(W0930-11-70) Assessment of Bio(in)Equivalence of Metronidazole Topical Formulations Using Stimulated Raman Scattering Microscopy

Purpose: Assessment of the bioequivalence (BE) of locally acting topical drug products at or near the site of action in the skin can be challenging. The principal challenges are (a) to identify techniques to quantify drug concentrations at or near the site of action in the skin, and (b) to characterise the metamorphosis of the drug product following its application and the nature and role of the residual film that remains thereafter. Recently, confocal Raman spectroscopy has successfully tracked the permeation of metronidazole (MTZ) into the epidermal skin layers beneath the stratum corneum ex vivo [1]. This has been achieved even though the Raman signals from MTZ overlap with the background spectral features from the skin. The BE of marketed MTZ gels was confirmed in this study, and two different laboratory-made formulations were clearly shown to be inequivalent to one another. The aim of the present investigation was to take advantage of the significantly faster acquisition time of stimulated Raman scattering (SRS) microscopy to confirm the confocal results and to shed light on the transformation of the MTZ formulations at the drug product-skin interface post-application

Methods: Saturated MTZ solutions in 90:10 or 30:70 v/v water:propylene glycol (PG) were prepared. In vitro skin permeation experiments were performed in thermostatted (32°C) Franz cells, using abdominal porcine skin (dermatomed at a nominal thickness of 750 μm) and with phosphate buffered saline (pH 7.4) in the receptor chamber. Each formulation (300 μL) was applied for 6 hours without occlusion. The skin surface was then gently cleaned with absorbent paper and the tissue was stored at -80°C before analysis. For imaging, small, thin slices (SC + epidermis) were sectioned horizontally with a scalpel from the skin samples. SRS images were acquired with a Leica SP8 microscope coupled to a PicoEmerald-S tuneable laser source. The sample stage was maintained at 10°C permitting an increased laser power to be used and preventing sample dehydration. Spurious signals were identified by spectroscopic data acquisition at off-resonance frequencies and subtracted in a pixel-by-pixel manner. SRS λ scans were performed to identify frequency shifts between dissolved and crystalline drug in the residual film at or near the skin surface. Then, the C=N signal from MTZ at 1195 cm^-1 and that from Amide I (C=O at 1666 cm^-1) were recorded as a function of skin depth. Data analysis was performed with ImageJ, and the MTZ signals were normalised by the corresponding Amide I values to correct for signal attenuation with depth. Experiments were performed in duplicate and, in each skin sample, measurements from 12 different positions were analysed.

Results: The earlier confocal Raman data showed that at 6 hours the 90:10 v/v water:PG vehicle (but not the 30:70) had evaporated and/or absorbed into the skin leaving a white solid on the skin surface [1]. SRS imaging confirmed this substantial MTZ crystallisation; furthermore, a characteristic shift in the peak MTZ signal frequency clearly differentiated between dissolved and crystalline drug (Figure 1). SRS imaging tracked drug distribution in the tissue and (as observed before) revealed that at 6 hours the 90:10 vehicle delivered more MTZ into the skin compared to the 30:70 v/v (Figure 2). One possible explanation for this difference is that the faster evaporation/absorption of the 90:10 vehicle created a transient state of MTZ supersaturation that temporarily enhanced drug uptake in a manner that was not achieved with the more slowly evaporating/absorbing 30:70 formulation. Finally, image analysis confirmed the appearance of MTZ in the intercellular lipids of the SC independent of the formulation used (Figure 3).

Conclusion: SRS imaging confirmed the inequivalence of two laboratory-made MTZ formulations and suggested a mechanism by which this observation might be explained. The added value of SRS microscopy is that the transformation of the vehicle at the interface with the skin can be clearly visualized and that the greater axial resolution permits both skin topology and drug localisation to be identified. Further work now targets examination of skin treated with commercially available drug products.

References: 1. Confocal Raman spectroscopic assessment of the topical bioavailability of metronidazole: comparison of laboratory-made formulations and approved drug products. P. Zarmpi et al. Available from https://www.eventscribe.net/2022/PharmSci360/, AAPS PharmSci 360 annual meeting, USA, 2022

Acknowledgements: This research is supported by the U.S. Department of Health & Human Services, Food & Drug Administration (1-U01-FD006533 and 1-U01-FD004947). The views expressed in this presentation do not reflect the official policies of the U.S. FDA or the U.S. DHHS; nor does any mention of trade names, commercial practices, or organization imply endorsement by the U.S. Government.

(a) Tile view of the skin surface after treatment with the 90:10 v/v water:PG MTZ formulation for 6 hours. (b) SRS spectra of MTZ-treated skin in areas where crystals are either absent (blue) or present (green).

MTZ signal (normalised by that from Amide I) as a function of skin depth after 6-hr application of two water:PG formulations (mean +/- SD, n=12 from each of two skin samples).

SRS images of the distribution of MTZ (shown in red) in the SC post-application of the 90:10 (left) and 30:70 (right) water:PG vehicles.