Polyethylene glycol based prodrug of adrenomedullin and use thereof

Abstract

The invention relates to novel polyethylene glycol (PEG) based prodrug of Adrenomedullin, to processes for preparation thereof, to the use thereof for treatment and/or prevention of diseases, and to the use thereof for producing medicaments for treatment and/or prevention of diseases, especially of cardiovascular, edematous and/or inflammatory disorders.

Claims

1. A compound of the formula (IV) ##STR00031## wherein n represents 0, 1, 2, or 3; and R.sup.1 represents hydrogen, methyl, ethyl, n-propyl or isopropyl.

2. The compound of claim 1 wherein n represents 1 or 2; and R.sup.1 is hydrogen or methyl.

3. The compound of claim 1 wherein the is a compound of formula (III) ##STR00032##

Description

A. EXAMPLES

Abbreviations

(1) AA amino acid Acm acetamidomethyl ADM adrenomedullin (human) ADM(2-52) Peptide sequence of ADM AA 2 to AA 52, including disulfide bond and C-terminal amide approx. approximately Boc tert-butyloxycarbonyl CDI carbonyldiimidazole d day(s), doublet (in NMR) TLC thin-layer chromatography DCI direct chemical ionization (in MS) dd doublet of doublets (in NMR) DIEA N,N-diisopropylethylamine DMAP 4-dimethylaminopyridine DMF N,N-dimethylformamide DMSO dimethyl sulfoxide of theory of theory (in yield) eq. equivalent(s) ESI electrospray ionization (in MS) Fmoc (9H-fluoren-9-ylmethoxy)carbonyl h hour(s) HATU O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate HPLC high pressure, high performance liquid chromatography LC-MS liquid chromatography-coupled mass spectroscopy m multiplet (in NMR) min minute(s) MS mass spectroscopy NMR nuclear magnetic resonance spectroscopy pbf 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl PEG polyethylene glycol RP reversed phase (in HPLC) RT room temperature R.sub.t retention time (in HPLC) s singulet (in NMR) TBTU benzotriazol-1-yl-N-tetramethyl-uronium tetrafluoroborate tBu tert-butyl TFA trifluoroacetic acid THF tetrahydrofuran Trt trityl

(2) Nomenclature of amino acids and peptide sequences is according to:

(3) International Union of Pure and Applied Chemistry and International Union of Biochemistry: Nomenclature and Symbolism for Amino Acids and Peptides (Recommendations 1983). In: Pure & Appl. Chem. 56, Vol. 5, 1984, p. 595-624

(4) TABLE-US-00001 Trivial Name Symbol One-letter Symbol Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamic acid Glu E Glutamine Gln Q Glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V

(5) LC-MS and MS Methods

(6) Method 1 (LC-MS):

(7) Instrument type: Waters ACQUITY SQD UPLC System; column: Waters Aequity UPLC HSS T3 1.8 50 mm1 mm; mobile phase A: 1 l water+0.25 ml 99% strength formic acid, mobile phase B: 1 l acetonitrile+0.25 ml 99% strength formic acid; gradient: 0.0 min 90% A.fwdarw.1.2 min 5% A.fwdarw.2.0 min 5% A; oven: 50 C.; flow: 0.40 ml/min; UV-detection: 210-400 nm.

(8) Method 2 (LC-MS):

(9) MS instrument: type: Waters (Micromass) Quattro Micro; HPLC instrument type: Agilent 1100 series; column: Thermo Hypersil GOLD 3 20 mm4 mm; mobile phase A: 1 l water+0.5 ml 50% strength formic acid, mobile phase B: 1 l acetonitrile+0.5 ml 50% strength formic acid; gradient: 0.0 min 100% A.fwdarw.3.0 min 10% A.fwdarw.4.0 min 10% A; oven: 50 C.; flow: 2.0 ml/min; UV-detection: 210 nm.

(10) Method 3 (HPLC):

(11) Instrument type: HP 1200 Series; UV DAD; column: Phenomenex Luna 5 m C5 100 , 150 mm4.6 mm; mobile phase A: 1 l water+0.5 ml 50% strength formic acid, mobile phase B: 1 l acetonitrile+0.5 ml 50% strength formic acid; gradient: 0.0 min 95% A.fwdarw.5 min 5% A; .fwdarw.5.8 min 95% A.fwdarw.6.2 min 95% A; flow rate: 2.5 ml/min; oven: RT; UV detection: 210 nm.

(12) Method 4 (HPLC):

(13) Instrument type: HP 1200 Series; UV DAD; column: Merck Chromolith Fastgradient RP18 50 mm2 mm; mobile phase A: 1 l water+0.5 ml 50% strength formic acid, mobile phase B: 1 l acetonitrile+0.5 ml 50% strength formic acid; gradient: 0.0 min 95% A.fwdarw.2.9 min 5% A.fwdarw.3.2 min 5% A; flow rate: 3 ml/min; oven: RT; UV detection: 210 nm.

(14) Method 5 (DCI MS):

(15) Instrument type: Thermo Fisher-Scientific DSQ; chemical ionization; reactant ammonia gas; source temperature: 200 C.; ionization energy 70 eV.

(16) Method 6 (MALDI MS):

(17) Instrument type Kratos PC-Kompact SEQ V1.2.2 MALDI TOF MS, positive ionization mode, Linear high, Power: 75.

(18) Microwave Synthesizer:

(19) Biotage Emrys Initiator II synthesizer, with variable vial size up to 20 ml reaction volume and Robot 60 sample processor

(20) pH 4 Citrate Buffer:

(21) Fluka No 82566; Citrate buffer pH 4, stabilized with sodium azide composition: citric acid, 0.056 M; sodium azide, 0.05%; sodium chloride, 0.044 M; sodium hydroxide, 0.068 M.

(22) 40 kDa methoxy poly(ethylene glycol) maleimido propionamide (linear 40 k mPEG maleimide); CAS No 724722-89-8; From Dr. Reddys Inc., Lot No 233101301; Weight average molecular weight, Mw (GPC) 40500 Da; Polydispersity (GPC) 1.08.

(23) Starting Compounds

Example 1A

Allyl-N-(tert-butoxycarbonyl)-O-[(4-nitrophenoxy)carbonyl]-L-tyrosinate

(24) ##STR00011##

(25) 36.7 g (114.3 mmol) N-Boc-L-tyrosine allyl ester, 23.0 g (114.3 mmol) 4-nitrophenyl chloroformate, 17.5 ml (125.7 mmol) triethylamine and 1.40 g (11.4 mmol) 4-dimethylamino pyridine were combined in 1000 ml dichloromethane and stirred at room temperature for 2 h. The reaction mixture was extracted with approx. 500 ml water and with approx. 250 ml brine and dried over approx. 100 g sodium sulfate. The solvent was removed by rotary evaporation (approx. 40 C., approx. 200 mbar, approx. 30 min.) and the product was dissolved in warm diethyl ether and crystallized over night at 4 C. The crystals were filtered of, washed with cold diethyl ether and dried in high vacuum (approx. 0.1 mbar, 18 h). The yield was 29.86 g, (59.6 mmol, 52% of theory) of the desired product.

(26) LC-MS (method 1): R.sub.t=1.23 min., m/z=487 (M+H).sup.+

Example 2A

(2S)-4-{[(4-{(2S)-3-(Allyloxy)-2-[(tert-butoxycarbonyl)amino]-3-oxopropyl}phenoxy)carbonyl]-amino}-2-[(tert-butoxycarbonyl)amino]butanoic acid

(27) ##STR00012##

(28) 4.0 g (8.22 mmol) of the compound from example 1A was dissolved in 60 ml dichloromethane. 1.795 (8.22 mmol) (2S)-4-Amino-2-[(tert-butoxycarbonyl)amino]butanoic acid and 1.43 ml (8.22 mmol) N,N-diisopropylethylamine were added. The reaction mixture was split into 3 portions. The portions were heated for 30 min in a sealed tube at 75 C. in a microwave synthesizer. From the combined reaction mixture the solvent was removed by rotary evaporation (approx. 40 C., approx. 200 mbar, approx. 30 min.). The raw product was dissolved in dichloromethane and chromatographed over approx. 600 ml silica gel. Solvents used were dichloromethane/ethyl acetate 4/1, dichloromethane/ethyl acetate 1/1, dichloromethane/methanol 4/1 and dichloromethane/methanol 1/1. The product-containing fractions were combined and concentrated to dryness under reduced pressure. This gave 4.02 g (6.54 mmol, 80% of theory) of the desired product.

(29) LC-MS (method 1): R.sub.t=1.07 min., m/z=564 (MH).sup.

Example 3A

Allyl O-({(3S)-4-{[(2R)-1-amino-1-oxo-3-(tritylsulfanyl)propan-2-yl]amino}-3-[(tert-butoxy-carbonyl)amino]-4-oxobutyl}carbamoyl)-N-(tert-butoxycarbonyl)-L-tyrosinate

(30) ##STR00013##

(31) 2.50 g (4.42 mmol) of the compound from example 2A was dissolved in 100 ml dichloromethane. 1.602 g (4.42 mmol) S-Trityl-L-cysteinamide, 0.77 ml (4.42 mmol) N,N-diisopropylethylamine and 1.68 g (4.42 mmol) HATU were added. The reaction mixture was split into 5 portions. The portions were heated for 30 min in a sealed tube at 60 C. in a microwave synthesizer. From the combined reaction mixture the solvent was removed by rotary evaporation (approx. 40 C., approx. 200 mbar, approx. 30 min.). The raw product was dissolved in dichloromethane and chromatographed over approx. 600 ml silica gel. Solvents used were dichloromethane/ethyl acetate 2/1, dichloromethane/ethyl acetate 1/1, dichloromethane/methanol 20/1 and dichloromethane/methanol 10/1. The product-containing fractions were combined and concentrated to dryness under reduced pressure. This gave 4.12 g (3.30 mmol, 75% of theory, 73% purity) of the desired product.

(32) LC-MS (method 1): R.sub.t=1.36 min., m/z=911 (M+H).sup.+

Example 4A

O-({(3S)-4-{[(2R)-1-Amino-1-oxo-3-(tritylsulfanyl)propan-2-yl]amino}-3-[(tert-butoxycarbonyl)-amino]-4-oxobutyl}carbamoyl)-N-(tert-butoxycarbonyl)-L-tyrosine

(33) ##STR00014##

(34) 4.14 g (4.55 mmol) of the compound from example 3A was dissolved in 90 ml tetrahydrofuran. 3.17 ml (22.8 mmol) triethylamine, 0.86 ml (22.8 mmol) formic acid and 0.526 g (0.455 mmol) tetrakis(triphenylphosphin)palladium(0) were added. The reaction mixture was stirred over night at room temperature. The reaction was diluted with approx. 100 ml water, and twice extracted with approx. 100 ml dichloromethane. The combined organic phases were extracted with brine, dried over sodium sulfate and concentrated to dryness under reduced pressure. The raw product was dissolved in dichloromethane and chromatographed over approx. 500 ml silica gel. Solvents used were dichloromethane, dichloromethane/methanol 20/1 and dichloromethane/methanol 1/1. The product-containing fractions were combined and concentrated to dryness under reduced pressure. This gave 2.62 g raw product of 94.5% purity. The product was further purified by preparative RP-HPLC on a C18 with a water/methanol gradient to yield 2.35 g (2.70 mmol, 59% of theory) pure product.

(35) LC-MS (method 1): R.sub.t=1.22 min., m/z=871 (M+H).sup.+

(36) .sup.1H-NMR (400 MHz, DMSO-d.sub.6, /ppm): =7.92 (d, 1H), 7.65 (t, 1H), 7.28-7.35 (m, 12H), 7.25-7.28 (t, 3H), 7.15-7.20 (m, 4H), 6.95 (d, 2H), 4.29 (q, 1H), 4.00 (m, 1H), 3.92 (m, 1H), 3.11 (m, 3H), 2.90 (m, 1H), 2.36 (m, 2H), 1.84 (m, 1H), 1.68 (m, 1H), 1.34 (d, 18H).

Example 5A

tert-Butyl-methyl(2-oxotetrahydrofuran-3-yl)carbamate

(37) ##STR00015##

(38) The compound was synthesized according to Alberico, Dino; Paquin, Jean-Francois; Lautens, Mark; Tetrahedron, 2005, vol. 61, p. 6283-6297.

(39) 5.18 g (25.7 mmol) tert-Butyl(tetrahydro-2-oxo-3-furanyl)carbamate, 4.81 ml (77.2 mmol) iodomethane were dissolved in 100 ml of dry dimethyl fomamide. The solution was cooled to 0 C. and 1.34 g (60% in mineral oil, 33.5 mmol) sodium hydride was added. The reaction was warmed to room temperature and stirred over night. The reaction mixture was added to approx. 400 ml water and the mixture was extracted three times with approx. 300 ml ethyl acetate. The combined organic phases were dried over sodium sulfate and concentrated to dryness under reduced pressure. This gave 8.70 g (25.7 mmol, 100% of theory, 63% purity) of the desired product.

(40) The analytic data was in accordance with the literature. The product was used in the next synthetic step without further purification.

Example 6A

2-[(tert-Butoxycarbonyl)(methyl)amino]-4-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)butanoic acid

(41) ##STR00016##

(42) 8.70 g (approx. 25 mmol, approx. 63% purity) of the compound from example 5A was dissolved in 560 ml dimethyl formamide. 8.23 g (44.4 mmol) potassium ophtalimide were added and the reaction mixture was heated to 150 C. for 7 h. Approx. 400 ml of the solvent was removed by rotary evaporation (approx. 60 C., approx. 10 mbar, approx. 30 min.). The reaction mixture was poured onto a mixture of approx. 100 ml water, 200 g ice and 15 ml acetic acid. After melting of the remaining ice the reaction mixture was filtered and the filtrate was extracted 3 times with approx. 100 ml dichloromethane. The combined organic phases were dried over sodium sulfate and concentrated to dryness under reduced pressure. The raw product was dissolved in dichloromethane and chromatographed over approx. 70 ml silica gel. Solvents used were dichloromethane/ethyl acetate 9/1 to dichloromethane/ethyl acetate 6/4. The product-containing fractions were combined and concentrated to dryness under reduced pressure. This gave 2.39 g (6.04 mmol, 24% of theory) product.

(43) LC-MS (method 1): R.sub.t=0.92 min., m/z=363 (M+H).sup.+

Example 7A

4-Amino-2-[(tert-butoxycarbonyl)(methyl)amino]butanoic acid

(44) ##STR00017##

(45) 11.8 g (32.6 mmol) of the compound from example 6A was dissolved in approx. 640 ml ethanol and 23.8 ml (488 mmol) hydrazine hydrate was added to the reaction mixture. After stirring over night, the reaction mixture was filtered and the filtrate was concentrated to dryness under reduced pressure. The raw product was dissolved in ethanol and approx. 50 g silica gel was added, the solvent was removed under reduced pressure. The resulting solid was added onto a approx. 500 g silica gel column and chromatographed. Solvents used were dichloromethane/methanol 9/1 to dichloromethane/methanol 1/1. The product-containing fractions were combined and concentrated to dryness under reduced pressure. This gave 2.98 g (12.8 mmol, 39% of theory) product.

(46) LC-MS (method 2): R.sub.t=0.21 min., m/z=233 (M+H).sup.+

(47) DCI MS (method 5): m/z=233 (M+H).sup.+

Example 8A

4-{[(4-{(2S)-3-(Allyloxy)-2-[(tert-butoxycarbonyl)amino]-3-oxopropyl}phenoxy)carbonyl]-amino}-2-[(tert-butoxycarbonyl)(methyl)amino]butanoic acid

(48) ##STR00018##

(49) 0.931 g (1.92 mmol) of the compound from example 1A was dissolved in 30 ml dichloromethane. 0.455 g (1.92 mmol) of the compound from example 7A was added. The reaction mixture was split into 2 portions. The portions were heated for 30 min in a sealed tube at 80 C. in a microwave synthesizer. From the combined reaction mixture the solvent was removed under reduced pressure. The raw product was purified by preparative RP-HPLC on a C18 column with a water methanol gradient from 9/1 to 1/9. The product-containing fractions were combined and concentrated to dryness under reduced pressure. This gave 0.523 g (0.85 mmol, 44% of theory) of the desired product as a mixture of 2 diastereomers.

(50) LC-MS (method 1): R.sub.t=1.08 and 1.11 min., m/z=578 (MH).sup.

Example 9A

Allyl O-[(4-{[(2R)-1-amino-1-oxo-3-(tritylsulfanyl)propan-2-yl]amino}-3-[(tert-butoxycarbonyl)-(methyl)amino]-4-oxobutyl)carbamoyl]-N-(tert-butoxycarbonyl)-L-tyrosinate

(51) ##STR00019##

(52) 2.24 g (3.86 mmol) of the compound from example 8A was dissolved in 100 ml dichloromethane. 1.401 g (3.86 mmol) S-Trityl-L-cysteinamide, 0.67 ml (3.86 mmol) N,N-diisopropylethylamine and 1.47 g (3.86 mmol) HATU were added. The reaction mixture was split into 5 portions. The portions were heated for 30 min in a sealed tube at 60 C. in a microwave synthesizer. From the combined reaction mixture the solvent was removed by rotary evaporation (approx. 40 C., approx. 200 mbar, approx. 30 min.). The raw product was purified by preparative RP-HPLC on a C18 column with a water methanol gradient from 9/1 to 1/9. The product-containing fractions were combined and concentrated to dryness under reduced pressure. This gave 3.26 g (2.75 mmol, 71% of theory, 78% purity) of the desired product as a mixture of diastereomers.

(53) LC-MS (method 1): R.sub.t=1.41 and 1.43 min., m/z=924 (M+H)+

Example 10A

O-[(4-{[(2R)-1-Amino-1-oxo-3-(tritylsulfanyl)propan-2-yl]amino}-3-[(tert-butoxycarbonyl)-(methyl)amino]-4-oxobutyl)carbamoyl]-N-(tert-butoxycarbonyl)-L-tyrosine

(54) ##STR00020##

(55) 2.2 g (2.38 mmol) of the compound from example 9A was dissolved in 48 ml tetrahydrofuran. 1.66 ml (11.9 mmol) triethylamine, 0.45 ml (11.9 mmol) formic acid and 0.275 g (0.238 mmol) tetrakis(triphenylphosphin)palladium(0) were added. The reaction mixture was stirred over night at room temperature. The reaction was diluted with approx. 50 ml water and twice extracted with approx. 50 ml dichloromethane. The combined organic phases were extracted with brine, dried over sodium sulfate and concentrated to dryness under reduced pressure. The raw product was dissolved in dichloromethane and chromatographed over approx. 100 g silica gel. Solvents used were dichloromethane, dichloromethane/methanol 50/1 and dichloromethane/methanol 4/1. The product-containing fractions were combined and concentrated to dryness under reduced pressure. This gave 1.44 g (1.61 mmol, 68% of theory) product as a mixture of diastereomers.

(56) LC-MS (method 1): R.sub.t=1.20 and 1.24 min., m/z=884 (M+H).sup.+

(57) .sup.1H-NMR (400 MHz, DMSO-d.sub.6, /ppm): =8.00 (m, 1H), 7.65-7.90 (m, 4H), 7.18-7.35 (m, 18H), 7.10 (m, 2H), 6.96 (m, 4H), 4.60 (m, 1H), 4.46 (m, 1H), 4.30 (m, 2H), 4.05 (m, 2H), 3.00 (m, 4H), 2.75 (m, 6H), 2.36 (m, 3H), 2.00 (m, 2H), 1.82 (m, 2H), 1.40 (m, 3H), 1.35 (s, 18H).

Example 11A

N5-[(4-{(2S)-3-(Allyloxy)-2-[(tert-butoxycarbonyl)amino]-3-oxopropyl}phenoxy)carbonyl]-N2-(tert-butoxycarbonyl)-L-ornithine

(58) ##STR00021##

(59) 6.00 g (12.33 mmol) of the compound from example 1A was dissolved in 120 ml dichloromethane. 2.57 g (12.33 mmol) N.sup.2-(tert-Butoxycarbonyl)-L-ornithine was added. The reaction mixture was split into 6 portions. The portions were heated for 90 min in a sealed tube at 75 C. in a microwave synthesizer. The combined reaction mixture was extracted with approx. 100 ml saturated ammonium chloride solution. The aqueous phase was twice back extracted with approx. 30 ml dichloromethane each. The combined organic phases were extracted with approx. 50 ml brine and dried over sodium sulfate. The solvent was removed under reduced pressure. The raw product was dissolved in dichloromethane and chromatographed over approx. 600 ml silica gel. Solvents used were dichloromethane, dichloromethane/methanol 40/1 to dichloromethane/methanol 1/1. The product-containing fractions were combined and concentrated to dryness under reduced pressure. This gave 2.63 g (4.06 mmol, 33% of theory, 89% purity) of the desired product.

(60) LC-MS (method 1): R.sub.t=1.03 min., m/z=578 (MH).sup.

Example 12A

N5-[(4-{(2S)-3-(Allyloxy)-2-[(tert-butoxycarbonyl)amino]-3-oxopropyl}phenoxy)carbonyl]-N2-(tert-butoxycarbonyl)-L-ornithyl-S-trityl-L-cysteinamide

(61) ##STR00022##

(62) 1.20 g (2.07 mmol) of the compound from example 11A was dissolved in 48 ml dichloromethane. 0.750 g (2.07 mmol) S-Trityl-L-cysteinamide, 0.36 ml (2.07 mmol) N,N-diisopropylethylamine and 0.787 g (2.07 mmol) HATU were added. The reaction mixture was split into 3 portions. The portions were heated for 30 min in a sealed tube at 60 C. in a microwave synthesizer. From the combined reaction mixture the solvent was removed by rotary evaporation (approx. 40 C., approx. 200 mbar, approx. 30 min.). The raw product was dissolved in dichloromethane and chromatographed over approx. 400 ml silica gel. Solvents used were dichloromethane/ethyl acetate 2/1, dichloromethane/ethyl acetate 1/1. The product-containing fractions were combined and concentrated to dryness under reduced pressure. This gave 1.30 g (1.5 mmol, 56% of theory, 82% purity) of the desired product.

(63) LC-MS (method 1): R.sub.t=1.35 min., m/z=924 (M+H).sup.+

Example 13A

N2-(tert-Butoxycarbonyl)-N5-[(4-{(2S)-2-[(tert-butoxycarbonyl)amino]-2-carboxyethyl}phenoxy)-carbonyl]-L-ornithyl-S-trityl-L-cysteinamide

(64) ##STR00023##

(65) 3.06 g (2.33 mmol) of the compound from example 12A was dissolved in 46 ml tetrahydrofuran. 1.63 ml (11.6 mmol) triethylamine, 0.44 ml (11.6 mmol) formic acid and 0.265 g (0.233 mmol) tetrakis(triphenylphosphin)palladium(0) were added. The reaction mixture was stirred over night at room temperature. The reaction was diluted with approx. 50 ml water and twice extracted with approx. 50 ml dichloromethane. The combined organic phases were extracted with brine, dried over sodium sulfate and concentrated to dryness under reduced pressure. The raw product was dissolved in dichloromethane and chromatographed over approx. 500 ml silica gel. Solvents used were dichloromethane, dichloromethane/methanol 40/1 and dichloromethane/methanol 1/1. The product-containing fractions were combined and concentrated to dryness under reduced pressure. This gave 1.40 g raw product of 86% purity. The product was further purified by preparative RP-HPLC on a C18 column with a water/methanol gradient to yield 2 fractions: 0.93 g product (45% of theory).

(66) LC-MS (method 1): R.sub.t=1.18 min., m/z=885 (M+H).sup.+

(67) .sup.1H-NMR (400 MHz, DMSO-d.sub.6, /ppm): =7.89 (d, 1H), 7.65 (t, 1H), 7.25-7.35 (m, 12H), 7.20-7.25 (m, 6H), 7.10-7.20 (m, 3H), 6.95 (d, 2H), 4.29 (m, 1H), 4.05 (m, 1H), 3.88 (m, 1H), 3.11 (d, 1H), 3.00 (m, 4H), 2.75 (m, 2H), 2.36 (m, 3H), 1.64 (m, 1H), 1.51 (m, 3H), 1.36 (s, 9H), 1.32 (s, 9H).

Example 14A

Tentagel Based Amide Resin Bound ADM (2-52)

(68) ##STR00024##

(69) The peptide was assembled stepwise on a Tentagel based amide resin on an automated peptide synthesizer (Protein Technologies Inc. Symphony). 8 poly-propylene reaction vessels were used in parallel performing the identical chemistry. Each vessel was loaded with 0.05 mmol Tentagel based Rink resin for a total batch size of 0.4 mmol.

(70) Each amino acid is added in 8 fold molar access with regard to the loading of the resin. The amino acids were Fmoc protected as the N-terminal protecting group and the protecting groups indicated below were used for side chain functionalities. Also 188 mg (0.59 mmol, 7.8 eq.) TBTU and 0.21 ml (1.2 mmol, 16 eq.) DIEA were added. Reactions were performed in DMF as solvent, whereas DMF was used in an amount sufficient to swell the resin and agitate it freely. Reaction time per amino acid was approx. 1 hour. Cleavage of the Fmoc protecting groups was achieved using 20% piperidine/DMF, whereas 20% piperidine/DMF was used in an amount sufficient to swell the resin and agitate it freely.

(71) The coupling sequence was as follows: 1. Tyr(tBu) (Tyr=Y=AA 52 of human ADM) 2. Gly (Gly=G=AA 51 of human ADM) 3. Gln(Trt) (Gln=Q=AA 50 of human ADM) 4. Pro (Pro=P=AA 49 of human ADM) 5. Ser(tBu) (Ser=S=AA 48 of human ADM) 6. Ile (Ile=I=AA 47 of human ADM) 7. Lys(Boc) (Lys=K=AA 46 of human ADM) 8. Ser(tBu) (Ser=S=AA 45 of human ADM) 9. Arg(pbf) (Arg=R=AA 44 of human ADM) 10. Pro (Pro=P=AA 43 of human ADM) 11. Ala (Ala=A=AA 42 of human ADM) 12. Val (Val=V=AA 41 of human ADM) 13. Asn(Trt) (Asn=N=AA 40 of human ADM) 14. Asp(OtBu) (Asp=D=AA 39 of human ADM) 15. Lys(Boc) (Lys=K=AA 38 of human ADM) 16. Asp(OtBu) (Asp=D=AA 37 of human ADM) 17. Lys(Boc) (Lys=K=AA 36 of human ADM) 18. Asp(OtBu) (Asp=D=AA 35 of human ADM) 19. Thr(tBu) (Thr=T=AA 34 of human ADM) 20. Phe (Phe=F=AA 33 of human ADM) 21. Gn(Trt) (Gn=Q=AA 32 of human ADM) 22. Tyr(tBu) (Tyr=Y=AA 31 of human ADM) 23. Ile (Ile=I=AA 30 of human ADM) 24. Gln(Trt) (Gln=Q=AA 29 of human ADM) 25. His(Trt) (His=H=AA 28 of human ADM) 26. Ala (Ala=A=AA 27 of human ADM) 27. Leu (Leu=L=AA 26 of human ADM) 28. Lys(Boc) (Lys=K=AA 25 of human ADM) 29. Gln(Trt) (Gln=Q=AA 24 of human ADM) 30. Val (Val=V=AA 23 of human ADM) 31. Thr(tBu) (Thr=T=AA 22 of human ADM) 32. Cys(Trt) (Cys=C=AA 21 of human ADM) 33. Thr(tBu) (Thr=T=AA 20 of human ADM) 34. Gly (Gly=G=AA 19 of human ADM) 35. Phe (Phe=F=AA 18 of human ADM) 36. Arg(pbf) (Arg=R=AA 17 of human ADM) 37. Cys(Acm) (Cys=C=AA 16 of human ADM) 38. Gly (Gly=G=AA 15 of human ADM) 39. Phe (Phe=F=AA 14 of human ADM) 40. Ser(tBu) (Ser=S=AA 13 of human ADM) 41. Arg(pbf) (Arg=R=AA 12 of human ADM) 42. Leu (Leu=L=AA 11 of human ADM) 43. Gly (Gly=G=AA 10 of human ADM) 44. Gln(Trt) (Gln=Q=AA 9 of human ADM) 45. Phe (Phe=F=AA 8 of human ADM) 46. Asn(Trt) (Asn=N=AA 7 of human ADM) 47. Asn(Trt) (Asn=N=AA 6 of human ADM) 48. Met (Met=M=AA 5 of human ADM) 49. Ser(tBu) (Ser=S=AA 4 of human ADM) 50. Gln(Trt) (Gln=Q=AA 3 of human ADM) 51. Arg(pbf) (Arg=R=AA 2 of human ADM)

(72) On-resin oxidation was achieved using Cys(Trt) and Cys(Acm) protection with concomitant cleavage of protecting groups and oxidation to a disulfide bond using Iodine (8 equivalents of Iodine plus 8 equivalents of DIEA with a reaction time of 30 minutes). Oxidation was confirmed by sample cleavage and analysis using HPLC and MALDI-MS.

(73) The 8 batches were pooled for further use.

Example 15A

O-{[(3 S)-3-Amino-4-{[(2R)-1-amino-1-oxo-3-sulfanylpropan-2-yl]amino}-4-oxobutyl]-carbamoyl}-L-tyrosyl-adrenomedullin(2-52)

(74) ##STR00025##

(75) To 0.075 mmol of the compound of example 14A 520 mg (0.6 mmol, 8 eq.) of the compound of example 4A were added. Also 188 mg (0.59 mmol, 7.8 eq.) TBTU and 0.21 ml (1.2 mmol, 16 eq.) DIEA were added. The reaction was performed with DMF as solvent, whereas DMF was used in an amount sufficient to swell the resin and agitate it freely. Reaction time was approx. 1 hour at room temperature. The peptide was cleaved from the resin with concomitant global deprotection using concentrated TFA in an amount sufficient to swell the resin and agitate it freely, whereas TFA contains scavengers (1-5% each of water, phenol, thioanisole and 1,2-ethanediol), with a reaction time of 2 hrs. The crude product was lyophilised and purified by RP-chromatography using 0.1% TFA in water and 0.1% TFA in acetonitrile as mobile phases to ensure that the pH remains below 4 at all times during the purification and lyophilisation process. All fractions containing the correct ion by MALDI-MS analysis were pooled. The yield was 44.0 mg of partially purified peptide (approx. 0.0035 mmol, approx. 4.7% of theory; estimated purity: approx. 50%, main impurity: ADM (2-52)).

(76) MALDI MS (method 6): m/z=6275 (M+H).sup.+ and 5866 (impurity: (ADM(2-52)+H).sup.+)

Example 16A

O-{[4-{[(2R)-1-Amino-1-oxo-3-sulfanylpropan-2-yl]amino}-3-(methylamino)-4-oxobutyl]-carbamoyl}-L-tyrosyl-adrenomedullin(2-52)

(77) ##STR00026##

(78) To 0.075 mmol of the compound of example 14A 530 mg (0.6 mmol, 8 eq.) of the compound of example 10A were added. Also 188 mg (0.59 mmol, 7.8 eq.) TBTU and 0.21 ml (1.2 mmol, 16 eq.) DIEA were added. The reaction was performed with DMF as solvent, whereas DMF was used in an amount sufficient to swell the resin and agitate it freely. Reaction time was approx. 1 hour at room temperature. The peptide was cleaved from the resin with concomitant global deprotection using concentrated TFA in an amount sufficient to swell the resin and agitate it freely, whereas TFA contains scavengers (1-5% each of water, phenol, thioanisole and 1,2-ethanediol), with a reaction time of 2 hrs. The crude product was lyophilised and purified by RP-chromatography using 0.1% TFA in water and 0.1% TFA in acetonitrile as mobile phases to ensure that the pH remains below 4 at all times during the purification and lyophilisation process. All fractions containing the correct ion by MALDI-MS analysis were pooled. The yield was 34.0 mg of partially purified peptide (approx. 0.0026 mmol, approx. 3.5% of theory; estimated purity: approx. 50%, main impurity: ADM (2-52)).

(79) MALDI MS (method 6): m/z=6289 (M+H).sup.+ and 5866 (impurity: (ADM(2-52)+H).sup.+)

Example 17A

O-{[(4R)-4-Amino-5-{[(2R)-1-amino-1-oxo-3-sulfanylpropan-2-yl]amino}-5-oxopentyl]-carbamoyl}-L-tyrosyl-adrenomedullin(2-52)

(80) ##STR00027##

(81) To 0.075 mmol of the compound of example 14A 530 mg (0.6 mmol, 8 eq.) of the compound of example 13A were added. Also 188 mg (0.59 mmol, 7.8 eq.) TBTU and 0.21 ml (1.2 mmol, 16 eq.) DIEA were added. The reaction was performed with DMF as solvent, whereas DMF was used in an amount sufficient to swell the resin and agitate it freely. Reaction time was approx. 1 hour at room temperature. The peptide was cleaved from the resin with concomitant global deprotection using concentrated TFA in an amount sufficient to swell the resin and agitate it freely, whereas TFA contains scavengers (1-5% each of water, phenol, thioanisole and 1,2-ethanediol), with a reaction time of 2 hrs. The crude product was lyophilised and purified by RP-chromatography using 0.1% TFA in water and 0.1% TFA in acetonitrile as mobile phases to ensure that the pH remains below 4 at all times during the purification and lyophilisation process. All fractions containing the correct ion by MALDI-MS analysis were pooled. The yield was 47 mg of partially purified peptide (approx. 0.0037 mmol, approx. 5.0% of theory; estimated purity: approx. 50%, main impurity: ADM (2-52)).

(82) MALDI MS (method 6): m/z=6289 (M+H).sup.+ and 5866 (impurity: (ADM(2-52)+H).sup.+)

Working Examples

Example 1

O-{[(3 S)-3-Amino-4-({(2R)-1-amino-3-[(2,5-dioxo-1-{3-oxo-3-[(2-{-methoxy-poly-oxyethylen[40 kDa]}ethyl)amino]propyl}pyrrolidin-3-yl)sulfanyl]-1-oxopropan-2-yl}amino)-4-oxobutyl]carbamoyl}-L-tyrosyl-adrenomedullin(2-52)

(83) ##STR00028##

(84) 44 mg of the crude peptide of example 15A were stirred with 426 mg (10.5 mol, 1.5 eq, sourced from Dr. Reddys) 40 kDa methoxy poly(ethylene glycol) maleimido propionamide in 9 ml citrate buffer of pH 4 over night at room temperature. The crude reaction mixture was injected in two portions onto a preparative HPLC system with a Phenomenex Luna 10 Proteo C5 100 A AXIA 250 mm21.2 mm column and chromatographed with a water/acetonitrile (both with 0.1% TFA) gradient. The fractions were collected in test tubes of 20 ml on an automated fraction collector. To ensure sufficient acidity each vial was filled with 0.5 ml acetic acid prior to collection.

(85) ADM(2-52), which is the side product of example 15A and which did not undergo PEGylation in this reaction, as well as unreacted PEG were removed completely.

(86) All fractions containing example 1 were combined. Acetonitrile was partially removed on a rotary evaporator at 30 C. water bath temperature and approx. 50 mbar for approx. 30 min.

(87) After addition of 0.5 ml acetic acid, the remaining solution was lyophilized. The total yield of example 1 was 109 mg (2.35 mol, 33% of theory).

(88) HPLC (method 3): R.sub.t=4.23-4.30 min

Example 2

O-{[(3-N-Methyl-amino-4-({(2R)-1-amino-3-[(2,5-dioxo-1-{3-oxo-3-[(2-{-methoxy-poly-oxyethylen[40 kDa]}ethyl)amino]propyl}pyrrolidin-3-yl)sulfanyl]-1-oxopropan-2-yl}amino)-4-oxobutyl]carbamoyl}-L-tyrosyl-adrenomedullin(2-52)

(89) ##STR00029##

(90) 15 mg of the crude peptide of example 16A were stirred with 145 mg (3.58 mol, 1.5 eq, sourced from Dr. Reddys) 40 kDa methoxy poly(ethylene glycol) maleimido propionamide in 5 ml citrate buffer of pH 4 over night at room temperature. The crude reaction mixture was injected onto a preparative HPLC system with a Phenomenex Jupiter 10 C18 300 A 250 mm21.2 mm column and chromatographed with a water/acetonitrile (both with 0.1% TFA) gradient. The fractions were collected in test tubes of 20 ml on an automated fraction collector. To ensure sufficient acidity each vial was filled with 0.5 ml acetic acid prior to collection.

(91) ADM(2-52), which is the side product of example 16A and which did not undergo PEGylation in this reaction, as well as unreacted PEG were removed completely.

(92) All fractions containing example 2 were combined. Acetonitrile was partially removed on a rotary evaporator at 30 C. water bath temperature and approx. 50 mbar for approx. 30 min.

(93) After addition of 0.5 ml acetic acid, the remaining solution was lyophilized. The total yield of example 2 was 50 mg (1.08 mol, 43% of theory).

(94) HPLC (method 4): R.sub.t=2.02-2.08 min

Example 3

O-{[(4S)-4-Amino-5-({(2R)-1-amino-3-[(2,5-dioxo-1-{3-oxo-3-[(2-{-methoxy-poly-oxyethylen[40 kDa]}ethyl)amino]propyl}pyrrolidin-3-yl)sulfanyl]-1-oxopropan-2-yl}amino)-5-oxopentyl]carbamoyl}-L-tyrosyl-adrenomedullin(2-52)

(95) ##STR00030##

(96) 15 mg of the crude peptide of example 17A were stirred with 145 mg (3.58 mol, 1.5 eq, sourced from Dr. Reddys) 40 kDa methoxy poly(ethylene glycol) maleimido propionamide in 5 ml citrate buffer of pH 4 over night at room temperature. The crude reaction mixture was injected onto a preparative HPLC system with a Phenomenex Jupiter 10 Proteo 90A AXIA 250 mm21.2 mm column and chromatographed with a water/acetonitrile (both with 0.1% TFA) gradient. The fractions were collected in test tubes of 20 ml on an automated fraction collector. To ensure sufficient acidity each vial was filled with 0.5 ml acetic acid prior to collection.

(97) ADM(2-52), which is the side product of example 17A and which did not undergo PEGylation in this reaction, as well as unreacted PEG were removed completely.

(98) All fractions containing example 3 were combined. Acetonitrile was partially removed on a rotary evaporator at 30 C. water bath temperature and approx. 50 mbar for approx. 30 min.

(99) After addition of 0.5 ml acetic acid, the remaining solution was lyophilized. The total yield of example 3 was 19.5 mg (0.42 mol, 17% of theory).

(100) HPLC (method 4): R.sub.t-2.02-2.08 min

B. ASSESSMENT OF PHARMACOLOGICAL ACTIVITY

(101) The suitability of the compounds according to the invention for treatment of diseases can be demonstrated using the following assay systems:

(102) 1) Test Descriptions (In Vitro)

(103) 1a) Tests on a Recombinant Adrenomedullin-Receptor Reporter Cell

(104) The activity of the compounds according to the invention is quantified with the aid of a recombinant Chinese hamster ovary (CHO) cell line that carries the human adrenomedullin-receptor. Activation of the receptor by ligands can be measured by aequorin luminescence. Construction of the cell line and measurement procedure has been described in detail [Wunder F., Rebmann A., Geerts A, and Kalthof B., Mol Pharmacol, 73, 1235-1243 (2008)]. In brief: Cells are seeded on opaque 384-well microtiter plates at a density of 4000 cells/well and are grown for 24 b. After removal of culture medium, cells are loaded for 3 h with 0.6 g/ml coelenterazine in Ca.sup.2+-free Tyrode solution (130 mM sodium chloride, 5 mM potassium chloride, 20 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 1 mM magnesium chloride, and 4.8 mM sodium hydrogen carbonate, pH 7.4) supplemented with 0.2 mM 3-Isobutyl-1-methylxanthine (IBMX) in a cell culture incubator. Compounds are added for 6 min in calcium.sup.2+-free Tyrode solution containing 0.1% bovine serum albumin. Immediately before adding calcium.sup.2+ to a final concentration of 3 mM measurement of the aequorin luminescence is started by use of a suitable luminometer. Luminescence is measured for 60 s. In a typical experiment compounds are tested in a concentration range of 110.sup.13 to 310.sup.6 M.

(105) In order to determine the release of active adrenomedullin from compounds according to the invention, compounds are incubated at different concentrations for different time spans up to 24 h in Tyrode solution supplemented with fetal calf serum, cell culture medium or plasma from different species at pH 7.4. Calcium.sup.2+ content of the respective incubation media is buffered by addition of 4 mM EDTA (ethylene diamine tetraacetic acid) before adding samples to the adrenomedullin-receptor reporter cell.

(106) After appropriate preincubation, the embodiment examples activate the adrenomedullin-receptor reporter cell more potently than before preincubation. This is indicated by the fact that EC.sub.50 values are determined by a factor of up to 10 smaller after preincubation than before and is explainable by the release of active adrenomedullin from the compounds.

(107) Representative EC.sub.50 values for the embodiment examples before and after incubation for 24 h in buffer supplemented with 2.5% fetal calf serum are given in the following Table 1:

(108) TABLE-US-00002 TABLE 1 Example no. EC.sub.50 T = 0 h [nM] EC.sub.50 T = 24 h [nM] ADM 0.5 2.5 1 110 8.4 2 >1000 161 3 124 12.3

(109) 1b) Transcellular Electrical Resistance Assays in Endothelial Cells

(110) The activity of the compounds according to the invention is characterized in in vitro-permeability assays in human umbilical venous cells (HUVEC, Lonza). By use of the ECIS apparatus (ECIS: Electric Cell-substrate Impedance Sensing; Applied Biophysics Inc; Troy, N.Y.) changes of transendothelial electrical resistance (TEER) over an endothelial monolayer are continuously measured by use of a small gold electrode on which the cells have been seeded. HUVEC are grown on the 96-well sensor electrode plates (96W1E, Ibidi GmbH, Martinsried) to confluent monolayers and hyperpermeability can be induced by inflammatory stimuli such as Thrombin, TNF-, IL-1, VEGF, Histamine and hydrogen peroxide which all have been demonstrated to cause break down of endothelial cell contacts and reduction of TEER. Thrombin is used at a final concentration of 0.5 U/ml. Test compounds are added before or after addition of thrombin. In a typical experiment compounds are tested in a concentration range of 110.sup.10 to 110.sup.6 M.

(111) The embodiment examples inhibit the thrombin induced hyperpermeability in this test at concentrations of 10.sup.6 M.

(112) 1c) In Vitro-Permeability Assays in Endothelial Cells

(113) In another in vitro model of endothelial hyperpermeability the activity of compounds according to the invention is examined with respect to modulation of macromolecular permeability. Human umbilical vein endothelial cells (HUVECS) are grown to confluency on fibronectin-coated Transwell filter membranes (24-well plates, 6.5 mm-inserts with 0.4 M polycarbonate membrane; Costar #3413) which separate an upper from a lower tissue culture chamber with endothelial cells growing on the bottom of the upper chamber. The medium of the upper chamber is supplemented with 250 g/ml of 40 kDa FITC-Dextran (Invitrogen, D1844). Hyperpermeability of the monolayer is induced by addition of thrombin to a final concentration of 0.5 U/ml. Medium samples are collected from the lower chamber every 30 min and relative fluorescence as a parameter for changes of macromolecular permeability over time is measured in a suitable fluorimeter. Thrombin challenge induces almost a doubling of FITC-dextran transition across the endothelial monolayers. In a typical experiment compounds are tested in a concentration range of 110.sup.10 to 110.sup.6 M.

(114) The embodiment examples inhibit the thrombin induced hyperpermeability in this test at concentrations of 10.sup.6 M.

(115) 2. Test Descriptions (In Vivo)

(116) 2a) Measurement of Blood Pressure and Heart Rate in Telemetered, Normotensive Wistar Rats

(117) The cardiovascular effects induced by compounds according to the invention are investigated in freely moving conscious female Wistar rats (body weight>200 g) by radiotelemetric measurement of blood pressure and heart rate. Briefly, the telemetric system (DSI Data Science International, MN, USA) is composed on 3 basic elements: implantable transmitters (TA11PA-C40), receivers (RA1010) and a computer-based acquisition software (Dataquest A.R.T. 4.1 for Windows). Rats are instrumented with pressure implants for chronic use at least 14 days prior to the experiments. The sensor catheter is tied with 4-0 suture several times to produce a stopper 0.5 cm from the tip of the catheter. During catheter implantation rats are anesthetized with pentobabital (Nembutal, Sanofi: 50 mg/kg i.p.). After shaving the abdominal skin, a midline abdominal incision is made, and the fluid-filled sensor catheter is inserted upstream into the exposed descending aorta between the iliac bifurcation and the renal arteries. The catheter is tied several times at the stopper. The tip of the telemetric catheter is located just caudal to the renal arteries and secured by tissue adhesive. The transmitter body is affixed to the inner peritoneal wall before closure of abdomen. A two-layer closure of the abdominal incision is used, with individual suturing of the peritoneum and the muscle wall followed by closure of the outer skin. For postsurgical protection against infections and pain a single dosage of an antibiotic (Oxytetracyclin 10% R, 5.0 ml/kg s.c., beta-pharma GmbH&Co, Germany) and analgesic is injected (Rimadyl R, 5.0 ml/kg s.c., Pfizer, Germany). The hardware configuration is equipped for 24 animals. Each rat cage is positioned on top of an individual receiver platform. After activation of the implanted transmitters, an on-line data acquisition system, samples data and converts telemetric pressure signals to mm Hg. A barometric pressure reference allows for relation of absolute pressure (relative to vacuum) to ambient atmospheric pressure. Data acquisition software is predefined to sample hemodynamic data for 10-intervals every 5 minutes. Data collection to file is started 2 hours before administration of test compounds and finished after completion of 24-h cycles. In a typical experiment test compounds are administered as bolus either subcutaneously or intravenously at does of 1 to 1000 g/kg body weight (as referred to the peptide component).

(118) Wild type adrenomedullin (Bachem, H-2932) induces blood pressure reduction in this test with duration of 4 h when tested at doses of 300 j g/kg body weight [FIG. 1].

(119) FIG. 1: 24 hour profiles of mean arterial blood pressure (MABP) recorded from telemeterd normotensive female Wistar rats after subcutaneous administration of ADM or vehicle as indicated (dotted line). Data points were plotted as means f SEM of averaged 30 min intervals from 4 animals per group. One hour after administration animals treated with ADM showed a mean reduction of MABP of almost 20% at peak (filled circles). After about 3.5 hours MABP had returned to base line levels and was in the range of that of vehicle treated animals (open circles).

(120) In this test substances according to the present invention induce blood pressure reduction of up to 10 h at doses of 500 g/kg body weight (as referred to the peptide component) [FIG. 2].

(121) FIG. 2: 24 hour profiles of mean arterial blood pressure (MABP) recorded from telemeterd normotensive female Wistar rats after subcutaneous administration of example 1 or vehicle as indicated (dotted line). Data points were plotted as meansSEM of averaged 30 min intervals from 6 animals per group. Administration of example 1 at a dose of 150 g/kg (as referred to the peptide component) reduced MABP by about 15 to 19% until 6 h after administration (filled circles). Between 6 h and 14 h after administration MABP gradually returned to baseline values and finally was in the range of that of vehicle treated animals.

(122) 2b) Skin Vascular Leak Assay in Wistar Rats

(123) An intracutaneous histamine challenge test is employed to assess the effect of compounds according to the invention on vascular barrier function in healthy animals. Male Sprague Dawley rats (body weight>200 g) are anesthetized with isoflurane (2%-3% in ambient air) and brought into supine position. The abdomen is shaved and a catheter is inserted into the femoral vein. Vehicle only (0.5 ml PBS+0.1% bovine serum albumin) or test compounds at appropriate doses are administered as i.v. bolus injections. After 15 min a second injection of 100 l/kg 2% Evans blue (Sigma) solution is administered and immediately thereafter 100 l of histamine solutions of appropriate concentrations (for example 0-2.5-5-10-20-40 g/ml) are injected intracutaneously into the abdominal skin. Evans blue is a highly plasma protein bound dye and therefore used as an indicator for protein-rich fluid extravasation and vascular leakage. 30 min after this procedure the rats are sacrificed by an overdose of isoflurane and subsequent neck dislocation and the abdominal skin is excised. The wheals are excised by use of an 8 mm biopsy punch, the tissue samples are weighted and transferred to formamide for 48 h in order to extract the Evans blue. Samples are measured at 620 nM and 750 nM wavelength on a suitable photometer and Evans blue content of the samples is corrected for heme pigments according to the formula A620 (corrected)=A620(1.426A750+0.030) and calculated against a standard curve. [method adapted from Wang L. F., Patel M., Razavi H. M., Weicker S., Joseph M. G., McCormack D. G., Mehta S., Am. Respir Crit Care Med, 165(12), 1634-9 (2002)].

(124) Substances according to the present invention reduce extravasation of protein rich plasma fluid induced by histamine challenge in this test.

(125) 2c) Intra-Tracheal Instillation of LPS in Mice

(126) An intra-tracheal challenge with lipopolysaccharide (LPS) is employed to examine the effects of compounds according to the invention on acute lung injury. Male BALB/c mice (average animal weight 20-23 g) are anesthetized with isoflurane (7%) and LPS from E. coli (e.g. serotype 055:B5; Sigma) is instilled in 100 l saline by use of a micropipette. Typical doses of LPS used for challenge are in the range of 1 to 10 mg/kg body weight. At different time points before and after instillation test compounds are administered by the subcutaneous route. Typical doses are in the range of 1 to 300 g/kg body weight. In this test typical time points of administration of test compounds are 15 min before or 1 h after LPS challenge. 48 hours after instillation of LPS mice are deeply anesthetized with isoflurane and sacrificed by dislocation of the neck. After cannulation of the trachea lavage of the bronchoalveolar space with 0.5 ml ice-cold saline is performed. Lungs are prepared and weighted. Cells in the bronchoalveolar lavage fluid (BALF) are counted on a cell counter (Cell Dyn 3700, Abbott). In this test lung weight as a measure for lung edema is reproducibly found to be increased by about 50% or more over sham controls 48 hours after LPS challenge. As lung weights show only very low variability in the groups, the absolute lung weight is used as parameter. The counts for white blood cells are always found to be significantly increased over control in the BALF after LPS challenge.

(127) Administration of substances according to the present invention resulted in significantly reduced lung weight and white blood cell counts in the BALF after 48 h when administered as bolus at doses300 g/kg body weight (as referred to the peptide component).

(128) 2d) Induction of Acute Lung Injury in Mini Pigs

(129) Acute lung injury is induced in anesthetized mini pigs by use of lipopolysaccharide (LPS) or oleic acid as challenges. In detail: female Gttingen minipigs of ca. 3.5 to 5.5 kg body weight (Ellegaard, Denmark) are kept anesthetized by an continuous i.v.-infusion of Ketavet, Dormicum and Pancuronium after premedication with an intramuscular injection of Ketavet/Stresnil. After intratracheal intubation animals are artificially ventilated using a pediatric respirator (Sulla 808V; Drger, Germany) with an oxygen air mixture at a tidal volume of 30 to 50 ml and constant frequency of 25 min.sup.1. Arterial PaCO.sub.2 is adjusted to about 40 mmHg by regulating the fraction of inspired oxygen (FiO.sub.2) via the ratio of oxygen air mixture. Routinely the following cardiovascular and respiratory parameters are measured after placement of necessary probes and catheters fitted to appropriate pressure transducers and recording equipment: central venous pressure (via left jugular vein), arterial blood pressure and heart rate (BP and HR; via left carotid artery), left ventricular pressure (LVP; using a Millar catheter [FMI, Mod.:SPC-340S, REF: 800-2019-1, 4F] introduced into the left ventricle via right carotid artery), pulmonary arterial pressure (PAP; using ARROW Berman angiographic balloon catheter [REF.: AI-07134 4 Fr. 50 cm] placed into the pulmonary artery via left jugular vein), cardiac output (CO) and extravascular lung water index (EVWLI) by use of the PiCCO system (Pulsion, Germany) connected to a Pulsion 4F Thermodilution-catheter (PV2014L08N) placed into the right femoral artery. Catheters for measurement of CVP, BP, HR, LVP, and PAP are fitted to a Ponemah recording system. Arterial blood gas analysis is performed to determine the PaO.sub.2/FiO.sub.2. According to the American-European Consensus Conference on ARDS a PaO.sub.2/FiO.sub.2<300 mmHg is considered as indicative for the presence of acute lung injury. Dependent on the applied protocol duration of experiments varied between 4 and 5 hours after administration of lung injury inducing challenge. At the end of experimentation pigs are sacrificed by exsanguination and bronchoalveolar lavage fluid (BALF) is collected from lungs. Cellular content of BALF is determined by use of a blood cell counter (Cell DYN 3700).

(130) In a typical setting acute lung injury is induced by intratracheal instillation of Lipopolysaccharide (LPS; E. coli 0111:B4; Sigma L2630) in saline at a dose of 5 mg/kg body weight into each lung via the endotracheal tube. PAP and EVWLI increased while PaO.sub.2/FiO.sub.2 decreased below 300 mmHg in response to the challenge. The cellular content of BALF is significantly increased. Administration of compound 1 of this invention as i.v.-bolus 15 min before the LPS challenge ameliorated or prevented the LPS induced changes.

(131) In an other protocol oleic acid (OA; Sigma-Aldrich, 01008) diluted with ethanol (1:1) is infused i.v. over 15 min at a final dose of 100 mg/kg body weight. Challenge with OA led to increase of PAP and EVLWI and decrease of PaO.sub.2/FiO.sub.2 below 300 mmHg. Changes are ameliorated or prevented by administration of compound 1 of this invention 15 min before start of the OA infusion.

(132) Doses of example 130 g/kg body weight (as referred to the peptide component) were found to be active in the described test systems.

C. EXEMPLARY EMBODIMENTS OF PHARMACEUTICAL COMPOSITIONS

(133) The compounds according to the invention can be converted into pharmaceutical preparations in the following ways:

(134) i.v. Solution:

(135) A compound according to the invention is dissolved at a concentration below saturation solubility in a physiologically acceptable solvent (for example buffers of pH 4 to pH 7, isotonic sodium chloride solution, glucose solution 5% and/or PEG 400 solution 30%). The solution is sterilized by filtration and filled into sterile and pyrogen-free injection containers.

(136) s.c. Solution:

(137) A compound according to the invention is dissolved at a concentration below saturation solubility in a physiologically acceptable solvent (for example for example buffers of pH 4 to pH 7, isotonic sodium chloride solution, glucose solution 5% and/or PEG 400 solution 30%). The solution is sterilized by filtration and filled into sterile and pyrogen-free injection containers.