Mass-spectrometry-imaging-wholebody

Biodistribution, Drug Metabolism and Biomarkers Hunting in Drug Discovery: What can mass spectrometry imaging provide?

Introduction:

In vivo Drug efficacy and toxicity evaluations are key successes of drug development. It is crucial to evaluate how much drug arrived to the site of action; and what are the positive and adverse effects on organs or cells functions ? Several in or ex vivo techniques are used to obtain these Pharmacokinetics/Pharmacodynamics (PK/PD), Target Engagement and Toxicity effects such as the MRI (Magnetic Resonance Imaging) which gives morphological and anatomical information about tissues changes after a drug treatment or a disease but doesn’t provide specific drug effects with pathway activations. Positron emission tomography (PET) is another technique to obtain PK and PD information but need some costly radiolabeling step as well as whole-body autoradiography (WBA) which can’t distinguish the drug to its metabolites. Recently, a new molecular imaging technology called Mass Spectrometry Imaging (MSI) combines unmatched skills in preclinical imaging landscape (MRI, PET, Autoradiography, Bioluminescence imaging, Computational Tomography), upon 4 levels of studies:

  • PK information: Specific distribution of drug and its metabolites – Quantification of these molecules – Metabolism, clearance, exposure studies of drug candidate in tissue without labeling in combination with histology.
  • Target engagement information: Drug distribution and quantification with its target using MSI technique.
  • PD information: Tracking biomarkers of efficacy or toxicity – Can be combined with PK and target engagement studies.
  • Toxicity information: Drug distribution and metabolites
    correlated to pathologist information (histology) to validate mechanism of action

MSI is a translational analytical technique which offers the unique capability to provide a broad range of information for drug discovery process in a single fast and reliable experiment. This application note focuses PK/PD capabilities of a psychotropic drug, the clozapine (CLZ). In this study, the MSI provides the in-situ characterization of drug and drug’s metabolites within tissue section which allows the global understanding and interpretation of ADME studies. Moreover, targeted and untargeted analysis of molecular modulation within tissue section and relate metabolite signature to histological area of the whole body will be highlighted.

Experimental Section:

  • Animal: Male C57Bl6 wild type mouse was dosed dosed with
    clozapine P.O. (100mg/kg) and sacrificed 30 min post dosing; snap
    frozen and stored at -80°C.
  • Sectioning: Mouse whole body was sectioned following sagittal
    plan (20 microns of thickness) using Microm HM560 cryostat (Thermo Scientific, Germany) at -20°C and mounted on adhesive double side tape and ITO conductive glass slides (Delta Technology USA).
  • Matrix: 2,5 DHB (40mg/ml Methanol/TFA 0.1% 1:1; v:v) was
    chosen and deposited using SunCollect device (Sunchrom,
    Germany).
  • Mass spectrometry imaging: Solarix 7.0T FTICR (Bruker Daltonik, Germany) with SmartBeam II laser. Positive fullscan mode
    (100-800 Da), 300 shots at 300 μm spatial resolution (18135 voxels
    in 8 hours).
  • Software: All presented MS images are from QuantinetixTM
    software 1.7 (ImaBiotech, France)

Results: Clozapine (CLZ) is a tricylic dibenzodiazepine belonging to the chemical class of benzisoxazole derivatives and is indicated for the treatment of schizophrenia. As selective monoaminergic antagonist, CLZ displays a high affinity for the serotonin Type 2 (5HT2), dopamine Type 2 (D2), 1 and 2 adrenergic, and H1 histaminergic receptors. The mechanism
of action of Clozapine is based on the modulation of these receptors especially for D2 and 5HT2 which are strongly involved in psychotic disorder. Figure 1 highlights the localization of CLZ in discrete organs of the whole-body (more than 15 tissues). Full characterization of CLZ was established directly on tissue section using MS/MS experiment on CLZ related ion, m/z 327.1367. Figure 2 shows the corresponding mass spectrum obtain using CID fragmentation method, several daughter ions are observed in agreement with fragmentation pathway of CLZ. In combination with high mass accuracy measurement of FTICR analyzer (low ppm level), the univocal identification of CLZ was achieved (Figure 3). Moreover, the spectral resolution power of the instrument (up to 200 000) avoids potential interferences due to surrounding biological matrix. Relative quantification of the drug can be then obtained, CLZ shows higher signal in liver, kidney and lung (stomach excluded) than in brain, its targeted tissue. Biotransformation products of CLZ have also been monitored. The two main CLZ metabolites, N-desmethyl-Clozapine (NDCLZ) also known as norclozapine and Clozapine N-oxide (CLZ-NO) are observed in Figure 1 (right panel). Interestingly, metabolite distributions are quite different from the parent drug distribution. CLZ NO or NDCLZ was not detected at the level of the brain at this particular time point. Some differences between NDCLZ and CLZ-NO distributions have been observed localized in the whole kidney and only in the inner medulla region, respectively. These results are consistent with previous published studies on Clozapine distribution using MSI [1, 2, 3] and cross-validated with LC/MS analyses. Thus, reliable and robust pharmacokinetics data can be obtained from a single MSI experiment to ensure the right targeting of the drug and of its metabolites within tissue section.

Whole-body imaging using MSI gives access to a wide range of endogenous molecule classes specific to histological regions of the tissue. Figure 3 listed several molecules of interest with their corresponding adduct form and mass accuracy measurement. We can observe on Figure 4 some preferential histological distributions of these molecules within tissue section which can be used to obtain a molecular histology of the sample. Heme b is a robust marker of blood mainly distributed in surrounded tissue and in the heart. Lipids such as PCs are well ionized in mass spectrometry and in MSI their distribution are often correlated with specific tissue type. PCs are detected in spinal cord and in the brain with some differences concerning saturated one more concentrated in the cerebral cortex than is the spinal cord. Ceramide phosphate ion (CerP) is mainly distributed in skin layer around the whole-body as shown in Figure 4. CerP is involved in skin disease disorders (in psoriasis for example) as mediator of the inflammatory response. Carnitine plays a vital role in muscle cell metabolism, because it serves as a carrier for energy substrates through mitochondrial membranes. It may be modulated in disease such as diabetes, cardiomyopathy, cirrhosis… The tracking of carnitine regulation in tissue after the administration of a drug could give information about its efficiency of action. Aspartate which is localized in the brain as well as glutamate (data not shown) serves as marker of brain disorder involving the modulation of Aspartate transaminase especially in Alzheimer disease. Gluthatione (GSH) which is spatially distributed within eye tissues plays a key role in maintaining normal hydration level and ensuring cellular membrane integrity. Changes of GSH activity in eye tissues can occur with some disorders such as ageing, cataract, diabetes, but also following the administration of some drugs. In this case, GSH can act as biomarkers of dysregulation of biological process in tissue. In comparison with a vehicle-control whole body, without drug administration, it is possible to follow up or down regulations of these potential markers within tissue and thus to gain precious information about the pharmacodynamics of your drug candidate.

Conclusion:

With this example of CLZ biodistribution in a mouse whole body, we describe the potential of MSI to provide PK/PD information. As opposed to classical preclinical imaging techniques, MSI gives access, in one single experiment, to multiple information about the behavior of your drug candidate in an organism. MSI o’ers di’erential localization and relative concentration of the drug and its metabolites. MSI also permits to follow disease state biomarker modulation or evaluate readout molecules changes depending on treatment applied

Drug discovery

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