Toxicokinetic studies are a key aspect of preclinical drug safety studies. Regulatory documents describe methodological approaches to the toxicokinetic study, but do not answer many questions that arise during their planning. The purpose of this work was the analysis of literature data, domestic and foreign methodological documents related to the preclinical study of drugs toxicokinetic, own experience in conducting such studies to propose possible designs for experiments on the study of toxicokinetic in concomitant studies and in studies with the formation of satellite groups. The study materials were regulatory documents, literature sources, and guidelines for the preclinical study of toxicokinetics of drugs in the public domain (including, according to the PubMed, Web of Science, RISC (eLibrary.ru), Google scholar electronic databases), which were subjected to further general scientific analysis and generalization. The toxicokinetic data as part of the drug registration dossier are necessary to establish the relationship between the detected toxicity and the administered dose of the drug, are important for planning clinical studies, contributing to the determination of threshold values of the substance in blood plasma to assess early exposure to humans. The features of planning and experimental toxicokinetic study in various formats are considered, designs of such studies are proposed. A review of the literature data, regulatory documents, and experimental experience related to the study of toxicokinetics, which is an important element of drug development, was carried out.

Lithium salts are known to treat bipolar disorder. Their high potential as neuroprotective agents in cerebral ischemia determines relevance for preclinical studies for the registration of new drugs based on them. Aim of the study was to investigate the toxic properties and local tolerability with an assessment of the toxicokinetics of the developed lithium chloride preparation for injection in dwarf pigs with repeated intravenous administration. Material and methods. Lithium chloride (solution, for intravenous administration 4.2 %) has been administered to minipigs (control and 3 experimental groups of animals of 3 males and 3 females) for 28 days at doses of 12.6; 29.4 and 63 mg/kg. General toxicity, local irritation, and basic pharmacokinetic parameters (Cmax, AUC0-24, MRT, T1/2, Vss, and Cl) were evaluated. Serum lithium ion concentration was estimated colorimetrically using quinizarin. Results and discussion. Toxic properties of the test drug were revealed, expressed in a change in the clinical state (vomiting after administration, inhibition of behavior and feed refusal), body weight negative dynamics, clinical and laboratory changes, shortening of the “QT” interval, accompanied by abnormalities according to the pathomorphological study results (focal infiltration with single renal tubular necrosis areas, kidneys fibrosis, replacement of thyroid tissue with adipose tissue). There were no signs of locally irritating effects of the test product. To evaluate toxicokinetic parameters, a bioanalytical assay (calibration range – from 0.17 to 5.45 μg/mL) was developed and validated, which is not inferior by its characteristics to the commercial reagent kits. According to the results of biomaterial analysis no lithium accumulation in the pig’s body was found with repeated use of the test drug. Doses of the drug that provide toxic concentrations of lithium (above 3 μg/ml) in the minipig’s serum were identified. The NOAEL was 12.6 mg/kg and the LOAEL was 29.4 mg/kg. Conclusions. The comprehensive approach to the consideration of toxic manifestations and toxicokinetics, including its analytical component of studies of this kind, was noted. The obtained results should be taken into account to assess the benefit/risk ratio in the clinical use of lithium chloride injection.

Relevance. The evaluation of control samples contamination is a necessary element for confirming the correctness of toxicokinetic studies (TK), concomitant with the study of toxicity of drugs and products. At the same time, insufficient elaboration of existing regulatory documents necessitates discussion of practical aspects and improvement of the regulatory framework. Purpose. The work is devoted to the consideration of experimental experience of control samples contamination in TK studies. Materials and methods. As experimental examples, two studies of TK drugs performed using rabbits with oral administration of study drugs for 28 (Study 1) or 90 (Study 2) days were considered. Results. Study 1 revealed a contamination level of 17–25 % (on the first and last days of study drug administration, respectively). Contamination of bioassays can occur in vivo (contamination of animals) and ex vivo (at the stage of bioassay sampling, at the pre-analytical stage and analytical stage). The nature of the analyte detection in the samples made it possible to exclude contamination in vivo, and the results of an internal investigation suggested that it occurred at the stage of biosample selection. In Study 2, a number of preventive measures were implemented and a slight level of contamination was obtained (0.83 %), which did not affect the completeness and correctness of results interpretation. Conclusion. Based on experimental experience, recommendations for minimizing and preventing contamination of control samples during the biological part, pre-analytical and analytical stages of studies were formulated. Presumably, in the presence of a random nature of contamination indicating its ex vivo origin, the level of contamination not exceeding 25 % can be considered insignificant, i.e. not leading to further deviation and cancellation of the results of the entire study

Background. Pembrolizumab belongs to a fundamentally new class of antitumor agents with biological source – monoclonal antibodies. when creating generic biological medicinal products, it is necessary to assess the comparability of the pharmacokinetics of the developed drug and the original (reference) drug product in relevant animal species.Aim. To compare the pharmacokinetics of two drugs with INN pembrolizumab, concentrate for solution for infusion, administered once intravenously to Macaca fascicularis.Materials and methods. Biosimilar RPH-075 (INN pembrolizumab, JSC R-Pharm, Russia) and reference drug Keytruda® (INN pembrolizumab, MSD International GmbH) were administered once intravenously to male monkeys (2 groups of 4 males each) at a dose of 30 mg/kg. Macaque blood samples were collected for analysis before administration and at 1, 2, 4, 8, 24, 48, 72, 144, 312, 480, 648, 984, 1320 h after administration. Plasma concentrations of the active ingredient were determined by bridging ELISA using commercially available antibodies, followed by calculation of the main pharmacokinetic parameters (Cmax, AUC, MRT, Vss, T1/2, Cl).Results. Using antibodies more available than commercial reagent kits, the pembrolizumab assay method recommended by the antibody manufacturer has been replicated. The method has been validated and applied to the analysis of biosamples obtained in the preclinical study. There was no effect of the test drug and the reference drug upon single intravenous administration to monkeys condition and animals body weigh; drugs were found to have comparable pharmacokinetic profiles.Conclusion. The creation of a biosimilar drug in the Russian Federation will improve the treatment of patients with cancer, reduce the cost of treatment and increase the number of patients receiving high-quality medical care.

In pharmacokinetics (PK) studies of medicinal products with small laboratory animals models, primarily rodents, the design of the animal-point experiment is often used, involves the selection of biological material after euthanasia of the animal. The question of experimental data processing and the PK parameters calculation method in a situation where all concentration values are obtained from different individuals is relevant. Purpose of the study . Comparison of pharmacokinetic parameters calculation methods in studies with the animal-point design. Materials and methods . For a number of previously conducted studies with male outbred rats test systems, a retrospective data analysis was performed and PK parameters were calculated in three different ways: from the average concentration values at each time point (method 1): from data obtained for animals with the same sequence numbers in subgroups corresponding to time points (method 2); using resempling based on modeling of individual PK profiles (method 3). Pharmacokinetic parameters (maximum concentration — Cmax, time to reach maximum concentration — Tmax, area under the curve "concentration-time" — AUC0-t, average time to stay in the body — MRT, half-life — T1/2) were calculated by non-compartment method of statistical moments using the validated PKSolver application for Microsoft Office Excel. Results . The comparison of the obtained results did not reveal any patterns and preferences for the use of a particular method of calculating PK parameters depending on the studied drugs, route and administration way. For all evaluated PK parameters (Cmax, Tmax, AUC0-t, MRT, T1/2), similar values and/or intervals were obtained, which indicated the correctness of all considered calculation methods. Conclusion . Based on advantages and disadvantages of the calculation methods comparison it is shown that it is optimal to use method 2, which is a special case of reception (method 3) with a minimum number of replications. It is important to emphasis the method of PK parameters calculation when describing the methodology of studies to improve their quality.

Introduction. Pharmaceutical development of an innovative highly effective and competitive drug is a long and expensive process, the result of which is quite difficult to predict in advance. To speed up the entry of a new drug to the treatment and reduce the developer's material costs, it is advisable to include preclinical experiments in the process of creating a drug.Text. The purpose of this work is to create a justified approach to the implementation of laboratory pharmaceutical development involving in vivo studies. The inclusion of preclinical studies in the process of laboratory pharmaceutical development will eliminate the negative impact of pharmaceutical factors on the bioavailability of a drugs and avoid errors in the selection of excipients, as well as reduce material and time costs. The review presents examples that demonstrate the relevance of conducting preclinical experiments at different stages of pharmaceutical development. These examples made it possible to describe a clearer algorithm of actions in the laboratory pharmaceutical development of a new drug from the moment a drug candidate molecule is selected.Conclusion. Due to increase the probability of successful pharmaceutical development at initial stage, it is necessary to carry out pharmacokinetic and/or pharmacodynamic experiments to make it possible to develop a drug with an optimal pharmacokinetic profile, reduce the number of preclinical studies, the cost of development, and ensure successful translation of data into clinical practice.

The high prevalence of fungal skin infections motivates expanding the range of sertaconazole products for ex-ternal use. The aim of the study was a preclinical comparison of the safety, antifungal activity, and pharmacokinetics of Sertaverin® 2% medicated shampoo (VERTEX JSC, Russia) with those of Sertamicol® 2% solution for external use (Glenmark Pharmaceuticals Ltd, India) and Nizoral® 2% shampoo (Janssen Pharmaceuticals N.V., Belgium) approved in the Russian Federation. Materials and methods. In the toxicity study, the medicinal products were applied to the skin of male and female outbred rats at doses of 0.5 or 1.5 mL/animal for 28 days. The authors evaluated the pharmacokinetics of two sertaconazole formulations (shampoo and solution) following a single administration to adult male rats at the same dose. Nizoral® was not used in the pharmacokinetics study because it contains a different active substance, ketoconazole. The minimum inhibitory concentration (MIC) was determined using the serial microdilution method in a wide range of concentrations. Results. The medicinal products did not exhibit any significant toxic effects in laboratory animals after 28 days of repeated dermal application. Plasma sertaconazole concentrations were negligible. Sertaconazole was intensively distributed in the liver, which is a highly vascularised organ, and in the target organ (skin at the site of application). The relative bioavailability of sertaconazole from the shampoo relative to that from the solution for external use was approximately 30% in liver tissues and approximately 363% in skin tissues at the application site. Sertaverin® was comparable to sertaconazole in the active substance form in terms of inhibiting the growth of Malassezia furfur strains. The MICs calculated on the active substance basis were ≤16–64 μg/mL. Conclusions. With its synergistic dual mechanism of action, broad-spectrum antifungal activity, lipophilic properties, and low systemic absorption, Sertaverin® may provide a more effective and safe alternative to marketed medicinal products for scalp diseases.

Esomeprazole, the S-isomer of omeprazole, is a second-generation proton pump inhibitor widely used for acid-related diseases of the oesophagus, stomach, and duodenum (peptic ulcer, gastro-oesophageal reflux disease, etc.). Studies on esomeprazole safety and toxicokinetics (TK) are essential for increasing the number of modified-release esomeprazole products manufactured in Russia. The aim of the study was to compare the safety and toxicokinetics of a new esomeprazole product, 40 mg modifiedrelease capsules (Valenta Pharm JSC, Russia), and Nexium ® 40 mg film-coated tablets (AstraZeneca AB, Sweden). Materials and methods. This toxicity study involved oral administration of esomeprazole 40 mg modified-release capsules (Valenta Pharm JSC, Russia) and Nexium ® 40 mg film-coated tablets (AstraZeneca AB, Sweden) to 5 groups of rabbits (8 males and 8 females per group) for 28 days at a dose of 1 or 3 capsules, or tablets, corresponding to approximately 4.8 or 14.3 maximum human therapeutic doses (MHTDs), respectively. Comparisons included general toxicity, local tolerance, safety pharmacology, effects on immune system organs, reproductive toxicity, and basic TK parameters ( C max , T max , AUC 0-24 , MRT , and T 1/2 ). Results. No toxic effects, including local irritation and immunotoxicity, were observed for the test product. The safety pharmacology testing demonstrated the safety of repeated oral administration of the test product for the cardiovascular, excretory, respiratory systems and the liver. The test product did not affect the reproductive system of male and female rabbits. The No Observed Adverse Effect Level (NOAEL) was determined to be 14.3 MHTDs. According to the TK parameters evaluated after single and repeated oral administration, the test product and Nexium ® demonstrated comparable TK profiles. Conclusions. The study demonstrated a favourable safety profile for the test product. All the test product parameters studied were comparable with those of Nexium ® . Positive clinical experience with Nexium ® supports the data obtained for the new esomeprazole product. Thea safety of these medicinal products may be considered similar.

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 (Amax or Cmax), time to maximum activity or concentration (Tmax), area under the activity–time (or concentration–time) curve (AUC), and half-life period (T1/2), by means of the 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 (Amax or Cmax, AUC0-24, AUC0-∞, Tmax, T1/2) 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.

The high prevalence of fungal skin infections motivates expanding the range of sertaconazole products for external use.The aim of the study was a preclinical comparison of the safety, antifungal activity, and pharmacokinetics of Sertaverin® 2% medicated shampoo (VERTEX JSC, Russia) with those of Sertamicol® 2% solution for external use (Glenmark Pharmaceuticals Ltd, India) and Nizoral® 2% shampoo (Janssen Pharmaceuticals N.V., Belgium) approved in the Russian Federation.Materials and methods. In the toxicity study, the medicinal products were applied to the skin of male and female outbred rats at doses of 0.5 or 1.5 mL/animal for 28 days. The authors evaluated the pharmacokinetics of two sertaconazole formulations (shampoo and solution) following a single administration to adult male rats at the same dose. Nizoral® was not used in the pharmacokinetics study because it contains a different active substance, ketoconazole. The minimum inhibitory concentration (MIC) was determined using the serial microdilution method in a wide range of concentrations.Results. The medicinal products did not exhibit any significant toxic effects in laboratory animals after 28 days of repeated dermal application. Plasma sertaconazole concentrations were negligible. Sertaconazole was intensively distributed in the liver, which is a highly vascularised organ, and in the target organ (skin at the site of application). The relative bioavailability of sertaconazole from the shampoo relative to that from the solution for external use was approximately 30% in liver tissues and approximately 363% in skin tissues at the application site. Sertaverin® was comparable to sertaconazole in the active substance form in terms of inhibiting the growth of Malassezia furfur strains. The MICs calculated on the active substance basis were ≤16–64 μg/mL.Conclusions. With its synergistic dual mechanism of action, broad-spectrum antifungal activity, lipophilic properties, and low systemic absorption, Sertaverin® may provide a more effective and safe alternative to marketed medicinal products for scalp diseases.

Phenosanic acid prevents convulsions, reduces the frequency of epileptic seizures, and improves cognitive, intellectual and mnestic functions in patients with epilepsy. Therefore, phenosanic acid-based medicinal products are promising candidates for inclusion in combination antiepileptic therapy. In order to combine medicinal products rationally and ensure that the therapy is safe, it is useful to study the pharmacokinetic interaction of medicinal products planned for clinical co-administration.The aim of the study was to examine single-dose pharmacokinetic interactions between Dibufelon® 200 mg capsules (PIQ-PHARMA LLC, Russia) and two medicinal products planned for clinical co-application with it, namely, valproic acid and carbamazepine, in sexually mature dogs.Materials and methods: the study included medicinal products of phenosanic acid (Dibufelon® 200 mg capsules by PIQ-PHARMA LLC, Russia), valproic acid (300 mg prolonged-release film-coated tablets), and carbamazepine (200 mg tablets). The medicinal products were administered to beagle dogs (2 groups of 9 males each) as a single oral dose separately and in the following combinations: phenosanic acid with valproic acid and phenosanic acid with carbamazepine. Dose selection involved adjusting maximum human therapeutic doses using interspecies conversion factors. Phenosanic acid was administered at a dose of 24 mg/kg; valproic acid and carbamazepine were administered at a dose of 60 mg/kg. Blood sampling took place at baseline and in 0.5, 0.75, 1, 2, 4, 6, 8, 10, and 24 h after dosing. Plasma concentrations of active substances were determined by HPLC-UV. Pharmacokinetic interactions were evaluated by changes in the main pharmacokinetic parameters (Сmax, Тmax, AUC0-24, MRT, Т1/2).Results: the study demonstrated rapid gastrointestinal absorption and prolonged systemic circulation of phenosanic acid administered separately (Tmax 2–4 h, T1/2 13–28 h) and combined with valproic acid (Tmax 2 h, T1/2 22 h). When administered with carbamazepine, phenosanic acid was eliminated from the systemic blood flow faster (T1/2 7.4 h).Conclusions: co-administration of phenosanic acid and valproic acid medicinal products had no significant effect on their respective pharmacokinetics. Whereas, the combination of phenosanic acid and carbamazepine demonstrated a significant decrease in the Tmax values of phenosanic acid and the MRT values of carbamazepine. The pharmacokinetic changes suggestive of a possible interaction between phenosanic acid and carbamazepine need further clinical investigation.

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 μ M 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.

Инграмон представляет собой пептидный фрагмент (65 – 76) моноцитарного хемотаксического белка-1 (МСР-1) человека, обладающий противовоспалительной активностью. Фармакокинетические параметры инграмона после однократного внутримышечного введения кроликам в дозе 0,5 мг/кг составляют: Cmax = 840 ± 68 нг/мл, Tmax = 0,08 ± 0,00 ч, AUC0-8 = 981 ± 17 ч  нг/мл, AUC0-∞ = 1052 ± 16 ч  нг/мл, MRT = = 1,04 ± 0,04 ч, Т1/2 = 0,70 ± 0,02 ч.

The pharmacokinetic profiling of active compounds is necessary for drug development and application. Approaches to a pharmacokinetic study based on biological markers are alternatives to traditional approaches based on chromatographic methods. The aim of the study was to compare two analytical approaches to pharmacokinetics investigation for an example of sitagliptin in rabbits after one dose oral administration. The method for sitagliptin quantification in rabbit plasma samples based on a correlation between its concentration and dipeptidyl peptidase IV activity was proposed, validated, and applied. The high-performance liquid chromatography (HPLC)-ultraviolet (UV) method was also validated and applied for the same sample analysis. The plasma pharmacokinetics of sitagliptin after oral administration to the rabbits in one dose was characterized after two analytical assays. The close values of the main pharmacokinetic parameters were obtained after two approaches. The nontraditional approach based on correlation of special marker activity and active substance concentration appears to be more sensitive than HPLC-UV. Thus, the sitagliptin concentrations determined by biomarker assay were higher than the lower limit of quantification (LLOQ) for a longer period (more timepoints) than after the HPLC-UV assay. This feature may influence the values of some calculated concentration-dependent (area under the curve [AUC]0-t , etc.) and time-dependent parameters (mean residence time [MRT], T1/2 , etc.). The values of Tmax obtained by the two approaches were similar and adequate for oral drug administration that confirms the correctness of biomarker selection for pharmacokinetics assessment. The obtained results on the example of sitagliptin confirms that the biomarker approach is adequate and applicable for a pharmacokinetics study. Similar approaches may be effective for individual compounds and complex mixtures when it is difficult or impossible to analyze them traditionally by chromatographic methods.

Abstract. Medicinal products of natural origin are widely used by virtue of their pharmacological efficacy and relative safety. Chemical composition of such medicines is usually complex, they may be represented by heteropolymers or mixtures containing peptides, polysaccharides, and other compounds which are endogenous and/or rapidly metabolised in a living organism. Conventional, chromatography-based approaches to evaluation of such medicines are often not applicable. Pharmacokinetics of medicinal products of natural origin may be studied by methods based on assessment of biological action and pharmacodynamic properties of such medicines, which involves determination of biological marker (biomarker) levels. The aim of the study was to summarise the accumulated experimental data on the use of biomarkers in pharmacokinetics studies as illustrated by a few medicinal products of natural origin. Material and methods. The authors studied fucoidan from Fucus vesiculosus , as well as a complex of bioactive compounds and a glycopeptide—both isolated from gonads of green sea urchins ( Strongylocentrotus droebachiensis) . In vitro/ex vivo experiments were used to establish correlation between the concentrations of the test mixtures and the activity/concentration of potential biomarkers. Experiments showing the biomarker concentration in plasma or serum ( in vitro ) and whole blood ( ex vivo ) before and after spiking with the studied products were performed in order to assess specificity, calibration (linear) range of the biomarker response, and its native concentration. The analytical procedures were based on the chromogenic (optical) anti-factor Xa activity (AXA) assay, and determination of dipeptidyl peptidase 4 and lactate dehydrogenase activity by kinetic analysis with spectrophotometric detection of enzymatic reaction products. Results. The analysis of the results of studies of a number of natural products (fucoidan from Fucus vesiculosus ; a complex of bioactive compounds isolated from gonads of green sea urchins S. droebachiensis ; a glycopeptide isolated from internal organs of S. droebachiensis ) demonstrated the feasibility of using biomarkers in pharmacokinetics studies of such products. This approach allowed for accurate calculation of pharmacokinetic parameters. Conclusion. The discussed approach may be used for various biological models and is an effective means of studying compounds that are difficult or impossible to detect by conventional bioassays in pharmacokinetics and bioequivalence studies.