A randomised placebo-controlled, double-blind phase II study to explore the safety, efficacy, and pharmacokinetics of sonlicromanol in children with genetically confirmed mitochondrial disease and motor symptoms ("KHENERGYC").

BACKGROUND: METHODS: The KHENERGYC trial will be a phase II, randomised, double-blinded, placebo-controlled (DBPC), parallel-group study in the paediatric population (birth up to and including 17 years). The study will be recruiting 24 patients suffering from motor symptoms due to genetically confirmed PMD. The trial will be divided into two phases. The first phase of the study will be an adaptive pharmacokinetic (PK) study with four days of treatment, while the second phase will include randomisation of the participants and evaluating the efficacy and safety of sonlicromanol over 6 months. DISCUSSION: Effective novel therapies for treating PMDs in children are an unmet need. This study will assess the pharmacokinetics, efficacy, and safety of sonlicromanol in children with genetically confirmed PMDs, suffering from motor symptoms. TRIAL REGISTRATION: clinicaltrials.gov: NCT04846036 , registered April 15, 2021. European Union Clinical Trial Register (EUDRACT number: 2020-003124-16 ), registered October 20, 2020. CCMO registration: NL75221.091.20, registered on October 7, 2020.

Sonlicromanol's active metabolite KH176m normalizes prostate cancer stem cell mPGES-1 overexpression and inhibits cancer spheroid growth.

Aggressiveness of cancers, like prostate cancer, has been found to be associated with elevated expression of the microsomal prostaglandin E synthase-1 (mPGES-1). Here, we investigated whether KH176m (the active metabolite of sonlicromanol), a recently discovered selective mPGES-1 inhibitor, could affect prostate cancer cells-derived spheroid growth. We demonstrated that KH176m suppressed mPGES-1 expression and growth of DU145 (high mPGES-1 expression)-derived spheroids, while it had no effect on the LNCaP cell line, which has low mPGES-1 expression. By addition of exogenous PGE2, we found that the effect of KH176m on mPGES-1 expression and spheroid growth is due to the inhibition of a PGE2-driven positive feedback control-loop of mPGES-1 transcriptional regulation. Cancer stem cells (CSCs) are a subset of cancer cells exhibiting the ability of self-renewal, plasticity, and initiating and maintaining tumor growth. Our data shows that mPGES-1 is specifically expressed in this CSCs subpopulation (CD44+CD24-). KH176m inhibited the expression of mPGES-1 and reduced the growth of spheroids derived from the CSC. Based on the results obtained we propose selective mPGES-1 targeting by the sonlicromanol metabolite KH176m as a potential novel treatment approach for cancer patients with high mPGES-1 expression.

Sonlicromanol improves neuronal network dysfunction and transcriptome changes linked to m.3243A>G heteroplasmy in iPSC-derived neurons.

Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) is often caused by an adenine to guanine variant at m.3243 (m.3243A>G) of the MT-TL1 gene. To understand how this pathogenic variant affects the nervous system, we differentiated human induced pluripotent stem cells (iPSCs) into excitatory neurons with normal (low heteroplasmy) and impaired (high heteroplasmy) mitochondrial function from MELAS patients with the m.3243A>G pathogenic variant. We combined micro-electrode array (MEA) measurements with RNA sequencing (MEA-seq) and found reduced expression of genes involved in mitochondrial respiration and presynaptic function, as well as non-cell autonomous processes in co-cultured astrocytes. Finally, we show that the clinical phase II drug sonlicromanol can improve neuronal network activity when treatment is initiated early in development. This was intricately linked with changes in the neuronal transcriptome. Overall, we provide insight in transcriptomic changes in iPSC-derived neurons with high m.3243A>G heteroplasmy, and show the pathology is partially reversible by sonlicromanol.

The Redox Modulating Sonlicromanol Active Metabolite KH176m and the Antioxidant MPG Protect Against Short-Duration Cardiac Ischemia-Reperfusion Injury.

PURPOSE: Sonlicromanol is a phase IIB clinical stage compound developed for treatment of mitochondrial diseases. Its active component, KH176m, functions as an antioxidant, directly scavenging reactive oxygen species (ROS), and redox activator, boosting the peroxiredoxin-thioredoxin system. Here, we examined KH176m's potential to protect against acute cardiac ischemia-reperfusion injury (IRI), compare it with the classic antioxidant N-(2-mercaptopropionyl)-glycine (MPG), and determine whether protection depends on duration (severity) of ischemia. METHODS: Isolated C56Bl/6N mouse hearts were Langendorff-perfused and subjected to short (20 min) or long (30 min) ischemia, followed by reperfusion. During perfusion, hearts were treated with saline, 10 µM KH176m, or 1 mM MPG. Cardiac function, cell death (necrosis), and mitochondrial damage (cytochrome c (CytC) release) were evaluated. In additional series, the effect of KH176m treatment on the irreversible oxidative stress marker 4-hydroxy-2-nonenal (4-HNE), formed during ischemia only, was determined at 30-min reperfusion. RESULTS: During baseline conditions, both drugs reduced cardiac performance, with opposing effects on vascular resistance (increased with KH176m, decreased with MPG). For short ischemia, KH176m robustly reduced all cell death parameters: LDH release (0.2 ± 0.2 vs 0.8 ± 0.5 U/min/GWW), infarct size (15 ± 8 vs 31 ± 20%), and CytC release (168.0 ± 151.9 vs 790.8 ± 453.6 ng/min/GWW). Protection by KH176m was associated with decreased cardiac 4-HNE. MPG only reduced CytC release. Following long ischemia, IRI was doubled, and KH176m and MPG now only reduced LDH release. The reduced protection against long ischemia was associated with the inability to reduce cardiac 4-HNE. CONCLUSION: Protection against cardiac IRI by the antioxidant KH176m is critically dependent on duration of ischemia. The data suggest that with longer ischemia, the capacity of KH176m to reduce cardiac oxidative stress is rate-limiting, irreversible ischemic oxidative damage maximally accumulates, and antioxidant protection is strongly diminished.

Live-Imaging Readouts and Cell Models for Phenotypic Profiling of Mitochondrial Function.

Mitochondria are best known as the powerhouses of the cells but their cellular role goes far beyond energy production; among others, they have a pivotal function in cellular calcium and redox homeostasis. Mitochondrial dysfunction is often associated with severe and relatively rare disorders with an unmet therapeutic need. Given their central integrating role in multiple cellular pathways, mitochondrial dysfunction is also relevant in the pathogenesis of various other, more common, human pathologies. Here we discuss how live-cell high content microscopy can be used for image-based phenotypic profiling to assess mitochondrial (dys) function. From this perspective, we discuss a selection of live-cell fluorescent reporters and imaging strategies and discuss the pros/cons of human cell models in mitochondrial research. We also present an overview of live-cell high content microscopy applications used to detect disease-associated cellular phenotypes and perform cell-based drug screening.

KH176 Safeguards Mitochondrial Diseased Cells from Redox Stress-Induced Cell Death by Interacting with the Thioredoxin System/Peroxiredoxin Enzyme Machinery.

A deficient activity of one or more of the mitochondrial oxidative phosphorylation (OXPHOS) enzyme complexes leads to devastating diseases, with high unmet medical needs. Mitochondria, and more specifically the OXPHOS system, are the main cellular production sites of Reactive Oxygen Species (ROS). Increased ROS production, ultimately leading to irreversible oxidative damage of macromolecules or to more selective and reversible redox modulation of cell signalling, is a causative hallmark of mitochondrial diseases. Here we report on the development of a new clinical-stage drug KH176 acting as a ROS-Redox modulator. Patient-derived primary skin fibroblasts were used to assess the potency of a new library of chromanyl-based compounds to reduce ROS levels and protect cells against redox-stress. The lead compound KH176 was studied in cell-based and enzymatic assays and in silico. Additionally, the metabolism, pharmacokinetics and toxicokinetics of KH176 were assessed in vivo in different animal species. We demonstrate that KH176 can effectively reduce increased cellular ROS levels and protect OXPHOS deficient primary cells against redox perturbation by targeting the Thioredoxin/Peroxiredoxin system. Due to its dual activity as antioxidant and redox modulator, KH176 offers a novel approach to the treatment of mitochondrial (-related) diseases. KH176 efficacy and safety are currently being evaluated in a Phase 2 clinical trial.

Therapeutic effects of the mitochondrial ROS-redox modulator KH176 in a mammalian model of Leigh Disease.

Leigh Disease is a progressive neurometabolic disorder for which a clinical effective treatment is currently still lacking. Here, we report on the therapeutic efficacy of KH176, a new chemical entity derivative of Trolox, in Ndufs4 -/- mice, a mammalian model for Leigh Disease. Using in vivo brain diffusion tensor imaging, we show a loss of brain microstructural coherence in Ndufs4 -/- mice in the cerebral cortex, external capsule and cerebral peduncle. These findings are in line with the white matter diffusivity changes described in mitochondrial disease patients. Long-term KH176 treatment retained brain microstructural coherence in the external capsule in Ndufs4 -/- mice and normalized the increased lipid peroxidation in this area and the cerebral cortex. Furthermore, KH176 treatment was able to significantly improve rotarod and gait performance and reduced the degeneration of retinal ganglion cells in Ndufs4 -/- mice. These in vivo findings show that further development of KH176 as a potential treatment for mitochondrial disorders is worthwhile to pursue. Clinical trial studies to explore the potency, safety and efficacy of KH176 are ongoing.

NANS-mediated synthesis of sialic acid is required for brain and skeletal development.

We identified biallelic mutations in NANS, the gene encoding the synthase for N-acetylneuraminic acid (NeuNAc; sialic acid), in nine individuals with infantile-onset severe developmental delay and skeletal dysplasia. Patient body fluids showed an elevation in N-acetyl-D-mannosamine levels, and patient-derived fibroblasts had reduced NANS activity and were unable to incorporate sialic acid precursors into sialylated glycoproteins. Knockdown of nansa in zebrafish embryos resulted in abnormal skeletal development, and exogenously added sialic acid partially rescued the skeletal phenotype. Thus, NANS-mediated synthesis of sialic acid is required for early brain development and skeletal growth. Normal sialylation of plasma proteins was observed in spite of NANS deficiency. Exploration of endogenous synthesis, nutritional absorption, and rescue pathways for sialic acid in different tissues and developmental phases is warranted to design therapeutic strategies to counteract NANS deficiency and to shed light on sialic acid metabolism and its implications for human nutrition.