Mass spectrometry Imaging skin tissue compared to histological tissue

Multimaging™ Applied to Skin Distribution Study: Applications in Dermatology for Pharmaceuticals and Cosmetics from Discovery to Clinical

Multimaging Skin

Introduction:

In pharmacology, drugs can be applied to skin for two purposes: 1) to directly treat disorders of the skin 2) to deliver drugs to other tissues (i.e transdermal application). Cosmetics (care substances) can also be applied to skin; however, they are typically used to enhance the appearance of the human skin. Care substances are generally mixtures of chemical compounds; with some being derived from natural sources and many being synthetics. Knowledge of different forms and physicochemical properties of compounds, as well as understanding the nature of the skin, are important as all these parameters can affect percu-taneous compound absorption (shunt diffusion, permea-tion or penetration). Understanding the biological principles and metabolism is essential for their effective and safe use. In addition, understanding the proper molecular formulation will not only improve the efficacy of these compounds, but it will also save money.

Effective and safe use of topical agents requires appreciation of the physiological variables that influence the interactions of drugs and the skin, impacting absorption and transport. The skin is a multi-compartment tissue affected in numerous ways by both diseases and their treatments. The bulk of percutaneous absorption for most agents is through the stratum corneum. Epidermal structure
and the role of hair follicles and sweat glands, as pathways for absorption, are also crucial for the absorption of drugs and care substances.
In order to study the diffusion of compounds in the skin, an in vitro diffusion system called the Franz diffusion cell (FD-C) is the primary reference of today.

Nevertheless, this approach gives only some information about the compound quantity which has passed through the tissue. In addition, the impact of the absorbed compounds on histological regions is not clearly correlated and determined. Quantitative predictions of molecular transport rates through the skin are key to the development of topically applied and transdermally delivered drugs, as well as risk assessment associated with dermal exposure. That is why liquid chromatography coupled to mass spectrometry (LC-MS) approaches are also performed on skin tissues: compound concentrations are calculated for the whole homogenized tissue or on isolated tissue slices from the epidermis to the hypodermis (which is timeconsuming). Once again, it is difficult to correlate the compound accumulation within a specific histological structure; as well as, to generate an understanding of the molecular distribution.

For this reason, the combination of images from standard histology, immunohistochemistry (IHC), microscopy, and quantitative mass spectrometry imaging (QMSI) allows for an individual to correlate the compound distribution and concentration within histological structures. We apply Multimaging™, a multi-imaging approach, to several dermatological applications such as penetration pathway studies, target engagement validations, screening of compounds and/or formulations, and pharmacodynamics studies which monitors the effects of the compound
(cf: ImaBiotech’s application note #MSI-03).

Penetration Pathway and Target Engagement
Two of the main questions in compound development
are the following:
1) What is the route of penetration used by the compound?
2) How long does it take for the compound to reach its
target?

Compounds can penetrate the skin tissue by using three
routes of penetration:
1) across the stratum corneum,
2) via the hair follicles, and/or
3) through the sweat ducts.
The route will primarily depend on the formulation preparation. For example, the stratum corneum is the limiting barrier and consists of 40% protein, 40% water and 20% lipids. Skin with a disrupted epidermal layer allows up to 80% of hydrocortisone to pass into the dermis but only 1% is absorbed if the skin is intact. Hydration of the skin also increases drug penetration as hydration causes swelling of the stratum corneum rendering it more permeable to drugs or care substances. The use of occlusive dressings can increase the hydration by preventing water loss due to perspiration. ImaBiotech’s team developed a specific sample preparation and sectioning process for the stratum corneum visualization and analysis. Thus fresh tissue section can be analyzed by MSI at 15m spatial resolution and the distribution study can be easily performed on the stratum corneum as illustrated in the figure 1 with an endogenous molecule in yellow.

Figure 2 shows an example of the multi-imaging approach obtained during a pharmacological study on Minipig skin
(example on one tissue section analysis). Based on the H&E staining (figure 2.a), each histological region of the tissue was
identified. Then the QMSI results obtained on the same section allowed for the visualization of the drug distribution (figure 2.b). Thus it was possible to conclude that the penetration of the compound in the tissues was via the hair follicle. With the same data, the quantification of the drug was calculated. The molecular image generated in figure 2.a presents the molecular concentration per pixel, and so per histological region. Thus, the target engagement was confirmed with the accumulation of the
compound in the sebaceous glands.

Compound Screening & Formulation Selection:
The choice of the compound and the selection of the formulation composition, for obtaining the expected penetration, are two major challenges in dermatology. Currently, the slicing approach applied on skin tissue is time-consuming (150 LCMS runs, with each run being 15 minutes, will be needed for 150 slices of 20m obtained from one 3mm biopsy) and doesn’t provide the impact to
the histological regions. By QMSI, one tissue section will be
analyzed in 2 hours at 20m of spatial resolution.

Then it is possible to generate a penetration profile of the
targeted compounds as illustrated in the figure 3. The
penetration profile in green was generated from the entire
data set. We can also observe the impact of the sebaceous
gland drug accumulation on this result. Indeed if a region
without sebaceous gland is selected (in red), a different
profile of penetration is observed.
Conclusion:

Today, Mass Spectrometry Imaging (MSI), combined with classical histological techniques, is an essential multimodal approach in dermatology and cosmetics development. Indeed the Multimaging™ platform allows for the study of compound distribution and quantification in histological structures of a tissue section. It is thus possible to determine the penetration pathway, the compound concentration in each histological region and the target engagement directly within the tissue without any labeling (radioactivity or fluorescence).

Comparison of metabolites profiles and localization in genetically predisposed mice
This approach based on the histology is useful if multiple formulations are compared. As detailed in figure 4, it is possible to compare and select a formulation depending on the penetration profiles if similar histological regions are selected
and compared. This advantage of QMSI allows for a quick comparison and a confident determination of a compound or formulation of choice. Depending on the need, the penetration profile can be generated based on qualitative data (relative intensity) or quantitative data (concentration in g/g or pmol/mm).
Experimental Section:
Tissues: Fresh Yorkshire pig and Human skin biopsies were snap
frozen, embedded in carboxymethyl cellulose (CMC) and stored at
-80°C until analysis.
Sectioning: Frozen tissue were sectioned at 10 m using Microm
HM560 cryostat (Thermo Scientific, Germany) at -20°C and
mounted on indium tin oxide (ITO) conductive glass slides (Delta
Technology USA). H&E staining was performed on adjacent tissue
sections or on the same sections as MSI aer a methanol washing
for better visualization of histological regions.
Matrix: 2,5 DHB ACN/TFA 0.1% 6:4 v:v was sprayed with a Suncollect system (Sunchrome, Germany)
Mass Spectrometry Imaging: Solarix MALDI-FTICR 7T (Bruker
Daltonik, Germany) with SmartBeam II laser. Positive mode of
ionization at 15 or 20 m spatial resolution.
Software: All presented images and graphs were generated with Quantinetix™ v1.7.1 and Mutlimaging™ v1.1 (ImaBiotech, France), except for figure 1 image which was created with Fleximaging v4.1 (Bruker Daltonics, Germany)

Conclusion:
Today, Mass Spectrometry Imaging (MSI), combined with classical histological techniques, is an essential multimodal approach in dermatology and cosmetics development. Indeed the Multimaging™ platform allows for the study of compound distribution and quantification in histological structures of a tissue section. It is thus possible to determine the penetration pathway, the compound concentration in each histological region and the target engagement directly within the tissue without any labeling (radioactivity or fluorescence).

Benefits:
– Label free molecular imaging combined with histological techniques
– Visualization of the compound distribution in each histological structure (R&D, screening, PK/ PD, toxicity)
– Penetration pathway, molecular concentration per histological region and target engagement information in each generated
image
– Possibility to follow the drug and its related metabolites in each image acquisition
-Tissues: rodent, Yorkshire pig and human fresh skin biopsy or necropsy

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