Concentrations Of Artesunate Research Articles

Population Pharmacokinetic and Pharmacodynamic Modeling of Artemisinin Resistance in Southeast Asia

Orally administered artemisinin-based combination therapy is the first-line treatment against uncomplicated P. falciparum malaria worldwide. However, the increasing prevalence of artemisinin resistance is threatening efforts to treat and eliminate malaria in Southeast Asia. This study aimed to characterize the exposure-response relationship of artesunate in patients with artemisinin sensitive and resistant malaria infections. Patients were recruited in Pailin, Cambodia (n = 39), and Wang Pha, Thailand (n = 40), and received either 2 mg/kg/day of artesunate mono-therapy for 7 consecutive days or 4 mg/kg/day of artesunate monotherapy for 3 consecutive days followed by mefloquine 15 and 10 mg/kg for 2 consecutive days. Plasma concentrations of artesunate and its active metabolite, dihydroartemisinin, and microscopy-based parasite densities were measured and evaluated using nonlinear mixed-effects modeling. All treatments were well tolerated with minor and transient adverse reactions. Patients in Cambodia had substantially slower parasite clearance compared to patients in Thailand. The pharmacokinetic properties of artesunate and dihydroartemisinin were well described by transit-compartment absorption followed by one-compartment disposition models. Parasite density was a significant covariate, and higher parasite densities were associated with increased absorption. Dihydroartemisinin-dependent parasite killing was described by a delayed sigmoidal Emax model, and a mixture function was implemented to differentiate between sensitive and resistant infections. This predicted that 84% and 16% of infections in Cambodia and Thailand, respectively, were artemisinin resistant. The final model was used to develop a simple diagnostic nomogram to identify patients with artemisinin-resistant infections. The nomogram showed > 80% specificity and sensitivity, and outperformed the current practice of day 3 positivity testing.

Light and scanning electron microscopy study of in vitro effects of artesunate in newly excysted metacercariae of Echinostoma paraensei (Trematoda: Digenea)

Chemotherapy of food-borne trematodes relies on two drugs, praziquantel and tricabendazole, and there is growing interest in finding alternative therapies. Plant oil extracts have long been used in traditional Chinese medicine as sources of bioactive compounds with antiparasitic activity. Species of the genus Echinostoma are used as good models to test effective compounds against food-borne trematodes. This study evaluated the anthelmintic activity of crude artesunate extracts in vitro on newly excysted metacercariae of Echinostoma paraensei by light and scanning electron microscopy (SEM). The flukes were incubated with 1 μg/mL, 10 μg/mL, 25 μg/mL, 50 μg/mL and 100 μg/mL of artesunate for 4, 12, 24, 48 and 72 h. When the exposure time and concentration of artesunate increased, there were changes in motor activity, tegument damage and death. Blebs and swelling were the most common damages quantified on the tegument. The in vitro study reproduced results described for other immature flukes incubated with artemisinin derivatives. Excysted metacercariae of E. paraensei constitute a good model to study in vitro drug effects.

Artesunate Decreases β-Catenin Expression, Cell Proliferation and Apoptosis Resistance in the MG-63 Human Osteosarcoma Cell Line

Background: This study aims to determine the effects of artesunate on proliferation, apoptosis and β-catenin expression in the human osteosarcoma cell line MG-63. Methods: MG-63 cells in the logarithmic growth phase were collected and cultured with different concentrations of artesunate (12.5 µg/mL, 25 µg/mL and 50 µg/mL) for 24 h, 48 h and 72 h. The total number of MG-63 cells and the morphological changes were observed under an inverted microscope. The MTT assay was adopted to test the inhibition rate (IR) of cell growth. The apoptosis rate was detected using annexin V/propidium iodide (PI) staining. Cell cycle distribution was identified by flow cytometry (FCM), and the expression levels of β-catenin, cyclins and cyclin dependent kinases (CDKs) were measured using Western blotting. Results: The results of the MTT assay indicated that artesunate could remarkably inhibit MG-63 cell proliferation compared with the rates in the untreated control group (0 µg/mL artesunate), and the inhibitory effect was dose-dependent. The apoptosis rate of MG-63 cells was elevated as the concentration of artesunate increased, and all the rates were significantly higher than that in the control group. Additionally, as the artesunate concentration increased, the proportion of MG-63 cells in G0/G1 phase gradually declined whereas that of cells in the G2/M and S phases increased. Western blotting confirmed that a higher concentration of artesunate reduced the expression levels of β-catenin, cyclin A, cyclin D1 and CDK1 and increased the expression levels of cyclin B1; however, artesunate had no impact on CDK2 expression in MG-63 cells. Conclusion: These results demonstrated that artesunate can inhibit β-catenin expression and cell proliferation as well as promote cell apoptosis in MG-63 cells, which indicates that artesunate may serve as a promising drug in the clinical treatment of osteosarcoma.

Low-cost, high-speed identification of counterfeit antimalarial drugs on paper

With the emergence of artesunate antimalarial counterfeiting in Southeast Asia and sub-Saharan Africa, we present the production of a rapid, inexpensive and simple colorimetric-based testing kit for the detection of counterfeit artesunate in order to preserve life and prevent the development of multi-drug resistant malaria. The kit works based on paper microfluidics which offer several advantages over conventional microfluidics, and has great potential to generate inexpensive, easy-to-use, rapid and disposable diagnostic devices. Here, we have developed a colorimetric assay that is specific to artesunate and turns yellow upon addition of the sample. The test can be done within minutes, and allows for a semi-quantitative analysis of the artesunate tablets by comparing the developed yellow color on the paper test to a color-coded key chart that comes with the kit. A more accurate and precise analysis is done by utilizing a color analyzer on an iPhone camera that measures the color intensity of the developed color on the paper chip. A digital image of the chip was taken and analyzed by measuring the average gray intensity of the color developed on the paper circle. A plot of the artesunate concentration versus the average gray scale intensity was generated. Results show that the intensity of the yellow color developed on the paper test was consistent and proportional to the amount of artesunate present in the sample. With artesunate concentrations ranging from 0.0 to 20mg/mL, a linear calibration plot was obtained with a detection limit of 0.98mg/mL.

Preliminary investigation of the contribution of CYP2A6, CYP2B6, and UGT1A9 polymorphisms on artesunate-mefloquine treatment response in Burmese patients with Plasmodium falciparum malaria.

CYP2A6, CYP2B6, and UGT1A9 genetic polymorphisms and treatment response after a three-day course of artesunate-mefloquine was investigated in 71 Burmese patients with uncomplicated Plasmodium falciparum malaria. Results provide evidence for the possible link between CYP2A6 and CYP2B6 polymorphisms and plasma concentrations of artesunate/dihydroartemisinin and treatment response. In one patient who had the CYP2A6*1A/*4C genotype (decreased enzyme activity), plasma concentration of artesunate at one hour appeared to be higher, and the concentration of dihydroartemisinin was lower than for those carrying other genotypes (415 versus 320 ng/mL). The proportion of patients with adequate clinical and parasitologic response who had the CYP2B6*9/*9 genotype (mutant genotype) was significantly lower compared with those with late parasitologic failure (14.0% versus 19.0%). Confirmation through a larger study in various malaria-endemic areas is required before a definite conclusion on the role of genetic polymorphisms of these drug-metabolizing enzymes on treatment response after artesunate-based combination therapy can be made.

Hypothesized cause of delayed hemolysis associated with intravenous artesunate

In recent publications, investigators described cases in which there was a delayed hemolysis following intravenous (IV) artesunate treatment. The delayed hemolysis event occurred at the nadir of blood hemoglobin concentration, i.e., at the time when blood hemoglobin concentration was switching from a progressive decline to a progressive increase. It is hypothesized that this nadir indicates the time when red cell production is resuming after having been arrested, the delayed hemolysis event is due to lysis of the first (aberrant) reticulocytes released once production is resumed and, therefore, that the hemolysis signals the resumption of red cell production. Since this delayed hemolysis has not been associated with a significant decrease in blood hemoglobin, the hemolytic event is not of particular concern even if it could be attributed to artesunate. More important than this hemolysis event was the preceding progressive anemia that lasted for up to 19days. Both a decrease in reticulocyte production and a shortened life span of previously infected red cells likely contributed to the anemia. The question that remains to be answered is whether the progressive anemia that lasted 2–3weeks in these patients was attributable solely to their severe malaria or was possibly enhanced and prolonged by the high plasma concentrations of artesunate associated with IV administration. Controlled clinical studies addressing this question may be needed.

OP221: In vitro and in vivo inhibition effects of artesunate on a novel human palatal salivary gland adenoid cystic carcinoma cell line

To investigate the effects of artesunate on adenoid cystic carcinoma cell line named NACC. Cell growth inhibition was detected by methyl thiazolyl tetrazolium and clone formation assaies after NACC cell line were treated with various concentrations of artesunate in vitroly, morphological changes were observed by inverted microscopy and electron microscopy. Wound healing and flow cytometry assay was used to analyze NACC cell migration, cell cycles and cell apoptosis after artesunate treated. NACC cells were inoculated subcutaneous into BABL-C nude mice for xenografts and treatment started on the 7th day when solid tumors were palpated, grouping as: control, artesunates, cialpatin and artesunate plus cialpatin treated groups. Drugs were given everyother days for eight times. On the 23th day, the animals were sacrificed then tumors were excised and weighted. Tumors were also investigated by immunohistochemistry and western bloting. The 25–200 μg/ml artesunate produced proliferation inhibition and apoptosis induction on NACC cell line in a time-and does-dependent manner. IC 50 of artesunate in 24, 48 and 72 h were 113.09, 74.80, 35.61 μg/ml respectively. Cell cycle of NACC cell could be arrested the in G1 phase by 12.5–50.0 μg/ml and G2 phase by 50.0–200.0 μg/ml of artesunate. Artesunate could change the morphological structure and significantly inhibited the migration and clone formation of NACC cell. Artesunate 200 mg/kg groups efficiently inhibited the growth of NACC xenograft; artesunate 50 mg/kg combined with cisplatin 1.0 mg/kg also resulted in significant growth inhibit of the tumors. VEGF, Bcl-2, ERK2 were down-regulated in ART 100 mg/kg, ART 200 mg/kg and ART plus DDP groups. In artesunate 200 mg/kg and ART plus DDP groups had different degrees of down-regulation of CXCR4, CD44, CD133 express. ART might significantly inhibit the growth of NACC cell in vitro and in vivo, by adjusting the cell cycle, inducing apoptosis and inhibiting neovascularization.

Optimizing the HRP-2 in vitro malaria drug susceptibility assay using a reference clone to improve comparisons of Plasmodium falciparum field isolates

BackgroundApparent emerging artemisinin-resistant Plasmodium falciparum malaria in Southeast Asia requires development of practical tools to monitor for resistant parasites. Although in vitro anti-malarial susceptibility tests are widely used, uncertainties remain regarding interpretation of P. falciparum field isolate values.MethodsPerformance parameters of the W2 P. falciparum clone (considered artemisinin “sensitive”) were evaluated as a reference for the HRP-2 immediate ex vivo assay. Variability in W2 IC50s was assessed, including intra- and inter-assay variability among and between technicians in multiple experiments, over five freeze-thaw cycles, over five months of continuous culture, and before and after transport of drug-coated plates to remote field sites. Nominal drug plate concentrations of artesunate (AS) and dihydroartemisinin (DHA) were verified by LC-MS analysis. Plasmodium falciparum field isolate IC50s for DHA from subjects in an artemisinin-resistant area in Cambodia were compared with W2 susceptibility.ResultsPlate drug concentrations and day-to-day technical assay performance among technicians were important sources of variability for W2 IC50s within and between assays. Freeze-thaw cycles, long-term continuous culture, and transport to and from remote sites had less influence. Despite variability in W2 susceptibility, the median IC50s for DHA for Cambodian field isolates were higher (p <0.0001) than the W2 clone (3.9 nM), both for subjects with expected (less than 72 hours; 6.3 nM) and prolonged (greater or equal to 72 hours; 9.6 nM) parasite clearance times during treatment with artesunate monotherapy.ConclusionThe W2 reference clone improved the interpretability of field isolate susceptibility from the immediate ex vivo HRP-2 assay from areas of artemisinin resistance. Methods to increase the reproducibility of plate coating may improve overall assay interpretability and utility.

A radiosensitizing effect of artesunate in glioblastoma cells is associated with a diminished expression of the inhibitor of apoptosis protein survivin

Background and purposeNovel strategies to overcome an irradiation resistant phenotype may help to increase therapeutic efficacy in glioblastoma multiforme. The present study aimed to elucidate radiation sensitizing properties of artesunate, a semi synthetic derivate of artemisinin and to assess factors involved in this effect. Materials and methodsLN229 and U87MG cells were treated with various concentrations of artesunate and radiation response was determined by a colony forming assay. Cell numbers, apoptosis induction, cell cycle distribution, and DNA repair following combined modality treatment were monitored by MTT-, caspase 3/7 assay, cytofluorometry, and γ-H2AX foci formation. Expression of survivin, survivin–GFP fusion protein, XIAP, cellular (c)IAP1 and cIAP2 was monitored by Western immunoblotting. ResultsTreatment of glioma cells with artesunate and irradiation resulted in an increased apoptotic fraction, pronounced G2/M arrest and increased DNA damage as demonstrated by an elevated amount of γ-H2AX foci/nucleus. Incubation with artesunate lowers survivin expression in a time and dose-dependent manner, whereas expression of XIAP, cIAP1 and cIAP2 was not affected. In clonogenic assays, treatment with artesunate revealed a significantly reduced surviving fraction, whereas stable over expression of a survivin–GFP protein reversed artesunate-mediated radiosensitization. ConclusionArtesunate selectively down regulates survivin that contributes to a radio-sensitization of glioma cells by an increased induction of apoptosis, cell cycle arrest, and a hampered DNA damage response.

Pharmacokinetics and pharmacodynamics of intravenous artesunate during severe malaria treatment in Ugandan adults

BackgroundSevere malaria is a medical emergency with high mortality. Prompt achievement of therapeutic concentrations of highly effective anti-malarial drugs reduces the risk of death. The aim of this study was to assess the pharmacokinetics and pharmacodynamics of intravenous artesunate in Ugandan adults with severe malaria.MethodsFourteen adults with severe falciparum malaria requiring parenteral therapy were treated with 2.4 mg/kg intravenous artesunate. Blood samples were collected after the initial dose and plasma concentrations of artesunate and dihydroartemisinin measured by solid-phase extraction and liquid chromatography-tandem mass spectrometry. The study was approved by the Makerere University Faculty of Medicine Research and Ethics Committee (Ref2010-015) and Uganda National Council of Science and Technology (HS605) and registered with ClinicalTrials.gov (NCT01122134).ResultsAll study participants achieved prompt resolution of symptoms and complete parasite clearance with median (range) parasite clearance time of 17 (8–24) hours. Median (range) maximal artesunate concentration (Cmax) was 3260 (1020–164000) ng/mL, terminal elimination half-life (T1/2) was 0.25 (0.1-1.8) hours and total artesunate exposure (AUC) was 727 (290–111256) ng·h/mL. Median (range) dihydroartemisinin Cmax was 3140 (1670–9530) ng/mL, with Tmax of 0.14 (0.6 – 6.07) hours and T1/2 of 1.31 (0.8–2.8) hours. Dihydroartemisinin AUC was 3492 (2183–6338) ng·h/mL. None of the participants reported adverse events.ConclusionsPlasma concentrations of artesunate and dihydroartemisinin were achieved rapidly with rapid and complete symptom resolution and parasite clearance with no adverse events.

Impact of artesunate on the expression and secretion of transforming growth factor-b1 of primary rat hepatic stellate cells

To investigate the impact of the Artemisia annua plant-derived drug, artesunate, on proliferation of primary rat hepatic stellate cells (HSCs), and to analyze the underlying molecular mechanisms of its anti-fibrogenic effects involving the inhibition of transforming growth factor-beta 1 (TGF-b1) expression and secretion in liver. Isolated, cultured, and activated primary rat HSCs were divided into sixteen groups, including one untreated control group and fifteen artesunate-treated experimental groups with 125, 150, 175, 200 or 225 mumol/L for 24, 48 or 72 hours. The rate of cellular proliferation was measured using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. TGF-b1 mRNA expression was evaluated by reverse transcription-polymerase chain reaction and protein expression was evaluated by Western blotting. Enzyme-linked immunosorbent assay was used to evaluate secreted levels of TGF-b1 protein. Artesunate significantly inhibited proliferation of cultured HSCs in a dose- and time-dependent manner (all, P less than 0.01). After 24 hours of exposure, the inhibition ratios of the various artesunate concentrations were: 6.06%+/-1.44% (125 mumol/L), 21.47%+/-5.57% (150 mumol/L), 42.00%+/-7.36% (175 mumol/L), 67.12%+/-4.55% (200 mumol/L), and 79.83%+/-3.67% (225 mumol/L). Artesunate significantly inhibited the TGF-b1 mRNA expression in HSCs, and the higher the drug concentration, the higher the degree of inhibition (all, P less than 0.01). In addition, artesunate significantly inhibited the expression of intracellular and secreted TGF-b1 protein (all, P less than 0.01). In response to artesunate (mumol/L concentrations), the TGF-b1 levels were (164.24+/-6.88) pg/ml (0μmol/L), (102.68+/-4.45) pg/ml (150μmol/L), (86.54+/-5.56) pg/ml (175μmol/L), and (56.55+/-5.66) pg/ml (200μmol/L). Artesunate exerts anti-fibrogenic effects on HSCs in vitro, possibly by reducing the expression, translation and secretion of TGF-b1.

Expression of p53 in the Effects of Artesunate on Induction of Apoptosis and Inhibition of Proliferation in Rat Primary Hepatic Stellate Cells

BackgroundActivation of hepatic stellate cells (HSCs) plays an important role in the development of cirrhosis through the increased production of collagen. p53, the “guardian of the genome”, is a transcription factor that can bind to promoter regions of hundreds of genes where it either activates or suppresses gene expression. Thereby, p53 serves as a tumor suppressor by inducing cell cycle arrest, apoptosis, senescence and DNA repair. Artesunate is a derivative of Artemisinin, Scholars had found it had more extensive pharmacological effects past 10 years. However, little is known about the expression of p53 in the effects of Artesunate on induction of apoptosis and inhibition of proliferation in rat HSCs.Methodology/Principal FindingsIsolated and cultured rat primary HSCs in the flask for 10 days to make cells activated. HSCs were divided into two groups: experimental groups and control groups, experimental groups included with various concentrations of Artesunate (125, 150, 175, 200, 225 µmol/L) for 24, 48 and 72 hours. Analysis of MTT revealed that activated HSCs treated with various concentrations of Artesunate (150–225 µmol/L) were inhibited on dose and time-effect relationships; Concentration of hydroxyproline in supernatant was detected by digestive method; Analysis of flow cytometry demonstrated that Artesunate could arrest cell cycle in G1 and induce apoptosis; The nuclear morphological changes in apoptotic cells were evaluated with DNA staining by Hoechst 33258 dye; The expression of p53 were up-regulated showed by western blotting and RT-PCR.ConclusionArtesunate could inhibit HSCs proliferation in dose-dependent and time-dependent manners in vitro through increase the expression of p53.

In vitro evaluation of the genotoxic and cytotoxic effects of artesunate, an antimalarial drug, in human lymphocytes

Artesunate is one of the main antimalarial drugs used in several countries. It is a semisynthetic compound derived from artemisinin, a substance extracted from the Chinese plant, Artemisia annua L. Despite the widespread use of artesunate as an antimalarial drug, there is a lack of data regarding its genotoxic effects in human lymphocytes. Therefore, in this study, we used the comet assay and micronucleus test to evaluate the possible genotoxic effects of artesunate in cultured human lymphocytes. In addition, cell death by necrosis and apoptosis was also assessed. Cells exposed to different concentrations of artesunate showed a significant concentration-dependent increase (P < 0.05) in DNA damage index and micronuclei frequency. A significant increase in the frequency of apoptotic and necrotic cells was also observed. Our results showed that artesunate is a genotoxic and cytotoxic compound in cultured human lymphocytes.

The Presence of Leukocytes in Ex Vivo Assays Significantly Increases the 50-Percent Inhibitory Concentrations of Artesunate and Chloroquine against Plasmodium vivax and Plasmodium falciparum

Plasmodium species ex vivo sensitivity assay protocols differ in the requirement for leukocyte removal before culturing. This study shows that the presence of leukocytes significantly increases the 50% inhibitory concentration (IC₅₀) of P. vivax and P. falciparum to artesunate and chloroquine relative to results with the paired leukocyte-free treatment. Although leukocyte removal is not an essential requirement for the conduct of ex vivo assays, its use has important implications for the interpretation of temporal and spatial antimalarial sensitivity data.

Localization of artesunate and its derivatives in the pregnant rat and fetus following oral administration and relationship to developmental toxicity

The antimalarial drug artesunate affects erythroid cells leading to developmental toxicity and adult reticulocytopenia. We report on a kinetic study in rats and the tissue distribution of radioactivity following oral administration of [(3)H]-artesunate to pregnant rats using quantitative whole-body autoradiography (QWBA). Rats were dosed orally with chlorproguanil/dapsone/artesunate (including 11.8 mg/kg artesunate) and plasma concentrations of artesunate and the active metabolite dihydroartemisinin (DHA) were determined. In the QWBA study, 6 rats received 13 mg/kg [(3)H]-artesunate on day 18 of gestation. Groups of 2 rats were euthanized at 1, 6, and 24 hours after dosing, rapidly frozen, and sectioned in a cryostat. Sagittal sections were freeze-dried and placed in contact with imaging plates. Tissue concentrations of radioactivity were quantified. Systemic exposure to DHA was up to 22-fold higher than the parent compound and was higher in non-pregnant females than males. In the QWBA study, high concentrations of radioactivity were seen in maternal tissues involved in absorption and excretion, the bone marrow and spleen. Fetal blood and liver levels were 3.8- to 8.8-fold higher than maternal blood levels at all timepoints. Excluding tissues involved in absorption and excretion, the highest concentrations of radioactivity were observed in tissues involved in hemoglobin synthesis and/or destruction in both the mother and the fetus and likely account for the maternal reticulocytopenia and embryotoxicity. Radioactivity concentrations in the fetal blood were 2.1- to 2.8-fold higher than maternal bone marrow at all timepoints and this difference could contribute to the lower dose threshold for embryotoxicity.

The sensitivity of artesunate against Schistosoma japonicum decreased after 10 years of use in China

Schistosomiasis due to Schistosoma japonicum is a major public health problem in China. Since 1995, artesunate has been used to treat and prevent schistosome infections in China. Artesunate previously showed a high prophylactic efficacy against schistosome infection, with a protection rate of 100%. However, recent clinical trials and animal experiments have found that the sensitivity of many schistosomes to artesunate, including Schistosoma mekongi and Schistosoma mansoni, decreased. Whether the prophylactic efficacy of artesunate on Schistosomiasis japonica decreased after being used over 10-year period was still unknown. In the current study, we conducted a double-blind trial and found that the protection rate of artesunate was only 13.5% in the Administration I group, whose dosage schedule was identical to schedules used in previous studies. Therefore, the sensitivity of S. japonicum to artesunate was confirmed to have decreased after being using for over 10 years. Moreover, when we increased the concentration of artesunate during the first week and third week, the protection reached 74.8%.

Effects of Artemisinin Derivative on the Growth Metabolism of Tetrahymena thermophila BF5 Based on Expression of Thermokinetics

The toxic effects of artesunate and dihydroartemisinin on the growth metabolism of Tetrahymena thermophila BF5 were studied by microcalorimetry. The results showed that: (1) low concentrations of artesunate (<or=1 mg L(-1)) and dihydroartemisinin (<or= 2 mg L(-1)) promoted the growth metabolism of T. thermophila BF5, whereas high concentrations of artesunate (1-60 mg L(-1)) and dihydroartemisinin (2-60 mg L(-1)) inhibited its growth; (2) the half inhibition concentrations IC(50) of artesunate and dihydroartemisinin were 17.5817 and 9.5089 mg L(-1), respectively. It was concluded that the inhibition of dihydroartemisinin was stronger than that of artesunate.

Artesunate induced morphological and mechanical changes of Jurkat cell studied by AFM

In this study, we have used atomic force microscopy (AFM) to study the morphology and mechanical property changes of Jurkat cells exposed to different concentrations of Artesunate (ART) for 24 h at single cellular level. Cell viability and proliferation assays were performed by using the Cell Counting Kit-8. The concentration of ART, which resulted in the inhibition rate >50% was selected. The AFM images revealed that the cell membrane changed and the ultrastructure also became complex. Mechanical properties of individual cell were tracked with AFM-based force spectroscopy. The force curves revealed that when a cell was exposed to the ART, the mechanical properties changed obviously. Treated cells had a lower adhesion force of 416.8+/-37.9 pN, whereas control group had a higher adhesion force of 1064.2+/-97.0 pN. The Young's modulus decreased to nearly one-third, from control group of 0.648+/-0.037 kPa to treated group of 0.254+/-0.035 kPa and the stiffness increased to nearly 1.5 times, from control group of 1.231+/-0.084 mN/m to treated group of 1.917+/-0.137 mN/m. These results suggest that ART can inhibit the proliferation of Jurkat and induce changes in the morphological structure and mechanical properties of Jurkat cells. The high resolution and high sensitivity of AFM can be used to detect morphological and mechanical properties of cells exposed to ART. The AFM may be developed to be a useful tool for detecting the cell death and evaluating the anti-carcinogen efficacy against tumor cell. SCANNING 31: 83-89, 2009. (c) 2009 Wiley Periodicals, Inc.

Study on the Change of Iron Transporter Expression in k562 Cells Apoptosis Induced by Artesunate

Background and objective The antitumor effect of artemisinin was widely reported in vivo and ex vivo, but the possible mechanisms have yet been poorly understood. As is the anti-malaria effect of artemisinin, the tumour cells have the richer iron than the normal cells, which maybe result to be sensitive to artemisinins. Moreover, it is a very interesting question whether artesininin and its derivates can affect the iron metabolism of tumor cells and finally result to the antitumor effect. So in this study, we investigate the expression change of iron transporter including TfR1 (transferrin receptor-1, CD71), DMT1(Divalent metal transporter 1) and FPN1(Ferroportin 1) in the wild-type K562 cell(K562-W) and imatinib-resistant K562 cell(K562-R) treated with artesunates.Methods The K562-W cells and K562-R cells were treated with 4×10−5 mol/L, 2×10−4 mol/L and 1×10−3 mol/L artesunate for 24, 48 and 72 hours respectively. The control groups include the untreated group and the group treated with desferrioxamine(20 umol/L). The different expressions of TfR1, DMT1 and FPN1 between the groups treated with artesunates and the control groups were analyzed by Semi-quantitative RT-PCR, Western-Blotting or flow cytometry. Apoptosis was measured by Annexin V-PI and Hochest 33258 staining.Results The artesunate can induce K562-W and K562-R cells apoptosis in time- and concentration-dependent manner. When K562-W cells treated with 4×10−5 mol/L, 2×10−4 mol/L and 1×10−3 mol/L of artesunate for 48 hours, the mRNA expression of TfR1 is lower in artesunate groups than in the untreated groups. DMT1 mRNA expression in low concentration of artesunate and desferrioxamine group is lower than untreated group, and there is no expression of DMT1 mRNA in middle and high concentration artesunate group. The expression of FPN1 mRNA in artesunate group and desferrioxamine group is lower than the untreared group. The positive rate of CD71 is lower in artesunate groups than untreated group by flow cytometry analysis. The expression of DMT1 protein in desferrioxamine group is lower than untreated group and no expression of DMT1 protein in artesunate groups by Western blot. The TfR1 mRNA expression of K562-R cells treated with 4×10−5mol/L, 2×10−4mol/L and 1×10−3mol/L artesunate for 48 hours is lower in artesunate groups than untreated group by Semi-quantity RT-PCR. DMT1 mRNA expression in low concentration artesunate group is lower than untreated group, and there is no expression of +IRE-DMT1 mRNA in middle and high concentration artesunate group. The expression of FPN1 mRNA in artesunate group is lower than that in the untreated group. The positive rate of CD71 is lower in artesunate groups than untreated group by flow cytometry analysis. There is no expression of DMT1 protein in middle concentration artesunate groups by Western blot.Conclusions Artesunate may reduce TfR1 and DMT1 expression in mRNA and/or protein level in K562-W and K562-R and lead to reduce the iron intake, iron transportation from the endosome to the cytoplasm and iron utilization in the mitochondria. Furthermore, Artesunate also reduce the expression of FPN1 mRNA to inhibit the outflow of iron. In addition to the generation of free radicals inducing to cell death, Artesunate can inhibit cell proliferation and induce cell apoptosis by the regulation of iron transporter.

Absence of time-dependent artesunate pharmacokinetics in healthy subjects during 5-day oral administration

The aim of the present study was to investigate the existence of time-dependent pharmacokinetics of artesunate (ARS) during 5 consecutive days of oral administration to 10 healthy Vietnamese subjects (aged 21-52 years and weighing 49-90 kg). Each volunteer received 200 mg oral doses of ARS once daily for 5 consecutive days. Blood samples (3 ml each) were collected on days 1 and 5 at 0 (before dosing), 0.5, 1, 2, 3, 4, and 6 h after drug administration. During days 2, 3, and 4, the same volumes of blood were collected at 0, 1, 2, and 4 h after dosing. Plasma ARS and dihydroartemisinin (DHA) were measured using high-performance liquid chromatography-mass spectrometry (LC-MS). Results did not show evidence of time-dependency for ARS or the active plasma metabolite DHA. There were no differences in the concentrations of ARS and DHA at all sampling times on days 1 and 5. In addition, the pharmacokinetics of both compounds were similar on days 1 and 5. This finding confirms that the enzyme auto-induction in drug metabolism may not be characteristic of the endoperoxide sesquiterpene antimalarial group.