Treatment of mitochondrial diseases

Abstract

The current invention concerns an innovative treatment for mitochondrial disorders and diseases or conditions associated with mitochondrial dysfunction. In particular, effective and safe dosages of compounds suitable for the treatment of mitochondrial disorders have been established, providing for new treatment regimens and patient populations.

Claims

1.-15. (canceled)

16. A method of treating, preventing, or suppressing symptoms associated with a mitochondrial disorder or with a disease or condition associated with mitochondrial dysfunction by administration of a total daily dose in the range of about 10 to 1000 mg of a compound represented by general structure (I): ##STR00042## wherein, T is a water-soluble vitamin E derivative having a core chromanyl or chromanyl quinone framework and a carboxylic acid moiety substituted at the 2-position, wherein T is connected to nitrogen via the carboxylic acid moiety, as such forming an amide moiety; N* is represented by structure (IIa) or (IIb) ##STR00043## R.sup.1 and R.sup.2 are each independently selected from hydrogen (H), C.sub.1-C.sub.6 alkyl or C.sub.1-C.sub.6 alkenyl, or R.sup.1 and R.sup.2 are joined together and thus form a second linker between the amide nitrogen atom and the distal nitrogen atom, or R.sup.1 is joined with a backbone atom of the linker L in a cyclic structure and/or R.sup.2 is joined with a backbone atom of the linker L in a cyclic structure; R.sup.3 is selected from hydrogen (H), C.sub.1-C.sub.6 alkyl or C.sub.1-C.sub.6 alkenyl, wherein the alkyl or alkenyl moiety may be substituted with one or more halogen atoms, hydroxyl moieties or (halo)alkoxy moieties, or R.sup.3 is absent when the distal nitrogen atom is part of an imine moiety; and R.sup.4 is selected from hydrogen (H) or C.sub.1-C.sub.6 alkyl, wherein the alkyl moiety may be substituted with one or more halogen atoms or (halo)alkoxy moieties; X is an anion, preferably a pharmaceutically acceptable anion.

17. The method of claim 16, wherein the compound is represented by structure (VI): ##STR00044## wherein, N* is —NR.sup.3 or —N.sup.+R.sup.3R.sup.4X.sup.−.

18. The method of claim 16, wherein T is represented by structure (IIIa) or (IIIb): ##STR00045## wherein R.sup.7 is individually a C.sub.1-C.sub.6 alkyl moiety, preferably each R.sup.7 is methyl.

18. The method of claim 16, wherein the total daily dose that is administered is in the range of about 20 to 800 mg, wherein preferably the total daily dose is in the range of about 30 to 700 mg, and wherein more preferably the total daily dose is in the range of between about 30 to 400 mg and wherein more preferably the total daily dose is in the range of between about 30 to 300 mg and wherein even more preferably the total daily dose is in the range of about 150 to 250 mg and wherein most preferably the total daily dose that is administered is about 200 mg.

19. The method of claim 16, wherein the mitochondrial disorder is a disorder selected from the group consisting of: Myoclonic epilepsy; Myoclonic Epilepsy with Ragged Red Fibers (MERRF); Leber's Hereditary Optic Neuropathy (LHON); neuropathy ataxia and retinitis pigmentosa (NARP); Mitochondrial Myopathy, Encephalopathy, Lactic acidosis, Stroke-like episodes (MELAS); Leigh syndrome; Leigh-like syndrome; Dominant Optic atrophy (DOA); Kearns-Sayre Syndrome (KSS); Maternally Inherited Diabetes and Deafness (MIDD); Alpers-Huttenlocher syndrome; Ataxia Neuropathy spectrum; Chronic Progressive External Ophthalmoplegia (CPEO); Pearson syndrome; Mitochondrial Neuro-Gastro-Intestinal Encephalopathy (MNGIE); Sengers syndrome; 3-methylglutaconic aciduria, sensorineural deafness, encephalopathy and neuro-radiological findings of Leigh-like syndrome (MEGDEL); myopathy; mitochondrial myopathy; cardiomyopathy; and encephalomyopathy, SURF1 (COX deficient Leigh syndrome due to complex IV surfeit protein deficiency) and isolated or combined OXPHOS deficiencies with so far unsolved genetic defect including disturbed pyruvate oxidation and ATP plus PCr production rates, wherein preferably the mitochondrial disorder is associated with a m.3242A>G mutation of the mitochondrial tRNA(leu) gene.

20. The method of claim 16, wherein the disease or condition associated with mitochondrial dysfunction preferably is a disease or condition selected from the group consisting of: Friedreich's Ataxia (FRDA); renal tubular acidosis; Parkinson's disease; Alzheimer's disease; amyotrophic lateral sclerosis (ALS); Huntington's disease; developmental pervasive disorders; hearing loss; deafness; diabetes; ageing; and adverse drug effects hampering mitochondrial function.

21. The method of claim 16, wherein a measurable biomarker is used to assess the efficacy of the therapy, wherein preferably the biomarker is selected from the group consisting of FGF21, GDF15, PRDX1 and oxidized glutathione/reduced glutathione ratio, wherein preferably the biomarker is measured ex vivo in serum.

22. The method of claim 16, wherein the compound is administered orally.

23. The method of claim 16, wherein the compound is administered in a solid form or in a liquid form, wherein preferably the compound is admixed with an aqueous solution prior to administration, wherein more preferably the aqueous solution is an isotonic aqueous solution and wherein even more preferably the isotonic aqueous solution is saline.

24. The method of claim 16, wherein the compound is administered at least twice daily, preferably wherein the compound is administered twice daily, wherein more preferably the compound is administered twice daily in two similar or equal doses.

25. The method of claim 24, wherein the interval between two administrations is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hours.

26. The method of claim 16, wherein the subject to be treated is a primate, wherein preferably the subject is a human.

27. The method of claim 16, wherein the subject to be treated has a clinically relevant normal ECG and/or a normal cardiac functioning, wherein preferably the subject to be treated does not have an abnormal QTc of more than 500 ms and/or an abnormal T-wave morphology and/or wherein preferably the subject to be treated does not have a condition selected from the group consisting of cardiovascular disease, alcoholic liver disease, obesity, hypertension and electrolyte disturbances.

28. The method of claim 16, wherein the subject to be treated does not have a concomitant medication that is known to inhibit CYP3A4 and/or PgP.

29. The method of claim 16, wherein the subject to be treated is a human of 17 years or younger.

Description

DESCRIPTION OF THE FIGURES

[0248] FIG. 1. Effect of L-buthionine-(S,R)-sulfoximine (BSO), an inhibitor of glutathione synthesis, on the viability of primary human fibroblasts derived from healthy individuals or derived from patients with Leigh syndrome, MELAS or LHON diseases. One day after treatment with 100 μM BSO, cells were washed and stained with Calcein-AM. Cell viability was determined as a function of fluorescence intensity.

[0249] FIG. 2A, FIG. 2B, FIG. 2C Effect of the compounds on oxidative stress-induced cell death. Primary human fibroblasts derived from patients with Leigh syndrome FIG. 2A, MELAS FIG. 2B or LHON FIG. 2C diseases, were treated with increasing concentrations of the compounds in combination with 100 μM BSO. The following day the cells were washed, stained with Calcein-AM and the fluorescence was measured. Cell viability is depicted as normalized against untreated cells. Each graph depicts the potency of the compound (filled square) and the corresponding metabolite (open square) to prevent cell death at selected concentrations.

[0250] FIG. 3. Posthoc ECG assessment methodology.

[0251] A. Example of an ECG adapted on the Global Superimposed Median Beat (GSMB). B. T wave symmetry index was computed by modeling the T wave in two independent half-Gaussian curves. The standard deviations of these functions (σ1 and σ2) and indicators of the ascending/descending speed.

[0252] FIG. 4A, FIG. 4B, FIG. 4C, FIG. 4D. Mean plasma concentrations of KH176 and its metabolite KH176m after single and multiple dose administration to healthy subjects. FIG. 4A Plasma-concentration/time curve of KH176 for the SAD study (fasted state), loglinear scale. FIG. 4B Plasma-concentration/time curve of KH176m for the SAD study (fasted state), loglinear scale. FIG. 4C. Plasma-concentration/time curve of KH176 for the MAD study, loglinear scale and FIG. 4D Plasma-concentration/time curve of KH176m for the MAD study, loglinear scale.

[0253] FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D. Dose-normalized individual values for Cmax and AUC0-inf of KH176 for the SAD and the MAD study. FIG. 5A Dose normalized Cmax for the SAD study, the horizontal lines depict the geometric mean value. FIG. 5B. Dose normalized AUC0-inf for the SAD study, the horizontal lines depict the geometric mean value. FIG. 5C. Dose normalized Cmax for the MAD study, the horizontal lines depict the geometric mean value and FIG. 5D. Dose normalized AUC0-inf for the MAD study, the horizontal lines depict the geometric mean value.

[0254] FIG. 6A, FIG. 6B, FIG. 6C, FIG. 6D, FIG. 6E, FIG. 6F Posthoc ECG assessment results. FIG. 6A. Change in QTcF (median increase from baseline; SAD study). FIG. 6B. Change in TpTe (median increase from baseline; SAD study). FIG. 6C. Change in the T-wave symmetry index (median increase from baseline; SAD study). FIG. 6D. Change in QTcF (median increase from baseline; MAD study). FIG. 6E. Change in TpTe (median increase from baseline; MAD study). FIG. 6F Change in the T-wave symmetry index (median increase from baseline; MAD study).

[0255] FIG. 7. Exposure-response analysis of KH176 plasma concentrations and a change from baseline for ECG derived TpTe intervals. The upper limit of normal (23.4 ms) is derived from the 95% confidence interval of the predose values. In healthy subjects a dose of 100 mg BID resulted in maximum concentrations ranging from 303-458 ng/mL.

[0256] FIG. 8. Representative example of the changes in the QRS interval, QT-time and the T-wave morphology in an individual in the 2000 mg group.

EXAMPLES

Methods

In Vitro: Oxidative Stress-Induced Cell Death

[0257] To determine the effective concentration of the compounds (the concentrations that protect patient cells against oxidative stress-induced cell death) the applicant established an assay using stressed primary human fibroblasts from patients with Leigh syndrome, MELAS or LHON diseases. Utilising the inherent oxidative stress of fibroblasts from patients with mitochondrial disease, their oxidative burden was further increased by depleting cellular glutathione with an inhibitor of glutathione synthesis, L-buthionine-(S,R)-sulfoximine (BSO). As a result, while fibroblasts from healthy individuals retained full viability, patient fibroblasts exhibited complete cell death within 24 hr of the BSO insult (100 μM) (FIG. 1). Cells were seeded at a density of 3000 cells/well in a 96-well format and incubated with increasing concentrations of compounds in combination with BSO (100 μM, Sigma-aldrich). One day after treatment, the cells were washed twice and stained with a solution of 5 μM calcein-AM (Life technologies C3100MP) during 25 min in 199 medium without phenol red (Life technologies, 11043-023) at room temperature. After 2 washes with PBS (Phosphate buffered saline) the plate was read on a fluorescence plate reader (Fluostar Omega, BMG labtech) and the percentage of cell viability determined as a function of fluorescence intensity.

In Vivo: Placebo-Controlled, Single-Centre Study

[0258] The compounds as described herein were tested in a double-blinded, randomized, placebo-controlled, single-centre study in healthy male volunteers. The single ascending dose (SAD) part has a partial alternating crossover design and the multiple-ascending dose (MAD) part has a sequential group design. Randomization was 2:1 (2 active for each placebo).

Study Population

[0259] For both the SAD and MAD study, healthy men between 18 and 55 years of age with a body mass index (BMI) of 18.0-30.0 kg/m.sup.2 were recruited. Good physical and mental health was established by medical history, physical examination, electrocardiogram (ECG) and vital signs recording, and results of clinical chemistry, hematology and urinalysis testing within 4 weeks prior to the first dose. Participants agreed to stay in the clinic during the first 24 hours after dosing (SAD) and during Day 8 (MAD) and refrain from multivitamins and dietary supplements and grapefruit juice at least 14 Days prior to the first dosing, from alcohol 7 Days prior to the first dosing, from strenuous exercise, beverages containing quinine and from xanthine-derivates (e.g. caffeine) 48 hours prior to the clinical admission and during the study. Only non-smokers (at least 3 months) were eligible for inclusion. Exclusion criteria included: clinically significant allergies, positive serology for hepatitis B surface antigen, hepatitis C antibodies, HIV1 of HIV2, history of alcohol or drug abuse in the past 2 years, history of cancer, surgery or active illness of the gastro-intestinal tract that might interfere with absorption, intake of any enzyme-affecting drugs in the 30 days prior to the first dosing period, use of any medication, herbal medicine or dietary supplement from 14 days prior to the first dosing (except for occasional paracetamol intake), participation in a trial of an investigational product in the 2 months prior to the first dosing, blood donation in the 2 months prior to the first dosing, history of hypersensitivity or idiosyncrasy to any of the components of the investigational drug, positive drug, alcohol or cotinine test at screening or admission, clinically relevant abnormal laboratory findings, ECG recordings, vital signs or physical or mental findings at screening, and/or major surgery and/or prolonged immobilisation (more than 2 weeks) within the 3 months prior to the screening.

Study Drug

[0260] A representative compound was used for testing in humans. More precisely, KH176 (or ((S)-6-hydroxy-2,5,7,8-tetramethyl-N—((R)-piperidin-3-yl)chroman-2-carboxamide hydrochloride; Patent application WO2014011047 A1) was available as a powder for reconstitution with saline. Placebo was a NaCl salt/bitrex powder for reconstitution with saline.

Study Design

[0261] The trial had a double-blinded, randomized, placebo-controlled, single-centre design. For the SAD part, a partial alternating crossover design and for the MAD part a sequential group design was applied.

[0262] In the SAD part the effects of 6 single orally administered ascending doses of KH176 or placebo were investigated alternately dosed to two groups of 6 healthy male subjects (4 active; 2 placebo per group). Dose escalation to the next dose level was done after evaluation of the safety and the first 24-hour pharmacokinetic results of the previous dose. The single dose part included a food effect investigation; the 100-mg dose was administered following the intake of a high calorie/high fat breakfast to the same subjects as who received this dose in fasting conditions. In the MAD study 3 multiple ascending doses of KH176 were administered for 7 Days to 3 sequential groups of 6 healthy male subjects each (4 active; 2 placebo per group).

Formulation and Administration

[0263] KH176 (solution) was administered orally. Single doses of 10, 30, 100, 300, 800 and 2000 mg were administered in the SAD part. Multiple oral doses of 100, 200 and 400 mg were administered b.i.d. for 7 Days in the MAD part. The starting dose for the SAD was ˜150 fold lower than the no observable adverse effect level (NOAEL) of both dogs and rats. Anticipated exposures at this starting dose were just below the Minimum Anticipated Biological Effect Level (MABEL). Placebo (an in taste and appearance matching oral liquid) was also administered orally on one occasion in the single-ascending dose part and b.i.d. for 7 Days in the MAD part.

Safety Assessment

[0264] Safety was assessed using standard vital signs, clinical laboratory results for blood chemistry, haematology and urinalysis, continuous cardiac telemetry and a 12-lead ECG at 1, 2, 4, 6, 8, 12 and 24 h post dosing. The changes from baseline (pre-dose assessment on Day 1) for body weight, physical examinations, vital signs, ECG-variables and clinical laboratory variables were determined for each time point. Treatment-emergent laboratory and ECG abnormalities as well as adverse events were monitored throughout the study, up to 28 Days after the last study drug intake.

[0265] Preclinical studies of the compounds of the invention in rats demonstrated the occurrence of phospholipidosis (data not shown). Therefore, the presence of phospholipidosis was determined by the concentration of di-docosahexaenoyl (22:6)-bis(monoacylglycerol) phosphate (di-22:6-BMP) in a midstream urine sample and by electron microscopy of peripheral leucocytes at Day 1 and 7 in the MAD study (Pospischil A. et al, Exp. Toxicol Pathol 2010, 62:567-571).

Pharmacokinetic Analysis

[0266] Plasma samples for pharmacokinetic analyses were taken pre-dose and at 0.5, 1, 1.5, 2, 3, 6, 8, 12, and 24 h after dosing (SAD) and pre-dose at Day 1, 2, 4, 7, and post-dose at Day 1 and Day 7 at 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12 h after dosing (MAD). All samples were stored at <−70° C. until analysis. Quantification of KH176 and its metabolite KH176m were performed using a validated LC-MS/MS method with good (max 15-20% CV) selectivity, precision and accuracy, little carry over effect, good stability of both the solutions and the samples.

For mean value calculations, all values below the limit of quantification (LOQ) were set to zero. If <50% of the values at a given time point were below the LOQ (BLQ), these values were set to zero for calculation of the mean value. If >50% of the values at a given time point were BLQ, no mean value was calculated. The non-compartmental pharmacokinetic analysis was performed using Phoenix, Version 6.3 (Pharsight Corporation, Mountain View, Calif., USA). Plasma concentration-time profiles of KH176 and its metabolite KH176m were determined for the SAD and MAD part and for trough concentrations in the MAD part. The PK parameters were calculated on the basis of the actual blood sampling time points relative to dosing. Selected pharmacokinetic parameters (C.sub.max, t.sub.max, t.sub.1/2, AUC.sub.last and AUC.sub.0-inf) following single-dose administration and selected pharmacokinetic parameters C.sub.max, t.sub.max, t.sub.1/2, AUC.sub.tau, the accumulation factor (R.sub.acc) and time to reach steady state) following multiple dose administration were determined. In urine, the percentage of dose excreted in urine was determined for the single- and multiple-dose administration.

Pharmacodynamic Analysis

[0267] Blood samples for pharmacodynamic analyses were taken pre-dose and at 3, 6 and 24 h post dosing at Day 7 (MAD). All samples were stored in −80° C. until analysis. Quantification of oxidized and reduced glutathione was performed at York Bioanalytical solutions using a previously described validated method (Moore T et al, J Chromatogr B Analyt Technol Biomed Life Sci 2013, 929:51-55). Change from baseline in the concentrations of oxidized and reduced glutathione (GSH/GSSG) was calculated.

ECG Post-Hoc Analysis

[0268] During the interim evaluations at dose escalation, machine-read ECGs and telemetry in the clinic indicated a QT prolongation of KH176, as indicated by QTcB, the Bazett corrected QT interval, particularly strong at high dosages of the compound. The goal of this post-hoc optimization study was to repeat the ECG assessment with an highly automated computer-assisted approach where in addition to the re-evaluation of standard intervals (PR, QRS and QT intervals), a set of parameters describing repolarization morphology were considered. The morphology indices were the TpTe interval (interval from the T wave apex to the end of the T wave), the TpTe/QT index (the ratio between the TpTe interval and the QT interval), Tamp (the amplitude in microvolt units of the T wave) and TSym (an index of repolarization morphology based on the symmetry of the T wave).

[0269] ECGs were digitally recorded using a Schiller AT104 ECG machine (500 Hz, 1 μV). Cardiac interval measurements were performed on the Global Superimposed Median Beat (GSMB), a methodology that allows measurements that consider each of the 12 individual median beats [15]. This measurement methodology ensures that the PR, QRS, and QT interval are measured from the earliest onset in any lead to the latest deflection in any lead. Cardiologist overview of the computer-based measurements is based on the superimposed (overlapped) display of the individual median beats, to assure measurements are performed at the earliest onset of any viable lead to the latest offset of any viable lead.

[0270] The RR interval used for heart-rate correction of the QT interval (QTcB) was based on all the beats in the ten second recording.

[0271] Other cardiac parameters were computed from the 12-lead vector magnitude VM (e.g. the square root of the sum of squares at each digital sample) computed from the individual median beats. On the VM lead, the apex of the T wave is placed and used to compute the TpTe interval (using the end of the T wave from the GSMB) and the T wave amplitude (height in microvolt of the VM T peak from the isoelectric line). FIG. 3A shows an example with all the calipers involved: the GSMB leads are drawn in black and the VM lead is drawn in green. T wave symmetry index was computed using a proprietary approach based on Gaussian Mesa function modeling (GMF) of the repolarization waves (Badilini F et al. J Electrocardiol 2008, 41:588-594). Briefly, the ascending and descending phases of the VM T wave are modeled by two independent half-Gaussian curves (see FIG. 3). The standard deviations of these functions (σ1 and σ2) and indicators of the ascending/descending speed and their ratio is an index of symmetry (TSym=σ1/σ2; TSym=1 for a perfectly symmetric T wave, TSym<1 for slow-ascending/fast-descending T waves, T>1 for fast-ascending/slow-decending T waves).

[0272] For the pre-dose (baseline) ECGs, all parameters per time point were calculated as the averages of the triplicate ECGs. Computation of QTcB and QTcF was performed using the RR interval averaged from the total ECG acquisition duration (10 seconds) and in the case of the triplicate ECGs is based on the average QT and average HR of the replicate ECGs.

[0273] The over-reading cardiologist provided a clinical interpretation for each ECG at each time point. Each ECG was classified as Normal, Abnormal Clinically Insignificant (ACI), or Abnormal Clinically Significant (ACS). Results of this post-hoc analysis were used for an exposure-response evaluation for the changes in the electrophysiological parameters as a function of the concentration of KH176.

Ethics

[0274] All studies were conducted at the Drug Research Unit Ghent in accordance with the Declaration of Helsinki and the Good Clinical Practice guidelines established by the International Conference on Harmonization (ICH). An independent medical ethical committee approved the protocol (University Hospital of Ghent). All participants have signed informed consent prior to their enrolment.

Statistical Analysis

[0275] The tolerability and safety data were compared between KH176 and placebo using descriptive summary statistics. Pharmacokinetic and pharmacodynamic parameters were analyzed by treatment group on the per-protocol set and summarized using descriptive statistics. Dose proportionality of log transformed C.sub.max and AUC values was explored graphically. The effect of food on the pharmacokinetics was explored by calculating the geometric means and 90% confidence interval of the ratio (fed/fasted) for AUC and C.sub.max. The pharmacodynamic endpoints were analyzed descriptively by treatment group on the per-protocol set as a change from baseline. Pharmacokinetic-effect modelling of PK/ECG data was explored visually and by an exposure-response evaluation for the changes in the electrophysiological parameters as a function of the concentration of KH176.

Results

In Vitro: Oxidative Stress-Induced Cell Death

[0276] The cellular oxidative-stress protectant potency of KH176 and its metabolite KH176m was assessed in 3 different mitochondrial disease patient-derived fibroblasts (FIG. 2). As shown in FIG. 2, the compound KH176 and its metabolite KH176m were able to protect fibroblasts of mitochondrial patients from redox-induced toxicity. For fibroblasts derived from Leigh patients, the EC.sub.50 (half maximum effective concentration) was 182 nM for KH176 and 16 nM for KH176m. Similarly, the EC.sub.50 for KH176 and KH176m in fibroblasts derived from MELAS patients was respectively 182 nm and 14 nM, The EC.sub.50 for KH176 and KH176m in fibroblasts derived from LHON patients was respectively 49 nM and 4 nM,

In Vivo: Placebo-Controlled, Single-Centre Study

Study Population

[0277] For the SAD part of the study, 14 healthy male subjects divided in two groups of 7 subjects were included. For the MAD part of the study, 18 healthy male subjects divided in 3 groups of 6 subjects were included. Two subjects prematurely withdrew from the study for non-medical reasons and were replaced. There were no protocol violations in either part of the study that excluded subjects from the analysis set(s) and, therefore, all subjects included in this study were evaluable for pharmacokinetics, pharmacodynamics, safety, and tolerability. For demographics, see Table 3 below.

TABLE-US-00004 TABLE 3 Summary of demographic characteristics for subjects included in the SAD and MAD study. MAD SAD Group III Group IV Group V Group I Group II 100 mg 200 mg 400 mg Placebo (N = 7)* (N = 7)* BID (N = 4) BID (N = 4) BID (N = 4) (N = 6) Age (years) Mean 30.4 32.0 40.8 34.5 44.0 44.7 (SD) (10.5) (11.3) (13.0) (11.6) (8.2) (9.8) Median 28.0 31.0 44.0 35.0 43.0 48.5 Range (22-52) (18-45) (24-51) (23-45) (37-53) (28-54) Height (cm) at screening Mean 178.07 176.01 184.63 181.75 175.70 175.55 (SD) (6.56) (8.11) (2.81) (5.25) (3.18) (8.25) Median 177.50 177.00 184.90 183.75 176.70 171.60 Range (165.7- (163.0- (181.5- (174.0- (171.1- (170.4- 184.5) 187.5) 187.2) 185.5) 178.3) 191.5) Weight (kg) at screening Mean 82.86 74.29 89.05 77.85 81.10 72.03 (SD) (10.56) (12.61) (11.84) (11.96) (4.82) (6.06) Median 80.00 78.60 89.30 80.30 82.80 72.30 Range (69.8- (55.2- (77.2- (61.2- (74.2- (62.6- 100.0) 88.6) 100.4) 89.6) 84.6) 78.8) BMI (kg/m.sup.2) at screening Mean 26.09 24.01 26.10 23.60 26.28 23.43 (SD) (2.51) (4.01) (2.74) (3.82) (1.67) (1.98) Median 26.10 22.80 26.25 24.90 26.70 23.50 Range (22.5- (19.0- (23.1- (18.2- (23.9- (21.2- 29.4) 29.6) 28.8) 26.4) 27.8) 26.6) Race White 7 7 4 4 4 6 (100.0%) (100.0%) (100.0%) (100.0%) (100.0%) (100.0%) *6 subjects were enrolled of whom 1 withdrew and has been replaced

Safety and Tolerability

[0278] Following administration of a single dose of KH176 to subjects in the fasted state (SAD study), a total of 43 adverse events (AEs) were reported by 14 subjects (56%; Table 4A). The majority of AEs (35) was rated as mild in severity whereas there were 6 moderate and 2 severe AEs. Up to and including the 800 mg dose, no relationship to dose in the number of reported AEs or severity could be discerned. However, 28 of the 43 reported AEs were reported by subjects in the highest dose group of which 6 and 2 were of moderate and severe severity, respectively. The 2 severe AEs were nausea and headache. Following administration of placebo, a total of 7 AEs were reported by 3 subjects (25%). Of note, 4 of these AEs were rated as moderate in severity. Headache was the most frequently reported AE with a total of 7 subjects including 2 placebo subjects. The majority of AEs occurred incidentally, i.e., was reported by only 1 or 2 subjects. Adverse events reported by more than 2 subjects and not by placebo subjects were psychiatric symptoms, dizziness, oral paraesthesia and prolonged QT at the electrocardiogram and telemetry. All these events occurred in the highest dose group (2000 mg dose) and were reported by 3 subjects each.

[0279] Following administration of a multiple doses of KH176 (MAD study), a total of 29 AEs were reported by 10 subjects (83.3%; Table 4B). The majority of AEs (27) was rated as mild in severity whereas there were 2 moderate AEs, one each in the 200- and 400-mg dose groups. No relationship to dose in the number of reported AEs or severity could be discerned. Headache was the most frequently reported AE with a total of 11 subjects including 5 placebo subjects reporting this AE. Except for skin irritation, all AEs occurred incidentally, i.e., were reported by only 1 or 2 subjects in the KH176 treated subjects. Skin irritation was also reported by 2 placebo subjects and, therefore, no AE was reported by more than 2 KH176-treated subjects and not by placebo subjects.

Table 4. Summary of Treatment-Emergent Adverse Events by System Organ Class and Preferred Term

[0280]

TABLE-US-00005 TABLE 4A SAD study Group I Group II Group I Group II Group I Group II G Placebo Placebo 10 mg 30 mg 100 mg 300 mg 8( System organ class (N = 6) (N = 6) (N = 4) (N = 4) (N = 4) (N = 4) (N Preferred term n (%) n (%) n (%) n (%) n (%) n (%) n (%) Any TEAE 2 (33.3%) 1 (16.7%) 2 (50.0%) 3 (75.0%) 4 (100.0%) 1 (25.0%) 0 Blood and lymphatic system 0 0 0 1 (25.0%) 0 0 0 disorders Lymphadenopathy 0 0 0 1 (25.0%) 0 0 0 Psychiatric disorders 0 0 0 0 0 0 0 Bradyphrenia 0 0 0 0 0 0 0 Depersonalisation 0 0 0 0 0 0 0 Hallucination, visual 0 0 0 0 0 0 0 Nervous system disorders 2 (33.3%) 0 0 2 (50.0%) 3 (75.0%) 1 (25.0%) 0 Dizziness 0 0 0 0 0 0 0 Dysgeusia 0 0 0 0 0 1 (25.0%) 0 Headache 2 (33.3%) 0 0 1 (25.0%) 3 (75.0%) 0 0 Presyncope 0 0 0 1 (25.0%) 0 0 0 Cardiac Disorders 0 0 0 0 0 0 0 Bundle branch block right 0 0 0 0 0 0 0 Respiratory, thoracic and mediastinal 0 0 1 (25.0%) 0 1 (25.0%) 0 0 disorders Oropharyngeal discomfort 0 0 0 0 1 (25.0%) 0 0 Rhinorrhoea 0 0 1 (25.0%) 0 1 (25.0%) 0 0 Gastrointestinal disorders 1 (16.7%) 1 (16.7%) 0 1 (25.0%) 1 (25.0%) 0 0 Abdominal pain 0 1 (16.7%) 0 0 1 (25.0%) 0 0 Diarrhoea 0 1 (16.7%) 0 0 0 0 0 Nausea 1 (16.7%) 0 0 0 0 0 0 Odynophagia 0 0 0 1 (25.0%) 0 0 0 Paraesthesia oral 0 0 0 0 0 0 0 Retching 0 0 0 0 0 0 0 Vomiting 0 0 0 0 0 0 0 Musculoskeletal and connective 1 (16.7%) 0 1 (25.0%) 1 (25.0%) 0 0 0 tissue disorders Arthralgia 0 0 0 1 (25.0%) 0 0 0 Musculoskeletal stiffness 0 0 1 (25.0%) 0 0 0 0 Pain in extremity 1 (16.7%) 0 0 0 0 0 0 General disorders and administration 1 (16.7%) 0 0 0 1 (25.0%) 0 0 site conditions Catheter site pain 0 0 0 0 0 0 0 Chills 0 0 0 0 0 0 0 Fatigue 0 0 0 0 1 (25.0%) 0 0 Influenza like illness 1 (16.7%) 0 0 0 0 0 0 Malaise 0 0 0 0 0 0 0 Investigations 0 0 0 0 0 0 0 Blood pressure diastolic 0 0 0 0 0 0 0 increased Blood pressure systolic 0 0 0 0 0 0 0 increased Electrocardiogram QT prolonged 0 0 0 0 0 0 0 Eosinophil count increased 0 0 0 0 0 0 0 n = number of subjects; TEAE = Treatment-Emergent Adverse Event indicates data missing or illegible when filed

TABLE-US-00006 TABLE 4B MAD study Group III Group IV Group V 100 mg 200 mg 400 mg BID BID BID Placebo (N = 4) (N = 4) (N = 4) (N = 6) n (%) n (%) n (%) n (%) Any TEAE 3 (75.0%) 3 (75.0%) 4 (100.0%) 5 (83.3%) Infections and infestations 1 (25.0%) 0 0 0 Nasopharyngitis 1 (25.0%) 0 0 0 Psychiatric disorders 0 0 1 (25.0%) 0 Nightmare 0 0 1 (25.0%) 0 Nervous system disorders 2 (50.0%) 2 (50.0%) 1 (25.0%) 3 (50.0%) Dizziness 0 0 0 1 (16.7%) Head discomfort 0 1 (25.0%) 0 1 (16.7%) Headache 2 (50.0%) 1 (25.0%) 1 (25.0%) 3 (50.0%) Eye disorders 1 (25.0%) 1 (25.0%) 0 0 Conjunctival haemorrhage 1 (25.0%) 0 0 0 Eye irritation 1 (25.0%) 0 0 0 Vision blurred 0 1 (25.0%) 0 0 Gastrointestinal disorders 1 (25.0%) 0 2 (50.0%) 2 (33.3%) Abdominal pain 1 (25.0%) 0 0 0 Diarrhoea 0 0 1 (25.0%) 0 Nausea 0 0 1 (25.0%) 2 (33.3%) Skin and subcutaneous tissue disorders 0 1 (25.0%) 2 (50.0%) 2 (33.3%) Rash macular 0 1 (25.0%) 0 0 Skin irritation 0 1 (25.0%) 2 (50.0%) 2 (33.3%) Renal and urinary disorders 0 0 1 (25.0%) 0 Polyuria 0 0 1 (25.0%) 0 General disorders and administration 1 (25.0%) 0 0 1 (16.7%) site conditions Feeling cold 0 0 0 1 (16.7%) Feeling hot 1 (25.0%) 0 0 0 Investigations 0 0 4 (100.0%) 0 Blood creatine phosphokinase 0 0 1 (25.0%) 0 increased Electrocardiogram QT prolonged 0 0 1 (25.0%) 0 Lipase increased 0 0 2 (50.0%) 0 Injury, poisoning and procedural 0 0 1 (25.0%) 1 (16.7%) complications Wound 0 0 1 (25.0%) 1 (16.7%) n = number of subjects; TEAE = Treatment-Emergent Adverse Event

[0281] The level of di-22:6-BMP was variable at baseline. There was no increase in di-22:6-BMP for the treatment groups versus placebo (change from Day 1 to Day 7 0.74-2.50; −1.44-1.32 and −0.69-1.46 ng/mg creatinine for group III, IV and V respectively and −0.92-4.31 ng/mg creatinine for placebo). No increased prevalence of the presence of phospholipidosis in either granulocytes or monocytes as evaluated by electron microscopy was present in the KH176-treated group versus the placebo-treated group. Hence, in contrast to preclinical studies in rats, we did not observe an increased prevalence of the presence of phospholipidosis.

Pharmacokinetic Analysis

[0282] SAD study: The plasma concentrations-time profiles of KH176 (FIG. 4A) showed that the median t.sub.max was between 0.75 and 1.5 h after administration and varied little with dose. Thereafter, the KH176 concentrations decreased in a biphasic way. The t.sub.1/2 was approximately 10 h and varied little among dose groups (Table 5). The elimination phase following administration of a dose of 10 mg was not well characterized and, therefore, t.sub.1/2 could not be estimated for this dose only.

[0283] The shape of the plasma concentration-time profiles of KH176m (FIG. 4B) resembled that of the parent compound but concentrations were lower. C.sub.max was reached at the same time or slightly later when compared to KH176 (table 6). Thereafter, KH176m plasma concentrations decreased in a biphasic way and the elimination was characterized by a t.sub.1/2 of approximately 16 h without any notable effect of dose.

[0284] In the presence of food, the absorption of KH176 was slower as indicated by a median t.sub.max that shifted from approximately 1 h in fasted condition to 2.5 h in fed condition for both analytes (Table 5). Exposure under fed conditions in terms of AUC.sub.0-inf increased slightly for KH176 whereas that to KH176m decreased slightly (AUC.sub.0-inf 1.28 (90% Cl 1.12-1.45) for KH176 and 0.90 (90% Cl 0.57-1.41) for KH176m). The t.sub.1/2 was not affected by food.

[0285] A graphical exploration for dose-proportionality of the pharmacokinetics of KH176 indicated that with increasing single dose there was a more than proportional increase in C.sub.max and AUC.sub.0-inf (FIG. 5).

[0286] Regardless of the dose and when combining KH176 and KH176m, approximately 16% of the administered dose was excreted in urine. Unchanged KH176 accounted for approximately 12%.

Table 5. Summary of Plasma Pharmacokinetic Variables of KH176.

[0287]

TABLE-US-00007 TABLE 5A SAD study SAD study Dose 10 mg 30 mg 100 mg 100 mg 300 mg Food (N = 4) (N = 4) (N = 4) (N = 4) (N = 4) status fasted fasted fasted fed fasted C.sub.max (ng/mL) Geomean 12.9 56.2 167 165 766 CV % geomean 21.3 21.0 27.2 7.40 53.9 t.sub.max* (h) Geomean 1.25 1.25 1.00 2.50 0.992 CV % geomean (0.500-1.50) (0.500-1.50) (0.500-2.00) (2.00-3.00) (0.50 AUC.sub.last (h*ng/mL) Geomean 75.0 389 1310 1650 6320 CV % geomean 14.8 8.16 16.7 1.67 20.2 AUC.sub.0-inf (h*ng/mL) Geomean 474 1540 1970 7500 CV % geomean 8.39 13.1 1.04 19.1 t.sub.1/2 (h) Geomean NA 10.3 9.10 9.09 9.64 CV % geomean 14.6 15.2 3.83 4.29 Geomean = geometric mean; h = hour; NA = not assessable; R = accumulation ratio; *median (range) indicates data missing or illegible when filed

TABLE-US-00008 TABLE 5B MAD study MAD study Dose 100 mg b.i.d (n = 4) 200 mg b.i.d (n = 4) Day 1 7 1 7 C.sub.max (ng/mL) Geomean 184 353 313 748 CV % geomean 57.0 19.1 20.0 29.3 t.sub.max* (h) Geomean 1.25 1.00 1.75 2.00 CV % geomean (0.500-8.00) (0.500-1.50) (1.50-2.00) (1.50-2.00) AUC.sub.tau (h*ng/mL) Geomean 1090 2760 2250 5960 CV % geomean 49.3 22.6 26.1 26.8 Racc Geomean 2.52 2.65 CV % geomean 31.2 2.5 Geomean = geometric mean; h = hour; R = accumulation ratio; *median (range) indicates data missing or illegible when filed

Table 6. Summary of Plasma Pharmacokinetic Variables of KH176m

[0288]

TABLE-US-00009 TABLE 6A SAD study SAD study Dose 10 mg 30 mg 100 mg 100 mg 300 mg 800 Food (N = 4) (N = 4) (N = 4) (N = 4) (N = 4) (N = 4) status fasted fasted fasted fed fasted fasted C.sub.max (ng/mL) Geomean 14.2 49.4 168 117 497 1100 CV % 49.5 9.00 29.6 20.0 17.6 9.58 geomean t.sub.max* (h) Geomean 1.25 1.50 1.25 2.50 1.24 1.50 CV % (1.00-2.00) (1.00-2.00) (1.00-2.00) (2.00-3.00) (1.00-2.00) (1.50-2.00) geomean AUC.sub.last (h*ng/mL) Geomean 151 547 1810 1590 4830 11700 CV % 26.7 12.4 37.4 26.1 11.8 20.2 geomean AUC.sub.0-inf (h*ng/mL) Geomean 234 881 2690 2410 6890 18700 CV % geomean 26.7 19.6 38.4 29.1 11.0 26.7 t.sub.1/2 (h) Geomean 16.6 17.8 15.4 14.9 14.3 17.2 CV % 5.86 19.0 8.44 7.86 18.9 18.5 geomean Geomean = geometric mean; h = hour; R = accumulation ratio; *median (range)

TABLE-US-00010 TABLE 6B MAD study MAD study Dose 100 mg b.i.d 200 mg b.i.d 400 mg b.i.d (n = 4) (n = 4) (n = 4) Day 1 7 1 7 1 7 C.sub.max (ng/mL) Geomean 99.4 152 251 250 550 450 CV % 20.9 37.5 18.0 10.6 14.1 28.3 geomean t.sub.max* (h) Geomean 1.50 1.00 1.75 1.50 1.25 1.50 CV % (1.00- (1.00- (1.00- (1.00- (1.00- (1.00- geomean 8.00) 2.03) 2.00) 2.00) 1.50) 3.00) AUC.sub.tau (h*ng/mL) Geomean 702 1310 1680 2220 3600 4270 CV % 29.2 29.5 21.0 12.7 11.1 30.5 geomean Racc Geomean 1.86 1.32 1.19 CV % 46.7 13.1 24.7 geomean Geomean = geometric mean; h = hour; R = accumulation ratio; *median (range)

[0289] MAD study: Visual inspection of the mean trough concentration-time curves indicated that steady concentrations of KH176 were reached by Day 4 of dosing (of note: Day 4 was the first time point of measurement of trough values; FIG. 4C).

[0290] The plasma concentration-time profiles of KH176 after single- and multiple-dose administration were similar. Peak concentrations were attained between 1 and 2 h after drug administration (Table 5). Following attainment of C.sub.max, the KH176 plasma concentrations declined rapidly. KH176 accumulated as shown by values for the accumulation index between doses varying from 2.17 to 2.65.

[0291] After multiple dosing, the shape of the plasma concentration-time profiles of KH176m resembled that of parent compound but concentrations were lower (FIG. 4D). On Day 7, C.sub.max was reached at approximately the same time when compared to KH176. After multiple dosing, the accumulation of the metabolite KH176m was less pronounced when compared to that of KH176 as indicated by values for the accumulation index varying from 1.19 to 1.86.

[0292] A graphical exploration for dose-proportionality of the pharmacokinetics of KH176 after multiple dosing indicated that with increasing multiple doses there was a more than proportional increase in C.sub.max and AUC.sub.tau, which was most pronounced at the 400 mg dose (FIG. 5).

[0293] On Day 7 and when combining KH176 and KH176m, the % dose excreted in urine varied from 18.0 to 25.1% between doses. Unchanged KH176 accounted for 14.0 to 18.1%.

Pharmacodynamic Analysis

[0294] No significant alterations in the GSH/GSSG ratio were observed.

Posthoc Analysis QT Prolongation

[0295] A QTcF prolongation was present after single-dose administration of 800 and 2000 mg KH176 (see FIG. 8 fora representative example). The largest median baseline increase observed was 46.8 ms (1 h post-dose), whereas the largest individual change was 64.7 ms (2000 mg group; FIG. 6). This QTcF prolongation was associated with moderate but clear changes of morphology, namely a reduction of the T wave amplitude, a prolongation of the TpTe interval (in both absolute terms and relatively to the QT interval), and the symmetry (shape) of the T wave. At lower doses, KH176 does not seem to affect repolarization. Changes were also observed on other cardiac intervals: the QRS interval increased but only at a dose of 2000 mg, whereas the PR interval increased progressively also at lower doses of KH176.

[0296] The MAD part of the study showed the same effects, particularly for the 400 mg dose group (FIG. 6). A dose of 100 mg b.i.d. had no effect on QTcF and the time curve for this dose was indistinguishable from placebo. On Day 4 and later, administration of 200 mg b.i.d. increased QTcF and a median maximum change from baseline was observed on Day 5 of 13 msec. A further increase in QTcF was observed with a dose of 400 mg b.i.d. and the median maximum change from baseline at trough of 29 msec was observed on Day 6. Table 7 shows the largest median an largest individual increase in the ECG parameters for the SAD and the MAD study.

[0297] Clinically relevant changes were observed in at the peak concentrations in 2 individuals in the 2000 mg subgroup and did not include appearance of notched and/or bumps on any of the T-waves (all leads).

[0298] When the change in the TpTe interval is correlated to the KH176-exposure, a clear dose-dependency is observed (FIG. 7). In exposures lower than 500 ng/ml, no significant prolongation of the TpTe interval compared to placebo was observed. When increasing the dose and the plasma concentration, the TpTe interval increases. When the highest (non-tolerated) dose was administered (2000 mg), all subjects have a prolonged TpTe interval.

TABLE-US-00011 TABLE 7 Largest median an largest individual increase in the ECG parameters for the SAD and the MAD study SAD study MAD study 800 mg 2000 mg 200 mg 400 mg Largest Largest Largest Largest median Largest median Largest median Largest median Largest baseline individual baseline individual baseline individual baseline individual increase increase increase increase increase increase increase increase QTcF 9 26 46.8 64.7 13.3 28.3 29.2 43.7 HR bpm 10.3 23.3 PR interval ms 10.3 19.3 15.3 20.7 12 19 QRS interval ms 5.3 8 25.7 30 9.7 15.3 TpTe interval ms 21.7 36.7 58.7 76 24 46 42.3 62.7 T-wave amplitude MV 600 706 734 994 T-wave symmetry 0.42 0.85 0.87 1.1 0.45 0.6 0.63 0.99 index

CONCLUSIONS

[0299] Mitochondrial disorders are a devastating group of disorders for which there is an urgent need for treatment development. We demonstrated that the compound KH176 showed promising properties in ameliorating the viability and phenotype of cells and mice affected by mitochondrial disease. In particular, in vitro assays with KH176 and KH176m demonstrated a low EC.sub.50 of 182 nM (around 60 ng/mL) for KH176 and 16 nM (around 5.8 ng/mL) for KH176m. In the same in vitro setting, the EC.sub.50 of the well-known drug Idebenone (a CoQ.sub.10 variant) was 1470 nM.

[0300] We further evaluated the tolerability, safety, pharmacokinetics and pharmacodynamics of single- and multiple-ascending doses of KH176 in healthy male subjects. KH176 was well tolerated in doses up to 800 mg SD and 400 mg b.i.d. Headache was the most frequently reported AE in both the KH176—and the placebo treated groups. Although there was no clear dose-response relationship for any of the adverse events after single and multiple dose administration, unforeseen markedly more severe adverse events were reported after a single dose of 2000 mg. At this dose, nausea, vomiting, dizziness and psychiatric disturbances were reported, along with a prolonged corrected QT time.

[0301] The shape of the plasma concentration-time profiles of KH176 and its metabolite KH176m were similar after single- and multiple-dose administration. The pharmacokinetics of KH176 showed several surprising aspects. In particular, the pharmacodynamics of KH176 were characterized by a i) median t.sub.max between 0.75 and 2.0 h, ii) terminal t.sub.1/2 of about 10 h, iii) biphasic elimination, and a iv) more than dose proportional increases in both C.sub.max and AUC. These pharmacodynamics could not have been predicted. In particular, the unexpected more than dose proportional increases in both C.sub.max and AUC should be considered when administrating the compound. We discovered that when a threshold concentration of the compound is reached, an increasing amount of the compound becomes available in the blood stream, which may cause side effects as described herein. Hence, a particular safe dose of the compound is a dose that does not show a more than dose-proportional increase in at least one of C.sub.max and AUC (see FIG. 5), e.g. a single dose should preferably stay below 300 mg.

[0302] In vitro, KH176 is metabolized by CYP3A4 and excreted by the PgP efflux pump. We did not observe signs for auto-induction in this study such as a decline in pre-dose concentrations. Without wishing to be bound by any theory, the more than proportional increase with dose could be caused by saturation of the PgP efflux pump for which KH176 is a substrate, as evidenced by the fact that half-life and clearance doesn't change with dose, but rather the availability seems to change with dose.

[0303] Up to 25.1% of an administered dose is excreted via urine after multiple dosing. Based on the results of the multiple-dose administration, steady state was reached at the first time point of measurement of trough concentrations (Day 4). Based on the estimated t.sub.1/2, it is expected that steady state will be reached after 2 to 3 Days of dosing.

[0304] In steady state conditions at 100 mg BID dosing the maximum concentration reached ranged within 303-458 ng/mL for KH176 and 89.4-204 ng/mL for KH167m (hence the maximum concentrations of the active moieties ranged within 392.4-662 ng/mL). The area under the curve for one dosing interval (i.e. 12 hours) at steady state (AUCtau) ranged within 2130-3680 h.Math.ng/mL for KH176 and 851-1570 h.Math.ng/mL for KH183, meaning that average concentrations ranged within 177.5-306.6 ng/mL for KH176 and 70.9-130.8 ng/mL for KH176m.

[0305] KH176 was well tolerated in the presence of food, and the tolerability and safety profile in the presence and absence of food was similar. Although the point estimates (as well as the sample covariance) for both C.sub.max and AUC.sub.0-inf did not completely remain within the usually accepted range of 0.8 to 1.25 (Table 5), no special measures are warranted regarding the intake of KH176.

[0306] KH176 clearly modifies cardiac repolarisation in a dose-dependent manner. QTcF prolongation was present after single-dose administration of 800 and 2000 mg and after multiple doses of 400 mg of KH176 b.i.d. Post-hoc studies showed that this QTcF prolongation was associated with changes in morphology and other cardiac intervals. Detailed analysis of the ECGs during the single-dose administration of 200, 100, 30 and 10 mg and multiple oral doses of 100 and 200 mg b.i.d. showed no cardiac electrophysiological abnormalities. The KH176 related changes in cardiac repolarisation include a reduction of T wave amplitude, a prolongation of the TpTe interval and a reduction of the T wave symmetry index. The reduction in T wave amplitude is largely explained by changes in heart rate and also seen in the placebo group. The prolongation of the TpTe interval and the reduction on the symmetry index indicate a prolongation of the descending phase of the T wave. With pure hERG blockade, there is a direct relationship between increasing plasma drug concentrations and the risk for torsades. Importantly, although the specific T wave morphology changes observed in this study have been associated with the hERG potassium channel blockade (Johannesen L, et al. Clin Pharmacol Ther 2014, 96:549-558). The IC.sub.50 of hERG blocking for KH176 was 3 times higher than the C.sub.max for the highest dose.

[0307] Notably, there were 2 subjects with a potentially clinically relevant change (>60 ms) at the peak concentration after administration of 2000 mg but no signs of arrhythmias or severe morphologic changes (such as T wave bumps, notches, etc) were observed. Since the sensitivity to torsades de pointes also depends on factors including cardiovascular disease, alcoholic liver disease, obesity, hypertension, and/or electrolyte disturbances (Drew B J et al, Circulation 2010, 121:1047-1060; Isbister G K et al. Br J Clin Pharmacol 2013, 76:48-57), high concentrations (e.g. more than a total daily dose of 1000 mg) of KH176 preferably should not be given to patients with any of these risk factors. Moreover, until dose adjustments for patients with concomitant medication (also metabolized by CYP3A4) are determined, high concentrations (e.g. more than a total daily dose of 1000 mg) of KH176 should preferably not be given to these patients either since unpredictable pharmacokinetics will possibly lead to plasma concentrations of KH176 above the currently defined safety threshold.

[0308] Since the mechanism of KH176 is based on correction of an abnormal redox balance, as expected, we did not observed any pharmacodynamics changes in the healthy male volunteers.

[0309] Benchmarking human exposure to in vitro activity of KH176 and metabolites indicates that 100 mg b.i.d. dosing results in an efficacious exposure.

[0310] We conclude that administration of single doses up to and including 800 mg or multiple doses up to 400 mg b.i.d. for 7 days of KH176, a new small redox-modulating molecule developed to treat mitochondrial(-related) diseases and conditions, is safe and well tolerated. Although doses above the anticipated human efficacious dose could lead to prolongation of the QTc interval with T-wave abnormalities, the administration of the anticipated efficacious doses (100 mg b.i.d.) did not lead to changes in cardiac electrophysiology. More precisely, an exposure-response analysis demonstrated that there is a concentration range available without effects on repolarization, and the 100 mg BID dose with maximum concentrations ranging within 303-458 ng/mL is within that range. Furthermore, effects on repolarization seem to start at plasma concentrations roughly a factor 2 higher and upwards (500-1000 ng/mL, see FIG. 7).

[0311] Compared to Idebenone (approved for the treatment of Friedreich's ataxia), we show that KH176 has a favourable pharmacokinetic profile with higher exposures at 100 mg BID, while KH176 is also more active in the fibroblast assays. Compared to EPI-743 (100 mg tid is a pediatric study in Leigh's disease), KH176 100 mg BID has a slightly higher exposure (due to a slightly longer half live). Fibroblast assays (Redox assay) with KH176 and KH176m demonstrate an EC50 of 182 nM (around 60 ng/mL) for KH176 and 16 nM (around 5.8 ng/mL) for KH176m. Therefore at 100 mg BID of KH176, average concentrations of the parent KH176 are a factor 3-5 above EC50's and average concentrations of KH176m are a factor 10-20 above EC50's. Of note, protein binding of KH176 is limited, around 50-60% and has limited influence on the calculation, however, free concentrations are half of the total.