Eva Liliemark

High-performance liquid chromatography with fluorometric detection for monitoring of etoposide and its cis-isomer in plasma and leukaemic cells

The podophyllotoxin derivative etoposide, extensively used in anticancer therapy, is highly protein-bound (95%) in plasma. It is a chiral drug and only the trans-isomer is pharmacologically active. Isomerisation to the inactive cis-lactone occurs in plasma. The cis-lactone is often present in ultrafiltrates of plasma from patients treated with etoposide, therefore it is important to separate the isomers when free etoposide concentrations are assayed. There is reason to believe that free and cellular concentrations are more important for the effect of etoposide therapy than total plasma concentrations. A high-performance liquid chromatographic (HPLC) method for quantification of etoposide and its cis-isomer in plasma, total and non-protein-bound concentrations, and in leukaemic cells is described. After addition of teniposide as internal standard the drugs were extracted with chloroform. Etoposide, its cis-isomer, teniposide and endogenous substances were separated isocratically on a Spherisorb phenyl reversed-phase column. Detection was performed fluorometrically, lambda ex/em = 230/330 nm. Non-protein-bound concentrations were determined after ultrafiltration. The detection limit for etoposide was 10 ng/ml plasma, 25 ng/ml ultrafiltrate and 10 ng/50 x 10(6) cells. The sensitivity of the assay for the cis-lactone was twice as high due to higher fluorescence. The protein binding of the cis-lactone in plasma from ten healthy blood donors was 54.5 +/- 4.8% (mean +/- S.D.). Thus, the free fraction was about ten-fold higher than that of the mother compound. The assay is convenient and sensitive enough for the determination of free and cellular fractions of etoposide.

Plasma pharmacokinetics of etoposide (VP-16) after i.v. administration to children.

The pharmacokinetics of etoposide (VP-16), a semi-synthetic derivative of podophyllotoxin, were studied in 16 pediatric patients (median age 8.3 years; range 4 months to 22 years) including two girls with Downs syndrome (DS). The drug was administered as infusions (1-3 h) in a wide range of doses (9-322 mg, corresponding to 32-210 mg/m2). The area under the plasma concentration versus time curve (AUC), dose normalized by the body surface area, was independent of age, while AUC normalized by the dose in mg/kg increased with increasing age of the patients. The interpatient variability of AUC, normalized for the dose in mg/m2, was 23% (CV) compared to 32% (CV) normalized for the dose in mg/kg. The terminal half-life time was 4.1 h (median value; range 2.0-7.8 h). The pharmacokinetics of etopside in children with DS and chromosomally normal children were very similar with regard to systemic drug exposure and plasma half-life time. From the pharmacokinetic point of view it was therefore not necessary to make any dose modifications in the two girls with DS. The two DS patients did not experience any enhanced degree of toxicity from their etoposide treatments. The results support that dosing of etoposide to children should be based on body surface area.

DNA damage induced by etoposide; a comparison of two different methods for determination of strand breaks in DNA

Etoposide induces DNA damage to cells by interacting with the nuclear enzyme topoisomerase II. In this investigation the human lymphoblastic leukemia cell line (CEM) was used to study induction of DNA-strand breaks and cellular drug uptake after treatment with etoposide at a concentration of 0.5-2 micrograms/ml. High performance liquid chromatography was used for determination of etoposide concentrations. The alkaline elution assay and the DNA unwinding technique were compared for quantifying strand breaks in DNA induced by etoposide. The concentrations required to increase the level of DNA damage significantly was as follows: the DNA unwinding technique, 0.20 microgram/ml; the alkaline elution assay with proteinase K, 0.45 microgram/ml; the alkaline elution assay without proteinase K, 0.60 microgram/ml. When the half-life was adjusted, considering the efflux time of etoposide from cells, it was found to be only a few minutes. The present data show that the DNA unwinding technique is to be preferred for the screening of DNA damage. This technique is easier and quicker to perform than the alkaline elution technique.

In Vivo Accumulation of Etoposide in Peripheral Leukemic Cells in Patients Treated for Acute Myeloblastic Leukemia; Relation to Plasma Concentrations and Protein Binding

Since etoposide interacts with the nuclear enzyme topoisomerase II, the drug concentrations in the malignant cells during chemotherapy may have clinical correlates. Plasma protein binding of etoposide is extensive (94%) and alterations of the non-proteinbound fraction affect pharmacokinetic behavior of the drug. The pharmacokinetics of etoposide was therefore studied in plasma, total and non-proteinbound concentrations, and in leukemic cells isolated from peripheral blood samples from 22 patients after the first dose of the induction treatment for acute myelocytic leukemia. Fourteen patients received 100 mg/m2 and eight patients 200 mg/m2 as a 1 h infusion. The mean area under the concentration versus time curve AUC(0-infinity) in plasma was at the lower dose level 78.4 +/- 29.1 (mean +/- S.D.) micrograms/ml x h and 201.0 +/- 56.5 micrograms/ml x h at the higher dose level. The fraction of non-proteinbound etoposide in plasma was 5.2 +/- 3.4 and 5.4 +/- 2.1% in the two treatment groups. AUC(0-16h) in leukemic cells was 8.4 +/- 8.7 and 22.4 +/- 12.1 micrograms/ml x h at the two dose levels, respectively. The cellular etoposide concentration was 12.1 +/- 7.9 and 14.7 +/- 5.1% of the plasma concentration at the end of the infusion. The interpatient variability in cellular drug levels was considerable and exceeded the variability in plasma concentrations. Cellular accumulation of etoposide could be important for treatment outcome.

Characterization of Drug-Resistant Cell Lines by Comparative Genomic Hybridization

The development of resistance to cytostatic agents is a serious obstacle to the success of cancer therapy and has been the focus of many research efforts. Traditionally, cell lines are selectively cultured in the presence of cytostatic agents and the biochemical and cytogenetic properties of the cell lines are then analyzed. In order to better understand the mechanisms by which drug resistance is mediated, we have analyzed three cell lines, each derived from the parent line K562, which are resistant to vincristine, mitoxantrone, or idarubicin, using comparative genomic hybridization (CGH). In each case, CGH successfully identified amplifications and/or deletions unique to the drug-resistant selected cell lines. Further characterization of the genetic regions identified in the CGH analysis could greatly contribute to our understanding of acquired drug resistance, and could potentially impact the clinical management of cancer.

Ultrafiltration and subsequent high performance liquid chromatography for in vivo determinations of the protein binding of etoposide

Etoposide is extensively (approximately 94%) bound to plasma proteins and the free non-protein-bound levels have been shown to correlate more closely to toxicity than total drug concentrations. A rapid and easily performed method, compared to the time consuming equilibrium dialysis, to obtain the free fraction is needed. The aim of this study was to evaluate ultrafiltration and subsequent high performance liquid chromatography (HPLC) for the determination of protein binding of etoposide. Spiked plasma from healthy, drug-free volunteers was used to compare ultrafiltration, using Amicon Centrifree filters, with equilibrium dialysis at 37 degrees C. The variability (CV) of the ultrafiltration method was 6.1 and 13.5% (n = 6) at 37 degrees C and room temperature (RT), respectively. The relative size of the free fraction obtained by ultrafiltration at 37 degrees C and RT was 1.22 (P = 0.0005) and 0.37 (P = 0.0001), respectively, compared with equilibrium dialysis at 37 degrees C. The chromatographic separation of metabolites from the mother compound when free etoposide is analyzed is crucial. It is shown that a hydroxy-acid metabolite of etoposide is quite dominant in a protein-free plasma fraction. The free concentrations were determined throughout a dose interval of 24 h in a patient receiving etoposide 100 mg/m2 daily. Ultrafiltration and subsequent HPLC is considered convenient and suitable for in vivo pharmacokinetic investigations.

In vitro topo II-DNA complex accumulation and cytotoxicity of etoposide in leukaemic cells from patients with acute myelogenous and chronic lymphocytic leukaemia

Topoisomerase II (topo II) is the target enzyme of etoposide, and DNA--topo II complex accumulation is considered crucial for the cytotoxic effect. We used a SDS--KCl precipitation assay to determine the complex accumulation induced by etoposide in leukaemic cells isolated from 58 patients, 31 with acute myelogenous leukaemia (AML), and 27 with chronic lymphocytic leukaemia (CLL). To investigate whether the sensitivity towards etoposide was dependent on the complex accumulation in the cells, we investigated the drug-induced DNA damage using a DNA unwinding assay and the in vitro cytotoxicity of etoposide using the MTT assay. AML cells had higher complex accumulation (P=0.006) and more DNA damage (P=0.029) compared with CLL cells. The data support a relationship between etoposide-induced complex accumulation and DNA damage in leukaemic cells from AML and CLL patients. However, the induced DNA damage did not translate to in vitro cytotoxicity, suggesting that other factors, such as DNA repair and apoptosis functions, also play important roles to determine the etoposide sensitivity.

Topoisomerase II-mediated alterations of K562 drug resistant sublines.

In order to further elucidate the, roles of DNA topoisomerase II (topo II) subtypes, α and β, as drug targets in chemotherapy, we have determined the enzyme levels in K562 cells selected for resistance to mitoxantrone (K562/Mxn), daunorubicin (K562/Dnr) and idarubicin (K562/Ida 20 and K562/Ida 60), as well as topo II-DNA complex formation, DNA damage and cytotoxicity, induced by topo II interactive agents, for example etoposide, teniposide, mitoxantrone and amsacrine. As compared to the parental cells, topo IIα/β protein levels in K562/Mxn, K562/Dnr, K562/Ida 20 and 60 lines, measured with Western blot, were 17/67%, 85/88, 24/31% and 10/7% respectively. DNA damage, determined by DNA unwinding technique, induced by teniposide and amsacrine correlated with both topo IIα/β protein levels (r2=0.8/0.9,P=0.03/0.01 andr2=0.8/0.9,P=0.04/0.01, respectively). Topo II-DNA complex formation induced by all studied drugs correlated with topo IIβ protein levels (r2-range 0.8–0.9,P-range 0.01–0.04), while the correlation with topo IIα was weaker. Topo IIα/β protein levels tended to show an inverse correlation with the cytotoxicity of etoposide (r2=−0.9/−0.7,P=0.01/0.06). The overall topo II-DNA complex formation correlated with drug-induced DNA damage (r2=0.9,P=0.0001), whilst not with the cytotoxicity.Our findings indicate that both topo II isozymes are the targets of the antitumor agents studied, and of potential clinical relevance for prediction of treatment efficacy. They could play a role in tailored chemotherapy.

Determination of etoposide in human plasma and leukemic cells by high-performance liquid chromatography with electrochemical detection

This paper describes a high-performance liquid chromatographic method with electrochemical detection for the determination of etoposide levels in plasma, total and non-protein bound concentration, and in leukemic cells. The precision for between-runs (n=6) was 7.0, 4.9, and 9.5%, the accuracy was 3.7, 7.1 and 6.3%, and within-runs precision (n=6) was 3.9, 2.9 and 5.1% for total plasma, non-protein bound plasma fraction and leukemic cells, respectively. The correlation coefficients (R2) were 1.00 for all calibration curves. These assays have been applied to analyze samples from one patient with acute myelogenous leukemia during 24 h after i.v. infusion of etoposide (100 mg/m2).

Real-time RT-PCR for the determination of topoisomerase II mRNA level in leukaemic cells

We developed a real-time RT-PCR assay for the quantification of topoisomerase II (topo II) mRNA level. It was applied on peripheral leukaemic cells from 23 patients with acute myelogenous leukaemia (AML) and 23 with chronic lymphocytic leukaemia (CLL). RNA template dilutions from 0.25 to 25ng per reaction were used as standard curves for topo IIalpha, beta and the internal control 18S rRNA. About 57% (26/46) and 26% (12/46) of the specimens had detectable topo IIbeta and alpha mRNA, respectively. The correlation between these two factors was rho=0.7 and P=0.0001. No relationship between topo IIalpha or beta mRNA level and response to chemotherapy was found in AML patients (n=19 assessable for response). Our method is rapid and convenient for quantification of topo IIalpha and beta mRNA levels, and could be suitable for investigation in a larger population.