Henk-Jan Guchelaar

Quantitative determination of the macrolide antibiotics erythromycin, roxithromycin, azithromycin and clarithromycin in human serum by high-performance liquid chromatography using pre-column derivatization with 9-fluorenylmethyloxycarbonyl chloride and fluorescence detection

A validated, highly sensitive and precise high-performance liquid chromatographic (HPLC) method for the determination of the macrolides erythromycin, azithromycin, clarithromycin and roxithromycin in human serum is described. A diethyl ether extract, obtained from serum using a saturated sodium carbonate solution, was treated with 9-fluorenylmethyl-oxycarbonyl chloride (FMOC-Cl) for 40 min at 40 degrees C and chromatographed on a base-deactivated octadecyl column, maintained at 50 degrees C during elution, using an eluent composed of acetonitrile-hydrogenphosphate buffer, pH 7.5, with 0.125% triethylamine (3:2, v/v). Fluorescence detection was used at an excitation wavelength of 255 nm and an emission wavelength of 315 nm. Erythromycin, clarithromycin, roxithromycin and azithromycin were found to have retention times of 8.8, 15.7, 17.1 and 20.7 min, respectively. Recoveries ranging from 93 to 104% were found with reproducibility coefficients of variation of 1.1-5%. Mean correlation coefficients of 0.9997, 0.9998, 0.9996 and 0.9994 were found for the linear calibration curves (n = 2) of erythromycin (0.320-16.1 mg/l), roxithromycin (3.24-19.4 mg/l), clarithromycin (0.190-19.4 mg/l) and azithromycin (0.0988-4.94 mg/l), respectively.

Apoptosis: molecular mechanisms and implications for cancer chemotherapy

Apoptosis, or programmed cell death, is an orderly and genetically controlled form of cell death. In a morphological sense, it differs from necrosis in that cellular shrinkage and chromatin condensation occurs, followed by fragmentation of nuclear components within membrane-bound vesicles which are cleared by phagocytosis without damage to adjacent tissue. The molecular pathway includes an initiating phase, which starts after signalling by external triggers, such as ligation to distinct receptors or by endogenous mechanisms related to aging or to exogenous irreversible cellular or nuclear damage. The initiation phase is followed by a decision phase. During this phase transduction occurs of the apoptotic signal to nuclear and cytoplasmatic target enzymes, which includes activation of endonucleases and enzymatic alterations of the cytoskeleton. There are numerous proteins and lipid-derived moieties which modulate the apoptotic mechanism in positive or negative direction. The execution phase is started when the cell has arrived at a stage of no return. The nuclear DNA is cleaved into multiples of 180-200 basepairs, the plasma membrane integrity and the mitochondria remain initially intact, the cell splits up into apoptotic bodies, small vesicles which enclose the nuclear and cellular remnants. Finally, the clearing phase is arrived, when the apoptotic bodies are phagocytosed by adjacent cells and macrophages. It is thought that the pharmacodynamics of anticancer drugs consists of two distinct steps. The first step includes the interaction with its cellular target; which is not lethal per se. The commitment of the cell to undergo apoptosis forms the second step. The efficacy of anticancer drugs is determined by the ability to selectively sensitize tumor cells to apoptosis, which depends to a large extent from the expression of various oncogenes, such as bcl-2, p53, bax, ras, c-myc and others, and from endogenous factors. It is a challenge in pharmacological research to explore apoptosis by modulating the extrinsic and intrinsic regulators in a positive or negative direction in order to improve the efficacy of anticancer treatment.

Apoptosis- and necrosis-inducing potential of cladribine, cytarabine, cisplatin, and 5-fluorouracil in vitro: a quantitative pharmacodynamic model.

Purpose: The purpose of this study was to characterize the concentration-dependent induction of apoptosis by anticancer drugs in vitro. Methods: The apoptosis- and necrosis-inducing potential of the anticancer drugs cladribine (CDA), cytarabine (ARA-C), cisplatin (CDDP), and 5-fluorouracil (5FU) were studied in vitro in the human leukemia cell lines HSB2 and Jurkat using a flow-cytometry assay that permits the simultaneous quantification of vital, apoptotic, and necrotic cells by double-staining with fluorescein isothiocyanate (FITC)-labeled Annexin-V and propidium iodide. The results were fit to different multicompartmental models and the sensitivity of the cell lines to apoptosis and necrosis was estimated. Results: A time- and dose-dependent decrease in vital cells as well as an increase in apoptotic and necrotic cells was observed in HSB2 cells upon continuous incubation with 10−5–10−7M CDA, 10−5–10−8M ARA-C, 5 × 10−5–5 × 10−6M CDDP, and 10−4–10−5M 5FU, whereas no effect was observed relative to controls upon incubation with 10−8–10−9M CDA, 10−9M ARA-C, 10−7–10−8M CDDP, or 10−6–10−9M 5FU. In Jurkat cells, apoptosis- and necrosis-inducing effects were observed at 10−4–5 × 10−6M CDA, 10−5–10−7M ARA-C, 5 × 10−5–5 × 10−6M CDDP, and 10−4–10−5M 5FU. In all experiments, apoptotic cells reached a peak after 6–48 h of drug exposure. These data were best fit by a model in which vital cells became irreversibly apoptotic by a direct pathway and necrotic by an irreversible indirect pathway following the apoptotic state (mean R=0.9876; range 0.9510–0.9993; mean modified Akaikes information criterion 3.88; range 1.86–5.82) and the rate constants of either pathway (Kva and Kan, respectively) were assessed. The sensitivity of both cell lines to apoptosis and necrosis (expressed as EC50 and Emax values) induced by the anticancer drugs could be calculated from the sigmoidal concentration-effect curves. Furthermore, it was shown that drug treatment (10−6M CDA or 10−6M ARA-C) potentiated the apoptosis-inducing effects of irradiation (6 Gy) but not its necrosis-inducing potential. Conclusion: This study demonstrates that CDA, ARA-C, CDDP, and 5FU possess concentration-dependent apoptosis-inducing potential in the cell lines studied. The cytotoxic mechanism and cell-killing potential of these drugs is different, which is reflected by different EC50 and Emax values. Furthermore, a method for pharmacodynamic modeling is introduced that permits a quantitative approach for the assessment of the sensitivity of tumor cells to anticancer drugs and combined treatments.

Flucytosine: Correlation between Toxicity and Pharmacokinetic Parameters

Flucytosine (5-fluorocytosine, 5-FC) is a systemic antimycotic drug the major toxicities of which are bone marrow depression and hepatotoxicity. The purpose of this observational and retrospective study was to assess a possible relationship between toxicity and 5-FC pharmacokinetics within a group of 53 intensive care unit patients. The presented results reveal that thrombocytopenia is associated with a decreased 5-FC clearance and that the thrombocyte nadir is linearly related to the 5-FC clearance. Furthermore, patients experiencing 5-FC levels exceeding 100 mg 5-FC/l were found to be at a higher risk of developing thrombocytopenia and hepatotoxicity as compared to those not exceeding this level.

Implementation of a computerized physician medication order entry system at the Academic Medical Centre in Amsterdam.

In the period 1997–2001 the Academic Medical Centre in Amsterdam implemented the computerized physician medication order entry (CPmOE) system Medicator®. This article describes several important aspects of this program: technological architecture, features, implementation project, authentication and training, continuous support, human resource investments, route of prescription, logistics and administration. Furthermore important advantages and disadvantages of the CPmOE system are discussed. Advantages mainly concern patient safety and drug logistics, while disadvantages are related to access to a computer, user friendliness of the software and printer problems.

Quantitative determination of thalidomide in human serum with high-performance liquid chromatography using protein precipitation with trichloroacetic acid and ultraviolet detection.

A validated and precise reversed-phase high-performance liquid chromatographic method for the determination of thalidomide in serum, with phenacetin as an internal standard, is described. Protein precipitation, using trichloroacetic acid, was used for clean-up. The aliquot was chromatographed on a octadecyl column, using an eluent composed of 250 ml 0.01 M potassium dihydrogenphosphate, adjusted to a pH of 3.0 with a 43% phosphoric acid solution, mixed with 750 ml methanol. Ultraviolet detection was used at an operation wavelength of 220 nm. Hydrolytic degradation was prevented during analysis by acidification of samples with the precipitation reagent. Thalidomide and phenacetin were found to have retention times of 7.9 and 15.0 min, respectively. Recoveries ranging from 79 to 84% were found for both components, with reproducibility relative standard deviations of 0.8-3% and repeatability coefficients of 1.2-3%. A mean correlation coefficient of 0.9995 was found for the linear calibration curve (n=2) of thalidomide with limits of quantitation of 0.222-21 mg/l. The method appeared to be feasible for pharmacokinetic studies with thalidomide.

An in vitro Study on the Active Conversion of Flucytosine to Fluorouracil by Microorganisms in the Human Intestinal Microflora

Background: Investigation of the rate of active conversion of flucytosine to fluorouracil by microorganisms in the intestinal microflora. Methods: Active conversion of flucytosine was investigated using viable and nonviable Escherichia coli at different flucytosine concentrations. Additionally, flucytosine conversion was studied in fecal specimens from 3 neutropenic patients at the start of the antimicrobial/antifungal prophylaxis (C/A regimen) and 1 week later. Results: Flucytosine levels decreased by an average of 72, 71 and 72% flucytosine after incubation for 48 h of 1010 viable E. coli/ml suspension in broth containing 13, 130 and 1,300 mg/l flucytosine, respectively. The decreasing flucytosine levels corresponded approximately to an identical increase in fluorouracil levels. Also, a 44% decrease of flucytosine levels occurred when nonviable E. coli were used, indicating that bacterial viability is not necessary for this conversion. When fecal specimens of 2 patients were investigated prior to the C/A regimen, significant flucytosine conversion occurred, whereas this conversion was not observed in the corresponding fecal specimens after 1 week of C/A regimen. Conclusion: These in vitro experiments showed that extensive flucytosine conversion can occur in the human intestinal microflora by E. coli. Consequently, fluorouracil exposure and fluorouracil-related toxicity may occur in the flucytosine-treated patient.

Quantitative determination of melatonin in human plasma and cerebrospinal fluid with high-performance liquid chromatography and fluorescence detection.

A validated new and precise reversed-phase high-performance liquid chromatographic method for the determination of melatonin in human plasma and cerebrospinal fluid, with 5-fluorotryptamine as internal standard, is described. Liquid-liquid extraction with dichloromethane was performed under alkaline conditions. After evaporation of the organic solvent, the extract was dissolved in eluent and chromatographed on a base-deactivated octadecyl column, using an eluent composed of 650 mL potassium dihydrogenphosphate solution (0.07 mol/L water), adjusted to a pH of 3.0 with a 43% phosphoric acid solution, mixed with 350 mL methanol. Fluorescence detection at an excitation wavelength of 224 nm and an emission wavelength of 348 nm was used for quantitation. Melatonin and 5-fluorotryptamine chromatographed with retention times of 5.3 and 9. 3 min, respectively. Mean recoveries of 96% (n = 10) and 95% (n = 5) were found for melatonin in plasma and cerebrospinal fluid respectively. 5-Fluorotryptamine was found to have a mean recovery of 90% (n = 10) and 82% (n = 5) in plasma and cerebrospinal fluid, respectively. The repeatability coefficients of variation for both melatonin and 5-fluorotryptamine in plasma were 4-5% [five different samples (r = 5) on two consecutive days (n = 2)], with reproducibility coefficients of 1.6-7% (n = 2, r = 5) and 0.9-4% (n = 2, r = 5) for melatonin and internal standard, respectively. In cerebrospinal fluid the repeatability coefficient of variation of the extraction procedure was 5% (n = 1, r = 5) for melatonin and 7% (n = 1, r = 5) for 5-fluorotryptamine. The correlation coefficients of the calibration curves were 0.9998 (n = 2) in plasma at a concentration range of 0.108-25.9 ng/mL and 0.9994 (n = 2) at a concentration range of 0.108-25.9 ng/mL in cerebrospinal fluid. The limit of detection was determined at 8 pg/mL which enables to measure melatonin concentrations at physiological concentrations reached during daytime.

Validation of a high-performance liquid chromatography assay for quantification of caffeine and paraxanthine in human serum in the context of CYP1A2 phenotyping

In this study the validation of a reversed-phase high-performance liquid chromatography (HPLC) method, with UV-detection, for both caffeine and paraxanthine in human serum is described. This method is feasible for cytochrome P450 1A2 (CYP1A2) phenotyping, according to the results of a pilot study. With this HPLC method caffeine and paraxanthine can be determined selectively and specifically. In the expected concentration range, caffeine recoveries were 98-108% (within-run variation 4.0-6.4%, between-run variation 6.4-8.8%), paraxanthine recoveries were 96.6-97.5% (within-run variation 5.0-7.2%, between-run variation 7.2-10.8%). The limits of detection for caffeine and paraxanthine using this HPLC system were 0.3 and 0.1 mg/L, respectively. Linear calibration curves for both caffeine and paraxanthine were obtained in the concentration range 0.5-30 mg/L (r > 0.9999. Serum samples were stable for a week, when stored at -20 and +4 degrees C.

Acute hepatitis in a patient using a Chinese herbal tea--a case report

A case is presented of reversible acute hepatitis in a patient using a Chinese herbal tea. Upon identification of the tea mixture Aristolochia species, including A. debilis, which contains the highly toxic aristolochic acid, could be identified. We conclude that the acute hepatitis as described in this patient is most likely to be caused by (one of) the active ingredients of the Chinese herbal tea. Furthermore, this case illustrates that so‐called natural products can cause unexpected severe adverse reactions.