Treatment of mitochondrial diseases using the CNS-permeable sGC stimulator zagociguat

Abstract

The present invention relates to a method for treating mitochondrial disease in a patient in need thereof by administering a compound (I), which is a stimulator of soluble guanylate cyclase (sGC), at a specific dosage, alone or in combination therapy. JPEG2025519489000012.jpg52112

Description

Technical Field

[0001] Related Applications

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 350,708, filed on June 9, 2022, and U.S. Provisional Application No. 63 / 410,829, filed on September 28, 2022. The entire content of each of the above-referenced applications is hereby incorporated by reference into this specification.

[0002]

[0002] The present invention provides a method of treating a particular mitochondrial disease in a human subject by administering a specific dosing regimen of a CNS-penetrant stimulator of soluble guanylate cyclase (sGC), either alone or in combination therapy.

Background Art

[0003] Mitochondrial Diseases and MELAS

[0003] Mitochondrial diseases or mitochondrial abnormalities are a group of rare genetic disorders that occur when mitochondria cannot produce enough energy for the body to function properly. Such diseases have clinically heterogeneous symptom presentations and can manifest as cerebral blood flow (CBF) disorders, oxidative stress, inflammation, and metabolic crises. Such diseases can affect most parts of the body, including cells of the brain, nerves, muscles, kidneys, heart, liver, eyes, ears, or pancreas. Such diseases cause debilitating physical, developmental, and cognitive impairments with symptoms including growth retardation, loss of muscle tone, muscle weakness and pain, fatigue, seizures, vision loss and / or hearing loss, gastrointestinal problems, cognitive impairment, learning disabilities, and organ failure. The average lifespan of mitochondrial patients is significantly shortened. Mitochondrial abnormalities are usually progressive. It is estimated that 1 in 4,000 people has a mitochondrial abnormality. 80% of patients with mitochondrial diseases present with CNS symptoms.

[0004]

[0004] Currently, there is no effective treatment or cure for these disorders. Management of such disorders is mainly supportive therapy and may include nutritional management, exercise, and / or supplements of vitamins or amino acids.

[0005]

[0005] For example, mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) is a specific mitochondrial disease that affects many body systems, particularly the brain and nervous system (encephalopathy) and muscles (myopathy). MELAS is the most common form of primary mitochondrial disease (https: / / www.mitoaction.org / resources / primary-mitochondrial-disease-and-secondary-mitochondrial-dysfunction-importance-of-distinction-for-diagnosis-and-treatment / ). The signs and symptoms of this disorder usually appear in childhood after a period of normal development, but can occur at any age. Initial symptoms may include muscle weakness and pain, fatigue, recurrent headaches, loss of appetite, vomiting, and seizures. Most affected individuals experience stroke-like episodes (SLE) that begin before the age of 40. These episodes are often accompanied by temporary muscle weakness (hemiparesis) on one side of the body, altered consciousness, visual disturbances, seizures, and severe headache similar to migraine. Repeated SLE can gradually damage the brain, leading to vision loss, movement problems, and loss of intellectual or cognitive function.

[0006]

[0006] Most MELAS patients have a buildup of lactic acid in their bodies and are in a state called lactic acidosis. An increase in the acidity of the blood can lead to vomiting, abdominal pain, extreme fatigue and weakness, muscle weakness, and difficulty breathing. Although not common, MELAS patients may experience involuntary muscle spasms (myoclonus), lack of muscle coordination (ataxia), hearing loss, heart and kidney problems, diabetes, and hormonal imbalance.

[0007]

[0007] In the absence of an approved treatment for MELAS, citrulline and L-arginine, precursors of nitric oxide (NO), are hypothesized to benefit this patient population. Consensus guidelines from the Mitochondrial Medicine Society recommend acute arginine administration to improve clinical symptoms associated with SLE in MELAS patients. Mechanistically, L-arginine is directly converted to NO, which is the starting point of the nitric oxide-soluble guanylate cyclase-cyclic guanosine monophosphate (NO-sGC-cGMP) pathway.

Summary of the Invention

Problems to be Solved by the Invention

[0008]

[0008] Treatment options for mitochondrial diseases are extremely limited; thus, there remains a need to develop new therapies to improve the many clinical signs associated with these diseases.

Means for Solving the Problems

[0009]

[0009] In a first aspect of the present invention, disclosed herein is a method of treating mitochondrial disease in a patient by administering to the patient a total daily oral dose of 15 mg to 60 mg of Compound I or a pharmaceutically acceptable salt of Compound I in an equimolar amount.

[0010]

[0010] In a second aspect of the present invention, disclosed herein is Compound I or a pharmaceutically acceptable salt thereof for use in the treatment of mitochondrial disease in a patient by administering to the patient a total daily oral dose of 15 mg to 60 mg of Compound I or a pharmaceutically acceptable salt of Compound I in an equimolar amount.

[0011]

[0011] In a third aspect of the present invention, disclosed herein is the use of compound I or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for treating mitochondrial disease in a patient by administering to the patient an oral daily dose of 15 mg to 60 mg of compound I or an equimolar amount of a pharmaceutically acceptable salt of compound I.

[0012]

[0012] In a fourth aspect, the methods and uses of the present invention involve a treatment that is combined with one or more additional therapeutic agents.

Brief Description of the Drawings

[0013]

Figure 1

[0013] Figure 1 shows the correlation between GDF-15 and FGF-21, which are biomarkers of mitochondrial dysfunction at baseline.

Figure 2

[0014] Figure 2 shows the increase in CBF on day 29 compared to baseline (day 1) observed across all brain regions of interest. CI = confidence interval.

Modes for Carrying Out the Invention

[0014] The NO-sGC-cGMP Pathway, sGC Stimulation, and Mitochondrial Disease

[0015] In vivo, NO is synthesized from arginine and oxygen by various groups of nitric oxide synthase (NOS) enzymes and by the sequential reduction of inorganic nitrates. Three distinct isoforms of NOS have been identified: inducible NOS (iNOS or NOS II), which is found in activated macrophage cells; constitutive neuronal NOS (nNOS or NOS I), which is involved in neurotransmission and long-term potentiation; and constitutive endothelial NOS (eNOS or NOS III), which regulates smooth muscle relaxation and blood pressure.

[0015]

[0016] sGC is the primary receptor enzyme for NO in vivo. sGC can be activated via both NO-dependent and NO-independent mechanisms. In response to this activation, sGC converts guanosine-5'-triphosphate (GTP) to the secondary messenger cyclic guanosine 3',5'-monophosphate (cGMP). When cGMP levels increase, they similarly regulate the activity of downstream effectors including protein kinases, phosphodiesterases (PDEs), and ion channels. Intracellular cGMP regulates vasoconstriction and local blood flow, fibrosis, and inflammation by activating cGMP-dependent protein kinase (PKG) and other downstream modulators.

[0016]

[0017] The NO signaling pathway is also important for the regulation of mitochondrial function and biogenesis. Dysregulation of the NO pathway is recognized as a major contributing factor in mitochondrial disease, leading to cerebral blood flow (CBF) impairment, oxidative stress, inflammation, and metabolic crisis. There is a clear association observed between NO signaling, stroke-like episodes (SLE), and dysregulated CBF in patients with mitochondrial disease. NO bioavailability may be reduced in these patients via several mechanisms, including endothelial dysfunction and associated decreases in endothelial nitric oxide synthase, increased levels of the NOS inhibitor asymmetric dimethylarginine (ADMA), and increased oxidative stress and reactive oxygen species (ROS) that react with NO.

[0017]

[0018] In the CNS, the NO-sGC-cGMP signaling pathway underlies a number of physiological processes that contribute to overall brain health, including neurotransmission, neurovascular function, cellular bioenergetics, and inflammation, and is involved in neuronal survival and cognitive function.

[0018]

[0019] sGC stimulators are a group of heme-dependent agonists of the sGC enzyme that act synergistically with various amounts of NO to enhance the enzymatic conversion of GTP to cGMP. sGC stimulators are clearly distinct from and structurally unrelated to another group of NO-independent and heme-independent agonists of sGC known as sGC activators. The benzylindazole compound YC-1 was the first sGC stimulator to be identified. Several sGC stimulators have been identified, including BAY 41-2272, BAY 41-8543, riociguat (BAY 63-2521), vericiguat, olinciguat (IW-1701), and praliciguat (IW-1973), and have since been pharmacologically characterized. To date, as far as the inventors are aware, there are no sGC stimulators that have been approved for manufacture and sale in the field of the CNS, and the following Compound I is the only CNS-penetrant sGC stimulator currently in clinical development for the treatment of CNS diseases and mitochondrial diseases.

[0019]

[0020] sGC stimulators may target abnormal NO pathways or offer significant advantages over other potential therapies that upregulate the NO pathway in other ways. For example, sGC stimulation is a more potent approach than either the use of NO supplementation (associated with tachyphylaxis) or inhibition of cGMP breakdown (via phosphodiesterase inhibitors [PDEi]), and the effect is limited when cGMP levels are very low. Furthermore, the wide distribution of sGC, including in various regions of the brain, can enhance signaling throughout the tissue, while PDEi targets have more limited cell and tissue expression.

[0020]

[0021] In the consensus guidelines from the Mitochondrial Medicine Society, acute L-arginine administration is recommended to improve the clinical symptoms associated with stroke-like episodes in patients with MELAS. Mechanistically, L-arginine is directly converted to NO, which is the starting point of the NO-sGC-cGMP pathway. As a central node in the NO-sGC-cGMP pathway, sGC stimulators are hypothesized to have a positive effect on mitochondrial diseases, including those that potentially promote mitochondrial function and biosynthesis and affect the CNS. Compound I (CY6463)

[0022] Compound I (also known as CY6463, IW-6463, or IWP-247) is an orally administered CNS-penetrant sGC stimulator being studied for the treatment of CNS diseases and mitochondrial diseases (clinical trials.gov identifiers NCT03856827, NCT04240158, NCT04475549, NCT04798989, NCT04972227). To the best of the inventors' knowledge, this is the only CNS-penetrant stimulator that has been tested in human subjects to date.

[0021]

[0023] As an sGC stimulator, Compound I acts as a positive allosteric modulator of sGC by binding to sGC and thereby amplifying downstream signaling.

[0022]

Chemical Structure

[0023]

[0024] In vitro studies of cells from patients with mitochondrial diseases have shown that Compound I may improve the cellular energy characteristics in these cells by increasing the abundance of available adenosine triphosphate (ATP), and may reduce mitochondrial dysfunction by enhancing the expression of genes involved in mitochondrial function, ATP synthesis, metabolism, and reduction of reactive oxygen species (ROS) (see WO2020 / 014504). Experiments on cells from patients with mitochondrial diseases have shown that the expression levels of mitochondrial genes such as TFAM and DDAH2 are lower in patient cells than in healthy cells. DDAH2 encodes an enzyme that degrades asymmetric dimethylarginine (ADMA). TFAM is a highly expressed protein present in mitochondria, is necessary for mitochondrial transcription, regulates the mtDNA copy number, and is important for maintaining ATP production. An increase in ADMA may cause mitochondrial dysfunction and has been observed to increase in patients with mitochondrial diseases. Consistent with the increase in ATP levels, treatment with Compound I increased the expression levels of TFAM and DDAH2 in patient cells. In an in vivo model of mitochondrial dysfunction-induced retinal degeneration, mice pretreated with Compound I had lower rotenone-induced astrogliosis compared to vehicle-treated mice, suggesting that Compound I may provide protection against tissue damage induced by mitochondrial dysfunction.

[0024]

[0025] In rodent studies, in contrast to CNS-restricted sGC stimulants that show lack of target disease engagement and clear pharmacological effects in the CNS, a single dose of Compound I increased the fMRI-BOLD signal, enhanced qEEG gamma band oscillatory power, and increased cGMP levels in the CNS. In a rat model of CNS injury, chronic administration of Compound I improved dendritic spine density, reversed the deficit of the brain metabolite N-acetylaspartate (NAA) + N-acetylaspartylglutamate (NAAG), and restored hippocampal long-term potentiation (LTP, a form of synaptic plasticity underlying memory formation). Compound I also increased neurotrophic factors such as phosphorylated cAMP response element-binding (pCREB) and brain-derived neurotrophic factor (BDNF), and improved behavioral task performance in pharmacologically impaired rats (see Correia, Susana S; Iyengar, Rajesh R; Germano, Peter; Tang, Kim; Bernier, Sylvie G; Schwartzkopf, Chad D; Tobin, Jenny; Lee, Thomas W-H; Liu, Guang; Jacobson, Sarah; Carvalho, Andrew; Rennie, Glen R; Jung, Joon; Renhowe, Paul A; Lonie, Elisabeth; Winrow, C; Hadcock, J; Jones, J; Currie, MG. The CNS-Penetrant Soluble Guanylate Cyclase Stimulator CY6463 Reveals its Therapeutic Potential in Neurodegenerative Diseases. Front Pharmacol. 24 May 2021|https: / / doi.org / 10.3389 / fphar.2021.656561).

[0025]

[0026] Safety and pharmacokinetic (PK) data from the Phase 1 trial in healthy adults, together with safety, PK, and pharmacodynamic (PD) data from a second Phase 1 trial in healthy elderly subjects, generally supported clinical studies of Compound I in the potential treatment of patients with mitochondrial diseases, particularly MELAS. Definitions and General Terms

[0027] Unless otherwise defined, all 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, which is the field of medicine, particularly the fields of mitochondrial diseases and / or brain medicine. Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0026]

[0028] As used herein, the word "a" before a noun represents one or more of that particular noun. As used herein, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise.

[0027]

[0029] As used herein, the terms "subject" and "patient" are used interchangeably. The subject or patient is a human patient or human subject.

[0028]

[0030] For the terms "for example" and "such as", and their grammatical equivalents, the phrase "without limitation" or "and without limitation" is understood to follow unless explicitly stated otherwise.

[0029]

[0031] Cognitive function naturally declines with age and can also become pathological. "Cognitive impairment" generally refers to deficits in one or more higher brain functions involving aspects of thinking and information processing (i.e., cognition).

[0030]

[0032] As used herein, the term "therapeutically effective amount" means the amount of an active compound or pharmaceutical agent that elicits a drug response in a human, which a physician or other clinician is seeking. The therapeutically effective amount of a compound is at least the minimum amount necessary to ameliorate, soothe, relieve, delay, reduce, mitigate, or cure a disease, disorder, or syndrome or one or more of its symptoms, signs, or causes. In another embodiment, the therapeutically effective amount is the amount necessary to bring an abnormal level of a particular clinical marker of a disease, disorder, or syndrome closer to a normal value or level. In another embodiment, the therapeutically effective amount is the amount necessary to bring the level of a particular clinical marker presented by an affected subject closer to that of a normal subject of the same age (normalize), or closer to that of a patient with a less severe medical condition, or a patient in the early stages of disease progression. The effective amount can be administered in one or more doses throughout the day.

[0031]

[0033] As used herein, when excessive orthostatic hypotension, severe dizziness, severe postural dizziness, excessive pre-syncope, or excessive syncope does not occur in a patient, the dose is "not associated with a significant occurrence of adverse events (AE) or serious adverse events (sAE) related to symptomatic hypotension." Excessive orthostatic hypotension, severe dizziness, severe postural dizziness, excessive pre-syncope, or excessive syncope in a patient warrants interruption of treatment by the patient or recommendation for interruption by an expert.

[0032]

[0034] The terms "administer", "administering", or "administration" with respect to a compound or pharmaceutical agent mean introducing the compound into the body of a patient in need of treatment. When Compound I or a pharmaceutically acceptable salt thereof is used in combination with one or more other therapeutic agents, "administration" and its variations are understood to include the simultaneous and / or sequential introduction of Compound I and the other therapeutic agents to the patient, respectively.

[0033]

[0035] As used herein, the term "disorder" refers to either a deviation from or an interruption of the normal structure or function of any body part, organ, or system, as indicated by a characteristic collection of signs and symptoms, whether or not its cause, pathology, and prognosis are known. The term "disorder" encompasses diseases and conditions (or states of health) and other related terms such as syndromes, which are defined as arising from a single cause or as a combination of symptoms that generally occur together to constitute a distinct clinical picture. In some embodiments, the term "disorder" refers to mitochondrial abnormalities. The terms "disorder", "disease", "condition", or "syndrome" as used herein are used interchangeably.

[0034]

[0036] "Mitochondrial abnormalities" refer to a group of genetic disorders that affect mitochondria (structures within each cell of the body responsible for energy production). These disorders can occur at any age and affect almost all organs, including the brain, muscles, heart, liver, nerves, eyes, ears, and kidneys. Some disorders affect only one organ or tissue, while many involve multiple organ systems, often including the brain, muscles, heart, liver, nerves, eyes, ears, and / or kidneys. There are various symptoms associated with mitochondrial abnormalities.

[0035]

[0037] Mitochondrial genetic diseases can be caused by mutations in either mitochondrial DNA or nuclear DNA, leading to mitochondrial dysfunction and insufficient production of cellular ATP. Those caused by mutations in mitochondrial DNA are maternally inherited, while those caused by mutations in nuclear DNA may follow autosomal dominant, autosomal recessive, or X-linked inheritance patterns. (See: https: / / rarediseases.info.nih.gov / diseases / 7048 / mitochondrial-genetic-disorders, last accessed on June 3, 2022, the teachings of which are incorporated herein by reference). Mitochondrial diseases contemplated throughout the present disclosure are primary mitochondrial diseases or mitochondrial abnormalities. As used herein, the term mitochondrial abnormality is equivalent to the term primary mitochondrial abnormality. In some cases, mitochondrial dysfunction may also be secondary to other diseases. However, the treatment of such secondary mitochondrial dysfunction is not considered or contemplated herein. For the definition and distinction between primary mitochondrial abnormalities or diseases and secondary mitochondrial dysfunction, see https: / / www.mitoaction.org / resources / primary-mitochondrial-disease-and-secondary-mitochondrial-dysfunction-importance-of-distinction-for-diagnosis-and-treatment / (last accessed on June 7, 2022).

[0036]

[0038] Mitochondrial diseases mainly manifest as chronic loss of cellular ATP, resulting in a variety of clinical phenotypes and relative symptoms. In addition to ATP crisis, mitochondrial respiratory chain dysfunction also causes excessive ROS production, increases oxidative stress, and leads to cell damage and inflammation.

[0037]

[0039] Specific mitochondrial diseases that can be treated and / or prevented by administering Compound I, or an equivalent amount of a pharmaceutically acceptable salt thereof, at a specific dosage (total oral daily dosage of 15 mg to 60 mg) disclosed herein include, but are not limited to, the following:

[0040] Alpers' disease, autosomal dominant optic atrophy (ADOA), Barth syndrome / LIC (lethal infantile cardiomyopathy), beta-oxidation deficiency, long-chain fatty acid transport deficiency, coenzyme Q10 deficiency, complex I, II, III, IV, V deficiency, chronic progressive external ophthalmoplegia (CPEO), Friedreich's ataxia, Kearns-Sayre syndrome, leukodystrophy, Leigh disease or Leigh syndrome, LHON, LHON Plus, MELAS (mitochondrial myopathy, encephalomyopathy, lactic acidosis, stroke-like episodes), ragged red fiber-myoclonus epilepsy (MERRF), mitochondrial recessive ataxia syndrome (MIRAS), mitochondrial cytopathy, mitochondrial DNA depletion, mitochondrial encephalopathy, mitochondrial myopathy, multiple mitochondrial dysfunction syndrome, MNGIE (myoneurogastrointestinal encephalopathy), NARP (neuropathy, ataxia, retinitis pigmentosa, and ptosis), Pearson syndrome, pyruvate carboxylase deficiency, pyruvate dehydrogenase deficiency or pyruvate dehydrogenase complex deficiency (PDCD / PDH), and POLG mutation.

[0038]

[0041] In one embodiment, the mitochondrial disease is selected from Alpers', complex I, II, III, IV deficiency, CPEO, KSS, LCHAD, Leigh syndrome, leukodystrophy, LHON, MELAS, MEPAN, MERRF, MIRAS, mitochondrial DNA depletion, MNGIE, NARP, Pearson syndrome, and POLG mutation. In one embodiment, the mitochondrial disease is complex I mitochondrial disease. In another embodiment, the mitochondrial disease is MELAS. In yet another embodiment, the mitochondrial disease is Leigh syndrome.

[0039]

[0042] As used herein, "treat," "treating," or "treatment" with respect to a disorder, disease, condition, symptom, or syndrome refers to invalidating or ameliorating a cause and / or an effect associated with the disorder, disease, condition, or syndrome (i.e., any of symptoms, physiological, physical, psychological, cognitive, emotional, or functional manifestations, or clinical parameters or observations). As used herein, the terms "treat," "treatment," and "treating" also refer to delaying or ameliorating or blunting or preventing the progression, severity, and / or duration of a disease (i.e., the known or predicted progression of the disease), or delaying or ameliorating or blunting or preventing the progression of one or more clinical parameters associated with the disease (i.e., "managing" the condition without "curing") resulting from the administration of one or more therapies.

[0040]

[0043] As used herein, the phrase "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable organic or inorganic salt of Compound I. Pharmaceutically acceptable salts of Compound I can be used in drugs. However, salts that are not pharmaceutically acceptable are useful in the preparation of Compound I or other pharmaceutically acceptable salts of Compound I. Pharmaceutically acceptable salts may involve the inclusion of another molecule such as an acetate ion, a succinate ion, or other counterions. A counterion can be any organic or inorganic moiety that stabilizes the charge in the parent compound. Further, a pharmaceutically acceptable salt may have more than one charged atom within its structure. Examples where multiple charged atoms are part of a pharmaceutically acceptable salt can have multiple counterions. Thus, a pharmaceutically acceptable salt can have one or more charged atoms and / or one or more counterions.

[0041]

[0044] The pharmaceutically acceptable salts of Compound I described herein include those derived from Compound I using inorganic acids, organic acids or bases. In some embodiments, the salts can be prepared in situ during the final isolation and purification of the compound. In other embodiments, the salts can be prepared from the free form of Compound I in a separate synthetic step.

[0042]

[0045] When a compound such as Compound I is acidic or contains a sufficiently acidic moiety, suitable "pharmaceutically acceptable salts" refer to salts prepared from pharmaceutically acceptable non-toxic bases including inorganic bases and organic bases. Salts derived from inorganic bases include those of aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic, manganous, potassium, sodium, zinc, etc. Specific embodiments include ammonium salts, calcium salts, magnesium salts, potassium salts and sodium salts. Salts derived from pharmaceutically acceptable non-toxic organic bases include salts of primary amines, secondary amines and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as salts of arginine, betaine, caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, etc.

[0043]

[0046] When a compound such as Compound I is basic or contains a sufficiently basic moiety, the salt can be prepared from pharmaceutically acceptable non-toxic acids including inorganic acids and organic acids. Such acids include acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, fumaric acid, gluconic acid, glutamic acid, hydrobromic acid, hydrochloric acid, isethionic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, mucic acid, nitric acid, pamoic acid, pantothenic acid, phosphoric acid, succinic acid, sulfuric acid, tartaric acid, p-toluenesulfonic acid, and the like. Specific embodiments include citric acid, hydrobromic acid, hydrochloric acid, maleic acid, phosphoric acid, sulfuric acid, and tartaric acid. Other representative salts include sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and salts of pamoic acid (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)), but are not limited thereto.

[0044]

[0047] The preparation of the above-mentioned pharmaceutically acceptable salts and other typical pharmaceutically acceptable salts is more fully described by Berg et al., "Pharmaceutical Salts", J. Pharm. Sci., 1977:66:1-19, which is hereby incorporated by reference in its entirety. Clinical Evaluation and Patient Reported Outcome Tools

[0048] The evaluation of the health status in patients with mitochondrial diseases and the evaluation of the corresponding pathology underlying the observed dysfunction, debilitation, or symptoms are known in the art and can be performed using a number of various evaluation tools or clinical measurements that are in use.

[0045]

[0049] These range from imaging tools (e.g., magnetic resonance imaging (MRI) such as using arterial spin labeling (ASL) or functional fMRI - BOLD modality), to assay measurements (e.g., body fluid biomarkers measured in blood, cerebrospinal fluid (CSF), urine, plasma, serum, skin, saliva), clinical outcome assessment tools or means (e.g., patient - reported outcome means or clinician - reported outcome means or performance outcome scales, e.g., cognitive assessments using PROMIS questionnaires, MFIS scoring as described herein or known in the art), digital assessments (e.g., those obtained by assessment with wearable devices, sensors or cameras) and electrophysiological assessments (e.g., EEG). Some of these are described in the Examples section below and were used in the clinical trials described in Example 1. Others are known in the art and can be used in hospital, clinical or community settings. For example, the American Academy of Family Physicians (AAFP) describes on its web page a number of potential cognitive assessment tools such as the Mini - Cog, MoCA, SLUMS test, CPCoG, MIS and MMSE etc. and provides links to them (https: / / www.aafp.org / pubs / afp / issues / 2019 / 0115 / p101.html, last accessed on June 3, 2022).

[0046]

[0050] Some measurements are performed to assist in diagnosis and / or patient selection. Others are performed to assist in prognostic assessment. Others may be performed to evaluate the pharmacological response to a particular intervention as described herein (pharmacodynamic assessment or PD assessment). Others may be performed to evaluate the susceptibility to or the risk of rejection or reaction to a particular intervention (e.g., evaluation of genetic markers or other biomarkers) or to evaluate the progression of a disease in a patient. Past clinical data on Compound I in healthy subjects

[0051] Electroencephalography (EEG) is a technique for measuring the electrical activity (electroencephalophysiology) in the brain by using electrodes placed on the scalp. The signals of the EEG power spectrum may be analyzed in various frequencies or frequency bands. This was analyzed in the following frequency bands: delta - 1 - 4 Hz (typically associated with sleep), theta - 4 - 7.5 Hz (associated with wakefulness / falling asleep, some related to cognition), alpha - 8 - 12 Hz (associated with passive wakefulness and cognitive processing), beta - 12 - 25 Hz (associated with attention and concentration), and gamma - 25 - 45 Hz (associated with higher cognitive functions) in two previous Phase 1 clinical trials using Compound I described below. qEEG represents quantitative electroencephalography.

[0047]

[0052] Event - related potential (ERP) is, for example, "a time - synchronous measurement of the electrical activity on the cerebral surface representing different phases of cortical processing" in response to auditory or visual stimuli (Patel and Azzam (2005), Characterization of N200 and P300: Selected Studies of the Event - Related Potential. International Journal of Medical Sciences 2(4):147 - 154). ERP is time - synchronous and represents the average of electrical responses observed after multiple trials. ERP is an objective non - invasive approach for studying information processing and cognitive functions in the brain.

[0048]

[0053] During EEG - ERP experiments, several major waveforms can be evaluated according to the stimuli: N200 (related to stimulus discrimination and differentiation), P300 (related to selective attention, information processing, and cognitive speed / ability), P200 (related to aspects of selective attention or stimulus encoding), P50 (related to sensory gating or a decrease in the neurophysiological response to unnecessary stimuli), and N100 (related to the initial perceptual process).

[0049]

[0054] Two main parameters: latency (how much time it is from the stimulus to the peak signal) and amplitude (how strong the peak signal is) are used to quantify each response.

[0050]

[0055] In preclinical models, stimulation of sGC by Compound I has been shown to result in changes in qEEG signals (see Meeting abstracts from the 9th International Conference on cGMP: Generators, Effectors and Therapeutic Implications, Journal of Translational Medicine, Volume 17, Article number: 254 (2019) S1-02 Evaluating soluble guanylate cyclase stimulation for serious central nervous system diseases; Correia, Susana S; Iyengar, Rajesh R; Germano, Peter; Tang, Kim; Bernier, Sylvie G; Schwartzkopf, Chad D; Tobin, Jenny; Lee, Thomas W-H; Liu, Guang; Jacobson, Sarah; Carvalho, Andrew; Rennie, Glen R; Jung, Joon; Renhowe, Paul A; Lonie, Elisabeth; Winrow, C; Hadcock, J; Jones, J; Currie, MG. The CNS-Penetrant Soluble Guanylate Cyclase Stimulator CY6463 Reveals its Therapeutic Potential in Neurodegenerative Diseases. Front Pharmacol. May 24, 2021 | https: / / doi.org / 10.3389 / fphar.2021.656561). These studies were performed in rats using a telemetry device implanted in the prefrontal cortex and fronto-parietal regions of the brain. Rats were administered orally with a suspension of Compound I, orally with a suspension of a peripherally restricted sGC stimulator, or subcutaneously with a solution of donepezil. Compound I altered qEEG measurements, such as increasing gamma oscillations, while the peripherally restricted sGC stimulator decreased gamma power compared to vehicle administration.Compound I administered orally to rats at 10 mg / kg increased gamma power, and the signal was further enhanced 1-2 hours after administration when combined with 1 mg / kg of donepezil.

[0051]

[0056] Subsequently, in a Phase I clinical trial (ClinicalTrials.gov identifier NCT03856827), after administering Compound I at 15 mg QD, changes were also observed in the brains of healthy participants aged 18-62 years by EEG. More specifically, in that study, the effect of Compound I on the amplitude of the ERP P300 was observed, and as the amplitude increased, the response, which was modulated by the dose level and the time from dosing, increased. Improvements in alpha power were also observed on day 14 across all dose levels tested in the NCT03856827 study and were compared to placebo.

[0052]

[0057] The second Phase I clinical trial (ClinicalTrials.gov identifier NCT04240158) was conducted in the elderly [described in a prior patent application publication (WO2022 / 081610) and A Phase 1 Translational Pharmacology Study in Healthy Elderly Volunteers Evaluating the Safety, Tolerability, Pharmacokinetics, and CNS Activity of IW-6463, a CNS-penetrant, Soluble Guanylate Cyclase Stimulator, Chad Glasser, Jacob Donoghue, Phillip Alday, Alex Arslan, Emily Florine, Chris Winrow, Chris Wright Neurology April 2021, 96(15 Supplement) 4701)]. In this trial, an increase in posterior alpha power, a tendency for an increase in gamma power, and a shortening of the N200 auditory stimulus event-related potential (ERP) latency were observed via EEG. Improvements in saccade reaction time and saccade maximum velocity in the saccadic eye movement (SEM) assessment were also observed. Furthermore, in exploratory CSF biomarker analysis, a positive trend for the compound I in important CSF neuroinflammatory markers was observed after 2 weeks of treatment with 15 mg QD compared to placebo. In particular, potentially important decreases in the concentrations of the alpha-2 macroglobulin (A2M) and complement C3 (C3) biomarkers were observed. Large decreases in the concentrations of these biomarkers were observed in subjects older than 70 years. An increase in A2M is associated with cerebrovascular disease and predicts cognitive decline and the onset of AD. This is reported to lead to hyperphosphorylation of tau. C3 is associated with Aβ and tau and may be involved in synaptic degeneration.

[0053]

[0058] Furthermore, several functional neuroimaging techniques: magnetic resonance imaging-arterial spin labeling (MRI-ASL) and functional magnetic resonance imaging (fMRI) were used in the NCT04240158 clinical trial in healthy elderly participants. Brain neuron metabolic profiles (as additional indicators of cell bioenergy therapy) were also measured by magnetic resonance spectroscopy (1H-MRS).

[0054]

[0059] MRI-ASL is used to quantify regional CBF during rest. fMRI is a relative measure based on the BOLD effect described below and is used to measure changes in CBF in certain regions of the brain as a result of increased brain activity.

[0055]

[0060] The BOLD effect is based on the fact that blood flow in the brain is highly locally regulated in response to the partial pressures of oxygen and carbon dioxide in the cortical tissue. When a specific area of the cortex increases its activity in response to a task, the oxygen uptake rate from local capillaries leads to an initial decrease in oxygenated hemoglobin (oxyHb), increasing local carbon dioxide (CO2) and deoxygenated hemoglobin (deoxyHb). Following a delay of 2 - 6 seconds, cerebral blood flow (CBF) increases, an excess of oxygenated hemoglobin is delivered, and deoxyhemoglobin is washed away. What is imaged is a large rebound in local tissue oxygenation. fMRI can detect this change due to the fundamental difference in the paramagnetism of oxyHb and deoxyHb. The NeuroCart® battery of assessments was also conducted in healthy elderly subjects in the NCT04240158 clinical trial. NeuroCart® is a full battery of tests developed at the Center for Human Drug Research (CHDR) to measure a wide range of CNS functions. NeuroCart can be used to correlate the CNS effects of a compound with drug concentration and helps to determine whether the effects are specifically due to the compound. NeuroCart provides both objective (e.g., neurophysiological, brain performance) and subjective (e.g., cognitive function, memory, mood, etc.) measures of CNS function. NeuroCart included electroencephalogram (EEG) and spectral entropy mapping (SEM) examinations, in addition to a diverse set of other assessments of CNS function described in WO2022 / 081610.

[0056]

[0061] In the NCT04240158 trial, positive effects were observed in EEG and SEM evaluations, supporting the beneficial effects of Compound I and its potential use for the treatment of cognitive impairment in patients in need thereof, as described in WO2022 / 081610, in some aspects of cognition. No changes were observed in ASL and fMRI BOLD, or MRS measurements, and in neuroimaging evaluations by CNS function assessment of NeuroCart in that trial. The results in EEG and SEM of that trial supported the treatment of cognitive impairment or decline in patients suffering from a diverse set of diseases, including mitochondrial diseases. Compound I in patients with MELAS

[0062] In the two previous Phase 1 clinical trials described above, changes in EEG parameters (alpha power and N200 ERP), changes in SEM, and a tendency towards improvement in two neuroinflammatory parameters were observed in healthy elderly subjects treated with a 14-day continuous administration of 15 mg QD.

[0057]

[0063] The goal of the study described in the Examples section of the present disclosure (Example 1) was to evaluate the effect of a 15 mg once-daily oral dose (QD) of Compound I on a number of parameters or scales related to brain and overall health status in patients with the mitochondrial disease MELAS. Enrolled patients were genetically confirmed to have MELAS and had a history of CNS symptoms such as headache, seizures and stroke. Patients were able to take stable medications including NO precursors (e.g., arginine and citrulline).

[0058]

[0064] The various evaluations and measurements performed are described in detail in the Examples section.

[0065] The primary objective of the trial was to evaluate the safety and tolerability of Compound I by measuring adverse events in the form of AEs, SAEs and TEAEs (defined in the Experimental section) that lead to drug discontinuation when administered to the target patient population with MELAS.

[0059]

[0066] The exploratory objectives included the evaluation of the plasma PK of the compounds. Further, other exploratory evaluation items were aimed at evaluating the effects of Compound I on physiology, neurophysiology, and cognitive and health status. In particular, changes from the baseline on Day 29 in plasma biomarker concentrations of a number of inflammation-related biomarkers and biomarkers related to mitochondrial dysfunction, changes from the baseline on Day 29 in CBF measured by ASL, changes from the baseline on Day 29 in fMRI-BOLD signal between rest and visual stimulation, changes from the baseline on Day 29 in brain metabolite levels by 1H-NMR, changes from the baseline on Day 29 in MFIS scores (total subscore, physical subscore, cognitive subscore, and psychosocial subscore), and changes from the baseline on Day 29 in the total score of the PROMIS item bank v2.0 - cognitive function were measured.

[0060]

[0067] The present invention is based on the surprising finding that Compound I administered at a total oral daily dose of 15 mg per day to a patient population with mitochondrial disease MELAS was safe, showed good tolerability, and demonstrated evidence of effects on CBF, fMRI-BOLD, and biomarkers of mitochondrial dysfunction and a number of inflammatory biomarkers. The biomarkers of mitochondrial dysfunction studied are known biomarkers of bioenergy and metabolism and have been found to be elevated in patients with mitochondrial disease. CBF and fMRI-BOLD are neuroimaging measures of cerebral perfusion and neural function / connectivity, respectively.

[0061]

[0068] The inflammatory process in patients with mitochondrial dysfunction is known to be upregulated both peripherally and centrally. The overall improvement in the inflammation panel described below suggests that Compound I has a beneficial effect on oxidative stress.

[0062]

[0069] Neuronal and / or glial injury due to mitochondrial dysfunction, nitric oxide deficiency, and cerebrovascular vascular disorders affects cerebral perfusion (CBF). Dysregulated cerebral perfusion is associated with stroke-like episodes and CNS symptoms.

[0063]

[0070] Furthermore, improvements in the patients' self-reported health status, fatigue, and cognitive assessment were observed in several subjects of the clinical trial, and some of these improvements were consistent with improvements in CBF, fMRI, and biomarkers in the same patients.

[0064]

[0071] Therefore, the results of the test described in Example 1 in the Examples section showed a positive effect across a number of biomarkers, patient-reported outcomes, increased cerebral blood flow, and improved functional connectivity within neural networks related to cognition, memory, and executive function.

[0065]

[0072] Therefore, Compound I may be used to treat additional aspects of mitochondrial disease, including the physical aspects of the disease, and to improve the general health of patients, in addition to the potential to improve some of the cognitive aspects as previously reported.

[0066]

[0073] The results of this trial using Compound I support the possibility that by using a specific dosing regimen of Compound I in human subjects, the activation of sGC can benefit several major pathological mechanisms of mitochondrial diseases and dysfunctions. These include effects on cellular bioenergetics, inflammation, neurological / cognitive function, and impairments in cerebral perfusion or cerebral blood flow. In particular, the fact that the lactate levels, which were elevated at baseline, and the known biomarkers of mitochondrial dysfunction such as FGF-21 and GDF-15 decreased, and that such decreases correlated with the plasma levels of Compound I, support the possibility that Compound I could be a disease-modifying treatment that can improve many aspects of the phenotype or symptoms of mitochondrial diseases. Furthermore, in addition to MELAS, it may act on other mitochondrial diseases presenting with related symptoms. Importantly, the greatest improvements were observed in patients with a more severe disease phenotype at baseline.

[0067]

[0074] Multiple plasma inflammation and mitochondrial dysfunction biomarkers were found to be elevated at baseline across participants, and multiple plasma inflammation and mitochondrial dysfunction biomarkers showed a directional improvement with treatment, many of which correlated with the plasma exposure to Compound I (i.e., higher exposure at the end of dosing corresponded to greater improvement in the biomarkers).

[0068]

[0075] Fibroblast growth factor-21 (FGF-21) is a hormone-like cytokine involved in the intermediate metabolism of carbohydrates and lipids. FGF-21 expression is driven by mitochondrial reactive oxygen species, and the concentration of FGF-21 is known to be dramatically higher in mitochondrial disease patients compared to controls.

[0069]

[0076] Growth differentiation factor 15 (GDF-15) is a member of the transforming growth factor-beta family and was first selected as a marker for mitochondrial dysfunction through gene expression studies of skeletal muscle from patients with mitochondrial disease caused by thymidine kinase 2 mutation, compared to healthy skeletal muscle. These revealed that GDF-15 was significantly upregulated in both the skeletal muscle and serum of patients with mitochondrial dysfunction. GDF-15 expression is known to be induced in response to stress such as mitochondrial dysfunction through upregulation of activating transcription factor 4.

[0070]

[0077] Lactate is a product of anaerobic production of ATP and is the most commonly used marker for detecting mitochondrial dysfunction in the general diagnosis of MD. Although not particularly sensitive (sensitivity of 34 - 62%), an elevated lactate concentration is estimated to have a specificity of 83 - 100% for detecting mitochondrial disease.

[0071]

[0078] CBF increased with treatment across all participants in the entire region of interest and was consistent with the change in the patient's general condition (PGIC) (i.e., better PGIC was associated with a greater increase in CBF), and was consistent with the improvement in the concentration of inflammatory biomarkers.

[0072]

[0079] The fMRI BOLD signals observed both during rest and during visual tasks suggested an improvement in functional connectivity within neural networks related to treatment-induced cognition, memory, executive function, and sensorimotor processing, which was consistent with the improvement in CBF. The fMRI-BOLD response to visual stimuli is known to be significantly reduced in symptomatic MELAS compared to controls (Rodan et al., 2020). Treatment with Compound I increased the activation of the occipital lobe brain region in response to visual stimuli, and the activation was greater on day 29 compared to day 1.

[0073]

[0080] Clinical outcomes of fatigue (MFIS) and cognition (PROMIS) did not show improvement by treatment at the study level, but a tendency towards improvement in PGIC was observed. In several patients, these evaluations at baseline were more adverse, but improvement was observed. Improvements in patient-reported outcomes, including fatigue, cognition, and overall disease recognition, were reported in patients with the most severe disease.

[0074]

[0081] Therefore, pharmacokinetic signals were observed throughout this trial in four disease domains: CBF (measured via ASL), inflammation (measured via plasma inflammatory biomarker levels), cellular bioenergetics (measured via mitochondrial dysfunction biomarker levels related to metabolism and bioenergetics), and neurofunction and connectivity (measured by fMRI and cognitive patient-reported outcome assessments).

[0075]

[0082] Furthermore, in another clinical trial conducted concurrently with the MELAS trial in patients with a high level of pathology (patients with stable schizophrenia), PK was achieved with a total oral daily dose of up to 60 mg per day, which was found to be safe and showed good tolerability. Therefore, it is highly reasonable that a dose of up to 60 mg per day is safe and shows good tolerability in patients with mitochondrial disease and may lead to a higher pharmacological response to those described herein.

[0076]

[0083] Furthermore, the fact that this trial was conducted in patients with MELAS and a positive effect was observed at the study level despite the high heterogeneity of the enrolled population makes it highly reasonable that a similar positive response may be observed in other patients with mitochondrial disease who share similar phenotypes or in whom the disease appears as a result of similar mechanisms. Treatment / Embodiment

[0084] In some embodiments of the methods and uses of the present invention, a therapeutically effective amount of Compound I is a total oral daily dose of 15 to 60 mg of Compound I. In some embodiments, the therapeutically effective amount is a total oral daily dose of 15 mg. In other embodiments, the therapeutically effective amount is a total oral daily dose of 20 mg. In other embodiments, the therapeutically effective amount is a total oral daily dose of 25 mg. In still other embodiments, the therapeutically effective amount is a total oral daily dose of 30 mg. In yet some embodiments, the therapeutically effective amount is a total oral daily dose of 45 mg. In still other embodiments, the therapeutically effective amount is a total oral daily dose of 60 mg.

[0077]

[0085] In some embodiments, a pharmaceutically acceptable salt of Compound I can be used in the methods and uses of the present invention described herein. When a pharmaceutically acceptable salt of Compound I is used, the dosage for the pharmaceutically acceptable salt is determined according to the molecular weight of the salt and has an equimolar amount with the dosage of Compound I described herein. Thus, in some embodiments, the present invention is a method of treating a patient with mitochondrial disease by administering to the patient a total oral daily dose of 15 mg to 60 mg of Compound I or an equimolar amount of a pharmaceutically acceptable salt of Compound I.

[0078]

[0086] In some embodiments, Compound I is applied to the treatment of mitochondrial diseases. In some embodiments, mitochondrial diseases that may be most suitable for treatment with Compound I include, but are not limited to, those showing a phenotype similar to MELAS. Mitochondrial disease patients with a phenotype similar to MELAS include a confirmed mitochondrial disease mutation and the following: a history of at least one SLE; a history of at least one encephalopathy episode defined as one or more episodes of personality or behavioral changes, disorientation or misidentification of time, place or person; a history of symptomatic seizures; a history of migraine headaches where the individual is prevented from functioning normally at school, work or home for at least one day per month on average over the past three months; or those having at least two of cognitive impairments defined as consistent amnesia of partial memory of events, memory loss and / or difficulty in problem-solving.

[0079]

[0087] In some embodiments of the methods and uses of the present invention described above, Compound I or a pharmaceutically acceptable salt thereof is adapted for the treatment of patients with mitochondrial diseases selected from the following.

[0080]

[0088] Alzheimer's disease, autosomal dominant optic atrophy (ADOA), Barth syndrome / LIC (lethal infantile cardiomyopathy), beta-oxidation deficiency, coenzyme Q10 deficiency, complex I, II, III, IV, V deficiency, chronic progressive external ophthalmoplegia (CPEO), Friedreich's ataxia, Kearns-Sayre syndrome, leukodystrophy, Leigh disease or Leigh syndrome, LHON, LHON Plus, MELAS (mitochondrial myopathy, encephalomyopathy, lactic acidosis, stroke-like episodes), ragged red fiber-myoclonic epilepsy (MERRF), mitochondrial recessive ataxia syndrome (MIRAS), mitochondrial cytopathy, mitochondrial DNA depletion, mitochondrial encephalopathy, mitochondrial myopathy, multiple mitochondrial dysfunction syndrome, MNGIE (myoneurogastrointestinal encephalopathy), NARP (neuropathy, ataxia, retinitis pigmentosa, and ptosis), Pearson syndrome, pyruvate carboxylase deficiency, pyruvate dehydrogenase deficiency or pyruvate dehydrogenase complex deficiency (PDCD / PDH), and POLG mutations.

[0081]

[0089] In some embodiments, the mitochondrial disease is selected from Alzheimer's, complex I, II, III, IV deficiency, CPEO, KSS, LCHAD, Leigh syndrome, leukodystrophy, LHON, MELAS, MEPAN, MERRF, MIRAS, mitochondrial DNA depletion, MNGIE, NARP, Pearson syndrome, and POLG mutations.

[0082]

[0090] In some embodiments, the mitochondrial disease is complex I mitochondrial disease. In some embodiments, the mitochondrial disease is MELAS. In other embodiments, the mitochondrial disease is Leigh syndrome.

[0083]

[0091] In some embodiments of the methods and uses of the invention described above, treatment with Compound I or a pharmaceutically acceptable salt thereof does not result in adverse events (AE) or serious adverse events (SAE) associated with excessive symptomatic hypotension or orthostatic hypotension.

[0084]

[0092] In some embodiments of the methods and uses of the present invention, the human patient is between 16 and 75 years old. In other embodiments, the patient is between 16 and 70 years old. In other embodiments, the patient is between 16 and 65 years old. In still other embodiments, the patient is between 16 and 60 years old. In some embodiments, the patient is between 16 and 55 years old, 16 and 50 years old, 16 and 40 years old, or 16 and 30 years old. In some embodiments, the human patient is 16 years old or older. In other embodiments, the human patient is 18 years old or older. In still other embodiments, the patient is less than 65 years old, less than 60 years old, less than 50 years old, less than 40 years old, less than 30 years old or less than 20 years old. In still other embodiments, the patient is pediatric. In still other embodiments, the patient is adult. In still other embodiments, the patient is adolescent. In still other embodiments, the patient is less than 16 years old. In other embodiments, the patient is 12 years old or older. In some embodiments, the patient is 3 years old or older. In still other embodiments, the patient is 12 years old or younger. In other embodiments, the patient is 10 years old or younger. In other embodiments, the patient is 5 years old or younger. In some embodiments, the patient is between 3 and 18 years old, 3 and 12 years old, 5 and 18 years old, 5 and 12 years old, or 3 and 5 years old.

[0085]

[0093] In some embodiments of the methods and uses of the present invention, the human patient has been treated with one or more other therapeutic agents used to treat mitochondrial disease prior to treatment with Compound I or a pharmaceutically acceptable salt thereof. In one embodiment, the other therapeutic agent used to treat mitochondrial disease is selected from citrulline and arginine. In another embodiment, the other therapeutic agent is the mito cocktail described herein.

[0086]

[0094] In some embodiments of the methods and uses described above, the total oral daily dose is given as a single dose (QD). In other embodiments, the total oral daily dose can be divided into two equal oral daily doses of 7.5 mg to 30 mg (BID).

[0087]

[0095] In certain embodiments, the methods and uses of the invention described herein comprise administering to a patient a single oral daily dose of 15 to 60 mg of Compound I or a pharmaceutically acceptable salt of Compound I in an equimolar amount.

[0088]

[0096] In certain embodiments, the methods and uses of the invention described herein comprise administering to a patient a single oral daily dose of 15 mg of Compound I or a pharmaceutically acceptable salt of Compound I in an equimolar amount.

[0089]

[0097] In certain embodiments, the methods and uses of the invention described herein comprise administering to a patient a single oral daily dose of 20 mg of Compound I or a pharmaceutically acceptable salt of Compound I in an equimolar amount.

[0090]

[0098] In certain embodiments, the methods and uses of the invention described herein comprise administering to a patient a single oral daily dose of 25 mg of Compound I or a pharmaceutically acceptable salt of Compound I in an equimolar amount.

[0091]

[0099] In certain embodiments, the methods and uses of the invention described herein comprise administering to a patient a single oral daily dose of 30 mg of Compound I or a pharmaceutically acceptable salt of Compound I in an equimolar amount.

[0092]

[0100] In certain embodiments, the methods and uses of the invention described herein comprise administering to a patient a single oral daily dose of 45 mg of Compound I or a pharmaceutically acceptable salt of Compound I in an equimolar amount.

[0093]

[0101] In certain embodiments, the methods and uses of the invention described herein comprise administering to a patient a single oral daily dose of 60 mg of Compound I or a pharmaceutically acceptable salt of Compound I in an equimolar amount.

[0094]

[0102] In some embodiments, the methods and uses of the invention described herein involve administering to a patient an initial total oral daily dose of 30 to 60 mg of Compound I or a pharmaceutically acceptable salt of Compound I in an equimolar amount, when the patient is evaluated by a physician as unable to tolerate a daily dose exceeding 30 mg, followed by tapering down to a total oral daily dose of 15 to 30 mg of Compound I or a pharmaceutically acceptable salt of Compound I in an equimolar amount. The patient can return to a higher dose at any time after a period of adaptation to a lower dose, when the patient is evaluated by the physician as having tolerated the lower dose for a reasonable period.

[0095]

[0103] In certain embodiments, the methods and uses of the invention described herein involve administering to a patient an oral dose of 7.5 to 30 mg of Compound I or a pharmaceutically acceptable salt of Compound I in an equimolar amount twice daily (BID). In one embodiment, the methods and uses of the invention described herein involve administering to a patient an oral dose of 7.5 mg, 10 mg, 12.5 mg, 15 mg, 22.5 mg, or 30 mg of Compound I or a pharmaceutically acceptable salt of Compound I in an equimolar amount twice daily (BID). In one embodiment, the methods and uses involve administering to a patient a first oral dose of 7.5 to 30 mg (e.g., 7.5 mg, 10 mg, 12.5 mg, 15 mg, 22.5 mg, or 30 mg of Compound I) or a pharmaceutically acceptable salt of Compound I in an equimolar amount, and a second oral dose of 7.5 to 30 mg (e.g., 7.5 mg, 10 mg, 12.5 mg, 15 mg, 22.5 mg, or 30 mg of Compound I) or a pharmaceutically acceptable salt of Compound I in an equimolar amount, wherein the first dose and the second dose are separated by a period of 5 to 15 hours, 8 to 15 hours, or 10 to 15 hours. In another embodiment, the first dose and the second dose are separated by 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 hours.

[0096]

[0104] In some embodiments, the maintenance dose is maintained indefinitely as long as the patient continues to experience clinical utility. In some embodiments, treatment with Compound I is chronic.

[0097]

[0105] In some embodiments of the above methods and uses, Compound I is administered to the patient before the symptoms of mitochondrial abnormalities are fully developed. In other embodiments of the above methods and uses, Compound I is administered to the patient after one or more symptoms of mitochondrial abnormalities have developed in the patient.

[0098]

[0106] In some embodiments, a patient with mitochondrial abnormalities is a patient diagnosed with having mitochondrial abnormalities or a patient with a genetic predisposition to the onset of the disorder. In other embodiments, a patient in need thereof has been genetically tested and has been found to have a mutation in a gene that predisposes to the disorder, even if the patient may not yet exhibit any physical symptoms of the disorder (or disease) or may exhibit only minimal symptoms. In still other embodiments, the patient exhibits symptoms of the disorder (or disease), even if a formal diagnosis has not yet been made. Some common signs and symptoms of mitochondrial abnormalities include growth retardation, loss of muscle tone, muscle weakness, fatigue, exercise intolerance, lactic acidosis, seizures, stroke-like episodes (SLE), headache, cognitive impairment, mental fatigue, lethargy, autism, visual and / or hearing problems, developmental delay, learning disability, heart, liver, and / or kidney disease, gastrointestinal disorder, diabetes, increased risk of infection, thyroid and / or adrenal abnormalities, autonomic dysfunction, and dementia.

[0099]

[0107] In some embodiments, treatment results in improvement of at least one measurable physical parameter of mitochondrial abnormalities. In other embodiments, treatment results in a decrease, inhibition, or attenuation of the progression of mitochondrial abnormalities, either by stabilization of measurable symptoms or a series of symptoms, or by stabilization of measurable biomarkers, for example.

[0100]

[0108] In some embodiments, treatment results in a measurable improvement in neural function and connectivity. In one embodiment, the improvement in neural function and connectivity is measured by functional magnetic resonance imaging (fMRI).

[0101]

[0109] In some embodiments, treatment results in an increase in cerebral blood flow (CBF). In one embodiment, treatment results in an increase in cerebral blood flow (CBF) in a brain region selected from the temporal lobe, parietal lobe, occipital lobe, frontal lobe, corpus callosum, cingulate gyrus, cerebral white matter and cerebellar white matter, and combinations of one or more of the foregoing regions.

[0102]

[0110] In some embodiments, treatment results in a decrease in one or more inflammatory biomarkers. In some embodiments, the inflammatory biomarkers described herein are selected from VCAM-1 (vascular cell adhesion molecule-1), ICAM (intercellular adhesion molecule), vWF (von Willebrand factor), and TNFR2 (tumor necrosis factor receptor 2). In other embodiments, treatment results in a decrease in one or more biomarkers of mitochondrial dysfunction. In still other embodiments, treatment results in a decrease in one or more inflammatory biomarkers and one or more biomarkers of mitochondrial dysfunction. In some embodiments, the biomarkers of mitochondrial dysfunction described herein are selected from lactate, GDF-15 and FGF-21. Combination therapy

[0111] The treatment of mitochondrial diseases and related symptoms using Compound I or a pharmaceutically acceptable salt thereof can be carried out using the compound alone or in combination therapy with other therapeutic agents. In some embodiments, Compound I or a pharmaceutically acceptable salt thereof can be used in combination with one or more agents independently selected from citrulline and arginine for the treatment of mitochondrial diseases.

[0103]

[0112] In some embodiments, Compound I or a pharmaceutically acceptable salt thereof can be used in combination with Mitocare for the treatment of mitochondrial diseases. As used herein, "Mitocare" refers to a combination of a variety of vitamins and supplements commonly used by adults and children diagnosed with mitochondrial diseases.

[0104]

[0113] Supplements and vitamins used by mitochondrial disease patients are often at high doses, and patients may need to take up to 50 pills per day. Pharmacists who graduated from the International Academy of Compounding Pharmacists (IACP) can combine pure powdered prescribed supplements and vitamins to make a "cocktail" in liquid, capsule or other forms. The final drug is usually in a much smaller amount than taken by other methods and can be further flavored to improve palatability. By avoiding common fillers in commercially available tablets, it is possible to accommodate an individual's allergies or dietary restrictions. The exact formulation, including dosage and ingredients, is determined by the patient's attending physician and varies according to the individual patient's diagnosis, clinical symptoms, and weight. The most common ingredients include the following. 1) Coenzyme Q-10

[0114] Coenzyme Q-10 (Coenzyme Q10, CoQ10, CoQ-10, CoQ, ubiquinone, Q-Gel (registered trademark)) is a fat-soluble vitamin-like substance present in all cells of the body and functions as a coenzyme for several important enzymatic steps in the production of energy within cells. It also functions as an antioxidant that protects against the accumulation of harmful free radicals, which is important in its clinical effects. Many patients report increased energy while using coenzyme Q-10, and thus it is a common "first-line" approach for assisting children and adults with mitochondrial disease. Since frequently reported side effects include gastrointestinal upset and sleep disturbances, pharmacists recommend taking the CoQ-10 dose with a meal early in the day. Since it may take time to establish therapeutic levels, patients may not experience beneficial effects immediately. Additionally, since excess substances not used are stored in fat cells, appropriate dosing is important. 2) Multivitamins

[0115] Some B vitamins are cofactors that participate in important mitochondrial reactions. Most of the B vitamins are bitter and are more palatable when flavored. The B vitamins are water-soluble; that is, they are excreted if not used, and the benefits of taking these vitamins should be felt immediately.

[0105]

[0116] a. Vitamin B1 (thiamine). This is a water-soluble vitamin that helps break down carbohydrates so that the body can use them better, helps with muscle growth and maintenance of muscle tone, and helps with memory. The possible side effect that is sometimes noted is only drowsiness.

[0106]

[0117] b. Vitamin B2 (riboflavin). Also, B2, a water-soluble vitamin, is necessary for energy production in mitochondria, enhances muscle performance, and similarly helps maintain healthy mucous membranes, skin, hair, and nails. The only side effect noted is a tendency for urine to turn orange. When given in the form of riboflavin diphosphate, the flavor of this vitamin can be improved.

[0107]

[0118] c. Vitamin B3 (niacin). B3, which is sometimes used, often causes facial flushing, so it is generally given separately first to check for any side effects before being added to cocktails.

[0108]

[0119] d. Vitamin B6 (pyridoxine) and vitamin B12 (cobalamin). These are other B vitamins that are often part of the formulation mix that patients with mitochondrial diseases may use.

[0109]

[0120] e. Vitamin C. This is used to assist the healing process and prevent infections, but increasing the dose may cause some stomach upset or sometimes headaches.

[0110]

[0121] f. Vitamin E. This protects cell membranes and improves nerve function. Usually, the dose is 400 - 600 mg or less per day for adults. Vitamin E may interfere with coumadin / warfarin drugs, and caution is required when using vitamin E in mitococktails.

[0111]

[0122] g. Vitamin K1. Another vitamin that may be added (however, caution is required as the safe range for the dosage of this vitamin is very small) needs to be prescribed by a doctor and cannot be purchased over the counter. 3) Other antioxidants

[0123] Antioxidants reduce the accumulation of free radicals within cells and are therefore also used in patients with mitochondrial diseases. Alpha-lipoic acid is perhaps the most commonly prescribed antioxidant used in mitococktails. 4) L-carnitine

[0124] L-carnitine helps transport fatty acids and improves muscle strength and tone. Side effects may include diarrhea and a fishy odor that may be excreted through the sweat glands. Some patients have reported a decrease in fatigue and an improvement in energy by taking L-carnitine. It is taken in either tablet or liquid form and is usually taken separately from the compounded cocktail. 5) Creatine

[0125] Creatine helps maintain muscle mass and increases energy for cells. Its side effects include diarrhea and drowsiness; its dosage ranges from 5 grams per day for children to 10 grams per day for adults and is generally formulated in liquid or capsule form.

[0112]

[0126] All of the above vitamins and supplements are added or not added to the cocktail as specified according to the patient's needs. Each cocktail varies from patient to patient and can be determined by a physician.

[0113]

[0127] As used herein, the terms "in combination" (as "in combination therapy" in the text) or "co-administration" can be used interchangeably and refer to the use of multiple therapies. The use of the terms does not limit the order in which the therapies are administered to the subject.

[0114]

[0128] For combination therapies using separate dosage forms or dosage forms of multiple therapeutic agents, the therapeutic agents can be administered separately or in combination (i.e., simultaneously). Further, when administered separately, the administration of one therapeutic agent can be before or after the administration of the other agent.

[0115]

[0129] When Compound I or a pharmaceutically acceptable salt thereof is used in combination therapy with other therapeutic agents, the other therapeutic agent or the therapeutically effective amount of each of the other therapeutic agents is determined according to the type of drug used. Appropriate dosages are known for approved therapeutic agents and can be adjusted by those skilled in the art according to the condition of the subject, the type of condition being treated, and the amount of Compound I or a pharmaceutically acceptable salt thereof used. In one embodiment of the present invention, Compound I or a pharmaceutically acceptable salt thereof, and the additional therapeutic agent are each administered in a therapeutically effective amount (i.e., an amount that is therapeutically effective when administered alone). In other embodiments, Compound I or a pharmaceutically acceptable salt thereof and the additional therapeutic agent are each administered in an amount that is not therapeutically effective alone (a sub-therapeutic dosage). In yet another embodiment, Compound I or a pharmaceutically acceptable salt thereof can be administered in a therapeutically effective amount, while the additional therapeutic agent is administered in a sub-therapeutic dosage. In yet another embodiment, Compound I or a pharmaceutically acceptable salt thereof can be administered in a sub-therapeutic dosage, while the additional therapeutic agent is administered in a therapeutically effective amount.

[0116]

[0130] If co-administration involves the separate administration of a first amount of Compound I or a pharmaceutically acceptable salt thereof and a second amount of the additional therapeutic agent, the compounds are administered in times that are close enough to exert the desired therapeutic effect. For example, the period between each administration that can bring about the desired therapeutic effect can range from several minutes to several hours and can be determined considering the properties of each compound such as potency, solubility, bioavailability, plasma half-life, and pharmacokinetic profile. For example, Compound I or a pharmaceutically acceptable salt thereof and the second therapeutic agent can be administered in any order, within 24 hours of each other, within 16 hours of each other, within 8 hours of each other, within 4 hours of each other, within 1 hour of each other, within 30 minutes of each other, within 5 minutes of each other, simultaneously or concomitantly.

[0117]

[0131] Furthermore, specifically, the first treatment can be administered to the subject before the administration of the second treatment (e.g., 5 minutes before, 15 minutes before, 30 minutes before, 45 minutes before, 1 hour before, 2 hours before, 4 hours before, 6 hours before, or 12 hours before), concomitantly with the administration of the second treatment, or following the administration of the second treatment (e.g., 5 minutes after, 15 minutes after, 30 minutes after, 45 minutes after, 1 hour after, 2 hours after, 4 hours after, 6 hours after, 12 hours after).

Example

[0118]

[0132] To better understand the present invention, the following examples are described. These examples are for illustrative purposes only and should not be construed as limiting the scope of the present invention in any way. All references given in the examples are incorporated herein by reference.

[0119] Example 1: A signal discovery study to evaluate the safety, tolerability, and effect of IW-6463 on CNS diseases Definition of abbreviations / terms

[0120]

Table 1

[0121] Study objectives and outcome measures

[0133] This study was completed at 5 investigative sites in the United States. Eight participants completed the study. This was an open-label single-group study to evaluate the safety, tolerability, PK, and pharmacodynamics (PD) of Compound I when administered to participants diagnosed with MELAS for up to 29 days. Since this was the first study to evaluate Compound I in the patient population, the primary objectives were safety and tolerability. The total study period for participants, including screening, treatment, and follow-up, was up to 75 days (ClinicalTrials.gov identifier NCT04475549).

[0122]

[0134] The goal of the study described in the Examples section (Example 1) was to evaluate the effect of a daily dose of 15 mg of Compound I on a number of parameters or measures related to the brain and overall health in patients with the mitochondrial disease MELAS. The various evaluations and measurements performed are described in detail below.

[0123]

[0135] The primary objective of the clinical trial was to evaluate the safety and tolerability of Compound I when administered to a population of target patients with MELAS by measuring AEs, SAEs, and TEAEs leading to drug discontinuation.

[0124]

[0136] The exploratory objectives included evaluating the plasma PK of the compound. Additionally, the other exploratory evaluation items were aimed at evaluating the effect of Compound I on physiology, neurophysiology, and cognitive and health status. Study design:

[0137] To complete the study, each participant was to proceed through three distinct periods as described below. A schematic diagram of the study is also shown below. Screening phase:

[0138] The screening period began with the signing of the informed consent form (ICF) at the screening visit. After signing the ICF, the eligibility of each participant was evaluated according to the inclusion and exclusion criteria described below. The collection of adverse events (AEs) started after signing the ICF and continued during the follow-up period / until the study was discontinued (whichever occurred first).

[0125]

[0139] After the screening visit, eligible participants also had to begin to comply with the lifestyle restrictions detailed below. Participants also began to complete a daily diary for the study. The end of the screening period coincided with the start of the in-hospital period. Treatment period:

[0140] The treatment period started on Day 1 (there was no "Day 0") when the participant returned to the research center after experiencing the baseline treatment and receiving the first daily dose of the study drug. The participant returned to the research center on Day 29 (-4 days) for the end-of-treatment (EOT) visit; all other visits scheduled during this period were completed at the participant's option, either at home or within an acceptable time frame at the research center. Throughout the treatment period, the participant continued to complete a daily diary.

[0126]

[0141] The end of the treatment period coincided with the start of the follow-up period. Follow-up period:

[0142] The follow-up period started immediately after the EOT visit and continued until the follow-up visit was conducted. During this period, the participant had to continue to comply with all lifestyle restrictions and complete the diary. Schematic diagram of the study

[0127]

Chemical

[0128] Participant's diary for recording changes in health status, daily dose, and concomitant medications

[0143] At the screening visit, each participant was given a paper (source) diary, and the participant (or their legal representative / guardian) was asked to record daily in the diary the name of all other medications taken, along with the date, time, and dose strength of the medications; as well as any changes in health status (including date, time, and a brief description).

[0129]

[0144] From Day 1 of the treatment period, the participant was instructed to also record the date and time of each study drug administration taken at home, as well as any other medications taken (including name, date, time, and dose strength), and any changes in health status (including date, time, and a brief description).

[0130]

[0145] Participants were required to keep diaries during the treatment period, at scheduled home and hospital visits, for monitoring by the research staff.

[0146] During the follow-up period, participants were required to continue recording any changes in concomitant medications and health status in the same manner. Diaries were collected from the participants at follow-up visits. Clinical Outcome Assessment / Participant Questionnaire Patient-Reported Outcome Measurement Information System (PROMIS)-Cognitive Function

[0147] The Patient-Reported Outcome Measurement Information System (PROMIS) Item Bank v2.0 - Cognitive Function is a self-administered questionnaire that assesses mental fatigue and multiple aspects of cognitive function over the past 7 days prior to questionnaire completion. It uses a Likert-type rating scale (e.g., from "not at all" to "very often"). Patient Global Impression of Change (PGIC)

[0148] The Patient Global Impression of Change (PGIC) is a single-item questionnaire that uses a Likert-type rating scale (e.g., from "much improved" to "much worse") to assess participants' perception of change in their overall health status since the start of the study. Modified Fatigue Impact Scale (MFIS)

[0149] The Modified Fatigue Impact Scale (MFIS) is a self-administered questionnaire that assesses the impact of fatigue in terms of physical, cognitive, and psychosocial function over the past 4 weeks. Patients respond using a Likert-type rating scale. (e.g., from "not at all" to "almost always"). PD Assessment and Parameters (Neuroimaging)

[0150] The potential PD effects of Compound I in the brain were measured using two exploratory functional neuroimaging modalities, arterial spin labeling (ASL) and functional magnetic resonance imaging (fMRI). During rest, ASL quantifies regional CBF, while fMRI is a relative measure based on the blood oxygenation level-dependent (BOLD) effect and was performed during both rest and visual stimulation to measure brain activity.

[0131]

[0151] Each individual MRI scan session took approximately 45 minutes (up to about 1 hour). Columbia Suicide Severity Rating Scale (C-SSRS)

[0152] Compound I is a CNS-active investigational drug. Compound I and other similar drugs in this class have not been associated with an increased risk of suicidal thoughts or suicidal behavior, but it was considered important to monitor for such thoughts or behavior before and during this clinical study. Therefore, participants were appropriately monitored and carefully observed for suicidal thoughts and suicidal behavior or any other abnormal changes in behavior. The Columbia Suicide Severity Rating Scale (C-SSRS) was initiated on the day before dosing on Day 1 of the treatment period and was performed at all subsequent visits, including any unscheduled visits, at which a clinical evaluation was performed. Participants who experienced signs of suicidal thoughts or suicidal behavior were required to have immediate medical evaluation using a medical monitor, and discontinuation of the study drug was considered. Physical examination and vital signs

[0153] At screening and follow-up, a complete physical examination was performed by the study physician. All other tests were performed at the discretion of the study physician, as indicated by symptoms.

[0132]

[0154] The complete physical examination was designed to include the following: general appearance, lymph nodes, nervous system; cardiovascular system; head, both eyes, both ears, nose, throat and skin; respiratory system, neck, mental status, abdomen / liver / spleen and musculoskeletal system. Breast, genitourinary, and rectal examinations were optional and were performed at the discretion of the study physician. Clinically relevant findings present prior to the start of the investigational product on Day 1 were recorded as part of the participant's treatment history. From after the start of the study product on Day 1, new clinically relevant findings or worsening of previous findings were recorded as AEs. Height (cm) was measured only at screening. Weight (kg) was recorded throughout the study. At screening, the body mass index was calculated and recorded.

[0133]

[0155] The measured vital signs included respiratory rate, oxygen saturation, and temperature. Respiratory rate and oral temperature (°C) were measured after the participant had rested supine / on a chair for ≥5 minutes. Oxygen saturation measurement needed to be performed by a pulse oximeter in room air.

[0134]

[0156] All BP and pulse measurements were obtained using an automated BP monitor (preferably the left arm) before blood sampling (or ≥10 minutes after blood sampling if necessary). Supine BP was measured only at screening and was recorded as the average of two measurements obtained at 2-minute intervals after the participant had rested quietly in the semi-recumbent / supine position for ≥5 minutes. Orthostatic vital signs were measured at all scheduled visits. The participant was allowed to rest quietly in the supine / semi-recumbent position for ≥5 minutes before the supine BP and pulse measurements were recorded. Then, it was assumed that the participant would sit for ≥1 minute, and finally, it was assumed that the participant would stand for 2 (±1) minutes before the standing measurements were recorded. Values from these measurements were used to calculate and record orthostatic BP and pulse. Electrocardiogram (ECG)

[0157] All ECGs and screening ECHOs (if there is no record of treatment history within 3 months prior to Day 1) were obtained after the participant had rested in the supine position for ≥5 minutes and were obtained before blood sampling (or, if necessary, ≥10 minutes after blood sampling). When the timing was appropriate, the ECG and vital signs could be evaluated together.

[0135]

[0158] If the QTc result (corrected using the Fridericia formula) was outside the normal range (>450 ms), the ECG was repeated twice and the average of the three results was calculated.

[0136]

[0159] All ECGs were evaluated by the investigator or a qualified designee for the presence of abnormalities. The results were recorded as "normal", "clinically significant abnormality", or "abnormality not clinically significant". Clinically significant abnormalities were recorded as AEs.

[0137]

[0160] If a confirmed clinically significant abnormal result was obtained, the research site was to follow standard institutionalized procedures until the result was resolved at baseline. If concerns remained, the issue was escalated to the sponsor's medical monitor. Adverse Events, SAEs, and Other Safety Reports

[0161] Adverse events (AEs) were monitored and recorded throughout the study from ICF signature through follow-up visits. Care was taken to avoid introducing bias when detecting AEs and / or SAEs. An open-ended, non-leading oral question of the participant was the preferred method for asking about the occurrence of AEs.

[0138]

[0162] The investigator was not obligated to actively collect AE information after the conclusion of the participant's study participation. However, if the investigator became aware of any SAE or death at any time after the participant was discharged and the investigator considered the event to be reasonably related to the investigational drug or study participation, the investigator was to promptly notify the sponsor. Pregnancy Reporting and Monitoring

[0163] Female participants who reported pregnancy before the start of the study drug administration on Day 1 withdrew from the study participation; the withdrawal was to be reported as a screening failure.

[0139]

[0164] If pregnancy occurred in either the participant or the participant's partner after the start of the study drug, it was reported and recorded on the study-specific pregnancy form. The study center was required to make reasonable efforts to follow up from pregnancy to delivery.

[0140]

[0165] All reports of congenital anomalies / congenital defects and stillbirths were to be considered SAEs. Spontaneous abortions, elective terminations without complications, and normal deliveries without congenital anomalies were neither to be reported nor handled as SAEs, but were to be reported as the outcome of each pregnancy. Other Evaluations

[0166] A variety of clinical laboratory evaluations for safety, drug testing, and other requirements in the protocol were performed on all subjects. The genetic evaluation in this study was limited to the confirmation of MELAS (by treatment history) at screening for eligibility determination. Plasma and CSF PK and Biomarker Evaluations

[0167] PK samples were collected. Each sample was divided into two aliquots (one for PK and one for backup). Approximately 2 mL of thin whole blood samples were collected for the measurement of the plasma concentration of Compound I using a validated liquid chromatography-tandem mass spectrometry-based bioanalytical method.

[0141]

[0168] Approximately 4 mL of CSF samples were collected from each participant who did not withhold consent for the procedure for the measurement of the CSF concentration of Compound I using a validated liquid chromatography-tandem mass spectrometry-based bioanalytical method.

[0142]

[0169] Plasma and serum samples for biomarkers were collected from each participant. These biomarkers were tested for the target disease involvement of Compound I and the effect of the compound on the disease. Plasma or serum samples were analyzed for the concentrations of growth differentiation factor 15 (GDF-15), lactate, pyruvate, alanine, neurofilament light chain (NFL), vascular cell adhesion molecule 1, interleukin-1 beta, asymmetric dimethylarginine (ADMA), L-arginine, and other biomarkers associated with the pathophysiology of mitochondrial disease, as well as their responses to Compound I.

[0143]

[0170] The effect of Compound I on the neuronal profile of the brain was also measured in participants by proton magnetic resonance spectroscopy ( 1 1H-MRS). Biomarkers measured using this imaging technique include, among others, the concentrations of ventricular lactate and N-acetylaspartate. Study drug, dosing level and administration

[0171] All eligible participants received open-label Compound I at an initial dose of 15 mg QD. Participants were instructed to take Compound I once daily from Day 2 to Day 28 at a time consistent (preferably ±1 hour) with the in-hospital study drug administration on Day 1.

[0144]

[0172] Exception / Note: On Day 8, Day 15, and Day 29 / EOT visit, participants took their dose during the visit to allow for pre- and post-dose evaluations at appropriate times. The visits on Day 8 and Day 15 (at home or in-hospital) were scheduled so that dosing occurred at approximately the same time as on Day 1 and subsequent administrations. Participants were asked to record the date and time of their dose administration at home in a diary.

[0145]

[0173] Permitted concomitant medications could be taken with the study drug. Participants were asked to record in a diary the date, time, and dose strength of any concomitant medications taken on each dosing day.

[0146]

[0174] Compound I can be taken with food or without food, but participants were required to fast for 3 to 4 hours before clinical safety and PD test sample collection both in the hospital and at home.

[0147]

[0175] The 15 mg QD dose of Compound I was selected based on safety, tolerability, PK, and PD data from repeated dosing at this level in two Phase 1 trials conducted in healthy adults aged 18 - 79 years. Among healthy participants who received 15 mg QD of Compound I for up to 15 days, there were no discontinuations due to AE and no SAE was reported. All AEs were considered mild or moderate by the principal investigator of the trial. Throughout the Phase 1 trials, no safety concerns were identified. The PK data were linear and dose - dependent, supporting QD dosing and showing no effect of food. Additionally, 14 days after dosing in healthy elderly participants, 15 mg QD of Compound I showed some effect on neurophysiological parameters by EEG. No effect on cognitive performance scales was observed. Participant Selection and Lifestyle Restrictions Inclusion Criteria

[0176] Each participant had to meet each of the following criteria to be eligible to participate in this study. 1. Had signed the ICF (either the participant or their legal representative / guardian as required) before all study - specific procedures were performed 2. Was 18 years of age or older on the date of consent 3. Had undergone prior genetic confirmation of known mitochondrial disease mutations 4. Had shown neurological features of MELAS (which may be based on treatment history) 5. Had elevated plasma lactate during screening (≥1.0 mmol / L) 6. Had agreed to comply with all study requirements, including lifestyle restrictions 7. Had agreed to refrain from any major lifestyle changes (e.g., starting a new diet or changing exercise patterns) from the time of ICF signature until after follow - up visits (and earlier for the need for contraception) 8. When receiving chronic medication, there was no change in the regimen from at least 4 weeks before the first day of administration and no plan to change the regimen during the study. 9. In the case of women, one of the following two criteria was met: postmenopausal (≥1 year or no menstruation for ≥12 consecutive months) or surgically infertile (bilateral oophorectomy, hysterectomy, or tubal infertility surgery [tying, clipping, binding, or cauterizing]) was confirmed - or - if fertile, not pregnant at the screening visit, not breastfeeding, negative pregnancy test results at the screening visit and before dosing on Day 1, and agreed to contraception during the study period and for ≥90 days after the last study drug administration. 10. Male and female participants of reproductive potential were required to agree to use one or more of a series of effective contraceptive methods from the date of ICF signature until ≥90 days after their last study drug administration. 11. Male participants were required to agree to refrain from providing sperm from the screening visit until 90 days after the last administration of the study drug. 12. Agreed not to receive investigational treatment or any other study device while participating in this study through follow-up visits. 13. There were no clinically significant findings on ECG (as evaluated at screening and before dosing on Day 1) and echocardiogram (ECHO; within 3 months from Day 1). Exclusion Criteria

[0177] Participants who met any of the following criteria were excluded from participating in this study. 1. Demonstrated severe visual impairment, hearing impairment, or cognitive impairment that could potentially affect the ability to comply with protocol requirements or complete the necessary evaluations, as determined by the principal investigator of the clinical trial. 2. Used any nicotine-containing products (e.g., cigarettes, e-cigarettes, vape pens, cigars, chewing tobacco, gum, patches) within 1 month of enrollment. 3. Had a positive pregnancy test at screening or on Day 1 (or any other time during the study period). 4. Hospitalized for alcohol dependence or drug intoxication during the 12 months prior to the screening visit and / or had a positive drug or alcohol test result at screening or prior to dosing on Day 1. Drug screening included amphetamines, cocaine, opioids, and cannabinoids. Note that in this study, use of cannabis and cannabidiol products for medical purposes was permitted except for 24 hours prior to screening, Day 1, and EOT visits, and 4 hours prior to all other visits. Participants were excluded from study participation if they had known cannabis abuse or dependence and, in the opinion of the study's responsible physician, the individual's ability to comply with the protocol was affected or there was a potential for harm to the individual. 5. Demonstrated a clinically significant hypersensitivity or allergy to any of the inactive ingredients contained in the formulation of Compound I 6. Demonstrated hypotension defined as systolic BP ≤ 90 mmHg or diastolic BP ≤ 60 mmHg at screening or prior to dosing on Day 1 7. Demonstrated hypertension defined as systolic BP > 160 mmHg or diastolic BP > 100 mmHg at screening or prior to dosing on Day 1 8. Demonstrated orthostatic hypotension defined as a ≥ 20 mmHg decrease in systolic BP or a ≥ 10 mmHg decrease in diastolic BP at screening or prior to dosing on Day 1 when measured assuming a transition from semi-recumbent / supine to standing 9. Had HbA1c > 11% or had uncontrolled diabetes as determined by the study's responsible physician 10. Had a lymphoma, leukemia, or any malignancy within the past 5 years, except for basal cell carcinoma or squamous cell carcinoma of the skin that had been excised and had no evidence of metastatic disease for 3 years. 11. Had a severe gastrointestinal motility disorder (e.g., history of dyspepsia, abdominal pain, nausea, vomiting, recurrent pancreatitis, constipation) that, in the opinion of the study's responsible physician, could potentially affect compliance and / or the administration, absorption, and exposure to oral medications 12. It was impossible to fast (i.e., no food or liquid; water was possible as needed) 3 to 4 hours after a meal. 13. There was a family history of QT shortening syndrome or QT prolongation syndrome. 14. There was a clinically significant heart disease complication, or an electrocardiogram (ECG) with a corrected QT interval (QTcF interval) > 500 ms using the Fridericia formula. 15. There was a current or past history of clinically significant cardiomyopathy and / or cardiac conduction abnormalities. 16. Had a history of platelet dysfunction, hemophilia, von Willebrand disease, coagulation abnormalities, other bleeding diathesis states, or significant non-traumatic bleeding episodes. 17. There was use of ≥ 325 mg / day of aspirin, any P2Y12 inhibitor, any anticoagulant, a specific inhibitor of phosphodiesterase 5 (PDE5), a non-specific inhibitor of PDE5 (including dipyridamole and theophylline), any supplement for the treatment of erectile dysfunction, riociguat, and / or any nitrate. These drugs were prohibited from the screening visit throughout the study period. Note: Participants taking arginine or citrulline for the treatment of mitochondrial disease were eligible and could continue these treatments. 18. Participated in any study of investigational treatment for mitochondrial disease and / or symptoms of mitochondrial disease within 1 month prior to Day 1. 19. There was any contraindication to MRI procedures. 20. Hospitalized due to any disease, mental trauma, surgical procedure, or mitochondrial disease-related complication within 4 weeks prior to screening. 21. Unable to comply with or unwilling to comply with the study schedule, lifestyle restrictions, and assessment requirements, or otherwise deemed by the clinical judgment of the principal investigator not suitable for study participation. 22. The patient or their legal representative / guardian (as needed) was unable to provide or unwilling to provide written informed consent to participate in this study. Lifestyle Restrictions

[0178] Participants were required to follow the following lifestyle restrictions throughout the study, starting from the screening visit to the follow-up visit, unless otherwise instructed. Diet and dietary restrictions

[0179] Fasting (i.e., no food or liquids; water was allowed as needed) was required for 3 - 4 hours before clinical safety and PD test sample collection at each scheduled visit, including all home visits.

[0148]

[0180] Since participants were at risk of developing metabolic decompensation during any catabolic state, on the day of each visit, participants fasted upon waking (water and light snacks were allowed if pre-approved), and then a standardized low - glycemic index meal was provided in the hospital 3 - 4 hours before clinical safety and PD test sample collection. For home visits, participants received counseling on what to consume before the fasting period or were provided with a standardized meal by home healthcare services.

[0149]

[0181] On the days of neuroimaging (screening, day 1, and day 29 visits): After collecting safety and PD test samples, participants were permitted to eat a light standardized snack; however, heavy meals had to be avoided until after all image scans were performed. Caffeine, alcohol, and tobacco

[0182] Caffeine - containing or xanthine - containing products (e.g., coffee, tea, cola beverages, and chocolate) could not be consumed within 24 hours before screening, day 1, and EOT visits, and within 4 hours before all other scheduled visits. These products may have affected the evaluations conducted in this study.

[0150]

[0183] The use of alcohol was not permitted within 24 hours prior to all scheduled visits. Tobacco-containing products or nicotine-containing products (e.g., e-cigarettes, patches) could not be used during the study until after the final study visit. Drugs, vitamins, supplements, and other substances

[0184] Participants were to be informed that the following were prohibited, starting from screening through follow-up visits: any medical treatment for erectile dysfunction; "illegal" drugs starting one month prior to screening through follow-up visits; the use of medical marijuana and cannabidiol products was permitted except within 24 hours prior to screening, Day 1, and the EOT visit, and 4 hours prior to all other visits; foods containing poppy seeds were avoided throughout the study as they could cause a positive result for "drugs". Concomitant medications and treatments

[0185] All drugs, vaccines, or other treatments / procedures that the participant had received at the time of screening, including over-the-counter or prescription drugs, vitamins, and / or herbal supplements, were recorded along with the reason for use, date of administration, route of administration, and frequency, as well as dosage information. The following drugs were prohibited or permitted with caution: specific inhibitors of PDE5 including sildenafil, tadalafil, vardenafil; non-specific inhibitors of PDE5 including dipyridamole, theophylline; any supplements for the treatment of erectile dysfunction, other sGC stimulants including riociguat and vericiguat; nitrates such as nitroglycerin, isosorbide mononitrate, isosorbide dinitrate, sodium nitroprusside, amyl nitrite; aspirin ≥325 mg / day; any anticoagulants; any P2Y12 inhibitors including cangrelol, clopidogrel, prasugrel, ticagrelor, ticlopidine; the use of any "illegal drugs" was not permitted starting one month before screening until the follow-up visit, except for the use of medical cannabis and cannabidiol products for medical purposes, excluding screening, 24 hours before the Day 1 and EOT visits, and 4 hours before all other study visits; and BCRP substrates, as compound I may have the potential to increase the exposure to these drugs; inhibitors of BCRP transporters including curcumin, cyclosporine A, and eltrombopag. Examples of BCRP substrates include azidothymidine, bisantrene, camptothecin derivatives, canertinib, cimetidine, difluoromethotrexate, flavopiridol, gefitinib, glyburide, imatinib mesylate, indolocarbazole, irinotecan, lamivudine, lapatinib, methotrexate, mitoxantrone, nilotinib, nitrofurantoin, pantoprazole, prazosin, rosuvastatin, SN38, sulfasalazine, and topotecan.

[0151]

[0186] Participants who were taking arginine or citrulline for the treatment of mitochondrial disease were permitted to continue these treatments. Basic characteristics

[0187] Of the 8 patients who completed the trial, 5 took arginine and 1 took citrulline before and during the trial. The use of arginine or citrulline was more common in patients with more progressive diseases.

[0152]

[0188] The enrolled patients were 19 - 54 years old. Among them, 5 were female and 3 were male. All had a history of one or more CNS symptoms such as stroke-like episodes, seizures or headaches. The MFIS score at baseline was 3 - 66 (on a 0 - 84 scale), and the PROMIS score was 148 - 77 on a 160 - 0 scale. These patients had elevated inflammatory biomarkers of 6 - 19 at baseline.

[0153]

[0189] Furthermore, a correlation between GDF - 15 and FGF - 21, biomarkers of mitochondrial dysfunction, was observed at baseline (see Figure 1), which further supports the evidence of the disease distribution among the 8 patients.

[0154]

[0190] Therefore, although the trial was completed with a small number of patients, it showed the distribution from patients with milder conditions to those with a more severe mitochondrial disease phenotype that can be considered.

[0155]

[0191] The plasma lactate level at baseline was in the range of 1.7 - 5.6 mmol / L. The normal lactate level is less than 2 mmol / L. The level of GDF - 15 at baseline was in the range of 0.49 - 4.1 ng / mL. The normal value of this biomarker is 0.14 - 0.46 ng / mL. The level of FGF - 21 at baseline was in the range of 0.055 - 0.72 ng / mL. The normal range of FGF is less than 0.44 ng / mL. Therefore, plasma biomarkers related to mitochondrial dysfunction were elevated at baseline in all participants. Study Evaluation / Results a) Safety

[0192] Fifteen milligrams once daily (QD) of Compound I for 29 days showed good tolerability, with no serious adverse events (SAEs) observed and no adverse events leading to treatment discontinuation in patients with mitochondrial disease MELAS. No safety signals, vital signs, electrocardiograms (ECGs), or suicide assessment scales were obtained from clinical laboratory tests. b) Pharmacokinetics

[0193] The PK profile and CNS penetration of Compound I in this patient population were similar to those observed in previous Phase 1 trials. c) Pharmacodynamics i) Neuroimaging

[0194] Throughout the study, a positive trend was observed as an increase in cerebral blood flow (CBF) on Day 29 compared to baseline (Day 1), as shown in Figure 2. This change was observed in all brain regions of interest. The effect size across the present disclosure is defined as the mean change from baseline in the specific parameter being measured, divided by the standard deviation. An effect size close to 1 or -1 (plus or minus depending on the direction of movement of the specific parameter, i.e., +0.8 to +1.0 or -0.8 to -1.0) is typically considered large. An effect size of 0.5 to 0.8 or -0.5 and -0.8 (plus or minus depending on the direction of movement of the specific parameter) is typically considered moderate. An effect size close to less than 0.5 or less than -0.5 (plus or minus depending on the direction of movement of the specific parameter) is typically considered small.

[0156]

[0195] Analysis of individual patient data showed that CBF increased in 5 out of 8 patients between Day 1 and Day 29, and patients with low CBF at baseline contributed to the overall change.

[0157]

[0196] During task-free functional MRI in the resting state, enhanced connectivity was shown on day 29 compared to screening and day 1. Increased signals were observed in several resting-state networks, including those involved in executive function and sensorimotor processing. Task-based functional MRI (visual activation with a flickering checkerboard pattern) showed an increase in voxel activation in the occipital region after using Compound I on day 29 compared to screening and the day 1 visit. ii) Biomarker

[0197] Of the 40 inflammatory biomarkers evaluated in plasma as part of the InflammationMAP® panel, 26 (65%) decreased with an effect size of -0.3 or less. Only 3 (7.5%) of the 40 inflammatory biomarkers evaluated in plasma increased with an effect size of 0.3 or greater. The changes in concentration and effect sizes for the inflammatory markers measured in plasma are summarized in the following three tables (Table 1: Large effect sizes; Table 2: Moderate effect sizes; and Table 3: Small effect sizes). The concentration values in Tables 1 - 3 were measured in standard units for each of these test measurements as known in the art. Many of these biomarkers tend to increase again to baseline levels after treatment with Compound I is stopped, supporting the efficacy of the drug.

[0158]

Table 2

[0159]

Table 3

[0160]

Table 4

[0161]

[0198] Beta-2-microglobulin (B2M) is a circulating factor that negatively regulates age-dependently the cognitive regeneration function in the adult hippocampus.

[0199] Compound I improved biomarkers known to be associated with MELAS. The expression of VCAM-1 (vascular cell adhesion molecule-1), ICAM (intercellular adhesion molecule), and vWF (von Willebrand factor) is higher in endothelial cells derived from MELAS patients. VCAM and ICAM are endothelial adhesion molecules that can promote pathological plaque formation and may contribute to stroke-like episodes. vWF is a marker of endothelial activation and injury and is elevated in patients with overt vascular damage. The expression of TNFR2 (tumor necrosis factor receptor) is higher in muscle biopsies from patients with mitochondrial respiratory chain dysfunction. The biological effects of TNF-alpha in mitochondrial diseases are mainly mediated by TNFR2.

[0162]

[0200] In this study, as confirmed in Table 4 below, the changes in CBF were strongly correlated with the changes in B2M (a correlation of 0.8 or above is considered very strong; a correlation between 0.6 and 0.8 or equal to it is considered strong).

[0163]

Table 5

[0164]

[0201] Serum amyloid P component is a small glycoprotein found in normal serum and all amyloid deposits. It functions as an acute-phase protein, modulates the immune response, inhibits elastase, and has been suggested as an indicator of liver disease or neuropathy.

[0165]

[0202] Tumor necrosis factor receptor 2 (TNFR2) is expressed in muscle fibers with abnormal focal accumulations of mitochondria and is delivered to the mitochondria where the receptor localizes.

[0203] Several biomarkers of mitochondrial dysfunction were also measured in plasma. The lactate concentration, which is known to be elevated in mitochondrial disease patients, showed a tendency to decrease with an effect size of -0.5. GDF-15, a biomarker of mitochondrial disease, was also measured in plasma and showed a tendency to decrease with an effect size of -0.5. FGF-21, another biomarker of mitochondrial disease, was also measured in plasma and showed a tendency to decrease with a smaller effect size of -0.2. Using treatment, the directionality was improved, and the changes in the biomarkers of mitochondrial dysfunction were correlated with the plasma trough concentration of Compound I on Day 29, as can be confirmed in Table 5 below. That is, the higher trough concentration of Compound I on Day 29, as confirmed in Table 5 below, was associated with a greater decrease in mitochondrial dysfunction (a correlation of 0.8 or more is considered very strong; a correlation between or equal to 0.6 and 0.8 is considered strong). Furthermore, on Day 29, a strong correlation was observed among the changes in GDF-15, FGF-21, and lactate, which are biomarkers of mitochondrial dysfunction, and the serum amyloid P component.

[0166]

[0204] A decrease in lactate was observed in 6 out of 8 participants and ranged from 7% to 46%. A decrease in GDF-15 was observed in 4 out of 8 participants, with the largest decrease (up to 39%) in the participant with the highest baseline concentration. The changes in these biomarkers of mitochondrial dysfunction were strongly correlated with each other and also correlated with the plasma concentration of CY6463 at the end of dosing.

[0167]

Table 6

[0168] iii) Subjective evaluation / PRO Of the 8 registered participants, 3 reported improvement in their disease (1 participant had minimal improvement, 1 participant had significant improvement, and 1 participant had very significant improvement), 4 participants reported no change in their disease, and 1 participant reported worsening of their disease. Changes at the study level were not observed with the MFIS and PROMIS assessment tools, but some relevant observations could be made at the patient level. Participants who had a good response to these PROs at baseline and those with mild disease did not show improvement on day 29, while participants who had a worse response to these evaluations and those with more progressive disease at baseline reported more improvement.

[0169]

[0199] As confirmed in Table 6 below, interesting corrections were also observed between CBF and clinical improvement as evaluated by changes in the patient's overall condition (PGIC). A correlation of 0.8 or higher is considered very strong, and 0.6 - 0.8 is considered strong.

[0170]

Table 7

Claims

1. A method for treating mitochondrial disease in a patient in need of treatment for mitochondrial disease, the method comprising the step of administering to the patient a total daily oral dose of 15 mg to 60 mg of Compound I. 【Chemical 1】

2. The method according to claim 1, wherein the mitochondrial disease is selected from Alzheimer's disease, autosomal dominant optic atrophy (ADOA), Barth syndrome / LIC (lethal infantile cardiomyopathy), beta-oxidation deficiency, long-chain fatty acid transport deficiency, coenzyme Q10 deficiency, complex I, II, III, IV, V deficiency, chronic progressive external ophthalmoplegia (CPEO), Friedreich's ataxia, Kearns-Sayre syndrome, leukodystrophy, Leigh disease or Leigh syndrome, LHON, LHON Plus, MELAS (mitochondrial myopathy, encephalomyopathy, lactic acidosis, stroke-like symptoms), ragged red fibers / myoclonic epilepsy (MERRF), mitochondrial recessive ataxia syndrome (MIRAS), mitochondrial cytopathy, mitochondrial DNA depletion, mitochondrial encephalopathy, mitochondrial myopathy, multiple mitochondrial dysfunction syndrome, MNGIE (myoneurogastrointestinal encephalopathy), NARP (neuropathy, ataxia, retinitis pigmentosa, and ptosis), Pearson syndrome, pyruvate carboxylase deficiency, pyruvate dehydrogenase deficiency or pyruvate dehydrogenase complex deficiency (PDCD / PDH), and POLG mutation.

3. The method according to claim 1, wherein the mitochondrial disease is selected from Alzheimer's, complex I, II, III, IV deficiency, CPEO, KSS, LCHAD, Leigh syndrome, leukodystrophy, LHON, MELAS, MEPAN, MERRF, MIRAS, mitochondrial DNA depletion, MNGIE, NARP, Pearson syndrome, and POLG mutation.

4. The method according to claim 1, wherein the mitochondrial disease is complex I mitochondrial disease.

5. The method according to claim 1, wherein the mitochondrial disease is MELAS.

6. The method according to claim 1, wherein the mitochondrial disease is Leigh syndrome.

7. The method according to any one of claims 1 to 6, wherein the patient is 16 years of age or older, 18 years of age or older, or 65 years of age or older.

8. The method according to any one of claims 1 to 6, wherein the patient is 16 to 75 years old, 16 to 70 years old, 16 to 65 years old, 16 to 60 years old, 16 to 55 years old, 16 to 50 years old, 16 to 40 years old, or 16 to 30 years old.

9. The method according to any one of claims 1 to 6, wherein the patient is less than 65 years old, less than 60 years old, less than 50 years old, less than 40 years old, less than 30 years old or less than 20 years old.

10. The method according to any one of claims 1 to 6, wherein the patient is 16 years old or less, 12 years old or less, 10 years old or less, or 5 years old or less.

11. The method according to any one of claims 1 to 6, wherein the patient is 3 to 18 years old, 3 to 12 years old, 5 to 18 years old, 5 to 12 years old, or 3 to 5 years old.

12. The method according to any one of claims 1 to 11, wherein the patient is administered a total oral daily dose of 15 mg of Compound I.

13. The method according to claim 12, wherein the patient is administered a single oral daily dose of 15 mg of Compound I.

14. The method according to claim 12, wherein the patient is administered two oral daily doses of 7.5 mg of Compound I.

15. The method according to any one of claims 1 to 11, wherein the patient is administered a total oral daily dose of 20 mg of Compound I.

16. The method according to claim 15, wherein the patient is administered a single oral daily dose of 20 mg of Compound I.

17. The method according to claim 15, wherein the patient is administered two oral daily doses of 10 mg of Compound I.

18. The method according to any one of claims 1 to 11, wherein the patient is administered a total oral daily dose of 25 mg of Compound I.

19. The method according to claim 18, wherein the patient is administered a single oral daily dose of 25 mg of Compound I.

20. The method according to claim 18, wherein the patient is administered two oral daily doses of 12.5 mg of Compound I.

21. The method according to any one of claims 1 to 11, wherein the patient is administered a total oral daily dose of 30 mg of Compound I.

22. The method according to claim 21, wherein the patient is administered a single oral daily dose of 30 mg of Compound I.

23. The method according to claim 21, wherein the patient is administered two oral daily doses of 15 mg of Compound I.

24. The method according to any one of claims 1 to 11, wherein the patient is administered a total oral daily dose of 45 mg of Compound I.

25. The method according to claim 24, wherein the patient is administered a single oral daily dose of 45 mg of Compound I.

26. The method according to claim 24, wherein the patient is administered two oral daily doses of 22.5 mg of Compound I.

27. The method according to any one of claims 1 to 11, wherein the patient is administered a total oral daily dose of 60 mg of Compound I.

28. The method according to claim 27, wherein the patient is administered a single oral daily dose of 60 mg of Compound I.

29. The method according to claim 24, wherein the patient is administered two oral daily doses of 30 mg of Compound I.

30. The method according to claim 14, 17, 20, 23, 26 or 29, wherein the patient is administered a first dose and a second dose, and the first dose and the second dose are separated by a period of 5 to 15 hours, 8 to 15 hours, or 10 to 15 hours.

31. The method according to any one of claims 1 to 30, wherein treatment with Compound I results in a measurable improvement in neurological function and connectivity.

32. The method according to claim 31, wherein the improvement in neurological function and connectivity is measured by functional magnetic resonance imaging (fMRI).

33. The method according to any one of claims 1 to 32, wherein treatment with Compound I results in an increase in cerebral blood flow (CBF).

34. The method according to claim 33, wherein the treatment with Compound I results in an increase in cerebral blood flow (CBF) within a brain region selected from the temporal lobe, parietal lobe, occipital lobe, frontal lobe, corpus callosum, cingulate gyrus, cerebral white matter and cerebellar white matter, or a combination of one or more of the aforementioned regions.

35. The method according to any one of claims 1 to 34, wherein treatment with Compound I results in a decrease in one or more biomarkers of mitochondrial dysfunction.

36. The method according to claim 35, wherein the one or more biomarkers of mitochondrial dysfunction are selected from lactate, GDF-15 and FGF-21.

37. The method according to any one of claims 1 to 36, wherein treatment with compound I results in a decrease in one or more inflammatory biomarkers.

38. The method according to claim 37, wherein the one or more inflammatory biomarkers are selected from VCAM-1, ICAM, vWF, and TNFR2.

39. The method according to any one of claims 1 to 38, further comprising the step of administering to the patient one or more additional therapeutic agents.

40. The method according to claim 39, wherein the additional therapeutic agent is selected from citrulline and arginine.

41. The method according to claim 39, wherein the additional therapeutic agent is mitocactide.

42. The method according to any one of claims 1 to 41, wherein the patient is treated with one or more other therapeutic agents used to treat mitochondrial disease.

43. The method according to claim 42, wherein the other therapeutic agent used to treat mitochondrial disease is selected from citrulline and arginine.

44. The method according to claim 42, wherein the other therapeutic agent is mitocactide.

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