Biomedical subjects

Christian R Noe

Publications and source records attributed to Christian R Noe.

15 recordsLinked to original sources

PubMed2006

New polyamine-sensitive inhibitors of the NMDA receptor: syntheses and pharmacological evaluation.

Derivatives of 5-(4-aminobutyl)-2-thiophene-octylamine, a potent polyamine-sensitive inhibitor of the NMDA receptor, were synthesized and evaluated as inhibitors of [(3)H]MK-801 binding to rat brain membranes. Alkylations of the terminal amino groups reduced inhibitory potency; only incorporation of the amino group of the short 4-aminobutyl arm into a piperidine ring was tolerated. Substitution of the thiophene nucleus with methyl or ethyl, and its replacement by a benzene nucleus, was of minor influence. The corresponding diguanidines exhibited high potency independent of chain length, whereas their sensitivity to spermine was sharply dependent on chain length. Insertion of an amide bond into the long octylamine arm increased sensitivity to spermine and to Tris buffer. Our results indicate that spermine sensitivity of [(3)H]MK-801 binding inhibition is responsive to subtle changes in inhibitor structure and represents a promising target for pharmaceutical research.

Animals↗

PubMed2006

A novel tool to characterize paracellular transport: the APTS-dextran ladder.

PURPOSE: The aim of this work was to develop an easy, manageable, and precise analytic tool to describe the tightness of cell layers by a molecular weight ladder. METHODS: Dextrans were labeled by reductive amination with fluorescent 8-aminopyrene-1,3,6-trisulfonate (APTS). This mixture, including the internal standard diazepam, was used for transport studies in Transwell models using Caco-2, ECV304, and PBMEC/C1-2 cell lines. Samples were analyzed by fluorimetry, capillary electrophoresis, and reverse-phase high-performance liquid chromatography. RESULTS: Following this approach, a logarithm correlation of R2 = 0.8958 between transepithelial electrical resistance (TEER) and APTS-dextran permeability was shown. In addition, a TEER-dependent permeability pattern could be observed including each single fraction from free APTS, APTS-glucose up to APTS-dextran consisting of 35 glucose units. The TEER-independent permeability coefficients of diazepam and confocal laser scanning microscopy images confirmed the paracellular transport of APTS-dextran. CONCLUSIONS: All in all, the developed APTS-dextran ladder is a useful tool to characterize cell layer tightness and especially to describe paracellular transport ways and the extent of leakiness of cell layers (for blood-brain barrier or intestinal studies) over time--applying a wide array from smaller to larger molecules at the same time to refine TEER, sucrose, or Evans blue measurements.

Animals↗

PubMed2006

A novel flow based hollow-fiber blood-brain barrier in vitro model with immortalised cell line PBMEC/C1-2.

A flow based hollow-fiber in vitro model of the blood-brain barrier (BBB) was established. The immortalised porcine brain microvascular endothelial cell line PBMEC/C1-2 was cultured in a pulsatile hollow-fiber cartridge system (Cellmax Quad). The usability of PBMEC/C1-2 in the flow based hollow-fiber model was increased from three days in the originally used Transwell model up to four months due to the application of shear stress and co-culturing with glioma cell line C6. It was shown that the tightness of PBMEC/C1-2 layers was enhanced significantly in astrocyte conditioned medium (ACM) and in co-culture. The morphology of PBMEC/C1-2 and C6 was visualised by environmental scanning electron microscopy (ESEM). Permeation studies were accomplished with a set of benzodiazepines. The raw data were processed with three different calculation models and the results were compared with permeability coefficients obtained with an established Transwell model. In summary a flow based hollow-fiber BBB in vitro model was developed, which can be used to perform experiments with physiological (e.g., regulation of BBB permeability), pharmacological (e.g., pharmacokinetics and dynamics) and pathophysiological (e.g., effects of diseases on BBB permeability and vice versa) objectives.

Algorithms↗

PubMed2006

Synthesis of thieno[2,3-b]pyridinones acting as cytoprotectants and as inhibitors of [3H]glycine binding to the N-methyl-D-aspartate (NMDA) receptor.

The standard glycine site antagonist of the N-methyl-D-aspartate (NMDA) receptor, 3-phenyl-4-hydroxyquinolin-2(1H)-one (21), was used as a template for bioisostere benzene/thiophene exchange. Phenylacetylation of aminothiophene carboxylic acid methyl esters and subsequent cyclization delivered the three possible thienopyridinone isomers. 4-Hydroxy-5-phenylthieno[2,3-b]pyridin-6(7H)-one (3a), with the shortest distance between the sulfur and the nitrogen atom, was the most potent isomer (K(i) against the binding of [(3)H]glycine to rat membranes 16 microM), comparable in potency to the model quinolinone (21, 12 microM). Replacement of the phenyl substituent of 21 by a 2-thienyl residue resulted in a 2-5-fold loss in potency and was abandoned. In the thieno part of the thienopyridinone nucleus, the most successful substituents were halogen (Cl or Br) close to the sulfur atom and short alkyl chains at the other position, resulting in 7h, 8h, 8i, and 8m, with K(i) values between 5.8 and 10.5 nM. Introduction of a 3'-phenoxy moiety yielded several compounds with still higher potencies (18h, 18i, 18l, and 18m; K(i) between 1.1 and 2.0 nM). Quantitative structure-activity relationship (QSAR) calculations resulted in a consistent interpretation of the potencies of most compounds. Several of these 3'-phenoxy derivatives protected mouse fibroblast cell lines with transfected NMDA receptors from glutamate-induced toxicity. In addition, we report in vivo results for four of these compounds.

Animals↗

PubMed2005

APTS-labeled dextran ladder: a novel tool to characterize cell layer tightness.

The aim of this work was the development of an easy manageable analytic system for describing tightness of cell layers in a molecular size dependent manner, which is more precise than currently used ones. Dextrans were labeled by reductive amination with fluorescent 1-aminopyrene-3,6,8-trisulfonate (APTS). This mixture, including internal standard diazepam, was used for transport studies, which were accomplished with an established transwell blood-brain barrier model culturing an immortalized porcine brain microvascular endothelial cell line (PBMEC/C1-2). Samples were analyzed by fluorescence measurements, capillary electrophoresis and RP-LC. Following this approach, a permeability pattern could be achieved including each single fraction from APTS, APTS-glucose to APTS-dextran consisting of 31 glucose units. Permeability coefficients were calculated and ranged from 16.38+/-3.79 microm/min for APTS to 6.07+/-1.23 microm/min for the APTS-dextran with 31 glucose units (diazepam: 67.97+/-7.32 microm/min). All in all, the developed APTS-dextran ladder is an useful tool to characterize cell layer tightness--especially to describe paracellular transport ways and leakiness status of the blood-brain barrier over time--applying a wide range from smaller to larger molecules at the same time in order to refine, e.g. TEER, sucrose or Evans blue measurements.

Animals↗

PubMed2005

Towards validating a method for two-dimensional electrophoresis/silver staining.

Two-dimensional electrophoresis (2-DE) is a technique involving numerous steps, many of them to be performed manually. Hence, some operator dependency must be taken into account. An attempt to elucidate the reliability of 2-DE combined with silver staining is presented, employing the general practice to validate a method in pharmaceutical analysis. Most proteomic studies employing 2-DE aim at qualitative or quantitative differences in protein expression. One of the most sensitive and broadly applied staining techniques is silver staining. In order to gain information on accuracy, precision, linearity, and ruggedness of this technique, gels were run in replicates with different amounts of protein from a complex standard sample. In addition, sets of gels were repeated by two different operators in a second independent laboratory equipped with identical hardware and software. Our results show that reliable qualitative data on differential protein expression can be obtained by 2-DE, nevertheless replicate gels should be run and experimental conditions have to be kept stringently to a standardized protocol. Quantitative data are just achievable with spots, which are well-resolved, of high quality, with an optical density (OD) above a certain threshold (OD > 10), and which show a linear response. Quantitative differences occurring due to method-derived deviations may easily be misinterpreted as true changes in protein expression. After normalization, relative standard deviation (RSD) values of approximately 30% (n = 4) could be obtained, therefore minor changes (< 50%) should be critically reviewed.

Electrophoresis, Gel, Two-Dimensional↗

PubMed2005

Zwitterionic oligonucleotides: a study on binding properties of 2'-O-aminohexyl modifications.

2'-O-Aminohexyl side chains provide excellent conditions for zwitterionic interstrand and intrastrand interactions of oligonucleotides. 2'-O-Aminoalkylated phosphoramidites of adenosine and uridine were synthesized and incorporated in increasing number into homo adenosine and homo uridine/thymidine dodecamers, respectively. CD spectra of these dodecamers with complementary sense DNA exhibited a B-DNA type structure. While duplex stability values of all tested oligonucleotides were lower than those of the native oligonucleotides, they were significantly higher than those of 2'-O-heptyl modified oligonucleotides. The destabilization amounted to 0.9, 1.5, and 2.7 degrees C per modification for 2'-O-aminohexyl adenosine, 2'-O-aminohexyl uridine, and 2'-O-heptyl adenosine substitutions. These findings are pointing to a duplex stabilizing effect of the interaction of side chain amino groups with backbone phosphoric acid.

Amines↗

PubMed2004

A novel concept for ligand attachment to oligonucleotides via a 2'-succinyl linker.

Conjugation of ligands to antisense oligonucleotides is a promising approach for enhancing their effects. In this report, a new method for synthesizing oligonucleotide conjugates is described. 2'-Amino-2'-deoxy-5'-dimethoxytrityl-uridine was select ively acylated with a succinic acid linker at the 2' position. This compound was incorporated at the 3' end of an oligonucleotide corresponding to the sequence of Oblimersen. The carboxyl group was protected for oligonucleotide synthesis as a benzyl ester, which could be selectively cleaved at the solid phase by a catalytic phase transfer reaction using palladium nanoparticles as catalyst. An oligonucleotide-fluorescein conjugate was prepared by condensation of aminofluorescein. Circular dichroism spectroscopic experiments showed a B-DNA type structure. The melting temperature of the duplex was only slightly lower than that of Oblimersen. Biological activity measured by western blotting resulted in a Bcl-2 target downregulation nearly identical to that of control Oblimersen on human melanoma cells, proving that this method is attractive for the binding of ligands located in the minor groove.

Base Sequence↗

PubMed2004

Development of an in vitro blood-brain barrier model based on immortalized porcine brain microvascular endothelial cells.

Immortalized porcine brain microvessel endothelial cells (PBMEC/C1-2) were used to develop a model for measurement of blood-brain barrier permeation of central nervous system active drugs. Previous studies showed that a system using C6 astrocyte glioma conditioned medium leads to cell layers with transendothelial electrical resistance values up to 300 Omega cm(2) and a permeability coefficient P(e) of 3.24 +/- 0.14 x 10(-4) cm/min for U-[(14)C]sucrose, which is in good agreement to published values and thus indicates the formation of tight junctions in vitro. However, commercially available inserts for the Transwell system were not permeable for highly lipophilic compounds, such as diazepam. Systematic studies with different insert showed, that inserts with a pore width of 1 microm proved to be optimal for permeation studies of lipophilic compounds. Permeability studies with a set of three benzodiazepines further supported this finding.

Animals↗

PubMed2004

In silico prediction models for blood-brain barrier permeation.

The ability to permeate across the blood brain barrier (BBB) is essential for drugs acting on the central nervous system (CNS). Thus, for speeding up the drug discovery process in the CNS-area, it is of great importance to develop systems that allow rapid and inexpensive screening of the BBB-permeability properties of novel lead compounds or at least small subsets of combinatorial CNS-libraries. In this field, in silico prediction methods gain increasing importance. Starting with simple regression models based on calculation of lipophilicity and polar surface area, the field developed via PLS methods to grid based approaches (e.g. VolSurf). Additionally, the use of artificial neural networks gain increasing importance. However, permeation through the BBB is also influenced by active transport systems. For nutrients and endogenous compounds, such as amino acids, monocarboxylic acids, amines, hexoses, thyroid hormones, purine bases and nucleosides, several transport systems regulating the entry of the respective compound classes into the brain have been identified. The other way round there is striking evidence that expression of active efflux pumps like the multidrug transporter P-glycoprotein (P-gp) on the luminal membrane of the brain capillary endothelial cells accounts for poor BBB permeability of certain drugs. Undoubtedly, P-gp is an important impediment for the entry of hydrophobic drugs into the brain. Thus, proper prediction models should also take into account the active transport phenomena.

Biological Transport↗

PubMed2003

Induction of apoptosis by vitamin D metabolites and analogs in a glioma cell line.

Gliomas are the most common malignant tumors in brain. Recent studies demonstrate the capacity of 1alpha,25(OH)2D3 to specifically induce cell death (apoptosis) in model glioma cell lines and in primary cultures from tumor tissue, but not in primary astrocytes. In spite of this promising activity, a broad therapeutic application of vitamin D metabolites and analogs is still restricted because of their poor bioavailability and their hypercalcemic actions. Compared to 1alpha,25(OH)2D3, its natural 3alpha-epimer exhibits far higher metabolic stability and a reduced calcemic effect. Focusing on a possible therapeutic advantage of the 3alpha-conformation, we have examined the apoptotic potential of a representative set of vitamin D analogs, each of them in the 3alpha- and 3beta-conformation, and of natural vitamin D metabolites in the rat C6 glioma cell line. Exposure of these cells to the synthetic analogs resulted in all cases in a pronounced reduction of cell density (tested by incorporation of neutral red) and induction of apoptosis, monitored by staining nuclei with Hoechst 33258 dye and by following DNA fragmentation by capillary electrophoresis. The 3alpha-epimers showed equivalent or even higher activity on C6 cells than their respective 3beta forms. For their potent effects on growth and apoptosis of tumor cells and their high metabolic stability combined with a low calcemic potential, we speculate that these 3a-epimers could provide advantages for a prospective treatment of glioma.

Animals↗

PubMed2003

In silico screening with benzofurane- and benzopyrane-type MDR-modulators.

Development of inhibitors of the drug efflux pump P-glycoprotein is a versatile approach to overcome multi drug resistance (MDR) in tumor therapy. In an approach to lower the conformational flexibility of the lead compound propafenone, we synthesized a set of dihydrobenzofuranes and benzopyranones. In the case of the 4 diastereomeric dihydrobenzofuranes, no significant differences in activity regarding the configuration on the side-chains at the dihydrofurane moiety (cis or trans) was observed. This may be due to the high flexibility of the side-chains, which still allow mutually overlap of pharmacophores. The benzopyranones showed a good correlation between lipophilicity and activity with gnerally lower logpotency/logP ratios. This decrease may be due to the rigidization of the molecules. In an in silico screening approach, a set of diverse propafenone-type compounds was used to establish a pharmacophore model, which was used to screen the world drug index. Among the hits retrieved there are several compounds, which were previously described as MDR-modulators. This demonstrates the validity of the model.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2003

Structural modifications of antisense oligonucleotides.

Antisense oligonucleotides are efficient tools for the inhibition of gene expression in a sequence specific way. Natural oligonucleotides are decomposed rapidly in biological systems, which strongly restrict their application. In contrast, artificial oligonucleotides are designed to be more stable against degradation than the target mRNA, which results in a catalytic effect of the drug. Modification of the phosphate linkage has been the first successful strategy for antisense drug developments and Fomivirsene the first antisense drug in therapy. The launch of Fomivirsene has resulted in a revolutionary spin off to antisense research leading to a second generation of antisense oligonucleotides, which are stable against oligonucleotide cleaving enzymes. Among these, oligonucleotides bearing an alkoxy substituent in position 2' were the most successful ones. The third generation of antisense oligonucleotides contains structure elements, which enhance the antisense action. Zwitterionic oligonucleotides show remarkable results, first, because the stability against ribozymes is largely increased, and secondly, because the electrostatic repulsion between the anionic sense and the zwitterionic antisense cords is minimized. Promising new target molecules in antisense research are oligonucleotide chimäres, which enhance the antisense action (chimäres with intercalators, chelators or polyamines) or enable an application as sequence specific detectors (chimäres with biotin, fluorescein or radioligands).

Animals↗

PubMed2002

Two-dimensional electrophoresis of recombinant human erythropoietin: a future method for the European Pharmacopoeia?

Quality assurance of recombinant protein drugs concerning identity and purity represents a difficult task, in particular, when post-translational modifications lead to a heterogeneous mixture of biomolecules. We chose Neorecormon (rh-EPO, Roche) for our studies to demonstrate the efficiency of two-dimensional electrophoresis (2-DE) to analyse post-translationally modified recombinant drugs. More than 40 protein spots in the range from isoelectric point (pI) 3.5-4.5 and 32-45 kDa could be separated. Enzymatic deglycosylation revealed that the heterogeneity of the protein pattern is mainly caused by variations in glycosylation. In comparison to the separately performed isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, as requested by the European Pharmacopoeia, we see a great synergy to use 2-DE for the analysis of rh-EPO. A by far higher resolution can be achieved, allowing an improved differentiation of the various rh-EPO glycoforms. Sequential deglycosylation of sialic acids, N-glycosides and the O-glycoside lead to significant shifts both in apparent relative molecular mass and pI. Comparing the 2-DE patterns of rh-EPO before and after deglycosylation allows on the one hand valuable information to be gained on the glycosylation of the recombinant protein and shows on the other hand how significantly the 2-DE protein pattern can be influenced by the glycosylation. As the equipment for the performance of 2-DE has improved significantly over the last decade, we see 2-DE as a reliable method, which should be approved for the routine quality assurance of recombinant drugs and also recommended for the European Pharmacopoeia.

Amino Acid Sequence↗

PubMedDate not supplied

Oligonucleotides conjugated to short lysine chains.

A new method for synthesizing oligonucleotide peptide conjugates by an in-line approach is presented. A phosphorothioate oligonucleotide with the sequence of bcl-2 targeted Oblimersen by employing a modified 2'-amino-2'-desoxy-uridine nucleotide bearing a succinyl linker at the 2' position was prepared. The carboxyl group was protected for solid-phase synthesis as the benzyl ester. Ester cleavage was afforded by a phase transfer reaction using palladium nanoparticles as catalyst and cyclohexadiene as hydrogen donor. Short tails of up to three lysyl residues were conjugated to the oligonucleotide by an inverse stepwise peptide synthesis. The conjugates were characterized by HPLC, mass spectrometry, and circular dichroism. Influence of lysyl tails on CD spectra were minimal. Melting profiles revealed only minimal destabilizing effects on duplexes by conjugation of peptides.

Chromatography, High Pressure Liquid↗