Pharmacological Research 159 (2020) 104999 Contents lists available at ScienceDirect Pharmacological Research journal homepage: www.elsevier.com/locate/yphrs Rimeporide as a first- in-class NHE-1 inhibitor: Results of a phase Ib trial in T young patients with Duchenne Muscular Dystrophy Stefano C. Previtalia, Teresa Gidarob, Jordi Díaz-Manerac,d, Alberto Zambona, Stephanie Carnesecchie, Pascale Roux-Lombardf, Pietro Spitalig, Mirko Signorellig, Cristina Al-Khalili Szigyartoh, Camilla Johanssoni, Julian Grayj, Delphine Labollej, Florence Porte Thoméj,*, Jacqueline Pitchforthk, Joana Domingosk,1, Francesco Muntonik,l a IRCCS San Raffaele Scientific Institute, Department of Neurology and INSPE, Milan, Italy b Institute of Myology, Hopital Trousseau, I- Motion, Paris, France c Hospital de la Santa Creu i Sant Pau de Barcelona Servei de Neurologia, Barcelona, Spain d Centro de Investigación Biomédica en Red en Enfermedades Raras (CIBERER), Spain e Geneva University (UNIGE), Geneva, Switzerland f Geneva University Hospital (HUG), Immunology and Allergology Department, Geneva, Switzerland g Leiden University Medical Center, Netherlands h KTH, School of Chemistry, Biotechnology and Health, Royal Institute of Technology, Stockolm, Sweden i Science for Life Laboratory, Department of Protein Science, Division of Systems Biology, Solna, Sweden j EspeRare, Geneva, Switzerland k UCL Great Ormond Street Institute of Child Health & Great Ormond Street Hospital Dubowitz Neuromuscular Centre, London, UK l NIHR Great Ormond Street Hospital Biomedical Research Centre, Great Ormond Street Institute of Child Health, Great Ormond Street Hospital Trust, University College London, London, UK A R T I C LE I N FO A B S T R A C T Keywords: Rimeporide, a first-in-class sodium/proton exchanger Type 1 inhibitor (NHE-1 inhibitor) is repositioned by Duchenne Muscular Dystrophy EspeRare for patients with Duchenne Muscular Dystrophy (DMD). Historically, NHE-1 inhibitors were developed NHE-1 for cardiac therapeutic interventions. There is considerable overlap in the pathophysiological mechanisms in Rimeporide Congestive Heart Failure (CHF) and in cardiomyopathy in DMD, therefore NHE-1 inhibition could be a pro- Safety mising pharmacological approach to the cardiac dysfunctions observed in DMD. Extensive preclinical data was Pharmacokinetic collected in various animal models including dystrophin-deficient (mdx) mice to characterise Rimeporide’s anti- Cardiomyopathy fibrotic and anti-inflammatory properties and there is evidence that NHE-1 inhibitors could play a significant role in modifying DMD cardiac and also skeletal pathologies, as the NHE-1 isoform is ubiquitous. We report here the first study with Rimeporide in DMD patients. This 4-week treatment, open label phase Ib, multiple oral ascending dose study, enrolled 20 ambulant boys with DMD (6–11 years), with outcomes including safety, pharmacokinetic (PK) and pharmacodynamic (PD) biomarkers. Rimeporide was safe and well-tolerated at all doses. PK evaluations showed that Rimeporide was well absorbed orally reaching pharmacological concentra- tions from the lowest dose, with exposure increasing linearly with dose and with no evidence of accumulation upon repeated dosing. Exploratory PD biomarkers showed positive effect upon a 4-week treatment, supporting its therapeutic potential in patients with DMD, primarily as a cardioprotective treatment, and provide rationale for further efficacy studies. ⁎ Corresponding author at: EspeRare, Campus Biotech, Innovation Park, 15 Avenue Sécheron, CH-1202, Geneva. E-mail address: porte.florence@esperare.org (F. Porte Thomé). 1 Deceased. https://doi.org/10.1016/j.phrs.2020.104999 Received 20 March 2020; Received in revised form 2 June 2020; Accepted 4 June 2020 Available online 12 June 2020 1043-6618/ © 2020 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). S.C. Previtali, et al. Pharmacological Research 159 (2020) 104999 1. Introduction skeletal and cardiac muscle fibers fragility, tears in the sarcolemma, influx of extracellular Ca2+ [15], subsequent inflammation and 1.1. Disease’s overview Na + overload [16], and alkalization [17]. Na + is presumed to accu- mulate in dystrophic muscle fibers through microtears and channels Duchenne Muscular Dystrophy (DMD) is a paediatric orphan disease leading to enhanced Ca2+ entry through the NCX. Cytosolic Na + with an incidence of approximately 1 in 5000 male births worldwide levels have been described to be elevated in myofibers from dystrophin- [1,2]. It is a rapidly progressing and severe form of muscular dystrophy deficient (mdx) mice, the most used animal model of DMD [18] and in where children typically display skeletal muscle weakness by the age of skeletal muscles in DMD patients [4,19]. 2–6 years, followed by progressive loss of their ability to walk between Also, pH is increased in resting muscle tissue of patients with DMD the ages of 7–13 years. By the age of 15–18 years, most patients start to [20]. Interestingly, pH increase is seen in descending order in DMD, develop progressive respiratory muscle weakness leading to respiratory Becker muscular dystrophy, and female carriers, when compared to insufficiency requiring ventilator support. While the widespread use of healthy controls [21], i.e. pH increases with the severity of the genetic corticosteroids improves muscle function and delay age at loss of am- alteration. An increase of pH in muscle tissue has also been reported in bulation, there continues to be a high unmet need for treatments for mdx mice [18]. In addition, Na + influx via the NHE-1 is increased in cardiorespiratory complications in these patients. The improved re- mdx myocytes compared to wild-type myocytes [22]. spiratory intervention has also improved the respiratory outcomes for NHE-1 inhibition by Cariporide, another selective Na affected patients. However, cardiac disease (cardiomyopathy leading to +/H + exchange inhibitor discontinued in late stage clinical develop- heart failure and arrhythmias) has become an increasingly important ment for safety reasons, has been shown to have significant beneficial cause of morbidity and mortality [3]. Pathological cellular hallmarks of effects on skeletal muscle function, intracellular ion homeostasis and DMD include, downstream the lack of dystrophin, abnormal ionic pH in mdx mice [22]. In these mice, treatment with Cariporide mark- homeostasis. Cytoplasmic sodium overload was shown to cause severe edly reduced creatine kinase (CK) levels, inflammation, fibrosis and osmotic oedema in DMD patients [4]. The dual overload in sodium ion histological skeletal muscle damage, whilst preserving muscle perfor- [5] and water precedes the dystrophic process and persists until fatty mance. In the BIO 14.6 hamster (delta sarcoglycan deficiency related muscle degeneration is complete. In addition, intracellular overload of dystrophy, also relevant to dystrophinopathies), Cariporide normalised calcium is well described as a major cause of muscle cell dysfunction in intracellular Ca2+ levels and Na + influx and decreased stretch in- DMD [6] and necrosis. duced CK release, strongly supporting that inhibition of NHE-1 can lead to regulation of Ca2+ via normalisation of NCX activity [22]. 1.2. Scientific rationale of Rimeporide repositioning in DMD Rimeporide, another NHE-1 inhibitor which showed a good safety and tolerability profile so far in adults (tested in 166 subjects in 7 phase Rimeporide (initial code EMD 87580), is a benzoyl-guanidine de- I studies) proved to provide potent anti-inflammatory and anti-fibrotic rivative. It is a potent and selective inhibitor of the sodium-proton ex- effects in short and long term studies in mdx mice both in skeletal and changer isoform 1 (NHE-1) which is found ubiquitously in mammalian cardiac muscles activity [23]. In UMX 101 hamsters, also a delta sar- cell membranes throughout the body [7]. There are ten NHE isoforms coglycan deficiency related dystrophy and a well-established and ac- but NHE-1 is the predominant isoform in the heart and in skeletal cepted model of dilated cardiomyopathy activity [14], lifelong pre- muscles [8]. This ubiquitous protein, encoded by the SLC9A1 gene, has ventive treatment with Rimeporide led to a dramatic reduction in several important physiological and pathological influences on mam- mortality and prevented myocardial necrosis and hypertrophy, as well malian cells as a result of its activity. In normal conditions, NHE-1 as Ca2+ accumulation [14,24]. Present preclinical findings suggest maintains intracellular pH (pHi) and volume by removing one in- that Rimeporide has a potential to be a successful pharmacological tracellular proton (H+) ion in exchange for a single extracellular so- approach to the cardiac and skeletal dysfunction observed in DMD. dium (Na+) ion [9]. In certain pathological conditions, NHE-1 is ac- tivated, leading to a rapid accumulation of sodium in cells [8] and an 1.3. Summary of clinical data with Rimeporide in adults acidification of the extracellular space. The high sodium concentration drives an increase in calcium (Ca2+) via direct interaction and reversal Rimeporide was developed up to clinical Phase I for the treatment of of the Na+/Ca2+ exchanger (NCX). The resulting accumulation of advanced congestive heart failure (CHF) by Merck KGaA (Germany). calcium triggers various pathways leading to cell death. As one ex- Development was discontinued for strategic reasons by Merck KGaA ample, NHE-1 is known to contribute to cardiac hypertrophy [10]. The despite a data package including preclinical pharmacology studies, 7 concept of NHE-1 involvement in cardiac pathology has been adopted clinical pharmacology studies involving 145 healthy adults and a clin- for decades and is supported by a plethora of experimental studies ical study in 21 patients with CHF, with oral and/or intravenous (IV) demonstrating effective NHE-1 inhibition in protecting the myocardium dosing. In 2013, EspeRare entered in a license agreement with Merck against ischemic and reperfusion injury as well as attenuating myo- KGaA for the repositioning and development of Rimeporide. cardial remodeling and heart failure [11]. Rimeporide was well tolerated in healthy male subjects as a single The cardioprotective effects of NHE-1 inhibitors, including oral dose up to 600 mg, as multiple oral doses three times a day (TID) Rimeporide, have been extensively studied in various animal models of up to 600 mg daily for seven days, and as a single IV dose of 350 mg. No myocardial infarction and dystrophic cardiomyopathy including DMD. dose limiting toxicity was observed in these studies and a maximum A recently completed study in Golden Retriever muscular dystrophy tolerated dose could not be defined. Adverse events (AEs) included (GRMD) dogs has confirmed the cardioprotective role of Rimeporide chest discomfort, abdominal discomfort, postural dizziness, vaso-vagal after a treatment in a preventing setting [12]. Other preclinical ex- episodes, dizziness, headache, light-headedness, paraesthesia of mouth, periments [13,14] have underlined the significance of myocardial ne- tongue, hands, rash, respiratory tract disorders, diarrhoea, and elevated crosis, due to pH abnormalities as well as calcium and sodium im- liver enzymes (in IV study only) and were equally reported in placebo balances in the pathophysiology of heart failure and have demonstrated and treated patients. The intensity of the AEs was mild to moderate. No the beneficial effects of NHE-1 inhibition using Rimeporide in pre- serious AEs were reported. In general, vital signs and laboratory in- venting the deleterious effects of Ca2+ and Na + overload [14]. vestigations did not reveal any dose-limiting effects of Rimeporide. There is also clear scientific rationale supporting the concept that Rimeporide was also well tolerated in patients with CHF when ad- NHE-1 could play an important role in several pathophysiological ministered at 300 mg daily for 8 days on top of standard heart failure processes involved in DMD. In skeletal muscles of DMD patients, due to medications. One serious AE of worsening heart failure was reported, the lack of dystrophin, mechanical stress during contraction leads to which was not related to the study drug but to the fact that this patient 2 S.C. Previtali, et al. Pharmacological Research 159 (2020) 104999 had stopped his heart failure medication. cohort and subsequently received Rimeporide for a total of 4 weeks as Based on an extensive data package including anti-inflammatory shown in Fig. 1. and antifibrotic effects in mdx mice, improved survival in cardiomyo- The primary objective of this study was to evaluate the safety of pathic hamsters and cardioprotection in GRMD dogs [12, 13, 14, 23] Rimeporide in ambulant DMD boys. Secondary objective was to eval- and good safety and tolerability in adults, EspeRare has commenced the uate PK profile of Rimeporide. Exploratory objectives aimed to quantify clinical development of Rimeporide for boys with DMD, with the main biomarkers of cardiac and skeletal muscle inflammation, metabolism or goal of preventing or slowing the progression of myocardial damage in injury, as well as other markers of efficacy mechanistically related to these children. Rimeporide mode of action or to DMD pathophysiology. These bio- This Phase 1b open label study (Esperare RIM4DMD, clinical- markers included serum/plasma and imaging assessments. trials.gov NCT02710591, EudraCT number 2015-002530-50) aimed to evaluate the safety, tolerability, pharmacokinetics and to explore bio- 2.3. Selection of doses in the study markers of efficacy of multiple ascending oral doses of Rimeporide in young ambulant patients with DMD. Given the rarity and morbidity of DMD, it was aimed to start dosing at a safe, yet pharmacologically effective dose. Dose finding studies in 2. Materials and methods animal models indicated that the exposure/concentration range that would have an efficacious response were ranging from 500 to 2500 ng/ 2.1. Investigational medicinal product mL [13]. Thus, simulated plasma concentrations for all cohorts were selected to fall within this range. The active drug substance was manufactured by Merck KGaA. The The dose-escalation procedure also included the fewest dose levels investigational medicinal product (IMP) Rimeporide was provided as without escalating up to exposures of overt toxicity. It was intended to hard gelatine 25 mg or 50 mg capsules, in bottles of 50 capsules each. proceed cautiously to take into account both the safety profile known to Rimeporide capsules were manufactured by GLATT Pharmaceutical date and emerging safety data in this young and vulnerable patient Services GmbH. Rimeporide hard gelatine capsules were then supplied population. Further dose escalation commenced only after safety and to study sites by Clinical Supplies Management Holdings, Inc. (CSM). PK data from all patients of the previous cohort had been reviewed by The capsules are immediate release formulations. The capsules contain the independent Safety Monitoring Committee (SMC). The re- mannitol, hydroxypropyl methylcellulose, low-substituted hydro- commendation to escalate to the next cohort was given by the SMC. xypropyl cellulose, and magnesium stearate as excipients. 2.4. Study assessments 2.2. Study design and conduct Safety assessments were conducted at the screening visit and after The study was designed as an open-label, multiple dose study with 4 one, two and four weeks of treatment, as well as within 1–2 weeks ascending dose cohorts. Scientific Advice was received from the following the end of treatment period. The clinical safety in these young European Medicines Agency (EMA) on the study design before study patients was carefully evaluated throughout the study by experienced initiation. It was a multicenter European study (UK, France, Italy, clinical staff. The safety assessments included: physical examinations, Spain). AEs/serious AEs, vital signs, diastolic and systolic blood pressure in The study protocol was approved by the Ethics Committees at each supine and standing positions, heart rate in same position (supine and participating site and National Competent Authority from each country standing) and respiratory rate; 12-lead electrocardiogram (ECG) in- prior to the study initiation. The legal guardian for each study parti- cluding QT (reviewed both locally by a cardiologist and centrally by an cipant signed informed consent prior to any study procedures. Main independent cardiologist). Laboratory parameters (from blood and criteria for inclusion were male subjects between 6–14 years of age at urine samples) included haematology and biochemistry panel. Gastrin study entry, genetically confirmed DMD, able to walk at least 75 m was also measured in serum as it was shown in some animal studies to independently and on a stable dose of corticosteroids for at least 6 increase and anecdotal and moderate elevations in blood gastrin were months prior to baseline. observed in humans in one of the previous phase I study. Finally, the Oral doses of Rimeporide from 50 mg to 300 mg three times daily number of patients withdrawn for safety issues was also captured. (TID) were administered. Each subject participated in only 1 dose For the PK assessments, as advised by the EMA Guideline on the Fig. 1. Dose escalation process in RIM4DMD phase I trial. Abbreviation: kg: kilograms; mg: milligrams TID: three time a day, 4w: 4weeks, N: number of subjects, PK: Pharmacokinetic data. 3 S.C. Previtali, et al. Pharmacological Research 159 (2020) 104999 clinical investigation of medicinal products for the treatment of commercially available multiplex beads immunoassay (Luminex Duchenne and Becker muscular dystrophy [25], a population PK ana- Performance assay, Human Obesity Panel, R&D Systems, Minneapolis, lysis model developed by Calvagone Sarl (France) was used based on USA) according to supplier’s instruction. prior PK data obtained with Rimeporide in adults. A 3-compartment Creatine kinase total (CK T) and isoenzymes (CK MM and MB) were disposition model with an absorption phase and a first order linear measured by the central laboratory Eurofins. CK total and isoenzymes elimination from the central compartment was designed. Using this 3- to monitor muscle damage were measured from serum by Roche compartment distribution population PK model, a sparse sampling ap- Imidazole buffered NADPH rate reaction for CK total and by Sebia proach including 6 time points over the study was derived from simu- Agarose electrophoresis for CK isoenzymes. lated PK profiles in children to best fit to the model. On day 1 of treatment, one time point in the absorption phase, one time point near 2.6. Quality assurance the maximum plasma concentration (Cmax) and two time points in the biphasic decay were deemed appropriate to document the PK model in Quality assurance and quality control systems were implemented DMD boys while minimizing burden of blood sampling. At the end of and maintained using written standard operating procedures to ensure the 4-week treatment, only 2 points were needed to confirm the lack of that the trial was conducted and data were generated, documented time dependency of Rimeporide PK profile. Given that 20 patients were (recorded), and reported in compliance with the protocol, Good Clinical planned to be enrolled, the amount of data for validation of the po- Practice, and the applicable regulatory requirement(s). pulation PK model was 120 concentration time points. On Study Day 1, samples were taken pre-dose and within the following time intervals 2.7. Statistical methods and analysis (0.5–1 h), (1–2 h), (2.5–3.5 h) and approximately 6 h after the first or the second dose. On study day 28, 2 samples were taken at the fol- Since this study was descriptive and exploratory in nature, no lowing time interval (0.5–1 h) post dose and approximately 6 h after the formal sample size calculation was performed. A sample size of 20 last dose. For the purpose of the population PK model, real sampling evaluable patients receiving active drug was proposed for this phase 1b times were considered for the analysis. study with no placebo control. This was discussed and deemed accep- For the PD imaging, all patients were proposed to have a Nuclear table by the Committee for Medicinal Products for Human Use during Magnetic Resonance Imaging (NMRI) at baseline and week 4 to explore the EMA scientific advice meeting (EMA/CHMP/SAWP/376863/2015). changes in NMRI indices in skeletal muscle (water T2, fat fraction). The The Intention to treat (ITT) population consisted of all patients who acquisitions were performed locally in both lower limbs of the patients. received at least one dose of study drug. This population was defined For the PD biomarker analysis, two serum/plasma samples were also as the Safety population. The PK and exploratory PD variables were analysed for each subject: baseline (pre-treatment), and after four analysed on the ITT population. weeks of daily treatment with Rimeporide at the indicated dose level For the safety data, descriptive statistics were provided for all (post treatment). The selection of biomarkers was based on a semi- variables in summary tables by cohort according to the type of variable targeted and targeted analysis of circulating proteins according to summarised. AEs were tabulated by body system and by preferred term Rimeporide’s mode of action, DMD pathological processes (inflamma- for each dose level. AEs were also tabulated by severity and relationship tion, fibrosis and muscle injury) and already established biomarkers in to the study medication. Summaries were produced for Serious AEs DMD patients. (SAEs). For each clinical laboratory test, individual patient values were listed and changes from baseline were calculated and summarised. 2.5. Sample management and analysis 2.7.1. Population PK analysis Collection kits, shipment material and documentation, and a la- Using population PK (PPK), PK data were acquired by analyzing boratory manual were provided by the Sponsor to each clinical site data pooled from healthy volunteers, CHF patients and DMD patients. before any patient inclusion in the study. Safety blood samples were Model development, model diagnostics and simulations were per- handled and analysed locally. Remaining plasma/serum aliquots were formed using NONMEM 7.3 and PsN 4.6.0 by Calvagone Sarl [28]. stored on site in secured and controlled freezers until shipment by an Primary PK parameters like clearance and volume of distribution were authorised carrier to Eurofins central laboratory in the Netherlands. directly obtained from the final NONMEM run. The following PK Temperature was monitored during shipment. parameters such as the Cmax, the area under the curve at 24 h (AUC24), PK plasma samples were transferred to Nuvisan GmbH, Germany for the time at which Cmax is observed (Tmax), the drug elimination half- the analysis of plasma concentrations of Rimeporide using a previously life (T1/2) and the time spent above the pharmacological targeted validated LC–MS/MS method. concentration (defined as > 500 ng/mL) over 24 h (TAT) were obtained PD samples (serum and plasma) were transferred to the Serum bank by a non-compartmental analysis (NCA) from simulated PK profiles of of Geneva University Hospital or to KTH Royal Institute of Technology, the first day and the last day of administration. NCA was performed Sweden or to Olink for analysis on behalf of the Department of Human using SAS software (version 9.4). An empirical Bayesian feedback es- Genetics, Leiden University Medical Center, The Netherlands. Two- timation (MAXEVAL = 0) was performed on the simulated dataset (on hundred and thirty protein biomarkers were measured with four dif- data points every 0.05 h) using the final PK parameter estimates from ferent technologies: Olink’s Proximity Extension Assay (PEA) [26], the final Rimeporide PK model and the actual dosing schedule for each Electrophoresis, ELISA and bead based immuno-Luminex assay [27] in DMD patient. In order to evaluate the predictive performance of the two types of biological matrix (serum and plasma). The Olink PEA final model, a prediction corrected visual predictive check (VPC) was immunoassay technology was used to measure 2 cardiometabolic pa- performed on the concentration-time data of the different studies [29]. nels of 92 protein biomarkers each. Another 48 biomarkers were se- The PPK model explored the influence of the following covariates: lected and analysed at KTH by suspension bead array using antibodies alanine transaminase (ALT), aspartate transaminase (AST), bilirubin, validated by western blot, immunoassay and immunochemistry. Active body mass index (BMI), bodyweight (BW), glomerular filtration rate TGF-β1, MMP-9, and PINP were measured from serum by enzyme- (GFR), serum creatinine, and lean body mass (LBM). linked immunosorbent assay ELISA kits (R&D Systems, Abingdon, UK) Descriptive statistics were also used for the main PK parameters and using the protocol recommended by the manufacturer. For the measure presented by dose cohorts and by day of administration. of active TGF-β1 (free + latent TGF-β1), serum samples were activated with reagents as recommended by manufacturer. 2.7.2. PD exploratory analysis TNFα, IL-6, IL-34 and CXCL10/IP-10 were measured by a Descriptive statistics were calculated for all parameters, separately 4 S.C. Previtali, et al. Pharmacological Research 159 (2020) 104999 for each cohort. Graphical and tabular presentations were produced schedule of Rimeporide was excellent: mean compliance was 97.1 %. when appropriate. An additional exploratory statistical analysis was performed for the plasma/serum biomarkers with the aim of identifying candidate biomarkers associated with Rimeporide treatment in a dose- 3.2. Primary objective: safety of Rimeporide after repeated dosing in young dependent or dose-independent manner. DMD boys For treatment association analyses, the following linear mixed effect models [30] were fitted to data: As already established in previous studies in healthy adults, Rimeporide had a good safety profile in young boys with DMD over a B ∼ TRT + Age + BMI + (1|SUBJID) wide range of doses. No SAE related to study medication was reported Where B is the biomarker expression (at Day 0 or 28), TRT is a dummy and no withdrawal due to adverse events or for any other reasons was variable equal to 0 at Day 0 and 1 at Day 28, Age and BMI (Body Mass observed. Index) are covariates, and SUBJID is a subject-specific random intercept Twelve patients reported a total of 37 treatment emergent adverse that accounts for correlation between repeated measurements from the events (TEAEs). Thirty-five TEAEs were of mild intensity and 2 were same patient. A type II analysis of deviance was performed. Wald chi- reported as severe. All were self-limiting. There was no evidence for an square p-values of the TRT terms were corrected using the Benjamini- increase in frequency or severity of AEs with increasing dose. The most Hochberg method [31] and biomarkers with False Discovery Rate frequently reported TEAEs were headache (6 events reported in 5 pa- (FDR) ≤10 % were reported. tients); diarrhoea (4 events reported in 3 patients); and nasopharyngitis (4 events reported in 4 patients) as shown in Table 3. The only SAEs reported were diarrhoea and vomiting, in one patient 3. Results in Cohort 3 not reported as related to study drug. The symptoms oc- curred simultaneously in a single episode considered to be due to food 3.1. Disposition of subjects and demographics poisoning. This was corroborated by the fact that the patient ś mother, experienced similar symptoms on the same day after eating the same A total of 24 patients were screened and 20 enrolled in 4 clinical (fish) meal. centres (Fig. 2): one patient was a screened failure as he was previously The only events which were reported as adverse drug reactions involved in another research study (less than 4 weeks before IMP ad- (ADRs) were an event of urticaria in one subject in Cohort 2, two events ministration) and the 3 other patients did not receive the study drug of flushing in one subject in Cohort 4 and one episode of nasophar- within 1 month of screening due to the unavailability of the IMP. yngitis in the same subject. All these events were reported as possibly All enrolled patients completed the study. The analysis set consisted related to the study drug and none as probably related. Therefore, apart of 20 DMD patients, aged from 6–11 years with a confirmed diagnosis from transient flushing which was observed in one subject in Cohort 3 of DMD. The demographic and clinical information for the subjects on one occasion and in one subject in Cohort 4 on two occasions, there enrolled are reported in Table 1. was no emergent pattern of AEs. A focus was given to concomitant medications that were relevant for There were no consistent or clinically significant effects on vital DMD throughout the study duration as shown in Table 2. All patients signs (data not shown). Standing and supine systolic and diastolic blood but one in cohort 3 (no corticosteroid at screening and during the entire pressure as well as heart rate appeared to be unaffected by the treat- study duration) were on a stable dose of corticosteroids. In addition, ment. Clinical laboratory parameters (biochemistry and haematology another patient in cohort 3 received omeprazole for the treatment of parameters) revealed no clinically significant changes over treatment heartburn at a stable dose of 10 mg daily. Treatment was started three period. No modifications of gastrin levels, a surrogate marker for signs months before inclusion in the study and continued throughout the of adverse effect on gastric parietal cells [32], were observed after a study period. four-week treatment. In general, vital signs and laboratory investiga- Finally, the overall compliance and adherence to the dosing tions did not reveal any adverse effects of Rimeporide. Most notably, Fig. 2. Disposition of subjects. Abbreviation: N: number of subjects: TID: three time a day. 5 S.C. Previtali, et al. Pharmacological Research 159 (2020) 104999 Table 1 Patient demographic and characteristics. Characteristics Cohort 1 Cohort 2 Cohort 3 Cohort 4 All N=5 N=5 N=5 N=5 N = 20 Age (years) Mean (SD) 8.4 (1.7) 8.2 (1.5) 8.8 (1.6) 9.2 (0.4) 8.7 (1.3) Median 8.0 8.0 8.0 9.0 9.0 Range 6; 10 6; 10 7; 11 9; 10 6; 11 Height (cm) Mean (SD) 126.4 (6.9) 127.6 (2.3) 125.4 (6.8) 121.2 (9.3) 125.2 (6.7) Median 127.0 127.0 125.0 120.0 125.5 Range 119; 135 125; 130 117; 134 110; 135 110; 135 Weight (kg) Mean (SD) 28.8 (9.0) 29.0 (5.2) 29.0 (4.9) 25.8 (6.9) 28.1 (6.3) Median 24.6 27.0 27.7 23.5 25.7 Range 20.4; 39.0 24.5; 37.6 24.0; 35.9 21.0; 37.9 20.4; 39.0 BMI (kg/m2) Mean (SD) 17.7 (3.9) 17.8 (3.2) 18.3 (1.9) 17.3 (2.2) 17.8 (2.7) Median 17.1 17.3 17.7 17.2 17.4 Range 13.6; 22.2 15.4; 23.3 16.3; 21.3 14.6; 20.8 13.6; 23.3 Time since DMD diagnosis (years) Mean (SD) 4.5 (2.7) 4.9 (1.5) 5.5 (2.5) 6.6 (2.7) 5.4 (2.3) Median 3.4 4.6 4.8 6.4 4.7 Range 2.9; 9.1 3.2; 7.2 2.2; 8.3 3.2; 9.6 2.2; 9.6 Abbreviation: BMI: body mass index; cm: centimetres; DMD: Duchenne muscular dystrophy; kg: kilograms; mg: milligrams; N: number of subjects; SD: standard deviation. Table 2 goodness of fit (GOF) plots showed a good agreement between the Relevant concomitant medications for DMD by patients throughout the study observed and predicted Rimeporide concentrations. Epsilon shrinkage duration. was acceptable, indicating that the GOF plots are reliable. A prediction- Anatomical Main Cohort 1 Cohort 2 Cohort 3 Cohort 4 All patients corrected visual predictive check for the final PPK model showed that Group N=5 N=5 N=5 N=5 N = 20 most of the observed concentrations fell within the 95 % confidence Chemical interval of the simulated data. The final PPK model described the data Subgroup well and was able to explain part of the variability in exposure on the Preferred Name basis of some covariates (Glomerular filtration rate, serum creatinine Systemic hormonal preparations, excl. sex hormones and insulins and food status). Finally, the PK model had an adequate predictive Glucocorticoids 5 (100 %) 5 (100 %) 4 (80 %) 5 (100 %) 19 (95 %) performance to simulate PK profiles in DMD children and to derive Deflazacort 0 3 (60 %) 2 (40 %) 5 (100 %) 10 (50 %) individual PK parameters by NCA. Prednisone 5 (100 %) 1 (20 %) 0 0 6 (30 %) Prednisolone 0 1 (20 %) 3 (60 %) 0 4 (20 %) Overall, Rimeporide PK in DMD patients was dose linear with no Alimentary tract and metabolism sign of any accumulation with time. Rate of absorption was slowed Proton pump 0 0 1 (20 %) 0 1 (5 %) down under fed conditions without changing the drug exposure (AUC). inhibitors Based on the identified covariates, dose adjustment did not seem to be Omeprazole 0 0 1 (20 %) 0 1 (5 %) necessary according to body weight. Oral dosing with immediate re- Abbreviations: %: percentage; N: number of subjects. In cohort 3, one patient lease capsules resulted in a quick and extensive absorption from the did not receive any glucocorticoid while another one received prednisolone and gastrointestinal tract. Cmax occurred within 1–2 h post dose on both deflazacort. Days 1 and 28 as shown in Table 4. The drug elimination half-life across all doses was short and unchanged between Day 1 and Day 28, aver- Rimeporide was devoid of any blood pressure or heart rate lowering aging about 3–4 h and justifying a three times daily administration to effects. maintain plasma levels above pharmacological levels. Cmax and AUC24 There were no clinically important changes in 12-lead ECGs. All values were generally comparable on Days 1 and 28 at all dose levels values for QTcB (corrected QT interval based on Bazett formula) or suggesting no accumulation occurs upon multiple dosing. PK para- QTcF (corrected QT interval based on Fridericia formula) were below meters increased linearly with increasing doses. When administered 450 msec, apart from two values in two subjects, for whom the eleva- under fed condition, a decrease of Cmax and a shift of Tmax are ob- tions were not confirmed by the central reading. Some increases in served while no impact on AUC24 is noticed. Regarding the time spent QTcB intervals above 30 msec were seen in a few patients, but these above targeted pharmacological concentration (e.g. > 500 ng/mL) were either not present on both the local and central readings or were over 24 h, TAT in cohort 1 was around 6 h suggesting that doses in not accompanied by changes in QTcF, except for one value at week 2 in cohort 1 might be too low to maintain plasma levels above the phar- a subject in cohort 3 receiving 450 mg/day. macological target concentration throughout the day. In cohorts 2, 3 and 4, TAT increased to 17 h suggesting that doses above 100 mg TID 3.3. Secondary objective: pharmacokinetic profile of Rimeporide in young were more suitable to lead to a sustained 24h-exposure to Rimeporide DMD boys and potentially to a sustained pharmacological effect. Finally, PK pro- files in children were compared to previous PK studies in healthy The objectives of this PK analysis were 1) to investigate the PK adults. It was observed that PK profiles in children result in a slightly profile of Rimeporide after single- and multiple-dose, and 2) to explore higher exposure than in adults. the linearity of the different doses administered in boys ages 6–11 years with DMD. The PK full dataset consisted in a pool of over 3400 Rimeporide concentrations, among healthy adults, CHF patients and 20 DMD children. The model qualification on the final model through the 6 S.C. Previtali, et al. Pharmacological Research 159 (2020) 104999 Table 3 Treatment-Emergent Adverse Events classified by System Organ Class and Preferred Term defined in term of number of patients and of events. System Organ Class Cohort 1 Cohort 2 Cohort 3 Cohort 4 All patients Preferred Term N=5 N=5 N=5 N=5 N = 20 Any treatment-emergent adverse event 2 (6 events) 1 (3 events) 5 (15 events) 4 (13 events) 12 (37 events) Nervous system disorders 1 (1 event) 0 3 (3 events) 3 (4 events) 7 (8 events) Headache 1 (1 event) 0 2 (2 events) 2 (3 events) 5 (6 events) Dizziness 0 0 1 (1 event) 0 1 (1 event) Presyncope 0 0 0 1 (1 event) 1 (1 event) Gastrointestinal disorders 0 1 (1 event) 4 (8 events) 1 (1 event) 6 (10 events) Diarrhoea 0 0 3 (4 events) 0 3 (4 events) Abdominal pain upper 0 0 1 (2 events) 1 (1 event) 2 (3 events) Vomiting 0 0 2 (2 events) 0 2 (2 events) Mouth ulceration 0 1 (1 event) 0 0 1 (1 event) Infections and infestations 0 1 (1 event) 2 (2 events) 1 (1 event) 4 (4 events) Nasopharyngitis 0 1 (1 event) 2 (2 events) 1 (1 event) 4 (4 events) Respiratory, thoracic and mediastinal disorders 0 1 (1 event) 1 (1 event) 2 (2 events) 4 (4 events) Cough 0 1 (1 event) 1 (1 event) 1 (1 event) 3 (3 events) Oropharyngeal pain 0 0 0 1 (1 event) 1 (1 event) General disorders and administration site conditions 1 (2 events) 0 0 1 (1 event) 2 (3 events) Chills 1 (2 events) 0 0 0 1 (2 events) Pyrexia 0 0 0 1 (1 event) 1 (1 event) Vascular disorders 0 0 1 (1 event) 1 (2 events) 2 (3 events) Flushing 0 0 1 (1 event) 1 (2 events) 2 (3 events) Ear and labyrinth disorders 0 0 0 1 (1 event) 1 (1 event) Tympanic membrane 0 0 0 1 (1 event) 1 (1 event) perforation Injury, poisoning and procedural complications 1 (1 event) 0 0 1 (1 event) Fall 1 (1 event) 0 0 1 (1 event) Abbreviation: N: number of subjects. Table 4 indices over the treatment period (data not shown). PK parameters after oral administration of Rimeporide from 150 to 900 mg daily obtained with non-compartmental analysis at Day 1 and Day 28 by dose group. 3.4.2. Serum biomarkers From the total of 230 biomarkers that were assessed in this study, Cohort/ Dose (mg) Day Statistics T1/2 Cmax AUC24 TAT 10 proteins were analysed with two or more assays. Cross-correlations (h) (ng/mL) (ng h/mL) (h) across technology platforms showed lack of correlation, probably due to Cohort 1 1 N 5 5 5 5 the use of different antibodies and sample matrices (serum and plasma). 150 to 225 Mean 3.3 1286 8857 6.6 Twelve proteins were significantly modulated by 4-week treatment SD 0.5 568 3868 4 with Rimeporide, as assessed by the comparison of baseline and 4-week 28 N 4 4 4 4 Mean 3.4 1361 10423 8.4 follow-up samples. There was no association between biomarkers levels SD 0.6 431 1208 1.7 and dose, age and BMI, which was not surprising considering the small Cohort 2 1 N 5 5 5 5 range of variations in these covariates. These 12 biomarkers, listed in 300 to 450 Mean 4.6 1987 19996 17 Table 5, represent potential new candidate biomarkers to evaluate the SD 1.4 650 4903 3 biological efficacy of Rimeporide non-invasively. 28 N 5 5 5 5 Mean 4.66 2077 18667 15 All proteins showed down-regulation upon treatment with SD 1.46 974 5669 3 Rimeporide with the exception of TR showing an upregulation. Among Cohort 3 1 N 5 5 5 5 modulated biomarkers, IGFBP1 (p = 1.7e-8; FDR = 4e-6) is of parti- 450 to 600 Mean 3.9 3013 29390 18 cular statistical relevance (Fig. 3). Of note, the concentration of the SD 0.9 730 4510 1 28 N 5 5 5 5 well-known CK T (p = 0,0007; FDR = 0,24) and its CK MM Mean 3.76 2817 26332 16 (p = 0,0009; FDR = 0,24) were reduced by the treatment. SD 0.991 675 3026 2.3 From these proteins significantly associated to Rimeporide treat- Cohort 4 1 N 5 5 5 5 ment, 8 were already studied in large scale protein biomarker discovery 600 to 900 Mean 3.9 3819 36179 19.0 longitudinal studies in DMD patients using SOMAscan and described in SD 1 743 7647 2 28 N 5 5 5 5 case control comparisons between DMD patients and aged-matched Mean 3.75 3909 35987 18 controls [33,34]. These already studied biomarkers are CK MM, SD 0.910 846 6981 1.1 IGFBP6, IGFBP1, MB, KLK6, CCL15, FAS and TNFα. In these long- itudinal studies, all these proteins plus TR in Spitali’s study [34] were Abbreviations: AUC24: area under the curve at 24 h; Cmax: maximal con- shown to be stable over time in DMD patients except for CK, which is centration over 24 h after the first administration of the day; N: number; SD: known to be variable in such a population. standard deviation; TAT: time spent above the target concentration of 500 ng/ ml over 24 h; T1/2: terminal elimination half-life. 4. Discussion 3.4. Exploratory pharmacodynamic biomarkers Seven phase I studies have been conducted in healthy adults with 3.4.1. Imaging biomarkers Rimeporide during its initial development for patients with congestive No notable change (worsening or improvement) in the NMRI indices heart failure and have shown no safety concern, a good tolerability and was observed following Rimeporide dose escalation and during the 4- a good PK profile. No safety and PK data were available in pediatrics week treatment. There was however no worsening of skeletal muscles nor in young DMD boys. The objectives of this phase I study were to 7 S.C. Previtali, et al. Pharmacological Research 159 (2020) 104999 Table 5 Circulating biomarkers associated with Rimeporide treatment. Rank Biomarker Biomarker name Source Term p-value FDR 1 IGFBP1 Insulin Growth Factor Binding Protein 1 OLink −2.7123 1.7e-8 4e-6 2 TR Transferrin receptor (also called TFRC) OLink 0.5252 3.0e-6 0.0004 3 SCGB3A2 Secretoglobulin family 3A-member-2 OLink −0.5466 1.3e-5 0.001 4 CCL15 C-C motif chemokine ligand 15 OLink −0.4489 0.0003 0.17 5 KLK6 Kallikrein 6 OLink −0.2612 0.0004 0.20 6 FAS Fas Ligand OLink −0.2823 0.0007 0.24 7 CK T Creatine Kinase total Electrophoresis −0.3851 0.0007 0.24 8 TNFα Tumor Necrosis Factor α Luminex −0.1726 0.0009 0.24 9 CK MM Creatine Kinase MM isoform Electrophoresis −0.3886 0.0009 0.24 10 MCP1 Monocyte Chemoattractant Protein OLink −0.5675 0.0016 0.39 11 MB Myoglobin OLink −0.6763 0.0036 0.79 12 IGFBP6 Insulin Growth Factor Binding Protein 6 OLink −0.3144 0.0044 0.89 Abbreviations: FDR (False discovery rate): p-value corrected for multiple testing. provide complementary data to the existing data package on Decreased levels of IGF binding proteins are expected to lead to in- Rimeporide safety, tolerability, PK and biomarker(s) of biological re- creased levels of free IGF1 which appeared to be effective in mdx mice sponse in pediatric patients with DMD. in increasing muscle strength and preventing muscle necrosis The safety and tolerability profile of Rimeporide in children was [36,38,39]. IGF1 has also been shown to have cardioprotective effect on good and similar to that observed in healthy adults. No treatment-re- hypertensive rats by inhibiting NHE-1 activity necrosis [40]. Such lated adverse events, nor dose limiting toxicity were observed as well as biomarker signature nicely correlates with the recent cardioprotective no consistent pattern of non-serious adverse events. There was also no effect observed in GRMD dogs treated with Rimeporide [12]. significant modification in the laboratory values. ECGs and QT intervals A significant reduction in total Creatine Kinase CK T (p < 0,0007; did not present any significant change from baseline as confirmed by a FDR 0.24) and its isoform CK MM (p < 0,0009; FDR 0.24) were also central reading. No clinically relevant change in blood pressure was reported. Both enzymes are known to be increased in boys with DMD observed. owing to muscle damage [27]. Downregulation of both enzymes in such A PPK model was applied and showed that the PK profiles in chil- young patients (6–11 years old) could be suggestive of a muscle pro- dren were increasing linearly with rising doses, with no sign of accu- tective effect by Rimeporide. mulation upon multiple dosing and that interaction with food did not Although still exploratory, these biomarkers results could prove to significantly impact exposure. Concentrations above targeted pharma- be helpful in a confirmatory phase II study for dose/dosing regimen(s) cological exposure (500 ng/mL) were achieved in all cohorts although adjustment. Positive findings on these biomarkers may help to predict in cohort 1, TAT was significantly shorter than in the other cohorts. clinical benefit non-invasively at early stage of clinical development, Particular efforts were also dedicated to the identification of ima- and may also confirm the pharmacological efficacy of Rimeporide while ging and serum candidate biomarkers. While skeletal NMRI indices did further understanding its mechanism of action. not change significantly over the 4-week treatment period (no im- provement and no worsening), this was expected given the short 5. Conclusions treatment duration. Prior studies [35] showed that therapeutic effect of corticosteroids can be observed no earlier than 3 months of treatment This phase Ib study establishes the safety of Rimeporide in young by an initiation of a decrease in muscle T2, consistent with the anti- boys with DMD along with encouraging early changes in PD markers of inflammatory action of corticosteroids. efficacy and biological effects. There was however, preliminary evidence for biological effect on Cardiac disease is one of the leading causes of death in DMD pa- several serum/plasma biomarkers relevant for Rimeporide mode of tients. There is today no drug that specifically treats the cardiac pa- action including inflammation, muscular repair and injury. The ob- thogenesis related to DMD. Heart Failure drugs such as ACE inhibitors, served biomarker changes provide encouraging evidence of the biolo- beta blockers, angiotensin II receptor blockers and eplerenone are gical activity of Rimeporide at all doses tested over a 4-week period in prescribed despite the certainty of evidence being very low. children with DMD. Although the biomarker changes were not dose- Interestingly, Rimeporide working through NHE-1 inhibition, is a first dependent, it is notable that even at the lowest dose, Rimeporide in class treatment and addresses some of the pathways that likely are plasma peak concentrations were within the expected range of phar- unique and target a portion of the DMD pathology, regardless of the macological concentration. However, TAT in cohort 1 was much shorter mutation, attributable to its more directly related mechanism of action. (6 h) than that in cohorts 2, 3 and 4 where TAT was around 17 h. It is A larger and longer clinical study with a placebo control is under not known at this stage whether a continuous inhibition of NHE-1 is preparation to confirm the beneficial effect of Rimeporide to prevent needed to achieve an optimal efficacy. the cardiac and skeletal degeneration. The rationale for future studies is For this study, we selected 230 candidate serum biomarkers based strongly underpinned both by the positive effects on function and sur- on the disease’s hallmarks and Rimeporide mode of action. 12 protein vival consistently seen in dystrophic animal models and the good tol- biomarkers were found to be significantly correlated with treatment. erability and the encouraging comprehensive biomarkers efficacy pro- For example, a significant reduction in circulating levels of IGFBP1 and file in patients, suggesting that Rimeporide has potential to provide a IGFBP6 was observed. The levels of circulating IGFBP1 & 6 are not much-needed therapeutic approach for young boys with DMD. known to be modulated over time in natural history studies in DMD patients [33,34], suggesting that the decrease of IGFBPs level observed Funding in DMD patients may be associated to Rimeporide and may reflect positive changes in muscle regeneration by different mechanisms. This work was supported by EspeRare as well as AFM Telethon IGFBP1 binding to endogenous insulin-like growth factor-1 (IGF1) was (grants 19163 & 17724), SwissLife, Innosuisse grant (25946.1 PFLS-LS), reported to prevent its regenerative action in dystrophic muscles [36]. PPMD Spain, PPMD Italy and Duchenne UK. This work was also sup- IGFBP1 knockout mice show increased muscle mass and force [37]. ported in part by The Muscular Dystrophy UK Central Grant to the DNC 8 S.C. Previtali, et al. Pharmacological Research 159 (2020) 104999 Fig. 3. Evolution of key biomarkers after a 4-week treatment with Rimeporide. Dot plot showing the reduction of selected individual biomarkers in the 4 cohorts between Week 0 (baseline) and Week4 (end of treatment period). Log transformed concentration data are plotted on the y-axis. 9 S.C. Previtali, et al. Pharmacological Research 159 (2020) 104999 and by the NIHR Great Ormond Street Hospital Biomedical Research [12] B. Ghaleh, et al., Protective effects of rimeporide on left ventricular function in golden Centre. retriever muscular dystrophy dogs, Int. J. Cardiol. S0167–5273 (19) (2020), https://doi. org/10.1016/j.ijcard.2020.03.031 35776-6. [13] M. Chahine, et al., NHE-1 dependent intracellular sodium overload in hypertrophic Declaration of Competing Interest hereditary cardiomyopathy: prevention by NHE-1 inhibitor, J. Mol. Cell. Cardiol. 38 (2005) 571–582, https://doi.org/10.1016/j.yjmcc.2005.01.003. [14] G. Bkaily, D. 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