RelA;Plasmodium;Autophagy

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].

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].

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].

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].

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.

Absorption, Distribution and Excretion
The reported oral bioavailability of Artenimol was reported to be 45% in healthy adults. The observed Tmax was 1-2 h This is known to increase in malaria infected patients which could be attributed to reduced metabolism by the liver or the drug's collection in infected erythrocytes. Artenimol was obeserved to have flip-flop kinetics with an overall absorption half-life of 1.04 h. When administered with food the AUC for Artenimol increases by 144%. Cmax was observed to increase by 129% but was not found to be statistically significant. Food was observed to delay Tmax by 1 h.
Artenimol is eliminated via metabolism to glucuronide conjugates. There is little data on elimination of Artenimol but elimination of unchanged artemisinin compounds in feces and urine has been reported to be negligible.
Artenimol was observed to have a mean apparent volume of distribution of 0.801 L/kg in adult patients and 0.705 L/kg in pediatric patients wit *P. falciparum
malaria.
Artenimol was observed to have a mean apparent clearance of 1.340 L/h/kg in adult patients and 1.450 L/h/kg in pediatric patients with *P. falciparum
malaria.

---

Metabolism / Metabolites
The primary metabolite of Artenimol is the glucuronide conjugate, α-artenimol-β-glucuronide. It is largely metabolized by UGT1A9 with some contribution by UGT2B7.
Dihydroqinghaosu (DQHS) is a known human metabolite of beta-artelinic acid.

---

Biological Half-Life
Artenimol was reported to have a half life of elimination of approximately 1 h.

Protein Binding
Artenimol has been reported to be 44-93% bound to plasma proteins. The identity of these proteins has not been reported.

[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

Artenimol is an artemisinin derivative and antimalarial agent used in the treatment of uncomplicated *Plasmodium falciparum* infections. It was first authorized for market by the European Medicines Agency in October 2011 in combination with [DB13941] as the product Eurartesim. Artemisinin combination therapy is highly effective against malaria and stongly recommended by the World Health Organization.
Artenimol is an active metabolite of artesunate, with anti-malarial activity, and potential insulin sensitivity-improving, anti-inflammatory, immunomodulating and antineoplastic activities. Upon administration of artenimol and the hydrolysis of its active endoperoxide bridge moiety by liberated heme in parasite-infected red blood cells (RBCs), reactive oxygen species (ROS) and carbon-centered radicals form, which damage and kill parasitic organisms. Artenimol may also increase insulin sensitivity and improve insulin resistance. In addition, artenimol induces the 26S proteasome-mediated degradation of the androgen receptor (AR), thereby lowering AR expression, which may prevent androgen-responsive cellular proliferation. It also reduces luteinizing hormone LH) and testosterone levels, and may improve polycystic ovary syndrome (PCOS). In addition, artenimol may modulate the immune system and may inhibit tumor cell proliferation through various apoptotic and non-apoptotic pathways.

---

Drug Indication
For the treatment of uncomplicated *Plasmodium falciparum* infection in adults, children, and infants aged 6 months and up weighing over 5 kg. Used in combination with [DB13941].
FDA Label

---

Mechanism of Action
Artemisinins, including Artenimol which is a major active metabolite of many artemisinins, are thought to act via a common mechanism. While the exact mechanism of action is not certain, theories exist as to how artemisinins produce their antimalarial effect. Artemisinins are believed to bind to haem within the *P. falciparum* parasite. The source of this haem varies with the life stage of the parasite. When the parasite is in the early ring stage artemisinins are believed to bind haem produced by the parasite's haem biosynthesis pathway. In later stages artemisinins likely bind to haem released by haemoglobin digestion. Once bound to haem, artemisinins are thought to undergo activation involving ferrous iron via reductive scission which splits the endoperoxide bridge to produce a reactive oxygen. This reactive oxygen is thought to undergo a subsequent intramolecular hydrogen abstraction to produce a reactive carbon radical. The carbon radical is believed to be the source of the drugs potent activity against *P. falciparum* by alkylating a wide array of protein targets. The nature and magnitude of the effect on specific protein function as a result of this alkylation is unknown. One target which has been the focus of research is the sarco/endoplasmic reticulum Ca2+ ATPase pump of *P. falciparum*. Artemisinins have been found to irreversably bind to and inhibit this protein at a binding site similar to that of Thapsigargin. The mechanism is likely the same as for other proteins, namely alkylation via the carbon radical intermediate. Artemisinins appear to preferentially collect in infected erythrocytes, concentrating the drug by several hundred-fold compared to uninfected cells. This may play a role in why little alkylation is seen in uninfected erythrocytes.

---

Pharmacodynamics
Artenimol is thought to form a reactive carbon radical intermediate which kills *P. falciparum* through alkylation of a wide array of proteins.

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.

---

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.

---

View More

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.

---

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.

---

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.

---

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.

---

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

---

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