MXPA06004505A - Use of xenon for the prevention of programmed cell death - Google Patents

Abstract

Described is the use of xenon for preventing or reducing cellular death, preferably aberrant apoptosis. Preferred embodiments relate to the use of xenon for preventing (a) cellular damage for tissue and organs to be transplanted, (b) apoptotic cell death after eye laser surgery, and (c) for protecting endothelial cells of the intestine in sepsis.

Description

USE OF XENON FOR THE PREVENTION OF PROGRAMMED CELLULAR DEATH FIELD OF THE INVENTION The present invention relates to the use of xenon to prevent or reduce cell death, preferably aberrant apoptosis. In particular, the present invention relates to the use of xenon to prevent (a) cell damage to tissues and organs to be transplanted, (b) apoptotic cell death after laser surgery of the eye, and (c) to protect endothelial cells of the intestine. in sepsis.

BACKGROUND OF THE INVENTION Cell death occurs by necrosis or apoptosis. In necrosis, death stimuli directly induce the death of the cell (for example, by ischemia), whereas in apoptosis, the death stimulus initiates a cascade of events that eventually lead, after a considerable time, to cell death . Necrosis is always a pathological process, while apoptosis is part of normal development and still essential for the normal physiological function of the organism. However, in addition to this, apoptosis originates in a variety of diseases and is then called aberrant apoptosis. However, until now, the specific treatment of diseases characterized by an aberrant, pathologically induced apoptosis which is based on the prevention / reduction of aberrant apoptosis is not possible.

SUMMARY OF THE INVENTION Therefore, it is the object of the present invention to provide a therapeutic means for the prevention / reduction of aberrant, pathologically induced apoptosis. According to the invention, this is achieved by the subject matter defined in claim 1. Additional advantageous embodiments and aspects of the present invention follow from the dependent claims, the description and the drawings. During the experiments leading to the present invention, it was unexpectedly found that xenon protects neurons from apoptotic cell death induced by substances that induce apoptosis, ie, under normoxic conditions. However, it was found by the present inventors, that such protective property of xenon is not limited to neurons. If, for example, human HeLa cells are incubated for several hours with a substance that induces apoptosis, most of the cells will be by this, committed to apoptotic death and will die after a few hours. If xenon is present during such incubation, cell death is almost completely prevented. Thus, this property of xenon can be used to protect cells from aberrant, pathologically induced apoptosis. Xenon is currently known as an anesthetic gas (EP-A-0 864 328; EP-A-0 864 329). Furthermore, it has been reported that xenon may provide some cellular protective effects against excesses of neurotransmitter (WO-A-00/53192). In addition, it has been reported that the administration of xenon during early reperfusion reduces infarct size after regional ischemia in the rabbit heart (Preckel et al., Anesthesia and analgesia, Dec. 2000, 91 (6), pages 1327 -1332).

BRIEF DESCRIPTION OF THE FIGURES Figure 1. Induction of apoptosis by staurosporine in cortical neurons Figure 2: Induction of apoptosis by staurosporine in HeLa cells Figure 3: Effect of pretreatment of HeLa cells with xenon to prevent apoptosis, subsequently induced by staurosporine under normoxic conditions Figure 4. Activation of caspase 3/7 in HeLa cells after treatment with staurosporine.

DETAILED DESCRIPTION OF THE INVENTION Thus, the present invention relates generally to the use of xenon or a mixture of xenon gas for the preparation of a pharmaceutical composition for treating (a) aberrant or unwanted apoptosis or (b) diseases associated with aberrant apoptosis. The finding that aberrant or unwanted apoptosis can be prevented or at least reduced by xenon, opens a new field of application for this noble gas which has been used until now mainly as an inhalation agent in the field of anesthesia. The prevention or reduction of apoptosis, for example, which characterizes the diseases discussed below, can be carried out in accordance with the present invention based on a simple inhalation therapy. The absorption of xenon via the respiratory system and transport in the brain are already proven by the use of xenon as an anesthetic agent. It can also be assumed that the use of xenon has no harmful effect on an organism, since many corresponding experiences could already be made by its use as an anesthetic agent. Xenon can be applied by several techniques which can be chosen depending on the particular use. For example, an inhalation device can be used in clinics, which is easily used for inhalation anesthesia. If a cardiopulmonary bypass machine or other artificial respiration device is used, xenon can be directly added to the machine and does not require additional devices. On a mobile basis, for example, in the case of an emergency, it is still possible to use simple inhalers, which mix the xenon with the ambient air during the inhalation process. In this context, it is also possible to adapt xenon concentrations and the time of application of xenon in a simple manner to the therapeutic requirements. For example, it may be advantageous to use mixtures of xenon with other gases harmless to humans, for example, oxygen, nitrogen, air, etc. Examples of preferred uses of xenon are described in the following sections. If "xenon" is mentioned, it also includes mixtures of xenon gas and is not intended to restrict the invention to pure xenon.

(A) Transplantation of organs and tissue (Preservation of intact organ / tissue from the beginning of removal, during transport, to re-implantation in the patient) During tissue transplantation, the apoptotic process that causes (a a varying degree) death in a given cell population. This can reach up to 95% of all cells, for example, the transfer of human embryonic nigral tissues in Parkinson's therapy, intracerebral transplants, etc. In the following examples, an injury rate of 10-30% of the tissue is until the present day unavoidable, and causes considerable increase in the proportion of transplant failures. (i) Ischemia / reperfusion injury (I / R) is a major problem in liver transplantation or hepatic resection with ischemic procedure, in addition to hypoxemic hepatocyte damage. A burst in the production of hydrogen peroxide (H202) that originates during the reperfusion phase can have a detrimental effect on the organ to be reperfused. Immediately after reperfusion, hepatocytes and Kupffer cells generate reactive oxygen species (EOR), which include H202. Subsequently, activated neutrophils, which infiltrate liver tissue, also produce EOR during the last reperfusion phase. Cells treated with H20 lead to apoptotic or necrotic death. (ii) Transplantation of embryonic nigral tissue decreases functional deficiencies in Parkinson's disease. The main practical restrictions of neural grafting are the scarcity of human donor tissue and the poor survival of dopamine neurons grafted onto patients, which is estimated to be 5-10%. The required amount of human tissue can be considerably reduced if neuronal survival is increased. Studies in rats indicate that most implanted embryonic neurons die within 1 week of transplantation and that most of this cell death is apoptotic. The reduction in cell death immediately after donor tissue preparation and increased long-term graft survival will thus dramatically improve the efficiency of neuronal transplantation. (iii) Apoptosis of T cells, monocytes / macrophages and endothelial cells after heart transplantation. (Transplant associated with coronary artery disease; Induction of apoptosis in lung transplant). It is therefore advantageous if the transport of the tissue to be transplanted and the transplant itself is carried out in the presence of xenon. Xenon can be administered either by being present in a surrounding atmosphere or in a buffer solution saturated with xenon. The considerable reduction of cell damage for tissues and organs to be transplanted and the improved transplantation, resulting itself, are the main developments based on this invention. Thus, a preferred use of xenon or a mixture of xenon gas is the prevention or reduction of cell damage of tissue or organs to be transplanted.

(B) Prevention of apoptotic cell death after eye laser surgery Photoreactive keratectomy (PRK) and laser-assisted keratectomy are widely used in ophthalmology to correct and adjust defects and deviations of the cornea. Although these techniques are widely used and accepted as safe, almost 30% of patients experience irregularities after several months, caused by programmed cell death of keratocytes. Unavoidable epithelial lesions associated with the use of laser-assisted eye surgery induce keratocyte apoptosis. Such loss of keratocyte causes malfunction of the cornea and requires immediate medical intervention. Currently, only the additional therapeutic surgery can alleviate or cure such a pathological condition of the cornea, the repopulation of the stroma of the anterior cornea after de-epithelialization, is a process too slow to be considered useful. Such induction of keratocyte apoptosis is also often observed in transplanted human corneasWhen the epithelium of the intact cornea is regenerated, the negative consequences eventually result in a considerable percentage of failure to successfully accept the transplant. As a preventive measure, xenon can be applied immediately after surgery in the form of a compressed air chamber filled with xenon or mixtures of xenon gas that is fixed to the eye for preferably a few hours (= treatment after surgery). Alternatively and using the same camera equipment, the eye can be incubated in a xenon atmosphere immediately before surgery for a period of 1-4 hours to reduce the subsequent proportion of apoptosis. The beneficial effect of such pre-treatment is shown in Fig. 3. Thus, an additional preferred use of xenon or xenon gas mixture is the prevention or reduction of apoptotic cell death after laser surgery.

(C) Protection of endothelial cells of the intestine in sepsis Sepsis represents a catastrophic collapse of the organism, often characterized by multiple organ failure, fulminating inflammation and general collapse of immune defense mechanisms. Mortality is extremely high and the therapeutic possibilities are severely limited. A typical feature is a rapid induction of apoptosis in the endothelial cells of the intestine. As a consequence, this important barrier collapses, resulting in the flooding of the organism with toxins and immunogens, in a situation where the defense mechanisms are easily weakened. If in such a situation, the induction of apoptosis can be prevented or is reduced in proportion, valuable time can be gained for additional therapies and mortality can be reduced. Even in a first stage of sepsis, the patient is in a critical situation where it is important to earn hours for additional treatment. Therefore, an immediate exposure of the intestine endothelium will be started to prevent the induction of programmed cell death, respectively, the further progress of programmed cell death. Exposure to xenon can be carried out either by induction of xenon directly in the intestine as gas (due to the high local concentration, only a small volume of gas will be required) or by using a salt solution saturated with xenon as described down. In the latter case, the application can be achieved either directly parenterally or indirectly via the stomach.

Thus, an additional preferred use of xenon or xenon gas mixture is the protection of endothelial cells of the intestine in sepsis. In the case of acute life-threatening conditions, for example, sepsis, respiration can be carried out salefully with a xenon mixture of 90: 10% by volume, preferably 80: 20% by volume, more preferably 75- 70: 25-30% by volume, for several hours to a day. Compared to this, intermittent breathing by a mixture of xenon-air to which less xenon has been added, for example 5 to 30% by volume of xenon, preferably 10 to 20% by volume of xenon, can be considered in chronic progression of a disease. Various methods for the inhalation of xenon and mixtures of xenon, respectively, can be used depending on the respective intended use. In clinics, it is possible to use an anesthetic device, in which pressure vessels containing pre-fabricated xenon-oxygen mixtures can be connected to the corresponding inputs of the apparatus. The breath is then carried out in accordance with a common procedure for such an apparatus. The same applies analogously to a cardiopulmonary bypass machine. As an alternative, xenon can be mixed with ambient air instead of oxygen in mobile use, which due to the smaller size of the pressure vessels required, increases the mobility of the apparatus. For example, it is possible to use an inhaler which provides xenon from a pressure vessel and is accommodated in a holder together with the latter in a mixing chamber. On one side, this mixing chamber contains a nozzle for inhaling the xenon and on the other side in which the xenon is supplied to the mixing chamber, it has at least one additional check valve which enables the entry of ambient air. The xenon pressure container can be equipped with a valve that reduces the pressure, for example, a valve which reduces the amount of xenon gas supplied to a suitable valve. When the patient breathes it, it sucks air from the air valves. In the mixing chamber, this air is mixed with the xenon supplied at the desired ratio and then inhaled by the patient. An advantageous inhaler suitable for mobile use and which serves to inhale xenon and its mixtures is, for example, described in EP-B1-0 560 928. For self-medication, a nozzle can be connected directly to the xenon pressure vessel. During inhalation, the patient opens the pressure valve and simultaneously inhales the xenon with the ambient air. When the patient breathes, he / she releases the valve, so that no more xenon reaches the mouthpiece. In this way, at least a sufficient regulation of the amount of inhaled xenon is possible. Alternatively, for example, to prevent cellular damage of tissues / organs to be transplanted, xenon can be administered as a saturated solution of xenon. A buffered physiological saline solution (Petzelt et al., Life Sci. 72, 1909-1918 (2003)), is exposed to 100% xenon, or alternatively, to 80% xenon / 20% oxygen, in a air-tight plastic bag and mixed for one hour in a shaker. The gas atmosphere is changed at least once and the mixing procedure is repeated. Afterwards, a complete saturation of the buffer is achieved with the gas (mixture). This solution is particularly useful for transplant purposes. If the tissue / organ is maintained during transport or during the pre-operation phase in such a solution, a considerable reduction in the proportion of apoptosis in the organ / tissue can be observed. To the xenon and xenon gas mixtures mentioned above, helium can also be added. Since helium is a small molecule, it can function as a carrier for the most voluminous xenon. In addition, additional gases that have medical effects can be added, for example NO, CO or C02. In addition, depending on the disease to be treated, other drugs can be added which are preferably inhalable, for example, cortisone, antibiotics, etc.

The following is a more extensive description of Figures 1 to 4. Figure 1. Induction of staurosporine apoptosis in cortical neurons While apoptosis originates under normoxic conditions, as measured by the release of LDH, xenon completely prevents death cell phone. Figure 2: Induction of apoptosis by staurosporine in HeLa cells While apoptosis originates under normoxic conditions, as measured by the release of LDH, xenon completely prevents cell death. Figure 3: Effect of pretreatment of HeLa cells with xenon to prevent apoptosis, subsequently induced by staurosporine under normoxic conditions ct: 4 hours control medium, 1 hour of saline; ct / stauro: 4 hours of control medium + 1 μM of staurosporine, 1 hour of saline solution + 1 μM of staurosporine; xenon 1: 1 hours xe-medium in xenon, 3 hours of control medium + 1 μM of staurosporine, 1 hour of saline solution + 1 μM of staurosporine; xenon 2: 2 hours xe-medium in xenon, 2 hours of control medium + 1 μM of staurosporine, 1 hour of saline solution + 1 μM of staurosporine; xenon 3: 3 hours xe-medium in xenon, 1 hour of control medium + 1 μM of staurosporine, 1 hour of saline solution + 1 μM of staurosporine; xenon 4: 2 hours xe-medium in xenon, 2 hours of xe-medium + 1 μM of staurosporine, 1 hour of saline solution + 1 μM of staurosporine. Figure 4. Activation of caspase 3/7 in HeLa cells after treatment with staurosporine Control: 5 hours of control medium, 1 hour of saline solution Staurosporine: 5 hours, 1 μM of staurosporine Xenon: 5 hours Xenon-saturated medium, in Xe Xenon + Staurosporine: 5 hours of xenon-saturated medium + 1 μM of staurosporine Nitrogen: 5 hours N2-medium saturated in N2 Nitrogen + Staurosporine: 5 hours N2-medium saturated in N2 + 1 μM of staurosporine The following examples illustrate the invention .

Example 1 Methods (A) Cells Rat cortical neurons were obtained from 15-day-old embryos and were maintained for 6 days in vitro as described (Petzelt et al., 2003, Life Esci. 12_ (2003), 1909-1918). Human HeLa cells were routinely maintained as monolayer cultures in MEM medium, supplemented with 10% fetal calf serum, 2 mM glutamine, 1% non-essential amino acids. The cultures were subcultured every two to three days. The absence of mycoplasma was verified every two weeks.

(B) Induction of apoptosis Apoptosis was induced using staurosporine. Staurosporine is an antibiotic originally discovered by Omura et al., J. Antibiot. 30 (1977), 275. It is generally considered an apoptosis inducing model, when present in micromolar concentration (Tamoaki et al., BBRC 135 (1986), 397; Nakano et al., J. Antibiot. 40 (1987), 706; Ruegg and Burges, TIPS 10 (1989), 218; Bertrand et al., Exp. Cell Res. 211 (1994), 314; Wiesner and Da son, CLAO J. 24 (1996), 1418; Boix et al. , Neuropharmacology 3_6 (1997), 811; Kirsch et al., J. Biol.

Chem. 274 (1999), 21155; Chae et al., Pharmacol. Res. 42 (2000), 373; Heerdt et al., Cancer Res. 60 (2000), 6704; Bijur et al., J. Biol. Chem. 275 (2000), 7583; Scarlett et al., FEBS Lett. 475 (2000), 267; Tainton et al., BBRC 276 (2000), 231; Tang et al., J. Biol. Chem. 275 (2000), 9303; Hill et al., J. Biol. Chem. 276 (2001), 25643). The cells were plated in 24-well plates at 6 days before the experiment (for cortical neurons), respectively two days (for HeLa cells) and incubated for several hours in the respective medium containing 1 μM of staurosporine, followed by incubation for an additional hour in a physiological saline solution (Petzelt et al., 2003). Cell damage was assessed after the experiment by spectrophotometrically measuring the concentration of LDH in the original supernatant, before the addition of perchloric acid (Roche Diagnostics, Mannheim, Germany). For the determination of the effect of xenon, the cells were maintained for the period of time indicated in xenon-saturated solution (medium or saline solution) within a gas-tight incubator filled with xenon (Petzelt et al., 2003).

Example 2 Xenon completely prevents staurosporin-induced apoptosis in cortical neurons Cortical neurons were incubated for four hours in medium containing 1 μM of staurosporine, followed by an additional 1 hour incubation in saline, also containing staurosporine. The control preparations were treated in exactly the same way, except that staurosporine is not present. The xenon incubation was performed as described above. As seen in Figure 1, the control cells survive well under the experimental conditions, the amount of LDH released is not appreciable. However, if staurosporine is present, considerable cell damage measured by the release of LDH is observed. If the cells are maintained in xenon-saturated medium, respectively the saline solution, inside a saturated atmosphere of xenon, they can survive well to the treatment, no differences were found in cells maintained in a normoxic atmosphere. Surprisingly, if the same incubation was performed in the medium containing xenon but in the presence of 1 μM of staurosporine, no cell damage was found, contrary to the cells maintained under normoxic conditions. The entry into apoptosis is prevented.

Example 3 Xenon completely prevents staurosporin-induced apoptosis in HeLa cells To test whether the effect reduces apoptosis 1 The xenon described in Example 2 is restricted to neurons or if it can be considered as a more general phenomenon, the HeLa cells were tested under identical conditions as described in Example 2. HeLa cells are cells that are derived from a carcinoma of Human uterus, therefore, is given a sufficient basis of discrimination (different species, completely unrelated tissue). As seen in Figure 2, basically the same results are obtained as with cortical neurons. Apoptotic cell death is induced by staurosporine under normoxic conditions, whereas it is completely suppressed in the presence of xenon. In a more discriminatory analysis, the activation of terminal effector caspases 3/7 was analyzed after treatment with staurosporine. Caspases are universal proteases, their intracellular activation cascade forms the central component of apoptosis (Slee, E.A. et al. (1999), Cell SEAT and FIFEC, 6: 1067-1074). Basically, the "initiating" caspases of signaling and the "effector" caspases can be differentiated. In addition, individual caspases can be identified by their specificity for a given substrate consisting of four to five amino acid sequences (Kumar, S. (1999), Cell Death and Differ, 6: 1060-1066, Thornberry, NA et al. 1997) J. Biol. Chem. 272: 17907-17911).

In the following experiment, the activation of caspases 3/7 is investigated using a specific and highly sensitive fluorogenic inhibitor of a given activated caspase (Ekert, P. G et al (1999), Cell Death and Differ., 6: 1081-1086). . The resulting fluorescent signal is a direct measure of the amount of activated caspase and can be analyzed by conventional fluorometry. The HeLa cells were treated for 5 hours with 1 μM of staurosporine and the activation of the resulting 3/7 caspase was determined using the in situ caspase detection kit of CHEMICON (cat No. APT403). The activity was expressed in RFU (relative fluorescence units). Fig. 4 shows that staurosporine induces an excessive increase in activated caspase 3/7 which is almost completely suppressed in the presence of xenon. Without the untreated cells are incubated for five hours under nitrogen, apoptosis - expressed by the activation of caspase 3 / 7-, is induced and such activation is further increased by staurosporine. No effect of the same incubation was found for 5 hours (see control and xenon).

Example 4 Xenon Pretreatment Reduces Apoptosis Caused by Subsequent Staurosporine Exposure A further important extension of the findings of Examples 2 and 3 were made when investigating whether a pretreatment with xenon that can reduce cell damage caused by subsequent exposure to staurosporine under normoxic conditions. Such a situation provides xenon as a truly preventative agent for apoptosis, since it can be applied before the apoptotic damage is expected to occur. As shown in Figure 3, already a 1 hour exposure to the medium containing xenon within a xenon atmosphere, is sufficient to protect the cells from a subsequent exposure to staurosporine under normoxic conditions. A prolonged pretreatment of the cells with durable xenon, better preventive effect of apoptosis comes to manifest itself. If the xenon is not present (= ct / stauro), considerable cell damage is found.

Claims (9)

NOVELTY OF THE INVENTION Having described the present is considered as a novelty, and therefore, the content of the following is claimed as property: CLAIMS

1. Use of xenon or xenon gas mixture for the preparation of an inhalable or liquid pharmaceutical composition to prevent or reduce unwanted or aberrant apoptosis, to reduce cell damage of tissues or organs to be transplanted.

2. Use according to claim 1, by means of which said pharmaceutical composition is administered from the beginning of the removal, during transport and / or until the re-implantation of said tissue or organ in a patient.

3. Use according to claim 1 or 2, by means of which, the pharmaceutical composition is administered before the apoptotic damage is expected to occur, to obtain a preventive effect of apoptosis.

4. Use according to claim 1 or 2, by means of which, the pharmaceutical composition is administered when the apoptotic damage originates.

5. Use according to any of claims 1 to 4, wherein the pharmaceutical preparation contains from 5 to 90% of the volume of xenon.

6. Use according to claim 5, wherein the pharmaceutical preparation contains from 5 to 30% by volume of xenon.

7. Use according to any of claims 1 to 6, wherein the pharmaceutical preparation additionally contains oxygen, nitrogen and / or air.

8. Use according to any of claims 1 to 6, wherein the pharmaceutical preparation additionally contains helium, NO, CO, C02, other gaseous compounds and / or inhalable medicaments.

9. Use according to claim 5, wherein the pharmaceutical preparation has a xenon to oxygen ratio of 80 to 20% by volume.