WO2015121500A1

WO2015121500A1 - Pharmaceutical compositions for use in the treatment of ventilator-induced diaphragmatic dysfunction

Info

Publication number: WO2015121500A1
Application number: PCT/EP2015/053332
Authority: WO
Inventors: Alain Lacampagne; Stefan MATECKI; Boris JUNG; Haikel DRIDI; Samir JABER
Original assignee: Institut National de la Sante et de la Recherche Medicale INSERM; Centre Hospitalier Universitaire de Montpellier; Universite de Montpellier
Current assignee: Institut National de la Sante et de la Recherche Medicale INSERM; Centre Hospitalier Universitaire de Montpellier; Universite de Montpellier
Priority date: 2014-02-17
Filing date: 2015-02-17
Publication date: 2015-08-20
Anticipated expiration: 2016-08-17

Abstract

The present invention relates to pharmaceutical compositions for use in a method of treatment of ventilator-induced diaphragmatic dysfunction. In particular, the present invention relates to a therapeutically effective amount of a beta2-adrenergic receptor antagonist for use in a method for the treatment of ventilator-induced diaphragmatic dysfunction in a subject in need thereof.

Description

PHARMACEUTICAL COMPOSITIONS FOR USE IN THE TREATMENT OF VENTILATOR-INDUCED DIAPHRAGMATIC DYSFUNCTION

FIELD OF THE INVENTION:

The present invention relates to methods and pharmaceutical compositions for the treatment of ventilator-induced diaphragmatic dysfunction.

BACKGROUND OF THE INVENTION:

Diaphragm muscle is the main respiratory muscle, which can be impaired by unloading induced by mechanical ventilation (MV). Mechanical ventilation is the most common life saving procedure that is done in critically ill patients and represents 25% of the patients admitted worldwide in the intensive care units¹. Although it is mandatory to save the live in most of the severe patients, MV has also been implicated in serious side effects, pulmonary, but also more recently muscular. Indeed, controlled MV (a mode in which, patients are deeply anesthetized and their respiratory muscles are unloaded) has been suspected to induce Ventilator-Induced Diaphragmatic Dysfunction (VIDD)². VIDD can impair the weaning process from the ventilator which already represents 40-50% of the time spent under MV ³ thus increasing the ICU and hospital length of stay, nosocomial infections risk, morbidity and mortality. VIDD prevention and/or treatment are therefore a need in order to decrease the costs of hospital stay but also to improve the morbidity risk. Indeed, Intensive care hospitalization is associated with a median daily cost in Europe of 2200€ ⁴. Delay to wean patient from MV is associated with a longer ICU length of stay but also a longer hospital length of stay which will increase the cost but also nosocomial complications occurrence.

Thus any pharmacological agent able decrease the incidence of VIDD will have a major economic issue with the decrease of the major impact on clinical practice and the utilization of health care resources.

VIDD pathophysiology has been explored in animal models and basically although nervous impulse transmission at the levels of the phrenic nerve and the neuromuscular junction remains normal it combines an impairment of intrinsic force production, expressed as N/cm² and a lower force developed during a fatigue resistance test. Beyond decreased diaphragmatic strength, a number of histological and biochemical changes have been described in the diaphragms of animals with VIDD. These include muscle fiber atrophy ^{5 8} , which appears to be the result of decreased protein synthesis ⁹' ¹⁰ as well as increased protein breakdown ^{11 14}; muscle fiber remodeling, as indicated by changes in the expression of multiple structural ¹⁵ as myosin heavy chain and muscle-specific proteins transcription factor involved in muscle development, myogenesis and repair such as myogenic determination factor 1 (MyoD), and myogenin ⁸' ¹⁶; and signs of muscle fiber injury, including disrupted myofibrils, increased numbers of vacuolar structures, and abnormal mitochondria ⁶' ¹⁴' ¹⁷' ¹⁸. Our team reported for the first time presence of sarcomeric disruptions evaluated by electronic microscopy in a rabbit model of VIDD ⁶. Several of these histological and biochemical changes have been linked to an increased level of oxidative stress in the diaphragm, which can be observed 6 hours after MV initiation in rats ¹⁹ and after 72 hours in piglets ²⁰. But sources of reactive oxygen production in VIDD are still poorly known. Although several antioxidant therapies have been tested with mitigate results, including xanthine oxidase or NADPH inhibitors ²¹ ' ²² , SS-31, a dedicated anti-oxidative molecule which targets specifically the mitochondrial production of superoxide seems to be promising in a rat model ²³ and strongy suggests the mitochondria as the main source of excessive ROS production in the diaphragm during mechanical ventilation. In this regard, mitochondria isolated from mechanically ventilated rats release significantly more ROS and also exhibit biochemical evidence of oxidative damage ²⁴. Furthermore, our team and others found abnormalities of diaphragmatic mitochondrial respiration in several animal species during mechanical ventilation ^{6 25 24}.

In the last 5 years, animal studies have been confirmed by a few Human studies ²' ^26~29. Those studies were performed on brain dead patients ventilated 3-10 days in the ICU before their death (long term MV group). During the organ harvesting, diaphragm samples were removed and compared to patients in whom diaphragm samples were removed during pneumectomy for cancer (short term MV group). In a landmark study, Levine et al reported in the long term MV group a decreased cross-sectional area of slow-twitch and fast-twitch fibers (atrophy), decreased glutathione levels (suggesting increased oxidative stress), and greater expression of active caspase-3 and the E3 ubiquitin ligases atrogin-1, and muscle RING- finger protein- 1 (MuRF-1) (implicated in muscle proteolysis) ² in comparison with the short term MV group. A study from our group reported similar results and added to the previous results an activation of NFKB pathway, calpain 1 , 2 and 3 activation, and for the first time sarcomeric lesions evaluated with electronic microscopy ²⁶. Furthermore, we could evaluate the trends in diaphragm force production in critically ill patients, mechanically ventilated, during their ICU stay with a non-invasive device, which stimulates with a magnetic field the phrenic nerves and consequently triggers diaphragm contraction. Interestingly, by the measurement of airway pressure (a surrogate of transdiaphragmatic pressure production during phrenic nerve stimulation), we could demonstrate a decrease of diaphragmatic force production during the ICU stay and a partial recovery of diaphragm force once the critically ill patient finally got better. Others reported the increase of autophagy ²⁸ but also a Fos/Fox01/Stat3-Bim dependent activation of intrinsic apoptosis ³⁰.

Although calpains and caspases, known to play a main role in VIDD are activated by dysregulation of intracellular calcium, calcium homeostasis has never been evaluated in animal model of VIDD models.

During skeletal muscle excitation-contraction coupling, sarcolemmal depolarization propagates down the transverse tubules (T -tubules), activates voltage-gated Ca²⁺ channels (L- type Ca²⁺ channels) which conformational changes activate Ca²⁺ release channels through ryanodine receptors (RyR) on the sarcoplasmic reticulum ³¹. RyR is a multiprotein complex, which contains a specific protein, FKBP12 (calstabinl), which stabilizes the channel and maintain it in a closed conformation without "calcium leak" ^{32 33}.

It has been recently showed in mice that phosphorylation on a protein kinase A (PKA) dependent residue, oxidation or nitrosylation of this complex can promote FKBP12 (calstabin 1) dissociation ³⁴. The consequence is a destabilization of the channel and reticulum calcium leak. This phenomenon can be observed with confocal microscopy, with evaluation of Calcium sparks, which show brief and highly localized releases of calcium from the reticulum

35

SUMMARY OF THE INVENTION:

The present invention relates to methods and pharmaceutical compositions for the treatment of ventilator-induced diaphragmatic dysfunction. In particular, the present invention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION:

In ICU, several pathophysiological events will drastically stimulate sympathetic nerve tonus with burst of catecholamine release in the blood or directly through neural termination, which is called catecholamine stress. Indeed hypotension induced by anesthetics commonly used in ICU to sedate patients stimulates sympathetic tonus at the induction of the sedation, through a feed back control. Moreover mechanical ventilation, through the positive pressure induced in the thorax, will decrease the venous blood return and the right heart telediastolic volume. The result is a decrease cardiac contractility with hypotension, which similarly stimulates sympathetic tonus. Moreover, burst of catecholamine in ICU are also frequently induced by pain and stress. The main downstream target of sympathetic stimulation in the muscle is protein kinase A (PKA) through beta-receptors present in the muscle fiber. Interestingly PKA can also be stimulated by oxidative stress ³⁶. Moreover, we and other teams have observed that stimulation of PKA activity though oxidative stress or catecholamine stress with beta-stimulation pathway may phosphorylate protein of interest in the excitation- contraction coupling such as Tnlc or CaVl . l channels ³⁷ ' ³⁸ with negative effect on muscular function. We demonstrated that that both oxidative and catecholamine stress, present under mechanical ventilation, synergistically converge to phosphorylation and oxidation of diaphragm ryanodine receptor (RyR), depletion of FKBP12 (calstabinl), SR calcium leak and diaphragm muscular weakness. We secondly demonstrated that the specific inhibition of the P2-adrenoreceptor with ICI 118,551³⁹ decreases the level of RyRl phosphorylation and depletion of calstabinl in the diaphragm mechanically ventilated and prevent the reduction in force production.

Accordingly, the present invention relates to a method for the treatment of ventilator- induced diaphragmatic dysfunction in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a p2-adrenergic receptor antagonist.

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. 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.

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 pathologic condition or disorder. A subject is successfully "treated" for ventilator-induced diaphragmatic dysfunction, if after receiving a therapeutic amount of the p2-adrenergic receptor antagonist according to the invention, the subject shows observable and/or measurable reduction in or absence of one or more signs and symptoms of ventilator-induced diaphragmatic dysfunction, such as, e.g., a significant reduction in diaphragmatic contractile function, or a decrease of diaphragm atrophy evaluated by CT scan or ultra sound. 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. In some embodiments, the p2-adrenergic receptor antagonist is administered before MV, immediately after MV initiation, during MV, and/or immediately after MV. In some embodiments, administration of the p2-adrenergic receptor antagonist according to the invention is provided at any time during MV.

The p2-adrenergic receptor antagonist 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 "P2-adrenergic receptor" or "β2 adrenoreceptor" has its general meaning in the art. The term "β -adrenergic receptor" refers to a family of G protein- coupled receptors that are targets of catecholamines. Adrenergic receptors are agonized by their endogenous ligands, epinephrine and norepinephrine. The term "P2-adrenergic receptor antagonist", refers to the ability of a compound to alter the function of p2-adrenergic receptor, i.e. to inhibit the activity of a b p2-adrenergic receptor. Typically, modulation of β-adrenergic receptors may be assessed using the method described in U.S. Pat. No. 5,223,510; U.S. Pat. No. 4,311,708; Phan et al, J. Ocul. Pharmacol. 1991, 7(3), 243-52; and Smith et al, Cardiovascular Drugs and Therapy 1999, 13, 123-126.

In some embodiments, the p2-adrenergic receptor antagonist is a non-selective β2- adrenergic receptor antagonist or a selective p2-adrenergic receptor antagonist. By "selective" it is meant that the inhibiting activity of the antagonist for the p2-adrenergic receptor is at least 10-fold, preferably 25-fold, more preferably 100-fold, still preferably 300-fold higher than the inhibiting activity for the βΐ -adrenergic receptor.

Examples of non-selective p2-adrenergic receptor antagonists include but are not limited to Alprenolol, Bucindolol, Carteolol, Carvedilol, Labetalol, Nadolol, Oxprenolol, Penbutolol, Pindolol, Propranolol, Sotalol and Timolol.

Examples of selective p2-adrenergic receptor antagonist include but are not limited to Butaxamine and ICI-118,551 ((±)-l-[2,3-(Dihydro-7-methyl-lH-inden-4-yl)oxy]-3-[(l- methylethyl)amino] -2-butano 1) .

As used herein, the terms "effective amount" or "therapeutically effective amount" or "pharmaceutically effective amount" refer to a quantity sufficient to achieve a desired therapeutic (including prophylactic effect), e.g., an amount which results in the prevention of, or a decrease in, ventilator-induced diaphragmatic dysfunction, or symptoms associated therewith. In the context of therapeutic or prophylactic applications, the amount of a composition administered to the subject will depend on the type and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. It will also depend on the degree, severity and type of disease. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. The compositions can also be administered in combination with one or more additional therapeutic compounds. A suitable amount of the p2-adrenergic receptor antagonists of the invention effective in the subject ranges from about 0.01 mg/kg/day to about 20 mg/kg/day, and/or is an amount sufficient to achieve plasma levels ranging from about 300 ng/ml to about 1000 ng/ml. Alternatively, the amount of compounds from the invention ranges from about 10 mg/kg/day to about 20 mg/kg/day. Also included are amounts of from about 0.01 mg/kg/day or 0.05 mg/kg/day to about 5 mg/kg/day or about 10 mg/kg/day which, can be administered.

The p2-adrenergic receptor antagonists of the invention are formulated into pharmaceutical compositions for administration to human subjects in a biologically compatible form suitable for administration in vivo. Accordingly, a further aspect of the invention relates a pharmaceutical composition comprising at least one agent of the invention in admixture with a pharmaceutically acceptable diluent and/or carrier. The pharmaceutically-acceptable carrier must be "acceptable" in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipient thereof. The pharmaceutically-acceptable carrier employed herein is selected from various organic or inorganic materials that are used as materials for pharmaceutical compositions and which are incorporated as analgesic agents, buffers, binders, disintegrants, diluents, emulsifiers, excipients, extenders, gellants, glidants, skin-penetration enhancers, solubilizers, stabilizers, suspending agents, tonicity agents, vehicles and viscosity-increasing agents. If necessary, pharmaceutical additives, such as antioxidants, aromatics, colorants, flavor- improving agents, preservatives, and sweeteners, are also added. Examples of acceptable pharmaceutical carriers include carboxymethyl cellulose, crystalline cellulose, glycerin, gum arabic, lactose, magnesium stearate, methyl cellulose, powders, saline, sodium alginate, sucrose, starch, talc and water, among others.

The pharmaceutical compositions of the present invention are prepared by methods well-known in the pharmaceutical arts. For example, the p2-adrenergic receptor antagonists of the invention are brought into association with a carrier and/or diluent, as a suspension or solution. Optionally, one or more accessory ingredients (e.g., buffers, flavoring agents, surface active agents, and the like) also are added. The choice of carrier is determined by the solubility and chemical nature of the p2-adrenergic receptor antagonist, chosen route of administration and standard pharmaceutical practice.

Additionally, the compounds of the present invention are administered to the subject by known procedures including, without limitation, oral administration, sublingual or buccal administration, parenteral administration, transdermal administration, via inhalation or intranasally, vaginally, rectally, and intramuscularly. The p2-adrenergic receptor antagonists of the invention are administered parenterally, by epifascial, intracapsular, intracranial, intracutaneous, intrathecal, intramuscular, intraorbital, intraperitoneal, intraspinal, intrasternal, intravascular, intravenous, parenchymatous, subcutaneous or sublingual injection, or by way of catheter. For oral administration, a formulation of the p2-adrenergic receptor antagonists of the invention may be presented as capsules, tablets, powders, granules, or as a suspension or solution. The formulation has conventional additives, such as lactose, mannitol, cornstarch or potato starch. The formulation also is presented with binders, such as crystalline cellulose, cellulose derivatives, acacia, cornstarch or gelatins. Additionally, the formulation is presented with disintegrators, such as cornstarch, potato starch or sodium carboxymethylcellulose. The formulation also is presented with dibasic calcium phosphate anhydrous or sodium starch glycolate. Finally, the formulation is presented with lubricants, such as talc or magnesium stearate.

For parenteral administration (i.e., administration by injection through a route other than the alimentary canal), the p2-adrenergic receptor antagonists of the invention are combined with a sterile aqueous solution that is isotonic with the blood of the subject. Such a formulation is prepared by dissolving a solid active ingredient in water containing physiologically-compatible substances, such as sodium chloride, glycine and the like, and having a buffered pH compatible with physiological conditions, so as to produce an aqueous solution, then rendering said solution sterile. The formulation is presented in unit or multi- dose containers, such as sealed ampoules or vials. The formulation is delivered by any mode of injection, including, without limitation, epifascial, intracapsular, intracranial, intracutaneous, intrathecal, intramuscular, intraorbital, intraperitoneal, intraspinal, intrasternal, intravascular, intravenous, parenchymatous, subcutaneous, or sublingual or by way of catheter into the subject's heart. For transdermal administration, the p2-adrenergic receptor antagonists of the invention are combined with skin penetration enhancers, such as propylene glycol, polyethylene glycol, isopropanol, ethanol, oleic acid, JV-methylpyrrolidone and the like, which increase the permeability of the skin to the p2-adrenergic receptor antagonists of the invention and permit the compounds to penetrate through the skin and into the bloodstream. The compound/enhancer compositions also may be further combined with a polymeric substance, such as ethylcellulose, hydroxypropyl cellulose, ethylene/vinylacetate, polyvinyl pyrrolidone, and the like, to provide the composition in gel form, which are dissolved in a solvent, such as methylene chloride, evaporated to the desired viscosity and then applied to backing material to provide a patch.

The composition may be provided in unit dose form such as a tablet, capsule or single- dose vial. Suitable unit doses, i.e., therapeutically effective amounts, can be determined during clinical trials designed appropriately for each of the conditions for which administration of a chosen compound is indicated and will, of course, vary depending on the desired clinical endpoint. The present invention also provides articles of manufacture for treating and preventing disorders, such as cardiac disorders, in a subject. The articles of manufacture comprise a pharmaceutical composition of one or more of the p2-adrenergic receptor antagonists of the invention.

A further aspect of the invention relates to a method for screening a plurality of test substances useful for treatment of ventilator-induced diaphragmatic dysfunction comprising the steps consisting of (a) testing each of the test substances for its ability to inhibit β2- adrenergic receptor and (b) and positively selecting the test substances capable of said inhibition.

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: VM-induced diaphragm RyRl post-translational modifications in the four groups of mice. Oxidation of RyRl (A) PKA-dependent phosphorylation on Ser-2844 (B) and depletion of calstabinl (C).

Figure 2 shows diaphragm force production during the 6 hours of mechanical ventilation between the 3 ventilated groups. CTL: control. MV: mechanical ventilation. MV PRO: mechanical ventilation + treatment with propranolol. MV ICI: mechanical ventilation + treatment with ICI 118,551.

Figure 3 shows the mean heart rate during the 6 hours of mechanical ventilation between the 3 ventilated groups. CTL: control. MV: mechanical ventilation. MV PRO: mechanical ventilation + treatment with propranolol. MV ICI: mechanical ventilation + treatment with ICI 118,551. Figure 4: Expression of βΐ, β2 and β3 receptors in mice diaphragm. Expression of β receptors isotypes was determined by Western Bolt analysis in control mice diaphragm (N= 6) with antibodies directed specifically against Bl, B2 and B3 receptors. Expression was normalized to GADPH. ***' p <0.001 vs. βΐ expression.

Figure 5: PKA activity in diaphragm muscle increases with mechanical ventilation. PKA (Protein Kinase A) activity was determined in muscle extracts with a Colorimetric Activity Assay Kit. PKA activity increases with mechanical ventilation (MV) to reach a steady state after 2 hours of MV. This is fully abolished when mice are treated with the B2 receptor antagonist ICI11851 prior to starting mechanical ventilation. These values where compared to control mice treated with 1 μΜ isoproterenol (iso) as a positif control and control mice treated with ICI118551 as a negative control. (N=6 per group) *, ** p <0.05, vs CTRL +iso an MV6 hours+ICIl 18551 respectively. EXAMPLE

Methods:

Protocol: 3 groups of 10 mice 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 4 cm 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 first group (MV-pro) received a priming dose of propranolol, a non-specific beta antagonist lOmg/kg, 20 minutes before start of MV, and during MV, a constant IV infusion 5 mg/kg per hours was maintained. The second groups received ICI (ICI-118,551 : (±)-l-[2,3-(Dihydro-7- methyl-lH-inden-4-yl)oxy]-3-[(l-methylethyl)amino]-2-butanol) at a dose of 10 mg/kg, intravenously (IV) infused over a 5 -minute period, 20 minutes before start of MV, and during MV, a constant IV infusion of ICI 118 551 at a rate of 0.7 mg/kg per hours was maintained. The third group of untreated mice (MV) received same volume of IV saline 20 minutes before starting and during MV. The mice drank about 3 ml per day (water consumption was variable, and we recorded water bottle and body weight to monitor consumption) for a daily dose of ~0.75 mg (~37.5 mg/kg/day). The fourth group served as control and received any treatment and was not ventilated (CTL). The 3 groups mechanically ventilated received the same general care. Mice were anesthetized with intraperitoneal injection of pentobarbital sodium (50 mg/kg body weight) and orally intubated with a 22-gauge angiocatheter. General care applied during the experiments also included continuous reheating using a homeothermic blanket (Homeothermic Blanket Control unit, Harvard Apparatus, Saint-Laurent, Canada, set at 35 °C), and hourly intraperitoneal injection of 0.05 ml of Ringers Lactate solution to maintain hemodynamic stability and compensate insensible losses, as well as bladder expression, eye lubrication and passive limb movements. Mice during the protocol were monitored in regard to heart rate with electrocardiograms by telemetry (DSI, St Paul, MN, USA, and EMKA Technologies, France).

Functional analysis: At the end of the protocol of MV, mice were euthanized, by exsanguination. The entire diaphragm surgically excised and a diaphragm strip was mounted into a 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, 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 used for biochemical analysis of RyR.

Biochemical analysis: Muscle biopsies were homogenized in 150 μΐ of buffer containing 5% SDS, 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

2 μg/μl. RyRl was immunoprecipitated from 250 μg of homogenate using an anti-RyR antibody (4 μg RyRl -1327) in 0.5 ml of a modified RIPA buffer (50 mM Tris-HCl pH 7.4, 0.9% NaCl, 5.0 mM NaF, 1.0 mM Na3V04, 1% Triton-XlOO, and protease inhibitors) for 1 hour at 4°C. The immune complexes were incubated with protein A Sepharose beads (Amersham Pharmacia) at 4°C for 1 hour 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 2 hour at 200 mA (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 with the amino acid sequence CAEPDTDYENLRRS, 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); phosphor epitope-specific antibody to human RyR2 phosphorylated on Ser-2808 (1 :5,000), which detects both 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 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:

Figure 1 present VM-induced diaphragm RyRl post-translational modifications in the four groups of mice. RyRl co-immunoprecipitation after mechanical ventilation induced an oxidation of RyRl (Figure 1A), a PKA-dependent phosphorylation on Ser-2844 (Fig IB) and a depletion of calstabinl (Figure 1C). The treatments inhibiting non- selectively β1/β2 adrenoreceptor by propranolol or selectively β2 adrenoreceptor by ICI118 551 did not reduced RyRl oxidation (Figure 1A) but was sufficient to prevent both PKA-dependent phosphorylation and calstabinl depletion (Figures IB & C).

As previously reported by our team, six hours of mechanical ventilation in mice is sufficient to reduce the diaphragm force production⁴⁰ (Figure 2). Both propranolol and ICI 118551 prevented this muscle weakness. As showed in figure 3, we did not observed any significant difference in mean heart rate during the 6 hours of mechanical ventilation between the 3 ventilated groups. Both of three presented a drastic decrease of mean heart rate compared to control, due to sedation, which overlaps the chronotropic effect of βΐ stimulation.

A Western blot analysis performed in diaphragm muscle from control mice was realized to dertemined the profil of B-receptors expression (Figure 4). This analysis indicates that the major isoform present in diaphragm muscle is B2. This strengthens the pertinence of the use of specific B2 receptors antagonist in VIDD.

PKA activity was increased after 2 hour of mechanical ventilation. This may account for RyRl phosphorylation on the PKA site (serine 2844). PKA activity was fully abolished when mice were treated specifically with the B2 receptor antagonist ICI 1 18551 (Figure 5). Discussion

We demonstrate here for the first time that reducing RyRl phosphorylation on a context of oxidative stress with antagonists of the β-receptors (propranolol and ICI1 18 551) can prevent muscle weakness induced by MV in mice. However, among the subtypes of β- receptors expressed in mammals, there is a different pattern of expression according to the tissues. For instance, cardiac tissue expresses both βΐ and 2-receptors while skeletal muscle preferentially expresses β2 -receptors. Therefore, non-specific β-blocker could have negative impact on hemodynamic and cardiac function, which may limit its usefulness in ICU. Here we observed a similar effect with ICI, a selective

receptor, which presents the advantage to have no negative effect on hemodynamic function, and could be more easily used in ICU, in patients without any antecedent of asthma.

As mentioned earlier, the RyR complex is a converging pathophysiological target in many pathophysiological situations, and all of them have certainly not been reported yet. This can be explained in part by the ubiquitous function of Ca²⁺ in cellular processes but also in the complexity and fragility of the RyR macromolecular complex. Therefore, the fact that RyRl is a potential mediator of muscle weakness in VIDD suggests that patients with comorbidities and/or confounding factors that may stimulate the β-adrenergic pathway, might have a greater vulnerability to VIDD. Our results emphasis the pathophysiological role of RyRl in VIDD and strongly supports the hypothesis that preventing the remodeling of RyRl induced by MV with a specific p2-receptor antagonist, may provide a new therapeutic approach to prevent diaphragm muscle dysfunction in patients who require artificial respiratory support.

REFERENCES:

All publications, references, patents and patent applications cited herein are incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety. While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be appreciated by one skilled in the art, from a reading of the disclosure, that various changes in form and detail can be made without departing from the true scope of the invention in the appended claims.

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Claims

CLAIMS:

1. A method for the treatment of ventilator-induced diaphragmatic dysfunction in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a p2-adrenergic receptor antagonist.

2. The method of claim 1 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 postsurgical recovery.

3. The method of claim 1 wherein the subject suffers from a disease for which the worsening of the symptoms has led the subject to need mechanical ventilation.

4. The method of claim 3 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).

5. The method of claim 1 wherein the subject suffers from a trauma.

6. The method of claim 1 wherein the ventilator-induced diaphragmatic dysfunction results from prolonged controlled mechanical ventilation (MV) greater than 12 hours.

7. The method of claim 1 wherein the p2-adrenergic receptor antagonist is administered before MV, immediately after MV initiation, during MV, and/or immediately after MV.

8. The method of claim 1 wherein the p2-adrenergic receptor antagonist is a nonselective p2-adrenergic receptor antagonist or a selective p2-adrenergic receptor antagonist.

9. The method of claim 8 wherein the non -selective p2-adrenergic receptor antagonist is selected from the group consisting of Alprenolol, Bucindolol, Carteolol, Carvedilol, Labetalol, Nadolol, Oxprenolol, Penbutolol, Pindolol, Propranolol, Sotalol and Timolol.

10. The method of claim 8 wherein the selective p2-adrenergic receptor antagonist is selected from the group consisting ofButaxamine and ICI-118,551.

11. A method for screening a plurality of test substances useful for treatment of ventilator- induced diaphragmatic dysfunction comprising the steps consisting of (a) testing each of the test substances for its ability to inhibit p2-adrenergic receptor and (b) and positively selecting the test substances capable of said inhibition.