Abstract: Green analytical techniques aim to replace or minimize the use of hazardous organic modifiers with environmentally friendly solvents without compromising chromatographic performance. This shift enables industries and research laboratories to adopt sustainable analytical methods. Metformin HCl (MF) and Sitagliptin (SITA) are widely used in managing type 2 diabetes mellitus to maintain blood sugar levels in adults when combined with a proper diet and regular exercise. The present study focuses on the Analytical Quality by Design (AQbD)-established development of an easy, quick, precise, accurate, cost-effective, and eco-friendly reversed-phase high-performance liquid chromatography (RP-HPLC) method for concurrent estimation of MF and SITA. A Design of Experiments (DoE) strategy was employed for multivariate optimization of chromatographic conditions, while risk assessment using an Ishikawa diagram helped identify the critical method parameters (CMPs). Factor screening was initially executed by the one-factor-at-a-time (OFAT) approach through trial runs under varying chromatographic conditions. Subsequently, a Box-Behnken Design (BBD) was used to study response surface methodology, enabling detailed evaluation of critical analytical attributes (CAAs)- retention time, resolution, symmetry factor, and peak area as indicators of robustness. Optimization was achieved using a desirability function, yielding mobile phase composition with 25 mM CH₃COONH₄ buffer (pH adjusted to 4.5 using 1 M OPA solution) and ethanol (45:55, v/v) at a flow rate of 1.157 mL/min. Separation was carried out on a HiQ sil C18HS column (4.6 mm I.D. x 250 mm length, 5 µm, 100Å), with the column oven temperature maintained at 25 °C and the detection wavelength at 256 nm. The optimized method was validated according to ICH (Q2R2) guidelines. Overall, the outcomes demonstrate that the AQbD approach effectively facilitates the development and optimization of an eco-friendly RP-HPLC method for the concurrent estimation of MF and SITA in prolonged-release tablets.

Introduction: Toxoplasmosis remains globally relevant because of its consequences on pregnancy outcomes, mental disorders, and opportunistic tendencies in immunocompromised individuals. Current medications have severe side effects on the host, low efficacy against the parasites, and potential development of resistance. This emphasizes the need to discover better and safer drugs to treat toxoplasmosis. This study evaluated the in vitro effects of ethanolic leaf extracts of Andrographis paniculata (EEAP) on protein kinase G (PKG) involved in cell invasion by T. gondii. Methods: EEAP was obtained through the maceration of dried leaf powder and then subjected to qualitative and quantitative screening of phytochemicals. Vero cells infected with the RH strain of T. gondii were used to evaluate the cytotoxicity and antiparasitic potential of EEAP, andrographolide, and clindamycin using the MTT assay. Microscopy was used to determine the cell invasion and intracellular replication of tachyzoites in the treated infected Vero cells at 24 h using 4 h post-infection treatment models. Gene expression profiling of TgPKG was performed using RT-qPCR. The expression of microneme proteins was determined using immunoblotting. Results: EEAP was found to contain more terpenoids. EEAP, andrographolide, and clindamycin were safe for host cells. EEAP and andrographolide exhibited good anti-parasitic activities against T. gondii. Microscopic assessment revealed a low infection index and intracellular replication by EEAP and andrographolide. RT-qPCR revealed a significant (P=0.002) downregulation of the TgPKG gene by 0.114-fold (88.6% decrease) and 0.068-fold (93.2% decrease), respectively, compared with the control. The expression of TgMIC1(0.442-fold, 55.8% decrease) and TgMIC2 (0.385-fold, 61.5% decrease) proteins significantly (P= 0.0001) decreased compared to the control. Conclusion: This study showed that EEAP and andrographolide are promising drug candidates effective against T. gondii, safe for the host cells, and can be used for the development of a potent anti-Toxoplasma drug to target the TgPKG gene involved in cell invasion to prevent disease progression.

Dry Eye Disease (DED) is a multifactorial condition characterized by tear film instability, leading to discomfort, visual disturbances, and potential ocular damage. Conventional treatments, such as topical eye drops, suffer from poor bioavailability due to rapid clearance, low drug penetration, and frequent dosing requirements, which negatively impact patient compliance. This study aimed to develop and evaluate a novel ethosome-based formulation for the trans-eyelid delivery of rebamipide, a mucoprotective and anti-inflammatory drug, to enhance its solubility, stability, therapeutic efficacy, and patient adherence.

The optimized ethosomal formulation (E6) exhibited a particle size of 242 ±11.8 nm polydispersity index (PDI) of 0.221 ± 0.003, and an entrapment efficiency (EE%) of 94.5 ± 0.6%, confirming its suitability for drug delivery. Fourier Transform Infrared Spectroscopy (FTIR) and Differential Scanning Calorimetry (DSC) confirmed the stability of rebamipide within the ethosomal system, while X-ray Diffraction (XRD) revealed a transition to an amorphous state, enhancing drug solubility. Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) demonstrated a smooth, spherical morphology with uniform particle distribution. Stability studies showed that E6 maintained its physicochemical properties over 180 days at both 4°C and ambient temperature.

In vitro drug release studies indicated a sustained release profile over 12 hours, with a controlled release pattern fitting the zero-order kinetic model. Ex vivo permeation studies revealed that E6 significantly enhanced drug penetration through the eyelid skin, achieving a flux of 0.4285 mg·cm⁻¹·h⁻¹ and a permeability coefficient of 0.1071 cm·h⁻¹, approximately three times higher than the control formulation. These findings highlight the potential of ethosome-based trans-eyelid delivery as an effective alternative to conventional ocular drug delivery, providing prolonged drug retention, reduced dosing frequency, and improved patient compliance.

Overall, this study supports the development of rebamipide-loaded ethosomes as a promising strategy for DED management. Further in vivo evaluations and clinical trials are recommended to establish their therapeutic efficacy, safety, and long-term adherence benefits.

SARS-CoV-2 is an emerging pathogen that caused the largest pandemic in modern history in 2020-2023. The surface S-glycoprotein, namely its RBD, significantly determines the pathogenesis of this virus: it allows SARS-CoV-2 to target human cells and ensures its internalization. Thus, the search for RBD inhibitors is currently considered a promising strategy for the drug development against COVID-19. In this work, we identified a new pocket on the RBD surface that could potentially bind small drug-like ligands. The FDA-approved drug compound library obtained from e-Drug3D was used as a source of drug-like chemical structures. The compounds were initially filtered by molecular weight (100-500 Da) using the Open Babel GUI. This allowed to reduce the size of the library from 2,118 to 1,749 compounds. Next, all filtered structures were docked to the RBD (PDB ID: 7T9L) using AutoDock Vina 1.1.2. The docking area was the entire RBD. Results and conclusions: We obtained a map of the uneven placement of ligands with different affinities to the target. The pocket formed by amino acids R454, R457, K458, S459, D467, S469, E471, I472, Y473, Q474, P479, N481, G482 and P491 was identified close to the functionally important ACE2-binding site of the RBD. It was able to interact with 128 ligands with the best estimated affinity of -7.8 kcal/mol for Oxymetholone. The latter interacted with the pocket through its hydroxyl and keto groups.

The study was funded under grant № 0125U002921 (contract 37/10-2025).

Combination therapy for metabolic diseases has traditionally been associated with an increased risk of chronic kidney disease (CKD). However, the co-administration of sodium-glucose cotransporter 2 (SGLT2) inhibitors and angiotensin II type I receptor antagonist (AT1) have demonstrated a protective effect against CKD progression. To build upon this therapeutic potential, the present study introduces an in silico design of a hybrid molecule as an innovative pharmacological strategy.

This approach integrates the key pharmacophores of empagliflozin (SGLT2 inhibitor) and telmisartan (ARB) into a single molecular entity, aiming to enhance nephroprotective synergy while streamlining the treatment regimen. The computational workflow began with the generation of a bioisostere library comprising 2017 compounds sourced from the Zinc20 database. Molecular docking simulations were conducted using the VINA 1.1.2 algorithm to assess binding affinity and stability toward both target receptors: SGLT2 (PDB: 7VSI) and AT1 (PDB: 4ZUD).

The lead candidate was identified based on its optimal Gibbs free energy values for both receptors and its favorable pharmacokinetic and safety profile, as predicted by ADMETlab 3.0. Visualization and analysis of docking interactions were performed using PyMOL and Discovery Studio.

This study not only confirms the feasibility of combining two complementary mechanisms of action within a single compound but also proposes a promising candidate for subsequent development stages, including chemical synthesis and preclinical evaluation (in vitro and in vivo). These findings lay the groundwork for a new generation of targeted therapies that are safer and more effective in protecting kidney and cardiovascular health.

Introduction: Senile plaques (SP), composed mainly of β-amyloid (βA) peptide fibers, are a distinctive feature of Alzheimer's disease (AD). Visualizing SP using magnetic resonance imaging (MRI) poses a challenge. MRI requires specific contrast agents (CAs) to recognize SP, thereby increasing detection sensitivity. This paper describes the synthesis of two types of CAs: positive or T1 (gadolinium oxide nanoparticles (Gd₂O₃NPs) or metal complexes supported on silica nanoparticles (SiO₂NPs)) and negative or T2 (iron oxide nanoparticles (IONPs). These CAs are functionalized with Amilovys compounds as target ligands (TLs) related to βA in order to design smart probes for the specific diagnosis of AD using MRI. Method: Gd₂O₃NPs, SiO₂NPs and IONPs were obtained by polyol solvothermal, sol-gel and thermal decomposition methods, respectively. For Gd₂O₃NPs some experimental conditions were optimized (molar ratio of precursors, solvent, and reaction time). After the functionalization with carboxyl or amino terminal groups, Amylovis compound was grafted to the SiO₂NPs and IONPs using carbodiimidation reaction. The grafting percentage was indirectly determined by means of the quantification of free Amylovis (HPLC). Results: Optimal conditions for the synthesis of Gd₂O₃NPs were 1:3 as precursor molar ratio, ethylenglicol as solvent and 4 h at 180 ˚C. All NPs were characterized using FT-IR (evidenced the metal-oxygen bonds and ligands at the surface of NPs), microscopic (TEM sizes of Gd₂O₃: 28 nm; SiO₂: 128 nm; IONPs: 12 nm), and granulometric techniques. For IONPs, the grafting efficiency (30-100 %) do not impact on the colloidal stability and relaxivity properties of the so-functionalized NPs. Conclusion: Three dispersions of Amylovis-functionalized metal nanoparticles (Gd₂O₃, metal-complexed SiO₂, and IONPs) were successfully synthesized. Their surface functionalization and nanometric sizes (all under 200 nm) were confirmed. This work successfully lays the groundwork for developing targeted MRI contrast agents to improve the detection of senile plaques in Alzheimer's disease.

Background:
Preliminary phytochemical analysis of the hydroalcoholic extract of Tecoma undulata (Sm.) Seem. revealed a high flavonoid content (88.66 ± 0.57 QE/g), suggesting wound-healing potential. To further elucidate its therapeutic efficacy, ointment formulations containing 5% and 10% of the extract were developed using Jathyadi Thailam as a herbal base (oil) and evaluated for wound-healing activity in Swiss albino mice.

Methods:
The wound-healing potential of the formulations was assessed using burn and excision wound models. The ointments were evaluated for their physicochemical properties, including pH, spreadability, viscosity, and stability. Parameters, including wound contraction rate, epithelialization time, collagen deposition, and tensile strength, were measured. Comparative assessments were conducted between topical and oral administration, with standard povidone-iodine ointment serving as the standard reference.

Results:
Both 5% and 10% formulations complied with standard physicochemical criteria and demonstrated significant wound-healing activity (p < 0.001) in both models compared to the control. Enhanced wound contraction, shortened epithelialization period, and increased tensile strength were observed, particularly in the burn wound model. Topical application produced superior results relative to oral administration.

Conclusion:
The hydroalcoholic extract of T. undulata, when formulated with Jathyadi Thailam, exhibits pronounced wound-healing efficacy, especially in burn wound management. These findings support its potential as a safe, natural, and effective topical therapeutic agent for treating burn injuries.

The incessant global burden of cancer, particularly breast cancer, drives the urgent need for innovative therapeutic strategies that improve efficacy while reducing the enfeebling side effects of conventional chemotherapy. Drug repurposing, combined with advanced pharmaceutical engineering, presents a promising avenue for rapid clinical translation. This study investigates the anti-cancer potential of a novel pharmaceutical drug-drug co-crystal (DDC) synthesised from Aspirin, a known chemo preventive agent, and Simvastatin, a statin with emerging anti-tumour properties

The co-crystal was successfully synthesised via mechanochemical grinding and thoroughly characterised. Its enhanced aqueous solubility and unique thermal profile confirmed the formation of a new solid-state phase. In vitro biological evaluation (with assays like DPPH, H₂O₂ scavenging, NO scavenging, Protein denaturation and HRBC) demonstrated superior free radical scavenging and anti-inflammatory activities compared to the parent drugs alone. Crucially, the co-crystal exhibited potent anti-proliferative effects against MCF-7 breast cancer cells, significantly reducing cell viability and migration and inducing apoptosis. The IC₅₀ values of DDC, Aspirin and Simvastatin alone were scored to be 49.3987, 68.86, 19.49 µg/ml respectively. Mechanistic insights from RT-PCR analysis revealed profound downregulation of key oncogenic markers (BCL-2, COX-2) and upregulation of the pro-apoptotic BAX gene. Furthermore, the co-crystal remained stable and bioavailable under simulated gastrointestinal conditions, supporting its potential for oral administration. Complementary molecular docking studies confirmed strong binding affinities of the parent molecules to the active sites of the target proteins, rationalising the observed biological activity.

These compelling results position the Aspirin-Simvastatin co-crystal as a highly promising candidate for breast cancer therapy. It exemplifies a rational approach to drug development by enhancing the therapeutic profile of existing drugs through crystal engineering, offering a faster and potentially safer route to novel oncology treatments.

Keywords: Drug-Drug Co-crystal, Drug Repurposing, Breast Cancer (MCF-7), Apoptosis, Molecular Docking, Bioavailability, Solid-State Chemistry.

Bacillus cereus is a widespread Gram-positive bacterium that can cause severe foodborne illnesses, opportunistic infections, and even life-threatening conditions such as endophthalmitis and bacteremia. Its pathogenic potential, combined with the ability to produce toxins and form resistant spores, makes it a serious public health concern. In recent years, the problem has been exacerbated by the increasing occurrence of antibiotic resistance in B. cereus, which reduces the effectiveness of conventional treatments. Therefore, the discovery and development of new drug candidates with strong antibacterial activity against B. cereus is of great importance. Since thiosemicarbazones and their metal coordination complexes, especially copper(II) complexes are frequently associated with pronounced antibacterial properties, the objective of the present study was the synthesis of novel thiosemicarbazone-based complexes and the evaluation of their antibacterial efficacy against Bacillus cereus.

In the present study, we report the synthesis of 2-benzoylpyridine N-(bicyclo[2.2.1]hept-2-yl)thiosemicarbazone (HL) and three corresponding copper(II) complexes: [Cu(L)NO₃], [Cu(L)Cl], and [Cu(L)CHCl₂COO]. The design of the ligand was based on a camphor-derived structural fragment, chosen due to the ability of such derivatives to display enhanced biological activity compared to their parent natural products. The thiosemicarbazone was prepared through a two-step synthetic procedure, followed by complexation reactions with appropriate copper(II) salts in ethanol in 1:1 molar ratio.

To evaluate the antibacterial activity of the obtained compounds against Bacillus cereus, the broth microdilution method was used. The HL did not exhibit activity, however, its copper(II) complexes demonstrated strong results, surpassing the activity of Furacillinum, an antimicrobial drug used in medicine. The most active compound is complex [Cu(L)NO₃], with MIC and MBC values of 0.24 µg/mL, which is 20 times higher than the activity of Furacillinum.

This work was fulfilled with the financial support of the ANCD project 24.80012.5007.14TC.

According to recent data from the WHO and the International Agency for Research on Cancer, cancer remains one of the leading public health challenges worldwide. Projections suggest that, due to demographic factors such as population growth and aging, the annual number of new cases could reach around 35 million by 2050. The WHO report also highlights that while progress has been made in prevention and treatment, access to effective diagnostic and therapeutic options remains uneven across different regions, particularly in low- and middle-income countries. Leukemia represents a significant cause of cancer-related morbidity and mortality, and the THP-1 cell line, derived from human acute monocytic leukemia, is frequently used as an established in vitro model to evaluate the anticancer potential of new compounds. Thiosemicarbazones and their metal complexes are compounds that have long been recognized in the literature as biological agents, including those with anticancer properties.

Based on the above, for our study we synthesized a series of 4-allylthiosemicarbazones of pyruvamides: 1-(piperidin-1-yl)propane-1,2-dione 4-allylthiosemicarbazone (HL¹), 3-(morpholin-4-yl)propane-2,3-dione 4-allylthiosemicarbazone (HL²), and N-(4-methoxyphenyl)-2-oxopropanamide 4-allylthiosemicarbazone (HL³), along with their copper(II) chloride complexes [Cu(L^1-3)Cl]. The physicochemical properties of all synthesized compounds were investigated to confirm their structure and composition.

The anticancer activity of the synthesized compounds was evaluated against the THP-1 cell line. All copper(II) complexes exhibited higher activity than the corresponding thiosemicarbazones from which they were obtained. HL¹ and HL³ showed no activity, while only 3-(morpholin-4-yl)propane-2,3-dione 4-allylthiosemicarbazone demonstrated activity with an IC₅₀ value of 2.8 µM. The most active compound was [Cu(L²)Cl], with an activity value of 0.14 µM, which is 2.2 times higher than that of Doxorubicin, used as a reference standard in this study.

The work was performed with financial support from subprogram 010602 of the institutional project.