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 50C 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

emISSIon wavelength of 410 nm. 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].
Pharmacokinetic analyses were performed using the EST RIP computer program [16].
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. Therefore modifications were required to yield 66.5 % 1.8 %
fluorescing product in the optimized system
using [8-3H]F-ara-A. Spectroscopic analysis of the fluorescent derivative confirmed
its identity with arabinosyl-1,N6-ethenoisoguanine. The reaction was shown to be
specific for F-ara-A and its respective
nucleotides, which could be derivatized
with a comparable yield. Among the physiological nucleosides of adenine and cytosine which are known to form fluorescent
etheno-derivatives [22], only adenosine and
deoxyadenosine were detected at considerably lower relative sensitivities.
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, derivatization, and separation of the
reaction products by HPLC.
Table 1. 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 - 2 nmoVml
plasma
3.0 % relative SD
0.5 % decay/h
100 % (range 0.05-2
nmoVml)
<0.03%

10

::E

<1
IC

t-

IC

1
1.1..

-------

.1

-----0

.01

20

40

hours after treatment

60

Fig. 1. Pharmacokinetics of F-ara-A in plasma

after a 30-min i.v. infusion (closed circles, one
patient) or oral application (open circles, one
patient) of 60 mg fludarabine phosphate. Plasma
(0.5-4 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.v. infusion or
as an aqueous solution orally. The pharmacokinetics of F-ara-A in plasma up to 72 h
after treatment are shown in Fig. 1. F-ara-A
was detected in plasma at 2 min following
oral administration. After a 1.5- to 2-h
accumulation phase, F-ara-A concentrations were comparable to those obtained by
i.v. infusion. The triexponential elimination
phases essentially paralleled after both
routes of administration with similar terminal half-lives (31 and 32 h, n = 5 for each
treatment group). F-ara-A plasma levels
measured at 72 h were about 20-40 times
above the quantitation limit of the fluorescence assay. Comparing the mean AVC
values of five patients per treatment group,
an oral bioavailability of about 80 % was
determined.
Levels of F-ara-ATP in circulating leukemia cells (Fig. 2) showed similar accumulation and elimination kinetics after both
routes of administration [tIll = 36 h (i.v.) and
32 h (oral)]. After oral ingestion of the
drug, however, maximum F-ara-ATP con-

100

h were found after doses ranging from 18 to

80 mg/m2 per day [11,13, 14].

::E

""

a:t-

<
I

10

'"''"

I
lL.

20
40
60
hours after treatment

Fig. 2. Pharmacokinetics of F-ara-ATP in circulating leukemia cells after a 30-min i.v. infusion
(closed circles, one patient) or oral application
(open circle, one patient) of 60 mg fludarabine
phosphate. 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 %. The
cellular "bioavailability" measured as nucleoside triphosphate was about 40 % less
after oral administration.
The derivatization reaction to a fluorescent compound may also be applied to the
F-ara-A mono- and triphosphates and to
arabinosyl-isoguanine, thereby providing
several potential applications.

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2.
centrations were generally lower, resulting
in about 40 % lower area under curve
(AUe) values.

3.

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. With a limit of quantitation of 2
pmol F-ara-A/ml plasma, 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
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