Combined tyrosine and serine/threonine kinase inhibition by sorafenib prevents progression of experimental pulmonary hypertension and myocardial remodeling.

BACKGROUND: Inhibition of tyrosine kinases, including platelet-derived growth factor receptor, can reduce pulmonary arterial pressure in experimental and clinical pulmonary hypertension. We hypothesized that inhibition of the serine/threonine kinases Raf-1 (also termed c-Raf) and b-Raf in addition to inhibition of tyrosine kinases effectively controls pulmonary vascular and right heart remodeling in pulmonary hypertension. METHODS AND RESULTS: We investigated the effects of the novel multikinase inhibitor sorafenib, which inhibits tyrosine kinases as well as serine/threonine kinases, in comparison to imatinib, a tyrosine kinase inhibitor, on hemodynamics, pulmonary and right ventricular (RV) remodeling, and downstream signaling in experimental pulmonary hypertension. Fourteen days after monocrotaline injection, male rats were treated orally for another 14 days with sorafenib (10 mg/kg per day), imatinib (50 mg/kg per day), or vehicle (n=12 to 16 per group). RV systolic pressure was decreased to 35.0+/-1.5 mm Hg by sorafenib and to 54.0+/-4.4 mm Hg by imatinib compared with placebo (82.9+/-6.0 mm Hg). In parallel, both sorafenib and imatinib reduced RV hypertrophy and pulmonary arterial muscularization. The effects of sorafenib on RV systolic pressure and RV mass were significantly greater than those of imatinib. Sorafenib prevented phosphorylation of Raf-1 and suppressed activation of the downstream ERK1/2 signaling pathway in RV myocardium and the lungs. In addition, sorafenib but not imatinib antagonized vasopressin-induced hypertrophy of the cardiomyoblast cell line H9c2. CONCLUSIONS: The multikinase inhibitor sorafenib prevents pulmonary remodeling and improves cardiac and pulmonary function in experimental pulmonary hypertension. Sorafenib exerts direct myocardial antihypertrophic effects, which appear to be mediated via inhibition of the Raf kinase pathway. The combined inhibition of tyrosine and serine/threonine kinases may provide an option to treat pulmonary arterial hypertension and associated right heart remodeling.

Chromanol 293B binding in KCNQ1 (Kv7.1) channels involves electrostatic interactions with a potassium ion in the selectivity filter.

The chromanol 293B (293B, trans-6-cyano-4-(N-ethylsulfonyl-N-methylamino)-3-hydroxy-2,2-dimethyl-chroman) is a lead compound of potential class III antiarrhythmics that inhibit cardiac I(Ks) potassium channels. These channels are formed by the coassembly of KCNQ1 (Kv7.1, KvLQT1) and KCNE1 subunits. Although homomeric KCNQ1 channels are the principal molecular targets, entry of KCNE1 to the channel complex enhances the chromanol block. Because closely related neuronal KCNQ2 potassium channels are insensitive to the drug, we used KCNQ1/KCNQ2 chimeras to identify the binding site of the inhibitor. We localized the putative drug receptor to the H5 selectivity filter and the S6 transmembrane segment. Single residues affecting 293B inhibition were subsequently identified through systematic exchange of amino acids that were either different in KCNQ1 and KCNQ2 or predicted by a docking model of 293B in the open and closed conformation of KCNQ1. Mutant channel proteins T312S, I337V, and F340Y displayed dramatically lowered sensitivity to chromanol block. The predicted drug binding receptor lies in the inner pore vestibule containing the lower part of the selectivity filter, and the S6 transmembrane domain also reported to be important for binding of benzodiazepines. We propose that the block of the ion permeation pathway involves hydrophobic interactions with the S6 transmembrane residues Ile337 and Phe340, and stabilization of chromanol 293B binding through electrostatic interactions of its oxygen atoms with the most internal potassium ion within the selectivity filter.

Impaired left ventricular relaxation in type 2 diabetic rats is related to myocardial accumulation of N(epsilon)-(carboxymethyl) lysine.

Myocardial dysfunction in the absence of myocardial ischemia is frequent in patients with diabetes mellitus but the underlying pathomechanism is unclear. We investigated whether accumulation of advanced glycation end products (AGEs) in the diabetic myocardium is related to its functional abnormalities. In 11 male homozygous Zucker diabetic fatty rats (ZDF/Gmi-fa/fa) aged 37 weeks (OBESE) and 11 non-obese, non-diabetic littermates (LEAN), we measured left ventricular function (pressure-volume catheter) and levels of N(epsilon)-(carboxymethyl) lysine (CML), a prototypical AGE, in serum and the left ventricle (competitive enzyme linked immuno-assay). Overt diabetes mellitus (HbA1c > 9%) was present in all OBESE animals but not in LEAN. Systolic left ventricular function was not different between the groups, but the markers of left ventricular relaxation, dP/dt(min) and the relaxation constant tau, were impaired in OBESE. In parallel, CML levels were increased in serum (273 +/- 15 vs. 197 +/- 10 ng/ml, p<0.05) and in the left ventricle (18.4 +/- 1.1 vs. 12.5 +/- 2.0 ng/mg protein, p < 0.05) in OBESE compared to LEAN. There was a linear correlation between tau and the left ventricular CML levels (r = 0.65; p < 0.05). We conclude that type 2 diabetes is associated with predominant left ventricular diastolic dysfunction. Myocardial accumulation of advanced glycation end products may contribute to relaxation abnormalities in type 2 diabetes.

Biochemistry and pharmacology of novel anthranilic acid derivatives activating heme-oxidized soluble guanylyl cyclase.

The heme-enzyme soluble guanylyl cyclase (sGC) is an ubiquitous NO receptor, which mediates NO downstream signaling by the generation of cGMP. We studied the mechanism of action of the anthranilic acid derivatives 5-chloro-2-(5-chloro-thiophene-2-sulfonylamino-N-(4-(morpholine-4-sulfonyl)-phenyl)-benzamide sodium salt (HMR1766) (proposed international nonproprietary name, ataciguat sodium) and 2-(4-chloro-phenylsulfonylamino)-4,5-dimethoxy-N-(4-(thiomorpholine-4-sulfonyl)-phenyl)-benzamide (S3448) as a new class of sGC agonists. Both compounds activated different sGC preparations (purified from bovine lung, or crude from human corpus cavernosum) in a concentration-dependent and quickly reversible fashion (EC50 = 0.5-10 microM), with mixed-type activation kinetics. Activation of sGC by these compounds was additive to activation by NO donors, but instead of being inhibited, it was potentiated by the heme-iron oxidants 1H-[1,2,4]-oxdiazolo[3,4-a]quinoxalin-1-one (ODQ) and 4H-8-bromo-1,2,4-oxadiazolo(3,4-d) benz(b)(1,4)oxazin-1-one (NS2028), suggesting that the new compounds target the ferric heme sGC isoform. Protoporphyrin IX acted as a competitive activator, and zinc-protoporphyrin IX inhibited activation of heme-oxidized sGC by HMR1766 and S3448, whereas heme depletion of sGC by Tween 20 treatment reduced activation. Both compounds increased cGMP levels in cultured rat aortic smooth muscle cells; induced vasorelaxation of isolated endothelium-denuded rat aorta, porcine coronary arteries, and human corpus cavernosum (EC50 1 to 10 microM); and elicited phosphorylation of the cGMP kinase substrate vasodilator-stimulated phosphoprotein at Ser239. HMR1766 intravenous bolus injection decreased arterial blood pressure in anesthetized pigs. All of these pharmacological responses to the new compounds were enhanced by ODQ and NS2028. Our findings suggest that HMR1766 and S3448 preferentially activate the NO-insensitive heme-oxidized form of sGC, which exists to a variable extent in vascular tissues, and is a pharmacological target for these new vasodilator drugs.

Effects of combined inhibition of the Na+-H+ exchanger and angiotensin-converting enzyme in rats with congestive heart failure after myocardial infarction.

1 We investigated the single vs the combined long-term inhibition of Na(+)-H(+) exchanger-1 (NHE-1) and ACE in rats with congestive heart failure induced by myocardial infarction (MI). 2 Rats with MI were randomized to receive either placebo, cariporide (3000 p.p.m. via chow), ramipril (1 mg kg(-1) day(-1) via drinking water) or their combination for 18 weeks starting on day 3 after surgery. 3 Cardiac morphology and function was assessed by echocardiography and by means of a 2.0 F conductance catheter to determine left ventricular (LV) pressure volume relationships. 4 MI for 18 weeks resulted in an increase in LV end-diastolic diameter (LVDed) in the placebo-treated group when compared to sham (placebo: 1.1+/-0.04 cm; sham: 0.86+/-0.01; P<0.05). Combined inhibition of NHE-1 and ACE, but not the monotherapies, significantly reduced LVDed (1.02+/-0.02 cm). 5 Preload recruitable stroke work (PRSW), dp/dt(max) (parameter of systolic function) and end-diastolic pressure volume relationship (EDPVR, diastolic function) were significantly impaired in placebo-treated MI group (PRSW: 39+/-7 mmHg; dp/dt(max): 5185+/-363 mmHg s(-1); EDPVR: 0.042+/-0.001 mmHg microl(-1); all P<0.05). Cariporide treatment significantly improved PRSW (64+/-7 mmHg), dp/dt(max) (8077+/-525 mmHg s(-1)) and EDPVR (0.026+/-0.014 mmHg microl(-1)), and reduced cardiac hypertrophy in rats with MI. Combined inhibition of NHE-1 and ACE had even a more pronounced effect on PRSW (72+/-5 mmHg) and EDPVR (0.026+/-0.014 mmHg microl(-1)), as well as cardiac hypertrophy that, however, did not reach statistical significance compared to cariporide treatment alone. 6 The NHE-1 inhibitor cariporide significantly improved LV remodeling and function in rats with congestive heart failure induced by MI. The effect of cariporide was comparable or tended to be stronger (e.g. systolic function) compared to ramipril. Combined treatment with cariporide and ramipril tended to be more effective on LV remodeling in rats with heart failure than the single treatments. Thus, inhibition of the NHE-1 may be a promising novel therapeutic approach for the treatment of congestive heart failure.

Dietary bread crust advanced glycation end products bind to the receptor for AGEs in HEK-293 kidney cells but are rapidly excreted after oral administration to healthy and subtotally nephrectomized rats.

In renal HEK-293 cells, the dietary Maillard reaction compounds casein-linked Nepsilon-carboxymethyllysine (CML), CML, bread crust (BC), and pronyl-glycine (a key compound formed in association with the process-induced heat impact applied to bread dough) all showed activation of p38-MAP kinase. Expression of the C-terminus truncated receptor for advanced glycation end products (RAGE) resulted in a reduction of HEK-293-MAP kinase activation. As these findings suggested a RAGE-mediated activating effect of CML, BC, and pronyl-glycine on kidney cellular signal transduction pathways, an in vivo study was performed. Male Wistar rats were subjected to a sham operation (CTRL, n = 20) or to 5/6 nephrectomy (NX, n = 20). Both groups were randomized into two subgroups and fed 20 g of a diet containing either 25% by weight BC or wheat starch (WS). GC-MS analyses of CML, carboxyethyllysine (CEL), and pentosidine revealed increased levels of CML and CEL in the liver but decreased levels of CML in the kidneys of CTRL and NX rats fed the BC diet compared to those on the WS diet. However, urinary levels of CML were also elevated in the CTRL and NX rats on the BC diet, pointing to enhanced excretion of AGEs after BC administration. Although renal insufficiency in the NX rats was reflected by proteinuria, the renal handling of CML and, presumably, other AGEs was not impaired.

Long term Rho-kinase inhibition ameliorates endothelial dysfunction in LDL-Receptor deficient mice.

Chronic inhibition of Rho-kinase has been recently implicated in retardation of atherogenesis induced by high-fat diet in low-density lipoprotein receptor deficient (LDLR-/-) mice. However, it remains to be examined whether long-term Rho-kinase inhibition will reduce vascular dysfunction in this model. LDLR-/- mice on a high-fat diet were treated either with saline (LDLR-/-) or with the Rho-kinase inhibitor Fasudil (HA1077, 5-Isoquinolinesulfonyl homopiperazine, 100 mg/kg/day by gavage, LDLR-/- +Fasudil) for 10 weeks. Fasudil-treatment normalized endothelial function (measured by means of endothelium-dependent vasorelaxation) in LDLR-/- +Fasudil, to the level of controls (C57BL/6J). No tolerance toward Rho-kinase inhibition has been detected in Fasudil-treated animals. We conclude that long-term Rho-kinase inhibition normalizes endothelial function without development of tolerance.

Down-regulation of calpain 9 is linked to hypertensive heart and kidney disease.

Calpains are a family of 14 intracellular calcium-dependent proteases, which have been implicated in cardiovascular diseases. We aimed to analyze specifically the expressional regulation of the different calpain isoforms in hypertensive target organ damage. Using real-time PCR, we found calpain 6 and 9 down-regulated by more than 50% and the endogenous calpain inhibitor calpastatin up-regulated by 225%, respectively, in the hearts of Dahl salt-sensitive rats on a high salt (4% NaCl) compared to normal salt diet. On the protein level, calpain 9 but not calpastatin was regulated in the hypertensive target organs heart and kidney. Moreover, the myocardial expression of calpain 9 protein was inversely linked to left ventricular mass (r= -0.93, p<0.01), and renal expression of calpain 9 protein correlated inversely with albuminuria (r= -0.82, p<0.05). In the aorta, there was no regulation of calpain 9 on the protein level. We conclude that differential regulation of calpain 9 may play a role in hypertensive target organ damage.

Inhibition of Rho-kinase stimulates nitric oxide-independent vasorelaxation.

Vasoconstrictor factors, like urotensin, angiotensin and catecholamines, activate Rho-dependent serine-threonine kinase (Rho-kinase) and inhibition of this pathway represents a novel therapy for cardiovascular diseases with hypertensive syndrome. The disbalance of relaxing endothelial nitric oxide (NO)-producing and vasoconstrictive pathways can be especially important in diseases where hypertension is accompanied by endothelial dysfunction that compromises NO generation. However, a recent study reported that the efficacy of the Rho-kinase inhibitor (R)-(+)-trans-N-(4-Pyridyl)-4-(1-aminoethyl)cyclohexanecarboxamide (Y27632) is dramatically attenuated upon removal of endothelium or inhibition of endothelial NO synthase (eNOS). This raises the question whether Rho-kinase inhibition could be an effective treatment in case of hypertension associated with endothelial dysfunction. The purpose of the present study was to determine whether the vasorelaxing effect of Rho-kinase inhibition is mediated through eNOS-dependent mechanisms. We show here that in the models of genetically reduced endothelial NO production (eNOS-/- mice and spontaneous hypertensive rats (SHR)) or in models of pharmacologically reduced endogenous NO production (N(omega)-nitro-L-arginine methyl ester (LNAME) treatment), Rho-kinase inhibition induced a strong vasodilation and reduction of blood pressure indicating independence of Rho-kinase pathway from eNOS. An additional important finding of our study is that Rho-kinase inhibitors induce a strong vasorelaxation and blood pressure reduction upon intravenous injection not only in hypertensive but in normotensive animals, as well. Inhibition of Rho-kinase represents a promising possibility to treat hypertension that is accompanied by endothelial dysfunction.

Nephroprotection in Zucker diabetic fatty rats by vasopeptidase inhibition is partly bradykinin B2 receptor dependent.

1. Vasopeptidase inhibition (i.e., the simultaneous inhibition of both angiotensin-converting enzyme (ACE) and neutral endopeptidase) can ameliorate diabetic nephropathy. We investigated whether this nephroprotection is mediated by the bradykinin B2 receptor. 2. In all, 43 obese Zucker diabetic fatty (ZDF/Gmi-fa/fa) rats aged 21 weeks were separated into four groups and treated for 26 weeks with either placebo, the bradykinin B2 receptor antagonist icatibant (500 microg kg(-1) day(-1) s.c. infusion), the vasopeptidase inhibitor AVE7688 (45 mg kg(-1) day(-1) in chow), or AVE7688 plus icatibant. Nephropathy was assessed as albuminuria at age 31 and 39 weeks, and by histopathologic scoring at the end of the treatment period. 3. All animals had established diabetes mellitus (blood glucose >20 mmol l(-1)) and marked albuminuria at baseline. Blood glucose was not influenced by any treatment. Icatibant alone did not influence albuminuria (8.6+/-1.6 vs placebo 9.5+/-1.3 mg kg(-1) h(-1)). AVE7688 reduced albuminuria at week 31 markedly to 1.1+/-0.1 mg kg(-1) h(-1) and reduced glomerular and tubulo-interstitial kidney damage at week 47. In the AVE7688 plus icatibant group, proteinuria was significantly higher than in the AVE7688 only group (2.0+/-0.6 mg kg(-1) h(-1)), but still reduced compared to placebo. In addition, icatibant partly antagonized the tubulo-interstitial protection mediated by AVE7688. 4. We conclude that vasopeptidase inhibition provides nephroprotection in rats with type II diabetic nephropathy, which is partly mediated by bradykinin B2 receptor activation.

Inhibition of Na+-H+ exchange by cariporide reduces inflammation and heart failure in rabbits with myocardial infarction.

The aim of this study was to assess the effects of the Na+-H+ exchange inhibitor cariporide on left ventricular (LV) morphology and function as well as inflammation in rabbits with heart failure. Rabbits with myocardial infarction (MI) and sham controls were randomized to receive either standard chow or chow supplemented with cariporide for 9 weeks. LV morphology was determined by echocardiography. LV systolic and diastolic function was assessed under load-dependent and -independent conditions by analysis of LV pressure-volume loops using piezo-electric crystals. Plasma concentrations of C-reactive protein and aldosterone were measured. Rabbits with MI developed LV dilatation that was reduced by cariporide. Systolic and diastolic LV function was impaired in rabbits with MI when compared to sham, as indicated by a decreased dP/dtmax (MI: 3537 +/- 718 mmHg s(-1), sham: 5839 +/- 247 mmHg s(-1), P < 0.05), the load-independent preload recruitable stroke work (PRSW)(MI: 22 +/-7 mmHg, sham: 81 +/- 23 mmHg, P < 0.05) and a reduction in the time constant of relaxation tau (tau) (MI: 27+/-1 ms, sham: 17+/-1 ms, P < 0.05), and significantly improved by cariporide (dP/dtmax: 4586 +/- 374 mmHg s(-1), PRSW: 67 +/- 18 mmHg, tau: 20 +/- 2 ms; P < 0.05 vs MI/control). Induction of MI was associated with an increase in aldosterone and CRP, indicating activation of the neurohormonal and the inflammatory system that were largely reduced by cariporide. Cariporide improves LV morphology and function post MI and suppresses inflammation and neurohormonal activation in congestive heart failure (CHF). Na+-H+ exchange inhibition may represent a new pharmaceutical approach for the treatment of CHF.

Effects of acute and chronic treatment with the sodium hydrogen exchanger 1 (NHE-1) inhibitor cariporide on myocardial infarct mass in rabbits with hypercholesterolaemia.

We investigated the cardioprotective effect of acute and chronic sodium hydrogen exchanger 1 (NHE-1) inhibition with cariporide under pathological conditions in rabbits fed an atherogenic diet (0.25% cholesterol, 3% coconut oil), an experimental model of atherosclerosis. New Zealand White rabbits were fed over 4 weeks with normal diet or with atherogenic diet and randomized in 3 subgroups (n=7 in each group); placebo, acute cariporide (0.3 mg/kg, 10 min. before occlusion of left anterior descending coronary artery and chronic cariporide (4 weeks 0.1% in chow). In the final infarction experiments the animals were subjected to 30 min. of myocardial ischaemia by occlusion of a branch of the left anterior descending coronary artery followed by 2 hr of reperfusion. Infarct mass was evaluated by triphenyl-tetrazolium chloride staining and the infarct size expressed as a percentage of area at risk. Besides the assessment of aortic endothelium-dependent function aortic and cardiac vessels were inspected for atherosclerotic lesions. In cholesterol-fed rabbits, the infarct size was significantly increased when compared with normal diet animals (63+/-3% versus 41+/-3%). Acute cariporide treatment reduced the infarct size in normal diet rabbits to 14%+/-3% (66% decrease, P<0.05) as well as in atherogenic diet rabbits to 22+/-3% (65% decrease, P<0.05). Chronic treatment with cariporide also reduced the infarct size significantly: normal diet 19+/-2% (53% decrease, P<0.05), atherogenic diet 32+/-3% (49% decrease, P<0.05). Total cholesterol serum levels in rabbits with atherogenic diet were significantly higher (15.3+/-2.7 mmol/l) than those on a standard diet (0.65+/-0.08 mmol/l). Chronic cariporide treatment significantly attenuated the increase of serum cholesterol (7.9+/-1.9 mmol/l) and improved the lipoprotein pattern. Although the aortas and heart vessels of hypercholesterolaemic animals were without any histological evidence of atherosclerosis they developed endothelial dysfunction (reduced endothelium-dependent relaxation by ACh), which was prevented by chronic cariporide treatment. Acute and chronic treatment with the NHE-1 inhibitor cariporide significantly reduced infarct mass. This effect was associated with improved endothelial function.

Atrial-selective antiarrhythmic actions of novel Ikur vs. Ikr, Iks, and IKAch class Ic drugs and beta blockers in pigs.

BACKGROUND: The Kv1.5 channel, underlying IKur, is supposed to be atrial selective in pigs and humans. We investigated the effects of different potassium channel blockers, i.e. the IKur blockers AVE 0118, S9947 and S20951, with amiodarone (AM), dofetilide (DO), azimilide (AZ), ibutilide (IB), the IKs blocker HMR 1556, atropine (ATR), flecainide (FL), propafenone (PR), d,l-sotalol (SO), atenolol (ATE), and esmolol (ES), on the left and right atrial and ventricular refractoriness and left atrial vulnerability (LAV) in vivo in pigs. MATERIAL/METHODS: In pentobarbital-anesthetized pigs (n=81), atrial and ventricular effective refractory periods (ERPs) were measured with the S1-S2-extrastimulus-method and QTc time from electrocardiograms. LAV was assessed after S2-extrastimulus to the left atrium. RESULTS: All IKur blockers prolonged left stronger than right atrial ERP and did not change QTc. All IKr blockers predominantly prolonged the right vs. left atria. AM prolonged both atria equally, and ATR the left only. Pure beta blockers acted predominantly on the left atrium, as did FL and PR, while d,l-sotalol acted predominantly on the right. AVE 0118, S9947, S20951, ibutilide, and d,l-sotalol significantly decreased LAV (-100%, -100%, -82%, -53%, -42%; p<0.05), in contrast to all other drugs. CONCLUSIONS: The IKur blockers exhibited stronger effects on the left atrium, which itself has shorter refractoriness, but strikingly with no effect on ventricular repolarization, while IKr blockers, IKs blockers, and d,l-sotalol exerted predominantly right atrial effects and known ventricular effects. IKur blockers inhibited atrial tachyarrhythmias stronger than all available drugs. Therefore, IKur blockers seem to be promising new atrial-selective antiarrhythmic drugs.

Chronic vasopeptidase inhibition normalizes diabetic endothelial dysfunction.

Type 2 diabetes mellitus is a major cause of vascular morbidity but animal models for this disease have not been adequately characterized. We demonstrate that endothelial dysfunction is present in the Zucker diabetic fatty (ZDF) rat. Vasopeptidase inhibition with AVE7688 (7-[[(2S)-2-(acetylthio)-1-oxo-3-methylpropyl]amino]-1,2,3,4,6,7,8,12b-octahydro-6-oxo-(4S,7S,12bR)-pyrido[2,1-a][2]benzazepine-4-carboxic acid), 45 mg/kg/day in chow for 6 weeks, normalized acetylcholine mediated relaxation of mesenteric artery rings. Thus, chronic vasopeptidase inhibition may prevent vascular complications related to type 2 diabetes mellitus.

Molecular basis for Kv1.5 channel block: conservation of drug binding sites among voltage-gated K+ channels.

Kv1.5 channels conduct the ultrarapid delayed rectifier current (IKur) that contributes to action potential repolarization of human atrial myocytes. Block of these channels has been proposed as a treatment for atrial arrhythmias. Here we report a novel and potent inhibitor of Kv1.5 potassium channels, N-benzyl-N-pyridin-3-yl-methyl-2-(toluene-4-sulfonylamino)-benzamide hydrochloride (S0100176), which exhibits features consistent with preferential block of the open state. The IC50 of S0100176 for Kv1.5 expressed in Xenopus oocytes was 0.7 microm. Ala-scanning mutagenesis within the pore helix and the S6 segment, regions that form the walls of the central cavity, was combined with voltage clamp analysis to identify point mutations that altered drug affinity. This approach identified Thr-479, Thr-480, Val-505, Ile-508, and Val-512 as the most important residues for block by S0100176. Mutations of these key residues to Ala or other amino acids caused marked changes in the IC50 of S0100176 (p<0.01). For example, the IC50 of S0100176 increased 362-fold for T480A, 26-fold for V505A, 150-fold for I508A, and 99-fold for V512A. We used modeling to dock S0100176 into the inner cavity of a Kv1.5 pore homology model that was generated based on the crystal structure of KcsA. The docking predicted that the five residues identified by the Ala scan were positioned less than 4.5 A from the compound. Based on the homology models, the positions of the five amino acids identified to interact with S0100176 face toward the central cavity and overlap with putative binding sites for other blockers and voltage-gated potassium channels.

Atrial effects of the novel K(+)-channel-blocker AVE0118 in anesthetized pigs.

OBJECTIVES: AVE0118 is a novel blocker of the K(+) channels K(v)1.5 and K(v)4.3 which are the molecular basis for the human cardiac ultrarapid delayed rectifier potassium current (I(Kur)) and the transient outward current (I(to)). The objective of this study was to investigate the effect of AVE0118 on atrial refractoriness (ERP), left atrial vulnerability (LAV) and on left atrial monophasic action potentials (MAP) in pentobarbital anesthetized pigs in comparison to the selective I(Kr) blocker dofetilide in order to assess the therapeutic potential of the novel K(+) channel blocker for atrial fibrillation. METHODS: Atrial ERP was determined with the S1-S2-stimulus method in the free walls of left and right atrium at 240, 300 and 400 ms basic cycle length (BCL). The inducibility of mostly nonsustained atrial tachyarrhythmias by the premature S2 extrastimulus, which is very high in the left pig atrium and referred to as LAV, was evaluated before and after drugs. Left atrial epicardial MAP was recorded to study the influence of the potassium channel blockers on the time course of repolarization. Left ventricular epicardial MAP, ERP and QT interval were measured to investigate a possible effect of AVE0118 on ventricular repolarization. RESULTS: ERPs determined at 240, 300 and 400 ms BCL were significantly shorter in the left vs. right atrium (99+/-3, 106+/-4 and 113+/-3 ms vs. 133+/-4 ms, 142+/-4 and 149+/-5, respectively; p<0.001; n=21). AVE0118 administered i.v. dose-dependently prolonged the atrial ERP independent from rate and inhibited LAV (100% at 0.5 and 1 mg/kg) while having no effect at all on the corrected QT (QTc) interval. At 1 mg/kg (n=5) AVE0118 prolonged left vs. right atrial ERP by 49.6+/-4.1 ms vs. 37.7+/-9.7 ms (means+/-SEM of changes at 240, 300, and 400 ms BCL), respectively, corresponding to a relative increase of 53.2+/-6.2% vs. 27.6+/-6.8% (p<0.05 for percent increase of left vs. right atrial ERP). In a separate group of pigs (n=5) AVE0118 had no effect on left ventricular ERP at 333, 400 and 500 ms BCL and no effect on MAP duration and QT at 600 ms BCL. After 1 mg/kg of AVE0118 the atrial MAP was significantly prolonged already at 10% repolarization (P<0.05; n=7) reaching the maximum at 40% repolarization. In contrast to AVE0118 the effect of dofetilide (10 microg/kg) on atrial MAP started to become significant only at 60% repolarization (n=6) with a maximum increase at 90%. Dofetilide, which prolonged the QTc interval by 16.9% (P<0.001), had a significantly stronger effect on right (34.7+/-5 ms) vs. left atrial ERP (23.5+/-7 ms) at 300 ms BCL, respectively, but did not significantly inhibit LAV (14%; n=6). CONCLUSION: The novel K(+) channel blocker AVE0118 prolonged atrial ERP and showed strong atrial antiarrhythmic efficacy with no apparent effect on ventricular repolarization in pigs in vivo.

Vasopeptidase inhibition prevents nephropathy in Zucker diabetic fatty rats.

BACKGROUND: Blocking the renin-angiotensin system is an established therapeutic principle in diabetic nephropathy. We investigated whether inhibition of both neutral endopeptidase and ACE (vasopeptidase inhibition) can prevent functional and morphological features of nephropathy in the Zucker diabetic fatty (ZDF) rat, an animal model of type II diabetes. METHODS: Homozygous (fa/fa) ZDF rats (each n=15) aged 10 weeks were treated with placebo, ramipril (1 mg/kg/day in drinking water), or the vasopeptidase inhibitor AVE7688 (45 mg/kg/day in chow). Metabolic parameters and renal function (metabolic cages) were assessed at baseline (age 10 weeks), and at age 17, 27, and 37 weeks. Twenty heterozygous animals (fa/-) served as lean, nondiabetic controls. At age 37 weeks, the animals were sacrificed and the kidneys analyzed histopathologically. RESULTS: Overt diabetes mellitus (blood glucose >20 mmol/l) was established at age 17 weeks in all homozygous ZDF rats. In the placebo group, urinary protein excretion increased progressively from 8+/-1 (baseline) to 342+/-56 mg/kg/day (week 37) whereas diabetes and proteinuria were absent in the lean control group. Ramipril tended to reduce albuminuria and morphological damage (p=ns) but AVE7688 virtually prevented albuminuria (33+/-12 mg/kg/day, p<0.05 vs. ZDF placebo) and drastically reduced the incidence and severity of glomerulosclerosis and tubulointerstitial damage. CONCLUSIONS: In ZDF rats, development of diabetes mellitus is accompanied by functional and morphological kidney damage that resembles human diabetic nephropathy. Diabetic nephropathy can be prevented by chronic vasopeptidase inhibition.

Effect of chronic treatment with the vasopeptidase inhibitor AVE 7688 and ramipril on endothelial function in atherogenic diet rabbits.

Cardiovascular disease is the major cause of death in Western nations, although improved possibilities regarding diagnosis and therapy now exist. Endothelial dysfunction is triggered by cardiovascular risk factors such as hypercholesterolaemia, hypertension, adiposity and smoking, contributing to the common endpoint of atherosclerosis. This study examined the pharmacological effects of angiotensin-converting enzyme (ACE) and combined ACE-neutral endopeptidase (NEP) (vasopeptidase) inhibitors on endothelial dysfunction in the model of hyperlipidaemic rabbits. The focus of the study was to assess endothelial function after treatment with the ACE-NEP inhibitor AVE 7688 (30 mg/kg/day) in comparison to the ACE inhibitor (ACE-I) ramipril (1 mg/kg/day). Different parameters, such as endothelial function, blood pressure (BP), expansion of plaques, endothelial nitric oxide (NO) and superoxide (O2-) release and plasma levels of various lipidaemic parameters were analysed. Control groups consisted of one group fed only with normal diet, one group fed only with atherogenic diet and the direct control group fed with varied diets (six weeks atherogenic diet followed by 12 weeks normal diet). Since for the treatment of atherosclerosis, a change in feeding is absolutely necessary, in the present study, at the start of the treatments with AVE 7688 and ramipril, the rabbits food was changed to a normal diet. At the end of the study, mean arterial blood pressure (MAP) was measured in the anaesthetised animals. The values in standard, atherogenic and varied diet-fed rabbits were around 73 2 mmHg. Angiotensin I (Ang I) given intravenous (i.v.) induced a strong increase in MAP of about 20%. In both the treated groups Ang I-induced BP increase was inhibited. In contrast, i.v. bradykinin led to a strong reduction in MAP in both the treated groups of around 50%. Six weeks feeding with an atherogenic diet in the rabbits induced an enduring endothelial dysfunction despite the food subsequently being changed to a normal chow. All measured parameters indicated a significant favourable effect on endothelial dysfunction as a result of the two treatment regimens. Endothelial function measured in the organ chamber showed somewhat greater improvement in the ACE-NEP treated group than in the ACE-I treated group. The treatment with ramipril, as well as with AVE 7688, restored endothelial function by increasing the ratio of NO to O2- concentration and bioavailability of NO. In this study, a similar protective effect on endothelial function was shown by ACE-NEP inhibition as already seen with ACE inhibitors in an animal model of atherosclerosis.

NHE-1 inhibition: from protection during acute ischaemia/reperfusion to prevention/reversal of myocardial remodelling.

The myocardial Na(+)/H(+) exchanger isoform 1 (NHE-1) represents a major H(+) extrusion mechanism for intracellular pH (pH(i)) regulation especially during ischaemia and early reperfusion. Paradoxically, however, its activation contributes to induction of cell injury because Na(+)/H(+) exchange is coupled closely to elevations in intracellular [Ca(2+)] through the Na(+)/Ca(2+) exchanger. NHE-1 is exquisitely sensitive to intracellular acidosis but other factors may have also stimulatory effects via phosphorylation-dependent processes, like autocrine and paracrine agents as well as hormonal factors such as endothelin-1, angiotensin II and alpha-1-adrenoceptor agonists. In addition, phosphorylation-independent NHE-1 activation mechanisms are known, e.g. cell shrinkage. To date at least 8 NHE isoforms have been identified and designated as NHE-1-8. All, except NHE-6 and NHE-7, which are located intracellularly, are restricted to the sarcolemmal membrane. The NHE-1 subtype is the predominant isoform in the heart, but NHE-6 is also expressed in the heart. Newly developed, selective NHE-1 inhibitors possess potent cardioprotective properties. The efficacy of NHE-1 inhibitors in experimental studies with ischaemia/reperfusion has led to clinical trials for the evaluation of these agents in high-risk patients with coronary artery disease (GUARDIAN Trial) and acute myocardial infarction (ESCAMI Trial). The GUARDIAN trial demonstrated only for the coronary artery by-pass graft (CABG) patient population a reduction in the primary cardiovascular endpoint (death and reoccurring myocardial infarction). However, recent evidence also suggests that NHE-1 inhibition may be conducive to attenuation of remodelling processes after myocardial infarction, independently of infarct size reduction and blood pressure. In addition, in separate preclinical studies, the NHE-1 inhibitor cariporide also prevented and/or caused regression of age-related and hypertension-induced myocardial fibrosis and hypertrophy. NHE-1 inhibitors thus offer substantial promise for clinical development for attenuation of both a) acute responses to myocardial injury, b) chronic post-infarct and hypertension- and age-related responses resulting in the development of heart failure.

Fluid shear stress differentially regulates gpr3, gpr6, and gpr12 expression in human umbilical vein endothelial cells.

Fluid shear stress is a major factor involved in the control of gene expression in vascular endothelial cells. Sphingosine 1-phosphate has emerged as a multifaceted regulator of endothelial cell function and its high affinity S1P1 receptor is one among the many shear stress regulated genes. We recently identified the orphan G protein coupled receptors gpr3, gpr6 and gpr12 as additional S1P receptors. Here, we investigated their expression in various human endothelial and vascular smooth muscle cell lines via RT-PCR and western blot analysis. We next sought to determine the role of fluid shear stress in the regulation of expression of gpr3, gpr6 and gpr12 by using human umbilical vein endothelial cells (HUVECs). Laminar shear stress (12dyne/cm2) did not significantly increase gpr3, gpr6 and gpr12 mRNA after 1, 2, 4, 6, and 8 hrs of application of elevated pressure as determined by quantitative Taqman RT-PCR analysis. In contrast, gpr3 and gpr12 protein were increased after 12 hrs of shear stress by 95% and 40%, respectively. gpr6 mRNA and protein were absent in HUVECs as determined by Taqman and western blot techniques. Our results suggest that shear stress regulates gpr3 and gpr12 but not gpr6 expression and that regulation does not occur transcriptionally but posttranslationally. gpr3 and gpr12 may therefore add to the repertoire of S1P receptors, translating extracellular S1P effects into intracellular signals in human endothelial cells.