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Rapid and sensitive LC/MS/MS analysis of the novel tyrosine kinase inhibitor ZD6474 in mouse plasma and tissues

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Abstract

ZD6474 (N-(4-Bromo-2-fluorophenyl)-6-methoxy-7-[(1-methylpiperidin-4-yl)methoxy] quinazolin-4-amine) is a tyrosine kinase inhibitor with anti-angiogenic and anti-tumor activity that is currently undergoing human trials for cancer treatment. Pharmacokinetic studies in animal models are an important component in clinical development of this agent to relate pre-clinical models to patient treatment. A liquid chromatography tandem mass spectrometry method was developed for the determination of ZD6474 levels in mouse plasma and tissues. Plasma (0.05 mL) and tissue homogenates (0.1 mL of 10 mg/mL) were extracted under alkaline conditions with ethyl acetate:pentane (1:1, v/v) after addition of the internal standard (trazodone, 2-[3-[4-(3-chlorophenyl)-1-piperazinyl]propyl]-1,2,4-triazolo[4,3-a]pyridine-3(2H)-one). Separation was achieved on a C18, 50 mm × 2 mm column with quantitation by internal standard reference and multiple reaction monitoring of the ion transitions m/z 475  112 (ZD6474) and m/z 372  176 (trazodone). The calibration curve was linear from a range spanning 20–20,000 ng/mL in plasma and 10–320 ng/mg in tissue homogenates. Mean recoveries from plasma and tissue homogenates were 88 and 90%, respectively. The accuracy in plasma was 88% at the lower limit of quantitation (20 ng/mL with a 50 μL plasma sample) with high precision (R.S.D.% < 10%). Assay performance in liver and other tissue homogenates is also reported. The assay was applied to a pharmacokinetic study in mice to determine dosing schedules that would approximate therapeutic ZD6474 levels determined in humans.

Introduction

Cancer progression: tumor growth, invasion, angiogenesis and metastasis, is largely regulated by autocrine and paracrine signaling through growth factors and their binding to receptor tyrosine kinases (RTKs). Upon binding of growth factors to respective RTKs, dimerization and subsequent phosphorylation of the intracellular kinase domain occurs. Resulting phosphotyrosines serve as docking sites that recruit signal transduction intermediates, ultimately leading to cell proliferation [1]. Approaches to therapies designed to target the signal transduction pathway to inhibit tumor growth, angiogenesis or both include: anti-growth factor antibodies, receptor antagonism, anti-receptor monoclonal antibodies, anti-sense and small molecule tyrosine kinase inhibitors (TKIs) [2], [3].

4-Anilinoquinazolines are a class of orally available synthetic small molecules designed to bind to the intracellular kinase domain of RTKs, preventing phosphorylation and disrupting the necessary signal transduction for cell proliferation [4], [5]. ZD6474 (N-(4-Bromo-2-fluorophenyl)-6-methoxy-7-[(1-methylpiperidin-4-yl)methoxy] quinazolin-4-amine) has demonstrated potent and selective activity against the vascular endothelial cell growth factor receptor (VEGFR2, Flk1/KDR) [4], [5], which plays a key role in tumor angiogenesis [6] and the epidermal growth-factor receptor (EGFR) [4], [5], which is overexpressed in several cancer types [7]. The dual activity of ZD6474 allows a single molecularly targeted agent to be effective against the dividing tumor vasculature as well as the tumor cell population. ZD6474, administered orally as a once daily treatment, has demonstrated anti-tumor and anti-angiogenic capability in vitro and in vivo [8], [9] and is currently in Phase II clinical trials.

Pharmacokinetic studies conducted in pre-clinical models provide useful information about absorption, distribution, elimination and efficacy that can be translated to the clinical setting. Presently, there are no available or published methods for the measurement of ZD6474. The 4-anilinoquinazolines with basic side chains demonstrate good tandem mass spectrometric performance. Therefore, we developed a simple, rapid and sensitive LC/MS/MS assay to measure ZD6474 from mouse plasma and tissue.

Section snippets

Chemicals and reagents

ZD6474 was a generous gift from AstraZeneca (Macclesfield, UK) and trazodone was obtained from Sigma (St. Louis, MO). All other chemicals and solvents were of reagent or higher grade and obtained from Fisher Scientific (Pittsburgh, PA).

Standards preparation

Standard dilutions of ZD6474 were prepared in acetonitrile. For analysis in plasma and tissue, ZD6474 was added to 10 mg/mL control tissue homogenate (10–320 ng/mg tissue) and plasma (20–20,000 ng/mL) and them extracted as described in Section 2.3. Each dilution

Chromatography

ZD6474 and the internal standard trazodone were eluted at 2.3 and 2.4 min, respectively (Fig. 3, Fig. 4). Peaks were detected by monitoring the transition from m/z 475  112 for ZD6474 and from m/z 372  176 for trazodone. No interfering peaks were detected at either monitored ion transition in extracted matrix (plasma or tissue homogenates). Chromatographic conditions were optimized for peak shape.

Inclusion of trazodone as an internal standard

Trazodone (2-[3-[4-(3-chlorophenyl)-1-piperazinyl]propyl]-1,2,4-triazolo[4,3-a]pyridine-3(2H)-one) was

Conclusions

Quantitation of ZD6474 is necessary for the investigation of pharmacokinetics in animal models, which is a fundamental component for clinical development. The LC/MS/MS assay developed provides clean chromatography with a low limit of quantitation. The hydrophobicity of ZD6474 allows for simple and rapid extraction in alkaline ethyl acetate:pentane. The use of trazodone as the internal standard is advantageous since trazodone demonstrates similar physico-chemical analytical properties as well as

Acknowledgements

The authors would like to thank AstraZeneca for supplying ZD6474 and for financial support. We would also like to thank Andrea L. Merz for help in carrying out the animal studies and for animal dosing.

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