Antiviral Combination Approach as a Perspective to Combat Enterovirus Infections.

Human enteroviruses distributed worldwide are causative agents of a broad spectrum of diseases with extremely high morbidity, including a series of severe illnesses of the central nervous system, heart, endocrine pancreas, skeleton muscles, etc., as well as the common cold contributing to the development of chronic respiratory diseases, including the chronic obstructive pulmonary disease. The above mentioned diseases along with the significantly high morbidity and mortality in children, as well as in the high-risk populations (immunodeficiencies, neonates) definitely formulate the chemotherapy as the main tool for the control of enterovirus infections. At present, clinically effective antivirals for use in the treatment of enteroviral infection do not exist, in spite of the large amount of work carried out in this field. The main reason for this is the development of drug resistance. We studied the process of development of resistance to the strongest inhibitors of enteroviruses, WIN compounds (VP1 protein hydrophobic pocket blockers), especially in the models in vivo, Coxsackievirus B (CV-B) infections in mice. We introduced the tracing of a panel of phenotypic markers (MIC50 value, plaque shape and size, stability at 50℃, pathogenicity in mice) for characterization of the drug-mutants (resistant and dependent) as a very important stage in the study of enterovirus inhibitors. Moreover, as a result of VP1 RNA sequence analysis performed on the model of disoxaril mutants of CVB1, we determined the molecular basis of the drug-resistance. The monotherapy courses were the only approach used till now. For the first time in the research for anti-enterovirus antivirals our team introduced the testing of combination effect of the selective inhibitors of enterovirus replication with different mode of action. This study resulted in the selection of a number of very effective in vitro double combinations with synergistic effect and a broad spectrum of sensitive enteroviruses. The most prospective attainment in our examinations in this field was the development of a novel scheme for the combined application of anti-enteroviral substances in coxsackievirus B1 neuroinfection in newborn mice. It consisted of a consecutive, alternating and non simultaneous administration of the substances in the combination. The triple combination - disoxaril- guanidine. HCl-oxoglaucine (DGO) showed a high effectiveness expressed in the marked reduction of the mortality rate in infected mice as compared both to the placebo group, and to the partner compounds used alone every day, and to the same combination applied simultaneously every day. The studies of the drug sensitivity of viral brain isolates from mice treated with DGO combination showed not only preserved, but even increased sensitivity to the drugs included in the combination. Obviously, the consecutive alternating administration of anti-enteroviral substances hinders the occurrence of drug-resistance in the course of the experimental enteroviral infections in mice.

Avoiding drug-resistance development by novel approach of combining anti-enteroviral substances against coxsackievirus B1 infection in mice.

Current study presents a novel scheme for combined application of anti-enteroviral substances in coxsackievirus B1 neuroinfection in newborn mice. It consists of a consecutive alternating, not simultaneous, administration of the substances in combination. A triple combination showing good efficacy was selected as a result of a screening of double, triple and quadruple combinations of enteroviral inhibitors. Its effectiveness is expressed in lengthening of the mean survival time and about 50% reduction of mortality rate in infected newborns as compared both to the placebo group, individual compounds used alone every day, and to the same combination applied simultaneously every day. Chronology of alternation of the individual drug administration plays a key role in the efficacy of the combination. Studies of the drug sensitivity of viral brain isolates from mice, treated with the drug combination indicate that virus isolates from the group treated with the alternating combination not only preserve, but even increase their sensitivity to the drugs. MIC(50) values of virus isolates from groups treated with monotherapies of the compounds manifested development of drug resistance. Obviously, the consecutive alternating administration of anti-enteroviral substances hinders the occurrence of drug resistance in the course of experimental coxsackievirus B1 infection in mice.