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Application of a liquid chromatography–tandem mass spectrometry (LC/MS/MS) method to the pharmacokinetics of ON01910 in brain tumor-bearing mice

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Abstract

ON01910 is a small molecular weight benzyl styryl sulfone currently under investigation as a novel anticancer agent. The purpose of the investigation was to develop a sensitive and reproducible liquid chromatography–tandem mass spectrometry (LC/MS/MS) method to quantitate levels of ON01910 in small amounts of five biological matrices; mouse plasma, feces, urine, normal brain and brain tumor. For all matrices, protein precipitation sample preparation was used that led to linear calibration curves with coefficients of determination greater than 0.99. The lower limit of quantitation (LLOQ) for all matrices was 5 ng/ml except that for mouse urine which was 10 ng/ml. The calibration standard curves were reproducible for all matrices with inter- and intra-day variability in precision and accuracy being less than 15% at all quality control concentrations except for the LLOQ in mouse plasma for which the accuracy was within 17%. The assay was successfully applied to characterize the systemic pharmacokinetics of ON01910 as well as its disposition in brain and brain tumor in mice. ON01910 exhibited a clearance of 3.61 ± 0.85 l/h/kg and a half life of 8.66 ± 3.30 h at 50 mg/kg dose given I.V.

Introduction

ON01910 (also referred to as ON 01910.Na and Estybon™) is a synthetic low molecular-weight anticancer agent (473.47 Da) that belongs to the class of benzyl styryl sulfones (Fig. 1). ON01910 causes a G2/M phase cell cycle arrest leading to mitotic inhibition [1]. ON01910 has exhibited both antitumor activity [1] and antiangiogenic activity [6] with a low toxicity profile in various preclinical tumor xenograft models, and is currently in Phase II clinical trials as a single agent and in combination with conventional chemotherapy in advanced and metastatic tumors.

Analysis of ON01910 in animal and human plasma using LC/MS/MS has been reported [2], [3] based on sample sizes of 0.1 ml although for our studies, a sample size of 10 μl was the limit and a key consideration in the development of the assay. In addition, the LC/MS/MS method had to be applicable to normal brain and brain tumor, the latter being the intended site of action, which had not been previously investigated.

Section snippets

Chemicals and standards

ON01910 and ON012380, the internal standard (Fig. 2), were synthesized in-house. Details on other chemicals, solvents and preparation of stock solutions, standards and quality control (QC) samples are presented in Supplementary information.

Chromatographic and mass-spectroscopic conditions

Method development and validation were performed with an LC/MS/MS (Agilent 1100 series HPLC, AB SCIEX API 4000 tandem mass spectrometer, Foster City, CA) using an ESI interface and operated in positive ion mode. Instrument control, data acquisition and

Chromatographic and mass spectrometric conditions

The isocratic mobile phase condition produced peaks for ON01910 and the internal standard with a total run time well within 3 min. Representative chromatograms of extracted blanks and ON01910 pharmacokinetic study samples in mouse plasma, urine, feces, brain and brain tumor are shown in Supplementary Fig. A.

The MS was operated in positive ion scan mode with ESI-SRM ion transitions of the protonated molecular ions (M+) at m/z of 469.00 and 466.00 for ON01910 and the internal standard,

Conclusions

In conclusion, we have developed a sensitive, specific and robust method for the quantitation of ON01910 in mouse plasma, urine, feces, brain and brain tumor that allowed the use of small plasma volumes of 10 μl and a serial sampling design that minimizes the number of animals and inter-animal variability. In addition, the method was successfully applied to brain and brain tumor samples that were used to assess the ability of ON01910 to penetrate the BBB, which under the conditions of this

Conflict of interest

Dr. E.P. Reddy is the scientific founder, stock holder, board member and paid consultant of Onconova Therapeutics Inc. He is also one of the inventors of the patents that describe the compounds described here.

Acknowledgement

This work was supported by NIH grant RO1CA127963.

References (6)

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Present address: Bioanalytical Services, Frontage Laboratories, 105 Great Valley Parkway, Malvern, PA 19355, USA.

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