Advanced Glycated End-products (AGEs)

Abstract

Advanced glycated end-products (AGEs) form as a result of a non-enzymatic reaction in which glucose forms adducts with proteins, lipids and nucleic acids. AGEs accumulate naturally as a result of chronological aging, but this process is greatly accelerated under conditions of hyperglycemia and oxidative stress. AGEs directly induce cross-linking of long-lived proteins, such as collagen and elastin in the extracellular matrix, thus promoting stiffness and compromising vascular structure and function. In addition, AGEs interact with specific AGE receptors on virtually all cell types (RAGE), inducing intracellular signaling that increases oxidative stress and the production of key pro-inflammatory and pro-sclerotic cytokines. AGEs therefore play a central role in diabetes-related pathology, cardiovascular disease, the deterioration associated with chronological aging. This review summarizes endogenous and exogenous factors involved in AGE accumulation; AGEs' involvement in cardiovascular disease, diabetic complications, and Alzheimer's disease; and the research on new anti-AGE-ing therapeutic agents, among which alagebrium (ALT-711) appears to be the safest and most effective.

ALT711: Cross Link Breaker Delivers Effective Anti-AGE-ing Therapy

Introduction

As we age, cumulative exposure of the body's amino groups on proteins, lipids and nucleic acids to reducing sugars in the blood and lymph allows a non-enzymatic glycation reaction that initiates the production of cross-linked proteins called advanced glycation end-products (AGEs).

AGE formation occurs slowly over months or even years, which is why the long-lived proteins of the extracellular matrix, collagen and elastin, are most vulnerable to AGE accumulation. (Kass, editorial Circ Res 2003; Vasan S, Foiles P et al. Arch Biochem and Biophys 2003)

Once formed, AGE cross-links, both within modified proteins and between these and adjacent proteins, drastically alter affected proteins' structure and ability to function, reducing tissue flexibility and elasticity. AGE cross-links also render collagen less digestible by metalloproteinases, further promoting its accumulation and exacerbating disorders in which collagen synthesis is already excessively stimulated by inflammatory cytokines, neurohormones, or mechanical stress.

AGEs form adducts not only on collagen and elastin, but cross-link other proteins, enzymes, lipids, and DNA or RNA, changing their structure, and disrupting enzyme activity, hormone regulation and immune function. In the vasculature, for example, atherosclerotic plaque formation involves AGE cross-links between the proteins of the endothelium and oxidized LDL cholesterol, which is likely to itself have been modified by glycosylation into advanced lipoxidation products or ALEs. (Meerwaldt R, van der Vaart MG, et al. Eur J Vasc Endovasc Surg. 2008)

Besides chronological aging, AGE accumulation is promoted by hyperglycemia, insulin resistance, hyperlipidemia, oxidative stress, decreased anti-oxidant activity, (e.g., insufficient dietary antioxidants, vitamin and mineral co-factors) and decreased renal clearance of AGE-precursors. (Meerwaldt R, van der Vaart MG, et al. Eur J Vasc Endovasc Surg. 2008)

In addition to endogenous formation, AGEs are also derived from exogenous sources, primarily diet and tobacco smoke. Food processing, especially prolonged heating, accelerates generation of glyco- and lipo-oxidation products. A significant proportion of ingested AGEs are absorbed from foods exposed to high temperatures. AGE levels are notably higher in smokers and individuals on high-AGE diets. (Goh SY, Cooper ME. J Clin Endocrinol Metab. 2008)

Cellular RAGE: the Cell's Receptors for AGE

The protein distortion resulting from AGE cross-links is only one feature of AGE pathology. AGEs also bind to AGE-specific receptors (RAGEs) on cells, stimulating the release of factors that create excessive fibrous tissue, increase inflammation, and ultimately lead to tissue thickening, scarring and/or dysfunction in the heart, blood vessels, kidneys, eyes and brain. Again, the acronym is apt: an AGE-ing cell is en-RAGED.

The AGE-ing Aspect of Cardiovascular Pathologies

AGEs' pathological effects on the vasculature are widespread and numerous. AGE-ing reduces elasticity in the myocardium and large arteries. In the myocardial extracellular matrix proteins, of which nearly 80% is Type I collagen, AGE-ing results in decreased proteolytic turnover and lessened heart muscle flexibility. The aorta stiffens, leading to systolic hypertension and eventual heart failure. (Vasan S, Foiles P, Arch Biochem Biophys 2003.)

By binding to RAGEs on endothelial cells, AGEs induce a signaling cascade via nuclear factor kappa beta (NF-kB) that increases transcription of pro-inflammatory proteins, including the endothelial adhesion molecules (endothelin-1, ICAM, VCAM, TNF-alpha and interleukins). This cascade aggravates vascular inflammation and increases the production of reactive oxygen species.

The AGE–RAGE interaction also induces endothelial dysfunction by suppressing production of the endothelial isoform of NO synthase (NOS-III) through two mechanisms: one rapid, involving suppression of its serine phosphorylation, and another slower, involving a decrease in its expression. Without sufficient NO, the vasculature does not dilate properly, so blood pressure increases, resulting in micro-injuries to the endothelium that lead to a cascade of inflammatory responses. (Xu B, Ji Y, et al. Clin Sci 2005; Jeyabal PV, Kumar R, et al. Neurogastroenterol Motil 2007)

AGEs also activate monocytes, causing increased expression of CD36 receptors, which bind adhesion molecules, leading to increased AGE-lipid uptake and foam cell formation. (Meerwaldt R,l van der Caart MG, Eur J Vasc & Endovasc Surg, 2008)

AGEs accumulate in atherosclerotic plaques in the aorta where concentrations are related to arterial wall stiffness, especially in hypertensive individuals, independent of age, diabetes or renal failure. (Meerwaldt R,l van der Caart MG, Eur J Vasc & Endovasc Surg, 2008)

Serum concentrations of AGEs are positively associated with carotid intima-media thickness (IMT), and carotid plaque inflammation and vulnerability to rupture. (Meerwaldt R,l van der Caart MG, Eur J Vasc & Endovasc Surg, 2008)

Increased serum AGE levels are predictive of adverse cardiac events after cardiac surgery; all cause, cardiovascular and coronary mortality in women with type 2 diabetes; and lower probability of survival in both diabetic and hemodialysis patients. (Meerwaldt R,l van der Caart MG, Eur J Vasc & Endovasc Surg, 2008)

Accelerated AGE-ing in Diabetes

Since AGE formation is initiated by a glycation reaction, the hyperglycemic state characteristic of diabetes provides ideal conditions for AGE development. AGEs accumulate most rapidly in diabetes, are a major factor in all diabetic sequelae, and are also known to play a key role in all age-associated pathologies. (Cooper ME. Am J Hypertens. 2004 Dec;17(12 Pt 2):31S-38S.)

Prolonged hyperglycemia, dyslipidemia and oxidative stress, all promote AGE production and accumulation in the kidney (with concomitant impaired renal function since the kidney is the major site of AGE clearance), retina, and in atherosclerotic plaques. (Goh SY, Cooper ME. J Clin Endocrinol Metab. 2008; Thomas MC, Baynes JW, et al. Curr Drug Targets. 2005)

Endothelial cells, along with pericytes, compose the microvasculature of the eye and kidneys. As AGEs not only disrupt endothelial cells, but retard the growth of and are acutely toxic to pericytes, it is not surprising that retinopathy and nephropathy are frequent diabetic complications. (Sakurai S, Yonekura H, et al. J Am Soc Nephrol, 2003)

AGEs play a pivotal role in atherosclerosis in diabetics. In addition to their direct effects on the vascular endothelium, AGEs increase oxidative stress by upregulating the expression and activity of NADPH (the reduced form of nicotinamide adenine dinucleotide phosphate), an important source of vascular oxidative stress in diabetes;  enhancing the formation of free radicals; depleting and glycating cellular antioxidants, such as glutathione peroxidase and superoxide dismutase; and inducing RAGE-mediated signaling that causes mitochondrial dysfunction, further increasing oxidative stress. (Thomas MC, Baynes JW, et al. Curr Drug Targets. 2005)

AGEs have been detected within atherosclerotic lesions in both extra- and intra-cellular locations and correlate with lesion size and complexity. Clinical studies in patients with type 1 diabetes show strong correlations between AGE accumulation and severity of diabetic complications, including peripheral artery occlusive disease and coronary heart disease. (Thomas MC, Baynes JW, et al. Curr Drug Targets. 2005)

RAGE expression is greatly increased in coronary lesions from diabetic victims of sudden cardiac death, and is associated with apoptotic smooth muscle cells and macrophages. (Apoptosis is a late hallmark of atherosclerosis associated with plaque instability and/or rupture.) (Meerwaldt R, van der Vaart MG, et al. Eur J Vasc Endovasc Surg. 2008)

In diabetic patients, AGEs have also been shown to deposit in peripheral nerves, to induce neuronal apoptosis, and to correlate with the severity of diabetic neuropathy and foot ulceration.

Alzheimer's: a Correlation with AGE-ing?

AGE receptors (RAGE) are found in the brain where they have the ability to bind β-amyloid protein and the neuroregulatory protein amphoterin, and mediate the latter's effects on microglia, the blood-brain barrier, and neurons through activating different signaling pathways. (Chen X, Walker DG, et al. Curr Mol Med.  2007; Thomas MC, Baynes JW, et al. Curr Drug Targets 2005)

Data from autopsy brain tissues, in vitro cell cultures and transgenic mouse models suggest that the β-amyloid-RAGE interaction increases neuronal stress, β-amyloid accumulation, the impairment of learning memory, and neuro-inflammation. Blocking RAGE has been shown to protect against β-amyloid-mediated cellular disturbance. AGE reduction is likely to have therapeutic benefit for Alzheimer's Disease and other neurodegenerative disorders. (Chen X, Walker DG, Curr Mol Med. 2007 Dec)

Anti-AGE-ing Interventions

Diet and Exercise

A whole-foods, low-glycemic-index diet is essential to minimize the extent and duration of postprandial surges in blood sugar and triglycerides, and resulting likelihood of AGE formation. Exercise to improve and maintain a healthful BMI, with its positive effects on glucose metabolism, is the second baseline strategy.

Older Established Therapies

A number of established therapies have been shown to decrease AGE formation in vitro including ACE inhibitors, angiotensin receptor antagonists, metformin, peroxisome proliferators receptor agonists, metal chelators and some antioxidants. (Thomas MC, Baynes JW, et al.  Curr Drug Targets 2005)

New Therapeutic Agents

Several new therapeutic agents that reduce AGE formation and/or accumulation have been developed. ALT 711 (alagebrium), the best researched with positive in vitro, in vivo, and human clinical trials, appears to provide the highest efficacy and safety of these compounds. Preliminary research on pyridoxamine (an active form of B6), benfotiamine (a thiamine derivative) and LR-90 (methylene bis) suggests they may also provide benefit.

Alt711 (Alagebrium)

The AGE-reducing actions of the prototype of ALT711, PTB (N-phenacylthiazolium bromide), were first reported over ten years ago. PTB was shown to cleave cross-links in vitro, and to decrease AGE accumulation in diabetic rat models.

ALT711 or alagebrium (3-phenyacyl-4,5-dimethylthiazolium chloride), was developed as a more stable derivative of PTB. Numerous animal and human studies have now confirmed ALT-711's effectiveness in preventing and reversing AGE-related pathology.

Mechanism of Crosslink Breaking Alagebrium

See figure III

ALT711 Animal Research

In animals with diabetic renal disease, alagebrium greatly delayed development of albuminuria, lowered blood pressure, decreased renal AGE accumulation, and increased urinary AGE excretion.  (Peppa M, Brem H, et al. Am J Nephrol. 2006; Forbes JM, Thallas V, et al. FASEB J. 2003)

In diabetic apoE-/- mice (a well established model of atherogenesis), alagebrium attenuated atherosclerosis by more than 30%. In animals with diabetic-associated cardiac abnormalities, ALT711 reduced cardiac AGE levels, restored LV collagen solubility to the same level seen in control animals, reduced LV weight and abrogated the increase in RAGE and expression of collagen III (involved in pathogenic cardiac remodeling). ((Forbes JM, Yee LT, et al. Diabetes 2004; Susic D, Varagic J, et al. Curr Opin Cardiol 2004; Candido R, Forbes JM, et al. Circ Res 2003)

In older, spontaneously hypertensive rats, treatment with ALT711 reversed aortic stiffening and restored LV elasticity. In older diabetic dogs, ALT711 reduced aortic stiffness and LV mass, increased LV ejection fraction and myocardial collagen solubility, and reversed upregulation of (pro-fibrotic) collagen I and III production. (Susic D 2004; Liu J, Masurekar MR, et al. Am J Physiol Heart Circ Physiol. 2003)

Within 6 weeks, treatment with ALT711 significantly reduced AGE levels, increased penile neuronal nitric oxide synthase (nNos) and reversed diabetic erectile dysfunction in rats. (Usta MF, Kendirci M, et al. J Sex Med 2006)

Applied topically, 5% ALT711 cream greatly improved hydration and elasticity in the skin of aged rats, compared to controls. (Vasan S, Foiles P, et al., Arch. Biochem. Biophys.,2003)

ALT711 Human Clinical Trials

Results of human clinical trials using ALT-711 have been consistently positive.  In men with high blood pressure on antihypertensive therapy (systolic blood pressure >140 mmHg, diastolic blood pressure <90 mmHg or pulse pressure >60 mmHg), ALT-711 (210 mg twice daily for 8 weeks) decreased arterial stiffness by 37% and reduced blood pressure by an average of 6.8 mmHg. Flow-mediated dilation increased from an average of 4.6 to 7.1, while markers of blood vessel fibrosis all decreased significantly. (Bakris GL, Bank AJ, et al. Am J Hypertens 2004; Zieman SJ, et al. J Hypertens. 2007 Mar;25(3):577-83.)

In a phase 2 double-blind trial, 62 patients with isolated systolic hypertension (resting arterial pulse pressures >60 mm Hg and systolic pressures >140 mm Hg) were given ALT-711 (210 mg per day for 8 weeks); 31 subjects received placebo. In ALT711-treated subjects, total arterial compliance rose 15% versus no change in placebo, and arterial pulse pressure dropped -5.3 in those given ALT711 versus -0.6 mm Hg for placebo. (Kass DA, Shapiro EP, et al. Circulation 2001)

In elderly patients (mean age 71) with diastolic heart failure, ALT711 (420 mg/day for a period of 16 weeks) significantly decreased LV mass (from 124 +/- 35 g at baseline to 119 +/- 34 g at follow up). Patients' quality of life greatly increased; the Minnesota Living with Heart Failure total score improved almost 25% (from 41 +/- 21 to 32 +/- 21). (Little WC, et al. J Card Fail. 2005 Apr;11(3):191-5.)

ALT711 has also produced significant improvements in endothelial function in patients with isolated systolic hypertension. In a double-blind, placebo-controlled trial, older adults (9 men, aged 65 +/- 2 years) with isolated systolic hypertension (systolic blood pressure > 140 mmHg, diastolic blood pressure < 90 mmHg or pulse pressure > 60 mmHg) on stable antihypertensive therapy received placebo (2 weeks) then oral alagebrium (210 mg bid for 8 weeks). Alagebrium reduced carotid artery stiffness by 37% and increased endothelial flow-mediated dilation from 4.6 +/- 1.1 to 7.1 +/- 1.1%. These positive changes inversely correlated with drops in markers of fibrotic collagen synthesis and the inflammatory markers, p-selectin and intracellular adhesion molecule. (Zieman SJ, Melenovsky V, et al. J Hypertens 2007)

ALT711 Contraindications, Side Effects and Drug Interactions

Approximately 800 patients have received alagebrium in Phase 2 clinical trials. ALT711 has been extremely well-tolerated with fewer adverse events reported in subjects receiving ALT711 than in those given placebo. (Vasan S, Foiles P, et al., Arch. Biochem. Biophys.,2003; Synvista Scientific Publications: http://www.alteon.com/scientific_publications/index.htm)

No negative drug interactions have been noted. In fact, animal research using ALT711 along with the angiotensin converting enzyme inhibitor, ramipril, indicates that alagebrium and ACE inhibitors share beneficial downstream effects ( i.e., reduction of  protein kinase C activity, oxidative stress and free radical production). (Coughlan MT, Thallas-Bonke V, et al. Endocrinology 2007)

Pyridoxamine

Pyridoxamine, one of the three active forms of vitamin B6, has been shown to inhibit the formation of ALEs (lipoxidation end products) and the development of renal and vascular complications in obese rats, and to inhibit AGE formation and delay the onset of end-stage renal disease in patients with type 1 and type 2 diabetes and overt nephropathy. (Metz TO, Alderson NL, Arch Biochem Biophys 2003; Williams ME, Bolton, WK, et al. Am J Nephrol 2007)

Benfotiamine

Benfotiamine, a lipid-soluble thiamine (vitamin B1) derivative, prevents activation of three major pathways of hyperglycaemic damage (hexosamine pathway, intracellular AGE formation, and the diacylglycerol-protein kinase C pathway), by increasing the activity of transketolase, the rate-limiting enzyme of the non-oxidative branch of the pentose phosphate pathway. (Goh SY, Cooper ME. J Clin Endocrinol Metab. 2008)

In a small, three-week study, benfotiamine alleviated painful neuropathy in diabetic patients, and other small studies have shown that benfotiamine prevents macro- and microvascular endothelial dysfunction and oxidative stress after a high-AGE meal. (Goh SY, Cooper ME. J Clin Endocrinol Metab. 2008) 2008)

LR-90

LR-90 has been shown to block RAGE expression in monocytes, reduce AGE/ALE accumulation and RAGE expression in the kidney in streptozotocin-induced diabetic rats, and slow the progression of diabetic retinopathy in rats. (Rahbar S. Cell Biochem Biophys 2007; Figarola JL, Diabetologia 2008; Bhatwadekar AD, Glenn JV, et al. Br J Opthalmol 2008)

Conclusion

AGE-related contributions to age- and diabetic-related pathologies are myriad and significant. The results of pre-clinical and clinical studies demonstrate that the crosslink breaker, ALT-711 offers significant therapeutic potential in the prevention, treatment and reversal of AGE-related cardiovascular, renal, retinal, neuronal, erectile dysfunction and dermatological alterations associated with aging and diabetes.

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