Hammes HP, Du X, Edelstein D, Taguchi T, Matsumura T, Ju Q, Lin J,
Bierhaus A, Nawroth P, Hannak D, Neumaier M, Bergfeld R, Giardino I, Brownlee M.

Medical Clinic V
School of Clinical Medicine
Mannheim, Germany.
Nat Med. 2003 Mar;9(3):294-9

Abstract

Three of the major biochemical pathways implicated in the pathogenesis of hyperglycemia induced vascular damage (the hexosamine pathway, the advanced glycation end product (AGE) formation pathway and the diacylglycerol (DAG)-protein kinase C (PKC) pathway) are activated by increased availability of the glycolytic metabolites glyceraldehyde-3-phosphate and fructose-6-phosphate. We have discovered that the lipid-soluble thiamine derivative benfotiamine can inhibit these three pathways, as well as hyperglycemia-associated NF-kappaB activation, by activating the pentose phosphate pathway enzyme transketolase, which converts glyceraldehyde-3-phosphate and fructose-6-phosphate into pentose-5-phosphates and other sugars. In retinas of diabetic animals, benfotiamine treatment inhibited these three pathways and NF-kappaB activation by activating transketolase, and also prevented experimental diabetic retinopathy. The ability of benfotiamine to inhibit three major pathways simultaneously might be clinically useful in preventing the development and progression of diabetic complications.

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March 3, 2003

Maintaining lower blood glucose levels is a critical and effective part of preventing diabetic retinopathy. Diabetes researchers would like to find ways to protect the retina even more. This study reveals a promising new possibility.
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Diabetic retinopathy is a serious long-term complication of diabetes that can impair vision or even result in blindness. The condition develops when the retina of the eye is repeatedly exposed to high blood glucose levels (hyperglycemia). Research has shown that lower blood glucose levels can prevent or delay diabetic retinopathy. However, since even the best efforts may not prevent hyperglycemia completely, diabetes researchers are exploring ways to protect the retina when hyperglycemia occurs. A study published recently in the journal Nature Medicine was designed to find out whether a synthetic version of thiamine (vitamin B1) could prevent diabetic retinopathy despite high blood glucose levels.

The retina receives oxygen and other nutrients from the blood through a network of very small vessels. The inner lining of these vessels is composed of cells that can’t block the entry of glucose even when they have absorbed enough to meet their energy needs. When blood glucose levels are high, these cells continue to burn unneeded glucose, and waste products build up. The accumulation of waste sets off chemical reactions within the cells that damage, and eventually destroy, the retina’s blood vessels.

The first part of the study involved laboratory experiments to observe what happens when cells are exposed to excess glucose. The researchers focused on an enzyme called transketolase. Transketolase blocks the absorption of too much glucose into cells. Since transketolase requires thiamine to work, the researchers conducted tests to see whether extra thiamine could boost the effect of transketolase. It did, but with only minimal success. Next the team turned to a synthetic form of thiamine called benfotiamine, and repeated the experiments. This time transkeolase activity more than doubled, and prevented cell damage from exposure to high glucose levels.

These experiments suggested that benfotiamine might stop the process that leads to diabetic retinopathy. To find out, the researchers studied three groups of rats. Two of the groups included rats with diabetes and persistent hyperglycemia. Average weight and HbA1c levels were similar in both groups. The rats in only one of the two groups were fed benfotiamine. Treatment with benfotiamine did not affect weight or HbA1c levels. Both groups of rats with diabetes were compared to a group of rats without diabetes. All three groups were studied for 36 weeks.

At the end of the study, the research team examined all of the rats. The rats without diabetes had normal blood vessels in their retinas. The rats with diabetes that were not fed benfotiamine had severely damaged blood vessels that were clear evidence of diabetic retinopathy. The rats with diabetes that had received benfotiamine did not have retinopathy. In fact, their retinas appeared as healthy as those of the rats without diabetes.

The researchers conclude that benfotiamine prevented diabetic retinopathy in the experimental rats. These promising results cannot be applied to humans without additional research to determine effectiveness, safety and appropriate dosages. The authors recommend that such research begin promptly. Based on the findings of their study, the team is optimistic that prevention of diabetic retinopathy may become easier in the near future.

Reference:

Hammes HP et al. Benfotiamine blocks three major pathways of hyperglycemic damage and prevents experimental diabetic retinopathy. Nature Medicine 2003; published online: 18 February 2003; doi:10.1038/nm834

Pomero F, Molinar Min A, La Selva M, Allione A, Molinatti GM, Porta M.

WHO Collaborating Centre for Diabetes-Related Blindness, Department of Internal Medicine,
University of Turin, Italy.
Acta Diabetol. 2001;38(3):135-8.

Abstract

We investigated the hypothesis that benfotiamine, a lipophilic derivative of thiamine, affects replication delay and generation of advanced glycosylation end-products (AGE) in human umbilical vein endothelial cells cultured in the presence of high glucose. Cells were grown in physiological (5.6 mM) and high (28.0 mM) concentrations of D-glucose, with and without 150 microM thiamine or benfotiamine. Cell proliferation was measured by mitochondrial dehydrogenase activity. AGE generation after 20 days was assessed fluorimetrically. Cell replication was impaired by high glucose (72.3%+/-5.1% of that in physiological glucose, p=0.001). This was corrected by the addition of either thiamine (80.6%+/-2.4%, p=0.005) or benfotiamine (87.5%+/-8.9%, p=0.006), although it not was completely normalized (p=0.001 and p=0.008, respectively) to that in physiological glucose. Increased AGE production in high glucose (159.7%+/-38.9% of fluorescence in physiological glucose, p=0.003) was reduced by thiamine (113.2%+/-16.3%, p=0.008 vs. high glucose alone) or benfotiamine (135.6%+/-49.8%, p=0.03 vs. high glucose alone) to levels similar to those observed in physiological glucose. Benfotiamine, a derivative of thiamine with better bioavailability, corrects defective replication and increased AGE generation in endothelial cells cultured in high glucose, to a similar extent as thiamine. These effects may result from normalization of accelerated glycolysis and the consequent decrease in metabolites that are extremely active in generating nonenzymatic protein glycation. The potential role of thiamine administration in the prevention or treatment of vascular complications of diabetes deserves further investigation.

Winkler G, Pal B, Nagybeganyi E, Ory I, Porochnavec M, Kempler P.

2nd Department of Internal Medicine,
Municipal St. John’s Hospital,
Budapest, Hungary.
Arzneimittelforschung. 1999 Mar;49(3):220-4.

Abstract

The therapeutic effectiveness of a benfotiamine vitamin B combination, administered in high (4 x 2 capsules/day, = 320 mg benfotiamine/day) and medium doses (3 x 1 capsules/day), was compared to a monotherapy with benfotiamine (3 x 1 tablets/day, = 150 mg benfotiamine/day) in diabetic patients suffering from painful peripheral diabetic neuropathy (DNP). In a 6-week open clinical trial, 36 patients (aged 40 to 70 yrs) having acceptable metabolic control (HbA1c < 8.0%) were randomly assigned to three groups, each of them comprising 12 participants. Neuropathy was assessed by five parameters: the pain sensation (evaluated by a modified analogue visual scale), the vibration sensation (measured with a tuning fork using the Riedel-Seyfert method) and the current perception threshold (CPT) on the peroneal nerve at 3 frequencies: 5, 250 and 2000 Hz). Parameters were registered at the beginning of the study and at the end of the 3rd and 6th week of therapy. An overall beneficial therapeutic effect on the neuropathy status was observed in all three groups during the study, and a significant improvement in most of the parameters studied appeared already at the 3rd week of therapy (p < 0.01). The greatest change occurred in the group of patients receiving the high dose of benfotiamine (p < 0.01 and 0.05, resp., compared to the other groups). Metabolic control did not change over the study. It is concluded that benfotiamine is most effective in large doses, although even in smaller daily dosages, either in combination or in monotherapy, it is effective.

[Article in Russian]

Sadekov RA, Danilov AB, Vein AM.

2nd Department of Internal Medicine,
Municipal St. John’s Hospital,
Budapest, Hungary.
Arzneimittelforschung. 1999 Mar;49(3):220-4.

Abstract

Efficiency of benfothiamine was studied in treatment of diabetic polyneuropathy in 14 patients with diabetes mellitus type II (1 dragee 3 times a day, within 6 weeks). After the course of treatment the intensity of pains was decreased according to visual analogous scale on the average from 8.2 to 2.3 scores, the indices of vibratory sensitivity improved significantly as well as the data of cardiovascular tests characterizing parasympathetic control of heart rhythm. Meanwhile latent periods of the evoked sympathetic potentials on arms and legs which were initially lengthened became significantly shorter. A clear-cut tendency was also found to increasing conduction rate for excitation through the motor nerves. The treatment resulted in the improvement of the condition in 93% of the cases. The conclusion was made about efficiency and safety of benfothiamine application in therapy of patients with diabetic polyneuropathy.

Simeonov S, Pavlova M, Mitkov M, Mincheva L, Troev D.

Endocrinology and Metabolic Disorders Clinic
Higher Medical Institute
Plovdiv, Bulgaria.
Folia Med (Plovdiv). 1997;39(4):5-10.

Abstract

Forty-five diabetes patients with painful peripheral polyneuropathy were enrolled in a 3-month observational study comparing the therapeutic efficacy of 50 mg benfothiamine with parallel randomized treatment assignment with the conventional vitamin B complex treatment regimen Neurobex (B1 10mg, B2 10mg, B6 2mg, B12 5mg, Niacinamide 50mg, Zinc sulfate 61.8mg). Thirty patients in group one were randomized to receive two benfothiamine tablets qid for three weeks followed by 1 benfothiamine tablet tid for 9 weeks. In group two 15 patients received two Neurobex tablets tid for the entire 3-month study period. Therapeutic efficacy was assessed on the basis of within-patient differences in pain severity between benfothiamine and Neurobex-treated patients and in vibration perception thresholds using the Rydel-Seiffer biothesiometer at baseline and at the end of the study. Statistically significant relief of both background and peak neuropathic pain was achieved in all of the benfothiamine-treated patients and vibration perception thresholds dramatically improved with a median of 1.56 measured on the biothesiometer scale (t = 3.24, P < 0.01). The sensory symptoms improvement was insignificant in the Neurobex-treated patient group and the changes in the vibration perception thresholds failed to reach statistical significance. The therapeutic efficacy of benfothiamine was greater in patients with early-stage diabetes as compared with those with advanced diabetic neuropathy. No adverse reactions were observed following the administration of the medication. Our results underscore the importance of benfothiamine tablets as an indispensable element in the therapeutic regimen of patients with painful diabetic polyneuropathy.

Stracke H, Lindemann A, Federlin K.

Third Medical Department,
University of Giessen, Germany.
Exp Clin Endocrinol Diabetes. 1996;104(4):311-6.

Abstract

In a double-blind, randomized, controlled study, the effectiveness of treatment with a combination of Benfotiamine (a lipid-soluble derivative of vitamin B1 with high bioavailability) plus vitamin B6,B12 on objective parameters of neuropathy was studied over a period of 12 weeks on 24 diabetic patients with diabetic polyneuropathy. The results showed a significant improvement (p = 0.006) of nerve conduction velocity in the peroneal nerve and a statistical trend toward improvement of the vibration perception threshold. Long-term observation of 9 patients with verum over a period of 9 months support the results. Therapy-specific adverse effects were not seen. The results of this double-blind investigation, of the long-term observation and of the reports in the literature support the contention that the neurotropic benfotiamine-vitamin B combination represents a starting point in the treatment of diabetic polyneuropathy.

Frank T, Bitsch R, Maiwald J, Stein G.

Department of Human Nutrition
Institute of Nutrition
Friedrich Schiller University of Jena, Germany.
Int J Clin Pharmacol Ther. 1999 Sep;37(9):449-55.

Abstract

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 benfotiamine 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 benfotiamine 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 benfotiamine 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 benfotiamine 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 benfotiamine as well as TN groups, respectively. In addition, a high transfer rate to thiamine diphosphate (TDP) was observed in the patients after ingestion of benfotiamine. The TDP concentration in whole blood increased by 2.6 and 1.4 times from baseline levels to Cmax in the benfotiamine and TN groups, respectively. The AUC0-24h of TDP in whole blood after benfotiamine ingestion exceeded those after TN ingestion by 420%.

CONCLUSION: These findings justify the therapeutic application of benfotiamine in end-stage renal disease, because a high intracellular concentration of TDP may protect against numerous adverse effects of uremia in the long run.

R. Bergfeld, T. Matsumara, X. Du, M. Brownlee

University Hospital Mannheim,
University of Heidelberg, German,
Justus-Liebig-University Giessen, Germany
Albert-Einstein College of Medicine, New York, USA

Abstract

Background and Aims: Vascular complications are the main cause of morbidity and mortality in diabetes mellitus. Four seemingly independent biochemical pathways are involved in the pathogenesis: glucose-induced activation of protein kinase C (PKC) isoforms, increased formation of glucose-derived advanced glycation end products; increased glucose flux through the aldose reductase pathway, and increased flux through the hexosamine pathway. Hyperglycemia increases reactive oxygen species (ROS) production inside cultured bovine aortic endothelial cells. ROS activate aldose reductase, activate PKC, induce advanced glycation end product formation, activate the hexosamine pathway, and activate the pleiotropic transcription factor nuclear factor-kappa B (NFkB). The thiamine pro-drug benfotiamine inhibits the formation of AGEs in target tissues of diabetic microangiopathy.

Materials and Methods: Using bovine aortic endothelial cells, we studied the effect of benfotiamin on intracellular AGE-formation, flux through the hexosamine pathway, activation of protein kinase C, and activation of NFkB. Benfotiamine was added to cells in high glucose media at a final concentration of 50 µM. Media was changed daily for 7 days. AGE-formation was determined Cell by dot blot technique, and complexes were visualized using an ECF kit (Amersham). Cell extracts were analysed on an HPLC system as described previously (PNAS 2000 97: 12222-12226). For NFkB determination cells were incubated in low glucose, high glucose, high glucose + benfotiamine for 6 hrs. NFkB was determined by a fluorescence in situ DNA-protein binding assay and fluorescence/cell was determined using Scanalytics. PKC assay was performed after cells were incubated for 7 days as described above. The cells were analysed using a PKC assay system from Life Technologies. Additionally, diabetic rats (i.v. injection of streptozotocin 65 mg/kg body weight) were treated with benfotiamin (80 mg/kg weight) for 36 weeks, and diabetic retinopathy was assessed using quantitative retinal analysis of digest preparations for the development of acellular capillaries. Age-matched non-diabetic and untreated diabetic rats served as controls.

Results: Benfotiamine decreases AGE formation by 60% using quantitative immunoblotting. UDP-GlcNAc was decreased 50% by benfotiamine using the previous conditions. NFKB was decreased 85% to below control levels. Benfotiamine decreased membrane PKC 55% to control levels. Chronic treatment of benfotiamine was well tolerated and did not result in significant changes of metabolic parameters. The development of acellular capillaries was reduced by benfotiamine-treatment from 72.5 ± 11.16 acellular capillary segments/mm2 of retinal area in diabetic rats to 29.64 ± 4.48 acellular capillary segments/mm2 in benfotiamine-treated rats (p < 0.001).

Conclusion: These data suggest that treatment with benfotiamine may be an effective approach to prevent the development of diabetic complications.

JIHONG LIN, ALEX ALT, JUTTA LIERSCH, REINHARD G. BRETZEL,
MICHAEL BROWNLEE, HANS-PETER HAMMES

Third Medical Department,
Justus-Liebig-University Giessen, Germany
Albert-Einstein College of Medicine, New York, USA

Abstract

We have demonstrated previously that intracellular formation of the advanced glycation end product (AGE) N-epsilon-(carboxymethyl)-lysine (CML) inversely correlates with diabetic vascular complications independently from glycemia (Diabetologia 42, 603, 1999).Here, we studied the effect of benfotiamine, a lipid-soluble thiamine derivative with known AGE-inhibiting properties in-vitro on the intracellular formation of (CML) and methylglyoxal-derived AGE in red blood cells. Blood was collected from 6 Type 1 diabetic patients(2 m, 4 f, age 31.8 ± 5.5 years; diabetes duration 15.3 ± 7.0 years) before and after treatment with 600 mg/day benfotiamine for 28 days. In addition to HbA1c (HULK), CML and methylglyoxal were measured using specific antibodies and a quantitative dot blot technique While treatment with benfotiamine did not affect HbA1c levels (at entry: 7.18±0.86%; at conclusion 6.88±0.88%; p not significant), levels of CML decreased by 40 % (737 ± 51 arbitray unit/mg protein (AU) vs. 470 ± 86 AU; p<0.001). The levels of intracellular methylglyoxal-derived AGE were reduced by almost 70% (1628 ±1136 AU vs. 500 ± 343 AU; p < 0.01). The data indicate that thiamine derivatives are effective inhibitors of both intracellular glycoxidation and AGE formation.

Introduction

Intracellular formation of the advanced glycation end product (AGE) Ne-(carboxymethyl)lysine (CML) inversely correlates with diabetic vascular complications independently from glycemia. (1) Intracellular CML is generated by the oxidation of Amadori products or, alternatively, by lipid peroxidation (2,3). The dicarbonylmethylglyoxal is formed by non-oxidative fragmentation of glycolysis-derived triosephosphates and is the most important intracellular AGE (4,5).

Thiamine is a potent AGE-inhibitor in-vitro (6), and benfotiamine, the lipid-soluble pro-drug of thiamine was shown to reduce CML and other AGE in target tissues of diabetic complications in-vivo (7). We studied the effect of benfotiamine, a lipid-soluble thiamine derivative with known AGE-inhibiting properties in-vitro on the intracellular formation of (CML) and methylglyoxal-derived AGE in red blood cells of patients with type 1 diabetes.

Methods

Study group: six patients (2 males, 4 females), age31.8 ± 5.5 years; diabetes duration 15.3 ± 7.0 years. Treatment with 600 mg/day benfotiamine for 28days after informed consent and approval by the local ethics committee. Venous EDTA-blood (3 ml) drawn before and at the end of the study, samples lysed and centrifuged, adjusted to identical hemoglobin concentrations. Quantitative immunoblotting carried out essentially as described before (1). Statistical analysis was performed using the alternate Welsh t test.

Conclusion

Thiamine derivatives, in particular the lipid-soluble pro-drug benfotiamine, are effective inhibitor of intracellular formation of AGE and CML.

References

1. Hammes HP et al.: Diabetologia 42, 603-607, 1999
2. Ahmed MU et al.: J Biol Chem 261, 4889-4894, 1986
3. Fu MX et al.: J Biol Chem 271, 9982-9986, 1995
4. Thornalley PJ: Biochem J 269, 1-11, 1990
5. Shinohara M et al.: J Clin Invest 101, 1142-1147, 1998
6. Booth AA et al.: Biochem Biophys Res Commun 220, 113-119, 1996
7. Hammes HP et al.: Diabetologia 41, Suppl. 1, A310, 1998