Problems of Biological Medical and Pharmaceutical Chemistry

Introduction. Multi-resistance of Klebsiella pneumoniae to known antimicrobial agents determines the need for urgent development of new drugs exhibiting an antibacterial effect. Compounds used in the treatment of non-infectious pathologies, affecting various protein targets and realizing the "antimicrobial potential", seem promising in the search for new derivatives. Of particular interest is the predicted high probability of inhibition of sterol-27-hydroxylase cytochrome P-450 (CYP27A1) by new derivatives of quinazoline-4(3H)-one and quinazoline-2,4(1H,3H)-dione with the possibility of preventing the processes of conversion of cholesterol, as the main component of macrophage lipid rafts on which pathogen adhesion occurs. Activation of phagocytosis, as well as disruption of the formation of the polysaccharide capsule of K. pneumoniae, is considered one of the probable mechanisms of the antibacterial effect of the studied substances.

The aim – determination of reactivity parameters and potential pharmacological activity of quinazolin-4(3H)-one and quinazolin-2,4(1H,3H)-dione derivatives; prediction of the mechanism of antimicrobial action of new quinazolinones against K. pneumoniae based on the formation of a complex with CYP27A1.

Material and methods. The probability of CYP27A1 inhibition by new quinazolinone derivatives was predicted using the PASS program. Quantum chemical parameters were calculated using the parameterized PM7 method in the MOPAC 2016 program. The structural characteristics of the studied substances were determined using ProTox 3.0. Some pharmacokinetic parameters were assessed using the admetSAR software tool. A three-dimensional model of the protein structure of CYP27A1 was generated on the AlphaFold platform; optimal conformation and further clustering of protein sequences using the MMseqs2 and Foldseek algorithms. The determination of the energy parameters of the intermolecular complexes “quinazolinone CYP27A1”, as well as the identification of the amino acid sites to which the quinzolinone derivative binds, was carried out using the Swiss Dock system.

Results. Quantum-chemical and structural parameters of quinazoline-4(3H)-one and quinazoline-2,4(1H,3H)-dione derivatives, as well as their intermolecular complexes with CYP27A1, were established, and some pharmacokinetic parameters of the studied substances were predicted.

Conclusions. Control of the interaction of the compound with the lipid raft of the macrophage and an increase in the degree of penetration of the pathogen into its cell can be considered as a probable mechanism of the antibacterial action of quinazoline-4(3H)-one and quinazoline-2,4(1H,3H)-dione derivatives against Klebsiella pneumoniae. An increase in the number of naphthyl radicals in their molecule leads to a decrease in reactivity. Naphthyl radicals do not act as a pharmacophore of antimicrobial action against K. pneumoniae.

Introduction. Tricyclic antidepressants (TCAs) are historically the first group of medications used to treat depression. They work by affecting the duration of monoamines presents in the presynaptic cleft in the brain and blocking certain receptors, such as m-cholinergic and alpha-adrenergic. TCAs can cause a toxicological effect and lead to fatal poisoning. This group of drugs is often used among drug addicts, as well as in the criminal environment.

The purpose of the study. Determination of an optimal and effective method for TCAs extraction from biological material using solid-phase (SPE) and liquid-liquid extraction (LLE) for further chemical-toxicological studies.

Materials and methods. The objects of the study were representatives of the TCAs group: imipramine, amitriptyline and clomipramine. Urine was used as a biological material. In the study of SPE method columns produced by Agilent Technologies company were used. These columns contained Evidex and ODS-C18 sorbents. Analutes isolation using LLE were realised according to the one of the most common methods. Analisys were conducted on gas-chromatography with a mass-selective detector (GC/MS).

Results. The method of extraction on a solid sorbent was developed using phosphate buffer with pH 4.8. After using the LLE method, samples with high contamination of co-extracted impurities of biological origin were observed. The error in the results of TCAs extraction using SPE was less than 10%, while the error for the LLE method was 25-35%. A comparison of two different extraction methods was studied using various TCAs concentrations. The advantages of using the ODS-C18 as a sorbent for SPE were determined. Calibration curves were constructed for samples using the SPE method. Limits of detection and quantification for all TCAs were determined.

Conclusions. The advantages of isolating TCAs using the SPE method over the LLE method were revealed.

Introduction. Glioblastoma is one of the most aggressive malignant brain tumors characterized by low sensitivity to drug therapy, frequent recurrence after surgical removal of the tumor, and an extremely unfavorable prognosis.

Aim. To analyze the overall survival of patients with primary glioblastoma of the brain based on the glial fibrillary acidic protein (GFAP) content of in blood serum.

Material and methods. 176 patients with primary malignancy grade IV glioblastoma confirmed by morphological and molecular genetic studies (99 men and 77 women) aged 18–82 years who underwent examination and treatment at the N.N. Blokhin National Medical Research Center of Oncology in the period from 2013 to 2024 were enclosed in the study. GFAP concentration in the pre-treatment blood serum was determined by enzyme immunoassay using Human GFAP ELISA kit (Biovendor, Czech Republic). The obtained data were processed using Statistica 10 (StatSoft) and SPSS (IBM) programs. Overall survival analysis was performed using the Kaplan–Meier method with the log-rank test for comparisons. Differences were considered statistically significant at p < 0.05.

Results. 72 of 176 patients were alive at the end of the study, the median follow-up period being 7.8 months, while 104 patients with died within a period of 1 to 103 months the median survival being 9.1 months. The median lifespan of all patients with glioblastoma was 14.8 months. 1-year overall survival rate comprised 56.7±4.2%, 2-year – 39.2±4.3%, 3-year – 30.0±4.1%, 4and 5-year – 26.8±4.0%, 10-year – 15.0±4.4%. The risk of death in patients with primary glioblastoma of the brain was the greatest in the first two years of follow-up from the time of diagnosis. No associations were found with patients’ age and gender, or the side of the tumor lesion. To study the relationship between long-term treatment results and serum GFAP concentrations patients were divided into two groups: 1) patients in whom GFAP was not detected (GFAP-negative) and 2) those with detectable GFAP concentrations (GFAP-positive). Median survival time of 46 GFAP-negative glioblastoma patients was 24.8 months, while in 130 GFAP-positive patients it comprised only 13.7 months (p = 0.047). No differences in the overall survival in GFAP-positive glioblastoma group depending on the serum GFAP concentration were found. The differences in the overall survival depending on the presence or absence of GFAP in blood serum were mostly pronounced in the female group and in patients below 50 years of age. Thus, median survival of GFAP-negative glioblastoma patients below 50 years was several fold higher than in GFAP-positive patients of this age group (70.8 and 12.4 months respectively).

Conclusion. The overall survival of patients with grade IV primary glioblastoma is significantly higher in GFAP-negative than in GFAP-positive group. Hence, the presence of GFAP in blood serum can be regarded as an unfavorable prognostic factor for this disease.

Introduction. The study of soluble forms of the receptor and immune checkpoint ligand (ICP) PD-1/PD-L1 is considered one of the promising areas in renal cell carcinoma (RCC). This system is actively involved in the regulation of antitumor immunity and includes the programmed cell death protein PD-1 (programmed cell death protein 1) and two ligands PD-L1, PD-L2. It is known that activation of the PD-1/PD-L1 system simultaneously stimulates apoptosis of antigen-specific T cells and suppresses apoptosis of regulatory suppressor T cells, which allows tumors to "escape" the host immune system.

The aim of the work is to analyze serum levels of sPD-1 and sPD-L1 in patients with renal cell carcinoma, their relationship with clinical and morphological characteristics of the disease.

Material and methods. We studied the levels of sPD-1 and sPD-L1 in the blood serum before treatment in 117 patients with kidney tumors, including 105 patients with renal cell carcinoma (RCC) (63 men and 42 women) aged 33 to 81 years (median 60 years) at various stages of the disease. Benign renal tumor (DOP) was detected in 12 patients (3 men, 9 women) aged 29 to 84 years (median 48.5 years). The control group consisted of 67 healthy individuals (27 men, 40 women) aged 22 to 82 years (median 53 years). The concentration of sPD-L1 and sPD-1 was determined in the blood serum before treatment using the Human PD-L1 Platinum ELISA and Human PD-1 ELISA kits (Affimetrix, eBioscience, USA). Statistical analysis of the distributions of the studied features was performed using the Kolmogorov-Smirnov goodness-of-fit test. To identify differences between values, single-factor and multifactorial variance analysis were used. All calculations were performed on a personal computer using the statistical software package "Statistica for Windows" and SPSS.

Results. A significantly higher median sPD-1 concentration was found in patients with RCC compared to the control; in patients with DOP, the median marker did not differ from the control. The median serum sPD-L1 content in RCC was significantly higher than in the control and did not differ from the group of patients with DOP. Analysis of variance of sPD-1, sPD-L1, sPD-1/sPD-L1 in the blood serum in the control and in patients with RCC did not show a relationship with age. The sPD-L1 index in RCC patients was significantly higher in men than in women. The T index reflected the sPD-L1 index and its concentrations were significantly higher in RCC patients with metastases in regional lymph nodes. The M index in RCC patients reflected serum sPD-L1 levels to the greatest extent. The lowest receptor median was found in clear cell RCC. ROC analysis indicates the prospects for further studies of sPD-L1 as a biomarker in the diagnosis of RCC.

Conclusions. The medians of sPD-1 and sPD-L1 are significantly higher in patients with RCC than in the control. Analysis of variance of the values of the studied parameters in the control and in patients with RCC did not show a relationship with age. The T and M index are closely related to sPD-L levels in RCC. In patients with RCC, significant differences in PD-1 receptor levels were found between different morphological variants of the tumor. Analysis of the diagnostic significance of sPD-L1 in patients with RCC indicates the prospect of further research of the biomarker in the diagnosis of RCC.

Introduction. Non-alcoholic fatty liver disease (NAFLD) is a major public health issue characterized by a rapidly increasing prevalence worldwide. NAFLD is associated with excessive lipid accumulation and the development of inflammation in the liver. To study the pathogenic mechanisms of the disease, a steatosis model using immortalized cell lines is widely employed.

The aim of this study was to improve the in vitro steatosis model using HepG2 cells by conducting a comparative analysis of two methods of steatosis induction, utilizing a fatty acid (FA) complex with bovine serum albumin (BSA) prepared by simple dissolution or conjugation.

Material and methods. Cell viability was assessed using the XTT assay. Lipid accumulation in HepG2 cells was measured by the GPO-PAP method, with triglyceride (TG) levels normalized to cellular protein content. Production of the pro-inflammatory cytokine IL-8, a marker of inflammation, was quantitatively determined using an enzyme-linked immunosorbent assay (ELISA). Statistical significance was evaluated using Student's t-test, with p-values < 0.05 considered statistically significant.

Results. The optimal FA concentration that promoted lipid accumulation and inflammation without a marked cytotoxic effect in HepG2 cells was 0.75 mM. The conjugated FA-BSA complex led to a higher TG accumulation compared to the FA-BSA complex prepared by dissolution. However, IL-8 levels were significantly lower in the culture medium of HepG2 cells treated with the conjugated complex compared to those treated with the FA-BSA complex prepared by dissolution.

Conclusions. The use of the conjugated FA-BSA complex allowed for the development of an improved in vitro steatosis model that more closely resembles the physiological mechanisms of NAFLD progression.