Efficient analysis of adverse drug events and toxicological mechanisms of newly marketed drugs by integrating pharmacovigilance and network toxicology: selumetinib as an example.

Objective: To integrate pharmacovigilance and network toxicology methods to explore the potential adverse drug events (ADEs) and toxic mechanisms of selumetinib, and to provide a reference for quickly understanding the safety and toxicological mechanisms of newly marketed drugs. Methods: Taking selumetinib as an example, this study integrated pharmacovigilance methods based on real-world data and network toxicology methods to analyze its ADE and its potential toxicological mechanism. First, the ADE reports of selumetinib were extracted from the US Food and Drug Administration (FDA) adverse event reporting system (FAERS), and the ADE signals were detected by reporting odds ratio (ROR) and UK medicines and healthcare products regulatory agency (MHRA) methods. The ADE signals were classified and described according to the preferred terms (PTs) and system organ class (SOC) derived from the Medical Dictionary for Regulatory Activities (MedDRA). The network toxicology method was used to analyze the toxicological mechanism of the interested SOCs. The specific steps included predicting the potential targets of selumetinib using TOXRIC, STITCH, ChEMBL, CTD, SwissTargetPreditcion, and Super-PRED databases, collecting the targets of SOC using GeneCards database, conducting protein-protein interaction (PPI) analysis through STRING database, conducting gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) analysis through DAVID database, and testing the molecular affinity using AutoDock software. Results: A total of 1388 ADE reports related to selumetinib were extracted, and 53 positive signals were detected by ROR and MHRA methods, of which 20 signals were not mentioned in the package insert, including ingrowing nail, hyperphosphatemia, cardiac valve disease, hematuria, neutropenia, etc. Analysis of the toxicological mechanism of six SOCs involved in positive ADE signals revealed that the key targets included EGFR, STAT3, AKT1, IL6, BCL2, etc., and the key pathways included PI3K/Akt pathway, apoptosis, ErbB signaling pathway, and EGFR tyrosine kinase inhibitor resistance, etc. Molecular docking assays showed spontaneous binding of selumetinib to key targets in these pathways. Conclusion: The pharmacovigilance analysis identified some new potential ADEs of selumetinib, and the network toxicology analysis showed that the toxic effects of selumetinib may be related to PI3K/Akt pathway, apoptosis, ErbB signaling pathway, EGFR tyrosine kinase inhibitor resistance and other pathways.

Post-marketing safety surveillance of dalfampridine for multiple sclerosis using FDA adverse event reporting system.

Objective: To investigate and analyze the post-marketing adverse event (AE) data of multiple sclerosis (MS) therapeutic drug dalfampridine using the US Food and Drug Administration Adverse Event Reporting System (FAERS) for its clinical safety application. Methods: Use OpenVigil2.1 platform to obtain AE data of dalfampridine from FAERS from February 2010 to September 2022. Match "adverse drug reaction" with preferred term (PT) and system organ class (SOC) according to the Medical Dictionary for Regulatory Activities (MedDRA), then merge the same PT and delete non-AE PT. Positive signals were identified by the reporting odds ratio (ROR), proportional reporting ratio (PRR), and Bayesian confidence propagation neural network (BCPNN) methods. When AE signals met the criteria of those three methods, they were identified as positive signals. Results: A total of 44,092 dalfampridine-related AE reports were obtained, and 335 AE signals were identified, including 11,889 AE reports. AEs were more common in females and in the 45-65 age group, which is consistent with the epidemiological characteristics of MS. The 335 AE signals involved 21 SOCs, including investigations, infections and infestations, eye disorders, etc. Among the top 20 PTs in signal strength, 10 were associated with abnormal lymphocyte percentage and count, and 5 were associated with abnormal urine tests, some of which were not described in the instruction, such as spinal cord injury cauda equina, haemoglobin urine present, urinary sediment abnormal and so on. The most frequently reported AE signals were urinary tract infection, dizziness, condition aggravated. In addition, 23 AE signals with death outcomes were identified, with an incidence of less than 0.1%. Conclusion: Data mining of FAERS was conducted to analyze the AEs of dalfampridine, and new AE signals were found. This study provides a reference for the safe use of dalfampridine in the treatment of MS.

Graphene quantum dots disturbed the energy homeostasis by influencing lipid metabolism of macrophages.

To investigate the potential factors of graphene quantum dots (GQDs), the assessment impact on the innate immune system is one of the most important. As the innate immune cell, macrophages possess phagocytosis activity and affect immunomodulation. Higher oxygen consumption rates (OCR) are used to gain insight into GQDs' effects on macrophages. Metabolomics profiling also revealed that GQDs exposure provoked an increase in phosphoglycerides, sphingolipids, and oxidized lipids in macrophages. The molecular pathways disrupted by GQDs were associated with lipid and energy metabolisms. Metabolite flux analysis was used to evaluate changes in the lipid metabolism of macrophages exposed to 100 µg mL-1 GQDs for 24 and 48 h. A combination of 13C-flux analysis and metabolomics revealed the regulation of lipid biosynthesis influenced the balance of energy metabolism. Integrated proteomics and metabolomics analyses showed that nicotinic acid adenine dinucleotide and coenzyme Q10 were significantly increased under GQDs treatment, alongside upregulated protein activity (e.g., Cox5b and Cd36). The experimental evidences were expected to be provided in this study to reveal the potential harmful effect from exposure to GQDs.

Identification of epigenetic dysregulation gene markers and immune landscape in kidney renal clear cell carcinoma by comprehensive genomic analysis.

Kidney cancer is one the most lethal cancers of the urinary system, but current treatments are limited and its prognosis is poor. This study focused on kidney renal clear cell carcinoma (KIRC) and analyzed the relationship between epigenetic alterations and KIRC prognosis, and explored the prognostic significance of these findings in KIRC patients. Based on multi-omics data, differentially expressed histone-modified genes were identified using the R package limma package. Gene enhancers were detected from data in the FANTOM5 database. Gene promoters were screened using the R package ChIPseeker, and the Bumphunter in the R package CHAMP was applied to screen differentially methylated regions (DMR). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and Gene Ontology (GO) functional enrichment analysis of genes was performed using the R package clusterProfiler. We identified 51 dysregulated epigenetic protein coding genes (epi-PCGs) from 872 epi-PCGs, and categorized three molecular subtypes (C1, C2, and C3) of KIRC samples with significantly different prognosis. Notably, among the three molecular subtypes, we found a markedly differential immune features in immune checkpoints, cytokines, immune signatures, and immune cell distribution. C2 subtype had significantly lower enrichment score of IFNγ, cytotoxic score (CYT), and angiogenesis. In addition, an 8-gene signature containing 8 epi-PCGs (ETV4, SH2B3, FATE1, GRK5, MALL, HRH2, SEMA3G, and SLC10A6) was developed for predicting KIRC prognosis. Prognosis of patients with a high 8-gene signature score was significantly worse than those with a low 8-gene signature score, which was also validated by the independent validation data. The 8-gene signature had a better performance compared with previous signatures of KIRC. Overall, this study highlighted the important role of epigenetic regulation in KIRC development, and explored prognostic epi-PCGs, which may provide a guidance for exploiting further pathological mechanisms of KIRC and for developing novel drug targets.

Quercetin inhibits Pseudomonas aeruginosa biofilm formation via the vfr-mediated lasIR system.

Pseudomonas aeruginosa is one of the most common opportunistic pathogens that cause biofilm-associated infections. Biofilm formation is partially regulated by the quorum sensing (QS) system, and quercetin can inhibit QS, biofilm formation and virulence factors. We therefore speculated that quercetin would inhibit the formation of P. aeruginosa biofilm via the QS system. In this study, we successfully constructed lasI, rhlI and lasI/rhlI gene-knockout strains. The knockout of the lasI and lasI/rhlI genes resulted in decreases in adhesion, biofilm formation, swarming motility and the expression of biofilm-associated genes, whereas deletion of the rhlI gene had no obvious influence on these biofilm-related indicators with the exception of the swarming motility. After treatment with quercetin, the lasI- and lasI/rhlI-mutant strains exhibited increased adhesion, biofilm formation, swarming motility and biofilm-associated gene expression compared with the control group. However, quercetin still exerted an inhibitory effect on these physiological factors and the biofilm-associated gene expression in the rhlI-mutant strain. The knockout of QS genes reduced the production of pyocyanin and protease activity, but after the virulence factors of the QS-mutant strains treated with quercetin showed almost no differences compared with those of the control group. In addition, quercetin could significantly inhibit vfr gene expression regardless of the presence of QS genes. The results indicated that quercetin might inhibit the lasIR system through the vfr gene and ultimately the formation of P. aeruginosa biofilms.

Effects of Ginsenoside Rg3 on fatigue resistance and SIRT1 in aged rats.

BACKGROUND: Ginsenoside Rg3 (Rg3) is one of the key components of a frequently used herbal tonic panax ginseng for fatigue treatment. However, the molecular mechanisms of Rg3 on anti-fatigue effects have not been completely understood yet. METHODS AND MATERIALS: We built a postoperative fatigue syndrome (POFS) model and tried to elucidate the molecular mechanisms responsible for anti-fatigue effects of Rg3. 160 aged male rats were randomly divided into four groups (n = 40/group): normal group, Rg3-treated normal group (Rg3 group), postoperative fatigue syndrome model group (POFS group) and Rg3-treated postoperative fatigue syndrome model group (POFS + Rg3 group). The open field test (OFT) was used to assess general activity and exploratory behavior of rats in different groups. We then analyzed total cholesterol (TC), serum triglyceride (TG) and lactate dehydrogenase (LDH) in the blood, as well as superoxide dismutase (SOD), malondialdehyde (MDA), peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) and phosphoenolpyruvate carboxykinase (PEPCK) mRNA expression in skeletal muscles of rats. We also detected the influence of Rg3 on silent information regulator of transcription 1 (sirtuin1, SIRT1) activity and protein 53 (p53) transcriptional activity in vitro. RESULTS: Rg3 significantly increased the journey distance and rearing frequency, while slowed down the rest time. The serum concentrations of TC, TG and LDH were all up-regulated by Rg3. Meanwhile, Rg3 increased concentrations of SOD, but also decreased MDA release out of skeletal muscles. The mRNA expressions of PGC-1α and PEPCK were also enhanced by Rg3. Besides, Rg3 could activate SIRT1 and suppress p53 transcriptional activity in the biological process. DISCUSSION AND CONCLUSION: Rg3 could improve exercise performance and resist fatigue possibly through elevating SIRT1 deacetylase activity.

Effect of chronic heat stress on some physiological and immunological parameters in different breed of broilers.

The differences in physiological and immunological parameters and pathological damage to organ tissues exposed to chronic heat stress provide the basis for evaluating heat resistance of different chicken breeds (white recessive rock [WRR] and The Lingshan [LS]). Ninety broilers of each breed were divided equally into a chronic heat stress group and a no heat stress group. The effects of chronic heat stress on the physiological and immunological parameters of broilers were analyzed using flow cytometry, ELISA, RT-qPCR, etc. Under heat stress conditions: (1) H and H/L values were significantly increased (P < 0.01) in the 2 breeds, and were higher in the WRR broilers than in the LS broilers at a late stage (P < 0.05). Although the corticosterone levels were also significantly increased (P < 0.01) in both breeds, they were lower in the 49 d WRR broilers than in the LS broilers (P < 0.01). The number of leukocytes were significantly increased in the 49 d WRR broilers (P < 0.01), whereas the number of CD3+, CD8+ cells, and erythrocytes were significantly reduced (P < 0.05). A significantly (P < 0.01) lower number of CD3+, CD4+ T-lymphocytes, and CD4+/CD8+ were present in WRR compared to that in the LS broilers. (2) The HSP70 transcript was significantly increased in the WRR broilers (P < 0.01), and was higher than the level in the LS broilers. The expression level of HSP70 protein was significantly (P < 0.05) increased in WRR broilers. (3) The WRR broilers developed cardiac and leg muscle inflammatory cellular hyperplasia and local inflammatory lesions, as well as cerebral meningitis and inflammatory hyperplasia of the brain tissue. The LS broilers developed mild cerebral inflammatory hyperplasia and mild inflammatory cellular proliferation in the leg muscle. In conclusion, under heat stress conditions, the relative physiological and immunological parameters were worse in the WRR broilers than in the LS broilers. The WRR broilers showed poor heat tolerance as evidenced from the expression of HSP70 and the extent of histopathological damages.

Quantitative determination of sarsasapogenin in rat plasma using liquid chromatography-tandem mass spectrometry.

Sarsasapogenin, a natural compound from Chinese medical herb Anemarrhena asphodeloides Bge., has recently received a great deal of attention due to its various bioactivities. In this study, an easy and applicable liquid chromatography tandem mass spectrometry method for the quantification of sarsasapogenin in rat plasma was developed. Sample preparation was accomplished through a simple one-step protein precipitation procedure with methanol. Negative electrospray ionization was performed using multiple reactions monitoring (MRM) mode with transitions of m/z 417.4/273.2 for sarsasapogenin, and 415.2/271.4 for diosgenin (internal standard). The calibration curve was linear over the range of 0.5-500ng/mL (r=0.9994), with a lower limit of quantification at 0.5ng/mL. The RSD of intra- and inter-day precision was below 6.41%, and accuracy ranged from 87.60% to 99.20%. The RSD of matrix effect and recovery yield was within ±15% of nominal concentrations and sarsasapogenin was stable during stability tests. This validated method had been successfully applied to the preclinical pharmacokinetic studies of sarsasapogenin in rats. The half-life (t1/2) was (15.1±2.3), (16.1±3.0) and (15.4±3.9) h after single intragastric administration of 25, 50 and 100mg/kg sarsasapogenin, respectively. And it was found that, the area under the plasma concentration versus time curve (AUC0-72h) and the maximum plasma concentration (Cmax) were linearly related to dose.