WO2010035074A1

WO2010035074A1 - Use of nitrous oxide, argon, xenon, helium, or neon, for the manufacture of a pharmaceutical composition for treating ischemic insults in patients who cannot be treated with thrombolytic agents

Info

Publication number: WO2010035074A1
Application number: PCT/IB2008/055624
Authority: WO
Inventors: Jacques Abraini
Original assignee: Nnoxe Pharmaceutiques Inc
Priority date: 2008-09-25
Filing date: 2008-09-25
Publication date: 2010-04-01

Abstract

The present invention relates to the use of at least one gas selected from the group consisting of nitrous oxide, argon, xenon, helium, neon, and mixtures thereof, for the manufacture of a pharmaceutical composition for treating ischemic insults in patients who cannot be treated with thrombolytic agents, such as the human recombinant form of tissue-type plasminogen activator (rt-PA).

Description

Use of nitrous oxide, argon, xenon, helium, or neon, for the manufacture of a pharmaceutical composition for treating ischemic insults in patients who cannot be treated with thrombolytic agents

The present invention relates to a method for treating ischemic insults in patients who cannot be treated with thrombolytic agents, especially in humans or animals. In particular, the present invention relates to the use of at least one gas selected from the group consisting of nitrous oxide, argon, xenon, helium, neon, and mixtures thereof, for the manufacture of a pharmaceutical composition for treating ischemic insults such as, but not limited to, cerebral ischemia, cardiac ischemia, renal ischemia, retinal ischemia, or lower limb's ischemia, in patients who cannot be treated or are not treated with a thrombolytic agent, such as the human recombinant form of tissue-type plasminogen activator (rt-PA).

Ischemia is a restriction in blood supply generally due to factors in the blood vessels, particularly thromboembolism (blood clots), which lead to tissue dysfunction and cell death through necrotic and apoptotic mechanisms. Ischemia is an absolute or relative shortage of the blood supply to an organ. Relative shortage means the mismatch of blood supply and blood request for adequate oxygen (and glucose) delivery in tissue. The extent of tissue damage mainly depends on the level and duration of ischemia. The heart, the kidneys, and the brain are among the organs that are the most sensitive to inadequate blood supply. For instance, ischemic stroke (also called brain attack or acute cerebral ischemia) and myocardial infarction (also called heart attack or acute cardiac ischemia) are with cancer the major causes of death in humans. It is estimated that global cardiovascular deaths will increase from 17 million deaths to more than 23 million deaths in 2030, and that cerebral stroke will represent more than 6 % of the diseases' global impact in 2020-2025 with nearly 25 % of males and 20% of females who will suffer a brain attack before reaching 85-year old.

Acute cerebral ischemia is caused by a reduction of blood flow in the brain because of the production of a fibrin (blood) clot. This leads more or less to brain dysfunctions and damage and neuronal death. The extent of brain injury mainly depends on the level and duration of ischemia. The physiological processes involved in ischemia-induced neuronal death are complex. Briefly, the reduction in cerebral blood flow compromises tissue energy stores and leads to a deficit in oxygen and glucose. At the cellular level, a critical consequence of this metabolic deprivation is an increase of the intracellular sodium concentration. This leads to an exaggerated efflux and uptake failure of many neurotransmitters, among them is glutamate (Dirnagl et al, Trends Neurosci. 22: 391, 1999). The excessive release of glutamate over-activates N-methyl- D-aspartate (NMDA) receptors. This results in a NMDA receptor-mediated neuronal depolarization and intraneuronal calcium influx that overstep the physiological bounds and lead to neuronal death through necrotic and apoptotic mechanisms (Choi et al., J. Neurosci., 8: 185, 1988; Sattler et al., J. Neurochem., 71 : 2349, 1998). Therefore, two strategies have been pursued for the treatment of ischemic stroke: a limitation of the vascular insult by early reperfusion through fibrinolysis-thrombolysis and/or a blockade of the neurotoxic cascade initiated by glutamate.

Reperfusion after ischemia remains the major therapeutic goal to be reached. Fibrinolysis is a particular case of proteolysis. Proteolysis can be defined as the directed (oriented) degradation of proteins by cellular enzymes called proteases through catalytic processes. Fibrinolysis is the physiological process wherein a fibrin (blood) clot is broken down. This occurs through endothelial cells that release a serine protease called tissue-type plasminogen activator (t-PA) that converts the proenzyme plasminogen to plasmin, the main enzyme of fibrin, which cuts the fibrin mesh. In healthy subjects, this process allows avoiding excessive clot formation and ischemic accidents. In patients suffering ischemia, fibrinolysis may allow individuals to break down endogenously a fibrin (blood) clot. Fibrinolysis can be also stimulated exogenously through administration of analogs of tissue-type plasminogen activator, such as the human recombinant form of tissue-type plasminogen activator (rt-PA). Exogenously-stimulated fibrinolysis is generally called thrombolysis. Today, the intravenous or intra-arterial injection of rt-PA is the only therapy approved by the Food and Drug Administration and the European Medical Agencies for treating ischemic stroke, i.e. acute cerebral ischemia. However, under certain conditions, thrombolytic therapy is associated with a risk of hemorrhagic transformation and neuronal death potentiation that is due to the general proteolytic properties of plasmin (Tsirka et al, Nature, 377: 340-344, 1995; Wang et al., Nature Med., 4: 228-231, 1998; Kaur et al., J. Cereb. Blood Flow Metab. 24: 945, 2004). In order to avoid these adverse side effects, rt-PA has to be administered to the patient within an appropriate period, called "therapeutic window", typically of up to 3 hours following the occurrence of the symptoms induced by ischemia according to the current medical practice and knowledge. Because of this, thrombolysis therapy is restricted to about only 5% of the patients suffering ischemic insults, those who can be treated within an appropriate therapeutic window. In contrast, blockade of the neurotoxic cascade initiated by glutamate by the use of NMDA glutamate receptor antagonists yet has not been proven being efficient in humans, because prototypical (high-affinity) NMDA receptor antagonists possess an intrinsic behavioral toxicity, which is believed to be related to the occurrence of vacuolizations in neurons of the posterior cingulated and retro-splenial cortices (Olney et al., Science, 244:1360, 1989; 254: 1515, 1991; Davis et al., Stroke, 31 :347, 2000). In order to resolve this problem, the development and the use of low-affinity (atypical) NMDA receptor antagonists is now considered as a major therapeutic strategy (Parsons et al., Drug News Perspect. 11: 523, 1998; Smith, Curr. Opin. Investig. Drugs, 4:826, 2003). Interestingly, the anesthetic gases xenon and nitrous oxide possess a pharmacological profile that is similar to that of the low-affinity NMDA receptor, with antagonistic properties at both the NMDA receptor and the nicotinic cholinergic receptor (Franks et al., Nature 396: 324, 1998; Jevtovic et al., Nature Med. 4: 460, 1998; Yamakura and Harris, Anesthesiology 93: 1095, 2000; David et al., Biol. Psychiatry, 60:49, 2006). Both xenon and nitrous oxide exhibit neuroprotective properties against ischemia with no proven adverse side effects when used at non- anesthetic concentrations (David et al., J. Cereb. Blood Flow Metab., 23:1168, 2003; FASEB J., 22:1275, 2008; Homi et al., Anesthesiology, 99:876, 2003; Ma et al., Ann. Neurol, 58:182, 2005; Martin et al., Br. J. Anaesth., 98:236, 2007; Rajakumaraswamy et al., Neurosci. Lett., 409:128, 2006; Haelewyn et al., Crit. Care Med., 36:2651, 2008). In addition, uniquely among the few molecules that show low-affinity antagonistic activity at the NMDA glutamatergic receptor, xenon and nitrous oxide readily cross the blood-brain barrier and have low blood/gas solubility that is advantageous in terms of rapid inflow and wash-out (Goto et al., Br. J. Anaesth, 880:255, 1998), conditions that favor treatment and reduce the risk of adverse side effects such as the occurrence of behavioral toxicity. Argon, helium, and neon have also been shown to be neuroprotective (Yarin et al., Hear Res., 201 :1, 2005; Pan et al., Exp Neurol, 205:587, 2007; Pagel et al., Anesth Analg., 105:562, 2007). Thus, some neuroprotective properties of nitrous oxide, xenon and argon have been patented. See for instance U.S. Patents n° 6,274,633 and 6,653,354, which relate to the use of xenon as an NMDA antagonist, in particular for providing neuroprotection, or European patent EP 1 158 992, which teaches the use of xenon or of a mixture of xenon and oxygen, nitrogen or air, to treat neurointoxications. See also French patent FR 2 863 169, which relates to the use of argon or of gas mixtures containing argon for treating neurointoxications.

The inventors discovered that the neuroprotective gases nitrous oxide, xenon, argon, helium, neon, and mixtures thereof, when administered at specific concentration ranges, inhibit the catalytic activity of t-PA and plasmin and further block or reduce thrombolysis and blood flow reperfusion. Interestingly, the inventors also discovered that those neuroprotective gases and mixtures thereof, when given at different concentration ranges than those that inhibit the t-PA-plasmin pathway, do not alter the catalytic activity of t-PA and plasmin, and thus do not block or reduce thrombolysis and blood flow reperfusion.

Consequently, based on these findings, the applicant patented a specific method for treating ischemic insults with xenon, nitrous oxide, argon, neon, helium, and mixtures thereof in patients who can be given thrombolytic agents, such as rt-PA, without blocking, reducing or inhibiting thrombolysis and thereby reperfusion (Patent Application PCT/EP2008/055392).

Nevertheless, nobody either disclosed or suggested a specific method for treating ischemic insults in patients who cannot be treated or are not treated with thrombolytic agents. Therefore, there was a need for a new specific method for treating ischemic insults in patients who cannot be treated on time with thrombolytic agents, such as rt-PA, within the appropriate therapeutic window mentioned above. The present invention thus relates to a method for providing neuroprotection for the treatment of ischemia in humans or animals, i.e. in human and veterinary medicine, without blocking, reducing or inhibiting fibrinolysis, and thereby without blocking, reducing or inhibiting possible spontaneous blood flow reperfusion, comprising the administration of the neuroprotective gases selected from nitrous oxide, xenon, argon, helium, neon, and mixtures thereof.

In particular, the present invention relates to the use of at least one gas selected from the group consisting of nitrous oxide, argon, xenon, helium, neon, and mixtures thereof, for the manufacture of a pharmaceutical composition for treating ischemic insults in patients who cannot be treated on time with thrombolytic agents, such as rt-PA, or in patients who are not treated with thrombolytic agents.

Advantageously according to the present invention, the pharmaceutical composition is for treating ischemic insults such as, but not limited to, cerebral ischemia, cardiac ischemia, renal ischemia, retinal ischemia, or lower limb's ischemia.

According to a first advantageous embodiment of the invention, said at least one gas is administered to patients at concentrations that do not block, reduce or inhibit fibrinolysis. Thus, said at least one gas is administered advantageously to patients who cannot be treated with thrombolytic agents, in order to provide neuroprotection without blocking, reducing or inhibiting fibrinolysis.

According to a particular embodiment of the invention, the pharmaceutical composition of the present invention comprises only one gas selected from nitrous oxide, xenon, argon, helium, and neon.

Particularly advantageously according to the present invention, the gas is xenon in a volume proportion between 1 % and 40 %, more advantageously between 1 % and 35 %, more advantageously between 10 % and 35 %, most advantageously between 15 % and 25 %. Or, particularly advantageously according to the present invention, the gas is nitrous oxide in a volume proportion between 1 % and 40 %, more advantageously between 1 % and 35 %, more advantageously between 10 % and 35 %, more advantageously between 15 % and 35 %, most advantageously between 20 % and 30 %.

Or, particularly advantageously according to the present invention, the gas is helium in a volume proportion between 1 % and 40 %, more advantageously between 1 % and 35 %, more advantageously between 10 % and 35 %, more advantageously between 15 and 35 %, most advantageously between 25 % and 30 %.

Or, particularly advantageously according to the present invention, the gas is neon in a volume proportion between 1 % and 40 %, more advantageously between 1 % and 35 %, more advantageously between 10 % and 35 %, more advantageously between 15 and 35 %, most advantageously between 25 % and 30 %.

Or, particularly advantageously according to the present invention, the gas is argon in a volume proportion between 46 % and 99 %, more advantageously between 50 % and 80 %, most advantageously between 50 % and 75 %.

According to another particular embodiment of the invention, the pharmaceutical composition of the present invention comprises a mixture of gases selected from nitrous oxide, xenon, argon, helium, and neon. Preferably, it comprises a mixture of two gases selected from nitrous oxide, xenon, argon, helium, and neon. Gases are in equimolar or non-equimolar volume proportions. Particularly advantageously according to the present invention, the gas mixture is a mixture of xenon and nitrous oxide, the volume proportion of xenon being between 1 % and 40 %, more advantageously between 5 % and 20 %, most advantageously between 5 % and 10 %, and the volume proportion of nitrous oxide being between 1 % and 40 %, more advantageously between 5 % and 20 %, most advantageously between 5 % and 10 %.

Or, particularly advantageously according to the present invention, the gas mixture is a mixture of xenon and helium, the volume proportion of xenon being between 1 % and 40 %, more advantageously between 5 % and 20 %, most advantageously between 5 % and 10 %, and the volume proportion of helium being between 1 % and 40 %, more advantageously between 5 % and 20 %, most advantageously between 5 % and 10 %. Or, particularly advantageously according to the present invention, the gas mixture is a mixture of xenon and argon, the volume proportion of xenon being between 1 % and 50 %, more advantageously between 5 % and 25 %, most advantageously between 10 % and 15 %, and the volume proportion of argon being between 1 % and 25 %, more advantageously between 5 % and 25 %, most advantageously between 10 % and 15 %.

Or, particularly advantageously according to the present invention, the gas mixture is a mixture of nitrous oxide and argon, the volume proportion of nitrous oxide being between 1 % and 40 %, more advantageously between 5 % and 20 %, most advantageously between 5 % and 10 %, and the volume proportion of argon being between 1 % and 40 %, more advantageously between 5 % and 20 %, most advantageously between 5 % and 10 %.

Or, particularly advantageously according to the present invention, the gas mixture is a mixture of nitrous oxide and helium, the volume proportion of nitrous oxide being between 1 % and 40 %, more advantageously between 5 % and 20 %, most advantageously between 5 % and 10 %, and the volume proportion of helium being between 1 % and 40 %, more advantageously between 5 % and 20 %, most advantageously between 5 % and 10 %.

Or, particularly advantageously according to the present invention, the gas mixture is a mixture of helium and argon, the volume proportion of helium being between 1 % and 50 %, more advantageously between 5 % and 30 %, most advantageously between 10 % and 20 %, and the volume proportion of argon being between 1 % and 50 %, more advantageously between 5 % and 30 %, most advantageously between 10 % and 20 %. Or, particularly advantageously according to the present invention, the gas mixture is a mixture of xenon and neon, the volume proportion of xenon being between 1 % and 40 %, more advantageously between 5 % and 20 %, most advantageously between 5 % and 10 %, and the volume proportion of neon being between 1 % and 40 %, more advantageously between 5 % and 20 %, most advantageously between 5 % and 10 %.

Or, particularly advantageously according to the present invention, the gas mixture is a mixture of nitrous oxide and neon, the volume proportion of nitrous oxide being between 1 % and 40 %, more advantageously between 5 % and 20 %, most advantageously between 5 % and 10 %, and the volume proportion of neon being between 1 % and 40 %, more advantageously between 5 % and 20 %, most advantageously between 5 % and 10 %. Or, particularly advantageously according to the present invention, the gas mixture is a mixture of neon and argon, the volume proportion of neon being between 1 % and 50 %, more advantageously between 5 % and 30 %, most advantageously between 10 % and 20 %, and the volume proportion of argon being between 1 % and 50 %, more advantageously between 5 % and 30 %, most advantageously between 10 % and 20 %.

Advantageously according to the present invention, for all the volume proportions of gases indicated above, the remainder of gases is oxygen alone or oxygen completed with nitrogen. Typically, the volume proportion of oxygen is comprised between 18 vol% and 30 vol%, more advantageously between 21 vol% and 25 vol%.

According to a second advantageous embodiment of the invention, once blood flow has been restored endogenously through fibrinolysis, at least one gas selected from the group consisting of nitrous oxide, xenon, argon, helium, neon, and mixtures thereof, is administered to the patient.

Once blood flow has been restored, said at least one gas can be administered at the same concentration as before (see above) or, if necessary, can be given at other concentrations advantageously in order to increase neuroprotection.

According to a particular embodiment of the invention, once blood flow has been restored, only one gas selected from nitrous oxide, xenon, helium, and neon is administered at the same concentration as before (see above) or, if necessary, is administered advantageously at higher concentrations to increase neuroprotection.

Particularly advantageously according to the present invention, the gas is xenon in a volume proportion between 30 % and 99 %, more advantageously between 36 % and 99 %, more advantageously between 40 % and 80 %, most advantageously between 35 % and 50 %. Or, particularly advantageously according to the present invention, the gas is nitrous oxide in a volume proportion between 30 % and 99 %, more advantageously between 36 % and 99 %, more advantageously between 40 % and 80 %, most advantageously between 35 % and 50 %. Or, particularly advantageously according to the present invention, the gas is helium in a volume proportion between 30 % and 99 %, more advantageously between 36 % and 99 %, more advantageously between 40 % and 80 %, more advantageously between 50 and 80 %, most advantageously between 50 % and 75 %.

Or, particularly advantageously according to the present invention, the gas is neon in a volume proportion between 30 % and 99 %, more advantageously between 36 % and 99 %, more advantageously between 40 % and 80 %, more advantageously between 50 and 80 %, most advantageously between 50 % and 75 %.

According to another particular embodiment of the invention, once blood flow has been restored, argon is administered at the same concentration as before (see above) or, if necessary, is given at lower concentrations in order to increase neuroprotection.

Particularly advantageously according to the present invention, the gas is argon in a volume proportion between 1 % and 45 %, more advantageously between 10 % and 40 %, more advantageously between 15 % and 40 %, most advantageously between 25 % and 35 %.

According to a third embodiment of the invention, once blood flow has been restored endogenous Iy through fibrinolysis, a mixture of gases selected from nitrous oxide, xenon, argon, helium, and neon is administered to the patient. Preferably, it comprises a mixture of two gases selected from nitrous oxide, xenon, argon, helium, and neon, wherein gases are at the same concentration as before (see above), or, if necessary, are given at higher concentrations in order to increase neuroprotection. Gases are in equimolar or non-equimolar volume proportions. Particularly advantageously according to the present invention, the gas mixture is a mixture of xenon and nitrous oxide, the volume proportion of xenon being between 1 % and 80 %, more advantageously between 10 % and 40 %, most advantageously between 15 % and 35 %, and the volume proportion of nitrous oxide being between 1 % and 80 %, more advantageously between 10 % and 40 %, most advantageously between 15 % and 35 %.

Or, particularly advantageously according to the present invention, the gas mixture is a mixture of xenon and helium, the volume proportion of xenon being between 1 % and 80 %, more advantageously between 10 % and 40 %, most advantageously between 15 % and 35 %, and the volume proportion of helium being between 1 % and 80 %, more advantageously between 10 % and 40 %, most advantageously between 15 % and 35 %. Or, particularly advantageously according to the present invention, the gas mixture is a mixture of xenon and argon, the volume proportion of xenon being between 1 % and 80 %, more advantageously between 10 % and 40 %, most advantageously between 20 % and 35 %, and the volume proportion of argon being between 1 % and 80 %, more advantageously between 10 % and 40 %, most advantageously between 20 % and 35 %.

Or, particularly advantageously according to the present invention, the gas mixture is a mixture of nitrous oxide and argon, the volume proportion of nitrous oxide being between 1 % and 80 %, more advantageously between 10 % and 40 %, most advantageously between 15 % and 35 %, and the volume proportion of argon being between 1 % and 80 %, more advantageously between 10 % and 40 %, most advantageously between 15 % and 35 %.

Or, particularly advantageously according to the present invention, the gas mixture is a mixture of nitrous oxide and helium, the volume proportion of nitrous oxide being between 1 % and 80 %, more advantageously between 10 % and 40 %, most advantageously between 15 % and 35 %, and the volume proportion of helium being between 1 % and 80 %, more advantageously between 10 % and 40 %, most advantageously between 15 % and 35 %.

Or, particularly advantageously according to the present invention, the gas mixture is a mixture of helium and argon, the volume proportion of helium being between 1 % and 80 %, more advantageously between 10 % and 40 %, most advantageously between 15 % and 35 %, and the volume proportion of argon being between 1 % and 80 %, more advantageously between 10 % and 40 %, most advantageously between 15 % and 35 %.

Or, particularly advantageously according to the present invention, the gas mixture is a mixture of xenon and neon, the volume proportion of xenon being between 1 % and 80 %, more advantageously between 10 % and 40 %, most advantageously between 15 % and 35 %, and the volume proportion of neon being between 1 % and 80 %, more advantageously between 10 % and 40 %, most advantageously between 15 % and 35 %.

Or, particularly advantageously according to the present invention, the gas mixture is a mixture of nitrous oxide and neon, the volume proportion of nitrous oxide being between 1 % and 80 %, more advantageously between 10 % and 40 %, most advantageously between 15 % and 35 %, and the volume proportion of neon being between 1 % and 80 %, more advantageously between 10 % and 40 %, most advantageously between 15 % and 35 %. Or, particularly advantageously according to the present invention, the gas mixture is a mixture of neon and argon, the volume proportion of neon being between 1 % and 80 %, more advantageously between 10 % and 40 %, most advantageously between 15 % and 35 %, and the volume proportion of argon being between 1 % and 80 %, more advantageously between 10 % and 40 %, most advantageously between 15 % and 35 %.

Also advantageously according to the present invention, once blood flow has been restored, when at least one gas or a mixture of gases selected from the neuroprotective gases nitrous oxide, xenon, argon, helium, and neon is used at concentrations that reduce fibrinolysis, it is administered to the patient with an appropriate delay in order not to favor re-occlusion since those gases were found to inhibit the catalytic activity of t-PA and plasmin and thereby fϊbrinolyis (ethical principle of caution). Advantageously, the delay is comprised between 5 and 180 min (3 h), more advantageously between 60 and 180 min, most advantageously between 120 and 180 min, after reperfusion has occurred (i.e. once blood flow has been restored).

According to a specific advantageous embodiment of the invention, at least one gas selected from the neuroprotective gases nitrous oxide, xenon, argon, helium, neon, and mixtures thereof, is first administered to the patient at concentrations that do not block or reduce fibrinolysis, advantageously within an appropriate therapeutic window following the occurrence of the symptoms of ischemia. Then, once blood flow has been restored, said at least one gas or a mixture of gases selected from the neuroprotective gases nitrous oxide, xenon, argon, helium, and neon can be administered at the same concentration as before or, if necessary as described above, can be given at other concentrations in order to increase neuroprotection, advantageously with an appropriate delay in order not to favor re-occlusion.

According to a fourth advantageous embodiment of the invention, said at least one gas selected from the neuroprotective gases nitrous oxide, xenon, argon, helium, neon, and mixtures thereof, is administered simultaneously, separately or sequentially with at least one other drug and/or any particular condition possessing or not neuroprotective properties by itself, which may enhance, increase, or potentiate the neuroprotective action of said at least one gas. Said at least one gas is thus advantageously administered before, together with, and/or after said at least one other drug and/or any particular condition. Such drugs can be for instance, but are not limited to, alpha(2)-adrenoceptor agonists, such as dexmedetomidine (Rajakumaraswamy et al, Neurosci Lett. 409:128, 2006), carbon monoxide, nitric oxide, garlic extracts or other natural compound extracts, and/or hydrogen which is a gas shown to possess therapeutic antioxidant properties (Osawa et al., Nature Med. 13:688, 2007). Typically, carbon monoxide or nitric oxide is administered at concentrations of a few ppm. Typically, hydrogen is administered in a volume proportion of 0.5 to 4.7 %. Such particular conditions are for instance hypothermia (Ma et al., Ann Neurol.,58:182, 2005; Hobbs et al., Stroke, 39:1307, 2008). According to another advantageous embodiment, said at least one gas selected from the neuroprotective gases nitrous oxide, xenon, argon, helium and neon, and mixtures thereof, is intended for inhalable administration, such as oral inhalation or nasal inhalation, or any other appropriate route of administration. If inhaled, the pharmaceutical composition according to the invention is administered to the patient via his upper respiratory pathways, i.e. by inhalation via the nose and/or the mouth, using any suitable administration device comprising a patient respiratory interface, such as a respiratory mask or a tracheal probe, one or more feed pipes serving to convey the gaseous pharmaceutical composition from a source containing the said pharmaceutical composition to the interface, and a regulator and/or a medical or an anesthesia ventilator serving to deliver and/or extract the patient's respiratory gas.

The following figures and examples describe and illustrate the present invention, but do not restrict the present invention.

FIGURE 1 illustrates the inhibiting effect of various concentrations of gases selected from nitrous oxide, xenon, helium, and argon on the catalytic activity of t-PA.

Figure 1 A to 1 C shows the inhibiting effect of various concentrations of nitrous oxide (Fig. 1 A), xenon (Fig. 1 B), and helium (Fig. 1C) on the catalytic activity of t-PA.

Figure 1 D shows the effects of xenon, argon, and helium at 75 vol% on the catalytic activity of plasmin.

Figure 1 E shows the effects of various gas mixtures containing xenon, nitrous oxide, helium, and/or argon at various concentrations on the catalytic activity of t-PA.

Figure 2 illustrates the inhibiting effect of xenon on fibrinolysis in vivo in rats subjected to middle cerebral artery occlusion using an autologous blood clot.

EXAMPLES:

All animal-use procedures were in accordance with the guidelines of the National Institute of Health (USA) and The European Communities Council Directive of 24 November 1986 (86/609/EEC) for the care and use of laboratory animals. The inventor was fully authorized (agreement n° 14-27).

EXAMPLE 1: EFFECT OF VARIOUS CONCENTRATIONS OF NITROUS OXIDE, XENON, ARGON, AND HELIUM ON THE CATALYTIC ACTIVITY OF T-PA AND PLASMIN EX VIVO (FIGURE 1).

All experiments were performed as follows: Fifty (50) μL of human recombinant t-PA was incubated with 50 μL of its substrate: methylsulfonyl-D-phenyl- glycil-arginine-7-amino-4-methylcoumarin acetate. For plasmin, twenty five (25) μL of human recombinant plasmin was incubated with 25 μL of its substrate: H-D- norleucyl-hexahydrotyrosol-lysine-para-nitroanilide diacetate. The kinetics of the catalytic activity of t-PA or plasmin was immediately measured using a spectrophotometer, and then estimated using the initial rate method. Solutions of t-PA or plasmin and their substrates were saturated with air (control), or with nitrous oxide, argon, xenon, or helium at concentrations of 15 vol% to 75 vol%, the remainder being oxygen at 25 vol%, completed with nitrogen when necessary.

Figure 1 A to 1 C shows the inhibiting effect of various concentrations of nitrous oxide (Fig. 1 A), xenon (Fig. 1 B), and helium (Fig. 1C) on the catalytic activity of t-PA. Figure 1 D shows the effects of xenon, argon, and helium at 75 vol% on the catalytic activity of plasmin. Figure 1 E shows the effects of various gas mixtures containing xenon, nitrous oxide, helium, and/or argon at various concentrations on the catalytic activity of t-PA. Similar results were obtained with neon (data not shown).

For all gas mixtures in Figure 1, the remainder is 25 vol% oxygen completed with nitrogen if necessary.

It can be concluded that the catalytic activity of t-PA and plasmin can be reduced by nitrous oxide, xenon, argon, neon, helium, and mixtures thereof when given at specific concentration ranges.

EXAMPLE 2: EFFECTS OF XENON ON FIBRINOLYSIS IN VIVO (FIGURE 2)

In vivo experiments were performed in Male adult Sprague-Dawley rats (290 ± 40 g). The animals had free access to food and water in an animal room at constant temperature and humidity. Rats monitored for their physiological functions were anesthesized, and subjected to cerebral ischemia by occlusion of the middle cerebral artery using an autologus blood clot obtained from whole blood withdrawn from the rat 24 h prior surgery, allowed to clot at 37°C for 1 h 30, and stored at 4°C for 22 h. Embolic occlusion of the middle cerebral artery was induced using an autologous blood clot. Briefly, a blood clot of 2.5 cm long was injected with a volume of 50μL of saline into the middle cerebral artery. In contrast with stronger autologous blood clot measuring 4 cm long, obtained from blood samples allowed to clot for 2 h at 37°C (see Patent Application PCT/EP2008/055392), this allows blood flow reperfusion to occur spontaneously through fibrinolysis, i.e. without performing thrombolysis through administration of rt-PA. Rats were treated either with medical air (controls) or xenon at 50 vol% or 75 vol%. As shown in Figure 2 A, rats treated with xenon at 50 vol% exhibit a lower reperfusion rate through fibrinolysis as compared to control rats treated with medical air. In addition, as shown in Figure 2B, reperfusion through fibrinolysis in rats breathing medical air is stopped and thereby delayed when rats are administered xenon at 75 vol% instead of medical air.

It can be concluded from these in vivo experiments that fibrinolysis can be reduced by xenon.

Claims

1. Use of at least one gas selected from the group consisting of nitrous oxide, argon, xenon, helium, neon, and mixtures thereof, for the manufacture of a pharmaceutical composition for treating ischemic insults in patients who cannot be treated with thrombolytic agents, such as the human recombinant form of tissue-type plasminogen activator (rt-PA).

2. Use as claimed in claim 1, characterized in that the pharmaceutical composition is for treating cerebral ischemia, cardiac ischemia, renal ischemia, retinal ischemia, or lower limb's ischemia.

3. Use as claimed in any of claims 1 and 2, characterized in that said at least one gas is at concentrations that do not block, reduce or inhibit fibrinolysis.

4. Use as claimed in claim 3, characterized in that said at least one gas is xenon, nitrous oxide, helium, or neon, in a volume proportion between 1 % and 35 %, advantageously between 10 and 35%.

5. Use as claimed in claim 3, characterized in that said at least one gas is argon in a volume proportion between 46% and 99 %, advantageously between 50 and 80%.

6. Use as claimed in claim 3, characterized in that said at least one gas is selected from the group consisting of a mixture of xenon and nitrous oxide, the volume proportion of xenon and of nitrous oxide being each between 1 % and 40 %; a mixture of xenon and helium, the volume proportion of xenon and of helium being each between 1 % and 40 %; a mixture of nitrous oxide and argon, the volume proportion of nitrous oxide and of argon being each between 1 % and 40 %; a mixture of nitrous oxide and helium, the volume proportion of nitrous oxide and of helium being each between 1 % and 40 %; a mixture of argon and helium, the volume proportion of argon and of helium being each between 1 % and 50 %; and a mixture of xenon and argon, the volume proportion of xenon being between 1 % and 50 % and the volume proportion of argon being between 1 % and 25 %.

7. Use as claimed in any of claims 3 to 6, characterized in that said at least one gas is first administered to the patient in order to provide neuroprotection without blocking, reducing or inhibiting fibrinolysis, and then, once blood flow has been restored, said at least one gas is administered at the same concentration as the ones described in any of claims 3 to 6, or, if necessary, is given at other concentrations in order to increase neuroprotection.

8. Use as claimed in claim 7, characterized in that, once blood flow has been restored, said at least one gas is xenon, nitrous oxide, helium, or neon at concentrations described in any of claims 3 and 4, or at higher concentrations.

9. Use as claimed in claim 8, characterized in that, once blood flow has been restored, said at least one gas is in a volume proportion between 36% and 99 %, advantageously between 40 % and 80%.

10. Use as claimed in claim 7, characterized in that, once blood flow has been restored, said at least one gas is argon at concentrations described in any of claims 3 and 5, or at lower concentrations.

11. Use as claimed in claim 10, characterized in that, once blood flow has been restored, argon is in a volume proportion between 1 % and 45 %, advantageously between 10 % and 40%.

12. Use as claimed in claim 7, characterized in that, once blood flow has been restored, said at least one gas is a mixture of two gases selected from the group consisting of a mixture of xenon and nitrous oxide, the volume proportion of xenon and of nitrous oxide being each between 1 % and 80 %; a mixture of xenon and helium, the volume proportion of xenon and of helium being each between 1 % and 80 %; a mixture of nitrous oxide and argon, the volume proportion of nitrous oxide and of argon being each between 1 % and 80 %; a mixture of nitrous oxide and helium, the volume proportion of nitrous oxide and of helium being each between 1 % and 80 %; a mixture of argon and helium, the volume proportion of argon and of helium being each between 1 % and 80 %; and a mixture of xenon and argon, the volume proportion of xenon and of argon being each between 1 % and 80 %.

13. Use as claimed in any of the preceding claims, characterized in that said at least one gas is administered before, together with, and/or after at least one other drug and/or any particular condition possessing or not a neuroprotective action by itself that may enhance, increase or potentiate the neuroprotective action of said at least one gas.

14. Use as claimed in any of the preceding claims, characterized in that the pharmaceutical composition is intended for inhalable administration.