Role of NKCC1 Activity in Glioma K+ Homeostasis and Cell Growth: New Insights With the Bumetanide-Derivative STS66.

Introduction: Na+-K+-2Cl- cotransporter isoform 1 (NKCC1) is important in regulating intracellular K+ and Cl- homeostasis and cell volume. In this study, we investigated a role of NKCC1 in regulating glioma K+ influx and proliferation in response to apoptosis inducing chemotherapeutic drug temozolomide (TMZ). The efficacy of a new bumetanide (BMT)-derivative NKCC1 inhibitor STS66 [3-(butylamino)-2-phenoxy-5-[(2, 2, 2-trifluoroethylamino) methyl] benzenesulfonamide] in blocking NKCC1 activity was compared with well-established NKCC1 inhibitor BMT. Methods: NKCC1 activity in cultured mouse GL26 and SB28-GFP glioma cells was measured by Rb+ (K+) influx. The WNK1-SPAK/OSR1-NKCC1 signaling and AKT/ERK-mTOR signaling protein expression and activation were assessed by immunoblotting. Cell growth was determined by bromodeoxyuridine (BrdU) incorporation assay, MTT proliferation assay, and cell cycle analysis. Impact of STS66 and BMT on cell Rb+ influx and growth was measured in glioma cells treated with or without TMZ. Results: Rb+ influx assay showed that 10 µM BMT markedly decreased the total Rb+ influx and no additional inhibition detected at >10 µM BMT. In contrast, the maximum effects of STS66 on Rb+ influx inhibition were at 40-60 µM. Both BMT and STS66 reduced TMZ-mediated NKCC1 activation and protein upregulation. Glioma cell growth can be reduced by STS66. The most robust inhibition of glioma growth, cell cycle, and AKT/ERK signaling was achieved by the TMZ + STS66 treatment. Conclusion: The new BMT-derivative NKCC1 inhibitor STS66 is more effective than BMT in reducing glioma cell growth in part by inhibiting NKCC1-mediated K+ influx. TMZ + STS66 combination treatment reduces glioma cell growth via inhibiting cell cycle and AKT-ERK signaling.

Functional characterization of novel bumetanide derivatives for epilepsy treatment.

Temporal lobe epilepsy (TLE) is the most common type of focal epilepsies, affecting approximately 35 million people worldwide. Despite the introduction of numerous novel antiepileptic drugs during the last decades, the proportion of patients with therapy-resistant TLE is still high. As an impaired cellular chloride homeostasis appears involved in disease pathophysiology, bumetanide, an antagonist to Na-K-Cl cotransporters, gained interest as potential therapeutic option. However, bumetanide induces a strong diuretic effect and displays poor penetration across the blood-brain barrier (BBB). To reduce these unwanted effects, we modified the already described BUM690 by exchanging the allyl-into a trifluoro-ethyl group to yield BUM532. Furthermore, we exchanged the nitrogen for oxygen in the trifluoro-ethyl group to yield BUM97. In the intrahippocampal kainic acid mouse model of TLE BUM532 ±â€¯phenobarbital (PB), bumetanide ±â€¯PB and PB alone significantly reduced hippocampal paroxysmal discharges (HPDs) but not spike trains. By contrast, treatment with BUM97 suppressed HPDs as well as spike trains dose-dependently, more pronounced compared to the other tested compounds and exerted a synergistic anticonvulsant effect with PB. Moreover, at higher doses BUM97 achieved long-lasting reduction of spike trains. In pentylenetetrazole-induced acute seizures only BUM532 combined with a sub-effective dose of PB increased the seizure threshold. No diuretic effects were observed at any dose of the three derivatives. Our data demonstrate the successful optimization of the pharmacological profile of bumetanide and the potential of the improved derivative BUM97 for the treatment of therapy-resistant TLE, in particular in combinatorial drug regimens with a GABA mimetic.

Impaired chloride homeostasis in epilepsy: Molecular basis, impact on treatment, and current treatment approaches.

Epilepsies represent one of the most common neurological diseases worldwide. They are characterized by recurrent spontaneous seizures with severe impact on a patient's life. An imbalance in excitatory and inhibitory signalling is considered the main underlying pathophysiological mechanism. Therefore, GABA-mimetic drugs, strengthening the main inhibitory signalling system in the CNS, are frequently used as antiepileptic or anticonvulsant drugs. However, the therapeutic effect of such treatment depends on the chloride gradient along the plasma membrane. Impairment of chloride homeostasis, caused by alterations in the functional balance of chloride transporters, was implicated in the pathophysiology of epilepsy and numerous other diseases. Breakdown or even inversion of the chloride gradient may result in ineffective or in worst cases proconvulsant effects of GABA-mimetics. Unfortunately, such situations are reported in considerable number. Consequently, bumetanide, an inhibitor of Na-K-Cl cotransporters gained interest as potential add-on therapy re-establishing the chloride gradient and thereby the hyperpolarizing effects of GABA-mimetic drugs. Indeed, preclinical studies yielded encouraging results, especially when applied in combination with GABA-mimetics in epilepsy models. However, bumetanide induces a strong diuretic effect and displays poor penetration across the blood-brain barrier, two adverse features for chronic antiepileptic treatment. Therefore, new compounds overcoming these limitations are under development. This review focuses on alterations in chloride homeostasis and its underlying molecular mechanisms in epilepsy, on the potential impact of impaired chloride homeostasis on the treatment of epilepsy and on concepts to overcome this problem including recent development of bumetanide derivatives with improved pharmacological profile.

A Novel Na+-K+-Cl- Cotransporter 1 Inhibitor STS66* Reduces Brain Damage in Mice After Ischemic Stroke.

Background and Purpose- Inhibition of brain NKCC1 (Na+-K+-Cl- cotransporter 1) with bumetanide (BMT) is of interest in ischemic stroke therapy. However, its poor brain penetration limits the application. In this study, we investigated the efficacy of 2 novel NKCC1 inhibitors, a lipophilic BMT prodrug STS5 (2-(Dimethylamino)ethyl 3-(butylamino)-4-phenoxy-5-sulfamoyl-benzoate;hydrochloride) and a novel NKCC1 inhibitor STS66 (3-(Butylamino)-2-phenoxy-5-[(2,2,2-trifluoroethylamino)methyl]benzenesulfonamide), on reducing ischemic brain injury. Methods- Large-vessel transient ischemic stroke in normotensive C57BL/6J mice was induced with 50-min occlusion of the middle cerebral artery and reperfusion. Focal, permanent ischemic stroke in angiotensin II (Ang II)-induced hypertensive C57BL/6J mice was induced by permanent occlusion of distal branches of middle cerebral artery. A total of 206 mice were randomly assigned to receive vehicle DMSO, BMT, STS5, or STS66. Results- Poststroke BMT, STS5, or STS66 treatment significantly decreased infarct volume and cerebral swelling by ≈40% to 50% in normotensive mice after transient middle cerebral artery occlusion, but STS66-treated mice displayed better survival and sensorimotor functional recovery. STS5 treatment increased the mortality. Ang II-induced hypertensive mice exhibited increased phosphorylatory activation of SPAK (Ste20-related proline alanine-rich kinase) and NKCC1, as well as worsened infarct and neurological deficit after permanent distal middle cerebral artery occlusion. Conclusions- The novel NKCC1 inhibitor STS66 is superior to BMT and STS5 in reducing ischemic infarction, swelling, and neurological deficits in large-vessel transient ischemic stroke, as well as in permanent focal ischemic stroke with hypertension comorbidity.

The role of oxidative stress in 63 T-induced cytotoxicity against human lung cancer and normal lung fibroblast cell lines.

It has been shown previously that molecules built on benzanilide and thiobenzanilide scaffolds possess differential biological properties including selective anticancer activity. In our previous study, we examined the cytotoxic activity and mechanism of action of the thiobenzanilide derivative N,N'-(1,2-phenylene)bis3,4,5-trifluorobenzothioamide (63 T) as a potential chemotherapeutic compound in an experimental model employing A549 lung adenocarcinoma cells and CCD39Lu non-tumorigenic lung fibroblasts. Since the results suggested oxidative stress as a co-existing mechanism of the cytotoxic effect exerted by 63 T on tested cells, studies involving the analysis of reactive oxygen species (ROS) generation and markers of oxidative stress in cells incubated with 63 T were carried out. It may be concluded that the selective activity of 63 T against cancer cells shown in our experiments is caused, at least in part, by the response of the tested cells to 63 T mediated oxidative stress in both tested cell lines.

Multiple blood-brain barrier transport mechanisms limit bumetanide accumulation, and therapeutic potential, in the mammalian brain.

There is accumulating evidence that bumetanide, which has been used over decades as a potent loop diuretic, also exerts effects on brain disorders, including autism, neonatal seizures, and epilepsy, which are not related to its effects on the kidney but rather mediated by inhibition of the neuronal Na-K-Cl cotransporter isoform NKCC1. However, following systemic administration, brain levels of bumetanide are typically below those needed to inhibit NKCC1, which critically limits its clinical use for treating brain disorders. Recently, active efflux transport at the blood-brain barrier (BBB) has been suggested as a process involved in the low brain:plasma ratio of bumetanide, but it is presently not clear which transporters are involved. Understanding the processes explaining the poor brain penetration of bumetanide is needed for developing strategies to improve the brain delivery of this drug. In the present study, we administered probenecid and more selective inhibitors of active transport carriers at the BBB directly into the brain of mice to minimize the contribution of peripheral effects on the brain penetration of bumetanide. Furthermore, in vitro experiments with mouse organic anion transporter 3 (Oat3)-overexpressing Chinese hamster ovary cells were performed to study the interaction of bumetanide, bumetanide derivatives, and several known inhibitors of Oats on Oat3-mediated transport. The in vivo experiments demonstrated that the uptake and efflux of bumetanide at the BBB is much more complex than previously thought. It seems that both restricted passive diffusion and active efflux transport, mediated by Oat3 but also organic anion-transporting polypeptide (Oatp) Oatp1a4 and multidrug resistance protein 4 explain the extremely low brain concentrations that are achieved after systemic administration of bumetanide, limiting the use of this drug for targeting abnormal expression of neuronal NKCC1 in brain diseases.

Selective anticancer activity of the novel thiobenzanilide 63T against human lung adenocarcinoma cells.

Previously, it has been reported that molecules built on the benzanilide and thiobenzanilide scaffold are the promising groups of compounds with several biological activities including antifungal, antimycotic, antibacterial, spasmolytic, and anticancer ones. In this study the mechanism of action of one selected thiobenzanilide derivative N,N'-(1,2-phenylene)bis3,4,5-trifluorobenzothioamide (63T) with strongest cytotoxic activity has been investigated for the first time in human lung adenocarcinoma (A549) and normal lung derived fibroblast (CCD39Lu) in a cell culture model. The results demonstrated, that 63T can be considered a selective anticancer compound. Based on these results, several experiments including the analysis of cellular morphology, cell phase distribution, cytoplasmic histone-associated DNA fragmentation, apoptosis, necrosis, and autophagy detection were performed to understand better the mechanism underlying the anticancer activity. The data showed that 63T is a small molecule compound, which selectively induces cancer cell death in a caspase independent pathway; moreover, the autophagic dose-dependent processes may be involved in the mechanism of cell death.

The search for NKCC1-selective drugs for the treatment of epilepsy: Structure-function relationship of bumetanide and various bumetanide derivatives in inhibiting the human cation-chloride cotransporter NKCC1A.

The Na(+)-K(+)-Cl(-) cotransporter NKCC1 plays a major role in the regulation of intraneuronal Cl(-) concentration. Abnormal functionality of NKCC1 has been implicated in several brain disorders, including epilepsy. Bumetanide is the only available selective NKCC1 inhibitor, but also inhibits NKCC2, which can cause severe adverse effects during treatment of brain disorders. A NKCC1-selective bumetanide derivative would therefore be a desirable option. In the present study, we used the Xenopus oocyte heterologous expression system to compare the effects of bumetanide and several derivatives on the two major human splice variants of NKCCs, hNKCC1A and hNKCC2A. The derivatives were selected from a series of ~5000 3-amino-5-sulfamoylbenzoic acid derivatives, covering a wide range of structural modifications and diuretic potencies. To our knowledge, such structure-function relationships have not been performed before for NKCC1. Half maximal inhibitory concentrations (IC50s) of bumetanide were 0.68 (hNKCC1A) and 4.0µM (hNKCC2A), respectively, indicating that this drug is 6-times more potent to inhibit hNKCC1A than hNKCC2A. Side chain substitutions in the bumetanide molecule variably affected the potency to inhibit hNKCC1A. This allowed defining the minimal structural requirements necessary for ligand interaction. Unexpectedly, only a few of the bumetanide derivatives examined were more potent than bumetanide to inhibit hNKCC1A, and most of them also inhibited hNKCC2A, with a highly significant correlation between IC50s for the two NKCC isoforms. These data indicate that the structural requirements for inhibition of NKCC1 and NKCC2 are similar, which complicates development of bumetanide-related compounds with high selectivity for NKCC1.

The bumetanide prodrug BUM5, but not bumetanide, potentiates the antiseizure effect of phenobarbital in adult epileptic mice.

OBJECTIVE: The loop diuretic bumetanide has been reported to potentiate the antiseizure activity of phenobarbital in rodent models of neonatal seizures, most likely as a result of inhibition of the chloride importer Na-K-Cl cotransporter isoform 1 (NKCC1) in the brain. In view of the intractability of neonatal seizures, the preclinical findings prompted a clinical trial in neonates on bumetanide as an add-on to phenobarbital, which, however, had to be terminated because of ototoxicity and lack of efficacy. We have recently shown that bumetanide penetrates only poorly into the brain, so that we developed lipophilic prodrugs such as BUM5, the N,N-dimethylaminoethylester of bumetanide, which penetrate more easily into the brain and are converted to bumetanide. METHODS: In the present study, we used a new strategy to test whether BUM5 is more potent than bumetanide in potentiating the antiseizure effect of phenobarbital. Adult mice were made epileptic by pilocarpine, and the antiseizure effects of bumetanide, BUM5, and phenobarbital alone or in combination were determined by the maximal electroshock seizure threshold test. RESULTS: In nonepileptic mice, only phenobarbital exerted seizure threshold-increasing activity, and this was not potentiated by the NKCC1 inhibitors. In contrast, a marked potentiation of phenobarbital by BUM5, but not bumetanide, was determined in epileptic mice. SIGNIFICANCE: Thus, bumetanide is not capable of potentiating phenobarbital's antiseizure effect in an adult mouse model, which, however, can be overcome by using the prodrug BUM5. These data substantiate that BUM5 is a promising tool compound for target validation and proof-of-concept studies on the role of NKCC1 in brain diseases.

Antiproliferative and Pro-apoptotic Activities of a Novel Resveratrol Prodrug Against Jurkat CD4+ T-Cells.

BACKGROUND/AIM: Resveratrol, a natural polyphenol, possesses many beneficial health properties but its therapeutic application is limited due to its low water solubility and instability against oxidative processes. To improve the stability and lipophilicity of the natural compound, we synthesized a resveratrol prodrug, termed FEHH4-1. In the present study, we compared the antiproliferative and pro-apoptotic effects of resveratrol with FEHH4-1 on Jurkat T-cells. MATERIALS AND METHODS: Cell proliferation and viability were monitored by 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide assay, annexin-V/7-amino-actinomycin D staining and western blot. To induce interleukin-2 (IL2) expression, cells were stimulated with phorbol 12-myristate 13-acetate/phytohemagglutinin. IL2 production was quantified by enzyme-linked immunosorbent assay. IL2 promoter activity was studied by a Jurkat T-cell line containing an IL2 promoter luciferase reporter construct. RESULTS: Both polyphenols inhibited proliferation, induced apoptotic cell death and blocked IL2 synthesis in Jurkat T-cells. Most importantly, FEHH4-1 was three-to four-times more potent than resveratrol. CONCLUSION: FEHH4-1 had improved antiproliferative and pro-apoptotic potential against Jurkat T-cells compared to resveratrol.

Novel Promising Estrogenic Receptor Modulators: Cytotoxic and Estrogenic Activity of Benzanilides and Dithiobenzanilides.

The cytotoxicity of 27 benzanilides and dithiobenzanilides built on a stilbene scaffold and possessing various functional groups in aromatic rings previously described for their spasmolytic properties was assayed on three human cancer cell lines (A549 -lung adenocarcinoma, MCF-7 estrogen dependent breast adenocarcinoma and MDA-MB-231 estrogen independent breast adenocarcinoma) and 2 non-tumorigenic cell lines (CCD39Lu-lung fibroblasts, MCF-12A - breast epithelial). Three compounds (6, 15 and 18) showed selective antiproliferative activity against estrogen dependent MCF-7 cancer cells and their estrogenic activity was further confirmed in MCF-7 transfected with an estrogen receptor reporter plasmid and in HEK239 cells over-expressing the estrogen receptor alpha (ERα). Compound 18 is especially interesting as a potential candidate for therapy since it is highly toxic and selective towards estrogen dependent MCF7 cell lines (IC50 = 5.07 µM versus more than 100 µM for MDA-MB-231) and almost innocuous for normal breast cells (IC50 = 91.46 µM for MCF-12A). Docking studies have shown that compound 18 interacts with the receptor in the same cavity as estradiol although the extra aromatic ring is involved in additional binding interactions with residue W383. The role of W383 and the extended binding mode were confirmed by site-directed mutagenesis.

Drug trapping in hERG K+ channels: (not) a matter of drug size?

Inhibition of hERG K+ channels by structurally diverse drugs prolongs the ventricular action potential and increases the risk of torsade de pointes arrhythmias and sudden cardiac death. The capture of drugs behind closed channel gates, so-called drug trapping, is suggested to harbor an increased pro-arrhythmic risk. In this study, the trapping mechanisms of a trapped hERG blocker propafenone and a bulky derivative (MW: 647.24 g mol-1) were studied by making use of electrophysiological measurements in combination with molecular dynamics simulations. Our study suggests that the hERG cavity is able to accommodate very bulky compounds without disturbing gate closure.

A Novel Genistein Prodrug: Design, Synthesis and Bioactivity on Mouse RAW264.7 Macrophages.

Genistein, a naturally occurring isoflavone, possesses many beneficial health effects. To improve the bioactivity of the natural compound, we designed and synthesized the genistein prodrug FEHH6-1. In the present study, we evaluated the biological effects of FEHH6-I on mouse RAW264.7 macrophages and compared them with those obtained with the parent drug genistein. The characteristics of FEHH6-1 were determined by melting point, nuclear magnetic resonance spectroscopy (NMR), and mass spectrometric analysis. The effects of FEHH6-I on cell proliferation, apoptosis, and pro-inflammatory cytokine expression were monitored by XTT-assay, Annexin-V/7-AAD staining, Western blotting, and ELISA. FEHH6-1 showed NMR spectra and relative molecular mass in agreement with the designed structure. In mouse RAW264.7 macrophages, FEHH6-1 inhibited proliferation, induced apoptotic cell death and blocked interleukin 6 and tumor necrosis factor alpha synthesis. At low concentrations, FEHH6-1 induced phosphorylation of AKT1, a kinase involved in cell proliferation and survival. Our data demonstrate that the genistein prodrug FEHH6-1 is a bioactive molecule but its solubility and therefore also its efficacy was significantly lower compared with genistein.

Factors Governing P-Glycoprotein-Mediated Drug-Drug Interactions at the Blood-Brain Barrier Measured with Positron Emission Tomography.

The adenosine triphosphate-binding cassette transporter P-glycoprotein (ABCB1/Abcb1a) restricts at the blood-brain barrier (BBB) brain distribution of many drugs. ABCB1 may be involved in drug-drug interactions (DDIs) at the BBB, which may lead to changes in brain distribution and central nervous system side effects of drugs. Positron emission tomography (PET) with the ABCB1 substrates (R)-[(11)C]verapamil and [(11)C]-N-desmethyl-loperamide and the ABCB1 inhibitor tariquidar has allowed direct comparison of ABCB1-mediated DDIs at the rodent and human BBB. In this work we evaluated different factors which could influence the magnitude of the interaction between tariquidar and (R)-[(11)C]verapamil or [(11)C]-N-desmethyl-loperamide at the BBB and thereby contribute to previously observed species differences between rodents and humans. We performed in vitro transport experiments with [(3)H]verapamil and [(3)H]-N-desmethyl-loperamide in ABCB1 and Abcb1a overexpressing cell lines. Moreover we conducted in vivo PET experiments and biodistribution studies with (R)-[(11)C]verapamil and [(11)C]-N-desmethyl-loperamide in wild-type mice without and with tariquidar pretreatment and in homozygous Abcb1a/1b((-/-)) and heterozygous Abcb1a/1b((+/-)) mice. We found no differences for in vitro transport of [(3)H]verapamil and [(3)H]-N-desmethyl-loperamide by ABCB1 and Abcb1a and its inhibition by tariquidar. [(3)H]-N-Desmethyl-loperamide was transported with a 5 to 9 times higher transport ratio than [(3)H]verapamil in ABCB1- and Abcb1a-transfected cells. In vivo, brain radioactivity concentrations were lower for [(11)C]-N-desmethyl-loperamide than for (R)-[(11)C]verapamil. Both radiotracers showed tariquidar dose dependent increases in brain distribution with tariquidar half-maximum inhibitory concentrations (IC50) of 1052 nM (95% confidence interval CI: 930-1189) for (R)-[(11)C]verapamil and 1329 nM (95% CI: 980-1801) for [(11)C]-N-desmethyl-loperamide. In homozygous Abcb1a/1b((-/-)) mice brain radioactivity distribution was increased by 3.9- and 2.8-fold and in heterozygous Abcb1a/1b((+/-)) mice by 1.5- and 1.1-fold, for (R)-[(11)C]verapamil and [(11)C]-N-desmethyl-loperamide, respectively, as compared with wild-type mice. For both radiotracers radiolabeled metabolites were detected in plasma and brain. When brain and plasma radioactivity concentrations were corrected for radiolabeled metabolites, brain distribution of (R)-[(11)C]verapamil and [(11)C]-N-desmethyl-loperamide was increased in tariquidar (15 mg/kg) treated animals by 14.1- and 18.3-fold, respectively, as compared with vehicle group. Isoflurane anesthesia altered [(11)C]-N-desmethyl-loperamide but not (R)-[(11)C]verapamil metabolism, and this had a direct effect on the magnitude of the increase in brain distribution following ABCB1 inhibition. Our data furthermore suggest that in the absence of ABCB1 function brain distribution of [(11)C]-N-desmethyl-loperamide but not (R)-[(11)C]verapamil may depend on cerebral blood flow. In conclusion, we have identified a number of important factors, i.e., substrate affinity to ABCB1, brain uptake of radiolabeled metabolites, anesthesia, and cerebral blood flow, which can directly influence the magnitude of ABCB1-mediated DDIs at the BBB and should therefore be taken into consideration when interpreting PET results.

Structure-activity relationships of bumetanide derivatives: correlation between diuretic activity in dogs and inhibition of the human NKCC2A transporter.

BACKGROUND AND PURPOSE: The N-K-Cl cotransporters (NKCCs) mediate the coupled, electroneutral movement of Na+ , K+ and Cl- ions across cell membranes. There are two isoforms of this cation co-transporter, NKCC1 and NKCC2. NKCC2 is expressed primarily in the kidney and is the target of diuretics such as bumetanide. Bumetanide was discovered by screening ∼5000 3-amino-5-sulfamoylbenzoic acid derivatives, long before NKCC2 was identified in the kidney. Therefore, structure-activity studies on effects of bumetanide derivatives on NKCC2 are not available. EXPERIMENTAL APPROACH: In this study, the effect of a series of diuretically active bumetanide derivatives was investigated on human NKCC2 variant A (hNKCC2A) expressed in Xenopus laevis oocytes. KEY RESULTS: Bumetanide blocked hNKCC2A transport with an IC50 of 4 µM. There was good correlation between the diuretic potency of bumetanide and its derivatives in dogs and their inhibition of hNKCC2A (r2 = 0.817; P < 0.01). Replacement of the carboxylic group of bumetanide by a non-ionic residue, for example, an anilinomethyl group, decreased inhibition of hNKCC2A, indicating that an acidic group was required for transporter inhibition. Exchange of the phenoxy group of bumetanide for a 4-chloroanilino group or the sulfamoyl group by a methylsulfonyl group resulted in compounds with higher potency to inhibit hNKCC2A than bumetanide. CONCLUSIONS AND IMPLICATIONS: The X. laevis oocyte expression system used in these experiments allowed analysis of the structural requirements that determine relative potency of loop diuretics on human NKCC2 splice variants, and may lead to the discovery of novel high-ceiling diuretics.

Discovery of small molecules with vasodilating characteristics and adjustable hydrolytic behavior.

In this contribution the development of a new class of vasodilating compounds obtained by lead structure optimization is described. Three groups of compounds were synthesized and tested for their activity on various smooth muscle preparations of the guinea pig. Beside the lead compound 3a, the most interesting derivative was 1H-imidazole-1-carbothioic acid O-cyclohexyl ester hydrochloride (5b) with a good selective vasodilating potential on aorta and pulmonary artery rings (EC50 14 µM and 24 µM, respectively). Due to the properties of small molecules the hydrolysis behavior of the compounds can be easily adapted hence opening a new route in terms of duration of the agent's effect. With the aid of structure-activity relationship studies, structural motifs influencing the biological activity on isolated smooth muscle cell preparations of the synthesized compounds were proposed. The presented compounds offer good tools in identifying promising molecules as emergency therapy in myocardial infarction.

Enhanced Antiproliferative and Pro-apoptotic Activities of a Novel Curcumin-related Compound in Jurkat Leukemia T-Cells.

BACKGROUND/AIM: Inhibition of arachidonic acid metabolism by curcumin has been suggested to be a key mechanism for its anti-carcinogenic action. Recently, we reported on the synthesis of curcumin analogues and their evaluation as selective COX1 inhibitors. Two compounds (HP109/HP102) were selected for evaluation of their anti-proliferative and pro-apoptotic potential in Jurkat T-cells. MATERIALS AND METHODS: Jurkat T-cells were stimulated with phorbol 12-myristate 13-acetate/phytohemagglutinin (PMA/PHA) in the absence and presence of different concentrations of curcumin or HP109/HP102. Interleukin 2 (IL2) production and IL2 promoter activity were analyzed by enzyme-linked immunosorbent assay and a luciferase reporter assay, respectively. Proliferation and cell viability were monitored by 2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide assay, annexin -V/7-amino-actinomycin D staining and western blotting. RESULTS: HP102 was about 10-times more effective in blocking IL2 synthesis compared to curcumin. Enhanced effects of HP102 were also observed in reducing the proliferation rate and cell viability. In contrast to HP102, HP109 did not exhibit enhanced effects compared to curcumin. CONCLUSION: The curcumin analog HP102 had strongly improved the anti-proliferative and pro-apoptotic potential in Jurkat T-cells compared to curcumin.

Hydrogen sulphide induces HIF-1α and Nrf2 in THP-1 macrophages.

The transcription factor HIF-1α regulates the adaptive response of cells to hypoxia and oxidative stress. In addition, an important regulatory role for HIF-1α in immune reactions and inflammation is suggested. The present study attempts to investigate the effect of the gaseous signalling molecule hydrogen sulphide (H2S) on HIF-1α in THP-1 macrophages using the slow H2S releasing donor GYY4137. We found that H2S induced HIF-1α protein accumulation in THP-1 macrophages in a concentration-dependent manner. Western blot analysis of cell fractions showed that HIF-1α protein translocates into the nucleus and leads to an increase of its target protein glucose transporter-1 (GLUT-1). Activation of nuclear factor-κB (NF-κB), as well as secretion of the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), were reduced in the presence of H2S. These findings indicate that HIF-1α accumulation due to H2S was not triggered by the NF-κB pathway. The antioxidant pathway Nrf2/HO-1 (nuclear factor erythroid 2-related factor 2/heme oxygenase-1) was activated by H2S. Inhibition of the p38 mitogen-activated protein kinase (MAPK) reversed H2S mediated effects, suggesting that the p38 MAPK pathway may be involved in H2S induced HIF-1α/Nrf2 signalling pathways.

SAR-guided development and characterization of a potent antitumor compound toward B-cell neoplasms with no detectable cytotoxicity toward healthy cells.

Acute hematological diseases (leukemias and aggressive lymphomas) can be cured in approximately half of the patients, while the other patients die from their disease. Chronic leukemias and indolent lymphomas can be well controlled for years in most cases. However, the cure rate of these patients is low and the course of the disease is characterized by frequent recurrence. Therefore, novel agents for monotherapies or combination therapies still need to be explored. The presented study describes the identification of the chalcone derivative 15 on different types of human malignant cells of the lymphoid and myeloid lineage. Further experiments performed with compound 15 on peripheral blood mononuclear cells (PBMCs) of chronic lymphocytic leukemia (CLL) patients clearly stated a higher cytotoxicity in PBMC from CLL patients compared to healthy donors (HD). The newly identified chalcone derivative 15 showed a higher therapeutic potential than fludarabine, a drug already in use in lymphoma treatment.

Bumetanide is not capable of terminating status epilepticus but enhances phenobarbital efficacy in different rat models.

In about 20-40% of patients, status epilepticus (SE) is refractory to standard treatment with benzodiazepines, necessitating second- and third-line treatments that are not always successful, resulting in increased mortality. Rat models of refractory SE are instrumental in studying the changes underlying refractoriness and to develop more effective treatments for this severe medical emergency. Failure of GABAergic inhibition is a likely cause of the development of benzodiazepine resistance during SE. In addition to changes in GABAA receptor expression, trafficking, and function, alterations in Cl(-) homeostasis with increased intraneuronal Cl(-) levels may be involved. Bumetanide, which reduces intraneuronal Cl(-) by inhibiting the Cl(-) intruding Na(+), K(+), Cl(-) cotransporter NKCC1, has been reported to interrupt SE induced by kainate in urethane-anesthetized rats, indicating that this diuretic drug may be an interesting candidate for treatment of refractory SE. In this study, we evaluated the effects of bumetanide in the kainate and lithium-pilocarpine models of SE as well as a model in which SE is induced by sustained electrical stimulation of the basolateral amygdala. Unexpectedly, bumetanide alone was ineffective to terminate SE in both conscious and anesthetized adult rats. However, it potentiated the anticonvulsant effect of low doses of phenobarbital, although this was only seen in part of the animals; higher doses of phenobarbital, particularly in combination with diazepam, were more effective to terminate SE than bumetanide/phenobarbital combinations. These data do not suggest that bumetanide, alone or in combination with phenobarbital, is a valuable option in the treatment of refractory SE in adult patients.