Pharmaceutics

Current tuberculosis (TB) treatment suffers from drawbacks such as long regimens, high pill burden and side effects leading to non-adherence and poor treatment outcomes. Dissolution-controlled drug depot formulation with high drug loading is a clinically successful drug delivery strategy. Such depots reduce the dosing frequency for treatments requiring daily administration, thereby improving treatment adherence and compliance. However, dissolution-controlled depots for first-line TB drugs have not been demonstrated due to their high solubility and high dose requirements. In this study, we overcame this challenge by developing injectable, extended-release, dissolution-controlled depots of nanocrystalline rifapentine (NCRPT), microcrystalline rifapentine (MCRPT) and amorphous rifapentine microparticles (ARPT) with more than 75% loading. Crystalline formulations resulted in much slower depot dissolution compared to amorphous formulations. A single intramuscular (IM) injection of NCRPT in mice resulted in therapeutic serum concentrations for over a week. We then demonstrated the efficacy of NCRPT in both pre-exposure prophylaxis and therapeutic models of mice TB. NCRPT administered at 60 mg/kg once every two weeks demonstrated excellent efficacy in a mouse model of TB infection. In each case, a [~] 4-log-fold reduction in lung bacterial load compared to untreated mice was observed. These results open new avenues for developing LAI formulations of TB drugs and could improve patient compliance and TB management.

Edible plant derived extracellular vesicles (PDEVs) are emerging as biocompatible, orally deliverable nanocarriers with therapeutic potential; however, their phytometabolite cargo, gastrointestinal stability, and source specific biological functions remain poorly characterized. Here, PDEVs were isolated from four phytochemically distinct plant based foods (black carrot, ginger, garlic, and turmeric), selected for their diverse bioactivity, and characterized by transmission electron microscopy, nanoparticle tracking analysis, and zeta potential. Gastrointestinal stability was evaluated in simulated digestion model. Source specific phytometabolites were profiled by untargeted LC MS MS metabolomics. Functionally validated in ammonia stressed epithelial cells and steatotic hepatocytes. PDEVs exhibited characteristic cup shaped morphology with particle sizes ranging from 60 to 214 nm and zeta potentials of -6.0 to -49.0 mV. PDEVs retained colloidal stability, supporting their suitability for oral delivery. We identified 572 phytometabolites with distinct source specific signatures, including lignin and quercetin in carrot EVs, [6] gingerol and silymarin in ginger EVs, diosgenin in garlic EVs, and curcumin in turmeric EVs. These metabolites found associated to antioxidant, anti inflammatory, epithelial barrier, lipid metabolic, and apoptotic pathways. Functional validation demonstrated carrot EVs significantly enhanced epithelial barrier integrity by increasing claudin (>8-fold, p<0.05), occludin (>2-fold, p<0.05). Ginger EVs restored ZO 1 while suppressing cyclin D1 and MMP9(p<0.05). Garlic and turmeric EVs attenuated inflammatory signaling by reducing STAT3, AKT1, and TNF , whereas turmeric EVs additionally decreased caspase 3 and PTGS2(p<0.01). In steatotic hepatocytes, garlic EVs significantly reduced PNPLA3 (p<0.001) and SREBP 1c while increasing PPAR- (p=0.002). Hence, our results indicate that edible PDEVs are gastrointestinally stable, phytometabolite enriched nanocarriers with distinct source specific functional properties, supporting their potential as orally deliverable nutraceuticals for improving gut liver functions.

BackgroundCannabis derivatives have been reported to possess antinociceptive properties. However, oral delivery is limited by poor bioavailability, stability, and reliability of effects. Previously, we reported an analgesic effect of the aqueous complex {Delta}9-tetrahydrocannabinol/2-hydroxypropyl-{beta}-cyclodextrin (THC/HP{beta}CD) after intracerebroventricular administration in male rats. MethodsHere, we investigated the analgesic effects of the THC/HP{beta}CD complex after oral administration (0.3 and 3 mg/kg) by the tail flick test after both acute and chronic administration (15 days) in female rats. Locomotor activity and anxiety-like behavior were also evaluated at the same experimental conditions. Moreover, dopamine and glutamate content in the periaqueductal gray (PAG), a key area for the antinociceptive action of THC, were also measured by HPLC. ResultsAfter acute administration, the antinociceptive effect of the complex was seen at 3 but not 0.3 mg/kg THC, with a maximum effect observed at 30 min (MPE 60%). Similar results were obtained after 15 days of treatment, although partially reduced (max MPE 20%). Reductions in locomotor activity with the dose of 3 mg/kg and a slight biphasic effect of the two doses on anxiety-like behavior were also observed. Finally, neurochemical analyses revealed that the dose of 3 mg/kg significantly increased dopamine and glutamate content in the PAG, an effect no longer present after 15 days of treatment. ConclusionsOur results highlight the antinociceptive efficacy of the THC/HP{beta}CD complex also after oral administration, notably higher than that previously seen with other carriers, although with some degree of tolerance after chronic administration. From a translational point of view, these results are relevant for the development of THC-based oral formulations with analgesic properties for the treatment of pain in humans. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=193 SRC="FIGDIR/small/742765v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@fff791org.highwire.dtl.DTLVardef@d672f4org.highwire.dtl.DTLVardef@1150b3forg.highwire.dtl.DTLVardef@956403_HPS_FORMAT_FIGEXP M_FIG C_FIG

Background and aims Model systems for bile duct disorders are needed for testing therapeutic interventions. Current models have poor human relevance or limited potential for recreating the complex bile duct microenvironment at scale. We aimed to generate a humanized microphysiological system to model and treat cholangiopathies. Methods An in vitro bile duct was created using 3D printed microfluidic chips containing a collagen-embedded canal seeded with patient-derived primary human cholangiocytes. Barrier permeability and compound transport across the epithelium was measured, and disruption of the barrier was performed with lipopolysaccharide treatment. The duct was challenged with the known hepatotoxicant Chlorpromazine. Biliatresone was used to model biliary-atresia and treated using N-acetyl-L-cysteine. Results Cholangiocytes in the bile duct chip established a tight, polarized epithelial barrier. Verapamil and Linerixibat inhibited transport of rhodamine 123 and cholyl-lys-fluorescein respectively with 66 % (p = 0.0004) and 57 % (p = 0.03) reduction. 10 g/mL lipopolysaccharide led to a loss of epithelial barrier integrity, measured by an increase of over 1000 % in leakage of both 3 kDa (p = 0.0002) and 10 kDa dextran (p = 0.0001) along with upregulation of cytokines. Chlorpromazine displayed dose-dependent toxicity with EC50 values of 84, 140 and 96 M for three patient lines. Biliatresone induced a dose-dependent abnormal phenotype with loss of viability. The induced phenotype could be treated with N-acetyl-L-cysteine, improving viability from 23 % to 59 % (p < 0.0001) with treatment of 2 g/mL Biliatresone. Conclusions Our novel platform allows complex studies of bile duct biology, testing of off-target effects from drugs and treatment of a disease phenotype.

Alzheimers disease (AD) is a progressive neurodegenerative disorder characterized by the accumulation of amyloid-{beta} (A{beta}) plaques, neurodegeneration, and cognitive decline. {beta}-Site amyloid precursor protein cleaving enzyme 1 (BACE1) catalyzes the rate-limiting step in A{beta} production and remains a therapeutic target for AD. However, effective delivery of RNA therapeutics to the brain remains challenging due to the blood-brain barrier (BBB). Here, we evaluated the feasibility of using clinical-grade mesenchymal stem cell-derived extracellular vesicles (EVs) as systemic carriers for Bace1-targeting small interfering RNA (siRNA) in the 5xFAD mouse model of AD. Engineered EVs crossed the BBB and delivered siRNA cargo to the brain, with uptake observed in both neurons and astrocytes. Systemic therapy with EVs engineered to encapsulate Bace1 siRNA resulted in reduced brain Bace1 protein levels and a decrease in amyloid plaque burden compared with control EVs carrying scrambled siRNA. The reduction was most pronounced in larger, high-intensity plaques, suggesting that Bace1 suppression may preferentially limit plaque growth and maturation. Repeated systemic administration was well tolerated, with no evidence of treatment-associated toxicity. These findings establish a proof-of-concept feasibility for EV-mediated delivery of Bace1-targeting siRNA to the brain and support further development of engineered EVs as a therapeutic platform for neurodegenerative diseases. Future studies incorporating behavioral, molecular, and mechanistic analyses will be required to determine the extent to which Bace1 suppression delivered through EVs can modify disease progression and improve functional outcomes in AD.

Chronic pain is frequently accompanied by negative affect and motivational deficits due to dysregulated mesocorticolimbic dopamine and kappa opioid receptor (KOR) signalling. Although intracranial KOR antagonism prevents pain-induced negative affect in preclinical models, systemic KOR antagonists can produce adverse off-target effects in the periphery, thereby limiting its clinical utility. Consistent with this, we found that systemic administration of KOR antagonist norbinaltorphimine (NorBNI), exacerbated motivational deficits in rats with persistent inflammatory pain. We hypothesized that maximizing central and minimizing peripheral KOR antagonism could overcome these limitations. To test this, we engineered an intranasal liposomal NorBNI formulation incorporated into an in-situ forming mucoadhesive hydrogel to enable selective nose-to-brain delivery (Nor-BNILV-HG). We characterized its physicochemical properties and functional efficacy in rats with inflammatory pain produced by Complete Freund's Adjuvant (CFA). NorBNI-loaded liposomes exhibited high drug entrapment efficiency, nanometric size, and suitable surface charge for intranasal administration. The selected thermosensitive hydrogel demonstrated appropriate gelation properties and sustained drug release. Intranasal administration of NorBNI-LV-HG produced negligible systemic NorBNI levels compared with intraperitoneal delivery. In vivo microdialysis showed that NorBNI-LV-HG prevented KOR agonist-induced reductions in nucleus accumbens (NAc) dopamine release, confirming functional central KOR blockade. Behaviourally, intranasal NorBNI-LV-HG attenuated pain-induced impairments in sucrose motivation. Importantly, unlike systemic NorBNI, repeated intranasal NorBNI-LV-HG did not alter mechanical nociceptive thresholds in pain-naive animals, suggesting this strategy mitigates unwanted peripheral nociceptive effects. Together, these findings demonstrate that intranasal NorBNI-LV-HG achieves functional brain KOR antagonism while minimizing systemic exposure and off-target effects. Selective nose-to-brain delivery of KOR antagonists therefore represents a promising therapeutic strategy to prevent and potentially reverse the affective and motivational consequences of pain and may overcome key translational barriers associated with systemic KOR treatments.

Nisin is a promising antimicrobial peptide widely used in food preservation due to its efficacy against Gram-positive spoilage and pathogenic bacteria. Although Nisin is increasingly applied in the food sector, the biopeptide suffers from instability within food matrixes and can rapidly lose its antimicrobial potential following interaction with food biomolecules. Thus, it is necessary to investigate approaches that can be employed to extend the stability and activity of Nisin. Hence, the aim of this study was to develop and characterise a chitosan-alginate polyelectrolyte microencapsulation system capable of enhancing Nisin stability while retaining antimicrobial activity. The microencapsulation of Nisin was achieved by pre-gelation of alginate using calcium chloride and subsequent direct electrostatic interaction between cationic Nisin and chitosan with pre-gelled anionic alginate at pH 5.0. Following microcapsule formation, physicochemical and structural characterisation was performed using Zeta potential determination and measurement of the polydispersity index (PDI) via dynamic light scattering. SEM micrographs were used to confirm morphology, while Fourier-transform infrared (FTIR) spectroscopy and high-performance liquid chromatography (HPLC) were utilised to assess chemical integrity and functional group preservation of encapsulated Nisin. Following this, stable microcapsules with diameters ranging from 150-200 nm and smooth surface morphology were obtained. Microcapsule formation was strongly influenced by formulation parameters, particularly pH, calcium ion concentration, and chitosan content, with deviations from optimal acidic conditions (< pH 5.0) resulting in aggregation, increased polydispersity, and reduced encapsulation efficiency. The microcapsules were monodispersed (PDI {approx} 0.30) and electrostatically stable, exhibiting a Zeta potential of approximately +36 mV. These microcapsules remained stable over a prolonged storage period of 21 days under refrigerated conditions while retaining antimicrobial activity against Bacillus cereus. Encapsulation efficiency reached approximately 65%, confirming effective retention of Nisin within the polymer matrix. Overall, the findings demonstrate that chitosan-alginate ionic gelation is a non-denaturing and effective encapsulation strategy for extending the functional stability of Nisin. These microcapsules show strong potential as natural antimicrobial delivery systems for food and beverage applications, particularly in acidic food matrices, with implications for improved food safety and shelf-life extension.

Valproic acid/sodium valproate (VPA) is a widely prescribed anticonvulsant and has also been used against certain tumor cells. It is a potent modulator of gene expression. Its ability to induce apoptosis has been well documented in HeLa cells. However, another form of cell death - mitotic catastrophe - has not yet been explored in VPA-treated HeLa cells. Here, we investigated the effects of VPA treatment on mitotic catastrophe characteristics, including morphological features and their frequencies, fluorescence intensity signals of caspase-2 and p53, and the expression and abundance of DNMT1 and DNMT3B. An increased frequency of mitotic catastrophe was observed not only morphologically, but also through enhanced induction of caspase-2, involvement of p53, at least under more drastic VPA treatment, but without a decrease in DNMT1 or DNMT3B levels. Additionally, enhancement of mitotic catastrophe coincided with a reduction in mitotic chromosome abnormalities. Increased DNMT3B expression following VPA action, may be favored by previously reported chromatin decondensation induced by this drug. Enhanced CpG methylation of specific DNA sites could thus be promoted. In conclusion, VPA was shown to trigger metabolic pathways linked to different forms of cell death in HeLa cells, supporting its oncosuppressive potential.

mRNA-based therapeutics have emerged as a transformative class of medicines, yet their translation beyond infectious disease vaccines remains challenged by the absence of an integrated pharmacological framework accounting for the tri-component nature of these therapies - the lipid nanoparticle, the mRNA, and the expressed protein. Here, we present a modular, multiscale computational platform integrating two complementary mechanistic models covering the full pharmacological cascade of mRNA-based immunotherapies. The first is a Quantitative Systems Pharmacology (QSP) model describing the immunological response to mRNA vaccines, from antigen expression in antigen-presenting cells through B cell activation and circulating antibody production. The second is a Physiologically Based Pharmacokinetic (PBPK) model tracking whole-body disposition of mRNA-encoded therapeutic antibodies, incorporating a molecular layer resolving LNP uptake, endosomal mRNA escape, and intracellular translation. Both models are informed by a machine learning pipeline that maps IVT-mRNA nucleotide sequences directly onto kinetic parameters, enabling product-specific model simulations. We propose this platform as a step toward the quantitative pharmacological framework that mRNA therapeutics currently lack, and as a practical tool for model-informed design and development of this therapeutic class.

Several potentially potent anticancer drugs have been identified by in vitro evaluation, such as Andrographolide. These compounds show strong anticancer activity in vitro, but struggle to reach effective concentrations in the bloodstream when taken orally because they dissolve poorly in water or break down rapidly in the body. Bioenhancers, which are compounds that have potential to improve drug stability in the body, offer an alternative solution to overcome this limitation. Naringin and Quercetin have been identified as candidate bioenhancers, and have been hypothesized to potentially slow rapid first pass metabolism of poorly bioavailable drugs. Our work focuses on testing Naringin and Quercetin because they are flavonoids with therapeutic potential, due to their anti-inflammatory and antioxidant properties. Data from the hepatic microsomal assays performed on Naringin and Quercetin suggest moderate to proficient periods of stability in the body, with Naringin having 91.86% remaining, while Quercetin had 74.84% remaining. When administered alongside Andrographolide, a drug known to rapidly degrade in the body, Naringin raised its metabolic stability from 38.93% to 80.77% and on the other hand, Quercetin raised Andrographolide metabolic stability from 38.93% to 86.70%. In addition, plasma protein binding assays show the percentage of compounds available at the target site where Naringin was observed to be 49.32% bound and Quercetin found to be 50.14% bound, implying 50.68% of Naringin, and 49.86% of Quercetin available at the target site, respectively. This preliminary study explores whether Quercetin and Naringin could act as bioenhancers by remaining stable and available in plasma and by slowing the metabolism of poorly bioavailable drugs such as Andrographolide.

The outer blood-retinal barrier (OBRB) is the primary interface through which systemically circulating drugs reach the retina. A tool that measures delivery across this barrier would support the development of targeted therapies as alternatives to repeated intravitreal injection, and the screening of drugs that reach the retina as an off-target toxicity. Such a tool should deliver drugs fluidically through a vascular compartment, measure transport across the retinal pigment epithelium (RPE), and display disease phenotypes relevant to efficacy. Here we adapt the SiM platform, which places epithelium and endothelium in direct juxtaposition across a permeable, optically transparent silicon nitride nanomembrane. ARPE-19 and human umbilical vein endothelial cells (HUVECs) were used as development cell sources. ARPE-19 monocultures reached a transepithelial electrical resistance of 68 {+/-} 26 {Omega} cm2 by 28 days, and ARPE-19 + HUVEC co-cultures reached a small-molecule permeability of 6.34 {+/-} 1.3 x 10-4 cm min-1 within 14 days, a state reported elsewhere only after longer culture. The barriers developed an intervening basement membrane. Drugs perfused through the basal vascular channel crossed into an open apical well, where sampling and mass spectrometry showed transport correlating with lipophilicity, as reported in vivo. The device also displayed two clinically relevant phenotypes. Digoxin at a clinically toxic concentration reduced viability in the co-barrier by about half and doubled permeability. In a vascularized configuration, VEGF drove endothelial invasion of the RPE layer, as seen in neovascular AMD. The SiM-OBRB therefore satisfies basic design criteria for measurement of drug bioavailability, toxicity, and efficacy.

Background: Oral mucositis is a clinically significant, potentially severe side effect of systemic chemotherapy in children with cancer. Understanding genetic predisposition to this side effect may assist in development of stratified prophylactic and treatment strategies. However, existing literature primarily focuses on children with haematological malignancies. Methods: We performed a candidate gene study of 101 children with solid tumours enrolled in the MARVEL-PIC study at the Royal Children's Hospital, Melbourne. Clinical data were extracted from the electronic medical record, with NCI-CTCAE v6.0 grade >2 oral mucositis defined as the primary outcome. Genetic variants previously associated with oral mucositis were analysed under an additive genetic model to identify significant associations. Exploratory gene-drug interactions were identified based on chemotherapy exposure. Results: 29 patients (28.7%) developed grade >2 oral mucositis. MTHFR A1298C (rs1801131) was associated with lower odds of grade >2 oral mucositis, lower peak mucositis grade, and lower odds of opioid use for oral mucositis. 25 exploratory gene-drug interaction signals were identified, including miR-1206 rs2114358 with methotrexate exposure and ABCB1 rs1045642 with anthracycline exposure. Conclusions: MTHFR A1298C (rs1801131) demonstrated a protective effect against chemotherapy-induced oral mucositis in our cohort of children with solid tumours. Larger, ancestry-informed studies are required to validate our findings.

Background Therapy resistance in pancreatic ductal adenocarcinoma (PDAC) is facilitated by the desmoplastic tumor microenvironment (TME) orchestrated by cancer associated fibroblasts (CAFs). Upon activation, pancreatic stellate cells (PSCs) deplete their intracellular retinoic acid (RA)-containing lipid droplets and secrete stromal remodeling proteins like pentraxin 3 (PTX3), leading to cancer progression. Preclinical evidence indicates that all-trans RA (ATRA) reprograms the TME, while circulating vitamin A and PTX3 were proposed as biomarkers for ATRA response in PDAC. To support further clinical development of RA-based therapies in PDAC, we studied the effects of ATRA on CAFs and patient-derived organoids (PDO) and evaluated the clinical relevance of these biomarkers in PDAC patients. Methods We employed viability assays in human and murine organoid mono- and co-culture models to explore the efficacy of adding ATRA to gemcitabine (GEM). In parallel, we conducted a prospective observational study and assessed vitamin A and PTX3 as response biomarkers in peripheral blood collected before first treatment and at cycles 2 and 4 of treatment among patients with advanced PDAC receiving GEM with or without nab-paclitaxel (NAB-P). Results In PDO monocultures, a significant additive effect of ATRA in combination with GEM on viability was observed in 5 (41%) of 12 PDOs and this effect was numerically more frequent in organoids from patients who had clinically responded to GEM. In human and murine 3D PDO+PSC/CAF co-cultures, ATRA demonstrated an additional direct impact on the viability of stromal cells. Clinically, among 18 patients with PDAC treated with GEM+/-NAB-P, patients with no treatment response (n=10) showed an increase in PTX3 and concomitant decrease in vitamin A levels under therapy. In contrast, response was associated with stable vitamin A levels and a trend towards lower PTX3 levels during chemotherapy. Conclusions Our preclinical data support the repurposing of ATRA, an agent with favorable toxicity profile, to potentiate the efficacy of GEM in PDAC treatment. Complementing these results, our clinical data suggest vitamin A and PTX3 as promising response biomarkers in PDAC treatment, not restricted to ATRA containing regimens.

Adipose-derived mesenchymal stem cells (gAD-MSCs) are promising candidates for veterinary regenerative medicine, yet the characterization of gAD-MSCs from locally adapted Indonesian goat breeds remains limited. This study aimed to isolate and characterize gAD-MSCs from Peranakan Ettawa (PE) goats using tissue explant culture. Subcutaneous adipose tissue was collected from the base of the tail of healthy PE goats (n=3). Primary cell outgrowth from explants was observed by Day 5, displaying characteristic fibroblast-like, spindle-shaped morphology and strong plastic adherence. Serial passaging to Passage 3 (P3) yielded a morphologically stable, homogeneous cell population. Assessment of cellular metabolic activity via the resazurin assay demonstrated sustained cell viability and a statistically significant increase in metabolic activity between Day 3 and Day 5 (p < 0.05). Furthermore, functional clonogenic capacity, evaluated using the colony-forming unit (CFU) assay, showed continuous temporal expansion of colonies over 14 days, yielding an average of 52.0 + - 4.1 colonies per dish. These findings confirm that expanded gAD-MSCs P3from PE goats maintain characteristic mesenchymal morphology, sustained metabolic activity, and clonogenic capacity. This work provides a baseline cellular profile of PE goat gAD-MSCs, supporting their potential use in veterinary regenerative medicine and tissue engineering.

Background: Prostate cancer enzalutamide treatment is approved at a standard dose of 160 mg daily. Concerns for real-world patients -- older and more fragile than those enrolled in clinical trials -- have prompted consideration of initiating treatment with lower doses, but the long-term efficacy of this approach remains unknown. We evaluate the long-term survival and longevity in patients treated with standard versus upfront low-dose enzalutamide. Methods: Retrospective analysis of 151 patients treated with enzalutamide (102 receiving 160 mg; 49 receiving [&le;]80 mg) between 2014--2021 at the Centre Hospitalier Universitaire de Martinique, with complete follow-up through end of life (98.7% completeness of follow-up). Primary outcomes were overall survival (OS), progression-free survival (PFS), and longevity (attained age). Results: Doses [&le;]80 mg were associated with longer median OS (36.3 vs. 20.7 months), improved restricted mean OS (difference of 0.7 years, p=0.05), and enhanced longevity (median 82.5 vs. 78.3 years, p=0.004). PSA response rate at 12 weeks was higher with lower-dose (71.4% vs. 48.8%, p=0.016). In multivariable models adjusted for prognostic factors, [&le;]40 mg compared with 160 mg was non-inferior regarding OS (HR=0.61, 95% CI 0.36--1.06), superior regarding PFS (HR=0.59, 95% CI 0.35--0.99), and superior regarding longevity (HR=0.48, 95% CI 0.28--0.84). Bone metastasis, poor performance status, PSA response, time to PSA nadir, and disease duration were independent predictors of outcomes. A post-hoc analysis revealed a strong association between dose and physician-prescribing profiles, ranging from "endorse-lowest-dose" to "never-deviate-from-full-dose". Conclusions: Lower doses of enzalutamide were non-inferior to full-dose. Dose-adapted strategies warrant further investigation.

Vanadium (V) is a potential insulinomimetic that can modulate carbohydrate metabolism, but its biological effects are sensitive to chemical form, concentration, and sex. Chelation of vanadium with organic ligands, in particular citrate, allows to increase its bioavailability and optimize pharmacokinetic properties. The aim of the study was to evaluate tissue-, dose-, and sex-dependent changes in physiological parameters and activity of the key glycolytic enzyme - lactate dehydrogenase (LDH) - under the influence of vanadium citrate. The study was conducted on 6-week-old Wistar rats of both sexes. The animals received vanadium citrate orally for 36-38 days at doses of 3, 12.5, and 50 g VCit/kg body weight. LDH activity in skeletal muscle, liver, kidney, and pancreas was investigated. No pronounced toxic effect on physiological parameters was detected: body weight dynamics corresponded to age norms, no behavioral changes were observed. LDH activity demonstrated pronounced sexual dimorphism and depended on the dose received. It was established that the optimal dose, which provides a modulating effect without signs of metabolic stress, for females is 12.5 g VCit/kg, while for males - 3 g VCit/kg. The most significant changes in LDH activity were recorded in the pancreas at a dose of 50 g V/kg, where the indicators decreased from 0.81 to 0.31 mol/(min x mg protein) in females and from 1.02 to 0.28 mol/(min x mg protein) in males. The effect of vanadium citrate on carbohydrate metabolism, as well as its dose-, tissue- and sex-specific nature, is likely determined by a dual action: the insulin-like effect of vanadium (redirecting pyruvate to oxidation) and the allosteric inhibition of glycolysis by the citrate ligand (substrate limitation for LDH). The obtained results emphasize the importance of considering sex and dose in the research and development of metabolically active compounds.

Background: Natural compounds from avocado fruit (avocadene, avocadyne, and acetate derivatives) exhibit notable biological activity, although their molecular mechanisms remain unclear. The avocado-derived lipids exert potent nematocidal activity against several parasitic nematodes. In Caenorhabditis elegans (C. elegans), those compounds caused concentration-dependent toxicity, impairing first stage larval growth, egg hatching, and adult survival. Treated worms exhibited impaired mitochondrial respiration, reduced oxygen consumption, and elevated reactive oxygen species. These effects suggest that avocado lipids disrupt mitochondrial function and lipid metabolism, in part by inhibiting acetyl-CoA carboxylase, the rate-limiting enzyme of fatty acid biosynthesis. Methods: We investigated the effects of these compounds on the lipid profile of C. elegans and their association with endogenous lipid pools using NMR spectroscopy, click-chemistry-based fluorescence labeling, thin-layer chromatography (TLC), and microscopy. Results: Lipidomic analysis of stage 4 larvae (L4) and embryos treated with avocadene acetate revealed increased lipid NMR signals. Fluorescence-assisted TLC and NMR further suggested that avocadyne preferentially associates with triglyceride-linked fatty acids, particularly monounsaturated and flexible polyunsaturated chains, without detectable interactions with conformationally-constrained polyunsaturated species. Fluorescent avocadyne derivatives were efficiently internalized with distinct localization patterns in L4 larvae and embryonic cells. Conclusions: Overall, the lipid homeostasis remodeling of L4 larvae in response to lipotoxic shock was associated with phospholipid increase and remarkable lipid droplets onset, whereas embryos showed accumulation of lipids in enlarged droplets and developmental arrest.

Background The number of novel cancer therapies approved for use in England by the National Institute for Health and Care Excellence is increasing. Monitoring the adoption of new therapies is important to assess equity of access and evaluate real-world prescribing practices. OpenPrescribing Hospitals has recently been launched to facilitate analysis of open secondary care medicines data in England. Using this platform, we set out to describe the use of cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors, including the frequency of dose reductions, within National Health Service (NHS) hospitals in England between January 2019 and December 2024. Methods The monthly proportion of each CDK4/6 inhibitor relative to total CDK4/6 inhibitor use was calculated at hospital level. Regional variation was assessed across Cancer Alliances by comparing the proportions of each CDK4/6 inhibitor used within each alliance in 2021 and 2024. Use of lower strength palbociclib and abemaciclib was used as a proxy for dose reductions. Findings There was more than a 3-fold increase in the use of CDK4/6 inhibitors between 2019 and 2024. In 2019, 78.6%, 11.9% and 9.5% of CDK4/6 inhibitors used were palbociclib, abemaciclib and ribociclib, compared with 40.2%, 41.2% and 18.6% in 2024. There was variation in the relative percentage change in use of each agent by Cancer Alliance. Use of lower strengths was common for both palbociclib (60%) and abemaciclib (63%). Interpretation Changes in usage appeared responsive to publication of key evidence and regulatory milestones. There was a higher apparent frequency of dose reductions than reported in clinical trials. OpenPrescribing Hospitals is an accessible, publicly available tool for understanding uptake and use of medicines in NHS hospitals in England.

Traumatic optic neuropathy (TON) occurs due to direct or indirect injury to the optic nerve and is a significant cause of visual disability. So far, there is no effective treatment. The lack of understanding of the cellular mechanisms by which trauma induces inflammation and damage in retinal neurons is a critical knowledge gap in developing effective therapies. We have studied the role of the arginase 1 (A1) enzyme in this pathology. We have found previously that treatment with a long-acting form of human recombinant A1, pegylated A1 (PEG-A1) after optic nerve crush limits activation of retinal microglia and macrophages (M{Phi}) and reduces inflammation, thereby decreasing injury and protecting visual function. Here we report on studies designed to demonstrate the therapeutic efficacy of PEG-A1 in mouse models of direct and indirect TON and to elucidate the underlying mechanisms. We used ONC to model direct TON and sonication-induced trauma to the supraorbital rim to model indirect TON (SI-TON). At different times after injury, mice were treated with PEG-A1 which was delivered systemically by i.p. injection or locally by intravitreal injection. In order to assess the role of A1-induced activation of the ornithine/polyamine pathway in the protective effects of PEG-A1, some mice were treated with the ornithine decarboxylase (ODC) inhibitor, difluoromethylornithine (DFMO) immediately after the PEG-A1 treatment. Retinal function was determined by OptoMotry and electroretinography. Retinal injury and microglia/M{Phi} activation were assessed by immunofluorescence imaging. Expression of inflammatory cytokines was determined by Western blotting and quantitative RT PCR. Liquid chromatography mass spectrometry was used to analyze changes in arginase/ODC pathway metabolites. Results showed that PEG-A1 treatment improved neuronal survival and visual function whether delivered systemically or intravitreally. This neuroprotection was associated with decreased microglia/M{Phi} activation, decreased inflammatory cytokine expression, and increased formation of L-ornithine and putrescine. Furthermore, DFMO treatment blocked these effects, indicating that PEG-A1 limits retinal injury and preserves vision after ocular injury by activating ODC. ODC processes the arginase product L-ornithine to form polyamines which are known to promote reparative functions. Thus, PEG-A1 therapy offers a new strategy to limit trauma-induced vision loss and promote repair after TON.

Graphene oxide (GO) has primarily been investigated as a carrier for intracellular delivery of therapeutic molecules. In previous work, we identified a cell type-dependent interaction pattern in which GO remained predominantly associated with the plasma membrane of cancer cells but was internalised by non-cancerous epithelial cells. Here, we explored whether plasma membrane-associated GO can be used as a platform to present bioactive ligands and influence cell-surface receptor signalling in cancer cells. To test this hypothesis, we targeted integrin receptors at the plasma membrane in glioblastoma cell models using an RGD-containing peptide non-covalently complexed with GO. We assessed GO-peptide interactions, cellular interactions/uptake, motility, and focal adhesion signalling readouts. Peptide association was quantified using a 2,4,6-trinitrobenzene sulfonic acid (TNBSA) assay, and GO was characterised by atomic force microscopy, X-ray photoelectron spectroscopy, X-ray diffraction, and colloidal measurements. Immediately after complexation, ~70% of RGD was associated with GO. Peptide association increased the nitrogen signal and shifted the principal GO XRD peak while retaining nanosheet morphology. Biological responses were examined in U87 and U251 glioblastoma cells with different integrin-positive fractions, and in non-cancerous BEAS-2B bronchial epithelial cells. Confocal microscopy showed that GO and GO:RGD remained predominantly localised on the plasma membrane in U87 and U251 cells, whereas greater intracellular localisation was observed in BEAS-2B cells. Importantly, GO:RGD significantly reduced key indicators of cell motility: cell velocity in U87 and U251 cells, with trajectory and mean-square-displacement analyses supporting restricted cellular movement. Free RGD had no significant effect, while GO alone produced a smaller reduction in motility only in U251 cells. No treatment significantly altered BEAS-2B motility. Flow cytometry also showed a reduced pFAK-associated signal in GO:RGD-treated U87 cells. These findings establish a proof of concept that the cell-line-dependent plasma membrane localisation of GO can be exploited as a membrane-associated nano-bio interface for cell-surface-active ligands, opening the way for the development of GO-based platforms that modulate receptor-mediated signalling and cell behaviour.