Bioanalysis and preliminary pharmacokinetics of the acridonecarboxamide derivative GF120918 in plasma of mice and humans by ion-pairing reversed-phase high-performance liquid chromatography with fluorescence detection

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Abstract

We have developed and validated a sensitive and selective method for the determination of the P-glycoprotein modulator GF120918 in murine and human plasma. Chlorpromazine is used as internal standard. Sample pretreatment involves liquid–liquid extraction with tert-butyl methyl ether. Chromatographic separation is achieved by reversed-phase high-performance liquid chromatography using a Symmetry C18 column and detection was accomplished with a fluorescence detector set at excitation and emission wavelengths of 260 and 460 nm, respectively. The mobile phase consists of acetonitrile–50 mM ammonium acetate buffer, pH 4.2 (35:65, v/v). To achieve good separation from endogenous compounds and to improve the peak shape the counter-ion 1-octane sulfonic acid (final concentration 0.005 M) was added to the mobile phase. The lower limit of quantitation was 5.7 ng/ml using 200 μl of human plasma and 23 ng/ml using 50 μl of murine plasma. Within the dynamic range of the calibration curve (5.7–571 ng/ml) the accuracy was close to 100% and within-day and between-day precision were within the generally accepted 15% range. The stability of GF120918 was tested in plasma and blood from mice and humans incubated at 4°C, room temperature, and 37°C for up to 4 h. No losses were observed under these conditions. This method was applied to study the pharmacokinetics of orally administered GF120918 in humans and mice. The sensitivity of the assay was sufficient to determine the concentration in plasma samples obtained up to 24 h after drug administration.

Introduction

GF120918 (Fig. 1) is an acridonecarboxamide derivative which has been developed in the search for more potent and selective inhibitors of P-glycoprotein (P-gp). P-gp is a product of the MDR1 gene and is located in the cell membrane where it can extrude a wide variety of many different substances from the cell [1]. Its expression in drug-selected or transfected mammalian cancer cell lines is known to confer the multidrug resistance phenotype to these cells. The presence of P-gp in tumors of patients is believed to be associated with the clinical resistance of these tumors [2]. Besides its expression in tumor cells, P-gp is also expressed in a number of normal tissues, especially at barrier sites, such as the gut epithelium, brain endothelial cells and in the placenta where it plays a prominent role in the protection of the host [3], [4], [5].

Since the observation that non-cytotoxic dose levels of verapamil were able to inhibit P-gp functioning and restore the cytotoxicity of vinca alkaloids in a vincristine-resistant murine leukemia cell-line both in vitro and in vivo [6], numerous other so called reversal agents have been identified with higher potency and less toxicity, including GF120918 [7]. By using in vitro models it has been shown that GF120918 was able to block P-gp completely at concentrations of 0.05–0.1 μM, rendering this compound about 100-fold more potent than the P-gp modulator cyclosporin A [7]. This level of GF120918 can be achieved in plasma of animals without significant systemic toxicity’s, although the plasma concentration required for in vivo inhibition of P-gp may be higher due to protein binding [8], [9].

In order to support the development of GF120918, an HPLC assay has been developed. Some of the technical details of this method are briefly described by Hyafil et al. [7]. Following alkaline extraction with diethyl ether, GF120918 was separated on a non-silica based PRPI column using a mobile phase with a pH of 11.5. These alkaline conditions were chosen because of an improvement in fluorescence signal at higher pH allowing quantification as low as 10 ng/ml.

In a later paper, Witherspoon et al. [10] described the use of liquid–liquid extraction with tert-butyl methyl ether. The separation of GF120918 and the internal standard (a structural analog) from endogenous substances was achieved using a Hypersil ODS column and a mobile phase at a pH of 4.0. In addition the pH of the column effluent was increased by adding 0.1 M NaOH as a post-column reagent.

For the support of our pharmacokinetic studies with GF120918 in wild-type and P-gp deficient mice and cancer patients we developed and validated a simple and sensitive assay requiring only 50 μl of mouse plasma or 200 μl of human plasma. To avoid the use of post-column reagents or alkaline mobile phases we decided to explore the usefulness of fluorescence detection at pH values compatible with a standard silica based column. Since structural analogs of GF120918 are not readily available we successfully tried to use chlorpromazine as internal standard. We also tested the stability of GF120918 in biological matrices (plasma, whole blood) to establish handling and storage conditions during pharmacokinetic studies.

Section snippets

Chemicals

GF120918·HCl (Batch CD-0030, purity 97.5%) was kindly provided as a powder by Glaxo Wellcome (Research Triangle Park, USA). Chlorpromazine⋅HCl originated from BUFA (Uitgeest, The Netherlands). PIC B-8 (1-octane sulfonic acid) was purchased from Waters (Milford, MA, USA). 1-Chlorobutane (HPLC-grade) originated from Sigma–Aldrich, Steinheim, Germany. All other chemicals were of analytical or Lichrosolv gradient grade and were purchased from E. Merck (Darmstadt, Germany). Water was purified by the

Detection

We investigated the fluorescence properties of GF120918 by recording emission and excitation spectra using the FP920 detector at pH values ranging from 3.6 to 13 because we wanted to eliminate the need for using an alkaline mobile phase or make-up flow (Fig. 2). When diluted in 0.1 M NaOH the emission maximum was found at 560 nm with excitation at 280 nm. When the pH of the solvent was decreased to values compatible with regular HPLC conditions the emission maximum shifted to a lower

Acknowledgments

This work was supported by grant NKB992033 from the Dutch Cancer Society.

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