Drug activity against Onchocerca gutturosa males in vitro: a model for chemotherapeutic research on onchocerciasis

Abstract

An in vitro system for chemotherapeutic research using adult male Onchocerca gutturosa has been developed as a model for O. volvulus. Using a culture system consisting of medium MEM+10% heat inactivated foetal calf serum (IFCS)+LLCMK2 (monkey kidney) feeder cells in an atmosphere of 5% CO2 in air, we examined the effects of a range of antiparasitic drugs on worm motility. Ivermectin, levamisole, furapyrimidone, Mel W, chloroquine, metrifonate, flubendazole, amoscanate and the Ciba-Geigy compounds CGP 6140, CGP 20′376 and CGI 17658 either immobilized or significantly reduced motility levels at a concentration of 5x10−5M or less within a 7-day period. Worms were affected at very low concentrations by ivermectin (effective conc. to reduce motility levels to 50% of controls, 3.14x10−8M), levamisole (7.95x10−8M), CGP 6140 (8.87x10−9M) and CGP 20′376 (2.78x10−8M). Difficulties were experienced in accurately repeating the immotile endpoint for levamisole due to an inconsistent partial recovery of motility. Over a 7-day period diethylcarbamazine had little effect on motility levels, while suramin caused a slight increase in activity compared to controls at some timepoints. Subsequent experiments demonstrated some differences in drug efficacy depending on the presence or absence of serum and feeder cells in the culture system probably because of drug avidly binding to serum proteins. However, serum and cells were found to be essential ingredients of the culture system to maintain worms in good condition, indicating that new drugs should be evaluated both in the presence and absence of serum and cells. Comparisons were made between the responses of O. gutturosa and Brugia pahangi to certain drugs and these species were found to significantly differ in their sensitivities to ivermectin and a novel compound (Wellcome), indicating that Onchocerca parasites should be used wherever possible for compound identification and development intended for the treatment of onchocerciasis. The in vitro system described here, using male O. gutturosa, provides a basis for further research and a practical alternative to O. volvulus.

Onchocerca parasites and Wolbachia endosymbionts: evaluation of a spectrum of antibiotic types for activity against Onchocerca gutturosa in vitro

“…Worm viability was measured by the motility levels and MTT colorimetry. Motility scores were assessed on an inverted microscope on a scale of 0 (immotile) to 10 (maximum) [ 22 ] at regular intervals up to 40 days. The biochemical evaluation of worm viability was carried out by MTT/formazan colorimetry on day 40.…”

Challenges in drug discovery for novel antifilarials

“…The development and further optimisation of this assay are described in [81][82][83]86] . This assay has been used in the identification of compounds with anti-Onchocerca adult worm activity.…”

“…NPIMR has had a long history and experience in the optimisation of culture conditions for O. gutturosa , a parasite from cattle [52,73,[79][80][81][82] . In addition, the application of MTT-formazan colorimetry (MTT is a tetrazole, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to determine filarial worm viability was developed at The Wellcome Foundation [83] .…”

The anterior central nervous system of male Onchocerca volvulus (Nematoda: Filarioidea) as a target for chemotherapy: results of an electron microscopy study

“…This heterogeneity complicates the distinction of changes in worm tissues and worm metabolism that are due to age processes from those caused by treatment (Striebel 1988) or other intervention steps ). Since the histological evaluation of drug ecacy is by far the most important and useful parameter in phase II clinical trials involving the target organism Onchocerca volvulus (BuÈ ttner 1985; Awadzi et al 1991Awadzi et al , 1995Duke 1991) and is of comparable importance in preclinical experiments with rodent, cattle, and in vitro models (Franz et al 1987(Franz et al , 1990Townson et al 1987Townson et al , 1989Franz 1988;Townson 1988;Strote et al 1990aStrote et al , 1993, a distinction of these eects is decisive. Working at the light microscope level with the predictive cattle model O. gibsoni, Striebel and Copeman (1990) emphasized that tissues in the most anterior part of these ®larial worms are those best preserved as compared with the tissues and organs in the posterior part.…”

“…We therefore decided to study the ®ne structure of the anterior nerve ring after incubation of viable adult worms with known and new compounds in vitro. This inevitable experimental situation with the host-speci®c target parasites has been proven to be of predictive value in terms of the activity of standard compounds and new promising therapeuticals (Nowak et al 1987;Townson et al 1987Townson et al , 1989Strote et al 1990aStrote et al , b, 1993.…”