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Biomedical subjects

G Hempel

Publications and source records attributed to G Hempel.

At least 19 recordsLinked to original sources
PubMed2005

Effects and interaction of 7-hydroxy methotrexate and methotrexate in leukaemic cells ex vivo measured by the thymidylate synthase inhibition assay.

In high dose therapy with methotrexate (MTX) the main metabolite 7-hydroxy-methotrexate (7-OH MTX) exceeds the plasma concentration of MTX achieving about tenfold higher levels. To investigate the interaction between 7-OH MTX and MTX ex vivo, the thymidylate synthase inhibition assay was used to quantify antifolate effects in patient blast samples, measuring the inhibition of the key enzyme thymidylate synthase (TS). In 18 leukemic samples (7 ALL, 11 AML) no dose-dependent TS inhibition was observed for 7-OH MTX. However, a statistically significant increase of TS inhibition (p<0.05) was observed for a 1:1 mixture of MTX and 7-OH MTX as compared to the effect of MTX alone. The half-maximal inhibitory concentrations in the short-exposure assay were 0.857 microM for MTX alone versus 0.088 microM for the 1:1 mixture with 7-OH MTX, respectively (p< or =0.05). This interaction was not observed with an excess of 7-OH MTX. Similar results were obtained in long exposure experiments. We conclude that there is a dose-dependent interaction between 7-OHMTX and MTX, despite the lack of TS inhibitory effects of the metabolite alone.

Antimetabolites, Antineoplastic↗
PubMed2001

Quantification of daunorubicin and daunorubicinol in plasma by capillary electrophoresis.

Capillary electrophoresis (CE) with laser-induced fluorescence detection was applied to quantify daunorubicin and daunorubicinol in plasma. Separation was carried out in a 47 cm x 50 microm I.D. fused-silica capillary, with a running buffer. pH 5 containing 60 microM spermine and 70% acetonitrile. Sample preparation was done either by protein precipitation with acetonitrile or by liquid-liquid extraction. The assay can be applied in a concentration range from 40 mg/l down to 2 microg/l for daunorubicin and from 1 mg/l to 2 microg/l for daunorubicinol. Precision and accuracy were between 2.9 and 14.5% (n=6) on 1 day and between 1.0 and 14.7% from day to day (n=6) for both analytes. Thus, the CE method enables precise and accurate quantification of daunorubicin and daunorubicinol in small sample volumes over a wide concentration range.

Antibiotics, Antineoplastic↗
PubMed2001

Simultaneous quantification of etoposide and etoposide phosphate in human plasma by capillary electrophoresis using laser-induced native fluorescence detection.

A method using capillary electrophoresis with UV laser-induced native fluorescence detection was developed as a sensitive and selective assay for the simultaneous determination of etoposide and etoposide phosphate in human plasma. Laser-induced native fluorescence detection with a frequency-doubled argon ion laser at an excitation wavelength of 257 nm was used for the simultaneous assay of etoposide and etoposide phosphate in plasma to improve the sensitivity compared to that obtained with UV absorption. The detection system consists of an imaging spectrograph and an intensified CCD camera which views an illuminated 1.5-mm section of the capillary. This setup is able to record the whole emission spectra of the analytes to achieve additional wavelength-resolved electropherograms. In the concentration range of 200 microg/L-50 mg/L in plasma for etoposide and 100 microg/L-20 mg/L for etoposide phosphate, coefficients of correlation were better than 0.998. Within-day variation determined with three different concentrations showed accuracies ranging from 91.0 to 109.3% for etoposide and from 91.2 to 109.9% for etoposide phosphate (n = 6) with a precision of about 8%. Day-to-day variation presented accuracies ranging from 91.8 to 107.9% for etoposide and from 94.4 to 109.3% for etoposide phosphate with a relative standard deviation less than 6% (n = 5). To our knowledge, this is the first method for the simultaneous quantification of etoposide and etoposide phosphate in plasma samples.

Antineoplastic Agents↗
PubMed2001

Quantitative analysis of specific Th1/Th2 helper cell responses and IgG subtype antibodies in interferon-alpha-treated patients with chronic hepatitis C.

This study aimed to characterise the immune mechanisms relevant to viral clearance in interferon (IFN)-alpha-treated chronic hepatitis C virus (HCV) infection. Proliferative responses of peripheral blood mononuclear cells from sustained complete IFN-alpha therapy responders (n = 8), nonresponders (n = 13), untreated patients (n = 10), and healthy controls (n = 5) were measured retrospectively upon stimulation with recombinant HCV-antigens (core, helicase, NS3, NS4, and NS5) and the secretion of IFN-gamma and interleukins (IL-4, IL-5, IL-10, and IL-12) were tested by ELISA. Furthermore, IFN-gamma as well as IL-10 secreting CD4+ T cells were quantitated by intracellular cytokine staining. Anti-HCV core and NS3-specific IgG subclass antibodies were quantitated in the corresponding patient sera. Sustained therapy responders had more frequent and stronger NS3 and helicase-specific cellular immune responses than nonresponders, untreated HCV patients and healthy controls. Independent from therapy outcome HCV-stimulated T cells in IFN-alpha treated patients secreted preferentially IFN-gamma The Th2 cytokines IL-4 and IL-10 were even decreased in nonresponders, while the IL-12 secretion was not influenced. With respect to the humoral immune response sustained complete responders showed significantly reduced IFN-gamma independent anti-HCV-core and -NS3 IgG1 antibody synthesis. In conclusion, vigorous NS3-specific T-helper cell responses were associated with viral clearance in IFN-alpha recipients; however, the cytokine and antibody analysis argues against a Th1/Th2 imbalance as a major factor that influence the therapy outcome.

Adult↗
PubMed2000

Determination of purines including 2,8-dihydroxyadenine in urine using capillary electrophoresis.

For the purpose of rapid drug monitoring, methods have been developed for the determination of 2,8-dihydroxyadenine, allopurinol, oxypurinol, adenine, hypoxanthine, hippuric acid and xanthine in urine with and without sodium dodecyl sulfate as additive in sodium tetraborate running buffer. No sample preparation is necessary. 6-methylmercaptopurine and etofylline have been used as the internal standards. The limit of detection is 5 microM and the range of quantification stretches from 20 to 2000 microM. The capillary electrophoresis methods are simple, fast and robust.

Adenine↗
PubMed2000

Strategies to improve the sensitivity in capillary electrophoresis for the analysis of drugs in biological fluids.

Capillary electrophoresis (CE) is a useful method to quantify drugs in biological fluids. However, especially for blood or plasma samples, the sensitivity is not sufficient to quantify drugs and their metabolites as they often need to be quantified in the lower microg/L range. To overcome this limitation and to increase the sensitivity, two strategies are applied: first, to increase the amount of analyte added to the capillary and, second, to increase the sensitivity on the detector site. To improve the sensitivity on the detector site, alternative detection techniques to UV detection, e.g., laser-induced fluorescence detection (LIF) or mass spectroscopy (MS), can be applied. However, LIF detection can only be used for fluorescent analytes and the current equipment for CE-MS coupling provides only small improvements in sensitivity compared to UV detection. The detection window for UV detection can be enhanced using capillaries with an extended light path (bubble cell) or Z-shaped capillaries. Sensitivity improvements up to a factor of 10 have been reported. Increasing the amount of analyte in the capillary can be done either by chromatographic or by electrokinetic methods. Chromatographic methods such as on-capillary membrane preconcentration have been used for several analytes. However, no validated application has been reported to date. In contrast, several validated examples can be found in which electrokinetic techniques like sample stacking have been applied to achieve limits of quantification in the lower microg/L range. In conclusion, to date, electrokinetic techniques such as field-amplified sample injection offer the most promising results in achieving a sufficient sensitivity to quantify drugs in biological fluids.

Body Fluids↗
PubMed2000

PATROS-A new MAS exchange method using sideband separation: application to Poly(n-butylmethacrylate)

The tr-ODESSA method (Reichert et al., J. Magn. Reson. 125, 245 (1997)), which is a 1D MAS experiment designed to monitor spin exchange involving both equivalent and inequivalent sites, is extended to situations where the spectrum consists of several spinning side band (ssb) manifolds with small chemical shift anisotropies. To increase the spectral resolution in such situations, the tr-ODESSA sequence is combined with that of PASS to a single experiment, which we term PATROS. In this hybrid experiment, magnetization transfer is monitored by the tr-ODESSA part, while the increase in resolution is provided by the separation of the ssb according to their order, during the PASS part. We demonstrate the feasibility of the method on a standard solid dimethylsulfone (DMS) sample and then apply it to monitor separately the ultraslow motions of the main- and side-chains in the polymer poly(n-butylmethacrylate). Theoretical expressions for the ssb intensities in PATROS experiments are derived and the merits and limitations of the method are discussed. Copyright 2000 Academic Press.

Journal Article↗
PubMed2000

Dynamic carbon-13 MAS NMR: application to benzene ring flips in polyaryl ethers.

Carbon-13 dynamic MAS NMR is used to determine the pi-flip rates of the phenyl rings in the low-molecular-weight members of the polyaryl ethers series (phenyl-O(-phenylene-O)n-phenyl). The first member in the series (diphenyl ether, n = 0) does not undergo measurable dynamic processes up to its melting point (28 degrees C). The second and third members (n = 1 and 2) exhibit, above room temperature, line broadening effects due to fast pi-flips of the terminal rings, while the spectra of the n = 1 homologue also exhibit line broadening for the inner phenylene ring. Kinetic parameters for the various pi-flip processes were derived by a detailed lineshape analysis of the MAS spectra. The measurements were extended to lower temperatures by time-reverse ODESSA experiments. The kinetic parameters derived from these experiments are, k(t)(300 K) = 31 s(-1), E(t) = 84 kJ/mol, and k(i)(300 K) = 1.3 s(-1), Ei = 77 kJ/mol for the n = 1 homologue and k(t)(300 K) = 3.2 s(-1), E(i) = 78 kJ/mol, for the n = 2 homologue, where the subscripts t and i refer to the terminal and inner benzene rings, respectively. For the simulation of the dynamic MAS spectra the Floquet expansion method was used. In an introductory chapter the Floquet method is reviewed with emphasis on the practical aspects of the computation procedure, on the sensitivity of the results to the isotropic and anisotropic chemical shift parameters, and on the form of the results in the limiting fast and slow exchange regimes.

Carbon Isotopes↗
PubMed1999

Enhanced expression of ELAM-1 on endothelium of renal cell carcinoma compared to the corresponding normal renal tissue.

Renal cell carcinoma (RCC) has been shown to respond to an immunological therapy with tumor infiltrating lymphocytes (TIL), which accumulate in RCC at a higher density than in normal renal tissue, suggesting that there is selective tumor invasion. Since invasion of TIL into the malignant tissue is mediated by adhesion molecules, we examined the different expression of the adhesion molecule endothelial-leukocyte-adhesion-molecule-1 (ELAM-1) on endothelial cells of RCC versus normal renal tissue. For a specific quantification, the level of ELAM-1 mRNA was investigated by both semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot analysis and referred to the content of endothelial cells in the tissue, determined by endothelium specific staining. Quantification of mRNA was evaluated by computer-aided integration. We observed a significantly lower amount of endothelial cells in RCC compared to normal renal tissue. The specific transcription rate of ELAM-1 in RCC, determined by RT-PCR was about 5.2 times that of normal tissue, while Northern blot analysis indicated an approximately 11.8 times increase. Our investigations show a significantly increased expression of ELAM-1 in tumor tissue compared to normal renal tissue, presumably caused by a higher amount of cytokines in the tumor tissue. This enhanced expression may be responsible for the high concentration of TIL in renal tumors.

Blotting, Northern↗
PubMed1999

Determination of (E)-5-(2-bromovinyl)-2'-deoxyuridine in plasma and urine by capillary electrophoresis.

(E)-5-(2-Bromovinyl)-2'-deoxyuridine is an antiviral drug used for treatment of infections with Herpes simplex virus type 1 as well as Varicella zoster virus. Two fast methods for the determination of the drug and its metabolite in plasma and urine by capillary electrophoresis have been developed. The plasma method can be used for measurement of total as well as unbound drug and metabolite. Plasma and urine samples are prepared for measuring by liquid/liquid extraction resulting in a limit of quantification of 40 ng/ml for total and 10 ng/ml for free BVdU in plasma and 170 ng/ml in urine. Inter- as well as intra-day precision were found to be better than 10% and both methods have been used for drug monitoring of patients.

Antiviral Agents↗
PubMed1999

Superslow backbone protein dynamics as studied by 1D solid-state MAS exchange NMR spectroscopy.

Superslow backbone dynamics of the protein barstar and the polypeptide polyglycine was studied by means of a solid-state MAS 1D exchange NMR method (time-reverse ODESSA) that can detect reorientation of nuclei carrying anisotropic chemical shift tensors. Experiments were performed on carbonyl 13C in polyglycine (natural abundance) and backbone 15N nuclei in uniformly 15N-enriched barstar within a wide range of temperatures in dry and wet powders for both samples. Two exchange processes were observed in the experiments: molecular reorientation and spin diffusion. Experimental conditions that are necessary to separate these two processes are discussed on a quantitative level. It was revealed that the wet protein undergoes molecular motion in the millisecond range of correlation times, whereas in dry protein and polyglycine molecular reorientations could not be detected. The correlation time of the motion in the wet barstar at room temperature is 50-100 ms; the activation energy is about 80 kJ/mol. Previously, protein motions with such a long correlation time could be observed only by methods detecting chemical exchange in solution (e.g., hydrogen exchange). The application of solid-state MAS exchange spectroscopy provides new opportunities in studying slow biomolecular dynamics that is important for the biological function of proteins.

Anisotropy↗
PubMed1999

Pharmacokinetics of anticancer drugs in vitro.

It is generally assumed that drug concentration does not change significantly under cell culture conditions. Nevertheless, most of the therapeutic trials in acute leukemia that were based on in vitro drug sensitivity assays of patient samples have been disappointing. In order to show possible pitfalls of unphysiological alterations in vitro we investigated concentration versus time curves, metabolism and effects on the culture media for some antineoplastic drugs. Oxazaphosphorines and cytarabine were incubated in RPMI and in established cell lines and measured by HPLC. HPLC also served to measure enzyme activity and levels of related amino acids at various concentrations of asparaginase, ammonia release was photometrically determined. Etoposide was monitored by HPLC relative to different contents of FCS in RPMI. All oxazaphosphorines showed a rapid decrease of in vitro activity down to about 10% within 4-6 h, and 2% within 72 h. The level of cytarabine, when incubated in RPMI, was stable over 24h, and no change was seen with K562, while a rapid decrease to below 50% occurred within 6h in the presence of HL 60 and BLIN. 2 U/L of asparaginase led to asparagine depletion of the medium within 4h, while 200 U/L were associated with a preferential increase of glutamic acid and ammonia. Further, there was evidence of instability by rapid adsorption to plastic surfaces (paclitaxel) or isomerisation (etoposide) in RPMI with low FCS content. The instability of drugs in vitro is attributed to a variety of different factors: i.e. physico-chemical instability results in inactivation of oxazaphosphorines, cytarabine disappears by cellular metabolism without saturation depending on the cell-line. Epiphenomena like adsorption and isomerisation in vitro are unphysiological. Results of drug sensitivity assays should be interpreted with great caution.

Amino Acids↗
PubMed1999

Investigation of the major human hepatic cytochrome P450 involved in 4-hydroxylation and N-dechloroethylation of trofosfamide.

Trofosfamide and its congeners ifosfamide and cyclophosphamide are cell-cycle-nonspecific alkylating agents that undergo bioactivation catalyzed by liver cytochrome P450 (CYP) enzymes. Two NADPH-dependent metabolic routes for the anticancer drug trofosfamide, i.e., 4-hydroxylation and N-dechloroethylation, were studied in human liver microsomes and in seven recombinant human CYP isoforms (i.e., CYP1A1, 1A2, 2A6, 2B6, 2D6, 2E1, and 3A4-OR) to identify the CYP enzymes involved. Recombinant human CYP3A4 and CYP2B6 exhibited catalytic activity with respect to both pathways of trofosfamide. Enzyme kinetic analyses revealed the dominant role of human CYP3A4 in 4-hydroxylation and N-dechloroethylation of trofosfamide. This was confirmed by the observation that only the CYP3A4 contents of five samples of human liver microsomes correlated with both pathways of trofosfamide. Furthermore, ketoconazole, a selective inhibitor of CYP3A4, substantially inhibited microsomal trofosfamide 4-hydroxylation and N-dechloroethylation (50% inhibitory concentration < 1 microM for both reactions). The present study indicates that human liver microsomal CYP3A4 preferentially catalyzes the two NADPH- dependent metabolic routes of trofosfamide, which emphasizes the necessity for awareness of potential interactions with any coadministered drugs that are CYP3A4 substrates.

Alkylation↗
PubMed1999

Solid-state photodimerization of 4-aryl-1,4-dihydropyridines studied by 13C CPMAS NMR spectroscopy.

13C CPMAS NMR spectroscopy has been applied to monitor the solid-state reaction of two different photodimerizing 4-phenyl-1,4-dihydropyridines yielding a cage dimer in one case and an anti-dimer in the other case. The spectra of the reacting monomers exhibit a magnetical inequivalence of chemically equivalent CO and C2/4 carbon atoms caused by a rotation of the pseudoaxially oriented 4-phenyl substituent out off the plane through N1, C3, C8 which could be determined by X-ray crystal structure analyses of the centrosymmetrically arranged monomers. The 13C CPMAS NMR monitoring of the cage dimer formation proves that the reaction takes place in two steps via a syn-dimer for which a non-symmetrical structure was derived from the spectrum. The non-symmetrical structure was confirmed by X-ray crystal structure analysis of one structurally related derivative. A centrosymmetric structure for both the finally formed cage dimer and the anti-dimer of the other monitored photoreaction was proved by their spectra with one set of signals for each half of the dimers. respectively. Thus, conformational properties of the molecules as well as the symmetry of the products can be directly derived from the 13C CPMAS NMR spectra.

Carbon Isotopes↗
PubMed1999

Pharmacokinetic dose adjustment of Erwinia asparaginase in protocol II of the paediatric ALL/NHL-BFM treatment protocols.

Native forms of asparaginase stem from different biological sources. Previously reported data from children treated with Erwinase showed significantly lower trough levels and pharmacokinetic dose intensity than after E. coli-derived preparations. Hence, schedule optimization was initiated to achieve relevant serum activities. 21 children on reinduction therapy received Erwinase on Mondays, Wednesdays and Fridays for 3 weeks (9 x 20000 IU/m2 i.v.) instead of 4x 10 000IU/m2 of E. coli asparaginase (twice weekly for 2 weeks). Asparaginase trough activities were measured as the primary parameter, targeting 100-200 IU/I after 2 d and >50 IU/l after 3 d. Concurrently, asparagine trough concentrations were monitored. The mean trough activity was 156+/-99 IU/l, with 2/108 samples showing no detectable activity. Regarding trough levels per individual (three or more measurements/patient), means ranged from 52+/-29 to 276+/-114 IU/l (20 patients, 106 samples), with nine, six, and five children inside, below, and above the target range, respectively. The mean 3 d trough activity was 50+/-39 IU/l (20 patients, 51 samples). In 11 of these samples no activity was measurable. Mean trough activities calculated per individual ranged from < 20-84+/-30 IU/l (14 patients, 42 samples) with seven children below the target limit of 50 IU/l and asparagine concentrations <0.2 - 1.5microM. We concluded that an increased dose of 9x20000 IU/m2 of Erwinia asparaginase within 3 weeks resulted in a pharmacokinetic dose intensity comparable to former observations made with 4 x 10 000IU/m2 of the E. coli product Crasnitin which is no longer marketed. High interindividual variability and the phenomenon of 'silent' inactivation necessitate monitoring wherever possible.

Antineoplastic Agents↗
PubMed1998

Capillary electrophoretic drug monitoring of methotrexate and leucovorin and their metabolites.

High-dose methotrexate is an important element of treatment protocols in childhood acute lymphocytic leukemia or osteosarcoma. A capillary electrophoretic method has been developed to measure peak levels and the metabolite pattern in patients with delayed methotrexate elimination. It serves to determine plasma levels of methotrexate, leucovorin and their metabolites, 7-hydroxymethotrexate, 2,4-diamino-N10 -methylpteroic acid and 5-methyltetrahydrofolic acid. For the determination of high concentrations (>10 microM) protein precipitation by acetonitrile will suffice for sample preparation. All other samples undergo solid-phase extraction and upgrading on C18 columns. Aminopterin, a therapeutic antecedent of methotrexate, serves as internal standard. Detection is done on a UV detector with a 300 nm filter. Five hundred microl of serum are needed to determine 0.2 microM of a specified substance (0.5 microM 5-methylterahydrofolic acid) with good precision and accuracy. For peak levels, 20 microl of capillary serum are sufficient.

Antimetabolites, Antineoplastic↗
PubMed1998

Therapeutic drug monitoring of doxorubicin in paediatric oncology using capillary electrophoresis.

A method for the determination of doxorubicin and its main metabolite doxorubicinol in human plasma is described. Two different sample preparation procedures are applied depending on the expected concentration: To monitor the peak plasma levels, 10 microL of plasma are deproteinated with acetonitrile. After centrifugation, the supernatant is directly applied to the capillary by hydrodynamic injection. For the determination of lower amounts of doxorubicin and its main metabolite doxorubicinol 100 microL of plasma is extracted by liquid-/liquid extraction with chloroform. After evaporation of the organic phase, the sample is reconstituted in acetonitrile/water (95/5 v/v) and injected into the capillary by electrokinetic injection. Idarubicin serves as the internal standard. Laser-induced fluorescence detection with an Ar-ion laser emitting at 488 nm and a 520 nm cut-off filter is used for detection. The accuracy of the method was calculated to be 3.0% at higher concentrations and 15.0% at the limit of quantification. Reproducibility data are in accordance to the generally accepted criteria for bioanalytical methods. The limit of quantification is 2 microg/L, enabling us to monitor doxorubicin plasma levels for several days after application. Noninvasive blood sampling (from the fingertip) using heparinized capillaries was found to be a simple and convenient procedure and provides reproducible data. Initial results show high interindividual variability in doxorubicin peak plasma levels.

Antibiotics, Antineoplastic↗