• View in gallery

    Total radioactivity (ng equivalents per ml) of [14C] artesunate in whole blood (dashed line with triangular markers) and in plasma (solid line with circular markers), and plasma concentration of unchanged artesunate (AS) (dotted line with dot makers in re-plot) and unchanged dihydroartemisinin (DHA, dashed line empty circle in re-plot) following a single 5 mg/20 μCi/kg intravenous injection of [14C] AS in rats (N = 6). This figure appears in color at www.ajtmh.org.

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    Mean distribution profile of total radioactivity (ng equivalents per gram tissue) of radiolabeled artesunate (AS) in various tissues (column) at 1, 6, 24, 48, 72, 96, and 192 hours following a single 5 mg/20 μCi/kg intravenous injection of [14C]AS in rats (n = 5). This figure appears in color at www.ajtmh.org.

  • View in gallery

    Individual elimination profiles of total radioactivity (μg equivalents per rat) of radiolabeled artesunate (circular markers) in urine (top) and feces (bottom), as well as computer fitted curves (solid line) by pharmacokinetic parameters following a single 5 mg/20 μCi/kg intravenous injection of [14C]AS in rats during 8 days collection (N = 5).

  • View in gallery

    Individual protein binding of [14C]AS in male humans (top) and male rats (bottom) plasma (triangle markers) and mean value in the humans and rats plasma (solid line) after incubation with variable concentrations of AS (0.2, 1, 5, 25, 125, 625, 3125, 15625, and 78125 ng/ml) at 37°C for 5 hours (N = 3).

  • View in gallery

    Metabolic patterns (solid line) of radiolabel distribution with standard references (dotted line) of alpha-dihydroartemisinin (α-DHA), β-dihydroartemisinin (β-DHA), and artesunate (AS) in plasma free fraction at 1 min (A), 5 min (B), plasma glucuronidate conjugation fraction at 5 min (C), and urine glucuronidate conjugation fraction at 8 hr (D) after single intravenous injection at dose of 5 mg/20μCi/kg of [14C]AS in rats displayed together in radiochromatography.

  • 1

    World Health Organization, 1990. Control of tropical diseases. Severe and complicated malaria. Trans R Soc Trop Med Hyg 84 (Suppl 2): 1–65.

    • Search Google Scholar
    • Export Citation
  • 2

    de Vries PJ, Dien TK, 1996. Clinical pharmacology and therapeutic potential of artemisinin and its derivatives in the treatment of malaria. Drugs 52 :818–836.

    • Search Google Scholar
    • Export Citation
  • 3

    Li QG, Brewer TG, Peggins JO, 1998. Anorectic toxicity of di-hydroartemisinin, arteether and artemether in rats following multiple intramuscular doses. Int J Toxicol 17 :663–676.

    • Search Google Scholar
    • Export Citation
  • 4

    Li QG, Mog SR, Si YZ, Kyle DE, Gettayacamin M, Milhous WK, 2002. Neurotoxicity and efficacy of arteether related to its exposure times and exposure levels in rodents. Am J Trop Med Hyg 66 :516–525.

    • Search Google Scholar
    • Export Citation
  • 5

    McLean WG, Ward SA, 1998. In vitro neurotoxicity of artemisinin derivatives. Med Trop 58 (Suppl 3):28S–31S.

  • 6

    Wesche DL, DeCoster MA, Tortella FC, Brewer TG, 1994. Neurotoxicity of artemisinin analogs in vitro. Antimicrob Agents Chemother 38 :1813–1819.

    • Search Google Scholar
    • Export Citation
  • 7

    Skinner TS, Manning LS, Johnston W, Davis TME, 1996. In vitro stage-specific sensitivity of Plasmodium falciparum to quinine and artemisinin drugs. Int J Parasit 26 :519–525.

    • Search Google Scholar
    • Export Citation
  • 8

    Davis TM, Phuong HL, Ilett KF, Hung NC, Batty KT, Phuong VD, Powell SM, Thien HV, Binh TQ, 2001. Pharmacokinetics and pharmacodynamics of IV artesunate in severe falciparum malaria. Antimicrob Agents Chemother 45 :181–186.

    • Search Google Scholar
    • Export Citation
  • 9

    Batty KT, Le AT, Ilett KF, Nguyen PT, Powell SM, Nguyen CH, Truong XM, Vuong VC, Huynh VT, Tran QB, Nguyen VM, Davis TM, 1998. A pharmacokinetic and pharmacodynamic study of artesunate for vivax malaria. Am J Trop Med Hyg 59 :823–827.

    • Search Google Scholar
    • Export Citation
  • 10

    Newton P, Suputtamongkol Y, Teja-Isavadiharm P, Pukrittay-akamee S, Navaratnam V, Bates I, White N, 2000. Antimalarial bioavailability and disposition of artesunate in acute falciparum malaria. Antimicrob Agents Chemother 44 :972–977.

    • Search Google Scholar
    • Export Citation
  • 11

    Bethell DB, Teja-Isavadharm P, Cao XT, Pham TT, Ta TT, Tran TN, Nguyen TT, Pham TP, Kyle D, Day NP, White NJ, 1997. Pharmacokinetics of oral artesunate in children with moderately severe Plasmodium falciparum malaria. Trans R Soc Trop Med Hyg 91 :195–198.

    • Search Google Scholar
    • Export Citation
  • 12

    Benakis A, Paris M, Loutan L, Plessas CT, Plessas ST, 1997. Pharmacokinetics of artemisinin and artesunate after oral administration in healthy volunteers. Am J Trop Med Hyg 56 :17–23.

    • Search Google Scholar
    • Export Citation
  • 13

    Na-Bangchang K, Karbwang J, Congpoung K, Thanavibul A, Ubalee R, 1998. Pharmacokinetics and bioequivalence evaluation of two generic formulations of oral artesunate. Eur J Clin Pharmacol 53 :375–376.

    • Search Google Scholar
    • Export Citation
  • 14

    Li QG, Peggins JO, Lin AJ, Masonic K, Trotman KM, Brewer TG, 1998. Pharmacology and toxicology of artelinic acid: Pre-clinical investigation on pharmacokinetics, metabolism, protein and RBC binding, acute and anorectic toxicities. Trans R Soc Trop Med Hyg 92 :332–340.

    • Search Google Scholar
    • Export Citation
  • 15

    Titulaer HA, Eling WM, Zuidema J, 1993. Pharmacokinetic and pharmacodynamic aspects of artelinic acid in rodents. J Pharm Pharmacol 45 :830–835.

    • Search Google Scholar
    • Export Citation
  • 16

    Batty KT, Thu TA, Davis TM, Ilett KF, Mai TX, Hung NC, Tien NP, Powell MS, Thien HV, Binh TQ, Kim NV, 1998. A pharmacokinetic and pharmacodynamic study of IV vs oral AS in uncomplicated falciparum malaria. Br J Clin Pharmacol 45 :123–129.

    • Search Google Scholar
    • Export Citation
  • 17

    Li QG, Peggins JO, Fleckenstein L, Masonic K, Heiffer MH, Brewer TG, 1998. Bioavailability and pharmacokinetics of di-hydroartemisinin, arteether, artemether, artesunic acid and artelinic acid in rats. J Pharm Pharmacol 50 :173–182.

    • Search Google Scholar
    • Export Citation
  • 18

    Li QG, Carpenter C, Trotman C, Kathcart AK, Donahue R, Milhous WK, 1999. Pharmacokinetic comparison of artesunate and dihydroartemisinin in beagle dogs. Am J Trol Med Hyg 61 :274.

    • Search Google Scholar
    • Export Citation
  • 19

    Brewer TG, Grate SJ, Peggins JO, Weina PJ, Petras JM, Levine BS, Heiffer MH, Schuster BG, 1994. Fatal neurotoxicity of arteether and artemether. Am J Trop Med Hyg 51 :251–259.

    • Search Google Scholar
    • Export Citation
  • 20

    Classen W, Altmann B, Gretener P, Souppart C, Skelton-Stroud P, Krinke G, 1999. Differential effects of orally versus parenterally administered qinghaosu derivative artemether in dogs. Exp Toxicol Pathol 51 :507–516.

    • Search Google Scholar
    • Export Citation
  • 21

    Genovese RF, Newman DB, Petras JM, Brewer TG, 1998. Behavioral and neural toxicity of arteether in rats. Pharmacol Biochem Behav 60 :449–458.

    • Search Google Scholar
    • Export Citation
  • 22

    Petras JM, Kyle DE, Gettayacamin M, Young GD, Bauman RA, Webster HK, Corcoran KD, Peggins JO, Vane MA, Brewer TG, 1997. Arteether: Risks of two-week administration in Macaca mulatta. Am J Trop Med Hyg 56 :390–396.

    • Search Google Scholar
    • Export Citation
  • 23

    Smith SL, Sadler CJ, Dodd CC, Edwards G, Ward SA, Park BK, McLean WG, 2001. The role of glutathione in the neurotoxicity of artemisinin derivatives in vitro. Biochem Pharmacol 61 :409–416.

    • Search Google Scholar
    • Export Citation
  • 24

    Nui XY, Ho LY, Ren ZH, Song ZY, 1985. Metabolic fate of qinghaosu in rats: a new TLC densitometric method for its determination in biological material. Eur J Drug Metab Pharmacokinet 10 :55–59.

    • Search Google Scholar
    • Export Citation
  • 25

    Kearney BP, Aweeka FT, 1999. The penetration of anti-infectives into the central nervous system. Neurol Clin 17 :883–900.

  • 26

    Zhao KC, Song ZY, 1989. Distribution and excretion of artesunate in rats. Proc Chin Acad Med Sci 4 :186–188.

  • 27

    Tian F, Zhu Y, Long H, Cregor M, Xie F, Kissinger CB, Kissinger PT, 2002. Liquid chromatography coupled with multi-channel electrochemical detection for the determination of daidzein in rat blood sampled by an automated blood sampling system. J Chromatogr B Analyt Technol Biomed Life Sci 772 :173–177.

    • Search Google Scholar
    • Export Citation
  • 28

    Li QG, Hümpel M, 1990. Serum protein binding characteristics of cyproterone acetate, gestodene, levonorgestrel and norethisterone in rat, rabbit, dog, monkey and man. J Steroid Biochem 35 :319–326.

    • Search Google Scholar
    • Export Citation
  • 29

    China Cooperative Research Group on Qinghaosu and its Derivatives as Antimalarials, 1982. Metabolism and pharmacokinetics of qinghaosu and its derivatives. J Trad Chin Med 2 :25–30.

    • Search Google Scholar
    • Export Citation
  • 30

    Maggs JL, Madden S, Bishop LP, O’Neill PM, Park BK, 1997. The rat biliary metabolites of dihydroartemisinin, an antimalarial endoperoxide. Drug Metab Dispos 10 :1200–1204.

    • Search Google Scholar
    • Export Citation
  • 31

    Pollack GM, Brouwer KLR, Demby KB, Jones JA, 1990. Determination of hepatic blood flow in the rat using sequential infusions of indocyanine green and galactose. Drug Metab Dispos 18 :197–202.

    • Search Google Scholar
    • Export Citation

 

 

 

 

THE EVALUATION OF RADIOLABELED ARTESUNATE ON TISSUE DISTRIBUTION IN RATS AND PROTEIN BINDING IN HUMANS

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  • 1 Division of Experimental Therapeutics, Walter Reed Army Institute of Research, Silver Spring, Maryland

The present study reports the tissue distribution, pharmacokinetics, mass balance, and elimination of [14C] artesunate (AS) following single intravenous administration in rats. Protein binding was performed with rat and human plasma. Radioactivity and drug levels in blood, plasma, tissues, urine, and feces up to 192 hours were collected and measured. The mean terminal half-life of plasma (76 h) and blood (105 h) radioactivity was prolonged compared with that of unchanged AS (0.43 h) and dihydroartemisinin (0.75 h), an active metabolite of AS. Drug was widely distributed after 1 hour in select tissues. After 24 hours, the radioactivity rapidly declined in all tissues except spleen until 96 hours. Only 1% of total radioactivity was detected in brain tissue. AS revealed a higher binding capacity with human and rat plasma proteins (73–81%). The radioactivity in whole blood was higher (two to fourfold) than that in plasma throughout the period of the treatment, suggesting that AS binding to RBCs may relate to its powerful antimalarial activity.

INTRODUCTION

Artesunate (AS), an intravenous (IV) drug for the management of severe and complicated malaria, has been used for decades in malaria endemic parts of the world. Clinical data suggests the drug is both safe and highly effective.1,2 The effectiveness of AS has been attributed to its rapid and extensive hydrolysis to dihydroartemisinin (DHA), which is threefold to fivefold more active, and toxic, than the parent compound.36 DHA rapidly and effectively fights all stages of the parasite life cycle and completely inhibits parasite growth within the shortest exposure time as compared with all other artemisinin and conventional antimalarial drugs.7

The pharmacokinetic parameters of AS in humans are very well known. Detailed pharmacokinetic data for AS and its active metabolite DHA have been reported in adults810 and children11 infected with malaria, and in healthy volunteers.12,13 Published work has shown AS to have an elimination half-life (t1/2) of 2–5 minutes and DHA to have a t1/2 on the order of 40–90 minutes. In animals the pharmacokinetic (PK) profile of both drugs has been clearly defined. The conversion of AS to DHA in human pharmacokinetic studies has been shown to be much more efficient than in animal species. The ratio of AUCDHA to AUCAS in humans had a range of 4.3–9.7, compared with rats and dogs with a ratio of only 0.5–1.1.9,1418 The ratio in humans was 4.5–13.5 times higher than that in animals.

Artemisinin derivatives are potent antimalarial drugs, but concerns have been raised as to their neurotoxic potential. Brain stem injury associated with the artemisinin derivatives, arteether (AE) and artemether (AM), has been shown in laboratory animals under experimental conditions, but despite their widespread use evidence of neurotoxicity at clinical doses in humans is weak.1922 In vitro testing has shown inhibition of neuronal development by artemisinin drugs at concentrations achieved in plasma during the management of malaria.23

An understanding of in vivo neurotoxicity secondary to certain artemisinin derivatives requires knowledge of the penetration of the cerebrospinal fluid (CSF) by these drugs. It has been shown that artemisinin derivatives cross the blood–brain barrier in rats;24 however, concentrations of artemether, another artemisinin derivative, in the CSF of dogs were low and there was no detectable penetration by its active metabolite, DHA.20 Artesunate is converted stoichiometrically to DHA with peak plasma concentrations occurring after 10 minutes.8,16 DHA is highly lipid soluble and has a low molecular mass (284 Da), favoring penetration of CSF.25 DHA has a relatively low solubility in water and would be able to cross cell membranes. After AS treatments in human subjects no AS was detected in CSF; DHA levels in CSF increased with time while DHA levels in plasma fell, suggesting a continuing influx but a slower efflux of DHA. DHA may accumulate in CSF during frequent artemisinin dosing.25

There is little or no information on the distribution and excretion of AS and DHA using radiolabeled drug and specific analytical techniques. For tissue distribution, a radioimmunoassay method was used to study distribution and excretion of AS in rats in China.26 This method detected the parent compound and its metabolites in various tissues. Ten minutes after IV administration of AS the greatest to least drug concentration in the organs and tissues were found to be in the: intestines > brain > liver > kidney > testicle > muscle > fat > heart > serum > eyeball > spleen > lung. After 60 minutes, the concentration of drug and metabolites significantly decreased in all tissues; data showed brain, fat, intestine, and serum maintained relatively high concentrations. Drug and metabolites detected in other tissues was very low. Less than 1% of the injected dose was found in the urine and feces collected during the first 24 hours after administration. This method did not discriminate between AS and DHA, but measured total drug and metabolites.

A previously published paper has shown preliminary results of AS tissue distribution in rats, but did not reveal quantitative and kinetic profiles of AS in each organ.26 There have been no published reports of pharmacokinetics, mass balance and elimination data for AS or DHA, including no distribution data on AS in the brain and red blood cells. The present study was undertaken to ascertain the tissue distribution, pharmacokinetics, mass balance, and elimination of radiolabeled AS, allowing separation and measurement of AS and DHA in rats.

MATERIALS AND METHODS

Chemicals.

The test unlabeled compound used in this study is artesunic acid (4-(10′dihydroartemisinin-oxymethyl) succinate; AS), which was manufactured as a phosphate salt with 0.3 M PBS and obtained from the Walter Reed Chemical Inventory System. Unlabeled AS (WR256283, BN: BQ38841, Lot N# 2.03) was synthesized and manufactured by Knoll AG and rebottled by BASF Pharmaceuticals. It was supplied through the Division of Experimental Therapeutics at Walter Reed Army Institute of Research (WRAIR, Silver Spring, MD). One lot of [16-14C] artesunic acid (Lot No. 10839-113) was used in this study and it was synthesized by Research Triangle Institute (Research Triangle Park, NC). The stated specific activity and radiochemical purity was 17.63 mCi/mmol (45.86 μCi/mg) and 97.0%, respectively. Unlabeled AS and radiolabeled compounds were stored at approximately −20°C.

Animals.

Male Sprague-Dawley rats obtained from Charles River Laboratories were used in this study. On arrival, the animals were acclimated for 7 days (quarantine). The animals were housed individually and maintained in a room with a temperature range of 64–79°F, 34–68% relative humidity, and 12-h light:dark cycles. Food and water were supplied ad libitum during quarantine and throughout the study. The animals were fed a standard rodent maintenance diet. The animal protocol was approved by IACUC, WRAIR, and the research was conducted in compliance with the Animal Welfare Act and other federal statutes. Regulations relating to animals and experiments involving animals adhered to principles stated in the Guide for the Care and Use of Laboratory Animals, NRC Publication, 1996 edition.

Pharmacokinetic (PK) study.

Traditional PK studies involve intermittent blood sampling and subsequent determination of drug concentrations in plasma. The Culex automated blood sampler provides a way for blood withdrawal at preprogrammed intervals, which is more accurate and less labor intensive.27 Six rats in the PK group were implanted with a jugular vein catheter under an isoflurane anesthesia. After the surgery, rats were allowed to recover for 96 hours with free access to food and water. The rats were dosed single IV at 5 mg/ml/kg with [14C] AS (20 μCi/kg) through the tail vein. A 100–200 μL blood sample was withdrawn from the jugular vein catheter into a cooled vial, containing powdered heparin, at 5, 10, 30 minutes and 1, 1.5, 3, 6, 12, 24, 48, 72, 96, and 192 hours post-dosing according to a preset schedule.

Tissue distribution of [14C] artesunate study.

Rats treated with a single [14C] AS dose of 5 mg/20 μCi/kg intravenously (5 rats per group) were anesthetized by isoflurane and euthanized via cardiac puncture at 1, 6, 24, 48, 72, 96, and 192 hours, post-dosing. Blood, liver, brain, eyes, adrenals, muscle, lungs, heart, liver, kidneys, spleen, stomach, small intestine, large intestine, perirenal fat, and bone marrow samples were removed from each animal by gross dissection. The gastrointestinal tract (GI) was separated into the stomach (esophagus and stomach), small intestine, and large intestine. Contents were procured from each GI segment. After flushing with saline, these and other tissues were stored. Tissues were rinsed gently, but thoroughly with water to remove remaining traces of blood before storage. Dissecting instruments were also washed between tissue procurements to avoid cross-contamination.

For most of the tissues 20% (w/w) aqueous homogenates of the tissue were prepared. Aqueous homogenates of the fecal and GI content samples were also prepared. Metabolism cages were washed with a solution of 2% Count-off® and the volume measured. Radioactivity in the plasma samples and in aliquots (approx. 0.02–1.0 mL) of urine and cage washings was determined. The blood cells (primarily erythrocytes), perirenal fat (approx. 0.2 g), bone marrow samples (approx. 0.1 mL), and approximately 300-μL aliquots of the homogenates of feces, liver, kidneys, heart, brain, stomach, and carcass were solubilized with SolvableTM tissue solubilizer (Perkin-Elmer Life and Analytical Sciences, Boston, MA). The homogenates of feces, stomach content, and intestinal contents were extracted with 50% methanol for 24 hours. Aliquots of blood and pooled urine samples from selected intervals were also analyzed for AS content directly.

Total radioactivity in the collected tissue samples were quantified in duplicate. Weighed tissue aliquots (~0.2 g) were digested overnight in 2 mL of Solvable® tissue solubilizer (Perkin-Elmer Life and Analytical Sciences, Boston, MA) at 50°C. Colored samples were decolorized by adding a maximum of 0.2 mL of 30% H2O2 for 4 hours. Scintillation cock-tail (Hyonic Fluor; Perkin-Elmer Boston, MA) was then added and radioactivity determined in a Perkin-Elmer Tri-Carb 3100 TR scintillation spectrophotometer (Packard Instrument Co., Downers Grove, IL). Feces and intestinal contents were thoroughly homogenized before taking aliquots. Plasma was also analyzed using the HPLC-ECD system to measure the concentration of unchanged AS, as described by Li et al.,17,18 to assay AS and its main and active metabolite, dihydroartemisinin (DHA).

Mass balance and elimination of [14C] artesunate study.

Samples of urine and feces were collected for a period of 192 hours after a single IV injection at 1, 8, 24, 48, 72, 96, 120, 144, 168, and 192 hours from individual stainless steel Nalgene metabolic cages (Nalgene Company, Rochester, New York) designed for separate collection of urine and feces. Metabolic cages were washed with a solution of 2% Count-off® (Perkin-Elmer Life and Analytical Sciences, Boston, MA) and the volume measured. The volume of each urine and cage rinse was measured. Duplicate portions (0.1 mL urine; 1 mL cage rinse) of each sample were radioassayed after the addition of scintillation cocktail. The weights of individual fecal samples were obtained and the samples were subsequently homogenized in 4 volumes of deionized water. Quadruplicate portions (1.0 mL) of each homogenate were decolorized with 30% hydrogen peroxide, and digested with Solvable® tissue solubilizer. After acidification with glacial acetic acid and addition of scintillation cocktail, samples were assayed for radioactivity. In addition, urine samples collected from each time point after dosing were assayed by HPLC-ECD to obtain a metabolite profile of the radioactivity in each sample. The samples were kept in at −20°C for 2 weeks prior to analysis.

Metabolic profile of [14C] artesunate study.

Metabolic profile evaluation of [14C] AS in plasma and whole blood, as well as unchanged AS in plasma was conducted in individual rats. Blood samples (7–8 mL) were collected from animals at 5, 10, 30 minutes and at 1, 1.5, 3, 6, 12, 24, 48, 72, 96, and 192 hours post-dosing when rats were euthanized. About 2 mL of plasma and urine, and 1–2 g of feces were used. Conjugates were hydrolyzed by glucuronidase (1000 FU/ml) at pH 4.6, 38°C for 24 hours. Active enzyme was checked by pheno-phthaleinglucuronide at the end of incubation. Urine and fecal samples were incubated for 48 hours and fresh enzyme was added after 24 hours. Total radioactivity in free, conjugated, and the remaining aqueous fractions were measured with liquid scintillation counters. Protein precipitates were dissolved in Solvable® (200 mg in 1 mL) and counted for radioactivity after addition of scintillation cocktail.

Protein binding of [14C] artesunate study.

Human blood samples (5 men and 5 women) were freshly obtained from subject volunteers at the Department of Clinical Trials, WRAIR. Rat samples were freshly collected from 5 male and 5 female rats. Equilibrium dialysis was performed using Teflon dialysis chambers and Diachema standard membranes as described by Li et al.28 The GD-4/90 Equilibrium Dialyzer (MM Developments, Ottawa) contained 20 micro-dialysis cells (2.0 mL/chamber). The molecular weight cutoff for dialysis membranes was 5000 D. Equilibrium was reached after 4 hours when cells were rotated at 16 rpm and 37°C. Drugs were dissolved in plasma samples with various concentrations (0.2–78125 ng/ml) and all experiments were carried out for 5 hours.

HPLC-ECD method.

HPLC with reductive electrochemical detection (HPLC-ECD) was performed utilizing a model BAS 200B liquid chromatography system (Bioanalytical Systems, West Lafayette, IN). The system had 3 mobile phase reservoirs, solenoid proportioning valves, a dual piston pump, a pulse dampener, a column and detector oven, dual thin-layer electrodes with Ag/AgCI reference electrode, and a Rheodyne injector for manual injection that was modified for reductive work. The system was also equipped for mobile phase heating and sparging. Stainless steel connectors and tubing were used throughout the system. For simultaneous determination of AS and its major metabolite DHA (artemisinin as an internal standard), the Waters μBondapack (Waters Associates, Milford, MA), CN column (4.6 mm × 30 cm), and a mobile phase consisting of 30% acetonitrile: 70% 0.1 M acetic acid/NaOH buffer (pH 4) was used. Compounds were detected via reductive electrochemical detection. Data was acquired and analyzed using a Waters model 820-chromatography data system, Maxima program (Waters Associates, Milford, MA). Reproducibility was good with a lower limit of quantitation of 2.5 ng/ml for AS and 4 ng/ml for DHA. The inter- and intra-day coefficient of variation for accuracy and precision was ± 10%.

Detector response linearity studies were performed by preparing 6 duplicate calibrations covering a range of 2.5–1000 ng/ml for AS and DHA. Linear regression lines were obtained by plotting the peak area ratios (target peak areas divided by internal standard peak areas). To evaluate the reproducibility (within and between-days precision) of the method, duplicate analyses (N = 6) of plasma spiked with AS were carried out. The absolute recoveries of AS were determined by comparing the peak areas of spiked plasma samples and reference samples. The reference samples were injected directly into the HPLC for both detections. The limit of detection for AS and DHA was determined as the lowest concentration giving a response with 1.0–2.5 ng/ml plasma in the spiked serum sample for AS and DHA (signal-to-noise ratio of 3). The limit of quantitation (lowest concentration of the calibration curve) was 1.8 ng/ml in spiked plasma for AS.

Data evaluation.

For PK, the concentration-time data of [14C] AS in plasma and whole blood collected during the 192-hours treatment period was fitted to a 3-compartment open model using a nonlinear, extended least-square fitting procedure (WinNonlin 4.1, Scientific Consulting, Inc. Apex, NC) using weighted (1/concentration) nonlinear regression. The area under the curve (AUC) was determined by the linear trapezoidal rule with extrapolation to infinity based on the concentration of the last time point divided by the terminal rate constant. Extrapolations to time zero were done using zero concentration for intravenous dosing and using C0 values determined from the 3-compartment model equation at time zero by IV route. Mean clearance rate (CL) was determined by dividing the dose by the AUCinf for IV injection. Mean residence time (MRT) was determined by dividing the area under the first moment curve (AUMC) by the AUC. The volume of distribution at steady state (Vss) was calculated as the product of CL and MRT. The concentration-time data of unchanged AS was fitted to a 2-compartment open model. The ratio of DHA to AS was calculated by AUCDHA/AUCAS. Statistical analysis was conducted with Microsoft Excel using a Student’s t test for dependent samples to compare means of paired and unpaired samples between the two groups.

RESULTS

Dose formulation analysis.

The results of the analysis of the IV formulations of [14C] AS are designated as 5 mg/20 μCi/ml in this study. Chemical concentration and radioactivity analysis of samples taken from each dosing solution indicated the formulation was homogeneous. The mean concentration of AS in the IV formulation was 4.77–5.20 mg/ml. The radioactivity content of the IV formulation was detected between 19.87 and 21.68 μCi/ml. The radiochemical purity of [14C] AS was determined to be greater than 97% while cold AS was greater than 99%.

Pharmacokinetics of [14C] artesunate, unchanged artesunate, and dihydroartemisinin.

Pharmacokinetic parameters calculated from mean levels of total radioactivity, unchanged AS, and unchanged DHA in either whole blood or plasma after single IV administration of [14C] AS are shown in Table 1. The concentration-time data of [14C] AS in plasma and blood (Figure 1) collected during the 192 hours of the treatment period were fitted to a 3-compartment open model. Radioactivity derived from [14C]AS was eliminated from plasma in 3 phases with half-lives of 0.12, 13.2, and 76.2 hours, and from blood in 3 phases with half-lives of 0.1, 26.5, and 104.7 hours. Unchanged AS was eliminated from plasma in 2 apparent phases with half-lives of 0.14 and 0.43 hours. Unchanged DHA, the active metabolite of AS, was eliminated from the plasma in 2 apparent phases with half-lives of 0.21 and 0.75 hours (Table 1).

Peak concentrations (Cmax) of [14C] AS and unchanged AS in plasma at 0.1 hour were 4,271 ng equivalents/ml and 1,627 ng/ml, respectively. The corresponding AUCs of [14C] AS and unchanged AS were 21,702 ng equivalents·h/ml and 392 ng·h/ml, suggesting AS is extensively metabolized in rats. Similar to the AUC, the volume of distribution at steady state (Vss) was 16,081 mL/kg for radiolabeled drug and 3,982 mL/kg for unchanged AS. Calculated clearance values for radioactivity and unchanged AS in plasma were 237 and 12,719 mL/hr/kg, respectively (Table 1).

Following the IV administration of [14C] AS two peaks of radioactivity were detected (Figure 1, re-plot). The first peak concentration of radioactivity, 2,631 and 2,999 ng equivalents/g, was present in plasma and whole blood, respectively, at 5 minutes (earliest measured time point) after dosing. The second peak formed at 3.3 hours with plasma at 744 ng equivalents/ml and at 3.8 hours for whole blood with 1316 ng equivalents/g. The peak concentration of unchanged AS was 1026 ng/ml, but a second peak was not detected in the unchanged AS. Levels of radioactivity in blood decreased at a slower rate than in plasma, indicating a majority of the radioactivity in blood was associated with red blood cells, thus reducing drug clearance and distribution (Figure 1).

The individual and mean plasma concentration-time curves following the single IV injections of unchanged AS and its active metabolite, DHA, are shown in Figure 1. The conversion of AS to DHA is presented in Table 1. DHA had a peak plasma drug concentration (Cmax) of 468 ng/ml at 0.27 hours after dosing. The AUC ratio for unchanged DHA (376 ng·h/ml) to unchanged AS (392 ng·h/ml) was 0.98 following the single dose.

Tissue distribution of [14C] artesunate.

In tissues collected at 1 hour, approximately 68% of radioactivity in the total measured tissues was amassed in the small intestine and its content. Lesser amounts of radioactivity were distributed in other tissues (Table 2). The total amount of radioactivity in all measured tissues per gram was 90,502 ng equivalents (Table 2). Six hours after the injection, high levels of radioactivity were still present in the intestine, colon, and in their contents with slow loss; however, drug distribution was significantly increased in other tissues such as kidney, spleen, liver, heart, adrenals, blood, muscle, colon, and colon content (Figure 2). The total amount of radioactivity in all measured tissues per gram at 6 hours increased to 107,210 ng equivalents. The remarkable increases of radioactivity in other tissues at 6 hours may relate to re-distribution from tissues.

From 24–192 hours, measured radioactivity rapidly declined in all tissues except for the spleen (Figure 2). The total amount of radioactivity in all measured tissues per gram was 40,278, 30,406, 22,581, 16,704, and 8,117 ng equivalent at 24, 48, 72, 96, and 192 hours, respectively (Table 2). At 192 hours after dosing, residual activity (close to 2.57% of total amount collected) was still detected in some tissues and the highest levels of radioactivity were measured in spleen, kidney, adrenals, and heart; trace amounts were found in other tissues. Radioactivity in the blood and plasma was measured in quantifiable levels at 192 hours after the single IV injection (Figure 1). Radioactivity in whole blood was always higher (2.1–4.2 fold) than in plasma throughout the period measured (Figure 1). Unchanged AS was eliminated within 2 hours (Table 1 and Figure 1), indicating long-lasting radioactivity to be potentially resultant of metabolites.

During the treatment period, measured levels of radioactivity were more than twofold higher in the brain than in plasma; the AUC of radioactivity from 0–192 hours was 70,723 ng·h/g in the brain and 29,425 ng·h/ml in the plasma. The highest levels of the radioactivity were detected in the spleen. The measured amount was 3,707 ng equivalents/g at 1 hour and rapidly increased to 6,448 ng equivalents/g at 6 hours after dosing. In the post 6-hour period the amount remained between 5,283 to 6,539 ng/g until 96 hours (Table 2). At 192 hours the radioactive amount was measured at 3,524 ng equivalent/g. During the 192-hours treatment period the radioactivity in spleen was the highest with an AUC of 1,860,779 ng·h/g and the highest percentage of total radioactivity (28.0%) when compared with other tissues (Table 1). In other measured tissues the highest to lowest AUCs were found as follows: small intestine content (1,310,061 ng·h/g), kidneys (832,224 ng·h/g), large intestine content (462,496 ng·h/g), adrenals (374,210 ng·h/g), liver (363,288 ng·h/g), heart (251,987 ng·h/g), small intestine (249,033 ng·h/g), large intestine (246,379 ng·h/g), and lung (160,034 ng·h/g).

The longest half-life of radioactivity was measured in the spleen at 154.3 hours, followed by the lungs (123.7 hours), fat (115.4 hours), adrenals (109.6 hours), heart (108.6 hours), muscle (105.8 hours), and blood (98.7 hours). In contrast, radioactivity found in the small intestine, large intestine, and their contents had short half-lives of 29.4–74.8 hours.

Mass balance determination.

The total recovery of radioactivity in excreta (urine and feces) and cage rinses collected from animals through 192 hours after single IV administration is shown in Table 3. Through 192 hours after injection, approximately 56.1%, 38.5%, and 1.6% of the dose was eliminated in urine, feces, and cage rinses, respectively. The urinary excretion was the major route of elimination, accounting for 48–66% of the administered dose. Feces excretion was the secondary route of elimination, accounting for 37–44% of the administered dose. The recovery of [14C] AS from urine, feces and cage washing solution was 96% of total dose during the 9 days of collection.

Urinary and fecal elimination.

The elimination parameter estimates after intravenous dosing of [14C] AS at 5 mg/kg are presented in Table 3. Results showed that greater than 73% of the total dose was excreted within first 24 hours. The maximum [14C] radioactivity concentration in urine was observed at 0.48 days with 381 μg equivalents per rat (Figure 3). The elimination of [14C] label from urine occurred in 2 phases, fast and slow, with half-lives of 0.37 and 2.06 days (Table 3). Similar to urine, fast and slow elimination phases were also exhibited in feces. A peak concentration of 301 μg equivalents per rat was seen in fecal matter at 0.53 days after dosing (Figure 3). The half-lives of [14C] radioactivity were 0.57 and 2.54 days for fast and slow phases in feces, respectively. The elimination of radioactivity in feces was delayed with a lag time of 0.29 days (Table 3), while the lag time for urine was 0.02 days (Figure 3).

Metabolite profiles of [14C] artesunate.

Unchanged AS was rapidly and completely eliminated from rats within 2–3 hours following single IV administration. The total radioactivity (metabolites) lasted longer than 192 hours in plasma, urine, and feces. Conjugation studies showed drug conjugation (up to 90% of the dose in plasma and urine) is a major metabolic pathway of [14C] AS, and that conjugation is time-dependent in rats (Table 4). Metabolites of AS were quantified before and after hydrolysis with beta-glucuronidase to give free and conjugated fractions of plasma, urine, and feces. Metabolites arising from AS in the free fraction were 12.3 ± 4.4% and 10.4 ± 2.5% of the total administered dose in plasma and urine; those arising from the total conjugated fractions (glucuronide and other conjugations) were 88.6% and 89.6% of administered dose in plasma and urine, respectively. Glucuronide conjugation was 8% in plasma and 18% in the urine (Table 4). Within 30 minutes after dosing the drug conjugated fraction was less than the free fraction; the ratio of conjugated to free ranged from 0.57–0.97 in plasma. From 0.5 hours post dosing, the ratio increased to 1.97 at 1 hour and then rapidly increased to 19.36 at 48 hours. After 48 hours the ratio increased moderately to 25.11 at 192 hours.

Protein binding of [14C] artesunate with rat and human plasma.

[14C] AS revealed a higher binding percentage with human and rat plasma proteins (73–81%) when incubated with undiluted plasma samples at concentrations of 0.2–78125 ng/ml at 37°C for 5 hours. The binding percent of [14C] AS was shown to be concentration-dependent. At higher concentrations (> 125 ng/ml) the binding percentage declined from 75–81% to 62–66%, indicating that the maximum binding percentage occurred in the concentration range of 0.2–125 ng/ml (Figure 4). The binding capacity of AS was not significantly different between male and female in human plasma (P < 0.15), but was significantly different between with male and female in rat plasma (P < 0.05). When compared with human plasma, the bound portion (75%) of radiolabeled AS was about 7% less than that (82%) in rat plasma (Figure 4).

DISCUSSION

Previous studies have established the role of biliary excretion in the elimination of artemisinin metabolites from rats16 and mice.29 As early reports on tissue distribution and metabolism indicate, AS was transformed rapidly into DHA when it was intravenously injected in rats. The order of distribution in tissues was: heart, muscle, lung, spleen, kidney, brain, blood, and liver. After 2 hours, no DHA was detected in the organs.26,29

In the present studies, the tissue distribution and metabolic profiles of AS revealed similar data to the published literature. The outcomes of this study indicate very little AS is eliminated by direct excretion from kidneys (< 1% of dose); rather most (99%) occurs via metabolism (hydrolysis and oxidation). Data presented in this study allows a comprehensive characterization of AS with tissue distribution and its metabolic profiles.

Artesunate underwent rapid hydrolysis, glucuronylation, biliary excretion, and likely enterohepatic circulation. The results of the tissue distribution studies show 65–82% of total radioactivity to be found in the content of small intestine within the first hour after IV dosing. At 1 hour after dosing most of the [14C] was present in the liver, where it was excreted into the intestines via bile ductules; this suggests the higher hepatic biotransformation of AS is similar to metabolic profiles of other artemisinin compounds.16,30 The biliary metabolites of AS in rats were predominantly from the hydrolysis pathway (first to DHA), followed by glucuronylation and other conjugated products from multiple oxidative pathways for both compounds (AS and DHA). DHA is the major metabolite of AS. Figure 5A shows DHA presented almost immediately in the free fraction of plasma at 1 minute post-dosing. AS and DHA were rapid and extensively metabolized into various polar metabolites; the unchanged AS disappeared from the plasma within minutes. DHA is the metabolite found in the greatest quantities (Figure 5B). In the glucuronide conjugated fraction of the plasma, unchanged AS, unchanged DHA, a few less polar and a greater number of more polar metabolites were detected. The unchanged AS and DHA were the major components of the conjugated plasma fraction (Figure 5C), indicating conjugation is also a major metabolic pathway of AS and DHA. In addition, the two peaks of radioactivity were detected in blood and plasma and the most of radioactivity was excreted into the intestines, showing a potential enterohepatic circulation of [14C] AS in rats.

Urinary and fecal excretion data obtained after IV administration of [14C] AS demonstrated that approximately 73% of total radioactivity was eliminated in the first 24 hours. Fifty-six percent of the dose was eliminated in urine and 40% in feces within 192 hours after dosing; radioactivity recovered in the cage rinses (approximately 2%) may have been derived from urine due to reduced fecal contamination in the new metabolic cage system. The finding that fecal elimination of radioactivity after an IV dose was less than that observed in urine suggests a majority of [14C] AS may be first excreted in bile and re-absorbed from intestines.

Pharmacokinetic parameters were calculated from the blood and plasma levels of radioactivity after IV injection of [14C] AS. The studies indicated the terminal half-life of radioactivity was approximately 1.4 times longer in whole blood (t1/2 = 104.7 hours) than in plasma (t1/2 = 76.2 hours). The calculated steady state volume of distribution (Vss) of radioactivity in whole blood and in plasma was 6,183 and 16,081 mL/kg, respectively; this is an indication of wide-extra-vascular distribution of the radiolabel or tissue binding. The clearance of radioactivity from whole blood was estimated to be 81 mL/hr/kg, which was lower than the rate of hepatic blood flow.31

Unchanged AS was eliminated from plasma with a terminal half-life of 0.43 hours after IV administration. The Vss of unchanged AS was estimated to be 3,982 mL/kg, indicating AS has a relatively small volume of distribution. The calculated plasma clearance of AS was 12,719 mL/hr/kg, which is higher than hepatic blood flow (4,800 mL/hr/kg). This is probably related to uptake and binding of AS in red blood cells. When corrected for an AS blood/plasma ratio of 2.44, the hepatic blood clearance is 5,213 mL/hr/kg, which is reasonably close to hepatic blood flow. The results indicate even though the distribution of unchanged AS is apparently limited to total body water, the radioactivity (metabolites of AS) derived from [14C]AS was extensively distributed throughout rats and lasted for an extended period (192 hours).

The average ratio of blood to plasma was 2.44 during the 192-hour period. When calculated with rat hematocrit, the ratios of the concentrations in red blood cells (RBC) to plasma (ml/ml) were 1.2–3.1 during the 0–1.5 hours, and from 3.4–6.4 during the 3–192 hour period after dosing. The median ratio of RBC to plasma was 4.03 during the treatment time of 192 hours. High concentrations of [14C] AS present in RBC is very important for the antimalarial agent. The drug concentration in RBC is about 4 times higher than that in plasma, indicating that the powerful antimalarial potency of AS in animal species and humans may relate to the high drug levels in the RBC.

An understanding of artemisinin-associated neurotoxicity in vivo requires knowledge of the penetration of the cerebro-spinal fluid (CSF) by these drugs. Such data is sparse. Artemisinin derivatives cross the blood–brain barrier in rats.24 Artesunate is converted stoichiometrically to DHA, which is highly lipid soluble and has a low molecular mass (284 Da), favoring penetration of CSF.25 Since DHA has relatively low solubility in water, it should be able to cross cell membranes and be conjugated. After AS treatments in patients, no AS was detected in CSF but DHA levels in CSF increased with time while DHA levels in plasma fell, suggesting continued influx, with a slower efflux of DHA. DHA may accumulate in CSF during frequent AS dosing.25 In our study, the radioactivity in brain tissues (AUC: 70,723 ng·h/g) was more than 2 times higher than that in plasma (AUC: 29,425 ng·h/g). The half-life of [14C] AS in brain tissue was 94.2 hours, which was also longer than that in plasma, 63.7 hours. The results indicated that the resident time of [14C] AS is longer in the brain than in plasma. Also, there was 1% of total dose [14C] AS detected in rat brain tissue. This may reflect a sink effect of DHA and/or other metabolites and an uptake transfer by lipid-rich brain structures.25 In this time, the other metabolites present in the brain tissue are unknown. In addition, whether the metabolites in the brain are associated with the neurotoxicity is unknown.

The present study showed AS to be completely hydrolyzed to DHA within 2–3 hours following the intravenous administration in rats. As a result of approximately 73% of total dose was eliminated in the first 24 hours, and 90% of the dose appears as glucuronide and other conjugations in blood, urine, and feces. However, the majority of elimination of [14C] AS is through urinary excretion (56% dose) due to a possible drug re-absorption in the intestines and enterohepatic circulation as suggested by multiple concentration peaks in blood, plasma, and tissues. The long-lasting metabolites of AS and DHA (> 192 hours) may be also related to enterohepatic circulation. The binding capacities of [14C] AS with human and rat plasma at higher percentages, 73–81%, are seen in a concentration-dependent manner. High tissue distribution was found in rat spleen, followed by bone marrow, kidney, adrenals, and heart during the 192-hour period. In rat brain, the total concentration of [14C] was 2.4-fold higher than that in plasma, indicating the radioactivity could easily penetrate the brain–blood barrier. Whether the metabolites in the brain are associated with the neurotoxicity is unknown. Total radioactivity distributed in RBC was about twofold to fourfold higher than that in plasma, suggesting that the powerful antimalarial potency of AS in treatment of blood stage malaria may relate to the high RBC binding.

Table 1

Comparison of main pharmacokinetic parameters of [14C] artesunate in plasma and blood, as well as unchanged artesunate (AS) and dihydroartemisinin (DHA) in plasma following a single intravenous dose at 5 mg/20 μCi/kg in rats*

Parameters Dose14C-Artesunate 5 mg/20 μCi/kgUnchanged AS 5 mg/kgUnchanged DHA (metabolite of AS)
Sample PK fittingPlasma 3-compartmentBlood 3-compartmentPlasma 2-compartmentPlasma 2-compartment
* DHA = dihydroartemisinin, a main and more active metabolite of artesunate; PK = pharmacokinetics; MRT = mean residence time; MAT = mean arrival time; – = no data is available. Mean ± SD (n = 6).
Cmax (ng/ml4271 ± 14824527 ± 10161627 ± 415468 ± 92
Tmax (hr)0.00.00.00.27 ± 0.04
AUCinf. (ng·g.h/ml)21702 ± 294966213 ± 5918392.2 ± 63.3376.1 ± 53.8
t½-alpha (hr)0.12 ± 0.050.10 ± 0.010.14 ± 0.020.21 ± 0.03
t½-beta (hr)13.24 ± 6.1426.54 ± 2.950.43 ± 0.040.75 ± 0.20
t½-gamma (hr)76.17 ± 16.68104.7 ± 24.48
CL (ml/hr/kg)237.2 ± 35.281.46 ± 7.9312719.3 ± 1751.0
Vss (ml/kg)16081 ± 41916183 ± 17323982 ± 1022
MRT (hr)66.98 ± 8.6079.01 ± 24.170.31 ± 0.051.00 ± 0.31
Ratio of DHA/AS0.98 ± 0.22
Cmax at 1st peak (ng/ml)2631 ± 6922999 ± 7891026 ± 183468 ± 92
Tmax at 1st peak (hr)0.0830.0830.0830.27 ± 0.04
Cmax at 2nd peak (ng/ml)744.4 ± 200.21316 ± 178
Tmax at 2nd peak (hr)3.25 ± 1.473.75 ± 1.84
Table 2

Distribution (ng equivalents/tissue gram), half-lives, and area under the curve (AUC of [14C]-AS in main organs and tissues of the rats following single intravenous dose at 5 mg/20 μCi/kg and animals were euthanized at 1, 6, 24, 48, 72, 96, and 192 hr after dosing (n = 5)

1 hr6 hr24 hr48 hr72 hr96 hr192 hr
Organs(ng equivalents per gram of organ and tissue)Half-lives (hr)Tissue AUC (ng·hr/g)% of Total AUC
Mean ± SD (n = 5).
Blood687.93990.04868.38291.10298.86145.2851.1798.8 ± 25.7104303 ± 810991.57
Plasma499.05740.54317.41133.25114.8359.0322.9763.7 ± 10.729425 ± 24550.44
Brain691.86591.37735.46365.69257.87168.75123.5294.2 ± 11.470723 ± 128281.07
Eyes1373.05560.03461.82208.62248.9544.9760.0062.1 ± 12.942505 ± 60120.64
Adrenals2688.053122.011874.821602.521372.771049.10754.01109.6 ± 11.7374210 ± 297585.64
Fat473.38501.3896.45122.27132.8770.8157.48115.5 ± 10.432176 ± 45330.48
Muscle404.29815.55242.76214.15252.51129.71100.12105.8 ± 6.354555 ± 96930.82
Lungs612.841568.242579.44458.43452.96335.15191.87123.8 ± 6.7160034 ± 151472.41
Heart811.251967.074313.07626.10696.07581.53338.66108.6 ± 6.6251987 ± 101293.80
Spleen3707.276448.145282.396132.076096.566539.263524.37154.3 ± 15.71860779 ± 31546228.04
Kidneys2401.767234.394975.956643.953209.112341.821372.3294.2 ± 13.5832224 ± 18292212.54
Liver3638.565417.102088.161866.091787.881261.59544.3978.9 ± 3.3363288 ± 360995.47
Stomach756.991753.69782.51483.68495.14356.16209.3093.5 ± 5.4122124 ± 66831.84
Stomach content327.622171.11213.63187.00116.7028.8656.8377.9 ± 4.148694 ± 144800.73
Large intestine (LI)1429.326006.642548.931194.681463.99443.89193.9869.5 ± 10.6246379 ± 636493.71
LI content1186.589421.076601.613831.901378.43842.04255.7765.6 ± 11.9462496 ± 1477056.97
Small intestine (SI)11463.67453.891484.20967.61733.54417.34202.4074.9 ± 11.6249033 ± 102333.75
SI content55906.550448.04192.945077.143471.981734.6257.4729.4 ± 3.81310061 ± 81963519.74
Bone marrow1441.56617.65154.1640.0 ± 9.552186 ± 8680.790.81
Total amount90501.51107210.2840277.5630406.2522581.0316704.088116.6375.37 ± 5.646635878.91100.00
% of total recovery28.6633.9512.759.637.155.292.57
Table 3

Percentages of renal and fecal excretion of total radiolabel following single intravenous administration of [14C] artesunate with phosphate buffer (pH 8.1) formulation at 5 mg/20 μCi/kg to rats (n = 5)*

Duration (days)Urine (%)Feces (%)Cage wash (%)
* All values expressed as % of dose, mean ± SD, n = 5.
    0–0.041.61 ± 1.07
0.04–0.3319.19 ± 2.731.10 ± 0.58
0.33–126.76 ± 6.9424.63 ± 5.45
    1–25.07 ± 0.967.50 ± 1.98
    2–31.50 ± 0.332.60 ± 0.60
    3–40.83 ± 0.131.25 ± 0.34
    4–50.47 ± 0.170.56 ± 0.11
    5–60.33 ± 0.090.36 ± 0.11
    6–70.20 ± 0.110.27 ± 0.08
    7–80.13 ± 0.060.18 ± 0.09
Total56.06 ± 7.7038.46 ± 6.261.55 ± 0.29
Ratio of excretion (urine/feces)1.48 ± 0.26
Balance (% of dose)96.07 ± 11.03
Elimination parameters
    Peak height (μg/rat)381.06 ± 46.72300.96 ± 89.58
    Peak time (days)0.48 ± 0.130.53 ± 0.04
    Fast elimination phase (days)0.37 ± 0.030.57 ± 0.07
    Slow elimination phase (days)2.06 ± 0.462.54 ± 0.46
Table 4

Percentage distribution, concentration, and half-lives of [14C] artesunate in total, free, glucuronide and other conjugation fractions in plasma or urine following a single intravenous dose at 5 mg/20 μCi/kg in rats*

TimeTotalFree fractionGlucuronide conjugation fractionOther conjugation fraction
* AUC = area under curve; t1/2 = half-life.
Plasma (n = 3)
03030.1 ± 77.6245.5 ± 16.4144.2 ± 21.3658.4 ± 106.1
1540.9 ± 27.691.3 ± 13.143.1 ± 5.7204.5 ± 68.3
61070.1 ± 219.2187.4 ± 29.2113.9 ± 14.9585.8 ± 148.5
24255.7 ± 21.924.8 ± 9.615.8 ± 1.9205.7 ± 27.1
48135.5 ± 19.68.8 ± 6.36.9 ± 0.6103.6 ± 3.2
7292.4 ± 12.86.5 ± 5.05.1 ± 0.479.1 ± 2.0
9663.4 ± 8.64.9 ± 3.93.8 ± 0.261.8 ± 2.0
16820.6 ± 3.32.1 ± 1.91.6 ± 0.129.5 ± 1.7
19214.2 ± 2.51.6 ± 1.51.2 ± 0.123.1 ± 1.5
AUC (ng·h/ml)28101 ± 32863491.1 ± 1255.62252.2 ± 193.522577.8 ± 2615.2
t1/2 (h)44.47 ± 3.6856.61 ± 7.4558.09 ± 4.8667.58 ± 2.53
% of total99.22 ± 11.6012.33 ± 4.437.95 ± 0.6879.72 ± 9.23
Urine (n = 5)
182995 ± 258997969 ± 187217041 ± 421362694 ± 10648
649996 ± 88194820 ± 11199758 ± 257838967 ± 5507
2419118 ± 20971836 ± 4283235 ± 96515300 ± 2476
485265 ± 1809512 ± 132654 ± 2284169 ± 1101
721793 ± 763193 ± 62154 ± 631399 ± 443
96774 ± 29597 ± 3848 ± 24620 ± 190
120413 ± 13557 ± 2621 ± 12349 ± 101
144256 ± 8936 ± 1911 ± 7226 ± 64
168172 ± 7224 ± 137 ± 5156 ± 45
192120 ± 6116 ± 104 ± 3110 ± 33
AUC (ng·h/ml)1671670 ± 212023173616 ± 41760298884 ± 871231189805 ± 194459
t1/2 (h)48.51 ± 13.2438.16 ± 7.5135.27 ± 10.9750.59 ± 8.82
% of total100.56 ± 12.7510.44 ± 2.5117.98 ± 5.2471.58 ± 5.24
Figure 1.
Figure 1.

Total radioactivity (ng equivalents per ml) of [14C] artesunate in whole blood (dashed line with triangular markers) and in plasma (solid line with circular markers), and plasma concentration of unchanged artesunate (AS) (dotted line with dot makers in re-plot) and unchanged dihydroartemisinin (DHA, dashed line empty circle in re-plot) following a single 5 mg/20 μCi/kg intravenous injection of [14C] AS in rats (N = 6). This figure appears in color at www.ajtmh.org.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 75, 5; 10.4269/ajtmh.2006.75.817

Figure 2.
Figure 2.

Mean distribution profile of total radioactivity (ng equivalents per gram tissue) of radiolabeled artesunate (AS) in various tissues (column) at 1, 6, 24, 48, 72, 96, and 192 hours following a single 5 mg/20 μCi/kg intravenous injection of [14C]AS in rats (n = 5). This figure appears in color at www.ajtmh.org.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 75, 5; 10.4269/ajtmh.2006.75.817

Figure 3.
Figure 3.

Individual elimination profiles of total radioactivity (μg equivalents per rat) of radiolabeled artesunate (circular markers) in urine (top) and feces (bottom), as well as computer fitted curves (solid line) by pharmacokinetic parameters following a single 5 mg/20 μCi/kg intravenous injection of [14C]AS in rats during 8 days collection (N = 5).

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 75, 5; 10.4269/ajtmh.2006.75.817

Figure 4.
Figure 4.

Individual protein binding of [14C]AS in male humans (top) and male rats (bottom) plasma (triangle markers) and mean value in the humans and rats plasma (solid line) after incubation with variable concentrations of AS (0.2, 1, 5, 25, 125, 625, 3125, 15625, and 78125 ng/ml) at 37°C for 5 hours (N = 3).

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 75, 5; 10.4269/ajtmh.2006.75.817

Figure 5.
Figure 5.

Metabolic patterns (solid line) of radiolabel distribution with standard references (dotted line) of alpha-dihydroartemisinin (α-DHA), β-dihydroartemisinin (β-DHA), and artesunate (AS) in plasma free fraction at 1 min (A), 5 min (B), plasma glucuronidate conjugation fraction at 5 min (C), and urine glucuronidate conjugation fraction at 8 hr (D) after single intravenous injection at dose of 5 mg/20μCi/kg of [14C]AS in rats displayed together in radiochromatography.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 75, 5; 10.4269/ajtmh.2006.75.817

*

Address correspondence to Qigui Li, Division of Experimental Therapeutics, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, MD 20307. E-mail: qigui.li@na.amedd.army.mil

Authors’ addresses: Qigui Li, Lisa H. Xie, Adam Haeberle, Jing Zhang, and Peter Weina, Division of Experimental Therapeutics, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, MD 20910-7500, Tel: (301) 319-9351, Fax: (301) 319-7360, E-mails: qigui.li@na.amedd.army.mil; lisa.xie@na.amedd.army.mil; adam.haeberle@na.amedd.army.mil; jing.zhang@na.amedd.army.mil; peter.weina@na.amedd.army.mil.

Disclaimer: The opinions or assertions contained herein are the private views of the author and are not to be construed as official, or as reflecting true views of the Department of the Army or the Department of Defense.

Financial support: This study was supported by the United States Army Research and Materiel Command.

REFERENCES

  • 1

    World Health Organization, 1990. Control of tropical diseases. Severe and complicated malaria. Trans R Soc Trop Med Hyg 84 (Suppl 2): 1–65.

    • Search Google Scholar
    • Export Citation
  • 2

    de Vries PJ, Dien TK, 1996. Clinical pharmacology and therapeutic potential of artemisinin and its derivatives in the treatment of malaria. Drugs 52 :818–836.

    • Search Google Scholar
    • Export Citation
  • 3

    Li QG, Brewer TG, Peggins JO, 1998. Anorectic toxicity of di-hydroartemisinin, arteether and artemether in rats following multiple intramuscular doses. Int J Toxicol 17 :663–676.

    • Search Google Scholar
    • Export Citation
  • 4

    Li QG, Mog SR, Si YZ, Kyle DE, Gettayacamin M, Milhous WK, 2002. Neurotoxicity and efficacy of arteether related to its exposure times and exposure levels in rodents. Am J Trop Med Hyg 66 :516–525.

    • Search Google Scholar
    • Export Citation
  • 5

    McLean WG, Ward SA, 1998. In vitro neurotoxicity of artemisinin derivatives. Med Trop 58 (Suppl 3):28S–31S.

  • 6

    Wesche DL, DeCoster MA, Tortella FC, Brewer TG, 1994. Neurotoxicity of artemisinin analogs in vitro. Antimicrob Agents Chemother 38 :1813–1819.

    • Search Google Scholar
    • Export Citation
  • 7

    Skinner TS, Manning LS, Johnston W, Davis TME, 1996. In vitro stage-specific sensitivity of Plasmodium falciparum to quinine and artemisinin drugs. Int J Parasit 26 :519–525.

    • Search Google Scholar
    • Export Citation
  • 8

    Davis TM, Phuong HL, Ilett KF, Hung NC, Batty KT, Phuong VD, Powell SM, Thien HV, Binh TQ, 2001. Pharmacokinetics and pharmacodynamics of IV artesunate in severe falciparum malaria. Antimicrob Agents Chemother 45 :181–186.

    • Search Google Scholar
    • Export Citation
  • 9

    Batty KT, Le AT, Ilett KF, Nguyen PT, Powell SM, Nguyen CH, Truong XM, Vuong VC, Huynh VT, Tran QB, Nguyen VM, Davis TM, 1998. A pharmacokinetic and pharmacodynamic study of artesunate for vivax malaria. Am J Trop Med Hyg 59 :823–827.

    • Search Google Scholar
    • Export Citation
  • 10

    Newton P, Suputtamongkol Y, Teja-Isavadiharm P, Pukrittay-akamee S, Navaratnam V, Bates I, White N, 2000. Antimalarial bioavailability and disposition of artesunate in acute falciparum malaria. Antimicrob Agents Chemother 44 :972–977.

    • Search Google Scholar
    • Export Citation
  • 11

    Bethell DB, Teja-Isavadharm P, Cao XT, Pham TT, Ta TT, Tran TN, Nguyen TT, Pham TP, Kyle D, Day NP, White NJ, 1997. Pharmacokinetics of oral artesunate in children with moderately severe Plasmodium falciparum malaria. Trans R Soc Trop Med Hyg 91 :195–198.

    • Search Google Scholar
    • Export Citation
  • 12

    Benakis A, Paris M, Loutan L, Plessas CT, Plessas ST, 1997. Pharmacokinetics of artemisinin and artesunate after oral administration in healthy volunteers. Am J Trop Med Hyg 56 :17–23.

    • Search Google Scholar
    • Export Citation
  • 13

    Na-Bangchang K, Karbwang J, Congpoung K, Thanavibul A, Ubalee R, 1998. Pharmacokinetics and bioequivalence evaluation of two generic formulations of oral artesunate. Eur J Clin Pharmacol 53 :375–376.

    • Search Google Scholar
    • Export Citation
  • 14

    Li QG, Peggins JO, Lin AJ, Masonic K, Trotman KM, Brewer TG, 1998. Pharmacology and toxicology of artelinic acid: Pre-clinical investigation on pharmacokinetics, metabolism, protein and RBC binding, acute and anorectic toxicities. Trans R Soc Trop Med Hyg 92 :332–340.

    • Search Google Scholar
    • Export Citation
  • 15

    Titulaer HA, Eling WM, Zuidema J, 1993. Pharmacokinetic and pharmacodynamic aspects of artelinic acid in rodents. J Pharm Pharmacol 45 :830–835.

    • Search Google Scholar
    • Export Citation
  • 16

    Batty KT, Thu TA, Davis TM, Ilett KF, Mai TX, Hung NC, Tien NP, Powell MS, Thien HV, Binh TQ, Kim NV, 1998. A pharmacokinetic and pharmacodynamic study of IV vs oral AS in uncomplicated falciparum malaria. Br J Clin Pharmacol 45 :123–129.

    • Search Google Scholar
    • Export Citation
  • 17

    Li QG, Peggins JO, Fleckenstein L, Masonic K, Heiffer MH, Brewer TG, 1998. Bioavailability and pharmacokinetics of di-hydroartemisinin, arteether, artemether, artesunic acid and artelinic acid in rats. J Pharm Pharmacol 50 :173–182.

    • Search Google Scholar
    • Export Citation
  • 18

    Li QG, Carpenter C, Trotman C, Kathcart AK, Donahue R, Milhous WK, 1999. Pharmacokinetic comparison of artesunate and dihydroartemisinin in beagle dogs. Am J Trol Med Hyg 61 :274.

    • Search Google Scholar
    • Export Citation
  • 19

    Brewer TG, Grate SJ, Peggins JO, Weina PJ, Petras JM, Levine BS, Heiffer MH, Schuster BG, 1994. Fatal neurotoxicity of arteether and artemether. Am J Trop Med Hyg 51 :251–259.

    • Search Google Scholar
    • Export Citation
  • 20

    Classen W, Altmann B, Gretener P, Souppart C, Skelton-Stroud P, Krinke G, 1999. Differential effects of orally versus parenterally administered qinghaosu derivative artemether in dogs. Exp Toxicol Pathol 51 :507–516.

    • Search Google Scholar
    • Export Citation
  • 21

    Genovese RF, Newman DB, Petras JM, Brewer TG, 1998. Behavioral and neural toxicity of arteether in rats. Pharmacol Biochem Behav 60 :449–458.

    • Search Google Scholar
    • Export Citation
  • 22

    Petras JM, Kyle DE, Gettayacamin M, Young GD, Bauman RA, Webster HK, Corcoran KD, Peggins JO, Vane MA, Brewer TG, 1997. Arteether: Risks of two-week administration in Macaca mulatta. Am J Trop Med Hyg 56 :390–396.

    • Search Google Scholar
    • Export Citation
  • 23

    Smith SL, Sadler CJ, Dodd CC, Edwards G, Ward SA, Park BK, McLean WG, 2001. The role of glutathione in the neurotoxicity of artemisinin derivatives in vitro. Biochem Pharmacol 61 :409–416.

    • Search Google Scholar
    • Export Citation
  • 24

    Nui XY, Ho LY, Ren ZH, Song ZY, 1985. Metabolic fate of qinghaosu in rats: a new TLC densitometric method for its determination in biological material. Eur J Drug Metab Pharmacokinet 10 :55–59.

    • Search Google Scholar
    • Export Citation
  • 25

    Kearney BP, Aweeka FT, 1999. The penetration of anti-infectives into the central nervous system. Neurol Clin 17 :883–900.

  • 26

    Zhao KC, Song ZY, 1989. Distribution and excretion of artesunate in rats. Proc Chin Acad Med Sci 4 :186–188.

  • 27

    Tian F, Zhu Y, Long H, Cregor M, Xie F, Kissinger CB, Kissinger PT, 2002. Liquid chromatography coupled with multi-channel electrochemical detection for the determination of daidzein in rat blood sampled by an automated blood sampling system. J Chromatogr B Analyt Technol Biomed Life Sci 772 :173–177.

    • Search Google Scholar
    • Export Citation
  • 28

    Li QG, Hümpel M, 1990. Serum protein binding characteristics of cyproterone acetate, gestodene, levonorgestrel and norethisterone in rat, rabbit, dog, monkey and man. J Steroid Biochem 35 :319–326.

    • Search Google Scholar
    • Export Citation
  • 29

    China Cooperative Research Group on Qinghaosu and its Derivatives as Antimalarials, 1982. Metabolism and pharmacokinetics of qinghaosu and its derivatives. J Trad Chin Med 2 :25–30.

    • Search Google Scholar
    • Export Citation
  • 30

    Maggs JL, Madden S, Bishop LP, O’Neill PM, Park BK, 1997. The rat biliary metabolites of dihydroartemisinin, an antimalarial endoperoxide. Drug Metab Dispos 10 :1200–1204.

    • Search Google Scholar
    • Export Citation
  • 31

    Pollack GM, Brouwer KLR, Demby KB, Jones JA, 1990. Determination of hepatic blood flow in the rat using sequential infusions of indocyanine green and galactose. Drug Metab Dispos 18 :197–202.

    • Search Google Scholar
    • Export Citation

Author Notes

Reprint requests: Dr. Qigui Li, Division of Experimental Therapeutics, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, MD 20910-7500. Tel: (301) 319-9351, Fax: (301) 319-7360, E-mail: qigui.li@na.amedd.army.mil.
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