MODIFIED ANTIBIOTIC PEPTIDES HAVING VARIABLE SYSTEMIC RELEASE

Abstract

The invention relates to modified antibiotic peptides, in particular derivatives of apidaecin and oncocin, preferably having increased stability, reduced immunoreaction, and improved pharmacokinetics. In the invention, the peptide antibiotics are reversibly protected by means of a linker having the polymer polyethylene glycol (PEG). The peptide linker contains a recognition sequence for trypsin-like serum proteases. In the apidaecin derivatives, the linker and the PEG are bonded to a side chain. In the serum, the linker is cut by serum proteases and PEG is separated off. The released peptide still contains remnants of the linker, which are still bonded to the amino group in the side chain. Astonishingly, said remaining remnants of the linker impair the activity of the antimicrobial peptide only a little or not at all.

Claims

1. A modified peptide containing one of the following sequences
NT-X.sub.1X.sub.2NX.sub.3PVYIPX.sub.4X.sub.5RPPHP-CT(formula 1)
NT-X.sub.1NX.sub.2X.sub.3PVYIPX.sub.4X.sub.5RPPHP-CT(formula 2)
NT-X.sub.2NNX.sub.3PVYIPX.sub.4X.sub.5RPPHP-CT(formula 3) wherein X.sub.1 is an amino acid residue whose side chain is positively charged under physiological conditions, preferably O, wherein X.sub.2 is an amino acid residue having an amino group in the side chain, wherein X.sub.3 is an amino acid residue whose side chain is positively charged under physiological conditions, preferably R, wherein X.sub.4 is an amino acid residue whose side chain is positively charged under physiological conditions, preferably R, wherein X.sub.5 is proline or a proline derivative, wherein CT is the C-terminus or a peptide having from 1 to 4 amino acid residues, preferably a dipeptide having the sequence RL, wherein NT is the N-terminus, which is preferably guanidated, and wherein a linear or branched polyethylene glycol polymer chain is bonded via a peptide linker to the amino group in the side chain of X.sub.1 or X.sub.2, and the peptide linker is from 3 to 10 amino acid residues long and contains at least one arginine or lysine.

2. The modified peptide according to claim 1, wherein X.sub.1 and/or X.sub.2 is an L-ornithine residue.

3. The modified peptide according to claim 1, wherein the other amino acid residues in the peptide linker are selected from glycine, alanine and serine.

4. The modified peptide according to claim 1 containing one of the following sequences: TABLE-US-00010 (SEQIDNo.9) ONORPVYIPRPRPPHPRL, (SEQIDNo.10) OONRPVYIPRPRPPHPRL, or (SEQIDNo.11) ONNRPVYIPRPRPPHPRL whereO= L-ornithine, wherein the alpha-amino group of the first ornithine is guanidated and a linear or branched polyethylene glycol polymer chain is bonded via a peptide linker, preferably selected from GRSG, GARSG, GAARSG, GAAARSG and GAAAARSG, to the delta-amino group of the first or second ornithine.

5-10. (canceled)

11. The modified peptide according to claim 1, wherein the polyethylene glycol polymer chain has a molecular weight of from 500 to 40,000 Da.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0143] The invention is explained below by the following exemplary embodiments and figures, without limiting the invention thereto:

[0144] FIG. 1 shows the serum stability of Api 300 (SG), Api 301 (SG) (left), Onc 72 and Onc110 (right) in mouse serum (100%). The respective starting products (solid line) and the degradation products (broken line), where detected, are shown.

[0145] FIG. 2 shows the serum stability of Api 137 (sPEG.sup.750-GRSG) (left, solid line) and Api 137 (PEG.sup.750-GARSG) (right, solid line) in 25% aqueous mouse serum (100%). The active ingredient released Api 137 (SG) (broken line) und and the resulting degradation product, shortened at the C-terminus, Api 137 (SG) 1-16 (dotted line).

[0146] FIG. 3 shows the serum stability of Api 300 (tPEG.sup.750-GRSG) (left) and Api 300 (tPEG.sup.5000-GRSG) (right) in mouse serum (100%). The respective starting products (triangle), the active ingredient released Api 300 (SG) (lozenge) and its degradation product shortened at the C-terminus Api300 (SG) 1-16 (asterisk) are shown.

[0147] FIG. 4 shows the serum stability of Api 301 (tPEG.sup.750-GRSG) (left) and Api 301 (tPEG.sup.5000-GRSG) (right) in mouse serum (100%). The respective starting products (triangle), the active ingredient released Api 301 (SG) (lozenge) and its degradation product shortened at the C-terminus Api301 (SG) 1-16 (asterisk) are shown.

[0148] FIG. 5 shows the serum stability of Api 300 (tPEG.sup.750-GARSG) (left) and Api 300 (tPEG.sup.5000-GARSG) (right) in mouse serum (100%). The respective starting products (triangle), the active ingredient released Api 300 (SG) (lozenge) and its degradation product shortened at the C-terminus Api300 (SG) 1-16 (asterisk) are shown.

[0149] FIG. 6 shows the serum stability of Api 301 (tPEG.sup.750-GARSG) (left) and Api 301 (tPEG.sup.5000-GARSG) (right) in mouse serum (100%). The respective starting products (triangle), the active ingredient released Api 301 (SG) (lozenge) and its degradation product shortened at the C-terminus Api301 (SG) 1-16 (asterisk) are shown.

[0150] FIG. 7 shows the serum stability of Api 300 (tPEG.sup.750-GAARSG) (left) and Api 300 (tPEG.sup.5000-GAARSG) (right) in mouse serum (100%). The respective starting products (triangle), the active ingredient released Api 300 (SG) (lozenge) and its degradation product shortened at the C-terminus Api300 (SG) 1-16 (asterisk) are shown.

[0151] FIG. 8 shows the serum stability of Api 301 (tPEG.sup.750-GAARSG) (left) and Api 301 (tPEG.sup.5000-GAARSG) (right) in mouse serum (100%). The respective starting products (triangle), the active ingredient released Api 301 (SG) (lozenge) and its degradation product shortened at the C-terminus Api301 (SG) 1-16 (asterisk) are shown.

[0152] FIG. 9 shows the serum stability of tPEG.sup.750-GAR-Onc 72 (left) and tPEG.sup.5000-GAR-Onc 72 (right) in mouse serum (100%). The respective starting products (triangle) and the active ingredient released Onc72 (lozenge) are shown. Degradation products of Onc72 were not detected.

[0153] FIG. 10 shows the serum stability of tPEG.sup.750-GAR-Onc 110 (left) and tPEG.sup.5000-GAR-Onc 110 (right) in mouse serum (100%). The respective starting products (triangle) and the active ingredient released Onc 110 (lozenge) are shown. Degradation products of Onc 110 were not detected.

DESCRIPTION OF PREFERRED EMBODIMENTS

Example 1 Peptide Synthesis and Modification

1. Peptide Synthesis

[0154] All the chemicals for the peptide synthesis were obtained, unless otherwise indicated, from Fluka Chemie GmbH (Buchs, Switzerland) in the greatest possible purity.

[0155] The peptides were synthesised by means of conventional solid-phase peptide synthesis using the Fmoc/Bu strategy. All standard Fmoc amino acids were obtained from MultiSynTech GmbH (Witten, Germany) or Orpegen Pharma GmbH (Heidelberg, Germany). Trans-4-hydroxyproline (t-4-Hyp) and tert-leucine were obtained from Novabiochem (Merck Biosciences GmbH, Darmstadt, Germany).

[0156] The peptides were synthesised on a 25 mol scale with the aid of a SYRO 2000 peptide synthesis robot (MultiSynTech GmbH, Witten, Germany). To that end, 42 mg of Leu-Wang resin (load: 0.6 mmol/g) were weighed out per synthesis reactor and swollen for 30 minutes in DMF. After cleavage of the N-terminal Fmoc protecting group, the peptides were synthesised with the following synthesis cycle (Table 2).

TABLE-US-00005 TABLE 2 Synthesis cycle of the automatic multiple solid-phase peptide synthesis (SYRO 2000) for coupling of an amino acid. Reaction Synthesis step Reagents time Amino acid 8 eq. amino acid in HOBt/DMF 60 min coupling (0.5 mol/l) and DIC/DMF (2.0 mol/l) Washing 3 x with 600 l DMF each time 1 min Fmoc cleavage 400 l 40% piperidine/DMF 3 min 500 l 20% piperidine/DMF 10 min Washing 6 x with 600 l DMF each time 1 min

[0157] For synthesis on a 100 mol scale with the aid of a Liberty microwave peptide synthesis robot (CEM GmbH, Kamp-Lintfort, Germany), 156.3 mg of commercially available Leu-Wang resin from NovaBiochem (load: 0.64 mmol/g) were weighed out per synthesis reactor and swollen for 30 minutes in DMF. After cleavage of the N-terminal Fmoc protecting group, the peptides were inter alia synthesised with the following synthesis cycle (Table 3).

TABLE-US-00006 TABLE 3 Synthesis cycle of the automatic multiple solid-phase synthesis (Liberty) for coupling of the individual amino acids. Reaction Cycle Synthesis step Reagents time Amino acid Washing 7.0 ml DMF (0.10-single) Fmoc cleavage 7.0 ml 20% piperidine 0.5 min 35 watts at 75 C. Washing 5.0 ml DMF Fmoc cleavage 7.0 ml 20% piperidine 3 min 35 watts at 75 C. Washing 4 x with 7.0 ml DMF each time Coupling 2.5 ml 0.2 mol/l amino acid 10 min in DMF 1.0 ml 0.5 mol/l HBTU 25 watts at 75 C. Washing 3 x with 7.0 ml DMF each time

[0158] The peptides were synthesised either as acid on Wang resin or Leu-Wang resin or as acid amide on Rink amide 4-methylbenzylhydrylamine (MBHA) resin (0.67 mmol/g) from MultiSynTech GmbH (Witten, Germany).

[0159] There were used as side chain protecting groups triphenylmethyl (trityl) for Cys, Asn, His and Gln, tert-butyl ether (Su) for Tyr, Ser and Thr, tert-butyl ester (O.sup.tBu) for Asp and Glu, -N-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) for Arg, tert-butyloxy-carbonyl (Boc) for Lys and Orn. The temporary Fmoc protecting group was cleaved with 40% piperidine (Biosolve BV, Valkenswaard, Netherlands) in DMF (v/v) for 5 minutes and again with fresh 20% piperidine in DMF (v/v) for 10 minutes.

2. Modification of the Peptides

[0160] a.) Guanidation of the N-Terminus:

[0161] The resin (1 eq.) was first swollen for 20 minutes in DMF. It was then incubated for 3 hours at room temperature and with shaking with HBTU in DMF (0.5 mol/l, 10 eq.) and NMM (10 eq.). The reaction solution was then renewed and the suspension was incubated for a further 4 hours. When the reaction was complete, the reaction solution was separated off and the resin was washed six times with DMF or DCM.

[0162] b.) Iodoacetylation

[0163] The resin (1 eq.) was first swollen for 20 minutes in DMF. Iodoacetic acid (8 eq.) and HOBt in DMF (0.5 mol/l, 8 eq.) were then added, and the reaction was activated with DIC (8 eq.). The reaction mixture was then incubated for 16 hours at room temperature in the dark and with shaking. When the reaction was complete, the reaction solution was separated off and the resin was washed six times with DMF or DCM and then dried.

[0164] The completeness of the modification at the N-terminus of the peptide or of the side chain was checked by means of the Kaiser test. To that end, some resin was incubated at 95 C. with 0.28 mol/l ninhydrin (Riedel de Haen, Seelze, Germany) in ethanol (Can Roth GmbH+Co. KG, Karlsruhe, Germany), 0.2 mmol/l potassium cyanide in pyridine and 76% phenol in ethanol in the ratio (1:1:2). If a blue colouration occurred, which indicates free primary amino groups, the coupling was repeated.

3. Coupling of the Polyethylene Glycol

[0165] Because polyethylene glycol has hygroscopic properties, it was dried under a high vacuum before each coupling reaction.

[0166] The following polyethylene glycol derivatives (all Iris Biotech GmbH, Marktredwitz, Germany, >95% purity) were used as starting materials for the couplings described below:

-Methoxy--carboxylic acid polyethylene glycol (20000 Da) MeO-PEG.sup.20000-COOH
-Methoxy--carboxylic acid polyethylene glycol (750 Da) MeO-PEG.sup.750-COOH
-Methoxy--carboxylic acid polyethylene glycol (5000 Da) MeO-PEG.sup.5000-COOH
-Methoxy-w-mercapto polyethylene glycol (750 Da) MeO-PEG.sup.750-SH
-Methoxy-w-mercapto polyethylene glycol (5000 Da) MeO-PEG.sup.5000-SH

[0167] a.) Coupling of polyethylene glycol with a free acid group (sPEG, e.g. for MeO-PEG-COOH) via DIC and HOBt:

[0168] The resin (1 eq.) was first swollen for 20 minutes in DMF. The polyethylene glycol derivative (5 eq.) dissolved in HOBt/DMF (0.5 mol/l, 5 eq.) was then added, and the reaction was activated with DIC (5 eq.). The reaction mixture was then incubated for one hour at RT and with shaking. DIC (2.5 eq.) was then added again, and incubation was carried out for a further 30 minutes. When the reaction was complete, the reaction solution was separated off and the resin was washed six times with DMF. Complete coupling was checked by means of the Kaiser test.

[0169] b.) Coupling after activation of the polyethylene glycol, in particular for PEG.sup.5000 and greater:

[0170] Here, the polyethylene glycol derivative with a free acid group was first activated as N-hydroxysuccinimide (NHS) ester and then coupled in solution.

[0171] To form the active ester, the polyethylene glycol (1 eq.) was incubated at room temperature, with shaking, with in each case 1 eq. of the DCC and N-hydroxysuccinimide previously dissolved in DMF until, after three hours, a fine precipitate formed. The PEG active ester (1 eq.) dissolved in the supernatant was added to the peptide (0.9 eq.) and incubated at RT, with shaking, in PBS (pH=7.4) or DMF. The reaction was monitored by means of RP-HPLC and MALDI-TOF-MS or by LC-MS.

[0172] c.) Thioether ligation with thiol-modified polyethylene glycol (tPEG):

[0173] A thiol-derivatised polyethylene glycol (e.g. MeO-PEG-SH) (1 eq.) was added to the iodoacetylated peptide (4 eq.) in PBS (pH=7.4), the reaction solution was degassed, and incubation was carried out under an N.sub.2 atmosphere at 4 C. for 18 hours. The progress of the reaction was monitored by means of RP-HPLC and MALDI-TOF-MS.

[0174] The PEGylated peptides were obtained in a yield of from 25 to 40% and characterised by means of RP-HPLC and MALDI-MS.

TABLE-US-00007 TABLE4 Overviewofthesynthesisedpeptidesequences SEQ IDNo. Name Sequence 2 Api1b* GNNRPVYIPQPRPPHPRL-OH 14 Api88* Guan-ONNRPVYIPRPRPPHPRL-NH.sub.2 15 Api134* Guan-ONNRPVYIPRPRPPHPOL-NH.sub.2 16 Api137* Guan-ONNRPVYIPRPRPPHPRL-OH 17 Api137(sPEG.sup.750) Guan-O(sPEG.sup.750)NNRPVYIPRPRPPHPRL-OH 18 Api137(SG)* Guan-O(SG)NNRPVYIPRPRPPHPRL-OH 19 Api137(GRSG)* Guan-O(GRSG)NNRPVYIPRPRPPHPRL-OH 20 Api137(GARSG)* Guan-O(GARSG)NNRPVYIPRPRPPHPRL-OH 21 Api137(sPEG.sup.750-GRSG) Guan-O(sPEG.sup.750-GRSG)NNRPVYIPRPRPPHPRL-OH 22 Api137(sPEG.sup.750-GARSG) Guan-O(sPEG.sup.750-GARSG)NNRPVYIPRPRPPHPRL-OH 23 Api137(sPEG.sup.5000-GARSG) Guan-O(sPEG.sup.5000-GARSG)NNRPVYIPRPRPPHPRL-OH 24 Api137(sPEG.sup.20000-GARSG) Guan-O(sPEG.sup.20000-GARSG)NNRPVYIPRPRPPHPRL-OH 25 Api300(SG).sup.+ Guan-OO(SG)NRPVYIPRPRPPHPRL-OH 26 Api300(GRSG)* Guan-OO(GRSG)NRPVYIPRPRPPHPRL-OH 27 Api300(GARSG)* Guan-OO(GARSG)NRPVYIPRPRPPHPRL-OH 28 Api300(GAARSG)* Guan-OO(GAARSG)NRPVYIPRPRPPHPRL-OH 29 Api300(sPEG.sup.750-GRSG) Guan-OO(sPEG.sup.750-GRSG)NRPVYIPRPRPPHPRL-OH 30 Api300(sPEG.sup.750-GARSG) Guan-OO(sPEG.sup.750-GARSG)NRPVYIPRPRPPHPRL-OH 31 Api300(sPEG.sup.750-GAARSG) Guan-OO(sPEG.sup.750-GAARSG)NRPVYIPRPRPPHPRL-OH 32 Api300(tPEG.sup.750-GRSG) Guan-OO(tPEG.sup.750-GRSG)NRPVYIPRPRPPHPRL-OH 33 Api300(tPEG.sup.750-GARSG) Guan-OO(tPEG.sup.750-GARSG)NRPVYIPRPRPPHPRL-OH 34 Api300(tPEG.sup.750-GAARSG) Guan-OO(tPEG.sup.750-GAARSG)NRPVYIPRPRPPHPRL-OH 35 Api300(tPEG.sup.5000-GRSG) Guan-OO(tPEG.sup.5000-GRSG)NRPVYIPRPRPPHPRL-OH 36 Api300(tPEG.sup.5000-GARSG) Guan-OO(tPEG.sup.5000-GARSG)NRPVYIPRPRPPHPRL-OH 37 Api300(tPEG.sup.5000-GAARSG) Guan-OO(tPEG.sup.5000-GAARSG)NRPVYIPRPRPPHPRL-OH 38 Api301(SG).sup.+ Guan-ONO(SG)RPVYIPRPRPPHPRL-OH 39 Api301(GRSG)* Guan-ONO(GRSG)RPVYIPRPRPPHPRL-OH 40 Api301(GARSG)* Guan-ONO(GARSG)RPVYIPRPRPPHPRL-OH 41 Api301(GAARSG)* Guan-ONO(GAARSG)RPVYIPRPRPPHPRL-OH 42 Api301(sPEG.sup.750-GRSG) Guan-ONO(sPEG.sup.750-GRSG)RPVYIPRPRPPHPRL-OH 43 Api301(sPEG.sup.750-GARSG) Guan-ONO(sPEG.sup.750-GARSG)RPVYIPRPRPPHPRL-OH 44 Api301(sPEG.sup.750-GAARSG) Guan-ONO(sPEG.sup.750-GAARSG)RPVYIPRPRPPHPRL-OH 45 Api301(tPEG.sup.750-GRSG) Guan-ONO(tPEG.sup.750-GRSG)RPVYIPRPRPPHPRL-OH 46 Api301(tPEG.sup.750-GARSG) Guan-ONO(tPEG.sup.750-GARSG)RPVYIPRPRPPHPRL-OH 47 Api301(tPEG.sup.750-GAARSG) Guan-ONO(tPEG.sup.750-GAARSG)RPVYIPRPRPPHPRL-OH 48 Api301(tPEG.sup.5000-GRSG) Guan-ONO(tPEG.sup.5000-GRSG)RPVYIPRPRPPHPRL-OH 49 Api301(tPEG.sup.5000-GARSG) Guan-ONO(tPEG.sup.5000-GARSG)RPVYIPRPRPPHPRL-OH 50 Api301(tPEG.sup.5000-GAARSG) Guan-ONO(tPEG.sup.5000-GAARSG)RPVYIPRPRPPHPRL-OH 51 Onc72.sup.+ VDKPPYLPRPRPPROIYNO-NH.sub.2 52 Onc110.sup.+ VDKPPYLPRPRPHypRHypTleYNO-NH.sub.2 53 GAR-Onc72* GAR-VDKPPYLPRPRPPROIYNO-NH.sub.2 54 GAR-Onc110* GAR-VDKPPYLPRPRPHypRHypTleYNO-NH.sub.2 55 sPEG.sup.750-GAR-Onc72 sPEG.sup.750-GAR-VDKPPYLPRPRPPROIYNO-NH.sub.2 56 sPEG.sup.750-GAR-Onc110 sPEG.sup.750-GAR-VDKPPYLPRPRPHypRHypTleYNO-NH.sub.2 57 tPEG.sup.750-GAR-Onc72* tPEG.sup.750-GAR-VDKPPYLPRPRPPROIYNO-NH.sub.2 58 tPEG.sup.750-GAR-Onc110 tPEG.sup.750-GAR-VDKPPYLPRPRPHypRHypTleYNO-NH.sub.2 59 tPEG.sup.5000-GAR-Onc72 tPEG.sup.5000-GAR-VDKPPYLPRPRPPROIYNO-NH.sub.2 60 tPEG.sup.5000-GAR-Onc110 tPEG.sup.5000-GAR-VDKPPYLPRPRPHypRHypTleYNO-NH.sub.2 Guan = guanidino group at the N-terminus, Hyp = trans-4-hydroxyproline, L(N.sub.3) = N-(9-fluorenylmethyloxycarbonyl)--azido-L-lysine, O = ornithine, Pra = N-(9-fluorenyl-methyloxycarbonyl)-L-progargylglycine, Tie = L-tertiary-leucine (L-tertiary-butylglycine), sPEG: PEG linked via acid group, tPEG: PEG linked via thiol group. comparison examples are marked with *, cleavage products are marked with +.

[0175] c.) Cleavage of protecting groups and purification:

[0176] When the synthesis of the peptides and optional modification were complete, the resins were washed carefully with DMF and DCM and dried. The resin-bonded peptides were cleaved for 4 hours at room temperature with a mixture of water, m-cresol, thioanisole and ethanedithiol (5:5:5:2.5) in 87.5% trifluoroacetic acid (TFA), and at the same time the side chains were deprotected. The peptides and peptide derivatives were precipitated with cold diethyl ether and centrifuged off at 3000g. The pellet was washed twice with cold ether, dried and dissolved in 0.1% aqueous TFA (UV spectroscopy). The samples were stored at 20 C.

[0177] The cleaved (optionally modified) peptides were purified by means of RP-HPLC on an Akta HPLC System (Amersham Bioscience GmbH, Freiburg, Germany) with a Jupiter C.sub.18 5 m 300 , 25010 mm or Jupiter C.sub.18 15 m, 300 , 25021 mm column (Phenomenex Inc., Torrance, USA).

[0178] As eluent there was used in each case 0.1% aqueous TFA (eluent A) and 60% aqueous acetonitrile (Biosolve BV, Valkenswaard, Netherlands) with 0.1% TFA (eluent B). A typical linear gradient began at 5% B and the elution took place at a gradient of 1% B per minute with a flow rate of 10 ml/min (25021 mm column) or 5 ml/min (25010 mm column). Detection was at 220, 230 and 240 nm. Analysis of the purified peptides was carried out with the same HPLC system with a Jupiter C.sub.18 5 m, 300 , 1504.6 mm column (Phenomenex Inc., Torrance, USA). Elution was carried out at a flow rate of 1 ml/min with a linear gradient of 5-95% B in 30 minutes, and detection was at 220 nm. In addition, the purity was determined by means of matrix-assisted laser desorption/ionisation with time-of-flight mass spectrometry (MALDI-TOF-MS; 4700 Proteomic Analyzer, Applied Biosystems GmbH, Darmstadt, Germany). To that end, 0.5 l of peptide solution was co-crystallised with 0.5 l of -cyanohydroxycinnamic acid (Bruker Daltonik GmbH; Bremen, Germany) as matrix (4 mg/ml in 60% acetonitrile in 0.1% aqueous TFA).

Example 2: Determination of the Minimum Inhibitory Concentrations and Growth Kinetics

1. Minimum Inhibitory Concentrations

[0179] The minimum inhibitory concentrations (MIC) of the peptides were determined in a double determination of triplicates with a positive control (gentamycin) and a negative control (0.9% NaCl solution).

[0180] To that end, the peptides were dissolved in water and diluted in a two-fold dilution series with 1% aqueous soybean medium (TSB) in sterile 96-well plates (Greiner Bio-One GmbH) from 128 g/ml in twelve dilution steps to 62.5 ng/ml. Overnight cultures of Escherichia coli strain BL21AI were adjusted with 1% TSB to about 1.510.sup.7 colony forming units per ml. In each case 50 l of peptide solution per well were then mixed with in each case 50 l of the bacteria solution, in order to achieve a starting concentration of 410.sup.5 bacteria per well. After incubation for 20 hours at 37 C., the absorption at 595 nm was determined (microplate reader, TECAN Trading AG). The minimum inhibitory concentration was identified as the lowest peptide concentration at which no bacterial growth was demonstrated.

[0181] In addition, the antibacterial activity was also determined in the presence of 25% mouse serum. To that end, the MIC values were determined as described above, but 25 l of mouse serum were also added to each well before the incubation (25% final concentration).

TABLE-US-00008 TABLE 5 Minimum inhibitory concentrations MIC [mol/l] MIC [mol/l] in TSB/mouse Seq. ID Peptide derivative in TSB serum 2 Apidaecin 1b* 0.48 1.75 14 Api 88* 0.44 1.19 15 Api 134* 1.78 7.12 16 Api 137* 0.46 0.23 18 Api 137 (SG).sup.+ 0.2 19 Api137(GRSG)* 0.7 20 Api 137(GARSG)* 0.9 22 Api 137 (sPEG.sup.750-GARSG) 1.1 23 Api 137 (sPEG.sup.5000-GARSG) 3.7 24 Api 137 (sPEG.sup.20000-GARSG) 10.2 25 Api 300 (SG).sup.+ 0.8 0.21 29 Api 300 (sPEG.sup.750-GRSG) 2.9 0.37 30 Api 300 (sPEG.sup.750-GARSG) 2.9 0.54 31 Api 300 (sPEG.sup.750-GAARSG) 2.8 32 Api 300 (tPEG.sup.750-GRSG) 2.9 33 Api 300 (tPEG.sup.750-GARSG) 1.5 34 Api 300 (tPEG.sup.750-GAARSG) 2.8 35 Api 300 (tPEG.sup.5000-GRSG) >44.8 36 Api 300 (tPEG.sup.5000-GARSG) 17.4 0.44 37 Api 300 (tPEG.sup.5000-GAARSG) 25.4 38 Api 301 (SG).sup.+ 1.6 42 Api 301 (sPEG.sup.750-GRSG) 1.5 43 Api 301 (sPEG.sup.750-GARSG) 2.9 0.27 44 Api 301 (sPEG.sup.750-GAARSG) 5.6 45 Api 301 (tPEG.sup.750-GRSG) 2.9 46 Api 301 (tPEG.sup.750-GARSG) 1.5 47 Api 301 (tPEG.sup.750-GAARSG) 2.8 48 Api 301 (tPEG.sup.5000-GRSG) >38 49 Api 301 (tPEG.sup.5000-GARSG) 9.2 50 Api 301 (tPEG.sup.5000-GAARSG) >47.5 51 Onc 72.sup.+ 1.7 1.7 53 GAR-Onc 72* 3.1 55 sPEG750-GAR-Onc 72 12.2 6.1 57 tPEG750-GAR-Onc 72 24.3 52 Onc 110.sup.+ 6.9 54 GAR-Onc 110* 1.5 0.65 56 sPEG.sup.750-GAR-Onc 110 24.3 9.2 58 tPEG.sup.750-GAR-Onc 110 48.4 comparison examples are marked with *, cleavage products are marked with .sup.+.

[0182] Owing to the large difference in mass between the unmodified and the PEG-coupled peptides, the MIC is not given in the conventional unit g/ml but in mol/l.

[0183] The results show that the presence of the peptide linker (GAR-Onc 72 and GAR-Onc 120) or residues thereof remaining in the cleavage product (Api 137 (SG), Api 300 (SG) and Api 301 (SG)) does not increase the MIC or increases it only insignificantly. In the PEG-modified peptides, the MIC in TSB increases with the size of the PEG. Without serum (TSB), the PEG-modified peptides exhibit an increased MIC. In the serum, the PEG-modified peptides have, however, a MIC which is comparable with the unpegylated peptides.

Example 3: Release of the Peptide Active Ingredients in Serum

1. Analysis in Mouse Serum

[0184] 30 l of a 1 mg/ml peptide solution were diluted with 120 l of water and, after addition of 50 of serum, incubated at 37 C. for 0 hours, 0.5 hour, 1 hour, 2 hours or 4 hours. Alternatively, the peptide derivatives having a starting concentration of 3 mg/ml were diluted with serum to a final concentration of 15 g/ml. According to the stability of the peptides, the samples were analysed at the times mentioned above or after 0 hours, 4 hours and 6 hours. The serum proteins were precipitated by addition of 50 l of 15% TCA, and the supernatant was neutralised with 25 l of 1 mol/l NaOH. The samples were made up to 250 l with 5% B, 230 l of the solution were transferred into a HPLC vial, and 220 l were injected.

[0185] RP-HPLC analysis was carried out with a linear acetonitrile gradient (Biosolve BV, Valkenswaard, Netherlands) in the presence of 0.1% trifluoroacetic acid (TFA, UV grade, Fluka Chemie GmbH, Buchs, Switzerland) as ion-pair reagent. The fractions were co-crystallised with -cyanohydroxycinnamic acid (Bruker Daltonik GmbH; Bremen, Germany) as matrix (4 mg/ml in 60% acetonitrile in 0.1% aqueous TFA) and analysed with a tandem mass spectrometer (MALDI-TOF/TOF-MS, 4700 Proteomics Analyzer; Applied Biosystems GmbH, Weiterstadt, Germany) in positive-ion reflector mode. The amount of intact peptides and their degradation products or metabolites could thus be identified and quantified at the individual times. 25% aqueous mouse serum, which was analysed in parallel for the same time intervals, was used as control.

TABLE-US-00009 TABLE 6 Overview of the half-lives in 25% (v/v) and pure mouse serum, the different side-chain modifications in combination with PEG750 were compared; primary (red) and secondary cleavage site (blue), the half-lives marked with * were quantified by means of MALDI-TOF MS. Half-life (t.sub.1/2) Seq. ID Peptide derivative in 25% serum in serum 16 Api 137* >360 min 18 Api 137 (GRSG)* 60 min 20 Api 137 (GARSG)* 30 min 21 Api 137 (sPEG.sup.750-GRSG) 120 min 22 Api 137 (sPEG.sup.750-GARSG) 60 min 25 Api 300 (SG).sup.+ 240 min 29 Api 300 (sPEG.sup.750-GRSG) 50 min 32 Api 300 (tPEG.sup.750-GRSG) 40 min 35 Api 300 (tPEG.sup.5000-GRSG) 75% (6 h)** 30 Api 300 (sPEG.sup.750-GARSG) 35 min 33 Api 300 (tPEG.sup.750-GARSG) 45 min 36 Api 300 (tPEG.sup.5000-GARSG) 69% (6 h)** 31 Api 300 (sPEG.sup.750-GAARSG) 45 min 34 Api 300 (tPEG.sup.750-GAARSG) 35 min 37 Api 300 (tPEG.sup.5000-GAARSG) 76% (6 h)** 38 Api 301 (SG).sup.+ 60% (4 h)** 42 Api 301 (sPEG.sup.750-GRSG) 30 min 45 Api 301 (tPEG.sup.750-GRSG) 55 min 48 Api 301 (tPEG.sup.5000-GRSG) 75% (6 h)** 43 Api 301 (sPEG.sup.750-GARSG) 45 min 46 Api 301 (tPEG.sup.750-GARSG) 45 min 49 Api 301 (tPEG.sup.5000-GARSG) 62% (6 h)** 44 Api 301 (sPEG.sup.750-GAARSG) 55 min 47 Api 301 (tPEG.sup.750-GAARSG) 50 min 50 Api 301 (tPEG.sup.5000-GAARSG) 78% (5 h)** 51 GAR-Onc 72* 85% (4 h)** 52 GAR-Onc 110* 75% (4 h)** 55 sPEG.sup.750-GAR-Onc 72 6 h 56 sPEG.sup.750-GAR-Onc 110 73% (6 h)** 57 tPEG.sup.750-GAR-Onc 72 55% (6 h)** 58 tPEG.sup.750-GAR-Onc 110 64% (6 h)** 59 tPEG.sup.5000-GAR-Onc 72 86% (6 h)** 60 tPEG.sup.5000-GAR-Onc 110 96% (6 h)** comparison examples are marked with *, cleavage products are marked with .sup.+. **uncleaved peptide remaining after the indicated time (instead of half-life)

[0186] The half-lives (or uncleaved peptide remaining after the indicated time) relate in the case of the free peptides (Api 137, Api300 (SG) and Api 301 (SG)) to the cleavage site in the peptide (downstream of Arg-16). In the case of the remaining derivatives, the values relate to the cleavage in the linker in the side chain with release of Api 137 (SG), Api300 (SG) and Api 301 (SG) or of Onc 72 and Onc 110.

[0187] The following abbreviations are used in the description of the invention:

Agp 2-amino-3-guanidinopropionic acid
BOC tert-butyloxy-carbonyl
.sup.tBu tert-butyl ether
Dap 2,3-diaminopropionic acid
DCC dicyclohexylcarbodiimide
DCM dichloromethane
DIC diisopropylcarbodiimide
DMF dimethylformamide
E. coli Escherichia coli
eq. equivalents per mol
Fmoc fluorenylmethoxycarbonyl
Guan guanidino group (at the N-terminus)
Hyp trans-4-hydroxyproline
HBTU 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate
HOBt 1-hydroxybenzotriazole
LC-MS liquid chromatography with mass spectrometry coupling
MALDI-TOF matrix assisted laser desorption/ionization with time of flight analysis
MIC minimum inhibitory concentration
MS mass spectrometry
Mtt 4-methyltrityl

NHS N-hydroxysuccinimide

NMM N-methylmorpholine

[0188] O ornithine
O.sup.tBu tert-butyl ester
PBS phosphate-buffered saline
PEG polyethylene glycol
sPEG PEG linked via an acid group
tPEG PEG linked via a thiol group
Pra N-(9-fluorenylmethyloxycarbonyl)-L-progargylglycine
RP-HPLC reversed phase high performance liquid chromatography
RT room temperature
TCA trichloroacetic acid
TFA trifluoroacetic acid
Tle L-tertiary-leucine (L-tertiary-butylglycine)
Tris tris(hydroxymethyl)-aminomethane
TSB tryptic soy broth