Molecular Pharmaceutics最新文献

Kisspeptins (KPs) and their receptor (KISS1R) promote metastasis and tumor progression in various cancers such as triple-negative breast cancer (TNBC). Targeting KISS1R holds great promise for molecular imaging and targeted radionuclide therapy of aggressively disseminated cancers. First ligand-based approaches using Ga-68/Lu-177-labeled KPs (KP-10, KP-54) have demonstrated feasibility but suffer from proteolytic degradation and low uptake in KISS1R positive tumors. However, lead structure optimization alone is insufficient, as KISS1R biology remains unexplored in a radiotheranostic context. In this study, N-terminally functionalized conjugates of KP-10, KP-54, and the hybrid peptide KiSS-34 (AMBA-2-Nal-Gly-Leu-Arg-Trp-NH₂), including scrambled controls, were synthesized in high purity (≥95%) for comparative studies. The conjugation to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and Alexa-Fluor-488 (AF-488) functionalities preserved biological activity, confirmed by (sub)nanomolar EC₅₀-values (0.05-0.85 nM) in calcium mobilization assays in transfected CHO-KISS1R cells. Conventional target detection methods using antibodies (Abs) and AF-488-KPs failed to visualize KISS1R in both model (CHO-KISS1R) and native cancer cell lines, likely due to unspecific Abs and rapid KISS1R internalization upon agonist stimulation. However, rapid KISS1R internalization was successfully visualized via live-cell imaging using AF-488-KP-10 and novel analogue AF-488-KiSS-34. Furthermore, DOTA-KPs were radiolabeled with Lu-177 in high efficiencies (≥95%) and examined in internalization assays, showing highest uptake (4.8%) and internalization rate (45.9%) for [¹⁷⁷Lu]Lu-DOTA-KiSS-34 in CHO-KISS1R cells compared to its KP-10 analogue (total uptake: 1.3%; internalization rate: 37.6%). Higher uptakes likely derive from faster binding kinetics, improved KISS1R targeting, and/or slower dissociation as evidenced by oil-based kinetics assays showing higher total uptake for [¹⁷⁷Lu]Lu-DOTA-KiSS-34 (15.3%) compared to KP-10 (3.8%) and KP-54 (4.5%) counterparts after 30 min. Positron emission tomography/computerized tomography (PET/CT) imaging, urine analysis, and all in vitro studies indicate that Ga-68/Lu-177-labeled DOTA-KiSS-34 exhibits superior pharmacodynamics, pharmacokinetics, and in vivo stability compared to its KP-10 and KP-54 analogues, which are critically suffering from rapid in vivo degradation. These results position DOTA-KiSS-34 as a strong structural lead for KISS1R-based radiotheranostics. Nevertheless, the dynamics between KPs and KISS1R need to be further investigated to fully harness the radiotheranostic potential of KISS1R for TNBC and other cancers.

Anaplastic large cell lymphoma (ALCL) is an aggressive T-cell malignancy for which improved therapeutic strategies are urgently needed. CD25, the α-chain of the interleukin-2 receptor, is abundantly expressed on malignant T cells and represents a promising target for antibody (Ab)-based radiotherapeutics. We previously developed a cysteine site-specifically labeled ¹⁷⁷Lu-CD25 Ab that induced complete regression of SUDHL1 tumors but caused dose-limiting bone-marrow suppression at high activities. In the present study, we investigated whether combining reduced-dose ¹⁷⁷Lu-CD25 Ab radioimmunotherapy (RIT) with molecularly targeted inhibitors of ALK (crizotinib) or mTOR (everolimus) could enhance antitumor efficacy while minimizing systemic toxicity. In SUDHL1 cells, crizotinib markedly suppressed ALK phosphorylation, while everolimus potently inhibited phosphorylation of p70S6K, confirming effective pathway blockade. Furthermore, both agents dose-dependently suppressed SUDHL1 cell survival. In SUDHL1 xenografts, treatment with low dose (4.625 MBq) ¹⁷⁷Lu-CD25 Ab alone induced tumor apoptosis and reduced p70S6K activation, while combination therapy with either crizotinib or everolimus further increased cleaved PARP levels, indicating enhanced apoptosis. Longitudinal tumor-growth analysis demonstrated that low-dose ¹⁷⁷Lu-CD25 Ab combined with crizotinib or everolimus produced significantly greater tumor-growth suppression than monotherapies, yielding the lowest tumor burdens and smallest area-under-the-curve values. Kaplan-Meier analysis confirmed prolonged survival in all RIT groups, with the strongest benefit observed in the combination cohorts. Importantly, hematologic, hepatic, and renal parameters remained within normal ranges following combination therapy, reflecting the favorable safety profile of reduced-dose ¹⁷⁷Lu-CD25 Ab. These findings demonstrate that targeted inhibition of ALK or mTOR synergizes with CD25-directed ¹⁷⁷Lu RIT to enhance therapeutic efficacy without increasing toxicity. This combinatorial approach enables radiation-dose reduction while preserving antitumor potency, supporting further preclinical and translational development of ¹⁷⁷Lu-CD25-based combination RIT for ALCL and other CD25-expressing malignancies.

We report the design and synthesis of new conjugates (H₂L^1-4) consisting of a bifunctional bis(thiosemicarbazone) chelator conjugated to prostate-specific membrane antigen pharmacophores. Radiolabeling H₂L^1-4 with the ^99mTc nitrido core in a one-pot synthesis at 95 °C for 10 min produced the complexes [^99mTc][TcN(L^1-4)] with high radiochemical purity (RCP > 95%) and excellent stability. In vitro evaluation of the [^99mTc]Tc PSMA complexes demonstrated high cell-surface receptor binding and internalization in cancer cells expressing PSMA (PSMA+), which was inhibited by an excess of a PSMA-specific inhibitor. The most lipophilic of the complexes, [^99mTc][TcN(L²)], also showed significant nonspecific uptake in the control cell line (PSMA-). SPECT-CT imaging of the four complexes, at 1 and 4 h postinjection and biodistribution studies at 5 h postinjection in BALB/c nude mice with both PSMA+ and PSMA- tumors, demonstrated significant differences in the receptor-specific tumor uptake and clearance pathways. [^99mTc][TcN(L³)] and the bivalent [^99mTc][TcN(L⁴)] showed predominantly renal clearance and the highest PSMA+ tumor uptake and retention. Conversely, [^99mTc][TcN(L¹)] and [^99mTc][TcN(L²)] displayed gastrointestinal clearance and lower PSMA+ tumor uptake. The work demonstrates that bis(thiosemicarbazones) are effective bifunctional chelators for the development of ^99mTc radiopharmaceuticals targeting PSMA with the potential to be easily modified to target other diseases.

The rational design of high-concentration protein formulations remains a significant challenge in biopharmaceutical development, which requires the screening of effective excipients to suppress aggregation. This study presents a combined experimental and modeling approach to systematically evaluate the effects of key excipients (salts, sugars, polyols, and amino acids) on protein interactions through the measurements of the osmotic second virial coefficient (B₂₂) of bovine serum albumin (BSA) using static light scattering. The results demonstrate that the type and concentration of excipient significantly modulate intermolecular interactions, with repulsive interactions generally enhanced by sugars, polyols, and amino acids, while electrostatic shielding by salts reduces B₂₂. The exclusion volume (dominant)-water competition (secondary) mechanism was proposed to explain the distinct stabilizing behaviors of the sugars and polyols. In addition, the xDLVO model accurately captures the trends in B₂₂ as a function of excipient type and concentration. This framework provides a powerful predictive method for the early stage screening of excipients, facilitating the development of a stable high-concentration protein formulation.

Astatine-211 (²¹¹At) is a promising alpha emitter for cancer treatment, wherein tumor-selective accumulation is pivotal due to its short path length. While sodium ascorbate (SA) successfully protects radioactive antibodies from reactive oxygen species (ROS)-induced denaturation, it does not reduce ²¹¹At distribution in normal organs or mitigate body weight loss. Here, we aimed to attenuate this normal organ uptake. We demonstrated that sodium perchlorate (SP), a competitive inhibitor of the sodium iodide symporter (NIS) expressed in thyroid and gastric mucosal cells, significantly reduced ²¹¹At uptake in the stomach and thyroid in ²¹¹At-radioimmunotherapy (RIT) under SA protection. This favorable biodistribution resulted in significantly milder body weight loss without attenuating the antitumor effect. The combined strategy proved feasible, with no renal toxicity and no exacerbation of transient hematotoxicity or hepatotoxicity. Crucially, NIS inhibition significantly reduced DNA double-strand breaks in stomach and thyroid tissues and helped maintain the thyroid's follicular structure. Overall, we demonstrate that combining SA protection to prevent antibody denaturation with competitive NIS inhibition by SP for greater tumor-selective ²¹¹At delivery is feasible, broadens the therapeutic window, and facilitates the clinical application of ²¹¹At-RIT in cancer treatment.

The treatment of superficial tumors is often limited by poor drug targeting and a lack of real-time visualization of drug distribution. To address these challenges, we developed an RGD-modified microbubble system (TF/ICG-MB@RGD) for ultrasound/near-infrared (NIR) dual-modal imaging-guided chemo-photothermal-photodynamic combination therapy. The microbubbles coloaded tegafur (TF), a chemotherapeutic agent, and indocyanine green (ICG), a photothermal and photosensitizing agent, within a lipid shell surrounding a sulfur hexafluoride (SF₆) core. The surface was functionalized with cyclic RGD peptides to achieve active targeting to tumor vasculature. Upon intravenous administration, the system allowed real-time visualization of accumulation in tumor tissue via Doppler ultrasound and NIR imaging. Based on the imaging feedback, an 808 nm laser was applied to trigger rapid release of TF and ICG, simultaneously inducing chemotherapy, hyperthermia, and reactive oxygen species generation. In a 4T1 murine breast cancer model, this strategy resulted in a tumor inhibition rate of 72.76% with minimal systemic toxicity. This integrated approach provides a promising theranostic platform for precision treatment of superficial tumors.

The low aqueous solubility of crystalline enzalutamide (ENZ) and the limited physical stability of amorphous ENZ present significant formulation challenges. In this study, we explore binary co-amorphous systems of ENZ with octaacetyl maltose (acMAL), focusing on the system having eutectic concentration (ENZ + 75 wt % acMAL) as a potential strategy to enhance both stability and solubility. Based on differential scanning calorimetry (DSC) studies of crystalline materials, the eutectic point was identified, while analysis of DSC thermograms of co-amorphous systems revealed pronounced deviations in values of glass transition temperature (T_g) from Gordon-Taylor predictions, implicating the existence of strong specific intermolecular interactions. FTIR studies confirmed the presence of heteromolecular bonding within the mixtures. Broadband dielectric spectroscopy (BDS) showed that, although acMAL increases ENZ molecular mobility, the eutectic co-amorphous formulation significantly suppresses recrystallization under isothermal conditions (T = 413 K), delaying crystallization onset by over 30 h and limiting crystallinity to ≤2% after 55 h. The eutectic ENZ + acMAL composition exhibited sustained supersaturation in both aqueous and biorelevant media, demonstrating a balanced combination of efficient drug release and superior stabilization against recrystallization. These results confirm that eutectic formation followed by co-amorphization of ENZ with acMAL effectively addresses the dual challenges of limited physical stability and poor aqueous solubility. This approach provides a mechanistically rational and transferable strategy for improving the performance of poorly water-soluble APIs in pharmaceutical formulations.

NAFLD is a rising health problem worldwide with unsatisfied therapies. Curcumin has an ameliorative but limited effect on NAFLD due to its low water solubility. In this study, we innovatively establish the high-PC-content liposome-loaded curcumin (hPLipo/Cur) with liver-targeting properties for NAFLD therapy. hPLipo/Cur, composed of DSPC, cholesterol, and DSPE-PEG₂₀₀₀, has better biocompatibility and water solubility and is loaded with curcumin with high efficiency. hPLipo/Cur is superior to curcumin in improving hepatic histology, as evidenced by reducing lipid deposition and macrophage infiltration in steatohepatitis. Mechanistically, hPLipo/Cur reduces NRF2 degradation and promotes the nuclear translocation of NRF2, as well as the expression of downstream antioxidant genes. The activated NRF2 pathway reduces cellular oxidative stress and the generation of mitochondrial ROS, thereby reducing the accumulation of lipid peroxides and inhibiting ferroptosis in steatohepatitis. In conclusion, hPLipo/Cur reduces mitochondrial ROS-mediated ferroptosis by enhancing the NRF2 pathway to alleviate steatohepatitis, providing a promising strategy for NAFLD treatment.

Cancer stem-like cells (CSCs) are a key driving factor of tumor heterogeneity, metastasis, and chemoresistance. All-trans retinoic acid (ATRA) shows strong potential for inducing CSC differentiation and reducing stemness; however, its clinical translation is limited by the lack of effective targeting and favorable biodistribution under physiological conditions. Here, we report a hypoxia-responsive ATRA-phospholipid conjugate (RAPC) as a delivery and differentiation-priming strategy, enabling the fabrication of ATRA-loaded liposomes (ATRA@Lip) with high drug loading and microenvironment-activated release. RAPC was synthesized via a modular route incorporating an azobenzene-based hypoxia-cleavable linker between ATRA and a phosphatidylcholine analog. By coassembling RAPC with cholesterol and phospholipid, stable liposomes were obtained with a maximal ATRA loading of 17 wt % at 50 mol % RAPC content. Under normoxia, ATRA@Lip exhibited minimal leakage, whereas hypoxic conditions triggered rapid azo bond cleavage and accelerated ATRA release. Functionally, hypoxia-triggered ATRA@Lip promoted CSC differentiation and enhanced the chemosensitivity of breast cancer cells to albumin-bound paclitaxel (Nab-PTX) in vitro. Collectively, this hypoxia-responsive lipid-drug conjugate platform enables functional CSC priming and improved combination chemotherapy, offering a promising strategy for microenvironment-activated modulation of CSC-associated chemoresistance in metastatic breast cancer.

Active compounds derived from traditional Chinese medicine have attracted considerable interest as potential strategies for alleviating inflammation. However, their clinical translation is often impeded by limitations, including poor bioavailability and unsatisfactory therapeutic efficacy. To overcome these challenges, a novel fullerene-based nanoantioxidant was developed to enable oral administration and achieve site-specific targeting of inflammatory regions, thereby improving the therapeutic performance of curcumin (CUR) in ulcerative colitis. HA-C60 was synthesized through amidation between amino groups on aminated fullerene and carboxyl groups on hyaluronic acid, yielding a carrier with intrinsic anti-inflammatory properties. In vitro studies demonstrated that the carrier possessed free radical scavenging activity. After encapsulation within HA-C60, the resulting CUR nanoparticle formulation (CUR@HA-C60) exhibited both targeted and synergistic anti-inflammatory effects. This carrier markedly enhanced CUR stability in the upper gastrointestinal tract and facilitated site-specific drug accumulation at inflamed colonic sites. Anti-inflammatory efficacy was confirmed by reduced levels of nitric oxide, tumor necrosis factor-α, interleukin-1β, myeloperoxidase, and malondialdehyde, alongside increased expression of interleukin-10, superoxide dismutase, and glutathione, thereby mitigating inflammatory responses and oxidative stress (OS). Furthermore, CUR@HA-C60 demonstrated favorable biosafety profiles in murine models and significantly improved CUR bioavailability by suppressing inflammation, reducing OS-related damage, and restoring intestinal epithelial integrity. These findings indicate that CUR@HA-C60 represents a promising oral therapeutic strategy for the treatment of inflammatory diseases.