The therapeutic use of Abs in cancer, autoimmunity, transplantation, and other fields is among the major biopharmaceutical advances of the 20th century. Broader use of Ab-based drugs is constrained because of their high production costs and frequent side effects. One promising approach to overcome these limitations is the use of highly diluted Abs, which are produced by gradual reduction of an Ab concentration to an extremely low level. This technology was used to create a group of drugs for the treatment of various diseases, depending on the specificity of the used Abs. Highly diluted Abs to IFN-γ (hd-anti–IFN-γ) have been demonstrated to be efficacious against influenza and other respiratory infections in a variety of preclinical and clinical studies. In the current study, we provide evidence for a possible mechanism of action of hd-anti–IFN-γ. Using high-resolution solution nuclear magnetic resonance spectroscopy, we show that the drug induced conformational changes in the IFN-γ molecule. Chemical shift changes occurred in the amino acids located primarily at the dimer interface and at the C-terminal region of IFN-γ. These molecular changes could be crucial for the function of the protein, as evidenced by an observed hd-anti–IFN-γ–induced increase in the specific binding of IFN-γ to its receptor in U937 cells, enhanced induced production of IFN-γ in human PBMC culture, and increased survival of influenza A–infected mice.

The study was aimed to investigate the effect of technologically treated diclofenac (release-active dilutions of diclofenac (RAD of diclofenac)) on anti-inflammatory activity of diclofenac in carrageenan-induced rat paw edema model. Ninety male Wistar albino rats (6–8 weeks) divided into nine groups (n = 10) were used. Anti-inflammatory activity was assessed at 1, 2, 3, 4, and 6 h after subplantar injection of carrageenan (0.1 ml of a 1 % solution in normal saline). Diclofenac alone was studied at 5 and 20 mg/kg, RAD of diclofenac alone at 7.5 ml/kg and their combination at 5 and 7.5 ml/kg, respectively. Diclofenac reduced (p < 0.05 at least) paw edema at all time points. RAD of diclofenac enhanced (p < 0.05) anti-inflammatory effect of diclofenac (5 mg/kg) at 2, 4, and 6 h on concurrent and at 2 and 4 h on sequential administration. Moreover at 2 h, anti-inflammatory effect of combination treatment reached values comparable to those of diclofenac (20 mg/kg). In conclusion, RAD of diclofenac enhanced anti-inflammatory effect of diclofenac.

During the preparation of high dilutions, repeated external vibration (shaking) is used. We hypothesized that it was the vibration treatment, and not the negligible content of the initial substance, that underlies the activity of highly diluted preparations. In order to test this, the vibration was separated from the dilution process. After vibrating two tubes together on a vortex mixer (one containing water and the other the initial substance) the electrical conductivity and radio frequency radiation intensity of water differed from the unvibrated control, and the ability to exert a modifying effect on the target solution appeared, as assessed using ELISA and terahertz spectroscopy, appeared. Thus, the properties of the neutral carrier (water) changed after non-contact exposure to the initial substance. We have named this process ‘crossing’ and its products ‘aqueous iterations of the initial substance’. Several aqueous iterations with different physical properties were obtained, some of which have a modifying effect and others cause various chemical (catalytic) and biological (antiviral) effects similar to those of the initial substance. This indicates that during crossing, substances enter into post-vibration supramolecular interactions. At the nanoscale level, aqueous iterations and the initial substance are structurally symmetrical, which allows us to assume that the preservation of the symmetry of substances subjected to vibration treatment is the basis of the post-vibration interaction phenomenon.

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% CO₂for 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. Thesein vitroresults give first insight on possible mechanism of action of Subetta and serve as a background for further studies.

The aim of the present study was to evaluate the potential antidiabetic effects of two-component drug Subetta and its components (release-active dilutions of antibodies toβ-subunit insulin receptor (RAD of Abs toβ-InsR) and to endothelial nitric oxide synthase (RAD of Abs to eNOS)) in Goto-Kakizaki (Paris colony) (GK/Par) diabetic rats. Subetta was administered orally for 28 days once daily (5 mL/kg) and compared to its two components (2.5 mL/kg), Rosiglitazone (5 mg/kg), and vehicle (5 mL water/kg). At day 28, fasting plasma glucose levels were significantly decreased only in Subetta and Rosiglitazone groups as compared to vehicle (P<0.01):147±4 mg/dL and145±4 mg/dL and165±4 mg/dL, respectively. The data of glucose tolerance test showed that Subetta and RAD of Abs toβ-InsR (similar to Rosiglitazone) prevented significantly (P<0.01) the age-related spontaneous deterioration of glucose tolerance as seen in the control group. Subetta and RAD of Abs toβ-InsR did not significantly modify the glucose-induced insulin secretion. Chronic administration of Subetta and RAD of Abs toβ-InsR improves glucose control, to an extent similar to that of Rosiglitazone. We hypothesize that Subetta and RAD of Abs toβ-InsR mostly act via an insulin-sensitizing effect upon target tissues.

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.