Interplay between endothelin and erythropoietin in astroglia: the role in protection against hypoxia.

We show that, under in vitro conditions, the vulnerability of astroglia to hypoxia is reflected by alterations in endothelin (ET)-1 release and capacity of erythropoietin (EPO) to regulate ET-1 levels. Exposure of cells to 24 h hypoxia did not induce changes in ET-1 release, while 48-72 h hypoxia resulted in increase of ET-1 release from astrocytes that could be abolished by EPO. The endothelin receptor type A (ETA) antagonist BQ123 increased extracellular levels of ET-1 in human fetal astroglial cell line (SV-FHAS). The survival and proliferation of rat primary astrocytes, neural precursors, and neurons upon hypoxic conditions were increased upon administration of BQ123. Hypoxic injury and aging affected the interaction between the EPO and ET systems. Under hypoxia EPO decreased ET-1 release from astrocytes, while ETA receptor blockade enhanced the expression of EPO mRNA and EPO receptor in culture-aged rat astroglia. The blockade of ETA receptor can increase the availability of ET-1 to the ETB receptor and can potentiate the neuroprotective effects of EPO. Thus, the new therapeutic use of combined administration of EPO and ETA receptor antagonists during hypoxia-associated neurodegenerative disorders of the central nervous system (CNS) can be suggested.

Losartan Improves Memory, Neurogenesis and Cell Motility in Transgenic Alzheimer's Mice.

Angiotensin receptor blockers (ARBs) have demonstrated multiple neuroprotective benefits in Alzheimer's disease (AD) models. However, their beneficial effects on memory deficits, cholinergic activity, neurogenesis and Amyloid beta (Aß) clearance reveal significant interstudy variability. The delivery route can impact not only delivery but also targeting and therapeutic efficacy of ARBs. Our previous findings on the beneficial effects of intranasally delivered losartan in the APP/PS1 model of AD prompted us to explore the influence of the delivery route by employing here the systemic administration of losartan. Consistent with our previous results with intranasal losartan, repeated intraperitoneal administration (10 mg/kg) resulted in a remarkable decrease in Aß plaques and soluble Aß42, as well as inflammatory cytokines (IL-2, IL-6 and TNFα). The Aß reduction can be ascribed to its facilitated degradation by neprilysin and diminished generation by BACE1. Losartan increased neurogenesis in vivo and in vitro and improved migratory properties of astrocytes isolated from adult transgenic AD mice. In summary, this data together with our previous results suggest therapeutic features of losartan which are independent of delivery route. The improvement of cell motility of Aß-affected astrocytes by losartan deserves further in vivo investigation, which may lead to new strategies for AD treatment.

Pharmacokinetics of natural mistletoe lectins after subcutaneous injection.

PURPOSE: Knowledge of natural mistletoe lectins (nML) pharmacokinetics can be regarded as essential for further rational studies with mistletoe preparations. Studies with intravenous application of a recombinant type II ribosome inactivating protein (rML) analogous to nML revealed a short half-life of about 13 min in cancer patients. This open-label, phase I, monocenter clinical trial was performed in order to describe the pharmacokinetics of nML. METHODS: In 15 healthy male volunteers aged 18-42 years, nML were detected with a modified sandwich immuno-polymerase chain reaction (PCR) technique (Imperacer, Chimera Biotec) after single subcutaneous injection of a mistletoe extract (abnobaVISCUM(R) Fraxini 20 mg) with marketing authorization containing about 20 microg nML/ml. Secondary objectives were safety and the number of activated natural killer cells (CD54(+)/CD94(+)). RESULTS: In none of the volunteers were nML detectable before the injection, and in all volunteers, nML were detected in serum samples after injection. Individual variability, however, was large. Mean and median peak concentrations were reached 1 and 2 h after injection, respectively. In some volunteers, nML were still detectable at the final investigation 2 weeks after injection. The injection resulted in fever and flu-like symptoms in all volunteers, but no serious adverse events occurred. All symptoms and local reactions at the injection site completely disappeared within a range of 4-95 days. The number of activated natural killer (NK) cells did not change. CONCLUSIONS: Natural ML from abnobaVISCUM Fraxini 20 mg are detectable in serum after a single subcutaneous injection. Detectability is considerably longer compared with intravenously administered rML. The subcutaneous injection of this preparation without usual pretreatment with lower doses results in short-lasting fever and other flu-like symptoms.

Antifibrotic Effects of Amyloid-Beta and Its Loss in Cirrhotic Liver.

The function and regulation of amyloid-beta (Aß) in healthy and diseased liver remains unexplored. Because Aß reduces the integrity of the blood-brain barrier we have examined its potential role in regulating the sinusoidal permeability of normal and cirrhotic liver. Aß and key proteins that generate (beta-secretase 1 and presenilin-1) and degrade it (neprilysin and myelin basic protein) were decreased in human cirrhotic liver. In culture, activated hepatic stellate cells (HSC) internalized Aß more efficiently than astrocytes and HSC degraded Aß leading to suppressed expression of α-smooth muscle actin (α-SMA), collagen 1 and transforming growth factor ß (TGFß). Aß also upregulated sinusoidal permeability marker endothelial NO synthase (eNOS) and decreased TGFß in cultured human liver sinusoidal endothelial cells (hLSEC). Liver Aß levels also correlate with the expression of eNOS in transgenic Alzheimer's disease mice and in human and rodent cirrhosis/fibrosis. These findings suggest a previously unexplored role of Aß in the maintenance of liver sinusoidal permeability and in protection against cirrhosis/fibrosis via attenuation of HSC activation.

Cell motility and migration as determinants of stem cell efficacy.

BACKGROUND: Stem cells` (SC) functional heterogeneity and its poorly understood aetiology impedes clinical development of cell-based therapies in regenerative medicine and oncology. Recent studies suggest a strong correlation between the SC migration potential and their therapeutic efficacy in humans. Designating SC migration as a denominator of functional SC heterogeneity, we sought to identify highly migrating subpopulations within different SC classes and evaluate their therapeutic properties in comparison to the parental non-selected cells. METHODS: We selected highly migrating subpopulations from mesenchymal and neural SC (sMSC and sNSC), characterized their features including but not limited to migratory potential, trophic factor release and transcriptomic signature. To assess lesion-targeted migration and therapeutic properties of isolated subpopulations in vivo, surgical transplantation and intranasal administration of MSCs in mouse models of glioblastoma and Alzheimer's disease respectively were performed. FINDINGS: Comparison of parental non-selected cells with isolated subpopulations revealed superior motility and migratory potential of sMSC and sNSC in vitro. We identified podoplanin as a major regulator of migratory features of sMSC/sNSC. Podoplanin engineering improved oncovirolytic activity of virus-loaded NSC on distantly located glioblastoma cells. Finally, sMSC displayed more targeted migration to the tumour site in a mouse glioblastoma model and remarkably higher potency to reduce pathological hallmarks and memory deficits in transgenic Alzheimer's disease mice. INTERPRETATION: Functional heterogeneity of SC is associated with their motility and migration potential which can serve as predictors of SC therapeutic efficacy. FUNDING: This work was supported in part by the Robert Bosch Stiftung (Stuttgart, Germany) and by the IZEPHA grant.

Prediction of breast cancer by profiling of urinary RNA metabolites using Support Vector Machine-based feature selection.

BACKGROUND: Breast cancer belongs to the most frequent and severe cancer types in human. Since excretion of modified nucleosides from increased RNA metabolism has been proposed as a potential target in pathogenesis of breast cancer, the aim of the present study was to elucidate the predictability of breast cancer by means of urinary excreted nucleosides. METHODS: We analyzed urine samples from 85 breast cancer women and respective healthy controls to assess the metabolic profiles of nucleosides by a comprehensive bioinformatic approach. All included nucleosides/ribosylated metabolites were isolated by cis-diol specific affinity chromatography and measured with liquid chromatography ion trap mass spectrometry (LC-ITMS). A valid set of urinary metabolites was selected by exclusion of all candidates with poor linearity and/or reproducibility in the analytical setting. The bioinformatic tool of Oscillating Search Algorithm for Feature Selection (OSAF) was applied to iteratively improve features for training of Support Vector Machines (SVM) to better predict breast cancer. RESULTS: After identification of 51 nucleosides/ribosylated metabolites in the urine of breast cancer women and/or controls by LC- ITMS coupling, a valid set of 35 candidates was selected for subsequent computational analyses. OSAF resulted in 44 pairwise ratios of metabolite features by iterative optimization. Based on this approach ultimately estimates for sensitivity and specificity of 83.5% and 90.6% were obtained for best prediction of breast cancer. The classification performance was dominated by metabolite pairs with SAH which highlights its importance for RNA methylation in cancer pathogenesis. CONCLUSION: Extensive RNA-pathway analysis based on mass spectrometric analysis of metabolites and subsequent bioinformatic feature selection allowed for the identification of significant metabolic features related to breast cancer pathogenesis. The combination of mass spectrometric analysis and subsequent SVM-based feature selection represents a promising tool for the development of a non-invasive prediction system.

The targeted oral, once-daily phosphodiesterase 4 inhibitor roflumilast and the leukotriene receptor antagonist montelukast do not exhibit significant pharmacokinetic interactions.

This nonrandomized, fixed-sequence, 3-period study investigated potential pharmacokinetic interactions between the leukotriene receptor antagonist montelukast, approved for the treatment of asthma, and roflumilast, an oral, once-daily phosphodiesterase 4 inhibitor in clinical development for asthma and chronic obstructive pulmonary disease. Pharmacokinetic interactions are of interest because both drugs may be coadministered and share a common metabolic pathway via cytochrome P450 3A. Single-dose montelukast (10 mg, po) was administered alone in period 1, followed by repeated once-daily roflumilast alone (500 microg, po) for 12 days (period 2). In period 3, 500 microg qd roflumilast was coadministered with 10 mg qd montelukast for 8 days. Different pharmacokinetic parameters were evaluated for montelukast alone, for steady-state roflumilast and its pharmacologically active metabolite roflumilast N-oxide alone, for single-dose montelukast when coadministered with steady-state roflumilast, and for steady-state roflumilast and its N-oxide metabolite when coadministered with steady-state montelukast. The AUC and Cmax of montelukast were modestly increased by 9% and 8%, respectively, when single-dose montelukast was coadministered with steady-state roflumilast. The pharmacokinetics of roflumilast and roflumilast N-oxide in steady state remained unchanged when repeat-dose montelukast was coadministered at steady-state. Concomitant administration of both drugs was well tolerated. These findings suggest that no dose adjustment is warranted for either drug when roflumilast and montelukast are coadministered.

Intranasal Losartan Decreases Perivascular Beta Amyloid, Inflammation, and the Decline of Neurogenesis in Hypertensive Rats.

The contribution of the local angiotensin receptor system to neuroinflammation, impaired neurogenesis, and amyloid beta (Aß) accumulation in Alzheimer's disease (AD) and in hypertension is consistent with the remarkable neuroprotection provided by angiotensin receptor blockers (ARBs) independent of their blood pressure-lowering effect. Considering the causal relationship between hypertension and AD and that targeting cerebrovascular pathology with ARBs does not necessarily require their systemic effects, we tested intranasal losartan in the rat model of chronic hypertension (spontaneously hypertensive stroke-prone rats, SHRSP). Intranasal losartan at a subdepressor dose decreased mortality, neuroinflammation, and perivascular content of Aß by enhancing key players in its metabolism and clearance, including insulin-degrading enzyme, neprilysin, and transthyretin. Furthermore, this treatment improved neurologic deficits and increased brain IL-10 concentration, hippocampal cell survival, neurogenesis, and choroid plexus cell proliferation in SHRSP. Losartan (1 µM) also reduced LDH release from cultured astroglial cells in response to toxic glutamate concentrations. This effect was completely blunted by IL-10 antibodies. These findings suggest that intranasal ARB treatment is a neuroprotective, neurogenesis-inducing, and Aß-decreasing strategy for the treatment of hypertensive stroke and cerebral amyloid angiopathy acting at least partly through the IL-10 pathway.

Effect of coadministered ketoconazole, a strong cytochrome P450 3A4 enzyme inhibitor, on the pharmacokinetics of ciclesonide and its active metabolite desisobutyryl-ciclesonide.

BACKGROUND AND OBJECTIVES: Cytochrome P450 (CYP) 3A4 isoenzyme has been identified in vitro as the key enzyme to metabolize desisobutyryl-ciclesonide (des-CIC), the pharmacologically active metabolite of the inhaled corticosteroid ciclesonide. This pharmacokinetic drug-drug interaction study was conducted to confirm this major metabolic pathway in vivo by using the strong CYP3A4 inhibitor ketoconazole, and to assess the effect of ketoconazole on the pharmacokinetics of ciclesonide and des-CIC. METHODS: Fourteen healthy adults participated in this open-label, nonrandomized, fixed sequence, two-period, repeated-dose pharmacokinetic study. During the first 7-day treatment period, the subjects orally inhaled ciclesonide 320 microg once daily. During the second 7-day treatment period, the subjects continued with the same dose of orally inhaled ciclesonide and concomitantly received oral ketoconazole 400 mg once daily. Pharmacokinetic profiles for ciclesonide and des-CIC were obtained on day 7 of each study period. RESULTS: For the parent compound, ciclesonide, no changes in the pharmacokinetic parameter estimates--the area under the serum concentration-time curve during the dosage interval (AUC(tau)), maximum serum concentration (C(max)) and time to reach the C(max)--were observed. In contrast, the AUC(tau) and C(max) of des-CIC increased approximately 3.5-fold and 2-fold under the influence of the CYP3A4 inhibitor ketoconazole. CONCLUSIONS: The CYP3A4 pathway is the major pathway for biotransformation of the active metabolite of ciclesonide in humans. While elimination of des-CIC was reduced by strong CYP3A4 inhibitor coadministration in vivo, activation of the parent compound ciclesonide to des-CIC was not affected. Dose adjustment is not necessary when ciclesonide needs to be coadministered with ketoconazole, because the potency of an inhaled corticosteroid is mediated by topical concentrations in the lung and because ciclesonide has a very low potential to produce systemic adverse effects.

Identification of urinary modified nucleosides and ribosylated metabolites in humans via combined ESI-FTICR MS and ESI-IT MS analysis.

The physiological response of the human body to several diseases can be reflected by the metabolite pattern in biological fluids. Cancer, like other diseases accompanied by metabolic disorders, causes characteristic effects on cell turnover rate, activity of modifying enzymes, and RNA/DNA modifications. This results in an altered excretion of modified nucleosides and biochemically related compounds. In the course of our metabolic profiling project, we screened 24-h urine of patients suffering from lung, rectal, or head and neck cancer for previously unknown ribosylated metabolites. Therefore, we developed a sample preparation procedure based on boronate affinity chromatography followed by additional prepurification with preparative TLC. The isolated metabolites were analyzed by ion trap mass spectrometry (IT MS) and Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS). IT MS was applied for LC-auto MS(3) screening runs and MS(n(n=4-6)) syringe pump infusion experiments, yielding characteristic fragmentation patterns. FTICR MS measurements enabled the calculation of corresponding molecular formulae based on accurate mass determination (mass accuracy: 1-5 ppm for external and sub-ppm values for internal calibration). We were able to identify 22 metabolites deriving from cellular RNA metabolism and related metabolic pathways like histidine metabolism, purine biosynthesis, methionine/polyamine cycle, and nicotinate/nicotinamide metabolism. The compounds 1-ribosyl-3-hydroxypyridinium, 1-ribosyl-pyridinium, and 3-ribosyl-1-methyl-l-histidinium as well as a series of ribosylated histamines, conjugated to carboxylic acids at the N(omega)-position were found as novel urinary constituents. The occurrence of the modified nucleosides 2-methylthio-N(6)-(cis-hydroxyisopentenyl)-adenosine, 5-methoxycarbonylmethyl-2-thiouridine, N(6)-methyl-N(6)-threonylcarbamoyladenosine, and 2-methylthio-N(6)-threonylcarbamoyladenosine in human urine is verified for the first time.

Metabolic signature of breast cancer cell line MCF-7: profiling of modified nucleosides via LC-IT MS coupling.

BACKGROUND: Cancer, like other diseases accompanied by strong metabolic disorders, shows characteristic effects on cell turnover rate, activity of modifying enzymes and DNA/RNA modifications, resulting also in elevated amounts of excreted modified nucleosides. For a better understanding of the impaired RNA metabolism in breast cancer cells, we screened these metabolites in the cell culture supernatants of the breast cancer cell line MCF-7 and compared it to the human mammary epithelial cells MCF-10A. The nucleosides were isolated and analyzed via 2D-chromatographic techniques: In the first dimension by cis-diol specific boronate affinity extraction and subsequently by reversed phase chromatography coupled to an ion trap mass spectrometer. RESULTS: Besides the determination of ribonucleosides, additional compounds with cis-diol structure, deriving from cross-linked biochemical pathways, like purine-, histidine- and polyamine metabolism were detected. In total, 36 metabolites were identified by comparison of fragmentation patterns and retention time. Relation to the internal standard isoguanosine yielded normalized area ratios for each identified compound and enabled a semi-quantitative metabolic signature of both analyzed cell lines.13 of the identified 26 modified ribonucleosides were elevated in the cell culture supernatants of MCF-7 cells, with 5-methyluridine, N2,N2,7-trimethylguanosine, N6-methyl-N6-threonylcarbamoyladenosine and 3-(3-aminocarboxypropyl)-uridine showing the most significant differences. 1-ribosylimidazole-4-acetic acid, a histamine metabolite, was solely found in the supernatants of MCF-10A cells, whereas 1-ribosyl-4-carboxamido-5-aminoimidazole and S-adenosylmethionine occurred only in supernatants of MCF-7 cells. CONCLUSION: The obtained results are discussed against the background of pathological changes in cell metabolism, resulting in new perspectives for modified nucleosides and related metabolites as possible biomedical markers for breast carcinoma in vivo.

Effect of high-dose metronidazole on pharmacokinetics of oral budesonide and vice versa: a double drug interaction study.

Recent case reports suggest that addition of high-dose metronidazole might be associated with elevated plasma concentrations of substrates of cytochrome P450 (CYP) 3A. Because patients with fistulizing Crohn's disease benefit by using high doses of metronidazole for prolonged periods, this study's primary aim was to evaluate the effect of high-dose metronidazole on the pharmacokinetics of oral budesonide, a sensitive substrate of CYP3A commonly prescribed in acute inflammatory bowel disease. Twelve healthy adults received 1.5 g metronidazole per day over 1 week. The CYP3A-dependent metabolic profile of an oral dose of budesonide (3 mg) and that of endogenous cortisol were compared intraindividually before and after administration of metronidazole. There was neither a significant effect of high-dose metronidazole on the area under the plasma concentration-time curve (AUC) of budesonide (90% confidence interval, 79%-115%) nor on the AUC ratios of 6beta-hydroxybudesonide/budesonide and 16alpha-hydroxyprednisolone/budesonide. In parallel, metronidazole did not significantly alter formation of 6beta-hydroxycortisol. Vice versa, budesonide did not affect the AUC of metronidazole (90% confidence interval, 91%-100%). The authors conclude that in contrast to concomitant intake of other imidazoles such as ketoconazole, concomitant intake of metronidazole may not lead to serious safety concerns due to elevated systemic concentrations of the glucocorticoid budesonide.

Dose-proportional intraindividual single- and repeated-dose pharmacokinetics of roflumilast, an oral, once-daily phosphodiesterase 4 inhibitor.

The dose-proportional, intraindividual, single- and repeated-dose pharmacokinetics of roflumilast, an oral, once-daily phosphodiesterase 4 inhibitor under investigation for chronic obstructive pulmonary disease and asthma, was investigated in healthy subjects. In an open, randomized, 2-period, 2-sequence crossover study, 15 subjects received immediate-release tablets of roflumilast 250 or 500 microg as single (day 1) and as repeated, once-daily doses for 8 days (days 5-12). Dose-adjusted point estimates and 90% confidence intervals of test (500 microg)/reference (250 microg) ratios for AUC and Cmax of roflumilast and its pharmacologically active N-oxide metabolite after single and repeated dosing were all within the standard equivalence acceptance range (0.80, 1.25) indicating dose proportionality. The pharmacokinetic properties of both roflumilast dosage forms provide clinically relevant evidence of predictable, intraindividual total (AUC) and maximum (Cmax) exposure of roflumilast and roflumilast N-oxide. Repeated oral dosing with roflumilast 250 and 500 microg once daily was well tolerated.

Cyclooxygenase-2 expression in human colorectal cancer is unrelated to overall patient survival.

PURPOSE: Cyclooxygenase-2 (COX-2) expression in human colorectal cancer and adenoma tissue seems to be higher than in normal mucosa. However, data about the relation between COX-2 expression and patient survival are inconclusive as yet. Therefore, we studied COX-2 expression in surgery tissue and survival time in a cohort of 747 colorectal cancer patients. EXPERIMENTAL DESIGN: Surgical specimens of primary colorectal cancer from 747 individuals were immunostained for COX-2 and evaluated under a transmission light microscope. COX-2 expression was scored according to intensity and extent of staining, resulting in the COX-2 immunoreactivity score (IRS-COX2). All possible cutoff points for IRS-COX2 were analyzed for a relation between COX-2 expression and patient survival. RESULTS: Both univariable and multivariable analysis have shown that the COX-2 expression in human tumor epithelial cells was unrelated to overall patient survival and to disease-free survival, irrespectively of the cutoff point for IRS-COX2. The survival rates for 1, 3, 5, and 10 years were 81.0%, 66.8%, 60.2%, and 49.8% (median: 117.3 months; 95% confidence interval, 102.3-132.0), respectively. In the multivariable analysis, only node and metastasis were significantly related to overall patient survival. Similar results were obtained when stage IV and rectal cancer patients were excluded from the analysis. CONCLUSIONS: COX-2 expression in tumor epithelial cells does not seem to be related to survival of colorectal cancer patients. Besides COX-2, there are several targets, such as the peroxisome proliferator-activated receptors, that are involved in carcinogenesis and may be modulated by nonsteroidal anti-inflammatory drugs. Further studies are needed to determine their prognostic relevance.

The blockade of endothelin A receptor protects astrocytes against hypoxic injury: common effects of BQ-123 anderythropoietin on the rejuvenation of the astrocyte population.

In the present study the role of endothelin (ET) and its receptors (ETA-R and ETB-R) in cellular mechanisms underlying the resistance of astroglial cells to low oxygen level and development of hypoxia has been investigated. To define the influences of ET and its receptors on survival and on antigenic as well as morphologic differentiation of rat astroglial cells in normoxic (NC) and hypoxic culture (HC) the selective antagonists of ETA-R (BQ-123) and ETB-R (BQ-788) were used. Treatment of HC with BQ-123 caused an increase in cell number and inhibited the hypoxia-induced apoptosis by 37%. BQ-123 decreased the hypoxia-induced cytotoxicity in HC. These effects of BQ-123 were abolished in cultures simultaneously treated with BQ-123 and BQ-788. Administration of BQ-788 alone decreased the number of living cells in NC, but not in HC. The activity of caspase-3/-7 was not changed by exposure of NC and HC to BQ-788. The protection provided by BQ-123 to astroglial cells against cytotoxicity in NC and HC was similar to that of erythropoietin (EPO), a cytokine with established neuroprotective effects. The functional improvement of astroglial cells and slowing down of their differentiation under exposure to BQ-123, or EPO, or BQ-123 + EPO has been evidenced by an increased number of nestin+/glial fibrillary acidic protein-positive (GFAP+) astrocytes accompanied by decrease of nestin-/GFAP+ cells. The simultaneous treatment with BQ-123 and EPO additionally decreased the activities of caspase-3/-7 (64%) and release of LDH into the medium (94%). The benefits in the functional states of astrocytes obtained by combined treatment of HC with BQ-123 and EPO suggest a new therapeutic strategy in treatment of hypoxic brain injury.

Similar protective effects of BQ-123 and erythropoietin on survival of neural cells and generation of neurons upon hypoxic injury.

Our recent study [Danielyan et al., 2005. Eur. J. Cell Biol. 84, 567-579] showed an additive protective effect of endothelin (ET) receptor A (ETA-R) blockade and erythropoietin (EPO) on the survival and rejuvenation of rat astroglial cells exposed to hypoxia. Whether the effects observed with rodent astroglial cells can be reproduced in human astrocytes and whether these effects of ETA-R blockade and EPO on astrocytes are associated with neuronal survival remained open. Therefore, in the present study, the effects of the ETA-R antagonist BQ-123 and EPO on the maintenance of the neuronal population and survival of the human fetal astroglial cell line (SV-FHAS) under normoxic and hypoxic conditions (NC and HC, respectively) were investigated. Rat brain primary cultures exposed to BQ-123 and/or EPO revealed an increase in the number of beta-III tubulin-positive neurons under NC. The hypoxia-caused loss of neurons was abolished by administration of BQ-123 or EPO. Simultaneous application of EPO and BQ-123 led to an additive protective effect on the generation of neurons under NC only. By contrast, BQ-788, the selective ETB-R antagonist, diminished the neuronal population both in NC and HC. Both under NC and HC the number of non-differentiated nestin+/GFAP- neural cells increased upon application of EPO or BQ-123. SV-FHAS responded to BQ-123 or EPO by a decrease in LDH activity in the culture medium under NC (35%) and HC (26% LDH decrease). Concomitant effects of EPO and BQ-123 were illustrated in an additional increase in the survival of human astrocytes (33% under NC and 17% under HC). These data hint at a neuroprotective therapeutic potency of ETA-R blockade, which either alone or in combination with EPO may improve the survival of astroglial and neuronal cells upon hypoxic injury.

Impact of CYP2D6 genotype on adverse effects during treatment with metoprolol: a prospective clinical study.

OBJECTIVE: Our objective was to study the impact of the cytochrome P450 (CYP) 2D6 polymorphism on the tolerability of metoprolol in a real-life primary care setting. The adverse effects studied comprised effects related to the central nervous system, cardiovascular effects, and sexual dysfunction. METHODS: Patients in whom treatment with metoprolol was considered were enrolled into this prospective, 6-week multicenter study. The dosage of metoprolol was determined on an individual basis and could be freely adjusted on clinical grounds. The indication for treatment was hypertension in about 90% of cases. Systolic and diastolic blood pressure, resting heart rate, and plasma metoprolol and alpha-hydroxymetoprolol concentrations were measured. CYP2D6 genotyping covered alleles *3 to *10 and *41 and the duplications. Possible adverse effects of metoprolol were systematically assessed over a 6-week period by means of standardized rating scales and questionnaires. RESULTS: The final study population comprised 121 evaluable patients (all white patients); among them, there were 5 ultrarapid metabolizers (UMs) (4.1%), 91 extensive metabolizers (EMs) (75%), 21 intermediate metabolizers (IMs) (17%), and 4 poor metabolizers (PMs) (3.3%). Plasma metoprolol concentrations normalized for the daily dose and metoprolol/alpha-hydroxymetoprolol ratios at steady state were markedly influenced by CYP2D6 genotype and displayed a gene-dose effect. The median of the dose-normalized metoprolol concentration was 0.0088 ng/mL, 0.047 ng/mL, 0.34 ng/mL, and 1.34 ng/mL among UMs, EMs, IMs, and PMs, respectively (P<.0001). There was no significant association between CYP2D6 genotype-derived phenotype (EMs and UMs combined versus PMs and IMs combined) and adverse effects during treatment with metoprolol. There was a tendency toward a more frequent occurrence of cold extremities in the PM plus IM group as compared with the EM plus UM group (16.0% versus 4.2%, P=.056; relative risk, 3.8 [95% confidence interval, 1.03--14.3]). CONCLUSIONS: CYP2D6 genotype-derived phenotype was not significantly associated with a propensity for adverse effects to develop during treatment with metoprolol. However, the results concerning tolerability of metoprolol in PMs were inconclusive because of the small number of PMs enrolled.

MALDI-TOF MS analysis of urinary nucleosides.

As RNA turnover seems to be impaired in cancer patients, modified nucleosides have been evaluated as potential tumor markers. Modified nucleosides are mainly formed post-transcriptionally in tRNA, set free during RNA metabolism, and excreted in urine. Especially methylated nucleosides play an important role, as their levels are higher in urine from cancer patients. For structural elucidation of known and unknown nucleosides from urine samples of cancer patients, MALDI-TOF MS and MALDI-PSD were used for the first time. This technique generally ensures high sensitivity, mass resolution, and accuracy. In our analytical approach we prepurified nucleosides from urine by affinity chromatography and subsequently separated them by semipreparative high performance liquid chromatography. The different fractions were collected separately and analyzed by MALDI-TOF MS and PSD-MALDI using a mixture of six low molecular weight calibrants for internal or external calibration. The molecular totals formulas based on a mass accuracy of 10 ppm and below were calculated and a systematic data base search was performed. The inherent problem of the MALDI-technique, the reduced sensitivity for low molecular weight substances caused by matrix suppression effects, was reduced by our technique. We identified several nucleosides in urine, which were previously identified via retention times and UV spectra of standards after HPLC analysis. Eight further nucleosides were observed. This work demonstrates for the first time the potential of MALDI-TOF and PSD-MALDI in combination with semipreparative HPLC for assignment of nucleosides in urine. The particularly high mass accuracy of this mass spectrometric method provides opportunities for identifying unknown compounds.

Adaptation of a high-performance liquid chromatographic method for quantitative determination of homocysteine in urine.

A modification of the Bio-Rad total homocysteine HPLC-test is presented in order to enable not only plasma homocysteine measurements but also the quantification of homocysteine in urine samples using the same principle of measurement. Coelution of the internal standard provided in the test kit with an endogenous compound in urine demands for an alternative analytical procedure. Therefore, we introduced 3-mercaptopropionic acid as a substitute for the internal standard. The analytical method validation was performed for the matrix of urine specimens. The applicability of this method was demonstrated in a clinical study with volunteers after homocysteine thiolactone hydrochloride loading.

Using trade names: a risk factor for accidental drug overdose.

Medication errors due to the exclusive use of trade names of drugs may lead to life-threatening complications. We report the case of a patient with verapamil overdose as a result of this. This case illustrates that the use of trade names, omitting the international nonproprietary names of the active moiety, carries the risk of serious adverse drug events by overdose.