Classification: Biological sciences – Pharmacology

Repurposing isoxazoline veterinary drugs for control of vector-borne human diseases

Short title: Isoxazolines for drug-based vector control

Marie Miglianico1, Maarten Eldering1, Hannah Slater2, Neil Ferguson2, Pauline Ambrose3, Rosemary S. Lees3, Karin Koolen1, Katerina Pruzinova4, Magdalena Jancarova4, Petr Volf4, Constantianus J. M. Koenraadt5, Graham Trevitt6, Peter G. Schultz7, Baiyuan Yang7, Arnab K. Chatterjee7, John Wisler7, Angelika Sturm1, Teun Bousema8, Robert W. Sauerwein1, 8, Matthew S. Tremblay7*, Koen J. Dechering1*

1 TropIQ Health Sciences, Nijmegen, The Netherlands

2 MRC Centre for Outbreak Analysis and Modelling, Department of Infectious Disease Epidemiology, Imperial College London, UK

3 The Liverpool Insect Testing Establishment (LITE), Liverpool School of Tropical Medicine, Liverpool, UK

4 Department of Parasitology, Faculty of Science, Charles University, Prague, Czech Republic

5 Laboratory of Entomology, Wageningen University and Research, Wageningen, The Netherlands

6 XenoGesis Ltd., Nottingham, UK

7 California Institute for Biomedical Research (Calibr), La Jolla, California, USA

8 Department of Medical Microbiology, Radboud University Medical Center, Nijmegen, The Netherlands

* Correspondence and requests for materials should be addressed to K.J. D. (k.dechering@tropiq.nl) or M. S. T. (mtremblay@calibr.org)

Keywords: Vector-control, Insecticide, Malaria, Zika fever, Isoxazoline

Abstract

Isoxazolines are oral insecticidal drugs currently licensed for ectoparasite control in companion animals. Here we propose their use in humans for reduction of vector-borne disease incidence. Fluralaner and afoxolaner rapidly killed Anopheles, Aedes, and Culex mosquitoes and Phlebotomus sandflies following feeding on a drug-supplemented bloodmeal, with IC50s ranging from 33-575 nM. The compounds were fully active against strains with pre-existing resistance to common insecticides. Based on allometric scaling of preclinical pharmacokinetics data, we predict that a single human dose of 260-410 mg of isoxazoline could provide a robust insecticidal effect lasting for 74 to 90 days against mosquitoes and up to 50 days against Phlebotomus sandflies. Computational modeling showed that seasonal mass drug administration of such a single dose to a fraction of a regional population would dramatically reduce Zika and malaria clinical cases in endemic settings. Isoxazolines therefore represent a promising, fundamentally new component of drug-based vector control.

Significance statement

Reduction in clinical cases of vector-borne diseases (e.g. malaria or Zika fever) is strongly dependent on the ability to reduce the number of infectious insect bites. Here we describe a novel treatment concept based on single dose administration of a mosquitocidal isoxazoline drug to a human population, which leads to killing of blood-fed mosquitoes and a sharp decline in disease transmission. Importantly, we show that isoxazolines are active against a range of vector species, which holds promise to expand the concept of drug-based vector control from malaria to arboviral diseases like dengue, Zika and West Nile fever.

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Introduction

Vector-borne diseases including malaria, Zika fever and leishmaniasis remain a major cause of mortality and morbidity in (sub)tropical regions (1, 2). Temperate areas are also at risk of such diseases, for instance due to the reintroduction of West Nile Virus in Europe (3). Elimination of these diseases will not only require clinical development of new drugs and vaccines, but also effective control of vector populations (4, 5). Drug-based vector control represents a new strategy that consists of administration of an oral insecticidal drug to an at risk human population, to kill the insect vector upon blood feeding, thereby reducing the vector population and preventing disease transmission (6). This approach has the advantage of being effective against mosquito populations feeding outdoors, which are increasingly important for malaria transmission (7) and escape the killing effects of traditional vector control methods such as insecticide-treated bed nets and indoor residual spraying. An oral insecticidal drug should preferentially have a long-lasting effect at a single dose to minimize logistical challenges and cost of mass drug administration (8), and show activity against a broad range of disease vector species.

Isoxazolines are a class of compounds recently licensed as veterinary drugs for protection of companion animals against fleas and ticks (9, 10), with very long in vivo half-lives that provide weeks to months of protection after a single oral administration (11, 12). Even though these compounds have neuronal targets (Figure 1A), they remain safe in mammals as they show limited brain penetration (13) and significant selectivity for insect over mammalian receptors (11, 14). Herein we evaluate two representatives of this compound class in multiple disease-carrying vector species and support the case for their potential use as human oral vector-control drugs.

Results and Discussion

Insecticidal activity of fluralaner and afoxolaner

The isoxazolines afoxolaner and fluralaner (Figure 1B) were tested on a number of strains of Anopheles, Aedes aegypti and Culex pipiens, which are important vectors for malaria, Zika/dengue and West Nile virus, respectively. Mosquitoes were fed on drug-supplemented human blood by membrane feeding. Twenty-four hours after the blood meal, fluralaner showed IC50 values in the range of 33 to 92 nM against all mosquito strains tested, whereas afoxolaner was slightly less active with IC50 values ranging from 90 to 177 nM (Figure 1C, Table 1). Isoxazolines occupy a binding site that is distinct from the targets of known modulators of ionotropic GABA receptors (Figure 1A) (15, 16). In line with this notion, fluralaner and afoxolaner were fully active against the Anopheles gambiae Tiassalé 13 strain that carries the resistance-to-dieldrin (rdl) mutation in the GABA receptor (Figure 1C, Supplemental Data Table 1). In addition, they were equipotent against pyrethroid- and carbamate-resistant strains carrying mutations in the kdr sodium channel and acetylcholine esterase (Ace-1) genes (Figure 1C, Supplemental Data Table 1).

The compounds were further tested by membrane feeding of sand flies, which are important vectors of Leishmania. Afoxolaner and fluralaner showed IC50s of 305 and 575 nM respectively, against Phlebotomus argentipes (Figure 1C, Table 1), a vector of Leishmania donovani on the Indian subcontinent (2). Both compounds were less active against the South American vector, Lutzomyia longipalpis, with IC50s in the range of 1-3 µM (Figure 1C, Table 1). The difference in isoxazoline activity seen between sand flies and mosquitoes suggests that the targeted pocket in the GABA receptor is not conserved among insects.

Human dose prediction

Neither fluralaner nor afoxolaner were measurably metabolized when incubated with dog or human hepatocytes, suggesting that the low in vivo intrinsic clearance observed in dogs (11, 12) could be similar in humans (Table 2). Both compounds were highly bound to plasma proteins (Table 2), which could further contribute to a long in vivo half-life. Based on allometric scaling of published dog pharmacokinetic parameters (Supplemental Data Table 2) (11, 17), we estimated that a single total human dose of 260 mg and 410 mg for afoxolaner and fluralaner, respectively, administered to a 70 kg subject, will result in circulating drug concentrations above the mosquitocidal IC99 for 90 days in the case of Anopheles and Aedes and for 74 days for Culex. In the case of afoxolaner, the resulting plasma concentration would also be above IC99 for Phlebotomus sand flies for 50 days. Since these doses are reasonable amounts to be formulated and delivered in a single-dose mass drug administration, we used this 90-day period of efficacy on Aedes and Anopheles mosquitoes to model the potential effect on two mosquito-borne diseases.

Modeled impact on Zika fever incidence

Zika is an immunizing infection, meaning that once an individual has been infected, they are no longer susceptible to new infections. We modeled the effect of an intervention in a population with historical exposure to Zika at a moment in time where herd immunity had declined to a degree that permits a new epidemic (18). Administration of an isoxazoline drug once a year is predicted to prevent nearly all clinical cases during the years of administration, even if only 30% of the population is treated (Figure 2A). As soon as the intervention ceases, transmission restarts. This rebound may result in a higher cumulative number of cases in the years post intervention compared to a scenario without treatment. This is explained by a reduction in the herd immunity and an increase in the susceptible population (due to births) during the years of intervention. Thus, mass drug administration of a mosquitocidal drug is very efficient in delaying Zika transmission in populations but would need to be maintained to sustainably prevent outbreaks. This risk of disease rebound is well known from mass drug administration programs (19, 20) and the length and coverage of any such campaign have to be carefully chosen to optimize the risk-benefit ratio.

Modeled impact on malaria incidence

Naturally acquired immunity to malaria is mainly non-sterilizing but reduces the severity of infections, and any infectious mosquito bite could potentially cause a new infection. Therefore, a transient intervention such as afoxolaner/fluralaner administration would result in a temporary reduction in malaria incidence and a large reduction in cumulative incidence over a specified time period. For example, in a transmission setting with 17% malaria parasite prevalence by microscopy and a short transmission season (~5-6 months), 80% coverage of the population with the drug would result in a 74% reduction in malaria cases in the intervention year (Figure 2B). In the same conditions, 64% reduction in cases is achieved with population coverage of only 30% (Figure 2B). To further study the impact of prevalence and seasonality, we estimated the reduction in clinical malaria cases for the malaria endemic areas of sub-Saharan Africa based on previously used parameterization of malaria transmission heterogeneity (21) (Supplemental Data Figure 1). The results show that the intervention has the highest impact (>80% reduction in clinical cases) in areas with low and very seasonal transmission, such as Senegal, Sudan, Ethiopia, Madagascar, Namibia, Botswana and Zimbabwe (Figure 3). In these countries, a large proportion of annual transmission occurs in a short period of time. Therefore, administration of a single dose of isoxazoline drug at the beginning of this season would dramatically decrease the number of cases over the full year. In the rest of the continent, isoxazoline administration is predicted to have a lower impact but still to result in a minimum of 30% reduction in clinical cases. From this simplified but illustrative approach (see Materials & Methods for limitations), the key message is that this intervention is predicted to have a significant impact on malaria transmission, with the greatest efficacy in areas with low transmission and a short transmission season.

Preliminary assessment of human safety

Nonclinical safety studies of fluralaner (22, 23) and afoxolaner (24-26) have been conducted for veterinary indications and can be leveraged to offer a preliminary assessment of the human safety of a single dose of these isoxazoline molecules (27). Anticipated single doses of fluralaner (410 mg) and afoxolaner (260 mg) in humans are comparable or lower than equivalent doses considered to be no-adverse effect levels (NOAEL) based on acute and repeat-dose toxicity studies in rats and dogs (Supplemental Data Table 3). Moreover, both fluralaner and afoxolaner were negative in genotoxicity studies and embryo-fetal development in rats was similarly unaffected. The veterinary application of these drugs provides therefore a first assessment of their safety, but supplemental toxicology studies will be required before administering these drugs to humans. Interestingly, veterinary formulations of afoxolaner and fluralaner are based on racemic mixtures, whereas the S-enantiomer has been reported to be the active component against ectoparasites (14, 28). Future development of a human application of these molecules will therefore benefit from further characterization of the activity and toxicology profile of the active enantiomer, which may lead to a 50% reduction in the required dose.

Conclusion

Drug-based vector control holds great promise for reducing disease burden. Pioneering work with ivermectin, a broadly used anti-helminthic that shows adulticidal activity against Anopheles mosquitoes, has delivered important proof of concept by demonstrating reduced survival of blood-fed mosquitoes and likelihood of malaria transmission (29, 30). However, ivermectin's half-life (18 hours in human) necessitates the use of multiple doses, a limitation that poses challenges in terms of logistics and drug compliance, and long-lasting formulations are being sought (6, 8). Moreover, the doses of ivermectin used today to impact anopheline mosquitoes are insufficient for control of Aedes or Culex mosquitoes that transmit arboviral diseases (31, 32). In contrast, our data show potent effects of isoxazolines against a range of disease vectors. The impact of isoxazoline mass drug administration may be highest on diseases where human is the single vertebrate host, as opposed to diseases that cycle between a human and an animal reservoir (e.g. West Nile Virus and leishmaniasis) (3, 33). As shown in our modeling data, even a 30% population coverage could lead to substantial reduction in clinical incidence of malaria or Zika fever. In conclusion, repurposing of isoxazoline compounds offers substantial promise for development of a single-dose vector-control drug, based on a novel mode of action compared to commonly used insecticides, and active against a broad range of relevant disease vectors.

Figure legends

Figure 1: Insecticidal activity of fluralaner and afoxolaner on vectors.

(A) Cartoon of the binding pockets of insecticides on the GABA or the glutamate-gated chloride channel (15, 16). The putative binding sites of ivermectin, isoxazolines and dieldrin are depicted on a subunit of the pentameric structure of the channel, in purple, orange and red respectively. (B) Chemical structures of fluralaner and afoxolaner. (C) Survival (expressed as percentage of engorged individuals) of Anopheles, Aedes aegypti and Culex pipiens mosquitoes and Phlebotomus argentipes and Lutzomyia longipalpis sand flies 24 hours after feeding on fluralaner (left) and afoxolaner (right) supplemented blood meals. The species or strain is indicated in the legend of each graph with the known resistance mutation(s) written in superscript. Error bars on the Anopheles and Aedes data indicate standard errors based on duplicate experiments with about 30 mosquitoes each. On the Culex data, they indicate standard errors based on 4 replicate experiments with about 15 mosquitoes each. On the Phlebotomus data, they indicate standard errors based on duplicate experiments with 100 flies each. Data points of the Lutzomyia data indicate results of a single experiment with 100 flies per concentration and per compound.

Figure 2: Modeled impact of mass drug administration of an isoxazoline drug.

Reduction in infection incidence (both symptomatic (clinical) and asymptomatic infections) in Zika (A) and clinical incidence and cumulative clinical incidence in malaria (B) after two years of fluralaner/afoxolaner mass drug administration (MDA) during the transmission season (indicated by the pink shaded areas) where either 30% or 80% of the population over the age of 5 received the drug each year. The model assumes a mosquitocidal drug dose resulting in blood levels >IC99 for 90 days.

Figure 3: Predicted impact of mass administration of an isoxazoline drug on malaria incidence in Africa.

The figure shows cumulative reduction in incidence during 2 years of fluralaner/afoxolaner mass drug administration covering 80% of the population over the age of 5, dosed once per year optimally timed in relation to the start of the transmission season. The model integrates available data on regional disease prevalence and seasonality profile as illustrated in Supplemental Data Figure 1.

Table 1: Insecticidal activity of fluralaner and afoxolaner against disease vectors

IC50 values (with 95% confidence intervals in brackets) determined from the curves shown in Figures 1C which represent survival of mosquito and sand fly strains in the presence of fluralaner or afoxolaner.

Table 2: Physicochemical properties of fluralaner and afoxolaner

(Left) In vitro degradation of fluralaner and afoxolaner, as well as ethoxycoumarin as a control, by human, dog and rat primary hepatocytes. The results are shown as the half-life (t1/2) and intrinsic in vitro clearance (CLint) of each compound. The results are based on 5-point time courses. (Right) Percentage of afoxolaner and fluralaner unbound to human plasma. The results are based on 3 replicate experiments. * ND: not detected, no peak was observed in the buffer sample, indicating that the molecule may be highly bound to plasma proteins.

Acknowledgements

Geert-Jan van Gemert and Laura Pelser-Posthumus (Radboud University Medical Center) are gratefully acknowledged for their help with Anopheles stephensi experiments. We also express our gratitude to David Malone from the Innovative Vector Control Consortium (IVCC), and to Helen S. Williams and the other members from the Liverpool Insect Testing Establishment (LITE) for help with mosquito experiments. Finally, the DuPont company is gratefully acknowledged for supporting the initiation of our research on isoxazolines. K.P., M.J. and P.V. were supported by the projects UNCE (No. 204072) and INFRAVEC 2 (H2020, No. 731060).

Author contributions

M. M., A. S., M. S. T., P. G. S. and K. J. D. conceived the study. B. Y. extracted and purified the isoxazoline compounds. M. M., M. E., P. A., R. S. L., K. K., K. P., M. J., P. V., C. J. M. K, A. K. C. and A. S. contributed to the data collection. M. M., M. E., R. S. L., A. K. C., G. T., J. W. and A. S. analyzed the data. H. S. and N. F. performed the modeling study. M. M., H. S., N. F., J. W., T. B., R. W. S. and K. J. D. wrote the manuscript and prepared the figures. All authors proofread and commented on the final draft of the manuscript.

Competing interests

K. J. D. and R.W.S. hold stock in TropIQ Health Sciences. Patent for the application described herein is pending.

Methods

Chemical extraction and purification

Fluralaner and afoxolaner were obtained by extraction and purification from Bravecto (Merck Animal Health) and Nexgard (Merial) pills, respectively. The pills were smashed into fine powder by mortar and pestle. A solvent of dichloromethane and methanol (1:1) was then added to the powder. The mixture was stirred at room temperature for 1h and filtered. The filtrate was concentrated under rotary evaporator, and the product was purified by silica gel chromatography. LCMS and NMR matched literature values (34, 35).

Mosquito colonies

The colony of Anopheles stephensi (Sind-Kasur Nijmegen strain) (36) was maintained at the Radboud University Medical Center, Nijmegen, The Netherlands, at 30oC, 70-80% humidity and on a 12/12 hour day/night cycle. The Anopheles gambiae strains Kisumu and Tiassalé 13 and the Aedes aegypti strains New Orleans and Cayman were reared at the Liverpool Insect Testing Establishment, Liverpool School of Tropical Medicine, Liverpool, UK. Kisumu and New Orleans is an insecticide susceptible lab strain (37). New Orleans was originally colonized by the Centers for Disease Control and Prevention (38). The Tiassalé 13 strain was colonized from Southern Côte D’Ivoire where resistance to all classes of insecticide is found (37) and the Cayman strain was colonized from Grand Cayman, where Aedes aegypti are highly resistant to DDT and pyrethroid insecticides (38). Both resistant strains are routinely selected with insecticides to ensure the maintenance of resistance, 0.75% permethrin for Cayman and 0.05% deltamethrin for Tiassalé, and profiled for resistance to a range of insecticides, including 4% dieldrin, to which Tiassalé is resistant but Cayman is susceptible (Supplemental Data Table 1). The colony of Culex pipiens biotype pipiens originated from egg rafts collected from above-ground habitats in 2015 in Best, The Netherlands, and was maintained at Wageningen University, The Netherlands, on 16/8 hour day/night cycle, at 23 oC and 60% humidity (39).

Systemic test of insecticidal activity on mosquitoes

Blood meals containing 50% human red blood cells and 50% human serum were supplemented with different doses of either isoxazoline (3.16 µM, 1µM, 316 nM, 100 nM, 31.6 nM, 10 nM, 3.16 nM or 1 nM) diluted first in DMSO and then in serum to reach a final DMSO concentration of 0.1%. A vehicle control (0.1% DMSO) was included in all experiments. The supplemented blood meals were then fed to 3 to 10-day old female mosquitoes in a membrane feeder system previously described for standard membrane feeding assays (40). Twenty-four hours after feeding, the numbers of dead and live mosquitoes among the fed population were recorded. Survival was calculated as the percentage of live mosquitoes 24 hours post feeding of the total number of fed mosquitoes per treatment.

Sand fly colonies and feeding

Phlebotomus argentipes (originating from India, 2008) and Lutzomyia longipalpis (originating from Brazil, 1991) have been reared at Laboratory of Vector Biology, Charles University, Prague, for many generations. Sand flies were maintained under standard conditions as was described previously (41). For evaluation of insecticidal activity of isoxazolines on sand flies, we tested different doses of fluralaner and afoxolaner (31.6 nM, 100 nM, 316 nM, 1µM, 3.16 µM, 10 µM) diluted in DMSO and mixed with sterile defibrinated rabbit blood. One hundred females (3–5 days old) of both species were fed through a chick-skin membrane on rabbit blood with different doses of insecticides. Fully blood fed females were separated and mortality was recorded 24 h post bloodmeal. The experiments with both isoxazolines were done twice in P. argentipes and once in L. longipalpis. A negative control (0.1% DMSO diluted in rabbit blood) was used in all experiments. The displayed results are normalized to control mortality (less than 3% in all experiments).

Data analysis for viability assays

IC50 values were calculated by applying a four-parameter logistic regression model using a least-squares method to find the best fit using the Graphpad Prism 5.0 software package.

WHO insecticide susceptibility test

Two to five day old female mosquitoes were routinely profiled for resistance to insecticides and colonies selected using the methodology recommended by the World Health Organization (42), and using test kits and insecticide impregnated papers supplied by Universiti Sains Malaysia (USM), Penang.

Hepatocyte metabolism assay

Isoxazolines and control compounds (7-Ethoxycoumarin and 7-Hydroxycoumarin [Sigma]) were dissolved in DMSO at 10 and 30 mM respectively, then diluted first 20-fold with 45% methanol in water and a further 10-fold in pre-warmed Williams’ Medium E. Cryopreserved human, dog and rat hepatocytes (In Vitro Technologies) were thawed, isolated by percoll gradient and suspended in Williams’ Medium E. They were then dispensed into the wells of 96-well plates containing 10 µL of diluted compounds, to reach a final concentration of 0.5x106 cells/mL and either 1 µM of isoxazolines or 3 µM of control. After an incubation at 37°C of 0, 15, 30, 60 or 90 minutes, the reaction was stopped with acetonitrile. The samples were then shaken for 10 minutes at 500 rpm and centrifuged at 3220 g for 20 minutes. Supernatants were transferred and stored at 4°C until LC-MS-MS analysis.

Determination of human doses

Total plasma clearance and volume of distribution measured in dogs have been scaled to humans using single species allometry with typical exponents of 0.75 for clearance and 1.0 for volume assuming a bodyweight of 11kg for dog and 70kg for human (43, 44). As the predicted clearance in man and the measured clearance in dog is much lower than hepatic blood flow, negligible first pass extraction by the liver is expected and oral bioavailability will be a function of absorption. Considering that the reported bioavailability for fluralaner was moderate-low in dogs and non-linear with dose, a bioavailability of 25% was assumed in man for this compound. By contrast, the bioavailability of afoxolaner in dogs being relatively high and dose-proportional, the same value of 74% was used for man. Predictions of exposure in man have been made using these estimated parameters (reported in Supplemental Data Table 4) and a single compartment PK model assuming first order rate of absorption at a rate of 1h-1. The doses reported herein are predicted to achieve a total plasma concentration above the whole blood IC99 for Aedes and Anopheles mosquitoes (129 nM=81 ng/mL for afoxolaner and 65 nM=36 ng/mL for fluralaner, based on maximal value obtained with the Aedes and Anopheles data displayed in Figure 1C) for 90 days following a single administration. For Culex mosquitoes and Phlebotomus sand flies, the same PK model and doses were used to estimate the time frame with plasma concentration above IC99 (for Culex mosquitoes, 200 nM=125 ng/mL for afoxolaner and 105 nM=58 ng/mL for fluralaner; for Phlebotomus, 407 nM=255 ng/mL for afoxolaner and 1,445 nM=804ng/mL for fluralaner). For Lutzomyia sand flies, as well as for Phlebotomus sand flies in the case of fluralaner, these doses were not sufficient to obtain a plasma concentration above IC99 at any time point.

Modeling of malaria incidence

An existing transmission model describing the impact of another mosquitocidal drug, ivermectin, on malaria (45) was extended to simulate the impact of afoxolaner or fluralaner with a 90-day efficacy period, and a 2-year intervention scheme (1 intervention per year at the beginning of the transmission period). The model assumes that mosquitoes taking a bloodmeal containing either drug on any day during the 90 day efficacy window will experience reduced survival – mean lifespan is reduced from 7.6 days pre-intervention (46) to 2 days during the intervention. This translates to less than 1% of the mosquitoes being able to survive until they complete sporogony. We assume that a proportion (c) of the human population over the age of 5 is treated, and only bloodmeals taken from treated individuals result in reduced survivorship. We simultaneously track the infectious state of the mosquito (susceptible, latently infected or infectious) to link the increase in vector mortality to a reduction in the infectious vector density and thus the rate of infections in humans.

The impact of afoxolaner/fluralaner was estimated in all malaria endemic areas in Africa. Each 1st administrative unit (top-level regional divisions) has a specific transmission intensity based on a prevalence-incidence relationship in the Imperial College malaria model and the Malaria Atlas Project estimates for 2015 prevalence, and seasonality profile based on rainfall data (21) (Supplemental Data Figure 1). Mass drug administration (MDA) with a mosquitocidal drug efficacious for 90 days and with a coverage of 80% of the population over the age of five was simulated in each 1st administrative unit. The MDA was conducted at an optimal time based on the specific seasonality profile of each administrative unit. The map presented in Figure 3 is a simplified and illustrative approach to estimating the true impact of this intervention across Africa – in reality a wider range of complexities would need to be considered, such as the vector species in each location (currently assumed to be all Anopheles gambiae-like), movement of individuals between locations, each individual country’s national malaria strategy (in terms of planned increases in current interventions such as distribution of long-lasting insecticide-treated bed nets and access to treatment) and true achievable coverage and compliance in each area.

Modeling of Zika incidence

An existing Zika transmission model (18) was adapted to include an increased rate of vector mortality during the 90 day efficacy period of the drug of 0.5cp/day where 0.5 translates into a mean lifespan of 2 days for mosquitoes biting a treated subject, c represents drug coverage in individuals over 5 years of age, p is the proportion of the population over 5 years of age (=0.908 for the demography assumed) and is the biting rate per adult female mosquito (=0.5/day). We simulate treatment occurring in one of twenty spatially coupled geographic regions (parameterized to represent Latin America) in a population with historical prior exposure to Zika but at a point in time where herd immunity has declined to the point where a new epidemic is able to occur. Treatment started within 2 months of the start of the new epidemic and is repeated exactly one year later. We track the annualized incidence of infection, and the cumulative infection incidence since the start of the epidemic.

Estimation of NOAEL for humans

Mouse, rat and dog no adverse effect levels (NOAEL) obtained from publicly available safety studies on fluralaner (22, 23) and afoxolaner (24-26) have been scaled to human values using allometry factors of 0.08, 0.16 and 0.54 respectively, assuming a bodyweight of 60 kg for human (27).

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