GAS MIXTURES CONTAINING LOW CONCENTRATIONS OF XENON AND ARGON PROVIDE NEUROPROTECTION WITHOUT INHIBITING THE CATALYTIC ACTIVITY OF THROMBOLYTIC AGENTS

Abstract

The present invention relates to low concentration synergistic Xenon-Argon gas mixtures argon for use in a method for preventing and/or treating ischemic insults, wherein —the volume proportion of xenon is between 5 and 20%; —the volume proportion of argon is between 5 and 20%; and —the pharmaceutical composition further comprises a gas complement to reach 100% volume proportion; —the method includes comprises restoration of blood flow and the gas mixture is administered to the patient before, during, or after blood flow restoration.

Claims

1-13. (canceled)

14. A method for treating ischemic insults, which includes: restoring blood flow and administering a pharmaceutical composition to the patient after restoring blood flow, said pharmaceutical composition comprising a gas mixture of xenon and argon wherein the volume proportion of xenon is between 10 and 15%, the volume proportion of argon is between 10 and 15%, and a gas complement to reach 100% volume proportion, wherein the ischemic insult is cerebral ischemia, wherein restoring blood flow is achieved by medical thrombolysis induction and/or by thrombectomy, wherein thrombolysis is induced by administering a pharmaceutical agent with thrombolytic properties.

15. The method of claim 14, wherein the pharmaceutical agent with thrombolytic properties is a thrombolytic agent such as streptokinase, urokinase, human recombinant tissue-type plasminogen activator or rtPA, reteplase or tenecteplase.

16. The method of claim 14, wherein the xenon/argon volume ratio is between 4/1 and 1/4, preferably 1/1.

17. The method of claim 14, wherein the volume proportion of xenon is 15%, and the volume proportion of argon is 15%.

18. The method of claim 14, wherein the gas complement comprises oxygen, nitrogen, helium, neon, or a mixture thereof.

19. The method of claim 14, wherein the gas complement comprises oxygen in a volume proportion of between 20% and 50%, and wherein the remainder of the gas in the gas complement is nitrogen, helium, hydrogen, neon, or a mixture thereof, preferably helium.

20. The method of claim 14, wherein the pharmaceutical composition is preconditioned as a compressed gas mixture.

21. The method of claim 14, wherein the pharmaceutical composition administered to the patient is obtained by mixing argon, xenon and the gas complement in a gas mixer.

Description

DESCRIPTION OF THE FIGURES

[0058] FIG. 1. Effects of gas mixtures containing equimolar concentrations of xenon and argon of 15 vol % to 37.5 vol % on the release of lactate dehydrogenase (LDH) induced by oxygen-glucose deprivation (OGD) expressed as a percentage of pre-OGD values. Xe—Ar-15: gas mixture with equimolar concentration of xenon and argon of 15 vol %; Xe—Ar-25: gas mixture with equimolar concentration of xenon and argon of 25 vol %; Xe—Ar-37.5: gas mixture with equimolar concentration of xenon and argon of 37.5 vol %. Xe-Ar-15 approximately reduced OGD-induced LDH release in a manner similar to that of xenon at 50 vol %. Significant differences are marked by stars and sharp. *P<0.0001 vs OGD slices; .sup.#P<0.0001 vs xenon or argon alone at 15 vol % or 25 vol %.

[0059] FIG. 2. Effects of gas mixtures containing equimolar concentrations of xenon and argon of 15 vol % to 37.5 vol % on brain damage induced by intracerebral injection of N-methyl-D-aspartate (NMDA) expressed in mm.sup.3. Xe—Ar-15: gas mixture with equimolar concentration of xenon and argon of 15 vol %; Xe—Ar-25: gas mixture with equimolar concentration of xenon and argon of 25 vol %; Xe-Ar-37.5: gas mixture with equimolar concentration of xenon and argon of 37.5 vol %. Xe—Ar-15 approximately reduced NMDA-induced brain damage in a manner similar to that of xenon at 50 vol %. Significant differences are marked by stars, sharps, or plus symbols. *P<0.0001 vs OGD slices; .sup.#P<0.002 vs xenon at 15 vol %; .sup.++P<0.02 vs argon at 15 vol %; P<0.05 vs argon at 25 vol %.

[0060] FIG. 3. Effects of gas mixtures containing equimolar concentrations of xenon and argon of 15 vol % to 37.5 vol % on the catalytic efficiency of tissue plasminogen activator as expressed in percentage of air controls values. Xe—Ar-15: gas mixture with equimolar concentration of xenon and argon of 15 vol %; Xe—Ar-25: gas mixture with equimolar concentration of xenon and argon of 25 vol %; Xe—Ar-37.5: gas mixture with equimolar concentration of xenon and argon of 37.5 vol %. Xe—Ar-15, unlike Xe—Ar-25 and Xe—Ar-37.5, does not reduce the catalytic efficiency of rtPA. Significant differences are marked by star, sharp or plus symbol. *P<0.0001 vs OGD slices; .sup.++P<0.0001 vs xenon at 25 vol %; .sup.+P<0.002 vs xenon at 25 vol %. .sup.#P<0.0001 vs xenon at 37.5 vol % and argon at 37.5 vol %.

EXAMPLES

[0061] The present invention is illustrated by the following examples, which are not to be construed as limiting the invention in any way.

MATERIALS AND METHODS

Animals

[0062] All animal-use procedures were performed in accordance with the Declaration of Helsinki and were within the framework of the French legislation for the use of animals in biomedical experimentation. Adult male Sprague-Dawley rats (Janvier, Le Genest Saint-Isle, France) weighing 250-280 g were used. Before being used, rats were housed at 21±0.5° C. in Perspex home cages with free access to food and water. Light was maintained on a light/dark reverse cycle, with lights on from 8:00 PM to 8:00 AM.

Preparation and Incubation of Brain Slices

[0063] Rats were killed by decapitation under halothane anesthesia. The brains were removed and placed in ice-cold freshly prepared artificial cerebrospinal fluid (aCSF). Coronal brain slices (400 μm thickness) including the striatum (anteriority: from +1.2 to +2 mm from bregma) were cut using a tissue chopper (Mickie Laboratory Engineering Co., Gomshall, Surrey, UK).

Measurement of Cell Injury with Lactate Dehydrogenase Activity Assay

[0064] The effects of gas mixtures containing xenon and argon on the release of lactate dehydrogenase (LDH) induced by OGD were assessed as described previously [6]. Before being used, brain slices were transferred into individual vials with 1.3 ml of freshly prepared oxygenated aCSF containing 120 mM NaCl, 2 mM KCl, 2 mM CaCl.sub.2, 26 mM NaHCO.sub.3, 1.19 mM MgSO.sub.4, 1.18 mM KH.sub.2PO.sub.4, 11 mM d-glucose, and 30 mM HEPES and allowed to recover at room temperature for 45 min. Then, brain slices were placed at 36±0.5° C. into individual vials containing 1.3 ml of freshly prepared aCSF, saturated, and continuously bubbled with 100% oxygen (25 ml/min per vial). After a 30-min period, the incubation aCSF solution was renewed with oxygenated aCSF maintained at 36° C., and the slices were then incubated for 1 h to allow recording of basal levels of LDH. Whereas control slices were incubated for an additional 20-min period in the same conditions, those corresponding to the ischemic group were incubated in a glucose-free solution, saturated, and continuously bubbled with 100% nitrogen (OGD slices). After this 20-min period of OGD, to mimic reperfusion and treatment, the medium was replaced in all groups with freshly prepared aCSF solution, saturated and continuously bubbled with either medical air or gas mixtures containing xenon and argon in volume proportions as described below (see gas pharmacology section; n=20-36).

NMDA-Induced Neuronal Death In Vivo

[0065] The effects of of gas mixtures containing xenon and argon on the catalytic activity of tPA were assessed as described previously [6]. On the day of surgery, rats were anesthetized with 1.5% halothane in oxygen alone in an anesthesia box and mounted on a stereotactic apparatus with the incisor bar set at 3.9 mm below the horizontal zero. A burr hole was drilled and a micropipette (less than 10 pm at the tip) was lowered into the right striatum (anterior, 0.6 mm; lateral, 3.0 mm; ventral, 5.8 mm, from bregma) to allow injection of 70 nmol of NMDA in 1 μl of phosphate buffered solution (pH 7.4) over a 2-min period. After an additional 5-min period, the micropipette was removed, and the wounds were then sutured. During surgery, body temperature was kept at 37 ±0.5° C. with a feedback-controlled thermostatic heating pad (Harvard Apparatus Limited, Edenbridge, UK). The animals woke up in their home cage after about 10 min, where they were given free access to food and water. Sixty minutes after NMDA administration, rats were treated with medical air or gas mixtures containing xenon and argon (see gas pharmacological section). The number of rats per group was n=7-11.

In Vitro tPA Catalytic Activity Assay

[0066] The effects of gas mixtures containing xenon and argon on the catalytic activity of tPA were assessed as described previously [6]. The recombinant form of human tPA (Actilyse®; Boehringer Ingelheim, Ingelheim am Rhein, Germany) and its specific chromogenic substrate methylsulfonyl-D-phenyl-glycil-arginine-7-amino-4-methylcoumarin acetate (Spectrozyme® XF, product 444; American Diagnostica, Stamford, CT) were diluted separately in 1 ml distilled water in 1.5-ml sterile tubes. Each tube containing 0.4 μM tPA or 10 μM tPA substrate was saturated for 20 min at a flow rate of 60-80 ml/min with medical air (controls) or gas mixtures containing xenon and argon in volume proportions as described below (n=9-14 per concentration). The catalytic efficiency of tPA was assessed by the initial rate method by incubating 50 μl tPA with 50 μl substrate in a spectrofluorometer microplate reader set at 37° C.

Gas Pharmacology

[0067] Xenon, argon, nitrogen, and oxygen were purchased from Air Liquide Sante (Paris, France). Medical air composed of 75 vol % nitrogen and 25 vol % oxygen, and xenon and argon mixtures containing 37.5 vol % xenon and 37.5 vol % argon (Xe—Ar-37.5), 25 vol % xenon and 25 vol % argon (Xe—Ar-25) or 15 vol % xenon and 15 vol % argon (Xe—Ar-15)—with 25 vol % oxygen and the remainder being nitrogen when needed—were obtained using computer-driven gas mass flowmeters, and an oxygen analyzer to check that the gas mixtures used were not hypoxic.

Statistical Analysis

[0068] Data are given as the mean±the standard error to the mean. The effects of gas mixtures containing xenon and argon were analyzed with Statview software (SAS Institute, Cary, N.C.) and compared to those of control experiments, and xenon and argon alone, using non-parametric Mann-Whitney U-test.

RESULTS

[0069] First, the neuroprotective effects of gas mixtures containing xenon and argon at equimolar concentrations of 15% to 37.5% were investigated in brain slices exposed to OGD, a model of brain ischemia, and in rats that were administered an intracerebral injection of NMDA.

[0070] OGD induced an increase in LDH release compared to control slices (P<0.0001). As illustrated in FIG. 1, we found Xe—Ar-15>Xe—Ar-25>Xe—Ar-37.5 at reducing the release of LDH, a marker of cell injury, in brain slices exposed to OGD. Xe—Ar-15 decreased LDH (P<0.0001 vs Air-treated OGD slices) in a manner similar to that of xenon at 50%. This led to a significant difference between Xe—Ar-15 and xenon at 15 vol % (P<0.0001), which had no effect by itself on OGD-induced LDH release, and Xe—Ar-15 and argon at 15 vol % (P<0.0001), which also had no effect by itself on OGD-induced LDH release. Xe—Ar-25 decreased OGD-induced LDH release (P<0.0001) with similar amplitude than xenon alone at 25-37.5 vol %. This led to a significant difference between Xe—Ar-25 and argon at 25 vol % (P<0.0001), which had no effect by itself on OGD-induced LDH release, but not with xenon at 25 vol %, thereby indicating that the neuroprotective effect of Xe—Ar-25 mainly resulted from the presence of xenon in the gas mixture. In contrast with Xe—Ar-15 and Xe—Ar-25, we found that Xe—Ar-37.5 failed to decrease LDH release in OGD slices.

[0071] Alternatively, intracerebral injection of NMDA in rats treated with air induced brain damage compared to air-treated controls injected with saline (P<0.0001). As shown in FIG. 2, it was found Xe—Ar-15>Xe—Ar-25>Xe—Ar-37.5 at reducing brain damage in rats injected with NMDA. Xe—Ar-15 reduced NMDA-induced brain damage (P<0.001) in a manner similar to that of xenon alone at 50 vol %. This led to a significant difference between Xe—Ar-15 and xenon at 15 vol % (P<0.002), which had no effect by itself on NMDA-induced brain damage, and Xe—Ar-15 and argon at 15 vol % (P<0.02), which also had no effect by itself on NMDA-induced brain damage. Likewise, Xe—Ar-25 reduced NMDA-induced brain damage (P<0.02) with similar amplitude than xenon alone at 25-37.5 vol %. This led to a significant difference between Xe-Ar-25 and argon at 25 vol % (P<0.05), which had no effect by itself on NMDA-induced brain damage, but not with xenon at 25 vol %, thereby indicating that the neuroprotective effect of Xe—Ar-25 mainly resulted from the presence of xenon in the gas mixture. In contrast with Xe-Ar-15 and Xe—Ar-25, Xe—Ar-37.5 failed to show neuroprotection in rats injected with intracerebral NMDA.

[0072] Next, because previous data have demonstrated that xenon and argon both interact with tPA [10,11], the effects of gas mixtures containing xenon and argon at equimolar concentration of 15 vol % to 37.5 vol % on the catalytic activity of tPA were studied. As illustrated in FIG. 3, it was found Xe—Ar-37.5>Xe—Ar-25>Xe—Ar-15 at reducing the catalytic efficiency of tPA. Xe—Ar-37.5 decreased the catalytic efficiency of tPA (P<0.0001) to a similar extent than xenon alone at 75 vol %. This led to a significant difference between Xe—Ar-37.5 and xenon at 37.5 vol % (P<0.0001), and Xe—Ar-37.5 and argon at 37.5 vol % (P<0.0001). Likewise, Xe—Ar-25 also decreased the catalytic efficiency of tPA (P<0.0001) to a similar extent than xenon alone at 50 vol %. This led to a significant difference between Xe—Ar-25 and xenon at 25 vol % (P<0.0001) and between Xe—Ar-25 and argon at 25 vol % (P<0.002). While xenon alone at 25 vol % had no effect on the catalytic efficiency of tPA, argon at 25 vol % decreased it significantly, thereby indicating that the reducing effect of Xe—Ar-25 on the catalytic efficiency of tPA mainly resulted from the effect of argon at 25 vol %. In contrast, we found that Xe—Ar-15 had no effect on the catalytic efficiency of tPA.

DISCUSSION

[0073] In the present study, it was shown that gas mixtures containing low concentrations of xenon and argon of 15 vol % (Xe—Ar-15) or 25 vol % (Xe—Ar-25) of each gas can provide neuroprotection. Particularly, it was found that gas mixtures containing Xe—Ar-15 reduced OGD-induced cell injury and NMDA-induced brain damage to a similar extent than xenon alone at 50 vol %, thereby indicating that Xe—Ar-15 has potent neuroprotective effects. These potent neuroprotective effects of Xe—Ar-15, together with the fact that neither xenon alone at 15 vol % nor argon alone at 15 vol % (data not shown) had neuroprotective properties, is believed to be due to a synergism between inhibition by xenon of the NMDA receptor [8], which is the main excitatory receptor in the brain, and activation by argon of the GABA-A receptor [9], which is the main inhibitory receptor in the brain. Because Xe—Ar-15 is as potent as xenon alone at 50 vol %, and much more potent than argon alone at any concentration, at providing neuroprotection, it is likely that the mechanisms by which Xe—Ar-15 acts is mainly mediated through the NMDA receptor, which is the main neuronal target of xenon [10]. In contrast with the potent neuroprotective effects of Xe—Ar-15, it was found that Xe—Ar-37.5 failed to provide neuroprotection. The more plausible cause for this lack of effect of Xe—Ar-37.5 is a too potent general reduction in neuronal activity, which could be equivalent to narcosis or anesthesia in vivo. Support for this is the fact that neuroprotection decreased as a function of the concentration of Xe—Ar used (Xe—Ar-15>Xe—Ar-25>Xe—Ar-37.5). Further support for this are previous data that have shown that xenon provided neuroprotection at subanesthetic concentrations of 37.5 vol % or 50 vol % but not at high anesthetic concentrations of or above 75 vol % [5,6].

[0074] Alternatively, it was further shown that gas mixtures containing Xe—Ar-37.5 and Xe—Ar-25, but not Xe—Ar-15, reduced the catalytic efficiency of tPA to a similar extent than xenon at high concentrations of 75 vol % and 50 vol %, thereby suggesting the presence of another synergistic mechanism between xenon and argon at the tPA level [10,11]. Interestingly, structural biophysical studies have shown that both xenon and argon bind to the active site of elastase, a serine protease used as a model of tPA [12]. This is believed to explain the synergistic effect of Xe—Ar-25 and Xe—Ar-37.5 at inhibiting tPA-induced catalytic activity, which could be mainly mediated through xenon since argon alone has no effect of tPA-induced catalytic activity and further bind to elastase with a lower affinity than xenon [12].

[0075] Previous data in a rat model of thromboembolic stroke have shown that intraischemic xenon dose-dependently inhibits tPA-induced thrombolysis and subsequent reduction of ischemic brain damage [10], thereby leading to the conclusion that xenon should only be administered after, but not before or together, tPA-induced thrombolysis. In order to not favor reocclusion, a phenomenon shown to occur in 10% to 15% of ischemic stroke patients 2-3 h after tPA-induced reperfusion [13], it was further suggested that the use of xenon could require to be delayed according to a benefit—risk medical evaluation for the patient. Although, such a delay should not hamper dramatically the neuroprotective potential of xenon, suggested to have a possible therapeutic window of about 8 h [6], there is a general consensus that time is critical for treating ischemic stroke. Therefore, because as shown in the present study Xe—Ar-15 has no interaction with tPA and is as potent as xenon at 50 vol % at providing neuroprotection, Xe—Ar-15 is a promising alternative, cost-efficient, neuroprotective strategy to xenon alone for treating acute ischemic stroke.

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