Antileishmanial Efficacy Of Nitazoxanide Against Leishmania Donovani And General Toxicity

Leishmania is a kind of obligate end trophic protozoan, which can infect both humans and animals,causing leishmaniasis or Kala-azar. leishmaniasis is distributed worldwide with more than 12million patients,and there are 367million are under threaten. Since 1950s,leishmaniasis was once widespread in northern areas of Yangtze River, it is one of the five major parasitosis in China.Traditional chemotherapeutic agent including multivalence antimonium, Pentamidine and amphotericin B,however, due to various reasons their application were greatly restricted, therefore a new kind of anti-leishmaniasis agent is in great need.Nitazoxanide,2-acetolyloxy-N-(5-nitro 2-thiazolyl) benzamide (NTZ),was first described in 1976 by Romark laboratory. In 2002 NTZ was approved by the US FDA for the treatment of diarrhea caused by Cryptosporidiosis and Giardiasis. Besides, lots of researches have demonstrated the NTZ could also against the activity of bacteria and virus, but few reports on the usage against leishmania or the toxicology researches on NTZ were found, therefore we have carried out experiments of NTZ activity to evaluate its general toxicology and its effect against leishmania for its preliminary assessment of security and make a foundation for its clinical application.We have microscope observation and MTT method to investigate the effect of the NTZ on leishmania in vitro under six different concentrations (200μg/mL, 100μg/mL,50μg/mL, 12.5μg/mL,25μg/mL, and 6.25μg/mL) and different time (48h,72h),3μg/mL of amphotericin B was set as positive control group,DMSO was regarded as negative control group. The results showed that 48h after drug action, the inhibition ratio of different concentrations using microscope observation were95.85%,87.17%,66.81%,53.10%,42.45%,28.13% and in MTT method were 94.81%,87.23%,59.26%,45.98%,38%,25.14%,72h after drug action,the inhibition ratio of different concentrations using microscope observation were 98.69%,90.52%, 73.56%,54.74%,45.86%,31.22% and in MTT method were 98.75%,91.08%,68.53%,55.79%,42.67%,31.80%,the results showed significant deviation compared to the negative group and similar inhibition ratio were found in the positive group.Gimesa staining and electron microscope showed that promastigotes distorted with disintegration of nucleus and kinetoplasts, appearance of vacuoles in cytopiasma and exfoliation of flagella. In control group, peomastigotes were active, most of which were fusiform.In vivo tests,limiting dilution assay, Real-time PRC and pathological section were carried out to detect protective effect of NTZ against leishmania under three concentrations(400mg/mL,200mg/mL and 100mg/mL),the results showed that treatment with NTZ(400mg/mL) successively for 10 days could reduce polypide burden in both liver and spleen compared to the negative group, with a inhibition ratio more than 90%,different inhibition ratio were also found in 200mg/kg and 100mg/kg groups, and showed dosage effect correlation. We have successfully established a SYBR-GreenⅠfluorescent quantitative PCR method to detect Leishmania,which can be used in the diagnosis and epidemiological surveillance of Leishmaniasis.Toxicity tests of NTZ were carried strictly under the conduction of.After the mice were drenched a toxic dose of NTZ, they were found less active, moist clothing hair, degraded appetite, lighten body weight, most were died between 1-3days after administration, and no sex differences. Pathological changes including yellow abdominal cavity mucous,intestine wall attenuated, hemorrhagic and enteritis necroticans,the survivals became normal gradually from 2 to 6 day. The mice of corn oil group were under good condition. The LD50 is 6.295g/kg, with a 95% confidence interval 4.889～8.105g/kg through calculation; NTZ was proved to be weak or without cumulative toxicity under accumulation toxicity test, with a accumulation coefficient K>5.26,and a good toleration under tolerated test.On the whole, NTZ showed a good inhibition and killing effect against leishmania under routine drug concentration with a dosage positive correlation effect. It has good protective effect against leishmania infection, with a low side effect, good tolerance and no cumulative toxicity.