EP4687904A1 - Pde5 inhibitor for use in the treatment of medical conditions associated with mitochondrial complex iv deficiency - Google Patents

Abstract

The invention relates to a PDE5 inhibitor for use in the treatment and/or prevention of a medical condition associated with mitochondrial Complex IV (COX) deficiency in a human subject. In embodiments, the mitochondrial Complex IV deficiency comprises at least one mutation within a structural subunit gene, or within an assembly gene, of the mitochondrial Complex IV (COX). In embodiments, the invention relates to PDE5 inhibitors, such as sildenafil or tadalafil, in the treatment and/or prevention of a Leigh syndrome mitochondrial Complex IV deficiency type 1 to 23, or a neurological syndrome associated with a mitochondrial Complex IV deficiency. In embodiments, the invention relates to treatment of mitochondrial Complex IV deficiency caused by at least one mutation in nuclear or mitochondrial DNA, preferably DNA mutations in the SURF1 gene causing Leigh Syndrome. The invention further relates to a pharmaceutical composition comprising a PDE5 inhibitor for use in the treatment and/or prevention of a medical condition associated with mitochondrial Complex IV deficiency.

Description

PDE5 INHIBITOR FOR USE IN THE TREATMENT OF MEDICAL CONDITIONS ASSOCIATED WITH MITOCHONDRIAL COMPLEX IV DEFICIENCY

DESCRIPTION

The invention relates to the field of pharmaceutical compositions, combinations, and the treatment of medical conditions.

The invention relates to a phosphodiesterase 5 (PDE5) inhibitor for use in the treatment and/or prevention of a medical condition associated with mitochondrial Complex IV (ATP synthase) deficiency in a human subject. In preferred embodiments, the mitochondrial Complex IV deficiency comprises at least one mutation within a structural subunit gene, or within an assembly gene, of the mitochondrial Complex IV (ATP synthase).

In embodiments, the invention relates to PDE5 inhibitors, such as sildenafil or tadalafil, in the treatment and/or prevention of a Maternally Inherited Leigh syndrome (MILS), a Neuropathy, an Ataxia or a Retinitis Pigmentosa (NARP) syndrome, or a neurological syndrome associated with a mitochondrial Complex IV deficiency. In embodiments, the invention relates to treatment of mitochondrial Complex IV deficiency caused by at least one mutation in nuclear or mitochondrial DNA, preferably DNA mutations in the SURF1 gene causing Leigh Syndrome. The invention further relates to a pharmaceutical composition comprising a PDE5 inhibitor for use in the treatment and/or prevention of a medical condition associated with mitochondrial Complex IV deficiency.

BACKGROUND OF THE INVENTION

Mitochondrial Complex IV is the terminal enzyme of the respiratory chain. Mitochondrial Complex IV deficiencies also refer to Cytochrome C Oxidase (COX) deficiency that is a very rare inherited metabolic disorder characterized by deficiency of the enzyme cytochrome C oxidase (COX), or Complex IV, an essential enzyme that is active in the subcellular structures that help to regulate energy production (mitochondria). COX consists of 13 subunits, 3 of which (named COX1 , COX2 and COX3) are encoded by mitochondrial DNA. Mitochondrial Complex IV deficiency includes a lack of a Complex IV protein, a loss of a Complex IV protein, its limited function, or a complete loss of its function. Mitochondrial Complex IV deficiency can cause a variety of signs and symptoms with heterogeneous clinical manifestations, ranging from isolated myopathy to severe multisystem disease affecting several tissues and organs, especially the nervous system and heart. Features include hypertrophic cardiomyopathy, hepatomegaly and liver dysfunction, hypotonia, muscle weakness, exercise intolerance, developmental delay, delayed motor development and intellectual disability. Such disorders can be life-threatening in both infancy and later life and often leads to premature death in patients. Some affected individuals manifest a fatal hypertrophic cardiomyopathy resulting in neonatal death. A subset of patients manifest Leigh syndrome. Mitochondrial Complex IV related diseases comprise Leigh Syndrome, and mitochondrial Complex IV deficiencies, Nuclear Type 1 to Type 23. [1] Leigh syndrome is a rare (incidence <1 :2,000) and a severe developmental disorder of the nervous system, causing lactic acidosis and symmetric lesions in the central nervous system (CNS), primarily in the basal ganglia and brainstem, leading to mental retardation and muscle weakness, with peak mortality before age three. It can be based on numerous genetic defects in more than 75 genes, which can be located either on nuclear DNA (inherited according to Mendelian rules) or on mitochondrial DNA (mtDNA, maternal inheritance only). The most commonly affected mitochondrial complexes in LS are complex I and complex IV.

Numerous mitochondrial genetic defects have been described so far. However, mutations in nuclear-encoded genes have been reported as the most common cause of Complex IV deficiency. Approximately 15% of LS cases worldwide are attributed to isolated COX deficiency, with mutations in the SURF1 gene accounting for at least a third of these cases. [6] The SURF1 gene, located in genomic DNA, encodes an assembly factor of mitochondrial complex IV (COX), the terminal component of the mitochondrial respiratory chain, in the mitochondria, the body's universal source of energy. Genes located on the mitochondrial DNA comprising mitochondrial Complex IV deficiencies comprise MT-CO1 , MT-CO2 and MT-CO3. Because each cell contains numerous copies of mtDNA, a certain percentage of the mtDNA copies must be mutated (mutation load) before clinical symptoms occur. [1-3, 6]

Mitochondriopathies are among the most difficult diseases to study and to treat. The cell types affected are difficult or impossible to access for the development of a therapy. In many cases, there are no suitable animal models, as in the case of diseases caused by mutations in mitochondrial genes. Moreover, mitochondrial diseases usually affect a large number of organs, are heterogeneous in expression, and are in many cases systemic, so that research into a therapy appears to be almost hopeless. Despite some initial hope-giving results, it has not yet been possible to treat groups of patients with mitochondrial complex V (ATP synthase) deficiency, such as Leigh syndrome. The results from these studies often come from mouse studies, which either cannot be applied in humans at all or then have been shown to be ineffective there. [11 ,12] [8] For example, SD rats were infused subcutaneously with sodium azide to establish a mitochondrial deficiency animal model. The activity of mitochondrial respiratory chain complex IV (i.e. cytochrome C oxidase, COX) was decreased and was ameliorated by an PDE5 inhibitor. However, sodium azide is an unspecific inhibitor and affects beside the mitochondrial respiratory chain Complex IV also, for example, lactoperoxidases.

Other PDE5 inhibitors have been shown to be effective in adipocytes for the treatment of diabetes 2 and other related cardiovascular diseases. [10,13] The first study focussed on the effects of the phosphodiesterase type 5 inhibitor udenafil on insulin sensitivity and mitochondrial function in 3T3-L1 adipocytes. Udenafil was found to improve insulin signalling pathways by enhancing mitochondrial function, resulting in increased oxygen consumption rate (OCR) and increased expression of mitochondrial oxidative phosphorylation (OxPhos) genes. In addition, Udenafil promoted lipid metabolism by increasing beta-oxidation and could thus offer a new therapeutic approach for the treatment of type 2 diabetes and related cardiovascular diseases. The study does not offer direct therapeutic approaches for Leigh syndrome and Complex IV deficiency as it focusses on metabolic diseases such as type 2 diabetes. Another study investigated the effects of the phosphodiesterase 5 inhibitor tadalafil on the hearts of mice with type 2 diabetes. The study shows that chronic treatment with tadalafil can reverse proteomic changes in the heart associated with cytoskeletal remodelling and redox regulation. This may explain why PDE-5 inhibition is beneficial in diabetes. No specific therapeutic approaches for Complex IV mutations or Leigh syndrome are mentioned as the focus is on cardiovascular effects in diabetes. [13] Nevertheless, a specific animal model for Complex IV, and treatments having an effect on Complex IV deficient target cells in Leigh Syndrome, are missing in the prior art.

Treatments of mitochondrial disorders have been suggested as combination therapy or specifically for disorders affecting a particular organ, such as ocular disorders, or for treatment targeting only a particular part or organ, but not for syndromes or disorders or syndromes caused by mitochondrial gene mutations. [11 ,12,14,15]

Treatment of mitochondrial diseases, including those caused by mutations in mitochondrial complex IV, with soluble guanylate cyclase (sGC) stimulators has been proposed but shows no data, and despite many years, there is still no successful treatment of patients with mitochondrial complex IV deficiency. [14] Inhibitors of cGMP phosphodiesterase, among others, have been proposed to improve blood flow in the eye to treat various ocular disorders. [15]

For example, studies using the Friedreich's Ataxia mouse model describe how phosphodiesterase (PDE) inhibitors can reverse axonal dystrophy. The studies shows that PDE inhibitors such as sildenafil, rolipram and nicardipine can reduce intracellular calcium levels and improve the morphology of the mitochondrial network, leading to the reversal of axonal damage. PDE inhibitors have been proposed as a potential therapeutic treatment for Friedreich's ataxia. [11 ,12] However, for Leigh syndrome with its multiple subgroups, which is characterised by mutations in mitochondrial complex IV, the study does not offer any direct therapeutic approaches. Compared to these studies, the specific treatment of genetic mutations in complex IV itself remains an unsolved technical problem. Frataxin, mutated in Friedreich's Ataxia (FRDA), interacts with the FeS cluster-containing respiratory Complexes I, II and III, but not Complex IV and V. [11 ,12] PDE5 inhibitors have been investigated but were not effective in FRDA and therefore never reached clinical trial status. It must be noted, that neuron elongation deficiency has not been shown before for Leigh Syndrom with Complex IV deficiency. Models and treatments developed for FRDA do not fall under the treatment options for Leigh Syndrome and its sub-groups and has been shown not be affective for multiple mitochondrial conditions, including Leigh Syndrome.

ATP deficiency results in impaired function, especially of organs that have a high energy demand, i.e. the nervous system, muscles, liver, but also endocrine glands and sensory organs. Common to all subtypes is selective damage to dopaminergic neurons in the brainstem and basal ganglia, which may be particularly apparent in the context of metabolic crises.

The prior art discloses a treatment for mitochondrial DNA-associated Leigh syndrome (MILS), a previously incurable brain disease that affects 1 in 100,000 newborns. The disease is typically caused by mutations in mitochondrial DNA in the MT-ATP6 gene, which is important for ATP production. Drug discovery for MILS is difficult because access to neuronal tissue from patients is limited and mtDNA cannot be easily manipulated, making the development of cell and animal models difficult. [7, 9, 16] The CureMILS consortium utilized cellular reprogramming techniques to generate neural cells from the cells of MILS patients to identify potential therapeutic approaches. However, Leigh Syndrome can be caused by a variety of genetic defects, both in mitochondrial DNA (mtDNA) and nuclear DNA that codes for proteins playing different roles in the various complexes I to V of the mitochondrial respiratory chain. CureMILS is limited to a specific subgroup of Leigh Syndrome and does not cover all genetic variants of the syndrome. [7, 9, 16] Patients with Leigh Syndrome caused by genetic mutations other than MT-ATP6 are thus excluded from this particular research approach and remain an unresolved issue in the current state of the art, which actually states that the drug used for MILS is not effective for the Leigh Syndrome patients carrying mutations in Complex IV. [9, 16]

Patients with mitochondrial Complex IV deficiency related diseases, such as the Leigh syndrome, often die in early childhood, commonly from heart or respiratory failure. Some patients with a less severe course or late manifestation also reach teenage or early adulthood.

Currently available treatments include vitamin B, coenzyme Q or L-carnitine, and oral sodium bicarbonate or sodium citrate to treat lactic acidosis. [4] These treatments are poorly effective and the prognosis for these patients is extremely poor. None of these treatments may reverse existing and progressed symptoms. Gene therapy measures are not accessible especially for patients with mutations in mitochondrial DNA due to the high copy number of mitochondrial DNA per cell with occasional variable percentage of mutations distributed among mitochondrial DNA copies. To date, there are no therapeutic options for Leigh syndrome that influence the pathophysiological process. The prior art teaches that membrane potential is not affected in mitochondrial Complex IV deficiency.

Although numerous endeavours and studies have been made over many years, no effective treatment has yet been found for Leigh Syndrome caused by mitochondrial Complex IV deficiency. Thus, there is a critical and currently unmet need for effective and/or symptomreversing treatments for mitochondrial Complex IV deficiency-associated diseases, including MILS, NARP, Leigh syndrome, and Leigh-like syndrome.

SUMMARY

In light of the prior art, the technical problem underlying the present invention is to provide alternative or improved means for the treatment and/or prevention of a medical condition associated with mitochondrial Complex IV deficiency.

The technical problem may also be viewed as the provision of means for the treatment and/or reduction of risk and/or reversing symptoms and/or delaying progress of symptoms of medical conditions associated with a mitochondrial Complex IV deficiency, more particular Leigh syndrome, COX deficiency - benign infantile mitochondrial myopathy type; COX deficiency - severe infantile mitochondrial myopathy type; COX deficiency associated with fatal infantile cardio-encephalomyopathy; COX deficiency associated with Leigh’s syndrome; COX deficiency associated with Leigh syndrome - French-Canadian type; COX deficiency - adult form; De Toni-Fanconi-Debre syndrome, and mitochondrial Complex IV deficiency, nuclear type 1-23 .

The technical problem may also be viewed as the provision of means for the prevention and/or treatment of mitochondrial Complex IV deficiency associated increase of neuronal branching, decrease or increase in number of branching points, decrease or increase the length of neuronal branches, decrease or increase of intracellular calcium signalling and/or decrease of mitochondrial energy supply in the cell of a human subject.

The technical problem may further be viewed as the provision of means for the prevention and/or treatment and/or reversion of mitochondrial Complex IV deficiency-associated muscular hypotonia, hypertrophic cardiomyopathy, psychomotor delay, encephalopathy, peripheral neuropathy, lactic acidosis, brain stem and basal ganglia degeneration, hepatomegaly, and palliative care.

These problems are solved by the features of the independent claims. Preferred embodiments of the present invention are provided by the dependent claims.

The invention therefore relates to a phosphodiesterase 5 (PDE5) inhibitor for use in the treatment and/or prevention of a medical condition associated with mitochondrial Complex IV (Cytochrom-c-Oxidase (COX)) deficiency in a human subject.

The invention also relates to a method for treating, preventing and/or reducing the risk of a medical condition associated with mitochondrial Complex IV (Cytochrom-c-Oxidase (COX)) deficiency in a human subject comprising administering a PDE5 inhibitor to said subject.

In embodiments, mitochondrial Complex IV deficiency-associated (or related) diseases are commonly caused by or comprise at least one DNA mutation in a structural subunit gene, or in an assembly gene, of the mitochondrial Complex IV (Cytochrom-c-Oxidase (COX)).

The present invention provides means and methods for the use of PDE5 inhibitors for treatment of mitochondrial Complex IV deficiency and related diseases in a human subject. In one embodiment, for example, a method of the present invention is described for treating mitochondrial Complex IV deficiency-associated diseases in a human subject, comprising treating the patient with an effective amount of PDE5 inhibitor, or a pharmaceutically acceptable salt, derivative, or composition thereof.

In one embodiment, the human subject is a patient suffering from the mitochondrial Complex IV deficiency-related disease COX deficiency - benign infantile mitochondrial myopathy type.

In one embodiment, the human subject is a patient suffering from the mitochondrial Complex IV deficiency-related disease COX deficiency - severe infantile mitochondrial myopathy type.

In one embodiment, the human subject is a patient suffering from the mitochondrial Complex IV deficiency-related disease Leigh-Syndrome.

In one embodiment, the human subject is a patient suffering from the mitochondrial Complex IV deficiency-related disease COX deficiency associated with fatal infantile cardio- encephalomyopathy.

In one embodiment, the human subject is a patient suffering from the mitochondrial Complex IV deficiency-related disease COX deficiency associated with Leigh syndrome - French- Canadian type.

In one embodiment, the human subject is a patient suffering from the mitochondrial Complex IV deficiency-related disease COX deficiency - adult form. In one embodiment, the human subject is a patient suffering from the mitochondrial Complex IV deficiency-related disease De Toni-Fanconi-Debre syndrome.

In one embodiment, the human subject is a patient suffering from the mitochondrial Complex IV deficiency-related disease mitochondrial Complex IV deficiency, nuclear type 1-23.

In some embodiments, the present invention further provides means and methods for the use of PDE5 inhibitors for treating, preventing, reducing and/or reversing symptoms of medical conditions associated with mitochondrial Complex IV deficiency, wherein said symptoms comprise paralytic events, motor and sensory neuropathy, metabolic crises events, lactic acidosis, cardiomyopathy, seizures, encephalopathy, stroke-like episodes, endocrine abnormalities, , hepatomegaly and liver dysfunction, anaemia, exercise insufficiency, muscle weakness, loss of muscle tone and/or muscular hypotonia, respiratory insufficiency, retinitis pigmentosa, ataxia, dystonia, deafness, apnoeic episodes, ataxia, tremor, and brisk tendon reflexes, ptosis, increased susceptibility to infections, ophthalmologic abnormalities, such as nystagmus or optic atrophy, developmental delay, delayed motor development, developmental regression, and intellectual disability in a human subject compared to control.

In a preferred embodiment, the PDE5 inhibitor for use as a medicament as described herein, is characterized in that the treatment of a medical condition associated with a mitochondrial Complex IV deficiency comprises the treatment of a subject with detectable mutation in target cells compared to a control, such as healthy controls.

In one embodiment, for example, a method of the present invention for the use of PDE5 inhibitors is described for treating, preventing and/or reducing the risk of said subject for metabolic crises, palliative care or mortality, or resolves the need of the subject to receive palliative care.

The present invention provides means having a surprisingly beneficial effect on patient survival in mitochondrial Complex IV deficiency associated mortality and risk for receiving palliative care.

As evident from the experimental support provided (refer Examples), mitochondrial Complex IV deficiency causes a significantly decreased number of branching points and/or decreased length of neuronal branches and disturbed intracellular calcium levels in neuronal progenitor cells from patients suffering from Leigh Syndrome. Treatment with a PDE5 inhibitor, including tadalafil and sildenafil, increased the number of branching points and increased the length of neuronal branches and/or intracellular calcium disturbance to levels comparable to healthy controls. PDE5 inhibitors prevent cGMP degradation and neither corrects the mutation nor directly interferes with the mitochondrial Complex IV. The experimental results obtained in vitro are surprising and could not have been expected by a skilled person from prior art. Particularly, the skilled person would derive from the prior art that PDE5 inhibitors are not suitable for treating Leigh Syndrome.

In one embodiment, said treatment prevents patient suffering from mitochondrial Complex IV deficiency-related disease from palliative care and reverses existing symptoms, such as exercise insufficiency, muscle weakness, cardiomyopathy, ptosis, muscular hypotonia, respiratory insufficiency, and developmental delay. As further being evident from individual therapy trials (refer Examples), patients suffering from Leigh Syndrome have been successfully treated with the PDE5 inhibitor sildenafil. The treatment shows tremendous and unexpected therapeutic success including improvement and reversion of symptoms and episodes of Leigh Syndrome. For example, patient A, suffering from Leigh Syndrome, presented with ptosis. The ptosis has reversed after treatment.

In all individual therapy trials provided (refer to Examples), sildenafil was found generally well tolerated and with a safety profile acceptable to patients, even for long term use. Treatment, reversion, or prevention, as used herein, is most advantageous for efficacy, gentle on the patient, and is also associated with fewer side effects, if any.

In some embodiments, the subject is or has been treated additionally with one or more therapeutic drugs, i.e., biotin, riboflavin and/or CoQ10.

In some embodiments, the invention relates to a PDE5 inhibitor for use in the treatment and/or prevention of a medical condition associated with mitochondrial Complex IV deficiency, wherein said medical condition is a mitochondrial Complex IV deficiency-related disease.

In some embodiments, the invention relates to a PDE5 inhibitor for use in the treatment and/or prevention of medical condition associated with mitochondrial Complex IV deficiency, wherein the condition comprises or is caused by at least one DNA mutation within a mitochondrial Complex IV structural subunit gene or within an assembly gene of a mitochondrial Complex IV, wherein said gene is located on chromosomal DNA in the nucleus of the human subject.

In some embodiments, the invention relates to a PDE5 inhibitor for use in the treatment and/or prevention of medical condition associated with mitochondrial Complex IV deficiency, wherein the condition comprises or is caused by at least one DNA mutation within a mitochondrial Complex IV structural subunit gene or within an assembly gene of a mitochondrial Complex IV, wherein said gene is located extra chromosomally on mitochondrial DNA in the mitochondria of the human subject.

In one embodiment, the mitochondrial Complex IV deficiency related disease is selected from MILS, NARP, Leigh syndrome, and Leigh like syndrome, preferably MILS and NARP.

In one embodiment, the PDE5 inhibitor is used in the treatment and/or prevention of the medical condition associated with mitochondrial Complex IV deficiency in the human subject, wherein said human subject suffer from, Leigh syndrome, COX deficiency - benign infantile mitochondrial myopathy type; COX deficiency - severe infantile mitochondrial myopathy type; COX deficiency associated with fatal infantile cardio-encephalomyopathy; COX deficiency associated with Leigh’s syndrome; COX deficiency associated with Leigh syndrome - French- Canadian type; COX deficiency - adult form; De Toni-Fanconi-Debre syndrome, or mitochondrial Complex IV deficiency, nuclear type 1-23, preferably Leigh Syndrome.

In one embodiment, the human subject suffering from mitochondrial Complex IV deficiency has at least one mutation or two or more mutations in one gene or in at least one gene or in two or more genes, wherein said mutation(s) affect Complex IV of the mitochondrial electron transport chain.

Genes that express proteins of the mitochondrial Complex IV structural subunits and the assembly proteins of the mitochondrial Cytochrom-c-Oxidase (COX) can be easily selected from public data bases, such as OMIM, malacards.org, NCBI or Orphan.net, by a skilled person.

In some embodiments, the patient suffering from mitochondrial Complex IV deficiency has at least one mutation or two or more mutations in one gene, or in at least one gene, or in two or more genes, wherein said gene can be selected from a group of C0X14, C0X15, COX20, C0X6B1 , FARS2, FASTKD2, LRPPRC, MT-C01 , MT-C02, MT-C03, MTTL1 , MTTS1 , PET100, POLG, SC01 , SC02, SURF1 , and/or TAC01.

In one embodiment, the patient suffering from Leigh syndrome has at least one mutation or two or more mutations in one gene or in at least one gene or in two or more genes, wherein said mutation(s) affect Complex IV of the mitochondrial electron transport chain.

In another embodiment, the patient suffering from mitochondrial Complex IV deficiency, nuclear type 1-23 has at least one mutation or two or more mutations in a gene or in at least one gene or in at least two genes, wherein said mutation(s) affect Complex IV of the mitochondrial electron transport chain.

In one embodiment, the patient suffering from COX deficiency - benign infantile mitochondrial myopathy type has a mutation or at least one mutation or two or more mutations in one gene or in at least one gene or in two or more genes, wherein said mutation(s) affect Complex IV of the mitochondrial electron transport chain.

In one embodiment, the patient suffering from COX deficiency - severe infantile mitochondrial myopathy type has at least one mutation or two or more mutations in one gene or in at least one gene or in two or more genes, wherein said mutation(s) affect Complex IV of the mitochondrial electron transport chain.

In one embodiment, the patient suffering from COX deficiency associated with fatal infantile cardio-encephalomyopathy has at least one mutation or two or more mutations in one gene or in at least one gene or in two or more genes, wherein said mutation(s) affect Complex IV of the mitochondrial electron transport chain.

In one embodiment, the patient suffering from COX deficiency associated with Leigh syndrome - French-Canadian type has at least one mutation or two or more mutations in one gene or in at least one gene or in two or more genes, wherein said mutation(s) affect Complex IV of the mitochondrial electron transport chain.

In one embodiment, the patient suffering from COX deficiency - adult form has at least one mutation or two or more mutations in one gene or in at least one gene or in two or more genes, wherein said mutation(s) affect Complex IV of the mitochondrial electron transport chain.

In one embodiment, the patient suffering from De Toni-Fanconi-Debre syndrome has at least one mutation or two or more mutations in one gene or in at least one gene or in two or more genes, wherein said mutation(s) affect Complex IV of the mitochondrial electron transport chain.

In preferred embodiments, the patient suffering from Leigh Syndrome has at least one mutation or two or more mutations within the nuclear SURF1 gene. In preferred embodiments, the patient suffering from Leigh Syndrome has at least one mutation or two or more mutations within the mitochondrial MT-CO1 or MT-CO2 or MT-CO3 gene.

As described at length herein, the composition comprising a PDE5 inhibitor, represents a novel approach towards developing an effective measure to treat and/or prevent a medical condition associated with mitochondrial Complex IV deficiency, particularly in subjects suffering from Leigh Syndrome. At present, said patients are not appropriately treated and have a high mortality as no effective medication is available for the patient group described herein. As evident from the experimental support provided (refer Examples), decreased number of branching points, decreased length of neuronal branches, and disturbed intracellular calcium levels significantly correlates with a medical condition associated with mitochondrial Complex IV deficiency, including Leigh Syndrome.

Furthermore, the inventors are able to demonstrate a surprising and beneficial effect in human subjects that could not have been expected by a skilled person, e.g., therapeutic effect in Leigh Syndrome patients with mutations in the SURF1 gene, as shown in the Examples. In some cases, the means and treatment as described here were the only and final solution that allowed patients to survive. Most surprisingly, however, treatment with means and methods described herein reversed pre-existing symptoms and associated organ changes, such as ptosis, cardiomyopathy and developmental delay.

In one embodiment, a mitochondrial mutation is heteroplasmic.

In one embodiment, prior to initiating treatment of said subjects, a sample is collected prior to treatment. Said sample is preferably used for screening of mutations in said genes and/or for measuring of the Complex IV activity, length of neuronal branches, number of neuronal branching points, mitochondrial membrane potential and/or metabolic parameters (e.g. lactate and alanine in the serum) described herein in cells, blood, plasma, cerebral fluid or serum according to the standard analytics of the prior art and by means of methods described in the Examples. More details for sample handling and measurement are described in the Examples.

In some embodiments, a target cell for the treatment is a cell carrying at least one mutation causing a medical condition associated with mitochondrial Complex IV deficiency.

In embodiment, the mitochondrial gene resulting in a medical condition associated with mitochondrial Complex IV deficiency can be SURF1 , SCO1 , SCO2, COX10, COX15, COX20, COA5, LRPPRC, MT-CO1 , MT-CO2, or MT-CO3.

In one embodiment, the MT-CO1 , MT-CO2, or MT-CO3 gene mutation refers to any respective MT-CO1 , MT-CO2, or MT-CO3 gene sequence differing to the reference sequences SEQ ID No: 1 described herein.

In one embodiment, the MT-CO1 , MT-CO2, or MT-CO3 gene mutation is heteroplasmic.

In one embodiment, the SURF1 gene mutation refers to any SURF1 gene sequence differing to the reference sequence SEQ ID No: 2 described herein.

Heteroplasmic mitochondrial mutations in a mitochondrial gene occur in a specific proportion, i.e. , less than 100%, of all mitochondrial gene copies present on mitochondrial DNA copies in a patient cell. In one embodiment, within the same cell of the patient, the mitochondrial DNA carries gene copies, such as MT-CO1 gene copies or MT-CO2 gene copies or MT-CO3 gene copies, that have a mutation as well as gene copies that do not carry a mutation. In one embodiment, the specific proportion of mutated mitochondrial gene copies can be about or at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%.

The specific proportion of mutated mitochondrial MT-CO1 gene copies or MT-CO2 gene copies or MT-CO3 gene copies can be determined by methods known in the art.

In one embodiment, the PDE5 inhibitor is used for the treatment of a medical condition associated with mitochondrial Complex IV deficiency caused by at least one heteroplasmic mutation in mitochondrial DNA.

In one embodiment, a mitochondrial mutation is homoplasmic.

In one embodiment, a cell of a subject has identical copies of mitochondrial DNA at any given locus, in all mitochondria of the cell. For example, homoplasmic MT-CO1 , MT-CO2, or MT- CO3 mutations occur in all cellular copies of the mitochondrial MT-CO1 , MT-CO2, or MT-C03 gene on the mitochondrial DNA of a subject.

In one embodiment, the MT-CO1 , MT-CO2, MT-CO3 gene mutation is homoplasmic. In one embodiment, the MT-ATP8 gene mutation is homoplasmic.

In one embodiment, the mitochondrial DNA mutation in MT-CO1 , MT-CO2 or MT-CO3 is homoplasmic or heteroplasmic, wherein said heteroplasmic mitochondrial DNA mutation is present in at least 30% of mitochondria per cell, preferably 60% or more, more preferably 70% or more.

In one embodiment, Leigh Syndrome is caused by mutations within the MT-CO1 , MT-CO2, or MT-CO3 gene. Leigh Syndrome differs in the number of mutated mitochondrial DNA copy numbers in a patient cell.

In one embodiment, Leigh Syndrome is caused by mutations within the SURF1 gene.

In one embodiment, patients having at least 90% mutated MT-CO1 , MT-CO2 or MT- C03copies per cell are classified as Leigh Syndrome patients.

In one embodiment, patients having 80% or less than 80% mutated MT-CO1 , MT-CO2 or MT- CO3 copies per cell are classified as Leigh Syndrome.

In one embodiment, the mutation in the mitochondrial Complex IV structural subunit or assembly gene comprises a variant at one or more nucleic acid positions in mitochondrial DNA or the nuclear genome. These positions can be for example selected from Table 3. As provided in detail below, various mutations are known to be related to mitochondrial Complex IV deficiency, and these are provided in Table 3.

In one embodiment, the patient suffering from COX deficiency - benign infantile mitochondrial myopathy type; COX deficiency - severe infantile mitochondrial myopathy type; COX deficiency associated with fatal infantile cardio-encephalomyopathy; COX deficiency associated with Leigh’s syndrome; COX deficiency associated with Leigh syndrome - French- Canadian type; COX deficiency - adult form; De Toni-Fanconi-Debre syndrome, or mitochondrial Complex IV deficiency, nuclear type 1-23 has at least one mutation or two or more mutations in one gene or in at least one gene or in two or more genes, wherein said mutation(s) affect mitochondrial Complex IV structural or assembly genes.

In one embodiment, DNA mutations in genes causing medical condition associated with mitochondrial Complex IV deficiency can be identified by genetic screening of patients at risk for disease or presenting symptom of the disease, wherein said genes gene can be selected from a group of APOPT1 , C12ORF62, COA3, COA5, COA6, COX10, COX14, COX15, COX20, COX6B1 , FARS2, FASTKD2, LRPPRC, MT-CO1 , MT-CO2, MT-CO3, MTTL1 , MTTS1 , PET100, POLG, SCO1 , SCO2, SURF1 , and/or TACO1.

In preferred embodiments, the patient suffering from Leigh Syndrome has at least one mutation or two or more mutations within the mitochondrial MT-CO1 , MT-CO2, or MT-CO3 gene.

In preferred embodiments, the patient suffering from Leigh Syndrome has at least one mutation or two or more mutations within the nuclear SURF1 gene

The skilled person is capable of identifying mitochondrial and nuclear genes and mutations within these genes causing the Complex IV deficiency and/or Leigh syndrome. For example, publically available databases can be used, such as ClinVar database, HGMD database, OMIM database, and MitoMap for screening and verifying genes and mutations, particularly pathogenic and disease-causing mutations. A mutation can be in frame, out of frame, located within a gene, located downstream of a gene and/or located upstream of a gene.

In one embodiment the DNA mutation is a nuclear mutation and is homozygous or compound heterozygous for a structural subunit or an assembly factor of the mitochondrial Complex IV.

In one embodiment, the medical conditions associated with mitochondrial Complex IV deficiency can be caused by at least one DNA mutation on nuclear DNA located genes, wherein said genes comprise APOPT1 , C12ORF62, COA3, COA5, COA6, COX10, COX14, COX15, COX20, COX6B1 , FARS2, FASTKD2, LRPPRC, MT-CO1 , MT-CO2, MT-CO3, MTTL1 , MTTS1 , PET100, POLG, SCO1 , SCO2, SURF1 , and/or TACO1.

In one embodiment, the medical condition comprises a COX deficiency - benign infantile mitochondrial myopathy type; COX deficiency - severe infantile mitochondrial myopathy type; COX deficiency associated with fatal infantile cardio-encephalomyopathy; COX deficiency associated with Leigh’s syndrome; COX deficiency associated with Leigh syndrome - French- Canadian type; COX deficiency - adult form; De Toni-Fanconi-Debre syndrome, mitochondrial Complex IV deficiency, nuclear type 1-23or a neurological syndrome associated with a mitochondrial Complex IV deficiency.

In one embodiment, the invention comprises a method of treating a subject suffering from mitochondrial Complex IV deficiency related disease, wherein the subject has a pre-treatment plasma lactate level of greater than or equal to about 2 mmol/liter, greater than or equal to about 3 mmol/liter, greater than or equal to about 4 mmol/liter, or greater than or equal to about 5 mmol/liter. The subject may be treated with PDE5 inhibitor, i.e., sildenafil.

In one embodiment, the invention comprises a method of treating a subject suffering from mitochondrial Complex IV deficiency related disease, wherein the subject has a pre-treatment cerebrospinal fluid lactate level of greater than or equal to about 2 mmol/liter, greater than or equal to about 3 mmol/liter, greater than or equal to about 4 mmol/liter, or greater than or equal to about 5 mmol/liter. The subject may be treated with PDE5 inhibitor, i.e., sildenafil.

In one embodiment, the invention comprises a method of treating a subject suffering from Leigh syndrome or Leigh-like syndrome, wherein the subject has a pre-treatment plasma lactate level greater than or equal to about 2 mmol/liter and a cerebrospinal fluid lactate level greater than or equal to about 2 mmol/liter. The subject may be treated with PDE5 inhibitor, i.e. sildenafil.

In one embodiment, the PDE-5 inhibitor is avanafil.

Several PDE5 inhibitors are known in the prior art. In one embodiment, the compound for use in treating mitochondrial Complex IV deficiency related disease is selected from the group consisting of sildenafil, avanafil, vardenafil, tadalafil, mirodenafil, udenafil, lodenafil E-8010, Zaprinast, and E-4021.

For all compounds and methods described herein that use a PDE5 inhibitor, a derivative, salt or metabolite thereof may be used, if desired.

For all compounds and methods described herein, the invention also encompasses the use of the compounds described herein for the manufacture of a medicament for use in the treatment of mitochondrial Complex IV deficiency-related diseases.

In preferred embodiments, the compounds are administered in concentrations or amounts, or according to dosage regimes, already established in the art, such as those for which regulatory approval has been issued (e.g. by the FDA or EMA), or in doses currently being assessed during phase 2 clinical trials, and/or according to the maximum allowed dose according to a phase I trial.

The above embodiments, regarding particular amounts, are based on clinically allowed or currently trialed doses of the various compounds described herein, being combined into a pharmaceutical composition as described herein.

For employing the treatments of the present invention, the mutation in genes within a mitochondrial Complex IV structural subunit or within an assembly gene of a mitochondrial ATP synthase in a human subject are not only used to identify patients who may need this treatment, but the treatment addresses mechanisms related to disease progression, i.e. by the inhibition or reduction of endogenous cGMP degradation. The spatiotemporal dynamics of cAMP and cGMP pathways depends upon PDE activity, which by breaking phosphodiesteric bonds terminate cyclic nucleotides signaling. PDE5 is an enzyme found, for example, in smooth muscle and neurons that selectively cleaves cGMP and degrades it to 5 -GMP. PDE5 inhibitors are similar in structure to cGMP; they bind competitively to PDE5 and inhibit cGMP hydrolysis, thereby enhancing the effect of NO.

In one embodiment, the PDE5 inhibitor competitively binds to PDE5 and inhibits cGMP hydrolysis in a target cell, such as smooth muscle cells, liver, retina, skeletal muscle, and neurons. Through this inhibiting effect, PDE5 inhibitors reduce or interrupt cGMP degradation leading to increased NO concentrations by each dose administered, wherein NO promotes intracellular calcium excretion from mitochondrial striatal neurons improving the impaired calcium signaling cascade, increasing number of branching points and increasing length of neuronal branches potential. In preferred embodiments, said treatment comprises administering avanafil, a derivative or metabolite thereof at 1-500 mg/day to a human subject, preferably at 3-200 mg/day.

In preferred embodiment, avanafil, a derivative or metabolite thereof is administered to a human subject in an amount of 1 mg to 500 mg, preferably 2 mg to 400 mg, more preferably 3 mg to 300 mg, or 4 mg to 200 mg, or 5 mg to 150 mg, preferably about 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110mg,120 mg, 130 mg, 140 mg or 150 mg or 160 mg or 170 mg or 180 mg or 190 mg or 200 mg per day. In preferred embodiments, the dose of avanafil, a derivative or metabolite thereof is an oral, daily dose of between 1-500 mg, preferably between 50 mg to 200 mg.

In one embodiment, said PDE-5 inhibitor is sildenafil.

In preferred embodiments, said treatment comprises administering sildenafil, a derivative or metabolite thereof at 1-500 mg/day to a human subject, preferably at 3-200 mg/day.

In preferred embodiment, sildenafil, a derivative or metabolite thereof is administered to a human subject in an amount of 1 mg to 500 mg, preferably 2 mg to 400 mg, more preferably 3 mg to 300 mg, or 4 mg to 200 mg, or 5 mg to 150 mg, preferably about 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110mg,120 mg, 130 mg, 140 mg or 150 mg per day. In preferred embodiments, the dose of sildenafil, a derivative or metabolite thereof is an oral, daily dose of between 1-500 mg, preferably between 25 mg to 100 mg.

In one embodiment, said PDE-5 inhibitor is tadalafil.

In preferred embodiments, said treatment comprises administering tadalafil, a derivative or metabolite thereof at 1-500 mg/day to a human subject, preferably at 3-200 mg/day.

In preferred embodiment, tadalafil, a derivative or metabolite thereof is administered to a human subject in an amount of 1 mg to 500 mg, preferably 2 mg to 400 mg, more preferably 3 mg to 300 mg, or 4 mg to 200 mg, or 5 mg to 150 mg, preferably about 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110mg,120 mg, 130 mg, 140 mg or 150 mg per day. In preferred embodiments, the dose of tadalafil, a derivative or metabolite thereof is an oral, daily dose of between 1-500 mg, preferably between 5 mg to 20 mg.

In one embodiment, said PDE-5 inhibitor is selected from the group consisting of vardenafil, mirodenafil, udenafil, and lodenafil.

In one embodiment, said PDE5 inhibitor is administered: a) At a dosage of 0.1-10 mg/kg/day to a human subject, preferably at 0.5-5 mg/kg/day, more preferably at 1-2 mg/kg/day.

In one embodiment, said PDE5 inhibitor is administered: b) At a frequency of 2 to 4 times per day, preferably 4 times daily, wherein the route of administration is subcutaneously, intravenously or orally, preferably orally via a tablet of 1 mg to 10Omg per dose, more preferably 2mg to 80mg per dose, even more preferably more preferably 4mg to 40mg per dose.

In some embodiments, said treatment comprises administering PDE5 inhibitor, a derivative or metabolite thereof at 1-500 mg/day to a human subject, preferably at 3-200 mg/day. In one embodiment, PDE5 inhibitor, a derivative or metabolite thereof is administered to a human subject in an amount of 1 mg to 500 mg, preferably 2 mg to 400 mg, more preferably 3 mg to 300 mg, or 4 mg to 200 mg, or 5 mg to 150 mg, preferably about 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110mg,120 mg, 130 mg, 140 mg or 150 mg per day. In preferred embodiments, the dose of PDE5 inhibitor, a derivative or metabolite thereof is an oral, daily dose of between 1-500 mg, preferably between 5 mg to 150 mg.

In some embodiments, the treatment comprises administration of PDE5 inhibitor, a derivative or metabolite thereof 3 times daily for more than one day, more than one week, more than one month, more than one year, preferably 3 times daily lifelong, wherein the route of administration can be a tablet, chewable tablets, meltable tablets or a drinking solution, preferably via a tablet 1 to 100 mg, preferably 1 to 50 mg per dose, via oral delivery or via a gastric tube.

In some embodiments, the PDE5 inhibitor, a derivative or metabolite thereof, the solid or the liquid formulation contain 50 mg active pharmaceutical ingredient per ml solution or per tablet.

In one embodiment, for example, the PDE5 inhibitor and the method of the present invention is described for use treatment and/or prevention of mitochondrial Complex IV deficiency related diseases in the human subject by administering the PDE5 inhibitor, or a pharmaceutically acceptable salt, solvate, or composition thereof. In one embodiment, the PDE5 inhibitor may be administered orally.

The PDE5 inhibitor may be administered orally and may be administered together with one or more therapeutic drugs and/or symptomatic measures to mitigate the suffering of the patients. In one embodiment, the PDE5 inhibitor is administered together with one or more therapeutic drugs, i.e. biotin, riboflavin and/or CoQ10. In one embodiment, administration of the PDE5 inhibitor can be carried out before, after or at the same time as symptomatic measures described herein.

The invention also relates to the combined administration of a therapeutically effective amount of the PDE5 inhibitor and one or more therapeutic drugs described herein.

Another major advantage of treatment described herein, is the minimal incidence of side effects, if any.

In one embodiment, said dosage and frequency is configured to prevent and/or to reduce cGMP degradation to decrease intracellular calcium levels in striatal neuron mitochondria and/or to increase in number of branching points and/or to increase the length of neuronal branches above a level at treatment initiation.

In some embodiments, disturbed intracellular calcium levels in striatal neuron mitochondria and increased decreased the number of branching points and/or decreased length of neuronal branches can lead to a manifestation, progression and/or severe course of mitochondrial Complex IV deficiency, particularly for Leigh Syndrome.

It has proven challenging to elucidate the significance of mitochondrial Complex IV deficiency in patients since the mitochondrial membrane potential can be normal in patient-derived cells, such as blood cells and fibroblasts, and striatal neurons are not accessible from patients and these cells are not suitable to study neuronal branching. Further, PDE5 inhibitor treatment of patient-derived neuronal cells developed from iPS cells carrying a mutation in a Complex IV gene has been shown in prior art being ineffective in decreasing mitochondrial membrane potential. Therefore, so far, no compounds have been found that have a therapeutic effect on intracellular calcium levels in striatal neuron mitochondria and/or neuronal branching.

As shown in the Examples, the inventors have surprisingly succeeded in generating induced pluripotent stem cells from fibroblast cells of patients with mutations in the SURF1 gene (Leigh Syndrome patients) and differentiating them into neuronal progenitor cells. The number of neuronal branching points and length of neuronal branches is significantly decreased in these neuronal precursor cells from Leigh Syndrome patients, caused by the lack of function of the cytochrom-c-Oxidase (COX). Unexpectedly, the treatment of Leigh Syndrome patient neuronal progenitor cells in vitro with the PDE5 inhibitor, such as sildenafil, increased the number of branching points and increased the length of neuronal branches to levels comparable with controls. Measuring increased number of branching points and increased length of neuronal branches in neuronal progenitors of MILS patients, the inventors were able to demonstrate this in neuronal progenitors derived from Leigh Syndrome patients with SURF1 mutations.

In one embodiment, said treatment with PDE5 inhibitors sildenafil, avanafil or tadalafil increased number of branching points and increased length of neuronal branchesin Leigh Syndrome patients to levels comparable with controls, such as neuronal progenitor cells derived from Leigh Snydrome.

In one embodiment, said treatment is administered at a dosage and frequency configured to prevent and/or to reduce cGMP degradation to decrease intracellular calcium levels in striatal neuron mitochondria to a level of at least 5%, 10%, 20%, 30%, 40% or at least 50% below a level at treatment initiation.

In one embodiment, the physiological response of competitively binding to PDE5 can be determined by quantifying neuronal branching potential , intracellular calcium level in striated neurons, and/or lactate levels in cells, blood, plasma, serum, cerebral fluid from a mitochondrial Complex IV deficient patient, preferably neuronal progenitor cells derived from iPS transformed fibroblasts from Leigh Syndrome subject, treated with PDE5 inhibitor.

In one embodiment, the physiological response to competitively binding to PDE5 can be determined by evaluating clinical parameters of a mitochondrial Complex IV deficient patient treated with PDE5 inhibitor. In some embodiments, the evaluation of clinical parameters includes monitoring the progression of medical disease associated with mitochondrial Complex IV deficieny, such as Leigh Syndrome, and the clinical status of said patient. In some embodiments, the clinical parameters for monitoring physiological response comprise ECHO cardiography, need for oxygen/invasive ventilation, need for gastric tube, determining walking distance, regaining of abilities (such as independently sitting, moving, speaking), reaching developmental milestones, reversing cardiomyopathy, gaining muscle strength and/or muscle tone.

In one embodiment, increase the number of branching points, increase the length of neuronal branches and intracellular calcium levels are determined as described in the Examples. Other means are known to one skilled in the art.

In one embodiment, said treatment is administered at a dosage and frequency configured to prevent and/or to reduce cGMP degradation to increase the number of branching points and/or increase the length of neuronal branches to a level of at least 5%, 10%, 20%, 30%, 40% or of at least 50% difference to a level at treatment initiation. Increase mitochondrial oxygen consumption by a level of at least 5%, 10%, 20%, 30%, 40% or at least 50% above the level at treatment initiation.

Increase mitochondrial ATP production by a level of at least 5%, 10%, 20%, 30%, 40% or at least 50% above the level at treatment initiation.

In one embodiment, the preferred administered dosage and frequency of PDE5 inhibitor (e.g. 20 mg per dose and 60 mg per day) has an inhibitory effect on PDE5 and reducing cGMP degradation, wherein said dosage increases the number of branching points, increases the length of neuronal branches before treatment initiation, preferably increases the number of branching points and/or length of neuronal branches to at least 20% above the levels before treatment initiation, and/or wherein said dosage reduces the level of intracellular calcium levels in striatal neuron mitochondria, lower than before treatment initiation, preferably a level of intracellular calcium levels in striatal neuron mitochondria at least 20% lower than before treatment initiation.

A skilled person is capable, based on the experimental and written support provided herein, to carry out the invention, in combination with common knowledge in the art.

By employing the quantitative measures described in the examples, or by using alternative quantitative means for determining increase in number of neuronal branching point and/or increase in length of neuronal branches in levels of neuronal branching potential and/or intracellular calcium levels (i.e. in striatal neuron mitochondria, neuronal progenitors), the PDE5 inhibitor and their necessary doses can be determined and adjusted in order to obtain beneficial effects due to the inventive application of the composition.

In one embodiment, the effect of said PDE5 inhibitor to said subjects comprises:

Preventing and/or inhibiting cGMP degradation and increasing the number of branching points, increasing the length of neuronal branches to levels comparable to healthy subjects,

Reducing the number of paralytic events, motor and sensory neuropathy, metabolic crises events, lactic acidosis, cardiomyopathy, seizures, encephalopathy, stroke-like episodes, endocrine abnormalities, , hepatomegaly and liver dysfunction, anemia, exercise insufficiency, muscle weakness, loss of muscle tone and/or muscular hypotonia, respiratory insufficiency, retinitis pigmentosa, ataxia, dystonia, deafness, apneic episodes, ataxia, tremor, and brisk tendon reflexes, ptosis, increased susceptibility to infections, ophthalmologic abnormalities, such as nystagmus or optic atrophy, developmental delay, delayed motor development, developmental regression, and intellectual disability compared to treatment initiation, and/or

Preventing and/or reducing the risk of said subject for repeated metabolic crises, palliative care or mortality, or resolves the need of the subject to receive palliative care.

As described in Example 4, the treatment of a Leigh Syndrome patient carrying mutations in the SURF1 gene using an PDE5 inhibitor is characterized by an excellent tolerability profile, as evidenced by the complete absence of observable side effects during the administration period. A further technical advantage of the treatment is its ability to maintain clinical and neurological stability over an extended period, specifically demonstrated during a continuous treatment duration of several months. The treatment surprisingly provides a significant improvement in developmental milestones, which includes but is not limited to, (i) enhancement in linguistic capabilities, (ii) advancement in communicative constructs, (iii) progression in motor skills, notably the subject's unexpected newfound capacity to ambulate independently for a few steps without support, (iv) extended mobility achievements, as observed by the subject's surprising ability to walk longer distances with assistance.

An additional unexpected technical effect of the treatment is its contribution to maintaining metabolic stability even during episodes of several and even severe infections, conditions typically associated with metabolic crisis in Leigh Syndrome.

These technical advantages and effects are non-obvious and surprising to the person skilled in the art that have not been previously disclosed or anticipated in the prior art.

In one embodiment, the effect of said PDE5 inhibitor to said subjects comprises improvement in developmental milestones.

In one embodiment, the effect of said PDE5 inhibitor to said subjects comprises one or more of enhanced linguistic capabilities, improved in communicative constructs, progression in motor skills, extended mobility achievements, and metabolic stability even during episodes of infections.

In one embodiment, abnormal neuronal branching potential and intracellular calcium levels in striatal neuron mitochondria may be effectively treated by the administration of from about 1 mg to about 500 mg, preferably 2 mg to about 400 mg, more preferably 3 mg to about 100 mg with PDE5 inhibitor per patient per day.

In one embodiment, paralytic events, motor and sensory neuropathy, metabolic crises events, lactic acidosis, cardiomyopathy, seizures, encephalopathy, stroke-like episodes, endocrine abnormalities, , hepatomegaly and liver dysfunction, anaemia, exercise insufficiency, muscle weakness, loss of muscle tone and/or muscular hypotonia, respiratory insufficiency, retinitis pigmentosa, ataxia, dystonia, deafness, apnoeic episodes, ataxia, tremor, and brisk tendon reflexes, ptosis, increased susceptibility to infections, ophthalmologic abnormalities, such as nystagmus or optic atrophy, developmental delay, delayed motor development, developmental regression, intellectual disability, may be effectively treated by the administration of from about 1 mg to about 500 mg, preferably 2 mg to about 400 mg, more preferably 3 mg to about 100 mg with PDE5 inhibitor per patient per day.

In one embodiment, repeated metabolic crises, palliative care or mortality, or resolves the need of the subject to receive palliative care may be effectively treated by the administration of from about 1 mg to about 500 mg, preferably 2 mg to about 400 mg, more preferably 3 mg to about 100 mg with PDE5 inhibitor per patient per day.

The invention further relates to a pharmaceutical composition comprising a PDE5 inhibitor for use as a medicament in the treatment and/or prevention of a medical condition as described herein, wherein said composition is in admixture with a pharmaceutically acceptable carrier and/or formulated in a pharmaceutically buffered solution. According to embodiments of the present invention, a composition comprising PDE5 inhibitor or salt thereof as well as a drug product comprising said composition have a therapeutically relevant effect on neuronal branching potential, intracellular calcium signaling, mitochondrial energy supply in cells of a subject, muscular hypotonia, hypertrophic cardiomyopathy, psychomotor delay, encephalopathy, peripheral neuropathy, lactic acidosis, brain stem degeneration, hepatomegaly, feeding ability, developmental delay, walking distance, walking speed, demyelinating neuropathy, urinary incontinence, chronic metabolic acidosis, exercise- induced insufficiency, metabolic crisis, cerebral seizures, respiratory failure, lifespan and survival of a subject.

Single preparations, combinational preparations or compositions are known to the skilled person who is able to evaluate compatible carrier materials and formulation forms suitable for both active compounds in the combination. In some embodiments, the quantity of active ingredient that can be combined with the carrier materials to produce a single dosage varies depending on the patient being treated and the particular route of administration.

Further examples of pharmaceutical compositions, pharmaceutical combinations, treatments, subjects, target genes, and targets are described in detail below.

The features of the invention regarding methods of treatment and diagnosis, and descriptions of the composition comprising PDE5 inhibitors, particularly sildenafil and tadalfil, for use in the treatment of various medical conditions, apply to the composition itself, and vice versa.

The present invention includes multiple aspects, features, and embodiments, wherein these multiple aspects, features, and embodiments may be combined and permuted in any desired manner. These and other aspects, features, and embodiments of the present invention will become apparent by reference to the remainder of this application, including the following detailed description. In addition, various references are provided herein that describe specific compositions and/or methods in greater detail; all such references are incorporated herein by reference in their entirety.

DETAILED DESCRIPTION

The present invention addresses the problem of inadequate pharmacological treatments for genetically caused mitochondrial Complex IV deficiency related disorder or a Leigh Syndrome. Current treatments are ineffective or can only slightly alleviate some symptoms and often resulting in palliative care being the last option for these patients. The present invention therefore relates to the treatment or prevention of a mitochondrial Complex IV deficiency related disorder or a Leigh Syndrome in a human subject.

All words and terms used herein shall have the same meaning commonly given to them by the person skilled in the art, unless the context indicates a different meaning. All terms used in the singular shall include the plural of that term and vice versa.

In accordance with the present disclosure, there is provided herein a composition comprising a PDE5 inhibitor, preferably sildenafil or tadalafil, and in the form of a medicament, having an effect on mitochondrial Complex IV gene mutation associated medical conditions and in particular mitochondrial Complex IV deficiency associated medical conditions, such as Leigh syndrome; COX deficiency - benign infantile mitochondrial myopathy type; COX deficiency - severe infantile mitochondrial myopathy type; COX deficiency associated with fatal infantile cardio-encephalomyopathy; COX deficiency associated with Leigh’s syndrome; COX deficiency associated with Leigh syndrome - French-Canadian type; COX deficiency - adult form; or De Toni-Fanconi-Debre syndrome. It has proven difficult in the art to identify treatments of said medical condition that have a therapeutic effect on neuronal branching potential and intracellular calcium levels in striated neurons.

If such an effect is possible, it may help delay or stop disease progression and possibly reverse an associated symptom, risk for symptom or medical condition, such as a cardiomyopathy, repeated metabolic crises, and symptoms, including paralytic events, motor and sensory neuropathy, metabolic crises events, lactic acidosis, cardiomyopathy, seizures, encephalopathy, stroke-like episodes, endocrine abnormalities, anemia, exercise insufficiency, muscle weakness, loss of muscle tone and/or muscular hypotonia, respiratory insufficiency, Retinitis pigmentosa, ataxia, ptosis, increased susceptibility to infections, developmental delay, and/or intellectual disability and even enabling and prolong said patients survival. Such an effect is found herein for a composition according to the disclosure, which is further described below.

Mitochondrial respiratory chain and oxygen reduction to water by Complex IV

A basic mechanism responsible for cellular ATP production is mitochondrial respiration, which generates ATP by utilizing the energy released during the oxidation from food. ATP is used in turn as the primary energy source for most biochemical and physiological processes, such as growth, movement and homeostasis The transfer of electrons by the specific components of the electron transport chain also leads to the transfer of protons from the mitochondrial matrix into the intermembrane space, creating a proton gradient. This proton gradient is then used to boost the production of metabolic energy in the form of adenosine triphosphate (ATP). ATP production by oxidative phosphorylation in functionally respiring mitochondria is associated with the re-oxidation of NADH via the activity of the electron transport system. In cases where the mitochondrial electron transport chain is impaired (as in mitochondrial diseases), there is thought to be a decrease in ATP production and, under certain conditions, a concomitant accumulation of NADH.

This bioenergetic metabolic pathway consists of five enzyme complexes: NADH:CoQ oxidoreductase (complex I, also referred to as NADH dehydrogenase), succinate:CoQ oxidoreductase (complex II), CoQ:cytochrome c oxidoreductase (complex III, also referred to as cytochrome b-c1 complex), cytochrome c oxidase (complex IV, also referred to as COX), and H+-ATPase (Complex V, also referred to as F0F1-ATP synthetase or simply ATP synthase). Both the nuclear and mitochondrial genomes are required for the assembly of oxidative phosphorylation enzyme complexes I, III, IV, and V, whereas complex II is exclusively nuclear-encoded.

As mentioned, the respiratory enzymes, complexes I, III, and IV, pump protons out of the inner mitochondrial matrix, building up proton pressure outside the "dam" (i.e. , membrane). Complex V uses the energy of the proton flow into the matrix to synthesize ATP.

The enzyme cytochrome c oxidase or complex IV (formerly EC 1 .9.3.1 , now reclassified as translocase EC 7.1.1.9) is a large transmembrane protein complex found in bacteria, archaea and mitochondria of eukaryotes. It is the last enzyme in the respiratory electron transport chain of cells and is located in the membrane. It accepts one electron from each of the four cytochrome c molecules and transfers it to an oxygen molecule and four protons, forming two water molecules. In addition to binding the four protons from the inner aqueous phase, it transports another four protons across the membrane, increasing the transmembrane difference in the electrochemical potential of the protons, which ATP synthase then uses to synthesize ATP.

Various mitochondrial disorders result from partial dysfunction of mitochondrial oxidative phosphorylation. Respiratory chain disorders include complex I: NADH dehydrogenase deficiency (NADH-CoQ reductase), complex II: succinate dehydrogenase deficiency, complex III: ubiquinone cytochrome c oxidoreductase deficiency, complex IV: cytochrome c oxidase (COX) deficiency, and Complex V: ATP synthase deficiency. See, e.g., Smeitink, J. A., "Mitochondrial disorders: clinical presentation and diagnostic dilemmas," J. Inherit. Metab. Dis. 2003; 26(2-3):199-207.

The complex is a large integral membrane protein comprisingmultiple metalloprotein sites and 14 protein subunits in mammals, wherein eleven subunits are of nuclear origin, and three are synthesized in mitochondria. The complex contains two heme, a cytochrome a and a cytochrome a3, and two copper centers, the CuA and CuB centers. The cytochrome a3 and CuB form a binuclear center that performs oxygen reduction. Cytochrome c, which is reduced by the preceding component of the respiratory chain (cytochrome bc1 complex, complex III), docks near the binuclear CuA center and donates an electron to it, oxidizing it back to cytochrome c, which contains Fe3+. The reduced binuclear CuA center now donates an electron to cytochrome a, which in turn donates an electron to the binuclear cytochrome a3>- CuB center. The two metal ions in this binuclear center are 4.5 A apart and coordinate a hydroxide ion in the fully oxidized state.

Cytochrome c oxidase comprises an unusual posttranslational modification linking C6 of Tyr(244) and the E-N of His(240) (bovine enzyme numbering), having a role in allowing the binuclear center of cytochrome a3-CuB to accept four electrons in the reduction of molecular oxygen and four protons to water. Rapid four-electron reduction involves immediate cleavage of the oxygen-oxygen bond and avoids an intermediate that could lead to superoxide formation.

COX assembly is not fully elucidated because the hydrophobic subunits that form the holoenzyme complex aggregate rapidly and irreversibly, and mutant subunits with exposed hydrophobic domains also aggregate. The COX subunits are encoded in both the nuclear and mitochondrial genomes. The three subunits that form the COX catalytic core are encoded in the mitochondrial genome.

Table 1 below comprises genes encoding proteins within Complex IV.

Table 1 : Genes encoding proteins within Complex IV, with database entry numbers for UniProt (https://www.uniprot.org/) and Online Mendelian Inheritance in Man (OMIM, h ftps ://www. o m i m . o rg/) .

Genes listed in the Table above are part of the mitochondrial Complex IV having a structural, transporting and/or enzymatic function within said Complex IV. In one embodiment, mitochondrial Complex IV deficiency may be caused by an increased enzymatic activity, decreased enzymatic activity, absent enzymatic activity, an increased transporting activity, decreased transporting activity, absent transporting activity, structural changes of the mitochondrial Complex IV, absence of mitochondrial Complex IV, and/or absence of one or more proteins within mitochondrial Complex IV.

In one embodiment, genes carrying a mitochondrial Complex IV deficiency causing sequence variant may be selected from APOPT1 , C12ORF62, COA3, COA5, COA6, COX10, COX14, COX15, COX20, COX6B1 , FARS2, FASTKD2, LRPPRC, MT-CO1 , MT-CO2, MT-CO3, MTTL1 , MTTS1 , PET100, POLG, SCO1 , SCO2, SURF1 , and/or TACO1 , wherein one or more genes are carrying at least one mutation within said gene. In one embodiment, one or more mutations may be located outside of at least one of said genes that affects an expression regulatory element of said gene.

There are three conformational states in which COX can occur: (1) fully oxidized (pulsed), (2) partially reduced, and (3) fully reduced. Each inhibitor has a high affinity for a different state. In the pulsed state, both the heme a3 and the CuB core centers are oxidized; this is the conformation of the enzyme that has the highest activity. Reduction with two electrons initiates a conformational change that allows oxygen to bind to the partially reduced enzyme at the active site. Four electrons bind to COX to fully reduce the enzyme. The fully reduced state of the enzyme, consisting of a reduced Fe2+ at the cytochrome a3 heme group and a reduced CuB+ binuclear center, is considered the inactive or resting state of the enzyme.

Nucleic acid

The term "nucleic acid" as used herein refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in single- or double-stranded form, and includes DNA, RNA, and hybrids thereof. DNA may be in the form of, for example, antisense molecules, RNA-DNA duplexes, a PCR product, chimeric sequences, derivatives, and combinations of these groups. RNA can be in the form of small interfering RNA (siRNA), dicer substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), mRNA and combinations thereof. DNA and RNA can form DNA-RNA hybrids. As used herein, the terms "polynucleotide" or "nucleic acid molecule" refers to messenger RNA (mRNA), RNA, genomic RNA (gRNA), plus strand RNA (RNA(+)), minus strand RNA (RNA(-)), genomic DNA (gDNA), complementary DNA (cDNA) or recombinant DNA. Polynucleotides include single and double stranded polynucleotides. Preferably, polynucleotides of the invention include polynucleotides or variants having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to any of the reference sequences described herein, typically where the variant maintains at least one biological activity of the reference sequence.

A "gene" refers to a DNA region that encodes a gene product including regions regulating the production of the gene product, regardless of whether these sequences are adjacent to coding and/or transcribed sequences. A gene comprises, but is not limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites, and locus control regions.

“Gene expression" is the conversion of the information encoded by a gene into a gene product. A gene product can be the direct transcription product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA or any other type of RNA) or a protein produced by translation of a mRNA. Gene products also include RNAs modified by processes such as capping, polyadenylation, methylation, and editing, as well as proteins modified by processes such as methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristoylation, and glycosylation. Gene expression is regulated by “expression regulatory element” or “regulatory sequence”.

An “expression regulatory element” or “regulatory sequence”, as used herein, refer to a segment of a nucleic acid molecule that is capable of increasing or decreasing the expression of certain genes in a cell of an organism. Expression regulatory element are known by a skilled person and comprise, for example, promoter, enhancer, silencer, poly-adenylation signal, histone binding sequences, and CpG islands. “Increased expression” of a certain gene refers to a number of protein molecules expressed from said gene being higher in the cell as compared to without elevated gene expression. “Decreased expression” of a certain gene refers to a number of protein molecules expressed from said gene being lower in the cell as compared to without lowered gene expression.

Sequence Variation:

“Sequence variant” or “gene mutation” “or “mutation”, as used herein, refers to a change in the nucleotide sequence of the genome of an organism, a virus, mitochondrial DNA, and/or extrachromosomal DNA. Mutations result from errors during DNA or viral replication, mitosis, or meiosis, or from other types of DNA damage (e.g., pyrimidine dimers caused by ultraviolet radiation), which can then undergo error-prone repair (especially microhomology-mediated end joining), cause an error in other forms of repair, or cause an error during replication (translesion synthesis). Mutations can also result from insertion or deletion of DNA segments due to mobile genetic elements.

Mutations can result in detectable changes in the observable characteristics (phenotype) of an organism. Mutations in genes can also have no effect, alter the product of a gene, or prevent the gene from functioning properly or completely. They can also occur in non-genetic regions. “Pathogenic” sequence variants refer to disease causing gene mutations. A skilled person knows how to find and identify a sequence variant, in particular the pathogenic sequence variant.

As used herein, a “SURF1 gene mutation” refers to any SURF1 gene sequence differing to the reference (wt) sequences SEQ ID No: 2 described herein or known to a skilled person.

Homoplasmic or heteroplasmic

“Homoplasmic” as used herein, describes a eukaryotic cell having all identical copies of mitochondrial DNA. In particular, homoplasmic MT-CO1 or MT-CO2 or MT-CO3- mutations occur in all cellular copies of the mitochondrial MT-CO1 or MT-CO2 or MT-CO3 gene on the mitochondrial DNA of a subjects. “Heteroplasmic” as used herein, describes a eukaryotic cell with copies of mitochondrial DNA varying in their nucleic acid sequence. As an example, an MT-CO1 or MT-CO2 or MT-CO3gene mutation, which is heteroplasmic, occurs in a specific proportion of all mitochondrial MT-CO1 or MT-CO2 or MT-CO3 gene copies present on mitochondrial DNA in an individual's cell. Meaning that in the same cell of a subject mitochondrial DNA with MT-CO1 or MT-CO2 or MT-CO3 gene copies that have a mutation are present as well as MT-CO1 or MT-CO2 or MT-CO3 gene copies that do not carry a mutation. The specific proportion of mutated mitochondrial MT-CO1 or MT-CO2 or MT-CO3 gene copies can be 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%.

“MT-CO1 gene mutation” refers to any MT-CO1 gene sequence differing to the reference (wt) sequences SEQ ID No: 1 described herein, or known to a skilled person.

“MT-CO2 gene mutation” refers to any MT-CO2 gene sequence differing to the reference (wt) sequences SEQ ID No: 1 described herein, or known to a skilled person.

“MT-CO3 gene mutation” refers to any MT-CO3 gene sequence differing to the reference (wt) sequences SEQ ID No: 1 described herein, or known to a skilled person.

Upstream/ downstream

“Upstream” and “downstream” as used herein, are employed in the context of the 5'— >3' direction of a nucleic acid strand. Upstream refers to nucleotides towards the 5' end and downstream towards the 3' end of any given nucleic acid.

In-frame, out-of-frame

A fusion product is defined as ‘in-frame’ when an open reading frame remains intact in the 3 - region downstream of the nucleic acid integration site, regardless of the number of inserted or deleted amino acids at the fusion junction point. A shift in the open reading frame of a protein coding gene sequence refers to “out-of-frame”.

Polypeptides

"Peptide" "polypeptide", "polypeptide fragment" and "protein" are used interchangeably, unless specified to the contrary, and according to conventional meaning, i.e., as a sequence of amino acids. Polypeptides are not limited to a specific length, e.g., they may comprise a full-length protein sequence or a fragment of a full-length protein, and may include post-translational modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like, as well as other modifications known in the art, both naturally occurring and non- naturally occurring.

Medical Indications

Genetic mutations that alter the function or structure of cytochrome c oxidase (COX) can lead to severe, often fatal metabolic disorders. Such disorders usually occur in early childhood and primarily affect tissues with high energy demands (brain, heart, muscles). Among the numerous classified mitochondrial diseases, mitochondrial diseases in which COX assembly is disturbed are considered the most severe.

The vast majority of COX disorders are associated with mutations in nuclear-encoded proteins called assembly factors or assembly proteins. These assembly factors contribute to COX structure and functionality and are involved in several essential processes, including transcription and translation of mitochondrion-encoded subunits, preprotein processing and membrane insertion, and cofactor biosynthesis and insertion.

Currently, mutations have been mainly identified in seven COX assembly factors: SURF1 , SCO1 , SCO2, COX10, C0X15, COX20, C0A5, and LRPPRC as well as MT-CO1 , MT-CO2, MT-CO3. Mutations in these proteins can lead to altered functionality of subcomplex assembly, copper transport, or translational regulation. Each gene mutation is associated with the etiology of a particular disease, with some mutations playing a role in multiple diseases. Diseases in which COX assembly is disrupted by gene mutations include Leigh syndrome, cardiomyopathy, leukodystrophy, anemia, and sensorineural deafness.

In one aspect of the present invention there is provided a pharmaceutical composition according to the invention as herein described for use in the treatment of a medical condition associated with mitochondrial Complex IV deficiency. Such deficiency may be caused by a gene mutation, wherein the medical condition associated with mitochondrial Complex IV deficiency is preferably a Leigh syndrome, COX deficiency - benign infantile mitochondrial myopathy type; COX deficiency - severe infantile mitochondrial myopathy type; COX deficiency associated with fatal infantile cardio-encephalomyopathy; COX deficiency associated with Leigh’s syndrome; COX deficiency associated with Leigh syndrome - French- Canadian type; COX deficiency - adult form; De Toni-Fanconi-Debre syndrome or mitochondrial Complex IV deficiency, nuclear type 1-23. The syndromes are defined by their symptoms and/or gene mutation.

In humans, gene mutations in genes of the mitochondrial Complex IV can cause mitochondrial Complex IV deficiency that can be mild, progressive, or can be lethal.

“Mitochondrial Complex IV deficiency” or “COX deficiency” refers to a lack of mitochondrial Complex IV or a loss or reduction of its function compared to normal, wt, healthy levels. Mitochondrial Complex IV deficiency associated diseases comprise COX deficiency - benign infantile mitochondrial myopathy type; COX deficiency - severe infantile mitochondrial myopathy type; COX deficiency associated with fatal infantile cardio-encephalomyopathy; COX deficiency associated with Leigh’s syndrome; COX deficiency associated with Leigh syndrome - French-Canadian type; COX deficiency - adult form; or De Toni-Fanconi-Debre syndrome and mitochondrial Complex IV deficiency, nuclear type 1-23. Complex IV is the last enzyme in the respiratory electron transport chain of cells located in the membrane. It receives an electron from each of four cytochrome c molecules and transfers them to one oxygen molecule and four protons, producing two molecules of water. Disease gene mutations causing mitochondrial Complex IV deficiency impair Complex IV formation or function. As a result, Complex IV activity is reduced and mitochondrial electron transfer system with reducing oxygen to water is impaired. This particularly affects cells, tissues, and organs having a high energy demand, such as skeletal muscles, the brain, or the heart. Genes involved in mitochondrial Complex IV deficiency are located on the nuclear DNA, such as SURF1 , SCO1 , SCO2, COX10, COX15, COX20, COA5 and LRPPRC. Depending on location and/or affected gene, various mutations may lead to differences in disease severity, syndromes, signs and symptoms of the disease, and the risk of exitus lethalis.

One of the most frequently mutated nuclear genes in Leigh Syndrome is the SURF1 gene (Surfeit locus protein 1). The SURF1 gene contains 9 exons, is located on chromosome 9q34, and encodes a protein that is highly conserved in eukaryotes and prokaryotes. The SURF1 protein is located in the inner membrane of mitochondria and is involved in the assembly of complex IV, i.e. cytochrome c oxidase (COX). SURF1 appears to be particularly important for the assembly of the mtDNA-encoded proteins MT-CO1 , MT-CO2, and MT-CO3, which form the catalytic core of COX. If the stability of this COX active site is compromised, the entire COX assembly may be at risk. Accordingly, SURF1 mutations lead to defective COX assembly, the absence of SURF1 leads to severe COX deficiency.

In humans, mitochondrial Complex IV deficiency can cause a variety of signs and symptoms affecting many organs and systems of the body, especially the nervous system and the heart. The disorder can occur in infancy or early childhood and can also be life-threatening. It is usually characterized by rapidly progressive neurodegeneration. Affected individuals may have problems with feeding, slow growth, low muscle tone (hypotonia), extreme fatigue (lethargy), encephalopathy with loss of motor and cognitive skills, hypotonia, failure to thrive, loss of the ability to sit or walk, poor communication, poor eye contact, hypotonia, ataxia, dystonia, and ophthalmologic abnormalities, such as nystagmus or optic atrophy, developmental regression and/or developmental delays. Other features may include oculomotor abnormalities, including slow saccades, strabismus, ophthalmoplegia, and nystagmus, as well as deafness, apnoeic episodes, ataxia, tremor, and brisk tendon reflexes. Brain imaging shows bilateral symmetric lesions in the basal ganglia. Affected individuals usually present increased serum, CSF pyruvate, and lactate levels and decreased levels and activity of mitochondrial respiratory complex IV. They tend to have elevated levels of lactic acid in the blood (lactic acidosis), which can cause nausea, vomiting, weakness, and rapid breathing. Affected individuals may also experience high levels of ammonia in the blood (hyperammonemia), leading in some cases to impaired brain function (encephalopathy) and damage to other organs.

Features in mitochondrial complex IV deficiency related diseases and Leigh Syndrome comprise hypertrophic cardiomyopathy, hepatomegaly and liver dysfunction, hypotonia, muscle weakness, exercise intolerance, developmental delay, delayed motor development and intellectual disability.

Hypertrophic cardiomyopathy is also a common feature of mitochondrial Complex IV deficiency and is defined as the thickening (hypertrophy) of the heart muscle, potentially leading to heart failure. People with mitochondrial Complex IV deficiency may also have a characteristic pattern of facial features, including a high forehead, arched eyebrows, downturned outer corners of the eyes (downturned palpebral fissures), a prominent bridge of the nose, low-set ears, thin lips, and a small chin (micrognathia).

Patients with mitochondrial Complex IV deficiency have specific groups of signs and symptoms that are classified as a specific syndrome, such as COX deficiency - benign infantile mitochondrial myopathy type; COX deficiency - severe infantile mitochondrial myopathy type; COX deficiency associated with fatal infantile cardio-encephalomyopathy; COX deficiency associated with Leigh’s syndrome; COX deficiency associated with Leigh syndrome - French-Canadian type; COX deficiency - adult form; or De Toni-Fanconi-Debre syndrome, mitochondrial Complex IV deficiency, nuclear type 1-23.

In one embodiment, the mitochondrial Complex IV deficiency comprise Leigh syndrome, COX deficiency - benign infantile mitochondrial myopathy type; COX deficiency - severe infantile mitochondrial myopathy type; COX deficiency associated with fatal infantile cardio- encephalomyopathy; COX deficiency associated with Leigh’s syndrome; COX deficiency associated with Leigh syndrome - French-Canadian type; COX deficiency - adult form; or De Toni-Fanconi-Debre syndrome, mitochondrial Complex IV deficiency, nuclear type 1-23. In one embodiment, pharmaceutical composition for use in the treatment of a medical condition associated with mitochondrial Complex IV deficiency comprise treatment with a PDE5 inhibitor.

Neuronal branching

The term “Neuronal branching” refers to a coordinated process involving dramatic plasma membrane expansion and dynamic cytoskeletal reorganization of neuronal cells. The process requires extensive reorganization of actin and microtubule (MT) filaments and expansion of the plasma membrane. These fundamental events are controlled in a spatially and temporally coordinated manner by numerous signaling pathways and mechanical processes. Mature neuronal circuits exhibit an astonishing density of synapses between axons and dendrites or between neurons and effector cells. The ability of a single axon to form multiple synapses is facilitated by axonal branching, which occurs by branching the tip of the propagating axon or by collateral branching from the axon shaft. Dendritic branching increases synaptic capacity, while being essential for sensory perception. Patterns of dendritic branching differ markedly between neuron types and are determined by the number and type of synaptic or sensory inputs received and by the geometry and size of receptive fields. The widely branching phenotypes of neurons were described in detail by Santiago Ramon y Cajal in the 19th century. Failure of proper branching patterns and circuits leads to various neurodevelopmental disorders and neuro psychiatric diseases. The number of branch points and the length of the branches are decisive. The skilled person in the art knows how to detect and analyse neuronal branching.

Guideline on Mitochondrial DNA-Associated Leigh Syndrome and NARP

A guideline is known to be constantly modified and adapted to the current state of medical and scientific knowledge. The guideline for mitochondrial DNA-Associated Leigh Syndrome and NARP covers the entire patient care setting comprising clinical characteristics, diagnostics, current recommendations on therapy management and palliative treatment as well as genetic counselling. (Thorburn DR, Rahman J, Rahman S. Mitochondrial DNA-Associated Leigh Syndrome and NARP. 2003 Oct 30 [Updated 2017 Sep 28], In: Adam MP, Ardinger HH, Pagon RA, et al., editors. GeneReviews® [Internet], Seattle (WA): University of Washington, Seattle; 1993-2021. Available from: https://www.ncbi.nlm.nih.gov/books/NBK1173/)

Leigh Syndrome

Certain forms of Leigh syndrome may be caused by mitochondrial Complex IV deficiency. "Leigh syndrome" is defined by characteristic changes in the brain (necrotizing encephalitis), particularly affecting the basal ganglia and brainstem, which are visualized as T2 signalintense areas on MRI. The disease frequently manifests in the first year of life, usually associated with a viral or bacterial infection. However, later ages of disease onset are also possible. Clinical symptoms of Leigh syndrome comprise muscle weakness, mental and motor developmental impairment, cerebral seizures, circumscribed paralysis, involuntary movements (dystonia, tremor, and chorea), disturbances in nerve function (neuropathy), and thickening of the heart muscle (hypertrophic cardiomyopathy). It is a progressive disease and 50% of affected children die before reaching the age of 3.

The stringent diagnostic criteria according to Rahman et al (1996) for Leigh syndrome are: (i) progressive neurological disease with motor and mental developmental delay, (ii) clinical signs of brainstem or basal ganglia dysfunction, (iii) elevated lactic acid (lactate) concentrations in the blood and/or cerebrospinal fluid, and (iv) at least one of the following abnormalities:

- Characteristic neuroradiological abnormalities (signal abnormalities in basal ganglia, thalamus, brainstem, dentate nucleus, optic nerve) and/or

- Post mortem: Characteristic neuropathological abnormalities (spongiform degeneration) and demyelination in the above designated areas, and/or

- Characteristic radiological or neuropathological abnormalities in a sibling.

Although a variety of diseases exhibit mitochondrial dysfunction, they fundamentally differ in their genetic and metabolic causes, as well as lifestyle influences, pathogenesis, and the resulting biochemical defects.

Friedreich’s Ataxia (FRDA) is a genetically caused neurodegenerative disease that results from mutations in the FXN gene, leading to a deficiency of frataxin. This protein is crucial for the proper function of mitochondria, and its absence leads to impaired mitochondrial energy production and an accumulation of iron within the mitochondria. Symptoms of FRDA include ataxia, muscle weakness, and heart problems.

Type 2 diabetes is a metabolic disorder characterized by insulin resistance and impaired glucose regulation. Mitochondrial dysfunction may contribute to its pathogenesis by impairing cells' ability to respond to insulin signals and effectively process glucose. The main features of type 2 diabetes are hyperglycemia and associated metabolic complications.

In contrast, Leigh syndrome with Complex IV deficiency is a rare inherited neurodegenerative disease caused by mutations that directly affect the mitochondrial respiratory chain.

Symptoms are typically severe and manifest early in life, including progressive neurological impairments and developmental delays.

While type 2 diabetes is primarily a metabolic disease that is also associated with lifestyle factors, Leigh syndrome is a primarily genetically determined mitochondrial disease with serious implications for the nervous system. The Complex V deficiency form of Leigh Syndrome, including MILS, and the Leigh Syndrome form with Complex IV deficiency represent distinct patient groups. On one hand, the MILS form of Leigh Syndrome is characterized by maternal inheritance due to mitochondrial DNA mutation, while the Leigh Syndrome form with Complex IV deficiency is typically attributed to mutations in nuclear DNA that code for subunits of the cytochrome c oxidase complex. The data available in the state of the art indicate that both patient groups exhibit distinct biochemical profiles; in particular, the specific defects in the mitochondrial respiratory chain differ. Clinical observations suggest that the presentation and progression of Leigh Syndrome vary between the two groups. For instance, patients with MILS may exhibit a different spectrum of symptoms and disease progression compared to patients with Complex IV deficiency. These differences usually point to distinct and targeted therapeutic approaches for these two forms of Leigh Syndrome due to their divergent pathophysiological mechanisms, as evidenced by the failed attempts with PDE5 inhibitors in other mitochondrial diseases such as FRDA, DMD, or Type 2 Diabetes.

In embodiments, the treatment of a medical condition associated with mitochondrial Complex IV (cytochrome c oxidase) deficiency represents the treatment of a novel patient subgroup compared to those treatments described in the art. For example, a skilled person is capable of delineating between Complex V and Complex IV deficiencies in Leigh Syndrome patients, as the different underlying pathomechanisms can be objectively determined and do not necessarily present as identical diseases with the same symptoms or detriments. The identification of Complex IV deficiencies as being treatable with the drug class of the present invention represents an unexpected and beneficial finding. A skilled person would not have concluded from an earlier disclosure regarding treatment of Complex V deficiencies, that also Complex IV deficiencies could be addressed using the same drug class. Thus, the present invention preferably relates to the discovery of a novel patient collective not previously treatable according to the state-of-the-art means. The discovery of the novel therapeutic effect, namely on Complex IV deficiencies, thus relates to a novel clinical situation that would not have arisen without knowledge of the present invention. In embodiments, the addressing of complex IV deficiency represent a novel technical effect that could not have been predicted from the art, which enables treatment of a new patient class by novel means.

Regarding the differences between Complex IV and V defects, Leigh syndrome is itself a heterogeneous condition, with various clinical phenotypes, although it can be clearly identified and Complex IV and V defects may be clearly distinguished based on pathophysiological aspects of the condition. For example, a defect in Complex IV usually causes depolarization of the mitochondrial membrane potential (Reference 17). This is typically not the case for Complex V defects, which instead are typically associated with an abnormal increase in mitochondrial membrane potential. Thus, the initial insults to the mitochondria are different in Complex IV and V deficiencies and will typically cause a different pathological cascade of events with respect to differences in the type of ROS produced and imbalance of calcium homeostasis and ATP production.

Leigh Syndrome caused by Cytochrome C oxidase deficiency

Cytochrome C oxidase deficiency is a very rare inherited metabolic disorder characterized by a deficiency of the enzyme cytochrome C oxidase (COX) or complex IV. COX is an essential enzyme being active in the subcellular structures that help regulating energy production (mitochondria). COX deficiency may be confined (localized) to skeletal muscle tissue or affect multiple tissues, such as the heart, kidney, liver, brain, and/or connective tissue (fibroblasts); in other cases, COX deficiency may be generalized (systemic).

A defect in complex IV leads to reduced ATP production and an accumulation of reactive oxygen species (ROS), which in turn can lead to cell damage and cell death. PRKG1 (protein kinase G 1) is an enzyme that functions as a serine/threonine kinase and regulates various cellular processes, including vasodilation, cell growth, apoptosis and neuronal plasticity. PRKG1 is activated by cyclic guanosine monophosphate (cGMP) and plays an important role in signalling within cells (Example 5, Fig 4A). The direct link between PRKG1 expression and Leigh syndrome due to a complex IV defect has not been well established nor generally recognised. However, the inventors found that alterations in signalling or energy metabolism due to mitochondrial dysfunction indirectly affect PRKG1 activity or expression. For example, impaired mitochondrial function caused by mutations in Complex IV genes led to changes in the levels of PRKG1 expression, as shown in Example 5 and Fig. 4B.

There are four different forms of cytochrome c oxidase deficiency:

(1) COX deficiency, a benign infantile mitochondrial myopathy. Symptoms in children affected by COX deficiency ranges from a benign infantile mitochondrial myopathy to a more severe infantile form of the disease. Since COX deficiency is mainly localized to skeletal muscle tissue, cardiac or renal dysfunction usually do not occur.

(2) Mitochondrial COX myopathy. Symptoms ranges from generalized weakness of skeletal muscle (myotonia), abnormalities of the heart and kidneys, and/or abnormally high levels of lactic acid in the blood (lactic acidosis). This type affects skeletal muscle and several other tissues. A subtype, the De Toni-Fanconi-Debre syndrome, is characterized by excessive thirst, excessive urination, and excessive excretion of glucose, phosphates, amino acids, bicarbonate, calcium, and water in the urine.

(3) Leigh syndrome - subacute necrotizing encephalomyelopathy. This form is a rather generalized (systemic) form of COX deficiency, characterized by progressive degeneration of the brain and dysfunction of other body organs, including, but not limited to, heart, kidneys, muscles, and liver. Symptoms may include loss of previously acquired motor skills, loss of appetite, vomiting, irritability, and/or seizures. As Leigh's syndrome progresses, general weakness, loss of muscle tone (hypotension), and/or attacks of lactic acidosis may occur.

(4) French-Canadian type COX deficiency. This form affects skeletal muscles, connective tissues, and especially brain (Leigh syndrome) and liver. Symptoms in children being affected comprises developmental delays, decreased muscle tone (hypotonia), squinting of the eyes (strabismus), Leigh disease, and/or episodes of lactic acidosis.

COX deficiency is usually inherited in an autosomal recessive manner. A summary of Mitochondrial complex IV deficiency, nuclear-types are shown in Table 2.

Table 2: Mitochondrial complex IV deficiency, nuclear-types - with Online Mendelian Entry number (Phenotype MIM number) and affected gene (AR = autosomal recessive).

Mitochondrial complex IV deficiency causing mutations:

Complex IV - also known as cytochrome c oxidase - is the last enzyme in the respiratory chain and consists of 14 subunits, three of which (COX1 , COX2, and COX3) are encoded by mitochondrial DNA. Pathogenic mutations causing COX deficiency are associated with autosomal recessive inheritance. COX deficiency can occur in isolation (when caused by mutations in any of the above genes) or as part of a chromosomal disorder.

Mitochondrial Complex IV deficiency related diseases are caused by genetic mutations in mitochondrial Complex IV and can be identified by genetic screening of patients at risk for disease.

The compounds disclosed herein can be administered to asymptomatic patients with mutations in genes encoding structural subunits or assembly factors of Complex IV who are at risk for developing the clinical symptoms of the disease, and the methods of the invention disclosed herein can be used to treat these patients to suppress the onset of adverse symptoms or reduce the severity of symptoms that may occur. The compounds disclosed herein may be administered to symptomatic patients with mutations in Complex IV to treat the disease, and the methods of the invention disclosed herein may be used to treat such patients.

The diagnosis of (i) Leigh syndrome; (ii) COX deficiency, benign infantile mitochondrial myopathy type; (iii) COX deficiency, severe infantile mitochondrial myopathy type; (iv) COX deficiency associated with fatal infantile cardioencephalomyopathy; (v) COX deficiency associated with Leigh’s syndrome; (vi) COX deficiency associated with Leigh syndrome, French-Canadian type; (vii) COX deficiency, adult form; (viii) De Toni-Fanconi-Debre syndrome, and other disorders characterized by mitochondrial Complex IV deficiency must be performed by a skilled person trained in this field. The skilled person includes physicians, especially those who have experience with this type of syndromes and identification of characteristic symptoms, as well as human geneticists being trained to identify a mutation causing the syndrome and diagnose it through molecular genetic testing. Mutations on nuclear DNA can be performed using any body cell that has a nucleus and chromosomal DNA contained within it, for example, blood cells containing nuclei. The mutations on the mitochondrial DNA associated with said syndromes can be detected in white blood cells (leukocytes), for example, but also other tissue samples such as skin, skeletal muscle, hair follicles or urinary sediment.

The skilled person knows how to identify mitochondrial and nuclear genes and mutations within these genes causing the Complex IV deficiency and/or Leigh syndrome. For example, publically available databases can be used, such as ClinVar database, HGMD database, OMIM database, and MitoMap for screening and verifying genes and mutations, particularly pathogenic and disease-causing mutations. Genes that cause mitochondrial Complex IV deficiency and/or Leigh syndrome comprise APOPT1 , C12ORF62, COA3, COA5, COA6, COX10, COX14, COX15, COX20, COX6B1 , FARS2, FASTKD2, LRPPRC, PET100, POLG, SCO1 , SCO2, SURF1 , and TACO1 .

In the below Table 3 genes with known disease-causing genetic variation, gene location, and Online Mendelian Entry number of these variation are exemplarily listed.

Table 3: Nuclear and mitochondrial genes involved in Leigh Syndrome. The most prevalent mutations are shown with the genetic defect and corresponding biochemical and clinical manifestations. [Bakare AB, Lesnefsky EJ, lyer S. Leigh Syndrome: A Tale of Two Genomes. Front Physiol. 2021 Aug 11 ;12:693734] As with all mitochondrial diseases, there is no cure for complex IV deficiency. Treatment with non-PDE5 inhibitor drugs and/or symptomatic measures depends on symptoms and the syndrome caused by the mutation. Non-PDE5 inhibitor drugs administered in the art to Leigh syndrome patients comprise biotin, carnitine, riboflavin, CoQ10, dichloroacetate, mitoQ, decanoic acid, resveratrol, nicotinamide, alpha-ketoglutarate, and/or aspartate. Therapies for the infantile multisystem form have been to date unsuccessful.

Standard therapy in the art include biotin administration, ketogenic diet, treatment of seizures and anti-seizure prophylaxis, treatment of dystonia with benzhexol, baclofen, gabapentin and other similar drugs such as botulinum toxin, supportive care for congestive heart failure in cardiomyopathy, advice on an appropriate nutritious diet and necessary feeding techniques to achieve adequate daily intake, acidosis that may be treated acutely with sodium bicarbonate or sodium citrate, and/or visual disability assistance. Patient at palliative care may also be treated with lifesaving measures including antiepileptic drugs, barbiturates, anaesthetics, full condition monitoring, and/or emergency treatment during dyspnea or respiratory failure.

In particular, the treatment described herein relates to either reducing or preventing a progressive course or symptoms thereof of the mitochondrial Complex IV related diseases described herein by inhibiting cGMP degradation within the cytosol of patients cells, in particular encephalopathy, cerebral seizures, severe leg-related motor-sensory neuropathy, demyelinating neuropathy, mechanical ventilation due to respiratory failure, cardiomyopathy, metabolic derailments, loss of ambulation, sitting, and autonomic care abilities, muscle hypotonia, palliative care, muscle weakness, chronic metabolic acidosis, exercise-induced failure.

The prophylactic therapy as described herein is intended to encompass prevention or reduction of risk of developing or progression of disease course and symptoms thereof in mitochondrial Complex IV deficiency related diseases described herein, preferably due to decrease in mitochondrial membrane potential, increase in intracellular calcium release and increase in cellular energy demand in patients cells to levels comparable to normal controls described herein via inhibiting cGMP degradation by PDE5.

In embodiments, the interventional therapy as described herein is intended to encompass prevention or reduction of developing or progressing disease course and symptoms thereof in mitochondrial Complex IV deficiency related diseases and death, preferably due to decrease in mitochondrial membrane potential, increase in intracellular calcium release, and increase in cellular energy demand in patients cells to levels comparable to normal controls described herein via inhibiting cGMP degradation by PDE5, such as palliative care, encephalopathy, cerebral seizures, severe leg-related motor-sensory neuropathy, demyelinating neuropathy, mechanical ventilation due to respiratory failure, cardiomyopathy, metabolic derailments, loss of ambulation, sitting, and autonomic care abilities, muscle hypotonia, muscle weakness, chronic metabolic acidosis, exercise-induced failure.

In one embodiment, the treatment described herein also relates to reversing an existing disease progression or existing symptoms thereof of the mitochondrial Complex IV deficiency related diseases, preferably by decrease in mitochondrial membrane potential, increase in intracellular calcium release, and increase in cellular energy demand in patients cells to levels comparable to normal controls described via inhibiting cGMP degradation by PDE5, such as palliative care, encephalopathy, cerebral seizures, severe leg-related motor-sensory neuropathy, demyelinating neuropathy, mechanical ventilation due to respiratory failure, cardiomyopathy, metabolic derailments, loss of ambulation, sitting, and autonomic care abilities, muscle hypotonia, muscle weakness, chronic metabolic acidosis, exercise-induced failure.

As used herein, a “patient with symptoms of a mitochondrial Complex IV deficiency” is a subject who presents with one or more of, without limitation, characteristic changes in the brain necrotizing encephalitis, muscle weakness, ataxia, mental and/or motor developmental impairment, psychomotor regression, mental and/or motor developmental delay, cerebral seizures, circumscribed paralysis, involuntary movements (dystonia, tremor, chorea), lesions in the basal ganglia, lesions in the white matter, disturbances in nerve function (neuropathy), peripheral neuropathy, progressive weakness of the arms and legs, hypertrophic cardiomyopathy, asymmetric septal hypertrophy progressive neurological disease with motor and mental developmental delay, clinical signs of brainstem or basal ganglia dysfunction, demyelination symptoms, cerebral atrophies, cerebellar atrophies, neurogenic muscle atrophy, elevated lactic acid (lactate) concentrations in the blood, brain and/or cerebrospinal fluid, neuroradiological abnormalities (signal abnormalities in basal ganglia, thalamus, brainstem, dentate nucleus, and/or optic nerve).

Further symptoms may be difficulty articulating words (dysarthria), loss of previously acquired intellectual skills, poor sucking ability, loss of head control, marked loss of appetite, recurrent vomiting, irritability, constant crying, possibly seizures, breathing abnormalities, including temporary cessation of spontaneous breathing (apnea), shortness of breath (dyspnea), abnormally rapid breathing (hyperventilation), abnormal breathing patterns, difficulty swallowing (dysphagia, abnormally rapid eye movements (nystagmus), sluggish pupils, squinting (strabismus), paralysis of certain eye muscles (ophthalmoplegia), damage to the nerves of the eye (optic atrophy), and/or visual disturbances including blindness. Additional symptoms such as generalized muscle weakness, lack of muscle tone (hypotonia), tremors, movement disorders such as chorea (rapid, involuntary, jerky movements), seizures, infantile spasms, and spasticity, a condition characterized by involuntary muscle spasms that result in slow, or stiff movements of the legs, dystonia and/or painful movements may also occur. Other characteristics and symptoms may be disease worsening and/or progression through or after a viral or bacterial infection. Life-threatening complications, often due to cardiac or respiratory problems, occur frequently in patients with symptoms of mitochondrial Complex IV deficiency.

As used herein, a “patient with symptoms of a Leigh syndrome” is a subject who presents with one or more of, without limitation, necrotizing encephalitis (particularly affecting the basal ganglia and brainstem), disease manifestation and progression associated with a viral or bacterial infection, muscle weakness, mental and motor developmental impairment, cerebral seizures, circumscribed paralysis, involuntary movements (dystonia, tremor, and chorea), disturbances in nerve function (neuropathy), and thickening of the heart muscle (hypertrophic cardiomyopathy, progressive neurological disease with motor and mental developmental delay, clinical signs of brainstem or basal ganglia dysfunction, elevated lactic acid (lactate) concentrations in the blood and/or cerebrospinal fluid, neuroradiological abnormalities (signal abnormalities in basal ganglia, thalamus. Brainstem, dentate nucleus, optic nerve), neuropathological abnormalities (spongiform degeneration) and/or demyelination (in basal ganglia, thalamus, brainstem, dentate nucleus, and/or optic nerve). As used herein, a “patient with symptoms of a Leigh like syndrome” is a subject who presents with one or more of, without limitation, a milder phenotype of Leigh syndrome in which not all of the diagnostic criteria listed for Leigh syndrome are met, lesions in the basal ganglia, lesions in the white matter, demyelination, cerebral atrophies, cerebellar atrophies, demyelination of peripheral nerves, neurogenic muscle atrophy, The course of the disease may be lethal due to cardiac or liver failure.

As used herein, a “patient with symptoms of a COX deficiency - benign infantile mitochondrial myopathy type” is a subject who presents with one or more, without limitation, primarily generalized weakness of skeletal muscles (myotonia), with no other tissues/organs affected.

As used herein, a “patient with symptoms of a COX deficiency - severe infantile mitochondrial myopathy type” is a subject who presents with one or more, without limitation, of a general weakness of skeletal muscles, abnormalities of the heart and kidneys, and/or lactic acidosis.

As used herein, a “patient with symptoms of a COX deficiency associated with fatal infantile cardio-encephalomyopathy [CEMCOX]” is a subject who presents with one or more, without limitation, cardiomyopathy, which may occur either in utero or in the first days of life. The following neurologic stigmata are also commonly observed: abnormal breathing, nystagmus, and gyral abnormalities. The disorder is usually fatal in early infancy. In people with mutations in the SCO2 gene, the disease is usually more severe.

As used herein, a “patient with symptoms of a French-Canadian type; COX deficiency” is a subject who presents with one or more, without limitation, early-onset progressive neurodegenerative disorder with delayed psychomotor development and mental retardation, dysmorphic facial features, hypotonia, and ataxia. MRI shows lesions in the brainstem and basal ganglia. Metabolic and/or neurologic crises can often lead to early death. Strabismus may occur in patients with COX deficiency associated with Leigh syndrome, French-Canadian type.

As used herein, a “patient with symptoms of a COX deficiency associated with Leigh syndrome” is a subject who presents with one or more, without limitation, Leigh disease is a general clinical syndrome characterized by progressive degeneration of the brain, heart, kidneys, muscles, and liver. Typical symptoms include regression of motor skills, generalized weakness with hypotonia, irritability, vomiting, seizures, and lactic acidosis. The disease begins in infancy or early childhood with encephalopathy and failure to thrive (usually between three months and two years of age). Children with earlier disease onset, especially those with mutations in SURF1 , tend to have more severe disease. More than half of patients die in infancy, often within the first 18 months of life.

As used herein, a “patient with symptoms of a De Toni-Fanconi-Debre syndrome” ”, is a subject who presents with one or more, without limitation, a renal syndrome caused by transport defects of amino acids, monosaccharides, sodium, potassium, phosphorus, calcium, bicarbonate, uric acid, and proteins in the proximal renal tubule, as well as excessive thirst and urination.

As used herein, a “patient with symptoms of an adult form of Leigh Syndrome”, is a subject who presents with one or more, without limitation, generalized muscle pain, hypotonia, and occasional muscle twitching and stiffness. Diabetes, hearing loss, hyperlipidemia, hyperuricemia, arterial hypertension, polyarthrosis, hypogonadism, and hypothyroidism may be present in these patients. Family history may include weakness, myalgias, CK elevation, and diabetes. Clinical examination may reveal postural tremor, decreased tendon reflexes, and elevated serum CK levels. A muscle biopsy is nonspecific, but biochemistry of the muscle homogenate may reveal an isolated complex IV defect and decreased levels of coenzyme Q (CoQ). In patients with the adult form, coenzyme Q supplementation, a I ow-carbo hydrate diet, and a gluten-free diet may have a beneficial effect on at least some of the symptoms.

As used herein, a “patient with symptoms of a mitochondrial Complex IV deficiency, nuclear type 1-23” is subject who presents with one or more, without limitation, global developmental delay or developmental regression, hypotonia, ataxia, dystonia, and ophthalmologic abnormalities, such as nystagmus or optic atrophy, mainly a Leigh Syndrome is developed. This is group of often progressive and severe neurodegenerative disorders with onset within the first months or years of life, and may result in early death.

In some embodiments, symptoms of Leigh syndrome are psychomotor retardation, seizures, muscle hypotonia, muscle weakness, ataxia, eye abnormalities including vision loss, difficulty in swallowing, lactic acidosis, lesions of the basal ganglia, thalamus, brain stem, and spinal cord, degeneration of the basal ganglia, thalamus, brain stem, and spinal cord, and/or lifethreatening cardiac complications.

In the present invention "treatment" or “therapy” generally means to obtain a desired pharmacological effect and/or physiological effect. The effect may be prophylactic in view of completely or partially preventing a disease and/or a symptom, for example by reducing the risk of a subject having a disease or symptom or may be therapeutic in view of partially or completely curing a disease and/or adverse effect of the disease.

In the present invention, "therapy" includes arbitrary treatments of diseases or conditions in mammals, in particular, humans, for example, the following treatments (a) to (c): (a) Prevention of onset of a disease, condition or symptom in a patient; (b) Inhibition of a symptom of a condition, that is, prevention of progression of the symptom; (c) Amelioration of a symptom of a condition, that is, induction of regression of the disease or symptom.

Subject

As used herein, the “patient” or "subject" may be a vertebrate. In the context of the present invention, the term "subject" includes both humans and animals, particularly mammals, and other organisms. The term "patient" refers to animals, preferably mammals, especially humans and includes adults and children, males and females. Children include neonates, infants, and adolescents. The term "patient" also refers to a "subject" suffering from or suspected of suffering from Leigh syndrome COX deficiency - benign infantile mitochondrial myopathy type; COX deficiency - severe infantile mitochondrial myopathy type; COX deficiency associated with fatal infantile cardio-encephalomyopathy; COX deficiency associated with Leigh’s syndrome; COX deficiency associated with Leigh syndrome - French-Canadian type; COX deficiency - adult form; or De Toni-Fanconi-Debre syndromeor mitochondrial Complex IV deficiency nuclear type 1-23.

As used herein, the term "subject" refers to a mammal, such as humans, but can also be another animal, such as a domestic animal (e.g. a dog, cat or the like), a farm animal (e.g. a cow, sheep, pig, horse or the like) or a laboratory animal (e.g. a monkey, rat, mouse, rabbit, guinea pig or the like). The term "patient" refers to a "subject" suffering from or suspected of suffering from the Leigh syndrome, COX deficiency - benign infantile mitochondrial myopathy type; COX deficiency - severe infantile mitochondrial myopathy type; COX deficiency associated with fatal infantile cardio-encephalomyopathy; COX deficiency associated with Leigh’s syndrome; COX deficiency associated with Leigh syndrome - French-Canadian type; COX deficiency - adult form; or De T oni-Fanconi-Debre syndrome, or the medical condition associated with mitochondrial Complex IV deficiency nuclear type 1-23.

In one embodiment, treatment criteria comprise a positive laboratory-confirmed mutation within a mitochondrial Complex IV gene and/or a patient being diagnosed with Leigh Syndrome before treatment. In one embodiment, the onset of Leigh syndrome, COX deficiency - benign infantile mitochondrial myopathy type; COX deficiency - severe infantile mitochondrial myopathy type; COX deficiency associated with fatal infantile cardio- encephalomyopathy; COX deficiency associated with Leigh’s syndrome; COX deficiency associated with Leigh syndrome - French-Canadian type; COX deficiency - adult form; or De Toni-Fanconi-Debre syndrome, , or the medical condition associated with mitochondrial Complex IV deficiency nuclear type 1-23 may be at birth, <1 , <2, <3, <4, <5, <6, <7, <8, <9, <10, <11 , <12, <13, <14, <15, <16, <17, <18, <19, <20 years or above, preferably <5 years.

Inhibitors

According to the present invention, an "inhibitor" in the context of "PDE5 inhibitor" is considered to be any agent, substance, compound, molecule, or other means that results in slowing, repressing, blocking, or otherwise interfering with or negatively affecting the activity, function, expression, or signalling that said target induces, performs, or exhibits in the absence of the inhibitor.

The terms “agent”, “substance”, “compound”, “molecule” can be used interchangeably. For example, phosphodiesterase 5 enzyme inhibitors, i.e. sildenafil and tadalafil, may effect activity of a target enzyme involved in cleaving the phosphoric ester bond into cGMP for 5'- GMP production within a cell, either directly or indirectly. The inhibitors as described herein may also be termed “agents”. References to the “agents” in the context of the combinations and methods described herein are to be understood as PDE5 enzymatic activity and cGMP degradation inhibitor. Preferred inhibitors are those described herein.

PDE and PDE5 Inhibitors

Phosphodiesterases (PDEs) are intracellular enzymes that specifically catalyze the hydrolysis of the second messengers cAMP and cGMP to the inactive metabolites AMP and GMP. PDEs can be divided into Class I and Class II, which have no apparent sequence similarity. Class I includes all known mammalian PDEs and consists of at least 10 families that have arisen from separate genes. Most families contain more than one gene and most genes encode more than one messenger RNA (mRNA) by alternative splicing or alternative transcription start sites. PDE4, PDE7, and PDE8 are highly specific for hydrolysis of cAMP, while PDE5, PDE6, and PDE9 are highly specific for cGMP. PDE1 , PDE2, PDE3, and PDE10 have mixed specificity.

Each PDE has a conserved catalytic domain of approximately 270 amino acids with a high degree of conservation (25-30%) of amino acid sequence between PDE families that is carboxyl-terminal to its regulatory domain. Cyclic nucleotides are degraded by PDE-catalyzed hydrolytic cleavage of the 3'-phosphodiester bond, resulting in the formation of the corresponding inactive 5'-monophosphate. Only PDE5 exclusively catalyses the breakdown of cGMP. By balancing cGMP production through guanylate cyclases, PDE5 is able to lower cGMP levels very effectively. As a result, PDE5 inhibition increases intracellular cGMP levels and initiates a cGMP-driven response cascade. Three different isoforms of PDE5A are known, PDE5A1 , PDE5A2 and PDE5A3. All PDE5 variants differ only at the AZ-terminal end. PDE5A1 appears to be the predominant form expressed in most PDE5-containing tissues. PDE5A2 contains a much shorter AZ-terminal amino acid fragment and has also been detected in several animal species. PDE5A3 has only been detected in human tissues, based on RT-PCR data. See Rybalkin et al, Circ Res, vol 93, pp 280-291 (2003). PDE5 is highly specific for cGMP hydrolysis and contains two homologous AZ-terminal regulatory domains, recently defined as GAF A and GAF B. See Martinez et al, Proc Natl Acad Sci USA, vol 99, pp 13260-13265 (2002). PDE5 is activated directly by the binding of cGMP to its GAF A domain. Without cGMP binding, PDE5 is in a non-activated state. Only activated PDE5 is phosphorylated by cGMP-dependent protein kinase (PKG) (Ser- 92).

"PDE 5 inhibitors" refers to cyclic guanosine-3', 5'-monophosphate type 5 cGMP inhibitors (or phosphodiesterase 5 (PDE5) inhibitors), sometimes referred to herein as PDE V or PDE5 inhibitors. Suitable PDE5 inhibitors for use according to the present invention include sildenafil, tadalafil, and vardenafil. These PDE5 inhibitors are currently approved for the treatment of erectile dysfunction. PDE5 inhibitors increase cyclic guanosine monophosphate (cGMP) levels, which has a neuroprotective effect via activation of protein kinase 1 (PKG1) and improves long-term potentiation.

Table 4: Exemplary PDE5 inhibitors

PDE5 inhibitors increase intracellular cGMP concentrations by decreasing degradation of the molecule and thus lead to vasodilatation. cGMP leads to increased NO concentrations, NO- itself promotes intracellular calcium excretion from mitochondrial striatal neurons and thus may at least partially improve the impaired calcium signaling cascade. (Horn TFW, Wolf G, Duffy S, Weiss S, Keilhoff G, MacVICAR BA. Nitric oxide promotes intracellular calcium re-lease from mitochondria in striatal neurons. FASEB J 2002; 16:1611-22.) Avanafil belongs to the larger group of substances known as phosphodiesterase 5 (PDE5) inhibitors. Avanafil has so far only been used in male erectile dysfunction and no dose data and experience exist for long-term use. In some embodiments, the treatment comprises treatment of Leigh syndrome, COX deficiency - benign infantile mitochondrial myopathy type; COX deficiency - severe infantile mitochondrial myopathy type; COX deficiency associated with fatal infantile cardio-encephalomyopathy; COX deficiency associated with Leigh’s syndrome; COX deficiency associated with Leigh syndrome - French-Canadian type; COX deficiency - adult form; or De Toni-Fanconi-Debre syndrome, mitochondrial Complex IV deficiency, nuclear type 1-23, and disorders caused by or associated with a mutation in a functional subunit of mitochondrial Complex IV (COX) with a PDE5 inhibitor.

Sildenafil citrate and its metabolites are the first selective PDE5 inhibitor. It is a potent inhibitor of PDE5 (IC50 of 3.9 nM), with a high selectivity (>1 OOO-fold) for human PDE5 over humanPDE2, PDE3, and PDE4 and moderate selectivity (>80-fold) over PDE1 . Sildenafil, however, is only approximately 10-fold as potent for PDE5 as for PDE6, which is found in the photoreceptors of the human retina. This lower selectivity toward PDE6 is presumed to be the cause for color vision abnormalities observed with high doses or plasma levels of sildenafil. Both sildenafil and its major active metabolite (N-desmethyl sildenafil) are highly bound to plasma proteins (=96%), and the protein binding is independent of drug concentrations. Plasma concentrations of sildenafil and its N-desmethyl metabolite were re-ported to decline bi-exponentially, with a mean terminal half-life of 3 to 5 hours for both of them, independent of the route of administration.

The substance sildenafil (Pfizer, Revatio®) has been approved for the German market since October 28, 2005; among other things, for the treatment of erectile dysfunction in men. The approval relates to the treatment of adult patients with pulmonary arterial hypertension (PAH) in WHO functional classes II and III for the improvement of exercise capacity. The dosage forms are additionally approved for the treatment of pediatric patients aged 1 to 17 years with pulmonary arterial hypertension. Several studies exist on the use of sildenafil for the treatment of persistent pulmonary hypertension of the premature or newborn infant. There is no approval for this indication in Germany. Other studies with positive outcome of treatment of children with sildenafil are bronchiopulmonary dysplasia, congenital diaphragmatic hernia, Eisenmenger complex, and lymphatic malformations.

Tadalafil is currently approved only for the treatment of erectile dysfunction and for the treatment of benign prostatic syndrome in adult men. Tadalafil is a selective and potent inhibitor of PDE5 with an IC50 of 0.94 nm. It exhibits high selectivity toward PDE5 compared to other PDEs: >700-fold relative to PDE6, >10 OOO-fold relative to PDE1-4 and 7-10, and >5- fold relative to PDE11 . Tadalafil is structurally different from both sildenafil and vardenafil, and the different structures are reflected in distinct differences in the clinical pharmacology profiles of these drugs. Like sildenafil, tadalafil was developed initially for use in cardiovascular disease. Metabolites of tadalafil, such as methylcatechol and methylcatechol glucuronide metabolites are clinically inactive at observed metabolite concentrations.

Vardenafil hydrochloride was the first second-generation PDE5 inhibitor. Vardenafil has a high potency in vitro (IC50= 0.1 nm-0.7 nm) and a high selectivity for the inhibition of PDE5 compared with the otherknown phosphodiesterases (>15-fold relative to PDE6,>130-fold relative to PDE-1 , >300-fold relative toPDE11 , and >1 OOO-fold relative to PDE2, 3, 4, 7, 8, 9, and 10). Unlike sildenafil and tadalafil, vardenafil was developed from the outset specifically for use as an erectogenic agent. Oral doses of 5, 10, and 20 mg vardenafil given no morethan once daily have been efficacious in clinical trials. Vardenafil is extensively metabolized, with more than14 metabolites identified. The major metabolite, M1 ,and 2 minor metabolites, M4 and M5, as well as their respective glucuronides, are all a result of the degradation of vardenafil’s piperazine ringThe major circulating metabolite, M1 ,has 28% of vardenafil’s potency for PDE5 inhibition, while M4 and M5 possess 5.6% and 4.9%, respectively.

Treatment and therapeutic methods

As used herein "treatment" or "treating," includes any beneficial or desirable effect on the symptoms or pathology of a disease or pathological condition, and may include even minimal reductions in one or more measurable markers of the disease or condition being treated.

T reatment can involve optionally either the reduction or amelioration of symptoms of the disease or condition, or the delaying of the progression of the disease or condition.

"Treatment" does not necessarily indicate complete eradication or cure of the disease or condition, or associated symptoms thereof.

As used herein, "prevent," and similar words such as "prevented," "preventing" or "prophylactic" etc., indicate an approach for preventing, inhibiting, or reducing the likelihood of the occurrence or recurrence of, a disease or condition. It also refers to delaying the onset or recurrence of a disease or condition or delaying the occurrence or recurrence of the symptoms of a disease or condition. As used herein, "prevention" and similar words also includes reducing the intensity, effect, symptoms and/or burden of a disease or condition prior to onset or recurrence of the disease or condition.

In some embodiments, a time interval between treatment or prevention comprises a period of several hours (2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12) several days (2, 3, 4, 5, 6, or 7), several weeks (1 , 2, 3, 4, 5, 6, 7, or 8), or months (2, 3, 4, 5, or 6), preferably 4 weeks to 3 months. In some embodiments, the time interval from one treatment or prevention to the next subsequent treatment can be the same, nearly the same or can change.

In some embodiments, the treatment comprises administration of PDE5 inhibitor, a derivative or metabolite thereof, one or two or three or four times daily for more than one day, more than one week, more than one month, more than one year, preferably 3 times daily, lifelong.

In one embodiment, a method of treating a Leigh syndrome or mitochondrial Complex IV deficiency related condition in a subject in need thereof comprises administering an effective amount, e.g., therapeutically effective amount of a composition comprising the PDE5 inhibitor contemplated herein. The quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease, although appropriate dosages may be determined by clinical trials.

The administration of the compositions contemplated herein may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. In a preferred embodiment, compositions are administered orally in tablet form or drinking solution.

Host cell and target cell

The term "host cell" and “target cell”, as used herein, refers to a single cell or cell culture that may be or has been a recipient of at least one of the agents described herein, individually or in combination. Host cells include progeny of a single host cell, which progeny may not necessarily be completely identical (in morphology or overall DNA complement) to the original parent cell due to natural, random or intentional mutations and/or changes. In one embodiment, the host cell also refers to a cell into which an infectious agent (e.g. a virus) has invaded or is capable of invading.

Pharmaceutical Compositions and Methods of administration

The present invention also relates to a pharmaceutical composition comprising the compounds described herein. The invention also relates to pharmaceutically acceptable salts of the compounds described herein, in addition to enantiomers and/or tautomers of the compounds described.

The term “pharmaceutical composition” refers to a combination of the agent as described herein with a pharmaceutically acceptable carrier. The phrase "pharmaceutically-acceptable" refers to molecular entities and compositions that do not produce a severe allergic or similar untoward reaction when administered to a human. As used herein, "carrier" or “carrier substance” includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Supplementary active ingredients can also be incorporated into the compositions.

The pharmaceutical composition containing the active ingredient may be in a form suitable for oral use, for example, as tablets, chewing tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.

In one embodiment, the pharmaceutical composition comprising the PDE5 inhibitor, preferably sildenafil, described herein is supplied to the subject three times daily as tablet or liquid formulation, preferably 1-2 mg/kg/day sildenafil, preferably administered either as tablet or drinking solution orally or via a gastric tube.

Administration of the compounds of the invention, or their pharmaceutically acceptable salts, in pure form or in an appropriate pharmaceutical composition, can be carried out via any of the accepted modes of administration or agents for serving similar utilities. Thus, administration can be, for example, orally, nasally, parenterally, topically, transdermally, or rectally, sublingually, intramuscular, subcutaneously, or intravenously in the form of solid, semi-solid, lyophilized powder, or liquid dosage forms, such as for example, tablets, suppositories, pills, soft elastic and hard gelatin capsules, powders, solutions, suspensions, or aerosols, or the like, preferably in unit dosage forms suitable for simple administration of precise dosages. The compositions will include a conventional pharmaceutical carrier or excipient and a compound of the invention as the/an active agent, and, in addition, may include other medicinal agents, pharmaceutical agents, carriers, adjuvants, etc.

Dosage levels of the order of from about 0.1 mg to about 10 mg per kilogram of body weight per day are useful in the treatment of the indicated conditions. For example, MILS and NARP may be effectively treated by the administration of from about 1 mg to about 500mg, preferably 2 mg to about 400 mg, more preferably 3 mg to about 100 mg, even more preferably 12 mg to about 120 mg, of the PDE5 inhibitor, preferably sildenafil, per patient per day.

Dosage levels of the order of from about 0.1 mg to about 10 mg per kilogram of body weight to a human subject, preferably at 0.5-5 mg/kg/day, more preferably at 1-2 mg/kg/day, are useful in the treatment of the indicated conditions.

The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. For example, a formulation intended for the oral administration of humans may vary from about 5 to about 95% of the total composition. Dosage unit forms will generally contain between from about 1 mg to about 500 mg of active ingredient. It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease undergoing therapy. The dosage effective amount of compounds according to the invention will vary depending upon factors including the particular compound, toxicity, and inhibitory activity, the condition treated, and whether the compound is administered alone or with other therapies.

The invention relates also to a process or a method for the treatment of the mentioned pathological conditions. The compounds of the present invention can be administered prophylactically or therapeutically, preferably in an amount that is effective against the mentioned disorders, to a warm-blooded animal, for example a human, requiring such treatment, the compounds preferably being used in the form of pharmaceutical compositions.

In some embodiments, a patient may receive therapy for the treatment and/or management of the medical condition associated with mitochondrial Complex IV deficiency before, during or after the administration of the therapeutically effective regimen of the compound of the invention, or a pharmaceutically acceptable salt thereof. Non-limiting examples of such a therapy include biotin, CoQ10, pain management, anti-inflammatory drugs, oxygen supply, immunotherapy, targeted therapy (i.e. therapy directed toward a specific target or pathway), and any combination thereof. In some embodiments, the patient has not previously received a therapy for the treatment and/or management of mitochondrial Complex IV deficiency.

Therapeutically effective and therapeutically relevant

The phrase "therapeutically effective" is intended to include, within the scope of sound medical judgment, excessive toxicity, irritation, allergic reactions, and/or other problems or complications, but commensurate with a reasonable benefit/risk ratio. As used herein to refer to the compounds, compositions, combinations and/or dosage forms suitable for use in contact with a subject that produces a result that in and of itself helps to treat and/or cure a disease.

A "therapeutically relevant amount", “therapeutically relevant dosage” or "therapeutically effective amount" of an agent or therapeutic means, such as an PDE5 inhibitor, i.e. sildenafil and tadalafil, is an amount sufficient to produce the desired effect, e.g., inhibition of cGMP degradation relative to the cGMP levels detected in the absence of an PDE5 inhibitor. Inhibition of cGMP degradation is achieved when the level obtained with an PDE5 inhibitor relative to the control is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%. Suitable assays for measuring cGMP degradation include, for example, assaying cGMP using techniques known to those skilled in the art, such as ELISA, enzyme function, mass spectrometry, LC/GC, and phenotypic assays known to those skilled in the art.

By "decrease," "decreasing," "reducing," “lower” or "diminishing" of cGMP degradation by an PDE5 inhibitor, i.e. sildenafil and/or tadalafil, is meant as a detectable rise in intracellular cGMP concentration to a given PDE5 inhibitor, i.e. sildenafil and/or tadalafil. The extent of decrease in intracellular cGMP degradation by a PDE5 inhibitor, i.e. sildenafil and/or tadalafil, may be determined relative to the intracellular cGMP level without the presence of a PDE5 inhibitor, i.e. sildenafil and/or tadalafil, preferably before initiation of treatment with a PDE5 inhibitor, i.e. sildenafil and/or tadalafil. A detectable increase intracellular cGMP level may be about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% than the intracellular cGMP level detected before initiation of treatment with PDE5 inhibitor, i.e. sildenafil and/or tadalafil. A decrease in mitochondrial membrane potential and increase of cellular cGMP levels by the PDE5 inhibitor is typically understood and measured by a decrease in cGMP degradation by a responder cell in vitro or in vivo.

"Systemic administration," as used herein, refers to the administration of a composition that results in broad biodistribution of PDE5 inhibitor, preferably sildenafil, within an organism. Systemic administration means exposing a therapeutic amount of an agent to preferred parts of the body. Systemic administration of the composition may be accomplished by any means known in the art, e.g., intravenously, subcutaneously, intraperitoneally.

Combined administration:

According to the present invention, the term “combined administration”, otherwise known as co-administration or joint treatment, encompasses in some embodiments the administration of separate formulations of the compounds described herein, whereby treatment may occur together, within minutes of each other, in the same hour, on the same day, in the same week or in the same month as one another. Alternating administration of two agents is considered as one embodiment of combined administration. Staggered administration is encompassed by the term combined administration, whereby one agent may be administered, followed by the later administration of a second agent, optionally followed by administration of the first agent, again, and so forth. Simultaneous administration of multiple agents is considered as one embodiment of combined administration. Simultaneous administration encompasses in some embodiments, for example the taking of multiple compositions comprising the multiple agents at the same time, e.g. orally by ingesting separate tablets simultaneously. A combination medicament, such as a single formulation comprising multiple agents disclosed herein, and optionally additional anti-viral and/or anti-inflammatory medicaments, may also be used in order to co-administer the various components in a single administration or dosage.

A combined therapy or combined administration of one agent may occur together or precede or follow treatment with the other agent to be combined, by intervals ranging from minutes to weeks. In embodiments where the second agent and the first agent are administered separately, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the first and second agents would still be able to exert an advantageously combined synergistic effect on a treatment site. In such instances, it is contemplated that one would contact the subject with both modalities within about 12-24 h of each other and, more preferably, within about 3-12 h of each other, with a delay time of only about 6 h being most preferred. In some situations, it may be desirable to extend the time period for treatment significantly, however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1 , 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations.

In the meaning of the invention, any form of administration of the multiple agents described herein is encompassed by combined administration, such that a beneficial additional therapeutic effect, preferably a synergistic effect, is achieved through the combined administration of the two agents.

SEQUENCES

Table 5: Preferred mitochondrial nucleic acid sequences of the invention:

FIGURES

The invention is further described by the figures. These are not intended to limit the scope of theinvention.

Short description of the figures:

Figure 1 : Schematic drawing of the development of a human model of Leigh syndrome using patient-derived induced pluripotent stem cells (carrying a SURF1 mutation) and CRISPR/Cas9 engineering.

Figure 2: High-throughput microscopy assay for measuring branching outgrowth in iPSC derived neurons.

Figure 3: Quantified neuronal branching in untreated neurons.

Figure 4: Mechanism of action of PDE5 inhibition in SURF1 defects.

Detailed description of the figures:

Figure 1 : Schematic drawing of the development of a human model of Leigh syndrome using patient-derived induced pluripotent stem cells (carring a SURF1 mutation) and CRISPR/Cas9 engineering. Single-cell RNA sequencing and multi-omics analyses revealed impaired neuronal morphogenesis in mutant neuronal cultures and brain organoids. The defects occurred at the level of neural progenitor cells (NPCs), which maintained a glycolytic proliferation state that did not instruct neuronal morphogenesis. SURF1 gene propagation supported metabolic programming of Leigh syndrome NPCs and resulted in restored neuronal morphogenesis. Induced pluripotent stem cells (iPSCs) from patients with SURF1 mutations and engineered with CRISPR/Cas9 as a model for Leigh syndrome.

Figure 2: High-throughput microscopy assay for measuring branching outgrowth in iPSC derived neurons. High-content analysis (HCA) protocol for monitoring the growth capacity of human neurons derived from induced pluripotent stem cells (iPSCs). The steps comprise performing HCA imaging followed by quantification of dendrite and axon morphology in a high-throughput system to evaluate neurons obtained by differentiation approaches. This method can be applied to patient-derived iPSCs.

Figure 3: Neuronal branching in iPSCs derived neurons from SURF1 patients (A) Assay for quantifying branching and length of branches in iPSCs derived neurons from Leigh Syndrome patients. (B) Fluorescent staining for nucleic acid (HOECHST, nuclei), green fluorescent protein (GFP, vector control), and microtubule associated protein 2 (MAP2, microtubule assembly) in neurons from patients carrying SURF1 mutations. Isogenic control left, with SURF1 mutation right, [microscopy bar = 100 pm] (C) Quantified neuronal branching in untreated neurons as exemplarily shown in Figure 3B. The fold change of branching of neurons carrying SURF1 mutation is relatively calculated to isogenic control. (D) Quantified neuronal branching in neurons carrying SURF1 mutations treated with sildenafil at concentrations of 1 pM, 10 pM, and 50 pM or with DMSO (vehicle). The fold change of branching of neurons carrying SURF1 mutation is relatively calculated to vehicle-treated control carrying SURF1 mutation.

Figure 4: Mechanism of action of PDE5 inhibition in SURF1 defects. (A) Downstream effect of Sildenafil as a PDE5 inhibitor. (B) Expression level of the Sildenafil target PRKG1 in human neurons and brain organoids carrying SURF1 mutations.

EXAMPLES

The invention is demonstrated through the examples disclosed herein. The examples provided represent particular embodiments and are not intended to limit the scope of the invention. The examples are to be considered as providing a non-limiting illustration and technical support for carrying out the invention.

Described in more detail below is:

Development of a human model of Leigh syndrome.

Investigating effectiveness of PDE5 inhibitors in patients with Complex IV deficiency.

- Quantification of neuronal branching in iPSC-derived neurons from Leigh Syndrome patients.

- Validation of a PDE5 inhibitor therapeutic effect in iPSC-derived neurons (NPC cells) carrying a mutation within the SURF1 gene by measuring neuronal branching.

Example 1 : Schematic drawing of the development of a human model of Leigh syndrome using patient-derived induced pluripotent stem cells (carrying a SURF1 mutation) and CRISPR/Cas9 engineering. Single-cell RNA sequencing and multi-omics analyses revealed impaired neuronal morphogenesis in mutant neuronal cultures and brain organoids. The defects occurred at the level of neural progenitor cells (NPCs), which maintained a glycolytic proliferation state that did not instruct neuronal morphogenesis. SURF1 gene propagation supported metabolic programming of Leigh syndrome NPCs and resulted in restored neuronal morphogenesis. Induced pluripotent stem cells (iPSCs) from patients with SURF1 mutations and engineered with CRISPR/Cas9 as a model for Leigh syndrome. (Figure 1)

Example 2: Investigation of the PDE5 inhibitor class effectivity for Leigh Syndrome.

High-throughput microscopy assay for measuring branching outgrowth in iPSC derived neurons. High-content analysis (HCA) protocol for monitoring the growth capacity of human neurons derived from induced pluripotent stem cells (iPSCs). The steps comprise performing HCA imaging followed by quantification of dendrite and axon morphology in a high-throughput system to evaluate neurons obtained by differentiation approaches. This method can be applied to patient-derived iPSCs. This method was used to investigate effectiveness of PDE5 inhibitors in patients with Complex IV deficiency using quantification of neuronal branching in iPSC-derived neurons from Leigh Syndrome patients as outcome measure.

HCA analysis pipeline allows quantification of both axonal and dendritic length, number of branch points, staining signal areas, and other parameters. We employed HCA to quantify the neuronal branching outgrowth and complexity using Celli nsight CX7 microscope (Thermo Fisher Scientific, Waltham, MA, USA). Briefly, we split NPCs or DNs at 4 or 8 weeks of differentiation using Accutase and seeded them at a density of 10,000 cells/well on Matrigel- coated 96-well plates with black-wall and clear-bottom (Corning, Corning, NY, USA, #353219). We then stained the cells with TUJ1 antibody (Sigma-Aldrich, St. Louis, MO, USA, #T8578; 1 :3000) using 4% PFA (EMS, Thermo Fisher Scientific, Waltham, MA, USA, #50980487) for 20 min at RT and washed two times with PBS. For permeabilization, we incubated the fixed cells with a blocking solution containing 10% normal donkey serum (DNS) and 1 % Triton X- 100 (Sigma-Aldrich, St. Louis, MO, USA, #T8787) in PBS with 0.05% Tween 20 (Sigma- Aldrich, St. Louis, MO, USA, #P9416) (PBS-T) for 1 h at RT. We diluted primary antibodies in blocking solution, incubated them overnight at 4 °C on a shaker, and performed counterstaining with Hoechst. The morphogenesis of TU J 1 -positive cells within NPCs or DN cultures were quantified using the “Cellomics Neuronal Profiling v4 BioApplication” (Cell Insight CX7, High Content Platform, Thermo Fisher Scientific, Waltham, MA, USA). We used a similar HCA-based approach to quantify the number of TU J 1 -positive neurons and TH-positive neurons within DNs.

Example 3: Validation of therapeutic effect in NPC cell lines derived from patients with different SURF1 mutation by quantification of neuronal branching.

Figure 3 A shows the assay used for quantifying branching and length of branches in iPSCs derived neurons from Leigh Syndrome patients. The inventors generated neurons from patient-derived iPSCs (with SURF1 mutation or without mutation following CRISPR/Cas9 correction). These neuronal cells were grown in high-content microscopy plates for 5 days and the ability of these neurons from patient-derived iPSCs to generate branches (branches naturally occurring in neuronal cells) was quantified. Defective neuronal branching was found in iPSC-derived neurons with SURF1 mutations (Figure 3B, C) The SURF1 mutant neurons developed less branched and shorter branches. (Figure 3B, C). The SURF1 mutant neurons were exposed to either DMSO or sildenafil at various doses. The defects in the branching outgrowth was rescued by sildenafil in a significant manner (Figure 3D).

Example 4: Case report SURF1, compassionate use treatment with sildenafil

A very first female child carrying a homozygous variant in the SURF1 gene with healthy non- consanguineous parents with German ancestry was treated sildenafil.

Clinical data: Pregnancy, birth and first months of life were uneventful. At the age of six months, her parents noted recurrent episodes with vomiting. Moreover, there was a stagnation in developmental progress. Subsequently, swallowing difficulties and failure to thrive became evident. Growth parameters at the age of 1 % years: length 73 cm (SDS -3.05, P <0.5), weight 7.23 kg (SDS -3.9, P <0.5). During diagnostic work-up, elevated lactate levels were noted (4-7 mmol/l). Brain MRI showed symmetrical signal alterations in the brain stem compatible with Leigh syndrome. Under the suspicion of a mitochondrial disease, genetic testing via exome sequencing was performed. Analysis revealed a pathogenic, homozygeous variant in the SURF1 gene. Because of feeding problems a gastric tube was placed.

Treatment: At the age of 1 8/12 years, a compassionate use treatment with sildenafil was initiated (starting dose 3 x 0.5 mg/d; 0.15 mg/kg/day). Sildenafil was increased weekly up to a dose of 3 x 3 mg/day (1 mg/kg/day). Treatment was tolerated well and no obvious side effects were noted. Currently, the child is treated for a duration of 6 months. During this time period, her clinical and neurological status was stable. The child made significant developmental progress (speaks more than 50 words, speaks 2-word sentences, is able to take a few steps without support, walks for longer distances holding hands). The child had several febrile infections and a Norovirus gastroenteritis without signs of metabolic decompensation. She gained significant weight (currently 10.6 kg, P 12).

Example 5: Reduced PRKG1 expression in neurons and brain organoids carrying SURF1 mutations.

Sildenafil impacts on the expression of PRKG1 due to inhibition of PDE5 (Figure 4A) but has not been shown in neurons and brain organoids carrying SURF1 . The inventors found that PRKG1 expression is significantly lower in neurons and brain organoids carrying SURF1 mutations compared to isogenic controls (Figure 4B). According to these first findings of the inventors, normalization of PRKG1 levels may be a beneficial effect of Sildenafil in SURF1 associated diseases.

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Claims

1 . A phosphodiesterase 5 (PDE5) inhibitor for use in the treatment and/or prevention of a medical condition associated with mitochondrial Complex IV (cytochrome c oxidase) deficiency in a human subject.

2. The PDE5 inhibitor for use according to claim 1 , wherein the mitochondrial Complex IV deficiency comprises at least one DNA mutation in a structural subunit gene, or in an assembly gene, of the mitochondrial Complex IV (cytochrome c oxidase).

3. The PDE5 inhibitor for use according to any of the preceding claims, wherein the mutation in the mitochondrial Complex IV structural subunit or assembly gene comprises a variant at one or more nucleic acid positions in the nuclear genome or in the mitochondrial DNA.

4. The PDE5 inhibitor for use according to any of the preceding claims, wherein the DNA mutation is a nuclear mutation and is homozygous or compound heterozygous for a structural subunit or an assembly factor of the mitochondrial Complex IV.

5. The PDE5 inhibitor for use according to any of the preceding claims, wherein the DNA mutation occurs in the assembly factor gene of mitochondrial complex IV SURFEIT 1 (SURF1).

6. The PDE5 inhibitor for use according to any one of claims 1 -3, wherein a mitochondrial DNA mutation in MT-CO1 , MT-CO2 or MT-CO3 is homoplasmic or heteroplasmic, wherein said heteroplasmic mitochondrial DNA mutation is present in at least 30% of mitochondria per cell, preferably 60% or more, more preferably 70% or more.

7. The PDE5 inhibitor for use according to any of the preceding claims, wherein the medical condition comprises a Leigh Syndrome, Mitochondrial complex IV deficiency, nuclear type 1-23 or a neurological syndrome associated with a mitochondrial Complex IV deficiency.

8. The PDE5 inhibitor for use according to any of the preceding claims, wherein the PDE- 5 inhibitor is avanafil.

9. The PDE5 inhibitor for use according to any of the preceding claims, wherein the PDE- 5 inhibitor is sildenafil.

10. The PDE5 inhibitor for use according to any of the preceding claims, wherein the PDE- 5 inhibitor is tadalafil.

11 . The PDE5 inhibitor for use according to any of the preceding claims, wherein the PDE- 5 inhibitor is selected from the group consisting of vardenafil, mirodenafil, udenafil, and lodenafil.

12. The PDE5 inhibitor for use according to any of the preceding claims, wherein the inhibitor is administered: a) At a dosage of 0.1-10 mg/kg/day to a human subject, preferably at 0.5-5 mg/kg/day, more preferably at 1-2 mg/kg/day, and/or b) At a frequency of 2 to 4 times per day, preferably 4 times daily, wherein the route of administration is subcutaneously, intravenously or orally, preferably orally via a tablet of 1 mg to 10Omg per dose, more preferably 2mg to 80mg per dose, even more preferably 4mg to 40mg per dose.

13. The PDE5 inhibitor for use according to any of the preceding claims, wherein said dosage and frequency is configured to prevent and/or to reduce cGMP degradation to decrease intracellular calcium levels in striatal neuron mitochondria, to increase the number of branching points, and/or increase the length of neuronal branches different to a level at treatment initiation.

14. The PDE5 inhibitor for use according to any of the preceding claims, wherein the effect of said inhibitor to said subjects comprises:

Preventing and/or inhibiting cGMP degradation and increasing the number of branching points and increasing the length of neuronal branches to levels comparable to healthy subjects,

Reducing the number of paralytic events, motor and sensory neuropathy, metabolic crises events, lactic acidosis, cardiomyopathy, seizures, encephalopathy, stroke-like episodes, endocrine abnormalities, anaemia, exercise insufficiency, muscle weakness, loss of muscle tone and/or muscular hypotonia, respiratory insufficiency, retinitis pigmentosa, ataxia, increased susceptibility to infections, developmental delay, intellectual disability, compared to treatment initiation, and/or

Preventing and/or reducing the risk of said subject for repeated metabolic crises, palliative care or mortality, or resolves the need of the subject to receive palliative care.

15. A pharmaceutical composition comprising a PDE5 inhibitor for use as a medicament in the treatment and/or prevention of a medical condition according to any of the preceding claims, wherein said composition is in admixture with a pharmaceutically acceptable carrier and/or formulated in a pharmaceutically buffered solution.