WO2010111789A1 - Inhibition of formation of advanced glycation endproducts - Google Patents

Abstract

The present invention provides compositions and methods that inhibit non-enzymatic protein glycation and/or inhibit the formation of advanced glycation endproducts (AGEs). The compositions comprise peptides derived from the Aktl protein, said peptides comprise an ammo acid sequence selected from CLQ or CLQWTTVER. Furthermore, the compositions are useful for treating advanced glycation endproduct (AGE)-related diseases.

Description

INHIBITION OF FORMATION OF ADVANCED GLYCATION ENDPRODUCTS

RELATED APPLICATIONS

This application claims priority to United States Application number US 61/166,692 filed April 3, 2009, the contents of which are all hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

[0001] The field of the invention generally relates to compositions and methods for inhibiting non-enzymatic glycation of proteins, which often results in formation of advanced glycation endproducts (AGEs) and cross links.

BACKGROUND OF THE INVENTION

[0002] Non-enzymatic glycation (also known as the Maillard reaction) is a complex series of reactions between reducing sugars and the amino groups of proteins, lipids and DNA which leads to cross linking. This complex series of reactions is cascade of condensations, rearrangements and oxidation produces heterogeneous and irreversible products known as advanced glycation endproducts (AGEs).

[0003] AGEs are heterogeneous groups of products formed between the free side chains of residues in proteins and reactive carbonyl groups through oxidation, degradation or rearrangement[l, 2]. Examples of AGEs include Ne-(carboxymethyl)lysine (CML), Ne- (carboxyethyl)lysine (CEL), S-(carboxymethyl)cysteine (CMC), S-(carboxyethyl)cysteine (CEC), argpyrimidine, pentosidine and the imidazaolium crosslines methylglyoxal-lysine dimer (MOLD, l,3-di(N^e-lysino)-4-methyl-imidazolium salt), glyoxal-lysine dimer (GOLD and l,3-di(Λ^-lysino)imidazolium salt). Increased formation of AGEs occurs naturally with aging and is greatly enhanced by hyperglycemia in diabetes. Furthermore, through the binding to specific receptors on endothelial cells, monocytes, macrophages and other cells, AGEs are demonstrated to signal the expressions of inflammatory cytokines and growth factors, increase oxidative stress, induce extracellular matrix expansion and angiogenesis [3-5]. AGEs formation had been suggested to be involved in the development of chronic clinical diseases such as diabetes mellitus and its complications, macrovascular diseases and ageing. [0004] Formation of AGEs interrupts the proper functions, and/or changes the morphological properties of glycated proteins, lipids and/or DNA. Some AGEs can crosslink with adjacent proteins by covalent bonds to reduce the flexibility, elasticity, and functionality of the proteins. For example, accumulated glycation of extracellular collagen and elastin in cardiovascular tissues reduces the arterial flexibility or elasticity and increases myocardial and vascular stiffness, which in turn confers cardiovascular risks and aggravates the adverse effects of aging and diabetes[6, 7].

[0005] Intracellular AGEs accumulation is countered by high turnover and short half- life of many cellular proteins. However, slow turnover and long half-life of extracellular proteins such as collagen and elastin favour extracellular AGEs accumulation [8]. AGEs are mainly removed by degradation requiring specific AGEs receptors, internalization and proteolytic processing, while some may be removed by enzymatic repair mechanisms [3]. [0006] Accumulation of AGEs in tissues and plasma has also been associated with debilitating diseases such as the development of cardiovascular diseases (including hypertension, stroke, ventricular hypertrophy, atherosclerosis), diabetes mellitus and its complications including nephropathy, retinopathy and neuropathy[9-13]. AGEs have also been implicated in the pathogenesis of Alzheimer's disease and rheumatoid arthritis as well as the normal aging process. Because hyperlipidemia, hyperglycemia, diabetes and metabolic syndrome are common causes of morbidity and mortality, methods to counteract the symptoms and consequences of these metabolic states are needed.

[0007] Age-dependent increases in AGEs were detected in aortic and kidney tissues from spontaneously hypertensive rats (SHR) from 8 weeks on [10, 14]. Normalization of AGE level in aortic tissues from SHR with chronic administration of aminoguanidine, an AGE inhibitor, was accompanied with decreased blood pressure [15]. Increased AGEs in aortic tissues was associated with elevation of blood pressure in chronically fructose-fed Sprague Dawley (SD) rats. Application of the AGEs inhibitor metformin decreased fructose- fed induced AGEs accumulation, as well as elevation in blood pressure [9]. [0008] Precursors to AGE formation include reactive carbonyl species, including but not limited to methylglyoxal (MG), glyoxal, and 3-deoxyglucosone (3-DG). Increased reactive dicarbonyl molecules, MG, glyoxal, and 3-DG are reported in pathological conditions including hypertension, diabeties and its complications. Among these reactive dicarbonyl molecules, MG is the most active one and believed to be the major generation of AGEs. [0009] MG is generated during the metabolism of glucose, triglyceride, and protein through both non-enzymatic and enzymatic pathways in mammalian cells, including vascular smooth muscle cells (VSMCs) [16]. MG is degraded inside cells by glyoxalase system using glutathione (GSH) as the co-factor. MG interacts, first reversibly and then irreversibly, with the side chains of arginine, lysine, and cysteine residues through the dicarbonyl groups in proteins to form different types of adducts called AGEs [17]. As noted above, reaction of MG with arginine to form argpyrimidine, and reaction with lysine to form CEL. As compared with the reaction with the side chains of arginine or lysine residues, the thiol groups of cysteine residues with low pKa values are favorable nucleophiles to react with MG or glyoxal to produce AGEs such as carboxymethyl cysteine (CMC) and carboxyethyl cysteine (CEC) [18]. [0010] Increased MG production and MG-induced AGEs have been reported in diabetes and hypertension [10, 17, 19]. Elevated cellular MG level has been reported in human red blood cells, cultured bovine endothelial cells and rat VSMCs, which were exposed to hyperglycemic conditions or with increased availability of MG precursors including glucose, fructose, acetol, sucrose, and aminoacetone [20-23].

[0011] Accumulation of endogenous MG has been observed and extensively studied in diabetes mellitus and diabetic complications for its potential pathogenic role in these pathologic situations [24-26] For example, physiological concentration of plasma MG in streptozotocin-induced diabetic SD rats is approximately 5 μM in contrast to 2 μM in non- diabetic control [27] In humans, plasma levels of MG was increased from 1.4 μM in healthy humans (average age=51.6±17.6 years) to 3.6 μM in diabetic patients (average age = 52.6±17.4 years) [28] It has also been shown that plasma levels of MG in type 2 diabetic patients (average age=61.4±1.6 years) were 5.9 ± 0.7 μM, 77% higher than non-diabetic subjects (average age=61.9±3.2 years) with a MG level of 3.3 ± 0.4 μM [19]. Plasma level of MG in young type 1 diabetic patients (average age=15.0±4.2 years) was elevated to 0.84 ± 0.24 μM from 0.44 ± 0.09 μM in non-diabetic group (average age=14.6±4.6 years) [29]. [0012] As noted above, it is thought that elevated MG may constitute one of the causative factors in the development of insulin resistance, which is a hallmark of diabetes and its complications.

[0013] It has recently been shown that hypertension development in spontaneously hypertensive rats (SHR) was related to increased MG levels in plasma and vascular tissues in an age-dependent fashion [10, 14]. The plasma MG levels were 33.6 μM in 20-week-old SHR and 14.2 μM in age-matched non-hypertensive Wista-Kyoto (WKY) rats [10]. Elevated cellular MG level was also observed in cultured VSMC from SHR [30]. [0014] Along the same line, a rise in blood pressure was reported in SD rats and normotensive WKY rats treated with the MG precursor fructose or MG itself [9, 31]. Application of metformin, a drug used to decrease blood glucose level, reduced MG levels in plasma and vascular tissues in fructose-treated SD rats, which was associated with a decrease in blood pressure and reversed mesenteric artery wall thickness [9].

[0015] The foregoing observations suggest that MG participates in the proliferative vascular disease including hypertension in addition to the development of diabetes and its complications.

[0016] Being an important risk factor for hypertension and diabetes, obesity is well recognized as a result of excessive consumption of dietary fat and carbohydrates, which are both major precursors of MG and AGEs. The development of obesity involves both adipocyte hypertrophy and hyperplasia [33, 34]. While imbalanced energy intake-induced adipocyte hypertrophy contributes to the typically adult-onset obesity, the development of hyperplastic adipose tissue is thought mainly associated with the obesity in children [35, 36](18c, 19c). However, proliferation of adipocytes is also observed in adult obesity. Recently, the role of PI3K/Akt and its downstream molecules such as p21 and p27 in regulating each step of adipogenesis, including the proliferation of preadipocytes [37-42] has been disclosed. It has been found that Akt phosphorylates cyclin-dependent kinase inhibitors p21 and p27, prevents the localization of these proteins in nucleus, and thus attenuates their inhibitory effect on CDK2 and the cell cycle progression from Gl to S phase [43-45]. Loss of cyclin-dependent kinase inhibitors produces adipocyte hyperplasia and obesity [44]. In addition, the degradation of cyclin-dependent kinase inhibitors is also required for the cellular transition from quiescence to the proliferative state.

[0017] As noted above, increased MG production has been reported in human red blood cells and cultured bovine endothelial cells and rat VSMCs under hyperglycemic conditions or with increased availability MG precursors such as fructose[20, 21]. These observations suggest that MG may also participate in the proliferative vascular diseases including hypertension [32].

[0018] VSMCs in vivo are in a quiescent state under physiological conditions.

Transformation of VSMCs from contractile to proliferative phenotype was observed in pathophysiological conditions such as hypertension and atherosclerosis [46-48]. Increased proliferation rate and DNA synthesis were reported in VSMC from SHR compared with normotensive WKY rats [49, 50]. However, little is known about the molecular mechanisms underlying the abnormal proliferation of VSMCs in cardiovascular diseases. Cell proliferation is controlled by cell cycle regulators composed of cyclins, cyclin-dependent kinases (CDKs), and cyclin-dependent kinase inhibitors (CKIs) [51]. Coordination of the activities of these proteins directs cell cycle towards either proliferation or growth arrest [51-53]. One of these "coordinators" is serine/threonine kinase B (Akt), also known as protein kinase B. For example, p21 is a CKI that negatively controls cell proliferation. Over-expressed Akt decreases[54], but a dominant-negative Akt mutant increases p21 protein levels [55]. Phosphorylation of p21 by Akt results in exporting phospho-p21 from nucleus to cytoplasm where it is subjected to phosphorylation-dependent proteolysis [42]. Expression of a constitutively activated Akt increased cell proliferation and inhibited apoptosis, but blocking

Akt activity resulted in cell cycle arrest and apoptosis [54, 55].

[0019] Akt is a key node in cell signaling downstream of growth factors, cytokines, and other cellular stimuli controlling cell proliferation, growth, survival and metabolism [54,

56]. There are 3 isoforms of Akt, i.e. Aktl, Akt2, and Akt3, each with multiple targets and different functional specificity. Gene knockout studies have revealed decreased size of all organs in Aktl-/- mice [[57]], impaired glucose homeostasis in Akt2-/- mice [58, 59] and an uniformly reduced brain size in Akt3-/- mice [60]. Increased phospho-Akt levels were reported in aortic and mesenteric arteries from Ang II-induced hypertensive SD rats [61].

Elevated levels of Aktl protein and activity were detected in aortic tissues from hypertensive

SHR compared with age-matched normotensive WKY rats [62]. In moderate hypertensive

Zucker fa/fa rats, increased Aktl protein level and activity in aortic tissues were also reported

[62].

[0020] Whether MG-caused VSMC over-proliferation in vasculoproliferative diseases is mediated by Aktl signaling has been unclear.

[0021] It would be desirable to reduced AGE formation in cells and/or tissues.

Alternatively, or in addition to, it would be desirable to inhibit those signaling protein(s) through which MG exerts a pathologic effect.

[0022] Currently no specific or effective agent or scavenger/trapping-agent against

MG is available.

[0023] In additional to MG, other reactive dicarbonyl molecules such as glyoxal and

3 -DG are also produced in physiological systems.

[0024] As mentioned before, MG is an intrinsic component of glycolysis, lipolysis and protein catabolism. In addition, MG, glyoxal, and 3-DG can be produced during the slow spontaneous oxidative degradation of glucose and the early glycation adducts through a retroaldol reaction followed by oxidation of the resulted glycolaldehyde [63, 64]. Glyoxal is also generated by exposure of DNA or deoxynucleosides to oxygen free radicals, and lipid peroxidation [65, 66]. Fructose, specifically fructose-3-phosphate, has been indicated as another potential precursor of 3-DG [67].

[0025] It was reported that plasma glyoxal levels in type 1 diabetic patients were increased to 1.05 ± 0.52 μM as compared with non-diabetic patients with a glyoxal level of

0.33 ± 0.21 μM [29].

[0026] It was also reported that fasting serum 3-DG level in diabetic patients (0.353 ±

0.11 μM) was significantly higher than that in control subjects (0.199 ± 0.053 μM) [68].

[0027] In addition to MG, it is believed that elevated levels of glyoxal and 3-DG may also be involved in the development of diabetes and its complications. Since reactive dicarbonyl molecules such as MG and glyoxal are 20,000 times more reactive than glucose in

AGEs formation [69][31], MG, together with glyoxal and 3-DG, is believed to be a major source of intracellular and extracellular AGEs [2].

[0028] It has been reported that inhibiting AGE formation or breaking AGE crosslink could improve or reverse the deleterious effects induced by AGEs accumulation [9, 70-73].

An AGEs inhibitor, OPB-9195, was demonstrated to lower glycated albumin in plasma, reduce systolic blood pressure and oxidative damage in stroke-prone spontaneously hypertensive rats [72].

[0029] AGEs inhibitors including aminoguanidine, pyridoxamine, metformin and alagebrium are effective in reserving normal HDL function in diabetes [74]. It has also been shown that metformin prevented fructose-induced hypertension, vascular remodelling and

AGEs formation [9].

[0030] Reversing of aortic stiffening along with a restoration of left ventricular elasticity by alagebrium treatment was observed in SHR [73]. Studies using several animal models also indicated the beneficial effect of alagebrium in reversing the cardiovascular complications of aging and diabetes [70, 71, 73].

[0031] Thus, reducing or preventing AGEs formation has emerged as a strategy for the treatment of AGEs-related diseases. As AGEs is formed mainly and rapidly from reactive carbonyl molecules such as MG, eliminating these reactive carbonyl molecules in tissues and plasma would be a more direct strategy to prevent AGEs formation as compared to breaking the already formed AGEs.

[0032] Currently, a compound that can specifically react with, and effectively scavenge, reactive carbonyl molecules without harmful effects is absent. Commonly used pharmacological compounds are either non-specific or having harmful side effects. Clinical trials for aminoguanidine have proved disappointing due to its adverse reactions [75]. Higher doses of metformin are needed to achieve AGEs lowering effects [2, 24]. Although promising results from preclinical [70, 76, 77] and clinical trials [7, 73, 78] conducted and being conducted for alagebrium chloride, a claimed AGEs breaker, are reported, the therapeutic mechanism of alagebrium is poorly understood.

[0033] The presence of reactive dicarbonyls, such as MG and glyoxal, and the resulting formation of AGEs constitutes a great risk factor to human health. It is, therefore, desirable to provide compositions and/or methods for reducing the deleterious effects of reactive carbonyls, such as MG.

[0034] Mitochondria are the powerhouse of mammalian cells. When electrons pass through complexes I - IV of the electron transport chain (ETC), 2-5% of electrons leak out of the ETC and interact with oxygen to form superoxide (O₂ ^") in mitochondria, which accounts for about 85% of total intracellular O₂ ^" [ld,2d]. Electron leakage most often occurs at complex I and complex III of the ETC, and the amount of O₂ ^" increases dramatically if these complexes are inhibited [3d]. Under physiological condition, O₂ ^" is converted to hydrogen peroxide (H₂O₂) by manganese superoxide dismutase (MnSOD), which is the primary antioxidant defensive enzyme in mitochondria [4d]. This anti-oxidant system ensures the clearance of free radicals and protects cells against oxidative damage. Mitochondria also contain specific nitric oxide synthase (mtNOS), which catalyzes the production of nitric oxide (NO) [5d]. A considerable amount of NO generated from mtNOS reacts with O₂ ^" to form peroxynitrite (ONOO^") [6d]. ONOO^" is a highly reactive oxidant, damaging proteins, DNA, and lipids [7d]. Mitochondrial oxidative stress is tightly related to the pathophysiology of type 2 diabetes and associated complications [8d]. [0035] It has been reported that MG [14d] or fructose (a precursor of MG) [15d] induced the production of ONOO^" in cultured rat thoracic aortic smooth muscle cells (A-IO cells).

[0036] The role of MG in the regulation of mitochondrial function is unclear.

[0037] This background information is provided for the purpose of making know information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should it be construed, that any of the preceding information constitutes prior art against the present invention.

SUMMARY OF THE INVENTION

[0038] An object of the present invention is to provide composition(s) and/or method(s) for preventing, reducing, treating, ameliorating and/or abrogating complications resulting from reactive diacarbonyls and/or AGEs.

[0039] In accordance with one aspect of the present invention there is provided An isolated peptide comprising an amino acid sequence selected from CLQ (SEQ. ID NO: lor

CLQWTTVIER (SEQ. ID. NO: 2). In one example, said peptide binds and/or scavenges a reactive carbonyl. In one example, said peptide inhibits formation of glycation endproducts in vitro or in vivo. In a specific example, said reactive carbonyl is methylglyoxal (MG) or glyoxal.

[0040] In another aspect of the present invention there is provided a pharmaceutical composition for treating, preventing, or ameliorating an AGE related condition or disease in a mammal, comprising an effective amount of an isolated peptide or a pharmaceutically acceptable salt thereof comprising an amino acid sequence selected from CLQ (SEQ. ID NO: lor CLQWTTVIER (SEQ. ID. NO: 2), and a pharmaceutical carrier.

[0041] In one example, said condition or disease is vascular disease; insulin resistance; diabetes mellitus; hyperlipidemia; hyperglycemia; metabolic syndrome; nephropathy; retinopathy; neuropathy; heart and artery disease; neurodegenerative diseases; endocrine, renal, respiratory, reproductive conditions; skin ageing; premature aging; rheumatoid arthritis; Alzheimer's disease; uremia; neurotoxicity or discolouration of teeth. In a specific example, said vascular disease is hypertension, stroke, ventricular hypertrophy, atherosclerosis, restenosis or stroke.

[0042] Said pharmaceutical composition is formulated for administration by bolus injection, intravenous infusion, subcutaneous infusion, oral administration, pulmonary administration, nasal administration, transdermal administration, parenteral administration, rectal administration or topical administration.

[0043] In another aspect of the present invention there is provided a use of an isolated peptide comprising an amino acid sequence selected from CLQ (SEQ. ID NO: lor

CLQWTTVIER (SEQ. ID. NO: 2) for treating, preventing and/or ameliorating an AGE related condition or disease in a mammal.

[0044] In one example, said condition or disease is vascular disease; insulin resistance; diabetes mellitus; hyperlipidemia; hyperglycemia; metabolic syndrome; nephropathy; retinopathy; neuropathy; heart and artery disease; neurodegenerative diseases; endocrine, renal, respiratory, reproductive conditions; skin ageing; premature aging; rheumatoid arthritis; Alzheimer's disease; uremia; neurotoxicity or discolouration of teeth. In a specific example, said vascular disease is hypertension, stroke, ventricular hypertrophy, atherosclerosis, restenosis or stroke. In another example, said peptide inhibits formation of glycation endproducts. Said peptide is suitable for administration by a single dosage or a variable dosage. In a specific example, said mammal is a human.

[0045] In another aspect of the present invention there is provided a use of an isolated peptide comprising an amino acid sequence selected from CLQ (SEQ. ID NO: lor

CLQWTTVIER (SEQ. ID. NO: 2) for preventing and/or reducing spoilage of proteins in food.

[0046] In another aspect of the present invention there is provided A kit for inhibiting formation of glycation endproducts in an organism, said kit comprising: a peptide comprising isolated peptide comprising an amino acid sequence selected from CLQ (SEQ. ID NO: lor CLQWTTVIER (SEQ. ID. NO: 2); and instructions for the use thereof. In one example, said peptide binds and/or scavenges a reactive carbonyl. In another example, said peptide inhibits formation of glycation endproducts in vitro or in vivo. In a specific example, said reactive carbonyl is MG or glyoxal.

[0047] In accordance with another aspect of the present invention there is provided a method of slowing progress in a patient of complications resulting from diabetes, wherein said complications results from formation of glycation endproducts or protein crosslinking, said method comprises administering an effective amount of a compound or pharmaceutically acceptable salt of said compound to said organism wherein said compound comprises an isolated peptide comprising an amino acid sequence selected from CLQ (SEQ. ID NO: lor CLQWTTVIER (SEQ. ID. NO: 2)

[0048] In accordance with one aspect of the present invention there is provided a method of slowing progress in a patient of complication resulting from hypertension, wherein said complications results from formation of glycation endproducts or protein crosslinking, said method comprises administering an effective amount of a compound or pharmaceutically acceptable salt of said compound to said organism wherein said compound comprises an isolated peptide comprising an amino acid sequence selected from CLQ (SEQ. ID NO: lor CLQWTTVIER (SEQ. ID. NO: 2)

[0049] In accordance with one aspect of the present invention there is provided a method of treating complications resulting from diabetes which results from elevated levels of methylglyoxal or glyoxal or both methylglyoxal and glyoxal, the method comprising administering an effective amount of a compound comprises an isolated peptide comprising an amino acid sequence selected from CLQ (SEQ. ID NO: lor CLQWTTVIER (SEQ. ID. NO: 2).

[0050] In accordance with one aspect of the present invention there is provided a method of preventing spoilage of proteins in foodstuffs wherein said method comprises mixing an effective amount of a compound or a pharmaceutically acceptable salt of said compound with said foodstuffs, wherein said effective amount inhibits formation of glycation endproducts or protein crosslinking, wherein said compound comprises an isolated peptide comprising an amino acid sequence selected from CLQ (SEQ. ID NO: lor CLQ WTTVIER (SEQ. ID. NO: 2).

[0051] In accordance with another aspect of the present invention, there is provided a method of slowing progress of complications in a patient resulting from a deleterious condition wherein said complications result from formation of glycation endproducts or protein crosslinking resulting from glycation, wherein said method comprises administering an effective amount of a compound or a pharmaceutically acceptable salt of said compound to said patient wherein said compound comprises an isolated peptide comprising an amino acid sequence selected from CLQ (SEQ. ID NO: lor CLQ WTTVIER (SEQ. ID. NO: 2). In one example, said deleterious condition is vascular disease; insulin resistance; diabetes mellitus; hyperlipidemia; hyperglycemia; metabolic syndrome; nephropathy; retinopathy; neuropathy; heart and artery disease; neurodegenerative diseases; endocrine, renal, respiratory, reproductive conditions; skin ageing; premature aging; rheumatoid arthritis; Alzheimer's disease; uremia; neurotoxicity or discolouration of teeth. In one example, said vascular disease is hypertension, stroke, ventricular hypertrophy, atherosclerosis, restenosis or stroke. [0052] In accordance with another aspect of the present invention, there is provided a method for inhibiting MG-induced Aktl activation in a cell population, the method comprising the steps: (a) providing a therapeutically effective amount of an Aktl inhibiting composition to a subject in need thereof, (b) contacting at least one MG-induced Aktl cell in the cell population with the kinase inhibitory composition such that said Aktl inhibiting composition associates with the at least one MG-induced Aktl cell; and (c) inhibiting the MG-induced Aktl activation of the at least one MG-induced Aktl cell. [0053] In accordance with another aspect of the present invention, said Aktl inhibiting composition comprises an inhibitory amount of a SH-6.

[0054] In accordance with another aspect of the preset invention, said Aktl inhibiting composition comprises an inhibitory amount of Akt siRNA. [0055] In accordance with a specific example, said Akt siRNA is D-OOl 810-01-05 from Dharmacon.

[0056] In accordance with another specific example, said Akt inhibitor is Akt inhibitor

I, II, III, IV, V, VI, VII, VIII, IX, XI, XII or XIII.

[0057] In accordance with another aspect of the present invention, there is provided a method of slowing progress in a patient of complications resulting from a deleterious condition wherein said complications result from Aktl activation in a cell population, wherein said method comprises administering an effective amount of a compound or a pharmaceutically acceptable salt of said compound to said patient wherein said compound comprises a therapeutically effective amount of an Aktl inhibiting composition wherein said deleterious condition is vascular disease; insulin resistance; diabetes mellitus; hyperlipidemia; hyperglycemia; metabolic syndrome; nephropathy; retinopathy; neuropathy; heart and artery disease; neurodegenerative diseases; endocrine, renal, respiratory, reproductive conditions; skin ageing; premature aging; rheumatoid arthritis; Alzheimer's disease; uremia; neurotoxicity or discolouration of teeth. In one example, said vascular disease is hypertension, stroke, ventricular hypertrophy, atherosclerosis, restenosis or stroke. [0058] In accordance with another aspect of the present invention there is provided a kit for inhibiting formation of glycation endproducts in an organism, said kit comprising (a) an Aktl inhibiting composition and (b)instructions for the use thereof. In one example, said Aktl inhibiting composition comprises an inhibitory amount of a SH-6. In another example, said Aktl inhibiting composition comprises an inhibitory amount of Akt siRNA. In one example, said Akt siRNA is D-001810-01 -05 from Dharmacon. In another example, said Akt inhibiting composition is Akt inhibitor I, II, III, IV, V, VI, VII, VIII, IX, XI, XII or XIII.

BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 depicts the effects of MG treatment on cell proliferation and DNA synthesis. (Panel A) depicts the effect of MG at different concentrations on cell proliferation and blockage of MG effect by SH-6. n= 8-16 for each group. * P<0.05 vs. control. # P<0.05 vs. MG treatment at the same concentration. Cell proliferation was determined by a colorimetric method. (Panels B & C) depict the effect of MG on DNA synthesis and blockage of MG effect by SH-6. n=4~8 for each group. * P<0.05 vs. control. # P<0.05 vs. MG treatment at the same concentration. [³H]-thymidine incorporation was determined by liquid scintillation counting and expressed as disintegrations per minute (DPM).; [0060] Figure 2 depicts the effect of MG treatment on cell apoptosis. (Panel A) shows representative result of stained nuclear chromatins with Hoechst 33258. Arrows indicate apoptotic cells (Panel B) shows percentage of apoptotic cell in MG treated groups. n= 9 for each group. ** P<0.01 vs. control.;

[0061] Figure 3 depicts Aktl silencing abolished MG effect on cell proliferation.

(Panel A & B) are representative Western blot result and summary of Aktl expression level in Aktl siRNA transfected cells. NT: non-transfected cells, CT (-): negative control siRNA transfection. Mock: mocked transfection. Data were from 4 independent experiments. ** P<0.01 vs. control (-). (Panels C & D) depict the effects of MG treatment on cell proliferation and DNA synthesis in Aktl silenced cells. n=8 for each group. * P<0.05 vs. control (-); # P<0.05 or ## P<0.01 vs. MG treatment at the same concentration.;

[0062] Figure 4 shows that MG treatment increased the levels of phospho-Akt and phospho-GSK-3. (Panel A) depicts representative Western blot result of phospho-Akt in MG- treated cells. CT: control cells. (Panel B) is a graph depicting the summary of phospho-Akt levels in MG-treated cells. Data were from 3 independent experiments. * P<0.05 vs. control, # P<0.05 vs. MG treatment at the same concentration. (Panel C) depicts representative Western blot result of phospho-GSK-3 in MG-treated cells. (Panel D) is a graph depicting a summary of phospho-GSK-3 levels in MG-treated cells. Data were from 3 independent experiments. * P<0.05 vs. control; # P<0.05 vs. MG treatment at the same concentration.; [0063] Figure 5 shows that MG treatment increased the level of phospho-Akt 1 and

Aktl kinase activity. (Panel A) depicts representative Western blot result of phospho-Akt in MG-treated cells. (Panel B) is a graph depicting a summary of phospho-Akt 1 levels in MG- treated cells. Data were from 3 independent experiments. * P<0.05 vs. control; # P<0.05 vs. MG treatment at the same concentration. (Panel C) depicts representative result of Aktl kinase activity in MG-treated cells. (Panel D) is a graph depicting a summary of Aktl kinase activity in MG-treated cells. Data were from 3 independent experiments. * P<0.05 vs. control; # P<0.05 vs. MG treatment at the same concentration.;

[0064] Figure 6 depicts effects of MG treatment on the levels of p21 and phospho-p21 proteins. (Panel A) depicts representative Western blot result of p21 and phospho-p21 in MG- treated cells. CT: control cells; p-p21: phospho-p21. (Panels B & C) are graphs depicting a summary of p21 and phospho-p21 levels in MG-treated cells. Data were from 3 independent experiments. * P<0.05 vs. control ** P<0.01 vs. control # P<0.05 vs. MG treatment at the same concentration.;

[0065] Figure 7 depicts the effects of MG treatment on the levels of p27 and phospho- p27 proteins. (Panel A) depicts a representative Western blot result of p27 and phospho-p27 in MG-treated cells. CT: control cells; p-p27: phospho-p27. (Panels B & C) are graphs depicting a summary of p27 and phospho-p27 levels in MG-treated cells. Data were from 3 independent experiments. * P<0.05 vs. control.;

[0066] Figure 8 depicts the effects of MG treatment on the levels of CDK2 protein and its activity. (Panel A) depicts a representative Western blot result of CDK2 proteins in MG treated VSMCs and a summary of CDK2 levels in MG-treated cells normalized by the loading of β-actin proteins determined by Western blot. (Panel B) is a graph depicting representative autoradiography of immunoprecipitated CDK2 protein activity using histone Hl and ³²P-ATP as the substrates and a summary result of CDK2 activity in MG-treated cells based on the density of autoradiography. Data were from 3 independent experiments. * P<0.05 vs. control. # P<0.05 vs. MG treatment at the same concentration.; [0067] Figure 9 depicts MALDI-TOF MS and MS/MS analysis of MG-treated Atkl proteins. (Panel A) MALDI-TOF MS analysis of phospho-Aktl after treatment with or without MG for 24 hours. In the PMF spectrum of untreated and MG-treated (50μmol/L) Aktl proteins, the peak at m/z 1248.7 corresponds to tryptic peptide T_n (CLQ WTTVIER), and the peak at m/z 1302.7 corresponds to TI l with a mass increase of 54 Da. (Panel B) MS/MS spectra of peptide Tn phospho-Aktl with or without MG treatment. The masses of observed b-ions (b₂, b₃, b₄, bs) are increased by 54 Da, while the masses of observed y'-ions (y'u y'2, y'₃, y'₄, y'5, y'ό) remained as predicted, indicating the modification of Cys77 residue by MG. MALDI-TOF MS analysis of unphospho-Aktl treated with or without MG (50μmol/L) in the presence (C) or absence (D) of PIP3 (5μmol/L). The peak at m/z 1302.7 corresponds to the tryptic peptide TI l with a mass increase of 54 Da, indicating the modification of unphospho-Aktl by MG in the presence of PIP3. The peak at m/z 3039.7 corresponds to the tryptic peptides of m/z 1248.7 (Cys77) and 1792.8 (Cys60) connected by a disulfide bond.;

[0068] Figure 10 depicts MALDI-TOF MS and Q-TOF MS/MS analysis of MG- treated synthetic peptide Tl 1. (Panel A) shows MALDI-TOF MS analysis of MG-treated and untreated peptide TI l. The peak at m/z 1290.7 corresponding to N-acetylated synthetic peptide Tl 1 was shifted to m/z 1362.7, indicating an addition of an intact MG (72 Da) to the synthetic peptide after MG (50 μM) treatment. (Panel B) shows MS/MS spectra of MG- treated and untreated synthetic peptide TI l. The mass of the observed b-ions (b2, b3, b4, b5) was increased by 72 Da, but the mass of observed y'-ions (y' l, y'2, y'3, y'4, y'5, y'6, y'7) was not changed after MG treatment.;

[0069] Figure 11 depicts MG treatment increased the phosphorylation and activity of phospho- and unphospho-Aktl in cell-free in vitro study. (Panel A) depicts the representative Western blot and summary of levels of Aktl activity (determined by the levels of p-GSK) and phospho-Aktl (Ser473) determined after unphospho-Aktl (5 μg) was treated with and without MG (10, 30 μmol/L) for 24 hrs followed by exposure to MAPKAPK2 (125 mU) and PDKl (0.1 μg) activation for 30 mins, respectively. (Panels B) show the representative Western blot and summary of levels of phospho-Aktl, and Aktl activity (p-GSK) determined after phospho-Aktl (5 μg) was treated with or without MG (10, 30 μmol/L) for 24 hrs. p-GSK: phospho-GSK3; p-Aktl(Ser473): phospho-Aktl (Ser473). Data were from at least 3 experiments. * P<0.05 vs. control.; [0070] Figure 12 is a schematic model for MG-induced activation of Aktl and cell proliferation. Modification of Cys77 residue in Aktl by MG breaks the disulfide bond between Cys77 and Cys60 in Aktl PH domain, which may result in a conformational change(s) that favors Aktl phosphorylation and its activity. Enhanced activation of Aktl causes the phosphorylation of p21, followed by disrupting its association with CDK2/cyclin complex, and resulting in CDK2 activation to favor cell proliferation. Phosphorylation of GSK-3 by Aktl blocks its negative impact on cell cycle progression, thus also mediating MG- enhanced cell proliferation. Increased VSMC proliferation may contribute to vascular remodeling, dysfunction and the development of vascular diseases.;

[0071] Figure 13 depicts a schematic structure of (A) SMG08 and (B) methylglyoxal.

[0072] Figure 14 depicts the effect of SMG08 on scavenging of methylglyoxal (MG) in which SMG08 (10 μM) was incubated with MG (10 μM) in PBS at 37°C from 2 minutes to 24 hours. MG levels were determined by HPLC (data were from three independent experiments. ** P<0.01 vs. MG at 10 μM).;

[0073] Figure 15 is a graph depicting the effect of GSH, cysteine, NAC or metformin on scavenging of methyl gloxal, in which GSH, L-cysteine, NAC or metformin (each at 10 μM) were incubated with MG (10 μM) in PBS at 37⁰C from 2 minutes to 24 hours. MG levels were determined by HPLC (data were from three independent experiments. * P<0.05 vs. MG at 10 μM).

[0074] Figure 16 is a graph depicting the effect of aminoguanidine on scavenging of

MG in vitro, in which aminoguanidine (10 μM) was incubated with MG (10 μM) in PBS at 37⁰C from 2 minutes to 24 hours. MG levels were determined by HPLC (data were from three independent experiments. ** P<0.01 vs. MG at 10 μM).;

[0075] Figure 17 is a graph depicting the effect of alagebrium on scavenging MG in vitro, in which alagebrium chloride (10 μM) was incubated with MG (10 μM) in PBS at 37⁰C from 2 minutes to 24 hours. MG levels were determined by HPLC (data were from three independent experiments. ** P<0.01 vs. MG at 10 μM).; [0076] Figure 18 is a graph depicting the effect of SMG08 on scavenging of MG in cultured VSMCs (A-IO cell line) starved in FBS-free DMEM for 24 hours followed by 18 hours of pre-treatment with SMG08 at different concentrations (1, 5, 10, 25, 100 μM). After SMG08 pre-treatment, SMG08 was removed and medium was replaced with DMEM containing 10% FBS followed by 24 hours of MG (10 μM) treatment. Cells were harvested and the supernatant of cell lysis was subjected to MG concentration analysis using HPLC. Panel (A) shows the dose-dependent effect of SMG08 on scavenging cellular MG (data are from three independent experiments. * p<0.05 vs. MG at 10 μM. # p<0.05 vs. SMG08 (1 uM) pre-treatment. + p<0.05 vs. SMG08 (5, 25 and 50 uM) pre-treatment); Panel (B) shows the effect of SMG08 (100 μM) pre-treatment on scavenging cellular MG (data were from three independent experiments. * p<0.05 and ** P<0.01 vs. control. # p<0.05 vs. MG at 10 μM.; [0077] Figure 19 is a graph depicting the effects of aminoguanidine and alagebrium on cellular level of MG in cultured VSMCs starved in FBS-free DMEM for 24 hours followed by 18 hours of pre-treatment with aminoguanidine (100 μM) or alagebrium (100 μM). After pre-treatment, aminoguanidine or alagebrium was removed and medium was replaced with DMEM containing 10% FBS followed by 24 hours of MG (10 μM) treatment. Treated cells were harvested and the supernatant of cell lysis was subjected to MG concentration analysis using HPLC (data were from 3 independent experiments. * P<0.05 vs. control. # p<0.05 vs. MG at 10 μM. + p<0.05 vs. aminoguanidine pre-treatment).; [0078] Figure 20 is a graph depicting the effect of SMG08 on MG-induced VSMC proliferation. Cells seeded in 96-well plates with equal number of cells (5^χ10³) per well were starved in FBS-free DMEM for 24 hrs. Starved cells were pre-treated with SMG08 for about 8 hours and then treated with MG (10 μM) or glucose (25 mM) for 24 hours as described above. Cell proliferation was determined by a colorimetric method using One Solution cell proliferation assay kit (Promega) following manufacture's procedure. The quantity of colored formazan product measured by its absorbance at 492 nm is directly proportional to the number of living cells in culture (data were from 3 independent experiments. * P<0.05 vs. control. # p<0.05 vs. corresponding MG or glucose treatment).; [0079] Figure 21 is a graph depicting the effect of SMG08 on MG-induced reactive oxygen species (ROS) production in which cultured VSMCs were starved in FBS-free DMEM for 24 hours followed by 8 hour of pre-treatment with SMG08 at 100 μM. After pre- treatment with SMG08, cells were treated with MG (10 μM) for 24 hours. Cells were then loaded with DCFH-DA for two hour to generate DCFH-D A-loaded cells. The media was then removed, thereby removing unincorporated DCFH-DA. DCFH-DA-loaded cells were then incubated in media containing MG for another four hours. Intensity of fluorescence was recorded in the presence of 100 μl PBS/well after cells were washed 3 times with PBS (data were from 3 independent experiments. * P<0.05 vs. control. # P<0.05 vs. MG treatment).; [0080] Figure 22 shows the staining of FITC labeled SMG08 in VSMC. Cells seeded on glass cover slips were starved in FBS-free DMEM (phenol red free) for 24 hours, then were incubated in FBS-containing DMEM together with 100 μM of FITC-labelled SMG08 (SMG08-FITC) for 4 hours or 18 hours. Some of the overnight pre-treated cover slips were transferred to FBS-containing DMEM without SMG08-FITC and further incubated for 24 hours. Finally, treated and untreated cells on glass cover slips were washed with FBS-free DMEM at least 5 times and subjected to analysis using fluorescence microscope (Olympus 1X70, Tokyo, Japan).;

[0081] Figure 23 depicts the abolishing of MG-induced AGEs by SMG08 treatment in

VSMCsCells seeded on glass cover slips were starved in FBS-free DMEM for 24 hours before pre-treated with SMG08 (100 μM) for about 18 hours. Pre-treated cells were exposed to MG treatments for 24 hours. MG-treated and untreated cells were fixed and stained with anti-CML antibody. Cells were subjected to detection using a confocal fluorescence microscope (Olympus confocal microscope, FV5-PSU).;

[0082] Figure 24 is a graph depicting the effect of SMG08 on scavenging of glyoxal in vitro. SMG08 (10 μM) was incubated with glyoxal (10 μM) in PBS at 37⁰C from 2 minutes to 24 hours. Glyoxal levels were determined by HPLC. Data were from 3 independent experiments. ** P<0.01 vs. glyoxal at 10 μM.; [0083] Figure 25 is a graph depicting the effect of alagebrium on scavenging of glyoxal in vitro. Alagebrium (50 μM) was incubated with glyoxal (10 μM) in PBS at about 37°C from 2 minutes to 24 hour. Glyoxal levels were determined by HPLC. Data were from 3 independent experiments.;

[0084] Figure 26 is a graph depicting the effect of alagebrium on scavenging of 3 -DG in vitro. Alagebrium (50 μM) was incubated with 3-DG (10 μM) in PBS at 37⁰C from 2 minutes to 24 hours. 3-DG levels were determined by HPLC. Data are from 3 independent experiments.;

[0085] Figure 27 is a graph depicting the effect of SMG08 on scavenging of 3-DG in vitro SMG08 (10 μM) was incubated with 3-DG (10 μM) in PBS at 37⁰C from 2 minutes to 24 hours. 3-DG levels were determined by HPLC. Data are from 3 independent experiments. [0086] Figure 28 depict the correlation between MG level and body weight. The increase of plasma MG concentration is closely correlated to BMI value of patients (Panel A, r=0.606, P=0.0046, n=20) and the increase of body weight of Zucker rats (Panel B, r=0-772, P=0.0054, n=l l);

[0087] Figure 29 depicts basic parameters and MG levels in Zucker rats. The MG levels in kidney, fat, liver (Panel A) and serum (Panel B) were measured using HPLC. At 10, 12, 14 and 16 weeks of age, serum MG levels in obese and lean Zucker rats were measured. The value shown in (Panel C) is presented as percentage of MG level in age-matched lean Zucker rats. *P<0.05, **P<0.01 vs lean rats, n=4 in each group;

[0088] Figure 30 depicts the activity of PI3K/Akt pathway in the adipose tissue of lean and obese Zucker rats. Western blotting shows the phosphorylation (Thr308) and expression level of Akt protein (Panel A). The phosphorylation and the expression of Akt (pAkt) in lean and obese Zucker rats were quantified by the Chemigenus2 Bio imaging system (PerkinElmer, ON, Canada) and presented as the percentage of that in lean Zucker rats (Panel B). The PI3K activity in the adipose tissue of lean and obese Zucker rats was measured using a competitive ELISA kit and presented as the amount of PI(3,4,5)P3 produced (Panel C). *P<0.05, **P<0.05 vs lean Zucker rats, n=3-4 in each group; [0089] Figure 31 depicts the effect of MG, SH-6 or alagebrium (ALT-711) on 3T3-L1 cell proliferation. The proliferation of 3T3-L1 cells was determined by a Celltiter 96® nonradioactive cell proliferation assay kit. The relative cell proliferation of each group was presented as the ratio between arbitrary absorbance on 570nm of each group and that from the control group without treatment. The effect of different MG concentrations on cell proliferation was shown in (Panel A) and the effect of 10 μM MG with/without SH-6 and ALT-711 was shown in (Panel B). *P<0.05 vs control cells; +PO.05 vs MG treated cells; n=48 in each group;

[0090] Figure 32 depicts the effect of MG treatment on the cell cycle progression of 3T3-L1 cells. After 12, 16 and 20 hour of MG treatment, cellular DNA content was determined by a flow cytometry and percentages of Gl, S and Gl phases (a, b and c graph in panel A, respectively) were analyzed using FlowJo software (Panel A). The effect of MG treatment together with/without SH-6 or ALT-711 on cell phase distribution was shown in a, b and c graph in panel A, for percentages of Gl, S and Gl phases, respectively (Panel B). *P<0.05 vs control group; +P<0.05 vs MG treated group. The indicated percentages of the cell phases were an average of three experiments.;

[0091] Figure 33 depicts the effect of MG, SH-6 or ALT-711 on the phosphorylation and expression of Akt and its downstream targets in cultured 3T3-L1 cells. After 24 hours treatment with MG and/or SH-6/ALT-711 , the phosphorylation and expression of Akt, p21 and p27 were determined by Western blotting (Panel A). The results of Western blotting were quantified by Chemigenus2 Bio imaging system and presented as the percentage of that from control cells (Panel B). The activity (Panel C) of CDK2 in 3T3-L1 adipocytes treated with 10 or 20 μM MG was determined by measuring ATP consumption with a PKLight Assay Kit. *P<0.05 vs control (CT) cells; +P<0.05 vs MG treated cells. The results were based on data from three experiments.;

[0092] Figure 34 is a table ("Table V) depicting basic parameters of lean/obese Zucker rats. [0093] Figure 35 depicts MG levels in mitochondria of A-10 cells with MG (30 μM) treatment for 18 hours. ** p < 0.01 vs. untreated control cells, n = 4.; [0094] Figure 36 depicts the effect of MG on the fluorescence intensity of CEL in A-IO cells. (Panel A) MG increased the staining of CEL in A-IO cells, which was decreased by alagebrium. (Panel B) Cells were treated with MG (5-30 μM). (Panel C) Cells were co- treated with alagebrium (10-100 μM) and MG (30 μM). After treated with different agents for 18 hours, cells were stained using anti-CEL (1:100 at 4°C overnight) and secondary fluorescent antibody (FITC-IgG, 1:200 at room temperature for 3 hours) and read under Confocal microscope. Fluorescence intensity was analyzed using Image J program. ** p < 0.01 vs. cells without any treatment; ## p < 0.01 vs. cells treated with MG (30 μM) alone, n = 12.;

[0095] Figure 37 depicts the effect of MG on mitochondrial ROS generation in A- 10 cells. (Panel A) MG enhanced mitochondrial ROS generation, which was decreased by n-acetyl-1- cysteine (NAC), alagebrium, and uric acid. (Panel B) Cells were treated with MG (5-100 μM) in the presence or absence of NAC (600 μM). (Panel C) Cells were co-treated with alagebrium (10-100 μM) and MG (30 μM). (Panel D) Cells were co-treated with uric acid (50 μM) and MG (30 μM). After 18 hours treatment with different agents, cells were loaded with molecular probe MitoTracker Red (300 μM, 15 min) and read under Confocal microscope. Fluorescence intensity was analyzed using Image J program. * p < 0.05 and ** p < 0.01 vs. cells without any treatment; + p < 0.01 vs. MG treatment alone at the same concentration; ## p < 0.01 vs. cells treated with MG (30 μM) alone, n = 10-14.; [0096] Figure 38 depicts the effect of MG on NO production in A- 10 cells. Cells were treated with different agents for 18 hours. Molecular probe DAF-FM (5 μM, 2hours) was used to detect cellular levels of NO. ** p < 0.01 vs. control; # p < 0.05 and ## p < 0.01 vs. cells treated with MG (30 μM) alone, n = 8. NAC, n-acetyl-1-cysteine.;

[0097] Figure 39 depicts the effect of MG on the fluorescence intensity of nitrotyrosine in A- 10 cells. (Panel A) MG increased nitrotyrosine staining, which was inhibited by alagebrium and n-acetyl-1-cysteine (NAC). (Panel B) Cells were co-stained with anti-nitrotyrosine and MitoTracker Red to determine whether increased nitrotyrosine was located in mitochondria. (Panel C) Cells were treated with MG (5-30 μM) in the presence or absence of NAC (600 μM). (Panel D) Cells were co-treated with alagebrium (10-100 μM) and MG (30 μM). Cells were treated with different agents for 18 hours. Double cell staining of MitoTracker Red (300 μM, 15 min) and nitrotyrosine (anti-nitrotyrosine 1: 200 at 4°C overnight; FITC-IgG 1:200 at room temperature for 3 hours) were conducted. Cells were read under Confocal microscope. Fluorescence intensity was measured using Image J program. ** p < 0.01 vs. cells without any treatment; + p < 0.05 and ++ p < 0.01 vs. MG treatment alone at the same concentration; # p < 0.05 vs. cells treated with MG (30 μM) alone, n = 10-14.;

[0098] Figure 40 depicts the effect of MG on mitochondrial 02.- generation in A- 10 cells. (Panel A) MG increased MitoSOX signal in mitochondria, which was decreased by alagebrium and 4-hydroxy-tempo (Tempol). (Panel B) Mitochondrial 02.- generation in A- 10 cells. After treated with different agents for 18 hours, cells were loaded with molecular probe MitoSOX (2 μM, 20 min) and read under Confocal microscope. Fluorescence intensity was measured using Image J program. ** p < 0.01 vs. control; ## p < 0.01 vs. cells treated with MG (30 μM) alone, n = 12.;

[0099] Figure 41 depicts the effect of MG on MnSOD activity in A- 10 cells. (Panel A) MG (5-30 μM) decreased MnSOD activity in A-10 cells. (Panel B) MnSOD activity in A-10 cells co-treated with alagebrium (10-100 μM) and MG (30 μM). Cells were treated with different agents for 18 hours. SOD Assay Kit was used to detect SOD activity. KCN at 3 mM was used to inhibit the activity of Cu/Zn SOD, leaving only MnSOD activity to be measured. * p < 0.05 vs. cells without any treatment; # p < 0.05 vs. cells treated with MG (30 μM) alone, n = 4.;

[00100] Figure 42 depicts the effect of MG on mitochondrial complexes in A-10 cells.

(Panel A) Effect of MG on activities of complex I, complex III and complex IV in A-10 cells. Cells were treated with different agents for 18 hours. * p < 0.05 vs. control; # p < 0.05 vs. cells treated with MG (30 μM) alone, n = 4. (Panel B) Effect of MG on mitochondrial 02.- generation in the presence of different inhibitors of respiratory complexes. A-10 cells were treated with different agents for 2 hours. Rotenone, thenoyltrifluoroacetone (TTFA), antimycin A and KCN are inhibitors of complexs I, II, III and IV, respectively. * p < 0.05 and ** p < 0.01 vs. control; # p < 0.05 and ## p < 0.01 vs. inhibitor alone treated cells, n = 12.;

[00101] Figure 43 depicts the effect of MG on ATP synthesis (30 min) in mitochondria of A- 10 cells. Cells were treated with different agents for 18 hours, and ATP levels were determined using ATP Bioluminescent Assay Kit. ** p < 0.01 vs. control; # p < 0.05 vs. cells treated with MG (30 μM) alone, n = 4.;

[00102] Figure 44 depicts elevation of phospho-Aktl levels in aortic tissue from SD rats chronically fed with fructose or treated with MG. (Panel A). Representative Western blot result and summary of phospho-Aktl levels from rats (4 weeks old) fed with normal diet, fructose (Fruc, 60%), metformin (Met, 500 mg/kg per day) fructose + metformin (Met), and Met for 16 weeks. (Panel B). Representative Western blot result and summary of phospho- Aktl levels from the rats (12 weeks old) implanted with or without MG minipump in the absence or presence of alagebrium for 4 weeks. CT: untreated animal in A and rats implanted with 0.9% saline osmotic minipump in B; MG pump: rats implanted with MG-minipump (60 mg/kg/day). ALA: alagebrium, 20 mg/kg/day in drinking water. Data were from 3 independent experiments. n=3-6 for each group in A and B. * P<0.05 vs. control; # P<0.01 vs. fructose-treated or MG-pump rats.;

[00103] Figure 45 depicts the effects of Aktl(C/S) mutation and MG treatment on

DNA synthesis and cell proliferation in HEK-293 cells. HEK-293 Cells stably transfected with vector, wild type Aktl or Aktl(C/S) mutant were treated with MG (10, 30 μmol/L) for 24 hours. Effects of MG treatment on cell proliferation (Panel A) and DNA synthesis (Panel B) in cells transfected with different plasmid constructs. Vector: empty vector; WT: wild type Aktl; Mutant: Aktl(C/S) mutation. C. Effects of MG treatment on cell number in cells transfected with different plasmid constructs. Equal numbers of cells were seeded in culture dish for each group. Cells were treated with MG (10, 30 μmol/L) for 5 days and culture medium was changed every 24 hrs during the treatment. Final cell number was determined using a Beckman Coulter counter. n= 8—16 for each group in A & B, n=5~9 for each group in C. * P<0.05, ** P<0.01 vs. untreated empty vector transfected cells; # P<0.05 vs. untreated wild type Aktl transfected cells.;

[00104] Figure 46 depicts Aktl(C/S) mutation and MG treatment increased the levels of phospho-Aktl and phospho-GSK-3α/β in transfected HEK-293 cells, and the activity of Aktl mutant protein. (Panel A). Representative Western blot result and summary of phospho- Aktl in cells transfected with different plasmid constructs. (Panel B). Representative Western blot result and summary of phospho-GSK-3α/β in transfected cells. (Panel C). Representative Western blot result and summary of Flag-Aktl activity. Flag-Aktl proteins were immunoprecipitated from cells after MG treatment and Aktl activity was directly measured using exogenous GSK-3 fusion protein as the substrate. V: empty vector transfection; W: wild type Aktl transfection; M: Aktl(C/S) mutant transfection; p-Aktl: phospho-Aktl; p-GSK- 3α/β: phospho-GSK-3α/β. Data were from 3 independent experiments for each group. * P<0.05 vs. untreated wild type Aktl transfected cells; # P<0.05 vs. untreated wild type Aktl transfected cells.;

[00105] Figure 47 depicts effects of Aktl(C/S) mutation and MG treatment on the levels of p21, phospho-p21 and CDK2 activity in HEK-293 cells. A. Representative Western blot result and summary of p21 and phospho-p21. B. Representative result and summary of CDK2 activity in transfected cells. V: empty vector transfection; W: wild type Aktl transfection; M: Aktl(C/S) mutant transfection; p-p21: phospho-p21. Data were from 3 independent experiments for each group. * P<0.05 vs. untreated empty vector transfected cells; # P<0.05 vs. untreated wild type Aktl transfected cells;

[00106] Figure 48 depicts MALDI-TOF MS analysis of the reaction of SMG08 with

MG in vitro. SMG08 (100 μM) was incubated with or without MG (100 μM) in PBS at 37°C for 24 hrs. Analysis of the peptide samples were carried out on a 4800 MALDI TOF/TOF AnalyzerTM (Applied Biosystems) operating in the negative ion and reflector modes, using the default calibration. In the top panel, the peak at m/z 361.2 corresponds to SMG08 (-H+). The peaks (bottom panel) at m/z 415.2, 433.2 and 487.3 correspond to SMG08 (361.2) reacted with MG, with masses shift of +54, +72, and +126 Da. Peaks at m/z 415.2 and 433.2 are likely representing SMG08 reacted with MG with or without loss of H2O; Peak at m/z 487.3 is likely corresponding to the reacted SMG08 at m/z 415.2 with another MG molecule attached on the N-terminal of SMG08; and [00107] Figure 49 depicts CLQ (SEQ. ID NO: 1) and CLQWTTVIER (SEQ. ID. NO:

2).

DETAILED DESCRIPTION

[00108] As will be described in more detail below, the present invention relates to compounds, compositions, methods, kits and the like, capable of reacting with highly reactive carbonyl intermediates of an early glycation product, thereby preventing those highly reactive carbonyl products from later forming advanced glycation endproducts

[00109] In one aspect of the present invention, the compounds of the present invention have inhibitory effects on reactive carbonyls, such as dicarbonyls, and are potent inhibitors at concentrations lower than an equal inhibitory concentration of know and/or putative inhibitors of MG and/or glyoxal.

[00110] In one aspect of the present invention, the compounds of the present invention and their useful compositions are capable of reacting with highly active carbonyl intermediate of an early glycation product thereby preventing those early products from later forming the advanced glycation endproducts (AGEs) which lead to protein cross-linking, and the resulting deleterious effects.

[00111] Also, as will be described in more detailed below, the present invention relates to compounds, compositions, methods, kits and the like, capable of reducing the induced effects of a carbonyl molecules.

[00112] In another aspect of the present invention, the compounds of the present invention have an inhibitory effect on Aktl activity. In a specific example, the compounds of the present invention have an inhibitory effect on MG-induced Aktl activity. [00113] Accordingly, the compounds of the present invention can be used to inhibit and/or reduce nonenzymatic glycation and therefore inhibit, treat, reduce, prevent, ameliorate and/or abrogate the adverse effect of glycation and AGEs.

[00114] The compounds, compositions and/or peptides of the present invention are useful in the prevention, reduction, treating, amelioration and/or abrogation of AGE related conditions and/or disease(s).

[00115] The compounds, compositions and/or peptides of the present invention are a useful for preventing, treating, reducing, ameliorating and/or abrogating cardio vascular disease including, but not limited to hypertension, stroke, ventricular hypertrophy, atherosclerosis, restenosis, stroke; insulin resistance; diabetes mellitus; hyperlipidemia; hyperglycemia; metabolic syndrome; nephropathy; retinopathy; neuropathy; heart and artery disease; neurodegenerative diseases; endocrine, renal, respiratory, reproductive conditions; skin ageing (i.e. anti-wrinkling); premature aging; rheumatoid arthritis; Alzheimer's disease; uremia; neurotoxicity, and spoilage of proteins in food and/or discolouration of teeth. [00116] The term "peptide", as used herein, includes peptides, polypeptides, consensus molecules, analogs, derivatives, mimetic or combinations thereof. Accordingly, proteins, fusion-proteins or -peptides oligopeptides and polypeptides are included. If required, peptides according to the invention can be modified in vivo or in vitro, for example by glycosylation, amidation, carboxylation or phosphorylation. Functional variants like, for example, acid addition salts, amides, esters, C-terrminal esters, and N-acyl derivatives of the peptides according to the invention are therefore also considered part of the present invention. Peptides are prepared using well known methods of chemical synthesis, or are purchased from commercial sources and/or produced by recombinant means, well known to the skilled worker.

[00117] The term "organism" as used herein, includes mammals and non-mammals, humans, non-human primates, cultured cell lines and primary cells. [00118] Composition(s) [00119] Compositions of the present invention are suitable for reducing, inhibiting and/or preventing nonenzymatic glycation of protein(s) which often results in formation of advanced glycation endproducts.

In accordance with one aspect of the present invention, a composition of the present invention comprises an isolated peptide N-cysteine-leucine-glutamine-COOH (CLQ) (SEQ. ID. NO. 1) (Figure 49).

In accordance with one aspect of the present invention, a composition of the present invention comprises an isolated peptide N-_acetyi-- cysteine-leucine-glutamine-tryptophan- threonine-threonine-valine-isoleucine-glutamic acid-arginine-COOH (CLQWTTVIER) (SEQ. ID. NO. 2) (Figure 49).

[00120] The present invention also encompasses nucleic acid sequences encoding a peptide comprising CLQ and CLQWTTVIER.

[00121] The term "nucleic acid sequence" as used herein refers to a polymeric form of nucleotides of any length, both to ribonucleic acid sequences and to deoxy ribonucleic acid sequences. In principle, this term refers to the primary structure of the molecule. Thus, this term includes double and single stranded DNA, as well as double and single stranded RNA, and modifications thereof.

[00122] A nucleic acid sequence according to the present invention can be ligated to various replication effecting DNA sequences with which it is not associated or linked in nature resulting in a so called recombinant vector molecule which can be used for the transformation or transfection of a suitable host. Useful recombinant vector molecules, are preferably derived from, for example plasmids, bacteriophages, cosmids or viruses. [00123] Specific vectors or cloning vehicles which can be used to clone nucleic acid sequences according to the invention are known in the art and include inter alia plasmid vectors, M 13 derived phages or viral vectors. The methods to be used for the construction of a recombinant vector molecule according to the present invention are known to those of ordinarily skill in the art and are inter alia set forth in Maniatis, T. et al. (Molecular Cloning A Laboratory Manual, second edition; Cold Spring Harbor Laboratory, 1989). [00124] For example, the insertion of the nucleic acid sequence according to the present invention into a cloning vector is achieved when both the nucleic acid sequence and the desired cloning vehicle have been cut with the same restriction enzyme(s) as complementary DNA termini are thereby produced.

[00125] The recombinant vector molecules according to the invention may additionally contain one or more marker activities that may be used to select for desired transformants, such as ampicillin, tetracycline resistance, and the like.

[00126] The present invention also comprises (a) host cell(s) transformed or transfected with a nucleic acid sequence or recombinant expression vector molecule described above, capable of producing the peptides according to the invention by expression of the corresponding nucleic acid sequence. A suitable host cell is a microorganism or cell which can be transformed by a nucleic acid sequence encoding a peptide or by a recombinant vector molecule comprising such a nucleic acid sequence and which can if desired be used to express said peptide encoded by said nucleic acid sequence. The host cell can be of procaryotic origin, e.g. bacteria such as Escherichia coli, Bacillus subtilis and Pseudomonas species; or of eucaryotic origin such as yeasts, e.g. Saccharomyces cerevisiae or higher eucaryotic cells such as insect, plant or mammalian cells, including A- 10 VSMC cells and 3T3-L1 cell line. For expression nucleic acid sequences of the present invention are introduced into an expression vector, i.e. said sequences are operably linked to expression control sequences. Such control sequences may comprise promoters, enhancers, operators, inducers, ribosome binding sites etc. Therefore, the present invention provides a recombinant vector molecule comprising a nucleic acid sequence encoding the peptides identified above operably linked to expression control sequences, capable of expressing the DNA sequences contained therein in (a) transformed or transfected host cell(s).

[00127] It should, of course, be understood that the nucleotide sequences inserted at the selected site of the cloning vector may include only a fragment of the complete nucleic acid sequence encoding for the peptides according to the invention as long as the transformed or tranfected host will produce a desired polypeptide. [00128] In accordance with another aspect of the inventions, a composition of the present invention is an inhibitor of Aktl. In a specific example, the inhibitor of Aktl comprises SH-6. In another specific example, the inhibitor of Aktl comprises Aktl siRNA effective to silence Aktl expression.

[00129] The term "siRNA" as used herein means short interfering RNAs and refers to short double stranded ribonucleic acids useful for RNA interference.

[00130] Selection of the specific components of the composition is made based on various criteria, include, for example, the stability of the composition when used as intended, the cost, safety of to the subject and/or laboratory worker, availability, and/or compatibility with downstream application. The choice of the components and their concentrations should be appropriate for reducing, inhibiting and/or preventing nonenzymatic glycation of protein which results in formation of advanced glycation endproducts.

[00131] Method(s)

[00132] It is desirable to initiate intervention against the long-term consequences of glycation and crosslinking to prevent the development of severe late complications of glycation-related disease. Is it also desirable to initiate early intervention. The development of nontoxic and effective compounds and methods that prevent reactive carbonyls, including but not limited to MG and glyoxal, mediated glycation in the tissues and bodily fluids is desirable.

[00133] As described herein, and as seen in the Examples, compounds and compositions of the present invention are beneficial in reducing formation of nonenzymatic glycation products (early and late products) and protein-protein crosslinking, and therefore reducing the pathology associated with glycation related disease.

[00134] As discussed below, this type of therapy has benefits in reducing the deleterious effects and/or severity associated with the formation of early glycation endproducts, a preliminary step in the advanced glycation end product formation.

[00135] By the term "reducing the deleterious effect and/or severity", it is to be understood that any reduction via the methods, compounds and compositions disclosed herein, is to be considered encompassed by the invention. Reduction in the deleterious effects and/or severity, may, in one embodiment comprise enhancement of survival, or in another embodiment, halting disease progression, or in another embodiment, delay in disease progression, or in another embodiment, diminishment of pain, or in another embodiment, delay in disease spread to alternate sites, organs or systems. It is to be understood that any clinically beneficial effect that arises from the methods, compounds and compositions disclosed herein, is to be considered encompassed by the invention.

[00136] In accordance with another aspect of the present invention, there is provided a method for reducing, inhibiting and/or preventing nonenzymatic glycation of proteins which results in formation of advanced glycation endproducts.

[00137] In a specific example, inhibiting and/or preventing nonenzymatic glycation of proteins which results in formation of advanced glycation endproducts is achieved by inhibiting/scavenging/trapping reactive carbonyls including, but not limited to MG and glyoxal.

[00138] In a specific example, inhibition of reactive carbonyls is carried out in vitro, including but not limited to, in test tube, in cultured cells (both adherent cells and nonadherent cells), and the like.

[00139] In another specific example, inhibition of reactive carbonyls is carried out in vivo in organisms, including but not limited to, mammals, including humans, including non- human primates and including rats or mice

[00140] In a specific example, the method of inhibiting of reactive carbonyls is carried out using an isolated peptide comprising an isolated peptide of CLQ (SEQ. ID. NO. 1). In another specific example, the method is carried out using an isolated peptide comprising a peptide of CLQWTTVIER (SEQ. ID. NO. 2)..

[00141] In accordance with another aspect of the present invention, there is provided a method for reducing, inhibiting and/or preventing the effect of MG-induced Aktl activity. [00142] In accordance a specific example of the present invention, the method for reducing, inhibiting and/or preventing the effect of MG-induced Aktl activity comprises contacting a cell with, or administering, SH-6. In another specific example, the method comprises administering Aktl siRNA effective to silence Aktl expression.

[00143] In yet another aspect of the present invention, there are provided pharmaceutical compositions and methods of treatment using such pharmaceutical compositions for therapeutic uses. Such pharmaceutical compositions may be for administration by bolus injection or by infusion (e.g., intravenous or subcutaneous), or for oral, pulmonary, nasal, transdermal, parenterally, rectally, topically or other forms of administration. The pharmaceutical compositions may be administered at variable dosage, depending on the activity of each agent in a single or individual amount.

[00144] Pharmaceutical compositions comprising effective amounts of compounds and compositions of the present invention together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers. Such pharmaceutical compositions include diluents of various buffer content, pH and ionic strength; additives such as detergents and solubilizing agents, anti-oxidants, preservatives and bulking substances; incorporation of the material into particulate preparations of polymeric compounds, etc. or into liposomes, as would be readily appreciated by the skilled worker.

[00145] Compositions may include lotions, ointments, gels, creams, suppositories, drops, liquids, sprays powders or granules, suspensions or solutions in water or non-aqueous media, sachets, capsules or tablets. Thickeners, carriers, buffers, diluents, surface active agents, preservatives, flavorings, dispersing aids, emulsifiers or binders may also be included, all as well other suitable additives, all of which are well known in the art.

[00146] Methods of the invention are conveniently practiced by providing the compositions used in such method in the form of a kit. Such a kit preferably contains the composition. Such a kit preferably contains instructions for the use thereof.

[00147] EXAMPLES

[00148] EXAMPLE I

[00149] Materials, methods and data analysis

[00150] VSMC preparation [00151] Rat thoracic aortic smooth muscle cell line (A-IO) was obtained from

American Type Culture Collection. A-IO cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) as previously described [79]. Cultured cells were grown to 60-80% of confluence before starved in FBS-free DMEM for about 24-48 hours and then exposed to DMEM containing 10% FBS alone or together with MG at different concentrations for about 24 hours. Cells were washed with ice-cold phosphate-buffered saline (PBS), and then harvested by trypsinization. For [³H]-thymidine incorporation assay and cell number counting, cells were seeded in 24-well plates with equal number of cells per well.

[00152] Measurement of cell proliferation

[00153] Cell proliferation was determined by a colorimetric method using One Solution

Cell Proliferation Assay kit (Promega) as well as DNA synthesis assay. The quantity of colored formazan product measured by its absorbance at 492 nm is directly proportional to the number of living cells in culture. DNA synthesis was examined by determining incorporation of [³H]-thymidine into cellular nucleic acids [80]. [³H]-thymidine (1 μCi/ml) was added to each well after treatment started and radioactivity was quantified using a liquid scintillation spectrometer (Beckman LS3801) and expressed as disintegrations per minute (DPM). Hoechst 33258 staining was used to determine cell apoptosis as reported [[8I]. Apoptotic cells were identified by condensation and fragmentation of nuclei under a fluorescence microscope (Olympus 1X70, Tokyo, Japan). Percentage of apoptotic cells was calculated based on the number of condensed and fragmented nuclei from about 15-20 random fields of each coverslip at x 200 magnification. [00154] Aktl siRNA transfection

[00155] ON-TARGETplus SMARTpool siRNA (L-099648-00, Dharmacon, Chicago,

IL) targeted against rat Aktl or ON-TARGETplus non-targeting control siRNA (D-001810- 01-05, Dharmacon) was transfected into VSMCs using DFECT2 transfection reagents following manufacture's protocol. Briefly, 1.5* 10⁴ cells in 500 μl or 4>< 10³ cells in 100 μl of antibiotic-free complete medium were plated in each well of 24-well or 96-well plates and incubated overnight before transfection. Aktl siRNA and non-targeting control siRNA at a final concentration of 100 nM was used to transfect cells. DharmaFECT2 reagents are used at 0.05 μl/well for 24-well plates or 0.01 μl/well for 96-well plates. For transfection, siRNA and DharmaFECT2 reagents are separately diluted in serum-free and antibiotic-free medium, incubated for 5 min at room temperature, then siRNA are combined with transfection regents and incubated for 20 min at room temperature. Cells are transfected with above mixture for 48 hours before starving in FBS-free medium for 24 hours and then subjected to MG treatments. Transfected cells were treated with MG (10 and 50 μM) for 24 hours, and then cell proliferation was analyzed according to the colorimetric method described previously. [00156] Western blot analysis

[00157] Total proteins were extracted from harvested cells with 200 μl of lysis buffer

(20 mM Tris-HCl at pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerolphosphate, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, and 1 μg/ml aprotinin). Proteins (40 μg) were subject to Western blot analysis according to the procedure reported [82]. The primary antibody dilutions were 1:500 for antibodies against p21, p27, and CDK2; 1:1000 for antibodies against Akt, Aktl, phospho-Akt, phospho-Aktl, and phospho-GSK; and 1:5000 for β-actin. Western blots were digitized with Chemi Genius2 Bio Imaging System (SynGene), quantitated using software of GeneTools from SynGene and normalized against the quantity of loaded β-actin.

[00158] Aktl activity assay

[00159] Aktl activity was determined using Aktl assay kit (Cell Signaling) following manufacturer's instruction with minor modification. Briefly, after 200 μg of proteins was incubated with 2 μg of Aktl antibody in cell lysis buffer for 4 houra at 4⁰C with shaking, protein A/G plus agarose beads (40 μl) were added to the mixture and further incubated overnight at 4⁰C with shaking. Agarose beads were pelleted and firstly washed 3 times with cell lysis buffer, then 2 times with kinase buffer. Washed pellet was suspended in 50 μl of kinase buffer supplemented with 1 μl of 10 niM ATP and 1 μg of glycogen synthase kinase 3 (GSK-3) fusion proteins. Reaction mixture was incubated for 30 min at 30⁰C, then the reaction was terminated by adding 25 μl of 3 x SDS sample loading buffer and heated for 5 minutes at 95°C. Sample was subjected to Western blot analysis using anti-phospho-GSK-3 antibody to detect Aktl activity. [00160] CDK2 activity assay

[00161] To study CDK2 activity, CDK2 proteins were immunoprecipitated from cell extracts and subjected to kinase activity assay using histone Hl as the substrate as reported [83]. Briefly, after incubation of 100 μg of proteins with 2 μg of primary anti-CDK2 antibodies in cell lysis buffer for 4 hours at 4°C, protein A/G plus agarose beads (20 μl) were added and the mixture was incubated overnight at 4⁰C with shaking. Washed beads were suspended in 20 μl of CDK2 assay buffer (containing 20 μM ATP and 0.1 μg/μl histone Hl) with 5 μCi [γ-³²P]ATP. The mixture was incubated at 30⁰C for 30 minutes. The reaction was terminated by adding 10 μl of 3 x SDS sample loading buffer and heated for 5 minutes at 95°C. Samples were resolved in 7.5% sodium dodecyl sulfate-polyacrylamide gel. The gel was fixed in methanol: glacial acetic acid:water = 45:10:45 for 1 hr at room temperature with shaking, dried and exposed to X-ray film. Density of autoradiography was digitized with Chemi Genius2 Bio Imaging System and quantitated using software of GeneTools from SynGene.

[00162] Mass spectrometry analysis

[00163] Aktl proteins (3 μg) or synthetic peptides (CLQWTTVIER) (10 μM) were incubated with MG (10, 30, 50 μM) in 100 μl PBS at 37°C for 24 hours. Analysis of trypsin- digested peptide samples was carried out on an Applied Biosystems 4800 TOF/TOF (Foster City, CA US) equipped with a Nd: YAG laser 355 nm wavelength, 3 ns pulse width, 200 Hz firing rate. Prior to analysis, plate modeling and default calibration are carried out utilizing a peptide mixture consisting of angiotensin 1 (M+H 1296.6853), adrenocorticotropic hormone (ACTH) peptide 1-17 (M+H 2093.0867), ACTH peptide 18-39 (M+H 2465.1989) and ACTH peptide 7-38 (M+H 3657.9294) mixed on plate in defined calibration spots with alpha-cyano- 4-hydroxy cinnamic acid (CHCA) matrix (5 mg/ml in 75% acetonitrile, 0.1% TFA, 10 mM ammonium phosphate). Calibration for MS/MS mode is carried out using fragment ion masses from glufibrinopeptide 1 (M+H 1570.6774). Protein digestion is mixed on the plate with the above matrix and air dried under a gentle stream of warm air. Analysis of the digested proteins is carried out in positive ion reflectron mode, and the default calibration is used. Eight hundred of laser shots are collected for peptide mass fingerprint (PMF) analysis scanning from 8OO~3OOO mass units, and precursor ion is manually selected for Q-TOF MS/MS analysis. Q-TOF MS/MS analysis is performed with air as the collision induced dissociation (CID) gas, and 2000 laser shots are collected per sample scanning from mass 10 to the mass of precursor ion using the IkV MS/MS instrument parameters. Fragment ions are assigned using the ion fragment calculator within the Data Explorer software (Applied Biosystems).

[00164] Determination of the effect of MG on Aktl proteins in cell-free preparations

[00165] Phospho-Aktl proteins (5 μg) were treated with MG (10 & 30 μmol/L) in 100 μl of PBS for 24 hrs at 37°C. Unphospho-Aktl proteins (5 μg) were similarly treated with MG in the presence of PIP3 (5 μmol/L). Treated and untreated unphospho-Aktl proteins were further activated by mitogen-activated protein kinase-activated protein kinase-2 (MAPKAPK2) and phosphoinositide-dependent kinase-1 (PDKl) following the manufacturer's protocol. Briefly, unphospho-Aktl proteins (5 μg) with or without MG treatment were activated with 0.5 μl of MAPKAPK2 (250 mU/μl) for 30 mins at 30⁰C in 40 μl of activation buffer (50 mmol/L Tris-HCl at pH7.5, 0.1 mmol/L EGTA, 0.2 mmol/L NaCl, 0.1% β-mercaptoethanol, 0.01% Brij-35, 0.1 mg/ml BSA, 0.4 mmol/L Mg/ATP), followed by adding 0.5 μl PDKl (0.2mg/ml) and another incubation of 30 mins at 30⁰C. The reaction was stopped by adding EDTA to a final concentration of 25 mmol/L. For the kinase activity assay, 2 μl of sample was used in kinase assay as described above. [00166] Materials and data analysis

[00167] MG, Hoechst 33258, and other chemicals were purchased from Sigma. [³H]- thymidine, [γ-³²P]ATP, nitrocellular membrane and ECL kit were purchased from Amersham. Histone Hl, active and inactive Aktl proteins were obtained from Upstate Biotechnology. Antibodies against p21 (M- 19), p27 (F-8), CDK2 (M2), phospho-p21, phospho-p27, protein A/G plus agarose were obtained from Santa Cruz Biotechnology. Antibodies against Akt, Aktl, phospho-Akt (Ser473), phospho-Aktl (Ser473) were purchased from Cell Signaling Technology (Danvers, MA, US) and Chemincon (Temecula, CA, US). Aktl siRNA and DFECT2 transfection regents were purchased from Dharmacon (Chicago, IL, US). N- acetylated peptide (Ac-CLQ WTTVIER-OH) with a sequence corresponding to tryptic peptide TI l of Aktl protein was synthesized by Biopeptide Co. Inc. (San Diego, CA, US). All data were expressed as mean ± SEM from at least 3 independent experiments unless otherwise stated. Statistical analyses were performed using Student's t test or ANOVA. [00168] Results I

[00169] MG increased VSMC proliferation

[00170] To investigate the effect of MG on the proliferation of VSMCs, A-IO cells were treated with MG at different concentrations for 24 hours. As shown in Figure IA, B & C, cell proliferation and DNA synthesis in VSMCs were significantly increased by MG treatments (0.1-50 μM) compared to untreated control cells. DNA synthesis responded in a dose-dependent manner to MG treatment at or lower than 10 μM. MG at 30 and 50 μM still increased DNA synthesis, but showing no significant difference as compared with that of MG at 10 μM. However, MG at 100 μM had no significant effect on DNA synthesis as compared with untreated cells. Therefore, MG at the concentrations of 10, 30 and 50 μM was selected for the experiments in current study.

[00171] The effects of MG on cell proliferation and DNA synthesis were abolished by co-application of SH-6, an inhibitor for Akt (Figure IB & C), indicating that the effect of MG is dependant on Akt pathway. Application of SH-6 (10 μM) alone had no significant effect on cell proliferation or DNA synthesis (Figure IB & C). In the present study, MG at concentrations lower than 50 μM did not increase cell apoptosis. However, MG at higher concentration (100 μM) significantly increased cell apoptosis (Figure 2A & B). [00172] Silencing Aktl reduced MG effect on cell proliferation

[00173] To examine the dependence of MG effect on Aktl, cultured VSMCs were transfected with Aktl siRNA in order to silence Aktl expression. Western blots confirmed the knockdown of Aktl protein levels by 80% as compared with untransfected cells, or cells transfected with negative control siRNA (Figure 3A & B). In cells transfected with negative control siRNA, MG promoted cell proliferation as demonstrated by the results from colorimetric assay and DNA synthesis (Figure 3C & D). However, decreased cell proliferation and DNA synthesis were detected in Aktl siRNA transfected cells compared with negative control transfected cells. Aktl silencing reduced the effects of MG on cell proliferation and DNA synthesis (Figure 3 C & D).

[00174] MG increased phosphorylation and activity of Akt and Aktl.

[00175] Activation of Akt is the consequence of its phosphorylation, especially the phosphorylation of Ser473 residue in C-terminal hydrophobic motif. Accordingly, the level of phospho-Akt (Ser473) in cells treated with MG was studied. The levels of total phospho-Akt were elevated in MG-treated cells as compared with untreated control cells, while no change in total Akt protein levels was observed. Application of the Akt inhibitor SH-6 attenuated MG effect on Akt phosphorylation, as well as the base level of phospho-Akt (Figure 4A & B). Glycogen synthase kinase-3 (GSK-3) is a phosphorylation target of Akt. Consistent with increased levels of phospho-Akt, increased levels of phospho-GSK-3 were detected in MG- treated cells compared with untreated control cells (Figure 4C & D), which imply an increase in Akt activity. SH-6 attenuated MG-induced increase in, as well as the basal level of phospho-GSK-3 (Figure 4C & D).

[00176] Aktl is an isoform of Akt, required for cell proliferation and therefore the change of phospho-Akt 1 was studied. MG increased phospho-Akt 1 levels without affecting total protein level of Aktl (Figure 5 A & B). Application of SH-6 attenuated the effect of MG on Aktl phosphorylation. Though SH-6 alone slightly decreased phospho-Akt 1 level, but not statistically significant (Figure 5A & B). The activity of Aktl proteins immunoprecipitated from cells with or without MG treatment was further studied. Increased Aktl activities were detected in MG-treated cells, which were prevented by application of SH-6. SH-6 alone had no significant effect on Aktl activity (Figure 5C & D). [00177] Effect of MG on p21 and p27

[00178] To understand the mechanisms of MG-induced VSMC proliferation, the levels of CKIs p21 and p27 in MG-treated cells were investigated. Total p21 levels were decreased in MG-treated cells as compared with that of control cells (Figure 6A & B). MG-induced decrease in p21 level was prevented by Akt inhibitor SH-6. SH-6 alone had no significant effect on p21 level (Figure 6A & B). The levels of phospho-p21 normalized by total p21 were significantly increased in MG-treated cells, which were abolished by SH-6 application. These results indicate that increased phospho-p21 is mediated through the up-regulation of Akt activity. Though SH-6 alone had no effect of p21 , but it significantly decreased p-p21 level (Figure 6A & C).

[00179] For the protein level of p27, no difference was detected in groups treated with or without MG (Figure 7 A & B). Phospho-p27 levels were increased in cells treated with MG, and application of SH-6 did not prevent the change in phospho-p27 level (Figure 7A & C). SH-6 alone had no effect on the levels of p27 or phospho-p27 (Figure 7 A, B & C). [00180] MG increased CDK2 activity

[00181] Activation of CDK2 is required for Gl/S phase transition and DNA duplication. The protein level of CDK2 was not affected by MG or SH-6 treatment (Figure 8A). To analyze CDK2 activity, CDK2 proteins were immunoprecipitated from cell extracts and subjected to activity assay using histone Hl as the substrate. CDK2 activity was significantly elevated in cells treated with MG. This is consistent with the decrease in p21 level in MG-treated cells, because CDK2 activity is negatively regulated by p21. Application of SH-6 blocked MG-induced increase in CDK2 activity. In addition, no significant effect on CDK2 activity was observed in cells treated with SH-6 alone (Figure 8B). [00182] Mass spectrum analysis of MG-treated Aktl proteins [00183] In view of the known interactions of MG with selective amino acids, the interaction between MG and Aktl protein was studied. Aktl proteins were treated with MG and analyzed using mass spectrometry. MALDI-TOF mass spectrum of the untreated Aktl protein contained a peak at m/z 1248.7, which corresponds to peptide Tl 1 (CLQWTTVIER) from tryptic digestion of Aktl. This m/z 1248.7 peak disappeared and a new peak at m/z 1302.7 emerged after the native Aktl protein was incubated with MG (50 μM) (Figure 9A). None of the peptides released from native Aktl protein after tryptic digestion has a predicted peak at m/z of 1302.7. This new peak of m/z 1302.7 represents a summation of 1248.7 and 54 Da. The molecular weight of intact MG is 72 Da. Therefore, the most reasonable interpretation of this net increase of 54 Da is that MG undergoes a condensation reaction with peptide Tl 1 with concomitant loss of a single water molecule (18 Da). The same results were observed in preparations of purified Aktl protein incubated with MG at 10 and 30 μM (data not shown). Two residues, cysteine and arginine, in peptide TI l could possibly react with MG. To confirm the actual site of MG attachment on Aktl, tryptic peptide Tl 1 was further analyzed with Q-TOF MS/MS technique. The mass of all observed b-ions were shifted by + 54 Da in Q-TOF MS/MS spectrum of MG-treated Aktl proteins relative to the corresponding spectrum for the untreated Aktl proteins (Figure 9B). The smallest b-ion observed was b2, confirming attachment of MG at either cysteine (C) or leucine (L). Since leucine has a non- reactive aliphatic side-chain, the only possible site of MG attachment is the thiol group of cysteine residue. In contrast, the mass of all observed y'-ions remained unchanged, a result consistent with the attachment of MG via loss of a H₂O (18 Da) to the side chain of cysteine in peptide Tl 1 (Figure 9B).

[00184] The formation of a disulfide bond between residues Cys77 and CysόO in Aktl has previously been reported 36. PMF (peptide mass fingerprint) spectra of unphospho-Aktl incubated with or without PIP3 did not include peaks corresponding to the two tryptic peptides (at m/z 1248.7, 1792.8) containing Cys77 and CysόO, respectively; whereas, a peak (at m/z 3039.1) corresponding to the two tryptic peptides linked by a disulfide bond was observed (Fig. 9D). These results confirmed the disulfide bond between residues Cys77 and CysόO in unphospho-Aktl. The ability of MG to break this disulfide bond via modification of Cys77 is demonstrated by the appearance of a modified TI l peptide peak (m/z 1302.7), especially in the presence of PIP3 (Fig. 9C). This result further supports the existence of a disulfide bond between Cys77 and CysόO in the unphosphorylated form of Aktl and proves the breakage of this covalent bond upon modification of Cys77 by MG. In contrast, the PMF spectrum of phospho-Aktl contains peaks corresponding to tryptic peptides that contain Cys77 (m/z 1248.7; Fig. 9A) and CysόO (m/z 1792.8), revealing the absence of a disulfide bond between these two cysteine residues in the phosphorylated form of Aktl. We conclude that the disulfide bond between Cys77 and CysόO is present only in unphospho-Aktl, and that binding of PIP3 to the Aktl PH domain promotes MG-induced modification of Cys77. [00185] To test whether the structural MG-induced modification of Akt happens regardless of the phosphorylation status of Aktl, unphospho-Aktl proteins were treated with MG (50 μmol/L) for 24 hrs in the presence or absence of PIP3 (which binds the Akt pleckstrin homology (PH) domain), then digested with trypsin and subjected to MALDI-TOF MS analysis. The peak at m/z 1248.7 was not observed in the mass spectrum of unphospho- Aktl protein treated with or without MG in the absence or presence of PIP3 (Fig. 9C & 9D). A weak signal of m/z 1302.7 was detectable in the spectrum of unphospho-Aktl after incubation with MG (50 μmol/L) (Fig. 9D). Interestingly, the presence of PIP3 dramatically increased the intensity of the peak at m/z 1302.7 (Fig 9C). These results indicated that MG also modifies unphospho-Aktl, especially in the presence of PIP3.

[00186] To confirm the reaction of MG with the side chain of cysteine residue, synthesized N-acetylated peptide TI l with the same amino acids sequence as the tryptic TI l peptide of Aktl, corresponding to a peak at m/z 1290.7 on mass spectrum, was incubated with MG (10, 30 and 50 μM) for 24 hours (Figure 10A). The presence of the m/z 1290.7 peak in all samples suggests that reaction of synthesized TI l peptide with MG is less extensive than that between intact Aktl proteins and MG under the same conditions. However, a peak at m/z 1362.7, corresponding to the addition of an intact MG (72 Da) to the synthetic peptide, was observed in all incubates except the control (Figure 10A). The intensity of this peak relative to m/z 1290.7 was greater in the 50 μM MG incubate than in the 10 or 30 μM MG incubate (data now shown). A peak at m/z 1344.7, which would correspond to the addition of one MG (72 Da) and loss of one H2O (18 Da), was not observed in any of the samples, unlike the results with the Tl 1 peptide from the tryptic digestion of Aktl treated with MG. Further comparison of Q-TOF MS/MS spectra for N-acetylated TI l peptide before (m/z 1290.7) and after (m/z 1362.7) incubation with MG showed that the mass of all observed b-ions were shifted by 72 Da, whereas the entire observed y'-ions remained unchanged, including the y' l-ion (i.e. the C-terminal Arg residue) (Figure 10B). Similar to tryptic peptide TI l of Aktl, the smallest b- ion in observed MS/MS of MG-treated synthetic peptide was b2, confirming the attachment of MG at cysteine residue.

[00187] Effects of MG on the phosphorylation and activity of Aktl

[00188] The effects of MG on Aktl phosphorylation and kinase activity were tested in vitro. Unphospho-Aktl was pre-incubated with or without MG for 24 hours with PIP3 (5 μmol/L) and then exposed to MAPKAPK2 and PDKl for Aktl activation for 30 minutes, respectively. As shown in Fig. 44 A, kinase-induced levels of phospho-Aktl(Ser473) were significantly increased ~2-fold when Aktl was pre-treated with MG (10 or 30 μmol/L). Consistently, kinase-induced Aktl activity was significantly enhanced ~2-fold with MG- pretreatment (Fig. HA).

[00189] To further confirm the facilitating effect of MG on Aktl activity, phospho-

Aktl was directly treated with MG (10 or 30 μmol/L) for 24 hours. Fig. 44B shows that MG treatment significantly increased Aktl activity ~2-fold over control levels. However, the level of phospho-Aktl(Ser473) was not changed by treatment with MG (Fig. 1 IB). [00190] Discussion - Example 1

[00191] Accumulation of endogenous MG was one of the hallmarks of diabetes mellitus and its complications [24-26]. Elevated MG levels in plasma and aortic tissues in an age-dependent fashion were reported in spontaneously hypertensive rats during the development of hypertension [9, 10, 14]. Feeding SD rats with fructose for 16 weeks increased MG levels in plasma and vascular tissues accompanied with vascular remodeling and hypertension [9]. Decreasing MG levels by metformin in fructose-fed SD rats is parallel with a reverse in vascular remodeling and a drop in blood pressure. This suggests that MG may play an important role in the development of vascular diseases including hypertension, in addition to diabetes mellitus and its complications.

[00192] To understand the effect of MG on vascular diseases, the proliferation of

VSMCs in the presence of MG at physiologically relevant concentration and the underlying molecular mechanisms was examined. As described herein, MG at low concentration range (0.1-50 μM) stimulated the proliferation of VSMCs as demonstrated by cell proliferation and DNA synthesis data (Figure 1).

[00193] The basal concentration of plasma MG in streptozotocin-induced diabetic SD rats is approximately 5 μM in contrast to 2 μM in non-diabetic control rats [27]. A previous study detected plasma MG levels of 33.6 μM in 20- week-old SHR and 14.2 μM in age- matched WKY rats [10]. Plasma levels of MG range from 1.4 to 3.3 μM in healthy humans and from 3.6 to 5.9 μM in diabetic patients [19, 28]. Taken these reports together, physiological and pathophysiological concentrations of plasma MG should be in the micromolar range. However, most previous studies used MG at milimolar range to treat cells and different results were reported. For example, MG at 0.1~1 mM concentration-dependently inhibited platelet-derived growth factor-induced DNA synthesis, and MG at 0.5-1 mM decreased cell viability in rabbit femoral smooth muscle cells [84]. It was reported that MG (2.5-5 mM) decreased the stimulating effects of insulin on glucose uptake, pRyr-IRS-1, PI3K activity, IRS/p85 complex formation, ERKl /2 phosphorylation, and Akt phosphorylation in L6 skeletal muscle cells [85]. MG at 0.25 mM induced apoptosis of Jurkat leukemia T cells [86], and inhibited proliferation of NIH 3T3 cells [87]. Considering the physiologically irrelevant high concentrations of MG used in above studies, effects of MG observed by these authors would be due to its unselective damage to the tested cells [86, 88, 89]. Previous studies using MG at non-physiological concentrations (>100 μM) reported apoptotic effects of MG on several types of cultured cells [84, 86, 87]. As described herein, significant increase in cell apoptosis with MG at the concentrations lower than 50 μM was not observed, until MG reached 100 μM (Figure 3). Instead, MG at lower micromolar concentration range stimulated cell proliferation. The opposite effects of MG at higher (>100 μM) and lower (<50 μM) concentrations suggest that While not wishing to be bound by theory, MG may be serving as a molecular switch regulating different cellular functions including proliferation and apoptosis. [00194] Akt is a versatile protein kinase that regulates various cellular processes including cell proliferation, growth, survival, glucose uptake, metabolism and angiogenesis [54, 56]. Previous studies have shown that Aktl isoform is required for cell proliferation [90, 91]. Accumulated evidence indicated that inhibited Akt activity decreased 41b, but increased Aktl activity favored cell proliferation [92, 93]. As described herein, it was found that MG promoted phosphorylation of Akt as well as Akt substrate GSK-3 (Figure. 4). Phosphorylation of Aktl and its activity in VSMCs were also increased by MG treatment (Figure.5). Blockade of MG-induced increases in Aktl phosphorylation and its activity by Akt inhibitor, together with the subsequent normalization of cell proliferation, indicates that the effect of MG on VSMC proliferation is mediated through increased Aktl activity. Finally, silencing Aktl significantly inhibited cell proliferation and DNA synthesis, and abolished the effects of MG on Aktl-siRNA transfected cells (Figure. 3). The foregoin demonstrated the pro-proliferative effect of Aktl and its mediating role for MG-stimulated cell proliferation (Figure. 2). [00195] Cell cycle negative regulators CKIs (such as p21 and p27) are phosphorylation substrates of Akt [42, 95, 96]. Phosphorylation of CKIs disrupts its association with CDKs including CDK2, which results in CDKs activation to favor cell proliferation [97]. Phosphorylated CKIs are also translocated from nucleus to cytoplasm and eventually subjected to phosphorylation-dependent proteolysis [42, 92]. In rat aortic VSMCs transfected with a dominant-negative Aktl mutant (AA-Aktl), cell proliferation rate was reduced 48 hrs after transfection [92]. AA-Aktl transfection increased p21 protein expression more than 2- fold compared with control. In addition, cell proliferation was not affected by AA-Aktl transfection in p21-null mouse VSMC [92]. In Swiss mouse 3T3 fibroblasts, silencing Aktl resulted in an increased nuclear localization of p21 [97]. On the other hand, over-expression of Aktl in 3T3 fibroblasts was correlated with decreased nuclear p21 and increased cytoplasmic p21 [97]. Similarly, higher Aktl protein level in both transformed HEK-293 cells and human tumoral colon tissue was correlated with low or undetectable levels of p21 [97]. As described herein, a decrease in total p21 level accompanied with an increase in phospho- p21 level was observed in MG-treated cells (Figure. 6). These changes in p21 and phospho- p21 levels induced by MG were reversed by application of Akt inhibitor. Similarly, AA- Aktl transfection decreased phospho-p21, but increased total p21 levels in balloon-injured arterial wall [92]. It was also reported that Aktl directly phosphorylated p21 and decreased p21 level [97]. While not wishing to be bound by theory, it is thought that MG-induced decrease in total p21 may be the result of Aktl -mediated phosphorylation-dependent proteolysis of p21. [00196] In chromatin, DNA is packed and locked on nucleosomes by histone Hl proteins. Phosphorylation of histone Hl by CDK2 to unwind DNA from nucleosomes is one of the steps required for DNA replication in cell proliferation. CDK2 activity is inhibited by CKIs such as p21. In addition to the relief on CDK2 inhibition by the decrease in p21 protein, phosphorylation of p21 also disrupts its association with CDK2 complex and results in CDK2 activation [97]. Consistent with decreased total p21 and increased phospho-p21 levels in MG- treated cells, CDK2 activity was significantly enhanced by MG treatment, which favors cell proliferation (Figure. 8). Accordingly, blocking MG-induced decrease in p21 and increase in phospho-p21 level by Akt inhibitor also abolished the increase in CDK2 activity. Again, while not wishing to be bound by theory, these results suggest that CDK2 is located at the downstream of Aktl and p21 pathway in MG-induced cell proliferation. [00197] Unlike p21, total p27 shows no significant change, though phospho-p27 level was increased, in MG-treated cells (Figure. 7). Consistent with these results, no change in p27 level after blocking Aktl signaling has been reported in VSMC [92]. However, Akt inhibitor failed to block the increased phospho-p27 level, and this indicates that other mechanism may be involved in the regulation of phospho-p27 level. These data support that p21, rather than p27, is involved in cell proliferation regulated by Aktl pathway in MG treatments. While note wishing to be bound by theory, the results suggest that MG at physiologically relevant concentrations may promote VSMC proliferation through Aktl -mediated decrease of p21, and the subsequently activation of CDK2. Of note, it was also observed an increase in phospho- GSK-3 as a result of increased Akt activity. It was reported that phosphorylation of GSK-3 by Akt inactivates its kinase activity and releases its suppression on cyclin D and CDK4/6 [98], which are the positive regulators for cell cycle progression. Therefore, GSK-3 may also contribute to MG-induced cell proliferation.

[00198] MG is a chemically reactive dicarbonyl molecule and a contributor for AGE formation 2b. MG-derived AGEs was reported in vascular tissues and kidney from hypertensive rats [9, 10]. Reactions of MG with proteins such as insulin [99], platelet-derived growth factor receptor β (PDGFR) [84], hemoglobin [100], and alphaA-crystallin [101] were reported. For example, incubation of MG with insulin in vitro results in the formation of insulin-MG adduct at the N-terminus and arginine residue in β-chain via Schiff base formation, and MG-modifϊed adduct shows reduced ability to induce glucose uptake [99]. MG-derived CML (^-(carboxymethytylysine) was detected in PDGFR from rabbit femoral SMCs, and this modification resulted in dysfunction of PDGFR [84]. Carboxymethyl cysteine (CMC) formed from reaction of MG with cysteine residue was reported in NAC, peptides and plasma proteins from diabetic patients [18, 102]. Results from MALDI-TOF MS and Q-TOF MS/MS analysis revealed that MG reacted with residue Cys₇₇ in pleckstrin homology (PH) domain of Aktl (Figure. 9). Although reaction of MG with cysteine was reported previously [18, 102], it is believed, the present study is the first one reporting a reaction of MG with a defined cysteine residue in a given protein. The reaction of MG with this cysteine residue was also confirmed with a synthetic peptide corresponding to the tryptic peptide (CLQ WTTVIER) from Aktl (Figure. 10). It was demonstrated that reaction of MG with the cysteine residue in the synthetic peptide resulted in a mass increase of 72 Da. However, attachment of MG to CyS₇₇ Ui intact Aktl protein led to a mass increase by 54 Da. It is believed that the formation of Aktl -MG adduct was coincided with the loss of one H₂O molecule (18 Da) due to the three-dimensional structure of Aktl protein, which is obviously absent in the synthetic peptide. There is a disulfide bond between CyS₇₇ and CyS₆₀ in Aktl PH domain [103]. The formation of Aktl-MG adduct at the site of CyS₇₇ may break the disulfide bond and result in a conformational change(s) in Aktl protein. This may explain MG-induced Aktl phosphorylation and activation.

[00199] The formation of a disulfide bond between residues Cys77 and Cys60 in Aktl has previously been reported 130. PMF spectra of unphospho-Aktl incubated with or without PIP3 did not include peaks corresponding to the two tryptic peptides (at m/z 1248.7, 1792.8) containing Cys77 and Cys60, respectively; whereas, a peak (at m/z 3039.1) corresponding to the two tryptic peptides linked by a disulfide bond was observed (Fig. 9D). These results confirmed the disulfide bond between residues Cys77 and CysόO in unphospho-Aktl. The ability of MG to break this disulfide bond via modification of Cys77 is demonstrated by the appearance of a modified Tl 1 peptide peak (m/z 1302.7), especially in the presence of PIP3 (Fig. 9C). This result further supports the existence of a disulfide bond between Cys77 and CysόO in the unphosphorylated form of Aktl and proves the breakage of this covalent bond upon modification of Cys77 by MG. In contrast, the PMF spectrum of phospho-Aktl contains peaks corresponding to tryptic peptides that contain Cys77 (m/z 1248.7; Fig. 9A) and CysόO (m/z 1792.8), revealing the absence of a disulfide bond between these two cysteine residues in the phosphorylated form of Aktl. We conclude that the disulfide bond between Cys77 and CysόO is present only in unphospho-Aktl, and that binding of PIP3 to the Aktl PH domain promotes MG-induced modification of Cys77

[00200] It was found that incubation of MG with inactive Aktl promoted Aktl activation and increased Aktl activity following the activation by MAPKAP kinase 2 and PDKl (Figure. 11). While not wishing to be bound by theory, it was thus reasoned that glycation of Cys₇₇ by MG may induce a conformational change(s), favoring AkI phosphorylative activation.

[00201] MG at physiologically relevant concentrations enhanced VSMC proliferation by increasing Aktl activity, which subsequently down-regulated p21, up-regulated CDK2 activity and inhibited GSK-3 (Figure 12). Blockade of MG effect on cell proliferation by inhibiting Akt activity or Aktl silencing suggests that Aktl is an important mediator of MG- promoted cell proliferation. MS analysis proved the glycation of CyS₇₇ in Aktl protein by

MG. While not wishing to be bound by theory, it is thought that MG-induced glycation of

CyS₇₇ may induce a conformational change(s) which favors the phosphorylative activation of

Aktl protein. Glyation of Aktl by MG may constitute a mechanism of the role of MG in vasculoproliferative diseases including hypertension, diabetes and its vascular complications.

[00202] EXAMPLE II

[00203] Materials, methods and data analysis

[00204] MG, glyoxal, o-phenylenediamine (o-PD) and 5 -methyl quinoxaline, N-acetyl-

1-cysteine (NAC), reduced glutathione (GSH), metformin, aminoguanidine, alagebrium chloride were purchased from Sigma. 3 -DG was from Toronto Research Chemicals Inc

(North York, ON Canada). One Solution Cell Proliferation assay kit was purchased from

Promega. 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) was from Invitrogen. Anti-

CEL and anti-CML primary antibodies were from Novo Nordisk (AJS, Denmark) Anti-mouse

IgG- FITC secondary antibody was from Sigma (Oakville, Ontario, Canada).

[00205] SMG08 and SMG08-FITC were synthesized by ChemPep Inc (Miami, FL

USA).

[00206] All data were expressed as mean ± SEM from at least 3 independent experiments unless otherwise stated. Statistical analyses were performed using Student's t test or one-way ANOVA.

[00207] Cell culture

[00208] The Rat thoracic aortic smooth muscle cell line (A- 10) was obtained from

American Type Culture Collection. A- 10 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) as previously described [48].

[00209] When used in the experiments as described herein, cultured cells were grown in FBS-containing DMEM to about 60-80% of confluence. FBS-containing DMEM was removed and cells were then cultured in FBS-free DMEM (i.e. starved) for about 24-48 hours. Cells were subsequently exposed to DMEM containing 10% FBS alone or together with MG for 24 hours. [00210] Incubation ofSMG 08 with MG, glyoxal and 3-DG in test-tube

[00211] To evaluate the scavenging ability of SMG08 against MG, glyoxal and 3-DG,

SMG08 (10 μM) was incubated with MG (10 μM), glyoxal (10 μM) and 3-DG (10 μM) in PBS at 37⁰C from 2 min to 24 hrs, respectively. Samples were kept in -80⁰C before HPLC measurement. Following incubation with SMG08, the concentration of free (i.e., that which was not scavenged by SMG08) MG, glyoxal or 3-DG was measured by HPLC (high pressure liquid chromatography) method as described below. Using same condition, incubation of other chemical compounds (L-cysteine, GSH, NAC, metformin, aminoguanidine and alagebrium choloride) with MG was carried out similarly unless stated. [00212] Incubation of SMG08 with cultured VSMCs

[00213] To determine the effect of SMG08 on cell proliferation, reactive oxygen species (ROS) production and scavenging endogenous MG, cultured VSMCs were pre-treated with SMG08. For SMG08 pre-treatment, 24 hours starved cells were incubated with SMG08 (100 μM) in FBS-free DMEM for about 4-8 hours (for proliferation and ROS production assays) or 18 hours (for MG measurement, staining). After pre-treatment, medium containing SMG08 was replaced with DMEM containing 10% FBS and was followed by MG treatment for 24 hours. For MG measurement, treated cells were washed with ice-cold phosphate- buffered saline (PBS), and then were harvested by gently scraping with rubber policeman. Collected cells were suspended in PBS and sonicated for 5 seconds 3 times. Resulted cell lysis was centrifuged at 12,000 rpm for 15 mins. Supernatant of cell lysis was used for MG measurement by HPLC as decribed below. [00214] Measurement of MG, glyoxal, 3-DG by HPLC

[00215] MG levels in samples of test-tubes and supernatant of cell lysis, and levels of glyoxal and 3-DG in test-tubes were determined by HPLC method are reported [13]. Briefly, samples were incubated with 100 mM o-phenylenediamine (o-PD, derivatizing agent) for 3 hours at room temperature protected from light. The quinoxaline formed between dicarbonyl compounds and o-phenylenediamine, and internal standard (5-methylquinoxaline) were measured using a Hitachi D-7000 high-performance liquid chromatography (HPLC) system (Hitachi Ltd., Mississauga, Ontario, Canada) [13]. The column was a Nova-Pak C18 column

(3.9 x 15 mm, and 4 ml particle diameter; Waters, MA, USA). The mobile phase for MG and glyoxal measurements was composed of 8 % (vol/vol) of 50 mM NaH₂PO₄ (pH 4.5), 17%

(vol/vol) of HPLC grade acetonitrile and 75% of water, for 3 -DG measurement the mobile phase was composed of 8 % (vol/vol) of 50 mM NaH₂PO₄ (pH 4.5), 14% (vol/vol) of HPLC grade acetonitrile and 78% of water. Triplicate of each sample were made for each measurement.

[00216] Measurement of cell proliferation and ROS production

[00217] For cell proliferation and ROS production assays [79, 104] , VSMCs (A-IO cells) were seeded in 96-well plates with an equal number of per well cells (about 5^χ 10³ cells per well) and incubated 24 hours for cells to attach. Cells were starved in FBS-free medium for about 24 hours before SMG08 pre-treatment. SMG08 pre-treatment and MG treatment were described above.

[00218] Cell proliferation was determined by a colorimetric method using One Solution cell proliferation assay kit (Promega) following manufacture's procedure. The quantity of colored formazan product as measured by its absorbance at 492 nm is directly proportional to the number of living cells in culture [104].

[00219] ROS production in cultured cells was measured using DCFH-DA probe as reported before [79]. DCFH-DA is oxidized by H₂O₂ and peroxynitrite (derived from superoxide anion and nitric oxide) to form DCF with detectable fluorescence. Fluorescence of oxidized-DCF was recorded with excitation/emission wavelength at 485/527 nm using fluorescence plate-reader, respectively. Cells were plated in 96-well plate and treated with

MG or SMG08 as described above. Treated cells were loaded with DCFH-DA (5 μM) probe for about 1 hours. Excess probe was washed away and probe-loaded cells were incubated in media containing MG for another 4 hours. Intensity of fluorescence was recorded in the presence of 100 μl PBS/well after cells were washed 3 times with PBS.

[00220] FITC staining

[00221] To determine the membrane permeability of SMG08, fluorescein isothiocyanate (FITC) labeled SMG08 (SMG08-FITC) was used to stain cultured VSMCs (A- 10). A-IO cells in FBS-containing DMEM media (phenol red free) were first allowed to adhere to glass cover slips. Then, the FBS-containing DMEM medium was removed and cells were then cultured in FBS-free (phenol red free) DMEM (ie. starved) for about 24 hours. The FBS-free DMEM (phenol red free) media was then remove and the cells were incubated in FBS-containing DMEM (phenol red free) with (i.e. treated) or without (i.e. untreated) SMG08-FITC (100 μM) for about 4 hours or 18 hours. Some of 18-hour treated cells were washed of SMG08-FITC and incubated in FBS-containing DMEM (phenol red free) for another 24 hours. Thereafter, the treated and untreated cells on glass cover slips were washed with FBS-free DMEM at least 5 times and subjected to analysis using fluorescence microscope (Olympus 1X70, Tokyo, Japan). [00222] Immunocytochemistry staining of AGEs

[00223] VSMCs seeded on glass cover slips was starved in FBS-free DMEM for about

24 hours followed by about 8 hours of pre-treatment with SMG08 (100 μM). After washing with FBS-free medium, pre-treated cells were exposed to MG treatments for about 24 hours and subjected to AGEs staining. Briefly, treated cells were fixed in 4% formalin for 30 minutes at room temperature (about 22~25°C). All incubation were conducted at room temperature. After permeation with 0.1% Triton X-100 for 30 minutes, fixed cells were incubated with blocking solution (1 vol of goat serum:30 vol of PBS) for about 1 hour, and then incubated with anti-CEL or anti-CML antibody (1:100; Novo Nordisk, AJS, Denmark) at room temperature for about 2 hours. Cells were washed in PBS for 5 min and incubated with Anti-mouse IgG- FITC (1:200; Sigma, Oakville, Ontario, Canada) secondary antibody for 1 hour. After washing with PBS, cells were subjected to detection using a confocal fluorescence microscope (Olympus confocal microscope, FV5-PSU). [00224] Results II

[00225] SMG08 is composed of 3 amino acids with a sequence of CLQ (N-cysteine- leucine-glutamine-COOH). The molecular weight of SMG08 is 362.4 Da with a formula of C14H26N4O5S. The purity of synthesized peptides was equal to or greater than 98%. Figure 13 shows the schematic structure of SMG08 (A) and MG (B). [00226] Scavenging MG by SMG08 in vitro (test-tube)

[00227] To test the ability of SMG08 on scavenging MG, SMG08 (10 μM) was incubated with MG (10 μM) in phosphate buffered saline (PBS) at 37⁰C from about 2 min to about 24 hours. As shown in Figure 14, SMG08 significantly decreased MG levels at all incubation times. SMG08 reacted with MG rapidly and efficiently as indicated by a high MG scavenging efficiency of 82.2%, 82.3%, 85.7%, 84.9%, 82.3%, 78.5% and 72.7% at the incubation time of 2 min, 30 min, 1 hour, 2 hours, 4 hours, 8 hours, 16 hours and 24 hours, respectively.

[00228] Since the reaction of MG with NAC is reported [102], we compared the scavenging ability of SMG08 as well as L-cysteine, GSH, NAC, or metformin on MG in vitro under the same experimental condition as SMG08. As shown in Figure 15, the highest MG scavenging efficiency after 24 hours of incubation was 17.8 % with L-cysteine (10 μM) and a 10% with NAC (10 μM). GSH (10 μM) and metformin (10 μM) did not induce any significant changes.

[00229] Recent studies suggest that aminoguanidine is an AGE inhibitor [2], and alagebrium is an AGE breaker [2, 73]. We, therefore, also tested the effect of aminoguanidine or alagebrium on scavenging MG using the same experimental conditions for SMG08. [00230] As shown in Figure 16, aminoguanidine (10 μM) significantly reduced the amount of MG by 9.1, 10.8, 13.4 and 32.2% at the incubation time of 2 minute, 30 minute, 1 hour and 24 hours, respectively.

[00231] As shown in Figure 17, alagebrium (10 μM) failed to scavenge MG at the incubation times tested (2 minute, 30 minute, 1 hour and 24 hours). Higher concentration of alagebrium (50 μM) reduced MG (10 μM) level by 55.5% at 24 hours of incubation. [00232] Cell membrane permeability of SMG08

[00233] The ability of fluorescein isothiocyanate labelled SMG08 (SMG08-FITC) to cross cell membrane(s) was examined. Cultured VSMCs were incubated with or without SMG08-FITC as described in methods. As shown in Figure 22, control cells without SMG08- FITC showed a low level of background fluorescence. Cells incubated with SMG08-FITC for 4 hours showed an increased intensity of fluorescence as compared to control cells lacking SMG08-FITC. A greater increase in fluorescence intensity was observed in cells incubated with SMG08-FITC for 18 hours. Additionally, some of fluorescence in overnight-stained cells was still observed 24 hours after the removal of SMG08-FITC from media, even though the intensity was decreased. These data indicate that SMG08 is cell membrane permeable and can exist inside living cells in its native form for at least 24 hours with the concentration tested.

[00234] Scavenging cellular MG by SMG08 in cultured VSMC

[00235] To further investigate MG scavenging ability of SMG08, 24-hour starved

VSMCs were pre-treated with SMG08 at different concentations (1, 5, 25, 50 and 100 μM) for 18 hours. After removal of SMG08 from medium, cells were exposed to MG (10 μM) treatment in FBS-containing DMEM for about 24 hours. As shown in Figure 18 A, SMG08 significantly decreased cellular MG levels in VSMC in a concentration-dependent manner with a maximum effect at 100 μM of SMG08. SMG08 at 100 μM was used to pre-treat cells in following experiments. The basal level of cellular MG in VSMCs was decreased by 74.8% after cells were pretreated with SMG08 (100 μM). As compared to cell treated with MG (10 μM) alone, for example, increase in cellular MG level was significantly inhibited by 70.1 % by SMG08 (100 μM) pre-treatment (Figure 18B).

[00236] The effect of aminoguanidine or alagebrium on scavenging cellular MG was examined, and compared with SMG08 under same experimental conditions. As shown in Figure 19, pre-treatment with aminoguanidine (100 μM) or alagebrium (100 μM) decreased cellular MG level by 31.4% or 30.8%, in comparison with that from cells treated with MG alone. Aminoguanidine, but not alagebrium (100 μM), decreased basal level of cellular MG in cultured cells.

[00237] Results from in vitro and cultured cell experiments indicates that SMG08 directly reacts with MG and is a powerful scavenger of cellular MG as compared to other reported MG scavenging chemicals. [00238] Effect of SMG08 on MG-induced cell proliferation

[00239] As shown above, MG at physiologically relevant concentration range stimulated the proliferation of VSMCs (Figure 1). The effect of SMG08 on cell proliferation was studied in cultured VSMCs. As shown in Figure 20, when cells were treated with MG (10 μM) for 24 hours, a 14.4% increase in cell proliferation was observed, in comparison with that from untreated cells. When cells were first pre-treated with SMG08 for 8 hours, followed by removal of SMG08 from media and cell exposure to MG (10 μM) treatment for about 24 hours, SMG08 pre-treatment effectively prevented MG-induced cell proliferation. These results indicate that SMG08 is effective in preventing MG-induced cell proliferation. [00240] Since MG is a metabolite of glucose (a precursor), we tested the effect of

SMG08 on glucose-induced cell proliferation. As shown in Figure 20, 25 mM glucose stimulated cell proliferation by 11.7%, compared with that from cells cultured in normal levels of glucose (5 mM). Pre-treatment of SMG08 significantly prevented 25 mM glucose- induced cell proliferation and SMG08 also decreased basal level of cell proliferation by 13.7%, compared with that from untreated cells [00241] Effect of SMG08 on MG-induced ROS production

[00242] An MG-increased ROS generation in VSMC has previously been reported

[48]. To further examine the scavenging ability of MG, we tested the effect of SMG08 on ROS production in VSMC treated with MG. As shown in Figure 21, MG (10 μM) treatment significantly increased oxidized-DCF level, indicating an increase in ROS production. Pre- treatment of cells with SMG08 effectively blocked MG-induced ROS production, suggesting that SMG08 is effective in scavenging MG and MG-induced ROS production. There was also a decrease in basal level of ROS I SMG08 treated cells, in comparison with that from untreated cells.

[00243] Effect of SMG08 on MG-induced AGE formation

[00244] Cells were stained with anti-CML antibody. As shown in Figure 23, MG (30 μM) treatment for 24 hours increased CML staining as compared to untreated control cells. Cells was pre-treated with SMG08 (100 μM) for 18 hours, then SMG08 was removed and subjected to MG (30 μM) treatment for 24 hours. As shown in Figure 23, SMG08 pre- treatment prevented MG-increased CML staining, which was similar to untreated control cells. In addition, a weaker staining of CML was observed in control cells after SMG08 pre- treatment as compared to that of control cells without SMG08 pre-treatment. [00245] Effect of SMG08 on glyoxal or 3-deoxyglucoson in vitro (test-tube)

[00246] Since glyoxal is also a family member of reactive carbonyl molecules structurally similar to MG, we tested the effect of SMG08 (10 μM) on scavenging glyoxal (10 μM) in vitro (test-tube). As shown in Figure 24, SMG08 (10 μM) efficiently scavenges glyoxal (10 μM) by 29.7, 40.8, 42.0% at the incubation time of 30 minutes, 1 hour, and 24 hours, respectively. No significant change was detected at the incubation time of 2 minutes. The ability of SMG08 to scavenge glyoxals was much lower as compared with its scavenging efficiency of MG. As shown in Figure 25, alagebrium at 50 μM failed to scavenge glyoxal after the incubation with glyoxal for different times tested (2 minutes, 30 minutes, 1 hour, 24 hours).

[00247] As 3-DG contains reactive carbonyl groups, we also the effect of SMG08 (10 μM) and alagebrium on scavenging 3-DG (10 μM) in vitro (test-tube). As shown in Figures 26 and 27 neither SMG08 nor alagebrium, respectively, induce changes of 3-DG level after incubation with 3-DG for different periods of time (2 min, 30 min, 1 hours or 24 hours). [00248] Reaction of SMG08 with MG in vitro

[00249] The reaction of SMG08 with MG was also confirmed using MALDI-TOF MS analysis. MG08 (100 μM) was incubated with or without MG (100 μM) in PBS at 37°C for 24 hours. In the top panel of Figure 48, the peak at m/z 361.2 corresponds to the theoretical molecular weight of SMG08 (-H+). The peaks (bottom panel) at m/z 415.2, 433.2 and 487.3 correspond to SMG08 reacted with MG, with masses shift of +54, +72, and +126 Da (54+72 Da). Peaks at m/z 415.2 and 433.2 are likely representing SMG08 reacted with MG with or without loss of H2O; Peak at m/z 487.3 is likely corresponding to the reacted SMG08 at m/z 415.2 with another MG molecule attached on the N-terminal of SMG08. Results of MS analysis revealed the reaction of SMG08 with MG in vitro. [00250] Discussion - Example II

[00251] MG is a highly reactive dicarbonyl molecule which is inevitably produced during the metabolism of glucose, triglyceride, and protein through both non-enzymatic and enzymatic pathways in mammalian cells. Under pathological conditions such as metabolic syndrome and increased uptake of sugars such as fructose enhances the accumulation of intracellular and extracellular MG. Elevated levels of MG in blood stream and tissues (including but not restricted to vascular tissues - responsible for diabetes's complications including nephropathy, retinopathy, neuropathy, insulin resistance, obesity, atherosclerorosis, stroke and ventricular hypertrophy heart and peripheral circulatory diseases, neural degenerative diseases, endocrinal diseases, renal diseases, retinal diseases, respiratory and digestive diseases and aging ) have been reported in hypertensive animals, diabetic patients and animals [19, 24-28]. Similarly, increased levels of glyoxal and 3-DG have also also reported in plasma from diabetic patients[29, 68].

[00252] Increased MG levels, as well as glyoxal and 3-DG, affects almost every type of cell due to the increased AGEs formation and ROS production. Although the underlying mechanism is still unclear, MG, glyoxal and 3-DG are believed to be involved in the development of hypertension, diabetes and its complications and other diseases. [00253] Two of the most frequently studied consequences of elevated MG, glyoxal and

3-DG are the formation of AGEs and production of ROS, which are widely explored in cardiovascular diseases including hypertension, atherosclerosis, stroke, diabetes and its complications. Previous studies have demonstrated that inhibiting AGEs formation or breaking AGEs crosslink could improve or reverse the deleterious effects induced by AGEs accumulation. Therefore, reducing level of MG, glyoxal and/or 3-DG is a strategy to treat, prevent, reduce and/or ameliorate the deleterious effects of these reactive dicarbonyl molecules.

[00254] In mammalian cells, the glyoxalase system is responsible the degradation of

MG. To treat, reduce, prevent and/or ameliorate the hazards of elevated dicarbonyl molecules, compounds that scavenge both intracellular and extracellular MG and/or glyoxal are needed.

[00255] There is described herein a compound designated SMG08 which rapidly and effectively scavengeed MG and glyoxal. The MG scavenging efficiency of SMG08 is over

70%, even an incubation time is only 2 minutes.

[00256] None of cysteine, NAC, metformin or GSH was found to scavenge MG as effectively as SMG08 in experiments conducted under the same condition as that of SMG08.

Cysteine and NAC scavenged MG by 17.8 % and 10%, respectively, after 24 hours of incubation, respectively.

[00257] The scavenging efficiency of aminoguanidine was 32.2% at an incubation time of 24 hours.

[00258] Alagebrium at 10 μM did not scavenge MG, whereas scavenging efficiency of

55.5% was observed at a concentration of 50 μM after 24 hour of incubation.

[00259] These data support that the reaction of SMG08 with MG is rapid and efficient, as compared to the other compounds tested. While note wishing to be bound by theory, it is thought that the efficiency and specificity of SMG08 in scavenging MG are related to its sequence and structure.

[00260] It was found that SMG08 did scavenge glyoxal by 42% after 24 hours of incubation.

[00261] Alagebrium (50 μM) did not scavenge glyoxal, though it did scavenge MG.

These results indicated SMG08 could efficiently scavenge glyoxal.

[00262] The ability of SMG08 to scavenge 3-DG was not observed in the experiments conducted herein. However, based on the structure of 3-DG, SMG08 might be able to react with the carbonyl in 3-DG. Therefore, the scavenging ability of SMG08 to scanvage 3-DG needs to be investigated in the future using different methods.

[00263] Using cultured VSMC, the MG scavenging ability of SMG08 was further examined. SMG08 treatment scavenges MG in a dose-dependent manner in cultured VSMCs treated with MG as shown in Figure 18 A As shown in Figure 18 B, MG (10 μM) treatment increased cellular MG level by 50.8% in cultured VSMC compared with that in control cells. Pre-treatment with SMG08 decreased cellular MG levels by 70.1%, compared to MG-treated control cells. Pre-treatment with SMG08 decreased cellular MG levels by 54.8% compared to untreated control cells. Pre-treatment with SMG08 decreased MG level by 74.8% compared with untreated control group. These results demonstrate that SMG08 effectively scavenges and decreases intracellular MG level in cultured cells.

[00264] Pre-treatment with aminoguanidine or alagebrium (100 μM) prevented the increase of MG level in MG-treated cells, and decreased cellular MG to a level similar to that of untreated control cells. As noted above, SMG08-induced decrease of MG to a level lower than that in untreated control cells.

[00265] SMG08 was found to be more potent in scavenging intracellular MG than either aminoguanidine or alagebrium. SMG08 is an efficient and possibly specific scavenger for MG and glyoxal.

[00266] As shown in Figure 20, MG (10 μM) treatment stimulated VSMC proliferation. High level of glucose (25 mM) increased cell proliferation as compared to control cells cultured in normal level of glucose (5 mM), which is consistent with the reported results [105, 106]. Pre-treatment with SMG08 prevented the effects of MG treatment and high level of glucose on cell proliferation. While not wishing to be bound by theory, this may be due to a scavenging of glucose-induced increase in MG by SMG08. Published data have shown that high level of glucose induced an increase of cellular MG level in cultured VSMCs [22]. Therefore, while not wishing to be bound by theory, blocking of MG and high glucose- induced cell proliferation may be explained by the MG scavenging ability of SMG08. These results also support that SMG08 is biologically effective in blocking MG-induced cell proliferation in cultured VSMC. Again, while not wishing to be bound by theory, the data that demonstrated pre-treatment with SMG08 alone decreased cell proliferation suggests that endogenous MG may be required for a normal cell proliferation. Therefore, increased MG levels may be a causative factor in vasculoproliferative diseases including hypertension and atherosclerosis. Scavenging endogenous MG with SMG08 may be a new strategy for the treatment of proliferative vascular diseases involved with elevated MG levels. [00267] It has previously been reported that ROS production was increased by MG treatment in cultured VSMC [79]. As shown in Figure 21, pre-treatment of VSMC with

SMG08 blocked MG-induced ROS production. A correlation of increased MG level, ROS production and enhanced cell proliferation was found.

[00268] ROS has been implicated in cellular signaling pathways regulating cellular functions including cell proliferation. The results herein suggest that ROS, at least partially, may be involved in MG-induced cell proliferation in cultured VSMC. Scavenging of MG and the subsequent decrease of ROS production may be one of the underlying mechanism(s) of

SMG08 inhibited cell proliferation.

[00269] Although there is not much data about the effect of alagebrium on ROS production, it was recently reported that mice treated with alagebrium shown a reduced level of oxidative stress in cardiac tissues as well as inflammation, in parallel with a reduction in cardiac AGEs [107]. Whether alagebrium can attenuate MG-induced ROS production is not clear.

[00270] MG treatment increased AEGs (CML) staining was abolished with SMG08 pre-treatment. In addition, SMG08 pretreatment also could decrease basal level of CML staining in untreated control cells as shown in Figure 23.

[00271] The results herein support that SMG08 is a powerful compounds in scavenging both extracellular and intracellular MG, blocking MG-induced ROS production and AGEs formation and VSMC proliferation.

[00272] Another property of SMG08 is that it is cell membrane permeable, which was demonstrated by cell staining using FITC labeled SMG08. The half-life of SMG08-FITCI was at least 24 hours, as evidenced in cultured VSMCs.

[00273] EXAMPLE - III

[00274] Methods and materials

[00275] Patients

[00276] Blood samples were obtained from 20 untreated patients with mild to moderate hypertension from the clinical research units at Sacre-Coeur Hospital or at Hόtel- Dieu Hospital in Montreal. Based on the body mass index (BMI, =body weight in kg/ square of the height in meter) value, the patients were divided into obese (BMI ^ 30) and non-obese (BMI<30) groups. Both the two groups comprised males and females ranging in age from 46 to 68 years. All subjects gave informed written consent approved by the ethics committees of both hospitals. Venous blood was drawn into pre-chilled tubes (BD, Franklin Lakes, NJ) containing EDTA (K₃). The samples were vortexed and centrifuged immediately at 4 ⁰C for 20 min at 3000 rpm and the plasma samples were stored at -80 ⁰C. [00277] Animals

[00278] Eight 8-week-old male obese Zucker rats and eight age-matched lean Zucker rats were purchased from Charles River laboratories, Inc. (Wilmington, MA), housed in temperature-regulated animal facility and maintained at 22-23°C. These animals were exposed to a 12 hour light/dark cycle with free access to water and different diet recipes. Rats were treated in accordance with guidelines of the Canadian Council on Animal Care and the experimental protocols were approved by the Animal Care Committee of the University of Saskatchewan. Body weight, blood glucose level were measured weekly using a Quantichrom glucose assay kit (Bioassay Systems, CA, USA). Blood samples (0.5 mL) were obtained from tail vein at the age of 10, 12 and 14 weeks for serum MG measurement. Intraperitoneal glucose tolerance test (IPGTT) was carried out after overnight fasting at the age of 16 weeks. At the end of week 16, different tissues were isolated and frozen under -80 ⁰C after anaesthetization of rats by intraperitoneal injection of sodium pentobarbital (50 mg/kg body weight).

[00279] MG measurement

[00280] Quantitation of MG was done by the widely accepted o-phenylenediamine (o-

PD)-based assay as described by Chaplen et al [108], with some modifications. Briefly, the supernatant of tissue homogenate or serum was incubated with 100 mmol/L o-PD (derivatizing agent) for 3 hours at room temperature (about 22-25⁰C). The quinoxaline derivative of MG (2-methylquinoxaline) and the quinoxaline internal standard (5- methylquinoxaline) were measured using a Hitachi D-7000 high-performance liquid chromatography (HPLC) system (Hitachi Ltd., Mississauga, Ontario, Canada). The column was a Nova-Pak® Cl 8 column (3.9 x 15 mm, and 4 um particle diameter; Waters, MA, USA). The mobile phase was composed of 8% (vol/vol) of 50 mmol/L NaH₂PO₄ (pH 4.5), 20% (vol/vol) of HPLC grade acetonitrile and 72% of water. Duplicate injections of each sample were made. Samples were calibrated by comparison with a 2-MQ standard. [00281] Western blotting

[00282] Isolated rat adipose tissue or 3T3-L1 cells were sonicated using a sonicator.

The supernatants containing crude cellular proteins were resolved on a 12% SDS-PAGE gel, and transferred onto the PVDF membrane (PALL Corporation, Ontario, Canada). The membrane was blocked with 5% skim milk solution in PBS containing 0.05% tween-20 (PBS-T) at room temperature for 1 hour and incubated with primary antibody (1:500 for p21, p27, phospho-p21 (pp21) and phospho-p27 (pp27) antibodies, Santa Cruz, MO, USA; 1: 1000 for Akt and phospho-Akt (p-Akt Thr308) and β-actin antibodies for about 18 hours at 4⁰C with shaking, Cell Signaling Technology, MA, USA). After washed 3 times with the PBS-T for 30 minutes, the membrane was incubated with the HRP-conjugated secondary antibody (1:10000) for 1~2 hours at room temperature. The immunoreactions were visualized by ECL and exposed to X-ray film (Kodak Scientific Imaging film, X-omat Blue XB-I). [00283] PI3K activity assay

[00284] The PI3K activity was measured using a competitive ELISA kit (Echelon

Biosciences, Salt Lake City, UT) following the manufacturer's instruction. Briefly, 5 μL of 1OX reaction buffer and 10 μL of PI(4,5)P2 substrate solution were added into immunoprecipitated PI3K from 250 μg of total protein. The kinase reaction was allowed to proceed for 3 h and then diluted PI(3,4,5)P3 detector was added. After that, the absorbance was read on a plate reader at 450 nm. The amount of PI(3,4,5)P₃ produced is proportional to PI3K activity.

[00285] Cell proliferation assay

[00286] The proliferation of 3T3-L1 cells was measured by the Celltiter 96® non- radioactive cell proliferation assay kit (Promega, WI, USA). Briefly, cells were seeded onto 96-well plates (about 5000 cells per well) and cultured in Dulbeco's Modified Eagle's Medium (DMEM, HyClone, Ontario, Canada). When they reached about 50% of confluence in medium, the medium was removed and the cells were washed with serum-free medium and incubated in serum-free medium for 48 hours. The cells were then treated with or without MG, SH-6 (10 μM) or ALT-711 (50 μM) for 48 hours in serum-containing DMEM medium supplemented. After that, the cells were incubated with dye solution (15 μL for each well) in medium at 37⁰C for 4 hours and then with solubilization solution at room temperature for 1 hour. The spectrophotometric absorbance of the samples was determined by using a plate reader (Thermo Labsystems, Finland) at 570 nm. [00287] Cell cycle assay

[00288] Cell cycle analysis was performed by propidium iodide (PI) staining. Briefly,

3T3-L1 cells were firstly seeded into 10 cm dishes. When they reached about 50% of confluence, the cells were incubated in serum-free medium for 48 hours and then treated with MG, SH-6 (10 μM) or ALT-711 (100 μM) for 12, 16 or 20 hours. Subsequently, the cells were harvested and re- suspended in PBS at 1 x lOVmL and fixed with 70% cool ethanol for 1 hour. After the cells were washed and centrifuged, the pelleted cells were re-suspended in 1 mL PBS and added with 50 mL of RNase A stock solution (10 g/mL). Followed a 3 hour incubation at 4°C, the cells were then pelleted and added with 1 mL of PI staining solution (3.8 mM sodium citrate, 50 mg/mL PI in PBS) and analyzed by flow cytometry on an Beckman Coulter Epics XL flow cytometer (Beckman Coulter Canada Inc, Ontario, Canada). [00289] CDK2 activity assay

[00290] CDK2 activity was determined by measuring ATP consumption with PKLight

Assay Kit (LT07-500, Cambrex Bio Science, Rockland, ME, US). Briefly, after incubation of 200 mg of proteins with 2 mg of anti-CDK2 antibody (Santa Cruz) in cell lysis buffer for 4 hours at 4⁰C, protein A/G plus agarose beads (20 mL) were added and the mixture was incubated overnight at 4°C with shaking. Beads were washed 3 times and suspended in 40 mL of CDK2 kinase assay buffer containing 20 mM ATP and 0.1 mg/mL histone Hl. The above mixture was reacted at 30⁰C for 30 min in 96-well plate before kinase stop solution and ATP detection reagent were added according to the manufacture's protocol. Bioluminescent signal in each well was detected using a microplate spectrofluorometer (NovoStar, BMG LABTECH

Inc., Durham, NC). CDK2 activity was expressed as ATP consumption from 3 experiments.

[00291] Data analysis

[00292] Data are expressed as mean ± SEM and analyzed using one way ANNOVA in conjunction with t test where applicable. Significant difference between treatments was defined at a level of P < 0.05.

[00293] Results III

[00294] Increased MG accumulation in obese patients

[00295] There was no difference between the age and sex distribution in the obese and non-obese patient groups. The body weight of non-obese and obese group was 73.78±2.70 kg and 98.31±7.38 kg, respectively. The mean BMI value for non-obese group and obese group was 25.23±1.05 and 33.5±3.0. The mean plasma MG level in obese patients was 3.5 ±0.4 μM, which was significantly higher than that of the non-obese group (2.1 ±0.1 μM). Correlation analysis indicated a strong correlation between plasma MG level and BMI value (r=0.606,

P=0.0046, Fig. 28A) in the tested people.

[00296] Increased MG accumulation in obese Zucker rats

[00297] To examine the correlation between MG accumulation and the development of obesity observed in human samples, we compared the MG levels in lean and obese Zucker rats. As in the patients, increased MG level were also detected in different tissues of the obese rats. Moreover, a strong correlation was shown between serum MG level and the body weight of the Zucker rats (r=0.772, P=0.0054, Fig. 28B). At the end of 16 weeks, the body weight of the obese rats was significantly higher than that of the lean rats (Figure 34). The obese rats exhibited higher plasma triglyceride, total cholesterol and high density cholesterol (HDL) level comparing with the lean rats (Figure 34). Although the fasting glucose level did not show significant difference between lean and obese rats during the age of 10 to 16 weeks (Figure 34), a markedly increased MG accumulation was observed in serum, kidney, and particularly in fat tissue from obese rats (Fig. 29A, B). Furthermore, it was found that the serum MG level increased with the aging of experimental rats. While in 10 and 12 week-old obese rats, it was 144±50% and 171±15% of the age-matched lean rats, this value increased significantly to 241±7 % and 329±10% at the end of 14 and 16 weeks (Fig. 29C). [00298] Increased Akt but not PI3K activity observed in adipose tissue of obese

Zucker rats

[00299] Activation of PI3K/Akt pathway is considered a critical factor for adipogenesis, including adipocyte proliferation [109, 110] and differentiation [43, 111-113]. In the present study, the expression of Akt protein in adipose tissue from 16 week-old obese Zucker rats was significantly enhanced compared with the lean rats (P<0.05 vs lean Zucker rats, n=4, Fig. 30A, B). Additionally, a 2.8 fold increase was observed on the phosphorylation (T308) of Akt protein from obese rats (P<0.005 vs lean Zucker rats, n=4, Fig. 30A, B). However, as a key upstream regulator, the PI3K activity in adipose tissue did not show significant difference between the lean and obese Zucker rats (P>0.05, n=4, Fig. 30C). [00300] MG stimulated proliferation of cultured adipose cells

[00301] To investigate whether MG induce the proliferation of adipose cells, a cell proliferation assay was carried out with or without MG. The results showed that 5, 10 and 20 μM MG significantly increased the proliferation rate of 3T3-L1 cells to 115±2.1%, 126±3.6% and 119±3.3% of the untreated cells (P<0.05 vs. Control; n=48 in each group, Fig. 31-A). While 10 μM of MG significantly increased the 3T3-L1 cell proliferation, co-treatment of Akt inhibitor SH-6 (10 μM) or AGE breaker ALT-711 (50 μM) alleviated the cell proliferation to the control level (Fig. 31B).

[00302] The effect of MG on cell proliferation was further confirmed by the cell cycle phase distribution after MG treatment (Fig. 32). Comparing the percentage of cells in Gl, S and G2 phase at different time point, it was found that the MG-treatment always lead to a faster cell cycle progression (Fig. 32A), which represented as increased cell number in S phase after 16 or 20 hours MG treatment (Fig. 32A-b) and increased cell number in G2 phase after 20 hours MG treatment (Fig. 32A-c). 10 μM of MG treatment for 20 hours increased the proportion of cells in S and G2 phase while the co-administration of SH-6 (10 μM) and significantly reversed this MG-induced proliferation (Fig. 32B-b, c). The anti-MG effect of ALT-711 was not as strong as SH-6. SH-6 co-treatment reversed the MG effect in both S and Gl phase but ALT-711 did not (Fig. 32A-a, b).

[00303] Effect of MG on the expression and activity of Akt and its downstream effectors

[00304] As Akt plays an important role in regulating cell growth by phosphorylating p21 and p27 [42], we also examined the effect of MG on these molecules (Fig. 33). After treated 3T3-L1 cells with MG and/or SH-6/ ALT-711 for 24 hours, the phosphorylation and expression of Akt protein were examined by Western blotting. With lOμM MG treatment for 24 hours, an increased phosphorylation of Akt as well as p21 and p27 were observed (Fig. 33- A, B). The co-administration of SH-6 or ALT-711 significantly reversed the phosphorylation on these molecules. Furthermore, the expression of p21 protein was attenuated in cells co- treated with SH-6 or ALT-711 (P<0.05 vs MG-treated cells, n=3). However, the expression of p27 protein was not affected by MG treatment.

[00305] As a molecule that directly regulate the progression of cell cycle from Gl to S phase, the activity of CDK2 was stimulated to about 4 fold of the control level by 10 μM of MG (Fig. 33C). The increased CDK2 activity was reversed by co-administration of either SH- 6 or ALT-711. However, no significant change was observed on its expression in MG treated 3T3-L1 cells (data not shown). [00306] Discussion - III

[00307] The effects of MG in diabetes has long been recognized [85, 114, 115]. In the study of Example III, the effect of MG on the development of obesity, another form of metabolic syndrome is reported. The increased MG levels in both obese patients and Zucker rats (Fig. 28, 29) strongly indicated a physiological relevance of MG accumulation and obesity. Indeed, further study suggested that accumulated MG stimulates the phosphorylation of Akt and its effectors including p21 and p27, accelerates the cell cycle progression and proliferation of preadipocytes, and therefore contribute to the development of obesity. [00308] Adult-onset obesity has been considered mainly due to adipocyte hypertrophy.

According to a previous study, the mean size of fat cells from obese Zucker rats is dramatically larger than that from lean Zucker rats [116], even at 8 weeks old. However, based on the same study, the ratio between the fat cell size of obese and lean rats did not change from 8 weeks to 16 weeks old. In contrast to cell size, the total fat cell number in obese rats increased 3 fold from 4 weeks old to 6 month old while in lean rats, only about 1.4 fold [116]. This result indicates that the increased fat cell number plays a more critical role in the development of obesity than cell size, especially in severe forms of adult-onset obesity. In the experiment of Example III, it was demonstrated that MG increased the fat cell number by promoting growth and proliferation of preadipocytes (3T3-L1 cells). After 16 to 20 hours of treatment, 10 μM of MG increased the cell number in S and G2 cell cycle phase (Fig. 32A) which indicated a stimulated progression of cell cycle. As a result, treatment of 10-20 μM MG for 48 hours increased the number of 3T3 -Ll cells in cell proliferation assay (Fig. 31). While the inhibitive effect of MG has been extensively studied [84, 117, 118], however, a decreased proliferative effect of MG with a concentration reached 50 μM was not observed. [00309] This might indicate a biphasic effect of MG on cell proliferation. While not wishing to be bound by theory, the inhibitive effect of MG might due to the acute effect of high MG concentration, but not the effect of the physiological dose, which is around 0.2-5 μM based on previous [27, 119] and the present study (Figure 29, Example III). [00310] The observation on MG-treated 3T3-L1 cells not only showed the effects of

MG on cell proliferation and cell cycle regulation, but also implied a possible pathway that mediated these effects. The PI3K/ Akt signal cascade plays an important role in regulating cell proliferation. Based on the present results, the effect of MG on cell proliferation was at least to some extend due to the MG-stimulated Akt activity and its downstream effectors including p21 and p27, lOμM of MG in cultured 3T3-L1 cells increased the phosphorylation of Akt protein. Furthermore, MG treatment increased the phosphorylation of p21 and p27 (Fig. 33 A, B), the major regulators that arrest the cells at Gl /S checkpoint. The increased phosphorylation of p21 and p27 activates the degradation of these proteins and lead to the entry of cells into S phase from Gl phase. This explains the MG-activated cell proliferation detected. Again, while not wishing to be bound by theory, further observation of the increased activity of CDK2 in MG-treated cells supported this theory (Fig. 33C). Unlike in cultured cells, the total amount of Akt protein was shown increased in the adipose tissue of obese Zucker rats. However, the increase of phosphorylation on Akt protein was more striking (Fig. 30 A, B), which suggests that the increased activity of Akt protein is a more critical factor to stimulate the adipocyte proliferation than the increased protein quantity in the fat tissue of obese rats.

[00311] ALT-711, also known as alagebrium, is the first drug to be clinically tested for the purpose of breaking the crosslinks caused by AGEs [120]. It is designed to reverse the stiffening of blood vessel walls that contributes to hypertension and cardiovascular disease, as well as many other forms of degradation associated with protein cross linking. In experiments of Example III, ALT-711 was used as a specific inhibitor to block the effect of MG. The similar role of ALT-711 in reversing the MG-induced cell proliferation and attenuating the activity of Akt and its downstream effectors (Fig. 31, 32 and 33) confirmed the observation that MG mediates adipocyte proliferation by stimulating Akt activity. In the cell cycle assay, however, ALT-711 showed less strong effect in reversing MG' s effect comparing with SH-6. While not wishing to be bound by theory, this may be because SH-6 directly works on Akt while ALT-711 does not.

[00312] The experiments of Example III did not show significant difference on the

PI3K activity between the lean and obese rats. Until now, two modes of Akt activation were found: PI3K-dependent pathway [121-123] and PI3K-independent pathway [124, 125]. Since PI3K did not show different activity in obese and lean rats as the Akt did in our experiment, the possibility cannot be excluded that the MG-induced cell proliferation might be regulated via other pathway instead of PI3K pathway. Increased accumulation of MG and AGEs observed in diabetic animals and patients [10, 19, 126] gives people an impression that the increased MG accumulation is a result of hyperglycemia. However, the finding of increased MG level in hypertensive animals without hyperglycemia [10, 30, 31] led to a re-evaluation of the synthesis and biological effect of MG. In the present study (Example III), the serum MG level increased with the ageing of obese Zucker rats from 14 weeks on ward. However, according to the present study (Fig. 29C) and previous data [127], the fasting glucose level during 10 to 16 weeks did not show significant increase in obese Zucker rats compared with the age-matched lean rats. This result, together with our observation in hypertensive animals

[10] suggest that increased MG accumulation may exist under normal range of blood glucose level. Thus, while not wishing to be bound by theory, the MG-stimulated adipocyte proliferation might occur long before hyperglycemia is noticed.

[00313] EXAMPLE IV

[00314] Materials and Methods

[00315] Chemicals and antibodies

[00316] Anti-nitrotyrosine antibody and bovine serum were purchased from Invitrogen

Corporation (Burlington, ON, Canada). Anti-CEL antibody was obtained from Novo Nordisk

(AJS, Denmark). Alagebrium (ALT-711) was from Alteon Inc. (Parsippany, NJ, USA). Cell culture medium, FITC IgG fluorescent antibody, MG, NAC, o-phenylenediamine (o-PD), 2- methylquinoxaline, 5-methylquinoxaline, KCN, 2,6-dichlorophenolindophenol (DCPIP), rotenone, thenoyltrifluoroacetone (TTFA), antimycin A, coenzyme Ql, cytochrome C, NaN3, tween, NADH, decylubiquinol, digitonin, sucrose, MOPS, EDTA, NaPO₃, fatty acid-free

BSA, ATP-free ADP, glutamate and malate were purchased from Sigma-Aldrich (Oakville,

ON, Canada).

[00317] Cell culture

[00318] A- 10 cell, which is a aortic smooth muscle cell line from rats, was obtained from American Type Culture Collection and cultured in Dulbecco's Modified Eagle's Medium

(DMEM) containing 10% bovine serum at 37°C in a humidified atmosphere of 95% air and

5% CO2, as described in our previous study [15d]. Cells of passages 3 to 8 were used in this study.

[00319] Isolation of mitochondria [00320] Following the instruction of Mitochondrion Isolation Kit from Sigma- Aldrich

(Oakville, ON, Canada), cells were lysed using cell lysis solution (1: 150, 5 min) and suspended in extraction buffer A. Unbroken cells and nuclei were pelleted by centrifugation at 600 g for 10 min. The supernatant was centrifuged at 15,000 g for 15 min, and the mitochondrial pellet was resuspended in celLytic M cell lysis reagent for MG measurement. The mitochondrial pellet was resuspended in extraction buffer A and freeze-thaw twice for mitochondrial complexes activity determination. Cytochrome C Oxidase Assay Kit from Sigma-Aldrich (Oakville, ON, Canada) was used to determine the integrity of isolated mitochondria. Cytochrome C oxidase is located on the inner mitochondrial membrane and has traditionally been used as a marker for this membrane [16d]. The activity of cytochrome C oxidase in isolated mitochodria was high, indicating the high integrity and purity of the preparation.

[00321] MG content determination

[00322] MG content was determined using an o-PD method as described previously

[15d]. In brief, mitochondria isolated from A- 10 cells were incubated on ice for 10 min with 1/4 volume of perchloric acid (PCA) and centrifuged (12,000rpm, 15min) to remove the PCA- precipitated mitochondrial debris. The supernatant was supplemented with 100 mM o-PD and incubated for 3 hours at room temperature. The quinoxaline derivative of MG (2- methylquinoxaline) and the quinoxaline internal standard (5-methylquinoxaline) were measured using a Nova-Pak ® Cl 8 column (3.9 >< 150 mm, and 4 μm particle diameter, MA, USA) equipped with a Hitachi high-performance liquid chromatography (HPLC) system (Hitachi Ltd., Mississauga, ON, Canada).

[00323] Detection of mitochondrial ROS (mtROS) and mitochondrial O₂

[00324] Mitochondria produce a variety of ROS, such as ONOO^", nitric oxide (NO) and O₂ ^". MitoTracker Red CM-H2XRos and MitoSOX from Invitrogen Corporation (Burlington, ON, Canada) were used to detect the levels of mtROS and mitochondrial O₂ ^" [17d,18d]. A-IO cells were seeded on 35 mm glass-bottom dishes and treated with different agents for 18 h. Then, cells were labeled with MitoTracker Red (300 μM, 15 min) or MitoSOX (2 μM, 20 min). After washing, cells were bathed in DMEM again and subjected to examination under a Confocal Laser Scanning Biological Microscope (Olympus Fluoview 300, Olympus America Inc., Melville, NY, USA) coupled with 40^χ objective lens. The exposure time of camera, the gain of amplifier and the aperture were fixed at 4.57s/scan, 4.0^χ and 3 respectively, to allow quantitative comparisons of the relative fluorescence intensity of the cells between groups. 10-14 cells were randomly collected from 4 different pictures of each group. The average fluorescence intensity of each cell was measured using Image J program (NIH, USA). Data were expressed as mean ± SEM of the fluorescence intensity of those cells.

[00325] Measurement of MnSOD activity and NO level

[00326] SOD activity of A- 10 cells was detected following the instruction of SOD

Assay Kit from Cayman Chemical (Ann Arbor, MI, USA). KCN at 3 mM was used to inhibit the activity of Cu/Zn SOD, leaving only MnSOD activity to be measured. For NO detection cells were preloaded with 5 μM membrane permeable DAF-FM (Invitrogen Corporation, Burlington, ON, Canada) in Kreb's buffer for 2 hours at 37°C. After removal of the excess probe and with different treatments, DAF-fluorescence intensity, reflecting intracellular NO level, was measured with excitation at 495 nm and emission at 515 nm in a Fluoroskan Ascent plate reader (Thermo Labsystem, Helsinki, Finland). [00327] Immunocytochemistry staining

[00328] A- 10 cells were seeded on glass cover slips with different treatments for 18 hours, and subjected to immuno-staining. As described previously [15d], cells were fixed in 4% formalin for 1 hour at room temperature. After permeation with 0.1% Triton X-100 for 5 min, fixed cells were incubated with 3% goat serum for 1 hour, and then incubated with primary antibody (anti-CEL, 1:100; anti-nitrotyrosine, 1:200) at 4°C about 12 hours. Cells were washed in PBS (0.01 M) for 15 min and incubated with diluted fluorescent secondary antibody (FITC-IgG, 1: 200) for 3 hours at room temperature (about 22 ⁰C). After washed with PBS, cells were mounted on glass slides and observed under a confocal microscope. Fluorescence intensity was measured using Image J program. [00329] Detection of the activities of complex I , complex III, and complex IV

[00330] Mitochondrial complex I activity was determined by monitoring the reduction of DCPIP at 600 nm with the addition of assay buffer (10 * buffer containing 0.5 M Tris-HCl at pH 8.1, 1% BSA, 10 μM antimycin A, 3 niM KCN, 0.5 mM coenzyme Ql) [19d]. Mitochondrial proteins (25 μg/ml) and DCPIP (64 μM) were added to the assay buffer before using. The reaction was started by adding 200 μM NADH and scanned at 600 nm with the reference wavelength of 620 nm for 2 minutes. Mitochondrial complex III activity was detected by monitoring the reduction of cytochrome C at 550 nm upon the addition of assay buffer (10x buffer contains 0.5 M Tris-HCl at pH 7.8, 2 mM NaN3, 0.8% Tween-20, 1% BSA, 2 mM decylubiquinol) with 40 μM cytochrome C [19d]. The reaction was started by adding 20 μg/ml mitochondria proteins to the assay buffer and scanned at 550 nm with the reference wavelength of 540 nm for 2 minutes. Mitochondrial complex IV activity was measured by monitoring the reduction of reduced cytochrome C at 550 nm with the addition of assay buffer (0.5 M phosphate buffer at pH 8.0, 1% BSA and 2% tween) [19d]. Freshly prepared reduced cytochrome C (80 μM) was added to the assay buffer before using. The reaction was started by adding mitochondria protein (20 μg/ml) and scanned at 550 nm with the reference wavelength of 540 nm for 2 min. All assays were performed at 37 ⁰C. [00331] Determination of ATP synthesis

[00332] ATP synthesis was assayed based on a modified method of Atorino et al [2Od].

Briefly, cells were incubated at 37⁰C for 30 min in a respiratory buffer (0.02% digitonin, 0.25 M sucrose, 20 mM MOPS, 1 mM EDTA, 5 mM NaPO₃, 0.1 % fatty acid-free BSA, 1 mM ATP-free ADP, 5 mM glutamate, and 5 mM malate, pH 7.4). Thereafter, 3% PCA was used to precipitate proteins, and samples were centrifuged at 13,000 rpm for 2 min. Supernatants were taken out to measure ATP after pH adjusted to 7.8 using 10 M KOH. ATP was detected using ATP Bioluminescent Assay Kit from Sigma-Aldrich (Oakville, ON, Canada). Data were expressed as nanomoles of ATP per milligram of protein. [00333] Statistical analysis

[00334] Data were expressed as mean±SEM from at least three independent experiments. Statistical analysis was performed by one-way analysis of variance (ANOVA). Differences between groups were examined by Student's unpaired t-Test. Values are considered to be statistically significant when p < 0.05. [00335] Results - IV

[00336] Effect of MG on mtROS generation

[00337] After A-IO cells were treated with exogenous MG (30 μM) for 18 hours, mitochondrial MG content increased by 50.7% (0.205 ± 0.012 vs. 0.136 ± 0.014 nmol/mg mitochondrial protein, p < 0.01, n = 4 for each group) (Figure 35). Alagebrium (50 μM) had no effect on basal content of mitochondrial MG but its presence decreased the effect of exogenous MG on mitochondrial MG content (0.14 ± 0.009 vs. 0.205 ± 0.01 nmol/mg mitochondrial protein, p < 0.01, n = 4 for each group). NAC (600 μM) had no effect on mitochondrial MG content.

[00338] MG increased the fluorescence intensity of CEL in a concentration-dependent manner. At 30 μM, MG increased the fluorescence intensity of CEL by 321% (Figure 36, A and B). Co-treatment with alagebrium (50 and 100 μM) decreased the effect of 30 μM MG (Figure 36, A and C). NAC (600 μM) did not show any effect on the staining of CEL (data not shown).

[00339] Exposure of cells to MG (5 to 100 μM) caused a significant concentration- dependent increase in mtROS generation. The production of mtROS increased dramatically with 30 μM MG and reached a plateau with 100 μM MG (Figure 37, A and B). Co- incubation of NAC (600 μM) significantly decreased mtROS generation induced by MG (Figure 37, A and B). Alagebrium (50 and 100 μM) and ONOO- specific scavenger uric acid (50 μM) inhibited mtROS generation induced by 30 μM MG (Figure 37, A, C and D). [00340] Effects of MG on NO and nitrotyrosine generation

[00341] The effect of MG on NO generation was evaluated by DAF-FM, a specific probe used for quantitating low concentration of NO. As shown in Figure 38, MG (30 μM) increased the production of NO by 48% (p < 0.01). Alagebrium (50 μM), NAC (600 μM), and mtNOS inhibitor 7-nitroindazole (50 μM) significantly reduced MG-increased NO generation.

[00342] Nitrotyrosine is formed by ONOO -mediated nitration of tyrosine residues of proteins. As shown in Fig. 39A and 39C, MG (20 and 30 μM) significantly increased the fluorescence intensity of nitrotyrosine in A- 10 cells by 176-191%. The addition of NAC (600 μM) significantly inhibited the formation of nitrotyrosine induced by MG. Co-incubation of alagebrium (50 μM) also significantly reduced the fluorescence intensity of nitrotyrosine induced by MG (30 μM) (Figure 39, A and D). Nitrotyrosine and mitotracker were co- localized in the tested cells as indicated by the overlap of yellow and red-green images (Figure 39B).

[00343] Effect of MG on mitochondrial O₂ ^" generation

[00344] MitoSOX, a specific probe to detect mitochondrial O₂ ^" level, was used in this assay. MG (30 μM) increased mitochondrial O₂ ^" production by 69.9% (p < 0.01), compared with untreated cells. Co-incubation of alagebrium (50 μM) and SOD mimic 4-hydroxy-tempo (Tempol, 500 μM) decreased mitochondrial O₂ ^" production induced by MG treatment by 57% (p < 0.01) and 85.8% (p < 0.01), respectively (Figure 40, A and B). [00345] Effect of MG on MnSOD activity

[00346] MG (5-30 μM) decreased the activity of MnSOD, the first line enzyme to scavenge O₂ ^" in mitochondria. MG at 30 μM decreased MnSOD activity by 24.5% (p < 0.05) (Figure 41A). Alagebrium (10-100 μM) normalized MG-decreased MnSOD activity (Figure 41B). NAC (600 μM) had no effect on MnSOD activity (data not shown). [00347] Effect of MG on mitochondrial functions

[00348] MG (30 μM) treatment for 18 hours had no obvious effect on the activity of complex I or complex IV, but significantly decreased complex III activity by 11.7% (p < 0.05), as shown in Figure 42A. Alagebrium (50 μM) inhibited the effect of MG on complex III by 64.61% (p < 0.05). NAC (600 μM) did not have effect on complex III activity (data not shown).

[00349] In order to confirm the effect of MG on mitochondrial ETC complexes, complex inhibitors were used to treat cells for 2 hours in the absence or presence of MG. Mitochondrial O₂ ^" generation was thereafter determined using the specific probe MitoSOX. Rotenone (0.5 μM and 1 μM), TTFA (5 μM and 10 μM), antimycin A (3 μM and 5 μM) and KCN (0.5 niM and 1 niM), which are respective blockers of complex I, complex II, complex III and Complex IV, significantly increased production of mitochondrial O₂ ^" in A-IO cells (Figure 42B). No difference was observed between effects of two concentrations of each blocker. Therefore, these inhibitors appear to maximally inhibit the respective complexes. MG (30 μM) further increased rotenone (1 μM), TTFA (10 μM) and KCN (1 mM)-induced mitochondrial O₂ ^" generation by 48.11%, 52.6% and 40.2%, respectively, in comparison with the cells treated with the inhibitor alone. However, the addition of MG (30 μM) did not change complex III inhibitor (antimycin A)-induced mitochondrial O₂ ^" generation. While not wishing to be bound by theory, these results suggested that MG targeted on complex III to induce mitochondrial O₂ ^" generation (Figure 42B). MG (30 μM) significantly lowered ATP production by 44.8% (4.76 ± 0.74 vs. 8.62 ± 0.24 nmol/mg protein, p<0.01). Alagebrium (50 μM) restored ATP synthesis inhibited by MG by 78.0% (Figure 43). [00350] Discussion - IV

[00351] MG causes crosslink among lysine, cysteine, and arginine residues of selective proteins to form AGEs, like CEL, altering the structure of proteins and their functions [9d]. Higher levels of MG have been found in diabetic patients than in healthy controls [2Id]. As discussed above, it was shown herein that mitochondrial MG content was significantly increased after the cells were treated with exogenous MG. While not wishing to be bound by theory, it appears that MG can move across plasmalemma and mitochondrial membrane to attack different molecular targets. Once inside the cells, MG induces glycation of many proteins in the cytosol, mitochondria and other vesicles. The formation of CEL in mitochondria may result in the dysfunction of mitochondrial proteins, and furthermore, increase mtROS generation. Alagebrium, an AGEs crosslink breaker [22d], not only decreased CEL formation, but also diminished MG levels in mitochondria. While not wishing to be bound by theory, the result indicates that alagebrium scavenges MG and inhibits glycation directly, although the mechanism is unknown. This discovery also echoes the observation obtained by Nobecourt et al [23d].

[00352] The physiological concentration of plasma MG in rats is approximately 5 μM

[24d]. A previous study detected the plasma MG levels of 33.6 μM in 20-week-old SHR and 14.2 μM in age-matched WKY rats [25d]. Plasma levels of MG increased from 3.3 μM in healthy humans to 5.9 μM in type 2 diabetic patients [2Id]. In addition, cultured cells may produce more MG since MG concentration up to 310 μM was detected in cultured Chinese hamster ovary cells [26d]. Furthermore, up to 10 niM MG had been used to investigate its effect on insulin secreting cells and insulin signaling pathways in rat L6 myoblasts [27d,28d]. Thus, MG (30 μM) used in the present study is not only the physiological relevant concentration, but also suitable to mimic the insulin resistance environment in rat aortic smooth muscle cells.

[00353] Previous work has shown that MG induced overproduction of O₂ ^', NO, and

ONOO^" in rat VSMCs [14d]. It is shown herein that mitochondria are targets of MG for this pro-oxidative action. More specifically, MG increased mitochondrial ONOO^" production in VSMCs. Several lines of evidence support this conclusion. (1) Uric acid, a specific scavenger of ONOO^", significantly decreased MG-induced mtROS generation. (2) Increased staining of nitrotyrosine was observed in MG treated A-IO cells, and the expression of nitrotyrosine was mostly co-localized with mitochondrial marker staining. (3) MnSOD is the major enzyme which catalyzes O₂ ^" degredation in mitochondria and protects mitochondria against oxidative stress. The results herein show that MG reduced the activity of MnSOD in mitochondria of VSMCs. (4) MG-induced mitochondrial O₂ ^" production was inhibited by Tempol. As a SOD mimic, Tempol is more stable and membrane-permeable than MnSOD itself [29d]. (5) Located on the inner mitochondrial membrane, mtNOS is considered as the alpha-isoform of neuronal nitric oxide synthase (nNOS) and is responsible for NO production in mitochondria [30d,31d]. MG-induced intracellular NO was decreased by 7-nitroindazole. The latter is the specific inhibitor of mtNOS [32d] and can prevent mitochondrial structural damage mediated by increased mitochondria NO generation in the developing brain [33d]. Together with MG-induced mitochondrial O₂ ^", the stimulation of mtNOS by MG also contributes to ONOO^" formation.

[00354] It is shown herein MG selectively damaged complex III activity, not complex I or complex IV. While not wishing to be bound by theory, this effect may underlie MG- inhibited ATP synthesis and MG-enhanced ROS production. Further evidence for the inhibition of complex III by MG was derived from the failure of MG to increase mitochondria O₂ ^" generation in the presence of antimycin A, a specific blocker of complex III. That alagebrium restored MG-inhibited complex III activity suggests that the complex III is glycated by MG. Complex III, which is also called cytochrome C reductase, transfers electrons from ubiquinone to cytochrome C. The inhibition of complex III by MG may disrupt the ETC, rendering more electrons leaking out to form O₂ ^". Consequently, hydrogen electrochemical gradient across the inner mitochondrial membrane is weakened, and the driving force for ATPase to synthesize ATP provided by hydrogen influx across the inner mitochondrial membrane is reduced. Cellular integrity and function are therefore compromised. The experiments herein with complex II inhibitor, TTFA, indicate that complex II is not a major site of mitochondrial O₂ ^" generation in A-IO cells. Furthermore, after complex II is maximally inhibited by TTFA, MG still induced mitochondrial O₂ ^" production. This shows that the effect of MG on superoxide production does not depend on complex II. Evidence shows that mitochondrial dysfunction, especially elevated production of mtROS resulted from Complex III inhibition, is closely linked with the pathogenesis of insulin resistance [8d]. Moreover, normalization of mitochondrial superoxide production blocked the diabetic hyperglycemia damage in bovine aortic endothelial cells [34d]. Therefore, complex III dysfunction-induced mitochondrial oxidative stress plays an important role in the pathophysiology of insulin resistance syndrome.

[00355] In addition, the experiments herein demonstrated that alagebrium reversed all harmful effects of MG on mitochondria of cultured cells. Compared with alagebrium, the beneficial effect of NAC is limited. It reduced MG-induced ROS, NO, and nitrotyrosine production, but did not affect mitochondrial functions. [00356] EXAMPLE - V [00357] Methods and materials

[00358] Sprague-Dawley (SD) rats (4-week old, male) were purchased from Charles

River Laboratories (St-Constant, Quebec, Canada). The rats were treated with fructose or MG in accordance with the guidelines of the Canadian Council on Animal Care, and the experimental protocols were approved by the Animal Care Committee at the University of Saskatchewan as previously reported 5. Briefly, 4 groups of SD rats were fed with normal rat chow, 60% fructose (in chow), metformin (500 mg/kg per day in drinking water), or fructose plus metformin (60% fructose in chow, metformin 500mg/kg per day in drinking water) for 16 weeks. For chronic treatment with MG, subcutaneous osmotic minipumps were used; SD rats (12 weeks old) were implanted for 4 weeks with either a 0.9% saline-minipump, a MG- minipump (60 mg/kg/day), or a MG-minipump (60 mg/kg/day) (this last group also received alagebrium (-20 mg/kg/day) in their drinking water). Aortic tissues were cleaned in ice-cold phosphate buffer saline and then were promptly snap-frozen in liquid nitrogen and stored at 8O⁰C. Proteins were extracted by homogenization for Western blot analysis as described below.

[00359] MG, Hoechst 33258, the anti-FLAG antibody and other chemicals were purchased from Sigma-Aldrich Ltd. [³H]-thymidine, [γ-³²P]ATP, nitrocellular membrane, and ECL kit were purchased from Amersham. Histone Hl, phospho- (active) and unphospho- (unactivated) Aktl proteins were obtained from Upstate Biotechnology. Antibodies against p21 (M-19), p27 (F-8), CDK2 (M2), _Phospho-p21(Thrl45, sc-20220-r), _Phospho-p27(Thrl87, sc-16324-r), and protein A/G plus agarose were obtained from Santa Cruz Biotechnology. Antibodies against Akt, Aktl, phospho-Akt(Ser473), phospho- Aktl (Ser473), and phospho- GSK-3α/β(Ser21/9) were from Cell Signaling Technology (Danvers, MA, US) and Chemicon (Temecula, CA, US). Aktl siRNA and DFECT2 transfection regents were purchased from Dharmacon (Chicago, IL, US). The QuikChange® Site-Directed Mutagenesis Kit was from Stratagene (La Jolla, CA, US). The N-acetylated peptide (Ac-CLQWTTVIER-OH, Ac-TI l), which has a sequence corresponding to tryptic peptide TI l of Aktl protein, was synthesized by Biopeptide Co. Inc. (San Diego, CA, US). All data were expressed as the mean±SE of at least 3 independent experiments unless otherwise stated. Statistical analyses were performed using Student's t test or ANOVA.

[00360] Cell lines

[00361] HEK-293 human embryonic kidney cell line was obtained from American

Type Culture Collection. HEK-293 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) as previously described (2Ie).

Cultured cells in log-phase were starved in FBS-free DMEM for 24 hours. The medium was then replaced with DMEM containing 10% FBS containing either the vehicle or MG at different concentrations for an additional 24 hours. Cells were washed with ice-cold phosphate-buffered saline (PBS), and then harvested by trypsinization. For [³H]-thymidine incorporation and cell counting, cells were seeded in 24-well plates with an equal number of cells per well.

[00362] Measurement of cell proliferation

[00363] Cell proliferation was determined by colorimetry using the One Solution Cell

Proliferation Assay kit (Promega). The quantity of colored formazan product measured by its absorbance at 490 nm is directly proportional to the number of living cells in culture. Direct cell counting was performed with a Beckman Coulter counter. DNA synthesis was examined by [ H]-thymidine incorporation into cellular nucleic acids (80). [ H] -thymidine (1 μCi/ml) was added to each well; following treatment, the cells were harvested and radioactive content was quantified using a liquid scintillation spectrometer (Beckman LS3801). Apoptotic cells were identified by Hoechst 33258 23 and condensation and fragmentation of nuclei under a fluorescence microscope (Olympus 1X70, Tokyo, Japan). Percentage of apoptotic cells was calculated based on 15-20 random fields per culture at ^χ200 magnification.

[00364] Plasmid construction and stable expression of Aktl variants in HEK-293 cells

[00365] The full-length Aktl coding sequence was PCR-amplified from the pCS2+- myr-Aktl plasmid (provided by Dr. A.B. Vojtek, University of Michigan, Ann Arbor) (24e,

25e). The Aktl cDNA (without the myristoylation sequence) was subcloned in-frame with an N-terminal triple-FLAG epitope in pFLAG3 vector (a gift from Dr. D.H. Anderson,

University of Saskatchewan). The subcloning was confirmed by DNA sequencing. This plasmid was subsequently used as a template to mutate residue Cys77 in Aktl to serine using the QuikChange® Site-Directed Mutagenesis Kit (Stratagene, Catalog # 200519). Point mutation was also confirmed by DNA sequencing. HEK-293 cells were transfected with empty vector, wild-type Aktl or Aktl(Cys77Ser) (Aktl(C/S)) expression plasmids using the

FuGENE 6 transfection reagent (Roche Applied Science). Transfected cells were selected with antibiotics G418 (800 μg/ml) for 4 weeks, and G418-resistent cells were passaged in culture medium containing 200 μg/ml of G418, and treated with or without MG as described above.

[00366] Western blot analysis

[00367] Total proteins were extracted from harvested cells with 200 μl of lysis buffer

(20 mmol/L Tris-HCl at pH 7.5, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1%

Triton X-100, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L β-glycerolphosphate, 1 mmol/L

Na₃ VO4, 1 mmol/L phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, and 1 μg/ml aprotinin).

Resolved proteins (40 μg) were subjected to Western blot analysis 82 with primary antibodies as follows: at a 1:500 dilution for antibodies against p21, p27, phospho-p21(Thrl45), phospho-p27(Thrl87) and CDK2; at 1:1000 for antibodies against Akt, Aktl, phospho-

Akt(Ser473), phospho-Aktl(Ser473), and phospho-GSK-3α/β; and at 1:5000 for anti-β-actin.

Western blots were digitized with Chemi Genius2 Bio Imaging System (SynGene), quantitated using GeneTools (SynGene) and normalized against the quantity of loaded β- actin.

[00368] Aktl activity assay

[00369] Aktl activity was determined using Aktl assay kit (Cell Signaling) following the manufacturer's instruction with minor modification. Briefly, 200 μg of precleared proteins from cultured A-10 cells or HEK-293 cells expressing Aktl variants was incubated with 2 μg of anti-Aktl or anti-Flag antibodies in cell lysis buffer for 4 hours at 4⁰C with shaking. The immunocomplex was then precipitated overnight with protein A/G agarose beads (40 μl) at 4°C with rocking. Agarose beads were pelleted and washed 3 times with cell lysis buffer, then 2 times with kinase buffer. Washed pellet was suspended in 50 μl of kinase buffer supplemented with 1 μl of 10 mmol/L ATP and 1 μg of glycogen synthase kinase 3 fusion proteins (GSK) as substrates. The reaction was allowed to proceed for 30 min at 3O⁰C, and was then terminated by addition of 25 μl of 3 x sodium dodecyl sulfate (SDS) sample-loading buffer and heating for 5 mins at 95°C. The sample was subjected to Western blot analysis using anti-phospho-GSK antibody to detect Aktl activity.

[00370] Determination of the effect of MG on Aktl proteins in cell-free preparations

[00371] Phospho-Aktl proteins (5 μg) were treated with MG (10 & 30 μmol/L) in 100 μl of PBS for 24 hours at 37°C. Unphospho-Aktl proteins (5 μg) were similarly treated with MG in the presence of PIP3 (5 μmol/L). Treated and untreated unphospho-Aktl proteins were further activated by mitogen-activated protein kinase-activated protein kinase-2 (MAPKAPK2) and phosphoinositide-dependent kinase- 1 (PDKl) following the manufacturer's protocol. Briefly, unphospho-Aktl proteins (5 μg) with or without MG treatment were activated with 0.5 μl of MAPKAPK2 (250 mU/μl) for 30 mins at 30⁰C in 40 μl of activation buffer (50 mmol/L Tris-HCl at pH7.5, 0.1 mmol/L EGTA, 0.2 mmol/L NaCl, 0.1% β-mercaptoethanol, 0.01% Brij-35, 0.1 mg/ml BSA, 0.4 mmol/L Mg/ ATP), followed by adding 0.5 μl PDKl (0.2mg/ml) and another incubation of 30 mins at 3O⁰C. The reaction was stopped by adding EDTA to a final concentration of 25 mmol/L. For the kinase activity assay, 2 μl of sample was used in kinase assay as described above. [00372] CDK2 activity assay

[00373] CDK2 proteins were immunoprecipitated from cell extracts and subjected to kinase activity assay using histone Hl as the substrate (27). Briefly, 100 μg of precleared protein was immunoprecipitated with 2 μg of anti-CDK2 antibody and agarose beads. Washed beads were suspended in 20 μl of CDK2 assay buffer (containing 20 μmol/L ATP and 0.1 μg/μl histone Hl) with 5 μCi [γ-³²P]ATP. The mixture was incubated at 30⁰C for 30 mins and the reaction was terminated by adding 10 μl of 3><SDS sample loading buffer and heating for 5 mins at 95⁰C. Samples were resolved on a 7.5% sodium dodecyl sulfate-polyacrylamide gel. The gel was fixed in methanol:glacial acetic acid:water = 45:10:45 for 1 hour at room temperature with shaking, then dried and exposed to X-ray film. Density of autoradiography was digitized with Chemi Genius2 Bio Imaging System and quantitated using software of GeneTools from SynGene. [00374] RESULTS - V

[00375] Increased phospho-Aktl (Ser473) level in aorta from fructose- and MG- treated SD rats

[00376] MG levels in plasma and aorta from fructose-fed SD rats (for 16 weeks) were significantly elevated compared with age-matched control SD rats. Consistently, the level of phospho-Aktl (Ser473) in aorta was also significantly increased in fructose-fed rats (Fig. 44A). Normalization of MG levels in plasma and aorta by co-treatment with metformin with fructose was paralleled with a decrease in phospho-Aktl (Ser473) level (Fig. 44A). Metformin itself also decreased phospho-Aktl (Ser473) level. Furthermore, the level of phospho- Aktl (Ser473) in aorta from SD rats treated with MG (4 weeks; Alzet® minipump) was augmented compared with control animals, while co-treatment with alagebrium (a MG scavenger administered via the drinking water) prevented the effects of MG treatment on phospho-Aktl (Ser473) level (Fig. 44B).

[00377] Effects of the Aktl(C/S) mutation on cell proliferation and DNA synthesis in transfected HEK-293 cells

[00378] MG treatment (10, 30 μmol/L) of vector- transfected cells exhibited increased cell proliferation (evidenced by MTT reduction and DNA incorporation assays), however not to the same degree as seen in cells transfected with wild-type Aktl (Fig. 45 A & 45B). Cell proliferation was highest in cells expressing the Aktl(C/S) mutant and MG treatment did not increase this any further. Cell number over a period of 5 days was determined using a Coulter counter, and similar trends as observed in the MTT reduction and DNA incorporation assays were observed (Fig. 45C). These data confirm that MG-induced cell proliferation results from the interaction of MG and Cys77 in Aktl.

[00379] Altered activities of different regulatory proteins in HEK-293 cells over- expressing the Aktl(C/S) mutant

[00380] The mechanism underlying the effect of the Aktl(C/S) mutant on cell proliferation was further investigated. The phosphorylation of Aktl(Ser473) was significantly increased in the Aktl(C/S) mutant transfected cells as compared with that of vector- or wild- type Aktl-transfected cells. MG treatment (30 μmol/L) significantly increased phospho- Aktl(Ser473) level in vector or wild-type Aktl transfected cells (Fig. 46A). The potentiating effect of MG on phospho-Aktl(Ser473) level was not observed in Aktl(C/S)-expressing cells. Furthermore, increased phospho-GSK-3α/β levels induced by MG treatment were observed in vector- or wild-type Aktl-transfected cells. However, the over-expression of Aktl(C/S) enhanced GSK-3α/β phosphorylation on its own an effect that was not influenced by MG treatment (Fig. 46B). Flag- Aktl proteins were isolated by immunoprecipitation from the MG- treated cells (30 μmol/L, 24 hours) and then Aktl kinase activity was directly measured using exogenous GSK-3 as the substrate. The activity of Flag- Aktl was significantly increased in Aktl(C/S)-transfected cells in comparison with that of wild-type Aktl-transfected cells in the absence of MG treatment, and the effect of Aktl(C/S) remained unaffected by treatment with MG (Fig. 46C).

[00381] The basal level of phospho-p21 was significantly higher in Aktl(C/S)- expressing cells than that in wild-type Aktl or vector transfected cells (Fig, 47A). The increased phospho-p21 level in Aktl(C/S)-expressing cells in the absence of MG reached the same level as seen in wild type Aktl- and vector- transfected cells in the presence of MG (30 μmol/L, 24 hours). Furthermore, this increase in phospho-p21 level in Aktl(C/S)-transfected cells was not augmented by MG treatment (30 μmol/L, 24 hours) (Fig, 47A). MG treatment significantly decreased total p21 level in vector-transfected cells as compared with untreated cells. A similar decrease of total p21 was also observed in wild-type Aktl- and Aktl(C/S)- transfected cells with or without MG treatment (Fig. 47A). [00382] Consistent with the changes of phospho-p21/p-21, basal CDK2 activity was significantly increased by virtue of expression of wild-type Aktl and further increased by MG treatment (30 μmol/L) in both vector- and wild-type Aktl-transfected cells. Basal CDK2 activity was increased the most by simple expression of Aktl(C/S) in the absence of MG and was not affected by MG treatment (Fig. 47B). [00383] Discussion - V

[00384] Akt is a multi-function protein kinase that regulates cellular processes including cell proliferation, growth, survival, glucose uptake, metabolism and angiogenesis. Deregulation of Akt signaling is involved in human diseases such as cardiovascular diseases, diabetes, or cancer.

[00385] MG is a chemically reactive dicarbonyl molecule that can modify the lysine or arginine residue in proteins including insulin, platelet-derived growth factor receptor β (PDGFR), hemoglobin, and alphaA-crystallin. As shown herein, Aktl (both phosphorylated and unphosphorylated forms) can be modified by MG at Cys77, which lies within the PH domain. In the PMF spectrum of untreated Aktl protein, the tryptic peptide containing Cys77 (Tl 1) corresponds to a peak at m/z 1248.7.

[00386] Irreversible modification of Cys77 by MG will prevent the re-formation of the disulfide bond with CysόO, which may favor the phosphorylation-dependent activation or long-lasting activity of Aktl.

[00387] MG-targeted Cys77 in Aktl was investigated by mutating Cys77 to serine

(C/S) in order to avoid the modification by MG and to prevent the formation of a disulfide bond with CysόO. The Aktl (C/S) mutation mimics the effect of MG treatment by showing elevated levels of phospho-Aktl(Ser473) and activity in Aktl(C/S)-transfected HEK-293 cells or immunoprecipitated Flag-Aktl(C/S) proteins (Fig. 46). MG treatment could not further augment the levels of phosphorylation and activity of the Aktl (C/S) variant (Fig 47C). Moreover, unphospho-Aktl proteins were incubated with MG in the presence of PIP3, followed by exposure to MAPKAPK2 and PDKl, and both Aktl phosphorylation and activity were significantly increased (Fig. 1 IA). MG treatment of phospho-Aktl also augmented Aktl activity, without affecting the level of phospho-Aktl(Ser473) (this may due to the fact that the Aktl protein used is already fully phosphorylated) (Fig. 1 IB).

[00388] Various previous studies assessing deregulation of Akt in pathological conditions have resulted in inconsistent findings. The present data revealed an augmented phospho-Aktl level in aortic tissue from SD rats either fed with fructose (a precursor for MG) or chronically administered with MG (using osmotic minipumps for 4 weeks). The changes in Aktl activation (Fig. 44) was accompanied by elevated blood pressure and mesenteric artery remodeling 2 (decreased lumen diameter, increased tunica width and medium-to-lumen ratio), or decreased vascular function. Furthermore, the augmented levels of phospho-Aktl (Ser473) were sensitive to metformin or alagebrium (two MG scavengers).

[00389] The change of phospho-Aktl (Ser473) level is positively correlated with MG levels in these animal models. These results indicate that elevated MG level and abnormal Aktl signaling may play an important role in the development of vascular diseases including hypertension.

[00390] As noted supra, at pathophysiological^ relevant concentrations (0.1-50 μmol/L), MG was found to enhance VSMC proliferation and DNA synthesis in an Aktl- dependent manner. Inhibition of Aktl by pharmacological inhibitor or gene-silencing effectively prevented the effects of MG on cell proliferation and on the targets downstream of Aktl (e.g. phospho-GSK, p21, phsopho-p21, CDK2 activity). The Aktl(C/S) mutation also mimics the effects of MG on cell proliferation and the targets downstream of Aktl (Fig. 47). MG treatment could not further augment the effects of Aktl(C/S) on cell proliferation and aforementioned proteins (Fig. 45 & 46). [00391] References:

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[00392] All publications, patents and patent applications mentioned in this

Specification are indicative of the level of skill those skilled in the art to which this invention pertains and are herein incorporated by reference to the same extent as if each individual publication patent, or patent application was specifically and individually indicated to be incorporated by reference.

[00393] The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modification as would be obvious to one skilled in the art are intended to be included within the scope of the following claims. .

Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. An isolated peptide comprising an amino acid sequence selected from CLQ (SEQ. ID NO: lor CLQWTTVIER (SEQ. ID. NO: 2).

2. The isolated peptide of claim 1, wherein said peptide binds and/or scavenges a reactive carbonyl.

3. The isolated peptide of claim 1 or 2, wherein said peptide inhibits formation of glycation endproducts in vitro or in vivo.

4. The isolated peptide of claim 2 or 3, wherein said reactive carbonyl is methylglyoxal (MG).

5. The isolated peptide of claim 2 or 3, wherein said reactive carbonyl is glyoxal.

6. A pharmaceutical composition for treating, preventing, or ameliorating an AGE related condition or disease in a mammal, comprising an effective amount of an isolated peptide or a pharmaceutically acceptable salt thereof comprising an amino acid sequence selected from CLQ (SEQ. ID NO: lor CLQWTTVIER (SEQ. ID. NO: T), and a pharmaceutical carrier.

7. The pharmaceutical composition of claim 6, wherein said condition or disease is vascular disease; insulin resistance; diabetes mellitus; hyperlipidemia; hyperglycemia; metabolic syndrome; nephropathy; retinopathy; neuropathy; heart and artery disease; neurodegenerative diseases; endocrine, renal, respiratory, reproductive conditions; skin ageing; premature aging; rheumatoid arthritis; Alzheimer's disease; uremia; neurotoxicity or discolouration of teeth.

8. The pharmaceutical composition of claim 7, wherein said vascular disease is hypertension, stroke, ventricular hypertrophy, atherosclerosis, restenosis or stroke.

9. The pharmaceutical composition of any one of claims 6 to 8, wherein said pharmaceutical composition is formulated for administration by bolus injection, intravenous infusion, subcutaneous infusion, oral administration, pulmonary administration, nasal administration, transdermal administration, parenteral administration, rectal administration or topical administration

10. Use of an isolated peptide comprising an amino acid sequence selected from CLQ (SEQ. ID NO: l)or CLQ WTTVIER (SEQ. ID. NO: 2) for treating, preventing and/or ameliorating an AGE related condition or disease in a mammal.

11. The use of claim 10 wherein, said condition or disease is vascular disease; insulin resistance; diabetes mellitus; hyperlipidemia; hyperglycemia; metabolic syndrome; nephropathy; retinopathy; neuropathy; heart and artery disease; neurodegenerative diseases; endocrine, renal, respiratory, reproductive conditions; skin ageing; premature aging; rheumatoid arthritis; Alzheimer's disease; uremia; neurotoxicity or discolouration of teeth.

12. The use of claim 11 wherein, said vascular disease is hypertension, stroke, ventricular hypertrophy, atherosclerosis, restenosis or stroke

13. The use of claim 10, wherein said peptide inhibits formation of glycation endproducts.

14. The use of any one of claim 11 to 13, wherein said peptide is suitable for administration by a single dosage or a variable dosage.

15. The use of any one of claims 11 to 14, wherein said mammal is a human.

16. Use of an isolated peptide comprising an amino acid sequence selected from CLQ (SEQ. ID NO: lor CLQ WTTVIER (SEQ. ID. NO: 2) for preventing and/or reducing spoilage of proteins in food.

17. A kit for inhibiting formation of glycation endproducts in an organism, said kit comprising: a peptide comprising isolated peptide comprising an amino acid sequence selected from CLQ (SEQ. ID NO: lor CLQ WTTVIER (SEQ. ID. NO: 2); and instructions for the use thereof.

18. The kit of claim 17, wherein said peptide binds and/or scavenges a reactive carbonyl.

19. The kit of claim 17 or 18, wherein said peptide inhibits formation of glycation endproducts in vitro or in vivo.

20. The kit of claim 17 or 18, wherein said reactive carbonyl is MG or glyoxal.