Extended Bibliography

Ahmed, N; Karachalias, N; Babaei-Jadidi, R; Thornalley, PJ.. 2005 . Degradation products of proteins damaged by glycation and oxidation increase markedly in experimental diabetes and are suppressed by high dose therapy with thiamine and benfotiamine. Diabetologia. 48(Suppl 1): A417-8.

Armett CJ, & Cooper JR. 1965 . Effect of thiamine analogs on electrical activity of rabbit vagus nerve. Experientia. 21(10): 605-7.
Babaei-Jadidi R, Karachalias N, Ahmed N, Battah S, & Thornalley PJ. 2003 . Prevention of incipient diabetic nephropathy by high-dose thiamine and benfotiamine. Diabetes. 52(8): 2110-20. Accumulation of triosephosphates arising from high cytosolic glucose concentrations in hyperglycemia is the trigger for biochemical dysfunction leading to the development of diabetic nephropathy-a common complication of diabetes associated with a high risk of cardiovascular disease and mortality. Here we report that stimulation of the reductive pentosephosphate pathway by high-dose therapy with thiamine and the thiamine monophosphate derivative benfotiamine countered the accumulation of triosephosphates in experimental diabetes and inhibited the development of incipient nephropathy. High-dose thiamine and benfotiamine therapy increased transketolase expression in renal glomeruli, increased the conversion of triosephosphates to ribose-5-phosphate, and strongly inhibited the development of microalbuminuria. This was associated with decreased activation of protein kinase C and decreased protein glycation and oxidative stress-three major pathways of biochemical dysfunction in hyperglycemia. Benfotiamine also inhibited diabetes-induced hyperfiltration. This was achieved without change in elevated plasma glucose concentration and glycated hemoglobin in the diabetic state. High-dose thiamine and benfotiamine therapy is a potential novel strategy for the prevention of clinical diabetic nephropathy.

Babaei-Jadidi, R; Karachalias, N; Ahmed, N; Thornalley, PJ.. 2005 . Prevention of incipient nephropathy by high dose thiamine and benfotiamine in streptozotocin-induced diabetic rats: Effect on markers of oxidative stress. Free Radic Res. 39: S81.

Babaei-Jadidi R, Karachalias N, Kupich C, Ahmed N, & Thornalley PJ. 2004 . High-dose thiamine therapy counters dyslipidaemia in streptozotocin-induced diabetic rats. Diabetologia. 47(12): 2235-46. AIMS/HYPOTHESIS: Cardiovascular disease in diabetes is linked to increased risk of atherosclerosis, increased levels of triglyceride-rich lipoproteins and enhanced hepatic lipogenesis. The hepatic hexosamine pathway has been implicated in signalling for de novo lipogenesis by the liver. In this study, we assessed if decrease of flux through the hexosamine pathway induced by high-dose thiamine therapy counters diabetic dyslipidaemia. METHODS: The model of diabetes used was the streptozotocin-induced diabetic rat with maintenance insulin therapy. Normal control and diabetic rats were studied for 24 weeks with and without oral high-dose therapy (7 and 70 mg/kg) with thiamine and benfotiamine. Plasma total cholesterol, HDL cholesterol and triglycerides were determined at 6-week intervals and hepatic metabolites and transketolase activity after death of the rats at 24 weeks. RESULTS: We found that thiamine therapy (70 mg/kg) prevented diabetes-induced increases in plasma cholesterol and triglycerides in diabetic rats but did not reverse the diabetes-induced decrease of HDL. This was achieved by prevention of thiamine depletion and decreased transketolase activity in the liver of diabetic rats. There was a concomitant decrease in hepatic UDP-N-acetylglucosamine and fatty acid synthase activity. Thiamine also normalised food intake of diabetic rats. A lower dose of thiamine (7 mg/kg) and the thiamine monophosphate prodrug benfotiamine (7 and 70 mg/kg) were ineffective. CONCLUSIONS/INTERPRETATION: High-dose thiamine therapy prevented diabetic dyslipidaemia in experimental diabetes probably by suppression of food intake and hexosamine pathway signalling but other factors may also be involved. Benfotiamine was ineffective.

Beltramo, E; Berrone, E; Buttiglieri, S; Porta, M.. 2003 . Thiamine and benfotiamine are able to correct increased apoptosis and advanced glycation-end products formation in endothelial cells and pericytes cultured under high glucose conditions.. Diabetologia. 46 (Suppl 2): A13.

Beltramo E, Berrone E, Buttiglieri S, & Porta M. 2004 . Thiamine and benfotiamine prevent increased apoptosis in endothelial cells and pericytes cultured in high glucose. Diabetes Metab Res Rev. 20(4): 330-6. BACKGROUND: High glucose induces pathological alterations in small and large vessels, possibly through increased formation of AGE, activation of aldose reductase and protein kinase C, and increased flux through the hexosamine pathway. We showed previously that thiamine and benfotiamine correct delayed replication and increase lactate production in endothelial cells subjected to high glucose. We now aim at verifying the effects of thiamine and benfotiamine on cell cycle, apoptosis, and expression of adhesion molecules in endothelial cells and pericytes, under high ambient glucose. METHODS: Human umbilical vein endothelial cells and bovine retinal pericytes were cultured in normal (5.6 mmol/L) or high (28 mmol/L) glucose, with or without thiamine or benfotiamine, 50 or 100 micro mol/L. Apoptosis was determined by two separate ELISA methods, measuring DNA fragmentation and caspase-3 activity, respectively. Cell cycleand integrin subunits alpha3, alpha5, and beta1 concentration were measured by flow cytometry. RESULTS: Apoptosis was increased in high glucose after 3 days of culture, both in endothelium and pericytes. Thiamine and benfotiamine reversed such effects. Neither cell cycle traversal nor integrin concentrations were modified in these experimental conditions. CONCLUSIONS: Thiamine and benfotiamine correct increased apoptosis due to high glucose in cultured vascular cells. Further elucidations of the mechanisms through which they work could help set the basis for clinical use of this vitamin in the prevention and/or treatment of diabetic microangiopathy.

Berrone E, Beltramo E, Solimine C, Ape AU, & Porta M. 2006 . Regulation of intracellular glucose and polyol pathway by thiamine and benfotiamine in vascular cells cultured in high glucose. J Biol Chem. 281(14): 9307-13. Hyperglycemia is a causal factor in the development of the vascular complications of diabetes. One of the biochemical mechanisms activated by excess glucose is the polyol pathway, the key enzyme of which, aldose reductase, transforms d-glucose into d-sorbitol, leading to imbalances of intracellular homeostasis. We aimed at verifying the effects of thiamine and benfotiamine on the polyol pathway, transketolase activity, and intracellular glucose in endothelial cells and pericytes under high ambient glucose. Human umbilical vein endothelial cells and bovine retinal pericytes were cultured in normal (5.6 mmol/liter) or high (28 mmol/liter) glucose, with or without thiamine or benfotiamine 50 or 100 mumol/liter. Transketolase and aldose reductase mRNA expression was determined by reverse transcription-PCR, and their activity was measured spectrophotometrically; sorbitol concentrations were quantified by gas chromatography-mass spectrometry and intracellular glucose concentrations by fluorescent enzyme-linked immunosorbent assay method. Thiamine and benfotiamine reduce aldose reductase mRNA expression, activity, sorbitol concentrations, and intracellular glucose while increasing the expression and activity of transketolase, for which it is a coenzyme, in human endothelial cells and bovine retinal pericytes cultured in high glucose. Thiamine and benfotiamine correct polyol pathway activation induced by high glucose in vascular cells. Activation of transketolase may shift excess glycolytic metabolites into the pentose phosphate cycle, accelerate the glycolytic flux, and reduce intracellular free glucose, thereby preventing its conversion to sorbitol. This effect on the polyol pathway, together with other beneficial effects reported for thiamine in high glucose, could justify testing thiamine as a potential approach to the prevention and/or treatment of diabetic complications.

Berrone E, Beltramo E, Solimine C, Ubertalli A, & Porta M. 2004 . Thiamine and benfotiamine inhibit the polyol pathway and increase transketolase mRNA in endothelial cells and retinal pericytes cultured in high glucose. Diabetologia. 47: A382-A383.
Bettendorff L, & Kolb HA. 1993 . Thiamine derivatives as modulators of anion channels in neuroblastoma cells. J Neurochem. 61: S214.

Bitsch R, Wolf M, Moller J, Heuzeroth L, & Gruneklee D. 1991 . Bioavailability assessment of the lipophilic benfotiamine as compared to a water-soluble thiamin derivative. Ann Nutr Metab. 35(5): 292-6. The bioequivalence of thiamin in 2 therapeutically used preparations was tested in 10 healthy young men. Thiamin was orally administered either as lipophilic benfotiamine or as water-soluble thiamin mononitrate. Biokinetic data, measured as area under the curve and maximal concentration in plasma and hemolysate after ingestion, demonstrated a significantly improved bioavailability from the lipophilic derivative despite an ingested dose of only 40% as compared with the water-soluble salt. A superior cellular efficacy of benfotiamine was also concluded from the short-term stimulation of the thiamin-dependent transketolase activity in erythrocytes.

Bontemps J, Bettendorff L, Schoffeniels E, Schmartz D, Dandrifosse G, Nevejans F, Yang Y, & Verzele M. 1985 . Recent advances in the analysis of thiamine derivatives by high-performance liquid-chromatography. Arch Int Physiol Biochim Biophys. 93(1): B4-B5.

Booth, AA; Khalifah, RG; Todd, P; Hudson, BG.. 1997 . In vitro kinetic studies of formation of antigenic advanced glycation end products (AGEs) - Novel inhibition of post-Amadori glycation pathways. J Biol Chem. 272(9): 5430-5437. Nonenzymatic protein glycation (Maillard reaction) leads to heterogeneous, toxic, and antigenic advanced glycation end products (''AGEs'') and reactive precursors that have been implicated in the pathogenesis of diabetes, Alzheimer's disease, and normal aging, In vitro inhibition studies of AGE formation in the presence of high sugar concentrations are difficult to interpret, since AGE-forming intermediates may oxidatively arise from free sugar or from Schiff base condensation products with protein amino groups, rather than from just their classical Amadori rearrangement products. We recently succeeded in isolating an Amadori intermediate in the reaction of ribonuclease A (RNase) with ribose (Khalifah, R. G., Todd, P., Booth, A. A., Yang, S. X., Mott, J. D., and Hudson, B. G. (1996) Biochemistry 35, 4645-4654) for rapid studies of post-Amadori AGE formation in absence of free sugar or reversibly formed Schiff base precursors to Amadori products. This provides a new strategy for a better understanding of the mechanism of AGE inhibition by established inhibitors, such as aminoguanidine, and for searching for novel inhibitors specifically acting on post-Amadori pathways of AGE formation, Aminoguanidine shows little inhibition of post-Amadori AGE formation in RNase and bovine serum albumin, in contrast to its apparently effective inhibition of initial (although not late) stages of glycation in the presence of high concentrations of sugar, Of several derivatives of vitamins B-1 and B-6 recently studied for possible AGE inhibition in the presence of glucose (Booth, A. A., Khalifah, R. G., and Hudson, B. G. (1996) Biochem. Biophys. Res. Commun. 220, 113-119), pyridoxamine and, to a lesser extent, thiamine pyrophosphate proved to be novel and effective post-Amadori inhibitors that decrease the final levels of AGEs formed, Our mechanism-based approach to the study of AGE inhibition appears promising for the design and discovery of novel post-Amadori AGE inhibitors of therapeutic potential that may complement others, such as aminoguanidine, known to either prevent initial sugar attachment or to scavenge highly reactive dicarbonyl intermediates.

Cameron NE, Gibson TM, Nangle MR, & Cotter MA. 2005 . Inhibitors of advanced glycation end product formation and neurovascular dysfunction in experimental diabetes. Ann N Y Acad Sci. 1043: 784-92. Advanced glycation and lipoxidation end products (AGEs/ALEs) have been implicated in the pathogenesis of the major microvascular complications of diabetes mellitus: nephropathy, neuropathy, and retinopathy. This article reviews the evidence regarding the peripheral nerve and its vascular supply. Most investigations done to assess the role of AGEs/ALEs in animal models of diabetic neuropathy have used aminoguanidine as a prototypic inhibitor. Preventive or intervention experiments have shown treatment benefits for motor and sensory nerve conduction velocity, autonomic nitrergic neurotransmission, nerve morphometry, and nerve blood flow. The latter depends on improvements in nitric oxide-mediated endothelium-dependent vasodilation and is responsible for conduction velocity improvements. A mechanistic interpretation of aminoguanidine's action in terms of AGE/ALE inhibition is made problematic by the relative lack of specificity. However, other unrelated compounds, such as pyridoxamine and pyridoxamine analogues, have recently been shown to have beneficial effects similar to aminoguanidine, as well as to improve pain-related measures of thermal hyperalgesia and tactile allodynia. These data also stress the importance of redox metal ion-catalyzed AGE/ALE formation. A further approach is to decrease substrate availability by reducing the elevated levels of hexose and triose phosphates found in diabetes. Benfotiamine is a transketolase activator that directs these substrates to the pentose phosphate pathway, thus reducing tissue AGEs. A similar spectrum of improvements in nerve and vascular function were noted when using benfotiamine in diabetic rats. Taken together, the data provide strong support for an important role for AGEs/ALEs in the etiology of diabetic neuropathy.

Cameron NE, Nangle MR, Gibson TM, & Cotter MA. 2004 . Effects of benfotiamine on vascular endothelium and nerve function and perfusion in diabetic rats. Diabetologia. 47: A367.

Cameron NE, Nangle MR, Gibson TM, & Cotter MA. 2004 . Benfotiamine treatment improves vascular endothelium and nerve function in diabetic rats. Diabetes. 53: A35.

Ceylan-Isik AF, Wu S, Li Q, Li SY, & Ren J. 2006 . High-dose benfotiamine rescues cardiomyocyte contractile dysfunction in streptozotocin-induced diabetes mellitus. J Appl Physiol. 100(1): 150-6. Diabetic cardiomyopathy is characterized by cardiac dysfunction. This study was designed to examine the effect of benfotiamine, a lipophilic derivative of thiamine, on streptozotocin (STZ)-induced cardiac contractile dysfunction in mouse cardiomyocytes. Adult male FVB mice were made diabetic with a single injection of STZ (200 mg/kg ip). Fourteen days later, control and diabetic (fasting plasma glucose > 13.9 mM) mice were put on benfotiamine therapy (100 mg.kg(-1).day(-1) ip) for another 14 days. Mechanical and intracellular Ca2+ properties were evaluated in left ventricular myocytes using an IonOptix MyoCam system. The following indexes were evaluated: peak shortening (PS), time to PS (TPS), time to 90% relengthening (TR90), maximal velocity of shortening/relengthening, resting and rise of intracellular Ca2+ in response to electrical stimulus, sarcoplasmic reticulum (SR) Ca2+ load, and intracellular Ca2+ decay rate (tau). Two- or four-week STZ treatment led to hyperglycemia, prolonged TPS and TR90, reduced SR Ca2+ load, elevated resting intracellular Ca2+ level and prolonged tau associated with normal PS, maximal velocity of shortening/relengthening, and intracellular Ca2+ rise in response to electrical stimulus. Benfotiamine treatment abolished prolongation in TPS, TR90, and tau, as well as reduction in SR Ca2+ load without affecting hyperglycemia and elevated resting intracellular Ca2+. Diabetes triggered oxidative stress, measured by GSH-to-GSSG ratio and formation of advanced glycation end product (AGE) in the hearts. Benfotiamine treatment alleviated oxidative stress without affecting AGE or protein carbonyl formation. Collectively, our results indicated that benfotiamine may rescue STZ-induced cardiomyocyte dysfunction but not AGE formation in short-term diabetes.

Di Rocco M, Patrini C, Rimini A, & Rindi G. 1997 . A 6-month-old girl with cardiomyopathy who nearly died. Lancet. 349(9052): 
616.
Edwards TE, & Ferre-D'Amare AR. 2006 . Crystal structures of the thi-box riboswitch bound to thiamine pyrophosphate analogs reveal adaptive RNA-small molecule recognition. Structure. 14(9): 1459-68. Riboswitches are noncoding mRNA elements that bind small-molecule metabolites with high affinity and specificity, and they regulate the expression of associated genes. The thi-box riboswitch can exhibit a 1000- fold higher affinity for thiamine pyrophosphate over closely related noncognate compounds such as thiamine monophosphate. To understand the chemical basis of thi-box pyrophosphate specificity, we have determined crystal structures of an E. coli thi-box bound to thiamine pyrophosphate, thiamine monophosphate, and the structural analogs benfotiamine and pyrithiamine. When bound to monophosphorylated compounds, the RNA elements that recognize the thiamine and phosphate moieties of the ligand move closer together. This allows the riboswitch to recognize the monophosphate in a manner similar to how it recognizes the beta-phosphate of thiamine pyrophosphate. In the pyrithiamine complex, the pyrophosphate binding site is largely unstructured. These results show how the riboswitch can bind to various metabolites, and why the thi-box preferentially binds thiamine pyrophosphate.

Fox JM, & Duppel W. 1975 . The action of thiamine and its di- and triphosphates on the slow exponential decline of the ionic currents in the node of Ranvier. Brain Res. 89(2): 287-302. Sodium and potassium currents in the node of Ranvier decrease exponentially with time during long lasting voltage clamp experiments. This decline is strongly dependent on temperature (Q10 approximately 3). Thiamine and, particularly, its diand triphosphoric acid esters are shown to prevent this exponential decline of the ionic currents. Thiamine acts from the outside and from the inside of the nodal membrane, but more potently from the inside. Thiamine diphosphate prevents the exponential decline of the ionic currents only when applied internally. Thiamine triphosphate, the most effective thiamine derivative was tested form the inside only. Bacterial thiaminases applied externally were not effective, presumably because they do not permeate the nodal membrane. Tetrodotoxin, that has been shown by other investigators to induce a release of thiamine from nerve membranes, does not alter the action of thiamine on the exponential decline of current and vice versa. It is concluded that: (1) thiamine diphosphate or thiamine triphosphate are the active thiamine compounds in nerve membranes; (2) the site of action is located at the internal suface of the membrane; (3) the reduction of the thiamine concentration in the membrane or in the axoplasm could cause the exponetial decline of currents; (4) the release of thiamine from nerve membranes induced by tetrodotoxin is interpreted as a side effect not even related to the mechanism by which tetrodotoxin blocks the sodium channels; (5) thiamine polyphosphates appear to stabilise the intrinsic electric field strength of the nodal membrane in the resing state. Threfore, as a working hypothesis, it is suggested that the thiamine derivatives control the number of functioning ionic channels by stabilising the density of negative surface charges at the inner side of the nerve membrane.

Frank T, Bitsch R, Maiwald J, & Stein G. 1999 . Alteration of thiamine pharmacokinetics by end-stage renal disease (ESRD). Int J Clin Pharmacol Ther. 37(9): 449-55. OBJECTIVE: In a comparative study with 20 end-stage renal disease (ESRD) patients the pharmacokinetics of two therapeutically used thiamine (vitamin B1) preparations were assessed. SUBJECTS, MATERIAL AND METHODS: After a single oral dose of either 100 mg benfotiamin (S-benzoylthiamine-o-monophosphate, BTMP) or 100 mg thiamine mononitrate (TN), blood levels of thiamine phosphate esters were analyzed by HPLC after precolumn derivatization to thiochrome phosphate esters for a 24-hour period. RESULTS: The pharmacokinetic parameters AUC0-24h, Cmax and tmax of the benfotiamin group in whole blood and plasma exceeded significantly those in the TN group. Only 1.0 vs. 0.6% of the administered dose were excreted in urine in the BTMP group and TN group, respectively. A high cellular efficacy, as was concluded from the short-term stimulation of the thiamine- dependent transketolase activity in erythrocytes (ETKA), was assessed for BTMP as well as TN. The activation coefficient (ETK-AC) decreased significantly from 1.10 to 1.04 vs. 1.12 to 1.07 in both the BTMP as well as TN groups, respectively. In addition, a high transfer rate to thiamine diphosphate (TDP) was observed in the patients after ingestion of BTMP. The TDP concentration in whole blood increased by 2.6 and 1.4 times from baseline levels to Cmax in the BTMP and TN groups, respectively. The AUC0-24h of TDP in whole blood after BTMP ingestion exceeded those after TN ingestion by 420%. CONCLUSION: These findings justify the therapeutic application of BTMP in ESRD, because a high intracellular concentration of TDP may protect against numerous adverse effects of uremia in the long run.

Frank T, Bitsch R, Maiwald J, & Stein G. 2000 . High thiamine diphosphate concentrations in erythrocytes can be achieved in dialysis patients by oral administration of benfontiamine. Eur J Clin Pharmacol. 56(3): 251-7. OBJECTIVE: The influence of either orally administered S-benzoylthiamine-O-monophosphate (benfotiamine) or thiamine nitrate on the thiamine status was tested in a randomised, two-group comparison study in 20 end-stage renal disease (ESRD) patients. Main outcome measures were the pharmacokinetics of thiamine diphosphate (TDP) in blood, the in vitro erythrocyte transketolase activity, its activation coefficient (alpha-ETK) and the TDP concentration in erythrocytes. METHODS: After ingestion of a single dose of either 100 mg thiamine nitrate (corresponding to 305 micromol thiamine) or 100 mg benfotiamine (corresponding to 214 micromol thiamine), the blood levels of thiamine phosphate esters were analysed by means of high-performance liquid chromatography for a 24-h period. The TDP concentration in erythrocytes was calculated using the haematocrit and TDP concentration in blood. Erythrocyte transketolase activity and alpha-ETK were measured before and 10 h after administration. The pharmacokinetics of TDP in blood were compared with healthy subjects of other studies retrieved from database query. RESULTS: Regarding the blood concentrations of TDP, the patients with ESRD had a 4.3 times higher area under the concentration time curve after benfotiamine administration than after thiamine nitrate. After benfotiamine administration, the peak plasma concentration of TDP exceeded that in healthy subjects by 51%. In the ESRD patients, after 24 h, the mean TDP concentration in erythrocytes increased from 158.7+/-30.9 ng/ml initially to 325.8+/-50.9 ng/ml after administration of benfotiamine and from 166.2+/-51.9 ng/ml to 200.5+/- 50.0 ng/ml after thiamine nitrate administration. The ratio between the maximum erythrocyte TDP concentration and basal concentration was 2.66+/-0.6 in the benfotiamine group and 1.44+/-0.2 in the group receiving thiamine nitrate (P < 0.001). After 24 h, it was 2.11+/-0.4 and 1.23+/-0.2, respectively. The transketolase activity increased from 3.54+/-0.7 microkat/l initially to 3.84+/-0.6 microkat/l after benfotiamine intake (P = 0.02) and from 3.71+/-0.8 microkat/l to 4.02+/-0.7 microkat/l after thiamine nitrate intake (P = 0.08). Likewise, alpha-ETK decreased from initially 1.10+/-0.07 to 1.04+/-0.04 (P = 0.04) and from 1.12+/- 0.05 to 1.08+/-0.06 (P = 0.09). After 24 h, the phosphorylation ratio in whole blood decreased from 12.9+/- 6.9 initially to 5.6+/-3.2 after benfotiamine administration (P = 0.02) and from 13.5+/-7.3 to 9.0+/-4.8 (P = 0.03) after administration of thiamine nitrate. No correlation between erythrocyte TDP concentration and transketolase activity and/or alpha-ETK was observed in ESRD patients, either before or 10 h after administration. CONCLUSION: Compared with thiamine nitrate, the oral administration of benfotiamine leads to higher TDP concentrations in erythrocytes accompanied with a significant improvement of the erythrocyte transketolase activity in ESRD patients.

Gadau S, Emanueli C, Van Linthout S, Graiani G, Todaro M, Meloni M, Campesi I, Invernici G, Spillmann F, Ward K, & Madeddu P. 2006 . Benfotiamine accelerates the healing of ischaemic diabetic limbs in mice through protein kinase B/Akt-mediated potentiation of angiogenesis and inhibition of apoptosis. Diabetologia. 49(2): 405-20. AIMS/HYPOTHESIS: Benfotiamine, a vitamin B1 analogue, reportedly prevents diabetic microangiopathy. The aim of this study was to evaluate whether benfotiamine is of benefit in reparative neovascularisation using a type I diabetes model of hindlimb ischaemia. We also investigated the involvement of protein kinase B (PKB)/Akt in the therapeutic effects of benfotiamine. METHODS: Streptozotocin-induced diabetic mice, given oral benfotiamine or vehicle, were subjected to unilateral limb ischaemia. Reparative neovascularisation was analysed by histology. The expression of Nos3 and Casp3 was evaluated by real-time PCR, and the activation state of PKB/Akt was assessed by western blot analysis and immunohistochemistry. The functional importance of PKB/Akt in benfotiamine-induced effects was investigated using a dominant-negative construct. RESULTS: Diabetic muscles showed reduced transketolase activity, which was corrected by benfotiamine. Importantly, benfotiamine prevented ischaemia-induced toe necrosis, improved hindlimb perfusion and oxygenation, and restored endothelium- dependent vasodilation. Histological studies revealed the improvement of reparative neovascularisation and the inhibition of endothelial and skeletal muscle cell apoptosis. In addition, benfotiamine prevented the vascular accumulation of advanced glycation end products and the induction of pro-apoptotic caspase-3, while restoring proper expression of Nos3 and Akt in ischaemic muscles. The benefits of benfotiamine were nullified by dominant-negative PKB/Akt. In vitro, benfotiamine stimulated the proliferation of human EPCs, while inhibiting apoptosis induced by high glucose. In diabetic mice, the number of circulating EPCs was reduced, with the deficit being corrected by benfotiamine. CONCLUSIONS/INTERPRETATION: We have demonstrated, for the first time, that benfotiamine aids the post-ischaemic healing of diabetic animals via PKB/Akt-mediated potentiation of angiogenesis and inhibition of apoptosis. In addition, benfotiamine combats the diabetes-induced deficit in endothelial progenitor cells.

Godhwani JL, Madan BR, & Vyas DS. 1975 . Anti-inflammatory and related activities of some recently synthesized thiamine derivatives: an experimental study. Indian J Med Res. 63(7): 1010-9.

Greb A, & Bitsch R. 1998 . Comparative bioavailability of various thiamine derivatives after oral administration. Int J Clin Pharmacol Ther. 36(4): 216-21. In a multiple change-over study the bioequivalence of 3 thiamine preparations, used therapeutically as neurotropic agents for the treatment of polyneuropathies, was tested in a collective of 7 volunteers. After ingestion of a single dose of either 100 mg benfotiamin CS-benzoylthiamine-o-monophosphate), fursultiamin (thiamintetrahydrofurfuryldisulfide) or thiaminedisulfide, thiamine blood levels were analyzed for a 10-hour period. Thiamine was measured by HPLC after precolumn derivatization to thiochrome. The maximal thiamine concentration Cmax and its time (tmax) in plasma and hemolysate, the area under concentration time curve (AUC), and thiamine excretion in 24-hour urine were assessed as criteria of bioavailability. Additionally the erythrocytic transketolase activity (ETK) and alphaETK were determined as indicators of the cellular thiamine availability. After benfotiamin ingestion a more rapid and earlier increase of thiamine in plasma and hemolysate was observed in contrast to fursultiamin and the disulfide. All biokinetic data demonstrated a significantly improved thiamine bioavailability from benfotiamin compared with the other preparations. The lowest bioavailability was detected with thiamindisulfide. From our results it can be concluded that oral administration of benfotiamin is best suitable for therapeutical purposes owing to its excellent absorption characteristics.

Greenwood J, & Pratt OE. 1981 . Inhibition of thiamine transport across the blood-brain barrier by thiamine analogs. J Physiol. 317(Supp): P65.

Halsted CH. 2004 . Nutrition and alcoholic liver disease. Semin Liver Dis. 24(3): 289-304. Malnutrition is a common finding in chronic alcoholics, and protein calorie malnutrition (PCM) is universal and predictive of survival in patients with established alcoholic liver disease (ALD). These patients also demonstrate frequent deficiencies