Molecular Pharmaceutics最新文献

Chimeric antigen receptor-T (CAR-T) cell therapy is a cellular immunotherapy that has emerged in recent years, and increasing studies showed that therapeutic resistance to CAR-T cell therapy presents in colorectal cancer patients. Lipopolysaccharide (LPS), a component of the cell wall of Gram-negative bacteria, is known to preserve a high concentration in the colon. Whether LPS is a contributing factor to the development of resistance in colorectal cancer cells against CAR-T cell therapy remains unclear. For in vivo experiments, colorectal cancer cells COLO205 were pretreated with LPS for 24 h and then were injected into nude mice through the tail vein, followed by CAR-T cells transplantation one day later. Later, the number of tumors in the lung tissues of the mice was observed. The in vitro experiments were performed on COLO205 cells, which were treated with LPS for 24 h. The effect of LPS on the stemness of COLO205 and SW620 cells was observed by using the colony formation assay and spheroidization experiments. The effect of LPS on the expression of stemness-related genes, including CD44, SOX2, and NANOG, was observed by qRT-PCR assay, Western blotting assay, and immunofluorescence staining. Inhibitors of TGF-β and the MYD88 inhibitor were used to study the mechanisms by which LPS induces the stemness enhancement and resistance to CAR-T cell therapy of COLO205 cells. The correlation between MYD88 and TGFB1, as well as the correlation between TGFB1 and stemness-related genes was analyzed using the TCGA database. Both the in vivo assay of nude mice and the in vitro assay showed that LPS pretreatment could induce resistance to CAR-T cell therapy of colorectal cancer cells. LPS could enhance COLO205 and SW620 cells stemness presented by upregulation of CD44, SOX2, and NANOG. The reverse interfering assay using the TGF-β inhibitor indicated that the autosecretion of TGF-β induced by LPS played a critical role in the stemness enhancement of colorectal cancer cells. The TCGA database analysis revealed a strong positive correlation between MYD88 and TGFB1. Additionally, TGFB1 has been found to upregulate the expression of genes associated with stemness. Further mechanism studies showed that the TLR4/MYD88 pathway medicates LPS-induced TGF-β expression. Our results suggested that LPS-induced resistance to CAR-T cell therapy of colorectal cancer cells through stemness enhancement. TLR4/MYD88 signal pathway-dependent TGF-β expression was involved in stemness enhancement and CAR-T cell therapy resistance. In conclusion, our findings help us to understand the underlying mechanisms of CAR-T cell therapy resistance and indicate that inhibitors of TGF-β and MYD88 are promising targeting candidates to promote a therapeutic effect of CAR-T cell therapy in colorectal cancer in the clinic.

This study aimed to elucidate the drug absorption mechanisms after oral administration of lipid-based formulations (LBFs), emphasizing the impact of their composition on first-pass drug metabolism. Ketoconazole (KTZ), a CYP3A substrate, was loaded into two types of LBFs: a long-chain LBF (type II-LC) and a lipid-free formulation (type IV). Following oral administration of type II-LC, the systemic exposure of KTZ was lower compared to that for the type IV and a control suspension. However, pretreatment with 1-aminobenzotriazole, a nonspecific CYP inhibitor, revealed equivalent in vivo exposure among the formulations tested. The absorption of KTZ from type II-LC in the early period was slower than that from the suspension and type IV. Experiments on in vitro digestion in sequence with in vitro permeation across a dialysis membrane showed that the drug permeation rate for type II-LC was extremely low. This was probably due to the reduction in free drug molecules in the donor compartment via the incorporation of KTZ into mixed micelles comprising digestion products derived from type II-LC and bile components. Furthermore, luminal concentration measurements revealed that gastric emptying was delayed when a type II-LC was administered. The reduced free drug concentration and transient delay in gastric emptying of KTZ resulted in the slower absorption of KTZ for type II-LC. The product of the fraction of drug absorbed and fraction of the drug not metabolized in the gut wall (Fa × Fg) calculated from the systemic and portal plasma concentration-time courses of KTZ was 0.185 for type II-LC and 0.327 for suspension. Since the luminal concentration measurement demonstrated complete absorption of KTZ from the gastrointestinal tract (Fa ≅ 1), the Fa × Fg values can be regarded as Fg. In conclusion, the lower in vivo exposure following oral administration of type II-LC was attributed to reduced Fg, that is, slower drug absorption from the jejunum resulted in low KTZ concentration in enterocytes, leading to enhanced metabolic efficiency. Our findings can be valuable when selecting excipients for designing LBFs with the preferred in vivo performance for highly metabolized drugs.

Human Organic Anion Transporter 4 (OAT4) is predominantly expressed in the kidneys, particularly in the apical membrane of the proximal tubule cells. This transporter is involved in the renal handling of endogenous and exogenous organic anions (OAs), making it an important transporter for drug-drug interactions (DDIs). To better understand OAT4-compound interactions, we generated single concentration (25 μM) in vitro inhibition data for over 1400 small molecules against the uptake of the fluorescent OA 6-carboxyfluorescein (6-CF) in Chinese hamster ovary (CHO) cells. Several drugs exhibiting higher than 50% inhibition in this initial screen were selected to determine IC₅₀ values against three structurally distinct OAT4 substrates: estrone sulfate (ES), ochratoxin A (OTA), and 6-CF. These IC₅₀ values were then compared to the drug plasma concentration as per the 2020 FDA drug-drug interaction (DDI) guidance. Several screened compounds, including some not previously reported, emerged as novel inhibitors of OAT4. These data were also used to build machine learning classification models to predict the activity of potential OAT4 inhibitors. We compared multiple machine learning algorithms and data cleaning techniques to model these screening data and investigated the utility of conformal predictors to predict OAT4 inhibition of a leave-out set. These experimental and computational approaches allowed us to model diverse and unbalanced data to enable predictions for DDIs mediated by this transporter.

ID93 + GLA-LSQ is an adjuvanted recombinant protein vaccine candidate that has demonstrated robust T-cell immunity and reduced bacterial burden in preclinical studies. Here, we explored the strategy of lyophilization by introducing 10% Trehalose as a bulking agent and cryoprotectant to develop a thermostable single-vial formulation of ID93 + GLA-LSQ. We further examined the stability of lyophilized formulations stored at 4 and 37 °C in the research laboratory and field stability across five study sites. Co-mixed (CoVL) and conjugated (ConjVL) formulations were prepared and assessed for various stability parameters including cake quality, melting point, liposome reformation, particle size, GLA/QS-21 concentration, presence of ID93, and biological activity for three months in the research laboratory and nine months at ambient temperature in five health centers. Stability assessment for both formulations stored in the research laboratory for three months showed that they were physically stable and biologically active. The field-based ambient stability assessment showed that the formulations maintained physical integrity, liposomal structure, and antigen integrity, with limited chemical degradation of GLA and QS-21 adjuvants observed. ConjVL retains GLA slightly better than the CoVL formulation, and a moderate increase in particle size was observed after nine months. These findings showed that the formulations demonstrate a promising stability profile after extended storage at ambient temperature, suggesting the potential for real-world application without strict refrigeration requirements.

Statins have been reported for diverse pleiotropic activities, including antimicrobial and antibiofilm. However, due to the limited understanding of their mode of action, none of the statins have gained approval for antimicrobial or antibiofilm applications. In a recent drug repurposing study, we observed that two statins (i.e., Simvastatin and Lovastatin) interact stably with TasA_(28-261), the principal extracellular matrix protein of Bacillus subtilis, and also induce inhibition of biofilm formation. Nevertheless, the underlying mechanism remained elusive. In the present study, we examined the impact of these statins on the physiological activity of TasA_(28-261), specifically its interaction with TapA_(33-253) and aggregation into the amyloid-like structure using purified recombinant TasA_(28-261) and TapA_(33-253) in amyloid detection-specific in vitro assays (i.e., CR binding and ThT staining assays). Results revealed that both statins interfered with amyloid formation by the TasA_(28-261)-TapA_(33-253) complex, while neither statin inhibited amyloid formation by lysozyme, a model amyloid-forming protein. Moreover, neither statin significantly altered the expressions of terminal regulatory genes (viz, sinR, sinI) and terminal effector genes (viz, tasA, tapA, and bslA) involved in biofilm formation by B. subtilis. While the intricate interplay between Simvastatin and Lovastatin with the diverse molecular constituents of B. subtilis biofilm remains to be elucidated conclusively, the findings obtained during the present study suggest that the underlying mechanism for Simvastatin- and Lovastatin-mediated inhibition of B. subtilis biofilm formation is manifested by interfering with the aggregation and amyloid formation by TasA_(28-261)-TapA_(33-253). These results represent one of the first experimental evidence for the underlying mechanism of antibiofilm activity of statins and offer valuable directions for future research to harness statins as antibiofilm therapeutics.

Neurotensin receptor subtype 1 (NTSR1) is overexpressed in numerous cancers. Our laboratory is exploring the utilization of covalent cysteine protease inhibitors (e.g., E-64) to increase tumor retention of targeted radionuclide therapeutics (TRTs) through protein adduct formation. Using this approach, we reported [¹⁷⁷Lu]Lu-NA-ET1, an NTSR1-targeted construct. In this work, we continue the exploration of [¹⁷⁷Lu]Lu-NA-ET1 in three different NTSR1-positive cancer models. [¹⁷⁷Lu]Lu-3BP-227, a clinically investigated NTSR1-targeted construct, was utilized as a comparative benchmark. Both [¹⁷⁷Lu]Lu-NA-ET1 and [¹⁷⁷Lu]Lu-3BP-227 underwent in vitro investigation, including internalization and autoradiographic sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) studies, in NTSR1-positive AsPC-1, HT-29, and PC-3 cell lines. Biodistribution, human radiation dosimetry, and in vivo autoradiographic SDS-PAGE studies were performed by using the same models. A dose escalation study using 585 MBq (15.8 mCi) of [¹⁷⁷Lu]Lu-NA-ET1 was implemented in immunocompetent CF-1 mice. In all three cell lines, [¹⁷⁷Lu]Lu-NA-ET1 demonstrated similar cellular uptake profiles relative to those of [¹⁷⁷Lu]Lu-3BP-227. Biodistribution studies of [¹⁷⁷Lu]Lu-NA-ET1 revealed increased (1.9-4.4-fold) tumor retention and radiation dose delivery relative to the control. Analysis of the in vitro and in vivo cellular and tissue lysates showed protein adducts that ranged from approximately 25-35 kDa, consistent with cysteine cathepsins, the speculative protein binding partner. A total of 585 MBq (15.8 mCi) of [¹⁷⁷Lu]Lu-NA-ET1 was administered and found to be well-tolerated. Incorporating the covalent inhibitor in [¹⁷⁷Lu]Lu-NA-ET1 resulted in an improved retention and radiation dose delivery profile compared to [¹⁷⁷Lu]Lu-3BP-227. Examination of the therapeutic potential of [¹⁷⁷Lu]Lu-NA-ET1 and further exploration of the chemical biology of this approach is underway.

Delivering drugs to the posterior eye segment is a complex task, particularly for treating retinal diseases. Neuroprotective approaches to maintain neuronal integrity have garnered significant attention in recent research. Here, we developed a mucoadhesive nanoparticulate system based on thiolated hyaluronic acid-modified cationic liposomes (HA-SH@liposomes) for topical administration. To fabricate these liposomes, we utilized microfluidic technology with a toroidal mixer to ensure consistent size and stability. Cationic liposomes were prepared by using the microfluidic method, and Epoetin-β (EPOβ), a neuroprotective agent, was loaded into the liposomes. Following this, HA-SH was conjugated to the EPOβ/HA-SH@liposomes using a postmicrofluidics conjugation method, wherein HA-SH was added dropwise to facilitate electrostatic interactions between the cationic liposomes and the anionic polymer. The resulting liposomes exhibited a mean size of 144 ± 1.3 nm and a polydispersity index (PDI) of 0.09 ± 0.01, indicating their uniformity. We evaluated the biocompatibility of the EPOβ/HA-SH@liposomes in vitro using live/dead and MTS assays on the RGC-5 cell line, demonstrating no notable cytotoxicity compared to the controls. To assess the in vivo performance, we conducted extensive ophthalmological examinations in C57/BL6 mice, including immunofluorescence staining to track the distribution of EPOβ and EPOβ/HA-SH@liposomes within the eyeball. Additionally, we quantified EPOβ levels in the retina using an enzyme-linked immunosorbent assay (ELISA) kit after the topical application of free EPOβ and the EPOβ/HA-SH@liposome formulation. The immunofluorescence staining revealed efficient delivery of EPOβ into the retina and choroid via the transcorneal route when administered as EPOβ/HA-SH@liposomes. ELISA results showed that the liposomal formulation achieved approximately 1.9× greater penetration efficiency than free EPOβ. Furthermore, optokinetic response (OKR) assays indicated that animals treated with EPOβ/HA-SH@liposomes exhibited slightly improved visual acuity compared with those treated with free EPOβ, though the difference was not statistically significant. In conclusion, the topical ocular administration of EPOβ/HA-SH@liposomes facilitated the efficient delivery of EPOβ to the retina, promoting retinal recovery and confirming its neuroprotective properties. This preclinical study provides a foundation for innovative strategies in the topical delivery of neuroprotective agents in ocular therapy.

A nanoplatform based on self-assembling peptides was developed with the ability to effectively transport and deliver a wide range of moieties across the blood-brain barrier (BBB) for the treatment of glioblastoma. Its surface was functionalized to have a targeted release of TMZ thanks to the targeting peptide that binds to EGFRvIII, which is overexpressed on tumor cells, and gH625, which acts as an enhancer of penetration. Furthermore, the on-demand release of TMZ was achieved through matrix metalloproteinase-9 (MMP-9) cleavage. Nanofibers were characterized for their stability, critical aggregation concentration, and morphology. Next, the effect on both 2D and 3D glioblastoma/astrocytoma (U-87) and glioma (U-118) cell lines was evaluated. The Annexin V/Propidium iodide showed an increase in necrotic and apoptotic cells, and the morphological analysis allowed to discover that both U-118 and U-87 spheroids are smaller in surface, perimeter, and Feret's diameter when treated with NF-TMZ. The developed nanofiber was demonstrated to permeate the BBB in vitro in a 3D spheroidal biodynamic BBB model. Finally, there were no cytotoxic effects of nanofibers without the drug on spheroids, while a significant decrease in viability was observed when NF-TMZ was used. Overall, these results open new opportunities for the evaluation of the efficacy and safety of this nanoplatform in in vivo studies.

Cadherin 17 (CDH17) is highly expressed in digestive system cancers, and the potential of nanobodies targeting CDH17 as imaging probes and delivery vehicles for radioactive β-particles warrants exploration for their theranostic potential in CDH17-overexpressing gastric cancer (GC). In this study, we screened an anti-CDH17 nanobody library and constructed two antibodies: anti-CDH17 VHH (recombinant nanobody fused with a polyhistidine tag) and anti-CDH17 VHH-ABD (recombinant nanobody fused with an albumin-binding domain). VHH targeting CDH17 and its derivative VHH-ABD were conjugated with DOTA and labeled with radionuclide ¹⁷⁷Lu. The pharmacokinetics and theranostic efficacy of these agents were evaluated in the GC xenograft models. [¹⁷⁷Lu]Lu-VHH and [¹⁷⁷Lu]Lu-VHH-ABD exhibited high radiochemical purity (>99%, n = 3) and successfully delineated CDH17-positive gastric cancer tissues on SPECT/CT imaging. Compared with the rapid renal clearance of [¹⁷⁷Lu]Lu-VHH, [¹⁷⁷Lu]Lu-VHH-ABD demonstrated prolonged circulation times with increased and sustained tumor accumulation. Survival experiments in the MKN-45 tumor model revealed that two doses of [¹⁷⁷Lu]Lu-VHH-ABD effectively suppressed tumor growth, with limited systemic biotoxicity. Histological analysis using hematoxylin and eosin (H&E) staining and Ki67 immunohistochemistry confirmed structural disruption and low tumor cell proliferative activity in the tumor tissue. In preclinical studies, [¹⁷⁷Lu]Lu-anti-CDH17 VHH-ABD demonstrated substantial antitumor efficacy with manageable toxicity, offering promising clinical potential as a viable therapeutic option for CDH17-overexpressing GC.

Peptides are highly receptor-affine molecules exhibiting suitable pharmacokinetics. Additionally, low-cost production, simple protocols allowing easy modifications, and tolerance toward harsh reaction conditions make peptides ideal ligands for preparation of radiopharmaceuticals for cancer detection and treatment. However, natural peptides being substrates for enzymes are susceptible to proteolysis, which limits the in vivo lifetime and the target uptake. Therefore, the majority of peptides are not able to progress beyond preclinical research. Advancement of peptides for clinical analysis needs modification to instill improved features. Continuous increase and further expected rise in cancer cases in the next decade require development of more disease-directed and promising radiopharmaceuticals. Redesigned peptide, mimicking the original peptide with similar or improved affinity and high metabolic stability, shall have significant edge. This review outlines the design of peptidomimetics by incorporation of D-amino acids (inverso); reversal of D-amino acid sequence (retro-inverso), and reversal of L-amino acid sequence (retro). Clinically successful radiopeptidomimetics prepared using the three approaches have been elaborated to elucidate the important role of peptidomimetics in cancer management.