Talarozole for use in the treatment of a disease caused by mitochondrial complex iv deficiency

Abstract

Talarozole for use in the treatment of a disease caused by mitochondrial complex IV deficiency The present invention relates to talarozole, or a pharmaceutically acceptable salt thereof, for use in the treatment of a disease caused by mitochondrial complex IV deficiency, particularly of SURF1- dependent Leigh syndrome. The present invention further relates to a pharmaceutical composition comprising as an active ingredient talarozole, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier for use in the treatment of a disease caused by mitochondrial complex IV deficiency.

Description

[0001] Talarozole for use in the treatment of a disease caused by mitochondrial complex IV deficiency

[0002] D e s c r i p t i o n

[0003] The present invention relates to the field of treatment of medical conditions associated with mitochondrial complex IV deficiency.

[0004] Mitochondria generate the majority of cellular adenosine triphosphate (ATP) by mitochondrial oxidative phosphorylation (OXPHOS), and genetic alterations affecting mitochondrial proteins can cause severe and often fatal disorders. Mitochondrial diseases represent a large class of inborn errors of metabolism. Leigh syndrome, also denoted Leigh disease, is a severe developmental disorder of the nervous system that can be based on numerous genetic defects, which can be located either on nuclear DNA (inherited according to Mendelian rules) or on mitochondrial DNA (mtDNA, maternal inheritance only). Common to all subtypes is selective damage to dopaminergic neurons in the brainstem and basal ganglia, which may be particularly apparent in the context of metabolic crises. Leigh syndrome causes lactic acidosis and symmetric lesions in the central nervous system (CNS), predominantly of basal ganglia and brainstem, leading to intellectual disability and muscle weakness. The commonly most affected mitochondrial complexes in Leigh syndrome are complex I and complex IV. One of the most frequently mutated nuclear genes in Leigh syndrome is the SURF 1 gene (Surfeit locus protein 1, NM_003172.2). SURFl-Leigh syndrome patients typically exhibit increased lactate in the liquor and midbrain neurodegeneration. Leigh syndrome affecting children is currently considered an untreatable neurologic mitochondrial disorder. To date, there are no therapeutic options for Leigh syndrome that influence the pathophysiological process.

[0005] Tripathy S, et al. "All-Trans-Retinoic Acid Enhances Mitochondrial Function in Models of Human Liver” Mol Pharmacol. 2016 May;89(5):560-74 (doi: 10.1124 / mol. 116.103697) disclose that inhibition of all-tra s -retinoic acid (atRA) metabolism by talarozole, a cytochrome P450 (CYP) 26 specific inhibitor, increased the effects of atRA on mitochondrial biogenesis markers in HepG2 cells and in vivo in mice. It is suggested that atRA regulates mitochondrial function and lipid metabolism and that increasing atRA concentrations in human liver via CYP26 inhibition may increase mitochondrial biogenesis and fatty acid -oxidation and provide therapeutic benefit in diseases associated with mitochondrial dysfunction. However, the publication has been retracted and the conclusion that atRA regulates mitochondrial biogenesis and that an enhancement of atRA levels by talarozole treatment may provide therapeutic benefit in diseases associated with mitochondrial dysfunction was mainly based on the retracted figures.

[0006] Jacob Morgan Leatherwood “Investigations on novel CYP26A1 / B1 Inhibitor, DX308: Using atRA response as a therapeutic target for traumatic brain injury and Parkinson ’s disease". University of Montana Graduate Student Theses, 2021, discloses that retinoic acid metabolism blocking agents (RAMBAs) are emerging as new therapeutic interventions with the goal of increasing endogenous atRA brain concentration, for the treatment of traumatic brain injury and Parkinson’s disease.

[0007] Hummel R, Ulbrich S, Appel D, et al., “Administration of all-trans retinoic acid after experimental traumatic brain injury is brain protective" Br J Pharmacol. 2020; 177:5208-5223 (https: / / doi.org / 10.1111 / bph. 15259) describe that atRA acts neuroprotective in traumatic brain injury.

[0008] Hirano M et al., “Emerging Therapies for Mitochondrial Diseases" Essays Biochem. 2018 July 20; 62(3): 467-481. (doi: 10.1042 / EBC20170114) describes that retinoic acid has been used to stimulate the retinoid X receptor-alfa (RXRalfa) in cybrid containing a m.3243A>G mutation, ameliorating the respiratory chain defect.

[0009] Inak et al. “Defective metabolic programming impairs early neuronal morphogenesis in neural cultures and an organoid model of Leigh syndrome”, Nature Communications (2021) 12: 1929 (https: / / doi.org / 10.1038 / s41467-021-22117-z) showed that SURF1 mutations impaired neuronal morphogenesis in induced pluripotent stem cell (iPSC)-derived neurons and cerebral organoids. It was shown that prime defects emerged at the level of neural progenitor cells (NPCs), which were unable to perform the metabolic switch required for neural commitment, and thus showed inefficient neurite outgrowth. Although some progress in discovering the pathophysiological mechanisms of mitochondrial diseases causing Leigh syndrome was made, major improvements in treatment are still lacking.

[0010] Therefore, the object underlying the present invention was to provide a means being usable in the treatment of diseases caused by mitochondrial complex IV deficiency, and particularly in the treatment of Leigh syndrome.

[0011] The problem is solved by talarozole, or a pharmaceutically acceptable salt thereof, for use in the treatment of a disease caused by mitochondrial complex IV deficiency.

[0012] Surprisingly it was found that talarozole provided a dose-dependent improvement in neurite outgrowth capacity in human neurons having a mutation in the mitochondrial complex IV gene SURF1, which is the most frequent cause of Leigh syndrome (Leigh disease) in children. These findings indicate that talarozole is usable in the treatment and / or prevention of a disease caused by mitochondrial complex IV deficiency, particularly in the treatment and / or prevention of Leigh syndrome.

[0013] As used herein, the term “mitochondrial complex IV” refers to a transmembrane protein complex found in bacteria and the mitochondrion of eukaryotes, which also is denoted cytochrome c oxidase and catalyzes the final step in the mitochondrial electron transfer chain. This enzyme is regarded as one of the major regulation sites for oxidative phosphorylation, and its dysfunction is associated with a wide range of human disorders.

[0014] As used herein, the term “mitochondrial complex IV deficiency”, also referred to as complex IV mitochondrial respiratory chain deficiency, COX deficiency or cytochrome c oxidase deficiency, refers to a defect and / or malfunction in the complex IV enzymatic activity. Such deficiency results in a defect and / or malfunction in the mitochondrial oxidative phosphorylation system causing severe and often fatal disorders. Disorders of the mitochondrial respiratory chain cause heterogeneous clinical manifestations, ranging from isolated myopathy to severe multisystem disease affecting several tissues and organs. Features include hypertrophic cardiomyopathy, hepatomegaly and liver dysfunction, hypotonia, muscle weakness, exercise intolerance, developmental delay, delayed motor development and intellectual disability. A subset of affected individuals manifest Leigh syndrome. In embodiments, the mitochondrial complex IV deficiency is Leigh syndrome (Leigh disease). In embodiments, talarozole, or the pharmaceutically acceptable salt thereof, is for use in the treatment of Leigh syndrome. Advantageously, talarozole has been shown to be able to reduce key disease-specific features of Leigh syndrome, such as increased lactate released in the media, decreased number of tyrosine hydroxylase (TH)-positive neurons and aberrant neuronal branching. It is thus assumed that talarozole is usable in the treatment of Leigh syndrome.

[0015] As used herein, the term “Leigh syndrome” (LS), also frequently denoted Leigh disease, refers to a severe developmental disorder of the nervous system that can be based on numerous genetic defects, which can be located either on nuclear DNA (inherited according to Mendelian rules) or on mitochondrial DNA (mtDNA, maternal inheritance only). Common to all subtypes is selective damage to dopaminergic neurons in the brainstem and basal ganglia, which may be particularly apparent in the context of metabolic crises. Leigh syndrome causes lactic acidosis and symmetric lesions in the central nervous system (CNS), predominantly of basal ganglia and brainstem, leading to intellectual disability and muscle weakness.

[0016] The commonly most affected mitochondrial complexes in Leigh syndrome are complex I and complex IV. One of the most frequently mutated nuclear genes in Leigh syndrome is the SURF1 gene (Surfeit locus protein 1, NM_003172.2). In embodiments, the mitochondrial complex IV deficiency is based in whole or in part on the presence of at least one mutation or variant in the surfeit locus protein 1 (SURF1) gene. In embodiments, talarozole, or the pharmaceutically acceptable salt thereof, is for use in the treatment of a mitochondrial complex IV deficiency that is based in whole or in part on the presence of at least one mutation or variant in the surfeit locus protein 1 (SURF1) gene.

[0017] In embodiments, the disease caused by mitochondrial complex IV deficiency is SURF 1 -dependent Leigh syndrome. SURF1 mutations are the most frequent cause of Leigh syndrome. In embodiments, talarozole, or the pharmaceutically acceptable salt thereof, is for use in the treatment of SURF1- dependent Leigh syndrome. It has been shown in SURF 1 -mutant midbrain organoids, which showed key disease-specific features, such as increased lactate released in the media, decreased number of TH positive neurons and aberrant neuronal branching, that talarozole reduced the aberrant release of lactate in the media, increased the amount of TH positive dopaminergic neurons, and rescued the overall neuronal branching organization.

[0018] As used herein, the term “SURF 1 -dependent Leigh syndrome” refers to Leigh syndrome, where the disease is caused by one or more mutation or variant in the surfeit locus protein 1 (SURF1) gene. SURF 1 -dependent Leigh syndrome patients typically exhibit increased lactate in the liquor and midbrain neurodegeneration.

[0019] In embodiments, talarozole, or the pharmaceutically acceptable salt thereof, is for use in the treatment of a disease caused by mitochondrial complex IV deficiency selected from the group of myopathy, hypertrophic cardiomyopathy, hepatomegaly, liver dysfunction, hypotonia, muscle weakness, exercise intolerance, developmental delay, delayed motor development and intellectual disability.

[0020] The term “treatment” as used herein refers to alleviating at least one of the symptoms associated with the disease, decreasing disease-caused mortality, or decelerating, impeding or suspending the progress of the disease. The term “treatment” as used herein does not refer to complete curing of the disease, as it does not change the mutated genetics causing the disease.

[0021] As used herein, the term “talarozole” refers to the International Nonproprietary Name (INN) of a compound, CAS number 201410-53-9, formerly R115866, of the following formula (1) or to a pharmaceutically acceptable salt thereof:

[0022] The IUPAC name of talarozole is N-{4-[2-ethyl-l-(l,2,4-triazol-l-yl)butyl]phenyl}-l,3-benzothiazol- 2-amine. The term “talarozole” as used herein encompasses any enantiomers, tautomers, pharmaceutically acceptable salts, as well as solvates, hydrates and solvates of pharmaceutically acceptable salts thereof. In embodiments, the racemate of talarozole may be used or a specific enantiomer of talarozole, such as the (R)- enantiomer. As used herein, “pharmaceutically acceptable salts” refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids. The term pharmaceutically acceptable salts as used herein includes both acid and base addition salts. Pharmaceutically acceptable salts refer to salts which retain the biological effectiveness and properties of the compound, and which are not biologically or otherwise undesirable. A pharmaceutically acceptable salt can be conveniently prepared from pharmaceutically acceptable non-toxic bases, including inorganic bases and organic bases. The term pharmaceutically acceptable salt includes mineral or organic acid salts of basic residues such as amines, alkali metal salts, organic salts of acidic residues such as carboxylic acids, and addition salts of free acids or free bases. Suitable pharmaceutically acceptable salts include metallic salts and organic salts, such as salts from benzoic acid, citric acid, fumaric acid, maleic acid, and tartaric acid.

[0023] In embodiments, the effect of talarozole is an improvement of neuronal growth and connectivity. It has been shown in SURF 1 -mutant neural progenitor cells (NPCs) that talarozole provided a dosedependent rescue of neurite outgrowth capacity. It is assumed that talarozole may improve post-natal brain wiring, which is the process that regulates the establishment of connections between developing neurons in various part of the brain. When this process does not occur properly then a child may experience mental retardation of developmental delay.

[0024] Talarozole can be used or included in a composition. Particularly for use as a medicament talarozole can be formulated as a pharmaceutical composition. A further aspect of the present invention relates to a pharmaceutical composition comprising as an active ingredient talarozole, or a pharmaceutically acceptable salt thereof, for use in the treatment of a disease caused by mitochondrial complex IV deficiency. In an embodiment, the pharmaceutical composition comprises talarozole, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0025] The pharmaceutical carrier can be, for example, a solid, liquid, or gas. Suitable carriers and adjuvants can be solid or liquid and correspond to the substances ordinarily employed in formulation technology for pharmaceutical formulations. For example, water, salt solutions such as NaCl, saline, buffered saline, ethanol, alcohols, glycerol, glycols, oils, polyethylene glycols, hydroxymethylcellulose, or combinations thereof and the like may be used to form liquid preparations such as solutions. Examples of solid carriers include lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid. Examples of liquid carriers are sugar syrup, peanut oil, olive oil, and water. Examples of gaseous carriers include carbon dioxide and nitrogen. The pharmaceutical composition can optionally comprise other therapeutic ingredients or adjuvants if desired, such as preservatives, stabilizers, wetting agents, emulsifiers.

[0026] The composition can be suitable for oral, dermal, rectal, topical, and parenteral administration. In embodiments, the pharmaceutical composition for use is formulated for topical or local application such as intraneural or periradicular application, or systemic application such as intramuscular, intraperitoneal, intravenous, subcutaneous, intrathecal or oral application.

[0027] The pharmaceutical composition can be suitable for oral or parenteral administration. Parenteral administration includes subcutaneous, intramuscular, intravenous, intraneural, periradicular, intraperitoneal, and local administration. Specifically, the pharmaceutical composition may be administered systemically, for example, orally.

[0028] The composition can be formulated in accordance with the routine procedures as a pharmaceutical composition adapted for administration to human beings. The pharmaceutical compositions may be conveniently presented in unit dosage form. The pharmaceutical composition may be produced under sterile conditions using standard pharmaceutical techniques well known to those skilled in the art.

[0029] In embodiments of the pharmaceutical composition for use, the mitochondrial complex IV deficiency is Leigh syndrome, preferably SURF 1 -dependent Leigh syndrome. For the description of mitochondrial complex IV deficiency, Leigh syndrome, and SURF 1 -dependent Leigh syndrome, reference is made to the description above.

[0030] A further aspect relates to a use of talarozole, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of a disease caused by mitochondrial complex IV deficiency. In embodiments, the mitochondrial complex IV deficiency is Leigh syndrome, preferably SURF 1 -dependent Leigh syndrome. For the description of mitochondrial complex IV deficiency, Leigh syndrome, and SURF 1 -dependent Leigh syndrome, reference is made to the description above.

[0031] A further aspect relates to a method of treating a disease caused by mitochondrial complex IV deficiency, the method comprising the step of administering to a subject a therapeutically effective amount of talarozole, or a pharmaceutically acceptable salt thereof. The term “therapeutically effective amount” is used herein to mean an amount or dose sufficient to cause an improvement in a clinically significant condition in the subject. For medical purposes, subjects include human subjects and animal subjects, particularly mammalian subjects such as human subjects. The treatment may be a continuous prolonged treatment.

[0032] In embodiments, the mitochondrial complex IV deficiency is Leigh syndrome, optionally SURF1- dependent Leigh syndrome. For the description of mitochondrial complex IV deficiency, Leigh syndrome, and SURF 1 -dependent Leigh syndrome, reference is made to the description above.

[0033] Talarozole, or the pharmaceutically acceptable salt thereof, may be administered as a liquid formulation, optionally by oral application, such as in the form of a tablet.

[0034] In embodiments of the pharmaceutical composition for use, the use of talarozole, or the method, the mitochondrial complex IV deficiency is selected from the group of myopathy, hypertrophic cardiomyopathy, hepatomegaly, liver dysfunction, hypotonia, muscle weakness, exercise intolerance, developmental delay, delayed motor development and intellectual disability.

[0035] Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0036] The Examples which follow serve to illustrate the invention in more detail but do not constitute a limitation thereof.

[0037] The figures show:

[0038] Figure 1: the quantification of neurite area in Fig. 1A and number of neurites in Fig. IB from control and SURFl-mutant neurons. Each value in Figure 1A represents the total neurite area and each value in Figure IB represents the total number of neurites in each well of a 96-well plate. Significant values were calculated using the nonparametric Mann-Whitney test. * * * *p<0.0001.

[0039] Figure 2: the effect of talarozole at concentrations of IpM and lOpM in 0. 1% DMSO for 48h on neurite area (Fig. 2A and B) and number of neurites (Fig. 2C and D) in two independent experiments. Shown are the total values in each well of a 96-well plate, having a total of 5 technical replicates per compounds per experiment. Significant values were calculated using the non-parametric Mann-Whitney test.

[0040] Figure 3 : the effect of 1 pM talarozole on the amount of dopaminergic neurons (TH staining) and neuronal processes (MAP2 and SMI312 staining, respectively) in midbrain organoids generated from SURF 1 -mutant NPCs carrying a mutation in the gene SURF1 causative of Leigh syndrome.

[0041] Figure 4: the quantification of dopaminergic neurons positive for Tyrosine hydroxylase (TH) in midbrain organoids after treatment with talarozole.

[0042] Figure 5 : the lactate amount released by midbrain organoids after treatment with talarozole .

[0043] Figure 6: the dose-dependent effect of talarozole on the survival of yeasts defective for the homologous of SURF 1 gene.

[0044] Figure 7: the effect of talarozole treatment on membrane-associated cholesterol in iPSC-derived neural progenitor cells (NPCs).

[0045] Figure 8: the effect of talarozole treatment on the regulation of the expression of the CYP26A1 enzyme of the retinoic acid pathway in iPSC-derived NPCs.

[0046] Figure 9: the effect of talarozole treatment on the regulation of the expression of the PPRE enzyme of the PPARG pathway in iPSC-derived NPCs.

[0047] Example 1: confirmation of neuromorphogenesis defect in SURF 1 -mutant induced neurons

[0048] Neuromorphogenesis defect in SURF 1 -mutant induced neurons were determined in induced neurons (iN) derived from neuronal progenitor cells of control cells and SURF 1 -mutant cells. CRISPR / Cas9 engineered isogenic SURF1 mutant NPCs were generated as described in Inak et al., Nature Communications (2021) 12: 1929. On these cells neural commitment was forced via overexpression of neurogenin 2 (NGN2).

[0049] Cell nuclei were stained with Hoechst33142 dye and neurites were stained against microtubule- associated protein 2 (MAP2). Neurite area and number of neurites from control and SURF 1 -mutant neurons were qunatified. Neurite area in induced neurons (iN) was measured after five days after induction of NGN2 overexpression, using high-content image analysis (HCA) of MAP2-positive stained cells. Figure 1 shows the results. Each value in Figure 1A represents the total neurite area and each value in Figure IB represents the total number of neurites in each well of a 96-well plate. Significant values were calculated using the non-parametric Mann-Whitney test. ****p<0.0001. as can be taken from Figure 1, neurite area and number of neurites was significantly lower in SURF 1 -mutant induced neurons.

[0050] The results confirm that SURF1 mutations impaired neuronal morphogenesis in induced pluripotent stem cell (iPSC)-derived neurons and cerebral organoids. The prime defects emerged at the level of neural progenitor cells (NPCs), which were unable to perform the metabolic switch required for neural commitment, and thus showed inefficient neurite outgrowth.

[0051] Example 2: Determination of effects of talarozole in SURF 1 -mutant induced neurons

[0052] CRISPR / Cas9 engineered isogenic SURF 1 mutant NPCs, on which neural commitment via overexpression of NGN2 was forced as described above, were used to measure the impact of talarozole on neurite outgrowth capacity. SURF 1 -mutant NGN2-derived induced neurons (SURFl_iN) were treated with 0.1% DMSO (vehicle) or lOpM Talarozole. It was seen that talarozole had a positive effect in stimulating neurite development (MAP2+) in SURF 1 -mutant cells.

[0053] The effect of talarozole at increasing concentrations of IpM and lOpM in 0. 1% DMSO for 48h on neurite area and number of neurites was determined in two independent experiments. Figure 2A and 2B illustrates the effect of talarozole at concentrations of IpM and lOpM in 0.1% DMSO for 48h on neurite area determined in two independent experiments. Figure 2C and 2D illustrates the effect of talarozole at concentrations of IpM and lOpM in 0. 1% DMSO for 48h on the number of neurites determined in two independent experiments. Shown are the total values in each well of a 96-well plate, having a total of 5 technical replicates per compounds per experiment. Significant values were calculated using the non-parametric Mann-Whitney test.

[0054] As can be seen in Figure 2, talarozole exerted a dose-dependent response in increasing both neurite area and number of neurites in SURF 1 -mutant NGN2-derived induced neurons. Example 3: Determination of effects of talarozole in midbrain organoids generated from SURF1- mutant NPCs

[0055] The effects of talarozole were further determined in SURF1 -mutant midbrain organoids (SURF1-MO), which were generated from SURFl-mutant NPCs as described in Inak et al., Nature Communications (2021) 12: 1929. These organoids showed key disease-specific features, such as increased lactate released in the media, decreased number of TH neurons and aberrant neuronal branching.

[0056] Organoids were treated every 2 days with either vehicle DMSO or IpM Talarozole for 30 days. The organoids were then fixed and stained with different antibodies against Tyrosine Hydroxylase (TH) which is a marker of dopaminergic neurons, MAP2 which is a marker for neuronal dendrites, and SMI312 which is a marker for neuronal axons.

[0057] Figure 3 shows the stained organoids. As can be taken from Figure 3, talarozole treatment increased the amount of dopaminergic neurons (TH) and neuronal processes (MAP2 and SMI312, respectively) in human midbrain organoids carrying a mutation in the gene SURF1 causative of Leigh syndrome.

[0058] These results show that talarozole reduced the aberrant release of lactate in the media, increased the amount of TH positive dopaminergic neurons, and rescued the overall neuronal branching organization.

[0059] Example 4: Quantification of dopaminergic neurons after treatment with talarozole

[0060] Free-floating midbrain organoids generated from SURFl-mutant NPCs were treated for 30 days every 2 days with either vehicle DMSO or 1 pM talarozole. After treatment, free-floating midbrain organoids were fixed with 4 % PFA for 20 min at RT, followed by three 10 min washes with PBS. Organoids were blocked and permeabilized with a solution consisting of 3 % BSA, 0.5 % Triton-X- 100 and 0.05 % sodium azide in PBS, for 1 h at RT. Anti-Tyrosine Hydroxylase (TH) rabbit polyclonal antibody (Millipore; #AB152) was diluted 1:500 in blocking solution and incubated overnight at 4 °C. Afterwards, they were washed 3 times with PBS and incubated overnight at 4 °C with secondary antibodies at a 1: 1000 dilution in blocking solution. Next, organoids were incubated for 1 h at RT with Hoechst 33342 (1 : 1000) and washed three times with PBS. Images were acquired using the ZEISS Axio Observer microscope with an apotome 3 as a z-stack, which were then deconvoluted using the Zeiss blue software default settings and z-projected with maximum intensity.

[0061] For quantification of TH, immunostained MO z-projected images were analyzed using Columbus software (v. 2.9.0). Prior to analysis, images were downscaled using the greyscale function in Photoshop (v. 12.1 x64) and resized from 300 to 72 pixels per inch. The images were converted from 8-bit to 16-bit channels to retain image information and saved as .tif files. An image analysis pipeline was established using different building blocks. Organoid objects were identified based on the TH channel using the “Find Image Region” block. The "Fill Region" and "Border" functions within the "Select Region" block were utilized to cover the entire organoid area. Within this defined area, the intensities of each channel were calculated using the “Calculate Intensity Properties” block and given as the average pixel intensity. Furthermore, morphological parameters such as area, width, length (all measured in pm), and roundness were assessed using the "Calculate Morphology Properties" block. For the comparison of patient and control midbrain organoids, we calculated the ratio of intensities of respective channels and the area of the individual organoids.

[0062] Figure 4 illustrates the quantification of dopaminergic neurons positive for tyrosine hydroxylase (TH) in midbrain organoids after treatment with 1 pM talarozole. As can be taken from Figure 4, treatment with talarozole increased the amount of dopaminergic neurons positive for tyrosine hydroxylase.

[0063] Example 5 : Determination of lactate released by midbrain organoids after treatment with talarozole

[0064] Free-floating midbrain organoids generated from SURF 1 -mutant NPCs were treated for 30 days every 2 days with either vehicle DMSO or 1 pM talarozole. Healthy control organoids were treated with only DMSO.

[0065] Extracellular lactate released by midbrain organoid in the media was measured by collecting 100 pl of maturation media at day 9 of generation. Media from at least 5 individual midbrain organoids from 3 different batches was collected for analysis and stored at -20 °C until used. The manufacturer’s instruction from the Lactate Assay commercial kit was followed for measuring the absorbance of each sample using a clear 96-well microplate assay and Tecan microplate to read the absorbance at 580 nm. The calculated amount of lactate per well was normalized to the respective organoid size. Measurements are expressed as fold-change compared to control (when untreated) or to DMSO condition (when treated) of their respective batch.

[0066] Figure 5 illustrates the quantification of lactate amount released by midbrain organoids after treatment with 1 pM talarozole. As can be taken from Figure 5, SURF 1 -mutant midbrain organoids in normal conditions (DMSO treatment) produced abnormally high level of lactate compared to control midbrain organoids, which level is indicated as horizontal line. Figure 5 further shows that treatment with talarozole in SURF 1 -mutant midbrain organoids lead to a decrease in lactate production.

[0067] Example 6: Determination of effect of talarozole on the survival of yeasts defective for the homologous of SURF 1 gene

[0068] Wild-type (WT) and SURF 1 -KO mutant (ASHY) strains were assessed for growth deficiency in a non-fermentable growth medium (YP 2 % lactate) at 30 °C. A 384-well optimization assay was developed: 1) to identify the right starting density of cells that accentuates the growth difference phenotype and best assay time, 2) to establish that the assay has a good Z’, which is a measure of assay quality and the likelihood of false positives and negatives in the screen. In this assay, the Z’ score was 0.7 indicating that the assay quality is excellent. Mutant and WT cell suspensions were adjusted to an OD of 0.025, and 25 pl of mutant cells were added to the plates. The plates were spun at 250 x g for 1 min, sealed with a gas-permeable seal, and covered with a water-filled lid, then incubated at 30 °C for 24 h with 2.5 pM, 7.5 pM, 20 pM or 40 pM of talarozole or DMSO. After incubation, seals were removed and plates were equilibrated at RT for 15 min. 25 pl of Bactiter-glo solution was added to each well, plates were shaken for 2 min, incubated for 10 min, and luminescence was read using an Envision plate reader.

[0069] Figure 6 illustrates the dose-dependent effect of talarozole on the survival of yeasts defective for the homologous of SURF1 gene. As can be taken from Figure 6, SURF1 knock-out yeast ae not able to grow. Figure 6 further shows that the addition of talarozole reverted this defect and allowed yeast to a growth rate similar to that of wild-type (WT) yeasts.

[0070] Example 7 : Determination of effect of talarozole on membrane-associated cholesterol For cholesterol analysis, 40,000 iPSC-derived neural progenitor cells (NPCs) per well were seeded onto matrigel-coated 96-well black ibidi plates. The next day, NPCs were either fed with normal media (untreated condition) or treated with either 0.1 % (v / v) DMSO, 10 pM Talarozole for 48 h. For the 24 h treatment, NPCs were treated with the above-mentioned conditions two days after. Both 24 h and 48 h treatment conditions were fixed on the same day using 4% PFA for 15 min at RT and washed 3 times for 10 min with PBS. For PFO-GST labelling of membrane -bound cholesterol, fixed NPCs were blocked and permeabilized with 10 % goat serum in 0. 1% Triton X-100, PBS (0. 1 % PBST) for Ih and incubated with 15 pg / ml recombinant PFO-GST for 3 h.104After washing the cells three times with 0.1 % PBST, anti-GST (1:200) was applied in 10% goat serum 0.1% PBST, overnight at 4 °C. After three washes with 0.1 % PBST, Alexa Fluor™ 488 goat anti-mouse IgG (H+L) secondary antibody was applied (1:500) for 1 h at RT. Lastly, wells were washed three times in PBS and cells were preserved in Mounting Medium with DAPI. Fluorescent images were acquired with a LSM 900 Zeiss confocal microscope. Laser power, gain and offset parameters were kept constant for each experiment, and any subsequent adjustments to contrast and brilliance were applied equally to all images. The image analysis was performed using Fji hnageJ software using custom-made macros.

[0071] Figure 7 illustrates the intensity of membrane-associated cholesterol in iPSC-derived neural progenitor cells (NPCs). As can be taken from Figure 7, SURF1 mutant NPCs had lower amount of membrane- associated cholesterol. Figure 7 further shows that treatment with talarozole for 24h improved this defect.

[0072] Example 8: Determination of effect of talarozole on enzyme expression in iPSC-derived neural progenitor cells (NPCs)

[0073] The expression of the CYP26A1 enzyme of the retinoic acid pathway and of the PPRE enzyme of the PPARG pathway in iPSC-derived NPCs was determined using luciferase activity. The CYP26A1 gene encodes cytochrome P450 26A1, a member of the cytochrome P450 proteins. This enzyme metabolises all-trans-retinoic acid. Cis-acting peroxisome proliferator responsive elements (PPREs) are regions in some genes inducible by peroxisome proliferator and therefore their expression indicates engagement of the PPARG pathway. CYP26A1-Luc was a gift from Andrea Rossi. PPAR-Luc and Renilla-Luciferase plasmids were obtained as described in Puighermanal et al., Nature Communications 2024, 15:7730. Briefly, the 3XDR1 sites from the PPREX3-TK-Luc plasmid, a gift from Bruce Spiegelman (Addgene plasmid #1015; http: / / n2t.net / addgene: 1015; RRID:Addgene_1015), were removed with Mlul and Hindlll and subcloned it into a minimal fos promoter-driven luciferase reporter vector. This fos promoter was obtained from plasmid p5xATF6-GL3, a gift from Ron Prywes (Addgene plasmid

[0074] #11976; http: / / n2t.net / addgene: 11976; RRID:Addgene_l 1976), by removing ATF6 binding sites with Xhol and Hindlll. Renilla-Luciferase plasmid was generated by obtaining the RLuc coding sequence from pGL4.73 [hRLuc / SV40] (Promega) plasmid (digested with Hindlll and Xbal) and cloning in pEFl plasmid (Invitrogen) in EcoRV and Xbal sites.

[0075] NPCs were transfected using ScreenFect®A-plus (ScreenFect; # S-6001) the day after plating them, in antibody-free media. Twenty-four hours after transfection, cells were treated with DMSO or lOpM Talarozole for additional 24h before luciferase assays were performed using the Dual Gio Luciferase Assay system (Promega) with firefly luciferase-based reporter gene activity normalized to the RLuc control.

[0076] The expression is regulated with the treatment. Basically, the luciferase reporter is expressed when the promoter for CYP26A1 or PPAR is activated, and thus the assay gives information about the regulation of the expression.

[0077] Figure 8 illustrates the luciferase activity showing the regulation of the expression of the CYP26A1 enzyme of the retinoic acid pathway in iPSC-derived NPCs. As can be taken from Figure 8, SURF1 mutant NPCs at basal level (DMSO treatment) have lower CYP26A1 expression compared to control NPCs, which level is indicated as dotted line. Figure 8 further shows that treatment with talarozole in SURF1 mutant NPCs restored the expression of the CYP26A1 enzyme of the retinoic acid pathway.

[0078] Figure 9 illustrates the luciferase activity showing the regulation of the expression of the PPRE enzyme of the PPARG pathway in iPSC-derived NPCs. As can be taken from Figure 9, SURF1 mutant NPCs at basal level (DMSO) have lower PPRE expression compared to control NPCs, which level is indicated as dotted line. Figure 9 further shows that treatment with talarozole in SURF1 mutant NPCs restored the expression of the PPRE enzyme of the PPARG pathway. This suggests that talarazoel may also engage the PPARG pathway whose modulation was shown to be associated with extended lifespan in mice models of Leigh syndrome (Puighermanal et al., Nature Communications 2024, 15:7730). In summary, these findings indicate that talarozole is usable in the treatment of a disease caused by mitochondrial complex IV deficiency, such as Leigh syndrome, and particularly SURF 1 -dependent Leigh syndrome.

Claims

C l a i m s1. Talarozole, or a pharmaceutically acceptable salt thereof, for use in the treatment of a disease caused by mitochondrial complex IV deficiency.

2. Talarozole, or the pharmaceutically acceptable salt thereof, for use according to claim 1, wherein the mitochondrial complex IV deficiency is Leigh syndrome.

3. Talarozole, or the pharmaceutically acceptable salt thereof, for use according to any one of the preceding claims, wherein the disease caused by mitochondrial complex IV deficiency is SURF 1 -dependent Leigh syndrome.

4. Talarozole, or the pharmaceutically acceptable salt thereof, for use according to any one of the preceding claims, wherein the disease caused by mitochondrial complex IV deficiency is selected from the group of myopathy, hypertrophic cardiomyopathy, hepatomegaly, liver dysfunction, hypotonia, muscle weakness, exercise intolerance, developmental delay, delayed motor development and intellectual disability.

5. Talarozole, or the pharmaceutically acceptable salt thereof, for use according to any one of the preceding claims, wherein the effect of talarozole is an improvement of neuronal growth and connectivity.

6. A pharmaceutical composition comprising as an active ingredient talarozole, or a pharmaceutically acceptable salt thereof, for use in the treatment of a disease caused by mitochondrial complex IV deficiency.

7. The pharmaceutical composition for use according to claim 6, wherein the pharmaceutical composition is formulated for topical or local application such as intraneural or periradicular application, or systemic application such as intramuscular, intraperitoneal, intravenous, subcutaneous, intrathecal or oral application.

8. The pharmaceutical composition for use according to claim 6 or 7, wherein the mitochondrial complex IV deficiency is Leigh syndrome, optionally SURF 1 -dependent Leigh syndrome.

9. The pharmaceutical composition for use according to claim 6 or 7, wherein the disease caused by mitochondrial complex IV deficiency is selected from the group of myopathy, hypertrophic cardiomyopathy, hepatomegaly, liver dysfunction, hypotonia, muscle weakness, exercise intolerance, developmental delay, delayed motor development and intellectual disability.

10. Use of talarozole, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of a disease caused by mitochondrial complex IV deficiency.

11. The use according to claim 10, wherein the mitochondrial complex IV deficiency is Leigh syndrome, optionally SURF 1 -dependent Leigh syndrome.

12. The use according to claim 10, wherein the disease caused by mitochondrial complex IV deficiency is selected from the group of myopathy, hypertrophic cardiomyopathy, hepatomegaly, liver dysfunction, hypotonia, muscle weakness, exercise intolerance, developmental delay, delayed motor development and intellectual disability.

13. A method of treating a disease caused by mitochondrial complex IV deficiency, the method comprising the step of administering to a subject a therapeutically effective amount of talarozole, or a pharmaceutically acceptable salt thereof.

14. The method according to claim 13, wherein the mitochondrial complex IV deficiency is Leigh syndrome, optionally SURF 1 -dependent Leigh syndrome.

15. The method according to claim 13, wherein the disease caused by mitochondrial complex IV deficiency is selected from the group of myopathy, hypertrophic cardiomyopathy, hepatomegaly, liver dysfunction, hypotonia, muscle weakness, exercise intolerance, developmental delay, delayed motor development and intellectual disability.