Efficacy and safety of single-dose artesunate plus sulfalene/pyrimethamine combined with praziquantel for the treatment of children with Schistosoma mansoni or Schistosoma haematobium in western Kenya: a randomised, open-label controlled trial.

BACKGROUND: Reliance on praziquantel for the treatment and control of schistosomiasis is likely to facilitate the emergence of drug resistance. Combination therapy targeting adult and juvenile schistosome worms is urgently needed to improve praziquantel efficacy and delay the potential development of drug resistance. We assessed the efficacy and safety of single-dose praziquantel combined with single-dose artesunate plus sulfalene-pyrimethamine in the treatment of Kenyan children with schistosomiasis. METHODS: This was an open-label, randomised clinical trial involving 426 school-aged children (7-15 years old) diagnosed with Schistosoma mansoni (by Kato-Katz) or S. haematobium (by urine filtration). They were randomly assigned (1:1:1) to receive a single dose of praziquantel (40 mg/kg), a single dose of artesunate plus sulfalene-pyrimethamine (12 mg/kg artesunate) or combination therapy using a single dose of praziquantel (40 mg/kg) combined with a single dose of artesunate plus sulfalene-pyrimethamine (12 mg/kg artesunate). The primary outcome was cure and egg reduction rates at 6 weeks post-treatment in the available case population. Adverse events were assessed within 3 h after treatment. RESULTS: Of the 426 children enrolled, 135 received praziquantel, 150 received artesunate plus sulfalene-pyrimethamine, and 141 received combination therapy. Outcome data were available for 348 (81.7%) children. For S. mansoni-infected children (n = 335), the cure rates were 75.6%, 60.7%, and 77.8%, and the egg reduction rates were 80.1%, 85.0%, and 88.4% for praziquantel, artesunate plus sulfalene-pyrimethamine, and combination therapy, respectively. For S. haematobium-infected children (n = 145), the corresponding cure rates were 81.4%, 71.1%, and 82.2%, and the egg reduction rates were 95.6%, 97.1%, and 97.7%, respectively. Seventy-one (16.7%) children reported mild-intensity adverse events. The drugs were well tolerated and no serious adverse events were reported. CONCLUSIONS: A single oral dose of praziquantel combined with artesunate plus sulfalene-pyrimethamine cured a high proportion of children with S. haematobium but did not significantly improve the treatment efficacy for either urinary or intestinal schistosomiasis. Sequential administration of praziquantel and artesunate plus sulfalene-pyrimethamine may enhance the efficacy and safety outcomes.

Antitumour effects of artesunate via cell cycle checkpoint controls in human oesophageal squamous carcinoma cells.

Artesunate (ART), an effective antimalarial semisynthetic derivative of artemisinin, exhibits antitumour properties, but the mechanism(s) involved remain elusive. In this study, we investigated the antitumour effects of ART on human oesophageal squamous cell carcinoma (ESCC) cell lines. Treatment of ESCC cell lines with ART resulted in the production of excessive reactive oxygen species (ROS) that induced DNA damage, reduced cell proliferation and inhibited clonogenicity via G1-S cell cycle arrest and/or apoptosis in vitro. The administration of ART to nude mice with ESCC cell xenografts inhibited tumour formation in vivo. However, the cytotoxicity of ART strongly differed among the ESCC cell lines tested. Transcriptomic profiling revealed that although the expression of large numbers of genes in ESCC cell lines was affected by ART treatment, these genes could be functionally clustered into pathways involved in regulating cell cycle progression, DNA metabolism and apoptosis. We revealed that p53 and Cdk4/6-p16-Rb cell cycle checkpoint controls were critical determinants required for mediating ART cytotoxicity in ESCC cell lines. Specifically, KYSE30 cells with p53Mut/p16Mut were the most sensitive to ART, KYSE150 and KYSE180 cells with p53Mut/p16Nor exhibited intermediate responses to ART, and Eca109 cells with p53Nor/p16Nor exhibited the most resistance to ATR. Consistently, perturbation of p53 expression using RNA interference (RNAi) and/or Cdk4/6 activity using the inhibitor palbociclib altered ART cytotoxicity in KYSE30 cells. Given that the p53 and Cdk4/6-cyclin D1-p16-Rb genes are commonly mutated in ESCC, our results potentially shed new light on neoadjuvant chemotherapy strategies for ESCC.

3D printing of multi-unit gastro-retentive tablets for the pulsatile release of artesunate.

Pulsatile drug delivery is hardly achieved by conventional gastro-retentive dosage forms. Artesunate as a typical anti-malaria medicine needs oral pulsatile release. Here, artesunate-loaded pulsatile-release multi-unit gastro-retentive tablets (APGTs) were prepared with a semi-solid extrusion three-dimensional (3D) printing method. An APGT was composed of three units: artesunate-loaded immediate and delayed release units and a block unit. The matrix of the immediate/delayed release units consisted of polyvinylpyrrolidone (PVP) K30 and croscarmellose sodium, which improved the rapid release of artesunate when contacting water. The block unit consisted of octadecanol, hydroxypropyl methyl cellulose K15M, PVP K30, and poloxamer F68. APGTs showed multi-phase release in simulated gastric liquids (SGLs). The first immediate release phase continued for 1 h followed by a long block phase for 7 h. The second rapid release phase was initiated when the eroded holes in the block unit extended to the inner delayed release unit, and this phase continued for about 14 h. Low-density APGTs could ensure their long-term floating in the stomach. Oral APGTs remained in the rabbit stomach for about 20 h. 3D printing provides a new strategy for the preparation of oral pulsatile-release tablets.

Exploring the neuroprotective role of artesunate in mouse models of anti-NMDAR encephalitis: insights from molecular mechanisms and transmission electron microscopy.

BACKGROUND: The pathway involving PTEN-induced putative kinase 1 (PINK1) and PARKIN plays a crucial role in mitophagy, a process activated by artesunate (ART). We propose that patients with anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis exhibit insufficient mitophagy, and ART enhances mitophagy via the PINK1/PARKIN pathway, thereby providing neuroprotection. METHODS: Adult female mice aged 8-10 weeks were selected to create a passive transfer model of anti-NMDAR encephalitis. We conducted behavioral tests on these mice within a set timeframe. Techniques such as immunohistochemistry, immunofluorescence, and western blotting were employed to assess markers including PINK1, PARKIN, LC3B, p62, caspase3, and cleaved caspase3. The TUNEL assay was utilized to detect neuronal apoptosis, while transmission electron microscopy (TEM) was used to examine mitochondrial autophagosomes. Primary hippocampal neurons were cultured, treated, and then analyzed through immunofluorescence for mtDNA, mtROS, TMRM. RESULTS: In comparison to the control group, mitophagy levels in the experimental group were not significantly altered, yet there was a notable increase in apoptotic neurons. Furthermore, markers indicative of mitochondrial leakage and damage were found to be elevated in the experimental group compared to the control group, but these markers showed improvement following ART treatment. ART was effective in activating the PINK1/PARKIN pathway, enhancing mitophagy, and diminishing neuronal apoptosis. Behavioral assessments revealed that ART ameliorated symptoms in mice with anti-NMDAR encephalitis in the passive transfer model (PTM). The knockdown of PINK1 led to a reduction in mitophagy levels, and subsequent ART intervention did not alleviate symptoms in the anti-NMDAR encephalitis PTM mice, indicating that ART's therapeutic efficacy is mediated through the activation of the PINK1/PARKIN pathway. CONCLUSIONS: At the onset of anti-NMDAR encephalitis, mitochondrial damage is observed; however, this damage is mitigated by the activation of mitophagy via the PINK1/PARKIN pathway. This regulatory feedback mechanism facilitates the removal of damaged mitochondria, prevents neuronal apoptosis, and consequently safeguards neural tissue. ART activates the PINK1/PARKIN pathway to enhance mitophagy, thereby exerting neuroprotective effects and may achieve therapeutic goals in treating anti-NMDAR encephalitis.

Lacrimal gland Alterations and the Effect of artesunate on experimental induced diabetes rat models and related mechanisms.

Diabetic patients are at high risk of developing lacrimal gland dysfunction, and the antimalarial drug artesunate (ART) was recently used to induce experimental-induced diabetes mellitus. This study's objective is to investigate the lacrimal gland alteration and the effect of ART on experimentally induced diabetes rat models and its related mechanisms. Forty rats were divided into five groups (8 rats/group): healthy control group (HC), diabetic group (DM), 50 mg/kg ART intervention diabetic group [DM + ART (50 mg/kg)], 100 mg/kg ART intervention diabetic group [DM + ART (100 mg/kg)] and 6 U/kg Insulin intervention diabetic group (DM + INS). The morphology of the eyeball and lacrimal gland tissues was determined using hematoxylin and eosin staining. In addition, external lacrimal glands were harvested for electronic microscopic examination, NFκB1, and TNF-α protein expression evaluation by immunohistochemistry and mRNA expression analysis by RT-PCR. Histopathological and ultrastructural changes suggest ART intervention has an improved structural effect. Protein expression of NFκB1 in the DM + ART (100 mg/kg) group was decreased. TNF-α significantly decreased in the DM + ART (50 mg/kg) and insulin groups. We concluded that ART improves structural changes in a lacrimal gland in diabetic rats. The present study provides further evidence of the therapeutic effect of ART on the lacrimal gland of diabetic rats by decreasing the expression of NFκB1 and TNF-α.

Artesunate attenuates the tumorigenesis of choroidal melanoma via inhibiting EFNA3 through Stat3/Akt signaling pathway.

PURPOSE: Choroidal melanoma (CM), a kind of malignant tumor, is the main type of Uveal melanoma and one half of CM patients develop metastases. As a member of Eph/ephrin pathway that plays vital role in tumors, EphrinA3 (EFNA3) has been proved to promote tumorigenesis in many tumors. But the effect of EFNA3 in CM has not been studied yet. Through inhibiting angiogenesis, inducing apoptosis and autophagy and so on, Artesunate (ART) plays a key anti-tumor role in many tumors, including CM. However, the exact mechanisms of anti-tumor in CM remain unclear. METHODS: The UALCAN and TIMER v2.0 database analyzed the role of EFNA3 in CM patients. Quantitative real time polymerase chain reaction (qPCR) and Western blot were used to detect the expression of EFNA3 in CM. The growth ability of CM was tested by clonogenic assay and Cell counting kit-8 assay, and the migration ability using Transwell assay. RESULTS: Our results found EFNA3 boosted CM cells' growth and migration through activating Stat3/Akt signaling pathway, while ART inhibited the tumor promoting effect of CM via downregulating EFNA3. In xenograft tumor model, EFNA3 knockdown and ART significantly inhibited tumor growth. CONCLUSION: EFNA3 could be a valuable prognostic factor in CM.

Anti-Opisthorchis felineus effects of artemisinin derivatives: An in vitro study.

BACKGROUND: The drug of choice for the treatment of opisthorchiasis caused by trematodes Opisthorchis viverrini and O. felineus is praziquantel (PZQ), but there is a constant search for new anthelmintics, including those of plant origin. Positive results on the use of artemisinin derivatives against O. viverrini opisthorchiasis have been shown previously, but the effect of these compounds on O. felineus has not been studied. Therefore, here, a comparative analysis of anthelmintic properties of artemisinin derivatives (artesunate [AS], artemether [AM], and dihydroartemisinin [DHA]) was carried out in vitro in relation to PZQ. Experiments were performed on newly excysted metacercariae (NEMs) and adult flukes of O. felineus. RESULTS: Dose- and time-dependent effects of artemisinin derivatives and of PZQ were assessed in terms of motility and mortality of both NEMs and adult flukes. The most pronounced anthelmintic action was exerted by DHA, whose half-maximal inhibitory concentrations (IC50) of 1.9 (NEMs) and 2.02 µg/mL (adult flukes) were lower than those of PZQ (0.56 and 0.25 µg/mL, respectively). In contrast to PZQ, the effects of DHA and AS were similar when we compared the two developmental stages of O. felineus (NEMs and adult flukes). In addition, AM, AS, and especially DHA at doses of 100 µg/mL disrupted tegument integrity in adult flukes, which was not observed with PZQ. CONCLUSIONS: Artemisinin derivatives (AS, AM, and DHA) have good anthelmintic efficacy against the trematode O. felineus, and the action of these substances is comparable to (and sometimes better than) the effects of PZQ.

Efficacy of a Single Oral Dose of Artesunate plus Sulfalene-Pyrimethamineversus Praziquantel in the Treatment of Schistosoma mansoni in Kenyan Children: An Open-Label, Randomized, Exploratory Trial.

Unlike praziquantel, artemisinin derivatives are effective against juvenile schistosome worms. We assessed the efficacy and safety of a single oral dose of artesunate plus sulfalene-pyrimethamine versus praziquantel in the treatment of Schistosoma mansoni. Seventy-three schoolchildren (aged 9-15 years) with confirmed S. mansoni infection in Rarieda, western Kenya, were randomly assigned to receive either a single oral dose of artesunate plus sulfalene-pyrimethamine (n = 39) or a single dose of praziquantel (n = 34). The cure and egg reduction rates at 4 weeks posttreatment were 69.4% (25/36) versus 80.6% (25/31) (P = 0.297) and 99.1% versus 97.5% (P = 0.607) in the artesunate plus sulfalene-pyrimethamine group versus praziquantel group, respectively. Fourteen children developed adverse events, and there were no serious adverse events. A single oral dose of artesunate plus sulfalene-pyrimethamine has efficacy comparable to that of praziquantel in the treatment of S. mansoni, but these results should be confirmed in larger randomized controlled trials.

Artesunate improves glucose and lipid metabolism in db/db mice by regulating the metabolic profile and the MAPK/PI3K/Akt signalling pathway.

BACKGROUND: Diabetes is a metabolic disorder characterized by chronic hyperglycaemia. Chronic metabolic abnormalities and long-term hyperglycaemia may result in a wide range of acute and chronic consequences. Previous studies have demonstrated that artesunate(ART) has antidiabetic, anti-inflammatory, antiatherosclerotic, and other beneficial effects, but the specific regulatory mechanism is not completely clear. AIM: This study investigated the effects of ART on metabolic disorders in type 2 diabetes mellitus (T2DM) model db/db mice and explored the underlying mechanisms involved. METHODS: C57BL/KsJ-db/db mice were used to identify the targets and molecular mechanism of ART. Metabolomic methods were used to evaluate the efficacy of ART in improving T2DM-related metabolic disorders. Network pharmacology and transcriptomic sequencing were used to analyse the targets and pathways of ART in T2DM. Finally, molecular biology experiments were performed to verify the key targets and pathways selected by network pharmacology and transcriptomic analyses. RESULTS: After a 7-week ART intervention (160 mg/kg), the glucose and lipid metabolism levels of the db/db mice improved. Additionally, the oxidative stress indices, namely, the MDA and SOD levels, significantly improved (p<0.01). Linoleic acid and glycerophospholipid metabolism, amino acid metabolism, bile acid synthesis, and purine metabolism disorders in db/db mice were partially corrected after ART treatment. Network pharmacology analysis identified important targets of ART for the treatment of metabolic disorders in T2DM . These targets are involved in key signalling pathways, including the highest scores observed for the PI3K/Akt signalling pathway. Transcriptomic analysis revealed that ART could activate the MAPK signalling pathway and two key gene targets, HGK and GADD45. Immunoblotting revealed that ART increases p-PI3K, p-AKT, Glut2, and IRS1 protein expression and suppresses the phosphorylation of p38, ERK1/2, and JNK, returning HGK and GADD45 to their preartesunate levels. CONCLUSION: Treatment of db/db mice with 160 mg/kg ART for 7 weeks significantly reduced fasting blood glucose and lipid levels. It also improved metabolic imbalances in amino acids, lipids, purines, and bile acids, thereby improving metabolic disorders. These effects are achieved by activating the PI3K/AKT pathway and inhibiting the MAPK pathway, thus demonstrating the efficacy of the drug.

Engineered Artesunate-Naphthalimide Hybrid Dual Drug for Synergistic Multimodal Therapy against Experimental Murine Lymphoma.

Lymphoma can effectively be treated with a chemotherapy regimen that is associated with adverse side effects due to increasing drug resistance, so there is an emergent need for alternative small-molecule inhibitors to overcome the resistance that occurs in lymphoma management and overall increase the prognosis rate. A new series of substituted naphthalimide moieties conjugated via ester and amide linkages with artesunate were designed, synthesized, and characterized. In addition to the conjugates, to further achieve a theranostic molecule, FITC was incorporated via a multistep synthesis process. DNA binding studies of these selected derivatives by ultraviolet-visible (UV-vis), fluorescence spectroscopy, intercalating dye (EtBr, acridine orange)-DNA competitive assay, and minor groove binding dye Hoechst 33342-DNA competitive assay suggested that the synthesized novel molecules intercalated between the two strands of DNA due to its naphthalimide moiety and its counterpart artesunate binds with the minor groove of DNA. Napthalimide-artesunate conjugates inhibit the growth of lymphoma and induce apoptosis, including ready incorporation and reduction in cell viability. The remodeled drug has a significant tumoricidal effect against solid DL tumors developed in BALB/c mice in a dose-dependent manner. The novel drug appears to inhibit metastasis and increase the survival of the treated animals compared with untreated littermates.

Artesunate inhibits macrophage-like phenotype switching of vascular smooth muscle cells and attenuates vascular inflammatory injury in atherosclerosis via NLRP3.

Inflammation is one of the main pathogenic factors of atherosclerosis (AS), and the phenotypic transformation of macrophages in human vascular smooth muscle cells (HVSMCs) contributes to the inflammatory injury of blood vessels and the formation of atherosclerotic plaques. Artesunate reportedly exerts anti-inflammatory activity against AS. Herein, we aimed to explore the artesunate-mediated anti-inflammatory and HVSMC phenotypic switch effects against AS and elucidate potential underlying mechanisms. In vitro, artesunate decreased expression of NLRP3, caspase-1, and interleukin (IL)- 1ß. Artesunate significantly inhibited low-density lipoprotein (LDL) expression in HVSMCs and macrophages. In vivo, artesunate reduced atherosclerotic plaque formation in high-fat diet (HFD)-fed ApoE-/- mice, as well as decreased NLRP3 and CD68 expression in atherosclerotic plaques. Artesunate decreased serum levels of triglycerides and increased high-density lipoprotein levels in HFD-med mice; however, serum levels of total cholesterol and LDL were unaltered. Treatment with artesunate substantially increased α-smooth muscle actin expression in aortic tissues while inhibiting expression levels of NLRP3, IL-1ß, heparinase, matrix metalloproteinase 9, and Krüppel-like factor 4 (KLF4). Collectively, our findings suggest that artesunate-mediated effects may involve inhibition of the ERK1/2/NF-κB/IL-1ß pathway in HVSMCs via the downregulation of NLRP3 expression. Thus, artesunate could serve as a novel strategy to treat AS by inhibiting AS plaque formation and suppressing macrophage-like phenotype switching of HVSMCs.

Artesunate Inhibits Lipid Accumulation and Inflammation by Regulating the NLRP3 Inflammasome in Nonalcoholic Fatty Liver Disease.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD), recognized as a chronic liver condition, has emerged as one of the most prevalent worldwide. This study explores the impact of artesunate (ART) on lipid accumulation and inflammatory factors within NAFLD model cells. METHODS: LO2 cells were subjected to treatment with oleic acid (OA) to establish NAFLD cell model. Subsequently, these cells were categorized into distinct groups: a control group, an OA group, an OA + 2.5 µm ART group, and an OA + 5 µm ART group. The activity of LO2 cells was determined using the Cell Counting Kit-8 (CCK-8) method. The presence of intracellular lipid droplets was examined through oil red O staining. Levels of triglycerides (TG), total cholesterol (TC), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) were evaluated using enzyme-linked immunosorbent assay (ELISA). Additionally, the protein expressions of nucleotide-binding oligomerization domain (NOD), leucine-rich repeat (LRR), and pyrin domain-containing protein 3 (NLRP3), Cleaved caspase-1, N-terminus of Gasdermin-D (GSDMD-N), and apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC) were measured via Western blot assay. RESULTS: In comparison to the control group, the OA group exhibited a significant increase in the contents of lipid droplets, TC, and TG (p < 0.01). Notably, ART effectively reversed the impact of OA (p < 0.01). Following OA stimulation, there was a pronounced elevation in the levels of IL-6 (p < 0.01), IL-1ß (p < 0.01), and TNF-α (p < 0.05). In comparison to the OA group, the 2.5 µm ART group showed no significant difference in TNF-α content (p > 0.05), while the 5 µm ART group significantly reduced TNF-α content (p < 0.05). Furthermore, both the 2.5 µm ART (p < 0.05) and 5 µm ART (p < 0.01) groups notably reduced IL-1ß and IL-6 content. When compared to the control group, the expressions of NLRP3, ASC, GSDMD-N, and Cleaved caspase-1 in the OA group significantly increased (p < 0.01). ART, however, mitigated this heightened expression trend (p < 0.05). CONCLUSIONS: ART demonstrated a reduction in TC and TG content, improvement in the deposit of intracellular lipid droplets, and a decrease in the release of inflammatory factors in LO2 cells. This effect was achieved through the regulation of the NLRP3 inflammasome, presenting a novel approach to the treatment of NAFLD.

Artesunate-loaded solid lipid nanoparticles resist esophageal squamous cell carcinoma by inducing Ferroptosis through inhibiting the AKT/mTOR signaling.

Esophageal squamous cell carcinoma (ESCC) is a severe malignancy with high incidence and mortality rate in China, while the application of standard chemotherapeutic drugs for ESCC meets the barriers of high toxicity and multiple drug resistance (MDR). In recent years, the anticancer effects of artesunate (ART), a Chinese medicine monomer have gained extensive attentions due to its characteristics of low toxicity, high potency, and reversal of MDR. In this study, we develop the artesunate-loaded solid lipid nanoparticles (SLNART) to overcome the poor water solubility and bioavailability of ART, further improving the efficiency of ART on ESCC treatment. Especially mentioned, SLNART is shown to present marked inhibitory effects on ESCC development based on the induction of ferroptosis by two pathways included upregulating TFR to increase Fe2+ ions and inhibiting the AKT/mTOR signaling to downregulate GPX4. Collectively, this study is the first to pave a promising approach for ESCC therapy based on a strategy of developing SLNART to induce ferroptosis by mediating Fe2+ ions and AKT/mTOR signaling.

Artesunate treats obesity in male mice and non-human primates through GDF15/GFRAL signalling axis.

Obesity, a global health challenge, is a major risk factor for multiple life-threatening diseases, including diabetes, fatty liver, and cancer. There is an ongoing need to identify safe and tolerable therapeutics for obesity management. Herein, we show that treatment with artesunate, an artemisinin derivative approved by the FDA for the treatment of severe malaria, effectively reduces body weight and improves metabolic profiles in preclinical models of obesity, including male mice with overnutrition-induced obesity and male cynomolgus macaques with spontaneous obesity, without inducing nausea and malaise. Artesunate promotes weight loss and reduces food intake in obese mice and cynomolgus macaques by increasing circulating levels of Growth Differentiation Factor 15 (GDF15), an appetite-regulating hormone with a brainstem-restricted receptor, the GDNF family receptor α-like (GFRAL). Mechanistically, artesunate induces the expression of GDF15 in multiple organs, especially the liver, in mice through a C/EBP homologous protein (CHOP)-directed integrated stress response. Inhibition of GDF15/GFRAL signalling by genetic ablation of GFRAL or tissue-specific knockdown of GDF15 abrogates the anti-obesity effect of artesunate in mice with diet-induced obesity, suggesting that artesunate controls bodyweight and appetite in a GDF15/GFRAL signalling-dependent manner. These data highlight the therapeutic benefits of artesunate in the treatment of obesity and related comorbidities.

Artesunate Inhibits the Growth of Insulinoma Cells via SLC7A11/ GPX4-mediated Ferroptosis.

BACKGROUND: Artesunate (ART) has been recognized to induce ferroptosis in various tumor phenotypes, including neuroendocrine tumors. We aimed to investigate the effects of ART on insulinoma and the underlying mechanisms by focusing on the process of ferroptosis. METHODS: The CCK8 and colony formation assays were conducted to assess the effectiveness of ART. Lipid peroxidation, glutathione, and intracellular iron content were determined to validate the process of ferroptosis, while ferrostatin-1 (Fer-1) was employed as the inhibitor of ferroptosis. Subcutaneous tumor models were established and treated with ART. The ferroptosis-associated proteins were determined by western blot and immunohistochemistry assays. Pathological structures of the liver were examined by hematoxylin-eosin staining. RESULTS: ART suppressed the growth of insulinoma both in vitro and in vivo. Insulinoma cells treated by ART revealed signs of ferroptosis, including increased lipid peroxidation, diminished glutathione levels, and ascending intracellular iron. Notably, ART-treated insulinoma cells exhibited a decline in the expressions of catalytic component solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4). These alterations were negated by Fer-1. Moreover, no hepatotoxicity was observed upon the therapeutic dose of ART. CONCLUSION: Artesunate might regulate ferroptosis of insulinoma cells through the SLC7A11/GPX4 pathway.

Mesenchymal-epithelial transition and AXL inhibitor TP-0903 sensitise triple-negative breast cancer cells to the antimalarial compound, artesunate.

Triple-negative breast cancer (TNBC) is a difficult-to-treat, aggressive cancer type. TNBC is often associated with the cellular program of epithelial-mesenchymal transition (EMT) that confers drug resistance and metastasis. EMT and reverse mesenchymal-epithelial transition (MET) programs are regulated by several signaling pathways which converge on a group of transcription factors, EMT- TFs. Therapy approaches could rely on the EMT reversal to sensitise mesenchymal tumours to compounds effective against epithelial cancers. Here, we show that the antimalarial ROS-generating compound artesunate (ART) exhibits higher cytotoxicity in epithelial than mesenchymal breast cancer cell lines. Ectopic expression of EMT-TF ZEB1 in epithelial or ZEB1 depletion in mesenchymal cells, respectively, reduced or increased ART-generated ROS levels, DNA damage and apoptotic cell death. In epithelial cells, ZEB1 enhanced expression of superoxide dismutase 2 (SOD2) and glutathione peroxidase 8 (GPX8) implicated in ROS scavenging. Although SOD2 or GPX8 levels were unaffected in mesenchymal cells in response to ZEB1 depletion, stable ZEB1 knockdown enhanced total ROS. Receptor tyrosine kinase AXL maintains a mesenchymal phenotype and is overexpressed in TNBC. The clinically-relevant AXL inhibitor TP-0903 induced MET and synergised with ART to generate ROS, DNA damage and apoptosis in TNBC cells. TP-0903 reduced the expression of GPX8 and SOD2. Thus, TP-0903 and ZEB1 knockdown sensitised TNBC cells to ART, likely via different pathways. Synergistic interactions between TP-0903 and ART indicate that combination approaches involving these compounds can have therapeutic prospects for TNBC treatment.

Antimalarial artesunate-mefloquine versus praziquantel in African children with schistosomiasis: an open-label, randomized controlled trial.

Schistosomiasis treatment entirely relies on a single drug, praziquantel, prompting research into alternative therapeutics. Here we evaluated the efficacy and safety of the antimalarial combination artesunate-mefloquine for the treatment of schistosomiasis in a proof-of-concept, pragmatic, open-label, randomized controlled trial in primary schools of six villages endemic for schistosomiasis in northern Senegal. Children (6-14 years) were eligible if Schistosoma eggs were detected by microscopy in urine and/or stool. In total, 726 children were randomized 1:1 to praziquantel (standard care: 40 mg kg-1 single dose; n = 364) or to artesunate-mefloquine (antimalarial dosage: artesunate 4 mg kg-1 and mefloquine 8 mg kg-1 daily for three consecutive days; n = 362). Eight children not meeting the inclusion criteria were excluded from efficacy analysis. Median age of the remaining 718 participants was 9 years; 399 (55.6%) were male, and 319 (44.4%) female; 99.3% were infected with Schistosoma haematobium and 15.2% with S. mansoni. Primary outcomes were cure rate, assessed by microscopy, and frequency of drug-related adverse effects of artesunate-mefloquine versus praziquantel at 4 weeks after treatment. Cure rate was 59.6% (208/349) in the artesunate-mefloquine arm versus 62.1% (211/340) in the praziquantel arm. The difference of -2.5% (95% confidence interval (CI) -9.8 to 4.8) met the predefined criteria of noninferiority (margin set at 10%). All drug-related adverse events were mild or moderate, and reported in 28/361 children receiving artesunate-mefloquine (7.8%; 95% CI 5.4 to 11.0) versus 8/363 (2.2%; 95% CI 1.1 to 4.3) receiving praziquantel (P < 0.001). Artesunate-mefloquine at antimalarial dosage was moderately safe and noninferior to standard-care praziquantel for the treatment of schistosomiasis, predominantly due to S. haematobium. Multicentric trials in different populations and epidemiological settings are needed to confirm these findings. ClinicalTrials.gov identifier: NCT03893097 .

Systematic Review and Meta-Analysis of Seasonal Malaria Chemoprevention.

Seasonal malaria chemoprevention (SMC) for children under 5 years of age for up to four monthly cycles during malaria transmission season was recommended by the WHO in 2012 and has been implemented in 13 countries in the Sahel, reaching more than 30 million children annually. Malaria control programs implementing SMC have asked the WHO to consider expanding the age range or number of monthly cycles. We conducted a systematic review and meta-analysis of SMC among children up to 15 years of age and up to six monthly cycles. Twelve randomized studies were included, with outcomes stratified by age (< 5/≥ 5 years), by three or four versus five or six cycles, and by drug where possible. Drug regimens included sulfadoxine-pyrimethamine + amodiaquine, amodiaquine-artesunate, and sulfadoxine-pyrimethamine + artesunate. Included studies were all conducted in Sahelian countries in which high-grade resistance to sulfadoxine-pyrimethamine was rare and in zones with parasite prevalence ranging from 1% to 79%. Seasonal malaria chemoprevention resulted in substantial reductions in uncomplicated malaria incidence measured during that transmission season (rate ratio: 0.27, 95% CI: 0.25-0.29 among children < 5 years; rate ratio: 0.27, 95% CI: 0.25-0.30 among children ≥ 5 years) and in the prevalence of malaria parasitemia measured within 4-6 weeks from the final SMC cycle (risk ratio: 0.38, 95% CI: 0.34-0.43 among children < 5 years; risk ratio: 0.23, 95% CI: 0.11-0.48 among children ≥ 5 years). In high-transmission zones, SMC resulted in a moderately reduced risk of any anemia (risk ratio: 0.77, 95% CI: 0.72-0.83 among children < 5 years; risk ratio: 0.70, 95% CI: 0.52-0.95 among children ≥ 5 years [one study]). Children < 10 years of age had a moderate reduction in severe malaria (risk ratio: 0.53, 95% CI: 0.37-0.76) but no evidence of a mortality reduction. The evidence suggests that in areas in which sulfadoxine-pyrimethamine and amodiaquine remained efficacious, SMC effectively reduced malaria disease burden among children both < 5 and ≥ 5 years old and that the number of cycles should be commensurate with the length of the transmission season, up to six cycles.

Artesunate Sensitizes human hepatocellular carcinoma to sorafenib via exacerbating AFAP1L2-SRC-FUNDC1 axis-dependent mitophagy.

Sorafenib is the most widely used first-line drug for the treatment of the advanced hepatocellular carcinoma (HCC). Unfortunately, sorafenib resistance often limits its therapeutic efficacy. To evaluate the efficacy of artesunate against sorafenib-resistant HCC and to investigate its underlying pharmacological mechanisms, a "sorafenib resistance related gene-ART candidate target" interaction network was constructed, and a signaling axis consisting with artesunate candidate target AFAP1L2 and sorafenib target SRC, and the downstream FUNDC1-dependent mitophagy was identified as a major contributor to the sorafenib resistance and a potential way of artesunate to mitigate resistance. Notably, our clinical data demonstrated that AFAP1L2 expression in HCC tissues was markedly higher than that in adjacent non-cancerous liver tissues (P < 0.05), and high AFAP1L2 expression was also significantly associated with an unfavorable overall survival of HCC patients (P < 0.05). Experimentally, AFAP1L2 was overexpressed in sorafenib resistant cells, leading to the activation of downstream SRC-FUNDC1 signaling axis, further blocking the FUNDC1 recruitment of LC3B to mitochondria and inhibiting the activation of mitophagy, based on both in vitro and in vivo systems. Moreover, artesunate significantly enhanced the inhibitory effects of sorafenib on resistant cells and tumors by inducing excessive mitophagy. Mechanically, artesunate reduced the expression of AFAP1L2 protein, suppressed the phosphorylation levels of SRC and FUNDC1 proteins, promoted the FUNDC1 recruitment of massive LC3B to mitochondria, and further overactivated the mitophagy and subsequent cell apoptosis of sorafenib resistant cells. In conclusion, artesunate may be a promising strategy to mitigate sorafenib resistance in HCC via exacerbating AFAP1L2-SRC-FUNDC1 axis-dependent mitophagy.Abbreviations: AFAP1L2, actin filament associated protein 1 like 2; ANOVA, analysis of variance; ANXA5, annexin V; ART: artesunate; CETSA, cellular thermal shift assay; CI: combination index; CO-IP: co-immunoprecipitation; CQ: chloroquine; CT, computed tomography; [18F]-FDG, fluoro-2-D-deoxyglucose F18; FUNDC1: FUN14 domain containing 1; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HCC, hepatocellular carcinoma; H&E Staining: hematoxylin - eosin staining; HepG2R, sorafenib resistant HepG2; IF, immunofluorescence; IHC, immunohistochemistry; LAMP1: lysosomal associated membrane protein 1; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; miR, microRNA; mRNA: messenger RNA; OE, overexpression; OS, overall survival; PET, positron emission tomography; qRT-PCR: quantitative real-time PCR; sh, short hairpin; shNC: negative control shRNA; shAFAP1L2: short hairpin AFAP1L2; SORA, sorafenib; SPR, surface plasmon resonance; SRC, SRC proto-oncogene, non-receptor tyrosine kinase; SUV, standardized uptake value; TEM, transmission electron microscopy; TOMM20: translocase of outer mitochondrial membrane 20.

Chitosan-coated artesunate protects against ulcerative colitis via STAT6-mediated macrophage M2 polarization and intestinal barrier protection.

Oral delivery of chitosan-coated artesunate (CPA) has been proven to be effective at preventing ulcerative colitis (UC) in mice. However, the anti-inflammatory mechanism is not fully understood. STAT6 is a key transcription factor that promotes anti-inflammatory effects by inducing M2 and Th2 dominant phenotypes, therefore we hypothesized STAT6 might play a key role in the process. To prove it, a STAT6 gene knockout macrophage cell line (STAT6-/- RAW264.7, by CRISPR/Cas9 method), and its corresponding Caco-2/RAW264.7 co-culture system combined with the STAT6 inhibitor (AS1517499, AS) in a mouse UC model were established and studied. The results showed that CPA remarkably suppressed the activation of TLR-4/NF-κB pathway and the mRNA levels of proinflammatory cytokines, while increased the IL-10 levels in RAW264.7. This effect of CPA contributed to the protection of the ZO-1 in Caco-2 which was disrupted upon the stimulation to macrophages. Simultaneously, CPA reduced the expression of CD86 but increase the expression of CD206 and p-STAT6 in LPS-stimulated RAW264.7 cells. However, above alterations were not obvious as in STAT6-/- RAW264.7 and its co-culture system, suggesting STAT6 plays a key role. Furthermore, CPA treatment significantly inhibited TLR-4/NF-κB activation, intestinal macrophage M1 polarization and mucosal barrier injury induced by DSS while promoted STAT6 phosphorylation in the UC mouse model, but this effect was also prominently counteracted by AS. Therefore, our data indicate that STAT6 is a major regulator in the balance of M1/M2 polarization, intestinal barrier integrity and then anti-colitis effects of CPA. These findings broaden our understanding of how CPA fights against UC and imply an alternative treatment strategy for UC via this pathway.