Dmitriy V. Gusev

Background. The development of innovative national antitumor drugs remains an ultimately important task. Among the new drugs, which selectively affect tumors, indolo [2,3-a] carbazole – N-glycosides LCS-1208 and LCS-1269, synthesized using an original technique at the Blokhin National Medical Research Center of Oncology of the Ministry of Health of the Russian Federation, are of essential interest. The paper presents possible options of instrumental analytical methods for identification of differences in the physical chemical features of structurally similar compounds LCS-1208 and LC S-1269. These methods may be used for establishing experimentally justified standards of quality for the standardization of the compounds as pharmaceutical substances. Aim. To study the physical chemical features and to develop methods for the qualitative and quantitative analysis of LCS-1208 and LCS-1269. Materials and methods. The study involved modern instrumental methods of analysis, such as polarimetry, ultraviolet spectroscopy, infrared spectroscopy, proton magnetic resonance spectroscopy spectroscopy, high performance liquid chromatography (HPLC), and HPLC with the tandem mass spectrometric detection (HPLC–MS / MS). Results. Significant differences in the physical chemical features of the original N-glycosides LCS-1208 and LCS-1269 were determined using modern instrumental analytical methods. The authors present the results verifying the structure of each of these compounds. The methods for qualitative and quantitative analysis of these compounds were developed in the study. The HPLC validation was performed for the LCS-1269 quantification method. The study showed the compliance of the obtained validation characteristics with the criteria approved by the Russian State Pharmacopoeia XV Ed and Pharmacopoeia of the Eurasian Economic Union. The authors determined the structure of impurities in LCS-1269 samples and proposed a method for their quantitative determination. Conclusion. Structures of indolo [2,3-a] carbazole derivatives LCS-1208 and LCS-1269 were confirmed and shown to be reliably distinguishable. Impurity structures from the final LHS-1269 synthesis step were identified. HPLC methods for quantifying LCS-1208 and LCS-1269 were developed.

Introduction. Infection of canids with canine parvovirus (CPV), especially in puppies, leads to serious damage to the gastrointestinal tract in a short period of time, up to a fatal outcome. In this regard, early and rapid diagnosis is crucial for initiating therapy and preventing the spread of infection. In this connection, immunochromatographic analysis (ICA) is the preferred method because it allows rapid detection of virus infection. In addition, this method is highly specific and does not require complex laboratory equipment, which greatly simplifies its use in comparison with other common methods for detecting canine parvovirus. The aim of this study is the development of an ICA test system for the detection of the VP2 protein of canine parvovirus. Materials and methods. Monoclonal antibodies to the recombinant VP2 protein of canine parvovirus were obtained using hybridomic technology. Colloidal gold is synthesized by the citrate method. Results. Monoclonal antibodies (MABs) against the VP2 protein of canine parvovirus were obtained and characterized, colloidal gold (AuNPs) was synthesized, the conditions for conjugation of MABs with AuNPs were selected, and the optimal ratios of the components of the test system were determined. The visual limit detection (vLOD) was 1.56 ng/mL. The developed ICA test system has shown high diagnostic efficiency in model experiments with a known concentration of VP2, as well as in sick animals with a confirmed diagnosis. Conclusion. An ICA test system has been developed to determine the VP2 protein of canine parvovirus. The resulting test system can be used as a fast and effective diagnostic tool for detecting CVP in the canine clinical specimens.

Background. Ferroptosis, a non-apoptotic, iron-dependent form of regulated cell death in which membrane phospholipid peroxidation products accumulate within the cell, was discovered relatively recently. Numerous scientific publications in recent years indicate that ferroptosis can lead to the tumor cells death that survive chemotherapy. Therefore, ferroptosis as an alternative pathway for cell death raises hope for new opportunities for the modern anticancer drug therapy development and improvement. 3-Hydroxyquinazoline derivatives as ferroptosis inducers are promising pharmacological substances with pronounced ferroptotic activity. Aim. To conduct a preliminary assessment of the new domestic derivative of 3-hydroxyquinazoline (OVF-009), antitumor activity, a potential ferroptosis inducer, in mouse tumor growth models administered via two routes of administration (intraperitoneal and oral). Materials and methods. Original substance OVF-009, excipients (Kolliphor ELP, ethanol 95 %, Kollidon 17 PF, sodium hydroxide, phosphate buffer, etc.); technological methods; immunocompetent mice, tumor models (Lewis lung epidermoid carcinoma, melanoma B-16, cervical cancer CC-5, breast adenocarcinoma Ca-755); biological methods described in the guidelines for drugs antitumor activity preclinical studies. The antitumor effect was assessed by tumor growth inhibition (TGI) of animals treated with OVF-009, compared with animals in the control group. Results. During the technological stage of the work, an OVF-009 oral dosage form based on corn oil was obtained. For intraperitoneal administration, a previously developed solution based on hydrogenated castor oil was used. The result of the experiments on mice tumor models showed the maximum antitumor effect on melanoma B-16 of 75 % TGI on the 8th day after the end of administration, with Lewis lung epidermoid carcinoma, a high but short-term effect of TGI = 84 % on the 1st day with intraperitoneal administration. With per os administration, the best results were obtained with Ca-755 and CC-5: with Ca-755, a significant antitumor effect was shown with TGI = 76–77 % (p 0.05) up to the 8th day after the end of administration; with CC-5, TGI = 73–68 % (p 0.05) was noted from the 8th to the 16th day of observation. Conclusion. The obtained results on the OVF-009 antitumor activity in mouse tumor models indicate the promise of this original substance continuing in-depth preclinical studies.

BACKGROUND: STING pathway activators with targeted delivery to tumor nodules are a promising option in cancer immunotherapy, especially as immunoconjugates. Existing compounds, such as MSA-2, are insufficiently effective as active immunoconjugate components, necessitating the development of new, more active compounds. AIM: The work aimed to examine the biological activity of the new compound SAD-2 and assess its potential as a new immunobiological drug for cancer therapy. METHODS: The work used contemporary methods of fine organic synthesis and analysis of the resulting compounds. MSA-2 was obtained from veratrole by multistage synthesis. SAD-2 was synthesized from MSA-2 by esterification with isopropyl alcohol in the presence of thionyl chloride. The antiproliferative activity of the compounds was assessed by the MTT method using colorectal cancer cell lines and human peripheral blood mononuclear cells. The induction of the interferon beta gene was assessed by real-time polymerase chain reaction using the human monocytic cell line THP-1. RESULTS: The new compound SAD-2 had 200–500 times higher antiproliferative activity according to IC50 than the existing compound MSA-2. Both MSA-2 and SAD-2 are active only in the presence of immune cells. SAD-2 showed 5–60 times higher relative induction of the IFNB1 gene than MSA-2, depending on the incubation time. CONCLUSION: SAD-2 is a promising new compound for developing immunoconjugates for targeted STING pathway activation in tumor nodules.

BACKGROUND: Monoclonal antibody (mAb)–drug immunoconjugates are a promising option in cancer immunotherapy. However, developing effective techniques of their synthesis remains relevant, especially when a small molecule has no mAb binding sites. AIM: The work aimed to synthesize immunoconjugates based on two modified rhodamine B molecules and four mAbs, and to examine their molecular and functional properties. METHODS: mAbs were conjugated to Rod-SMCC via reduced disulfide groups of immunoglobulin G (IgG). Rod-piperazine was added to oxidized IgG oligosaccharides. The synthesis was assessed using spectrophotometry. The activity of the resulting conjugates was assessed using enzyme-linked immunosorbent assay. The molecular heterogeneity of the resulting conjugates was assessed using high-performance chromatography. RESULTS: Rod-SMCC produced conjugates with all examined antibodies via thiol groups, with maximum saturation for each antibody. The maximum incorporation rate of approximately 10.96 ± 1.3 mol Rod per 1 mol IgG was observed for ICO 204, with approximately 5.19 ± 1.75 for trastuzumab. These Rod incorporation rates decreased after additional fractionation of the resulting immunoconjugates on a separating gel, amounting to 7.94 ± 1.8 for ICO 204 and 3.32 ± 1.2 for trastuzumab. The antigen-binding activity of ICO 204 immunoconjugates was comparable to that of native antibodies. The incorporation of Rod in immunoconjugates depended on the mAb fractionation technique. Acid elution during mAb fractionation on protein G decreased the incorporation of Rod in immunoconjugates. Rod-piperazine produced immunoconjugates with ICO 204 and trastuzumab via oxidized carbohydrate residues of antibodies. The maximum incorporation rate of rhodamine B in ICO 204 was 2.3 ± 0.3 mol per 1 mol IgG, with 0.2 ± 0.12 for trastuzumab. CONCLUSION: Chemical synthesis was used to produce two derivatives of Rod, which initially had no IgG binding sites. These compounds were used to synthesize model immunoconjugates with high Rod incorporation rates while maintaining antigen-binding activity. This work demonstrated that immunoconjugates can be produced using pharmaceutical substances with carboxyl groups that are unsuitable for such synthesis. Furthermore, it was shown that immunoconjugates can be synthesized through carboxyl group modification of pharmaceutical substances using piperazine and SMCC.

The Federal Law No. 323-FZ of November 21, 2011 establishes the requirements for the content and format of the “Informed Voluntary Consent” (IVC) form for medical interventions. This document serves as a legal guarantee of protecting the rights of both patients and healthcare professionals. The article discusses the procedure of signing the IVC and highlights its key sections that require special consideration. The most important are the sections “Indications” and “Contraindications for medical intervention.” Indications provide patients with information about the necessity of a particular procedure and the possible consequences of refusal, ensuring awareness and informed decision-making. The section on contraindications reflects potential health and life risks related to the patient’s condition or to the specifics of the intervention, including possible complications. The patient’s signature confirms awareness and consent, while also providing legal protection for the healthcare institution and staff in case of adverse outcomes or legal disputes. Special attention is paid to the section “Risks associated with medical care.” The list of potential adverse events (for example, during colonoscopy) can be extensive, which makes it impossible to include every detail. Therefore, the authors propose standardization and adaptation of such risks for inclusion in the IVC form. This approach allows achieving a balance between comprehensive patient information and practical usability of the document.

Palladium-catalyzed arylation of tris(3-aminopropyl)amine (TRPN) with a number of aryl halides, including 1- and 2-bromonaphthalenes, 1- and 2-chloroanthracenes, 9-bromoanthracene, 9-bromophenanthrene, and 1-bromopyrene, afforded the corresponding N,N′,N″-triaryl derivatives. Almost quantitative yields of the target products were obtained in the reactions with 1-chloro- and 9-bromoanthracenes. Study of the fluorescence spectra of the synthesized compounds in the presence of different metal salts revealed complete or very strong fluorescence quenching of naphthalen-1-yl, anthracen-2-yl, and phenanthren-9-yl derivatives upon addition of Cu(II), Al(III), and Cr(III) perchlorates. Complete fluorescence quenching of pyren-1-yl derivative was observed on in the presence of copper ions. Some other metal cations induced small changes in the emission intensity with a blue shift of the fluorescence maximum by about 10 nm. Characteristic changes in the electronic absorption spectra of napththalen-1-yl, anthracen-9-yl, and pyren-1-yl derivatives in the presence of Cu(II), Al(III), and Cr(III) ions make these compounds promising for fluorescent and spectrophotometric detection of Cu(II), Al(III), and Cr(III) ions.

Background. Ferroptosis is a type of non-apoptotic iron-dependent form of regulated cell death. Ferroportin is the only exporter of iron from cells, however, the involvement of ferroportin in cell ferroptosisis poorly understood. Activation or blocking of ferroptosis pathways may provide a therapeutic strategy for the treatment of a number of diseases. Control of ferroportin expression will make it possible to select a more effective tumor therapy. Obtaining monoclonal antibodies to human ferroportin will help to further study its role in ferroptosis of tumor cells.Aim. Obtaining and characterization of monoclonal antibodies to human ferroportin.Materials and methods. Mouse monoclonal antibodies against a synthetic peptide corresponding to the ferroportin protein region were obtained using hybridomic technology. The peptide was synthesized by the method of solid-phase automatic synthesis.Results. A 15-membered peptide with the sequence ELKQLNLHKDTEPKP (ELK) was selected and synthesized. Conjugates of the synthetic peptide with carrier proteins were obtained. 4 stable hybridomic clones were obtained, and samples of monoclonal antibodies were obtained. The samples were successfully tested using methods of flow cytometry, enzyme immunoassay, and polyacrylamide gel electrophoresis.Conclusion. Four antibodies against ferroportin binding the ELK peptide were obtained and characterized. The antibodies can be used in further studies of iron transport in tumor cells.

Background. The study of new representatives of indolo[2,3-a]carbazole N-glycosides with a biological activity broad spectrum remains a relevant direction in the highly effective antitumor drugs creation.Aim. To evaluate the efficacy of the lyophilized dosage form of a new derivative of indolocarbazole N-glycoside 6-picolinamido-12-(β-D-xylopyranosyl)indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7-dione (LCS-1269-lyo) on transplantable mice colon adenocarcinoma (CAC) and on SW620 human colon cancer xenografts.Materials and methods. In studies on female Balb / c mice with subcutaneously (s.c.) transplanted CAC, a single intravenous (i.v.) administration of LCS-1269-lyo was used at doses of 80, 90, 100, 120 mg / kg and five-time administrataion daily at doses of 80 and 90 mg / kg. The LCS-1269-lyo study on SW620 effectiveness was carried out at doses of 60 and 90 mg / kg with five-time daily intraperitoneal (i.p.) administration to male Balb / c nude mice with subcutaneously transplanted SW620 xenografts. The antitumor effect was assessed by tumor growth inhibition and an increase in the lifespan of animals receiving LCS-1269-lyo, compared to the controls.Results. A dose-dependent reliable antitumor effect of LCS-1269-lyo was observed on CAС after a single exposure at doses of 80, 90, 100 and 120 mg / kg compared to the control group of mice. LCS-1269-lyo, when administered intravenously five times at doses of 80 and 90 mg / kg (total doses of 400 and 450 mg / kg), significantly inhibited CAC growth by 89–88 and 86–84 % (p 0,05) over 30 days of observation and increased the life span of Balb / c mice by 36 and 54 %, respectively. The LCS-1269-lyo effectiveness in a total dose of 450 mg / kg on CAC has been confirmed by the activity of LCS-1269-lyo on SW620 xenografts in Balb / c nude mice with tumor growth inhibition 72,9–71,0 % (p 0,05) during 8 days of observation.Conclusion. The results of the antitumor activity evaluation of LCS-1269 in a lyophilized dosage form on the model of CAC mice transplantable colon adenocarcinoma and on human colon cancer SW620 subcutaneous xenografts allow to continue preclinical studies of the drug.

One of the promising approaches to eliminate the disadvantages of gemcitabine chemotherapy is its encapsulation in the liposomal delivery system. However, loading gemcitabine into liposomes is fraught with a number of difficulties, which requires rational formulation selection and optimization of the conditions of the production process. This study is devoted to studying the influence of technological factors of formulation on the effectiveness of passive loading of gemcitabine into liposomes. A thin-film method was used to obtain gemcitabine liposomes. The chloroform lipid solution was evaporated on a rotary evaporator to form a film, which was dried at low pressure and then hydrated with an aqueous gemcitabine solution. The resulting liposomal dispersion was extruded by passing through Nuclepore polycarbonate filters with a pore diameter of 0.4 and 0.2 microns. The concentration of the drug in the dispersion was determined by spectrophotometry using a standard sample at a wavelength of (268±2) nm. Gel filtration method using NAP-5 columns filled with sephadex G-25 was used to purify liposomal dispersion from non-activated gemcitabine. As a result, 20 model liposomal compositions were obtained, differing in qualitative and quantitative composition. By comparing the effectiveness of passive gemcitabine loading, the effect of the following potentially critical formulation parameters was studied: total lipid concentration, drug concentration, cholesterol and pegylated lipid content, phospholipid type (origin, degree of saturation, length of the hydrocarbon chain, charge). It has been shown that with an increase in lipid concentration, there is an increase in the efficiency of drug loading. At the same time, cholesterol and pegylated lipid, depending on the type of structurally forming phospholipid, can contribute to both an increase and decrease in the level of encapsulation. The properties of the structure-forming phospholipid have a significant effect on the efficiency of gemcitabine loading. The maximum switching efficiency (over 80%) and loading capacity (over 3%mas) is achieved when using compositions based on saturated phospholipids, characterized by a high phase transition temperature. Thus, the effect of liposomal composition on the effectiveness of gemcitabine encapsulation was studied. The data obtained can be used as a basis for the development of a liposomal dosage form of gemcitabine.