| Objective: Artemisinin and its derivatives have been used not only as the first-linetreatment drugs for malaria, but also as drugs for a variety of protozoan infections.Besides, they are also confirmed to be effective in anti-inflammation, anti-tumor andimmune regulation based on the results of in vivo and in vitro experiments. However,up to now a long-term preclinical safety study to support their clinical indicationsother than malaria are not available yet. The objective of this study was to furthercharacterize the toxicological profile of two artemisinin derivatives administered indogs at low dose for13weeks, to find their toxicity organs and elucidate thepathomechanisms of their potential cardiac toxicity. We hope this study could throwsome lights on the safe and rational use of these drugs.Materials and Methods: Sixty-four (32of each sex) Beagle dogs were randomlyassigned by weight and sex to one of the four groups (8dogs of each sex per group):the ART vehicle control group, the ART group (administered orally once daily at6mg/kg/d), the ARM vehicle control group (peanut oil, intramuscular injection [i.m.])and the ARM group (6mg/kg/d, i.m.,). Euthanized termination of the animals wasscheduled at week4,8(two interim necropsies) and13of drug administration, withtwo recovery periods of2and4weeks after drug withdrawal. Parameters to bemeasured as follows: general clinical observation (body weight, food intake, bodytemperature, ECG and neurobehavioral performance), eye examinations,hematological and biochemical analysis, blood smears examination, urineexamination, histopathological examination, ultrastructural examination and theaccompanying toxicokinetic studies. In addition, automated patch clamp techniquewere applied to study the effects of ART and ARM on the hERG potassium channel.Results:1) The results of ophthalmoscopic examinations on the dogs did notindicate any treatment-related effects. Neurobehavioral abnormaity, such as tremor, paralysis, vomiting, gait impairment, and auditory or vision impairment, were notfound in this study.2) Positive findings from clinical observations as follows: in the ARM-injected group,the main clinical signs were skin thickening at the injection site and myonecrosis-likefeatures after treatment for4days up to the end of the treatment period. Signs likeredness, stiff and swelling were found in the injection site. Those lesions at theinjection sites were markedly severer in the ARM group than in the ARM-vehiclecontrol group, which alleviated significantly after4weeks of recovery.3) ECG was recorded for each dog while conscious every2weeks from W-1up to theend of recovery period. As the PQ and QT intervals vary with heart rate, they wereadjusted to a RR-interval of500ms, corresponding to a heart rate of120bpm usingthe following equation: QT500=QT–0.084×(RR–500). ECG showed a trend towardprolonged QT intervals in the ARM-treated dogs and a decreased heart rate in theART-treated dogs.4) Injection related inflammatory reaction was found by hematologic analysis in theARM and the vehicle control group, which was demonstrated as significant increasesin WBC, NE%, and MO%. The RBC counts were found decreased in the dogstreated with ART or ARM at week2, with a corresponding decrease in Hb levels.5) Serum chemistry results showed that significant increases in serum AST and CKin the ARM group at week4and6post treatment compared to the vehicle controlgroup, as well as decreased Na+, K+and A/G ratio in ARM intramuscular injectiongroup.6) Bone marrow smear reaveled that hematopoietic cells mildly increased proportionof granulocytes and a decrease of erythrocytes, while the percentage of middle–latestage erythroblasts showed a mild increase within the erythrocyte series comparedwith the control group.7) Histopathological examination showed fascial inflammation, pseudocysts, musclenecrosis and hemorrhage at the injection sites, and histopathological datademonstrated that the drug exerted a cytotoxic effect on muscle cells close to the injection site, and muscle tissue at the site of the ARM injection was more severelyinflamed than that in the vehicle control group.8) Histological examination of sternal bone marrow revealed mild inhibition oferythropoiesis and a decreased count of mature erythrocytes. Basophilic andpolychromatophilic erythroblasts were the most frequently encountered erythroblastcell type, and extramedullary hematopoiesis was observed in the spleen. Thesechanges were observed both in the ART-and ARM-treated groups with nosignificant differences.Neuropathological alterations like neuron degeneration and necrosis in the cerebralcortex and the grey matter of the spinal cord were observed exclusively in the ARMgroup. The lesions occurred8weeks after treatment with ARM were found in two ofthe four dogs at week8, four of the four dogs examined at week13, and three of thefour dogs at the end of the4-week recovery period, when mild damage could still befound.9) Examination of the SAN and AVN of the cardiac conducting system in the ARTgroup revealed that inflammatory and fatty infiltrations in the SAN of one dog atweek8and vocuolar degeneration of the P cells and T cells in the AVN of two out offour dogs at week13. No obvious abnormalities were found in the CCS of the dogsin ARM group.10) Ultra-structural studies were carried out on the liver, kidney and heart followingtreatment with ART and ARM. Mitochondrial bending, distortion and vacuolationwere found in the hepatocytes and myocardial cells as well as the epithelial cells inthe proximal tubules of kidney at week4,8and13.11) Toxicokinetic study showed that both ARM and ART were finally metablized invivo as dihydroartemisinin (DHA). The mean concentration–time profile of ARMwas shown. The maximum concentrations after dosing of ARM were90.63±24.99μg l-1on day1,219.10±41.30μg l-1at week4,46.20±13.15μg l-1at week8and43.85±10.79μg l-1at week13. Using a40.92ng ml-1concentration as the lowestobserved neurotoxic effect level (LONEL), as reported by Li et al.(2006), the dosage of ARM supplied up to week4may be of toxicological relevance, after that, theplasma concentrations of the drug began to decrease.12) Automated patch clamp technique were used to observe the effects of differentconcentrations of ART and ARM on the hERG potassium channel using CHO cellsexpressing the hERG potassium channel. The results showed that IC50of ART were5.51±5.40mM (n=3), therefore concluded that ART had no obvious blocking effecton hERG potassium channel; Whereas ARM displayed a ready blocking effect onhERG potassium channel in that the IC50of ARM was as high as124.9155.19μM(n=3).Conclusion:(1) An oral administration of ART and intramuscular injection of ARMcould result in bone marrow suppression, red blood cell counts reduction, as well ashematopoiesis in spleen after2weeks’ administration, which could be used as toxicmarker.(2) ART and ARM have different effects on cardiac morphology andfunction. ART could induce vacuolar degeneration of the P-cells and the T-cells andinflammatory infiltration in cardiac conducting system, resulting in decreased heartrate as shown by ECG. However, ARM could lead to QT prolongation, which couldbe interpreted by it’s inhibition on hERG potassium channel based on the results ofpatch clamp analysis.(3) Mitochondrial swelling and vacuolization were found inmyocardial cells, hepatocytes and the epithelial cells in the proximal tubules of thekidney after ART and ARM administration, suggesting mitochondria was a sensitivetoxic target.(4) Histopathological examination revealed that skeletal muscle tissue atthe injection site of the ARM group was more severely inflamed than that in thevehicle control group.(5) Neuropathological alterations were found exclusively inARM group. The involved neurons in the cerebral cortex and the grey matter of thespinal cord in the ARM group were found with degeneration and necrosis, with Nisslbodies decreased or disappeared.(6) The skeletal muscle injury at the injection sitemight affect the absorption of ARM, which resulted in decreased AUC andneurotoxicity. |
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