Drugs that target early stages of Onchocerca volvulus: A revisited means to facilitate the elimination goals for onchocerciasis

Several issues have been identified with the current programs for the elimination of onchocerciasis that target only transmission by using mass drug administration (MDA) of the drug ivermectin. Alternative and/or complementary treatment regimens as part of a more comprehensive strategy to eliminate onchocerciasis are needed. We posit that the addition of “prophylactic” drugs or therapeutic drugs that can be utilized in a prophylactic strategy to the toolbox of present microfilaricidal drugs and/or future macrofilaricidal treatment regimens will not only improve the chances of meeting the elimination goals but may hasten the time to elimination and also will support achieving a sustained elimination of onchocerciasis. These “prophylactic” drugs will target the infective third- (L3) and fourth-stage (L4) larvae of Onchocerca volvulus and consequently prevent the establishment of new infections not only in uninfected individuals but also in already infected individuals and thus reduce the overall adult worm burden and transmission. Importantly, an effective prophylactic treatment regimen can utilize drugs that are already part of the onchocerciasis elimination program (ivermectin), those being considered for MDA (moxidectin), and/or the potential macrofilaricidal drugs (oxfendazole and emodepside) currently under clinical development. Prophylaxis of onchocerciasis is not a new concept. We present new data showing that these drugs can inhibit L3 molting and/or inhibit motility of L4 at IC50 and IC90 that are covered by the concentration of these drugs in plasma based on the corresponding pharmacological profiles obtained in human clinical trials when these drugs were tested using various doses for the therapeutic treatments of various helminth infections.

Onchocerca volvulus is an obligate human parasite and the causative agent for onchocerciasis, which is a chronic neglected tropical disease prevalent mostly in the sub-Saharan Africa. In 2017, 20.9 million people were infected, with 14.6 million having skin pathologies and 1.15 million having vision loss [1]. The socioeconomic impact of onchocerciasis and the debilitating morbidity caused by the disease prompted the World Health Organization (WHO) to a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 initiate control programs that were first focused on reducing onchocerciasis as a public health problem, and since 2012, the ultimate goal is to eliminate it by 2030 [2]. Over the years, WHO sponsored and coordinated 3 major programs: The Onchocerciasis Control Programme (OCP), the African Programme for Onchocerciasis Control (APOC), and the Onchocerciasis Elimination Program of the Americas (OEPA). Since 1989, the control measures depended on mass drug administration (MDA) annually or biannually with ivermectin, which targets the transmitting stage of parasite, the microfilariae [3][4][5]. However, several issues have been identified with the current MDA programs including the need to expand the treatment to more populations depending on baseline endemicity and transmission rates [2,6]. Moreover, it became apparent that alternative and/or complementary treatment regimens as part of a more comprehensive strategy to eliminate onchocerciasis are needed [2]. Ivermectin has only mild to moderate effects on the adult stages of the parasite [7-9], and there are communities in Africa where the effects of ivermectin are suboptimal [10]. It is also contraindicated in areas of Loa loa co-endemicity [11], as well as in children under the age of 5 and in pregnant women. By relying only on MDA with ivermectin, the most optimistic mathematical modeling predicts that elimination will occur only in 2045 [12].
To support the elimination agenda, much of the recent focus has been on improving efficacy outcomes through improved microfilariae control with moxidectin and the discovery of macrofilaricidal drugs that target the adult O. volvulus parasites [13][14][15][16][17][18]. We posit that the addition of "prophylactic" drugs or therapeutic drugs that can be utilized in a prophylactic strategy to the toolbox of present microfilaricidal drugs and/or future macrofilaricidal treatment regimens will not only improve the chances of meeting the elimination goals but may also hasten the time for elimination and support achieving a sustained elimination of onchocerciasis. These "prophylactic" drugs will target the infective third-(L3) and fourth-stage (L4) larvae of O. volvulus and consequently prevent the establishment of new infections not only in the uninfected individuals but also in the already infected individuals and thus reduce the overall adult worm burden and transmission. Importantly, an effective prophylactic treatment regimen can utilize drugs that are already part of the onchocerciasis elimination program (ivermectin), those being considered for MDA (moxidectin) [19,20], and/or the potential macrofilaricidal drugs (oxfendazole and emodepside) currently under clinical development [21].
Prophylaxis of onchocerciasis is not a new concept. In the 1980s, once ivermectin was introduced as a "prophylactic" drug against the filarial dog heartworm, Dirofilaria immitis [22], its prophylactic effects were also examined in Onchocerca spp. In chimpanzees, a single dose of ivermectin (200 μg/kg) was highly protective (83% reduction in patent infections) when given at the time of the experimental infection and tracked for development of patency over 30 months. It was, however, much less effective (33% reduction in patent infections) when given 1 month postinfection with the L3s, at which time the L4s had already developed [23]. Moreover, monthly treatment with ivermectin at either 200 μg/kg or 500 μg/kg for 21 months completely protected naïve calves against the development of O. ochengi infection as compared to untreated controls, which were 83% positive for nodules and 100% positive for patency [24]. When naïve calves exposed to natural infection were treated with either ivermectin (150 μg/ kg) or with moxidectin (200 μg/kg) monthly or quarterly, none of the animals developed detectable infections after 22 months of exposure, except 2 animals in the quarterly ivermectin treated group which had 1 nodule each; in the non-treated control group, the nodule prevalence was 78.6% [25]. These prophylactic studies in calves exposed to natural infections clearly demonstrated that monthly or quarterly treatments with ivermectin and/or moxidectin over 22 months were highly efficacious against the development of new infections. When ivermectin was administered in a highly endemic region of onchocerciasis in Cameroon every 3 months over a 4-year period, it resulted in reduced numbers of new nodules (17.7%) when compared to individuals who were treated annually. This recent study suggests that ivermectin may have also a better prophylactic effect in humans when administered quarterly [26].
Importantly, moxidectin, a member of the macrocyclic lactone family of anthelmintic drugs, also used in veterinary medicine like ivermectin [20], was recently approved for the treatment of onchocerciasis as a microfilaricidal drug in individuals over the age of 12 [20]. In humans, a single dose of moxidectin (8 mg) appeared to be more efficacious than a single dose of ivermectin (150 μg/kg) in terms of lowering microfilarial loads [17]. Modeling has shown that an annual treatment with moxidectin and a biannual treatment with ivermectin would achieve similar reductions in the duration of the MDA programs when compared to an annual treatment with ivermectin [27].
In our efforts to identify macrofilaricidal drugs, we tested a selection of drugs for their ability to inhibit the molting of O. volvulus L3 to L4 as part of the in vitro drug screening funnel [13,28-31]. With some being highly effective, we decided to also examine the effects of the known MDA drugs and those already in clinical development for macrofilaricidal effects on molting of L3 and the motility of L4 (S1 Text) as potential "prophylactic" drugs. When ivermectin and moxidectin were evaluated, we found that both drugs were highly effective as inhibitors of molting: IC 50  Interestingly, when the treatment of L4 with both drugs was prolonged, the IC 50 values for the inhibition of L4 motility on day 11 with ivermectin and moxidectin were 0.444 μM and 0.380 μM, respectively. Significantly, from the prospect of employing both drugs for prophylaxis against new infections with O. volvulus, moxidectin (8 mg) has an advantage as it achieves a maximum plasma concentration of 77.2 ± 17.8 ng/ml, is metabolized minimally, and has a half-life time of 40.9 ± 18.25 days with an area under the curve (AUC) of 4,717 ± 1,494 ng � h/ ml in healthy individuals [32], which covers the experimental IC 50 achieved by moxidectin for inhibiting both L3 molting and L4 motility, and the IC 90 for L3s. In comparison, ivermectin reaches a maximum plasma concentration of 54.4 ± 12.2 ng/ml with a half-life of 1.5 ± 0.43 days and an AUC of 3,180 ± 1,390 ng � h/ml in healthy humans [33], which only covers the IC 50 for inhibiting molting of L3 and motility of L4. We therefore reason that based on the significantly improved pharmacokinetic profile of moxidectin and its efficacy against both L3 and L4 larvae in vitro (Table 1), it might have a better "prophylactic" profile than ivermectin for its potential to interrupt the development of new O. volvulus infections, and thus ultimately affect transmission and further support the elimination of onchocerciasis. Adding to moxidectin's significance, in dogs, it is a highly effective prophylactic drug against ivermectin-resistant D. immitis strains [19], an important attribute in the event that a suboptimal responsiveness to ivermectin treatment becomes more widespread in the onchocerciasis endemic regions of Africa. Testing the potential effect of moxidectin on the viability or development of transmitted L3 larvae was already recommended by Awadzi and colleagues in 2014 [34], when the excellent half-life of moxidectin in patients with onchocerciasis was realized. We have to acknowledge, however, that the key parameters that can predict the potency of a drug is actually a combination of exposure (drug concentrations) at the site of action and the duration of that exposure that is above the determined IC 50 /IC 90 . As we have access to only the AUC, halflife, and Cmax data for each of the in vitro-tested drugs, the use of plasma concentrations for predicting the anticipated potency of these putative "prophylactic" drugs in vivo has to be further assessed with care during clinical trials.
The prospects for identifying additional "prophylactic" drugs against O. volvulus increased when we tested 3 other drugs: albendazole, already in use for controlling helminth infections in humans; and oxfendazole and emodepside, being tested by the Drugs for Neglected Diseases initiative (DNDi) as potential repurposed macrofilaricidal drugs for human indications [21]. O. volvulus L3 obtained from infected Simulium sp. were washed and distributed at n = approximately 10 larvae per well and cocultured in contact with naïve human peripheral blood mononuclear cells for a period of 6 days with or without the respective drugs in vitro (S1 Text) and as previously described [13,30]. Ivermectin   S1 Fig), which is also more potent than the other drugs. In the ascending dose (1 to 40 mg) human clinical trial (NCT02661178), emodepside achieved a maximum plasma concentration in the range of 18.6 to 595 ng/ml, AUC of 100 to 4,112 ng � h/ml, and half-life of 1.7 to 24.6 days depending on the dose administered, and all doses were well-tolerated (Table 1) [43]. Considering that the IC 90 for inhibition of L3 molting and L4 motility in vitro are 2 nM and 78 nM (Table 1 and S1 Fig), respectively, these values are already covered by the PK profile of the drug starting at 2.5 mg. Hence, the clinical trials for emodepside as a macrofilaricidal drug, if efficacious at 2.5 mg or above, could have additional implications in terms of utilizing emodepside for prophylactic potential.
We propose that all 5 drugs are effective against the early stages of O. volvulus based on their efficacy (IC 50 /IC 90 ) in vitro. However, based on their known pharmacokinetic profiles in humans, they can be prioritized for future evaluation for their utility for prophylactic activity in humans as follows: emodepside > moxidectin > albendazole > oxfendazole > ivermectin.
Moreover, we believe that the addition of some of these putative "prophylactic" drugs individually or in combination with the current MDA regimens against onchocerciasis would also align well with the integrated goals of the Expanded Special Project for Elimination of Neglected Tropical Diseases and possibly also expedite the elimination goals of one of the other 6 neglected tropical diseases amenable to MDA: the STH [44]. All 5 of these drugs are broad-spectrum anthelmintic drugs that are effective against STH infections [45][46][47][48][49], and thus may also benefit MDA programs aimed at controlling STH infections. The effects of MDA with ivermectin or albendazole on STHs (hookworms, Ascaris lumbricoides, and Trichuris trichiura) have already been explored in clinical studies [45,47,50] and were shown to have a significant impact on the STH infection rates in the treated communities. One dose of moxidectin (8 mg) in combination with albendazole (400 mg) was as effective as a combination of albendazole and oxantel pamoate (currently the most efficacious treatment against T. trichiura) in reducing fecal T. trichiura egg counts [46]. Notably, oxfendazole is also being tested for its effectiveness in humans against trichuriasis (NCT03435718). Additionally, emodepside was shown to not only have a strong inhibitory activity against adult STH worms in animal models with an ED 50 of less than 1.5 mg/kg, but also against STH larval stages in vitro with IC 50 <4 μM for L3s [49].
We could envision that a single drug, a combination of any of these 5 drugs, or just those we have prioritized (moxidectin and emodepside), when administered also for prophylaxis against the development of new O. volvulus infection, would also protect against new STH infections. Broad-spectrum chemoprophylaxis of nematode infections in humans could potentially also save on costs and time invested toward elimination of co-endemic parasites through the administration of a combination of drugs. Moreover, considering the time-consuming process of drug discovery, the heavy costs incurred, and the excessive failure rates, the prospect of repurposing commercially available drugs used for other human or veterinary diseases for the prophylaxis of O. volvulus infection is an attractive one [31, [51][52][53][54]. Repurposing of drugs could also accelerate the approval timeline for new drug indications since information regarding mechanism, dosing, toxicity, and metabolism would be readily available.
In summary, our O. volvulus in vitro drug testing studies reinforce the "old" proposition of employing MDA drugs for prophylactic strategies as well, inhibiting the development of new infections with O. volvulus in the endemic regions under MDA. We report for the first time that in vitro, emodepside, moxidectin, and ivermectin have very promising inhibitory effect on both L3s and L4s, with albendazole and oxfendazole for additional consideration. Importantly, considering that the L4 larvae are longer lived as compared to the L3 stage, and hence the more feasible target against the establishment of new infections, we believe that targeting the L4 stage would be an invaluable tool toward advancing sustainable elimination goals for onchocerciasis. Moxidectin and emodepside with their superior half-life and pharmacokinetic profiles in humans and their efficacy in vitro against both L3 and L4 stages of the parasite seem to show the most promise for this purpose. Of significance, the doses required to provide exposures that would cover the IC 90 achieved by these 2 drugs in vitro against L3 and emodepside against L4 have been shown to be well-tolerated in humans (Table 1). Crucially, as these new drugs are rolled out for human use as microfilaricidal and/or macrofilaricidal drugs, it would be important to add to the clinical protocols to also observe their effects on the development of new infections in populations that are exposed to active transmission using serological assays that can predict new infections and distinguish them from earlier infections [55]. This could potentially reveal valuable information to foster the development of more complementary elimination programs that not only target the microfilariae (moxidectin) and the adult worms (emodepside) but also the other infectious stages of the parasite, with their effects on STH being an added advantage.
Mathematical modeling has long influenced the design of intervention policies for onchocerciasis and predicted the potential outcomes of various regimens used by the elimination programs and the feasibility of elimination [56][57][58][59][60]. We believe that a revised mathematical model that also takes into account the additional aspect of targeting L3 and L4 stages could be helpful to assess the enhanced impact this complementary tool might have in advancing the goal of elimination, and accordingly support a revised policy for operational intervention programs first for onchocerciasis, and perhaps also as a pan-nematode control measure, by the decision-making bodies [7, 61,62]. Given that in human clinical trials in which infected people were treated quarterly with ivermectin, there was an indication of a considerable trend of reduced number of newly formed nodules, it becomes apparent that the recommendation for such a revised regimen might also support protection from new infections. Clinical trials to assess the efficacy of biannual doses of ivermectin or moxidectin versus annual doses of these drugs against onchocerciasis have been already initiated (NCT03876262). Alternatively, increasing the frequency of future treatments with moxidectin and/or emodepside to biannual or quarterly treatment and/or using them in combinations could also improve their chemotherapeutic potential by targeting multiple stages of the parasite, thus increasing all the control potential of these new MDA drugs on multiple stages of the parasite and ultimately support not only a faster timeline but also sustained elimination.