A UPLC-MS/MS method for quantification of perindopril and perindoprilat and applied in a bioequivalence study for their pharmacokinetic parameter measurement
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OBJECTIVES: To investigate the pharmacokinetic parameters of perindopril and perindoprilat in healthy volunteers, a simple and sensitive UPLC-MS/MS method with isotope-labeled internal standards of perindopril-d4 and perindoprilat-d4 was established and further applied in a bioequivalence study. MATERIALS AND METHODS: A simple and sensitive UPLC-MS/MS method with isotope-labeled internal standards of perindopril-d4 and perindoprilat-d4 was validated and applied in a single-center, randomized, cross-over, and two-period bioequivalence study. 20 healthy Chinese subjects (16 males and 4 females) were enrolled and had their plasma concentrations of perindopril and perindoprilat quantified and calculated for the pharmacokinetic parameters. After acetonitrile precipitation, the analytes and internal standards were gradient eluted with methanol-acetonitrile-ammonium acetate on an Acquity UPLC BEH C18 (2.1 × 50 mm, 1.7 µm) column. Detection was carried out in a multireaction monitoring mode using positive ionization electrospray mass spectrometry. RESULTS: The total chromatographic run time was 4 minutes with retention time for perindopril and perindopril-d4 of ~ 1.86 minutes, whereas perindoprilat and perindoprilat-d4 was ~ 1.79 minutes. The calibration curves of perindopril and perindoprilat were linear over 0.4 - 80 ng/mL and 0.2 - 40 ng/mL, respectively. The method was fully validated to meet the requirement for bioassay in accuracy (89.6 - 112.4%), precision (coefficient of variation (CV) ≤ 13.8%), recovery (79.65 - 97.83%), matrix effect (CV ≤ 5.9%), and stability (CV ≤ 10.0%). The 90% confidence intervals (CIs) for the geometric mean ratios of Cmax, AUC0-tlast, and AUC0-∞ of perindopril and perindoprilat all fell within the bioequivalence acceptance criteria (80 - 125%). There were no significant differences between the two formulations in terms of tmax and T1/2 of perindopril and perindoprilat. There was no adverse event in this clinical study. Interestingly, it was found that the pharmacokinetics of perindoprilat in 1 subject were significantly different from that of the others which may be associated with genetic diversity. CONCLUSION: This method was successfully applied to the bioequivalence test of two perindopril tert-butylamine tablets. The two one-sided t-tests showed that these two products were bioequivalent.

The effects of endoplasmic reticulum stress inducer thapsigargin on the toxicity of ZnO or TiO2 nanoparticles to human endothelial cells.

It was recently shown that ZnO nanoparticles (NPs) could induce endoplasmic reticulum (ER) stress in human umbilical vein endothelial cells (HUVECs). If ER stress is associated the toxicity of ZnO NPs, the presence of ER stress inducer thapsigargin (TG) should alter the response of HUVECs to ZnO NP exposure. In this study, we addressed this issue by assessing cytotoxicity, oxidative stress and inflammatory responses in ZnO NP exposed HUVECs with or without the presence of TG. Moreover, TiO2 NPs were used to compare the effects. Exposure to 32 µg/mL ZnO NPs (p < 0.05), but not TiO2 NPs (p > 0.05), significantly induced cytotoxicity as assessed by WST-1 and neutral red uptake assay, as well as intracellular ROS. ZnO NPs dose-dependently increased the accumulation of intracellular Zn ions, and ZnSO4 induced similar cytotoxic effects as ZnO NPs, which indicated a role of Zn ions. The release of inflammatory proteins tumor necrosis factor α (TNFα) and interleukin-6 (IL-6) or the adhesion of THP-1 monocytes to HUVECs was not significantly affected by ZnO or TiO2 NP exposure (p > 0.05). The presence of 250 nM TG significantly induced cytotoxicity, release of IL-6 and THP-1 monocyte adhesion (p < 0.01), but did not significantly affect intracellular ROS or release of TNFα (p > 0.05). ANOVA analysis indicated no interaction between exposure to ZnO NPs and the presence of TG on almost all the endpoints (p > 0.05) except neutral red uptake assay (p < 0.01). We concluded ER stress is probably not associated with ZnO NP exposure induced oxidative stress and inflammatory responses in HUVECs.

Olanzapine-induced lipid disturbances: A potential mechanism through the gut microbiota-brain axis.

Objective: Long-term use of olanzapine can induce various side effects such as lipid metabolic disorders, but the mechanism remains to be elucidated. The gut microbiota-brain axis plays an important role in lipid metabolism, and may be related to the metabolic side effects of olanzapine. Therefore, we explored the mechanism by which olanzapine-induced lipid disturbances through the gut microbiota-brain axis. Methods: Sprague Dawley rats were randomly divided into two groups, which underwent subphrenic vagotomy and sham surgery. Then the two groups were further randomly divided into two subgroups, one was administered olanzapine (10 mg/kg/day) by intragastric administration, and the other was administered normal saline by intragastric administration (4 ml/kg/day) for 2 weeks. The final changes in lipid parameters, gut microbes and their metabolites, and orexin-related neuropeptides in the hypothalamus were investigated among the different groups. Results: Olanzapine induced lipid disturbances as indicated by increased weight gain, elevated ratio of white adipose tissue to brown adipose tissue, as well as increased triglyceride and total cholesterol. Olanzapine also increased the Firmicutes/Bacteroides (F/B) ratio in the gut, which was even aggravated by subphrenic vagotomy. In addition, olanzapine reduced the abundance of short-chain fatty acids (SCFAs) metabolism related microbiome and 5-hydroxytryptamine (5-HT) levels in the rat cecum, and increased the gene and protein expression of the appetite-related neuropeptide Y/agouti-related peptide (NPY/AgRP) in the hypothalamus. Conclusion: The abnormal lipid metabolism caused by olanzapine may be closely related to the vagus nerve-mediated gut microbiota-brain axis.

Gut microbiota: An intermediary between metabolic syndrome and cognitive deficits in schizophrenia.

Gut microbiome interacts with the central nervous system tract through the gut-brain axis. Such communication involves neuronal, endocrine, and immunological mechanisms, which allows for the microbiota to affect and respond to various behaviors and psychiatric conditions. In addition, the use of atypical antipsychotic drugs (AAPDs) may interact with and even change the abundance of microbiome to potentially cause adverse effects or aggravate the disorders inherent in the disease. The regulate effects of gut microbiome has been described in several psychiatric disorders including anxiety and depression, but only a few reports have discussed the role of microbiota in AAPDs-induced Metabolic syndrome (MetS) and cognitive disorders. The following review systematically summarizes current knowledge about the gut microbiota in behavior and psychiatric illness, with the emphasis of an important role of the microbiome in the metabolism of schizophrenia and the potential for AAPDs to change the gut microbiota to promote adverse events. Prebiotics and probiotics are microbiota-management tools with documented efficacy for metabolic disturbances and cognitive deficits. Novel therapies for targeting microbiota for alleviating AAPDs-induced adverse effects are also under fast development.

The endoplasmic reticulum stress inducer thapsigargin enhances the toxicity of ZnO nanoparticles to macrophages and macrophage-endothelial co-culture.

It was recently shown that exposure to ZnO nanoparticles (NPs) could induce endoplasmic reticulum (ER) stress both in vivo and in vitro, but the role of ER stress in ZnO NP induced toxicity remains unclear. Because macrophages are sensitive to ER stress, we hypothesized that stressing macrophages with ER stress inducer could enhance the toxicity of ZnO NPs. In this study, the effects of ER stress inducer thapsigargin (TG) on the toxicity of ZnO NPs to THP-1 macrophages were investigated. The results showed that TG enhanced ZnO NP induced cytotoxicity as revealed by water soluble tetrazolium-1 (WST-1) and neutral red uptake assays, but not lactate dehydrogenase (LDH) assay. ZnO NPs dose-dependently enhanced the accumulation of intracellular Zn ions without the induction of reactive oxygen species (ROS), and the presence of TG did not significantly affect these effects. In the co-culture, exposure of THP-1 macrophages in the upper chamber to ZnO NPs and TG significantly reduced the viability of human umbilical vein endothelial cells (HUVECs) in the lower chamber, but the release of tumor necrosis factor α (TNFα) was not induced. In summary, our data showed that stressing THP-1 macrophages with TG enhanced the cytotoxicity of ZnO NPs to macrophages and macrophage-endothelial co-cultures.

Combined effects of low levels of palmitate on toxicity of ZnO nanoparticles to THP-1 macrophages.

We have recently proposed that the interaction between food components and nanoparticles (NPs) should be considered when evaluating the toxicity of NPs. In the present study, we used THP-1 differentiated macrophages as a model for immune cells and investigated the combined toxicity of low levels of palmitate (PA; 10 or 50µM) and ZnO NPs. The results showed that PA especially at 50µM changed the size, Zeta potential and UV-vis spectra of ZnO NPs, indicating a possible coating effect. Up to 32µg/mL ZnO NPs did not significantly affect mitochondrial activity, intracellular reactive oxygen species (ROS) or release of interleukin 6 (IL-6), but significantly impaired lysosomal function as assessed by neutral red uptake assay and acridine orange staining. The presence of 50µM PA, but not 10µM PA, further promoted the toxic effects of ZnO NPs to lysosomes but did not significantly affect other endpoints. In addition, ZnO NPs dose-dependently increased intracellular Zn ions in THP-1 macrophages, which was not significantly affected by PA. Taken together, the results of the present study showed a combined toxicity of low levels of PA and ZnO NPs especially to lysosomes in THP-1 macrophages.

Consideration of interaction between nanoparticles and food components for the safety assessment of nanoparticles following oral exposure: A review.

Nanoparticles (NPs) are increasingly used in food, and the toxicity of NPs following oral exposure should be carefully assessed to ensure the safety. Indeed, a number of studies have shown that oral exposure to NPs, especially solid NPs, may induce toxicological responses both in vivo and in vitro. However, most of the toxicological studies only used NPs for oral exposure, and the potential interaction between NPs and food components in real life was ignored. In this review, we summarized the relevant studies and suggested that the interaction between NPs and food components may exist by that 1) NPs directly affect nutrients absorption through disruption of microvilli or alteration in expression of nutrient transporter genes; 2) food components directly affect NP absorption through physico-chemical modification; 3) the presence of food components affect oxidative stress induced by NPs. All of these interactions may eventually enhance or reduce the toxicological responses induced by NPs following oral exposure. Studies only using NPs for oral exposure may therefore lead to misinterpretation and underestimation/overestimation of toxicity of NPs, and it is necessary to assess the synergistic effects of NPs in a complex system when considering the safety of NPs used in food.