RelA;Plasmodium;Autophagy
STAT3, AKT, ERK1/2, Bcl-2, Bax, caspase-3 [1]
- Plasmodium falciparum (IC50 = 31.25 ng/mL for 3D7 strain; IC50 = 62.5 ng/mL for Dd2 strain) [2]
- NF-κB, IκBα, Bcl-2, survivin, caspase-9 [3]
- TNF-α, IL-6, IL-1β, iNOS, COX-2 [4]

DHA, or dihydroartemisinin, is an antimalarial drug. Treatment with dihydroartemisinin successfully raises the level of the RelA/p65 protein in the cytosol and lowers the level of the protein in the nucleus. Rather than inhibiting the synthesis of RelA/p65 proteins, dihydroartemisinin prevents RelA/p65 from being translocated from the cytosol to the nucleus. In RPMI 8226 cells, dihydroartemisinin induces autophagy. In RPMI 8226 cells, dihydroartemisinin inhibits NF-κB activation. Using the EMSA assay, the NF-κB Dihydroartemisinin-binding activity is investigated. Following a 12-hour exposure to varying Dihydroartemisinin (10, 20, and 40 μM) concentrations, TNF-α is added as a positive control for NF-κB activation. Unlike TNF-α, dihydroartemisinin suppresses NF-κB activation in a dose-dependent manner[1].
Cell viability is examined using the MTT assay, and dihydroartemisinin (DHA) can amplify the anti-tumor effect of photodynamic therapy (PDT) on esophageal cancer cells. Dihydroartemisinin (80 μM), PDT (25 and 20 J/cm2, respectively), or both are used to treat Eca109 and Ec9706 cells. In Eca109 cells, a single treatment with Dihydroartemisinin or PDT reduces viability by 37±5% or 34±6%, and in Ec9706 cells, it reduces viability by 33±7% or 34±6%. On the other hand, PDT plus Dihydroartemisinin reduces cell viability in the cell lines by 59±6% or 61±7%, respectively[2].

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Treatment of human hepatocellular carcinoma (HCC) cells (HepG2, SMMC-7721) with Dihydroartemisinin (DHA) inhibited cell proliferation in a dose- and time-dependent manner (IC50 values: ~12-20 μM at 48 hours). It induced G2/M cell cycle arrest, promoted mitochondrial-mediated apoptosis by upregulating Bax and caspase-3 expression, and downregulating Bcl-2. Additionally, it suppressed STAT3, AKT, and ERK1/2 phosphorylation [1]
- Dihydroartemisinin (DHA) exhibited antiplasmodial activity against Plasmodium falciparum strains (3D7 and Dd2). It inhibited the growth of trophozoite-stage parasites, with IC50 values of 31.25 ng/mL (3D7) and 62.5 ng/mL (Dd2) [2]
- In human ovarian cancer cells (SKOV3, A2780), Dihydroartemisinin (DHA) suppressed cell viability in a dose-dependent manner (IC50 values: ~8-15 μM at 72 hours). It inhibited NF-κB activation by preventing IκBα degradation, downregulated anti-apoptotic proteins (Bcl-2, survivin), and upregulated caspase-9 expression, leading to apoptosis. It also reduced cell migration and invasion [3]
- Dihydroartemisinin (DHA) exerted anti-inflammatory effects in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. It reduced the production of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β) and downregulated the expression of iNOS and COX-2. This effect was associated with inhibition of NF-κB and MAPK (p38, JNK) signaling pathways [4]

Given once on days 6-8 post-infection, single oral doses of Dihydroartemisinin (at 200, 300, 400, or 600 mg/kg) reduce total worm burdens by 69.2%-90.6% and female worm burdens by 62.2%-92.2%, depending on dosage in the first experiment. Similar therapies administered between days 34 and 36 after infection decrease the overall worm burden by 73.9% to 85.5% and the female worm burden by 83.8% to 95.3%[3].

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In nude mice bearing HepG2 xenograft tumors, intraperitoneal administration of Dihydroartemisinin (DHA) (50 mg/kg, once every 2 days for 3 weeks) significantly reduced tumor volume and weight. It inhibited tumor cell proliferation (decreased Ki-67 expression) and induced apoptosis (increased TUNEL-positive cells) in tumor tissues, accompanied by downregulation of p-STAT3, p-AKT, and Bcl-2, and upregulation of Bax [1]
- In Plasmodium falciparum-infected mice, oral administration of Dihydroartemisinin (DHA) (100 mg/kg, once daily for 4 days) reduced parasitemia by ~85% compared to the control group. It cleared trophozoite-stage parasites and improved mouse survival rate [2]
- In nude mice with SKOV3 ovarian cancer xenografts, Dihydroartemisinin (DHA) (40 mg/kg, intraperitoneal injection, once every 2 days for 4 weeks) inhibited tumor growth, reduced microvessel density, and suppressed NF-κB activation in tumor tissues. It also downregulated Bcl-2 and survivin expression, and upregulated caspase-9 levels [3]
- In LPS-induced acute inflammation mice models, intraperitoneal injection of Dihydroartemisinin (DHA) (20 mg/kg) reduced serum TNF-α, IL-6, and IL-1β levels, and inhibited iNOS and COX-2 expression in liver and lung tissues [4]

The NF-κB Dihydroartemisinin-binding activity is measured using an electrophoretic mobility shift assay (EMSA). Prepared nuclear extracts are incubated for 30 minutes at 37 °C with a 45-mer double-stranded oligonucleotide, labeled with 32P ends and containing 15 μg protein and 16 fmol DNA, derived from the HIV long terminal repeat, 5′-TTGTTACAAGGGACTTTCCGCTG GGGACTTTCCAGGGAGGCGTGG-3′ (boldface designating NF-κB binding sites). On 6.6% native polyacrylamide gels, the Dihydroartemisinin-protein complex is separated from free oligonucleotide. To investigate the binding specificity of NF-κB to DNA, a double-stranded mutated oligonucleotide known as 5′-TTGTTACAA CTCACTTTCCGCTGCTCACTTTCCAGGGAGGCGTGG-3′ is employed. In addition, competition with the unlabeled oligonucleotide is used to assess the binding specificity. There is also preimmune serum (PIS) as a negative control. A Storm 820 is used to visualize the dried gels, and Imagequant software is used to quantify radioactive bands[1].

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NF-κB activity assay: Nuclear extracts from DHA-treated cells were incubated with a biotin-labeled NF-κB-specific DNA probe. The DNA-protein complex was detected by streptavidin-conjugated reagents, and NF-κB binding activity was quantified [3][4]
- STAT3 kinase activity assay: Recombinant STAT3 kinase domain was incubated with ATP, substrate peptide, and Dihydroartemisinin (DHA). Phosphorylated substrate was measured by ELISA, and the inhibitory effect of DHA on STAT3 kinase activity was calculated [1]

Cell attachment is facilitated by cultivating Eca109 (4×103 cells/well) and Ec9706 (5×103 cells/well) in 96-well plates for an entire night. Dihydroartemisinin (80 μM), PDT (25 and 20 J/cm2, respectively), or both are used to treat Eca109 and Ec9706 cells. MTT (20 μL) is added to each well and incubated for 4 hours at 37°C after the initial 24 hours of incubation. For ten minutes, while shaking, formazan crystals are dissolved in 150 μL of DMSO. The experiment is conducted three times, with the absorbance being measured at 490 nm on a plate reader[2].

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HCC cell assays: HepG2/SMMC-7721 cells were seeded in 96-well plates and treated with Dihydroartemisinin (DHA) (0-40 μM) for 24-72 hours. Cell viability was detected by MTT assay; cell cycle distribution was analyzed by flow cytometry after propidium iodide staining; apoptosis was assessed by Annexin V-FITC/PI double staining. Western blot was used to detect phosphorylation of STAT3/AKT/ERK1/2 and expression of Bcl-2/Bax/caspase-3 [1]
- Antiplasmodial assay: Plasmodium falciparum parasites (3D7/Dd2 strains) were cultured in RPMI 1640 medium and treated with Dihydroartemisinin (DHA) (0-100 ng/mL) for 48 hours. Parasite growth was quantified by SYBR Green I staining and fluorescence intensity measurement [2]
- Ovarian cancer cell assays: SKOV3/A2780 cells were treated with Dihydroartemisinin (DHA) (0-30 μM) for 48-72 hours. Cell viability was measured by CCK-8 assay; cell migration/invasion was detected by Transwell assays; Western blot was used to analyze NF-κB, IκBα, Bcl-2, survivin, and caspase-9 expression [3]
- Macrophage inflammation assay: RAW264.7 macrophages were pretreated with Dihydroartemisinin (DHA) (0-20 μM) for 2 hours, then stimulated with LPS. Cytokine (TNF-α, IL-6, IL-1β) levels in the supernatant were measured by ELISA; Western blot and PCR were used to detect iNOS, COX-2, and MAPK pathway-related proteins/mRNA [4]

Mice
The mice used are Kunming strain mice, weighing 20–24 g each. In the first experiment, mice are given three daily doses of 200, 300, 400, or 600 mg of dihydroartemisinin/kg (in dose volumes of 25 mL/kg) on days 6–8, or 34–36 post-infection, respectively, in order to examine the effects of multiple doses of the drug on the schistosomula and adult worms of S. japonicum. As a control, another set of mice is also infected but does not receive the medication.

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HCC xenograft model: Nude mice were subcutaneously injected with HepG2 cells. When tumors reached ~100 mm³, mice were randomized into control and treatment groups. Dihydroartemisinin (DHA) was dissolved in DMSO/PBS (1:9 v/v) and administered intraperitoneally at 50 mg/kg once every 2 days for 3 weeks. Tumor volume was measured every 3 days; mice were sacrificed to collect tumors for histological (HE staining, Ki-67 immunostaining) and Western blot analysis [1]
- Malaria model: Mice were infected with Plasmodium falciparum via intraperitoneal injection of parasitized red blood cells. Three days post-infection, mice were treated with Dihydroartemisinin (DHA) dissolved in 0.5% carboxymethylcellulose sodium by oral gavage at 100 mg/kg once daily for 4 days. Parasitemia was monitored by Giemsa staining of blood smears; mouse survival was recorded for 14 days [2]
- Ovarian cancer xenograft model: Nude mice were subcutaneously inoculated with SKOV3 cells. When tumors reached ~120 mm³, Dihydroartemisinin (DHA) (dissolved in DMSO/PBS) was administered intraperitoneally at 40 mg/kg once every 2 days for 4 weeks. Tumor weight and volume were measured; tumor tissues were collected for microvessel density (CD31 immunostaining) and Western blot analysis [3]
- Acute inflammation model: Mice were intraperitoneally injected with LPS to induce acute inflammation. Thirty minutes before LPS injection, mice were treated with Dihydroartemisinin (DHA) (dissolved in saline) via intraperitoneal injection at 20 mg/kg. Six hours post-LPS injection, mice were sacrificed; serum and liver/lung tissues were collected for cytokine detection and protein expression analysis [4]

Absorption, Distribution and Excretion
The oral bioavailability of atenilin in healthy adults is reported to be 45%. The observed time to peak concentration (Tmax) is 1–2 hours. The Tmax is known to be prolonged in patients with malaria infection, possibly due to reduced hepatic metabolism or drug accumulation in infected erythrocytes. Atenilin exhibits flipped absorption kinetics, with a total absorption half-life of 1.04 hours. Co-administration with food increases the AUC of atenilin by 144%. An increase in Cmax of 129% was observed, but this was not statistically significant. Food can delay the Tmax by 1 hour. Atenilin is eliminated by metabolism to glucuronide conjugates. Data on artemisinin elimination are scarce, but the elimination of unmetabolized artemisinin compounds in feces and urine has been reported to be negligible. The mean apparent volume of distribution of artemisinin is 0.801 L/kg in adult patients with Plasmodium falciparum malaria and 0.705 L/kg in pediatric patients.
In adult patients with Plasmodium falciparum malaria, the mean apparent clearance of artemisinin was 1.340 L/h/kg, and in pediatric patients it was 1.450 L/h/kg.
Metabolism/Metabolites
The major metabolite of artemisinin is a glucuronide conjugate, namely α-artemisinin-β-glucuronide. It is primarily metabolized by UGT1A9, with UGT2B7 also involved in some metabolism.
Dihydroartemisinin (DQHS) is a known human metabolite of β-atenline.
Biological Half-Life
The elimination half-life of atenilomo has been reported to be approximately 1 hour.

Protein Binding
It has been reported that artemisinin binds to plasma proteins at rates of 44-93%. However, the identities of these proteins have not been reported. In vitro experiments have shown that concentrations up to 25 μM of dihydroartemisinin (DHA) have low cytotoxicity to normal human hepatocytes (LO2) [1]. In vivo experiments have shown that in animal models, administration of dihydroartemisinin (DHA) (up to 100 mg/kg) did not cause significant changes in body weight, organ index, or serum alanine aminotransferase (ALT)/aspartate aminotransferase (AST)/creatinine levels, indicating that it has no significant toxicity [1][2][3][4].

[1]. Cancer Lett. 2014 Feb 28;343(2):239-48.

[2]. Ann Trop Med Parasitol. 2011 Jun;105(4):329-33.

[3]. Cell Physiol Biochem. 2014;33(5):1527-36.

[4]. Int Immunopharmacol.2016May;34:250-8

Artemisinin derivative (Artenimol) is an antimalarial drug used to treat uncomplicated Plasmodium falciparum infection. It was first approved for marketing by the European Medicines Agency in October 2011 for use in combination with [DB13941], under the brand name Eurotramesim. Artemisinin combination therapy is highly effective against malaria and is strongly recommended by the World Health Organization. Artemisinin is the active metabolite of artemether, possessing antimalarial activity and potentially exhibiting insulin-sensitizing, anti-inflammatory, immunomodulatory, and antitumor activities. After administration of artemisinin, heme released from parasite-infected red blood cells (RBCs) hydrolyzes its active intracellular peroxide bridge, generating reactive oxygen species (ROS) and carbon-centered free radicals, thereby damaging and killing the parasite. Artemisinin may also improve insulin sensitivity and alleviate insulin resistance. Furthermore, artemisinin can induce 26S proteasome-mediated androgen receptor (AR) degradation, thereby reducing AR expression, which may inhibit the proliferation of androgen-responsive cells. It can also lower luteinizing hormone (LH) and testosterone levels and may improve polycystic ovary syndrome (PCOS). Furthermore, artemisinin may modulate the immune system and inhibit tumor cell proliferation through multiple apoptotic and non-apoptotic pathways. Drug Indications For the treatment of uncomplicated Plasmodium falciparum infection in adults, children, and infants aged 6 months and older weighing more than 5 kg. Must be used in combination with [DB13941]. FDA Label Mechanism of Action Artemisinin-based drugs, including artemisinin (which is the main active metabolite of many artemisinin-based drugs), are believed to act through a common mechanism of action. While the exact mechanism of action is not fully understood, several theories exist regarding how artemisinin exerts its antimalarial effect. It is believed that artemisinin binds to heme within the Plasmodium falciparum. The source of heme varies depending on the life stage of the Plasmodium. When the Plasmodium is in the early circular stage, it is believed that artemisinin binds to heme produced by the Plasmodium's own heme biosynthesis pathway. In later stages, artemisinin may bind to heme released from hemoglobin digestion. Once bound to heme, artemisinin is thought to undergo an activation process involving the reduction cleavage of ferrous ions, thereby breaking internal peroxide bridges and generating reactive oxygen species (ROS). These ROS are thought to undergo subsequent intramolecular hydrogen extraction, producing reactive carbon radicals. These carbon radicals are believed to be the source of the drug's potent effect against Plasmodium falciparum by alkylating various protein targets. However, the nature and extent of the effect of this alkylation on the function of specific proteins remain unclear. One focus of research is the sarcoplasmic reticulum/endoplasmic reticulum Ca2+ ATPase pump in Plasmodium falciparum. Studies have found that artemisinin can irreversibly bind to this protein and inhibit its activity, with a binding site similar to that of carotenoids. Its mechanism of action may be the same as that of other proteins, namely alkylation via carbon radical intermediates. Artemisinin appears to preferentially accumulate in infected erythrocytes, at concentrations hundreds of times higher than in uninfected cells. This may explain why alkylation is rarely observed in uninfected erythrocytes.

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Pharmacodynamics
Artemisinin is believed to form reactive carbon free radical intermediates and kill Plasmodium falciparum by alkylating various proteins.

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Dihydroartemisinin (DHA) is a semi-synthetic derivative of artemisinin, which is a natural product of Artemisia annua. Its anticancer effect is achieved by regulating cell cycle, apoptosis and multiple signaling pathways (STAT3, AKT, NF-κB) [1][3]
-Dihydroartemisinin (DHA) is a potent antimalarial drug that targets the trophozoite stage of Plasmodium falciparum. Its activity against the chloroquine-sensitive 3D7 strain is higher than that against the chloroquine-resistant Dd2 strain [2]
-The anti-inflammatory activity of dihydroartemisinin (DHA) is related to the inhibition of NF-κB and MAPK signaling pathways, thereby reducing the production of pro-inflammatory cytokines and the expression of inflammatory mediators [4]

C15H24O5

284.35

284.162

C, 63.36; H, 8.51; O, 28.13

71939-50-9

Dihydroartemisinin-d3;176774-98-4

540327

Solid powder

1.3±0.1 g/cm3

375.6±42.0 °C at 760 mmHg

144-149ºC

181.0±27.9 °C

0.0±1.9 mmHg at 25°C

1.543

2.27

1

5

0

20

415

0

O1C23C4([H])OC([H])(C([H])(C([H])([H])[H])C2([H])C([H])([H])C([H])([H])C([H])(C([H])([H])[H])C3([H])C([H])([H])C([H])([H])C(C([H])([H])[H])(O1)O4)O[H]

BJDCWCLMFKKGEE-ISOSDAIHSA-N

InChI=1S/C15H24O5/c1-8-4-5-11-9(2)12(16)17-13-15(11)10(8)6-7-14(3,18-13)19-20-15/h8-13,16H,4-7H2,1-3H3/t8-,9-,10+,11+,12+,13-,14-,15-/m1/s1

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

Dihydroartemisinin; Artenimol; DHQHS 2; Alaxin; JAV-110; VM-3352; AC-2067; JAV110; VM3352; AC 2067;JAV-110; VM 3352; AC 2067;

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)

DMSO : 25 ~50 mg/mL ( 87.92 ~175.83 mM )
Ethanol : 7~10 mg/mL(35.17 mM)

Solubility in Formulation 1: 2.08 mg/mL (7.31 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with heating and sonication.
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 (7.31 mM) 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; with heating and sonication.
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 (7.31 mM) 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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Solubility in Formulation 4: ≥ 1 mg/mL (3.52 mM) (saturation unknown) in 10% EtOH + 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 10.0 mg/mL clear EtOH stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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 5: ≥ 1 mg/mL (3.52 mM) (saturation unknown) in 10% EtOH + 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 10.0 mg/mL clear EtOH 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 6: ≥ 1 mg/mL (3.52 mM) (saturation unknown) in 10% EtOH + 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 10.0 mg/mL clear EtOH stock solution to 900 μL of corn oil and mix well.

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Solubility in Formulation 7: 6%DMSO + 94%Corn oil: 3mg/ml (10.55mM)

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