Use Of Xenon For The Prevention Of Programmed Cell Death

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

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Induction of apoptosis by staurosporine in cortical neurons

• • Whereas under normoxic conditions apoptosis occurs, as measured by the release of LDH, xenon prevents completely cell death.

FIG. 2: Induction of apoptosis by staurosporine in HeLa cells

• • Whereas under normoxic conditions apoptosis occurs, as measured by the release of LDH, xenon prevents completely cell death.

FIG. 3: Effect of pretreating HeLa cells with xenon in order to prevent apoptosis, subsequently induced by staurosporine under normoxic conditions

• • ct: 4 hrs control medium, 1 hr salt solution;
  • ct/stauro: 4 hrs control-medium+1 μM staurosporine, 1 hr salt solution+1 μM staurosporine;
  • xenon 1: 1 hr xe-medium in xenon, 3 hrs control medium+1 μM staurosporine, 1 hr salt solution+1 μM staurosporine;
  • xenon 2: 2 hrs xe-medium in xenon, 2 hrs control medium+1 μM staurosporine, 1 hr salt solution+1 μM staurosporine;
  • xenon 3: 3 hrs xe-medium in xenon, 1 hr control medium+1 μM staurosporine, 1 hr salt solution+1 μM staurosporine;
  • xenon 4: 2 hrs xe-medium in xenon, 2 hrs xe-medium+1 μM Staurosporine, 1 hr salt solution+1 μM staurosporine.

FIG. 4: Activation of caspase 3/7 in HeLa cells after treatment with staurosporin

• • Control: 5 hrs control medium, 1 hr salt solution
  • Staurosporine: 5 hours 1 μM Staurosporine.
  • Xenon: 5 hours Xenon-saturated medium, in Xe
  • Xenon+Staurosporine: 5 hours Xenon-saturated medium+1 μM staurosporine
  • Nitrogen: 5 hours N₂-saturated medium in N₂
  • Nitrogen+Staurosporine: 5 hours N₂-saturated medium in N₂+1 μM staurosporine

The following Examples illustrate the invention.

EXAMPLE 1

Methods

(A) Cells

Rat cortical neurons were obtained from 15-old embryos and maintained for 6 days in vitro as described (Petzelt et al., 2003, Life Sci. 72 (2003), 1909-1918). Human HeLa cells were maintained routinely as monolayer cultures in MEM medium, supplemented with 10% fetal calf serum, 2 mM glutamine, 1% non-essential amino acids. Cultures were subcultivated every two to three days. 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 a model apoptosis inducer when present in micromolar concentration (Tamaoki et al., BBRC 135 (1986), 397; Nakano et al., J. Antibiot. 40 (1987), 706; Ruegg and Burgess, TIPS 10 (1989), 218; Bertrand et al., Exp. Cell Res. 211 (1994), 314; Wiesner and Dawson, CLAO J. 24 (1996), 1418; Boix et al., Neuropharmacology 36 (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). Cells were seeded 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 staurosporine, followed by a further 1-hour incubation in a physiologic salt solution (Petzelt et al., 2003). Cellular damage after the experiment was assessed by measuring spectrophotometrically 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, cells were maintained for the time period indicated in xenon-saturated solution (medium or salt solution) within a gas-tight incubator filled with xenon (Petzelt et al., 2003).

EXAMPLE 2

Xenon Completely Prevents Staurosporine Induced Apoptosis in Cortical Neurons

Cortical neurons were incubated for four hours in medium containing 1 μM staurosporine, followed by an additional 1-hour incubation in salt solution, also containing staurosporine. Control preparations were treated in exactly the same way, except that no staurosporine was present. Xenon incubation was performed as described above. As seen in FIG. 1, the control cells survive well under the experimental conditions, no appreciable amount of LDH is released. However, if staurosporine is present, considerable cellular damage is observed as measured by the release of LDH. If cells are maintained in xenon-saturated medium, respectively salt solution, within a xenon-saturated atmosphere, they also survive well the treatment, no difference to cells maintained in a normoxic atmosphere is found. Surprisingly, if the same incubation is performed in the xenon-containing environment but in the presence of 1 μM staurosporine, no cellular damage is found, in contrast to the cells maintained under normoxic conditions. The entrance into apoptosis is prevented.

EXAMPLE 3

Xenon Completely Prevents Staurosporine Induced Apoptosis in HeLa Cells

In order to test if the apoptosis-reducing effect of xenon described in Example 2 is restricted to neurons or if it may be considered as a more general phenomenon, human HeLa cells were tested under identical conditions as described in Example 2. HeLa cells are cells derived from a human uterus carcinoma, therefore a sufficient basis for discrimination was given (different species, completely unrelated tissue).

As seen in FIG. 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 discriminating analysis the activation of the terminal effector caspases 3/7 were analysed after treatment with staurosporine. Caspases are universal proteases, their intracelllular cascade of activation form the central component of apoptosis (Slee, E. A. et al. (1999). Cell Death and Fiffer. 6 : 1067-1074). Basically, the signalling “initiator” caspases and the “effector” caspases can be differentiated. Furthermore, individual caspases can be identified by their specificity for a given substrate consisting of a four to five amino acid sequence (Kumar, S. (1999). Cell Death and Differ. 6: 1060-1066; Thornberry, N. A. et al. (1997). J. Biol. Chem. 272: 17907-17911)

In the following experiment the activation of caspase 3/7 is investigated using a highly sensitive and specific fluorogenic inhibitor of a given activated caspase (Ekert, P. G et al (1999), Cell Death and Differ. 6: 1081-1086). The resulting fluorescence signal is a direct measure of the amount of active caspase and can be analyzed by conventional fluorometry.

HeLa cells were treated for 5 hours with 1 μM staurosporine and the resulting caspase 3/7-activation was determined using the in situ caspase detection kit of CHEMICON (cat.no. APT403). The activity is expressed in RFU (relative fluorescence units).

FIG. 4 shows that staurosporine induces a steep increase in activated caspase 3/7 that is almost completely suppressed in the presence of xenon. If untreated cells are incubated for five hours in nitrogen, apoptosis—as expressed by the activation of caspase 3/7—is induced and that activation is increased even further by staurosporine. No effect of the 5-hour-incubation itself is found (see control and xenon).

EXAMPLE 4

Pretreatment with Xenon Reduces Apoptosis caused by a Subsequent Exposure to Staurosporine

A further important extension of the findings of Examples 2 and 3 was made when it was investigated if a pretreatment with xenon may reduce cellular damage caused by a subsequent exposure to staurosporine under normoxic conditions. Such a situation would render xenon a truly apoptosis-preventive agent since it could be applied before the apoptotic damage was expected to occur.

As shown in FIG. 3, already a 1-hour exposure to xenon-containing medium within a xenon-atmosphere suffices to protect the cells from a subsequent exposure to staurosporine under normoxic conditions. The longer the pretreatment of the cells with xenon lasts, the better the apoptotosis-preventive effect becomes manifest. If xenon is not present (=ct/stauro), considerable cell damage is found.

Claims

1-11. (canceled)

12. A method for treating a human having an: (a) aberrant or undesired apoptosis or(b) a disease associated with aberrant apoptosiscomprising administering to said human an effective amount of a pharmaceutical preparation comprising xenon or a xenon gas mixture.

13. The method according to claim 12 comprising, preventing or reducing cellular damage of tissue or organs to be transplanted in a human.

14. The method according to claim 12 comprising preventing or reducing apoptotic cell death after eye laser surgery.

15. The method according to claim 12 comprising protecting endothelial cells of the intestine in sepsis.

16. The method according to any one of claims 12 to 15 wherein the pharmaceutical preparation contains 5 to 90% by volume of xenon.

17. The method according to claims 16 wherein the pharmaceutical preparation contains 5 to 30% by volume of xenon.

18. The method according to any one of claims 12 to 15 wherein the pharmaceutical preparation additionally contains oxygen, nitrogen and/or air.

19. The method according to any of claims 12 to 15 wherein the pharmaceutical preparation additionally contains helium, NO, CO, CO2, other gaseous compounds and/or inhalable medicaments.

20. The method according to claim 16 wherein the pharmaceutical preparation has a ratio of xenon to oxygen of 80 to 20% by volume.

21. A method of preparing a pharmaceutical preparation by mixing xenon with another gas harmless to humans.

22. The method of claim 21 wherein xenon is mixed with an oxygen-containing gas.

23. A pharmaceutical preparation comprising a xenon gas mixture, wherein the xenon to oxygen ratio is 80 to 20% by volume.

24. The pharmaceutical preparation of claim 23 which additionally contains helium, NO, CO, CO2, and other gaseous compounds and/or inhalable medicaments.