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Plasma Pharmacology of Fludarabine and Cellular Bioavailability

of Fludarabine Triphosphate*
A. Kemena, M. Keating, and W. Plunkett

Introduction

Fludarabine
phosphate
(F-ara-AMP)
shows excellent response rates at low doses
(18-30 mg/m2 per day) in indolent lymphoid malignancies without major nonhematologic toxicity [1-5]. In high-dose
regimens, however, it caused severe neurotoxic side effects [6-8]. This comparatively
narrow therapeutic window requires precise pharmacokinetic studies for optimal
dose scheduling. Further, as chronic lymphocytic leukemia (CLL) is the most frequent in this group of malignancies, elderly
patients are an important target popUlation
for treatment with fludarabine phosphate
and might especially benefit from an oral
formulation. Therefore studies of its bioavailability are needed. Finally, fludarabine
phosphate is currently investigated as a
biochemical modulator of cytosine arabinoside [9, 10]. As these nucleoside analogues
exert close schedule dependent interaction,
a careful pharmacokinetic analysis is essential. At low fludarabine phosphate doses
comprehensive pharmacokinetic studies
were limited by the sensitivity of assays
based on UV detection [11-14]. We therefore developed a sensitive method based on
the chemical condensation of F-ara-A with
chloroacetaldehyde to a fluorescent deriva-

* This study is supported by a grant from the
German Research Association (DFG) and by
grant CA 32839 from the National Cancer Institute DHHS
W. Hiddemann et al. (eds.), Acute Leukemias
© Springer-Verlag Berlin Heidelberg 1992

tive. Combined with a solid-phase extraction prior to derivatization, the quantitation limit was 2 pmol/ml plasma. In a
clinical study designed to evaluate the oral
bioavailability of fludarabine phosphate, we
determined plasma F-ara-A levels over a
period of 72 h in patients with relapsed
CLL. Simultaneously the corresponding
concentrations of the active metabolite fludarabine triphosphate (F-ara-ATP) were
measured in circulating leukemic cells.
Materials and Methods

The chemical structure analysis of the fluorescent derivative of F-ara-A was carried
out by Berlex Biosciences, Alameda, CA
(United States).
Plasma samples were prepared from
heparinized blood which contained 1 !J,M
erythro-9-(2-hydroxy-3-nonyl)adenine to
inhibit adenosine deaminase. Protein removal and sample concentration was
accomplished by solid-phase extraction
(SEP-PAK CIS cartridge). The derivatization of F-ara-A was carried out in citrate
buffer, pH 4.0 (final concentration 0.2 M),
in the presence of 5.2 M chloroacetaldehyde. After incubation for 24 h at 50°C the
reaction products were separated by reverse-phase high-performance liquid chromatography (HPLC) (!J,Bondapak CIS)
under isocratic conditions. The mobile
phase consisted of 2 % methanol and 5 %
N,N-dimethylformamide in water. The
fluorescent F-ara-A derivative (retention
time about 10 min) was detected at an
excitation wavelength of 296 nm and an
355

Spectroscopic analysis of the fluorescent derivative confirmed its identity with arabinosyl-1. Comparing the mean AVC values of five patients per treatment group. 2) showed similar accumulation and elimination kinetics after both routes of administration [tIll = 36 h (i.v. The triexponential elimination phases essentially paralleled after both routes of administration with similar terminal half-lives (31 and 32 h. ------- . F-ara-A plasma levels measured at 72 h were about 20-40 times above the quantitation limit of the fluorescence assay.1. infusion. an oral bioavailability of about 80 % was determined. Validation of the fluorescence assay for F-ara-A quantitation in plasma Limit of quantitation Linearity Precision Stability Recovery Protein binding 356 2 pmoVml plasma 2 pmol . 1. Table 1.emISSIon wavelength of 410 nm.8 % fluorescing product in the optimized system using [8-3H]F-ara-A. Plasma (0. however. maximum F-ara-ATP con- . Pharmacokinetic analyses were performed using the EST RIP computer program [16].05-2 nmoVml) <0.1 -----0 .2 nmoVml plasma 3.5-4 ml) was processed at each time point according to the procedure described in "Methods" Quantitation of F-ara-A and Its Triphosphate in Patient Samples Patients with refractory chronic lymphocytic leukemia received 60 mg fludarabine phosphate either as a 30-min i. Levels of F-ara-ATP in circulating leukemia cells (Fig. only adenosine and deoxyadenosine were detected at considerably lower relative sensitivities.to 2-h accumulation phase.5. The reaction was shown to be specific for F-ara-A and its respective nucleotides.01 0 20 40 hours after treatment 60 Fig.v. Pharmacokinetics of F-ara-A in plasma after a 30-min i. one patient) of 60 mg fludarabine phosphate.N6-ethenoisoguanine. F-ara-A was detected in plasma at 2 min following oral administration. Therefore modifications were required to yield 66. which could be derivatized with a comparable yield.v.) and 32 h (oral)]. Results Optimization and Validation of the Test System Published reaction conditions for the synthesis of etheno-adenine compounds [17 -21] did not generate a fluorescent signal with the relatively inert fluorinated arabinosyladenine. and separation of the reaction products by HPLC. n = 5 for each treatment group). Table 1 shows the results of the validation of the fluorescence assay for F-ara-A quantitation in plasma.. Processing (5 ml plasma per sample) consisted of solid-phase extraction. infusion or as an aqueous solution orally. one patient) or oral application (open circles. derivatization. infusion (closed circles. 1.5 % decay/h 100 % (range 0.03% 10 ::E ~ <1 IC t- IC 1 1. After a 1.v.0 % relative SD 0. After oral ingestion of the drug. The methodology is described in detail elsewhere [15]. Intracellular nucleotide concentrations were determined by anion exchange HPLC in perchloric acid extracts of blood mononuclear cells [9]. The pharmacokinetics of F-ara-A in plasma up to 72 h after treatment are shown in Fig.5 % ± 1. F-ara-A concentrations were comparable to those obtained by i. Among the physiological nucleosides of adenine and cytosine which are known to form fluorescent etheno-derivatives [22].

Dahlberg S. 2. thereby providing several potential applications. o 20 40 60 hours after treatment Fig. Alexanian R. Kraut EH. Eyre HJ (1990) Activity of fludarabine monophosphate in patients with advanced mycosis fungoides: a southwest Oncology Group study. Discussion Conventional UV-based methods for the quantitation of F-ara-A reach their limit of detection at 3-24 h after treatment with low-dose regimes of fludarabine phosphate [11-14]. We therefore developed an assay that employs fluorescence detection after HPLC. Grever MR (1987) Phase II trial of 9-~-D-arabinofuranosyl-2-fluoroadenine 5' -monophosphate in non-Hodgkin's lymphoma: prospective comparison of response with deoxycytidine kinase activity. Our preliminary pharmacokinetic data from a study designed to evaluate the oral bioavailability of fludarabine phosphate suggest a triexponential elimination profile of F-ara-A with a terminal half-life of about 32 h. centrations were generally lower. Schoene W. Blood 74: 19-25 Leiby JM. Pharmacokinetics of F-ara-ATP in circulating leukemia cells after a 30-min i. Talpaz M et al. Circulating mononuclear cells were processed at each time point according to the procedure described in "Methods" The oral bioavailability of the drug in plasma seems to be around 80 %. infusion or orally.and triphosphates and to arabinosyl-isoguanine. one patient) of 60 mg fludarabine phosphate. With a limit of quantitation of 2 pmol F-ara-A/ml plasma. 6.13. Cancer Res 47: 2719-2722 Von Hoff DD. Leyland-Jones BR. The cellular "bioavailability" measured as nucleoside triphosphate was about 40 % less after oral administration. 14]. 7. 9. Keating MJ (1990) Fludarabine therapy in macroglobulinemic lymphoma. J Nat! Cancer Inst 82: 1353-1355 Kantarjian HM. Coltman CA et al. Kufe DW (1986) Fludarabine phosphate (NSC 312878) infusions for the treatment of acute leukemia: phase I and neuropathological study. Kopecky KJ. 2. Caryk SM. resulting in about 40 % lower area under curve (AUe) values. 5. Kurzrock R. it enables elimination kinetics to be monitored over a 72-h period after treatment with a fludarabine phosphate dose of 60 mg/m2 per day given either by short-term i.v. Snider KM. biphasic or triphasic elimination kinetics with a terminal half-life of about 10 4. Hartstock RJ. one patient) or oral application (open circle. in which the less sensitive UV detection method was employed. (1989) Fludarabine: a new agent with major activity against chronic lymphocytic leukemia. Mayer RJ. Koller CA. Hoth DF (1986) Central nervous system toxicity of fludarabine phosphate. Cancer Treat Rep 70: 1225-1228 Spriggs DR. (1988) Fludarabine monophosphate: a potentially useful agent in chronic lymphocytic leukemia. Nouv Rev Fr Hematol 30: 457-459 Keating MJ. Blood 75: 1928-1931 Chun HG. Malspeis L. The derivatization reaction to a fluorescent compound may also be applied to the F-ara-A mono.v. Grever MR. Measuring accumulation kinetics during absorption from the gastrointestinal tract. Metz EN. 8. Kantarjian H. Cancer Res 46: 5953-5958 Warrell RP Jr. infusion (closed circles. ::E "" a:t- < I 10 '"''" I lL. J Clin Oncol 4: 74-79 Gandhi V. Plunkett W (1988) Modulation of arabinosylnucleoside metabolism by arabi- 357 . Berman E (1986) Phase I and II study of fludarabine phosphate in leukemia: therapeutic efficacy with delayed central nervous system toxicity. In previous reports. Stopa E. References 1.100 h were found after doses ranging from 18 to 80 mg/m2 per day [11. 3. F-ara-A was detected 2 min following drug intake.

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