key: cord-103567-nh9i28lu
authors: Vanachayangkul, Pattaraporn; Im-erbsin, Rawiwan; Tungtaeng, Anchalee; Kodchakorn, Chanikarn; Roth, Alison; Adams, John; Chaisatit, Chaiyaporn; Saingam, Piyaporn; Sciotti, Richard J.; Reichard, Gregory A.; Nolan, Christina K.; Pybus, Brandon S.; Black, Chad C.; Lugo, Luis A.; Wegner, Matthew D.; Smith, Philip L.; Wojnarski, Mariusz; Vesely, Brian A.; Kobylinski, Kevin C.
title: Safety, pharmacokinetics, and liver-stage Plasmodium cynomolgi effect of high-dose ivermectin and chloroquine in Rhesus Macaques
date: 2020-04-29
journal: bioRxiv
DOI: 10.1101/2020.04.27.065409
sha: 
doc_id: 103567
cord_uid: nh9i28lu

Previously, ivermectin (1–10 mg/kg) was shown to inhibit liver-stage development of Plasmodium berghei in orally dosed mice. Here, ivermectin showed inhibition of the in vitro development of Plasmodium cynomolgi schizonts (IC50 = 10.42 μM) and hypnozoites (IC50 = 29.24 μM) in primary macaque hepatocytes when administered in 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 seven consecutive days was evaluated for prophylaxis or radical cure of Plasmodium 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 seven 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.

Novel chemoprophylactic therapeutics and vector control interventions could support and 91 accelerate malaria elimination efforts. Ivermectin mass drug administration (MDA) has been 92 proposed as a malaria control tool since it makes the blood of treated persons lethal to 93

Anopheles mosquitoes, the vectors of malaria (1-5), and repeated ivermectin MDAs in Burkina 94

Faso were able to reduce malaria transmission to humans (6). Ivermectin is a safe and well-95 tolerated endectocidal drug used widely in veterinary and human medicine to combat both 96 internal and external parasites. 97

Ivermectin has been shown to inhibit liver-stage development of Plasmodium berghei in 98 both an in vitro Huh7 human hepatoma cell line model (7) and an in vivo C57BL/6 mouse model 99 (8). The in vitro half maximal inhibitory concentration (IC 50 ) for ivermectin P. berghei inhibition, 100 IC 50 = 1.8 µg/ml (2.1 µM), was higher than blood levels that can be achieved in treated humans. 101

However, mice that were orally dosed with ivermectin at 1-10 mg/kg at 24 and 12 hours before 102 and 12 hours after sporozoite challenge demonstrated liver-stage inhibition equal to primaquine 103 (10 mg/kg) under the same dosing schedule (8). Human equivalent dosing (HED) that was 104 evaluated in mice would correlate to ivermectin doses in the range of 0.08 -0.81 mg/kg (9). 105

Thus, ivermectin is promising for human malaria chemoprophylaxis as ivermectin doses as high 106 as 2 mg/kg have been safely administered to humans (10). If ivermectin can prevent 107

Plasmodium liver-stage infection, then ivermectin chemoprophylaxis could be considered in high 108 risk groups such as forest-goers in the Greater Mekong Subregion or naïve soldiers deployed to 109 malaria endemic areas. Furthermore, if ivermectin MDA is deployed for community-wide malaria 110 vector control, and ivermectin is chemoprophylactic, then there would be direct benefits to MDA 111 participants in preventing malaria infections. 112 model can evaluate both the causal prophylaxis, (i.e. protection from developing liver schizonts), 115 and the hypnozoiticidal (i.e. radical cure of liver hypnozoites) efficacy of compounds (11) . 116

Ivermectin has been used in Rhesus macaque colonies to treat mites (12), lice (13), and 117

intestinal helminths, such as Ascaris, Trichuris, and Strongyloides fulleborni (14) (15) (16) . studies demonstrated that oral ivermectin was safe in macaques at doses up to 1.2 mg/kg for 14 119 days and that macaques are an ideal animal model for ivermectin human treatment (17, 18) . 120

However, no study to date has evaluated the pharmacokinetics of repeated ivermectin treatment 121

in Rhesus macaques or in combination with chloroquine. 122

Here we evaluate the in vitro and in vivo liver-stage effect of ivermectin against P. 123 cynomolgi in Rhesus macaque liver hepatocytes and infected macaques. The safety and 124 pharmacokinetics of repeated oral ivermectin dosing with and without chloroquine in macaques 125 is also presented. 126 127

Ivermectin efficacy against liver-stage parasites was initially evaluated using an in vitro P. 130 cynomolgi liver model which utilizes primary Rhesus macaque hepatocytes in order to closely 131 resemble the in vivo anti-relapse mode. The drugging regimen was defined by treatment mode, 132 either prophylactic mode (i.e. drug administered with sporozoites and 3 days thereafter) or 133 radical cure mode (i.e. drug administered from days 4 to 7 post sporozoite infection) similar to 134 previously described methods (19) . In prophylactic mode, ivermectin showed marginal in vitro 135 causal protection against the development of P. cynomolgi-infected rhesus macaque hepatocyte 136 liver schizonts IC 50 = 9.12 μg/ml (10.42 μM) and hypnozoites IC 50 = 25.59 μg/ml (29.24 μM) 137 ( Figure 1 ). However, in radical cure mode, ivermectin had no activity on developing P. 138 cynomolgi liver schizonts or established hypnozoites, even when dosed at a high initial 139 concentration of 100 µg/ml (114.26 μM). inhibition of liver schizonts (IC 50 = 9.12 μg/ml) and hypnozoites (IC 50 = 25.59 μg/ml). LS = liver-144 stage. Graph bars represent means with standard deviation of biological replicates (n = 3) with 145 experimental replicates (n = 2). 146 147

There was only one adverse event in a single macaque (R1435) that vomited three hours after 149 the first oral dose of ivermectin (1.2 mg/kg) when administered as monotherapy one day prior to 150 P. cynomolgi sporozoite injection. No adverse events occurred when ivermectin (0.6 or 1.2 151 mg/kg) was co-administered with chloroquine. No abnormal hematology outcomes were 152 observed for ivermectin alone or ivermectin plus chloroquine co-administration. 153 154

Primary blood-stage parasitemia greater than 5,000/μl was detected ten days post inoculation 156 for negative and positive control groups and for 2 of 3 macaques in both ivermectin high (1.2 157 mg/kg)-and low (0.3 mg/kg)-dose groups, with remaining macaques from each group reaching 158 greater than 5,000/µl eleven days post inoculation which was 5 and 6 days after the last 159 ivermectin administration. Primary infection blood-stage parasitemia was cleared from the 160 negative control group with ten days of chloroquine (10 mg/kg) and both blood-and liver-stage 161 parasites from positive control group with seven days of chloroquine (10 mg/kg) and primaquine 162

(1.78 mg/kg). Blood-stage parasitemia was cleared from the three macaques in the low-dose 163 ivermectin group with seven days ivermectin (0.6 mg/kg) and ten days chloroquine (10 mg/kg). 164

Two of three macaques were cleared of primary infection blood-stage parasitemia in the high-165 dose group with ivermectin (1.2 mg/kg) for seven days and chloroquine (10 mg/kg) for ten days, 166 while one macaque was cleared with ivermectin (1.2 mg/kg) and chloroquine (10 mg/kg) for 167 seven days. However, the first relapse occurred within 3 weeks, at approximately the same time 168

for negative control and both ivermectin groups with no significant differences for time to blood-169 stage parasitemia or treatment (Log-Rank (Mantel Cox) test P > 0.05). The first relapse infection 170 blood-stage parasitemia was cleared from the negative control with chloroquine (10 mg/kg) 171 alone for seven days. First relapse infection blood-stage parasitemia was cleared from both high 172

(1.2 mg/kg)-and low (1.2 mg/kg)-dose ivermectin groups when given in combination with 173 chloroquine (10 mg/kg) for seven days. Approximately 3 weeks later, a second relapse occurred 174 in all negative control and ivermectin high-and low-dose treated macaques with no significant 175 differences for time to blood-stage parasitemia or treatment (Log-Rank (Mantel Cox) test P > 176 0.05). At the point of second relapse, all ivermectin-group macaques were treated with 177 primaquine (1.78 mg/kg) and chloroquine (10 mg/kg) for seven days. The positive control group 178 was treated with primaquine (1.78 mg/kg) and chloroquine (10 mg/kg) for seven days at point of 179 primary infection and had no relapses for the remainder of the study (Figure 2 ). The negative 180 control group was treated with primaquine (1.78 mg/kg) and chloroquine (10 mg/kg) for seven 181 days at the point of third relapse (data not shown). Table 1 illustrates the pharmacokinetic parameters of ivermectin when administered alone after 213 the first and seventh (last) doses. AUC %Extrap is the percentage of area-under-the-curve infinity 214 due to extrapolation from the last collection time point to infinity, AUC 24hr is the exposure 215 through 24 hours, AUC INF is the total exposure, Cl/F is the apparent clearance, Vz/F is the 216 apparent volume of distribution, C max is the maximum concentration, C max /Dose is the maximum 217 concentration divided by the dose administered, t 1/2 is the elimination half-life, and T max is the 218 time to reach the maximum concentration. 219 220 Table 2 illustrates the pharmacokinetic parameters of ivermectin when administered with 224 chloroquine (10 mg/kg) after the first and seventh (last) doses. AUC %Extrap is the percentage of 225 area-under-the-curve infinity due to extrapolation from the last collection time point to infinity, 226 AUC 24hr is the exposure through 24 hours, AUC INF is the total exposure, Cl/F is the apparent 227 clearance, Vz/F is the apparent volume of distribution, C max is the maximum concentration, 228 C max /Dose is the maximum concentration divided by the dose administered, t 1/2 is the elimination There was no delay to patency of first blood-stage P. cynomolgi infection in either low-or 285 high-dose ivermectin groups (Figure 2 ). Ivermectin displayed µM levels of liver schizont efficacy 286 in vitro, however, a lack of delay to blood-stage patency suggests minimal impact of ivermectin 287 on liver schizont development. Admittedly, the injection of one million P. cynomolgi sporozoites 288 into the macaque sets a very high bar for any drug as it only requires one sporozoite to develop 289 into a liver schizont to continue the blood-stage malaria infection. This is in contrast to a single 290 mosquito that is predicted to deliver <100 sporozoites during blood feeding (21) To the best of our knowledge this is highest repeated dose ivermectin pharmacokinetic 300 investigation in any mammal species. There were no significant changes in the Cl/F or T 1/2 . It 301 should be noted that this study had a small sample size, only three macaques per ivermectin-302 treated group, and thus ivermectin autoinhibition warrants further evaluation in future trials. In 303 humans, three repeated doses of ivermectin (30 or 60 mg) every third day did not inhibit C max 304 when comparing the first and third dose, suggesting a lack of autoinhibition (10). In FVB mice 305 administered oral ivermectin (0.2 mg/kg) twice a week for five weeks there was a 1.7-fold 306 reduction in 24 hour post-dose plasma ivermectin concentrations, while increasing the major 307 metabolite concentration by 1.7-fold (23), suggesting induction of metabolism. 308

In macaques, co-administration of ivermectin (0.6 or 1.2 mg/kg) and chloroquine (10 309 mg/kg) for seven days was safe and well-tolerated. Co-administration of chloroquine and 310 ivermectin did not have an effect on the C max or AUC of ivermectin or chloroquine (Tables 1 and  311 2; Figure 5 ). The 1.2 and 0.6 mg/kg dose in macaques has an approximate HEDs of 0.55 mg/kg 312 (total 3.85 mg/kg) and 0.27 mg/kg (total 1.89 mg/kg) respectively. This suggests that repeated 313 daily dosing of ivermectin at 0.6 or 0.3 mg/kg could be used in combination with chloroquine in 314 humans. While billions of ivermectin and chloroquine treatments have been administered to 315 humans, there is very limited safety evidence for their co-administration. Only one study, on 316

Plasmodium vivax, has co-administered ivermectin (0.2 mg/kg single-dose) and chloroquine 317 (0.6 mg/kg first day, 0.45 mg/kg second and third day), in ten persons with no adverse events 318 passively reported (20). 319

Ivermectin (24), chloroquine (25), and hydroxychloroquine (26, 27) have been shown in 320 vitro to inhibit replication of the novel Severe Acute Respiratory Syndrome Coronavirus 2 321 (SARS-CoV-2). All three drugs distribute into lung tissues at higher concentrations than plasma 322 for chloroquine and hydroxychloroquine in rats (28) 

Anopheles dirus mosquitoes were used to produce P. cynomolgi (B strain) sporozoites, from a 375 donor macaque infected with blood-stage P. cynomolgi parasites. For liver-stage challenge, 376 each macaque was injected intravenously with 1 x 10 6 P. cynomolgi sporozoites in a 1ml 377 inoculum of PBS and 0.5% bovine serum albumin. USAMD-AFRIMS colony-born Rhesus 378 macaques of Indian origin were used in this study. Ten healthy macaques, five male and five 379 female, 3-5 years old and ranging in weight from 4.5-6.4 kg were selected for this study. All 380 macaques were negative for simian retroviruses and simian herpes B virus. Two macaques 381 served as negative controls and were treated initially with seven days of vehicle controls and 382 treated with seven days of chloroquine (10 mg/kg) when parasitemia reached >5,000 parasites 383 per µl at primary infection and first relapse, and with seven days chloroquine (10 mg/kg) plus 384 primaquine (1.78 mg/kg) at second relapse. Two macaques served as positive causal 385

prophylaxis controls and were treated initially with seven days of vehicle controls and treated 386 with seven days of chloroquine (10 mg/kg) plus primaquine (1.78 mg/kg) at point of primary 387 infection when parasites reached >5,000 parasites per µl. All study drugs were administered to 388 restrained conscious macaques via nasogastric intubation at 1 ml/kg body weight. 389

Sparmectin-E (Sparhawk Laboratories, Inc., Lenexa, KS, USA) is a water-soluble 390 formulation of ivermectin developed for oral use in horses. Ivermectin was diluted in sterile 391 water and administered via nasogastric route. Six macaques received ivermectin; three low-392 dose (0.3 mg/kg) and three high-dose (1.2 mg/kg) for seven consecutive days starting one day 393 before sporozoite challenge. If a primary blood-stage infection occurs, and blood-stage 394 parasitemia reaches >5,000 parasites per µl, then the macaques receive seven days of 395 chloroquine (10 mg/kg) plus ivermectin (1.2 mg/kg) for the high-dose group, and seven days of 396 chloroquine (10 mg/kg) plus ivermectin (0.6 mg/kg) for the low-dose group. If a relapse occurs, 397 and blood-stage parasitemia reaches >5,000 parasites per µl, then macaques received seven 398 days of chloroquine (10mg/kg) plus ivermectin (1.2 mg/kg) for both the low-and high-dose 399 groups. If a second relapse occurs, then the macaques were treated with seven days of 400 chloroquine (10 mg/kg) and primaquine (1.78 mg/kg), terminating the experiment. Both the 401 negative and positive control group macaques were treated with seven days of chloroquine (10 402 mg/kg) and primaquine (1.78 mg/kg) at the third relapse and first infection, respectively. 403

Macaques were observed several times in the first few hours post dosing, and at least 404 three times a day for the remainder of the study for any clinical signs of neurological (e.g. ataxia, 405 lethargy, imbalance) or gastroenterological (e.g. diarrhea, vomiting, weight loss) complications. 406

Venous blood was collected at select time points and after macaques become blood smear 407 positive for hematocrit, white and red blood cell count was determined. 408 409

Thick and thin blood smear samples were made and examined daily to quantify malaria 412 parasitemia. Samples were fixed in methanol and stained with Giemsa stain. Blood smears 413

were examined for the presence or absence of blood-stage parasites under oil-immersion 414 objective. If no parasites were found in 50 microscopic oil-immersion thick fields or 415 approximately 1,000 white blood cells (WBCs), the smear was considered negative. The 416 parasitemia level was reported as number of parasites per 1µl or mm 3 of whole blood. Parasites 417 were counted per number of WBCs or red blood cells (RBCs) (i.e., per 1,000 WBCs or 1,000-418 10,000 RBCs). Parasitemia levels were calculated by the appropriate total blood cell count 419 (white or red) per mm 3 . 420 421

Blood samples (0.2 ml) were collected on days 5, 6, and 7 after sporozoite injection. The same 423 sampling schedule occurred in control macaques with the addition of sampling days 8, 9, and 10 424 (1ml) to obtain infected blood for controls used for method development. Blood was collected, 425 stored in EDTA tubes, and kept frozen at -80°C. Parasite DNA was extracted from 200 ul from 426 EDTA whole blood using EZ1 DNA blood kit with automated EZ1 Advanced XL purification 427 system (Qiagen, Hilden, Germany). Real Time PCR for P. cynomolgi detection was performed 428 by using Rotor Gene Q 5plex HRM platform (Qiagen, Hilden, Germany). Primer and probe were 429 designed to target P. cynomolgi small subunit rRNA of blood-stage parasites (GenBank 430 accession number L08242.1). Primer and probe sequences are as follows; P. cynomolgi Fwd: 

Blood sampling (1ml) for pharmacokinetic time points: just prior to first ivermectin dose, and 444 after first dose 1, 2, 4, 8, 12 hours, then each consecutive day just before dosing, then after the 445 7 th dose at 1, 2, 4, 8, 12 hours, and days 1, 2, 5, 12, 19. If a primary infection occurred, then the 446 same blood sampling schedule was repeated, but no blood for pharmacokinetics were collected 447 at first or second relapses. Blood was collected in heparinized sodium Vacutainer tubes and 448 centrifuged at 2,500 rpm for 20 min and then the supernatant (plasma) was transferred and kept 

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We thank the AFRIMS Department of Veterinary Medicine for conducting the macaque trial 497 especially Laksanee Inamnuay, Kesara Chumpolkulwong, Natthasorn Komchareon, Chardchai