WO2015197562A1 - Methods and pharmaceutical compositions for the treatment of disorders or diseases associated with ryanodine receptor dysfunction - Google Patents

Abstract

The present invention relates to method and pharmaceutical composition for the treatment of disorders or diseases associated with Ryanodine Receptor (RyR) dysfunction. In particular, the present invention relates to a method of treating a disease associated with a RyR dysfunction in a subject in need thereof comprising administering the subject with a therapeutically effective amount of F4-neuroprostane (F4-NeuroP).

Description

METHODS AND PHARMACEUTICAL COMPOSITIONS FOR THE TREATMENT OF DISORDERS OR DISEASES ASSOCIATED WITH RYANODINE RECEPTOR

DYSFUNCTION

FIELD OF THE INVENTION:

The present invention relates to method and pharmaceutical composition for the treatment of disorders or diseases associated with Ryanodine Receptor (RyR) dysfunction. BACKGROUND OF THE INVENTION:

Skeletal muscle contraction is activated by SR Ca²⁺ release via Ryanodine receptor calcium release channels. Depolarization of the transverse (T)-tubule membrane activates the dihydropyridine receptor voltage sensor (Cavl. l) that in turn activates RyRl channels via a direct protein-protein interaction causing the release of SR Ca²⁺ stores. Ca²⁺ binds to troponin C allowing actin-myosin cross-bridging to occur and sarcomere shortening.

RyRs are channels in the SR, which open and close to regulate the release of Ca²⁺ from the SR into the intracellular cytoplasm of the cell. Release of Ca²⁺ into the cytoplasm from the SR increases cytoplasmic Ca²⁺ concentration. Open probability of RyRs refers to the likelihood that a RyR is open at any given moment, and therefore capable of releasing Ca²⁺ into the cytoplasm from the SR. There are three types of RyR, all of which are highly homologous: RyRl, RyR2, and RyR3. RyRl is found predominantly in skeletal muscle as well as other tissues. The RyRl macromolecular complex consists of a tetramer of the 560-kDa RyRl subunit that forms a scaffold for proteins that regulate channel function including PKA and the phosphodiesterase 4D3 (PDE4D3), protein phosphatase 1 (PPl) and calstabinl . A-kinase anchor protein (mAKAP) targets PKA and PDE4D3 to RyRl, whereas spinophilin targets PPl to the channel (Marx et al. 2000; Brillantes et al, Cell, 1994, 77, 513-523; Bellinger et al. J. Clin. Invest. 2008, 118, 445-53). The catalytic and regulatory subunits of PKA, PPl, and PDE4D3 regulate PKA-mediated phosphorylation of RyRl at Ser2843 (Ser2844 in the mouse). It has been shown that PKA-mediated phosphorylation of RyRl at Ser2844 increases the sensitivity of the channel to cytoplasmic Ca²⁺, reduces the binding affinity of calstabinl for RyRl, and destabilizes the closed state of the channel (Reiken et al., 2003; Marx, S.O. et al, Science, 1998, 281 :818-821). Calstabinl concentrations in skeletal muscle are reported to be approximately 200 nM and that PKA phosphorylation of RyRl reduces the binding affinity of calstabinl for RyRl from approximately 100-200 nM to more than 600 nM. Thus, under physiologic conditions, reduction in the binding affinity of calstabinl for RyRl , resulting from PKA phosphorylation of RyRl at Ser2843, is sufficient to substantially reduce the amount of calstabinl present in the RyRl complex. Chronic PKA hyperphosphorylation of RyRl at Ser2843 (defined as PKA phosphorylation of 3 or 4 of the 4 PKA Ser2843 sites present in each RyRl homotetramer) results in "leaky" channels (i.e., channels prone to opening at rest), which contribute to the skeletal muscle dysfunction that is associated with persistent hyperadrenergic states such as occurs in individuals with heart failure (Reiken et al, 2003). Moreover, regulation of RyRl by posttranslational modifications other than phosphorylation, such as by nitrosylation of free sulfhydryl groups on cysteine residues (S-nitrosylation), as well as channel oxidation, have been reported to increase RyRl channel activity. S-nitrosylation and oxidation of RyRl have each been shown to reduce calstabinl binding to RyRl .

Accordingly, RyRl dysfunction is a hallmark of various muscular conditions. For example, it was recently demonstrated that excessive oxidation or nitrosylation of RyRl can disrupt the interaction of calstabinl with the RyRl complex, leading to RyRl leakiness and muscle weakness in a mouse model of muscular dystrophy (mdx) and that treatment with S 107 improves indices of muscle function in this mouse model (Bellinger, A. et al. 2009, Nature Medicine, 15 :325-330). Age-related loss of muscle mass and force (sarcopenia) contributes to disability and increased mortality. Andersson, D. et al. (Cell Metab. 201 1 Aug 3; 14(2): 196- 207) reported that RyRl from aged (24 months) mice is oxidized, cysteine-nitrosylated, and depleted of calstabinl, compared to RyRl from younger (3-6 months) adults. This RyRl channel complex remodelling resulted in "leaky" channels with increased open probability, leading to intracellular calcium leak in skeletal muscle. Treating aged mice with SI 07 stabilized binding of calstabinl to RyRl , reduced intracellular calcium leak, decreased reactive oxygen species (ROS), and enhanced tetanic Ca²⁺ release, muscle-specific force, and exercise capacity.

There is a need to identify new compounds effective for treating disorders and diseases associated with RyR dysfunction. More particularly, a need remains to identify new agents that can be used to treat RyR-associated disorders by, for example, preventing posttranslational modification such as PKA phosphorylation oxidation or nitrosylation and/or enhancing binding of calstabins to PKA-phosphorylated/oxidized/nitrosylated RyRs, and to mutant RyRs that otherwise have reduced affinity for, or do not bind to, calstabins.

SUMMARY OF THE INVENTION: The present invention relates to a method of treating a disease associated with a RyR dysfunction in a subject in need thereof comprising administering the subject with a therapeutically effective amount of F4-neuroprostane (F4-NeuroP).

DETAILED DESCRIPTION OF THE INVENTION:

The present invention provides compounds that are capable of treating diseases associated with a RyR dysfunction, especially with RyRl dysfunction. In particular, the present invention relates to a method of treating a disease associated with a RyR dysfunction in a subject in need thereof comprising administering the subject with a therapeutically effective amount of F4-neuroprostane (F4-NeuroP).

As used herein, the term "subject" denotes a mammal, such as a rodent, a feline, a canine, and a primate. Preferably, a subject according to the invention is a human.

As used herein, the terms "treating" or "treatment" or "alleviation" refers to therapeutic treatment, wherein the object is to prevent or slow down (lessen) the targeted disease. A subject is successfully "treated" for a particular disease, if after receiving a therapeutic amount of the F4-neuroprostane according to the invention; the subject shows observable and/or measurable reduction in or absence of one or more signs and symptoms of said disease, such as, e.g., a significant reduction in contractile dysfunction. In a particular embodiment the treatment is a prophylactic treatment. The term "prophylactic treatment" as used herein, refers to any medical or public health procedure whose purpose is to prevent a disease. As used herein, the terms "prevent", "prevention" and "preventing" refer to the reduction in the risk of acquiring or developing a given condition, or the reduction or inhibition of the recurrence or said condition in a subject who is not ill, but who has been or may be near a subject with the disease. It is also to be appreciated that the various modes of treatment or prevention of medical conditions as described are intended to mean "substantial," which includes total but also less than total treatment or prevention, and wherein some biologically or medically relevant result is achieved.

As used herein the term "disorder or diseases associated with RyR dysfunction" means any disorder and disease that can be treated and/or prevented by modulating the RyR receptors that regulate calcium channel functioning in cells. "Disorders and diseases associated with RyR dysfunction" include, without limitation, cardiac disorders and diseases, skeletal muscular disorders and diseases, cognitive disorders and diseases, malignant hyperthermia, diabetes, and sudden infant death syndrome. In some embodiments, the disorder or disease is associated with an abnormal function of RyR 1 .

In some embodiments, the method of the present invention is particularly suitable for treating a condition selected from, the group consisting of cardiac disorders and diseases, muscle fatigue, musculoskeletal disorders and diseases, Central Nervous System (CNS) disorders and diseases, cognitive dysfunction, bone disorders and diseases, malignant hyperthermia, diabetes, sudden cardiac death, and sudden infant death syndrome, or for improving cognitive function. Cardiac disorders and diseases include, but are not limited to, irregular heartbeat disorders and diseases, exercise-induced irregular heartbeat disorders and diseases, heart failure, congestive heart failure, chronic heart failure, acute heart failure, systolic heart failure, diastolic heart failure, acute decompensated heart failure, cardiac ischemia reperfusion (I/R.) injury ( including 1 R injury following coronary angioplasty or following thrombolysis during myocardial infarction (MI)), chronic obstructiv e pulmonary disease, and high blood pressure. Irregular heartbeat disorders and diseases include, but are not limited to atrial and ventricular arrhythmia, atrial and ventricular fibrillation, atrial and ventricular tachyarrhythmia, atrial and ventricular tachycardia, catechoiaminergic polymorphic ventricular tachycardia (CPVT), and exercise-induced variants thereof.

The method of the present invention is also particularly suitable for the treatment of muscle fatigue, which may be due to prolonged exercise or high-intensity exercise, or may be caused by a muscular disorder or disease. Examples of muscular disorders and diseases include, but are not limited to, skeletal muscle fatigue, central core diseases, exercise-induced skeletal muscle fatigue, bladder disorders, incontinence, age-associated muscle fatigue, sarcopenia, congenital myopathies, cancer cachexia, myopathy ith cores and rods, mitochondrial myopathies [e.g., Kearns-Sayre syndrome, MELA.S (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke) syndrome, and MERRF (myoclonus epilepsy with ragged-red fibers) syndrome], endocrine myopathies, muscular glycogen storage diseases [e.g., Pompe's disease, Andersen's disease, and Cori's diseases], myoglobinurias [e.g., McArdie's disease, Tarui disease, and DiMauro disease], dermatomyositis, myositis ossificans, familial periodic paralysis, polymyositis, inclusion body myositis, neuromyotonia, stiff- man syndrome, mal ignant hyperthermia, common muscle cramps, tetany, myasthenia gravis, and muscular dystrophy. Examples of muscular dystrophy include, but are not limited to, Duchenne Muscular Dystrophy (DMD), Becker's Muscular Dystrophy (BMD), Limb Girdle Muscular Dystrophy

(LGMD), Congenital Muscular Dystrophy (CMD), distal muscular dystrophy, facioscapulohumeral dystrophy, myotonic muscular dystrophy, Emery-Dreifuss muscular dystrophy, and oculopharyngeal muscular dystrophy. Congenital muscular dystrophy as used herein refers to muscular dystrophy that is present at birth. CMD is classified based on genetic mutations: 1) genes encoding for structural proteins of the basal membrane or extracellular matri o the skeletal muscle fibres; 2) genes encoding for putative or demonstrated glycosyltransf erases, that in turn affect the glycosylation of dystroglycan, an external membrane protein of the basal membrane; and 3) other. Examples of CMD include, but are not limited to Laminin-a2-deficient CMD (MDC1A), Ullrich CMC (UCMDs 1 , 2 and 3), Walker- Warburg syndrome (WWS), Muscle-eye-brain disease (MEB), Fukuyama CMD (FCMD), CMD plus secondary lam in in deficiency 1 (MDC1B), CMD plus secondary laminin deficiency 2 (MDC 1C), CMD with mental retardation and pachygyria (MDC 1D), and Rigid spine with muscular dystrophy Type 1 (RSMD1).

Cognitive disorders, diseases or dysfunction include, but are not limited to, Alzheimer's Disease, memory loss, age-dependent memory loss, post-traumatic stress disorder (PTSD), a neuropathy and seizures. The cognitive dysfunct ion may be stress-related, age-related or a combination thereof. Alternatively, the cognitive dysfunction is associated with a disease or disorder, including but not limited to, Alzheimer's disease (AD), attention deficit hyperactiv ity disorder (ADHD), autism spectrum disorder ( ASD), generalized anxiety disorder (GAD), obsessive compulsive disorder (OCD), Parkinson's Disease (PD), post-traumatic stress disorder (PTSD), Schizophrenia, Bi olar disorder; and major depression. In some embodiments, the method of the present invention is particularly suitable for the treatment of ventilator-induced diaphragmatic dysfunction. As used herein, the expression "ventilator-induced diaphragmatic dysfunction" or "VIDD" has its general meaning in the art and refers to the condition wherein diaphragmatic atrophy and contractile dysfunction occur after prolonged controlled mechanical ventilation (Powers SK, Wiggs MP, Sollanek KJ, Smuder AJ. Invited Review: Ventilator-induced diaphragm dysfunction: cause and effect. Am J Physiol Regul Integr Comp Physiol. 2013 Jul 10.). Ventilator-induced diaphragmatic dysfunction may result from prolonged controlled mechanical ventilation (MV), e.g., greater than 12 hours. However, such prolonged MV is not limited to any specific time-length. For example, in some embodiments, prolonged MV includes a time from at least about 0.5, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 50, or 100 hours, to from at least about 1, 10, 20, 50, 75, 100 or greater hours, days, or years. In another embodiment, prolonged MV includes a time from at least about 5, 6, 7, 8, 9 or 10 hours, to from at least about 10, 20 or 50 hours. In some embodiments, prolonged MV is from about at least 10-12 hours to any time greater than the 10- 12 hour period.

In some embodiment, the subject needs artificial respiratory support because he suffers from respiratory failure and/or heart failure, which can be aggravated by sepsis, metabolic disorder, neuromuscular diseases, or surgery along with post-surgical recovery. Typically, the subject suffers from a disease for which the worsening of the symptoms has led the subject to need artificial respiratory support (i.e. mechanical ventilation). For example, some lung diseases, such as Chronic Obstructive Pulmonary Disease (COPD), pneumonia, sepsis (including severe sepsis and septic shock), Acute Respiratory Distress Syndrome (ARDS), Severe Acute Respiratory Syndrome (SARS) and cystic fibrosis (CF) usually require some form of ventilation assistance in order to clinically improve the subject. In some embodiments the subject suffers from a trauma. Pulmonary dysfunction in trauma patients is multifactorial and may be the result of direct contusion of the lung tissue, lung injury by fractured ribs, loss of chest wall function, fat embolism to the lung from long bone fractures, aspiration of blood or gastric contents and the consequences of the activation of the systemic inflammatory response syndrome (SIRS) of shock, reperfusion, and transfusion therapy.

In some embodiments, the F4-neuroprostane is administered before MV, immediately after MV initiation, during MV, and/or immediately after MV. In some embodiments, administration of the F4-neuroprostane according to the invention is provided at any time during MV.

The F4-neuroprostane according to the invention is also suitable for preventing risks associated with ventilator-induced diaphragmatic dysfunction. Risks associated with ventilator dependence include increased discomfort and risk of secondary diseases for the patient (such as pneumonia, pulmonary fibrosis, aspiration, acute renal failure, cardiac arrhythmias, sepsis, vocal fold dysfunction, and acute lung injury secondary to barotrauma or volotrauma), increased morbidity and mortality, high health care costs, and longer treatment duration times. Although patients with chronic ventilator dependency (CVD) comprise only 5% to 10% of patients in intensive care units, they consume approximately 50%> of all ICU resources, as measured in staff time and equipment usage. Specifically, it has been estimated that weaning patients consumed about 41% of total ventilation time in intensive care unit patients. The economic cost of long term MV dependence is enormous. Episodes of long term MV dependency can financially devastate families and health care institutions and are a financial drain on private insurers and government health care resources.

As used herein, the term "F4-neuroprostane" has its general meaning in the art and refers to the class of lipid oxidation metabolites derived from docosahexanoic acid (F4-isoprostanes: a novel class of prostanoids formed during peroxidation of docosahexaenoic acid (DHA). Nourooz-Zadeh J., Liu E., Anggard E., Halliwell B,. Biochem. Biophys. Res. Com., 1998, 242, 338.; Regiochemistry of neuroprostanes generated from the peroxidation of docosahexaenoic acid in vitro and in vivo. Yin H., Musiek E., Gao L., Porter N. & Morrow J.. J. Biol. Chem. 2005, 280: 2600). In particular, F₄-NeuroPs include but are not limited to 4-F₄-NeuroPs, 7-F₄- NeuroPs, 1 l-F₄-NeuroPs, 10-F₄-NeuroPs, 14-F₄-NeuroPs, 13-F₄-NeuroPs, 17-F₄-NeuroPs and 20-F₄-NeuroPs (Figure 1). F₄-NeuroPs may be synthesised through any method well known in the art. Typically, the compounds may be synthesized by the method described in: The handy use of Brown's catalyst for a skipped diyne deuteration: application to the synthesis of a d4- labelled-F4t-neuroprostane. Oger C, Bultel-Ponce V., Guy A., Balas L., Rossi J-C, Durand T., Galano J-M. Chem. Eur. J. 2010, 16, 13976. In a particular embodiment, the epimers of configuration (S) of the allylic hydroxyl or F₄-NeuroPs with a (S) absolute configuration of the allylic hydroxyl are used.

According to the invention, the F₄-NeuroP is administered in a therapeutically effective amount. By a "therapeutically effective amount" is meant a sufficient amount of the F4-NeuroP to treat the target disorder or disease at a reasonable benefit/risk ratio applicable to any medical treatment. It is understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, gender and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. In particular, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, in particular from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.

The F4-neuroprostane of the invention is typically combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to be administered in the form of a pharmaceutical composition. "Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms. Typically, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The antibody can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active antibody in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum- drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES: Figure 1: biosynthesis of F4-neuroprostanes

Figure 2: Calcium sparks frequency recorded in saponin skinned EDL muscle fibres from control (n=5), post myocardial infarction (PMI, n=2) and post myocardial infarction incubated with 4-(RS)-4F_4t-neuroprostane (4F4T) ΙμΜ (PMI + 4F4T, n=3) mice. *, p<0.05.

Figure 3: 4F4T prevents diaphragm weakness induced after 6h of mechanical ventilation in anesthetized mice. A/ Force-frequency relationships in diaphragms after 6 h of mechanical ventilation with or without iv injection of 4F4T. B/ Mean maximal force production recorded at 120Hz in control diaphragms, after 6h mechanical ventilation (VIDD) and VIDD treated with 4F4T. Data expressed as mean±SEM, *, p<0.05.

Figure 4: Dose effect of 4F4T on VIDD. Mice were ventilated for 6 hours and treated with 4F4T at different concentration ranging from 0.1 to 100 μΜ. The dashed line VIDD correspond to the force production in the absence of treatment. Data were fitted with a standard Hill equation (line) giving an EC50 of 0.39μΜ. Each data point (mean±SEM) corresponds to a group of 6 mice ventilated for 6 hours and treated with the corresponding dose of 4F4T. Figure 5: Post-translational modification of RyRl after VIDD: impact of 4F4T. Mean values of DNP/RyRl, P-RyRl/RyRl and Calstabinl/RyRl reflecting respectively RyRl oxidation, phosphorylation on ser2844 and calstabinl interaction. VIDD affects negatively all parameters and post-translational remodeling is prevented by 4F4T treatment. EXAMPLE:

Material & Methods

Murine model of Heart failure:

Seven week-old male C57B1/6 mice (Janvier, France) were subjected to a post myocardial infarction after left coronary artery ligation (PMI mice). Briefly, a left thoracotomy was performed under anesthesia and cardiac monitoring (2% isoflurane/02 , Aerrane®, Baxter, France). The artery was ligated 1-2 mm beyond the emergence from the top of the left atrium, using an 8-0 suture. A subcutaneous injection of 0.01 ml buprenorphine solution (0.3 mg.ml-1) for post-operative analgesia was administered. An echocardiography was systematically realized before the inclusion of animal to ensure that ligation was correctly performed. Only animals surviving at day -5 post surgery and with comparable echocardiography parameters at this time were included in the study to limit bias due to differences in size infract. All procedures conformed to European Parliament Directive 2010/63/EU and the 22 September 2010 Council on the protection of animals, and were approved by the institutional animal research committee (Departmental Directorate of protecting populations and animal health (ethics for animal welfare and environmental protection, N° A 34 -485) and by our Ethics committee for animal experiments, Languedoc Roussillon, N° CE -LR-0714). 10 to 12 weeks after ligation, mice were euthanized by cervical dislocation and the EDL muscle dissected for calcium sparks recording.

Calcium sparks measurements.

Diaphragm muscles samples were dissected and stored in a HEPES buffered physiological medium (in mM: 119 NaCl, 5 KC1, 1.25 CaCl₂, 1 MgS0₄, 10 glucose, 1.1 mannitol, 10 HEPES, pH 7.4). Muscles were then rapidly placed in a dissecting chamber and the solution exchanged with a relaxing solution (in mM: 140 K-glutamate, 10 HEPES, 10 MgCl₂, 0.1 EGTA, pH 7.0). Bundles of 5 to 10 EDL fibers were manually dissected, mounted as described previously [13, 14, 16] and permeabilized in a relaxing solution containing 0.01% saponin for 30 s. After washing with saponin free solution, the solution was changed to an internal medium for imaging: (in mM) 140 K-glutamate, 5 Na₂ATP, 10 glucose, 10 HEPES, 4.4 MgCl₂, 1.1 EGTA, 0.3 CaCl₂, Fluo-4 0.05 pentapotassium salt (Invitrogen), pH 7.0, for sparks acquisition as previously reported [14]. Potential sparks were empirically identified using an autodetection algorithm [14]. The mean fluorescence (F0) value for the image was calculated by summing and averaging the temporal F at each spatial location, while ignoring potential spark areas. This F0 value was then used to create a smoothing routine, potential spark locations were visualized and analyzed for spatiotemporal properties as described previously [15]. Image analysis was performed using IDL (v5.5, Research System, Inc.). Statistical comparisons were performed using an ANOVA test with a significance level set at P<0.05 (Sigmastat v3.5).

Murine model of VIDD

The experimental design has been described in recent study [17]. In brief, C57/BL6 mice (10 to 12 weeks old, 25 to 30g) were intubated with a 22-gauge angio- catheter and mechanically ventilated for 6 consecutive hours using a volume-driven small-animal ventilator (Minivent®, Harvard Apparatus, Saint-Laurent, Canada). Tidal volume was established at ΙΟμΙ/mg body weight with a respiratory rate of 150 breaths/min, a positive end-expiratory pressure (PEEP) level from 2 to 4cm H₂0 and a fraction of inspired oxygen of 0.21. Non- spontaneous ventilation was defined as a lack of diaphragm contractile activity attested by repetitive stereotypical deflections observed in the airway pressure curve.

The mice were divided into two groups. The first group (VIDD) received a single injection of 4F4T (300μ1 of a ΙΟΟμΜ solution) intravenously (IV) infused over a 5-minute period, before start of MV. The second group received same volume of IV saline 20 minutes before starting and during MV.

Contractile function in murine muscle samples

At the end of the protocol of MV, the entire diaphragm was surgically excised and mice were euthanized, by exsanguination. Isometric contractile properties were assessed as described previously in detail [17]. The excised diaphragm strip was mounted into jacketed tissue bath chambers filled with equilibrated and oxygenated Krebs solution. The muscles were supra- maximally stimulated using square wave pulses (Model S48; Grass Instruments, West Warwick, RI). The force-frequency relationship was determined by sequentially stimulating the muscles for 600 ms at 10, 20, 30, 50, 60, 80, 100 and 120Hz with 1 minute between each stimulation train. After measurement of contractile properties, muscles were measured at Lo (the length at which the muscle produced maximal isometric tension), dried and weighted. For comparative purposes, diaphragmatic force production was normalized for total muscle strip cross-sectional area and expressed in N.cm^"2. The total muscle strip cross-sectional area was determined by dividing muscle weight by its length and tissue density (1.056 g/cm3).

The rest of the diaphragm was partitioned, one part was quick frozen in liquid nitrogen and secondarily used for biochemical analysis, and the other part was used freshly for Ca²⁺ spark measurements.

RyRl biochemical analysis

Muscle biopsies were homogenized in 150μ1 of buffer containing 5% SDS, 5% beta- mercaptoethanol, 10% glycerol, 10 mM EDTA and 50 mM Tris/HCl buffer (pH= 8.0). Each sample was immediately denatured at 90°C for 4 min. After centrifugation (5000 rpm) at 4°C, supernatant protein concentrations were measured in duplicate using the BCA protein assay, equilibrated at the same concentration by dilution with loading buffer and aliquoted at 2μg/μl. RyRl was immunoprecipitated from 250μg of homogenate using an anti-RyR antibody (4μg RyRl -1327) in 0.5ml of a modified RIPA buffer (50mM Tris-HCl pH 7.4, 0.9% NaCl, 5.0mM NaF, l .OmM Na₃V0₄, 1% Triton-X100, and protease inhibitors) for lhr at 4°C. The immune complexes were incubated with protein A Sepharose beads (Amersham Pharmacia) at 4°C for lhr and the beads were washed three times with buffer. Proteins were separated on SDS-PAGE gels (4-20% gradient) and transferred onto nitrocellulose membranes for 2hr at 200mA (SemiDry transfer blot, Bio-Rad). To prevent non-specific antibody binding, the membranes were incubated with blocking solution (LICOR Biosciences) and washed with Tris-buffered saline with 0.1% Tween-20.

Blots were respectively incubated with primary antibody to RyRl (RyRl -1327, an affinity-purified rabbit polyclonal antibody raised against a KLH-conjugated peptide corresponding to residues 1327-1339 of mouse skeletal RyRl, with an additional cysteine residue added to the amino terminus), and affinity purified with the unconjugated peptide. We also used antibody to calstabinl (1 : 2500 in blocking buffer, LICOR Biosciences); phospho- epitope-specific antibody to human RyR2 phosphorylated on Ser-2808 (1 :5,000), which detects PKA-phosphorylated mouse RyRl (on Ser-2844) and RyR2 (on Ser-2808); antibody to S- nitrosylated cysteine residues (1 : 1000, Sigma). To determine RyRl oxidation, the immunoprecipitate was treated with 2, 4-dinitrophenyl hydrazine, and the derivatized carbonyls were detected using an OxyBlot protein oxidation detection Kit (catalog S7150, Chemicon International Inc.). After three washes, membranes were incubated with infrared-labeled secondary antibodies. Control samples were analyzed on each gel for normalization and total levels of RyRl were not different between groups.

Results

Accute application of 4F4T prevents RyRl mediated Ca²⁺ leak

The term of spontaneous Ca²⁺ release events (i.e. Ca²⁺ sparks) refers to local Ca²⁺ release events arising spontaneously from a single Ca²⁺ release unit (CRU) in a resting muscle fiber [12]. Each CRU corresponds to a cluster of RyRs. Depending on the type of muscle and species, a CRU may contain a variable number of RyRs, remaining approximate (from a few tens up to a few hundred). Therefore, by measuring Ca²⁺ sparks in a muscle fiber, we have a direct measurement in situ of the gating behavior of RyRs belonging to a single CRU. Most particularly, the frequency of Ca²⁺ sparks gives a good approximation of the open probability of RyRl and represents an index of RyR-mediated SR calcium leak.

We have previously reported a leaky beaviour of RyRl in experimental models of heart failure [13, 14]. In figure 2, we confirm, in a mice model of heart failure obtained after permanent ligature (PMI) of the cardiac left anterior descending coronary artery, an impaired function of RyRl in the fast twitch Extensor digitorum longus (EDL) muscle as shown by the significant increase in sparks frequency reflecting SR calcium leak. When PMI muscle fibers were incubated with 1 μΜ 4(RS)-4F4t-neuroprostanes (4F4T), one could observe a full prevention of this RyRl -dependent leaky behavior.

4F4T in vivo prevents muscle weakness induced by mechanical ventilation

Ventilator-induced diaphragmatic dysfunction (VIDD) refers to the diaphragm muscle weakness following prolonged controlled mechanical ventilation (MV). The presence of VIDD impedes recovery from respiratory failure, but the pathophysiological mechanisms accounting for VIDD are still not fully understood. We have developed in our laboratory the first murine model of VIDD that exhibit after 6 hours of ventilation a 30% decrease in force production without any histological signs of damages or necrosis. Using this model (Figure 3) we can observe that the muscle weakness induced by mechanical ventilation is fully prevented by a single IV injection of 4F4T (300μ1 of a ΙΟΟμΜ solution). A dose response of the inhibitory effect of 4F4T on VIDD was further performed (Figure 4). It indicates an EC50 of about 0.4μΜ and a maximal effect reached at about ΙμΜ.

Post-translational modification of RyRl during VIDD: effect of 4F4T

Diaphragm samples from the experiments above were frozen and solubilized to further investigate the biochemical properties of the RyRl macromolecular complex. After 6 hours of mechanical ventilation, VIDD is responsible for an oxidation of the channel as well as a phosphorylation on ser2844 and depletion from calstabinl (Figure 5). This is prevented by 4F4T. This is however important to note that in contrast with Rycal effects on RyR remodeling, 4F4T is able to rebind calstabinl but also to prevent oxidation and phosphorylation of the channel. This observation is similar to that previously reported by our group [15] where in the heart after ischemia reperfusion such post-translational modifications of RyR2 was not prevented by the Rycal S 107 while it was prevented by caspase-8 inhibitors suggesting an effect of an upstream mechanism.

Therefore this experiment, strongly supports the hypothesis that the beneficial effect of 4FT4 on RyRl is unlikely to be direct on the channel itself as it is the case for Rycals, but due to an upstream signaling pathway involved in RyR remodeling and post-translational modifications.

REFERENCES: Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

[7] W.L. Song, J.A. Lawson, D. Reilly, J. Rokach, C.T. Chang, B. Giasson, G.A. FitzGerald, Neurofurans, novel indices of oxidant stress derived from docosahexaenoic acid, J. Biol. Chem. 283 (2008) 6-16.

[8] A. De La Torre, Y.Y. Lee, C. Oger, P.T. Sangild, T. Durand, C.Y.J. Lee, J.-M. Galano, Synthesis, Discovery and Quantitation of dihomo-Isofurans: Novel Biomarkers of in Vivo Adrenic Acid Peroxidation, Angew. Chem. Int. Ed. in press (2014) DOI: 10.1002/anie.201402440Rl . [9] J. Nourooz-Zadeh, B. Halliwell, E.E. anggard, Evidence for the Formation of F3-Isoprostanes during Peroxidation of Eicosapentaenoic Acid, Biochem. Biophys. Res. Commun. 236 (1997) 467-472.

[10] L.J. Roberts, T.J. Montine, W.R. Markesbery, A.R. Tapper, P. Hardy, S. Chemtob, W.D. Dettbarn, J.D. Morrow, Formation of Isoprostane-like Compounds (Neuroprostanes) in Vivo from Docosahexaenoic Acid, J. Biol. Chem. 273 (1998) 13605- 13612.

[11] H. Yin, E.S. Musiek, L. Gao, N.A. Porter, J.D. Morrow, Regiochemistry of neuroprostanes generated from the peroxidation of docosahexaenoic acid in vitro and in vivo, J Biol Chem. 280 (2005) 26600-11.

[12] Cheng H & Lederer WJ (2008) Calcium sparks. Physiological reviews 88(4): 1491-1545.

[13] Reiken S, et al. (2003) PKA phosphorylation activates the calcium release channel (ryanodine receptor) in skeletal muscle: defective regulation in heart failure. The Journal of cell biology 160(6):919-928.

[14] Ward CW, et al. (2003) Defects in ryanodine receptor calcium release in skeletal muscle from post-myocardial infarct rats. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 17(11): 1517-1519.

[15] Fauconnier J, et al. (2011) Ryanodine receptor leak mediated by caspase-8 activation leads to left ventricular injury after myocardial ischemia-reperfusion. Proceedings of the National Academy of Sciences of the United States of America 108(32): 13258-13263.

[16] Bellinger AM, et al. (2009) Hypernitrosylated ryanodine receptor calcium release channels are leaky in dystrophic muscle. Nat Med 15(3):325-330.

[17] Mrozek S, et al. (2012) Rapid onset of specific diaphragm weakness in a healthy murine model of ventilator-induced diaphragmatic dysfunction. Anesthesiology 117(3):560- 567.

Claims

CLAIMS:

1. A method of treating a disease associated with a Ryanodine receptor (RyR) dysfunction in a subject in need thereof comprising administering the subject with a therapeutically effective amount of F4-neuroprostane (F4-NeuroP).

2. The method of claim 1 wherein the disease is selected from the group consisting of cardiac disorders and diseases, skeletal muscular disorders and diseases, cognitive disorders, diseases or dysfunction, malignant hyperthermia, diabetes, and sudden infant death syndrome.

3. The method of claim 1 wherein the disease is selected from the group consisting of cardiac disorders and diseases, muscle fatigue, musculoskeletal disorders and diseases, CNS disorders and diseases, cognitive dysfunction, bone disorders and diseases, malignant hyperthermia, diabetes, sudden cardiac death, and sudden infant death syndrome, or for improving cognitive function.

4. The method of claim 3 wherein the cardiac disorders is selected from the group consisting of irregular heartbeat disorders and diseases, exercise-induced irregular heartbeat disorders and diseases, heart failure, congestive heart failure, chronic heart failure, acute heart failure, systolic heart failure, diastolic heart failure, acute decompensated heart failure, cardiac ischemia/reperfusion (I/R) injury, chronic obstructive pulmonary disease, high blood pressure and irregular heartbeat disorders and diseases including but not limited to atrial and ventricular arrhythmia, atrial and ventricular fibrillation, atrial and ventricular tachyarrhythmia, atrial and ventricular tachycardia, catecholaminergic polymorphic ventricular tachycardia (CPVT), and exercise-induced variants thereof.

5. The method of claim 2 wherein the musculoskeletal disorder is selected from the group consisting of muscle fatigue, central core diseases, exercise-induced skeletal muscle fatigue, bladder disorders, incontinence, age-associated muscle fatigue, sarcopenia, congenital myopathies, cancer cachexia, myopathy with cores and rods, mitochondrial myopathies, endocrine myopathies, muscular glycogen storage diseases, myoglobinurias, dermatomyositis, myositis ossificans, familial periodic paralysis, polymyositis, inclusion body myositis, neuromyotonia, stiff-man syndrome, malignant hyperthermia, common muscle cramps, tetany, myasthenia gravis, and muscular dystrophy.

6. The method of claim 5 wherein the muscular dystrophy is selected the group consisting of Duchenne Muscular Dystrophy (DMD), Becker's Muscular Dystrophy (BMD), Limb Girdle Muscular Dystrophy (LGMD), Congenital Muscular Dystrophy (CMD), distal muscular dystrophy, facioscapulohumeral dystrophy, myotonic muscular dystrophy, Emery-Dreifuss muscular dystrophy, and oculopharyngeal muscular dystrophy.

7. The method of claim 2 wherein the cognitive disorders, diseases or dysfunction is selected from the group consisting of Alzheimer's Disease, memory loss, age-dependent memory loss, post-traumatic stress disorder (PTSD), neuropathy and seizures, attention deficit hyperactivity disorder (ADHD), autism spectrum disorder (ASD), generalized anxiety disorder (GAD), obsessive compulsive disorder (OCD), Parkinson's Disease (PD), post-traumatic stress disorder (PTSD), Schizophrenia, Bipolar disorder; and major depression.

8. The method of claim 2 wherein the disease is ventilator-induced diaphragmatic dysfunction.

9. The method of claim 8 wherein the subject needs artificial respiratory support because he suffers from respiratory failure and/or heart failure which, can be aggravated by sepsis, metabolic disorder, neuromuscular diseases, or surgery along with post-surgical recovery.

10. The method of claim 8 wherein the subject suffers from a disease for which the worsening of the symptoms has led the subject to need mechanical ventilation.

11. The method of claim 10 wherein the disease is selected from the group consisting of Chronic Obstructive Pulmonary Disease (COPD), pneumonia, sepsis, Acute Respiratory Distress Syndrome (ARDS), Severe Acute Respiratory Syndrome (SARS) and cystic fibrosis (CF).

12. The method of claim 8 wherein the subject suffers from a trauma.

13. The method of claim 8 wherein the ventilator-induced diaphragmatic dysfunction results from prolonged controlled mechanical ventilation (MV) greater than 12 hours.

14. The method of claim 1 wherein the F4-neuroprostane is administered before MV, immediately after MV initiation, during MV, and/or immediately after MV.

15. The method of claim 1 wherein the F4-neuroprostane is selected from the group of epimers of configuration (S) of the allylic hydroxyl or F4-NeuroPs with a (S) absolute configuration of the allylic hydroxyl.