Mass spectrometry Imaging brain images analyses drug efficacy

Mapping Molecules in Central Nervous System (CNS) using Mass Spectrometry Imaging (MSI): Pharmacokinetics & Pharmacodynamics Applications.

Does the drug candidate reach amyloid plaques or does a repeat dose of drug candidate decrease the amyloid plaques formation? Is my drug candidate molecule reaching specific region of the brain and what are the drug and metabolite concentrations at different time points?

Introduction:
To answer these questions, ImaBiotech has developed different approaches to look at your drug candidate and to evaluate its distribution, its quantification, as well as for its drug metabolites[1] and biomarker in a single experiment, providing a fast and effective way to assess efficacy or potential toxicity of your molecules. Mass Spectrometry Imaging (MSI) is widely used to study the brain and consequently all central nervous system processes or dysfunctions. Numbers of pathologies target the brain and the central nervous system have been studied using MSI such as Alzheimer’s [2], Parkinson’s [3], Gaucher’s [4], Fabry’s [5] diseases, Migraine, Pain, Psychiatric Diseases, Sleep Disorders…In this context, specific molecular variation closely related to pathological development also known as disease biomarkers can be assessed by this emerging molecular imaging
technique. In preclinical drug development, MSI provides pharmacokinetics and pharmacodynamics information about a drug targeting specific anatomical regions of the brain using high definition images.

Drug and its metabolites can be localized and can be discriminated
on the same molecular image, as well as some biomarkers of
toxicity, inflammation or disease state. Several applications will
illustrate the benefits given by MSI in CNS studies and are listed
below:

  • Amyloid Beta (A) peptide localization in Alzheimer’s
    brain model: Study of efficacy by Mass Spectrometry
    Imaging
  • Distribution & quantification of a Alzheimer’s drug candidate and its metabolites in brain substructures: Quantitative mass spectrometry application (QMSI)
  • Molecular histology of the brain substructures with biologi
    cal implications: High lateral Resolution Mass Spectrometry Imaging (HR-MSI)

Amyloid Beta peptide localization in Alzheimer brain model:
study of efficacy by Mass Spectrometry Imaging

Experimental Section:
Animal: Brain from transgenic mouse model (Tg2576, 7 months) was removed; snap frozen and stored at -80°C.

Sectionning: Brain was sectioned following the coronal plan (10 microns)  using Microm HM560 cryostat (Thermo Scientific, Germany) at -20°C and mounted on ITO conductive glass slides (Bruker Daltonik, Germany). Prior to analyzes, washing step protocol [6] was applied on tissue section to increase detection of peptides. HE Staining was performed after MSI acquisition for better visualization of histological regions and amyloid plaques.

Matrix: SA (30 mg/ml AcN/W+TFA 0.2% 1:1; v:v) was chosen and
deposited manually using TLC sprayer (Sigma, France).

Mass spectrometry imaging: Solarix 7.0T FTICR (Bruker Daltonik,
Germany) with SmartBeam II laser. Full scan Positive mode (800-6000 Da) at 100 μm spatial resolution.

Software: FlexImaging 3.0 (Bruker Daltonik, Germany) & Quantinetix 1.4 (ImaBiotech, France).

Results & Discussion:

  • Amyloid beta peptides are the main component of amyloid
    plaques which are found in the brains of patients with Alzheimer’s
    disease. A Beta could therefore be hypothesized associated to Alzheimer disease state biomarkers or pathology’ consequences. The
    accurate localization of Ab may be useful to monitor the efficiency
    of a drug targeted specific Alzheimer’s disease regions in the brain.
  • Amyloid beta peptides were observed with sporadic (hot spots)
    distribution in transgenic brain section at the level of cortex and
    hippocampus region as shown in figure 1. Several A! forms
    including (1-37, 1-38, 1-39, 1-40 and 1-42) have been detected on
    mass spectra and followed on molecular images from figure 1. A!
    was also detected on sagittal brain section from transgenic mouse
    (data not shown).
  • High degree of confidence in the identification of amyloid beta
    peptides was achieved thanks to high mass accuracy measurement
    capacity of FTICR mass spectrometer. Mass measurement accuracy was calculated at low ppm level (between 0.4 & 3.8) on 5Abéta forms and reported in table from figure 1.
  • Co-localization between A béta 1-42 peptides and amyloid plaques was observed on optical and molecular images (black arrows on HE staining indicate amyloid plaques). This result highlights two MSI
    capabilities: the complementarity of MS imaging and histological (or
    immunohistochemistry) imaging techniques for disease biomarkers
    hunting and capacity of drug distribution with biomarkers of efficacy.
  • In combination with classical LC-MSMS, MS imaging provides a
    fast answer to your questions of drug distribution, biomarker
    distribution and quantification at a high resolution. ImaBiotech has
    developed a dedicated service to assess drug efficacy or toxicity by
    combining drug distribution and biomarkers directly from an in vivo
    study.

 

 

Distribution & quantification of a Alzheimer’s drug candidate and its metabolites in brain substructures: Quantitative mass spectrometry application (QMSI)

Experimental Section:

Animal: C57 Black6 wild type mice were dosed with the drug and brains
were removed, snap frozen and stored at -80°C.

Sectioning: Brains were sectioned following the saggital plan
thickness) using Microm HM560 cryostat (Thermo Scientific, Germany) at
-20°C and mounted on ITO conductive glass slides (Bruker Daltonik, Germany). Washing of sections with chloroform was performed (30 seconds) for better visualization of histological regions.

Matrix: 2,5 DHB (40mg/ml Methanol/TFA 0.1% 1:1; v:v) was chosen and
deposited using SunCollect device (Sunchrom, Germany).

TEC Determination & Calibration: Dilution range of drug between 0.02 – 10 μM, 1 μL of standards deposited near dosed section on the slide. Drug concentration for TEC experiment was set at 10 μM and deposited on control section. TEC was calculated according methodology from Hamm et al [1].

Mass spectrometry imaging: Solarix 7.0T FTICR (Bruker Daltonik,
Germany) with SmartBeam II laser. Positive CASI mode (50 Da Window) at 80 μm spatial resolution.

Software: FlexImaging 3.0 (Bruker Daltonik, Germany) & Quantinetix 1.4
(ImaBiotech, France).

Results & Discussion:

  • The second MSI application is focused on a candidate drug against Alzheimer’s disease targeting specific regions of the brain.
    This study is closely related to the previous example (n°1) on Amyloid beta peptides localization; in fact, it is possible to evaluate the action of a drug on its environment or on potential disease state biomarkers such as A#s. Thereby MSI may play a significant role in understanding ‘target engagement’ in early phases of drug discovery. This example deals with a sponsor candidate drug study, which is still under development and therefore requires confidentiality.
  • The candidate drug shows a specific distribution in the hippocampus, the 3rd and 4th ventricles, the septum, the plexus choroid and in the medio-ventral hypothalamus. The drug identification was validated by on tissue mass spectrometric fragmentation experiment (MS2) highlighted attended mass fragments detection. These histological regions of the brain could be involved in Alzheimer’s disease development and consequently could demonstrate the efficiency of drug targeting in CNS.
  • MSI provides a global approach: gives information not only on drug distribution as previously explained, but also provides their metabolites. Based on preliminary microsomal assays of drug
    metabolism, we are able to discover some mass to charge ratio (m/z)
    related to potential metabolites of our drug, directly from the original
    MSI data set (no additional experiment needed). Therefore, a drug
    metabolite was detected in the choroid plexus and the medio-ventral
    hypothalamus, which closely co-localized with its parent compound.
    This example shows MSI benefits compared to classical molecular
    imaging techniques such as QWBA, which cannot discriminate the
    parent drug and its metabolite.
  • Finally, using quantitative mass spectrometry imaging [1] and
    Quantinetix software, we are able to determine the amount of the
    drug directly in small histological structures of the brain. In this
    example, the drug is quantified in the medio-ventral hypothalamus
    and in the plexus choroid at some μg/g of tissue level whereas other
    histological regions have weak concentration. As for example the
    hippocampus, the amount of drug was calculated at 119 pmol/g of
    tissue which corresponds to approximately 50 ng/g of tissue. This
    value was cross validated by LC-MS quantitative measurement on
    corresponding hippocampus homogenate of the same animal (139
    pmol/g of tissue).

Distribution & quantification of a Alzheimer’s drug candidate and its metabolites in brain substructures: Quantitative mass spectrometry application (QMSI)

 

Experimental Section:

Animal: Brains from control wild type rat were removed; snap frozen and
stored at -80°C.

Sectioning: Brains were sectioned following sagittal and coronal plan (10
microns of thickness)using Microm HM560 cryostat (Thermo Scientific, Germany)  at -20°C and mounted on ITO conductive glass slides (Delta
Technology USA).

Mass spectrometry imaging: Autoflex Speed LRF MALDI-TOF (Bruker, Daltonik, Germany) with SmartBeam II Laser. Positive mode at 150/300 or 20 μm spatial resolution.

Software: FlexImaging 4.0 (Bruker Daltonik, Germany) & Quantinetix 1.4 (ImaBiotech, France)

Results & Discussion:

  • Instrumental development for mass spectrometry imaging allows
    improving spatial resolution of the generated molecular image
    especially thanks to laser beam  spot diameter decreasing and new matrix deposition device. The sublimation process allows
    achieving a thin matrix layer (1micron of thickness) with small matrix  crystals (less than 1 μm of diameter). These parameters induce a minimized analyte spreading within tissue and combining with high focused laser, creates a high spatial resolution mass spectrometric images.
  • Two examples of a coronal and sagittal brain sections images are
    presented at low spatial resolution, 150 and 300 microns respectively, in figure 3. Some contrast ionic species are used to differentiate simply large histological structures of the brain. From one hand, white (green filter) and grey (red filter) matters are easily discriminated on overlay molecular image from coronal section. From the other hand, cerebellum region is well localized from the rest of the  sagittal brain section (red filter). Low spatial resolution images allow observing only large histological regions while high spatial resolution can give higher level of interpretation.

Conclusion:
Showing different applications related to Alzheimer’s disease, we demonstrate the utility of MSI to answer specific CNS
questions The combination candidate drug, metabolites and disease state biomarker (A!) study demonstrates the type of reliable information provided by MSI. Moreover, biological processes can be assessed directly on tissue sections, such as the formation of amyloid plaques correlated with Amyloid beta peptides detection. Quantitative measurement of the drug candidate in small histological regions using MSI prevents the challenging and time consuming dissection steps. Finally, high resolution molecular imaging allows following molecules at the cellular level such as neurons or other cells, which could be related to some specific brain diseases.

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