Цель. Изучение механизма противовирусного действия и оценка клинической эффективности, безопасности и переносимости терапии препаратом Фортепрен у пациентов с хронической рецидивирующей герпесвирусной инфекцией генитальной локализации. Материалы и методы. Клинические исследования проходил лекарственный препарат Фортепрен (натрия полипренилфосфат) раствор для инъекций, 4 мг/мл, который вводили пациентам, прошедшим базовый курс препарата Ацикловир-Акри для снятия острой фазы заболевания. Исследование проводили на 80 пациентах мужского и женского пола, отобранных в процессе скрининга с подтвержденным диагнозом хроническая рецидивирующая герпесвирусная инфекция генитальной локализации (ХРГВИ). Было сформировано 2 группы. Пациентам 1 группы (экспериментальной) Фортепрен в дозе 2 мл (8 мг) вводили внутримышечно на стадии ремиссии трехкратно с интервалом в 21 день на 3±2, 24±2 и 45±2 сутки после окончания 10-дневного базового курса лечения острой фазы заболевания с применением препарата ацикловир таблетки по 400 мг — 13±2, 34±2 и 55±2 день от начала исследования. Пациентам 2 группы (контрольной) раствор плацебо в объеме 2 мл вводили внутримышечно на стадии ремиссии трехкратно на 3±2, 24±2 и 45±2 сутки после окончания базового курса лечения острой фазы заболевания с применением препарата ацикловир таблетки по 400 мг — 13±2, 34±2 и 55±2 день от начала исследования. Для оценки эффективности применения препарата Фортепрен использовали такие критерии как: увеличение продолжительности межрецидивного периода, уменьшение частоты рецидивов за весь период наблюдения; снижение выраженности рецидивов, оцененное в баллах, изменение иммунологических показателей по динамике изменения продукции основных цитокинов. Результаты. В 1 группе пациентов межрецидивный период за весь период проведения исследования статистически значимо увеличился с 29,36±2,16 до 42,98±3,29 суток. В то время как во 2 группе изменения этого показателя не отмечено. Соответственно у пациентов 1 группы отмечено статистически достоверное сокращение частоты рецидивов ХРГВИ с 3,03±0,02 до начала лечения до 1,94±0,19 во время лечения при отсутствии снижения частоты рецидивов в контроле. Оценка в баллах выраженности рецидивов ХРГВИ у пациентов группы 1 указывает на эффективность данной схемы лечения. Сумма баллов средних значений балла выраженности признаков ХРГВИ статистически достоверно снизилась в 1 группе с 7,36±0,35 баллов на 1 визите до начала лечения до 4,75±0,35 баллов во время лечения. Во 2 группе изменений не отмечено. Уровень лейкоцитарного вирус-индуцированного интерферона (ЛВИ—ИФН) у пациентов экспериментальной группы повысился к завершению клинического исследования с 36 до 64% по сравнению с контрольной группой, в которой не наблюдалось прироста титров ЛВИ-ИФН. Оценка продукции ИФНα, ИФНγ, ИЛ-10, ИЛ-12p40, ИЛ-12p70, ИЛ-15, ИЛ-2, ИЛ-4, МИФ-1α, ФНОα показала увеличение их количества к завершению исследования у пациентов, получавших препарат Фортепрен, по сравнению с контрольной группой. Заключение. Показана эффективность применения препарата Фортепрен в дозе 2 мл (8 мг) при внутримышечном введении пациентам с хронической рецидивирующей герпесвирусной инфекцией генитальной локализации на стадии ремиссии трехкратно с интервалом в 21 день на 3±2, 24±2 и 45±2 сутки после окончания 10-дневного базового курса лечения острой фазы заболевания с применением препарата ацикловир.

Aim. The study of the mechanism of antiviral action and evaluation of the clinical efficacy, safety and tolerability of therapy with Fortepren® in patients with chronic recurrent herpesvirus infection of genital localization (CRHVI). Materials and methods. Clinical studies were carried out of a drug Fortepren® (0.4% sodium polyprenyl phosphate solution), which was administered to patients who underwent a basic therapeutic course of the drug Acyclovir-Acry® to relieve the acute phase of the disease. The study was performed on 80 male and female patients selected during the screening with a confirmed diagnosis of CRHVI. Two groups were formed. Patients of group 1 (experimental) were intramuscularly injected with Fortepren® at a dose of 2 ml (8 mg) three times at intervals of 21 days by 3 ± 2, 24 ± 2 and 45 ± 2 days following the 10-day basic course of treatment of the acute phase of diseases with the use of the acyclovir tablets of 400 mg - 13 ± 2, 34 ± 2 and 55 ± 2 days from the beginning of the study. Patients of the 2nd group (control) were intramuscularly injected with placebo solution at a volume of 2 ml instead of Fortepren®. To evaluate Fortepren® efficacy, the following criteria were used: increase in the duration of the inter-recessive period, a decrease in the frequency of relapses over the entire observation period; decrease in the severity of relapses, estimated in points, changes in immunological parameters according to the dynamics of changes in the production of the main cytokines. Results. In patients treated with Fortepren®, the inter-recurrence period for the entire study period increased statistically from 29.36 ± 2.16 to 42.98 ± 3.29 days, while in the control group this indicator have not changed. Accordingly, in patients treated with Fortepren®, a statistically significant reduction in the incidence of recurrence of CRHVI from 3.03 ± 0.02 before treatment to 1.94 ± 0.19 was observed during treatment in the absence of a decrease in the frequency of relapses in the control. Evaluation of the severity of CRHVI relapses in patients treated with Fortepren® indicates the efficacy of this protocol. The sum of the scores of the mean values of CRHVI symptoms signs was statistically significantly decreased in the group 1 from 7.36 ± 0.35 points at the 1 st visit before the start of treatment to 4.75 ± 0.35 points during the treatment. No changes were seen in the control group. The level of leukocyte virus-induced interferon (LVI-IFN) in the patients of the group 1 increased from 36% to 64% in the end of the clinical trial compared to the control group, in which the increase in LVI-IFN titers was not observed. To further justify the possibility of increasing the immune response of cells, establishing possible mechanisms that determine the efficacy of treatment for CRHVI with Fortepren®, evaluation of the production of IFNz, IFNy, IL-10, IL-12p40, IL-12p70, IL-15, IL-2, IL-4, MIF-Fz, TNFа was made. In the of the study levels of all these cytokines was increased in patients treated with Fortepren® compared with the control group. Conclusion. The efficacy of using Fortepren® in a dose of 2 ml (8 mg) with intramuscular administration to patients with chronic recurrent herpesviral infection of genital localization at the stage of remission three times with an interval of 21 days by 3 ± 2, 24 ± 2 and 45 ± 2 days after the end of 10 day basic course of treatment of the acute phase of the disease with the use of the drug acyclovir.

Aim. The study of the mechanism of antiviral action and evaluation of the clinical efficacy, safety and tolerability of therapy with Fortepren ® in patients with chronic recurrent herpesvirus infection of genital localization (CRHVI). Materials and methods. Clinical studies were carried out of a drug Fortepren ® (0.4% sodium polyprenyl phosphate solution), which was administered to patients who underwent a basic therapeutic course of the drug Acyclovir-Acry® to relieve the acute phase of the disease. The study was performed on 80 male and female patients selected during the screening with a confirmed diagnosis of CRHVI. Two groups were formed. Patients of group 1 (experimental) were intramuscularly injected with Fortepren ® at a dose of 2 ml (8 mg) three times at intervals of 21 days by 3 ± 2, 24 ± 2 and 45 ± 2 days following the 10-day basic course of treatment of the acute phase of diseases with the use of the acyclovir tablets of 400 mg - 13 ± 2, 34 ± 2 and 55 ± 2 days from the beginning of the study. Patients of the 2nd group (control) were intramuscularly injected with placebo solution at a volume of 2 ml instead of Fortepren ® . To evaluate Fortepren ® efficacy, the following criteria were used: increase in the duration of the inter-recessive period, a decrease in the frequency of relapses over the entire observation period; decrease in the severity of relapses, estimated in points, changes in immunological parameters according to the dynamics of changes in the production of the main cytokines. Results. In patients treated with Fortepren ® , the inter-recurrence period for the entire study period increased statistically from 29.36 ± 2.16 to 42.98 ± 3.29 days, while in the control group this indicator have not changed. Accordingly, in patients treated with Fortepren ® , a statistically significant reduction in the incidence of recurrence of CRHVI from 3.03 ± 0.02 before treatment to 1.94 ± 0.19 was observed during treatment in the absence of a decrease in the frequency of relapses in the control. Evaluation of the severity of CRHVI relapses in patients treated with Fortepren ® indicates the efficacy of this protocol. The sum of the scores of the mean values of CRHVI symptoms signs was statistically significantly decreased in the group 1 from 7.36 ± 0.35 points at the 1 st visit before the start of treatment to 4.75 ± 0.35 points during the treatment. No changes were seen in the control group. The level of leukocyte virus-induced interferon (LVI-IFN) in the patients of the group 1 increased from 36% to 64% in the end of the clinical trial compared to the control group, in which the increase in LVI-IFN titers was not observed. To further justify the possibility of increasing the immune response of cells, establishing possible mechanisms that determine the efficacy of treatment for CRHVI with Fortepren®, evaluation of the production of IFNz, IFNy, IL-10, IL-12p40, IL-12p70, IL-15, IL-2, IL-4, MIF-Fz, TNFа was made. In the of the study levels of all these cytokines was increased in patients treated with Fortepren® compared with the control group. Conclusion. The efficacy of using Fortepren ® in a dose of 2 ml (8 mg) with intramuscular administration to patients with chronic recurrent herpesviral infection of genital localization at the stage of remission three times with an interval of 21 days by 3 ± 2, 24 ± 2 and 45 ± 2 days after the end of 10 day basic course of treatment of the acute phase of the disease with the use of the drug acyclovir.

AIMSelection of optimal dosage regimen, length of treatment course (frequency of administration), safety, tolerance and clinical effectiveness evaluation of the medical preparation fortepren in patients with chronical recurrent herpes virus infection of genital localization.MATERIALS AND METHODSThe medical product of antiviral and immune modulating effect--fortepren (sodium polyprenyl phosphate) as a 4 mg/ml solution for injections combined with the base course of acyclic nucleoside acyclovir, 400 mg tablets, held studies. 40 male and female patients participated in the study. After a 10-day acyclovir course (400 mg x 3 times a day) for removing the acute phase, 4 groups of 10 individuals were formed: 1--5 ml (20 mg) of fortepren i/m once at day 13 ± 2 after the start of the study after the completion of the treatment of the acute phase of the disease; 2--5 ml (20 mg) fortepren i/m 3 times at an interval of 21 days; 3--2 ml (8 mg) fortepren i/m 3 times at an interval of 21 days; 4 (control)--5 ml of placebo i/m at remission stage 3 times at an interval of 21 days. Increase of the duration of inter-recurrence period, decrease of the severity of the recurrences, state of skin and mucous damage elements, improvements of immunologic parameters were considered during effectiveness evaluation.RESULTSSignificant differences in the frequency of recurrences of genital herpes were shown for 3 months of observation in experimental and control groups. A significant reduction of genital herpes recurrence frequency from 3.52 ± 0.09 (before treatment) to 2.89 ± 0.08 (after treatment) was noted in patients of group 3 (p < 0.001). The frequency of recurrences in the control group was 3.84 ± 0.10, that was higher than the parameters in all the experimental groups. A significant reduction of the rash area was noted in group 3, moreover, a redution of frequency of detection of clinical manifestations of genital herpes in the form of vesicle elements after treatment in groups 2 (p = 0.02) and 3 (p = 0.005) was found. Evaluation of local symptoms has established that burning have caused minimal discomfort for patients of groups 3 and 4 and itch and soreness--of groups 1 and 3. The least pronounced exacerbations were noted in patients of group 3. Intramuscular administration of fortepren preparation was established to result in the increase of titers of leukocyte virus-induced interferon for the whole duration of treatment.CONCLUSIONAn intramuscular dose of 2 ml (8 mg) at recurrence stage 3 times at an interval of 21 days after the completion of the 10-day base course of treatment of the acute phase of chronical recurrent herpes virus infection of genital localization using acyclovir was accepted as an optimal dosage regimen. Analysis of the obtained results has shown an acceptable safety profile and a good level of tolerance for fortepren preparation.

Aim. Evaluation of an antiviral effect of miRNA in the nanoparticles of a polycationic compound against mRNA of vp16 protein (UL48 gene) of herpes simplex virus type 2 (HSV-2) in vitro. Materials and methods. 50% aqueous solution of polyethyleneimine (BDH, Great Britain), chitosan, containing approximately 15% of N-acetylated glucosamine chains (Sonat, Russia), hydrazine-hydrate and other chemical reagents (Chimmed, Russia); Vero continuous cell line, MS HSV-2 virus were used. Vero cells were cultivated in DMEM medium supplemented by 10% fetal bovine serum at 37°C in the atmosphere of 5% CO2. Cell viability was evaluated by using Neutral Red vital stain and MTT-test. Primers and probes for RT-PCR were modeled in Vector NTI 8.0 computer program according to the mRNA sequences of the studied genes (the sequences were obtained from GenBank) and synthesized in Sintol (Russia). RT-PCR tests were set using a standard procedure. Synthesis of PEI-PG-chitosan was carried out by Krivtsov G.G. et al. (2010). Results. A design and synthesis of nucleotide sequences, that have interfering activity against this virus, was carried out to study the effect of siRNA on HSV-2 virus replication. During simultaneous addition of HSV-2 and specific siRNA to Vero cells in cell culture, a significant (by 4 lg) reduction of virus yield was observed. A level of UL48 mRNA expression level was determined after the influence of various siRNA variants. A S2 siRNA variant was shown to cause the most pronounced virus-inhibiting effect, aiming for the center of RNA-target (the level of expression of the studied gene decreased by 0.5 lg). Conclusion. siRNA in the PEI-PG-chitosan complexes were established to possess in vitro pronounced suppressive HSV-2 replication activity. The results obtained could be used in creation of new therapeutic preparation against herpes viruses.

AIM:Study of antiviral activity of moraprenil phosphates (MPP) against herpes simplex type 1 virus (HSV1) in vitro and during experimental infection caused by HSV1 in mice.MATERIALS AND METHODS:Activity of MPP in vitro was tested by the ability to suppress formation of symplasts in VERO cells infected with HSV1, strain VR-3. A series of MPP that suppress virus-induced symplast-formation by 30 times was selected for in vivo experiments. Anti-viral activity of MPP in vivo was studied in HSV-1 infected mice after administration of either prophylaxis or therapy regimens.RESULTS:MPP at the dose of 20 microg/mice during s/c administration exhibited a pronounced prophylactic-therapeutic effect. Effectiveness of MPP during clinically evident herpes against the background of developing neurologic symptoms was demonstrated for the first time. Visual observation of the mice, that had received MPP as the first clinical symptoms of the disease appeared, has shown that against the background of preparation injection the clinical signs have ceased after 2 - 3 days and did not registered at least for the whole duration of the observation period (14 days).CONCLUSION:Active herpes infection is accompanied by the increase of FoxP3 expression in-thymus was shown. Possible mechanisms of anti-viral effect of MPP are discussed.

Treatment of malignant tumors is among challenging issues of clinical medicine. Dissemination and matestasis of tumor cells plays a major role in oncology, being immediately dependent on chemotaxis of tumor cells. Hence, the aim of our study is to evaluate migration of tumor cells, in terms of ligand-mediated chemotaxis of a human U 937 lymphoma cell line. Synthetic TLR ligands (DNA_lig, RNA_lig) were used as chemoattractants. Assessment of time-dependent TLR expression by the migrating cells (including TLR2, TLR3, TLR7, TLR9) was carried out by means of real-time PCR, using Boyden’s chamber system for the migration assays. We have revealed an increased cell migration towards DNA_ lig and TNFα gradient. Expression of TLR2, TLR7 and TLR9 was found to be increased under the influence of TNFα (respectively 1.5-, 4- and 19-fold). Under the influence of DNA_lig, TLR9 expression was 105-fold increased, whereas TLR3 expression was 2-fold higher, along with decrease of TLR2 expression. Expression of TLR 3 was shown to be 3-times higher under the influence of RNA_lig. The effects observed could be potentially applied for suppression of tumor cell chemotaxis by means of these ligands and by influencing different pathways ofchemotaxis regulation.

Целью данной работы являлось изучение механизмов противовирусной и иммуномоду-лирующей активности препаратов на модели герпесвирусной инфекции in vitro. В качестве модели была использована перевиваемая культура клеток Vero, инфицированная вирусом простого герпеса 1 типа (ВПГ-1). Исследование проводили с использованием 3-х схем опытов. Оценку молекулярных механизмов вируснейтрализующей и иммуномодулирующей активности проводили с использованием ОТ-ПЦР для определения уровней экспрессии а и в генов вируса герпеса простого, а также экспрессии распознающих структур врожденного иммунитета. Впервые показано, что природный комплекс цитокинов вызывает отсрочку синтеза вирусной ДНК на 6 часов и способствует повышению уровня экспрессии вирусной тимидинкиназы (ТК), что увеличивает эффективность лечения герпеса ацикловиром. Комплекс флавоноидов вызывал эффект достоверного снижения уровня экспрессии генов U L48 и U L30, белковые продукты которых (вирусная полимераза и VP16) активно участвуют в жизненном цикле вируса простого герпеса. Изучение экспрессии генов, кодирующих распознающие (сигнальные) структуры врожденного иммунитета (Toll-подобные рецепторы - TLR) при добавлении в культуру клеток рабочих доз препаратов показало, что под влиянием данных препаратов отмечается увеличение уровня экспрессии гена TLR2 в 14 - 29 раз. Экспрессия эндосомального рецептора TLR9, при этом, изменяется незначительно. Эксперименты, направленные на изучение на молекулярном уровне противовирусного и иммуномодулирующего действия препаратов, по-видимому, смогут найти применение в практической сфере.

Expressed antiviral activity of Fortepren (FP) and Gamapren (GP), polyprenyl phosphate (PPP)-contaning agents, was demonstrated in experiments on mice infected with the human herpes simplex virus, type 1 (HSV1) or the vernal encephalitis virus (VEV). Since both the viral infections are of great social significance, the PPP-containing agents should be considered prospective for the medical practice. The experimental data suggested that both the drugs considerably inhibited the VEV infectiousness in the susceptible cell culture. The quantity of protein E, the main immunogen of VEV, in the culture fluid of the VEV infected cells was shown to be markedly lowered under the effect of FP and GP. It was demonstrated for the first time that FP and GP significantly inhibited evolution of the VEV protein E in the cell culture J-96. The experiments with the infectious rhinotracheitis virus (IRTV) of the corned cattle revealed that FP and GP greatly retarded the HSV1 development in the susceptible cell culture. One of the mechanisms of the antiviral action of the PPP-containing agents was likely the effect on the evolution of the virus proteins in the cells. The impact of FP on production of some key cytokines (CT) was studied on mice with experimental vernal encephalitis (IFN-gamma, IL-4 and IL-12). The content of the above mentioned CT in blood of the mice was determined by the IFA test. Under the normal conditions and in the mice infected with VEV, production of IL-12 and IFN-gamma was shown to be stimulated during the first 3-5 days after the FP administration, whereas in the animals not exposed to FP there was observed stimulation of the IL-4 production during the first 3 days after the contamination, followed by increased production of IL-12 and IFN-gamma.