Anti-malarial; STAT-3; exported protein 1 (EXP1).

Artesunate inhibits exportin 1 (EXP1)[2] and STAT-3[1]. Both cell lines showed a notable dose-dependent rise in reactive oxygen species (ROS) following a 24-hour treatment with artesunate. Furthermore, artesunate treatment of cancer cells at greater doses for 24 hours was found to considerably raise γ-H2AX levels, as demonstrated by Western blotting. Additionally, in A2780 and HO8910 cells, artesunate exhibited time-dependent impacts on RAD51 levels. Two types of non-malignant cells (normal human fibroblasts and immortalized epithelial cells FTE-187) showed no change in RAD51 levels in response to artesunate. In fact, artesunate decreased RAD51 mRNA levels in A2780 cells in a dose-dependent way. Correspondingly, artesunate markedly reduced the promoter activity of RAD51. In contrast, artesunate had no effect on the amounts of RAD51 mRNA in H8910 cells [3].

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Our virtual analysis of the STL candidates revealed Artesunate (ATS) as the best potential inhibitor of STAT-3 with comparable potency to specific inhibitor S3I-201. We also observed that ATS inhibited IL-6 driven STAT-3-DNA binding activity with comparable potency to S3I-201 in a cell free system. Furthermore ATS was observed to interfere with STAT-3 dimerization and suppression of both constitutive and IL-6 inducible STAT-3 in vitro. Nevertheless, we also observed that ATS modulated STAT-3 dependent targets (procaspase-3, Bcl-xl and survivin) favoring occurrence of apoptosis in vitro. Overall, the putative inhibition of STAT-3 by ATS suggested its capacity to interfere with STAT-3 dimerization by binding to the SH2 domain of STAT-3 monomer. It resulted in suppression of STAT-3 and also favored promotion of in vitro cells towards apoptosis. Consequently, ATS also exhibited selective cytotoxicity of cancer cells over normal cells in vitro.[1]

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EXP1 efficiently degrades cytotoxic hematin, is potently inhibited by artesunate, and is associated with artesunate metabolism and susceptibility in drug-pressured malaria parasites. These data implicate EXP1 in the mode of action of a frontline antimalarial drug.[2]

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Artesunate induces ROS and DNA double-strand breaks in ovarian cancer cells. Artesunate downregulates RAD51 in ovarian cancer cells. Artesunate inhibits the formation of RAD51 foci and HRR. Artesunate sensitizes ovarian cancer cells to cisplatin. Ectopic expression of RAD51 attenuates the increased chemosensitivity conferred by artesunate. [3]

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Artesunate has potent anti-lymphoma activity in vitro [4]
To characterize the efficacy of artesunate, inhibitors currently in clinical testing for treatment of B-cell lymphoma were compared with artesunate in a drug sensitivity screen. Artesunate was identified as a promising candidate, due to its potent reduction of cell growth across all B lymphoma cell lines tested, which contrasted more variable efficacy of the other drugs (Fig. 1). To broaden the testing of artesunate, a dose-response experiment was performed in 18 different B-cell lymphoma cell lines, representing various histological types. This revealed that artesunate exhibited broad activity across the lymphoma cell lines and potently affected cell growth, whereas it had limited effect in normal B-cells activated with CD40L alone or in combination with IL21(Fig. 2a). Artesunate reduced cell growth with IC50 values from 0.189 to 3.72 μM, and a high sensitivity to artesunate (IC50 < 1 μM) was observed in 11 out of 18 cell lines tested (Fig. 2a, Additional file 2: Figure S1). A pronounced cell death was detected with PI staining after 72 h, with only minor effects in normal B-cells (Additional file 2: Figure S1B). Detection of DNA fragmentation by TUNEL assay demonstrated potent artesunate-induced apoptosis with percentage apoptotic cells ranging from 55 to 96 after 72 h exposure to artesunate, whereas normal B-cells were not affected (Fig. 2b). Induction of apoptosis was an early event, with prominent increase of active caspase 3+ cells after 24 h of artesunate treatment (Additional file 2: Figure S1C). The increase of active caspase 3+ cells by artesunate was counteracted by the presence of the pan caspase inhibitor Z-VAD-FMK (Fig. 2c), indicating that artesunate induces apoptosis in tumor cells via a caspase-dependent pathway. Cell cycle analysis did not reveal any overall changes induced by artesunate (Additional file 3: Figure S2). Together, these results indicated that induction of apoptosis represents the main mechanism of the anti-lymphoma activity of artesunate.

In the group receiving combined treatment with artesunate and cisplatin, tumor development was considerably inhibited (P<0.01). On the other hand, neither cell line's tumor xenografts grew significantly when artesunate was used alone [3].

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Artesunate in combination with cisplatin inhibits ovarian cancer xenograft tumor growth [3]
We next tested the effects of artesunate and cisplatin, administered alone or in combination, on the growth of ovarian cancer cells in vivo. A2780 and HO8910 cells, 5×106 cells/ in 0.2 ml PBS, were injected subcutaneously into the left inguinal area of the female nude mice. Tumor-bearing mice were randomly divided into 4 groups and were administered daily via i.p. injection of artesunate (50 mg/kg), cisplatin (2 mg/kg), or in combination of the 2 for 16 days. As shown in Figure 6 , tumor growth was significantly reduced in the group receiving combined treatment of artesunate and cisplatin (P < 0.01). In comparison, artesunate alone had no significant effect on the growth of tumor xenografts for both cell lines. Cisplatin alone appeared to have some inhibitory effect only on HO8910 cells, but not on A2780 cells. These results indicate that artesunate can render ovarian cancer cells sensitive to cisplatin in vivo.

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Artesunate has potent anti-lymphoma activity in a lymphoma xenograft model [4]
To test the efficacy of artesunate as a potent anti-lymphoma drug in vivo, NSG mice were subcutaneously inoculated with 2 × 106 BL-41-luc lymphoma cells and subsequently treated with artesunate (200 mg/kg/day, n = 10) or left untreated (control, n = 10). Mice with varying tumor loads were evenly distributed among the groups (Additional file 4: Figure S3A and B), and treatment was initiated at day 4 after inoculation. Tumor growth was significantly reduced in mice treated with artesunate, and statistically significant differences were reached at day 12 and day 16 after start of treatment (P = .0045, day 12 and P < .0050, day 16; Fig. 3a). IVIS images of the mice at day 16 showed a distinct reduction in tumor growth in the artesunate-treated mice compared to the control group (Fig. 3b). In one of the artesunate-treated mice, the tumor was undetectable (Fig. 3b). At day 16, half of the control mice were euthanized due to maximum tumor size limitation (2 cm3). In comparison, the first artesunate-treated mouse was euthanized due to its tumor size 6 days later. All remaining mice were monitored after end of treatment (day 19), and a delayed tumor growth was observed in artesunate-treated mice. Survival analysis showed that the median time to reach maximum tumor size criteria was 17.5 versus 30.5 days for control and artesunate-treated mice, respectively (p < .0001; Fig. 3c). The artesunate dose (200 mg/kg/day) was well tolerated with no body weight loss during treatment (Additional file 4: Figure S3C). Taken together, artesunate showed potent anti-tumor response in an aggressive B-cell lymphoma xenograft model.

EXP1 artesunate inhibition assay [2]
Artesunate(ART) and atovaquone were dissolved in 100% ethanol was added to 100 nM WT EXP1 hematin reactions to determine hematin degradation inhibition. WT EXP1 was pre-incubated in GST assay buffer pH 6.5 with 0.1% Triton X-100 and ART on ice for 30 minutes followed by the addition of 2 mM GSH and hematin. The reaction was monitored at 395 nm, every second for 3 minutes.

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EXP1 GST enzyme assay [2]
Purified EXP1 was pre-incubated at pH 6.5 with 0.1% Triton X-100 and 1 mM reduced GSH on ice for 90 minutes, followed by the addition CDNB. Absorbance of the reaction was monitored at 340 nm, every 15 seconds for a period of 10 minutes. GST activity towards hematin was assayed by pre-incubating 100 nM WT protein with 0.1% Triton X-100, 2 mM GSH in pH 6.5 assay buffer for 30 minutes on ice followed by the addition of hematin to initiate hematin degradation. Absorbance was monitored at 395 nm every second for 3 minutes.

Generation of DHA/ART-tolerant parasites [2]
3b1 parasites were selected from the Dd2 strain by culturing asexual blood stage parasites (∼108 on average) in the presence of the artemisinin derivative dihydroartemisinin (DHA) at sub-IC50 concentrations (2.8 nM, as compared to a parental IC50 value of 6.4 nM) for 55 days, followed by progressive increases in drug concentration (up to 28 nM) over the next 200 days. Acquired tolerance to DHA and to Artesunate (ART) was evidenced as an increased frequency of recrudescence over a 30-day period following parasite exposure to high concentrations of DHA/ART for four days (up to 112 nM; Eastman et al., manuscript in preparation).

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Flow cytometric analysis of ROS [3]
The ROS production following Artesunate treatment was determined by membrane permeable CM-H2DCFDA. Briefly, cells were loaded with 5 μM of CM-H2DCFDA and incubated at 37°C for 20 min after treatment with artesunate. Cells were resuspended using preserving fluid and analyzed with a FACSCanto II (Becton Dickinson). The peak excitation wavelength for oxidized CM-H2DCFDA was 490 nm and emission was 530 nm.

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Immunofluorescence staining of γ-H2AX and RAD51 [3]
Cells grown on coverslips in 6-well plates were treated with Artesunate for 24 h before they were processed for determination of the level of γ-H2AX. For the immunofluorescence staining of RAD51, cells were pre-treated with 10 μg/ml Artesunate for 24 h, and then 30 μM cisplatin for 4 h to induce RAD51 foci. Cells were fixed with 4% formaldehyde, followed by treatment with 0.2% Triton X-100 in PBS for 5 minutes, then blocked with 5% bovine serum albumin in PBS containing 0.3% Triton X-100 for 30 minutes. Mouse anti-γ-H2AX antibody was used at a dilution of 1:600 in 5% bovine serum albumin in PBS. Rabbit anti-Rad51 antibody was used at a dilution of 1:600. The specimens were incubated overnight at 4°C. Cells were then washed thrice in PBS before incubating in the dark with a Rhodamine-labeled secondary antibody for 60 minutes. After washing with PBS containing 0.3% Triton X-100 for 3 times, cells were then counterstained with 4,6-diamidino-2-phenylindole for 5 minutes. The coverslips were mounted to slides with an antifade solution. Slides were then examined under a fluorescence microscope. At least 500 nuclei were scored for nuclear foci in each of 3 experimental repeats.

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Western blot analysis [3]
After treatment with Artesunate for 24 h, cells were harvested and lysed in 1×cell lysis buffer. Total proteins of 15–25 μg were separated by SDS–PAGE and transferred to polyvinylidenedifluoride (PVDF) membranes. Membranes were blocked with 5% non-fat milk for 1-2 h at room temperature and then probed with primary antibodies and incubated at 4°C overnight. After extensive washing with TBS-T, membranes were incubated with appropriate HRP-conjugated secondary antibody for 1 h at room temperature, and then were detected by Western ECL− enhanced luminol reagen.Antibodies against the following proteins were as RAD51, β-actin and His-tag.

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Luciferase reporter assay [3]
Cells were transfected with pGL3-basic reporter plasmids carrying a series of truncated RAD51 promoters using Lipofectamine 2000 (Invitrogen). Cells were incubated with/without Artesunate (10 μg/ml) for 24 h and were then harvested for luciferase assay using the Dual-Luciferase Reporter Assay system with a multilabel counter (Victor 1420; PerkinElmer). The firefly luciferase activity was normalized to Renilla luciferase activity of the pRL-TK reporter (co-transfected internal control). Transfections were performed in 3 independent experiments, and assayed in quadruplicates

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Clonogenic survival assay [3]
Ovarian cells were divided into 4 groups, the Artesunate group (5 μg /ml for 24 h), the cisplatin group (0.3 μM for 24 h), the combination group (a sequential treatment of Artesunate for 24 h and cisplatin for 24 h) and the control group (DMSO). Cells were seeded into 6 cm dishes. 10–12 days later, colonies were fixed with methanol and stained with 1.25% Giemsa for counting. Only those colonies containing at least 50 cells in size were counted.

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HR reporter assay [3]
Plasmids containing HR reporter cassette were kindly provided by Dr. Gorbunova. 13 The reporter cassette consists of 2 mutated copies of GFP. The first copy contains an artificial intron and a deletion of 22 nt and an insertion of 2 I-SceI recognition sites in inverted orientation in the first exon. The second copy contains the first exon but lacks a promoter and a start codon. Upon induction of DSBs by I-SceI, gene conversion events reconstitute a functional GFP gene. The plasmids were linearized by I-SceI restriction enzymes and purified using TIANGEN Universal DNA Purification Kit. Exponentially growing A2780 and HO8910 cells were transfected with 2 μg of the HR reporter constructs, and 0.1 μg of pDsRed-N1 as the internal control. Then, cells were treated with Artesunate for 24h, replaced with fresh medium, and sequentially cultured for 48h. Cells were harvested, resuspended in 0.4 ml of PBS, pH 7.4, and analyzed by FACS. The DNA repair efficiency expressed as GFP+/DsRed+ ratio, which was independent of the transfection conditions.

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Viability and apoptosis assays [4]
Cells were grown in 96-well plates (10,000 cells/well) for 72 h with or without Artesunate (0.125–8 μM) and small molecular inhibitors (Additional file 1: Table S1). The CellTiterGlo Assay was used for measuring relative cell growth (ATP levels), using Modulus microplate reader. Measurements were related to control and reported as relative luminescence units (RLU). Viability was measured by propidium iodide (PI) assay (1 μg/ml) after treatment with Artesunate (5–10 μM) for 72 h. Apoptosis was detected by active caspase-3 assay (Alexa-647-rabbit anti-active caspase-3 clone C92-605) after 24 h treatment with Artesunate (1 and 5 μM) with and without Z-VAD-FMK (1 μl/ml). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was determined by the In Situ Cell Death Detection Kit after 72 h treatment with artesunate (10 μM). For both apoptosis assays, cells were fixed in PFA for 5 min and permeabilized with ice cold methanol. All assays were analyzed using FACS Canto II or LSR II flow cytometer. Flow cytometry data were analyzed using the online Cytobank flow cytometry software (www.cytobank.org) [18]. DL-buthionine sulfoximine (BSO) was used at 50 μM for 18–24 h for reducing the cellular glutathione levels by inhibiting γ-glutamylcysteine synthetase. Glutathione levels were measured by GSH-Glo Glutathione assay (Promega).

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Gene expression profiling [4]
Total RNA was isolated using MiRNeasy after 4 and 12 h treatment with Artesunate (5 μM). The microarray analyses were performed on Illumina’s HumanHT-12 v4 Expression BeadChip platform. The differential gene expression analysis was done using the limma package in R (version 3.3.1). Two technical replicates were run per cell line and time point, and the results were averaged together. The differentially expressed genes were selected based on a log fold change larger than the absolute value of 0.5, and an adjusted p value (FDR) of less than 0.01. The probes were collapsed according to gene symbol, using the annotation file for the Illumina’s HumanHT-12 v4 Expression BeadChip platform. When several probes mapped to the same gene, the probe with lowest log fold change was selected. The pathways and networks most enriched for the differential expressed genes were identified by Ingenuity Pathway Analysis (IPA) software with default settings.

Four to 6 weeks old female athymic nude mice (BALB/c, nu/nu) were purchased from Beijing Experimental Animal Center (Beijing, China). A2780 and HO8910 cells were harvested and resuspended in 0.1 ml of PBS, 5 × 106 cells/0.2 ml were injected subcutaneously into the left inguinal area of the mice. Two weeks later, mice bearing tumors (∼70 mm3 for A2780 and HO8910) were randomly divided into 4 groups. Artesunate was administered daily via i.p. injection at doses of 50 mg/kg alone or in combination with cisplatin (2 mg/kg) for 16 days. The tumor growth was monitored every other day. Tumor volume was determined by the formula 1/2a × b2 where a is the long diameter (mm) and b is the short diameter (mm).

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Four to 6 weeks old female athymic nude mice (BALB/c, nu/nu) were used. A2780 and HO8910 cells were harvested and resuspended in 0.1 ml of PBS, 5 × 106 cells/0.2 ml were injected subcutaneously into the left inguinal area of the mice. Two weeks later, mice bearing tumors (∼70 mm3 for A2780 and HO8910) were randomly divided into 4 groups. Artesunate was administered daily via i.p. injection at doses of 50 mg/kg alone or in combination with cisplatin (2 mg/kg) for 16 days. The tumor growth was monitored every other day. Tumor volume was determined by the formula 1/2a × b2 where a is the long diameter (mm) and b is the short diameter (mm).[3]

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NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice were bred in-house. Pilot experiments were performed with three mice in each group. Based on the results, n = 10 for treatment and control group was chosen. The mice (6–10 weeks old) were injected subcutaneously with 2 × 106 BL-41 cells expressing firefly luciferase (BL-41-luc). Tumor take was measured by IVIS at day 4 before mice were divided into control and treatment groups. The mice were divided according to tumor size within each cage for either treatment or control group to have non-biased, comparable groups. Artesunate was dissolved in EtOH/DMSO (1:1) to 370 mg/ml and diluted 1:10 in 5% Na3CO3 before injections. Mice were injected daily from day 4 with 200 mg/kg Artesunate intraperitoneally or control (5% EtOH and 5% DMSO in Na3CO3). Treatment was given for 12 days, then 2 days off, followed by treatment every day until day 19 (17 injections total). Tumor growth was monitored by bioluminescent imaging at regular intervals. Mice were injected intraperitoneally with 150 mg/kg d-luciferin and imaged after 10 min. Inhalation anesthetic sevofluran supplemented with O2 and N2O was used during imaging. Caliper measurement was used to determine if the tumor size had reached maximum 2 cm in one direction or 2 cm3 that was the limit for euthanasia.[4]

Absorption, Distribution and Excretion
The peak plasma concentration (Cmax) of artesunate is 3.3 µg/mL, and the peak plasma concentration (Cmax) of its active metabolite DHA is 3.1 µg/mL. The AUC of artesunate is 0.7 µgh/mL, and the AUC of DHA is 3.5 µgh/mL. After intravenous injection of artesunate, the time to peak DHA concentration (Tmax) in adult patients is 0.5–15 minutes, and in pediatric patients it is 21–64 minutes. The Tmax of intramuscular injection of artesunate is 8–12 minutes. Infants under 6 months of age have higher AUC values because their UGT metabolic pathway is not yet fully developed. The main elimination pathway of artesunate in humans is not yet clear. In rats, 56.1% of the artesunate dose is excreted in the urine and 38.5% in the feces.
The volume of distribution (VOD) of artesunate is 68.5 L, while that of dihydroartemisinin is 59.7 L.
The clearance rate of artesunate is 180 L/h, while that of dihydroartemisinin is 32.3 L/h.
Artesunate is rapidly hydrolyzed to its major active metabolite, dihydroartemisinin, after human administration. The pharmacokinetic characteristics of artesunate show significant inter-individual variability, with significant differences between healthy volunteers and infected patients, as well as between patients with different disease severities.
The pharmacokinetic characteristics of artesunate and dihydroartemisinin show significant inter-individual variability. Pharmacokinetic parameters of artesunate and dihydroartemisinin differ significantly between healthy volunteers and infected patients, as well as between patients with different disease severities. Because the drugs selectively accumulate in parasite-infected erythrocytes, pharmacokinetic data from free plasma concentrations of artesunate or dihydroartemisinin should be interpreted with caution. In vitro studies showed that dihydroartemisinin accumulated at concentrations approximately 300 times higher in infected erythrocytes than in plasma. This study investigated the pharmacokinetics of oral dihydroartemisinin (DHA) at 2 mg/kg and 4 mg/kg body weight, and oral artesunate (AS) at 4 mg/kg body weight, in 20 healthy Thai volunteers (10 men and 10 women). All formulations were generally well-tolerated. Oral DHA was rapidly absorbed from the gastrointestinal tract, but inter-individual variability was significant. The pharmacokinetic characteristics of DHA were similar at both dose levels and linear. Based on model-independent pharmacokinetic analysis, the median Cmax (95% CI) of DHA at 1.5 hours after administration of 2 mg/kg and 4 mg/kg body weight were 181 (120–306) ng/ml and 360 (181–658) ng/ml, respectively. The corresponding AUC_0-∞, t_1/2z, CL/f, and V_z/f values were 377 (199-1,128) vs 907 (324-2,289) ng·hr/mL, 0.96 (0.70-1.81) vs 1.2 (0.75-1.44) h, 7.7 (4.3-12.3) vs 6.6 (3.1-10.1) L/kg, and 90.5 (28.6-178.2) vs 6.6 (3.1-10.1) mL/min/kg (at doses of 2 mg/kg and 4 mg/kg, respectively). Oral administration of AS rapidly bioconverts to DHA, which is detectable in plasma within 15 minutes of administration. Following a 4 mg/kg dose, the median (95% CI) Cmax was 519 (236–284) ng/mL, with a time to reach of 0.7 (0.25–1.5) hours. AUC0-∞ and t1/2z were 657 (362–2,079) ng·hr/mL and 0.74 (0.34–1.42) hours, respectively. The Cmax of DHA after oral artesunate administration was significantly higher than that after rectal administration, but at the same dose level (4 mg/kg body weight), the total systemic exposure of oral DHA was higher. There were no significant gender differences in the pharmacokinetics of DHA. This study aimed to determine the pharmacokinetic parameters of a single oral and rectal administration of 200 mg artesunate in healthy volunteers and to propose a reasonable rectal administration regimen. This study employed a randomized, open-label, crossover design, enrolling 12 healthy volunteers… Due to the rapid clearance of artesunate from plasma, pharmacokinetic parameters were derived from data on the major metabolite α-dihydroartemisinin. Following oral administration of artesunate, the AUC (0–∞) (P<0.05; 95% confidence interval (CI) -1168.73, -667.61 ng·hr/mL⁻¹) and Cmax (P<0.05; 95% CI -419.73, -171.44 ng/mL⁻¹) of dihydroartemisinin were significantly higher than those of rectal administration, and the tmax (P<0.05; 95% CI -0.97, -0.10 hr) was significantly shortened. There was no statistically significant difference in elimination half-life between the two routes of administration (P>0.05; 95% CI -0.14, 0.53 hours). The relative bioavailability of artesunate administered rectally was [mean (coefficient of variation) 54.9 (24.8%) %]. For more complete data on the absorption, distribution, and excretion of artesunate (8 items), please visit the HSDB record page. Metabolites/Metabolites Artesunate is rapidly metabolized to dihydroartemisinin (DHA) by plasma esterases. DHA is then glucuronidated via UGT1A9 and UGT2B7 to DHA-glucuronide. DHA-glucuronide can be converted to furanoic acid derivatives of DHA-glucuronide via secondary metabolic pathways. CYP2A6 may make a slight contribution to the metabolism of artesunate. Following administration to the human body, artesunate is rapidly hydrolyzed to its major active metabolite, dihydroartemisinin. Data from in vitro human liver microsomal studies and clinical trials indicate that DHA-glucuronide (position 10) is the major phase II metabolite of DHA, and uridine diphosphate glucuronide transferase isoenzymes 1A1, 1A8-9, or 2B7 are likely the major binding enzymes.
In rats, artemisinin is completely and rapidly absorbed after oral administration. However, even at doses up to 300 mg/kg, plasma drug concentrations are extremely low. The liver is the primary site of inactivation. Significant and more sustained plasma drug concentrations were detected after intramuscular injection of artemisinin. Following intravenous administration, artemisinin can cross the blood-brain barrier and the blood-placental barrier. Regardless of the route of administration, little unmetabolized artemisinin was detected in urine or feces within 48 hours. Metabolites identified after human administration include deoxyartemisinin, deoxydihydroartemisinin, and 9,10-dihydroxydeoxyartemisinin.
The known human metabolites of artesunate include (1S,4S,5R,8S,9R,10R,12R,13R)-1,5,9-trimethyl-11,14,15,16-tetraoxatetracyclo[10.3.1.04,13.08,13]hexadecane-10-ol.
Biological half-life
The elimination half-life of artesunate is 0.3 hours, ranging from 0.1 to 1.8 hours. The elimination half-life of dihydroartemisinin is 1.3 hours, ranging from 0.9 to 2.9 hours. After intramuscular administration, the half-life in children is 48 minutes, and in adults it is 41 minutes.
In volunteer studies, artesunate was rapidly eliminated (within minutes) via bioconversion to dihydroartemisinin, which has a half-life of approximately 45 minutes.

Hepatotoxicity
In an open-label study of 104 patients with severe malaria in the United States, 27% of participants experienced elevated ALT, 49% experienced elevated AST, and 17% experienced elevated bilirubin. However, these abnormalities were all attributed to hemolysis and liver involvement common in patients with acute severe malaria. All elevations were not related to drug-induced liver injury. Since artesunate injection was approved for marketing and widespread clinical use in the United States, there have been no reports of acute liver injury due to artesunate use. Probability score: E (unlikely to be the cause of clinically significant liver injury). Pregnancy and Lactation Effects ◉ Overview of Use During Lactation Limited information suggests that after a mother takes 200 mg of artesunate orally, the drug concentration in breast milk is low and is not expected to have any adverse effects on breastfed infants, especially those older than 2 months. Discontinuing breastfeeding within 6 hours of administration significantly reduces the amount of drug ingested by the infant. Normally, the amount of antimalarial drug secreted in the breast milk of lactating women is very small. Because the amount of antimalarial drugs in breast milk is insufficient to provide adequate protection against malaria, infants requiring chemoprevention must receive the recommended dose of antimalarial drugs.
◉ Effects on breastfed infants
Breastfed infants receiving dihydroartemisinin and piperaquine for malaria vomited more frequently than non-breastfed infants receiving these drugs. Whether this finding applies to infants who ingest dihydroartemisinin through breast milk has not been studied.
◉ Effects on lactation and breast milk
No relevant published information was found as of the revision date.

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Protein binding
Artemisinin and its metabolite DHA have approximately 93% protein binding in plasma. Artemisinin can bind to serum albumin.

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Interactions
The activity of artemisinin in combination with other antimalarial drugs against Plasmodium falciparum and Plasmodium berghei was determined in vitro and in vivo. Both in vitro and in vivo experiments showed that artemisinin had a synergistic effect with mefloquine and an antagonistic effect with pyrimethamine. In vivo studies have also shown that artemisinin exhibits antagonistic effects when used in combination with other antimalarial drugs (sulfadiazine, sulfadoxine, sulfadoxine-pyrimethamine, cycloguanidine, and dapsone). There are concerns that the use of antipyretics might weaken the host's defenses against malaria because it delays parasite clearance. However, this appears to be due to delayed cell adhesion, which may be beneficial. Therefore, there is no reason to discontinue antipyretics in malaria treatment. Acetaminophen (paracetamol) and ibuprofen are the first-line drugs for reducing fever.

[1]. Artesunate as an Anti-Cancer Agent Targets Stat-3 and Favorably Suppresses Hepatocellular Carcinoma. Curr Top Med Chem. 2016;16(22):2453-63.
[2]. Supergenomic network compression and the discovery of EXP1 as a glutathione transferase inhibited by artesunate. Cell. 2014 Aug 14;158(4):916-928.
[3]. Artesunate sensitizes ovarian cancer cells to cisplatin by downregulating RAD51. Cancer Biol Ther. 2015;16(10):1548-56.
[4]. Artesunate shows potent anti-tumor activity in B-cell lymphoma. J Hematol Oncol. 2018 Feb 20;11:23.

Therapeutic Uses
Treatment Category: Antimalarial Drugs Artesunate rectal capsules are indicated for the initial treatment of acute malaria patients who cannot take oral medication or undergo parenteral therapy. To address the threat of resistance to monotherapy by Plasmodium falciparum and to improve treatment efficacy, the World Health Organization currently recommends the use of combination antimalarial drugs for the treatment of falciparum malaria. Currently recommended artemisinin-based combination therapies (ACTs) are as follows (in alphabetical order): AS+AQ (artesunate + amodiaquine), AS+MQ (artesunate + mefloquine), AS+SP (artesunate + sulfadoxine-pyrimethamine). Artemisinin and its derivatives (artesunate, artemether, artemether, dihydroartemisinin) rapidly clear parasitemia and quickly relieve symptoms. They can reduce the number of parasites by approximately 10,000 times per asexual reproductive cycle, which is more effective than other existing antimalarial drugs (which can reduce the number of parasites by 100 to 1000 times per cycle). Artemisinin and its derivatives are rapidly metabolized. When used in combination with rapidly metabolized compounds (tetracyclines, clindamycin), a 7-day course of artemisinin-based therapy is required; however, when used in combination with slowly metabolized antimalarial drugs, a shorter course (3 days) is effective. Evidence of their superiority over monotherapy is well-established. For more complete data on the therapeutic uses of artemisinic acids (14 in total), please visit the HSDB record page. Drug Warnings Artemisinin-based compounds should not be used in patients with a history of allergic reactions to artemisinin-based compounds or who develop urticaria during treatment. Patients with a history of allergy to artemisinin-based drugs should be advised against taking any artemisinin derivatives. The efficacy of artesunate rectal capsules as a monotherapy for malaria has not been evaluated; therefore, all patients initially treated with artesunate rectal capsules should be immediately referred to the nearest healthcare facility capable of providing a complete course of malaria cure. Adverse reactions to artesunate include bitter taste, mild pain at the injection site, bradycardia, paroxysmal premature ventricular contractions, incomplete right bundle branch block, first-degree atrioventricular block, and urticaria. The most common reported adverse reactions (incidence <1%) are mild gastrointestinal reactions (nausea, vomiting, diarrhea, abdominal pain). For more complete data on drug warnings for artesunate (25 in total), please visit the HSDB record page. Pharmacodynamics Artesunate is an artemisinin derivative that is metabolized to DHA, which generates free radicals that inhibit normal function. It is used to treat Plasmodium falciparum. Due to its short half-life and short duration of action, it has a moderate therapeutic index. Patients should be informed of the risk of hemolytic anemia and hypersensitivity reactions after treatment. One core problem in biology is identifying gene function. One approach is to infer the function of gene interactions and ancestral relationships through large supergenomic networks; however, the computational cost of such analyses can be enormous. Here we demonstrate that these biological networks are compressible. By eliminating redundant evolutionary relationships, these networks can be significantly reduced, and the process is highly efficient because the number of compressible elements in these networks grows linearly rather than exponentially as in other complex networks. Compression enables global network analysis to utilize hundreds of interconnected genomes for computation and to generate functional predictions. As an example, we demonstrate that EXP1, an essential but functionally undefined antigen of Plasmodium falciparum, is a membrane glutathione S-transferase. EXP1 effectively degrades cytotoxic heme, is potently inhibited by artesunate, and is associated with artesunate metabolism and sensitivity in Plasmodium under drug stress. These data suggest that EXP1 is involved in the mechanism of action of first-line antimalarial drugs. [2] Artesunate is a semi-synthetic artemisinin derivative that was originally used to treat malaria, but recent studies have shown that it has antitumor properties. One of the cytotoxic effects of artesunate on cancer cells is mediated by inducing oxidative stress and DNA double-strand breaks (DSBs). Here we report that, in addition to inducing oxidative stress and DSBs, artesunate can also downregulate RAD51 and impair DSB repair in ovarian cancer cells. We observed that the formation of RAD51 aggregates and homologous recombination repair (HRR) were significantly reduced in artesunate-treated cells. Thus, artesunate and cisplatin synergistically induced DNA double-strand breaks (DSBs) and inhibited clonal formation in ovarian cancer cells. Ectopic expression of RAD51 rescued the chemosensitization enhanced by artesunate, confirming that the chemosensitizing effect of artesunate is at least partially mediated by downregulation of RAD51. Our results suggest that artesunate can impair DSB repair in ovarian cancer cells and thus can be used as a chemosensitizer. [3]

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Although chemotherapy combined with immunotherapy has significantly improved overall survival in most patients with B-cell lymphoma, relapse and refractory disease remain a challenge. The antimalarial drug artesunate has previously been shown to inhibit the growth of certain cancer types and has been tested as a new treatment option for B-cell lymphoma. Methods: We included artesunate in the screening of cancer-sensitive drugs for B-cell lymphoma cell lines. This study tested the preclinical properties of artesunate as a single drug in vitro (using 18 B-cell lymphoma cell lines representing different histological types) and in vivo (using an aggressive B-cell lymphoma xenograft model constructed from NSG mice). Functional analysis, gene expression profiling, and protein expression analysis were performed on artesunate-treated B-cell lymphoma cell lines to elucidate its mechanism of action. Results: Drug screening revealed artesunate to be a highly potent anti-lymphoma drug. Artesunate exhibited significant growth inhibition against most B-cell lymphoma cells, with an IC50 value comparable to that found in the serum of malaria patients treated with artesunate, and had no effect on normal B cells. In the xenograft model, artesunate significantly inhibited the growth of highly aggressive tumors. Gene expression analysis revealed that endoplasmic reticulum (ER) stress and unfolded protein responses were the most significantly affected pathways. The expression of artesunate-induced ER stress markers ATF-4 and DDIT3 was specifically upregulated in malignant B cells, but not in normal B cells. In addition, artesunate significantly inhibited overall cell metabolism, affecting respiration and glycolysis. Conclusion: Artesunate showed strong apoptosis-inducing effects on various B-cell lymphoma cell lines in vitro and significant anti-lymphoma activity in vivo, suggesting that it may be an effective drug for the treatment of B-cell lymphoma. [4]

C19H28O8

384.42

384.178

C, 59.36; H, 7.34; O, 33.29

88495-63-0

Artesunate-d3;1316303-44-2;Artesunate-d4;1316753-15-7; 82864-68-4 (Sodium salt)

6917864

Fine white crystalline powder

1.3±0.1 g/cm3

507.1±50.0 °C at 760 mmHg

132-135ºC

175.6±23.6 °C

0.0±2.8 mmHg at 25°C

1.544

2.94

1

8

5

27

623

8

C[C@H]1[C@H](OC(=O)CCC(=O)O)O[C@@H]2O[C@]3(CC[C@@H]4[C@@]2(OO3)[C@H]1CC[C@H]4C)C

FIHJKUPKCHIPAT-AHIGJZGOSA-N

InChI=1S/C19H28O8/c1-10-4-5-13-11(2)16(23-15(22)7-6-14(20)21)24-17-19(13)12(10)8-9-18(3,25-17)26-27-19/h10-13,16-17H,4-9H2,1-3H3,(H,20,21)/t10-,11-,12+,13+,16-,17-,18-,19-/m1/s1

4-oxo-4-(((3R,5aS,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)oxy)butanoic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.41 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (5.41 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (5.41 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)