Spatial profiling of stapled α–helical peptide ATSP-7041 in mouse whole-body thin tissue sections using droplet-based liquid microjunction surface sampling-HPLC-ESI–MS/MS

Highlights

• The application of an autosampler/HPLC-ESI–MS/MS system for spatially resolved quantitative surface sampling/profiling of ATSP-7041.

• The observed ATSP-7041 concentration was lower than that measured by the tissue punch workflow at the same tissue location of a serial section.

• Calculated extraction efficiencies were reproducible within a given organ.

• Reproducibility of the approach could provide a non-labor intensive and high-throughput means to acquire spatially resolved quantitative data.

Abstract

The application of a fully automated autosampler/HPLC-ESI–MS/MS system for spatially resolved quantitative droplet-based liquid extraction surface sampling/profiling of stapled α–helical peptide ATSP-7041 in mouse whole-body thin tissue sections is reported. 20-μm-thick serial tissue sections of an ATSP-7041 dosed mouse were prepared and the absolute concentration of the targeted peptide was first determined in different organs using 2.3-mm diameter tissue punches, standard bulk tissue extraction protocols, and subsequent HPLC separation and tandem mass spectrometric analysis. The same organs/locations were then analyzed in neighboring tissue sections using the droplet-based surface sampling approach. The observed ATSP-7041 concentration using this method was always significantly lower than that measured by the tissue punch workflow at the same tissue location of a serial section. Calculated extraction efficiencies were 10.7 ± 0.5% (brain), 11.0 ± 3.2% (liver spot 1), 10.7 ± 2.6% (liver spot 2), 15.0 ± 0.6% (lung) and 12.9 ± 0.7% (blood). While these extraction efficiency values were low, they were reproducible within a given organ. This suggests that once the extraction efficiency is established for a given tissue type and drug, the reproducibility of the droplet-based approach could provide a non-labor intensive and high-throughput means to acquire spatially resolved quantitative analysis of multiple samples of the same type.

Introduction

Peptides are increasingly being utilized as therapeutics for various extra- and intra-cellular targets. Pharmaceutical discovery and development would benefit from a label-free method to probe biodistribution, metabolic and elimination pathways, as well as query pharmacokinetic and pharmacodynamic correlations in preclinical species. Thus, spatially resolved quantitation of targeted pharmaceuticals including the dosed therapeutic and its metabolites in preclinical tissues via a label-free method with minimal sample preparation is desirable.

The two methods most often used to obtain spatially resolved chemical information in biological tissue samples are quantitative whole-body autoradiography (QWBA) [[1], [2]] and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) [[3], [4], [5]]. Unfortunately, QWBA is unable to distinguish between a parent drug and its metabolites and thus is not well suited alone for detailed drug and metabolite distribution studies. Quantitative MALDI-MS has been the subject of numerous studies and reports and its ability to reliably provide absolute quantitation is continuing to advance [[6], [7], [8], [9]]. However, the technique requires coating the sample with a chemical matrix prior to MS analysis. In addition, MALDI-MS tends to fragment fragile molecules like phase II metabolites thus it is not well-suited for their detection [4]. Finally, sampling and ionization processes are not separated in MALDI-MS thus it lacks the capability to transfer the sampled material for post-sampling processing. For example, HPLC separation of sampled material can be advantageous in dealing with complex sample matrices and/or for separation and identification of isomeric compounds not distinguishable by MS methods alone.

Liquid extraction based surface sampling probes of different types [[10], [11], [12], [13], [14], [15], [16], [17], [18]] are getting increased attention due to their enhanced sensitivity compared to other ambient surface sampling techniques [[19], [20], [21], [22]] and due to their ability to integrate with HPLC/MS systems [[23], [24], [25], [26], [27], [28], [29], [30], [31], [32]]. These techniques for spatially resolved sampling rely on liquid-solid extraction of material from the tissue. No modifications to existing tissue sections are required to employ the technique. Typical sampling spatial resolution is 1–3 mm, or roughly equivalent to the size of traditional punch samples [[32], [33]], and thereby adequate for many relevant applications for examining tissue distributions of drugs and metabolites.

Importantly, relative quantitation of drug and metabolite distributions from dosed tissue sections determined by liquid extraction based surface sampling/mass spectrometry approaches has been shown to compare well with other studies using QWBA and HPLC/MS [24], QWBA and radio thin-layer chromatography [25] and HPLC/MS of tissue homogenate [28]. Even more significant, the ability for absolute quantitation with the liquid extraction based surface sampling/mass spectrometry approaches has been demonstrated. That work, quantifying propranolol [32], employed a workflow that included calibration of the extraction efficiency of the targeted molecules from the different tissue organs of a dosed mouse. That was accomplished by comparing quantitative results from the analysis of serial tissue sections using tissue punches (considered to provide 100% extraction) and the liquid extraction based surface sampling methods. These established extraction efficiency values were then used to correct the experimentally obtained concentration values for subsequent sections to provide absolute quantitation.

The human transcription factor protein p53 plays a critical role in protecting cells from cancerous transformation [[34], [35]]. Inactivation of this biomacromolecule by inhibitory proteins is the most common defect in human cancers. In one type of cancer proteins MDM2 and MDMX are overexpressed and inhibit p53 [36]. Thus, disruption of the interactions between p53 and these proteins offers the pharmacological opportunity to restore p53-dependent cell-cycle arrest and apoptosis. Stapled α–helical peptide ATSP-7041 (under development by Aileron Therapeutics, Cambridge, MA) has emerged as a highly potent and specific dual inhibitor of MDM2 and MDMX that possesses robust drug-like properties and on-mechanism in vitro and in vivo activity [37]. It is of interest to study the tissue distribution of ATSP-7041 on a time scale that is consistent with its rapid absorption and elimination [37]. In this communication, the spatial distribution and extraction efficiency for ATSP-7041 from mouse whole-body thin tissue sections utilizing a droplet-based liquid microjunction surface sampling probe (LMJ-SSP)-HPLC–MS/MS system [[23], [24], [25], [26], [27], [28], [31], [32]] is reported. Extraction efficiency varied with tissue type (∼10 to 15%), but was reproducible (∼5 to 30% relative standard deviation) for the individual organs. The potential advantages of using droplet-based liquid microjunction surface sampling mass spectrometry as a label-free, non-labor intensive, and relatively high-throughput means to acquire spatially resolved quantitative analysis of dosed pharmaceuticals in tissue samples is discussed.