Short Communication Measurement of Fraction Unbound Paclitaxel in Human Plasma

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-ml aliquots of plasma containing a tracer amount of [G-H]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/m 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. 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). It has recently been shown that Cremophor EL (CrEL), 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. Materials and Methods Chemicals. Paclitaxel powder (batch 484034; purity 98.3% by reversed phase HPLC) was obtained from Bristol-Myers Squibb (Woerden, The Netherlands). [G-H]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 mand p-positions of the aromatic rings, with minor amounts in the 10-, 39-, 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-ml inside recess in the lids (Reinard and Jacobsen, 1989). Before incubation, 2 ml of a [G-H]paclitaxel solution (500-fold diluted in ethanol) was added to 300 ml of plasma, followed by mixing for 10 s. Dialysis was 1 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. 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: [email protected] 0090-9556/00/2810-1141–1145$03.00/0 DRUG METABOLISM AND DISPOSITION Vol. 28, No. 10 Copyright © 2000 by The American Society for Pharmacology and Experimental Therapeutics 5/851840 DMD 28:1141–1145, 2000 Printed in U.S.A.