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 Cmax of artesunate is 3.3µg/mL while the Cmax of the active metabolite DHA is 3.1µg/mL. The AUC of artesunate is 0.7µg\*h/mL while the AUC of DHA is 3.5µg\*h/mL. After intravenous artesunate, DHA has a Tmax of 0.5-15 minutes in adult patients and 21-64 minutes in pediatric patients. Intramuscular artesunate has a Tmax of 8-12 minutes. Infants less than 6 months old will have a higher AUC due to an undeveloped UGT metabolic pathway.
The main route of elimination in humans is unknown. In rats, a dose of artesunate is 56.1% eliminated in the urine and 38.5% in the feces.
The volume of distribution of artesunate is 68.5L while the volume of distribution of DHA is 59.7L.
The clearance of artesunate is 180L/h while the clearance of DHA is 32.3L/h.
Following administration to humans, artesunate is rapidly hydrolyzed to its principal active metabolite, dihydroartemisinin. The pharmacokinetics of artesunate are characterized by marked inter-subject variability, differing significantly between healthy volunteers and infected patients, and among patients with different disease severity.
The pharmacokinetic of artesunate and dihydroartemisin are characterized by marked inter-subject variability. The pharmacokinetic parameters of artesunate and dihydroartemisinin differ significantly between healthy volunteers and infected patients, and among patients with different disease severity. Pharmacokinetic data from unbound plasma concentrations of artesunate or dihydroartemisinin should be interpreted with caution because the drug accumulates selectively in parasitized RBC's In in vitro experiments, accumulation of dihydroartemisinin in infected RBC's is in concentrations approximately 300-fold higher than those in plasma .
The pharmacokinetics of oral dihydroartemisinin (DHA) following the dose of 2 and 4 mg/ kg body weight dihydroartemisinin and 4 mg/kg body weight oral artesunate (AS) were investigated in 20 healthy Thai volunteers (10 males, 10 females). All formulations were generally well tolerated. Oral DHA was rapidly absorbed from gastrointestinal tract with marked inter-individual variation. The pharmacokinetics of DHA following the two dose levels were similar and linearity in its kinetics was observed. Based on the model-independent pharmacokinetic analysis, median (95% CI) values for Cmax of 181 (120-306) and 360 (181-658) ng/ml were achieved at 1.5 hours following 2 and 4 mg/kg body weight dose, respectively. The corresponding values for AUC0-infinity, t1/2z, CL/f and Vz/f 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) hours, 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, respectively (2 vs 4 mg/kg dose). Oral AS was rapidly biotransformed to DHA, which was detectable in plasma as early as 15 minutes of AS dosing. Following 4 mg/kg dose, median (95% CI) value for Cmax of 519 (236-284) ng/mL was achieved at 0.7 (0.25-1.5) hours. AUC0-infinity, and t1/2z were 657 (362-2,079) ng.hr/mL, 0.74 (0.34-1.42) hours, respectively. Cmax of DHA following oral AS were significantly higher, but total systemic exposure was greater following oral DHA at the same dose level (4 mg/kg body weight). There was no significant sex difference in pharmacokinetics of DHA
The aims of this study were to determine the pharmacokinetic parameters of a single dose of 200 mg oral and rectal artesunate in healthy volunteers, and to suggest a rational dosage regimen for rectal administration. The study design was a randomized open cross-over study of 12 healthy volunteers... Pharmacokinetic parameters were derived from the main metabolite alpha-dihydroartemisinin data due to the rapid disappearance of artesunate from the plasma. Dihydroartemisinin following oral administration of artesunate had a significantly higher AUC(0-infinity) (P<0.05 95% confidence interval (CI) -1168.73, -667.61 ng x hr/mL(-1)) and Cmax (P<0.05; 95% CI -419.73, -171.44 ng/mL(-1)), and had shorter tmax (P<0.05; 95% CI -0.97, -0.10 hr) than that following rectal artesunate. There was no statistically significant difference in the elimination half-life between both routes of administration (P>0.05; 95% CI -0.14, 0.53 hr). The relative bioavailability of rectal artesunate was [mean (coefficient of variation %) 54.9 (24.8%) %].
For more Absorption, Distribution and Excretion (Complete) data for ARTESUNIC ACID (8 total), please visit the HSDB record page.

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Metabolism / Metabolites
Artesunate is rapidly metabolized to dihydroartemisinin (DHA) by plasma esterases. DHA is glucuronidated by UGT1A9 and UGT2B7 to DHA-glucuronide. DHA-glucuronide can undergo a minor metabolic pathway to for a furano acetate derivative of DHA-glucuronide. CYP2A6 may minorly contribute to the metabolism of artesunate.
Following administration to humans, artesunate is rapidly hydrolyzed to its principle active metabolite, dihydroartemisinin. Data from in vitro studies with human liver microsomes and from clinical studies suggest that DHA-glucoronide (10-position) is the principal Phase II metabolite of DHA and that uridine diphosphate glucuronyl transferase isoforms 1A1, 1A8-9, or 2B7 may be the main conjugating enzyme.
Artemisinin is completely and rapidly absorbed after oral administration in rats. However, a very low plasma level was obtained even after a dose of 300 mg/kg. Liver was found to be the chief site of inactivation. When artemisinin was given i.m., significant and more persistent plasma levels were detected. Artemisinin was shown to pass the blood-brain and blood-placenta barriers after i.v. injection. Very little unchanged artemisinin was found in the urine or feces in 48 hours regardless of the route of administration. Metabolites identified after administration to humans include deoxyartemisinin, deoxydihydroartemisinin, and 9,10-dihydroxydeoxyartemisinin.
Artesunate has known human metabolites that 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]hexadecan-10-ol.

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Biological Half-Life
The elimination half life of artesunate is 0.3h with a range of 0.1-1.8h. The elimination half life of DHA is 1.3h with a range of 0.9-2.9h. Half life after intramuscular administration is 48 min in children and 41 min in adults.
In volunteer studies, artsunate was cleared very rapidly (within minutes) by biotransformation to dihydroartemisinin, which was eliminated by with a half-life of approximately 45 minutes.

Hepatotoxicity
In an open label study of severe malaria in 104 patients in the United States, elevations in ALT occurred in 27% of subjects, AST in 49%, and bilirubin in 17%. These abnormalities, however, were attributable to the hemolysis and liver involvement that are common in patients with acute, severe malaria. None of the elevations were attributed to drug induced liver injury. Since licensure of artesunate for injection and its more widescale clinical use in the United States, there have been no reports of acute liver injury attributed to its use.
Likelihood score: E (unlikely cause of clinically apparent liver injury).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
Limited information indicates that a maternal dose of 200 mg orally produced low levels in milk and would not be expected to cause any adverse effects in breastfed infants, especially if the infant is older than 2 months. Withholding breastfeeding for 6 hours after a dose should markedly reduce the dose the infant receives.
In general, very small amounts of antimalarial drugs are excreted in the breast milk of lactating women. Because the quantity of antimalarial drugs transferred in breast milk is insufficient to provide adequate protection against malaria, infants who require chemoprophylaxis must receive the recommended dosages of antimalarial drugs.
◉ Effects in Breastfed Infants
Breastfed infants who were given dihydroartemisinin and piperaquine as a treatment for malaria had a higher frequency of vomiting than non-breastfed infants given the drugs. Whether this finding applies to infants who receive dihydroartemisinin via breastmilk has not been studied.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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Protein Binding
Artesunate and its metabolite DHA are approximately 93% protein bound in plasma. Artesunate can bind to serum albumin.

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Interactions
The activity of artemisinin in combination with other antimalarial drugs against P. falciparum was measured in vitro and against P. berghei in vivo. A combination of artemisinin with mefloquine was synergistic whereas that with pyrimethamine was antagonistic in vitro and in vivo. A combination of artemisinin with other antimalarials (sulfadiazine, sulfadoxine, sulfadoxine-pyrimethamine, cycloguanil, and dapsone) was also shown to be antagonistic in vivo.
There has been some concern that antipyretics might attenuate the host defense against malaria, as their use is associated with delayed parasite clearance. However, this appears to result from delaying cytoadherence, which is likely to be beneficial. There is no reason to withhold antipyretics in malaria. ...Paracetamol (acetaminophen) and ibuprofen are the preferred options 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
Therap Cat: Antimalarial
Artesunate Rectal Capsules is indicated for the initial management of acute malaria in patients who cannot take medication by mouth and for whom parenteral treatment is not available.
To counter the threat of resistance of P. falciparum to monotherapies, and to improve treatment outcome, combinations of antimalarials are now recommended by WHO for the treatment of falciparum malaria. The following ACTs are currently recommended (alphabetical order): AS+AQ artesunate + amodiaquine combination, AS+MQ artesunate + mefloquine combination, AS+SP artesunate + sulfadoxine-pyrimethamine combination.
Artemisinin and its derivatives (artesunate, artemether, artemotil, dihydroartemisinin) produce rapid clearance of parasitaemia and rapid resolution of symptoms. They reduce parasite numbers by a factor of approximately 10,000 in each asexual cycle, which is more than other current antimalarials (which reduce parasite numbers 100- to 1000-fold per cycle). Artemisinin and its derivatives are eliminated rapidly. When given in combination with rapidly eliminated compounds (tetracyclines, clindamycin), a 7-day course of treatment with an artemisinin compound is required; but when given in combination with slowly eliminated antimalarials, shorter courses of treatment (3 days) are effective. The evidence of their superiority in comparison to monotherapies has been clearly documented.
For more Therapeutic Uses (Complete) data for ARTESUNIC ACID (14 total), please visit the HSDB record page.

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Drug Warnings
Artemisinin congeners should not be given to patients with a previous history of an allergic reaction following their consumption or if an urticarial rash develops during treatment. Patient with a history of hypersensitivity reaction to one of the artemsinins should be advised not to take any of the derivatives again.
Artesunate rectal capsules have not been evaluated as sole therapy for malaria; consequently all patient who are initially treated with artesunate rectal capsules should be promptly referred and evaluated at the nearest health care facility able to provide a full curative course of treatment for malaria.
Adverse events described /following artesunate/ included bitter taste, mild pain at the injection site, bradycardia, paroxysmal ventricular premature beat, incomplete right bundle branch block, first-degree atrio-ventricular block, and urticaria.
... The most commonly reported adverse events (in the order of <1%) to be mild gastrointestinal (nausea, vomiting, diarrhea, abdominal pain) events.
For more Drug Warnings (Complete) data for ARTESUNIC ACID (25 total), please visit the HSDB record page.

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Pharmacodynamics
Artesunate is an artemisinin derivative that is metabolized to DHA, which generates free radicals to inhibit normal function of _Plasmodium_ parasites. It has a short duration of action due to its short half life, and a moderate therapeutic index. Patients should be counselled regarding the risk of post treatment hemolytic anemia and hypersenstivity.

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A central problem in biology is to identify gene function. One approach is to infer function in large supergenomic networks of interactions and ancestral relationships among genes; however, their analysis can be computationally prohibitive. We show here that these biological networks are compressible. They can be shrunk dramatically by eliminating redundant evolutionary relationships, and this process is efficient because in these networks the number of compressible elements rises linearly rather than exponentially as in other complex networks. Compression enables global network analysis to computationally harness hundreds of interconnected genomes and to produce functional predictions. As a demonstration, we show that the essential, but functionally uncharacterized Plasmodium falciparum antigen EXP1 is a membrane glutathione S-transferase. 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, a semi-synthetic derivative of arteminisin originally developed for the treatment of malaria, has recently been shown to possess antitumor properties. One of the cytotoxic effects of artesunate on cancer cells is mediated by induction of oxidative stress and DNA double-strand breaks (DSBs). We report here 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 foci and homologous recombination repair (HRR) were significantly reduced in artesunate-treated cells. As a consequence, artesunate and cisplatin synergistically induced DSBs and inhibited the clonogenic formation of ovarian cancer cells. Ectopic expression of RAD51 was able to rescue the increased chemosensitivity conferred by artesunate, confirming that the chemosensitizing effect of artesuante is at least partially mediated by the downregulation of RAD51. Our results indicated that artesunatecan compromise the repair of DSBs in ovarian cancer cells, and thus could be employed as a sensitizing agent in chemotherapy.[3]

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Although chemo-immunotherapy has led to an improved overall survival for most B-cell lymphoma types, relapsed and refractory disease remains a challenge. The malaria drug artesunate has previously been identified as a growth suppressor in some cancer types and was tested as a new treatment option in B-cell lymphoma. Methods: We included artesunate in a cancer sensitivity drug screen in B lymphoma cell lines. The preclinical properties of artesunate was tested as single agent in vitro in 18 B-cell lymphoma cell lines representing different histologies and in vivo in an aggressive B-cell lymphoma xenograft model, using NSG mice. Artesunate-treated B lymphoma cell lines were analyzed by functional assays, gene expression profiling, and protein expression to identify the mechanism of action. Results: Drug screening identified artesunate as a highly potent anti-lymphoma drug. Artesunate induced potent growth suppression in most B lymphoma cells with an IC50 comparable to concentrations measured in serum from artesunate-treated malaria patients, while leaving normal B-cells unaffected. Artesunate markedly inhibited highly aggressive tumor growth in a xenograft model. Gene expression analysis identified endoplasmic reticulum (ER) stress and the unfolded protein response as the most affected pathways and artesunate-induced expression of the ER stress markers ATF-4 and DDIT3 was specifically upregulated in malignant B-cells, but not in normal B-cells. In addition, artesunate significantly suppressed the overall cell metabolism, affecting both respiration and glycolysis. Conclusions: Artesunate demonstrated potent apoptosis-inducing effects across a broad range of B-cell lymphoma cell lines in vitro, and a prominent anti-lymphoma activity in vivo, suggesting it to be a relevant drug for 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.)