Gates Open Research Gates Open Research 2022, 6:62 Last updated: 28 SEP 2022 STUDY PROTOCOL An adaptive phase II/III safety and efficacy randomized controlled trial of single day or three-day fixed-dose albendazole-ivermectin co-formulation versus albendazole for the treatment of Trichuris trichiura and other STH infections. ALIVE trial protocol [version 1; peer review: 2 approved] Alejandro Krolewiecki 1,2, Wendemagegn Enbiale3,4, Javier Gandasegui 1, Lisette van Lieshout 5, Stella Kepha6, Augusto Messa Junior 7, Michel Bengtson5, Woyneshet Gelaye 3, Valdemiro Escola 7, María Martinez-Valladares8,9, María Cambra-Pellejà 8,9, Jaime Algorta10, Helena Martí-Soler1, Pedro Fleitas1,2, Maria Rosa Ballester 11-13, Stephen R. Doyle 14, Nana Aba Williams1, Almudena Legarda 1, Inácio Mandomando 7, Charles Mwandawiro6, José Muñoz1 1Barcelona Institute for Global Health (ISGlobal), Hospital Clínic—Universitat de Barcelona, Barcelona, Spain 2Instituto de Investigaciones de Enfermedades Tropicales, Universidad Nacional de Salta, Oran, Salta, 4530, Argentina 3College of Medicine and Health Science, Bahir Dar University, Bahir Dar, Ethiopia 4Department of Dermatology,, Amsterdam Institute for Infection and Immunity, Academic Medical Centre, Amsterdam, The Netherlands 5Department of Parasitology, Centre of Infectious Diseases, Leiden University Medical Centre (LUMC), Leiden, The Netherlands 6Eastern and Southern Africa Centre of International Parasite Control, Kenya Medical Research Institute (KEMRI), Nairobi, Kenya 7Centro de Investigação em Saúde de Manhiça (CISM), Maputo, Mozambique 8Instituto de Ganadería de Montaña, CSIC-Universidad de León, Grulleros, León, Spain 9Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad de León, Campus de Vegazana, León, Spain 10Liconsa SA, Azuqueca de Henares, Guadalajara, Spain 11Institut d’Investigació Biomèdica Sant Pau (IIB SANT PAU), Barcelona, Spain 12Faculty of Health Sciences Blanquerna,, University Ramon Llull, Barcelona, Spain 13Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM), Barcelona, Spain 14Wellcome Sanger Institute, Hinxton, Cambridgeshire, UK v1 First published: 05 May 2022, 6:62 Open Peer Review https://doi.org/10.12688/gatesopenres.13615.1 Latest published: 05 May 2022, 6:62 https://doi.org/10.12688/gatesopenres.13615.1 Approval Status 1 2 Abstract Background: Soil-transmitted helminths (STH) are targeted for version 1 control through mass drug-administration campaigns to prevent 05 May 2022 view view morbidity affecting at-risk groups in endemic regions. Although broadly successful, the use of albendazole and mebendazole achieved Page 1 of 18 Gates Open Research Gates Open Research 2022, 6:62 Last updated: 28 SEP 2022 variable progress, with deficiencies against Trichuris trichiura and a predictable low efficacy against Strongyloides stercoralis. Novel drug 1. Antonio Montresor, World Health combinations offer a potential solution, providing they can be Organization, Geneva, Switzerland delivered safely and maintain efficacy against all STH species. Here we present the protocol of a clinical trial to evaluate a fixed-dose 2. Kevin Kobylinski , Armed Forces Research combination (FDC) tablet containing albendazole and ivermectin that Institute of Medical Sciences, Bangkok, will be compared against albendazole against STH. Methods: An adaptive phase II/III randomized controlled trial will be Thailand undertaken in STH endemic sites in Ethiopia, Kenya and Mozambique Any reports and responses or comments on the to evaluate an oral FDC of 400 mg albendazole and either 9- or 18 mg ivermectin. FDC will be administered as a single dose or single doses article can be found at the end of the article. over three-consecutive days and assessed against a single dose of 400 mg albendazole. In the phase II trial, 126 T. trichiura-infected children weighting 15 to 45 kg will be treated in a dose-escalation manner to determine safety objectives. In the phase III trial, 1097 participants aged 5 to 18 years old infected with T. trichiura, hookworm and S. stercoralis will be recruited to determine safety and efficacy. The trial will be open-label with blinded outcome assessors. Cure rate measured 21-days after-treatment in duplicate Kato-Katz is the primary efficacy outcome. Secondary objectives include efficacy evaluation by quantitative polymerase chain reaction (PCR) as an outcome measurement, description of pharmacokinetic parameters, palatability and acceptability evaluations, and monitoring of anthelmintic resistance. Conclusions: This trial with registrational goals seeks to evaluate an innovative fixed-dose combination of albendazole and ivermectin co- formulated tablets, with the goal of providing an anthelmintic regimen with improved efficacy and spectrum of coverage against STH. ClinicalTrials.gov registration: NCT05124691 (18/11/2021). Keywords Antihelmintics, STH, Trichuris trichiura, Strongyloides, Hookworm, Albendazole, Ivermectin Page 2 of 18 Gates Open Research Gates Open Research 2022, 6:62 Last updated: 28 SEP 2022 Corresponding author: Alejandro Krolewiecki (alekrol@hotmail.com) Author roles: Krolewiecki A: Conceptualization, Funding Acquisition, Methodology, Supervision, Writing – Original Draft Preparation, Writing – Review & Editing; Enbiale W: Conceptualization, Project Administration, Supervision, Writing – Review & Editing; Gandasegui J: Formal Analysis, Investigation, Methodology, Writing – Original Draft Preparation, Writing – Review & Editing; van Lieshout L: Conceptualization, Methodology, Supervision, Writing – Review & Editing; Kepha S: Investigation, Methodology, Project Administration, Supervision, Writing – Review & Editing; Messa Junior A: Investigation, Methodology, Writing – Review & Editing; Bengtson M: Data Curation, Methodology, Project Administration, Software, Supervision, Validation, Writing – Review & Editing; Gelaye W: Investigation, Methodology, Writing – Review & Editing; Escola V: Investigation, Project Administration, Supervision, Writing – Review & Editing; Martinez-Valladares M: Conceptualization, Methodology, Supervision, Validation, Writing – Review & Editing; Cambra-Pellejà M: Investigation, Methodology, Writing – Review & Editing; Algorta J: Conceptualization, Investigation, Supervision, Writing – Review & Editing; Martí-Soler H: Formal Analysis, Methodology, Resources, Writing – Review & Editing; Fleitas P: Data Curation, Formal Analysis, Methodology, Software, Writing – Original Draft Preparation, Writing – Review & Editing; Ballester MR: Methodology, Writing – Review & Editing; Doyle SR: Conceptualization, Methodology, Supervision, Writing – Review & Editing; Williams NA: Project Administration, Resources, Validation, Writing – Review & Editing; Legarda A: Data Curation, Resources, Software, Supervision, Validation, Writing – Review & Editing; Mandomando I: Conceptualization, Supervision, Writing – Review & Editing; Mwandawiro C: Conceptualization, Supervision, Writing – Review & Editing; Muñoz J: Conceptualization, Funding Acquisition, Investigation, Methodology, Supervision, Writing – Review & Editing Competing interests: Jaime Algorta is employee of Laboratorios Liconsa SA, an organization of the STOP consortium. Grant information: This project is funded by the EDCTP2 program supported by the European Union (grant number RIA2017NCT-1845 –STOP and PSIA2020-3072 STOP-2; www.stoptheworm.org) Horizon 2020 European Union Funding for Research and Innovation. For the purpose of Open Access, the author has applied a CC BY public copyright license to any Author Accepted Manuscript version arising from this submission. MC-P is funded by Junta de Castilla y León and Fondo Social Europeo (grant number LE-135-19). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Copyright: © 2022 Krolewiecki A et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. How to cite this article: Krolewiecki A, Enbiale W, Gandasegui J et al. An adaptive phase II/III safety and efficacy randomized controlled trial of single day or three-day fixed-dose albendazole-ivermectin co-formulation versus albendazole for the treatment of Trichuris trichiura and other STH infections. ALIVE trial protocol [version 1; peer review: 2 approved] Gates Open Research 2022, 6:62 https://doi.org/10.12688/gatesopenres.13615.1 First published: 05 May 2022, 6:62 https://doi.org/10.12688/gatesopenres.13615.1 Page 3 of 18 Gates Open Research 2022, 6:62 Last updated: 28 SEP 2022 Introduction reviews as one that combines adequate safety, lack of signifi- Neglected tropical diseases (NTDs) are a group of infectious cant drug-drug interactions and importantly, efficacy against and non-infectious diseases that affect pediatric and adult popu- all STH, including both T. trichiura and S. stercoralis12–15. Due lations in the world’s poorest communities1. Among the NTDs to the different mechanisms of action of both drugs, this combi- that cause significant global morbidity and mortality, soil- nation has a theoretical lower risk for the selection of resistant transmitted helminths (STH) infections (Ascaris lumbricoides, parasites than a single drug treatment. Trichuris trichiura, and the hookworms Ancylostoma duodenale and Necator americanus) are associated with malnutrition, In the search for safe, efficacious and logistically simple to impaired growth and cognitive development in children2. It has distribute and administer therapeutic alternatives, the “Stop been estimated that almost 900 million people were infected Transmission of intestinal Parasites” (STOP) Project Consortium with at least one of these STH species in 2017, correspond- has developed a public-private international collaboration for ing to 1.7 million years lived with disability (YLDs) attribut- the design and validation of innovative products and strategies able to STH infection3. Strongyloides stercoralis, also a STH for the control of STH as a public health problem16. The study but with distinctive biological and life-history features, has been protocol presented in this report summarizes the progress and traditionally excluded from control initiatives but has recently achievements leading to the registrational clinical trial that aims been included in the World Health Organization (WHO) tar- to evaluate the efficacy and safety of an ALB-IVM fixed-dose gets for 20304,5. It is estimated that over 350 million people combination (FDC), co-formulated for the treatment of STH. are infected by S. stercoralis worldwide6. The current WHO mandated control strategy for STH emphasizes morbidity control Study rationale and hypothesis through mass drug administration (MDA) of benzimidazole-class We hypothesize that a FDC of 400 mg ALB together with anthelmintics – primarily albendazole (ALB) or mebendazole 9 mg or 18 mg IVM (the specific dose chosen according to (MEB) – targeting pre-school and school-aged children (SAC), body weight), as a single dose or three-day regimen, will be women of childbearing age and adults in certain high-risk more effective against STH compared to the current strategy occupations7. Treatment with ALB or MEB is given as a single of a single dose of 400 mg ALB while maintaining an excellent dose once or twice a year (depending on the estimated safety profile. To test this hypothesis, we designed an adap- prevalence of STH in the community) and both have excellent tive phase II/III superiority trial (called the ALIVE trial) that safety profiles7. With this approach, the global target is to achieve will primarily target safety objectives in the phase II component elimination of STH as a public health problem in 96% of the and safety and efficacy in the phase III component. 101 currently endemic countries by 20308. Despite the use of registered drugs for the medical indication While improvements in water and sanitation, and mass adminis- under evaluation, the rationale for a phase II safety component tration of benzimidazoles remain the cornerstone for reducing is based on the use of a novel co-formulation and the conse- STH morbidity, there are increasing concerns that the ongoing quent administration of a higher dose of IVM per kg than what success of these programs may be hindered, among other is currently approved. These co-formulations consist of 9 mg or reasons, by the reliance on a single class of drug9. A recent 18 mg IVM with 400 mg ALB; and with these two formulations systematic review and network meta-analysis identified worrisome trial participants between 15 and 90 kg of body weight would be efficacy indicators against T. trichiura (30.7% CR, and 49.9% treated with a dose of IVM between 200 μg/kg and 600 μg/kg ERR), with even lower efficacy in more recent studies, fall- (Figure 1). Preliminary studies, including a systematic review ing from 38.6% in 1995 to 16.4% in 2015; for hookworm (79.5 and meta-analysis of the safety of high dose IVM, identi- CR and 89.6 ERR) the indicators were more satisfactory and fied no concerns despite the limitations of the available data17, for A. lumbricoides efficacy remained adequate (95.7 CR and and a study on high fixed-dose IVM in healthy adult volunteers 98.5 ERR)10. Neither ABZ nor MEB in single doses have any contributed data on safety and pharmacokinetic parameters18. significant activity against S. stercoralis4. In parallel, there is Moreover, a trial of the co-administration of 400 mg ALB and growing concern that widespread monotherapy could lead to 600 μg/kg IVM against T. trichiura infection in Honduran selection pressures resulting in the emergence of drug resistant children reported non-significant safety findings (and a promising populations of parasites surviving treatment. Although there is efficacy response)15. Concerning this FDC, a study conducted little evidence to suggest this is a problem for STH control in healthy adult volunteers with the aim of characterizing now, there is clear and widespread evidence of drug resistance its pharmacokinetic profile, showed that the FDC was well in helminths of veterinary importance; therefore, represents a tolerated, with no safety concerns (submitted for publication). credible risk to global STH control programs in the near future11. While the overall objective of the ALIVE trial is to identify a Considering the low efficacy of treatment against some STH therapeutic approach with superior efficacy against STH as a species together with the risk of anthelmintic resistance by group, the study design focuses on those species with subop- reliance on a monotherapy, a combination therapy using existing timal (hookworms), poor (T. trichiura) or no (S. stercoralis) drugs with complementary but distinct modes of action is a responses to benzimidazoles in monotherapy but not on potential solution. The combination of ALB and ivermectin A. lumbricoides, which is currently adequately treated with ALB (IVM) has been identified in different trials and systematic or MEB monotherapy19. Page 4 of 18 Gates Open Research 2022, 6:62 Last updated: 28 SEP 2022 Figure 1. Flow diagram of the phase II & III trials, including participant sample size and randomization in three treatment study arms, dose escalation in phase II based on weight stratification, and targeted soil-transmitted helminth (STH) species in both trials. Abbreviations: ALB, albendazole; FDC, fixed-dose albendazole-ivermectin combination; DSMB, Data Safety Monitoring Board. Protocol System in the area which covers the entire district (2,380 km2), Adaptive phase II/III randomized, superiority trial to com- with a population of approximately 204,000 inhabitants under pare the safety and efficacy of the active control arm (current surveillance, 44% of which are <15 years of age. Every house- standard of care, single dose of 400 mg ALB) against two hold from the study area is geo-localized and each individual experimental arms (single day or three-day single dose FDC has a unique an identification number22. The district has 93 of ALB+IVM) will be performed in pediatric and young primary and 9 secondary schools with a student population adult populations in Bahir Dar (Ethiopia), Kwale (Kenya) and of 72,829. The area has been identified to harbor areas with Manhiça (Mozambique). significant prevalence of STH, including S. stercoralis23. Study sites Objectives and endpoints In Kenya, the study will take place in Kwale county situ- Phase II component. The phase II component of the trial ated on the south coast, bordering Tanzania to the South-West. will be held in Kwale, Kenya. The primary objective of this The total area of the county is approximately 8,270 km2 with phase is an open label evaluation of the safety of the FDC as a a population of about 760,897. The southern region of the single dose or a single dose delivered on three consecutive county is on the shoreline of the Indian Ocean. The area has days for the treatment of T. trichiura in participants between been identified as a hotspot for infections with T. trichiura and 15 and 45 kg of body weight. Secondary objectives include: hookworms despite MDA campaigns through the National (i) measuring the efficacy of treatment against T. trichiura as School-based Deworming Programme (NSBDP), active since well as other STH species in those subjects co-infected with the 201220. The full recruitment for the phase II trial will be target parasites, (ii) the pharmacokinetics analysis, (iii) accept- performed at this site, as well as part of phase III. ability and palatability evaluations of the FDC. The efficacy data from the participants of the phase II component will be In Ethiopia, the study will be conducted in the Bahir Dar incorporated into the efficacy data of the phase III component Zurya woreda (district) at a height of 1,900 m above sea level, for a joint analysis. in the West Gojam zone of the Amhara region, which has a population of approximately 230,000 people. The district Phase III component. The phase III component of the trial will has 36 primary (grade 1 to 8) and six secondary (grade 9 and be held in all three study sites. The primary objective of the 10) schools. The student population is 49,013 with an aver- Phase III trial is to evaluate the efficacy assessed as CR rate at age of 1,160 students in each school. The area has a high day-21 post-treatment by Kato-Katz of the above-mentioned prevalence of hookworm and S. stercoralis infections21. experimental and control arms used in the phase II for the treatment of T. trichiura in pediatric and young adult The Manhiça district in Southern Mozambique, a peri-urban populations. Secondary objectives include: (i) determining the setting with a predominantly young population, will be the CR against hookworms and S. stercoralis and ERRs of T. trichiura third site; it runs a Health and Demographic Surveillance and hookworms determined by stool microscopy, (ii) evaluation Page 5 of 18 Gates Open Research 2022, 6:62 Last updated: 28 SEP 2022 by quantitative polymerase chain reaction (qPCR) of parasite C. Treatment Arm 3: Daily dose of FDC tablet for three specific DNA in calculating the primary outcome measurement consecutive days: (efficacy) compared to stool microscopy, and (iii) evaluation of the frequency of known ALB resistant alleles in hookworm • F  or participants <45 kg of body weight at baseline: FDC and T. trichiura in the three treatment arms before and after of 400 mg ALB + 9 mg IVM treatment. Efficacy against A. lumbricoides is included as an • F  or participants ≥45 kg of body weight at baseline: FDC exploratory objective. of 400 mg ALB + 18 mg IVM Reporting of the study protocol has been verified in accord- Recruitment to both phase II and phase III components of the ance with the SPIRIT (Standard Protocol Items for trial will be done at school and community levels through invi- Randomized Trials) recommendations24. tations by the local teams to group meetings, followed by indi- vidual meetings with parents willing to participate. Parents of Trial design children participating will be explained the process of informed Phase II component. The phase II component is a randomized, consent, which will be completed by signing the informed controlled, parallel-group, open-label (outcome assessor blinded), consent form and written assent for participants 12 to 17 dose-escalation, trial. Participants (n=126) will be stratified by years old (Table 1). Allocation of participants to the study weight into three groups (Figure 1). Within each weight group, arms will be done by block randomization and stratified by there are three treatment arms, consisting of: (i) an active control STH species. We will ensure balanced allocation to the three arm receiving a single dose of 400 mg ALB, (ii) an experimen- arms in the three study countries. The randomization list will tal arm receiving a single dose FDC, and (iii) an experimental be generated with the R software by the trial statistician25,26. arm receiving three consecutive days of the single dose FDC. All participants will be allocated by simple randomiza- The main eligibility criteria include: (i) infections with T. trichiura tion to one of the three study arms with unequal probability for the phase II component and T. trichiura, hookworms (ALB: 20%, FDCx1: 40%, and FDCx3: 40%). The randomiza- and/or S. stercoralis for the phase III trial; (ii) participant tion list will be generated using R software (RRID:SCR_001905), age between 5 to 18 years-old, and (iii) participant weight package blockrand25,26 by the trial statistician. ≥15 kg and < 45 kg for the phase II component specific weight strata are described in Figure 1. Exclusion criteria include: Progression to the next weight group will be granted by the (i) treatment with benzimidazoles within the previous three Data Security and Monitoring Board (DSMB), provided months, (ii) pregnancy or first week postpartum, or (iii) the the safety stopping rules are not met. The predefined stop- active use of warfarin. ping rules that would result in suspension of dose escalation in the FDC arm are the occurrence of ≥1 serious adverse reac- Diagnostics tion having a reasonable possibility of a causal relationship At baseline and on day-21 (+/- 7 days), all participants will be with the study drug in any of the weight groups in phase II asked to provide a stool sample. The sample will subsequently and the overall assessment of the trial by the DSMB. Upon be divided into aliquots, which will be used for Kato-Katz thick evaluation of the safety data of the whole phase II study smears, modified Baermann, qPCR (ethanol preserved), resist- population, the DSMB will assess clearance for the phase III ance testing and back-up storage. Duplicate Kato-Katz thick component of the trial. smears (41.7 mg each) will be prepared for examination under a microscope for eggs of T. trichiura, A. lumbricoides and hook- Phase III component. The phase III component is a rand- worms by experienced technicians. For quality control, 10% of omized, multi-center, parallel-group, active-controlled, outcome the slides will be randomly selected and re-read by an independ- assessor blinded (laboratory personnel performing Kato Katz ent reader. Discrepant/inconsistent results will be considered; if and Baermann methods), superiority trial that will seek to there is a difference in presence/absence of a specific helminth recruit 1097 participants. These participants, together with the species; or if differences in egg counts exceed 10 eggs for Kato- 126 participants of the phase II trial, bring the total to 1223 trial Katz slides with ≤100 eggs or exceed 20% for Kato-Katz slides participants (Figure 1). with >100 eggs. If a discrepancy is detected between the first readings and the quality control (QC) reading, a third techni- The three treatments arms (Figure 1) consist of: cian will read the slide and that reading will be considered the valid one. Modified Baermann will be performed with 3 A.Treatment Arm 1: Single dose tablet of 400 mg ALB (active grams of stool samples in all samples for identification of the control arm). presence of S. stercoralis larvae27, and a second microscopist will be required to confirm the identification for QC. B. Treatment Arm 2: Single dose of the FDC tablet. Multiplex real-time quantitative PCR (qPCR) will be used • F  or participants <45 kg of body weight at baseline: FDC for the detection of parasite-specific DNA in stool28. The out- of 400 mg ALB + 9 mg IVM come of the qPCR is the cycle-threshold (Ct)-value, which is the PCR cycle in which the level of fluorescent signal represent- • F  or participants ≥45 kg of body weight at baseline: FDC ing DNA amplification exceeds the background fluorescence; the of 400 mg ALB + 18 mg IVM Ct value is inversely proportional to the DNA concentration of Page 6 of 18 Gates Open Research 2022, 6:62 Last updated: 28 SEP 2022 Table 1. Schedule of procedures for the phase II and III components of the ALIVE trial.. Screening Baseline Follow-up Enrolment/ Day 21+/- Pre-screening Screening Procedures Baseline Day 1 Day 2 Day 3 Day 7 7-day post (up to -3 months) Day -7 to -1 Day 0 treatment Informed consent/assent X X Stool analysis (Baermann and Kato-Katz) X X Inclusion and Exclusion criteria X X History & Physical exam X X X Urine Pregnancy test X Randomization and treatment X assignation Study drug administration single-day X arms Study drug administration Arm 3 X X X (FDCx3) Acceptability questionnaire single-day X arms * Acceptability questionnaire Arm 3 X (FDCx3) * Pharmacokinetics blood sampling * ** X X X Concomitant medication & disease X X X X X X X review Adverse event monitoring X X X X X X X Abbreviations: FDC, fixed-dose albendazole-ivermectin co-formulation. *: only for the phase II participants. **: Population pharmacokinetics: two timepoints per participant for ALB arm at 1, 2, 3, 4, 5, 6, 7, 8, or 24 h and for the single dose FDC arm at 1, 2, 3, 4, 5, 6, 7, 8, 24, 48 or 72 h; two timepoints for three-dose FDC arm at 1, 2, 3, 4, 5, 6, 7, 8, 24, 48 or 72h post-administration and one additional timepoint at pre-dose Day 3. the target, and therefore, can be used to quantify the amount of ALB will be provided as a chewable tablet (Eskazole 400 mg parasite-specific DNA present in the sample tested. In addition, tabets, Allen Pharmaceuticals, Spain). Both FDC and ALB will qPCR will allow the identification of hookworm species be taken after a light meal, followed by visual observation of (N. americanus and A. duodenale) to evaluate if there are the participant by the study physician at each site. A light meal differences in the response to treatment for these species. is considered to be the equivalent of a soft breakfast with a con- Molecular diagnostic testing in the context of the ALIVE trial tribution of 15 g of fat. Participants that vomit within one hour will include participation in the Helminth External Molecular after treatment will require re-dosing. The participants will not External Assessment Scheme (HEMQAS) for quality assess- be allowed more than one repeated dose. In case a participant ment to ensure delivery of reliable and comparable results29. vomits following redosing during the enrollment visit, the For an extended description of all laboratory procedures and subject will be withdrawn from the trial and standard treatment handling of samples, standard operating procedures (SOPs) of will be offered according to national guidelines. the ALIVE trial and STOP Consortium are publicly available at https://stoptheworm.org/resources/protocols/. Outcomes Safety. Safety is the primary outcome in the phase II Study medication component. After signing informed consent, evaluations and The FDC (400 mg ALB + 9 mg or 18 mg IVM) will be pro- measurements, including adverse events, vital signs, physical vided as an oro-dispersible and chewable tablet manufactured examination, weight, height and body mass index (BMI) will under Good Manufacturing Practices (GMP) by Laboratorios be assessed through post-treatment follow-up visit (Table 1). Liconsa SA (Guadalajara, Spain), which will be responsible All adverse events during all study visits, will be noted and for the pharmaceutical development and subsequent produc- surveillance 3 hours post-treatment will be conducted each tion, labeling, packaging and distribution to the trial sites; while day a participant receives study treatment. Type, frequency, Page 7 of 18 Gates Open Research 2022, 6:62 Last updated: 28 SEP 2022 duration, severity and relatedness to study drug will be be generated by pooled sample whole-genome sequencing. recorded for every adverse event and severe adverse event. By comparing genetic diversity analysis before and after Any clinically significant abnormalities persisting at the end treatment, we aim to identify genomic regions and genes under of the study will be followed up by the study physician until positive selection potentially due to ALB and/or FDC treat- resolution or until a clinically stable endpoint is reached. Safety ment. The second output will be a list of single nucleotide poly- will also be evaluated as a secondary objective in the Phase III morphisms (SNPs) with statistical associations with treatment trial. efficacy by both logistic and linear regression models. By these two outputs, we will define the genetics of treatment response Efficacy. Efficacy estimated by CR for T. trichiura is the and, therefore, potential markers of anthelmintic resistance in primary outcome of the phase III component. Anthelmintic T. trichiura and hookworm. Unfortunately, we will exclude efficacy, measured by CR for each STH target species, will be S. stercoralis from the resistance assessment due to the com- determined by comparative analysis of a stool sample taken plexity of larvae culture and isolation for genomics analysis before and 21 (+/-7) days after treatment by Kato Katz or the as previously described30, which hamper its implementation Baermann method19. Cure is defined as absence of the STH in the context of a RCT. species in participants who had a positive egg count and/or larvae count for that STH at baseline. CR is defined as the Data handling proportion of individuals cured to the total of those infected Data will be collected in password protected electronic case at baseline with each particular species of STH. report forms (eCRF) containing internal quality checks. Paper- based study visit worksheets for each participant containing Efficacy measured through ERR will be determined by using all the relevant demographic and clinical data for the trial will geometric means, calculated for hookworms, T. trichiura be used as source documents. Clinical data will be entered and A. lumbricoides from the findings on duplicate Kato- from the source documents and no direct data entry in the Katz samples taken both before treatment and again at day-21 eCRF will be performed. Laboratory results will be recorded post-treatment. The Baermann method will also be performed in an ad-hoc database at each site. All data storage will be in parallel to Kato-Katz for the identification of S. stercoralis. encrypted and password protected. All the data generated at CR and variation in Ct values will be assessed by qPCR the trial sites will run quality management procedures inter- analysis between baseline and day-21 samples. All molecular nally. Anomalies and missing data identified at the centralized testing in pre- and post-treatment samples will be performed database will be clarified and resolved with the site. Using blinded from the microscopy data. SOPs developed ad-hoc, monitors will verify compliance with the approved protocol (including local regulations for each Pharmacokinetics. Samples for population pharmacokinetic site) and with International Conference on Harmonization analysis will be collected by finger prick using Mitra sticks Good Clinical Practice (ICH GCP). Regulatory authorities and (Neoteryx, Toronto CA) (20 µL). After drying, the samples the sponsor will have access to all data for monitoring, auditing will be kept at room temperature for up to 21 days and at -80℃ and inspection activities. until transport and processing at Kymos labs, Barcelona, Spain. Quantification of the study drugs will be performed by HPLC- Statistical methods MS/MS, with has a lower limit of detection of 5 ng/mL for Sample size calculation. Sample size was calculated based on IVM B1a and 5 ng/mL for ALB sulfoxide. available data from peer-reviewed publications complemented with reasonable estimates of efficacy for those experimental The 126 subjects included in the phase II trial will be ran- groups that have not been previously tested. Given the pub- domly assigned to different sampling times (two blood samples lic health relevance of generating data for each species of STH for each participant in the single dose arms, plus a pre-dose species of interest to this trial, sample size calculations are made on the 3rd dose for arm three) to adjust a population pharma- to provide adequate power for each of these species, including cokinetic model. A simultaneous pharmacokinetic analysis of that in the primary endpoint (T. trichiura) and those in the all concentration-time data of either IVM or ALB will be secondary endpoints (hookworms and S. stercoralis). For performed by a nonlinear mixed-effects modeling approach. these calculations, the efficacy of the control arm (400 mg ALB in a single dose), was obtained for T. trichiura and Participant acceptability/palatability. The acceptability of the hookworms from a systematic review and meta-analysis where new FDC will be investigated in children during the phase II temporary trends in efficacy (with the corresponding confidence following standard procedures. For children 5-8 years-old, a intervals (CIs)) were incorporated (Table 2)10. Facial Hedonic Scale will be applied after drug administration. For older participants and parents, a Numerical Rating Scale, For the efficacy of the control arm against S. stercoralis, a including specific questions on taste, smell, and texture, clinical trial that included an arm of 400 mg ALB for three will be used. consecutive days was used, assuming a “best case scenario” for the efficacy of the control group. This estimated efficacy is Resistance outcome. We will evaluate genotypic resistance asso- also in the range of a systematic review assessing the efficacy ciated with treatment failures for T. trichiura and hookworms of ALB at various (but not single) doses31. For the FDC at sin- using genome-wide approaches on data which will primarily gle dose, the calculations were based on the estimated efficacies Page 8 of 18 Gates Open Research 2022, 6:62 Last updated: 28 SEP 2022 Table 2. Sample size according to the expected efficacy (CR) of the different treatment. The estimated sample size was calculated for pairwise comparisons of the expected CR for three study groups with an overall significance level of 5% adjusted for multiple tests by Bonferroni’s correction, and 80% power. The total sample size was calculated by inflating the estimated calculation by 10% considering losses to follow-up. Phase II* Phase III Phase II/III Group Expected Cure Rate (%) N estimated N total phase II N estimated N total phase III N Total T. trichiura ALB 23.7 23 26 93 103 129 FDCx1 43.7 45 50 178 198 248 FDCx3 59.0 45 50 178 198 248 Total 113 126 449 499 625 Hookworms ALB 79.5 91 101 101 FDCx1 79.5 91 101 101 FDCx3 95.0 99 110 110 Total 281 312 312 S. stercoralis ALB 45.0 42 47 47 FDCx1 79.0 108 120 120 FDCx3 94.0 107 119 119 Total 257 286 286 Total trial 126 1097 1223 *The phase II sample size corresponds to 20% of the total sample size for T. trichiura. (and their corresponding CIs) in a systematic review The study design has a blinded assessment for the primary end- that calculated the Relative Risks of cure of diverse drug point, which is a laboratory-based measurement to be per- regimens against a single 400 mg ALB dose (Table 2)14. For formed by blinded operators who will receive coded samples. S. stercoralis, the estimated efficacy of FDC was calculated Although a double-blind design would be methodologically based on a recent clinical trial using IVM single and multiple- superior, this would imply that the participants in two of the dose regimens32. Finally, for the FDC in three-dose regimens, three treatment arms would have to swallow multiple placebo considering its use in public health, deployment logistics in tablets, which could lead to unnecessary complications and MDA campaigns and expected impact of the FDC, we estimated interfere with the evaluation of several safety outcomes. that an improvement of at least 15 percentage points would be the minimum improvement in efficacy to be demonstrated in Statistical analysis of the main outcomes. For the safety analy- order to make the FDCx3 regimen worth considering in public sis, an intent to treat (ITT) analysis (those participants who have health interventions. The sample size was calculated for received at least one dose of study intervention) will be used pairwise comparisons of the expected CR for three study and participants will be considered by arm and by both FDC groups with an overall significance level of 5% adjusted for arms pooled (overall) and by number of doses of FDC received multiple tests by Bonferroni’s correction, 80% power and to explore dose-responses. First, all adverse events will be inflated for 10% lost-to-follow-up. described and classified by causality, severity, seriousness and expectedness. Drug-related adverse events will be analyzed using ordinal logistic regression with the untoward effect The estimated total number of participants for the adaptive design classified as absent, mild, moderate, or severe and the factorial is 1223 (T. trichiura 625, hookworm 312 and S. stercoralis treatment regimens (without interaction term) as predictor 286). The sample size for the phase II component is 20% of variables. For a count outcome such as the number of adverse the total participants for T. trichiura (126 participants), with events or severe adverse events incidence rate ratio (IRR) the remaining 80% of T. trichiura participants randomized and its 95% CI will be computed using Poisson or negative in the phase III component (Table 1). binomial regression. Page 9 of 18 Gates Open Research 2022, 6:62 Last updated: 28 SEP 2022 The primary efficacy analysis will be based on the ITT pop- pre-treatment vs post-treatment) in the three treatment arms ulation. Efficacy for each target STH species will be ana- separately and in combination. We will also perform genome- lyzed separately. A participant with multiple infections will wide association to identify variants in the parasite species be included in the analysis of each species that the participant associated with differences in treatment response and drug is infected with. Cochran–Mantel–Haenszel (CMH) test, con- efficacy. Two different association tests will be performed: trolling the effect of site if that is appropriate (i.e., sufficient (i) a logistic regression genome-wide association between participants), will be used to compare the CRs for the three samples from participants with good treatment response phe- treatment groups. Differences among CRs will be assessed by notypes, and post-treatment samples from children with poor using marginal modeling. treatment response phenotypes; and (ii) a linear regression genome-wide association analysis with the ERR estimates as a The difference in ERR between the different treatment arms continuous variable for all samples collected before treatment. will be assessed using three different approaches. First by an We will characterize the predicted effects of genetic variation analysis of covariance (ANCOVA), in which the logarithm of we uncover on genes and identify relationships between genes the egg counts at post-treatment is the dependent variable, site associated with the outlier variation by functional enrichment (if appropriate) and treatment as fixed effect, and the loga- analyses. rithm of the egg count at pre-treatment will be used as the covariate. Baseline egg counts will also be analyzed as an inde- Pharmacokinetic analysis. A simultaneous pharmacokinetic pendent variable that conditions treatment response. The analysis of all concentration-time data of either IVM or ALB second and third approaches will be to model ERR through will be performed by means of the non-linear mixed effects marginal models and mixed models, respectively33. modeling approach implemented in Phoenix program (Phoe- nix NLME version 8.2, Pharsight, RRID:SCR_003163. A Correlation between Kato-Katz counts and qPCR Ct values Certara Company. Princeton, NJ, USA). The base model will will be explored through linear regression tests of Pearson’s or be developed by fitting the one, two or three open compartment Spearman´s (according to the underlying distribution). Kappa models with first-order elimination. Interindividual variability test will be used for the evaluation of both tests in the calcula- (IIV) will be tested in all the pharmacokinetic parameters tion of CRs. Relevant covariates will also be included in the assuming a log-normal distribution. Proportional and combined data analysis. error will be applied to describe the residual error associated with the concentrations. Once the base model is developed, the All the analyzes will be carried out with the R software, effect of all covariates, physiologically reasonable (body weight, packages geepack and lme426,34,35. height, sex and age) and co-medication will be investigated on model parameters, graphically and by means of statistical Anthelmintic resistance. Anthelmintic resistance analysis will multivariate models. focus on T. trichiura and hookworms separately. In the case of hookworms (which comprise more than one species), Covariates will be tested firstly univariate and then by the cumu- anthelmintic resistance will be evaluated for each hookworm lative forward inclusion/backward elimination procedures. species if more than one species is present; and we predicted Significance levels of 5% (reduction in the minimum objec- that Necator americanus will be the predominant species tive function value (MOFV) of >3.841 units) and 0.1% based on previous data23,36. Samples determined to be posi- (increase in the MOFV of >10.8 units) will be employed tive for these parasites by microscopy (pre- and post-treatment) during the forward addition and backward elimination steps. will be included in the resistance evaluation and grouped by The decrease in the minimum objective function value (MOFV; treatment arm. -2xlog likelihood), parameter precision expressed as rela- To identify genetic variation associated with the treatment tive standard error (RSE%), reductions in IIV associated with response, we will measure distribution of genome-wide genetic a specific pharmacokinetic parameter, model completion status diversity between treatment groups and throughout the genome, (e.g., successful convergence or termination) and visual inspec- consistent with recent studies on anthelmintic resistance tion of goodness-of-fit plots will also be considered for human- and veterinary-infective helminth species37. Genome- model selection. wide measures of within sample genetic diversity (for example, nucleotide diversity (pi), Waterson estimator, Tajima’s D, Fu’s) The final model evaluation will be performed through a predic- and between sample genetic diversity (e.g. FST, Dxy, between tion corrected visual predictive check to investigate the predic- pre- and post-treatment groups) will be estimated in non-over- tive capability of the model. Parameter precision will be assessed lapping sliding windows throughout the genome. Statistical through a non-parametric bootstrap. Once the final model significance will be inferred by the distribution of data points, has been evaluated, simulations of different scenarios will be from which data points that lie greater than three standard performed to establish the best dose for each body weight range deviations from the genome-wide mean will indicate outliers of of children. interest. We will identify genes in these outlier regions of genetic differentiation between parasite populations showing differences Graphical diagnostics will be assessed using Phoenix version in treatment response (i.e., good responders vs poor responders, 8.2 or Xpose version 4.2.1 implemented in R version 3.6.0)26,38. Page 10 of 18 Gates Open Research 2022, 6:62 Last updated: 28 SEP 2022 Exploratory analyses. Exploratory analysis will be performed interpretation will be shared through peer-reviewed, open-access to understand the potentially large number of unknown con- scientific journals and at scientific conferences. A final study founders/effect modifiers in this study. A bivariate analysis of report will be presented to regulatory agencies and the data baseline characteristics between included and excluded children made available unless required to be concealed by IRBs or will be performed. Subgroup safety and efficacy analyzes will regulatory agencies. also be performed in the ITT population. The subgroups to be analyzed will be: Discussion This adaptive randomized clinical trial introduces an innovative • I VM drug exposure: categorized by >400 µg/Kg vs combination of ALB and IVM with registrational goals and the ≤400 µg/Kg. aim of contributing to STH recommendations and control activi- ties, as well as WHO prequalification. Besides STH, the safety, • A  ge: categorized by SAC (5 to 14 years-old) and young acceptability and pharmacokinetic data generated by this trial adults (15 to 18 years-old). can also open possibilities of expanding its use to other NTD • C  o-infection: categorized by mono-infected vs co-infected. control programs currently using either or both drugs. • W  orm burden: categorized by WHO categories of egg The main challenge in the development of a fixed combina- burden categories measured by Eggs Per Gram (EPG) tion of ALB and IVM was the different posology of both drugs; through Kato-Katz method39. then, while ALB is well established with a single authorized fixed dosage of 400 mg for children older than 12 months as Monitoring, ethics and regulatory per WHO´s STH control guidelines7, at present IVM has a This study will be conducted in conformity with the principles flexible dosage regime of between 200 - 400 µg per kg body- set forth in the Declaration of Helsinki in its current weight. The objectives of the ALIVE trial and its design are sup- version, the requirements of Good Clinical Practice (GCP) ported by a development program carried by members of the as defined in Guidelines and The International Council for STOP Project Consortium, that have demonstrated that IVM Harmonization of Technical Requirements for Registration of up to 600 µg/kg can be safely administered. This high dos- Pharmaceuticals for Human Use (ICH) E6(R2), and all national and age profile allows a fixed-dose, rather than commonly used local regulations and guidance applicable at each site. Each weight or height based dosing, to be used here18. Despite not institution engaged in this research will hold registered IRBs/ECs strictly a high-dose regimen of IVM, since participants will that will review and approve this protocol, associated informed receive a dose between 200- and 600 µg/kg of IVM, the data consent documents, recruitment material, and handouts or generated here will identify pharmacokinetic / pharmacodynamic surveys intended for the participants, prior to the recruitment, parameters that will contribute towards an evidence-based screening, and enrolment of participants. The IRB/IEC review therapeutic index for IVM in a combination regimen with ALB. shall be in accordance with ICH E6 (R2), and other regula- tions and policies, as applicable. The trial is registered at Existing limitations in diagnostic methodologies for the detec- ClinicalTrials.gov, Identifier: NCT0512469; first posted on tion of STH in general and in particular for S. stercoralis, November 18, 2021. pose a significant challenge towards determining the efficacy of treatment in the context of a randomized clinical trial. In The regulatory goal for the use of the FDC formulations this context, a more precise assessment is required to identify (9 mg/400 mg and 18 mg/ 400 mg of IVM and ALB, meaningful significant differences between treatment arms, respectively) against STH are subject to review and evaluation and are affected by sensitivity, specificity, reproducibility by the European Medicines Agency (EMA) for use outside the and operator dependency, the later emphasized in multi- European Union through the EU-Medicines for all procedure centric trials41. The contribution of qPCR into solving these “EU-M4all” (previously known as the Article 58 procedure). This weaknesses will be explored in our trial and will add to efforts program, operated by EMA in cooperation with WHO, provides initiated by other groups to evaluate molecular biology tools scientific opinions on high priority human medicines, that are as a potential improvement in the assessment of key outcomes intended for markets outside of the European Union with the in randomized clinical trials of anthelmintic drugs42. While the aims to facilitate prequalification of the medicine by WHO current trial includes the implementation of this technology in and registration in the target countries40. As part of this process, all three clinical sites, the efficacy outcome will be processed in the whole development of the products and the trial protocols a centralized manner at KEMRI in Kilifi. have been discussed and adapted based on the results of three Scientific Advice rounds with EMA’s Committee for Medicinal Although the drivers and mechanisms underlying anthelmintic Products for Human Use (CHMP). drug resistance in STH of human interest are incompletely understood, there is extensive data from the veterinary world Study status and timelines where it is a widespread problem43. Moreover, a meta-analysis The phase II component of the trial began recruitment of of randomized clinical trials against T. trichiura showed a participants in February 2022 and is expected to progress to temporal trend towards decreased efficacy of benzimidazole recruitment for the phase III component of the trial in all three drugs and a recently published trial found lower efficacy of field sites between May and July 2022 and complete recruitment ALB in communities that have received MDA activities by May 2023. Upon completion of the study, its results and for more prolonged periods, suggesting drug resistance as Page 11 of 18 Gates Open Research 2022, 6:62 Last updated: 28 SEP 2022 a potential mechanism10,44. By using different mechanisms program managers as an adjunct to the trials, which should of action, ALB/IVM combinations provide regimens with a shed light on the possibilities and limitations of three-day theoretical lower risk for the emergence of resistance. As in regimens and provide a comprehensive outlook into the poten- HIV and tuberculosis, co-formulations further contribute to it tial impact of these regimens in the control and transmission by ensuring compliance with a combination regimen. We also interruption aims against STH. aim to identify markers of resistance through the comprehen- sive evaluation of pre- and post-treatment samples of T. trichiura From a programmatic perspective, a fixed-dose co-formulation through whole-genome sequencing. While the trial is pow- provides advantages in packaging, transportation, storage and ered for each STH species of interest (T. trichiura, hookworms drug administration, each of which also positively impacts and S. stercoralis), it is powered for the entire study popula- the cost of medical products and programs that deliver them. tion rather than for each of the three study sites, which might constitute a limitation should significant differences in either Conclusion or both treatment response and genetic differences unrelated to This trial seeks to provide evidence for the registration of a treatment response (i.e., population genetic differences) between fixed-dose coformulation of ALB and IVM through the demon- study sites be found. stration of its safety, efficacy and acceptability for the treatment of all STH species of interest for WHO-guided control activi- The trial also seeks to understand the potential benefit of three- ties. With an adaptive design, it will first define the safety of day treatment regimens, which depart from the current dogma a FDC in phase II, followed by a phase III safety and efficacy of single-dose regimens for MDA strategies against STH. trial. The data generated hopes to provide important advance- While multiple-day regimens are in place for other indications ments towards the safe and effective control of all STH in line affecting communities endemic for STH45, the suboptimal with 2030 elimination of morbidity goals as a public health efficacy of current regimens against T. trichiura calls for an tool towards relieving the burden of STH on more than a billion evaluation of new approaches to improve the efficacy of treat- people worldwide. ment. To assess its feasibility in control programs, the trial includes palatability evaluations in the phase II trial. 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Hatz C, Marti H, Chwaya HM, et al.: A Comparative Trial of a Single-Dose PubMed Abstract | Publisher Full Text Page 13 of 18 Gates Open Research Gates Open Research 2022, 6:62 Last updated: 28 SEP 2022 Open Peer Review Current Peer Review Status: Version 1 Reviewer Report 05 July 2022 https://doi.org/10.21956/gatesopenres.14889.r32202 © 2022 Kobylinski K. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Kevin Kobylinski Department of Entomology, Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand This is an important trial which is well designed and will provide critical information for the use of a novel co-formulation of ivermectin and albendazole, which are likely the ideal drugs for soil- transmitted helminth control and elimination. There are a few points that could be explained better to make the trial design clearer and the rationale better justified. Dose escalation (and weights) for Phase II Groups 2 and 3 should be explained better in the Trial Design text, but figure 1 is helpful. It is not clear how many children in which weight class will be provided Arms 1, 2, or 3 as Table 2 suggests a 1:2:2 ratio distribution for treatment arms but there are different numbers listed for the Phase II weight classes in Figure 1, and the target weight distribution is not clearly explained for Phase III. Is there a rationale for performing Phase II Group 2 before Group 3? One assumes that the rationale is to evaluate the ivermectin dose range of 400-600 ug/kg in larger/older children prior to administering the same dose range 391-600 ug/kg to smaller/younger children, but it would be ideal to see rationale explained. The Phase II ivermectin pharmacokinetic output will provide excellent evidence for the effect of age on ivermectin bioavailablity. It has been shown that children <12 years old reach half the peak and total bioavailability of ivermectin compared to adults (Schulz et al., 20191) and two ivermectin pharmacokinetic models predict that higher doses of ivermectin are necessary in young children to reach the same levels as adults (Brussee et al., 20192, Gwee et al. 20203). Some discussion as to the potential contribution of the ALIVE trial to understanding the influence of age on ivermectin pharmacokinetics in children is warranted. References 1. Schulz J, Coulibaly J, Schindler C, Wimmersberger D, et al.: Pharmacokinetics of ascending doses of ivermectin in Trichuris trichiura-infected children aged 2–12 years. Journal of Antimicrobial Chemotherapy. 2019; 74 (6): 1642-1647 Publisher Full Text 2. Brussee JM, Schulz JD, Coulibaly JT, Keiser J, et al.: Ivermectin Dosing Strategy to Achieve Equivalent Exposure Coverage in Children and Adults.Clin Pharmacol Ther. 106 (3): 661-667 Page 14 of 18 Gates Open Research Gates Open Research 2022, 6:62 Last updated: 28 SEP 2022 PubMed Abstract | Publisher Full Text 3. Gwee A, Duffull S, Zhu X, Tong SYC, et al.: Population pharmacokinetics of ivermectin for the treatment of scabies in Indigenous Australian children.PLoS Negl Trop Dis. 14 (12): e0008886 PubMed Abstract | Publisher Full Text Is the rationale for, and objectives of, the study clearly described? Yes Is the study design appropriate for the research question? Yes Are sufficient details of the methods provided to allow replication by others? Yes Are the datasets clearly presented in a useable and accessible format? Not applicable Competing Interests: No competing interests were disclosed. Reviewer Expertise: Neglected Tropical Diseases, Entomology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Author Response 11 Aug 2022 ALEJANDRO JAVIER KROLEWIECKI, Hospital Clínic—Universitat de Barcelona, Barcelona, Spain We thank Kevin Kobilinsky for his review and comments on our protocol, and take the opportunity to further clarify the following concepts: Dose escalation: among the study candidates of the Phase II trial, which recruits children between 15 and 45 Kg of body weight, the design is to start with the subgroup between 23 and 30 Kg, receiving doses of IVM between 300 and 391 µg/Kg and only upon DSMB clearance, to recruit the cohort including participants receiving a higher dose of IVM of 400 to 600 µg/Kg (children weighting 30 to 45Kg), followed by, after DSMB approval, the lighter group of children (15 to 23Kg), who received 391 to 600µ/Kg. The rationale for this study design is to progress to higher doses and younger/lighter participants progressively upon confirmation of adequate safety in each subgroup. Sample size in Phase II: in this trial, the 1:2:2 randomization between the 3 study arms is applied in the 3 sequential weight sub-groups; therefore within each sub-group the number of participants per study arms would be distributed approximately in a 1:2:2 manner. Weight distribution in Phase III: for this trial participants receive FDC 400/9mg if the weight is below 45Kg and 400/18 in those 45Kg and up, without any dose escalation included. PK: there is a scarcity of data on the pharmacokinetics of IVM in pediatric populations further worsened by an almost complete absence of evidence of PK/PD parameters related to antihelmintic drugs against STH. The effect of age on these parameters is another Page 15 of 18 Gates Open Research Gates Open Research 2022, 6:62 Last updated: 28 SEP 2022 variable of consideration given variations in the expression of cytochrome P450s at different pediatric ages (1) References 1. Upreti VV, Wahlstrom JL. Meta-analysis of hepatic cytochrome P450 ontogeny to underwrite the prediction of pediatric pharmacokinetics using physiologically based pharmacokinetic modeling. J Clin Pharmacol. 2016 Mar;56(3):266–83. Competing Interests: No competing interests were disclosed. Reviewer Report 16 May 2022 https://doi.org/10.21956/gatesopenres.14889.r32064 © 2022 Montresor A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Antonio Montresor Department of Control of Neglected Tropical Diseases, World Health Organization, Geneva, Switzerland The protocol is well written, complete and appropriate to reply to the research question. I have however some concerns on the relevancy of the research question: There are several studies that investigated the efficacy and safety of albendazole ivermectin co- administration (Hürlimann et al., 20221, Welsche et al., 20212, Keller et al., 20213, Palmeirim et al., 20184) and as a consequence, there is no doubt, in my opinion, that a combination of albendazole plus ivermectin (in Fixed Dose Combination or dosed according to weight) is more efficacious than albendazole alone against T. trichiura and S. stercoralis (on the other side, no additional efficacy of the Fixed Dose Combination should be expected on hookworms). In my opinion a much more relevant question is if the Fixed Dose Combination (that has clear advantages in terms of logistics and ease of administration) is as efficacious as albendazole and ivermectin dosed according to weight). In my opinion the protocol is a missed opportunity because it will provide already known replies and will not address an important research question that would allow simpliying the logistics of mass drug administration for the control of Soil Transmitted helminthiasis. References 1. Hürlimann E, Keller L, Patel C, Welsche S, et al.: Efficacy and safety of co-administered ivermectin and albendazole in school-aged children and adults infected with Trichuris trichiura in Côte d'Ivoire, Laos, and Pemba Island, Tanzania: a double-blind, parallel-group, phase 3, randomised controlled trial.Lancet Infect Dis. 22 (1): 123-135 PubMed Abstract | Publisher Full Text 2. Welsche S, Mrimi EC, Keller L, Hürlimann E, et al.: Efficacy and safety of moxidectin and albendazole compared to ivermectin and albendazole co-administration in adolescents infected Page 16 of 18 Gates Open Research Gates Open Research 2022, 6:62 Last updated: 28 SEP 2022 with Trichuris trichiura: a randomized controlled trial protocol.Gates Open Res. 2021; 5: 106 PubMed Abstract | Publisher Full Text 3. Keller L, Welsche S, Patel C, Sayasone S, et al.: Long-term outcomes of ivermectin-albendazole versus albendazole alone against soil-transmitted helminths: Results from randomized controlled trials in Lao PDR and Pemba Island, Tanzania.PLoS Negl Trop Dis. 15 (6): e0009561 PubMed Abstract | Publisher Full Text 4. Palmeirim MS, Hürlimann E, Knopp S, Speich B, et al.: Efficacy and safety of co-administered ivermectin plus albendazole for treating soil-transmitted helminths: A systematic review, meta- analysis and individual patient data analysis.PLoS Negl Trop Dis. 12 (4): e0006458 PubMed Abstract | Publisher Full Text Is the rationale for, and objectives of, the study clearly described? Yes Is the study design appropriate for the research question? Yes Are sufficient details of the methods provided to allow replication by others? Yes Are the datasets clearly presented in a useable and accessible format? Yes Competing Interests: No competing interests were disclosed. Reviewer Expertise: Neglected Tropical Diseases control I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Author Response 26 May 2022 ALEJANDRO JAVIER KROLEWIECKI, Hospital Clínic—Universitat de Barcelona, Barcelona, Spain Response to Dr. Montresor. We appreciate the comments and insightful opinions from Dr. Antonio Montresor on our protocol and would like to highlight that we fully agree that a regimen with improved efficacy against T. trichiura than what is achieved with albendazole is feasible through its combination with available medications, as is the case for ivermectin. Besides the available data on its efficacy, safety is also encouraging even at high doses of ivermectin, as demonstrated in preliminary trials performed by our groups in healthy adult volunteers and infected children (1,2). With the same ultimate implementation goals in mind as those expressed in the review, our Phase II/III adaptive trial is relevant and necessary. The ALIVE trial contains innovations in the strategy (a fixed-dose approach to the use of ivermectin along with albendazole) and in the product (the fixed-dose combinations (FDC)); as such, this is a registrational trial with Page 17 of 18 Gates Open Research Gates Open Research 2022, 6:62 Last updated: 28 SEP 2022 primary safety and efficacy objectives. For that, the protocol has been developed with the scientific advice from the European Medicines Agency (EMA). The selection of albendazole as the control arm is based on the rationale of using the current standard of care as the proper comparator. The ALIVE trial is a key step in the development and registration of the FDC, which includes prior developments and trials as the acquisition of capacities for the industrial fermentation of ivermectin and the WHO prequalification of a generic ivermectin by Liconsa Labs, producer of the FDC and member of the STOP Consortium (https://extranet.who.int/pqweb/news/lab-liconsa%E2%80%99s- ivermectin-prequalified), as well as trials showing the pharmacokinetic behavior of high dose ivermectin and the bioavailability of each component of the FDC in comparison to the reference products (manuscript under review); the later being the standard approach to assess the comparability of a drug combination to each of its components administered separately (1). As described in the manuscript, that bioavailability trial provided data supporting the design of the ALIVE trial. In full agreement with the opinion of Dr Montresor regarding the logistic advantages foreseen for the FDC, the STOP project includes further studies assessing the acceptability and implementation strategy for these innovations. References 1. Muñoz J, Ballester MR, Antonijoan RM, Gich I, Rodríguez M, Colli E, et al. Safety and pharmacokinetic profile of fixed-dose ivermectin with an innovative 18mg tablet in healthy adult volunteers. Prichard RK, editor. PLoS Negl Trop Dis. PLoS Negl Trop Dis; 2018;12:e0006020. 2. Matamoros G, Sánchez A, Gabrie JA, Juárez M, Ceballos L, Escalada A, et al. Efficacy and Safety of Albendazole and High-Dose Ivermectin Coadministration in School- Aged Children Infected With Trichuris trichiura in Honduras: A Randomized Controlled Trial. Clin Infect Dis; 2021;73:1203–10.Response to Dr. Montresor. Competing Interests: No competing interests were disclosed. Page 18 of 18