Features of ZED1227: The First-In-Class Tissue Transglutaminase Inhibitor Undergoing Clinical Evaluation for the Treatment of Celiac Disease.

ZED1227 is a small molecule tissue transglutaminase (TG2) inhibitor. The compound selectively binds to the active state of TG2, forming a stable covalent bond with the cysteine in its catalytic center. The molecule was designed for the treatment of celiac disease. Celiac disease is an autoimmune-mediated chronic inflammatory condition of the small intestine affecting about 1-2% of people in Caucasian populations. The autoimmune disease is triggered by dietary gluten. Consumption of staple foods containing wheat, barley, or rye leads to destruction of the small intestinal mucosa in genetically susceptible individuals, and this is accompanied by the generation of characteristic TG2 autoantibodies. TG2 plays a causative role in the pathogenesis of celiac disease. Upon activation by Ca2+, it catalyzes the deamidation of gliadin peptides as well as the crosslinking of gliadin peptides to TG2 itself. These modified biological structures trigger breaking of oral tolerance to gluten, self-tolerance to TG2, and the activation of cytotoxic immune cells in the gut mucosa. Recently, in an exploratory proof-of-concept study, ZED1227 administration clinically validated TG2 as a "druggable" target in celiac disease. Here, we describe the specific features and profiling data of the drug candidate ZED1227. Further, we give an outlook on TG2 inhibition as a therapeutic approach in indications beyond celiac disease.

Novel inhibitor ZED3197 as potential drug candidate in anticoagulation targeting coagulation FXIIIa (F13a).

BACKGROUND: Factor XIII (FXIII) is the final enzyme of the coagulation cascade. While the other enzymatic coagulation factors are proteases, FXIII belongs to the transglutaminase family. FXIIIa covalently crosslinks the fibrin clot and represents a promising target for drug development to facilitate fibrinolysis. However, no FXIII-inhibiting compound has entered clinical trials. Here, we introduce the features of a peptidomimetic inhibitor of FXIIIa (ZED3197) as a potential drug candidate. METHODS: The potency of ZED3197 against FXIIIa and the selectivity against other human transglutaminases were characterized using transamidation and isopeptidase assays. The inhibition of fibrin crosslinking was evaluated by biochemical methods and thromboelastometry. Further, the pharmacology of the compound was explored in a rabbit model of venous stasis and reperfusion. RESULTS: ZED3197 proved to be a potent and selective inhibitor of human FXIIIa. Further, the compound showed broad inhibitory activity against cellular FXIIIA from various animal species. Rotational thromboelastometry in whole human blood indicated that the inhibitor, in a dose-dependent manner, prolonged clot formation, reduced clot firmness, and facilitated clot lysis without affecting the clotting time, indicating minimal impact on hemostasis. In vivo, the novel FXIIIa inhibitor effectively decreased the weight of clots and facilitated flow restoration without prolongation of the bleeding time. CONCLUSIONS: ZED3197 is the first drug-like potent compound targeting FXIIIa, a yet untapped target in anticoagulation. Due to the function of FXIII downstream of thrombin the approach provides minimal impact on hemostasis. In vivo data imply that the inhibitor dissociates an antithrombotic effect from increased bleeding tendency.

Probing the contribution of IKs to canine ventricular repolarization: key role for beta-adrenergic receptor stimulation.

BACKGROUND: In large mammals and humans, the contribution of IKs to ventricular repolarization is still incompletely understood. METHODS AND RESULTS: In vivo and cellular electrophysiological experiments were conducted to study IKs in canine ventricular repolarization. In conscious dogs, administration of the selective IKs blocker HMR 1556 (3, 10, or 30 mg/kg PO) caused substantial dose-dependent QT prolongations with broad-based T waves. In isolated ventricular myocytes under baseline conditions, however, IKs block (chromanols HMR 1556 and 293B) did not significantly prolong action potential duration (APD) at fast or slow steady-state pacing rates. This was because of the limited activation of IKs in the voltage and time domains of the AP, although at seconds-long depolarizations, the current was substantial. Isoproterenol increased and accelerated IKs activation to promote APD95 shortening. This shortening was importantly reversed by HMR 1556 and 293B. Quantitatively similar effects were obtained in ventricular-tissue preparations. Finally, when cellular repolarization was impaired by IKr block, IKs block exaggerated repolarization instability with further prolongation of APD. CONCLUSIONS: Ventricular repolarization in conscious dogs is importantly dependent on IKs. IKs function becomes prominent during beta-adrenergic receptor stimulation, when it promotes AP shortening by increased activation, and during IKr block, when it limits repolarization instability by time-dependent activation. Unstimulated IKs does not contribute to cellular APD at baseline. These data highlight the importance of the synergism between an intact basal IKs and the sympathetic nervous system in vivo.

In vivo electrophysiological effects of a selective slow delayed-rectifier potassium channel blocker in anesthetized dogs: potential insights into class III actions.

OBJECTIVES: This study evaluated the in vivo electrophysiological effects of a highly selective slow delayed-rectifier K+-current blocker, HMR 1556, to gain insights into the consequences of selectively inhibiting the slow delayed-rectifier current in vivo. METHODS: Atrial and ventricular effective refractory periods, sinus node recovery time, Wenckebach cycle-length, atrial fibrillation duration and electrocardiographic intervals were measured before and after intravenous HMR 1556. RESULTS: HMR 1556 increased atrial and ventricular refractory periods (e.g. by 6 +/- 4% and 27 +/- 6% at cycle lengths of 360 and 400 ms, respectively), QT intervals and sinus-node recovery times. Beta-adrenoceptor blockade with nadolol abolished all effects except those on ventricular refractoriness and changed positive use-dependent effects on refractoriness to reverse use-dependent ones. In the presence of dofetilide to block rapid delayed-rectifier current, HMR 1556 effects were potentiated (e.g. atrial and ventricular refractory periods increased by 26 +/- 3% and 34 +/- 3% at cycle lengths of 360 and 400 ms, respectively). HMR 1556 reduced vagal atrial fibrillation duration from 1077 +/- 81 to 471 +/- 38 s, an effect abolished by nadolol and greatly potentiated by dofetilide (duration 77 +/- 30 s). HMR 1556 increased Wenckebach cycle length only in the presence of dofetilide. CONCLUSIONS: Slowed delayed-rectifier current inhibition affects atrial repolarization, sinus node function and atrial fibrillation in vivo, but only in the presence of intact beta-adrenergic tone, and delays ventricular repolarization even when beta-adrenoceptors are blocked. The slow delayed-rectifier current is particularly important when rapid delayed-rectifier current is suppressed, illustrating the importance of repolarization reserve.

Inhibition of cardiac potassium currents by pentobarbital.

The inhibitory effects of the anesthetic barbiturate pentobarbital on the slow ( I(Ks)) and fast component ( I(Kr)) of cardiac delayed rectifier potassium currents ( I(K)) and on the inward rectifier potassium currents ( I(K1)) were examined in Xenopus oocytes expressing the human minK, human ether-á-go-go related gene (HERG) and guinea pig Kir2.2, respectively. Block of native I(K) ( I(Ks) and I(Kr)) and inward rectifier potassium current ( I(K1)) by pentobarbital was examined in guinea pig ventricular myocytes. In oocytes using the two electrode voltage clamp technique potassium currents of hminK-, HERG- and Kir2.2-expressing oocytes were inhibited by pentobarbital with IC50 values of 0.20, 1.58 and 0.54 mM, respectively. I(Ks) block was time- and voltage-independent and had no influence on activation at positive voltages although it shifted voltage-dependent activation to more positive voltages. Pentobarbital-induced HERG inhibition was not dependent on voltage but influenced the deactivation kinetics and shifted half-maximal activation to more negative voltages. In guinea pig cardiomyocytes, using the patch clamp technique, I(Ks) and I(Kr) were inhibited by pentobarbital with IC50 values of 0.18 mM and 2.75 mM, respectively. I(Kr) deactivation and I(Ks) activation kinetics were only slightly influenced by pentobarbital, if at all. Block of I(K1) was weakly voltage-dependent with IC(50) values of 0.26 mM (-40 mV) and 0.91 mM (-120 mV). The data show that pentobarbital suppresses both cloned ( I(K), I(Kir2.2)) and native ( I(K), I(K1)) cardiac potassium currents with the highest affinity for I(Ks).

Effects of the chromanol HMR 1556 on potassium currents in atrial myocytes.

PURPOSE: The chromanol HMR 1556 is a potent blocker of KvLQT1/minK potassium channels expressed in Xenopus oocytes. The compound is therefore a new class III antiarrhythmic drug with a distinct mechanism of action. However, the effect of HMR 1556 on atrial ion channels and the selectivity of block in the human heart has not been investigated. We tested the effects of HMR 1556 on repolarizing potassium currents in human and guinea pig atrial myocytes. METHODS AND RESULTS: Single atrial myocytes were isolated by enzymatic dissociation. Atrial potassium currents (I(Ks), I(Kr), in guinea pig, I(to), I(Kur), I(K1) in humans) were recorded at 36 degrees C in the whole cell mode of the patch clamp technique. HMR 1556 produced a concentration-dependent and reversible block of I(Ks) with a half maximal concentration (EC(50)) of 6.8 nmol/l. 10 micromol/l HMR 1556 almost completely inhibited I(Ks) (97.2+/-3.2%, n=6). Steady-state activation as well as kinetic properties of the current were not altered by HMR 1556. I(Kr) currents were not affected up to concentrations of 10 micromol/l. HMR 1556 did not inhibit other potassium currents in human atrium: I(to), I(Kur) and the classical inward rectifier potassium current I(K1) were not significantly affected up to concentrations that completely blocked I(Ks) (10 micromol/l). CONCLUSIONS: HMR 1556 is a highly-potent blocker of I(Ks) channels without exerting effects on other potassium currents involved in atrial repolarization. Given the potential advantages of I(Ks) vs. I(Kr) blockade, the drug's new mechanism of action warrants further investigation to clarify its role as an antiarrhythmic agent.

Blockers of the ATP-sensitive potassium channel SUR2A/Kir6.2: a new approach to prevent sudden cardiac death.

The cardiac ATP sensitive potassium channel (K(ATP) channel) SUR2A/Kir6.2 is an emerging target for antiarrhythmic intervention. This channel accounts for known electrophysiological derangements soon after the onset of myocardial ischemia. Consequently, blockers of this channel have the potential to prevent ischemic malignant arrhythmias and sudden cardiac death in humans. Since cardiac K(ATP) channels are closed at physiological intracellular ATP concentrations (ATP(i)) and open only when ATP(i) falls below a critical value, these agents do not affect the normal cardiac action potential and should be devoid of proarrhythmic side effects. Due to the existence of isoforms of this channel, mainly in vascular smooth muscle cells, pancreatic beta-cells and cardiac mitochondria, only specific blockers of SUR2A/Kir6.2 will offer a reasonable option for the treatment of cardiovascular patients at risk of sudden cardiac death. Presently known K(ATP) blockers are derived from diverse classes of compounds with antidiabetic sulfonylureas being their most prominent members. Retrospective evaluations of clinical studies with the sulfonylurea glibenclamide in diabetics revealed antifibrillatory activity to be an important additional effect of this class of compounds. However, for the safe treatment of arrhythmias nearly all presently known blockers lack sufficient selectivity, either within the target family or with respect to other ion channels modulating the cardiac action potential. The present article illustrates the new principle in terms of molecular biology and electrophysiology and summarizes all presently known K(ATP) blockers. As a highlight, first strategies to come to selective SUR2A/Kir6.2 blockers, such as HMR 1883, are reviewed.

Ototoxic side-effects of the I(Ks)-channel blocker HMR1556.

The inhibitor of I(Ks)-channels, HMR1556, a potentially antiarrhythmic drug, might possess ototoxic side-effects. The I(Ks)-channels are not only expressed in the heart but also in the stria vascularis of the inner ear and in the dark cells of the vestibular organ. Therefore possible effects of HMR1556 on hearing were studied in cats. Thresholds and intensity function of the cochlear action potential (CAP) were used as criteria. In addition to effects of the drug on heart rate and ECG, a substantial elevation of hearing thresholds and a shift in CAP intensity functions were observed. There was a clear dose-effect relationship. The hearing impairment observed showed a tendency for recovery. It is concluded that inhibitors of I(Ks)-channels may generally exert ototoxic effects provided they can reach the cochlear spaces.

KCNQ1-dependent transport in renal and gastrointestinal epithelia.

Mutations in the gene encoding for the K+ channel alpha-subunit KCNQ1 have been associated with long QT syndrome and deafness. Besides heart and inner ear epithelial cells, KCNQ1 is expressed in a variety of epithelial cells including renal proximal tubule and gastrointestinal tract epithelial cells. At these sites, cellular K+ ions exit through KCNQ1 channel complexes, which may serve to recycle K+ or to maintain cell membrane potential and thus the driving force for electrogenic transepithelial transport, e.g., Na+/glucose cotransport. Employing pharmacologic inhibition and gene knockout, the present study demonstrates the importance of KCNQ1 K+ channel complexes for the maintenance of the driving force for proximal tubular and intestinal Na+ absorption, gastric acid secretion, and cAMP-induced jejunal Cl- secretion. In the kidney, KCNQ1 appears dispensable under basal conditions because of limited substrate delivery for electrogenic Na+ reabsorption to KCNQ1-expressing mid to late proximal tubule. During conditions of increased substrate load, however, luminal KCNQ1 serves to repolarize the proximal tubule and stabilize the driving force for Na+ reabsorption. In mice lacking functional KCNQ1, impaired intestinal absorption is associated with reduced serum vitamin B12 concentrations, mild macrocytic anemia, and fecal loss of Na+ and K+, the latter affecting K+ homeostasis.

The new selective I(Ks)-blocking agent HMR 1556 restores sinus rhythm and prevents heart failure in pigs with persistent atrial fibrillation.

BACKGROUND: Antiarrhythmic drugs for treatment of atrial fibrillation in patients with heart failure are limited by proarrhythmia and low efficacy. Experimental studies indicate that the pure I(Ks) blocking agents chromanol 293b and HMR 1556 prolong repolarization more markedly at fast than at slow heart rates and during beta-adrenergic stimulation. These properties may overcome some of the above quoted limitations. METHODS AND RESULTS: Ten domestic swine underwent pacemaker implantation (PM) and atrial burst pacing to induce persistent AF. Four days after onset of persistent AF, pigs were randomized to HMR 1556 (30 mg/kg, p.o., 10 days) or placebo. All animals receiving HMR 1556 converted to SR (5.2 +/- 1.9 days), whereas placebo pigs remained in AF. Pigs treated with placebo developed high ventricular rates (297 +/- 5 bpm) and severe heart failure, whereas pigs treated with HMR 1556 remained hemodynamically stable. Left ventricular ejection fraction on the day of euthanization was significantly lower in the placebo compared to the HMR 1556 group (30 +/- 4% vs. 69 +/- 5%, p < 0.005). Similar results were seen with epinephrine levels (placebo 1563 +/- 193 pmol/l vs. HMR 613 +/-196 pmol/l, p < 0.05). Right atrial monophasic action potentials were significantly longer in the HMR 1556 compared to the placebo group (230 +/- 7 ms vs. 174 +/- 13 ms, p < 0.05). CONCLUSIONS: The new I(Ks) blocker HMR 1556 efficiently and safely restores SR and prevents CHF in a model of persistent AF. Restoration of SR is most likely linked to a marked prolongation of atrial repolarization even at high heart rates.

Effects of I(Ks) channel inhibitors in insulin-secreting INS-1 cells.

Potassium channels regulate insulin secretion. The closure of K(ATP) channels leads to membrane depolarisation, which triggers Ca(2+) influx and stimulates insulin secretion. The subsequent activation of K(+) channels terminates secretion. We examined whether KCNQ1 channels are expressed in pancreatic beta-cells and analysed their functional role. Using RT/PCR cellular mRNA of KCNQ1 but not of KCNE1 channels was detected in INS-1 cells. Effects of two sulfonamide analogues, 293B and HMR1556, inhibitors of KCNQ1 channels, were examined on voltage-activated outwardly rectifying K(+) currents using the patch-clamp method. It was found that 293B inhibited 60% of whole-cell outward currents induced by voltage pulses from -70 to +50 mV with a concentration for half-maximal inhibition (IC(50)) of 37 microM. The other sulfonamide analogue HMR1556 inhibited 48% of the outward current with an IC(50) of 7 microM. The chromanol 293B had no effect on tolbutamide-sensitive K(ATP) channels. Action potentials induced by current injections were broadened and after-repolarisation was attenuated by 293B. Insulin secretion in the presence but not in the absence of tolbutamide was significantly increased by 293B. These results suggest that 293B- and HMR1556-sensitive channels, probably in concert with other voltage-activated K(+) channels, influence action potential duration and frequency and thus insulin secretion.

Inhibitory effect of somatostatin on secretin-induced augmentation of the slowly activating K+ current (IKs) in the rat pancreatic acinar cell.

Recently, the slowly-activating voltage-dependent K+ channel current (IKs) has been reported in the rat pancreatic acinus (RPA). IKs is modulated positively by ACh and secretin, Ca2+ - and cAMP-mediated secretagogues, respectively. In this study, we investigated the effect of somatostatin (SS), a well-known inhibitory hormone of pancreatic fluid secretion, on I(Ks) in RPAs. The whole-cell patch clamp technique was applied to intact RPAs. Step-like depolarizations from -60 mV to above -40 mV induced IKs, a response blocked by the chromanols 293B [concentration for half-maximal inhibition (IC50) 5.3 microM), HMR-1556 (IC50 0.17 microM) and IKs 420 (IC50 2 nM). The application of secretin (5 nM), forskolin (5 microM) or 8-Br-cAMP (0.3 mM) increased the amplitude of IKs two- to fourfold. The addition of SS (1-100 nM) markedly suppressed the augmentation of IKs by secretin or forskolin but had no effect on IKs stimulated by 8-Br-cAMP, nor did SS block the Ca2+ -mediated augmentation of IKs by ACh. These results suggest that the effect of SS on IKs in the rat pancreatic acinar cell is mediated by inhibition of cAMP production, which may play a role in the negative regulation of the exocrine pancreas by SS.

HMR 1556, a potent and selective blocker of slowly activating delayed rectifier potassium current.

The slowly activating delayed rectifier potassium current (IKs) contributes prominently to ventricular repolarization of the cardiac action potential. Development of a selective IKs blocker is important for the elucidation of the physiologic and pathophysiologic relevance of IKs and the development of antiarrhythmic strategies. HMR 1556 [(3R,4S)-(+)-N-[3-hydroxy-2,2-dimethyl-6-(4,4,4-trifluorobutoxy) chroman-4-yl]-N-methylmethanesulfonamide] is a new chromanol derivative developed as a selective IKs blocker. Chromanol 293B, the most specific IKs blocker currently available, also inhibits the transient outward current (Ito). HMR 1556 was examined for its effects on IKs compared with rapidly activating delayed rectifier (IKr), inward rectifier (IK1), Ito, and L-type calcium (ICa.L) currents in canine left ventricular myocytes. HMR 1556 (0.5-500 nM ) inhibited IKs in a concentration-dependent manner (IC50 of 10.5 nM, compared with chromanol 293B's IC50 of 1.8microM). Inhibition of Ito was observed only at relatively high concentrations (IC50 of 33.9 microM comparable to chromanol 293B's IC of 38 microM). High concentrations of HMR 1556 also inhibited ICa.L (IC of 27.5 microM) and IKr (IC50 of 12.6 microM) while IK1 was unaffected. Our results indicate that HMR 1556 is superior to chromanol 293B in its potency and specificity for inhibition of IKs, making it a valuable experimental tool and a potential therapeutic agent.

Effects of chromanol 293B on transient outward and ultra-rapid delayed rectifier potassium currents in human atrial myocytes.

It is unclear whether chromanol 293B, a selective inhibitor of slow component of delayed rectifier K(+) current (I(Ks)), may affect other K(+) currents in human atrium. With whole-cell patch configuration, we evaluated effects of 293B on transient outward K(+) current (I(to1)) and ultra-rapid delayed rectifier K(+) current (I(Kur)) in isolated human atrial myocytes. It was found that 293B inhibited I(to1) and I(Kur) in a concentration-dependent manner. At 10 microM 293B suppressed I(to1) to 3.4 +/- 0.4 from 5.1 +/- 0.3 pA/pF (P < 0.01), and I(Kur) to 1.5 +/- 0.2 from 2.1 +/- 0.3 pA/pF (P < 0.01) at +50 mV. The inhibition of I(to1) and I(Kur) was independent of depolarizing voltage, and the concentration of 50% inhibition was 31.2 microM for I(to1), and 30.9 microM for I(Kur). 293B blocked I(to1) and I(Kur) with the same concentration range, and the significant effect was observed from the concentration of 1 microM. The maximum inhibitive effect was 88% for I(to1) and 96% for I(Kur) at 250 microM. Voltage dependence of activation and inactivation, and time-dependent recovery from inactivation of I(to1) were not altered by 293B; however, time to peak and time-dependent inactivation of I(to1) was significantly accelerated. The results indicate that 293B significantly inhibits the major repolarization K(+) currents I(to1) and I(Kur) in human atrial myocytes.

Heteromeric KCNE2/KCNQ1 potassium channels in the luminal membrane of gastric parietal cells.

Recently, we and others have shown that luminal K+ recycling via KCNQ1 K+ channels is required for gastric H+ secretion. Inhibition of KCNQ1 by the chromanol 293B strongly diminished H+ secretion. The present study aims at clarifying KCNQ1 subunit composition, subcellular localization, regulation and pharmacology in parietal cells. Using in situ hybridization and immunofluorescence techniques, we identified KCNE2 as the beta subunit of KCNQ1 in the luminal membrane compartment of parietal cells. Expressed in COS cells, hKCNE2/hKCNQ1 channels were activated by acidic pH, PIP2, cAMP and purinergic receptor stimulation. Qualitatively similar results were obtained in mouse parietal cells. Confocal microscopy revealed stimulation-induced translocation of H+,K+-ATPase from tubulovesicles towards the luminal pole of parietal cells, whereas distribution of KCNQ1 K+ channels did not change to the same extent. In COS cells the 293B-related substance IKs124 blocked hKCNE2/hKCNQ1 with an IC50 of 8 nM. Inhibition of hKCNE1- and hKCNE3-containing channels was weaker with IC50 values of 370 and 440 nM, respectively. In conclusion, KCNQ1 coassembles with KCNE2 to form acid-activated luminal K+ channels of parietal cells. KCNQ1/KCNE2 is activated during acid secretion via several pathways but probably not by targeting of the channel to the membrane. IKs124 could serve as a leading compound in the development of subunit-specific KCNE2/KCNQ1 blockers to treat peptic ulcers.

Tratamiento paliativo de estenosis tumorales inoperables de esófago y estómago con prótesis metálicas autoexpandibles / Paliativo treatment of inoperables esophageal and stomach tumoral stenosis with autoexpandable metalic prosthesis

En los pacientes con cáncer de esófago avanzado, el síntoma más importante es la disfagia. Una vez establecida la inoperabilidad de un cáncer de esófago, esposible ofrecer al paciente diferentes alternativas de paliación. Por vía endoscópica podemos implantar prótesis plásticas. Desde 1991 se dispone de prótesis metálicas autoexpandibles, las cuales tienen un sistema de implante pequeño y se expanden espontáneamente, siendo menos traumáticas. Sin embargo, la oclusión de la prótesis por crecimiento tumoral a través de la malla, es la complicación y desventaja más importante. Se presenta nuestra experiencia en 20 pacientes en los cuales se implantaron 22 prótesis. La indicación más frecuente fue por cáncer de esófago. Hubo mejoría significativa de la disfagia y 26 complicaciones que requirieron algún tipo de intervención terapéutica. La sobrevida promedio fue de 2,8 meses