Rainer Boer

(+)-Niguldipine binds with very high affinity to Ca2+ channels and to a subtype of α1-adrenoceptors

Abstract The enantiomers of the 1,4-dihydropyridine (DHP) niguldipine (3-methyl-5-[3-(4,4-diphenyl-1-piperidinyl)-propyl]-1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-pyridine-3,5-dicarboxylate-hydrochloride) were investigated with respect to their interaction with 1,4-DHP receptors on L-type Ca2+ channels and α-adrenoceptors. The Ki values for niguldipine were dependent on the membrane protein concentrations in the radioligand binding assay. ‘True’ Ki values (at extrapolated ‘zero’ membrane protein) were determined with guinea-pig membranes for (+)-niguldipine and were found to be 85 pmol/l for the 1,4-DHP receptor of skeletal muscle, 140 pmol/l for that of brain and 45 pmol/l for that of heart. (−)-Niguldipine was approximately 40 times less potent. (+)-Niguldipine (Ki: 78 nmol/l) and (−)-niguldipine (Ki: 58 nmol/l) bound with approximately equal affinity to the α1-adrenoceptors (‘α 1 B ’) in liver cell membranes. The (+)-niguldipine α1-adrenoceptor inhibition data for rat brain cortex membranes were better fitted by a two-site model. The high-affinity component (‘α 1 A ’) had a Ki value of 52 pmol/l in competition experiments with [3H]prazosin. The low-affinity site (α1B) had 200- to 600-fold less affinity. (−)-Niguldipine was > 40-fold less potent at α1A- but was nearly equipotent to the (+)-enantiomer at α1B-sites. (+)-Niguldipine was the most selective compound for discriminating α1A- from α1B-adrenoceptors and is a novel prototype for 1,4-DHPs which bind with nearly equal affinity to skeletal muscle and brain or heart 1,4-DHP receptors.

Inhibition of inducible nitric oxide synthase in respiratory diseases.

Nitric oxide (NO) is a key physiological mediator and disturbed regulation of NO release is associated with the pathophysiology of almost all inflammatory diseases. A multitude of inhibitors of NOSs (nitric oxide synthases) have been developed, initially with low or even no selectivity against the constitutively expressed NOS isoforms, eNOS (endothelial NOS) and nNOS (neuronal NOS). In the meanwhile these efforts yielded potent and highly selective iNOS (inducible NOS) inhibitors. Moreover, iNOS inhibitors have been shown to exert beneficial anti-inflammatory effects in a wide variety of acute and chronic animal models of inflammation. In the present mini-review, we summarize some of our current knowledge of inhibitors of the iNOS isoenzyme, their biochemical properties and efficacy in animal models of pulmonary diseases and in human disease itself. Moreover, the potential benefit of iNOS inhibition in animal models of COPD (chronic obstructive pulmonary disease), such as cigarette smoke-induced pulmonary inflammation, has not been explicitly studied so far. In this context, we demonstrated recently that both a semi-selective iNOS inhibitor {L-NIL [N6-(1-iminoethyl)-L-lysine hydrochloride]} and highly selective iNOS inhibitors (GW274150 and BYK402750) potently diminished inflammation in a cigarette smoke mouse model mimicking certain aspects of human COPD. Therefore, despite the disappointing results from recent asthma trials, iNOS inhibition could still be of therapeutic utility in COPD, a concept which needs to be challenged and validated in human disease.

Vasodilatation elicited by 5-HT1A receptor agonists in constant-pressore-perfused rat kidney is mediated by blockade of α1A-adrenoceptors

The vasodilator mechanism of the putative serotonin1A (5-HT) receptor agonists, urapidil, 5-methyl-urapidil, ipsapirone, flesinoxan and 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) was investigated in constant-pressure perfused rat kidneys. The compounds (10(-12)-10(-7) mol bolus injection) neither enhanced basal flow nor evoked vasodilatation in kidneys preconstricted by 27 mM KCl, 1.5 mM BaCl2 or 10(-6) M prostaglandin (PG)F2 alpha, but evoked a dose-dependent, reversible and spiroxatrine-resistant increase in vasodilatation of organs preconstricted by 6 x 10(-7) M noradrenaline. 5-Carboxamidotryptamine and sumatriptan did not reverse the vasoconstriction induced by all stimuli or that induced by noradrenaline in the presence of 5-HT2 plus 5-HT3 receptor blockade. No correlation for the vasorelaxant drugs was found between their -log ED50 in rat kidney and pKi values at 5-HT1A binding sites in pig cortex as determined in radioligand experiments. The relaxation in rat kidney induced by 5-HT1A receptor agonists and alpha 1A-adrenoceptor-selective antagonists (WB 4101 and (+)-niguldipine) was significantly correlated with pKi values at alpha 1A binding sites in rat cortex and the pA2 values derived from contraction studies for competitive antagonism at alpha 1-adrenoceptors in prostatic portions of the rat vas deferens, but differed from pKi values for alpha 1B binding sites in rat cortex. Thus, the vasodilator effect of the 5-HT1A receptor agonists urapidil, 5-methyl-urapidil, ipsapirone, flesinoxan and 8-OH-DPAT in the noradrenaline-perfused rat kidney appears to be mediated by their concomitant alpha 1A-adrenoceptor blockade. No evidence for a vasodilator effect mediated through 5-HT1A receptors was found under our experimental conditions.

Imidazoquinolinone, Imidazopyridine, and Isoquinolindione Derivatives as Novel and Potent Inhibitors of the Poly(ADP-ribose) Polymerase (PARP) : A Comparison with Standard PARP Inhibitors

We have identified three novel structures for inhibitors of the poly(ADP-ribose) polymerase (PARP), a nuclear enzyme activated by strand breaks in DNA and implicated in DNA repair, apoptosis, organ dysfunction or necrosis. 2-[4-(5-Methyl-1H-imidazol-4-yl)-piperidin-1-yl]-4,5-dihydro-imidazo[4,5,1-i,j]quinolin-6-one (BYK49187), 2-(4-pyridin-2-yl-phenyl)-4,5-dihydro-imidazo[4,5,1-i,j]quinolin-6-one (BYK236864), 6-chloro-8-hydroxy-2,3-dimethyl-imidazo-[1,2-α]-pyridine (BYK20370), and 4-(1-methyl-1H-pyrrol-2-ylmethylene)-4H-isoquinolin-1,3-dione (BYK204165) inhibited cell-free recombinant human PARP-1 with pIC50 values of 8.36, 7.81, 6.40, and 7.35 (pKi 7.97, 7.43, 5.90, and 7.05), and murine PARP-2 with pIC50 values of 7.50, 7.55, 5.71, and 5.38, respectively. BYK49187, BYK236864, and BYK20370 displayed no selectivity for PARP-1/2, whereas BYK204165 displayed 100-fold selectivity for PARP-1. The IC50 values for inhibition of poly(ADP-ribose) synthesis in human lung epithelial A549 and cervical carcinoma C4I cells as well in rat cardiac myoblast H9c2 cells after PARP activation by H2O2 were highly significantly correlated with those at cell-free PARP-1 (r2 = 0.89-0.96, P < 0.001) but less with those at PARP-2 (r2 = 0.78-0.84, P < 0.01). The infarct size caused by coronary artery occlusion and reperfusion in the anesthetized rat was reduced by 22% (P < 0.05) by treatment with BYK49187 (3 mg/kg i.v. bolus and 3 mg/kg/h i.v. during 2-h reperfusion), whereas the weaker PARP inhibitors, BYK236864 and BYK20370, were not cardioprotective. In conclusion, the imidazoquinolinone BYK49187 is a potent inhibitor of human PARP-1 activity in cell-free and cellular assays in vitro and reduces myocardial infarct size in vivo. The isoquinolindione BYK204165 was found to be 100-fold more selective for PARP-1. Thus, both compounds might be novel and valuable tools for investigating PARP-1-mediated effects.

The adrenoceptor agonist, SDZ NVI 085, discriminates between α1A- and α1B-adrenoceptor subtypes in vas deferens, kidney and aorta of the rat

Abstract The potency of the α 1 -adrenoceptor agonist (−)-(4aR, 10aR)-3,4,4a,5,10,10a-hexahydro-6-methoxy-4-methyl-9-methylthio-2H-naphth [2,3-b]-1,4-oxazine (SDZ NVI 085) was investigated both in isolated vas deferens and perfused kidney of the rat, two tissues with α 1 A -adrenoceptor subtype characteristics, and in the rat thoracic aorta, in which the contribution of different α 1 -adrenoceptor subtypes mediating contraction is controversial. In vas deferens and kidney, SDZ NVI 085 evoked smooth muscle contraction and vascular constriction and was of similar potency to L-phenylephrine. Contractions of vas deferens in response to (−)-noradrenaline and SDZ NVI 085 were resistant to chloroethylclonidine treatment (3 × 10 −5 M), sensitive to (±)-isradipine (10 −8 M) and competitively antagonized by 5-methyl-urapidil (pA 2 = 9.04 and 8.82, respectively). The potencies of a number of α 1A -/ α 1B -adrenoceptor-discriminating antagonists to reverse renal vasoconstriction due to either (−)-nora or SDZ NVI 085, and their affinities in vas deferens correlated significantly with their pK i values at α 1A binding sites in rat cortex. In rat aorta, SDZ NVI 085 up to 5 × 10 −4 M failed to evoke contraction. The affinities of subtype-selective antagonists determined in aorta correlated significantly with the pK i values at α 1B binding sites but differed from pK i values at α 1A sites in rat cortex. Thus, the contractile α 1 -adrenoceptor in rat aorta can be best characterized as B subtype. SDZ NVI 085 might be a selective α 1A -adrenoceptor agonist and thus be used as a new tool either to detect (rat vas deferens and kidney) or exclude (rat aorta) a contribution of α 1A -adrenoceptors functionally involved in smooth muscle contraction.

Dexniguldipine-HCl is a potent allosteric inhibitor of [3H]vinblastine binding to P-glycoprotein of CCRF ADR 5000 cells

Cell membranes were prepared from the multidrug resistant, P-glycoprotein expressing human lymphoblastoid cell line CCRF-ADR 5000. The P-glycoprotein of these membranes possessed high affinity binding sites for [3H]vinblastine, with a Kd of 8 +/- 2 nM and Bmax of 17 +/- 8 pmol/mg of protein. The binding of [3H]vinblastine to P-glycoprotein was not ATP-dependent, and was inhibited by cytotoxic drugs with the following potency order; vincristine > doxorubicin > etoposide. The 1,4-dihydropyridine and multidrug resistance reversing agent, dexniguldipine-HCl, inhibited binding with a Ki value of 37 nM. The multidrug resistance reversing agent cyclosporin A, and the cytotoxics doxorubicin and etoposide did not alter the kinetics of [3H]vinblastine dissociation from P-glycoprotein; however, the 1,4-dihydropyridines dexniguldipine-HCl and nicardipine accelerated dissociation of [3H]vinblastine. These data suggest that P-glycoprotein possesses at least two allosterically coupled drug acceptor sites; receptor site 1 which binds vinblastine, doxorubucin, etoposide and cyclosporin A, and receptor site 2 which binds dexniguldipine-HCl and other 1,4-dihydropyridines.

The Novel Imidazopyridine 2-[2-(4-Methoxy-pyridin-2-yl)-ethyl]-3H-imidazo[4,5-b]pyridine (BYK191023) Is a Highly Selective Inhibitor of the Inducible Nitric-Oxide Synthase

We have identified imidazopyridine derivatives as a novel class of NO synthase inhibitors with high selectivity for the inducible isoform. 2-[2-(4-Methoxy-pyridin-2-yl)-ethyl]-3H-imidazo[4,5-b]pyridine (BYK191023) showed half-maximal inhibition of crudely purified human inducible (iNOS), neuronal (nNOS), and endothelial (eNOS) NO synthases at 86 nM, 17 μM, and 162 μM, respectively. Inhibition of inducible NO synthase was competitive with l-arginine, pointing to an interaction of BYK191023 with the catalytic center of the enzyme. In radioligand and surface plasmon resonance experiments, BYK191023 exhibited an affinity for iNOS, nNOS, and eNOS of 450 nM, 30 μM, and >500 μM, respectively. Inhibition of cellular nitrate/nitrite synthesis in RAW, rat mesangium, and human embryonic kidney 293 cells after iNOS induction showed 40- to 100-fold higher IC50 values than at the isolated enzyme, in agreement with the much higher l-arginine concentrations in cell culture media and inside intact cells. BYK191023 did not show any toxicity in various rodent and human cell lines up to high micromolar concentrations. The inhibitory potency of BYK191023 was tested in isolated organ models of iNOS (lipopolysaccharide-treated and phenylephrine-precontracted rat aorta; IC50 = 7 μM), eNOS (arecaidine propargyl ester-induced relaxation of phenylephrine-precontracted rat aorta; IC50 > 100 μM), and nNOS (field-stimulated relaxation of phenylephrine-precontracted rabbit corpus cavernosum; IC50 > 100 μM). These data confirm the high selectivity of BYK191023 for iNOS over eNOS and nNOS found at isolated enzymes. In summary, we have identified a new highly selective iNOS inhibitor structurally unrelated to known compounds and l-arginine. BYK191023 is a valuable tool for the investigation of iNOS-mediated effects in vitro and in vivo.

Effect of nitric oxide synthase (NOS) inhibition on macro- and microcirculation in a model of rat endotoxic shock

Treatment of hemodynamic instability in septic shock often demands the administration of vasopressor agents, although these may have deleterious effects on microcirculatory homeostasis. Inhibition of nitric oxide synthase (NOS) has been suggested as an alternative therapeutic approach, as NO formation may be excessively increased in sepsis. To compare the effects of epinephrine titration, non-selective NOS inhibition by L-NMMA and selective inhibition of inducible NOS (iNOS) by 1400W on hemodynamics and on the regulation of microcirculation in a rat model of endotoxic shock, we intravenously injected endotoxin (LPS) or saline to male Wistar rats and after 2 hours randomized LPS treated rats into four different groups that received either saline, norepinephrine, L-NMMA or 1400W (n = 6 per group). Three hours after LPS administration, rats presented with severe systemic arterial hypotension (64 +/- 3 vs. 115 +/- 4 mmHg, p < 0.001), unresponsiveness to volume treatment, lactate acidosis and a marked increase in plasmatic nitrite and nitrate levels (15 +/- 8 vs. 263 +/- 47 microM, p < 0.001). Measurement of the tissue oxygenation in the ileum mucosal layer by the Erlangen micro-lightguide spectrophotometer (EMPHO) technique demonstrated marked heterogeneity of hemoglobin saturation, with appearance of low oxygenated areas. Norepinephrine, usually stabilizing blood pressure (99 +/- 7 vs. 67 +/- 4 mmHg 60 min after infusion, p < 0.01), increased lactate formation (7.9 +/- 0.2 vs. 3.7 +/- 0.5 mM, p < 0.001) and drastically increased low oxygenated regions in the ileum mucosal layer. L-NMMA similarly increased blood pressure (92 +/- 6 vs. 67 +/- 4 mmHg 60 min after infusion, p < 0.05), but did not enhance lactate acidosis. However, some further deterioration of mucosa oxygenation was again noted. 1400W forwarded stabilization of blood pressure (88 +/- 5 vs. 67 +/- 4 mmHg 60 min after injection, p < 0.05), reduced plasmatic nitrite and nitrate levels similar to L-NMMA, without an aggravation of lactate acidosis. In addition, mucosal oxygenation did not deteriorate in response to this agent. Thereby, we conclude that in a rat model of endotoxic shock selective iNOS inhibitors are superior to non-specific NOS inhibitors and in particular to norepinephrine for the treatment of macro- and microcirculatory abnormalities in experimental septic shock.

Novel nanomolar imidazo[4,5-b]pyridines as selective nitric oxide synthase (iNOS) inhibitors: SAR and structural insights

Inducible arginine oxidation and subsequent NO production by correspondent synthase (iNOS) are important cellular answers to proinflammatory signals. Prolonged NO production has been proved in higher organisms to cause stroke or septic shock. Several classes of potent NOS inhibitors have been reported, most of them targeting the arginine binding site of the oxygenase domain. Here we disclose the SAR and the rational design of potent and selective iNOS inhibitors which may be useful as anti-inflammatory drugs.

Interaction of cytostatics and chemosensitizers with the dexniguldipine binding site on P-glycoprotein

The interaction of cytostatics and chemosensitizers with the dexniguldipine binding site of P-glycoprotein was investigated in photoaffinity labeling experiments. A tritiated azidoderivative of the chemosensitizer dexniguldipine with dihydropyridine structure, [3H]B9209-005, was used to irreversibly label P-glycoprotein. The apparent affinity of cytostatics and chemosensitizers to this binding site was estimated from labeling experiments in the presence of increasing concentrations of compounds. From the cytostatics tested, the vinca alkaloids and taxol showed the highest affinity, anthracyclins possessed moderate affinity while methotrexate, ara C and camptothecin, cytostatics not involved in P-glycoprotein-mediated multidrug resistance, were almost inactive. The chemosensitizers GF 120918, cyclosporin A and SDZ PSC-833 inhibited photoincorporation with the highest potency. Steep dose-inhibition curves were obtained with the cyclic peptides and S9788, indicating that these compounds may bind allosterically to a separate binding site. Compounds with dihydropyridine structure with or without chemosensitizing potency were also tested and some structure-activity relationships could be derived from the data. Our data show that inhibition of photoaffinity labeling by [3H]B9209-005 is a valuable and reliable system for measuring the interaction with and potency of chemosensitizing compounds at P-glycoprotein. Furthermore, data obtained in this test system are well suited to investigate structure-activity relationships for chemosensitizers at P-glycoprotein. In addition cytostatics underlying P-glycoprotein-mediated multidrug resistance can be identified.