In-vitro efficacy of moxidectin versus ivermectin against Sarcoptes scabiei

AAC Accepted Manuscript Posted Online 30 May 2017 Antimicrob. Agents Chemother. doi:10.1128/aac.00381-17 Copyright 2017 American Society for Microbiology. All Rights Reserved. 1 In-vitro efficacy of moxidectin versus ivermectin against Sarcoptes scabiei 2 3 Running title: ivermectin and moxidectin for scabies 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Kate E Mounsey 1#, Shelley F Walton 1, Ashlee Innes 1, Skye Cash-Deans 1 and James S McCarthy 2,3 1 Inflammation and Healing Cluster, School of Health & Sport Sciences, Faculty of Science, Health, Education and Engineering, University of the Sunshine Coast, Sippy Downs, QLD 4556 Australia 2 QIMR Berghofer Medical Research Institute, Herston QLD 4006 Australia 3 University of Queensland, Herston QLD 4006 Australia # Corresponding author kmounsey@usc.edu.au 1

19 Abstract 20 21 22 23 24 25 26 27 28 Moxidectin is under consideration for development as a treatment for human scabies. As some arthropods show decreased sensitivity to moxidectin relative to ivermectin, it was important to assess this for Sarcoptes scabiei. In-vitro assays showed that the concentration of moxidectin required to kill 50% of mites was lower compared to ivermectin (0.5 µm vs 1.8 µm at 24 hours, p <0.0001). This finding provides further support for moxidectin as a candidate for the treatment of human scabies. Downloaded from http://aac.asm.org/ on December 30, 2018 by guest 2

29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 The macrocyclic lactones ivermectin and moxidectin have been widely utilised in veterinary practice for the treatment of Sarcoptes scabiei infestation. For human scabies, ivermectin is the only licenced oral acaricide, with moxidectin under recent consideration for development as an alternative to ivermectin (1). As moxidectin has a prolonged plasma-half life in humans (29-47 days versus 14 hours) (2-4), it is anticipated that it may be more suitable than ivermectin as a single oral dose, providing coverage over the entire mite life cycle. Ivermectin is administered at a concentration of approximately 200 µg/kg for scabies, with rationale for this based on its activity against nematodes. There have been no empirical dosefinding studies for scabies. It is acknowledged that doses 150 µg/kg have reduced efficacy (5-7), and it has been suggested that higher doses may be advantageous (8). Suboptimal responses to ivermectin in some groups (9-11) indicate that a more detailed investigation of optimal therapeutic doses in scabies is warranted. This is particularly relevant with the utilisation of ivermectin for scabies mass treatment, where a single dose regimen is desirable (12, 13). Moxidectin has demonstrated activity against sarcoptic mange, although several studies suggest a single 200 µg/kg dose is still insufficient to achieve cure (14, 15), with long acting formulations or higher doses required (16). Other reports show 100% efficacy following a single 200-300 µg/kg dose (17, 18). Differences are apparent between ivermectin and moxidectin activity, especially in arthropods (19-22). From this, questions emerge regarding the threshold for acaricidal activity of both drugs over the mite life cycle and how this relates to therapeutically relevant concentrations, bioavailability and safety margins. In-vitro studies are a useful pre-clinical measure of relative toxicity, and are routinely used to measure acaricide activity in S. scabiei. In this study, we compared the in-vitro toxicity of ivermectin and moxidectin in S. scabiei var suis. 3

53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 Mites were harvested from pigs maintained at the Queensland Agricultural Science Precinct (QASP), University of Queensland, Australia, with ethical approval from the Queensland Department of Agriculture, Forestry and Fisheries (SA 2015-03-504). Scabies mites in this colony were originally obtained from naturally infested pigs and have had no known acaricide exposure. Establishment of this model and protocols for mite collection have been described in detail previously (23). Briefly, skin scrapings were obtained from the ear of infested pigs, and transported to laboratory. To isolate mites, skin crusts were placed on glass petri dishes and incubated at 28 o C, which encourages mites to move out of the crusts towards the heat source. In-vitro assays were commenced within 4 hours of collecting skin crusts. For all assays, mortality was defined as the absence of any movement when gently touched with a probe. We used injectable solutions of ivermectin (Ivomec, Merial) and moxidectin (Cydectin, Virbac). An initial analysis was performed to compare the effect of diluent on mite survival, using ten adult female mites exposed to 50 µm and 100 µm of acaricides diluted in phosphate buffered saline (PBS), mineral oil or Polyethylene Glycol (PEG). Negative controls (n=10) consisted of diluent in the absence of acaricide. Mortality was measured at hourly intervals up to five hours, then again at 24 hours. Kaplan-Meier survival curves were generated and curves compared statistically using the Log-rank (Mantel-Cox) test (Graphpad prism 7). Results from these assays were used to confirm the most appropriate diluent for subsequent assays. Next, assays to determine the concentration required to kill 50% of mites (LC 50 ) were conducted using female mites as previously described (24). Stock solutions (200 µm) and two fold serial dilutions (1.6-100 µm) were prepared in PBS immediately prior to use. Mortality was recorded at one, three and 24 hours post exposure. Each compound was assayed in duplicate with ten mites per concentration, and assays repeated on three separate 4

78 79 80 81 occasions (n = 60 per concentration). LC 50 values were determined by normalised dose response analysis and best fit curves generated by non-linear regression. The two regression models and LC 50s were compared statistically using the extra sum-of-squares F test (Graphpad prism 7). 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 The LC 50 assays were restricted to adult female mites to limit variation in susceptibility due to differences in activity associated with developmental stages. To assess this we then compared the survival of adult females to juvenile and adult male mites when exposed to a fixed concentration of acaricide (6.5 µm moxidectin or 12.5 µm ivermectin) diluted in PBS. 15 mites per life stage were assessed in duplicate assays (n = 30). These assays revealed that moxidectin was significantly more active than ivermectin at all time-points tested (Figure 1, Table 1). At one and three hours post exposure, the reductions in moxidectin micromolar LC 50 relative to ivermectin were 6.2-fold and 7.5-fold respectively. LC 50 values decreased for both acaricides over time of exposure, and the magnitude of difference in LC 50 of the drugs at 24 hours was smaller (3.6-fold), but still significant (Table 1). Survival analysis revealed significant differences associated with developmental stages. For ivermectin (12.5 µm), median survival was 30 minutes for larvae, 1 hour for nymphs and adult males, and 2 hours for adult females (Figure 2A). For moxidectin (6.25 µm), median survival time was 30 minutes for juvenile and male mites compared to females with 1 hour (Figure 2B). We also found that survival times of adult female mites to 50 µm ivermectin were significantly different depending on the diluent (1-2 hours in PBS or PEG versus 4 hours in mineral oil, p = 0.0003). For 50 µm moxidectin no significant differences between diluents were apparent. Notably in all diluents, moxidectin showed a significantly reduced survival 5

102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 time compared to ivermectin (p < 0.0001). Median survival time in negative controls for all assays exceeded 20 hours, with no statistical differences between diluents. These findings suggest that differences in diluent should be considered when comparing data from other in vitro studies, at least for ivermectin. Considering this, the median survival time for ivermectin in mineral oil (1 hour at 100µM) was consistent with previously reported in-vitro data from S. scabiei var. hominis mites prior to ivermectin exposure (for these var. hominis assays, ointment containing 100 µm ivermectin in paraffin oil was used). Following ten years of ivermectin use, median in-vitro survival times to ivermectin doubled in these mites (11). Precise LC 50 estimates have not yet been undertaken in var. hominis, nor has moxidectin been assessed in this population. The in-vitro LC 50 values reported here are in the range documented for other parasitic arthropods and nematodes. The LC 50 for ivermectin at 24 hours (1.8 µm) is similar to Cooperia sp. (1.7 µm), Haemonchus contortus and Strongyloides ratti (~1.14 µm) (25-27), although in the latter two nematodes a high-throughput motility assay demonstrated lower IC 50 (< 0.34 µm) (28). Where the filarial nematodes Brugia malayi and Dirofilaria immitis are concerned, the ivermectin IC 50 ranged from 4.6-28.2 µm depending on life stage (27, 29). These concentrations are generally higher than has been documented for arthropods, where ivermectin LC 50 values range from 23 nm (Cimex lectularius, Musca domestica, Anopheles gambiae) to 0.69 µm (Aedes aegypti) (19, 20, 22). There have been few studies on mites, although one recent report shows extremely high activity of ivermectin against spider mites (T. cinnabarinus 10.3 nm) (30). Variation in reported values are likely due to different assay methodologies, particularly in arthropod assays which involve assessment of mortality after direct feeding (from a spiked blood meal for example), whereas our S. scabiei in-vitro assays are primarily contact based. 6

126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 The finding that moxidectin was more toxic than ivermectin to S. scabiei is in contrast to that reported for other arthropods. The LC 50 for moxidectin at 24 hours in A. gambiae, (4.2 µm) is over 100-fold higher than for ivermectin (20). In spider mites ivermectin was 2-fold more active than milbemycin (30), although newly developed synthetic milbemycins had higher activity (0.03-0.17 µm) (31). Equivalent concentrations of moxidectin were less active than ivermectin against bed bugs (C. lactuarius) (22). Ivermectin was approximately 10-times more potent than moxidectin in a variety of fly species (19). Differences in toxicity between the macrocyclic lactones are less apparent in nematodes (27), however studies of B. malayi and Caenorhabditis elegans show differences between phenotypic effects of the drugs, suggesting different target sites exist (32, 33). While moxidectin and ivermectin have a similar mechanism of action, the existence of different binding targets is further reflected by differences in ABC-transporter mediated efflux for example (21). It is important to note that in other parasites considerable variation exists between the in-vitro susceptibility and plasma drug concentrations, with the latter invariably lower (29). This is likely due to aforementioned differences in mode of drug delivery (direct ingestion of skin and sera versus absorption of the drug through the mite cuticle). Thus, while these in-vitro studies are a useful measure of relative toxicity which may aid clinical decision making, the LC 50 values cannot be directly translated to the in-vivo setting. Notwithstanding the above, when considering the clinical relevance of these results, how do these in-vitro concentrations compare to bioavailability in skin? A recent study in pig skin measured a moxidectin C max of 0.94 µm and T ½ of 8.6 days, compared to only 0.069 µm and one day for ivermectin (17). While the moxidectin levels are within our in-vitro susceptibility range, it is possible that these levels of ivermectin might be insufficient to kill all mites. In the above study ivermectin could not be detected in pig skin beyond 9-12 days post treatment, 7

151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 suggesting little activity against newly hatched eggs, although it is seen in our data that juvenile mites would be susceptible to lower drug concentrations. Our results explain in part why single dose moxidectin achieved 100% efficacy in pigs at day 14, compared to only 62% for ivermectin (17). While moxidectin has excellent retention in the skin of pigs and cattle (34), different pharmacokinetics may relate to observed differences in treatment efficacy in other species. There have been few reports regarding the skin concentrations of ivermectin and moxidectin in humans. One study of five participants measured an ivermectin C max of 0.023 µm on the skin surface, with levels declining after 24 hours (35). Determination of the concentrations of moxidectin in human skin would be of great benefit, complementing the work herein and informing future dose finding studies. This work contributes important preclinical data towards recently funded human Phase II efficacy and dose finding studies for moxidectin in scabies (36). Our findings confirm that S. scabiei are highly susceptible to moxidectin, and that moxidectin is superior to ivermectin invitro. This increased susceptibility, combined with enhanced bioavailability (4), provide strong support for the development of moxidectin for human scabies. Acknowledgments We thank the staff of the Queensland Animal Science Precinct, Meredith Johnson and Mallory King for assistance with mite collections and in-vitro assays. This work was supported by a University of the Sunshine Coast Faculty grant. 172 173 References 8

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284 Figure legends 285 286 287 288 289 290 291 292 Figure 1: Dose-response curves of S. scabiei mortality after 1 hour of in-vitro exposure to serial dilutions of 0 200 µm ivermectin and moxidectin. Points show median mortality, bars standard error. n = 60 mites per concentration. Figure 2: In-vitro survival of S. scabiei life stages upon exposure to 12.5 µm ivermectin (A) or 6.25 µm moxidectin (B). n = 30 mites per life stage. Downloaded from http://aac.asm.org/ on December 30, 2018 by guest 14

293 294 Table 1. Concentrations of ivermectin and moxidectin required to kill 50% of S. scabiei at one, three and 24 hours post-exposure. n = 60 mites per concentration tested. 295 296 297 298 299 300 1 hr 3 hr 24 hr Drug (µm) LC 50 95% CI LC 50 95% CI LC 50 95% CI Ivermectin 50.5 45.4-56.4 10.5 8.2-13.5 1.8 1.18-2.68 Moxidectin 8.2 a 7.6-8.9 1.4 a 1.1-1.9 0.5 a 0.34-0.77 a p<0.0001 15