Highly Diluted Antibodies to eNOS Restore Endothelium Function in Aortic Rings From Hypertensive Rats.

Background: Nitric oxide (NO) as a vaso- and cardio-protective agent could reduce vasomotor dysfunction in different cardiovascular diseases. One of the current therapeutics targeted at NO availability in the vascular wall are highly diluted antibodies to endothelial NO-synthase (eNOS). This drug has previously shown its endothelium-protective effect and effectiveness in reducing hypertension. Current study was dedicated to evaluate the direct impact of highly diluted antibodies to eNOS on the vessel constriction and dilation ex vivo. Methods: For that purpose, we used thoracic aortas dissected from spontaneously hypertensive (SHR) rats. Endothelium-dependent relaxation in the presence of highly diluted antibodies to eNOS (1 mL) was examined after phenylephrine-induced pre-constriction of the aorta rings in response to gradually increased acetylcholine concentration (1 nM to 10 µM). Results: Highly diluted antibodies to eNOS enhanced acetylcholine-induced relaxation in a statistically significant manner. Moreover, it was demonstrated that observed effect was similar to perindopril, a well-known angiotensin-converting-enzyme inhibitor, which works through relaxing and widening blood vessels. Conclusions: Our findings indicate that highly diluted antibodies to eNOS restored impaired endothelium function, as demonstrated by increased relaxation of SHR rats aorta rings. The revealed results suggest beneficial effect of highly diluted antibodies to eNOS to ameliorate hypertension and related diseases.

Insights into the Mechanism of Action of Highly Diluted Biologics.

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.

Dose-Response Effect of Antibodies to S100 Protein and Cannabinoid Receptor Type 1 in Released-Active Form in the Light-Dark Test in Mice.

Earlier studies have shown that combination of antibodies to S100 protein and to cannabinoid receptor type 1 in released-active form (Brizantin) may possess anxiolytic properties and decrease nicotine dependence. Released-active form of antibodies is a novel approach that permits to modify natural functions of the target molecule (antigen) under investigation. The aim of the present study was to evaluate the anxiolytic-like effect of Brizantin in the light-dark test in mice, according to its ability to influence the number of entries into the lit compartment and the total time spent there. Three doses of Brizantin (2.5, 5, and 10 mL/kg) were compared with diazepam (1 mg/kg), placebo, and vehicle control. Anxiolytic-like effect of the tested drug was shown to be dose dependent, with an increasing trend from 2.5 to 10 mL/kg. Brizantin in its highest dose significantly increased studied behavioral parameters, although its effect was less pronounced than that of the reference drug diazepam (1 mg/kg).

Hydration of the Carboxylate Group in Anti-Inflammatory Drugs: ATR-IR and Computational Studies of Aqueous Solution of Sodium Diclofenac.

Diclofenac (active ingredient of Voltaren) has a significant, multifaceted role in medicine, pharmacy, and biochemistry. Its physical properties and impact on biomolecular structures still attract essential scientific interest. However, its interaction with water has not been described yet at the molecular level. In the present study, we shed light on the interaction between the steric hindrance (the intramolecular N-H···O bond, etc.) carboxylate group (-CO2-) with water. Aqueous solution of sodium declofenac is investigated using attenuated total reflection-infrared (ATR-IR) and computational approaches, i.e., classical molecular dynamics (MD) simulations and density functional theory (DFT). Our coupled classical MD simulations, DFT calculations, and ATR-IR spectroscopy results indicated that the -CO2- group of the diclofenac anion undergoes strong specific interactions with the water molecules. The combined experimental and theoretical techniques provide significant insights into the spectroscopic manifestation of these interactions and the structure of the hydration shell of the -CO2- group. Moreover, the developed methodology for the theoretical analysis of the ATR-IR spectrum could serve as a template for the future IR/Raman studies of the strong interaction between the steric hindrance -CO2- group of bioactive molecules with the water molecules in dilute aqueous solutions.

Efficacy of novel antibody-based drugs against rhinovirus infection: In vitro and in vivo results.

Rhinoviruses (RVs) cause the common cold and are associated with exacerbations of chronic inflammatory respiratory diseases, especially asthma and chronic obstructive pulmonary disease (COPD). We have assessed the antiviral drugs Anaferon for Children (AC) and Ergoferon (containing AC as one of the active pharmaceutical ingredients) in in vitro and in vivo experimental models, in order to evaluate their anti-rhinoviral and immunomodulatory potential. HeLa cells were pretreated with AC, and levels of the interferon-stimulated gene (ISG), 2'-5'-oligoadenylate synthetase 1 (OAS1-A) and viral replication were analyzed. In a mouse model of RV-induced exacerbation of allergic airway inflammation we administered Ergoferon and analyzed its effect on type I (IFN-ß), type II (IFN-γ) and type III (IFN-λ) IFNs induction, cell counts in bronchoalveolar lavage (BAL), cytokine (interleukin (IL)-4; IL-6) and chemokine (CXCL10/IP-10; CXCL1/KC) levels. It was shown that AC increased OAS1-А production and significantly decreased viral replication in vitro. Increased IFNs expression together with reduced neutrophils/lymphocytes recruitment and correlated IL-4/IL-6 declination was demonstrated for Ergoferon in vivo. However, there was no effect on examined chemokines. We conclude that AC and Ergoferon possess effects against RV infection and may have potential as novel therapies against RV-induced exacerbations of asthma.

Activity of ergoferon against lethal influenza A (H3N2) virus infection in mice.

BACKGROUND: The influenza A virus accounts for serious annual viral upper respiratory tract infections. It is constantly able to modify its antigenic structure, thus evading host defence mechanisms. Moreover, currently available anti-influenza agents have a rather limited application, emphasizing the further need for new effective treatments. One of them is ergoferon, a drug containing combined polyclonal antibodies - anti-interferon gamma, anti-CD4 receptor and anti-histamine - in released-active form. The purpose of the study was to assess ergoferon antiviral efficacy in mice challenged with the A/Aichi/2/68 (H3N2) influenza virus. METHODS: The virus was inoculated intranasally at a 90% lethal dose. Ergoferon was administered at 0.4 ml/day per os in a preventive and therapeutic regimen - daily for 5 days prior to and for 16 days after the challenge. The reference product, Tamiflu (oseltamivir), was used as a positive control treatment - at 20 mg/kg/day for 5 days after the challenge. Mice in the negative control group received distilled water which had been utilized for test sample preparation; untreated control animals received no treatment. Antiviral efficacy was assessed by an increase in survival rate, average life expectancy and virus titre reduction in the challenged mouse lungs. RESULTS: Survival rate and average life expectancy values were increased significantly in groups treated with ergoferon and Tamiflu, as compared with controls. Lung virus titres were reduced in these groups as observed on days 2 and 4 post-inoculation. CONCLUSIONS: Ergoferon demonstrated antiviral activity by reducing the severity and duration of the major signs of induced influenza infection.

In vitro screening of major neurotransmitter systems possibly involved in the mechanism of action of antibodies to S100 protein in released-active form.

Experimentally and clinically, it was shown that released-active form of antibodies to S100 protein (RAF of Abs to S100) exerts a wide range of pharmacological activities: anxiolytic, antiasthenic, antiaggressive, stress-protective, antihypoxic, antiischemic, neuroprotective, and nootropic. The purpose of this study was to determine the influence of RAF of Abs to S100 on major neurotransmitter systems (serotoninergic, GABAergic, dopaminergic, and on sigma receptors as well) which are possibly involved in its mechanism of pharmacological activity. Radioligand binding assays were used for assessment of the drug influence on ligand-receptor interaction. [(35)S]GTPγS binding assay, cyclic adenosine monophosphate HTRF™, cellular dielectric spectroscopy assays, and assays based on measurement of intracellular concentration of Ca(2+) ions were used for assessment of agonist or antagonist properties of the drug toward receptors. RAF of Abs to S100 increased radioligand binding to 5-HT1F, 5-HT2B, 5-HT2Cedited, 5-HT3, and to D3 receptors by 142.0%, 131.9%, 149.3%, 120.7%, and 126.3%, respectively. Also, the drug significantly inhibited specific binding of radioligands to GABAB1A/B2 receptors by 25.8%, and to both native and recombinant human sigma1 receptors by 75.3% and 40.32%, respectively. In the functional assays, it was shown that the drug exerted antagonism at 5-HT1B, D3, and GABAB1A/B2 receptors inhibiting agonist-induced responses by 23.24%, 32.76%, and 30.2%, respectively. On the contrary, the drug exerted an agonist effect at 5-HT1A receptors enhancing receptor functional activity by 28.0%. The pharmacological profiling of RAF of Abs to S100 among 27 receptor provides evidence for drug-related modification of major neurotransmitter systems.

Novel approach to activity evaluation for release-active forms of anti-interferon-gamma antibodies based on enzyme-linked immunoassay.

Selection of a suitable assay to measure the activity of drug agents based on release-active forms of anti-interferon-gamma antibodies (RA forms of Abs) is an important step forward in the investigation of such agents. In this study, the enzyme-linked immunosorbent assay was utilized to examine the effect of RA forms of Abs specific for human interferon gamma on the interaction between monoclonal anti-interferon gamma antibodies and recombinant human interferon gamma. The experimental data and the results obtained by using relevant mathematical analysis showed that such RA forms of Abs are able to modulate the monoclonal antibody interaction with both soluble and immobilized (to the assay plate well) interferon gamma. These data demonstrated the importance of using relatively low concentrations of both soluble and plate-immobilized interferon gamma to detect the effects of RA forms of Abs to interferon gamma on the binding of monoclonal antibodies to interferon gamma. It has been suggested that the observed influence of RA forms of Abs on 'antibody-antigen' interaction could be used to detect and analyze the activity of drugs containing RA forms of Abs.

Release-active dilutions of diclofenac enhance anti-inflammatory effect of diclofenac in carrageenan-induced rat paw edema model.

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.

Subetta treatment increases adiponectin secretion by mature human adipocytes in vitro.

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.

Activity of ultra-low doses of antibodies to gamma-interferon against lethal influenza A(H1N1)2009 virus infection in mice.

BACKGROUND: The influenza A virus is a highly infective agent that causes acute pulmonary diseases. In serious cases, it causes pneumonia which is particularly fatal in patients with cardiopulmonary diseases, obesity, young children and elderly people. The present study shows a protective effect of ultra-low doses of purified antibodies to gamma-interferon (Anaferon for children®, AC®) against lethal influenza virus infection caused by pandemic influenza virus A(H1N1) in mice. METHODS: Balb/c mice were infected with mouse-adapted pandemic influenza virus A/California/07/09 (H1N1)v. Mortality, weight loss, infectious titer of the virus in lungs and lung morphology were monitored in the groups of AC®-, oseltamivir- and placebo-treated animals. RESULTS: The protective action of AC® was demonstrated by prolongation of life of the infected animals, reduction of infectious titer of the virus in the lung tissue, normalization of weight dynamics in the course of disease, decrease in mortality of treated animals compared to a placebo control and normalization of lung tissue structure. The protective activity of AC® was similar to that of the reference compound oseltamivir. Combination of AC® with oseltamivir resulted in a higher protective effect comparing to oseltamivir alone. CONCLUSION: Based on the results obtained, AC® should be considered as an important part of anti-influenza prophylaxis and therapy, in particular in severe cases of the disease.