Molecular Pharmaceutics最新文献

Magnetic resonance imaging (MRI) of dry or solid materials in the gastrointestinal (GI) tract requires the use of contrast agents to enhance visualization of the dosage forms. In this study, we explore the novel use of manganese gluconate added to tablets. Manganese was released during tablet dissolution, generating a bright "halo" effect around the tablets, consistent with shortening of the longitudinal relaxation time of the bulk water surrounding the tablet. This is the first study to use MRI to directly image tablet dissolution in the fed stomach using a manganese gluconate contrast agent as dissolution marker.

Radioiodine has been exploited in nuclear medicine for diagnostic and therapeutic purposes in various diseases. There are two radioiodination methods for biomolecules, that is, (1) direct radioiodination of tyrosine or histidine residue in a biomolecule and (2) indirect radioiodination by using a prosthetic group, which bridges radioiodine and the biomolecule. While directly radioiodinated biomolecules suffer from deiodination in vivo, the most commonly used indirect labeling method based on N-succinimidyl-3-[*I]iodobenzoate has a problem of inconvenience due to an high-performance liquid chromatography (HPLC) purification process. To tackle both issues, a novel prosthetic click-linker-antibody conjugate (3-[^123/125I]iodobenzoyl-PEG₄-tetrazine-TCO-PEG₄-trastuzumab (3-[^123/125I]IBTTT)) with favorable radiochemical yield (>57%) and purity (>99%) was developed using a fluorous tin-based organotin precursor with streamlined purification process utilizing fluorous solid-phase extraction (FSPE) cartridge and spin column. In vitro binding studies demonstrated that 3-[¹²⁵I]IBTTT maintained its biological activity with a K_D value (5.606 nM) comparable to that of unmodified trastuzumab (5.0 nM). In vivo imaging of 3-[¹²³I]IBTTT in a human epidermal growth factor receptor 2 (HER2)-expressing gastric cancer mouse model revealed favorable tumor accumulation and negligible thyroid uptake compared to directly radioiodinated trastuzumab ([¹²³I]trastuzumab). It was also confirmed, by blocking experiments and a biodistribution study, that the tumor accumulation of 3-[¹²³I]IBTTT was attributed to HER2-specific binding. In summary, we developed a novel radioiodinated prosthetic click-linker agent (3-[^123/125I]IBTTT) with favorable radiochemical yield, purity, stability, and in vivo behavior, providing a highly promising tool for targeted imaging and potential therapy of HER2-positive cancers.

This study aims to develop an innovative microencapsulation method for coated Polymyxin B, utilizing various polysaccharides such as hydroxypropyl β-cyclodextrin, alginate, and chitosan, implemented through a three-fluid nozzle (3FN) spray drying process. High-performance liquid chromatography (HPLC) analysis revealed that formulations with a high ratio of sugar cage, hydroxypropyl β-cyclodextrin (HPβCD), and sodium alginate (coded as ALG_HCD_HP_L^PM) resulted in a notable 16-fold increase in Polymyxin B recovery compared to chitosan microparticles. Morphological assessments using fluorescence labeling confirmed successful microparticle formation with core/shell structures. Alginate-based formulations exhibited distinct layers, while chitosan formulations showed uniform fluorescence throughout the microparticles. Focused beam reflectance and histograms from fluorescence microscopic measurements provided insights into physical size analysis, indicating consistent sizes of 6.8 ± 1.2 μm. Fourier-transform infrared (FTIR) spectra unveiled hydrogen bonding between Polymyxin B and other components within the microparticle structures. The drug release study showed sodium alginate's sustained release capability, reaching 26 ± 3% compared to 94 ± 3% from the free solution at the 24 h time point. Furthermore, the antimicrobial properties of the prepared microparticles against two Gram-negative bacteria, Escherichia coli and Pseudomonas aeruginosa, were investigated. The influence of various key excipients on the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values was evaluated. Results demonstrated effective bactericidal effects of ALG_HCD_HP_L^PM against both E. coli and P. aeruginosa. Additionally, the antibiofilm assay highlighted the potential efficacy of ALG_HCD_HP_L^PM against the biofilm viability of E. coli and P. aeruginosa, with concentrations ranging from 3.9 to 500 μg/m. This signifies a significant advancement in antimicrobial drug delivery systems, promising improved precision and efficacy in combating bacterial infections.

The adsorption of plasma proteins (human serum albumin, immunoglobulin γ-1, apolipoproteins A-I and E-III) onto polystyrene surfaces grafted with polyethylene glycol (PEG) at different grafting densities is simulated using an all-atom PEG model validated by comparing the conformations of isolated PEG chains with previous simulation and theoretical values. At high PEG density, the grafted PEG chains extend like brushes, while at low density, they significantly adsorb to the surface due to electrostatic attraction between polystyrene amines and PEG oxygens, forming a PEG layer much thinner than its Flory radius. Free energy calculations show that PEGylation can either increase or decrease the binding strength between proteins and surfaces, to an extent dependent on PEG density and specific proteins involved, in agreement with experiments. In particular, grafted PEG chains not only sterically block the binding between proteins and surfaces but also strongly interact with proteins via hydrogen bonds and electrostatic and hydrophobic interactions, with apolipoproteins exhibiting stronger hydrophobic interactions with PEG than other proteins, implying that these specific protein-PEG interactions help certain proteins remain on the PEGylated surface. These simulation findings help explain experimental observations regarding the abundance of specific plasma proteins adsorbed onto nanoparticles grafted with PEG at different densities.

Oral peptide therapeutics are increasingly favored in the pharmaceutical industry for their ease of use and better patient adherence. However, they face challenges with poor oral bioavailability due to their high molecular weight and surface polarity. Permeation enhancers (PEs) like salcaprozate sodium (SNAC) have shown promise in clinical trials, achieving about 1% bioavailability. One proposed mechanism for enhancing permeation is membrane perturbation or fluidization, though direct experimental proof and quantitative analysis of these effects are still needed. This study employs solid-state NMR (ssNMR) to investigate how SNAC interacts with hydrated DMPC liposomes, measuring enhancements in membrane fluidity across interfacial and transmembrane regions. The methodology involves analyzing phosphate lipid headgroups and acyl chains using static ³¹P chemical shift anisotropy and ²H quadrupolar coupling measurements alongside ¹H and ¹³C magic angle spinning NMR for motional averaging of ¹H-¹H and ¹H-¹³C dipolar couplings. Our findings indicate an overall increase in the uniaxial motion of phospholipids with SNAC in a PE concentration-dependent manner. It boosts lipid headgroup dynamics and enhancement plateaus at 25% between 24 and 72 mM concentrations. SNAC effectively enhances the fluidity of the hydrophobic center by 43% at 72 mM PE concentration, more significantly than the interfacial region. It is worth noting that the extent of liposome dissolution and conversion to micelles increases as SNAC concentration rises. Including a model peptide drug, octreotide, introduces a competitive equilibrium in this complex PE-lipid-peptide system, further influencing membrane dynamics for peptide permeation. Interestingly, the membrane enhancement does not show the expected plateau, and a less significant lipid mobility increase is observed in the presence of octreotide, suggesting a less substantial impact compared to peptide-free systems, which is likely due to peptide-PE interactions that consume monomeric SNAC, reducing its interaction with the lipid membrane. This study provides the first quantitative and site-specific ssNMR measurements of membrane mobility influenced by one representative PE as a snapshot of PE lipid interaction in a liposome model, demonstrating how peptide drugs modulate competitive equilibria and PE-induced lipid dynamics.

In the pharmaceutical industry, the Chinese hamster ovary cell, a type of mammalian cell, is extensively employed for the production of conventional full-length monoclonal antibodies. Nanobody is one of the most attractive directions for the development of next-generation antibody drugs. However, a suitable expression system for its manufacture has not yet been comprehensively evaluated. Previously, we proposed that the immunoglobulin constant CH2 domain could be a promising scaffold for developing C-type nanoantibodies (C-Nabs) as candidate therapeutics. Here, we used an antiviral C-Nab, which we identified previously (under review), as a model for investigation. We expressed C-Nabs without a tag in different systems, including a bacterium (C-Nab_bac), yeast (C-Nab_yeast), and mammalian cell (C-Nab_mam). After purification, the binding and neutralizing activities of C-Nabs from different expression systems are similar. Their secondary structures are rich in β-strand. The melting temperatures of C-Nab_bac (71.5 °C) and C-Nab_mam (70.2 °C) are similar, which are slightly higher than that of C-Nab_yeast (65.6 °C), while C-Nab_yeast and C-Nab_mam are more resistant to urea-induced unfolding than C-Nab_bac. C-Nab_yeast and C-Nab_mam demonstrate higher resistance to aggregation compared to C-Nab_bac. C-Nab_yeast exhibits greater resistance to enzyme digestion compared to C-Nab_bac and C-Nab_mam. Notably, when administered via intraperitoneal injection in mice, C-Nab_yeast shows superior pharmacokinetics. Overall, after comparing C-Nab proteins from various expression systems, we determined that yeast is the most suitable host for producing C-Nabs. This finding is beneficial for the production of nanobodies as potential drug candidates.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an attractive candidate for anticancer therapeutics due to its efficient pro-apoptotic activity against tumor cells and its well-tolerated safety profile. However, the in vivo antitumor efficacy of TRAIL is often limited by its poor tumor targeting capacity. Nowadays, the B7 homologue 3 (B7-H3) immune checkpoint has emerged as a promising target for tumor immunotherapy and drug delivery. Here, we report the achievement of tumor-targeted delivery of TRAIL by genetically fusing it with a B7H3-antagonistic affibody. The affibody-TRAIL fusion protein, named ACT, was easily expressed in Escherichia coli with a high yield and could form the active trimeric state. In vitro ACT showed significantly increased cellular binding to multiple B7H3-positive tumor cells and improved cytotoxicity by 2-3 times compared to the parent TRAIL. In vivo ACT demonstrated a 2.4-fold higher tumor uptake than TRAIL in mice bearing B7H3-positive A431 tumor grafts. More importantly, ACT exhibited significantly improved antitumor efficacy against tumors in vivo. In addition, ACT treatment did not cause body weight loss or histopathological changes in the major organs of mice, indicating its good safety profile. Overall, our findings demonstrate that targeting B7H3 to enhance TRAIL delivery is a viable approach to improve its therapeutic efficacy, and ACT may be a potential agent for targeted therapy of B7H3-positive tumors.

Alzheimer's disease (AD) is a prevalent neurodegenerative condition characterized by the aggregation of amyloid-β plaques and neurofibrillary tangles in the brain, leading to synaptic dysfunction and neuronal degeneration. Recently, new treatment approaches involving drugs such as donanemab and lecanemab have been introduced for AD. However, these drug regimens have been associated with adverse effects, leading to the exploration of gene therapy as a potential treatment option. The apolipoprotein E (ApoE) isoforms (ApoE2, ApoE3, and ApoE4) play pivotal roles in AD pathology, with ApoE2 known for its protective effects against AD, making it a promising candidate for gene therapy interventions. However, delivering therapeutics across the blood-brain barrier (BBB) remains a crucial challenge in treating neurological disorders. Liposomes, lipid-based vesicles, are effective nanocarriers due to their ability to shield therapeutics from degradation, though they often lack specificity for brain delivery. To address this issue, liposomes were functionalized with cell-penetrating peptides such as penetratin (Pen), cingulin (Cgn), and a targeting ligand transferrin (T_f). This modification strategy aimed to enhance the delivery of therapeutic ApoE2 plasmids across the BBB to neurons, thereby increasing the level of ApoE2 protein expression. Experimental findings demonstrated that dual-functionalized liposomes (CgnT_f and PenT_f) exhibited higher cellular uptake, biodistribution, and transfection efficiency than single-functionalized (Pen, Cgn, or T_f) and nonfunctionalized liposomes. In vitro studies using primary neuronal cells, bEnd.3 cells, and primary astrocytes consistently supported these findings. Following a single dose treatment via tail vein administration in C57BL6/J mice, in vivo biodistribution results showed significantly higher biodistribution levels in the brain (∼12% ID/gram of tissue) for dual-functionalized liposomes. Notably, treatment with dual-functionalized liposomes resulted in a 2-fold increase in ApoE2 expression levels compared to baseline levels. These findings highlight the potential of dual-functionalized liposomes as an efficacious delivery system for ApoE2 gene therapy in AD, highlighting a promising strategy to address the disease's underlying mechanisms.

A framework for the rational selection of a minimal suite of nondegenerate developability assays (DAs) that maximize insight into candidate developability or storage stability is lacking. To address this, we subjected nine formulation:mAbs to 12 mechanistically distinct DAs together with measurement of their accelerated and long-term storage stability. We show that it is possible to identify a reduced set of key variables from this suite of DAs by using orthogonal statistical methods. We exemplify our approach by predicting the rank formulation:mAb degradation rate at 25 °C (determined over 6 months) using just five DAs that can be measured in less than 1 day, spanning a range of physicochemical features. Implementing such approaches focuses on resources, thus increasing sustainability and decreasing development costs.