Pyridoxamine, an inhibitor of advanced glycation and lipoxidation reactions: a novel therapy for treatment of diabetic complications.
Pyridoxamine (PM), originally described as a post-Amadori inhibitor of formation of advanced glycation end-products (AGEs), also inhibits the formation of advanced lipoxidation end-products (ALEs) on protein during lipid peroxidation reactions. In addition to inhibition of AGE/ALE formation, PM has a strong lipid-lowering effect in streptozotocin (STZ)-induced diabetic and Zucker obese rats, and protects against the development of nephropathy in both animal models. PM also inhibits the development of retinopathy and neuropathy in the STZ-diabetic rat. Several products of reaction of PM with intermediates in lipid autoxidation have been identified in model reactions in vitro and in the urine of diabetic and obese rats, confirming the action of PM as an AGE/ALE inhibitor. PM appears to act by a mechanism analogous to that of AGE-breakers, by reaction with dicarbonyl intermediates in AGE/ALE formation. This review summarizes current knowledge on the mechanism of formation of AGE/ALEs, proposes a mechanism of action of PM, and summarizes the results of animal model studies on the use of PM for inhibiting AGE/ALE formation and development of complications of diabetes and hyperlipidemia.
Arch Biochem Biophys. 2003 Nov 1;419(1):41-9
A post-Amadori inhibitor pyridoxamine also inhibits chemical modification of proteins by scavenging carbonyl intermediates of carbohydrate and lipid degradation.
Reactive carbonyl compounds are formed during autoxidation of carbohydrates and peroxidation of lipids. These compounds are intermediates in the formation of advanced glycation end products (AGE) and advanced lipoxidation end products (ALE) in tissue proteins during aging and in chronic disease. We studied the reaction of carbonyl compounds glyoxal (GO) and glycolaldehyde (GLA) with pyridoxamine (PM), a potent post-Amadori inhibitor of AGE formation in vitro and of development of renal and retinal pathology in diabetic animals. PM reacted rapidly with GO and GLA in neutral, aqueous buffer, forming a Schiff base intermediate that cyclized to a hemiaminal adduct by intramolecular reaction with the phenolic hydroxyl group of PM. This bicyclic intermediate dimerized to form a five-ring compound with a central piperazine ring, which was characterized by electrospray ionization-liquid chromatography/mass spectrometry, NMR, and x-ray crystallography. PM also inhibited the modification of lysine residues and loss of enzymatic activity of RNase in the presence of GO and GLA and inhibited formation of the AGE/ALE N(epsilon)-(carboxymethyl)lysine during reaction of GO and GLA with bovine serum albumin. Our data suggest that the AGE/ALE inhibitory activity and the therapeutic effects of PM observed in diabetic animal models depend, at least in part, on its ability to trap reactive carbonyl intermediates in AGE/ALE formation, thereby inhibiting the chemical modification of tissue proteins.
J Biol Chem. 2002 Feb 1;277(5):3397-403
Propagation of protein glycation damage involves modification of tryptophan residues via reactive oxygen species: inhibition by pyridoxamine.
Nonenzymatic modification of proteins is one of the key pathogenic factors in diabetic complications. Uncovering the mechanisms of protein damage caused by glucose is fundamental to understanding this pathogenesis and in the development of new therapies. We investigated whether the mechanism involving reactive oxygen species can propagate protein damage in glycation reactions beyond the classical modifications of lysine and arginine residues. We have demonstrated that glucose can cause specific oxidative modification of tryptophan residues in lysozyme and inhibit lysozyme activity. Furthermore, modification of tryptophan residues was also induced by purified albumin-Amadori, a ribose-derived model glycation intermediate. The AGE inhibitor pyridoxamine (PM) prevented the tryptophan modification, whereas another AGE inhibitor and strong carbonyl scavenger, aminoguanidine, was ineffective. PM specifically inhibited generation of hydroxyl radical from albumin-Amadori and protected tryptophan from oxidation by hydroxyl radical species. We conclude that oxidative degradation of either glucose or the protein-Amadori intermediate causes oxidative modification of protein tryptophan residues via hydroxyl radical and can affect protein function under physiologically relevant conditions. This oxidative stress-induced structural and functional protein damage can be ameliorated by PM via sequestration of catalytic metal ions and scavenging of hydroxyl radical, a mechanism that may contribute to the reported therapeutic effects of PM in the complications of diabetes.
Free Radic Biol Med. 2008 Apr 1;44(7):1276-85
Thiamine pyrophosphate and pyridoxamine inhibit the formation of antigenic advanced glycation end-products: comparison with aminoguanidine.
Nonenzymatic glycation of proteins by glucose leading to the formation of toxic and immunogenic advanced glycation end products (AGEs) may be a major contributor to the pathological manifestations of diabetes mellitus, aging, and, possibly, neurodegenerative diseases such as Alzheimer’s. We tested the in vitro inhibition of antigenic AGE formation on bovine serum albumin, ribonuclease A, and human hemoglobin by various vitamin B1 and B6 derivatives. Among the inhibitors, pyridoxamine and thiamine pyrophosphate potently inhibited AGE formation and were more effective than aminoguanidine, suggesting that these two compounds may have novel therapeutic potential in preventing vascular complications of diabetes. An unexpected finding was that aminoguanidine inhibited the late kinetic stages of glycation much more weakly than the early phase.
Biochem Biophys Res Commun. 1996 Mar 7;220(1):113-9
Pyridoxamine protects proteins from functional damage by 3-deoxyglucosone: mechanism of action of pyridoxamine.
Pyridoxamine (PM) is a promising drug candidate for treatment of diabetic nephropathy. The therapeutic effect of PM has been demonstrated in multiple animal models of diabetes and in phase II clinical trials. However, the mechanism of PM therapeutic action is poorly understood. One potential mechanism is scavenging of pathogenic reactive carbonyl species (RCS) found to be elevated in diabetes. We have suggested previously that the pathogenicity of RCS methylglyoxal (MGO) may be due to modification of critical arginine residues in matrix proteins and interference with renal cell-matrix interactions. We have also shown that this MGO effect can be inhibited by PM (Pedchenko et al. (2005) Diabetes 54, 2952-2960). These findings raised the questions of whether the effect is specific to MGO, whether other structurally different physiological RCS can act via the same mechanism, and whether their action is amenable to PM protection. In the present study, we have shown that the important physiological RCS 3-deoxyglucosone (3-DG) can damage protein functionality, including the ability of collagen IV to interact with glomerular mesangial cells. We have also demonstrated that PM can protect against 3-DG-induced protein damage via a novel mechanism that includes transient adduction of 3-DG by PM followed by irreversible PM-mediated oxidative cleavage of 3-DG. Our results suggest that, in diabetic nephropathy, the therapeutic effect of PM is achieved, in part, via protection of renal cell-matrix interactions from damage by a variety of RCS. Our data emphasize the potential importance of the contribution by 3-DG, along with other more reactive RCS, to this pathogenic mechanism.
Biochemistry. 2008 Jan 22;47(3):997-1006
Pyridoxine and pyridoxamine inhibits superoxide radicals and prevents lipid peroxidation, protein glycosylation, and (Na+ + K+)-ATPase activity reduction in high glucose-treated human erythrocytes.
Vitamin B(6) (pyridoxine) supplementation has been found beneficial in preventing diabetic neuropathy and retinopathy, and the glycosylation of proteins. Oxygen radicals and oxidative damage have been implicated in the cellular dysfunction and complications of diabetes. This study was undertaken to test the hypothesis that pyridoxine (P) and pyridoxamine (PM) inhibit superoxide radical production, reduce lipid peroxidation and glycosylation, and increase the (Na+ + K+)-ATPase activity in high glucose-exposed red blood cells (RBC). Superoxide radical production was assessed by the reduction of cytochrome C by glucose in the presence and absence of P or PM in a cell-free buffered solution. To examine cellular effects, washed normal human RBC were treated with control and high glucose concentrations with and without P or PM. Both P and PM significantly lowered lipid peroxidation and glycated hemoglobin (HbA(1)) formation in high glucose-exposed RBC. P and PM significantly prevented the reduction in (Na+ + K+)-ATPase activity in high glucose-treated RBC. Thus, P or PM can inhibit oxygen radical production, which in turn prevents the lipid peroxidation, protein glycosylation, and (Na+ + K+)-ATPase activity reduction induced by the hyperglycemia. This study describes a new biochemical mechanism by which P or PM supplementation may delay or inhibit the development of complications in diabetes.
Free Radic Biol Med. 2001 Feb 1;30(3):232-7
The AGE inhibitor pyridoxamine inhibits lipemia and development of renal and vascular disease in Zucker obese rats.
BACKGROUND: In previous studies, pyridoxamine (PM) limited the formation of advanced glycation end products (AGEs) and development of nephropathy in streptozotocin-diabetic rats without affecting glycemic control. However, the lipid-lowering effects of PM and the correlation of plasma cholesterol and triglycerides with AGEs in skin collagen suggested that lipids might be an important source of AGEs in the diabetic rat. This study addresses the effects of hyperlipidemia on formation of advanced glycation and lipoxidation end products (AGE/ALEs) and the effects of PM on hyperlipidemia, hypertension, AGE/ALE formation, and development of nephropathy in the nondiabetic, Zucker obese rat. METHODS: Three groups of Zucker rats were studied: lean (Fa/fa), untreated fatty (fa/fa), and fa/fa treated with PM (2 g/L drinking water). Blood pressure, plasma lipids and creatinine, and urinary albumin were measured monthly. AGE/ALEs were measured in skin collagen by high-performance liquid chromatography (HPLC) and gas chromatography/mass spectrometry (GC/MS). Changes in wall thickness of the aorta and renal arterioles were evaluated by light microscopy. RESULTS: AGE/ALEs formation was increased two- to threefold in skin collagen of obese versus lean rats. PM inhibited the increases in AGE/ALEs in collagen, and significantly decreased the rise in plasma triglycerides, cholesterol, and creatinine, corrected hypertension and thickening of the vascular wall, and nearly normalized urinary protein and albumin excretion in Zucker fa/fa rats. CONCLUSION: Lipids are an important source of chemical modification of tissue proteins, even in the absence of hyperglycemia. PM inhibited AGE/ALE formation and hyperlipidemia and protected against renal and vascular pathology in a nondiabetic model.
Kidney Int. 2003 Jun;63(6):2123-33
Pyridoxamine lowers kidney crystals in experimental hyperoxaluria: a potential therapy for primary hyperoxaluria.
BACKGROUND: Primary hyperoxaluria is a rare genetic disorder of glyoxylate metabolism that results in overproduction of oxalate. The disease is characterized by severe calcium oxalate nephrolithiasis and nephrocalcinosis, resulting in end-stage renal disease (ESRD) early in life. Most patients eventually require dialysis and kidney transplantation, usually in combination with the replacement of the liver. Reduction of urinary oxalate levels can efficiently decrease calcium oxalate depositions; yet, no treatment is available that targets oxalate biosynthesis. In previous in vitro studies, we demonstrated that pyridoxamine can trap reactive carbonyl compounds, including intermediates of oxalate biosynthesis. METHODS: The effect of PM on urinary oxalate excretion and kidney crystal formation was determined using the ethylene glycol rat model of hyperoxaluria. Animals were given 0.75% to 0.8% ethylene glycol in drinking water to establish and maintain hyperoxaluria. After 2 weeks, pyridoxamine treatment (180 mg/day/kg body weight) started and continued for an additional 2 weeks. Urinary creatinine, glycolate, oxalate, and calcium were measured along with the microscopic analysis of kidney tissues for the presence of calcium oxalate crystals. RESULTS: Pyridoxamine treatment resulted in significantly lower (by approximately 50%) levels of urinary glycolate and oxalate excretion compared to untreated hyperoxaluric animals. This was accompanied by a significant reduction in calcium oxalate crystal formation in papillary and medullary areas of the kidney. CONCLUSION: These results, coupled with favorable toxicity profiles of pyridoxamine in humans, show promise for therapeutic use of pyridoxamine in primary hyperoxaluria and other kidney stone diseases.
Kidney Int. 2005 Jan;67(1):53-60
Renoprotective effects of the AGE-inhibitor pyridoxamine in experimental chronic allograft nephropathy in rats.
BACKGROUND: Advanced glycation end products (AGEs) are involved in diabetic nephropathy (DN). The AGE formation inhibitor pyridoxamine (PM) is renoprotective in DN and in normoglycaemic obese Zucker rats. In chronic allograft nephropathy (CAN), renal AGE accumulation occurs as well. METHODS: To investigate whether inhibition of AGE formation is renoprotective in CAN, we studied the Fisher 344 to Lewis (F-L) allograft rat model of experimental CAN. Fisher to Fisher (F-F) isografts served as controls. Proteinuria, renal function and renal histology of untreated transplanted rats (F-L n = 8, F-F n = 8) were compared to rats receiving PM 2 g/l in drinking water for 20 weeks starting at transplantation (F-L n = 5, F-F n = 10). All rats received cyclosporin A (1.5 mg/kg/day) for 10 days after transplantation to prevent early acute rejection. RESULTS: Compared to untreated allografts, PM significantly decreased proteinuria (76 +/- 18 vs 29 +/- 3 mg/day), serum creatinine (130 +/- 12 vs 98 +/- 5 micromol/l), focal glomerulosclerosis (116 +/- 27 vs 16 +/- 5 AU), glomerular macrophage influx (5.6 +/- 0.6 vs 3.3 +/- 1.0), interstitial fibrosis (132 +/- 24 vs 76 +/- 2 AU) and interstitial macrophage influx (47.0 +/- 8.7 vs 15.4 +/- 5.0. Moreover, PM significantly ameliorated tubular accumulation of pentosidine, compared to untreated allografts (2.5 +/- 0.6 vs 0.3 +/- 0.3, all p < 0.05). In the isograft controls, these values did not differ between untreated and PM treated rats. CONCLUSION: PM exerts renoprotective effects and decreases renal pentosidine accumulation in experimental CAN, suggesting a detrimental role for renal AGE accumulation in the pathogenesis of renal damage in this non-diabetic model. These results indicate that inhibition of AGE formation might be a useful adjunct therapy to attenuate CAN.
Nephrol Dial Transplant. 2008 Feb;23(2):518-24
Inhibition of advanced glycation and absence of galectin-3 prevent blood-retinal barrier dysfunction during short-term diabetes.
Breakdown of the inner blood-retinal barrier (iBRB) occurs early in diabetes and is central to the development of sight-threatening diabetic macular edema (DME) as retinopathy progresses. In the current study, we examined how advanced glycation end products (AGEs) forming early in diabetes could modulate vasopermeability factor expression in the diabetic retina and alter inter-endothelial cell tight junction (TJ) integrity leading to iBRB dysfunction. We also investigated the potential for an AGE inhibitor to prevent this acute pathology and examined a role of the AGE-binding protein galectin-3 (Gal-3) in AGE-mediated cell retinal pathophysiology. Diabetes was induced in C57/BL6 wild-type (WT) mice and in Gal-3(-/-) transgenic mice. Blood glucose was monitored and AGE levels were quantified by ELISA and immunohistochemistry. The diabetic groups were subdivided, and one group was treated with the AGE-inhibitor pyridoxamine (PM) while separate groups of WT and Gal-3(-/-) mice were maintained as nondiabetic controls. iBRB integrity was assessed by Evans blue assay alongside visualisation of TJ protein complexes via occludin-1 immunolocalization in retinal flat mounts. Retinal expression levels of the vasopermeability factor VEGF were quantified using real-time RT-PCR and ELISA. WT diabetic mice showed significant AGE -immunoreactivity in the retinal microvasculature and also showed significant iBRB breakdown (P < .005). These diabetics had higher VEGF mRNA and protein expression in comparison to controls (P < .01). PM-treated diabetics had normal iBRB function and significantly reduced diabetes-mediated VEGF expression. Diabetic retinal vessels showed disrupted TJ integrity when compared to controls, while PM-treated diabetics demonstrated near-normal configuration. Gal-3(-/-) mice showed significantly less diabetes-mediated iBRB dysfunction, junctional disruption, and VEGF expression changes than their WT counterparts. The data suggests an AGE-mediated disruption of iBRB via upregulation of VEGF in the diabetic retina, possibly modulating disruption of TJ integrity, even after acute diabetes. Prevention of AGE formation or genetic deletion of Gal-3 can effectively prevent these acute diabetic retinopathy changes.
Exp Diabetes Res. 2007;2007:51837
The AGE inhibitor pyridoxamine inhibits development of retinopathy in experimental diabetes.
We examined the ability of pyridoxamine (PM), an inhibitor of formation of advanced glycation end products (AGEs) and lipoxidation end products (ALEs), to protect against diabetes-induced retinal vascular lesions. The effects of PM were compared with the antioxidants vitamin E (VE) and R-alpha-lipoic acid (LA) in streptozotocin-induced diabetic rats. Animals were given either PM (1 g/l drinking water), VE (2,000 IU/kg diet), or LA (0.05%/kg diet). After 29 weeks of diabetes, retinas were examined for pathogenic changes, alterations in extracellular matrix (ECM) gene expression, and accumulation of the immunoreactive AGE/ALE N( epsilon )-(carboxymethyl)lysine (CML). Acellular capillaries were increased more than threefold, accompanied by significant upregulation of laminin immunoreactivity in the retinal microvasculature. Diabetes also increased mRNA expression for fibronectin (2-fold), collagen IV (1.6-fold), and laminin beta chain (2.6-fold) in untreated diabetic rats compared with nondiabetic rats. PM treatment protected against capillary drop-out and limited laminin protein upregulation and ECM mRNA expression and the increase in CML in the retinal vasculature. VE and LA failed to protect against retinal capillary closure and had inconsistent effects on diabetes-related upregulation of ECM mRNAs. These results indicate that the AGE/ALE inhibitor PM protected against a range of pathological changes in the diabetic retina and may be useful for treating diabetic retinopathy.
Diabetes. 2002 Sep;51(9):2826-32
Effects of pyridoxamine in combined phase 2 studies of patients with type 1 and type 2 diabetes and overt nephropathy.
BACKGROUND/AIMS: Treatments of diabetic nephropathy (DN) delay the onset of end-stage renal disease. We report the results of safety/tolerability studies in patients with overt nephropathy and type 1/type 2 diabetes treated with pyridoxamine, a broad inhibitor of advanced glycation. METHODS: The two 24-week studies were multicenter Phase 2 trials in patients under standard-of-care. In PYR-206, patients were randomized 1:1 and had baseline serum creatinine (bSCr) <or=2.0 mg/dl. In PYR-205/207, randomization was 2:1 and bSCr was <or=2.0 for PYR-205 and >or=2.0 but <or=3.5 mg/dl for PYR-207. Treated patients (122 active, 90 placebo) received 50 mg pyridoxamine twice daily in PYR-206; PYR-205/207 patients were escalated to 250 mg twice daily. RESULTS: Adverse events were balanced between the groups (p = NS). Slight imbalances, mainly in the PYR-205/207 groups, were noted in deaths (from diverse causes, p = NS) and serious adverse events (p = 0.05) that were attributed to pre-existing conditions. In a merged data set, pyridoxamine significantly reduced the change from baseline in serum creatinine (p < 0.03). In patients similar to the RENAAL/IDNT studies (bSCr >or=1.3 mg/dl, type 2 diabetes), a treatment effect was observed on the rise in serum creatinine (p = 0.007). No differences in urinary albumin excretion were seen. Urinary TGF-beta1 also tended to decrease with pyridoxamine (p = 0.049) as did the CML and CEL AGEs. CONCLUSION: These data provide a foundation for further evaluation of this AGE inhibitor in DN.
Am J Nephrol. 2007;27(6):605-14
Pyridoxamine, an inhibitor of advanced glycation reactions, also inhibits advanced lipoxidation reactions. Mechanism of action of pyridoxamine.
Maillard or browning reactions lead to formation of advanced glycation end products (AGEs) on protein and contribute to the increase in chemical modification of proteins during aging and in diabetes. AGE inhibitors such as aminoguanidine and pyridoxamine (PM) have proven effective in animal model and clinical studies as inhibitors of AGE formation and development of diabetic complications. We report here that PM also inhibits the chemical modification of proteins during lipid peroxidation (lipoxidation) reactions in vitro, and we show that it traps reactive intermediates formed during lipid peroxidation. In reactions of arachidonate with the model protein RNase, PM prevented modification of lysine residues and formation of the advanced lipoxidation end products (ALEs) N(epsilon)-(carboxymethyl)lysine, N(epsilon)-(carboxyethyl)lysine, malondialdehyde-lysine, and 4-hydroxynonenal-lysine. PM also inhibited lysine modification and formation of ALEs during copper-catalyzed oxidation of low density lipoprotein. Hexanoic acid amide and nonanedioic acid monoamide derivatives of PM were identified as major products formed during oxidation of linoleic acid in the presence of PM. We propose a mechanism for formation of these products from the 9- and 13-oxo-decadienoic acid intermediates formed during peroxidation of linoleic acid. PM, as a potent inhibitor of both AGE and ALE formation, may prove useful for limiting the increased chemical modification of tissue proteins and associated pathology in aging and chronic diseases, including both diabetes and atherosclerosis.
J Biol Chem. 2000 Jul 14;275(28):21177-84