The invention relates to the use of Vitamin E and, particularly, isomers thereof to treat respiratory diseases such as, but not limited to, obstructive/restrictive airway disorders, asthma, chronic obstructive pulmonary disease, bronchitis, fibrosis, respiratory distress syndrome, and other inflammatory lung diseases; a method of treating said diseases employing the use of Vitamin E and, particularly, isomers thereof; a combination therapeutic comprising said Vitamin E and, particularly, isomers thereof, and at least one other therapeutic or bioactive molecule, for treating respiratory diseases; a composition or supplement comprising Vitamin E and, particularly, isomers thereof; and an inhaler comprising Vitamin E and, particularly, isomers thereof for treating respiratory diseases.

Introduction

Vitamin E is a group of eight fat-soluble compounds that includes both tocopherols and tocotrienols. Tocopherols are the predominant forms of vitamin E. There are four different tocopherol homologues/isomers (alpha, beta, gamma, delta) and four different tocotrienol homologues/isomers (alpha, beta, gamma, delta). The generic formula of the tocotrienol homologues/isomers is shown below along with the chemical structure of each honiologue/isomer.

Chemical structure of tocotrienols. a: R ¹ = R ² = R ³ = CH ₃ ; β: R ¹ = R ³ = CH ₃ , R ² = H; γ. R ¹ = H, R ² = R ³ = CH ₃ ; δ: R = R ² = H, R ³ = CH ₃ .

All of the tocopherols and tocotrienols have antioxidant activity to varying extents. However, more recently it has been discovered that the different vitamin E homologues also have other biological activities unrelated to their antioxidant activity. -These other-aetivities inelude-m gene expression, and neurological function(s). It has also been suggested that the most important function of vitamin E is in cell signalling.

Vitamin E is incorporated into cell membranes where it protects from oxidative damage. As an antioxidant, vitamin E acts as a peroxyl radical scavenger, preventing the propagation of free radicals in tissues, by reacting with them to form a tocopheryl radical, which is then reduced by a hydrogen donor (such as vitamin C) and thus returned to its reduced state. a-Tocopherol is an important lipid-soluble antioxidant. As an enzymatic activity regulator, Vitamin E has been implicated in protein kinase C (PKC) activity which can be inhibited by oc-tocopherol. oc-Tocopherol has a stimuJator-y.effect n--the-dephosphor lation--enzyme;-protein-phosphatase ^' 2A, ^" Which cleaves phosphate groups from PKC, leading to its deactivation.

Vitamin E also has an effect on gene expression. Treatment with oc-tocopherol was found to downregulate the expression of the CD36 scavenger receptor gene and the scavenger receptor class A (SR-A) gene and modulate expression of the connective tissue growth factor (CTGF) gene. Thus Vitamin E has been implicated in both atherogenetic tissue (down regulation of CD36) and the repair of wounds and regeneration of the extracellular tissue lost or damaged during atherosclerosis (CTGF modulation).

Vitamin E also has a role to play in neurological functions and the inhibition of platelet aggregation. Moreover, Vitamin E also protects lipids and prevents the oxidation of polyunsaturated fatty acids.

So far, most human dietary supplement studies have used only oc-tocopherol. This can affect levels of other forms of vitamin E, e.g. reducing serum γ- and δ- tocopherol concentrations.

Vitamin E isomers are usually derived from palm oil but can also be derived from rice bran, annatto, barley and other natural sources or synthesized from precursors or metabolites of Vitamin E. The isomers of the invention can be derived from any of these natuFal-sources-Alternativelyrsynthetic isomers rmay be manufactured.

While it was initially hoped that vitamin E supplements would have a positive effect on health, research has not supported these expectations. Vitamin E does not decrease mortality in adults, even at large doses and may even slightly increase it. Vitamin E does not improve blood sugar control in diabetes mellitus or decrease the risk of stroke. Daily supplementation of vitamin E does not decrease the risk of prostate cancer and may even increase it. A Japanese study in 2012 found that vitamin E may contribute to osteoporosis. Moreover, a 2007 clinical study involving a-tocopherol concluded that this supplement did not reduce the risk of major cardiovascular events in middle-aged and older men.

Compared with the tocopherols, the tocotrienols are sparsely studied. Less than 1 % of PubMed papers on vitamin E relate to tocotrienols (1 ). Some studies have suggested that tocotrienols have specialized roles in protecting neurons from damage (1 ), others have shown a reduction in cholesterol (2) by inhibiting the activity of HMG-CoA reductase; δ-tocotrienol blocks processing of sterol regulatory element-binding proteins (SREBPs); others have suggested certain isomers may have a role to play in asthma treatment (4) although, notably, this latter study was undertaken in a non-asthmatic human cancer lung cell model and so the suggestion is highly speculative. Oral consumption of tocotrienols is also thought to protect against stroke-associated brain damage in vivo (3).

Others have developed combination therapeutics, where a tocotrienol isomer is combined with a plant extract to promote general health and well-being; see WO201 1 150312. Others have stated tocotrienol isomers can be used to treat or prevent conditions such as fever, edema, diabetes mellitus, cancer, signs of aging, pain, rheumatoid diseases, septic shock, chronic fatigue syndrome, functio laesa, reduce lipogenesis, increase the HDULDL cholesterol ratio, reduce total serum cholesterol, low density lipoprotein-cholesterol, coronary artery disease, aggregation of blood platelets, blood coagulation, fibrinolysis, chronic inflammation, such as that associated with rheumatoid disease, and immunoregulatory diseases, such as autoimmune diseases and so, given this breadth of action, also promote general health and well-being; see US6239171. We have recently tested the potential beneficial effects of tocotrienol isomers in a house dust mite (HDM)-induced mouse asthma model. HDM is a major allergen that causes allergic responses in allergic asthma in patients. We observed a strong inhibition of HDM-induced inflammatory cell counts, especially eosinophil count, in bronchoalveolar lavage (BAL) fluid brought about by tocotrienol isomers, especially the γ-tocotrienol isomer and the δ-tocotrienol isomer. In addition, tocotrienol isomers, specifically the a-tocotrienol isomer, the γ-tocotrienol isomer and the δ- tocotrienol isomer significantly suppressed HDM-induced reactive oxygen species escalation in the BAL fluid. More specifically, γ-tocotrienol significantly suppressed HDM-induced increases in allergic cytokines like IL-4, IL-5 and KC, G-CSF and RANTES in BAL fluid; inflammatory cell infiltration in the airways, and oxidative damage markers in the airways. Further, γ-tocotrienol was found to upregulate the expression-of- anti-oxidative -enzyme -superoxide- dismutase (SOD) to- ameliorate oxidative stress in the airways. In addition, using a cigarette induced COPD mouse model, we observed a similar inhibition of inflammatory cell count and observation of markers of oxidative damage.

Taken together our findings show for the first time that the tocotrienol isomers, especially γ-tocotrienol and δ-tocotrienol, are therapeutically active as anti- inflammatory and anti-oxidative agents, notably, in a clinically relevant HDM allergen-induced and oxidative stress-induced obstructive airway disorder model such as asthma and COPD. Moreover, given the underlying mechanisms of action, our findings strongly imply the tocotrienol isomers will be effective in treating bronchitis, fibrosis and other obstructive and restrictive airway disorders.

Current standard treatments for inflammatory lung diseases include inhaled bronchodilators (e.g. salbutamol) and inhaled steroids. These medications must be prescribed by a trained physician as they have known adverse side effects. Vitamin E isomers including tocotrienols are commonly found in our diet such as in rice bran, barley and palm oil etc. They are therefore proven to be safe for consumption over long periods of time and through multiple generations. This is a major advantage of using tocotrienol isomers to treat respiratory diseases compared to prescription medicines and even over natural products which are not commonly consumed or found in our diet. Statements of Invention

According to a first aspect of the invention there is provided at least one tocotrienol isomer for use in the treatment or prevention of a respiratory disease.

Alternatively, according to a first aspect of the invention there is provided the use of at least one tocotrienol isomer in the manufacture of a medicament to treat or prevent a respiratory disease.

Preferably said disease is a reactive/obstructive airway disorder.

Reference herein to a reactive/obstructive airway disorder is to a condition characterised by inflammation and/or oxidative stress in lung or airway tissue and it is typically, but not exclusively, initiated by allergens, pollutants, radiation, cigarette smoke, chemicals and medications.

In a further preferred embodiment of the invention said respiratory disease is selected from the group comprising reactive/obstructive airway disorder, asthma, COPD, bronchitis, fibrosis, respiratory distress syndrome, and other airway disorders.

In a preferred embodiment of the invention said isomer is selected from the group comprising the cr-tocotrienol isomer, the γ-tocotrienol isomer and the δ-tocotrienol isomer.

In yet a further preferred embodiment of the invention said isomer is either, or both, the γ-tocotrienol isomer and the δ-tocotrienol isomer. Ideally said isomer is the γ- tocotrienol isomer.

In yet a further preferred embodiment of the invention said isomer is any selected combination of two or more of the following three isomers: ar-tocotrienol isomer, γ- tocotrienol isomer and δ-tocotrienol isomer. In a further preferred embodiment of the invention said isomer is provided as a prodrug or in a derivatised form. Thus, in the former instance the isomer is included in a prodrug that exists in a relatively inactive form and is converted into an active form, i.e. the isomer can perform its beneficial effects on airways tissue, following activation of said prodrug, typically but not exclusively, as a result of the body's normal metabolic processes. In the latter instance, the isomer is provided in a derivatised form and so in the form of an ester or a synthetic molecule.

More preferably said isomer is in the form of Vitamin E and, optionally, an acceptable carrier.

The carrier, or, if more than one be present, each of the carriers, must be acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient.

In yet a further preferred embodiment of the invention said Vitamin E or said isomers are usually derived from palm oil but it or they can also be derived from rice bran, annatto, barley and other natural sources, alternatively, it or they is/are synthesized from precursors or metabolites of Vitamin E.

Yet more preferably still said isomer is formulated for oral administration or inhalation. Alternatively, the isomers are formulated for intravenous or transdermal administration.

The formulations may be prepared by any methods well known in the art of pharmacy.

Preferred compositions are formulated for oral, nasal or bronchial administration.

The composition may be prepared by bringing into association the isomer of the invention and the carrier. In general, the formulations are prepared by uniformly and intimately bringing into association the isomers with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product. The invention extends to methods for preparing a pharmaceutical composition comprising bringing an isomer of the invention in conjunction or association with a pharmaceutically or veterinarily acceptable carrier or vehicle.

Formulations for oral administration in the present invention may be presented as: discrete units such as capsules, sachets or tablets each containing a predetermined amount of the isomer; as a powder or granules; as a solution or a suspension of the active agent in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water in oil liquid emulsion; or as a bolus etc.

For compositions for oral administration (e.g. tablets and capsules), the term "acceptable carrier" includes vehicles such as common excipients e.g. binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone (Povidone), methylcellulose, ethylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, sucrose and starch; fillers and carriers, for example corn starch, gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride and alginic acid; and lubricants such as magnesium stearate, sodium stearate and other metallic stearates, glycerol stearate stearic acid, silicone fluid, talc waxes, oils and colloidal silica. Flavouring agents such as peppermint, oil of wintergreen, cherry flavouring and the like can also be used. It may be desirable to add a colouring agent to make the dosage form readily identifiable. Tablets may also be coated by methods well known in the art.

A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine said isomer in a free flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface-active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active agent. Other formulations suitable for oral administration include lozenges comprising the active agent in a flavoured base, usually sucrose and acacia or tragacanth; and pastilles comprising the active agent in an inert base such as gelatin and glycerin, or sucrose and acacia.

Isomers of the invention may be used for the treatment of the respiratory tract by nasal, bronchial or buccal administration of, for example, aerosols or sprays which can disperse the pharmacological active ingredient in the form of a powder or in the form of drops of a solution or suspension. Pharmaceutical compositions with powder-dispersing properties usually contain, in addition to the active ingredient i.e. said isomers, a liquid propellant with a boiling point below room temperature and, if desired, adjuncts, such as liquid or solid non-ionic or anionic surfactants and/or diluents. Pharmaceutical compositions in which the pharmacological active ingredient, i.e. said isomers, is in solution contain, in addition to this, a suitable propellant, and furthermore, if necessary, an additional solvent and/or a stabiliser. Instead of the propellant, compressed air can also be used, it being possible for this to be produced as required by means of a suitable compression and expansion device.

For topical application to the skin, isomers or compositions comprising same may be made up into a cream, ointment, jelly, solution or suspension etc. Cream or ointment formulations that may be used for the isomers are conventional formulations well known in the art, for example, as described in standard text books of pharmaceutics such as the British Pharmacopoeia.

According to a further aspect of the invention there is provided a method of treatment comprising administering to an individual, or patient with a respiratory disease, at least one tocotrienol isomer. According to a further aspect of the invention there is provided a combination therapeutic comprising either at least one tocotrienol isomer in combination with at least one further therapeutic or bioactive molecule that is effective at treating or preventing a respiratory disease. In a preferred embodiment of this aspect of the invention said at least one further therapeutic is effective at treating at least one symptom of, or aspect of the physiological condition giving rise to, said respiratory disease.

Most preferably said symptom is selected from the group comprising: wheezing, shortness of breath, chest tightness, coughing and sputum production.

Most preferably said aspect is selected from the group comprising: increase in inflammatory cell count, especially eosinophil and neutrophil counts; increase in reactive oxygen and nitrogen species; increase in inflammatory cytokines such as IL-4, IL-5 and KC, G-CSF; increase in chemokines such as RANTES; increase in inflammatory cell infiltration in the airways, increase in oxidative damage markers in the airways and a decrease in anti-oxidative enzymes such as superoxide dismutase (SOD).

According to a further aspect of the invention there is provided an inhalation device comrising or including said at least one tocotrienol isomer, or Vitamin E, or said combination therapeutic.

Any of the aforementioned aspects of the invention may, in preferred embodiments, include or be characterised by any of the aforementioned features.

In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprises", or variations such as "comprised" or "comprising" is used in an inclusive sense i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention. All references, including any patent or patent application, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. Further, no admission is made that any of the prior art constitutes part of the common general knowledge in the art. Pjreferred_features-of-eaGh-aspeet— o heHnvention ^{^} may be ^{^} as ^{^} described in connection with any of the other aspects.

Other features of the present invention will become apparent from the following examples. Generally speaking, the invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including the accompanying claims and drawings). Thus, features, integers, characteristics, compounds or chemical moieties described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein, unless incompatible therewith.

Moreover, unless stated otherwise, any feature disclosed herein may be replaced by an alternative feature serving the same or a similar purpose.,

The invention will now be described by way of example only with reference to the following figures, wherein:-

Figure 1 shows the effects of tocotrienols onHDM-lnduced inflammatory cell counts in BAL fluid;

Figure 2 shows the effect of varying concentrations of γ-tocotrienol on HDM-lnduced inflammatory cell counts in BAL fluid;

Figure 3 shows the effects of tocotrienols on HDM-induced Reactive Oxygen Species level in BAL fluid;

Figure 4 shows the effects of γ-tocotrienol on HDM-induced inflammatory cell infiltration and airway epithelial swelling in the mouse airways;

Figure 5 shows the effects of γ-tocotrienol HDM-induced mucus secretion in the lungs ; Figure 6 shows the effects of γ-tocotrienol on HDM-induced cytokine and chemokine levels in BAL fluid levels of IL-4, IL-5, IL-17, IL-1a, IL-12, IFN-γ, G-CSF and RANTES KC is a mouse analogue of IL-8 in human;

Figure 7 shows the effects of γ-tocotrienol on oxidative damage marker levels in the HDM-induced asthmatic airways. [A] Effect upon the lipid oxidative marker 8-isoprostane. [B] Effect upon the nucleic acid oxidative marker 8-hydroxy-2-deoxyguanosine. [C] effect upon the protein oxidative marker 3-nitrotyrosine;

Figure 8 shows the effects of γ-tocotrienol on superoxide dismutase expression in HDM- induced asthmatic airways;

Figure 9 shows the effects of γ-tocotrienol on airway hyperresonsiveness assessed using

[A] airway resistance and [B] dynamic compliance induced by methacholine;

Figure 10 shows the effect of γ-tocotrienol upon the expression of the transcription factors

NF- B and Nuclear factor-erythroid 2 related factor 2 (Nrf2);

Figure 1 1 shows the effects of γ-tocotrienol on cigarette smoke induced inflammatory cell counts in BAL fluid; and

Figure 12 shows the effects of γ-tocotrienol on oxidative damage marker levels in the cigarette smoke-induced COPD model lung airways. [A] Effect upon the lipid oxidative marker 8-isoprostane. [B] Effect upon the nucleic acid oxidative marker 8- hydroxy-2-deoxyguanosine. [C] Effect upon the protein oxidative marker 3- nitrotyrosine

Materials & Methods

HDM-lnduced Animal Asthma Model

The development of house dust mite mouse asthma model used female BALB/c mice, which were anaesthetized using isoflurane (Halocarbon Products Corporation, River Edge, NJ, USA) and then administered through intratracheal route with either 40 μΙ of Dermatophagoides pteronyssinus extracts (100 mg, Greer Laboratories, Lenoir, NC, USA) or saline as a negative control on days 0, 7 and 14 as described

[5]. Tocotrienol (30, 100, or 300 mg/kg), vehicle emulsifier for tocotrienol (3%) or prednisolone (10 mg/kg) in 0.2 ml reverse-osmosis water was administered via oral gavage on days 7, 8, 9, 14, 15 and 16. Mice were sacrificed on day 17 and BAL fluid was collected for total and differential cell counts as described below. Treatment groups were: naive (background control); saline (negative control); house dust mite [HDM] (positive control); house dust mite/vehicle [HDM/Veh] (vehicle control); house dust mite/yT3 [HDM/yT3] (drug treatment); and house dust mite/prednisolone [HDM/Pred] (positive drug control). Cigarette smoke (CS)-lnduced Animal COPD Model

The development of cigarette smoke (CS) induced mouse COPD model used female BALB/c mice, which were anaesthetized using isoflurane (Halocarbon Products Corporation, River Edge, NJ, USA) and then administered through

Health Research Institute, University of Kentucky, Lexington, KY, USA] 3 sticks three times daily at 2 hour intervals or sham air as a negative control on days 0 and 1 1. Tocotrienol (30, 100 or 300 mg/kg), vehicle emulsifier for tocotrienol (3%), prednisolone (5 or 10 mg/kg) or tocotrienol and prednisolone (30 + 5 mg/kg) was administered via air on days 9, 10 and 1 1. Mice were sacrificed on day 12 and BAL fluid was collected for total and differential cell counts as described below. Treatment groups were: naive (background control); saline (negative control); cigarette smoke [CS] (positive control); cigarette smoke/vehicle [CS/Veh] (vehicle control); cigarette smoke/yT3 [CS/yT3] (drug treatment); cigarette smoke/prednisolone [CS/Pred] (positive drug control); and cigarette smoke/ vT3+prednisolone [CS/ yT3+Pred] (combinatorial treatment).

Bronchoalveolar lavage fluid

Mice were anesthetized 24 h after the last dose of drug or water administration and bronchoalveolar lavage (BAL) was performed as previously described [6]. Briefly, tracheotomy was performed, and a cannula was inserted into the trachea. Ice-cold PBS (0.5 ml X 3) was instilled into the lungs, and BAL fluid was collected. BAL fluid total and differential cell counts were determined. BAL fluid supernatants were stored at - 80 °C for subsequent analysis.

Levels of dichlorofluorescin diacetate (DCF-DA) in BAL fluid

BAL fluid cells suspended in RPMI was transferred into a new tube, which contained 20 μΜ of DCF-DA (Sigma-aldrich; St. Louis, MO, USA). The tube was left in a 37 °C incubator for 20 mins and then subjected to centrifugation of 10,000 rpm for 5 mins. Supernatant was removed and the cell pellet was homogenized via medium vortex after the addition of 100 μΙ RPMI in the tube. Subsequently, the solution was pipetted into a black flat-based Greiner 96-well plate and the readings measured with a fluorescence plate reader (492 nm excitation and 525 nm emission).

Histologic analysis

Lungs were fixed in 10% neutral formalin, paraffinized, cut into 5 μιη sections, and stained with hematoxylin and eosin (H&E) for examining cell infiltration. Fluorescence Microscopy

Lungs were collected 24 h after last HDM challenge, fixed in 10% neutral formalin, paraffinized, cut into 5-μ ^ηη sections, and stained with periodic acid-fluorescence Schiff stain (PAFS) for mucus production. PAFS allows visualization of mucus through covalent bonding of sulfited acriflavine to mucin glycoconjugates. Mucin granules emit red fluorescence when excited at 380-580 nm and observed at 600- 650 nm using a confocal microscope. Levels of cytokines and chemokines in BAL fluid

BAL fluid levels of IL-4, IL-5, IL-17, IL-1a, IL-12, IFN-γ, G-CSF and RANTES were measured using a multiplex ELISA, obtained from Biorad (Hercules, CA, USA).

Levels of 3-Nitrotyrosine (3-NT), 8-hvdroxy-deoxy-quanosine (8-OHdG) and 8- isoprostane in lung tissues and BAL fluid

Lung levels of 3-NT were measured using an enzyme immunoassay (Cell Biolabs Inc.; San Diego, CA, USA) according to the manufacturer's instructions. Briefly, lung tissues were snap-frozen in liquid nitrogen and lyophilized using a freeze-dryer (Labconco Corp., Kansas City, MO, USA) at - 85 °C. Lyophilized lung tissues were homogenized in hepes solution using a glass bead-based homogenizer (Tissuelyser LT; Qiagen, Valencia, CA, USA). The supernatants were assayed for 3-NT levels. BAL fluid levels of 8-OHdG and 8-isoprostane were measured using enzyme immunoassay (Cayman Chemical, Ann Arbor, Ml, USA) according to the manufacturer's instructions.

Measurements of superoxide dismutase (SOD) activity in lung tissues

SOD activity in lung tissues was determined using enzymatic assay kits (Cayman Chemical) according to the manufacturer's instructions. Briefly, lung tissues were snap-frozen in liquid nitrogen, lyophilized, and homogenized. Supernatants were determined for enzymatic activities.

Measurement of Airway Hyper-responsiveness Mice were-anesthetized^and-traeh^ The trachea was intubated with a cannula that was connected to the pneumotach, ventilator and nebulizer. Lung resistance (Rl) and dynamic compliance (Cdyn) in response to nebulized methacholine (0.5-8.0 mg/ml) were recorded using FinePointe™ data acquisition and analysis software (Buxco, Wilmington, NC, USA). Results are expressed as a percentage of the respective basal values in response to PBS.

Immunoblottinq of NF- Β and Nrf2 To determine NF-κΒ nuclear translocation, lung nuclear extracts (10 μg per lane) were separated by 10% SDS-PAGE, and immunoblots were probed with anti-p65 (Cell Signaling, Beverly, MA, USA) and anti-TATA binding protein (TBP, Abeam, Cambridge, UK). For Nrf2 protein analysis, nuclear proteins (30 μg per lane) were separated by 10% SDS-PAGE and immunoblots were probed with anti-Nrf2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), or anti-TATA box binding protein (TBP, Abeam) antibody. Band intensity was quantitated using ImageJ software. Results

Figure 1 shows that house dust mite induced inflammatory cell counts, in BAL fluid, can be ameliorated using the α, γ and δ tocotrienol isomers. Moreover, the eosinophil content of the induced inflammatory cell count is particularly reduced using these isomers, especially the γ and δ tocotrienol isomers.

Figure 2 shows that the effect of tocotrienol isomers on house dust mite induced inflammatory cell counts, as exemplified using γ-tocotrienol, is dose dependent.

Figure 3 shows that effects of tocotrienols on house dust mite induced Reactive Oxygen Species levels in BAL fluid is reduced following treatment with the α, γ and δ tocotrienol isomers, especially the γ and δ tocotrienol isomers. Figure 4 sjTpjwsJhaLhouse^dust-mite-indueedHnflamm

airway epithelial swelling in the mouse airways is reversed by treatment with γ- tocotrienol. Figure 5 shows that house dust mite induced mucus secretion in mouse airways is reduced by treatment with γ-tocotrienol.

Figure 6 shows that house dust mite induced cytokine levels in BAL fluid, particularly the levels of IL-4, IL-5, and G-CSF are reduced by treatment with γ- tocotrienol, further the levels of the chemokine RANTES KC [a mouse analogue of IL-8 in human] is also reduced by treatment with γ-tocotrienol.

Figure 7 shows that oxidative damage ^ markerjevels.in .the house dust- mite-induced asthmatic airways is reduced by treatment with γ-tocotrienol.

Figure 8 shows that superoxide dismutase expression [an oxidative damage marker] in HDM-induced asthmatic airways is reduced by treatment with γ- tocotrienol. Figure 9 shows that γ-tocotrienol alleviates airway hyperresponsiveness in HDM- induced airway models, with reduced airway resistance [figure 9a] and improved dynanmic compliance [figure 9b].

Figure 10 shows that γ-tocotrienol reduces the expression of NF-κΒ, a pro- inflammatory transcription factor [figure 10a], and increases expression of Nuclear factor-erythroid 2 [figure related factor 2 (Nrf2) which increases expression of antioxidant enzyme and cytoprotective proteins [figure 10b].

Figure 11 shows using a cigarette induced COPD model that inflammatory cell counts in BAL fluid can be ameliorated using tocotrienol isomers as exemplified using γ-tocotrienol, and also with improved synergy combined with prednisolone. Figure-J-2a^G-shows-that-Gxidatwe-damage ^~ marker levels in the cigarette induced COPD airways is reduced by treatment with γ-tocotrienol.

DISCUSSION

Our findings show for the first time that the tocotrienol isomers, especially γ- tocotrienol and δ-tocotrienol, are therapeutically active as anti-inflammatory and anti-oxidative agents for allergen-induced inflammatory- and oxidative stress- induced reactive/obstructive airway disorders such as asthma, COPD, bronchitis and, indeed, other obstructive airway disorders.

References

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3. Khanna, S.; Roy, S.; Slivka, A.; Craft, T. K.S.; Chaki, S.; Rink, C; Notestine, M. A.; Devries, A. C. et al. (2005). "Neuroprotective Properties of The Natural Vitamin E a-Tocotrienol". Stroke 36 (10): 2258-64.

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