Repurposing Isoxazoline Veterinary Drugs For Control of Vector-Borne Human Diseases
Repurposing Isoxazoline Veterinary Drugs For Control of Vector-Borne Human Diseases
Repurposing Isoxazoline Veterinary Drugs For Control of Vector-Borne Human Diseases
Contributed by Peter G. Schultz, June 4, 2018 (sent for review January 31, 2018; reviewed by Michael H. Gelb and Timothy Wells)
Isoxazolines are oral insecticidal drugs currently licensed for ministration (12, 13). Even though these compounds have neu-
ectoparasite control in companion animals. Here we propose their ronal targets (Fig. 1A), they have been generally shown to be safe
use in humans for the reduction of vector-borne disease incidence. in mammals, as they show limited brain penetration (14) and
Fluralaner and afoxolaner rapidly killed Anopheles, Aedes, and significant selectivity for insect over mammalian receptors (12,
Culex mosquitoes and Phlebotomus sand flies after feeding on a 15). Herein we evaluate two representatives of this compound
drug-supplemented blood meal, with IC50 values ranging from class in multiple disease-carrying vector species and support the
33 to 575 nM, and were fully active against strains with preexist- case for their potential use as human oral vector-control drugs.
ing resistance to common insecticides. Based on allometric scaling
of preclinical pharmacokinetics data, we predict that a single hu- Results and Discussion
man median dose of 260 mg (IQR, 177–407 mg) for afoxolaner, or Insecticidal Activity of Fluralaner and Afoxolaner. The isoxazolines
410 mg (IQR, 278–648 mg) for fluralaner, could provide an insecti- afoxolaner and fluralaner (Fig. 1B) were tested on various strains
cidal effect lasting 50–90 days against mosquitoes and Phleboto- of Anopheles, Aedes aegypti, and Culex pipiens, which are im-
mus sand flies. Computational modeling showed that seasonal
portant vectors for malaria, Zika/dengue, and West Nile virus,
mass drug administration of such a single dose to a fraction of a
respectively. Mosquitoes were fed on drug-supplemented human
regional population would dramatically reduce clinical cases of
blood by membrane feeding. At 24 h after the blood meal,
Zika and malaria in endemic settings. Isoxazolines therefore rep-
resent a promising new component of drug-based vector control.
fluralaner showed IC50 values in the range of 33–92 nM against
all mosquito strains tested, whereas afoxolaner was slightly less
active, with IC50 values ranging from 90 to 177 nM (Fig. 1C and
vector control | insecticide | malaria | zika fever | isoxazoline Table 1). Isoxazolines occupy a binding site that is distinct from
the targets of known modulators of ionotropic GABA receptors
V ector-borne diseases, including malaria, Zika fever, and
leishmaniasis, remain major causes of mortality and mor-
bidity in (sub)tropical regions (1, 2). Temperate areas are also at Significance
risk for such diseases, for instance, due to the reintroduction of
West Nile Virus in Europe (3). Elimination of these diseases will Reduction in clinical cases of vector-borne diseases is strongly
require not only clinical development of new drugs and vaccines, dependent on the ability to reduce the number of infectious
but also effective control of vector populations (4, 5). Drug- insect bites. Here we describe a treatment concept based on
based vector control is a new strategy that involves administra- single-dose administration of an insecticidal isoxazoline drug
tion of an oral insecticidal drug to a human population at risk to to a human population, which leads to killing of blood-fed in-
kill the insect vector on blood feeding, thereby reducing the sect vectors and a predicted sharp decline in disease trans-
vector population and preventing disease transmission (6). This mission. Based on the long half-life observed in preclinical
approach has the advantage of being effective against mosquito species, a single human dose of <500 mg is predicted to pro-
populations feeding outdoors, which are increasingly important vide plasma exposure above the insecticidal threshold for
for malaria transmission (7) and escape the killing effects of longer than 2 months. Importantly, we show that isoxazolines
traditional vector control methods such as insecticide-treated are active against a range of vector species, which holds
bed nets and indoor residual spraying. In contrast to approaches promise for expanding the concept of drug-based vector con-
targeting livestock, administering an insecticidal drug to a human trol from malaria to leishmaniasis and arboviral diseases.
population would directly prevent onward transmission of hu-
Author contributions: M.M., H.S., N.F., P.V., A.K.C., J.W., A.S., R.W.S., P.G.S., M.S.T., and
man vector-borne pathogens and reduce the population size of K.J.D. designed research; M.M., M.E., N.F., P.A., R.S.L., K.M.J.K., K.P., M.J., P.V., C.J.M.K.,
fully anthropophilic mosquitoes and sand flies, which play major and A.S. performed research; B.Y. contributed new reagents/analytic tools; M.M., M.E.,
roles in the transmission of malaria in Africa and of visceral H.S., N.F., R.S.L., H.-P.D., G.T., A.K.C., J.W., A.S., and K.J.D. analyzed data; and M.M., H.S.,
leishmaniasis in India, respectively (8). An oral insecticidal drug N.F., A.K.C., J.W., T.B., R.W.S., M.S.T., and K.J.D. wrote the paper.
should preferentially have a long-lasting effect at a single dose to Reviewers: M.H.G., University of Washington; and T.W., Medicines for Malaria Venture.
minimize logistical challenges and cost of mass drug adminis- Conflict of interest statement: K.J.D. and R.W.S. hold stock in TropIQ Health Sciences.
tration (9), have a very broad safety window, and show activity Published under the PNAS license.
against a wide range of disease vector species. 1
To whom correspondence may be addressed. Email: [email protected], mtremblay@
Isoxazolines are a class of compounds recently licensed as calibr.org, or [email protected].
veterinary drugs for protection of companion animals against This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
fleas and ticks (10, 11), with very long in vivo half-lives that 1073/pnas.1801338115/-/DCSupplemental.
provide weeks to months of protection after a single oral ad- Published online July 2, 2018.
PHARMACOLOGY
Anopheles and Aedes data indicate SEs based on duplicate experiments with approximately 30 mosquitoes each; on the Culex data, they indicate SEs based on
four replicate experiments with approximately 15 mosquitoes each; on the Phlebotomus data, they indicate SEs 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.
(Fig. 1A) (16, 17). In line with this notion, fluralaner and afoxolaner Using published dog pharmacokinetic parameters (SI Appendix,
were fully active against the Anopheles gambiae Tiassalé 13 strain Table S2) (12, 18), we performed allometric scaling to predict
that carries the resistance-to-dieldrin (rdl) mutation in the GABA human plasma exposure following oral dosing. Obviously, plasma
receptor (Fig. 1C and SI Appendix, Table S1). In addition, they were concentrations after dosing of an eventual drug product will vary
equipotent against pyrethroid- and carbamate-resistant strains car- among human subjects due to variations in absorption rates,
rying mutations in the kdr sodium channel and acetylcholine es- genotypes and expression of CYP450 drug metabolizing en-
terase (ace-1) genes (Fig. 1C and SI Appendix, Table S1). zymes, etc. The exact distribution of human pharmacokinetic
The compounds were further tested by membrane feeding of parameters will not be known until human population pharma-
sand flies, which are important vectors of Leishmania. Afox- cokinetics data become available. However, to estimate vari-
olaner and fluralaner showed IC50 values of 305 and 575 nM, ability, a stochastic simulation approach was adopted using a
respectively, against Phlebotomus argentipes (Fig. 1C and Table fixed coefficient of variation (CV) of 20% with a log-normal
1), a vector of Leishmania donovani on the Indian subcontinent distribution for each parameter used in the single compart-
(2). Both compounds were less active against the South Ameri- ment model, i.e., clearance (Cl), volume of distribution (V),
can vector, Lutzomyia longipalpis, with IC50 values of 1–3 μM absorption rate (Ka) and bioavailability (F). This variation is in
(Fig. 1C and Table 1). The difference in isoxazoline activity seen line with data reported for the dog studies, where CV values
between sand flies and mosquitoes suggests that the targeted ranged from 14 to 24% (12, 18). Using this approach, we pre-
pocket in the GABA receptor is not conserved among insects. dicted the human dose resulting in circulating drug concentra-
tions above the mosquitocidal IC99 of Anopheles and Aedes for
Human Dose Prediction. Neither fluralaner nor afoxolaner was 90 d (SI Appendix, Fig. S1). The results indicate an estimated
measurably metabolized when incubated with dog or human single total human median dose level of 260 mg (IQR, 177–
hepatocytes, suggesting that the low in vivo intrinsic clearance 407 mg) of afoxolaner or 410 mg (IQR, 278–648 mg) of flur-
observed in dogs (12, 13) could be similar in humans (Table 2). alaner. For Culex, this dose would lead to circulating drug levels
Both compounds were highly bound to plasma proteins (Table above IC99 for 74 d. As sand flies appeared less sensitive to the
2), which could further contribute to a long in vivo half-life. compounds (Table 1), a 410-mg dose of fluralaner would not
The table shows in vitro degradation of fluralaner and afoxolaner and, as a control, ethoxycoumarin, 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 data are based on 5-point time courses. The last column
shows the 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.
Fig. 2. Modeled impact of mass drug administration of an isoxazoline drug. Reduction in the incidence of both symptomatic (clinical) and asymptomatic
infections in Zika (Top) and clinical incidence and cumulative clinical incidence in malaria (Bottom) after 2 y of fluralaner/afoxolaner mass drug administration
(MDA) during the transmission season (indicated by the pink shaded areas), with either 30% or 80% of the population age >5 y receiving the drug each year.
The model assumes a mosquitocidal drug dose resulting in blood levels >IC99 for 90 d. The two initial years of treatment are followed by three transmission
seasons without further intervention.
impact (>70% reduction in clinical cases) in areas with low and terms of absolute impact, a 30% reduction in cases in countries
very seasonal transmission, such as Senegal, Sudan, Madagascar, like the Democratic Republic of the Congo, where the World
Namibia, Botswana, and Zimbabwe (Fig. 3). In these countries, a Health Organization estimated 16–26 million cases in 2015,
large proportion of annual transmission occurs in a short period. may be more significant than a 70% reduction in, for example,
Therefore, administration of a single dose of an isoxazoline drug Senegal, where the estimated number of cases was 1.1–2.8
at the beginning of this season would dramatically decrease the million (28).
number of cases over the full year. In the rest of the continent, Because up-to-date information on population size at the
isoxazoline administration is predicted to have less impact but administrative unit scale is lacking (29), data on the absolute
still to result in a minimum 30% reduction in clinical cases. In reduction in cases cannot be provided here. Nevertheless, from
this simplified but illustrative approach (see Materials and were negative in mutagenicity/genotoxicity studies, and no effects
Methods for limitations), the key message is that this intervention on embryo-fetal development in rats were observed below ma-
is predicted to have a significant impact on malaria transmission, ternal toxic levels.
with the greatest efficacy in areas with low transmission and a To assess a maximum human tolerated dose, we used the
short transmission season. guidelines provided by the US Food and Drug Administration to
translate the reported rat and dog no-adverse effect levels
Preliminary Assessment of Human Safety. Nonclinical safety studies (NOAELs) to cognate human dose levels. These allometry
of fluralaner (30, 31) and afoxolaner (32–34) have been con- guidelines are based on data showing that toxic endpoints scale
ducted for veterinary indications and can be leveraged to offer a well between species when doses are normalized to body surface
preliminary assessment of the human safety of a single dose of area (35). The results show that anticipated median single doses
these isoxazoline molecules. The reported oral toxicity profile of of fluralaner (410 mg) and afoxolaner (260 mg) in humans are
afoxolaner consists of a diuretic effect (rats only), effects sec- comparable to or lower than equivalent doses considered to be
ondary to a reduction in food consumption (rats and rabbits NOAELs based on acute and repeat-dose toxicity studies in rats
only), and occasional vomiting and/or diarrhea in dogs following and dogs (SI Appendix, Table S3). Interestingly, veterinary for-
high oral doses. No treatment-related effects on vomiting or mulations of afoxolaner and fluralaner are based on racemic
diarrhea were noted in dogs following oral afoxolaner doses of mixtures, whereas the S-enantiomer has been reported to be the
up to 31.5 mg/kg once monthly for 3 mo (33). Fluralaner showed active component against ectoparasites (15, 36). Thus, future
similar mild gastrointestinal events (diarrhea, vomiting, in- development of a human application of these molecules will
appetence, and drooling) in dogs, no adverse events in a single- benefit from further characterization of the activity and toxi-
dose toxicity study in rats, and some histopathological abnor- cology profile of the active enantiomer, which may lead to a 50%
malities in lung, thymus, and liver in a repeat dose study in rats in reduction in the required dose. The veterinary application of the
the highest dose groups (400–600 mg/kg) with liver as the main racemic form of these drugs provides a first assessment of their
target, showing hepatocellular fatty changes (30). Importantly, safety. Given the indirect clinical benefit of an isoxazoline-based
there was no significant neurotoxicity findings in rats or beagle intervention, an extremely favorable human safety margin would
dogs. This is consistent with both in vitro and in vivo data be essential, and detailed additional nonclinical studies (toxi-
showing that these compounds have no significant interaction cology, pharmacokinetics, and metabolism) will be required be-
with the mammalian GABA receptor (12, 15). Overall, isoxazo- fore a first-in-human isoxazoline drug application can be filed
lines were well tolerated and showed mild and clinically moni- with a regulatory authority. Alternatively, administration to
torable adverse events. Moreover, both fluralaner and afoxolaner livestock could be considered as a means of controlling the
PHARMACOLOGY
a broad range of relevant disease vectors. Cryopreserved human, dog, and rat hepatocytes (In Vitro Technologies) were
thawed, isolated by Percoll gradient, and suspended in Williams’ Medium E.
Materials and Methods They were then dispensed into the wells of 96-well plates containing 10 μL of
Chemical Extraction and Purification. Fluralaner and afoxolaner were diluted compounds, to reach a final concentration of 0.5 × 106 cells/mL and
obtained by extraction and purification from Bravecto (Merck Animal Health) either 1 μM isoxazolines or 3 μM control. After an incubation at 37 °C of 0, 15,
and Nexgard (Merial) pills, respectively. The pills were smashed into fine 30, 60 or 90 min, the reaction was stopped with acetonitrile. The samples were
powder by mortar and pestle. A solvent of dichloromethane and methanol then shaken for 10 min at 500 rpm and then centrifuged at 3,220 × g for 20 min.
(1:1) was then added to the powder. The mixture was stirred at room Supernatants were transferred and stored at 4 °C until LC-MS-MS analysis.
temperature for 1 h and then filtered. The filtrate was concentrated under a
rotary evaporator, and the product was purified by silica gel chromatogra- Human Dose Prediction. Dog pharmacokinetic parameters reported previously
(12, 18) were used to estimate corresponding human parameters and derive
phy. LCMS and NMR matched values reported in the literature (43, 44).
drug doses necessary to reach human plasma concentrations with insecticidal
activity. Details of the allometry methods are provided in SI Appendix.
Mosquito Colonies. The colony of Anopheles stephensi (Sind-Kasur Nijmegen
strain) (45) was maintained at the Radboud University Medical Center at
Modeling of Mosquito-Borne Disease Incidence. The models of malaria and
30 °C and 70–80% humidity and on a 12/12-h day/night cycle. The A. gam-
Zika fever incidence were adapted from published transmission models (19,
biae strains Kisumu and Tiassalé 13 and the A. aegypti strains New Orleans
24) to include yearly administration of an isoxazoline drug efficacious
and Cayman were reared at the Liverpool Insect Testing Establishment. against Anopheles and Aedes mosquitoes for 90 d. Details of the model
Kisumu and New Orleans are insecticide-susceptible laboratory strains (46). parameters are provided in SI Appendix.
The New Orleans strain was originally colonized by the Centers for Disease
Control and Prevention (47). The Tiassalé 13 strain was colonized from Estimation of Human No Adverse Effect Levels. Mouse, rat, and dog NOAELs
southern Côte D’Ivoire, where resistance to all classes of insecticide is found obtained from publicly available safety studies on fluralaner (30, 31) and afoxolaner
(46), and the Cayman strain was colonized from Grand Cayman, where A. (32–34) have been scaled to human values using allometry factors of 0.08, 0.16,
aegypti are highly resistant to DDT and pyrethroid insecticides (47). Both and 0.54 respectively, assuming a body weight of 60 kg for humans (35).
resistant strains are routinely selected with insecticides to ensure the
maintenance of resistance (0.75% permethrin for Cayman and 0.05% del- ACKNOWLEDGMENTS. We thank Geert-Jan van Gemert and Laura Pelser-
tamethrin for Tiassalé), and profiled for resistance to a range of insecticides, Posthumus (Radboud University Medical Center) for their help with the A.
including 4% dieldrin, to which Tiassalé is resistant but Cayman is susceptible stephensi experiments. We also thank David Malone from the Innovative
Vector Control Consortium and Helen S. Williams and the other members
(SI Appendix, Table S1). The colony of C. pipiens biotype pipiens originated
of the Liverpool Insect Testing Establishment for help with mosquito exper-
from egg rafts collected from aboveground habitats in 2015 in Best, The iments. Finally, we thank the DuPont Company for supporting the initiation
Netherlands, and was maintained at Wageningen University, The Nether- of our research on isoxazolines. K.P., M.J., and P.V. were supported by the
lands, on a 16/8-h day/night cycle at 23 °C and 60% humidity (48). UNCE (204072) and Infravec2 (H2020 and 731060) Projects.