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Biomedical subjects

Ulrike Holzgrabe

Publications and source records attributed to Ulrike Holzgrabe.

At least 19 recordsLinked to original sources
PubMed2003

Mechanism of action of the diazabicyclononanone-type kappa-agonists.

The 2,4-di-2-pyridyl-3,7-dimethyl-3,7-diazabicyclo[3.3.1]nonan-9-one 1,5-diester HZ2 was recently found to exhibit high affinity and selectivity to the kappa-opioid receptor (KOR) in combination with an unusually long duration of action. Docking of HZ2 to the putative binding site model of the KOR revealed HZ2 to be tightly sitting in the binding pocket. Strong interactions, especially salts bridges between the protonated nitrogens of HZ2 and the glutamic acids 209 and 297, nicely explain the high affinity of HZ2 to the KOR. A formation of a hemiaminal bond between the keto carbonyl group of HZ2 and a lysine residue (Lys200) may explain the long duration of action.

Amino Acid Sequence↗
PubMed2003

Systematic development of high affinity bis(ammonio)alkane-type allosteric enhancers of muscarinic ligand binding.

Bis(ammonio)alkane compounds carrying lateral phthalimidopropyl substituents on the nitrogen atoms belong to the archetypal muscarinic allosteric agents. Herein, a series of symmetrical and nonsymmetrical compounds was synthesized in which the phthalimide residues were replaced by differently substituted imide moieties. The allosteric action was measured in porcine heart muscarinic M(2) receptors using [(3)H]N-methylscopolamine (NMS) as a ligand for the orthosteric receptor site in equilibrium binding and dissociation experiments. 1,8-Naphthalimido residues conferred an up to 100-fold gain in affinity leading into the low nanomolar range, while the inhibition of NMS binding was maintained. Additional propyl chain methylation was accompanied by an allosteric elevation of orthosteric ligand binding. In general, the gain in allosteric activity achieved by ring variation plus propyl chain methylation on one side of the molecule could not be augmented by symmetrical variations. The elevation of the ligand binding can be explained by different quantitative structure-activity relationships for the affinities to the free and the orthoster-liganded receptor.

Alkanes↗
PubMed2003

Determination of clotrimazole in mice plasma by capillary electrophoresis.

In addition to its antifungal activity, clotrimazole attracts interest as an anti-inflammatory drug. In order to correlate this effect with plasma concentrations in mice, a capillary electrophoretic method was developed. Sample preparation was carried out by protein precipitation using methanol. Quantification of clotrimazole was achieved by means of capillary electrophoresis using ketoconazole as an internal standard (IS). The background electrolyte (BGE) composed of a Tris buffer solution (100 mM, pH 3.0, adjusted with acetic acid) and methanol (8:2, v/v). Injection was carried out electrokinetically with 10 kV over a time period of 20 s. A special rinsing procedure utilizing a sequence of a SDS/methanol solution, a sodium hydroxide solution, water and BGE, was applied to enhance the reproducibility. With this procedure, an intermediate precision (day-to-day precision) of the area ratios of clotrimazole and IS of 5.0% for 0.5 microg ml(-1) and 2.6% for 10 microg ml(-1) was obtained. In summary, with the described capillary zone electrophoresis (CZE) method it is possible to handle small sample volumes of 60 microl, to detect clotrimazole concentrations of 0.3 microg ml(-1) (limit of detection), and to quantify clotrimazole down to concentrations of 0.5 microg ml(-1) (limit of quantification).

Animals↗
PubMed2003

Tautomerism of 4-hydroxy-2,5-dimethyl-3(2H)-furanone: evidence for its enantioselective biosynthesis.

Chiral natural flavor compounds exhibit characteristic enantiomeric excesses due to stereoselective, enzymatically catalyzed reactions during biogenesis. Although the enzymatic formation of the strawberry key flavor compound 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF; Furaneol(R)) is anticipated, the naturally occurring compound is racemic. As racemization due to keto-enol-tautomerism of HDMF could account for this observation, HDMF was investigated by (1)H-NMR spectroscopy tracing the exchange of the proton bound to the furanone-ring at C2 with deuteron from the medium (D(2)O). In addition, the racemization rate of HDMF was directly determined by cyclodextrin-modified capillary electrophoresis of enantiomerically enriched HDMF stored at different pH values. Tautomerism and the racemization rate of HDMF was lowest at pH values between 4 and 5. However, tautomerism and thus racemization was catalyzed under stronger acidic conditions (pH 2) and especially at pH values greater than 7, the value published for plant cell cytosol. Approximately 50% of the protons at C2 were exchanged with deuteron within 1 h at pH 7.2. Therefore, in order to demonstrate the enzymatic formation of HDMF, incubation experiments with Zygosaccharomyces rouxii as well as strawberry protein extract were carried out under slightly acidic conditions (pH 5), the most suitable pH value for studies on the enantiomeric ratio of HDMF. In both experiments the formation of enantiomerically enriched HDMF could be demonstrated for the first time, whereas incubation experiments under neutral conditions resulted in the detection of racemic HDMF.

Flavoring Agents↗
PubMed2003

Quantitation of talinolol and other beta-blockers by capillary electrophoresis for in vitro drug absorption studies.

A capillary zone electrophoresis method is described for the enantioseparation of talinolol using heptakis(2,3-diacetyl-6-sulfo)-beta-cyclodextrin (HDAS-beta-CD) as a chiral selector. After liquid-liquid extraction of talinolol from physiological solution, electrokinetic injection was employed to improve the sensitivity. The use of a coated capillary was necessary to achieve stable and reproducible enantioseparations. A baseline separation of the talinolol enantiomers was achieved in less than 10 min using 100 mM phosphate solution as background electrolyte and pH 3.5, at the presence of 3.0 mM HDAS-beta-CD and at 20 degrees C. In addition, this analytical condition proved to be useful for the enantioseparation of a number of other beta-blocking agents such as alprenolol, atenolol, bisoprolol, celiprolol, metipranolol, oxprenolol, and sotalol. For determining talinolol, the method could be validated in terms of precision, accuracy and linearity, and was found to be suitable in determination of talinolol enantiomers in highly diluted samples obtained from in vitro experiments.

Absorption↗
PubMed2003

A new micellar electrokinetic capillary chromatography method for separation of the components of the aminoglycoside antibiotics.

Aminoglycoside antibiotics are always a mixture of structurally related amino sugars, which do not have a chromophore or fluorophore. The aim of the study was to find one method for evaluation of the components and impurities of the antibiotics. Derivatization with o-phthaldialdehyde and thioglycolic acid is found to be appropriate for all antibiotics. The components of gentamicin (GM), sisomicin, netilmicin, kanamycin, amikacin, and tobramycin were tried to separate by means of micellar electrokinetic chromatography. The background electrolyte was composed of sodium tetraborate (100 mM, pH 10.0), sodium deoxycholate (20 mM), and beta-cyclodextrin (15 mM). This method is valid for evaluation of GM, kanamycin, and tobramycin. It has to be improved for amikacin and netilmicin. In addition, 46 bulk samples of GM of different manufacturer or pharmaceutical companies were investigated. Many samples were found to contain many minor products and different amounts. Beside different patterns of the main compounds GM C1, GM C1a, GM C2a, and GM C2, many lots were found consisting of a substantial number of minor products. The appearance of a high number of minor products is always associated with the existence of sisomicin, which is not found in "pure" samples. However, almost all samples met the requirements of the European Pharmacopoeia (EP) and United States Pharmacopoeia (USP).

Amikacin↗
PubMed2003

[Antimycotics].

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Antifungal Agents↗
PubMed2003

[Arrays overall].

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GTP-Binding Proteins↗
PubMed2003

Cooperative interactions at M2 muscarinic acetylcholine receptors: structure/activity relationships in stepwise shortened bispyridinium- and bis(ammonio)alkane-type allosteric modulators.

Muscarinic M2-receptors allow for divergent modes of allosteric action, depending on the structure of the allosteric modulator. Phthalimido-substituted bis(ammonio)alkane-type modulators belong to the common mode allosteric agents, whereas a physicochemically closely related bispyridinium-oxime with dichlorobenzyl-substituents at both ends is an atypical agent. Here, we compared the actions of stepwise shortened compounds composed of the phthalimido moiety and middle chains of either the bispyridinium- or the bis(ammonio)alkane-type. Allosteric interactions were measured in pig M2 receptors with the orthosteric probe [3H]N-methylscopolamine ([3H]NMS) to label the acetylcholine binding site of the receptors. Dissociation and equilibrium binding experiments revealed parallel structure/activity-relationships in both series of compounds with regard to the cooperativity of interaction with [3H]NMS and to the underlying binding affinities in radioligand-occupied and free receptors. In conclusion, the findings are in line with the hypothesis that the phthalimido-moiety, but not the middle chain, is pivotal for the topology of interaction with the M2-receptor protein.

Alkanes↗
PubMed2002

Elevation of ligand binding to muscarinic M(2) acetylcholine receptors by bis(ammonio)alkane-type allosteric modulators.

Bis(ammonio)alkane-type compounds are archetypal muscarinic allosteric modulators. Phthalimido-substituted hexane-bis-ammonium agents were methylated in the phthalimide moieties and the lateral propyl side chains. All compounds retarded allosterically the dissociation of the orthosteric ligand [(3)H]N-methylscopolamine ([(3)H]NMS) from porcine heart M(2) receptors. [(3)H]NMS equilibrium binding was reduced, left unaltered, or elevated, depending on the degree and position of methylation. This is the first time that an allosteric elevation of ligand binding is demonstrated for bis(ammonio)alkane-type compounds.

Allosteric Regulation↗
PubMed2002

Photocatalyzed [2 + 2 + 2]-cycloaddition of nitriles with acetylene: an effective method for the synthesis of 2-pyridines under mild conditions.

The photocatalyzed [2 + 2 + 2]-cycloaddition of nitriles with 2 equiv of acetylene to 2-pyridines can be carried out under mild conditions and represents a valuable extension to common synthetical methods. For the ideal wavelength range (350-500 nm), lamps as well as sunlight can be used. Working at room temperature and in organic solvents such as toluene or hexane as well as in water gives satisfying results in many cases. However, it is also possible to vary the solvent and the reaction temperature of the photocatalyzed synthesis and to choose, with respect to the specific substrate, specific requirements for this particular reaction and general requirements of the method. This simple and selective method derives its potential mainly from the large variety of applicable nitriles. Suitable substrates include (functionalized) aliphatic and aromatic nitriles as well as cyanamides derived from secondary amines.

Journal Article↗
PubMed2002

Evaluation of the impurity profile of alcuronium by means of capillary electrophoresis.

Alcuronium, a neuromuscular blocking drug, was recently introduced to the European Pharmacopoeia. A HPLC method is described to limit the impurities of alcuronium, namely the diallylcaracurine (DAC) and the allyl-Wieland-Gumlich-aldehyde (WCA), to less than 0.5%. Since alcuronium and all impurities are quaternary salts, capillary electrophoresis (CE) is highly suitable to evaluate the impurity profile. Using 12 mM heptakis-(2,6-di-O-methyl)-beta-cyclodextrin in a 50 mM phosphate buffer at pH 5.5 or 50 mM diethanolamine buffer (pH 9.2)-acetonitrile 19:1 containing heptakis-(2,3-O-diacetyl-6-sulfo)-beta-cyclodextrin the impurities could be baseline separated and quantified. The limit of detection for DAC and WCA was found to be in the same range as found with HPLC; thus, less than 0.1% of both DAC and WCA could be detected in the solution for injection in presence of alcuronium. In injection solutions of alcuronium which were stored at higher temperatures three additional, unidentified impurities were detected. In addition, the conversion of alcuronium to DAC, occurring under acidic condition, was monitored by means of the CE method developed.

Alcuronium↗
PubMed2002

Evaluation of amino sugar, low molecular peptide and amino acid impurities of biotechnologically produced amino acids by means of CE.

As the second part of our studies on the impurity profiles of amino acids produced by biotechnological processes, a micellar electrokinetic chromatography (MEKC) was applied to determine amino sugars, low molecular peptides and amino acids as potential expected impurities at a level 0.1% w/w. 3-(4-Carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA) was found to be suitable as a labeling reagent for laser induced fluorescence (LIF) detection. The labeling reaction was optimized to achieve maximum reaction yields and reproducibility for all groups of substances. The 'level 0.1%' was chosen as quantities/concentrations in such a way that the major reagent peak becomes comparable with the impurities at this level, and the minor reagent peaks (<0.01%) are not able to cover other peaks at the level of interest. The reaction optimization and the validation studies were performed with model mixtures of representative amino sugars, low molecular peptides and amino acids. A linearity range of labeling of three orders of magnitude was achieved. The total precision of the method, including the labeling reproducibility was studied and for all test substances relative standard deviation less than 5% were obtained. The accuracy was evaluated by performing recovery experiments at three concentrations covered a 50-200% interval of the level 0.1%. Confidence intervals below +/-2.5% (lambda=0.05, n=9) of the target were found sufficient for purity tests.

Amino Acids↗
PubMed2002

Quantitative liquid chromatography-mass spectrometry determination of isatin in urine using automated on-line extraction.

Here we describe a simple, fast and sensitive liquid chromatography/mass spectrometry method with automated on-line extraction to quantify isatin, an endogenous monoamine oxidase, and atrial natriuretic peptide inhibitor, in urine. After derivatisation of isatin to isatinoxime with hydroxylamine hydrochloride and zinc sulfate precipitation, samples were loaded on the extraction column, washed and, after activation of the column-switching valve, backflushed onto the analytical column. Using electrospray ionisation, [M+H]+ ions could be detected in the selected ion monitoring mode. The assay was linear from 5 to 5000 ng/ml (r2>0.99) and analytical recovery was >80%. Inter-assay precision for the quality control samples was less than 3% and inter-assay accuracy was within +/- 5%.

Calibration↗
PubMed2002

Studies on the chiral recognition of peptide enantiomers by neutral and sulfated beta-cyclodextrin and heptakis-(2,3-di-O-acetyl)-beta-cyclodextrin using capillary electrophoresis and nuclear magnetic resonance.

The separation of dipeptide and tripeptide enantiomers using a neutral single isomer cyclodextrin (CD) derivative, heptakis-(2,3-di-O-acetyl)-beta-CD (DIAC-beta-CD), was investigated with respect to the amino acid sequence applying standard separation conditions. With only one exception the DD-enantiomers migrated faster than the LL-stereoisomers. Separations obtained for the same set of peptides using beta-CD and the sulfated single isomer derivatives heptakis-(2,3-di-O-acetyl-6-sulfo)-beta-CD (HDAS-beta-CD) and heptakis-6-sulfo-beta-CD (HS-beta-CD) revealed identical enantiomer migration order in the presence of the 2,3-disubstituted derivatives DIAC-beta-CD and HDAS-beta-CD. In contrast, reversed migration sequence was found for beta-CD and HS-beta-CD compared to DIAC-beta CD and HDAS-beta-CD indicating the importance of the substitution pattern on the wider rim of the CD cavity on the chiral recognition of the peptide enantiomers by the CDs. Nuclear magnetic resonance (NMR) experiments indicated different complexation modes between the enantiomers and the CDs depending on the presence of acetyl substituents on the wider rim of the CD torus. Thus, the CD-induced chemical shifts observed in samples containing Ala-Phe or Ala-Tyr and beta-CD or HS-beta-CD were consistent with an inclusion of the aromatic moiety into the CD cavity. Although the CD-induced chemical shifts in the presence of DIAC-beta-CD and HDAS-beta-CD did not allow direct conclusions on the complexation mode they substantially differed from those observed in the presence of 2,3-unsubstituted CDs indicating different structures of the peptide-CD complexes.

Chromatography, Micellar Electrokinetic Capillary↗
PubMed2002

Inhibition of cytochrome P450 3A4 by extracts and kavalactones of Piper methysticum (Kava-Kava).

Inhibitors of cytochrome P450 3A4 (CYP3A4) were identified in crude extracts from the rhizomes of Piper methysticum G. Forst. (Kava-Kava) using bioassay-guided fractionation. After preliminary purification of an ethyl acetate extract with solid phase extraction, the eluate was further fractionated by means of HPLC and fractions were tested for inhibitory potency using cDNA expressed CYP3A4. Positive fractions were analysed with LC/MS using electrospray ionisation and kavapyrones could be identified as the main CYP3A4 inhibitory components of Piper methysticum.

Anisoles↗