The Pentose Phosphate Pathway of Plasmodium parasites as a drug target

s of Keynote Talks 10 Antiparasitic drug discovery and research in Animal Health: Overview & development of Afoxolaner isoxazoline Loïc Le Hir de Fallois, Diane Larsen Merial Inc. now part of Boehringer Ingelheim, Duluth, GA-USA E-mail: loic.lehir@merial.com After a general overview of Antiparasitic Drug Discovery in Animal Health and its associated Research and Development process, we will review the work that led to the discovery and development of Afoxolaner isoxazoline used in NexGard. Afoxolaner is a new antiparasitic isoxazoline compound characterized by a good safety profile and extended effectiveness against fleas and ticks on dogs following a single oral administration [1]. The isoxazoline starting point seemed to have originated from the insecticidal phthalic diamides from Nissan and Nihon Nohyaku and the anthranilic diamides from DuPont [2]. The excellent ectoparasitic activity of the novel isoxazoline chemotype was demonstrated against flea and tick species both in laboratory on whole organism and in dose confirmation in dogs followed by comprehensive field studies [3a, b]. The flea and tick efficacies are directly related to the rapid absorption of afoxolaner and a plasma peak reaching the lethal concentration (LC90) in the blood, around 23 ng/mL for fleas and 100 ng/mL for ticks [4]. Extensive formulation optimization work led to the design of a highly palatable soft chew used in (NEXGARD). The speed of kill of afoxolaner against experimental infestations by Ctenocephalides felis was evaluated after oral administration of afoxolaner at a dose to achieve 2.5 mg/kg bodyweight. The efficacy of afoxolaner compared to control dogs increased to 99.5% at 8 h post-treatment and 100% at 12 and 24 h post-treatment [5]. [1] Wesley L. Shoop et al., Discovery and mode of action of afoxolaner, a new isoxazoline parasiticide for dogs, Vet. Parasitol. 2014, 201, 179-189 [2] George P. Lahm et al., 4-Azolyphenyl isoxazoline insecticides acting at the GABA gated chloride channel, Bioorg. Med. Chem. Lett. , 2013, 23, 3001-3006 [3a] James S. Hunter III et al., Evaluation of the curative and preventive efficacy of a single oral administration of afoxolaner against cat flea Ctenocephalides felis infestations on dogs, Vet. Parasitol. , 2014, 201, 207–211 [3b] Pascal Dumont, Curative and preventative efficacy of orally administered afoxolaner against dog flea Ctenocephalides canis (Curtis, 1826) infestation in dogs. Vet. Parasitol. 2014, 201, 212–215 [4] Laura Letendre et al., The intravenous and oral pharmacokinetics of afoxolaner used as a monthly chewable antiparasitic for dogs. Vet. Parasitlol. 2014, 201, 190–197 [5] B.N. Kunkle et al., Assessment of the onset of action of afoxolaner against existing adult flea (Ctenocephalides felis) infestations on dogs, Vet. Parasitol. 2014, 201, 204–206 Abstracts of Keynote Talkss of Keynote Talks 11 Parasitic helminth small RNAs: regulators of development, host-parasite interactions and as potential therapeutic targets Collette Britton, Alan Winter, Neil Marks, Henry Gu, Kirsty Maitland, Victoria Gillan, Eileen Devaney Institute of Biodiversity, Animal Health and Comparative Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK E-mail: Collette.Britton@glasgow.ac. uk The mechanisms regulating development and survival of parasitic helminths within their hosts are not well understood. We are examining microRNAs in parasitic nematodes to investigate their regulatory roles in development and immune modulation. miRNAs are small (22 nucleotide), non-coding RNAs that regulate gene expression post-transcriptionally and are important in controlling cell proliferation and differentiation in diverse organisms. We previously sequenced the miRNAs expressed by the highly pathogenic veterinary nematode Haemonchus contortus and the filarial nematode Brugia pahangi, which is closely related to filarial nematodes of humans and companion animals (1). Using microarray analysis we have profiled the expression pattern of miRNAs in the parasitic stages of both species. This identified miRNAs significantly enriched in the L3 stages preand post-infection, suggesting roles in regulating larval arrest and activation in response to host signals (2). Mimics and inhibitors of differentially expressed miRNAs are being tested for their effects on interfering with nematode development. miRNAs are also released in the excretory-secretory (ES) products of H. contortus and have been sequenced from both the ES supernatant and ES microvesicles. Secreted parasite miRNAs can be detected in gut tissue from H. contortus infected sheep and we speculate that these may modulate immune outcome. Our results are important in understanding parasite development and host-parasite interactions and highlight the potential of miRNAs and the pathways they regulate as novel drug targets for parasite control. [1] Winter, A.D. et al., 2012. BMC Genomics 13: 4 [2] Winter, A.D. et al., 2015. BMC Genomics 16: 331 Abstracts of Keynote Talkss of Keynote Talks 12 More complicated than imagined what mechanisms lead to benzimidazole resistance in helminths? Georg von Samson-Himmelstjerna and Jürgen Krücken Institute for Parasitology and Tropical Veterinary Medicine, Freie Universität Berlin, Germany E-mail: gvsamson@fu-berlin.de Only since the start of the area of broad spectrum anthelmintics in the 1960s safe, affordable and effective treatment and control of helminth infections has become available. As the first real broad spectrum antiparasiticides, the benzimidazoles (BZ) are in use against gastrointestinal nematodes (GIN) as well as some cestode and even protozoan parasite species for now more than five decades. The frequent and intensive use of anthelmintics can lead to drug resistance as also known for other antiinfectives. For the BZs this has already been reported during the early 1970s in small ruminant GIN and since then became a widespread problem in this important group of parasites as well as in equine cyathostomins. More recently, BZ resistance has been seen also in cattle GIN [1] and our own recent investigations indicate also a sub optimal response of Ascaris sp. in African school children. BZs act by binding with high affinity to the β-tubulins of pathogens which leads to a blocking of microtubule polymerization and due to the ongoing depolymerization thus subsequently to a complete loss of these essential cell structure. By comparison of the β-tubulin isotype 1 coding sequences from BZ-susceptible and –resistant populations of the sheep GIN Haemonchus contortus it was found that a single nucleotide polymorphism at the codon 200 leading to the expression of tyrosine instead of phenylalanine correlates with BZ resistance. Subsequently the similar P167Y and an E198A polymorphism were also described to be associated with BZ-resistance in this and other GIN species. In addition to these drug target related BZ resistance mechanisms, subsequently also unspecific resistance mechanisms involving an increased drug efflux mediated by P-glycoprotein transmembrane pumps were encountered. However, it is currently unclear which of the many P-glycoprotein genes of parasitic nematodes are involved in this process. Furthermore, also the modification of cytochrome P450 based drug metabolism was recently suggested to potentially be associated with BZ resistance in nematodes [2]. Noteworthy, parasite species and even population specific differences concerning the relevance of the various potential BZ resistance mechanisms have already been demonstrated. Future research should address the question if certain selectional factors contribute to the evolution of specific resistance mechanism, which fitness costs (if any) are associated with the different resistance mechanisms and if for example combinations of resistance mechanisms also occur. [1] Bullen et al. Aust Vet J. 2016; 94:35-41 [2] AlGusbi et al. Int. J. Parasitol. 2014; 44:647-58 Abstracts of Keynote Talkss of Keynote Talks 13 Targeting cell signalling and proteolysis for trypanosomatid drug discovery