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  5. Determining topical product bioequivalence with stimulated Raman scattering microscopy
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Determining topical product bioequivalence with stimulated Raman scattering microscopy

Historically, for topical drug products applied to the skin, bioequivalence (BE) has often been established based on comparative clinical endpoint BE studies. However, such studies are expensive and time-consuming, potentially involving hundreds to thousands of patients in studies with endpoints that take many weeks to months to manifest [1]. To assess more innovative and cost efficient scientifically valid approaches for BE studies of topical drug products, researchers at FDA’s Center for Drug Evaluation and Research (CDER) and researchers at Massachusetts General Hospital, Harvard Medical School, and Massachusetts Institute of Technology conducted a first-of-its-kind study that evaluated the use of noninvasive stimulated Raman scattering microscopy (SRS) to assess topical product bioavailability and BE. The study established both initial feasibility as well as methodologies to improve reliability and specificity when implementing SRS for assessment of topical product bioavailability and BE measurements. 

Background

Generic drugs, which are frequently available from multiple manufacturers, mitigate the risk of drug shortages that otherwise occur when individual supply chains are disrupted. Generic drugs also enhance patient access to affordable, safe, effective, and high-quality medicines by facilitating market competition to the supply of the same drug. Generally, the more generic drug competitors enter the market, the more the price of the generic drugs declines [2]. Affordable drugs, an essential part of equitable healthcare, are associated with broader accessibility to treatments, better patient medication adherence, and, thus, improved health outcomes.

A prospective generic product is generally expected to demonstrate BE to a reference listed drug (RLD), and to satisfy other criteria that are intended to ensure the therapeutic equivalence of an approved generic product relative to its RLD [3]. For topical products applied to the skin, historically BE has been established based on comparative clinical endpoint BE studies, which typically compare a prospective generic product and its RLD using pre-specified clinical endpoint(s) that reflect the therapeutic effectiveness of the drug at treating the disease. However, such studies are expensive and time-consuming, potentially involving hundreds to thousands of patients in studies with endpoints that take many weeks to months to manifest [1], and that may not be the most sensitive and discriminating method for identifying differences in bioavailability between a prospective generic product and the RLD [4]. These challenges have especially hindered topical drug product development and approval [1].

Study Discussion

For systemically acting drug products, the rate and extent of drug availability can be efficiently demonstrated by BE studies with pharmacokinetics (PK) endpoints. Because systemic PK BE studies are well established and straightforward to conduct by conducting blood draws or sampling other biological fluids, and measuring drug concentrations, many oral generic products have been approved through the ANDA pathway [4]. In contrast, for locally acting, topical drug products applied to the skin, the drug may not be measurable in the systemic circulation. Even if detectable it may not be assumed that the systemic PK would necessarily reflect the cutaneous PK, since the systemic circulation is often downstream of the site of action in the skin, and some of the drug from the skin may independently distribute to other biological compartments before reaching the systemic circulation. The challenges associated with assessing the cutaneous bioavailability or BE of locally acting topical generic products have been well-understood for decades, and the need for more efficient alternative BE approaches for these products has been widely discussed [5]. To date, various in vitro and in vivo cutaneous PK (cPK)-based techniques, such as in vitro permeation testing, dermal microdialysis, dermal open flow microperfusion and tape stripping have been proposed for evaluating topical BE but each of these methods also have challenges.

Noninvasive optical tools such as fluorescence microscopy, confocal Raman spectroscopy, and SRS microscopy show promise for use in cPK studies [4,6,7]. Confocal Raman spectroscopy (CRS) is able to detect drugs via intrinsic molecular vibrations that arise from a drug’s chemical structure and can detect drugs without the need for labels (e.g., radiolabeling). However, the spontaneous Raman signal of CRS is inherently weak, thereby limiting the capabilities for high-resolution imaging of dynamic changes occurring in the tissue structures. SRS, like CRS, is a non-destructive optical method that detects drugs via their chemical structures. SRS is based on the simultaneous use of two pulsed laser light sources that target a specific Raman-active vibration of a molecule. When the difference in the frequencies of the two sources matches a molecular vibrational resonance in the sample, a signal is generated that is greater by several orders of magnitude compared with the signal obtained by spontaneous Raman methodologies. SRS is characterized by high spatial and temporal resolution. Importantly, the signal is free from background interference (including auto-fluorescence), and the signal intensity is linearly proportional with the molecular concentration, thereby enabling quantitative imaging of drugs across the tissue [8-11].

In this study, CDER researchers presented a novel approach to assess topical BE based on SRS imaging and the use of a polymer standard reference for signal normalization. The proof of concept of the proposed method was investigated using commercially available formulations of tazarotene (a topical treatment of mild to moderate plaque psoriasis, acne vulgaris, and photoaging [12]). The formulations examined were Tazorac® cream 0.1% and a generic tazarotene cream 0.1%, which served as the RLD and approved generic drug product, respectively, and an alternative formulation (Tazorac® 0.1% gel). Statistical comparisons among the cPK parameters under the normalized intensity vs time curve (AUC) and peak drug concentration were performed to investigate (1) topical BE between the RLD and the generic product; (2) Tazarotene penetration from the RLD compared with the negative control, as well as with itself as a separate treatment group.

Two sets of experiments using Tazorac® 0.1% cream (RLD) were implemented, and their results were labeled as R1 and R2 (see figure B), respectively. Analysis between R1 and R2 helped to show the variance between experiments and how it affected an evaluation of BE. The values of R1 and R2 met the expected BE criteria. Further, comparing the RLD (R1) and the generic product, the values were also within the [0.8, 1.25] range, indicating the expected BE between the two products. Another formulation containing polyethylene glycol as the vehicle was demonstrated to be not bioequivalent to the RLD. Interestingly, comparing the RLD (R1) and the alternative formulation (Tazorac® 0.1% gel), values in both the lipid-rich intercellular lamellae region and the lipid-poor corneocytes space were within the [0.8, 1.25] range. This indicates that despite the differences in composition the cPK of the products in the upper skin layers were within the acceptable BE range. 

Schematic of skin layers with drug penetration shown via fluorescence imaging (10 min–7 hrs). Graphs compare SRS intensity over time (2–6 hrs) for various drug formulations, showing steady increases with differences by formulation.

Fig. X. (A) SRS images collected at different time points of the skin (at 8 μm depth). The images are colour-coded with the ‘viridis’ lookup table to display the regions of low (blue) and high (yellow) tazarotene uptake. The red outline shows the regions where the drug permeation is mainly via the intralamellar lipids. Normalized intensity-time profile of the (B) lipid-rich intralamellar region uptake, and (C) lipid-poor corneocytes region uptake of tazarotene. Reference product (R1): Tazorac® cream; Generic product: Taro Pharmaceuticals U.S.A., Inc. (cream); Reference product (R2): Tazorac® cream; Alternative formulations: Tazorac® gel & PEG-200 solution. Upper skin layers: 0-16 μm. The error bar showed the standard error of the mean (SEM) of 16 samples, 4 donors and 4 replicate skin sections from each donor. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

In BE assessment, it is critical to ensure the measurements are consistent and reproducible throughout the study duration and across different days. Reference concentration standards are often used in chemometric experiments to control for experiment-to-experiment variability [13-16]. Especially for microscope image-based techniques, concentration standards provide a means of converting measurements of relative intensity into concentration values [17-20]. However, this type of standard reference is missing in current SRS studies [21]. A polymer-based standard reference was developed and utilized to control for experiment-to-experiment variability in the current study.

These results collectively demonstrate the potential capability of SRS imaging for the assessment of topical product BE. Moreover, the developed SRS approach enables the study of BE cPK parameters calculated from different microstructures of the skin. The capability of establishing BE in specific microstructure regions has a huge potential for better and more rapid product development toward applications through specific routes of permeation. Current limitations for this technology include the depth of penetration of the analytical acumen, some of which is improved by this current method, but continues to remain an issue regarding practicality for many topical products.

How this research improves generic drug development and supports patient access to needed drugs

Using Stimulated Raman Scattering (SRS) microscopy, researchers at FDA’s Center for Drug Evaluation and Research and the Harvard Medical School investigated potential differences in cutaneous drug delivery based on SRS and a polymer-based standard reference for the evaluation of topical products bioavailability and BE in human skin ex vivo. SRS microscopy enabled the gathering of chemically-specific quantitative volumetric imaging over time within human skin samples to follow the permeation of tazarotene formulated in multiple products. The abundant information from this research may help to systematically identify key areas of study design that will enable a better comparison of topical products and support an assessment of BE. This, in turn, may help inform multiple manufacturers, mitigate the risk of drug shortages that otherwise occur when individual supply chains are disrupted, and enhance patient access to affordable, safe, effective, and high quality medicines by facilitating market competition among the supply of some topical generic drugs.

(This Impact Story is based on the Journal of Controlled Release article “Determining topical product bioequivalence with stimulated Raman scattering microscopy” by Fotis Iliopoulos, Dandan Tu, Isaac J. Pence, Xiaolei Li, Priyanka Ghosh, Markham C. Luke, Sam G. Raney, Elena Rantou, and Conor L. Evans, published 19 February, 2024 in the Journal of Controlled Release, https://doi.org/10.1016/j.jconrel.2024.02.010 ) 

References

1. J.J. Darrow, J. Avorn, A.S. Kesselheim, FDA approval and regulation of pharmaceuticals, 1983-2018, Jama 323 (2) (2020) 164–176.
2. S.G. Raney, M.C. Luke, A new paradigm for topical generic drug products: impact on therapeutic access, J. Am. Acad. Dermatol. 82 (6) (2020) 1570–1571.
3. R. Gupta, N.D. Shah, J.S. Ross, Generic drugs in the United States: policies to address pricing and competition, Clin. Pharmacol. Ther. (St. Louis, MO, U. S.) 105(2) (2019) 329–337.
4. T. Ilic, I. Pantelic, S. Savic, The implications of regulatory framework for topical semisolid drug products: from critical quality and performance attributes towards establishing bioequivalence, Pharmaceutics 13 (5) (2021) 710.
5. Yacobi A, Shah VP, Bashaw ED, et al. Current challenges in bioequivalence, quality, and novel assessment technologies for topical products. Pharm Res. 2014;31(4):837-846. doi:10.1007/s11095-013-1259-1
6. Raney SG, Ghosh P, Ramezanli T, Lehman PA, Franz TJ. Cutaneous Pharmacokinetic Approaches to Compare Bioavailability and/or Bioequivalence for Topical Drug Products. Dermatol Clin. 2022;40(3):319-332. doi:10.1016/j.det.2022.02.007
7. Jung N, Namjoshi S, Mohammed Y, et al. Application of Confocal Raman Microscopy for the Characterization of Topical Semisolid Formulations and their Penetration into Human Skin Ex Vivo. Pharm Res. 2022;39(5):935-948. doi:10.1007/s11095-022-03245-7
8. Saar BG, Contreras-Rojas LR, Xie XS, Guy RH. Imaging drug delivery to skin with stimulated Raman scattering microscopy. Mol Pharm. 2011;8(3):969-975. doi:10.1021/mp200122w
9. Pence IJ, Evans CL. Translational biophotonics with Raman imaging: clinical applications and beyond. Analyst. 2021;146(21):6379-6393. Published 2021 Oct 25. doi:10.1039/d1an00954k
10. Feizpour A, Marstrand T, Bastholm L, Eirefelt S, Evans CL. Label-Free Quantification of Pharmacokinetics in Skin with Stimulated Raman Scattering Microscopy and Deep Learning. J Invest Dermatol. 2021;141(2):395-403. doi:10.1016/j.jid.2020.06.027
11. Kuzma BA, Pence IJ, Ho A, Evans CL. Visualizing and Quantifying Pharmaceutical Compounds within Skin using Coherent Raman Scattering Imaging. J Vis Exp. 2021;(177):10.3791/63264. Published 2021 Nov 24. doi:10.3791/63264
12. Roeder A, Schaller M, Schäfer-Korting M, Korting HC. Tazarotene: therapeutic strategies in the treatment of psoriasis, acne and photoaging. Skin Pharmacol Physiol. 2004;17(3):111-118. doi:10.1159/000077236
13. Guo L, Zhang D, Xue ZJ, et al. Comparison of Artemisiae argyi Folium and Artemisiae lavandulaefoliae Folium by simultaneous determination of multi-components with single reference standard method and chemometric analysis. Phytochem Anal. 2019;30(1):14-25. doi:10.1002/pca.2786
14. Sørensen L, Groven AS, Hovsbakken IA, et al. UV degradation of natural and synthetic microfibers causes fragmentation and release of polymer degradation products and chemical additives. Sci Total Environ. 2021;755(Pt 2):143170. doi:10.1016/j.scitotenv.2020.143170
15. Cajka T, Fiehn O. Toward Merging Untargeted and Targeted Methods in Mass Spectrometry-Based Metabolomics and Lipidomics. Anal Chem. 2016;88(1):524-545. doi:10.1021/acs.analchem.5b04491
16. Wang Y, Zhu W, Levy DE. Nuclear and cytoplasmic mRNA quantification by SYBR green based real-time RT-PCR. Methods. 2006;39(4):356-362. doi:10.1016/j.ymeth.2006.06.010
17. He D, Wang H, Ho SL, et al. Total internal reflection-based single-vesicle in situ quantitative and stoichiometric analysis of tumor-derived exosomal microRNAs for diagnosis and treatment monitoring. Theranostics. 2019;9(15):4494-4507. Published 2019 Jun 9. doi:10.7150/thno.33683
18. Belykh E, Miller EJ, Patel AA, et al. Optical Characterization of Neurosurgical Operating Microscopes: Quantitative Fluorescence and Assessment of PpIX Photobleaching. Sci Rep. 2018;8(1):12543. Published 2018 Aug 22. doi:10.1038/s41598-018-30247-6
19. Vergalli J, Dumont E, Pajović J, et al. Spectrofluorimetric quantification of antibiotic drug concentration in bacterial cells for the characterization of translocation across bacterial membranes. Nat Protoc. 2018;13(6):1348-1361. doi:10.1038/nprot.2018.036
20. Model M. Intensity calibration and flat-field correction for fluorescence microscopes. Curr Protoc Cytom. 2014;68:10.14.1-10.14.10. Published 2014 Apr 1. doi:10.1002/0471142956.cy1014s68
21. Tsikritsis D, Legge EJ, Belsey NA. Practical considerations for quantitative and reproducible measurements with stimulated Raman scattering microscopy. Analyst. 2022;147(21):4642-4656. Published 2022 Oct 24. doi:10.1039/d2an00817c

 

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