Molecular Pharmaceutics最新文献

Tyrosine protein kinase c-Met, encoded by the Met gene, is a membrane-associated receptor tyrosine kinase that is often aberrantly expressed in a wide range of tumors. The development of imaging probes specifically targeting c-Met is critical for improving cancer diagnostics. In this study, we successfully designed and fabricated an aptamer molecular imaging probe ([⁶⁸Ga]Ga-NOTA-SL1) with high radiochemical purity (RCP), good stability in vitro, and high affinity for c-Met expressed tumors. As shown by the micro-PET/CT scanning, [⁶⁸Ga]Ga-NOTA-SL1 efficiently imaged tumor models with varying c-Met expression. The quantitative analysis of micro-PET/CT showed tumor uptake of [⁶⁸Ga]Ga-NOTA-SL1 in the HCC827 tumor models (30 min, 2.93 ± 0.64%ID/g; 60 min, 2.03 ± 0.67%ID/g; 90 min, 1.63 ± 0.61%ID/g), PC-9 tumor models (30 min, 2.1 ± 0.72%ID/g; 60 min, 1.7 ± 0.56%ID/g; 90 min, 1.33 ± 0.38%ID/g), and HCT116 tumor models (30 min, 1.4 ± 0.17%ID/g; 60 min, 1.23 ± 0.15%ID/g; 90 min, 0.97 ± 0.21%ID/g). The results of immunohistochemistry (IHC) further confirmed the targeting ability of [⁶⁸Ga]Ga-NOTA-SL1 to c-Met from a molecular pathological perspective. The probe effectively imaged c-Met-positive tumors and demonstrated a favorable metabolism profile and targeting performance in non-small cell lung cancer (NSCLC) or colorectal cancer tumor models. Consequently, this probe shows promise as an imaging agent capable of providing valuable diagnostic insights into tumors with aberrant c-Met expression.

Cancer-associated fibroblasts (CAFs) are essential components of the tumor microenvironment. Fibroblast activation protein (FAP) is overexpressed in CAFs. FAP-targeted molecular imaging agents, including the FAP inhibitors (FAPIs), have shown promising results in tumor diagnosis. We aimed to design a Gd-labeled FAPI Dimer, Gd-DOTA-Suc-Lys-(FAPI04)₂, to optimize the pharmacokinetics and evaluate its potential capacity for targeting FAP-positive solid tumors in vivo. The Gd-labeled FAPI Dimer was successfully synthesized with exceeding 98% purity. Preclinical pharmacokinetics were determined in assessed FAP-positive U87 cell-derived xenografts and FAP-negative C6-derived xenografts using small-animal T1-weighted 7.0T MR imaging. The longitudinal correlation coefficient (r1) of the agent was 3.813 mM^-1·S^-1. The administration of the Gd-FAPI04 Dimer probe showed a notable enhancement of tumor contrast on T1-weighted whole-body MRI. At 10 and 30 minutes post-injection, the U87 subcutaneous tumor demonstrated significantly greater contrast enhancement than the C6 subcutaneous tumor (P <0.05). In vivo, the safety of the Gd-FAPI-04 Dimer probe was evaluated, which showed no tissue damage in vital organs like the heart, liver, spleen, lung, and kidneys, as indicated by unchanged morphology compared to a normal saline control group. The novel Gd-FAPI04 Dimer molecular probe, Gd-DOTA-Suc-Lys-(FAPI-04)₂ specifically targeting FAP may serve as a safe and promising tool for the diagnostic imaging of solid tumors.

Nuclear protein localization 4 (NPL4) plays a key role in the ubiquitination pathway and has emerged as a promising target for cancer therapy. The ditiocarb-copper complex, Cu(DDC)₂, an anticancer metabolite derived from the antialcoholism drug disulfiram (DSF), exhibits a high affinity for NPL4. Thus, quantifying NPL4 expression in tumors is crucial for ubiquitination research and for developing NPL4-targeted diagnostic and therapeutic strategies. In this study, we replaced the cold copper ion in Cu(DDC)₂ with the positron-emitting isotope copper-64 and developed three methods for visualizing NPL4 in tumors in vivo using positron emission tomography/computed tomography (PET/CT): (1) an in vivo "synthesis-free" method for preparing [⁶⁴Cu]Cu(DDC)₂, (2) an in vitro synthesis method, and (3) a stabilization method using PEG5000-PLA5000 (PP) to enhance [⁶⁴Cu]Cu(DDC)₂'s hydrophilicity by preparing [⁶⁴Cu]Cu(DDC)₂ NPs. Micro-PET/CT imaging showed minimal uptake of [⁶⁴Cu]Cu(DDC)₂ in NPL4-positive tumors with the in vivo "synthesis-free" method, resulting in poor lesion visualization. However, in vitro synthesized [⁶⁴Cu]Cu(DDC)₂ and [⁶⁴Cu]Cu(DDC)₂ NPs successfully visualized NPL4-positive U87MG tumors. Compared to [⁶⁴Cu]Cu(DDC)₂, [⁶⁴Cu]Cu(DDC)₂NPs demonstrated significantly higher tumor uptake (7.2 ± 0.7% ID/g vs 3.8 ± 0.6% ID/g at 12 h postinjection, P = 0.001) and tumor-to-muscle (T/M) ratio (7.8 ± 1.2 vs. 3.2 ± 0.7, P = 0.001). Tumor uptake of [⁶⁴Cu] Cu (DDC)₂NPs was consistent with NPL4 expression levels and was inhibited by an excess of Cu(DDC)₂. The optimal PP stabilizer concentration was determined to be 0.0005%. This study successfully developed a PET probe, [⁶⁴Cu]Cu(DDC)₂NPs, and established a novel imaging modality for in vivo visualization of NPL4 expression, potentially guiding future NPL4-targeted therapies.

Intrinsic differential scanning fluorimetry (DSF) is essential for analyzing protein thermal stability. Until now, intrinsic DSF was characterized by medium throughput and high consumable costs. Here, we present a microplate-based intrinsic DSF approach that enables the measurement of up to 384 samples in parallel by consuming only 10 μL per sample. We systematically test and benchmark the new intrinsic DSF against gold-standard methods such as differential scanning microcalorimetry and circular dichroism. Using a range of model proteins and sample conditions, we demonstrate the robustness and versatility of the intrinsic DSF method for characterizing protein stability and ranking protein drug candidates. In addition, we demonstrate modulated scanning fluorimetry (MSF) capabilities on the intrinsic DSF hardware that enable simultaneous MSF measurements in 384-microwell plates. Overall, the presented technology is a powerful tool for the early stability analysis of various protein samples and drug candidates.

Breast cancer has the highest incidence rates among all cancers, which represent a global health concern. Effective chemotherapy for breast cancer must minimize adverse effects to improve patient outcomes. Palbociclib (PB), a CDK 4/6 inhibitor, restricts cell growth and suppresses DNA replication in the retinoblastoma tumor suppressor gene (RB). Despite its breakthrough status postapproval, PB is associated with severe side effects, including neutropenia, leukopenia, infections, and thrombocytopenia. The current study aims to develop and optimize a PB-loaded lipidic nanocarrier. The development method was solvent evaporation, and formulation optimization was performed using a central composite rotatable design. Characterization of the nanostructured lipid carrier (NLC) showed a particle size of 129.8 ± 7.6 nm with a PDI of 0.2694 ± 0.04 and a zeta potential of -29.8 ± 2.4 mV. Surface morphology was studied using transmission electron microscopy, which confirmed the particles' uniform and spherical shape. In vitro release studies in 0.1 N HCl and pH 6.8 phosphate buffer demonstrated cumulative drug releases of 91.23 ± 2.1% and 72.9 ± 2.0%, respectively. Intestinal permeation studies demonstrated a 3.76-fold increase in gut permeation with PB-NLC compared to that with PB-Sus. The lipolysis study indicated an enhanced drug availability at the site of absorption. Confocal studies revealed improved drug penetration depth in the intestine with PB-NLC compared to that with PB-Sus. In vivo pharmacokinetic studies demonstrated that incorporating PB into a lipidic nanocarrier (PB-NLC) significantly enhanced its bioavailability by approximately 5.9-fold (p < 0.05) compared to PB suspension. Additionally, acute toxicity studies in Wistar rats confirmed the safety of the developed NLC for oral administration in managing breast cancer. Therefore, the PB-loaded NLC shows significant promise for breast cancer treatment, providing improved drug delivery and minimized side effects.

Due to the increased expression of iron storage proteins in cancer cells, utilizing the endogenous iron-catalyzed Fenton reaction for cancer ferroptosis therapy has recently emerged as a prominent research focus. However, endogenous iron primarily exists within ferroxidase FTH1 in the Fe (III)-bound state, hindering the effective catalysis of the Fenton reaction. Herein, an endogenous iron(II) self-enriched Fenton nanocatalyst (BAI@cLANCs) is fabricated by encapsulating the FTH1 inhibitor baicalin (BAI) in cross-linked lipoic acid nanocarriers (cLANCs) to amplify endogenous ferroptosis. Once internalized, BAI@cLANCs are disrupted by glutathione (GSH) in tumor cells to release BAI, which inhibits FTH1 activity and hinders Fe²⁺ oxidation. Meanwhile, cLANCs degrade into dihydrolipoic acid (DHLA), which reduces Fe³⁺ to Fe²⁺, synergically enriching endogenous Fe²⁺. Simultaneously, both BAI and DHLA stimulate H₂O₂ production and facilitate the Fenton reaction to produce abundant ^·OH, thereby triggering lipid peroxidation and inducing tumor ferroptosis. Moreover, the reduction of Fe³⁺ to Fe²⁺ depletes GSH, facilitating ^·OH production and inactivating glutathione peroxidase-4, ultimately amplifying tumor ferroptosis. Overall, this work highlights the potential of an endogenous iron(II) self-enriched Fenton nanocatalyst for cancer ferroptosis therapy, providing a paradigm for amplifying endogenous ferroptosis by inhibiting FTH1 activity and reducing iron(III) to enrich endogenous iron(II).

The first-in-class oncolytic peptide LTX-315 has exhibited positive anticancer responses in multiple phase I/II clinical trials. Nevertheless, the linear peptide LTX-315 suffers from poor proteolytic stability and undesired toxicity, especially hemolysis, which may limit its widespread applications. Except for the direct structural modifications, drug delivery systems (DDSs) are expected to protect LTX-315 from degradation and shield its hemolytic properties. Therefore, the LTX-315 and zeolitic imidazolate framework (ZIF-8)-based nanoparticles (NPs) were constructed with a high LTX-315 encapsulation rate of 59.9%, utilizing the biomineralized "one-pot method" in an aqueous system. The release of LTX-315, in vitro anticancer potency, serum stability, anticancer durability, antimigration activity, hemolysis effect, subcellular localization, and the membrane disruption/permeation effects of LTX-315@ZIF-8 NPs were investigated. LTX-315@ZIF-8 NPs exhibited potent cytotoxicity against cancer cells. The serum stability experiment and time-inhibition curve assay indicated that ZIF-8 NPs could effectively improve the stability of LTX-315, prolong the duration of anticancer action, and enhance the cytostatic potency. Especially, the LTX-315@ZIF-8 NPs not only effectively attenuated the hemolytic toxicity of LTX-315 but also achieved the pH-responsive release of LTX-315. The mechanism investigation indicated that LTX-315@ZIF-8 NPs possessed potent membranolytic activity and reduced the mitochondrial membrane potential to trigger cell death. Collectively, this paper not only established a robust strategy to improve the stability and reduce the hemolytic properties of LTX-315 but also provided a reliable reference for the future delivery of oncolytic peptides.