Frontiers in Pharmacology

IntroductionCannabinoid receptor-2 (CB2) is an emerging therapeutic target for chronic and inflammatory pain, cancer, and neurological disorders. Understanding the efficacy of CB2 ligands is crucial for future drug design and development. AimsWe aimed to establish a simple and robust system to control CB2 expression using a tetracycline-regulated mammalian expression system (T-REx), to enable application of the Black and Leff operational model to measure the operational efficacy ({tau}) of CB2 ligands. MethodsLigand-induced hyperpolarisation of AtT20 cells transfected with T-REx and human CB2 was measured by FLIPR membrane potential assay. Maximal and submaximal responses of the CB2 ligands were produced by regulating CB2 expression using tetracycline. Data were fitted to the operational model of receptor depletion to quantify the efficacy of seven ligands. Additionally, the maximal initial rate of signalling (IRmax), another putative measure of ligand efficacy, was determined. ResultsAK-F-064, CP55940 and 2-AG exhibited similar efficacy with a {tau} values of 11.4, 11 and 10.4 respectively, while anandamide (AEA) had the lowest efficacy ({tau}=1.07) among the tested agonists. The rank order of operational efficacy and IRmax was similar and was estimated as: AK-F-064 = CP55940 = 2-AG > 5F-AB-PICA = WIN55212-2 > HU-308 = AEA. ConclusionThis inducible expression system provides a reliable platform for quantifying and comparing CB2 ligand efficacy using the operational model. This approach may facilitate more precise CB2-targeted drug development and can be readily extended to other GPCR targets.

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.

Polypharmacy, the concurrent use of multiple medications, is increasingly prevalent in older people and is associated with adverse outcomes such as falls, frailty, functional and cognitive decline, and increased hospitalization and mortality. The liver, as the primary site of metabolism, is exposed to varying drug concentrations during first pass metabolism, hepatic clearance and perfusion, potentially causing alterations in liver sinusoidal endothelial cells (LSEC). LSEC are specialized endothelial cells responsible for maintaining fenestrations - dynamic, transcellular pores that facilitate the exchange of substances between the blood and liver parenchyma. Disruption of fenestrations can compromise liver function, contributing to a variety of hepatic disorders. This study investigated the effects of four commonly prescribed drugs -- metoprolol, citalopram, oxybutynin and oxycodone -- on LSEC function. We examined their impact on LSEC viability, endocytosis, and fenestration morphology at both systemic steady-state and first-pass concentrations, separately and in a polypharmacy cocktail to model clinical exposure. All treatments induced sublethal metabolic changes, but effects on LSEC functions were drug- and concentration-dependent. Citalopram and oxybutynin caused dose-dependent defenestration, whereas metoprolol and oxycodone produced mild, non-dose-dependent effects. Endocytic activity was increased with oxybutynin, metoprolol, oxycodone, and the polypharmacy cocktail, while citalopram had no effect. The polypharmacy cocktail triggered synergistic defenestration at first-pass concentrations, but not at steady-state levels. These results highlight the concentration-dependent and combinatorial effects of polypharmacy on LSECs, emphasizing the need to consider endothelial responses in drug safety and pharmacokinetic assessments, particularly in patients exposed to multiple medications.

BackgroundReliable population pharmacokinetic (PopPK) parameter estimation can be compromised by outliers under Gaussian residual error models. A common mitigation strategy is post hoc filtering based on conditional weighted residuals (CWRES); however, this approach can be insensitive due to model "masking" driven by variance inflation. Practical barriers to implementing robust likelihoods in standard software have motivated interest in computationally simpler exponential-tail alternatives such as the Laplace and exponential power distribution (EPD). MethodsWe implemented a one-compartment PopPK model using a custom likelihood workaround in Monolix to benchmark four residual error distributions: Normal, Laplace, Generalized Error Distribution (GED), and Students t. We assessed CWRES sensitivity under extreme contamination and compared estimation performance using theoretical tail-behavior analysis, controlled simulation studies spanning multiple contamination severities, and a real-world caffeine PK case study with influential terminal-phase deviations. ResultsSimulations showed that CWRES-based diagnostics can be unreliable: extreme outliers frequently produced |CWRES| < 6 because the Normal model inflated residual variance, thereby masking contamination. Exponential-tail models (Laplace, GED) improved robustness for mild to moderate outliers but failed under extreme deviations due to insufficiently heavy tails compared to power-law decay. In contrast, the Students t model, via power-law tail behavior, maintained stable and minimally biased structural parameter estimates across contamination scenarios. Consistent patterns were observed in the caffeine case study, where the Students t model provided improved fit and physiologically plausible parameter estimates. ConclusionsCWRES-driven outlier handling is methodologically fragile because influential contamination can be masked by variance inflation and induce biased inference. Among robust residual error models, exponential-tail distributions may be insufficient for extreme outliers, whereas the Students t distribution provides more stable inference across contamination severities. These findings support adopting Students t residual modeling as a default robust option in routine PopPK workflows when outlier contamination is plausible.

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

The current investigation introduces single-cell physiologically-based pharmacokinetic (scPBPK) models to gain insight into drug disposition at the cellular scale. The transition from standard PBPK (sPBPK) models to scPBPK models required depiction of expression-dependent (ED) processes, such as drug metabolism or membrane transport. ED processes utilize weighting functions - a defined or data-driven distribution -that yield heterogeneity in individual cell kinetics. Two scPBPK model examples are provided, one involving a drug (AZD1775) subject to 3 ED blood-brain barrier transport processes, and another drug (midazolam) with a single ED process of metabolism by hepatocytes. For both examples, the weighting function for each ED process was defined by a negative binomial distribution that is often used in scRNAseq analytics. The AZD1775 model simulations indicated a large degree of single cell drug concentration heterogeneity, whereas those for midazolam did not, due to high membrane transport relative to metabolism. scPBPK models offer a means to probe cellular pharmacokinetics compatible with modern omic technologies and may be extended to pharmacodynamic models. TeaserThe modeling framework to predict drug concentrations in single cells is presented.

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.

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.

BackgroundPolycystic kidney disease (PKD) is a genetic disorder characterized by progressive cyst formation, epithelial disorganization, and impaired fluid transport, ultimately leading to renal failure. Disruption of cytoskeletal dynamics and epithelial polarity is central to PKD pathogenesis. Malpighian tubules (MTs) of Drosophila melanogaster serve as a conserved renal analog, and caspase-3/Drice-deficient flies exhibit a robust PKD-like tubular phenotype, providing a powerful in vivo model to investigate therapeutic interventions. PurposeThis study evaluates the therapeutic potential of the Brahmi Ghrita (BG) in ameliorating PKD-like defects in Drosophila Drice mutants and elucidates the underlying cellular and molecular mechanisms. MethodsDrice mutant flies were reared with dietary BG supplementation, and developmental viability (pupation and eclosion) was assessed. Tubule morphology was analyzed by measuring cyst formation and tubule dimensions. Stellate cell (SC) number, shape, and nuclear size were quantified. Cytoskeletal organization and epithelial polarity were examined using F-actin and polarity markers. Molecular analyses included assessment of Rho1 signaling, Gelsolin, and Rho kinase (Rok) localization. Tubule physiology was evaluated by uric acid crystal deposition and Na{square}/K{square}-ATPase expression. ResultsBG supplementation significantly improved pupation and eclosion rates in Drice mutants and markedly reduced cystic dilation by restoring tubule width without altering developmental length. BG selectively increased stellate cell number and normalized aberrant morphology, while principal cell number remained unchanged. Cytoskeletal disorganization and polarity defects were rescued, accompanied by normalization of elevated Rho1 levels and restoration of the actin-severing protein Gelsolin. BG enhanced Na{square}/K{square}-ATPase expression and reduced uric acid accumulation, consistent with improved epithelial transport function. Additionally, BG promoted nuclear enrichment of Rok, indicating altered Rho-associated signaling dynamics. ConclusionBrahmi Ghrita confers nephroprotective effects in a genetic PKD model by coordinately restoring cytoskeletal integrity, epithelial polarity, and ion transport machinery. Rather than broadly suppressing Rho signaling, BG appears to rebalance the Rho1-Gelsolin axis and re-establish transport competency, culminating in structural and functional rescue. These findings provide mechanistic evidence supporting BG as a multi-target modulator of epithelial homeostasis in PKD-relevant contexts. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=123 SRC="FIGDIR/small/705756v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@d909a9org.highwire.dtl.DTLVardef@10938bborg.highwire.dtl.DTLVardef@6ba065org.highwire.dtl.DTLVardef@1ef7857_HPS_FORMAT_FIGEXP M_FIG C_FIG

Voluntary ingestion is a refined method for substance administration that can replace oral gavage in rats and mice. It requires no physical restraint and has no associated risks of adverse effects, resulting in improved welfare and reduced distress for both animals and research staff. This method has been shown to be effective for a variety of compounds but is still not widely used due to concerns about accuracy and reliability. One potential issue is aversion to the taste of the compound being administered, including a common issue of bitter taste. In this study we tested compounds used in oral preparations for human medicines to mask bitter tasting drugs, including a commercial formulation designed for this purpose, Bitter Drug Powder (BDP). The masking agents were given in combination with a palatable vehicle (10% condensed milk) and the amount consumed and time to consume recorded. Animals were first habituated to the vehicle with reliable ingestion achieved within a few days. In the first studies, only BDP was fully effective at masking the bitter taste of quinine and preventing the progressive reduction in reliability of intake of the antidepressant, venlafaxine in mice and rats. We were able to replicate these effects using a combination of two different artificial sweeteners, saccharine and acesulfame K, and a thickening agent xanthan gum. These studies demonstrate that using a masking agent can improve the reliability of voluntary oral dosing in mice and rats and provide evidence to support a formulation which is readily available for researchers.

AimsNonsteroidal anti inflammatory drugs (NSAIDs) are a leading cause of drug hypersensitivity reactions (HRs), yet genetic determinants of individual susceptibility remain unclear. Growing evidence implicates oxidative stress in these reactions. This study aimed to identify genetic variants in redox and immune related pathways associated with cross reactive NSAID hypersensitivity (CR NSAIDs) and single NSAID induced urticaria/angioedema/anaphylaxis (SNIUAA), and to characterize their functional relevance. ResultsGenetic association analyses identified significant associations between NSAID HRs and single nucleotide variants (SNVs) in genes involved in redox homeostasis. In CR NSAIDs patients, SNV GSTM5 rs11101989 and GSTZ1 haplotype (rs1046428, rs7975, rs7972) were associated with increased risk of developing HRs, suggesting impaired glutathione dependent detoxification as the underlying factor. Functional analyses demonstrated that carriers of the GSTM5 rs11101989 CC genotype had significantly reduced serum GST activity. In SNIUAA patients, SOD1 variants (rs2070424 and rs2833483) were consistently associated with increased susceptibility and reduced serum superoxide dismutase activity. Additionally, AKR1C3 rs34186955 is also associated with increased susceptibility whereas the variants AKR1C3 rs12529 and IL4R rs1805016 are associated with decreased susceptibility, highlighting the interplay between prostaglandin metabolism, redox regulation, and immune signalling. InnovationIntegration of genetic and functional enzymatic validation provides mechanistic evidence that oxidative stress pathways modulate distinct NSAID hypersensitivity phenotypes. Identification of GSTM5 and SOD1 variants with measurable functional impact establishes promising biomarkers to guide personalized risk assessment. ConclusionGenetic variability in oxidativestress and immune regulatory pathways influences susceptibility to cross reactive and selective NSAID hypersensitivity, supporting biomarker based strategies for safer NSAID prescribing.

Hypertension (HTN) is the most prevalent risk factor for severe cardiovascular disease and can cause major renal damage, inflammation, and immune cell accumulation. Lymphatic endothelial cells (LECs) are involved in the removal of pro-inflammatory immune cells and cytokines and kidney-specific augmentation of lymphangiogenesis can prevent or reduce HTN. In our previous paper, we performed single-cell RNA sequencing (scRNAseq) on CD31+/podoplanin+ renal cells from mice that underwent angiotensin II-induced (A2HTN) or salt sensitive (SSHTN) models of HTN (and their respective controls) and identified populations of LECs, myeloid immune cells (MICs), and a novel multipotent population we dubbed support cells (SCs). Using NicheNet, we compared baseline signaling between these three cell types in control samples and differences in signaling between control and HTN samples in both LECs and SCs. Ligands with high regulatory potential were identified for all three cell types, with Tgfb1 having the strongest and most consistent activity across all cell types. When comparing control and HTN samples in both LECs and SCs, HTN samples consistently had a larger number of downstream targets enriched and targets that were enriched in HTN samples also corresponded to significantly increased differentially expressed genes (p<0.01) as reported previously. Significant GO terms (p<0.01) were identified from targets and showed a shift in HTN samples away from homeostatic processes and toward growth and proliferation in LECs and translation and metabolism in SCs. Validation and manipulation of the ligand-receptor-target links identified here may provide novel approaches to reduce renal inflammation and immune cell activation. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/701352v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@19bd1faorg.highwire.dtl.DTLVardef@f6a105org.highwire.dtl.DTLVardef@1c56b73org.highwire.dtl.DTLVardef@11897a_HPS_FORMAT_FIGEXP M_FIG C_FIG Created with BioRender.com

Gastric cancer is among the most common cancers and represents a major public health problem worldwide. New therapeutic strategies and drugs are needed. Anti-angiogenic agents targeting the Vascular Endothelial Growth Factor (VEGF) are used in combination therapy in the clinic, although their efficacy remains modest. We believe that these large anti-VEGF antibodies could be advantageously replaced by smaller peptides with better tissue penetration. In this study, we evaluate the efficacy of a previously described dimer peptide ligand of VEGF, D6, in inhibiting the proliferation of gastric cancer cells and the growth of the corresponding murine xenograft. The activity of the D6 peptide in these assays was comparable to that of bevacizumab, the positive control antibody, although the peptide required repeated injections at higher molar concentrations. These promising results justify the continued optimization of the peptide dimer, currently under investigation in our laboratory.

Ribociclib, a selective cyclin-dependent kinase (CDK) 4/6 inhibitor, is approved as a first-line therapy for HR-positive/HER2-negative advanced breast cancer. Emerging evidence suggests that Ribociclib may exert immunomodulatory effects. However, its role in cytokine regulation remains largely unexplored. This study presents a comprehensive in silico investigation of Ribociclibs interactions with eight key pro-inflammatory cytokines--IL-6, TNF-, IL-17A, IL-17F, IL-17A/F, IL-1{beta}, MCP-1, and IFN-{gamma}. Computational assessments included molecular docking, molecular dynamics (MD) simulations, MM-GBSA binding free energy calculations, principal component analysis (PCA), and dynamic cross-correlation matrix (DCCM) analyses. Molecular docking and MD simulations indicated strong and stable complex formation with TNF-, IL-6, MCP-1, IL-1{beta}, and IL-17A/F. MM-GBSA results further showed that Ribociclib formed the most stable complexes with IL-17A/F ({Delta}Gbind = -25.94 kcal/mol) and MCP-1 ({Delta}Gbind = -25.88 kcal/mol), comparable to binding with the CDK-6 ({Delta}Gbind = -36.23 kcal/mol) control protein. PCA and DCCM analyses further supported the stabilizing influence of Ribociclib on these cytokine conformations. Moderate interactions were observed with TNF-, IL-6, and IFN-{gamma}. Collectively, these findings suggest that Ribociclib may function as a multi-target inhibitor capable of modulating diverse inflammatory pathways, providing a computational foundation for its repurposing as a cost-effective anti-inflammatory therapeutic candidate.

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.

Whole-cell patch-clamp studies often fail to observe the expected effect of melatonin on the IK1 current in cardiomyocytes, which may be due to cytoplasmic dialysis and the loss of key components of the intracellular signaling system. The aim of this study was to develop a simple theoretical model to estimate the expected effect on the IK1 inward-rectifying potassium current in an experiment with intact melatonin signaling. The modeling was performed using a well-established model of rat cardiomyocyte electrophysiology (Pandit et al., 2001). The maximum conductance of IK1 (gK1) channels was chosen as the target for modulation, consistent with the established mechanism of direct receptor-mediated increase in potassium conductance under the action of melatonin.Realistic modulation values were used for the modeling. The -50% value for the antagonist effect of 1 M luzindole was obtained by direct calculation from our experimental data. The +20% value for the agonist effect (melatonin) was determined by generalizing literature data and reflects the typical expected strength of signaling pathway modulation, rather than being strictly tied to a specific concentration.It was shown that modulation of gK1 in the specified ranges leads to significant changes in IK1 amplitude in the physiologically important range of resting potentials. The developed model serves as a "computational benchmark" for validating experimental protocols, allowing one to distinguish methodological artifacts from a true lack of effect.

Opioid addiction and misuse are a serious national crisis that affects public health, as well as social and economic welfare. Mortality due to opioid misuse is further exasperated by the combination of opioids with non-opioid respiratory depressants such as xylazine that are resistant to mu opioid receptor antagonists such as naloxone. This study tested the hypothesis that oxytocin can mitigate the severe opioid induced respiratory depression (OIRD) and mortality induced by high doses of fentanyl or the combination of fentanyl with xylazine. Our results show OXT can improve survival and respiratory function in both male and female rats with opioid induced respiratory depression caused by fentanyl, as well as a combination of fentanyl and xylazine. The improvement in respiratory function by OXT post fentanyl-xylazine was significantly greater than the recovery using only naloxone. Chemogenetic activation of OXT receptor positive neurons in the ventral respiratory group (VRG) provided similar benefits to that of OXT administration in reversing OIRD. These results indicate OXT is a promising therapeutic target for reversing OIRD and the respiratory depression that occurs with the combination of opioids and xylazine, a situation where naloxone is only partially effective. Additional translational benefits of OXT include it can be repurposed as it is already a FDA approved drug for other uses, has a high safety profile, and is unlikely to induce the withdrawal or reversal of analgesia that occurs with naloxone. Key PointsO_LIOxytocin (OXT) improves survival and respiratory function in both male and female rats with opioid induced respiratory depression (OIRD) caused by fentanyl C_LIO_LIOXT also reverses OIRD induced by the combination of fentanyl and xylazine C_LIO_LIThe improvement in respiratory function by OXT post fentanyl-xylazine was significantly greater than the recovery using only naloxone C_LIO_LIChemogenetic activation of OXT receptor positive neurons in the ventral respiratory group (VRG) provided similar benefits to that of OXT administration in reversing OIRD C_LIO_LIThese results indicate OXT is a promising therapeutic target for reversing OIRD and the respiratory depression that occurs with the combination of opioids and xylazine C_LI

Topiramate (TPM) is approved for seizures and migraine prophylaxis and is used off-label for several neuropsychiatric conditions. The available dosage forms, including tablets and sprinkle capsules, are unsuitable for patients who may be unable to take medicine orally. The resulting potential treatment interruptions could have untoward consequences and underscores the importance of developing a parenteral formulation. In this study, we developed a population pharmacokinetic model of a novel, intravenous TPM formulation using data from a study in patients with epilepsy or migraine receiving a single intravenous dose of stable-labeled TPM. In total, 246 TPM concentrations from 20 adult patients were included for model development. A three-compartment pharmacokinetic model with linear elimination fit the concentration-time data best. Simulations for various loading and maintenance regimens for patients with and without enzyme-inducing comedications were performed. The final estimates(95% confidence interval (CI)) for CL (L/h), V1 (L), and the peripheral volumes, V2 and V3 for a 70 kg person were 1.31(1.01 - 1.53), 9.84 (8.49 - 11.0), 39.1 (36.5 - 41.8)L, and 9.01 (6.41 - 44.3) respectively. The use of enzyme-inducing co-medication was the only significant covariate, associated with a 63% increase in clearance .Goodness-of-fit plots and visual predictive checks indicate satisfactory model performance and prediction. The simulation results indicate that adjusting doses for patients receiving IV TPM can mitigate the changes in plasma TPM concentrations resulting from enzyme induction. This population pharmacokinetic model for intravenous topiramate can inform dosing decisions for patients with epilepsy when used as either initiation or bridging therapy.