Molecular Pharmaceutics最新文献

In this study, crystal engineering was employed to enhance the solubility and druggability of Chrysin (CHR). Four nitrogen heterocyclic compounds, including piperazine (PIP), 4,4′-bipyridine (BIP), imidazole (IMI), and sophoridine (SOP), were investigated using computational screening methodologies. Screening experiments were conducted to validate the computational screening results, and four CHR crystals were successfully prepared, three of which were reported for the first time. The structures of these cocrystals were characterized by using single-crystal X-ray diffraction (SXRD), powder X-ray diffraction (PXRD), and thermal analysis. The spatial structure, arrangement, interactions, and associations were analyzed. Additionally, physical stability, apparent solubility, and biological evaluation were performed to assess those cocrystals. Finally, the CHR-SOP cocrystal shows a significant improvement in solubility and dissolution rate, making it a promising candidate for further study.

Understanding the molecular basis that influences the viscosity behavior of antibodies is essential for the development of high-concentration antibody therapeutics. In this study, we investigated the pH-dependent viscosity behavior of four IgG₄ antibodies and dissected the structural and physicochemical factors contributing to their viscosity profiles. Two antibodies (mAb2 and mAb3) showed marked viscosity increases with rising pH, whereas the viscosity of mAb1 and mAb4 remained largely unchanged. To explore the mechanisms behind this divergence, we performed molecular dynamics simulations, surface charge mapping, and measurements of protein–protein interaction coefficients (k_D). Antibodies with increasing viscosity over pH displayed significantly larger solvent-accessible surface areas (SASA), prominent negative surface charge patches in the Fv region, and stronger intermolecular attraction at elevated pH. These trends were further validated by viscosity reduction under high-salt conditions, suggesting a key role of electrostatic interactions. These findings highlight that solvent exposure and localized charge distributions act synergistically to promote self-association, offering a framework to better understand and mitigate antibody viscosity in molecular design and formulation.

High-concentration protein formulations are increasingly demanded for the development of subcutaneous injections owing to its clinical and commercial advantages. However, their high protein concentrations can lead to dried residues, causing failures in manufacturing operations such as piston pump seizing and filling needle clogging. Transient dehydration during fill-finish holds can generate interfacial viscoelastic films that resist redissolution and perturb delivery performance. Here, we establish an operational framework that links dehydration exposure to film mechanics and reversibility for high-concentration biologics. Using a bench-scale apparatus with controlled airflow, we observe orders-of-magnitude increases in interfacial storage modulus (G′) with accumulated mass loss (m) and identify conditions where films fail to fully redissolve on minutes time scales. We define four portable readouts, maximum film strength (G_M^′), final film strength (G_F^′), imbibition time (t_Imb), and dissolving time (t_Dis)─to translate viscoelastic traces into process decision variables. Normalization by hydrodynamic volume fraction (ϕ_hyd) exposes two packing thresholds that organize mechanics and kinetics: a transition near random loose packing (RLP, ϕ_RLP ≈0.56), where percolation limits rearrangement and attenuates growth of G_M^′, and a second near random close packing (RCP, ϕ_RCP ≈0.64), where bulk jamming undercuts further strengthening and slows film disintegration. Consistently, t_Imb steeply scales once mass loss approaches ∼30% (mapping toward RCP), while t_Dis reflects a protein diffusion-dominated recovery. A critical ∼52–55% mass-loss window marks a sharp rise in G_F^′ and elastic persistence (G′ > G″) after imbibition, indicating conditional irreversibility on process time scales. Together, these results yield an actionable “no-film/at-risk” map indexed to exposure dehydration flux, time, and formulation, providing quantitative limits for downtime and rewet strategies in fill-finish.

Helicobacter pylori (H. pylori) infection affects about half the world population, and if left untreated, can cause painful sores in the stomach lining and intestinal bleeding, leading to peptic ulcer disease (PUD) and stomach cancer. Treatment of H. pylori infection is always a challenge to the treating doctor because of the treatment inefficiency resulting from the poor bioavailability of the drug at the inner layers of the gastric mucosa, where the bacteria reside. This also results in the development of antibiotic resistance. In this work, we developed a mucoadhesive gastroretentive drug delivery system (M-GRDDS) for the effective delivery of antibiotics and piperine to the gastric mucosa. The GRDDS system was formulated by using the ion-gelation method and was evaluated for its entrapment efficiency, particle size, swelling behavior, drug release, mucoadhesion property, and H. pylori eradication efficacy. The efficacy of the drug-loaded mucoadhesive GRDDS formulation was compared with that of the free drug. Results showed that the percentage entrapment efficiency was more than 80% for all the drugs. M-GRDDS beads showed controlled release at pH 1.2 and 7.4. The optimized mucoadhesive beads showed good in vitro mucoadhesion in X-ray photography, with a mean gastric residence time of more than 8 h in rabbits. Tissue distribution study in rats revealed local delivery of the drugs to the gastric mucosa from the M-GRDDS beads. The in vivo efficacy study performed on Sprague–Dawley rats showed that the colony-forming units in the group treated with the novel GRDDS formulation were fewer than those in the group treated with the free drugs. Biochemical tests, gene expression studies, and histopathology studies corroborated the enhanced efficacy of the M-GRDDS formulation in eradicating the infection and curing peptic ulcers. The results conclude that the developed M-GRDDS formulation holds significant potential for improving local bioavailability, contributing to the more effective eradication of H. pylori-based gastric ulcers.

Clopidogrel, a frequently used prodrug, is converted to its active metabolite through the intermediate 2-oxo-clopidogrel by cytochrome P450 (CYP) enzymes, which accounts for only 5%–15% of its metabolism. Majority of the clopidogrel dose (85%–90%) is extensively hydrolyzed to its inactive metabolite, clopidogrel carboxylic acid by carboxylesterase 1 (CES1). In vitro studies suggest the involvement of multiple CYP isoforms, with CYP1A2, CYP2C19, and CYP2B6 identified as major contributors to 2-oxo-clopidogrel formation. While CYP2C19 genetic polymorphisms are often highlighted as the primary factor contributing to variability in the clopidogrel response, the confounding role of CES1 variability on clopidogrel oxidation is less well understood. Our study utilizing proteomics-informed scaling highlights the importance of accurate estimation of the fraction metabolized (f_m) by CES1 and CYPs in clopidogrel metabolism. The results also indicate that differential subcellular localization of these enzymes and technical variability in sample preparation can influence f_m estimation, suggesting that HLM may not be an ideal model for investigating dual substrates of CYPs and CES. Quantitative proteomics and activity assays revealed significant variability in the absolute content and activities of CES1 and CYP enzymes across HLM donors (n = 10), which affected the estimation of f_mCES versus f_mCYP. Human hepatocyte assay, which represents a CYP versus CES abundance ratio similar to that in liver tissue, demonstrated the critical roles of CYP3A4 and CES1 abundance in the 2-oxo-clopidogrel formation rate. Further, enzyme kinetic studies identified CYP3A4 as the primary contributor to 2-oxo-clopidogrel formation, but multiple other enzymes, including CYP2C9, were identified as contributors. Overall, our findings emphasize the need for accounting for variability in both CES1 and CYP enzymes to improve f_m estimation in the in vitro to in vivo extrapolation of dual substrates of CYP/CES such as clopidogrel.

The freezing rate is a critical factor governing ice nucleation and growth during freeze-drying, which directly determines the microvoid architecture of the freeze-dried cake and its reconstitution properties. This study systematically evaluates the effects of controlled freezing methods on reconstitution time, formation of visible bubbles (VBs), and long-term stability of high-concentration monoclonal antibody (mAb) formulations─addressing a pivotal challenge in freeze-drying process development. Solid-state microstructure was analyzed using scanning electron microscopy and mercury intrusion porosimetry. Paradoxically, accelerated freezing rates had divergent effects on reconstitution: they shortened the reconstitution time but increased the level of generation of VBs. Nonetheless, protein stability─as assessed by size exclusion chromatography and ion-exchange chromatography, remained unaffected across different freezing rates. Importantly, employing an intermediate freezing rate significantly reduced VB formation and shortened the reconstitution time for high-concentration mAb formulations. These results underscore freezing rate modulation as an essential strategy for optimizing the reconstitution performance of freeze-dried high-concentration biologics without compromising stability, offering valuable insights for industrial pharmaceutical development.

Previous studies have demonstrated that induction of reactive oxygen species (ROS) significantly enhances the cytotoxicity of paclitaxel (Ptx). Tetrandrine (Tet), a potent ROS inducer, synergistically enhances the antitumor effects of Ptx. To codeliver Ptx and Tet, which have low solubility, high systemic toxicity, and poor tumor selectivity, we designed a tumor microenvironment-activatable prodrug-based delivery system. A ROS-responsive prodrug was developed by the conjugation of Ptx to a biocompatible polymer (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol), DSPE-PEG) via the ROS-cleavable thioketal (TK) linker, which could self-assemble into core–shell nanoparticles with Tet in the inner core to form redox-responsive Ptx/Tet-coloaded nanoparticles (P/T-NPs). The synergistic anticancer mechanism of Ptx and Tet was systematically investigated by cytotoxicity assays, ROS detection, mitochondrial tracing, transmission electron microscopy, and both in vitro and in vivo experiments. Cell uptake of P/T-NPs increased in a time-dependent manner, with partial accumulation observed in the mitochondria. The targeted release of Tet in tumor sites with high ROS levels could further elevate intracellular ROS, which in turn accelerated the cleavage of the TK linker and promoted the release of Ptx, thereby enhancing its antitumor effect. P/T-NPs showed superior cytotoxicity, which is strongly correlated to the superior autophagy-inducing ability of P/T-NPs. Moreover, P/T-NPs effectively inhibit the tumor growth compared to either the free drug or their combination therapy. This study integrates tumor microenvironment activation, self-amplified drug release, and ROS-enhanced chemotherapy into a single nanoplatform, offering a promising strategy for targeted cancer therapy.

Type 1 diabetes patients develop autoantibodies against pancreatic-islet-derived autoantigens, with proinsulin emerging as an early and dominant target. Existing antigen-specific immunotherapy (ASIT) strategies remain limited by poor antigen stability and rapid clearance, leading to reliance on global immunosuppression. To address these challenges, we engineered nonhormonal variants of proinsulin featuring cationic peptide “tails” at the C-terminus. All the expressed cationic proinsulin variants retained low-nM binding affinity to mouse anti-insulin mAb125 and exhibited low affinity for the insulin receptor. In addition, all variants exhibited physical stability profiles similar to those of proinsulin. A diffusion assay was performed in a viscous hyaluronic acid gel to simulate subcutaneous injection. The addition of a cationic binding motif to proinsulin slowed diffusion compared to that of unmodified proinsulin. Modifying autoantigens such as proinsulin with cationic “tails”, therefore, slows release, providing a depot-like effect with prolonged antigen presence at the injection site. This slow-release strategy also provided a steady accumulation of proinsulin in the draining lymph nodes. This strategy may enhance the efficacy of ASITs by negating insulin receptor activity, promoting autoimmune cell engagement, and sustaining exposure to draining lymph nodes.