Antiproliferative and Cytotoxic Effects of Betulinic Acid on Equine Melanoma Cells and Percutaneous Permeation of Betulinic Acid Through Equine Skin In Vitro

Objective Equine malignant melanoma (EMM) is a frequently occurring disease in grey horses. Current therapies are still not satisfying. Betulinic acid (BA), a naturally occurring substance, exhibits anticancer activity. With intent to develop a drug for the topical treatment of EMM BA was assessed for its cytotoxic and antiproliferative effects on primary equine melanoma cells and fibroblasts and evaluated for its percutaneous permeation in vitro.
Material and methods Cells were treated with BA in different concentrations ranging from 1 to 100 µmol/l. The proportion of treated cells relative to untreated controls was determined 5, 24, 48 and 96 hours after the beginning of drug exposure. Proliferation and viability were measured using crystal violet staining (CVS) and a CellTiter 96^® AQueous One Solution (MTS) assay. Half maximal inhibitory concentration (IC₅₀) values were determined. A pharmaceutical test formulation containing 1% BA was evaluated for its transdermal permeation on isolated equine skin (700 µm thickness) using Franz-type diffusion cells. Afterwards permeated skin was sliced with a cryostat and a depth profile of BA, extracted from the skin, was determined by HPLC analysis.
Results BA showed antiproliferative and cytotoxic activity on both equine melanoma cells and fibroblasts with the lowest IC₅₀ -values measured after 96 hours. BA was able to penetrate all epidermal and dermal layers of the tested equine skin. In all examined layers IC₅₀ -values were reached.
Conclusion These in vitro results identify BA as a promising compound for treatment of EMM. Further clinical studies in horses are necessary to assess safety and antitumoral effects of the formulation in vivo.