Safety, tolerability and pharmacokinetics of emodepside, a potential novel treatment for onchocerciasis (river blindness), in healthy male subjects

Aims Emodepside is an anthelmintic, originally developed for veterinary use. We investigated in healthy subjects the safety, and pharmacokinetics of a liquid service formulation (LSF) and immediate release (IR) tablet of emodepside in 2 randomised, parallel‐group, placebo‐controlled, Phase I studies. Methods Seventy‐nine subjects in 10 cohorts in the single ascending dose study and 24 subjects in 3 ascending‐dose cohorts in the multiple ascending dose study were enrolled. Emodepside as LSF was administered orally as single 1–40‐mg doses and for 10 days as 5 or 10 mg once daily and 10‐mg twice daily doses, respectively. Pharmacokinetics and safety were assessed up to 21 and 30 days, respectively. In addition, IR tablets containing 5 or 20 mg emodepside were tested in the single ascending dose study. Results Emodepside as LSF was rapidly absorbed under fasting conditions, with dose‐proportional increase in plasma concentrations at doses from 1 to 40 mg. Terminal half‐life was > 500 hours. In the fed state, emodepside was absorbed more slowly but overall plasma exposure was not significantly affected. Compared to the LSF, the rate and extent of absorption was significantly lower with the tablets. Conclusions Overall, emodepside had acceptable safety and tolerability profiles, no major safety concerns, after single oral administration of 20 mg as LSF and after multiple oral administration over 10 days at 5 and 10 mg OD and at 10 mg twice daily. For further clinical trials, the development of a tablet formulation overcoming the limitations observed in the present study with the IR tablet formulation is considered.


| INTRODUCTION
Onchocerciasis (river blindness) is a neglected tropical disease caused by Onchocerca volvulus, a parasitic nematode transmitted to humans through the bite of the blackfly. 1 The larvae mature into reproductively competent adults within 1 year. Adult worms have a lifespan of 9-11 years and reside primarily in subcutaneous and deep-tissue nodules where they produce offspring (microfilariae), which migrate to the skin awaiting uptake by another blackfly. The disease results from the death of the microfilariae, which prompts an inflammatory response, causing skin rash and lesions, including skin depigmentation, and unbearable itching. Microfilariae also migrate to the eye, causing local inflammation and other complications, including eye lesions, often leading to blindness. 2 Onchocerciasis is endemic in 27 countries mainly in sub-Saharan Africa, as well as in Yemen and Latin America. 1 Onchocerciasis treatment and control currently rely on mass drug administration (MDA) of ivermectin (Mectizan, Merck & Co. Inc.), which targets the microfilarial stage of the parasite and temporarily sterilises, but does not kill, the adult worms. MDA programmes must therefore be repeated at regular intervals for many years, which represents a considerable economic and logistical burden in endemic countries. 3 There is also mounting evidence of potential resistance to ivermectin. 4 Another avermectin parasiticide, moxidectin, with prolonged efficacy compared to ivermectin was approved in 2018.
Like ivermectin, however, it targets only microfilariae. 5 Thus, there is an urgent need for new agents against onchocerciasis. Ideally, such agents should have activity against multiple lifestages of the parasite, a good safety profile and a long-lasting effect with a relatively simple dosing regimen. With ivermectin in place, it is not feasible to aim for replacement of current MDA drugs. However, a new macrofilaricidal drug would be an asset in focused MDA treatment or test-and-treat strategies for patients in endemic areas, where repeated ivermectin distribution is difficult or remains ineffective. In addition, the access to a macrofilaricidal drug for individual case management is an important goal for drug development in onchocerciasis.
The generic Target Product Profile of such new agents is available on the website of Drugs for Neglected Diseases initiative (www.dndi.org/ diseases/filariariverblindness/targetproductprofile/). Emodepside, a semi-synthetic cyclo-octadepsipeptide, is active across multiple nematode species. Like ivermectin and moxidectin, 6,7 emodepside was originally developed as an anthelmintic for veterinary use. It was first marketed as Profender (Bayer AG, Leverkusen, Germany) in 2005, in combination with praziquantel, and subsequently as Procox (Bayer AG, Leverkusen, Germany), in combination with toltrazuril.
Because of its unique, although not fully elucidated mechanism of action relative to other anthelmintics, emodepside is active at various stages in the nematode life-cycle. 8 Emodepside interacts with SLO-1, 9 a calcium activated potassium channel, which finally results in flaccid paralysis of the parasites (inhibition of locomotion, feeding, egg-laying and slowed development). In gastrointestinal nematodes, it has been shown that emodepside also interacts with the g-protein coupled receptors latrophilin LAT-1, 10 which is responsible for the paralytic effects on the worm pharynx. 11 Preclinical pharmacology studies using in vitro and in vivo models of human filarial infections, including onchocerciasis, showed that emodepside was consistently active on parasites across several species and stages 12 and is thus a potential candidate for human use. It is clear from these examples, that the effect on microfilariae is expected to be related to C max , whereas macrofilaricidal effects require a certain time over threshold for efficacy, as is the case for other tissue dwelling parasites. Additional nonclinical pharmacology information for emodepside efficacy against gastrointestinal nematodes is available in the literature. [12][13][14][15][16] Also, emodepside meets criteria of the Target Product Profile for river blindness. Based on the evidence in animals, including favourable pharmacokinetics (PK) in various species and efficacy on filarial parasites, 15,16 evaluation of emodepside in human was considered in the perspective of developing it as a macro-and microfilaricidal treatment of onchocerciasis.
Here we report the basic PK including biopharmaceutical features and safety of emodepside in healthy male subjects after single (NCT02661178) and multiple (NCT03383614) oral doses as a liquid service formulation (LSF). As such a formulation would not be practical for use in the field in countries where river blindness is endemic, the safety and relative bioavailability of a standard immediate release tablet containing crystalline emodepside were also investigated. In addition, preliminary data on the effect of a standard Food and Drug Administration meal on the PK of emodepside are also described.
What is already known about the subject • Emodepside is an anthelmintic agent, currently registered for veterinary use in combination with other drug substances.
• Emodepside may have the potential to treat parasitic infections in humans, including onchocerciasis.

What this study adds
• No major safety concerns were identified in 103 healthy male subjects exposed to emodepside orally as a liquid service formulation up to 40-mg single dose and 10 mg twice daily for 10 days.
• Emodepside applied as liquid service formulation had a favourable pharmacokinetics profile with roughly doseproportional C max and AUC after up to 40-mg dose.
• Tissue distribution was relatively rapid, which, with a long terminal half-life, are expected to be beneficial for treatment of onchocerciasis.

| General
The study designs are presented in Table 1

| Investigational products
Emodepside and placebo were supplied as a LSF and a standard immediate-release (IR) tablet. The LSF was a 0.1% (w/v) solution con- Randomisation, using a predetermined randomisation list and investigational product (IP) allocation, was performed by research personnel not involved in any other study-related activity.

| Subjects
At screening, subjects were deemed healthy based on medical history, physical examination, electrocardiography, vital signs and laboratory tests. Key exclusion criteria included presence or history of severe allergies, recent use of any prescription medicine, blood loss >400 mL or participation in another clinical study in the past 3 months.
To preclude any dietary effects on the PK of emodepside, subjects in the fasting cohorts fasted for 9 hours before receiving the IP. Subjects in the fed cohort fasted for 10 hours prior to dosing and received a standard high-calorie, high-fat breakfast 30 minutes prior to dosing.

| PK analyses
Blood samples (5 mL) were collected by venepuncture or via a cannula into EDTA tubes and immediately placed on ice. Samples were centrifuged at 1500 g for 10 minutes at 4 C. The plasma was aliquoted into 2 polypropylene tubes, which were stored at −20 C. Blood samples for PK analysis were taken at the following time points: • SAD: predose and 0. were not used to calculate the PK parameters, except values that were below the limit of quantification before C max , which were set to zero.
The highest observed plasma concentration (C max ) was deter-

| Statistical analyses
Statistical analyses were performed using SAS version 9.3 (Cary, NC, USA). Demographic data and baseline characteristics were listed and summarised. Safety data did not undergo formal statistical analysis. PK parameters were derived from plasma concentration vs. time data using a noncompartmental analysis in Phoenix WinNonlin version 7 (Certara Inc., Princeton, NJ, USA). Plasma concentration vs. time data and PK parameters were listed, and summarised by treatment, using descriptive statistics. Mean concentrations were calculated only if at least 2 /3 of the individual concentrations were above the lower limit of quantification. Individual subject and mean plasma concentrations were displayed graphically.
Planned sampling times were used to summarise plasmaconcentration data; actual sampling times were used in the derivation of PK parameters.

| Nomenclature of targets and ligands
Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to Pharmacology.

| Subject disposition
Demographic characteristics were consistent across the 2 studies (Table 2). In the SAD study, 1 subject in the 1 mg LSF cohort was withdrawn from the study due to an AE. He received an incomplete dose of 0.1 mg emodepside in error. He was included in the safety assessment, but not the PK assessments. Only 5 of the planned 6 subjects were included in the 5-mg IR tablet cohort. In the MAD study, all subjects received the IP as intended. The initial doses were selected based on the predicted human PK and therapeutic dose, derived from data obtained from in vitro and in vivo studies after administration of emodepside to rats and dogs and from in vitro data on plasma-protein binding and blood-plasma partitioning.

| SAD and food effect study
Mean plasma emodepside concentration-time profiles are shown in Figure 1 and PK parameters are presented in Table 3. Across all doses and for both formulations, after single administration, emodepside concentrations were rapidly quantifiable in the plasma, starting with the first timepoint at 0.5 hours postdose. Median t max in subjects in the fasting state was shorter for the LSF than for the IR tablet. Exposure, based on C max and AUC 0-24 was dose-proportional with the LSF up to the 40-mg dose, but less than dose proportional with the IR tablet. The relative bioavailability of the tablet vs. the LSF was 35.0% for the 5-mg dose and 11.7% for the 20-mg dose (Table 4).
In the fed state, after a single 10-mg dose of the LSF, geometric mean C max and AUC 0-24 were lower and median t max was longer than after the same dose in the fasting state, indicating delayed absorption of emodepside; However, AUC 0-last was not statistically different in fed relative to fasting conditions (Table 3).
Geometric mean elimination t 1/2 at all dose levels and for both formulations was very long, while geometric mean t 1/2 during the first

| MAD study
Only LSF formulation was used in that study. Mean plasma emodepside concentration-time profiles are shown in Figure 2 and PK parameters are presented in Table 5. Rapid absorption of emodepside and the median t max seen in the SAD study were confirmed across all dosing groups and regimens in the MAD study.
Emodepside levels were still quantifiable in all subjects at the final sampling timepoint, 507 hours after the last morning dose, which was consistent with the findings in the SAD study.
C max and AUC increased in a dose-proportional manner after once daily (OD) dosing of 5 and 10 mg emodepside LSF. Exposure was higher after dosing with 10 mg twice daily (BID) than with 10 mg   (Table 5). Plasma concentrations declined from C max more rapidly during the 24 hours postdose than subsequently, again consistent with the findings in the SAD study.
The increase in plasma emodepside concentrations after the last dose on Day 10 was lower after 10 mg BID than after 10 mg OD, with C max /C trough ratios of 1.9 and 3.0, respectively. Although the total daily dose in the 10 mg BID group was double that in the 10 mg OD group on Days 1-9, geometric mean C max on Day 10 was only 1.2-fold higher in the 10 mg BID group than in the 10 mg OD group.

| Safety
Safety monitoring across all 2 studies did not identify any major con-   After oral administration of 10 mg emodepside as LSF in fed condition, absorption was delayed with a longer median t max compared to that after the same dose in fasting condition (2.5 vs 1.0 h), however food did not significantly alter total exposure as AUC 0-tlast were 3390 and 3070 h.ng/mL, respectively.

COMPETING INTERESTS
The authors declare that they have no conflicts of interest.
T A B L E 7 Treatment-emergent adverse events (TEAEs) reported with emodepside and placebo in the multiple ascending dose study, presented by system organ class  LSF: liquid service formulation; OD: once daily; BID: twice daily. Subjects with ≥1 adverse event are counted only once per system organ class and preferred term.