Development and validation of a method to determine the unbound paclitaxel fraction in human plasma

doi:10.1016/j.ab.2003.07.024

Analytical Biochemistry

Volume 324, Issue 1, 1 January 2004, Pages 11-15

https://doi.org/10.1016/j.ab.2003.07.024 Get rights and content

Abstract

Paclitaxel is pharmaceutically formulated in a mixture of Cremophor EL and ethanol (1:1, v/v). The unbound fraction of the anticancer drug paclitaxel in plasma is dependent on both plasma protein binding and entrapment in Cremophor EL micelles. We have developed a simple and reproducible method for the quantification of the unbound paclitaxel fraction in human plasma. Human plasma was spiked with [³H]paclitaxel and [¹⁴C]glucose (unbound reference) and incubated at 37 °C for 30 min. Plasma ultrafiltrate was prepared by a micropartition system (MPS-1) and collected in a sample cup containing 100 μl of plasma to prevent the loss of paclitaxel due to adsorption. The radionuclides were separated after combustion of the biological samples using a sample oxidizer and the radioactivity was determined by liquid scintillation counting. The unbound fraction of paclitaxel was calculated by dividing the ratios of ³H and ¹⁴C in plasma ultrafiltrate and in plasma. The method was thoroughly validated using human plasma spiked with pharmacologically relevant concentrations of paclitaxel (10–1000 ng/ml) and Cremophor EL (0.25–2.0%). The method was precise, with a within-day precision ranging from 3.9 to 11.0% and a between-day precision ranging from 5.8 to 13.1%. In patient plasma with low serum albumin values containing 1% of Cremophor EL, the unbound fraction appeared to be significantly higher than that in plasma with normal albumin values. The determination of the unbound fraction of paclitaxel proved to be stable during a 10-week storage at −20 °C. Furthermore, the assay was applicable in patient samples. This assay can be used to determine the unbound fraction of paclitaxel in plasma. Moreover, its design should allow the determination of the unbound concentrations of other hydrophobic drugs.

Section snippets

Chemicals and reagents

A stock solution of paclitaxel (Bristol Myers Squibb, Syracuse, NY, USA) at a concentration of 1 mg/ml in methanol was used to spike control human plasma (Central Laboratory for the Blood Transfusion Services, Amsterdam, The Netherlands) to obtain a concentration of 10,000 ng/ml of the drug. This plasma sample was stored at −20 °C. Cremophor EL was obtained from Sigma Chemical Co. (St. Louis, MO, USA).

Tritriated paclitaxel ([³H]paclitaxel; specific activity 17.4 MBq/mmol) was manufactured by

Method development

The MPS-1 system is a generally accepted method to determine the protein binding of hydrophilic drugs such as platinum agents [15]. However, adsorption of the hydrophobic drug paclitaxel occurred rapidly when this compound was present in a protein-free environment such as ultrafiltrate. The adsorption of paclitaxel to the collection cups of the Amicon ultrafiltration device could be prevented by prefilling these vials with 100 μl of blank plasma before ultrafiltration. However, since the yield

Conclusions

The presented method to determine the unbound fraction of paclitaxel in plasma proved to be a precise method at pharmacologically relevant paclitaxel and Cremophor EL concentrations. In agreement with the equilibrium dialysis method, the unbound fraction decreased at higher Cremophor EL concentrations in plasma [4], [5], [11]. Furthermore, it was shown that the unbound fraction was significantly increased at lower plasma albumin levels, but only when the sample contained Cremophor EL. The

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Development and validation of a method to determine the unbound paclitaxel fraction in human plasma

Abstract

Section snippets

Chemicals and reagents

Method development

Conclusions

J. Chromatogr. B

Taxanes: a new class of antitumor agents

Cancer Invest.

Pharmacokinetics of the taxanes

Pharmacotherapy

Nonlinear pharmacokinetics of paclitaxel in mice results from the pharmaceutical vehicle Cremophor EL

Cancer Res.

Cremophor EL-mediated alteration of paclitaxel distribution in human blood: Clinical pharmacokinetic implications

Cancer Res.

Cremophor EL causes (pseudo-) non-linear pharmacokinetics of paclitaxel in patients

Br. J. Cancer

Pharmacokinetic modelling of paclitaxel encapsulation in Cremophor EL micelles

Cancer Chemother. Pharmacol.