WO2005053718A1

WO2005053718A1 - Methods and compositions for treating inflammatory and dermatological conditions

Info

Publication number: WO2005053718A1
Application number: PCT/AU2004/001708
Authority: WO
Inventors: Nirdosh Kumar Puri; Peter Hugh Hull
Original assignee: Naturaleaf, Inc.
Priority date: 2003-12-05
Filing date: 2004-12-06
Publication date: 2005-06-16

Abstract

The present invention relates to a method for the treatment or prophylaxis of a condition selected from a non-arthritic inflammatory condition (e.g., dermatitis, psoriasis, allergic rhinitis, acne, angina, asthma, emphysema, pelvic inflammatory disease, pharyngitis, raw or sore throat, redness or rubor), an arthritic condition, an aesthetic skin condition (e.g., skin aging, wrinkles, skin texture, skin cracking, skin blemishes, skin colour, acne, photo aging or symptom of sun exposure) and a muscle injury in a subject, said method comprising administering to said subject oil from a Canarium nut or other part of a Canarium plant or horticultural relative thereof or one or more pharmacologically active fractions, extracts or components thereof or a formulation comprising the oil or an active fraction, extract or component thereof, wherein the inflammatory condition is treated by the administration of an active fraction, extract or component of the oil.

Description

METHODS AND COMPOSITIONS FOR TREATING INFLAMMATORY AND DERMATOLOGICAL CONDITIONS

BACKGROUND OF THE INVENTION

FIELD OF THE INVENTION

The present invention relates generally to a method for the treatment and/or prophylaxis of inflammatory conditions in a subject. The present invention further contemplates cosmetic treatment or prophylaxis protocols. The present invention also provides compositions including pharmaceutical compositions and cosmetic compositions for use in the treatment and/or prophylaxis of inflammation conditions and skin conditions.

DESCRIPTION OF THE PRIOR ART

Bibliographic details of the publications referred to in this specification are also collected at the end of the description.

Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.

Nuts from the Canarium species from the Burseraceae family are widely distributed throughout tropical Asia. Of particular interest, are the nuts from several Canarium species harvested in the Solomon Islands (known as Ngali), which are used as foods and a source of cooking oils (Duke, Handbook of Nuts, Boca Raton FL, CRC Press, 1989, Stevens et al. (Eds), South Pacific Indigenous Nuts ACIAR Proceedings 69: 176, 1995).

Greater than 95 per cent of the Ngali oil from Canarium indicum, Canarium harveyi, and Canarium salomonense, constitute triglycerides. These include significant amounts of unsaturated fatty acids, including oleic acid (26-42%) and linolenic acid (10-25%), but only very small quantities of linolenic acid (not more than 0.4 per cent). The proportion of saturated fatty acids (palmitic and stearic) is of the order 45-50 percent. Interestingly, vegetable oils of approximately similar composition (e.g. olive, sunflower, peanut) have little or no medicinal value.

The treatment of many medical ailments is frequently initiated by the administration of analgesics (eg. aspirin, paracetamol) to relieve pain, followed by more powerful non- steroid anti-inflammatory drugs (NSAID), usually given orally. These treatments, particularly if prolonged, carry significant risks. For example, aspirin and NSAIDs can induce gastric bleeding, paracetamol can cause liver damage and both NSAIDs and paracetamol can provoke kidney injury after prolonged usage (Borda and Koff, NSAIDs: A profile of adverse effects, Hanley & Belfus Inc, Philadelphia, 1992).

Alternative non-oral medication is based on applying analgesics in a "topical" formulation. Examples are creams or gels for rubbing into the skin that contain methyl salicylate ('oil of wintergreen') or capsaicin formulated with a high proportion of excipient(s) to reduce local irritancy. These are essentially palliative, rather than curative treatments, as they rarely remove the basic source of the pain.

It has now been surprisingly determined that oil from Canarium plants and its active fractions, extracts and components thereof are efficacious in the treatment and prophylaxis of inflaminatory conditions including dermatological conditions and are also useful for the treatment of cosmetic conditions as well as damage due to UV exposure. SUMMARY OF THE INVENTION

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

The present invention provides a method for the treatment and prophylaxis of inflammatory conditions including dermatological conditions in a subject. The present invention further contemplates methods of cosmetic treatment or prophylaxis such as of aesthetic skin conditions and/or to prevent or reduce wrinkles and/or the effects of UV exposure. In addition, the conditions treatable in accordance with the present invention include muscle injuries such as sporting injuries. The method contemplated by the present invention comprises administering to the subject the oil from a Canarium nut or other part of a Canarium plant or a horticultural relative thereof (referred to herein as "Ngali oil"), and/or a pharmacologically active fractions, extracts or components thereof. The oil, fractions, extracts and components of the present invention or formulations containing same including free-flowing oil powder forms may be administered to the subject by any convenient means, although the present invention identifies the oral, transdermal and topical routes of administration as being particularly useful. The present invention also contemplates pharmaceutical and cosmetic compositions comprising Ngali oil and/or pharmacologically active fractions, extracts or components of the oil, when used for the treatment of inflammatory and/or aesthetic skin conditions.

Accordingly, one aspect of the present invention contemplates a method for the treatment or prophylaxis of a condition selected from a non-arthritic inflammatory condition, an arthritic condition, an aesthetic skin condition and a muscle injury in a subject, said method comprising administering to said subject oil from a Canarium nut or other part of a Canarium plant or a horticultural relative thereof or one or more pharmacologically active fractions, extracts or components thereof or a formulation comprising the oil or an active fraction, extract or component thereof, wherein the arthritic condition is treated by the administration of an active fraction, extract or component of the oil. The methods of the present invention contemplate the use of Ngali oil and pharmacologically active fractions, extracts or components thereof or a formulation comprising the oil or an active fraction, extract or component thereof as both prophylactic and therapeutic treatments of inflammatory conditions or aesthetic skin conditions. A "formulation" includes free-flowing oil powder forms.

For the purposes of the present invention, "Ngali oil" is defined as the oil obtained from any one of several varieties of Canarium plants or trees or horticultural relatives thereof. Those include tree species from Burma, Indonesia and Timor, Malaysia, and Western and Northern Australia including the Torres Strait Islands, Papua New Guinea, the Philippines) and many of the Pacific Islands (Fiji, New Caledonia, Samoa, The Solomons, Tonga, Vanuatu, etc). Well over 50 species are known and considered of economic importance (timber, gum, bark extracts and nuts). They include Canarium album, Canarium australasicum, Canarium copaliferum, Canarium decumannum, Canarium harveyi, Canarium indicum, Canarium kinaiense, Canarium lamii, Canarium luzonicum, Canarium mehenbethani, Canarium muelleri, Canarium nigrum, Canarium odontophyllu, Canarium ovalum, Canarium pimelai, Canarium polyphyllum, Canarium salomonense, Canarium schweinfurtheri, Canarium vitientise, Canarium vulgare. Particularly preferred trees are those grown in the Solomon Islands, Vanuatu, Papua New Guinea and/or the Philippines. Three particularly useful varieties of Ngali nut trees include Canarium indicium, Canarium solomonesis and Canarium harveyi. The present invention also contemplates Canarium plants not assigned to any of the above species, hybrids of Canarium tree varieties, and horticultural relatives of the Canarium tree.

In a particularly preferred embodiment of the present invention, the "Ngali oil" is "Ngali nut oil" or oil from a Canarium nut or other part of the plant or from a horticultural relative thereof. However, the present invention also contemplates Ngali oil derived from other tissue types including: bark, woody tissues, leaves, fruit, roots, nut skin, nut kernel or nut shell. The term "tree" and "plant" are used interchangeably. Active fractions, extracts and components of the Ngali oil of the present invention may be generated by any number of means readily ascertained by those of skill in the art. Exemplary, and particularly useful, methods include column chromatographic techniques, thin layer chromatography, liquid chromatography, high performance liquid chromatography, gas chromatography, ion exchange chromatography, size-exclusion chromatography, affinity chromatography and the like. Another suitable method for producing extracts, or fractions of Ngali oil is supercritical fluid extraction. Yet other useful methods include NMR (¹³C, 1H, ³¹P) and various solvent or aqueous extraction methods. Alternatively, a combination of any of the above methods may be employed.

The Ngali oil or a pharmacologically active fraction, extracts or components thereof or a formulation comprising the oil or an active fraction, extract or component thereof may be administered by any convenient means. Oral, transdermal and topical routes of administration have been identified as being particularly useful. In the case of transdermal administration, the active agent is typically administered with a percutaneous enhancer which increases the absorption of the agent across the skin.

The present invention also provides pharmaceutical and cosmetic compositions comprising Ngali oil or one or more pharmacologically active fractions, extracts or components thereof or a formulation comprising the oil or an active fraction, extract or component thereof together with one or more pharmaceutically acceptable carriers, diluents and/or excipients. Such compositions are particularly useful when used for therapeutic or prophylactic purposes such as in the treatment of an inflammatory condition or in the treatment of an aesthetic condition. Reference to an aesthetic skin condition includes wrinkles, skin abnormalities or loss of firmness such as resulting from aging or weight loss and the effects of UV exposure to skin. The present invention further contemplates sun lotions and creams for use before, during or after exposure to UV rays. The compositions of the present invention further represent anti-photoaging and sun repair formulations.

In one particular embodiment, the composition is useful in the treatment of a dermatological or aesthetic condition. The composition may be in a bland oil or sorbolene cream diluent and may comprise other agents such as emulsifiers, preservatives and antioxidants (such as Vitamin E).

A list of abbreviations used herein is provided in Table 1.

TABLE 1. Abbreviations

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a graphical representation depicting a schematic of the protocol used to isolate and identify pharmacologically active fractions or extracts from a raw or native Oil from a Ngali plant or horticultural relative thereof.

Figure 2 is a graphical representation showing the relative efficacies of some Non- Steroidal Anti-Inflammatory Drugs (NSAIDs) compared to Ngali nut oil extracts applied both transdermally and orally.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides a method for treating inflammatory and/or including aesthetic conditions such as aesthetic skin conditions in a subject. The method contemplated by the present invention comprises administering to the subject the oil from a Canarium plant or a horticultural relative thereof, referred to herein as "Ngali oil", and/or a pharmacologically active fractions, extracts or components of the oil or a formulation comprising the oil or an active fraction, extract or component thereof. The oil, particularly oil from a nut or other part of a Canarium plant, and pharmacologically active fractions, extracts or components thereof or a formulation comprising the oil or an active fraction, extract or component thereof may be administered to the subject by any convenient means with oral, transdermal and topical routes of administration being particularly useful. The present invention also contemplates pharmaceutical or cosmetic compositions comprising Ngali oil and pharmacologically active fractions, extracts or components thereof or a formulation comprising the oil or an active fraction, extract or component thereof when used for the treatment of inflammatory and/or aesthetic skin conditions. Such conditions include muscle inflammatory conditions such as resulting from a sporting injury. In addition, the Ngali oil and pharmacologically active fractions, extracts or components thereof are proposed to be useful in treating wrinkles, loss of firmness of the skin due to aging or weight loss, and the effects of sun (i.e. UV) exposure. With respect to the latter, one effect of UV exposure is DNA damage with the potential for cancerous skin conditions to develop the formulations of the present invention are proposed to facilitate DNA repair, alone or in combination with other agents.

Before describing the present invention in detail, it is to be understood that unless otherwise indicated, the subject invention is not limited to specific combinations of oils, extracts, fractions, components, methodologies and the like, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

As used in the subject specification, the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "a Ngali oil" includes a single Ngali oil preparation or a combination of oils from different sources from the same or different plants; reference to a pharmacologically active fraction, extract or component includes a single fraction, extract or component or two or more fractions, extracts or components from the same or different oils. For example, multiple (i.e. two or more) fractions, extracts or components may be prepared from the one oil or from oils of different species of Canarium and admixed together.

The term "pharmacological" is not to be construed, especially with respect to cosmetic compositions, and requiring that the oil or fraction, extract or component or a formulation comprising the oil or an active fraction, extract or component thereof have medical therapeutic or prophylactic potential. The oil or its fractions, extracts or components may be used, therefore, for cosmetic purposes including being admixed with cosmetic agents such as neurological toxins including botulinum toxin. The formulation may also be in the form of a sun lotion or sun cream used to rejuvenate skin after, during or prior to exposure to the sun or other harsh environmental conditions.

Accordingly, one aspect of the present invention contemplates a method for the treatment or prophylaxis of a condition selected from a non-arthritic inflammatory condition, an arthritic condition, an aesthetic skin condition and a muscle injury in a subject, said method comprising administering to said subject oil from a Canarium nut or other part of a Canarium plant or a horticultural relative thereof or one or more pharmacologically active fractions, extracts or components thereof or a formulation comprising the oil or an active fraction, extract or component thereof, wherein the arthritic condition is treated by the administration of an active fraction, extract or component of the oil.

The term "administering" includes administering to, into or onto a subject.

"Subject" as used herein refers to an animal, preferably a mammal and more preferably a primate including a lower primate and even more preferably, a human who can benefit from the formulations and methods of the present invention. A subject regardless of whether a human or non-human animal may be referred to as an individual, subject, animal, patient, host or recipient. The compounds and methods of the present invention have applications in human medicine, the cosmetic or aesthetic industry, veterinary medicine as well as in general, domestic and wild animal husbandry and the agricultural industry. For convenience, an "animal" specifically includes livestock species such as cattle, horses, sheep, pigs, goats and donkeys. With respect to horses, these include horses used in the racing industry as well as those used recreationally or in the livestock industry. Any animal or human subject requiring treatment or prophylaxis of inflammatory condition or requiring aesthetic treatment or prophylaxis may benefit from the method and formulation of the present invention.

A human is the most preferred target. However, the method of the present invention is suitable for the treatment of inflammatory conditions in other non-human animals, including laboratory test animals.

Examples of laboratory test animals include mice, rats, rabbits, guinea pigs and hamsters. Rabbits and rodent animals, such as rats and mice, provide a convenient test system or animal model as do primates and lower primates. Non-mammalian animals include animals such as avian species, zebrafish and amphibians (including cane toads). The latter animals are particularly useful in generating animal models to test the oil fractions, extracts and components.

As used herein "inflammatory conditions" or "inflammatory diseases and disorders" encompass those disease and disorders which result in a response of redness, swelling, pain, and a feeling of heat in certain areas that is meant to protect tissues affected by injury or disease. Inflammatory diseases which can be treated using the methods of the present invention, include, without being limited to, asthma, dermatitis, allergic rhinitis, psoriasis, acne, arthritis, empysema, inflammation, pelvic inflammatory disease, pharyngitis, raw or sore throat, redness and rubor. The use of novel oil, from a species of Canarium or a fraction, extract or component from any Canarium oil, whether the oil was previously known or not, is contemplated for the treatment of any inflammation condition in accordance with the present invention. The treatment of arthritis is generally by fraction, extract or component of the oil. The methods of the present invention, therefore, contemplate the use of Ngali oil and pharmacologically active fractions, extracts or components thereof or a formulation comprising the oil or an active fraction, extract or component thereof in both the prophylactic and therapeutic treatment of inflammatory and/or aesthetic skin conditions.

For the purposes of the present invention, the term "prophylactic treatment" of inflammation should be understood to refer to administration of the Ngali oil and/or pharmacologically active fractions, extracts or components thereof or a formulation comprising the oil or an active fraction, extract or component thereof for the purpose of preventing, delaying or inhibiting the onset of an inflammatory condition or an aesthetic skin condition. This should be understood as encompassing both treatment before the onset of any symptoms and also treatment after the onset of symptoms wherein the prophylactic treatment is designed to slow or delay progression of the inflammatory or aesthetic skin condition. An "aesthetic" skin condition includes wrinkling, loss of firmness of the skin due to, for example, aging or weight loss and damage due to exposure to sun (i.e. UV), cold or wind. An example of "damage" includes DNA damage and the potential for skin carcinomas to develop. An "aesthetic" skin condition includes, therefore, skin aging, wrinkles, skin texture, skin cracking, skin blemishes, skin color, acne, amongst many others which are treatable or have symptoms which can be alleviated by the use of an anti- inflammatory agent.

The formulation of the present invention have a role, therefore, in the treatment or prophylaxis of photoaging and as sun repair agents.

Accordingly, another aspect of the present invention contemplates a method for the prophylactic treatment, prevention or delay of an inflammatory condition in a subject, wherein the method comprises administering Ngali oil and/or pharmacologically active fractions, extracts or components thereof or a formulation comprising the oil or an active fraction, extract or component thereof to the subject. Generally, the inflammatory condition includes non-arthritic conditions treated using oil or on extract thereof and arthritis treated using an extract of the oil. An inflammatory condition includes a muscle injury such as a sporting injury. A further embodiment provides a method for the prophylactic treatment, prevention or delay of an aesthetic skin condition in a subject, wherein the method comprises administering Ngali oil and/or pharmacologically active fractions, extracts or components thereof or a formulation comprising the oil or an active fraction, extract or component thereof to the subject.

The term "treatment" should be understood to refer to the amelioration of symptoms of an inflammatory or aesthetic skin condition and/or treatment of the underlying causes of inflammation or an aesthetic skin condition in a subject. Typically, treatment is instigated after the appearance of symptoms and/or diagnosis of an inflammatory or aesthetic skin condition. The treatment may delay or halt progression of the condition, repair existing damage as a result of the condition and alleviate symptoms of the condition. In some cases the concepts of "treatment" and "prophylaxis" treatment may intersect, such as where treatment of an existing condition may also prevent recurrence of that condition.

For the purposes of the present invention, "Ngali oil" is defined as the oil obtained from any one of several varieties of Canarium plants or trees such as but not limited to trees or plant, grown in the Solomon Islands, Vanuatu, Papua New Guinea and the Philippines. Three particularly useful Canarium trees are Canarium indicium, Canarium solomonesis and Canarium harveyi. The present invention contemplates Canarium plants not assigned to any of the above species, hybrids of Canarium plants and horticultural relatives of the Canarium plants.

In a preferred embodiment of the present invention, the Ngali oil is the oil derived from the nut of a Canarium plant or tree or a horticultural relative thereof, and may be referred to herein as "Ngali oil". However, the present invention also contemplates Ngali oil derived from other parts of a Ngali nut plant or horticultural relative thereof. Examples of these other tissue types from which oil may be derived include, but are not limited to bark, woody tissues, leaves, fruit, roots, outer husk, nut kernel or nut shell.

Typically, Ngali oil is obtained from a Canarium tree such as from a nut by harvesting the selected part of the tree, removing and drying it and finally, in the case of nuts, cracking of the dried nut to access the kernel which is then pressed to extract the Ngali nut oil. However, the method used to obtain the Ngali nut oil from the Ngali nut does not define or limit the invention in any way. The present invention contemplates the harvesting and extraction of the Ngali nut oil by any means.

As disclosed herein the present invention contemplates the use of Ngali oil and/or pharmacologically active extracts, fractions or components thereof or a formulation comprising the oil or an active fraction, extract or component thereof. The term "pharmacologically active" should be understood to refer to the activities in oil or its fraction, extract or component or a formulation comprising the oil or an active fraction, extract or component thereof which is useful in the treatment or prophylaxis of inflammatory or aesthetic skin conditions. For example, a Ngali oil or fraction, extract or component thereof or a formulation comprising the oil or an active fraction, extract or component thereof is "pharmacologically active" if it had greater therapeutic or prophylactic activity than olive oil, with respect to treating inflammation or an aesthetic skin condition. The terms "extracts", "fractions" and "components" or their singular forms should be understood to refer to any compound or mixture of compounds which comprises fewer species of chemical entities than "native" or "raw" Ngali oil or more concentrated chemical species than "native" or "raw" Ngali oil. Native or natural Ngali oils may be fractionated by any convenient means to yield one or more fractions or extracts or component which are relatively enriched in one or more components of the "native" Ngali oil.

One form of fractionation is solvent partitioning or solvent extraction. In this method, one or more particular compounds from the native Ngali extract preferentially dissolve into one of two or more immiscible solvents on the basis of polarity or lack of polarity. For example, in a two phase system using a polar and a non-polar solvent, the polar compounds preferentially dissolve into the polar solvent and the non-polar compounds preferentially dissolve into the non-polar solvent. As the two solvents used are immiscible (ie. form two distinct phases), the two solvents are easily separated by any convenient means. Any number of useful solvents would be readily ascertained by one of skill in the art, and the choice of solvent will depend on the polarity of the compounds that are to be extracted. However, Table 2 shows examples of commonly used solvents for partitioning or extraction and their relative polarities.

TABLE 2 Common Solvents and Their Relative Polarities

Alkanes (hexane, cyclohexane, pet ether) Carbon tetrachloride Toluene INCREASING Dichloromethane POLARITY Chloroform Ethyl Acetate Isopropyl alcohol Acetone Ethanol Water

DMSO is another example of an organic solvent useful to separate various fractions.

Chromatographic techniques may also foe used to fractionate Ngali oils. Chromatography is defined as the separation of a mixture of two or more compounds by distribution between two phases, one of which is stationary and one of which is moving. Chromatography is so called because it was originally used to separate substances of different colors . Various types of chromatography are possible depending on the nature of the two phases involved: solid-liquid, liquid-liquid, and gas-liquid chromatographic methods are commonly available.

TLC and column chromatography are often used in conjunction with each other. It is often convenient to investigate a suitable so vent system for the best mixture separation by TLC, since TLC is much quicker than running a column. The solvent system may then be used to elute the column and should give approximately the same types of separation, if the adsorbents are of similar activity. Both methods rely on a stationary solid phase and a mobile liquid phase. Examples of useful solid phases include silica gel (Siθ2 x H₂O), alumina (Al₂O₃ x H₂O) and cellulose. The relative movement of a compound through a column or TLC plate is determined by the solubility of the compound in the particular solvent used, which, as mentioned supra, is determined by the relative polarities of the compound and solvent. For example, a compound will have higher mobility in a solvent of similar polarity, than in a solvent of dissimilar polarity. In the case of column chromatography, compounds are eluted from a column using a series of washes of increasing or decreasing polarity, and fractions are collected at each elution, thereby separating the mixture applied to the column into fractions on the basis of polarity or lack of polarity. For thin layer chromatography separation occurs on the TLC plate, with high mobility compounds running closer to the solvent front than lower mobility compounds. Detection of particular compounds on a TLC plate may be performed using any convenient means, as would be readily ascertained by a person of skill in the art. Some examples of commonly used detection methods include: visual inspection (if a compound is coloured), visualization under ultraviolet light and spray reagents which facilitate the generation of a detectable signal. Table 3 shows examples of suitable TLC spray reagents for the detection of a number of different chemical classes.

TABLE 3 TLC Spray Reagents

In addition to column chromatographic techniques and thin layer chromatography, many other solid-liquid, liquid-liquid and gas-liquid chromatographic techniques may be used to separate, fractionate or extract Ngali oils into active and inactive fractions. These techniques include liquid chromatography, high performance liquid chromatography, gas chromatography and the like.

Another method for fractionating and/or preparing extracts from native Ngali oil can include supercritical fluid extraction (SFE). Supercritical fluids (SCFs) replace the organic solvents that are used in industrial purification and recrystallization operations.

The critical point (CP) marks the end of the vapor-liquid coexistence curve. A fluid is termed supercritical when the temperature and pressure are higher than the corresponding critical values. Above the critical temperature, there is no phase transition in that the fluid cannot undergo a transition to a liquid phase, regardless of the applied pressure. A SCF is characterized by physical and thermal properties that are between those of the pure liquid and gas. The fluid density is a strong function of the temperature and pressure. The diffusivity of SCF is much higher than for a liquid and SCF readily penetrates porous and fibrous solids.

SFE is based on the fact that, near the critical point of the solvent, its properties change rapidly with only slight variations of pressure. Supercritical fluids can be used to extract analytes from samples. The SCF, CO , is the reagent widely used as the supercritical solvent.

SCFs have solvating powers similar to liquid organic solvents, but with higher diffusivities, lower viscosity, and lower surface tension. Since the solvating power can be adjusted by changing the pressure or temperature separation of analytes from solvent is fast and easy. By adding modifiers to a SCF (like methanol to CO₂) its polarity can be changed for having more selective separation power. In industrial processes involving food or pharmaceuticals, one does not have to worry about solvent residuals as you would if a "typical" organic solvent were used. Candidate SCFs, such as carbon dioxide and water, are generally cheap, simple and safe.

In one useful method, Ngali nut lipids are extracted by TLC with the lipid classes being roughly quantitated by densitometry and the fatty acid composition analyzed by GC.

In one embodiment, active fractions comprised triglyceride, free fatty acid (FFA), sterols, triglycerides and polarlipids. Another useful fraction comprised non-polar fatty acids and non-polarlipids. Yet another active fraction comprises a 75% triglyceride-free extract (see Example 5 samples E4 and E5). Types of fatty acids identified included palmitic acid, oleic acid, linoleic acid and stearic acid. When rubbed into the skin, Ngali nut oil pharmacological activity was in part associated with the presence of FFA, e.g. oleic acid which promotes skin permeation.

Accordingly, another aspect of the present invention contemplates a method for identifying a fraction, extract or compound in Ngali oil having anti-inflammatory or pro-aesthetic activity said method comprising subjecting said oil to fractionation means and assessing one or more fractions to functional and/or biochemical analysis.

By "fractionation means" includes solvent separation and chromatographic separation amongst others.

The Ngali oil or pharmacologically active fraction, extract or component thereof may be administered by any convenient means. Oral, transdermal and topical routes of administration are particularly useful. In the case of transdermal administration, the active agent is preferably administered with a percutaneous enhancer which increases the absorption of the agent across the skin. Furthermore, transdermal administration may be performed on any region of the body of the subject. Preferably, this region is the site of inflammation or the face or hands. Administration of the Ngali oil or a pharmacologically active fraction, extract or component thereof to the hands is one preferred method where the site of inflammation is near this part of the body. Administration of the Ngali oil or pharmacologically active fraction, extract or component thereof to the hands may be used to treat inflammation in a distal region of the body.

Ngali oil or pharmacologically active fractions, extracts or components thereof, for the treatment of inflammatory or aesthetic skin conditions in accordance with the present invention, are conveniently supplied in pharmaceutical compositions or other formulations.

Accordingly, in another aspect, the present invention provides a pharmaceutical composition or cosmetic formulation comprising a Ngali oil or a pharmacologically active fraction, extract or component thereof or a formulation comprising the oil or an active fraction, extract or component thereof together with a pharmaceutically or cosmetically acceptable carrier, excipient or diluent.

Such a composition or formulation generally is, but not necessarily, defined as being when used for the treatment or prophylaxis of a particular inflammatory or cosmetic condition.

Compositions are generally stable under the conditions of manufaxture and storage and may be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dilution medium comprising, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof and vegetable oils. Fluidity or pliability can be maintained, for example, by the use of superfactants. Various anti-bacterial and anti- fungal agents may also be employed such as, for example, parabens, chlorobutanol, phenol, sorbic acid, thirmerosal and the like. In addition, the composition may be in the sun lotion or sun cream formulation or as a lotion or cream to be applied after, during or prior to exposure to harsh environmental conditions such as the sun.

Pharmaceutically acceptable carriers and/or diluents include a_ny and all solvents, dispersion media, coatings, anti-bacterial and anti-fungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art and except insofar as any conventional media or agent is incompatible with the modulator, their use in the therapeutic or co smetic compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions. This is particularly the case where the oil or its fractions, extracts or components are admixed with various neurological agents or other components useful for cosmetic purposes.

Formulations including free-flowing oil powder forms and creams comprise the oils or their components or extracts are also contemplated herein.

In the case of dried powders for the preparation of creams, methods of preparation include vacuum drying and the freeze-drying technique which yield a powder of active ingredient plus any additionally desired ingredient.

When the active liquid, extract or powder agent is suitably protected, it may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food or the diet. For oral therapeutic administration, the active ingredient may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, soft or hard gel capsules, elixers, suspensions, syrups, wafers and the like. Such compositions and preparations should contain at least 1% by weight of active agent. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit. The amount of active agent in such therapeutically useful compositions is such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 0.1 μg and 200 mg of modulator. Alternative dosage amounts include from about 1 μg to about 1000 mg and from about 10 μg to about 500 mg. These dosages may be per individual or per kg body weight. Administration may be per hour, day, week, month or year.

In one preferred embodiment, the pharmaceutical composition is a dermatological composition or formulation.

Dermatological formulations include solutions (i.e., collodions, liniments, aqueous and oleaginous solutions), suspensions and gels, emulsions, lotions, and creams. Lotions can be either suspensions or emulsions but are fluid liquids that are typically used for their lubricating effect. Creams are emulsions and are typically opaque, thick liquids or soft solids used for their emollient properties. Creams also have the added feature that they tend to "vanish" or disappear with rubbing. Distinctions between lotions and creams are open to individual interpretation. Other dermatological formulations include aerosols, dusting powders and devices such as transdermal patches, tapes, and gauzes. Regardless of the formulation, most dermatological formulations are applied to the skin.

The skin is composed of three layers of stratified tissue: epidermis, dermis, and subcutaneous tissue. In normal skin, the epidermal cells are continually replenished by the formation of initially viable cells from the basal germinative layer. As the new cells develop, they displace the outer epidermal cells. The outer layer is called the stratum corneum and these cells are sloughed off to the environment. As the cells migrate to become the stratum corneum, they become flattened, lose their nuclei, and the organized cell contents becomes replaced with keratin fibrils. The turnover time from germination to sloughing is about 21 days.

It is the stratum comeum that is the barrier to drug penetration through the skin. The stratum comeum is approximately 10 microns thick. It can swell to approximately three times its original thickness and absorb about five times its weight in water. When the stratum corneum hydrates, it becomes more permeable. Therefore, occlusive dressings are often used to hydrate the stratum comeum and increase the penetration of certain drugs. Dermatoses such as eczema and psoriasis can also hydrate the stratum comeum and increase the absorption of some drugs.

Dermatological formulations produce a local drag effect either on or in the skin. Besides the specific therapeutic action of incorporated active drags, the formulations also as serve as protectants, lubricants, emollients, or drying agents.

Some dermatological formulations are intended to systemically deliver a drug. Percutaneous (through the skin) absorption is the result of three competing processes:

(i) The potential of the drug to cross the stratum comeum.

(ii) The potential of the drag to leave the formulation.

(iii) The influence of the formulation on the stratum comeum. Although the percutaneous absorption of drugs is a complex process, several generalizations are possible:

(i) More drag is absorbed when the formulation is applied to a larger surface area.

(ii) Formulations or dressings that increase the hydration of the skin generally enhance percutaneous absorption. (iii) The greater the amount of rubbing in or inunction of the formulation, the greater the absorption.

(iv) The longer the formulation remains in contract with the skin, the greater will be the absorption.

Percutaneous administration affords an ease of administration not found in other routes, and patient compliance is generally good with dermatological formulations. There is also the possibility of continuous drug administration exemplified by transdermal patches. In addition, dermatological formulations can be easily removed if necessary.

In a further preferred embodiment, the present invention provides a dermatological composition comprising oil from a Canarium plant, or a pharmacologically active fraction, extract or component thereof, in a bland oil diluent. As used herein, the term "bland oil" refers to oils with little or no free fatty acids and/or peroxides. In a more preferred embodiment, the bland oil diluent is a bland olive oil. In an alternate preferred embodiment, the topical cream, lotion, ointment or gel comprises Ngali oil or pharmacologically active fractions, extracts or components thereof in sorbolene cream.

In particularly preferred embodiments, for native or unfractionated Ngali oil, the compositions may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95% oil from a Canarium plant on a weight/weight, weight/volume, or volume/volume basis. In a further preferred embodiment, the dermatological composition may further comprise a percutaneous enhancer. "Percutaneous enhancers" are agents which improve the solubility of the active drag in the stratum comeum and facilitate the diffusion of the drug through this barrier into the systemic circulation. Commonly used percutaneous enhancers include:

Alcohols - eg. methanol, ethanol, propanol, isopropanol, octanol Fatty Alcohols - eg. myristyl alcohol, cetyl alcohol, stearyl alcohol Fatty Acids - eg. myristic acid, stearic acid, oleic acid ^■ Fatty Acid Ester - eg. isopropyl myristate, isopropyl palmitate Polyols - eg. propylene glycol, polyethylene glycol, glycerol Anionic surfactants - eg. sodium lauryl sulfate Cationic surfactants - eg. benzalkonium chloride, cetylpyridinium chloride Amphoteric surfactants - eg. lecithins ^■ Nonionic surfactants - eg. Spans (registered trade mark), Tweens (registered trademark), poloxamers, Miglyol (registered trade mark)

Other useful enhancers include DMSO (dimethyl sulfoxide), urea, triethanolamide, salicylate and azaleate esters, N-methyl pyrrolidine, plant-sourced hydrocarbons and ceraphyls.

In a particularly preferred embodiment, the percutaneous enhancer is a some plant-sourced hydrocarbon, such as cineole, limonene, tea-tree oil or sandalwood oil. In an even more preferred embodiment, the percutaneous enhancer is cineole.

In further preferred embodiments, the percutaneous enhancer(s) comprise 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50% of the weight or volume of the dermatological composition or formulation.

The dermatological compositions of the present invention may further comprise other agents such as emulsifiers, preservatives and antioxidants (such as Vitamin E), as would be readily ascertained by a skilled artisan.

It is also proposed that the dermatological compositions of the present invention are useful in preventing or repairing DNA damage, especially to cells of the skin. Consequently, the compositions of the present invention have a role in preventing or reducing the risk of skin cancers and carcinomas. In addition, the compositions have a role in ameliorating the effects of UV exposure to the immune system.

The present invention further contemplates a business model comprising the wholesale supply of Ngali nut oil or its active fractions for use in an appropriate formulation.

Accordingly, the present invention provides a method of conducting business said method comprising receiving an order for Ngali nut oil or an active fraction thereof or an isolated component therefrom, supplying the Ngali nut oil or fraction or component for use in a formulation prepared by a third party and receiving payment for said Ngali nut oil or fraction or component and optionally a fee component from the sale of the formulation by the third party.

In accordance with this business model, Ngali nut oil or its fractions or components is regarded as a high value ingredient which is supplied to third party retailers for incorporation into a formulation.

The present invention is further described by the following non-limiting examples:

EXAMPLE 1 Ngali Oil

Antigen-induced polyarthritis in rats has been a well accepted and widely used small animal model pharmacoassay for testing the efficacy (of oral or topical preparations) of NSAID's, corticosteroids, or other drags or natural products. In particular the assay has been used as a means to assess an agents efficacy in controlling chronic inflammation and in modulating some of the other symptoms related to common musculoskeletal complains such as rheumatism and arthritis (Billingham Pharmacol. Ther. 21: 89-417, 1983).

Measurable outcomes can be attributed to either the anti-inflammatory and/or other arthritis-suppressing activity of a given test agent. The rat adjuvant model of inflammation/arthritis also responds beneficially to known anti-inflammatory drags. The use of standard drags where the class and mode of action is known, is particularly important when evaluating new compounds to enable meaningful conclusions to be reached. In the following Examples, this model is used to evaluate the beneficial anti- inflammatory and arthritis-suppressing effects of topical and oral formulations of Ngali oil, relative to a number of representative drags with known anti-inflammatory activity as well as a number of well-known natural product remedies.

The model employed can be used to measure both the therapeutic, or disease/symptom modifying effects of a substance (by commencing treatment after the first signs of arthritis/inflammation are manifest) or the prophylactic or disease-preventing effects (by commencing treatment coincident with the administration of the arthritigen/inflammation inducing adjuvent).

For the therapeutic use of the model rats from a susceptible strain such as female hooded wistar (160-200g) are injected with an arthritic adjuvant (0.25-1.0 mg Mycobacterium tuberculosis in 5-100 μl of squalane) into the base of the tail at day 0. The disease is allowed to develop until the first sign's of arthritic inflammation are clearly expressed 10- 12 days layer. The polyarthritis is usually manifested as local inflammation and ulceration in the tail and inflammation in all paws. At this time (day 10) the size of the rear paws and tail thickness are measured (in ml) using a screw gauge micrometer, the severity of inflammation in the front paws is given a score on an arbitrary scale (0-4+) and the body weight is measured with or without shaving an area of skin on the upper part of the rat (approximately 6cm) just below the neck. Animals (5-7 per group) are treated with either oral or topically administered test formulations, once per day, for 4 days (days 10-»13) and on the fifth day (day 14) the above quantitative and other parameters of inflammation and arthritis are re-measured and re-scored. Note that for certain experiments with NS AID's treatment was for up to 7 days (i.e. days 7— 13) and the relevant measurements and scoring therefore commenced on day 7 and were re-evaluated on day 14.

For the prophylactic use of the model, test treatments commenced at day 0, coincident with injection of the arthritis adjuvant (as above) and continued once daily for 15 days (i.e. days 0-»14 inclusive). All the above measurements and scoring were therefore done on day 0 and then on day 14. The oral doses administered were relatively high, being based in x mg/kg/day, where x = approximately half the cumulative human daily dose. Topically administered agents (1-2 ml/kg) were typically composed of 85% Ngali oil and 15% cineole (eucalyptol), a penetration enhancer. Negative controls contained 85% olive oil and 15% cineole. On day 14, treatment was suspended, changes in tail thickness and rear paw thickness quantitatively assessed, the animal re-weighed and the severity of arthritis in the front paws re-scored by an independent assessor (0-no arthritis to 4-severe arthritis). The blinded, independent observer also assessed and assigned an overall arthritis score, on a scale of 0-4+ based on clinical impressions (piloerection, grooming, mobility, sensitivity to handling) and overall inflammatory signs in ears, tail and paws. Results are presented as the change (Δ value) in the various parameters and scores over the treatment period. Suppression of the rear paw swelling and of the disease-associated arthritis score indicates successful treatment. Animals were typically observed for a further 3-4 days after suspending treatment; nearly always there is a rebound in the signs of arthritis in the successfully treated group. This indicates that the drag is truly anti-inflammatory and that these treated animals are not "false-positives" in the sense of having failed to respond adequately to the original arthritigen (typically n<15%). To allow direct assessment of the ngali oil data and comparison with the results of extensive published testing of other natural products and drags with the rat inflammation/arthritis model the results are presented in the format shown in Tables 4 (therapeutic study) and 5 (prophylactic study). Data relating to: 1) mode of administration (p.o. = oral; t.d. = topical); 2) dosage - mg/kg; 3) mean change in rear paw thickness (mm); 4) mean change in forepaw inflammation score and 5) change in overall mean "arthritis score", were derived as described above. In general, the positive efficacy of test agents is shown by a reduction in inflammation (in particular, rear-paw thickness) and the other signs of experimental arthritis over a 4 or 15 day period. These measurements and overall arthritis score, permit the recognition and quantification of putative therapeutic agents exhibiting significant anti-inflammatory activity and/or ability to modulate the symptoms of an experimentally induced polyarthritis; both relative to each other and against well known natural therapies or standard anti-inflammatory and pain relieving drags currently on the market.

The key findings/conclusions from the results shown in Table 4 (Therapeutic testing) can be summarized as follows:

1. Ngali oil and certain purified extracts were therapeutically (relative to a placebo control) extremely efficacious in treating inflammation and arthritis in the rat model. This is particularly evident when looking at the relative changes in mean rear paw thickness and the overall arthritis score relative to the untreated control. Ngali oil samples from two independent suppliers (designated Lot 1 and Lot 2) both exhibited this activity to an equal extent. Moreover, the two oil samples were several years apart in age, suggesting that the ngali oil therapeutic activity is relatively stable during extended storage.

2. It is scientifically possible to purify and extract and concentrate the efficacious or "active fraction" (which interestingly is low in triglyceride content) from ngali oil as shown by the results with ngali extracts. 3. Ngali oil and extracts thereof, were effective in treating arthritis when administered both topically and orally (based on similar changes in rear paw thickness, forepaw inflammation and arthritis score). However the topical application appears superior on a dose basis; since topical ngali oil at 400 mg/kg gave the same effect in rats as orally administration nglai oil at 1400 mg/kg.

4. The rat model of inflammation/arthritis does respond to known NSAID's such as Naproxen, Celebrex and Vioxx.

5. On a relative basis, Ngali oil (at a high dose) or an extract thereof (at a lower dose), when administered orally, appears in this model to be a more effective anti- inflammatory agent than aspirin, ibuprofen, Naproxen, Celebrex or Vioxx and a more generally effective arthritis suppressant.

6. Ngali oil extract when administered orally shows similar anti-inflammatory and arthritis suppressing activity as a natural (nutraceutical) product such as Lyprinol (purified extract or "oil" from New Zealand green-lipped mussels).

The key findings/conclusions from the results shown in Table 5 (prophylactic testing) can be summarized as follows:

1. Ngali oil, administered orally over a 15 day period, shows outstanding efficacy in the rat model when tested for disease prevention capabilities i.e. ability to prevent the onset of inflammation and arthritis-like symptoms in rats. This suggests that ingredients within ngali oil can suppress the adjuvant-induced inflammation and polyarthritis symptoms in rats.

2. Ngali oil's disease and inflammation prevention capabilities at high dosages were greatly superior to those of aspirin or ibuprofen (at recommended dosages) both of which showed little prophylactic activity and also superior to those of other natural products and treatments promoted to prevent/treat inflammation and arthritis. TABLE 4 Therapeutic efficacy of ngali nut oil and purified extracts (after oral=p.o. or tpical=t.d. administration) in treating inflammation in rats, compared to no treatment or a placebo (olive oil) and various common OTC anti-inflammatory drugs and NSAID's

TABLE 5 Prophylactic efficacy of ngali nut oil in treating inflammation in rats, compared to no treatment or a placebo (olive oil) and various common OTC anti-inflammatory drugs and NSAID's after oral=p.o. administration

EXAMPLE 2 Anti-Inflammatory Activity of three Ngali Nut Oils

A bioassay of three different Ngali nut oils (Stocks: Bl, B2 and B3) was performed as follows:

Assessment of anti-inflammatory activity

Anti-inflammatory activity was assessed in Wistar rats with pre-established arthritis, induced by inoculating with M. tuberculosis in squalene 10 days previously. Signs of arthritis were measured on days 10, 14 and 17. Oils were administered on days 10 through 13 in two modes: a) transdermally after dilution with olive oil and adding 15% v/v cineole, a penetration enhancer. The volume applied to shaved upper dorsal skin (6 cm²) was 2.5 ml/kg/day, equivalent to 0.53 oil/kg from a Canarium plant for four days; b) orally as 2 ml/kg/day with no diluent or additive.

Table 6 shows the results of the bioassay comparing the three samples of Ngali nut oil as potential therapeutic agents to treat developing polyarthritis over the critical time period from when the polyarthritis is first expressed (day 10) until it is almost fully developed (day 14). Observations were also made to assess the rebound after ceasing treatment on day 13. This ensures that any animals that had failed to respond to the original arthritigen were not included as false-positives for response to the therapy.

At least four times the dose was needed to demonstrate oral efficacy (2 ml/kg); a far lower dose being required for an effective oil (0.5 ml/kg) to demonstrate sufficient activity to significantly arrest polyarthritis development when applied dermally. TABLE 6 Anti-Inflammatory Activity of Three Ngali Nut Oils

EXAMPLE 3 Bioassay Guided Fractionation of Ngali Nut Oil

A schematic diagram showing a plan for the extraction, identification and purification of pharmacologically active fractions of Ngali nut oil is shown in Figure 1.

The pharmacologically active molecules in Ngali nut oil are extracted and concentrated from the raw oil by one or more of rounds of solvent extraction and/or solid phase adsorption on magnesium silicate or its equivalent. Supercritical fluid extractions and chromatography steps are also used as required.

Pharmacologically active Ngali nut oils and pharmacologically active fractions and extracts thereof are characterized by the presence of, wter alia:

(i) endogenous antioxidant(s); (ii) mono- and poly- hydroxy fatty acids; (iii) hydroperoxides of mono- and poly- hydroxy fatty acids; (iv) keto and epoxy derivatives of fatty acids and combinations thereof; (v) cyclic fatty acids and prostaglandin-like molecules; and (vi) aliphatic esters of the same fatty acids, where the fatty acids contain both odd and even numbers of carbon atoms.

EXAMPLE 4 Solvent Extraction of Ngali Nut Solids

If the active component of the oil is found to be a minor compound, or one which would have low solubility in the non-polar oil, other parts of the Ngali nut (eg. skin, shell, kernel) would be investigated. To extract these compounds, a solvent which is more or less polar than the Ngali nut oil itself is used. The extract is then analysed using a method such as TLC or HPLC to look for the active compound. Extracts putatively containing the active compound are then bioassayed for anti-inflammatory activity. EXAMPLE 5 Preparation of Ngali oil extracts

Materials Various Ngali nut lipid extracts were prepared according to the Scheme in Figure 1.

Thin Layer Chromatography of Extracts

Samples of the extracts were dissolved in solvent (30-40 mg/ml: petroleum ether or ethanol) and spotted onto Silica Gel plates (MERCK, 60 F₂₅₄). Non-polar lipid classes were analyzed by running the plates with a petroleum etheπdiethyl ether:acetic acid (80:20:1) solvent mixture. The plates were then dipped in a solution of sulfuric acid (2% w/v in ethanol) and the spots developed by charring with a heat gun. Quantification was performed by densitometry using an Alphalmager [Trademark] (Alpha Innotech Corp). Response factors for the various lipid classes were calculated with the use of standards (triolein, linoleic acid, cholesterol, dilaurin and phosphatidylethanolamine).

Fatty Acid Composition of Extracts

Fatty acids in the extracts (free and bound) were converted to methyl esters with H SO₄ in methanol (Luddy et al., J. Am. Oil Chem. Soc. 37:447-451, 1960). GC analysis was performed on a HP-5890 gas chromatograph using an Alltech EC- Wax column (0.25mm x 30m). The temperature program was 165°C for 3 min, 4°C/min to 195°C for 10 min, 4°C/min to 225°C for 12 min. Peaks were identified by comparison to well characterized oils as external standards (eg. cod liver oil). Nonadecanoic acid (19:)0) was used as an internal standard for quantification. Results

The TLC separation of non-polar lipid classes is quantified in Table 7. There is a clear similarity between the lipid class composition of the whole oil samples (lot 1, and lot 2) and that of the first three extracts (El, E2 and E3). All have high levels of triglycerides (>94%), with lower levels of FFA (1-4%), sterols (.04-0.8%), diglycerides (0.2-0.6%) and polar lipids (0.3-0.6%). It is clear that Lot 2 has a higher level of FFA than that of Lot 1.

There are very clear differences between the two purified extracts (E4 E5) from that seen in the other extracts. Extract E4 is predominantly free fatty acid (78%) and also contains significantly lower levels of polar compounds (21%). Extract E5, consists of mainly polar compounds (82%) and FFA (16%). Both extracts are notably >95% triglyceride-free.

Table 7: Major lipid classes in Ngali Nut extracts as determined by TLC

Sample Lipid Class (% wt/wt) Trij ξlyceride FFA Sterol Dig lyceride Polar Lipids lot l 98.1 1.1 0.4 0.2 0.3 lot 2 94.3 4.0 0.8 0.6 0.4

El 95.7 2.9 0.7 0.4 0.4

E2 96.4 2.1 0.6 0.4 0.6

E3 95.2 3.2 0.7 0.5 0.4

E4 0.3 78.0 nd 1.1 20.7

E5 nd 16.2 nd 2.1 81.7 nd = not detected

The nature of polar compounds in Extract E5 was investigated further with TLC using a more polar solvent system (chloroform:methanol:water 65:30:5). Most of the material ran with the solvent front (non polar material), though there was a faint spot indicative of phosphatidylethanolamine. The sample was analyzed by 1H NMR which showed the presence of hydrocarbon chains (as expected in compounds containing fatty acids) and other peaks characteristic of phospholipids (3.1 ppm and 4.0 ppm). Fatty acid profiles by GC showed the four major fatty acids in Ngali nut oil to be oleic acid (33%), palmitic acid (40%), stearic acid (12%) and linoleic acid (13%). The similarity between the whole oil Lot 2 and Extracts El, E2 and E3 is striking. There is a littler more variation between these samples and that of Lot 1. Literature values for the fatty acid composition of Ngali nut oil from the Solomon Islands (Canarium salomonense) (Martin and Harris, J. Sci. FoodAgric. (57:383-384, 1993) are quite similar to the fatty acid profiles of these samples.

Extract E4, has a higher level of oleic acid than the other samples. It also has a significant amount of palmitoleic acid (5%) as well as the presence of some unknown compounds and a much reduced level of linoleic acid.

Extract E5, has a very high level of palmitic acid (53%), and like E4 also contains some unidentified compounds and a much reduced level of linoleic acid (no linoleic acid was detected in this extract). Unlike the other extracts which all had a fatty acid content close to 100%) (measured wit the use of an internal standard), the material in E4 only consisted of 56% fatty acid.

The fatty acid compositions are shown in Table 8.

Table 8: Fatty Acid Composition of Ngali Nut Extracts

Fatty Acid lit* lot l lot 2 El E2 E3 E4 E5 myristic (14:0) 0.09 0.10 0.10 0.10 0.10 0.90 unknown 1.10 unknown 0.77 0.88 unknown 1.61 4.74 palmitic (16:0) 34.9 39.99 39.30 39.62 39.87 39.61 33.98 53.18 palmitoleic (16:1 n-7) 0.44 0.49 0.50 0.53 0.49 5.21 0.75 heptadecanoic (17:0) 0.12 0.13 0.11 0.12 0.12 stearic (18:0) 12.6 11.45 12.29 12.30 12.33 12.30 9.97 14.50 oleic (18:1 n-9) 41.6 30.20 33.23 32.94 32.92 33.00 42.99 23.78

(18:1 n-7) 1.23 1.25 1.25 1.26 1.24 2.32 1.06 linoleic (18:2 n-6) 10.3 15.68 12.68 12.64 12.36 12.61 1.85 α-linolenic (18:3 n-3) 0.4 0.31 0.29 0.29 0.28 0.29 arachidic (20:0) 0.2 0.22 0.24 0.25 0.25 0.24 gondoic (20:l n-9) 0.41 lit*: Martin and Harris, Supra 1993

EXAMPLE 6 Treatment of Inflammation using Ngali Nut Oil and Extracts

When rubbed into the skin, without any additive, Ngali nut oils showed variable pharmacological activity, which was partly attributed to the presence of free fatty acids (triglyceride decomposition products). These free fatty acids, which include oleic acid, are known to promote skin permeation. Adding other, more acceptable, penetration enhancers was found to consistently raise the anti-inflammatory potency of certain Ngali nut oils, particularly those without evidence of some prior decomposition. These enhancers included salicylate esters (methyl, ethyl, isopropyl), some plant sourced hydrocarbons (cineole, limonene, tea-tree oil, sandalwood oil) and isopropanol. All of these percutaneous enhancers when mixed with olive oil or lard oil in proportions of 5-20% v/v, of themselves failed to show any anti-inflammatory activity (dose of oil = 2 ml/kg, maximum dose of enhancer 0.5ml/kg applied dermally once daily for four days). By contrast some, but not all, Ngali nut oils tested were highly active in the anti-inflammatory bioassay when delivered transdermally at 0.4 ml/kg in an olive oil carrier supplemented with 15% (v/v) cineole.

The most potent Ngali nut oils were fractionated to obtain specific subfractions which were almost triglyceride free. These subfractions consistently showed anti-inflammatory activity at a dose of 5 mg/kg or less when applied transdermally in olive oil-cineole (85:15 v/v). For oral application, somewhat higher doses were required (10 mg/kg) to obtain maximal activity. These doses are nevertheless more effective than corresponding doses of standard NSAIDs, including Cox II inhibitors (Figure 2).

EXAMPLE 7 Typical dermatological formulation

Dermatological formulations were prepared based on either an olive oil or a Ngali nut oil dispersed into stable aqueous emulsion. Typical cream formulae consist of:

5-30.0%) w/w Of either a pharmacologically active Ngali nut oil or a carrier oil such as refined (non-rancidifying) olive oil containing one or more pharmacologically active fractions or extracts of Ngali nut oil (0.5- 5.0% w/w).

6.0-10.0% w/w Emulsifier(s)

0.0-10.0% w/w Percutaneous enhancer(s)

0.0-1.0% w/w Suitable preservative

0.0-1.0% w/w Antioxidant such as Vitamin E acetate or equivalent

Appropriate quantity of triethanolamine to adjust viscosity and remainder water to 100% w/w. EXAMPLE 8 Veterinary Applications

'Rub on' lotions/cream formulations or gels are prepared based on either a pharmacologically active Ngali nut oil or a concentrated pharmacologically active extract or fraction of a Ngali nut oil dispersed in an oil based carrier, such as refined olive oil.

Several different creams, lotions and oral preparations were tested on a number of dogs and horses for a range of inflammatory conditions such as arthritis, muscle injuries, slow- healing wounds as an adjunct to other modes of pain management; all with a high degree of success.

EXAMPLE 9 Development of anti-inflammatory formulation

Ngali oil or fractions thereof are assayed as follows:

(i) Suppression of superoxide production by human neutorphils. This assay is a cell microtitre-plate based assay and only requires a few hours to perform but only tests a single aspect of inflammation. It involves incubating extracted human neutrophils in the presence of the compound under test and determining how that affects the production of superoxides when the cells receive an inflammatory stimulus (Tan and Berridge, J. Immunol. Methods, 238:59-6 , 2000). Aspirin is used as a positive control.

(ii) COX I/COX II assay. This assays specifically for suppression of the major enzyme involved in inflammation. Blood is incubated with the test materials and LPS is added to the incubation mixture. At the conclusion of the incubation, the concentration of Thromboxane B2 (TxB2) and of Prostaglandin E2 (PGE2) are determined by immunoassay as markers of COX-I and COX-II respectively (Brideau et al, Inflammation Research, 45:68-74, 1996). The levels of these in the presence of the test materials relative to controls gives a measure of the inhibitory activity. Indomethacin is used as a positive control.

(iii) Carageenan induced footpad swelling assay. This is an assay for activity against acute inflammation. It involves producing a localised swelling in the hind paw of a rat by injection of carageenan and monitoring the effect on the amount of swelling of the compound under test, which may be administered orally, by injection or topically (Onnrod and Miller, Pharmaceutical Research 8: 1270-1273, 1991). While this assay does involve the use of animals, it only requires a few hours to perform (depending on the time allowed to let circulating levels of the material under test build up).

(iv) Adjuvant induced Arthritis. This is a long term assay of arthritis, not just inflammation. Arthritis is induced in the rates by the injection of Freund's complete adjuvant containing mycobacterium tuberculosis (Zhang et al, J. Immunol, 145:2489-2493, 1990). Rats with induced arthritis will have Ngali oil applied topically and the extent of joint swelling compared to control animals.

The active components of the Ngali nut are proposed to include neutral lipids (including terpenoids), polar or non-lipids (including free polyunsaturated fatty acids and phospholipids) and saponins (Belch and Muir, Proceedings of the Nutrition Society, 57:563-569, 1998; Gill and Valivety, Tibtech. 75:401-409, 1997; Mahato et al, Phytochemistry 21:959-918, 1982 and Mahato et al, Phytochemistry 27:3037-3067, 1988). The broad fatty acid composition of Ngali nut oil is known (Martin and Harris, 1993 supra).

To separate the above classes of compound, Ngali oil or extracts thereof are assayed for biological activity using one or more of the above assays. The fraction of highest activity is further purified using bioassay guided chromatography. The active fractions are examined by a selection of the following analytical techniques:

GC-MS LC-MS NMR (¹³C, 1H, ³¹P) HPLC TLC

EXAMPLE 10 Reduction of effects of UV exposure by Ngali nut oil

The effects of Ngali nut oil or its fractions or components is tested on animals following, during or prior to exposure to sunlight. Hairless mice are divided into three cohorts. One cohort is not treated; a second cohort is treated with a small amount of cream containing Ngali nut oil or its fraction or component; a third cohort is initially not treated but then after one hour of exposure, the mice are then treated with the Ngali nut oil cream. The cohorts of mice are then removed from the sunlight and examined on a three to five day period for:

(1) immune sensitivity in skin;

(2) thickness of skin;

(3) the presence of wrinkling in skin; and

(4) pain sensitivity of skin.

The experiment is also conducted using mice susceptible to cancer development. In this case, the rate of development of cancer after UV exposure, with or without the Ngali nut oil cream are less likely to become wrinkled, tend to maintain a normal immune response, and are generally less sensitive to pain. EXAMPLE 11 Clinical trial

(1) Subject Selection

A sufficient number of male and female subjects, 18-65 years of age (inclusive) and in general good health, are empaneled so that at least 90 will finish (30 subjects in each cell).

(i) Inclusion Criteria

1. Males and females, 18-65 years of age (inclusive), in general good health.

2. Individuals having at least 1 joint (finger, hand, elbow, foot or knee) with chronic pain.

3. Individuals with anticipated ability to complete the course of the study and to comply with instructions.

4. Individuals willing to refrain from taking oral or topical pain medications, including, but not limited to, aspirin, tylenol, ibuprofen, etc., during the course of the study.

(ii) Exclusion Criteria

1. Women who are pregnant, planning a pregnancy, lactating and/or nursing a child.

2. Individuals with any visible skin disease that might interfere with the evaluations.

3. Individuals engaged in a concurrent research project of a topical product involving the fingers, hands, elbows, feet or knees. 4. Individuals taking medications which might interfere with the test results including the use of steroidal/non-steroidal anti-inflammatory drags or antihistamines.

5. Individuals with acne, active atopic dermatitis/eczema or psoriasis.

6. Individuals who are currently under treatment for asthma or diabetes.

7. Individuals with a known sensitivity to topical creams and/or ointments or pain relief medications (oral or topical).

8. Individuals with a known sensitivity to the ingredients of the test materials or an allergy to any nuts or nut products.

(2) Test Procedure

The study is designed as a 2-week, double-blind, placebo-controlled study in which the test article is used by each of the test panelists according to the method of the present invention. Subjects are given the product to use according to a randomization schedule ensuring that at least one-third of the test population use the active and at least one third use a placenta.

Subjects indicate at least one joint on their body (finger, hand, elbow, foot or knee) where they are suffering from chronic pain. One joint is selected to receive treatment. Subjects receive the test material and a daily diary to record product use. On Days 1, 7 and 14, subjects also complete a 10cm visual analog scales (VAS) to rate their pain at different time intervals (immediately upon waking and 15 and 30 minutes and 1 and 3 hours post- application), to rate any changes in joint stiffness or mobility and to rate their quality of life or general well being. Additionally, an irritation evaluation is conducted at the treated site for safety purposes and will not be used to determine efficacy.

Evaluations of efficacy are based upon a comparison of each evaluation time versus baseline for each of the three treatment cells and a comparison between the three treatment cells at each time point. Additionally, if a subject has a second joint where they suffer from chronic pain, it will not receive treatment, but it will be monitored to determine if there are any systemic effects from application of the test materials. The second, untreated joint, is not compared to the treated joint in any manner.

(3) Directions

To relieve pain and discomfort, the Ngali nut oil or fraction or component is rubbed into the affected area 2 times daily.

(4) Pain Assessments

On Days 1, 7 and 14 of product use, subjects are required to complete a VAS to evaluate the pain they feel in the treated and non-treated joints. This VAS will be included as part of the subjects daily diary. The VAS is completed on the required days, immediately upon waking and 15 and 30 minutes and 1 and 3 hours post-application. Subjects do not need to return to the Testing Facility on Days 1 or 7, but they are required to return on Day 14.

(5) Clinical Evaluation Procedures

Evaluations are conducted according to the scales outlined below:

(i) Visual Analog Scales (VAS) On Days 1, 7 and 14, subjects complete VAS scales evaluating parameters.

(ii) VAS - Level of Pain

On Days 1, 7 and 14, subjects will rate their level of pain according to a 10cm VAS (0=no pain and 10=severe pain). (iii) VAS - Joint Stiffness/Increased Mobility

On Days 1, 7 and 14, subjects will assess their level of joint stiffness/increased mobility according to a 10cm VAS (0=no improvement and 10=most improvement).

(iv) VAS - Quality of Life

On Days 1, 7 and 14, subjects will assess their quality of life according to a 10cm Vas (0=no improvement and 10=most improvement) or (SF-36 Questionnaire).

(v) Irritation Evaluation

At each visit, a trained technician evaluates the treated joint of each subject for irritation. Evaluations are conducted according to the following scale:

Scale for Scoring Irritation 0 = No evidence of any irritation + = Barely perceptible irritation present 1 = Mild irritation present 2 = Moderate irritation present 3 = Marked irritation present 4 = Severe irritation present

(6) Statistical Analyses

All data points collected at Days 1, 7 and 14 are compared to the baseline of each subject for differences between the time points. The summation of the difference is analyzed using the Wilcoxon Signed-Rank Test. A response is considered significantly different from the baseline when the p-value is <0.05.

Statistical analysis is also conducted to determine difference between the test products (ANOVA) at Days 1, 7 and 14.

The data from the clinical trial are showing VAS pain and VAS joint stiffness/mobility score improvements are shown in Tables 9 and 10. No statistical analyses /significance data has been done yet since it seems to me that Essex may possibly/will exclude some of the patients based on raw data scores. Either way data shows good improvements in VAS pain relief score and increased joint mobility scores of treated groups.

Table 9: Improvement in VAS pain score

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Table 10: Improvement in VAS joint stiffness/increase in mobility

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 to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to, or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

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BIBLIOGRAPHY

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Claims

48CLAIMS

1. A method for the treatment or prophylaxis of a condition selected from a non-arthritic inflammatory condition, an arthritic condition, an aesthetic skin condition and a muscle injury in a subject, said method comprising administering to said subject oil from a Canarium nut or other part of a Canarium plant or a horticultural relative thereof or one or more pharmacologically active fractions, extracts or components thereof or a formulation comprising the oil or an active fraction, extract or component thereof, wherein the inflammatory condition is treated by the administration of an active fraction, extract or component of the oil.

2. The method of Claim 1 wherein said oil or pharmacologically active fraction, extract or component thereof or a formulation comprising the oil or an active fraction, extract or component thereof is administered topically to the skin within or distal to the area to be treated.

3. The method of Claim 2 wherein said oil or pharmacologically active fraction, extract or component thereof or a formulation comprising the oil or an active fraction, extract or component thereof is transdermally absorbed.

4. The method of Claim 1 wherein said oil or pharmacologically active fraction or extract or component thereof or a formulation comprising the oil or an active fraction, extract or component thereof is orally administered.

5. The method of Claim 1 wherein said oil or pharmacologically active fraction or extract or compound thereof or a formulation comprising the oil or an active fraction, extract or component thereof is in the form of a dried oil powder

6. The method of Claim 1 wherein said oil or pharmacologically active fraction or extract or compound thereof or a formulation comprising the oil or an active fraction, extract or component thereof is in the form of a topical preparation. 49

7. The method of Claim 1 wherein said oil or pharmacologically active fraction or extract or compound thereof or a formulation comprising the oil or an active fraction, extract or component thereof is in the form of a topical cream.

8. The method of Claim 1 wherein the inflammatory condition is selected from the list consisting of: dermatitis, psoriasis, allergic rhinitis, acne, angina, asthma, empysema, pelvic inflammatory disease, pharyngitis, raw or sore throat, redness and rubor.

9. The method of Claim 1 wherein the aesthetic condition is selected from the list consisting of skin aging, wrinkles, skin texture, skin cracking, skin blemishes, skin color, acne, amongst many others which are treatable or have symptoms which can be alleviated by the use of an anti-inflammatory agent.

10. The method of Claim 1 wherein the aesthetic condition is photoaging or a symptom of sun exposure.

11. The method of any one of Claims 1 to 5 wherein said oil is from a nut of the Canarium tree.

12. A composition comprising an oil from a Canarium tree or horticultural relative thereof, and/or one or more pharmacologically active fractions, extracts or components of said oil or a formulation comprising the oil or an active fraction, extract or component thereof, together with a pharmaceutically acceptable carrier, diluent and/or excipient when used for the treatment of a condition selected from a non-arthritic inflammatory condition, an arthritic condition, an aesthetic skin condition and a muscle injury.

13. The composition of Claim 12 wherein said composition is a dermatological composition. - 50

14. The composition of Claim 12 wherein the composition is a sun lotion or sun cream.

15. The composition of Claim 12 wherein the composition is a formulation for use after, during or prior to exposure to UV.

16. The pharmaceutical composition of Claim 12 wherein said pharmaceutically acceptable carrier, diluent or excipient is a bland oil.

17. The pharmaceutical composition of Claim 16 wherein said bland oil is olive oil.

18. The pharmaceutical composition of Claim 12 wherein said pharmaceutically acceptable carrier, diluent or excipient is sorbolene cream.

19. The pharmaceutical composition of Claim 12 wherein the oil or fraction, extract or component is in a free flowing dried powder form.

20. The pharmaceutical composition of Claim 12, wherein said composition further comprises a percutaneous enhancer.

21. The pharmaceutical composition of Claim 20, wherein said percutaneous enhancer is selected from the list of salicylate and azaleate esters, N-methyl pyrrolidine, ceraphyls, isopropanol and cineole.

22. The pharmaceutical composition of Claim 21, wherein said percutaneous enhancer is cineole.

23. The pharmaceutical composition of any one of Claims 12 to 22, wherein said composition further comprises an emulsifier. 51

24. The composition of any one of Claims 12 to 22, wherein said pharmaceutical composition further comprises a preservative.

25. The composition of any one of Claims 12 to 22, wherein said pharmaceutical composition further comprises an antioxidant.

26. The composition of Claim 25 wherein the antioxidant is vitamin E acetate.

27. The composition of any one of Claims 12 to 26 wherein said oil is from Canariumi nut oil.

28. A method for treating a condition selected from a non-arthritic inflammatory condition, an arthritic condition, an aesthetic skin condition and a muscle injury, an inflammatory or aesthetic condition in a subject, said method comprising administering to said subject the pharmaceutical composition of any one of Claims 12 to 27.

29. The method of Claim 25 wherein the inflammatory condition is selected from the list consisting of: dermatitis, psoriasis, allergic rhinitis, acne, photoaging, angina, asthma, empysema, pelvic inflammatory disease, pharyngitis, raw or sore throat, redness and rubor.

30. The method of Claim 25 wherein the aesthetic condition is selected from wrinkles, blemishes, skin cracking, skin coloration and skin texture.

31. Use of active fraction of Ngali nut oil in the manufacture of a medicament for the treatment of inflammation.

32. Use of Ngali nut oil or an active fraction thereof in the manufacture of a medicament for repairing or preventing DNA damage due to sun exposure. 52

33. A method of conducting business said method comprising receiving an order for Ngali nut oil or an active fraction thereof or an isolated component therefrom, supplying the Ngali nut oil or fraction or component for use in a formulation prepared by a third party and receiving payment for said Ngali nut oil or fraction or component and optionally a fee component from the sale of the formulation by the third party.