Search Results (612)

Background/Objectives: Monkeypox, twice declared a public health emergency of international concern by the WHO, currently lacks approved targeted therapeutics. This study focused on the development of monkeypox virus (MPXV) E8-specific human monoclonal antibodies (mAbs) derived from recipients of the recombinant vaccinia vaccine (rTV), with subsequent evaluation of their cross-neutralizing activity against orthopoxviruses, including the vaccinia virus (VACV) and MPXV. Methods: Three mAbs (C5, C9, and F8) were isolated from rTV vaccinees. Structural mapping characterized their binding domains on the MPXV E8 and VACV D8 proteins. Neutralization potency was assessed against the VACV TianTan strain and MPXV clade IIb. A combo was further evaluated in a VACV-infected mice model for clinical recovery and viral load reduction. Complement-dependent enhancement mechanisms were also investigated in vitro. Results: C9 targets the virion surface region of E8 and both the virion surface region and intravirion region of D8, showing cross-neutralization activity against the MPXV (IC₅₀ = 3.0 μg/mL) and VACV (IC₅₀ = 51.1 ng/mL) in vitro. All three antibodies demonstrated potent neutralization against the VACV in vitro: C5 (IC₅₀ = 3.9 ng/mL), C9 (IC₅₀ = 51.1 ng/mL), and F8 (IC₅₀ = 101.1 ng/mL). Notably, complement enhanced neutralization against the VACV by >50-fold, although no enhancement was observed for the MPXV. In vivo administration accelerated clinical recovery by 24 h and achieved significant viral clearance (0.9-log reduction). Conclusions: E8-targeting mAbs exhibited broad-spectrum neutralization against orthopoxviruses, demonstrating therapeutic potential against both historical (VACV) and emerging (MPXV) pathogens. However, MPXV’s resistance to complement-dependent enhancement highlights the necessity for pathogen-adapted optimization. These findings establish E8 as a critical conserved target for pan-poxvirus VACV and MPXV countermeasure development. Full article

(1) Background: Doxorubicin (DOX) is a frontline chemotherapeutic, but its side-effects from oxidative stress, leading to cardiotoxicity, pose significant challenges to its clinical use. We recently discovered a novel family of proteolysis-resistant, cystine-dense, and cell-penetrating microproteins from Panax ginseng that we term ginsentides. Ginsentides, such as the 31-residue TP1, coordinate multiple biological systems to prevent vascular dysfunction and endoplasmic reticulum stress induced by internal and external stressors. (2) Methods: We assessed the protective effects of ginsentide TP1 on DOX-induced cardiotoxicity using both in vitro functional studies on H9c2 cardiomyocytes and in vivo animal models by zebrafish and ICR mouse models. In these models, we examined oxidative stress, apoptosis, intracellular calcium levels, mitochondrial function, inflammatory responses, and cardiac function. (3) Results: We show that ginsentide TP1 protects against DOX-induced cytotoxicity in the mitochondria-rich H9c2 cardiomyocytes and reduces myocardial injury in zebrafish and mice by mitigating oxidative stress, inflammation, calcium, and mitochondrial dysfunction, as well as apoptosis-mediated cell death. Importantly, TP1 preserves cellular homeostasis without compromising the anticancer potency of DOX in breast cancer cells. (4) Conclusions: our findings highlight a specific antioxidative function of ginsentide TP1 in managing DOX-induced cardiotoxicity during cancer treatment and provide a promising lead for developing cardioprotective peptides and microproteins against oxidative stress. Full article

Endoscopic investigations reveal that a significant majority of individuals taking NSAIDs exhibit acute hemorrhages and mucosal erosions within the gastroduodenal lining. Ketoprofen acid (KA) is a potent NSAID with established efficacy and cardiovascular tolerability, but its gastric tolerability is a recognized limitation. To mitigate this, ketoprofen lysine salt (KLS) was developed. This review evaluates the pharmacological advantages of KLS over KA. While both KA and KLS maintain similar potency, KLS offers distinct advantages. Firstly, KLS demonstrates superior gastrointestinal protection through enhanced antioxidant properties and upregulation of mucosal defenses, as evidenced by both in vitro and in vivo studies. Secondly, KLS exhibits significantly faster absorption, leading to a more rapid onset of analgesic effects; this is attributed to its increased solubility and faster achievement of therapeutic concentrations. In essence, KLS addresses the gastric tolerability issues of KA while providing a quicker onset of action, making it a valuable alternative for patients requiring NSAID therapy, particularly those with gastric sensitivities or in need of rapid pain relief. Full article

Neglected tropical diseases (NTDs), including Chagas disease, human African trypanosomiasis (HAT), leishmaniasis, and malaria, remain a major global health challenge, disproportionately affecting low-income populations. Current therapies for these diseases suffer from significant limitations, such as reduced efficacy, high toxicity, and emerging parasite resistance, highlighting the urgent need for new therapeutic strategies. In response, substantial efforts have been directed toward the synthesis of new molecules with improved potency, selectivity, and pharmacokinetic profiles. However, despite many of these compounds exhibiting favorable ADMET (absorption, distribution, metabolism, excretion, and toxicity) profiles and strong in vitro activity, their translation into in vivo models remains limited. Key challenges include the lack of investment, the absence of fully representative experimental models, and difficulties in extrapolating cell-based assay results to more complex biological systems. In this review, we analyzed the latest advancements (2019–2024) in the development of these compound classes, correlating predictive parameters with their observed biological activity. Among these parameters, we highlighted the partition coefficient (LogP), which measures a compound’s lipophilicity and influences its ability to cross biological membranes, and Caco-2 cell permeability, an in vitro model widely used to predict intestinal drug absorption. Additionally, we prioritized the most promising molecules and structural classes for pharmaceutical development, discussing structure–activity relationships (SARs) and the remaining challenges that must be overcome to enable the clinical application of these compounds in the treatment of NTDs. Full article

Background: The three commercial Enterovirus 71 (EV71) inactivated vaccines which have effectively controlled the EV71 pandemic currently rely on inherent variable in vivo potency methods for batch release. To align with 3R (Replacement, Reduction, Refinement) principles and enhance quality control, this study referred to WHO guidelines and the European Pharmacopoeia to develop in vitro relative potency (IVRP) methods. Methods: Working standards tracing to phase 3 clinical vaccines were established. Manufacture-specific IVRP methods were developed and validated per ICH Q14/Q2(R2), utilizing conformational epitope-targeting neutralizing monoclonal antibodies (MAbs). One of the MAbs (CT11F9) recognition sites was clarified with Cryo-EM. Subsequently, the performance of IVRP was assessed using varied concentrations and heat-treated vaccines. The correlation between IVRP and in vivo methods was analyzed, followed by setting IVRP specifications. Results: The manufacturer-specific working standard exhibited ED50 values comparable to those of related phase 3 clinical vaccines. All IVRP methods achieved a relative bias/precision/total error ≤ 15%. The IVRP methods correlated with in vivo methods (p < 0.05, r > 0.9) can discriminate EV71 antigen concentrations (p < 0.01, r > 0.99) and indicate the stability of the vaccines. Cryo-EM was adopted to identify the epitopes recognized by CT11F9, revealing that this neutralizing antibody recognizes a conformational epitope spanning VP1-3 of the same protomer. Using 31–47 batches of commercial vaccines, IVRP specifications were proposed as 0.56–1.35, 0.58–1.40, and 0.54–1.50. Conclusions: Based on conformational epitope-targeting neutralizing MAbs, manufacturer-specific IVRP methods, which were sensitive to process variations and correlated with in vivo results, have been established. IVRP methods provide a reliable, animal-free alternative for EV71 vaccine batch release. Full article

Background: Nrf2 plays a key role in regulating the antioxidant response against oxidative stress. Therefore, it is imperative to examine the advantages of Nrf2 activation by new small molecules capable of inhibiting the Nrf2-Keap1 protein interaction that do not present electrophilic sites, since electrophilic compounds have intrinsic toxicity. The bixin pigment has been used as a form of treatment and prevention of several pathological conditions in animal models since it was described as an Nrf2 activator without electrophilic sites. This study aims to synthetize a soluble derivate KBx (potassium bixinate) and evaluate its ability to activate Nrf2/ARE in a model of exposure to cigarette smoke extract (CSE; in vitro) and intranasal LPS administration (in vivo). Methods: In the in vivo study, C57BL/6 mice were pretreated with 200 mg/kg of KBx (gavage) during 5 consecutive days and then challenged with 60 µg of LPS i.n. for 16 h. Bronchoalveolar lavage was collected to examine cytokines dosage. In the in vitro study, RAW 264.7 macrophages were exposed to CSE and post-treated with KBx to evaluate their ability to revert the redox imbalance caused by the stressor. Results: KBx was characterized using mass spectrometry (433.1778 m/z). KC levels were increased in the LPS group (p = 0.021), and KBx inhibited this (p = 0.001). IL-10 levels were decreased (p = 0.055) in the LPS group that was prevented when pretreated with KBx (p = 0.037). The in vitro study showed KBx to be a more potent derivate of bixin through its ability to intercept ROS formation with three-fold more potency, and it showed an anti-inflammatory propriety by reducing the nuclear translocation of p65 (p < 0.001). Conclusions: In conclusion, these data suggest that KBx was able to activate the Nrf2/ARE pathway and intercept ROS formation induced by CSE and LPS in both in vivo and in vitro studies. Full article

Hepatocellular carcinoma (HCC) causes the third highest mortality worldwide. Liver ablation, surgery, and embolization are conventional methods for treatment. However, these methods have limitations. To overcome these issues, nanomedicines have potential due to their high stability, high drug load capacity, and controlled release. Thus, we prepared quercetin-loaded polylactic-co-glycolic acid (PLGA) nanoparticles coated with folic acid-chitosan (QPCF-NPs) to improve drug delivery and targetability applications of quercetin for the treatment of HCC. We prepared QPCF-NPs by solvent evaporation and coated them with chitosan-folic acid (CS-FA). QPCF-NPs were examined using Fourier-Transform infrared (FTIR), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and X-ray diffraction (XRD). In addition, the drug release rate and cytotoxicity were studied. Moreover, in vivo HCC studies such as histopathology and biochemical parameters were conducted. Subsequently, QPCF-NPs with a spherical shape and an average size of 200–290 nm have been demonstrated to have formed by FTIR, XRD, SEM, and TEM. Further, we observed sustained drug release from QPCF-NPs compared to quercetin. Cellular cytotoxicity showed significant inhibition in the HEPG2-cell line with QPCF-NPs treatment. Biochemical estimate and oxidative stress regulation were considerably more regulated in the treatment groups than the HCC group in a dose-dependent way after subcutaneous administration of QPCF-NPs. ELISA of interleukin and caspase-3 demonstrated the anticipated results in comparison to the carcinogen control group. Compared to earlier preparations, the QPCF-NPs generated demonstrated better drug targetability and potency for treating HCC. Full article

Background: Lipid nanoparticles (LNPs) have proven effective in delivering RNA-based modalities. Rapid approval of the COVID-19 vaccines highlights the promise of LNPs as a delivery platform for nucleic acid-based therapies and vaccines. Nevertheless, improved LNP designs are needed to advance next-generation vaccines and other gene therapies toward greater clinical success. Lipid components and LNP formulation excipients play a central role in biodistribution, immunogenicity, and stability. Therefore, it is important to understand, identify, and assess the appropriate lipid components for developing a safe and effective formulation. Herein, this study focused on developing a novel Polysorbate-80 (PS-80)-based LNP. We hypothesized that substituting conventional linear PEG-lipids with PS-80, a widely used, biocompatible injectable surfactant featuring a branched PEG-like structure, may change the LNPs biodistribution pattern and enhance long-term stability. By leveraging PS-80’s unique structural properties, this study aimed to develop an mRNA-LNP platform with improved extrahepatic delivery and robust freeze/thaw tolerance. Methods: We employed a stepwise optimization to establish both the lipid composition and formulation buffer to yield a stable, high-performing PS-80-based SARS-CoV-2 mRNA-LNP (SC2-PS80 LNP). We compared phosphate- versus tris-based buffers for long-term stability, examined multiple lipid ratios, and evaluated the impact of incorporating PS-80 (a branched PEG-lipid) on in vivo biodistribution. Various analytical assays were performed to assess particle size, encapsulation efficiency, mRNA purity, and in vitro potency of the developed formulation and a humanized mouse model was used to measure its immunogenicity over six months of storage at −80 °C. Results: Replacing the standard 1,2-dimyristoyl-rac-glycero-3-methoxy polyethylene glycol-2000 (PEG-DMG) lipid with PS-80 increased spleen-specific expression of the mRNA-LNPs after intramuscular injection. Formulating in a tris-sucrose-salt (TSS) buffer preserved the LNP’s physicochemical properties and in vitro potency over six months at −80 °C, whereas a conventional PBS-sucrose (PSS) buffer was less protective under frozen conditions. Notably, TSS-based SC2-PS80 LNPs elicited potent humoral immunity in mice, including high anti-spike IgG titers and robust pseudovirus neutralization, comparable to freshly prepared formulations. Conclusions: A PS-80-based mRNA-LNP platform formulated in TSS buffer confers improved extrahepatic delivery, long-term frozen stability, and strong immunogenicity against SARS-CoV-2 following six months. These findings offer a promising pathway for the design of next-generation mRNA vaccines and therapeutics with enhanced stability and clinical potential. Full article

The control of socioeconomically important parasitic roundworms (nematodes) of animals has become challenging or ineffective due to problems associated with widespread resistance in these worms to most classes of chemotherapeutic drugs (anthelmintics) currently available. Thus, there is an urgent need to discover and develop novel compounds with unique mechanisms of action to underpin effective parasite control programmes. Here, we evaluated an in silico (computational) approach to accelerate the discovery of new anthelmintics against the parasitic nematode Haemonchus contortus (barber’s pole worm) as a model system. Using a supervised machine learning workflow, we trained and assessed a multi-layer perceptron classifier on a labelled dataset of 15,000 small-molecule compounds, for which extensive bioactivity data were previously obtained for H. contortus via high-throughput screening, as well as evidence-based datasets from the peer-reviewed literature. This model achieved 83% precision and 81% recall on the class of ‘active’ compounds during testing, despite a high imbalance in the training data, with only 1% of compounds carrying this label. The trained model was then used to infer nematocidal candidates by in silico screening of 14.2 million compounds from the ZINC15 database. An experimental assessment of 10 of these candidates showed significant inhibitory effects on the motility and development of H. contortus larvae and adults in vitro, with two compounds exhibiting high potency for further exploration as lead candidates. These findings indicate that the present machine learning-based approach could accelerate the in silico prediction and prioritisation of anthelmintic small molecules for subsequent in vitro and in vivo validations. Full article

The aim of this study was to evaluate an in vitro method using the EpiOral^TM model, a three-dimensional cultured human buccal epithelium, for assessing the oral irritation potential of various products. We evaluated different concentrations of nine chemicals commonly found in over-the-counter (OTC) products and medical devices, including chlorhexidine digluconate, sodium hypochlorite, phosphoric acid, hydrogen peroxide, lactic acid, ethanol, sodium dodecyl sulfate, 1-decanol and methyl methacrylate. The method was able to identify the irritants with a clear dose–response relationship between cell viability and an increasing concentration of the chemicals in the tested solutions. Using three exposure times (1, 4 and 18 h) and calculating the ET-50 (time required to induce a 50% reduction in cell viability), the solutions were classified according to their irritant potency (strong, moderate, mild or non-irritant). The results showed excellent correlation with historical in vivo data by matching the potency classifications in most cases. This study highlighted the importance of multiple exposure times for accurate assessment, as some solutions with irritant chemicals require longer exposure to produce effects. By providing information on both the irritant potential and potency, this method proved useful for toxicologists in the risk assessment of OTC products and medical devices that come into contact with the oral cavity. Full article

Despite advances in vaccination and early detection, the total number of cases and deaths from cervical cancer has risen steadily in recent decades, making it the fourth most common type of cancer in women worldwide. Low-income countries in particular struggle with limited resources and treatment limitations for cervical cancer. Thus, effective medicines that are simple to manufacture are needed. The newly developed dual topoisomerase inhibitor P8-D6, with its outstanding ability to induce apoptosis, could be a promising option. In this study, the efficacy of P8-D6 in combination with radiochemotherapy against cervical carcinoma was investigated in established cell lines and in a translational approach in ex vivo patient cells by measuring the cytotoxicity, cell viability and caspase activity in vitro in 2D and 3D cell cultures. Treatment with P8-D6 resulted in significantly greater cytotoxicity and apoptosis induction compared to standard therapeutic cisplatin in both 2D and 3D cell cultures. Specifically, a considerably stronger anti-proliferative effect was observed. The treatment also led to morphological changes and a loss of membrane integrity in the 3D spheroids. Radiotherapy also benefited greatly from P8-D6 treatment. In fact, P8-D6 was a more potent radiosensitizer than cisplatin. Simple synthesis, favorable physicochemical properties and high potency make P8-D6 a promising cervical cancer drug candidate. Full article

Background/Objectives: Potency is a critical quality attribute for vaccine development as well as clinical drug product (DP) lot release and stability testing. Animal studies have the potential to offer conclusive insights about the potency of vaccines by demonstrating technical relevance with respect to the hypothesized vaccine mode of action. However, animal studies are expensive, time-consuming, labor intensive, and, most importantly, involve the use of animals. Therefore, alternative in vitro potency assays should be explored. Methods: In this study, female BALB/c mice were immunized intramuscularly with various doses of a respiratory syncytial virus (RSV) mRNA vaccine V171 lots at day 0 and day 21. Vaccine-elicited immune responses were determined by ELISA (post-dose 1) and neutralizing assay (post-dose 2). These vaccine lots were also tested in a cell-based relative potency assay in which the ability of each lot to express the RSV F protein in Hep G2 cells was measured against a reference standard. Results: Effective Dose 50s (ED50s) of the vaccine lots were determined with probit models based on dichotomized ELISA or neutralizing titers. Statistical analysis demonstrated that the post-dose 2 neutralizing ED50 correlates with cell-based relative potency (Pearson’s correlation test ln (RP) and ln (ED₅₀): correlation coefficient = −0.82; p-value = 0.047). Conclusions: These data merit the use of a cell-based potency assay to replace the animal study to support V171 vaccine development and to use for DP lot release and stability testing. This study also establishes proof-of-concept of using cell-based potency assays as an alternative to animal immunogenicity studies for mRNA-based vaccines. Full article

Background/Objectives: Antibody–drug conjugates (ADCs) show significant promise in oncology but often suffer from a narrow therapeutic window. Introducing a positive charge on the antibody is one proposed strategy to enhance tumor distribution and efficacy of ADC. Accordingly, this study evaluates the pharmacokinetics (PK) and pharmacology of an ADC developed using a positively charged (+5) version of anti-HER2 antibody trastuzumab conjugated with vc-MMAE linker-payload. Methods: A positively charged variant of trastuzumab was generated and conjugated to vc-MMAE. In vitro cytotoxicity assays were performed in cell lines with varying HER2 expression levels: N87 (high), MCF-7 (low), and MDA-MB-468 (non-expressing). In vivo biodistribution of wild-type (WT) and positively charged (+5) ADC was investigated in plasma, tumors, liver, and spleen. A pilot efficacy and toxicity study was also conducted in N87 tumor-bearing mice. Results: The charged ADC showed differential potency and PK behavior compared to the WT ADC. The charged ADC had similar potency in N87 cells but demonstrated ~20-fold and ~60-fold higher potency in MCF-7 and MDA-MB-468 cells. Plasma exposures of all the analytes were found to be reduced following the administration of charged ADC. However, total antibody exposure was found to increase in liver, spleen, and low antigen-expressing MCF-7 tumors. Tumor payload exposures were found to be significantly reduced for the charged ADCs, but liver and spleen displayed higher peak concentrations and increased tissue-to-plasma exposure ratios for the payload, suggesting preferential distribution of ADC with high drug–antibody ratio (DAR) to liver and spleen. Consistent with reduced tumor exposures, charged ADC showed lower efficacy in N87 tumor-bearing mice. No overt toxicity was observed for the charged ADC. Conclusions: Our findings suggest that while positively charged ADCs may be more potent in vitro, their efficacy in vivo may be compromised due to altered PK behavior. Thus, introducing a positive charge into the antibody framework may not be a viable strategy for improving the therapeutic potential of ADCs. Full article

Dietary and environmental exposure to aflatoxins via contaminated food items can pose major health challenges to both humans and animals. Studies have reported the coexistence of aflatoxins and other environmental toxins. This emphasizes the urgent need for efficient and effective mitigation strategies for aflatoxins. Previous reports from our laboratory have demonstrated the potency of the green-engineered clays (GECs) on ochratoxin and other toxic chemicals. Therefore, this study sought to investigate the binding and detoxification potential of chlorophyll (CMCH and SMCH) and chlorophyllin (CMCHin and SMCHin)-amended montmorillonite clays for aflatoxin B1 (AFB1). In addition to analyzing binding metrics including affinity, capacity, free energy, and enthalpy, the sorption mechanisms of AFB1 onto the surfaces of engineered clays were also investigated. Computational and experimental studies were performed to validate the efficacy and safety of the clays. CMCH showed the highest binding capacity (Qmax) of 0.43 mol/kg compared to the parent clays CM (0.34 mol/kg) and SM (0.32 mol/kg). Interestingly, there were no significant changes in the binding capacity of the clays at pH2 and pH6, suggesting that the clays can bind to AFB1 throughout the gastrointestinal track. In silico investigations employing molecular dynamics simulations also demonstrated that CMCH enhanced AFB1 binding as compared to parent clay and predicted hydrophobic interactions as the main mode of interaction between the AFB1 and CMCH. This was corroborated by the kinetic results which indicated that the interaction was best defined by chemosorption with favorable thermodynamics and Gibbs free energy (∆G) being negative. In vitro experiments in Hep G2 cells showed that clay treatment mitigated AFB1-induced cytotoxicity, with the exception of 0.5% (w/v) SMCH. Finally, the in vivo results validated the protection of all the clays against AFB1-induced toxicities in Hydra vulgaris. This study showed that these clays significantly detoxified AFB1 (86% to 100%) and provided complete protection at levels as low as 0.1%, suggesting that they may be used as AFB1 binders in feed and food. Full article