Purpose. To investigate the mechanism of action in peripheral tissues of novel complex drug containing release-active dilutions of antibodies to the beta subunit of the insulin receptor and antibodies to endothelial nitric oxide synthase (Subetta), which has shown efficacy in animal models of diabetes. Methods. Human mature adipocytes were incubated either with Subetta, with one of negative controls (placebo or vehicle), with one of nonspecific controls (release-active dilutions of antibodies to cannabinoid receptor type I or release-active dilutions of rabbit nonimmune serum), or with dimethyl sulfoxide (DMSO) at 37°C in a humidified incubator at 5% CO2 for three days. Rosiglitazone was used as reference drug. Secretion of adiponectin was measured by quantitative enzyme-linked immunosorbent assay (ELISA). Results. Only Subetta significantly stimulates adiponectin production by mature human adipocytes. Nonspecific controls did not significantly affect adiponectin secretion, resulting in adiponectin levels comparable to background values of the negative controls and DMSO. Conclusion. Increasing adiponectin production in absence of insulin by Subetta probably via modulating effect on the beta subunit of the insulin receptor might serve as one of the mechanisms of the antidiabetic effect of this drug. These in vitro results give first insight on possible mechanism of action of Subetta and serve as a background for further studies.

It has been previously shown that Subetta (a drug containing released-active forms of antibodies to the insulin receptor β-subunit and antibodies to endothelial nitric oxide synthase) stimulated insulin-induced adiponectin production by mature human adipocytes in the absence of insulin. Therefore, it was assumed that Subetta could activate the insulin receptor. To confirm this hypothesis, the capacity of Subetta to activate the insulin receptor in mature human adipocytes in the absence or presence of the insulin was investigated. Cells were incubated either with Subetta or with vehicle, or with basal medium for 3 days. Then, adipocytes were treated with water or insulin (100 nm) for 15 min. Following treatment, lysates were prepared and phosphorylation of insulin receptor β-subunits was analyzed by western blot analysis. It was shown that Subetta significantly increased (P<0.001) the ‘phosphorylated-insulin receptor β-subunit/total insulin receptor β-subunit' ratios in both the presence and the absence of insulin. These results support previously published data and indicate that Subetta could activate the insulin receptor through the effect on its β-subunits, whose conformational state is essential for insulin receptor activation. This action might serve as one of the primary mechanisms of the drug's antidiabetic effect.

Background: Solar ultraviolet radiation (UV) induces DNA damages in skin via direct absorption of UVB or indirectly by photosensitization mediated through UVA. Recent findings have revealed that UVA induces cyclobutane pyrimidine dimer (CPD) generation via chemiexcitation in melanocytes hours after the exposure. This UVA-induced delayed CPD (dark CPD) constitutes the majority of CPD in melanocytes. These findings indicate that sun light can damage the skin hours after the exposure, suggesting the need for skin care products post sun exposure. The main objective of this study was to investigate whether a blend of Chrysanthemum Morifolium flower extract (Chrys) and vitamin C derivative, Ascorbic Acid-2-Glucoside (AA2G), can provide protective effects against reactive oxygen species, melanin formation and UVA-induced dark CPD. Methods: Intracellular ROS levels were measured in epidermal keratinocytes using DHR123 dye. Melanogenesis inhibition efficacy was determined using B16 cells. As for the dark CPD measurement, Melan-a cells were treated with or without actives for 6 days, then irradiated with UVA at various doses. Cells were exposed with anti-CPD mAb followed by secondary Ab. CPD levels were determined by measuring fluorescent intensity using a high content imaging analysis. Results: Chrys, AA2G and their blend at various concentrations demonstrated ROS scavenging activity. Though Chrys alone did not show significant melanogenesis inhibition in B16 assay, the blend of Chrys with AA2G demonstrated additive effects in comparison with AA2G alone. The blend of AA2G and Chrys at various concentrations exhibited enhanced efficacy for inhibiting dark CPD compared to AA2G alone. Conclusion:The results from this study indicate that the use of natural antioxidant, Chrys in combination with AA2G, provides protection against UVA-induced delayed CPD formation by enhancing ROS scavenging activity and melanogenesis inhibition. These findings could potentially be applied for formulating post-sun exposure skin care products, possibly extending to evening-after care products.

Addition of Subetta to insulin (10 nM) increased insulin-stimulated glucose uptake 43% (p<0.001). Moreover, glucose uptake stimulated by insulin (10 nM) in the presence of Subetta was similar to that stimulated by 300 nM insulin. These findings suggest that Subetta significantly enhanced insulin sensitivity of tissues through stimulation of glucose transport to myocytes mediated by glucose transporter 4.

Transformation of white adipose tissue (WAT) to a brown adipose tissue-like (BAT-like) phenotype has emerged as an attractive approach against obesity e.g. using g ß3 adrenergic receptor agonists. These could however, produce side-effects following systemic exposure. The present study explored the possibility of local use of CL-316,243 - a selective ß3 agonist - to circumvent this problem. Rats treated s.c. for 2 weeks (0.3 and 1 mg/kg) showed decreased inguinal fat pad (IFP) weight/volume, increased UCP-1 staining and expressed BAT-like features in H&E stained micrographs. Interscapular BAT increased in weight/volume. In contrast, local treatment into the IFP was not efficacious in terms of weight/volume, despite slight increases in UCP-1 staining and changes in histological features. After local treatment, the exposure of the IFP was lower than after systemic treatment. In turn higher local doses (0.5 and 5 mg/ml) were then tested which produced a strong trend for decreased volume of the IFP, a significant increase in UCP-1 staining, and also a decrease in adipocytes size but increased number. However, after this treatment the systemic exposure was in the same range as following systemic treatment. In conclusion, we saw no evidence for the possibility of converting inguinal WAT to a BAT-phenotype solely through local activation of ß3 receptors. This is in concert with our in vitro experiments which detected direct effects of PPARγ agonists at the gene/protein expression and functional level, but were unable to detect any effect of CL-316,243.