High-throughput screening of small molecules identifies hepcidin antagonists.

Anemia of inflammation (AI) is common in patients with infection, autoimmune diseases, cancer, and chronic kidney disease. Unless the underlying condition can be reversed, treatment options are limited to erythropoiesis-stimulating agents with or without intravenous iron therapy, modalities that are not always effective and can cause serious adverse effects. Hepcidin, the iron regulatory hormone, has been identified as a pathogenic factor in the development of AI. To explore new therapeutic options for AI and other iron-related disorders caused by hepcidin excess, we developed a cell-based screen to identify hepcidin antagonists. Of the 70,000 small molecules in the library, we identified 14 compounds that antagonized the hepcidin effect on ferroportin. One of these was fursultiamine, a Food and Drug Administration (FDA)-approved thiamine derivative. Fursultiamine directly interfered with hepcidin binding to its receptor, ferroportin, by blocking ferroportin C326 thiol residue essential for hepcidin binding. Consequently, fursultiamine prevented hepcidin-induced ferroportin ubiquitination, endocytosis, and degradation in vitro and allowed continuous cellular iron export despite the presence of hepcidin, with IC(50) in the submicromolar range. Thiamine, the fursultiamine metabolite, and benfotiamine, another thiamine derivative, did not interfere with the effect of hepcidin on ferroportin. Other FDA-approved thiol-reactive compounds were at least 1000-fold less potent than fursultiamine in antagonizing hepcidin. In vivo, fursultiamine did not reproducibly antagonize the effect of hepcidin on serum iron, likely because of its rapid conversion to inactive metabolites. Fursultiamine is a unique antagonist of hepcidin in vitro that could serve as a template for the development of drug candidates that inhibit the hepcidin-ferroportin interaction.

Powerful beneficial effects of benfotiamine on cognitive impairment and beta-amyloid deposition in amyloid precursor protein/presenilin-1 transgenic mice.

Reduction of glucose metabolism in brain is one of the main features of Alzheimer's disease. Thiamine (vitamin B1)-dependent processes are critical in glucose metabolism and have been found to be impaired in brains from patients with Alzheimer's disease. However, thiamine treatment exerts little beneficial effect in these patients. Here, we tested the effect of benfotiamine, a thiamine derivative with better bioavailability than thiamine, on cognitive impairment and pathology alterations in a mouse model of Alzheimer's disease, the amyloid precursor protein/presenilin-1 transgenic mouse. We show that after a chronic 8 week treatment, benfotiamine dose-dependently enhanced the spatial memory of amyloid precursor protein/presenilin-1 mice in the Morris water maze test. Furthermore, benfotiamine effectively reduced both amyloid plaque numbers and phosphorylated tau levels in cortical areas of the transgenic mice brains. Unexpectedly, these effects were not mimicked by another lipophilic thiamine derivative, fursultiamine, although both benfotiamine and fursultiamine were effective in increasing the levels of free thiamine in the brain. Most notably, benfotiamine, but not fursultiamine, significantly elevated the phosphorylation level of glycogen synthase kinase-3alpha and -3beta, and reduced their enzymatic activities in the amyloid precursor protein/presenilin-1 transgenic brain. Therefore, in the animal Alzheimer's disease model, benfotiamine appears to improve the cognitive function and reduce amyloid deposition via thiamine-independent mechanisms, which are likely to include the suppression of glycogen synthase kinase-3 activities. These results suggest that, unlike many other thiamine-related drugs, benfotiamine may be beneficial for clinical Alzheimer's disease treatment.

Comparison of the effects of pantethine and fursultiamine on plasma gastrointestinal peptide levels in healthy volunteers.

Pantethine and fursultiamine have been evaluated for their clinical usefulness in the treatment and prevention of uncomplicated postoperative adhesive intestinal obstruction. In recent years, the actions of drugs used to treat gastrointestinal diseases have been elucidated pharmacologically from the viewpoints of gastrointestinal peptide levels. We examined the effects of pantethine and fursultiamine on plasma levels of calcitonin gene-related peptide (CGRP)-, vasoactive intestinal polypeptide (VIP)-, motilin- and substance P (SP)-like immunoreactive substances (IS) in healthy subjects. An open-labeled study was conducted on five healthy volunteers. Each subject was administered a single oral dose of pantethine, fursultiamine and placebo at intervals of one month. Venous blood samples were collected before and at 20, 40, 60, 90, 120, 180 and 240 min after each administration. Plasma peptide levels were measured using a highly sensitive enzyme immunoassay. A single oral dose of pantethine resulted in significant increases of plasma CGRP- and VIP-IS levels compared to placebo. Furthermore, areas under the plasma concentration-time curves (AUC(0-240)) of CGRP- and VIP-IS were significantly higher after pantethine administration compared with placebo. On the other hand, fursultiamine had no effect on plasma levels and AUC(0-240) of CGRP-, VIP-, motilin- and SP-IS. This study demonstrated the different effects of pantethine and fursultiamine from the viewpoint of plasma gastrointestinal peptide changes. The pharmacological effects of pantethine may be closely related to the changes in plasma CGRP- and VIP-IS levels.

Thiamine tetrahydrofurfuryl disulfide promotes voluntary activity through dopaminergic activation in the medial prefrontal cortex.

A physically active lifestyle is associated with better health in body and mind, and it is urgent that supporting agents for such lifestyles be developed. In rodents, voluntary locomotor activity as an active physical behavior may be mediated by dopaminergic neurons (DNs). Thiamine phosphate esters can stimulate DNs, and we thus hypothesized that thiamine tetrahydrofurfuryl disulfide (TTFD), a thiamine derivative, promotes locomotor activity via DNs in rats. Acute i.p. administration of TTFD enhanced rat locomotor activity in a normal cage. In vivo microdialysis revealed that TTFD-enhanced locomotor activity was synchronized with dopamine release in the medial prefrontal cortex (mPFC). Antagonism of the dopamine D1 receptor, but not D2 receptor, in the mPFC fully suppressed TTFD-enhanced locomotor activity. Finally, we found a TTFD dose-dependent increase in voluntary wheel running. Our findings demonstrate that DNs in the mPFC mediates TTFD-enhanced locomotor activity, suggesting the potential of TTFD to induce active physical behavior.

Identification and Validation of an Antivirulence Agent Targeting HlyU-Regulated Virulence in Vibrio vulnificus.

Antimicrobial resistance (AMR) in pathogens is the result of indiscriminate use of antibiotics and consequent metabolic/genetic modulation to evolve survival strategies and clonal-selection in AMR strains. As an alternative to antibiotic treatment, antivirulence strategies are being developed, not only to combat bacterial pathogenesis, but also to avoid emerging antibiotic resistance. Vibrio vulnificus is a foodborne pathogen that causes gastroenteritis, necrotizing wound infections, and sepsis with a high rate of mortality. Here, we developed an inhibitor-screening reporter platform to target HlyU, a master transcriptional regulator of virulence factors in V. vulnificus by assessing rtxA1 transcription under its control. The inhibitor-screening platform includes wild type and ΔhlyU mutant strains of V. vulnificus harboring the reporter construct P rtxA1::luxCDABE for desired luminescence signal detection and control background luminescence, respectively. Using the inhibitor-screening platform, we identified a small molecule, fursultiamine hydrochloride (FTH), that inhibits the transcription of the highly invasive repeat-in-toxin (rtxA1) and hemolysin (vvhA) along with other HlyU regulated virulence genes. FTH has no cytotoxic effects on either host cells or pathogen at the tested concentrations. FTH rescues host cells from the necrotic cell-death induced by RtxA1 and decreases the hemolytic activity under in vitro conditions. The most important point is that FTH treatment does not induce the antivirulence resistance. Current study validated the antivirulence strategy targeting the HlyU virulence transcription factor and toxin-network of V. vulnificus and demonstrated that FTH, exhibits a potential to inhibit the pathogenesis of deadly, opportunistic human pathogen, V. vulnificus without inducing AMR.

Human brain activation in response to olfactory stimulation by intravenous administration of odorants.

To identify the BOLD effects related to olfaction in humans, we recorded functional magnetic resonance imaging (fMRI) scans in response intravenously instilled thiamine propyl disulfide (TPD) and thiamine tetrahydrofurfuryl disulfide monohydrochloride (TTFD). TPD and TTFD evoked a strong and weak odor sensation, respectively. Since we did not spray the odor stimuli directly, this method is expected to reduce the effect caused by direct stimulation of the trigeminal nerve. For the analysis of fMRI data, statistical parametric mapping (SPM2) was employed and the areas significantly activated during olfactory processing were located. Both strong and weak odorants induced brain activities mainly in the orbitofrontal gyrus (Brodmann's area: BA 11) in the left hemisphere. TPD (a strong odorant) induced activity in the subthalamic nucleus in the left hemisphere and the precentral gyrus (BA 6) and insula in the right hemisphere. TTFD (a weak odorant) induced activity in the superior frontal gyrus (BA 11) in the right hemisphere. In both circumstances, there was an increase in blood flow at the secondary olfactory cortex (SOC) but not the primary olfactory cortex (POC), probably due to a habituation effect in the POC. From the present results, we found brain activity in not only odor-specific regions but also regions whose levels of activity were changed by an intensity difference of odor stimuli.

Thiamine and its derivatives inhibit delayed rectifier potassium channels of rat cultured cortical neurons.

We examined the effects of thiamine and its derivatives on voltage-gated ion channels of neuronal cells isolated from fetal forebrain cortex and cultured for 6-14 days. Under the whole-cell voltage clamp, thiamine tetrahydrofurfuryl disulfide (TTFD), a membrane-permeable derivative of thiamine, inhibited the delayed rectifier K+ current (IK) in a concentration-dependent manner (10(-4)-10(-3) M). The IK-suppressing effect was also observed by internal perfusion with 1 mM thiamine, but not by the external application of thiamine, indicating the poor permeability of thiamine through the cell membrane. However, thiamine which was applied directly to the intracellular side of patch membranes in the inside-out configuration failed to decrease the open probability of the single IK channel. In contrast, thiamine diphosphate decreased both the open probability and the open-time of the channel without changing the single channel conductance. These results suggest that phosphorylated thiamine can function as an endogenous K+ channel blocker in neuronal cells. TTFD, when applied extracellularly at a concentration of 1 mM, prolonged the action potential (AP) duration of neurons (172.8 +/- 6.6%) without changing the resting membrane potential or AP amplitude, while the same concentration of thiamine did not influence any parameters of the AP, implying that TTFD may cause the potentiation of neuronal AP through the inhibition of IK.

Inhibition of the delayed rectifier K current in guinea-pig cardiomyocytes by thiamine tetrahydrofurfuryl disulfide.

We examined effect of thiamine tetrahydrofurfuryl disulfide on electrophysiological characteristics of single atrial myocytes, obtained by digestion of guinea-pig heart, using collagenase. Membrane potential and ion channel current in the atrial myocytes were recorded by the patch clamp method. Thiamine tetrahydrofurfuryl disulfide prolonged action potentials at cycle lengths from 250 to 10,000 ms. The degree of thiamine tetrahydrofurfuryl disulfide-induced prolongation was similar among these cycle lengths. Thiamine tetrahydrofurfuryl disulfide inhibited the delayed rectifier K+ current, without affecting Ca2+ current and inward-rectifier K+ current. Thiamine tetrahydrofurfuryl disulfide blocked the delayed rectifier K+ current in voltage- and time-independent manner, indicating that thiamine tetrahydrofurfuryl disulfide blocked both subtypes of the delayed rectifier K+ current (rapid and slow components). Thiamine, the parent molecule of thiamine tetrahydrofurfuryl disulfide, blocked the delayed rectifier K+ current only when thiamine was applied intracellularly. Thiamine tetrahydrofurfuryl disulfide may be converted to thiamine in the cytoplasm, and then may block the the delayed rectifier K+ channel from the intracellular side. Although thiamine tetrahydrofurfuryl disulfide (or thiamine) has some of the properties of class III antiarrhythmics agents, thiamine tetrahydrofurfuryl disulfide did not exhibit reverse use-dependent prolongation of action potential.

Ultracytochemical studies on the effects of thiamine and thiamine tetrahydrofurfuryl disulfide on acetylcholinesterase activity in guinea pig atria.

As a result of histological and ultracytochemical investigations concerning the influence of thiamine and its disulfide derivative, thiamine tetrahydrofurfuryl disulfide (TTFD), on acetylcholinesterase (AChE) in extirpated guinea pig atria, it was found that there was a clear increase in the AChE activity by 10(-4) M TTFD, a weak increase with 10(-5) M and an extremely weak increase, about the same as that in only the nutrient (Locke's solution), with 5 x 10(-4) M. However, with 10(-4) M of thiamine there was a tendency for the activity to be the same degree as with Lock's solution alone or be even inhibited at times.

Cardiac action of thiamine derivatives in guinea pig atria.

Pharmacological studies were performed on allithiamine (TAD), thiamine propyl disulfide (TPD), and thiamine tetrahydrofurfuryl disulfide (TTFD) to investigate positive inotropic and negative chronotropic effects seen when they are applied to spontaneous beats in isolated guinea pig atria at concentrations higher than 10-5 g/ml. 1. The effects of thiamine 8-(methyl-6-acetyldihydrothioacetate) disulfide (TATD) and thiamine hydroxyethyl disulfide (TOED) at 5 x 10-4 were slight, and those of dibenzoyl thiamine (DBT) and thiamine were limited at any concentration. 2. Dimethyl propyl disulfide (DMPD) which has anti-thiamine activity, showed these effects at concentrations higher than 10-5. 3. The negative chronotropic effect of TAD, TPD, and TTFD was not influenced by the prior application of atropine, and the positive inotropic effect was not influenced by propranolol. 4. The effects of TTFD on the electrically driven left atrial muscle were remarkable when the muscle was driven at low frequency, while they were less remarkable at high frequency. 5. The decrease in tension of the electrically driven left atria induced by mersalyl at 5 x 10-4 g/ml was recovered by the subsequent addition of TTFD or TPD at 5 x 10-4 as well as dimercaprol at 5 x 10-5. From the results, it was assumed that (a) the effects of TAD, TPD and TTFD might relate to their common chemical structure of the disulfide, especially to the alkyldisulfide chain, (b) the effects are irrevelant to their common activity as an vitamin B1 and to either cholinergic or adrenergic effect, and (c) a mutual dependence is seen between the positive inotropic effect and the negative chronotropic effect.

Effects of low temperature and electrical (square wave) stimulation on spontaneous contractions in isolated guinea pig atria and influence of thiamine tetrahydrofurfuryl disulfide (TTFD) on these effects.

The spontaneous contractions of isolated guinea pig atria were arrested by a temperature change in the medium for 30 to 20 degrees C within 20 min. 2. When thiamine tetrahydrofurfuryl sulfide (TTFD) at concentration of 10-5 g/ml was added in the medium, the arrest was not seen for more than 30 min. 3. Arrhythmic contractions induced by the electrical square wave stimulation of threshold intensity were prevented by TTFD at 10-4 g/ml which was added to the medium. 4. These effects of TTFD at respective concentrations were seen even 30 min after the drug was removed from the medium, when the atria had been pre-incubated with the drug for one hour before the removal. 5. From these results, it was assumed that TTFD might show these effects against the extrinsic physical invasions through the stabilization of the tissue membrane.

A thiamin derivative inhibits oxidation of exogenous glucose at rest, but not during exercise.

Thiamin (vitamin B(1)) is known to activate carbohydrate metabolism in part through activation of pyruvate dehydrogenase. The purpose of this study was to investigate the effect of thiamin tetrahydrofurfuryl disulfide (TTFD), a thiamin derivative, on utilization of exogenous glucose by measuring oxidation of (13)C-glucose at rest and during prolonged exercise in mice under normal dietary conditions. Mice orally ingested TTFD (0.1 mg/g BW [body weight]) and (13)C-glucose (0.8 mg/g BW) or (13)C-lactate (0.1 mg/g BW) plus glucose (0.8 mg/g BW) at rest or before endurance running. The average percent of (13)C atoms in total (12)C+(13)C ((13)C atom%) in expired air after ingestion of (13)C-glucose at rest was significantly lower in the TTFD group than in the control group. No significant difference was found in (13)C atom% in expired air after ingestion of (13)C-glucose and prolonged exercise. In addition, no significant effect of TTFD was found in expired (13)C atom% after ingestion of (13)C-lactate plus glucose at rest. TTFD also had no effect on concentrations of muscle or liver glycogen at rest. These results suggest that TTFD, which is a thiamin derivative, decreases oxidation of exogenous glucose at rest, but not during exercise.

Thiamine tetrahydrofurfuryl disulfide improves energy metabolism and physical performance during physical-fatigue loading in rats.

Impaired energy metabolism is considered a possible cause of fatigue. The thiamine derivative, thiamine tetrahydrofurfuryl disulfide (TTFD), is prescribed and is also an over-the-counter drug for the attenuation of fatigue. It is readily absorbed from the intestinal tract and converted into thiamine pyrophosphate (TPP), which plays an important role as a cofactor for enzymes of metabolic pathways involved in the production of adenosine triphosphate (ATP). We postulated that TTFD has an anti-fatigue effect by improving energy metabolism during physical-fatigue loading. Here, we initially used the forced swimming test to determine whether daily TTFD or thiamine for 5 days has anti-fatigue effects on weight-loaded rats. The swimming duration of TTFD-, but not of thiamine-treated rats, was significantly longer than that of control rats (P < .05). Based on these findings, we examined changes in the levels of thiamine and its phosphate esters in various organs and the effect of TTFD on ATP levels in skeletal muscle after forced swimming, to determine the cellular mechanisms of the anti-fatigue effect of TTFD. Daily TTFD resulted in a characteristic distribution of thiamine and its phosphate esters in rat skeletal muscle, liver, kidney, heart, brain, and plasma. Furthermore, daily TTFD attenuated the decrease in ATP content in the skeletal muscle caused by forced swimming with a weight load for a defined period (150 s). These results indicate that TTFD exerts anti-fatigue effects by improving energy metabolism during physical fatigue.

Neuromagnetic changes of brain rhythm evoked by intravenous olfactory stimulation in humans.

To identify the changes in the respective frequency band and brain areas related to olfactory perception, we measured magnetoencephalographic (MEG) signals before and after instilling intravenously thiamine propyl disulfide (TPD) and thiamine tetrahydrofurfuryl disulfide monohydrochloride (TTFD), which evoked a strong and weak sensation of odor, respectively. For the frequency analysis of MEG, a beamformer program, synthetic aperture magnetometry (SAM), was employed and event-related desynchronization (ERD) or synchronization (ERS) was statistically determined. Both strong and weak odors induced ERD in (1) beta band (13-30 Hz) in the right precentral gyrus, and the superior and middle frontal gyri in both hemispheres, (2) low gamma band (30-60 Hz) in the left superior frontal gyrus and superior parietal lobule, and the middle frontal gyrus in both hemispheres, and (3) high gamma band 2 (100-200 Hz) in the right inferior frontal gyrus. TPD induced ERD in the left temporal, parietal and occipital lobes, while TTFD induced ERD in the right temporal, parietal and occipital lobes. The results indicate that physiological functions in several regions in the frontal lobe may change and the strength of the odor may play a different role in each hemisphere during olfactory perception in humans.

Fursultiamine, a vitamin B1 derivative, enhances chondroprotective effects of glucosamine hydrochloride and chondroitin sulfate in rabbit experimental osteoarthritis.

OBJECT AND DESIGN: The therapeutic effect of glucosamine hydrochloride (GH) and chondroitin sulfate (CS) in combination with fursultiamine, a vitamin B1 derivative, on the development of cartilage lesions was investigated in an animal model of osteoarthritis (OA). METHODS: The OA model was created by partial medial meniscectomy of the right knee joint (day 0). The rabbits were placed into three experimental groups: operated (OA) rabbits that received placebo treatment, OA rabbits that received GH (1000 mg/kg) + CS (800 mg/kg), and OA rabbits that received GH + CS + fursultiamine (100 mg/kg). Each treatment was initiated on day 3 and continued for 8 weeks. Macroscopic and histologic analyses were performed on the cartilage. The level of MMP-1 in OA cartilage chondrocytes was evaluated by immunohistochemistry. RESULTS: Only the group receiving combined treatment with GH + CS + fursultiamine showed a significant reduction in the severity of macroscopic and histologic lesions on tibial plateau, which is the weight bearing cartilage surface of the tibia, compared with placebo-treated OA rabbits. This treatment group also revealed a small, but significant, decrease in the body weight gain of the rabbits. In cartilage from placebo-treated OA rabbits, a significantly higher percentage of chondrocytes in superficial layer stained positive for MMP-1 compared with unoperated control. Rabbits treated with the GH + CS + fursultiamine revealed a significant reduction in the level of MMP-1. CONCLUSION: These results suggest that the chondroprotective effect of GH + CS is enhanced by the addition of fursultiamine in experimental OA. This effect was associated with a reduction in the level of MMP-1, which are known to play an important role in the pathophysiology of OA lesions.

The effect of a thiamin derivative on exercise performance.

The purpose of this study was to investigate the effect of a thiamin derivative, thiamin tetrahydrofurfuryl disulfide (TTFD), on oxygen uptake (VO2), lactate accumulation and cycling performance during exercise to exhaustion. Using a randomized, double-blind, cross-over design with a 10-day washout between trials, 14 subjects ingested either 1 g.day-1 of TTFD or a placebo (PL) for 4 days. On day 3, subjects performed a progressive exercise-test to exhaustion on a cycle ergometer for the determination of VO2submax, VO2peak, lactate concentration ([La-]), lactate threshold (ThLa) and heart rate (fc). On day 4, subjects performed a maximal 2000-m time trial on a cycle ergometer. A one-way analysis of variance (ANOVA) with repeated measures was used to determine significant differences between trials. There were no significant differences detected between trials for serial measures of VO2submax, [La-] or fc. Likewise, VO2peak [PL 4.06 (0.19) TTFD 4.12 (0.19) l.min-1, P = 0.83], ThLa [PL 2.47 (0.17), TTFD 2.43 (0.16) l.min-1, P = 0.86] and 2000-m performance time [PL 204.5 (5.5), TTFD 200.9 (4.3).s, P = 0.61] were not significantly different between trials. The results of this study suggest that thiamin derivative supplementation does not influence high-intensity exercise performance.

Inhibition by thiamine tetrahydrofurfuryl disulfide (TTFD) of the arachidonic acid cascade-line activation as evidenced in the heart-lung preparation of the dog.

The effects of thiamine tetrahydrofurfuryl disulfide (TTFD) on the gradual increase in the coronary blood flow (CBF) inherent in the canine heart-lung preparation were studied. TTFD is a disulfide-type derivative of thiamine reported to have an antiinflammatory effect in experimental animals. Since it was found that the substance could reverse the gradual increase in CBF, the possibility that the reversal was brought about through an inhibition of activation of the arachidonic acid cascade-line was tested, examining the effects of this substance on the CBF increase produced by arachidonic acid (AA) and prostacyclin (PGI2). The vasodilator response to AA, which was barely detectable at the start of the experiment at which CBF was at a physiological low level, became potentiated as the gradual increase in CBF occurred, returning to the initial magnitude after TTFD, while the vasodilator response to PGI2 remained essentially unchanged during the entire course of the experiment. It was concluded that TTFD reversed the gradual increase in CBF in the HLP through the inhibition of the arachidonic acid cascade-line activation.

Effects of thiamine supplementation on exercise-induced fatigue.

High-dose thiamine (vitamin B1) supplementation (100 mg/day) may be helpful in preventing or accelerating recovery from exercise-induced fatigue. Sixteen volunteer male athletes volunteer, 8 with a blood thiamine level of 40 ng/ml or more (normal thiamine group) and 8 with levels below that level (low thiamine group) were selected as subjects. They exercised on a bicycle ergometer and the effects of thiamine supplementation were compared with placebo. Blood thiamine level markedly increased following supplementation of thiamine for 3 days before exercise. Exercise-induced changes in hemodynamic parameters and cardiopulmonary function indicated the onset of fatigue. Thiamine supplementation significantly suppressed the increase in blood glucose in the normal thiamine group and significantly decreased the number of complaints shortly after exercise in the subjective fatigue assessment of 30 items.

Effect of thiamine tetrahydrofurfuryl disulfide on audiogenic seizures in DBA/2J mice.

Weanling mice of the DBA/2J strain are spontaneously audiogenically seizure prone between 19 and 24 days. Thereafter, susceptibility declines rapidly within the next 7-10 days. It was found that thiamine tetrahydrofurfuryl disulfide (TTFD) significantly delayed the natural disappearance of seizure proneness in male animals compared with controls treated with identical doses of saline. There was no significant difference between treated females and controls. Evidence is presented that suggests that TTFD is cholinergic in its action and has pharmacologic effects other than a simple vitamin replacement. The difference between males and females found in this strain of mouse in this experiment is consistent with previous information which indicates that sex-related differences exist in their response to stress.