TREATMENT OF DIABETES AND COMPLICATIONS THEREOF AND RELATED

DISORDERS

PRIORITY CLAIM

This application claims priority to Australian provisional patent application 2008902922, the contents of which are hereby incorporated by reference.

FIELD

The present invention relates to methods and compositions for inhibiting PKC enzymes, and to methods and compositions for inhibiting Nuclear Factor Kappa B. The present invention also relates to the treatment of PKC-β mediated diseases and Nuclear Factor Kappa B mediated diseases including, for example, diabetes and complications thereof and atherosclerosis.

BACKGROUND

PKC is a serine/ threonine protein kinase which regulates a diverse range of cellular processes in an isozyme-spedfic manner. It plays a role in receptor desensitization, membrane structure, regulation of transcription, immune responses, and cell growth.

Eleven isozymes have been identified which are classified into classical (a, 61,, β2, γ), novel (δ, ε, η, μ, θ), and atypical (L, λ, ι) forms depending on their sensitivity to diacylglycerol (DAG) and calcium. Classical PKCs are activated by calcium, DAG, phosphatidylserine (PS), unsaturated fatty acids and phorbol 12-myristate 13-acetate (PMA). Novel PKCs are activated by DAG, PS, unsaturated fatty acids and phorbol esters such as PMA. Atypical PKCs are activated by PS and phosphatidylinositides.

When PKC is in the inactive state, the kinase domain is masked by the folding back of a pseudosubstrate domain. Diacylglycerol (DAG) enhances PICC activity by removing

the pseudosubstrate from the kinase domain and increasing the affinity of PKC for the phospholipid cefaclors. DAG can be formed through the hydrolysis of phosphatidylinositol 4,5 bisphosphate (P1P2), or through the de nβvo synthesis via glycerol-3-phosphate and phosphatidic acid. In unstimulated cells, PKC is generally found in the soluble fractions and it translocates to the particulate fraction such as cellular membranes upon stimulation where it associates with proteins such as RACKs (receptor for activated C kinase) and STICKS (substrates that interact with C kinases).

Nuclear factor kappa B (NFKB) is a cytoplasmic transcription protein and is an important mediator of the inflammatory response. It regulates the expression of interleukins, growth factors and cytokines, cell adhesion receptors and genes which regulate apo ptosis.

Recent observations that both glucose and advanced gly cation end-products (ACJE) stimulate NFKB activation in a variety of cell-types implicate N FKB as a mediator of macrovascular and microvascular complications.

Both PKC and NFKB have been implicated in a number of diseases, conditions and states, such as diabetes and complications thereof, microvascular and macrovascular complications, retinopathy, nephropathy, and atherosclerosis. The effective prevention and/or treatment of such diseases, conditions and states remains problematic.

Accordingly, new approaches are required to preventing and/or treating these diseases, conditions and states.

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

DESCRIPTION

The present invention arises from the investigation of particular compounds in activation of protein kinase C enzymes and the activation of N FK[I

PKC is a serine/threonine protein kinase. Eleven isozymes have been identified which are classified into classical (a, βl, 62,, γ), novel (&,, ε, η, μ, θ), and atypical (C λ, ι) forms depending on their sensitivity to diacylglycerol (DAG) and calcium. Classical PKCs are activated by calcium, DAG,. phosphatidylserine (PS), unsaturated fatty acids and phorbol 12-τnyristate 13-acetate (PMA). Novel PKCs are activated by DAQ. PS, unsaturated fatty acids and phorbol esters such as PMA. Atypical PKCs are activated by PS and phosphatidyiinositides.

PKC regulates a diverse range of cellular processes in an isozyme-specifie manner. It plays a role in receptor desensitization, membrane structure, regulation of transcription, immune responses, and cell growth.

When PKC is in the inactive state, the kinase domain is masked by the folding back of a pseudosubstrate domain. Diacylglycerol (DAG) enhances PICC activity by removing the pseudosubstrate from the kinase domain and increasing the affinity of PKC" for the phospholipid cofaetors, DAG can be formed through the hydrolysis of phosphatidylinositol 4,5 bisphosphate (P1P2), or through the άc novo synthesis via glycerol-3-phosphate and phosphatidic acid. Dc novo synthesis is the major mechanism by which DAG is increased in response to hyperglycemia and it has been shown that this elevation remains despite a reduction in glucose concentration. In unstimulated cells, PKC is generally found in the soluble fractions and it translocates to the particulate fraction such as cellular membranes upon stimulation where it associates with proteins such as RACKs (receptor for activated C kinase) and STICKS (substrates that interact with C kinases).

PKC enzymes are generally as described in Protein Kinase C (Second Ed, ed. By Lodwijk

V. Dekker, KIu wer Academic/Plenum Publishers, 2004)

In some embodiments, the present invention is directed to inhibition of PKC-β enzymes. The term "PKC-β enzyme" as used throughout the specification is to be understood to mean a member of the classical isozymes of the PKC family of enzymes, being enzymes that are stimulated when diacylglycerol (DAC) and Ca ^2π accumulate in appropriately stimulated cells.

Examples of PKC-β enzymes include the βl and β2 isoform s. The human β1 isoform is provided in GenBank accession number NP_997700. The human β2 isoform is provided in GenBank accession number NP__002729, Other PKC-β enzymes may identified by a method known in the art. Generally, a PKC-β enzyme has greater than

90% similarity at the amino acid level to human PKC -βl. For example, the level of similarity with the human PKC-β 1 enzyme from the PKC-βl enzymes from other species is as follows: dog (XP_547088>- 94.7%; rat (NP_036845) - 98.7%; mouse

(NP _.. 032881)- 98.8%, Other PKC-β enzymes may be identified by methods known in the art.

In accordance with the present invention it has been found that particular compounds, salts or solvates thereof may inhibit activation of PKC enzymes.

Accordingly, in one aspect, the present invention provides a method of inhibiting activation of a PKC enzyme in a cell, the method including exposing the cell to an effective amount of a compound, or a salt or solvate thereof, selected from the group consisting of (i) a polyunsaturated fatty acid including an oxa and/or thia substitution at either or both of the β or γ position of the polyunsaturated fatty acid; (ii) a polyunsaturated fatty acid covalently coupled to an amino acid; (iii) a polyunsaturated nitroalkene or nitroalkyne; (iv) a n-3 polyunsaturated fatty acid; and (v) a n-6 polyunsaturated fatty acid.

In some embodiments, the PKC enzyme in the various embodiments of the present

invention is a β isoform, In some embodiments, the PKC-β enzyme is PKC-βl and/or PKC-βZ

In some embodiments, the cell is a human or an animal cell. Examples of animal cells include cells from the following: a mammal, a primate cell, a livestock animal (eg a horse, a cow, a sheep, a pig, or a goat), a companion animal (eg. a dog, a cat), a laboratory test animal (eg. a mouse,, a rat, a guinea pig, a bird, a rabbit), an animal of veterinary significance, or an animal of economic significance.

In some embodiments, the cell is a retinal cell, a kidney cell, a pancreatic cell, a vascular cell, or a cell lining a blood vessel. Examples of cell types include endothelial cells and mesangial ceils.

The cell in the various embodiments of the present invention may be a cell present in vivo or in vitro.

In some embodiments the cell may be part of a biological system, such as a cell present in vivo, for example a cell that is associated with a PKC-β mediated disease, condition or state. In this regard, the term "biological system" is to be understood to mean any multi-cellular system and includes isolated groups of cells to whole organisms. For example, the biological system may be a tissue or organ, or an entire subject.

The term ''subject" as used throughout the specification is to be understood to mean a human or animal subject. In this regard, it will be understood that the present invention includes within its scope veterinary applications. For example, the animal subject may be a mammal, a primate, a livestock animal (eg. a horse, a cow, a sheep, a pig, or a goat), a companion animal (eg. a dog, a cat), a laboratory test animal (eg. a mouse, a rat,, a guinea pig,, a bird, a rabbit), an animal of veterinary significance, or an animal of economic significance.

Known inhibitors of PKC-β act by binding to the ATP-binding site of the kinase.

However, without limiting the present invention to any particular mode of action, the compounds, salts or solvates of the present invention may act by reducing the association of PKC-Ji with the particulate fraction Accordingly, in some embodiments inhibiting activation of a PkC enzyme in a t ell mav include inhibiting transloc ation of a PKC enzyme in a cell from the cytoplasm to the particulate fraction

inhibiting translocation ot a PkC enzyme in a cell from the cytoplasm to the particulate fraction mav represent a new mode of therapeutic action for the treatment ol a number ot diseases, condition and states, including PkC mediated diseases conditions and states.

in this regard, it has also been recognised that the compounds, salts or solvates thereof may be used to prevent and/or treat a number of diseases, conditions and states, including PKC-β mediated diseases, conditions or states in a subject.

Fxamples of PKC-β mediated diseases, conditions or states include (i) diabetes, or a complication thereof, including diabetic microvascular complications and/or diabetic macrovascular complications, diabetic retinopathy, diabetic nephropathy and diabetic uremia, (ii) ischemia/reperfusion injury, and complications thereof, (iii) atherosclerosis; (iv) restenosis; (v) tumour growth; and (vi) undesired or uncontrolled angiogenesis, including tumour-induced angiogenesis.

The subject in the various embodiments of the present invention may be susceptible to, or suffering trom, a disease, condition or state that would benefit from inhibition of a PICC enzyme.

In another aspect, the present invention provides use of a compound, or a salt or solvate thereof, in the preparation ot a medicament tor preventing and/or treating a PKC-Ji mediated disease, condition or state in a subject, the compound being selected from the group consisting of (i) a polyunsaturated fatty acid including an oxa and/or thia substitution at either or both of the β or γ position ot the polyunsaturated fatty

acid, (ii) a polyunsaturated fatty acid covalently coupled to an amino acid; (iii) a polyunsaturated nitroalkene or nilroalkync, (iv) a n-3 polyunsaturated tally acid, and (v) a n-6 polyunsaturated fatty acid.

It has also been found that the compounds, salts or solvates thereof described herein have the ability to inhibit activation ot KF ^' κβ.

Nuclear tactor kappa B (KFKB) is a cytoplasmic transcription protein and is an important mediator of the inflammatory response. It regulates the expression of inter! eukins, growth factors and cytokines, cell adhesion receptors and genes which regulate apoptosis.

Nl'kJB exists as a heterodimer composed of 2 subunits. the most common being p50 (5OkDa) and p65 (65kDa). Other family members include e-rel ReIB and p52. Within the iytoplasm it remains inactive due to its interaction with the inhibirorv protein, Inhibitory kappa B (IκB-α, β or t). Several factors have been recognised m activating KFKB such as the inflammatory mediators I MF, inlerleukin-1 (IL-I), bacterial lipopolysaccharide (LPS), advanced glycalion end products (λGlis), and platelet- activating factor It is also activated by conditions such as hyperglycemia, shear stress, oxidi/ed lipids oxidant stress and hypoxia/reperfusion.

Upon activation, KFKB inducing kinase (KIK) is phosphorylated and activates IKB kinases (IKKl/IKKα and lKK2/lKkβ, XFMCVlKKy) which then phosphorylates IKB at two serine residues, ber 32 and 36. IKB is subsequently ubiquitinated and degraded by the 26S proteasome. MEKkI (rnitogen-activated protein kinase/extraiellular signal- regulated kinase kinase-]), transforming growth factor (TGF)-β-mducible kinase (TAKl), Akt, and protein kinase C-ς and 0 are also recognised to interact with IKKs in the XFKB signalling pathway. NFKB then translocates to the nucleus where it acts to regulate gene transcription (May and Ghosh, 1998)

N ₁ FKB ac tivation is transient due to intracellular regulatory mechanisms. The

resynthesis of IκBα after phosphorylation results in shuttling of NFKB back to the cytoplasm where it is re-coupled with IKB and inactivated.

Accordingly, the present invention also provides a method of inhibiting activation of NF iφ in a cell, the method including exposing the cell to an effective amount of a compound,, or a salt or solvate thereof, selected from the group consisting of (i) a polyunsaturated fatty acid including an oxa and/or tnia substitution at either or both of the β or γ position of the polyunsaturated fatty acid; (ii) a polyunsaturated fatty acid covalently coupled to an amino acid; (iii) a polyunsaturated nitroalkene or nitroalkyne; (iv) a n-3 polyunsaturated fatty acid; and (v) a n-6 polyunsaturated fatty acid.

in some embodiments, the ceil is selected from the group consisting of a retinal cell, a kidney cell, a pancreatic cell, and a vascular cell.

In some embodiments, the cell is a present in a biological system,, such as a human or animal subject, for example a human or animal subject is susceptible to, or suffering from a disease, condition or state associated with activation of NFiφ, or a disease, condition or state that would benefit from inhibition of activation of NF Kβ.

In some embodiments, the disease, condition or state is selected from one or more of the group consisting of (i) diabetes, or a complication thereof, including diabetic microvascular complications and/or diabetic macrovascular complications, diabetic retinopathy, diabetic nephropathy and diabetic uremia; (ii) ischemia/reperfusion injury, and complications thereof; (iii) atherosclerosis; (iv) restenosis; (v) tumour growth; (vi) undesired or uncontrolled angiogenesis, including tumour-induced angiogenesis; (vii) inflammatory disease; and (viii) cardiovascular disease.

The present invention also provides a method of preventing and/or treating in a subject one or more of (i) diabetes, or a complication thereof; (ii) ischemia/reperfusion injury, and complications thereof; (iii) atherosclerosis; (iv) restenosis; (v) tumour growth; and

(vi) undesired or uncontrolled angiogenesis; the method including administering to the

subject an effective amount of one or more compounds, or salts or solvates thereof, selected from the group consisting of (i) a polyunsaturated fatty acid including an oxa and/or thia substitution at either or both of the β or γ position of the polyunsaturated fatty acid; (ii) a polyunsaturated fatty acid eovalently coupled to an amino acid; (in) a polyunsaturated mtroaJkene or nitroalkyne; (iv) a n-3 polyunsaturated fatty acid; and (v) a n-6 polyunsaturated fatty acid.

As set out above, in some embodiments the present invention provides a method for preventing and/or treating diabetes and/or a complication thereof.

Diabetes is characterized by hyperglycemia and depressed metabolism of carbohydrate, protein and fat due to impairment of insulin secretion or resistance to insulin action. Diabetes may be classified into 'Type 1 and Type 2 diabetes, as well as other rarer forms of the disease. In contrast to type 1 diabetes, the onset of type 2 diabetes does not usually occur until adult years and there is no evidence of autoantibodies and no increase in frequency of HLA-DR3, or DR4. The onset of the disease is more insipid with individuals having milder symptoms of hyperglycemia with polyuria and polydipsia over a longer time period.

In some embodiments, preventing/or treating diabetes and/or a complication thereof in a subject includes a reduction in glucose production by the subject. In some embodiments, a reduction in glucose production by the subject may include a reduction in gluconeogensis in the subject.

Gluconeogenesis is a pathway consisting of eleven enzyme-catalyzed reactions. The pathway can begin in the mitochondria or cytoplasm, depending on the substrate being used. Many of the reactions are the reversible steps found in glycolysis. Gluconeogenesis begins in the mitochondria with the formation of oxaloacetate through carboxylation of pyruvate. This reaction also requires one molecule of ATP, and is catalyzed by pyruvate carboxylase. This enzyme is stimulated by high levels of acetyl -Co A (produced in β-oxidation in the liver) and inhibited by high levels of ADP.

Oxaloacetate is reduced to malate using NADH, a step required for transport out of the mitochondria. Malate is oxidized to oxaloacetate using NAD" in the cytoplasm, where the remaining steps of gluconeogenesis occur. Oxaloacetate is decarboxylatcd and phosphorylated to produce phosphoenolpyruvate by phosphoenolpyruvate carboxykinase. One molecule of GTP is hydrolyzed to GDP during this reaction. The next steps in the reaction are the same as reversed glycolysis. However, fructose-1,6- bisphosphatase converts fructose-l,6-bisphosphate to fructose 6-phosphate. Glucose-6- phosphate is formed from fructose 6-phosphate by phosphoglucoisomerase. Glucose-6- phosphate can be used in other metabolic pathways or dephosphorylated to free glucose. Whereas free glucose can easily diffuse in and out of the cell, the phosphorylated form (glυcose-6-phosphate) is locked in the cell, a mechanism by which intracellular glucose levels are controlled by cells. The final reaction of gluconeogenesis, the formation of glucose, occurs in the lumen of the endoplasmic reticulum where glucose-6-phosphate is hydrolyzed by glucose-6-phosphatase to produce glucose. Glucose is shuttled into the cytosol by glucose transporters located in the membrane of the endoplasmic reticulum.

Without limiting the present invention to any particular mode of action, in some embodiments, a reduction of glucose production by the subject and/or a reduction in gluconeogenesis in the subject may be via inhibition of one or more of the enzyme- catalysed reactions of gluconeogenesis as described above.

Significant morbidity and premature mortality occurs due to the complications of diabetes. Long term morbidity occurs due to microvascular and macrovascular complications of diabetes including cardiovascular disease, nephropathy, retinopathy and neuropathy. The degree of hyperglycemia has been shown be significant in the development and progression of clinical complications.

In some embodiments, complications of diabetes include polyuria and polydipsia. These conditions results in dehydration and physiologic stress with the elevation of stress hormones (adrenaline, Cortisol, growth hormone, glucagon). These counter-

regulatory hormones antagonise the action of insulin and promote glycogenosis, gluconeogenesis, lipolysis and ketogenesis resulting in acceleration of metabolic decompensation and progression to ketoacidosis.

Complications of diabetes may also include microalbuminuria. Microalbuminuria occurs when the kidney leaks small amounts of albumin into the urine. Microalbuminuria may be caused by an abnormally high permeability for albumin in the renal glomerulus. Microalbuminuria is diagnosed either from a 24-hour urine lolk'ction (20 to 200 μg/min) or, more commonly, from elevated concentrations (30 to 300 mg/T ) on at least two occasions. An albumin level above these values may be described as macroaJbuminuria or albuminuria.

Accordingly, in some embodiments, the complication of diabetes contemplated herein kuludes one or more ot polydypsia, polyuria and/or muroalbuminuria.

Hyperglycemia may also result in biochemical changes which lead to microvascular and macrovascular complications of diabetes. Macrovascuiar complications include coronary artery disease, atherosclerosis and peripheral vascular disease which can lead Io acute myocardial infarction, angina and poor wound healing. Microvascular comput ations irulude nephropathy, neuropathy, and retinopathy which can result in end-stage kidney disease, impaired nerve conduction and blindness. In some embodiments, the method ot the present invention is directed to preventing and/or treating microvascular and macrovascular complications of diabetes.

Long term monitoring and screening for vascular complications is also necessary so that medical therapies to reverse and treat conditions such as diabetic retinopathy, nephropathy, neuropathy, and cardiovascular disease can be commenced before irreversible damage occurs.

Individuals with diabetes also have an increased risk of i ardiovascular iom plication «. There i« an increased risk of coronary artery disease, in particular, myoi ardial

infarction, as well as cerebrovascular disease, such as stroke. Limb ischemia from peripheral arterial disease is also increased, contributing to the development of nonhealing ulcers and amputation.

Diabetics may also have abnormal lipid metabolism with elevated levels of free fatty acids and triglycerides. This lowers HDL (high density lipoprotein) levels. Both hypertriglyceridaemia and low HDL contribute to endothelial dysfunction. An elevated free fatty acid level in individuals is believed to contribute to insulin resistance in type 2 diabetes. Hyperglycemia induced biochemical changes along with the abnormal lipid metabolism and insulin resistance may result in endothelial cell damage, platelet activation and aggregation and increased coagulation factors which increases the risk of vascular thrombosis. This leads to coronary artery disease, cerebrovascular disease ie. stroke and peripheral vascular disease. In some embodiments, the present invention contemplates preventing and/or treating vascular compri cati ons of di abetes.

Hyperglycemia may also result in complications which alter blood vessel function, resulting in hacmodynamic changes and hyperfiltration through the glomeruli. Pathological changes in the kidneys occur characterised by widening of the glomerular basement membrane and thickening of the mesangium. The earliest indication of nephropathy is microalbuminuria which has a prevalence of 3.7-30.6% in type 1 diabetics. Approximately 1.6% of diabetics develop microalbuminuria per year. TMs progresses to overt proteinuria as glomerular function deteriorates and eventually results in end-stage renal failure. Diabetic nephropathy is the leading cause of death and disability in diabetes with 35% of type 1 diabetics and 15-60% of type 2 diabetics developing end-stage kidney disease. It is the major cause of end-stage renal disease. The present invention is also directed to preventing and/or treating complications of diabetes associated with changes in blood vessel function, including diabetic nephropathy.

Diabetic neuropathy is a complication that affects approximately 50% of individuals

with diabetes, it can manifest as a mono- or poly-neuropathy and is associated with significant morbidity. Peripheral neuropathy presents with varying degrees of numbness, paresthesia, hyperesthesia and severe neuropathic pain. Loss of peripheral sensation is often associated with impaired peripheral vascular function which contributes to the development of non-healing ulcers and the potential risk of limb amputation. Autonomic neuropathy can result in gastrointestinal dysfunction, orthostatic hypotension, syncope, bladder dysfunction and impotence. Neuronal damage is due to direct hyperglycaemic-induced damage to nerve parenchyma. Hyperglycemia-induced endothelial changes in the rni crovessels contribute to neuronal ischemia and damage. In some embodiments, the present invention contemplates preventing and/or treating complications of diabetes associated with neuropathy.

Diabetic retinopathy is a complication of diabetes and is the leading cause of blindness with the prevalence of retinopathy increasing with the duration of diabetes. 80%-90% of diabetics will have some evidence of retinopathy after 20 years of the disease with 30-40% having proliferative retinopathy. Blood vessel damage occurs due to hyperglycemia induced metabolic and chemical changes. Background retinopathy is characterised by capillary vasodilation, increased permeability, and capillary occlusion resulting in retinal ischemia. Neutrophils in diabetics are also less deformable than those in non-diabetics and neutrophils entering the microvessels become trapped in the vessels and adhere to the endothelium of choroidal and retinal capillaries. TMs contributes to retinal vasculature injury by increasing capillary dropout and blood vessel occlusion. Microaneurysms, retinal haemorrhages and exudates are also seen. Proliferative retinopathy results from hypoxia which stimulates endothelial cell migration and proliferation. These new vessels are fragile and frequently rupture; causing retinal haemorrhages that can lead to scarring, retinal detachment and blindness. Macular oedema due to the increase in vascular permeability contributes to the loss of visual acuity. Hie present invention also contemplates preventing and/or treating complications of diabetes associated with retinopathy.

The pathogenic effects of hyperglycemia are mediated by at least protein kinase C (PKC) and nuclear factor kappa B (NFKB) in tissues at risk of developing diabetic complications. Hyperglycemia results in activation of the polyol pathway. Hie subsequent increase in the NADH/NAD+ ratio increases free radical formation and DAG synthesis. Auto-oxidation of excess glucose and non-enzymatic glyeation results in oxidative cellular damage. These pathways have a complex interaction which results in the activation of PKC through DAC and reactive oxygen species, and of NFKB through AGE and reactive oxygen species.

It appears that hyperglycemia in diabetes activates PKC. However, not all PKC isozymes are activated by hyperglycemia. PKCβ mediates the action of glucose in the glomeruli, retina, aorta and heart of diabetic animals and cultured cells. PKC a, βl, βll and δ have been shown to be activated by high glucose conditions in bovine retinal endothelial cells, PKC a, β, and ε in the rat retina, and PKC a and ζ in rat mesangial cells, and PKC βl I in rat aorta.

Glucose induced PKC activation promotes the production of endothelin-1 from retinal endothelial cells. Endothelin-1 is a potent vasoconstrictor which has been implicated in causing a reduction in blood flow in the early diabetic eye. The resultant hypoxia stimulates the production of vascular endothelial growth factor (VEGF) which increases vascular permeability and endothelial cell proliferation and migration. This ultimately leads to retinal neovascularisation.

Recent observations that both glucose and advanced glyeation end-products stimulate NFKB activation in a variety of cell-types implicate N FKB as a mediator of macrovascular and microvascular complications.

Hyperglycemia and Advanced Glyeation Endproducts (AGEs) may also upregulate the expression of adhesion molecules such as ICAM-I (intercellular adhesion molecule), VCAM-1 (vascular cell adhesion molecule) and E-selectin and this is believed to play significant roles in increasing the adhesiveness of the endothelium and be associated

with complications of diabetes.

In some embodiments, method of the present invention is for preventing and/or treating atherosclerosis in a subject. As set out in the examples,, in a mouse model of atherosclerosis, treatment with a polyunsaturated fatty acid compound was found to reduce plaque area.

In further embodiments, the method of the present invention provides a method for preventing and/or treating undesired or uncontrolled angiogenesis including, for example, tumour induced angiogenesis.

As set out above, the present invention contemplates the administration of a compound, salt or solvate thereof to a subject, wherein the compound is selected from the group consisting of: (i) a polyunsaturated fatty acid including an oxa and/or thia substitution at either or both of the β or γ position of the polyunsaturated fatty acid; (ii) a polyunsaturated fatty acid eovalently coupled to an amino acid; (iii) a polyunsaturated nitroalkene or nitroalkyne; (iv) a n-3 polyunsaturated fatty acid; and (v) a n-6 polyunsaturated fatty acid.

The term "polyunsaturated" as used throughout the specification is to be understood to mean a molecule including a carbon chain which contains more than one double and/or triple valence bond. The term includes within its scope geometric isomers. The term "polyunsaturated fatty acid" as used throughout the specification is to be understood to mean a carboxylic acid, or a salt thereof, the carboxylic acid including a carbon chain of which contains more than one double and/or triple valence bond.

As will be appreciated, the compounds, salts or solvates thereof, contemplated for use in accordance with the present invention may fall into one or more of the classes defined above. For example, a compound may be both a polyunsaturated fatty acid including an oxa and/or thia substitution at either or both of the β or γ position of the polyunsaturated fatty acid and an n-3 polyunsaturated fatty acid or an n-6

polyunsaturated fatty acid.

In some embodiments, the compound, salt or solvate thereof includes a carbon chain containing 16 to 26 carbon atoms.

In some embodiments, the compound, salt or solvate thereof may have 1 to 6 carbon double bonds,, including 1, 2, 3, 4, 5 or 6 carbon double bonds. In some embodiments, the compound, salt or solvate thereof may have between 3 and 6 carbon double bonds.

In some embodiments, the compound has an polyunsaturated hydrocarbon chain having 18 to 26 carbon atoms and three double bonds separated by methylene groups, with the first double bond relative to the omega carbon atom being between the third and fourth or sixth and seventh carbon atoms.

In some embodiments the compound includes a polyunsaturated fatty acid including an oxa substitution at the β position of the polyunsaturated fatty acid.

Examples of β-oxa compounds include β-oxa-23:4n-6; β-oxa-21:3n-6; β-oxa-21:3n-3; β- oxa-25:6n-6; β-oxa-21:4n-3; 16-OH-β-oxa-21:3n-6; 16-OH-β-oxa-21:3n-3; β-oxa-18:3n-3, β-oxa-20:4n-6, β-oxa-20:5n-3, β-oxa-22:6n-3, β-oxa-23:4n-6, 15-OOH-β-oxa-20:4n-6, β- oxa-23:4n-ό, β-oxa-21:3n-6, β~oxa~21 :3n~3 _. , β-oxa-25:6n-3, β-oxa-21:4n-3, 16-OH-β-oxa- 21:3n-6, 16-OH-β-oxa-21:3n-3.

In some embodiments the compound includes a n-3 polyunsaturated fatty acid. In some embodiments, the compound is β-oxa 21:3 n-3. β-oxa 21:3 n-3 is also referred to herein as "MP5".

In some embodiments the compound includes a n-6 polyunsaturated fatty add. In some embodiments the compound is compound is β-oxa 23:4 n-6. β-oxa 23:4 n-6 is also referred to herein as "MP3".

In some embodiments, lhe compound includes a polyunsaturated fatty acid including a lhia substitution at the β position of the polyunsaturated fatty acid. Examples of β- thia compounds include β-thia-21:3n-6; β-thia-21:3n-3; β-thia-25:6n-3; β-thia-23:4n-6; α- i arboxymethyl-β-thia-23:4n-6.

In some embodiments, the compound includes a polyunsaturated fatty acid including a thia substitution at the γ position of the polyunsaturated tatty acid. Examples ot γ-ihia polyunsaturated fatty adds include γ-thia-22:3n-6; γ-thia-22:3n-3; γ-lhia-24:4n-fo; γ-thia- 25:6n-3.

The method of the present invention also specifically contemplates administering a combination ot a n-3 polyunsaturated fatty add as hereinbefore described and a n-6 polyunsaturated fatty add as hereinbefore described for the prevention and/or treatment of the diseases, conditions and states dest ribed herein.

As set out above, the compound may also include a polyunsaturated fatty acid covalently coupled to an amino acid. The amino acid may be a natural amino acid, or an amino add sequences modified either by natural processes, such as post- lranslational processing, or by a chemical modification technique known in the art. Naturally oα urring amino add" include alanine, arginine, asparagine, aspartu acid, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleudne, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine, in some embodiments, the amino acid is glycine or aspartic acid.

In some embodiments the polyunsaturated fatty acid may be coupled to the amino acid by way of an amide linkage, although it will be appreciated that the amino acid may be coupled to carboxylic by other means known in the art. Such compounds are generally described in US patent 5,998,476.

In some embodiments, the amino add conjugated compound may be γ-linυlenic acid-

glycine, α-linolenic add-glydne, arachidonic add-glycine, docosahexaenoic add- glydne, eicosapentaenoic glycine, γ-linolenic add-aspartic acid, α-linolenic acid- aspartic add, arachidonic add-aspartic acid, eicosapentaenoic add-aspartic acid and docosahexaenoic add-aspartic acid.

As set out above,, in some embodiments, the compound,, salt or solvate thereof may be a polyunsaturated nitroaikene or nitroalkyne. The term ''polyunsaturated nitroalkene or nitroalkyne" as used throughout the specification is to be understood to mean an alkene or alkyne including a nitro group, the molecule including a carbon chain of which contains more than one double and/or triple valence bond. In some embodiments the polyunsaturated nitroalkene or nitroalkyne may have the formula X- NO2, wherein X is a polyunsaturated hydrocarbon chain of 14 to 26 carbon atoms, and which may be optionally substituted. In some embodiments, the polyunsaturated nitroalkene or nitroalkyne has a formula of Ri-X-NO:, wherein X is an polyunsaturated hydrocarbon chain of 14 to 26 carbon atoms, and which may be optionally substituted, and Ri is (CH2),-.(COOH) _m , in which n is 0 to 2, and m is independently 0 to 2. Such compounds are as generally described in international patent application VVO 01/21575, In some embodiments, X is a hydrocarbon chain of 18 to 22 carbon atoms, and in one specific embodiment has 3-6 double bonds.

In some embodiments, the polyunsaturated nitroaikene or nitroalkyne is selected from the group consisting of (z,z,,z)-l-Nitro-9,12,15-octadecatriene, (z,z,z)-l-Nitro-6,9,12- octadecatriene, (ali-z)-l-Nitrø-5,8,ll,14-eico-satetraene, (ali-z)-l-Nitro-4,7,10,13,16,19- docosahexaene, (all-Z)-4-Nitrotricosa-8J 1,14,17-tetraenoic acid, 3-[{all-Z)-Nonadeca- 4,7,10, i3-tetraeϊψl]-3-nitropentane-1,5-dicarboxylk: acid.

it will be appreciated that the compounds, salts or solvates thereof described herein may also be optionally substituted. The term "substituted" means that a hydrogen atom on a molecule has been replaced with a different atom or molecule. The atom or molecule replacing the hydrogen atom is denoted as a "substituent". The term substituted specifically envisions and allows for substitutions that are common in the

art. However, it is generally understood by those skilled in the art that the substituents should be selected so as to not adversely affect the pharmacological characteristics of the compound or adversely interfere with the use of the medicament.

Sυbstituent groups may include, for example: halogen (F, Cl, Br, I), =O, =5, alkyl, alkenyl, alkynyl, haloalkyl, haloalkenyl, haloalkynyi, heteroalkyl, cycloalkyl, cycloalkenyl, heterocydoalkyi, heterocycloalkenyi, aryl, heteroaryl, cydoaikyialkyl, heterocycloalkylalkyl, heteroaiyfalkyl, arylalkyl, cydoafkyfalkenyl, heterocydoalkyi alkenyl, arylalkenyl, heferoarylalkenyl, cydoalkylheteroalkyl, heterocycloalkyiheteroalkyl, aryiheteroalkyl, heteroarylheteroalkyl, hydroxy, hydroxyalky], alkoxy, aikoxyaikyl, alkoxycycloalkyl, alkoxyheterocydoalkyl, alkoxyaryl, alkoxyheteroaryl, alkoxycarbonyl, alkylaminocarbonyl, alkenyloxy, alkynyloxy, cydoalkyloxy, cydoalkenyloxy, heterocycloalkyloxy, heterocydoalkenyloxy, aryloxy, phenoxy, benzyloxy, heteroaryloxy, arylalkyloxy, arylalkyl, heteroaryl alkyl, cydoaikyialkyl, heterocycloalkylalkyl, arylalkyloxy, alkylammo, acylamino, aminoaikyi, aryiamino, sυlfonyl amino, sυlfinyiamino, sυlfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyi, sulfinyl, alkylsuifinyl, arylsulfinyl, aminosulfinylairunoalkyl, cyano, nitro, amino, thio, thioalkyl, carboxyl, carboxyl ester, arnido, keto, acyl, -NHCOO-, -NHCONH-, and -Q=NOH)- Where appropriate, the substituent group may be a terminal group or a bridging group. Trie substituent group may include two or more of the aforementioned groups bonded to one another.

in this regard, the term "alkyl" as used throughout the specification is to be understood to mean a group or part of a group of saturated straight chain, branched or cyclic hydrocarbon groups, such as a Ct-C-* alkyl, a C1-C30 alkyl, or a Ct-Cr alkyl. Examples of straight chain and branched alkyl include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyi, n-pentyl and branched isomers thereof, n-hexyl and branched isomers thereof, n-heptyl and branched isomers thereof, n-octyl and branched isomers thereof, n-nonyl and branched isomers thereof, and n-decyl and branched isomers thereof. Examples of cyclic alkyl include mono-or polycyclic alkyl groups such as cydopropyl, cydobutyl, cydopentyl, cydohexyl, cydoheptyl,

cyclooctyl, cyciononyl, cydodecyl, and the like. An aikyi group may be further optionally substituted by one or more optional substituents as herein defined.

The term "alkenyl" as used throughout the specification is to he understood to mean a group or part of a group straight chain _. , branched or cyclic hydrocarbon residues containing at least one carbon to carbon double bond including ethyienically monounsaturated or polyunsaturated alkyl or cycloaikyl groups. Examples of alkenyl include vinyl, allyl, 1 -methyl vinyl, butenyl, iso-butenyl, 3-methyl-2-butenyL 1- pentenyl, cydopentenyl, 1-methyl-cydopentenyl, 1-hexenyl, 3-hexenyl,. cydohexenyl, 1-heptenyl, 3-heptenyl, 1-octenyl, cyclooctenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 1- decenyl, 3- decenyl, 1,3-butadienyl, 1-4, pentadienyl, 1,3-cydopentadieny], 1,3- hexadienyl, 1,4- hexadienyl, 1,3-cyclohexadienyl, 1,4-cydohexadienyl, 1,3- cycloheptadienyl, 1,3, 5- cydoheptatrienyl, 1,3, 5,7-cyclooctatetraenyl, and the like. An alkenyl group may be optionally substituted by one or more optional substituents as herein defined.

The term "alkynyl" as a group or part of a group as used throughout the specification is to be understood to mean straight chain, branched or cyclic hydrocarbon residues containing at least one carbon-carbon triple bond including ethynicaUy mono-, cli-or polyunsaturated alkyl or cycloaikyl groups as previously defined. Examples include ethynyl, 1- propynyl, 2-propynyl, burynyl isomers, pentynyl isomers, and the like. An alkynyl group may be further optionally substituted by one or more optional substituents as herein defined.

The term "heterocydyl" as a group or part of a group as used throughout the specification is to be understood to mean monocyclic, poly cyclic, fused or conjugated hydrocarbon residues wherein one or more carbon atoms (and where appropriate, hydrogen atoms attached thereto) are replaced by a heteroatom so as to provide a non- aromatic residue. Suitable heteroatoms include nitrogen, oxygen, sulphur and selenium. Where two or more carbon atoms are replaced, this may be by two or more of the same heteroatom or by different heteroatoms. Examples of heterocyclic groups

include pyrrolidinyl, pyrrolinyl, piperidyl, piperazinyl, morpholino, indolinyl, imiazolidinyl, pyrazolidinyl, thiomorpholino, dioxanyl, tetrahydrofuranyl, tetrahydropyranyl, tctrahydropyrrolyl, and the like. A heterocydyl group may be further optionally substituted by one or more optional suhstituents as herein defined.

The term "aryl" as a group or part of a group as used throughout the specification is to be understood to mean: (i) an optionally substituted monocyclic, or fused polycyciic, aromatic carbocycle (ring structure having ring atoms that are all carbon) preferably having from 5 to 12 atoms per ring. Examples of aryl groups include phenyl, naphthyl, and the like; and (ii) an optionally substituted partially saturated bi cyclic aromatic carbocyclic moiety in which a phenyl and a Cs-7 cycloalkyl or C?-7 cycJoalkenyl group are fused together to form a cyclic structure, such as fetrahydronaphthyi, indenyi or indanyl.

The term "heteroaryl" as a group or part of a group as used throughout the specification is to be understood to mean an optionally substituted aromatic ring having one or more heteroatoms as ring atoms in the aromatic ring with the remainder of the ring atoms being carbon atoms. Suitable heteroatoms include nitrogen, oxygen, sulphur and selenium. Examples of heteroaryl include thiophene, benzothiophene, benzofuran, benzimidazole, benzoxazole, benzofhiazole, benzisothiazole, naphtho[2,3- bjthiophene, furan, isoindolizine, xantholene, phenoxatine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, triazine, tetrazole, pyridazine, indole, isoindole, IH-indazoie, purine, quinoline, isoquinoline, phthalazine, naphthyridine, quinoxaline, cinnoline, carbazole, phenanthridine, acridine, phenazine, thiazole, isothiazole, phenothiazine, oxazole, isooxazole, furazane, phenoxazine, 2-,3- or 4- pyridyl, 2-, 3-, A-, 5-, or 8- quinolyl, 1-, 3-, A-, or 5- isoquinolinyll -, 2-, or 3- indolyl, and 2-, or 3-fhienyl.

The term "acyl" as a group or part of a group as used throughout the specification is to be understood to mean a group containing the moiety OO (and not being a earboxylic acid, ester or amide). Examples of acyl include forrnyl; straight chain or branched

alkanoyl such as, acetyl, propanoyl, bulanoyl, 2-methylpropanoyl, pentanoyl, 2,2- dimelhylpropanoyl, hexanoyl, heplanoyl. odanoyl, nonanoyl, decanoyl, undecanoyl, dodecanoyl, tridecanoyl, fetradecanoyl, pentadecanoyl, hexadecanoyf, heptadecanoyl, υi tadecanoyl, nonadecanoyl and icosanoyl; cycloalkylcarbonyl such a« cyclopropylcarbonyl cydobutylcarbony], cyclopentylcarbonyl and cyclohexykarbonyl; aroyl sudi as benzoyl, ioluoyl and naphlhoyl; aralkanoyl such as phenylalkanoyl (e. g phenyiacefyl, phenylpropanoyl, phenylbulanoyl, phenylisobutylyl, phenylpentanoyl and phcnylhexanoyl) and naphlhylalkanoyl (e g naphlhylacetyl. naphthylpropanoyl and naphthylbutanoyl]; aralki-noyl suth as phenylalkenoyl (c. g. phenylpropenoyl, phenylbυtenoyl, phenylmethacryloyl, pheriylpentenoyl and phenylhexenoyl and naphthylalkenoyl (e. g naphthylpropenoyl, naphthylbυtenoyl and naphthylpentenoyl); aryloxy alkanoyl such as phenoxyacelyl and phenovypropionyl; arylthiocarbamoyl such as phenyllhiocarbamoyl; arylglyoxyloyl such as phenylglyoxyloyl and naphthylglyoxyloyl: aryfculfonyl such as phonylsulfonyl and napthylsulfonyl; heterocydici arbonyl; hcttToi yclicalkanoyl such a« tliienylacetyl, thienylprcpanoyl, thienylbutanoyl, thienylpentanoyl, thienylhexancyl, thiazolyl acetyl, Ihiadiazolylacetyl and tetrazolylacelyl; heterocyclicalkenoyl such as heterocyclicpropenoyl, helcTocyclicbulenoyl, helerocydicpenlenoyl and heUTOcydichexenoyl, and hclerocyclicglyoxyloyl such as Ihiazolyglyoxyloyl and thicnylglyoxyloyl.

The terms alkoxy, alkenoxy aryloxy, heteroaryloxy, heterocyclyloxy and acyloxy respectively denote alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl and acyl groups as hereinbefore defined when linked by oxygen,

The term thioalkyl refers to an alkyl group when linked by sulfur.

The term "carboxyl" as a group or part of a group refers generally to the group COiH and "carboxyl ester" as a group or part ot a group refers generally to the group CO2R wherein R is any group not being ϊ I.

The term "amino" as a group or part of a group as used throughout the specification is to be understood to mean the group NRR' and "amido" as a group or part of a group refers generally to the group CONRR', wherein R and R' can independently be H, alkyl, alkenyl, alkynyi, aryl, acyl, heteroaryl, heteroeytiyl, or derivatives thereof.

The term ''halo" as used throughout the specification is to be understood to mean a halogen group, including fiuoro, chloro, bromo and iodo groups.

In some specific embodiments, the compound includes one or more substitutions selected from the group consisting of a hydroxy!, a hydroperoxy, a peroxy, and a carboxyalkyl substitution.

The compound, salt or solvate thereof may also be in the form of a pro-drug. The term "pro-drug" as used throughout the specification is to be understood to mean a precursor which upon administration to a biological system, undergoes chemical conversion by metabolic or chemical processes to yield the compound, salt or solvate thereof.

As set out above, the method of the present invention contemplates administration of the compound, salt or solvate thereof to a subject.

Any suitable method of administration known in the art may be used. For example, the compound,, salt or solvate thereof may be delivered to the desired site of action directly, or be delivered by administration of the compound, salt or solvate thereof to the subject. Administration and delivery of the compound, salt or solvate thereof may be, for example, by intravenous, intraperitoneal, subcutaneous, intramuscular, oral, or topical route, or by direct injection into the desired site of action. The mode and route of administration in most cases will depend on the type of disease, condition or state being treated.

The effective amount of the compound, salt or solvate thereof to be delivered is not

particularly limited, so long as it is within such an amount and in such a form that generally exhibits a useful or therapeutic effect. The term "effective amount" is the quantity which when delivered or administered, improves the prognosis of the subject. The amount to be delivered will depend on the particular characteristics of the condition being treated, the mode of delivery, and the characteristics of the subject, such as general health, other diseases,, age, sex, genotype, body weight and tolerance to drugs. A person skilled in the art will be able to determine appropriate dosages depending on these and other factors.

In some embodiments, the administration of the compound, salt or solvate thereof to the subject includes recurrent administration of the compound, salt or solvate thereof to the subject daily. For example,, the treatment regime may occur daily over a period of 12 weeks.

For example, effective amounts of the compound, salt or solvate thereof typically range between about 0.1 mg/kg body weight per day and about 1000 mg/kg body weight per day, and in some embodiments between 1 mg/kg body weight per day and 100 mg/kg body weight per clay.

As described later, the administration of the compound, salt or solvate thereof may also include the use of one or more pharmaceutically acceptable additives, including pharmaceutically acceptable salts, amino acids, polypeptides, polymers, solvents, buffers, excipients, preservatives and bulking agents, taking into consideration the particular physical, microbiological and chemical characteristics of the compound to be administered.

In some embodiments the method of the present invention may further include administering to the subject an Angiotensin-Converting Enzyme (ACE) inhibitor. For example, the ACE inhibitor may be selected from one or more of the group consisting of (i) a sulfhydryl-containing ACE inhibitor, including Captopril; (ii) a dicarboxylate- containing ACE inhibitors, Enalapril, Ramipril, Quinapril, Perindopril, Lisinopril, and

Benazepril; and (iii) a phosphonate-containing ACE inhibitor, including Fosinopril.

Methods, dosages and treatment regimes for administering ACE inhibitors are known in the art. ACE-inhibitors are generally as described in "ACE Inhibitors" (2001) ed. by P,D. Orleans-Juste and G.E. Plante, Birkhauser Verlag.

In another aspect, the present invention provides the use of one or more compounds, or salts or solvates thereof, in the preparation of a medicament for preventing and/or treating one or more of (i) diabetes, or a complication thereof; (ii) ischemia/reperfusion injury, and complications thereof; (iii) atherosclerosis; (iv) restenosis; (v) tumour growth; and (vi) un desired or uncontrolled angiogenesis in a subject; the compound being selected from the group consisting of (i) a polyunsaturated fatty acid including an oxa and/or thia substitution at either or both of the β or γ position of the polyunsaturated fatty add; (ii) a polyunsaturated fatty acid covalently coupled to an amino acid; (iii) a polyunsaturated nitroalkene or nitroalkyne; (iv) a n-3 polyunsaturated fatty acid; and (v) a n-6 polyunsaturated fatty acid.

Methods for producing fatty acids are known in the art, for example as described in international patent application VVO 96/11908.

The preparation of medicaments is known in the art, for example as described in

Remington's Pharmaceutical Sciences (18th ed.,. Mack Publishing Co., Easton, Pa., 1990) and U.S. Pharmacopeia: National Formulary (Mack Publishing Company, Easton, Pa.,

1984).

In some embodiments, the medicament is for preventing and/or treating diabetes and/or a complication thereof as hereinbefore described.

In some embodiments, preventing/or treating diabetes and/or a complication thereof in a subject includes a reduction in glucose production by the subject. In some embodiments, a reduction in glucose production by the subject may include a

reduction in gluconeogensis in the subject, as hereinbefore described.

In some embodiments, the medicament is for preventing and/or treating atherosclerosis in a subject as hereinbefore described.

In various embodiments, the compound, salt or solvate thereof used in the preparation of the medicament may be a compound, salt or solvate thereof, as hereinbefore described.

In some embodiments, the use may further include the use of an Angiotensin- Converting Enzyme (ACE) inhibitor, as hereinbefore described, in the preparation of the medicament.

In some embodiments, the medicament may take the form of a pharmaceutical composition. In some embodiments, the pharmaceutical composition may include the compound, salt or solvate thereof together with an ACE inhibitor as hereinbefore described.

Accordingly, in another aspect, the present invention provides a pharmaceutical composition including an ACE inhibitor and a compound, or a salt or solvate thereof, selected from the group consisting of (i) a polyunsaturated fatty acid including an oxa and/or thia substitution at either or both of the β or γ position of the polyunsaturated fatty acid; (ii) a polyunsaturated fatty acid covalently coupled to an amino acid; (iii) a polyunsaturated nitroalkenc or nitroalkyne; (iv) a n-3 polyunsaturated fatty acid; and (v) a n-6 polyunsaturated fatty acid.

In some embodiments, the ACE inhibitor used in the pharmaceutical composition may be an ACE inhibitor as hereinbefore described.

In some embodiments, the compound or a salt or solvate thereof, used in the pharmaceutical composition may be as hereinbefore described.

The pharmaceutical composition may be formulated into a composition that is suitable for the desired route and mode of delivery or administration. For example, an active agent may be admixed with a pharmaceutically acceptable carrier, diluent, excipient,. suspending agent, lubricating agent, adjuvant, vehicle, delivery system, emulsifier, disintegrant, absorbent, preservative, surfactant, colorant, giidant, anti-adherent, binder, flavourant or sweetener. A pharmaceutical carrier can be any compatible nontoxic substance suitable for delivery of the agent to a subject.

Examples of pharmaceutically acceptable additives include pharmaceutically acceptable salts, amino acids, polypeptides, polymers, solvents, buffers, excipients, preservatives and bulking agents, taking into consideration the particular physical, microbiological and chemical characteristics of the compound to be administered.

The preparation of pharmaceutical compositions is known in the art, for example as described in Remington's Pharmaceutical Science? (18th ed., Mack Publishing Co., Easton, Pa., 1990) and U.S. Pharmacopeia: National Formulary (Mack Publishing Company, Easton, Pa., 1984).

The dosage form, frequency and amount of dose will depend on the mode and route of delivery or administration. For example, the active compound can be prepared into a variety of pharmaceutical acceptable compositions in the form of, e.g., an aqueous solution, an oily preparation, a fatty emulsion, an emulsion, a lyophilised powder for reconstitute on, etc. and can be administered as a sterile and pyrogen free intramuscular or subcutaneous injection or as injection to an organ, or as an embedded preparation or as a transmucosal preparation through nasal cavity, rectum, uterus, vagina, lung, etc. The composition may be administered in the form of oral preparations (for example solid preparations such as tablets, caplets, capsules, granules or powders; liquid preparations such as syrup, emulsions, dispersions or suspensions).

Compositions containing the compound, salt or solvate thereof may also contain one or

more of a pharmaceutically acceptable preservative,, buffering agent, diluent, stabiliser, chelating agent, viscosity-enhancing agent, dispersing agent, pH controller, solubility modifying agent or isotonic agent. These excipients are well known to those skilled in the art. Examples of suitable preservatives are benzoic acid esters of para- hydroxybenzoic acid, propylene glycol, phenols, phenylethyl alchohol or benzyl alcohol. Examples of suitable buffers are sodium phosphate salts, citric acid, tartaric acid and the like. Examples of suitable stabilisers include antioxidants such as alpha- tocophero! acetate, alpha-thioglycerin, sodium metabisulphite, ascorbic acid, acetylcysteine, 8-hydroxyquinoline, and chelating agents such as disodiυm edetate. Examples of suitable viscosity enhancing agents, suspending or dispersing agents are substituted cellulose ethers, substituted cellulose esters, polyvinyl alcohol, polyvinylpyrrolidone, polyethylene glycols, carbomer, polyoxypropylene glycols, sorbitan monooleate, sorbitan sesquioleate, poly oxy ethylene hydrogenated castor oil 60. Examples of suitable pH controllers include hydrochloric acid, sodium hydroxide, buffers and the like. Examples of suitable isotonic agents are glucose, D-sorbitol or D- mannitol, sodium chloride.

The composition may be administered orally, parenterally, by inhalation spray, adsorption, absorption, topically, rectally, nasally, bucally, vaginally, intraventricularly, via an implanted reservoir in dosage formulations containing conventional non-toxic pharrnaceυticaUy-acceptable carriers, or by any other convenient dosage form. The term parenteral as used herein includes subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal, and intracranial injection or infusion techniques.

When administered parenterally, the composition will normally be in a unit dosage, sterile, pyrogen free injectable form (solution, suspension or emulsion, which may have been reconstituted prior to use) which is usually isotonic with the blood of the recipient with a pharmaceutically acceptable carrier. Examples of such sterile injectable forms are sterile injectable aqueous or oleaginous suspensions. These suspensions may be formulated according to techniques known in the art using suitable vehicles,

dispersing or wetting agents and suspending agents. The sterile injectable forms may also be sterile injectable solutions or suspensions in non-toxic parentcrally acceptable diluents or solvents, for example, as solutions in 1,3-butanediol. Among the pharmaceutically acceptable vehicles and solvents that may be employed are water,. ethanol, glycerol, saline, Ringer's solution, dextrose solution, isotonic sodium chloride solution, and Hanks' solution. In addition, sterile, fixed oils are conventionally employed as solvents or suspending mediums. For this purpose, any bland fixed oil may be employed including synthetic mono- or dϊ-glycerkles, corn, cottonseed, peanut, and sesame oil. Fatty acids such as ethyl oleate, isopropyl rnyristate, and oleic acid and its glyceride derivatives, including olive oil and castor oil, especially in their poiyoxyethylated versions, are useful in the preparation of injectables. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants.

The carrier may contain minor amounts of additives, such as substances that enhance solubility, isotonicity, and chemical stability, for example antioxidants, buffers and preservatives,

in addition, the composition may be in a form to be reconstituted prior to administration. Examples include lyophilization, spray drying and the like to produce a suitable solid form for reconstitute on with a pharmaceutically acceptable solvent prior to administration.

Compositions may include one or more buffer, bulking agent,, isotonic agent and cryoprotectant and lyoprotectant. Examples of excipients include, phosphate salts, citric add, non-reducing sugars such as sucrose or trehalose, polyhydroxy alcohols, amino acids, methyl amines, and lyotropic salts are preferred to the reducing sugars such as maltose or lactose.

In some embodiments, the composition may be suitable for oral administration. Suitable oral formulations may include unit dosage forms such as tablets, caplets, cachets, powder, granules, beads, chewahle lozenges, capsules, liquids, aqueous

suspensions or solutions, or similar dosage forms, using conventional equipment and techniques known in the art. Such formulations typically include a solid, semisolid, or liquid carrier. Exemplary carriers include excipients such as lactose, dextrose, sucrose, sorbitol, manrutol, starches, gum acacia, calcium phosphate, mineral oil, cocoa butter, oil of theobroma, alginates, tragacanth, gelatin, syrup, substituted cellulose ethers, polyoxyethylene sorbitan monoiaurafe, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium s tear ate, and the like.

A tablet may be made by compressing or moulding optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing, in a suitable machine, the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active, or dispersing agent. Moulded tablets may be made by moulding in a suitable machine, a mixture of the powdered active ingredient and a suitable carrier moistened with an inert liquid diluent.

Compositions may also be a sustained release composition. Such compositions may be formulated with additional components such as vegetable oil (for example soybean oil, sesame oil, camellia oil, castor oil, peanut oil, rape seed oil); middle fatty acid triglycerides; fatty add esters such as ethyl oleate; polysiloxane derivatives; alternatively, water-soluble high molecular weight compounds such as hyaluronic acid or salts thereof (weight average molecular weight: ca. 80,000 to 2,,000,,00O), carboxymethylcelluiose sodium (weight average molecular weight: ca. 20,000 to 400,000), hydroxypropylcellulose (viscosity in 2% aqueous solution: 3 to 4,000 cps), atherocollagen (weight average molecular weight: ca. 300,000), polyethylene glycol (weight average molecular weight: ca. 400 to 20,000), polyethylene oxide (weight average molecular weight: ca. 100,000 to 9,000,000), hydroxypropylmethylcellulose (viscosity in 1% aqueous solution: 4 to 100,000 cSt), methylcellulose (viscosity in 2% aqueous solution: 15 to 8,000 cSt), polyvinyl alcohol (viscosity: 2 to 100 cSt), polyvinylpyrrolidone (weight average molecular weight: 25,000 to 1,200,000).

In some embodiments, the sustained release composition may include, for example, a hydrophobic polymer matrix or biodegradable polymer for controlled release of the active over a period of days. Such compositions may be moulded into a solid implant, or externally applied patch, suitable for providing efficacious concentrations of the active over a prolonged period of time without the need for frequent re-dosing. Such sustained release films are well known to the art. Other examples of polymers commonly employed for this purpose that may be used include nondegradabie ethylene-vinyl acetate copolymer a degradable lactic acki-glycolic acid copolymers which may be used externally or internally. Certain hydrogels such as poly(hydroxyerhy]methacrylate) or poiy(vinylalcohol) also may be useful, but for shorter release cycles than the other polymer release systems, such as those mentioned above.

The present invention also contemplates topical pharmaceutical formulations. For topical administration, the composition may be in the form of a solution, spray, lotion, cream (for example a non-ionic cream), gel, paste or ointment. Alternatively, the composition may be delivered via a liposome, nanosome, ribosome, or nutri-diffuser vehicle.

The compound, salt or solvate thereof and the ACE inhibitor may also be produced in a combination product.

Accordingly, in another embodiment the present invention provides a combination product including the following components: an ACE inhibitor; and a compound, salt or solvate thereof selected from the group consisting of: (i) a polyunsaturated fatty acid including an oxa and/or thia substitution at either or both of the β or γ position of the polyunsaturated fatty acid; (ii) a polyunsaturated fatty acid covalently coupled to an amino acid; (iii) a polyunsaturated nitroalkene or nitroalkyne; (W) a n-3 polyunsaturated fatty acid; and (v) a n-6 polyunsaturated fatty add; wherein the components are provided in a form for separate administration to a

subject,, or in a form for co-administration to a subject.

In some embodiments, the subject is susceptible to, or suffering from a PKC-β mediated disease,, condition or state, or a disease, condition or state that would benefit from inhibition of activation of PKC-β.

In another embodiment, the subject is susceptible to, or suffering from an NFKB mediated disease, condition or state in a subject, or a disease, condition or state that would benefit from inhibition of activation of NFKB mediated disease, condition or state.

The present invention also provides a combination product including the following components: a n-3 polyunsaturated fatty acid; and an n-6 polyunsaturated fatty acid; wherein the components are provided in a form for separate administration to a subject, or in a form for co-administration to a subject.

In some embodiments, the subject is susceptible to, or suffering from a disease, condition or state selected from one or more of the group consisting of (i) diabetes, or a complication thereof, including diabetic microvascular complications and/or diabetic macrovascular complications, diabetic retinopathy, diabetic nephropathy and diabetic uremia; (ii) ischemia/reperf usion injury, and complications thereof; (iii) atherosclerosis;

(iv) restenosis; (v) tumour growth; (vi) undesired or uncontrolled angiogenesis, including tumour-induced angiogenesis; (vii) inflammatory disease; and (viii) cardiovascular disease.

The components of the combination product may packaged separately or together in suitably sterilized containers such as ampoules, bottles, or vials, either in multi-dose or in unit dosage forms. The containers are typically hermetically sealed. Methods are known in the art for the packaging of the components.

The present invention also provides screening assays lor identity ing polyunsaturated fatty adds that may be used as inhibitors of PkC -β activation, and lor identifying polyunsaturated fatty acids that are therapeutic i andidates for PKC-β mediated diseases, condition and states, and thus can be used in the preparation of a medicament for the prevention and/or treatment of such diseases, states or conditions.

Accordingly, the present invention also provides a method of identifying an inhibitor of activation of a PkC-β enzyme, the method ύu hading: providing a test compound, or a salt or solvate thereof, selected from the group consisting of (i) a polyunsaturated fatty acid including an o\a and/or thia substitution at either or both of the β or γ position of the polyunsaturated fatty acid; (ii) a polyunsaturated tally acid covalenlly coupled to an amino acid; (iii) a polyunsaturated nitrυalkene or nitroalkyne; (iv) a n-3 polyunsaturated fatty add; and (v) a n-6 polyunsaturated fatty acid; determining the ability of the test compound to inhibit translocation of a PkC-β enzyme from the cytoplasm to the particulate fraction in a cell; and identifying the test compound as an inhibitor ol activation ol a PKC-β enzyme by the ability ol the test compound Io inhibit translocation of a PKC-β enzyme from the cytoplasm to the particulate fraction.

Determination that a test compound inhibits translocation of a PkC-β enzyme from the cytoplasm to a particulate fraction may be achieved by a suitable method known in the art. For example, selection of a suitable compound, salt or solvate lhereol for inhibiting activation of a PKC" enzyme may be achieved by determining the ability ot a seleded compound, salt or solvate thereof to inhibit activation of a PkC enzyme. For example, the ability of a selected compound, salt or solvate thereof to inhibit translocation of a PKC enzyme from the cytoplasm Io a particulate fraction will be indicative thai the compound, salt or solvate thereof may be used in the present invention.

The method of identification may utilise a cell in a cell-free in vitro system, a cell

present in a cell in in vitro culture, or a cell present in a biological system, such as a subject.

Methods for screening agents involving the high-throughput screening of test compounds are specifically contemplated. For example, high throughput screening methods are as described in High Throughput Screening (Humana Press inc. edited by William P. Janzen., 2002).

As described above,, the present invention may also be used to identify an agent for preventing and/or treating a PKC-β mediated disease, condition or state.

The present invention also provides screening assays for identifying polyunsaturated fatty acids that may be used as inhibitors of NFκ:β activation.

Accordingly,, the present invention also provides a method of identifying an inhibitor of activation of a NFκ"β enzyme, the method including: providing a test compound, or a salt or solvate thereof, selected from the group consisting of (i) a polyunsaturated fatty add including an oxa and/or thia substitution at either or both of the β or γ position of the polyunsaturated fatty acid; (ii) a polyunsaturated fatty acid covalently coupled to an amino acid; (iii) a polyunsaturated nitroalkene or nitroaikyne; (iv) a n-3 polyunsaturated fatty acid; and (v) a n-6 polyunsaturated fatty acid; determining the ability of the test compound to inhibit activation of a NF Kβ enzyme; and identifying the test compound as an inhibitor of activation of a NFicβ enzyme.

Determination that a test compound inhibits activation of NFxβ may be achieved by a suitable method known in the art.

In the case of a compound, salt or solvate thereof for inhibiting activation of NFKB, selection of a suitable compound, salt or solvate thereof may be achieved by

determining the ability of a selected compound, salt or solvate thereof to inhibit activation of NFKB.

The method of identification may utilise a cell in a cell-free in vitro system, a cell present in a cell in in vitro culture, or a cell present in a biological system, such as a subject.

Methods for screening agents involving the high- throughput screening of test compounds are specifically contemplated. For example, high throughput screening methods are as described in High Throughput Screening (Humana Press Inc. edited by William P. ϊanzen, 2002).

It will be appreciate that the method of identification may utilise a cell in a cell-free in vitro system, a cell present in a cell in in viho culture, or a cell present in a biological system, such as a subject.

As described above, the present invention may also be used to identify an agent for preventing and/or treating a NF Kβ mediated disease, condition or state.

The present invention also contemplates an agent identified according to the above methods.

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

BRIEF DESCRIPTION OF THE FIGURES

Figure ^' ! shows monolayer of bovine retinal endothelial cells prepared by primary cell culture at the 4 ^th passage.

Figure 2 shows fluorescent antibody staining with Von Willebrand Factor antibody. Endothelial cells were shown to be >95% pure.

Figure 3 shows expression of PKC isozymes in bovine retinal endothelial cells (BREC).

BRECs were grown to confluence (3 x IQ" cells) in tissue culture plates and sonicated in buffer containing 2% Triton-XIOO to extract PKC from the particulate fraction of the cells. The presence of PKC isozymes in BREC was analysed by Western blotting and stained with antibodies for PKC a, (31, βll, ε and δ. Representative bands derived from

3 separate experiments are shown. Each antibody is specific for the intended isozyme and the isozymes are identified by their Mr in SDS gels and their alignment with brain

PKC. Each blot was stripped and reprobed with a different antibody for up to 2 times in a random order.

Figure 4 shows expression of PKC isozymes in human umbilical vein endothelial ceils (HUVEC). HUVECs were grown to confluence (3 x 10 ⁶ cells) in tissue culture plates and sonicated in buffer containing 2% Triton-XIOO to extract PKC from the particulate fraction of the cells. The presence of PICC isozymes was analysed by Western blotting and stained with antibodies for PKC a, βl, βlT, ε and δ. Representative bands derived from 3 separate experiments are shown.

Figure 5 shows expression of PKC isozymes in human T lymphocytes. Human T lymphocytes were prepared from healthy donors and adjusted to a concentration of 4 x 10 ⁶ ceils/ml. Cells (1 x 10 ⁷ ) were sonicated and the soluble fractions were analysed for the expression of PKC a, βl, βll, ε, 0 and δ by Western blotting. Representative bands derived from 3 separate experiments are shown.

Figure 6 shows expression of PKC isozymes in HL60 cells. HL60 cells were cultured at a density of <1 x 10 ^r cells/ml. Cells (1 x 10 ⁷ ) were sonicated and soluble fractions were analysed for the presence of PKC α, βl, βll, ε, 0 and δ by Western blotting. Representative bands derived from 3 separate experiments are shown.

Figure 7 shows translocation of PKC a in PMA-acfivated BREC. BREC were grown in tissue culture plates to confluence (1x10 ^h cells) supplemented with DMEM/plasrna.

After incubation with 10OnM PMA for 5 min, cells were sonicated and PKC was extracted from the particulate fraction by sonication in the presence of 2% Triton-XIOO. The amount of PKCα in the particulate fraction was determined by Western blotting and a representative blot is shown. The densities of the bands were quantified with Image Quant software and are expressed as percentage of control (100%). The data are expressed as mean + SEM derived from 3 separate experiments (*p<0.05).

Figure 8 shows translocation of PKC βl in PMA-activated BREC. BREC were grown in tissue culture plates to confluence (1x10 ^h cells) supplemented with DMEM/plasma. After incubation with 10OnM PMA for 5 min, cells were sonicated and PKC was extracted from the particulate fraction by sonication in the presence of 2% Triton-XIOO. The amount of PKCβl in the particulate fraction was determined by Western blotting and a representative blot is shown. The densities of the bands were quantified with Image Quant software and are expressed as percentage of control (100%). The data are expressed as mean + SEM derived from 5 separate experiments. ( ^* p<0.01 )

Figure 9 shows translocation of PKC δ in PMA-activated BREC. BREC were grown in tissue culture plates to confluence (IxIO ⁶ cells) supplemented with DMEM/plasma. After incubation with 10OnM PMA for 5 min, cells were sonicated and PKC was extracted from the particulate fraction by sonication in the presence of 2% Triton-XIOO. The amount of PKC5 in the particulate fraction was determined by Western blotting and a representative blot is shown. The densities of the bands were quantified with Image Quant software and are expressed as percentage of control (100%). The data are expressed as mean + SEM derived from 6 separate experiments.

Figure 10 shows translocation of PKC ε in PMA-activated BREC. BREC were grown in tissue culture plates to confluence (IxIO ⁶ cells) supplemented with DMEM/plasma. After incubation with 10OnM PMA for 5 min, cells were sonicated and PKC was extracted from the particulate fraction by sonication in the presence of 2% Triton-XIOO. The amount of PKO; in the particulate fraction was determined by Western blotting and a representative blot is shown. The densities of the bands were quantified with

Image Quant software and are expressed as percentage of control (100%). The data are expressed as mean + SEM derived from 3 separate experiments.

Figure 11 shows effect of Wgh glucose on PKCα translocation in BREC. BREC were grown in tissue culture plates to confluence and treated with normal or high glucose

DMEM/plasma for 3 days. Cells were sonicated and PKC was extracted from the particulate fraction by sonication in the presence of 2% Triton-XlOO. The amount of

PKCα in the particulate fraction was determined by Western blotting and a representative blot is shown. The densities of the bands were quantified with Image Quant software and are expressed as percentage of control (100%). The data are expressed as mean + SEM derived from 3 separate experiments. (*p<0.05)

Figure 13 shows effect of high glucose on PKCβl translocation in BREC. BREC were grown in tissue culture plates to confluence and treated with normal or high glucose DMEM/plasma for 3 days. Cells were sonicated and PKC was extracted from the particulate fraction by sonication in the presence of 2% Triton-XIOQ. The amount of PKCβl in the particulate fraction was determined by Western blotting and a representative blot is shown. The densities of the bands were quantified with Image Quant software and are expressed as percentage of control (100%). The data are expressed as mean + SEM derived from 5 separate experiments. ( ^* p<0.05)

Figure 14 shows effect of high glucose on PKCε translocation in BREC. BREC were grown in tissue culture plates to confluence and treated with normal or high glucose DMEM/plasma for 3 days. Cells were sonicated and PKC was extracted from the particulate fraction by sonication in the presence of 2% Triton-X100. The amount of PKCε in the particulate fraction was determined by Western blotting and a representative blot is shown. The densities of the bands were quantified with Image Quant software and are expressed as percentage of control (100%). The data is expressed as mean + SEM derived from 3 separate experiments.

Figure 15 shows effect of MP5 on PKC βl activation. BREC were pre-treated with 30 μM

MPi) for 2h before exposure to high glucose (25mM/2% plasma) for 3 clays and compared with control samples (25mM/2%plasma). At confluence (1x106 cells) cells were sonicated and PKC was extracted from the particulate fraction by sonication in the presence of 2% Triton-XIOO. The amount of PKC j.3I in the particulate fraction was determined by Western blotting and a representative blot is shown. The densities of the bands were quantified with Image Quant software and are expressed as percentage of control (100%). The data are expressed as mean + SEM derived from 3 separate experiments (*p<0.05)

Figure 16 shows effect of hyperglyeaemie conditions on IκBα degradation in short term culture, BREC were grown to confluence and treated with 25mM glucose for 1,2,3, and 4h and compared to 5.5mM glucose as a control. Cells were lysed and iysates analysed by Western blotting for IscBα. The membrane was stripped and re-probed with β-actin. The density of each band was quantified and the ratio of IscBα;: β-actin calculated. The ratios are expressed as percentage of control (100%). Representative blots are shown. The data are expressed as mean + SEM of 3 experiments.

Figure 17 shows effect of hyperglyeaemie conditions on lκBα degradation in long term culture. BREC were grown to confluence and treated with 5.5mM or 25mM glucose for 3 days. Cells were lysed and Iysates analysed by Western blotting for IκBα. The membrane was stripped and re-probed with β-actin. The density of each band was quantified and the ratio of lκBα: β-actin calculated. The ratios are expressed as percentage of control (100%). Representative blots are shown. The data are expressed as mean + SEM of 7 experiments.

Figure 18 shows concentration effect of TNFα on IKB α degradation under low (5.5mM) glucose conditions. BREC were grown to confluence in low glucose DMEM/"10%plasma and treated with increasing concentrations of TNF (0, 1, 10, lOOOU/ml) for 15min. Cytoplasmic extracts were analysed using Western blotting for IscBα. The membrane was stripped and re-probed with β-actin. The density of each band was quantified and the ratio of IκBα:β-actin calculated. The ratios are expressed as percentage of control

(100%). Representative blots are shown. The data are expressed as mean + SEM of 3 experiments. (*p<0.01)

Figure 19 shows concentration effect of TNF on TscBα degradation under high (25m M) glucose conditions. BREC were exposed to 25mM glucose for 3 days until confluent then treated with increasing concentrations of TNF (O ₇ I ₇ 1O ₇ lOOOU/ml) for 15min.

Cytoplasmic extracts were analysed using Western blotting for IκBα. The membrane was stripped and re-probed with β-actin. The density of each band was quantified and the ratio of IκBα:β-actin calculated. The ratios are expressed as percentage of control (100%). Representative blots are shown. The data are expressed as mean + SEM of 3 experiments. (*p<0.001 )

Figure 20 shows time course for TNF-induced lκBα degradation under low (5.5mM) glucose conditions. BREC" were grown to confluence in 5.5mM glucose and treated with 1000U/ml TNF for O ₇ 5, 10, 15, 30min. The cells were then lysed and analysed by Western blotting for IκBα. The membrane was stripped and re-probed with β-actin. The density of each band was quantified and the ratio of IκBα: β-actin calculated. The ratios are expressed as percentage of control (100%). Representative blots are shown. The data are expressed as mean + SEM of 2 (5 and 30min) to 5 experiments. Significance of difference between control and treated cells: p<0.0001 (ANOVA).

Figure 21 shows time course for TNF-induced IκBα degradation under high (25mM) glucose conditions. BREC were exposed to 25mM glucose for 3 days until confluent and treated with 1000U/ml TNF for O ₇ 5 ₇ 1O ₇ 15, 30min. Cells were lysed and analysed by Western blotting for IκBα. The membrane was stripped and re-probed with β-actin. The density of each band was quantified and the ratio of lκBα:β-actin calculated. The ratios are expressed as percentage of control (100%). Representative blots are shown. The data are expressed as mean + SEM of 2 (5 and 30mm) to 5 experiments. Significance of difference between control and treated cells: pO.OOOl (ANOVA).

Figure 22 shows suppression of TNF induced IκBα degradation by MP3. BREC were

exposed io 25mM glucose for 3 days until confluent (IxIO'' cells). Cells were treated with increasing concentrations of MP3 for 2h before exposure to TNF 10ϋ0U/ml. Cells were lysed and cytoplasmic extracts analysed for lκBα levels by Western blotting. Blots were stripped and re-probed with [3-actin. The density of each band was quantified and expressed as IκBα:β-actin as a percentage of control (100%). Results of ? separate experiments are shown.

Figure 23 shows suppression or FKC-I translocation in glucose-stimulated mesangiaf cells (a) and in the glomeruli of diabetic rats (b). Mesangial cells were pretreated with MP5 or vehicle (ethanol) for I hour before being incubated with 25 mM glucose for 5 days, Male rats were rendered diabetic with streptozotocin and MP5 or vehicle (ethanol) was administered for 7 days after confirmation of diabetes. The cells and glomeruli were sonicated and particulate traction associated PkC-βl was determined by Western Plot analysis. High glucose and diabetes increased PKC-βl in the particulate fraction. MP5 inhibited this effect.

Figure 24 shows the effect of β-oxa-21:3n-3 treatment of diabetic rats on water consumption, urinary output and urine albumin levels. Rats received 40 mg/kg of the fatly acid daily for two weeks. Significance of difference *p<0.05.

Figure 25 shows that in a mouse model for atherosclerosis, mice treated with MP? have significantly reduced plaque area as compared to control.

EXAMPLE 1 Materials and Methods

(i) Biochemical s

Collagenase type 1, collagen type Vl, and gelatin type B were from Sigma- Aldrich. Collagenase type IL deoxyribonuclease were from Worthington, Lakewood, NJ, USA.

Tumour Necrosis Factor α (TNF) was produced by Genentech, Inc (San Francisco, CA,

USA) Il had a specific activity of 6x10' U/mg as assessed lor cytotoxicity by lhe supplier on actinomycin- D -treated murine fibrosarcoma cell line (L929), and was >9S% pure using {he Limulus amoebocyte lysate assay. Lipopolysacchaiide (LPS) i ontaniination wa« < 0.125 1 PS L /ml. TN ₁ F was stored at a concentration of 5x107 L/ml in 5ul aliquots at -20 ⁰ C and was prepared tresh daily in HBSS.

(li) Serum, Albumin, Culture media and buffers

Bovine Scrum Albumin was from Boehringcr Manheim, VV Germany, l^eial calf serum

(FCS), Hank* balanced salt solution (HBSS), RPMI 1640, L-glutamine (20OmM) were from JRI I biosciences, Lenexa, KS, USA. DuJbecco's Minimal Essential Media (DMFM) was from Sigma-Aldrich Fungizone was from Bristol-Myers Squibb, Noble Pk, VTC Penicillin (5000U/ml)/streptomycin (5000μg/ml) was from Commonwealth Serum Laboratories (CbL), Parkville, VlC. Phosphate buffered saline (PBS) and medium 199 were from Cell Image, Adelaide SA.

(iii) Protease Inhibitors

Leupeptin, pepstatm A, benzamidine hydrochloride, PMSF (phenylmethylsultonyl fluoride) were from Sigma-Aldrich. Aprolinin (bovine lung, 100,000UzSmI) was from Calbiochem, La jolla, CA, USA

(iv) Antibodies and Conjugates

Rabbit anti-human to Von Willebrand Factor, anti-mouse Ig, (HRP conjugated) was from DAKO Corporation, Carpinleπa, CA, USA λnti rabbit Ig (HRP conjugated) was lrom Chemicon. Boronia, VlC. FITC conjugated rabbit IgG, PKC (31, fill, a, b, f, θ antibodies were from Santa Cruz Biotechnology Santa Cruz, CA, USA. β-actin antibody was from Sigma- Al drich.

(v) Preparation of Plasma

Human group AB scrum and pooled plasma: Serum was collected from group AB healthy donors or pooled from several donors. It was heat inactivated for 30rnin at 56°C. Serum was stored at -20 ^r C. Plasma was collected from healthy donors as described in section

4.)

2.5 and heat inactivated for 30min at 56°C. it was frozen at -20 ⁰ C, thawed, then centrifuged at 10,00Og for 2Qmin. This process was repeated to remove excess fibrin and plasma was stored in aliquots of 1OmL

(vi) Preparation of culture media for BREC

Various growth factors have been used to sustain long-term culture of bovine retinal endothelial ceils. In this project, C6 glioma cell-conditioned media and retinal crude extract were used to support the growth of these cells as these conditions were considered to be optimal for eulturing BREC.

Culture flasks were coated with type VI collagen for a minimum of 1h, then the collagen was removed and flasks allowed to air-dry prior to eulturing of cells. Endothelial cells seeded onto matrices such as collagen and fibrinogen have been shown to attach and proliferate more readily than those seeded onto laminin, fibronectin and gelatin.

Cb conditioned media: C6 glioma cells were obtained from American Type Culture Collection (ATCC), Rockville, MD, USA. They were grown in 75cm ² culture flasks in 5.5mM DMEM supplemented with 10% plasma, lOOU/rnl penicillin/100 μg/ml streptomycin, and 4rnM L-glutamine. The media were collected after 3-4 days, and centrifuged at 155Og for lOmin to remove cells. The media were then stored at -20°C to ensure death of any residual C6 glioma cells, and thawed for use in BREC culture media.

Retinal crude extract After isolation of retinal tissue from fresh cow eyes, the retinas were incubated at room temperature in PBS (1 retina per ml) for 2h. They were centrifuged at 45Og for 5min and the supernatant filtered through a 0.2μm filter and used as retinal crude extract.

BREC culture media: BREC were grown in culture media containing a combination of 20% FCS (heat inactivated in a 56°C water bath for 30min), 30% DMEM (5.5rnM

glucose supplemented with antibiotics and 4mM L-glutamine), 10% plasma, 40°/« Cb conditioned media, and 10jαi/ml retinal crude extract.

(vii) Primary Cell Culture of BRRC BRFC were prepared trorn tresh eyes (I obethal Abattoir, Adelaide, SA) using methods described by Wong et al (Invest Ophthalmol Vis Sci 28 1767-177% 19^7). The eyes were dissected removing excess fat and muscle from the surface and rinsed in ethanol and PBS I hey were bisected and the retina removed and rinsed in DMEM/HhPhb pH 7.4 Retinas were homogenised 3x at 200-3G0rpm (Mulfifix hornogeraser, Orpington, Kent, UR) and centrifuged at 70Og tor 10m in The resultant pellet was resuspended in PBS and filtered through an 83 μm filter. Blood vessel fragments were inverted onto collagenase type 1 (lmg/ml)/deoxyπbonuclease (300μg/ml) enzyme mixture and incubated on a rotary shaker lor 25min al 37°C until devoid of pericytes This was then filtered through a 53 um filter and inverted onto DMRM supplemented with 20% FCS. Cells were centrifuged at 45Og for iOmin, resuspended in culture medium supplemented with fungizone (2.5μg/ml) and transferred to collagen-coated culture flasks Cells were grown at 37°C in an atmosphere of 5°/« COz in air and high humidity. Culture media was changed every 2-3 days.

(viii) Primary Cell Culture of I IUVRC

HUVRCs were prepared using methods described previously (Bates et al., J Lcukoc Biol 54: 590-5%, 1993) Human umbilical cords were collected immediately after delivery and stored at 4°C tor less than 36h. The umbilical vein was cannulated and washed with HBSS. They were then filled with collagenase type Il {0.4mg/ml), clamped and incubated for 2mm in a 37°C water bath. The c ontent" of each vein were collected and the vein flushed through with HBSS to collect any remaining cells. Cells were centntuged at 70Og for 5mm and the pellet resuspended in RPMl 1640 (supplemented with antibiotics and 4mM L-glutamine) and 20% pooled heat inactivated human group AB serum Cells were grown in 0.2% gelatincoated culture flasks at 37°C in an atmosphere of 5% CCh in air and high humidity. The medium was changed every 2-3 days.

(ix) Determination of BREC and HUVEC purity

BREC were identified by their growth as a monolayer and their spindle shaped appearance, whereas HUVEC display a cobblestone appearance when confluent. Purity was assessed by Von Willebrand Factor staining (as described by Jaffe ci ai., J CHn Invest 52: 2757-2764, 1973), For this, BREC or HUVEC were grown to confluence on collagen- or gelatin-coated coverslips and fixed with ethanol for 5min at 4°C. Coverslips were incubated for 45min at room temperature with rabbit anti-human Von Willebrand Factor (dilution 1:150 in azide/PBS 1:100), washed 3x with azide/PBS (1 :100) before being incubated with FtTC-conjυ gated antirabbit IgG for 45m in (dilution 1:50 in azide/PBS 1:100). After washing to remove unbound secondary antibody, Von Willebrand Factor staining was assessed by fluorescent microscopy.

(x) Trypsinisation of Cells For passaging, BREC and HLJVEC" were trypsinised by incubating for 3-5min at 37°C in trypsin/EDTA diluted in PBS (1 :5), and centrifuged at 56Og for 5min. Cells were resuspended in culture media and plated into matrix-coated tissue culture dishes. Ceils were used in experiments up to passage 5.

(xi) Culture of T lymphocytes

Venous blood was collected from healthy donors and placed into tubes containing 25 lU/ml heparin. Blood (6ml) was layered onto 4ml Hypaque-Ficoll media (8% Ficoli 400, sodium diatrizoate and angiograffin, density 1.114) and centrifuged at 56Og for 35min. After centrif ligation, the leukocytes were resolved into two discrete bands with the mononuclear leukocytes (monocytes, T and B lymphocytes) in the top band and neutrophils in the lower band. Red blood cells were at the bottom of the tube and plasma on the top. The lymphocyte layer was gently aspirated and washed twice with Medium 199 for 5min at 56Og. The viability of the cells was >99%, assessed by their ability to exclude trypan blue.

T lymphocytes were purified using a modification of the method of Zhang et al. (J

Immunol 148: 177, 1992). Tissue culture plates (10cm) were coated with 3ml autologous plasma for 30min at 37°C in a humidified atmosphere of 5% CO: in air. The isolated mononuclear cells were resuspended in 40ml RPMl 1640, supplemented with 20% heat inactivated FCS and 5ml added to each plate. After incubation at 37°C in the CO?. incubator, monocytes adhered to the plastic tissue culture dishes and were therefore depleted. The plates were washed with 10ml RPMl 1640/10% FCS to remove nonadherent T and B lymphocytes and the media collected and centrifuged for 5min at 56Og. The cell pellet was resuspended in 0.8ml RPMl 1640/10% FCS and applied to a ImI syringe packed with nylon wool which was incubated at 37°C in the CO:, incubator. (The column had been pre-equilibrated by passing RPMI 1640/10% FCS through the column at 37°C). Ten ml of RPMI 1640/10% FCS was flushed through the column to remove non-adherent T lymphocytes and residual red blood cells were removed by centrif ligation over Lymphoprep gradient for 15rnin at 955g, T lymphocytes were removed and washed twice with serum-free RPMl 1640. The viability of purified T lymphocytes was >99%, assessed by their ability to exclude trypan blue. Cells were resuspended at a density of 4x10 ⁶ cells/ml.

(xii) Culture of HL 60 cells

HL-60 cells were obtained from American Type Culture Collection (ATCC), Rockville, MD, USA, and maintained in RPMI 1640 supplemented with 10% heat inactivated FCS, antibiotics and 4mM L-gJutamme. They were incubated at 37°C in a humidified atmosphere of 5% CO2 in air, and kept at a density of <lxlθ ⁶ . cells/ml.

(xiii) PKC isozyme expression in different cell types BREC and HIJVEC were grown in 10cm collagen- or gelatin-coated tissue culture plates until confluent (3 x 10 ⁶ cells). At confluence, the culture medium was aspirated from the plate and the adhered cells were rinsed with PBS. After removal of PBS, cells were scraped from the plate using a rubber policeman. Samples were disrupted in sonication buffer as described below.

T lymphocytes and HL60 cells (IxIO ⁷ cells) were centrifuged to remove the media and

disrupted in sonication buffer.

Total cell extracts were prepared by sonicating samples in 150ul of 2% TritonX- 100/sonication buffer (2OmM Tris-HCI, pH 7.4,. 5mM ECITA, 2mM EDTA,. 2mM dithiothreitoi (DTT), lOμg/ml leυpeptin, lOμg/ml aprotinin, lOμg/ml pepstatin A, 1OmM benzamidine hydrochloride, and 1OmM phenylmethylsufonyl fluoride (PMSF)), Samples were sonicated (3 x 10 sec) at 4°C on setting 7, tune 2 using a Heat Systems sonicator (Lab Supply, Adelaide, SA) and left on ice for 30min to allow PKC to dissociate from the membranous fraction of the cell. Samples were then centrifuged (100,000g) for 30m in at 4°C. The supernatant was collected and protein content determined using the Lowry method. Laemmli buffer (3x strength of 6OmM Tris-HO, pH 6,8, 40% sucrose, 6% SDS, 1OmM β-mercaptoethanol) was added to the supernatant in a ratio of Laemmli buffer:sample of 1:2. The samples were then boiled at 100 ⁰ C for 5min and stored at -20 ⁰ C. Samples were analysed using Western blotting for the presence of PKC isozymes a, βϊ, βll, δ, ε and θ, using isozyme specific antibodies. Identification was facilitated by running brain PKC on the same gel.

(xiv) PKC isozyme translocation in BREC

When PKC is activated it translocates from the cytosol to the membranous fraction of the cell. The amount of PKC on the membranous fraction was determined using Western Blot analysis with PKC isozyme specific antibodies.

BREC were grown in 6cm collagen-coated tissue culture plates in culture media. At 40- 50% confluence, cells were treated with DMEM containing either 5.5mM or 25mM glucose and 10% plasma for 3 days until confluent (1x106 cells/plate). Cells were then treated with PMA (10OnM) or vehicle (DMSO, 0.1% v/v) for 5mm before they were harvested. To each plate was added 300 μl of sonication buffer (see above for composition) and the cells scraped and transferred into centrifuge tubes. After sonication (3 x 10 sec) and centrif ligation (100,00Og x 30min, at 4°C) the pellets were resuspended in sonication buffer (lOOμl) containing 2% TritionX-lGO, re-sonicated (3 x 10 sees) and the samples were left to stand on ice for 30min. The samples were

centrifuged (100,00Og x 3Qmin, 4°C) and the supernatants were collected. An aliquot was used for protein estimation using the Lo wry method. Laemmli buffer was added to the remaining supernatants and the samples were boiled at 100 ⁰ C for 5min and stored at -20 ⁰ C until Western blotted.

(xv) lκBα degradation

BREC were grown to confluence (IxIO ⁶ ceils/plate) in 6cm collagen-coated tissue culture plates. After treatment with various concentrations of glucose or TNF for different time periods, cells were lysed. To each plate was added 300 μl of lysis buffer (NP40 0.5% v/v, 2OmM HEPES pH 7.2, 10OmM NaCl 1mM EDTA, 2mM dithiothreitol (DTT), lOμg/ml leυpeptin, lOμg/ml aprotmin/lOμg/ml Sigma 104, lOμg/ml pepstatin A, 1OmM benzamidine hydrochloride, and 1OmM PMSF) and cells scraped from the plate with a rubber policeman and transferred into eppendorf tubes. Samples were lysed on a rotary shaker at 4°C for 2h, then centrifuged. The supernatant was collected and protein content determined using the Lowry method. Laemmli buffer was added to the remaining supernatant. The samples were then boiled at ^' 100 ⁰ C for 5m in and stored at - 20 ⁰ C until Western blotted for IκBα.

(xvi) Western Blotting Western blotting was used to detect the presence of PKC isozymes or IκBα. Bio-Rad minigel apparatus was used and a 10% SDS-PAGE gel (for PKC) or 12% SDS-PAGE gel (for IκBα) prepared. The samples were boiled for 5min at 100 ⁰ C and 20 μg (PKC) or 40 μg (lκBα) protein were loaded to separate lanes. The volume required to load these amounts of protein was calculated using the protein concentration/5μl determined by the protein assay. A SDS-PACJE LOW Range marker was loaded to provide a range of molecular weight markers. The gel was run at 200V for approximately 45min until the dye front migrated off the gel.

Protein was transferred onto a nitrocellulose membrane using electrophoresis at 100V for 1.5h. Ponceau S staining (0.1% in 5% acetic acid) was used to confirm equal protein load and even transfer of protein to the membrane. The membrane was blocked for Ih

at 37°C in blocking buffer (5% skimmed milk, 25mM Tris/HCl pH 7.4,, 10OmM NaCl) then incubated with primary antibody (Table 2.1) diluted in Tris/HCl-Twccn20 (pH 7.4) for 45min. The membrane was washed (3x5min) in blocking buffer + 0.1% Tween- 20 before being incubated for 45m in with horse radish peroxidase conjugated secondary antibody (Table 2.1) diluted in blocking buffer. The membrane was washed again (3x5min) with blocking buffer. The protein bands were visualised using enhanced chemiluminescence. The relative density of each band was determined using Image Quant software, version 3.3 (Molecular Dynamics, Charlottesville, VA, USA),

Membranes which were probed for IκBα were stripped and reprobed (see 2.12) for β- actin as a protein loading control. Densitometry scanning facilitated the correction of any unequal protein loading and results were expressed as a ratio of lκBα:β-actin signal.

(xvii) Synthesis of Engineered Polyunsaturated Fatty Acids

Engineered polyunsaturated fatty acids were synthesized by a lipid biochemist using the method described by Pitt et ai. (Synthesis 1240-1242,, 1997). TMs is outlined briefly below. For synthesis of β-oxa 21:3n-3 (MP5), a solution of linolenyl alcohol (l.Oόg, 4.01mmol) and rhodium (11) acetate dimer (9mg, 0,5% mol equiv) was stirred in CH2C12 (15ml) at room temperature under dry nitrogen. A solution of teri-butyl diazoacetate (1.46g, 10.22mmol) in Cl-foCb. (5ml) was added dropwise and stirring continued at room temperature for 2h. The mixture was concentrated under a stream of dry nitrogen and the residue was purified by flash column chromatography, eiuted with hexane/Et2O (9:1), to afford tert-butyl (Z,Z,Z)-(Octacleca-9,12,15-trienyloxy) acetate. Trifluoroacetic acid (4ml) was added to afford tert-butyl (Z,Z,Z)-(Octadeca- 9,12,15-trienyloxy) acetate (728mg, 1.92mmol) in CH2C12 (1OmI) under N?.. The solution was stirred at room temperature for 2h. Flash column chromatography on silica was used to purify the crude reaction mixture, eiuted with hexane/Et2O/aceric acid (40:60:2, v,/v), affording (Z,Z,Z)-(Octadeca-9,12,15-trienyloxy) acetic acid as a colourless oil.

For synthesis of β-oxa:23:4n-6 (MP3), tert-butyl (rfL ^f -Z)-(eicosa-5,8/il/i4-tetraenyToxy)

acetate was used in the above procedure and (tf/J-Z)-(eicosa-5,8, 11,14- tetraenyloxy)acctic acid obtained as a colourless oil.

The β-oxa fatty acids were identified using ¹ H and ¹ Xl nuclear magnetic resonance spectroscopy. Engineered fatty acid purity was assessed using thin layer chromatography (TLC). 50 μi of a 2OmM stock solution of the fatty add was spotted onto a silica coated aluminium plate and allowed to dry. The plate was developed in a tank containing diethyl ether:hexane:acetic acid:H2O (65:35:0.5:0.5, v/v). After the solvent front had travelled 16cm, the plate was air dried and exposed to iodine vapour until the lipids could be visualised. There was no evidence of decomposition or degradation of the fatty acids.

(xviii) Presentation of fatty acids to cells

Stocks of β-oxa 23:4n-6 (MP3) and β-oxa 21:3n-3 (MP5) were prepared in chloroform. Chloroform was evaporated with N2 and the fatty acids were reconstituted with ethanol (100% purity) to the required concentration (2OmM) and stored at -20 ⁰ C in siliconized glassware.

The fatty add was diluted in serum-free DMEM to the required concentration (5- 3OuM). The final concentration of ethanol was 0.01-0.1%. Control cells were treated with equivalent amounts of ethanol. Cells were pre-treated with the fatty acid for l-2h prior to any other treatment and lysis of cells. After pre-incubation with the fatty acid, cells were maintained in culture media containing 2-10% serum.

(xix) Statistics

Statistics were calculated using Graph Pad lnStat V2.02 (Graph Pad Software). Difference between treatments were compared to control values which were set at 100%, using a one-sample t test with a hypothetical mean of 100. In some experiments, differences were analysed by analysis of variance (ANOVA). Where appropriate, paired t-tests were also used. Results are considered statistically significant when p<0.05.

EXAMPLE 2 Protein Kinase C Expression in Bovine Retinal Endothelial Cells

(i) Introduction

Bovine retinal endothelial cells (BREC) were used as a model to examine the ability of novel polyunsaturated fatty acids to target PKC activation under hyperglycaemic conditions. It is known that different cell types express a different spectrum of PKC isozymes. Thus it was essential to firstly establish which PKC isozymes are expressed in BREC and the ability of classical agonists such as PMA to translocate (ie activate) these isozymes from the cytosol to the membrane. Comparisons of PKC isozyme expression were made with different cell types, ie, human umbilical vein endothelial cells (HUVEC), human T lymphocytes and HL60 cells.

(ii) Isolation of BREC

Bovine retinal endothelial cells were prepared from fresh bovine eyes using methods modified from Wong et al. {Invest Ophthalmol Vis Sd 28:1767-1775, 1987). Cell culture and culture media preparation are described in Example 1. Isolated retinal endothelial cells were grown in collagen-coated culture flasks at 37°C with 5% CCh in air and high humidity in BREC culture media which was changed every 2-3 days. Stocks were stored for future use at -70 ⁰ C and cells were used in experiments up to passage 5,

BREC were recognized by their characteristic spindle-shaped appearance and their growth as a monolayer (Figure 1). Von Willebrand Factor fluorescent staining was used as confirmation that the preparations were endothelial cells and cells were shown to be >95% pure and devoid of pericytes which do not stain positively for Von Willebrand Factor (Figure 2).

(iii) PKC isozyme expression in endothelial cells BREC and HIJVEC were cultured in 10cm tissue culture plates for approximately 3 days in culture medium. After adherence to the collagen-coated plates (BREC) and

S ₁ I

gelatin coated plates (HUVEC) and reaching confluence (3x10'' ceils), the media was aspirated and the cells were rinsed with PBS. Cells were scraped from the plate using a rubber policeman. Total cell extracts were prepared by sonicating in sonication butter containing 2% Triton-XIGO to extract PKC from the particulate fraction. The soluble fraction was analyzed by Western blotting for the presence of PKC isozymes. This was partly based on the availability of antibodies to these isozymes and analyses were conducted for PKC α,βl, 611, h, and ε. PKC θ was not assessed as this isozyme is known to be expressed mainly in haemopoietic and skeletal muscle cells.

The expression of PKC isozymes in BREC are shown in Figure 3. The data show that PKC a, βl, δ, and ε are expressed in BREC shown by the dense bands. In comparison, there was no evidence of PKC βll expression in these cell preparations.

Figure 4 shows the expression of PKC isozymes in HIJVEC. The data demonstrate that HiJVEC express PKC a, βl,. βll, b, and ε shown by the dense bands on Western blotting,

(iv) PKC isozyme expression in human T lymphocytes

Venous blood was collected from healthy donors and mononuclear cells were obtained by density gradient centrifugarion as described in Example 1. Mononuclear cells were isolated and T lymphocytes purified by adherence to nylon wool. The cells were cultured in RPMl 1640 supplemented with 10%FCS and antibiotics to a concentration of 4x106 cells/ml, then sonicated in buffer containing and 2% Triton-XIOO. The PKC expression in these preparations was determined by Western blotting.

The results presented in Figure 5 show the presence of PKC a, βl, βll, δ, ε, and 0 in Tlymphoeytes as demonstrated by dense bands on Western blotting.

(v) PKC isozyme expression in myeloid HL60 cells HL60 cells were cultured in tissue culture flasks in RPMH 640 supplemented with 10% FCS and antibiotics. The cells were maintained at <lxlθ ⁶ cells/ml. The cells (Ix 10 ⁷ )

were sonicated in buffer containing 2% Triton-XIOO and the expression of PKC a, βl, βll, b, εand θ was determined by Western blotting.

The results show that HL60 cells express PKC a, βll, δ, ε and θ (Figure 3.6). PKCβl was not detected.

(Vi) Summary

PICC isozyme expression varies in different cell types which could account for differences in cellular responses to PKC! activation. BREC" were found to express PKC a, βl, 5 and ε whereas HUVECs contain PKC a, βl, βϊϊ, δ and ε. T lymphocytes express the greatest number of PKC isozymes, including PKC a, βl, βll, δ, ε, and 0. HL60 ceils express PKCα, βll, δ, ε, and θ.

In our studies, cell type and purity were confirmed by morphology and Von Willebrand factor staining, and our methods and reagents were validated by the demonstration that PKCβϊl could readily be detected in HUVEC _. , T cells and HL60 cells. These results imply that PKCβll is not expressed in BREC.

EXAMPLE 3 inhibition of Protein Kinase C Activation in Bovine Retinal Endothelial ("ells by β-QXA 21:3n-3

(i) introduction

The purpose of this study is to attempt to confirm the effect of glucose on PKC isozyme transkx:atk)n/ activation. The isozymes of interest are PKC a, βl, δ and ε as these have been shown in Example 2 to be expressed in BREC. The main objective of this study was to examine the effect of the engineered PUFA, β-oxa 21:3n-3 (MP5) on PKC activation in BREC.

(ii) Activation of PKC by PMA

The translocation of PKC" from the cvtosol to the cell membrane equates to PKC

activation. Thus, a convenient way to study PKC activation is to determine the degree of PKC translocation to the particulate fraction (containing cell membranes). To ensure that our experiments were conducted under optimal conditions for PKC translocation, 10OnM PMA,. a classical activator of PKC, was used to standardise the experimental conditions.

BREC were isolated from bovine eye retinal capillaries and cultured in tissue culture plates as described in Example 1. They were grown to confluence (ixlO ⁰ cells) and treated with either 10OnM of PMA or the equivalent amount of diluent for 5min at 37°C. The cells were detached from the plate and sonicated. The particulate (membranous) fraction was isolated from the soluble fraction by eentrifugation at 100,000g for 30min. PKC was extracted from the membranous fraction by sonication in 2% Triton -X 100 and after eentrifugation, the soluble fraction was subjected to Western blot analyses using a panel of PKC isozyme specific antibodies. The density of each band was quantified by Image Quant software (version 3.3).

PKC isozyme translocation is shown in Figures 7 - 10. Treatment of BREC with 10OnM PMA for 5min resulted in significant increases in PKC a (249+22.3%, p<0.05) and PKC βl (203+21.5%, p<0.01) in the particulate fraction. There was a modest increase in PKC δ (174+29.3%, p=0.054) and PKC ε (167+38%, p=0.2) but this was not found to be statistically significant from control. In all these blots, a certain amount of particulate fraction-associated PKC is seen in unstimulated BREC. This may reflect basal levels of PKC activation by serum in the medium,

(iii) Activation of PKC by hyperglycemic conditions

BREC were grown in tissue culture plates (1 x 10 ⁶ cells) and treated with high (25mM) glucose for 3 days and compared with low (5.5mM) glucose as a control (physiological glucose levels). Cells were scraped from the plate and sonicated. The membranous fraction was isolated and PKC extracted from the membranous fraction by sonication in 2% Triton-XlOO. Western blotting was used for detection of PICC α, βl, δ, and ε. The density of each band was quantified by Image Quant software (version 3.3).

¹ S ¹ S

Figure 11 shows that there is a 70% decrease in particulate fraction-associated PKCα after treatment with high (25mM) glucose tor 3 days. In contrast, there is a significant increase in PKCβϊ translocation (201+27.8%, p<0.05) and PKC δ translocation (155+12.3% _. , p<0.05) after treatment with high glucose for 3 days (Figure 12,13). PKC ε (Figure 14) translocation in response to high glucose was not significantly increased (133+13.5%, p=0.13).

(iv) Inhibition of PKCβl by (3-oxa 21 :3n-3 (MP5) MP5 was reconstituted in 100% ethanol to a concentration of 2OmM and diluted in serum free DMEM to a concentration of 30 μM. BREC were pre-treated with 30 μM MP5 for 2h before exposure to high glucose (DMEM 25mM/2% plasma) for 3 days. Control samples were pre-treated with equivalent amounts of vehicle (ethanol) and maintained in high glucose media (DMEM 25mM/2% plasma). At confluence (1 x 10 ^h cells) cells were scraped from the plate with a rubber policeman and sonicated. The amount of particulate fraction-associated PKC was analysed by Western blotting. The density of each band was quantified.

Figure 15 shows that 30 μM MP5 significantly inhibited the activation of PKCβl (37+11.8% reduction). No significant effects of MP5 were seen with respect to PKC a, δ, or ε.

(v) Summary

PKC isozymes vary in their responsiveness to activation by PMA, a known activator. In BREC, PKC a appeared to be the most responsive to activation by PMA followed by PKC β!. PKC δ and ε showed a trend towards activation but this was not statistically significant. Results show that PKC βl and δ are activated by hyperglycemic conditions in BREC, demonstrated by an increase in PKC translocation after 3 days of treatment with 25mM glucose. PKC βl showed the greatest increase in activation, supporting its role as the main mediator of the effects of hyperglycemia in these cells. PKC a and ε were not activated by hy perglycaemie conditions in these cells.

Pre-exposure with [J-oxa21 3n-3 (MPo) inhibited glucose-induced PKCβl activation in BREC No significant etlects ot MP3 were seen with respect to PKC «, & or e.

FXAMPI E 4

Inhibition of Nuclear Factor K b Activation in Bovine Retinal Endothelial Cells by β-oxa 23:4n-6

(i) Introduction Activation ot the transcriptional factor, NFKB, IS recognised to be important m the processes leading to endothelial dysfunction XFKB can be activated by both endogenous and exogenous mediators of inflammation including INF, IL-I, LPS, AGH, and platelet activating factor. It is also activated by conditions such as hyperglycemia, oxidized lipids, oxidant stress and hypoxia/reperfusion.

NFKP IS associated with IKB a, [3, or t in its inactive state. LTpon activation, IKB kinases are activated which phosphorylate lκBα Phosphorylated lκBα dissociates from NFKB and is degraded in the cytosol NFKB is thus free Io migrate to the nucleus where it exerts its action Therefore, loss of lκBα corresponds to \FκB activation.

Activation of NFKB leads to the upregulation ot cell adhesion molecules such as ICAM- 1, VCAM-I and L-selectin These changes lead to increased adhesiveness ot the endothelium which promotes binding of leukocytes including neutrophils to the endothelium ol choroidal and retinal capillaries This, together with the observation that in diabetics neutrophils entering the rnicrovessels can become trapped within the vessels has been proposed to contribute to increased capillary dropout and blood vessel occlusion which are associated with pathological changes seen in diabetic retinopathy

The aim ot this study was to examine the ettect ot hyperglvcaemic conditions on the v- \ FKP sv^tern.

(ii) Activation of NFKB by hy perglycacmic conditions

BREC were grown in όcm culture plates and treated with high (25mM) glucose for various time periods (10-30min, l-4h, 3 days). This was compared to low (5.5mM) glucose as a control (physiological glucose level). At the appropriate time periods, the cells were lysed, the cytosoi collected and analysed for lκBα levels by Western blotting. Assessing the loss of lκBα is a convenient and validated assay method for NFKB activation.

BREC incubated for 10 to 30min in the presence of 25mM glucose did not show any increase in IκBα degradation (data not shown). Similarly,, when the incubation period with high glucose was increased to 4h, the level of lκBα was not significantly affected (Figure 16) (p>0.05). When the incubation period was further increased to 3 days, no significant change in IκBα levels were observed (Figure 17) (p>0.05). These results therefore demonstrate that high glucose concentrations alone do not activate NFKB.

(Ill) Activation of NFKB by TNF in the presence of high ambient glucose levels

TNF has been shown to be involved in the pathogenesis of diabetic nephropathy. It was therefore of interest to examine the effect of TNF on NFKB activation in the presence of high glucose. To determine the optimum concentration of TNF which would activate NFKB, BREC were grown to confluence (1x10 ^b cells) under low (5.5m M) or high (25mM) glucose conditions for 3 days. They were then exposed to 0, 1, 10 and 1000 U/ml TNF for 15min. Cells were lysed and analysed by Western blotting for IκBα levels.

The results presented in Figure 18 show that that under low glucose conditions, lU/ml andl0U/ml TNF did not alter IκBα levels significantly but 1000U/ml caused a significant reduction (65%) indicating NFKB activation. Similar results were seen under high glucose conditions (Figure 19) with a greater reduction of IxBa levels (96%) than at 5.5mM glucose after treatment with lOOOU/ml TNF (p<0.05). These data demonstrate that BREC" respond to TNF in a manner similar to other cell types, including HIJVEC in

that TNF caused the degradation of lκBα. The data also suggest that the inability of hyperglycaemic conditions to reduce the intracellular levels ot IxBa (Figures 16 and 17) was not due to a technical problem. In fact, high ambient glucose levels amplified the effect of TNF.

To assess the optimum treatment time for TNF-induced activation of NFKB, BREC were cultured in 6cm culture plates until confluent (IxIO ⁶ cells). They were treated with TNF (lOOOU/ml), for 0, 5, 10, 15 and 30min under low (5.5mM) and high (2SmJVl) glucose conditions for 3 days. Cells were Jysed, the cytosol collected and analysed for the amount of IκBα by Western blotting.

Figure 20 shows that under low glucose conditions a dense band is seen in unstimulated BREC (0 time) indicating the presence of lκBα bound to NFKB in the cytosol. Treatment with lG00U/ml TNF caused a time dependent loss of IκPα which was reduced by 64.3+2.8% at lOmin, 82.8+4.4% at 15min, with some recovery of levels by 30m in (67.7+14.3% reduction). This reversal has also been seen previously in TNF- stimulated cells.

TNF exposure under hyperosmolar conditions (2SmJVI glucose) showed a similar effect with a time dependent loss of IκPα (Figure 21). Density scanning showed a 61.5+7% reduction at lOmin, a 95,7+0.7% reduction at 15min, with less recovery of IκBα levels by 30min (82.8^0.95% reduction) when compared to low glucose conditions. The loss of lκBα was greater at 15min than at lOmin under low and high glucose conditions

(p<0.01). This again demonstrates that high ambient glucose amplifies the effect of TNF on the NFKB pathway.

(iv) Inhibition of NFKB activation by 23:4n-6 (MP3)

BREC were grown to confluence (1x10° cells) and exposed to DMEM containing 23mM glucose and 10% plasma tor 3 clays. MP3 was reconstituted in 100% ethanol to a concentration of 2OmM and diluted in serum-free DMEM to concentrations of 5, 10,. 15 and 2OμJV3. BREC were rinsed twice in serum-free DMEM and pre-treafed with

increasing concentrations of MP3 lor 2h before exposure to 100UU/ml FNF for 10- 15min. Control samples were pre -treated with equivalent amounts of vehicle {ethanol, maximum ol 0.1%, v/v). Cells were scraped from the plate with a rubber policeman and ly^ed, the i ytosol colli -ι ted and analysed tor lκBα levels by Western blotting.

Figure 22 shows the results from 3 individual experiments. MP3 dose-dependently inhibited I MF-induced lκBα degradation Consistent with the above data, the effect ol TNiF per se was greater at 15min than at lOmin.

(vj Summary

The results shown in this study characterize the effects of TXF on NFKB activation in bovine retinal endothelial ceils Stimulation of cells for 10-30rrun with 1000 L /ml TKF under low and high glucose conditions results in a reduction ot lκBα in the cytosol, ie N ₁ FKB activation. Under high glucose conditions there is a greater reduc tion ot IKBO. levels at 15min and a slower recovery of levels than under low glut ose conditions. This suggests that TKF caused a greater and more persistent activation of NFKB under high glucose conditions than at low glucose conditions.

The β-oxa PLFA, MP3 (β-oxa 23 4n-6), suppressed {he TNF-induced activation of NFKB under high glucose c onditions in bovine retinal endothelial t ells. A concentration effect is demonstrated with 15μM and 2OuVI ot MP3 producing the greatest inhibitory effects of lκBα degradation.

EXAMPLE 5 Suppression of PKC-I translocation in glucose-stimulated mesangial cells and in the glQig _. llglJloi _. dl _' -jbgtjc rats

Preliminary data from glucose-stimulated mesangial ceils is shown in Figure 23A and the glomeruli of diabetic rats in Figure 23B have confirmed that MP5 (β-oxa-21 3n-3) inhibits the translocation ot PKC-J31, the mam β isotorm in mesangial cells to a partit ulate fraction.

Mesangial cells were pretreatcd with MP5 or vehicle (cthanol) for 1 hour before being incubated with 25 rnJVl glucose for 5 days. Male rats were rendered diabetic with streptozotocm and N4P5 or vehicle (ethanol) was administered for 7 days after confirmation of diabetes. The cells and glomeruli were sonicated and particulate fraction associated PKC-βl was determined by Western Blot analysis. High glucose and diabetes increased PKC-βl in the particulate fraction. MP5 inhibited this effect.

Data previously described herein has also shown that MP5 blocks glucose-stimulated activation of PKC-β in retinal endothelial cells, suggesting the potential use of such compounds in blocking diabetic retinopathy.

Long term in vivo experiments (up to 3 months) have shown that MP5 (up to 100 mg/'kg) has no visible adverse effects on the well-being of animals (eg coat appearance and activity/mobility),, and exerted no advise effects on liver and kidney function and electrolyte levels.

Our data indicate that the compounds have the hallmarks for blocking the actions of glucose. Our studies have also demonstrated that diabetic rats treated with MP5 showed a significant reduction in diabetic complication, suppressed polydypsia and polyuria, and microalbuminuria (Figure 24) without normalising blood glucose levels (Control diabetic: 22.8 + 0.8 niM; MP5- treated diabetic: 21.4 + 1.6 mM). The most likely reason for the decrease in polyuria is that MP5 normalises the glomerular filtration rate (GFR) in the diabetic rats. This would reduce the rate of glucose filtration and, although filtered glucose would still exceed the transport maximum for reabsorption, less would be excreted. This results in a reduced osmotic diuresis. Our data of a 47% increase in electrolyte (Na', K ^* , Ck) excretion support this. Because less glucose is excreted in the urine, the unchanged blood glucose levels in MP5-treated rats most likely represents a reduction in glucose production. Since the decrease in polyuria is similar in magnitude to the decrease in polydipsia, there is no net gain in body fluid.

EXAMPLE 6 Treatment with MP3 in a mouse model for atherosclerosis reduces plaque area

Previous data discussed herein indicates that MP3 would be effective in reducing and/or preventing the development of atherosclerosis. Studies to confirm this were undertaken using the apoE" ^" mouse model of atherosclerosis. The model was verified first in terms of aortic lesion development by assessing mice on a high fat/ cholesterol diet. To study the effect of MP3, 4-5 week old mice were treated with the fatty acid

(70mg/kg) or vehicle daily for 2 weeks prior to being placed on a high fat diet. The mice were then placed on the high fat diet and continued to receive the MP3 or vehicle every other day for 5 weeks. The mice were then sacrificed, heart isolated, sectioned and stained for examination.

Results showed that in the mice treated with MP3 the plaque area was significantly reduced in comparison to control (Figure 25A). It also showed a preventative effect of treatment on compensatory aortic enlargement in those treated with MP3 compared with control (Figure 25B).

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 ail 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 the steps or features.

Also, it must be noted that, as used herein, the singular forms "a", "an" and "the" include plural aspects unless the context already dictates otherwise.