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Metabolites and Sensitive Method To Determine FLR Kemena1991 PDF
Metabolites and Sensitive Method To Determine FLR Kemena1991 PDF
95
CCA 05023
Summary
Rapid and quantitative dephosphorylation
of the new anticancer nucleotide
analogue fludarabine phosphate to its nucleoside 9-/3-D-arabinofuranosyl-2-fluoroadenine (F-ara-A) renders this metabolite the target for pharmacologic investigations. At clinically effective doses of fludarabine phosphate (18-30 mg/m per
day) comprehensive pharmacokinetic analysis of F-ara-A has been limited by the
sensitivity of UV based HPLC assays. To address this problem we developed a
sensitive test based on the condensation of F-ara-A with chloroacetaldehyde
to
form the fluorescent derivative, arabinosyl-l,N6-etheno-isoguanine.
Combined with
a solid-phase extraction step prior to derivatization and separation of the reaction
products by reverse-phase
HPLC, this assay had a quantitation limit of 2 pmol
F-ara-A per ml plasma. Slightly modified, the system was also applicable to urine
specimens, with a quantitation limit of 1 nmol F-ara-A per ml urine.
Introduction
The nucleotide analogue 9-/3-D-arabinofuranosyl-2-fluoroadenine
5-monophosphate (fludarabine phosphate, F-ara-AMP, Fludara), appears to be effective in the
treatment of low grade lymphocytic malignancies at doses of 18-30 mg/m per
day for 5 days [l-5]. Nevertheless, clinical studies in acute leukemias which
Correspondence and requests for reprints to: William Plunkett, Department of Medical Oncology, Box
52, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA.
employed relatively high doses either by daily i.v.-infusions (196 mg/m* per day
for 5-7 days) or continuous infusions (> 40 mg/m* per day for 5 days) were
associated with severe neurotoxicity [6-81. This therapeutic window suggested that
pharmacokinetic
analyses may provide critical information concerning dose
scheduling and the rationale for the design of combination therapies [9,10].
Rapid and quantitative dephosphorylation
of fludarabine phosphate to its
nucleoside 9-P-D-arabinofuranosyl-2-fluoroadenine
(F-ara-A) [ill renders this
metabolite the target for pharmacologic investigations. Conventional methods of
quantitating F-ara-A in plasma consist of a deproteinization procedure prior to
HPLC separation and detection by UV absorbance [ll-141. The detection limits
reported were 0.05 [13] and 0.53 [ll] nmol F-ara-A/ml plasma, or approximately
15 pmol, given as an absolute value [12,14]. The similarity of the assay systems used
suggests that the sensitivities of all four are essentially comparable. After low-dose
i.v.-infusions of fludarabine phosphate (18-25 mg/m* over 30 min) the range of
the detection limit was reached in one case at 24 h [14]; in another F-ara-A was no
longer detectable at 3 h after the end of the infusion [13]. At a dose of 80 mg/m*
given as an i.v.-bolus the detection limit was approached 30 h after treatment [ll].
To achieve increased sensitivity, we combined a solid-phase extraction for
deproteinization
and concentration of the drug metabolite with a fluorescence
detection method after HPLC separation. The final validation of the method gave
a quantitation limit of 2 pmol F-ara-A per ml plasma.
Materials and methods
Materials
of the fluorescent
F-ara-A
97
and heated at 55C overnight. The mixture was concentrated under high vacuum to
obtain a glassy solid. This was triturated in 10 ml of acetonitrile and concentrated
under vacuum. The residue was resuspended in acetonitrile and allowed to stand
at room temperature overnight. The fine precipitate that formed was collected by
filtration and suspended in 3 ml of water before neutralization with saturated
aqueous sodium bicarbonate. After filtration the aqueous solution was concentrated to about 1 ml and refiltered prior to preparative HPLC. The sample was
divided in half, each portion was loaded onto a 10 X 250 mm Alltech Econosil C,,
column and eluted with 5:95 methanol: water at a flow rate of 5 ml/min and
monitored at 254 nm. Appropriate fractions were pooled, concentrated under
vacuum, and the resulting glassy residue was dissolved in 0.5 ml methanol and
diluted with 2 ml acetonitrile. The solvents were evaporated under vacuum to
obtain 40 mg of the fluorescent F-ara-A derivative as a dry but hygroscopic
powder. This material was approximately 95% pure by HPLC and was shown to be
identical to material prepared in the analytical scale experiments by HPLC
coelution and fluorescence characteristics. H-NMR (DMSO-d,, 500 MHz) 6 3.62
(dd, J = 5.0 and 11.6 Hz, H-5a), 3.69 (dd, J= 4.0 and 11.6 Hz, H-5b), 3.76 (q,
J = 4.4 Hz, H-4), 4.11 (t, J = 4.4 Hz, H-2 or H-3), 4.16 (t, J = 4.2 Hz, H-3 or
H-2), 6.16 (d, J= 4.8 Hz, H-l), 7.04 (bs, H-10 or H-111, 7.43 (bs, H-11 or H-101,
8.67 (s, H-8) (purine nucleoside numbering system, OH signals not listed); 13C-NMR
(DMSO-d,, 125 MHz) 61.3, 75.7, 76.0, 83.6, 88.3, 110.2, 112.2, 127.9, 134.1, 143.2,
144.3, 151.1 ppm; CI+-mass spectrometry (m/z) 308 (M + H)+; UV (methanol)
Amax 292 nm.
Plasma preparation and deriuatization
Blood samples (5-10 ml>, obtained through a peripheral catheter, were immediately transferred into a heparinized glass tube containing 100 ,ul 0.1 mmol/l
erythro-9-(2-hydroxy-3-nonyljadenine
to inhibit adenosine deaminase, and placed
in an ice-bath. After centrifugation for 20 min at 500 X g and 4C the supernatant
was removed and stored at -20C. Standard samples were prepared by spiking 5
ml of thawed fresh frozen plasma with a known amount of F-ara-A.
Protein removal was accomplished by solid-phase extraction. A SEP-PAR C,,
cartridge was preconditioned
with 2 ml alkaline methanol (1 ,& concentrated
ammonium hydroxide per 1 ml methanol) and subsequently rinsed with 10 ml
water. The thawed plasma was centrifuged to remove coagulates, and 1 to 5 ml was
passed through the cartridge, which was then rinsed with 5 ml water, forcing out
most of the liquid phase. The F-ara-A was eluted with 2 ml alkaline methanol. All
steps were carried out at a flow rate of about 5 ml/min using a Vat Elute SPS 24
(Analytichem International Inc.). The methanol was evaporated completely in a
Speed Vat Concentrator (Savant), and the sample was stored at - 20C. To the dry
sample 0.1 ml 0.6 mol/l citrate buffer, pH 4.0, was added and vortex-mixed to
dissolve. After addition of 0.2 ml 50% chloroacetaldehyde (final concentration 5.2
mol/l>, the sample was incubated for 24 h in a 50C water bath.
98
Urine fractions of 300-1,000 ml were collected at 8-h intervals in bags containing 100 ~1 1 mmol/l erythro-9-(2-hydroxy-3-nonyl)adenine.
Aliquots of about 2 ml
each were stored at -20C. For derivatization, thawed urine was centrifuged to
remove sediment and diluted 1: 10 with water; 0.1 ml was added to an equal
volume of 1 mol/l citrate buffer, pH 4.0 and 0.3 ml 50% chloroacetaldehyde was
added (final concentration 4.7 mol/l>. The sample was incubated for 24 h in a
50C water bath. Standard samples were prepared by spiking urine with a known
amount of F-ara-A.
Separation and detection
After derivatization, the samples were diluted 1: 10 with 0.1 mol/l citrate
buffer, pH 4.0, and 50 ~1 was injected into a Waters HPLC system consisting of 2
model 6000A pumps, a model 715 Ultra WISP sample processor, a model 470
fluorescence detector, a system interface module, and an NEC data capture system
equipped with Maxima 820 Chromatography
Software. As stationary phase a
PBondapak C,, column (Waters Associates, 3.9 mm X 30 cm, 10 km particle size)
connected with a precolumn filter cartridge (Waters Associates) was used. The
mobile phase consisted of a ternary solution of 2% methanol and 5% N,N-dimethylformamide in water. Under isocratic conditions at a flow rate of 1 ml/min the
fluorescent derivative of F-ara-A eluted with a retention time of about 10 min. An
excitation wavelength of 296 nm and an emission wavelength of 410 nm were
optimal for detection. Fluorescence signals of samples corresponding to more than
42 pmol or 12 ng of underivatized F-ara-A per injection were truncated by the
detector at the highest sensitivity setting, necessitating further dilution of these
samples. After 20-25 runs the column was regenerated with a gradient from the
ternary mobile phase to 50% methanol in water to maintain optimal separation.
Analysis of data
99
HO
HO
4
3
Fig. 1. Formation and structure of the fluorescent F-ara-A derivative.
100
TABLE I
Derivatization to fluorescent compounds *
compound
relative sensitivity
F-ara-A
F-ara-AMP
F-ara-ATP
F-Ade
Ado
dAdo
Ade
Cyd
dCyd
arabinosylisoguanine
9.8
4.0
3.1
19.7
18.3
24.3 and 14.3
14.4
no signal up to 20 PM
no signal up to 20 PM
1.oo
1.00
0.98
0.17
0.26
0.05 and 0.12
0.15
9.8
1.36
to the respective
101
2-deo~urine
nucleosides under acidic conditions. in addition, the excitation and
emission wavelengths (296 nm and 410 nm, respectively) have been optimized for
detection of the F-ara-A derivative and are clearly less than optimal for detection
of either the adenine or cytosine derivatives.
The relative sensitivity of this assay for F-ara-A and for its nucleotides was very
similar. The sensitivity for F-Ade, however, was considerably lower, perhaps due to
the fo~ation of multiple ChIoroacetaIdehyde derivatives which were not detected
by fluorescence.
Since both F-ara-A and arabinosyl-isoguanine form the same fluorescent derivative, these cannot be independently quantitated by this assay. The higher sensitivity
for arabinosyl-isoguanine
indicates that it is derivatized more efficiently than
F-ara-A. This is consistent with the 66.5% conversion rate observed for F-ara-A. It
is also consistent with the expectation that hydrolytic cleavage of F-ara-A to
arabinosyl-isoguanine is the iimiting step in the derivatization of F-ara-A.
Validation of the assay in biological samples
ooi
0
F-ara-A. nH
10
10
15
20
minutes
Fig. 2. Concentration-to-signal
relationship of the derivatization of F-ara-A in plasma in the low
concentration range, including the quantitation limit. Each data point represents the mean of triplicate
determinations&SD.
Standard deviations ranged from 8% tat 2 nmol/l) to 2% (at 10 nmol/l), and
therefore some SD values do not appear. 5 ml plasma was processed for each determination as
described in Methods.
Fig. 3. HPLC chromatograms of patient plasma samples obtained prior to treatment (lower panel), and
6 h after the end of a 30-min i.v.-infusion of 60 mg fludarabine phosphate (upper panel). Plasma (1 ml)
was processed in each case as described in Methods. The fluorescence signal at 9.8 min was equivalent
to an F-ara-A plasma concentration of 0.34 pmol/l.
102
103
.OlL
0
I
20
40
60
24
40
72
Fig. 5. Excretion of F-ara-A in urine after a 30-min i.v.-infusion of 60 mg fludarabine phosphate. The
data represent mean values f SD of three patients; 10 ~1 of urine from each 8-h collection period was
processed as described in Methods.
for the terminal phase. The F-ara-A plasma level was 62 & 19 nmol/l 72 h after
the end of the infusion. A total body clearance of 15.0 k 9.7 l/h was calculated
from these data. Quantitation of F-ara-A in urine collected in 8-h intervals for 24 h
after fludarabine phosphate infusion indicated that 37 f 5% of the dose had been
excreted during that time (Fig. 5). When values from the interval 64-72 h after
infusion were included, excretion of 57 f 7% of the dose was estimated with a
renal clearance of 5.4 k 1.6 I/h.
Discussion
The determination of F-ara-A in plasma at fludarabine phosphate doses in the
range of X3-30 mg/m2 per day has been limited by the sensitivity of UV based
HPLC assays [12-141. To study the F-ara-A pharmacokinetics more precisely at
these low doses, we developed a more sensitive assay based on fluorescence
detection after HPLC. The condensation of adenine nucleosides with chloroacetaldehyde produces fluorescent 1,N6-etheno-derivatives
[16-18,21-261, and assays employing the detection of these compounds by fluorescence have shown a loto 30-fold greater sensitivity than the corresponding UV based methods [l&19].
Treatment of F-ara-A with chloroacetaldehyde under conditions suitable for the
derivatization of other adenine nucleosides [16-201 produced no fluorescent
derivative. The unique structural characteristic of this adenine nucleoside analogue
104
is the fluorine attached to the 2-carbon of the adenine moiety (Fig. 1, (1)) which
renders it a poor target for degradation by adenosine deaminase [29,30]. Substitution of adenine nucleosides with a halogen atom at C-2 also considerably reduces
their reactivity with chloroacetaldehyde
[23]. It was therefore necessary to modify
the derivatization conditions. We found that by decreasing the pH and temperature of the reaction while increasing the reaction time and concentration of
chloroacetaldehyde,
a fluorescent derivative of F-ara-A could be produced in
66.5% yield. The use of bromoacetaldehyde,
which has been reported to provide
superior results in some cases 1251, led to the generation of two fluorescent
derivatives from F-ara-A and was therefore not suitable.
Purification and structural characterization revealed that the fluorine had been
replaced by a hydroxy group during derivatization, so that the fluorescent derivative was actually arabi~osyl-l,~~-etheno-isoguanine
(Fig. 1, (4)) rather than
etheno-F-ara-A (2). The identity of the compound was supported by conversion of
authentic arabinosyl-isoguanine to its etheno-derivative, which coeluted by HPLC
with the F-ara-A reaction product. Thus, the derivatization reaction to a fluorescent product would also be of value for investigations of arabinosyl-isoguanine 2311
and of the respective monophosphate.
The optimized assay for the quantitation of F-ara-A in plasma was sensitive
enough to monitor the pharmacokinetics of the drug metabolite over a 72-h period
after the end of a 30-min i.v.-infusion of 60 mg fludarabine phosphate (Fig. 4).
Minimum plasma levels during this time were still 20 to 40 times above the limit of
quantitation of the assay. The triphasic F-ara-A elimination profile with a terminal
half-life of 33.5 + 5 h was in contrast to a previous report, in which the less
sensitive UV detection method was employed and a biphasic elimination profile
with a mean terminal half-life of 9.2 h was found under comparable treatment
conditions (e.g. 18-25 mg/m infusion over 30 min) [14]. At 50 mg/m2 fludarabine phosphate infused over 30 min, similar pharmacokinetic parameters were
reported, but the terminal half-lives varied from 7.8 h to over 24 h [13]. After an
i.v.-bolus of 80 mg/m, plasma elimination was triphasic with a terminal half-life
of 10.9 + 2.9 h ill]. Additional studies utilizing this fluorescence assay should
clarify the long-term elimination kinetics of F-ara-A.
The reaction conditions which provide the maximum yield of the fluorescent
F-ara-A derivative may also be applied to the mono- and triphosphates and F-Ade
(Table I). There are several potential applications of the derivatization reaction for
these compounds. First, it may permit the quantitation of very low levels of
F-ara-AMP in human plasma (or tissue), because the nucleotide is very rapidly
dephosphorylated
and is not measurable by UV detection only minutes after the
end of i.v.-infusions [11,13,14]. Second, it may be possible to detect F-adenine, a
potential metabolite of F-ara-A, which is highly toxic [32,33], and has been found
in animal tissue and urine after treatment with 40-400 mg/m* [3H]F-ara-A
[12,34]. Third, quantitation of very small intracellular F-ara-ATP concentrations
might be achieved by this method. Finally, it is likely that this procedure has
adequate sensitivity to facilitate quantitation of F-ara-AMP incorporated into
DNA. The feasibility of all these applications is being pursued.
105
Acknowledgements
The authors thank Dr. Abdallah Cherif for his help in the synthesis of bromoacetaldehyde and for valuable discussions concerning the chemical aspects of this
work. We are grateful to Richard Willson and Jean Nowlin of Shell Westhollow
Research Center, Houston TX, for their assistance in obtaining the NMR and
mass spectrometry data of the fluorescent F-ara-A derivative. We would also like
to express our gratitude to Walter J. Page1 for excellent editorial assistance in the
preparation of the manuscript. This work was supported in part by grant CA32839
from the National Cancer Institute, DHHS. A. Kemena received a grant from the
German Research Association (DFG).
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