An Aza-michael Addition Product Causes Incompatibility Between Etacrynic Acid and Theophylline in a Paediatric Cardiological ICU.

In a compatibility study of parenteral drugs commonly used in paediatric cardiological intensive care units, an unknown reaction product was found in a mixture of etacrynic acid and theophylline. The conditions in terms of the concentration of etacrynic acid and theophylline as well as the materials used corresponded to the conditions in the intensive care unit. Initially, the reaction product appeared as a significant and increasing peak in the chromatograms when determining the content of etacrynic acid and theophylline via HPLC. At the same time, the concentrations of both drugs decreased. A literature search in the chemical databases Reaxys® and Scifinder ® revealed a patent from 1967 describing an aza-Michael addition between etacrynic acid and theophylline to either N-7 or N-9. Using LC-MS/MS experiments, we were able to confirm that Michael-like reaction between etacrynic acid and theophylline occurs. To elucidate the exact structure of the reaction product we performed NMR experiments (COSY, HSQC and HMBC). With the acquired data we were finally able to identify the unknown compound as the N-7 substituted adduct [2-(2,3-dichloro-4-{2-[(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)methyl]butanoyl}phenoxy)acetic acid]. Our findings show that etacrynic acid and theophylline should not be mixed and should be administered through separate venous lines when infused.

Temperature corrected transepithelial electrical resistance (TEER) measurement to quantify rapid changes in paracellular permeability.

Determining the transepithelial electrical resistance (TEER) is a widely used method to functionally analyze tight junction dynamics in cell culture models of physiological barriers. Changes in temperature are known to have strong effects on TEER and can pose problems during the process of TEER measurements in cell culture vessels, complicating comparisons of TEER data across different experiments and studies. Here, we set out to devise a strategy to obtain temperature-independent TEER values based on the physical correlation between parameters such as TEER, temperature, medium viscosity and pore size of the cell culture inserts. By measuring the impact of temperature and different electrode types on TEER measurements on Caco-2 and HPDE (normal human pancreatic ductal epithelium) monolayers, we were able to derive a mathematical method that is suitable for the correction of TEER values for temperature changes. Applying this method to raw TEER values yields temperature-corrected TEER (tcTEER) values. Validity of tcTEER was demonstrated by showing a direct correlation with permeability of monolayers as determined by flux of RITC dextran. Taken together, the mathematical solution presented here allows for a simple and accurate determination of paracellular permeability independent of temperature variation during the process of TEER recording.

[Coagulation and formation of malignant effusions]. / Die Rolle des Gerinnungssystems bei der Entstehung maligner Ergüsse.

Malignant effusions are a frequent problem for cancer patients. Due to the high resistance of tumor cells within these effusions, no effective treatment has been defined yet. Most patients exhibit additional phenomena related to hyper-coagulability such as elevated levels for d-dimers and prothrombin fragments f1.2; half of them suffer from manifest thrombosis or complications. We followed the hypothesis that the activated coagulation system contributes to the resistance of tumor cells and analyzed the effusions from cancer patients. The majority of isolated tumor cells aberrantly expressed PAR-1 thrombin receptors. In vitro pre-incubation of PAR-1 expressing human leukemia cells with thrombin resulted in a dose-dependent resistance to idarubicin. Within the effusions, we did not only find high concentrations of VEGF and tissue factor, but also all coagulation factors of the tissue factor pathway. Very high levels of prothrombin fragments f1.2 indicate constant thrombin generation. Upon the basis of these findings, we developed a multistep model elucidating the pathophysiological generation of malignant effusions, which might serve as a basis for further examinations.

Activation of the coagulation system in cancerogenesis and metastasation.

The activation of the coagulation system in cancer patients is a well-known phenomenon responsible for recurrent clinical problems. A number of fascinating molecular mechanisms have been recognized showing that the tumor not only activates the coagulation system, but vice versa, activated coagulation proteins are able to induce molecular effects in tumor cells. The molecular basis is the expression of defined membrane receptors by tumor cells that are activated, for example, by thrombin. As the liberation of thrombin from prothrombin is one of the key events in coagulation, it's impact upon biological processes, such as cancerogenesis and metastasation, seems to be a regular pathophysiological consequence. These perceptions are not only interesting for the comprehension of cancerogenesis, metastasation, and clinical phenomena, but they also have a high impact upon modern strategies of tumor therapy. Especially, the development of clinically useful coagulation inhibitors, such as modern low molecular weight heparins or melagatran, created the possibility of therapies that combine cell biological approaches with apoptosis-inducing principals such as chemotherapy. Several clinical studies that demonstrate the implication of these strategies have already been published recently. In this article the cell biological basics for these approaches are reviewed.

Phosphono analogs of glutathione: inhibition of glutathione transferases, metabolic stability, and uptake by cancer cells.

Glutathione transferases (GSTs) have been shown to play an important role in multiple drug resistance in cancer chemotherapy. The inactivation of GST isoforms could lead to an enhanced activity of cytotoxic drugs. Thus, we have developed glutathione phosphono analogs [(S)-gamma-glutamyl-(2RS)-(+/-)-2-amino-(dialkoxyphosphinyl)-ac etylgl ycines], which were previously shown to be inhibitors of GSTP1-1. In the present study, the inhibition characteristics of these analogs, including isoenzyme specificities, type of inhibition, and determination of K(i) values, were determined. The inhibition of class alpha GSTs was competitive towards GSH. A mixed-type, non-competitive inhibition of class mu and pi GSTs was observed. The K(i) values varied between 880 +/- 210 and 0.45 +/- 0.1 microM. The inhibitors were most effective towards class mu GSTs. In order to investigate the potential use of these GST inhibitors in intact cellular systems, two additional approaches were examined. Firstly, the metabolic stability was tested with purified gamma-glutamyl transpeptidase and cell homogenates as well as during incubation of cell lines. No appreciable degradation was observed in any of the tested systems. Secondly, to facilitate cellular uptake, three derivatives were synthesized in which the glycine carboxylic group was esterified. Uptake and a possible intracellular cleavage to the corresponding free acids were monitored by HPLC analysis. The esters were effectively transported into HT29 (colon cancer) and EPG85-257P (gastric cancer) cells, respectively, and readily converted into the more active free acids. In conclusion, the tested inhibitors may be regarded as model compounds for the development of modulating agents in cancer chemotherapy.

Model based classification of cardiovascular response patterns.

A comprehensive analysis of cardiovascular control (CVC) patterns with multiple subjects is presented. It became feasible by recent methodological advances. Simple computer models were generated automatically, reproducing only factors of the true model that are relevant to the focus if investigation. These models--named aspect-models--could in turn be used in model individualization, thus reducing the necessary computational amount. The achieved speedup by a factor of more than three thousand and the high numerical stability of the resulting method allows the unsupervised identification of a large body of experimental data. The analysis of tilt table experiments of 18 subjects revealed a remarkable variety of reaction patterns. Closer examination yielded different classes of subjects. Two main groups corresponding to basic types of CVC were observed. Three outliers could be assigned to the specific situation of some subjects.

Purification and characterization of class alpha and Mu glutathione S-transferases from porcine liver.

Six cytosolic GSTs from porcine liver were purified by a combination of glutathione affinity chromatography and ion-exchange HPLC. The isoenzymes were characterized by SDS-PAGE, gel filtration, isoelectric focusing, immunoblotting analysis and determination of substrate specificities and inhibition characteristics. The purified GSTs belong to the alpha and mu classes, respectively. No class pi isoenzyme was isolated or detected. The class alpha GST pA1-1* exists as a homodimer (M(r) = 25.3 kDa), whereas GST pA2-3* consists of two subunits with different M(r) values (27.0 and 25.3 kDa). The estimated pI values were 9.5 and 8.8, respectively. Furthermore, four class mu porcine GSTs, pM1-1*, pM1-2*, pM3-?* and pM4-?*, were isolated. The isoenzyme pM1-1* possesses a relative molecular mass of 27.2 kDa and a pI value of 6.2. Additional pM1 isoenzymes hybridize with the subunit pM2* (M(r) = 25.2) to furnish a heterodimer, which shows a pI value of 5.8. The other class mu isoenzymes are heterodimers with pI values of 5.45 and 5.05. Substrate specificities and inhibition characteristics correlate very well with those of the corresponding human isoenzymes. The results are discussed with regard to the usefulness of porcine GSTs as an in vitro testing model.

Phosphono analogues of glutathione as new inhibitors of glutathione S-transferases.

Phosphono-analogues of glutathione containing the O = P(OR)2 moiety in place of the cysteinyl residue CH2SH 1a-1d were prepared by solution phase peptide synthesis. Benzyl, benzyloxy-carbonyl, and tert-butyl protecting groups were used to mask the individual amino acid functional groups. The formation of peptide bonds was achieved by the usual peptide synthesis via activation of carboxylic functions with cyclohexylcarbodiimide and subsequent reaction with free amino groups. The thus obtained, fully-protected peptides were each purified by normal phase column chromatography. Deprotection was accomplished by hydrogenolysis and by treatment with HBr/acetic acid yielding the desired phosphonic acid diester 1a-1d. The inhibition of the glutathione conjugation of 1-chloro-2,4-dinitrobenzene by human placental glutathione S-transferase was studied by determining the IC50 values of the new glutathione analogues. The IC50 values were 291 microM, 139 microM, 64 microM, and 21 microM for the dimethyl, diethyl, diisopropyl, and di-n-butyl esters, respectively. The results clearly show that the formal substitution of the glutathione thiol function by phosphonic acid esters leads to a new class of glutathione S-transferase inhibitors. Further investigations directed at the question of whether or not these glutathione analogues are suitable for a modulation in chemotherapy are in progress.

In vitro oxygenation of N,N'-diphenylguanidines.

1. The enzymic C-oxygenation of N,N'-diphenylguanidine (DPG) to N-(4-hydroxyphenyl)-N'-phenylguanidine (4HPG) and the N-oxygenation of N,N'-bis-(pentafluorophenyl)-guanidine (BPG) to N"-hydroxy-N,N"-bis-(pentafluorophenyl)-guanidine (HBPG) is reported. 2. The metabolites were identified by t.l.c. and mass spectral analysis using synthetic reference compounds. 3. Rat and rabbit liver homogenates (9000 g supernatant and microsomes) were used as enzyme source. 4. The enzymic oxygenations were both O2 and NADPH dependent. NADPH could not be replaced by hydrogen peroxide. 5. 15N-n.m.r. spectroscopy was used to elucidate structure and tautomerism of BPG and HBPG.

Microsomal N-oxygenation of adenine to adenine 1-N-oxide.

During investigations on the N-oxygenation of adenine (1) the enzymatic formation of adenine 1-N-oxide 3 was demonstrated for the first time. The identity of this metabolite was confirmed by its chromatographic behaviour and UV-spectrum recorded after HPLC separation. Adenine 1-N-oxide (3) and similar oxygenated derivatives of adenine were synthesized as reference substances. The enzymatic formation of 3 exhibits the typical characteristics of a reaction catalysed by microsomal mono-oxygenases. In induction experiments, an increase in the rate of formation of 3 after pretreatment with phenobarbital was observed. A participation of those isoenzymes of the cytochrome P-450 enzyme system which can be induced by phenobarbital is assumed.

The reduction of 6-N-hydroxylaminopurine to adenine by xanthine oxidase.

The genotoxic and mutagenic compound 6-N-hydroxylaminopurine (HAP) can be detoxified in vitro by enzymatic N-reduction to adenine. This reaction is catalysed by both rat and rabbit liver cytosolic fractions. The formation of adenine was monitored using HPLC. Subcellular distribution of the activity, kinetic parameters and the influence of various cofactors and inhibitors were determined. The N-reduction required NADH or hypoxanthine or xanthine and was strongly inhibited by allopurinol. These observations suggested that the N-reductase activity is due to xanthine oxidase (EC 1.2.3.2). Moreover, the involvement of xanthine oxidase is supported by the observation that purified cow milk xanthine oxidase also catalysed this reaction. The N-reduction of HAP was inhibited only weakly by oxygen. In addition, the formation of adenine is catalysed by either the oxidase or dehydrogenase form of xanthine oxidase. Thus, this reaction should be significant for the in vivo detoxification of HAP.

Hepatic microsomal N-hydroxylation of adenine to 6-N-hydroxylaminopurine.

The enzymatic N-hydroxylation of the purine base adenine to the genotoxic and mutagenic compound 6-N-hydroxylaminopurine is reported for the first time. Adenine was N-oxygenated in vitro by aerobic incubations with 3-methylcholanthrene or isosafrole induced microsomal fractions of rat liver homogenates and NADPH. The formation of 6-N-hydroxylaminopurine in the incubation mixtures under widely differing conditions was assayed using newly-developed, high-performance liquid- and thin-layer chromatographic methods. Optimal reaction conditions and kinetic parameters were determined. Neither superoxide anion nor hydrogen peroxide was directly involved in the N-hydroxylation reaction. Oxidases like xanthine oxidase and peroxidase (in the presence of hydrogen peroxide) did not catalyse this N-hydroxylation. The involvement of cytochrome P-450 isoenzymes in this reaction is supported by the observation that the N-hydroxylation is only observed after pretreatment of the rats with 3-methylcholanthrene or isosafrole. Other inducers (phenobarbital, ethanol, 5-pregnen-3 beta ol-20-one-16 alpha-carbonitrile) were without effect. This is the first example of the microsomal transformation of an endogenous substance to a toxic derivative by usually foreign substances (xenobiotics) metabolizing cytochrome P-450 isoenzymes. The significance for the in vivo situation is discussed on the basis of the data obtained in this study.