Automatation of an analytical laboratory for the determination of drug levels in body fluids

Summary For the specific determination of drug levels in body fluids selective chromatographic methods are now widely used. Because these methods are time-consuming, there is a need for automatation, especially when many samples are to be run. This automatation needs special precautions so that the quality of analyses is controlled and maintained. Possibilities for automatation of chromatographic methods and solutions of the attendant problems are discussed.

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Automatisierung der Bestimmung von Arzneispiegeln in Körperflüssigkeiten

Zusammenfassung Die spezifische Bestimmung von Arzneimittelspiegeln in Körperflüssigkeiten erfolgt heute überwiegend mit chromatographischen Methoden. Besonders bei der Bestimmung größerer Serien wird es notwendig, diese zu automatisieren. Dazu sind jedoch spezielle Vorkehrungen zur Kontrolle und Aufrechterhaltung der Qualität der Analysenergebnisse zu treffen. Möglichkeiten zur Automatisierung chromatographischer Bestimmungsmethoden und Vorschläge zur Lösung der damit zusammenhängenden Probleme werden diskutiert.

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Herrn Prof. Dr. Eberhard von Wasielewski zu seinem 60. Geburtstag gewidmet.

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Presented at the 8th International Microchemical Symposium, Graz, August 25–30, 1980.     

Cytotoxicity of Amino-BODIPY Modulated via Conjugation with 2-Phenyl-3-Hydroxy-4(1H)-Quinolinones

The combination of cytotoxic amino-BODIPY dye and 2-phenyl-3-hydroxy-4(1H)-quinolinone (3-HQ) derivatives into one molecule gave rise to selective activity against lymphoblastic or myeloid leukemia and the simultaneous disappearance of the cytotoxicity against normal cells. Both species′ conjugation can be realized via a disulfide linker cleavable in the presence of glutathione characteristic for cancer cells. The cleavage liberating the free amino-BODIPY dye and 3-HQ derivative can be monitored by ratiometric fluorescence or by the OFF-ON effect of the amino-BODIPY dye. A similar cytotoxic activity is observed when the amino-BODIPY dye and 3-HQ derivative are connected through a non-cleavable maleimide linker. The work reports the synthesis of several conjugates, the study of their cleavage inside cells, and cytotoxic screening.     

Synthesis and anticancer activity of some 1,5-diaryl-3-(3,4,5-trihydroxyphenyl)-1H-pyrazolo[4,3-e][1,2,4]triazines

Abstract  

The deregulation of cell cycle components in cancer cells has provided a rationale for the development of small molecule inhibitors of cyclin-dependent kinases as novel anticancer drugs. A series of 1,5-diaryl-3-(3,4,5-trihydroxyphenyl)-1H-pyrazolo[4,3-e][1,2,4]triazines was synthesized and their kinase inhibitory activity and cytotoxicity against several cancer cell lines has been evaluated. Some of the compounds of the series exhibited induction of caspase-dependent cell death and inhibition of cyclin-dependent kinase 2 (CDK2).     

Batch immunoextraction method for efficient purification of aromatic cytokinins

A range of benzylaminopurines naturally occur in plants and exhibit high biological activity. Others have been synthesized, such as 6-(2-hydroxy-3-methoxybenzylamino)purine riboside (2OH3MeOBAPR), which has shown interesting anti-cancer activity under in vitro conditions. In order to study the biological activity of this interesting compound in more detail, a rapid and highly efficient method for its purification from complex samples (e.g. blood and plant extracts) is needed. Therefore, we prepared monoclonal antibodies against 2OH3MeOBAPR. The antibody had undetectable cross-reactivity with all natural isoprenoid cytokinins, but relatively high cross-reactivity with aromatic cytokinins as well as some synthetic di- and tri-substituted 6-benzylaminopurines and the corresponding ribosides. The antibody also showed strong responses and specificity in enzyme-linked immunoassays (ELISAs). In addition, it was used to prepare, for the first time, an immunoaffinity sorbent with high specificity and capacity for aromatic cytokinins. A batch immunoextraction method was then developed and optimized for the purification of 2OH3MeOBAPR from murine blood samples. The high efficacy and simplicity of this method (in off-line combination with HPLC-MS) for the isolation of target analytes from biological material is demonstrated in this study.     

Proteomics approach in classifying the biochemical basis of the anticancer activity of the new olomoucine-derived synthetic cyclin-dependent kinase inhibitor, bohemine

The aim of this study was to use two-dimensional electrophoresis (2-DE) coupled with multivariate principal component analysis (PCA) to characterize the quantitative changes in the protein composition of the CEM T-lymphoblastic leukemia cell line after treatment with bohemine (BOH), a synthetic olomoucin-derived cyclin-dependent kinase inhibitor (CDKI). Cell classification, reflecting protein patterns, clearly distinguished two main groups: one group consists of 9, 12 and 24 h treated BOH cells while the second is represented by the 0 and 24 h control untreated cells and the 6 h BOH-exposed CEM lymphoblasts. Discriminant protein spots differentially expressed in the BOH-treated CEM cells were selected for identification by matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) or electrospray ionization-tandem MS (ESI-MS/MS). Five of the selected protein spots were unequivocally identified as alpha-enolase, triosephosphate isomerase, eukaryotic initiation factor 5A, and alpha- and beta-subunits of Rho GDP-dissociation inhibitor 1. These proteins, all significantly downregulated in CEM T-lymphoblast leukemia in the course of BOH treatment, are known to play an important role in cellular functions such as glycolysis, protein biosynthesis, and cytoskeleton rearrangement. These results indicate that the cellular effects of olomoucine-derived CDKIs are not dependent on their ability to inhibit CDKs and could be mediated by several factors such as a decrease in protein synthesis and/or glycolysis which in turn diminishes the ability of cancer cells to function.     

Synthesis,characterization and biological activity of two nickel(II) complexes with 6-(2-chlorobenzylamino)purine

Compounds of composition [Ni(L)(H₂O)₂Cl] and [Ni(L)(H₂O)(NO₃)] · EtOH [HL = 6-(2-chlorobenzylamino)purine] have been synthesized and characterized by elemental analyses, i.r. and electronic spectra, magnetic measurements and mass spectroscopy as tetrahedral nickel(II) complexes. The geometry of both complexes has been optimized using molecular mechanics modelling. Although the monoanionic ligand L is potentially bidentate, we assume that the coordination to nickel is via the N₉ atom only. Cytokinin and anti-cancer activities of the complexes were also tested in an Amaranthus cytokinin bioassay and in an in vitro MTT-based cytotoxicity assay, respectively. In human T-lymphoblastic leukemia cell line CCRF-CEM both complexes showed potent cytotoxic activity.     

Identification of potential glutaminyl cyclase inhibitors from lead-like libraries by in silico and in vitro fragment-based screening

A glutaminyl cyclase (QC) fragment library was in silico selected by disconnection of the structure of known QC inhibitors and by lead-like 2D virtual screening of the same set. The resulting fragment library (204 compounds) was acquired from commercial suppliers and pre-screened by differential scanning fluorimetry followed by functional in vitro assays. In this way, 10 fragment hits were identified (\(\sim \)5 % hit rate, best inhibitory activity: 16 \(\upmu \hbox {M}\)). The in vitro hits were then docked to the active site of QC, and the best scoring compounds were analyzed for binding interactions. Two fragments bound to different regions in a complementary manner, and thus, linking those fragments offered a rational strategy to generate novel QC inhibitors. Based on the structure of the virtual linked fragment, a 77-membered QC target focused library was selected from vendor databases and docked to the active site of QC. A PubChem search confirmed that the best scoring analogues are novel, potential QC inhibitors.