WO2009051804A1 - Thiazolium compounds for treating or preventing diseases associated with insulin resistance - Google Patents

Abstract

Thiazolium derivates for treating a patient having a metabolic disorder related to insulin resistance and/or beta cell dysfunction, including patients with type I diabetes, type II diabetes, metabolic syndrome and/or pre-diabetes.

Description

THIAZOLIUM COMPOUNDS FOR TREATING OR PREVENTING DISEASES ASSOCIATED WITH INSULIN RESISTANCE

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to, and the benefit of, U.S. Patent Application No.

60/999,583, filed October 18, 2007 and U.S. Patent Application No. 61/070,306, filed March 21, 2008. The contents of each of these applications are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

A large number of people suffer, or are predisposed to suffer from disturbances in their metabolism. Such disturbances include insulin resistance and β cell dysfunction, which are characteristic of diseases such as metabolic syndrome (syndrome X), types I and II diabetes and pre-diabetes, diseases that are rapidly growing in number in the western world. These diseases are multi-factorial and their mechanism or physiology are, in the majority of cases, not well characterized or understood.

Diabetes is a disease derived from multiple causative factors and characterized by elevated levels of plasma glucose (hyperglycemia) in the fasting state or after administration of glucose during an oral glucose tolerance test. There are two generally recognized forms of diabetes. In type I diabetes, or insulin-dependent diabetes mellitus (IDDM), patients produce little or no insulin, the hormone which regulates glucose utilization. Type I diabetes, formerly known as insulin-dependent diabetes (IDDM), childhood diabetes or also known as juvenile diabetes, is characterized by loss of the insulin-producing beta cells of the islets of Langerhans of the pancreas leading to a deficiency of insulin. There is no known preventative measure that can be taken against type I diabetes. Most people affected by type I diabetes are otherwise healthy and of a healthy weight when onset occurs. Diet and exercise cannot reverse or prevent type I diabetes. Sensitivity and responsiveness to insulin are usually normal, especially in the early stages. This type of diabetes comprises up to 10% of total cases in North America and Europe, though this varies by geographical location. This type of diabetes can affect children or adults but was traditionally termed "juvenile diabetes" because it represents a majority of cases of diabetes affecting children. The main cause of beta cell loss leading to type I diabetes is a T-cell mediated autoimmune attack. The principal treatment of type I diabetes, even from the earliest stages, is replacement of insulin. Without insulin, ketosis and diabetic ketoacidosis can develop and coma or death will result. In type II diabetes, or noninsulin-dependent diabetes mellitus (NIDDM), insulin is still produced in the body. Patients having type II diabetes often have hyperinsulinemia (elevated plasma insulin levels); however, these patients are insulin resistant, which means that they have a resistance to the effect of insulin in stimulating glucose and lipid metabolism in the main insulin-sensitive tissues, which are muscle, liver and adipose tissues. Some patients are insulin resistant, but not diabetic. These patients compensate for the insulin resistance by secreting more insulin, so that serum glucose levels are not elevated enough to meet the criteria of type II diabetes. In patients with type II diabetes, even elevated plasma insulin levels are insufficient to overcome the pronounced insulin resistance. Persistent or uncontrolled hyperglycemia that occurs with diabetes is associated with increased and premature morbidity and mortality. Often abnormal glucose homeostasis is associated both directly and indirectly with obesity, hypertension, and alterations of the lipid, lipoprotein and apolipoprotein metabolism, as well as other metabolic and hemodynamic disease. Patients with type II diabetes have a significantly increased risk of macro vascular and microvascular complications, including atherosclerosis, coronary heart disease, stroke, peripheral vascular disease, hypertension, nephropathy, neuropathy, and retinopathy. Therefore, therapeutic control of glucose homeostasis, lipid metabolism, obesity, and hypertension are critically important in the clinical management and treatment of type II diabetes. Many patients who have insulin resistance or type II diabetes often have several symptoms that together are referred to as syndrome X, or the metabolic syndrome. A patient having this syndrome is characterized as having three or more symptoms selected from the following group of five symptoms: abdominal obesity, hypertriglyceridemia, low high- density lipoprotein cholesterol (HDL), high blood pressure, and elevated fasting glucose, which may be in the range characteristic of type II diabetes if the patient is also diabetic. Each of these symptoms is defined in the recently released Third Report of the National Cholesterol Education Program Expert Panel on Detection, Evaluation and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III, or ATP III), National Institutes of Health, 2001, NIH Publication No. 01-3670. Patients with metabolic syndrome, whether or not they have or develop overt diabetes mellitus, have an increased risk of developing the macrovascular and microvascular complications that are listed above that occur with type II diabetes, such as atherosclerosis and coronary heart disease.

Insulin resistance is not primarily caused by a diminished number of insulin receptors but by a post-insulin receptor binding defect that is not yet completely understood. This lack of responsiveness to insulin results in insufficient insulin-mediated activation of uptake, oxidation and storage of glucose in muscle and inadequate insulin-mediated repression of lipolysis in adipose tissue and of glucose production and secretion in the liver.

There are available treatments for type II diabetes, each of which has its own limitations and potential risks. Physical exercise and a reduction in dietary intake of calories often dramatically improve the diabetic condition and are the best first line treatment of type II diabetes. Compliance with this treatment is very poor because of well-entrenched sedentary lifestyles and excess food consumption, especially of foods containing high amounts of fat. A widely used drug treatment involves the administration of meglitinide or a sulfonylurea (e.g. tolbutamide or glipizide), which are insulin secretagogues. These drugs increase the plasma level of insulin by stimulating the pancreatic beta-cells to secrete more insulin. When administration of a sulfonylurea or meglitinide becomes ineffective, the amount of insulin in the body can be supplemented by the injection of insulin so that insulin concentrations are high enough to stimulate even the very insulin-resistant tissues. However, dangerously low levels of plasma glucose can result from administration of insulin and/or insulin secretagogues, and an increased level of insulin resistance due to the even higher plasma insulin levels can occur.

Pre-diabetes is a condition in which blood glucose levels are higher than normal but not high enough for a diagnosis of diabetes. Pre-diabetes is also called impaired fasting glucose or impaired glucose tolerance. Many people with pre-diabetes develop type II diabetes within 10 years. In addition, they are at risk for heart disease and stroke.

Advanced glycation, the biochemical non-enzymatic modification of proteins by reducing sugars [Fu, M.X. et al. Glycation, glycoxidation, and cross-linking of collagen by glucose. Kinetics, mechanisms, and inhibition of late stages of the Maillard reaction. Diabetes 43, 676-683 (1994)], has been extensively assessed as a promoter of the progressive complications seen in diabetes [Brownlee, M. Biochemistry and molecular cell biology of diabetic complications. Nature 414, 813-20 (2001)]. Within the human body, tissue and circulating advanced glycation end products (AGEs) accumulate over time in natural aging, however this is accelerated as the result of redox imbalances or hyperglycaemia seen in diabetes. In addition, an important exogenous source of AGEs in the absence of hyperglycaemia, is from the Western diet, primarily from pasteurised dairy foods, bakery products and "browned" foodstuffs such as coffee and meat [Koschinsky, T. et al. Orally absorbed reactive glycation products (glycotoxins): an environmental risk factor in diabetic nephropathy. Proc Natl Acad Sci USA 94, 6474-9 (1997); Vlassara, H. et al. Inflammatory mediators are induced by dietary glycotoxins, a major risk factor for diabetic angiopathy. Proc Natl Acad Sci USA 99, 15596-601 (2002)] or from smoking [Cerami, C. et al. Tobacco smoke is a source of toxic reactive glycation products. Proc Natl Acad Sci USA 94, 13915-20 (1997)]. Although Maillard reactions were traditionally considered to contribute to flavour, texture and color in food preparation (e.g. in roasted meat, coffee or toast), food technologists and manufacturers are now also using this reaction to add functional properties (e.g. improved emulsification and gel formation) to a wide variety of foods. Once consumed, dietary AGEs are absorbed through the intestine and are the largest contributor to the body's AGE pool. Evidence for a role of dietary AGEs in β cell dysfunction has been suggested by improvements in insulin sensitivity and secretion in type II diabetic lepr ⁺'C57KsJ (dbdb) mice [Hofmann, S.M. et al. Improved insulin sensitivity is associated with restricted intake of dietary glycoxidation products in the db/db mouse. Diabetes 51, 2082-9 (2002)] as well as a reduced incidence of diabetes in non obese type I diabetic (NOD) mice [Peppa, M. et al. Fetal or neonatal low-glycotoxin environment prevents autoimmune diabetes in NOD mice. Diabetes 52, 1441-8 (2003)] with dietary restriction of AGEs. Indeed, a diet high in fat content, a known contributor to insulin resistance and β cell secretory defects, loses its pathogenic effects if the AGE content is reduced [Sandu, O. et al. Insulin resistance and type 2 diabetes in high-fat-fed mice are linked to high glycotoxin intake. Diabetes 54, 2314-9 (2005)].

AGEs can exert their biological effects via receptors such as the receptor for advanced glycation end products (RAGE) [Chavakis, T. et al. The pattern recognition receptor (RAGE) is a counterreceptor for leukocyte integrins: a novel pathway for inflammatory cell recruitment. J Exp Med 198, 1507-15 (2003)]. RAGE is a multi-ligand receptor involved in the amplification of immune and inflammatory responses primarily via nuclear factor -KB

(NF-KB), leading to the subsequent production of chemokines and cytokines which ultimately recruit inflammatory cells. Indeed, a recent study has identified blockade of the late stages of autoimmune diabetes with the "decoy" soluble RAGE receptor [Bierhaus, A. et al. Diabetes- associated sustained activation of the transcription factor nuclear factor-kappaB. Diabetes 50, 2792-808 (2001)]. Another study has suggested that polymorphisms of the RAGE gene may be important to the heritability of insulin resistance [Sullivan, CM. et al. RAGE polymorphisms and the heritability of insulin resistance: the Leeds family study. Diab Vase Dis Res 2, 42-4 (2005). A need exists to identify and develop compounds and pharmaceutical compositions, which ameliorate insulin resistance and β cell dysfunction in diseases involving disturbances in metabolism such as metabolic syndrome, insulin resistance, and types I and II and prediabetes.

SUMMARY OF THE INVENTION

The present invention provides methods of treating, or ameliorating a symptom of, a disease, disorder or condition associated with insulin resistance or β-cell dysfunction in a patient in need thereof, comprising administering a pharmaceutical composition comprising a compound of Formula I, or a pharmaceutically acceptable salt of the compound of Formula I,

Wherein R¹ and R² are selected from the group consisting of hydrogen, hydroxy (lower) alkyl, acetoxy (lower) alkyl, lower alkyl, lower alkenyl; or R and R together with their ring carbons may be an aromatic fused ring, optionally substituted by one or more amino, halo or alkylenedioxy groups; Z is hydrogen or an amino group; Y is amino, a group of the formula:

O Il -CH₂C-R, wherein R is a lower alkyl, alkoxy, hydroxy, amino or an aryl group, said aryl group optionally substituted by one or more lower alkyl, lower alkoxy, halo, dialkylamino, hydroxy, nitro or alkylenedioxy groups; a group of the formula: -CH₂R' wherein R' is hydrogen, or a lower alkyl, lower alkenyl, or aryl group; or a group of the formula:

wherein R" is hydrogen and R" is a lower alkyl group, optionally substituted by an aryl group, or an aryl group, said aryl group optionally substituted by one or more lower alkyl, halo, or alkoxylcarbonyl groups; or R" and R'" are both lower alkyl groups; and

X is a pharmaceutically acceptable anion, and a pharmaceutically acceptable carrier, thereby treating said disease, disorder or condition associated with insulin resistance or β-cell dysfunction.

The method can further include administering a inhibitor of a receptor for advanced glycation end-products (RAGE). The RAGE inhibitor can be soluble RAGE.

Rl and R2 can be independently lower alkyl. Z can be hydrogen. R can be an aryl group. The compound of Formula I can be 3-(2-phenyl-2-oxoethyl)-4,5-dimethylthiazolium. Preferably, the compound of Formula I is 3-(2-phenyl-2-oxoethyl)-4,5-dimethylthiazolium chloride or 3-(2-phenyl-2-oxoethyl)-4,5-dimethylthiazolium bromide. The disease, disorder or condition associated with insulin resistance or β-cell dysfunction can be type I diabetes, non-insulin dependent (type II) diabetes, pre-diabetes or metabolic syndrome.

Administration of a pharmaceutical composition comprising a compound of Formula I can increase insulin sensitivity, can ameliorate insulin resistance, can ameliorate plasma insulin and glucose levels, can suppresses basal insulin secretion, can increase acute insulin secretion, can reduce plasma methylglyoxal levels and/or can reduce mitochondrial oxidative stress.

In accordance with the present invention, a method for the treatment or prevention of diseases associated with insulin resistance and/or β cell dysfunction using compounds and compositions of the invention is disclosed. Diseases associated with insulin resistance and/or β cell dysfunction include insulin resistance, type I diabetes, type II diabetes, pre-diabetes, and metabolic syndrome. In one aspect, the compositions of the invention include pharmaceutical compositions comprising compounds for inhibiting the formation of and reversing the pre-formed advanced glycosylation (glycation) endproducts and breaking the subsequent cross-links. While not wishing to be bound by any theory, it is believed that the breaking of the pre-formed advanced glycosylation (glycation) endproducts and cross-links is a result of the cleavage of a dicarbonyl-based protein crosslinks present in the advanced glycosylation endproducts. In one aspect, the methods and compositions of this invention are thus directed to compounds which, by their ability to affect such cleavage, can be utilized to break the pre-formed advanced glycosylation endproduct and cross-link, and the resultant deleterious effects thereof, both in vitro and in vivo. In one aspect of the invention, treatment directed against AGEs may be useful for the treatment, reduction of risk in the development and prevention of diseases related to insulin resistance and/or β cell dysfunction. The invention includes a method of treating or reducing the risk of developing or preventing one or more diseases, disorders, or conditions selected from the group consisting of insulin resistance, type I diabetes, non-insulin dependent diabetes (type II diabetes), prediabetes, and metabolic syndrome, the method comprising the administration of an effective amount of the compound of formula I, or a pharmaceutically acceptable salt thereof:

wherein R¹ and R² are independently selected from the group consisting of hydrogen, hydroxy(lower)alkyl, acetoxy(lower)alkyl, lower alkyl, lower alkenyl, or R¹ and R² together with their ring carbons may be an aromatic fused ring, optionally substituted by one or more amino, halo or alkylenedioxy groups; Z is hydrogen or an amino group;

Y is amino, a group of the formula:

O Il

-CH₂C-R wherein R is a lower alkyl, alkoxy, hydroxy, amino or an aryl group, said aryl group optionally substituted by one or more lower alkyl, lower alkoxy, halo, dialkylamino, hydroxy, nitro or alkylenedioxy groups; a group of the formula: -CH₂R' wherein R' is hydrogen, or a lower alkyl, lower alkynyl, or aryl group; or a group of the formula:

-CH₂C-N

R"¹ wherein R" is hydrogen and R'" is a lower alkyl group, optionally substituted by an aryl group, or an aryl group, said aryl group optionally substituted by one or more lower alkyl, halo, or alkoxylcarbonyl groups; or R" and R'" are both lower alkyl groups;

X is a halide, tosylate, methanesulfonate or mesitylenesulfonate ion; and mixtures thereof, and a carrier therefor.

The invention includes a method of treating or reducing the risk of developing or preventing non-insulin dependent (type II) diabetes in a patient in need of such treatment, by administering to the patient a therapeutically or prophylactically effective amount of the compound of formula I or a pharmaceutically acceptable salt thereof. The invention includes a method of treating or reducing the risk of developing or preventing type I diabetes in a patient in need of such treatment, by administering to said patient a therapeutically or prophylactically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof. The invention includes a method of treating or reducing the risk of developing or preventing insulin resistance in a patient in need of such treatment, by administering to said patient a therapeutically or prophylactically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof. The invention includes a method of treating or reducing the risk of developing or preventing pre-diabetes in a patient in need of such treatment, by administering to said patient a therapeutically or prophylactially effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof. The invention includes a method of treating or reducing the risk of developing or preventing metabolic syndrome in a patient in need of such treatment by administering to said patient a therapeutically or prophylactically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof. The invention includes the method of the invention, wherein the treatment increases the insulin sensitivity of the patient. The invention includes a compound of formula I, wherein R¹ is lower alkyl. The invention includes a compound, wherein R² is lower alkyl. The invention includes a compound, where in R¹ and R² are lower alkyl. The invention includes a compound, wherein Z is H. The invention includes a compound, wherein Y is a group of the formula - CH₂C(O)R. The invention includes a compound, wherein R is aryl group. The invention includes a compound, wherein R is phenyl. The invention includes a compound, wherein X is halo. The invention includes a compound, wherein halo is chloride.

The invention includes a method of treating or reducing the risk of developing or preventing one or more diseases, disorders, or conditions selected from the group consisting of insulin resistance, type I diabetes, non-insulin dependent diabetes (type π diabetes), pre- diabetes, and metabolic syndrome, the method comprising the administration of an effective amount of the compound alagebrium or a pharmaceutically acceptable salt thereof. The invention includes the compound alagebrium chloride.

The invention includes a method of ameliorating insulin resistance in a patient in need thereof, by administering a pharmaceutical composition of the invention, thereby ameliorating said insulin resistance. The invention includes a method of ameliorating plasma insulin and glucose levels in a patient in need thereof, by administering a pharmaceutical composition of the invention, thereby ameliorating said plasma insulin and glucose levels. The invention includes a method of suppressing basal insulin hypersecretion and stimulating acute insulin secretion in a patient in need thereof, by administering a pharmaceutical composition of the invention, thereby suppressing said basal insulin hypersecretion and stimulating said acute insulin secretion.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are , described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGURE 1 is a series of bar graphs that illustrate that exposure of MIN6N8 cells to advanced glycation end products causes insulin secretory defects independent to glucose concentrations. Figure Ia shows the effect on basal insulin secretion of MIN6N8 cells upon exposure to AGEs in normal and high glucose and the effect of alagebrium. Figure Ib shows the effect on glucose stimulated insulin secretion (GSIS) for cells grown in high glucose conditions as compared to cells grown in normal glucose and the effect of alagebrium. Figure Ic shows flow cytometry analysis for cell surface RAGE. Figure Id shows basal insulin secretion for MIN6N8 cells that were transiently transfected with human full length RAGE or the control vector pCIneo, for 7 days in 25 mM glucose. Figure Ie shows 2OmM GSIS for MIN6N8 cells that were transiently transfected with human full length RAGE or the control vector pCIneo, for 7 days in 25 mM glucose. FIGURE 2 is a series of bar graphs which illustrate that AGEs disrupt glucose stimulated insulin secretion by uncoupling, interruption of ATP production, Ca2+ flux and superoxide production. Figure 2a shows the effect on ATP content in cells grown in high glucose upon exposure to AGE-BSA, and alagebrium. Figure 2b shows the effect on calcium flux in MIN6N8 cells grown in high glucose and treated with AGE-BSA, alagebrium (ALT), and verapamil (VER). Figure 2c shows the effect on superoxide production in mitochondria isolated from MIN6N8 cells grown in high glucose and treated with AGE-BSA and alagebrium. Figure 2d shows the effect on UCP-2 mRNA expression in cells treated with AGE-BSA and alagebrium. Figure 2e shows the effect of siRNA to UCP-2 on insulin secretion in cells treated with AGE-BSA.

FIGURE 3 is a series of graphs which illustrate that short-term infusion of AGE-RSA into healthy rats induces early β cell decompensation. Figure 3 a shows the effect on plasma insulin in rodents following short term infusion of AGE-RSA. Figure 3b shows the effect on the gene expression of proinsulin within the pancreas in rodents following short term infusion of AGE-RSA. Figure 3c shows the effect on the number of proliferating β cells within islets in rodents following short term infusion of AGE-RSA. Figure 3d shows the effect on the islet AGE (CML) content within islets in rodents following short term infusion of AGE-RSA. Figure 3e shows the effect on islet RAGE expression in rodents following short term infusion of AGE-RSA. FIGURE 4 is a series of graphs which illustrate that long term infusion of AGE-RSA into healthy rats interrupts first phase insulin secretion and induces β cell death. Figure 4a shows the effect on plasma insulin in rats following long term infusion of AGE-RSA and treatment with alagebrium. Figure 4b shows the effect on proinsulin gene expression in rats following long term infusion of AGE-RSA and treatment with alagebrium. Figure 4c shows the effect on the islet AGE (CML) content within islets in rats following long term infusion of AGE-RSA and treatment with alagebrium. Figure 4d shows the effect on ED-I monocyte/macrophage cellular infiltration within pancreatic islets in rats following long term infusion of AGE-RSA and treatment with alagebrium. Figure 4e shows the effect on β cell death within islets in rats following long term infusion of AGE-RSA and treatment with alagebrium.

FIGURE 5 is a series of graphs which illustrate that a high dietary intake of AGEs causes insulin deficiency, secretory defects, and hyperglycaemia. Figure 5a shows fasting plasma insulin and glucose levels at 6 months. Figure 5b shows insulin levels before and during i.v. glucose challenge. Figure 5c shows proinsulin gene expression by real time RT- PCR. Figure 5d shows islet RAGE expression by immunohistochemistry. Figure 5e shows islet AGE (CML) content by immunohistochemistry.

*p<0.05 vs Low AGE fed group, tp<0.001 vs Low AGE, $p<0.01 vs Low AGE.

FIGURE 6 is a series of graphs which illustrate that a high dietary intake of AGEs causes insulin deficiency and hyperglycaemia. Figure 6a shows the difference in dietary AGE (CML) intake between low AGE and high AGE containing diets. Figure 6b shows the difference in circulating AGEs (CMLs) found in rats receiving a high AGE diet verses a low AGE diet. Figure 6c shows the difference in fasting plasma glucose found in rats receiving a high AGE diet verses a low AGE diet. Figure 6d shows the difference in fasting plasma insulin levels found in rats receiving a high AGE diet verses a low AGE diet.

FIGURE 7 is a series of bar graphs which illustrate an acute insulin secretory experiment using MIN6N8 cells treated with AGE-BSA, BSA only and AGE-BSA + ALT- 711 at different time points (30min, 60 min, 2h and 4 h). Figure 7A shows the effect on basal insulin secretion. Figure 7B shows the effect on acute insulin secretion. Figure 7C shows the effect on total insulin secretion. Figure 7D shows the effect on cellular insulin.

FIGURE 8 is a series of graphs which illustrate in vivo insulin resistance data. Figure 8A shows plasma insulin concentrations over time following an intraperitoneal bolus of glucose given at 16 weeks. Figure 8B shows AUC insulin to AUC glucose ratio. Figure 8C shows fasting plasma glucose and insulin data.

DETAILED DESCRIPTION OF THE INVENTION The details of one or more embodiments of the invention are set forth in the accompanying description below. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. The preferred methods and materials are now described herein. Other features, objects, and advantages of the invention will be apparent from the description. In the specification, the singular forms also include the plural unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the case of conflict, the present specification will control.

AGEs have been implicated as potential toxins for pancreatic β cells both in vitro [Kaneto, H. et al. Reducing sugars trigger oxidative modification and apoptosis in pancreatic beta-cells by provoking oxidative stress through the glycation reaction. Biochem J 320 (Pt3), 855-63 (1996); Matsuoka, T. et al. Glycation-dependent, reactive oxygen species-mediated suppression of the insulin gene promoter activity in HIT cells. J Clin Invest 99, 144-50 (1997)] and in vivo [Hofmann, M. A. et al. RAGE mediates a novel proinflammatory axis: a central cell surface receptor for SlOO/calgranulin polypeptides. Cell 97, 889-901 (1999); Peppa, M. et al. Fetal or neonatal low-glycotoxin environment prevents autoimmune diabetes in NOD mice. Diabetes 52, 1441-8 (2003); Sandu, O. et al. Insulin resistance and type 2 diabetes in high-fat-fed mice are linked to high glycotoxin intake. Diabetes 54, 2314-9 (2005)] although the cellular and molecular mechanism responsible for this damage has not been previously examined. In the present examples, specific effects of AGEs via the inflammatory RAGE receptor on the insulin secretory pathway and β cell dysfunction in cultured insulin secreting cells (MIN6N8) were identified which were independent of elevations in glucose. These abnormalities included oxidative stress, mitochondrial respiratory chain uncoupling, decreases in ATP production and altered calcium flux. Indeed, AGE induced insulin secretory defects were reproduced by over-expression of RAGE in these cells, and ameliorated with an antagonist of this reaction, soluble RAGE. In vitro AGE induced effects on cultured β cells, were confirmed in vivo in a simplified model of long term AGE-RSA infusion. Specifically, AGE infusion in healthy non-diabetic rats had profound and progressive effects on first phase insulin secretion and caused β cell decompensation, in association with activation of classical pathways invoked in the destruction of pancreatic islet β cells. This included recruitment of monocytes and macrophages which may have been mediated via RAGE. Each of these abnormalities was attenuated with an anti-AGE therapy, alagebrium. Furthermore, consumption of diets high in AGE content by healthy rats also caused insulin secretory defects in the context of insulin deficiency and hyperglycaemia. One aspect of the invention includes a method of ameliorating first phase insulin secretion in a patient in need thereof, by administering a pharmaceutical composition of the invention, thereby ameliorating said first phase insulin secretion. Another aspect of the invention includes a method of ameliorating the destruction of pancreatic islet β cells in a patient in need thereof, by administering a pharmaceutical composition of the invention, thereby ameliorating said destruction of pancreatic islet β cells. Another aspect of the invention includes a method of ameliorating the recruitment of monocytes and macrophages in a patient in need thereof, by administering a pharmaceutical composition of the invention, thereby ameliorating said recruitment of monocytes and macrophages. Another aspect of the invention includes a method of reducing levels of ED-I monocyte/macrophage infiltration in a patient in need thereof, by administering a pharmaceutical composition of the invention, thereby reducing said levels of ED-I monocyte/macrophate infiltration.

In MIN6 cells, secretory defects including elevated basal and decreased glucose stimulated insulin secretion, could be induced by AGE exposure in both the presence and absence of high glucose, in a time dependent manner. The abnormalities present in the absence of high glucose took at least four times longer (4 weeks) to manifest, however, were almost identical to those seen with high glucose exposure. In addition, glucose uptake in these cells was enhanced in the presence of AGEs which may have contributed to the elevated basal and decreased glucose stimulated insulin secretion seen in the present study. Indeed, recent studies in human subjects have identified a loss of β cell function even when fasting glucose is increasing within the normal range [Godsland, LF. , Jeffs, J.A. & Johnston, D.G. Loss of beta cell function as fasting glucose increases in the non-diabetic range. Diabetologia 47, 1157-66 (2004)]. Other clinical data have suggested that β cell defects progress more rapidly in the presence of hyperglycaemia, emphasizing the concept of glucotoxicity which supports the finding in high glucose conditions where AGE induced defects were prominently observed. Furthermore, the present studies show more advanced impairment of β cell function, namely insulin deficiency when hyperglycaemia was already present in high AGE fed rats when compared to AGE-RSA infused rats. It is important to note, however, that circulating AGE levels were also not elevated in AGE-RSA infused rats suggesting that a combination of hyperglycaemia and elevated circulating AGE levels in high AGE fed rats was accelerating β cell injury. This was also suggested in the rats which consumed the high dextrose/glucose diet who also had hyperglycaemia and high circulating AGE levels. Nevertheless, it is important to appreciate that progression occurs as a result of exogenous AGE exposure even in the absence of hyperglycaemia. One aspect of the invention includes a method of ameliorating insulin secretory defects in a patient in need thereof, by administering a pharmaceutical composition of the invention, thereby ameliorating said insulin secretory defects. In one aspect, the insulin secretory defect is basal hypersecretion of insulin. In another aspect the insulin secretory defect is decreased glucose stimulated insulin secretion (GSIS). Another aspect of the invention includes a method of ameliorating cellular uptake of glucose in a patient in need thereof, by administering a pharmaceutical composition of the invention, thereby ameliorating cellular uptake of glucose. In one aspect, the cellular uptake is AGE-induced.

Previous studies in HIT insulin secreting cells have shown that exposure to increased concentrations of the highly reactive sugar ribose, causes glycation of the insulin promoter, followed by a decrease in cellular insulin synthesis and content [Matsuoka, T. et al. Glycation-dependent, reactive oxygen species-mediated suppression of the insulin gene promoter activity in HIT cells. J Clin Invest 99, 144-50 (1997); Kaneto, H. et al. Reducing sugars trigger oxidative modification and apoptosis in pancreatic beta-cells by provoking oxidative stress through the glycation reaction. Biochem J 320 (Pt3), 855-63 (1996)]. This may also have contributed to the accelerated secretory defects and decreased insulin gene transcription seen in high glucose in MIN6 cells within the present examples. In addition, over-expression of the AGE receptor, RAGE, mimicked the β cell secretory defects seen with exposure to AGEs, suggesting that AGE induced elevations of RAGE may initiate these pathogenic pathways. Indeed, RAGE expression was elevated in AGE exposed MIN6 cells and this is consistent with other settings such as various inflammatory disorders, where increases in local AGE levels enhance RAGE expression [Hofmann, M.A. et al. RAGE mediates a novel proinflammatory axis: a central cell surface receptor for SlOO/calgranulin polypeptides. Cell 97, 889-901 (1999); Bierhaus, A. et al. Understanding RAGE, the receptor for advanced glycation end products. J MoI Med 83, 876-86 (2005)]. This is further supported in the present study by amelioration of AGE-induced defects by the soluble competitive RAGE receptor. Furthermore, RAGE expression in islets was found to be increased by both AGE-RSA infusion (in the absence of elevated circulating AGEs) and in rats which consumed a high AGE diet. Another aspect of the invention includes a method of ameliorating RAGE expression in a patient in need thereof, by administering a pharmaceutical composition of the invention, thereby ameliorating RAGE expression.

AGEs administered exogenously to rodents provided a simplified in vivo model, without potentially confounding effects of hyperglycaemia, to study the direct effects of AGEs per se on islet function. The exogenous concentrations given led to increases in β cell CML (a prevalent AGE) concentrations at both time-points studied. Within these studies, islet CML accumulation was associated with progressive loss of first phase insulin secretion and initially β cell compensation, but was subsequently followed by decompensation. In rats given AGE-RSA for 1 month, proliferation of pancreatic β cells was evident, in association with elevated proinsulin gene expression, in conjunction with up-regulation of islet RAGE expression. After four months of AGE-RSA infusion, acute 1^st phase insulin secretion had significantly declined. By this time, β cells also had reduced proinsulin and insulin expression and had elevations in RAGE expression. In addition, chronic AGE infusion had an impact on β cell death demonstrated by increases in TUNEL staining in some islets within the present study. These functional molecular and structural changes provide strong evidence of progressive β cell damage and importantly were all prevented with AGE-lowering therapy with alagebrium. This ability of alagebrium to attenuate AGE induced β cell injury suggests that the AGE-RAGE axis is an excellent target for therapeutic strategies to prevent, retard or reverse progressive β cell injury as the result of nutrient excess and in particular increased intake of dietary AGEs glycotoxins. One aspect of the invention includes a method of ameliorating β cell damage in a patient in need thereof, by administering a pharmaceutical composition of the invention, thereby ameliorating said β cell damage. Another aspect of the invention includes a method of ameliorating β cell CML levels in a patient in need thereof, by administering a pharmaceutical composition of the invention, thereby ameliorating said β cell CML levels. Another aspect of the invention includes a method of ameliorating proinsulin and/or insulin expression in a patient in need thereof, by administering a pharmaceutical composition of the invention, thereby ameliorating said proinsulin and insulin expression.

Previous studies in both dbdb^(+/+) mice and wild type C57/BL6 mice have shown that high fat diets induce insulin secretory defects and insulin resistance [Chen, D. & Wang, M. W. Development and application of rodent models for type 2 diabetes. Diabetes Obes Metab 7, 307-17 (2005)]. Further to this, a recent study has demonstrated that reducing the AGE content of high fat diets, attenuates insulin secretory defects, suggesting that the AGE component may be the pathogenic dietary contributor [Sandu, O. et al. Insulin resistance and type 2 diabetes in high-fat-fed mice are linked to high glycotoxin intake. Diabetes 54, 2314-9 (2005)]. Indeed, treatment of dbdb^(+/+) mice with diets low in AGEs also improves insulin sensitivity. [Hofmann, S.M. et al. Improved insulin sensitivity is associated with restricted intake of dietary glycoxidation products in the db/db mouse. Diabetes 51, 2082-9 (2002)]. The present examples provide further evidence that high dietary AGEs are an important pathogenic contributor to insulin secretory defects. The specific mechanisms which may explain the pathogenicity of diets high in AGE content for β cells has not been shown previously. One aspect of the invention includes a method of ameliorating insulin sensitivity in a patient in need thereof, by administering a pharmaceutical composition of the invention, thereby ameliorating said insulin sensitivity. Ligand engagement of RAGE is known to induce oxidative stress [Bierhaus, A. et al.

Understanding RAGE, the receptor for advanced glycation end products. JMo/ Med 83, 876- 86 (2005)] and specifically the production of superoxide [Basta, G. et al. Advanced glycation end products activate endothelium through signal-transduction receptor RAGE: a mechanism for amplification of inflammatory responses. Circulation 105, 816-22 (2002)]. In the present examples, treatment of MIN6 cells with AGEs caused mitochondrial superoxide production and increases in UCP-2 expression. Transfection with UCP-2 siRNA, restored insulin secretion to control levels. Furthermore, increased superoxide generation was not seen in the AGE treated group which concomitantly received anti-AGE therapy with alagebrium. These data are consistent with previous studies where superoxide mediated activation of UCP-2 has been shown to be a major contributor to progressive pancreatic β cell dysfunction [Krauss, S. et al. Superoxide-mediated activation of uncoupling protein 2 causes pancreatic beta cell dysfunction. J Clin Invest 112, 1831-42 (2003)]. In those experiments, there was a decline in cellular ATP production in damaged islets, which was improved by increasing anti-oxidant defences via MnSOD or prevented in UCP-2 deficient islets. These findings are consistent with the present examples which have shown that the AGE inhibitor, alagebrium prevented ATP depletion, and that certain AGE induced abnormalities could be ameliorated by UCP-2 inhibition. One aspect of the invention includes a method of ameliorating oxidative stress in cells in a patient in need thereof, by administering a pharmaceutical composition of the invention, thereby ameliorating said oxidative stress. Another aspect of the invention includes a method of ameliorating mitochondrial superoxide production in cells in a patient in need thereof, by administering a pharmaceutical composition of the invention, thereby ameliorating said mitochondrial superoxide production. Another aspect of the invention includes a method of ameliorating UCP-2 expression in a patient in need thereof, by administering a pharmaceutical composition of the invention, thereby ameliorating said UCP- 2 expression. Another aspect of the invention includes a method of ameliorating cellular ATP content in cells in a patient in need thereof, by administering a pharmaceutical composition of the invention, thereby ameliorating said cellular ATP content. Another aspect of the invention includes ameliorating ATP depletion in cells in a patient in need thereof, by administering a pharmaceutical composition of the invention, thereby ameliorating said ATP depletion.

Impaired calcium flux in β cells, has previously been implicated as contributing to insulin secretory dysfunction [Sakurada, M. et al. Relation between glucose-stimulated insulin secretion and intracellular calcium accumulation studied with a superfusion system of a glucose-responsive pancreatic beta-cell line MIN6. Endocrinology 132, 2659-65 (1993)] which is relevant to AGE induced β cell dysfunction since AGEs in the present examples were shown to interfere with cellular calcium flux. Indeed AGEs per se have been shown to interfere with cellular calcium flux in other settings [Mene, P. et al. Effects of advanced glycation end products on cytosolic Ca2+ signaling of cultured human mesangial cells. J Am Soc Nephrol 10, 1478-86 (1999)]. In addition, mice with transgenic over-expression of RAGE in cardiac myocytes have impaired calcium flux [Petrova, R. et al. Advanced glycation endproduct-induced calcium handling impairment in mouse cardiac myocytes. J MoI Cell Cardiol 34, 1425-31 (2002)]. These data further strengthen the view that the AGE- RAGE axis when activated, influences cellular calcium flux as identified in the present examples. One aspect of the invention includes a method of ameliorating cellular calcium flux in a patient in need thereof, by administering a pharmaceutical composition of the invention, thereby ameliorating said cellular calcium flux.

In the present examples, immunohistochemistry for RAGE revealed a modest increase in the number of RAGE positive β cells in AGE-RSA infused rodents which was not seen in sham or RSA infused groups. These data suggest that long-term infusion of AGEs caused significant defects in GSIS associated with decreased insulin production and increased islet β cell death. One aspect of the invention includes a method of ameliorating the number of RAGE positive β cells in a patient in need thereof, by administering a pharmaceutical composition of the invention, thereby ameliorating said number of RAGE positive β cells.

There is evidence from the present examples that glycotoxic AGEs, contribute to the pathogenesis of progressive insulin secretory defects and decline of insulin producing pancreatic islet β cells. Indeed, supporting data have been presented from in vitro studies in cells, rodent models and susceptible human populations. There is also compelling evidence from experiments using alagebrium, an AGE cross-link breaker which reduced islet AGE accumulation. Taken together, this series of studies presents a modifiable risk factor for type II diabetes and insulin secretory defects, which may be a novel therapeutic target able to be addressed using compounds and/or compositions of the invention which reduce AGE accumulation. The compounds and compositions of the invention may be used to treat a variety of diseases including these listed below: a method for treating or controlling or reducing the risk of developing non-insulin dependent diabetes mellitus (type II diabetes) in a human or other mammalian patient in need of such treatment by administering to the patient a therapeutically effective amount of a compound of the invention; a method for treating or controlling or reducing the risk of developing metabolic syndrome in a human or other mammalian patient in need of such treatment by administering to the patient a therapeutically effective amount of a compound of the invention; a method for treating or controlling or reducing the risk of developing insulin resistance in a human or other mammalian patient in need of such treatment by administering to the patient a therapeutically effective amount of a compound of the invention; a method for treating or controlling or reducing the risk of developing type I diabetes in a human or other mammalian patient in need of such treatment by administering to the patient a therapeutically effective amount of a compound of the invention; a method for treating or controlling or reducing the risk of developing pre-diabetes in a human or other mammalian patient in need of such treatment by administering to the patient a therapeutically effective amount of the compound of the invention.

The compounds and compositions of the invention may be used to prevent a variety of diseases from occurring, including the diseases listed below: a method for preventing non-insulin dependent diabetes mellitus (type II diabetes) in a human or other mammalian patient by administering to the patient a prophylatically effective amount of a compound of the invention; a method for preventing the metabolic syndrome in a human or other mammalian patient by administering to the patient a prophylactically effective amount of a compound of the invention; a method for preventing insulin resistance in a human or other mammalian patient by administering to the patient a prophylactically effective amount of a compound of the invention; a method for preventing type I diabetes in a human or other mammalian patient by administering to the patient a prophylactically effective amount of a compound of the invention; a method for preventing pre-diabetes in a human or other mammalian patient by administering to the patient a prophylactically effective amount of the compound of the invention.

The invention comprises the use of thiazolium compounds having the following structural formula:

wherein R¹ is selected from the group consisting of hydrogen, hydroxy (lower) alkyl, acetoxy (lower) alkyl, lower alkyl, lower alkenyl;

R² is selected from the group consisting of hydrogen, hydroxy (lower) alkyl, acetoxy (lower) alkyl, lower alkyl, lower alkenyl; or R¹ and R² together with their ring carbons may be an aromatic fused ring, optionally substituted by one or more amino, halo or alkylenedioxy groups;

Z is hydrogen or an amino group; Y is amino, a group of the formula: o

Il -CH₂C-R, wherein R is a lower alkyl, alkoxy, hydroxy, amino or an aryl group, said aryl group optionally substituted by one or more lower alkyl, lower alkoxy, halo, dialkylamino, hydroxy, nitro or alkylenedioxy groups; a group of the formula :

-CH₂R' wherein R' is hydrogen, or a lower alkyl, lower alkenyl, or aryl group; or a group of the formula:

-CH₂C-N

R"¹

wherein R" is hydrogen and R'" is a lower alkyl group, optionally substituted by an aryl group, or an aryl group, said aryl group optionally substituted by one or more lower alkyl, halo, or alkoxylcarbonyl groups; or R" and R'" are both lower alkyl groups; and X is a halide, tosylate, methanesulfonate or mesitylenesulfonate ion;

The preferred thiazolium compound of the instant invention comprises the structure of formula I, wherein R¹ and R² are lower alkyl, Z is hydrogen, Y is a group of the formula

O

Il

-CH₂C- R_s wherein R is an aryl group and X is halide. In more preferred embodiments, the compound of the invention is 3-(2-phenyl-2-oxoethyl)-4,5-dimethylthiazolium chloride or N- ρhenacyl-4,5-dimethylthiazolium chloride, also referred to as ALT-711 or alagebrium chloride herein. In other embodiments, the compound of the invention is 3-(2-phenyl-2- oxoethyl)-4,5-dimethylthiazolium bromide or N-phenacyl-4,5-dimethylthiazolium bromide, also referred to as DMPTB or PMTB.

The invention includes the use of a pharmaceutical composition comprising a compound of the formulae of the invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. The compounds, and their compositions, utilized in this invention appear to react with an early glycosylation product thereby preventing the same from later forming the advanced glycosylation end products which lead to cross-links, and thereby, to molecular or protein aging and other adverse molecular consequences. Additionally, the compounds react with already formed advanced glycosylation end products to reduce the amount of such products. The invention additionally comprises an analytic method for identifying compounds for the treatment or prevention of diseases such as type I and type II diabetes, metabolic syndrome, and insulin resistance. In one aspect, the method determines the "breaking" or reversal of the formation of non-enzymatic endproducts. In this connection, the invention further extends to the identification and use of a novel cross-link structure which is believed to represent a significant number of the molecular crosslinks that form in vitro and in vivo as a consequence of advanced glycation. More particularly, the cross-link structure includes a sugar-derived α-dicarbonyl segment or moiety, such as a diketone, that is capable of cleavage by a dinucleophilic, thiazolium-like compound. Specifically, the cross-link structure may be according to the formula shown below:

where A and B independently, are sites of attachment to the nucleophilic atom of a biomolecule.

Accordingly, it is one object of the present invention to provide a method for the treatment or prevention of diseases such as type I and type II diabetes, metabolic syndrome, and/or insulin resistance, e.g., diseases that arise from a reduction in nNOS expression, where the formation of advanced glycosylation endproducts and extensive cross-linking of molecules is inhibited, and cross-links that are formed from pre-existing advanced glycosylation endproducts and occur as a consequence of the reaction of susceptible molecules, such as proteins with glucose and other reactive sugars, by correspondingly inhibiting the formation of advanced glycosylation endproducts, are broken and the breaking of the advanced glycosylation mediated cross-linking has previously occurred.

It is a further object of the present invention to provide a method for the treatment or prevention of diseases such as type I and type II diabetes, metabolic syndrome, and/or insulin resistance, e.g., complications that arise from a reduction of nNOS expression which are characterized by a reaction with an initially glycosylated protein identified as an early glycosylation product.

It is a further object of the present invention to provide a method the treatment or prevention of diseases such as type I and type II diabetes, metabolic syndrome, and/or insulin resistance, e.g., complications that arise from a reduction of nNOS expression which prevents the rearrangement and cross-linking of early glycosylation products to form the advanced glycosylation endproducts.

It is a yet further object of the present invention to provide compounds capable of participating in the reaction with early glycosylation products in the method the treatment or prevention of diseases such as type I and type II diabetes, metabolic syndrome, and/or insulin resistance, e.g., complications that arise from a reduction of nNOS expression.

It is a yet further object of the present invention to provide compounds which break or reverse the advanced glycosylation endproducts formed as a consequence of the aforesaid advanced glycosylation reaction sequence by cleaving the α-dicarbonyl-based protein crosslinks present in the advanced glycosylation endproducts.

It is a further objective of the present invention to provide a method for the treatment or prevention of diseases such as type I and type II diabetes, metabolic syndrome, and/or insulin resistance, where the accumulation of AGEs within the small intestine, accompanied by the reduction in myenteric nNOS expression is reversed by a compound of the invention. The compound of the invention is administered prophylatically or therapeutically.

It is a still further object of the present invention to provide compositions, including pharmaceutical compositions, incorporating the compounds of the present invention.

It is still further object of the present invention to provide compounds, as well as processes for their preparation, for use in the method and compositions of the present invention.

It is a still further object of the present invention to provide assays which can be utilized to detect compounds having the ability to "break" or reverse the formation of non- enzymatic glycosylation endproducts and their subsequent cross-links in order to identify compounds that are useful for the treatment or prevention of diseases such as type I and type II diabetes, metabolic syndrome, and insulin resistance, e.g., complications that arise from a reduction of nNOS expression.

Other objects and advantages will become apparent to those skilled in the art from a consideration of the ensuing description which proceeds with reference to the following illustrative drawings.

DEFINITIONS

The term "lower alkyl" or "Ci^ linear alkyl" means that the group contains 1, 2, 3, 4, 5, or 6 carbon atoms and includes methyl, ethyl, propyl, butyl, pentyl, and hexyl and the corresponding branched and cycloalkyl isomers thereof. The term "Ci_-6 linear alkyl" means that the group contains 1, 2, 3, 4, 5, or 6 carbon atoms and includes methyl, ethyl, propyl, butyl, pentyl, and hexyl. The term "Ci -₆ branched alkyl" means that the group contains 1, 2, 3, 4, 5, or 6 carbon atoms in a branched arrangement, and includes e.g., isopropyl and isobutyl. The term "Ci_-6 cycloalkyl alkyl" means that the group contains 1, 2, 3, 4, 5, or 6 carbon atoms in a cyclic arrangement, and includes e.g., cyclopentyl and cyclohexyl

The term "lower alkynyl" means that the group contains from 2, 3, 4, 5, or 6 carbon atoms. Similarly, the term "lower alkoxy" means that the group contains from 1, 2, 3, 4, 5, or 6 carbon atoms, and includes methoxy, ethoxy, propoxy, butoxy, pentoxy, and hexoxy, and the corresponding branched-chain isomers thereof. These groups are optionally substituted by one or more halo, hydroxy, amino or lower alkylamino groups.

The term "lower acyloxy(lower)alkyl" means that the acyloxy portion contains from 2, 3, 4, 5, or 6 carbon atoms and the lower alkyl portion contains from 1, 2, 3, 4, 5, or 6 carbon atoms. Typical acyloxy portions are those such as acetoxy or ethanoyloxy, propanoyloxy, butanoyloxy, pentanoyloxy, hexanoyloxy, and the corresponding branched chain isomers thereof. Typical lower alkyl portions are as described hereinabove.

The aryl groups or "C₆-CiO aryl" encompassed by the formulae of the invention are those containing 6, 7, 8, 9, or 10 carbon atoms, such as naphthyl, phenyl and lower alkyl substituted-phenyl, e.g., tolyl and xylyl, and are optionally substituted by 1-2 halo, hydroxy, lower alkoxy or di (lower) alkylamino groups. Preferred aryl groups are phenyl, methoxyphenyl and 4-bromophenyl groups.

The halo atoms in the formulae of the invention may be fluoro, chloro, bromo or iodo. For the purposes of this invention, the compounds of the invention are formed as biologically and pharmaceutically acceptable salts. Useful salt forms are the halides, particularly the bromide and chloride, tosylate, methanesulfonate, and mesitylenesulfonate salts. Other related salts can be formed using similarly non-toxic, and biologically and pharmaceutically acceptable anions.

The preferred thiazolium compound of the instant invention comprises the structure of formula I, wherein R¹ and R² are lower alkyl, Z is hydrogen, Y is a group of the formula O Il

-CH₂C-R , wherein R is a phenyl group and X is halide. In more preferred embodiments, the compound of the invention is 3-(2-phenyl-2-oxoethyl)-4,5-dimethylthiazolium chloride or N- phenacyl-4,5-dimethylthiazolium chloride, also referred to as ALT-711 or algebrium chloride herein. In other embodiments, the compound of the invention is 3-(2-phenyl-2-oxoethyl)-4,5- dimethylthiazolium bromide or N-phenacyl-4,5-dimethylthiazolium bromide, also referred to as DMPTB or PMTB.

As used herein, "treating" or "treatment" includes any effect e.g., lessening, reducing, modulating, or eliminating, that results in the improvement of the condition, disease, disorder, etc. "Treating" or "treatment" of a disease state means the treatment of a disease- state in a mammal, particularly in a human, and include: (a) inhibiting an existing disease- state, i.e., arresting its development or its clinical symptoms; and/or (c) relieving the disease- state, i.e., causing regression of the disease state.

As used herein, "preventing" means causing the clinical symptoms of the disease state not to develop i.e., inhibiting the onset of disease, in a subject that may be exposed to or predisposed to the disease state, but does not yet experience or display symptoms of the disease state. Preventing the disease from developing means prophylatically treating the diease.

Of the compounds encompassed by formula I, certain substituents are preferred. For instance, the compounds wherein R¹ or R² are lower alkyl groups are preferred. Also highly preferred are the compounds wherein Y is 2-phenyl-2-oxoethyl or a 2-(substituted phenyl)-2- oxoethyl group.

Representative compounds of the present invention are: 3-aminothiazolium mesitylenesulfonate; 3-amino-4,5-dimethylaminothiazolium mesitylenesulfonate; 2,3-diaminothiazoliniurn mesitylenesulfonate; 3 -(2-methoxy-2-oxoethyl)-thiazolium bromide; 3-(2-methoxy-2-oxoethyl)-4,5-dimethylthiazolium bromide; 3 -(2-methoxy-2 -oxoethyl)-4-methylthiazolium bromide; 3-(2-phenyl-2-oxoethyl)-4-methylthiazolium bromide; 3-(2-phenyl-2-oxoethyl)-4,5-dimethylthiazolium bromide; 3-(2-phenyl-2-oxoethyl)-4,5-dimethylthiazolium chloride; 3-amino-4-methylthiazolium mesitylenesulfonate; 3-(2-methoxy-2-oxoethyl)-5-methylthiazolium bromide;

3-(3-(2-phenyl-2-oxoethyl)-5-methylthiazolium bromide; 3-[2-(4'-bromophenyl)-2-oxoethyl] thiazolium bromide;

3-[2-(4'-bromophenyl)-2-oxoethyl]-4-methylthiazolium bromide;

3-[2-(4'-bromophenylDhenyl)-2-oxoethyl]-5-methylthiazolium bromide;

3-[2- (4 'bromophenyl)-2-oxoethyl)-4,5-dimethylthiazolium bromide; 3-(2-methoxy-2-oxoethyl)-4-methyl-5-(2-hydroxyethyl) thiazolium bromide;

3-(2-phenyl-2-oxoethyl)-4-methyl-5-(2-hydroxyethyl) thiazolium bromide;

3-[2-(4'-bromophenyl)-2-oxoethyl]-4-methyl-5-(2-hydroxyethyl) thiazolium bromide;

3, 4-dimethyl-5-(2-hydroxyethyl) thiazolium iodide;

3-ethyl-5-(2-hydroxyethyl)-4-methylthiazolium bromide; 3-benzyl-5-(2-hydroxyethyl)-4-methylthiazolium chloride;

3-(2-methoxy-2-oxoethyl) benzothiazolium bromide;

3-(2-phenyl-2-oxoethyl) benzothiazolium bromide;

3-[2-(4'bromophenyl)-2-oxoethyl] benzothiazolium bromide;

3-(carboxymethyl) benzothiazolium bromide; 2,3-(diamino) benzothiazolium mesitylenesulfonate;

3-(2-amino-2-oxoethyl) thiazolium bromide;

3 -(2-amino-2-oxoethyl)-4-methylthiazolium bromide;

3-(2-amino-2-oxoethyl)-5-methylthiazolium bromide;

3-(2-amino-2-oxoethyl) 4,5-dimethylthiazolium bromide; 3-(2-amino-2-oxoethyl) benzothiazolium bromide;

3-(2-amino-2-oxoethyl) 4-methyl-5-(2-hydroxyethyl) thiazolium bromide;

3-amino-5-(2-hydroxyethyl)-4-methylthiazolium mesitylenesulfonate;

3-(2-methyl-2-oxoethyl) thiazolium chloride;

3-amino-4-methyl-5-(2-acetoxyethyl) thiazolium mesitylenesulfonate; 3-(2-phenyl-2-oxoethyl) thiazolium bromide;

3-(2-methoxy-2-oxoethyl)-4-methyl-5-(2-acetoxyethyl) thiazolium bromide;

3-(2-amino-2-oxoethyl)-4-methyl-5-(2-acetoxyethyl) thiazolium bromide;

2-amino-3-(2-methoxy-2-oxoethyl) thiazolium bromide;

2-amino-3-(2-methoxy-2-oxoethyl) benzothiazolium bromide; 2-amino-3-(2-amino-2-oxoethyl) thiazolium bromide;

2-amino-3-(2-amino-2-oxoethyl) benzothiazolium bromide;

3-(2-(3 '-methoxyphenyl)-2-oxoethyl)-thiazolium bromide;

3-(2-(3'-methoxy phenyl)-2-oxoethyl)-4 methyl-5-(2'-hydroxyethyl)-thiazolium bromide;

3-(2-(3 '-methoxyphenyl)-2-oxoethyl)-benzothiazolium bromide; 2,3-diamino-4-chlorobenzothiazolium mesitylenesulfonate;

2,3-diamino-4-methyl-thiazolium mesitylenesulfonate;

3-amino-4-methyl-5-vinyl-thiazolium mesitylenesulfonate;

2,3-diamino-6-chlorobenzothiazolium mesitylenesulfonate; 2,6-diamino-benzothiazole dihydrochloride;

2,6-diamino-3 [2-(4'-methoxyphenyl)-2-oxoethyl) benzothiazolium bromide;

2,6-diamino-3 [2-(3'-methoxyphenyl)-2-oxoethyl) benzothiazolium bromide;

2,6-diamino-3 [2-(4'-diethylaminophenyl)-2-oxoethyl] benzothiazolium bromide;

2,6-diamino-3 (2-(4'-bromophenyl)-2-oxoethyl] benzothiazolium bromide; 2,6-diamino-3 (2-(2-phenyl-2-oxoethyl) benzothiazolium bromide;

2,6-diamino-3 [2-(4'-fluorophenyl-2-oxoethyl) benzothiazolium bromide;

3-acetamido-4-methyl-5-thiazolyl-ethyl acetate mesitylenesulfonate;

2,3-diamino-5-methylthiazolium mesitylenesulfonate;

3 -[2-(2 ' -naphtyl)-2-oxoethyl] -4-methyl-5 -(2 ' -hydroxyethyl)-thiazolium bromide; 3-[2-(3',5'-di-ter-butyl-4'-hydroxyphenyl)-2-oxoethyl]-4-methyl-5-(2'-hydroxyethyl- thiazolium bromide;

3 - [2-(2 ' ,6 ' -dichlorophenethylamino)-2-oxoethyl] -4-methyl-5 -(2 ' -hydroxyethyl) -thiazolium- bromide;

3-[2-dibutylamino-2-oxoethyl)-4-methyl-5-(2'-hydroxyethyl)-thiazolium bromide; 3-[2-4'-carbethoxyanilino)-2-oxoethyl]-4-methyl-5- (2 '-hydroxyethyl) thiazolium bromide;

3-[2-(2',6'-diisopropylanilino)-2-oxoethyl]-4-methyl-5-(2'-hydroxyethyl) thiazolium bromide;

3-amino-4-methyl-5-(2- (T ,6'-dichlorobenzyloxy)ethyl]-thiazolium mesitylenesulfonate;

3-[2-(4'-carbmethoxy-3'-hydroxyanilino)-2-oxoethyl)-4-methyl-5-(2'-hydroxyethyl)- thiazolium bromide;

2,3-diamino-4,5-dimethylthiazolium mesitylenesulfonate;

2,3-diamino-4-methyl-5-hydroxyethyl-thiazolium mesitylene sulfonate;

2,3-diamino-5-(3',4'-trimethylenedioxy phenyl)-thiazolium mesitylene sulfonate;

3-[2-(r,4'-benzodioxan-6-yl)-2-oxoethyl]-4-methyl-5-(2'-hydroxyethyl)-thiazolium bromide;

3-[2-(3',4'-trimethylenedioxyphenyl)-2-oxoethyl]-4-methyl-5-(2'-hydroxyethyl)-thiazolium bromide;

3-(2-[l',4-benzodioxan-6-yl)-2-oxoethyl)-thiazolium bromide; 3-[2-(3',4'-trimethylenedioxyphenyl)-2-oxoethyl]-thiazolium bromide; 3-[2-(3 ',5 '-di-tert-butyl-4'-hydroxyphenyl)-2-oxoethyl]-thiazolium bromide;

3-[2- (3', 5'-di-tert-butyl-4'-hydroxyphenyl)-2-oxoethyl]-4-methyl-thiazolium bromide;

3-[2-(3', 5'-di-tert-butyl-4'-hydroxyphenyl)-2-oxoethyl)-5-methyl-thiazolium bromide;

3-[2-(3 ',5 '-di-tert-butyl-4'-hydroxyphenyl)-2-oxoethyl]-4,5-dimethyl-thiazolium bromide; 3-[2-(3',5'-di-tert-butyl-4'-hydroxyphenyl)-2-oxoethyl]-benzothiazolium bromide;

3-(2-phenyl-2-oxoethyl)-4-methyl-5-vinyl-thiazolium bromide;

3 - [2-(3 ' , 5 ' -tert-butyl-4 ' -hydroxyphenyl)-2-oxoethyl)-4-methyl-5 -vinyl-thiazolium bromide;

3-(2-tert-butyl-2-oxoethyl)-thiazolium bromide;

3-(2-tert-butyl-2-oxoethyl)-4-methyl-5-(2'-hydroxyethyl)-thiazolium bromide; 3-(3'-methoxybenzyl)-4-methyl-5-(2'-hydroxyethyl)-thiazolium chloride;

3 -(2 ' ,6 ' -dichlorobenzyl)-4-methyl-5-(2 ' -hydroxyethyl)-thiazolium chloride;

3-(2'-nitrobenzyl)-4-methyl-5-(2'-hydroxyethyl)-thiazolium bromide;

3 [2-(4'-chiorophenyl)-2-oxoethyl]-thiazolium bromide;

3[2-(4'-chlorophenyl)-2-oxoethyl]-4-methyl-5-(2'-hydroxyethyl)-thiazolium bromide; and 3 [2-(4'-methoxyphenyl)-2-oxoethyl]-4-methyl-5-(2'-hydroxyethyl)-thiazolium bromide.

Compounds of the invention further include those compounds represented by the formula Ia:

wherein R is independently selected from the group consisting of hydrogen, hydroxy(lower)alkyl, acetoxy(lower)alkyl, lower alkyl, lower alkenyl; R² is independently selected from the group consisting of hydrogen, hydroxy(lower)alkyl, acetoxy(lower)alkyl, lower alkyl, lower alkenyl, or R¹ and R² together with their ring carbons may be an aromatic fused ring, optionally substituted by one or more amino, halo or alkylenedioxy groups;

Z is hydrogen or an amino group; Y is amino, a group of the formula

O

Il -CH₂C-R wherein R is a lower alkyl, alkoxy, hydroxy, amino or an aryl group, said aryl group optionally substituted by one or more lower alkyl, lower alkoxy, halo, dialkylamino, hydroxy, nitro or alkylenedioxy groups; a group of the formula -CH₂R' wherein R' is hydrogen, or a 'lower alkyl, lower alkynyl, or aryl group; or a group of the formula

wherein R" is hydrogen and R'" is a lower alkyl group, optionally substituted by an aryl group, or an aryl group, said aryl group optionally substituted by one or more lower alkyl, halo, or alkoxylcarbonyl groups; or R" and R" are both lower alkyl groups; and X is a halide, tosylate, methanesulfonate or mesitylenesulfonate ion. In one aspect the invention includes a compound where at least one of Y and Z is an amino group. In another aspect the invention includes where Y is amino and R² and Z are both hydrogen, then R¹ is other than a lower alkyl group

Other compounds of the invention include those compounds of the formula Ib:

wherein R¹ is independently selected from the group consisting of hydroxy (lower) alkyl, acetoxy(lower)alkyl, lower acyloxy(lower)alkyl, lower alkyl;

R² is independently selected from the group consisting of hydroxy (lower) alkyl, acetoxy(lower)alkyl, lower acyloxy(lower)alkyl, lower alkyl, or R¹ and R² together with their ring carbons may be an aromatic fused ring;

Z is hydrogen or an amino group; Y is an alkynylmethyl group, or a group of the formula

-CH₂C-N

R" wherein R" is hydrogen and R'" is a lower alkyl group, optionally substituted by an aryl group, or an aryl group, the aryl group optionally substituted by one or more lower alkyl, halo, or alkoxylcarbonyl groups; or R" and R'" are both lower alkyl groups; and X is a halide, tosylate, methanesulfonate or mesitylenesulfonate ion.

Compounds of the invention include those compounds of formula (Ic):

wherein R¹ and R² are methyl; Z is hydrogen; Y is a group of the formula:

O Il

-CH₂C-R, wherein R is phenyl; and X is halide. Compounds of the invention include those of formula (Id)

wherein:

R is selected from hydrogen, C_]-6 linear or branched alkyl and cycloalkyl; or together with their ring carbons form a C₅-C₇ fused cycloalkyl ring having up to two double bonds including any fused double bond of the -olium containing ring, which cycloalkyl ring is optionally substituted by one or more substituents selected from alkyl and fluoro; R² is selected from hydrogen, C_1-6 linear or branched alkyl and cycloalkyl; or together with their ring carbons form a C₅-C₇ fused cycloalkyl ring having up to two double bonds including any fused double bond of the -olium containing ring, which cycloalkyl ring is optionally substituted by one or more substituents selected from alkyl and fluoro; Z is hydrogen or C_r6 linear or branched alkyl;

5 6

Y is a group of the formula -CH(R )-C(O)-R wherein

R is hydrogen, C_1-6 linear- or branched- alkyl, or cycloalkyl; and

6 6

R is a C₆ or C₁₀ aryl, wherein R is optionally substituted with one or more substituents selected from the group consisting of alkyl and halo;

Q is S; and X is a pharmaceutically acceptable anion.

The above compounds are capable of inhibiting the formation of advanced glycosylation endproducts on target molecules, including, for instance, proteins, as well as being capable of breaking or reversing already formed advanced glycosylation endproducts on such proteins. The compounds employed in accordance with this invention inhibit this late-stage Maillard effect and reduce the level of the advanced glycosylation endproducts already present in the protein material.

The rationale of the present invention is to use compounds which block, as well as possibly reverse, the post-glycosylation step, e.g., the formation of fluorescent chromophores and cross-links, the presence of which may be associated with, and leads to diseases such as types I and type II diabetes, metabolic syndrome, and/or insulin resistance. In one aspect of the invention, the compound would prevent the formation of such chromophores and of cross-links between protein strands and trapping of proteins onto other proteins and reverse the level of such cross-link formation already present. The chemical nature of the early glycosylation products with which the compounds of the present invention are believed to react may vary, and accordingly the term "early glycosylation product(s)" as used herein is intended to include any and all such variations within its scope. For example, early glycosylation products with carbonyl moieties that are involved in the formation of advanced glycosylation endproducts, and that may be blocked by reaction with the compounds of the present invention, have been postulated. In one embodiment, it is envisioned that the early glycosylation product may comprise the reactive carbonyl moieties of Amadori products or their further condensation, dehydration and/or rearrangement products, which may condense to form advanced glycosylation endproducts. In another scenario, reactive carbonyl compounds, containing one or more carbonyl moieties (such as glycolaldehyde, glyceraldehyde or 3-deoxyglucosone) may form from the cleavage of Amadori or other early glycosylation endproducts, and by subsequent reactions with an amine or Amadori product, may form carbonyl containing advanced glycosylation products such as alkylformyl-glycosylpyrroles.

Several investigators have studied the mechanism of advanced glycosylation product formation. In vitro studies by EbIe et al., (1983), "Nonenzymatic Glucosylation and Glucose- dependent Cross-linking of Protein", J. Biol. Chem., 258:9406-9412, concerned the cross- linking of glycosylated protein with nonglycosylated protein in the absence of glucose. EbIe et al. sought to elucidate the mechanism of the Maillard reaction and accordingly conducted controlled initial glycosylation of RNase as a model system, which was then examined under 'varying conditions. In one aspect, the glycosylated protein material was isolated and placed in a glucose-free environment and thereby observed to determine the extent of cross-linking.

EbIe et al. thereby observed that cross-linking continued to occur not only with the glycosylated protein but with non-glycosylated proteins as well. One of the observations noted by EbIe et al. was that the reaction between glycosylated protein and the protein material appeared to occur at the location on the amino acid side chain of the protein. Confirmatory experimentation conducted by EbIe et al. in this connection demonstrated that free lysine would compete with the lysine on RNase for the binding of glycosylated protein. Thus, it might be inferred from these data that lysine may serve as an inhibitor of advanced glycosylation; however, this conclusion and the underlying observations leading to it should be taken in the relatively limited context of the model system prepared and examined by EbIe et al. Clearly, EbIe et al. does not appreciate, nor is there a suggestion therein, of the discoveries that underlie the present invention, with respect to the inhibition of advanced glycosylation of proteins both in vitro and in vivo. The experiments of EbIe et al. do not suggest the reactive cleavage product mechanism or any other mechanism in the in vivo formation of advanced glycosylation endproducts in which glucose is always present. In fact, other investigators support this mechanism to explain the formation of advanced glycosylated endproducts in vivo (see for example, Hayase et al, J. Biol. Chem., 263:3758-3764 (1989); Sell and Monnier, J. Biol. Chem., 264:21597-21602 (1989); Oimomi et al., Agric. Biol. Chem., 53(6): 1727-1728

(1989); and Diabetes Research and Clinical Practice, 6:311-313 (1989). Accordingly, the use of lysine as an inhibitor in the EbIe et al. model system has no bearing upon the utility of the compounds of the present invention in the 'inhibition of advanced glycosylated endproducts formation' in the presence of glucose in vivo, and the amelioration of complications of diabetes and aging.

While not wishing to be bound by any particular theory as to the mechanism by which the compounds of the instant invention reverse already formed advanced glycosylation endproducts, studies have been structured to elucidate a possible mechanism. Earlier studies examining the fate of the Amadori product (AP) in vivo have identified one likely route that could lead to the formation of covalent, glucose-derived protein crosslinks. This pathway proceeds by dehydration of the AP via successive beta-eliminations as shown in the Scheme A below. Thus, loss of the 4-hydroxyl of the AP (I) gives a l,4-dideoxy-l-alkylamino-2,3- hexodiulose (AP-dione)(II). An AP-dione with the structure of an amino- 1,4-dideoxyosone has been isolated by trapping model APs with the AGE-inhibitor aminoguanidine. Subsequent elimination of the 5-hydroxyl gives a l,4,5-trideoxy-l-alkylamino-2, 3-hexulos-4- ene (AP-ene-dione) (III), which has been isolated as a triacetyl derivative of its 1,2-enol form. Amadori-diones, particularly the AP-ene-dione, would be expected to be highly reactive toward protein cross linking reactions by serving as targets for the addition of the amine (Lys, His)-, or sulfhydryl (Cys)-based nucleophiles that exist in proteins, thereby producing stable cross links of the form (IV).

X-[Protein]

Scheme A

Note that the linear AP-ene-dione of (III) and the stable 20 cross-link of (IV) may cyclize to form either 5- or 6-member lactol rings, although only the 6-member cyclic variant is shown in Scheme A set forth above.

The possibility that a major pathway of glucose-derived cross link formation proceeds through an AP-ene-dione intermediate was investigated by experiments designed to test the occurrence of this pathway in vivo as well as to effect the specific cleavage of the resultant α- dicarbonyl-based protein crosslinks. The thiazolium compounds of the instant invention are thus believed to act as novel "bidentate" nucleophiles, particularly designed to effect a carbon-carbon breaking reaction between the two carbonyls of the cross link, as shown in Scheme B below under physiological conditions. This scheme shows the reaction of a prototypic α-dione cleaving agent of the formula I, N-phenacylthiazolium bromide, with an AP-ene-dione derived cross link.

A further experiment to elucidate this reaction involves the reaction of a compound of the formula I, N-phenacyithiazolium bromide, with 1 -phenyl- 1, 2-propanedione to produce the predicted fission product, benzoic acid. The reaction between N-phenacylthiazolium bromide and l-phenyl-i,2-propanedione was rapid and readily proceeded, confirming this possible mechanism. Once early, glucose-derived addition products form on proteins, further reactions can ensue to effect a covalent, protein-protein crosslinking reaction. In this regard, a compound of the formula I, N-phenacylthiazolium bromide, was allowed to react with the AGE-crosslinks that form when AGE-modified BSA (AGE-BSA) is allowed to react with unmodified, native collagen. This resulted in a concentration-dependent release of BSA from the pre-formed AGE-mediated complexes. Again, this study confirmed that a significant portion of the

AGE-crosslinks that form under experimental conditions consist of an α-diketone or related structure that is susceptible to cleavage by the advantageous bidentate-type molecules of the compounds of formula I under physiological conditions.

To confirm that, the same situation occurs in vivo, isolated collagen from the tail tendons of rats which had been diabetic for 32 weeks were treated with a compound of the formula I, N-phenacylthiazolium bromide, prior to cyanogen bromide digestion and gel electrophoresis analysis. The subsequent electrophoresis revealed that the treated collagen was indistinguishable from untreated, non-diabetic (control) collagen, in marked contrast to the AGE-modified, highly cross linked, digestion-resistant collagen that is typically isolated from diabetic animals.

The present invention likewise relates to methods for inhibiting the formation of advanced glycosylation endproducts, and reversing the level of already formed advanced glycosylation endproducts, which comprise contacting the target molecules with a composition of the present invention.

As is apparent from a discussion of the environment of the present invention, the present methods and compositions hold the promise for arresting, and to some extent reversing, the aging of key proteins both in animals and plants, and concomitantly, conferring both economic and medical benefits as a result thereof.

The therapeutic implications of the present invention relate to the a method of treating or preventing diseases such as types I and II diabetes, metabolic syndrome, and insulin resistance. The present invention relates to a method of treating or preventing complications that arise from a reduction in nNOS expression. The present invention relates to a method of treating or preventing diseases such as types I and II diabetes, metabolic syndrome, and insulin resistance.

In the instance where the compositions of the present invention are utilized for in vivo or therapeutic purposes (e.g., acute or prophylactic treatment), it may be noted that the compounds used therein are biocompatible. Pharmaceutical compositions may be prepared with a therapeutically effective quantity of the compounds of the present invention and may include a pharmaceutically acceptable carrier, selected from known materials utilized for this purpose. Such compositions may be prepared in a variety of forms, depending on the method of administration. Also, various pharmaceutically acceptable addition salts of the compounds of the invention may be utilized.

A liquid form would be utilized in the instance where administration is by intravenous, intramuscular or intraperitoneal injection. When appropriate, solid dosage forms such as tablets, capsules, or liquid dosage formulations such as solutions and suspensions, etc., may be prepared for oral administration. For topical or dermal application to the skin or eye, a solution, a lotion or ointment may be formulated with the agent in a suitable vehicle such as water, ethanol, propylene glycol, perhaps including a carrier to aid in penetration into the skin or eye. For example, a topical preparation could include up to about 10% of the compound of the invention. Other suitable forms for administration to other body tissues are also contemplated. In the instance where the present method has therapeutic application, the animal host intended for treatment may have administered to it a quantity of one or more of the compounds, in a suitable pharmaceutical form. Administration may be accomplished by known techniques, such as oral, topical and parenteral techniques such as intradermal, subcutaneous, intravenous or intraperitoneal injection, as well as by other conventional means. Administration of the compounds may take place over an extended period of time.

The compound of the invention is formulated in compositions in an amount effective to inhibit and reverse the formation of advanced glycosylation endproducts. The compound of the invention is formulated in compositions in an amount effective to inhibit the expression of intestinal neuronal nitric oxide synthase nNOS. This amount will, of course, vary with the particular agent being utilized and the particular dosage form, but typically is in the range of 0.01% to 1.0%, by weight, of the particular formulation.

The compounds encompassed by the invention are conveniently prepared by chemical syntheses well-known in the art. Certain of the compounds encompassed by the invention are well-known compounds readily available from chemical supply houses and/or are prepared by synthetic methods specifically published therefor. For instance, 3,4-dimethyl-5-(2- hydroxyethyl) thiazolium iodide; 3-ethyl-5-(2-hydroxyethyl)-4-methylthiazolium bromide; 3- benzyl-5-(2-hydroxyethyl) -4-methylthiazolium chloride; and 3-(carboxymethyl) benzothiazolium bromide are obtainable from compounds described in the chemical and patent literature or directly prepared by methods described therein and encompassed by the present invention are those such as 3-(2-phenyl-2-oxoethyl)-4-methylthiazolium bromide and 3-benzyl-5- (2-hydroxyethyl) -4-methyl thiazolium chloride [Potts et al., J. Org. Chem., 41:187-191 (1976)]. Other features and advantages of the present invention are apparent from the additional descriptions provided herein including the different examples. The provided examples illustrate different components and methodology useful in practicing the present invention. The examples do not limit the claimed invention. Based on the present disclosure the skilled artisan can identify and employ other components and methodology useful for practicing the present invention.

EXAMPLES

The following methods below entitled Albumin Preparation, in vitro and in vivo Models apply to Examples 1-7: Albumin Preparation

Preparation of Exogenous AGE-albumin

Since the AGE, CML is a modification to the parent amino acid lysine, albumin was used as the protein source of lysine available for modification by advanced glycation. Albumin is also widely consumed constituent of dairy products which are high within western diets. Fatty acid free, cold ethanol precipitated fraction V of rat or bovine serum albumin (50mg/ml) was incubated in the presence and absence of 0.5M D-glucose in 0.4M phosphate buffer, pH 7.4 at 37⁰C for 12 weeks under aerobic conditions following filter sterilization through 0.2μm filters [Oldfϊeld, M.D. et al. Advanced glycation end products cause epithelial-myofibroblast transdifferentiation via the receptor for advanced glycation end products (RAGE). J Clin Invest 108, 1853-63. (2001)]. The preparations were then dialysed (MW cut off - 14kDa) against four changes of tissue culture grade phosphate buffered saline prepared in water at 4⁰C overnight. Endotoxin was then removed from dialysed preparations by Detoxigel columns, followed by filter sterilization through 0.2μm filters and stored dessicated at -80⁰C. Aliquots of each albumin preparation were dialysed against 5OmM DTPA in water and lyophilised for transportation. The AGEs/ALEs, N^ε(carboxymethyl)lysine (CML) and N^ε(carboxyethyl)lysine (CEL) were quantified by isotope dilution, selected ion monitoring gas chromatography-mass spectrometry (SIM- GC/MS) [Dyer, D.G. et al. Accumulation of Maillard reaction products in skin collagen in diabetes and aging. J Clin Invest 91, 2463-9 (1993)] and normalised to their parent amino acid lysine. Pentosidine was analysed by RP-HPLC and was also normalised to lysine content [Dyer, D.G. et al. Accumulation of Maillard reaction products in skin collagen in diabetes and aging. J Clin Invest 91, 2463-9 (1993)].

Dietary CML levels were determined by in house ELISA. ELISA in clear supematants obtained from rodent food following powdering in a mortar and pestle and overnight extraction.

Endotoxin levels in AGE-albumin preparations and plasma taken from AGE-RSA and RSA infused animals were assessed by LAL validation. Samples diluted 1 : 100 in pyrogen free water were pretreated at 80⁰C for 10 mins followed by a further dilution to 1 :500 for the assay. The LAL assay lower detection limit was 2.5 EU/ml. In vitro models - MIN6N8 cells Cell culture

MIN6N8 cells, SV40 transformed insulinoma cells derived from non-obese diabetic (NOD) mice [Miyazaki, J. et al. Establishment of a pancreatic beta cell line that retains glucose-inducible insulin secretion: special reference to expression of glucose transporter isoforms. Endocrinology 127, 126-32 (1990)], were grown in Dulbecco's modified eagle's medium containing 15% fetal bovine serum, 2mmol/L glutamine and penicillin-streptomycin with normal glucose (5mmol/L) or high glucose. Cells were incubated in the presence and absence of AGE-BSA (AGE-BSA) or BSA (BSA- lOOμg/mL) for 7 days in high glucose (HG, 25mmol/L) or for 28 days in normal glucose (NG, 5mmol/L). Soluble RAGE was administered at 1 μg/mL for 7 days. Insulin Secretion

MIN6 cells were seeded in 12-well plates and treated for 7d. Cells were washed once with modified Krebs-Ringer Bicarbonate HEPES buffer (KRBH; 110.8mM NaCl, 4.87mM KCL, 2.29mM CaCl₂.2H₂O, 1.22mM KH₂PO₄, 1.2ImM MgSO₄.7H₂O, 25.7mM NaHCO₃, 10.4mM HEPES, 0.1% BSA) containing 2.8mM glucose and pre-incubated with the same buffer for 30mins at 37°C. Cells were then incubated with KRBH containing either 2.8mM or 2OmM glucose for Ih at 37⁰C, followed by Ih at 37°C with KRBB containing non-glucose secretagogues (2OmM arginine, ImM isobutylmethylxanthine (IBMX) and 50μM carbachol). The incubation buffer was collected and the cells were extracted with 0.36M HCl/95% ethanol. Insulin content was expressed as the sum of insulin secreted during the first and second stimulation periods, and insulin extracted at the end of the glucose challenge, while insulin secretion was expressed as a percentage of total insulin content. The amount of insulin in the incubation buffer and cellular extracts was measured using the sensitive rat competitive insulin radioimmunoassay (RIA) kit.

MIN6 cells were loaded with ImM serotonin (5-hydroxtryptamine) for 16 hours before the glucose challenge as described above. The incubation buffer was collected and assayed for 5-HT by competitive serotonin EIA. Cellular Uptake of 2-Deoxyglucose

Glucose uptake was determined using ³H-2-deoxy-glucose (³H-2-DG). Non-specific uptake was assessed using cytochalasin-B, which was subtracted from total uptake. Cells were serum deprived for 4hrs, washed twice in warm phosphate buffered saline (PBS) containing 0.1% w/v bovine serum albumin (BSA) and incubated for 30mins at 37°C. Following this, cell treatments; insulin (10OnM), phenformin (ImM), HDL (50mg/ml), apoAI (40mg/ml) and cytochalasin-B (1OmM) were added to the PBS/BSA solution and incubated for lhr. Medium was aspirated and cells primed with 1OmM unlabelled 2-deoxy-glucose (2DG) in PBS/BSA for 5mins before incubation with 2OnM 3H-2DG in the priming medium for lOmins. Washing 4 times with ice-cold PBS terminated the uptake process and cell associated radioactivity was determined by lysing cells in 0.3M NaOH followed by liquid scintillation counting. Transient Transfection of human FL-RAGE

A day prior to transfection, MIN6 cells were plated onto 6 well plates at a density of 2 x 10⁵ cells/well in complete growth medium (10% fetal calf serum, 1% L-glutamine, 1% penicillin/streptomycin antibiotic and 25mM D-glucose) to reach 30-40% confluency on the day of transfection. One microgram of human FL-RAGE cDNA in PCIneo plasmid (pRAGE, [Yonekura, H. et al. Novel splice variants of the receptor for advanced glycation end-products expressed in human vascular endothelial cells and pericytes, and their putative roles in diabetes-induced vascular injury. Biochem J 370, 1097-109 (2003)] was added into each well after combination with Lipofectamine 2000 (1 : 2.5 ratio) in Opti-MEM. At the same time, cells were also co-transfected with 0.8ug pControl-SEAP to normalise for transfection efficiency. After 6 hours of incubation at 37°C, the cells were washed and cultured in fresh complete growth medium. Also, various treatments were initiated. After 3 days, cells were transfected for a second time as described above and the treatments continued for another 3 days before the cells were harvested. Following transfection, approximately lOOμl of medium was collected to assay using the SEAP Assay Kit. The fluorescence was then measured. UCP-2 gene silencing with siRNA UCP-2 gene silencing was performed using a siRNA target sequence. Using this sequence the siRNA was then constructed using a Silencer™ siRNA Construction Kit. The cell transfection conditions were similar to those utilised for RAGE except that the siRNA transfection was performed using TKO reagent, and 4nM of UCP-2 siRNA where 50% inhibition of UCP-2 expression was evident at a 2:1 ratio of TKO: siRNA. A concentration dependent inhibition from (2-12nM) was confirmed by real time RT-PCR and protein immunoblotting for UCP-2 (data not shown). Transfection efficency of siRNA was determined using fluorescence. MIN6N8 A TP production

Measurement of mitochondrial ATP content and production was performed in triplicate as previously described [Drew, B. & Leeuwenburgh, C. Method for measuring ATP production in isolated mitochondria: ATP production in brain and liver mitochondria of Fischer-344 rats with age and caloric restriction. Am J Physiol Regul Integr Comp Physiol 285, Rl 259-67 (2003)] using the bioluminescent ATP determination assay kit. Mitochondria were isolated from 25mm² flasks of cells using a mitochondrial isolation kit, immediately prior to analysis. Baseline measurements for each standard or sample were obtained every 5s for a total analysis time of 30s. Then, lOμl of sample or standard was added to the reaction mixture to determine the basal mitochondrial ATP content. Immediately after measurement of ATP content, lOμl of 2.5mM ADP was added to the wells containing the reaction mixture and mitochondria to determine the rate of ATP production. The values for ATP content and rate of production were normalized to the mitochondrial protein concentration present in each

1 Oμl aliquot.

Mitochondrial Superoxide production

Superoxide production in isolated mitochondria extracted from 75mm² flasks using a Mitochondrial Isolation Kit [Coughlan, M.T. et al. Combination therapy with the advanced glycation end product cross-link breaker, alagebrium, and angiotensin converting enzyme inhibitors in diabetes: synergy or redundancy? Endocrinology 148, 886-95 (2007)]. Briefly, pelleted mitochondria were resuspended in 50 μl of 10 mM K₂HPO₄ pH 10.5 and then diluted 1 in 4 with H₂O. One hundred μl of sample was added in duplicate to a 96 well plate and background chemiluminescence was measured [Pitkanen, S. & Robinson, B.H. Mitochondrial complex I deficiency leads to increased production of superoxide radicals and induction of superoxide dismutase. J Clin Invest 98, 345-51. (1996)]. The plate was incubated at 37°C for 15 min and then 50 μg of NADH was injected into each well. After 15 min at 37°C, 100 μM of lucigenin plus NADH (50 μg) in K₂HPO₄ was injected. The plate was counted 10 times with a 1 s read per well. The results were expressed as relative light units per μg of protein content of each mitochondrial fraction. Calcium Flux across MIN6 cells

MIN6 cells were cultivated on coverslips in various treatment conditions to confluence. On day 7, confluent cells were loaded with 2μM fura-2 /DMSO for 30mins at 37°C in the dark. The cells were then bathed with pre-warmed Krebs-Ringer Bicarbonate buffer (KRBB) and placed in a microchamber on the stage of a fluorescence microscope. Fura-2 loaded MIN6 cells were excited at IHz with 340nm and 380nm light, and the emission signals at 510nm were detected by a photomultiplier. Glucose (2OmM) was injected at t=30s while the calcium ionophore ionomycin (Ca²⁺ channel opener) was added at the end of fluorescence measurements. The 340 (Ca²⁺-bound chelator), 380 (Ca²⁺-free), and 340/380nm ratio signals were recorded continuously over a period of 300s with CCD video camera. The intracellular Ca²⁺ concentration ([Ca²⁺],) is expressed as the 340/380nm ratio. The calcium channel blocker, verapamil was used as a control. In vivo models Experimental model of Glycated Albumin Infusion

Groups of male Sprague Dawley rodents (n=10/group) were given daily intraperitoneal injections of either AGE-RSA (high AGE) or RSA (low AGE) at 20mg/kg/day, or saline (SHAM) and followed for a period of 1 or 4 months. Rat serum albumin (RSA) was used to minimise cross-species immune reactions in vivo. This level of glycotoxin has been shown previously in long-term studies to initiate disease in rodents [Vlassara, H. et al. Advanced glycation end products induce glomerular sclerosis and albuminuria in normal rats. Proc Natl Acad Sci USA 91, 11704-11708 (1994)]. No systemic response to the injections was noted by measurement of plasma TNF-α, IL-I β, endotoxin and anti-CML antibodies (data not shown). Two additional subgroups of rodents infused with AGE-RSA or RSA (n=10/group) were randomised to receive treatment with the AGE crosslink breaker, alagebrium, lOmg/kg/day by oral gavage for the study duration (AGE- RSA+ALT; RSA+ALT). Blood glucose by auto analyser and %GHb [Cefalu, W.T., Wang, Z.Q., Bell-Farrow, A., Kiger, F.D. & Izlar, C. Glycohemoglobin measured by automated affinity HPLC correlates with both short-term and long-term antecedent glycemia. Clin Chem 40, 1317-21. (1994)] were measured each month. Animals were culled by exsanguination at 1 or 4 months. Administration of diets high in AGE content to healthy rodents

Groups of male Sprague Dawley rats (n=10/group) were randomised to receive a diet containing low levels of the AGEs (unbaked AIN-93G, CML=I 7.05 nmol/mol lysine/mg protein) or high levels of AGEs, (baked AIN-93G, CML=87.05 nmol/mol lysine/mg protein) for 6 months. AIN-93G was baked for 1 hour at 165⁰C to increase the AGE content. Food intake was monitored by metabolic caging for 24 hours, performed bimonthly. All animals were given ad libitum access to food and water and maintained on 12 hour dark-light cycles. Intravenous and Intraperitoneal Glucose Tolerance Tests - IVGTT/IPGTT

IVGTTs were performed on rats. Groups of rodents (n=6/group) were anaesthetized and the left carotid artery cannulated. After an equilibration period of 15mins, a 0.5ml blood sample was taken (time 0) followed by the administration of a bolus glucose injection of lg/kg. Subsequent 0.5ml blood samples were taken at 2, 5, 10, 15, 30 and 45 mins for the measurement of plasma glucose and insulin concentrations. Whole blood was resuspended in heparinised saline after gentle centrifugation to obtain plasma and returned via the cannula to minimise blood loss. Area under the curve (AUC) was calculated by the Trapezoidal rule. CML concentrations by indirect ELJSA

A seven point standard curve was constructed using AGE-BSA. One hundred μl of sample (diluted at 1/10, 000 to 1/20, 000) or standard, diluted in 50 mM carbonate buffer (pH 9.6) was added to a microtitre plate and incubated overnight at 4°C. All of the following procedures were performed at room temperature. Wells were washed three times with phosphate-buffered saline (PBS, pH 7.4) containing 0.1% Tween-20. Each well was blocked for 1 h with 250 μl of blocking buffer (PBS, pH 7.4 containing 1% BSA, washed three times and then 100 μl of a rabbit polyclonal anti-CML antibody (5 μg/ml) diluted in blocking buffer was added to each well. After 2 h shaking on an orbital shaker, wells were washed three times and 100 μl of 0.2 μg/ml goat-anti-rabbit IgG biotinylated antibody diluted in blocking buffer was added to each well. After 1 h of shaking, and three washes, 100 μl of streptavidin horseradish peroxidase, diluted 1/5000 in blocking buffer was added to each well. After 30 min, the wells were washed three times and 100 μl of TMB was added and the reaction was terminated after 15 min using 100 μl 1.8 M H₂SO₄. The absorbance was quantitated using a microtitre plate reader at 450 run. Fluorescent Activated Cell Sorting Islets of Langerhans were isolated from rats as previously described [Thomas, H.E.,

Darwiche, R., Corbett, J.A. & Kay, T. W. Interleukin-1 plus gamma-interferon-induced pancreatic beta-cell dysfunction is mediated by beta-cell nitric oxide production. Diabetes 51, 311-6 (2002)]. Purified islets were washed, dispersed into single cells using trypsin (0.2% trypsin, 10 mM EDTA in Hanks's balanced salt solution) and stained using standard procedures. Antisera used were rabbit anti CML [Forbes, J.M. et al. Renoprotective effects of a novel inhibitor of advanced glycation. Diabetologia 44, 108-114 (2001)] followed by biotinylated anti rabbit (8.8.9) followed by phycoerythrin-conjugated streptavidin. β cells were identified based on their high autofluorescence [Thomas, H.E., Darwiche, R., Corbett, J.A. & Kay, T. W. Evidence that beta cell death in the nonobese diabetic mouse is Fas independent. J Immunol 163, 1562-9 (1999)]. Anti-RAGE was also used to confirm cell surface expression of RAGE by established methods. Islet Extraction Rat pancreatic islet isolation

The bile duct was cannulated and injected with 10ml of cold Hanks balanced salt solution (HBBS) containing 0.75mg/ml collagenase type V. The pancreas was then incubated at 37⁰C in a shaking water bath for 10-20 mins. Once the digestion was complete, the pancreas was disrupted by vigorous shaking and filtered through 500μm mesh. The pancreatic islets were separated from exocrine tissue by histopaque density gradient, which the islets were suspended in histopaque 1.119g/l, followed by layering of histopaque 1.083g/l and histopaque 1.077g/l (Sigma). The hand-picked islets were then rested overnight and RNA extracted as below. Real-time Reverse transcription-polymerase chain reaction

Three micrograms of total RNA extracted from pancreatic tissue collected immediately into RNA later or from MIN 6 cells, were used to synthesize cDNA with the Superscript First strand synthesis system for RT-PCR. Gene expression for each of the sequences listed below were analysed by real-time quantitative RT-PCR performed with the TaqMan system based on real-time detection of accumulated fluorescence [Candido, R. et al. A breaker of advanced glycation end products attenuates diabetes-induced myocardial structural changes. Circ Res 92, 785-92 (2003)]. For rat proinsulin (NM_019130) the forward primer was 5'-TGGTTCTCACTTGGTGGAAGCT-S' (SEQ ID NO: 1), the reverse primer, 5'-GGACATGGGTGTGTAGAAGAATCC-S' (SEQ ID NO: 2) and the probe was 6-FAM CCCACACACCAGGTAG-MGB (SEQ ID NO: 3), for mouse proinsulin (NM_008387) the probe was as for rat, however the forward primer was 5 'TC AAGCAGC ACCTTTGTGGTT- 3 ' (SEQ ID NO: 4) and the reverse primer 5 ' -GGGAC ATGGGTGTGTAGAAGAAG-3 ' (SEQ ID NO: 5). Fluorescence for each cycle was quantitatively analysed. To control for variation in the amount of DNA available for PCR in the different samples, gene expression of the target sequence was normalised in relation to the expression of an endogenous control, 18S ribosomal RNA (rRNA). The amplification was performed with the following time course: 50⁰C, 2 minutes and 10 minutes at 95°C; and 40 cycles of 94°C, 20 seconds, 60⁰C, 1 minute. Each sample was tested in triplicate with the average inter-assay coefficient of variation being 2.1%. Results were expressed relative to sham pancreatic samples, which were arbitrarily assigned a value of 1. Immunohistochemistry A modification of the ABC immunoglobulin enzyme bridge technique was used for immunohistochemistry as described previously [Forbes, J.M. et al. The breakdown of preexisting advanced glycation end products is associated with reduced renal fibrosis in experimental diabetes. Faseb J YI, \762A (2003)]. Formalin fixed paraffin sections of pancreas at 4μm were dewaxed and hydrated. Following incubation with 0.3% hydrogen peroxide for 20 mins, sections were incubated with protein blocking agent for 30 mins and then incubated with primary antibody overnight at 4⁰C. The primary antibodies used in this study were mouse anti insulin (1:2000), rabbit anti CML (1:500, Forbes, J.M. et al. Renoprotective effects of a novel inhibitor of advanced glycation. Diabetologia 44, 108-114 (2001), goat anti RAGE (1:500), mouse anti ED-I (monocyte/macrophages, 1:50) and mouse anti proliferating cell nuclear antigen (PCNA, 1 :50). Tissue sections were consecutively stained with biotinylated IgG for 10 mins and avidin-biotin horseradish peroxidase complex for 15 mins before a substrate solution of 3,3'-diaminobenzidine tetrahydrochloride was added. Sections were counterstained in Harris' haematoxylin and mounted in dePex. Negative control sections had the omission of the primary antibody. Positive control tissues were also included. Quantitation of islet immunostaining was completed by computer-aided densitometry, where a minimum of 20 islets (xlOO) were counted per section. Ten animals per group were assessed. Results were expressed as proportional area of positive staining within islets [Forbes, J.M., Hewitson, T.D., Becker, GJ. & Jones, CL. Ischemic acute renal failure: long-term histology of cell and matrix changes in the rat. Kidney Int 57, 2375-85. (2000)]. PCNA was analysed as the number of positive proliferating nuclei per islet. Terminal dUTP Nick End Labelling

DNA fragmentation is a hallmark of cells where endonucleases have been activated during the process of cell death. Cell death was identified by 3' in situ end labelling of fragmented DNA with biotinylated deoxyuridine-triphosphate. Terminal transferase labels the nicked DNA with labelled deoxy-uridine-triphosphate (dUTP), which is subsequently detected by immunohistochemical techniques as above. Sections of formalin fixed tissue were dewaxed and hydrated. Following digestion with Proteinase K (2μg/ml), sections were consecutively washed in TdT buffer (0.5M cacodylate pH6.8, ImM cobalt chloride, 0.15M NaCl) and then incubated at 37⁰C with TdT (25U) and biotinylated dUTP ( 1 nmol/μl). After washing in TB buffer (30OmM NaCl, 3OmM sodium citrate) to terminate the reaction, incorporation of biotinylated dUTP was detected by a modification of the ABC method. Horseradish peroxidase conjugated strepavidin-biotin complex was applied (Vector), the sections developed with DAB and counterstained in Harris haematoxylin. A mouse spleen with increased apoptosis was used as a positive control for TUNEL. The omission of TdT during dUTP nick end labelling provided a negative control.

The aim of the following examples was to determine if AGEs, directly contribute to insulin secretory defects, independent to hyperglycaemia. The insulinoma cell line MIN6N8 cells (derived from a β cell insulinoma) were exposed to AGEs and mitochondrial function, Ca²⁺ flux and insulin secretion assessed. In addition, the effects of strategies to reduce AGE accumulation, block AGE signalling via RAGE with the antagonist of AGE-RAGE interactions, soluble RAGE and gene silencing with UCP-2 siRNA were also determined on GSIS. In vivo, the effects of short and long term AGE exposure via intraperitoneal injection or dietary intake on pancreatic islet function, insulin secretion and islet integrity in the absence of high glucose concentrations in healthy non-diabetic rodents was examined. The efficacy of specific blockade of AGE accumulation with the AGE therapy, alagebrium chloride was also tested in some groups of rodents animals. EXAMPLE 1

Analysis of Exogenous AGE-albumin

AGE formation requires amino groups on proteins. In this example, albumin was used as representative example of a circulating AGE modified protein present in vivo. Endotoxin levels within all preparations were found to be below assay detectable levels (<2.5EU/ml). Analysis of the principal AGE modifications of lysine residues revealed that the major moiety in both AGE-RSA (rat serum albumin) and AGE-BSA (bovine serum albumin) was carboxymethyllysine (CML; 38.2±3.6 and 67.0+1.2 mmol/mol lysine respectively), although pentosidine (0.0037 and 0.0029 mmol/mol lysine) and carboxyethyllysine (CEL; 1.2±0.2 and 1.4±0.2 mmol/mol lysine) were also detected. There was also evidence for low levels of these AGE moieties detected in native BSA (CML- 0.5 mmol/mol lysine; pentosidine- 0.001 mmol/mol lysine) and RSA (CML-0.3 mmol/mol lysine; pentosidine- 0.001 mmol/mol lysine; CEL 0.02 mmol/mol lysine) corresponding to those produced during natural ageing.

EXAMPLE 2

Exposure ofMIN6N8 cells to AGEs causes insulin secretory defects independent to glucose concentrations in a time dependent manner.

Following 7 days of exposure to AGEs (AGE-BSA) in 25mmol/L glucose (HG),

MIN6N8 cells demonstrated basal hypersecretion of insulin, which was attenuated by treatment with the AGE cross-link breaker alagebrium (ALT; Fig Ia). There were modest changes in basal insulin secretion noted in normal glucose (NG; 5mmol/L) at 7 days

(ContNG-3.64+0.11 vs NG+AGE-1.33±0.16; PO.05). However, by 28 days of AGE exposure in normal glucose, basal insulin secretion was similar to that seen with high glucose at day 7 (Fig Ia) and these changes were attenuated by alagebrium. Alagebrium is also referred to as alagebrium chloride, ALT-711 or 3-(2-phenyl-2-oxoethyl)-4,5- dimethylthiazolium chloride. These terms are used interchangeably throughout.

Glucose stimulated insulin secretion (GSIS) for cells grown in high glucose conditions was elevated as compared to cells grown in normal glucose at day 7 (Fig Ib).

Exposure to AGEs for 7 days significantly reduced GSIS (HG+AGE, Fig Ib), however concomitant administration of alagebrium ameliorated GSIS to control levels. Under normal glucose conditions, 28 days of AGE exposure significantly decreased GSIS, which was partially attenuated by alagebrium (Fig Ib). There was no change in cellular insulin content under normal glucose conditions. Proinsulin gene expression in MIN6N8 cells at 7 days was unchanged by high glucose, however, concomitant AGE exposure in high glucose media markedly decreased its expression (Cont HG-0.86±0.11 vs HG AGE-BSA-0.28±0.05, PO.001) and this decrease was attenuated with alagebrium treatment (HG AGE-BSA vs HG AGE-BSA+ALT- 2.30±0.52; P<0.001). There was also a significant decrease in proinsulin gene expression seen with AGE exposure under normal glucose conditions at day 7 (Cont NG-1.02±0.11 vs NG AGE-BSA- 0.51 ±0.09; P<0.05). After 28 days of AGE exposure under normal glucose conditions, however, there was a marked increase in proinsulin gene expression (Cont NG- l.Ol±O.lO vs NG AGE-BSA- 1.62±0.05; P<0.05), which was attenuated with alagebrium (NG AGE-BSA vs NG AGE-BSA+ALT-0.41 ±0.49)

The gene expression of RAGE by real time RT-PCR was significantly increased following exposure to AGEs as compared to both control and BSA treated cells independent to glucose exposure. Increases in the cell surface expression of RAGE with AGE exposure was confirmed by FACS analysis at 7 days in high glucose (Fig. Ic). Further to this, MIN6N8 cells over-expressing human full length RAGE cDNA

(pRAGE) had increases in both the gene and surface expression of human RAGE. Over- expression of RAGE (pRAGE) in MIN6N8 cells significantly increased basal insulin secretion equivalent to that seen with AGE-BSA treatment (Fig Id). In addition, pRAGE transfected cells showed a decrease in GSIS, which was also consistent with that seen with AGE-BSA treatment (Fig Ie). Furthermore, AGE-induced decline in GSIS was ameliorated with the competitive inhibitor of RAGE, soluble RAGE (sRAGE, Fig Ie).

Figure 1: For Figures Ia-Ic, cells were exposed to either 25mM glucose (HG) for 7 days or 5mM glucose (NG) for 28 days in the presence and absence of AGE-BSA (lOOμg/ml). White bars - Glucose only, Black Bars - Glucose and AGE-BSA, Grey bars - Glucose and BSA, Checked bars - Glucose, AGE-BSA and the AGE inhibitor, alagebrium, (ALT - 1 μM). a) Basal insulin secretion, b) 2OmM glucose stimulated insulin secretion (GSIS). c) Flow cytometry analysis for cell surface RAGE. For d-e, MIN6N8 cells were transiently transfected with human full length RAGE or the control vector pCIneo, for 7 days in 25mM glucose (80% transfection efficiency determined by co-transfection with SEAP). d) Basal insulin secretion e) 2OmM GSIS in the presence of sRAGE (1 μg/mL) = the soluble isoform of the RAGE receptor and natural occurring competitive inhibitor. Open bars - HG control + Empty Vector (PCIneo), Black bars - PCIneo + lOOμg/ml AGE-BSA, Grey bars - pRAGE transfection, Spotted bars - pCIneo transfected cells + lOOμg/ml AGE-BSA + sRAGE. LPF - Lipofectamine transfection reagent only. *p<0.001 vs NG control, |p<0.05 vs AGE-BSA, $p<0.001 vs AGE-BSA, #p<0.05 vs HG control, §p<0.05 vs NG control, ¥p<0.05 vs PCIneo, $p<0.05 vs PCIneo+AGE-BSA.

EXAMPLE 3 AGEs disrupt glucose stimulated insulin secretion via by uncoupling, interruption of ATP production and Ca ^H flux resulting in mitochondrial superoxide production.

Although there was no difference between normal and high glucose at 7 days, AGE- BSA exposure significantly decreased cellular ATP content under both glucose conditions (Fig 2a for HG; NG Cont, 47.2±7.1 vs NG AGE-BSA, 34.6±1.9 pmol/mg protein; P<0.01), which was restored with alagebrium treatment (Fig 2a for HG; NG AGE-BSA vs NG AGE- BSA+ALT- 42.1±1.6 pmol/mg protein; PO.001).

Following acute stimulation with 20mmol/L glucose, MIN6N8 cells grown in normal glucose for 7 days in the presence of AGE-BSA, had a ~20% increase in [Ca²⁺]/ above that seen in the control group (81.2±0.6 vs 107.7±5.8 % above basal respectively; PO.001). This was normalised with alagebrium (85.92±12.1%; PO.01 vs NG AGE-BSA). In contrast, cells grown in high glucose and AGE-BSA for 7 days had a significant decline in [Ca²⁺]/ as compared to cell grown in high glucose alone. This AGE induced decline in [Ca²⁺]/ was attenuated by alagebrium (Fig 2b). Interestingly by 28 days, however, groups grown in normal glucose conditions showed an identical pattern Of [Ca²⁺]/ in response to glucose challenge, to that seen with high glucose at 7 days (NG, 261.5±55.0 vs NG AGE-BSA, 100.1±35.6 % above basal respectively; P<0.001). The dihydropyridine calcium channel blocker verapamil inhibited the glucose stimulated changes in [Ca²⁺]/. There were no changes at any time in [Ca²⁺]/ among groups in their response to the calcium ionophore, ionomycin. Superoxide production in mitochondria isolated from MIN6N8 cells was increased with exposure to AGE-BSA for 7 days in high glucose (Fig 2c). This mitochondrial superoxide production was attenuated with alagebrium. Indicative of respiratory chain uncoupling, UCP-2 gene (Fig 2d) and protein (data not shown) expression were increased in AGE-BSA treated cells which was ameliorated with concomitant alagebrium treatment (Fig 2d). Further to this, introduction of siRNA to UCP-2 to AGE-BSA treated cells, restored GSIS to those levels seen in untreated MIN6N8 cells (Fig 2e). Knock down efficiency of UCP-2 gene and protein was confirmed to be 50% at 4nmol/L of UCP-2 siRNA with a titration effect observed from 2nmol/L to 6nmol/L. Transection reagent (TKO) alone nor UCP-2 siRNA in the absence of TKO did not affect GSIS (Fig 2e). Figure 2: MIN6N8 cells were exposed to 25mM glucose (HG) for 7 days in the presence and absence of AGE-BSA (lOOμg/ml). White bars - Glucose (HG, 2OmM) only, Black Bars - HG+AGE-BSA, Grey bars - HG+BSA, Checked bars -HG+AGE- BSA+alagebrium (ALT, lμM), Spotted bars - HG+verapamil (VER, Ca²⁺ blocker), Diagonal striped bars - HG+AGE-BSA+UCP-2 siRNA 4μM, Vertical striped bars - HG+TKO transfection reagent, Horizontal striped bars - HG+UCP-2 siRNA 4nM. a) Cellular ATP content, b) Changes in intracellular calcium flux [Ca ⁺]i following 2OmM glucose challenge, c) Mitochondrial superoxide production, d) Cellular UCP-2 gene expression, e) Effects of siRNA inhibition of UCP-2 (4nM) on 2OmM glucose stimulated insulin secretion. *p<0.001 vs HG, tpO.OOl vs HG+AGE, Jp<0.001 HG+AGE vs HG+BSA with unpaired t- test, §p<0.01 vs HG, #p<0.01 vs HG+AGE.

EXAMPLE 4

Short term infusion of AGE-RSA into healthy rodents induces early βcell decompensation There were no changes in plasma glucose, body weight or glycated haemoglobin observed among groups at 1 month as shown below in Table 1. Circulating CML (AGE) levels remained low, suggesting clearance was normal via the kidneys and liver.

Plasma Glycated Body Plasma

Glucose Haemoglobin Weight CML mmol/L % g μmol/mol lysine

A GE infused rats

IM AGE-RSA 7.0±0.8 3.1+0.3 458+22 252.5124.8

IM RSA 7.1+1.0 3.0+0.7 439131 250.3128.3

IM Sham 7.2±0.8 2.910.4 431131 226.7121.8

4M AGE-RSA 7.8±0.5 4.710.6*1 548+37* 359.2+57.7

4M RSA 7.411.0 4.210.8*1 529148* 311.7138.42

4M AGE- 7.6±0.5 4.510.5* 567150* 217118.2J

RSA+ALT

4M Sham 7.410.5 3.010.3 629167* 304.6125.6

Table 1: Rodent physiological and metabolic parameters following AGE-RSA infusion . Plasma glucose and glycated haemoglobin are included as measures of glycaemic control at the study endpoints (1 or 4 months). Final body weight and plasma CML (AGE) levels are also shown.

*P<0.05 vs IM Sham, f p<0.05 vs same group at IM, $p<0.05 vs AGE-RSA. By 1 month, glucose (not shown) and insulin area under the curve (AUC) were increased in rats treated with AGE-RSA compared to the RSA treated group (Fig 3a). AUC insulin/ AUC glucose was modestly but significantly higher in the AGE-RSA infused group compared to RSA only (AGE-RSA, 0.59±0.02; RSA, 0.52±0.01; p=0.03). Real time RT-PCR revealed a significant increase in the gene expression of proinsulin within the pancreas from the AGE-RSA infused group as compared to the RSA group at 1 month (Fig 3b). This was also supported by immunohistochemistry demonstrating increased protein expression of proinsulin/insulin within islets of AGE-RSA treated rats as compared to RSA and sham groups (data not shown). Pancreatic gene expression of UCP-2 was significantly elevated with AGE-RSA infusion (16.09±6.33 AU) as compared to RSA infused and sham rats (RSA, 1.59±0.95; Sham, 1.09±0.32; p<0.001 vs AGE-RSA).

The number of proliferating β cells within islets was assessed using PCNA immunohistochemistry. At 1 month, there was a significant increase in the number of β cells proliferating cells within islets from animals which received AGE-RSA, as compared to both Sham and RSA groups (Fig 3 c). Confirmation of β cells as the proliferating cell type, was performed by concomitant immunostaining with insulin.

Immunohistochemistry revealed a significant increase in CML (AGE) within islets from AGE-RSA infused animals by 1 month as compared to both Sham and RSA infused rats (Fig 3d). RAGE protein expression within islets was also significantly increased following 1 month of AGE-RSA treatment as compared to both sham and RSA groups (Fig 3e). Together this data suggests that short-term infusion of AGEs results in insulin resistance that is associated with a concomitant increase in islet β cells, insulin production and secretion. Figure 3: 20mg/kg/day AGE-RSA or RSA was administered for 1 month in the absence of increased glucose elevations, a) Insulin levels before and during i.v. glucose challenge (lg/kg), □ AGE-RSA infused rats φ RSA infused rats, b) Proinsulin gene expression real time RT-PCR. c) Proliferation of β cells by PCNA morphometry d) Morphometric analysis of islet AGE (CML) content, e) Morphometric analysis of islet RAGE protein expression. ♦p<0.001 vs Sham, |p<0.001 vs AGE-RSA, JpO.Ol vs AGE-RSA, #p<0.05 vs AGE-RSA. EXAMPLE 5

Long term infusion of AGE-RSA into healthy rats interrupts first phase insulin secretion and induces βcell death

There were no significant differences in plasma glucose and body weight among the groups after 4 months. Glycated haemoglobin was increased in both AGE-RSA and RSA treated groups at 4 months and was not normalised with alagebrium therapy (Table 1). There was a significant increase in 4 month circulating AGE levels as compared to 1 month. Although there was a trend toward higher circulating CML levels in rats administered AGE- RSA for 4 months, this did not reach significance (Table 1). However, AGE-RSA rats treated with alagebrium for 4 months had a significantly lower level of circulating AGE as compared to those administered AGE-RSA alone.

By 4 months, the insulin AUC was significantly decreased in those rodents which received AGE-RSA (Fig 4a). Incremental first phase insulin secretion was also significantly reduced in this group (41.5±12.1 ng/mL x 10 mins; p<0.001 vs RSA and Sham) as compared to the sham and RSA groups (Sham, 90.9+20.0; RSA, 128.1+13.1 ng/mL x 10 mins) and normalised with alagebrium (133.5±18.7 ng/mL; pO.OOl vs AGE-RSA). In addition, AUC insulin/ AUC glucose was significantly lower in AGE-RSA infused rodents when compared to sham animals (AGE-RSA, 0.20±0.01; vs Sham, 0.37±0.01, pO.OOl). Those animals which received RSA only, had modest increases in first phase insulin secretion and in AUC insulin/AUC glucose (RSA, 0.57±0.20, p=0.049 vs Sham). Gene expression of proinsulin was significantly decreased by AGE-RSA infusion as compared to the sham and RSA treated groups (Fig 4b). Changes in insulin AUC, incremental first phase insulin secretion and pancreatic islet insulin expression seen with AGE-RSA, were each attenuated by treatment with alagebrium (Fig 4a-b). Immunohistochemistry demonstrated increased islet AGE (CML) staining by 4 months in AGE-RSA infused rodents (Fig 4c). There was also a modest but significant increase in CML staining in islets from rats infused with RSA as compared to Sham at 4 months. These increases in CML islet content were attenuated by alagebrium to levels seen in sham rats (Fig 4c). Flow cytometry confirmed a significant increase in CML staining within β cells from AGE-RSA infused rats as compared to RSA and sham groups (data not shown; PO.OOl AGE-RSA vs RSA) which was attenuated with alagebrium (AGE-RSA vs AGE-RSA+ALT, P<0.001). Flow cytometry also confirmed a modest increase in CML staining seen in rodents, which received RSA only for 4 months (RSA vs Sham, p<0.01). This staining was also attenuated with alagebrium (RSA vs RSA+ ALT, p<0.01). TUNEL revealed a significant increase in β cell death within islets from the AGE- RSA infused rats at 4 months (Fig 4e). This was normalised to levels seen within Sham and RSA only groups by treatment with alagebrium. Confirmation of β cell phenotype was performed by concomitant labelling with insulin. Analysis of monocytes/macrophages (ED-I) demonstrated a significant infiltration of these cells within pancreatic islets of AGE-RSA infused animals as compared to sham and RSA animals at 4 months (Fig 4d). Treatment with alagebrium led to reduced ED-I monocyte/macrophage cellular infiltration to levels seen in sham rats. Immunohistochemistry for RAGE revealed a modest increase in the number of RAGE positive β cells in AGE-RSA infused rodents which was not seen in sham or RSA infused groups. These data suggest that long-term infusion of AGEs caused significant defects in GSIS associated with decreased insulin production and increased islet β cell death.

Figure 4: 20mg/kg/day AGE-RSA or RSA was administered for 4 months in the presence and absence of the AGE cross-link breaker alagebrium (ALT, lOmg/kg/day) to 8 week old male Sprague Dawley rats. Sham-closed triangles; AGE-RSA-Open squares; RSA- Closed circles; AGE-RSA+ALT-Open circles, a) Insulin levels before and during i.v. glucose challenge (lg/kg). b) Real time RT-PCR for the gene expression of proinsulin in pancreata. c) CML (AGE) content of islets, d) Islet monocyte/macrophage infiltrate (ED-I) e) TUNEL analysis of β cell apoptosis. *p<0.05 Sham vs AGE-RSA at 2, 5 and 10 minutes. |p<0.05 AGE-RSA vs RSA at 2, 5, 10 . and 15 minutes, §p<0.05 vs Sham, #p<0.05 vs AGE-RSA.

EXAMPLE 6

A high dietary intake of AGEs causes insulin deficiency, secretory defects and hyperglycaemia.

To confirm that excess dietary intake of AGEs (CML) was analogous to the AGE- RSA infusion studies performed above, rats were fed diets with either high or low AGE content. Rats on the high AGE diet (22622±4042 nmol/mol lysine) had a daily AGE (CML) intake that was almost 5 times higher than those rodents ingesting the low AGE diet (4552±892 nmol/mol lysine). A subgroup of rats also received a high dextrose (glucose polymer) diet which also contained high levels of AGE comparable to those seen with a high AGE diet (Daily CML intake, 24232±6146 nmol/mol lysine). Over the time course of the study, the daily intake of energy was identical in groups receiving either the low or high AGE containing diet (Low AGE, 350+68.7; High AGE, 357.4+63.9 KJ/day) or high dextrose diet (High Dext, 385.8±97.8 KJ/day). Since the AGE diets only differed in baking, the macronutrient profiles were identical in both the high and low AGE containing diets. Consumption of the high dextrose diet resulted in an increase in carbohydrate intake (14.7±3.7g/day) when compared to the high or low AGE diets (High AGE, 2.2±0.4g/day) although the intake of all other macronutrients was identical. Circulating levels of CML were significantly increased in rats consuming both the High AGE (1246.0±284.3 nmol/mol lysine vs low AGE 523.0±74.8, p<0.05) and High Dextrose containing diets (806.2±125.2 nmol/mol lysine, p<0.05 vs low AGE).

Fasting glucose was elevated in those rats, which consumed diets high in AGE or glucose content when compared to the low AGE diet (Fig 5a). Furthermore, fasting insulin concentrations were significantly diminished with diets high in either AGE or glucose content (Fig 5a). In addition, insulin area under the curve from IVGTTs, was decreased compared with low AGE fed rats, with intake of diets high in either AGE or glucose content (Fig 5b). This apparent insulin deficiency with high AGE or glucose diets was further supported by islet proinsulin gene expression, which was markedly reduced in these groups compared with islets from rats which consumed a low AGE diet (Fig 5c). Islet immunohistochemistry also demonstrated increases in both RAGE (Fig 5d) and the AGE, CML (Fig 5e) within the islets of rats which consumed diets high in AGE or glucose content.

Figure 5: Groups of healthy SD rats were administered isocaloric diets, which differed only in AGE (High and Low AGE) or glucose content (High Dext) for 6 months, a) Fasting plasma insulin and glucose levels at 6 months, b) Insulin levels before and during i.v glucose challenge, c) Proinsulin Gene expression by real time RT-PCR. d) Islet RAGE expression by immunohistochemistry. e) Islet AGE (CML) content by immunohistochemistry. *p<0.05 vs Low AGE fed group, fpθ.001 vs Low AGE, Jp<0.01 vs Low AGE.

EXAMPLE 8

Acute Insulin Secretory Experiment

MIN6N8 cells were treated for 7 days in High Glucose (25mmol/L glucose) with either lOOug/ml AGE-BSA or BSA. Six replicates for each group were completed. Prior to commencement of insulin secretion testing at day 7, different groups were incubated with lumol/L alagebrium chloride (ALT-711) for varying times to complete the following groups:

(i) AGE-BSA only

(ii) BSA control group (iii) AGE-BSA + ALT-711 for 30 mins before insulin secretory testing, (iv) AGE-BSA + ALT-711 for 60 mins before insulin secretory testing, (v) AGE-BSA + ALT-711 for 2 hours before insulin secretory testing, (vi) AGE-BSA + ALT-711 for 4 hours before insulin secretory testing.

Acute insulin secretion involved transferring cells to 3mmol/L glucose to take basal insulin secretion. The cells were then challenged with 20mmol/L glucose (acute secretion is 1^st 20 mins in culture), and media collected for 1 hour. Insulin secretion during the various phases is expressed as % content (which includes intracellular insulin as well). Figure 7: Insulin secretory function of MIN6N8 cells exposed to AGE-BSA at day 7 expressed as a % of total content. Figure 7A) Basal insulin secretion. Figure 7B) Acute insulin secretion. Figure 7C) Total insulin secretion. Figure 7D) Intracellular insulin content. *p<0.05 vs AGE-BSA (AGE), #p<0.01 vs AGE-BSA (AGE).

EXAMPLE 9

Insulin Resistance Data

Figure 8 A IPGTT graph: Plasma insulin concentrations over time following an intraperitoneal bolus of glucose given at 16 weeks. LAGE group are wild type (C57BL/6J) mice which have been fed a control diet low in AGE content (LAGE) and followed for 16 weeks. HFAT+HAGE is a western style diet which is isocalorically identical to the LAGE diet except for fat and AGE content. Two groups were fed this diet for 16 weeks , namely wild type (C57BL/6J) and RAGE deficient (RAGE-/-) mice. A fourth group of wild type mice were fed the western (HFAT+HAGE) diet for 16 weeks and were concomitantly administered alagebrium (ALT-711; lmg/kg/day oral gavage). * is p<0.01 vs LAGE, the cross is p<0.01 vs the HAGE+HFAT C57BL6J group. This curve shows the body's ability to "deal" with a glucose load which is equivalent to what would be seen following a meal. The more stable the curve, the more capable the body is in handling glucose. Both the groups receiving the western diet had a constant increase in the amount of circulating insulin suggesting that they were not able to put in place appropriate mechanisms to control the plasma glucose levels and therefore hypersecreted insulin. Figure 8B. AUC

Insulin: Glucose: This is a ratio of the area under the curve of plasma insulin and glucose during the IPGTT testing. The higher the ratio of insulin to glucose in this test, the more insulin resistance is present. The groups are as above. Simplistically, it is clear however that there is insulin resistance in both the groups fed the western diet ie RAGE-/- mice are not protected against the devleopment of IR. Alagebrium therapy ameliorated the effects seen with the western diet in the wild type mice.

3. Figure 8C. Fasting plasma data: These values are measured following a 6 hour fast (equivalent to overnight for mice). Again it is obvious that mice fed a western style diet have poor glycaemic control with elevated fasting plasma glucose and insulin levels. The data is significant for the western diets in both strains compared to the LAGE group (p<0.01 vs LAGE). Alagebrium treatment also normalised plasma insulin and glucose (significant compared to western diet group p<0.05).

Figure 8: Intraperitoneal glucose tolerance testing in mice. Figure 8A) Plasma insulin curve over time Figure 8B) AUC insulin to AUC glucose ratio Figure 8C) Fasting plasma glucose and insulin data.

* is p<0.01 vs LAGE, t P<0.05 vs the HAGE+HFAT C57BL6J group, $p<0.01 vs HAGE+HFAT C57BL6J

EXAMPLE 10

Alagebrium Attenuation of Glucose Intolerance induced by Acute Elevation of Plasma Methylglyoxal

Advanced glycation endproducts (AGEs) are unavoidable byproducts of various metabolic pathways, such as glucose metabolism. They are formed by reactive metabolic intermediates such as methylglyoxal (MG). These reactive intermediates bind to proteins, DNA, and other molecules and disrupt their structures and functions, which leads to different diseases such as vascular complications of diabetes, atherosclerosis, hypertension, and aging. Plasma MG levels are elevated in diabetes. The effects of exogenous MG have been studied using very high doses in most in vivo and in vitro studies. Alagebrium was tested for its ability to inhibit MG, inhibit MG-induced AGE formation and assess its ability to treat or prevent disorders related to insulin resistance such as diabetes and hypertension.

The acute effects of a single intraperitoneal low dose (8.64 mg/kg) of MG were analysed in Sprague-Dawley rats. MG levels were measured by HPLC. An intravenous glucose tolerance test (IVGTT) was preformed 2 h after the administration of MG. Endothelium-dependent relaxation was tested in aortic rings. Plasma MG levels peaked 15 min after i.p. injection (2.50±0.20 μM vs. 1.60±0.18 μM control) and were significantly decreased (1.86±0.04 μM) after co-administration of alagebrium. The IVGTT area under curve for glucose was significantly greater after MG [4277±270 vs. 3413±108 control] and was attenuated by alagebrium (3823±93). MG levels increased significantly in liver (1.4 fold) and aorta (1.3 fold). Acetylcholine-induced relaxation was decreased in aortic rings from MG-treated group (EC50 - 0.59 μM vs 0.49 μM control). These results show that an acute increase in plasma MG induces glucose intolerance and causes endothelial dysfunction which is attenuated by alagebrium administration.

EXAMPLE 11

Alagebrium Attenuation of Mitochondrial Oxidative Stress induced by Methylglyoxal

Methylglyoxal (MG) is a highly reactive dicarbonyl compound that induces oxidative stress in vascular smooth muscle cells (A-10 cells). Since mitochondria are considered the most important source of free radical generation, the effect of MG on mitochondria of A-10 cells was investigated. Additionally, alagebrium was tested for its ability to inhibit the resultant effects of MG on mitochondria.

Mitochondria were prepared from A- 10 cells by lysis with the detergent, followed by low (600^χg) and high speed (1 l,000^χg) centrifugation. MG was measured by HPLC. Reactive oxygen species (ROS) and superoxide were detected by molecular probes and read under confocal microscopy. Nitrotyrosine (a marker for peroxynitrite formation), MG- induced advance glycation endproduct (AGE), N⁶ -(carboxyethyl) lysine (CEL), and mnSOD were measured by immunostaining.

After treatment of cells with MG (5-100 μM, 18 h), MG levels in mitochondria were significantly increased along with increased production of ROS compared to control. Alagebrium, antioxidant n-acetyl-cysteine (ΝAC), and peroxynitrite scavenger uric acid reversed the effects of MG. MG treatment significantly increased nitrotyrosine, which was decreased by co-treatment with alagebrium or ΝAC. Increased production of superoxide in mitochondria of MG treated cells was reduced by the co-application of alagebrium or superoxide dismutase (SOD) mimetic 4-hydroxy-tempo. MG treated cells also showed increased formation of CEL. At the same time, the activity and abundance of mnSOD were decreased, which were restored by alagebrium treatment. These results indicate that MG increased generation of peroxynitrite, superoxide and AGE formation, decreased activity of mnSOD and induced oxidative stress in mitochondria of A- 10 cells and that these effects were attenuated by alagebrium administration.

Claims

We claim:

1. A method of treating, or ameliorating a symptom of, a disease, disorder or condition associated with insulin resistance or β-cell dysfunction in a patient in need thereof, comprising administering a pharmaceutical composition comprising a compound of Formula I, or a pharmaceutically acceptable salt of the compound of Formula I,

wherein:

R¹ and R² are selected from the group consisting of hydrogen, hydroxy (lower) alkyl, acetoxy

(lower) alkyl, lower alkyl, lower alkenyl; or R¹ and R² together with their ring carbons may be an aromatic fused ring, optionally substituted by one or more amino, halo or alkylenedioxy groups;

Z is hydrogen or an amino group;

Y is amino, a group of the formula:

O Il

— CH2C — R wherein R is a lower alkyl, alkoxy, hydroxy, amino or an aryl group, said aryl group optionally substituted by one or more lower alkyl, lower alkoxy, halo, dialkylamino, hydroxy, nitro or alkylenedioxy groups; a group of the formula:

-CH₂R' wherein R' is hydrogen, or a lower alkyl, lower alkenyl, or aryl group; or a group of the formula:

wherein R" is hydrogen and R" is a lower alkyl group, optionally substituted by an aryl group, or an aryl group, said aryl group optionally substituted by one or more lower alkyl, halo, or alkoxylcarbonyl groups; or R" and R'" are both lower alkyl groups; and

X is a pharmaceutically acceptable anion, and a pharmaceutically acceptable carrier, thereby treating said disease, disorder or condition associated with insulin resistance or β-cell dysfunction.

2. The method of claim 1 , wherein Rl and R2 are independently lower alkyl.

3. The method of claim 1 , wherein Z is hydrogen.

4. The method of claim 1 , wherein R is an aryl group.

5. The method of claim 1 , wherein the compound of Formula I is 3-(2-phenyl-2- oxoethyl)-4,5-dimethylthiazolium.

6. The method of claim 1, wherein the compound of Formula I is 3-(2-phenyl-2- oxoethyl)-4,5-dimethylthiazolium chloride.

7. The method of claim 1, wherein the compound of Formula I is 3-(2-phenyl-2- oxoethyl)-4,5-dimethylthiazolium bromide.

8. The method of claim 1 , wherein said disease, disorder or condition associated with insulin resistance or β-cell dysfunction is type I diabetes.

9. The method of claim 1 , wherein said disease, disorder or condition associated with insulin resistance or β-cell dysfunction is non-insulin dependent (type II) diabetes.

10. The method of claim 1 , wherein said disease, disorder or condition associated with insulin resistance or β-cell dysfunction is pre-diabetes.

11. The method of claim 1 , wherein said disease, disorder or condition associated with insulin resistance or β-cell dysfunction is metabolic syndrome.

12. The method of claim 1 , wherein the administration of said pharmaceutical composition increases insulin sensitivity.

13. The method of claim 1 , wherein the administration of said pharmaceutical composition ameliorates insulin resistance.

14. The method of claim 1 , wherein the administration of said pharmaceutical composition ameliorates plasma insulin and glucose levels.

15. The method of claim 1 , wherein the administration of said pharmaceutical composition suppresses basal insulin secretion.

16. The method of claim 1 , wherein the administration of said pharmaceutical composition increases acute insulin secretion.

17. The method of claim 1 , wherein the administration of said pharmaceutical composition reduces plasma methylglyoxal levels.

18. The method of claim 1 , wherein the administration of said pharmaceutical composition reduces mitochondrial oxidative stress.

19. The method of claim 1 , further comprising administering a inhibitor of a receptor for advanced glycation end-products (RAGE).

20. The method of claim 18, wherein the RAGE inhibitor is soluble RAGE.