High expression of RelA/p65 is associated with activation of nuclear factor-kappaB-dependent signaling in pancreatic cancer and marks a patient population with poor prognosis.

Activation of nuclear factor-kappaB (NF-kappaB) signaling was observed in pancreatic adenocarcinoma cell lines and tumours. However, information on the expression of RelA/p65, the major transcription activating NF-kappaB subunit, in these carcinomas and possible correlations thereof with NF-kappaB activation and patient survival is not available. To provide this missing translational link, we analysed expression of RelA/p65 in 82 pancreatic adenocarcinomas by immunohistochemistry. Moreover, we measured activation of the NF-kappaB pathway in 11 tumours by quantitative PCR for NF-kappaB target genes. We observed strong cytoplasmic or nuclear expression of RelA/p65 in 42 and 37 carcinomas, respectively. High cytoplasmic and nuclear expression of RelA/p65 had negative prognostic impact with 2-year survival rates for patients without cytoplasmic or nuclear RelA/p65 positivity of 41 and 40% and rates for patients with strong cytoplasmic or nuclear RelA/p65 expression of 22 and 20%, respectively. High RelA/p65 expression was correlated to increased expression of NF-kappaB target genes. The observation that high expression of RelA/p65 is correlated to an activation of the NF-kappaB pathway and indicates poor patient survival identifies a patient subgroup that might particularly benefit from NF-kappaB-inhibiting agents in the treatment of pancreatic cancer. Based on our findings, this subgroup could be identified by applying simple immunohistochemical techniques.

[Harmonisation of regulatory requirements for clinical trials on medicinal products for human use in the Directive 2001/20/EC and complementary guidance. Implementation of the 12th Law Amending the German Drug Law]. / Angleichung der regulatorischen Anforderungen für die klinische Prüfung von Arzneimitteln am Menschen in der EU Die Richtlinie 2001/20/EG und die diese ergänzenden Verordnungen und Empfehlungen: die 12. Novelle zum AMG.

Directive 2001/20/EC of the European Parliament and the Council of 4 April 2001 aims to harmonise national regulations governing clinical trials of medicinal products for human use in the European Union. This aim is to be achieved by harmonising the legal regulations and administrative provisions in force in the Member States, especially with respect to the requirements for starting and conducting clinical trials, taking into account international ethical and scientific standards (Good Clinical Practice). The goal is to further improve the protection of participants in clinical trials and to promote clinical research within the European Union. In Germany, the necessary transposition into the national Drug Law has taken place in the form of the 12th Law Amending the Drug Law of 30 July 2004 as well as complementary implementation provisions. The amendments to the German Drug Law affect in particular the official authorisation procedure, the involvement of ethics committees as well as the conduct of clinical trials on minors. The Directive's requirements have been transposed into German law while maintaining the high level of protection for the participants in clinical trials which had already existed in German legislation.

Pyrrolylquinoxalinediones carrying a piperazine residue represent highly potent and selective ligands to the homomeric kainate receptor GluR5.

Pyrrolylquinoxalinediones carrying aminoalkyl residues were evaluated for affinity to the recombinant, homomeric kainate receptors GluR5, GluR6 and GluR7. Most derivatives preferred binding to GluR5. In particular, the piperazine 6e represents a highly potent and selective antagonist to GluR5.

Dexamethasone and cyclosporin A do not inhibit interleukin-15 expression in the human lung carcinoma cell line A549.

A549 cells constitutively expressed IL-15 mRNA which could be upregulated by stimulation with TNF-alpha- or IL-1beta. Constitutive and induced levels of IL-15 mRNA were not decreased in the presence of 10- 6 M dexamethasone. Control experiments revealed that 10- 6 M dexamethasone inhibited the TNF-alpha- or IL-1beta-mediated increase of IL-8 mRNA in A549 cells, which showed that the glucocorticoid was functional. A549 cells did not secrete relevant amounts of IL-15 protein. The constitutive expression and the TNF-alpha- or IL-1beta-mediated upregulation of intracellular IL-15 protein was not inhibited by dexamethasone, in contrast, the release of IL-8 protein was inhibited. Also, cyclosporin A at 250 ng/ml did not inhibit the TNF-alpha-induced upregulation of IL-15 mRNA and intracellular IL-15 protein. The data suggest that the synthesis of IL-15 mRNA and protein is not influenced by immunosuppressive glucocorticoids or by cyclosporin A.

Synthesis and biological evaluation of novel piperidine carboxamide derived calpain inhibitors.

Calpain inhibitors which are derived from piperidine carboxamides in the P2 region were prepared and evaluated for mu-calpain inhibition. In particular, the keto amides 11f and 11j have Ki of 30 and 9 nM and display a more than 100-fold selectivity over the closely related cysteine protease cathepsin B. Furthermore, these compounds inhibit NMDA induced convulsions in mice indicating that calpain inhibition in brain results in some anticonvulsive properties.

A new pyrrolyl-quinoxalinedione series of non-NMDA glutamate receptor antagonists: pharmacological characterization and comparison with NBQX and valproate in the kindling model of epilepsy.

Antagonists at the ionotropic non-NMDA [AMPA (amino-methyl proprionic acid)/kainate] type of glutamate receptors have been suggested to possess several advantages compared to NMDA (N-methyl-D-aspartate) receptor antagonists, particularly in terms of risk/benefit ratio, but the non-NMDA receptor antagonists available so far have not fulfilled this promise. From a large series of pyrrolyl-quinoxalinedione derivatives, we selected six new competitive non-NMDA receptor antagonists. The basis of selection was high potency and selectivity for AMPA and/or kainate receptors, high in vivo potency after systemic administration, and an acceptable ratio between neuroprotective or anticonvulsant effects and adverse effects. Pharmacological characteristics of these novel compounds are described in this study with special emphasis on their effects in the kindling model of temporal lobe epilepsy, the most common type of epilepsy in humans. In most experiments, NBQX and the major antiepileptic drug valproate were used for comparison with the novel compounds. The novel non-NMDA receptor antagonists markedly differed in their AMPA and kainate receptor affinities from NBQX. Thus, while NBQX essentially did not bind to kainate receptors at relevant concentrations, several of the novel compounds exhibited affinity to rat brain kainate receptors or recombinant kainate receptor subtypes in addition to AMPA receptors. One compound, LU 97175, bound to native high affinity kainate receptors and rat GluR5-GluR7 subunits, i.e. low affinity kainate binding sites, with much higher affinities than to AMPA receptors. All compounds potently blocked AMPA-induced cell death in vitro and, except LU 97175, AMPA-induced convulsions in vivo. In the kindling model, compounds with a high affinity for GluR7 (LU 97175) or compounds (LU 115455, LU 136541) which potently bind to AMPA receptors and low affinity kainate receptor subunits were potent anticonvulsants in the kindling model, whereas the AMPA receptor-selective LU 112313 was the least selective compound in this model, indicating that non-NMDA antagonists acting at both AMPA and kainate receptors are more effective in this model than AMPA receptor-selective drugs. Three of the novel compounds, i.e. LU 97175, LU 115455 and LU 136541, exerted potent anticonvulsant effects without inducing motor impairment in the rotarod test. This combination of actions is thought to be a prerequisite for selective anticonvulsant drug action.

LU 73068, a new non-NMDA and glycine/NMDA receptor antagonist: pharmacological characterization and comparison with NBQX and L-701,324 in the kindling model of epilepsy.

The aim of this study was to assess whether a drug which combines an antagonistic action at both NMDA and non-NMDA receptors offers advantages for treatment of epileptic seizures compared to drugs which antagonize only one of these ionotropic glutamate receptors. The novel glutamate receptor antagonist LU 73068 (4,5-dihydro-1-methyl-4-oxo-7-trifluoromethylimidazo[1,2a]quinoxal ine-2-carbonic acid) binds with high affinity to both the glycine site of the NMDA receptor (Ki 185 nM) and to the AMPA receptor (Ki 158 nM). Furthermore, binding experiments with recombinant kainate receptor subunits showed that LU 73068 binds to several of these subunits, particularly to rGluR7 (Ki 104 nM) and rGluR5 (Ki 271 nM). In comparison, the prototype non-NMDA receptor antagonist NBQX (2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo[f]quinoxaline) binds with high affinity to AMPA receptors only. Both NBQX and LU 73068 were about equieffective after i.p. injection in mice to block lethal convulsions induced by AMPA or NMDA. In the rat amygdala kindling model of temporal lobe epilepsy, LU 73068 dose-dependently increased the focal seizure threshold (afterdischarge threshold, ADT). When rats were stimulated with a current 20% above the individual control ADT, LU 73068 completely blocked seizures with an ED50 of 4.9 mg kg(-1). Up to 20 mg kg(-1), only moderate adverse effects, e.g. slight ataxia, were observed. NBQX, 10 mg kg(-1), and the glycine/NMDA site antagonist L-701,324 (7-chloro-4-hydroxy-3-(3-phenoxy)phenyl-quinoline-2(1H)one), 2.5 or 5 mg kg(-1), exerted no anticonvulsant effects in kindled rats when administered alone, but combined treatment with both drugs resulted in a significant ADT increase. The data indicate that combination of glycine/NMDA and non-NMDA receptor antagonism in a single drug is an effective means of developing a potent and effective anticonvulsant agent.

In vitro selected RNA molecules that bind to elongation factor Tu.

RNA molecules which bind to elongation factor Tu from T. thermophilus were isolated from a pool of ribooligonucleotides with a randomized sequence region. These RNAs interact with elongation factor Tu in both the GTP and the GDP form. A slight preference for the GTP form of the protein was observed. The isolated RNA aptamers compete with each other for a common binding site on elongation factor Tu. This binding site is different from the binding site for aminoacyl-tRNA or the binding site for elongation factor Ts and is located on domain II of elongation factor Tu. The selected RNAs do not bind to elongation factor G. The EF-Tu binding RNAs share a short consensus sequence, 5'-ACCGAAG-3', which was also found in the alpha-sarcin domain of T. thermophilus23S rRNA. The isolated RNAs have a hairpin structure with the 5'-ACCGAAG-3' sequence located in non-base-paired regions. Chemical probing and deletion experiments indicate that the consensus sequence is required for the interaction with elongation factor Tu.

Ni2+-binding RNA motifs with an asymmetric purine-rich internal loop and a G-A base pair.

RNA molecules with high affinity for immobilized Ni2+ were isolated from an RNA pool with 50 randomized positions by in vitro selection-amplification. The selected RNAs preferentially bind Ni2+ and Co2+ over other cations from first series transition metals. Conserved structure motifs, comprising about 15 nt, were identified that are likely to represent the Ni2+ binding sites. Two conserved motifs contain an asymmetric purine-rich internal loop and probably a mismatch G-A base pair. The structure of one of these motifs was studied with proton NMR spectroscopy and formation of the G-A pair at the junction of helix and internal loop was demonstrated. Using Ni2+ as a paramagnetic probe, a divalent metal ion binding site near this G-A base pair was identified. Ni2+ ions bound to this motif exert a specific stabilization effect. We propose that small asymmetric purine-rich loops that contain a G-A interaction may represent a divalent metal ion binding site in RNA.

The 3'-terminal end (NCCA) of tRNA determines the structure and stability of the aminoacyl acceptor stem.

We have done a systematic study on the contribution of the single-stranded NCCA end (where N is any nucleotide) to the stability of the aminoacyl stem of tRNA. A 7-bp RNA duplex with the single-strand ACCA 3' terminus derived from the aminoacyl stem of Escherichia coli tRNA(Ala) and several chemically synthesized sequence variants are characterized by proton NMR and thermodynamic parameters. The single-stranded 3' terminus noticeably stabilizes the duplex in a sequence-dependent manner. Though the largest contribution to the stability gain due to the ACCA end is provided by the first dangling 3' nucleotide, the influence of even the fourth nucleotide is measurable. The nature of the N73 discriminator base influences the stem structure and stability, which may be important for the recognition of tRNA by aminoacyl-tRNA synthetase. The stepwise attachment of the nucleotides to the 3' tail improves the stacking of the unpaired bases over the helix stem. Hence, the ACCA end appears to be structured. Replacing Mg2+ with Mn2+ causes broadening of certain imino proton peaks in the NMR spectrum, indicating a specific divalent metal ion binding site in the vicinity of the major identity element of the duplex (G3-U70) that is required for its recognition by the Ala-tRNA synthetase.

Dependency of growth hormone (GH) stimulation following releasing hormones on the spontaneous 24-hour GH secretion in healthy male and female subjects.

The purpose of this investigation was to evaluate whether in healthy subjects the GH response following stimulation with releasing hormones is dependent on the spontaneous GH secretion within 24 hr prior to the stimulation test. In 18 male and 9 female healthy subjects (21-59 years) GH was measured every 15 min over 26 hr. Twenty-four hours after the beginning of blood sampling, a GH stimulation test was performed by using a combined releasing hormone test. Sleep was recorded in three consecutive nights. A positive correlation was found between the AUCs of the 24-hr GH secretion and the AUCs of GH stimulation, which could not be explained by an age effect only. This study demonstrates that subjects with comparatively high amounts of GH secreted within 24 hr also show good GH secretory responses when immediately after the 24-hr sampling period a stimulation test is undertaken. Therefore, a low GH response to stimulation cannot be explained by feedback effects of high GH amounts secreted during the 24 hr before the test or by empty pituitary GH storages.

[Standards for infant clothing]. / Anforderungen an die Bekleidung des Säuglings.

The presence of clothing for infants does not reflect in quality and quantity the actual situation of medical knowledge. Economical conditions are not responsible for that fact, but especially uninformation of industry and physicians, too. Our contribution should inform doctors, especially from child welfare centres, about clothing to infants. Recommendations were given to a more proper clothing of infants.

(S)-emopamil, a novel calcium and serotonin antagonist for the treatment of cerebrovascular disorders. 1st communication: pharmacological profile.

(S)-Emopamil ((2S)-2-isopropyl-5-(methylphenethylamino)-2-phenylvaleronitril e hydrochloride) is a novel compound of the phenylalkylamine group of calcium antagonists. (S)-Emopamil was tested in comparison to verapamil and gallopamil for calcium and serotonin antagonism and for cerebroprotective activity in acute hypoxia/ischemia. In receptor binding studies with (S)-3H-devapamil, (S)-emopamil exhibited distinct affinity to the verapamil binding site of the calcium channel. In rat cerebrocortical membranes, its affinity (Ki = 38 nmol/l) equalled that of verapamil and gallopamil (Ki = 49 and 27 nmol/l, respectively), whereas it was somewhat weaker in guinea pig skeletal muscle membranes. Comparing (S)-emopamil to its (R)-enantiomer, there was no clear stereoselectivity. Additionally, (S)-emopamil showed very high affinity to the cerebral serotonin S2 receptor; its Ki value (4.4 nmol/l) for 3H-ketanserin displacement being substantially lower than that of verapamil and gallopamil (Ki = 177 and 242 nmol/l, respectively). This feature is clearly stereoselective; (S)-emopamil's affinity was distinctly higher than that of the (R)-enantiomer (Ki = 58 nmol/l). The functional significance of (S)-emopamil's receptor affinity was tested in rat aortic strips. (S)-Emopamil's serotonin antagonistic efficacy (EC50 = 4.5 nmol/l) was an order of magnitude higher than that of verapamil and gallopamil. (S)-Emopamil has a less potent calcium antagonistic effect (EC50 = 270 nmol/l) on the aorta than verapamil and gallopamil (EC50 = 35 and 14 nmol/l, respectively). In isolated electrically driven (1 Hz) left atria of guinea pigs, (S)-emopamil inhibited contractile force at a much higher concentration (EC50 = 29 mumol/l) than verapamil and gallopamil (EC50 = 1.1 and 0.19 mumol/l, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

(S)-emopamil, a novel calcium and serotonin antagonist for the treatment of cerebrovascular disorders. 3rd communication: effect on postischemic cerebral blood flow and metabolism, and ischemic neuronal cell death.

The effect of (S)-emopamil ((2S)-2-isopropyl-5-(methylphenethylamino)-2-phenylvaleronitril e hydrochloride) treatment on postischemic cerebral blood flow and metabolism was investigated in nitrous oxide anesthetized, artificially ventilated rats. Forebrain ischemia was induced and maintained for 20 min by lowering arterial blood pressure to approximately 40 mmHg and clamping both carotid arteries. Local cerebral blood flow and glucose utilization were evaluated autoradiographically in 34 cerebral regions. In the cerebral blood flow studies intravenous infusion of 0.1 mg/(kg min) (S)-emopamil was begun 5 min after the end of ischemia. Local cerebral blood flow was determined 60 min later using [14C]iodoantipyrine. When the animals were treated with saline only, postischemic blood flow of 22 affected forebrain areas fell on average to 42 +/- 13% of nonischemic control. Treatment with (S)-emopamil increased perfusion of the same areas in a region-dependent fashion by an average of 54 +/- 19%, resulting in 63 +/- 17% of control values. The rise of blood flow in structures not directly affected by ischemia amounted to 52 +/- 27% (134 +/- 23% of control). In the studies on cerebral metabolism, the experimental animals received a total of 6 mg/kg (S)-emopamil by slow intravenous infusion before and during the ischemic episode. Determination of local cerebral glucose utilization was initiated after 50 min of postischemic recirculation using [14C]deoxyglucose. In the placebo-treated experimental group average glucose utilization of 14 forebrain areas was significantly lower (74 +/- 9% of control) than in the nonischemic control group.(ABSTRACT TRUNCATED AT 250 WORDS)

[Pharmacological characterization of the new highly potent beta-adrenergic receptor blocker soquinolol]. / Pharmakologische Charakterisierung des neuen, hochwirksamen beta-Rezeptorenblockers Soquinolol.

The present pharmacological test results characterize soquinolol (5-[3-tertiary butylamino-2-hydroxypropoxy]-2-formyl-1,2,3,4- tetrahydroisoquinoline mucate, We 704, Sertum) as a highly potent non-subtype-selective beta-adrenergic receptor blocker, which is devoid of any intrinsic sympathomimetic activity. Its localanaesthetic activity (membrane stabilizing effect) is very weak. It also shows good enteral efficacy and long duration of action. In binding studies with heart (Ki beta 1 = 3.25 nmol/l) and lung membranes (Ki beta 2 = 0.85 nmol/l) its binding profile was found to be similar to that of propranolol. Soquinolol inhibits the isoprenaline-induced tachycardia (EC50% = 48 micrograms/l) in the guinea-pig Langendorff heart in vitro to the same degree as propranolol. However, in the conscious dog soquinolol's beta 1-adrenergic blocking activity (ED 50%) on intravenous injection (5.5 micrograms/kg) and oral administration (5.8 micrograms/kg) is about twice as great as that of pindolol and 19 times (i.v.) or 138 times (p.o.) greater than that of propranolol. These results suggest 95% enteral efficacy for soquinolol (pindolol 88%, propranolol 13%). The differences in soquinolol's and propranolol's efficacy detected in vitro and in vivo are partially attributable to differences in their kinetic properties namely the lower protein binding and the higher distribution volume of soquinolol. In the conscious dog, soquinolol inhibits beta 1-(ED 50% = 4.0 micrograms/kg) and beta 2-receptors (ED 50% = 2.7 micrograms/kg) at dose levels which do not differ significantly.(ABSTRACT TRUNCATED AT 250 WORDS)

Tricyclic epines. Novel (E)- and (Z)-11H-dibenz[b,e]azepines as potential central nervous system agents. Variation of the basic side chain.

The synthesis and pharmacological activity of new (E),(Z)-[6-(alkylamino)-11H-dibenz[b,e]azepin-11- ylidene]acetonitriles 12-45 and (E),(Z)-[6-(aminoalkoxy)-11H-dibenz[b,e]azepin-11-ylidene] acetonitriles 46-51 are described. The introduction of the cyanomethylene group into the 11-position of the 11H-dibenz[b,e]azepine framework has been carried out by a Wittig-Horner reaction under mild conditions. The (E),(Z) isomers were separated by fractional crystallization, assignment being achieved by X-ray analysis. A number of (E),(Z)-[6-(alkylamino)-11H-dibenz-[b,e]azepin-11-ylidene] acetonitriles (12, 14, 16, 20) show potent neuroleptic activity (2-7 times that of clozapine) in animal tests. The screening included tests for sedative and anticholinergic activity in mice, apomorphine and tryptamine antagonism in rats, and muscle-relaxing activity in rabbits. The divergence in the activity profile in the case of the separated (E),(Z) isomers has been observed as an interesting new aspect: the (Z) isomers show a significantly higher sedative and muscle-relaxant activity, whereas the (E) isomers possess a higher anticholinergic efficacy and somewhat greater apomorphine antagonism. Broad changes in the basic side chain were made in order to investigate structure-activity relationships. The important geometrical parameters for the molecules, obtained by X-ray analysis, were compared with the corresponding features in dopamine agonists and antagonists.