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Journal of Antimicrobial Chemotherapy (2009) 63, 543 - 549doi:10.1093/jac/dkn550Advance Access publication 24 January 2009 Pharmacokinetic parameters of artesunate and dihydroartemisinin Jennifer Keiser1*, Marie-Stella Gruyer2, Nancy Perrottet3, Boris Zanolari3, Thomas Mercier3 1Swiss Tropical Institute, PO Box, CH-4002 Basel, Switzerland; 2Hoffmann-La Roche Ltd, Grenzacherstrasse 124, CH-4070 Basel, Switzerland; 3Laboratory of Division of Clinical Pharmacology, BH18-218, CHUV, University Received 18 August 2008; returned 26 September 2008; revised 23 October 2008; accepted 19 December 2008 Objectives: The pharmacokinetic (PK) parameters of artesunate, recently discovered to possess prom-ising trematocidal activity, and its main metabolite dihydroartemisinin (DHA) were determined in ratsinfected with hepatic and biliary stages of Fasciola hepatica and compared with uninfected rats aftersingle intragastric and intravenous (iv) doses.
Methods: Rats infected with F. hepatica for 25 and 83 days and uninfected rats were cannulated in theright jugular vein and blood samples were withdrawn at selected timepoints following 10 mg/kg of ivand a single 100 mg/kg oral dose of artesunate. Plasma was analysed for artesunate and DHA by liquidchromatography coupled to tandem mass spectrometry.
Results: Rats harbouring juvenile and adult F. hepatica infections revealed considerable changes in PKparameters of artesunate and DHA. Following oral administration, maximum plasma concentrations(Cmax) of artesunate and DHA were 1.8-2.3-fold higher in infected rats [artesunate: 1334+ 1404 ng/mL(no infection) versus 2454+ 1494 ng/mL (acute infection) and 2768+ 538 ng/mL (chronic infection); DHA: 3802+ 2149 ng/mL (no infection) versus 6507+ 3283 ng/mL (acute infection) and 9093+ 884 ng/mL(chronic infection)]. The AUCs of artesunate and DHA were 2.1-4.4-fold greater in infected rats. An oppo-site trend was observed after iv injection. Cmax and AUC of artesunate and DHA following iv dosing were5784+ 3718 and 140 938+ 128 783 ng.min/mL and 3849+ 3060 and 86107+ 41863 ng.min/mL, respect-ively, in uninfected rats versus 2623+ 1554 and 21617+ 12230 ng.min/mL and 2835+ 980 and64290+ 29057 ng.min/mL, respectively, in rats harbouring a chronic infection. The elimination half-lives(t1/2) of artesunate and DHA were considerably altered in infected rats following oral and iv administrationof artesunate.
Conclusions: F. hepatica infections strongly influence the disposition kinetics of artesunate and itsmetabolite in rats. The clinical implications of this finding need to be carefully studied.
Keywords: food-borne trematodiasis, artemisinins, LC-MS/MS associated with fascioliasis are estimated to be $2 - 3 billionannually.1,2 Fascioliasis is an infection caused by the liver flukes Fasciola In the absence of vaccines, triclabendazole is the main drug hepatica and Fasciola gigantica. Fascioliasis is a zoonotic used to treat liver fluke infections as it is highly effective against disease of global distribution, which is of considerable public juvenile and adult Fasciola spp.3 However, resistance to the health significance and veterinary importance. It is estimated drug in fluke populations first developed in Australian livestock that 91 million people are at risk and between 2.4 and 17 in the mid-1990s and has since been reported from several million people are infected with F. hepatica and/or F. gigantica.
European countries. No confirmed cases of triclabendazole Approximately 300 million cattle and 250 million sheep are resistance in human Fasciola infections have been documented infected with liver flukes. Economic losses in livestock yet, but this may happen soon, given the spread of resistance in .
*Corresponding author. Tel: þ41-61-284-8218; Fax: þ41-61-284-8105; E-mail: jennifer.keiser@unibas.ch .
# The Author 2009. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.
For Permissions, please e-mail: journals.permissions@oxfordjournals.org livestock Fasciola populations.1,3 Hence, there is a pressing need dark cycle) and acclimatized for 1 week. They had free access to to develop novel trematocidal drugs.
water and rodent food (Rodent Blox from Eberle NAFAG; Gossau, The artemisinins, well known for their antimalarial4 and anti- Switzerland). Three groups were formed. Rats of groups 1 and 2 schistosomal5 properties, are also active against Fasciola spp.6 (n ¼12) were each infected intragastrically with 20 metacercarial Complete worm burden reductions were achieved in rats infected cysts of F. hepatica. F. hepatica metacercariae (Cullompton isolate) with adult F. hepatica after treatment with artesunate and arte- were purchased from Mr G. Graham (Addlestone, UK). Six rats mether at single oral doses of 400 and 200 mg/kg, respectively.
Administration of artesunate and artemether at a 200 mg/kg dose Twenty-five (group 1) and 83 (group 2) days post-infection with to rats harbouring juvenile F. hepatica resulted in worm burden F. hepatica, six rats each and also the six uninfected rats (group 3)were cannulated in the right jugular vein following standard pro- reductions of 46% and 82%, respectively.6 Artemether has also cedures. The animals were allowed to recover for 3 days. Three days shown promising fasciocidal properties in sheep.7 In addition, a after implantation of the catheter, rats in each group were further recent study in Vietnam has shown that artesunate might also divided into two subgroups of three rats each-receiving either oral play a role in the treatment of acute human fascioliasis.8 or iv artesunate. The iv doses were injected for 60 s via the jugular Infections with Fasciola spp. are well known to dramatically vein cannula, immediately followed by injection of 1 mL of saline impair the hepatic function, including the metabolism and phar- macokinetic (PK) parameters of drugs.9,10 Hence, with a poss- Blood samples (0.2 mL) were withdrawn from the cannulated ibly increasing role of the artemisinins in the treatment of jugular vein into heparinized tubes at 5, 15, 30, 45, 60, 90, 120, 240 fascioliasis, it is important to determine the PK parameters of and 300 min following the iv dose. After oral treatment, blood these drugs during the course of Fasciola infections.
samples were taken at 15, 30, 45, 60, 90, 120, 180, 240, 360 and Here, we compare the PK characteristics of artesunate and its 480 min. The blood volume was replaced with an equal volume of main metabolite dihydroartemisinin (DHA) (Figure 1) in rats 0.9% saline solution. The blood samples were centrifuged and the infected with juvenile and adult F. hepatica and in uninfected plasma was taken and stored at 2808C until analysed by LC-MS/ rats. Plasma drug levels were determined by liquid chromato- MS. At the end of the experiments, all rats were sacrificed by the graphy coupled to tandem mass spectrometry (LC-MS/MS). We CO2 method and the parasitic pathology was ascertained by examin- furthermore evaluated two different routes of drug adminis- tration, an oral and an intravenous (iv) drug administration.
Solvents and chemicals. DHA and artesunate were kind gifts fromAbbott (Baar, Switzerland). The internal standard (IS) artemisinin Germany). Ultrapure water was obtained from a Milli-Qw UF-Plus apparatus (Millipore Corp., Burlington, MA, USA). Ammonium Switzerland). For the oral administration (100 mg/kg), artesunate formate was purchased from Fluka (Buchs, Switzerland). Sodium was prepared in a homogeneous suspension in 7% (v/v) Tween-80 nitrite, acetic acid (glacial) 100% anhydrous, formic acid (98%), and 3% (v/v) ethanol. The iv preparation (10 mg/kg) was prepared methanol (MeOH) and acetonitrile (MeCN) for chromatography with 0.9% (v/v) sterile NaCl. The drug formulations were prepared were purchased from Merck (Darmstadt, Germany). All other Ethical clearance, animals, infection, study design and PK Instrumentation. The liquid chromatography system consisted of a sampling. All animal studies presented here were approved by the Rheos 2200 quaternary pump, equipped with an online degasser local government based on Swiss national regulations ( permission and a HTS PAL autosampler (CTC Analytics AG, Zwingen, Switzerland) and controlled by Janeiro-CNS software. Separations Female Wistar rats (n ¼18; age, 5 weeks; weight, $100 g) were were done on a 2.1 mmÂ50 mm Atlantisw dC18 3 mm analytical purchased from RCC (Itingen, Switzerland). Rats were kept in groups of five in macrolon cages in environmentally controlled con- 2.1 mmÂ10 mm guard column containing the same packing ditions (temperature, $258C; humidity, $70%; 12 h light and 12 h material and placed in a thermostated column heater at 258C (model Figure 1. Chemical structures of artesunate, DHA and artemisinin (IS).
PK parameters of artesunate and DHA in rats infected with F. hepatica Croco-Cil, Cluzeau, France). The chromatographic system was Sample preparation. A rat plasma sample aliquot (100 mL) onto coupled to a triple-stage quadrupole Quantum Ion Max mass spec- which 30 mL of 3 M sodium nitrite containing 1% acetic acid trometer (MS) from Thermo Scientific, Inc. (San Jose, CA, USA), ( pH %4.3) was added was placed in a water bath at 378C for equipped with an electrospray ionization (ESI) interface and oper- 30 min. Addition of sodium nitrite was found to successfully ated with Xcalibur 2.0 software (Thermo). The mobile phase used prevent the apparent degradation of artesunate and DHA in haemo- for chromatography was 20 mM ammonium formate in ultrapure lysed samples. The beneficial effect of sodium nitrite on DHA and water adjusted to pH 4.1 with formic acid (98%) (solution A) and artesunate recovery had been verified using plasma samples spiked acetonitrile (solution B). The mobile phase was delivered at a flow with DHA, artesunate and artemisinin onto which 0.5%, 1% or 2% rate of 0.3 mL/min using the following stepwise gradient elution (v/v) of haemolysed red blood cells was added. Conversely, sodium programme: 0 min, 75/25% of A/B; 4 min, 55/45%; 10.0 min, 40/ nitrite was found not to alter DHA and artesunate levels in non- 60%; and 12 min, 100% of B. The second part of the run included haemolysed plasma levels. A 100 mL volume of IS solution 4 min of intensive washing (100% B with a flow rate of 0.5 mL/ (2000 ng/mL) was then added and the resulting sample was sub- min) and re-equilibration step to the initial solvent up to 20 min (at jected to protein precipitation with acetonitrile (600 mL). Sample 16.01 with 0.5 mL/min and after 19 min with a flow rate of 0.3 mL/ was vortex-mixed and centrifuged for 10 min at 14 000 rpm min). The injection volume was 10 mL.
(18 620 g) at 48C (Benchtop Universal 16R centrifuge, Ba¨ch, The tuning of MS/MS parameters was performed by direct infu- Switzerland). The supernatant (800 mL) was transferred into a poly- sion of each compound solution in the ESI separately: DHA, artesu- propylene tube and evaporated to dryness under nitrogen at room nate and artemisinin (1 mg/mL in 50:50 of solution A buffer/ temperature. The solid residue was reconstituted in 130 mL of MeOH). The LC-MS/MS conditions were as follows: ESI in posi- MeOH, vortex-mixed and centrifuged again under the above- tive mode; capillary temperature, 3508C; in source collision-induced mentioned conditions. A 100 mL aliquot of supernatant was mixed dissociation, 10 V; tube lens range voltage, 41 - 56 V; spray voltage with 100 mL of buffer (solution A) to give the reconstituted extract 4 kV; and sheath and auxiliary gas (nitrogen) flow rate of 35 psi and placed in glass HPLC micro-vials kept at 48C in the autosampler 10 arbitrary units, respectively. The selected m/z transitions were 221.1!163.1 with a collision energy (CE) of 24 eV for DHA andartesunate and 283.1!247.2 with a CE of 20 eV for artemisinin(IS). Because of the specificity of product ions, no cross-talk was observed between MS transitions. The Q2 collision gas (argon) PK parameters for artesunate and DHA were determined by non- compartmental analysis using WinNonLin (Version 5.2, Pharsight The MS/MS acquired in selected reaction monitoring. MS acqui- Corporation, USA). Cmax and Tmax were the observed values. The sitions were done in centroid mode and the mass resolution was set AUC (from 0 to infinity) was estimated using the linear trapezoidal at full-width half-maximum equal to 0.7, corresponding to 1 amu rule. The elimination half-life (t1/2) was calculated by the equation: t1/2 ¼0.693/l, where l was estimated by performing a regression ofthe natural logarithm of the concentration values in this range onsampling time. Clearance (CL) was calculated as dose/AUC. The Calibration and quality control (QC) plasma samples. Standard stock solutions of DHA and artesunate were prepared in methanol.
The stock solutions were protected from light and stored at 2208C.
ss ¼ MRTINFÂCL, where MRTINF ¼ mean resi- dence time when the drug concentration profile is extrapolated to Appropriate quantities of stock solutions were diluted with H2O to infinity and CL ¼total body clearance. Absolute bioavailability (F) obtain working solutions of 40 mg/mL DHA and artesunate in for non-infected, acute infections and chronic infections of artesu- MeOH/H2O (1:1). Plasma calibration samples of 1, 5, 10, 100, 500, 1000 and 2000 ng/mL DHA and artesunate, and three plasma QC samples of 15, 150 and 1500 ng/mL were prepared by serial dilution The above indices were determined for each individual animal (with MeOH/H2O, 1:3) of working solution and diluted 1:20 with and their arithmetic mean (+SD) was calculated.
blank plasma from rat, in accordance with the recommendations ofbioanalytical method validation stating that the total added volumemust be 10% of the biological sample volume.11 Blank rat plasma pool for the preparation of matrix-matched calibration and controlsamples was collected by cardiac puncture of rats. Rat plasma PK parameters between infected and uninfected rats were compared samples were stored at 2808C until analysis.
using the Kruskal - Wallis test (KW). A P value ,0.05 was con- A 100 mg/mL stock solution for the IS (artemisinin) was also sidered statistically significant. Version 2.4.5 of Statsdirect statistical prepared in MeOH and kept at 2208C, and was diluted to 2000 ng/ software (Statsdirect Ltd, Cheshire, UK) was used for the statistical mL with MeOH on the day of analysis. The calibration curves were fitted by least-squares quadratic regression using 1/concentration (1/x) as weighting factor of the peak-area ratio of DHA and artesunateto IS versus the respective DHA and artesunate concentration in each standard samples. During the analysis, each level of the cali-bration curve was measured with two sets of calibrators: one at the beginning and the second at the end of the run. Control samples atthree concentrations levels (low, medium and high: i.e. 15, 150 and The lower limit of quantification was 1 ng/mL. The inter-assay 1500 ng/mL) were assayed throughout rat sample analysis. Samples precision obtained with the rat plasma QC samples of 15, 150 with levels exceeding 2000 ng/mL (as found in the early samples and 1500 ng/mL DHA and artesunate were 11.2%, 7.4% and just after artesunate administration) were appropriately diluted with 11.9% for DHA and 14.1%, 7.0% and 11.8% for artesunate.
blank rat plasma to yield concentration comprised within the cali- Mean absolute deviation from nominal values of QC samples bration range, prior to their extraction.
(15, 150 and 1500 ng/mL) during the series of analysis (n ¼15) Table 1. Artesunate and DHA parameters in control rats and rats infected with F. hepatica (acute and chronic infection) after iv and oral administration Data are presented as arithmetic mean (+SD).
aPlasma concentration at t¼15 could not be measured for one rat; hence, this value might be overestimated.
bSignificantly different from control.
Downloaded from http://jac.oxfordjournals.org/ PK parameters of artesunate and DHA in rats infected with F. hepatica 68.3 min) (KW ¼ 3.857, P ¼ 0.049), while t1/2 of DHA was slightly increased in rats with an acute infection (61.7 versus53.7 min).
Rats that had been infected with F. hepatica for 86 days (chronic, biliary infection) had a more than 2-fold increasedCmax of artesunate and DHA, which was statistically significantfor DHA [3802+2149 ng/mL (control) versus 9093+884 ng/mL(infected); KW ¼3.857, P¼0.049]. The AUCs of the parent 32 439+29808 ng.min/mL (control rats)] and the metabolite[691 102+336 175 ng.min/mL 155 592+66921 ng.min/mL (control rats)] were significantly elevated (KW ¼3.857, P¼0.049). Similar to the rats with anacute infection, t1/2 of artesunate was significantly shorter (42.6 versus 68.3 min) (KW ¼3.857, P¼0.049), while t1/2 of DHA was increased in these rats (74.6 versus 53.7 min).
The ratio of AUCDHA to AUCartesunate had a range of 4.7 - 8.5, the highest value determined in chronic F. hepatica infec- Figure 5. Arithmetic mean plasma concentration -time profiles of DHA tions. In infected rats, the oral bioavailability (F) of artesunate following a single iv dose of 10 mg/kg artesunate. Error bars represent SD.
was considerably greater than the controls (49.7% and 37.5% In chronically F. hepatica-infected rats, the terminal half-life of artesunate was increased (75.6 min compared with 34.5 min Intravenous administration. Following iv administration of in control rats), while t1/2 of DHA was only slightly longer 10 mg/kg artesunate to control and infected rats, the mean (43.5 min versus 31.6 min). Maximum plasma concentrations of plasma concentration - time profiles of artesunate and DHA are shown in Figures 4 and 5. The PK parameters are summarized Rats harbouring an acute F. hepatica infection showed the fol- 3849+3060 ng/mL (control)]. Finally, the AUCs of the parent lowing not significant changes in mean PK parameters of artesunate and DHA when compared with control rats. Cmax values were lower 140 938+128 783 ng.min/mL (control)] and the metabolite [artesunate: 1652+643 ng/mL (infected) versus 5784+3718 ng/ mL (control); and DHA: 2860+1269 ng/mL (infected) versus 86 107+41863 ng.min/mL (control)] were considerably, but not significantly, smaller translating into higher CL. No significant [13665+5573 ng.min/mL (infected) versus 140938+128783 infected rats and rats without infection.
(infected) versus 86 107+41863 ng.min/mL (control)] were There was evidence of a secondary artesunate peak in un- smaller, translating into higher CL. The t1/2 of artesunate and infected rats 3 h after artesunate administration, as shown in DHA were similar in acute infection and control rats [38.6 Figure 4, which points to an enterohepatic circulation.
(infected) versus 34.5 min (control) and 32.3 versus 31.6 min for The ratio of AUCDHA to AUCartesunate was 0.6 for the control artesunate and DHA, respectively]. In addition, there was no sig- rats and 3.8 and 2.9 for acute and chronic F. hepatica infections nificant difference in the Vss of DHA and artesunate in rats with an acute infection and uninfected rats.
Though several studies have analysed hepatic functions duringthe course of F. hepatica infections in rats,12 to our knowledge, this is the first PK study in F. hepatica-infected rats. Evaluation of pharmacological and PK properties at early stages of drug discovery is crucial as it can accelerate the conversion of hits and leads into candidates for further development. Promisingfasciocidal properties of the artemisinins and the synthetic per-oxide (trioxolane) OZ78 have been recently reported in rodent models,6,13 sheep7 and even humans;8 hence, further preclinicaland clinical investigations are warranted.
We have chosen to evaluate in a first step the PK properties of artesunate, as we were surprised that in our recent study this drug was not tolerated by rats harbouring adult F. hepatica, at single oral doses of 200 mg/kg and above.6 For comparison, in Figure 4. Arithmetic mean plasma concentration -time profiles of artesunate uninfected rats, the LD50 value of artesunate was reported to be following a single iv dose of 10 mg/kg artesunate. Error bars represent SD.
351 mg/kg following a single iv administration of the drug.14 PK data for artesunate have been reported in adults and chil- While most PK studies on the artemisinins have used HPLC dren with uncomplicated and severe malaria, and in healthy vol- using post-column alkali decomposition or electrochemical unteers. It was found that malaria, infection had a significant detection,22,23 we developed a sensitive and selective LC-tandem effect on the PK of the artemisinins. For example, peak plasma MS assay procedure for the specific and quantitative analysis of concentrations of DHA and the relative oral bioavailability of artesunate and DHA in plasma. Similar to a recently established DHA were higher in malaria patients.15,16 The PK properties of LC/MS method for artesunate and DHA in plasma,24 our artesunate have also been well studied in healthy rats.17,18 method is reproducible and accurate and has a detection limit of In our artesunate PK study in F. hepatica-infected rats, two study timepoints were selected during the course of the Fasciola Since several of our samples were haemolysed and first infection, one during the acute phase of infection (day 28), attempts of the analysis of these samples failed (only very when flukes migrate through and hence cause great damage to low, if any, DHA or artesunate levels could be detected), a the liver parenchyma (hepatic stage), and one during the chronic possible alteration of artesunate and DHA levels by haemo- stage (day 86), when adult flukes have moved to the central bile globin (Hb) during the extraction procedure was hypothesized.
duct and the liver tissue is regenerating (biliary stage). It was Since DHA is reported to react with ferrous (Fe2þ)-but not demonstrated that cytochrome P450 activities decrease in the ferric (Fe3þ)-haeme from Hb,25 sodium nitrite, a known acute phase of F. hepatica infection in rats and return to normal methaemoglobin-forming agent,26 was added to all haemo- values by week 9 of infection.12 In our study, the parasitic path- lysed plasma samples. Addition of sodium nitrite to haemo- ology was ascertained by autopsy, but it might be useful in lysed samples was able to successfully prevent most of DHA future studies to also integrate basic liver function tests and an or artesunate degradation and, importantly, proportionally to assessment of cytochrome P450 activities with PK studies to be artemisinin added as IS. Thus, the DHA/IS and artesunate/IS able to correlate the degree of parasite-induced metabolism ratios in haemolysed samples in the presence of sodium impairment with the changes observed in PK parameters.
nitrite were similar to those determined in non-haemolysed We found both in rats suffering from acute and chronic F. hepatica infections considerable changes in all PK parameters In conclusion, our study has shown that F. hepatica infec- of artesunate and DHA. Following oral administration, artesu- tions strongly influence the disposition kinetics of artesunate nate is rapidly hydrolysed to DHA in the stomach, as well as by and its metabolite in rats. The clinical significance of this blood esterases and the hepatic cytochrome CYP3A4. DHA is finding is unclear and should be studied in fasciolasis, schisto- cleared predominantly by hepatic biotransformation to glucuro- somiasis and other patients suffering from liver diseases that nides and metabolites, lacking the peroxide bridge.18,19 The con- are treated with artemisinins, since alterations in plasma con- siderably higher Cmax and AUC levels of artesunate and DHA centrations may lead to toxic adverse events or sub-curative we observed in infected rats following oral artesunate might be drug levels causing treatment failures. Drug levels of the arte- due to an impaired metabolism and elimination of DHA due to misinins should also be monitored in patients suffering from hepatic damages related to F. hepatica pathology. High and malaria and concurrent liver diseases or elderly patients.
toxic levels of DHA might explain the high mortality rate in Neutropenia, anaemia, haemolysis, elevated levels of liver infected rats observed in our previous experiments.6 enzymes, neurotoxicity and embryotoxic effects have been An opposite trend was observed after iv injection (consider- described following artesunate treatment in healthy volunteers ably lower Cmax and AUC of artesunate in infected rats, when and malaria patients,27 and there is concern that the severity compared with non-infected rats). The differences in Cmax levels of these adverse events might increase in patients suffering might be explained with an outlier datapoint in one of the rats from liver diseases. In addition, PK parameters of novel sampled; hence, follow-up studies are necessary to confirm this fasciocidal agents should be studied in the presence of hepatic finding. Lower AUC levels of artesunate following iv adminis- impairment at early phases of drug development to allow tration in infected rats might be due to an impaired enterohepatic identifying changes in drug disposition, which might lead to a circulation in infected rats. Extensive enterohepatic circulation decrease in therapeutic efficacy or drug-induced toxicity.
was demonstrated for artesunate following iv administration in Further studies on the PK properties of artemether and the healthy rats.20 In our study, a second plasma peak of artesunate synthetic trioxolane OZ78 in F. hepatica-infected rodents and was observed in uninfected rats also pointing to enterohepatic sheep are ongoing in our laboratories. These studies will circulation of this drug. To confirm whether enterohepatic circu- assist in the selection of an ideal peroxidic trematocidal drug lation is impaired in infected rats, plasma exposure levels should be studied in infected and uninfected bile duct cannulated rats.
The lower Cmax and AUC of DHA might be attributed to thedamage of CYP3A4, which has been described to metabolize Finally, interestingly we observed a very low oral bioavail- We thank Dr Franz Schuler, Hoffmann-La Roche, for his ability of artesunate in uninfected rats (2.3%), which is lower support with the rat catheterization.
than that in two previous studies, which reported mean oralbioavailability of 27% to 29.5% for artesunate.17,21 The oralbioavailability of artesunate was higher in infected rats, namely 37.5 and 49.7%. However, these results need to be interpretedwith care, as we used a Tween/alcohol suspension for the oral J. K. (Project No. PPOOA-114941) and L. D. (REQUIP Grant drug administration, which might have influenced the solubility No. 326000-121314/1) are grateful to the Swiss National and hence biodisposition of artesunate.
Science Foundation for financial support.
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