Analysis of the hypoxia-activated dinitrobenzamide mustard phosphate pre-prodrug PR-104 and its alcohol metabolite PR-104A in plasma and tissues by liquid chromatography–mass spectrometry

doi:10.1016/j.jchromb.2007.06.035

Journal of Chromatography B

Volume 856, Issues 1–2, 1 September 2007, Pages 302-311

https://doi.org/10.1016/j.jchromb.2007.06.035 Get rights and content

Abstract

PR-104 is a dinitrobenzamide mustard pre-prodrug that is activated by reduction to a cytotoxic hydroxylamine metabolite in hypoxic tumour cells; it has recently commenced Phase I clinical trial. Here, we report two validated methods for the determination of PR-104 and its alcohol hydrolysis product, PR-104A in plasma and tissues across species. A high pH LC/MS/MS method was optimised for rapid and sensitive analysis of both analytes in rat, dog and human plasma. This assay was linear over the concentration range 0.005–2.5 μg/ml for PR-104 and 0.05–25 μg/ml for PR-104A (0.005–2.5 μg/ml for rat). A second method, using a low pH LC separation, was designed to provide higher chromatographic resolution, facilitating identification of metabolites. Both methods were successfully applied to the plasma pharmacokinetics of PR-104 and PR-104A in rats. In addition, cytotoxic reduced metabolites of PR-104A were identified in human tumour xenografts by the higher chromatographic resolution method.

Introduction

Regions of very low oxygen levels (hypoxia) are unique features of solid tumours [1], [2], [3] and confer resistance to radiotherapy [4], [5], [6] and chemotherapy [7], [8], [9]. This has led to the development of strategies to exploit hypoxia as a basis for tumour selectivity, particularly the development of drugs that are toxic only under hypoxic conditions [10]. The dinitrobenzamide mustard (DNBM) prodrugs provide an opportunity to explore this strategy. DNBM prodrugs contain a latent nitrogen mustard moiety which becomes activated when either of the nitro groups is reduced to the corresponding hydroxylamine or amine [11]. This provides an “electronic switch” to activate the nitrogen mustard selectively in hypoxic cells, resulting in hypoxia-selective cytotoxicity [12], [13]. PR-104 is a water-soluble phosphate “pre-prodrug” that is converted efficiently to the more lipophilic DNBM alcohol PR-104A (Fig. 1A), which is a hypoxia-selective DNA cross-linking agent and cytotoxin [14]. PR-104 is currently in phase I clinical trial, which commenced in January 2006.

In order to assist pharmacokinetic evaluation of this new drug during toxicological and clinical studies, we developed two methods for simultaneous bioanalysis of both PR-104 and PR-104A. One method was optimised for sensitive and rapid quantitation of PR-104 and PR-104A, using a high pH, low chromatographic resolution HPLC method with triple quadrupole (LC/MS/MS) tandem mass spectrometry. The second method was optimised for separation of metabolites using a high chromatographic resolution HPLC method which can be interfaced with a variety of detectors (photodiode array, single stage or tandem MS). We report validated assays for both methods here, together with their application for pharmacokinetic analysis.

Section snippets

Chemicals and reagents

PR-104 (2-((2-bromoethyl)-2-{[(2-hydroxyethyl) amino] carbonyl}-4, 6-dinitroanalino) ethyl methanesulfonate phosphate ester, was synthesized as the free acid from PR-104A [15]. The batches used in this study varied in purity from 96 to 98% for PR-104 and 96–100% for PR-104A, based on HPLC with absorbance detection (254 nm). The analogue internal standards SN 29894 (99% purity) and SN 29893 (100% purity) were synthesized in this laboratory. Tetra-deuterated stable isotope standards of PR-104

Linearity

For the high pH LC/MS/MS method, calibration curves obtained by plotting peak height ratios (d₀/d₄) against nominal (spiked) concentrations were linear for rat, dog and human plasma over the range 0.005–2.5 μg/ml for PR-104 and 0.05–25 μg/ml for PR-104A (0.005–2.5 μg/ml for rat). Mean accuracy and precision values at each concentration within these ranges gave RSD values within 15.0 % and DEV within ±15.0% (Table 1). Analysis of mouse and rat plasma, and mouse tissues by the less sensitive LC/MS

Discussion and conclusion

The objective of this study was to validate two assays developed for the determination of PR-104 and PR-104A, and identification of their metabolites, in plasma and tissues from different species. The two assays were developed with different primary objectives. The high pH LC/MS/MS method is optimised for rapid and sensitive analysis of PR-104 and PR-104A, and uses either stable isotope (tetra-deuterated) internal standards or analogue standards (which co-elute in this system which has low

Acknowledgements

This study was supported by Proacta Inc. and by a grant from the Health Research Council of New Zealand. We thank Proacta Inc. for provision of GMP grade PR-104 and access to data, and LAB (Montreal) for conducting the rat pharmacokinetic study. We also thank Drs. William A. Denny and Graham J. Atwell for synthesis of internal standards.

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PR-104 is a dinitrobenzamide mustard currently in clinical trial as a hypoxia-activated prodrug. It is converted systemically to the corresponding alcohol, PR-104A, which is activated by nitroreduction to the hydroxylamine (PR-104H) and amine (PR-104M). PR-104A is also metabolised to the O-glucuronide (PR-104G), and by oxidative debromoethylation to the semi-mustard PR-104S. We now report a validated ultra-high-pressure liquid chromatography and tandem mass spectrometry (UHPLC–MS/MS) method for the determination of these metabolites in human plasma. Plasma proteins were precipitated with acidified methanol and the supernatant diluted into water. Aliquots were analysed by UHPLC–MS/MS using a Zorbax Eclipse XDB-C18 Rapid Resolution HT (50 mm × 2.1 mm, 1.8 μm) column and gradient of acetonitrile and 0.01% formic acid with a 6 min run time. The method had a linear range of 0.1–50 μM for PR-104, PR-104A and PR-104G, 0.05–5 μM for PR-104H, 0.025–2.5 μM for PR-104M and 0.01–1 μM for PR-104S. The intra-day and inter-day precision and accuracy were within 14%. The extraction recovery of all analytes was over 87%. The validated method was illustrated by using it to study the pharmacokinetics of PR-104 and its metabolites in a human patient.
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Thawed plasma was deproteinized and analyzed as were the cell metabolism samples discussed previously. Plasma PK of PR-104A by intraperitoneal injection of PR-104 at a dose of 562 μmol/kg was determined previously (10) using the recently reported analytic method (35). Noncompartmental pharmacokinetic parameters were estimated using WINNONLIN, version 5.
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The biotransformation of the electron-withdrawing nitro group to the electron-donating hydroxylamine and amine results in direct activation of the nitrogen mustard moiety, as previously described for the 2,4-dinitrobenzamide-5-mustard prototype SN 23862 [8,9], and results in hypoxia-selective cytotoxicity in cell culture [7]. PR-104A penetrates efficiently into hypoxic regions of tumours [10] where it forms the same reduced metabolites as in hypoxic cell cultures [11]. PR-104 provides very efficient killing of hypoxic cells in human tumour xenografts and is also surprisingly active against aerobic cells as demonstrated by its marked antitumour activity as a single agent [7].
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Analysis of the hypoxia-activated dinitrobenzamide mustard phosphate pre-prodrug PR-104 and its alcohol metabolite PR-104A in plasma and tissues by liquid chromatography–mass spectrometry

Abstract

Introduction

Section snippets

Chemicals and reagents

Linearity

Discussion and conclusion

Acknowledgements

Int. J. Radiat. Oncol. Biol. Phys.

Int. J. Radiat. Oncol. Biol. Phys.

Radiother. Oncol.

Cancer Res.

Acta Oncol.

Lancet

In Vivo