Development of MS Binding Assays targeting the binding site of MB327 at the nicotinic acetylcholine receptor.

The bispyridinium compound MB327 has been shown previously to have a positive pharmacological effect against poisoning with organophosphorous compounds (OPCs). The mechanism by which it exerts its therapeutic effect seems to be directly mediated by the nicotinic acetylcholine receptor (nAChR). In the present study, the development of mass spectrometry based binding assays (MS Binding Assays) for characterization of the binding site of MB327 at the nAChR from Torpedo californica is described. MS Binding Assays follow the principle of radioligand binding assays, but do not, in contrast to the latter, require a radiolabeled reporter ligand, as the readout is in this case based on mass spectrometric detection. For [2H6]MB327, a deuterated MB327 analogue employed as reporter ligand in the MS Binding Assays, an LC-ESI-MS/MS method was established allowing for its fast and reliable quantification in samples resulting from binding experiments. Using centrifugation for separation of non-bound [2H6]MB327 from target-bound [2H6]MB327 in saturation and autocompetition experiments (employing native MB327 as competitor) enabled reliable determination of specific binding. In this way, the affinities for [2H6]MB327 (Kd=15.5±0.9µmolL-1) and for MB327 (Ki=18.3±2.6µmolL-1) towards the nAChR could be determined for the first time. The almost exactly matching affinities for MB327 and [2H6]MB327 obtained in the MS Binding Assays are in agreement with potencies previously found in functional studies. In summary, our results demonstrate that the established MS Binding Assays represent a promising tool for affinity determination of test compounds towards the binding site of MB327 at the nAChR.

In vitro pharmacological characterization of the bispyridinium non-oxime compound MB327 and its 2- and 3-regioisomers.

The primary toxic mechanism of organophosphorus compounds, i.e. nerve agents or pesticides, is based on the irreversible inhibition of acetylcholinesterase. In consequence of the impaired hydrolysis, the neurotransmitter acetylcholine accumulates in cholinergic synapses and disturbs functional activity of nicotinic and muscarinic acetylcholine receptors by overstimulation and subsequent desensitization. The resulting cholinergic syndrome will become acute life-threatening, if not treated adequately. The current standard treatment, consisting of administration of a competitive mAChR antagonist (e.g. atropine) and an oxime (e.g. obidoxime, pralidoxime), is not sufficient in the case of soman or tabun intoxications. Consequently, alternative therapeutic options are necessary. An innovative approach comprises the use of compounds selectively targeting nAChRs, especially positive allosteric modulators, which increase the population of the conducting receptor state. MB327 (1,1'-(propane-1,3-diyl)bis(4-tert-butylpyridinium) di(iodide)) is able to restore soman-blocked muscle-force in preparations of various species including human and was recently identified as "resensitizer". In contrast to the well-studied MB327, the pharmacological efficacy of the 2- and 3-tert-butylpyridinium propane regioisomers is unknown. As a first step, MB327 and its 3-regioisomer (PTM0001) and 2-regioisomer (PTM0002) were pharmacologically characterized using [3H]epibatidine binding assays, functional studies by solid supported membranes based electrophysiology, and in vitro muscle-force investigations of soman-poisoned rat hemidiaphragm preparations by indirect field stimulation technique. The results obtained from targets of different complexity (receptor, muscle tissue) showed that the pharmacological profiles of the 2- and 3-regioisomers were relatively similar to those of MB327. Furthermore, high concentrations showed inhibitory effects, which might critically influence the application as an antidote. Thus, more effective drugs have to be developed. Nevertheless, the combination of the methods presented is an effective tool for clarifying structure-activity relationships.

Evaluation of GABA uptake in subcellular fractions of bovine frontal cortex and brainstem.

GABA uptake as well as the distribution of GAT-1, GAT-2 and GAT-3 were investigated in bovine brain membrane fractions. GABA uptake was characterised by kinetic constants and IC50-values for a series of known inhibitors in subcellular fractions of frontal cortex and brainstem obtained by subsequent centrifugations on sucrose gradients. Additionally, the immunoreactivity for rGAT-1, rGAT-2 and rGAT-3 antibodies was studied in these fractions. The pharmacological profile for GABA uptake inhibition as well as results from immunoblotting indicated that GABA uptake in a selected subcellular fraction of frontal cortex (P2B) is almost exclusively due to GAT-1 whereas GABA uptake performed with a selected subcellular fraction of brainstem (P2A) in the presence of NNC 711 is mainly attributable to GAT-3.

Synthesis and opioid-receptor binding of novel amino-substituted morphan analogues.

Starting with methyl 4,6-O-benzylidene-alpha-D-glucopyranoside (4), an optimized procedure is reported for preparation of the bromide 7, which is transformed into the N-acylated heptopyranosamine 9. After introduction of an axially positioned azido moiety in position 3 intramolecular N/O-acetal formation succeeds to provide the morphan analogue 17. In receptor binding studies with radioligands the amines 18b-18d reveal higher affinity for mu-receptors than for kappa-receptors. The most mu-active compound 18b (Ki = 14 nM) contains two aryl substituents, which presumably may occupy both aryl binding sites of mu-receptors.

[Balloon dilatation of the pulmonary valve. Short-, middle- and long-term results]. / Die Ballondilatation der Pulmonalklappe. Kurz-, mittel- und langfristige Ergebnisse.

BACKGROUND: Immediate and long-term results after balloon dilatation of pulmonary valve stenosis in our unit. METHODS AND PATIENTS: All 111 patients (1 day-18 years) who have had balloon dilatation of a pulmonary valvar stenosis between 12/1987 and 8/1997 were divided into 4 groups: Typical valvar pulmonary stenosis (group A; n = 78), stenosis with dysplastic pulmonary valve (group B; n = 10), critical pulmonary stenosis (group C; n = 16) and pulmonary atresia after transcatheter or operative opening of the valve (group D; n = 7). Patients with pulmonary stenosis and complex congenital heart disease were excluded. RESULTS: The average systolic transvalvular gradient was reduced from 68.5 to 27.2 mmHg (60%) immediately after balloon dilatation. After a follow up of 48.8 +/- 37 months 101 patients could be reevaluated. In group A (n = 69 at FU) and C (n = 16 at FU) 81% showed a systolic transvalvular gradient < 30 mmHg after one and 83% (A) respective 94% (C) after two balloon dilatations. In group B (n = 9 at FU) 44% exhibited a systolic gradient < 30 mmHg after one and 56% after two balloon dilatations. In group D (n = 7 at FU) 57% showed a systolic gradient < 30 mmHg with no further improvement by a second dilatation. Over all, 80% of our patients could be treated sufficiently by transcatheter means. The rate of major complications was 7.3% with no lasting residuals at follow up and no deaths. CONCLUSION: Balloon dilatation of the pulmonary valve is secure and effective. Best results are obtained in patients with typical pulmonary valve stenosis and in newborns and infants with critical pulmonary valve stenosis.

Azagrevellins, Part I. Grevellin analogs with affinity to the N-methyl-D-aspartate (glycine site) receptor, a novel lead structure.

A series of piperidine-2,3,5-triones (azagrevellins) has been prepared. A new synthesis has been introduced using the rearrangement of spiroepoxides in the presence of triethyloxonium tetrafluoroborate. The binding affinity toward the N-methyl-D-aspartate (glycine site) receptor has been measured to provide a basis for more detailed structure-activity studies. Azagrevellin 18d showed the highest binding potency.

Synthesis and resolution of racemic eliprodil and evaluation of the enantiomers of eliprodil as NMDA receptor antagonists.

A short synthesis of the NMDA receptor antagonist (rac)-Eliprodil (9) and its resolution into the enantiomers by chiral HPLC is described. The enantiomers (R)-9 and (S)-9 were found to exhibit markedly different affinities for NR2B subunit containing NMDA receptors.

Enteral nutritional support by percutaneous endoscopic gastrostomy in children with congenital heart disease.

One of the major problems of children with severe congenital heart disease (CHD) is their poor nutritional status. Among other consequences, it influences the surgical outcome. Retrospectively we present our experience with percutaneous endoscopic gastrostomy (PEG) in 15 children with CHD. This technique allows enteral nutritional support without the disadvantages related to long-term nasogastric tube feeding. Major complications were absent, and minor complications were rare both at PEG insertion, which was performed under deep sedation, and during feeding via PEG tube. In 4 of the 8 children who were followed for at least 6 months the age-matched body weight increased more than one standard deviation. In 2 other patients it increased more than 0.5 standard deviations. In 7 children the tube was removed after 2.5 to 42 months since enteral support was no longer necessary. Apart from initial reservations the parental acceptance of PEG was good. We conclude that the PEG is a safe and reliable technique to support enteral nutrition in children with severe CHD.

[3H]ifenprodil binding to NMDA receptors in porcine hippocampal brain membranes.

(+/-)-2-(4-Benzylpiperidino)-1-(4-hydroxyphenyl)propan-1-ol ([3H]ifenprodil) binding to a subcellular fraction of porcine hippocampus, which was obtained by centrifugation on a discontinuous sucrose gradient, was investigated with the objective to label selectively the ifenprodil recognition site of native NMDA receptors. Saturation experiments revealed high-affinity sites for [3H]ifenprodil in this membrane fraction which could be characterised by a K(d) value of 23.0+/-1.8 nM using a one-site model. Calculation of saturation isotherms on the basis of a two-site model yielded a K(d1) value of 10.4+/-2.4 nM and a K(d2) value of 2200+/-1300 nM, respectively. Inhibition of [3H]ifenprodil binding by NR2B subunit-selective NMDA receptor antagonists, by polyamines, by sigma receptor ligands, by a variety of ligands acting at different NMDA receptor recognition sites and by several cations was studied and compared with the effects of these compounds on (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5, 10-imine ([3H]MK-801) binding under non-equilibrium conditions. It turned out that sigma receptor ligands such as 1, 3-di(2-tolyl)-guanidine (DTG), (+)-3-(3-hydroxyphenyl)-N-propylpiperidine (R)-3-PPP, (S)-3-PPP and (1-¿2-[bis(4-fluorophenyl)methoxy]ethyl¿)(-4-[3-phenylpropyl]piperazi ne) (GBR-12909) did not affect [3H]ifenprodil binding in the nanomolar range or only slightly. In contrast, ifenprodil, eliprodil, nylidrin and haloperidol inhibited [3H]ifenprodil binding in the nanomolar range and in the same rank order and with the same potency as observed for the inhibition of the high-affinity fraction of [3H]MK-801 binding. The polyamines, which activate NMDA receptors, inhibited [3H]ifenprodil binding in a biphasic manner. Their potency to inhibit the high-affinity fraction of [3H]ifenprodil binding was found to be in the same range as their potency to enhance [3H]MK-801 binding. In the presence of 10 microM spermine a significantly enhanced (P=0.0097) rate of dissociation of [3H]ifenprodil binding was found, suggesting that inhibition of [3H]ifenprodil binding by spermine is not, or at least not exclusively mediated by a competitive interaction.

Pyrrolidine-2,4-diones with affinity to the N-methyl-D-aspartate (glycine site) receptor, Part II. 5-Arylidene-pyrrolidine-2,3,4-trione 3-oximes as NMDA receptor antagonists.

A series of oximes deriving from 5-arylidene-pyrrolidine-2,3,4-triones and pyridine-2,3,4-triones has been prepared. The presence of the tautomeric nitrosoenol was proven in solutions of alpha-ketooxime 7a. The binding affinity of the new oximes toward the N-methyl-D-aspartate (glycine site) receptor has been measured as a basis for more detailed structure-activity relationship studies. Oxime 13b showed the highest binding potency acting as glycine antagonist in nanomolar concentration.

Stereoselective synthesis and receptor binding of conformationally restricted and flexible 2,4-disubstituted 1,3-dioxanes derived from benzomorphans.

The key steps in the stereoselective synthesis of the tricyclic aminomethyl derivatives 19 and 20 and the aminoethyl substituted 1,3-dioxanes 24 and 25 are nucleophilic addition of aryllithium intermediates to the nitroalkene 13, intramolecular transacetalization of the addition products 15 and 16 (only for the tricyclic derivatives 19 and 20) and subsequent reduction of the nitro group. The affinities of the secondary and tertiary amines 19c,d, 20c,d, 24c,d, and 25c,d for the ion channel binding site of the NMDA receptor, for mu-, kappa-, and sigma-receptors have been investigated. In the group of tricyclic compounds only 19d shows remarkable sigma-receptor affinity (Ki = 21.6/1.10 microM). In the 1,3-dioxane series the moderate mu- (Ki = 27.8 microM) and kappa-receptor affinity (Ki = 36 microM) as well as the high sigma-receptor affinity (Ki = 3.3 microM) of the (S,S,S)-configurated methylamine 24c should be emphasized. The pentan-1-ol 26, the side product isolated during the synthesis of 24c, is of particular interest because of its considerable affinity to mu- (Ki = 16.0 microM), kappa- (Ki = 2.8 microM), and sigma-receptors (Ki = 14.5/1.26 microM). The biphasic competition curves obtained during sigma-receptor binding studies of 19d and 26 (two Ki values) may be explained by different interaction with sigma-receptor subtypes.

Syntheses of novel pyrazolomorphinans and their binding to mu- and kappa-opioid-receptors.

A number of novel pyrazolomorphinans have been synthesized in excellent yields by the reaction of the enolic morphinan diketones 6 with various hydrazines. Hydrazine dihydrochloride led to the N-unsubstituted tautomeric pyrazoles 7a in equilibrium with 9a and 8a in equilibrium with 10a which could not be separated. Arylhydrazines on the other hand furnished the regioisomeric pyrazolomorphinans 7 and 9 as well as 8 and 10, which could be isolated and characterized. The structures of the new compounds were clarified by their spectra, the assignment of the regioisomeres was achieved by determination of NOE enhancements. Compounds 6a, 7c, 8b and 10c have been evaluated for their affinity at mu and kappa opioid receptors in radioligand binding assays. Their ability to inhibit [3H] DAMGO binding to mu and [3H] U 69,593 binding to kappa opioid receptors has been found to be comparable with that of codeine.

5-Arylidene-3-aryl-pyrrolidine-2,4-diones with affinity to the N-methyl-D-aspartate (glycine site) receptor, Part I.

A series of new 5-arylidene 3-aryl-pyrrolidine-2,4-diones has been prepared. Their binding affinity toward the N-methyl-D-aspartate (glycine site) receptor has been measured as a basis for more detailed structure-activity relationship studies.

Noncompetitive NMDA antagonists: a novel synthesis of 1-phenyltetrahydro-3-benzazepines.

The key step in the synthesis of the pharmacologically interesting 1-phenyltetrahydro-3-benzazepine skeleton is the Michael addition of (2-lithiophenyl)acetaldehyde acetals, which are generated in situ upon treatment of the bromo acetals 5a,b with n-butyllithium, to beta-nitrostyrene (6). The reductive ring closure of the nitro acetals 7a,b succeeded with zinc dust and hydrochloric acid to give the 3-benzazepines 11a,b in good yields. The unsubstituted 3-benzazepine 11a showed a considerable affinity for the phencyclidine binding site of the NMDA receptor (Ki = 6.41 microM), whereas donor substituents in the aryl moiety (11b,c) reduce the affinity for the NMDA receptor.

[Intrapericardial hemorrhage as a manifestation of mycoplasma pneumoniae infection]. / Intraperikardiale Blutung als Manifestation einer Mycoplasma pneumoniae-Infektion.

Although carditis associated with Mycoplasma pneumoniae is infrequent it is an important cause of death in M. pneumoniae infections. We report on a 4-year-old boy with a M. pneumoniae infection, who developed a large hemorrhagic pericardial effusion and was successfully treated by percutaneous catheter drainage. Except in cancer patients, nontraumatic hemorrhagic pericardial effusions are rare. Our case strongly supports direct bacterial invasion into pericardial tissue as a cause of M. pneumoniae pericarditis rather than autoimmune phenomenon.

Syntheses of novel pyridazinomorphinans by inverse electron demand cycloaddition and their binding to mu and kappa receptors.

A number of novel pyridazinomorphinans have been synthesized by the inverse electron demand Diels-Alder reaction of various 3,6-disubstituted 1,2,4,5-tetrazines with enamines derived from dihydrocodeinone and with codeinone. Reduction of some of the pyridazinomorphinans did not furnish the expected pyrroloepoxymorphinans; in all cases investigated reductive cleavage of the epoxybridge was observed to yield dihydropyridazino- or pyrrolomorphinans. The structures of all new compounds were assigned by the spectral data, that of the cycloadduct of codeinone was additionally verified by X-ray crystallography. Compounds 5a, 8, 11a and 16 have been evaluated for their affinity at mu and kappa opioid receptors in radioligand binding assays. Their ability to inhibit [3H]DAMGO binding at mu and [3H]U 69.593 binding at kappa receptors, respectively as compared to codeine has been found to be lower.

[Functional tricuspid atresia in a newborn infant with cardiac rhabdomyoma]. / Funktionelle Trikuspidalatresie bei einem Neugeborenen mit kardialem Rhabdomyom.

Cardiac rhabdomyomas are benign tumours closely associated with tuberous sclerosis. We report on a neonate with a cardiac rhabdomyoma, which simulated tricuspid atresia with duct-depending pulmonary perfusion due to almost complete obliteration of right ventricular cavum. Under infusion of prostaglandin E1 the newborn stabilized and was successfully operated on the 3rd day of life. We conclude that successful tumour resection is possible in neonates with cardiac rhabdomyomas causing relevant hemodynamic obstruction.

Characterisation of the binding of [3H]MDL 105,519, a radiolabelled antagonist for the N-methyl-D-aspartate-associated glycine site, to pig cortical brain membranes.

Binding of [3H]MDL 105,519 to glycine sites on N-methyl-D-aspartate (NMDA) receptors of pig cortical brain membranes was evaluated. [3H]MDL 105,519 labelled a homogeneous population of binding sites with a Kd-value of 3.73 +/- 0.43 nM and a Bmax-value of 3030 +/- 330 fmol/mg protein. Clear correlations between the affinity (Ki) to [3H]MDL 105,519 labelled sites and the potency (EC50) to enhance or inhibit non-equilibrium [3H]MK-801 binding in the nominal absence of glycine were shown for a variety of glycine site agonists, partial agonists and antagonists. The ratio of Ki to EC50 was >1 for agonists and partial agonists and <1 for antagonists. Various cations as well as glutamate and polyamine site ligands were shown to be able to influence [3H]MDL 105,519 binding to pig cortical brain membranes substantially.

Stereoselective syntheses and CNS activity of novel tricyclic amines.

The enantiomerically pure amines (+)-5, (-)-9, (+)-11 and (+)-12 are stereoselectively prepared by reductive amination of the ketone (-)-4 [-->(+)-5], LiAlH4 reduction of the oxime (-)-7 followed by reductive methylation [-->(-)-9], SN2-substitution of the benzenesulfonate 10 [-->(+)-11] and reductive methylation of (+)-11 [-->(+)-12]. In the same way the racemic amines (+/-)-5, (+/-)-9, (+/-)-11 and (+/-)-12 are accessible starting from the racemic ketone (+/-)-4. Kinetic resolution of the racemic ketone (+/-)-4 with baker's yeast leads to the dextrorotatory ketone (+)-4 (86% ec), which is transformed via the methanesulfonate 16 into the tricyclic amines (-)-11 and (+)-17. Weak sedative effects are observed after application of the amines (+)-5, (+/-)-5, 0-9, (+/-)-9(+)-12, and (+/-)-12 to mice. Strong sedation is caused by (+/-)-11 with the dextrorotatory enantiomer (+/-)-11 being more effective than the levorotatory enantiomer (-)-11.