Development of a robust, sensitive and selective liquid chromatography-tandem mass spectrometry assay for the quantification of the novel macrocyclic peptide kappa opioid receptor antagonist [D-Trp]CJ-15,208 in plasma and application to an initial pharmacokinetic study
Introduction
The macrocyclic tetrapeptide CJ-15,208 (cyclo[Phe-d-Pro-Phe-Trp]) was first isolated from the fermentation broth of a fungus, Ctenomyces serratus, and reported to exhibit kappa opioid receptor (KOR) antagonism in vitro [1]. We synthesized both cyclo[Phe-d-Pro-Phe-Trp], which appears to be the natural product, and its D-Trp isomer [2], and found that both exhibited promising in vivo opioid activity following central (intracerebroventricular) administration to mice [3]. These peptides are also active after oral administration and appear to penetrate into the central nervous system [4], [5], and hence can serve as lead compounds for further development.
A growing body of preclinical evidence suggests that selective KOR antagonists may have therapeutic potential as treatments for drug abuse and mood disorders [6]. KOR antagonists have shown promising results in preclinical models, preventing stress-induced reinstatement of drug seeking behavior for cocaine and nicotine and also for increased ethanol consumption [7], [8], [9]. Since [D-Trp]CJ-15,208 is a potent KOR antagonist in vivo it is imperative to evaluate the pharmacokinetic properties of this lead compound to assist in the design and development of analogs as potential therapeutic agents. This necessitated the development and validation of a quantitative method for determining its plasma levels.
In recent years the combination of high performance liquid chromatography (HPLC) with tandem mass spectrometry (MS/MS) detection has become an important technique in bioanalytical research. The LC–MS/MS quantitation of macrocyclic peptides has not been extensively reported, with the only exceptions being the determination of cyclosporine A and apicidin plasma levels by LC–MS/MS [10], [11]. The quantitation of the macrocyclic tetrapeptide [D-Trp]CJ-15,208 in biological samples has not been reported. Therefore, the objective of this study was to develop a fully-validated method for the quantitation of [D-Trp]CJ-15,208 in plasma that could be applied to pharmacokinetic studies of the lead peptide. Herein we describe for the first time an LC–MS/MS method for quantification of [D-Trp]CJ-15,208 in plasma utilizing a simple one-step protein precipitation method that is sensitive, reproducible, and selective. This method was successfully applied to an initial pharmacokinetic study of the peptide following intravenous administration.
Section snippets
Materials
[D-Trp]CJ-15,208 and the structurally related analog [D-NMeAla2]CJ-15,208 (cyclo[Phe-d-NMeAla-Phe-Trp], Fig. 1) used as the internal standard were synthesized as reported previously [2], [12]. Drug-free (blank) mouse plasma was obtained from Bio-Reclamation Inc (Westbury, NY, USA). HPLC-grade acetonitrile was obtained from Fisher Scientific (Pittsburgh, PA, USA), and deionized water was obtained from a Millipore water purification system. Solutol HS 15 was a kind gift from BASF.
Instrumentation and LC–MS/MS conditions
Liquid
LC–MS/MS method development
A method was developed for the rapid and robust quantitation of [D-Trp]CJ-15,208 in mouse plasma. A one-step protein precipitation method was employed to facilitate rapid sample processing. Ice-cold acetonitrile containing the internal standard was added to the ice-cold plasma samples which were analyzed by LC–MS/MS following centrifugation and dilution with water. This simple, rapid sample processing method has advantages over solid-phase and tedious liquid-phase extraction methods. The
Conclusions
A robust and sensitive LC–MS/MS assay was developed to quantitate [D-Trp]CJ-15,208 in mouse plasma for the first time. The method has a wide linear dynamic range from 0.5 to 500 ng/mL. The method utilizes a simple one-step protein precipitation by acetonitrile and does not require solid-phase or time-consuming liquid–liquid sample extraction procedures. The method has high sensitivity, specificity and reasonable sample throughput. This simple, robust method was successfully applied to an initial
Acknowledgements
We wish to thank Mr. Robert Drake and Mr. Larry Seib of the University of Kansas Mass Spectrometry and Analytical Proteomics Laboratory for their assistance in acquiring the LC–MS/MS spectra. We also thank Mr. William McGuinness and Dr. Colleen Flynn of the University of Kansas Biotechnology Innovation and Optimization Center for their help in dosing and blood collection in mice, and the calculation of the pharmacokinetic parameters, respectively. This research was supported by the National
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Present address: Department of Pharmaceutical Chemistry, the University of Kansas, Lawrence, KS 66047, USA.