Original article
Utility of quantitative whole-body autoradiography (QWBA) and imaging mass spectrometry (IMS) by matrix-assisted laser desorption/ionization (MALDI) in the assessment of ocular distribution of drugs

https://doi.org/10.1016/j.vascn.2010.10.003Get rights and content

Abstract

Introduction

Assessment of drug candidate properties and potential liabilities can greatly benefit from issue driven studies that are designed to address specific toxicological effects such as ocular phototoxicity. If a compound absorbs light in the wavelength range of 290–700 nm (UV-A, UV-B, and visible light) and generates a positive response in a standard in vitro neutral red uptake phototoxicity assay in Balb/c 3T3 mouse fibroblasts, a single-dose in vivo study may be conducted to assess the potential for drug-induced phototoxicity in the eyes and skin of pigmented Long–Evans rats. Critical to ocular phototoxicity assessment is the hypothesis that the drug or drug-related material must be present in the affected substructures such as the uveal tract, retina, lens, or cornea. For compounds that induce a positive ocular response in the in vivo phototoxicity assay, data on distribution patterns to substructures of the eye can inform decisions regarding the nature of the ocular findings and possibly influence compound advancement.

Methods

Quantitative whole-body autoradiography (QWBA) and imaging mass spectrometry (IMS) by matrix-assisted laser desorption ionization (MALDI) on an ion trap mass spectrometer employing higher order mass spectrometric scanning functions were utilized for localization of dosed drug or metabolites in eye substructures.

Results

In investigative studies designed to simulate an in vivo phototoxicity study, rats were administered radio-labeled test article for QWBA analysis and un-labeled test article for IMS analysis. Autoradiograms from the QWBA study indicated that the radio-labeled analyte(s) preferentially distributed to the uveal tract and not the cornea. However, QWBA did not provide information on the nature of the detected analyte(s); i.e. intact parent drug versus potential metabolites or degradants. Multistage MS experiments performed directly on tissue sections demonstrated semi-quantitative localization in the uveal tract and unequivocal identification of the analyte as the dosed parent drug; no potential metabolites were detected.

Discussion

Image analysis by QWBA and IMS by MALDI proved complementary in the localization and identification of small molecule drug distribution within the eye.

Introduction

In the discovery and development process of novel chemical entities, potential compound liabilities are investigated and findings are used to iteratively improve the drug properties of the candidates and to better predict safety and efficacy in humans. In addition to the routine and standardized in silico, in vitro, and in vivo ADME (absorption, distribution, metabolism and elimination) assays, a selective approach might be chosen to assess specific toxicological effects. For example, if a compound absorbs light in the wavelength range of 290–700 nm (UV-A, UV-B, and visible light) and generates a positive response in a standard in vitro neutral red uptake phototoxicity assay in Balb/c 3T3 mouse fibroblasts, an in vivo study may be conducted to assess the potential for drug-induced phototoxicity in the eyes and skin of pigmented Long–Evans rats. Often, the in vivo phototoxicity assay is only conducted with systemically administered drugs that are known to distribute to the eyes and/or skin (FDA Guidances (Drugs) — Pharm/Tox — Guidance for Industry & Photosafety, 2003). For compounds that induce a positive ocular response in the in vivo phototoxicity assay, data on distribution patterns to substructures of the eye (such as the uveal tract, lens, and/or cornea) can inform decisions regarding the nature of the ocular findings and possibly influence compound advancement.

Quantitative whole-body autoradiography (QWBA) can detect the location and thus generate a tissue distribution image of the radio-labeled dosed analyte as well as provide quantitative information on tissue levels of the analyte; however, it cannot distinguish between the parent compound and possible metabolites or degradants (Solona and Kraus, 2002, Solona and Lee, 2002, Ullberg and Larsson, 1981, Trim et al., 2008, Solon et al., 2010). A complementary label-free technique to resolve compound identity is the imaging mass spectrometry (IMS) by matrix-assisted laser desorption/ionization (MALDI) (Trim et al., 2008, Solon et al., 2010, Drexler et al., 2007, Grey et al., 2009, Heeren et al., 2009, Hsieh et al., 2007, msimaging, 2010). The mass selective detection of analyte specific product ion spectra generated by multiple stage mass spectrometry (MS) can afford the identification (qualitative MS) and an ion density map (relative quantitative MS) of the analytes in the tissue section.

An in vivo phototoxicity study with a proprietary compound resulted in equivocal, corneal-specific lesions. Investigative studies were designed to assess tissue distribution (specifically looking at substructures of the eye) of systemically administered drug and its potential metabolites using complementary application of QWBA (radio-labeled drug) and IMS by MALDI ion trap MS (un-labeled drug). Data from QWBA analysis provided a quantitative distribution of a drug-related radio-labeled material in the tissue; however, the nature of the detected analyte(s), whether intact parent drug or potential metabolites or degradants could not be determined. Multistage MS experiments performed directly on eye tissue sections demonstrated semi-quantitative localization in the uveal tract and no accumulation in the cornea. The analyte was unequivocally identified as the dosed parent drug and no metabolites were detected.

Section snippets

Animals

These studies were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals (NIH publication No. 85–23, revised 1996) and were approved by the Bristol-Myers Squibb Company Site Institutional Animal Care and Use Committee. Female pigmented Long–Evans rats (approximately 14 weeks old at study initiation) were purchased from Charles River Laboratories, Wilmington, MA. All animals were housed individually in stainless-steel wire-bottom cages in environmentally controlled

Quantitative whole-body autoradiography (QWBA)

The autoradiograms of whole-body or eye sections (Fig. 1) indicated that the radio-labeled analyte(s) preferentially distributed to the back of the eye (retina and/or uveal tract), with very little present in the cornea and lens. Since QWBA actually detects only the radio-label itself, no information is obtainable on the molecular species of the analyte(s).

Imaging mass spectrometry (IMS) by matrix-assisted laser desorption/ionization (MALDI)

The IMS by MALDI analysis was performed by alternating a universal full scan MS method to survey the sample and targeted full scan MS/MS/MS

Discussion

The ocular distribution of a drug and its potential metabolites was assessed by employing the complementary technologies of quantitative whole-body autoradiography (QWBA) and imaging mass spectrometry (IMS) by matrix-assisted laser desorption/ionization (MALDI) in support of equivocal corneal-specific in vivo phototoxicity findings. While QWBA provided the quantitative localization of the drug-related radio-labeled species in the tissue samples, the use of IMS by MALDI confirmed the

References (11)

There are more references available in the full text version of this article.

Cited by (35)

  • Mass spectrometry imaging of diclofenac and its metabolites in tissues using nanospray desorption electrospray ionization

    2022, Analytica Chimica Acta
    Citation Excerpt :

    Therefore, the metabolites or possible degradation products cannot be distinguished from the precursor compound. Furthermore, if the radiolabel is lost during the metabolism process, these metabolites cannot be detected [1,2,5]. To overcome the limitations of requiring a radiotracer for QWBA, mass spectrometry imaging (MSI) has been implemented to determine drug and metabolite distributions in tissues [1,5–9].

  • An analysis of the biopharmaceutical behaviour of proton pump inhibitors with different physicochemical properties

    2021, Life Sciences
    Citation Excerpt :

    Then, two representative PPIs were radiolabelled with 14C. Their biodistribution in various tissues, especially in the stomach, was studied by radioactivity counts and quantitative whole-body autoradiography (QWBA) [17]. 14C-Ilaprazole(equaling 5.70 mCi/mmol) was synthesized by Shanghai Qizhen Environmental Technology Co., Ltd. 14C-Esomeprazole(equaling 2.84 mCi/mmol) was synthesized by Shanghai Qizhen Environmental Technology Co., Ltd.

  • MALDI imaging of the eye: Mapping lipid, protein and metabolite distributions in aging and ocular disease

    2016, International Journal of Mass Spectrometry
    Citation Excerpt :

    Not only was a novel methodology reported to co-register the MALDI IMS data with complementary optical imaging, and reconstruct the three dimensional volume from the series of two dimensional MALDI IMS data sets, but differences in protein signals from the disease model were also highlighted. With the view towards treating ocular pathologies with drug interventions, it is worth noting that some exogenous compounds and drugs have already been spatially mapped in ocular tissues with MALDI IMS [41–43]. In addition, benzalkonium chlorides (BACs), which are common antimicrobial compounds found in topically administered eye drops, have previously been identified and spatially mapped in human retina using on-tissue tandem mass spectrometry on a MALDI-QIT instrument [27], and in multiple ocular tissues in the rabbit eye on MALDI-TOF instrumentation [44].

  • MALDI imaging mass spectrometry: Spatial molecular analysis to enable a new age of discovery

    2014, Journal of Proteomics
    Citation Excerpt :

    In one example, the authors demonstrate that this method is comparable to similar analysis by LC MS/MS for rat liver from a rat dosed with the drug nevirapine (Fig. 7). Even in cases when robust methods for quantitation are established, additional challenges will certainly arise for special cases such as whole body imaging [48,49,56–59], where diverse tissue types are sampled simultaneously and 3D imaging [60–62], in which the signal intensities and variations in sample preparation must be normalized throughout several tissue sections. Finally, it should be noted that the development of quantitative methods in MALDI IMS is primarily driven by an interest to quantitate small molecule pharmaceuticals in tissue and therefore most quantitation methods are developed for analysis of exogenous compounds in tissue from dosed animals.

View all citing articles on Scopus
View full text