(T1330-02-11) Assessing Cutaneous Pharmacokinetics of Tazarotene Using Coherent Raman Imaging

Purpose: Skin permeation and distribution of drugs upon application of topical agents are still poorly understood. Thus, topical drug exposure in skin cannot be adequately predicted preclinically or evaluated in clinical studies, limiting the ability to translate preclinical compound properties into clinical efficacy. Coherent Raman imaging (CRI) is a chemically-sensitive imaging technique, which can quantitatively visualize active pharmaceutical ingredients (API) via their unique chemical structure in a label-free manner. CRI affords the ability to monitor dynamic changes such as the cutaneous pharmacokinetics after topical drug application. The prominent CRI modality utilized to quantify API cutaneous concentrations is stimulated Raman scattering (SRS) as the signal in SRS scales linearly with concentration. Computational approaches make it feasible to comprehend the permeation pathways for various topical APIs. The central hypothesis of this research is that CRI may offer a non-invasive, accurate, sensitive, and reproducible method for determination of both microscale permeation dynamics as well as cutaneous pharmacokinetics parameters of a topically administered drug. In addition, we aim to employ CRI to understand the influence of perfusion on dermal and transfollicular permeation.

Methods: This study utilized tazarotene (TAZ) dissolved into a transcutol:ethanol 70:30 evaporative vehicle at 2% concentration. To investigate the influence of perfusion on (trans)dermal/transfollicular permeation of TAZ, in vivo and ex vivo experiments were carried out using SKH1 hairless mice model setup. The model was proposed and approved by the MGH-IACUC (protocol number: 2021N000223). In the first experimental phase, the mouse is anesthetized and the topical formulation is applied to one of its ears. The mouse is then placed on the microscope and multiple regions of interest (ROIs) on the ear are volumetrically imaged (XYZ) over time (T) to create XYZT 4D datasets at different depths within the ear to visualize different layers of the skin: the stratum corneum (SC), the layer containing the sebaceous glands (SG), the layer containing the adipocytes (AD), and the subcutaneous fat layer (SCF). After a two-hour period, to allow for topical product permeation, the mouse is then sacrificed, and the untreated ear is removed. The topical product is then placed on the untreated ear and XYZT data is acquired from now fresh ex vivo tissue that is lacking perfusion. This matched-ear model allows for within-animal control of the observed pharmacokinetics. 5 µL/cm² of the formulation were applied to each mouse ear skin. TAZ was chosen for this study due as it is a commonly used topical API that also has a strong Raman signal (-C≡C vibration) at 2170 cm^-1 where skin is spectroscopically ‘quiet’. At the start of the imaging session, a single depth stack (from – 5 µm to 100 µm) was acquired at each tissue location with the system tuned into the lipid CH₂ symmetrical stretching vibration (2845 cm^-1) to record the tissue’s microscale lipid structure. This was carried out to define lipid-rich and lipid-poor regions of the epidermis and dermis to map and understand drug partitioning. The laser system was then tuned to visualize TAZ and multiple depth stacks were acquired.

Results: It was possible to monitor the uptake of TAZ into the skin stratifications (SC, SG, AD and SCF) over 2 hours during both in vivo and ex vivo experiments. Differences between the distributions of TAZ could be seen at all depths of the skin. Quantitative analysis using a machine-learning based image analysis pipeline showed differences in T_MAX values (time to reach the maximum observed concentration) between perfused in vivo ears and non-perfused ex vivo ears. It was also possible to observe differences in T_MAX between SC and SG layers, in the in vivo experiments, TAZ seems to preferentially permeate through the transfollicular route (TMAX-SC > TMAX-SG), whereas in the ex vivo experiments, the opposite was observed: TMAX-SC < T_MAX-SG (Figure 1).

Conclusion: Overall, the approaches presented here facilitate the direct observation and quantification of pharmacokinetics and they could be used to shed light on the mechanisms of drug kinetics into the skin.

Acknowledgments: The authors would like to acknowledge Pfizer for sponsoring this project.

Extracted TMAX values (in minutes) for the stratum corneum (SC, left panels) and sebaceous glands (SG, right panels) for 2% tazarotene in transcutol:ethanol (70:30, v/v). “In vivo” refers to experiments using perfused in vivo ears (blue) and “Fresh mouse ear” refers to the non-perfused ex vivo ears (green).