Current review of isoxazoline ectoparasiticides used in veterinary medicine

The isoxazolines are a novel class of ectoparasiticides with potent inhibitory activity on glutamate- and gamma- aminobutyric acid- gated chloride channel located in nervous system of invertebrates. In recent years, studies have been performed to evaluate the efficacy and safety of isoxazolines against various types of ectoparasites, including fleas, ticks, and mites. As more single and combined isoxazoline products have been approved by the United States Food and Drug Administration and European Medicines Agency, a more comprehensive understanding of isoxazolines becomes es-sential for veterinary clinical practitioners. This article provides a complete review of isoxazolines with respect to pharmacodynamics, pharmacokinetics,

and peripheral nervous system of invertebrates (Lunt, 1991). GABA receptors are members of the Cys-loop superfamily linked to chloride channels (Sine & Engel, 2006). They have five transmembrane subunits, each consisting of four transmembrane helices. Current research indicates the helical subunits are the target of isoxazolines, though the precise inhibition site has not yet been identified (Weber et al., 2016). Variation in arrangement and substitution of these subunits is ultimately responsible for the different pharmacological action of the drug targeting the receptor and the degree of safety in the use of a particular isoxazoline in vertebrates. A study utilizing fly and rat models has demonstrated a significant preference of isoxazolines for invertebrate GABA receptors over vertebrates (Gassel et al., 2014). The glutamate-gated chloride channel, a ligand-gated ion channel unique to invertebrates and the target of macrocyclic lactones, is also targeted by isoxazolines to a lesser degree (Gassel et al., 2014). GABA and glutamate exert their actions by stimulating chloride influx on the postsynaptic tissue, causing hyperpolarization, preventing the generation of an action potential. Isoxazolines inhibit this modulatory action by binding to the postsynaptic tissue, preventing chloride influx leading to depolarization/hyperexcitation, paralysis, and death of the parasite (Gassel et al., 2014;Ozoe et al., 2010).  (Canada, 2014a(Canada, , 2014b(Canada, , 2016(Canada, , 2017(Canada, , 2018(Canada, , 2020bFDA, 2014FDA, , 2018aFDA, , 2018b; ornate cow tick (D. reticulatus), castor bean tick (I. ricinus), Hedgehog tick (I. hexagonus), and brown dog tick in Member States of European Union (EU) (EMA, 2013(EMA, , 2014(EMA, , 2015a(EMA, , 2017; Australian paralysis ticks (I. holocyclus), brown dog tick, and Asian longhorned tick (Haemaphysalis longicornia) in Australia (APVMA, 2014(APVMA, , 2015(APVMA, , 2016(APVMA, , 2018a(APVMA, , 2018b(APVMA, , 2018c; and Asian longhorned tick in New Zealand (ACVM, 2014a(ACVM, , 2014b(ACVM, , 2015a(ACVM, , 2016. Other than common tick species, sarolaner has shown additional efficacy against Gulf Coast ticks (Amblyomma maculatum) in dogs (FDA, 2016b). In addition, most isoxazolines with single active substance are approved by EMA for treatment of demodicosis, scabies, and ear mite infestations (Table 1).

| APPROVED USAG E OF ISOX A ZOLINE S
Fluralaner differs from the other three isoxazolines with prolonged duration up to 12 weeks, which is three times longer than afoxolaner, sarolaner, and lotilaner (FDA, 2014). Fluralaner is also available as a topical solution for both dogs and cats; in cats, it is for the treatment and prevention of fleas and control of black-legged ticks (FDA, 2016a). Lotilaner has been approved by EMA as chewable tablets for treatment of fleas and ticks (castor bean tick) in cats (EMA, 2017). As for other detailed information of approved usage, for example, minimum dose and age of animal, please refer to Tables 1 and 2.
It is interesting to note that, although, lotilaner has the longest plasma half-life among isoxazolines, which leads to a moderate degree of accumulation (Kuntz & Kammanadiminti, 2017). Its recommended oral dosing interval is the same as afoxolaner and sarolaner of 4 weeks ( Table 1). Studies of a prolonged dosing interval (for instance, 8-12 weeks) of lotilaner are suggested to explore sustained efficacy longer than one month. Feeding of the target animal plays a critical role in the pharmacokinetics of lotilaner, especially bioavailability, which is decreased from 82% to 24% when administered to fasted dogs, and 100% to 8.4% in fasted cats (Toutain et al., 2017(Toutain et al., , 2018. Thus, it is strongly recommended to provide wet or dry food with lotilaner for an effective treatment and prevention of fleas and control of ticks. On the contrary, the plasma concentration of afoxolaner does not appear to be affected by food with a high bioavailability of 74% (Letendre et al., 2014). Isoxazolines have a high degree of plasma protein binding (≤99.9%), indicating that the clearance is likely largely hepatic rather than renal (Kilp et al., 2014;Letendre et al., 2014;McTier et al., 2016;Toutain et al., 2017Toutain et al., , 2018. The average volume of distribution (Vss) has been determined to be ~3 L/ kg for afoxolaner (Letendre et al., 2014), fluralaner (Kilp et al., 2014(Kilp et al., , 2016, and sarolaner (McTier et al., 2016), whereas lotilaner has a higher Vss value of ~6 L/kg, which implies a higher distribution into adipose tissue than other isoxazolines (Toutain et al., 2017(Toutain et al., , 2018.
Besides moderate to high distribution, low clearance rate (0.3% of canine daily hepatic blood flow) of isoxazolines is another factor that contributes to the prolonged plasma half-life of isoxazolines (Kilp et al., 2014(Kilp et al., , 2016Letendre et al., 2014;McTier et al., 2016;Toutain et al., 2017Toutain et al., , 2018 Not approved yet a Effective for 8 weeks.

| Ticks
Tick infestations have been implicated in the transmission of rickettsial diseases, babesiosis, theileriosis, anaplasmosis, Lyme disease, and ehrlichiosis, along with a number of bacterial, viral, and other pathogens (Breitschwerdt et al., 2010;Dantas-Torres et al., 2012;Durden et al., 2005;Ozoe et al., 2010;Pantchev et al., 2015). Isoxazolines have been approved by the USA and Canada against multiple tick species as indicated above and have been demonstrated to be effective against additional species experimentally (

| Mites
In our opinion, the most important future application of isoxazoline ectoparasiticides is in the treatment of mite infestations, including demodicosis and scabies. While not currently approved by the USA and Canada for use against mites, isoxazolines demonstrate high efficacy in the treatment of mites while offering greater ease of application, safety margin for the animal treated, and efficacy (Zhou et al., 2020), and are registered for treatment of mite infestations in EU, Australia, and New Zealand (Table 1).

| Demodex
Demodicosis is a common form of mange disease in canine patients, occasionally affecting cats. Conventional treatments including amitraz and macrocyclic lactones have been shown variable likelihood of clinical resolution, some may require prolonged treatment course with high probability of adverse effects (Arsenovic et al., 2015;Saari et al., 2009).
All four isoxazolines have shown efficacy in the treatment of generalized demodicosis Fourie et al., 2015;Snyder et al., 2017), with marked reduction of mites as early as day 14 with the sarolaner treatment . Afoxolaner, fluralaner, and sarolaner have been evaluated against weekly topical application of 2.5 mg/kg moxidectin plus 10 mg/kg imidacloprid (Advocate ® ), which demonstrated 99% reduction of Demodex in 4 weeks, compared with 90-98% efficacy with topical Advocate ® treatment Fourie et al., 2015;. Lotilaner treatment also killed mites 99.9% with monthly applications . While none of isoxazolines is currently labeled for use against demodicosis in the USA or Canada, they represent an excellent improvement in the field of veterinary dermatology (Zhou et al., 2020).  . Compared with sarolaner, the data of the fluralaner study suggest a more rapid resolution of canine sarcoptic mange by achieving 100% efficacy in a month, while sarolaner required 2 months to reach clinical cure with a monthly dosing interval . In addition, a study of six racoon dogs revealed 100% negative skin scraping in 7 days following a single oral dose of fluralaner; parasitological and clinical cure maintained up to 21 days (Hyun et al., 2019). Afoxolaner, given orally on days 0, 14, 28, and 56, appeared to exhibit a slower speed of killing against Sarcoptes scabiei in dogs, as it necessitated one more month than sarolaner to obtain 99% efficacy .

| Sarcoptes/Notoedres
To our best knowledge, no research evaluating isoxazolines against the parasite of feline scabies (Notoedres cati) has been published.

| Otodectes
Otodectes cynotis, an obligate parasite of the external ear canal, is a common cause of otitis externa, particularly in kittens, with zoonotic potential ( Van de Heyning & Thienpont, 1977). Current treatment methods involve topicals, both on-and off-label, and off-label oral moxidectin (0.2 mg/kg oral, twice a day for 10 days) .
Oral sarolaner at the recommended dose of 2 mg/kg yields a 98.2% mite reduction by day 28, followed by a 99.5% reduction by day 60 following a second monthly dose . Afoxolaner has similar efficacy with >98% mite reduction by day 28 following a single 2.5 mg/kg oral dose (Carithers et al., 2016). Fluralaner is 100% effective against O. cynotis in both dogs and cats by day 28 after treatment in dogs; both oral and topical applications are effective (Taenzler et al., 2017).

| Miscellaneous ectoparasites
In addition to common mites affecting cats and dogs, fluralaner was investigated against Psoroptes cuniculi in naturally infested rabbits.
P. cuniculi is a common ear mite in rabbits, causing otitis externa.

| SA FE T Y
As mentioned earlier, isoxazolines block glutamate-gated chloride channels, which exist in invertebrates, but not vertebrates (Gassel et al., 2014). In addition, isoxazolines selectively block invertebrate GABA-gated chloride channels. These modes of action of isoxazolines account for their high margin of safety for use in veterinary species (Gassel et al., 2014). Safety studies have been performed for all four isoxazoline compounds at a minimum of five times the recommended doses with no apparent adverse effects, except neurological effects observed Kuntz & Kammanadiminti, 2017, 2018McTier et al., 2016;Walther, Allan, et al., 2014b   have shown that isoxazolines are safe in cats Cavalleri et al., 2018b;Kuntz & Kammanadiminti, 2018) and MDR1-/-collies (Walther et al., 2014).
Safety in puppies or kittens on afoxolaner, fluralaner, and lotilaner was confirmed with oral or topical dose at 1, 3, or 5X the maximum FDA-approved dose, while mild and self-limiting ataxia and tremors with seizures were observed in 8-week-old puppies treated at elevated doses (3X and 5X the maximum FDA-approved dose) (Table 7; FDA , 2014, 2016a, 2016b, 2018a, 2018b

ANIMAL WELFARE S TATEMENTS
No animals were used in this investigation.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interests.