In vitro pharmacokinetic behavior in lung of harringtonine, an antagonist of SARS-CoV-2 associated proteins: New insights of inhalation therapy for COVID-19.

BACKGROUND: Recent studies have shown that harringtonine (HT) could specifically bind with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein and host cell transmembrane serine protease 2 (TMPRSS2) to block membrane fusion, which is an effective antagonist for SARS-CoV-2. PURPOSE: Our study focused on in-depth exploration of in vitro pharmacokinetic characteristics of HT in lung. METHODS: HPLC-fluorescence detection method was used to detect changes of HT content. Incubation systems of lung microsomes for phase I metabolism and UGT incubation systems for phase II metabolism were performed to elucidate metabolites and metabolic mechanisms of HT, and then the metabolic enzyme phenotypes for HT were clarified by chemical inhibition method and recombinant enzyme method. Through metabolomics, we comprehensively evaluated the physiological dynamic changes in SD rat and human lung microsomes, and revealed the relationship between metabolomics and pharmacological activity of HT. RESULTS: HPLC-fluorescence detection method showed strong specificity, high accuracy, and good stability for rapid quantification of HT. We confirmed that HT mainly underwent phase I metabolism, and the metabolites of HT in different species were all identified as 4'-demethyl HT, with metabolic pathway being hydrolysis reaction. CYP1A2 and CYP2E1 participated in HT metabolism, but as HT metabolism was not NADPH dependent, the esterase HCES1 in lung also played a role. The main KEGG pathways in SD rat and human lung microsomes were cortisol synthesis and secretion, steroid hormone biosynthesis and linoleic acid metabolism, respectively. The downregulated key biomarkers of 11-deoxycortisol, 21-deoxycortisol and 9(10)-EpOME suggested that HT could prevent immunosuppression and interfere with infection and replication of SARS-CoV-2. CONCLUSION: HT was mainly metabolized into 4'-demethyl HT through phase I reactions, which was mediated by CYP1A2, CYP2E1, and HCES1. The downregulation of 11-deoxycortisol, 21-deoxycortisol and 9(10)-EpOME were key ways of HT against SARS-CoV-2. Our study was of great significance for development and clinical application of HT in the treatment of COVID-19.

Dauricine Inhibits Non-small Cell Lung Cancer Development by Regulating PTEN/AKT/mTOR and Ras/MEK1/2/ERK1/2 Pathways in a FLT4-dependent Manner.

OBJECTIVE: Non-small cell lung cancer (NSCLC) is still a solid tumor with high malignancy and poor prognosis. Vascular endothelial growth factor receptor 3 (FLT4, VEGFR3) is overexpressed in NSCLC cells, making it a potential target for NSCLC treatment. In this study, we aimed to explore the anti-cancer effects of dauricine on NSCLC cells and its mechanism targeting FLT4. METHODS: We found that dauricine inhibited the growth of NCI-H1299 cells by blocking the cycle in the G2/M phase through flow cytometry analysis. In addition， dauricine also inhibited the migration of NCI-H1299 cells by wound healing assay and transwell migration assay. More importantly, our empirical analysis found the anti-cancer effect of dauricine on NCI-H1299 cells and the protein level of FLT4 had a distinctly positive correlation, and this effect was weakened after FLT4 knockdown. RESULTS: It is suggested that dauricine suppressed the growth and migration of NCI-H1299 cells by targeting FLT4. Furthermore, dauricine inhibited FLT4 downstream pathways, such as PTEN/AKT/mTOR and Ras/MEK1/2/ERK1/2, thereby regulating cell migration-related molecule MMP3 and cell cycle-related molecules (CDK1, pCDK1-T161, and cyclin B1). CONCLUSION: Dauricine may be a promising FLT4 inhibitor for the treatment of NSCLC.

Analysis of Fever Following Bronchoscopy and Endobronchial Ultrasound-Guided Transbronchial Needle Aspiration.

Objective: Bronchoscopy and endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) are two essential methods for obtaining the pathological diagnosis of central lung masses or hilar and mediastinal lymphadenopathy. We can observe that many patients have a fever after examinations, but the pathogenesis is not yet fully clear. We tried to comprehensively assess the occurrence of postoperative fever and bacterial infections in patients undergoing bronchoscopy and endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) procedures. Methods: We retrospectively analyzed 512 patients undergoing bronchoscopy or EBUS-TBNA examination. According to examination methods, all patients were classified into three groups: Only perform bronchoscopy examination (BO) group (122 cases)，both perform bronchoscopy and biopsy (BB) group (262 cases), and EBUS-TBNA after bronchoscopy (EBUS) group (128 cases). Peripheral blood leucocyte, neutrophil count, and serum IL-6 test results were obtained before and after the examination. A blood culture was performed when the body temperature was higher than 38.5°C. Results: Among the three groups, the onset time (5.5h), average duration (6h), and peak temperature (37.7°C) of fever in the BO group were lower than those in the BB and EBUS groups. Still, there was no significant difference in onset time (11.66h, 11.83h), average duration (12.86h, 13.56h), and peak temperature (39.1°C, 39.1°C) between the BB group and EBUS group. There was no significant difference in the peripheral blood leukocyte count, neutrophil count or IL-6 level before the operation (P > .05). Compared with the preoperative, the leukocyte count, neutrophil count and IL-6 level in the three groups were increased after the operation (P < .05). Positive blood cultures were diagnosed as normal oropharyngeal flora. Conclusions: Postoperative fever after bronchoscopy is a relatively common complication, most of which do not require special treatment. Individuals with concomitant diseases such as diabetes may have postoperative infections after EBUS-TBNA, and they should be emphatically observed. The findings could potentially extend to similar diagnostic procedures or situations in pulmonary medicine. Understanding the risk factors associated with postoperative fever can help healthcare providers manage patient expectations and monitor certain groups more closely.

HPLC-fluorescence detection for stability of harringtonine, and identification of degradation products by UPLC-Q-TOF-MS.

Harringtonine (HT) is an anticancer alkaloid early extracted and isolated from cephalotaxus fortunei Hook. f., also has various pharmacological activities such as antiviral, antibacterial, antimalarial, anti-inflammatory, antioxidant, herbicidal and insecticidal. However, the factors affecting the stability of HT, the main degradation sites and mechanisms involved in its disposal process in vivo have not yet been elucidated. This study utilized HPLC-fluorescence detection method to establish a simple quantitative detection method for HT with good accuracy, precision, and high sensitivity. Temperature and pH were the main factors affecting the stability of HT, which underwent significant degradation in high temperature and alkaline environments because of the occurrence of hydrolysis reactions. In isolated biological homogenates of SD rats, except gastrointestinal tract, HT was degraded in other sites, especially respiratory, mainly in airway and lungs, and systemic metabolism, mainly in livers, spleens, and kidneys. Through UPLC-Q-TOF-MS, three forced degradation products were identified as 4'-demethyl HT, cephalotaxine, and dehydrated HT, respectively. However, the degradation product in isolated biological homogenates of SD rats was only 4'-demethyl HT due to the relatively mild environment. Our findings contributed to a necessary study basis for HT in terms of structural optimization, dosage form selection, storage and transportation.

Thebaine induces anaphylactic reactions via the MRGPRX2 receptor pathway on mast cells.

Morphine derivatives are clinically important anesthetic and sedative drugs, which often show anaphylactic side effects. Mas-related G-protein coupled receptor member X2 (MRGPRX2) triggers mast cell degranulation, which is important process in anaphylactic reactions. MRGPRX2-HEK293 and LAD2 cell membrane chromatographic (CMC) models were used to screen morphine derivatives binding to MRGPRX2. Furthermore, most morphine derivatives significantly enhanced Ca2+ mobilization. More importantly, thebaine was found to effectively promote histamine release. Thebaine induced the increased release of ß-hexosaminidase and high secretion level of cytokines, confirming that thebaine could further trigger anaphylactic reactions and promote subsequent inflammatory reactions. Moreover, the ability of thebaine inducing degranulation and the release of allergenic mediators in mast cells was significantly decreased after MRGPRX2 knockdown, which proved that MRGPRX2 is the key media for thebaine-induced anaphylactic reactions. Significant hind paw swelling and hypothermia in mice after injecting thebaine suggested that thebaine could trigger anaphylactic reactions in vivo.

Fabrication of poly(t-butyl betaine carboxylate)-based nanoparticles and study on their in vivo biosecurity.

The purpose of this article was to fabricate the novel poly(t-butyl betaine carboxylate)-S-S-poly (1, 3-dioxan-2-one) nanoparticles (PCB-tBU-S-S-PDI NPs) and study their in vivo biosecurity. The poly(t-butyl betaine carboxylate) (PCB-tBU) segment was conjugated to the poly(1, 3-dioxan-2-one)(PDI) moity with a disulfide bond to obtain the copolymer PCB-tBU-S-S-PDI. Hydrogen nuclear magnetic resonance (1H NMR) and Fourier transform infrared spectroscopy (FTIR) spectra were applied to study the structure of PCB-tBU-S-S-PDI. The cargo-free NPs were administrated to Sprague-Dawley (SD) rats by intraperitoneal injection every 3 days for 30 days. Then, the blood routine examination, blood biochemistry, and histologic slides of rat's organs were carried out to monitor the in vivo biosecurity of cargo-free PCB-tBU-S-S-PDI NPs. 1H NMR and FTIR spectra confirmed the successfully synthesis of PCB-tBU-S-S-PDI. The cargo-free NPs showed spherical morphology with an average of 139.8 ± 0.26 nm. The results of blood biochemistry and blood routine examination suggested that the cargo-free PCB-tBU-S-S-PDI NPs did not show any influence on the liver and renal functions of treated rats. Significantly, the physiological slides of treated rat's organs did not show any physiological and pathological changes. These phenomena suggested that the PCB-tBU-S-S-PDI NPs had good biosecurity, and it could be used as a vehicle for antineoplastic drug delivery.