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Vol. 28, Issue 10, 1141-1145, October 2000
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results and Discussion
References
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The clinical pharmacokinetic behavior of paclitaxel (Taxol) is distinctly nonlinear, with disproportional increases in systemic exposure with an increase in dose. We have recently shown that Cremophor EL, the formulation vehicle used for i.v. administration of paclitaxel, alters drug distribution as a result of micellar entrapment of paclitaxel, and we speculated that the free drug fraction (fu) is dependent on dose and time-varying concentrations of Cremophor EL in the central plasma compartment. To test this hypothesis, a reproducible equilibrium dialysis method has been developed for the measurement of paclitaxel fu in plasma. Equilibrium dialysis was performed at 37°C in a humidified atmosphere of 5% CO2 using 2.0-ml polypropylene test tubes. Experiments were carried out with 260-µl aliquots of plasma containing a tracer amount of [G-3H]paclitaxel with high-specific activity against an equal volume of 0.01 M phosphate buffer (pH 7.4). Drug concentrations were measured by both reversed-phase HPLC and liquid scintillation counting. Using this method, fu has been measured in three patients receiving three consecutive 3-weekly courses of paclitaxel at dose levels of 135, 175, and 225 mg/m2 and found to range between 0.036 and 0.079. The method was also used to define concentration-time profiles of unbound drug, estimated from the product of the total plasma concentration and fu.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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Paclitaxel (Taxol; Fig. 1)
is a naturally occurring
taxane diterpenoid first extracted from the bark of the Western yew
tree, Taxus brevifolia (Wani et al., 1971). The compound is
a potent inhibitor of cell replication in malignant cells, a property
attributed to its ability to stabilize the microtubule cytoskeleton and
to block the transit of cycling cells from the
G2-phase to the M-phase (Verweij et al., 1994
;
Sparreboom et al., 1998c). The clinical pharmacokinetics of
paclitaxel (administered as a 3-h i.v. infusion) have been well
documented, and revealed a striking nonlinear disposition profile in
plasma with disproportional increases in systemic exposure resulting
from a given increase in dose (Schiller et al., 1994; Sonnichsen et
al., 1994; Gianni et al., 1995; Bhalla et al., 1999; Karlsson et al.,
1999).
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It has recently been shown that Cremophor EL
(CrEL1), the vehicle used for i.v. drug
administration, profoundly influences the cellular distribution of
paclitaxel in human blood (Sparreboom et al., 1999a). Cellular kinetic
experiments indicated that erythrocyte uptake of paclitaxel was
significantly reduced by polyoxyethyleneglycerol triricinoleate
(Fig. 1), the major compound present in CrEL, as a result of binding to
CrEL micelles which, in turn, can reduce the free drug fraction (fu)
available for cellular partitioning (Sparreboom et al., 1999a).
Furthermore, we found that the altered blood distribution is highly
dependent on dose and time-varying concentrations of CrEL in the
central blood compartment during paclitaxel administration (Sparreboom
et al., 1999b). This latter aspect of a concentration-dependent binding
of paclitaxel to CrEL micelles occurring after therapeutic doses
suggests that the total plasma concentration
(Cp), which is routinely measured, is not reflective of the unbound drug concentration (Cu). The rationale for
monitoring Cu is founded on the basic pharmacologic tenet that drug
bound to protein, or other (macro)molecules, is unable to cross cell
membranes and interact with the active site. Although this relationship
is intuitive, little has been published on this topic regarding
anticancer drugs, with the notable exception of etoposide (Stewart et
al., 1991). Knowledge of the extent of binding of paclitaxel is of
crucial importance for understanding the clinical pharmacologic
behavior of this drug, and could have significant clinical relevance in
view of the fact that relationships between drug exposure and effect
(i.e., toxicity and efficacy) are still poorly defined. To address
these issues, we set out to define a reliable equilibrium dialysis
method and demonstrate its application to binding measurements of
paclitaxel in plasma samples of cancer patients receiving multiple
courses of the drug administered by a 3-h i.v. infusion.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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Chemicals. Paclitaxel powder (batch 484034; purity 98.3% by reversed phase HPLC) was obtained from Bristol-Myers Squibb (Woerden, The Netherlands). [G-3H]Paclitaxel (batch 227-163-0024; radiochemical purity 99.7%), with a specific activity of 2.4 Ci/mmol was obtained from Moravek (Brea, CA). The majority of the tritium label is in the m- and p-positions of the aromatic rings, with minor amounts in the 10-, 3'-, and 2-positions of the taxane ring system. Ethanol absolute was purchased from Merck (Darmstadt, Germany), and phosphate-buffered saline from Oxoid (Unipath LTD, Basingstoke, Hampshire, UK). The CrEL reference material was obtained from Sigma Chemicals Co. (St. Louis, MO). Emulsifier-safe scintillation cocktail was purchased from Packard Instruments Co. (Groningen, The Netherlands).
Equilibrium Dialysis.
Paclitaxel fu was measured by equilibrium dialysis at 37°C in a
humidified atmosphere of 5% CO2 using test vials
made from 2.0-ml polypropylene Safe-Lock vials (Eppendorf, Hamburg,
Germany), carrying a 260-µl inside recess in the lids (Reinard and
Jacobsen, 1989). Before incubation, 2 µl of a
[G-3H]paclitaxel solution
(500-fold diluted in ethanol) was added to 300 µl of plasma, followed
by mixing for 10 s. Dialysis was carried out with 260 µl of this
sample against an equal volume of phosphate-buffered saline (pH
7.4) for 24 h in a moist chamber, which was shown previously to be
sufficiently long to attain equilibrium (Kumar et al., 1993; Sparreboom
et al., 1999a). Spectra/Por 3 dialysis tubing with a 12,500 molecular
weight cut-off (Spectrum Medical, Kitchener, Canada) was soaked in
phosphate-buffered saline (pH 7.4) before use. After establishment of
the equilibrium, 150 µl of the buffer solution, containing only
unbound paclitaxel, and 150 µl of the plasma fraction, containing
both bound and unbound drug, were transferred to separate 2-ml vials
(Eppendorf) and 1.9 ml of Emulsifier-Safe scintillation cocktail were
added. After manual mixing for 30 s, the
3H-labeled paclitaxel was quantified by liquid
scintillation counting using a Wallac 1409 liquid scintillation counter
(Turku, Finland). All samples were counted until a preset time of 20 min was reached. The ratio of drug concentrations measured in the
buffer and plasma after dialysis was taken as an estimate of paclitaxel
fu. Because the volume shift during dialysis was negligible (<10%),
the results were used directly without applying a correction factor.
Pharmacokinetic Studies.
The three patients studied were a 64-year-old female with non-small
cell lung cancer, a 62-year-old male with bladder cancer, and a
51-year-old female with a malignant solid tumor of unknown primary
origin for whom paclitaxel as monotherapy was a viable therapeutic
option (Table 1). Before treatment, the
patients had a World Health Organization performance status of <2, did not receive previous anticancer treatment with taxanes, and had adequate bone marrow function (white blood cell count, >3.0 × 109/liter; platelet count, >100 × 109/liter), renal function (serum creatinine,
140 µM and creatinine clearance >60 ml/min), and hepatic function
(serum bilirubin, alkaline phosphatase, aspartate aminotransferase, and
alanine aminotransferase levels within normal limits).
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20°C at the patient site, and the remaining sample was centrifuged for 5 min at
4000g. Plasma was transferred into a polystyrene container and snap-frozen at 20°C. All samples were stored at 80°C until analysis. The concentration of paclitaxel in total plasma
(Cp) and whole blood (Cb)
was determined by isocratic reversed-phase HPLC with UV detection at
= 230 nm, as described (Sparreboom et al., 1998a). The unbound
paclitaxel concentration (Cu) was estimated from the product of
Cp and fu in each individual pharmacokinetic sample. The analytical procedure for CrEL in plasma samples was based
on a colorimetric dye-binding microassay using Coomassie Brilliant Blue
G-250 (Sparreboom et al., 1998b), with modifications as described
(Brouwer et al., 1998).
Paclitaxel concentration-time profiles of Cu, Cp,
and Cb were analyzed using the Siphar software
package (version 4.0; SIMED, Créteil, France), by determination
of slopes and intercepts of the plotted curves with multiexponential
functions. The program determined initial parameter estimates, and
these were improved using an iterative numerical algorithm based on
Powell's method. Model discrimination was assessed by a variety of
considerations including visual inspection of the predicted curves,
dispersion of residuals, minimization of the sum of weighted squares
residuals, and the Akaike information criterion. Final values of the
iterated parameters of the best-fit equation were used to calculate
pharmacokinetic parameters, including the terminal disposition
half-life (t1/2, z), area under the
concentration-time curve (AUC) from zero to infinity, and clearance
(CL, defined as dose divided by AUC). The peak concentration (Cmax) was put on par with the observed drug
level at the end of infusion. Noncompartmental analysis of CrEL plasma
concentration data was performed as described previously (Sparreboom et
al., 1998d). All statistical tests were calculated using the Number Cruncher Statistical System package (version 5.X; Dr. J. Hintze, Kaysville, UT).
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Results and Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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In Vitro Binding Experiments.
We initially investigated ultrafiltration, as measurement of paclitaxel
in the ultrafiltrate would provide a direct measure of Cu (Bowers et
al., 1984). This technique, using a standard micro system (Amicon
Centrifree, Danvers, MA), however, gave nonreproducible results and
posed serious hurdles owing to a problem of sensitivity with currently
available analytical methods for the determination of paclitaxel
(reviewed in Sparreboom et al., 1998a). As a next step, we evaluated
the possibility of using equilibrium dialysis to determine the effects
of CrEL on paclitaxel fu in human plasma. The major prerequisite in
determining paclitaxel Cu is that the condition of the in vitro
measurement should simulate those existing in vivo when the blood
sample was taken. Hence, all measurements were carried out with
undiluted plasma and at 37°C to give meaningful results. It was
confirmed in all equilibrium dialysis experiments that the total drug
recovery from all the fractions was equal to the amount of
[G-3H]paclitaxel added to the
plasma samples (P > .29 versus hypothesized mean of
initial value).
1-acid glycoprotein (Kumar et al., 1993), with a mean (± S.D.) fu of 0.13 ± 0.016 (n = 14). This value is within the same range as
described previously in human plasma samples as determined by both
equilibrium dialysis and ultrafiltration techniques (Longnecker et al.,
1987; Wiernik et al., 1987; Jamis-Dow et al., 1993; Van Tellingen et
al., 1999). Experiments demonstrated that the radioactivity seen in the
protein-free fraction was not derived from any radioactive material
that might have been associated with the polypropylene tube wall or the
membrane. Very small amounts of 3H-labeled water
were formed, however, during the course of the 24-h incubation
experiments. This was determined in selected buffer samples as the
difference before and after drying (at 37°C) following equilibrium
dialysis in the absence or presence of 5.0 µl/ml (paclitaxel, 1.2 µM). It accounted for 4.7% of total radioactivity and was independent of the CrEL concentration applied. The buffer solutions collected after dialysis of paclitaxel (12 µM) were also analyzed by
a specific HPLC method (Sparreboom et al., 1998a) to exclude an
artifact due to alteration of the fraction of total radioactivity associated with unchanged compound (not shown).
As fu measurements were to be made on patient samples that contained
variable amounts of paclitaxel, fu was also determined in blank plasma
samples over the entire anticipated concentration range (0.03, 0.12, 0.30, 0.60, and 1.2 µM). Paclitaxel concentration had no influence on
fu as determined by unweighted ANOVA (P = .19;
n = 36), with an overall coefficient of variation of
4.2%, indicative of a lack in saturation of paclitaxel binding to
human plasma. In the presence of CrEL, however, a clear and
statistically significant decrease in fu of paclitaxel (1.2 µM) was
observed, which was distinctly concentration-dependent within the
clinical range (P < .00001, one-way ANOVA). At the
highest CrEL concentration tested, i.e., 5 µl/ml, a decrease of
(about) 60% was noted in fu as compared to fu in the absence of CrEL
(Fig. 2).
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), were always
less than 9.2% at the six CrEL concentrations analyzed (Table
2). These values are comparable with that
obtained for a variety of other compounds using equilibrium dialysis or
ultrafiltration techniques (Verbeeck and Cardinal, 1985; Legg and
Rowland, 1987), and were considered to be acceptable for analysis of
patient samples in support of pharmacokinetic studies.
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Patient Studies.
The developed method was applied to plasma samples of three patients
treated with paclitaxel at three different dose levels. Similar to our
in vitro experiments, a distinct CrEL concentration and time dependence
was noted for paclitaxel fu (Fig. 3).
Logarithmic concentration-time profiles of paclitaxel Cu, and total
paclitaxel in plasma and whole blood are shown in Fig.
4 (upper panel). Plasma AUC values of
total paclitaxel increased disproportional with dose from 10.2 ± 1.34 to 15.5 ± 1.38 and 31.8 ± 5.40 µM · h at dose
levels of 135, 175, and 225 mg/m2, respectively,
which is in excellent agreement with earlier studies (Gianni et al.,
1995; Bhalla et al., 1999). Disproportionality was less pronounced with
data based on fu and whole blood, as indicated by the respective
clearance values as a function of the dose administered (Fig. 4; lower
panel). The overall mean values for paclitaxel fu and whole blood
clearance [255 ± 33.1 l/h/m2 (coefficient
of variation: 13.0%) and 16.0 ± 3.22 l/h/m2 (coefficient of variation: 20.1%),
respectively] were relatively consistent in the three patients,
suggesting minor interindividual variability. In addition, preliminary
analysis showed that a linear two-compartment model could adequately
describe the Cu versus time curves based on the Akaike information
criterion (r2 = 0.99 ± 0.01;
P < .0001), whereas linear models for total paclitaxel plasma data were significantly biased (not shown). The terminal disposition half-life of paclitaxel was similar between dose levels and
analyzed matrices, with mean values of 6.54 ± 1.43 h (Cu), 7.10 ± 1.01 h (Cp), and 6.91 ± 0.97 h (Cb). In all, these findings, although preliminary, corroborate our hypothesis that the AUC of
unbound paclitaxel should be a linear function of the dose administered, in spite of the nonlinear disposition profile when only
total paclitaxel plasma levels are considered (Sparreboom et al.,
1999a). Mean plasma concentration-time profiles of CrEL following
paclitaxel administration are shown in Fig.
5. As expected, the apparent plasma
clearance of CrEL was dose-independent in each patient and averaged
331 ± 48.8 l/h/m2, with
Cmax levels progressively increasing from
2.56 ± 0.48 to 3.60 ± 0.67 and 4.14 ± 0.55 µl/ml at
the three consecutive dose levels, which is within the range described
earlier (Sparreboom et al., 1998d).
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Eric Brouwer
Jaap Verweij
Peter De Bruijn
Walter J. Loos
Marrimuthoo Pillay
Dirk Buijs
Alex Sparreboom
Departments of Medical Oncology
(E.B., J.V., P.d.B.,
W.J.L., A.S.),
and Nuclear Medicine (M.P., D.B.),
Rotterdam
Cancer Institute
(Daniel den Hoed Kliniek)
and University
Hospital
Rotterdam, The Netherlands
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Footnotes |
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Received January 11, 2000; accepted June 28, 2000.
Send reprint requests to: Alex Sparreboom, Ph.D., Department of Medical Oncology, Rotterdam Cancer Institute (Daniel den Hoed Kliniek) and University Hospital Rotterdam, P.O. Box 5201, 3008 AE Rotterdam, The Netherlands. E-mail: sparreboom{at}onch.azr.nl
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Abbreviations |
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Abbreviations used are: CrEL, Cremophor EL; fu, fraction unbound; Cp, total plasma concentration; Cu, unbound concentration; AUC, area under the concentration versus time curve; CL, clearance.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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1-acid glycoprotein.
Res Commun Chem Pathol Pharmacol
80:
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), 175 mg/m2 (
) and 225 mg/m2
(
).
