Molecular Pharmaceutics最新文献

Mucin 17 (MUC17), a transmembrane mucin, is overexpressed in pancreatic cancer and is associated with tumor proliferation and metastasis. CD3 is an indispensable molecule on the surface of T lymphocytes, which is associated with T cell activation and participates in immune responses. Here, we developed a bispecific T-cell engager radiotracer, ⁸⁹Zr-M17C3, targeting MUC17 and CD3, to enable noninvasive PET imaging of both tumor cells and T-cell infiltration in pancreatic cancer. ⁸⁹Zr-M17C3 was synthesized by conjugating AMG199 with zirconium-89 and verified for its radiochemical purity and in vitro stability. The ⁸⁹Zr-M17C3 probe demonstrated excellent radiochemical purity (>99%) and stability (maintained ≥99% over 120 h). Cellular uptake assays and binding affinity studies were conducted to evaluate the probe’s specificity for MUC17 and CD3. Micro-PET/CT imaging and biodistribution studies were performed in MUC17-expressing nude mice and CD3 humanized mice to assess probe uptake in tumors and T-cell-infiltrated tissues. In MUC17-expressing AsPC-1 tumors, probe uptake was significantly higher than in MUC17-negative PANC-1 tumors (SUVmax: 2.26 ± 0.18 vs 1.13 ± 0.14, P < 0.001) and was confirmed to be MUC17-dependent through blocking studies. In CD3 humanized mice, the probe was able to visualize both T-cell infiltration and MUC17-positive tumors, with peak uptake in AsPC-1 tumors (SUVmax: 2.35 ± 0.46) and spleen (SUVmax: 2.19 ± 0.40) at 216 h. Immunohistochemical analysis confirmed the spatial correlation between MUC17 expression and CD3-positive T-cell infiltration in AsPC-1 tumors but not in PANC-1 tumors. In summary, the ⁸⁹Zr-M17C3 radiotracer exhibited high affinity for MUC17 and CD3 and successfully differentiated MUC17-positive tumors from MUC17-negative tumors while simultaneously providing insight into the T-cell distribution. This study highlights the potential of ⁸⁹Zr-M17C3 as a versatile imaging tool to support patient stratification and therapeutic monitoring in tumor-targeted immunotherapy, particularly for bispecific T-cell engager-based approaches such as AMG199.

The aim of this work was to develop a physiologically based pharmacokinetic (PBPK) model for conversion of phosphate prodrugs to active drug via intestinal alkaline phosphatase (IAP) implementing a generalized modeling strategy. Fostemsavir and fostamatinib were chosen as model drugs since there is extensive clinical pharmacokinetic data following administration of oral formulations. LUA scripting was used to develop an “in vitro” to “in vivo” extrapolation of the conversion rate of prodrugs derived from Caco2 cell lines using an absolute IAP abundance approach. The Simcyp v23 platform was modified to generate a virtual population to reflect gastric emptying rates following administration of a moderate fat meal. The PBPK model predicted the results of three different extended-release (ER) tablets of fostemsavir under fasted and fed conditions as well as for powder in capsule and tablet immediate release (IR) formulations of fostamatinib. Retrospectively, the model was also able to assess the clinical relevance of the in vitro dissolution method to rate changes of different microcrystalline cellulose-based IR tablets of fostamatinib, observed in acidic media. All predictions were within 2-fold of the observed Cmax, AUC, and Tmax, with 81% being within 1.25-fold. The developed modeling strategy can be effectively adopted to increase the confidence of using PBPK modeling to prospectively assess the in vivo performance of phosphate prodrugs and support the development of optimal oral extended-release formulations for this class of drugs.

Glioblastoma (GBM) is a highly aggressive brain tumor with resistance to conventional therapies. Mithramycin (Mit-A), a potent antitumor agent, has shown promise in several tumor types including, GBM. However, its clinical application is limited by toxicity. To address this, we explored the use of milk-derived extracellular vesicles (mEVs) as a delivery system to enhance the therapeutic efficacy of Mit-A. In this study, mEVs were isolated using a 3000 PEG precipitation method and confirmed their size, morphology, and stability through dynamic light scattering (DLS), transmission electron microscopy (TEM), and atomic force microscopy (AFM). The isolated vesicles with a size of 125.6 ± 2.78 nm, a polydispersity index (PDI) of 0.083 ± 0.02, and a ζ-potential of 15 ± 0.57 mV. The presence of typical EV markers such as TSG101, HSP70, and CD63 confirmed their purity. Encapsulation of Mit-A within mEVs led to a slight increase in size to 131.8 ± 6.9 nm, a PDI of 0.081 ± 0.006, and a decrease in ζ-potential to -17 ± 2.0 mV, with an encapsulation efficiency of 58% by the freeze-thaw method. The in vitro transepithelial transport assay revealed that mEV(Mit-A) transported Mit-A more effectively than free Mit-A. The mEV(Mit-A) formulation demonstrated excellent stability in simulated salivary and gastrointestinal fluids, with a sustained release of Mit-A observed over 24 h in vitro in PBS (pH 6.8). Furthermore, mEV(Mit-A) formulations significantly inhibited glioma cell growth, and migration, and induced apoptosis, showing a 2-fold lower IC50 than free Mit-A, indicating superior efficacy. These findings suggest that mEVs represent a promising delivery vehicle for Mit-A, enhancing its potential as an effective treatment for glioblastoma.

Lipid nanoparticles (LNPs) have emerged as the premier drug delivery system for oligonucleotide vaccines and therapeutics in recent years. Despite their prosperous advancement in research and clinical applications, there is a significant lack of mechanistic understanding of the assembly of lipid particles at the molecular level. In our study, we utilized a combination of solution and solid-state NMR, together with molecular dynamics simulations, to elucidate local structures and interactions of chemical components across multiple motional regimes. Our results comprehensively evaluated the impact of formulation components and engineering process factors on the particle formation and identified the interplay of phospholipids (DSPC), poly(ethylene glycol) (PEG) lipid conjugates, and cholesterol in governing the particle size and lipid dynamics from a structural perspective, using static ³¹P NMR techniques. These studies provide novel insights into the impact of particle engineering on the molecular properties of the LNP envelope membrane. Additionally, molecular interactions and compositional distribution play a critical role in particle engineering and the consequent stability and potency. In this study, we have identified intermolecular contacts among the lipid components using one-dimensional ¹H–¹³C cross-polarization magic angle spinning experiments, ¹H relaxation measurements, and two-dimensional ¹H–¹H correlation methods, providing a structural basis for the lipid assembly. Interestingly, the cationic and ionizable lipids, conventionally regarded as stabilizing agents primarily located within the core of LNPs, were found to interact with PEG lipids and coexist in the outer layer of the particles. We suggest that LNPs examined here are comprised of an outer layer rich in lipid components surrounding a core region. Our high-resolution findings offer insightful structural and dynamic details pertaining to the individual chemical components in the lipid particles and their interactions influence lipid complex structure and stability in particle engineering.

Inflammation and oxidative stress are important features of traumatic ulcerative colitis (UC). Turmeric has been used as a dietary and functional ingredient for its potent anti-inflammatory effects in UC therapy. However, its practical effectiveness is hindered by limited reactive oxygen species (ROS) elimination properties. To address this, we constructed a unique treatment agent by growing cerium oxide (CeO₂) nanocrystals on the membranes of turmeric-derived nanovesicles (TNVs), named as TNV-Ce. The resulted TNV-Ce could suppress inflammation and exhibit exceptional ROS-scavenging activity, which was validated both in lipopolysaccharide-induced macrophages and dextran sulfate sodium salt-induced chronic colitis mouse model. Following oral administration, TNV-Ce significantly accumulated at inflamed sites, effectively eliminating ROS and inhibiting pro-inflammatory cytokines for synergistic action against UC.

To understand requirements for immunization via the oral mucosa, an in vitro model that recapitulates the physical barrier of the mouth, allows for quantification of antigen uptake and permeability and mounts an inflammatory response to antigen and adjuvant is needed. The physical structure of 4 models of the human oral mucosa was determined by histochemical staining and transepithelial electrical resistance (TEER) measurements. A TR146 based air-liquid interface (ALI) model most closely mimicked in vivo conditions. This was confirmed by validation studies using dextran and caffeine as diffusant molecules. Apparent permeability coefficients (P_app) of adenovirus (Ad) and adeno-associated virus (AAV) in this model were 4.3 × 10^-13 and 2.2 × 10^-10 respectively, while 100% of the total dose of H1N1 influenza remained in the epithelial layer. Sodium glycocholate and a hyperosmotic formulation improved the amount of Ad (p = 0.02) and AAV (p = 0.003) that entered the epithelium, respectively. Significant amounts of IL-6 (45.1 pg/mL), GM-CSF (94.7 pg/mL) and IFN-γ (4.3 pg/mL) were produced in response to influenza infection. Treatment with an AS03-like adjuvant induced production of IL-6 (34.9 pg/mL), TNF-∝ (43 pg/mL), GM-CSF (121.2 pg/mL) and IFN-γ (14.1 pg/mL). This highlights the contribution of differentiated epithelial cells to the immune response to vaccines and adjuvants.

Immunotherapy-induced tumor apoptosis is one of the crucial pathways in tumor cell death. This study aimed to explore the potential of PET imaging for noninvasively visualizing pivotal processes in immunotherapy, specifically immune activation and tumor apoptosis, by targeting granzyme-B and caspase-3. Bioinformatic analyses validated granzyme-B and caspase-3 expression in cancer tissues and their associations with immune infiltration and patient prognosis using the GEPIA and TIMER databases. Two radiolabeled probes, [⁶⁸Ga]Ga-GZP and [⁶⁸Ga]Ga-AC3, were used to specifically target granzyme-B and caspase-3 for PET imaging, respectively. CT26 xenograft tumor models were assigned to PD-1 inhibitor or PBS control groups to receive treatment every 3 days, with imaging conducted at baseline and after each treatment. Imaging results showed significantly increased tumor uptake of both [⁶⁸Ga]Ga-GZP and [⁶⁸Ga]Ga-AC3 in the ICB-treated group compared to controls, indicating early molecular changes in immune activation and tumor apoptosis. Immunofluorescence analysis further supported these findings, revealing upregulated granzyme-B and caspase-3 expression in treated tumor tissues. Immunohistochemistry also confirmed increased T-cell infiltration and elevated levels of effector molecules, such as IFN-γ and TNF-α, in the ICB group. This study demonstrates that granzyme-B and caspase-3 PET/CT can noninvasively visualize early molecular changes in immunotherapy-induced CD8⁺ T cell activation and tumor apoptosis. These noninvasive diagnostic techniques hold significant promise for future clinical applications, particularly for a more accurate evaluation of immunotherapy efficacy.

Ribonucleic acid (RNA)-based therapies represent a promising class of drugs for the treatment of non-small cell lung cancer (NSCLC) due to their ability to modulate gene expression. Therapies leveraging small interfering RNA (siRNA), messenger RNA (mRNA), microRNA (miRNA), and antisense oligonucleotides (ASOs) offer various advantages over conventional treatments, including the ability to target specific genetic mutations and the potential for personalized medicine approaches. However, the clinical translation of these therapeutics for the treatment of NSCLC faces challenges in delivery due to their immunogenicity, negative charge, and large size, which can be mitigated with delivery platforms. In this review, we provide a description of the pathophysiology of NSCLC and an overview of RNA-based therapeutics, specifically highlighting their potential application in the treatment of NSCLC. We discuss relevant classes of RNA and their therapeutic potential for NSCLC. We then discuss challenges in delivery and non-viral delivery strategies such as lipid- and polymer-based nanoparticles that have been developed to address these issues in preclinical models. Furthermore, we provide a summary table of clinical trials that leverage RNA therapies for NSCLC [which includes their National Clinical Trial (NCT) numbers] to highlight the current progress in NSCLC. We also discuss how these NSCLC therapies can be integrated with existing treatment modalities to enhance their efficacy and improve patient outcomes. Overall, we aim to highlight non-viral strategies that tackle RNA delivery challenges while showcasing RNA’s potential as a next-generation therapy for NSCLC treatment.

This study aimed to elucidate the drug absorption mechanisms after oral administration of lipid-based formulations (LBFs), emphasizing the impact of their composition on first-pass drug metabolism. Ketoconazole (KTZ), a CYP3A substrate, was loaded into two types of LBFs: a long-chain LBF (type II-LC) and a lipid-free formulation (type IV). Following oral administration of type II-LC, the systemic exposure of KTZ was lower compared to that for the type IV and a control suspension. However, pretreatment with 1-aminobenzotriazole, a nonspecific CYP inhibitor, revealed equivalent in vivo exposure among the formulations tested. The absorption of KTZ from type II-LC in the early period was slower than that from the suspension and type IV. Experiments on in vitro digestion in sequence with in vitro permeation across a dialysis membrane showed that the drug permeation rate for type II-LC was extremely low. This was probably due to the reduction in free drug molecules in the donor compartment via the incorporation of KTZ into mixed micelles comprising digestion products derived from type II-LC and bile components. Furthermore, luminal concentration measurements revealed that gastric emptying was delayed when a type II-LC was administered. The reduced free drug concentration and transient delay in gastric emptying of KTZ resulted in the slower absorption of KTZ for type II-LC. The product of the fraction of drug absorbed and fraction of the drug not metabolized in the gut wall (Fa × Fg) calculated from the systemic and portal plasma concentration–time courses of KTZ was 0.185 for type II-LC and 0.327 for suspension. Since the luminal concentration measurement demonstrated complete absorption of KTZ from the gastrointestinal tract (Fa ≅ 1), the Fa × Fg values can be regarded as Fg. In conclusion, the lower in vivo exposure following oral administration of type II-LC was attributed to reduced Fg, that is, slower drug absorption from the jejunum resulted in low KTZ concentration in enterocytes, leading to enhanced metabolic efficiency. Our findings can be valuable when selecting excipients for designing LBFs with the preferred in vivo performance for highly metabolized drugs.