Brendan Prideaux

High-sensitivity MALDI-MRM-MS imaging of moxifloxacin distribution in tuberculosis-infected rabbit lungs and granulomatous lesions.

MALDI-MSI is a powerful technology for localizing drug and metabolite distributions in biological tissues. To enhance our understanding of tuberculosis (TB) drug efficacy and how efficiently certain drugs reach their site of action, MALDI-MSI was applied to image the distribution of the second-line TB drug moxifloxacin at a range of time points after dosing. The ability to perform multiple monitoring of selected ion transitions in the same experiment enabled extremely sensitive imaging of moxifloxacin within tuberculosis-infected rabbit lung biopsies in less than 15 min per tissue section. Homogeneous application of a reference standard during the matrix spraying process enabled the ion-suppressing effects of the inhomogeneous lung tissue to be normalized. The drug was observed to accumulate in granulomatous lesions at levels higher than that in the surrounding lung tissue from 1.5 h postdose until the final time point. MALDI-MSI moxifloxacin distribution data were validated by quantitative LC/MS/MS analysis of lung and granuloma extracts from adjacent biopsies taken from the same animals. Drug distribution within the granulomas was observed to be inhomogeneous, and very low levels were observed in the caseum in comparison to the cellular granuloma regions. In this experiment the MALDI-MRM-MSI method was shown to be a rapid and sensitive method for analyzing the distribution of anti-TB compounds and will be applied to distribution studies of additional drugs in the future.

Mass spectrometry imaging for drug distribution studies.

Since its introduction mass spectrometry imaging (MSI) has proven to be a powerful tool for the localization of molecules in biological tissues. In drug discovery and development, understanding the distribution of both drug and its metabolites is of critical importance. Traditional methods suffer from a lack of spatial information (tissue extraction followed by LCMS) or lack of specificity resulting in the inability to resolve parent drug from its metabolites (whole body autoradiography). MSI is a sensitive and label-free approach for imaging drugs and metabolites in tissues. In this article we review the different MSI technologies that have been applied to the imaging of pharmaceuticals. Recent technical advances, applications and current analytical limitations are discussed.

Autoradiography, MALDI-MS, and SIMS-MS Imaging in Pharmaceutical Discovery and Development

Whole-body autoradiography ((WBA) or quantitative WBA (QWBA)), microautoradiography (MARG), matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI), and secondary ion mass spectrometric imaging (SIMS-MSI) are high-resolution, molecular imaging techniques used to study the tissue distribution of radiolabeled and nonlabeled compounds in ex vivo, in situ biological samples. WBA, which is the imaging of the whole-body of lab animals, and/or their organ systems; and MARG, which provides information on the localization of radioactivity in histological preparations and at the cellular level, are used to support drug discovery and development efforts. These studies enable the conduct of human radiolabeled metabolite studies and have provided pharmaceutical scientists with a high resolution and quantitative method of accessing tissue distribution. MALDI-MSI is a mass spectrometric imaging technique capable of label-free and simultaneous determination of the identity and distribution of xenobiotics and their metabolites as well as endogenous substances in biological samples. This makes it an interesting extension to WBA and MARG, eliminating the need for radiochemistry and providing molecular specific information. SIMS-MSI offers a complementary method to MALDI-MSI for the acquisition of images with higher spatial resolution directly from biological specimens. Although traditionally used for the analysis of surface films and polymers, SIMS has been used successfully for the study of biological tissues and cell types, thus enabling the acquisition of images at submicrometer resolution with a minimum of samples preparation.

Inflammatory signaling in human tuberculosis granulomas is spatially organized

Granulomas are the pathological hallmark of tuberculosis (TB). However, their function and mechanisms of formation remain poorly understood. To understand the role of granulomas in TB, we analyzed the proteomes of granulomas from subjects with tuberculosis in an unbiased manner. Using laser-capture microdissection, mass spectrometry and confocal microscopy, we generated detailed molecular maps of human granulomas. We found that the centers of granulomas have a pro-inflammatory environment that is characterized by the presence of antimicrobial peptides, reactive oxygen species and pro-inflammatory eicosanoids. Conversely, the tissue surrounding the caseum has a comparatively anti-inflammatory signature. These findings are consistent across a set of six human subjects and in rabbits. Although the balance between systemic pro- and anti-inflammatory signals is crucial to TB disease outcome, here we find that these signals are physically segregated within each granuloma. From the protein and lipid snapshots of human and rabbit lesions analyzed here, we hypothesize that the pathologic response to TB is shaped by the precise anatomical localization of these inflammatory pathways during the development of the granuloma.

Applications of MALDI-MSI to Pharmaceutical Research

MALDI-MSI has been demonstrated to be a suitable technique in pharmaceutical research for providing information of the distribution of low molecular weight compounds such as drugs and their metabolites within whole-body tissue sections. Important ADME information can be determined by MALDI-MSI analysis of the distribution of drugs and metabolites in whole-body tissue sections taken from animals killed at a range of time points postdose. In this example we applied MALDI-MSI to the localization of a compound and its primary metabolite in whole-body mouse sections.

Mass spectrometry imaging of biological tissue: an approach for multicenter studies

AbstractMass spectrometry imaging has become a popular tool for probing the chemical complexity of biological surfaces. This led to the development of a wide range of instrumentation and preparation protocols. It is thus desirable to evaluate and compare the data output from different methodologies and mass spectrometers. Here, we present an approach for the comparison of mass spectrometry imaging data from different laboratories (often referred to as multicenter studies). This is exemplified by the analysis of mouse brain sections in five laboratories in Europe and the USA. The instrumentation includes matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF), MALDI-QTOF, MALDI-Fourier transform ion cyclotron resonance (FTICR), atmospheric-pressure (AP)-MALDI-Orbitrap, and cluster TOF-secondary ion mass spectrometry (SIMS). Experimental parameters such as measurement speed, imaging bin width, and mass spectrometric parameters are discussed. All datasets were converted to the standard data format imzML and displayed in a common open-source software with identical parameters for visualization, which facilitates direct comparison of MS images. The imzML conversion also allowed exchange of fully functional MS imaging datasets between the different laboratories. The experiments ranged from overview measurements of the full mouse brain to detailed analysis of smaller features (depending on spatial resolution settings), but common histological features such as the corpus callosum were visible in all measurements. High spatial resolution measurements of AP-MALDI-Orbitrap and TOF-SIMS showed comparable structures in the low-micrometer range. We discuss general considerations for planning and performing multicenter studies in mass spectrometry imaging. This includes details on the selection, distribution, and preparation of tissue samples as well as on data handling. Such multicenter studies in combination with ongoing activities for reporting guidelines, a common data format (imzML) and a public data repository can contribute to more reliability and transparency of MS imaging studies. Comparison of MS imaging platforms in international multicenter study

Software tools of the Computis European project to process mass spectrometry images.

Among the needs usually expressed by teams using mass spectrometry imaging, one that often arises is that for user-friendly software able to manage huge data volumes quickly and to provide efficient assistance for the interpretation of data. To answer this need, the Computis European project developed several complementary software tools to process mass spectrometry imaging data. Data Cube Explorer provides a simple spatial and spectral exploration for matrix-assisted laser desorption/ionisation–time of flight (MALDI-ToF) and time of flight–secondary-ion mass spectrometry (ToF-SIMS) data. SpectViewer offers visualisation functions, assistance to the interpretation of data, classification functionalities, peak list extraction to interrogate biological database and image overlay, and it can process data issued from MALDI-ToF, ToF-SIMS and desorption electrospray ionisation (DESI) equipment. EasyReg2D is able to register two images, in American Standard Code for Information Interchange (ASCII) format, issued from different technologies. The collaboration between the teams was hampered by the multiplicity of equipment and data formats, so the project also developed a common data format (imzML) to facilitate the exchange of experimental data and their interpretation by the different software tools. The BioMap platform for visualisation and exploration of MALDI-ToF and DESI images was adapted to parse imzML files, enabling its access to all project partners and, more globally, to a larger community of users. Considering the huge advantages brought by the imzML standard format, a specific editor (vBrowser) for imzML files and converters from proprietary formats to imzML were developed to enable the use of the imzML format by a broad scientific community. This initiative paves the way toward the development of a large panel of software tools able to process mass spectrometry imaging datasets in the future.

Spatial Quantification of Drugs in Pulmonary Tuberculosis Lesions by Laser Capture Microdissection Liquid Chromatography Mass Spectrometry (LCM-LC/MS)

Tuberculosis is still a leading cause of morbidity and mortality worldwide. Improvements to existing drug regimens and the development of novel therapeutics are urgently required. The ability of dosed TB drugs to reach and sterilize bacteria within poorly-vascularized necrotic regions (caseum) of pulmonary granulomas is crucial for successful therapeutic intervention. Effective therapeutic regimens must therefore contain drugs with favorable caseum penetration properties. Current LC/MS methods for quantifying drug levels in biological tissues have limited spatial resolution capabilities, making it difficult to accurately determine absolute drug concentrations within small tissue compartments such as those found within necrotic granulomas. Here we present a protocol combining laser capture microdissection (LCM) of pathologically-distinct tissue regions with LC/MS quantification. This technique provides absolute quantification of drugs within granuloma caseum, surrounding cellular lesion and uninvolved lung tissue and, therefore, accurately determines whether bactericidal concentrations are being achieved. In addition to tuberculosis research, the technique has many potential applications for spatially-resolved quantification of drugs in diseased tissues.

Unraveling Drug Penetration of Echinocandin Antifungals at the Site of Infection in an Intra-Abdominal Abscess Model

Abstract Background Intra-abdominal candidiasis (IAC) is a prominent invasive fungal infection associated with high mortality. Prompt antifungal therapy and source control are crucial for successful treatment. Echinocandin antifungal drugs are first-line agents. Yet, their clinical effectiveness is highly variable with known potential for breakthrough resistance, and little is known about drug exposure at the site of infection. Using matrix-assisted desorption/ionization (MALDI) mass spectrometry imaging as well as standard analytical techniques, we investigated the spatial and quantitative distribution in tissue lesions for two echinocandin drugs, micafungin and CD101, in a clinically relevant IAC mouse model. Methods Female 6–8 week old CD1 mice weighing 18–22 g were infected intraperitoneally (IP) with 1 × 107 CFU of C. albicans SC5314 mixed with sterile stool matrix. Single IP doses of CD101 at 5 or 20 mg/kg (equivalent to humanized therapeutic dose) or micafungin at 5 mg/kg (therapeutic dose) were administered to mice at day 3 post-inoculation. Mice were sacrificed at just before antifungal treatment (n= 1), and at 1, 3, 6, 24, and 48 hours post-dose (n = 3 per group per time point). Liver and kidney lesions were collected for MALDI imaging. Laser capture microdissection (LCM) followed by liquid chromatography coupled tandem mass spectrometry (LC/MS-MS) was applied to 6 and 24 hours samples for drug exposure measurement. In a separate experiment, mice were treated with 2 or 3 doses of micafungin (5 mg/kg), or a single dose of CD101 (20 mg/kg). Drug accumulation was analyzed at 48 and 72hours post the first dose. Results Drug accumulation within lesions was observed with both drugs at their humanized therapeutic dose. However, micafungin, even at steady-state, failed to approach the mutant prevention concentration (MPC) (16 µg/mL) of the infecting strain. CD101 demonstrated extensive penetration into the lesions after a single dose administration and persisted in lesions at above MPC level of 29.7 µg/mL at 72 hours postdose. Conclusion These findings indicate that current echinocandin drugs may be limited by penetration at the site of infection, which have implications for clinical outcomes and emergence of resistance in patients with IAC. Disclosures C. J. Clancy, Merck: Received research funding, Research support; Astellas: Received research funding, Research support; Cidara: Received research funding, Research support; Astellas: Scientific Advisor, Advisory board; Merck: Scientific Advisor, Advisory board; Cidara: Scientific Advisor, Advisory board; Medicines Company: Scientific Advisor, Advisory board; D. Perlin, Cidara: Research Contractor and Scientific Advisor, Research grant; Amplyx: Research Contractor and Scientific Advisor, Research grant; Matinas: Scientific Advisor, Research support; Scynexis: Research Contractor and Scientific Advisor, Research grant; Merck: Research Contractor, Research grant; Astellas: Research Contractor, 
Research grant

Abstract C61: Non-homogeneous drug penetrance of veliparib measured in triple negative breast tumors

Background: Veliparib, an inhibitor of Poly(ADP-ribose) polymerase (PARPi), in combination with carboplatin showed efficacy in triple negative breast cancer (TNBC) patients in the I-SPY 2 TRIAL. However ∼42% of TNBC did not achieve pathologic complete response. Insufficient uptake of drug in TNBC may lead to inadequate response to PARPi. As a first step toward testing this hypothesis in patients, we quantified veliparib penetration in mouse xenograft models of TNBC. Methods: MDA-MB-231, HCC70 or MDA-MB-436 human TNBC cells were implanted in 41 beige SCID mice. Veliparib at low dose (20mg/kg) or high dose (60mg/kg) and carboplatin (60mg/kg) was given three times daily for three days. MR images were taken at day 1. Plasma, fresh frozen and OCT embedded tissues were analyzed using Matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI) Liquid chromatography–mass spectrometry (LC-MS). Drug penetration was compared among doses and cell lines. Results:Ex vivo veliparib concentrations quantified by LC-MS differed significantly among the tumors derived from the three cell lines. Liver and plasma concentrations were uniformly high in all mice compared to tumor and muscle tissues. Plasma pharmacokinetics in mice exhibited non-linear clearance resulting in prolonged high plasma levels at higher doses, while tumor and plasma concentrations were linearly correlated. MALDI-MSI images of tumor and muscle in 12 mice showed higher veliparib concentrations in necrotic areas compared to areas with viable tumor cells (P = 0.126, Table) and higher concentrations at the rim then in the center of the tumor (P = 0.046). Lower concentrations were found in MDA-MB-231 than in other cell lines (0.008). Contrast agent and veliparib accumulated near the rim of the tumors and a fast elimination of the contrast agent from the tumor correlated with relatively low veliparib tumor concentrations. Conclusions: The spatial distribution of veliparib in TNBC depends on the dose and tumor cell biology. We demonstrated that MALDI-MSI can be used to measure veliparib penetration tumor samples, which may have potential to monitor response to PARPi therapies. Table: Veliparib concentrations by LC-MS and its spatial distribution by MALDI varies by tissue, drug dose and TNBC cell line of origin. Citation Format: Imke H. Bartelink, Brendan Prideaux, Gregor Krings, Lisa Wilmes, Pei R.E. Lee, Byron Hann, Jean-Philippe Coppe, Diane Heditsian, Lamorna Swigart-Brown, Ella F. Jones, Sergey Magnitsky, Ron Keizer, Laura Esserman, Weiming Ruan, Alan Wu, Douglas Yee, Veronique Dartois, Denise Wolf, Rada Savic, Laura vantVeer. Non-homogeneous drug penetrance of veliparib measured in triple negative breast tumors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C61.