Determination of the heat shock protein 90 inhibitor 17-dimethylaminoethylamino-17-demethoxygeldanamycin in plasma by liquid chromatography–electrospray mass spectrometry

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Abstract

A rapid method was developed for the quantitative determination of the novel heat shock protein 90 inhibitor, 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG; NSC707545), in human plasma. Calibration curves were constructed, and were analyzed using a weight factor proportional to the nominal concentration. Sample pretreatment involved a one-step extraction with ethyl acetate of 0.5-ml samples. The analysis was performed in the range of 1–100 ng/ml on a column (75 mm × 2.1 mm internal diameter with 3.5 μm C18 particle size), using 55% methanol in water containing formic acid as the mobile phase. The column effluent was monitored by mass spectrometry with positive electrospray ionization. The values for precision and accuracy were always <8% and <10% relative error, respectively. The method was successfully applied to examine the pharmacokinetics of 17-DMAG in a cancer patient.

Introduction

The heat shock protein 90 (Hsp90) molecular chaperone has emerged as one of the most exciting targets for cancer drug development in recent years [1]. Hsp90 is overexpressed in many malignancies, possibly as a result of the stress that is induced both by the hostile cancer microenvironment and also by the mutation and aberrant expression of oncoproteins. A particularly attractive feature of Hsp90 as a cancer drug target is that it is required for the conformational stability and function of a wide range of oncogenic ‘client’ proteins, including c-Raf-1, Cdk4, ErbB2, mutant p53, c-Met, Polo-1 and telomerase hTERT, which processes are accompanied by dose-dependent inhibition of tumor growth [1]. Inhibition of Hsp90 should therefore block multiple critical oncogenic pathways in the cancer cell, leading to inhibition of all the hallmark traits of malignancy and induce a broad spectrum of antitumor activity across multiple cancer types. In the mid-1990s, it was discovered that certain natural products, including geldanamycin, exert their antitumor activity by inhibiting the essential ATPase activity associated with the N-terminal domain of the Hsp90 protein [2].

Although geldanamycin was not developed for clinical use because of excessive toxicity, the analogue 17-allylamino-17-demethoxygeldanamycin (17-AAG) is presently under evaluation as an anticancer drug in clinical trials [3], [4], [5], [6]. In an attempt to improve the physicochemical properties and biological characteristics of 17-AAG, a library of more than 60 structurally related agents was developed [7], of which 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG; NSC707545; Fig. 1) was considered the most promising. Both 17-AAG and 17-DMAG have similar patterns of activity in the National Cancer Institute (NCI) 60 cell line screen, suggesting that the two compounds have similar mechanisms of action (COMPARE analysis; http://www.dtp.nci.nih.gov/docs/dtp_search.html). 17-DMAG has shown antiproliferative activity in various in vitro human tumor models, including gynecologic cancer cell lines [8]. Furthermore, impressive in vivo antitumor activity in several human orthotopic tumor xenograft models with a remarkable lack of toxicity has been observed following 17-DMAG administration [9], [10]. Compared to 17-AAG, 17-DMAG offers a potential advantage because its aqueous solubility eliminates the need for complicated formulations that are currently used for administration of 17-AAG. In addition, 17-DMAG undergoes only limited metabolism compared to 17-AAG in preclinical models [11], [12], which may reduce drug clearance and interindividual pharmacokinetic variability in humans. Based on these promising data, we have initiated a Phase I clinical trial of 17-DMAG in patients with refractory solid tumors. Here, we report the development and validation of an analytical method for the quantitation of 17-DMAG in human plasma in support of a project to understand the clinical pharmacology of this agent.

Section snippets

Chemicals and materials

17-DMAG was supplied as a crystalline white powder by the Pharmaceutical Management Branch, Cancer Therapy Evaluation Program, Division of Cancer Treatment and Diagnosis, NCI (Bethesda, MD, USA). HPLC-grade methanol was obtained from J.T. Baker (Phillipsburg, NJ, USA). Formic acid was purchased from Sigma (St. Louis, MO, USA). Deionized water was generated with a Hydro-Reverse Osmosis system (Durham, NC, USA) connected to a Milli-Q UV Plus purifying system (Malbourough, MA, USA). Drug-free

Chromatography

The mass spectrum of 17-DMAG showed a protonated molecular ion ([MH+]) at m/z 617, in accordance with the NTP chemical repository database, and a sodium adduct at m/z 639 (MH+ + Na) (Fig. 2). Sample pretreatment was performed by a single solvent extraction using ethyl acetate, based on previous experience with extraction of the drug from biological matrices [11]. In spite of 17-DMAG's relatively high solubility in water (1.4 mg/ml) [7], which is more than 10-fold higher than that of 17-AAG, the

Conclusion

In conclusion, the method presented for the determination of 17-DMAG in human plasma is specific, accurate and precise, and is selective and sensitive enough to be used in clinical trials. The method permits the analysis of patient samples with low concentrations of 17-DMAG, and is currently being used in various Phase I clinical trials in patients with malignancies to further investigate the clinical pharmacologic profile of this agent.

Acknowledgment

This research was supported in part by the Intramural Research Program of the NIH, NCI, Bethesda, MD, USA.

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