Activity of Ivermectin and Its Metabolites against Asexual Blood Stage Plasmodium falciparum and Its Interactions with Antimalarial Drugs

ABSTRACT Ivermectin is an endectocide used widely to treat a variety of internal and external parasites. Field trials of ivermectin mass drug administration for malaria transmission control have demonstrated a reduction of Anopheles mosquito survival and human malaria incidence. Ivermectin will mostly be deployed together with artemisinin-based combination therapies (ACT), the first-line treatment of falciparum malaria. It has not been well established if ivermectin has activity against asexual stage Plasmodium falciparum or if it interacts with the parasiticidal activity of other antimalarial drugs. This study evaluated antimalarial activity of ivermectin and its metabolites in artemisinin-sensitive and artemisinin-resistant P. falciparum isolates and assessed in vitro drug-drug interaction with artemisinins and its partner drugs. The concentration of ivermectin causing half of the maximum inhibitory activity (IC50) on parasite survival was 0.81 μM with no significant difference between artemisinin-sensitive and artemisinin-resistant isolates (P = 0.574). The ivermectin metabolites were 2-fold to 4-fold less active than the ivermectin parent compound (P < 0.001). Potential pharmacodynamic drug-drug interactions of ivermectin with artemisinins, ACT-partner drugs, and atovaquone were studied in vitro using mixture assays providing isobolograms and derived fractional inhibitory concentrations. There were no synergistic or antagonistic pharmacodynamic interactions when combining ivermectin and antimalarial drugs. In conclusion, ivermectin does not have clinically relevant activity against the asexual blood stages of P. falciparum. It also does not affect the in vitro antimalarial activity of artemisinins or ACT-partner drugs against asexual blood stages of P. falciparum.

in ivermectin exposure of approximately 25% when combined with dihydroartemisininpiperaquine but with no effect of primaquine co-administration (24). The lack of substantial pharmacokinetic drug-drug interactions and safety signals are promising, but there is still a paucity on the information of possible pharmacodynamic drug-drug interactions. The current study investigated the effects of ivermectin and its metabolites against asexual blood stages of artemisinin-sensitive and artemisinin-resistant P. falciparum isolates and the pharmacodynamic interactions of ivermectin when combined with commonly used antimalarial drugs.

RESULTS
Ivermectin parent compound, ivermectin aglycone, ivermectin monosaccharide, M1, M3, and M6 ( Fig. S1) were selected to study antimalarial effects in this study. Antimalarial activity of ivermectin and its metabolites on asexual blood stage were investigated using a standard SYBR green I-based 72 h in vitro assay. Ivermectin parent compound showed antimalarial activity on asexual blood stage in a dose-dependent manner with mean (95% confidence interval) IC 50 of 0.81 (0.67 to 0.95) mM and 0.81 (0.75 to 0.88) mM when tested against two artemisinin-sensitive and five artemisinin-resistant isolates, respectively (Table 1; Fig. S2). There was no significant difference in IC 50 between artemisinin-sensitive and artemisinin-resistant isolates (P = 0.574). The IC 50 s of all ivermectin-related compounds, including its major metabolites, were 2-fold to 4fold higher than ivermectin parent compound (Table 1). Ivermectin aglycone had the highest IC 50 and all ivermectin metabolites showed less potency compared to ivermectin parent compound against all isolates (P , 0.001).
Ivermectin and antimalarial drug combination activity against P. falciparum was evaluated by a checkerboard analysis and presented as isobolograms and FIC indices. Eight combinations were tested with artemisinin-sensitive and artemisinin-resistant P. falciparum isolates. Isobologram analysis demonstrated no substantial interaction between ivermectin and amodiaquine, atovaquone, artesunate, dihydroartemisinin, lumefantrine, mefloquine, piperaquine, or pyronaridine ( P FIC . 0.5 and #4) ( Fig. 1; Table 2). The antimalarial activity of artesunate was further studied alone and in combination a All data are reported as mean IC 50 (95% confidence interval), with the unit mM.
Effect of Ivermectin and Metabolites on Plasmodium falciparum Antimicrobial Agents and Chemotherapy with a fixed dose of 50 ng/mL ivermectin by the trophozoite maturation assay. No difference was observed in IC 50 values of artesunate alone or in combination with ivermectin in both artemisinin-sensitive (P = 0.385) and artemisinin-resistant (P = 0.546) isolates (Fig. S3).

DISCUSSION
The World Health Organization (WHO) has recommended a range of interventions to achieve the elimination of malaria (36). Ivermectin has a significant mosquito-lethal effect on many species of Anopheline mosquitoes. (17-19, 21, 28, 37). It has been proposed that ivermectin be used as a complementary malaria vector control tool (38).
Although several studies have reported that ivermectin has a clear concentration-dependent mosquito-lethal effect, resulting in a reduced incidence of malaria, no study has evaluated the pharmacodynamic drug-drug interactions of ivermectin and the commonly used antimalarial drugs against the asexual blood stage of Plasmodium parasites. The in vitro activity of ivermectin was assessed against P. falciparum laboratory strains and isolates. Previous reports on the effect of ivermectin on the asexual blood stage of P. falciparum have reported IC 50 values ranging from approximately 0.021 mM to 9 mM (Table S1) (32-34, 37, 39). Differences in IC 50 values between studies may be due to variations in parasite strains, drug exposure times, and methods of assessment. In this study, the antimalarial activity of ivermectin was evaluated against artemisininsensitive and artemisinin-resistant P. falciparum isolates; however, there was no correlation between the artemisinin resistance status of the isolate and the ivermectin IC50 (;0.8 mM). A study from Gabon P. falciparum isolates reported relatively low IC50 of 0.14 mM in a chloroquine-sensitive isolate JH26, and 0.021 mM and 0.13 mM in chloroquine-resistant isolates JH1 and JH13, respectively. Also, there was no correlation between the effect of ivermectin and chloroquine resistance (32).
Previous work identified ivermectin metabolites generated by liver microsomes, primary human hepatocyte, and human blood after ivermectin administration (26). In this current study, we examined how three primary ivermectin metabolites (M1, M3, M6), as well as ivermectin monosaccharide and aglycone, affected both artemisinin-sensitive and resistant parasites. Ivermectin was more potent than ivermectin aglycone, ivermectin monosaccharide, and all ivermectin in vivo metabolites (M1, M3, and M6). The effect of ivermectin aglycone, which lacks the sugar moiety and has a hydroxy-group at C-13 position, presented .90% parasite growth at 1 mM concentrations in this study. The reduction of effect due to these molecular modifications has been shown previously (37,39).
A single dose of 5 mg/kg ivermectin had no effect on the blood stages of Plasmodium berghei in rodents, resulting in the same level of parasitemia, gametocytemia, and exflagellation as vehicle control (29). The impact of ivermectin on different Plasmodium developmental stages has been evaluated, and found to inhibit liver-stage development of P. berghei (30,37,39) and Plasmodium cynomolgi in vitro (31). Three doses of 10 mg/kg ivermectin inhibited approximately 80% of P. berghei liver infections and enhanced host survival in 80% of the treated mice (30). However, no causal prophylactic effect of ivermectin was observed for P. cynomolgi infections in macaques (0.3 to 1.2 mg/kg) (31) or P. falciparum infections in a controlled human malaria infection model of ivermectin administration at 400 mg/kg (40). Ivermectin inhibited the sporogony of P. falciparum (27) and P. vivax (19,21) at sublethal concentrations to Anopheles vectors by reducing oocyst prevalence and intensity. It remains unclear if the drug acts on mosquito midgut physiology or interferes with sporogony development. Ivermectin and avermectin derivatives showed no activity against gametes. However, they exhibited inhibitory effects against the late sporogony process of ookinete and oocyst formation in a mosquito-free in vitro assay to study the direct drug effect on sporogony in Plasmodium berghei (29).
The optimal dosage and regimen of ivermectin are key parameters to maintain plasma ivermectin concentration at effective levels (41). A standard dose of ivermectin for onchocerciasis and lymphatic filariasis were 150 mg/kg and 200 mg/kg. In comparison, the tested doses of ivermectin trials for malaria transmission control varied from 150 to 600 mg/kg (13-16, 22, 23). Peak concentrations of 56.8 ng/mL ivermectin was observed after a single dose of 400 mg/kg in healthy Thai adults (24). While peak concentrations of 64.1 ng/mL and 105.2 ng/mL were reported in malaria patients after receiving a 3-day treatment of dihydroartemisinin-piperaquine and ivermectin at 300 and 600 mg/kg/ day, respectively (42). At this dosage, plasma ivermectin showed potent mosquitocidal effects against Anopheline mosquitoes (24,43). In contrast, the asexual blood stage IC 50 observed in this study was approximately 0.8 mM (equal to 712 ng/mL), which is 6-fold to 12-fold higher than clinically relevant peak plasma ivermectin concentration at commonly used doses and regimens. Thus, it is unlikely that ivermectin MDA for malaria transmission control will impact asexual blood stage malaria parasites. In addition, ivermectin-treated parasites showed increasing trends of sexual commitment in a transgenic P. falciparum NF54 strain that expressed endogenous mScarlet-tagged AP2-G, a specific marker for sexually committed ring stages (44). During the malaria transmission season, ivermectin MDA alone (14,15) and in combination with artemether-lumefantrine (13) or dihydroartemisinin-piperaquine (16,22,24) or albendazole (15,23) significantly reduced mosquito survival and malaria cases. Although ivermectin MDA was designed to target mosquitoes that feed on humans, it is important to address the interaction between ivermectin and antimalarial drugs on asexual blood stages of P. falciparum. For instance, antagonistic interactions have been observed in vitro on P. falciparum when combining ivermectin and doxycycline (45). The results presented here demonstrate that the parasite killing effects of the commonly used antimalarial drugs is not altered when combined with ivermectin when evaluated in vitro. This suggests that there is no clinically important pharmacodynamic drug-drug interactions to consider for possible MDA administrations. However, limited data are available on pharmacokinetic drug-drug interactions between ivermectin and antimalarial drugs. A healthy volunteer trial in Thailand showed relatively minor increases in the exposure to both piperaquine and ivermectin when co-administered, but there was no drug-drug interaction reported for primaquine (24). The combination of ivermectin and ACTs in MDA campaigns need further monitoring of drug efficacy and pharmacokinetic drug interactions in endemic area.
In conclusion, ivermectin and its metabolites showed no antimalarial effects at clinically relevant concentrations, although ivermectin demonstrated stronger antimalarial activity than its metabolites. Furthermore, neither artemisinin-sensitive nor artemisinin-resistant P. falciparum isolates exhibited pharmacodynamic interactions between ivermectin and commonly used antimalarial drugs. These findings support that ivermectin is unlikely to interfere with the antimalarial activity of the commonly used antimalarial drugs.
Artesunate (Artesunate for Injection; registration no. 1C 3/35 [N], Guilin No.2 Pharmaceutical Factory) was dissolved in 5% NaHCO 3 at 60 mg/mL. Amodiaquine was dissolved in 70% ethanol at 1 mg/mL. Atovaquone and dihydroartemisinin were dissolved in DMSO at 1 mg/mL. Lumefantrine was dissolved in absolute ethanol at 1 mg/mL. Mefloquine and piperaquine were dissolved in 0.1 M H 3 PO 4 at 1 mg/mL. Pyronaridine was dissolved in RPMI 1640 medium at 1 mg/mL. All antimalarial drugs were kindly provided by Worldwide Antimalarial Resistance Network (WWARN). Stock solutions were kept at 280°C and diluted with culture medium before the assay was set up.
Drug sensitivity on asexual blood stage. Ivermectin and ivermectin metabolites were prepared by a 2-fold serial dilution (0.02 to 10 mM) in RPMI 1640 medium supplemented with 0.5% AlbuMAX II (Thermo Fisher Scientific catalog no.11021045) in flat bottom 96-well plates at 50 mL/well. Asexual blood stage parasites, predominantly at the ring stage, were prepared at 1% parasitemia and 2% hematocrit. In each well, 50 mL of parasite suspension was added and gently mixed with the compounds. After 72 h of incubation, SYBR green I staining was used to detect parasite growth (47,48 Pharmacodynamic drug-drug interactions with antimalarial drugs. The effects of ivermectin parent compound in combination with antimalarial drugs against the asexual blood stage of P. falciparum were evaluated using the checkerboard technique (49,50). Briefly, two-dimensional checkerboard titration was prepared in flat-bottom 96-well plates in a volume of 50 mL. The assay plate was prepared with combinations of ivermectin (0.05 to 10 mM) and individual antimalarial drugs (0.20 to 100 nM amodiaquine, 0.10 to 50 nM artesunate, 0.02 to 10 nM atovaquone, 0.1 to 50 nM dihydroartemisinin, 0.39 to 200 nM lumefantrine, 0.78 to 400 nM mefloquine, 0.39 to 200 nM piperaquine, and 0.20 to 100 nM pyronaridine). Asexual blood stage parasites, predominantly at the ring stage, were prepared at 1% parasitemia and 2% hematocrit. In each well, 50 mL of parasite suspension was added to a final volume of 100 mL and gently mixed with the compounds. After 72 h of incubation, parasite growth was assessed by DNA content using a SYBR green I-based fluorescence staining (47,48). Each well was filled with 100 mL of 2ÂSYBR-green I lysis buffer. Plates were incubated in the dark for 30 min before the fluorescence signal was measured on a microplate reader (Synergy H1, BioTek) using a 485-nm excitation filter and a 520-nm emission filter. The interactions between two compounds were evaluated using isobolograms and derived fractional inhibitory concentrations (FIC). The sum of FICs (RFIC) were calculated by a fraction of the IC50s in each drug combination and the IC50s of the single drug according to equation 1.
where IC50 A1B is the IC50 of drug A in combination with drug B, IC50 B+A is the IC50 of drug B in combination with drug A, IC 50 A and IC 50 B are the IC 50 of drug A and drug B alone, respectively. RFIC of #0.5, 0.5 , FIC # 4 and FIC . 4 indicated synergistic, indifferent, and antagonistic effects, respectively (51,52). The isobolograms and derived mean FIC values were calculated from at least three independent biological replicates with technical duplicates in each assay. The antimalarial effect of artesunate when combined with a fixed dose of ivermectin was further investigated by the trophozoite maturation assay (53). Two-fold serial dilutions of artesunate alone (0.001 to 1 mM) and artesunate combined with a fixed dose ivermectin at 50 ng/mL, the observed clinical peak concentration of ivermectin after administration of ivermectin (150 mg/kg) (54), were prepared in flat-bottom 96-well plates. Parasites, predominantly at the ring stage, were prepared at 1% parasitemia and 2% hematocrit, and then incubated with the drug for 24 h. Thick and thin blood films were harvested and stained with Field's stain. The staging of parasite development was investigated using a light microscope, and the numbers of trophozoites were counted per 100 parasitized red blood cells. Trophozoites were identified by morphology, size, nuclear/cytoplasm ratios, and visible pigment. IC 50 was evaluated from the inhibition of the parasite development from ring to trophozoites compared to parasites in drug-free control wells using GraphPad Prism version 8. Statistical significance was determined by Student's t test and nonparametric Mann-Whitney U tests.

SUPPLEMENTAL MATERIAL
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