Synergistically Interaction Between Indinavir And Chloroquine Against Malaria Parasites And The Construction Of Attenuated Malaria Parasites

Many malaria-endemic areas are also associated with high rates of human immunodeficiency virus (HIV) infection. An understanding of the chemotherapeutic interactions that occur during malaria and HIV co-infections is important. Our previous studies have demonstrated that some antiretroviral protease inhibitors are effective in inhibiting Plasmodium falciparum growth in vitro. Currently, studies examining the interactions between antiretroviral protease inhibitors and antimalarial drugs are being conducted, but the data are limited. In this study, we examined the synergistic interactions between the antiretroviral protease inhibitor indinavir and chloroquine (CQ) in chloroquine-resistant and chloroquine-sensitive malaria parasites in vitro and in vivo. In vitro antimalarial activity was determined using a malaria SYBR Green I-based fluorescence method. Antimalarial drug combinations against P. falciparum 3D7 and P. falciparum Dd2 were assessed by isobolographic analysis. The obtained results showed that indinavir in combination with CQ had a synergistic effect in vitro against the two strains. Next, we examined the synergistic activity between indinavir and CQ in a rodent model of infection. Female NIH mice (average body weight, ~25 g) were intraperitoneally inoculated with parasitized erythrocytes of either the chloroquine-sensitive line ASS or the chloroquine-resistant line ASCQ. The drugs were administered orally at 4, 24, 48, and 72 h post-inoculation. All experiments included a drug-free control group and groups treated with varying doses of indinavir administered alone or CQ administered alone or CQ in combination with varying doses of indinavir. On day 4, thin blood films were made to determine parasite densities. We examined in more detail the effect of indinavir on CQ-mediated clearance of asexual parasites. In this experiment, another set of 72 mice were randomly divided into 6 groups (12 animals per group) and intraperitoneally injected with 2×107 parasitized erythrocytes of the chloroquine-resistant line ASCQ. Oral treatments commenced on day 3 post-inoculation. Animals in Group 1 were given 10 mg/kg CQ daily for 4 days. In Groups 2-5, the animals were treated with CQ (10 mg/kg) in combination with the varying doses of indinavir (0.3 g/kg, 0.6 g/kg, 1.2 g/kg, 1.8 g/kg) daily for 4 days. Animals in Group 6 were treated with 100 mg/kg CQ daily for 4 days. Starting on day 3 post-inoculation, tail blood films were prepared daily until day 9. The results suggested that indinavir acted synergistically with CQ against P. chabaudi in vivo, but the synergistic activity is more evident in the chloroquine-resistant line P. chabaudi ASCQ than in the chloroquine-sensitive line P. chabaudi ASS. More importantly, 100% clearance of asexual parasites is achieved when CQ is used in combination with indinavir at an appropriate drug ratio without obvious toxicity in an in vivo infection model of P. chabaudi ASCQ. In conclusion, indinavir acted synergistically with CQ against malaria both in vitro and in vivo.Malaria remains a threat to human health. It is an effective way to control malaria or even to eradicate it through developing a safe, low cost and effective malaria vaccine. Attenuated live whole-parasite vaccine is an ideal malaria vaccine. Malaria parasites were genetically manipulated to achieve the purpose. Therefore, genetic engineering of malaria parasites has become increasingly important. More recently, research in this area has made great achievements. In our research, we genetically modified the Plasmodium berghei parasites in a rodent model. After being genetically manipulated, P. berghei parasites were virulence attenuated and could express MUC-1. Linearized plasmid containing the homologous sequence of toxicity related gene- Rhomboid 1 and expression sequence of MUC-1 was used to transform P. berghei parasites by AMAXA nucleofector. Western blot and integration-specific PCR were performed to confirm the expression of MUC-1 and the disruption of the toxicity related gene, respectively. RT-PCR was also used to confirm the disruption Rhomboid 1 gene. And limited dilution method was used to clone the transfected parasites. The result showed that MUC1can be successfully expressed by P. berghei. But unfortunately, we didn't get the virulence attenuated live whole-parasite. Rhomboid is a gene family and the slow-growth phenotype is specific to PbROM1 disruptants, so we speculate that the plasmid used for transfection may insert into other genes of the family.