Concentrations Of Dihydroartemisinin Research Articles

Micromolar Dihydroartemisinin Concentrations Elicit Lipoperoxidation in Plasmodium falciparum-Infected Erythrocytes.

Malaria is still the most important parasitic infectious disease. Numerous substances are known to have antimalarial activity; among them, artemisinin is the most widely used one, and artemisinin-based combination therapy (ACT) is recommended for the treatment of Plasmodium falciparum (P.f.) malaria. Antitumor, immunomodulatory, and other therapeutic applications of artemisinin are under extensive study. Several different mechanisms of action were proposed for dihydroartemisinin (DHA), the active metabolite of artemisinin, such as eliciting oxidative stress in target cells. The goal of this study is to monitor the generation of reactive oxygen species (ROS) and lipid peroxidation product 4-hydroxynonenal (4-HNE) by DHA in P.f.-infected human erythrocytes. Checking ROS and 4-HNE-protein adducts kinetics along the maturation of the parasite, we detected the highest level of 4-HNE in ring forms of P.f. due to DHA treatment. Low micromolar concentrations of DHA quickly induced levels of 4-HNE-adducts which are supposed to be damaging. Mass spectrometry identified the P.f. protein cysteine proteinase falcipain-1 as being heavily modified by 4-HNE, and plausibly, 4-HNE conjugation with vital P.f. proteins might contribute to DHA-elicited parasite death. In conclusion, significant 4-HNE accumulation was detectable after DHA treatment, though, at concentrations well above pharmacologically effective ranges in malaria treatment, but at concentrations described for antitumor activity. Thus, lipid peroxidation with consequent 4-HNE conjugation of functionally relevant proteins might be considered as a uniform mechanism for how DHA potentiates antimalarials' action in ACT and controls the progression of tumors.

Estimation of dihydroartemisinin in human plasma using a highly sensitive LTQ Orbitrap mass spectrometer with Xcalibur software.

Background: Artemether (ARM), the O-methyl ether prodrug of dihydroartemisinin (DHA), is considered a first-line antimalarial agent. Artemether is extensively metabolized in vivo to its active metabolite DHA, and therefore its determination offers considerable difficulties. In the present study, DHA identification and estimation were accurately performed by the mass spectrometric analysis, using a high-resolution liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS) LTQ Orbitrap hybrid mass spectrometer. Methods: The plasma samples were taken from healthy volunteers, and the spiked plasma was extracted by adding 1mL of a mixture of dichloromethane and tert.-methyl butyl ether (8:2v/v) to 0.5mL of plasma. The internal standard solution (artemisinin 500ng/mL) was added to the plasma samples. After vertexing and centrifugation, the organic layer was separated and transferred into another tube and dried under nitrogen. The residue was reconstituted in 100μL of acetonitrile and was injected onto the LC-MS system for analysis. Measurement of standards and samples was carried out isocratically on a Surveyor HPLC system combined with an LTQ Orbitrap mass spectrometer using an ACE 5 C18-PFP column. Mobile phase A consisted of 0.1% v/v formic acid in water, Mobile phase B consisted of acetonitrile only, and isocratic elution was carried out with A:B 20:80, v/v. The flow rate was 500μL/min. The ESI interface was operated in a positive ion mode with a spray voltage of 4.5kV. Results: Artemether is not a very biologically stable compound and is immediately metabolized to its active metabolite dihydroartemisinin, so no clear peak was observed for artemether. Both artemether and DHA after ionization undergo neutral losses of methanol and water, respectively, in the source of the mass spectrometer. The ions observed were (MH-H2O) m/z 267.15 for DHA and (MH-m/z 283.15 for internal standard artemisinin. The method was validated according to international guidelines. Discussion: The validated method was applied successfully for the determination and quantification of DHA in plasma samples. This method works well for the extraction of drugs, and the Orbitrap system with the help of Xcalibur software accurately and precisely determines the concentration of DHA in spiked as well as volunteer's plasma.

Transcriptome analysis of Tetrahymena thermophila response to exposure with dihydroartemisinin

Dihydroartemisinin (DHA) is a derivative of artemisinin and is toxic to parasites. We used the Tetrahymena thermophila (T. thermophila) as a model to explore DHA toxicity. Results showed that low concentration of DHA (20 μmol/L) promoted cell proliferation, whereas high concentrations of DHA (40–1280 μmol/L) inhibited that. Appearance of nucleus was pycnosis by laser scanning confocal microscope. DHA significantly elevated activities of SOD and GSH-Px (P < 0.01) and MDA was markedly increased at high level but decreased at low level (P < 0.01). Further results of transcriptome in T. thermophila treated with different concentration DHA group (0, 20, 160 μmol/L) showed that differentially expressed genes (DEGs) were involved in oxidation-reduction and metabolism of exogenous substances indicated oxidative stress stimulation. Kyoto Encyclopedia of Genes and Genomes showed that DEGs were involved in the cytochrome P450-mediated metabolism of exogenous substances, glutathione metabolism and ABC transport. Remarkably, DNA replication was significantly enriched in low concentration DHA, energy metabolism related pathways and necrotic process were considerably enriched in high concentration DHA. The results of RT-qPCR of 13 DEGs were the same as that of transcriptome, in which the expression of GST and GPx family genes were significantly altered after exposed to high-DHA group. DHA induced oxidative stress damage through disturbing with energy. However, detoxification pathways in T. thermophila to resist oxidative damage and cell alleviated low concentration DHA stress by regulating antioxidant enzyme. This study provides good practice on pharmacological mechanism of artemisinin-based drugs in antiparasitic.

Dihydroartemisinin Affects STAT3/DDA1 Signaling Pathway and Reverses Breast Cancer Resistance to Cisplatin.

Dihydroartemisinin (DHA) has anticancer effects on multiple tumors, including those associated with breast cancer. This study aimed to investigate the mechanism causing DHA-reversing cisplatin (DDP) resistance in breast cancer. Relative mRNA and protein levels were tested using a qRT-PCR and western blot assay. Cell proliferation, viability, and apoptosis were evaluated using colony formation, MTT, and flow cytometry assays, respectively. Interaction of STAT3 and DDA1 was measured via a dual-luciferase reporter assay. The results showed that DDA1 and p-STAT3 levels were dramatically elevated in DDP-resistant cells. DHA treatment repressed proliferation and induced apoptosis of DDP-resistant cells by suppressing STAT3 phosphorylation; the inhibition ability was positively proportional to the DHA concentration. DDA1 knockdown inhibited cyclin expression, promoted G0/G1 phase arrest, restrained cell proliferation, and induced apoptosis of DDP-resistant cells. Furthermore, knockdown of STAT3 restrained proliferation and induced apoptosis and G0/G1 cell cycle arrest of DDP-resistant cells by targeting DDA1. DHA could restrain tumor proliferation of breast cancer via enhancing drug sensitivity of DDP-resistant cells through the STAT3/DDA1 signaling pathway.

Antitumor Effect of Dihydroartemisinin on Diffuse Large B-Cell Lymphoma

To investigate the potential antitumor effect and its mechanism of dihydroartemisinin (DHA) on diffuse large B-cell lymphoma (DLBCL). OCI-Ly7 cells were respectively treated with different concentrations of DHA (0, 12.5, 25, 50 and 100 μmol/L) , CCK-8 was used to detect the cells viability. Subsequently, OCI-Ly7 cells were divided into 5 groups : DHA 0,25,50,100 μmol / L and DHA (100 μmol / L) + Colivelin (STAT3 activator). Aldehyde dehydrogenase (ALDH) positive cells were sorted by flow cytometry, the sphere-forming ability of stem cells was detected. Transwell assay and scratch test were used to analyze the invasion and migration of cells. Western blot was used to detect the expression of migration and invasion-related proteins, as well as the phosphorylation levels of Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3(STAT3). DHA induced obvious cytotoxicity to OCI-Ly7 cells. Compared with the control group, the stem cell-like properties, invasion and migration of OCI-Ly7 were significantly inhibited in DHA 50 μmol/L group and 100 μmol/L group, while the phosphorylation levels of JAK2 and STAT3 were significantly reduced. There was no significant difference in DHA 25 μmol/L group compared with the control group. Treated with Colivelin, the inhibition of DHA on OCI-Ly7 stem cell-like properties, invasion and migration was significantly reversed, and the expression of p-STAT3 was significantly up-regulated. DHA has antitumor effect on DLBCL, and its mechanism may be through inhibiting the activation of JAK2/STAT3 pathway to inhibit the stem cell-like properties, invasion and migration of DLBCL cells.

Anti-proliferation and apoptosis-inducing effects of dihydroartemisinin on SH-SY5Y cells and metabolomic analysis

BackgroundIn childhood, metastatic neuroblastoma (NB) is the most common extracranial solid tumor, but there are no appropriate drugs for its treatment. Dihydroartemisinin (DHA), a drug for malaria treatment, has therapeutic potential in several cancers; however, its mechanisms remain unclear. This study aimed to investigate the anti-proliferation effect of DHA on SH-SY5Y cells and to explore its mechanism in vitro.MethodsWe used 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to measure the half-maximal inhibitory concentration (IC50) of DHA; western blot was used to determine protein levels; propidium iodide (PI) staining was used to determine apoptotic cells; JC-1 staining to measure mitochondrial membrane potential; and dichloro-dihydro-fluorescein diacetate (DCFH-DA) staining was used to determine reactive oxygen species (ROS). Metabonomic analysis was performed by using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS)-based untargeted metabolomics. Multivariate statistical analysis was performed to screen potential metabolites associated with DHA treatment in SH-SY5Y cells.ResultsIt was shown that DHA inhibited SH-SY5Y cell proliferation and increased poly (ADP-ribose) polymerase (PARP-1) and caspase 3 in a dose-dependent manner. In Further, DHA promoted ROS generation and γH2AX expression. In addition, a total of 125 proposed metabolites in SH-SY5Y cells and 45 vital metabolic pathways were identified through UHPLC-MS/MS-based untargeted metabolomic analysis.ConclusionsThese data suggest that DHA could regulate taurine, linoleic acid, phenylalanine metabolism, and tryptophan metabolism, which are involved in the anti-proliferation effect of DHA in SH-SY5Y cells.

Dihydroartemisinin Attenuates Hypoxic Pulmonary Hypertension via the Downregulation of miR-335 Targeting Vangl2.

Dihydroartemisinin (DHA) is a traditional antimalarial drug. DHA plays a crucial role in preventing pulmonary hypertension (PH); however, its regulatory function on microRNAs (miRNAs) in PH remains unclear. This study aimed to investigate whether DHA exerts its protective functions by regulating miR-335 in PH. Hypoxia-induced PH models were induced both in vitro and in vivo. Mice were treated with various concentrations of DHA, and pulmonary arterial smooth muscle cells (PASMCs) were treated with DHA, miR-335 inhibitor, miR-335 mimic, or Van Gogh-like 2 (Vangl2) plasmid. The expression of miR-335 and Vangl2, pulmonary arterial remodeling index; right ventricular hypertrophy index; and proliferation and migration indexes were measured. DHA improved pulmonary vascular remodeling and alleviated PH in vivo. miRNA sequencing and real-time PCR results further show that the increase in hypoxia-induced miR-335 was avoided by DHA administration, and miR-335 increased the hypoxia-induced PASMC proliferation and migration. MiRNA databases and dual-luciferase reporter assay show that miR-335 directly targets Vangl2, and Vangl2 decreased the hypoxia-induced PASMC proliferation and migration. The miR-335 inhibitor failed to inhibit hypoxia-induced proliferation and migration upregulation in Vangl2 knockdown PASMCs, and the effect of DHA can be blocked by miR-335 upregulation. In hypoxic PH, MiR-335 is increased, whereas Vangl2 is decreased. MiR-335 can significantly promote the hypoxia-induced proliferation and migration of PASMCs by targeting the Vangl2 gene. DHA effectively reverses the hypoxia-induced upregulation of miR-335 expression, avoiding the miR-335-mediated downregulation of Vangl2 and thereby promoting the expression of Vangl2 to prevent PH.

Effects of dihydroartemisinin combined with cisplatin on proliferation, apoptosis and migration of HepG2 cells

Cisplatin (DDP) is a potent and widely applied chemotherapeutic agent. However, its clinical efficacy for the treatment of liver cancer is limited by adverse effects and the development of resistance. Combinatorial therapy may alleviate these issues. Dihydroartemisinin (DHA) is a first-generation derivative of artemisinin. The effects of DDP on liver cancer when applied in combination with DHA have not previously been studied. Therefore, the present study aimed to investigate the effects of DHA combined with DDP on HepG2 cells and their potential underlying molecular mechanisms. HepG2 cells were treated with different concentrations of DHA and/or DDP. Cell Counting Kit-8 assay was used to assess the cell viability. Cell proliferation and apoptosis were quantified using flow cytometry, acridine orange/ethidium bromide (AO/EB) fluorescent dual staining and the colony formation assay. Cell migration was quantified using the Transwell and wound healing assays. The HepG2 cell protein expression levels of Fas, Fas-associated death domain (FADD), procaspase-3, cleaved caspase-3, pro-caspase-8, cleaved caspase-8, Bax, Bcl-2, E-cadherin and N-cadherin, were detected via western blotting. Gelatin zymography was used to assess the levels of MMP-9 secreted by HepG2 cells into the supernatant. Following combined DHA and DDP treatment, the percentage of apoptotic cells was significantly increased, whereas cell proliferation and migration were significantly reduced, compared with cells treated with DDP only. DHA and DPP in combination significantly inhibited the expression of MMP-9, significantly increased the protein expression levels of Fas, FADD, Bax and E-cadherin, significantly increased the ratio of cleaved caspase-3 and cleaved caspase-8 to their precursor proteins and significantly decreased the protein expression levels of Bcl-2 and N-cadherin. The findings of the present study suggested that, DHA may confer synergistic effects with DDP in potentially promoting apoptosis and inhibiting the epithelial-mesenchymal transition for the treatment of liver cancer.

RETRACTED ARTICLE: Dihydroartemisinin targets fibroblast growth factor receptor 1 (FGFR1) to inhibit interleukin 17A (IL-17A)-induced hyperproliferation and inflammation of keratinocytes

Statement of Retraction Baojiang Chen, Chen Li, Guizheng Chang, and Huan Wang- 2022, Dihydroartemisinin targets fibroblast growth factor receptor 1 (FGFR1) to inhibit interleukin 17A (IL-17A)-induced hyperproliferation and inflammation of keratinocytes, Bioengineered 13(1), doi: 10.1080/21655979.2021.2021701. Since publication, significant concerns have been raised about the integrity of the data and reported results in the article. When approached for an explanation, the authors did not provide their original data or any necessary supporting information. As verifying the validity of published work is core to the integrity of the scholarly record, we are therefore retracting the article. All authors listed in this publication have been informed. We have been informed in our decision-making by our editorial policies and the COPE guidelines. The retracted article will remain online to maintain the scholarly record, but it will be digitally watermarked on each page as ‘Retracted’.

Validation of a Liquid Chromatography/Tandem Mass Spectrometry Assay for the Quantification of Plasma Dihydroartemisinin

Background: Insufficient plasma level of dihydroartemisinin (DHA) can select resistance and will further hinder malaria elimination program. We investigated clinical applicability of a validated liquid chromatography/tandem mass spectrometry (LC-MS/MS) assay to quantify plasma concentration of DHA in healthy subjects from a single oral administration of fixed dose combination of Dihydroartemisinin-Piperaquine.Materials and Methods: Micro-elution solid-phase extraction in a 96-well plate format was used to prepare the samples. DHA separation happened in Acquity UPLCTM BEH C18 column (50 × 2.1 mm, 1.7 µm). Mobile phase was a mixture of acetonitrile-ammonium acetate 10 mM pH 3.5 (50:50, v/v) at 0.3 mL/minute flow rate. Waters Acquity UPLC™ H-Class system coupled with triple quadruple mass spectrometry in positive electrospray ionization mode was used for detection. The internal standard was a stable isotope labelled DHA.Results: Calibration curve was linear with a correlation coefficient &gt;0.995 over a concentration range of 1–1,000 ng/mL. Bias and variation for accuracy and precision were in the range of 15% (20% at the lower limit of quantification). Using 5 µL sample, lower limit of quantification was 1 ng. Matrix effect was less than 15%. The method was successfully applied to investigate the pharmacokinetics of DHA from five healthy subjects, although carry over and the role of anticoagulants were not tested.Conclusion: The LC-MS/MS assay for the quantification of plasma DHA was validated for selectivity, linearity, lower limit of quantitation, accuracy, precision, matrix effect and stability. Although clinical applicability was demonstrated, this method was to be improved to address the not-tested validation parameters.Keywords: dihydroartemisinin, liquid chromatography/tandem mass spectrometry assay (LC-MS/MS), malaria, Indonesia 

Human Telomerase Reverse Transcriptase as a Therapeutic Target of Dihydroartemisinin for Esophageal Squamous Cancer

Objective: To elucidate the oncogenic role of human telomerase reverse transcriptase (hTERT) in esophageal squamous cancer and unravel the therapeutic role and molecular mechanism of dihydroartemisinin (DHA) by targeting hTERT. Methods: The expression of hTERT in esophageal squamous cancer and the patients prognosis were analyzed by bioinformatic analysis from TCGA database, and further validated with esophageal squamous cancer tissues in our cohort. The Cell Counting Kit-8 (CCK8) and colony formation assay were used to evaluate the proliferation of esophageal squamous cancer cell lines (Eca109, KYSE150, and TE1) after hTERT overexpression or treated with indicated concentrations of DHA. Transwell migration assay and scratch assay were employed to determine the migration abilities of cancer cells. Fluorescence microscopy and flow cytometry were conducted to measure the intracellular reactive oxygen species (ROS) levels in cancer cells after treated with DHA. Moreover, RT-PCR and Western blot were performed to test the alteration of associated genes on mRNA and protein level in DHA treated esophageal squamous cancer cell lines, respectively. Furthermore, tumor-bearing nude mice were employed to evaluate the anticancer effect of DHA in vivo. Results: We found that hTERT was significantly upregulated in esophageal squamous cancer both from TCGA database and our cohort also. Overexpression of hTERT evidently promoted the proliferation and migration of esophageal squamous cancer cells in vitro. Moreover, DHA could significantly inhibit the proliferation and migration of esophageal cancer cell lines Eca109, KYSE150, and TE1 in vitro, and significantly down-regulate the expression of hTERT on both mRNA and protein level in a time- and dose-dependent manner as well. Further studies showed that DHA could induce intracellular ROS production in esophageal cancer cells and down-regulate SP1 expression, a transcription factor that bound to the promoter region of hTERT gene. Moreover, overexpression of SP1 evidently promoted the proliferation and migration of Eca109 and TE1 cells. Intriguingly, rescue experiments showed that inhibiting ROS by NAC alleviated the downregulation of SP1 and hTERT in cells treated with DHA. Furthermore, overexpression of SP1 or hTERT could attenuate the inhibition effect of DHA on the proliferation and migration of Eca109 cells. In tumor-bearing nude mice model, DHA significantly inhibited the growth of esophageal squamous cancer xenografts, and downregulated the expression of SP1 and hTERT protein, while no side effects were observed from heart, kidney, liver, and lung tissues by HE stain. Conclusion: hTERT plays an oncogenic role in esophageal squamous cancer and might be a therapeutic target of DHA through regulating ROS/SP1 pathway.

Dihydroartemisinin triggers ferroptosis in primary liver cancer cells by promoting and unfolded protein response‑induced upregulation of CHAC1 expression.

Dihydroartemisinin (DHA), an artemisinin derivate, has been investigated as a potential antitumor drug in primary liver cancer (PLC). Ferroptosis is a form of iron-dependent cell death that can be driven by lipid peroxidation inducers. The present study aimed to determine whether and how DHA could promote the death of PLC cells by inducing ferroptosis. In total, four PLC cell lines with different p53 statuses, including Hep3B (p53 null), Huh7 (p53 mutant), PLC/PRF/5 (p53 mutant) and HepG2 (p53 wild-type), were treated with various concentrations of DHA. The effects of DHA on all three branches of the unfolded protein response (UPR) were evaluated. To deactivate the UPRs, small interfering RNA was used to knockdown the expression of activating transcription factor (ATF)4, X-box binding protein 1 (XBP1) or ATF6 in PLC cells. The effect of DHA on the promoter activity of Chac glutathione specific γ-glutamylcyclotransferase 1 (CHAC1) was evaluated using a dual luciferase reporter assay. The results revealed that DHA-induced death in PLC cells was irrelevant of the p53 status. PLC cells exposed to DHA displayed classic features of ferroptosis, such as increased lipid reactive oxygen species and malondialdehyde levels, an iron overload, and decreased activity or expression of glutathione (GSH), glutathione peroxidase 4, solute carrier family (SLC) 7 member 11 and SLC family 3 member 2. The antitumor effects of DHA in PLC cells were significantly weakened by two typical ferroptosis inhibitors, ferrostatin-1 and deferoxamine mesylate salt, whereas the antitumor effects were augmented following iron overload. Furthermore, DHA activated all three branches of the UPR (eukaryotic translation initiation factor 2 α kinase 3/eukaryotic translation initiation factor 2A/ATF4, inositol-requiring transmembrane kinase/endoribonuclease 1α/XBP1 and ATF6 branches) in vitro. Notably, DHA-induced ferroptosis was significantly attenuated following the knockdown of ATF4, XBP1 or ATF6 expression. In addition, the promoter activity of CHAC1, a gene capable of degrading GSH, was enhanced by DHA, but weakened when the aforementioned three UPR transcription factors were knocked down. In conclusion, the findings of the present study suggested that DHA may effectively induce ferroptosis in PLC cells through the activation of anti-survival UPRs and the upregulation of CHAC1 expression.

Dihydroartemisinin suppresses proliferation, migration, the Wnt/β-catenin pathway and EMT via TNKS in gastric cancer.

Gastric cancer is a common malignancy worldwide. However, the molecular mechanisms underlying this malignancy remain unclear and there are a lack of effective drugs. The present study aimed to investigate the antitumor effect of Dihydroartemisinin (DHA) or inhibition of Tankyrases (TNKS), and determine the underlying molecular mechanisms of gastric cancer. Immunohistochemistry and immunofluorescence analyses were performed to detect the expression levels of TNKS, epithelial-to-mesenchymal transition (EMT) and Wnt/β-catenin pathway-related proteins in gastric cancer tissues and adjacent normal tissues. The Cell Counting Kit-8 assay was performed to assess the viability of HGC-27 and AGS cells following treatment with different concentrations of HLY78 (a Wnt activator) or DHA. Following treatment with HLY78, DHA or small interfering (si)-TNKS1/si-TNKS2, colony formation and migratory abilities were assessed via the colony formation, wound healing and Transwell assays. Furthermore, western blot and immunofluorescence analyses were performed to detect the expression levels of TNKS, EMT- and Wnt/β-catenin-related proteins. The results demonstrated that the expression levels of TNKS, AXI2, β-catenin, N-cadherin and Vimentin were upregulated, whereas E-cadherin expression was downregulated in gastric cancer tissues compared with normal tissues. Furthermore, HLY78 and DHA suppressed the viability of HGC-27 and AGS cells, in a concentration-independent manner. Notably, TNKS knockdown or treatment with DHA suppressed colony formation, migration, TNKS expression, EMT and the Wnt/β-catenin pathway. Opposing effects were observed following treatment with HLY78, which were ameliorated following co-treatment with DHA. Taken together, these results suggest that DHA or inhibition of TNKS can suppress the proliferation and migration of gastric cancer cells, which is partly associated with inactivation of the Wnt/β-catenin pathway and EMT process.

Pharmacokinetics and antimalarial activities of reduction-responsive releasing dihydroartemisinin prodrug self-assembled nanoparticles in rodents

Artemisinin and its derivates (ARTs) exert antimalarial effect by virtue of unique endoperoxide bridge, which is generally considered to generate oxygen free radicals and then form carbon-centered free radicals to damage parasites. The known high level of glutathione (GSH) in parasites plays a key role in maintaining redox balance to protect parasites from oxidative stress damage, which may be a risk factor for the failure of ARTs-based combination therapy and drug resistance. Nevertheless, the highly reducing environment in cytoplasm of malaria parasites provides an available trigger for intracellular nanoparticle disassembly to responsively release drug. In this study, a reduction-responsive releasing dihydroartemisinin (DHA) prodrug (C14-SS-DHA) was synthesized by conjugating DHA and tetradecylamine (C14–NH2) with disulfide bond (–SS–) as a linker. As a control, a DHA carbon-carbon bond (–CC–) prodrug (C14-CC-DHA) was synthesized using succinic anhydride as a –CC– linker. To note, DHA prodrugs were prepared as self-assembled nanoparticles (C14-SS-DHA NPs and C14-CC-DHA NPs). The size and zeta potential of C14-SS-DHA NPs were 121 ± 1.582 nm and −29 ± 0.635 mV, and those of C14-CC-DHA NPs were 137.5 ± 0.5132 nm and −41 ± 0.377 mV, respectively. The C14-SS-DHA NPs can release DHA in a reductive media in vitro. The pharmacokinetics study showed that the area under the curve (AUC(0-t)) of plasma concentration versus time of DHA from C14-SS-DHA NPs was 20737.2 ± 6028.3 h μg L−1, which was much higher than that of C14-CC-DHA NPs (6254.5 ± 2578.8) or DHA solution (857.6 ± 103.7 h μg L−1). The pharmacodynamics study presented that the ED90 of C14-SS-DHA NPs was 3.81 ± 0.35 μmol/kg, which was lower than that of C14-CC-DHA NPs (12.70 ± 1.63 μmol/kg)) or DHA solution (17.67 ± 3.38 μmol/kg). C14-SS-DHA NPs showed significantly higher systemic exposure and better antimalarial activity in comparison with C14-CC-DHA NPs or DHA solution (P < 0.05).

Pharmacokinetic Study of Rectal Artesunate in Children with Severe Malaria in Africa.

When severe malaria is suspected in children, the WHO recommends pretreatment with a single rectal dose of artesunate before referral to an appropriate facility. This was an individually randomized, open-label, 2-arm, crossover clinical trial in 82 Congolese children with severe falciparum malaria to characterize the pharmacokinetics of rectal artesunate. At admission, children received a single dose of rectal artesunate (10 mg/kg of body weight) followed 12 h later by intravenous artesunate (2.4 mg/kg) or the reverse order. All children also received standard doses of intravenous quinine. Artesunate and dihydroartemisinin were measured at 11 fixed intervals, following 0- and 12-h drug administrations. Clinical, laboratory, and parasitological parameters were measured. After rectal artesunate, artesunate and dihydroartemisinin showed large interindividual variability (peak concentrations of dihydroartemisinin ranged from 5.63 to 8,090 nM). The majority of patients, however, reached previously suggested in vivo IC50 and IC90 values (98.7% and 92.5%, respectively) of combined concentrations of artesunate and dihydroartemisinin between 15 and 30 min after drug administration. The median (interquartile range [IQR]) time above IC50 and IC90 was 5.68 h (2.90 to 6.08) and 2.74 h (1.52 to 3.75), respectively. The absolute rectal bioavailability (IQR) was 25.6% (11.7 to 54.5) for artesunate and 19.8% (10.3 to 35.3) for dihydroartemisinin. The initial 12-h parasite reduction ratio was comparable between rectal and intravenous artesunate: median (IQR), 84.3% (50.0 to 95.4) versus 69.2% (45.7 to 93.6), respectively (P = 0.49). Despite large interindividual variability, rectal artesunate can initiate and sustain rapid parasiticidal activity in most children with severe falciparum malaria while they are transferred to a facility where parenteral artesunate is available. (This study has been registered at ClinicalTrials.gov under identifier NCT02492178.).

Application of LC-MS/MS method for determination of dihydroartemisin in human plasma in a pharmacokinetic study.

Aim: Dihydroartemisinin (DHA) was also found therapeutic potential for the treatment of systemic lupus erythematosus (SLE). To assess the pharmacokinetic profile of DHA, the concentration of DHA in plasma of SLE patients needed be accurately determined based on a rapid and reliable analytical method. Experimental method & results: Developed method utilizes stable isotope-labeled internal standards and SPEmethod for sample preparation, applied XBridge C18 column (2.1×50mm, 3.5μm) for chromatography separation. Detection of the analytes was achieved by an AB Sciex 4000 mass spectrometer under positive electrospray ionization mode. The method was validated in accordance with international guidelines on bioanalytical methods validations. Conclusion: DHA concentrations in human plasma of Chinese SLEpatients were quantified by developed LC-MS/MS (no. 2016L02562).

Antibacterial Photodynamic Treatment of Porphyromonas gingivalis with Toluidine Blue O and a NonLaser Red Light Source Enhanced by Dihydroartemisinin.

In vitro experiments confirmed that antibacterial photodynamic treatment (aPDT) inactivates periodontal pathogens. However, more effective sterilization is needed in the complex oral environment. This study tested whether dihydroartemisinin (DHA) enhanced the photokilling effect of aPDT on Porphyromonas gingivalis (P.gingivalis) in planktonic and biofilm states. aPDT combining toluidine blue O (TBO) with 630nm red light was performed on bacterial suspensions and biofilms in vitro with different final concentrations of DHA (10, 20 and 40μgmL-1 ). The sensitization mechanism was preliminarily investigated by uptake experiments. The above experiments were repeated with different incubation times (30, 60, 120s). Porphyromonas gingivalis biofilms exhibited significantly higher resistance to aPDT than P.gingivalis in suspension under the same experimental parameters. DHA alone had no cytotoxic effect on P.gingivalis with or without light irradiation. In either bacterial suspensions or biofilms, DHA concentration-dependently enhanced the photokilling effect of aPDT and increased TBO uptake by P.gingivalis. Prolonged incubation time enhanced the photokilling efficiency of aPDT until cellular TBO uptake reached saturation. DHA can enhance aPDT activity against P.gingivalis in planktonic and biofilm states. DHA also accelerated TBO uptake, reducing incubation time.

Dihydroartemisinin induces ER stress-dependent apoptosis of Echinococcus protoscoleces in vitro.

In this study, we investigated the effect of dihydroartemisinin on Echinococcus protoscoleces and explored the role of endoplasmic reticulum stress in this process. Echinococcus protoscoleces were collected and cultured in RPMI 1640 medium. Changes in the expressions of glucose-regulated protein 78 (GRP-78), caspase-12, and C/EBP homologous protein (CHOP) were assessed through confocal immunofluorescence and western blot analysis. Cell viability and morphological changes were observed under a light microscope. The ultrastructure of protoscoleces was observed by scanning electron microscopy and transmission electron microscopy. Caspase-3 activity was detected using an enzyme assay kit. After dihydroartemisinin treatment, the protoscoleces showed loss of viability, and morphological changes including soma contraction, blebs formation, hooks loss, microtrichia destruction, and development of lipid droplets was observed. The levels of caspase-12 and CHOP were increased within 2 days of dihydroartemisinin treatment. However, the levels of GRP-78, caspase-12, and CHOP were decreased in 4 days. Furthermore, caspase-3 activity was increased after treatment with different concentrations of dihydroartemisinin. Dihydroartemisinin can induce apoptosis in protoscoleces via the ER stress-caspase-3 apoptotic pathway in vitro. These results indicate that dihydroartemisinin is a potentially valuable therapeutic agent against echinococcosis.

Genetic screens reveal a central role for heme metabolism in artemisinin susceptibility

Artemisinins have revolutionized the treatment of Plasmodium falciparum malaria; however, resistance threatens to undermine global control efforts. To broadly explore artemisinin susceptibility in apicomplexan parasites, we employ genome-scale CRISPR screens recently developed for Toxoplasma gondii to discover sensitizing and desensitizing mutations. Using a sublethal concentration of dihydroartemisinin (DHA), we uncover the putative transporter Tmem14c whose disruption increases DHA susceptibility. Screens performed under high doses of DHA provide evidence that mitochondrial metabolism can modulate resistance. We show that disrupting a top candidate from the screens, the mitochondrial protease DegP2, lowers porphyrin levels and decreases DHA susceptibility, without significantly altering parasite fitness in culture. Deleting the homologous gene in P. falciparum, PfDegP, similarly lowers heme levels and DHA susceptibility. These results expose the vulnerability of heme metabolism to genetic perturbations that can lead to increased survival in the presence of DHA.

Effects and Mechanisms of Anlotinib and Dihydroartemisinin Combination Therapy in Ameliorating Malignant Biological Behavior of Gastric Cancer Cells.

Gastric Cancer (GC) is currently one of the major malignancies that threaten human lives and health. Anlotinib is a novel small-molecule that inhibits angiogenesis to exert antitumor effects. However, its function in gastric cancer is incompletely understood. The aim of the present study was to investigate the anti-tumor effects and molecular mechanisms of anlotinib combined with Dihydroartemisinin (DHA) in SGC7901 gastric cancer cells. Different concentrations of anlotinib and DHA were used to treat SGC7901 gastric cancer cells, after which cell proliferation was measured. Drug interactions of anlotinib and DHA were analyzed by the Chou-Talalay method with CompuSyn software. Proliferation, apoptosis, invasion, migration, and angiogenesis were measured using the Cell Counting Kit-8 (CCK8) assay, flow cytometry, Transwell invasion assays, scratch assays, and chicken Chorioallantoic Membrane (CAM) assays. Proliferation- associated protein (Ki67), apoptosis-related protein (Bcl-2), and Vascular Endothelial Growth Factor A (VEGF-A) were quantified by Western blotting. The combination of 2.5 μmol/L of anlotinib and 5 of μmol/L DHA was highly synergistic in inhibiting cell growth, significantly increased the apoptosis rate and suppressed obviously the invasion and migration capability and angiogenesis of gastric cancer cells. In addition, the expression levels of Ki67, Bcl-2, and VEGF-A, as well as angiogenesis, were significantly decreased in the Combination of drugs compared with control and either drug alone. The combination of anlotinib and DHA showed synergistic antitumor activity, suggesting their potential in treating patients with gastric cancer.