Lyophilisate containing a cyclic peptide of formula X.SUB.1.-GQRETPEGAEAKPWY-X.SUB.2
10711041 ยท 2020-07-14
Assignee
Inventors
Cpc classification
A61K9/0073
HUMAN NECESSITIES
A61K38/12
HUMAN NECESSITIES
A61K38/191
HUMAN NECESSITIES
International classification
A61K9/00
HUMAN NECESSITIES
C07K7/64
CHEMISTRY; METALLURGY
Abstract
Cyclic peptide of formula TABLE-US-00001 I X.sub.1-GQRETPEGAEAKPWY-X.sub.2,
wherein
X.sub.1 comprises an amino acid (sequence), with 1 to 4 members, comprising natural and unnatural amino acids, and X.sub.2 comprises a natural amino acid, and wherein X.sub.1 comprises the N-terminal amino acid left in position 1, and X.sub.2 the C-terminal amino acid in the ultimate, right position, in the form of a lyophilisate without additives and/or stabilizers and its use.
Claims
1. An aqueous aerosol for inhalation generated by nebulization, the aqueous aerosol consisting of: water; and a cyclic peptide of formula I, or a salt thereof with a proviso that the salt excludes a trifluoroacetate ion, dissolved in the water: TABLE-US-00018 I X.sub.1-GQRETPEGAEAKPWY-X.sub.2, wherein X.sub.1 comprises an amino acid sequence, with 1 to 4 members, comprising natural and unnatural amino acids, and X.sub.2 comprises a natural amino acid, and wherein X.sub.1 comprises the N-terminal amino acid in position 1 on the left in formula I, and X.sub.2 comprises the C-terminal amino acid in the ultimate, right position in formula I, wherein the cyclic peptide is in the form of a lyophilisate prior to mixing with the water, wherein the aqueous aerosol includes particles of the water and the cyclic peptide, the particles having a diameter less than or equal to 5 m, wherein the cyclic peptide, when in solution, remains fully active for a period of at least 7 days, and wherein the cyclic peptide, when in a lyophilized form, remains fully active at room temperature for a period of at least 6 months.
2. The aqueous aerosol of claim 1, wherein X.sub.1 in formula I is selected from the amino acid (sequence) C (Cys), KSP (Lys-Ser-Pro), K (Lys), ornithine, 4-amino butyric acid, and -alanine.
3. The aqueous aerosol of claim 1, wherein X.sub.2 in formula I is selected from the group C (Cys), D (Asp), G (Gly) and E (Glu).
4. The aqueous aerosol of claim 1, wherein the cyclic compound of formula I is selected from a peptide of the amino acid sequences TABLE-US-00019 SEQIDNO:1 Cyclo(CGQRETPEGAEAKPWYC) wherein a disulfide bridge is formed between both of the terminal cysteine residues; TABLE-US-00020 SEQIDNO:2 Cyclo(KSPGQRETPEGAEAKPWYE) wherein an amide bond is formed between the amine group which is attached to the -carbon atom of the N-terminal lysine residue and the side chain carboxy group, attached to the y-carbon atom of the C-terminal glutamic acid residue; TABLE-US-00021 SEQIDNO:3 Cyclo(KGQRETPEGAEAKPWYG) wherein an amide bond is formed between the amine group which is attached to the -carbon atom of the side chain of the N-terminal lysine residue with the carboxy group of the C-terminal glycine residue; TABLE-US-00022 SEQIDNO:4 Cyclo(ornithine-GQRETPEGAEAKPWYG) wherein an amide bond is formed between the amine group which is attached to the -carbon atom of the side chain of the N-terminal ornithine residue and the carboxy group of the C-terminal glycine residue; TABLE-US-00023 SEQIDNO:5 Cyclo(4-aminobutyricacid-GQRETPEGAEAKPWYD) wherein an amide bond is formed between the amine group of the N-terminal 4-amino butyric acid residue and the side chain carboxy group which is attached to the -carbon atom of the C-terminal aspartic acid residue; and TABLE-US-00024 SEQIDNO:6 Cyclo(-alanine-GQRETPEGAEAKPWYE), wherein an amide bond is formed between the amine group of the N-terminal -alanine residue (3-amino propanoic acid residue) and the side chain carboxy group, which is attached to the y-carbon atom of the C-terminal glutamic acid residue.
5. An extracorporeal method of conditioning/improving lung functions, comprising spraying a donor lung ex vivo with the aqueous aerosol of claim 1.
6. An aqueous aerosol for inhalation generated by nebulization, the aqueous aerosol consisting of: water; and a cyclic peptide of formula I in the form of a hydrochloride salt, dissolved in the water: TABLE-US-00025 I X.sub.1-GQRETPEGAEAKPWY-X.sub.2, wherein X.sub.1 comprises an amino acid sequence, with 1 to 4 members, comprising natural and unnatural amino acids, and X.sub.2 comprises a natural amino acid, and wherein X.sub.1 comprises the N-terminal amino acid in position 1 on the left in formula I, and X.sub.2 comprises the C-terminal amino acid in the ultimate, right position in formula I, wherein the cyclic peptide is in the form of a lyophilisate prior to mixing with the water, wherein the cyclic peptide remains fully active in solution for a period of at least 7 days and remains fully active when in a lyophilized form for a period of at least 6 months at room temperature, and wherein the aqueous aerosol includes particles of the water and the cyclic peptide, the particles having a diameter less than or equal to 5 m.
7. An aqueous aerosol for inhalation generated by nebulization, comprising: water, and a cyclic peptide of formula I, or a salt thereof with a proviso that the salt excludes a trifluoroacetate ion, dissolved in the water: TABLE-US-00026 I X.sub.1-GQRETPEGAEAKPWY-X.sub.2, wherein X.sub.1 comprises an amino acid sequence, with 1 to 4 members, comprising natural and unnatural amino acids, and X.sub.2 comprises a natural amino acid, and wherein X.sub.1 comprises the N-terminal amino acid in position 1 on the left in formula I, and X.sub.2 comprises the C-terminal amino acid in the ultimate, right position in formula I, wherein the cyclic peptide is in the form of a lyophilisate without additives and/or stabilizers prior to mixing with the water, the additives and/or stabilizers comprising one or more of tensides, buffer mixtures, lipid admixtures, albumin, osmotic agents, antioxidants, aggregation and/or precipitation prevention agents, liposomes, gelatine, alginates, or sugars, wherein the aqueous aerosol includes particles of the water and the cyclic peptide, the particles having a diameter less than or equal to 5 m, wherein the cyclic peptide when in a lyophilized form remains fully active for a period of at least 7 days, wherein the cyclic peptide when in a lyophilized form remains fully active at room temperature for a period of at least 6 months, and wherein the aqueous aerosol is without additives and/or stabilizers after mixing with the water.
8. The aqueous aerosol of claim 7, wherein X.sub.1 in formula I is selected from the amino acid (sequence) C (Cys), KSP (Lys-Ser-Pro), K (Lys), ornithine, 4-amino butyric acid, and -alanine.
9. The aqueous aerosol of claim 7, wherein X.sub.2 in formula I is selected from the group C (Cys), D (Asp), G (Gly) and E (Glu).
10. The aqueous aerosol of claim 7, wherein the cyclic compound of formula I is selected from a peptide of the amino acid sequences TABLE-US-00027 SEQIDNO:1 Cyclo(CGQRETPEGAEAKPWYC) wherein a disulfide bridge is formed between both of the terminal cysteine residues; TABLE-US-00028 SEQIDNO:2 Cyclo(KSPGQRETPEGAEAKPWYE) wherein an amide bond is formed between the amine group which is attached to the -carbon atom of the N-terminal lysine residue and the side chain carboxy group, attached to the y-carbon atom of the C-terminal glutamic acid residue; TABLE-US-00029 SEQIDNO:3 Cyclo(KGQRETPEGAEAKPWYG) wherein an amide bond is formed between the amine group which is attached to the -carbon atom of the side chain of the N-terminal lysine residue with the carboxy group of the C-terminal glycine residue; TABLE-US-00030 SEQIDNO:4 Cyclo(ornithine-GQRETPEGAEAKPWYG) wherein an amide bond is formed between the amine group which is attached to the -carbon atom of the side chain of the N-terminal ornithine residue and the carboxy group of the C-terminal glycine residue; TABLE-US-00031 SEQIDNO:5 Cyclo(4-aminobutyricacid-GQRETPEGAEAKPWYD) wherein an amide bond is formed between the amine group of the N-terminal 4-amino butyric acid residue and the side chain carboxy group which is attached to the -carbon atom of the C-terminal aspartic acid residue; and TABLE-US-00032 SEQIDNO:6 Cyclo(-alanine-GQRETPEGAEAKPWYE), wherein an amide bond is formed between the amine group of the N-terminal -alanine residue (3-amino propanoic acid residue) and the side chain carboxy group, which is attached to the y-carbon atom of the C-terminal glutamic acid residue.
11. A method of preparing an aqueous aerosol for inhalation wherein the aerosol includes particles of the water and a cyclic peptide of formula I, or a salt thereof with a proviso that the salt excludes a trifluoroacetate ion: TABLE-US-00033 I X.sub.1-GQRETPEGAEAKPWY-X.sub.2, wherein X.sub.1 comprises an amino acid sequence, with 1 to 4 members, comprising natural and unnatural amino acids, and X.sub.2 comprises a natural amino acid, and wherein X.sub.1 comprises the N-terminal amino acid in position 1 on the left in formula I, and X.sub.2 comprises the C-terminal amino acid in the ultimate, right position in formula I, comprising the steps of: preparing a lyophilisate of the cyclic peptide of formula I free of stabilizers and additives; mixing the lyophilisate with water; and generating the aqueous aerosol by nebulization, wherein the aqueous aerosol is generated without stabilizers and additives and includes particles of the water and the cyclic peptide, the particles having a diameter less than or equal to 5 m.
Description
DESCRIPTION OF THE FIGURES
(1)
(2)
(3)
(4)
(5) 1 denotes a polystyrene container filled with ice and salt,
(6) 2 denotes a small tube with the dissolved peptide (control substance),
(7) 3 denotes a nebulizer,
(8) 4 denotes a store room, and
(9) 5 a control module.
(10)
(11)
(12) 1=inhalation filter
(13) 2=exhalation filter
(14) 3=filter connection piece
(15) 4=nebulizer Y-piece (one-way valve inclusive)
(16) 5=residue in the nebulizer
(17) whereby 2 nebulizers, one from type (TYP) A and one from type (TYP) B were used.
EXAMPLE 1
Peptide Synthesis
(18) Peptides were prepared according to the following steps:
(19) Sequential coupling of the amino acids; selective cleavage from the solid phase; purification and lyophilisation, selective cyclisation; cleavage of the protecting groups; purification and lyophilisation; analytical investigation.
(20) All cyclic peptides of the present invention, the peptides of the amino acid sequences SEQ ID NO:1 to SEQ ID NO:6, as well as the peptide with the amino acid sequence SEQ ID NO:7, were prepared fully automated according to the fluorenylmethoxycarbonyl/t-butyl protection strategy in the form of a solid phase synthesis on a carrier (2-chlorotritylchloride resin). Diisopropyl carbodiimide and N-hydroxybenzotriazole were used as coupling reagents. All coupling steps were carried out in NN-dimethyl formamide as a solvent. The protected amino acids thereby were sequentially coupled onto the respective C-terminal amino acid which was used as a starting material. Deprotection of fluorenylmethoxycarbonyl was carried out in 20% piperidine in NN-dimethyl formamide. Cleavage of the completed, partially-protected peptide from the resin was carried out in a 1:1 mixture of acetic acid and dichloromethane.
(21) In the case of cysteine-containing peptides, side-chain deprotection took place after cleavage from the carrier (resin), in 95% trifluoroacetic acid, whereupon cyclisation was performed by oxidation of terminal cysteine residues, achieved by aeration of the crude linear peptide at basic pH (8.5) for 90 hours. The crude cyclic peptide was purified by reverse phase medium pressure liquid chromatography (RP-MPLC) on an RPC18-silica gel column with a gradient of 5%-40% acetonitril/water. Finally, the trifluoracetate-ion was replaced by acetate on a Lewatit MP64 column (acetate form). Following a wash in water, the purified peptide was lyophilized as an acetate salt and obtained as a white to off-white powder.
(22) In the case of cysteine-free peptides, the cyclisation step was carried out on the partially-protected linear peptide following cleavage from the carrier(resin). After selective cyclisation of the cysteine-free peptides, side-chain deprotection in trifluoroacetic acid was carried out, followed by preparative RP-MPLC chromatography, replacement of the trifluoroacetate ion by acetate and lyophilisation of the acetate form, as for the cysteine-containing peptides.
(23) Thereafter the cyclic proteins of SEQ ID NO:1 to SEQ ID NO:6 were analyzed regarding purity and mass by means of reverse HPLC.
(24) The purity of the cyclic protein of SEQ ID 1 was 96.3%. m/z (ESI) 1924.2 (M++1).
(25) The purity of the cyclic protein of SEQ ID 2 was 96.3%. m/z (ESI) 1924.2 (M++1).
(26) The purity of the cyclic protein of SEQ ID 3 was 98.8%. m/z (ESI) 1888.2 (M++1).
(27) The purity of the cyclic protein of SEQ ID 4 was 97.4%. m/z (ESI) 1873.4 (M++1).
(28) The purity of the cyclic protein of SEQ ID 5 war 99%. m/z (MALDI-TOF) 1901.6 (M++1).
(29) The purity of the cyclic protein of SEQ ID 6 was 99%. m/z (MALDI-TOF) 1902.7 (M++1).
(30) The purity of the cyclic protein of SEQ ID 7 was 95%. m/z (MALDI-TOF) 1778.02 (M++1).
(31) The compound having the amino acid sequence SEQ ID NO:7
(32) TABLE-US-00010 Cyclo(CGQREAPAGAAAKPWYC)
(33) wherein a disulfide bridge is formed between both of the terminal cysteine residues, became apparent to be biologically inactive and was used for comparison purposes.
EXAMPLE 2
Electrophysiological Investigations of the Amilorid-Sensitive Sodium Ion Channel (ENaC)
(34) Macroscopic Sodium Ion Currents were derived from human lung epithelian cells A549 in the whole cell configuration by means of the Patch-clamp technique (Hamill et al, Pflugers Arch. 1981, 391(2):85-100, 1981). For the currents in the whole cell configuration the following bath- and electrode solutions were used:
(35) Bath solution: 135 mM sodium methansulfonate, 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.
(36) Electrode solution: 120 mM potassium methansulfonate, 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).
(37) Cover glasses with thereon cultivated cells were transferred into a 1 ml containing trial bath, fixed onto the microscope table (Axiovert 100, 400-fold enlargement) and the cells were superfunded with the above described bath solution. Subsequent from an appropriate cell (sticking on the cover glass) the current was deduced. For that a microelectrode filled with an electrolyte solution (glass capillary with a defined heat-polished top opening of approximately 1-3 mcorresponds to an electric resistance of the electrode top of 3-5 M) was attached and the membrane sucked in, so that a Gigaohm-Seal between membrane and electrode was formed, in order to minimize the leakage current. At the whole cell-configuration the membrane under the electrode top was disrupted, in order to enable measurement of the current which flows through all ion channels of the cell. On receipt of a Gigaohm-Seal a defined membrane hold potential was created via a preamplifier (CV-4 Headstag, Axon Instruments) and amplifier (Axopatch 1D, Axon Instr.) and the current which thereby flows through the ion channels is measured.
(38) The pulse protocol consisted in a hyperpolarisation of the cell membrane to 100 mV for 5 seconds and subsequent stepwise depolarisation in 20 mV steps to +100 mV.
(39) That protocol was done before (control) and after addition of the cyclic proteins. The thus obtained currents were recorded and analyzed by means of the program PCLAMP 6.0. For that, the currents obtained under the presence of amilorid were subtracted from the previously recorded currents, with the result that the amilorid-sensitive sodium current by the epithelian sodium channels could be determined.
(40) The results of the measurements are outlined in Table 1, wherein there is indicated the activity of the cyclic proteins of SEQ ID 1 to SEQ ID 7 on the cellular amilorid-sensitive sodium ion current. The activity of the individual peptides is indicated as EC.sub.50 (in nM). The EC50 is the effective concentration, at which 50% of the maximum activity is measured (that means maximum increase of current intensity I).
(41) TABLE-US-00011 TABLE 1 Cyclic Protein EC.sub.50 (nM) SEQ ID NO: 1 54 SEQ ID NO: 2 56 SEQ ID NO: 3 38 SEQ ID NO: 4 45 SEQ ID NO: 5 24 SEQ ID NO: 6 19 SEQ ID NO: 7 no activity
(42) In
(43) The investigations shown in Table 1 and in
EXAMPLE 3
Preparation of the Lyophilisate
(44) Development of a stable storage form of the cyclic peptide of formula I was carried out on technical scale. For that the cyclic peptides of SEQ ID NO:1 to SEQ ID NO:6, as well as of the cyclic peptide of SEQ ID NO:7 were dissolved in pure water in amounts of 0.1 mg/ml to 100 mg/ml and filtered for removal of turbidities, contaminations and possible unsterility through a filter with a pore size of 0.2 m.
(45) After filtration the cyclic peptide, dissolved in pure water was portioned into glass or plastic ampoules and by means of freeze-drying (lyophilisation) converted into a stable powder. Thereby the lyophilisation parameters described in Table 2 and the lyophilisation cycle described in
(46) TABLE-US-00012 TABLE 2 Temperature shift ( C.) Shelf Holding during temperature time Holding Pressure Step Process ( C.) (min) Time (mTorr) 1 Load 5 60 5.0 n/a 2 Freezing 45 150 0.5 n/a 3 Primary 15 940 0.3 250 Drying 4 Secondary 20 630 0.5 75 Drying 5 Termination Vial closed under 95% pure nitrogen atmosphere
(47) As a result of lyophilisation a white powder each was obtained for the cyclic peptides having the amino acid sequence SEQ ID NO:1 to SEQ ID NO:6.
EXAMPLE 4
Stability Investigation of the Lyophilisate from Example 2 after Storage at Room Temperature and in the Refrigerator
(48) The stability investigation of the lyophilisates of the cyclic peptides of amino acid sequence SEQ ID NO:1 to SEQ ID NO:6. was done on technical scale. For that the lyophilisate was stored up to 24 months at 2 to 8 C. and up to 6 months at 25 C. at 60% relative humidity. Stability was investigated at different time points during that period. In particular the appearance, contents, as well as purity of the cyclic peptides were investigated. For that laboratory-common analytical processes were used, such as e.g. visual inspection and reverse HPLC.
(49) In addition, after a storage of 24 months at 2-8 C. the biological activity was determined by means of Patch Clamp experiments. Thereby macroscopic currents of A549 cells were deduced in the whole cell configuration of the Patch-clamp technique, as described under Electrophysiological Investigations of the amilorid-sensitive Sodium Ion Channel (ENaC).
(50) In Table 3 the results of the stability investigations of the respective lyophilisate of the cyclic peptides having the amino sequences SEQ ID NO:1 to SEQ ID NO:6 at time point T=0 and after 6 months (T=6M), or after 24 months (T=24M), respectively, are outlined. The appearance did not change during the whole time period. The contents on cyclic peptides, as well as purity are subject of only minor fluctuations.
(51) TABLE-US-00013 TABLE 3 Storage temperature Storage temperature Analytical 2-8 C. 25 C./60% rH Parameter T = 0 T = 24 M T = 0 T = 6 M Appearence SEQ ID 1 white powder white powder SEQ ID 2 white powder white powder SEQ ID 3 white powder white powder SEQ ID 4 white powder white powder SEQ ID 5 white powder white powder SEQ ID 6 white powder white powder Amount/Contents SEQ ID 1 100% 99% 100% 99% SEQ ID 2 100% 99% 100% 99% SEQ ID 3 100% 99% 100% 99% SEQ ID 4 100% 99% 100% 99% SEQ ID 5 100% 99% 100% 99% SEQ ID 6 100% 99% 100% 99% Purity SEQ ID 1 96% 96% 96% 95% SEQ ID 2 96% 96% 96% 95% SEQ ID 3 98% 98% 98% 97% SEQ ID 4 97% 97% 97% 96% SEQ ID 5 99% 99% 99% 98% SEQ ID 6 99% 99% 99% 98%
(52) The lyophilisate of the cyclic peptides of amino acid sequence SEQ ID NO:1 to SEQ ID NO:6 each is thus stable for up to 24 months at 2 to 8 C. and up to 6 months at 25 C./60% relative humidity.
(53) The biological activity measurement by means of Patch Clamp Experiments revealed that a lyophilisate, each with one of the cyclic peptides of amino acid sequence SEQ ID NO:1 to SEQ ID NO:6, still fully was active after 24 months of storing at 2 to 8 C.
EXAMPLE 5
Production of an Aqueous Solution of Cyclic Peptides of Amino Acid Sequence SEQ ID NO:1 to SEQ ID NO:6 Before Inhalation
(54) For pre-preparation for administration of the cyclic proteins of amino acid sequence SEQ ID NO:1 to SEQ ID NO:6, the stable white powders, obtained during lyophilisation according to example 2, were dissolved, each in a defined volume of pure water to obtain a concentration between 0.1 mg pro ml and 100 mg pro ml. The resultant solutions of the cyclic proteins of SEQ ID 1 to SEQ ID 6 were then transferred into the storage containers of the nebulizers. By dissolution of the cyclic peptides of SEQ ID NO:1 to SEQ ID NO: 6 in water clear solutions were obtained.
EXAMPLE 6
Investigations Regarding Stability of the Cyclic Peptides of Amino Acid Sequences SEQ ID NO:1 to SEQ ID NO:6 in Dissolved Preparation Before Inhalation
(55) For practical reasons, an aqueous solution of a cyclic peptide of amino acid sequences SEQ ID NO:1 to SEQ ID NO:6 not always can be used directly after its preparation for inhalation. For that reason the stability of an aqueous solution was investigated exemplarily. The ready to use-solution therefore was stored either in a syringe commonly used in laboratory, at 2 to 8 C. for 7 days, or in a container of a nebulizer at 25 C. for 24 hours. In particular the appearance, the contents of cyclic protein of SEQ ID NO:1, as well as the purity thereof were investigated. The methods used therefore were analytical methods commonly used in laboratory, as for example the visual inspection, as well as analysis by means of reverse HPLC.
(56) The results of the stability investigations of an aqueous solution of the cyclic peptide having the amino acid sequence of SEQ ID NO:1 are set out in Table 4. The appearance did not change during the whole time period. The contents on cyclic peptides, as well as purity were subject of only minor fluctuations.
(57) TABLE-US-00014 TABLE 4 Storage container of a Laboratory Syringe nebulizer, temperature 2 to 8 C. temperature 25 C. Parameter T = 0 T = 7 days T = 0 T = 24 hours Appearance clear solution clear solution Amount/contents 25 mg/ml 25 mg/ml Purity 96.3% 96.2% 96.6% 96.5%
(58) An aqueous solution of a cyclic peptide of amino acid sequence SEQ ID NO:1 to SEQ ID NO:6 for inhalation thus is stable in a laboratory commonly used syringe at 2 to 8 C. for 7 days, or in the container of a nebulizer at 25 C. for at least 24 hours.
EXAMPLE 7
Investigation Regarding Stability of the Cyclic Peptides of Amino Acid Sequence SEQ ID NO:1 to SEQ ID NO:6 in Dissolved Preparation During Conversion into an Aerosol
(59) Since proteins and peptides occasionally may be rather unstable, it was investigated, whether the cyclic peptides of amino acid sequence SEQ ID NO:1 to SEQ ID NO:6 in dissolved preparation during conversion into an aerosol remain stable. For that the cyclic peptides were dissolved in water as described in example 4. After filling into the storage container of a nebulizer the aqueous solution of the cyclic peptides were converted into an aerosol. For that a Mesh-Type nebulizer was used. The aerosol passing out from the nebulizer was collected in a cooling trap as shown in
(60) The chemical stability of the condensed aerosols was determined by means of reverse HPLC/MS.
(61) It could exemplarily be shown that the cyclic peptide of amino acid sequence SEQ ID NO:1 in dissolved preparation during the transformation into an aerosol keeps its biological, as well as its chemical stability.
EXAMPLE 8
Physicochemical Characterisation of the Aerosols of the Cyclic Peptides of Amino Acid Sequence SEQ ID NO:1 to SEQ ID NO:6
(62) By means of a nebulizer which converts aqueous solution into an aerosol, also the aqueous solutions of the cyclic peptides of amino acid sequence SEQ ID NO:1 to SEQ ID NO:6 may be converted into aerosols. Such aerosols can be characterized with respect to the medium droplet size, as well as to size distribution of the aerosol droplets. For that conventional methods are used, which are described also in pharmacopoeias. The one analytical method uses the cascade impactor in its actual construction, the Next Generation Impactor. Thereby the aerosol is conducted through a series of sieve plates, whereby the diameter of the holes is diminished with each plate and the amount of the holes is increased. In the other analytical method, the laser-diffraction measurement, the droplet sizes are determined by means of laser. The 2 important parameters which are determined during these measurements, one the one hand is the median of the diameter of all droplets, and on the other hand, the amount of droplets having a diameter of 5 m. In the literature that diameter is described as a limit, under which inhaled aerosol particles arrive in fact in the lung.
(63) It is ramblingly known, that in practice from a generated aerosol only a part is available for the patient. Thus, for determining the amount of aerosol which is available for a user, trials with a breathing simulator were carried out. For investigating the particle size and for the implementation of the breathing simulation an aerosol from an aqueous solution of the cyclic peptide of amino acid sequence SEQ ID NO:1 was used. For generating aerosol different nebulizer types were used. The nebulizers A and B were so called Mesh-Type nebulizers.
(64) The amount of droplets with a diameter of 5 m in the aerosol was in all nebulizers at least 50%, see Table 5 and
(65) TABLE-US-00015 TABLE 5 Nebulizer Median particle diameter Amount of particles with 5 m Type A 4.7 m 50% Type B 3.3 m 70% Type C 3.7 m 65%
(66) Furthermore it could be proven, that the predominant part of the aerosol of an aqueous solution of the cyclic peptide of amino acid sequence SEQ ID NO:1, generated by the nebulizers, is available for inhalation, as shown in
EXAMPLE 9
Proof of the Cyclic Peptides of Amino Acid Sequence SEQ ID NO:1 to SEQ ID NO:6 in the Blood after Parenteral Administration
(67) The exemplary proof of the cyclic peptides of amino acid sequence SEQ ID NO:1 to SEQ ID NO:6 in the blood after parenteral administration took place in dog and rat. For that exemplarily an aqueous solution of the cyclic peptide of amino acid sequence SEQ ID NO:1 as a bolus (25 mg/kg body weight) was administered intravenously into the experimental animals. Directly after termination of the intravenous application blood was harvested and the concentration of the cyclic protein of amino acid sequence SEQ ID NO:1 was determined by means of common laboratory reverse HPLC/MS. The results are set out in Table 6, wherein the plasma concentration of the cyclic peptide of amino acid sequence SEQ ID NO:1 after intravenous application is indicated.
(68) TABLE-US-00016 TABLE 6 Rat Dog Plasma concentration 28 42 (g/ml)
EXAMPLE 10
Proof of the Cyclic Peptides of Amino Acid Sequence SEQ ID NO:1 to SEQ ID NO:6 in Lung Tissue after Inhalation as an Aerosol
(69) The proof of a cyclic peptide of amino acid sequence SEQ ID NO:1 to SEQ ID NO:6 may take place in lung tissue of rats. For that exemplarily an aerosol was produced with a nebulizer from an aqueous solution of the cyclic peptide of amino acid sequence SEQ ID NO:1. The aerosol was inhaled by the experimental animal (72 mg/kg body weight). After termination of aerosol inhalation, the lung tissue, as well as the blood of the experimental animal was examined. By means of reverse HPLC/MS the concentration of the cyclic peptide of amino acid sequence SEQ ID NO:1 was determined Exemplarily, the cyclic peptide of amino acid sequence SEQ ID NO:1 could be detected in the lung tissue in a concentration of 1.2 m/g after inhalation of 72 mg/kg body weight in sum. In contrast to that the cyclic peptide of amino acid sequence SEQ ID NO:1 could not be detected in the blood up to a detection limit of 0.1 m/ml.
EXAMPLE 11
Effect of the Peptides of Amino Acid Sequence SEQ ID NO:1 to SEQ ID NO:7 onto Deglycolized Cell Surfaces
(70) In whole cell experiments A549 cells were incubated with the enzyme PNGase F (Peptide-N.sup.4(N-acetyl--D-glucosaminyl)asparagine Amidase F), 100 units for 1 to 5 minutes, directly before the Patch Clamp measurements and the cover glasses with the cultivated cells were rinsed with external solution, before transferring them into the chamber of the 1 mL bath. After control recordings, 240 nM of the peptide of amino acid sequence SEQ ID NO:1 was added to the bath solution. The total cell current was recorded of cells without any pre-treatment under controlled conditions and after addition of a peptide of amino acid sequence SEQ ID NO:1 to SEQ ID NO:7 at Eh=100 mV, also as pre-treatment with PNGase F. The results of the deglycosilation experiments under use of the Patch Clamp assays in the whole cell modus shows Table 7, wherein the effect of deglycosilation of A549 cells onto the activation of the sodium current by means of the peptides of amino acid sequence SEQ ID NO:1 to SEQ ID NO:7 is indicated. Whole cell currents were recorded at Eh=100 mV. The concentration of the peptides of amino acid sequence SEQ ID NO:1 to SEQ ID NO:7 in the bath solution was 240 nM.
(71) TABLE-US-00017 TABLE 7 Pre-treatment with No pretreatment Control/Peptide PNGase F with PNGase F control 25.4 pA (n = 16) SEQ ID NO: 1 19.6 pA (n = 3) 1073.3 15.1 pA SEQ ID NO: 2 21.3 pA (n = 3) (n = 10) SEQ ID NO: 3 20.6 pA (n = 3) SEQ ID NO: 4 22.5 pA (n = 3) SEQ ID NO: 5 22.4 pA (n = 3) SEQ ID NO: 6 19.9 pA (n = 3) SEQ ID NO: 7 No activity No activity
(72) The cells with PNGase F (treatment) before the Patch Clamp Assay reduced the ability of the peptides of amino acid sequence SEQ ID NO:1 to SEQ ID NO:6 to enhance the sodium current. Under control conditions without addition of peptide into the bath and at a Holding Potential of 100 mV, the sodium current was 25.4 pA, in untreated cells, as well as in cells which were pretreated with PNGase F. In untreated cells resulted the addition of peptides of amino acid sequence SEQ ID NO:1 to SEQ ID NO:6 (end concentration 240 nM) to the bath solution at a Holding Potential of 100 mV in a noticeable sodium current of more than 1,000 pA. In contrast, a peptide of amino acid sequence SEQ ID NO:7 showed no activity.
EXAMPLE 12
Lung Transplantation Experiments with Pigs
(73) Brain-dead pigs were brought into back position and a vertical sternotomy was carried out. The pericardium and both pleural cavities were opened. The lower and upper cavial venes were embraced. An inflow catheter was inserted into the lung artery by means of a purse-string suture in the right ventricular discharging tract. An inflow occlusion was obtained by connecting the lower and upper cavial venes, discharge occlusion by stapling off the aorta. The lungs then were protected by a prophylactic rinsing with cold, isotonic conservation solution (50 ml per kg body weight of the pig, comprising potassium ions, sodium ions, magnesium ions, calcium ions, chloride ions, dextran, glucose, buffer ions) through the inflow catheter. Incision of the left heart tube yields discharge. The lungs were aerated during that time period with 50% oxygen and ice sludge was given into both pleural cavities and into the mediastinum.
(74) The explanation technique in sum was the removal with heart and esophagus according to the following steps: a) Dissection of the soft tissue bridges to the thoracic cavity at both sides of the trachea. b) Transsection of both pulmonal ligaments (very deep, difficult exposure), then of VCI, of the low thoracic aorta ascendens, or the esophagus, respectively. c) Blunt-separation of residual mediastinal adhesions. d) Entire distension of the donor lung before the tracheal closure with a clip.
(75) After explantation the lungs were winded into gauze, put into an ice bag, filled with low-potassium-dextran-extracellular solution and stored at 4 C. for 18 to 24 hours.
(76) For the ex-vivo lung conditioning the EVLP-technique was used (extravascular lung perfusion). In the EVLP-technique donor lungs are put into a circulation, composed of a pump, aerator and filters. In the EVLP-technique the temperature may be increased up to 37 C. In the EVLP an aerator is used, in order to deliver oxygen to the lungs. The pump is used, to perfuse the lungs with an extracellular solution, containing human albumin and nutrient. During the EVLP the lung function may be assessed regularly regarding key indicators.
(77) For the experimental pig lung transplantation experiment, the EVLP circulation was primed with 2.0 litres human albumin solution. That extracellular solution had an optimal colloidal osmotic pressure. After the circulation was vented, the prime was circulated at 20 C. until it was connected with the lungs. Heparin, Cefuroxim, Methylprednisolon war added to the perfusate.
(78) The processing of the donor lung from the pig was started with sewing on a funnel-shaped tube from Silastic with a pressure-monitored catheter, implemented into the left atrial cuff (LA), for openly splinting the LA and for maintaining a closed perfusion circulation.
(79) That tube was secured anastomosed onto the LA cuff under use of an ongoing 5-0 mMonofilament yarn, in order to achieve a reliable and effective discharging drainage. The same type cannula was used for the transfixion of the lung artery (PA), all spruced up as required, to adjust onto the PA size. A retrograde Back-Table rinsing was carried out under use of 500 ml buffered, extracellular solution. Before fixing the donor lungs into the EVLP circulation, the trachea was opened and direct bronchial sucking off was carried out, in order to clean the airways. An endotracheal tube (size 8 mm I.D.) was introduced into the trachea and was firmly secured with a Numbilicale band. Thereafter the lungs were transferred onto the EVLP circulation unit. First, connection of the LA cannula with the circulation and initiation of a slow, retrograde flow, in order to vent the PA cannula. A soon as venting was complete, the PA cannula was connected with the circulation and anterograde flow of 150 ml/min was initiated with the perfusate at room temperature. In the next 30 minutes the temperature of the perfusate was increased stepwise up to 37 C. As soon as the temperature had reached 32-34 C., mechanical aeration of the donor lungs of the pigs was started, whereby the aeration rate and the rate of the perfusate flow were stepwise increased.
(80) Flow of the EVLP gas transports oxygen to the lungs and delivers carbon dioxide to the inflow perfusate (86% N.sub.2, 6% O.sub.2, 8% CO.sub.2), by means of the gas exchanger membrane it was initiated (start at 0.5 L/min of gas flow and titration based on the inflow perfusate pCO.sub.2), the inflow perfusate pressure (pCO.sub.2) between 35-45 mm Hg to maintain. At the moment where the lung was fully expanded, a single dose AP301 (1 mg/kg in 5 ml Aqua), under use of a simple liquid standard nebulizer system was given into the donor lung of a pig, ventilated and run through by the EVLP system switching.
(81) During the EVLP experiments that blood flow was determined constant. The following functional parameters were measured every hour and recorded: Flow Pulmonal artery (PAF): L/min (mean) Pulmonal artery pressure (PAP): mm Hg Left Atrial pressure(LAP): mm Hg Pulmonal vascular resistance (PVR=[PAPLAP]80/PAF): dynes/sec/cm-5 medium, top-und plateau pressure of the airway (mAwP, peak AwP, platAwP): cm H.sub.2O dynamic compliance (mL/cm H.sub.2O) perfusate gas analysis-inflow (PA) and effluent (PV) .sub.PO.sub.2, .sub.PCO.sub.2 und pH.
(82) Results
(83) This study assesses the effects of a peptide having the amino acid sequence SEQ ID NO:1 onto the lung function in an extracorporeal system simulating lung transplantation. Study results showed, that upon administration via inhalation both, the dynamic lung conformity, as well as the arterio-venous pO.sub.2 difference pO.sub.2 in lungs, which were treated with a peptide having the amino acid sequence SEQ ID NO:1 were improved, as shown in
(84) The use of a peptide having the amino acid sequence of SEQ ID NO:7 showed no improving effects regarding the lung.