RESEARCH ARTICLE Efficacy of subcutaneous doses and a new oral amorphous solid dispersion formulation of flubendazole on male jirds (Meriones unguiculatus) infected with the filarial nematode Brugia pahangi Chelsea Fischer1, Iosune Ibiricu Urriza1, Christina A. Bulman1, KC Lim1, Jiri Gut1, a1111111111 Sophie Lachau-Durand2, Marc Engelen2, Ludo Quirynen2, Fetene Tekle2, Benny Baeten2, 3 4 1 a1111111111 Brenda Beerntsen , Sara Lustigman , Judy SakanariID * a1111111111 a1111111111 1 Dept. of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, California, United States of America, 2 Janssen R&D, Janssen Pharmaceutica, Beerse, Belgium, 3 Veterinary a1111111111 Pathobiology, University of Missouri-Columbia, Columbia, Missouri, United States of America, 4 Laboratory of Molecular Parasitology, Lindsley F. Kimball Research Institute, New York Blood Center, New York, New York, United States of America * [email protected] OPEN ACCESS Citation: Fischer C, Ibiricu Urriza I, Bulman CA, Lim K, Gut J, Lachau-Durand S, et al. (2019) Efficacy of Abstract subcutaneous doses and a new oral amorphous solid dispersion formulation of flubendazole on River blindness and lymphatic filariasis are two filarial diseases that globally affect millions male jirds (Meriones unguiculatus) infected with of people mostly in impoverished countries. Current mass drug administration programs rely the filarial nematode Brugia pahangi. PLoS Negl on drugs that primarily target the microfilariae, which are released from adult female worms. Trop Dis 13(1): e0006787. https://doi.org/10.1371/ journal.pntd.0006787 The female worms can live for several years, releasing millions of microfilariae throughout the course of infection. Thus, to stop transmission of infection and shorten the time to elimi- Editor: Roger K. Prichard, McGill University, CANADA nation of these diseases, a safe and effective drug that kills the adult stage is needed. The benzimidazole anthelmintic flubendazole (FBZ) is 100% efficacious as a macrofilaricide in Received: February 9, 2018 experimental filarial rodent models but it must be administered subcutaneously (SC) due to Accepted: August 26, 2018 its low oral bioavailability. Studies were undertaken to assess the efficacy of a new oral Published: January 16, 2019 amorphous solid dispersion (ASD) formulation of FBZ on Brugia pahangi infected jirds (Mer- Copyright: © 2019 Fischer et al. This is an open iones unguiculatus) and compare it to a single or multiple doses of FBZ given subcutane- access article distributed under the terms of the ously. Results showed that worm burden was not significantly decreased in animals given Creative Commons Attribution License, which oral doses of ASD FBZ (0.2±15 mg/kg). Regardless, doses as low as 1.5 mg/kg caused permits unrestricted use, distribution, and reproduction in any medium, provided the original extensive ultrastructural damage to developing embryos and microfilariae (mf). SC injec- author and source are credited. tions of FBZ in suspension (10 mg/kg) given for 5 days however, eliminated all worms in all Data Availability Statement: All relevant data are animals, and a single SC injection reduced worm burden by 63% compared to the control within the paper and its Supporting Information group. In summary, oral doses of ASD formulated FBZ did not significantly reduce total files. worm burden but longer treatments, extended takedown times or a second dosing regimen, Funding: JS received partial funding from Janssen may decrease female fecundity and the number of mf shed by female worms. Pharmaceutica (http://www.janssen.com/) (Award Number A126468-333023). SLD, ME, LQ, FT and BBa are employed by Janssen Pharmaceutica and were involved in the study design, pharmacokinetic analysis, and manuscript preparation for PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0006787 January 16, 2019 1 / 16 Brugia pahangi infected jirds treated with oral ASD FBZ Experiments 1 and 2. JS received partial funding from the Bill & Melinda Gates Foundation (https:// Author summary www.gatesfoundation.org/) (Award Number A115808). This funder had no role in study design, Safe and effective macrofilaricidal drugs are critically needed to treat onchocerciasis and data collection and analysis, decision to publish, or lymphatic filariasis, which affect over 54 million people worldwide. Flubendazole (FBZ) in preparation of the manuscript. its current commercial formulations is an effective anthelminthic for intestinal soil trans- Competing interests: I have read the journal's mitted helminth (STH) infections but not for filarial infections due to its low bioavailabil- policy and the author of this manuscript have the ity. The purpose of this study was to assess the efficacy of a new amorphous solid following competing interests: SLD, ME, LQ, BBa dispersion (ASD) formulation of FBZ given orally to jirds (Meriones unguiculatus) and FT are employed by Janssen Pharmaceutica. infected with the filarial nematode Brugia pahangi and compare it to FBZ (in suspension) JS received funding from Janssen. given subcutaneously as a single or multiple dose. Our results indicated that treatment with ASD FBZ did not significantly reduce the total number of worms. However, doses as low as 1.5 mg/kg caused ultrastructural damage to the early stages of developing embryos. No worms were recovered from jirds given 10 mg/kg SC injections of FBZ (suspension) for 5 days when necropsied 68 days after the first-dose, and the number of mf at necropsy was significantly decreased compared to the control group. Animals given only a single 10 mg/kg SC injection had a 63% decrease in the number of adult worms. These animals also had fewer female worms and mf compared to the control group suggesting that even a sin- gle SC injection of FBZ had an effect on female survival and fecundity. TEM of worms recovered from jirds given low doses of the ASD formulated FBZ (1.5 mg/kg) suggested that female fecundity and mf production were reduced, but longer treatments, longer takedown times or a second dosing regimen of ASD FBZ may be needed to significantly decrease the total worm burden. Introduction River blindness (onchocerciasis) and lymphatic filariasis are two major neglected diseases caused by parasitic nematodes that together affect an estimated 54 million people worldwide in mostly poor, developing countries. With river blindness, approximately 12 million people suffer from skin disease and 1 mil- lion people have vision loss [1]. It is a chronic disease caused by the first larval stage, microfi- lariae (mf) of Onchocerca volvulus which are released from female worms residing in subcutaneous tissues. Microfilariae migrate throughout the skin causing severe itchiness, and in the eye, they induce an inflammatory response that eventually leads to blindness [2±5]. Lymphatic filariasis (LF) or elephantiasis is caused by Wuchereria bancrofti, Brugia malayi and B. timori whose adult worms infect and damage the lymphatic tissues. This debilitating disease is characterized by pain and severe lymphedema often involving the arms, legs, breasts and genitalia, leading to great economic losses as well as social suffering and the stigma associ- ated with elephantiasis [2, 6]. To date, there are no vaccines for these diseases and international control programs attempt to interrupt transmission of infection in Africa with mass drug administration (MDA) annu- ally or biannually using microfilaricidal drugs: ivermectin for onchocerciasis; albendazole plus ivermectin or albendazole plus diethylcarbamazine (DEC) for LF. Recently a triple-drug ther- apy with ivermectin, DEC and albendazole was explored for the treatment of LF outside of Africa with the assumption that it will accelerate the elimination of LF if a coverage of >65% of the population is achieved [7]. Notably, this new therapy significantly improves mf clear- ance and maintenance of amicrofilaremia compared to the two-drug MDA regimen with DEC and albendazole and offers great promise in eliminating LF [7, 8]. PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0006787 January 16, 2019 2 / 16 Brugia pahangi infected jirds treated with oral ASD FBZ The triple-drug therapy however, is not relevant for treatment of onchocerciasis due to the major adverse affects caused by DEC [9]. Although elimination of onchocerciasis has been achieved in a few foci in Africa and in the Americas [10±12], there has been only a 31% reduc- tion in the incidence of onchocerciasis in Africa since 1995 [13]. The African Program for Onchocerciasis Control (APOC) has therefore called for some 1.15 billion treatments by 2045 [14], though many neglected tropical disease experts doubt that onchocerciasis can ever be eliminated through MDA alone [15], especially given that MDA with ivermectin cannot be used in 11 Central African countries co-endemic with Loa loa infections due to the risk of severe adverse events [16, 17]. Given the longevity and high fecundity of the adult worms (macrofilariae) [18±22] and the current lack of macrofilaricidal drugs, it is unlikely that the WHO goal of eliminating onchocerciasis by 2025 will be met when microfilaricidal drugs alone are used [17, 23, 24]. To achieve the ultimate goal of onchocerciasis elimination, drugs that cure infections and thus stop transmission of infection and ultimately shorten the time to elimination, a safe and effective drug that kills adult worms is needed. The need for such alternate treatment strategies is further