Background: Equine malignant melanoma (EMM) is a frequently occurring dermoepidermal tumor in grey horses. Currently available therapies are either challenging or inefficient. Betulinic acid (BA), a naturally occurring triterpenoid, is a promising compound for cancer treatment. To evaluate the potential of BA as a topical therapy for EMM, its anticancer effects on primary equine melanoma cells and dermal fibroblasts and its percutaneous permeation through isolated equine skin were assessed in vitro. Results: BA showed antiproliferative and cytotoxic effects on both primary equine melanoma cells and fibroblasts in a time-and dose-dependent manner. The lowest half-maximal inhibitory concentrations were obtained 96 h after the beginning of drug exposure (12.7 μmol/L and 23.6 μmol/L for melanoma cells eRGO1 and MelDuWi, respectively, in cytotoxicity assay). High concentrations of the compound were reached in the required skin layers in vitro. Conclusion: BA is a promising substance for topical EMM treatment. Further clinical studies in horses are necessary to assess safety and antitumoral effects in vivo.

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.

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Background: Little is known about the implications of hyperinsulinemia on energy metabolism, and such knowledge might help understand the pathophysiology of insulin dysregulation. Objectives: Describe differences in the metabolic response to an oral glucose test, depending on the magnitude of the insulin response. Animals: Twelve Icelandic horses in various metabolic states. Methods: Horses were subjected to 3 oral glucose tests (OGT; 0.5 g/kg body weight glucose). Basal, 120 and 180 minutes samples were analyzed using a combined liquid chromatography tandem mass spectrometry and flow injection analysis tandem mass spectrometry metabolomic assay. Insulin concentrations were measured using an ELISA. Analysis was performed using linear models and partial least-squares regression. Results: The kynurenine : tryptophan ratio increased over time during the OGT (adjusted P-value = .001). A high insulin response was associated with lower arginine (adjusted P-value = .02) and carnitine (adjusted P-value = .03) concentrations. A predictive model using only baseline samples performed well with as few as 7 distinct metabolites (sensitivity, 86%; 95% confidence interval [CI], 81%-90%; specificity, 88%; 95% CI, 84%-92%). Conclusions and Clinical Importance: Our results suggest induction of low-grade inflammation during the OGT. Plasma arginine and carnitine concentrations were lower in horses with high insulin response and could constitute potential therapeutic targets. Development of screening tools to identify insulin-dysregulated horses using only baseline blood sample appears promising.

BackgroundInsulin dysregulation (ID) with basal or postprandial hyperinsulinemia is one of the key findings in horses and ponies suffering from the equine metabolic syndrome (EMS). Assessment of ID can easily be performed in clinical settings by the use of oral glucose challenge tests. Oral glucose test (OGT) performed with 1 g/kg bodyweight (BW) glucose administered via naso-gastric tube allows the exact administration of a defined glucose dosage in a short time. However, reliable cut-off values have not been available so far. Therefore, the aim of the study was to describe variations in insulin response to OGT via naso-gastric tubing and to provide a clinical useful cut-off value for ID when using the insulin quantification performed with an equine-optimized insulin enzyme-linked immunosorbent assay.ResultsData visualization revealed no clear separation in the serum insulin concentration of insulin sensitive and insulin dysregulated horses during OGT. Therefore, a model based clustering method was used to circumvent the use of an arbitrary limit for categorization. This method considered all data-points for the classification, taking into account the individual insulin trajectory during the OGT. With this method two clusters were differentiated, one with low and one with high insulin responses during OGT. The cluster of individuals with low insulin response was consistently detected, independently of the initialization parameters of the algorithm. In this cluster the 97.5% quantile of insulin is 110 µLU/mL at 120 min. We suggest using this insulin concentration of 110 µLU/mL as a cut-off value for samples obtained at 120 min in OGT.ConclusionOGT performed with 1 g/kg BW glucose and administration via naso-gastric tubing can easily be performed under clinical settings. Application of the cut-off value of 110 µLU/mL at 120 min allows assessment of ID in horses.