Cancer Letters, 9 (1980) 263-269 o Elsevier/North-Holland Scientific Publishers Ltd. 263 DETERMINATION OF DAUNORUBICIN AND ITS MAIN METABOLITES IN PLASMA, URINE AND LEUKAEMIC CELLS IN PATIENTS WITH ACUTE MYELOBLASTIC LEUKAEMIA CHRISTER PAULa, ROGER BAURAINb, GGSTA GAHRTONa and CURT PETERSONC Vection of Oncology and Haematology, Department of Medicine, Huddinge Hospital, S-141 86 Huddinge (Sweden), bZnternational Institute of Cellular and Molecular Pathology, Avenue Hippocrate 75, B-1200 Brussels (Belgium) and ‘Department of Pharmacology, Karolinska Institute& S-l 04 01, Stockholm (Sweden) (Received 20 September 1979) (Accepted 20 November 1979) SUMMARY The pharmacokinetics of daunorubicin were studied in 3 previously untreated patients with acute myeloblastic leukaemia by simultaneous monitoring of daunorubicin (DNR), daunorubicinol (DOL) and their aglycones in plasma, urine and leukaemic cells. The drug was given as an i.v. infusion in a dose of 1.5 mg/kg body wt. The plasma concentration of daunorubicin declined rapidly after the infusion. The concentration of daunorubicinol exceeded that of the parent compound only 5 min after the end of the infusion. Daunorubicin accumulated extensively in the leukaemic cells and reached concentrations there which exceeded the plasma concentra- tion 400 -4000 times. As compared to what was found in plasma, dauno- rubicinol appeared much slower in the leukaemic cells and the concentration ratio only reached 30 -200. The concentration of aglycones was low in the leukaemic cells as well as in plasma. Only about 15% of the administered dose of daunorubicin could be recovered in the urine within 4 days, most of it as daunorubicinol. The results demonstrate that the plasma concentration of daunorubicin and its metabolites provides little information on the drug concentration in the leukaemic cells. Direct determinations of drug concen- trations in the leukaemic cells might be of clinical value for optimization of the therapy in acute leukaemia. INTRODUCTION Daunorubicin (DNR) is one of the most effective drugs for induction Address all correspondence to: Dr. Christer Paul, Section of Oncology and Haematology, Department of Medicine, Huddinge Hospital, S-141 86 Huddinge, Sweden.
Transcript
Cancer Letters, 9 (1980) 263-269 o Elsevier/North-Holland Scientific Publishers Ltd.
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DETERMINATION OF DAUNORUBICIN AND ITS MAIN METABOLITES IN PLASMA, URINE AND LEUKAEMIC CELLS IN PATIENTS WITH ACUTE MYELOBLASTIC LEUKAEMIA
CHRISTER PAULa, ROGER BAURAINb, GGSTA GAHRTONa and CURT PETERSONC
Vection of Oncology and Haematology, Department of Medicine, Huddinge Hospital, S-141 86 Huddinge (Sweden), bZnternational Institute of Cellular and Molecular Pathology, Avenue Hippocrate 75, B-1200 Brussels (Belgium) and ‘Department of Pharmacology, Karolinska Institute& S-l 04 01, Stockholm (Sweden)
(Received 20 September 1979) (Accepted 20 November 1979)
SUMMARY
The pharmacokinetics of daunorubicin were studied in 3 previously untreated patients with acute myeloblastic leukaemia by simultaneous monitoring of daunorubicin (DNR), daunorubicinol (DOL) and their aglycones in plasma, urine and leukaemic cells. The drug was given as an i.v. infusion in a dose of 1.5 mg/kg body wt. The plasma concentration of daunorubicin declined rapidly after the infusion. The concentration of daunorubicinol exceeded that of the parent compound only 5 min after the end of the infusion. Daunorubicin accumulated extensively in the leukaemic cells and reached concentrations there which exceeded the plasma concentra- tion 400 -4000 times. As compared to what was found in plasma, dauno- rubicinol appeared much slower in the leukaemic cells and the concentration ratio only reached 30 -200. The concentration of aglycones was low in the leukaemic cells as well as in plasma. Only about 15% of the administered dose of daunorubicin could be recovered in the urine within 4 days, most of it as daunorubicinol. The results demonstrate that the plasma concentration of daunorubicin and its metabolites provides little information on the drug concentration in the leukaemic cells. Direct determinations of drug concen- trations in the leukaemic cells might be of clinical value for optimization of the therapy in acute leukaemia.
INTRODUCTION
Daunorubicin (DNR) is one of the most effective drugs for induction
Address all correspondence to: Dr. Christer Paul, Section of Oncology and Haematology, Department of Medicine, Huddinge Hospital, S-141 86 Huddinge, Sweden.
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treatment of acute leukaemias [ 151. The drug probably exerts its effects by intercalation between the base pairs in the DNA-helix in the cell nucleus, thereby preventing further replication [ 4,121.
In vitro experiments have demonstrated that the drug rapidly accumulates intracellularly and reaches a steady-state concentration which exceeds the extracellular level by a factor of 450-800 [9,13]. In human plasma, DNR is rapidly reduced to daunorubicinol (DOL) and after an iv. infusion, its plasma level decreases rapidly [ 5,7].
The determination of drug concentrations in blood plasma during steady- state conditions plays an important role to optimize the clinical use of many drugs. However, for cancer chemotherapeutics which, like DNR, are admini- stered as short infusions with long intervals, plasma pharmacokinetic data may not give sufficient information for that purpose. Since DNR exerts its effect intracellularly, patients with leukaemia offer a unique opportunity to study the pharmacokinetics in a pharmacological compartment which can be regarded as a target for the cytostatic therapy, namely the circulating leukaemic cells. We have therefore simultaneously monitored DNR, DOL and their aglycones in leukaemic cells and plasma in patients which acute myeloblastic leukaemia. The urinary excretion of DNR and its metabolites was also studied.
The results of this study were presented in part at the 10th Meeting of the Nordic Haematological Societies, Oslo, May 18 -19, 1979 [lo].
METHODS AND MATERIALS
Collection of blood and urine Three previously untreated patients with acute myeloblastic leukaemia
and 20 -100,000 myeloblasts/mm3 in the peripheral blood were studied after i.v. infusion of daunorubicin in a dose of 1.5 mg/kg body wt.
Venous blood samples (5 ml) were repeatedly collected and immediately cooled on ice. The leukaemic cells (80-90% pure) were isolated as previously described [ 111 and then suspended in 1 ml of phosphate buffered (pH 7.4) saline. Leukaemic cells, plasma and urine samples were stored at -20°C until the day of analysis.
High performance liquid chromatography After thawing, the cell samples were sonicated for 20 s at 50 W with a
Branson B-12 sonicator (Branson, CT, USA). A O.l-ml aliquot of cell sample, plasma or urine was added to 0.1 ml of 0.1 M borate buffer (pH 9.8) con- taining doxorubicin (3.5 PM) as internal standard. The drugs were extracted with 1.8 ml of chloroform/methanol (4 : 1 by vol.). The chromatographic procedure was performed as described by Baurain et al. [ 2,3].
Cell protein was assayed according to the method of Lowry et al. [ 81 using bovine serum albumin as standard.
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RESULTS
Figures l- -3 show the results obtained with samples from one of the patients (S.L., female, 42 years old).
Drug concentrations in plasma Immediately after the end of the infusion, the DNR concentration in
plasma rapidly declined; this was followed by a slow elimination phase. The concentration of DOL exceeded that of the parent compound already 5 min after the end of the infusion; it then remained relatively constant for more than 20 h. The aglycone levels were very low at all times.
Drug concentrations in leukaemic cells The pattern of cellular drug concentration changes with time was quite
different from that of the plasma concentrations. Thus, the DNR level exceeded that of DOL up to 6 h after the end of the infusion and even after 22 h it was only slightly lower. The aglycone levels were very low at all times. Assuming that 1 mg of cell protein corresponds to a cell vol. of 5 ~1, it can be calculated that the concentration of DNR in the leukaemic cells was about 400 times higher than that in plasma immediately after the end of the infusion. Thereafter the ratio increased to between 900 and 1600 and eventually reached more than 4000 22 h after the infusion. Similarly, it can be calculated that the intracellular concentration of DOL exceeded the plasma level by a factor of 30 immediately after the infusion. The ratio then increased to 175 after 22 h.
TABLE 1
CONCENTRATIONS OF DNR AND DOL IN PLASMA AND LEUKAEMIC CELLS OF 2 PATIENTS AFTER AN iv. INFUSION OF DNR 1.5 mg/kg BODY WEIGHT. THE INFUSION TIME WAS 45 min IN PATIENT U.H. AND 4 h IN PATIENT B.C.
Fig. 1. Plasma concentrations of DNR (filled circles) and DOL (open circles) in patient S.L. after an i.v. infusion of DNR, 1.5 mg/kg body wt during 45 min.
CONCENTRATION bxnd/mg protelnl
5 lo 1s 20 25 TIME lhwrsl
Fig. 2. Concentrations of DNR (filled circles) and DOL (open circles) in leukaemic cells isolated from peripheral blood of patient S.L.
267 UJMUL4TIVE AMOUNT
I
20 40 60 80 100
TIME (hours) Fig. 3. Cumulative excretion of DNR (filled circles) and DOL (open circles) in urine of patient S.L.
Urinary drug excretion Significant amounts of DNR only appeared in the urine during the first
12 h after the end of the infusion, whereas excretion of DOL continued for more than 60 h. Taken together, the excretion of DNR and DOL only accounted for about 15% of the administered dose. The urinary concentra- tions of aglycones were very low.
Table 1 shows the concentrations of DNR and DOL in plasma and in leukaemic cells from 2 other patients from which fewer samples were obtained. The results were very similar to those presented in Figs. 1 and 2. In all plasma samples the concentration of DOL exceeded that of DNR. In contrast, the cellular DOL concentration did not exceed that of DNR until several hours after the end of the infusion. The ratio between the cellular concentration of DNR and the plasma level varied between 600 and 3800, whereas the corresponding ratio for DOL was between 110 and 280.
DISCUSSION
Our results from the plasma and urine determinations of DNR and its metabolites are in accordance with those of others [ 5,6,7,14]. However, our study also demonstrates that after an i.v. infusion, DNR rapidly enters the leukaemic cells and reaches much higher concentrations there than in plasma. There was no direct relation between the plasma level and the intracellular concentration of DNR; the ratio varied considerably at different times after the infusion.
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Our study also demonstrates that the pattern of metabolism differs con- siderably between the leukaemic cells and the plasma. The ratio between the cellular concentration of DOL and the plasma level was lower than for DNR. The slow appearance of cellular DOL indicates that the activity of DNR reductase in the leukaemic cells is low. It is also possible that some of the DOL in the leukaemic cells is formed outside the myeloblasts and enters by diffusion. Since DOL is less lipophilic than DNR [13], diffusion of the former can be expected to be much slower.
The significance of DOL formation for the cytotoxic effects during treat- ment with DNR is not yet clear. In vitro, extracellularly applied DOL has a lower inhibitory effect than DNR on DNA and RNA biosynthesis [ 11. How- ever, this can partly be explained by a lower cellular accumulation. There- fore intracellularly formed DOL might have a greater cytotoxic effect than indicated by such in vitro experiments.
Only about 15% of the administered DNR was recovered as DNR and DOL in the urine within 96 h. This indicates that biliary excretion plays an important role for the elimination of the drug.
Although this report is based on a small material it demonstrates that the plasma concentration of DNR and its metabolites provides little information on the drug concentration in the leukaemic cells. Direct determinations of drug concentrations in the leukaemic cells is probably of greater clinical value for optimization of the therapy in leukaemia. Such studies are in progress.
ACKNOWLEDGEMENTS
This study was supported by grants from the Swedish Cancer Society (1015-1378-02x), Ake Wibergs Foundation and AB Leo’s Research Founda- tion. Gifts of DNR hydrochloride and DOL hydrochloride by Rhone Poulenc, S.A. Paris, France and of doxorubicin hydrochloride by Farmitalia, Milan, Italy are gratefully acknowledged.
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