WO2012010641A2

WO2012010641A2 - Pharmaceutical composition for increasing cellular hypoxic tolerance

Info

Publication number: WO2012010641A2
Application number: PCT/EP2011/062469
Authority: WO
Inventors: Christian Frelin; Paul Vigne; Michel Tauc; Isabelle Rubera; Christophe Duranton; Marc Cougnon; Nicolas Blondeau
Original assignee: Universite De Nice Sophia Antipolis; Centre National De La Recherche Scientifique (Cnrs)
Priority date: 2010-07-20
Filing date: 2011-07-20
Publication date: 2012-01-26
Also published as: FR2962906A1; WO2012010641A3

Abstract

The present inventors have discovered a new class of cryoprotective therapeutic agents which make it possible for mammalian cells to survive for longer periods of time under hypoxic conditions, and to prevent problems linked to oxygen deprivation in a model of renal failure due to ischemic lesion in rats and of cardiac stroke. The present invention is therefore directed towards a pharmaceutical composition for preventing and/or treating pathological conditions resulting from hypoxic conditions and/or from ischemic lesions in a mammal, said composition comprising, as cytoprotective agent, an elF5A-hypusination-inhibiting agent, such as, for example, N1-guanyl-1,7-diaminoheptane (GC7).

Description

PHARMACEUTICAL COMPOSITION FOR INCREASING THE

CELLULAR HYPOXIC TOLERANCE

The present invention aims to improve existing treatments to relieve patients who have suffered ischemic injury and / or hypoxia conditions, preventing in vivo the onset of disorders resulting from the deprivation of oxygen supply.

Hypoxia consists of insufficient oxygenation of the tissues. It can occur in several ways: on the one hand when one climbs aloft (natural hypoxia), on the other hand because of an ischemic lesion (pathological hypoxia).

Altitude hypoxia (due to the surrounding natural environment) is called hypobaric. It is the decrease of atmospheric air pressure (less than 760mmHg) which leads to the decrease of the partial pressure of each of the gases that compose it, including oxygen.

Ischemia is the decrease in arterial blood supply to an organ. This decrease leads to a decrease in tissue oxygenation and the accumulation of anaerobic metabolic products. These disturbances can lead to the cessation of tissue function. Ischemia can have various causes:

presence of a blood clot obstructing an artery (thrombosis),

- formation of an atheromatous plaque,

hemorrhage or hypo-infusion preventing certain tissues from being properly fed,

compression of an artery by an external object (crushing of a limb, tourniquet) or by an internal phenomenon (hematoma, tumor, or effusion of a liquid).

Ischemia can be reversible and cause only limited discomfort. It can be irreversible and can lead to infarction of the organ, that is to say to the death of part or all of it. The two most critical cases are obviously ischemia affecting the brain or the heart muscle. The acute ischemia of a limb, consecutive to the abrupt obliteration of the arterial axis of the latter, is a vascular emergency with vital prognosis (mortality of about 20%). It occurs most often after thrombosis or embolism. This results in tissue suffering due to oxygen deprivation.

The molecular mechanisms responsible for tissue damage are well known. They include in particular:

- a cellular acidosis and the consequences of the deregulation of the intracellular pH on the homeostasis of sodium and intracellular calcium,

metabolic disturbances, including decreased ATP stores and kinase activation,

- the production of free radicals, and

apoptosis according to the severity of the ischemic attack.

Numerous pharmaceutical developments and clinical studies have focused on the prevention of ischemic lesions by themselves (prevention of myocardial infarction and / or stroke), and the management by emergency departments of patients suffering from these pathologies have been significantly improved in recent years. However, pathological conditions resulting from these lesions, as well as disorders related to aging (cardiac and renal insufficiency, cerebral microangiopathies) have been little studied to date, and the pharmaceutical industry needs new molecular targets validated in this field. Such targets could advantageously increase the hypoxic tolerance of cells, allowing them to survive longer under hypoxic conditions, in order to delay the deleterious effects of oxygen deprivation in ischemic tissue.

A molecular target known to increase the hypoxic tolerance of cells is the hypoxia-induced protein HIF (Hypoxia Inducible Factor) and the associated enzymes HIF-prolyl hydroxylases. Inhibitors of these enzymes induce a state of hypoxic tolerance in vivo and in vitro. The use of these inhibitors in humans is however excluded because these molecules also promote tumor development and dissemination of metastases (Semenza GL et al, Science 2007).

In this context, the present inventors have discovered a new class of cytoprotective therapeutic agents allowing mammalian cells to survive longer under hypoxic conditions, and to prevent related disturbances. to oxygen deprivation in a model of renal failure due to ischemic injury in rats and in a model of cerebral ischemia in mice. These new agents make it possible to limit the consequences of cellular hypoxia and to reduce oxygen deficiency disorders in vivo in mammals. These disorders most often result from ischemic lesions or conditions of hypoxia. In particular, the agents identified in the present invention make it possible to reduce the symptoms of renal insufficiency caused by a 40-minute renal ischemia, and reduce the volume of the infarction induced by a focal cerebrovascular accident.

Moreover, these agents induce a significant decrease in the basal oxygen consumption of an individual, without modifying the level of activity.

SUMMARY OF THE INVENTION

The subject of the present invention is a pharmaceutical composition for its use for preventing and / or treating pathologies resulting from hypoxic and / or ischemic conditions in a mammal, said composition comprising as a cyto-protective agent an agent that inhibits hypusinylation. of eIF5A. According to a more specific aspect of the invention, said pathologies are chosen from: renal insufficiency due to ischemic injury, cardiac insufficiency due to an infarction, neurological sequelae due to a cerebrovascular accident, peripheral and central microangiopathies , or disruption of an organ such as the liver, intestine, heart, lung or kidney following transplantation.

The inventors have shown in particular that the pharmaceutical composition of the invention can be used to increase the hypoxic tolerance of mammalian cells subjected to ischemic injury or hypoxic conditions.

Preferably, said hypusinylation inhibiting agent according to the invention inhibits the synthesis of hypusin. In particular, said hypusinylation inhibiting agent inhibits deoxyhypusine synthase (DHS) or deoxyhypusine hydroxylase (DHH). Even more particularly, said hypusinylation inhibiting agent is chosen from 1,7-diaminoheptane (DAH) derivatives such as N-guanyl-1,7-diaminoheptane (GC7), or chosen from 1,3-diazacyclohexane and 1,3-diazacyclo-1-hexene, and is preferably N-Guanyl-1,7-diaminoheptane (GC7). Even more preferably, the subject of the invention is a pharmaceutical composition which can be used to prevent and / or treat renal failure due to ischemic injury and to reduce the consequences of a cerebrovascular accident, said composition comprising, as a Cytoprotective agent, an inhibitor of the hypusinylation of eIF5A, preferably Nl-Guanyl-1,7-diaminoheptane (GC7).

The composition of the invention can also be used to decrease the oxygen consumption of an individual.

LEGENDS OF FIGURES

Figure 1 depicts the model of renal ischemia by temporary arrest of the arterial flow and used to demonstrate the effects of GC7 on hypoxia / ischemia. The left renal artery of anesthetized rats is kept ligated for 40 minutes before being released. Prior to surgery, the rats were treated intraperitoneally with either placebo (0.9% NaCl) or GC7 (1 IP injection per day for 3 days at 3 mg / kg). Renal function parameters were analyzed 24 hours after restoration of the arterial flow.

FIG. 2 shows the determination of the Neutralophil Gelatinase Associated Lipocalin (NGAL) tissue marker in total urine before and 24 hours after the application of the ischemia / reperfusion protocol (FIG. 1) to the left renal artery, control rats (white bars) or treated with GC7 3 mg / kg intraperitoneal for 3 days (gray bars). The NGAL values are classically related to urinary creatinine. The averages are represented (+ sem) and the number of animals used is indicated (in parentheses). ** = P <0.01 with Student's "t" test. GC7 protects renal tissue integrity impaired by ischemia reperfusion.

Figure 3 shows the plasma NGAL (Neutrophil Gelatinase Associated

Lipocalin) dosed 24 hours after the application of the ischemia / reperfusion protocol (FIG. 1) on the left renal artery in control rats (white bar) or treated with GC7 3 mg / kg intraperitoneal for 3 days before ligature, (gray bar). The averages are represented (+ sem) and the number of animals used is indicated (in parentheses). * = P <0.05 with Student's "t" test. Figure 4 shows fractional excretion (as a% of glucose filtered) urinary glucose of the right kidneys (white bars) and left (gray bars) measured 24 hours after the application of the ischemia / reperfusion protocol (Figure 1) on the left renal artery in control or GC7-treated rats (3 mg / kg intraperitoneal for 3 days). The averages are represented (+ sem) and the number of animals used is indicated (n). * = P <0.05 with Student's "t" test. GC7 protects the renal reabsorption function of glucose impaired by hypoxia / ischemia.

Figure 5 is a graph showing fractional excretions (as a% of sodium and filtered phosphate) of sodium urinary (A) and phosphate (B) of right kidneys (white bars) and left (gray bars) measured 24 hours after the application of the ischemia / reperfusion protocol (Figure 1) to the left renal artery in control or GC7-treated rats (3 mg / kg intraperitoneal for 3 days before ligation). The averages are represented (+ sem) and the number of animals used is indicated (in parentheses). * = P <0.05 with Student's "t" test. GC7 protects the renal reabsorption functions of sodium and phosphate impaired by hypoxia / ischemia.

FIG. 6 describes the level of hypusinylated eIF5A (in% relative to total eIF5a) is determined on extracts of kidneys, from animals treated or not treated with GC7, by western blot (6A) with the aid of specific antibodies directed respectively against hypusinylated (eIF5a hyp) or total (eIF5a early) forms of eIF5A. The densitometric analysis in arbitrary units of the gels (6B) is carried out before (white bar) and after treatment with the GC7 (gray bar). In order to verify the total amount of proteins deposited per lane (80 μg), an antibody directed against β-actin was used (actin). Standard means and deviations from 4 animals in each category are shown (**: p <0.01, Student's "t" test). GC7 induces in vivo inhibition of hypusinylation of eIF5a.

Figure 7 is a graph showing the phosphate / creatinine ratio in urine of normoxia or hypoxia-treated (gray bars) or without (white bars) pretreatment with GC7. Mice pretreated daily (3 days) or not with GC7 at 3 mg / kg are maintained in normoxia (21% oxygen) or in hypoxia (8% oxygen) for 24 hours. The urines collected at t = 24 hours are analyzed for their phosphate and creatinine content. GC7 protects against hypoxia-induced phosphate loss

Figure 8 is a graph showing oxygen consumption (8A), intracellular ATP content (8B) glucose consumption (8C) and and lactate production (8D) of renal epithelial cells (proximal tubule). ) treated (gray bars) or not (white bars) with GC-7 at 30 μΜ. Measurements are taken 24 hours after the start of GC7 treatment. GC7 induces a metabolic shift toward anaerobic glycolysis, allowing cells to break free of oxygen while retaining ATP synthesis capabilities.

Figure 9 is a graph showing the basal oxygen consumption of male (A) or female (B) mice injected with placebo (0.9% NaCl, white bars) or with 3 mg GC-7 (gray bars) / kg in IP (1 injection per day during the 3 days preceding the measurement). The mice are maintained in a closed chamber of a volume of 500 cm and containing an expired C0 ₂ capture system based on barium hydroxide. The O ₂ content is measured every 5 minutes with an oxybaby® (Witt) oximeter. The values are expressed in ml of oxygen consumed per hour and per gram of body weight. The averages are represented (+ sem) and the number of animals used is indicated (n). * = P <0.05 with Student's "t" test.

FIG. 10 represents the quantification of viable neurons in primary cultures of cortical neurons of GC-7 treated and ungaged mice, having undergone (or not) oxygen and glucose deprivation (OGD) for 60 minutes. This in vitro model of concomitant oxygen and glucose deprivation is described as best reproducing the effects of ischemia observed during stroke. The glucose deprivation is total and hypoxia of the order of 75% (passage of 5% oxygen in the culture medium to 1.2%).

Figure 11 shows the volume of cerebral infarction (in mm ³ ) of stroke mice with or without pretreatment with GC-7. The ischemic lesion is quantified by histomorphometric analysis on coronal sections according to 12 defined stereotactic levels. The volume of the infarct is calculated by a mathematical integration of the surfaces of the lesions measured by computer imaging, with the introduction of a corrective factor of the cerebral edema. DETAILED DESCRIPTION OF THE INVENTION

The therapeutic agents identified by the inventors are molecules known to inhibit a post-translational modification called hypusinylation. In particular, these molecules of interest inhibit the hypusinylation of the eIF5A translation initiation factor (SEQ ID NOs: 1, 3 and 4, Al isoforms in humans, the mouse and the rat, respectively: SEQ ID NOs : 5, 6 and 7, isoforms A2 in humans, mice and rats, SEQ ID NO: 2, isoform B in humans).

Hypusinylation is a post-translational modification that results from the addition of a 4-aminobutyl residue (from spermidine) to a Lys residue. The eIF5A protein is the only protein known to be hypusinylated to date (Park M.H. et al, J. Biochem, 2006). This protein is a eukaryotic translation initiation factor whose structure is highly conserved between yeast and humans. Hypusine formation is generally known to render the eIF5A protein functional, and contributes to cell proliferation, so that it is associated with different forms of cancer. The inhibition of hypusinylation of eIF5A is therefore known for its antiapoptotic and cytostatic properties, favoring a quiescent state of the cells by blocking cells in the Gl / S phase (Balabanov S et al, Blood 2007, Jasiulionis MG et al. Biochem Funct 2007).

Several isoforms of eIF5A have been discovered: among them, eIF5Al (SEQ ID NO 1 to 4 in humans, mice and rats) would be specifically expressed in response to the induction of apoptosis, induced by a proteasome inhibitor. the MG132, another (designated eIF5A2, SEQ ID NO: 5-7 for humans, mice and rats) would likely be involved in translation and cell proliferation (Park MH et al, Amino Acids 2010).

The eIF5Al protein is presented as an apoptosis-specific protein having pro-apoptotic properties that can be used to induce cell apoptosis, in particular that of the tumor cells (see EP1959010 for example). The non-hypusinylated form of eIF5Al is active in apoptosis. Inhibition of eIF5Al hypusinylation is therefore generally associated with a pro-apoptotic effect (Sun Z, J. Cell Physiol 2010). Furthermore, WO 2009/144933 proposes to use inhibitors of the secreted form of eIF5A to diagnose or treat atherosclerosis induced by oxidative stress or by ischemic injury.

On the other hand, it is generally accepted that it is the hypusinylated form of eIF5A isoforms that is involved in cell proliferation. In this case, inhibition of hypusinylation blocks the cell cycle and induces apoptosis in many tumor cells (hence its use as a cytostatic agent) (Taylor CA, Exp Cell Res 2007, Li AL, J. Biol. Chem 2004). Similarly, Huang Y et al demonstrated that inhibition of eIF5A hypusinylation blocks neurite outgrowth and significantly reduces the survival of mammalian neurons.

Although eIF5A was originally described as a translation initiation factor, its inactivation is not actually associated with a decrease in protein synthesis, and a more complex role of nucleocytoplasmic transport of messenger RNAs has also been suggested (Park et al., Amino Acids, 2010).

Two enzymes, deoxyhypusine synthase DHS (EC No. 2.5.1.46; SEQ ID NO:

8 to 10 for humans (isoforms A, B, and C), SEQ ID NO: 11 in mice, and SEQ ID NO: 12 in rats) and deoxyhypusin hydroxylase DHH (EC No. 1.14.99.29; ID NO: 13 for humans, SEQ ID NO: 14 in mice, and SEQ ID NO: 15 in rats) are required for hypusinylation of eIF5A. Other enzymes also contribute: it is the enzymes responsible for the synthesis of polyamines, including spermidine, which is converted into hypusine by DHS and DHH. These enzymes are: ornithine decarboxylase (ODC), spermine synthase, spermidine synthase, spermidine / spermine acetyltransferase (SSAT) and polyamine oxidase, and S-adenosyl-L-methionine decarboxylase.

Inhibitors of hypusinylation of eIF5A have been identified. The best known are DFMO (R, S) -2- (difluoromethyl) ornithine) or α-difluoromethylornithine (CAS 70052-12-9), a specific inhibitor of ornithine decarboxylase and GC7 (Nl-guanyl-1, 7 diaminoheptane), an inhibitor of deoxyhypusine synthase DHS (CAS 150333-69-0). Many DHS inhibitors have also been tested and characterized (Lee YB and Folk JE, 1998). These inhibitors are used as powerful cytostatic agents, and have been proposed to inhibit cancerous pathologies as well as other infectious indications (HIV, malaria, leishmaniasis). (Jasiulionis MG et al, Cell Biochem Funct 2007, Kersher B et al, Amino Acids 2010). Some studies suggest a specific pro-apoptotic effect of GC7 and other DHS inhibitors (see EP1959010, Sun Z, J. Cell Physiol 2010). Thus, hypusinylation of eIF5A is considered a potential target in treatment. cancers and other infectious diseases (HIV, malaria, leishmaniasis).

Furthermore, WO 2003/106433 proposes to inhibit hypusinylation of eIF5A in order to limit the immune reactions that are often induced during organ transplantation. On the other hand, WO 2010/118224 mentions that the administration of agents that inhibit the hypusinylation of eIF5A makes it possible to prevent pancreatic islet degradation, to lower the level of glucose in the blood and to avoid the resistance to insulin in diabetic patients.

However, the link between hypusinylation and hypoxic tolerance has never been identified in a mammal.

Recent studies on the mechanisms involved in hypoxia tolerance mechanisms have been performed on flies of the Drosophila species (Vigne P, et al., Mech Ageing Dev 2007 and Vigne P. et al., BMC Physiology 2008). These studies demonstrate that sucrose increases the survival of Drosophila placed under conditions of chronic hypoxia (5% 0 ₂ ). Conversely, natural amino acids (in particular L-proline, L-arginine, L-glutamine, and L-asparagine), the amino acids of the urea cycle (L-citrulline, L-ornithine) and polyamines (putrescine, spermine, and spermidine) reduce the survival of hypoxia-fed and sucrose-solution-fed flies. The amino acids in the diet are beneficial under normal oxygenation conditions (21% of 0 ₂ ). Vigne et al. demonstrated in 2008 that the specific inhibitor of eIF5A hypusinylation, GC7, is capable of decreasing the toxic effect of the amino acids and polyamines mentioned above. In the absence of amino acids, treatment of flies with GC7 reduces their longevity under hypoxia conditions, suggesting that GC7 has a deleterious effect on hypoxiated cells. In other words, according to this study, in the absence of amino acids or polyamines, treatment with GC7 induces a decrease in hypoxic tolerance in Drosophila. On the other hand, under hypoxic conditions and in the presence of amino acids, GC7 increases the hypoxic tolerance of Drosophilae. In a first aspect, the present invention relates to a pharmaceutical composition for its use for preventing and / or treating disorders related to hypoxic conditions and / or ischemic injury in a mammal, said composition comprising, as a cytoplasmic agent, protective agent, an inhibitor of hypusinylation of eIF5A.

In other words, the present invention is directed to the use of a pharmaceutical composition comprising, as a cytopterive agent, an eIF5A hypusinylation inhibiting agent, for the manufacture of a medicament for preventing and / or treating disorders related to hypoxic conditions and / or ischemic injury in the mammal.

The pharmaceutical composition of the invention comprises, in addition to the cytoprotective agent, at least one pharmaceutically acceptable excipient.

More specifically, the present invention provides an eIF5A hypusinylation inhibiting agent for use in preventing and / or treating disorders related to hypoxic conditions and / or ischemic injury in a mammal, or the use of an inhibitor of eIF5A hypusinylation for the manufacture of a medicament for preventing and / or treating disorders related to hypoxic conditions and / or ischemic injury in a mammal.

Finally, the present invention also relates to a method for preventing and / or treating disorders related to hypoxic conditions and / or ischemic lesions in an individual suffering from them, comprising the step of administering a pharmaceutical composition comprising, in as a cytopterant agent, an eIF5A hypusinylation inhibitory agent, and a pharmaceutically acceptable excipient.

Preferably, in the context of the present invention, said hypusinylation inhibiting agent inhibits the synthesis of hypusin.

Hypusine (N ^e - (4-amino-2-hydroxybutyl) lysine) is a unique amino acid that only occurs for the modification of eIF5A, either in mammals or in Drosophila. It is formed in two stages. First, the DHS enzyme transfers the 4-aminobutyl group of spermidine to the ε group of the lysine residue of the inactive eIF5A protein. Then DHH hydroxylates the eIF5A-deoxyhypusin protein into eIF5A-hypusine which is the active form of eIF5A (Park MH et al., Amino Acids 2010). Thus, in a preferred embodiment, the hypusinylation inhibiting agent inhibits deoxyhypusine synthase (DHS) or deoxyhypusin hydroxylase (DHH).

In the context of the present invention, this hypusinylation inhibiting agent may be chosen from 1,7-diaminoheptane derivatives (DAH, CAS 646-19-5) such as N-guanyl-1,7-diaminoheptane ( GC7) (CAS 150333-69-0), 1,3 diazacyclohexane (CAS 505-21-5) and 1,3 diazacyclo-1-hexene (CAS 25377-66-6), the (R, S) - 2- (difluoromethyl) ornithine (DFMO, CAS 70052-12-9), trans-4-methylcyclohexylamine (CAS 2523-55-9), N- (3-aminopropyl) cyclohexylamine (CAS 3312-60-5), and is preferably Nl-Guanyl-1,7-diaminoheptane (GC7).

More generally, the hypusinylation inhibiting agent is chosen from 1,7-diaminoheptane derivatives, 1,3-diazacyclohexane derivatives (CAS 505-21-5) and 1,3-diazacyclo-1-hexene derivatives. (CAS 25377-66-6).

Preferably, the hypusinylation inhibiting agent is chosen from the derivatives of 1,7-diaminoheptane, 1,3-diazacyclohexane and 1,3-diazacyclo-1-hexene.

The derivatives of 1,7 diaminoheptane (C ₇ ) are advantageously branched derivatives such as N-guanyl-1,7-diaminoheptane (GC7), GC7G, GC6, GC8, GC6G, GC8G, CNI-1493 (or semapimod, N, N'-bis [3,5-bis [N- (diaminomethylideneamino) -C-methylcarbonimidoyl] phenyl] decanediamide

tetrahydrochloride) or the compounds 25b (1,7-diaminooctane, C ₈ H ₂ 0N _2), 26b (1,7-diguanidinooctane, CIOH ₂₄ N _6), 26c (1,7-diguanidinononane, CHE N ^ _O) and 7-amino-1-guanidinooctane (Compound 39, CefoN-) described in Lee and Folk (1998).

The derivatives of 1,7 diaminoheptane (C ₇ ) can also be unsaturated derivatives such as the compounds described in Lee and Folk (1998): 19a (1,7-diguanidinohept-3-yne, C ₇ H ₄ N ₂ ), 22a (1,7-diaminohept-3-yne, C ₈ H ₁₈ N ₆ ), 1,7-diamino-trans-hept-3-ene (compound 20a, C-7H ₁₆ N ₂ ), or 23 (1,7-diguanidinohept-1-ene). 3-ene, C9H ₂ 0N _6).

Most preferably, the hypusinylation inhibiting agent is N 1 -Guanyl-1, 7-diaminoheptane (GC7).

In the context of the present invention, the "hypoxic" conditions result either from an ischemic injury (pathological hypoxia), or when climbing at altitude or underwater diving (natural hypoxia). Preferably, the pharmaceutical composition is used to prevent the occurrence of disorders related to the occurrence of ischemic injury or hypoxia, specifically disorders resulting from the occurrence of ischemic injury or hypoxia affecting an organ, a tissue or cells. These disorders are, for example, renal failure or cortical necrosis (due to ischemic injury or hypoxic conditions), heart failure (due to infarction), neurological sequelae resulting from the death of neurons or reduced functioning (due to stroke or trauma), or organ disruption or tissue damage to the liver, intestine, heart, lung, kidney or other organs as a result of their transplantation, mountaineering, aeronautical activities or scuba diving.

Thus, preferably, the composition of the invention - or the hypusinylation inhibiting agent of the invention - is used to prevent and / or treat pathologies resulting from hypoxic conditions and / or ischemic lesions. , for example selected from: renal failure due to ischemic injury or hypoxic conditions, heart failure due to infarction, neurological sequelae due to stroke or trauma, or disturbances of a organ such as liver, intestine, heart, lung or kidney following transplantation.

The present inventors have demonstrated that the previously described hypusinylation inhibiting agents increase the hypoxic tolerance of different cell types, i.e., to increase their resistance to oxygen deprivation. In other words, the cells (or tissues) that are deprived of oxygen (due to ischemic injury or the occurrence of hypoxic conditions) are less affected (therefore more functional) when they have been previously treated with these inhibitors.

The pharmaceutical composition of the invention - or the hypusinylation inhibiting agent of the invention - can therefore be used to improve the remission of patients who have undergone ischemic injury, or who have been under hypoxic conditions (diving, mountaineering) for a while. It is thus perfectly indicated in the treatment of any pathology likely to induce ischemic lesions in order to reduce the harmful consequences. It is also perfectly indicated during physical activities likely to induce hypoxic conditions such as aeronautism, mountaineering or diving, in order to reduce the harmful consequences.

More particularly, the pharmaceutical composition of the invention can be used to prevent and / or treat disorders of a hypoxia organ, preferably for a period of at least 2 minutes, and even more preferably to at least 5 minutes.

Within the meaning of the present invention, the "conditions of hypoxia" likely to affect a cell, a tissue, an organ or an individual are such that the level of oxygen supplied to said cell, tissue, or organ (especially via the blood stream ) is decreased by 30%, preferably 50%, even more preferably 80% or even 100% compared to a normal situation in vivo. Decreasing this oxygen level can be easily measured by determining the oxygen level in the individual's blood (and comparing it to standard values), or the number of functional blood vessels that irrigate the target organ. (by comparing it with standard data).

Most preferably, "hypoxia" refers to a situation in which a cell, tissue, organ or individual is completely deprived of oxygen (for example, during a cut in the bloodstream or in a non-ventilated medium, for example in submarine or super-atmospheric environment) for at least 5 minutes, preferably at least 10 minutes, most preferably at least 15 minutes.

The term "disorders of an organ which has undergone hypoxia or ischemia" means the molecular mechanisms induced by hypoxia and ischemia known to those skilled in the art as well as the consequences, or sequelae, of these molecular mechanisms on the subject. general condition of the organ (tissue damage, malfunctions). As examples of sequelae / dysregulations, mention may again be made of renal insufficiency or cortical necrosis, cardiac insufficiency, neurological sequelae resulting from the necrosis of the neurons or the reduction of their functioning, or the disturbances organs or tissue damage affecting the liver, intestine, heart, lung or kidney or any organ after transplantation, mountaineering, aeronautical activities or scuba diving. Thus, preferably, the composition of the invention - or the hypusinylation inhibiting agent of the invention - is used to treat pathologies inducing hypoxic conditions and / or ischemic lesions, for example selected from: stroke, trauma, cardiac arrest, atrial fibrillation, angiopathies, peripheral and central microangiopathies , thrombosis, infarction or pulmonary embolism.

The results provided in this application demonstrate the protection induced by GC7 against the sequelae of hypoxia / ischemia on the kidney and against the sequelae of a stroke. These results can be reasonably extended to other organs likely to undergo ischemic or hypoxic or transplantable injury, since any organ is suffering as soon as the oxygen supply decreases with more or less serious functional consequences. Organs such as the heart, brain and kidney are the most sensitive because of their inability to regenerate under hypoxic / ischemic stress. The administration of GC7 or more broadly the inhibitor of deoxyhypusine synthase (DHS), allows the organ to better resist the deprivation of oxygen, and it will see its physiological functions preserved, improving its subsequent functioning .

Indeed, during the transplantation of organs such as liver, intestines, heart or lungs, these organs are sometimes transported for a certain distance and for a longer or shorter time. Classically, the grafts are stored at 4 ° C. in appropriate solutions for each organ.

The duration of cold ischemia (FID) of organs removed is the time during which the graft is refrigerated. It has been shown that the risk of transplant failure increases with increasing FID.

The ischemic lesion should not generally exceed a few hours, otherwise irreversible sequelae.

In a particular embodiment, the pharmaceutical composition of the invention can therefore be used to increase the hypoxic tolerance of mammalian cells subjected to ischemic injury or hypoxic conditions.

In particular, with regard to organ donation, the pharmaceutical composition of the invention makes it possible to increase the waiting time of the organ before its reoxygenation in the tissues of the transplanted individual (DIF), before that sequelae do not appear. In a particular embodiment, the pharmaceutical composition of the invention is therefore used to prevent and / or treat disorders of the organs following their transplantation. More specifically, the pharmaceutical composition of the invention is used to improve the resistance of the organ and to limit cell and tissue damage in the organ during the various stages of its transplantation. It thus makes it possible to improve the functions of the organ once after its transplantation and the remission of the patient who has undergone the transplant. It can therefore also be used to treat patients who have undergone organ transplantation.

In a preferred embodiment, the hypusinylation inhibiting agent is present in the organ preservation fluid prior to transplantation into the host individual. Thus, the cells are protected during the time when oxygen is lacking. The present invention therefore also relates to the use of the hypusinylation inhibiting agent described above as a cytoprotective agent used in an organ preservation fluid, as well as a liquid for the preservation of ex vivo organs containing the inhibiting agent of the organism. hypusinylation previously described.

Organ preservation fluids are well known to those skilled in the art, and the cyto-protective agent of the invention may therefore be added to any of them.

It is also possible to administer the hypusinylation inhibiting agent or the pharmaceutical composition of the invention in the individual to whom the organ will be removed, in order to impregnate the organ before it is cut off from the bloodstream.

Most preferably, in this particular embodiment, the composition of the invention comprises, as a cyto-protecting agent, Nl-Guanyl-1,7-diaminoheptane (GC7).

The results presented in this application also demonstrate that in vitro pretreatment of GC7 neurons results in an increase in their survival of more than 30% under hypoxic conditions. This protective effect of GC7 on neurons has been confirmed in vivo in animals with stroke: the volume of infarction in animals treated with GC7 is much lower compared to control animals. These experiments clearly demonstrate a protective effect of GC7 on neurons against hypoxic and ischemic phenomena. The composition of the invention thus also makes it possible to prevent neurological sequelae due to ischemic or hypoxic injury, for example due to a cerebrovascular accident or to a trauma. By "sequelae" is meant both direct and / or indirect damage to neurons (eg necrosis of neurons, reduction of their functioning) and their consequences on the individual and his physiology, in the short, medium and and long-term (for example, the occurrence of Alzheimer's disease, post-stroke depression, or the reduction of mental and motor faculties). The composition of the invention thus makes it possible to prevent the formation of cellular and / or tissue lesions in the brain, and thus to reduce or even prevent the appearance of functional, motor and mental sequelae (post-stroke depression or diabetic disease). Alzheimer for example) due to the oxygen deprivation of the patient's neurons.

The composition of the invention is also useful for treating any pathology likely to induce reduction and / or oxygen deprivation on neuronal cells, in particular to treat pathologies of purely neurological origin (neuropathologies), or neuro-or cerebrovascular (stroke type and angiopathy).

In a particular embodiment, the pharmaceutical composition of the invention is used to prevent and / or treat heart failure due to infarction in an animal. More specifically, the pharmaceutical composition of the invention is used to prevent heart failure or any other disorder or injury due to infarction. It can therefore also be used to treat patients who have suffered a heart attack.

Preferably, in this particular embodiment, the composition then comprises, as a cytopter protective agent, Nl-Guanyl-1,7-diaminoheptane (GC7).

Finally, the composition of the invention makes it possible to significantly reduce the basal oxygen consumption of the treated animals. Surprisingly, this decrease in oxygen consumption is not accompanied by a decrease in the level of activity of the treated animals.

The composition of the invention can therefore be used in any activity where the oxygen supply is limiting, for example for activities at altitude (first aid, mountaineering, aerial activities, etc.), or even for underwater activities ( diving). This discovery is important because there is no other way to limit the oxygen consumption of a mammal without reducing physical activity.

The present invention therefore also aims more particularly at the use of the pharmaceutical composition of the invention for reducing the oxygen consumption of an individual.

In a second aspect, the present invention is therefore directed to the pharmaceutical composition (or hypusinylation inhibiting agent) of the invention for use in decreasing the oxygen consumption of an individual in need thereof.

Also contemplated is the use of the pharmaceutical composition (or hypusinylation inhibiting agent) of the invention for the manufacture of a medicament for decreasing the oxygen consumption of an individual in need thereof, thereby a method for decreasing the oxygen consumption of an individual in need thereof, said method comprising administering to said individual the pharmaceutical composition (or hypusinylation inhibiting agent) of the invention.

In this aspect of the invention, said individual is preferably a mammal, even more preferably a human being.

The present inventors have demonstrated that GC7 protects a mammal from the adverse consequences of renal failure caused by ischemic injury (see examples).

The present invention therefore furthermore relates to a pharmaceutical composition for its use for preventing and / or treating ischemic injury-related renal failure, said composition comprising, as a cyto-protective agent, an inhibitory agent for hypusinylation of eIF5A, preferably Nl-Guanyl-1,7-diaminoheptane (GC7).

In other words, the present invention is directed to the use of a pharmaceutical composition comprising, as a cytopterive agent, an eIF5A hypusinylation inhibiting agent, for the manufacture of a medicament for preventing and / or treating renal failure due to ischemic injury in an animal, particularly in a mammal.

Preferably, the pharmaceutical composition is used to prevent the occurrence of disorders related to renal failure. Furthermore, the present invention is directed to the hypusinylation inhibiting agent of the invention, for its use for preventing and / or treating renal failure due to ischemic injury in an animal, or the use of this agent. inhibitor for the manufacture of a medicament for preventing and / or treating renal failure due to ischemic injury in an animal.

Preferably said animal is a mammal, in particular man.

Preferably, said hypusinylation inhibiting agent of the invention is N-Guanyl-1,7-diaminoheptane (GC7).

The present invention finally provides a method of preventing and / or treating renal failure due to ischemic injury in an individual suffering from it, comprising the step of administering the hypusinylation inhibiting agent of the invention. or a pharmaceutical composition comprising, as a cyto-protective agent, the hypusinylation inhibiting agent of the invention, and a pharmaceutically acceptable carrier.

The present invention also relates to the pharmaceutical composition of the invention, for its use for - or for the manufacture of a medicament for - preventing and / or treating neurological sequelae due to a stroke or trauma in an animal .

More specifically, the present invention relates to the hypusinylation inhibiting agent of the invention, for its use for - or for the manufacture of a medicament for - preventing and / or treating neurological sequelae due to a cerebrovascular accident or trauma in an animal.

Said animal is preferably a mammal, and in particular man.

The present invention finally provides a method for preventing and / or treating neurological sequelae due to a stroke or trauma in an animal, comprising the step of administering the hypusinylation inhibiting agent of the invention , or a pharmaceutical composition comprising it.

Preferably, said composition comprises, as a cyto-protective agent, N 1 -Guanyl-1, 7-diaminoheptane (GC7).

Said animal is preferably a mammal, and in particular man. EXAMPLES

The inventors have evaluated whether and to what extent deoxyhypusin synthase (DHS) could be used as a potential pharmacological target in hypoxia / ischemia indications in a mammalian species, for example the rat.

To do this, a model of renal ischemia by temporary arrest of the arterial flow

(Wenzel et al., 1992, Basile et al., 2005) was implemented (Figure 1). Ischemia was surgically performed by ligation of the left renal artery in adult female rats anesthetized with isoflurane. After 40 minutes of ischemia, the blood flow was restored. Renal impairment was assessed by measuring a renal sulfur marker (NGAL, Neutrophil Gelatinase-Associated Lipocalin, by ELISA) in rat urine. Renal function was assessed by the clearance method (Dworkin LD and Brenner BM 2000): Plasma and urine samples were collected and analyzed for fractional excretion of glucose, sodium and phosphate.

CG Summary 7

GC7 (Nl-guanyl-1,7 diaminoheptane), an inhibitor of deoxyhypusine synthase DHS, was synthesized according to the method described by Jalusionis et al. Cell Biochem Funct (2007). The structure of the product and its purity were evaluated by analysis methods known to those skilled in the art (NMR, mass spectrometry). The purity obtained is greater than 99%. GC7 is soluble in water. A 3 mg / ml solution was prepared in physiological saline.

Production of anti-hypusin antibodies

L-Hypusine was synthesized according to the technique described by Bergeron et al. (Med Chem 1998) and incorporated in the peptide: Cys-Thr-Gly-Hpu-His-Gly (Hpu = hypusine). A polyclonal antibody was produced in the rabbit after coupling the peptide to ovalbumin. It recognizes in Western blot experiments, a single protein with the expected molecular weight (17 kDa). The pre-immune serum is inactive. The dilution used is 1/1000. I. Measurement of NGAL, a marker of renal impairment

Neutrophil Gelatinase-Associated Lipocalin (NGAL) is considered a reliable marker of renal tissue hypoxia (Mishra et al., 2003, 2004, Haase et al., 2009). The urine levels of NGAL are determined by a commercial ELISA test (Bioporto) and reported at urinary creatinine concentrations.

The rats were divided into two groups, one treated with GC7 (3 mg / kg intraperitoneal), the other with the control vehicle (same amount of saline) for 4 days before the ischemic injury.

To achieve the ischemic injury, the rats were anesthetized with isoflurane, and the left renal artery was ligated. After 40 minutes of ischemia, the blood flow is restored. Urine and a blood sample are collected at time zero (before ischemia) and after 24 hours of recovery.

a) urinary NGAL assay

The NGAL marker was assayed in total urine before and 24 hours after 40-minute cross-clamping (temporal ligation) of the left renal artery. The results are shown in Figure 2. These results demonstrate that ischemia induces urine production of NGAL in control animals (confirming the existence of an ischemic state), and that this production is inhibited by GC7, indicating a protective effect of GC7.

FIG. 2 also shows that the administration of GC7 does not modify the urinary excretion of NGAL at time 0 before ischemia.

b) plasma NGAL assay

Plasma NGAL (from the ischemic kidney) was assayed 24 hours after 40-minute cross-clamping (temporal ligation) of the left renal artery in control or GC7-treated rats. The results are shown in Figure 3. These results demonstrate that the increase in plasma NGAL is more significantly reduced in the GC7-treated animals, indicating a protective effect.

c) Renal function

Renal function was assessed by fractional excretion of glucose, sodium, and phosphate. All rats underwent 40-minute ischemia of the left renal artery and were reperfused. After 24 hours of recovery the clearance experiments (Dworkin LD and BM Brenner, 2000) were carried out independently on the right (control) and left (ischemic) kidneys of anesthetized rats. Blood and urine glucose was measured by a spectrometric technique (Randox, Kit Gluc HK). Figure 4 shows an alteration of glucose reabsorption by ischemic kidneys. The GC7 corrects this default meaningfully.

Plasma (Na) and urinary sodium were measured by flame spectrometry. Plasma and urine phosphate were determined by ion chromatography (Dionex IonPac AS 11). Figure 5 shows an alteration of the fractional excretion of sodium (A) and phosphates (B) on the ischemic left kidneys. Here again, the animals treated with GC7 are protected from the harmful consequences induced by ischemia.

d) Hypusinylation of eIF5A

GC7 is a specific inhibitor of desoxyhypusine synthase, the enzyme that allows hypusinylation (and functionalization of eIF5A). It was important to verify that treatment of rats with GC7 effectively reduced hypusinylation of eIF5A. Hypusinylation of eIF5A was analyzed by "Western blot" experiments using two primary antibodies:

1. a commercial antibody (AbCam, Ref: ab32443) which recognizes all forms (hypusinylated or otherwise of eIF5A), and

2. A polyclonal antibody against the hypusinylated forms of eIF5A that was produced in rabbits from a synthetic peptide (Cys-Thr-Gly-Hpu-His-Gly) that mimics the eIF5A sequence around the hypusinylation site .

Animal kidneys controlled or treated with GC7 (3 mg / kg intraperitoneal for 3 days) were homogenized in solubilization buffer (10 mM Phosphate, 3% SDS, 3% glycerol). The expressions of the total and hypusynilic forms of eIF5A are analyzed by Western blot. After digitizing the gels (FIG. 6A), the ratio of the two signals obtained after densitometric measurement of the specific protein bands is calculated. Figure 6B shows that administration of GC7 significantly decreases the hypusinylated eIF5A fraction at the renal level. e) Conclusion

The results presented above indicate that GC7 inhibits hypusinylation of eIF5A at the renal level. Moreover, the present inventors have been able to show that GC7 protects renal damage induced by ischemia / reperfusion. The GC7 therefore largely protects animals against the renal consequences of ischemia.

II. GC7 protection of hypoxia-induced urinary phosphate loss

The kidney is the organ responsible for maintaining the ionic homeostasis of the body and especially the phospho-calcium balance. This balance is essential and is described in the literature as hypoxia-sensitive (Baines AD et al 1998, Mimura Y and Knox FG 1994, Brazy PC et al 1984). The latter induces a rapid loss of phosphate ions in the urine which can become problematic.

The urinary loss of phosphate was studied in mice treated with GC-7: Male and female mice were injected (3 times at 1 day intervals) either with a placebo or with GC7 at 3 mg / kg in IP . The ^3rd day, the animals were kept either in normoxic (21% 0 ₂₎ or hypoxia (6-8% 0 ₂₎ in normobariques conditions for 24 hours. Urine samples were then collected from each animal and analyzed for inorganic phosphate (PO ₄ ) and creatinine content. FIG. 7 gives for each animal category the average of the PO ₄ / creatinine ratios on (n) mice. The ratio to creatinine eliminates the urine dilution rate specific to each animal and its physiological condition at the moment of measurement.

It has been shown that:

hypoxia induces a massive urinary loss of phosphate,

GC7 does not modify the urinary excretion of phosphates under normoxic conditions, and

GC7 significantly attenuates hypoxia-induced urinary phosphate loss.

Since phosphate reabsorption in mammals is located mainly in the proximal segment of the nephron, these results prove that GC7 protects this essential function of the kidney in cases of hypoxia. III. GC7 decreases the basal oxygen consumption of a mammal.

III.1. In vitro results

Under physiological conditions in vitro, the glucose consumption of renal cells derived from the proximal tubule of GC7-exposed mice is greatly increased (FIG. 8C). At the same time, the production of lactate by these same cells increases (FIG. 8D). The oxygen consumption of the same cells is very significantly decreased (Figure 8A). Despite the drastic modification of these parameters, the intracellular level of ATP remains constant (FIG. 8B).

These data suggest that GC7 induces a "shift" of cells to anaerobic glycolytic metabolism, explaining why GC7-treated kidney cells suffer less from the absence of oxygen during ischemia.

III.2. In vivo results

These data have been validated on an in vivo model.

The basal oxygen consumption of mice injected with placebo (NaCl 0.9%) or with GC-7 at 3 mg / kg was investigated:

Male (Figure 9A) and female (Figure 9B) mice were treated with an IP injection of placebo NaCl 0.9% (control, n = 5) or GC7 at 3 mg / kg (GC7, n = 4). in 0.9% NaCl the day before and the day of measurement. For measurement the animals were placed individually in a 500 cm enclosure containing a device (barium hydroxide solution) capable of trapping the C0 ₂ exhaled by the animals. The oxygen consumption was recorded for about 30 minutes (1 point every 5 minutes) using an oxybaby® (Witt) oximeter. The indicated consumptions were expressed in ml of 0 ₂ per hour and per gram of body weight. The experiments were stopped when the oxygen level of the container reached 10% of 0 ₂ .

The summary of the results is illustrated in Figure 9.

They demonstrate that:

GC7 induces a significant decrease in the basal oxygen consumption of a male or female mammal; this effect of GC7 on oxygen consumption in vitro and in vivo is explained by a metabolic shift making the mammal less dependent on oxygen; this less dependence on oxygen explains the resistance of the kidney to a hypoxic / ischemic episode; and this decrease in oxygen consumption does not change the level of sleep of animals that maintain normal behavior.

IV. GC7 protects neurons from ischemia

IV.1. In vitro: GC7 increases neuronal resistance against ischemia reperfusion

Primary cultures called "mixed" were carried out: they contain 55% of neurons, grown on an astrocytic carpet representing 45% of the total number of cells. Under these conditions, neurons that are mature and differentiated have the same functionalities as cerebral neurons in vivo (Gouix et al., 2009). On these cultures, stroke was mimicked by oxygen and glucose deprivation ("OGD" model for "oxygen glucose deprivation") of 60 min (Goldberg et al., 1993). The cultures were then replaced in a normally glucose and oxygenated medium to reproduce the reperfusion for 24 hours and the number of neuronal cells was quantified. For quantification, four microscopic fields per well (one field = 1.6 mm) were randomly selected by an experimenter ignoring the experimental conditions. The results presented in Figure 10 come from 3 separate experiments (totally independent primary cultures) for a total of 24 wells per analyzed conditions. Before the OGD, the cultures were pretreated or not for 12 hours with 30 μl of GC7.

different from the control (p <0.001) and $$$, different from the untreated GMO condition at GC7, (p <0.001).

Figure 10 shows that deprivation of oxygen and glucose for 60 minutes results in the death of 32% of neurons. (Control condition: 90.28 + 2.48 versus OGD condition: 61.40 + 1.35 neurons per field). The OGD leads to only 11% neuronal loss when the cells were pre-treated with GC7 (Condition control + GC7: 86.74 + 1.58 versus condition OGD + pretreatment GC7: 77,27 + 1,7).

Pretreatment with GC7 therefore leads to an increase in neuronal survival of more than 30%. These experiments clearly demonstrate a protective effect of GC7 on neurons against ischemic phenomena. They also show that, under physiological conditions, pretreatment with GC7 has no deleterious effect on cultured neurons (control condition: 90.28 + 2.48 versus Condition control + GC7: 86.74 + 1, 58, NS - no significant difference). IV.2. In vivo: GC7 protects against transient ischemic stroke

The results presented in FIG. 11 were obtained with male C57B1 / 6 mice with a minimum weight of 20 g. The animals had free access to food and water and were kept in the circadian cycle (12 h of light / 12 h of darkness) in our licensed pet shop. To reproduce the focal ischemic stroke, the model of transient focal ischemia (so-called MCAO model) was used. It consists of the occlusion of the middle cerebral artery (MCA) by the intraluminal introduction of a nylon filament. The average cerebral artery corresponds to the sylvian artery in humans, whose occlusion is responsible for approximately 80% of strokes. The occlusion time was 60 min (Blondeau et al., 2009, Nguemeni et al., 2010). The size of the infarct, the mortality rate, the percentage of cases of spontaneous reperfusion were characterized according to a published method (Nguemeni et al., 2010). The preventive approach consisted of 3 repeated injections of GC7 (3 mg / kg body weight) spaced 24 hours apart, the last injection was performed 30 minutes before the induction of stroke. The same protocol was performed for placebo.

Table 1:

These results showed that GC7 pretreatment clearly decreases the animal mortality rate 24 hours after carotid occlusion by doubling the survival rate of animals on the first day post-stroke. Similarly, GC7 treatment increases spontaneous reperfusion rate after stroke. These two factors are the first indications of a GC7 increase in resistance to transient ischemic stroke. Indeed, mortality is the first consequence of stroke and non-reperfusion is the most unfavorable prognosis for stroke. Mortality is a consequence of excessive brain damage / infarction / neuronal loss and the absence of reperfusion results in an expansion of the lesional volume. The present results suggest that pretreatment with GC7 reduces infarct size by about 30%. The different brain regions have different sensitivities to stroke and the severity of the tissue injury is related to its proximity to vascular occlusion. Also, it is admitted in the ischemia of the middle cerebral artery that the ischemic heart mainly affects the subcortical regions, whereas the target ischemic penumbra of the therapeutics would be cortical. The finer analysis of GC7-induced protection suggests that GC7 protection also occurs at the cortical level (Figure 11). Since the effect of GC7 on reperfusion may affect protection, we analyzed the effects of GC7 only in reperfused animals. Pretreatment with GC7 also reduced cerebral infarction by 30% in reperfused animals (control: 26.4 + 4.2 mm versus GC7: 18.3 + 2.6, p <0.05).

In conclusion, pretreatment with GC7 increases neuronal survival by approximately 30% in in vivo and in vitro models reproducing ischemic stroke.

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Claims

A pharmaceutical composition for use in preventing and / or treating disorders related to hypoxic conditions and / or ischemic injury in a mammal, said composition comprising as cytopterant agent an eIF5A hypusinylation inhibitor capable of inhibiting the synthesis of hypusine.

Pharmaceutical composition according to Claim 1, characterized in that the said inhibitor of hypusinylation of eIF5A inhibits deoxyhypusine synthase (DHS) or deoxyhypusine hydrolase (DHH).

Pharmaceutical composition according to one of Claims 1 or 2, characterized in that the said agent for inhibiting the hypusinylation of eIF5A is chosen from the derivatives of 1,7-diaminoheptane (DAH), the 1,3-diazacyclohexane derivatives and the 1,3-diazacyclo-1-hexene derivatives.

Pharmaceutical composition according to any one of Claims 1 to 3, characterized in that the said agent for inhibiting hypusinylation of eIF5A is chosen from the derivatives of 1,7-diaminoheptane (DAH), 1,3-diazacyclohexane and 1-diazacyclohexane. , 3-diazacyclo-1-hexene.

A pharmaceutical composition according to any one of claims 1 to 4, wherein said eIF5A hypusinylation inhibiting agent is a 1,7-diaminoheptane (DAH) derivative selected from Nl-Guanyl-1,7-diaminoheptane ( GC7), 1,7-diguanidinoheptane (GC7G), 1,7 diaminoheptane (C7), 7-amino-1-guanidinooctane, 1,7-diamino-iran5-hept-3-ene, GC6, GC8, GC6G, GC8G, CNI-1493.

The pharmaceutical composition according to any one of claims 1 to 5, wherein said eIF5A hypusinylation inhibitory agent is N1-Guanyl-1,7-diaminoheptane (GC7).

7. Pharmaceutical composition according to any one of claims 1 to 6, characterized in that said disorders result from hypoxic conditions and / or ischemic lesions.

8. Pharmaceutical composition according to claim 7, characterized in that said disorders are chosen from: renal insufficiency, heart failure, neurological sequelae, or disorders of an organ such as the liver, intestine, intestines heart, lung or kidney following transplantation.

9. Pharmaceutical composition according to claim 1, characterized in that said disorders induce hypoxic conditions and / or ischemic lesions and are selected from cerebrovascular accident, cerebral trauma, cardiac arrest, atrial fibrillation, angiopathies. , peripheral and central microangiopathies, thromboses, infarcts or pulmonary embolisms.

10. The pharmaceutical composition as defined in any one of claims 1 to 6 for use in increasing the hypoxic tolerance of mammalian cells subject to ischemic injury or hypoxic conditions.

The pharmaceutical composition according to any one of claims 1 to 6 for use in preventing and / or treating renal failure due to ischemic injury in an animal.

12. Pharmaceutical composition according to any one of claims 1 to 6, for its use for preventing and / or treating neurological sequelae due to a stroke or trauma in an animal.

13. A pharmaceutical composition according to any one of claims 1 to 6 for use in preventing and / or treating heart failure due to infarction in an animal.

14. Pharmaceutical composition according to any one of claims 1 to 6, for its use for preventing and / or treating disorders of an organ following its transplantation.

15. Use of the hypusinylation inhibiting agent described in claims 1 to 6 as a cytoprotective agent in an organ preservation fluid.

16. Liquid for the preservation of ex vivo organs, containing the hypusinylation inhibiting agent described in claims 1 to 6.

17. Use of the composition as defined in claims 1 to 6, to reduce the oxygen consumption of an individual.

18. The use of claim 17, wherein said individual is a human being.

19. A pharmaceutical composition as defined in any one of claims 1 to 6 for use in preventing and / or treating disorders of a hypoxia-affected organ.