Development of a robust functional cell-based assay for replacing the rabbit blood sugar bioidentity test of insulin glargine drug substance.

A rabbit blood sugar bioidentity assay is required by the FDA to evaluate biological activity for all insulin and its analogs per USP<121> guideline. Not only are a large number of live animals used, but the rabbit blood sugar method is also highly variable and expensive. Our goal is to develop a functional cell-based assay to replace rabbit blood sugar method. An H4IIE G6P-Luc reporter assay was developed by utilizing insulin's role in regulating hepatic gluconeogenesis pathway. It is known that Glucose 6-phosphatase is a rate-limiting enzyme in the gluconeogenesis pathway, and the mRNA expression of its catalytic subunit, G6PC, is highly regulated by insulin. A G6P-Luc stable cell line in H4IIE hepatocytes was first generated by stably expressing luciferase reporter gene driven by human G6PC promoter via lentivirus technology. The cell-based assay was developed and optimized to demonstrate good dose-dependent responsiveness to insulin. We further qualified the assay with two analysts through multiple runs, and demonstrated excellent performance characteristics of linearity, accuracy, and precision. A robustness study was then conducted to define critical factors for assay performance. We compared this newly developed assay with a previously established cell-based pIR MSD assay, which measures insulin receptor phosphorylation (pIR) in HepG2 cell line using Meso-Scale Discovery (MSD) technology. The comparability study was conducted to compare the two assays using samples generated from forced degradation. This study showed high correlation between assays, and both are stability indicating. Compared with the pIR MSD assay, the G6P-Luc assay not only has a significantly lower variability in qualification studies, but also offers many other advantages, including ease of use in a quality control laboratory with fewer steps, lower cost, and does not depend on a single vendor. In conclusion, we have developed a physiologically relevant and robust functional cell-based assay that is suitable to replace rabbit blood sugar method.

Enhanced antitumor efficacy of folate targeted nanoparticles co-loaded with docetaxel and curcumin.

BACKGROUND: The current study aimed to investigate whether the novel folate (FT) modified nanoparticles (NPs) co-loaded with docetaxel (DT) and curcumin (CU) (named as FT-NPs) could enhance the delivery efficiency to tumor compared with the NPs without FT (non-targeted NPs). METHODS: FT-NPs were successfully formulated in this article. In vitro cytotoxic activity against A549 cells and in vivo antitumor activity of FT-NPs in S180 cell bearing mice were conducted. Cellular uptake test was used to evaluate uptake efficiency of FT-NPs. Histological observation was used to determine the lung security. Besides, the physical chemical properties such as stability, particle size, zeta potential, drug encapsulation efficiency, transmission electron microscopy (TEM) were also conducted. RESULTS: FT-NPs exhibited stronger growth inhibition effects on A549 cells compared with non-targeted NPs, moreover, the novel FT-NPs indicated more effective antitumor efficacy in inhibiting tumor growth. Confocal laser scanning microscopy indicated that the uptake of FT-NPs was facilitated and effective. Histological observation meant that FT-NPs were biocompatible and appropriate for pulmonary administration. CONCLUSION: These results confirmed that FT-NPs with relatively high drug loading capacity could effectively inhibit tumor growth and reduce toxicity. The novel FT-NPs could produce as an outstanding nanocarrier for the targeted treatment of cancers.

Extracts from Curcuma zedoaria Inhibit Proliferation of Human Breast Cancer Cell MDA-MB-231 In Vitro.

Objective. To evaluate the effect of petroleum ether extracts of Curcuma zedoaria on the proliferation of human triple negative breast cancer cell line MDA-MB-231. Methods. The reagents were isolated from Curcuma zedoaria by petroleum ether fraction. It was assayed by CCK8 for MDA-MB-231 cellular viability with various concentrations and days, cell cycle analyses, Western Blot analysis, and Realtime Reverse Transcriptase PCR analyses for chemokines molecules including E-cadherin, and E-selectin, and adhesion molecules including CCR7, SLC, SDF-1, and CXCR4. Epirubicin was used as control in the study. Results. MDA-MB-231 cells were inhibited by petroleum ether extracts of Curcuma zedoaria (P < 0.05), and the inhibition rate was dependent on concentrations and time. Petroleum ether extracts of Curcuma zedoaria as well as Epirubicin produce a significant G0/G1 cell cycle arrest. The level of expression of proteins E-cadherin and E-cadherin mRNA was significantly increased, while proteins SDF-1, CCR7, and CXCR4 mRNA were decreased after being incubated with petroleum ether extracts of Curcuma zedoaria at the concentrations of 300 µ g/mL than control (P < 0.05). The differences were that the protein CXCR4 mRNA expression level was higher than vehicle. Conclusions. MDA-MB-231 cells were inhibited by petroleum ether extracts of Curcuma zedoaria.

Ablation of central nervous system progenitor cells in transgenic rats using bacterial nitroreductase system.

Specific ablation of central nervous system (CNS) progenitor cells in the brain of live animals is a powerful method to determine the functions of these cells and to reveal novel avenues for the treatment of several CNS-related disorders. To achieve this goal, we generated a line of transgenic rats expressing a bacterial enzyme, Escherichia coli nitroreductase gene (NTR), under control of the nestin promoter. In this system, NTR(+) cells are selectively eliminated upon application of prodrug CB1954, through activation of programmed cell death machineries. At 5 days of age, which is a time when cerebellar development is occurring, transgenic rats bearing the nestin-NTR/green fluorescent protein (GFP) gene are overtly normal and express NTR/GFP in neuronal stem cells, without any toxicity in these cells. The functional consequence of progenitor cell ablation was demonstrated by administering prodrug CB1954 into the cerebellum at this 5-day time point. Stem cell ablation in these neonates resulted in sensorimotor abnormalities, cerebellar degeneration, overall reduction in cerebellar seize, and manifestation of ataxia. In adult rats, GFP expression was not seen in the hippocampal progenitor cells and seen only at very low levels in the lateral ventricles, indicating a different NTR/GFP expression pattern between neonates and adults. In addition, application of CB1954 by intraventricular delivery reduced the number of 5-bromo-2'-deoxyuridine-labeled proliferating cells in the lateral ventricle but not hippocampus of NTR/GFP rats. These findings shows that targeted expression of NTR under a specific promoter might be of significant value in addressing the function of distinct cell population in vivo.