关键词: 日本血吸虫, 抗血吸虫作用, 抗疟药, 疟色素, 氯化血红素, 体外试验, 体内试验

Abstract: Objective  To observe and compare the inhibition of hemozoin formation and the in vitro as well as in vivo antischistosomal activity induced by seven antimalarial drugs.  Methods  Inhibition of hemozoin formation displayed by chloroquine phosphate，　quinine hydrochloride，　quinidine，　mefloquine hydrochloride，　pyronaridine phosphate and lumefantrine at 25 μmol/L，　and artemether at 100 μmol/L was performed by assay of inhibition of β-hematin formation in 1 mol/L sodium acetate buffers containing hematin with various pH of 4.0， 4.2， 4.4， 4.6， 4.8，　and 5.0. In in vitro antischistosomal study， the medium of RPMI 1640 supplemented by 10% calf serum was used to maintain the adult Schistosoma japonicum， and the 50% and 95% lethal concentratrions（LC50 and LC95） to kill the adult worms of each drug were then determined. Meanwhile，　the interaction of quinine，　pyronaridine and chloroquine combined with hemin against adult schistosomes was also undertaken. As to in vivo test， the efficacy of seven antimalarial drugs administered orally or intraperitoneally to mice infected with adult schistosomes was observed.  Results  In the acidic acetate-hematin solution， 25 μmol/L pyronaridine showed significant inhibition of β-hematin formation at pH 4.4-5.0 with inhibition rates of 81.3%-97.0%. At pH 4.6， the inhibition rates of β-hematin formation in acetate-hematin solution induced by mefloquine， chloroquine or quinine at concentration of 25 μmol/L were 79.7%， 72.8% or 65.8%， respectively， and the β-hematin formation was continually inhibited by these 3 antimalarial drugs at pH 4.8 and 5.0 with inhibition rates of 83.1%-90.6%， 41.9%-49.0% or 53.2-62.0%. The inhibition rates of β-hematin formation at pH 4.6 and 4.8-5.0 induced by lumefantrine 25 μmol/L were 74.3% and 40.4%-40.5%， respectively. While under the same concentration of quinidine， 53.4% and 50.9% inhibition rates of β-hematin formation were observed at pH 4.8 and 5.0. As to artemether， higher concentration of 100 μmol/L only showed light inhibition of β-hematin formation at pH 4.4-4.8 with inhibition rates of 16.6%-25.0%. As regard to in vitro test， the LC50 and LC95 of mefloquine， pyronaridine， quinine and quinidine were 4.93 and 6.123 μg/ml， 37.278 and 75.703 μg/ml， 93.688 and 134.578 μg/ml， as well as 101.534 and 129.957 μg/ml，respectively. When adult schistosomes were exposed to the medium containing chloroquine， lumefantrine or artemether at higher concentrations of 100 or 120 μg/ml for 72 h， no or only individual worms died. Hence the LC50 and LC95 of these 3 drugs could not be determined. In other in vitro test， adult schistosomes exposed to quinine 50 μmol/L（20 μg/ml） in combination with 153.4 μmol/L（100 μg/ml） hemin， all worms died within 72 h post incubation. While the worms exposed to 50 μmol/L（26 μg/ml） chloroquine combined with the same concentration of hemin，　only 18.8%（3/16） of worm died at 72 h post exposure. Unexpectedly， in schistosomes exposed to pyronaridine at a toxic concentrations of 50 μmol/L（46 μg/ml） in combination with 153.4 mol/L（100 μg/ml） hemin for 72 h， all of the worms were protected from the toxic action induced by pyronaridine， which revealed in normal motor activity and appearance of morphology in majority of the worms. In in vivo test， mice infected with adult schistosomes were treated orally with chloroquine， pyronaridine or lumefantrine at a daily dose of 400 mg/kg for 3 days， or intraperitoneally with chloroquine or pyronaridine at a daily dose of 100 mg/kg for 2 or 3 days， no apparent efficacy was seen. When mefloquine，　quinine， quinidine or artemether were administered orally to infected mice at a single dose of 400 mg/kg or 200 mg/kg（mefloquine），　all groups of mice treated showed moderate or higher efficacy with worm burden reductions of 61.1%-98.1%.  Conclusion  Among the seven antimalarial drugs tested， their inhibitions of hemozoin（β-hematin） exhibit no definite correlation to their in vitro and in vivo antischistosomal activity. Quinine in combination with hemin shows synergistic effect against schistosomes in vitro. While antagonist effect is observed in pyronardine combined with hemin.

Key words: Schistosoma japonicum, Antischistosomal activity, Antimalarial drug, Hemozoin, Hemin, In vitro test, In vivo test