Petra Schulze-Westhoff

Monitoring of asparaginase activity and asparagine levels in children on different asparaginase preparations

The antileukaemic enzyme L-asparaginase is used to achieve the greatest possible reduction in blood levels of the amino acid asparagine, an essential factor for the growth of leukaemic blasts. There are two main sources of the enzyme, E. coli and Erwinia. Faced with increasing reports of treatment complications, we established a programme to monitor enzyme activity and asparagine levels in serum, in children receiving treatment for acute lymphoblastic leukaemia (ALL) and non-Hodgkins lymphoma (NHL). Trough asparagine and asparaginase levels were measured in 49 children on induction treatment with different E. coli preparations (Asparaginase medac, Crasnitin) and in 52 children on re-induction (Asparaginase medac, Crasnitin, and, in the event of allergic reactions, Erwinase) just prior to each sequential application of 10000 U/m2 of asparaginase. Measurements were made by an enzyme assay and an HPLC method. During induction, both Escherichia coli preparations induced the desired reduction in asparagine, but the asparaginase activity with Asparaginase medac was significantly higher than with Crasnitin (median of trough levels 475 versus 74 U/l). Under re-induction treatment (median, Asparaginase medac 528 U/l, Crasnitin 49 U/l, and Erwinase < 20 U/l) complete asparagine depletion was recorded on day 3 in more than 90% of Asparaginase medac samples, more than 60% of Crasnitin samples and in 26% of Erwinase samples. The latter two groups included some children with unchanged asparagine levels and no measurable enzyme activity. Different asparaginase preparations are not readily interchangeable. When Asparaginase medac is used instead of Crasnitin, and identical dose will be associated with significantly higher enzyme activity, well above the level required for complete asparagine depletion. Clinical studies will need to specify both the preparation and the dose to be used. When substitution of an alternative drug is mandatory owing to allergic reactions, monitoring is advisable.

Dose reduction of asparaginase under pharmacokinetic and pharmacodynamic control during induction therapy in children with acute lymphoblastic leukaemia

The enzyme asparaginase is an important element in the therapy of acute lymphoblastic leukaemia (ALL). The usual asparaginase dose as prescribed in the ALL‐BFM‐86/90 treatment protocol for the therapy of ALL is 10 000 IU/m2 at 3 d intervals and had been developed on the basis of the E. coli asparaginase preparation CrasnitinTM from the Bayer company. Using the described schedule the E. coli asparaginase preparation from the Medac company shows significantly higher biological activity than the Bayer preparation. These findings prompted an attempt to reduce the dose of the Asparaginase medacTM under careful pharmacokinetic and pharmacodynamic monitoring. At the first step of dose reduction in ALL treatment protocol I, 11 children received 5000 IU/m2 of Asparaginase medacTM. Another 15 children were given 2500 IU/m2 of the enzyme at the second step of dose reduction. Prior to each asparaginase dose, blood samples were taken to determine amino acids and trough enzyme activity. Concurrent with the asparaginase monitoring, the coagulation parameters were measured. 96% of samples from the first step of dose reduction (5000 IU/m2 every third day) showed complete L‐asparagine depletion (<0.1 μM), the median trough enzyme acitivity was 265 IU/l. At the second step of dose reduction (2500 IU/m2) complete L‐asparagine depletion was seen in 97% of samples, and the median trough enzyme acitivity was 102 IU/l. Cerebrospinal fluid (CSF) depletion was complete in all samples tested (11/11).  We concluded that an Asparaginase medacTM dose reduced from the usual 10000 IU/m2 down to 5000 IU/m2 or 2500 IU/m2, applied at 3 d intervals, was sufficient to achieve complete L‐asparagine depletion in serum. Changes of the fibrinogen levels was significantly less pronounced in the group on 2500 IU.

Asparagine synthetase activity in paediatric acute leukaemias: AML‐M5 subtype shows lowest activity

Lack of sufficient cellular activity of asparagine synthetase (AS) in blast cells compared with normal tissue is thought to be the basis of the antileukaemic effect of l‐asparaginase in acute lymphoblastic leukaemia (ALL). Although l‐asparaginase is routinely used in ALL, its role and value in the treatment of acute myelogenous leukaemia (AML) is still being discussed. To evaluate the pharmacological basis for l‐asparaginase treatment, we established pretreatment monitoring of the intracellular AS activity in blast cells of patients with AML and ALL. There was no general difference in AS activity between ALL and AML samples. Significantly lower AS activity, however, was found in the B‐lineage ALL subgroups as well as AML‐M5.

Intracellular retention of cytosine arabinoside triphosphate in blast cells from children with acute myelogenous and lymphoblastic leukemia

The importance of the cellular pharmacokinetics of cytarabine triphosphate (ara-CTP) with regard to therapeutic efficacy is well established. In vitro and in vivo monitoring of pharmacokinetic parameters of leukemic blast cells were initiated in order to contribute to the pharmacological basis of optimal ara-C treatment strategies. Peripheral or bone marrow blast cells from 66 leukemic patients [51 acute myelogenous leukemia (ALL), 15 acute lymphoblastic leukemia (AML) were separated and incubated with ara-C for 1 hour and in ara-C-free medium for another 3 hours, and the intracellular formation and retention of ara-CTP was measured. In eight children who received continuous ara-C infusion for induction treatment, the ara-CTP concentration in circulating blast cells was monitored in vivo. The in vitro values observed in this assay corresponded to the cellular levels monitored in vivo. The ara-CTP retention differed clearly among the individual groups, as classified by immunophenotype at the time of the initial diagnosis: non-T-ALL 67+/-25% (x+/-SD, n=33), T-ALL 37+/-15% (n=8), and AML 34+/-18% (n=14). The difference in ara-CTP retention between non-T-All and AML (P<0.05) as well as T-ALL (P<0.05) was significant. There was a tendency toward lower ara-CTP retention in relapsed as compared with newly diagnosed ALL, but the difference was not significant. The maximal accumulation of ara-CTP (after 1 hour incubation) was comparable in AML, T-ALL, non-T-ALL, and blast cells from children in relapse. The observed similarity of cellular accumulation in all groups and the significantly more rapid decrease in T-ALL and AML provide the pharmacokinetic rationale supporting the prolonged infusion duration for ara-C in these subgroups as an alternative to the intensification by high-dose ara-C schedules with short-term infusion.

Determination of idarubicin and idarubicinol in plasma by capillary electrophoresis

A rapid and sensitive capillary electrophoretic method for the determination of idarubicin and its metabolite idarubicinol in plasma has been developed and validated. Plasma is extracted by liquid-liquid extraction using chloroform. Idarubicin, idarubicinol and the internal standard daunorubicin can be separated in less than 5 min using a phosphate buffer of pH 5 with 70% acetonitrile. Laser-induced fluorescence detection with an Ar ion laser operated at 488 nm provides a sensitive and selective detection method without interferences from biological fluids. The small sample volume of 100 microl is of particular advantage for studies in pediatric oncology. The reproducibility of the method has been shown to be sufficient for drug monitoring or pharmacokinetic studies. The limit of quantification for idarubicin in plasma is 0.5 ng/ml.

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.

Asparagine Synthetase in Pediatric Acute Leukemias: AML-M5 Subtype Shows Lowest Activity

Objective The enzyme L-asparaginase is used to achieve maximum reduction of L-asparagine in blood and CSF. Lack of sufficient cellular activity of asparagine synthetase in blast cells compared to normal tissues is thought to be the basis of the antileukemic effect in ALL. While L-Asparaginase is routinely used in acute lymphoblastic leukemia its role and value in the treatment of acute myeloblastic leukemia is still being discussed. As the drug causes a number of relevant side effects, measuring the asparagine synthetase activity of individual patients’ blast cells might help to restrict the use to patients with low expression and to exclude others with high synthetase capacity from this treatment. We, therefore, established asparagine synthetase monitoring.

Drug Monitoring of Doxorubicin in Children

Anthracyclines show activity against both haematological malignancies and solid tumours. Doxorubicin is an important part of many treatment protocols for children with leukemia. A very serious side effect of anthracyclines is myocardial degeneration causing congestive heart failure. Apart from the cumulative dose the peak plasma levels seem to be an independent risk factor. In the literature fractional dosing and prolongation of infusion times have been suggested to reduce the risk of congestive heart failure [1,4]. This admits the presumption that high peak plasma levels are responsible for the cardiotoxicity by short infusions full dose. Therefore, a drug monitoring program has been established to measure the peak plasma levels of children receiving doxorubicin in different treatment protocols. Plenty of preanalytical problems had to be solved before.

In Vitro Monitoring of Asparaginase: Unphysiological Alteration of the Culture Medium

Purpose Drug sensitivity testing, in this instance of asparaginase (ASNase), is a major tool in developing target therapy concepts. It is generally assumed that the in vitro drug concentration and the culture medium do not change within the incubation time. We propose unphysiological alteration of the culture medium since enzyme products may accumulate whereas substrates may decline. ASNase catabolizes asparagine to aspartic acid. Besides, the inherent glutaminase activity, which is of minor importance in vivo, results in the desamination of glutamine to glutamic acid and ammonia. We addressed the question of a dose-response relationship between the ASNase activity and related substrates and products in vitro.