(M0930-09-59) Topical MEK Inhibitor Formulations Suppress HPV E6/E7 Expression and Promote Tumor Regression in Preclinical Models

Purpose: The objective of this study was to evaluate the antiviral efficacy of a newly developed topical MEKi formulation designed for localized treatment of HPV-associated lesions. This formulation incorporates an aqueous polymeric base and a mucosal absorption enhancer to facilitate targeted delivery of MEK inhibitors while minimizing systemic exposure. We aimed to assess its ability to suppress HPV oncogene expression in organotypic tissue cultures and to induce tumor regression in a preclinical murine papillomavirus model, with minimal toxicity and maximal therapeutic localization.

Methods: Base Formulation: Aqueous topical formulations were developed using a biocompatible polymeric base composed of hydroxypropyl cellulose (HPC, 2.5% w/v) and the tissue-permeation enhancer Transcutol® (15% v/v; diethylene glycol monoethyl ether, DEGEE). This vehicle was selected for its ability to solubilize MEK inhibitors and enhance epithelial penetration without relying on cytotoxic solvents such as DMSO. Multiple FDA-approved MEK inhibitors—including trametinib, cobimetinib, selumetinib, and mirdametinib—were evaluated across varying concentrations to identify optimal candidates for topical antiviral therapy. Organotypic Tissue Culture Treatments: HPV16-infected organotypic raft epithelial tissues were established and treated topically with MEKi formulations. Rafts were harvested at 24 hours and 5 days post-treatment for molecular and protein analysis. Outcome measures included suppression of phosphorylated ERK1/2 (p-ERK1/2), HPV16 E6/E7 mRNA expression (RT-qPCR), and HPV16 E7 protein levels (immunoblotting). Tumor Regression in MmuPV1-Infected Mice: Athymic nude mice bearing MmuPV1-induced tail tumors were treated topically with MEKi formulations every other day for up to 42 days. Tumor volumes were measured using calipers, and animals were monitored for changes in body weight or signs of toxicity. Tumor tissues were harvested at endpoint for quantification of viral genome and mRNA levels, histopathological analysis, and immune cell profiling. Vehicle Control and Dose Comparisons: Mice were randomized into treatment groups receiving vehicle alone, vehicle + 150 µM trametinib, or vehicle + 600 µM trametinib. Comparative outcomes between male and female mice were assessed to determine sex-based differences in therapeutic response. Plasma samples were collected for future LC-MS/MS analysis to evaluate systemic trametinib exposure.

Results: Topical application of MEK inhibitors to HPV16-infected organotypic raft cultures resulted in robust suppression of MEK/ERK signaling and HPV E6/E7 oncogene transcription within 24 hours, with effects sustained for at least 5 days following a single application. Immunoblot analysis confirmed reduction in phosphorylated ERK1/2 and E7 protein levels, while RT-qPCR demonstrated significant downregulation of E6/E7 mRNAs in treated tissues (Fig. 1). In vivo, repeated topical treatment with trametinib-containing formulations led to substantial MmuPV1 tumor regression in both male and female athymic mice. Tumor volume reductions ranged from 75–90% over a 42-day period (Figs. 2A and B), with no observable signs of systemic toxicity or weight loss (Fig. 3). Notably, higher trametinib concentrations (600 µM) were more effective across both sexes, while lower doses (150 µM) were sufficient in female mice but not in males. Endpoint analyses of regressing tumors revealed significant decreases in papillomaviral genome and mRNA levels, as well as reduced cellular proliferation. Preliminary immunohistochemical data indicated altered immune cell infiltration, particularly involving increased activation and presence of natural killer (NK) cells and macrophages. No signs of irritation or damage were observed at the treatment sites. Ongoing spatial transcriptomics and single-cell RNA sequencing analyses are being conducted to define the tissue-specific and cellular responses underlying MEKi-induced tumor regression.

Conclusion: The newly developed aqueous polymeric topical MEK inhibitor formulation effectively delivers antiviral and anti-neoplastic activity to HPV-infected epithelial tissues in both in vitro and in vivo models. This formulation achieves sustained suppression of MEK/ERK signaling and HPV E6/E7 transcription while minimizing systemic exposure and toxicity. In murine models, treatment led to significant tumor regression and favorable local immune modulation without adverse effects. These results highlight the therapeutic potential of formulation-driven localized MEKi delivery for HPV-associated diseases. Ongoing work is focused on further optimizing the formulation, characterizing immune responses, and extending application to cervico-vaginal neoplasia models to support future clinical translation.

References: [1] Luna, A. J., et al. (2021). PLOS Pathogens, 17(1). https://doi.org/10.1371/journal.ppat.1009216
[2] Luna, A. J., et al. (2023). Antiviral Research, 216, 105667. https://doi.org/10.1016/j.antiviral.2023.105667
[3] Topical Formulations for Targeted Delivery of Therapeutics: Provisional Application (US63/411,027), filed: Sep 28, 2022.

Acknowledgements: The authors would like to thank International Flavors & Fragrances (IFF) Pharma Solutions, and Gattefossé Corporation for their support of this project.

Funding: NCI NIH P30 CA118100; NIAID NIH R21 AI176571

HPV16+ rafts were grown for 12 days, left untreated, treated with topical vehicle, or vehicle plus the trametinib for 48 hours. Note that this vehicle formulation activates p-ERK1/2 and disrupts the tissue, which is an undesirable outcome.

Female (A) and male (B) mice bearing relatively large MmuPV1 tumors were randomized into treatment groups with similar average tumor volumes. Mice were treated every two days with a topical formulation of vehicle (2.5% HPC, 15% Tc), vehicle plus 150 µM trametinib, or vehicle plus 600 µM trametinib for 42 days.

No significant weight loss was noted in any of the animals.