Half Maximal Inhibitory Concentration Research Articles

Microwave-assisted synthesis of novel Ti/BTB-MOFs as porous anticancer and antibacterial agents

Nano compounds, especially metal-organic frameworks (MOFs), have significant properties. Among the most important properties of these compounds, which depend on their specific surface area and porosity, are biological properties, such as anticancer and antibacterial properties. In this study, a new titanium/BTB metal-organic framework (Ti/BTB-MOF) was synthesized by using titanium nitrate and 1,3,5-Tris(4-carboxyphenyl)benzene (BTB) under microwave radiation. The structure of the synthesized Ti/BTB-MOF was characterized and confirmed using X-ray diffraction (XRD) patterns, X-ray photoelectron spectroscopy (XPS) analysis, Fourier transform infrared (FT-IR) spectra, energy-dispersive X-ray (EDAX) analysis mapping, scanning electron microscope (SEM) images, thermogravimetric analysis (TGA) curves, and Brunauer–Emmett–Teller (BET) analysis. The in vitro anticancer properties of Ti/BTB-MOF were evaluated using the MTT method against MG-63/bone cancer cells and A-431/skin cancer cells. The in vitro antibacterial activity was tested using the Clinical and Laboratory Standards Institute (CLSI) guidelines. In the anticancer activity, IC50 (half-maximal inhibitory concentration) values of 152 μg/mL and 201 μg/mL for MG-63/bone cancer cells and A-431/skin cancer cells, respectively, were observed. In the antibacterial activity, minimum inhibitory concentrations (MICs) of 2–64 μg/mL were observed against studied pathogenic strains. The antimicrobial activity of Ti/BTB-MOF was higher than that of penicillin and gentamicin. Therefore, the synthesized Ti/BTB-MOF could be introduced as a suitable bioactive candidate.

Contribution of aldehyde oxidase to methotrexate-induced hepatotoxicity: in vitro and pharmacoepidemiological approaches

ABSTRACT Background Methotrexate (MTX) is partially metabolized by aldehyde oxidase (AOX) in the liver and its clinical impact remains unclear. In this study, we aimed to demonstrate how AOX contributes to MTX-induced hepatotoxicity in vitro and clarify the relationship between concomitant AOX inhibitor use and MTX-associated liver injury development using the U.S. Food and Drug Administration Adverse Event Reporting System (FAERS). Methods We assessed intracellular MTX accumulation and cytotoxicity using HepG2 cells. We used the FAERS database to detect reporting odds ratio (ROR)-based MTX-related hepatotoxicity event signals. Results AOX inhibition by AOX inhibitor raloxifene and siRNA increased the MTX accumulation in HepG2 cells and enhanced the MTX-induced cell viability reduction. In the FAERS analysis, the ROR for MTX-related hepatotoxicity increased with non-overlap of 95% confidence interval when co-administered with drugs with higher Imax, u (maximum unbound plasma concentration)/IC50 (half-maximal inhibitory concentration for inhibition of AOX) calculated based on reported pharmacokinetic data. Conclusion AOX inhibition contributed to MTX accumulation in the liver, resulting in increased hepatotoxicity. Our study raises concerns regarding MTX-related hepatotoxicity when co-administered with drugs that possibly inhibit AOX activity at clinical concentrations.

A novel disulfide death-related genes prognostic signature identifies the role of IPO4 in glioma progression

Background“Disulfide death,” a form of cellular demise, is triggered by the abnormal accumulation of intracellular disulfides under conditions of glucose deprivation. However, its role in the prognosis of glioma remains undetermined. Therefore, the main objective of this study is to establish prognostic signature based on disulfide death-related genes (DDRGs) and to provide new solutions in choosing the effective treatment of glioma.MethodsThe RNA transcriptome, clinical information, and mutation data of glioma samples were sourced from The Cancer Genome Atlas (TCGA) and the Chinese Glioma Genome Atlas (CGGA), while normal samples were obtained from the Genotype-Tissue Expression (GTEx). DDRGs were compiled from previous studies and selected through differential analysis and univariate Cox regression analysis. The molecular subtypes were determined through consensus clustering analysis. Further, LASSO analysis was employed to select characteristic genes, and subsequently, a risk model comprising seven DDRGs was constructed based on multivariable Cox analysis. Kaplan-Meier survival curves were employed to assess survival differences between high and low-risk groups. Additionally, functional analyses (GO, KEGG, GSEA) were conducted to explore the potential biological functions and signaling pathways of genes associated with the model. The study also explored immune checkpoint (ICP) genes, immune cell infiltration levels, and immune stromal scores. Finally, the effect of Importin-4(IPO4) on glioma has been further confirmed through RT-qPCR, Western blot, and cell functional experiments.Results7 genes associated with disulfide death were obtained and two subgroups of patients with different prognosis and clinical characteristics were identified. Risk signature was subsequently developed and proved to serve as an prognostic predictor. Notably, the high-risk group exhibited an immunosuppressive microenvironment characterized by a high concentration of M2 macrophages and regulatory T cells (Tregs). In contrast, the low-risk group showed lower half-maximal inhibitory concentration (IC50) values. Therefore, patients in the high-risk group may benefit more from immunotherapy, while patients in the low-risk group may benefit more from chemotherapy. In addition, in vitro experiments have shown that inhibition of the expression of IPO4 leads to a significant reduction in the proliferation, migration, and invasion of glioma cells.ConclusionThis study identified two glioma subtypes and constructed a prognostic signature based on DDRGs. The signature has the potential to optimize the selection of patients for immune- and chemotherapy and provided a potential therapeutic target for glioma.

Structural and Functional Analyses of Inhibition of Human Dihydroorotate Dehydrogenase by Antiviral Furocoumavirin.

Natural products are important sources of seed compounds for drug discovery. However, it has become difficult in recent years to discover new compounds with valuable pharmacological activities. On the other hand, among the vast number of natural products that have been isolated so far, a considerable number of compounds with specific biological activities are thought to be overlooked in screening that uses biological activity as an index. Therefore, it is conceivable that such overlooked useful compounds may be found by screening compound libraries that have been amassed previously through specific assays. Previously, NPD723, a member of the Natural Products Depository library comprised of a mixture of natural and non-natural products developed at RIKEN, and its metabolite H-006 were found to inhibit growth of various cancer cells at low nanomolar half-maximal inhibitory concentration. Subsequent analysis revealed that H-006 strongly inhibited human dihydroorotate dehydrogenase (DHODH), the rate-limiting enzyme in the de novo pyrimidine biosynthetic pathway. Here, we elucidated the crystal structure of the DHODH-flavin mononucleotide-orotic acid-H-006 complex at 1.7 Å resolution to determine that furocoumavirin, the S-enantiomer of H-006, was the actual inhibitor. The overall mode of interaction of furocoumavirin with the inhibitor binding pocket was similar to that described for previously reported tight-binding inhibitors. However, the structural information together with kinetic characterizations of site-specific mutants identified key unique features that are considered to contribute to the sub-nanomolar inhibition of DHODH by furocoumavirin. Our finding identified new chemical features that could improve the design of human DHODH inhibitors.

Amperometry approach curve profiling to understand the regulatory mechanisms governing the concentration of intestinal extracellular serotonin

Enterochromaffin (EC) cells located within the intestinal mucosal epithelium release serotonin (5-HT) to regulate motility tones, barrier function and the immune system. Electroanalytical methodologies have been able to monitor steady state basal extracellular 5-HT levels but are unable to provide insight into how these levels are influenced by key regulatory processes such as release and uptake. We established a new measurement approach, amperometry approach curve profiling, which monitors the extracellular 5-HT level at different electrode–tissue (E–T) distances. Analysis of the current profile can provide information on contributions of regulatory components on the observed extracellular 5-HT level. Measurements were conducted from ex vivo murine ileum and colon using a boron-doped diamond (BDD) microelectrode. Amperometry approach curve profiling coupled with classical pharmacology demonstrated that extracellular 5-HT levels were significantly lower in the colon when compared to the ileum. This difference was due to a greater degree of activity of the 5-HT transporter (SERT) and a reduced amount of 5-HT released from colonic EC cells. The presence of an inhibitory 5-HT4 autoreceptor was observed in the colon, where a 40% increase in extracellular 5-HT was the half maximal inhibitory concentration for activation of the autoreceptor. This novel electroanalytical approach allows estimates of release and re-uptake and their contribution to 5-HT extracellular concentration from intestinal tissue be obtained from a single series of measurements.

Design, Synthesis, and Biological Activity of Novel Triketone-Containing Phenoxy Nicotinyl Inhibitors of HPPD.

4-Hydroxyphenylpyruvate dioxygenase (HPPD) is a crucial target enzyme in albino herbicides. The inhibition of HPPD activity interferes with the synthesis of carotenoids, blocking photosynthesis and resulting in bleaching and necrosis. To develop herbicides with excellent activity, a series of 3-hydroxy-2-(6-substituted phenoxynicotinoyl)-2-cyclohexen-1-one derivatives were designed via active substructure combination. The title compounds were characterized via infrared spectroscopy, 1H and 13C nuclear magnetic resonance spectroscopies, and high-resolution mass spectrometry. The structure of compound III-17 was confirmed via single-crystal X-ray diffraction. Preliminary tests demonstrated that some compounds had good herbicidal activity. Crop safety tests revealed that compound III-29 was safer than the commercial herbicide mesotrione in wheat and peanuts. Moreover, the compound exhibited the highest inhibitory activity against Arabidopsis thaliana HPPD (AtHPPD), with a half-maximal inhibitory concentration of 0.19 μM, demonstrating superior activity compared with mesotrione (0.28 μM) in vitro. A three-dimensional quantitative structure-activity relationship study revealed that the introduction of smaller groups to the 5-position of cyclohexanedione and negative charges to the 3-position of the benzene ring enhanced the herbicidal activity. A molecular structure comparison demonstrated that compound III-29 was beneficial to plant absorption and conduction. Molecular docking and molecular dynamics simulations further verified the stability of the complex formed by compound III-29 and AtHPPD. Thus, this study may provide insights into the development of green and efficient herbicides.

Design, Synthesis, Bioactive Evaluation, and Molecular Dynamics Simulation of Novel 4H-Pyrano[3,2-c]pyridine Analogues as Potential Sterol 14α-Demethylase (CYP51) Inhibitors.

To discover potential sterol 14α-demethylase (CYP51) inhibitors, thirty-four unreported 4H-pyrano[3,2-c]pyridine derivatives were designed and synthesized. The assay results indicated that most compounds displayed significant fungicidal activity against Sclerotinia sclerotiorum, Colletotrichum lagenarium, Botrytis cinerea, Penicillium digitatum, and Fusarium oxysporum at 16 μg/mL. The half maximal effective concentration (EC50) values of compounds 7a, 7b, and 7f against B. cinerea were 0.326, 0.530, and 0.610, respectively. Namely, they had better antifungal activity than epoxiconazole (EC50 = 0.670 μg/mL). Meanwhile, their half maximal inhibitory concentration (IC50) values against CYP51 were 0.377, 0.611, and 0.748 μg/mL, respectively, representing that they also possessed better inhibitory activities than epoxiconazole (IC50 = 0.802 μg/mL). The fluorescent quenching tests of proteins showed that 7a and 7b had similar quenching patterns to epoxiconazole. The molecular dynamics simulations indicated that the binding free energy of 7a and epoxiconazole to CYP51 was -35.4 and -27.6 kcal/mol, respectively.

The cuproptosis-related gene UBE2D2 functions as an immunotherapeutic and prognostic biomarker in pan-cancer.

Cuproptosis, as a unique modality of regulated cell death, requires the involvement of ubiquitin-binding enzyme UBE2D2. However, the prognostic and immunotherapeutic values of UBE2D2 in pan-cancer remain largely unknown. Using UCSC Xena, TIMER, Clinical Proteomic Tumor Analysis Consortium (CPTAC), and Human Protein Atlas (HPA) databases, we aimed to explore the differential expression pattern of UBE2D2 across multiple cancer types and to evaluate its association with patient prognosis, clinical features, and genetic variations. The association between UBE2D2 and immunotherapy response was assessed by gene set enrichment analysis, tumor microenvironment, immune gene co-expression and drug half maximal inhibitory concentration (IC50) analysis. The mRNA and protein levels of UBE2D2 were markedly elevated in most cancer types, and UBE2D2 exhibited prognostic significance in liver hepatocellular carcinoma (LIHC), kidney chromophobe (KICH), uveal melanomas (UVM), cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), and kidney renal papillary cell carcinoma (KIRP). UBE2D2 expression was correlated with clinical features, tumor mutation burden, microsatellite instability, and anti-tumor drug resistance in several tumor types. Gene enrichment analysis showed that UBE2D2 was significantly associated with immune-related pathways. The expression level of UBE2D2 was correlated with immune cell infiltration, including CD4 + T cells、Macrophages M2、CD8 + T cells in pan-cancer. PDCD1, CD274 and CTLA4 expression levels were positively correlated with UBE2D2 level in multiple cancers. We comprehensively investigated the potential value of UBE2D2 as a prognostic and immunotherapeutic predictor for pan-cancer, providing a novel insight for cancer immunotherapy.

In vitro antileishmanial activities of hydro-methanolic crude extracts and solvent fractions of Clematis simensis fresen leaf, and Euphorbia abyssinica latex.

As a result of increasing drug resistance, crossover resistance development, prolonged therapy, and the absence of different agents with innovative methods for implementation, the efficacy of recent antileishmanial medications is severely declining. So, it is vital to look for other medications from botanical remedies that have antileishmanial activity. The latex of Euphorbia abyssinica (E abyssinica) and the leaves of Clematis simensis fresen (C simensis) were macerated in methanol (80%). In vitro antileishmanial activity of the preparation was tried on promastigotes of Leishmania aethiopica (L aethiopica) and Leishmania donovani (L donovani) using resazurin assay, and fluorescence intensity was measured. One percent of dimethyl sulfoxide (DMSO) and media as negative control and amphotericin B as positive control were used. Additionally, hemolytic & phytochemical tests of the preparation were done. The mean and standard errors of each extract were evaluated and interpreted for statistical significance using one-way analysis of variance. From sigmoidal dose-response curves of % inhibition, half maximal inhibitory concentration (IC50) values were determined by GraphPad Prism and Microsoft Excel; outcomes were presented as mean ± standard error of mean of triplicate trials. P < .05 was statistical significance. The phytochemical screening of C simensis and E abyssinica confirmed the existence of steroids, phenols, tannins, saponins, alkaloids, terpenoids, flavonoids and glycosides. C simensis possesses antileishmanial activity with IC50 outcomes of 46.12 ± 0.03 and 8.18 ± 0.10 µg/mL on the promastigotes of L aethiopica and L donovani, respectively. However, E abyssinica showed stronger activity with IC50 outcomes of 16.07 ± 0.05 µg/mL and 4.82 ± 0.07 µg/mL on L aethiopica and L donovani, respectively. C simensis and E abyssinica have a less hemolytic effect on human red blood cells at low concentrations. The outcomes from this investigation demonstrated that the preparation of C simensis and E abyssinica indicated significant antileishmanial activity. Therefore, further in vivo assessment of antileishmanial, cytotoxicity activity and quantitative identification of secondary metabolites are highly recommended.

Physical and Chemical Properties of Boiled Oil: A Traditional Method of Extracting Oil from Boiled Olive Fruits

Background In some villages in Northern Jordan, farmers boil part of their olive fruit harvest before oil extraction to increase the quantity of oil, get a desirable acquired taste, as they claim, and get additional health benefits. Locals call this oil Boiled oil (BO), and its price is about 20% higher than virgin olive oil (VOO) produced by the same farmer. Objective The hypothesis was that boiling olive fruits may affect the quality of the produced oil. Therefore, our study aimed to determine the chemical and physical characteristics of boiled oil in comparison with VOO and the effect of storage of both types of oils for one year. Methods Total phenolic compounds, ferric-reducing antioxidant power assay (FRAP), and radical scavenging activity (RSA) were evaluated. Moreover, the quality indices of BO and VOO were also evaluated. Results Total phenolic compounds decreased significantly (p &lt;0.05) from 8.7 mg GAE/100g in VOO to 2.47 mg GAE/100g in BO. The antioxidant activity measured using FRAP assay also decreased significantly (p &lt;0.05) from 962 to 379 micromole/kg. Half-maximum inhibitory concentration (IC50) of the RSA was significantly higher (p &lt;0.05) in BO samples (414 mg/ml) in comparison with VOO samples (38.9 mg/ml). Moreover, there was a significant increase (p &lt;0.05) in acid value in BO samples (0.943%) in comparison with VOO samples (0.518%). However, the increase in acid value after one year of storage was higher in VOO than in BO. The peroxide value also increased significantly in boiled oil (500 meq/kg) in comparison with VOO (19 meq/kg). Additionally, a significant increase in ultraviolet absorption was observed in BO at k232 and k270 (3.5), which is considered unsuitable for human consumption compared with VOO (2.43). Conclusion In conclusion, these results showed that boiling olive fruits before oil extraction deteriorates oil quality as expected, and consumers should be educated that this type of oil is hazardous to human health and is a waste of effort and money.

Chemoreactome screening of aquacobalamin and heptamethyl ester of cyanoaquacobyrinic acid cytotoxic effects on tumor cells with experimental confirmation on BT-474 and A549 cell

Background. The search for an effective and safe pharmacotherapy for tumor diseases includes the evaluation of the action of candidate molecules on various types of tumor cells. Vitamin B12 and its derivatives are promising molecules whose properties can be controlled through chemical modifications.Objective: conducting in silico chemoreactom screening and in vitro experimental study of aquacobalamin and heptamethyl ester of cyanoaquacobyrinic acid (HECСA).Material and methods. Chemoreactome screening was carried out on the basis of a problem-oriented theory of chemograph isomorphism analysis, which is an extension of the algebraic approach to machine learning and recognition problems. Trainable algorithms for calculating chemical distances between molecules were used, on the basis of which the values of half-maximal inhibitory concentration (IC50) were calculated. Screening was carried out for 470 cultures of human tumor cells, including SNB19 (astrocytoma), HCT116 (colon cancer), HeLa (cervical carcinoma), BT-474 (breast duct carcinoma), and A549 (lung carcinoma) cell lines. Dicyanocobyric acid heptamethyl ester ((CN)2Cby(OCH3)7) was obtained by boiling a solution of vitamin B12 in methanol with sulfuric acid (1.0 M) for 4 days. HECСA ((CN)(H2O)Cby(OCH3)7) was obtained by vacuum drying an aqueous solution of (CN)2Cby(OCH3)7 (pH 4.0 and 25 °С). The ester structure and purity were confirmed by 1H nuclear magnetic resonance data, elemental analysis, and MALDI-ToF (matrix-assisted laser desorption/ionization time of flight) mass spectroscopy. Experimental studies of tumor cell cultures were carried out using the MTT testwith aquacobalamin and HECСA on cell lines of immortalized (telomerized) fibroblasts (Fb-hTERT), lung carcinoma (A549), and breast duct cancer (BT-474).Results. Chemoreactome screening of the effects of molecules on tumor cells made it possible to obtain estimates of cell growth IC50 for 470 tumor cell lines. Depending on cell line and vitamin B12 derivative molecule, IC50 values varied in a fairly wide range: from 15 to 2000 nM. In vitro studies on cultures of two human tumor cell lines (BT-474 and A549) and telomerized Fb-hTERT fibroblasts confirmed the cytotoxic effect of aquacobalamin and its hydrophobic derivative HECСA. It was shown that aquacobalamin had weak cytotoxic properties in the concentration range of 3.125–200 µg/l (IC50 &gt; 200 nM), and HECСA significantly reduces the survival of BT-474 and A549 tumor cell lines at high concentrations (100–200 µg/l, IC50 about 100 nM).Conclusion. Correspondence was shown between the results of in silico chemoreactome screening and in vitro cell culture studies: IC50 values for HECСA were significantly lower than for aquacobalamin, and the conversion factor from chemoreactome estimates to experimental ones was almost the same (2.64 for BT-474, and 2.63 for A549). The results of chemoreactome screening for other tumor cell lines can be used to plan further cell experiments with vitamin B12 derivatives.

Preparation and characterization of artemether-loaded niosomes in Leishmania major-induced cutaneous leishmaniasis

Cutaneous leishmaniasis is the most prevalent form of leishmaniasis worldwide. Although various anti-leishmanial regimens have been considered, due to the lack of efficacy or occurrence of adverse reactions, design and development of novel topical delivery systems would be essential. This study aimed to prepare artemether (ART)-loaded niosomes and evaluate their anti-leishmanial effects against Leishmania major. ART-loaded niosomes were prepared through the thin-film hydration technique and characterized in terms of particle size, zeta potential, morphology, differential scanning calorimetry, drug loading, and drug release. Furthermore, anti-leishmanial effect of the preparation was assessed in vitro and in vivo. The prepared ART-loaded niosomes were spherical with an average diameter of about 100 and 300 nm with high encapsulation efficiencies of > 99%. The results of in vitro cytotoxicity revealed that ART-loaded niosomes had significantly higher anti-leishmanial activity, lower general toxicity, and higher selectivity index (SI). Half-maximal inhibitory concentration (IC50) values of ART, ART-loaded niosomes, and liposomal amphotericin B were 39.09, 15.12, and 20 µg/mL, respectively. Also, according to the in vivo study results, ART-loaded niosomes with an average size of 300 nm showed the highest anti-leishmanial effects in animal studies. ART-loaded niosomes would be promising topical drug delivery system for the management of cutaneous leishmaniasis.

Abstract PO5-27-07: A novel ROR1 inhibitor CPD86 suppresses Triple-Negative Breast Cancer cells via regulation of AKT/GSK3β pathway

Abstract Breast cancer is the second most commonly diagnosed cancer in women in the United States. The most aggressive subtype is triple negative breast cancer (TNBC), which accounts for 15-20% of new cases on average each year. Histologically, it is characterized by the absence of estrogen receptors (ER), progesterone receptors (PR) and human epidermal growth factor receptor 2 (HER2), which are typically targeted by hormone therapies such as tamoxifen, fulvestrant, or letrozole. Standard of care for TNBC includes traditional chemotherapy and radiation, which are detrimental to both cancerous and normal cells. Therefore, the lack of all hormone receptors in TNBC calls for the investigation of novel anti-cancer compounds that specifically target TNBC with minimal damage to non-cancerous tissues. Receptor orphan tyrosine kinase-like receptor 1 (ROR1) is an oncoprotein that is overexpressed in several human malignancies such as lung, breast, prostate and pancreatic cancers, but not in normal tissues. Inhibition of ROR1 signaling has shown to repress proliferation and induce apoptosis of cancer cells. Through in-silico docking and isothermal titration calorimetry, we identified a novel compound CPD86 that interacts with and inhibits ROR1. We hypothesize that CPD86 selectively targets TNBC cells by reducing the phosphorylation of AKT/GSK3β via the inhibition of ROR1. Our in vitro results suggest that CPD86 inhibits cell viability and induces intrinsic apoptosis of TNBC cells at a half-maximal inhibitory concentration of 2-5 µM, but not in normal breast epithelial cells with minimal ROR1 expression. CPD86 also represses TNBC cell migration and invasion while leaving non-malignant cells unharmed. This study highlights that ROR1 inhibitors could be developed as a potential therapeutic for TNBC and the data would serve as proof-of-concept justification for evaluation in other ROR1-upregulated cancers. Citation Format: Tram Ta, Victoria Reed, Nikhil Chandra, Nick Dwyer, Norman Fultang, Shradheya Gupta, Indrakant Singh, Bela Peethambaran. A novel ROR1 inhibitor CPD86 suppresses Triple-Negative Breast Cancer cells via regulation of AKT/GSK3β pathway [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-27-07.

Colorectal carcinoma peritoneal metastases-derived organoids: results and perspective of a model for tailoring hyperthermic intraperitoneal chemotherapy from bench-to-bedside

BackgroundPeritoneal metastases from colorectal cancer (CRCPM) are related to poor prognosis. Cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) have been reported to improve survival, but peritoneal recurrence rates are still high and there is no consensus on the drug of choice for HIPEC. The aim of this study was to use patient derived organoids (PDO) to build a relevant CRCPM model to improve HIPEC efficacy in a comprehensive bench-to-bedside strategy.MethodsOxaliplatin (L-OHP), cisplatin (CDDP), mitomycin-c (MMC) and doxorubicin (DOX) were used to mimic HIPEC on twelve PDO lines derived from twelve CRCPM patients, using clinically relevant concentrations. After chemotherapeutic interventions, cell viability was assessed with a luminescent assay, and the obtained dose–response curves were used to determine the half-maximal inhibitory concentrations. Also, induction of apoptosis by different HIPEC interventions on PDOs was studied by evaluating CASPASE3 cleavage.ResultsResponse to drug treatments varied considerably among PDOs. The two schemes with better response at clinically relevant concentrations included MMC alone or combined with CDDP. L-OHP showed relative efficacy only when administered at low concentrations over a long perfusion period. PDOs showed that the short course/high dose L-OHP scheme did not appear to be an effective choice for HIPEC in CRCPM. HIPEC administered under hyperthermia conditions enhanced the effect of chemotherapy drugs against cancer cells, affecting PDO viability and apoptosis. Finally, PDO co-cultured with cancer-associated fibroblast impacted HIPEC treatments by increasing PDO viability and reducing CASPASES activity.ConclusionsOur study suggests that PDOs could be a reliable in vitro model to evaluate HIPEC schemes at individual-patient level and to develop more effective treatment strategies for CRCPM.

Abstract PO2-24-10: Anti-Tumorigenic Immuno-Microenvironment by AB-329, a Potent and Selective AXL inhibitor, combined with paclitaxel in Triple-Negative Breast Cancer Preclinical Model

Abstract BACKGROUND The Gas6/AXL signaling pathway plays a substantial role in cancer cell survival, proliferation, immunosuppression, and metastasis. Elevated AXL expression is associated with advanced cancer stages and decreased overall survival rates, underscoring its potential as a therapeutic target for metastatic cancers. We investigated this possibility by utilizing AB-329, a selective small molecule AXL kinase inhibitor, to treat triple-negative breast cancer (TNBC). TNBC, typified by the lack of ER, PR, and HER2 expression, frequently displays mesenchymal characteristics and carries a significant risk of metastasis and an immunosuppressive tumor microenvironment. METHODS We assessed AXL protein expression in 28 human and 11 mouse breast cancer cell lines using Western blotting. The Microarray database was utilized to examine AXL gene expression in 40 human breast cancer cells. The Sulforhodamine B proliferation and soft agar assays measured the antiproliferative effect of AB-329 in TNBC cell lines. The in vitro anti-metastatic impact of AB-329 was evaluated through migration and invasion assays. We appraised the anti-tumor effects of AB-329 in combination with paclitaxel using 4T1 and E0771-LMB mouse TNBC xenograft models. We used FACS and CIBERSORTx to perform immune cell type profiling in E0771-LMB xenograft tumor tissues. To understand AB-329’s immunomodulatory effects in tumors, single-cell RNA sequencing was conducted in a humanized mouse model with SUM149 human TNBC xenografts. RESULTS Microarray data revealed higher AXL expression in TNBC cells compared to non-TNBC cells. Western blotting supported this, showing higher AXL protein expression, particularly in basal and mesenchymal subtypes of TNBC cells, compared to luminal breast cancer cells. AB-329 presented moderate antiproliferative effects as a monotherapy in the TNBC cell lines tested, with half-maximal inhibitory concentrations exceeding 5 µM. Nevertheless, combining AB-329 with paclitaxel resulted in significant growth inhibition in all tested TNBC cell lines (p &amp;lt; 0.05). Importantly, AB-329 alone could inhibit TNBC migration and invasion at a concentration of 1 µM (p &amp;lt; 0.01) and showed enhanced inhibition rates when paired with paclitaxel (p &amp;lt; 0.05). The combination of AB-329 and paclitaxel considerably reduced tumor growth in the 4T1 and E0771-LMB xenograft models compared to paclitaxel or AB-329. Total RNA sequencing and CIBERSORTx analysis indicated that the AB-329 and paclitaxel combination increased Natural Killer (NK) cells’ infiltration into E0771-LMB mouse TNBC xenograft tumors compared to single agent-treated tumors. A nearly 2.5-fold increase in NKT cell infiltration was also detected with AB-329 treatment compared to Control in SUM149 human TNBC xenografts. CONCLUSIONS Our preclinical data suggest that AB-329, an AXL inhibitor, could synergize effectively with paclitaxel in TNBC treatment, notably inhibiting tumor cell proliferation and migration while enhancing NK cell infiltration. Intriguingly, we observed an increased infiltration of NKT cells into tumors following AB-329 treatment, which may promote an anti-tumorigenic immuno-microenvironment. These results underscore the necessity for further investigation into the role of AB-329 in modulating the tumor immuno-microenvironment through AXL pathway inhibition and accentuate the potential for combining AB-329 with immunotherapy. Citation Format: Dileep Reddy Rampa, Jon Fuson, Huey Liu, Max Pan, Naoto Ueno, Jangsoon Lee. Anti-Tumorigenic Immuno-Microenvironment by AB-329, a Potent and Selective AXL inhibitor, combined with paclitaxel in Triple-Negative Breast Cancer Preclinical Model [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-24-10.

Discovery of 3-trifluoromethyl-substituted pyrazoles as selective phosphodiesterase 10A inhibitors for orally attenuating isoprenaline-induced cardiac hypertrophy

Recently, MP-10, a previous drug candidate with potent inhibition of phosphodiesterase 10A (PDE10A) in clinical phase II trials for schizophrenia or Alzheimer's disease, has shown significant potential in preventing and treating cardiovascular diseases. However, its poor metabolic stability and high permeability across the blood-brain barrier (BBB) make it unsuitable for preventing and treating peripheral cardiovascular diseases. Herein, the hit-to-lead optimization was performed to discover novel 3-trifluoromethyl-substituted pyrazole derivatives as potent and selective PDE10A inhibitors. The structure-activity relationships, biological characterization, molecular mechanism, and drug-like evaluation were discussed to identify compound C7 which showed potent inhibition against PDE10A (half maximal inhibitory concentration, IC50 = 11.9 nmol/L), more than 840-fold selectivity over other PDE subtypes, enhanced liver microsomes stability (T1/2 = 239 min) compared to MP-10 and low BBB permeability. Importantly, oral pretreatment with C7·3HCl at a dose of 5.0 mg/kg significantly attenuated the pathological and functional changes induced by isoprenaline (ISO)-induced pathological cardiac hypertrophy in mice, particularly suppressing increase of cardiac weight, atrial natriuretic peptide (ANP) and β-myosin heavy chain (β-MHC) hypertrophic markers along with cardiac fibrosis. These findings further support that targeting PDE10A provides an innovative therapeutic approach for preventing and treating cardiac diseases.

Development of a Rapid HPLC-Fluorescence Method for Monitoring Warfarin Metabolites Formation: In-vitro Studies for Evaluating the Effect of Piperine on Warfarin Metabolism and Plasma Coagulation

Warfarin, a widely prescribed anticoagulant, is highly effective for various coagulation disorders. However, its efficacy is limited by a narrow therapeutic index and frequent drug interactions, especially those involving metabolism by Cytochrome P450 (CYP450) enzymes. Piperine, found in black and long pepper, possesses blood-thinning properties and has been observed to inhibit CYP3A and CYP2C enzymes linked to warfarin metabolism. This study investigated the effect of piperine on warfarin metabolism in liver microsomes using a rapid and sensitive HPLC-Fluorescence method. The use of PFP (pentafluorophenyl) column with core shell particles provided the selectivity and resolution to resolve warfarin and its 4-, 6-, 7-, and 10-hydroxy metabolites in addition the internal standard naproxen in less than 3 minutes. This is the fastest analytical assay for warfarin and its major metabolites reported to date making it ideal for metabolic studies. The applicability of the method was demonstrated by monitoring the metabolism of S-warfarin in human and rat liver microsomes and evaluating the inhibitory effect of piperine on metabolite formation. The results showed that piperine inhibited the formation of the major metabolite, 7-hydroxywarfarin, with half-maximal inhibitory concentration (IC50) 14.2 μM and 3.2 μM in human and rat liver microsomes, respectively. Furthermore, coagulation studies in vitro using rat plasma showed that piperine does not affect prothrombin time (PT) and activated partial thromboplastin time (aPTT). This study suggested that piperine may present a potential drug interaction with warfarin at the metabolism level, but has no direct effect on the activation of the extrinsic or intrinsic coagulation cascades. Further clinical investigation is therefore required as piperine may increase the bioavailability of warfarin thus increasing risk of serious adverse events in patients.

Pharmacological Properties of White Mulberry (Morus alba L.) Leaves: Suppressing Migratory and Invasive Activities Against A549 Lung Cancer Cells.

Morus alba known as a white mulberry is a medicinal plant that has been used in food ingredients and traditional medicine. M. alba leaves contain various bioactive phenolic compounds, in particular chlorogenic acid (CGA), which is a major bioactive ingredient. Their anticancer potency of M. alba leaf extracts derived from Soxhlet extraction was evaluated based on cytotoxicity and antimigratory and antiinvasive properties. The dichloromethane extract exhibited the highest nitric oxide radical scavenging activity with a half-maximal inhibitory concentration (IC50) value of 780μg/mL, promising cytotoxicity against HuCCA-1, MCF-7, and A-549 cells with IC50 values of 59.18, 62.20, and 103.25μg/mL, respectively. CGA selectively inhibited the growth of MCF-7 cells with an IC50 value of 26.75μg/mL and showed potent radical scavenging activity against DPPH radicals (IC50 = 18.85μg/mL). An ethanolic extract derived from the gradient Soxhlet extraction suppressed A549 lung cancer cell migration and invasion more effectively than CGA with no migratory inhibition effect on noncancerous HaCaT cells. Furthermore, the ethanolic extract and CGA accelerated HaCaT wound closure at 20µg/mL, which was the same as allantoin. Bioactive ingredients including triterpenes, steroids, phenolics, and flavonoids were mainly detected in all extracts. The highest content of CGA (52.23g/100g dry weight) was found in the ethanolic extract derived from the gradient Soxhlet extraction. These findings show the potency of the dichloromethane extract as a cytotoxic agent against various cancer types and the ethanolic extract as an antimetastatic agent by their antimigratory and antiinvasive activities.

Enhancing the therapeutic landscape of cutaneous leishmaniasis: pegylated liposomal delivery of miltefosine for controlled release and improved efficacy

This study develops and evaluates two oral delivery systems, including miltefosine-loaded polyethylene glycol (PEG)ylated and non-PEGylated liposomal formulations (MT-Lip and MT-PEG-Lip, respectively), for cutaneous leishmaniasis (CL). MT-PEG-Lip nanoparticles displayed nanoscale sizes, negative zeta potentials, and homogenous populations (259 ± 13.6 and 248 ± 13.8 nm; -28 ± 1.6 and -13 ± 0.8 mV; polydispersity index (PDI) of 0.272 ± 0.014 and 0.279 ± 0.013 for MT-Lip and MT-PEG-Lip, respectively). Morphological evaluations confirmed uniform, spherical particles with smooth surfaces. PEGylation enhanced sustained drug release, vital for prolonged therapeutic effects. MT-Lip released 92.7% and 70.5% of the drug at pHs 1.2 and 6.8 after 72 h, while MT-PEG-Lip exhibited 69.8% and 57.3%, respectively. Stability tests over three months showed slight changes, affirming resilience. In vitro, MT-PEG-Lip demonstrated superior antileishmanial effects (half-maximal inhibitory concentration (IC50) for MT, MT-Lip, MT-PEG-Lip: 73.6, 23.7, and 9 μM against promastigote, and 86.1, 46.6, and 26.8 μM against amastigote, respectively). In vivo, MT-PEG-Lip significantly reduced lesion size (7.8- and 2.1-fold compared to MT and MT-Lip, respectively) and parasite burden (2.8- and 1.8-fold compared to MT and MT-Lip, respectively) in Leishmania major (L. major)-infected BALB/c mice. Histopathological assessments confirmed the safety of liposomal formulations, establishing MT-PEG-Lip as an efficient drug delivery platform for targeted CL treatment, addressing challenges in drug delivery and efficacy.

A novel sustainable immunoassay for sensitive detection of atrazine based on the anti-idiotypic nanobody and recombinant full-length antibody

Immunoassays have been widely used to determine small-molecule compounds in food and the environment, meeting the challenge of obtaining false positive or negative results because of the variance in the batches of antibodies and antigens. To resolve this problem, atrazine (ATR) was used as a target, and anti-idiotypic nanobodies for ATR (AI-Nbs) and a recombinant full-length antibody against ATR (ATR-rAb) were prepared for the development of a sustainable enzyme-linked immunosorbent assay (ELISA). AI-Nb-7, AI-Nb-58, and AI-Nb-66 were selected from an immune phage display library. ATR-rAb was produced in mammalian HEK293 (F) cells. Among the four detection methods explored, the assay using AI-Nb-66 as a coating antigen and ATR-rAb as a detection reagent yielded a half maximal inhibitory concentration (IC50) of 1.66 ng mL−1 for ATR and a linear range of 0.35 − 8.73 ng mL−1. The cross-reactivity of the assay to ametryn was 64.24 %, whereas that to terbutylazine was 38.20 %. Surface plasmon resonance (SPR) analysis illustrated that these cross-reactive triazine compounds can bind to ATR-rAb to varying degrees at high concentrations; however, the binding/dissociation kinetic curves and the response values at the same concentration are different, which results in differences in cross-reactivity. Homology modeling and molecular docking revealed that the triazine ring is vital in recognizing triazine compounds. The proposed immunoassay exhibited acceptable recoveries of 84.40 − 105.36 % for detecting fruit, vegetables, and black tea. In conclusion, this study highlights a new strategy for developing sustainable immunoassays for detecting trace pesticide contaminants.