Molecular Pharmaceutics最新文献

The thyroid-stimulating hormone receptor (TSHR) is a promising molecular target for thyroid cancer imaging and therapy. Our previous work has demonstrated that PET imaging with the radiolabeled anti-TSHR human monoclonal antibody K1-70 enables assessment of TSHR expression in thyroid cancer. However, full-length antibody-based radiopharmaceuticals exhibit delayed systemic clearance and increased off-target radiation burden, resulting in suboptimal pharmacokinetics for immuno-PET imaging. Herein, we report the synthesis and evaluation of ⁶⁴Cu-labeled anti-TSHR K1-70 antibody fragment antigen-binding (Fab) and single-chain variable fragment (scFv), for immuno-PET imaging of TSHR in thyroid cancer mouse models. These smaller formats enabled rapid tumor targeting and favorable pharmacokinetics with high tumor-to-background contrast. Two radiotracers, named ⁶⁴Cu-NOTA-TSHR-Fab and ⁶⁴Cu-NOTA-TSHR-scFv, were prepared by conjugating TSHR-Fab or TSHR-scFv to p-SCN-Bn-NOTA, followed by radiolabeling with ⁶⁴Cu, achieving high radiochemical purity (>99%). The specificity and binding affinity of each radiotracer were determined by cellular uptake and binding assays using TSHR-positive THJ529T cells and corresponding wild-type controls. Both radiotracers exhibited specific, nanomolar binding affinity to TSHR-positive cells. Immuno-PET imaging, ex vivo biodistribution, and blocking studies of each radiotracer were performed in NSG mice bearing subcutaneous TSHR-positive THJ529T tumor xenografts at various time points (1, 4, 18, and 24 h postinjection). In comparative in vivo evaluations, ⁶⁴Cu-NOTA-TSHR-Fab showed rapid and superior TSHR-specific tumor accumulation compared with ⁶⁴Cu-NOTA-TSHR-scFv, evident as early as 1 h postinjection. Both radiotracers demonstrated rapid pharmacokinetics and low background signal, but with high renal uptake. This head-to-head comparison of small-size antibody fragment-based radiotracers for TSHR-targeted immuno-PET imaging identifies ⁶⁴Cu-NOTA-TSHR-Fab as a promising immuno-PET radiotracer for in vivo detection of TSHR expression in thyroid cancer and for guiding TSHR-targeted therapy.

The intrinsic resistance of tumor cells to apoptosis frequently results in chemoresistance and therapeutic failure in clinical settings. The main active ingredients of traditional Chinese medicine, in combination with chemotherapy drugs, have become one of the effective treatment methods to overcome this kind of drug resistance by targeting double subcellular organelles to promote apoptosis. In this study, we developed a Cu²⁺-coordinated paeoniflorin (PF)/doxorubicin (DOX) biocomplex, referred to as PCD, with the aim of overcoming cellular apoptosis resistance for combinational lung cancer therapy. PCD demonstrates remarkable water solubility and superior in vivo biocompatibility. Owing to the coordination effect, the self-assembled PCD exhibits a nanoscale particle size, a narrow and homogeneous grain distribution, as well as exceptional dispersion stability. Furthermore, PCD has the potential to disassemble under conditions of high glutathione levels and low pH, thereby facilitating effective drug release. PF-mediated endoplasmic reticulum stress (ERS) can downregulate the expression of FDX1 and DLAT proteins. Ca²⁺ overload induced by ERS disrupts mitochondrial matrix ion balance, accelerates water efflux, and ultimately leads to mitochondrial volume reduction and a decrease in mitochondrial membrane potential. Ultimately, this process synergizes with DOX-induced reactive oxygen species production to enhance apoptosis in lung cancer cells. PCD exhibits significant superiority over monotherapy in inhibiting tumor growth while minimizing systemic toxicity via enhanced induction of lung cancer apoptosis. This study may provide a promising avenue for advancing self-delivery nanomedicine to overcome apoptosis resistance in lung cancer therapy.

Radiolabeled fibroblast activation protein inhibitors (FAPIs) face a critical challenge in radionuclide therapy due to their short tumor retention. While existing strategies to prolong retention are often limited in clinical translation, we hypothesized that targeting the endoplasmic reticulum (ER) could provide a solution. We therefore designed a novel agent for sequential targeting, first to fibroblast activation protein (FAP) and then to the ER, aiming to enhance both tumor retention and radiotherapy efficacy. The sequential targeting agent, FAPI-PEG₃-K-PTSA, was synthesized by conjugating a PTSA ER-targeting moiety to the FAPI-46 core via a PEG₃-K linker. The compound was radiolabeled with ¹⁷⁷Lu and synthesized with high radiochemical yield (>95%). Cellular assays demonstrated specific binding to FAP and successful ER localization. This sequential-targeting capability resulted in superior in vivo performance, demonstrating a 3-5-fold higher tumor uptake and a retention time exceeding 72 h compared to FAPI-46. Consequently, ¹⁷⁷Lu-FAPI-PEG₃-K-PTSA achieved remarkable tumor suppression. The developed compound pioneers a three-level active targeting mechanism. It utilizes FAP for primary tumor enrichment, followed by ER-mediated internalization and specific organelle localization. This strategy effectively circumvents rapid efflux, dramatically prolongs intratumoral retention, and significantly enhances radiotherapeutic outcomes. Our findings provide crucial insights into organelle-targeted radionuclide therapy (TRT) and highlight its strong translational potential.

In previous studies, we discovered that the exposure of IgG4-Fc and IgG1 to UV light resulted in the side chain cleavage of specific Tyr and Trp residues, converting these amino acids into a series of products, including Gly [Haywood, J. Mol. Pharmaceutics 2013, 10(3), 1146-1150; Kang, H. Mol. Pharmaceutics 2019, 16, 258-272]. In order to evaluate the physicochemical consequences of such photochemical transformations, we prepared a series of IgG4-Fc mutants, in which Trp and Tyr residues were replaced by Gly, i.e., Y300G, Y373G, Y436G, and W381G, for biophysical studies. As the expression yields of W381G IgG4-Fc were low, we also prepared W381A to achieve higher yields required for some of the studies. Among these mutants, Y373G displayed significantly lower melting temperatures compared to wild-type IgG4-Fc, as analyzed by differential scanning calorimetry and fluorescence spectroscopy, indicating a decrease in thermal stability of both the C_H2 and C_H3 domains. In contrast, W381A showed no thermal transitions, indicating a significant loss of overall thermal stability. As for binding affinity to FcγRIIIA, Y300G and Y436G displayed ca. 10-fold reduction compared to wild-type IgG4-Fc. Interestingly, W381A and W381G IgG4-Fc did not only contain N-linked glycans but also high levels of O-mannose (>60%) at Ser³⁷⁵. Therefore, we prepared additional mutants, S375A and S375A/W381A IgG4-Fc, to evaluate the effect of N- vs O-glycosylation on thermal stability and the susceptibility of Tyr and Trp residues to undergo photodegradation. The removal of N-glycans specifically destabilized the C_H2 domain of IgG4-Fc. The removal of O-glycans did not affect the overall thermal stability of S375A IgG4-Fc, and S371A/W381A shows no thermal transitions, suggesting that the W381A mutation alone is sufficient to perturb the protein structure irrespective of O-glycosylation. As for photostability, we observed a 2.6-13.5 fold reduction of yields of Tyr side chain fragmentation products for W381A IgG4-Fc, potentially due to a lower probability for electron transfer between Tyr and oxidized Trp, and photodegradation of alternative Trp residues in W381A IgG4-Fc.

Broadband Acoustic Resonance Dissolution Spectroscopy (BARDS) is a novel analytical technology based on the change in the acoustic phenomena observed when a material is added into a solvent. Addition of a solid material results in the introduction of air (gas) into the solvent, which changes the compressibility of the liquid system and reduces the velocity of the sound (resonance) contained therein. In this study, the behavior of tablets in water was monitored using the BARDS technique. Tablets investigated contained 10% metoclopramide HCl (model water-soluble drug) and a microcrystalline cellulose filler and were formulated with and without a lubricant (0.5% magnesium stearate) at a range of tensile strength and porosity properties. The BARDS frequency-time profile for lubricated tablets showed an extended profile, indicating a slower wetting and prolonged displacement of gas compared to those of tablets without a lubricant. BARDS frequency-time profiles were transformed into gas volume-time profiles from which the areas under the total gas volume and volume curve (AUVC) were calculated. Lubricated tablets displayed a greater AUVC compared with unlubricated tablets. A decrease in the AUVC was observed for tablets up to the yield pressure of the formulation. For unlubricated tablets compacted at lower pressures, the gas elimination phase displayed first-order rate elimination kinetics. However, for lubricated tablets and unlubricated tablets compacted at higher pressures, two phases of gas elimination were observed: an initial fast elimination phase followed by a slower terminal elimination phase. This preliminary analysis of BARDS profiles collected demonstrates the capability of BARDS to capture differences between tablet formulations manufactured under different conditions.

Nanostructured lipid carriers (NLCs) and lipid-polymeric hybrid nanoparticles (H-NPs) were developed for the local administration of paclitaxel (PTX) and breast cancer therapy. Here, we investigated how nanoparticle type and composition influence the molecular effects and in vivo antiangiogenic activity of PTX. Elevated BAX expression and PARP-1 cleavage in MCF-7 and MDA-MB-231 breast cancer cells treated with nanoencapsulated PTX indicate that apoptosis is the primary mechanism of cell death, regardless of the nanocarrier type. However, distinct molecular effects were observed for other markers. Both unloaded nanocarriers increased α-tubulin acetylation in MCF-7 cells, indicating an intrinsic ability of the carriers to modulate cytoskeletal organization. Upon PTX loading, these effects became carrier-dependent: NLC-PTX induced higher α-tubulin acetylation than H-NP-PTX compared to the PTX solution. Moreover, in MCF-7 cells, NLC-PTX, but not H-NP-PTX, markedly enhanced drug-induced DNA damage, increasing γH2AX expression by 13.4-fold compared to PTX as a solution. These findings suggest that the nanocarriers not only act as delivery systems but may also confer additional biological effects that may contribute to PTX cytotoxicity. In the chicken chorioallantoic membrane model, nanoencapsulation reduced PTX-induced irritation from moderately irritant (irritation score 6) to nonirritant while preserving its antiangiogenic activity, achieving a 6.1-7.8-fold inhibition of vessel growth at subcytotoxic doses. Collectively, these results highlight nanoencapsulation as a promising strategy to potentiate PTX activity while improving safety for local breast cancer therapy. The distinct molecular responses of lipid and hybrid systems demonstrate that nanocarrier composition and structure modulate biological outcomes, underscoring the importance of rational nanocarrier design to overcome current therapeutic challenges.

Although weakly basic drugs with specific properties (e.g., dose number >1-10, pK_a 5-9) are known to form supersaturated solutions and exhibit complex phase behavior in the gastrointestinal tract, key aspects of this process (namely, the physical stability of nanodroplets and the charge site) remain poorly characterized. This work employs the dual-pK_a anticancer drug crizotinib (CZT; pK_a 5.6, 9.4) as a model drug to establish a systematic methodological framework for investigating the complex behaviors of weakly basic drugs induced by pH shift. The results revealed that upon pH shift from 1.0 to above 5.4, a 1 mg/mL CZT solution underwent liquid-liquid phase separation with nanodroplets forming immediately. ¹H nuclear magnetic resonance (NMR), ¹³C solid-state NMR, and synchrotron single-crystal X-ray diffraction (SCXRD) data together confirmed that this phase transition is attributed to the deprotonation from a dicationic species (with both piperidinium and pyridinium protonated) to a monocationic species (with only the piperidinium protonated) in both the drug-rich and drug-lean phases. Ultraviolet-visible spectroscopy, dynamic light scattering, confocal laser scanning microscopy, and polarized optical microscopy together revealed that the resulting additive-free CZT droplets are unstable at pH 6.5 with a low zeta potential of 5.1 mV, and sedimented into a bulk gel within minutes. This sedimentation significantly reduces the surface area of the colloidal particles (drug reservoir) and, consequently, slows the dissolution of CZT from the drug-rich phase─but interestingly, it has little effect on transmembrane flux through a cellulose membrane, suggesting that for CZT, transport across the membrane, not interphase diffusion, is the rate-limiting step. By establishing the methodology, which integrates NMR spectroscopy and X-ray crystallography to precisely locate the charge site of drug in both drug-rich phase (droplets or gel-like precipitate) and drug-lean phase (bulk solution), this work bridges the critical gap between macroscopic phase behavior and molecular-level understanding. These insights provide a mechanistic foundation for the rational design of oral formulations for weakly basic drugs.

Noninvasive radionuclide imaging of epithelial cell adhesion molecule (EpCAM) expression in lung, ovarian, breast, kidney, and other cancers can stratify patients for EpCAM-targeted therapy. The constructed scaffold proteins, designed ankyrin repeat proteins (DARPins), are highly specific high-affinity probes for radionuclide imaging. A clinical study demonstrated that the anti-EpCAM DARPin [^99mTc]Tc-(HE)₃-Ec1 showed precise EpCAM imaging at 2, 4, and 6 h after injection in patients with nonsmall cell lung cancer. However, a noticeable accumulation in healthy organs has prompted the development of new Ec1-based agents with improved biodistribution properties. In addition, it would be desirable to substitute a labor-intensive labeling procedure. The purpose of this study was to test the hypothesis that the use of Gly-Gly-Gly-Cys (G₃C) or Glu-Glu-Glu-Cys (E₃C) peptide chelators placed at the C-terminus of DARPin for labeling with ^99mTc (V) could improve the image contrast and biodistribution of Ec1. The radiochemical yield of the new variants exceeded 95%. The labeled proteins specifically bound to human EpCAM-expressing cancer cell lines with affinities of 8-10 nM. The biodistribution of [^99mTc]Tc-Ec1-G₃C and [^99mTc]Tc-Ec1-E₃C in mice was compared with the biodistribution of clinically tested [^99mTc]Tc-(HE)₃-Ec1 in a Nu/j mouse model with SKOV-3 xenografts. The new variants specifically accumulate in human xenografts with EpCAM expression. The accumulation of new variants in healthy organs (liver, salivary glands, spleen, and stomach) was reduced compared to [^99mTc]Tc-(HE)₃-Ec1. [^99mTc]Tc-Ec1-G₃C provided the best imaging contrast and is suitable for clinical testing.

This study aimed to develop and evaluate reactive oxygen species (ROS)-responsive (Thioketal-grafted DSPE-PEG2000, DTP) liposomes loaded with dexamethasone (DTP@DEX-LP) for aerosol inhalation to achieve targeted drug delivery and controlled release in the pulmonary fibrosis microenvironment. DTP@DEX-LP was prepared and optimized. The liposomes were characterized for particle size, zeta potential, encapsulation efficiency (EE), and morphology. Their aerosol performance, ROS-responsive drug release, and cellular uptake were assessed in vitro. In vivo pulmonary deposition, pharmacokinetics, and antifibrotic efficacy were evaluated in a bleomycin-induced mouse model, alongside safety profiling. The optimized DTP@DEX-LP exhibited a uniform particle size of ∼ 115 nm, a high EE of >82%, and desirable aerosol properties (FPF ∼ 50%, MMAD ∼ 4.9 μm). The formulation demonstrated ROS-triggered drug release and enhanced cellular uptake in vitro. Following inhalation, DTP@DEX-LP significantly prolonged lung retention and reduced systemic exposure of DEX compared to controls. In the fibrosis model, DTP@DEX-LP treatment yielded superior therapeutic outcomes, markedly improving survival, and reducing collagen deposition. It also showed a notably improved safety profile, with reduced hepatotoxicity compared to intravenous DEX. The ROS-responsive liposomal system represents a promising inhaled platform for the precise treatment of pulmonary fibrosis, effectively enhancing the therapeutic index of dexamethasone by simultaneously improving its efficacy and mitigating systemic toxicity.

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.