In vitro assessment of triterpenoids NVX-207 and betulinyl-bis-sulfamate as a topical treatment for equine skin cancer

Abstract

Equine sarcoid (ES) is the most prevalent skin tumor in equids worldwide. Additionally, aging grey horses frequently suffer from equine malignant melanoma (EMM). Current local therapies targeting these skin tumors remain challenging. Therefore, more feasible topical treatment options should be considered. In order to develop a topical therapy against ES and EMM, betulinyl-bis-sulfamate and NVX-207, derivatives of the naturally occurring betulin and betulinic acid, respectively, were evaluated for their antiproliferative (crystal violet staining assay), cytotoxic (MTS assay) and apoptotic (AnnexinV staining, cell cycle investigations) effects on primary ES cells, EMM cells and equine dermal fibroblasts in vitro. The more potent derivative was assessed for its in vitro penetration and permeation on isolated equine skin within 30 min and 24 h using Franz-type diffusion cells and HPLC analysis. Betulinyl-bis-sulfamate and NVX-207 inhibited the proliferation and metabolism in ES cells, EMM cells and fibroblasts significantly (p < 0.001) in a time- and dose-dependent manner. NVX-207 had superior anticancer effects compared to betulinyl-bis-sulfamate. Both compounds led to the externalization of phosphatidylserines on the cell membrane and DNA fragmentation, demonstrating that the effective mode of action was apoptosis. After 48 h of treatment with NVX-207, the number of necrotic cells was less than 2% in all cell types. Detected amounts of NVX-207 in the different skin layers exceeded the half-maximal inhibitory concentrations calculated by far. Even though data obtained in vitro are auspicious, the results are not unconditionally applicable to the clinical situation. Consequently, in vivo studies are required to address the antitumoral effects of topically applied NVX-207 in ES and EMM patients.

Concentration profiles and safety of topically applied betulinic acid and NVX‐207 in eight healthy horses—A randomized, blinded, placebo‐controlled, crossover pilot study

“…The horses were topically treated with test formulations that had been previously tested in permeation studies with isolated equine skin (Weber, Meißner, et al, 2020; Weber, Funtan, et al, 2020, under review). They were assigned to the following treatment groups: Test formulation 1 (TF‐PLACEBO) contained “Basiscreme DAC” (amphiphilic cream as published in the German Drug Codex; Table 1) with 20% medium‐chained triglycerides; test formulation 2 (TF‐BA) contained “Basiscreme DAC” with 20% medium‐chained triglycerides and 1% BA; and test formulation 3 (TF‐NVX207) contained “Basiscreme DAC” with 1% NVX‐207.…”

“…The naturally occurring betulinic acid (BA) and synthetically modified BA derivative NVX‐207 have been demonstrated to exert antiproliferative and cytotoxic effects in equine melanoma cells in vitro, which is mediated by the induction of apoptosis (Liebscher et al, 2016; Weber, Meißner, et al, 2020; Weber, Funtan, et al, 2020, under review). Furthermore, a sufficient penetration and permeation of both compounds in isolated equine skin has been reported, as assessed by Franz‐type diffusion cell (FDC) experiments (Weber, Meißner, et al, 2020; Weber, Funtan, et al, 2020, under review).…”

“…The naturally occurring betulinic acid (BA) and synthetically modified BA derivative NVX‐207 have been demonstrated to exert antiproliferative and cytotoxic effects in equine melanoma cells in vitro, which is mediated by the induction of apoptosis (Liebscher et al, 2016; Weber, Meißner, et al, 2020; Weber, Funtan, et al, 2020, under review). Furthermore, a sufficient penetration and permeation of both compounds in isolated equine skin has been reported, as assessed by Franz‐type diffusion cell (FDC) experiments (Weber, Meißner, et al, 2020; Weber, Funtan, et al, 2020, under review). After application of 1% formulations, the in vitro concentration profiles determined in the integument exceeded the half‐maximal inhibitory concentrations (IC 50 ) for equine melanoma cells in the epidermal layers and superficial and partially deep dermal skin layers (Weber, Meißner, et al, 2020; Weber, Funtan, et al, 2020, under review).…”

“…Furthermore, a sufficient penetration and permeation of both compounds in isolated equine skin has been reported, as assessed by Franz‐type diffusion cell (FDC) experiments (Weber, Meißner, et al, 2020; Weber, Funtan, et al, 2020, under review). After application of 1% formulations, the in vitro concentration profiles determined in the integument exceeded the half‐maximal inhibitory concentrations (IC 50 ) for equine melanoma cells in the epidermal layers and superficial and partially deep dermal skin layers (Weber, Meißner, et al, 2020; Weber, Funtan, et al, 2020, under review). Both methods, cell culture experiments and FDC studies, are valuable tools for the evaluation of the in vitro efficacy and quality of topical formulations.…”

“…However, the whole complexity of a biological system, including the metabolism, distribution, and elimination of a therapeutic agent, cannot be reproduced by FDC experiments, and in vivo data may have to follow the initial evaluations (Luís et al, 2016; OECD, 2004; OECD/OCDE, 2004). Furthermore, as normal equine dermal fibroblasts are also sensitive toward BA and NVX‐207 in vitro, it has been indicated that time‐ and concentration‐dependent antiproliferative and cytotoxic effects cannot be ruled out for this cell type in vivo (Weber, Meißner, et al, 2020; Weber, Funtan, et al, 2020, under review). To the best of the authors’ knowledge, no further literature exists about the effects of either compound on equine keratinocytes or other unaltered equine skin cells but fibroblasts.…”