Onchocerciasis Drug Discovery: In vitro Evaluation of FDA- Approved Drugs against Onchocerca gutturosa in Gambia

1. Introduction

Onchocerciasis (river blindness) is caused by the tissue dwelling filarial worm Onchocerca volvulus. The infection is transmitted between humans, by the bite of the blackfly vector of the Simulium genus. Circulating microfilariae (mf) accumulate in the skin but in high intensity infections the mf can also enter the tissues of the eye. Death of the mf causes the pathologies of pruritus, skin atrophy, skin de-pigmentation, papular rash, eye-lesions and blindness in humans [1]. Related to high morbidity, is reduced work productivity which can then lead to social stigmatisation; this poverty promoting nematode infection has been included within the group of neglected tropical diseases (NTDs) [2].

Without an available vaccine, current preventive chemotherapy and elimination of onchocerciasis rely on mass drug administration (MDA) programs (large scale distribution without diagnosis and supervision of health care staff) that distribute ivermectin (Mectizan® Merck) either on an annual or biannual basis [2]. This drug, belonging to the family of macrocyclic lactones, targets only the mf stage of the parasite by killing mf and exerting embryostatic effects on the adult female worm, that is, mf release from the uterus is temporarily suppressed [3]. Both of these mechanisms have the effect of reducing the skin mf by up to 99% two months after treatment [4]. In order to have an impact on filarial transmission reduction, treatment needs to be administered for the duration of the reproductive life span of the long-lived (up to 15 years [5]), tissue dwelling adult worms, so program success is constrained by the absence of drugs with macrofilaricidal activity. There are now concerns of developing ivermectin resistance in Onchocerca parasites, as has already been reported in nematodes of veterinary importance [6,7,8], and construed in human onchocerciasis [9]. A study in endemic communities exposed to frequent rounds of treatment in Ghana, demonstrated that although ivermectin retained microfilarial activity, sub optimal responses to treatment could be due to development of resistance by O. volvulus, resulting in a decreased effect on the inhibition of mf release [9]. Similarly in Cameroon, studies on communities given multiple rounds of ivermectin therapy compared to those that were ivermectin naive, indicated that continuous exposure to this drug had a reduced embryostatic effect on the female adult worms [10]. The findings from these studies highlight the possible emerging problems of ivermectin resistance and the urgent need for alternative methods of treatment. Recent studies demonstrated that the closely related drug moxidectin (registered for use in humans by the United States Food and Drug Administration (US FDA) in 2018) may offer improved treatment over ivermectin, although no macrofilaricidal effect has been observed so far [11,12].

The Pharmakon 1600 Collection, a US FDA-approved library, consists of 1600 drugs that have reached clinical evaluation and demonstrated biological activity against known targets. By screening a selection of this drug set, the identification of any potential antifilarial candidates could be rapidly repurposed and prove useful in onchocerciasis elimination programs, as many of the drugs represented in the library are available on the market. Humans are the only viable hosts of O. volvulus and there are no laboratory models that support the complete life cycle of this parasite, as such drug discovery for onchocerciasis has to rely in part, on the use of surrogate parasites and animal hosts. Several in vitro and in vivo standard operating protocols for testing drugs against the adult stage of Onchocerca have been developed and optimized [13] and references therein. Using a World Health Organisation approved 5-day motility/MTT based assay with the cattle filarial nematode Onchocerca gutturosa, in this study we have assessed the activity of 106 selected drugs with a range of biological activities (see Table 1).

2. Materials and Methods

A workflow diagram for the experimental procedure used in this study is depicted in Figure 1.

2.1. Parasites - Onchocerca gutturosa

Adult male worms were obtained post mortem from naturally infected, freshly slaughtered cattle in Gambia, West Africa. The material used in this study was purchased from local butchers by the West Africa Livestock Innovation Centre (WALIC), Banjul, Gambia. The adult male worms were dissected from the cattle nuchal ligament connective tissue and transferred to each well of a sterile 24-well (2 ml) plate (Fisher Scientific, UK), and maintained for at least 24 h in culture before use. The culture medium used was Minimum Essential Medium (MEM) with Earl’s Salts and L-Glutamine (Life Technologies Ltd, UK) supplemented with 10% heat inactivated new-born calf serum (Life Technologies Ltd, UK) and 200 units/ml penicillin, 200 µg/ml streptomycin and 0.5 µg/ml amphotericin B (Life Technologies Ltd, UK). Only normally active worms were used for the test and all assays were conducted at 37°C under an atmosphere of 5% CO₂ in air.

2.2. Origin of drugs tested

The drugs of the Pharmakon 1600 Collection were supplied by MicroSource Discovery Systems Inc. (U.S.A) as 10 mM DMSO stock solutions in microtubes in a 96-well plate format and stored at -20°C. The positive control drug, Immiticide® (melarsomine dihydrochloride, Merial) was supplied as a dry solid, known amounts of this drug were solubilized in 1 ml DMSO.

2.3. In vitro drug activity against O. gutturosa adult worms as described by Townson et al. [13]

The selected Pharmakon drugs with the positive control Immiticide® were screened using a final concentration of 1.25 x 10^-5 M. At this concentration the drugs were not toxic to mammalian cells ( LLMCK2 monkey kidney cells, evaluated by microscopy [14]). Two trials were performed using different numbers of worms (according to availability) and drugs. Trial 1: 4 worms per group were used for the test and positive control drugs, and 8 worms for the untreated control group. Only 100 of the selected 106 drugs were tested due to an insufficient number of available worms. Trial 2: this was performed using three separate assays, 23 drugs were tested of which 17 were retested from Trial 1 (confirmation tests), and the 6 remaining drugs that could not be tested in Trial 1; 2 worms per group were used for the test and positive control drugs, 6 worms for the untreated control group. Worm viability was assessed using the following parameters: measurement of mean worm motility scores on a scale of 0 (immotile) to 10 (maximum) every 24 h, terminating at 120 h, using an Olympus inverted microscope. Biochemical evaluation of worm viability using MTT/formazan colorimetry was carried out after the last motility reading (120 h), outlined in Figure 1. Inhibition of formazan formation is correlated with worm damage or death (viability). The results of the test drugs were compared to the untreated controls. The results were calculated as motility reduction (%) and MTT reduction (%) compared to the untreated controls (Microsoft Office Excel, 2010) and designated: good activity, 100% motility and/or MTT reduction; moderate/marginal activity, 50-99% motility and/or MTT reduction; inactive, < 50% motility reduction and MTT reduction. The test drug is considered active when the motility and/or MTT reductions of ≥ 50% is observed by comparison to the untreated controls. Statistical analysis was performed on only the Trial 1 data, using the 2 tailed t-test for the comparison between the motility and optical density means of the test drug and untreated control (Microsoft Office Excel, 2010) with a significance level of p < 0.05.

3. Results

Of the 106 selected drugs tested, 39 were inactive (< 50% motility reduction and MTT reduction), 44 showed marginal/moderate activity (50-99% motility and/or MTT reduction) and 23 showed good activity (100% motility and/or MTT reduction) after 120 h drug exposure.

The results for the 44 drugs that showed moderate or marginal activity in Trial 1 are shown in Table 2. For the majority of these drugs, the MTT reductions correlated with the motility reductions, indicating that the worms did not only have reduced motility but were permanently damaged. Examination of the data shows that for 13 drugs with the greatest motility reductions of > 80%, the MTT reduction was ≥ 70% (p-values < 0.001), with the highest number of hits in the antibacterial (cefuroxime sodium, demeclocycline hydrochloride, methenamine), antiparasitic (amitraz, primaquine phosphate) and neurological (armodafinil, chlorpromazine, dopamine hydrochloride, rivastigmine tartrate) bioactivity groups.

The 23 drugs which demonstrated good activity are shown in Table 3 (includes the chemical structures), 17 of these drugs were retests from Trial 1, and 6 drugs were new tests in Trial 2. All of the drugs produced 100% motility reduction and high levels of MTT reduction, and for most drugs there was a good level of concordance between the results of Trial 1 when compared to Trial 2. The highest number of hits was found in the antibacterial (cefaclor, chlorhexidine dihydrochloride, gramicidin, lasalocid sodium, nitrofurantoin, nitrofurazone, nitroxoline, rifaximin), antiinfective (benzethonium chloride, dequalinium chloride, methylbenzethonium chloride, oxyquinoline hemisulfate) and antiparasitic (atovaquone, hexetidine, homidium bromide, iodoquinol, levamisole hydrochloride, pyrantel pamoate) bioactivity groups.