Parasitol Res DOI 10.1007/s00436-011-2427-z
ORIGINAL PAPER
Synergy of the antiretroviral protease inhibitor indinavir and chloroquine against malaria parasites in vitro and in vivo Xiaofen Li & Zhengxiang He & Lili Chen & Yayong Li & Qinyan Li & Siting Zhao & Zhu Tao & Wen Hu & Li Qin & Xiaoping Chen
Received: 4 March 2011 / Accepted: 8 April 2011 # Springer-Verlag 2011
Abstract 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, by using modified fixed-ratio isobologram method, fractional inhibitory concentrations index (FICI) was calculated to indicate the interaction between the two drugs. The results demonstrated that indinavir interacted synergistically with chloroquine against both chloroquine-sensitive P. falciparum clone 3D7 (mean FICI 0.784) and multidrugX. Li : Z. He : L. Chen : Y. Li : Q. Li : S. Zhao : Z. Tao : W. Hu : L. Qin : X. Chen (*) Laboratory of Pathogen Biology, State Key Laboratory of Respiratory Disease, Center for Infection and Immunity, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Science, Guangzhou 510530, China e-mail:
[email protected] X. Li : Z. Tao : X. Chen School of Life Sciences, University of Science and Technology of China, Hefei 230027, China L. Chen : Q. Li : W. Hu Graduate University of the Chinese Academy of Sciences, Beijing 100049, China
resistant P. falciparum clone Dd2 (mean FICI 0.599). In vivo drug interactions were measured using a 4-day suppressive test in a rodent malaria model infected with Plasmodium chabaudi. We observed that indinavir enhanced the antimalarial activity of chloroquine against both the chloroquinesensitive line P. chabaudi ASS and the chloroquine-resistant line P. chabaudi ASCQ. More importantly, chloroquine had a 100% clearance of asexual parasites when used in combination with indinavir at an appropriate dose ratio (10 mg/kg CQ + 1.8 g/kg indinavir) where there was no obvious toxicity. We conclude from this study that the combination of indinavir and chloroquine may become a novel antimalarial drug regimen.
Introduction Malaria is one of the most widespread diseases in the world, particularly in the Third World countries and especially in sub-Saharan Africa. The World Health Organization estimates that 300–500 million cases of malaria and one to two million deaths occur annually due to malaria, with most malaria-related morbidity and mortality occurring in children (Snow et al. 1999). Chloroquine (CQ) has been widely used as an antimalarial drug due to its efficacy, affordability, easy administration, and low toxicity (Ward and Bray 2001). However, the widespread emergence of drug resistance in strains of Plasmodium falciparum reduces the antimalarial efficacy of the drug and limits its use, which leads to the increased morbidity and mortality of malaria (Ridley 2002). Recent studies have shown that physiologically relevant concentrations of antiretroviral protease inhibitors (APIs), which block the action of the aspartyl protease of HIV, can directly inhibit the growth of P. falciparum in vitro
Parasitol Res
(Andrews et al. 2006; Parikh et al. 2005; Skinner-Adams et al. 2004; He et al. 2009). Other studies have demonstrated that APIs can also inhibit the growth in Plasmodium chabaudi in a murine model of infection (Andrews et al. 2006). Malaria is co-endemic with HIV in many regions of the developing world. Thus, many patients with acquired immune deficiency syndrome will inevitably be exposed to antimalarial agents, and conversely, many malaria patients will inevitably be exposed to antiretroviral drugs. Therefore, an understanding of the chemotherapeutic interactions between drugs during malaria and HIV co-infection is important. Although the mechanism of the antimalarial activity of APIs is still being debated, studies examining the interactions between APIs and antimalarial drugs are now being conducted. Our previous studies have suggested that APIs act synergistically (He et al. 2008; Skinner-Adams et al. 2007) or antagonistically (He et al. 2010) with the different antimalarial drugs. Indinavir, an HIV protease inhibitor, behaves antagonistically with artemisinine endoperoxides (He et al. 2010), but there are no data describing the interactions of indinavir and CQ against malaria. In this study, we examined the synergistic activity of the HIV protease inhibitor indinavir and CQ against the chloroquinesensitive clone 3D7 and the multidrug resistance clone Dd2 in vitro. We also examined the chloroquine-sensitive line P. chabaudi ASS and the chloroquine-resistant line P. chabaudi ASCQ in vivo.
Materials and methods In vitro cultivation of parasites The chloroquine-sensitive P. falciparum clone 3D7 and the multidrug-resistant P. falciparum clone Dd2, which were obtained from the Malaria Research and Reference Reagent Resource Center (MR4), were cultured and maintained as stocks on human O+ erythrocytes using the candle-jar method as previously described (Trager and Jensen 1976). Parasites were synchronized using serial treatments with 5% D-sorbitol (Lambros and Vanderberg 1979), and synchronized ring-stage-parasitized erythrocytes were used in all the experiments. Animals and in vivo parasite inoculation We purchased 8- to 10-week-old female NIH mice from Vital River Experiment Animal Limited Company (Beijing, China) and housed them in the Animal Center at the Guangzhou Institutes of Biomedicine and Health according to the Guide for the Care and Use of Laboratory Animals established by this institute. The animals were acclimated for 10 days in standard cages before the experiments, and standard feed and water
given ad libitum. The chloroquine-sensitive line P. chabaudi ASS and the chloroquine-resistant line P. chabaudi ASCQ (MR4), which were maintained by serial blood passage in mice, were the malaria parasites used in this study. Assay for in vitro drug interactions In vitro antimalarial activity was determined using a malaria SYBR Green I-based fluorescence method as described previously ( Kelly et al. 2009; He et al. 2010). Briefly, drug solutions were serially diluted and administered in quadruplicate to parasite cultures in 96-well plates to achieve 0.2% parasitemia with a 2% hematocrit. The plates were then incubated for 72 h at 37°C. Following incubation, 100 μL of lysis buffer containing 0.2 μL/mL SYBR Green I was added to each well. The plates were incubated for 1 h in the dark, and a 96-well fluorescence plate reader (Multilabel HTS Counter; PerkinElmer) was used to measure relative fluorescence. The 50% inhibitory concentration (IC50) was determined using a nonlinear regression analysis of the logistic dose response curves using the software GraphPad Prism (GraphPad Software Inc., La Jolla, CA). Drug interaction studies were performed using a modified fixed-ratio method as previously described (Deloron et al. 1991; Fivelman et al. 2004). Fractional inhibitory concentrations (FICs) (Canfield et al. 1995; Fivelman et al. 2004) were calculated using the following formula: FICA =IC50 of drug A in combination/IC50 of drug A alone, and FICB =IC50 of drug B in combination/IC50 of drug B alone. The FICI= FICA + FICB (Kelly et al. 2009). Isobolograms were constructed by plotting a pair of FICs for each API and CQ drug combination. A straight diagonal line (FICI=1) on the isobologram indicates an additive effect. A concave curve below the line (FICI1) indicates antagonism. In vivo drug interaction assay In vivo drug interactions were measured using a 4-day suppressive test in a P. chabaudi rodent malaria model of infection (Deloron et al. 1991; Fidock et al. 2004). Experimental groups of six female NIH mice (average body weight, ~25 g) were intraperitoneally inoculated with 2×107 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 postinoculation. All experiments included a drug-free control group and groups treated with varying doses of CQ administered alone or in combination with varying doses of indinavir. On day 4, thin blood films were made to determine parasite densities. The difference between the mean value of the control group (defined as 100%) and those of the
Parasitol Res
experimental groups was calculated and expressed as a percent reduction (=activity) using the following equation: activity=100−[(mean parasitemia treated/mean parasitemia control)×100]. Significance differences between groups were calculated using Student’s t test, and P values less than 0.05 were considered significant. All data were analyzed using the software GraphPad Prism (GraphPad Software Inc., La Jolla, CA). Treatment for parasite clearance In this experiment, another set of 72 mice were randomly divided into six 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, 0.6, 1.2, 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 postinoculation, tail blood films were prepared daily until day 9. For each group, the percent clearance of asexual parasites (%) was defined by dividing the number of the mice without peripheral parasitemia at the end of the experiment by the number of mice initially infected.
Results Synergy of indinavir and CQ against P. falciparum in vitro The interaction between indinavir and CQ against P. falciparum was expressed as an isobologram in Fig. 1.
Fig. 1 In vitro interactions of indinavir and CQ against the drugsensitive P. falciparum strain 3D7 (a) and the multidrug-resistant strain Dd2 (b). The combinatorial effect of indinavir and CQ against malaria parasites was tested by titrating the two drugs at fixed ratios proportional to their 50% effective concentrations. The mean (±standard error of the mean) FICIs were derived from three
The isobologram analysis showed that indinavir and CQ exerted a synergistic action against the drug-sensitive clone 3D7 (mean FICI 0.784; Fig. 1a). Similar synergistic characteristics were observed for the combination of indinavir and CQ against the multidrug-resistant clone Dd2 (mean FICI 0.599; Fig. 1b). These results suggest that the HIV protease inhibitor indinavir in combination with CQ has a synergistic effect in vitro against the chloroquinesensitive clone 3D7 and also against the multidrug resistance clone Dd2. Synergy of indinavir and CQ against P. chabaudi in vivo Next, we examined the synergistic activity between indinavir and CQ in a rodent model of infection. Figure 2a illustrates the synergy effect of the combination of indinavir and CQ on the growth of the chloroquinesensitive line P. chabaudi ASS. P. chabaudi ASS-infected mice treated with indinavir alone showed a mild suppression of infection. The combinatorial treatments of P. chabaudi ASS-infected mice (1.5 mg/kg CQ + 0.3 g/kg indinavir or 1.5 mg/kg CQ + 0.6 g/kg indinavir) were not significant compared to either CQ (1.5 mg/kg) or indinavir (0.3 and 0.6 g/kg) treatment alone. However, when indinavir (1.2 and 1.8 g/kg) was co-administered with 1.5 mg/kg CQ, significant parasite suppressive effects were observed compared to the CQ-alone control group (Fig. 2a). Furthermore, the synergistic effect between indinavir and CQ was also pronounced in the drug-resistant line ASCQ (Fig. 2b). We observed that when indinavir was administered alone at 0.3 to 1.2 g/kg, there was no effect on the growth of P. chabaudi ASCQ. When indinavir was co-administered with 2.5 mg/kg CQ, we observed significant parasite suppressive effects (>90% inhibition) compared to the chloroquine-alone
independent experiments. The diagonal (dashed) line (FICI=1) indicates the hypothetical additive drug effect. A concave curve (FICI1) above the diagonal line indicates an antagonistic effect
Parasitol Res
Fig. 2 Synergistic suppression activity of indinavir and CQ against the chloroquine-sensitive line P. chabaudi ASS (a) and the chloroquine-resistant line P. chabaudi ASCQ (b) in a rodent model of infection. Mice were treated orally with CQ alone, indinavir (I)
alone, or a combination of both drugs at different dosages after being infected with either P. chabaudi ASS or P. chabaudi ASCQ. The results are presented as means and standard errors (n=6). *P
