The first principle for ensuring patient safety is to do no harm (primum non nocere). It clearly justifies why the legislative requirements for the safety of medicinal products should be strict and meticulous. The aim of preclinical safety evaluation     is to protect public health by preventing the clinical use of medicinal products with   a potentially unfavourable risk–benefit profile. Traditionally, toxicology is defined as the study of the adverse effects of chemical substances on living organisms. Toxicity studies are integral to preclinical programmes for new medicinal products. The role of toxicology in preclinical studies is explained in this interview by Corresponding Member of the Russian Academy of Sciences, Doctor of Medical Sciences, and Full Professor Andrey D. Durnev. Dr. Durnev is a laureate of the State Prize of the Russian Federation. He holds the positions of Head of the Pharmaceutical Toxicology Department of V.V. Zakusov Institute of Pharmacology, Chairman of the Commission of the Ministry of Health of the Russian Federation for the Certification of Experts for the Right to Perform Regulatory Evaluation of Medicinal Products for Human Use, and Deputy Chairman of the Interdepartmental Council of the Russian Academy of Sciences on the Scientific Substantiation and Maintenance of Drug Policy of the Russian Federation.

The lack of approved paediatric medicines is a global problem. Since paediatric patients are a high-risk group, a developer of paediatric medicines needs to evaluate and minimise unjustified risks early in the development process.

The aim of the study was to analyse the current regulatory and methodological framework for non-clinical research and identify key risk factors that need addressing in paediatric drug development in order to substantiate the safety of children. One of the main objectives of non-clinical safety assessment of a medicinal product intended for children is to identify undesirable effects on child growth and development. According to national and international methodological  guidelines, the developer may obtain the most valuable information to decide whether it is   safe to enrol children into a clinical trial from the safety data collected in clinical studies in adult subjects. If the safety data available from studies in mature animals and adult subjects are insufficient to evaluate the safety of the clinical trial for paediatric patients, the developer may consider conducting non-clinical studies in immature (juvenile) animals. A paediatric non-clinical testing programme should be in line with a comprehensive significance assessment of key risk factors carried out on a case-by-case basis using weight-of-evidence (WoE) analysis. The conclusion on the safety of a medicinal product for children should be based on risk assessment in relevant clinical and non-clinical studies.

The endocrine system coordinates almost all organs and other systems in vertebrates. In particular, it regulates such important biological functions as metabolism, development, reproduction, and behaviour. To date, a significant amount of information has accumulated on endocrine disorders associated with chemical compounds (endocrine disruptors) used in various fields of human activity. The aim of this study was to evaluate the possibility of preclinical risk assessment for the endocrine function disorders attributable to new medicinal products. Endocrine disruptors are associated with a wide range of adverse events, including developmental problems arising from functional abnormalities of the endocrine system. Endocrine disorders caused by endocrine-disrupting chemicals are characterised by a long latency period between exposure and manifestation of a dysfunction; a nonlinear dose–response relationship; and a linear correlation of damage severity to exposure timing and duration. The chemicals influence the endocrine system through multiple mechanisms, the main of which being the interaction with cellular receptors sensitive to certain hormones and the influence on gene expression, intracellular signalling, and hormone transport, etc. This paper discusses the possibility of using hormone levels as indicators of endocrine disruption and presents the literature and authors’ own data on normal levels of relevant hormones in the blood of animals. An analysis of animal blood hormone levels in preclinical programmes will provide an opportunity to evaluate potential iatrogenic risks.

Selective kappa-opioid receptor (KOR) agonists are considered a promising group of substances for developing opioid analgesics characterised with an original mechanism of action without the risk of respiratory depression and drug addiction. Previous studies identified a fluorophenyl derivative of imidazo[1,2-a]benzimidazole (RU-1205) with a KOR-based mechanism of analgesic action established in in vitro and in vivo experiments.

The aim of the study was to assess the effect of 9-(2-morpholinoethyl)-2-(4-fluorophenyl)imidazo[1,2-a]benzimidazole dihydrochloride on the level of DNA damage in rats after a single subcutaneous injection.

Materials and methods. The study was conducted in adult white outbred laboratory rats of both sexes. DNA damage was estimated using the comet assay. The study involved a single subcutaneous injection of an aqueous solution of RU-1205 in three doses: 1, 10, and 100 mg/kg. The authors used intraperitoneal methyl methanesulfonate (40 mg per kg of animal body weight) as a positive control and 0.9% NaCl  (100 μL per 100 g of animal body weight) as a negative control.

Results. A single subcutaneous injection of RU-1205 to rats did not produce a significant dose-dependent increase in % tail DNA when compared with the state of   the corresponding organ/tissue cell genome in negative control animals after normal saline administration at the same time points. In the negative control groups, % tail DNA in cells of various organs/tissues ranged from 1.83% to 3.82% (median values [25–75%]). On the contrary, the administration of 40 mg/kg of genotoxic methyl methanesulfonate led to an increase in damaged DNA in all studied organs and tissues when compared with negative control animals.

Conclusions. The study of 9-(2-morpholinoethyl)-2-(4-fluorophenyl)imidazo[1,2-a] benzimidazole dihydrochloride genotoxicity demonstrated that a single subcutaneous injection of 1, 10, or 100 mg/kg of RU-1205 to rats did not damage the cell genome of the studied organs.

The humane treatment of laboratory animals is an integral part of good laboratory practice. It remains relevant to study the anaesthetic effects of various medicinal products helping to reduce pain and distress in laboratory animals.

The aim of the study was to compare the effects of the α₂ blockers proroxan and atipamezole on changes in electroencephalogram rhythm index ratios after dexmedetomidine administration.

Materials and methods. The study used male Soviet chinchilla rabbits weighing 3.0±0.3 kg (n=12). Study animals received single injections of 100 μg/kg dexmedetomidine subcutaneously, 50 μg/kg atipamezole intramuscularly, and 170 μg/kg proroxan intravenously (equimolar to the dose of dexmedetomidine). The effects of these medicinal products were evaluated by pharmacoelectroencephalography. The authors recorded electroencephalograms using cup electrodes and a Neuron-Spectrum-1 8-channel encephalograph (Neurosoft, Russia)  with  a  bandwidth of 0.5–35 Hz and a sampling frequency of 500 Hz. The distribution of quantitative characteristics was checked for normality using the Shapiro–Wilk W test. The authors used one-way ANOVA with Dunnett’s post hoc test to evaluate the significance of differences for the normal distribution of quantitative characteristics; they used the nonparametric Kruskal–Wallis test with  Dunn’s  post  hoc  test  for the non-normal one.

Results. Dexmedetomidine administration resulted in significant two-hour changes in the rabbit brain, and the authors observed an increase in the delta rhythm and     a decrease in the theta rhythm. At equimolar doses, atipamezole returned the ratios of the wave rhythm indices to the baseline values, whereas proroxan had no effect on the ratios.

Conclusions. As demonstrated by the neutralisation of dexmedetomidine sedative and hypnotic effects, atipamezole can be used in veterinary for recovery from anaesthesia. Proroxan, on the contrary, is not effective in reversing the sedative effect of dexmedetomidine.

Neuraminidase inhibitors are a class of antivirals used to  treat  influenza  infections. Screening assays for potential neuraminidase inhibitors would benefit from  the development of in vitro procedures that do not require handling viruses. The aim of the study was to  develop  and  validate  a  procedure  for  in  vitro  determination of inhibitory effects on neuraminidase (EC 3.2.1.18), using 2’-4(methylumbelliferyl)-α-D-N-acetylneuraminic acid (4MU-NANA) as a fluorogenic substrate and quinonoid pigments, potential neuraminidase inhibitors, as a case study. Materials and methods: the method is based on neuraminidase cleavage of 4MU-NANA   to release fluorescent 4-methylumbelliferone, which is detected at the excitation  and emission wavelengths of 360 and 450 nm, respectively. Results: the procedure was validated for specificity, range, accuracy, and precision. It remained linear over the range of 0.31–80 μM of 4-methylumbelliferone. The accuracy for four concentration levels (including the LLOQ) was 87–114%; i.e., the relative error of accuracy evaluation was less than 15%. The intra- and inter-day precision ranged from 1.5 to 10.4% and from 2.3 to 9.6%, respectively. Inhibitory effect evaluation using zanamivir hydrate (0.6–150 nM) demonstrated the accuracy of 89–120% and the precision  of 3.1–11.0%. The IC₅₀ values for positive controls (zanamivir hydrate and oseltamivir) were 27 ± 3 and 16 ± 2 nM, respectively. The following solvents may be used: 50% dimethyl sulfoxide, 5% Polysorbate 80, 50% ethanol, 50 and 100% methanol. If  a compound is insoluble in the solvents, it is possible to form inclusion complexes with 2-hydroxypropyl-β-cyclodextrin. For bisnaphthazarin, the natural quinonoid pigment used in the study, the IC₅₀ amounted to 273 ± 28 nМ. Conclusion: the procedure demonstrated adequate accuracy and reproducibility and is recommended  for screening for potential neuraminidase inhibitors. In order to use the procedure for insoluble substances, the authors suggest forming inclusion complexes with cyclodextrins.

Russian and international regulatory documents require that analytical procedures for establishing bioequivalence and comparability of quality attributes of biotechnological (biological) products, carrying out batch release of medicinal products, and conducting other equally  important in  vitro  studies must be  validated. These in vitro studies include molecule–receptor binding and product bioactivity assays. However, at present, there is no single approach to validation of in vitro bioanalytical methods not involving the determination of active ingredient concentrations in biological fluids. The aim of the work was to validate a procedure for assessing insulin-dependent glucose uptake and demonstrate the suitability of GOD-PAP GLUCOSE kits for glucose determination in culture media. Materials and methods: The study used RinFast® insulin aspart by Geropharm, Russia; a placebo for insulin aspart; the L6J1 rat myogenic cell line; and a GOD-PAP GLUCOSE kit for quantitative determination of glucose. The study was carried out on differentiated cells cultured for 7 days. To encourage L6J1 differentiation, the authors used DMEM with 4.5 g/L glucose and 2% horse serum. The statistical analysis of results was performed using Prism 9. Results: The study demonstrated the analytical procedure’s specificity, as the concentration of residual glucose in the culture medium observed with the placebo was 4 times higher than that with the maximum concentration of the medicinal product. The determination of precision showed the repeatability of 4–9% and the intralaboratory precision of 11–16%. The coefficient of variation for robustness amounted to 14% in 4 independent experiments comprising a total of 9 analytical runs. The authors compared insulin products (the insulin aspart and a genetically engineered human  insulin),  and the half-maximal inhibitory concentration (IC50) values differed by 1.5 times. For the GOD-PAP GLUCOSE kit, the linear regression coefficient  of  determination was 0.9983, the sensitivity amounted to 1 mmol/ L, and the accuracy ranged between 95% and 107%. Conclusions: the procedure using L6J1 rat myoblasts as  a test system may be considered specific, highly precise, and robust in assessing insulin-dependent glucose uptake and suitable for detecting biological activity   of insulin preparations in vitro.

According to current regulatory views, a comparative study of the pharmacodynamics (PD) of low molecular weight heparin (LMWH) products and confirmation of their equivalence require comparing three PD markers: the anti-Xa activity, the anti-IIa activity, and the tissue factor pathway inhibitor (TFPI) concentration. The aim of this study was to analyse the features specific to the bioanalytical part of an equivalence study of a nadroparin calcium biosimilar after single subcutaneous administration. Material and methods: the anti-Xa and anti-IIa activity values and TFPI content were determined in human plasma samples obtained after single subcutaneous administration of the test and the reference product in the same dose, using commercially available reagent kits and pre-validated assays. The authors calculated the main PD parameters (surrogate pharmacokinetic markers), namely the maximum activity or concentration (A_max or C_max), time to maximum activity or concentration (T_max), area under the activity–time (or concentration–time) curve (AUC ), and half-life period (T_1/2), by means of model-independent statistical moment analysis and carried out further statistical testing of the parameters. Results: the anti-Xa activity and TFPI concentration results provided for  the  possibility  of  calculating  and  comparing  the PD parameters (A_max or C_max, AUC_0-24, AUC_0-∞, Tmax, T_1/2) and estimating the confidence intervals that are necessary to confirm the bioequivalence of the studied products. The anti-IIa activity data had a characteristic pattern of slight fluctuations around one level, which prevented the calculation and comparison of PD parameters. Conclusion: the study identified specific features to consider when planning comparative PD studies of nadroparin calcium products. Firstly, it is feasible to divide samples into two test aliquots (one for anti-Xa and anti-IIa activity determination, the other for TFPI analysis) at the moment of collection in order to perform the analytical step correctly. Secondly, there is no need in full validation for the bioanalytical assays of the anti-Xa and anti-II activity and TFPI content in human plasma validated in the concentration ranges of 0.024–0.182 IU/mL, 0.0069–0.052 IU/mL and 1.56–100 ng/mL, respectively; a confirmation that the active ingredient does not interfere with the analytical procedure is adequate for the purpose. Finally, the data obtained may not allow for calculating PD parameters and comparing confidence intervals for all three markers. The listed considerations may be relevant for other LMWH products as well.

Mitoxantrone is a marker substrate of breast cancer resistance protein (BCRP). BCRP is involved in a number of pharmacokinetic drug-drug interactions. The transporter’s possible saturability makes it advisable to use low concentrations of mitoxantrone for in vitro studies. Consequently, mitoxantrone quantification requires   a method with high sensitivity.

The aim of the study was to develop and validate a procedure for mitoxantrone quantification in Caco-2 culture media by HPLC-MS/MS.

Materials and methods.  The  authors  used  an  Ultimate  3000  HPLC  system  and a TSQ Fortis triple quadrupole mass spectrometer by Thermo Fisher Scientific and a Selectra C18 column (4.6×100 mm, 5 μm, 100 Å) by United Chemical Technologies. The elution ran in a gradient mode with a mobile phase of 1% formic acid solution and methanol. Experimental parameters were as follows: eluent flow rate, 0.3 mL/min; separation column temperature, 35 °C; injection volume, 5  μL; ana lysis time, 10 min; approximate mitoxantrone retention time, 5.51 min. The sample preparation involved protein precipitation from the culture medium with methanol, followed by centrifugation at 13,000 g for 10 min. The detection was performed using electrospray ionisation in the positive ion mode. Detection parameters were   as follows: electrospray voltage, 3700 V; sheath gas flow rate, 50 L/min; auxiliary    gas flow rate, 10 L/min; sweep gas flow rate, 1 L/min; ion-transfer tube temperature, 300 °C; and evaporator temperature, 350 °C. The detection was set at mass transitions of m/z 455 to 88.2 and m/z 455 to 358.1, with the collision energy for these transitions amounting to 25 V and 18 V, respectively. The source fragmentation was at 0, and the CID gas pressure was at 2 mTorr.

Results. The analytical procedure showed selectivity, high sensitivity (limit of detection, 10 nmol/L; lower limit of quantification, 50 nmol/L), accuracy, precision, and linearity in the concentration range of 50–1000 nmol/L. The authors observed no carryover or matrix effects. A simulation of real-life storage conditions demonstrated high stability of mitoxantrone samples. Thus, the analytical procedure enables preclinical evaluation of medicinal product effects on the functional activity of BCRP, based on assessing the transcellular mitoxantrone transport in the presence of a test product.

Conclusion. The authors developed and validated the analytical procedure for mitoxantrone quantification in Caco-2 culture media by HPLC-MS/MS.

Being widely used, medicinal products based on vegetable oils require strict regulation and evaluation of quality attributes, determination of shelf-life periods, and monitoring of storage conditions. The most common testing method for unsaturated compounds in vegetable oils is the iodine value determination, which has a range     of limitations. An alternative method for determining the degree of unsaturation is based on epoxidation.

The aim of the study was to evaluate the possibility of determining the degree of unsaturation of terpenoids and essential oils using peroxycarboxylic acids.

Materials and methods. The authors performed epoxidation of linalool, myrcene, and lemon oil with peroxydecanoic and peroxyoctanoic acids, followed by iodometric titration of the excess acid.

Results. The study demonstrated the possibility of measuring the degree of unsaturation of the selected essential oil and terpenoids using peracid epoxidation.   The authors developed a procedure for determining the iodine value of essential oils and calculated the iodine values of linalool, myrcene, and lemon oil. Epoxidation proceeded as a second-order reaction. The authors obtained the following reaction rate constants: 3.9 L×mol^–1×min^–1 for linalool (298 К), 1.76 L×mol^–1×min^–1 for myrcene converting to monoxide (298 К), 0.044 L×mol^–1×min^–1 for myrcene epoxide converting to diepoxide (298 К), and 3.9 L×mol^–1×min^–1 for lemon oil (297 К).

Conclusions. The suggested procedure involving peracid titration for rapid and efficient determination of the degree of unsaturation (iodine value) provides a potential basis for developing a quantification method for total unsaturated bioactive compounds in essential oils.