Anders Andersen

High sensitivity assays for docetaxel and paclitaxel in plasma using solid-phase extraction and high-performance liquid chromatography with UV detection

BackgroundThe taxanes paclitaxel and docetaxel have traditionally been used in high doses every third week in the treatment of cancer. Lately there has been a trend towards giving weekly low doses to improve the therapeutic index. This article describes the development of high performance liquid chromatographic (HPLC) methods suitable for monitoring taxane levels in patients, focusing on patients receiving low-dose therapy.MethodsPaclitaxel and docetaxel were extracted from human plasma by solid phase extraction, and detected by absorbance at 227 nm after separation by reversed phase high performance liquid chromatography. The methods were validated and their performance were tested using samples from patients receiving paclitaxel or docetaxel.ResultsThe limits of quantitation were 1 nM for docetaxel and 1.2 nM for paclitaxel. For both compounds linearity was confirmed from the limit of quantitation up to 1000 nM in plasma. The recoveries ranged between 92% and 118% for docetaxel and between 76% and 104% for paclitaxel. Accuracy and precision were within international acceptance criteria, that is within ± 15%, except at the limit of quantitation where values within ± 20% are acceptable. Low-dose patients included in an on going clinical trial had a median docetaxel concentration of 2.8 nM at 72 hours post infusion. Patients receiving 100 mg/m2 of paclitaxel had a mean paclitaxel concentration of 21 nM 48 hours after the end of infusion.ConclusionWe have developed an HPLC method using UV detection capable of quantifying 1 nM of docetaxel in plasma samples. The method should be useful for pharmacokinetic determinations at all relevant doses of docetaxel. Using a similar methodology paclitaxel can be quantified down to a concentration of 1.2 nM in plasma with acceptable accuracy and precision. We further demonstrate that the previously reported negative influence of Cremophor EL on assay performance may be overcome by degradation of the detergent by incubation with lipase.

A sensitive and simple high-performance liquid chromatographic method for the determination of doxorubicin and its metabolites in plasma

A sensitive high-performance liquid chromatographic (HPLC) method for the measurement of doxorubicin and its metabolites in plasma is described. After precipitation with zinc sulphate and methanol, samples are resolved by isocratic elution from a C18 reverse phase support within 20 min and quantified by endogenous fluorescence. Recoveries over a concentration range from 5 to 1,000 nM of doxorubicin, doxorubicinol, doxorubicinone, doxo-rubicinolone, and 7-deoxydoxorubicinolone were 80–110%, while recovery for 7-deoxydoxorubicinone was –60%. At concentrations of 5 nM, within-run and between-day coefficients of variation for each compound were <8 and <16%, respectively. Limits of detection for the compounds were 1–2 nM and standard curves were linear up to at least 1,000 nM. The drug and its metabolites are stable in deproteinized plasma samples at room temperature and in the dark for at least 24 h. The method requires few manipulations and is readily adaptable to automated analysis of large series of samples.

Continuous intratumoral microdialysis during high-dose methotrexate therapy in a patient with malignant fibrous histiocytoma of the femur: a case report

Abstract We used a microdialysis technique to assay intratumoral methotrexate (MTX) levels during high-dose (12 g/m2 given as a 4-h infusion) therapy in a 43-year-old man with a malignant fibrous histiocytoma in the medial femoral condyle. Additional microdialysis probes were implanted in muscle tissue contralateral to the tumor and in an antecubital vein. Microdialysis was attempted during the initial two high-dose courses, but the two latter probes were removed at the start of the second treatment cycle due to leakage. No attempt to correct for microdialysis recovery was made. The intratumorally localized probe gave reproducible data on tumor MTX exposure of 9.3–14% of unbound systemic MTX. There was a close correlation between tumor and systemic levels for both MTX and its major extracellular metabolite 7-hydroxymethotrexate. Although limited to the study of MTX pharmacokinetics in a single subject, the experiment demonstrates that intratumoral microdialysis may provide data on tumor drug exposure, although of an indirect nature and dependent on the probe characteristics, the flow rate, and, possibly, the time after probe implantation. We propose that the application of microdialysis may prove useful for elucidation of the relationship between local drug exposure and the therapeutic response in normally inaccessible compartments during cancer pharmacotherapy.

Pharmacokinetics and metabolism of doxorubicin after short-term infusions in lymphoma patients

Purpose/Methods: Twenty-four patients (17 males and 7 females with a mean age of 54 years) with malignant lymphoma participated in a study of doxorubicin pharmacokinetics after 50 mg/m2 as 10-min infusions. In addition to plasma samples, serial leukocyte samples and – in one subject – serial biopsy specimens from lymphoma infiltrates were obtained. The samples were analysed by reversed-phase high-performance liquid chromatography. Results: In contrast to several previous studies, the data suggested that 7-deoxydoxorubicinolone, and not doxorubicinone, is a metabolite of doxorubicin in humans. Doxorubicin, but no metabolites, was present in significant and fairly constant concentrations in circulating leukocytes. These levels may reflect the drug levels in lymphoma infiltrates. The data further suggest that metabolism to 7-deoxydoxorubicinone is subject to large interindividual variation, possibly due to a genetic polymorphism, and that significant levels of a metabolic product which may be a doxorubicin glucuronide can be recovered from plasma of patients treated with doxorubicin.

The role of reflection in supporting dynamic QoS management functions

In previous papers, the authors have reported on the design and implementation of reflective middleware platforms. The aim of this paper is to consider the implications of such an architecture for the area of quality of service (QoS) management. More specifically, the paper describes how critical QoS management functions can be incorporated into our reflective middleware platform, with particular emphasis on the dynamic QoS management functions of QoS monitoring and adaptation. It is argued that the reflective approach leads to a natural and highly flexible implementation of such QoS management functions (and indeed the approach offers considerable benefits over more conventional approaches).

Renal and hepatic toxicity after high-dose 7-hydroxymethotrexate in the rat

To examine directly the hepatic and renal toxicity of 7-hydroxymethotrexate (7-OH-MTX) without interference of the parent compound methotrexate (MTX), we purified and gave 100 mg/kg 7-OH-MTX to rats, a dose resulting in serum levels of 7-OH-MTX comparable with those achieved in the clinic after the administration of high-dose MTX (HD-MTX). After only 5 h, the 7-OH-MTX-treated rats demonstrated 2.6-fold increases in serum creatinine values and 2-fold elevations in serum aspartate aminotransferase (ASAT) levels as compared with the controls. Morphologic evidence of toxicity, however, was apparent only in the kidneys. Intraluminal cellular debris containing membranous material and deteriorated organelles was seen, but no precipitate of the delivered drug. The peak serum concentration of 7-OH was up to 939 μM, and concentrations of 7-OH-MTX declined triphasically, showing at1/2α value of 2.45 min, at1/2β value of 30.5 min, and a terminal half-life (t1/2γ) of 240 min. The total clearance value was 14.5 ml min−1 kg, and the postdistributional volume of distribution (Vβ) was 5070 ml/kg. Our results may indicate a direct toxic effect of 7-OH-MTX on kidney and liver cells.

Determination of extracellular methotrexate tissue levels by microdialysis in a rat model

We used a microdialysis technique to determine tissue methotrexate (MTX) levels during steady state in a rodent model. Two different approaches were employed to measure the actual extracellular MTX concentrations in muscle, liver, and kidney tissues of anesthetized Wistar rats. With the reduced-perfusionrate technique, the flow in the microdialysis perfusate was gradually decreased toward zero to permit calculation of zero-flow intercepts. Using the net change technique, microdialysis probes were perfused with different MTX concentrations to allow an assessment of equilibrium drug levels. For these two methods to be used, drug concentrations in the matrix to be analyzed must remain unchanged during the experimental procedure. In the animal model, steady state was attained after 1.5 h and maintained throughout the rest of the experiments by the administration of MTX as continuous infusions through a venous catheter. In vitro and in vivo, both the reduced-perfusion-rate and net change techniques gave reproducible data that permitted the estimation of extracellular drug concentrations in the dialyzed tissue compartments. The data suggest that the level of unbound MTX in the circulation is fairly similar to the extracellular concentrations in the muscle and liver. In the kidney, MTX levels were measured to be 3–8 times higher than those of unbound, circulating MTX, and a considerable discrepancy between the two methods used for estimations was apparent. These results demonstrate that both the net change and reduced-flow microdialysis techniques can produce reproducible and precise data. The results may constitute a basis for determining recoveries and, thus, true extracellular drug levels during in vivo microdialysis of MTX. This may be of importance in delineation of the relationship between tissue MTX levels and outcome in a variety of normally inaccessible compartments during cancer pharmacotherapy.

Formal Support for Dynamic QoS Management in the Development of Open Component-Based Distributed Systems

An aspect-oriented specification technique that supports the specification of component-based distributed systems is presented. Importantly, this technique also supports the synthesis of quality of service management components from particular aspects of the specification. It is described how, by using a tool to support the aspect-oriented environment, one can first specify and verify QoS management subsystems and then synthesise components that can be placed into a running system. The focus is on dynamic QoS management functions, particularly monitoring and adaptation. The approach is illustrated by giving a simple example of the dynamic QoS management of an audio stream.

Pharmacokinetics of different doses of methotrexate at steady state by in situ microdialysis in a rat model

We used a microdialysis technique to monitor extracellular methotrexate (MTX) levels during the steady state in a rodent model. Microdialysis probes were implanted in the muscle, liver, and kidney of anesthetized male Wistar rats. MTX (18.75–500 mg/kg) was given as a continuous infusion through a venous catheter, and blood samples were obtained through a second venous catheter. Heparinized plasma, ultrafiltered plasma, microdialysis effluent from tissues, and tissue samples (obtained at the end of experiments) were analyzed for MTX content by high-performance liquid chromatography (HPLC). Steady state was demonstrated in the blood and tissues from 2 h until the end of the experiments (6 h). Extracellular drug levels in muscle and liver displayed a linear correlation with doses, whereas kidney levels reached a plateau at an MTX dose of 150 mg/kg per 6 h. Microdialysis-fluid endpoint levels for muscle, liver, and kidney were positively correlated to the endpoint total tissue levels (r2=0.80, 0.85, and 0.68, respectively). In the kidneys, the maximal relative tissue MTX accumulation was measured at a total dose of 75 mg/kg per 6 h. At higher doses, the relative drug sequestration declined to less than half of the values observed at this dose. This study demonstrates that the microdialysis technique can provide reproducible data on MTX tissue exposure in an animal model and that it offers a means of serial and reproducible monitoring of extracellular-tissue MTX levels at steady state and over a wide dose range. Pending additional studies, microdialysis may be a helpful technique for elucidating the kinetics of drug delivery to both targeted and toxicity-prone tissues during chemotherapy.

INTRATUMORAL DIFFERENCES IN METHOTREXATE LEVELS WITHIN HUMAN OSTEOSARCOMA XENOGRAFTS STUDIED BY MICRODIALYSIS

A central tenet in oncology is the assumed relationship between drug concentration and cytotoxicity. Determinations of drug levels in tumor tissues are, however, generally not undertaken. Microdialysis is a method where continuous drug monitoring may be achieved by sampling of low molecular weight substances from the extracellular space. By employing this technique it is possible to observe variable drug levels within tissues, including tumors, over time. Herein, we present results from a nude rat model where subcutaneous human osteosarcoma xenografts were established prior to the administration of the antifolate methotrexate as an intravenous infusion. Significant differences in drug exposure within single tumors were evident. Generally, peak drug concentrations were lower and drug efflux slower from the center of the tumors as compared to the periphery. The use of microdialysis could be an important tool for optimizing current strategies in anticancer chemotherapy.