quality control (QC) samples were prepared in KBR or blank mouse plasma at concentrations of 50, 500 and 5000 ng/mL and quantified against a calibration curve ranging from 50-5000 ng/mL.

[123]. Precision was expressed as the relative standard deviation and accuracy was determined as the ratio of the calculated concentration of the QC samples relative to the nominal concentration, expressed as a percentage.

EXAMPLE 4: HPLC analysis of pinocembrin

[124]. Chromatography was performed using a Shimadzu HPLC system consisting of two

LC-20AD pumps, a SIL-20 AHT autoinjector, a DGU-20A3 degasser, a DPD-20A UV-Vis detector (Shimadzu, Kyoto, Japan) with a Discovery™ Cl 8 HPLC Column (5 pm, 15 cm x 4.6 mm, Sigma-Aldrich, St Louis, MO) and a Discovery™ Guard cartridge holder (5 pm, 2 cm x 4 mm, Sigma-Aldrich, St Louis, MO). A gradient method was used to elute pinocembrin with a gradient program of 0.01 - 0.50 min: 20% B; 0.50 - 4.00 min: 20-60% B; 4.00 - 4.50 min: 60-95% B; 4.50 - 4.55 min: 95% B; 4.55 - 6.00 min: 95-20% B; and 6.00 - 7.50 min: 20% B, at a flow rate of 1.5 mL/min with mobile phase A being 0.1% (v/v) formic acid in water and mobile phase B being 0.1% (v/v) formic acid in acetonitrile. The column oven temperature was 40°C and pinocembrin eluted at 3.8 min with a detection at 288 nm.

EXAMPLE 5: Dose normalisation and statistical analysis

[125]. Given that it was not possible to dose every mouse with exactly 8 mg of gel, all plasma concentrations were normalised to a nominal dose of 40 mg/kg. The average amount of pinocembrin administered to mice was 38.8 mg/kg with chitosan-only gels and 39.1 mg/kg with chitosan/oleic acid/PEG400 gels.

[126]. Statistical analysis was performed using GraphPad Prism 9. Differences in the percentage of pinocembrin dose permeating the buccal mucosa from various gels to that from the chitosan-only gel were conducted using a two-way analysis of variance with a post-hoc Dunnett’s test. The area under the pinocembrin plasma concentration versus time profile from time zero to 8 h following application of buccal gels and their associated variances were calculated using Bailer’s method, and statistical differences determined using a z-test.

EXAMPLE 6: Development and validation of a HPLC assay for pinocembrin quantification in KBR

[127]. To determine pinocembrin concentrations in KBR for assessment of in vitro pinocembrin permeability, an HPLC-UV assay was developed and validated. As shown in Figure 1, the assay response was found to be linear (R2 = 0.9956) and met the prescribed acceptance criteria for accuracy and precision (Table 1).

Table 1. Precision and accuracy of the HPLC-UV assay for the quantification of pinocembrin in KBR (n=5).

[128]. The assay was therefore considered suitable for the quantification of pinocembrin in KBR collected from the receptor chamber of Ussing chambers following application of pinocembrin gel formulations. Representative chromatograms are shown in Figure 2.

EXAMPLE 7: Assessment of in vitro pinocembrin permeability across the buccal mucosa

[129]. To determine which chitosan formulation resulted in the highest permeability of pinocembrin across the buccal mucosa, gel formulations (5-15 mg) containing pinocembrin were applied to tissue membrane. As shown in Figure 3, pinocembrin was able to permeate the buccal mucosa when formulated in a chitosan-only gel (mean permeation of 0.45% of applied dose at 240 min), and the inclusion of either oleic acid or PEG400 did not significantly increase the permeability of pinocembrin, with 0.38% and 0.51% of pinocembrin dose permeating by 240 min, respectively. However, incorporation of both oleic acid and PEG400 improved permeability of pinocembrin compared to the chitosan base gel, with a significant increase in the % of dose permeating at 180 min (p<0.01) and 240 min (p<0.0001), at which time point, 1.01% of the pinocembrin dose had permeated into the receptor chamber.

EXAMPLE 8: Development and validation of a HPLC-UV assay for pinocembrin quantification in mouse plasma

[130]. To assess buccal absorption of pinocembrin in vivo, a HPLC-UV assay was developed and validated. As shown in Figure 4, the assay response was found to be linear (R2 = 0.9986) and met the prescribed acceptance criteria for accuracy and precision (Table 2).

Table 2. Precision and accuracy of the HPLC-UV assay for the quantification of pinocembrin in mouse plasma (n=4-6).

The assay was therefore considered suitable for the quantification of pinocembrin in mouse plasma. Representative chromatograms are shown in Figure 5.

EXAMPLE 9: Assessment of pinocembrin levels in mouse plasma following buccal application

[131]. The plasma concentrations of pinocembrin following buccal application of a chitosan gel with or without oleic acid and PEG400 are shown in Figure 6. When applied in a chitosan only gel, pinocembrin was not detected in the plasma at 15 and 30 min, however, detectable levels were observed from 1 h post-dose onwards. The maximum plasma concentrations of pinocembrin were observed 4 h post-application with concentrations of 6114.2 ± 1762.1 ng/mL (mean ± SD). When pinocembrin was administered in a chitosan gel containing oleic acid and PEG400, plasma concentrations were detected as early as 30 min post-dose, which also peaked at 4 h post-dose with concentrations of 10801.5 ± 2391.0 ng/mL (mean ± SD). Importantly, statistical analysis demonstrated that the area under the pinocembrin plasma concentration vs time curve was significantly higher when pinocembrin was administered in a chitosan gel containing oleic acid and PEG400 relative to the chitosan-only gel. The area under the pinocembrin plasma concentration vs time curve values increased 1.95-fold in the presence of oleic acid and PEG400 with values of 26856.6 ± 2550.1 ng.h/mL and 52403.8 ± 4118.4 ng.h/mL without and with oleic and PEG400, respectively.

[132]. The aim of this experimental work detailed supra was to investigate whether the permeation enhancers oleic acid and polyethylene glycol - 400 (PEG400), or a combination thereof, improve the in vitro and in vivo buccal permeability of pinocembrin formulated in a chitosan-based gel. Pinocembrin was applied to excised porcine buccal mucosa in a chitosan-based gel with or without oleic acid (5% w/w) and/or PEG400 (5% w/w) and the amount of pinocembrin permeating the mucosa quantified by a validated HPLC-UV assay. At 240 min post-application, the percentage of pinocembrin applied to the buccal mucosa that had permeated the mucosa was significantly (p<0.05) increased when the chitosan gel contained oleic acid and PEG400 in combination, relative to chitosan alone. Pinocembrin (40 mg/kg) was then applied to the buccal mucosa of anaesthetised C57BL/6 mice in a chitosan-based gel with or without oleic acid (5% w/w) and PEG400 (5% w/w), and plasma concentrations determined over 8 h post-application using a validated HPLC assay. Plasma concentrations of pinocembrin were detected from 30 min post-dose and peaked at 4 h (mean plasma concentrations of 6114.2 ± 1762.1 ng/mL (mean ± SD)). Incorporation of oleic acid and PEG400 to the chitosan buccal gel led to a 1.95- fold increase in area under the pinocembrin plasma concentration curve, with peak plasma concentrations also detected at 4 hpost-dose (10801.5 ± 2391.0 ng/mL, mean ± SD). These studies demonstrate that the buccal mucosa represents a suitable application site for the systemic delivery of pinocembrin, with oleic acid and PEG400 significantly improving buccal permeability of pinocembrin.

[133]. Those skilled in the art will appreciate that the invention described herein is susceptible to further variations and modifications other than those specifically described.

It is understood that the invention comprises all such variations and modifications which fall within the spirit and scope of the present invention.

[134]. Accordingly, the spirit and scope of the present invention is not to be limited by the foregoing examples, but is to be understood in the broadest sense allowable by law.

Claims

CLAIMS:

1. A composition for delivering a flavonoid to a subject, the composition comprising: a flavonoid in combination with one or more of: a fatty acid, a solubility enhancer configured to increase the aqueous solubility of the fatty acid, and a matrix.

2. The composition of claim 1, wherein the fatty acid has a chain length of: between about 13 to 23 carbon atoms, or about 14 to 22 carbon atoms, or about 15 to 21 carbon atoms, or about 16 to 20 carbon atoms, or about 17 to 19 carbon atoms, or 18 carbon atoms.

3. The composition of claim 1 or claim 2, wherein the fatty acid is a dietary fatty acid.

4. The composition of any one of claims 1 to 3, wherein the fatty acid is a monounsaturated fatty acid.

5. The composition of any one of claims 1 to 4, wherein the fatty acid is a cis fatty acid

6. The composition of claim 4 or claim 5, wherein the double bond of the monounsaturated fatty acid is at or about midway along the carbon chain.

7. The composition of any one of claims 1 to 6, wherein the fatty acid is an omega-9 fatty acid.

8. The composition of any one of claims 1 to 7, wherein the omega-9 fatty acid is oleic acid.

9. The composition of any one of claims 1 to 8, wherein the solubility enhancer is an emulsifier capable of emulsifying the fatty acid.

10. The composition of claim 9, wherein the solubility enhancer is a polymer.

11. The composition claim 9 or claim 10, wherein the polymer is a polymer of ethylene oxide.

12. The composition of any one of claims 1 to 11, wherein the solubility enhancer is polyethylene glycol (PEG).

13. The composition of claim 12, wherein the PEG has a molecular weight or an average molecular weight of: between about 10 and 1000 Da, or between about 50 and 900 Da, or between about 100 and 800 Da, or between about 150 and 700 Da, between about 200 and 600 Da, or between about 300 and 500 Da, or about 400 Da.

14. The composition of any one of claims 1 to 13, wherein the fatty acid and the solubility enhancer are provided as separate molecules, or combined in a single molecule.

15. The composition of any one of claims 1 to 14, wherein the single molecule is formed by reaction of a fatty acid with a solubility enhancer at a ratio of about 1: 1, or another suitable ratio.

16. The composition of claim 14 or claim 15, wherein the single molecule is a PEG oleate.

17. The composition of claim 16, wherein the PEG oleate is PEG-7 oleate, PEG-8 oleate, PEG-9 oleate, PEG- 10 oleate, or PEG-11 oleate,

18. The composition of any one of claims 1 to 17, wherein the matrix is a polymer or a biodegradable polymer.

19. The composition of claim 18, wherein the polymer or biodegradable polymer is an anionic or cationic polymer.

20. The composition of any one of claims 1 to 19, wherein the matrix is stable under a physiological condition for less than about 20 minutes, including or absent any enzyme capable of breaking down the matrix.

21. The composition of any one of claims 1 to 20, wherein the matrix is a gel or a hydrogel.

22. The composition of any one of claims 1 to 21, wherein the matrix is positively charged at a physiological pH, including a pH between about 5.0 and 8.5.

23. The composition of any one of claims 1 to 22, wherein the matrix is charged so as to bind a mucosal surface of a living animal or a cadaver animal, including the buccal, sublingual, pharyngeal, nasopharyngeal, pulmonary, or nasal mucosa of an animal.

24. The composition of any one of claims 1 to 23, wherein the matrix is configured to retain the flavonoid and the fatty acid therein and/or thereabout, and controllably release the flavonoid to a mucosa of an animal that is proximal to or in contact with the composition.

25. The composition of any one of claims 1 to 24, wherein the matrix comprises an organic polymer.

26. The composition of claim 25, wherein the organic polymer is a polysaccharide.

27. The composition of claim 26, wherein the polysaccharide is a linear polysaccharide

28. The composition of any one or claims 1 to 27, wherein the matrix is selected from a chitosan, an aminated cellulose, a starch, an alginate, a collagen, a gelatin, a chitin, a polylysine, a pollyallylamine, a polyethylenimine, a polyhydroxyalkanoate, a cellulose nanofiber, and a cellulose nanocrystal.

29. The composition of any one of claims 1 to 28, wherein the flavonoid is a flavanone.

30. The composition of claim 29, wherein the flavanone has a chemical structure according to formula 1

wherein R2’, R3, R3’, R4’, R5, R6, R7 are each independently:

H,

OH,

O-,

O-CH3, a glucoside (including a rhamnosidoglucoside), or any other organic functional group.

31. The composition of claim 30, wherein R2’, R3, R3’, R4’, R5, R6, R7 are as follows:

R3 R5 R6 R7 R2' R3' R4' Name

H H H H H H H Flavanone

H OCH3 H H H H H 5 -Methoxyflavanone

H H OH H H H H 6-Hydroxyflavanone

H H OCH₃ H H H H 6-Methoxyflavanone

H H H OH H H H 7-Hydroxyflavanone

H H H H OH H H 2'-Hydroxyflavanone

H H H H H H OH 4'-Hydroxyflavanone

H H H H H H OCH3 4'-Methoxyflavanone

H OH H OH H H H Pinocembrin

H OH H OCH₃ H H H Pinocembrin -7-methylether

H OH H OH H H OH Naringenin

H OH H OH H H OCH3 Isosakuranetin

H OH H OCH3 H H OH Sakuranetin

H OH H Gia H H OH Naringenin-7-glucoside

H OH H OH H OH OCH₃ Hesperetin

OH OH H OH H OH OH Taxifolin a; Gl=Glucoside. b; Rh-Gl=Rhamnosidoglucoside.

32. The composition of any one of claims 1 to 31, wherein the flavanone is dihydroxyflavanone and/or a (2S)-flavan-4-one, or a functional derivative thereof.

33. The composition of any one of claims 1 to 32, wherein the flavanone is (2S)-5,7- dihydroxy-2-phenyl-2,3-dihydrochromen-4-one, or a functional derivative thereof.

34. The composition of any one of claims 1 to 33, wherein the flavonoid is of the type naturally synthesized in a plant cell, although is not necessarily obtained from a plant cell.

35. The composition of any one of claims 1 to 34 formulated as a gel, a lozenge, a troche, a dragee, a viscous composition, a bioadhesive, a medication stick, a tablet, a capsule, a spray, or a slurry

36. The composition of any one of claims 1 to 35, comprising a taste-masking agent, and/or a taste modifying agent, and/or a sweetener.

37. A method of administering a flavonoid to a subject in need thereof, or desirous thereof, the method comprising contacting the composition of any one of claims 1 to 36 to a target issue of the body of the subject.

38. The method of claim 37, wherein the target tissue is an external surface or an internal surface.

39. The method of claim 37 or claim 38, wherein the external surface is a skin surface, and the internal surface is a mucosal surface.

40. The method of claim 39, wherein the mucosal surface is a buccal, sublingual, pharyngeal, nasopharyngeal, pulmonary or nasal mucosal surface.

41. The method of any one of claims 37 to 40, wherein the step of contacting is for a period of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 minutes, or until the matrix of the composition has mostly or fully degraded.

42. The method of any one of claims 37 to 41, wherein the step of contacting comprises actuation of a dispenser about the surface, the dispenser comprising an outlet configured to direct the composition toward or onto the surface.

43. The method of claim 42, comprising causing the dispenser to release the composition via the outlet.

44. The method of claim 43, wherein release of the composition is caused by decreasing the internal volume of a container holding the composition, compressing a flexible container holding the composition, or moving a plunger through a container holding the composition.