Keyhole limpet hemocyanin is a large metalloprotein present in hemolymph of the mollusk Megathura crenulata. This protein is highly immunogenic, making it very attractive to the biopharmaceutical industry, where it is used as a carrier protein for haptens, an adjuvant, or as a stand-alone biologic for the treatment of various cancer diseases. The structural complexity of such immunogenic protein makes its characterization and quality control very challenging. Here, we developed, optimized and validated a size exclusion chromatography approach for quality control of keyhole limpet hemocyanin products. The separation procedure was performed on an Acquity UPLC BEH450 SEC column with pore size of 450 Å in a mobile phase composed of 25 mmol L- 1 phosphate buffer (pH 6.8) with 250 mmol L- 1 NaCl. These conditions led to appropriate elution of the analysed protein. The developed method showed very promising values of validation parameters, such as coefficient of de termination (R 2 = 0.9997), sensitivity (limit of detection 10.21 μg mL-1, limit of quantitation 31.25 μg mL- 1), precision (repeatability 0.3-2.5 %, intermediate precision 1.1-4.2 %), and accuracy (100.4-102.8 %). The developed method was used to determine the content of keyhole limpet hemocyanin in three commercially available samples obtained from various suppliers. The determined content ranged from 20.08 to 22.91 mg per vial.

The application of nanotechnology in drug delivery systems has attracted considerable scientific interest, particularly with respect to improving the therapeutic efficacy and safety profiles of pharmacological agents. This review critically examines nanotechnology-based drug delivery systems for the management of rheumatoid arthritis (RA), a chronic systemic autoimmune disease characterized by persistent synovial inflammation, progressive joint destruction, and significant extraarticular sequelae. Conventional pharmacological strategies such as non-steroidal anti-inflammatory drugs (NSAIDs), corticosteroids, conventional synthetic disease-modifying antirheumatic drugs (csDMARDs), and biological disease-modifying anti-rheumatic drugs (bDMARDs) provide disease control but are associated with significant adverse effects upon prolonged use, inadequate joint specific drug concentrations, and limited responsiveness in a subset of patients. Nanotechnology offers a compelling therapeutic solution by enabling targeted drug delivery to inflamed synovial tissue, thereby enhancing bioavailability, reducing systemic toxicity, and extending drug half-life. This review discusses diverse nanotechnology platforms, such as liposomes, dendrimers, polymeric nanoparticles, hydrogels, metal, inorganic nanoparticles, nucleic acid-based carriers, and stimuli-responsive nanocarriers evaluated for RA treatment. The evidence underscores the potential of these systems to overcome the limitations of traditional therapies and to facilitate precision therapeutics in RA. The review also addresses existing challenges in nanomedicine translation, such as inter-patient variability, protein corona formation, endosomes (as a barrier), biocompatibility, and the need for rigorous clinical validation, while outlining future directions including exosome mediated delivery, biomimetic nano-carriers, and stimuli-responsive platforms.

This study aims to investigate the effects of syringin on insulin resistance (IR) and its mechanisms using an IR model in HepG2 cells. IR was induced by 10[4] mol L[-1] insulin for 24 h, followed by syringin treatment at various concentrations for 48 h. Levels of glucose, triglycerides, TNF-α, IL-6, and GRP78 were measured, and protein expression of IRS-1, p-IRS-1, PI3K, p-PI3K, AKT, p-AKT, FoxO1, and p-FoxO1 was analysed by Western blot. Compared with controls, the IR model showed significant increases in glucose, triglycerides, TNF-α, IL-6, GRP78, and decreases in p-IRS1/IRS1, p-PI3K/PI3K, and p-AKT/AKT, with increased p-FoxO1/FoxO1 (all p < 0.01). Syringin (50-200 μg mL[-1]) significantly lowered glucose, triglycerides, TNF-α, IL-6, and GRP78, and reversed these signalling changes by upregulating p-IRS1/IRS1, p-PI3K/PI3K, p-AKT/AKT and downregulating p-FoxO1/FoxO1 (p < 0.05). Syringin effectively ameliorates insulin resistance in HepG2 cells, lowers glucose and triglycerides, and improves glucose-lipid metabolism, likely via the PI3K/AKT pathway.

This study conducted a retrospective disproportionality analysis of 50,031 adverse drug event (ADE) reports from the FDA Adverse Event Reporting System (FAERS) database spanning Q2 2009 to Q3 2025, in which golimumab was identified as the primary suspect drug. Four disproportionality algorithms were simultaneously applied: the reporting odds ratio (ROR), proportional reporting ratio (PRR), Bayesian confidence propagation neural network (BCPNN), and multi-item gamma Poisson shrinker (MGPS). A total of 397 positive signals were identified across 25 system organ classes (SOCs). The SOCs with concordant positive signals across all four algorithms were identified for infections and infestations and surgical and medical procedures. High-frequency signals included pneumonia, lower respiratory tract infection, influenza, nasopharyngitis, sinusitis and cellulitis. Comparison with the current prescribing information revealed several previously undocumented ADEs, including urinary tract infection, ear infection, flavivirus infection, Zika virus infection and Dengue fever. Twenty-nine positive malignancy signals absent from the prescribing information were identified, including basal cell carcinoma, colorectal cancer, cervical cancer and breast cancer. Time-to-onset analysis revealed that the risk of ADEs was the highest within the first few months of treatment initiation. Clinicians should rigorously monitor for infectious complications and malignancies when prescribing golimumab to ensure patient safety.

This research introduces a first-in-class high-performance liquid chromatography with fluorescence detection (HPLC-FD) method for the reliable quantification of ROS in plasma. The method relies on the native fluorescence of ROS, with excitation and emission set at 280 nm and 340 nm, resp. Sample preparation involved a simple one-step protein precipitation with methanol. Chromatographic resolution of ROS and pemigatinib (PEM), which served as the internal standard (IS), was achieved using Zorbax Eclipse Plus C18 HPLC column. The mobile phase consisted of methanol and formate buffer solution. The method was validated in compliance with ICH M10 guidelines. It showed linearity across concentration range of 50-1,500 ng mL[-1] (R[2] = 0.9991), with a lower limit of quantitation of 50 ng mL[-1]. Accuracy ranged from 96.8 to 103.4 %, and intra- and inter-day precision (RSD) were between 3.2 and 6.1 %. The method was successfully applied to quantify ROS in plasma samples from rats following administration of a single oral dose (25 mg kg[-1]), enabling the determination of key pharmacokinetic parameters, including C max (361.31 ng mL[-1]), t max (1.75 h, t ½ (2.867 h), V d (0.077 L kg[-1]), CL (0.018 L h[-1] kg[-1]), AUC (ng h mL[-1]) and and MRT (h). The greenness and practical applicability of the method were confirmed using four comprehensive independent metric tools. In comparison to previously reported bioanalytical approaches, the proposed method offers several practical advantages, such as reduced complexity and cost, a shorter run time, full validation, established greenness, and suitability for the clinical laboratory setting.

Histone acetyltransferase (HAT) inhibitors are promising epigenetic anticancer therapeutics. However, challenges remain regarding tissue specificity and off-target toxicity. This study presents a preclinical safety assessment of the HAT inhibitor anacardic acid (AA) in erythrocytes, given that anaemia is a clinically significant complication of chemotherapy. Freshly isolated erythrocytes were cultured in vitro with and without 1-10 µmol L[-1] of AA, then markers and mechanisms of cell death were examined by fluorescence-activated cell sorting, spectrophotometry, and potentiometry. AA treatment induced marked hemolysis independently of oxidative stress, which was paralleled by Ca[2+] accumulation and phosphatidylserine (PS) trans location. Morphological abnormalities were also observed upon AA treatment, including eryptotic loss of volume and elliptocyte phenotype. Co-treatment of AA with ATP, acetylsalicylic acid, NSC23766, or polyethylene glycol conferred significant cytoprotective effects. AA is cytotoxic to erythrocytes, causing both hemolysis and eryptosis, through non-genomic mechanisms involving Ca[2+] signalling, metabolic exhaustion, K[+] depletion, shrinkage and elliptocytic morphological aberrations. This toxic effect is sensitive to cyclooxygenase and Rac1 GTPase inhibition.

The metabolic effects of ginger (Zingiber officinale) on type 2 diabetes have been increasingly assessed in clinical trials, but the results remain inconsistent. In this updated meta-analysis, the Cochrane Library, Scopus, PubMed, Web of Science, and Google Scholar were searched from the inception to November 12, 2025, to find randomised controlled trials comparing the effect of ginger and placebo on the metabolic indexes of patients with type 2 diabetes. Pooling data from 13 articles with 837 participants indicated that treatment with ginger significantly decreased fasting blood sugar (FBS) (weighted mean difference (WMD) -16.22 mg per 100 mL, 95 % confidence interval (CI) (-25.49, -6.95), I[2] = 85.67 %), hemoglobin A1c (HbA1c) (WMD -0.49 %, 95 % CI (-0.86, -0.12), I[2] = 98.14 %), triglyceride level (WMD -17.04 mg per 100 mL, 95 % CI (-28.61, -5.47), I[2] = 71.30 %), and low-density lipoprotein cholesterol (LDL-C) (WMD -5.5 mg per 100 mL, 95 % CI (-10.98, -0.01), I[2] = 83.99 %). However, the beneficial effects of ginger on body mass index (BMI), homeostasis model assessment for insulin resistance (HOMA-IR), total cholesterol, high-density lipo-protein-cholesterol (HDL-C), and systolic and diastolic blood pressure did not meet the threshold of statistical significance. Ginger was more effective at a daily dose of less than 2000 mg (powdered rhizome of Zingiber officinale administered orally) and in males, individuals younger than 54 years, and individuals with a baseline HbA1c of more than 8 %.

Conventional RP-HPLC methods in pharmaceutical industry still dominantly rely on hazardous solvents like methanol (MeOH) and acetonitrile (ACN), presenting significant environmental and safety challenges. Although ethanol (EtOH) is recognized as greener alternative, isoeluotropic relationships between EtOH and these conventional solvents will broaden its implementation in routine HPLC analyses. To address this issue, the study presents an empirical approach using volu-metric fraction (φ) equivalence to establish initial chromatographic conditions, enabling predictable substitution of MeOH and ACN with EtOH in the LC mobile phase without altering other chromatographic parameters. The approach was established using adsorption (log-log) retention model across multiple C18 stationary phases, applicable for lipophilic neutral and basic active pharmaceutical ingredients (APIs). For neutral APIs, φ MeOH ≈ 1.6 φ EtOH and φ ACN ≈ 1.1 φ EtOH, while for basic APIs φ MeOH ≈ 1.5 φ EtOH and φ ACN ≈ 1.0 φ EtOH. The general applicability was confirmed under isocratic conditions by successfully converting conventional RP-HPLC methods for an independent set of APIs into green, EtOH-based alternatives. Notably, this work extends previous research focused on lipophilic acidic to neutral and basic APIs, broadening its utility for the most widely used APIs in the pharmaceutical industry. The proposed framework supports sustainable analytical practices and improves laboratory safety without extensive method redevelopment.

This review presents a chronological analysis of the scientific contributions of Professor Janko N. Herak (1937-2025), a Croatian biophysicist whose research played a major role in the development of electron para-magnetic resonance (EPR) spectroscopy in Croatia and significantly advanced the study of free radicals in biological systems. Beginning with his early work at Duke University under the mentorship of Prof. Walter Gordy, Herak investigated radiation-induced radicals in nucleic acid bases, nucleosides, and related biomolecules using EPR spectroscopy. During subsequent decades at the Ruđer Bošković Institute and later at the University of Zagreb Faculty of Pharmacy and Biochemistry, he conducted extensive studies of free radicals in irradiated single crystals of nucleic acid constituents, elucidating mechanisms of radical formation, hydrogen transfer, and structural rearrangements. His research later expanded to the investigation of oxidative processes in biological macromolecules, particularly the slow oxidation of plasma lipoproteins, where EPR spectroscopy and kinetic modelling provided insight into radical-mediated lipid peroxidation and the physicochemical basis of lipoprotein modification. In parallel, Herak explored long-range charge migration in nucleobase crystals and investigated metal-nucleobase interactions, integrating experimental EPR and ENDOR spectroscopy with theoretical modelling. These studies contributed to a deeper understanding of radical stabilisation, electronic structure, and energy transfer in ordered molecular systems. By situating Herak's work within the broader development of radiation chemistry and magnetic resonance spectroscopy, this review highlights the methodological rigour, conceptual contributions, and lasting scientific impact of his research on free radicals and biologically relevant molecular systems.

Piperine had synergistic effects with other natural products in treating lung cancer. The synergistic effects of piperine combined with luteolin on anti-non-small cell lung cancer activity and their mechanism of action are studied using network pharmacology and molecular docking. Cell viability, wound-healing, colony-formation, cell-apoptosis, and cell-cycle tests on A549 cells were used to detect the in vitro synergistic effects. Tumour growth and hematoxylin and eosin (HE) staining were used to verify the synergistic effects in Lewis lung carcinoma (LLC) tumour-bearing mice. Their combination indexes ranged from 0.3 to 0.8 across different concentrations, and the combination of 175.3 µmol L-1 piperine and 87.32 µmol L-1 luteolin showed synergistic inhibition of A549 cell viability. Moreover, the combination induced a higher apoptosis rate (75.60 %) than luteolin (24.56 %) or piperine (17.53 %). It arrested more cells in the G0/G1 phase than luteolin or piperine did. The combination achieved a tumour inhibition rate of 75.28 %, with lower tumour density and more obvious apoptosis. Network pharmacology identified AKT1, EGFR, SRC, and MMP9 as core targets regulating the PI3K/AKT pathway. Multi-software molecular docking confirmed binding of both compounds to these targets, with MMP9 as the common primary target. In conclusion, piperine and luteolin acted synergistically against non-small cell lung cancer.

Atherosclerosis, the main pathological background of cardiovascular diseases, is not only characterized by lipid metabolism disorders but also chronic inflammation as a major driver of disease progression. As a key regulator of inflammatory immune responses, the NLRP3 inflamma-some is involved in the development and instability of atherosclerotic plaques. Although reducing low-density lipoprotein cholesterol (LDL-C) confers benefits, residual cardiovascular risk persists, necessitating novel anti-inflammatory therapies. This review provides a comprehensive overview of NLRP3 inflammasome activation mechanisms and its role in plaque inflammation. We systematically summarize current NLRP3 inhibitors under preclinical and clinical development, including small molecules and biologics, with emphasis on design strategies, structural characteristics and pharmacological activities. Challenges in clinical translation, such as tissue targeting and safety, are also discussed. By integrating recent advances in molecular mechanisms and medicinal chemistry, this article aims to provide a theoretical basis for targeted anti-inflammatory therapies beyond lipid lowering.

As global life expectancy continues to rise, urinary tract infections (UTIs) have become an increasing concern in older adults. The higher prevalence in this population is attributed to anatomical and physiological changes of the urinary tract, hormonal imbalances, immunosenescence, and the presence of comorbidities. These factors, combined with a distinct microbiological profile and rising antimicrobial resistance, create significant clinical challenges in diagnosis and treatment. We conducted a systematic review of clinical trials and observational studies on the epidemiology, pathogenesis, diagnosis, and management of recurrent urinary tract infections (rUTIs) in older adults. The prevalence of rUTIs increases with age, disproportionately affecting women, with 53 % of those over 55 years experiencing recurrences within one year. Healthcare-associated UTIs (HAUTIs) account for 20-30 % of nosocomial infections, primarily impacting older adults. The host microbiome seemed crucial in UTI pathogenesis, with Escherichia coli being the leading causative agent due to its ability to adhere, colonise, and evade the immune response. In elderly patients, atypical presentations - such as delirium, functional decline, or nonspecific abdominal symptoms - complicate diagnosis, underscoring the critical need to differentiate symptomatic infections from asymptomatic bacteriuria (ASB) to prevent misdiagnosis and overtreatment. Effective management requires accurate diagnosis, appropriate antibiotic selection, and careful monitoring of adverse effects, especially in patients with comorbidities. Emerging therapies, including faecal microbiota transplantation, bacteriophages, probiotics, and proanthocyanidins, offer promising adjuncts. While long-term antibiotic prophylaxis is effective, it increases the risk of bacterial resistance, particularly in catheterised patients. Behavioural modifications, such as increased fluid intake, aid pathogen clearance, and topical estrogen therapy in postmenopausal women provides additional preventive benefit. Managing recurrent UTIs in ageing populations requires addressing microbiological, diagnostic, and antimicrobial resistance challenges. Despite resistance levels, the first-line treatment, nitrofurantoin, remains a viable therapeutic option, particularly in developed countries. An integrated approach combining individualised care, healthcare provider training, and rational antimicrobial use is essential to improving patient outcomes and quality of life. Future strategies should focus on novel antimicrobials targeting bacterial virulence factors, vaccines against uropathogens, and advanced diagnostic technologies.

The aim of this study was to evaluate scanning electron microscopy (SEM) combined with energy-dispersive X-ray spectroscopy (EDS) as complementary analytical tools for supporting continuous improvement in pharmaceutical granule manufacturing. Pilot-scale cefixime granules for oral suspension were prepared as defined process scenarios, including placebo, reference, intermediate, stress-exposed, and optimised batches. SEM was used to compare granule morphology, surface integrity, and agglomeration behaviour, whereas EDS provided qualitative and semi-quantitative information on localised elemental composition, with emphasis on sulfur as an API-related marker and oxygen-to--sulfur trends as surface-sensitive indicators of process- or stress-related variability. Intermediate and stress-exposed batches showed increased surface roughness, microstructural deterioration, and higher oxygen-to-sulfur ratios, whereas reference and optimised batches showed more uniform morphology and comparable elemental profiles. The findings indicate that SEM/EDS can provide useful material-level insight into process-related variability and may support root--cause investigation and process refinement. Overall, SEM/EDS is proposed as a complementary, localised, and semi-quantitative approach for supporting continuous improvement in pharmaceutical granule manufacturing.

A series of thirteen novel β-carboline and chloroquine (CQ) hybrids was designed and synthesized. The compounds were evaluated for in vitro antiplasmodial activity against Plasmodium falciparum strains 3D7 (CQ-sensitive) and Dd2 (multidrug-resistant). All compounds exhibited potent activity, with IC 50 values in the nanomolar to low micromolar range, while retaining potency against the resistant strain (1.0 to 365.5 nmol L[-1] for 3D7 and from 2.2 to 2415.3 nmol L[-1] for Dd2 strain). Several compounds were more active against Dd2, then against 3D7 strain with resistance index (RI) lower than 1. Moreover, nearly all compounds showed lower RI values compared to CQ which additionally underscores their outstanding activity. At the highest tested concentration (250 μmol L[-1]), no cytotoxicity was observed in HepG2 cells, resulting in favourable selectivity indices expressed as lower limits. Overall, the obtained results confirm strong antiplasmodial activity of the β-carboline and chloroquine hybrids as promising antiplasmodial candidates, particularly due to their activity against resistant strains and improved selectivity.

Daratumumab is a fully human anti-CD38 monoclonal antibody with strong potential as a targeting vector for therapeutic radionuclides. This study aimed to develop a freeze-dried daratumumab immunoconjugate kit by selecting a suitable chelator (DOTA-NHS, p-SCN-Bn-DOTA, or p-SCN-Bn-1B4M-DTPA) for the 177Lu-labelling, optimising the freeze-drying formulation, and evaluating the physicochemical properties and purity profiles. Conjugation performed in carbonate buffer at elevated temperature enhanced chelator incorporation and supported the selection of daratumumab-p-SCN-Bn-DOTA as the most suitable candidate, achieving radiolabelling yield up to 99.8 % without additional purification. Among the evaluated freeze-dried formulations, a saline-based, buffer-free sucrose-mannitol formulation containing polysorbate 20 (S.F5) provided the most favourable characteristics, including minimal residual moisture and the highest monomer purity with non-detectable HMWS species under the applied SE-HPLC conditions. ATR-FTIR and Raman spectroscopy confirmed preservation of the antibody structural integrity after conjugation and freeze-drying. In an in vitro study using human serum, [177Lu]Lu-daratumumab-p-SCNBn-DOTA maintained higher radiochemical purity for 168 h than [177Lu]Lu-daratumumab-p-SCN-Bn-1B4MDTPA, indicating greater stability. These results support the feasibility of a ready-to-use freeze-dried daratumumab-p-SCN-Bn-DOTA kit for the 177Lu-labelling.