Optimization of isoxazoline amide benzoxaboroles for identification of a development candidate as an oral long acting animal ectoparasiticide.

Novel isoxazoline amide benzoxaboroles were designed and synthesized to optimize the ectoparasiticide activity of this chemistry series against ticks and fleas. The study identified an orally bioavailable molecule, (S)-N-((1-hydroxy-3,3-dimethyl-1,3-dihydrobenzo[c][1,2]oxaborol-6-yl)methyl)-2-methyl-4-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)benzamide (23), with a favorable pharmacodynamics profile in dogs (Cmax=7.42ng/mL; Tmax=26.0h; terminal half-life t1/2=127h). Compound 23, a development candidate, demonstrated 100% therapeutic effectiveness within 24h of treatment, with residual efficacy of 97% against American dog ticks (Dermacentor variabilis) on day 30 and 98% against cat fleas (Ctenocephalides felis) on day 32 after a single oral dose at 25mg/kg in dogs.

Discovery of an orally bioavailable isoxazoline benzoxaborole (AN8030) as a long acting animal ectoparasiticide.

A novel series of isoxazoline benzoxaborole small molecules was designed and synthesized for a structure-activity relationship (SAR) investigation to assess the ectoparasiticide activity against ticks and fleas. The study identified an orally bioavailable molecule, (S)-3,3-dimethyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)benzo[c][1,2]oxaborol-1(3H)-ol (38, AN8030), which was long lasting in dogs (t1/2=22 days). Compound 38 demonstrated 97.6% therapeutic effectiveness within 24 h of treatment, with residual efficacy of 95.3% against American dog ticks (Dermacentor variabilis) on day 30% and 100% against cat fleas (Ctenocephalides felis) on day 32 after a single oral dose at 50 mg/kg in dogs.

Efficacy of spinosad against acaricide-resistant and -susceptible Rhipicephalus (Boophilus) microplus and acaricide-susceptible Amblyomma americanum and Dermacentor variabilis.

Various acaricide-resistant strains of Rhipicephalus (Boophilus) microplus, representative of the major resistance mechanisms found in Mexico and Brazil, were exposed to spinosad using the Food and Agriculture Organization of the United Nations larval packet test and adult immersion test (AIT). Larvae of all strains tested were found to be susceptible to spinosad. Conversely, spinosad did not show toxic activity toward engorged females used in the AIT. In vitro tests against larvae, nymphs, and adults of acaricide-susceptible Amblyomma americanum and Dermacentor variabilis revealed differences in activity that were dependent on tick species and parasitic life stage. Spinosad seemed to be a viable alternative to current acaricides available for tick control in the species tested. The larval packet test should be used for future monitoring of resistance, as the AIT did not provide useful information with this chemical. The potential benefit for the use of spinosad in integrated pest management or eradication programs is discussed.

Evaluation of an 11.2% spinetoram topical spot-on solution for the control of experimental and natural flea (Ctenocephalides felis) infestations on cats in Europe.

Spinetoram is the newest member the spinosyn-class of natural products to be commercialized for flea control on cats in the United States under the tradename Cheristin® for cats. This report describes results from two laboratory studies and a multi-center clinical field trial designed to confirm the efficacy of a topical spot-on solution containing spinetoram (11.2% w/w, 130 mg/mL) against European strains of the cat flea, Ctenocephalides felis. In the laboratory studies, cats were allocated to one of two treatment groups of eight animals each: negative control (mineral oil) or spinetoram, both applied as a topical spot-on at the base of the skull on Day 0. Cats were infested with ∼100 newly emerged, unfed adult fleas on Days -2 or -1, 7, 14, 21, 28 and 35. To calculate efficacy, fleas were counted and removed 48 h after treatment, and 48 h after each subsequent infestation through week 5. Spinetoram treatments provided 100% efficacy through at least day 16 and ≥ 97% efficacy (arithmetic mean) for one month. For the field trial, 23 clinics from Northern and Southern Europe participated in the study that compared the effectiveness and safety of spinetoram and fipronil/(S)-methoprene treatments over a period of two months. There were 258 and 248 evaluable efficacy cases for month 1 and month 2, respectively, with 300 total evaluable cases for safety. Treatments were administered on Day 0 and again on Day 30 (±3 days). The effectiveness of treatments was calculated based on reduction in live flea counts on Days 14, 30, 44 and 60 (±3 days) relative to flea counts obtained on Day 0. Efficacy (geometric mean percent flea reduction) on Days 14, 30, 44 and 60 was 97.0%, 95.0%, 99.3% and 99.1% for spinetoram, respectively, and 86.1%, 80.9%, 92.4% and 92.3% for fipronil/(S)-methoprene, respectively. Spinetoram was deemed non-inferior at all intervals and superior to fipronil/(S)-methoprene at Days 30 and 60. Clinical signs of flea allergy dermatitis (FAD) were markedly improved following spinetoram treatment, as demonstrated through statistically significant reductions in severity of FAD scores for most of the clinical signs when compared to fipronil/(S)-methoprene treatment. There was a lower overall adverse event incidence rate for spinetoram (5.1%) versus fipronil/(S)-methoprene treatment (11.5%).

A comparison of three bioassay techniques to determine amitraz susceptibility in Boophilus microplus (Acari: Ixodidae).

The ability of the Miller, Soberanes, and White bioassay techniques to describe amitraz susceptibility in three different strains of Boophilus microplus (Canestrini) (Acari: Ixodidae) was compared. For a susceptible strain, all techniques adequately described amitraz susceptibility by producing a full range of mortality that corresponded with increasing concentration of amitraz. However, when resistant strains were evaluated, only the Miller and the Soberanes techniques adequately estimated the dose-response relationship. Lethal concentrations were not precisely estimated when all the data were included in the analyses for every strain and technique tested. Better estimates were obtained when subsets of data around the range of interest were subjected to probit analysis. For the Soberanes technique, the slope of the probit regression was steeper for the Brazilian resistant and Texan susceptible strains compared with the heterozygous Mexican strain. The pattern was different when the same strains were tested with the Miller technique. The slopes of the regressions for the Mexican and the Texan strains did not differ significantly, but the Brazilian strain had a steeper slope than the other strains. Resistance ratios were much greater when the Soberanes technique was used than when the Miller technique was used on the same strains. However, neither technique produced enough separation between susceptible and resistant strains to develop a traditional discriminating dose (DD) test that required a concentration of 2x LC99.9 estimate. A DD test at the LC99 would be possible for both techniques. We discuss the strengths and weaknesses of the three techniques, including potential improvements to the White technique. The White technique has the greatest potential to determine the mechanisms of amitraz resistance in detailed synergist studies. Currently, only the Miller method can fulfill this task. The Miller and Soberanes techniques are well suited for the study of the epidemiology of resistance worldwide, because they use commercially available, formulated amitraz that is easy and inexpensive to obtain.

Therapeutic and persistent efficacy of spinosad applied as a pour-on or a topical spray against natural infestations of chewing and sucking lice on cattle.

Studies were conducted in Wisconsin and Illinois, USA, to assess and compare the therapeutic and persistent efficacy of spinosad when applied as either a pour-on or topical spray and compared with cyfluthrin pour-on and coumaphos topical spray for controlling natural infestations of chewing (Bovicola bovis) and sucking (Linognathus vituli, Solenopotes capillatus and Haematopinus eurysternus) lice on cattle. Thirty-five animals at each trial site were blocked according to pre-treatment lice counts and randomly allocated to one of five treatment groups: single treatments of spinosad (25 g/L), diluted with water to 0.04% active ingredient and applied as a whole-body topical spray; spinosad (25 g/L) applied as a neat pour-on at 2 mg/kg body weight; Co-Ral Emulsifiable Livestock Insecticide (5.8% coumaphos), diluted to 0.03% active ingredient and applied as a whole-body topical spray; CyLence Pour-on Insecticide (1% cyfluthrin), applied as a neat pour-on at the manufacturer's recommended use rate for lice; and untreated control. Both spinosad treatments and cyfluthrin provided > or =96% control of B. bovis for up to 7 weeks, whereas the efficacy of coumaphos dropped to <90% after week 5 at one site. Spinosad spray had the best therapeutic and residual control of all treatments against L. vituli, > or =98% for at least 5 weeks at both sites, compared with 3 weeks for coumaphos at one site. Spinosad and coumaphos sprays provided 100% control of S. capillatus for at least 8 weeks compared with > or =97% control for spinosad and cyfluthrin pour-on treatments over the same interval. While H. eurysternus burden was low and limited to one study site, all four treatments provided 100% control for at least 6 weeks. These studies showed that topically applied spinosad provided a high degree of therapeutic and residual control against both sucking and chewing lice.

In vitro and in vivo characterization of p-amino-phenethyl-m-trifluoromethylphenyl piperazine (PAPP), a novel serotonergic agonist with anthelmintic activity against Haemonchus contortus, Teladorsagia circumcincta and Trichostrongylus colubriformis.

The neurotransmitter serotonin (5-hydroxy tryptamine or 5HT) regulates key physiological processes in nematodes such as locomotion and feeding. PAPP (p-amino-phenethyl-m-trifluoromethylphenyl piperazine) is a known agonist of the 5-HT(1Hc) receptor of the barber pole worm, Haemonchus contortus. In this study, PAPP was highly active against L3-stage larvae of H. contortus and Trichostrongylus colubriformis in an in vitro larval migration assay, with EC50 values of 9.36 and 11.8 microM, respectively, that were comparable to levamisole (10.2 microM) and superior to pyrantel (55.39 microM). When administered orally or subcutaneously to nematode infected gerbils, PAPP provided >99% efficacy against H. contortus and >98% efficacy against Teladorsagia circumcincta at 100 mg/kg, comparable to levamisole at 10 mg/kg. Drug titration revealed significant activity down to 50 mg/kg against these two species. Spectrum was limited, however, with somewhat lower efficacy (83%) in T. colubriformis infected gerbils at 100 mg/kg. Oral delivery of hydrochloride, acetate and phosphate salts of PAPP to nematode infected gerbils did not result in an increase in either potency or spectrum. The finding that PAPP exhibits significant anthelmintic activity suggests that the nematode-specific serotonergic system is a viable target for future anthelmintic discovery.

Knockdown and mortality comparisons among spinosad-, imidacloprid-, and methomyl-containing baits against susceptible Musca domestica (Diptera: Muscidae) under laboratory conditions.

The activity of spinosad, imidacloprid, and methomyl baits and technical actives were assessed against susceptible house flies, Musca domestica L. (Diptera: Muscidae). In a feeding assay, imidacloprid affected flies more rapidly than methomyl or spinosad, but spinosad was 2.7 times more potent than methomyl and 8 times more potent than imidacloprid. The profile of technical actives correlated with their respective fly bait formulations in laboratory assays. Although having the most rapid onset of activity in laboratory tests, up to 50% of flies remained alive after exposure to imidacloprid bait. In contrast, <5% of flies survived 24-h exposure to spinosad or methomyl baits. High temperature reduced the knockdown activity of imidacloprid bait and slowed the speed of kill for spinosad and methomyl baits over a 24-h exposure period. Spinosad and methomyl baits were also superior to imidacloprid when applied to the floors of environmentally controlled rooms at label recommended rates, providing good fly control for up to 21 d. The fact that a significant percentage of flies exposed to imidacloprid were rapidly knocked down but subsequently remained alive in all of the assays suggested that flies were recovering from initial exposure to this compound. Given its favorable safety profile, a high degree of initial and residual activity comparable with methomyl and lack of cross-resistance to other chemistries, spinosad bait may be a valuable component of house fly control programs to help control or delay the emergence of resistant populations.

Initial evaluations of the effectiveness of spinetoram as a long-acting, oral systemic pulicide for controlling cat flea (Ctenocephalides felis) infestations on dogs.

Spinetoram is a semi-synthetic, spinosyn class natural product derived from fermentation by the actinomycete, Saccharopolyspora spinosa. Based on LD50 (50% lethal dose) values against adult cat fleas (Ctenocephalides felis) using an in vitro contact assay, spinetoram was approximately 4-fold more potent than spinosad. Subsequently, two parallel-arm, randomized block design laboratory studies were conducted to evaluate the effectiveness of orally administered spinetoram against experimental C. felis infestations on dogs, when administered as a single dose or multiple doses over a 6-12h interval. In the first study, 16 mixed-breed dogs were allocated to two treatment groups of eight dogs each, based on pre-treatment flea retention rates: negative (placebo) control; and a single dose of spinetoram at 30mg/kg. In the second study, 32 mixed- and pure-breed dogs were allocated to four treatments groups of eight dogs each, based on pre-treatment flea retention rates: negative (placebo) control; a single dose of 60mg/kg; three sequential 20mg/kg oral doses evenly administered over a 6h period; and three sequential 20mg/kg oral doses evenly administered over a 12h period. In both studies, treatments were administered to dogs in a fed state in order to enhance absorption of spinetoram. Therapeutic efficacy was assessed 24h after treatment and persistent efficacy was assessed 48h after each subsequent flea infestation. The duration of effectiveness was assessed at approximate weekly intervals beginning on Day 5 through Day 56 in the first study, or through Day 105 in the second study. In both studies, treatment efficacy was ≥99% (geometric means) through 44 d, with ≥99% efficacy continuing through 72 d for all three treatments in the second study. Efficacy remained ≥90% for at least 8 weeks with a single 30mg/kg dose; through 13 weeks with three sequential 20mg/kg doses; and through 15 weeks with a single 60mg/kg dose. For all time points and in both studies, spinetoram-treated groups had significantly fewer live fleas relative to their respective negative control group (p<0.05). The pharmacokinetic profile in dogs revealed that the mean plasma concentration of spinetoram required for effectiveness against fleas was maintained for at least 3 months regardless of whether the 60mg/kg total body dose was administered as a single bolus or in three sequential 20mg/kg doses administered over a 6-12h period of time. The results of preliminary in vitro and in vivo studies demonstrate that orally administered spinetoram was well tolerated, and provides long lasting effectiveness against C. felis infestations on dogs.

An in vivo rodent model for identifying and characterizing acaricides.

Evaluation of candidate acaricides in livestock or companion animals is expensive, time-consuming, and usually requires large quantities of test material. To identify promising substances at the earliest possible stage of the development process, robust and predictive surrogate animal models, capable of rapidly characterizing potency with minimal compound requirements, are necessary. The objective of this study was to generate an in vivo surrogate animal bioassay capable of rapidly and accurately predicting the topical activity of acaricides emerging from in vitro acaricide bioassays. The rat acaricide test (RAT) requires adult rats, Rattus norvegicus (Berkenhout, 1769), a flexible tick containment device fastened to their dorso-thoracic region, and the nymphal stage of the lone star tick, Amblyomma americanum (L.). The feeding kinetics of A. americanum nymphs on rats was assessed, and compound efficacies were determined by measuring tick survivorship and engorgement weight on acaricide-treated animals. Results from this bioassay demonstrated efficacy with fipronil, ivermectin, permethrin, and chlorpyrifos, and dose-response relationships for each acaricide were determined. The rank order of potencies was fipronil > ivermectin > chlorpyrifos = permethrin for nymphal mortality and fipronil > ivermectin > chlorpyrifos > permethrin for inhibition of nymphal engorgement. The activity of permethrin against nymphs in the RAT was positively correlated with potency values for technical and commercial permethrin formulations against adult A. americanum infestations on cattle. The RAT proved to be an economical, rapid surrogate animal bioassay that together with the in vitro acaricide bioassay can be used for the rapid identification, characterization, and prioritization of candidate acaricides.

Specialization of function among aldehyde dehydrogenases: the ALD2 and ALD3 genes are required for beta-alanine biosynthesis in Saccharomyces cerevisiae.

The amino acid beta-alanine is an intermediate in pantothenic acid (vitamin B(5)) and coenzyme A (CoA) biosynthesis. In contrast to bacteria, yeast derive the beta-alanine required for pantothenic acid production via polyamine metabolism, mediated by the four SPE genes and by the FAD-dependent amine oxidase encoded by FMS1. Because amine oxidases generally produce aldehyde derivatives of amine compounds, we propose that an additional aldehyde-dehydrogenase-mediated step is required to make beta-alanine from the precursor aldehyde, 3-aminopropanal. This study presents evidence that the closely related aldehyde dehydrogenase genes ALD2 and ALD3 are required for pantothenic acid biosynthesis via conversion of 3-aminopropanal to beta-alanine in vivo. While deletion of the nuclear gene encoding the unrelated mitochondrial Ald5p resulted in an enhanced requirement for pantothenic acid pathway metabolites, we found no evidence to indicate that the Ald5p functions directly in the conversion of 3-aminopropanal to beta-alanine. Thus, in Saccharomyces cerevisiae, ALD2 and ALD3 are specialized for beta-alanine biosynthesis and are consequently involved in the cellular biosynthesis of coenzyme A.

Comparison of in vitro and in vivo ectoparasiticide activity of experimental benzimidazole-carbamate with permethrin and amitraz.

A series of in vitro and in vivo bioassays were conducted to assess the ectoparasiticide activity of isopropyl-4-nitro-2,6-bis(trifluoromethyl)-1-benzimidazole-carbamate, an experimental benzimidazole-carbamate class compound. This compound was less potent than permethrin against ectoparasiticide-susceptible larvae of the lone star tick, Amblyomma americanum (L.) (Acari: Ixodidae); larvae of the southern cattle tick, Boophilus microplus (Canestrini); and adult stable fly, Stomoxys calcitrans (L.) (Diptera: Muscidae) in vitro, but it was significantly more potent than permethrin against the Santa Luiza strain of B. microplus known to possess high-level resistance to amitraz and pyrethroids. In contrast, the benzimidazole-carbamate was substantially more efficacious than permethrin when applied topically onto rats that were infested with A. americanum nymphs. These results suggest that this experimental compound may be a viable candidate ectoparasiticide that retains significant activity against resistant B. microplus and also suggests that the benzimidazole-carbamate chemistry may be useful for addressing the growing problem of resistance in ectoparasites.

An in vitro larval immersion microassay for identifying and characterizing candidate acaricides.

We have optimized a larval immersion microassay (LIM) that offers superior sensitivity, flexibility to accommodate multiple formulations, and a robust capability for rapidly screening many compounds with a minimal requirement of test article for evaluation. Dose-response studies were conducted for representative members from the organophosphate, pyrethroid, pyrazole, carbamate, macrocyclic lactone, and formamidine chemistries against Amblyomma americanum (L.). Time-response experiments revealed that permethrin was the most rapid acting, whereas fipronil had the slowest speed-of-kill against A. americanum. Comparison of drug susceptibility profiles between multiple ixodid ticks suggests that A. americanum is an effective model for predicting compound potency against Boophilus spp. in this bioassay. The LIM is suitable for the identification and characterization of active molecules from small- and medium-sized compound or natural product libraries, and it can be a useful tool to prioritize molecules for further in vivo testing in animal models.