Molecular Pharmaceutics最新文献

Nectin-4 is highly expressed in several malignancies, including triple-negative breast cancer, and it represents an attractive target for molecular imaging and therapy. In this study, we report the first head-to-head comparison of two structural optimization strategies for Nectin-4-targeted peptide radiotracers: peptide dimerization (for the development of ⁶⁸Ga-DOTA-HTA-DM) and sulfonyl fluoride modification (for the development of ⁶⁸Ga-DOTA-HTA-SF). The dimeric peptide DOTA-HTA-DM achieved a pronounced affinity gain (SPR apparent K_D ≈ 0.37 nM), consistent with the bivalent binding mode predicted by docking and molecular dynamics simulations. In contrast to the lead compound N188, the sulfonyl fluoride-modified peptide DOTA-HTA-SF retained low-nanomolar affinity, and the ⁶⁸Ga radiolabeled probe exhibited markedly higher tumor uptake at early time postinjection (∼5% ID/g at 30 min) and sustained tumor retention (>4% ID/g at 2 h), resulting in superior tumor-to-background contrast. Both radiotracers were predominantly cleared through the renal system, and blocking studies confirmed their Nectin-4-mediated tumor accumulation. Taken together, these findings demonstrate that dimerization enhances molecular recognition through multivalency, while sulfonyl fluoride modification prolongs tumor residence and improves imaging contrast. The complementary advantages of these two strategies establish a rational framework for the design of next-generation Nectin-4-targeted radiotracers.

The goal of the current study was to assess the release rate of model high glass transition temperature (T_g) drugs from amorphous solid dispersions (ASDs) as a function of drug loading, and then to evaluate the permeation rate of the resultant solutions using Caco-2 cells. Ivacaftor and ARV-825 were selected as model drugs and were formulated as ASDs with hydroxypropyl methylcellulose acetate succinate (HPMCAS). Release was found to be very slow or not detectable at higher drug loadings. Dramatically improved release was observed upon addition of 10 wt % glyceryl tributyrate to the ASDs, which reduced T_g. Nanosized drug-rich amorphous droplets, generated upon dissolution of ivacaftor ASDs, enhanced the Caco-2 membrane permeation rate, likely by partitioning into the unstirred water layer (UWL) at the surface of the membrane and reducing the concentration gradient across the UWL. The extent of improvement was correlated with the size of droplets: smaller droplets resulted in faster permeation rates. Addition of glyceryl tributyrate, while increasing the release rate, decreased the permeation rate due to formation of larger droplets. In conclusion, the addition of a plasticizer to an ASD containing a high T_g drug led to an improvement in release rate but increased the size of drug-rich nanodroplets produced via the release process with unknown potential implications for in vivo performance.

Biparatopic antibodies (BpAbs) are an attractive format of engineered therapeutic antibodies that bind to two distinct epitopes of a single antigen. Accelerated cell internalization is anticipated in many developmental campaigns, and both bivalent and tetravalent forms have been developed for this purpose. However, their pharmacokinetic properties have not been understood systematically; thus, optimization approaches for BpAbs have been limited. In this study, we conducted a comparative biodistribution analysis of bivalent and tetravalent BpAbs using the same variable fragments. Two different pairs of epitopes of human epidermal growth factor receptor 2 (HER2) were targeted, and their distribution was evaluated in a tumor xenograft model by ¹¹¹In-labeling. Bivalent BpAbs showed higher accumulation in tumors than tetravalent BpAbs in both cases. However, epitope-dependent differences in biodistribution did not correlate with those of the original monoclonal antibodies. In addition, cell internalization during the early stages of incubation was higher for tetravalent BpAbs. These results suggest the advantage of bivalent BpAbs over tetravalent BpAbs in pharmacokinetics; however, the design may require optimization depending on the mechanism of action of the BpAb of interest.

Conventional nanomedicines frequently suffer from rapid systemic clearance via the mononuclear phagocytic system (MPS) and off-targeting, which severely limits their therapeutic index. Recently, new attempts using cell membrane-derived nanoparticles (CDNs) have been proposed as a novel platform of drug carriers. To address these challenges, we report a biomimetic drug delivery platform utilizing erythrocyte-derived nanoparticles (EDNs) that leverage the actual "self" signaling of erythrocyte cell membranes. But many ligand functionalizations often rely on monoclonal antibodies, which are frequently hampered by antidrug antibody (ADA) response and limited tumor penetration due to their bulky size. In this study, we engineered a next-generation biomimetic nanocarrier by conjugating anti-EGFR targeting aptamers onto EDNs. And we successfully encapsulated doxorubicin (DOX) into EDNs using an optimized phosphate gradient method, achieving a high loading efficiency of 38% while preserving the structural integrity of membrane proteins. The size of Apt-EDNs-DOX was measured by the dynamic light scattering (DLS) method, 215 nm in diameter. Furthermore, in vitro assays confirmed that the aptamer-mediated targeting significantly enhanced intracellular drug delivery and selective cytotoxicity in MDA-MB-231 (EGFR+) compared to MDA-MB-453 (EGFR-). In vivo therapeutic evaluation in a tumor xenograft mouse model demonstrated significant tumor growth inhibition and a favorable safety profile compared with the free drug. These findings suggest that substituting antibodies with aptamers represents a crucial advancement in developing more stable, less immunogenic, and highly efficient targeted nanomedicines for clinical translation in cancer therapy.

Human rhinovirus (HRV) is a highly widespread pathogen, the most frequent cause of the common cold, and often associated with asthma exacerbation. To date, attempts to develop direct-acting antivirals (DAAs) have proved unsuccessful, also due to their tendency to select resistant variants when challenged with HRV quasispecies. 27-hydroxycholesterol (27OHC), a cholesterol-derived host-targeting antiviral (HTA), inhibits HRV replication and is less prone to selecting resistant variants than the DAAs pleconaril and rupintrivir. In the present study, we developed and evaluated a lipid nanoparticle (LNP)-based formulation for the nasal delivery of 27OHC. The antiviral efficacy of 27OHC-loaded LNPs was assessed on HeLa cells by focus reduction assays and yield reduction assays. The effect on cell viability and the cytotoxicity were determined via MTS and LDH assays to calculate the 50% cytotoxic concentration (CC50). Efficacy and biocompatibility of 27OHC were further validated in a physiologically relevant 3D model of reconstituted human nasal epithelia derived from healthy donors. Cellular uptake and internalization kinetics of LNPs were assessed on HeLa cells with the use of fluorochrome-tagged LNPs and indirect immunofluorescence. Our results demonstrate that 27OHC-loaded LNPs strongly inhibit HRV infectivity at 50% effective concentration (EC50) in the low micromolar range and are characterized by a selectivity index (SIs = CC50/EC50) above 150. Importantly, the adopted formulation suppressed viral replication in the nasal epithelium without cytotoxic effects. The uptake experiments show that LNPs enter cells and are clearly detectable intracellularly at 24 h post-treatment. These findings highlight the therapeutic potential of 27OHC delivered via LNPs as a promising host-targeting strategy against HRV and provide a rationale for further studies aiming to explore its potential in a preclinical setting.

Covalent radiopharmaceuticals are emerging as a powerful new class of agents for cancer imaging and therapy, offering durable target engagement that overcomes the key limitations of conventional, reversible tracers. By forming covalent bonds with nucleophilic residues on or near disease-relevant proteins, covalent radiopharmaceuticals can achieve prolonged tumor retention, improved target selectivity, and enhanced imaging contrast, or therapeutic efficacy. This strategy is particularly well-suited to addressing biological challenges such as rapid internalization, low target abundance, and tumor heterogeneity, where noncovalent agents often underperform. Recent advances have demonstrated the versatility of covalent radiopharmaceuticals across a range of molecular formats, including small molecules, protein binders, and peptidomimetics. These agents have been engineered with diverse covalent targeting moieties that enable selective and stable binding under physiological conditions. In preclinical and early clinical studies, covalent tracers have shown superior tumor retention and, in some cases, improved performance over standard-of-care agents. Importantly, covalent design also allows for greater alignment between tracer pharmacokinetics and radionuclide decay, improving dosimetry and expanding therapeutic windows. While challenges remain in optimizing covalent handle reactivity and minimizing off-target effects, ongoing innovations in synthetic chemistry and protein engineering are rapidly advancing the field. As the mechanistic and translational advantages of covalency become increasingly clear, covalent radiopharmaceuticals are poised to redefine molecular imaging and therapy. They are not merely specialized tools but are foundational components of next-generation precision oncology.

Amorphous solid dispersions (ASDs) are a commonly used formulation approach for poorly water-soluble drugs to enhance release rates, generate supersaturated solutions, and improve oral absorption. Hypromellose (HPMC) has been used in commercial ASD formulations, but relatively little is known about drug release mechanisms from ASDs based on this polymer. Herein, confocal fluorescence microscopy (CFM) was employed to reveal the morphology of phase separation in tacrolimus/HPMC ASDs in aqueous media. Release and absorption performance of different drug loading (DL) ASDs were monitored in vitro and in vivo. CFM revealed that at 10% DL and above, a drug-rich network formed while the polymer diffused into the bulk solution. Surface area-normalized release rate measurements confirmed this observation revealing that HPMC was released 10× faster than tacrolimus at 10% DL, while at 5% DL, the release rates were congruent. Despite reduced drug release rates at 10% DL, sufficiently high surface area and moderate agitation enabled powder formulations to completely release within 40 min and achieve supersaturation under nonsink release conditions. At the commercial DL of 50%, a lower extent of supersaturation was achieved in comparison to the 10% DL. However, the surface area-normalized release rate was 100 times than that estimated for the crystalline form. In vivo absorption data in fasted dogs reflected this difference, with the 50% DL formulation significantly outperforming the crystalline drug with further improvements observed with the 10% DL formulation. This study connects the observations from CFM, surface area-normalized release rates, powder dissolution, and in vivo absorption data to the performance of ASDs at different drug loadings.

Targeted radionuclide therapy (TRT) has emerged as a promising strategy for cancer treatment. While TRT offers potent therapeutic efficacy, its clinical application requires careful control due to the risk of systemic toxicity arising from off-target distribution of radionuclides. A key strategy to minimize such toxicity and enhance targeting efficiency lies in selecting an appropriate chelator that forms highly stable complexes with radionuclides and maintains their integrity under physiological conditions. This study aimed to computationally assess the chelation compatibility of four clinically relevant chelators (DOTA, NOTA, NODAGA, and TETA) with various therapeutic and diagnostic radionuclides. Chelator-radionuclide interactions were evaluated using density functional theory (DFT) computational modeling. The thermodynamic stabilities of the chelator-radionuclide complexes were evaluated using interaction energies (E_int), with lower values indicating stronger coordination. Coordination geometries, compatibility between internal cavity volumes of chelators and radii of radionuclides, and charge distributions upon chelation were also assessed to provide a comprehensive evaluation. Computational predictions were validated against existing literature to assess their agreement in coordination geometries and chelator-radionuclide compatibility. DOTA exhibited moderate-to-strong chelation affinity across various radionuclides, generally forming stable 8-coordinate complexes, but with a marked preference for medium-sized ions (e.g., Lu³⁺: -4.99 eV vs Ac³⁺: -2.45 eV; E_int). NOTA and NODAGA, which possess smaller cavity volumes (7.29 ų and 4.56 ų, respectively; vs 15.29 ų for DOTA), showed strong size-selective affinity toward smaller metal ions. TETA, structurally flexible, preferentially formed stable 6-coordinate complexes with smaller trivalent ions such as Ga³⁺ (-4.32 eV; E_int). Charge neutrality was identified as a critical factor for chelation, as neutral complexes exhibited more homogeneous electrostatic environments and improved stability compared to charged complexes. This computational study identifies three key factors─cavity volume compatibility, structural rigidity/flexibility, and charge neutrality─as critical determinants of chelator-radionuclide stability. By providing predictive insights into chelation behavior, our validated DFT modeling supports the rational selection and optimization of chelators, with direct implications for the development of safer and more effective theranostic radiopharmaceuticals in TRT.

Nonsmall cell lung cancer (NSCLC) remains a leading cause of cancer-related mortality, with MYC oncogene overexpression driving tumor progression and immunosuppression. MYC has been deemed "undruggable" for a long period, and the impact of its silencing on the tumor associated macrophage polarization and circadian rhythm remains unexplored. Here, we developed a lipid nanoparticle (siMYC@Dmix) composed of cytidinyl lipid (DNCA), gemini-like cationic lipid (CLD), and DSPE-PEG2000 for efficient MYC siRNA delivery. In vitro, siMYC@Dmix showed robust cellular uptake, lysosomal escape, and ∼77% MYC mRNA silencing in Lewis Lung Carcinoma (LLC) cells. In vivo, siMYC@Dmix treatment significantly inhibited tumor growth in C57BL/6J mice and induced a profound remodeling of the tumor immune microenvironment. This was characterized by a shift in macrophage polarization toward the M1 phenotype, increased infiltration and cytotoxic function of CD8⁺ T cells, enhanced natural killer (NK) cell activity, and maturation of dendritic cells (DCs). Crucially, MYC silencing restored the expression of core circadian clock genes. Our findings unveil a promising RNAi-based strategy that concurrently targets MYC-driven tumorigenesis, corrects circadian dysfunction, and reinstates antitumor immunity, presenting a multifaceted therapeutic approach for NSCLC.