Integration of protein L-immobilized epoxy magnetic bead capture with LC-MS/MS for therapeutic monoclonal antibody quantification in serum.

In recent years, there has been a growing interest in the thriving monoclonal antibody (mAb) industry due to the wide utilization of mAbs in clinical therapies. Robust and accurate bioanalytical methods are required to enable fast quantification of mAbs in biological matrices, especially in the context of pharmacokinetics (PKs)/pharmacodynamics (PDs) and therapeutic drug monitoring (TDM) studies. In this investigation, we presented a novel immuno-magnetic capture coupled with a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method designed for the quantification of immunoglobulin G-kappa-based mAbs in biological fluids. The immunoaffinity absorbent for mAb drug purification was meticulously crafted by immobilizing protein L onto monosize, magnetic poly(glycidyl methacrylate) (m-pGMA) beads, synthesized through dispersion polymerization. The microspheres were acquired with an average size of 1.6 µm, and the optimal binding of mAbs from the aqueous mAb solution was determined to be 45.82 mg g-1. The quantification of mAbs in 10 µL serum samples was achieved through affinity purification using m-pGMA@protein L beads (employing rituximab as an internal standard (IS)), on-bead reduction, and rapid tryptic digestion. Remarkably, the entire process, taking less than 2.5 hours, held significant potential for simplifying pretreatment procedures and minimizing analytical time. Furthermore, the developed method underwent validation in accordance with the European Medicines Agency (EMA) guidelines. The assay demonstrated commendable linearity within the 2-400 µg mL-1 range for both daratumumab and pembrolizumab. Intra- and inter-assay coefficients of variation fell within the range of 0.7% to 13.4%, meeting established acceptance criteria. Other validation parameters also conformed to regulatory standards. Ultimately, the efficacy of the method was substantiated in a pharmacokinetic study following a single-dose intravenous administration to mice, underscoring its applicability and reliability in real-world scenarios.

Differential toxic and antiepileptic features of Vigabatrin raceme and its enantiomers.

BACKGROUND: The study aimed to investigate the potential pharmacological and toxicological differences between Vigabatrin (VGB) and its enantiomers S-VGB and R-VGB. The researchers focused on the toxic effects and antiepileptic activity of these compounds in a rat model. METHODS: The epileptic rat model was established by intraperitoneal injection of kainic acid, and the antiepileptic activity of VGB, S-VGB, and VGB was observed, focusing on the improvements in seizure latency, seizure frequency and sensory, motor, learning and memory deficits in epileptic rats, as well as the hippocampal expression of key molecular associated with synaptic plasticity and the Wnt/ß-catenin/GSK 3ß signaling pathway. The acute toxic test was carried out and the LD50 was calculated, and tretinal damages in epileptic rats were also evaluated. RESULT: The results showed that S-VGB exhibited stronger antiepileptic and neuroprotective effects with lower toxicity compared to VGB raceme. These findings suggest that S-VGB and VGB may modulate neuronal damage, glial cell activation, and synaptic plasticity related to epilepsy through the Wnt/ß-catenin/GSK 3ß signaling pathway. The study provides valuable insights into the potential differential effects of VGB enantiomers, highlighting the potential of S-VGB as an antiepileptic drug with reduced side effects. CONCLUSION: S-VGB has the highest antiepileptic effect and lowest toxicity compared to VGB and R-VGB.

Pharmacokinetics and tissue distribution of vigabatrin enantiomers in rats.

Purpose: To investigate the pharmacokinetics and tissue distribution of VGB racemate and its single enantiomers, and explore the potential of clinic development for single enantiomer S-VGB. Methods: In the pharmacokinetics study, male Sprague-Dawley rats were gavaged with VGB racemate or its single enantiomers dosing 50, 100 or 200 mg/kg, and the blood samples were collected during 12 h at regular intervals. In the experiment of tissue distribution, VGB and its single enantiomers were administered intravenously dosing 200 mg/kg, and the tissues including heart, liver, spleen, lung and kidney, eyes, hippocampus, and prefrontal cortex were separated at different times. The concentrations of R-VGB and S-VGB in the plasma and tissues were measured using HPLC. Results: Both S-VGB and R-VGB could be detected in the plasma of rats administered with VGB racemate, reaching Cmax at approximately 0.5 h with t1/2 2-3 h. There was no significant pharmacokinetic difference between the two enantiomers when VGB racemate was given 200 mg/kg and 100 mg/kg. However, when given at the dose of 50 mg/kg, S-VGB presented a shorter t1/2 and a higher Cl/F than R-VGB, indicating a faster metabolism of S-VGB. Furthermore, when single enantiomer was administered respectively, S-VGB presented a slower metabolism than R-VGB, as indicated by a longer t1/2 and MRT but a lower Cmax. Moreover, compared with the VGB racemate, the single enantiomers S-VGB and R-VGB had shorter t1/2 and MRT, higher Cmax and AUC/D, and lower Vz/F and Cl/F, indicating the stronger oral absorption and faster metabolism of single enantiomer. In addition, regardless of VGB racemate administration or single enantiomer administration, S-VGB and R-VGB had similar characteristics in tissue distribution, and the content of S-VGB in hippocampus, prefrontal cortex and liver was much higher than that of R-VGB. Conclusions: Although there is no transformation between S-VGB and R-VGB in vivo, those two enantiomers display certain disparities in the pharmacokinetics and tissue distribution, and interact with each other. These findings might be a possible interpretation for the pharmacological and toxic effects of VGB and a potential direction for the development and optimization of the single enantiomer S-VGB.

The complete mitochondrial genome of Pseudofabraea citricarpa (Dermateaceae: Helotiales) causing Citrus target spot.

Pseudofabraea citricarpa (Dermateaceae: Helotiales) is known as a significant pathogen causing Citrus target spot disease and results in profound yield loss. In the present study, the complete mitochondrial genome (mitogenome) determined based on next-generation sequencing technology. The circular mitogenome (56,935 bp) comprised 14 conserved protein-coding genes (PCGs), 16 ORFs, two ribosomal RNA genes (rns and rnl), one non-coding RNA gene (rnpB), one ribosomal protein S3 (rps3) and 28 transfer RNA (tRNA) genes. The overall base composition is as follows: 36.08% A, 35.25% T, 13.04% C, and 15.63% G, with a GC content of 28.70%. The phylogenetic analysis shows that P. citricarpa, belonging to Dermateaceae, forms a separate clade and is sister to Sclerotiniaceae. The mitogenome of P. citricarpa reported in this study provides more molecular data for further research on the evolutionary relationships of Helotiales.

Development and Validation of a Combined Hypoxia and Immune Prognostic Classifier for Lung Adenocarcinoma.

Lung cancer is the leading cause of cancer-related deaths worldwide. Hypoxia is a crucial microenvironmental factor in lung adenocarcinoma (LUAD). However, the prognostic value based on hypoxia and immune in LUAD remains to be further clarified. The hypoxia-related genes (HRGs) and immune-related genes (IRGs) were downloaded from the public database. The RNA-seq expression and matched complete clinical data for LUAD were retrieved from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) database. The least absolute shrinkage and selection operator (LASSO) Cox regression analysis was applied to model construction. Hypoxia expression profiles, immune cell infiltration, functional enrichment analysis, Tumor Immune Dysfunction and Exclusion (TIDE) score and the somatic mutation status were analyzed and compared based on the model. Moreover, immunofluorescence (IF) staining in human LUAD cases to explore the expression of hypoxia marker and immune checkpoint. A prognostic model of 9 genes was established, which can divide patients into two subgroups. There were obvious differences in hypoxia and immune characteristics in the two groups, the group with high-risk score value showed significantly high expression of hypoxia genes and programmed death ligand-1 (PD-L1), and maybe more sensitive to immunotherapy. Patients in the high-risk group had shorter overall survival (OS). This model has a good predictive value for the prognosis of LUAD. We constructed a new HRGs and IRGs model for prognostic prediction of LUAD. This model may benefit future immunotherapy for LUAD.

Construction of a circular RNA-microRNA-messenger RNA regulatory network of hsa_circ_0043256 in lung cancer by integrated analysis.

BACKGROUND: Patients with non-small cell lung cancer (NSCLC) are diagnosed in advanced stages and with a poor 5-year survival rate. There is a critical need to identify novel biomarkers to improve the therapy and overall prognosis of this disease. METHODS: Differentially expressed genes (DEGs) were identified from three profiles of GSE101586, GSE101684 and GSE112214 using Venn diagrams. hsa_circ_0043256 were validated using quantitative real-time polymerase chain reaction (RT-qPCR). The circular RNA-microRNA-messenger RNA (circRNA-miRNA-mRNA) regulatory network was constructed with Cytoscape 3.7.0. Hub genes were identified with protein interaction (PPI) and validated with the Gene Expression Profiling Interactive Analysis (GEPIA), Human Protein Atlas (HPA) databases, and immunohistochemistry. Survival analyses were also performed using a Kaplan-Meier (KM) plotter. The effects of hsa_circ_0043256 on cell proliferation and cell cycles were evaluated by EdU staining and flow cytometry, respectively. RESULTS: hsa_circ_0043256, hsa_circ_0029426 and hsa_circ_0049271 were obtained. Following RT-qPCR validation, hsa_circ_0043256 was selected for further analysis. In addition, functional experiment results indicated that hsa_circ_0043256 could inhibit cell proliferation and cell-cycle progression of NSCLC cells in vitro. Prediction by three online databases and combining with DEGs identified from The Cancer Genome Atlas (TCGA), a network containing one circRNAs, three miRNAs, and 209 mRNAs was developed. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated DEGs might be associated with lung cancer onset and progression. A PPI network based on the 209 genes was established, and five hub genes (BIRC5, SHCBP1, CCNA2, SKA3, and GINS1) were determined. Following verification of five hub genes using GEPIA database, HPA database, and immunohistochemistry. High expression of all five hub genes led to poor overall survival. CONCLUSION: Our study constructed a circRNA-miRNA-mRNA network of hsa_circ_0043256. hsa_circ_0043256 may be a potential therapeutic target for lung cancer.

ARID1A, ARID1B, and ARID2 Mutations Serve as Potential Biomarkers for Immune Checkpoint Blockade in Patients With Non-Small Cell Lung Cancer.

Worldwide, non-small cell lung cancer (NSCLC) has the highest morbidity and mortality of all malignancies. The lack of responsiveness to checkpoint inhibitors is a central problem in the modern era of cancer immunotherapy, with the rapid development of immune checkpoint inhibitors (ICIs) in recent years. The human switch/sucrose nonfermentable (SWI/SNF) chromatin-remodeling complex has been reported to be recurrently mutated in patients with cancer, and those with SWI/SNF mutations have been reported to be sensitive to ICIs. Six reported cohorts, a total of 3416 patients, were used to analyze the mutation status of ARID1A, ARID1B, ARID2 and SMARCA4 in patients with NSCLC and the effect of mutations on prognosis after ICIs. Finally, a nomogram was established to guide the clinical use of ICIs. The results show that patients with NSCLC who have ARID1A, ARID1B, and ARID2 mutations of the SWI/SNF complex were more likely to benefit from ICI therapy.

Arsenic Trioxide Combining Leflunomide Activates Nrf2-ARE-HO-1 Signaling Pathway and Protects Heart Xenografts.

OBJECTIVE: To investigate the molecular mechanisms underlying the effects of arsenic trioxide (As2O3) in combination with leflunomide on the hamster-to-rat heart xenotransplant. METHODS: Transplantation of LVG hamster hearts to Lewis rats was performed by anastomosis of vessels in the neck using end-to-end anastomosis with a non-suture cuff technique. Four groups of recipient rats (n=6 in each) were treated with normal saline (control), As2O3 [5 mg/(kg·day) intraperitoneally], leflunomide [5 mg/(kg·d) orally], or leflunomide [5 mg/(kg·d)+As2O3 [5 mg/(kg·d)] in combination. Donor hearts and/or rat spleens were harvested and analyzed 4 days after transplantation. Quantitative reverse-transcription polymerase chain reaction and Western blot analysis were performed to detect the expression of the nuclear factor erythroid-derived factor 2-related factor (Nrf2) and its target gene heme oxygenase-1 (HO-1), Treg cell marker fork-head Box P3 (FOXP3), apoptosis-associated proteins Bcl-2, Bax, and cleaved caspase-3. Immunohistochemical staining was used to detect the levels of inflammatory natural killer cell and macrophage infiltration, intercellular cell adhesion molecule-1 (ICAM-1) and complement C3. RESULTS: Expression of Nrf2-ARE-HO-1 signaling pathway was upregulated in heart xenografts in rats treated with As2O3 plus leflunomide compared with control rats or rats treated with either drug alone (P<0.01), and this was accompanied by an increased Treg cells in the recipient rat spleen (P<0.01). In contrast, the expressions of Bax, cleaved caspase-3, ICAM-1, and complement C3, and infiltration of inflammatory cells in the xenografts were inhibited by As2O3 plus leflunomide treatment (P<0.01). CONCLUSION: Combination treatment with As2O3 and leflunomide protected hamster heart-xenografts in recipient rats.

Mechanical property characterization of partially crystalline Poly-Ether-Ether-Ketone.

The present study investigates the mechanical properties of partially crystalline Poly-Ether-Ether-Ketone between the glass transition and the cold crystallization. Biaxial tension, uniaxial tension, and DMA experiments were conducted to investigate the influence of temperature-induced crystallization on mechanical properties, and three stiffening behaviors are observed. Firstly, a 'U' type mechanical property is observed for all three experiments with first softening and then significant stiffening behavior with increasing temperature. Secondly, stiffening also occurs during low strain rate tests but not in higher strain rate tests. Thirdly, the stiffening behavior of the anisotropic film shows orientation dependence. Crystallinity evolution is predicted by the Nakaruma non-isothermal crystallization kinetics with optimized parameters, with which we demonstrate and explain that the stiffening behaviors are connected to the onset of crystallization. Therefore, the conclusion provides a new tool to approach and distinguish extrinsic and intrinsic properties during characterization, promoting future implementation for constitutive modeling and corresponding simulation that could replicate the influence of temperature-induced crystallization.

Prognostic value of ferroptosis-related genes in patients with lung adenocarcinoma.

BACKGROUND: The prevalence of lung adenocarcinomas (LUADs) has dramatically increased in recent decades. Ferroptosis is a process of iron-dependent regulatory cell death. It is still unclear whether the expression of ferroptosis-related genes (FRGs) is involved in the pathogenesis and survival of patients with LUAD. METHODS: We retrieved LUAD data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases and used LASSO Cox regression analysis to select the gene signature suitable for modeling. The risk score was calculated according to the model, and the patients were divided into high- and low-risk groups according to the median risk score. Functional enrichment analysis was carried out by this group, and a model for predicting clinical prognosis was established by combining this group with clinical factors. RESULTS: Gene set enrichment analysis (GSEA) and single-sample gene set enrichment analysis (ssGSEA) analysis showed that there were several immune-related pathways and immune infiltration differences between high- and low-risk groups. A prognostic model integrating 10 ferroptosis-related genes (FR-DEGs), and clinical factors were constructed and validated in an external cohort. CONCLUSIONS: The FR-DEGs signature was related to immune infiltration, and a model based on FR-DEGs and clinical factors was established to predict the prognosis of patients with LUAD.

[Immune Microenvironment Comparation Study between EGFR Mutant and EGFR Wild Type Lung Adenocarcinoma Patients Based on TCGA Database].

BACKGROUND: Lung cancer is a malignant with high incidence and mortality and adenocarcinoma is among the most popular subtypes. Epidermal growth factor receptor (EGFR) mutation is one of the most important driver mutations for lung adenocarcinoma and EGFR-tyrosine kinase inhibitor (TKI) will benefit those patients with sensitive EGFR mutations. Recently, immune checkpoint inhibitor (ICI) therapy, provide a new breakthrough treatment for lung cancer patients. Whereas immunotherapy as an emerging treatment does not benefit patients with EGFR mutations, for which mechanistic studies are poorly defined and focused on the link of EGFR mutations and programmed cell death-ligand 1 (PD-L1) expression, we speculate that the different immune microenvironment associated with the two classes of patients. METHODS: Lung adenocarcinoma datasets were collected from the Cancer Genome Atlas (TCGA) database, and clinical information and gene expression profiles were downloaded. The immune related lymphocyte infiltration in TCGA database were generated through timer 2.0 GSEA was used to analyze the difference of pathway expression between EGFR mutant patients and wild type patients. RESULTS: EGFR mutation was more frequently among women and never smokers. Immunoinfiltration analysis showed that patients with EGFR mutation tends to have more tumor associated fibroblasts, common myeloid progenitor cells, hematopoietic stem cells, effector CD4⁺ T cells and natural killer T cells infiltration, and less memory B cells, naïve B cells, plasma B cells, plasmacytoid dendritic cells, memory CD4⁺ T cells, CD4⁺ helper T cells 2, naive CD8⁺ T cells, CD8⁺ T cells and central memory CD8⁺ T cells infiltration. Moreover, patients with more infiltration of CD8⁺ T cells, natural killer T cells, memory B cells and hematopoietic stem cells, tends have better prognosis (Log-rank test, P=0.017, 0.0093, 0.018, 0.016). However, the patients with more CD4⁺ T th2 infiltration in the tumor tends to have worse prognosis (Log-rank test, P=0.016). Furthermore, the results of gene set enrichment analysis showed that compared with the lung adenocarcinoma patients with EGFR wild type, the three pathways positive regulation of natural killer (NK) cell-mediated immune response to tumor cells, NK cell activation involved in immune response, and NK cell-mediated immune response to tumor cells related to natural killer cells in patients with EGFR mutation were down regulated, while the pathway the positive regulation of cytokine secretion involved in immune response was up-regulated in EGFR mutation patients. CONCLUSIONS: The tumour microenvironment of patients with EGFR mutations lacks potent tumour killing effector cells and appears dysfunctional with effector cells. This may be a potential reason for the poor efficacy of immunotherapy in patients with EGFR mutations.

Identification of small proline-rich protein 1B (SPRR1B) as a prognostically predictive biomarker for lung adenocarcinoma by integrative bioinformatic analysis.

BACKGROUND: With the ongoing development of targeted therapy and immunotherapy in recent years, the overall five-year survival rate of NSCLC patients has not improved, and the search for novel diagnostic and prognostic markers for lung adenocarcinoma continues. METHODS: Lung adenocarcinoma (LUAD) gene expression data and relevant clinical information were obtained from the TCGA. Hub genes were identified with weighted gene co-expression network analysis (WGCNA) and protein-protein interaction network (PPI). Survival analyses were also performed using GEPIA. The 536 LUAD patients were divided into two groups according to the SPRR1B expression level and analyzed by gene set enrichment analysis (GSEA) and verified by immunoblotting. The effects of SPRR1B on cell proliferation and cell metastasis and apoptosis were evaluated by 5-ethynyl-2'-deoxyuridine (EdU) staining, colony formation assay, transwell migration and invasion assay, and flow cytometry, respectively. RESULTS: A total of 2269 DEGs were analyzed by WGCNA and five hub genes (CCK, FETUB, PCSK9, SPRR1B, and SPRR2D) were identified. Among them, SPRR1B was selected as one of the most significant prognostic genes in LUAD. SPRR1B was found to be highly expressed in lung adenocarcinoma cells compared with that in normal bronchial epithelial cells. In addition, silencing of SPRR1B could inhibit the cell proliferation, invasion, and migration of lung adenocarcinoma cells, but induced cell apoptosis and G2/M phase arrest in vitro. The result of GSEA and immunoblotting revealed that SPRR1B activated the MAPK signaling pathway involved in the proliferation and metastasis of lung cancer. CONCLUSIONS: Our findings demonstrate that SPRR1B may function as a prognosis predictor in lung adenocarcinoma.

[Apatinib Combined with CCI-779 Inhibits the Proliferation and Migration of Small Cell Lung Cancer NCI-H446 Cells In Vitro].

BACKGROUND: Lung cancer is the most common malignancy world-wide. Small cell lung cancer is the deadliest subtype of lung cancer, which features such as rapid growth, early metastasis, and high vascularization. Apatinib is a vascular endothelial growth factor receptor 2 inhibitor independently developed in China, which has a significant inhibition in a variety of solid tumors. The purpose of this study is to investigate the effects of Apatinib alone or Apatinib combined with mammalian target of rapamycin (mTOR) inhibitor, CCI-779, on small cell lung cancer cell line NCI-H446 in vitro. METHODS: The small cell lung cancer cell line NCI-H446 was grew in vitro. The effects of Apatinib alone or Apatinib combined with CCI-779 on proliferation, apoptosis, cell cycle and migration of NCI-H446 small cell lung cancer cells were detected by CCK8; FACS and transwell assays were also carried out; Western blot assays were used to detect vascular endothelial growth factor and cell cycle related protein expression. RESULTS: CCK8 assays showed that high concentration of Apatinib could inhibit the proliferation of NCI-H446 cells. Apoptosis assays showed that high concentration of Apatinib could induce NCI-H446 cell apoptosis. Transwell assays showed that high concentration of Apatinib could inhibit NCI-H446 cell migration. After combined with mTOR inhibitor CCI-779, low concentration of Apatinib could inhibit the proliferation and migration of NCI-H446 small cell lung cancer cells and induce apoptosis. CONCLUSIONS: Apatinib has a concentration-dependent effect on the small cell lung cancer cell line NCI-H446. High concentration of Apatinib can inhibit the proliferation and migration of NCI-H446 small cell lung cancer cells, induce apoptosis. Apatinib combined with the mTOR inhibitor CCI-779 can sensitize the NCI-H446 cells to Apatinib.

Modification of the contact surfaces for improving the puncture resistance of laminar structures.

Uncovering energy absorption and surface effects of various penetrating velocities on laminar structures is essential for designing protective structures. In this study, both quasi-static and dynamic penetration tests were systematical conducted on the front surfaces of metal sheets coated with a graphene oxide (GO) solution and other media. The addition of a GO fluid film to the front impact surface aided in increasing the penetration strength, improving the failure extension and dissipating additional energy under a wide-range of indentation velocity, from 3.33 × 10-5 m/s to 4.42 m/s. The coated -surfaces improved the specific energy dissipation by approximately 15~40% relative to the dry-contact configuration for both single-layer and double-layer configurations, and specific energy dissipations of double-layer configurations were 20~30% higher than those of the single-layer configurations. This treatment provides a facile strategy in changing the contact state for improving the failure load and dissipate additional energy.

Evaluation of a Non-aqueous Ibuprofen-Phospholipid Complex Formulation in Rats.

AIM: In the present study, a non-aqueous ibuprofen-phospholipid complex was developed to reduce the gastrointestinal (GI) toxicity of ibuprofen. MATERIALS AND METHODS: A non-aqueous ibuprofen-phospholipid complex (IBU-PC) was prepared by mixing phosal-35SB and ibuprofen. In vitro release behavior was studied using a dissolution apparatus. Irritation to gastrointestinal (GI) tract and pharmacokinetics of IBU-PC were studied in rats. RESULTS: Rapid release of drug occurred with approximately 85% of ibuprofen released from the composition within the first 30 min. The GI injury in IBU-PC-treated rats was minimal compared to those of Advil Liqui-gels-treated group. There was no significant difference between IBU-PC and Motrin-treated groups. The area under the concentration-time curve (AUC0~24) of IBU-PC and Motrin were 366±115 and 391±105 µg/h/ml, respectively. The relative bioavailability of IBU-PC was 94.2%. CONCLUSION: IBU-PC can decrease GI adverse reaction induced by ibuprofen.

CD33-Targeted Lipid Nanoparticles (aCD33LNs) for Therapeutic Delivery of GTI-2040 to Acute Myelogenous Leukemia.

CD33-targeted lipid nanoparticles (aCD33LNs) were synthesized for delivery of GTI-2040, an antisense oligonucleotide (ASO) against the R2 subunit of ribonucleotide reductase, to acute myelogenous leukemia (AML). These LNs incorporated a deoxycholate-polyethylenimine (DOC-PEI) conjugate, which has shown significant activity to facilitate oligonucleotide delivery. Anti-CD33 scFv (aCD33) was added as a targeting ligand. The delivery efficiency of this system was investigated both in vitro and in vivo. When cells were treated with aCD33LN/GTI-2040, significant uptake was observed in CD33 positive Kasumi-1 cells. aCD33LNs loaded with GTI-2040 induced significant down-regulation of R2 mRNA and protein levels in AML cells. Moreover, aCD33LN/GTI-2040 showed a 15-fold reduction in the IC50 of antileukemic drug Ara-C in Kasumi-1 cells. In Kasumi-1 xenograft model, aCD33LN/GTI-2040 showed significant R2 downregulation compared to LN/GTI-2040. Furthermore, aCD33LN/GTI-2040 coadministered with Ara-C was shown to be highly effective in tumor growth inhibition and to greatly increase survival time of mice bearing Kasumi-1 xenograft tumors. The conjugate DOC-PEI has shown an ability to include calcein release from lipid nanoparticles, suggesting a potential mechanism contributing to efficient endosome release by DOC-PEI2K. These results indicate that aCD33LNs are a highly effective vehicle for the therapeutic delivery of antisense agents to AML.

A microfluidic method to synthesize transferrin-lipid nanoparticles loaded with siRNA LOR-1284 for therapy of acute myeloid leukemia.

The siRNA LOR-1284 targets the R2 subunit of ribonucleotide reductase (RRM2) and has shown promise in cancer therapy. In this study, transferrin (Tf) conjugated lipid nanoparticles (Tf-NP-LOR-1284) were synthesized by microfluidic hydrodynamic focusing (MHF) and evaluated for the targeted delivery of LOR-1284 siRNA into acute myeloid leukemia (AML) cells. The in vitro study showed that Tf-NP-LOR-1284 can protect LOR-1284 from serum nuclease degradation. Selective uptake of Tf-NP-LOR-1284 was observed in MV4-11 cells. In addition, qRT-PCR and Western blot results revealed that Tf-NP-LOR-1284 was more effective than the free LOR-1284 in reducing the R2 mRNA and protein levels. The Tf-NP-LOR-1284 showed prolonged circulation time and increased AUC after i.v. administration relative to the free LOR-1284. Furthermore, Tf-NP-LOR-1284 facilitated increased accumulation at the tumor site along with the decreased R2 mRNA and protein expression in a murine xenograft model. These results suggest that Tf-conjugated NPs prepared by MHF provide a suitable platform for efficient and specific therapeutic delivery of LOR-1284 into AML cells.

DABCO-based ionic liquids: recyclable catalysts for aza-Michael addition of α,ß-unsaturated amides under solvent-free conditions.

An array of novel 1,4-diazobicyclo[2.2.2]octane (DABCO) based ionic liquids were developed and used as recyclable catalysts for the aza-Michael addition at room temperature without any organic solvent. [DABCO-PDO][OAc] was found to be the most efficient catalyst, and the amount of catalyst was only 10 mol %. Various amines reacted with a wide range of α,ß-unsaturated amides, smoothly affording target products in good to excellent yields within hours. Moreover, the catalyst could be reused up to eight times, still maintaining a high catalytic activity. Finally, a plausible mechanism was proposed. FTIR and computational chemistry were used to verify the catalytic mechanism.

Development and validation of a rapid LC-MS/MS method for the determination of JCC76, a novel antitumor agent for breast cancer, in rat plasma and its application to a pharmacokinetics study.

JCC76 is a novel nimesulide analog that selectively inhibits the human epidermal growth factor receptor 2 (HER2) overexpressing breast cancer cell proliferation and tumor progression. To support further pharmacological and toxicological studies of JCC76, a novel and rapid method using liquid chromatography and electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) has been developed and validated for the quantification of the compound in rat plasma. A C18 column was used for chromatographic separation, and the mobile phase was aqueous ammonium formate (pH 3.7; 5 mm)-methanol (1:9, v/v) with an isocratic elution. With a simple liquid-liquid extraction procedure using the mixture of methyl tert-butyl ether-hexane (1:2, v/v), the mean extraction efficiency of JCC76 in rat plasma was determined as 89.5-97.3% and no obvious matrix effect was observed. This method demonstrated a linear calibration range from 0.3 to 100 ng/mL for JCC76 in rat plasma and a small volume of sample consumption. The intra- and inter-assay accuracy and precision were within ±10%. The pharmacokinetics of JCC76 was also profiled using this validated method in rats. In conclusion, this rapid and sensitive method has been proven to effectively quantify JCC76 for pharmacokinetics study.

Synthesis and evaluation of a novel lipophilic folate receptor targeting ligand.

BACKGROUND: Folate receptor (FR)-targeted liposomes have been investigated as delivery vehicles for anticancer drugs. A novel lipophilic FR ligand, folate-glutathione-polyethyleneglycol-distearoyl phosphatidylethanolamine (F-GSH-PEG-DSPE), was synthesized, incorporated into liposomes and evaluated for FR targeting efficiency. These liposomes were then evaluated as carriers of the chemotherapy agent vincristine (VIN). MATERIALS AND METHODS: F-GSH-PEG-DSPE was synthesized and FR-targeted liposomes loaded with either calcein (F-L-Calcein) or VIN (F-L-VIN) were prepared by thin film hydration followed by polycarbonate membrane extrusion and, in the case of VIN, by remote loading. To assess liposome stability, the uptake of F-L-VIN in KB (FR+) cancer cells was measured after storage under 4°C for 3 months. Comparative pharmacokinetic studies were carried out with F-L-VIN and L-VIN (non-targeted control liposomes). RESULTS: F-L-Calcein showed significantly higher cellular uptake in KB cells compared to non-targeted liposomes. In addition, F-L-VIN showed enhanced cytotoxicity in KB cells in vitro compared to control liposomes. Pharmacokinetic parameters indicated that both F-L-VIN and control liposomes had higher area under the curve (AUC), mean residence time (MRT), elimination half life (t1/2-ß) and lower total body clearance (CL) than those of free VIN, while there were no significant differences between these liposomal formulations. CONCLUSION: F-GSH-PEG-DSPE is effective as a novel ligand for the synthesis of FR-targeted liposomes.