Purpose: Poor wound healing outcomes are common in patients seeking gender-affirming surgery and our prior work implicates exogenous testosterone (T) as a contributor. However, cessation of testosterone is associated with impaired quality-of-life. Further, blockade of deleterious effects of endogenous T in cis-males at the wound site is desirable. Similarly, estradiol has been shown to exert pro-wound repair effects via estrogen receptor (ER). We sought to develop sustained-release topical therapeutics for the delivery of 17β-estradiol and flutamide (an ER agonist and AR antagonist respectively) to improve healing of cutaneous wounds.
Methods: Three different formulations of topical 17b-estrogen and one of flutamide were created. Active agents were mixed at different concentrations with propylene glycol monocaprylate (cosurfactant, humectant, solubilizer), stearyl alcohol (emulsifier/gelling agent), water, and coconut oil (vehicle). Topicals underwent stability and in vitro permeation/release tests using rat skin in a Franz vertical diffusion cell. Ointment was applied to rat skin in the donor compartment. Samples were taken from the receptor compartment, containing PBS plus methyl-β-cyclodextrin (MβCD), at timepoints of 30 minutes, 1-8h, and 24h then diluted in methanol, centrifuged, and underwent high-performance liquid chromatography. Rat skin samples were homogenized, centrifuged and supernatant subsequently analyzed using HPLC.
A flutamide and 17b-estradiol ointment were tested in our rat model of testosterone-influenced WH. Rats underwent surgical castration and were randomly allocated based on systemic hormone and topical therapy (no systemic hormone vs. systemic testosterone cypionate; topical 17b-Estradiol vs. Flutamide vs. vehicle applied daily). Animals underwent bilateral dorsal excisional wounding, were harvested on POD10, and analyzed with planimetry, histology, and immunofluorescence. Systemic testosterone and estradiol levels were measured with mass spectrometry.
Results: Estradiol levels were detected in all skin samples treated with 17b-estradiol formulations (55.2ug formulation A, 25.8ug formulation B, 37.7ug formulation C), showing wound delivery. Estradiol levels were undetectable on all nine Franz diffusion cell runs across all formulations, suggesting negligible systemic release.
Wounds from rats with exogenous T and topically treated with estradiol (OVX/E2 + 2T) or Flutamide (OVX/FLUT+2T) experienced faster wound closure rates at days 3 and 5 compared to OVX/2T (p=0.0019 and p=0.0069 respectively). Histology revealed increased granulation thickness in OVX/E2 + 2T and OVX/FLUT+2T rats compared to controls. Wounds from OVX/E2 + 2T had decreased epithelial gap and increased epithelial thickness compared to OVX/FLUT+2T (p<0.05). Immunofluorescence staining revealed higher expression of CD68 in OVX/FLUT+2T and OVX/E2 + 2T compared to OVX/PLAC (p=0.0077 and p=0.0461 respectively). ER biomarkers showed higher expression in the OVX/E2 + 2T group as compared to OVX/PLAC.
Conclusion: We developed proof-of-concept 17b-estradiol and flutamide sustained-release topical formulations to improve wound repair. Stability, permeation tests, and E2 levels on the skin and transdermal compartment revealed all three topical estradiol formulations were stable and permeated into the skin adequately with minimal transdermal penetration, suggesting these formulations may offer local delivery with minimal systemic delivery. Ongoing efforts include improvements in formulation (e.g., addition of stabilizers) and conduction of further animal efficacy experiments.