Evaluation of a method for measuring the radioprotective metabolite WR-1065 in plasma using chemical derivatization combined with UHPLC-MS/MS

Abstract

Hypotension is the dose-limiting side effect of the radio-protective drug Amifostine and results from relaxation of the vascular smooth muscle, which is directly mediated by the active metabolite, WR-1065, of Amifostine. The route of administration (currently FDA-approved only for intravenous administration) and the rapid metabolic conversion of Amifostine combine to yield high systemic levels of WR-1065 and facilitate the onset of hypotension. Research efforts aiming to optimize the delivery of WR-1065 to maintain efficacy while reducing its peak, systemic concentration below levels that induce hypotension are underway. To fully characterize the effect of reduced dose levels and alternative routes of administration of Amifostine on systemic WR-1065 concentrations, improved analytical techniques are needed. We have developed and evaluated a highly sensitive method for measuring WR-1065 in rat plasma that employs chemical derivatization, protein precipitation and UPLC-MS/MS analysis. The method exhibits a limit of quantification (LOQ) of 7.4 nM in plasma, which is a significant improvement over conventional approaches that utilize LC-electrochemical detection (ECD) (LOQ 150 nM or higher). The method was assessed in a pharmacokinetics study in rats administered Amifostine intravenously and via direct jejunal injection (10 mg/kg each route). The bioavailability of WR-1065 was 61.5% after direct jejunal injection indicating rapid conversion and absorption of the metabolite in the intestinal tract. This demonstrates that an oral formulation of Amifostine designed for site-specific release of the drug in the upper GI tract can deliver systemic absorption/conversion to WR-1065, provided that the formulation protects the therapeutic from gastric decomposition in the stomach.

Introduction

Amifostine (Ethyol®, WR-2721) is a cytoprotective drug that is FDA-approved for intravenous (IV) administration in cancer patients receiving radiotherapy and some forms of chemotherapy [1]. Amifostine is a phosphorothioate prodrug and is activated in vivo by alkaline phosphatase, forming the radioprotective aminothiol metabolite WR-1065 (Fig. 1) [2]. WR-1065 is an efficient scavenger of oxygen free radicals formed from ionizing radiation [3,4]. Oxidation of WR-1065 converts it to the intramolecular disulfide (WR-33278, Fig. 1) or intermolecular, protein-bound form [4]. As a cytoprotector, WR-1065 exhibits selective protection of healthy cells from reactive oxygen species. The selectivity of Amifostine has been attributed to a number of physiological differences between normal versus tumor cells. For example, alkaline phosphatase, a membrane-bound protein localized in the blood vessels of tissues, is present at reduced levels in tumors due primarily to reduced vascularity compared to normal, heathy tissue [2,5,6]. As a result, reduced local conversion of Amifostine to WR-1065 affords lesser protection of tumors from radiation. Evidence for other contributing factors includes the characteristically lower physiological pH of tumor cells and tissues, which is not optimal for alkaline phosphatase-dependent activation of Amifostine nor for intracellular uptake of WR-1065 via passive diffusion [6].

However, dose-limiting side effects have limited the utilization and patient compliance of Amifostine in cancer treatment [7]. Most notably, hypotension has been observed and linked to WR-1065, which mediates the relaxation of the vascular smooth muscle [7,8]. The high dose levels of Amifostine infused intravenously combined with rapid conversion by alkaline phosphatase in the vascular system lead to high initial, systemic concentrations of WR-1065 [9]. For the full potential of Amifostine as a cytoprotective therapeutic to be realized, as well as to achieve greater clinical acceptance, administration strategies that maintain its cytoprotective efficacy while mitigating side effects is necessary. In principle, this requires strategies that provide adequate, efficacious distribution of WR-1065 to multiple tissues while reducing peak exposure levels below the threshold that causes hypotension. Strategies that have been explored include alternative routes of administration [7,9,10], local administration [11] and sustained-release oral formulations [[12], [13], [14]]. In some cases, hypotension was partially mitigated but other side effects were introduced.

To fully characterize the pharmacokinetics/pharmacodynamics (PK/PD) relationship of alternative routes of administration of Amifostine, robust and sensitive analytical methods for measuring WR-1065 in plasma will be needed. In particular, strategies relying on local administration with reduced dose levels or sustained release formulations that provide reduced peak exposure levels will require methods with improved limits of quantification (LOQ) [14]. Initial analytical development for measuring WR-1065 in biological matrices predominately utilized liquid chromatography-electrochemical detection (LC-ECD), which has had LOQs reported in the range of 0.15 to 1 μM [10,11,[15], [16], [17]]. More recently developed methods using ECD have examined the use of coulorimetric electrodes [18] or the use of capillary electrophoresis as a substitute to LC in analyzing WR-1065 [19]. While such approaches exhibited improved efficiencies, they have not contributed to any improvement in detection sensitivity compared to the LC-ECD approach. Other methods have applied chemical alkylation of WR-1065 with alternative detection techniques, including UV [20] and fluorescence [21]. Other than the approach applying fluorescence detection, which reported an LOQ of 20 nM, none of the other approaches yields quantitation limits less than those afforded by conventional LC-ECD. For the large dose levels that have been evaluated from intravenous or subcutaneous administration (50 to 200 mg/kg in rats), LC-ECD has adequate sensitivity to measure the high exposure levels in plasma at early time points. However, as WR-1065 depletes rapidly from systemic circulation due to clearance and bio-distribution, plasma levels approach the LOQ after 2 to 4 h post-administration. A recent study evaluated orally-administered, enteric-coated Amifostine as a radioprotective formulation in mice [14]. While this formulation and route of administration exhibited effectiveness in protecting mice from whole body irradiation, the LC-ECD method employed was not sensitive enough to detect any WR-1065 in plasma at any time point after administration. Thus, methods with improved sensitivity are required to fully evaluate these alternative formulations and routes of administration in order to reliably characterize their associated PK/PD relationships.

According to the literature, one detection technique that has not been carefully evaluated in the measurement of WR-1065 is tandem mass spectrometry (MS/MS). MS/MS has been applied with greater frequency in recent years, mainly due to its high analytical sensitivity compared to spectroscopic detectors and its high-throughput capability [22]. As summarized in Fig. 2, we have developed and analytically characterized a highly sensitive method for measuring WR-1065 in rat plasma that employs chemical derivatization, protein precipitation and UPLC-MS/MS analysis. The MS/MS detection utilizes optimized multiple-reaction monitoring (MRM) settings to measure the derivatized WR-1065. The very low chemical background noise associated with the high detection specificity of MRM for the analyte results in an LOQ of 7.4 nM, which is a 20-fold improvement over LC-ECD methods reported in the literature [15,16]. In this study, we evaluated the analytical performance of this method and tested it in a PK study in rats administered comparatively low IV and direct jejunal injection dose levels (10 mg/kg each). The latter route of administration provides insight into the level of absorption and systemic exposure of WR-1065 that may be achievable for formulations that are designed for site-specific release in the upper GI tract after oral delivery and minimal stomach decomposition (e.g. enteric-coating formulations) [14].