Precise evaluation of plasma protein binding (PPB) (fu) is critical for predicting the pharmacokinetic behaviour of new chemical entities.In this study, we describe a rapid equilibrium dialysis (RED)-based flux dialysis approach for estimating PPB (fu) utilising pre-equilibration diffusion data.The empirical membrane permeability (Pmem) of the RED system was established using reference compounds spanning a wide range of binding affinities, including quinidine, imipramine, fluconazole and sertraline.The averaged Pmem value was subsequently applied to determine fu for test compounds such as efavirenz, mifepristone and warfarin, representing diverse physicochemical and binding properties.The current approach demonstrates immediate applicability for low- to moderately bound compounds, where reliable flux data can be obtained within a shorter timeframe.While early-phase diffusion provides useful kinetic information, accurate fu estimation for highly bound compounds requires extended sampling to obtain reliable flux measurements.Fu values obtained from the flux-based model showed close agreement with literature-reported equilibrium dialysis data, supporting the robustness and predictive validity of the approach.The results demonstrate proof-of-concept feasibility of a flux-based approach using RED device, while highlighting limitations related to signal sensitivity and assumptions associated with empirical permeability.

Multidrug resistance (MDR), often emerging after chemotherapy, has long restricted the therapeutic efficacy of cancer treatment in clinical practice. Among the various strategies developed to overcome MDR, the sequential delivery of P-glycoprotein inhibitors and anticancer drugs has shown great promise when compared with traditional co-delivery approaches. In this work, porous amino acid nanoparticles (NPs) are fabricated via a self-templating method and loaded with doxorubicin (Dox). NPs are coated with polydopamine (PDA), which is functionalized with quinidine, a P-glycoprotein inhibitor, via a pH-sensitive linker. The pH/glutathione (GSH) sensitivity of PDA allows a delayed release of Dox compared to quinidine, thereby enabling a sequential drug delivery. Specifically, the release profile of Dox enables to be precisely controlled through adjusting the number of the PDA layer. In addition, the incorporation of PDA imparts photothermal capabilities to NPs, allowing for their use in synergistic photothermal therapy (PTT) and chemotherapy. Finally, the antitumor effect of NPs is evaluated in vitro and in vivo. The viability of MDR EMT-6/AR1 cells is decreased to less than 5% after the treatment with NPs and a significant tumor regression is observed in MDR tumor-bearing mice after combining PTT and chemotherapy, providing a high antitumor efficacy.

Derivation of electrophysiologically mature cardiomyocytes from human pluripotent stem cells (hPSCs) remains a prerequisite for effective cardiac modeling and preclinical drug evaluation. However, current differentiation protocols often produce heterogeneous cell populations with limited maturity. In this study, we developed a CD47-based fluorescence-activated cell sorting strategy to purify ventricular-like cardiomyocytes from three-dimensionally differentiated hPSC-derived spheroids. CD47+ cardiomyocytes exhibited highly consistent contractile behavior and enhanced structural maturation, as confirmed using immunocytochemistry for myosin regulatory light chain 2 ventricular/cardiac muscle isoform and α-actinin. Electrophysiological analysis using a patch clamp revealed that the majority of CD47+ cells displayed ventricular-like action potential waveforms, characterized by prolonged repolarization duration, elevated amplitude, and a reduced negative maximum diastolic potential. Drug responsiveness was assessed using multi-electrode array recordings. CD47-enriched cardiomyocytes demonstrated reproducible and dose-dependent field potential alterations in response to known cardiotoxic agents, including remdesivir and quinidine, and outperformed metabolically purified controls in sensitivity and inter-replicate consistency analysis. These results establish CD47 as a reliable surface marker for isolating mature ventricular-like cardiomyocytes from hPSCs. The method enables the generation of functionally robust cardiomyocyte populations suitable for in vitro cardiac research and pharmacological testing.

Given the high prevalence of polypharmacy among patients with depression, this study systematically assesses the drug-drug interaction (DDI) risks of the investigational antidepressant ammoxetine (AMT) mediated by cytochrome P450 2D6 (CYP2D6) (metabolic enzyme) and multidrug and toxin extrusion protein 1 (MATE1) (renal transporter), and characterizes the resulting metabolism-transport interaction cascade. In vitro DDI risk screening for AMT was performed using human liver microsomes, recombinant CYP enzymes, and transporter-overexpressing cell lines. Static mechanistic models were used to predict human DDI risks, with sensitivity analyses incorporated to assess CYP2D6 poor metabolizers (PMs). Finally, in vivo validation was carried out in Institute of Cancer Research mice, humanized liver mice, and Sprague-Dawley rats. AMT was identified as a dual substrate and a reversible inhibitor of CYP2D6. Quinidine inhibition of CYP2D6-mediated AMT metabolism showed marked species differences (IC50, human < IC50, mouse). AMT inhibited MATE1 with an IC50 of 18.75 μM, with no significant activity against MATE2-K/organic cation transporter 1/2. In humanized liver mice, quinidine coadministration increased AMT exposure (AUCRobs = 2.87); in rats, AMT coadministration elevated metformin (MATE1 substrate) area under the curve by 68%. Static models predicted that CYP2D6 PMs may have 5- to 10-fold higher AMT exposure, amplifying MATE1 inhibition to increase metformin area under the curve ratio (AUCR) to 2.5-4.1. Coadministration of quinidine was projected to increase metformin AUCR to 2.2. AMT clinical development requires rigorous assessment of the elevated systemic exposure when coadministered with potent CYP2D6 inhibitors. Coadministration with MATE1 substrates (eg, metformin) may delay AMT renal excretion, with superimposed metabolism-transport DDIs posing heightened risk in patients with CYP2D6 PMs or renally impaired. This study highlights the importance of integrating metabolic and transporter pathways for comprehensive DDI risk assessment. SIGNIFICANCE STATEMENT: This study reveals that the novel antidepressant ammoxetine poses dual drug interaction risks as a cytochrome P450 2D6 (CYP2D6) substrate/inhibitor affecting metabolism and a multidrug and toxin extrusion protein 1 inhibitor impairing renal excretion. Using humanized models and static mechanistic models, the interactions were validate in vivo and characterize the proposed metabolism-transport interaction cascade, delineating clinically relevant risks, including amplified ammoxetine exposure with strong CYP2D6 inhibition and heightened vulnerability in CYP2D6 poor metabolizers.

The Nairoviridae family of segmented negative-sense RNA viruses includes the serious human pathogen Crimean-Congo haemorrhagic fever virus (CCHFV), associated with a case/fatality rate of up to 40% for which no approved vaccines or treatments exist. Nairoviruses internalize via endocytosis and pass through the endolysosomal network, exploiting the changing ionic environment to promote envelope fusion. Fusion is influenced by hydrogen (H[+]) and potassium ions (K[+]), which increase in concentration as endosomes mature, regulated by host ion channels. Using the model nairovirus Hazara virus (HAZV) of the CCHFV serogroup, we performed an siRNA screen to identify cellular ion channels involved in nairovirus infection. Most high-ranking hits belonged to K[+] and calcium (Ca[2+]) channel families. Consistent with this, we showed that clinically-approved K[+] channel blockers quinidine, quinine and dronedarone and clinically-approved Ca[2+] channel blockers tetrandrine and nifedipine significantly reduced HAZV activities. To further probe the role of K[+] in HAZV infection, we used time-of-addition studies, showing K[+] was required during entry. Biochemical experiments showed K[+] expanded the pH range that promoted entry, potentially allowing endosome escape deeper within the endolysosomal network. These results show clinically-approved channel blockers effectively inhibit HAZV replication, suggesting repurposing existing therapies may represent promising avenues to block nairovirus infection.

We show that solvent selection plays a decisive role in chiral resolution of RS-phenprocoumon-quinidine. In particular, formation of a bis-acetone solvate suppresses solid solution formation, and affords spontaneous enantioselective crystallization of S-phenprocoumon with enantiomeric excesses of up to 92% in a single resolution step.

Voltage-gated sodium channel NaV1.5 is essential for cardiac excitability, mediating the rapid depolarization phase of the cardiac action potential (AP) and ensuring proper electrical conduction in the heart. Dysfunction of NaV1.5 is implicated in life-threatening arrhythmias, making it a critical therapeutic target. Acting as a NaV1.5 open-state blocker, quinidine demonstrates efficacy in arrhythmia treatment, but its low specificity restricts its clinical application. Here, we report an optopharmacological strategy that enables a precise and optical control of NaV1.5 function by means of photoswitchable quinidine derivatives. Through systematic structural optimization, we identify azo-Q2a as a high-performance photoswitchable inhibitor, exhibiting low activity in the dark or under 480 nm light irradiation (trans isomer), while approximately 7-fold higher efficacy is observed under 365 nm light irradiation (cis isomer). Of note, azo-Q2a demonstrates exceptional selectivity for NaV1.5 over cardiac ion channels and other NaV1 subtypes, minimizing potential off-target effects. Furthermore, by solving the cryo-EM structure of the NaV1.5 in complex with the cis-active isomer azo-Q2a (3.0 Å resolution), we reveal the essential binding site that is responsible for the optical control of NaV1.5. Finally, azo-Q2a also attenuates the heart rate of living zebrafish larvae with light, showing its potential in cardiac-related research and treatment. Our work not only establishes azo-Q2a as a robust photoswitchable inhibitor for NaV1.5 but also provides a structural blueprint for the rational design of next-generation optopharmacological antiarrhythmic agents.

The ease of hydrogen-activation by platinum-group metals in combination with metal nanoparticle-halloysite support interactions impacts the catalytic hydrogenation of tetrasubstituted alkenes. Thus, Pd, Pt, and Ru nanoparticles were synthesized in a one-pot process using quinidine as a stabilizer, immobilized on halloysite nanotubes at low and high metal loadings. Structural analyses revealed that low metal loading favors small, well-dispersed nanoparticles, whereas higher loading induces aggregation except for Ru. Among all materials, Pd-A (4.1 wt% Pd) displayed the best balance between particle size and homogeneous distribution on the support, resulting in the highest catalytic activity and chemoselectivity. Moreover, Pd-A enables the hydrogenation of sterically demanding endocyclic and exocyclic oxazolidinones under mild conditions (3 bar H2, 80 °C-100 °C), affording the corresponding saturated products with complete syn-diastereoselectivity. Deuteration studies using multi-nuclear NMR demonstrate that hydrogenation of the endocyclic oxazolidinone proceeds via direct addition to the tetrasubstituted C═C bond, accompanied by benzylic H/D exchange arising from C-H activation. Overall, the study of the catalytic activity of the as-prepared metal nanocomposites in terms of reactivity and selectivity permitted to conclude that Pd-A, a zero-valent Pd-halloysite nanocomposite, is the best heterogeneous catalyst of the series in terms of activity and selectivity for the hydrogenation of sterically hindered alkenes.