Pharmaceutical composition for treatment of the pulmonary form of altitude sickness caused by lack of oxygen and reduced air pressure

Abstract

What is described is a peptide which consists of 7-20, especially 7-17, adjacent amino acids and comprises the hexamer TX.sub.1EX.sub.2X.sub.3E where X.sub.1, X.sub.2 and X.sub.3 may be any natural or unnatural amino acid, where the peptide does not have any TNF receptor binding activity and is cyclized, for use for the treatment and avoidance of the pulmonary form of altitude sickness.

Claims

1. A method for therapeutic treatment of the pulmonary form of altitude sickness, comprising administering to a patient having the pulmonary form of altitude sickness an effective amount of a peptide, wherein the peptide is 17-20 amino acids in length and comprises the amino acid sequence CGQREFPEGAEAKPWYC (SEQ ID NO: 1), wherein the peptide does not have tumor necrosis factor receptor binding activity, wherein the peptide is cyclized, and wherein the peptide is formulated in a nebulizble powder formulation.

2. The method according to claim 1, wherein the peptide is cyclized via the C residues.

3. The method according to claim 2, wherein the peptide is cyclized by a disulphide bridge between the C residues.

4. The method according to claim 1, wherein the nebulizable powder formulation further comprises a pharmaceutically acceptable carrier.

5. The method according to claim 4, wherein the peptide is administered to the patient in a quantity of 1 μg to 10 g.

6. The method according to claim 5, wherein the peptide is administered to the patient in a quantity of 10 μg to 1 g.

7. The method according to claim 5, wherein the peptide is administered to the patient in a quantity of 1 mg to 100 mg.

8. The method of claim 1, further comprising the step of obtaining an emergency pack for a mountaineer prior to said administering, the emergency pack comprising the effective amount of the peptide and a powder inhaler for said administering.

9. A method for therapeutic treatment of the pulmonary form of altitude sickness, comprising administering by inhalation an effective amount of a peptide to a patient having the pulmonary firm of altitude sickness, wherein the pulmonary form of altitude sickness is treated by said administering, wherein the peptide consists of the amino acid sequence CGQRETPEGAEAKPWYC (SEQ ID NO: 1) and is cyclized via the C residues, and wherein the peptide is formulated as a powder for inhalation.

10. The method of claim 9, further comprising the step of obtaining an emergency pack for a mountaineer prior to said administering, the emergency pack comprising the effective amount of the peptide and a powder inhaler for said administering.

Description

DESCRIPTION OF THE DRAWINGS

(1) Reference is now made to the accompanying figures and in which:

(2) FIG. 1: The intensity of the pulmonary form of altitude sickness in rats was determined 4 hours after intratracheal administration of saline solution or respectively peptide SEQ ID NO: 1. Control: Control rates under conditions of normal oxygen and air pressure values. PBS: Rats under conditions of reduced oxygen and air pressure and intratracheal administration of saline solution. Peptide SEQ ID NO: 1 100 μg: Rats under conditions of reduced oxygen and air pressure and intratracheal administration of 100 μg peptide SEQ ID NO: 1. Peptide SEQ ID NO: 1 300 μg: Rats under conditions of reduced oxygen and air pressure and intratracheal administration of 300 μg peptide Seq. ID NO: 1. Peptide SEQ ID NO: 1 600 μg: Rats under conditions of reduced oxygen and air pressure and intratracheal administration of 600 μg peptide SEQ ID NO: 1.

(3) FIG. 2: The protein content in the lung fluid in rats was determined 4 hours after intratracheal administration of saline solution or respectively peptide SEQ ID NO: 1. Control: Control rats under conditions of normal oxygen and air pressure values. PBS: Rats under conditions of reduced oxygen and air pressure and intratracheal administration of saline solution. Peptide SEQ ID NO: 1 100 μg: Rats under conditions of reduced oxygen and air pressure and intratracheal administration of 100 μg peptide SEQ ID NO: 1. Peptide SEQ ID NO: 1 300 μg: Rats under conditions of reduced oxygen and air pressure and intratracheal administration of 300 μg peptide SEQ ID NO: 1. Peptide SEQ ID NO: 1 600 μg: Rats under conditions of reduced oxygen and air pressure and intratracheal administration of 600 μg peptide SEQ ID NO: 1.

(4) FIG. 3: Histological appearance of lung tissue in rats 4 hours after intratracheal administration of saline solution or respectively peptide SEQ ID NO: 1. Control: Control rats under conditions of normal oxygen and air pressure values. PBS: Rats under conditions of reduced oxygen and air pressure and intratracheal administration of saline solution. Peptide SEQ ID NO: 1 100 μg: Rats under conditions of reduced oxygen and air pressure and intratracheal administration of 100 μg peptide Seq. ID NO: 1. Peptide SEQ ID NO: 1 300 μg: Rats under conditions of reduced oxygen and air pressure and intratracheal administration of 300 μg peptide SEQ ID NO: 1. Peptide SEQ ID NO: 1 600 μg: Rats under conditions of reduced oxygen and air pressure and intratracheal administration of 600 μg peptide SEQ ID NO: 1.

(5) FIG. 4: Mean values of the inwardly flowing Na.sup.+ flows in A549 cells, in a whole cell patch clamp test during the control phase at −100 mV clamped, after addition of PEPTIDE SEQ ID NO: 1 (“AP301”) (240 nM) and after addition of amiloride (100 mM) to the bath solution. The values are mean values+/−SE.

(6) FIG. 5: Action of the synthetic peptide QRETPEGAEAKPWY (SEQ ID NO: 3, described in the prior art as suitable for the treatment of oedemas, is, however, in contrast to the form according to the invention, not cyclized in this experiment) on the Na.sup.+ flow in an A549 cell patched in whole cell mode. Representative original recording of a cell clamped at a holding potential of −100 mV during the control phase and after addition of the peptide QRETPEGAEAKPWY (300 nM) in the bath solution.

(7) FIG. 6: Action of the synthetic peptide TKPIELGPDEPKAV (SEQ ID NO: 16; described in the prior art as suitable for the treatment of oedemas, is, however, in contrast to the peptides according to the invention, not cyclized and does not contain the core sequence TX.sub.1EX.sub.2X.sub.3E or respectively TPEGAE) on the Na.sup.+ flow in an A549 cell patched in the whole cell mode. Representative original recording of a cell clamped at a holding potential of −100 mV during the control phase and after addition of the peptide TKPIELGPDEPKAV (300 nM) in the bath solution.

(8) FIG. 7: Action of the synthetic cyclic peptide CGTKPIELGPDEPKAVC (SEQ ID NO: 17; described in the prior art as suitable for the treatment of oedemas, however, in contrast to the peptides according to the invention, does not contain the core sequence TX.sub.1EX.sub.2X.sub.3E or respectively TPEGAE) on the Na.sup.+ flow in an A549 cell patched in the whole cell mode. Representative original recording of a cell clamped at a holding potential of −100 mV during the control phase and after addition of the cyclic peptide CGTKPIELGPDEPKAVC (300 nM) in the bath solution.

(9) FIG. 8: Activity of the cyclic peptides SEQ ID NOS: 1 and 11 to 15 as a function of the concentration. On the x-axis the concentration is entered in a logarithmic scale in nM; on the y-axis the sodium ion flow is entered (in %).

EXAMPLE 1

(10) Use of the peptide with SEQ NO: 1 according to the invention for the treatment of the pulmonary form of altitude sickness.

(11) With the present example, in an experimental rat model of altitude sickness it is shown that the aim according to the invention was achieved by the synthetic peptide according to the invention (SEQ ID NO: 1) being administered to rats suffering from the pulmonary form of altitude sickness. Physical exertion under conditions of reduced oxygen and air pressure, such as occur at high altitudes, are the 2 main factors which lead to the development of the pulmonary form of altitude sickness. Therefore, the selected rat model, in which the rats carried out physical activity under conditions of reduced oxygen and reduced air pressure, simulates a physically strenuous ascent to high altitudes. This takes place without carrying out a prior acclimatization. This corresponds to the scenario such as is to be found in the case of mountaineers who suffer from the pulmonary form of altitude sickness at high altitudes. In the model which was used, the rats develop the typical symptoms for the pulmonary form of altitude sickness, as is to be read in the “intensity of the pulmonary form of altitude sickness”, the increased protein concentration in the lung fluid and the histological appearance of the lung tissue. It is further to be noted that the lung damage in this model is not caused by administration of endotoxins, microbes or other agents which are damaging to the lung. An intensified inflammation of the lung does not occur. Also, no specific strain of rat was used for this experiment. Therefore, this rat model is well suited to investigate a medicament for the treatment of the pulmonary form of altitude sickness.

(12) Method

(13) Laboratory rats (Sprague Dawley rats) carried out physical activity through external stimulation for 48 hours under conditions of reduced oxygen and air pressure. Here, the air pressure was reduced to a value below 430 Torr, so that a height of over 4500 m was simulated. A prior acclimatization of the rats to the height of over 4500 m was not carried out. During this time, the rats were able to undertake a 15-20 minute pause every 4 hours in order to take in water and food. After 48 h physical activity at the simulated height of over 4500 m the rats were treated intratracheally with 300 μl/animal subject peptide SEQ ID NO: 1 (100 μg, 300 μg and 600 μg) or 300 μl saline solution. The rats then spent a further 4 hours under conditions of reduced oxygen and air pressure at the simulated height of over 4500 m. The lungs were then removed and the intensity of the pulmonary form of altitude sickness was determined (FIG. 1), the protein content in the lung fluid was determined (FIG. 2) and the histological appearance of the lung tissue was determined (FIG. 3).

(14) Result

(15) The investigation showed that the intratracheal administration of peptide SEQ ID NO: 1 to laboratory rats which were exposed to the conditions of reduced air pressure and reduced oxygen concentration, led to reduction of the intensity of the pulmonary form of altitude sickness (FIG. 1). This was able to be demonstrated for 100 μg/laboratory rat and 600 μg/laboratory rat and especially for 300 μg/laboratory rat peptide SEQ ID NO: 1.

(16) The investigation showed, furthermore, that the intratracheal administration of peptide SEQ ID NO: 1 to laboratory rats which were exposed to the conditions of reduced air pressure and reduced oxygen concentration, led to reduction of the protein concentration in the lung fluid (FIG. 2). This was able to be demonstrated for 100 μg/laboratory rat and 600 μg/laboratory rat and especially for 300 μg/laboratory rat peptide SEQ ID NO: 1.

(17) The histological examination showed that the rats treated with saline solution exhibited swollen lung tissue with erythrocytes, with the lung tissue in rats after administration of peptide SEQ ID NO: 1 being comparable with healthy lung tissue of the control rats, which were not exposed to the conditions of reduced oxygen and air pressure.

EXAMPLE 2

(18) ex vivo assessment of the pro-inflammatory characteristics of the peptide according to the invention with SEQ ID NO: 1 in human whole blood.

(19) A pharmacological ex vivo safety study was carried out with regard to the peptide SEQ ID NO: 1 according to the invention in human full blood, in order to establish whether the peptide SEQ ID NO: 1 leads to the release of the pro-inflammatory marker interleukin-6 (IL-6) from fresh full blood (i.e. whether or not peptide SEQ ID NO: 1 shows TNF-specific inflammatory activity (i.e. TNF receptor binding activity)). In this study, fresh full blood was used; this is a recognized prediction model for the assessment of the inflammatory reaction in vivo.

(20) Summary of the Methodology

(21) The aim of this study was to determine the pro-inflammatory signal capacity of the peptide SEQ ID NO: 1. Here, full blood cultures were used and the secretion of interleukin-6 (IL-6), a very sensitive marker for pro-inflammatory stimulation, was quantified by means of ELISA. Test System 25 ml heparinised blood freshly taken from 5 healthy subjects (HS) was used in the tests. Test Object Identification: Peptide SEQ ID NO: 1 (Dose: 1 ng/ml to 10 μg/ml; single administration in solution) Description: White powder, purity 96%
Full Blood Cultures

(22) Full blood (FB) cultures were carried out by pipetting 1 ml FB in depressions of 24-well plates. In each experiment, unstimulated and stimulated control cultures were included.

(23) If possible, the substances and stimulants to be examined were always used in the same volume in each well in a given experiment, which is not greater than 10% of the total volume in a well. Unstimulated controls took place with PBS. Volume adjustment and dilutions for different treatments were likewise carried out with PBS.

(24) The content of each well was mixed and the plates were incubated at 37° C. and 5% CO.sub.2 for 24 hours. After incubation, the content of each well was transferred into a fresh 1.5 ml microtube and centrifuged at 8000 to 9000×g for 15 minutes. The supernatant of each sample was divided individually to two 1.5 ml reaction vessels and stored at −20° C. until use.

(25) Analysis of Interleukin-6

(26) Interleukin-6 was quantified by means of a specific ELISA (Human IL-6 ELISA-Set, BD Biosciences, Cat. No. 555220) using an anti-human-IL-6 antibody as capture antibody, a biotinylated anti-human IL-6 detection antibody, avidin horseradish peroxidise conjugate as enzyme reagent and recombinant IL-6 as standard. Absorption measurement at 450 nm was carried out with the Packard Fusion reader.

(27) Data Analysis

(28) The results of each plate were stored and evaluated with the fusion data analysis software.

(29) Summary of the Results of the Study

(30) The aim of this study was to determine the pro-inflammatory signalling capacity of the peptide SEQ ID NO: 1. Full blood cultures were used and the secretion of IL-6, a very sensitive marker for inflammatory pro-stimulation, was quantified by means of ELISA.

(31) Full blood samples of five healthy subjects were either left unstimulated (negative control), stimulated with high and low doses of LPS (positive controls) or incubated with the peptide in nine semi-logarithmic dilutions of 10 μg/ml to 1 ng/ml. The results are presented in the following table:

(32) Table: Release of Interleukin-6 from Fresh Full Blood with Addition of Peptide SEQ ID NO: 1 and LPS

(33) Peptide SEQ ID NO: 1 Positive control (LPS)

(34) Concentration Concentration of IL-6 (pg/ml, n=5)

(35) TABLE-US-00002 0 (Negative less than 0.5 less than 0.5 Control) 10 mg/ml less than 0.5 195.640  1 mg/ml less than 0.5 108.370  3 ng/ml less than 0.5  34.867  1 ng/ml less than 0.5 not determined

(36) The results clearly show that the peptide SEQ ID NO: 1 did not induce any detectable amount of IL-6 secretion in any of the tested concentrations. The positive controls (LPS) led to an intensive induction of the IL-6 secretion.

(37) Discussion

(38) The experiments were carried out in order to establish whether the peptide SEQ ID NO: 1 brings about the induction of a pro-inflammatory cascade. The readout parameter was the induced secretion of IL-6 in full blood cultures from five healthy donors. The results clearly showed that the peptide SEQ ID NO: 1 induced no detectable level of IL-6 in the donor cultures. It is therefore demonstrated that the peptide SEQ ID NO: 1 does not induce a pro-inflammatory response in the selected ex vivo model and therefore does not have TNF receptor binding activity. This test can be applied for any variants of the peptide according to the invention, in order to establish the feature of freedom from TNF receptor binding activity.

EXAMPLE 3

(39) Assessment of the bioactivity of the peptide according to the invention compared with the non-cyclized (and therefore not according to the invention) form of the peptide and other synthetic peptides which have been proposed in the prior art for the treatment of oedemas, in a patch clamp assay with A549 cells.

(40) Summary:

(41) In this example, the biological activity of the peptide according to the invention was assessed with three other synthetic peptides with regard to the capability for induction of the sodium flow. The synthetic comparative peptides were also proposed in European Patent Application EP 2 009 023 A1 as peptides for the treatment of oedemas. For these peptides, it was assumed in EP 2 009 023 A1 that they would be able to inhibit or reduce the accumulation of excess fluid in tissues. In EP 2 009 023 A1 this characteristic was investigated by means of the TEER test; within the present example, this biological activity is investigated in a whole cell patch clamp test with A549 cells.

(42) This measurement principle (whole cell patch clamp test) reflects the fluid balance in the human lung significantly better and is therefore a recognized test system for this question. The fluid balance in the healthy adult human lung depends on ion transport mechanisms which lead via the lung epithelium, with the participation of Na.sup.+ transporter in the clearance of alveolar fluid having been documented in several studies. In particular here, the amiloride-sensitive epithelial sodium ion channel (ENaC) of type II alveolar cells was identified as main regulator of the clearance of alveolar fluid.

(43) In order to assess the activity of the amiloride-sensitive epithelial sodium ion channel (ENaC) and to determine its activation by biological and chemical compounds, the whole cell patch clamp technique was established as the experimental methodology of choice for the measurement of the sodium ion movement via the apical membrane of alveolar cells to predict the clearance of alveolar fluid.

(44) Accordingly, in the present example the biological activity of the peptide according to the invention and of three synthetic peptides, QRETPEGAEAKPWY (SEQ ID No: 3, described in the prior art as suitable for the treatment of oedemas, is, however, in contrast to the form according to the invention, not cyclized in this experiment), TKPIELGPDEPKAV (SEQ ID NO: 16; described in the prior art as suitable for the treatment of oedemas, is, however, in contrast to the peptides according to the invention, not cyclized and does not contain the core sequence TX.sub.1EX.sub.2X.sub.3E or respectively TPEGAE) and CGTKPIELGPDEPKAVC (SEQ ID NO: 17; described in the prior art as suitable for the treatment of oedemas, however, in contrast to the peptides according to the invention, does not contain the core sequence TX.sub.1EX.sub.2X.sub.3E or respectively TPEGAE) was determined by means of whole cell patch clamp measurements on A549 cells, a continuous cell line of human alveolar type II cells.

(45) It was shown here that none of the peptides QRETPEGAEAKPWY (SEQ ID NO: 18), TKPIELGPDEPKAV (SEQ ID NO: 76) and CGTKPIELGPDEPKAVC (SEQ ID NO: 2), although related from their primary sequence with the peptides provided according to the invention, had any effect on the sodium flow and therefore also no activating effect on the amiloride-sensitive epithelial sodium ion channel (ENaC), whereas the peptide according to the invention induced an increase of the sodium flow over that of the control value, when it was added to the bath solution in a whole cell patch clamp test using A549 cells. As therefore the three comparative peptides showed no effect on the amiloride-sensitive epithelial sodium ion channel (ENaC), compared with the positive control (peptide according to the invention with SEQ ID NO: 1; CGQRETPEGAEAKPWYC (SEQ ID NO: 1)) in a whole cell patch clamp test using A549 cells, the clearance of alveolar fluid is, however, a consequence of this sodium ion movement over the alveolar epithelial cells, it can be concluded that these peptides according to the prior art—in contrast to the peptide according to the invention—are not able to reduce lung oedemas, although respectively particularly preferred variants both of the linear and also of the cyclic peptides which are described in EP 2 009 023 A1 were investigated in the present example (QRETPEGAEAKPWY (SEQ ID NO: 18), TKPIELGPDEPKAV (SEQ ID NO: 76) and CGTKPIELGPDEPKAVC (SEQ ID NO: 2), which are indicated as peptides SEQ ID NO: 18, SEQ ID NO: 76 and SEQ ID NO: 2 in EP 2 009 023 A1). This is all the more remarkable, since an activity in the combating of oedemas was attributed to the comparative peptides in EP 2 009 023 A1.

(46) This shows on the one hand that the features provided according to the invention, in particular the cyclizing and the core sequence TX.sub.1EX.sub.2X.sub.3E or respectively TPEGAE, are essential features of the present invention. On the other hand, the present investigations also justify scientifically corroborated doubts regarding the assumption that these peptides themselves are suitable for the oedema treatment proposed in the prior art. The present example, which was carried out by the test system of the whole cell patch clamp test, which is recognized in the specialist scientific world, shows namely that the investigation system (the TEER test) used in EP 2 009 023 A1 is evidently not suitable to prove this activity.

(47) Introduction:

(48) The fluid balance in the healthy adult human lung depends on ion transport mechanisms over the lung epithelium, wherein the participation of the Na.sup.+ transporters in the clearance of the alveolar fluid is well documented. In particular here the amiloride-sensitive epithelial Na.sup.+ channel (ENaC) represents a limiting step for the Na.sup.+ reception over the alveolar epithelium and plays the key role in fluid reabsorption in the lung. As an improved clearance of alveolar fluid leads directly to an improved prognosis and restoration in the case of a lung oedema, the improvement of the ENaC activity offers a promising therapeutic option for the treatment of lung oedemas.

(49) European patent application EP 2 009 023 A1 proposes for this peptides such as QRETPEGAEAKPWY, TKPIELGPDEPKAV and CGTKPIELGPDEPKAVC (described there as peptides SEQ ID NO: 18, SEQ ID NO: 76 and SEQ ID NO: 2) as new molecules which are to inhibit or reduce the accumulation of excess fluid in the tissue.

(50) According to patent application EP 2 009 023 A1, the so-called transepithelial electrical resistance (TEER) test was used for the screening of anti-lung oedema active ingredient candidates. The “TEER test” is not an established test for the prediction of fluid clearance in lung oedemas (this test can not be found in the relevant scientific literature and also has no relevance with regard to the cells being used (Calu-3 cells) in a model for gas exchange in the human lung). In the present example—in addition to the peptide according to the invention—the peptides QRETPEGAEAKPWY, TKPIELGPDEPKAV and (cyclized) CGTKPIELGPDEPKAVC were investigated by means of a whole cell patch clamp assay, an established methodology for the measurement of ion movement over the cell membranes and especially for the measuring of the sodium transport over the cell membrane of alveolar epithelial cells (Eaton et al., Fed. Proc. 45 (1986), 2707; Hamill et al., Pflugers Arch. 391 (1981) 85-100. Here it was to be tested whether or not the peptides can activate the amiloride-sensitive epithelial sodium flow in lung cells.

(51) The “TEER test”, as it has been described in EP 2 009 023 A1, uses cell layers of Calu-3 cells. However, Calu-3 cells are bronchial cells. Bronchial cells represent approximately 1% of the surface of the human lung for gas exchange and therefore do not represent an appropriate model for alveolar epithelial cells, which form approximately 99% of the surface of the human lung for gas exchange. In the present example, the human alveolar epithelial cell line A549 was used, because this cell line defines the generally accepted experimental standard and is regarded in the literature as the model of choice for alveolar epithelial cells (Lazrak et al., Am. J. Physiol. Lung Cell. Mol. Physiol. 278 (2000), 848-857).

(52) Experimental Procedure

(53) Peptides Investigated

(54) Peptide “AP301” (peptide according to the invention):

(55) Cyclo-H-Cys-Gly-Gln-Arg-Glu-Thr-Pro-Glu-Gly-Ala-Glu-Ala-Lys-Pro-Trp-Tyr-Cys-OH (SEQ ID NO: 1)

(56) Synthetic peptide QRETPEGAEAKPWY:

(57) H-Gln-Arg-Glu-Thr-Pro-Glu-Gly-Ala-Glu-Ala-Lys-Pro-Trp-Tyr-OH

(58) (SEQ ID NO: 3, described in the prior art as suitable for the treatment of oedemas, is, however, in contrast to the form according to the invention, not cyclized in this experiment)

(59) Synthetic peptide TKPIELGPDEPKAV:

(60) H-Thr-Lys-Pro-Ile-Glu-Leu-Gly-Pro-Asp-Glu-Pro-Lys-Ala-Val-OH

(61) (SEQ ID NO: 16; described in the prior art as suitable for the treatment of oedemas, is, however, in contrast to the peptides according to the invention, not cyclized and does not contain the core sequence TX.sub.1EX.sub.2X.sub.3E or respectively TPEGAE)

(62) Synthetic peptide CGTKPIELGPDEPKAVC:

(63) Cyclo-H-Cys-Gly-Thr-Lys-Pro-Ile-Glu-Leu-Gly-Pro-Asp-Glu-Pro-Lys-Ala-Val-Cys-OH (SEQ ID NO: 17; described in the prior art as suitable for the treatment of oedemas, however, in contrast to the peptides according to the invention, does not contain the core sequence TX.sub.1EX.sub.2X.sub.3E or respectively TPEGAE).
Peptide Synthesis

(64) All peptides in the present example were produced by solid phase peptide synthesis according to the fluorenylmethyloxycarbonyl/t-butyl protection strategy on 2-chlorotritylchloride resin. Diisopropylcarbodiimide and N-hydroxybenzotriazole were used as coupling reagents. All coupling steps were carried out in N—N-dimethylformamide. Protected amino acids were coupled in succession to the peptide chain, beginning with the C-terminal amino acid. Deprotection of the fluorenylmethyloxycarbonyl was carried out in 20% piperidine in N—N-dimethylformamide. Separation of the completed, partially protected peptide from the resin was carried out in a 1:1 mixture of acetic acid and dichloromethane.

(65) In the case of the peptide SEQ ID NO: 1, after separation from the resin, the side chain deprotection was carried out in 95% trifluoroacetic acid, 5% water, followed by cyclizing of the linear raw peptide by oxidation of the terminal cysteine residues by the supply of oxygen (O.sub.2 at 1.2 bar) at pH 8.5 for approximately 100 hours.

(66) The raw peptide product was purified by reverse phase medium pressure liquid chromatography (RP-MPLC) on a RP-C18 silica gel column with a gradient of 5%-40% acetonitrile. Finally, the trifluoroacetate counterion was replaced by acetate on a Lewatit MP64 column (acetate form). After a final washing step in water, the purified peptide was lyophilised as acetate salt and obtained as a white to cream-coloured powder. In the case of peptide 2, the intermolecular disulphide bridge caused problems in the separation from the Lewatit column, therefore this cyclic peptide was used in the trifluoroacetate form instead of the acetate form.

(67) Characterization of the Peptides

(68) The molecular masses of the peptides were confirmed by electrospray ionization mass spectrometry or MALDI-TOF-MS; the purity was determined by analytical high performance liquid chromatography.

(69) The peptides were stored at −20° C.

(70) Patch Clamp Protocol

(71) The whole cell patch clamp test using A549 cells took place as described in Hazemi et al. (J. Med. Chem. 53 (2010), 8021-8029). Solutions of the peptides were added to the external (bath) solution in the patch clamp test, so that a final concentration of 300 nM was reached. In cases where an increase of the flow after addition of a given peptide was observed, an amiloride solution (to 100 mM final concentration) was added to the bath solution—after the flow reached a stationary state —, in order to differentiate the amiloride-sensitive from the amiloride-insensitive flow. The amiloride-sensitive flow was then calculated by subtracting the flow value after addition of amiloride (amiloride-insensitive) from the flow value of the stationary state before the addition of amiloride. For each peptide, three experiments were carried out in different A549 cells (n=3).

(72) Results

(73) The peptide according to the invention; SEQ ID NO: 1 (“AP301”); positive control peptide) led, when it was added to the bath solution in a whole cell patch clamp test using A549 cells in a final concentration of 240 nM, to an increase of the active Na.sup.+ flow from a control value of 86 pA±5 pA (before addition of AP301) to a maximum of 1073±15 pA (after addition of AP301). The subsequent addition of amiloride caused a reversion of the flow to 36 pA±5 pA. This showed that the flow which has been increased by AP301 is the amiloride-sensitive NA.sup.+ flow (FIG. 4).

(74) When the three synthetic comparative peptides, QRETPEGAEAKPWY, TKPIELGPDEPKAV and CGTKPIELGPDEPKAVC were added in separate whole cell patch clamp experiments using A549 cells to the bath solution in a final concentration of 300 nM, no effect on the flow was able to be observed: the values remained in the range of the control value (FIGS. 5-7).

(75) Discussion of the Results

(76) In the present example, the capability was shown of the peptide according to the present invention (“AP301”) as positive control in the increase of the amiloride-sensitive Na.sup.+ flow in a whole cell patch clamp test with A549 cells. Addition of AP301 to the bath solution led to an increase of the flow proceeding from a control value of 86 pA±5 pA (before addition of AP301) to a maximum of 1073±15 pA (after addition of AP301). The subsequent addition of amiloride caused a reversion to 36 pA±SpA. This shows that AP301 increases the amiloride-sensitive Na.sup.+ flow of 50 pA to 1037 pA and therefore confirms the activating effect of AP301 on the amiloride-sensitive epithelial Na.sup.+ channel (ENaC) (cf. also Tzotzos et al., Pulm. Pharmacol. Ther. 26 (2013), 356-363), which is arranged in the lung apically in alveolar epithelial cells. Activation of ENaC leads to an increase of the Na.sup.+ reception from the alveolar fluid into the epithelial layer, so that the osmotic driving force is increased, which underpins the clearance of alveolar fluid and leads to water flowing from the alveoli into the interstitial layer under the epithelium. The mechanism which forms the basis of the observed alveolar fluid clearing effect of AP301 administered directly to the lung could be due to this.

(77) Each of the three other synthetic comparative peptides, QRETPEGAEAKPWY, TKPIELGPDEPKAV and CGTKPIELGPDEPKAVC was likewise tested for the capability of influencing the Na.sup.+ flow when it was added to the bath solution in a whole cell patch clamp test with A549 cells. However, unlike AP301, which showed an immediate intensifying effect, none of the other three peptides had an influence on the flow in these cells, even in a somewhat higher application concentration than the peptide AP301 according to the invention (300 nM for the three peptides, 240 nM for AP301).

EXAMPLE 4

Activation of the Amiloride-Sensitive Sodium Ion Channel (EnaC) by the Peptides According to the Invention

(78) The peptides SEQ ID NOS: 1 and 11 to 15 according to the invention were characterized extensively in cell-based studies. These cyclic peptides SEQ ID NOS: 1 and 11 to 15 activate the amiloride-sensitive sodium ion channel (ENaC) in lung cells. Thereby, the equivalency of these peptides with the previously investigated AP301 in the effect according to the present invention is clarified.

(79) Peptide Sequences

(80) SEQ ID NO: 1: CGQRETPEGAEAKPWYC: The cyclizing of the peptide was achieved in that the terminal cysteines (C) were oxidized with the development of a sulphur bridge.

(81) SEQ ID NO: 11: KSPGQRETPEGAEAKPWYE: In the cyclic peptide SEQ ID NO: 11 the amino acids are linked peptidically from the C-terminal amino acid glutamic acid (E) to the N-terminal amino acid lysine (K), whilst the N-terminal amino acid lysine (K) is connected with the C-terminal amino acid glutamic acid (E) by means of an amide bond between the nitrogen of the epsilon amino group of the side chain of the lysine and the gamma carbon in the side group of the glutamic acid.

(82) SEQ ID NO: 12: KGQRETPEGAEAKPWYG: In the cyclic peptide SEQ ID NO: 12 the amino acids are linked peptidically from the C-terminal amino acid glycine (G) to the N-terminal amino acid lysine (K), whilst the N-terminal amino acid lysine (K) is connected with the C-terminal amino acid glycine (G) by means of an amide bond between the nitrogen of the epsilon amino group of the side chain of the lysine and the carbon of the carboxyl group of the glycine.

(83) SEQ ID NO: 13: Ornithine-GQRETPEGAEAKPWYG: In the cyclic peptide SEQ ID NO: 13 the amino acids are linked peptidically from the C-terminal amino acid glycine (G) to the N-terminal amino acid ornithine (Orn), whilst the N-terminal amino acid ornithine (Orn) is connected with the C-terminal amino acid glycine (G) by means of an amide bond between the nitrogen of the delta amino group of the side chain of the ornithine and the carbon of the carboxyl group of the glycine.

(84) SEQ ID NO: 14: 4-aminobutanoic acid-GQRETPEGAEAKPWYD: In the cyclic peptide SEQ ID NO: 14 the amino acids are linked peptidically from the C-terminal aspartic acid (D) to the N-terminal amino acid glycine (G), whilst the C-terminal aspartic acid (D) is connected with the N-terminal amino acid glycine by means of an amide bond between the nitrogen of the amino group of the N-terminal glycine and the carbon C1 of the carboxyl group of the 4-aminobutyric acid on the one hand, and by means of an amide bond between the nitrogen of the amino group of the 4-aminobutyric acid and the carbon of the carboxyl group of the side chain of the C-terminal aspartic acid on the other hand.

(85) SEQ ID NO: 15: β-alanine-GQRETPEGAEAKPWYE: In the cyclic peptide SEQ ID NO: 15 the amino acids are linked peptidically from the C-terminal glutamic acid (E) to the N-terminal amino acid glycine (G), whilst the C-terminal glutamic acid (E) is connected with the N-terminal amino acid glycine by means of an amide bond between the nitrogen of the amino group of the N-terminal glycine and the carbon C1 of the carboxyl group of the β-alanine on the one hand, and by means of an amide bond between the nitrogen of the amino group of the β-alanine and the carbon of the carboxyl group of the side chain of the C-terminal glutamic acid on the other hand.

(86) SEQ ID NO: 19: CGQREAPAGAAAKPWYC (not according to the invention): The cyclizing of the peptide SEQ ID NO: 19 was achieved in that the terminal cysteines (C) were oxidized with the development of a sulphur bridge.

(87) Peptide Synthesis

(88) The cyclic peptides SEQ ID NOS: 1, 11 to 15 and 19 were produced by means of Fmoc solid phase synthesis fully automatically, with adherence to the following steps: sequential coupling of the amino acids; selective separating from the solid phase; purification and lyophilisation, selective cyclizing; separating of the protective groups; purification and lyophilisation; analytical examination.

(89) The cyclic peptides SEQ ID NOS: 1 and 11 to 15 (according to the invention) and 19 (not according to the invention) were then examined for purity and mass by means of reverse HPLC.

(90) The purity of the cyclic peptide SEQ ID NO: 1 was 96.3% m/z (ESI) 1924.2 (M++1). The purity of the cyclic peptide SEQ ID NO: 11 was 96.3%. m/z (ESI) 1924.1 (M++1). The purity of the cyclic peptide SEQ ID NO: 12 was 98.8%. m/z (ESI) 1888.2 (M++1). The purity of the cyclic peptide SEQ ID NO: 13 was 97.4%. m/z (ESI) 1873.4 (M++1). The purity of the cyclic peptide SEQ ID NO: 14 was 99%. m/z (MALDI-TOF) 1901.6 (M++1). The purity of the cyclic protein SEQ ID NO: 15 was 99%. m/z (MALDI-TOF) 1902.7 (M++1). The purity of the cyclic peptide SEQ ID NO: 19 was 95%. m/z (MALDI-TOF) 1778.02 (M++1).

(91) All the peptides according to the invention SEQ ID NOS: 1 and 11 to 15 have the following shared structural characteristic:

(92) Sequence: X.sub.1-GQRETPEGAEAKPWY-X.sub.2 (SEQ ID NO: 21)

(93) where X.sub.1 represents an amino acid or 1 to 4 amino acids, in particular 1 or 3 amino acids, where the amino acids are natural or unnatural amino acids,

(94) where X.sub.1 represents the amino acid C, K, ornithine, 4-aminobutyric acid, β-alanine, or the sequence KSP,

(95) where X.sub.2 may be a natural or unnatural amino acid,

(96) where X.sub.2 may be the amino acid C, D, G or E,

(97) and where X.sub.1 is the N-terminal amino acid and X.sub.2 is the C-terminal amino acid.

(98) Electrophysiological Investigations of the Amiloride-Sensitive Sodium Ion Channel (ENaC)

(99) Macroscopic sodium ion flows were derived from human lung epithelial cells A549 with the “whole cell” configuration by means of the “patch clamp” technique (Hamill et al., Pflugers Arch. 391 (1981), 85-100). For the flow derivations in the “whole cell” configurations the following bath- and electrode solutions were used:

(100) Bath solution: 135 mM sodium methanesulphonate, 10 mM NaCl, 2.7 mM KCl, 1.8 mM CaCl.sub.2, 2 mM MgCl.sub.2, 5.5 mM glucose, and 10 mM HEPES, pH 7.4.

(101) Electrode solution: 120 mM potassium methanesulphonate, 15 mM KCl, 6 mM NaCl, 1 mM Mg.sub.2ATP, 2 mM Na3ATP, 10 mM HEPES, and 0.5 mM EGTA (pH 7.2).

(102) Coverslips with the cells cultivated thereon were transferred into an experimental bath holding 1 ml, were fixed on the microscope table (Axiovert 100, 400× magnification) and the cells were superfused with the bath solution described above. The flow was then derived from a suitable cell (which adheres to the coverslip). For this, a microelectrode (glass capillary with a defined, heat-polished tip opening of approx. 1-3 μm, corresponds to a resistance of the electrode tip of 3-5 MΩ) filled with an electrolyte solution was placed onto the cell and the membrane was suctioned, so that a “Gigaohm seal” was formed between membrane and electrode, in order to minimize the leakage current. With the “whole cell configuration” the membrane was penetrated under the electrode tip, so that the flow, which flows through all ion channels of the cell, can be measured. On obtaining a “Gigaohm seal”, a defined membrane holding potential was applied via a pre-amplifier (CV-4 Headstage, Axon Instruments) and amplifier (Axopatch 1D, Axon Instr.) and the flow, which flows here through the ion channels, was measured.

(103) The pulse protocol consisted of a hyperpolarisation of the cell membrane to −100 mV for 5 s and subsequent incremental depolarisation in 20 mV stages to +100 mV.

(104) This protocol was carried out before (control) and after addition of the cyclic proteins. The flow derivations which were thus obtained were stored and analysed by means of the PCLAMP 6.0 programme. For this, the flow derivations obtained in the presence of amiloride were subtracted from the previously registered flows, so that the amiloride-sensitive sodium flow through the epithelial sodium channels was able to be determined.

(105) The results of the measurements are summarized in Table 1. The activity of the individual peptides is indicated as EC50 (in nM). The EC50 is the effective concentration at which 50% of the maximum activity (i.e. maximum increase of flow intensity, I) is measured.

(106) Table 1. Activity of the peptides according to the invention SEQ ID 1 and SEQ ID 11-15, and of the peptide SEQ ID NO: 19 not according to the invention, on the cellular amiloride-sensitive sodium ion flow. The activity is indicated as effective concentration at 50% of the maximum activity (EC.sub.50).

(107) TABLE-US-00003 Cyclic Peptide EC.sub.50 (nM) SEQ ID NO: 1 54 SEQ ID NO: 11 56 SEQ ID NO: 12 38 SEQ ID NO: 13 45 SEQ ID NO: 14 24 SEQ ID NO: 15 19 SEQ ID NO: 19 No activity

(108) The activity of the cycle peptides SEQ ID NOS: 1 and 11 to as a function of the concentration is presented in FIG. 8. The maximum activity was indicated by 100%.

(109) The illustrated investigations show that the peptides SEQ ID NOS: 1 and 11 to 15 according to the invention are biologically active, whereas the peptide SEQ ID NO: 19 not according to the invention is not active. The difference between the cyclic peptides SEQ ID NOS: 1 and 11 to 15 and the cyclic peptide SEQ ID NO: 19 consists in that within the general peptide sequence X.sub.1-GQRETPEGAEAKPWY-X.sub.2 the amino acid T (at 5.sup.th position) and the amino acid E (at 7.sup.th position) and the amino acid E (at 10.sup.th position) were exchanged by alanine. The sequence TPEGAE is therefore essential. The structure of X.sub.1 and X.sub.2 have no essential influence on the activity.

(110) Summary of the Sequences:

(111) TABLE-US-00004 SEQ ID NO: 1 CGQRETPEGAEAKPWYC SEQ ID NO: 2 TPEGAE SEQ ID NO: 3 QRETPEGAEAKPWY SEQ ID NO: 4 PKDTPEGAELKPWY SEQ ID NO: 5 CGPKDTPEGAELKPWYC SEQ ID NO: 6 CGQKETPEGAEAKPWYC SEQ ID NO: 7 CGQRETPEGAEARPWYC SEQ ID NO: 8 CGQRETPEGAEAKPC SEQ ID NO: 9 CQRETPEGAEAKPWYC SEQ ID NO: 10  CGQRETPEGAEAKFWYC SEQ ID NO: 11  KSPGQRETPEGAEAKPWYE SEQ ID NO: 12  KGQRETPEGAEAKPWYG SEQ ID NO: 13  Ornithine-GQRETPEGAEAKPWYG SEQ ID NO: 14  4-aminobutanoic acid-GQRETPEGAEAKPWYD SEQ ID NO: 15  β-alanine-GQRETPEGAEAKPWYE SEQ ID NO: 16  TKPIELGPDEPKAV SEQ ID NO: 17  CGTKPIELGPDEPKAVC SEQ ID NO: 18  GQRETPEGAEAKPWY SEQ ID NO: 19  CGQREAPAGAAAKPWYC SEQ ID NO: 20  TXEXXE SEQ ID NO: 21 X.sub.1-GQRETPEGAEAKPWY-X.sub.2