Application Note #MSI-10

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 di�erent forms and physicochemical properties of compounds, as well as understanding the nature of the skin, are important as all these parameters can a�ect percu-taneous compound absorption (shunt di�usion, permea-tion or penetration). Understanding the biological principles and metabolism is essential for their e�ective and safe use. In addition, understanding the proper molecular formulation will not only improve the e�icacy of these compounds, but it will also save money.

E�ective 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 a�ected 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 di�usion of compounds in the skin, an in vitro di�usion system called the Franz di�usion cell (FD-C) is the primary reference of today.

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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 time-consuming). Once again, it is di�icult 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 derma-tological applications such as penetration pathway studies, target engagement validations, screening of compounds and/or formulations, and pharmacodynamics studies which monitors the e�ects of the compound (cf: ImaBiotech’s application note #MSI-03).

MultimagingTM Applied to Skin Distribution Study: Applications in Dermatology for Pharmaceuticals and Cosmetics

from Discovery to Clinical

Penetration Pathway and Target Engagement

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Figure 1. Stratum corneum visualization by using endogenous species on fresh human skin section following MSI analysis at 15µm. a, Hematoxylin and Eosin (H&E) optical image obtained a�er MSI acquisition. b, endogenous species distribution obtained by MSI in the same data set (yellow: specific ion

of the stratum corneum, blue: specific ion of the epidermis).

Two of the main questions in compound develop-ment 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 prepara-tion. 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 15µm 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.

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Figure 2: 3D drug quantitation in a fresh Yorkshire pig skin section by QMSI. a, H&E optical image obtained a�er QMSI acquisition. b, Multi-parameters image of the drug in a fresh Yorkshire pig tissue section (depth, width, drug concentration and distribution correlated with a histological image).

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 20µm 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 20µm of spatial resolution.

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 concen-tration per pixel, and so per histological region. Thus, the target engagement was confirmed with the accumulation of the compound in the sebaceous glands.

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 di�erent profile of penetration is observed.

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Comparison of metabolites profiles and localization in genetically predisposed mice

Figure 3. Impact of the selected tissue areas on the penetration profile determination based on the average calculated concentrations as a function of the depth.

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 formu-lation of choice. Depending on the need, the penetration profile can be generated based on qualitative data (relative intensity) or quantitative data (concentration in µg/g or pmol/mm2).

Figure 4. Formulation selection based on the compound penetration profile calculated by MSI (graph: penetration profiles of the selected areas on each image)

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 μm 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 a�er 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 Suncol-lect 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 µm spatial resolution.- So�ware: 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).

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MS Imaging Department | 885 ave. Eugène Avinée - 59120 Loos - France | +33 (0) 320 164 091 | contact@imabiotech.com185 Alewife Brook Parkway - Cambridge - MA 02138 - USA

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Authors

Benefits

Label free molecular imaging combined with histological techniques

Penetration pathway, molecular concentration per histological region and target engagement information in each generated image

Visualization of the compound distribution in each histological structure (R&D, screening, PK/ PD, toxicity)

Possibility to follow the drug and its related metabolites in each image acquisition

Keywords

► Mass Spectrometry Imaging► Dermatology► Cosmetics

► Distribution

► Dermatology

► Yorkshire pig

► Skin

► Sebaceous Gland

► Histology

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Bonnel DavidPamelard Fabien

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).

Tissues: rodent, Yorkshire pig and human fresh skin biopsy or necropsy

► Dermis► Epidermis

► Quantification

► Human

► Hair Follicle

► Stratum Corneum

Legou�e RaphaëlPicard de Muller Gaël

► Penetration► Formulation

Stauber Jonathan