Treatment of mitochondrial disease Zagociguat using CNS-permeable sGC stimulators

Abstract

The present invention relates to a method of treating a mitochondrial disease in a patient in need thereof by administering a dose of compound (I), which is a soluble guanylate cyclase (sGc) stimulator, alone or in combination therapy. # imgabs0 #

Description

[0001] Related Applications

[0002] This application claims 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 contents of each of the above applications are incorporated herein by reference. Field of the Invention

[0003] The present invention provides methods for treating certain mitochondrial diseases in human subjects by administering specific dosage regimens of central nervous system (CNS) permeable soluble guanylate cyclase (sGC) stimulators, alone or in combination therapy. Background of the Invention

[0005] Mitochondrial diseases and MELAS

[0006] Mitochondrial diseases or disorders are a group of rare genetic diseases that occur when the mitochondria cannot produce enough energy for the body to function properly. They have different clinical manifestations and may manifest as impaired cerebral blood flow (CBF), oxidative stress, inflammation and metabolic crisis. They can affect almost any part of the body, including brain cells, nerves, muscles, kidneys, heart, liver, eyes, ears or pancreas. They can cause physical, developmental and cognitive disabilities, with symptoms including poor growth, loss of muscle coordination, muscle weakness and pain, fatigue, seizures, vision and / or hearing loss, gastrointestinal problems, cognitive impairment, learning disabilities and organ failure. The life expectancy of patients with mitochondrial diseases is greatly shortened. Mitochondrial diseases are usually progressive. It is estimated that 1 in 4,000 people have mitochondrial diseases. 80% of patients with mitochondrial diseases show CNS symptoms.

[0007] Currently, there is no effective treatment or cure for these conditions. Treatment is primarily supportive and may include nutritional management, exercise, and / or vitamin or amino acid supplementation.

[0008] 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 / ). Signs and symptoms of this condition most often appear in childhood after a period of normal development, although they can begin at any age. Early symptoms may include muscle weakness and pain, fatigue, recurring headaches, loss of appetite, vomiting, and seizures. Most affected individuals experience stroke-like episodes (SLE) before the age of 40. These episodes typically include temporary muscle weakness on one side of the body (hemiparesis), altered consciousness, abnormal vision, seizures, and severe headaches similar to migraines. Recurring episodes of SLE gradually damage the brain, leading to vision loss, movement problems, and loss of intellectual or cognitive function.

[0009] Most people with MELAS have a buildup of lactic acid in their bodies, a condition called lactic acidosis. Increased blood acidity can cause vomiting, abdominal pain, extreme tiredness and fatigue, muscle weakness, and difficulty breathing. Less commonly, people with MELAS may experience involuntary muscle spasms (myoclonus), impaired muscle coordination (ataxia), hearing loss, heart and kidney problems, diabetes, and hormone imbalances.

[0010] In the absence of approved therapies for MELAS, it has been hypothesized that citrulline and L-arginine, a precursor of nitric oxide (NO), could provide benefit in this patient population. Consensus guidelines from the Society for Mitochondrial Medicine recommend acute arginine administration to improve clinical symptoms associated with SLE in patients with MELAS. 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.

[0011] Treatment options for mitochondrial diseases remain extremely limited, and thus, there remains a need to develop new therapies to improve the many clinical manifestations associated with these diseases. Summary of the invention

[0012] In a first aspect of the present invention, disclosed herein is a method for treating a mitochondrial disease in a patient by administering to the patient a total oral daily dose of Compound I or an equivalent molar amount of a pharmaceutically acceptable salt of Compound I.

[0013] In the second aspect of the present invention, disclosed herein is Compound I or a pharmaceutically acceptable salt thereof, which is used to treat mitochondrial disease in a patient by administering to the patient a total oral daily dose of Compound I or an equivalent molar amount of a pharmaceutically acceptable salt of Compound I between 15 mg and 60 mg.

[0014] In the third aspect of the present invention, disclosed herein is the use of Compound I or a pharmaceutically acceptable salt thereof for preparing a medicament for treating a mitochondrial disease in a patient by administering to the patient a total oral daily dose of Compound I or an equivalent molar amount of a pharmaceutically acceptable salt of Compound I.

[0015] In a fourth aspect, the methods and uses of the invention relate to treatment in combination with one or more additional therapeutic agents. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Correlation of mitochondrial dysfunction biomarkers GDF-15 and FGF-21 at baseline is shown.

[0017] Figure 2 Shown are increases in CBF on day 29 compared to baseline (day 1) observed in all brain regions of interest. CI = confidence interval. DETAILED DESCRIPTION

[0018] NO-sGC-cGMP pathway, sGC stimulation and mitochondrial diseases

[0019] In vivo, NO is synthesized from arginine and oxygen by various nitric oxide synthases (NOS) and by the continuous reduction of inorganic nitrates. Three different NOS subtypes have been identified: inducible NOS (iNOS or NOS II) found in activated macrophages; constitutive neuronal NOS (nNOS or NOS I) involved in neurotransmission and long-term potentiation; and constitutive endothelial NOS (eNOS or NOS III) regulating smooth muscle relaxation and blood pressure.

[0020] sGC is the main receptor enzyme for NO in the body. sGC can be activated via both NO-dependent and NO-independent mechanisms. In response to this activation, sGC converts 5'-guanosine triphosphate (GTP) into the second messenger 3',5'-cyclic guanosine monophosphate (cGMP). The increase in cGMP levels in turn regulates the activity of downstream effectors including protein kinases, phosphodiesterases (PDEs) and ion channels. Intracellular cGMP regulates vascular tone and local blood flow, fibrosis and inflammation by activating cGMP-dependent protein kinases (PKG) and other downstream regulators.

[0021] The NO signaling pathway is also critical for the regulation of mitochondrial function and mitochondrial biogenesis. Dysregulation of the NO pathway is considered a major contributor to mitochondrial disease, leading to impaired cerebral blood flow (CBF), oxidative stress, inflammation, and metabolic crisis. In patients with mitochondrial disease, a clear link between NO signaling, stroke-like episodes (SLE), and dysregulation of CBF has been observed. NO bioavailability in these patients may be reduced through multiple mechanisms, including endothelial dysfunction and concomitant reduction in endothelial NOS, increased levels of the NOS inhibitor asymmetric dimethylarginine (ADMA), and increased oxidative stress and reactive oxygen species (ROS) that react with NO.

[0022] In the CNS, the NO-sGC-cGMP signaling pathway underlies multiple physiological processes that promote overall brain health, including neurotransmission, neurovascular function, cellular bioenergetics, and inflammation, and is associated with neuronal survival and cognitive function.

[0023] sGC stimulators are a class of heme-dependent agonists of the sGC enzyme that act synergistically with varying amounts of NO to increase the enzymatic conversion of GTP to cGMP. sGC stimulators are distinct and structurally unrelated to another class of NO-independent, heme-dependent sGC agonists, known as sGC activators. The benzyl indazole compound YC-1 was the first sGC stimulator to be identified. Since then, a variety of sGC stimulators have been identified and pharmacologically characterized, including BAY 41-2272, BAY 41-8543, riociguat (BAY 63-2521), vericiguat, olinciguat (IW-1701), and praliciguat (IW-1973). To date, no sGC stimulator has been approved for marketing in the CNS setting, and to our knowledge, Compound I (shown below) is the only CNS-penetrant sGC stimulator currently in clinical development for the treatment of CNS and mitochondrial diseases.

[0024] Compared with other potential therapies that target aberrant NO pathways or otherwise upregulate NO pathways, sGC stimulators may have considerable advantages. For example, sGC stimulation is a more effective approach compared with the use of NO supplements (which are associated with tachyphylaxis) or inhibition of cGMP breakdown (by phosphodiesterase inhibitors [PDEi], which have limited efficacy if cGMP levels are very low). In addition, the widespread distribution of sGC (including in different regions of the brain) could enhance cross-tissue signaling, whereas PDEi targets have more limited cellular and tissue expression.

[0025] Consensus guidelines from the Society for Mitochondrial Medicine recommend acute L-arginine administration to improve 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 core node in the NO-sGC-cGMP pathway, sGC stimulators are hypothesized to be in a critical position to potentially enhance mitochondrial function and biogenesis and have a positive effect on mitochondrial diseases, including those affecting the CNS.

[0026] Compound I (CY6463)

[0027] Compound I (also known as CY6463, IW-6463, or IWP-247) is an oral CNS-penetrant sGC stimulator that is being investigated for the treatment of CNS and mitochondrial diseases (clinical trials.gov identifiers NCT03856827, NCT04240158, NCT04475549, NCT04798989, NCT04972227). To our knowledge, it is the only CNS-penetrant stimulator tested in human subjects to date.

[0028] As an sGC stimulator, Compound I acts as a positive allosteric modulator of sGC by binding to sGC, thereby amplifying downstream signaling.

[0029]

[0030] In vitro studies in cells from patients with mitochondrial diseases have shown that compound I can improve cellular energetics in these cells by increasing the abundance of available adenosine triphosphate (ATP) and can reduce mitochondrial dysfunction by increasing the expression of genes involved in mitochondrial function, ATP synthesis, metabolism, and ROS reduction (see WO2020 / 014504). Experiments in cells from patients with mitochondrial diseases have shown that mitochondrial genes such as TFAM and DDAH2 are expressed at lower levels 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 that is required for mitochondrial transcription and regulates mtDNA copy number, and is therefore important for maintaining ATP production. Increased ADMA can lead to mitochondrial dysfunction, and elevated ADMA levels have been found in patients with mitochondrial diseases. Consistent with increased ATP levels, treatment with compound I increased the expression levels of TFAM and DDAH2 in patient cells. In an in vivo model of retinal degeneration caused by mitochondrial dysfunction, mice pretreated with compound I had less rotenone-induced astrogliosis compared with vehicle-treated mice, suggesting that compound I may provide protection against tissue damage caused by mitochondrial dysfunction.

[0031] In rodent studies, a single dose of Compound I increased fMRI-BOLD signal, enhanced qEEG gamma-band oscillation power, and increased cGMP levels in the CNS—in contrast to CNS-restricted sGC stimulators, which exhibit lack of target engagement and unique CNS pharmacology. In a rat CNS injury model, chronic administration of Compound I improved dendritic spine density, reversed deficits in the brain metabolites N-acetylaspartate (NAA) + N-acetylaspartate-glutamate (NAAG), and restored hippocampal long-term potentiation (LTP, a synaptic plasticity that underlies memory formation). Compound I also increases neurotrophic factors such as phosphorylated cAMP response element binding (pCREB) and brain-derived neurotrophic factor (BDNF), and improves 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 WH; 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.)

[0032] Safety and pharmacokinetic (PK) data from a Phase 1 study in healthy adults and safety, PK, and pharmacodynamic (PD) data from a second Phase 1 study in healthy elderly individuals also support clinical investigation of Compound 1 in the potential treatment of patients with mitochondrial diseases in general and MELAS in particular.

[0033] Definitions and General Terms

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs, which is the field of medicine, specifically the field 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 may also be used. The materials, methods, and examples are illustrative only and are 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 the event of a conflict, the present specification (including definitions) shall prevail.

[0035] As used herein, the word "a (kind)" before a noun represents one (kind) or more (kind) of a specific noun. As used herein, the singular forms "a (kind)" and "the" include plural referents unless the context clearly indicates otherwise.

[0036] As used herein, the terms "subject" and "patient" are used interchangeably. A subject or patient is a human patient or human subject.

[0037] The terms "for example" and "such as" and grammatical equivalents thereof, unless expressly stated otherwise, should be understood to be followed by the phrase "but not limited to" or "and not limited to."

[0038] Cognitive function declines naturally with age and in pathological conditions. "Cognitive impairment" refers to deficits in one or more higher-level brain functions, typically involving thinking and information processing (i.e., cognition).

[0039] The term "therapeutically effective amount" as used herein refers to the amount of active compounds or agents that a doctor or other clinical staff is seeking to cause a medical response in the human body. The therapeutically effective amount of a compound is at least the minimum amount required to improve, slow down, alleviate, delay, reduce, relieve or cure a disease, disorder or syndrome or one or more symptoms, signs or causes thereof. In another embodiment, it is the amount required to make the abnormal level of some clinical markers of a disease, disorder or syndrome closer to the normal value or level. In another embodiment, it is the amount required to make the level of some clinical markers displayed by the sick subject closer to the level (normalization) of normal subjects of the same age or closer to the level of patients with less severe disease performance or patients in the early stages of disease progression. The effective amount can be applied with one or more administrations throughout the day.

[0040] As used herein, a dose does not "result in a significant incidence of adverse events (AEs) or serious adverse events (sAEs) related to symptomatic hypotension" if it does not cause excessive orthostatic hypotension, excessive dizziness, excessive postural dizziness, excessive presyncope, or excessive syncope in the patient. Excessive orthostatic hypotension, excessive dizziness, excessive postural dizziness, excessive presyncope, or excessive syncope in the patient requires the patient to discontinue treatment or is advised by the physician to discontinue treatment.

[0041] The terms "administering," "administering," or "administration" when referring to a compound or agent means introducing the compound into the body of a patient in need of treatment. When Compound 1 or a pharmaceutically acceptable salt thereof is used in combination with one or more other therapeutic agents, "administering" and variations thereof are each understood to encompass the introduction of Compound 1 and the other therapeutic agent into the patient simultaneously and / or sequentially.

[0042] The term "disorder" as used herein refers to any deviation or interruption of the normal structure or function of any body part, organ or system, which is manifested by a set of characteristic symptoms and signs, and whose etiology, pathology and prognosis may be known or unknown. The term "disorder" encompasses other related terms, such as diseases and conditions (or medical conditions) and syndromes, which are defined as a combination of symptoms caused by a single cause or usually occur together to constitute a unique clinical manifestation. In some embodiments, the term disorder refers to a mitochondrial disorder. As used herein, the terms disorder, "disease", "condition" or "syndrome" are used interchangeably.

[0043] "Mitochondrial disease" refers to a group of genetic conditions that affect mitochondria, the structures in every cell of the body responsible for producing energy. These diseases can appear at any age and affect almost any organ, including the brain, muscles, heart, liver, nerves, eyes, ears, and kidneys. Some diseases affect only one organ or tissue, and many involve multiple organ systems, including the brain, muscles, heart, liver, nerves, eyes, ears, and / or kidneys. Mitochondrial diseases have a heterogeneous presentation.

[0044] Mitochondrial genetic diseases can be caused by mutations in mitochondrial DNA or nuclear DNA, leading to mitochondrial dysfunction and insufficient cellular ATP production. Diseases caused by mitochondrial DNA mutations are passed down through maternal inheritance, while diseases caused by nuclear DNA mutations may follow autosomal dominant, autosomal recessive, or X-linked inheritance patterns. (See: https: / / rarediseases.info.nih.gov / diseases / 7048 / mitochondrial-genic-disorders, last accessed on June 3, 2022, the teachings of which are incorporated herein by reference). The mitochondrial diseases contemplated throughout this disclosure are primary mitochondrial diseases or disorders. The term "mitochondrial disease" used here is equivalent to the term "primary mitochondrial disease." In some cases, mitochondrial dysfunction may also be secondary to other diseases. However, treatment of such secondary mitochondrial dysfunction is not discussed or considered herein. See https: / / www.mitoaction.org / resources / primary- mitochondrial-disease-and-secondary-mitochondrial-dysfunction-importance-of- distinction-for-diagnosis-and-treatment / (Last accessed: June 7, 2022) to understand the definition and distinction between primary mitochondrial disorders or diseases and secondary mitochondrial dysfunction.

[0045] Mitochondrial diseases manifest primarily due to chronic loss of cellular ATP, which results in a variety of clinical phenotypes and symptoms. In addition to ATP crisis, mitochondrial respiratory chain dysfunction leads to excessive ROS production and increased oxidative stress, resulting in cellular damage and inflammation.

[0046] 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 dose disclosed herein (total oral daily dose between 15 mg and 60 mg) include, but are not limited to, Alpers disease, autosomal dominant optic atrophy (ADOA), Barth syndrome / LIC (fatal infantile cardiomyopathy), beta-oxidation defect, long chain fatty acid transport defect, coenzyme Q10 deficiency, complex I, II, III, IV, V deficiency, chronic progressive external ophthalmoplegia (CPEO), Friedreich's ataxia, Kearns-Sell syndrome, leukodystrophy, Leigh disease or syndrome, LHON, LHON Plus, MELAS (mitochondrial myopathy, encephalomyopathy, lactic acidosis, stroke-like symptoms), myoclonic epilepsy with ragged red fibers (MERRF), mitochondrial recessive ataxia syndrome (MIRAS), Syndrome), mitochondrial cytopathy, mitochondrial DNA depletion, mitochondrial encephalopathy, mitochondrial myopathy, multiple mitochondrial dysfunction syndrome, MNGIE (mitochondrial neurogastrointestinal encephalomyopathy), 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.

[0047] In one embodiment, the mitochondrial disease is selected from Alpers disease, 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 a complex I mitochondrial disease. In another embodiment, the mitochondrial disease is MELAS. In yet another embodiment, the mitochondrial disease is Leigh syndrome.

[0048] "Treat", "treating" or "treatment" with respect to a disorder, disease, condition, symptom or syndrome refers to eliminating or ameliorating the cause and / or effects (i.e., symptoms, physiological, physical, psychological, cognitive, emotional or functional manifestations, or any clinical parameter or observation) associated with the disorder, disease, condition or syndrome. As used herein, the term "treat" also refers to delaying or ameliorating or slowing or preventing the progression of a disease (i.e., known or expected disease progression), severity and / or duration, or delaying or ameliorating or slowing or preventing the progression of one or more clinical parameters associated with the disease (i.e., "controlling" rather than "curing" a condition) resulting from the administration of one or more therapies.

[0049] The phrase "pharmaceutically acceptable salt" as used herein refers to a pharmaceutically acceptable organic or inorganic salt of Compound I. Pharmaceutically acceptable salts of Compound I can be used in medicines. However, non-pharmaceutically acceptable salts can be used to prepare 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 counter ions, etc. The counter ion can be any organic or inorganic part that stabilizes the charge on the parent compound. In addition, a pharmaceutically acceptable salt may have more than one charged atom in its structure. When multiple charged atoms are part of a pharmaceutically acceptable salt, there may be multiple counter ions. Therefore, a pharmaceutically acceptable salt may have one or more charged atoms and / or one or more counter ions.

[0050] Pharmaceutically acceptable salts of Compound 1 described herein include those derived from Compound 1 and 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 1 in a separate synthetic step.

[0051] 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 aluminum salts, ammonium salts, calcium salts, copper salts, iron salts, ferrous salts, lithium salts, magnesium salts, trivalent manganese salts, divalent manganese salts, potassium salts, sodium salts, zinc salts, etc. Specific embodiments include ammonium salts, calcium salts, magnesium salts, potassium salts and sodium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines (including naturally occurring substituted amines), cyclic amines, and basic ion exchange resins, such as arginine, betaine, caffeine, choline, N,N'dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glycamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like.

[0052] When a compound such as Compound I is alkaline or contains a sufficiently alkaline portion, a salt can be prepared from a pharmaceutically acceptable nontoxic acid (including inorganic and organic acids). These 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, etc. Specific embodiments include citric acid, hydrobromic acid, hydrochloric acid, maleic acid, phosphoric acid, sulfuric acid and tartaric acid. Other exemplary salts include, but are not limited to, 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 pamoate (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)).

[0053] The preparation of the above-mentioned pharmaceutically acceptable salts and other typical pharmaceutically acceptable salts is more fully described in Berg et al., "Pharmaceutical Salts," J. Pharm. Sci., 1977:66:1-19, the entire contents of which are incorporated herein by reference.

[0054] Clinical Assessment and Patient-Reported Outcome Tools

[0055] Assessment of the health status of a patient with mitochondrial disease and the corresponding pathology underlying observed dysfunction, decline, or symptoms can be performed using a number of different assessment tools or clinical measurements known and used in the art.

[0056] These range from imaging tools (e.g., magnetic resonance imaging (MRI), such as using arterial spin labeling (ASL) or functional fMRI-BOLD mode, etc.), to laboratory measurements (e.g., fluid biomarkers measured in blood, cerebrospinal fluid (CSF), urine, plasma, serum, skin, saliva), to clinical outcome assessment tools or instruments (e.g., patient or clinician reported outcome instruments or performance outcome measures, such as cognitive assessments using PROMIS questionnaires, MFIS scores, and other conditions described herein or known in the art), digital assessments (e.g., assessments obtained using wearable devices, sensor-based or camera-based assessments), and electrophysiological assessments (e.g., EEG). Some of these are described in the Examples section below and used in the clinical trials described in Example 1. Others are known in the art and may be used in hospital, clinical, or community settings. For example, the American Association of Family Physicians (AAFP) describes and provides links to many potential cognitive assessment tools on its webpage, such as the MiniCog, MoCA, SLUMS exam, CPCoG, MIS, and MMSE, etc. ( https: / / www.aafp.org / pubs / afp / issues / 2019 / 0115 / p101.html , last accessed on June 3, 2022).

[0057] Some measurements are performed to aid in diagnosis and / or patient selection. Other tests are to help assess prognosis. Other assessments may be performed to assess the pharmacological response to a certain intervention (pharmacodynamic or PD assessments), such as described herein. Others may be used to assess sensitivity to a certain intervention or risk of decline or the response to the intervention (e.g., assessing genetic markers or other biomarkers) or assessing disease progression in patients.

[0058] Previous clinical data of Compound I in healthy subjects

[0059] Electroencephalography (EEG) is a technique that measures the electrical activity of the brain (electrophysiology) by using electrodes placed on the scalp. The EEG power spectrum signal can be analyzed at different frequencies or frequency bands. In two previous Phase 1 clinical trials, Compound I was analyzed in the following frequency bands as described below: delta-1-4Hz (generally associated with sleep), theta-4-7.5Hz (associated with waking / falling asleep, with some association with cognition), alpha-8-12Hz (associated with passive wakefulness and cognitive processing), beta-12-25Hz (associated with alertness and attention), gamma-25-45Hz (associated with higher cognitive function). qEEG stands for quantitative electroencephalography.

[0060] Event-related potentials (ERPs) are "time-locked measures of electrical activity on the brain's surface that represent different stages of cortical processing," such as responses to auditory or visual stimuli (Patel and Azzam (2005), Characterization of N200and P300: Selected Studies of the Event-Related Potential. International Journal of Medical Sciences 2(4): 147-154). ERPs are time-locked and represent the average of electrical responses observed over multiple trials. ERPs are an objective, non-invasive method for studying information processing and cognitive function in the brain.

[0061] During an EEG-ERP experiment, several key waveforms can be assessed after a stimulus: the N200 (associated with stimulus identification and discrimination), the P300 (associated with selective attention, information processing, and cognitive speed / ability), the P200 (associated with aspects of selective attention or stimulus encoding), the P50 (associated with sensory gating or reduced neurophysiological responses to redundant stimuli), and the N100 (associated with early perceptual processes).

[0062] Two key parameters are used to quantify each response: latency (how long after the stimulus the spike occurs) and amplitude (how strong the spike is).

[0063] In preclinical models, sGC stimulation of 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 disorders; Correia, Susana S; Iyengar, Rajesh R; Germano, Peter; Tang, Kim; Bernier, Sylvie G; Schwartzkopf, Chad D; Tobin, Jenny; Lee, Thomas WH; Liu, Guang; Jacobson, Sarah; Carvalho, Andrew; Rennie, Glen R; Jung, Joon; Renhowe, Paul A; Lonie, Elisabeth; Winrow, C; Hadcock, J; Jones, J; Currie, MG. https: / / doi.org / 10.3389 / fphar.2021.656561 ). These studies were conducted in rats with telemetry devices implanted in the frontal cortex and frontoparietal regions of the brain. Rats were orally administered a suspension of Compound I, an oral suspension of a peripherally restricted sGC stimulator, or a subcutaneous injection of donepezil solution. Compound I altered qEEG measurements compared to vehicle administration, including increased gamma oscillations, while peripherally restricted sGC stimulators decreased gamma power. Oral administration of Compound I to rats at 10 mg / kg increased gamma power, and the signal was further increased when combined with 1 mg / kg donepezil 1-2 hours after administration.

[0064] Subsequently, in a Phase I clinical trial (clinicaltrial.gov identifier NCT03856827), brain changes were also observed via EEG following QD administration of 15 mg of Compound I to healthy participants aged 18 to 62 years. More specifically, effects of Compound I on ERP P300 amplitude were observed in this study, with amplitude increasing with increasing dose level, and responses modulated by time since administration. Improvements in alpha power were also observed at day 14 of the NCT03856827 study at all dose levels tested compared to placebo.

[0065] A second Phase I clinical trial (clinical trial.gov identifier NCT04240158) was conducted in elderly subjects [described in: Prior Patent Application Publication (WO 2022 / 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(15Supplement)4701)]. In this trial, an increase in posterior segment alpha power, a trend toward an increase in gamma power, and a decrease in N200 auditory event-related potential (ERP) latency were observed by EEG. Improvements in saccadic reaction time and saccadic peak velocity in the saccadic eye movement (SEM) assessment were also observed. Additionally, in an exploratory CSF biomarker analysis, positive trends in important CSF neuroinflammatory markers were observed in subjects after two weeks of treatment with 15 mg compound IQD compared to placebo. In particular, potentially important reductions in concentrations of alpha 2 macroglobulin (A2M) and complement C3 (C3) biomarkers were observed. Greater decreases in concentrations of these biomarkers were observed in subjects older than 70 years. Elevated A2M is associated with cerebrovascular disease and is predictive of cognitive decline and the development of AD. They have been reported to cause hyperphosphorylation of tau protein. C3 is associated with Aβ and tau and may play a role in synaptic degeneration.

[0066] In addition, multiple functional neuroimaging techniques were used in healthy elderly participants in the NCT04240158 trial: MRI arterial spin labeling (MRI-ASL) and functional magnetic resonance imaging (fMRI). The brain's neuronal metabolic signature (as an additional indicator of cellular bioenergetics) was also measured by magnetic resonance spectroscopy (1H-MRS).

[0067] MRI-ASL is used to quantify regional CBF in the resting state. fMRI is a relative measure based on the BOLD effect (described below) and is used to measure changes in CBF in certain brain regions due to increased brain activity.

[0068] The BOLD effect is based on the fact that blood flow in the brain is highly locally controlled by the oxygen and carbon dioxide tensions of the cortical tissue. When a specific area of ​​the cortex increases its activity in response to a task, the fraction of oxygen extracted from the local capillaries causes an initial drop in oxyhemoglobin (oxyHb) and an increase in local carbon dioxide (CO2) and deoxyhemoglobin (deoxyHb). After a lag of 2-6 seconds, CBF increases, delivering excess oxyhemoglobin and washing away deoxyhemoglobin. It is this large rebound in local tissue oxygenation that is imaged. The reason fMRI is able to detect this change is due to fundamental differences in the paramagnetic properties of oxyHb and deoxyHb. A series of evaluations are also being conducted in healthy elderly subjects in the NCT04240158 trial. NeuroCart is a complete set of tests developed by the Center for Human Drug Research (CHDR) to measure various CNS functions. NeuroCart can be used to correlate the CNS effects of a compound with drug concentration, helping to determine if the effect is caused by the compound. NeuroCart provides objective (e.g. neurophysiology, brain performance) and subjective (e.g. cognitive function, memory, mood, etc.) measures of CNS function. NeuroCart includes EEG and SEM tests as well as various other CNS function assessments described in WO2022 / 081610.

[0069] In the NCT04240158 trial, positive effects were observed in EEG and SEM assessments, which supports the beneficial effects of Compound I in some cognitive aspects and the potential use for the treatment of cognitive impairment in patients in need thereof as described in WO 2022 / 081610. In this trial, no changes were observed in neuroimaging assessments measured by ASL and fMRI BOLD or MRS, and NeuroCart CNS functional assessments. The EEG and SEM results of this trial support the treatment of cognitive impairment or decline in patients with a variety of diseases, including mitochondrial diseases.

[0070] Compound I in MELAS patients

[0071] In the two previous Phase 1 clinical trials mentioned above, changes in EGG parameters (alpha power and N200ERP), SEM changes, and trends for improvement in two neuroinflammatory parameters were observed in healthy elderly subjects treated with multiple doses of 15 mg QD for 14 days.

[0072] The goal of the study described in the Examples section of this disclosure (Example 1) is to evaluate the effects of an oral daily dose of 15 mg (QD) of Compound I on a number of parameters or measurements related to brain and general health in patients with the mitochondrial disease MELAS. The enrolled patients all had genetically confirmed MELAS with a history of CNS symptoms such as headaches, seizures, and stroke. They were allowed to receive stable medication, including nitric oxide precursors (e.g., arginine and citrulline).

[0073] The Examples section describes in detail the different assessments and measurements performed.

[0074] The primary objective of this trial was to evaluate the safety and tolerability of Compound I when administered to a patient population of MELAS subjects by measuring adverse events in the form of AEs leading to drug discontinuation, SAEs, and TEAEs (defined in the Experimental Section).

[0075] Exploratory objectives included evaluation of the plasma PK of the compound. In addition, other exploratory endpoints were designed to evaluate the effects of Compound I on physiology, neurophysiology, and cognition and health status. In particular, the following items were measured: changes from baseline in plasma biomarker concentrations of a number of relevant inflammatory biomarkers and biomarkers associated with mitochondrial dysfunction at day 29, changes from baseline in CBF measured by ASL at day 29, changes from baseline in fMRI-BOLD signal at rest and during visual stimulation at day 29, changes from baseline in brain metabolite levels measured by 1H-NMR at day 29, changes from baseline in MFIS scores (total, physical, cognitive, and psychosocial) at day 29, and changes from baseline in PROMIS Item Bank v2.0-cognitive function total score at day 29.

[0076] The present invention is based on the surprising discovery that administration of Compound I at a total oral daily dose of 15 mg per day to a patient population with the mitochondrial disease MELAS was safe and well tolerated and showed evidence of effects on CBF, fMRI-BOLD, and biomarkers of mitochondrial dysfunction and a number of inflammatory biomarkers. The mitochondrial dysfunction biomarkers investigated are known bioenergetics and metabolic biomarkers that have been found to be elevated in patients with mitochondrial disease. CBF and fMRI-BOLD are neuroimaging measures of brain perfusion and neuronal function / connectivity, respectively.

[0077] Inflammatory processes are known to be upregulated both peripherally and centrally in patients with mitochondrial dysfunction. The improvements in the following inflammatory groups suggest that compound I has a positive effect on oxidative stress.

[0078] Neuronal and / or glial damage caused by mitochondrial failure, nitric oxide deficiency, and cerebrovascular disease can affect cerebral perfusion (CBF). Dysregulation of CBF is associated with stroke-like episodes and CNS symptoms.

[0079] Additionally, improvements in patient well-being, fatigue, and cognitive assessments were observed for several subjects in the trial, some of which were consistent with improvements in CBF, fMRI, and biomarkers in those same patients.

[0080] Thus, the experimental results described in Example 1 of the Examples section demonstrate positive effects on multiple biomarkers, patient reported outcomes, increased cerebral blood flow, and increased functional connectivity within neural networks associated with cognition, memory, and executive function.

[0081] Compound I therefore has the potential to be used to treat other aspects of mitochondrial disease, including the physical aspects of the disease, and may improve the overall health of patients in addition to its potential to improve certain aspects of cognition as previously reported.

[0082] The results of this Compound I trial support the potential to exploit sGC stimulation to benefit several key pathological mechanisms of mitochondrial disease and dysfunction using a specific dosing regimen of Compound I in human subjects. These include effects on cellular bioenergetics, inflammation, neuronal / cognitive function, and impaired cerebral perfusion or cerebral blood flow. In particular, the fact that known biomarkers of mitochondrial dysfunction, such as elevated lactate levels at baseline and FGF-21 and GDF-15, were reduced and that this reduction correlated with Compound I plasma levels supports that Compound I is a disease-modifying therapy that can improve many aspects of the mitochondrial disease phenotype or manifestation. In addition, it has the potential to treat other mitochondrial diseases that display relevant symptoms in addition to MELAS. Importantly, the greatest improvements were observed in patients with a more severe disease phenotype at baseline.

[0083] Multiple plasma inflammatory and mitochondrial dysfunction biomarkers were found to be elevated at baseline in all participants, and multiple plasma inflammatory and mitochondrial dysfunction biomarkers showed treatment-directed improvements, many of which correlated with plasma exposure to Compound I (i.e., higher exposure at the end of treatment corresponded to higher improvements in biomarkers).

[0084] Fibroblast growth factor-21 (FGF-21) is a hormone-like cytokine involved in the intermediary metabolism of carbohydrates and lipids. FGF-21 expression is driven by mitochondrial reactive oxygen species, and FGF-21 concentrations in patients with known mitochondrial diseases are significantly higher than in controls.

[0085] Growth differentiation factor 15 (GDF-15), a member of the transforming growth factor beta family, was first selected as a marker of mitochondrial dysfunction by studying gene expression in skeletal muscle of patients with mitochondrial disease caused by thymidine kinase 2 mutations compared to healthy skeletal muscle. They found that GDF-15 was significantly upregulated in skeletal muscle and serum of patients with mitochondrial dysfunction. It is known that the expression of GDF-15 can be induced in response to stresses such as mitochondrial dysfunction by upregulating activating transcription factor 4.

[0086] Lactate, a product of anaerobic production of ATP, is the most commonly used marker for detecting mitochondrial dysfunction in the general diagnosis of MD. Although not particularly sensitive (sensitivity ranges from 34% to 62%), the estimated specificity of elevated lactate concentrations for detecting mitochondrial disease ranges from 83% to 100%.

[0087] CBF increased with treatment across participants across regions of interest and was consistent with Patient Global Impression of Change (PGIC) (i.e., better PGIC was associated with greater increases in CBF) and was associated with improvements in concentrations of inflammatory biomarkers.

[0088] fMRI BOLD signals observed in the resting state and during visual tasks indicated increased functional connectivity within neural networks associated with cognition, memory, executive function, and sensorimotor processing after treatment and were consistent with improvements in CBF. Symptomatic MELAS patients are known to have significantly reduced fMRI-BOLD responses to visual stimulation compared to controls (Rodan et al., 2020). Compound I treatment increased activation in occipital regions to visual stimulation, with greater activation on day 29 compared to day 1.

[0089] Although clinical outcomes for fatigue (MFIS) and cognition (PROMIS) did not demonstrate improvement with study-level treatment, trends toward improvement were observed for PGIC. However, improvements were observed in some patients who had poor responses to these assessments at baseline. Improvements were reported in outcomes reported by patients with the most severe disease, including fatigue, cognition, and global illness perception.

[0090] Thus, pharmacodynamic signals were observed in all four disease domains: CBF (measured by ASL), inflammation (measured by plasma levels of inflammatory biomarkers), cellular bioenergetics (measured by levels of mitochondrial dysfunction biomarkers related to metabolism and bioenergetics), and neural function and connectivity (measured by fMRI and cognitive patient-reported outcome assessments).

[0091] Furthermore, in another clinical trial conducted in parallel with the MELAS trial in patients with high levels of pathology (stable schizophrenia patients), total oral doses up to 60 mg per day were also found to produce proportional PK and were safe and well tolerated. Therefore, it is very plausible that doses up to 60 mg per day are also safe and well tolerated in patients with mitochondrial diseases, and they have the potential to produce a higher pharmacological response to those described herein.

[0092] Furthermore, although this trial was conducted in patients with MELAS and despite the highly heterogeneous population enrolled, the fact that a positive effect was observed at the study level makes it highly plausible that similar positive responses would be observed in patients with other mitochondrial diseases that present a similar phenotype or in whom the disease manifests as a result of similar mechanisms.

[0093] Treatment Methods / Examples

[0094] In some embodiments of the methods and uses of the present invention, the therapeutically effective amount of Compound I is a total oral daily dose of 15 mg to 60 mg of Compound I. In some embodiments, the total oral daily dose is 15 mg. In other embodiments, the total oral daily dose is 20 mg. In other embodiments, the total oral daily dose is 25 mg. In other embodiments, the total oral daily dose is 30 mg. In yet other embodiments, the total oral daily dose is 45 mg. In other embodiments, the total oral daily dose is 60 mg.

[0095] In some embodiments, a pharmaceutically acceptable salt of Compound I can be used in the methods and uses of the invention described herein. When a pharmaceutically acceptable salt of Compound I is used, the dosage of the pharmaceutically acceptable salt depends on the molecular weight of the salt and has the same molar amount as the dosage of Compound I described herein. Therefore, in some embodiments, the present invention is a method of treating a patient suffering from a mitochondrial disease by administering to the patient a total oral daily dose of Compound I or an equivalent molar amount of a pharmaceutically acceptable salt of Compound I.

[0096] In some embodiments, Compound I is suitable for treating mitochondrial diseases. In some embodiments, mitochondrial diseases most suitable for treatment with Compound I include, but are not limited to, those with a phenotype similar to MELAS. Patients with mitochondrial diseases with a phenotype similar to MELAS include those with confirmed mitochondrial disease mutations and at least two of the following: at least one history of SLE; at least one history of encephalopathy, defined as one or more personality or behavioral changes, confusion or disorientation with time, place or people; a history of symptomatic epileptic seizures; a history of migraines on an average of at least 1 day per month in the past 3 months, resulting in the individual being unable to function normally in school, work or home; or cognitive impairment defined as persistent forgetfulness with partial recall of events, memory loss and / or difficulty solving problems.

[0097] In some embodiments of the above methods and uses of the present invention, Compound I or a pharmaceutically acceptable salt thereof is suitable for treating a patient suffering from a mitochondrial disease selected from the group consisting of: Alpers disease, autosomal dominant optic atrophy (ADOA), Barth syndrome / LIC (fatal infantile cardiomyopathy), β-oxidation deficiency, coenzyme Q10 deficiency, complex I, II, III, IV, V deficiency, chronic progressive external ophthalmoplegia (CPEO), Friedreich's ataxia, Kearns-Sell syndrome, leukodystrophy, Leigh disease or syndrome, LHON, LHON Plus, MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, stroke-like symptoms), myoclonic epilepsy with shattered red fibers (MERRF), mitochondrial recessive ataxia syndrome (MIRAS), mitochondrial cytopathy, mitochondrial DNA depletion, mitochondrial encephalopathy, mitochondrial myopathy, multiple mitochondrial dysfunction syndrome, MNGIE (mitochondrial neurogastrointestinal 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.

[0098] In some embodiments, the mitochondrial disease is selected from Alpers disease, 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.

[0099] In some embodiments, the mitochondrial disease is a complex I mitochondrial disease. In some embodiments, the mitochondrial disease is MELAS. In other embodiments, the mitochondrial disease is Leigh syndrome.

[0100] In some embodiments of the above methods and uses of the present invention, treatment with Compound 1 or a pharmaceutically acceptable salt thereof does not result in adverse events (AEs) or serious adverse events (SAEs) associated with excessive symptomatic hypotension or orthostatic hypotension.

[0101] 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 other embodiments, the patient is between 16 and 60 years old. In some embodiments, the patient is between 16 and 55 years old, between 16 and 50 years old, between 16 and 40 years old, or between 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 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 other embodiments, the patient is a child. In other embodiments, the patient is an adult. In other embodiments, the patient is a teenager. In 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 other embodiments, the patient is 12 years old or younger. In other embodiments, the patient is 10 years older 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, between 3 and 12 years old, between 5 and 18 years old, between 5 and 12 years old, or between 3 and 5 years old.

[0102] 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 for treating mitochondrial disease prior to treatment with Compound I or a pharmaceutically acceptable salt thereof. In one embodiment, the other therapeutic agent for treating mitochondrial disease is selected from citrulline and arginine. In another embodiment, the other therapeutic agent is a mito cocktail as described herein.

[0103] In some embodiments of the above methods and uses, the total oral daily dose is administered as a single dose (QD). In other embodiments, the total oral daily dose can be divided into two equal oral daily doses (BID) of between 7.5 mg and 30 mg.

[0104] In certain embodiments, the methods and uses of the present invention described herein comprise administering to a patient a single oral daily dose of 15 to 60 mg of Compound I or an equivalent molar amount of a pharmaceutically acceptable salt of Compound I.

[0105] In certain embodiments, the methods and uses of the present invention described herein comprise administering to a patient a single oral daily dose of 15 mg of Compound I or an equivalent molar amount of a pharmaceutically acceptable salt of Compound I.

[0106] In certain embodiments, the methods and uses of the present invention described herein comprise administering to a patient a single oral daily dose of 20 mg of Compound I or an equivalent molar amount of a pharmaceutically acceptable salt of Compound I.

[0107] In certain embodiments, the methods and uses of the present invention described herein comprise administering to a patient a single oral daily dose of 25 mg of Compound I or an equivalent molar amount of a pharmaceutically acceptable salt of Compound I.

[0108] In certain embodiments, the methods and uses of the present invention described herein comprise administering to a patient a single oral daily dose of 30 mg of Compound I or an equivalent molar amount of a pharmaceutically acceptable salt of Compound I.

[0109] In certain embodiments, the methods and uses of the present invention described herein comprise administering to a patient a single oral daily dose of 45 mg of Compound I or an equivalent molar amount of a pharmaceutically acceptable salt of Compound I.

[0110] In certain embodiments, the methods and uses of the present invention described herein comprise administering to a patient a single oral daily dose of 60 mg of Compound I or an equivalent molar amount of a pharmaceutically acceptable salt of Compound I.

[0111] In some embodiments, the methods and uses of the present invention described herein comprise administering to a patient an initial total oral daily dose of 30 to 60 mg of Compound I or an equivalent molar amount of a pharmaceutically acceptable salt of Compound I, followed by a titration reduction in dose to a total oral daily dose of 15 to 30 mg of Compound I or an equivalent molar amount of a pharmaceutically acceptable salt of Compound I if, as assessed by a practicing physician, the patient does not tolerate a daily dose exceeding 30 mg. Sometimes, upon physician assessment, once a lower dose is tolerated over a relevant period of time, the patient may resume a higher dose after a period of adaptation to the lower dose.

[0112] In certain embodiments, the methods and uses of the present invention described herein comprise administering to a patient an oral dose of 7.5 to 30 mg of Compound I or an equivalent molar amount of a pharmaceutically acceptable salt of Compound I twice daily (BID). In one embodiment, the methods and uses of the present invention described herein comprise 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 an equivalent molar amount of a pharmaceutically acceptable salt of Compound I twice daily (BID). In one embodiment, the method and use comprises administering to the 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 an equivalent molar amount of Compound I, 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 an equivalent molar amount of a pharmaceutically acceptable salt of Compound I, wherein the first dose and the second dose are separated by 5 hours to 15 hours, 8 hours to 15 hours, or 10 hours to 15 hours. In another embodiment, the first dose and the second dose are separated by 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, or 15 hours.

[0113] In some embodiments, the maintenance dose is continued indefinitely as long as the patient continues to experience clinical benefit. In some embodiments, treatment with Compound I is chronic.

[0114] In some embodiments of the above methods and uses, Compound I is administered before symptoms of a mitochondrial disorder fully develop in the patient. In other embodiments of the above methods and uses, Compound I is administered after the patient develops one or more symptoms of a mitochondrial disorder.

[0115] In some embodiments, the patient with mitochondrial disorder is a patient who has been diagnosed with mitochondrial disorder or a patient who is genetically susceptible to the disorder. In other embodiments, the patient in need is a person who has been genetically tested and found to have a genetic mutation that makes him or her susceptible to the disorder, even if he or she may not yet show any physical symptoms of the disorder (or disease) or may show minimal symptoms. In other embodiments, even if a formal diagnosis has not yet been made, the patient also shows symptoms of the disorder (or disease). Some common signs and symptoms of mitochondrial disease include: poor growth, loss of muscle coordination, muscle weakness, fatigue, exercise intolerance, lactic acidosis, epileptic seizures, stroke-like attacks (SLE), headaches, cognitive impairment, mental fatigue, fatigue, autism, vision and / or hearing problems, developmental delay, learning disabilities, heart, liver and / or kidney disease, gastrointestinal disease, diabetes, increased risk of infection, thyroid and / or adrenal abnormalities, autonomic dysfunction, and dementia.

[0116] In some embodiments, treatment results in an improvement in at least one measurable physical parameter of a mitochondrial disorder. In other embodiments, treatment results in a reduction, inhibition, or slowing of the progression of a mitochondrial disorder, such as by stabilization of a measurable symptom or set of symptoms, or by stabilization of a measurable biomarker, such as a mitochondrial disorder.

[0117] In some embodiments, treatment results in a measurable improvement in neuronal function and connectivity. In one embodiment, the improvement in neuronal function and connectivity is measured by functional magnetic resonance imaging (fMRI).

[0118] 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 a combination of one or more of the foregoing regions.

[0119] In some embodiments, treatment results in a reduction 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 reduction in one or more biomarkers of mitochondrial dysfunction. In other embodiments, treatment results in a reduction in one or more inflammatory biomarkers and one or more biomarkers of mitochondrial dysfunction. In some embodiments, the mitochondrial dysfunction biomarkers described herein are selected from lactate, GDF-15 and FGF-21.

[0120] Combination therapy

[0121] Treatment of mitochondrial diseases and related symptoms with Compound I or a pharmaceutically acceptable salt thereof can be performed using the compound alone or in combination with other therapeutic agents. In some embodiments, Compound I or a pharmaceutically acceptable salt thereof can be used in combination with one or more drugs independently selected from citrulline and arginine for the treatment of mitochondrial diseases.

[0122] In some embodiments, Compound I or a pharmaceutically acceptable salt thereof can be used in combination with mito cocktail therapy to treat mitochondrial disease. As used herein, "mito cocktail therapy" refers to a combination of multiple vitamins and supplements commonly used by adults and children who have been diagnosed with mitochondrial disease.

[0123] The supplements and vitamins used by patients with mitochondrial disease are often in larger doses and may require patients to take up to 50 different pills per day. Through the International Association of Compounding Pharmacists (IACP), compounding pharmacists can create liquid, capsule, or other “cocktails” that combine prescription supplements and vitamins in pure powder form. The final medication amount is often much smaller than what would otherwise be taken and can even be flavored to improve palatability. By avoiding fillers commonly found in over-the-counter medications, individual allergies or dietary restrictions can be accommodated. The exact compound, including dosage and ingredients, is determined by the patient’s physician and varies based on the individual patient’s diagnosis, clinical symptoms, and weight. The most common ingredients include:

[0124] 1) Coenzyme Q-10

[0125] Coenzyme Q-10 (Coenzyme Q10, CoQ10, CoQ-10, CoQ, Ubiquinone, ) is a fat-soluble, vitamin-like substance that is present in every cell of the body and is a coenzyme in several key enzymatic steps in energy production within cells. It also acts as an antioxidant, preventing the accumulation of harmful free radicals, which is critical to its clinical effect. Many patients report increased energy after using CoQ-10, so it is a common "first-line" method of supporting children and adults with mitochondrial disease. Frequently reported side effects include stomach upset and sleep disturbances, so pharmacists recommend taking CoQ-10 doses early in the day with food. Therapeutic levels may take time to establish, so patients may not see beneficial effects immediately. In addition, unutilized material is stored in fat cells, so appropriate dosing is important.

[0126] 2) Multivitamins

[0127] Some B vitamins are cofactors involved in important mitochondrial reactions. Most B vitamins have a bitter taste and are more palatable if flavored. B vitamins are water-soluble; that is, they are excreted from the body if not used. The benefits of taking these vitamins should be felt immediately.

[0128] a. Vitamin B1 (Thiamine). This is a water-soluble vitamin that helps break down carbohydrates so the body can better use them, helps grow and maintain muscle tone, and aids in memory. The only possible side effect sometimes noticed is drowsiness.

[0129] b. Vitamin B2 (Riboflavin). B2 is also a water-soluble vitamin that is necessary for energy production in the mitochondria, improves muscle performance, and helps maintain healthy mucous membranes, skin, hair, and nails. The only side effect noted is a tendency for urine to turn orange. Giving it in the form of riboflavin diphosphate can improve the taste of this vitamin.

[0130] Vitamin B3 (Niacin). B3 can often cause facial flushing when used occasionally, so it is usually taken alone first to see if any side effects occur before adding it to a cocktail.

[0131] Vitamin B6 (pyridoxine) and vitamin B12 (cobalamin). These are other B vitamins that are often part of a multivitamin that people with Mito may use.

[0132] Vitamin C. Used to aid the healing process and prevent infection, but may cause stomach upset and occasional headaches at higher doses.

[0133] f. Vitamin E. It can protect cell membranes and improve nerve function. Usually the daily dose for adults does not exceed 400-600 mg. Vitamin E can interfere with coumarin / warfarin drugs, so caution should be used when using vitamin E in Mito cocktail therapy.

[0134] g. Vitamin K1. Another vitamin that can be added (but be careful as the safe range of dosage for this vitamin is very small) must be prescribed by a doctor and cannot be purchased over the counter.

[0135] 3) Other antioxidants

[0136] Antioxidants reduce the accumulation of free radicals in cells and therefore may also be used in patients with mitochondrial disease. Alpha-lipoic acid is probably the most commonly used antioxidant in Mito cocktail therapy.

[0137] 4) L-Carnitine

[0138] 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 sweat glands. Some patients report reduced fatigue and improved energy from taking L-Carnitine. It is taken in tablet or liquid form and is usually taken separately from a mixed cocktail.

[0139] 5) Creatine

[0140] Creatine helps maintain muscle mass and increases cellular energy. Side effects include diarrhea and drowsiness; dosages range from 5 g / day for children to 10 g / day for adults, usually in liquid or capsule form.

[0141] All of the above vitamins and supplements may or may not be added to the cocktail, depending on the patient's needs. Each cocktail is patient-specific and can be determined by the doctor.

[0142] As used herein, the terms "combination" (as in the sentence "combination therapy") or "co-administration" can be used interchangeably to refer to the use of more than one therapy. The use of these terms does not restrict the order in which the treatments are administered to a subject.

[0143] For combined treatment using more than one therapeutic agent, wherein the therapeutic agent is in separate dosage formulations or dosage forms, the therapeutic agent can be administered alone or in combination (i.e., simultaneously). In addition, when administered alone, the administration of one therapeutic agent can be before or after the administration of another agent.

[0144] When Compound I or its pharmaceutically acceptable salt is used for combination therapy with other therapeutic agents, the therapeutically effective amount of the other therapeutic agent or each other therapeutic agent will depend on the type of drug used. Suitable dosages for approved therapeutic agents are known and can be adjusted by a technician according to the condition of the subject, the type of condition treated, and the amount of Compound I or its pharmaceutically acceptable salt used. In one embodiment of the present invention, Compound I or its pharmaceutically acceptable salt and another therapeutic agent are each administered in a therapeutically effective amount (ie, each in an amount that would be therapeutically effective if administered alone). In other embodiments, Compound I or its pharmaceutically acceptable salt and another therapeutic agent are each administered in an amount that does not provide a therapeutic effect alone (subtherapeutic dose). In another embodiment, Compound I or its pharmaceutically acceptable salt may be administered in an effective therapeutic amount, while another therapeutic agent is administered in a subtherapeutic dose. In yet another embodiment, Compound I or its pharmaceutically acceptable salt may be administered in a subtherapeutic dose, while another therapeutic agent is administered in a therapeutically effective amount.

[0145] When co-administration involves separate administration of a first amount of Compound 1 or a pharmaceutically acceptable salt thereof and a second amount of an additional therapeutic agent, the administration of the compounds is close enough in time to have the desired therapeutic effect. For example, the time period between each administration that can produce the desired therapeutic effect can be in the range of a few minutes to a few hours, and can be determined by considering the properties of each compound, such as efficacy, solubility, bioavailability, plasma half-life, and pharmacokinetic characteristics. For example, Compound 1 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.

[0146] More specifically, administration of the first therapy can be performed before (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, or 12 hours before), simultaneously with, or after (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, or 12 hours after) administration of the second therapy.

[0147] Example

[0148] In order to better understand the present invention, the following examples are given. These examples are only for illustrative purposes and should not be construed as limiting the scope of the present invention in any way. All references provided in the examples are incorporated herein by reference.

[0149] Example 1: Signal seeking study to evaluate the safety, tolerability and effects of IW-6463 on CNS diseases

[0150] Abbreviations / Definition of Terms

[0151]

[0152]

[0153] Study objectives and outcome measures

[0154] This study was completed at 5 investigational sites in the United States. 8 participants completed the study. This study was an open-label, single-arm study evaluating the safety, tolerability, PK, and pharmacodynamics (PD) of Compound I administered for up to 29 days to participants diagnosed with MELAS. Because this was the first study to evaluate Compound I in this patient population, the primary objectives were safety and tolerability. The overall study duration for participants was up to 75 days, including screening, treatment, and follow-up periods (clinical trial.gov identifier NCT04475549).

[0155] The goal of the study described in the Examples section (Example 1) was to evaluate the effects of a daily dose of 15 mg of Compound I on a number of parameters or measurements related to brain and general health in patients with the mitochondrial disease MELAS. The different assessments and measurements performed are described in detail below.

[0156] The primary objective of the trial was to evaluate the safety and tolerability of Compound I when administered to a patient population of MELAS subjects by measuring AEs leading to drug discontinuation, SAEs, and TEAEs.

[0157] Exploratory objectives include evaluating the plasma PK of the compound. In addition, other exploratory endpoints are designed to evaluate the effects of Compound I on physiology, neurophysiology, and cognition and health status.

[0158] Study Design:

[0159] To complete this study, each participant was required to go through 3 different phases as described below. A schematic diagram of the study is also shown below.

[0160] Screening stage:

[0161] The screening period began with the signing of the Informed Consent Form (ICF) at the screening visit. After signing the ICF, each participant was assessed for study eligibility based on the inclusion and exclusion criteria described below. Collection of adverse events (AEs) began after the signing of the ICF and continued until the follow-up period / study termination (whichever occurred first).

[0162] After the screening visit, eligible participants must begin following the lifestyle restrictions detailed below. They also begin completing a daily diary for the study. The end of the screening period coincides with the start of the clinical period.

[0163] Treatment period:

[0164] The treatment period began on Day 1 (there was no "Day 0"), when participants returned to the study center for baseline procedures and received their first daily dose of study medication. Participants returned to the study center on Day 29 (-4 days) for an end-of-treatment (EOT) visit; all other scheduled visits during this period were completed at home or at the study center during the times allowed, depending on the participant's preference. Participants continued to complete their daily diaries throughout the treatment period.

[0165] The end of the treatment period coincided with the beginning of the follow-up period.

[0166] Follow-up period:

[0167] The follow-up period began immediately after the EOT visit and continued until the follow-up visit, during which time participants were required to continue to adhere to all lifestyle restrictions and to continue to complete the diary.

[0168] Research diagram

[0169]

[0170] During the treatment period, all participants will receive open-label IW-6463 once daily for up to 29 days.

[0171] *Screening assessments may be conducted over more than 1 day. However, all screening procedures must be completed at least 10 days but no more than 28 days prior to the start of the treatment period.

[0172] **Day 8, Day 15, and follow-up visits may be performed at home or at the study clinic based on participant / site preference.

[0173] Participant diary to record changes in health status, daily doses, and concomitant medications

[0174] At the screening visit, each participant was given a paper (source) diary in which they (or their legal representative / guardian) were asked to record daily the name of any other medications they took and the date, time, and dosage strength of the medication; and any changes in their health status (including date, time, and a brief description).

[0175] Beginning on day 1 of the treatment period, they were asked to record the date and time they took each dose of study medication at home, as well as any changes in their health status (including date, time, and a brief description), in addition to any other medications they took (including name, date, time, and dosage strength).

[0176] Participants were asked to provide a diary at each scheduled home and clinic visit during treatment to allow for monitoring by the research staff.

[0177] During the follow-up period, participants were asked to continue recording any concomitant medications and changes in health status in the same manner. These diaries were collected from participants at the follow-up visit.

[0178] Clinical Outcome Assessment / Participant Questionnaire

[0179] Patient-Reported Outcomes Measurement Information System (PROMIS) – Cognitive Function

[0180] The Patient-Reported Outcomes Measurement Information System (PROMIS) Item Bank v2.0-Cognitive Function is a self-administered questionnaire that assesses multiple aspects of mental fatigue and cognitive function in the 7 days prior to administering the questionnaire. It uses a Likert-type rating scale (e.g., “never” to “often”).

[0181] Patient Global Impression of Change (PGIC)

[0182] The Patient Global Impression of Change (PGIC) is a single-item questionnaire that uses a Likert-type rating scale to assess the participant's perception of changes in his / her overall health status since the start of the study (e.g., "much improved" to "very worse").

[0183] Modified Fatigue Impact Scale (MFIS)

[0184] The modified fatigue impact scale (MFIS) is a self-administered questionnaire that assesses the impact of fatigue on physical, cognitive, and psychosocial functioning over the past 4 weeks. Patients respond using a Likert-type rating scale (e.g., “never” to “almost always”).

[0185] PD Assessment and Parameters (Neuroimaging)

[0186] Two exploratory functional neuroimaging modalities, arterial spin labeling (ASL) and functional magnetic resonance imaging (fMRI), were used to measure potential PD effects of Compound I in the brain. ASL quantifies regional CBF during the resting state, while fMRI is a relative measure based on the blood oxygen level dependent (BOLD) effect, performed both at rest and during visual stimulation to measure brain activity.

[0187] Each MRI scan takes about 45 minutes (up to 1 hour).

[0188] Suicide Risk Surveillance (C-SSRS)

[0189] Compound I is a CNS-active investigational drug. Although Compound I and other similar drugs in this class have not been associated with an increased risk of suicidal thoughts or behaviors, monitoring for such thoughts or behaviors prior to and during this clinical study is considered important. Therefore, participants should be appropriately monitored and closely observed for suicidal thoughts and behaviors or any other unusual behavioral changes. Administer the Columbia-Suicidality Severity Rating Scale (C-SSRS), pre-dose, beginning on Day 1 of the treatment period, and at all subsequent visits for clinical evaluation, including any unscheduled visits. For participants who develop signs of suicidal thoughts or behaviors, consult the medical monitor promptly and consider discontinuation of study drug.

[0190] Physical examination and vital signs

[0191] A complete physical examination was performed by the investigator at screening and follow-up. All other examinations may be performed symptomatically at the investigator's discretion.

[0192] A complete physical examination included examination and assessment of the following: general appearance, lymph nodes, neurologic system; cardiovascular system; head, eyes, ears, nose, throat, and skin; respiratory system, neck, mental status, abdomen / liver / spleen, and musculoskeletal system. Breast, genitourinary, and rectal examinations were optional and performed at the discretion of the investigator. Clinically relevant findings that occurred before the start of study drug on Day 1 were recorded as part of the participant's medical history. New clinically relevant findings or worsening of earlier findings from the start of study drug on Day 1 were recorded as AEs. Height (cm) was measured only at Screening. Weight (kg) was recorded throughout the study. Body mass index was calculated and recorded at Screening.

[0193] Vital signs measured include respiratory rate, oxygen saturation, and body temperature. Respiratory rate and oral temperature (°C) should be measured after the participant rests in a supine / sitting position for ≥5 minutes. Oxygen saturation should be measured by pulse oximetry under room air conditions.

[0194] All blood pressure and pulse measurements were obtained using an automated blood pressure device (preferably the left arm) before blood draw (or ≥10 minutes after blood draw, if necessary). Supine blood pressure was measured at Screening (only) and recorded as the average of 2 measurements obtained at 2-minute intervals after the participant had rested quietly in a semirecumbent / supine position for ≥5 minutes. Orthostatic vital signs were collected at all scheduled visits, and participants were to rest quietly in a supine / semirecumbent position for ≥5 minutes, followed by a record of supine blood pressure and pulse measurements, then a seated position for ≥1 minute, and finally a standing position for 2 (±1) minutes, followed by a record of standing measurements. These measurements were used to calculate and record the orthostatic blood pressure and pulse.

[0195] Electrocardiogram (ECG)

[0196] All ECGs and screening ECHOs (if not documented in the medical history within 3 months before Day 1) were obtained after the participant had rested in the supine position for ≥5 minutes and before blood draw (or ≥10 minutes after blood draw, if necessary). When the timing was consistent, the ECG and vital signs could be assessed together.

[0197] If the QTc result (corrected using the Fridericia formula) is outside the normal range (>450 ms), the ECG is repeated twice and the average of the three results is calculated.

[0198] All ECGs were assessed by the investigator or qualified designee for abnormalities. Results were reported as “normal,” “clinically significant abnormal,” or “not clinically significant abnormal.” Clinically significant abnormalities were recorded as AEs.

[0199] If a confirmed clinically significant abnormal result is obtained, the site will follow standard institutional procedures until the result returns to baseline. If the problem persists, the issue will be escalated to the sponsor's medical monitoring.

[0200] Adverse Event, SAE and Other Safety Reports

[0201] Adverse events (AEs) were monitored and recorded throughout the study, from the time of ICF execution to follow-up. Care was taken not to introduce bias when detecting AEs and / or SAEs. Open-ended and non-leading verbal questioning of participants was the preferred method for inquiring about the incidence of AEs.

[0202] The investigator is under no obligation to proactively collect AE information after a participant's participation in the study has ended. However, if the investigator learns of any SAE or death at any time after a participant is discharged from the hospital, he or she should promptly notify the sponsor if the investigator believes the event is reasonably related to the study drug or participation in the study.

[0203] Pregnancy reporting and monitoring

[0204] Female participants who report pregnancy prior to initiation of study drug dosing on Day 1 will be withdrawn from study participation; discontinuation will be recorded as screening failure.

[0205] If a participant or the participant's partner becomes pregnant after starting a study drug, it will be reported and recorded on the study-specific pregnancy form. Sites should make reasonable efforts to follow the pregnancy to term.

[0206] All reports of congenital anomalies / birth defects and stillbirths should be considered SAEs. Spontaneous abortions, uncomplicated elective terminations of pregnancy, and normal deliveries without congenital anomalies should not be reported or treated as SAEs but rather reported as outcomes of the respective pregnancies.

[0207] Other assessments

[0208] All subjects underwent various safety, drug testing, and protocol-required clinical laboratory evaluations. Genetic evaluation in this study was limited to confirmed MELAS (by medical history) at screening eligibility.

[0209] Plasma and CSF PK and biomarker assessments

[0210] PK samples were collected. Each sample was divided into 2 portions (1 for PK and 1 for backup). Approximately 2 mL of dilute whole blood samples were collected for measurement of plasma concentrations of Compound I using a validated liquid chromatography-tandem mass spectrometry bioanalytical method.

[0211] From each participant who did not refuse consent for the procedure, approximately 4 mL of CSF sample was collected for measurement of compound ICSF concentrations using a validated liquid chromatography-tandem mass spectrometry bioanalytical method.

[0212] Plasma and serum samples for biomarkers were collected from each participant. These biomarkers test the target engagement 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-1β, asymmetric dimethylarginine (ADMA), L-arginine, and other biomarkers associated with the pathophysiology of mitochondrial disease, and their response to Compound I.

[0213] Also by proton magnetic resonance spectroscopy ( 1 H-MRS) was used in participants to measure the effects of Compound I on the properties of neurons in the brain. Biomarkers measured using this imaging technique included ventricular lactate and N-acetylaspartate concentrations, among others.

[0214] Study Drugs, Dose Levels, and Administration

[0215] All eligible participants received open-label Compound I, starting at 15 mg daily. Participants were asked to take Compound I once daily on Days 2 to 28, at the same time as the outpatient study drug administration on Day 1 (preferably ± 1 hour).

[0216] Exceptions / Notes: For Day 8, 15, and 29 / EOT visits, participants During the visit Doses were taken so that pre- and post-dose assessments could be performed at appropriate times. Day 8 and Day 15 visits (at home or in the clinic) were scheduled so that dosing could still be performed at approximately the same time as Day 1 and subsequent doses. Participants recorded the date and time of each home dose in a diary.

[0217] Permitted concomitant medications may be taken with the study drug. Participants were asked to record in a diary each dosing day, time, and dosage strength of any concomitant medication they took.

[0218] Although Compound I can be taken with or without food, participants were asked to fast for 3 to 4 hours prior to clinic and home clinical safety and PD laboratory sample collection.

[0219] The 15 mg Compound I QD dose was selected based on safety, tolerability, PK, and PD data from repeated dosing at this level in two Phase 1 studies conducted in healthy adults aged 18 to 79 years. There were no discontinuations due to AEs and no SAEs were reported in healthy participants who received 15 mg Compound I QD for up to 15 days. All AEs were considered by the investigators to be mild or moderate. No safety issues were identified in the Phase 1 studies. PK data were linear and dose-dependent, supporting QD dosing and were not affected by food. In addition, EEG showed that 15 mg Compound I QD had modest effects on neurophysiological parameters after 14 days of dosing in healthy elderly participants. No effects on cognitive performance measures were observed.

[0220] Participant Selection and Lifestyle Restrictions

[0221] Inclusion criteria

[0222] Each participant must meet the following Each Criteria to be eligible for this study:

[0223] 1. An ICF has been signed by the participant or his / her legal representative / guardian, as applicable, before any study-specific procedures are performed

[0224] 2. Be 18 years of age or older on the date of consent

[0225] 3. Genetic confirmation of previously known mitochondrial disease mutations

[0226] 4. Show neurological features of MELAS (can be based on medical history)

[0227] 5. Elevated plasma lactate during screening (≥1.0mmol / L)

[0228] 6. Agree to comply with all study requirements, including lifestyle restrictions

[0229] 7. Agree to refrain from any major lifestyle changes (e.g., starting a new diet or changing exercise patterns) from the time the ICF is signed until the end of follow-up (and thereafter, relative to contraception requirements)

[0230] 8. If receiving long-term medication, there was no change in treatment regimen ≥ 4 weeks before the first day of dosing, and there is no plan to change treatment regimen during the study

[0231] 9. If female, meets one of the following 2 criteria: confirmed postmenopausal (no menstruation for ≥1 year or ≥12 months) or has undergone sterilization surgery (bilateral oophorectomy, hysterectomy, or tubal sterilization [ligation, clip, band, or cauterization]) - or - if female of reproductive potential is not pregnant or breastfeeding at the screening visit and has a negative pregnancy test result at the screening visit and before dosing on Day 1, and agrees to contraception during the study and for ≥90 days after the final dose of study drug

[0232] 10. Male and female participants of reproductive potential must agree to use ≥1 of a range of effective contraceptive methods from the date of signing the ICF until ≥90 days after receiving their final dose of study drug.

[0233] 11. Male participants must agree not to donate sperm within 90 days from the screening to the final administration of study drugs.

[0234] 12. Agree through follow-up visits not to receive any other investigational treatment or device during the study period

[0235] 13. No clinically significant findings on ECG (screening and pre-dose assessment on Day 1) and echocardiogram (ECHO; within 3 months after Day 1) Exclusion criteria

[0236] satisfy any Participants with the following criteria were excluded from this study.

[0237] 1. As determined by the investigator, exhibits severe visual, hearing, or cognitive impairment that may affect the ability to comply with protocol requirements or complete required assessments

[0238] 2. Use of any nicotine-containing products (e.g., cigarettes, e-cigarettes, e-cigarette pens, cigars, chewing tobacco, gum, patches) within 1 month after enrollment

[0239] 3. Positive pregnancy test at Screening or Day 1 (or any other time point during the study)

[0240] 4. Inpatient treatment for alcohol or drug abuse within 12 months prior to the screening visit and / or positive drug or alcohol test results prior to screening or Day 1 dosing. Drug screen includes amphetamines, cocaine, opioids, and cannabinoids. Please note that the use of marijuana and cannabidiol products for medical purposes is permitted in this study, except for 24 hours prior to screening, Day 1 and EOT visits, and 4 hours prior to all other visits. Participants are excluded from the study if there is a known marijuana abuse or dependence that, in the opinion of the researcher, affects the individual's ability to comply with the protocol or may cause harm to the individual.

[0241] 5. Clinically significant hypersensitivity or allergy to any inactive ingredient contained in the Compound I drug product

[0242] 6. Display of hypotension, defined as systolic blood pressure ≤ 90 mmHg or diastolic blood pressure ≤ 60 mmHg at screening or before dosing on Day 1

[0243] 7. Hypertension, defined as systolic blood pressure > 160 mmHg or diastolic blood pressure > 100 mmHg at screening or before dosing on Day 1

[0244] 8. Orthostatic hypotension during screening or before dosing on Day 1, defined as a decrease in systolic blood pressure ≥ 20 mmHg or a decrease in diastolic blood pressure ≥ 10 mmHg after standing up from a semi-recumbent / supine position

[0245] 9. Uncontrolled diabetes, HbA1c>11% or determined by the investigator

[0246] 10. Patients with lymphoma, leukemia or any malignant tumor within the past 5 years, except for basal cell carcinoma or squamous cell carcinoma of the skin that has been resected and has no evidence of metastatic disease within 3 years.

[0247] 11. Severe gastrointestinal dysmotility (e.g., history of dyspepsia, stomach pain, nausea, vomiting, recurrent pancreatitis, constipation) determined by the investigator that may affect compliance and / or administration, absorption, and exposure of oral medications

[0248] 12. Inability to fast within 3 to 4 hours after a meal (i.e., no food or fluids; may drink water as needed)

[0249] 13. Family history of short QT syndrome or long QT syndrome

[0250] 14. ECG with clinically significant cardiac involvement or QT interval corrected by Fridericia formula (QTcF interval) > 500ms

[0251] 15. Current or past history of clinically significant cardiomyopathy and / or cardiac conduction abnormalities

[0252] 16. Have a history of platelet dysfunction, hemophilia, von Willebrand disease, coagulation disorders, other bleeding disorders, or major non-traumatic bleeding events

[0253] 17. Currently taking aspirin ≥ 325 mg / day, any P2Y12 inhibitor, any anticoagulant, phosphodiesterase 5 (PDE5) specific inhibitor, PDE5 nonspecific inhibitor (including dipyridamole and theophylline), any supplement for erectile dysfunction, riociguat, and / or any nitrate. Use of these medications is prohibited from the screening visit and throughout the study. Note: Participants taking arginine or citrulline for mitochondrial disease are eligible and allowed to continue these treatments.

[0254] 18. Participation in any study of investigational therapy for mitochondrial disease and / or mitochondrial disease symptoms within 1 month prior to Day 1

[0255] 19. Any contraindications to MRI surgery

[0256] 20. Hospitalization due to any illness, trauma, surgery or mitochondrial disease-related complications within 4 weeks before screening

[0257] 21. Unable or unwilling to comply with the study schedule, lifestyle restrictions, and assessment requirements, or not suitable for study participation based on the investigator's clinical judgment

[0258] 22. The patient or his / her legal representative / guardian (if applicable) is unable or unwilling to provide written informed consent to participate in this study.

[0259] Lifestyle restrictions

[0260] Unless otherwise stated, participants were to adhere to the following lifestyle restrictions throughout the study, from the screening visit to the follow-up visit.

[0261] Meals and dietary restrictions

[0262] Fasting (ie, no food or fluids; water is allowed as needed) for 3 to 4 hours is required prior to collection of clinical safety and PD laboratory samples at each scheduled visit (including any home visits).

[0263] Because participants may be susceptible to metabolic decompensation in any catabolic state, on the day of each visit, participants fasted upon waking (water and light snacks were permitted if approved in advance) and were then given a standardized meal with a low glycemic index in the clinic 3 to 4 hours before clinical safety and PD laboratory samples were collected. For home visits, participants either received dietary advice before the fasting period or were provided with a standardized meal by home health services.

[0264] Days of Neuroimaging (Screening, Day 1, and Day 29 Visit): After safety and PD laboratory samples were collected, participants were allowed to eat a standardized light snack; however, heavy meals must be avoided until all imaging scans were completed.

[0265] Caffeine, alcohol, and tobacco

[0266] Products containing caffeine or xanthines (e.g., coffee, tea, cola drinks, and chocolate) should not be consumed within 24 hours prior to the Screening, Day 1, and EOT visits and within 4 hours prior to all other scheduled visits. These products may affect the assessments performed in this study.

[0267] Do not consume alcohol within 24 hours before any scheduled visit. Do not use tobacco or nicotine-containing products (e.g., e-cigarettes, patches) during this study and until after the last study visit.

[0268] Medications, vitamins, supplements, and other substances

[0269] Participants should be informed that the following are Prohibited : Any medical treatment for erectile dysfunction from screening to follow-up; Use of "illegal" drugs starting 1 month before screening until follow-up; Marijuana and cannabidiol products are allowed for medical purposes, except within 24 hours before screening, Day 1 and EOT visits, and 4 hours before all other visits; Foods containing poppy seeds should be avoided throughout the study as they can cause a positive "drug" result.

[0270] Concomitant medications and procedures

[0271] Any medications received by the participant from screening until follow-up, including over-the-counter (OTC) or prescription medications, vitamins and / or herbal supplements, vaccines, or other treatments / procedures, were recorded, along with the reason for use, date of administration, route of administration, frequency, and dosage information. The following medications were prohibited or allowed with caution: specific inhibitors of PDE5, including sildenafil, tadalafil, and vardenafil; nonspecific inhibitors of PDE5, including dipyridamole and theophylline; any supplements used to treat erectile dysfunction, other sGC stimulants, including riociguat and vericipirat; nitrates, such as nitroglycerin, isosorbide mononitrate, isosorbide dinitrate, sodium nitroprusside, and amyl nitrate; aspirin ≥325 mg / day; any anticoagulant medication; any P2Y12 inhibitor, Including cangrelor, clopidogrel, prasugrel, ticagrelor, ticlopidine; starting from 1 month before screening until the follow-up period, no use of any "illicit drugs" was allowed, but the use of marijuana and cannabidiol products for medical purposes was allowed, except 24 hours before screening, 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 of these drugs; BCRP transporter inhibitors, including curcumin, cyclosporine A, and eltrombopag. Examples of BCRP substrates include azidothymidine, bisantrene, camptothecin derivatives, canertinib, cimetidine, diflutecan, flavopiridol, gefitinib, glyburide, imatinib mesylate, indolecarbazole, irinotecan, lamivudine, lapatinib, methotrexate, mitoxantrone, nilotinib, nitrofurantoin, pantoprazole, prazosin, rosuvastatin, SN 38, sulfasalazine, and topotecan.

[0272] Participants taking arginine or citrulline for treatment of mitochondrial disease were allowed to continue these treatments.

[0273] Baseline characteristics

[0274] Of the eight patients who completed the trial, five took arginine before and during the trial, and one took citrulline. Use of arginine or citrulline was more common in patients with more advanced disease.

[0275] The patients were aged between 19 and 54 years. Five were women and three were men. All of them had a history of one or more CNS symptoms, such as stroke-like episodes, seizures, or headaches. Baseline MFIS scores ranged from 3 to 66 (range 0-84). And PROMIS scores ranged from 148 to 77 (range 160 to 0). These patients had 6 to 19 elevated baseline inflammatory biomarkers.

[0276] In addition, a correlation was observed between the biomarkers of mitochondrial dysfunction GDF-15 and FGF-21 at baseline (see Figure 1 ), which further supports the evidence of disease distribution among the eight patients.

[0277] So even though the trial was done in a small number of patients, it shows the distribution of patients, from those who could be considered to have a milder manifestation of the disease to those who could be considered to have a more severe mitochondrial disease phenotype.

[0278] Baseline plasma lactate levels ranged from 1.7 to 5.6 mmol / L. Normal levels of lactate are less than 2 mmol / L. Baseline GDF-15 levels ranged from 0.49 to 4.1 ng / mL. Normal levels of this biomarker are between 0.14 and 0.46 ng / mL. Baseline FGF-21 levels ranged from 0.055 to 0.72 ng / mL. The normal range for FGF is less than 0.44 ng / mL. Thus, all participants had elevated plasma biomarkers associated with mitochondrial dysfunction at baseline.

[0279] Study Evaluation / Results

[0280] a) Safety

[0281] Compound I 15 mg QD for 29 days was well tolerated with no SAEs observed and no adverse events leading to treatment discontinuation in patients with mitochondrial disease MELAS. No safety signals from clinical laboratories, vital signs, ECGs, or suicide rating scales were obtained.

[0282] b) Pharmacokinetics

[0283] The PK profile and CNS penetration of Compound I in this patient population were similar to those observed in previous Phase 1 studies.

[0284] c) Pharmacodynamics

[0285] i) Neuroimaging

[0286] Compared with baseline (day 1), CBF increased on day 29, as shown in Figure 2As shown, a positive trend was observed throughout the study. This change was observed in all brain regions of interest. Throughout this disclosure, effect size is defined as the mean change relative to baseline of the specific parameter being measured divided by the standard deviation. Effect sizes close to 1 or -1 (positive or negative depending on the direction of movement of the specific parameter, i.e., +0.8 to +1.0 or -0.8 to -1.0) are generally considered large. Effect sizes between 0.5 and 0.8 or -0.5 to -0.8 (positive or negative depending on the direction of movement of the specific parameter) are generally considered medium. Effect sizes approaching less than 0.5 or -0.5 (positive or negative depending on the direction of movement of the specific parameter) are generally considered small.

[0287] When analyzing individual patient data, CBF increased in five of eight patients between days 1 and 29, whereas patients had lower baseline CBF, driving the overall change.

[0288] Task-free functional MRI at rest showed increased connectivity at Day 29 compared to screening and Day 1. Increased signal was observed for multiple resting-state networks, including those involved in executive function and sensorimotor processing. Task-based functional MRI (visual activation with a flickering checkerboard pattern) showed increased activation of voxels in the occipital region of Compound I at Day 29 compared to screening and Day 1 visits.

[0289] ii) Biomarkers

[0290] As As part of the group, 26 of the 40 inflammatory biomarkers assessed in plasma (65%) decreased with an effect size less than or equal to -0.3. Of the 40 inflammatory biomarkers assessed in plasma, only 3 (7.5%) increased with an effect size equal to or greater than 0.3. The changes in concentrations of inflammatory markers measured in plasma and the effect sizes are summarized in the following 3 tables (Table 1: large effect sizes; Table 2: medium effect sizes; Table 3: small effect sizes). The concentration values ​​in Tables 1-3 are measured in standard units for each of these laboratory measurements known in the art. After cessation of Compound I treatment, many of these biomarkers tended to increase again back to baseline levels, supporting the effect of the drug.

[0291] Table 1 (Changes in biomarker concentrations, biomarkers with large effect sizes)

[0292]

[0293] Table 2 (Changes in biomarker concentrations, biomarkers with medium effect sizes)

[0294]

[0295] Table 3 (Changes in biomarker concentrations, biomarkers with small effect sizes)

[0296]

[0297]

[0298] Beta-2-microglobulin (B2M) is a circulating factor that negatively regulates cognitive regeneration in the adult hippocampus in an age-dependent manner.

[0299] Compound I improved biomarkers known to be associated with MELAS. MELAS patients had higher expression of VCAM-1 (vascular cell adhesion molecule-1), ICAM (intercellular adhesion molecule), and vWF (von Willebrand factor) in endothelial cells. VCAM and ICAM are endothelial adhesion molecules that promote pathological plaque formation and may contribute to stroke-like episodes. vWF is a marker of endothelial activation and damage and is elevated in patients with significant vascular lesions. TNFR2 (tumor necrosis factor receptor) expression is higher in muscle biopsies from patients with mitochondrial respiratory chain dysfunction. The biological effects of TNF-α in mitochondrial disease are primarily mediated by TNFR2.

[0300] In this experiment, changes in CBF were closely correlated with changes in B2M, as can be seen in Table 4 below (correlations greater than or equal to 0.8 were considered extremely strong; correlations between or equal to 0.6 and 0.8 were considered strong).

[0301] Table 4. Correlation between increased ASL (CBF) changes and decreased B2M

[0302]

[0303]

[0304] Serum amyloid P component is a small glycoprotein that can be found in normal serum and in all amyloid deposits. It acts as an acute phase protein, regulates immune responses, inhibits elastase, and is considered an indicator of liver disease or neurological disorders.

[0305] Tumor necrosis factor receptor 2 (TNFR2) is expressed in myofibers with focal accumulation of abnormal mitochondria and is delivered to the mitochondria where the receptor is located.

[0306] Several mitochondrial dysfunction biomarkers in plasma were also measured. Lactate concentrations, which are known to be elevated in patients with mitochondrial disease, showed a trend toward decrease, with an effect size of -0.5. GDF-15 (a biomarker for mitochondrial disease) was also measured in plasma, showing a trend toward decrease, with an effect size of -0.5. FGF-21 (another biomarker for mitochondrial disease) was also measured in plasma, showing a trend toward decrease, with a smaller effect size of -0.2. Treatment had directional improvements, and changes in mitochondrial dysfunction biomarkers were associated with the trough plasma concentration of compound I on day 29, as shown in Table 5 below. That is, higher trough concentrations of compound I on day 29 were associated with greater reductions in mitochondrial dysfunction, as shown in Table 5 below (correlations greater than or equal to 0.8 were considered extremely strong; correlations between 0.6 and 0.8 were considered strong). In addition, on day 29, strong correlations were observed between changes in mitochondrial dysfunction biomarkers GDF-15, FGF-21, and lactate, as well as serum amyloid P components.

[0307] Lactate reductions were observed in 6 of 8 participants, ranging from 7% to 46%. GDF-15 reductions were observed in 4 of 8 participants, with the greatest reductions (up to 39%) in participants with higher baseline concentrations. Changes in these mitochondrial dysfunction biomarkers were strongly correlated with each other and also with CY6463 plasma concentrations at the end of treatment.

[0308] Table 5. Correlations between mitochondrial dysfunction biomarkers, inflammatory biomarkers, and Compound I concentrations

[0309]

[0310] iii) Subjective Assessment / Benefits

[0311] Three of the eight enrolled participants reported improvement (one participant was slightly improved, one participant was much improved, and one participant was very much improved), four participants reported no change in their condition, and one participant reported being much more severely ill. Although no study-level changes were observed on the MFIS and PROMIS assessment tools, some relevant observations could be made at the patient level. Participants who responded more positively to these benefits and had less severe disease at baseline did not improve at Day 29, whereas participants who responded less positively to these assessments and had more severe disease at baseline reported greater improvements.

[0312] Interesting correlations were also observed between CBF and clinical improvement, as assessed by the Patient Global Impression of Change (PGIC), as shown in Table 6. Correlations equal to or above 0.8 were considered very strong, and between 0.6 and 0.8 were considered strong.

[0313] Table 6. Correlation between CBF measured by PGIC and clinical improvement

[0314]

Claims

1. A method of treating a mitochondrial disease in a patient in need thereof, comprising administering to the patient a total oral daily dose of between 15 mg and 60 mg of Compound I:

2. The method of claim 1, wherein the mitochondrial disease is selected from the group consisting of: Alpers disease, autosomal dominant optic atrophy (ADOA), Barth syndrome / LIC (fatal infantile cardiomyopathy), beta-oxidation defects, long chain fatty acid transport defects, 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 syndrome, LHON, LHON Plus, MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, stroke-like symptoms), myoclonic epilepsy with shattered red fibers (MERRF), mitochondrial recessive ataxia syndrome (MIRAS), mitochondrial cytopathy, mitochondrial DNA depletion, mitochondrial encephalopathy, mitochondrial myopathy, multiple mitochondrial dysfunction syndrome, MNGIE (mitochondrial neurogastrointestinal 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.

3. The method of claim 1, wherein the mitochondrial disease is selected from the group consisting of: Alpers disease, 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 of claim 1, wherein the mitochondrial disease is a Complex I mitochondrial disease.

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

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

7. The method of any one of claims 1-6, 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 of any one of claims 1-6, wherein the patient is between 16 and 75 years old, between 16 and 70 years old, between 16 and 65 years old, between 16 and 60 years old, between 16 and 55 years old, between 16 and 50 years old, between 16 and 40 years old, or between 16 and 30 years old.

9. The method of any one of claims 1-6, 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 of any one of claims 1-6, wherein the patient is 16 years of age or younger, 12 years of age or younger, 10 years of age or younger, or 5 years of age or younger.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

31. The method of any one of claims 1 to 30, wherein treatment with Compound I results in measurable improvements in neuronal function and connectivity.

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

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

34. The method of claim 33, wherein treatment with Compound I 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, or a combination of one or more of the foregoing regions.

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

37. The method of 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 of claim 37, wherein the one or more inflammatory biomarkers are selected from the group consisting of VCAM-1, ICAM, vWF, and TNFR2.

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

40. The method of claim 39, wherein the additional therapeutic agent is selected from the group consisting of citrulline and arginine.

41. The method of claim 39, wherein the additional therapeutic agent is a mito cocktail.

42. The method of any one of claims 1 to 41, wherein the patient has been treated with one or more other therapeutic agents for treating mitochondrial disease.

43. The method of claim 42, wherein the additional therapeutic agent for treating mitochondrial disease is selected from citrulline and arginine.

44. The method of claim 42, wherein the additional therapeutic agent is a mito cocktail.

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