WO1998051291A1

WO1998051291A1 - Use of a pharmaceutical composition for treating and/or preventing ischemia

Info

Publication number: WO1998051291A1
Application number: PCT/BE1998/000067
Authority: WO
Inventors: José REMACLE; Carine Michiels
Original assignee: Remacle Jose; Carine Michiels
Priority date: 1997-05-13
Filing date: 1998-05-12
Publication date: 1998-11-19
Also published as: EP0981339A1; AU7327298A; CA2287363A1; BE1011151A3; JP2001526658A; NO995500D0; NO995500L

Abstract

The invention concerns the use of a pharmaceutical composition comprising a suitable pharmaceutical carrier and an active compound selected among the group consisting of bioflavonoids, rutin-garlic, troxerutin, coumarin, diosmin, o-(-hydroxyethyl) rutins, sweet clover and rutin extracts, tribenoside, methylchalcone hesperidin, Indian chestnut extract, naphtazone, esculoside, aescin, procyanidine oligomers, butcher's broom and methylchalcone hesperidine extracts, ruscosides, common holly and black currant extracts, blueberry anthocyanin extracts, the active principles of these compounds and/or a mixture of them, acting on a patient's mitochondrial membrane protein complexes, to prepare a medicine for treating and/or preventing ischemia and/or pathologies associated with ischemia or with energy deficiency.

Description

USE OF A PHARMACEUTICAL COMPOSITION IN THE TREATMENT AND / OR PREVENTION OF ISCHEMIA

Subject of the invention

The present invention relates to the therapeutic and / or prophylactic application of a pharmaceutical composition in the treatment and / or prevention of ischemia and of pathologies associated with

Ischemia.

State of the art and technological background

To treat certain varicose diseases, various active compounds have been proposed, most of which are present in plant extracts. Such compounds are described in particular in the following documents: French patent application FR-2692145, French patent application FR-2668705, European patent EP-0541874- B1, international patent application WO93 / 20046, international patent application O93 / 20045 , European patent EP-0566445-B1, European patent application EP-0210781-A1 and European patent application EP-0112770-A1.

Likewise, active compounds isolated from plant extracts have been used for the treatment of the consequences of ischemia or of the pathologies associated with ischemia. This is the case with the flavonoids which are known as antioxidants and can limit the damage caused by free radicals produced during reperfusion. Indeed, during reperfusion after a period of ischemia, a very significant production of free radicals derived from oxygen is observed which will cause damage to the various constituents of the cell weakened by the period of ischemia in particular by the lack of energy (ATP). This increased production of free radicals has been well demonstrated by Me Cord JM (1985, N. Engl. J. Med., 312: 159-163). It is due to the activation of xanthma oxidase during the period of ischemia and to its very high activity during reperfusion. Thus antioxidant molecules like flavonoids have a beneficial effect on the process of isperemia reperfusion since they protect the tissue against this excess of free radicals (Reddy DS et al., 1995, Drugs of today, 31: 335-349 ).

However, many plant extracts consist of a mixture of many complex macromolecules, and the presence on some of them of flavonoid type structures known for their antioxidant properties has obscured the possibility for these extracts or molecules of a possible presence of its own anti-ischemic activity, that is to say independent of the reoxygenation process.

The activity of flavonoids on ischemia and the decrease in the impact of the myocardium is due to this activity of control of active oxygen derivatives (Reddy et al., 1995, Drugs of today, 31: 335-349; DE-3623255, 0X0 Chemistry GMBH, 1988). Troxerutme has been used in particular as an anti-oxidant molecule to protect the heart during the process of ischemia reperfusion (Blasug IE et al., 1987, Biomed. Biochim. Acta, vol. 46: 5539-544; XP 002052079 and Olszenski AJ, 1991, Atherosclerosis, 88: 97-98).

Likewise diosmin blocking the formation of free radicals by Xanthine oxidase (which occur during reperfusion after ischemia) has been used in this sense as being able to protect during ischemia

(Bouskela E. et al., 1997,48: 33-37; Int. J. Microcirc.

Clin. Exp., 1995, 15: 293-300; Debbarre B. et al., 1995, Int. J. Microcirc. Clin. Exp., 15 suppl. I: 27-33).

Rutosides have also been successfully tested for their anti-ischemic activity. This is how they reduce the size of the impact in animals subject to arterial obstruction (Zale ski A. et al., Am. J. Cardiol., 1985, 56: 974-977), and also relieve patients with ischemia of the lower limbs (Milliken JC, Vasa, 1974, 3: 203-206).

Naftoquinone, which is known for its anti-aggregating activity of platelets, can be used to inhibit the formation of Thrombus during thromboses (EP-A-0631777).

The demonstration of this activity does not constitute an anti-ischemic activity as such but an action on one of the causes of thrombosis, namely the aggregation of platelets.

Aims of the invention

The present invention aims to provide a new therapeutic and / or prophylactic method of ischemia and / or pathologies associated with ischemia.

A particular object of the present invention aims to obtain a process using agents non-toxic or little toxic therapeutics, presenting little or no side effects, and the synthesis or extraction of which from living products, in particular from plants, is simple and inexpensive.

Character-defining elements of the invention

The present invention relates to the use of a pharmaceutical composition, comprising a pharmaceutical vehicle and a sufficient amount of an active compound chosen from the group consisting of citroflavonoids, garlic-rutoside, troxerutin, coumarin, diosmin, o- (-hydroxyethyl) rutosides, extracts of Melilot and rutoside, tribenoside, hesperidin methylchalcone, horse chestnut extract, naftazone, esculoside, aescin, procyanidolic oligomers, extracts of ruscus and hesperidin methylchalcone, ruscosides, extracts of small holly and blackcurrant, extracts of blueberry anthocyanin, active ingredients isolated from these compounds and / or a mixture thereof, for the preparation of a medicament intended for the treatment and / or prevention of ischemia and / or pathologies associated with ischemia.

The active compounds mentioned above are products generally isolated from plants and / or plant extracts, marketed by different pharmaceutical companies under different brands. These different active compounds, identified by their brand and the companies which market them, are listed in Table 1 below. Table 1: Active compounds of the composition according to the invention

Table 2 shows the active compounds of the composition according to the invention, which have already been shown to be able to protect in the process of isperemia reperfusion. Table 2; Active compounds of the composition according to the invention, which have been shown to be able to protect in the process of ischemia reperfusion

Such active compounds as well as their dosage and their preferred form of administration, are also described in the documents "Commented directory of medicaments" (Belgian Center for Pharmaco-Therapeutic Information, Brussels, 1994) and Vidal 1997 (ed. Du Vidal , 33 Av. De Wagram, Paris, France).

The term "active principle isolated from an active compound of the invention" means the active part having a therapeutic and / or prophylactic effect with respect to its biochemical target as described below, and capable of having properties comparable and / or improved in the therapeutic and / or prophylactic field to those of the active compound described below. The active principles and compounds of the invention are also characterized by a "protective effect" on the protein complexes of the internal mitochondrial membrane, that is to say that the active principles and compounds of the invention are capable of increasing RCR (Respiratory Control Ratio), which represents a patient's mitochondrial respiratory control. The RCR represents the relationship between the consumption of oxygen in the presence of endogenous substrates (glutamate / malate) and the consumption after phosphorylation of ADP to ATP, as will be illustrated below.

By this mechanism of action, the products of the invention make it possible to protect the patient from ischemia or from the consequences of ischemia. The compounds and active principles of the invention are therefore characterized both by a prophylactic and / or therapeutic effect.

Preferably, the products of the invention have a protective effect on the complex I or III of the electron transport chain of the mitochondria, and / or on the adenine translocase protein complex as illustrated below.

The preferred active compounds of the invention are hesperidin methylchalcone, aescin, procyanidolic oligomers and extracts of bilberry anthocyanodisics, which are characterized by particularly advantageous and unexpected properties in the treatment of ischemia, pathologies associated with ischemia, energy deficit and aging-related deficiencies. The term "sufficient quantity of an active principle or compound" means a sufficient quantity of this principle or of this active compound to treat, relieve, dispel or alleviate the symptoms or dysfunctions of the human or animal body associated with the aforementioned diseases and / or to prevent or reduce the possibility of having them. Consequently, the application of the abovementioned therapeutic treatment relates to a prophylactic treatment or a curative treatment of said diseases. The percentage of this active compound can vary according to very wide ranges, only limited by the tolerance and the level of acceptance of the compound by the patient. These limits are in particular determined by the frequency of administration.

Preferably, the dosages are the doses generally used for these products in the treatment of varicose diseases, as described in the document "Commented directory of medicines, Belgian Center for Pharmacotherapeutic Information, Brussels (1994)" and in the document "Medicines used for the symptomatic treatment of peripheral venous conditions,

Annex to the folia ^" pharmacotherapeutica, Belgian Ministry of Public Health and the Environment, Transparency Commission (June 1994)". For example, the product sold under the trademark aescin reappeared ^® is in the form of dragees of an active compound dosage of 20 mg in a pharmaceutical composition of 100 mg. The pharmaceutical vehicle used varies according to the mode of administration chosen (intravenous, intramuscular, oral, etc.) and may include different forms such as tablets, coated or uncoated, pills, capsules, solutions, syrups, etc. . The pharmaceutical compositions will be prepared according to methods generally used by galenicians and pharmacists, and may include any type of suitable pharmaceutical vehicle, solid, liquid and / or gaseous (including water), non-toxic or not very toxic.

The pharmaceutical composition according to the invention may also include an adjuvant or another pharmaceutical compound known to a person skilled in the art for its therapeutic and / or prophylactic effects on the abovementioned diseases or for its properties capable of reducing the side effects associated with the active compound present in the pharmaceutical composition of the invention.

In addition, the bioavailability of the active compounds of the invention at the level of the organism, in particular due to their possible absorption and their passage through target tissues to be protected, can be improved by packaging and coating techniques and / or "drug • targetting" well known to those skilled in the art.

The term “ischemia (partial or total) or pathologies associated with ischemia” is understood to mean vascular diseases chosen from the group consisting of myocardial infarction, cerebral ischemia, chronic venous insufficiency, atheropathies, i.e. lesions due to atherosclerosis affecting the arteries of patients, Raynaud's phenomenon linked to vasospasms leading to vasoconstriction of the arteries, ulcers, impaired capillary permeability, fragile capillaries, scarring, skin changes, retinal defects of ischemic origin, reduced hearing acuity of ischemic origin, disorders associated with stays at high altitude, angina caused by short moments of coronary obstruction , pulmonary hypertension, hepatic ischemia, Parkinson's disease, myopathies and syndromes associated with vascular problems such as diabetes, where hypertension and impaired blood flow appear in the lower limbs. These diseases and pathologies linked to ischemia are well known to clinicians and physicians, who can adapt the use of the pharmaceutical composition for the treatment and / or prevention of symptoms and dysfunctions of the human or animal body associated with the abovementioned diseases and / or to prevent or decrease the possibility of having it.

Another aspect of the present invention relates to the use of the pharmaceutical composition according to the invention in the case of a decrease in the production of energy by various and differentiated cells, associated with aging. This is the case of intellectual defects in an elderly subject, dizziness syndrome and the decrease in perspective adaptation due to a change in the regulation of metabolism.

A final aspect of the present invention relates to the use of the pharmaceutical composition according to the invention in the field of transplants.

Said pharmaceutical composition can be used directly on the patient or intended for ex vivo treatment of the patient, in which an organ, tissue and / or physiological fluid such as blood or serum, originating from the patient himself or from another human or animal patient is treated by adding said pharmaceutical composition according to the invention directly to the organ, tissue or physiological liquid before its administration to the patient. This application relates in particular to the field of heart transplants. The present invention also relates to a method of therapeutic and / or prophylactic treatment of ischemia and / or pathologies associated with ischemia or an energy deficit as well as the deficiencies linked to aging, such as intellectual defects in the elderly, vertiginous syndrome or the reduced adaptation in perspective due to a modification of the metabolic regulations, of a patient, in which the pharmaceutical composition according to the invention is administered to said patient so as to treat, relieve, dissipate or reduce the symptoms or dysfunctions of the human or animal body associated with the aforementioned diseases and / or to prevent or reduce the possibility of having it. This administration is carried out according to methods well known to those skilled in the art, in particular those applied for the treatment of varicose diseases.

These prophylactic and / or therapeutic effects are described in particular in the examples appended hereto, ^" with reference to the figures, given by way of nonlimiting illustration of the subject of the invention.

Brief description of the figures

Figure 1 schematically represents the activation cascade of the endothelial cell by hypoxia as well as its consequences in terms of adhesion and activation of neutrophils.

FIG. 2 schematically represents the activation cascade of endothelial cells by hypoxia and its consequences at the level of the vascular wall. FIG. 3 represents the measurement of the respiratory activity of the mitochondria as a function of the addition of different active compounds.

FIG. 4 schematically represents the chain of electron transporters at the level of the internal mitochondrial membrane.

Examples

Endothelial cells (EC), by virtue of their location at the blood-tissue interface, are responsible for maintaining vascular homeostasis. They thus fulfill a whole series of functions and constantly interact with circulating leukocytes and smooth muscle cells (SMCs) in the media. Any disturbance in their metabolism can therefore lead to alterations in the functioning of the underlying tissues.

Because they are localized at the blood-tissue interface, the EC are the first to suffer from any modification of the blood flow and in particular from a reduction of this during stasis, which lead to a depletion of the intake oxygen and nutrients to tissues (Hinsha et al., 1988; Tsao et al., 1990).

Hypoxia, which can in particular result from such a stasis, induces a significant activation of the ECs which release inflammatory mediators capable of activating neutrophils and inducing infiltration as well as growth factors for them. CML. This cascade of events ultimately leads to structural and functional changes in the venous wall. Effect of hypoxia on endothelial functions

In order to study changes in the metabolism of ECs when oxygen supply is reduced, ECs isolated from the human umbilical vein are incubated in hypo under hypoxia. Under these experimental conditions, no mortality of the EC is observed during the first two hours of incubation. On the other hand, important modifications of their metabolism are observed: the EC are strongly activated by hypoxia in a similar way to the activation initiated by thrombin or histamine.

The first sign of this activation is an increase in the cytosolic calcium concentration [Arnould et al. , 1992]. This increase is linked to a decrease in the ATP concentration. Calcium is an important secondary messenger in all cells.

In the EC, it is notably capable of activating phospholipase A2, the first enzyme in a metabolic pathway leading to the synthesis of mediators of inflammation. This activation leads to an increased synthesis of prostaglandins [Michiels et al. , 1993]. It also induces the synthesis of platelet activating factors (Platelet Activating Factor (PAF)), which is a very powerful inflammatory mediator [Arnould et al. , 1993].

Hypoxia therefore leads to significant activation of the ECs which then release prostaglandins and synthesize PAF in large quantities. This activation pathway is summarized in FIG. 1. This synthesis of inflammatory mediators can have significant consequences on vascular homeostasis by modulating the functions of the different cell types with which the EC are in contact.

In order to visualize in more detail what these consequences could be, we study the adhesion of a particular type of leukocyte, polymorphonuclear neutrophils (PMN). In vi tro, when ECs are subjected to hypoxia, their adhesiveness to PMNs increases sharply. This adhesion is partly due to the synthesis of PAF by the ECs activated by hypoxia [Arnould et al. , 1993]. Not only do PMNs become adherent to hypoxic ECs, but this adhesion is responsible for their activation [Arnould et al. , 1994] (Figure 1).

It is also known that the EC synthesize vasoactive molecules which modulate the functions of CML. Experiments suggest that EC activated by a lack of oxygen release different mitogenic factors for CML (prostaglandme F2a e basic fibrosbiast growth factor), which induces the proliferation of these cells [Michiels et al. , 1994].

Presumed origin of venous disease

In view of the results described using the experimental model where ECs are exposed m vi tro to hypoxia, the origin of the above-mentioned pathologies seems based on a cascade of events initiated by this hypoxia and which ultimately leads to the structural and functional modifications observed in vascular systems. Figure 2 illustrates this hypothesis. Venous stasis since it disrupts blood circulation causes a decrease in oxygen supply and therefore a hypoxia. This hypoxia can activate the ECs that form the first layer of the venous wall. These cells then release different inflammatory and mitogenic molecules. Inflammatory molecules are capable of inducing the adhesion of certain leukocytes. This is true not only for the EC in culture but also for the endothelium of a complete human vein whether it is an umbilical vein [Arnould et al. , 1995] or a saphenous vein. In addition, during the adhesion process, neutrophils are activated and can release proteases and free radicals. These molecules are known to have the ability to degrade many biological molecules including the components of the extracellular matrix such as collagen. On the other hand, ECs activated by hypoxia also synthesize mitogenic factors for SMCs which induce proliferation. In addition, we know that proliferating SMCs are in a synthetic phenotype unlike the contractile phenotype normally present in the wall of normal veins. When synthetic, SMCs synthesize more components of the extracellular matrix and lose the expression of contractile filaments. Proliferation and increased synthesis of the components of the extracellular matrix lead to thickening of the venous wall while the loss of actin filaments accounts for the loss of overall contractility of the vein.

From the experimental results obtained on the EC model exposed to hypoxia, a new hypothesis concerning the origin of the abovementioned pathologies is proposed: it would be a cascade of cellular interactions involving leukocytes and SML and initiated by activation of EC by venous stasis which would ultimately lead to the structural and functional changes observed.

Mechanism biochimi <d ¹ that action of the active compounds

With the compounds of the invention, there is for example an inhibition of the decrease in ATP content, of activation of phospholipase A2 and of the adhesion of PMNs induced by hypoxia. They are also capable of inhibiting the adhesion of PMNs to the endothelium of a complete human umbilical vein when the latter is incubated under hypoxic conditions. These results clearly indicate an important protective action of these compounds on the endothelium subjected to hypoxia. The inhibition by these drugs of

• CE activation cascade induced by hypoxia is due to their inhibitory effect on the fall in ATP content. This decrease is the initiating event for the activation of

CE because it is directly coupled to an entry of calcium ions into the cell.

These compounds can maintain the ATP content of CE under hypoxia according to two possible hypotheses: either the compounds activate glycolysis, or they preserve mitochondrial respiratory activity. The inventors have discovered that these compounds do not activate glycolysis in the EC subjected to hypoxia but rather that they delay the activation of glycolysis directly induced by hypoxia. These results suggest that they could act on the mitochondria by maintaining respiratory activity under hypoxia for longer. This hypothesis is confirmed by measuring the respiratory activity expressed by the control Respiratory (RCR) (Figure 3) of liver mitochondria from rats treated orally.

Effect of bilobalide on the mid tochondria of the brain under normal conditions

Different concentrations of active compounds of the invention (bilobalide) were tested on the RCR of the mitochondria isolated from the brains of rats. Five concentrations were used: 4, 6, 8, 10 and 12 mg / kg of patient (rat) treated. The rats received these doses of bilobalide orally for 14 days. The mitochondria were isolated according to the method described by Nowicki et al.

(J. Cérébral Blood Flow and Métabolism, 2, pp. 33-40

(1982)). Mitochondrial respiration is measured in a Clark oxygen electrode connected to a recorder. The CPR represents respiratory control. It represents the relationship between oxygen consumption in the presence of endogenous substrates (glutamate / malate) and consumption after phosphorylation of ADP to ATP. This technique was developed by Chance and Williams (Nature, 175, pp. 1120-1121 (1955)). Dose-dependent effect is obtained with an increase in the RCR of 3.7 for the controls to a RCR of 4.6 for concentrations of 8 and 10 mg / kg. A maximum of 24% increase is obtained at 10 mg / kg. These results clearly show that this product is protected against mitochondrial respiration.

Effect of bilobalide on the mid tochondria of the brain in the event of ischemia A 15 minute ischemia was carried out on control rats and rats treated with active compounds of the invention (bilobalides) for 14 days. Ischemia is achieved by decapitation. The rats were treated per os with doses of bilobalide of 10 mg / kg for 14 days. When the RCR is measured in the presence of glutamate / malate for a 15-minute ischemia, an RCR of 3 is observed for the controls for a RCR of 3.9 for the treated rats. The active compound therefore has a protective action on the decrease in respiratory activity induced by ischemia. This protection manifests itself at the level of the activity of complex I and of the transport chain of the mitochondria. The rate of respiratory control measured in the presence of glutamate / malate indirectly reflects the activity of complex I of the electron transfer chain.

The measurement on the complex I of the mitochondria was carried out by a prior sonication of the mitochondria in order to allow access of the assay substrates to the complex I. The latter is assayed according to the reduction of ferricytochrome C at 550 nm. The suspension of mitochondria being in a phosphate buffer of K at 25 nM, pH 7.4, containing MgCl2, 10 μM of cytochrome C and

2.5 mg / ml bovine albumin is sonicated for 30 seconds at

0 ° C. 2 mM KCW are added and the reaction is started by adding 7.5 mM NADH. The mitochondria are incubated at

37 ° C and the reduction in ferricyanide at 550 nm is followed. Correction is made for reduction of cytochrome

C in the presence of Rotenone which inhibits complex I. The results are expressed in μmoles of reduced ferricytochrome C per minute.

The activity of complex I was measured on mitochondria of rats treated for 14 days with 10 mg / kg of bilobalide per os and after a 15-minute ischemia of the brain. An activity of 36 mU / mg of protein is obtained for the control rats while the mitochondria of treated rats show an activity of 44 mU / mg of protein. There is therefore an important protection of the activity of the complex I of the mitochondria. The same protection of complex I is also observed on mitochondria isolated from the brains of rats which have not undergone a period of ischemia.

Effect of bilobalide on mi tochondria of the liver The active compound of the invention (bilobalide) at concentrations of 8 mg / kg of patient (rat) was administered orally to rats for 14 days. The mitochondria of the liver of these rats were isolated according to the method described by Remacle (J. Cell. Biol. 79, 291, 1978). The respiratory activity of the mitochondria of treated rats showed an RCR of 13.25 compared to a RCR of 7.6 for the control rats. A 10-minute ischemia by perfusion was carried out on the livers in a medium consisting of 0.137 M NaCl, KC1 5.4 mM, MgSO ₄ 0.8 mM, glucose 11 mM, Na ₂ HP0 ₃ 0.34 mM, NaHC0 ₃ 24.4 mM, KH ₂ P0 ₄ 6.35 mM and bilobalide 8 mg / 1. The medium is degassed beforehand under an atmosphere containing 95% of N2 and 5% of CO2. The mitochondria are isolated and their respiratory activity determined. For the controls, the rats were given water for 14 days and the livers infused with the same solution as the tests. An RCR of 5.24 was observed for the mitochondria of treated rats and 3.73 for the control rats. These results demonstrate that the active compound of the invention (bilobalide) has an anti- ischemic which is revealed both in vitro (EC subject to hypoxia) and in vivo (hepatic and cerebral ischemia in treated rats) and that this activity is due at least in part to protection of the mitochondria which increase their respiratory activity and so their ATP synthesis.

Each of the active compounds of the invention, such as bilobalide, can increase the RCR of mitochondria isolated from the liver of untreated rats when these are preincubated for 1 hour in the presence of these drugs. They can therefore act directly on the mitochondria. It is very interesting to note that from these results, we can separate the drugs into two different classes: sweet clover extract, Ruscus extract, procyanolic oligomers and hydroxyethylrutosides which increase RCR by increasing the stage 3 of breathing (class II) while the bilobalide, aescin, naftoquinone and diosmin which constitute the drugs of class I increase the

CPR by decreasing stage 4 of breathing (see Table 3).

Table 3; Effect of drugs on the protection of mitochondria

Different action sites therefore seem to be involved and lead to increased mitochondrial respiratory activity and thus to decrease the ATP content during hypoxia.

The process of oxidative phosphorylation of ATP is a complex process involving different enzyme complexes or electron transporters generating a proton gradient (complexes I, III, IV), ATP synthase (complex V) directly responsible for the synthesis of ATP and adenine translocase which is the transporter necessary for the import of ADP and for the export of ATP (FIG. 4). In order to highlight what could be the enzymatic target of these drugs, each complex was inhibited by a specific inhibitor and it was studied whether these preparations could detect this inhibition while measuring the RCR. The results show that none of these drugs has an effect on complexes IV and V. On the other hand, class I drugs which increase RCR by decreasing stage 4 strongly protect complex III and to a lesser extent complex I (see table 4). Table 4 Protective effect of class 3 drugs on mitochondria complexes and III

0.5 mM Amytal 0.75 μM Antimycin A

Mitochondria complex I was 58% inhibited by the presence of 0.5 mM Amytal. Under these conditions, the presence of the molecules at the indicated concentrations (incubation 60 minutes) shows a much less pronounced inhibition which was expressed as a percentage of protection compared to the mitochondria without drug, considered to have 0% of protection.

Furthermore, by directly measuring the activity of adenine translocase, it has been shown that class II drugs which increase stage 3 of respiration increase the activity of this transporter (see Table 5). Table 5 Effect of class II drugs on the activity of mitochondria adenine translocase

The effect of the drugs was achieved on the activity of the adenine translocase of the mitochondria measured by the transport of ADP (C ¹⁴ ) in the mitochondria for 45 seconds.

In addition, the protective action of cyclo 3 on the RCR of the mitochondria is linked to the increase in

'RC of mitochondria but probably comes from its protective effect on the decoupling of mitochondria. Indeed, in the presence of cyclo 3, better resistance of the mitochondria to decoupling by mCCP is observed at 0.5 and 1 μM, this protective effect of the order of 20%.

Another advantageous property of the compounds of the invention is that a synergistic effect can be obtained by the combination of different molecules acting on different targets, for example, a molecule acting on complexes I and III which results in a decrease in the stage 4 of mitochondrial respiration and one or more molecules acting on adenine translocase which results in an increase in stage 3 of mitochondrial respiration. The double protection thus obtained gives an advantageous and unexpected overall result, since it exceeds the maximum possible protection with each of the compounds. Two enzymatic targets of the active compounds have been identified. The action of the compounds on these two targets has the consequence of increasing the production of ATP by the mitochondria and of preventing this production from decreasing under ischemic conditions. Thus, they protect cells from the consequences of an energy deficit which for ECs can lead to their activation, recruitment, adhesion and activation of leukocytes and proliferation of CML (Figure 2). These molecules all have the property of maintaining the rate of production of ATP in the mitochondria high even in non-physiological situations such as periods of ischemia or reduction of these mitochondrial activities due to age or to pathologies associated with aging. , allowing their application in these different pathologies.

Claims

1. Use of a pharmaceutical composition comprising a suitable pharmaceutical vehicle and an active compound chosen from the group consisting of citroflavonoids, garlic-rutoside, troxerutin, coumarin, diosmin, o - (- hydroxyethyl) rutosides, extracts of Melilot and rutoside, tribenoside, hesperidin methylchalcone, horse chestnut extract, naftazone, esculoside, aescin, procyanidolic oligomers, extracts of ruscus and hesperidin methylchalcone, ruscosides, extracts of small holly and blackcurrant, extracts of blueberry anthocyanodisics, the active principles of these compounds and / or a mixture of them, acting on the protein complexes of the internal mitochondrial membrane of a patient, for the preparation of a medicament intended for the treatment and / or prevention of ischemia and / or pathologies associated with ischemia or with an energy deficit.

2. Use according to claim 1, characterized in that the pathologies associated with ischemia are chosen from the group consisting of myocardial infarction, cerebral ischemia, chronic venous insufficiency, atheropathies, that is i.e. lesions due to atherosclerosis affecting the arteries of patients, Raynaud's phenomenon linked to vasospasms leading to vasoconstriction of the arteries, retinal deficits of ischemic origin, reduced hearing acuity of ischemic origin, disorders due to a stay at high altitude, ulcers, impaired capillary permeability, capillary fragility, scarring, skin alterations, angina chest caused by short moments of coronary artery obstruction, pulmonary hypertension, hepatic ischemia, Parkinson's disease, diabetes and / or heart transplants.

3. A method of ex vivo treatment of a patient, preferably a human patient, in which an organ, tissue and / or physiological fluid originating from said patient or from another human or animal patient is treated by the addition, to the organ and / or to the tissue prior to its implantation on the patient, of a pharmaceutical composition comprising an adequate pharmaceutical vehicle and an active compound chosen from the group consisting of citroflavonoids, garlic-rutoside, troxerutin, coumarin, diosmin, o - (- hydroxyethyl) rutosides, Melilot extract and rutoside, tribenoside, hesperidin methylchalcone, horse chestnut extract, naftazone, 1 'esculoside, aescin, procyanidolic oligomers, extracts of ruscus and hesperidin methylchalcone, ruscosides, extracts of holly and blackcurrant, extracts of blueberry anthocyanodisics, the active principles of these compounds and / or a mixture of them acting on the protein complexes of the internal mitochondrial membrane of a patient.

4. Method of therapeutic and / or prophylactic treatment of ischemia and / or pathologies associated with ischemia or an energy deficit of a patient, preferably a human patient, characterized in that said patient is administered a pharmaceutical composition comprising a suitable pharmaceutical vehicle and an active compound chosen from the group consisting of citroflavonoids, garlic-rutoside, troxerutin, coumarin, diosmin, o - (- hydroxyethyl) rutosides, extracts of Melilot and rutoside, tribenoside, hesperidin methylchalcone, horse chestnut extract, naftazone, 1 esculoside, l aescin, procyanidolic oligomers, extracts of ruscus and hesperidin methylchalcone, ruscosides, extracts of holly and blackcurrant, extracts of blueberry anthocyanin, the active ingredients of these compounds and / or a mixture of them, acting on the protein complexes of the internal mitochondrial membrane of a patient, for the preparation of a medicament intended for the treatment and / or prevention of ischemia and / or pathologies associated with ischemia or with a energy deficit.

5. Method according to claim 4, characterized in that the pathologies associated with 1 ischemia are chosen from the group consisting of 1 myocardial infarction, cerebral ischemia, chronic venous insufficiency, atheropathies, that is i.e. lesions due to atherosclerosis affecting the arteries of patients, Raynaud's phenomenon linked to vasospasms leading to vasoconstriction of the arteries, retinal deficits of ischemic origin, reduced hearing acuity of ischemic origin, disorders due to a stay at high altitude, ulcers, impaired capillary permeability, capillary fragility, scarring, skin alterations, angina caused by short moments of coronary obstruction, l hypertension, hepatic ischemia, Parkinson's disease, diabetes and / or heart transplants.

6. Use of a pharmaceutical composition comprising a pharmaceutical vehicle adequate and an active compound chosen from the group consisting of citroflavonoids, garlic-rutoside, troxerutin, coumarin, diosmin, o - (- hydroxyethyl) rutosides, extracts of Melilot and rutoside, tribenoside, 1 hesperidin methylchalcone, horse chestnut extract, naftazone, esculoside, aescin, procyanidolic oligomers, ruscus and hesperidin methylchalcone extracts, ruscosides, small holly and blackcurrant extracts, extracts of blueberry anthocyanodisics, the active principles of these compounds and / or a mixture of them, acting on the protein complexes of the internal mitochondrial membrane of a patient, for the preparation of a medicament intended for the treatment and / or the prevention of ischemia and / or pathologies associated with ischemia or an energy deficit, for the preparation of a medicament intended for the treatment and / or prevention of deficiencies linked to aging such as intellectual defects in the elderly, dizziness - or reduced adaptation perspective due to a change in the regulation of metabolisms.

7. A method of therapeutic and / or prophylactic treatment of deficiencies linked to the aging of a patient, in particular of a human patient, such as intellectual defects in the elderly, dizziness syndrome or reduced adaptive perspective due to a modification of metabolic regulation, characterized in that a pharmaceutical composition comprising a suitable pharmaceutical vehicle and an active compound chosen from the group consisting of citroflavonoids, garlic-rutoside, troxerutin, coumarin, is administered to said patient diosmin, the o - (- hydroxyethyl) rutosides, extracts of Melilot and rutoside, tribenoside, hesperidin methylchalcone, horse chestnut extract, naftazone, esculoside, aescin, procyanidolic oligomers, extracts of ruscus and d hesperidin methylchalcone, ruscosides, extracts of holly and black currant, extracts of blueberry anthocyanin, the active ingredients of these compounds and / or a mixture of them, acting on the protein complexes of the inner mitochondrial membrane d a patient, for the preparation of a medicament intended for the treatment and / or prevention of ischemia and / or of the pathologies associated with ischemia or with an energy deficit.