bay-44-4400 and Filariasis

Filariae are parasitic nematodes that are transmitted to their definitive host as third-stage larvae by arthropod vectors like mosquitoes. Filariae cause diseases including: lymphatic filariasis with distressing and disturbing symptoms like elephantiasis; and river blindness. Filarial diseases affect millions of people in 73 countries throughout the topics and sub-tropics. The drugs available for mass drug administration, (ivermectin, albendazole and diethylcarbamazine), are ineffective against adult filariae (macrofilariae) at the registered dosing regimen; this generates a real and urgent need to identify effective macrofilaricides. Emodepside, a veterinary anthelmintic registered for treatment of nematode infections in cats and dogs, is reported to have macrofilaricidal effects. Here, we explore the mode of action of emodepside using adult Brugia malayi, one of the species that causes lymphatic filariasis. Whole-parasite motility measurement with Worminator and patch-clamp of single muscle cells show that emodepside potently inhibits motility by activating voltage-gated potassium channels and that the male is more sensitive than the female. RNAi knock down suggests that emodepside targets SLO-1 K channels. We expressed slo-1 isoforms, with alternatively spliced exons at the RCK1 (Regulator of Conductance of Potassium) domain, heterologously in Xenopus laevis oocytes. We discovered that the slo-1f isoform, found in muscles of males, is more sensitive to emodepside than the slo-1a isoform found in muscles of females; and selective RNAi of the slo-1a isoform in female worms increased emodepside potency. In Onchocerca volvulus, that causes river blindness, we found two isoforms in adult females with homology to Bma-SLO-1A and Bma-SLO-1F at the RCK1 domain. In silico modeling identified an emodepside binding pocket in the same RCK1 region of different species of filaria that is affected by these splice variations. Our observations show that emodepside has potent macrofilaricidal effects and alternative splicing in the RCK1 binding pocket affects potency. Therefore, the evaluation of potential sex-dependent effects of an anthelmintic compound is of importance to prevent any under-dosing of one or the other gender of nematodes once given to patients.

Topics: Alternative Splicing; Amino Acid Sequence; Animals; Binding Sites; Brugia malayi; Depsipeptides; Female; Filariasis; Filaricides; Gene Knockdown Techniques; Humans; Large-Conductance Calcium-Activated Potassium Channels; Male; Models, Molecular; Movement; Muscles; Peptides; Potassium Channel Blockers; Sequence Homology, Amino Acid; Sex Factors

Cytokine (interferon (IFN)-gamma, interleukin (IL)-2, IL-4, IL-5, IL-10) gene transcription in response to filarial antigens was determined in peripheral blood mononuclear cells of Brugia malayi-infected Mastomys coucha in the course of untreated and chemotherapeutically abbreviated infections. Transcript levels in infected untreated animals suggest particular time courses for the various cytokines with ongoing parasite development and differing efficacies of female, male, microfilarial, and L3 antigens in inducing cytokine gene transcription. Gene transcription of both of Th1- and Th2-associated cytokines were initiated in the course of the infection in a manner that does not fit in a simple Th1-Th2 paradigm. IFN-gamma and IL-4 gene transcripts prevailed during prepatency. In case of the other cytokine genes considered in the study, transcription in general peaked around beginning of patency. During the phase of increasing microfilaremia (approximately 120-180 days p. i.) cytokine gene transcription was generally decreased. Later on, when the parasitemia had leveled off, except IFN-gamma, transcript levels often tended to increase. In chemotherapeutically treated animals, the outcome varied with the different efficacies of the drugs employed. The highly microfilaricidal cyclodepsipeptide BAY 44-4400 eliminated circulating microfilariae and partially sterilized adult worms without killing them. This kind of treatment hardly affected cytokine responses. In contrast, the therapy with Flubendazole, a selectively macrofilaricidal benzimidazole, and particularly the application of CGP 20376, a highly efficient microfilaricidal and macrofilaricidal benzthiazole, resulted in enhanced transcription of the Th1-associated IFN-gamma and IL-2 genes as well as of the Th2-associated IL-5 gene 2-3 months after treatment. IL-10 gene transcription seemed transiently increased after 1 month. There was no effect of any treatment on the IL-4 gene transcription.

Topics: Animals; Anthelmintics; Brugia malayi; Cytokines; Filariasis; Lymphocytes; Mebendazole; Murinae; Peptides, Cyclic; Thiazoles; Time Factors

Bay 44-4400 was used as a spot on formulation and administered in single doses of 25 and 100 mg/kg to Acanthocheilonema viteae, Brugia malayi, and Litomosoides sigmodontis infected Mastomys coucha on various dates during prepatency, aiming to affect third stage larvae, fourth stage larvae or preadult worms. Microfilaraemia levels were controlled in comparison to untreated controls until necropsies were performed 100 days p.i. (A. viteae, L. sigmodontis) and 150 days p.i. (B. malayi) to determine the numbers of surviving worms and the condition of intrauterine developing stages. A significant proportion (86-100%) of larval and preadult stages of A. viteae were killed by Bay 44-4400 at a dose of 100 mg/kg. A dose of 25 mg/kg had only insignificant effects on the developing parasites, however, it strongly reduced microfilaraemia levels caused by surviving worms in the early phase of patency. Larval and preadult B. malayi and L. sigmodontis were not killed by Bay 44-4400 to a significant degree. Microfilaraemia developing by surviving parasites was generally and significantly reduced throughout the observation period when treatment was performed to affect the preadult parasites. In the other cases variable results were obtained. Intrauterine early embryonic stages were found to be pathologically altered in worms which had been treated at a preadult stage.

Topics: Administration, Cutaneous; Animals; Brugia malayi; Dipetalonema; Dipetalonema Infections; Disease Models, Animal; Filariasis; Filaricides; Filarioidea; Larva; Microfilariae; Muridae; Peptides, Cyclic

PF 1022A, a novel anthelmintically active cyclodepsipeptide, and Bay 44-4400, a semisynthetic derivative of PF 1022A were tested for filaricidal efficacy in Mastomys coucha infected with Litomosoides sigmodontis, Acanthocheilonema viteae and Brugia malayi. The parent compound PF 1022A showed limited anti-filarial efficacy in L. sigmodontis and B. malayi infected animals. Oral doses of 5 x 100 mg/kg on consecutive days caused only a temporary decrease of microfilariaemia levels. By contrast, Bay 44-4400 was highly effective against microfilariae of all three species in single oral, subcutaneous and cutaneously applied (spot on) doses. Minimum effective doses (MED, reducing parasitaemia density by > or =95%) determined 3 and 7 days after treatment were 3.125-6.25 and 6.25-12.5mg/kg, respectively. Using the spot on formulation, doses of 6.25mg/kg (L. sigmodontis), 12.5mg/kg (A. viteae) and 25mg/kg (B. malayi) were required to cause reductions of microfilaraemia levels by > or =95% until day 56. Adulticidal effects, determined as minimum curative doses (MCD, eliminating adult parasites within 56 days by >95%) after single dose treatment were limited to A. viteae (MCD, 100mg/kg independent of the route of administration). Repeated oral treatment (100mg/kg on 5 consecutive days) killed all adult L. sigmodontis but did not affect B. malayi. However, single doses of 6.25 and 25mg/kg resulted in severe pathological alterations of intrauterine stages of L. sigmodontis and B. malayi, respectively. These alterations may be responsible for long-lasting reductions of microfilaraemia even when curative effects could not be achieved.

Topics: Administration, Oral; Administration, Topical; Animals; Anthelmintics; Brugia malayi; Depsipeptides; Dipetalonema; Female; Filariasis; Filarioidea; Injections, Subcutaneous; Muridae; Parasitemia; Peptides, Cyclic; Rats