Frontiers in Pharmacology

IntroductionSystemic Lupus Erythematosus (SLE) is a chronic autoimmune disease characterized by loss of immune tolerance, autoantibody production, and multi-organ damage. Current therapies, including glucocorticoids and CAR-T/CAR-NK cell therapies, are limited by adverse effects, high cost, and safety concerns. ObjectivesTo develop engineered NK-92 cell-derived extracellular vesicles displaying CD19 single-chain variable fragment (V-CD19-Exo) and evaluate their therapeutic efficacy in an MRL/lpr mouse model of SLE. MethodsThe CD19scFv-LAMP-2B fusion construct was stably expressed in NK-92 cells via lentiviral transduction. Extracellular vesicles were isolated by differential centrifugation and characterized by NTA, TEM, and Western Blot. In vivo efficacy was assessed in MRL/lpr mice through B cell depletion analysis, renal function monitoring, cytokine profiling, autoantibody detection, and survival observation. ResultsV-CD19-Exo significantly reduced splenic CD19{square}CD20{square} B cells from 10.53% to 1.51% (p < 0.0001). Treatment attenuated proteinuria, ameliorated lupus nephritis pathology, reversed splenomegaly, and downregulated serum IgE, IL-17A, IFN-{gamma}, anti-dsDNA, and ANA levels. Notably, V-CD19-Exo improved survival to approximately 80% compared to 25% in untreated controls. ConclusionEngineered NK-92 cell-derived extracellular vesicles represent a novel, safe, and effective cell-free therapeutic strategy for SLE, offering advantages over conventional cell therapies including lower immunogenicity, scalable production, and no requirement for lymphodepletion.

Population pharmacokinetic (popPK) models are commonly developed using ordinary differential equations (ODEs) to describe deterministic concentration-time profiles, with unexplained variability typically attributed to interindividual variability or residual error. When model misspecification is present, system-level deviations may be absorbed into these conventional variability terms, making the source and magnitude of model inadequacy difficult to assess quantitatively. Stochastic differential equations (SDEs) provide an alternative framework by introducing an explicit system-noise component into the structural model, allowing model-data mismatch to be evaluated more directly. However, historical implementation of SDE-based models in NONMEM has been technically challenging. The availability of the Fortran plug-in subroutine SDE.f90 substantially lowers this barrier and enables more practical implementation of SDE-based models in NONMEM. In this work, SDE-based nonlinear mixed-effects models were evaluated as a quantitative diagnostic framework for probing popPK model misspecification. The SDE.f90 implementation was first verified using simulated one-compartment intravenous bolus datasets with stochastic process noise. Additional simulation-estimation scenarios were then conducted under intentionally misspecified structural or stochastic assumptions, including time-varying elimination, compartmental misspecification, and residual error misspecification. Across these scenarios, the estimated system-noise parameter was generally sensitive to misspecification, with larger values usually associated with greater structural or stochastic mismatch. SDE-based modeling also helped partially separate system-level variability from residual variability and, in selected settings, supported localization of misspecification to specific model components, thereby helping guide model refinement. Overall, SDE-based popPK modeling is a useful addition to the pharmacometric diagnostic toolbox, with system-noise estimates best interpreted together with structural model evaluation, residual diagnostics, parameter behavior, and pharmacologic plausibility.

TRPA1 is a non-selective cation channel that plays a crucial role in several pain and inflammatory conditions. Agents reducing membrane cholesterol decrease TRPA1 activation, but it remains unclear how cholesterol-lowering medications affect TRPA1 function. Given that TRPA1 is activated by a wide variety of chemicals, we explored whether statins have acute effects on this channel. We found that five commonly used statins activate human and mouse TRPA1 in a reversible and concentration-dependent manner. The effective concentrations were above the micromolar range, in the order: simvastatin {approx} lovastatin < fluvastatin < atorvastatin < pravastatin. Statin-induced activation was not correlated to changes in membrane order, nor mediated by N-terminal cysteine residues contributing to electrophilic compound agonism. Molecular docking calculations and the functional characterization of single-point mutants revealed two separate putative binding sites, one situated close to the kink of transmembrane segment 5 (TM5) and the other at the interface between TM4 and TM5. The mTRPA1 inhibitor A-967079 largely abrogated the response to the electrophilic agonist allyl isothiocyanate, but had weaker and varied effects across different statins and menthol. Mutation T877L strongly altered the effect of A-967079, also in an agonist-dependent manner, suggesting competitive binding between this antagonist and the non-electrophilic agonists. The identification of two distinct agonist binding sites may help explaining how TRPA1 is able to respond to a large variety of non-electrophilic compounds, while the finding of competitive interactions at one of these sites may help guide the development of agonist-specific antagonists of therapeutic relevance.

BackgroundChemokine receptor type 5 (CCR5) is expressed on hepatic stellate cells (HSCs), which, together with fibroblasts, are major producers of extracellular matrix during liver fibrosis. Leronlimab is a humanized IgG4{kappa} monoclonal antibody that binds to CCR5. The objective of the present study was to evaluate the antifibrotic effects of leronlimab in three independent preclinical studies using two mouse models of liver fibrosis. MethodsIn STAM (Stelic Animal Model) model 1, leronlimab was administered at doses of 5 or 10 mg/kg/week for 3 weeks. STAM model 2 was conducted as a confirmatory study to validate the antifibrotic effect observed with the 10 mg/kg/week dose in STAM model 1. In a third study, a carbon tetrachloride (CCl)-induced liver fibrosis mouse model was used to evaluate leronlimab administered at 10 mg/kg/week for 3 weeks. An isotype-matched control antibody was included in all studies for comparison. Evaluations included liver enzymes and histological assessment of liver fibrosis. ResultsIn STAM model 1, leronlimab at 10 mg/kg/week significantly reduced fibrosis area compared with the isotype control (p = 0.0005). These findings were confirmed in STAM model 2 (p < 0.0001). Consistent antifibrotic effects were also observed in the CCl-induced liver fibrosis model (p = 0.0006). ConclusionsCollectively, these preclinical results demonstrate that CCR5 blockade by leronlimab is associated with a significant reduction of established liver fibrosis in multiple mouse models and support further evaluation of leronlimab as a potential therapeutic option, either as monotherapy or in combination regimens, for chronic liver diseases with fibrosis.

BackgroundCardiac hypertrophy is a key pathological process in hypertensive heart failure, yet current antihypertensive therapies do not directly target it. Red yeast rice (RYR), rich in monacolin K {beta}-hydroxy acid (MKA), is known for lipid-lowering effects, but its potential to ameliorate cardiac hypertrophy is unreported. PurposeTo investigate the effects of RYR-derived MKA on cardiac hypertrophy in spontaneously hypertensive rats (SHR) and elucidate its molecular mechanisms. MethodsSpontaneously hypertensive rats (SHR) were treated with 0.6% red yeast rice for 8 weeks to assess its effects on blood pressure, cardiac function (echocardiography), cardiac hypertrophy and fibrosis (histopathology), and multi-organ toxicity (histopathology). A multigenerational study was conducted to evaluate protective effects in offspring. Network pharmacology and transcriptomic analysis were integrated to predict molecular targets, which were subsequently validated by molecular docking and experiments. ResultsEight-week RYR treatment significantly reduced blood pressure, inhibited cardiac hypertrophy and fibrosis, and improved cardiac function without gender differences. No pulmonary, hepatic, or renal toxicity was observed. Offspring from treated parents exhibited further reduced hypertrophy upon continued treatment. Mechanistically, MKA bound ERK1/2 with high affinity, inhibiting its phosphorylation and downstream c-Fos expression, thereby downregulating hypertrophy markers. ConclusionRed yeast rice improves hypertensive cardiac hypertrophy via MKA-mediated inhibition of the ERK1/2/c-Fos pathway. Its multi-organ safety and transgenerational effects offer a novel dual-therapy strategy for hypertension and cardiac hypertrophy. Graphic abstract O_FIG O_LINKSMALLFIG WIDTH=139 HEIGHT=200 SRC="FIGDIR/small/710945v1_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@cbb85org.highwire.dtl.DTLVardef@1eb399dorg.highwire.dtl.DTLVardef@13746dorg.highwire.dtl.DTLVardef@140c512_HPS_FORMAT_FIGEXP M_FIG C_FIG

Background and PurposeCardiotoxicity is a major cause for drug failure throughout the drug development process, with particular concern for action potential prolongation and arrhythmia. Hence, such liabilities are heavily considered during the early phases of drug design to pre vent dangerous compounds from progressing. New approach methodologies (NAMs) that efficiently examine this risk early in the discovery pipeline should help streamline drug development programs. We developed a cardiac NAM, a 384-well open bath platform consisting of cardiac tissue derived from human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes, enabling high-throughput drug screening while maintaining the structural and functional complexity of 3D cardiac micromuscles. MethodsWe dramatically increased throughput without compromising physiological relevance provided by the 3D micromuscle structure. Our 384-well open bath high-throughput platform allowed evaluation of multiple compounds at a time, enabling us to study the CiPA (comprehensive in vitro proarrhythmia assay) drug panel for proarrhythmia screening. We obtained phenotypic fingerprints of all 28 compounds (9 low, 11 intermediate, and 8 high arrhythmia risk; https://cipaproject.org) in dose-escalation studies around their respective clinical concentrations. The analysis was augmented with an in silico pipeline that used phenotypic biomarkers to invert data into a mathematical model of cellular currents to infer which ion channels were affected upon drug exposure, and then trained a ML model to predict channel block. Results and ConclusionsWe found accurate detection of arrhythmic potential for most of the compounds, and the in silico model inversions were consistent with published values of compound channel block. All the high risk compounds showed action potential duration (APD) prolongation coupled with either action potential abnormalities, early afterdepolarizations (EADs), or beat cessation. For the intermediate risk group, 9 out of 11 compounds caused APD prolongation alone or in combination with EADs while 2 others showed either beat cessation or beat rate change. Augmentation of APD analysis with detailed biophysical modeling and ML tools provided meaningful insight into the mechanisms involved in APD changes. Overall, our cardiac NAM allowed for fast and relevant screening for mechanistic understanding of APD prolongation and proarrhythmic activity, at massively increased throughput compared to other 3D micromuscle models. SummaryCardiotoxicity testing is critical in drug development to prevent arrhythmogenic side effects. Current stringent regulations have greatly reduced market withdrawals; however, these strict evaluations often lead to costly late-stage failures and loss of promising candidates as false positives. We developed a cardiac new approach methodology (NAM), a 384-well open bath cardiac micromuscle platform created from hiPSC-derived cardiomyocytes, enabling high-throughput drug screening while maintaining the structural and functional complexity of 3D cardiac micromuscles. Using the comprehensive in vitro proarrhythmia assay (CiPA) drug panel, we validated the system to accurately detect proarrhythmic potential. Our assay provided phenotypic fingerprints based on mechanical and electrophysiological biomarkers. Integration with computational modeling offered insights into multi-ion channel effects (MICE). Particularly, we identified sodium channel block contributions as a significant factor for poor risk prediction based on traditional parameters. The combined experimental and computational platform can enhance early drug screening, thereby reducing late-stage failures and promoting the progression of low-risk compounds with complex electrophysiological profiles.

BackgroundNeuropathic pain is a major source of disability and distress with few pharmacological options for treatment. Opioid drugs can be effective, but high doses are needed, leading to unwanted effects. BMS-986122 is a positive allosteric modulator of the mu opioid receptor that potentiates acute opioid antinociception without increasing opioid-induced constipation, reward, or respiratory depression. Therefore, we asked if BMS-986122 could increase the effects of low-dose opioid analgesics in chronic neuropathic pain. MethodsWe employed the spared nerve injury and tibial neuroma models in rats and assessed the tactile hypersensitivity of the hind paw and site of neuroma, respectively. ResultsAdministration of low doses of (R)-methadone, morphine, or buprenorphine slightly reduced the tactile hypersensitivity of the hind paw the in spared nerve injury model. Pretreatment with BMS-986122 significantly enhanced the reversal of hypersensitivity, reaching the effect of high-dose gabapentin, a standard of care in neuropathic pain. Pretreatment with BMS-986122 similarly increased the anti-allodynic effects of low dose (R)-methadone on neuroma pain. A similar effect of (R)-methadone in the absence of BMS-986122 was only observed at a dose where respiratory distress was seen. ConclusionsThese findings show that allosteric modulators of the mu opioid receptor such as BMS-986122 can enhance opioid activity that could translate to a safe and effective treatment for chronic neuropathic pain.

ObjectivesTo determine whether adding S-ketamine or dexmedetomidine to oxycodone affects the microbiological, physical, or chemical stability of patient-controlled analgesia (PCA) solutions prepared in a hospital pharmacy. MethodsOxycodone solution (1 mg/mL) and three oxycodone-S-ketamine mixtures (0.25, 0.50, 0.75 mg/mL) and three oxycodone-dexmedetomidine mixtures (2.5, 5.0, 10 {micro}g/mL) were compounded under validated EU GMP Class A/B aseptic conditions and filled into PCA reservoirs. Reservoirs (n=42 for physicochemical studies; n=21 for sterility; n=4 for antimicrobial activity testing) were stored at 2-8{degrees}C for 28 days, then at 20-25{degrees}C for 2 days. Sterility was assessed by membrane filtration according to Ph. Eur. 2.6.1. Physical stability was evaluated by visual inspection, pH, weight, and osmolality. Chemical stability was assessed using a validated HPLC-UV method developed in accordance with FDA and ICH Q2(R1) guidelines. ResultsAll antimicrobial activity tests showed growth of the six reference strains, indicating no inhibition by the drug mixtures. All 21 sterility-test reservoirs remained free of turbidity throughout 30 days. No visual changes, precipitation, or discolouration were observed. Weight loss was [&le;]0.3%, pH changes were between required range 4,5-7, and osmolality increased by <1.4% during the study. Measured oxycodone, S-ketamine, and dexmedetomidine concentrations remained within {+/-}5% of initial values, and no degradation products were detected. ConclusionsOxycodone PCA solutions containing S-ketamine or dexmedetomidine remained sterile, physically stable, and chemically stable for 28 days at 2-8{degrees}C followed by 2 days at room temperature at 20-25{degrees}C. These findings support extended shelf-life and centralized batch preparation of opioid-adjuvant PCA reservoirs in hospital pharmacy practice. Key MessagesO_ST_ABSWhat is already known on this topicC_ST_ABSOpioid-adjuvant combinations such as oxycodone with S-ketamine or dexmedetomidine are increasingly used in patient-controlled analgesia, but no commercial multi-agent formulations exist. Hospital pharmacies therefore prepare these mixtures as compounded sterile preparations, despite limited data on their chemical and microbiological stability. What this study addsThis study demonstrates that oxycodone PCA solutions containing S-ketamine or dexmedetomidine remain chemically stable and microbiologically sterile for 28 days at 2-8{degrees}C plus 2 days at 20-25{degrees}C, when prepared under validated aseptic conditions. Concentrations of all analytes remained within {+/-}5% of initial values, and no degradation products were detected using a validated HPLC-UV method. How this study might affect research, practice or policyThese stability data support the assignment of extended beyond-use dates and enable centralized batch compounding of PCA reservoirs in hospital pharmacies. The findings have the potential to reduce aseptic workload, improve production efficiency and decrease medication waste while ensuring product quality.

The development of novel therapeutics for infectious diseases remains a global health priority. To accelerate the treatment development, innovative strategies through new approach methodology (NAM) are needed to bridge speed of in vitro with predictive power of in vivo studies, while reducing mammalian experiments. The zebrafish (Danio rerio), particularly the embryo/larva, has been established as a valuable non-mammalian in vivo model in biomedical research. We developed a standardized and streamlined workflow for the zebrafish as NAM, which consisted of 3 steps: drug selection and efficacy evaluation, internal exposure assessment, and PKPD modelling. Compounds with higher tolerated doses than minimum inhibitory concentration were selected. Drug efficacy was quantified through longitudinal individual fluorescence microscopy at baseline and 24 and 48h on treatment. Drug exposure was quantified in larval homogenates and exposure medium from 0-48h on treatment. The PKPD relationship was quantified by non-linear mixed effects modelling. For case study bedaquiline, PKPD was quantified using a one-compartment model with age-depending elimination, and an Emax concentration-response relationship on the delayed logistic bacterial growth function, with an EC50 of 26.6 {micro}g/mL and an Emax of 1.07-1.37. In the case of clarithromycin, in contrast, negligible internal exposure after waterborne treatment were observed, illustrating the risk of false negatives without internal exposure assessments. Bactericidal efficacy was confirmed by intravenous drug injections, showing a clear dose dependent antimycobacterial effect. The standardized zebrafish NAM workflow presented here facilitates the translation of drug efficacy to higher vertebrates, reducing rodent studies to confirmatory or replacing them completely, thus accelerating drug development.

Background/ObjectivesThe quality and characteristics of approved medicines can vary substantially depending on manufacturing processes and standards within a given country. The aim of the study was to compare the available marketed brands of ademetionine tablets derived from various countries in order to identify potential differences between the different formulations. MethodsWe performed comprehensive analyses of the physical, chemical, and dissolution characteristics of different formulations of ademetionine tablets marketed in China, India, Russia, Ukraine, and Uzbekistan, using the originator formulation of Heptral(R) as the reference standard. The formulations were evaluated at initial analysis and after 3 months at 40{degrees}C/75% relative humidity. Clinical parameters such as ademetionine content, degradation products, S,S-isomer, and water content were assessed using HPLC, and a dissolution profile analysis performed in 2 hours of acid solution followed by 90 minutes in a buffer solution. ResultsThe Nusam (India) and Ximeixin (China) products were the two products most comparable to the Heptral products. Adenomak (Ukraine), the only food-grade product and only one with the tosylate salt showed the most significant quality variations compared to Heptral including dissolution failure as well as considerable variability between batches. ConclusionsThe study highlights the importance of using pharmaceutical-grade ademetionine products to maintain clinical efficacy and ensuring standards are maintained across global markets.

ObjectiveConventional pharmacodynamic (PD) modeling workflows require manual model selection, repeated equation rewriting, and empirical parameter adjustment, resulting in limited automation, high cross-scenario migration costs, and insufficient reproducibility. This study aims to develop PD Union, a unified, automated, and interpretable framework for mechanistic PD modeling. MethodsPD Union is built upon a unified continuous dynamical skeleton that organizes absorption and systemic exposure module, the receptor module, the drug input module, the first delay module, the primary pharmacodynamic function module, the primary pharmacodynamic state module, the downstream pharmacodynamic state module, the second delay module, the feedback module, the circadian modulation module, the biophase module, the direct effect module, the disease state module, the second PD axis first delay module, the second PD axis primary pharmacodynamic function module, the second PD axis primary pharmacodynamic state module, the second PD axis downstream pharmacodynamic state module, the second PD axis second delay module, and the second PD axis feedback module. A machine learning-based structure identification module is incorporated to recognize drug input modes and mechanism labels from population PK/PD time series, followed by constrained population parameter optimization, forming an integrated pipeline of structure identification, candidate generation, and parameter fitting. ResultsValidation was conducted at two levels. In standardized synthetic benchmarking across 14 representative single-endpoint scenarios, the structure identification model achieved an output mode accuracy(NRMSE) of 0.7600 and macro-average F1 of 0.6307; parameter fitting yielded an NRMSE mean of 0.146 and median of 0.117. In the unified reconstruction validation based on 15 population pharmacokinetics/pharmacodynamics (PK/PD) literature data, the mean NRMSE of PDUnion model for PD was 0.261, and the median was 0.228. Among the 15 studies, 14 performed better than the models provided in the original literature. ConclusionsPD Union demonstrates that interpretable mechanistic modularization combined with machine learning-assisted structure identification is feasible for automated PD modeling. The framework provides an executable methodological foundation for unified, reproducible, and extensible mechanistic PD modeling, with potential applicability to multi-endpoint and complex disease-state modeling scenarios.

1.Pharmacokinetic studies in critically ill patients are often constrained by small sample sizes, limiting the strength and generalizability of conclusions drawn solely from observed data. Bayesian inference offers a powerful strategy to address this challenge by incorporating prior knowledge. In this study, we evaluated two model-based approaches for characterizing the population pharmacokinetics of ceftolozane and tazobactam in critically ill patients, comparing nonlinear mixed-effects modeling with Bayesian hierarchical analyses. The Bayesian methods incorporated literature-derived prior information. The data was collected from 13 critically ill patients receiving 3.0 g of ceftolozane combined with tazobactam (2:1) via intravenous infusion. Pharmacokinetic modeling was performed using NONMEM and Stan software with the Torsten extension. Model diagnostics and graphical analyses were conducted in RStudio with relevant packages. In the absence of prior information, a one-compartment model with a limited set of parameters describing inter-individual variability adequately characterized the pharmacokinetics of ceftolozane and tazobactam. When prior information was incorporated, a two-compartment model became feasible and yielded a characterization of parameter variability and correlations that was more consistent with published literature. The application of Bayesian inference ensured alignment with existing literature on ceftolozane and tazobactam pharmacokinetics and mitigated some systematic biases observed in the data-driven approaches. Moreover, the Bayesian approach enables direct decision-making by incorporating uncertainty into the analysis, as demonstrated by probability of target attainment analysis. Collectively, these results underscore the utility of Bayesian methods in pharmacokinetic modeling for critically ill patients, offering a robust framework for optimizing dosing strategies in data-limited settings.

The neurosteroid allopregnanolone is a positive allosteric modulator of GABA(A) receptors, which has proved beneficial in the treatment of major depressive disorder and epilepsies. It also has a role in treating the mood swings that are associated with fluctuations in its level during the menstrual cycle. Nonetheless, a subset of women do not tolerate high levels of allopregnanolone. Iso-allopregnanolone, a negative allosteric modulator, as well as synthetic steroid antagonists are used to treat such conditions. However, steroid-based medications are difficult to deliver and their specificity of action can be unclear. Recently introduced novel nonsteroidal agents that, like iso-allopregnanolone, can reverse the action of positive allosteric modulators without changing the positive action of GABA, might provide an alternative. We surveyed a number of them on human 1{beta}3{delta} GABAARs using a [3H]muscimol binding assay. A 6-membered ring spiro-hydantoin, DKD99, allosterically reversed the positive allosteric action of allopregnanolone over a wide concentration range (6 to 1,000 nM). DKD99 shifted allopregnanolones modulation curve 10-fold to the right. Furthermore, it has a much lower affinity when exerting similar actions on 1{beta}3{gamma}2 receptors. Agents such as this have utility for elucidating underlying mechanisms and may offer an alternative pathway for the development of nonsteroidal therapies against the positive allosteric modulatory actions of neurosteroids.

BackgroundEpidemiological studies consistently report inverse associations between caffeinated coffee consumption and dementia risk. However, the molecular mechanisms linking coffee-derived phytochemicals to neuroprotection remain only partially understood. ObjectiveTo evaluate, through integrated in silico pharmacology, the relative contribution of adenosine receptor modulation versus direct amyloidogenic enzyme and kinase inhibition in mediating the putative neuroprotective effects of major coffee constituents. MethodsMolecular docking analyses were conducted for caffeine, paraxanthine, chlorogenic acid, trigonelline, cafestol, and kahweol against adenosine A2A and A1 receptors (A2AR, A1R), {beta}-secretase 1 (BACE1), glycogen synthase kinase-3{beta} (GSK-3{beta}), and NLRP3 inflammasome components. Docking was performed using the CB-Dock2 platform. Binding affinities, interaction patterns, and ligand efficiency metrics were assessed. Blood-brain barrier permeability and ADMET properties were predicted using pkCSM. ResultsCaffeine and paraxanthine demonstrated structurally coherent binding within the orthosteric pockets of A2AR and A1R, supported by favorable predicted blood-brain barrier penetration and high unbound fractions. Ligand efficiency analysis identified adenosine receptors as the most pharmacologically plausible targets for small xanthine derivatives. Although larger phytochemicals exhibited stronger absolute docking scores at BACE1, GSK-3{beta}, and NLRP3, predicted pharmacokinetic constraints suggest a small biological effect due to a limited central exposure. ConclusionsThese findings support an adenosine receptor-centered mechanism as the dominant molecular axis linking caffeinated coffee consumption to reduced dementia risk, favoring neuroinflammatory and signaling modulation over direct enzymatic inhibition. Experimental validation is warranted to confirm translational relevance. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=193 HEIGHT=200 SRC="FIGDIR/small/723029v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1a02629org.highwire.dtl.DTLVardef@129890dorg.highwire.dtl.DTLVardef@1e4c05corg.highwire.dtl.DTLVardef@110ec7a_HPS_FORMAT_FIGEXP M_FIG C_FIG

IntroductionImmune checkpoint inhibitors (ICIs) enhance antitumor responses by blocking inhibitory receptors, including PD-1 and CTLA-4. Overactivation can trigger systemic toxicity akin to autoimmune diseases, including kidney manifestations. We sought to 1) profile immune signaling and 2) interrogate potential mechanisms of ICI-related kidney injury in a Human Immune System (HIS) tumor-bearing mouse model treated with nivolumab and ipilimumab. MethodsImmunodeficient BRGS (BALB/c-Rag2nullIl2r{gamma}nullSirpNOD) neonates were engrafted with human CD34+ cells to generate HIS-BRGS mice. Human MDA-MB-231 tumor cells were implanted subcutaneously; once tumors reached [~]150 mm3, mice received weekly intraperitoneal vehicle (PBS) or ICI (nivolumab 20 mg/kg + ipilimumab 10 mg/kg) for 4 weeks (Veh BRGS n=4; ICI BRGS n=6; Veh HIS-BRGS n=7; ICI HIS-BRGS n=7). Kidneys were evaluated by histopathology (H&E, TEM), flow cytometry for human immune phenotypes, multiplex ELISA (80 human proteins; 10 injury biomarkers), bulk RNA sequencing, and targeted qPCR. Pearson correlations identified predictors of histopathological injury. ResultsRenal vasculitis and interstitial nephritis were observed only in ICI-treated HIS-BRGS mice. These kidneys showed a shift toward CD4+ T-cell enrichment with an increased TNF- production capacity compared to CD8+ counterparts. Toxicity was accompanied by increased renal concentrations of human cytokines, chemokines, and soluble receptors. ICI treatment significantly elevated serine proteases (Granzyme A/B) and NGF-{beta}, while decreasing IL-4. Interstitial nephritis correlated with renal PD-1 and MIF. Renal vasculitis correlated with kidney PD-1, CCL1, MIF, Granzyme A, IL-15, and BAFF. Traditional injury biomarkers (KIM-1, NGAL) remained unchanged; however, a trending decrease in EGF was observed. ConclusionsOur study suggests that shifts in human T-cell populations and specific immune proteins could serve as promising biomarkers and mechanistic targets for ICI nephrotoxicity. The tumor-bearing HIS-BRGS mouse model reproducibly recapitulates the histopathological and immunological features of human ICI-induced nephrotoxicity and represents a validated preclinical platform for testing novel therapeutic interventions to preserve kidney function during cancer immunotherapy. Translational StatementImmune checkpoint inhibitor (ICI)-associated nephrotoxicity occurs in up to 25% of treated patients, yet the immunological mechanisms driving renal injury remain poorly characterized due to the scarcity of human biopsy material and the absence of robust preclinical models that recapitulate human immune responses. This study demonstrates that tumor-bearing humanized immune system (HIS) mice treated with combined nivolumab and ipilimumab reproducibly develop renal vasculitis and interstitial nephritis mediated by a human CD4+ T cell-dominant infiltrate, mirroring the clinicopathological features reported in patients with ICI-associated acute kidney injury. By integrating histopathology, flow cytometry, multiplex proteomics, and transcriptomics, we identify a coordinated immune network, including IL-15, CCL1, MIF, GZMA, and BAFF, that correlates with the severity of renal pathology and represents tractable mechanistic targets and candidate biomarkers. These findings provide a validated preclinical platform for dissecting irAE mechanisms and testing novel therapeutic strategies to preserve kidney function during cancer immunotherapy.

BackgroundPharmacogenomic testing (PGx) can optimise drug efficacy and minimise toxicity, but the extent of prescriber adherence to PGx recommendations remains unclear. We aimed to quantify clinician adherence to international genotype-guided prescribing recommendations in a cohort of paediatric oncology patients. MethodsWe reviewed files of children enrolled in the MARVEL-PIC (NCT05667766) randomised control trial, who had PGx recommendations available. Patients were included if 12 weeks had passed since their PGx report was released to clinicians. Prescribing events were identified for actionable PGx recommendations, and classified as "explicitly followed", "inadvertently followed", or "not followed". Adherence was assessed by patient, drug, and recommendation. Results2,063 PGx recommendations were available for 216 patients. 64 (3.1%) recommendations were actionable for 44 patients and 10 drugs within the 12-week study period. Recommendations were explicitly followed in 57/288 (19.8%) of prescribing events, inadvertently followed in 145 (50.3%), and not followed in 86 (29.9%). Mercaptopurine demonstrated the highest rate of explicit adherence (87.5%). No significant associations were observed between adherence and age group, cancer type, drug type, or strength of recommendation. ConclusionAdherence to pharmacogenomic recommendations was very low, highlighting the need to understand barriers to PGx implementation, and consideration of clinical decision supports to facilitate adherence. Plain Language SummaryPharmacogenomic medicine (PGx) looks at how our genes affect our response to drugs, including their effectiveness and toxicity. Through genetic analysis we can create recommendations for drug dosing, avoidance, and monitoring. The MARVEL-PIC study aims to understand if having PGx recommendations decreases the rate of adverse events in children with cancer. We aimed to understand how often prescribers follow PGx recommendations after they are made available, in the MARVEL-PIC trial. To do this, we reviewed medical records and identified relevant prescribing events. We marked these as "recommendation explicitly followed", "recommendation not followed", or "recommendation inadvertently followed" (where the recommendation was followed, but it wasnt clear if this due to PGx). We found that when recommendations were available, they were only explicitly followed in around 20% of cases. In 50% of cases, they were followed but it was unclear whether this was due to PGx. In the remaining 30%, they were not followed. We also found that alerts on our electronic system were fired in about 80% of events where the recommendation was not followed, but did not change the outcome. These findings show that prescriber adherence to PGx recommendations is low. We need to better understand why this is the case and implement more specific tools to assist prescribers in following recommendations. Article HighlightsO_LIPharmacogenomic (PGx) testing can reduce adverse drug reactions by guiding drug choice, dosing, and monitoring. C_LIO_LI!Prescriber to PGx recommendation adherence has not been widely investigated. C_LIO_LIRetrospective analysis showed that explicit adherence to recommendations occurred in only 19.8% of relevant prescribing events. C_LIO_LIIn 50.1% of prescribing events, recommendations were followed, but there was no clear reference to PGx. C_LIO_LIMercaptopurine had the highest explicit adherence (87.5%) from the drugs analysed. C_LIO_LIThere were no statistically significant associations between adherence and age group, cancer type, drug type, or recommendation strength. C_LIO_LIRecommendations were explicitly followed in 29% of events where an interruptive alert was fired, and inadvertently followed in 8%. C_LIO_LITailored interruptive alerts have been shown to increase adherence in other studies, suggesting that the specific design of interruptive alerts may influence adherence. C_LIO_LIWe concluded that explicit prescriber adherence to PGx recommendations is very low (19.8%), and further research needs to be done to understand barriers to implementation. C_LI

BackgroundAutosomal dominant polycystic kidney disease (ADPKD) is a common inherited disorder marked by numerous renal cysts that impair kidney function, with about half of affected individuals progressing to kidney failure by midlife. Patients exhibit reduced circulating apelin, a ligand of the apelin receptor, known to regulate cardiovascular function including hypertension. We tested whether diminished apelin signaling contributes to cystogenesis and if exogenous apelin receptor activation can improve disease outcomes. MethodsPlasma samples from age- and sex-matched healthy controls and ADPKD participants were analyzed for circulating apelin peptides. To assess direct cystic effects, primary ADPKD renal epithelial cells were grown as 3D collagen-embedded cysts and treated with apelin agonists. Male and female Pkd1RC/RC; Pkd2+/- (PKD) mice were treated for 27 days with apelin agonists, vehicle, or the standard of care drug, Mozavaptan. Kidney and heart weight ratios, BUN, renal cAMP, and kidney transcriptional profiles were evaluated. ResultsCirculating apelin peptides were significantly reduced in ADPKD patients despite normal kidney function (eGFR, BUN, and creatinine). In vitro, both apelin and the small molecule apelin receptor agonist Azelaprag inhibited cyst growth. Apelin and Mozavaptan reduced kidney weight, cystic index, blood urea nitrogen and renal cAMP in PKD mice, whereas Azelaprag did not. Apelin downregulated expression of genes associated with cyst progression, including Lcn2 (Ngal), Postn, and Havcr1 (Kim-1). Mozavaptan, but not apelin, induced diuresis and reduced urinary concentration. ConclusionApelin receptor activation by exogenous apelin inhibited cAMP synthesis and cyst growth and improved kidney function in an orthologous mouse model of ADPKD. We propose that the apelin receptor may be a potential therapeutic target in ADPKD.

Background and AimsFenofibrate is widely prescribed for hyperlipidaemia and has been associated with rare but severe cases of drug-induced liver injury (DILI), yet its effects on liver sinusoidal endothelial cells (LSECs) remain to be investigated. LSECs maintain a highly permeable specialized sinusoidal barrier characterized by transcellular pores (fenestrations), regulating the bidirectional transfer of circulating compounds to and from the hepatocytes. As drug-induced alterations in fenestration architecture could influence xenobiotic access to hepatocytes, these changes may modulate pathways associated with DILI. Understanding the effects of fenofibrate on LSEC ultrastructure may therefore provide insights into previously underexplored endothelial contributions to hepatic drug responses. MethodsBoth fenofibrate and its active metabolite, fenofibric acid, were evaluated for their effects on LSEC ultrastructure, mechanical properties, and functional markers. Atomic force microscopy (AFM) and scanning electron microscopy (SEM) and were used to quantify fenestration architecture. AFM was additionally used to measure cellular mechanical properties, which were interpreted in the context of fluorescence-based quantification of cytoskeletal organization. Gene expression, viability, and cytotoxicity were assessed using PCR-based and biochemical assays. ResultsFenofibrate reduced fenestration number and porosity at both tested concentration (10, and 25 {micro}M). It also decreased the apparent Youngs modulus of LSECs, accompanied by changes in tubulin and actin architecture, without detectable cytotoxicity. In contrast, treatment with fenofibric acid did not result in significant structural or mechanical effects on LSECs, even at higher concentrations. ConclusionsTogether, these data identify LSECs as a drug-responsive hepatic cell type for fenofibrate, suggesting that LSECs could represent an underrecognized contributor to the complex, multifactorial processes underlying DILI. This work provides a framework for evaluating endothelial contributions to fenofibrate-associated liver effects in more complex models. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/718907v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@1f9ec6forg.highwire.dtl.DTLVardef@11174a1org.highwire.dtl.DTLVardef@1000e2borg.highwire.dtl.DTLVardef@a23b00_HPS_FORMAT_FIGEXP M_FIG Fenofibrate reduces LSEC fenestrations and metabolic activity at higher concentrations, while its metabolite, fenofibric acid, does not affect LSEC, regardless of its concentration. C_FIG

Severe forms of inflammation-induced acute and chronic myocarditis have a poor prognosis. Promising therapeutic efforts focused on monoclonal antibodies (mAbs) inhibiting inflammation-inducing molecules. However, most mAbs target only one or a limited number of such molecules. Since inflammation involves multiple redundant pathways, we postulated that an mAb inhibiting multiple inflammatory pathways would be a potent therapeutic agent. We initially tested the commercially available anti-natural killer (NK) cell mAb (anti-NK1.1), which binds a receptor expressed on NK cells and depletes them. Since NK cells are key cellular orchestrators of inflammation, by reducing their number, we aimed to inhibit multiple inflammatory pathways. Our initial studies demonstrated that administration of this antibody significantly improved myocardial outcomes in mouse models of acute myocardial infarction and of heart failure. Since NK1.1 is not expressed in human cells, we built on these promising preclinical results by developing a novel mAb targeting CD160 on human NK cells for evaluation as an immunosuppressive therapy. We found that the anti-CD160 mAb depletes both murine and human NK cells. We also found that, while CD160+ cells were largely present in the NK population, they also occurred among CD8+ and {gamma}/{delta} T cell subsets in human cells. Anti-CD160 therapy entirely prevented the deterioration of the myocardial function of mice with autoimmune-induced acute myocarditis. This outcome suggests our novel approach for inhibiting multiple inflammatory pathways may provide a potent strategy for improving outcomes of inflammation-driven myocarditis, as well as of other inflammation-driven diseases. Key PointsO_ST_ABSQuestionC_ST_ABSCan the depletion of CD160+ cells prevent autoimmune-induced myocarditis? FindingsIn this study we found that CD160 is expressed by mouse and human natural killer cells and other subtypes of cytotoxic T cells, and that a monoclonal antibody targeting CD160 depletes NK cells. In a preclinical model of experimental autoimmune myocarditis, administration of the anti-CD160 monoclonal antibody prevented myocardial dysfunction and systemic inflammation. MeaningOur results are compatible with the hypothesis that early autoimmune-induced myocardial dysfunction is promoted by CD160+ cells, which elevate inflammation-induced circulating factors (or factors released by tissue-resident cytotoxic immune cells) that cause myocardial dysfunction in the absence of myocardial necrosis or fibrosis, and further, that targeting CD160+cells with a mAb that depletes NK cells (and probably CD160 expressing cytotoxic T cells) entirely prevents the deterioration of myocardial function in such mice. This outcome suggests our novel approach for inhibiting multiple inflammatory pathways may provide a potent strategy for improving outcomes of inflammation-driven myocarditis, as well as of other inflammation-driven diseases.

ABSTRACT Background: Self medication with analgesics and non steroidal anti inflammatory drugs (NSAIDs) is common in low- and middle income countries and may expose users to preventable adverse outcomes. Evidence from Guinea remains scarce. This study aimed to estimate the prevalence of self medication with analgesics and NSAIDs among pharmacy clients in urban Conakry, identify associated factors, and describe clinical risk situations. Methods: We conducted a pharmacy based analytical cross sectional study in 30 private pharmacies across Conakry, Guinea. A total of 1,032 participants seeking analgesics or NSAIDs were enrolled between November 3, 2012, and April 5, 2013. Self-medication was defined as acquisition or use without a valid medical prescription. Factors associated with self-medication were analysed using multivariable logistic regression. Results: Among 1,032 participants, 603 reported self medication (prevalence 58.4%). Previous unsupervised use was reported by 78.7%. The most frequently used medicines were paracetamol (56.9%, n=587), diclofenac (21.3%, n=220), ibuprofen (17.9%, n=185), and aspirin (3.9%, n=40). Overall, 68.0% (n=702) reported no knowledge of potential adverse effects. Clinical risk situations were frequent: gastrointestinal disorders (41.3%, n=426), hypertension (9.2%, n=95), and pregnancy exposure among reproductive age women (26.0%). In multivariable analysis, self medication was independently associated with previous analgesic/NSAID use (aOR = 2.8, 95% CI: 2.1 to 3.6), lack of knowledge of adverse effects (aOR = 1.9, 95% CI: 1.4 to 2.5), informal occupation (aOR = 1.6, 95% CI: 1.2 to 2.2), and age 18 to 59 years (aOR = 1.5, 95% CI: 1.1 to 2.1). Conclusions: In this pharmacy based study conducted in urban Conakry, self medication with analgesics and NSAIDs was common and frequently associated with limited awareness of potential adverse effects. These findings support the need for strengthened pharmaceutical regulation, pharmacist-led counselling, health literacy interventions, and improved access to primary care. Keywords: self medication; analgesics; NSAIDs; paracetamol; diclofenac; ibuprofen; pharmacy; Guinea; Conakry; drug safety; public health.