Descripción del montaje de las piezas arqueológicas de las culturas prehispánicas de

Information regarding the distribution of drug-related material in target tissue may provide important biological information during the drug development process. Industry-standard methods used to look at total localisation of drug-related material can have limitations due to their dependence on labels, either fluorescent tags or radioactivity (e.g. quantitative whole body autoradiography (QWBA)). These approaches may have difficulty in distinguishing between the drug of interest and its metabolites. Mass spectrometry imaging (MSI) is a technique that has the potential to distinguish spatially between the drug of interest and its metabolites. A number of mass spectrometry-based imaging experiments have been described for localisation of drug compounds and metabolites in tissue. These experiments have been shown to provide data complementary to existing imaging techniques.

In this work, two-mass spectrometry based imaging approaches have been evaluated for the identification and localisation of two drug compounds. The first section focused on the identification and localisation of fenclozic acid, with evaluation and comparison of two imaging MS experiments. This work has been published in the peer-reviewed journal Xenobiotica (Blatherwick, Van Berkel et al. 2011).

The second section investigated other applications. Optimisation experiments were performed in order to determine the most selective method for the localisation of diclofenac (a common non-steroidal anti-inflammatory drug). These methods were evaluated for their ability to localise diclofenac in dosed whole body tissue sections and harvested tissue, for comparison with existing QWBA data.

The ionisation techniques used in the imaging MS experiments were Matrix-Assisted Laser Desorption/Ionisation (MALDI) and liquid extraction surface analysis (LESA). This was employed using a chip-based robotic nanoelectrospray platform (TriVersa NanoMate, Advion).

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3.1.1 Fenclozic Acid

Fenclozic acid is a low molecular weight acidic non-steroidal anti-inflammatory drug (NSAID) (ICI 54, 450: Myalex) (Hepworth, Newbould et al. 1969). The chemical structure of fenclozic acid is shown in Figure 3.1. This drug was withdrawn from the market following documented hepatotoxicity (Hart, Bain et al. 1970). It is important, therefore to gain a better understanding of its disposition in tissues and metabolic fate.

Figure 3.1 Chemical structure of fenclozic acid.

An original study into fenclozic acid metabolism showed five metabolites that were identified in rats and dogs. These metabolites included decarboxylated fenclozic acid and metabolites with oxidation and hydroxylation at the chlorine substituent (Foulkes 1970).

3.1.2 Diclofenac distribution in mice

Diclofenac is a common non-steroidal anti-inflammatory drug (NSAID), often used as a painkiller for arthritis. The chemical structure of diclofenac is shown in Figure 3.2 below.

86 Diclofenac has been shown to undergo hepatic metabolism in mice via both cytochrome P450 (CYP2C9) and uridine 5'-diphospho-glucuronosyltransferase (UGT) mediated pathways to produce potentially reactive intermediates shown in Figure 3.3. Other major metabolites of diclofenac have been shown to include 4’- hydroxydiclofenac and 5’-hydroxydiclofenac (Tang 2003).

Figure 3.3 Metabolism of diclofenac by CYP and UGT mediated pathways, forming potentially reactive intermediates.

A study was completed by AstraZeneca UK in order to assess the differences in disposition and hepatic effects of 14C-diclofenac in hepatic P450 null reductase (HRN) mice compared with wild-type mice. These mice do not have the cytochrome P450 enzyme in the liver. This prevents metabolism via the CYP-mediated pathway. The study used a combination of techniques: QWBA, for drug localisation and conventional metabolite identification from urine using LC-MS methods.

These metabolite identification results highlighted a lack of oxidative metabolites identified from the HRN mice. An increase in glucuronide and taurine conjugation of metabolites was also observed. This was expected based on known metabolism pathways.

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Figure 3.4 QWBA images of diclofenac distribution.

a) QWBA results showing radioactivity distribution in a 14C-diclofenac dosed wild- type mouse sacrificed 3 hours post-dose.

b) QWBA results showing radioactivity distribution in a 14C-diclofenac dosed hepatic P450 null reductase (HRN) mouse sacrificed 3 hours post-dose.

Darker regions represent higher levels of radioactivity. Pink areas represent over- exposure of the phosphor-imaging plate as a result of high concentrations of radioactivity. QWBA images reproduced with permission from AstraZeneca.

Significant differences in metabolism and rates of excretion were observed between the wild type and HRN strains. QWBA results showed widespread radioactivity distribution for wild type and HRN mice sacrificed 3 hours post-dose, these are shown in Figure 3.4 a) and b) respectively.

The QWBA results for the wild-type mouse in Figure 3.4 showed widespread radioactivity throughout the tissues, with a moderate presence in the liver and elevated presence in the caecum, kidneys, intestines and the stomach. The section shown on the right indicated high levels of radioactivity present in the urine and the faecal pellets. Noticeably the brain showed little presence of radioactivity.

88 QWBA results for HRN mice showed widespread radioactivity throughout the tissues, with a moderate presence observed in the liver, caecum, heart, lung, kidneys, intestines and faecal pellets. Over-exposure was observed in the urine and stomach. The QWBA results showed differences in the distribution of radioactivity between wild type and HRN mice, but they could not determine whether this was due to the presence of parent drug, or metabolite compounds within the tissue. Mass spectrometry imaging approaches may help to distinguish between parent drug and metabolites in tissue, and answer important questions surrounding the observed differences in distribution between the two groups of mice.

Here, optimisation experiments have been performed both on and off tissue using two different imaging mass spectrometry approaches, to identify the most selective method for localisation of diclofenac. The optimised MALDI-MS and LESA-MS methods have been used for the detection and localisation of diclofenac in whole body tissue sections and kidney sections from dosed mice. Both techniques were coupled to a travelling wave ion mobility (TWIM) Q-TOF (Synapt G2, Waters) mass spectrometer.

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