Pharmacology of all-trans-retinoic acid in children with acute promyelocytic leukemia.

BACKGROUND: Due to severe side effects in virtually all children treated with a standard dose of 45 mg/m(2)/day all-trans-retinoic acid (ATRA) for acute promyelocytic leukemia (APL) the AML-BFM study group reduced the dosage to 25 mg/m(2)/day. For the lack of data on the use of ATRA at this dosage in children with APL, the study group further decided to evaluate the pharmacokinetics and metabolism of ATRA in children. PROCEDURE: Twenty-three pharmacokinetic and metabolic profiles of ATRA were studied in 14 children (aged 0.9-18.4 years) with APL. Eleven plasma samples were collected over a period of 8 hr and analyzed for ATRA and its metabolites by high-performance liquid chromatography. RESULTS: Peak plasma concentrations of ATRA were characterized by wide interpatient variability (range: 28.6-513.0 nM). Compared to adults the same metabolic pathways were observed in children. Even though peak plasma concentrations were in the lower range of those considered effective in vitro, ATRA side effects, notably neurotoxicity, still required dose reduction, treatment break, or drug withdrawal in eight patients. In this small number of patients, neurotoxicity could not be related to age or any specific level of ATRA or metabolites in the plasma. Plasma concentrations of vitamin A, however, were significantly higher in those patients, who developed signs of neurotoxicity (P = 0.03, Mann-Whitney Rank Sum test). CONCLUSIONS: Considering the low plasma concentrations and the persistence of toxicity in spite of dose reduction intermittent dosing schedules might be considered as an alternative to further dose reduction of ATRA in the treatment of APL especially in children, who might be at risk of ATRA-induced neurotoxicity.

Radiation-induced changes of telomerase activity in a human Ewing xenograft tumor.

AIM: The effect of ionizing irradiation on telomerase activity and further associated biological factors was evaluated in a human Ewing tumor xenograft model on nude mice. MATERIAL AND METHODS: The human Ewing tumor cell line STA-ET-1 was established in a nude mouse model. Initially, the dose-response relationship for the tumor model was established. For the radiation experiments two dose levels were chosen: 5 Gy and 30 Gy. After 5 Gy, there was no significant growth delay whereas after 30 Gy there was a marked growth delay without the induction of a complete remission. Tumors were examined 6, 12, 24, 48, 72, and 96 hours post irradiation. After irradiation with 30 Gy further time points were 6, 9, 12 and 15 days. For each dose and time group, three tumors were evaluated. RESULTS: There was a reduction of telomerase activity after 5 Gy to 50% (not statistically significant) after 3 days; however, after 30 Gy there was a reduction of telomerase activity to 23% of the initial value after 6 days (p = 0.001). Telomerase activity correlated with the expression of human telomerase reverse transcriptase (hTERT), but not with the expression of telomerase-associated protein (TP1) and human telomerase RNA (hTR). The maximal amounts of necrosis or apoptosis after 30 Gy were 19% and 6.9%, respectively. CONCLUSIONS: Ionizing radiation reduces telomerase activity and the expression of hTERT which cannot be explained by the induction of necrosis or apoptosis alone. The reduction of telomerase activity may contribute to delayed cell death after radiotherapy. The combined use of radiation and specific telomerase inhibitors may be a potentially synergistic treatment strategy.

Analytical validation of a microplate reader-based method for the therapeutic drug monitoring of L-asparaginase in human serum.

The enzyme L-asparaginase (ASNASE), which hydrolyzes L-asparagine (L-Asn) to ammonia and L-aspartic acid (L-Asp), is commonly used for remission induction in acute lymphoblastic leukemia. To correlate ASNASE activity with L-Asn reduction in human serum, sensitive methods for the determination of ASNASE activity are required. Using L-aspartic beta-hydroxamate (AHA) as substrate we developed a sensitive plate reader-based method for the quantification of ASNASE derived from Escherichia coli and Erwinia chrysanthemi and of pegylated E. coli ASNASE in human serum. ASNASE hydrolyzed AHA to L-Asp and hydroxylamine, which was determined at 710 nm after condensation with 8-hydroxyquinoline and oxidation to indooxine. Measuring the indooxine formation allowed the detection of 2 x 10(-5)U ASNASE in 20 microl serum. Linearity was observed within 2.5-75 and 75-1,250 U/L with coefficients of correlation of r(2)>0.99. The coefficients of variation for intra- and interday variability for the three different ASNASE enzymes were 1.98 to 8.77 and 1.73 to 11.0%. The overall recovery was 101+/-9.92%. The coefficient of correlation for dilution linearity was determined as r(2)=0.986 for dilutions up to 1:20. This method combined with sensitive methods for the quantification of L-Asn will allow bioequivalence studies and individualized therapeutic drug monitoring of different ASNASE preparations.

Telomerase as a prognostic marker in breast cancer: high-throughput tissue microarray analysis of hTERT and hTR.

Telomerase activity (TA) has been shown to correlate with poor clinical outcome in various tumour entities, indicating that tumours expressing this enzyme may be more aggressive and that TA may be a useful prognostic marker. For breast cancer, however, TA is a controversial prognostic marker; whereas some studies suggest an association between TA and disease outcome, others do not find this association. This study used tissue microarrays (breast carcinoma prognosis arrays) containing 611 samples (each 0.6 mm in diameter) from the tumour centre of paraffin-embedded breast carcinomas to analyse the catalytic subunit of telomerase, human telomerase reverse-transcriptase (hTERT), and the internal RNA component (hTR), which are the core components of the telomerase holoenzyme complex. hTERT protein expression was obtained by immunohistochemistry (human anti-telomerase antibody Ab-2, Calbiochem), and hTR RNA was measured by radioactive in situ hybridization. hTERT and hTR expression were determined semi-quantitatively and graded (scores 1-4). Clinical data, such as histological subtype, pT stage, tumour diameter, pN stage, BRE grade, tumour-specific survival (in months), patient's age and others, were available for statistical analysis. A statistically significant correlation was found between tumour-specific survival (overall survival) and hTERT expression (p < 0.0001) or hTR expression (p = 0.00110). Tumours with higher scores (scores 3, 4) for hTR and/or hTERT were associated with a worse prognosis. In multivariate analysis, hTERT expression was an independent prognostic factor. Previous studies, focusing on analysis of TA in smaller numbers of fresh-frozen breast carcinomas by the TRAP assay, gave controversial results with respect to TA as a prognostic marker. Using tissue microarrays from 611 breast carcinomas, this study has demonstrated that increased expression levels of the telomerase core components, hTERT and hTR, are associated with lower overall survival. These findings suggest that TA should be included in future validation studies as a prognostic marker in breast cancer.

c-KIT-expressing Ewing tumour cells are insensitive to imatinib mesylate (STI571).

PURPOSE: In order to determine whether Ewing tumour patients may be potential candidates for imatinib mesylate therapy, we analysed the expression of the currently known imatinib mesylate-sensitive tyrosine kinases and tested sensitivity to imatinib mesylate in a panel of eight Ewing tumour cell lines in vitro. METHODS: Expression of the different tyrosine kinases was assessed by flow cytometry and RT-PCR. Sensitivity to imatinib mesylate was analysed using a standard MTT proliferation assay. RESULTS: Flow cytometric and RT-PCR analyses in a panel of eight Ewing tumour cell lines demonstrated expression of several imatinib mesylate-sensitive tyrosine kinases, including c-KIT, platelet-derived growth factor receptor, c-ABL and c-ARG. However, in the MTT proliferation assay, all eight Ewing tumour cell lines were found to be resistant to imatinib mesylate at concentrations ranging from 0.1 to 10 micro M. CONCLUSIONS: Despite the expression of imatinib mesylate-sensitive tyrosine kinases, Ewing tumour cells proved resistant to imatinib mesylate in vitro. This observation has implications for the selection of patients for experimental therapy with imatinib mesylate.

Population pharmacokinetics of high-dose etoposide in children receiving different conditioning regimens.

Pharmacokinetics after high-dose (HD) etoposide (Eto) (40 mg/kg i.v. once as 4-h infusion, one patient 20 mg/kg i.v. as 4-h infusion, for 3 consecutive days) were studied in 31 children and young adults (age 0.8-23.7 years, median: 8.0 years) undergoing bone marrow transplantation after different conditioning regimens. Blood samples were collected until 97 h after the end of infusion. The population analysis of the first part of data (112 samples/21 patients, well documented) served to establish the pharmacokinetic model. The same data combined with the second part of data (50 samples/10 patients, 'intention to treat') then served to calculate the final population model. Data were best described by a three-compartment model with t1/2alpha = 0.28 h +/- 3.2%, t1/2beta = 3.6 h +/- 16.9% and t1/2gamma = 44.2 h +/- 56.5%, respectively (mean(geom) +/- CV(geom)). Clearance (CL) was 15.5 ml/min/m2 +/- 30.6% (mean(geom) +/- CV(geom)) and thus at the lower range of data reported in the literature. The fraction of unbound Eto (fu) was 7.0% (4.3-11.9%) [median (range)], with high intra-individual variability. An increase in f(u) with increasing total Eto was observed. The question of a principally lower Eto CL in children, as compared to adults, after HD treatment remains open.

PEG-asparaginase (Oncaspar) 2500 U/m(2) BSA in reinduction and relapse treatment in the ALL/NHL-BFM protocols.

PURPOSE: As previous data had shown that only two-thirds of patients had the predicted activity time courses when PEG-asparaginase 1000 U/m(2) was used in reinduction after native E. coli asparaginase in induction treatment of acute lymphoblastic leukaemia (ALL), drug monitoring was performed with the use of a higher dose. METHODS: Because one-third of patients had insufficient serum asparaginase activity time courses after a single dose of 1000 U/m(2) PEG-asparaginase during reinduction treatment, a dose of 2500 U/m(2) PEG-asparaginase, which is the approved dosage in Germany, was used in 39 reinduction and 20 relapse patients to determine whether prolongation of the activity time course may be possible with this higher dose, and to look for significant differences between reinduction and relapse patients. RESULTS: After 1, 2 and 3 weeks, the mean activities were 1113 +/- 699 U/l, 231 +/- 259 U/l, and 13 +/- 35 U/l in the reinduction patients, and 1078 +/- 649 U/l, 165 +/- 195 U/l and 19 +/- 28 U/l in the relapse patients, respectively. There were a considerable number of patients with a substantially shortened activity time course in both groups. In 10 of 39 reinduction patients and in 7 of 24 doses during relapse treatment, only activities <100 U/l were found after 1 week with a further fast decline. No statistically significant differences between the two patient groups could be shown at any time-point. CONCLUSIONS: Comparison of these data with activities after 1000 U/m(2) PEG-asparaginase showed no prolongation of the time with activity in the therapeutic range with the higher dose. Therefore, for a longer duration of therapeutic activity, administration of further doses is mandatory.