Innovative Biosystems and Bioengineering

Inhibition of Cytochrome P450 Activities by Propoxazepam: Safety Assessment in Context for Potential Drug Interactions

Vitalii Larionov — 2025-02-05

Background. Propoxazepam is a new anelgetic agent of the benzodiazepine group, chemically known as 7-bromo-5-(o-chlorophenyl)-3-propyloxy-1,2-dihydro-3H-1,4-benzodiazepine-2-one. Propoxazepam is considered a possible substrate of the CYP system, so its effect of on the CYP3A4 enzyme activity was investigated in vitro using human liver microsomes.

Objective. To evaluate the effects of propoxazepam on CYP3A4 activity in vitro using testosterone and midazolam as markers of metabolic activity for CYP3A4 in human liver microsomes.

Methods. Midazolam (1′-hydroxylation reaction) and testosterone (6β-hydroxylation reaction) were used as markers for CYP3A4-mediated activity. Ketoconazole (0.2 μM) was used as a positive control for reversible inhibition, and troleandomycin (50 μM) for metabolism-dependent inhibition. For reversible inhibition, propoxazepam was added together with the corresponding substrate and cofactor (NADPH), while for metabolism-dependent inhibition, it was incubated with microsomes and cofactor for 30 minutes prior to substrate addition.

Results. Propoxazepam at various concentrations (0 to 100 μM) consistently inhibited CYP3A4 activities for both substrates, showing a similar "concentration–activity inhibition" dependence, with IC₅₀ values of 52.3 ± 4.9 μM for midazolam and 46.1 ± 9.2 μM for testosterone. For metabolism-dependent inhibition, IC₅₀ values were 36.6 ± 8.6 μM for midazolam and 28.3 ± 7.4 μM for testosterone. Given that the binding of propoxazepam to microsomal protein under the experimental conditions, which reflected those in the IC₅₀ experiments, was low, no microsomal binding correction factor was applied to the reported IC₅₀ values.

Conclusions. The highest predicted unbound C_max plasma concentration of propoxazepam, above which interactions can occur, is between 0.462 and 0.524 μM, or 462 and 524 nM. This corresponds to concentrations of 188 to 214 ng/mL (based on the molecular weight of propoxazepam, 414.73 g/mol). According to pharmacokinetic data, these concentrations are not achievable after a single oral administration. Further studies are required for multiple-dose administration.

Optimization of Parameters of Saline Sodium Citrate Buffer for Stability of Colloidal Gold Nanoparticles Used in DNA Hybridization Biosensor

Maksym Soboleskyi — 2025-03-16

Background. The use of optical biosensors based on surface plasmon resonance (SPR) spectrometry have long been established as a viable alternative to the traditional molecular biological methods, such as immunostaining or ELISA. Its capacity to perform real-time quantitative measurements is complemented with the possibility for the enhancement of the sensor signal with the use of optically active colloidal nanoparticles. On the other hand, few such nanoparticle-containing DNA biosensors have been developed, owing to poor colloidal stability of nanoparticles in chemical conditions suitable for hybridization of nucleic acid sequences.

Objective. This study investigates the possibility of using gold nanoparticles (AuNPs) modified with thiolated oligonucleotides, 6-mercapto-1-hexanol, and lipoic acid as part of a hybridization system for biosensor detection of DNA sequences in order to improve its main analytical characteristics.

Methods. A study of the colloidal stability of nanoparticles in SSC (saline sodium citrate) media of different multiplicity before and after modification was carried out in order to select the best environment for the operation of the biosensor system. Aggregation of modified AuNPs was facilitated by their centrifugation, after which pelleted nanoparticles were resuspended and investigated by spectrophotometry. The conclusions regarding the colloidal stability of AuNPs were based on the drop in concentration of colloidal AuNPs after each centrifugation.

Results. The possibility of using the studied NPs in biosensor analysis was shown, and 0.1×SSC buffer solution was determined to be the optimal medium for their operational stability. Approbation of the nanoparticle-containing DNA hybridization biosensor based on SPR spectrometry was carried out in 2.0×SSC medium.

Conclusions. The prospective use of the studied NPs as components of DNA hybridization systems for the detection of genetic markers has been proven.

Anti-adhesion Properties of 4-(adamantyl-1)-1-(1-aminobutyl)benzole Against Staphylococcus aureus

Nataliia Humeniuk — 2025-04-21

Background. Staphylococcus aureus is classified as a bacterium with a high level of antibiotic resistance, one of the contributing factors being its ability to form biofilms. Targeting the early stages of biofilm formation is a promising strategy for antimicrobial therapy in patients with biofilm-associated infections.

Objective. To determine the ability of 4-(adamantyl-1)-1-(1-aminobutyl)benzole to influence the formation of S. aureus biofilms and the expression of biofilm formation genes.

Methods. The minimum inhibitory concentration (MIC) of 4-(adamantyl-1)-1-(1-aminobutyl)benzole (code AM-166) against the methicillin-resistant S. aureus strain 222 was determined using the serial microdilution method. The antibiofilm activity of AM-166 was studied using O'Toole's method, and the intensity of S. aureus biofilm formation was assessed according to Stepanovic. The effect of AM-166 on gene expression was investigated using real-time PCR.

Results. The MIC of AM-166 against S. aureus 222 was determined to be 5 μg/ml. In the presence of AM-166 at 0.25 and 0.5 MIC, S. aureus biofilm formation decreased from moderate (control) to weak, while at 5.0 MIC, the strain completely lost its ability to form biofilms. It was found that at a concentration of 0.5 MIC, AM-166 increased the expression of the icaR gene while decreasing the transcriptional activity of the icaA, clfB, fib, fnbB, ebpS, and eno genes, which are involved in biofilm formation and adhesion.

Conclusions. The adamantane derivative AM-166 disrupts MRSA biofilm formation, alters the transcriptional activity of ica-locus genes, and inhibits S. aureus attachment to biotic surfaces by affecting gene expression.

Biosafety Aspects of Hybridoma Technology: Nature of Risks and Approaches to Their Management

Kateryna Shevchuk — 2025-04-23

This study investigates the biosafety aspects of hybridoma technology, focusing on the identification and management of associated risks. Monoclonal antibodies, essential tools in immunology, biotechnology, and medicine, are primarily produced through hybridoma technology. This process involves fusing B lymphocytes from immunized animals with myeloma cells to create hybridomas, which are then cultured to produce specific antibodies. The research highlights significant contamination risks, particularly from rodent-borne viruses and other pathogens, during both in vivo and in vitro cultivation. It systematically analyzes existing strategies for identifying and mitigating these risks at various stages of monoclonal antibody production, including hybridoma identification, cell fusion, and antibody purification. The study underscores the importance of stringent biosafety protocols and optimized purification methodologies to ensure the production of high-quality, contaminant-free monoclonal antibodies. Additionally, it emphasizes the necessity of comprehensive risk assessments and the implementation of advanced contamination control systems in laboratories. The conclusions drawn from this study provide valuable insights into enhancing the safety and efficacy of monoclonal antibody production. By addressing these biosafety concerns, the research supports the widespread application of monoclonal antibodies in scientific and medical fields, ensuring their reliability and effectiveness in various diagnostic and therapeutic contexts.

In silico the Ames Mutagenicity Predictive Model of Environment

Sergey Kislyak — 2025-05-07

Background. The classical in vitro and in vivo methods developed and widely used in the past decades to assess the genetic effects of environmental factors are complex in view of their implementation, are expensive, long-lasting, have the problem of reproducibility of the results of experiment in different laboratories and may face ethical problems of using warm-blooded animals in experiments.

Objective. Development, optimisation and testing of effective in silico models for assessment of Ames mutagenicity of environmental factors.

Methods. The genetic assessment of the impact of environmental factors was carried out in accordance with a set of chemical compounds for which information on potential mutagenic activity was obtained experimentally, using the in vitro Ames Salmonella/microsome test. Four machine learning models were developed to solve the problem of binary classification to form two classes of xenobiotics (mutagen/non-mutagen). The total sample is represented by a set of 8,083 xenobiotics.

Results. We developed four machine learning models with 85% accuracy, matching the reproducibility of Ames test data across laboratories. In addition, we have proposed a binary classifier that subject to dimensionality reduction of the input data, taking into account the qualitative composition of molecular descriptors, allows us to improve the accuracy of in silico prediction of genotoxicity of chemicals.

Conclusions. The necessity of updating and expanding the list of effective and more productive methods and approaches for assessing the genotoxic effects of environmental factors is substantiated, which allows avoiding the use of warm-blooded animals in the experiment, saving time and reducing the number of false-negative and false-positive results. The possibility of increase the accuracy of predictive machine learning models for assessing the genotoxic potential of environmental factors in conditions of dimensionality reduction of the data set is presented.

Biosafety Management: Emphasis on Medicine, Pharmacy, and Biotechnology

Alexander Galkin — 2025-05-07