Visualizing and Quantifying Pharmaceutical Compounds within Skin using Coherent Raman Scattering Imaging

Summary

A coherent Raman scattering imaging methodology to visualize and quantify pharmaceutical compounds within the skin is described. This paper describes skin tissue preparation (human and mouse) and topical formulation application, image acquisition to quantify spatiotemporal concentration profiles, and preliminary pharmacokinetic analysis to assess topical drug delivery.

Abstract

Cutaneous pharmacokinetics (cPK) after topical formulation application has been a research area of particular interest for regulatory and drug development scientists to mechanistically understand topical bioavailability (BA). Semi-invasive techniques, such as tape-stripping, dermal microdialysis, or dermal open-flow microperfusion, all quantify macroscale cPK. While these techniques have provided vast cPK knowledge, the community lacks a mechanistic understanding of active pharmaceutical ingredient (API) penetration and permeation at the cellular level.

One noninvasive approach to address microscale cPK is coherent Raman scattering imaging (CRI), which selectively targets intrinsic molecular vibrations without the need for extrinsic labels or chemical modification. CRI has two main methods-coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS)-that enable sensitive and selective quantification of APIs or inactive ingredients. CARS is typically utilized to derive structural skin information or visualize chemical contrast. In contrast, the SRS signal, which is linear with molecular concentration, is used to quantify APIs or inactive ingredients within skin stratifications.

Although mouse tissue has commonly been utilized for cPK with CRI, topical BA and bioequivalence (BE) must ultimately be assessed in human tissue before regulatory approval. This paper presents a methodology to prepare and image ex vivo skin to be used in quantitative pharmacokinetic CRI studies in the evaluation of topical BA and BE. This methodology enables reliable and reproducible API quantification within human and mouse skin over time. The concentrations within lipid-rich and lipid-poor compartments, as well as total API concentration over time are quantified; these are utilized for estimates of micro- and macroscale BA and, potentially, BE.

Introduction

Methodologies to assess cPK after topical drug product application have expanded from classical in vitro permeation testing (IVPT) studies^1,2,3,4,5 and tape-stripping^6,7,8 to additional methodologies such as open-flow microperfusion or dermal microdialysis^9,10,11,12,

Protocol

The use of nude mouse ear tissue was approved by Massachusetts General Hospital Institutional Animal Care and Use Committee (IACUC), while the use of human skin tissue was approved by the Massachusetts General Hospital Institutional Review Board (IRB). According to IACUC protocols, freshly euthanized mice were obtained from collaborators with nude mice colonies. Human tissue was procured from elective abdominoplasty procedures at Massachusetts General Hospital via an approved protocol. In addition, specific tissue types .......

Discussion

The evaluation of topical BA/BE is an area of research the requires a multifaceted approach as no single method can fully characterize in vivo cPK. This protocol presents a methodology for the evaluation of a topical drug product's BA/BE based on coherent Raman imaging. One of the first points that might be overlooked is how thin the skin samples must be, especially for quantitative transmission SRS imaging. If the skin is too thick (i.e., light cannot readily pass through), there is little to no si.......

Materials

Name	Company	Catalog Number	Comments
Tissue Preparation
Autoclavable Biohazard Bags	FisherBrand	22-044562	As refered to in text: biohazard bags https://www.fishersci.com/shop/products/fisherbrand-polyethylene-biohazard-autoclave-bags-without-sterilization-indicator-8/22044562?searchHijack=true&searchTerm= 22044562&searchType=RAPID& matchedCatNo=22044562
Cell Culture Buffers: Dulbecco's Phosphate-Buffered Salt Solution 1x	Corning	MT21030CV	As refered to in text: PBS https://www.fishersci.com/shop/products/corning-cellgro-cell-culture-buffers-dulbecco-s-phosphate-buffered-salt-solution-1x-8/MT21030CV?searchHijack=true&searchTerm= 21-030-cv&searchType= RAPID&matchedCatNo=21-030-cv
Disposable Scalpels	Exel International	14-840-00	As refered to in text: scalpel https://www.fishersci.com/shop/products/exel-international-disposable-scalpels-3/1484000?keyword=true
High Precision 45° Angle Broad Point Tweezers/Forceps	Fisherbrand	12-000-132	As refered to in text: forceps https://www.fishersci.com/shop/products/high-precision-45-angle-broad-point-tweezers-forceps/12000132#?keyword=
Kimwipes Delicate Task Wipers, 1-Ply	Kimberly-Clark Professional Kimtech Science	06-666	As refered to in text: task wiper https://www.fishersci.com/shop/products/kimberly-clark-kimtech-science-kimwipes-delicate-task-wipers-7/06666
Parafilm M Laboratory Wrapping Film	Bemis	13-374-12	As refered to in text: parafilm https://www.fishersci.com/shop/products/curwood-parafilm-m-laboratory-wrapping-film-4/1337412
Petri Dish (35 mm x 10 mm)	Fisherbrand	FB0875711YZ	As refered to in text: small petri dish https://www.fishersci.com/shop/products/fisherbrand-petri-dishes-specialty-6/FB0875711YZ?keyword=true
Petri Dish (60 mm x 15 mm)	Fisherbrand	FB0875713A	As refered to in text: large petri dish https://www.fishersci.com/shop/products/fisherbrand-petri-dishes-clear-lid-12/FB0875713A?keyword=true
Surgical Scissors	Roboz	NC9411473	As refered to in text: scissors https://www.fishersci.com/shop/products/scissors-327/NC9411473?searchHijack=true&searchTerm= RS-5915SC&searchType=RAPID& matchedCatNo=RS-5915SC
Laser/microscope
650/60 nm BrightLine single-band bandpass filter	Semrock		As refered to in text: CARS filter - CH2 vibrations (645nm/60nm filter)
Control box IX2-UCB	Olympus		As refered to in text: Control Box
D700/30m	Chroma		As refered to in text: CARS filter - deuterated band https://www.chroma.com/products/parts/d700-30m
DeepSee Insight	Spectra-Physics		As refered to in text: Laser https://www.spectra-physics.com/f/insight-x3-tunable-laser
Digital Handheld Optical Power and Energy Meter Console	ThorLabs	PM100D	As refered to in text: power meter https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=3341
Fluoview Software	Olympus		As refered to in text: Microscope Control software
Frosted Microscope Slides	FisherBrand		As refered to in text: microscope slides https://www.fishersci.com/shop/products/fisherbrand-frosted-microscope-slides-4/22265446
FV1000	Olympus		As refered to in text: Microscope
Incubation Chamber	Tokai Hit	GM-800	As refered to in text: incubation chamber
Integrating Sphere Photodiode Power Sensor	ThorLabs	S142C	As refered to in text: photodiode https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=3341
Power supply FV31-PSU	Olympus		As refered to in text: Power Supply
Precision 4063, 80MHz Dual Channel Function Generator	BK Precision		As refered to in text: function generator
ProScan – Precision Microscope Automation	Prior Scientific Instruments		As refered to in text: stage controller https://www.prior.com/microscope-automation/inverted-microscope-systems/proscan-linear-stage-highest-precision-microscope-automation
SecureSeal Imaging Spacers	Grace Biolabs	654004	As refered to in text: spacer https://gracebio.com/product/secureseal-imaging-spacers-654004/
SRS Detection Kit	APE		As refered to in text: SRS detector
UPLSAPO 20X NA:0.75	Olympus		As refered to in text: 20X Objective https://www.olympus-lifescience.com/en/objectives/uplsapo/
Lipid/Drug Imaging
35 mm Dish, No. 0 Uncoated Coverslip, 14 mm Glass Diameter	MatTek Corporation	NC9711297	As refered to in text: Glass bottom dish https://www.fishersci.com/shop/products/glass-bottom-mircrowell-dish/nc9711297
Cotton-tipped applicators	FisherBrand		As refered to in text: Cotton-tipped applicator
Distriman Postive Displacement Pipette	Gilson		As refered to in text: Postive Displacement Pipette https://www.fishersci.com/shop/products/gilson-distriman-positive-displacement-repetitive-pipette/F164001G#?keyword=
Distriman Postive Displacement Pipette Tips	Gilson		As refered to in text: Tips for pipette https://www.fishersci.com/shop/products/gilson-distritip-syringes-6/f164100g?keyword=true
Data Analysis
FIJI	Open-source		As refered to in text: FIJI/ImageJ https://imagej.net/software/fiji/
Jupyter-Lab	open-source		As refered to in text: JupyterLab https://jupyter.org/
Rstudio	Open-source		As refered to in text: Rstudio https://www.rstudio.com/