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W Jäger

Publications and source records attributed to W Jäger.

At least 37 records · Page 2Linked to original sources
PubMed2001

In vitro glucuronidation of the cyclin-dependent kinase inhibitor flavopiridol by rat and human liver microsomes: involvement of UDP-glucuronosyltransferases 1A1 and 1A9.

The metabolism of flavopiridol (FLAP), a novel anticancer drug currently undergoing clinical development, was investigated in rat and human liver microsomes. In the presence of uridine 5'-diphosphoglucuronic acid, two biotransformation products (M1 and M2) could be detected. Formation of metabolite M1 and M2 in terms of enzymatic efficacy (Vmax/K(M)) was about 50- and 5-fold higher in rat (1.58 +/- 2.23 and 7.22 +/- 1.17 microl/min/mg) as compared with human liver microsomes (0.032 +/- 0.016 and 1.52 +/- 0.93 microl/min/mg), indicating species-related differences in FLAP glucuronidation. Incubation in the presence of human recombinant UDP-glucuronosyltransferases (UGTs) demonstrated that M1 is almost exclusively catalyzed by UGT1A1, whereas M2 is formed by UGT1A9 and only to a minor extent by UGT1A1 and UGT1A10. Chemical inhibition experiments further prove the involvement of UGT1A1 and UGT1A9 in the formation of M1 and M2, as the UGT1A1 substrate bilirubin preferably inhibited M1 over M2 (K(i): 36 and 258 microM, respectively), whereas the UGT1A9 substrate propofol showed a more pronounced decrease in M2 but not in M1 formation (K(i): 47 and 142 microM, respectively). Both conjugates were purified from rat liver microsomes and analyzed by mass spectrometry, NMR, and UV experiments. On the basis of these results, M1 was identified as 5-O-beta-glucopyranuronosyl-flavopiridol and M2 as 7-O-beta-glucopyranuronosyl-flavopiridol. In conclusion, our results elucidate the enzymatic pathways of FLAP in rat and human liver, which must be considered during cancer therapy of patients.

Animals↗
PubMed2001

Metabolism and biliary excretion of the novel anticancer agent 10-hydroxycamptothecin in the isolated perfused rat liver.

10-hydroxycamptothecin (HCPT), a natural analog of the alkaloid camptothecin (CPT), is a promising anticancer agent currently undergoing preclinical trials. Though HCPT is less toxic and more active in various human cancer cell lines and in animal tumor models than the clinically approved CPT-analog topotecan, little is known about its biotransformation products and their route of elimination. To investigate the metabolism and biliary excretion, livers of male Wistar rats were perfused with HCPT (5 microM). Bile and perfusate samples were collected for 60 min and quantified by reversed-phase high-performance liquid chromatography (HPLC). Besides HCPT, three metabolites, namely HCPT glucuronide (M1), hydroxyHCPT glucuronide (M2), and hydroxyHCPT (M3) could be identified by enzymatic hydrolysis with beta-glucuronidase and mass spectroscopy. Biliary secretion of HCPT and M1-M3 reached a peak secretion of 1532+/-124, 75+/-16, 5.8+/-1.6 and 2.1+/-0.5 pmoles/g liver.min, respectively, after 25 min. The total amount of HCPT and M1-M3 excreted into bile during the time of perfusion (60 min) was low and represented a mean of 9.9+/-3.2%, 0.44+/-0.17%, 0.041+/-0.010%, and 0.022+/-0.004% of the initial HCPT dose, respectively. In the perfusate, besides HCPT M1 and M2 but not M3 could be detected (maximum concentrations after about 20 min: 3248+/-210, 16.8+/-2.8 and 1.0+/-0.4 pmoles/g liver.min, respectively). The cumulative efflux of HCPT and M1 and M2 into the perfusate was 21.1+/-3.9%, 0.145+/-0.036% and 0.018+/-0.004% of the initial dose, respectively, indicating a preferable non-biliary secretion for HCPT and a predominant biliary elimination for conjugated HCPT biotransformation products. In conclusion, HCPT is biotransformed in a rat liver model to three metabolites, mainly excreted into bile, which may be of clinical relevance during cancer therapy.

Animals↗
PubMed2000

Topology of double minutes (dmins) and homogeneously staining regions (HSRs) in nuclei of human neuroblastoma cell lines.

Amplification of the MYCN gene is a characteristic feature of many neuroblastomas and is correlated with aggressive tumor growth. Amplicons containing this gene form either double minutes (dmins) or homogeneously staining regions (HSRs). To study the nuclear topology of these tumor-specific and transcriptionally active chromatin structures in comparison to chromosome territories, we performed fluorescence in situ hybridization with a MYCN probe and various chromosome paint probes, confocal laser scanning microscopy, and quantitative three-dimensional image analysis. The dmins formed dot-like structures in interphase nuclei and were typically located at the periphery of complexly folded chromosome territories; dmins noted in the chromosome territory interior were often detected within an invagination of the territory surface. Interphase HSRs typically formed extremely expanded structures, which we have never observed for chromosome territories of normal and tumor cell nuclei. Stretches of HSR-chromatin often extended throughout a large part of the cell nucleus, but appeared well separated from neighboring chromosome territories. We hypothesize that dmins are located within the interchromosomal domain (ICD) space and that stretches of HSR-chromatin align along this space. Such a topology could facilitate access of amplified genes to transcription and splicing complexes that are assumed to localize in the ICD space.

Cell Nucleus↗
PubMed2000

Apoptosis, bcl-2 expression, and proliferation in benign and malignant endometrial epithelium: An approach using multiparameter flow cytometry.

OBJECTIVE: Disturbances in the regulation of cell proliferation and differentiation play an important role in the formation of neoplastic lesions. Consequently, abnormalities in apoptosis regulation may contribute to this process. Expression of a neoepitope on cytokeratin 18, unmasked by an early caspase cleavage event and recognized by the novel monoclonal antibody M30, is an indicator of early epithelial cell apoptosis. The purpose of this study was to evaluate the quantitative relation among apoptosis (M30), cell persistence (bcl-2), and proliferation (S-phase fraction; SPF) in malignant and benign endometrium. METHODS: Using multiparameter DNA flow cytometry on 54 formalin-fixed paraffin-embedded samples from benign (proliferative, secretory, inactive, and hyperplastic endometrium) and malignant (grades 1-3 endometrial adenocarcinoma) endometrial tissue, bcl-2 expression and M30 reactivity were assessed together with the SPF in the cytokeratin-positive epithelial cells. RESULTS: Benign cyclic endometrium showed a relatively high bcl-2 expression and low M30 reactivity in the proliferative phase whereas in the secretory phase this relation was inverse. In endometrial hyperplasia the expression of bcl-2 was increased compared to that in secretory and postmenopausal endometrium, but still below the level of proliferative samples. The expression of M30 also increased compared to normal proliferative endometrium but did not reach the level of endometrium in the secretory phase of the menstrual cycle. In cancer the expression of bcl-2 decreased with the progression of differentiation grade. For M30 expression this relation was inverse. Overall there was a significant increase of M30 reactivity in cancerous compared to hyperplasia and normal cyclic endometrium. CONCLUSION: Transition of endometrial epithelium from hyperplasia to cancer seems to involve both increased apoptosis and decreased bcl-2 expression. Flow cytometric evaluation of M30 and bcl-2 expression levels, with the SPF, in currettage specimens from postmenopausal patients complaining of bleeding provides a quantitative assessment of endometrial apoptosis, anti-apoptosis, and proliferation. Further studies are needed to determine the relationship among these three processes as indicators of the biological behavior of gynecological tumors.

Adult↗
PubMed2000

Metabolism of camptothecin, a potent topoisomerase I inhibitor, in the isolated perfused rat liver.

PURPOSE: Camptothecin (CPT) is a potent topoisomerase I inhibitor that has recently been undergoing phase I clinical trials. Though CPT shows high activity against various tumor cells, its biotransformation is still unknown. To investigate the metabolism and biliary excretion of CPT, an isolated perfused rat liver system was used. METHODS: CPT was added to the perfusion medium at a concentration of 20 microM, and bile and perfusate samples were collected for 90 min. CPT (lacton and carboxylate) and three novel metabolites were identified by mass spectroscopy and quantified by reversed-phase high-performance liquid chromatography (HPLC). Kinetic parameters of CPT and its biotransformation products were then estimated in bile and effluent perfusate. RESULTS: Biliary secretion of CPT and its three metabolites reached a peak secretion of 37.6 +/- 16.3, 0.94 +/- 0.29, 0.19 +/- 0.023 and 0.302 +/- 0.076 nmol/g liver/min, respectively, after 20 min. The total amount of CPT and M1-M3 excreted into bile during 90 min of perfusion was 63.5 +/- 15.4%, 1.8 +/- 0.37%, 0.43 +/- 0.06%, and 0.72 +/- 0.15% of CPT cleared from the perfusate during 90 min, respectively. In the perfusate, only one metabolite (M3) could be detected (cumulative release into the perfusion medium: 0.37 +/- 0.026 micromol/liver). Analysis of the biliary metabolites by mass spectroscopy supported the existence of dihydroxy-CPT derivatives (M1 and M2), whereas M3 appears to be a monohydroxy-analog. CONCLUSION: CPT is biotransformed to three novel metabolites, mainly excreted into bile. The possible pharmacological effects of these new metabolites need to be considered.

Animals↗
PubMed2000

Noise-induced aspartate and glutamate efflux in the guinea pig cochlea and hearing loss.

Aspartate and glutamate were monitored in the scala tympani of the guinea pig cochlea using in vivo microdialysis before and during noise exposure. Moderate level broad band noise [105 dB sound pressure level (SPL), 30 min] neither altered the levels of aspartate or glutamate, nor auditory brainstem response (ABR) thresholds. High level noise exposure (135 dB SPL, 30 min) caused a large increase in aspartate (330%), a smaller increase in glutamate (150%), and a permanent ABR threshold shift of 60-75 dB between 2.0 and 12.5 kHz. Morphological analysis of the cochlea revealed a collapse of supporting structures, swelling of the afferent dendrites under the inner hair cells, and outer hair cell loss. Pretreatment with the NMDA antagonist, MK 801 (1 mg/kg body weight, i.p.) 1 h before noise exposure protected the afferent dendrites from swelling but did not protect the collapse of supporting structures, outer hair cell loss, or auditory thresholds. In conclusion, the noise-induced increase in aspartate and glutamate release in the cochlea and the protective effect of NMDA antagonism suggest that these two neurotransmitters are involved in noise-induced hearing loss.

Acoustic Stimulation↗
PubMed2000

The patterns of expression of an apoptosis-related CK18 neoepitope, the bcl-2 proto-oncogene, and the Ki67 proliferation marker in normal, hyperplastic, and malignant endometrium.

Expression of a neoepitope on cytokeratin 18, recognized by the monoclonal antibody M30, is an early indicator of apoptosis in epithelial cells. The aim of this study was to determine the equilibrium between apoptosis (M30), anti-apoptosis (bcl-2), and proliferation (Ki-67) in different endometrial conditions. Paraffin-embedded samples (n = 107), representing proliferative endometrium (18), secretory endometrium (19), postmenopausal endometrium (15), disordered proliferative endometrium (6), simple hyperplasia (12), complex hyperplasia (8), and endometrial adenocarcinoma (29), were evaluated immunohistochemically. The indirect streptavidin-biotin-horseradish peroxidase technique, with 3-amino-9-ethylcarbazole as the chromogen, was used to visualize the reactions. Proliferative endometrium showed high bcl-2 and Ki-67 expression levels with no M30. In the secretory phase, the balance was tipped in favor of M30 with a decrease of bcl-2 and Ki-67. Postmenopausal endometrium revealed high Ki-67 and bcl-2 expression levels and no M30. In complex hyperplasia, M30, bcl-2, and Ki-67 showed increased expression. In endometrial carcinoma, an increasing reactivity for M30 and Ki-67 was seen as the grade progressed. bcl-2 reacted weakly and only in grade 1 cancer. Immunohistochemistry facilitates the study of the expression of proteins related to cyclic endometrial activity. Interruption of these cyclic events is associated with specific disturbances in the expression patterns of these proteins.

Apoptosis↗
PubMed2000

Stereoselective metabolism of the monoterpene carvone by rat and human liver microsomes.

The large amounts of carvone enantiomers consumed as food additives and in dental formulations justifies the evaluation of their biotransformation pathway. The in-vitro metabolism of R-(-)- and S-(+)-carvone was studied in rat and human liver microsomes using chiral gas chromatography. Stereoselective biotransformation was observed when each enantiomer was incubated separately with liver microsomes. 4R, 6S-(-)-Carveol was NADPH-dependently formed from R-(-)-carvone, whereas 4S, 6S-(+)-carveol was produced from S-(+)-carvone. Metabolite formation followed Michaelis-Menten kinetics exhibiting a significant lower apparent Km (Michaelis-Menten Constant) for 4R, 6S-(-)-carveol compared with 4S, 6S-(+)-carveol in rat and human liver microsomes (28.4+/-10.6 microM and 69.4+/-10.3 microM vs 33.6+/-8-55 microM and 98.3+/-22.4 microM). The maximal formation rate (Vmax) determined in the same microsomal preparations yielded 30.2+/-5.0 and 32.3+/-3.9 pmol (mg protein)(-1) min(-1) in rat liver and 55.3+/-5.7 and 65.2+/-4.3 pmol (mg protein)(-1) min(-1) in human liver microsomes. Phase II conjugation of the carveol isomers by rat and human liver microsomes in the presence of UDPGA (uridine S'-diphosphogluaronic acid) only revealed glucuronidation of 4R, 6S-(-)-carveol. Vmax for glucuronide formation was more than 4-fold higher in the rat liver compared with human liver preparations (185.9+/-34.5 and 42.6+/-7.1 pmol (mg protein)(-1) min(-1), respectively). Km values, however, showed no species-related difference (13.9+/-4.1 microM and 10.2+/-2.2 microM). This study demonstrated stereoselectivity in phase-I and phase-II metabolism for R-(-)- and S-(+)-carvone and might be predictive for carvone biotransformation in man.

Animals↗
PubMed2000

Cyclosporine metabolism in patients after kidney, bone marrow, heart-lung, and liver transplantation in the early and late posttransplant periods.

Cyclosporine is used in the prevention of allograft rejection. Owing to its narrow therapeutic index, regular monitoring of the whole blood levels of cyclosporine is required. We observed that immunoassays measured significantly higher cyclosporine levels than did high-performance liquid chromatography (HPLC) over time after transplantation. As cyclosporine metabolites cross-react even with immunoassays, this observation might be due to alterations of the cyclosporine metabolism. We analyzed cyclosporine metabolite concentrations in the early and in the late posttransplantation periods in 127 patients after kidney, bone marrow, heart-lung, and liver transplantation by HPLC and determined whole blood levels of cyclosporine by 4 immunoassays (enzyme-multiplied immunoassay [EMIT], cloned enzyme donor immunoassay [CEDIA], AxSYM [Abbott Laboratories, Chicago, IL], and TDx [Abbott Laboratories]). Despite reduced dose, we found significantly higher cyclosporine concentrations measured by the EMIT, AxSYM, and TDx assays in various patient groups. These results are due to the increased metabolite/cyclosporine ratio in the late posttransplantation period. In particular, the metabolites AM1 and AM19 increased significantly over time in bone marrow transplant recipients. Therefore, cyclosporine levels measured by immunoassays should be interpreted with caution.

Bone Marrow Transplantation↗
PubMed1999

Rotational Spectroscopic Investigation of the Weak Interaction between CO and N(2)O.

Pure rotational spectra of the CO-N(2)O complex are reported for the first time. Spectra of five isotopomers, i.e., (12)C(16)O-(14)N(14)NO, (13)C(16)O-(14)N(14)NO, (13)C(18)O-(14)N(14)NO, (13)C(16)O-(15)N(14)NO, and (13)C(16)O-(14)N(15)NO, were measured using a pulsed molecular beam Fourier transform microwave spectrometer. Rotational and quartic centrifugal distortion constants as well as the nuclear quadrupole coupling constants of the terminal and the central (14)N nuclei were determined. Comparisons of the nuclear quadrupole coupling constants with the corresponding values of the N(2)O monomer and with those of other N(2)O-containing complexes indicate significant electronic charge redistribution at the central nitrogen atom upon complex formation. Structural parameters based on the experimental rotational constants were derived. With the isotopic data available, the orientations of the CO and N(2)O subunits in the complex could be determined. Copyright 1999 Academic Press.

Journal Article↗
PubMed1999

Micromechanical effects in the cochlea of tetracaine.

Local anesthetics applied in the tympanic cavity have earlier been shown to affect the gross receptor potentials in reducing the cochlear microphonics and increasing the positive summating potential. To study the effects of this drug on the mechanical responses in the cochlea, vibrations were measured using laser heterodyne interferometry in an isolated in vitro temporal bone preparation from the guinea pig. Measurements were made at a set of frequencies in the fourth cochlear turn from the Hensen's cells and the outer hair cells in response to sound applied to the ear. The tuning curves of the fundamental and the second harmonic components of the vibratory responses were plotted. When 2 mM tetracaine was applied, the high frequency slope of the second harmonic curve shifted down in frequency, this caused the frequency of the maximum of second harmonic tuning to shift down. These changes were reversible when tetracaine was washed out. Observations were also made in the temporal bone preparation in vitro with a confocal microscope. Fluorescent probes were used to label various structures in the organ of Corti. Optical sections were obtained by tilting the organ permitting a view from the side like a radial section through the organ. Images were acquired before, during and after application of tetracaine and were later analyzed with a computer program. Simultaneously, cochlear microphonics and the summating potential were obtained to monitor the electrical response of the preparation. Although the cochlear microphonics and summating potential decreased when 2 mM tetracaine was applied, structural changes were not measurable in the organ of Corti. The decrease was reversible when tetracaine was washed out. It is concluded that tetracaine affected the high frequency part of the non-linear second harmonic component, possibly by lowering the stiffness of the stereocilia bundle or the body of the outer hair cells.

Acoustic Stimulation↗
PubMed1999

Ethynylestradiol-mediated induction of hepatic CYP3A9 in female rats: implication for cyclosporine metabolism.

Repeated treatment of female rats with the synthetic estrogen ethynylestradiol (EE(2)) increases the formation of the cyclosporine A (CyA) metabolites AM1c and AM9 by 3-fold, whereas the formation of AM1 and AM4N is not significantly enhanced. The formation of all four CyA metabolites was inhibited by greater than 80% by the CYP3A-selective substrate midazolam or polyclonal anti-rat CYP3A IgGs in liver microsomes of untreated and EE(2)-induced rats. In contrast, anti-rat CYP2C6 IgGs had little effect, indicating the involvement of a CYP3A but not 2C6 in this EE(2)-stimulated CyA metabolism. Semiquantitative reverse-transcriptase polymerase chain reaction was used to determine the mRNA content for four CYP3A genes (CYP3A2, CYP3A9, CYP3A18, and CYP3A23) in livers of control and EE(2)-treated female rats. EE(2) selectively induced CYP3A9 by 3.3-fold whereas the expression of CYP3A18 and CYP3A23 was slightly decreased; neither CYP3A2 mRNA nor CYP3A1 mRNA was detectable in these EE(2)-treated livers. To determine whether rat liver microsomal CYP3A9 was indeed responsible for the EE(2)-stimulated CyA metabolism, a recombinant CYP3A9 was heterologously expressed in Escherichia coli. When functionally reconstituted, this enzyme was active in metabolizing CyA preferentially to its AM9 and AM1c metabolites as compared with CYP3A4. These findings thus support the notion that the increased CyA-metabolizing capacity of EE(2)-treated female rat liver microsomes is due to the induction of the CYP3A9 enzyme.

Actins↗
PubMed1998

Study of the Rotational Spectrum of the Ne-N2O van der Waals Dimer with a Fourier Transform Microwave Spectrometer.

Rotational spectra of six isotopomers of the van der Waals dimer Ne-N2O, namely 20Ne-14N14NO, 22Ne-14N14NO, 20Ne-14N15NO, 22Ne-14N15NO, 20Ne-15N14NO, and 22Ne-15N14NO, were measured in the frequency range between 5 and 18 GHz using a pulsed beam cavity Fourier transform microwave spectrometer. The spectra are those of prolate asymmetric rotors and are in accord with a T-shaped structure of the complex. Both a- and b-type transitions were measured. Nuclear quadrupole hyperfine patterns of the rotational transitions due to the 14N nuclei were observed and analyzed. The rotational and centrifugal distortion constants were determined, as well as the quadrupole coupling constants chiaa and chibb, for both terminal and central 14N nuclei. The distance from the center of mass of the N2O subunit to the Ne atom and the angle between this distance and the N2O axis were derived from the rotational constants. The structural parameters indicate that the Ne atom is on average closer to the O atom than to the terminal N atom. Copyright 1998 Academic Press.

Journal Article↗
PubMed1998

Rotational Spectra of NeCO2 Isotopomers.

Rotational spectra of seven isotopomers of the weakly bound complex NeCO2, i.e., NeCO2, 22NeCO2, Ne13CO2, 22Ne13CO2, Ne18O13CO, Ne17OCO, and 22Ne17OCO, were measured using a pulsed molecular beam Fourier transform microwave spectrometer. Rotational and centrifugal distortion constants as well as the nuclear quadrupole coupling constants of 17O in Ne17OCO and 22Ne17OCO were determined. The obtained constants were used to derive structural and force field information. The parameters obtained are discussed, and compared with those of other, previously measured, rare gas-CO2 complexes. The 17O nuclear quadrupole coupling constants were analyzed and interpreted in terms of the angular anisotropy of the intermolecular potential energy surface. Copyright 1998 Academic Press.

Journal Article↗