Molecular Pharmaceutics最新文献

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype characterized by the absence of targetable receptors and limited therapeutic options. Its inherently low immunogenicity necessitates strategies to enhance its susceptibility to immune attack. This study aimed to enhance the antitumor immune response by inducing downregulation of the programmed death ligand 1 (PD-L1), thereby increasing cancer cell recognition and activating tumor-specific immunity. To this end, a targeted solid lipid nanoparticle (tSLN) system was developed for the precise delivery of CRISPR/Cas9 plasmids to achieve functional PD-L1 downregulation. The SLN formulation comprised a cetyl palmitate core stabilized with cationic and PEGylated lipids. Functionalization with the tumor-penetrating peptide iRGD yielded tSLN, which demonstrated good colloidal stability, serum protection of the CRISPR cargo, and minimal cytotoxicity. In vitro studies in two breast cancer subtypes, the luminal MCF-7 and the triple-negative 4T1 line, confirmed greater transfection efficiency and PD-L1 knockdown by tSLN:Crispr/PD-L1 relative to non-targeted controls. In an orthotopic 4T1 mouse model, systemic administration of tSLN:Crispr/PD-L1 significantly inhibited tumor growth and metastasis while enhancing T cell infiltration, and was more effective than standard chemotherapy (paclitaxel) in this model. The targeted approach also exhibited a favorable short-term safety profile, with no organ toxicity detected over the treatment period. These findings underscore the potential of SLN-mediated CRISPR delivery as an immunomodulatory strategy for breast cancer, and particularly for TNBC.

The effectiveness of liposome-based antitumor drugs is significantly limited by the poor tumor accumulation of liposomes. Herein, we report that resiquimod (R848) can selectively increase tumor accumulation of 7-Ethyl-10-hydroxycamptothecin (SN38)-loaded liposomes (SN38-LIPs) without significantly altering their pharmacokinetics. R848 enhanced the tumor distribution of SN38-LIPs and free SN38 by approximately 5-fold and 10-fold, respectively. This further resulted in greatly improved in vivo antitumor activity compared with those treated with R848 or SN38-LIPs alone (tumor inhibition rate: ∼95% versus ∼50%). The increased antitumor activity was associated with increased serum TNF-α and enhanced macrophage infiltration in the tumor. These results suggested that R848 held great potential to enhance the clinical antitumor effect of nanomedicine by increasing its tumor accumulation.

The solubility advantage of an amorphous drug can be realized only when it releases at a sufficiently rapid rate from its amorphous solid dispersion (ASD). Herein, we evaluated the impact of a plasticizer, glyceryl tributyrate (GTB), on the release of a poorly soluble drug with a high glass transition temperature (T_g). Cyclosporine has a T_g of 124 °C and was formulated as an ASD with hydroxypropyl methylcellulose acetate succinate (HPMCAS). Binary and ternary ASDs were prepared using solvent evaporation, their T_gs were measured using differential scanning calorimetry, and surface area normalized release rates were determined. The drug-rich phase formed above the amorphous solubility was characterized for composition and size. Drug flux following release from the ASD was measured across Caco-2 cell monolayers. Cyclosporine released at a substantially faster rate from ternary ASDs containing GTB as compared to the corresponding binary ASD. This improvement was attributed to a decrease in T_g upon plasticization due to GTB increasing molecular mobility. Addition of GTB, however, reduced the amorphous solubility of cyclosporine resulting in a decrease in the transport rate across the Caco-2 cell monolayer. These observations may have implications for drug absorption. Ultimately, it is expected that the in vivo performance of these ternary ASDs would be determined by the interplay between the enhanced release rate and the decreased permeation rate of the drug.

Bacteriophage therapy is being explored as an alternate therapeutic approach for treating drug-resistant bacteria, including mycobacteria. However, rational phage dosing remains limited by scarce pharmacokinetic (PK) data and an incomplete understanding of tissue distribution. We performed dose-ranging studies in mice of three therapeutic mycobacteriophages (BPsΔ, ZoeJΔ, Muddy) after intravenous (IV) and intratracheal (IT) administration. All phages behaved similarly. IV dosing produced biphasic kinetics with nonproportional exposure and declining tissue-to-plasma ratios, indicating saturable uptake and elimination. IT delivery yielded monophasic profiles with ∼390-fold higher lung exposure and ∼490-fold lower plasma exposure, supporting inhaled therapy for pulmonary mycobacterial infections. Using BPsΔ data, we developed a mechanistic PBPK model incorporating transcytosis, saturable host clearance, plasma elimination, and lymphatic transport. The model accurately predicted ZoeJΔ and Muddy PK, enabled cross-species extrapolation, and showed that phage morphology influences disposition. This framework advances phage therapy toward model-informed, exposure-guided dose and route selection for multidrug-resistant bacterial infections.

In high-drug-loaded amorphous solid dispersions, effective stabilization requires polymers that can hinder nucleation as well as crystal growth. We hypothesized that combining two polymers with complementary stabilization mechanisms would enhance physical stability more effectively than the individual polymers. Evaluation of single-polymer systems at a polymer loading of 20% w/w showed that HPMCAS (hydroxypropyl methylcellulose acetate succinate) had the highest thermodynamic miscibility with carbamazepine (CBZ), whereas PVP (polyvinylpyrrolidone) and PVPVA (PVP/vinyl acetate) dispersions had the longest α-relaxation time. Based on these findings, ternary systems combining 80% w/w CBZ, 10% w/w HPMCAS, and 10% w/w of either PVP or PVPVA were evaluated. The ternary systems exhibited longer crystallization onset times than the corresponding single-polymer systems at the same total polymer loading. The improved stability was due to a synergistic mechanism where HPMCAS provided resistance to nucleation by increasing the activation energy barrier, while PVP/PVPVA kinetically lowered the attempt jump frequency to suppress both nucleation and crystal growth. This dual-polymer approach provided a basis for designing amorphous solid dispersions with high drug loading and improved physical stability.

The lysosomal transporter SLC46A3 has been implicated in the pharmacology of antibody−drug conjugates (ADCs) by facilitating the transport of payloads from lysosomes to the cytosol. To date, two payload classes, maytansinoid catabolites (e.g., DM1 and Lys-SMCC-DM1) and the pyrrolobenzodiazepine (PBD) dimer SG3199, have been identified as SLC46A3 substrates; however, the transport mechanisms for other payloads remain to be elucidated. Auristatin derivatives (auristatins) are among the most widely deployed payloads in approved ADCs, yet the mechanism by which these highly potent peptides egress from the lysosome remains unclear. Here, we demonstrate that SLC46A3 directly transports auristatins. At pH 5.0, mimicking the lysosomal environment, SLC46A3 significantly facilitated the membrane transport of all tested auristatins, whereas transport via passive diffusion was negligible. Among the auristatins, monomethyl auristatin F (MMAF), which bears a negative charge, was a superior substrate for SLC46A3 compared to monomethyl auristatin E (MMAE), a finding consistent with the preference of SLC46A3 for organic anions. Interestingly, MMAF conjugated with a non-cleavable maleimidocaproyl (mc) linker, was also recognized as an SLC46A3 substrate, exhibiting higher transport activity than unmodified MMAF. Furthermore, depletion of SLC46A3 in HER2-positive cancer cells attenuated the cytotoxicity of trastuzumab-mcMMAF, suggesting that SLC46A3 mediates the lysosomal egress of its active metabolites. Taken together, our findings highlight lysosomal transporters including SLC46A3, as critical determinants of the efficient cytoplasmic release of auristatins, providing an opportunity to optimize ADC efficacy.

Drug-excipient incompatibility in solid oral dosage forms can generate toxic degradants and compromise potency without producing a visible macroscopic signal in early development. We reported, for the first time, a mechanistically resolved, temperature-sequenced characterization of the solid-state acid−base incompatibility between amlodipine besylate (AMLO; pK_a ≈ 9.4) and citric acid (CA; pK_a1 3.13; ΔpK_a = 6.27) using six orthogonal techniques: DSC, TGA, isothermal titration calorimetry (ITC), powder X-ray diffractometry with two-dimensional correlation spectroscopy (2D-COS-PXR), FTIR with 2D-COS (2D-COS-FTIR), and stability-indicating HPLC coupled to LC-QToF HRMS. Binary mixtures were thermally stressed at 40−90 °C (7 day isothermal, closed-vial). ITC confirmed a spontaneous, entropy-dominated interaction (ΔG = −5.14 kcal/mol; −TΔS = −3.78 kcal/mol; N ≈ 1:1; K_D = 197 μM) driven by proton transfer to the AMLO dihydropyridine nitrogen with concurrent besylate displacement. DSC showed complete loss of the AMLO Form-I endotherm (onset 201.01 °C), replaced by a single event at 155.57 °C with super-additive enthalpy (+19.50 J/g), consistent with amlodipine citrate salt formation. Application of Noda’s 2D-COS algorithm to temperature-resolved PXRD and FTIR datasets independently resolved a four-stage mechanism, viz., CA surface proton mobilization (40 °C) → amlodipine citrate nucleation (50−60 °C) → besylate counter-ion displacement and d-spacing contraction (∼70 °C) and → partial amorphization and acid-catalyzed covalent degradation (80−90 °C). Three sequential-order reversals in the 2D-COS-FTIR asynchronous map independently corroborated the Stage-3 crystallographic sequence. HPLC revealed a 4.48-fold enhancement of AMLO loss in the mixture (7.97%) versus pure drug (1.78%), with LC-QToF HRMS identifying four mixture-unique degradants consistent with the four-stage model. This is also the first pharmaceutical application of 2D-COS-PXRD as a mechanistic probe of sequential crystallographic events in drug-excipient incompatibility.

Gemcitabine (GEM) is a first-line therapeutic option for pancreatic cancer; however, it has low efficacy due to rapidly developed drug resistance and severe dose-limiting myelosuppression. To enhance its therapeutic effect, this study developed a novel peptide−drug conjugate using a CXCR4 antagonistic peptide as the targeting head and gemcitabine as the drug payload, based on the characteristics of pancreatic tumor cells highly expressing CXCR4 that mediates immunosuppression and tumor progression through interaction with its specific ligand CXCL12. The therapeutic effect of the conjugate (P12-GEM) was investigated using pancreatic ductal adenocarcinoma cell lines and an orthotopic pancreatic cancer mouse model. Its myelosuppressive effect was assessed from the perspective of hematological toxicity profiles. The results showed that P12-GEM maintained a cell-killing capability comparable to that of GEM while effectively inhibiting the phosphorylation of Erk and P38, thereby reducing CXCL12-mediated tumor cell migration and adhesion to stromal cells. In a tumor-bearing mouse model, P12-GEM demonstrated superior antitumor efficacy compared to GEM and significantly extended animal survival. Moreover, P12-GEM reduced the proportion of tumor-associated macrophages and increased the infiltration of CD8⁺ T cells in the tumor microenvironment without reducing platelet and white blood cell counts in the peripheral blood. In summary, P12-GEM possesses dual functions of CXCR4 antagonism and tumor cell killing, contributing to reversal of the immunosuppressive microenvironment and alleviation of myelotoxicity.

Fibroblast activation protein (FAP)-targeted radioligand therapy is often limited by insufficient tumor uptake and retention. We designed and synthesized two novel aryl fluorosulfate-based covalent FAP-targeted radioligands, [¹⁷⁷Lu]Lu-DOTA-mFS-FAP-2286 and [¹⁷⁷Lu]Lu-DOTA-mFS-KERERG-FAP-2286, to enhance tumor retention and therapeutic efficacy. Two FAP-targeted radioligands were synthesized, radiolabeled with ¹⁷⁷Lu, and evaluated for radiochemical stability. Cellular uptake and internalization were assessed in HEK293-huFAP cells. Biodistribution and serial SPECT imaging were performed in tumor-bearing mice to evaluate tumor targeting and retention. Therapeutic efficacy was investigated in murine xenograft models, and the toxicity of [¹⁷⁷Lu]Lu-DOTA-mFS-KERERG-FAP-2286 was assessed. A pilot clinical study including three patients with solid tumors was conducted to preliminarily evaluate the therapeutic potential. All radioligands were successfully labeled with radiochemical purity >95% and showed favorable stability. Cellular assays confirmed the high target specificity. In tumor-bearing mice, [¹⁷⁷Lu]Lu-DOTA-mFS-KERERG-FAP-2286 demonstrated significantly higher tumor uptake at 1 h after injection than [¹⁷⁷Lu]Lu-DOTA-FAP-2286 and [¹⁷⁷Lu]Lu-DOTA-mFS-FAP-2286 (p < 0.01). Treatment with [¹⁷⁷Lu]Lu-DOTA-mFS-KERERG-FAP-2286 significantly inhibited tumor growth and showed superior therapeutic efficacy compared with [¹⁷⁷Lu]Lu-DOTA-FAP-2286 (p < 0.05). No treatment-related adverse events were observed in the three patients. Primary tumors and metastatic lesions demonstrated high tracer uptake with prolonged retention up to day 10 after injection. [¹⁷⁷Lu]Lu-DOTA-mFS-KERERG-FAP-2286 showed favorable tumor targeting, prolonged retention, and potent therapeutic efficacy, supporting its further clinical development for FAP-targeted radioligand therapy (RLT).