Pharmaceutics最新文献

Background: Self-amplifying mRNA (sa-mRNA) represents a promising platform for vaccines and gene therapies, offering sustained protein expression at low doses through self-replication. For vaccines targeting respiratory pathogens, pulmonary delivery of sa-mRNA lipid nanoparticles (LNPs) is particularly advantageous, enabling direct delivery to the infection site and induction of mucosal immunity. Objective: In this study, we evaluated the stability of sa-mRNA-LNPs under refrigerated and frozen conditions and developed a dry powder formulation suitable for inhalation, produced by freeze-drying followed by cryomilling with leucine. Methods: sa-mRNA-LNPs formulated in HEPES buffer with 20% (w/v) sucrose were stored for up to 8 weeks as liquid or freeze-dried samples at various temperatures (-80 °C, -20 °C, 4 °C, and 20 °C). Biological stability was assessed by transfection efficiency in HeLa cells, while physical stability was characterized by encapsulation efficiency, zeta potential, particle size, and polydispersity index. Results: Liquid formulations remained stable for at least 8 weeks at -80 °C and -20 °C but rapidly lost stability at 4 °C and 20 °C. Freeze-drying effectively preserved sa-mRNA-LNP functionality and structural integrity for up to 8 weeks at 4 °C, with only minor structural changes. Subsequent cryomilling in the presence of 4 wt-% leucine produced a respirable dry powder while retaining approximately 60% of the original sa-mRNA-LNP functionality. Although cryomilling induced some structural alterations, the remaining functional fraction remained stable during storage. The resulting powders displayed favorable aerosol performance for deep lung delivery, as demonstrated by cascade impaction (MMAD = 4.13 ± 0.26 µm). Conclusions: In conclusion, freeze-drying effectively preserved sa-mRNA-LNP integrity at 4 °C, whereas cryomilling with leucine produced a respirable dry powder suitable for pulmonary delivery, providing a foundation for globally accessible, needle-free sa-mRNA vaccines against respiratory diseases.

Background and Objectives: Aggressive cancer development is characterized by rapid tumor growth and progressive immune dysfunction. Tumor-derived microRNAs (miRNAs) emerge as master regulators of both malignant transformation and immune evasion, making them promising therapeutic targets. Using the highly aggressive CT-26 peritoneal adenomatosis model, this study explored the potential of selective miRNA inhibition to simultaneously suppress tumor growth and overcome immunosuppression. Methods and Results: Our results revealed that inhibition of miR-155, miR-21, and miR-17 by methylsulfonyl phosphoramidate (mesyl) oligonucleotides exhibited markedly different therapeutic profiles. miR-155 inhibition demonstrated minimal efficacy. miR-21 suppression provided early tumor regression and prevented cancer-associated thymic atrophy, translating into extended survival. miR-17 inhibition displayed delayed but superior tumor growth inhibition, significantly reducing pathologically elevated polymorphonuclear myeloid-derived suppressor cell (MDSC) populations, and nearly doubled animal lifespan. Combination therapy targeting all three miRNAs integrated these complementary mechanisms, maintaining consistent anti-tumor efficacy across early and late stages while providing thymic protection and MDSC reduction. Importantly, therapeutic responses in vivo substantially exceeded predictions based on in vitro tumor cell proliferation and motility measurements, revealing critical contributions of systemic immunomodulation. Conclusions: These findings demonstrate that miRNA inhibition reshapes tumor-immune interactions, positioning anti-miRNA therapeutics as immunomodulatory agents for effective colorectal cancer treatment.

Background/Objectives: Rotavirus remains a major cause of severe acute gastroenteritis in infants worldwide. The suboptimal efficacy of current vaccines underscores the need for alternative microbiome-based interventions, including postbiotics. Extracellular vesicles (EVs) from probiotic and commensal E. coli strains have been shown to mitigate diarrhea and enhance immune responses in a suckling-rat model of rotavirus infection. Here, we investigate the regulatory mechanisms activated by EVs in rotavirus-infected enterocytes. Methods: Polarized Caco-2 monolayers were used as a model of mature enterocytes. Cells were pre-incubated with EVs from the probiotic E. coli Nissle 1917 (EcN) or the commensal EcoR12 strain before rotavirus infection. Intracellular Ca²⁺ concentration, ROS levels, and the expression of immune- and barrier-related genes and proteins were assessed at multiple time points post-infection. Results: EVs from both strains exerted broad protective effects against rotavirus-induced cellular dysregulation, with several responses being strain-specific. EVs interfered with viral replication by counteracting host cellular processes essential for rotavirus propagation. Specifically, EV treatment significantly reduced rotavirus-induced intracellular Ca²⁺ mobilization, ROS production, and COX-2 expression. In addition, both EV types reduced virus-induced mucin secretion and preserved tight junction organization, thereby limiting viral access to basolateral coreceptors. Additionally, EVs enhanced innate antiviral defenses via distinct, strain-dependent pathways: EcN EVs amplified IL-8-mediated responses, whereas EcoR12 EVs preserved the expression of interferon-related signaling genes. Conclusions: EVs from EcN and EcoR12 act through multiple complementary mechanisms to restrict rotavirus replication, spread, and immune evasion. These findings support their potential as effective postbiotic candidates for preventing or treating rotavirus infection.

This review summarizes the role of nuclear factor erythroid 2-related factor 2 (Nrf2) as a common link between aging, neurodegeneration, and neuropathic pain. Aging is characterized by oxidative stress and constant inflammation, which coincides with reduced Nrf2 activity and weaker antioxidant responses, increasing vulnerability to diseases. In neurodegenerative disorders-including Alzheimer's, Parkinson's, Huntington's disease, and amyotrophic lateral sclerosis-evidence indicates that impaired Nrf2 signaling contributes to oxidative damage, neuroinflammation, and mitochondrial dysfunction. Furthermore, in neuropathic pain, similar mechanisms are involved, and Nrf2 could play a role as a potential analgesic target because of its role in regulating cellular defense pathways. We also review natural Nrf2 modulators (e.g., flavonoids, other polyphenols, terpenoids, alkaloids), discussing their benefits alongside common translational limitations such as poor solubility, low oral bioavailability, rapid metabolism, and potential safety issues, including possible pro-oxidant effects and chemoresistance. We also outline future directions that should prioritize improving delivery systems, addressing NRF2/KEAP1 gene variations, evaluating combinations with standard therapies, exploring preventive applications, and defining dosing, treatment duration, and long-term safety. Overall, current evidence indicates that Nrf2 modulation is a practical, cross-cutting approach relevant to healthy aging and disease management.

Background/Objectives: Essential oils (EOs) from plants of the genus Cinnamomum have been widely used based on their antimicrobial, antioxidant, and anti-inflammatory properties. However, their elevated volatility and limited aqueous solubility restrict their use in pharmaceutical and food formulations. Cyclodextrins (CDs) have emerged as a promising strategy to overcome these limitations through the formation of inclusion complexes. Methods: In this study, inclusion complexes of essential oil from C. camphora L. (EOCNM) with β-cyclodextrin (β-CD) were developed using physical mixing (PM), ultrasonic treatment (US), and freeze-drying (FD). The inclusion complexes were physicochemically characterized by differential scanning calorimetry (DSC), thermogravimetric analysis (TG/DTG), X-ray diffraction (XRD), and scanning electron microscopy (SEM) to evaluate their physicochemical interactions and complexation efficiency. Results: Our results demonstrated successful complex formation, with the FD and US methods showing greater amorphization and stronger inclusion characteristics compared to the PM method. Thermal analysis confirmed improved physicochemical stability of the essential oil when complexed with β-CD. Furthermore, the cytotoxicity assay of the complexes was assessed using the MTT assay and J774 macrophage cells. The complexes exhibited low cytotoxicity, indicating their potential biocompatibility for biomedical and food applications. Conclusions: Overall, β-CD encapsulation effectively enhanced the physicochemical stability and safety profile of C. camphora essential oil, providing a promising strategy for its controlled delivery and protection against degradation.

Background: Gout is an inflammatory arthritis associated with increased bone anabolism and a higher risk of ectopic bone formation. Colchicine, used to prevent and treat acute gouty flares, inhibits microtubule polymerization and has been described to promote osteoblastogenesis. In bone disorders such as osteoporosis, disruption of the osteoblast-adipocyte balance contributes to pathology, yet no therapies directly target bone marrow adiposity. Thus, we decided to investigate the impact of colchicine on the osteoblast-adipocyte balance. Methods: C3H10T1/2 mesenchymal stem cells were differentiated to both cell fates in the presence or absence of colchicine. Differentiation was assessed by studying differentiation phenotypes as well as adipocytic and osteoblastic marker genes. Disrupting microtubule homeostasis through stathmin (STMN1) silencing was employed to mimic colchicine effects on differentiation. Proteomic analysis was performed to gain further insight into colchicine's effects on adipogenesis. Results: Colchicine promoted transcriptional changes consistent with osteoblastogenic commitment and inhibited adipogenesis, as evidenced by reduced intracellular lipid accumulation and downregulation of adipogenic marker genes. These effects were observed following both continuous and transient exposure (median fold change across adipogenic markers 0.41 and 0.59, respectively). Consistent with colchicine-induced microtubule destabilisation, microtubule disruption by STMN1 silencing also suppressed adipogenic differentiation (median fold change = 0.66), suggesting that colchicine's anti-adipogenic effect may be due to its impact on the cytoskeleton. Conclusions: These findings indicate that colchicine can suppress adipogenic differentiation while favouring osteoblast commitment in mesenchymal stem cells. Although further validation in relevant preclinical models is required, its efficacy following transient exposure supports the exploration of site-specific strategies that limit systemic toxicity.

Metabolic dysfunction-associated steatotic liver disease (MASLD) represents a multifaceted systemic condition, with the mechanisms linking intrahepatic lesions to systemic complications remaining a significant enigma in the field. This review posits that extracellular vesicles (EVs) serve as pivotal mediators facilitating communication between the liver and the entire organism. Within the hepatic environment, lipotoxic hepatocyte-derived EVs modulate macrophage populations and stellate cells, thereby promoting inflammatory and fibrotic processes. Systemically, the liver engages in bidirectional communication with adipose tissue, the intestinal tract, the cardiovascular system, and the pancreas via EVs, thus orchestrating metabolic homeostasis. Furthermore, we critically evaluate non-invasive diagnostic strategies and emerging therapies, including both natural and engineered EVs, based on EV-based interventions. We highlight the substantial potential and current challenges associated with achieving precision medicine in MASLD through targeted modulation of this specific communication network.

Background/Objectives: Flavonoids are widely used as lead structures in drug discovery, and their pharmacological and metabolic properties are strongly influenced by structural features such as positional isomerism. This study aimed to compare the metabolic profiles and underlying mechanisms of two isoflavone-based positional isomers, ACF-02 (2-(4-hydroxy-3-methoxyphenyl)-6,7-dimethoxy-3-(4-methoxyphenyl)-4H-chromen-4-one) and ACF-03 (2-(3-hydroxy-4-methoxyphenyl)-6,7-dimethoxy-3-(4-methoxyphenyl)-4H-chromen-4-one). Methods: The metabolic pathways of synthetically prepared ACF-02 and ACF-03 were investigated using an in vitro incubation system with human liver microsomes (HLMs) supplemented with an NADPH-regenerating system, followed by liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS/MS) analysis. Metabolites were identified based on LC-HRMS/MS data and molecular networking-based node connectivity with the parent compounds. Major metabolites were further characterized by CYP phenotyping using recombinant CYP450 isoforms, and the potential for drug-drug interactions of ACF-03 was evaluated using a CYP probe substrate cocktail approach. Results: HLM incubation of ACF-02 and ACF-03 produced both hydroxylated and O-demethylated metabolites, with O-demethylation as the predominant pathway; notably, the most abundant O-demethylated metabolite differed in an isomer-dependent manner, occurring at the B₂ ring for ACF-02 and at the A ring for ACF-03, with distinct CYP isoform involvement. Molecular networking supported the relationships between the parent compounds and their metabolites, and both compounds exhibited relatively high metabolic stability with limited CYP inhibition. Conclusions: Despite differing only in the position of a single methyl substituent, ACF-02 and ACF-03 exhibited distinct isomer-dependent metabolic profiles. These findings demonstrate that even subtle positional isomerism can significantly influence metabolic behavior and should be carefully considered during lead optimization and drug design.

Type 2 diabetes mellitus (T2DM) is a multifaceted metabolic disorder marked by impaired insulin action, pancreatic β-cell dysfunction, and the involvement of several interconnected mechanisms, including inflammation, oxidative stress, and epigenetic alterations. Despite progress in conventional therapies, achieving durable glycemic control and minimizing complications remain major challenges. This review discusses the emerging role of bioactive phytochemicals-such as curcumin, berberine, resveratrol, flavonoids, and polysaccharides-in modulating essential molecular pathways including AMPK, PI3K/AKT, and cAMP/PKA, which contribute to enhanced insulin sensitivity, glucose regulation, and β-cell protection. These natural compounds also influence gut microbiota modulation and epigenetic mechanisms, offering additional metabolic and anti-inflammatory benefits. This review synthesizes evidence from peer-reviewed studies published between 2000 and 2024, incorporating bibliometric trends showing an increasing research focus on phytochemicals for T2DM management. However, limitations such as low solubility, instability, and poor absorption restrict their clinical application. Advances in nanotechnology-based delivery systems, including nanoparticles, liposomes, and nanoemulsions, have shown potential to overcome these barriers by improving stability, bioavailability, and targeted delivery of phytochemicals. The integration of gut microbiota modulation with nanocarrier-enabled phytochemical therapy supports a precision medicine approach for managing T2DM. Preliminary clinical evidence highlights significant improvements in glycemic control and inflammatory status, yet further large-scale, well-controlled trials are essential to ensure safety, optimize dosages, and standardize combination regimens. Overall, phytochemical therapies, reinforced by nanotechnology and microbiota modulation, present a promising, safe, and holistic strategy for T2DM management. Continued interdisciplinary research and clinical validation are crucial for translating these advances into effective therapeutic applications and reducing the global diabetes burden.

Background: Infections caused by the protozoan Trichomonas vaginalis affect millions of people worldwide and are responsible for one of the most common sexually transmitted diseases. Despite the efficacy of 5-nitroimidazoles like metronidazole, concerns regarding widespread resistance and the absence of viable alternatives for specific patient populations necessitate the development of structurally diverse pharmacological agents. In this study, we investigated the antiparasitic activity of 1,3-thiazolidin-4-one derivatives against T. vaginalis. Methods: Thiazolidines were synthesized via multicomponent reaction (MCR) using one-pot methodology and tested in vitro against the parasite and mammalian cell lines. Results: Seventy percent of the compounds showed more than 80% antiparasitic activity at 100 μM, with compounds 4a, 4b, and 4f exhibiting IC₅₀ ≤ 20 µM. None of the molecules exhibited cytotoxic against Vero CCL-81 and HeLa cells. Evaluation of the structure-activity relationship (SAR) indicates that the substituent at the nitrogen position of the heterocycle may be involved in the antiparasitic effect of these compounds. In silico studies also revealed that the three compounds possess adequate oral bioavailability and do not present mutagenic, tumorigenic or irritating risks. Finally, molecular docking predicted strong interactions of compounds 4a, 4b, and 4f with T. vaginalis enzymes lactate dehydrogenase and purine nucleoside phosphorylase; compound 4f also interacted with methionine Ƴ-lyase. Conclusions: These preliminary results suggest that 1,3-thiazolidin-4-ones are promising scaffolds for developing new trichomonacidal agents.