Purpose:

Monoclonal antibodies (mAbs) are significant biotherapeutic agents for various diseases. Their oral administration is hindered by instability in the gastrointestinal tract (GIT). Lyophilization offers a viable method for stable mAb formulation for oral use. This research discusses the development of a stable oral formulation of mAbs through optimized freeze-drying process.

Method:

mAb formulations were created in buffers of pH 5 (mAb1) and pH 7.4 (mAb2). Bulking agents, stabilizers, and their concentrations were identified for lyophilization. EDTA served as a protease inhibitor to reduce mAb degradation. The lead formulation was established using optimized freezing and drying parameters. Modulated differential scanning calorimetry (MDSC) assessed the glass transition temperature (Tg) and eutectic temperature (Teu). Short-term stability up to 30 days was evaluated through assessments of cake appearance, reconstitution time, moisture content, purity, and related substances.

Results:

Trehalose showed superior cryoprotection as a bulking agent. Increasing trehalose concentration from 10% to 20% lowered the Tg of the lead formulation from -36˚C to -34˚C. The lyophilization cycle was optimized to 72 hours at freezing (-50°C), primary drying (-34°C), and secondary drying (20°C) with a pressure of 72mTorr. Methionine effectively stabilized the lyophilized mAbs, yielding minimal %Impurity. The addition of EDTA to the lead formulation with 20% trehalose did not influence Tg. MDSC findings indicated Tg's significance in maintaining low primary drying temperatures. Moisture content was below 3%. No significant differences in mAb concentrations were noted across various temperatures. However, Lyo-mAb1 exhibited greater stability compared to Lyo-mAb2, as indicated by %Purity data. Both formulations demonstrated %Impurity below 1%, indicating no process-related mAb degradation.

Conclusion:

This study provides critical insights for developing stable lyophilized mAb formulations, addressing stability issues related to enzymatic degradation and acidic pH in the GIT. Furthermore, the lyophilized formulation in enteric-coated capsules presents a promising method for oral mAb delivery.

Pomegranate (Punica granatum L.) is one of the richest fruits in polyphenols, compounds with valuable antioxidant and antimicrobial properties. In recent decades, there has been an ever-increasing demand for its fruits, primarily for juice production, accompanied by a growing generation of agri-food waste, estimated at 9 tons for each ton of pomegranate juice produced.

In this study, the peels of the pomegranates discarded for colour and shape, inappropriate for sale, granted by an Italian farm, were extracted with 95% ethanol. Subsequently, the enrichment of the phenolic compounds in the extract was achieved through solid phase extraction (SPE) by sequential elution with water, water/acetonitrile 1:1 v/v and acetonitrile as mobile phase. The phenol-rich extract was subjected to HPLC-PDA, revealing the presence of high amount of punicalagin (36.1 ± 3.2 %), along with ellagic acid and ellagic acid derivatives.

To evaluate the possible application of the punicalagin-rich extract within formulations for environmental or human/veterinary use, it was tested on six phytopathogenic fungi (from the culture collection of the Fungal Biodiversity Laboratory of Sapienza), reported also as pathogenic for humans or animals, assessing fungistatic and fungicidal activity through the microdilution broth method. MIC and MFC values for the extract and fluconazole, as drug reference, were determined. For four species (FBL number: 4, 57, 59, 659) MIC 90 was observed to be in the range of 500-1000 μg/mL, while the respective MFC was 2000 μg/mL; however, only three (FBL 4, 57, 659) showed MIC 100 at 1000 or 2000 μg/mL. Interestingly, FBL 4 was more sensitive to pomegranate extract than to fluconazole (MIC90 > 64 μg/mL).

In conclusion, in this study, the preparation of a pomegranate peel extract rich in compounds with valuable biological activities paves the way for an innovative valorisation of pomegranate waste products, representing an efficient example of circular economy.

Chronic inflammation and oxidative stress are major factors to the development and progression of degenerative, autoimmune and musculoskeletal diseases. The persistent creation of reactive oxygen species (ROS) and protracted inflammatory reactions cause cellular damage, lipid peroxidation, and DNA instability, demanding safer and more effective therapeutic alternatives to current medications. Traditional medicines describe various medicinal plants for treating inflammatory diseases their pharmacological qualities remain unknown. Hydroalcoholic extracts of Aloe barbadensis Mill., Chrysanthemum indicum L., Commiphora mukul Engl., Convolvulus scammonia L., Ipomoea turpethum L., and Merendera persica Boiss. was extracted using Soxhlet and tested for phytochemical composition, antioxidant potential, DNA protective effects, anti-inflammatory, antimicrobial activity and safety. Phytochemical screening revealed the presence of flavonoids, phenolics, carbohydrates, and alkaloids, suggesting a high concentration of bioactive metabolites. Antioxidant ability was determined using DPPH, ABTS, FRAP, nitric oxide scavenging, and malondialdehyde (MDA) tests, which revealed significant free radical neutralisation and prevention of lipid peroxidation. Among the extracts examined, A. barbadensis showed considerable activity in DPPH and FRAP assays, while Convolvulus scammonia showed significant activity in ABTS and nitric oxide scavenging assays. C. indicum had the lowest amounts of MDA, indicating excellent lipid peroxidation inhibition. Furthermore, C. scammonia proved very efficient in preventing protein denaturation. A. barbadensis showed strong antimicrobial activity against Escherichia coli at a concentration of 10 µg/mL. The FTIR study also identified hydroxyl, carbonyl, and phenolic functional groups that are related with antioxidant and anti-inflammatory bioactivity. In conclusion, the findings give scientific support to the traditional usage of these herbs and emphasise their potential, natural and multi-targeted therapeutic agents for oxidative stress and chronic inflammation, notably in arthritis and associated diseases.

Introduction: Malaria remains a significant public health concern in Ghana, underscoring the need to explore the antimalarial efficacy of medicinal plants, such as Bambusa vulgaris (bamboo), traditionally used for its management. This study investigates the in vivo antimalarial efficacy of aqueous (BVA) and hydroethanolic (BVE) leaf extracts of B. vulgaris against Plasmodium berghei-infected mice, alongside their antioxidant activity and pharmacognostic standards.

Method: Acute toxicity was assessed following OECD guidelines. Antimalarial activity was evaluated using Peter’s four-day suppressive and Rane’s curative tests. Antioxidant properties were investigated using the DPPH assay. Pharmacognostic standards were established following standard procedures.

Results: The LD₅₀ of the extracts was > 2000 mg/kg. In the four-day suppressive test, BVE exhibited parasitaemia suppression and an ED₅₀ of 90.68 ± 1.53% at 400 mg/kg and 177.83 ± 16.36 mg/kg, respectively. BVA, similarly, provided a suppression and an ED₅₀ of 84.98 ± 1.62% and 281.84 ± 22.31 mg/kg. In the curative test, BVA achieved a % suppression of 63.15 ± 1.53 at 400 mg/kg and an ED₅₀ of 234.42 ± 10.28 mg/kg. Both extracts prolonged survival and increased body weight of mice, with BVA reversing hypothermia and anaemia. Antioxidant assays revealed BVE’s stronger DPPH scavenging activity, with an IC₅₀ of 851.3 µg/mL, compared to BVA, which had an IC₅₀ of 1552 µg/mL, attributed to higher flavonoid (4.71 ± 0.039 mg quercetin/g) and phenolic (58.99 ± 0.039 mg GAE/g) contents. Pharmacognostic studies established macro-morphological, microscopic, physicochemical, chromatographic, and spectroscopic standards.

Conclusion: The study validates the traditional antimalarial use of B. vulgaris leaves and provides standard benchmarks for its quality control.

Introduction. Oncolytic viruses (OV), a novel class of anti-cancer therapies, selectively replicate in cancer cells and activate the immune response. The unique OV-targeted destruction of malignant cells sets OV-based therapies apart from the non-specific conventional chemo-, radio-, and even targeted antibody-based therapies. However, the sensitivity of cancer cell lines to rVSV and, hence, the success of virotherapy, may differ depending on cancer type and the strength of the anti-viral response. At the same time, the oncolytic potency of rVSV decreases as a result of the insertion of larger genes (such as interleukin-12, IL12, and granulocyte macrophage colony-stimulating factor, GMCSF, used to potently activate the dampened immune response) into the viral genome as compared to green fluorescent protein (GFP). Therefore, the combination of rVSV and mRNA-delivered immunostimulatory factors may increase the efficiency of anti-tumor therapy.
Methods. rVSV-mIL12-mGMCSF cytotoxicity was assessed in vitro on three murine cancer cell lines: B16-F10, LL/2, and CT26 24 hours post-infection by flow cytometry. In vivo experiments were performed on syngeneic models via intratumoral injection of rVSV. Quantitative PCR (qPCR) analysis was performed to assess the mRNA expression levels 12 hours post-infection in vitro.
Results. Based on the obtained data, Lewis lung carcinoma (LL/2) cells were more susceptible to oncolysis compared to colon carcinoma (CT26) or melanoma (B16-F10). Detection of expression levels of certain antiviral and pro-apoptotic genes in response to the rVSV infection by qPCR showed higher levels of IFNβ and p53 and lower levels of IFIT, RIG-I, and N-cadherin in LL/2 cells. Subsequently, C57BL/6 and BALB/c mice, infused subcutaneously with B16-F10 and LL/2 cells, respectively, were injected intratumorally with rVSV-mIL12-mGMCSF to assess the synergistic effect of rVSV and immunostimulatory factors. The in vivo study demonstrated tumor growth inhibition (TGII) of over 50% for both cancer models. Finally, we assessed the combined effect of two platforms: mRNA-LNP-based delivery of mIL-12 and mGMCSF and rVSV-mediated oncolysis, leading to TGII of 95% in animals with colon cancer.
Conclusions. The unique combination therapy approach significantly improved therapy outcome. These promising results are giving hope that this novel approach will also be successful for melanoma and lung carcinoma models in the near future.

Introduction:
The urgent demand for new antimicrobial strategies has highlighted transition metal complexes as promising therapeutic candidates. Among them, Co(III) complexes with diamine chelate ligands exhibit notable stability and biomolecular affinity. To elucidate their antimicrobial activity and mode of action, phosphorescent Ru(II)- and Pt(II)-based probes are particularly advantageous due to their oxygen-sensitive phosphorescence. Phosphorescence optical respirometry (POR) enables real-time monitoring of microbial respiration, providing a sensitive measure of metabolic inhibition induced by metal complexes.

Materials and Methods:
Co(III)-diamine complexes were synthesized and characterized by elemental analysis, an ATR technique, and a scan method. Their antimicrobial properties were examined against representative gram-positive (Staphylococcus aureus, Staphylococcus epidermidis) and gram-negative (Escherichia coli, Pseudomonas aeruginosa) reference and clinical bacterial strains. Ru(II) and Pt(II) phosphorescent probes were employed in POR assays to monitor oxygen consumption as a marker of cellular respiration.

Results:
The Co(III) complexes displayed strain-dependent antimicrobial effects, with more pronounced activity against gram-positive bacteria. POR measurements revealed a dose-dependent suppression of microbial respiration, consistent with metabolic inhibition. Changes in phosphorescence lifetimes of Ru(II) and Pt(II) probes confirmed oxygen depletion dynamics and allowed kinetic profiling of bacterial response to the complexes.

Conclusions:
Ru(II) and Pt(II) phosphorescent biosensors, applied through phosphorescence optical respirometry, provide a robust platform for investigating the antimicrobial activity of Co(III)-diamine complexes. as well as other antimicrobial compounds. This method not only enables sensitive detection of respiratory inhibition but also permits the determination of viable aerobic microbial cell counts through analysis of the phosphorescence value and offers mechanistic insights into the biological action of metal-based therapeutics and. These findings establish a foundation for further development the PRO method to investigate the interactions of antibiotics and antimycotics with newly developed metal complex–derived antimicrobial agents targeting microorganisms.

Oxidative stress plays a central role in the pathogenesis of neurodegenerative, cardiovascular, and inflammatory disorders, as well as cancer. In this sense, the discovery of novel antioxidant agents has a critical priority in medicinal chemistry. Organoselenium derivatives stand out in this context for their ability to modulate redox processes, frequently mimicking the activity of glutathione peroxidase (GPx). At the same time, the tetrazole nucleus functions as a bioisosteric moiety, widely employed in clinically approved drugs due to its favorable pharmacokinetic and pharmacodynamic properties. The strategic combination of these two pharmacophoric elements results in selenotetrazoles, molecules with promising biological potential and new perspectives for therapeutic development. The compounds were obtained via [3+2] cycloaddition between SeCN-containing precursors and sodium azide. In this work, the antioxidant activity of the newly synthesized selenotetrazoles was evaluated through radical scavenging assays (ABTS•+ and DPPH•) to investigate their redox-modulating potential. In the ABTS•+ assay, which measures the ability of molecules to neutralize radicals in aqueous medium, ethyl 3-(1H-tetrazol-5-yl)selanyl benzoate and ethyl 4-(1H-tetrazol-5-yl)selanyl benzoate at 200 µM showed significant antioxidant activity, comparable to ascorbic acid (positive control, 25 µM). Conversely, in the DPPH• assay, which evaluates electron transfer in apolar medium, the compounds showed no significant activity. This selectivity suggests that their antioxidant effect is not based on electron transfer in nonpolar systems, but rather occurs in aqueous environments, consistent with a GPx-like mechanism . In conclusion, the synthesized selenotetrazoles present selective antioxidant activity, redox stability and multitarget potential, consolidating them as promising scaffolds for the development of antioxidant, anti-inflammatory, neuroprotective and/or anticancer agents.

To develop pharmacologically active molecules, a series of analogues of 3-(1,2-dihydro-6-substituted phenyl-2-thioxopyrimidin-4-yl)-1-methylquinolin-2(1H)-one (compounds 4a–j) were synthesized through condensation and ring-closure reactions, yielding novel pyrimidine derivatives. These newly synthesized 2-quinolone-linked pyrimidin-2-thiol analogues were structurally characterized using various spectroscopic techniques. The compounds were evaluated for their antioxidant activity using the DPPH (2,2-diphenyl-1-picrylhydrazyl) assay, and for anti-Alzheimer potential through acetylcholinesterase (AChE) inhibition, assessed via Ellman’s method. Among the series, compounds 4c and 4e, containing electron-donating groups on the phenyl ring, showed excellent radical scavenging activity. In AChE inhibition studies, compound 4c [3-(1,2-dihydro-2-thioxo-6-p-tolylpyrimidin-4-yl)-1-methylquinolin-2(1H)-one] exhibited notable inhibitory activity, likely due to the influence of the p-tolyl moiety.Compound 4g [3-(1,2-dihydro-6-(3-nitrophenyl)-2-thioxopyrimidin-4-yl)-1-methylquinolin-2(1H)-one] also demonstrated considerable AChE inhibition.Kinetic studies provided further insights, compound 4e showed a competitive inhibition pattern at both low and high inhibitor concentrations. Compound 4g displayed mixed-type inhibition, suggesting its ability to interact with both the catalytic anionic site (CAS) and the peripheral anionic site (PAS) of AChE. Molecular docking of compounds 4a–j supported the experimental findings, with docking scores ranging from moderate to good compared to the reference drug. Notably, compound 4g had the best docking score of −10.134, forming a key hydrogen bond with PHE295, correlating well with its strong AChE inhibition. Further molecular dynamics simulations of compound 4g within the AChE binding site confirmed its stable interactions and potential binding mode. Based on these findings, compound 4g emerges as a promising lead molecule for the development of novel anti-Alzheimer agents. Finally, drug-likeness properties of all synthesized compounds were evaluated, supporting their potential for further development.

Endothelial dysfunction (ED) is currently recognized as a key pathogenic factor in the development of cardiovascular diseases. Reduced bioavailability of nitric oxide (NO), increased oxidative and nitrosative stress, and disruption of redox homeostasis contribute to vascular endothelial damage, leading to ischemic, hypertensive, and neurodegenerative conditions. Currently, there is no pharmacotherapy specifically targeting ED, highlighting the urgent need for novel therapeutic agents with endothelioprotective properties.

This study highlights the rationale and experimental basis for the creation of a new class of pharmacological agents—endothelioprotectors. Particular focus is given to a new compound, Angiolin (S)-2,6-diaminohexanoic acid 3-methyl-1,2,4-triazolyl-5-thioacetate, designed as an endothelioprotector with neuro- and cardioprotective capabilities. Structurally combining L-lysine and thiotriazoline fragments, Angiolin demonstrates a wide spectrum of pharmacological activities, including endothelioprotective, cardioprotective, neuroprotective, anti-ischemic, antioxidant, and anti-inflammatory effects.

Preclinical studies have shown that Angiolin enhances NO bioavailability, protects endothelial cells, preserves mitochondrial function, activates the glutathione system, and prevents oxidative damage in ischemic tissues. Furthermore, Angiolin improves cerebral and cardiac hemodynamics, promotes ATP production via the malate-aspartate shuttle, and modulates GABAergic neuroprotection by increasing pipecolic acid levels.

Toxicological assessment classifies Angiolin as a substance of low toxicity (Class VI), and Phase I clinical trials conducted with the approval of the Ministry of Health of Ukraine confirmed its safety profile. The compound represents a promising therapeutic strategy for complex treatment of chronic cerebral ischemia, myocardial infarction, heart failure, and prenatal hypoxia, offering advantages over current metabolitotropic drugs such as meldonium, piracetam, and trimetazidine.

In conclusion, Angiolin is a novel pharmacological agent with targeted endothelioprotective action and broad metabolic, cardio-, and neuroprotective potential, warranting further clinical investigation.

In recent decades, new methods of cancer treatment have been developed. One of these methods is photodynamic therapy (PDT), which is non-invasive and based on the generation of reactive oxygen species (ROS) when light interacts with photosensitizers (PS). However, photosensitizers often have low solubility and reduced bioavailability under physiological conditions. To address this issue, nanocarriers have been created to deliver photosensitizers more effectively, increasing the depth of penetration into cellular structures and enhancing the therapeutic effect of PDT.

The paper presents nanocarriers obtained through the nanoemulsion polymerization reaction between homologous methyluracil derivatives (4-(3,6-dimethyluracil)-butyl acrylate or 3-(3,6-dimethyluracil)-propyl acrylate) and N,N'-bis(acryloyl)cystamine. These methyluracil derivatives form the shells of the nanocarriers, while N,N'-bis(acryloyl)cystamine, dissolved in DMSO, acts as the dispersed phase to form a hydrophobic core. Porphyrin derivatives with various substituents at the meso-position are encapsulated within the core of these nanocarriers. According to transmission electron microscopy data, carrier sizes ranged from 50 to 200 nanometers (nm), and nanocarriers containing a glycoporphyrin derivative had the smallest hydrodynamic diameters.

The cores, formed by N,N'-bis(acryloyl)cystamine, contain disulfide bonds that can be reduced by intracellular antioxidants such as glutathione. These antioxidants are often found in tumor environments. The reaction of reducing the disulfide bond leads to the disintegration of the particles and the release of PS. This process has been confirmed by the fluorescence method, which shows an increase in emission intensity over time when glutathione is added to the carrier solution.

The formation of singlet oxygen upon irradiation of nanocarriers with PS was confirmed using 1,3-diphenylisobenzofuran (DPBF) and UV spectroscopy. PS in nanocarriers based on 4-(3,6-dimethyluracil)-butyl acrylate generated singlet oxygen at almost twice the rate of individual PS.