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.

BACKGROUND: Complications in diabetes mellitus are partially mediated by enhanced formation of reactive oxygen species. Among the factors involved in reactive oxygen species formation, advanced glycation end products play a key role. Owing to a reduced activity of the enzyme transketolase, which requires diphosphorylated thiamine (vitamin B(1)) as cofactor, an accumulation of those deleterious glucose metabolites especially in diabetic patients can be observed. Benfotiamine, a lipophilic thiamine diphosphate prodrug, prevented early renal and retinal changes in animal studies, and reduced neuropathic pain in clinical studies. Several mechanisms for these activities have been described. We investigated for the first time direct antioxidant abilities of benfotiamine. Additionally, a potential DNA protective effect of benfotiamine was analysed. METHODS: Oxidative stress was detected by flow cytometry, antioxidative capacity was measured with the ferric reducing ability of plasma (FRAP) assay, two endpoints for genomic damage were assessed: the comet assay and the micronucleus test, and the expression and activity of transketolase was quantified. RESULTS: Benfotiamine prevented oxidative stress induced by the mutagen 4-nitroquinoline-1-oxide (NQO), the uremic toxin indoxyl sulfate, and the peptide hormone angiotensin II in three different kidney cell lines. Cell-free experiments showed a direct antioxidant effect of benfotiamine, which might account for the protective effect. Oxidative DNA damage, induced by angiotensin II, was completely prevented by benfotiamine. Incubation with benfotiamine increased transketolase expression and activity in the cells. CONCLUSIONS: Benfotiamine shows a direct antioxidant action. This effect of benfotiamine may be involved in the improvement of diabetic late complications, including peripheral neuropathy. Copyright (c) 2008 John Wiley & Sons, Ltd.

AIM: Efficacy and safety of benfotiamine in treatment of diabetic polyneuropathy. METHODS: Double blind, placebo-controlled, phase-III-study. 181 patients were screened. 165 patients with symmetrical, distal diabetic polyneuropathy were randomised to one of three treatment groups entering the wash-out phase and 133/124 patients were analysed in the ITT/PP analysis: Benfotiamine 600 mg per day (n=47/43), benfotiamine 300 mg per day (n=45/42) or placebo (n=41/39). RESULTS: After 6 weeks of treatment, the primary outcome parameter NSS (Neuropathy Symptom Score) differed significantly between the treatment groups (p=0.033) in the PP (per protocol) population. In the ITT (intention to treat) population, the improvement of NSS was slightly above significance (p=0.055). The TSS (Total Symptom Score) showed no significant differences after 6 weeks of treatment. The improvement was more pronounced at the higher benfotiamine dose and increased with treatment duration. In the TSS, best results were obtained for the symptom "pain". Treatment was well tolerated in all groups. CONCLUSION: Benfotiamine may extend the treatment option for patients with diabetic polyneuropathy based on causal influence on impaired glucose metabolism. Further studies should confirm the positive experiences.

OBJECTIVE: Diabetes is characterized by marked postprandial endothelial dysfunction induced by hyperglycemia, hypertriglyceridemia, advanced glycation end products (AGEs), and dicarbonyls (e.g., methylglyoxal [MG]). In vitro hyperglycemia-induced MG formation and endothelial dysfunction could be blocked by benfotiamine, but in vivo effects of benfotiamine on postprandial endothelial dysfunction and MG synthesis have not been investigated in humans until now. RESEARCH DESIGN AND METHODS: Thirteen people with type 2 diabetes were given a heat-processed test meal with a high AGE content (HAGE; 15.100 AGE kU, 580 kcal, 54 g protein, 17 g lipids, and 48 g carbohydrates) before and after a 3-day therapy with benfotiamine (1,050 mg/day). Macrovascular flow-mediated dilatation (FMD) and microvascular reactive hyperemia, along with serum markers of endothelial disfunction (E-selectin, vascular cell adhesion molecule-1, and intracellular adhesion molecule-1), oxidative stress, AGE, and MG were measured during both test meal days after an overnight fast and then at 2, 4, and 6 h postprandially. RESULTS: The HAGE induced a maximum reactive hyperemia decrease of -60.0% after 2 h and a maximum FMD impairment of -35.1% after 4 h, without affecting endothelium-independent vasodilatation. The effects of HAGE on both FMD and reactive hyperemia were completely prevented by benfotiamine. Serum markers of endothelial dysfunction and oxidative stress, as well as AGE, increased after HAGE. These effects were significantly reduced by benfotiamine. CONCLUSIONS: Our study confirms micro- and macrovascular endothelial dysfunction accompanied by increased oxidative stress following a real-life, heat-processed, AGE-rich meal in individuals with type 2 diabetes and suggests benfotiamine as a potential treatment.