Safety, Pharmacokinetics, and Activity of High-Dose Ivermectin and Chloroquine against the Liver Stage of Plasmodium cynomolgi Infection in Rhesus Macaques
ABSTRACT Previously, ivermectin (1 to 10 mg/kg of body weight) was shown to inhibit the liver-stage development of Plasmodium berghei in orally dosed mice. Here, ivermectin showed inhibition of the in vitro development of Plasmodium cynomolgi schizonts (50% inhibitory concentration [IC50], 10.42 μM) and hypnozoites (IC50, 29.24 μM) in primary macaque hepatocytes when administered as a high dose prophylactically but not when administered in radical cure mode. The safety, pharmacokinetics, and efficacy of oral ivermectin (0.3, 0.6, and 1.2 mg/kg) with and without chloroquine (10 mg/kg) administered for 7 consecutive days were evaluated for prophylaxis or radical cure of P. cynomolgi liver stages in rhesus macaques. No inhibition or delay to blood-stage P. cynomolgi parasitemia was observed at any ivermectin dose (0.3, 0.6, and 1.2 mg/kg). Ivermectin (0.6 and 1.2 mg/kg) and chloroquine (10 mg/kg) in combination were well-tolerated with no adverse events and no significant pharmacokinetic drug-drug interactions observed. Repeated daily ivermectin administration for 7 days did not inhibit ivermectin bioavailability. It was recently demonstrated that both ivermectin and chloroquine inhibit replication of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in vitro. Further ivermectin and chloroquine trials in humans are warranted to evaluate their role in Plasmodium vivax control and as adjunctive therapies against COVID-19 infections.
Ivermectin efficacy against liver-stage parasites was initially evaluated using an in vitroP. cynomolgi liver model which utilizes primary rhesus macaque hepatocytes in order to closely resemble the in vivo antirelapse mode. The drugging regimen was defined by treatment mode, which was either a prophylactic mode (i.e., drug administered with sporozoites and 3 days thereafter) or a radical cure mode (i.e., drug administered from days 4 to 7 post-sporozoite infection) similar to that in previously described methods (19). In prophylactic mode, ivermectin showed marginal in vitro causal protection against the development of P. cynomolgi-infected rhesus macaque hepatocyte liver schizonts (IC50, 9.12 μg/ml; 10.42 μM) and hypnozoites (IC50, 25.59 μg/ml; 29.24 μM) (Fig. 1). However, in radical cure mode, ivermectin had no activity on developing P. cynomolgi liver schizonts or established hypnozoites, even when dosed at a high initial concentration of 100 μg/ml (114.26 μM)
There was only one adverse event in a single macaque (R1435) that vomited 3 hours after the first oral dose of ivermectin (1.2 mg/kg) when administered as monotherapy 1 day prior to P. cynomolgi sporozoite injection. No adverse events occurred when ivermectin (0.6 or 1.2 mg/kg) was coadministered with chloroquine. No abnormal hematology outcomes were observed for ivermectin alone or ivermectin plus chloroquine coadministration.
In vivo results for parasitemia.
Primary blood-stage parasitemia greater than 5,000/μl was detected 10 days postinoculation for negative- and positive-control groups and for 2 of 3 macaques in both ivermectin high-dose (1.2 mg/kg) and low-dose (0.3 mg/kg) groups, with the remaining macaques from each group reaching greater than 5,000/μl at 11 days postinoculation, which was 5 and 6 days after the last ivermectin administration, respectively. Primary infection blood-stage parasitemia was cleared from the negative-control group with 10 days of chloroquine (10 mg/kg), and both blood- and liver-stage parasites were cleared from the positive-control group with 7 days of chloroquine (10 mg/kg) and primaquine (1.78 mg/kg). Blood-stage parasitemia was cleared from the three macaques in the low-dose ivermectin group with 7 days ivermectin (0.6 mg/kg) and 10 days chloroquine (10 mg/kg). Two of three macaques were cleared of primary infection blood-stage parasitemia in the high-dose group with ivermectin (1.2 mg/kg) for 7 days and chloroquine (10 mg/kg) for 10 days, while one macaque was cleared with ivermectin (1.2 mg/kg) and chloroquine (10 mg/kg) for 7 days. However, the first relapse occurred within 3 weeks, at approximately the same time for negative-control and both ivermectin groups, with no significant differences for time to blood-stage parasitemia or treatment (log-rank [Mantel Cox] test, P > 0.05). The first relapse infection blood-stage parasitemia was cleared from the negative control with chloroquine (10 mg/kg) alone for 7 days. First relapse infection blood-stage parasitemia was cleared from both high-dose (1.2 mg/kg) and low-dose (1.2 mg/kg) ivermectin groups when given in combination with chloroquine (10 mg/kg) for 7 days. Approximately 3 weeks later, a second relapse occurred in all negative-control and ivermectin high- and low-dose-treated macaques with no significant differences for time to blood-stage parasitemia or treatment (log-rank [Mantel Cox] test, P > 0.05). At the point of second relapse, all ivermectin group macaques were treated with primaquine (1.78 mg/kg) and chloroquine (10 mg/kg) for 7 days. The positive-control group was treated with primaquine (1.78 mg/kg) and chloroquine (10 mg/kg) for 7 days at the point of primary infection and had no relapses for the remainder of the study (Fig. 2). The negative-control group was treated with primaquine (1.78 mg/kg) and chloroquine (10 mg/kg) for 7 days at the point of third relapse (data not shown).
Blood-stage P. cynomolgi parasitemia results and drug regimens for each treatment group. Displays the number of P. cynomolgi blood-stage parasites per μl of blood observed via microscopy. Shaded areas represent the duration of drug administration when daily drug dosing was administered, with yellow for ivermectin, peach for chloroquine, blue for ivermectin plus chloroquine, and green for primaquine plus chloroquine. Drug concentrations are stated as numbers, and all quantities displayed are in mg/kg. Numbers (i.e., Rxxx) in the legend denote the individual macaque identification number. The red arrows indicate when sporozoites were administered. The dashed red line denotes the 5,000 parasites per μl cutoff to trigger drug administration. IVM, ivermectin; CQ, chloroquine; PQ, primaquine.
The real-time PCR (RT-PCR) method detected primary blood-stage parasitemia 1 day earlier than microscopy at the point of first infection for the negative- and positive-control group macaques and in two out of three ivermectin low-dose (0.3 mg/kg) macaques. The remaining four ivermectin high- and low-dose macaques had blood-stage parasitemia detected by RT-PCR on the same day as microscopy
We thank the AFRIMS Department of Veterinary Medicine for conducting the macaque trial, especially Laksanee Inamnuay, Kesara Chumpolkulwong, Natthasorn Komchareon, Chardchai Burom, Noppon Popruk, Sujitra Tayamun, Mana Saithasao, Alongkorn Hanrujirakomjorn, Nuttawat Wongpim, Khrongsak Saengpha, Phakorn Wilaisri, Chakkapat Detpattanan, Rachata Jecksaeng, Siwakorn Sirisrisopa, Yongyuth Kongkaew, Sakda Wosawanonkul, Sonchai Jansuwan, Amnart Andaeng, Chaisit Pornkhunviwat, Manas Kaewsurind, Wuthichai Puenchompu, Sawaeng Sripakdee, Dejmongkol Onchompoo, Thanaphon Rattanathan, Paitoon Hintong, and Siwadol Samano. We also thank the Department of Entomology Malariology and Insectary Sections, especially Ratawan Ubalee and Siriporn Phasomkusolsil, for supporting the sporozoite production. Monoclonal antibody 7.2 (anti-GAPDH) was obtained from The European Malaria Reagent Repository (http://www.malariaresearch.eu).
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