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Advanced Glycation End products and benfotiamine

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Age final

  1. 1. Emerging therapy options Protein Glycation and Diabetes Dr. B. K. Iyer
  2. 2. Topics for discussion today <ul><li>The Hypotheses of damage in diabetes </li></ul><ul><li>The biochemical basis of AGE pathway </li></ul><ul><li>The chemistry of glycation </li></ul><ul><li>AGE & diabetes </li></ul><ul><li>Emerging concepts to AGE control </li></ul><ul><li>Benfotiamine </li></ul><ul><li>L carnosine </li></ul><ul><li>The product Offering </li></ul>
  3. 3. The Hypotheses of damage in diabetes
  4. 4. Hyperglycaemic hypothesis <ul><li>DM is associated with 2 types of complication - </li></ul><ul><ul><li>Macrovascular </li></ul></ul><ul><ul><ul><li>Accelerated atherosclerosis </li></ul></ul></ul><ul><ul><li>Microvascular </li></ul></ul><ul><ul><ul><li>Affecting predominantly the eye, nerves and kidneys </li></ul></ul></ul><ul><ul><ul><li>Specific to diabetes </li></ul></ul></ul><ul><ul><ul><li>Although subject to genetic influences, microvascular complications are related to the duration and quality of glucose control. </li></ul></ul></ul>
  5. 5. Hyperglycaemic hypothesis <ul><li>Due to changes in protein structure </li></ul><ul><ul><li>Changes in structure alter function </li></ul></ul><ul><ul><ul><li>Example, lens crystal is rendered opaque and collagen inflexible </li></ul></ul></ul><ul><li>Due to changes in metabolism </li></ul><ul><ul><li>Alterations in cellular metabolism affect cell function </li></ul></ul><ul><ul><ul><li>Example, disturbances in axoplasmic transport affect nerve function </li></ul></ul></ul><ul><li>Due to extracellular matrix structural changes </li></ul><ul><ul><li>Deposition of interstitial materials may affect function </li></ul></ul><ul><ul><ul><li>Example, glomerular function severely affected -> to renal failure . </li></ul></ul></ul><ul><li>Due to cellular proliferation </li></ul><ul><ul><li>Changes in cell signalling may lead to cell division </li></ul></ul><ul><ul><ul><li>Example, proliferation of new blood vessels in the retina lead to blindness </li></ul></ul></ul><ul><ul><ul><li>Changes in vascular permeability lead to production of exudates or haemorrhages in the retina causing blindness </li></ul></ul></ul>
  6. 6. Prevalence of diabetic complications in India Ramachandran A,.et al. Prevalence of vascular complications in type 2 diabetes. J Assoc Physicians India. 1999 Dec;47(12):1152-6. Peripheral neuropathy Retinopathy CHD Nephropathy PVD CVD % of patients diagnosed 27.5 23.7 19.7 11.4 4.0 0.9
  7. 7. Cause of diabetic complications <ul><li>There is an underlying Biochemical basis </li></ul>Why is an understanding of the biochemistry of diabetic complications important? Because it leads to therapeutic opportunities
  8. 8. The 4 hypotheses of diabetic complications* <ul><li>Increased activity of aldose reductase </li></ul><ul><ul><li>(sorbitol pathway) </li></ul></ul><ul><li>Formation of reactive oxygen species </li></ul><ul><ul><li>(‘free-radicals’) </li></ul></ul><ul><li>Activation of protein kinase C </li></ul><ul><ul><li>(PKC) </li></ul></ul><ul><li>Increased production of advanced glycation end-products (AGE) </li></ul>
  9. 9. The link in diabetic complications*
  10. 10. Principles of damage by the different pathways <ul><li>Polyol pathway </li></ul><ul><ul><li>Aldose reductase normally has the function of reducing toxic aldehydes in the cell to inactive alcohols; </li></ul></ul><ul><ul><li>During hyperglycaemia, in the process of reducing high intracellular glucose to Sorbitol, the Aldose reductase consumes the cofactor NADPH, which in turn reduces the amount of glutathione (endogenous antioxidant). </li></ul></ul><ul><ul><li>This in turn increases susceptibility to intracellular oxidative stress. </li></ul></ul>
  11. 11. Principles of damage by the different pathways <ul><li>Hexosamine pathway </li></ul><ul><ul><li>Intracellularly, most glucose is metabolized through glycolysis, going first to Glu-6 P, then Fruc-6 P, and then on through the rest of the glycolytic pathway. </li></ul></ul><ul><ul><li>However, some of that Fru-6-phosphate can get diverted into a signaling pathway [hexosamine] in which an enzyme called GFAT converts the Fruc-6 P to glucosamine-6 P and finally to UDP (uridine diphosphate) & N -acetyl glucosamine. </li></ul></ul><ul><ul><li>Activation of the hexosamine pathway causes deterioration of cell function by inducing oxidative stress, implying that this mechanism could be responsible for at least some of the cell glucose toxicity found in diabetes. </li></ul></ul>
  12. 12. Principles of damage by the different pathways <ul><li>Protein Kinase C pathway </li></ul><ul><ul><li>Hyperglycemia inside the cell increases the synthesis of a molecule called diacylglycerol, which is a critical activating cofactor for the classic isoforms of protein kinase-C, -β, -δ, and –α. </li></ul></ul><ul><ul><li>PKC activation leads to variety of effects on gene expression which in turn leads to tissue abnormalities </li></ul></ul>
  13. 13. General Introduction to The “A.G.E. Pathway”
  14. 14. What is AGE and how do they form? <ul><li>Sugars combine with proteins in several ways, both, outside the body in our food and inside our bodies through non-enzymatic processes. This process is called Glycation and these glycated protein products are labile and called Advance Glycation End products (AGE) </li></ul><ul><li>Understanding and tackling glycation is the single most important approach to controlling the complications of diabetes. </li></ul><ul><li>The big question - What in the world is glycation and what does glycation have to do with diabetes ? </li></ul>
  15. 15. So, what is Glycation? <ul><li>Glycation is the non-enzymatic joining of a sugar compound with a protein or amino acids. </li></ul><ul><ul><li>In contrast, Glycosylation is the enzymatic joining of a sugar with a protein or amino acids </li></ul></ul><ul><li>Glycation is a pathological process. </li></ul><ul><ul><li>Glycosylation is a physiological process. </li></ul></ul><ul><li>The end result of the process of glycation is the formation of AGE products that crosslink with other proteins and lipids and inactivate them, or make them essentially nonfunctional. </li></ul>
  16. 16. What is Glycation associated with? <ul><li>It is a process that is at the very root of most of the disorders and its end products AGE are associated with complications and degenerative diseases. </li></ul><ul><ul><li>Hyperglycemia & Diabetes mellitus </li></ul></ul><ul><ul><li>Protein Kinase C activation </li></ul></ul><ul><ul><li>Renal disorders </li></ul></ul><ul><ul><li>Collagen disorders </li></ul></ul><ul><ul><li>Age Receptor disorders </li></ul></ul><ul><ul><li>Atherosclerosis </li></ul></ul>
  17. 17. Principle of hyperglycaemic ‘memory’ <ul><li>Experimentally, the damage done to tissue during periods of hyperglycaemia is ‘remembered’. </li></ul><ul><li>Thus, after a period of poor diabetic control complications occur at an accelerated rate even though subsequent glycaemic control is excellent. </li></ul>
  18. 18. The chemistry of glycation in The “A.G.E. Pathway”
  19. 19. AGE Formation
  20. 20. AGE Formation Amino Acids Sugar Lipid Schiff Base Amadori Product (e.g. HbAlc) Serum and Tissue AGEs Dicarbonyl Intermediates + Polyol Pathway [ox] [ox] glu glu [ox]
  21. 21. Advanced Glycation End Products (AGE) <ul><li>After the bad news, the good news! </li></ul>
  22. 22. Advanced Glycation End Products (AGE) <ul><li>AGE formation & their proposed role in diabetes </li></ul>Sheetz MJ, King GL, JAMA 2002: 288; 2579=2588
  23. 23. AGEs in Subjects with & Without Diabetes Beiswenger,PJ et al.Diabetes, 44:824-29,1995 Skin AGEs (Units / mg Collagen
  24. 24. Sources of AGE <ul><li>Diet is the single greatest source of AGE products. </li></ul><ul><ul><li>It is not so much what we eat, but how it is prepared. </li></ul></ul><ul><ul><li>Foods higher in fat and protein, such as meat and cheese, will give higher AGE levels. </li></ul></ul><ul><ul><li>And, in general, cooking at a higher temperature creates higher levels of AGEs. </li></ul></ul><ul><li>Cigarette smoking is # 2. </li></ul><ul><li>Internal glycation is # 3 {Thus avoid high sugar levels} </li></ul>
  25. 25. Effects of AGE <ul><li>When there is increased AGE receptor formation, proteins modified by advanced glycation endproducts (AGE-proteins) act as photosensitizers of DNA damage. </li></ul><ul><ul><li>This leads to damaged microcirculation. </li></ul></ul><ul><li>AGE forms a glue like substance that makes the blood vessels inelastic and stenotic. </li></ul><ul><li>AGE promotes inflammation. </li></ul><ul><li>AGE affects fats, DNA, and other biological materials. </li></ul><ul><li>AGEs act on RAGE …. </li></ul>
  26. 26. What is RAGE and why are they harmful? <ul><li>RAGE = Receptor Advanced Glycation End Products. </li></ul><ul><li>They are receptors on cells that send wrong signals or messages to the DNA within </li></ul><ul><li>RAGEs are TLRs which are cellular pattern-recognition receptors that can identify molecular patterns of pathogens. </li></ul><ul><li>TRL= TOLL like receptor </li></ul><ul><li>When engaged, the cytosolic part of the TLR activates a transcription factor NF-kb which leads to the cell expressing inflammatory cytokines . </li></ul><ul><li>The net result is an inflammatory response . </li></ul><ul><ul><li>These inflammatory reactions have been implicated in complications of diabetes as well as CVD, neurodisorders, cancer, etc. </li></ul></ul>
  27. 27. How does AGE affects cells? <ul><li>High AGE containing foods + Mallaird reaction </li></ul>NFkb DNA RAGE Acts on DNA AGEs Signal Transmitted Multiple bad products AGE Products Body Cell RAGE Inflammation Cytokines
  28. 28. How do the RAGEs induce Inflammation?  IL-1  TNF   TGF β  NF κβ  eNOS <ul><li>Pathologies: </li></ul><ul><li>Vascular Stiffening </li></ul><ul><li>Chronic Heart Failure </li></ul><ul><li>Nephropathy, </li></ul><ul><li>Cataract, Retinopathy </li></ul>Source: Diabetes, Brownlee, Vol. 54, June 2005 Intracellular protein glycation AGE precursors Glucose Matrix Intracellular transducers Transcription factors Glucose DNA Transcription AGE receptor AGE plasma proteins AGE receptor ROS NF-  β Macrophage mesangial cell mRNA Proteins Integrins Endothelial cell RNA
  29. 29. Understanding “ A.G.E. role in diabetes”
  30. 30. AGE Consequences in Diabetics <ul><li>The chemical modifications of glycation and crosslinking initiates harmful inflammatory and autoimmune responses. </li></ul><ul><ul><li>Glycation has been found in connective tissue collagen, arterial collagen, kidney glomerular basement membrane, eye lens crystallins, nerve myelin proteins and in the circulating low-density lipoprotein (LDL) of the blood. </li></ul></ul><ul><ul><li>Crosslinking reduces the flexibility, elasticity and functionality of the proteins. </li></ul></ul>
  31. 31. Outcomes of AGE in Diabetics <ul><li>Thus, AGE effects various cells in the body including: </li></ul><ul><ul><li>Capillary endothelial cells in the retina </li></ul></ul><ul><ul><li>Mesangial cells in the renal glomerulus </li></ul></ul><ul><ul><li>Schwann cells in the peripheral nerves </li></ul></ul><ul><ul><li>Vascular endothelium </li></ul></ul>
  32. 32. Inhibition of AGE in Diabetics <ul><li>Benefits of AGE inhibtion include effects on various cells in the body such as: </li></ul><ul><ul><li>Normalization of endothelial cell proliferation & restoration of retinal neurons </li></ul></ul><ul><ul><li>Inhibition of basement membrane thickening & prevention of albuminuria </li></ul></ul><ul><ul><li>Reduction of vaso dilatory impairment and preservation of vascular elasticity </li></ul></ul>
  33. 33. Emerging approach to AGE control
  34. 34. Basis of therapy <ul><li>In people with diabetes, all cells are bathed in blood that contains elevated levels of glucose. </li></ul><ul><li>Most cells still manage to keep their internal glucose at normal levels. </li></ul><ul><li>But certain cells - particularly endothelial cells that line arteries and the capillaries of the retina and kidney - are unable to regulate glucose and instead develop high internal levels of the sugar, which they can’t completely metabolize. </li></ul><ul><li>As a result, glucose-derived &quot;intermediate&quot; metabolic products accumulate inside these cells, where they activate pathways of cellular damage that can eventually lead to blindness and other complications. </li></ul>
  35. 35. Current therapies for diabetes complications
  36. 36. Novel Therapeutic Approaches <ul><li>To activate the transketolases for tacking the polyol pathway </li></ul><ul><li>To counter the oxidative stress emerging out of the hexosamine and PKC pathways; </li></ul><ul><li>To prevent the formation of AGE and to limit the damages to the end organs due to AGE, already produced. </li></ul>
  37. 37. AGE inhibitors in diabetes
  38. 38. Novel Therapeutic Approaches Transketolase Activators PARP Inhibitors Catalytic Antioxidants
  39. 39. Transketolase activators Transketolase Activators PARP Inhibitors Catalytic Antioxidants <ul><li>The first new class of potential therapeutic agents. </li></ul><ul><ul><li>By activating Transketolase enzyme, we could decrease the concentration of glycolytic metabolites and thus divert their flux away from 3 of the damaging pathways normally activated by hyperglycemia. </li></ul></ul><ul><li>The big question </li></ul><ul><ul><li>What are transketolases and what is their role? </li></ul></ul>
  40. 40. Transketolase activators Transketolase Activators PARP Inhibitors Catalytic Antioxidants <ul><li>By activating Transketolase enzyme, we could decrease the concentration of glycolytic metabolites and thus divert their flux away from 3 of the damaging pathways normally activated by hyperglycemia. </li></ul><ul><li>But how could we activate Transketolase? </li></ul><ul><ul><li>Since Transketolase enzyme requires the vitamin thiamine as a cofactor, the thiamine derivative called Benfotiamine is known to activate Transketolase by 250% in arterial endothelial cells. </li></ul></ul><ul><li>Benfotiamine completely prevents hexosamine pathway activation. </li></ul>
  41. 41. Novel Therapeutic Approaches Transketolase Activators PARP Inhibitors Catalytic Antioxidants <ul><li>PARP inhibitor prevents hyperglycemia-induced activation of PKC, NFκB, intracellular AGE formation, and the hexosamine pathway. </li></ul><ul><li>Example of PARP inhibitor is </li></ul>
  42. 42. Novel Therapeutic Approaches Transketolase Activators PARP Inhibitors Catalytic Antioxidants <ul><li>Therapeutic correction of diabetes-induced superoxide production may be done by a powerful new approach for preventing diabetic complications </li></ul>
  43. 43. AGE inhibitors in diabetes
  44. 44. Benfotiamine, Carnosine & Pyridoxine in AGE Protein Glucose Schiff’s base Amadori products AGE + Adapted from Khalilah et al.1999 Benfotiamine Pyridoxine Carnosine Carnosine Benfotiamine AGE breakers after they are formed Carnosine Benfotiamine Pyridoxine
  45. 45. Studies with Benfotiamine in Humans
  46. 46. Benfotiamine <ul><li>Benfotiamine (S-benzyolthiamine-O-monophosphate) is a lipid soluble, biologically active form of thiamin (vit. B-1). </li></ul>
  47. 47. Benfotiamine <ul><li>Benfotiamine raises the levels of thiamine pyrophosphate (TPP) which is the co-enzymated form of thiamine and stimulates the enzyme Transkotelase </li></ul><ul><li>The enzyme, transketolase, like many enzymes, depends on a cofactor for its activity which happens to be thiamine and is responsible for maintenance of normal glucose metabolic pathways. </li></ul>
  48. 48. Benfotiamine instead of thiamine – why? <ul><li>Thiamine activates transketolase at level around 20%, which isn’t enough to stop the glucose-derived compounds accumulating in healthy cells. </li></ul><ul><li>In this regard, it is worthwhile to note </li></ul><ul><ul><li>Thiamine bioavailability from Benfotiamine is 3.6 times higher than other thiamine supplements. </li></ul></ul><ul><ul><li>Benfotiamine increases the levels of transketolase by 300%. </li></ul></ul><ul><ul><li>Benfotiamine prevents nerve and blood-vessel damage caused by a build-up of sugar in the tissues. </li></ul></ul><ul><ul><li>Benfotiamine thus helps to maintain integrity of cells and controls formation of Advanced Glycation End products (AGEs). </li></ul></ul>
  49. 49. Benfotiamine – mechanism of action <ul><li>Benfotiamine exerts it beneficial effects via a number of mechanisms impacting all pathways of potential damage. </li></ul><ul><ul><li>↑ transketolase activity, and as a result, blocks 3 of the major molecular pathways leading to hyperglycemic damage. </li></ul></ul><ul><ul><li>Prevents the increase in UDP-N-acetylglucosamine (UDP-GlcNAc) and enhances hexosamine pathway activity decreasing the buildup of detrimental glucose metabolites that can lead to advanced glycation end products (AGE). </li></ul></ul><ul><ul><li>Normalizes protein kinase (PKC) activity and prevents nuclear factor-kappaB (NF-κB) activation in the retina of diabetics. </li></ul></ul><ul><ul><li>Corrects imbalances in the polyol pathway by decreasing aldose reductase activity, sorbitol concentrations, and intracellular glucose, thereby protecting endothelial cells from glucose-induced damage. </li></ul></ul>
  50. 50. Benfotiamine – transketolase activity <ul><li>Fructose-6-phosphate & glyceraldehyde-3-phosphate) are 2 glucose-derived intermediates are the end products of a biochemical pathway mediated by the enzyme - transketolase. </li></ul><ul><ul><li>They activate 3 of the damaging biochemical pathways in diabetes </li></ul></ul><ul><ul><li>These 2 damage-triggering glucose metabolites can be essentially converted into harmless chemicals by boosting transketolase’s activity, thereby preventing all 3 damaging biochemical pathways from being activated. </li></ul></ul><ul><ul><li>The activation of the enzyme transketolase, turns these triose phosphates which are glucose-derived compounds into chemicals that are harmless. </li></ul></ul>
  51. 51. Benfotiamine – PPP
  52. 52. Benfotiamine – transketolase activity <ul><li>TDP acts as a coenzyme for transketolase </li></ul><ul><ul><li>(A) in the glycolytic pathway, and for the </li></ul></ul><ul><ul><li>(B) pyruvate dehydrogenase and </li></ul></ul><ul><ul><li>(C) a-ketoglutarate dehydrogenase complexes in the Krebs cycle, </li></ul></ul><ul><li>Transketolase also shifts excess fructose-6-phosphate & glycerhaldeyde-3-phosphate from glycolysis into the pentose-phosphate shunt, thus eliminating from he excess of these damaging metabolites. </li></ul>
  53. 53. Studies on AGE levels with benfotiamine <ul><li>The levels of Intracellular advanced glycation end product (AGE) were reduced by benfotiamine - </li></ul><ul><ul><li>Ne-(carboxymethyl)lysine (CML) by 40% </li></ul></ul><ul><ul><li>methylglyoxal-derived AGE by 70 % </li></ul></ul>
  54. 54. Studies on diabetic polyneuropathy <ul><li>No. of patients, n= 40, Randomized control study </li></ul><ul><li>History of Polyneuropathy not longer than 2 yrs </li></ul><ul><li>Duration of study =3 weeks. </li></ul><ul><li>Efficacy Parameters: </li></ul><ul><ul><li>Neuropathy score (Katzenwadel et al,1987) </li></ul></ul><ul><ul><li>Vibration perception threshold </li></ul></ul><ul><ul><li>Physician and patient assesment. </li></ul></ul>
  55. 55. Studies on diabetic polyneuropathy <ul><li>Results </li></ul><ul><ul><li>Significant improvement in neuropathy score with Benfotiamine. </li></ul></ul><ul><ul><li>Significant pain relief. </li></ul></ul><ul><ul><li>No change in Vibration perception threshold - Short study </li></ul></ul>
  56. 56. Studies on diabetic polyneuropathy <ul><li>Assessment of efficacy by physician </li></ul>0 8 14 Benfotiamine Placebo Improved Unchanged Patients (n) Worsened Haupt E,et al. Int J Clin Pharmacol Ther,43(2),71-77,2005
  57. 57. Studies on painful diabetic neuropathy <ul><li>Assessment of pain and vibration with benfotiamine 150 mg </li></ul>0 6.5 13 Week 0 Week 6 Vibration sensation Pain sensation Patients (n) 4.9* 5.9* 12.8 7.2 Arzneim-Forsch, 49(I),Nr 3(1999); 220-224 * Due to short duration of study
  58. 58. Studies on painful diabetic neuropathy <ul><li>Significant improvement in in vibratory sensitivities and conduction rate through motor nerves with benfotiamine 100 mg. tid. </li></ul>Week 0 Week 6 Before After Pain intensity – VAS 8.2 2.3 Arzneim-Forsch, 49(I),Nr 3(1999); 220-224 1 2 3 4 5 6 7 8 9 10
  59. 59. Studies on painful diabetic neuropathy <ul><li>Significant improvement with benfotiamine 100 mg + pyridoxine in 93% of the cases. </li></ul>Week 0 Week 6 Before After Pain intensity – VAS 8.2 2.3 Zh Nevrol Psikhiatr Im S S Korsakova. 1998;98(9):30-2 1 2 3 4 5 6 7 8 9 10
  60. 60. Benfotiamine + cyanocobalamine Vs Conventional Vitamin B Complex Regimen <ul><li>in Painful Diabetic Neuropathy </li></ul><ul><ul><li>Significant relief of pain was achieved in all Benfotiamine treated patients. </li></ul></ul><ul><ul><li>Vibration perception threshold improved. </li></ul></ul><ul><ul><li>No statistical significance Changes in the B complex regimen. </li></ul></ul>Folia Med (Plovdiv). 1997;39(4):5-10.
  61. 61. Benfotiamine - indications <ul><li>Benfotiamine </li></ul><ul><ul><li>counteracts glucose toxicity blocks damage by hyperglycemia and thus may help to prevent diabetic complications </li></ul></ul><ul><ul><li>usage is a new approach to preventing diabetic retinopathy </li></ul></ul><ul><ul><li>[blocks formation] inhibits / delays formation of advanced glycosylation end-products </li></ul></ul><ul><ul><li>is effective in diabetic neuropathy </li></ul></ul><ul><ul><li>is also indicated in end-stage renal disease </li></ul></ul>
  62. 62. Studies with L-Carnosine in Humans
  63. 63. L- Carnosine <ul><li>Carnosine’s structure is supposed to resemble glycation sites on proteins, there by accepting the damaging Advanced Glycation End products and disposing them off to be totally harmless. </li></ul><ul><ul><li>L-Carnosine is thus a potent & selective scavenger of alpha, beta-unsaturated aldehydes </li></ul></ul><ul><ul><ul><li>typical by-products of membrane lipids peroxidation and considered second messengers of the oxidative stress </li></ul></ul></ul><ul><ul><li>L-carnosine also inhibits aldehyde-induced protein-protein and DNA-protein cross-linking. </li></ul></ul>
  64. 64. L- Carnosine – mechanism of action <ul><li>Provides antioxidant activity </li></ul><ul><li>Inhibits glycation reaction by, </li></ul><ul><ul><li>quenching AGEs </li></ul></ul><ul><ul><li>quenching carbonyl compounds </li></ul></ul><ul><li>Prevents macromolecules cross-linking </li></ul><ul><li>Promotes modified protein degradation mediated by enzymes </li></ul>
  65. 65. L- Carnosine – dosage adherence <ul><li>Carnosine  is beta-alanyl-L-hystidine and so may convert into histidine which can convert into histamine. </li></ul><ul><li>A high dose of carnosine can shift the metabolic pathway towards histamine and so more than 100 mg of carnosine is not needed on a regular basis. </li></ul>
  66. 66. The product benefits
  67. 67. The product benefits
  68. 68. Thank you