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P Schelling

Publications and source records attributed to P Schelling.

At least 19 recordsLinked to original sources

A spectrophotometric assay for quantitative determination of kcat of herpes simplex virus type 1 thymidine kinase substrates.

A simple method to determine the in vitro catalytic turnover constant of several substrates of herpes simplex virus type 1 thymidine kinase is presented in this study. The method is based on a continuous spectroscopic enzyme-coupled assay and allows one to monitor the herpes simplex virus type 1 thymidine kinase activity in the presence of unlabeled substrates. A clear correlation between the catalytic turnover constant and the rate of decrease in absorbance over time during the assay has been demonstrated. Exploiting this correlation, this method has been used to determine rapidly and precisely the catalytic turnover constant of antiviral lead compounds not readily available in the radioactive labeled form.

Animals↗

The rational of catalytic activity of herpes simplex virus thymidine kinase. a combined biochemical and quantum chemical study.

Most antiherpes therapies exploit the large substrate acceptance of herpes simplex virus type 1 thymidine kinase (TK(HSV1)) relative to the human isoenzyme. The enzyme selectively phosphorylates nucleoside analogs that can either inhibit viral DNA polymerase or cause toxic effects when incorporated into viral DNA. To relate structural properties of TK(HSV1) ligands to their chemical reactivity we have carried out ab initio quantum chemistry calculations within the density functional theory framework in combination with biochemical studies. Calculations have focused on a set of ligands carrying a representative set of the large spectrum of sugar-mimicking moieties and for which structural information of the TK(HSV1)-ligand complex is available. The k(cat) values of these ligands have been measured under the same experimental conditions using an UV spectrophotometric assay. The calculations point to the crucial role of electric dipole moment of ligands and its interaction with the negatively charged residue Glu(225). A striking correlation is found between the energetics associated with this interaction and the k(cat) values measured under homogeneous conditions. This finding uncovers a fundamental aspect of the mechanism governing substrate diversity and catalytic turnover and thus represents a significant step toward the rational design of novel and powerful prodrugs for antiviral and TK(HSV1)-linked suicide gene therapies.

Antiviral Agents↗

Na(+)/H(+) exchange inhibition prevents endothelial dysfunction after I/R injury.

Whereas inhibition of the Na(+)/H(+) exchanger (NHE) has been demonstrated to reduce myocardial infarct size in response to ischemia-reperfusion injury, the ability of NHE inhibition to preserve endothelial cell function has not been examined. This study examined whether NHE inhibition could preserve endothelial cell function after 90 min of regional ischemia and 180 min of reperfusion and compared this inhibition with ischemic preconditioning (IPC). In a canine model either IPC, produced by one 5-min coronary artery occlusion (1 x 5'), or the specific NHE-1 inhibitor eniporide (EMD-96785, 3.0 mg/kg) was administered 15 min before a 90-min coronary artery occlusion followed by 3 h of reperfusion. Infarct size (IS) was determined by 2,3,5-triphenyl tetrazolium chloride staining and expressed as a percentage of the area-at-risk (IS/AAR). Endothelial cell function was assessed by measurement of coronary blood flow in response to intracoronary acetylcholine infusion at the end of reperfusion. Whereas neither control nor IPC-treated animals exhibited a significant reduction in IS/AAR or preservation of endothelial cell function, animals treated with the NHE inhibitor eniporide showed a marked reduction in IS/AAR and a significantly preserved endothelial cell function (P < 0.05). Thus NHE-1 inhibition is more efficacious than IPC at reducing IS/AAR and at preserving endothelial cell function in dogs.

Acetylcholine↗

Nucleoside binding site of herpes simplex type 1 thymidine kinase analyzed by X-ray crystallography.

The crystal structures of the full-length Herpes simplex virus type 1 thymidine kinase in its unligated form and in a complex with an adenine analogue have been determined at 1.9 A resolution. The unligated enzyme contains four water molecules in the thymidine pocket and reveals a small induced fit on substrate binding. The structure of the ligated enzyme shows for the first time a bound adenine analogue after numerous complexes with thymine and guanine analogues have been reported. The adenine analogue constitutes a new lead compound for enzyme-prodrug gene therapy. In addition, the structure of mutant Q125N modifying the binding site of the natural substrate thymidine in complex with this substrate has been established at 2.5 A resolution. It reveals that neither the binding mode of thymidine nor the polypeptide backbone conformation is altered, except that the two major hydrogen bonds to thymidine are replaced by a single water-mediated hydrogen bond, which improves the relative acceptance of the prodrugs aciclovir and ganciclovir compared with the natural substrate. Accordingly, the mutant structure represents a first step toward improving the virus-directed enzyme-prodrug gene therapy by enzyme engineering.

Adenine↗

Inhibitors of ischemic preconditioning do not attenuate Na+/H+ exchange inhibitor mediated cardioprotection.

Pharmacologic inhibition of the K(ATP) channel with sulfonylureas or the adenosine receptor with methylxanthines has been shown to attenuate ischemic preconditioning (IPC). Both classes of compounds are widely used clinically, and several reports have demonstrated adverse outcomes in patients taking sulfonylureas. Recently inhibition of the sodium/hydrogen exchanger isozyme-1 (NHE-1) has been shown to be equal to IPC at providing myocardial protection in dogs and may be an alternative to IPC in patients taking sulfonylureas or methylxanthines. However, no experiments have examined the pharmacologic overlap between IPC and NHE-1 inhibitor-mediated cardioprotection in dogs. With an in vivo canine infarct model in which the left anterior descending coronary artery was occluded for 60 min and reperfused for 3 h, neither the K(ATP) channel antagonist glibenclamide nor the adenosine-receptor antagonist PD 115199 attenuated NHE-1 inhibitor-mediated reduction in infarct size expressed as a percentage of the area at risk produced by EMD 85131 (Control, 24.2 +/- 3.6%; EMD 85131, 6.4 +/- 2.3%; PD 115199 + EMD 85131, 6.6 +/- 2.4%; glibenclamide + EMD 85131, 3.5 +/- 1.2%). NHE-1 inhibition and IPC do not overlap pharmacologically, and NHE-1 inhibition may be an alternative for cardioprotection in patients taking sulfonylureas or methylxanthines.

Animals↗

Endothelin antagonists: discovery of EMD 122946, a highly potent and orally active ETA selective antagonist.

The discovery, in vitro and in vivo studies of the highly potent ETA antagonist EMD 122946 are presented. This compound displayed high binding affinity and functional antagonism [IC50 = 3.2 x 10(-11) M, pA2 = 9.5 (ETA)] and inhibited the ET-1 induced pressor response in pithed rats with an ED50 of 0.3 mg/kg. In conscious spontaneously hypertensive rats and in DOCA-salt hypertensive rats the compound lowered mean blood pressure with an ED50 of 0.06 mg/kg. EMD 122946 exhibited high bioavailability in rats and monkeys.

Animals↗

A new sodium/hydrogen exchange inhibitor, EMD 85131, limits infarct size in dogs when administered before or after coronary artery occlusion.

Administration of inhibitors of the Na+/H+ exchanger (NHE) have been shown to produce cardioprotective effects in a number of animal models of ischemia-reperfusion injury; however, controversy still exists as to the efficacy of these agents when administered just before reperfusion. To address this question, the efficacy of several doses of a new selective NHE-1 isoform inhibitor (IC50 for inhibition of 22Na uptake in NHE-1 expressing mouse fibroblast cells = 10.4 +/- 1.0 nM), EMD 85131 (2-methyl-5-methylsulfonyl-1-(1-pyrrollyl)-benzoyl-guanidine), was tested in a canine infarct model in which the left anterior descending coronary artery was occluded for 60 min followed by 3 hr of reperfusion. EMD 85131 (0.75 or 3.0 mg/kg) was infused for 15 min before left anterior descending occlusion or 15 min before reperfusion. Infarct size was determined by use of the triphenyltetrazolium chloride histochemical stain and was expressed as a percent of the area at risk. EMD 85131 (0.75 or 3.0 mg/kg) administered before left anterior descending occlusion produced a marked (*P < .05) and dose-related reduction in IS/AAR (24.3 +/- 3.6, control; 9.3 +/- 3.4%, EMD 0.75; 6.4 +/- 2.3%, EMD 3.0). These two doses of EMD also produced significant (*P < .05) reductions in infarct size/area at risk (12.2 +/- 2.1%, EMD 0.75; 13.0 +/- 2.9%, EMD 3.0) when administered 15 min before reperfusion. These results suggest that selective NHE-1 inhibitors are able to markedly reduce infarct size when given before or during ischemia and also suggest that these compounds may have clinical utility when administered after the initiation of an ischemic insult.

Animals↗

In swine myocardium, the infarct size reduction induced by U-89232 is glibenclamide sensitive: evidence that U-89232 is a cardioselective opener of ATP-sensitive potassium channels.

We determined whether U-89232, a derivative of the ATP-sensitive potassium (KATP) channel opener cromakalim, is cardioselective and whether its action on the myocardium is still sensitive to glibenclamide. Experiments were performed in open-chest pigs subjected to a 60-min occlusion of the left anterior descending coronary artery (LADCA) and to 2 h of reperfusion. Four groups of animals were studied (n = 6 each). Animals received either U-89232, 3 mg/kg i.v. over a 15-min period (U), or glibenclamide, a selective KATP channel blocker, 1 mg/kg i.v. over a 15-min period (GLI) before the LADCA occlusion. In the GLI + U group, first glibenclamide (1 mg/kg/15 min) and then U-89232 (3 mg/kg/15 min) were infused before the 60 min of ischemia. Saline-treated animals served as controls (CON). Hemodynamic parameters were continuously monitored. Regional contractile wall function was quantified with ultrasonic crystals aligned to measure wall thickening. At the end of the protocol, infarct size (IS, as percentage of risk region) was determined by incubating the myocardium with p-nitrobluetetrazolium. With comparable myocardium at risk, infusion of U-89232 before 60 min of LADCA occlusion significantly reduced infarct size (IS, 18.5 +/- 3.7%; p < 0.001 vs. 63.2 +/- 3.3% for the controls), whereas glibenclamide had no effect on infarct size (IS, 69.5 +/- 4.4%). The administration of glibenclamide before U-89232 infusion blocked the infarct size-reducing effect of U-89232 [IS, 61.2 +/- 9.1 (NS) vs. controls and p < 0.001 vs. U]. Infusion of U-89232 had no effect on hemodynamic parameters or on regional wall function. At least in a pig model, U-89232 appears to be a cardioselective KATP channel opener, because in the absence of hemodynamic alterations, it exhibits a profound cardioprotective effect, which is fully reversible by blocking KATP channels.

Animals↗

In vivo evidence of positive inotropism of EMD 57033 through calcium sensitization.

The previous separation of the racemic cardiotonic thiadiazinone derivative EMD 53998 yielded two enantiomers with different pharmacologic properties: EMD 57,033, a potent Ca2+ sensitizer with some residual phosphodiesterase III (PDE III) inhibition, and EMD 57,439, a pure PDE III inhibitor. Although numerous in vitro studies demonstrated the ability of EMD 57,033 to increase the responsiveness of cardiac contractile proteins to Ca2+, in vivo evidence for such an action is lacking. Because there is no possibility of directly proving Ca2+ sensitization in vivo, we attempted to exclude PDE III inhibition as a major contributing component of the positive inotropic action of EMD 57,033. In anesthetized rats, EMD 57,033 increased left ventricular (LV) first derivative of change in systolic pressure over time (dP/dt max) without affecting blood pressure. In contrast, the PDE III-inhibitory enantiomer EMD 57,439 decreased blood pressure. The pattern of hemodynamic effects in anesthetized dogs revealed similar differences between EMD 57,033 and PDE inhibitors. Thus the increase in LV dP/dt max in response to EMD 57,033 was not accompanied by changes of heart rate and blood pressure. As expected for PDE inhibitors, pimobendan and milrinone increased cardiac contractile force in dogs, concomitant, however, with tachycardia, hypotension, and a decrease in total peripheral resistance. When regional contractility was measured separately in two different areas of the dog myocardium, the positive inotropic action of the PDE inhibitors pimobendan and milrinone was antagonized by local coronary infusion of acetylcholine. The cardiotonic effect of the Ca2+ sensitizer EMD 57,033 was entirely resistant to inhibition by acetylcholine. In conscious dogs, beta-blockade markedly attenuated the increase in LV dP/dt max produced by two different doses of the PDE III inhibitor EMD 57,439. In contrast, a dose of EMD 57,033 equieffective in positive inotropic action with the lower dose of EMD 57,439 remained unaffected by < b tau-blockade. We concluded (a) that EMD 57,033 increases cardiac contractile force in two species in vivo, (b) that this action is independent of the cardiac cyclic adenosine monophosphate (AMP) system, (c) that EMD 57,033 does not reduce blood pressure and increase heart rate, an action indicative of PDE inhibition, and (d) that, on the basis of numerous previous in vitro findings, the mechanism of action of EMD 57,033, also in vivo, is consistent with sensitization of the cardiac myofibrils to Ca2+. Of special importance is the finding that this Ca2+ sensitizer at appropriate doses may be able to improve systolic function without adverse effects on diastolic function, as indicated by a slight decrease in left ventricular end-diastolic pressure.

Anesthesia, General↗

Non-peptide angiotensin II receptor antagonists: synthesis and biological activity of a series of novel 4,5-dihydro-4-oxo-3H-imidazo[4,5-c]pyridine derivatives.

A series of novel non-peptide angiotensin II receptor antagonists containing a 2,3,5-trisubstituted 4,5-dihydro-4-oxo-3H-imidazo[4,5-c]pyridine was prepared via several synthetic routes. Their affinity for angiotensin II receptors was established in a binding assay experiment and in an isolated-organ test. Molecules with small alkyl groups at C-2 and the (methylbiphenylyl)tetrazole moiety at N-3 were the preferred compounds with affinities and potencies in the nanomolar range. Variations at the N-5 position modulate the activity. Substitution at N-5 with various benzyl groups led to derivatives with in vitro potencies in the nanomolar range, which were equivalent to those of losartan in these assays. Replacement of the N-5 hydrogen with acetic acid esters or, in particular, acetamides gave molecules with increased activity. The most potent was 2-butyl-4,5-dihydro-4-oxo-3-[[2'-(1H-tetrazol-5-yl)-4- biphenylyl]methyl]-3H-imidazo[4,5-c]pyridine-5- (N,N-diethylacetamide) (14u), which is superior to L-158,809 in vitro. Two prototypes were selected as their potassium salts for in vivo testing as antihypertensives. Compounds 14a (EMD 61,650) and 14q (EMD 66,684) reduced blood pressure dose dependently in spontaneously hypertensive rats when administered iv. In this assay, acetamide 14q is superior to losartan.

Angiotensin Receptor Antagonists↗

Effect of bimakalim (EMD 52692), an opener of ATP sensitive potassium channels, on infarct size, coronary blood flow, regional wall function, and oxygen consumption in swine.

OBJECTIVE: The aim was to assess whether bimakalim, an opener of ATP sensitive potassium channels, can reduce infarct size in swine myocardium. METHODS: Experiments were performed in open chest pigs subjected to a 60 min occlusion of a branch of the left anterior descending coronary artery and to 2 h reperfusion. Five groups of animals were studied. In seven animals bimakalim infusion (3 micrograms.kg-1 bolus over 5 min followed by 0.1 microgram.kg-1.min-1) was started at 45 min of coronary occlusion and continued until 60 min of reperfusion (group A), while in seven other animals the bimakalim infusion was started 15 min before occlusion and also ended at 60 min of reperfusion (group B). In a further seven animals bimakalim infusion was started 15 min before coronary occlusion, but was stopped at the onset of ischaemia (group C). In the fourth group of animals (n = 7), a hydralazine infusion (0.2 mg.kg-1 over 15 min) was started 15 min before the occlusion and also terminated at the start of occlusion. The dose of hydralazine was chosen such that it lowered arterial pressure to the same extent as bimakalim. A fifth group of animals (n = 7) received the vehicle and served as controls. At the end of the protocol, infarct size (as percent of risk region) was determined by incubating myocardium with p-nitrobluetetrazolium. Regional myocardial oxygen consumption (MVO2) was calculated as the product of coronary blood flow (electromagnetic flowmeter) and the difference in the oxygen contents of the aorta and the interventricular vein accompanying the left anterior descending coronary artery. Regional wall function was quantified with ultrasonic crystals aligned to measure wall thickening (% delta WT). RESULTS: In all pigs in which bimakalim treatment was started prior to the 60 min coronary occlusion, infarct size was significantly reduced [B: 22.4(SEM 4.5)%; C: 35.3(6.6)%] compared with 60.4(5.2)% in pigs subjected to 60 min of ischaemia only (p < 0.05); drug-induced potassium channel opening during reperfusion had no effect [A: 56.6(4.1)%]. Treatment with hydralazine did not reduce infarct size [59.4(4.3)%]. Neither drug altered % delta WT; however, they reduced MVO2 by 36.5% in B, by 27.1% in C, and by 14.6% in the hydralazine group. CONCLUSIONS: Bimakalim treatment prior to the onset of a 60 min coronary occlusion increases the tolerance of pig myocardium to ischaemia. The data are consistent with the hypothesis that bimakalim reduces infarct size by activation of cardiac ATP sensitive potassium channels and not through unloading of the heart because of its vasodilator effects.

Animals↗

Pharmacological basis for antihypertensive therapy with a novel dopamine agonist.

In the past, nearly all major mechanisms involved in the regulation of blood pressure have become targets of antihypertensive drugs. They include the brain stem with its neuronal circuits of central cardiovascular regulation, the sympathetic neuro-effector system, the kidney, the renin angiotensin aldosterone system and the vascular smooth muscle cell. There are various ways of influencing the function of the sympathetic nervous system, but the clinical potential of one mechanism of action has not yet been explored in detail. Drugs that inhibit noradrenaline release through stimulation of inhibitory receptors located at adrenergic nerve terminals in the cardiovascular system (inhibitory presynaptic receptors) are not available for the treatment of hypertension. Among the multiple presynaptic receptors, dopamine receptors which belong to the dopamine2 subtype, are of particular interest. Carmoxirole is a novel indole derivative with a potent agonist action selective for dopamine2-receptors of the periphery. Experimental evidence shows that carmoxirole lowers blood pressure in various models of hypertension mainly or exclusively through inhibition of noradrenaline release from sympathetic nerve endings. This effect of carmoxirole is mediated by presynaptic dopamine receptors with the characteristic that release inhibition is restricted to low rates of sympathetic nerve discharge.

Animals↗

An approach to measuring cardiac output with Doppler flow probes in conscious rats.

The continuous measurement of cardiac output in conscious rats is difficult. Previously, Doppler flow probes have been applied to the ascending aorta by means of silicone cuffs. The placement of these cuffs can conceivably introduce errors engendered by the destruction of nerve fibers that are important in cardiac autonomic innervation. We report a novel method of Doppler flow-probe attachment directly to the ascending aorta by means of a polyacrylic cement. When we compared the response with baroreceptor activation with methoxamine and nitroprusside in conscious rats equipped either with probes attached via cuffs or directly via cement, we observed that animals equipped with cuffs failed to give the expected cardiac output and heart rate responses after nitroprusside administration. In contrast, animals that had their probes attached with cement displayed the expected responses. The data not only show that probes applied with cement give a result superior to probes attached with cuffs but also verify the suspicion that the ascending aorta is associated with nerve fibers that are important in mediating baroreceptor responses.

Animals↗

High beta 1-selectivity and favourable pharmacokinetics as the outstanding properties of bisoprolol.

Bisoprolol, (+/-)1-(4-[(2-isopropoxyethoxy)-methyl]-phenoxy)-3-isopropyl-amino -2- propanol-hemifumarate, is a new, highly selective beta 1-adrenoceptor blocking agent without intrinsic sympathomimetic activity and low to moderate local anaesthetic activity. As demonstrated in binding experiments, and in classical pharmacological studies using rats, guinea pigs, cats, and dogs, bisoprolol markedly differentiated between beta 1-adrenoceptors of the heart, or the renal juxtaglomerular apparatus, and the beta 2-subtype in arterial blood vessels, bronchi, liver, or skeletal muscle. Up to concentrations nearly 100-fold higher than the therapeutic plasma levels in humans, bisoprolol did not affect the functional refractory period of the heart, and was devoid of a direct suppressive effect on myocardial contractility and of calcium antagonistic properties in heart and vascular muscle. The pattern of haemodynamic effects of bisoprolol was typical of beta-blockers and included decreases in blood pressure (BP), heart rate (HR), and cardiac output, concomitant with an increase in calculated total peripheral resistance. In contrast to other beta-blockers, bisoprolol increased renal blood flow in anaesthetized dogs. Bisoprolol lowered BP in hypertensive dogs and rats, attenuated the development of spontaneous hypertension in rats, decreased plasma renin activity and protected the heart from the sequelae of transient ischemia. It did not block presynaptic beta-adrenoceptors in blood vessels. Serum lipids and the serum lipoprotein profile remained unaltered after bisoprolol. Bisoprolol was devoid of affinity for autonomic receptors other than beta-adrenoceptors or for autacoid receptors. This is probably one of the reasons why bisoprolol did not affect the function of the central nervous, respiratory, and gastrointestinal systems in an obvious way. The high beta 1-selectivity of bisoprolol is linked with extremely favourable pharmacokinetic properties. These include nearly complete enteral absorption and virtual absence of liver first-pass metabolism, both resulting in high bioavailability, long plasma half-life, pharmacokinetics that are linear over a wide dose range and independent of age, food intake and hydroxylator status, low plasma protein binding, and a 1:1 ratio of hepatic metabolization to renal elimination of the unaltered substance. This sum of favourable pharmacological and pharmacokinetic properties characterize bisoprolol as an optimized beta-blocker.

Adrenergic beta-Antagonists↗

Pharmacodynamic profile of the selective beta 1-adrenoceptor antagonist bisoprolol.

The pharmacodynamic activity of (+/-)-1-[4-(2-isopropoxyethoxymethyl)-phenoxy]-3-isopropylamino-2- propranol- hemifumarate (bisoprolol, EMD 33 512) has been investigated under in vitro and in vivo conditions. Bisoprolol was found to be an effective beta-adrenoceptor antagonist, the pA2 values determined against isoprenaline in guinea pig atria and tracheal muscle being 7.45 and 6.41, respectively. Thus, the selectivity ratio of bisoprolol in favour of beta 1-adrenoceptors is 11. Inhibition of the isoprenaline-induced tachycardia in guinea pigs indicated a long duration of action for bisoprolol. The compound was devoid of intrinsic sympathomimetic activity as shown by the lack of effect on heart rate in anaesthetized and reserpine pretreated rats. Studies in rabbits and guinea pigs revealed a local anaesthetic activity of bisoprolol at high concentrations. Bisoprolol protected the hearts of anaesthetized dogs against the sequelae of intermittent coronary occlusions, as judged by the reduction of the ST-segment elevation in the epicardial ECG. Bisoprolol exerted a blood pressure lowering effect in conscious renal hypertensive dogs after oral administration of 30 micrograms/kg. There was no indication of any action on the CNS in monkeys following an oral dose of up to 8 mg/kg.

Adrenergic beta-Antagonists↗

Regional distribution of renin and angiotensinogen in the brain of normotensive (WKY) and spontaneously hypertensive (SHR) rats.

The distributions of angiotensinogen and specific renin activity were examined in the brains of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto controls (WKY). Specific renin activity was markedly elevated in the pituitary of SHR compared to WKY. Renin levels in other regions of SHR brain were either significantly lower or similar compared to WKY. In contrast, angiotensinogen was significantly elevated in several regions of SHR compared to WKY brain. These results indicate involvement of a brain renin-angiotensin system in the development of genetic hypertension.

Angiotensinogen↗