Larisa N. Shishkina

The antiviral activity of 12 nitrogenand oxygen-containing heterocyclic compounds was studied by screening against the vaccinia virus in Vero cell culture and the data obtained were compared with the results of ab initio quantum chemical calculations using the DFT (density functional theory) method. The index of selectivity was found to be contingent upon the toxicity of the compounds under study. Compounds containing a 2,3-dichlorobenzyl group were found to be more toxic and unpromising as antiviral compounds. In addition, biological testing showed that (E)-azomethines containing a fragment of 1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazole showed no antiviral activity. Compounds derived from vanillin and vanillal have proved to be the most promising in relation to the smallpox vaccine virus. The method of preliminary quantum chemical modeling can make it possible to identify unpromising, low-activity structures even at the planning stage of work on the synthesis and bioscreening of new chemical compounds.

Изучена фармакокинетика отечественного противооспенного препарата — НИОХ-14 (аналога разработанного в США препарата «Тековиримат») при применении внутрь в твердых желатиновых капсулах в клиническом исследовании у 90 добровольцев. Концентрацию активного метаболита — ST-246, образующегося из НИОХ-14 в организме млекопитающих, оценивали методом LC-MS/MS (тандемной масс-спектрометрии — MS/MS и жидкостной хроматографии — LC). Были определены основные фармакокинетические параметры препарата НИОХ-14 при его однократном и многократном (в течение 6 суток) применении в дозах 200, 600 и 1200 мг. Показано, что НИОХ-14 в капсулах является биодоступным, и его активный метаболит ST-246 достигает максимальных концентраций от 342 ± 146 до 1281 ± 550 нг/мл в период времени от 5,2 ± 2,4 до 85,8 ± 47,0 ч после первого введения НИОХ-14 добровольцам в разных схемах и дозах.

INTRODUCTION. Vaccination against smallpox was discontinued in 1980 following the success of the World Health Organisation’s Smallpox Eradication Programme. Consequently, more than half of the current world population lacks immunity to orthopoxviruses pathogenic to humans. Since 2022, several countries have been reporting outbreaks of mpox (previously known as monkeypox), which necessitates developing new small molecules active against orthopoxviruses.AIM. The aim was to evaluate the safety and tolerability of NIOCH-14, a Russian anti-smallpox medicinal product, after oral administration in a phase I clinical trial.MATERIALS AND METHODS. The clinical trial was conducted in 90 healthy volunteers at the Federal State Bud­getary Institution of Healthcare ‘Medical and Sanitary Unit No. 163 of the Federal Medical and Biological Agency’ in accordance with Protocol No. NIOCH-01/20 ‘An open, simple, randomised study of the safety, tolerability, pharmacokinetics of NIOCH-14 in volunteers aged 18–50 years in parallel groups’. Investigators used a standard set of clinical, laboratory, and instrument-based testing methods. The clinical trial included six experimental groups of 15 subjects each. Groups 1, 2, and 3 received NIOCH-14 as a single oral dose of 200, 600, and 1200 mg, respectively. Groups 4, 5, and 6 received NIOCH-14 at daily oral doses of 200, 600, and 1200 mg/day, respectively, for 6 days.RESULTS. After single and repeated administration of NIOCH-14 at doses of 200, 600, and 1200 mg to volunteers, the study did not demonstrate any changes in the blood test results (erythrocyte, leucocyte, platelet, and differential leucocyte counts; haemoglobin levels; erythrocyte sedimentation rate values; alanine transaminase, aspartate transaminase, and alkaline phosphatase activity levels; total protein, total bilirubin, glucose, creatinine, urea, and C-reactive protein levels; prothrombin time ratios; thymol turbidity). Similarly, the urinalysis results remained unaltered throughout the entire observation period. No adverse events were observed in volunteers receiving NIOCH-14 at all single doses or repeated doses of 200 and 600 mg. One volunteer receiving the investigational medicinal product at a repeated dose of 1200 mg/day experienced transient epigastric pain on days 2 and 5.CONCLUSIONS. The physical examination, haematology, blood chemistry, and urinalysis results did not reveal any pathological changes in healthy volunteers. The investigational medicinal product, formulated as hard gelatin capsules containing 200 mg of NIOCH-14 and excipients, demonstrated a favourable safety and tolerability profile.

In 2022, the number of mpox cases spiked worldwide, leading to a surge in scientific research on members of the Orthopoxvirus genus and the discovery of new compounds exhibiting anti-orthopoxvirus activity. This work is devoted to the synthesis of compounds containing an adamantane fragment and the evaluation of their activity against the vaccinia virus, offering a possible mechanism of the antiviral action of the synthesized agents. Among all the studied adamantane derivatives, three compounds (2, 4, and 12) were found to demonstrate the highest antiviral activity, with the most promising compound 2 (N-(adamantan-1-yl)isonicotinamide) having the lowest toxicity level with a selectivity index (SI) of 115. The pharmacophoric profiles of these compounds are similar to the pharmacophoric profile of tecovirimat, an inhibitor of the membrane viral protein p37. Analysis of the results of molecular modeling suggests that the investigated compounds can inhibit the vaccinia virus by suppressing the phospholipase activity of membrane viral protein p37.

   The aim of this work was to obtain and study antiviral properties of a drug for intranasal use containing yeast double-stranded RNA (dsRNA) and recombinant human interferon alpha-2b (IFN-α2b) incorporated into a delivery system (molecular construct).   Material and methods. Molecular constructs carrying IFN-α2b and dsRNA were obtained by our original method. For present study, the samples of intranasal dosage forms of three formulations were chosen: 50 µg of dsRNA and 10, 50 or 100 IU of IFN-α2b per one dose. The in vitro antiviral activity of the composition preparations was determined by inhibition of cytopathic effect (CPE) of murine encephalomyocarditis virus (EMCV), Columbia strain, in mouse L929 and L-68 cell cultures. The in vivo protective properties of the preparations were studied in white outbred ICR male mice intranasally infected with 10 lethal dose, 50 %, of influenza virus strain A/Aichi/2/68 (H3N2). The protection coefficient was calculated by average life expectancy and death of animals.   Results and discussion. All the composition preparations used in this study demonstrated the ability to inhibit the destructive effect of the test virus and increase the number of viable cells in L929 and L-68 cultures. The enhancement of the protective effect was observed with increasing IFN-α2b content in the drug formulation. The greatest antiviral activity was shown for the preparation containing 50 µg of dsRNA and 100 IU of IFN-α2b per dose. A composition preparation of the same formulation, administered intranasally three times at a dose of (2.5 mg dsRNA and 5000 IU IFN-α2b)/kg according to the therapeutic and prophylactic regimen, protected 50 % of animals from death, which was not observed in a group administered with dsRNA alone. IFN-α2b used at a dose equivalent to its content in the composition preparation had a similar but less pronounced effect.   Conclusions. The results of the in vitro and in vivo experiments have confirmed the fact that combining interferon and its inducer enhances the antiviral effect of the composition drug compared to that of its components used separately. The data obtained testify to the prospects for the development of intranasal forms of antiviral drugs using a combination of interferon and dsRNA incorporated in a delivery system.

A series of 7‐(het)aryl‐6‐benzyloxycarbonyl‐substituted dihydro(azolo)pyrimidines were synthesized by exploiting the multicomponent Biginelli reaction between benzyl acetoacetate, various aldehydes, aminoazoles (3‐aminopyrazole, 5‐aminotetrazole, and 3‐amino‐1,2,4‐triazole) or urea. The compounds obtained were assessed for their cytotoxicity and the antiviral activity in MDCK cell culture against the influenza virus strain A/California/04/2009 (H1N1)v (subtype A/H1N1pdm09) in comparison with the known anti‐influenza drugs rimantadine, oseltamivir, and triazide. In addition, using molecular docking, the free energies for binding of the polymerase proteins PA (polymerase acidic) and PB2 (polymerase basic) of the internal ribonucleoprotein (RNP) of influenza virus (IV) A/California/04/2009 (H1N1)v with dihydro(azolo)pyrimidines. Compounds 1a and 11 were found to be the most active against this strain of influenza virus, but the value of binding free energies of the PB2 protein for compound 11 was higher (12.55 µg/mL).

Introduction. Since early May 2022, more than 90,000 cases of monkeypox virus infection have been reported in more than 70 countries around the World. This is the largest outbreak of monkeypox ever recorded outside of Africa. The aim of the study is to confirm the first case of monkeypox in Russia, to isolate and sequence a new strain of monkeypox virus (MPXV), and to assess its sensitivity to the 7-[N-(4-trifluoromethylbenzoyl)-hydrazinocarbonyl]-tricyclo-[3.2.2.0^2,4]non-8-en-6-carboxylic acid (NIOCH-14) antipox drug. Materials and methods. The biological materials obtained from the affected area of the skin (contents of vesicles), a nasopharyngeal smear, sputum and venous blood from a patient with suspected monkeypox were used. The disease was confirmed by PCR followed by determination of the nucleotide sequence of viral DNA by sequencing. Isolation of the new MPXV strain from clinical samples was carried out in Vero E6 cells. The antiviral effectiveness of NIOCH-14 against the new MPXV strain was assessed using an adapted spectrophotometric method. Results. A diagnostic study of the biological samples of a patient who returned from a tourist trip to European countries with complaints of skin rashes all over the body revealed MPXV DNA. A new strain of MPXV was isolated from vesicles in Vero E6 cells, and the genomic sequence MPXV-pustule S45 was assembled using high-throughput parallel sequencing (NGS). Discussion. The effectiveness of the finished dosage form of NIOCH-14 against the new strain of MPXV based on the results of determining the 50% virus inhibitory concentration (IC50) was 0.02 μg/mL, and the selectivity index (SI) was 15,000. Conclusion. In this study, the pathogen of monkeypox was detected and identified using real-time PCR, NGS and electron microscopy, and the first imported case of this disease in Russia was confirmed. It has been proven that the drug NIOCH-14 exhibits high antiviral activity in vitro against the new MPXV strain.

Selective synthesis of polycarbonyl conjugates of (+)‐fenchone and (+)‐camphor was carried out (44–91 % yields) via the ring‐opening transformation of 5‐acyl‐4‐pyrones with hydrazones of the corresponding monoterpenoids. A strong influence of the hydrazone fragment on the observed tautomeric equilibrium of the tricarbonyl system was shown. Although the major tautomer of the conjugates is the acyclic polycarbonyl form, the camphor‐based conjugates undergo new type of ring‐chain tautomerism, diketoenaminone‐dihydropyridone equilibrium, and predominantly exist in the cyclic dihydropyridone form in DMSO‐d₆. The polyketones can undergo intramolecular cyclization to form N‐amino‐4‐pyridones in high selectivity. In vitro screening for activity against the influenza virus H1 N1 and vaccinia virus was estimated for the obtained conjugates. The (+)‐fenchone derivatives demonstrated the higher activity against vaccinia virus than camphor derivatives. The conjugate, which was prepared from diethyl isochelidonate and hydrazone (+)‐fenchone, showed the highest activity against vaccinia virus (SI=17).

High variability of influenza viruses requires the development of agents for nonspecific resistance stimulation, along with the development of new drugs for prevention and treatment. Among the antiviral drugs, interferons and their inducers are known to exhibit a universally wide spectrum of action. The Institute of Medical Biotechnology, a branch of SRC VB «Vector», Rospotrebnadzor, has developed the technology and obtained pharmaceutical compositions containing an interferon inducer — double stranded ribonucleic acid (dsRNA) from the killer strain of Saccharomyces cerevisiae yeast and recombinant human interferon-alpha-2b (IFN- alpha-2b). The specific antiviral activity of the preparations and synergistic effect of the components within the compositions were shown in L-68 and L-929 cell cultures. The aim of this work was to study antiviral activity of intranasal forms of the pharmaceutical compositions containing yeast dsRNA and recombinant human interferon-alpha-2b in a model of lethal influenza infection in mice. The outbred ICR/CD1 mice were intranasally infected with influenza A/Aichi/2/68 (H3N2) virus. The study compositions were intranasally administered 3 hours before infection with influenza virus, as well as 1 and 3 days post infection. The doses of active components in the administered compositions were as follows: for dsRNA — 2.5 mg/kg, for IFN-alpha-2b — 500 IU/kg, 2500 IU/kg, or 5000 IU/kg. The antiviral activity of the drugs was assessed based on the mortality rate and the average life expectancy of mice. It was shown that a three-time intranasal administration of the composition of dsRNA (2.5 mg/kg) and IFN-alpha-2b (2500 IU) into the infected mice according to therapeutic-prophylactic regimen led to an increase in the rates of survival and average life expectancy of animals, which were comparable to the effect of Tamiflu. The comparison preparations — dsRNA and IFN-alpha-2b — administered intranasally at the same doses and regimen exerted no antiviral effect in this mouse model of viral infection. The data obtained confirm the prospects for further development of new dosage forms of dsRNA and interferons for intranasal application as agents for prevention and treatment of influenza.