NZ618829B2 - Modified antibiotic peptides having variable systemic release - Google Patents
Modified antibiotic peptides having variable systemic release Download PDFInfo
- Publication number
- NZ618829B2 NZ618829B2 NZ618829A NZ61882912A NZ618829B2 NZ 618829 B2 NZ618829 B2 NZ 618829B2 NZ 618829 A NZ618829 A NZ 618829A NZ 61882912 A NZ61882912 A NZ 61882912A NZ 618829 B2 NZ618829 B2 NZ 618829B2
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- New Zealand
- Prior art keywords
- peptide
- side chain
- residue
- peptides
- amino acid
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- A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
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- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
- A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K11/00—Depsipeptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
- C07K14/43572—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from bees
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
Abstract
Disclosed is a modified peptide containing one of the following sequences: NT-X1-D2-K3-P4-P5-Y6-L7-P8-R9-P10-X2-P12-P13-R14-X3-I16-Y17-N18-X4-CT NT-X1-D2-K3-P4-P5-Y6-L7-P8-R9-P10-X2-P12-P13-R14-X3-I16-Y17-N18-N19-X4-CT NT-X1-D2-K3-P4-P5-Y6-L7-P8-R9-P10-X2-P12-P13-R14-X3-I16-P17-N18-X4-CT X1 is a residue having a non-polar, hydrophobic side chain or an amino acid residue whose side chain is positively charged under physiological conditions, having a positive net charge or a side chain that is positively charged under physiological conditions; D2 is an aspartic acid or glutamic acid residue, K3 is a residue having a positive net charge or a side chain that is positively charged under physiological conditions, preferably lysine or arginine, X2 and X4 are selected independently of one another from residues having a positive net charge or a side chain that is positively charged under physiological conditions; X3 is a residue having a positive net charge or a side chain that is positively charged under physiological conditions or proline or a proline derivative; L7 and I16 are selected independently of one another from residues having a non-polar, hydrophhobic side chain, preferably leucine, isoleucine and valine, Y6 and Y17 are each tyrosine, R9 and R14 are each arginine, N18 and N19 are each asparagine or glutamine, P4, P5, P8, P10, P12, P13 and P17 are selected independently of one another from proline and proline derivatives or hydroxyproline and hydroxyproline derivatives, wherein P13 and R14 are optionally interchanged, and/or optionally one or two of the residues selected from D2, P4, P5, P8, P10, P12, P13, P17 and Y17 are replaced by any desired residue, NT is the free or modified N-terminus of the amino acid X1, CT is the free or modified C-terminus of the amino acid X4, characterised in that a linear or branched polyethylene glycol polymer chain is bonded to NT via a peptide linker, and wherein the peptide linker is from 3 to 8 amino acid residues long and contains at least one arginine or lysine at the C-terminus of the peptide linker, that is to say upstream of X1. Also disclosed is its use in the manufacture of a medicament for treating infections by microorganisms including bacteria and fungi. X1 is a residue having a non-polar, hydrophobic side chain or an amino acid residue whose side chain is positively charged under physiological conditions, having a positive net charge or a side chain that is positively charged under physiological conditions; D2 is an aspartic acid or glutamic acid residue, K3 is a residue having a positive net charge or a side chain that is positively charged under physiological conditions, preferably lysine or arginine, X2 and X4 are selected independently of one another from residues having a positive net charge or a side chain that is positively charged under physiological conditions; X3 is a residue having a positive net charge or a side chain that is positively charged under physiological conditions or proline or a proline derivative; L7 and I16 are selected independently of one another from residues having a non-polar, hydrophhobic side chain, preferably leucine, isoleucine and valine, Y6 and Y17 are each tyrosine, R9 and R14 are each arginine, N18 and N19 are each asparagine or glutamine, P4, P5, P8, P10, P12, P13 and P17 are selected independently of one another from proline and proline derivatives or hydroxyproline and hydroxyproline derivatives, wherein P13 and R14 are optionally interchanged, and/or optionally one or two of the residues selected from D2, P4, P5, P8, P10, P12, P13, P17 and Y17 are replaced by any desired residue, NT is the free or modified N-terminus of the amino acid X1, CT is the free or modified C-terminus of the amino acid X4, characterised in that a linear or branched polyethylene glycol polymer chain is bonded to NT via a peptide linker, and wherein the peptide linker is from 3 to 8 amino acid residues long and contains at least one arginine or lysine at the C-terminus of the peptide linker, that is to say upstream of X1. Also disclosed is its use in the manufacture of a medicament for treating infections by microorganisms including bacteria and fungi.
Description
release
Modified antibiotic peptides having variable systemic
modified antibiotic peptides in particular for use in medicine. The
This invention s to
for killing microorganisms, such as bacteria,
invention relates also to compositions and methods
infections.
s or fungi. and to methods for treating microbial
of serious bacterial and fungal infections is an increasing problem despite
The occurrence
million
remarkable progress in antibiotic therapy. Each year there are more than 40
hospital
million of those patients become
stays in the United States of America, and more than 2
ed. it is
infected in hospital. in 50—60% of these cases. antibiotic-resistant bacteria are
deaths in the USA and
estimated that such diseases acquired in hospital lead to -70,000
up to 10.000 deaths in y.
for new antibiotics. Inducible antibacterial
This illustrates the necessity to continue the search
active
peptides represent a research area in which current biochemistry, immunology and
with a size of from 13 to more than a
ingredient research come together. Peptide antibiotics,
H.G.
hundred amino acids. have been isolated from plants. animals and es (Boman.
1995 Annu. Rev. Immunol. 13: 61—92).
each peptide often ting a
A single animal possesses about 6-10 antimicrobial peptides.
completely different activity spectrum . D. 9! at, 1998. FEBS Lett. 430: 130-134). is
the d defensins.
known that the majority of antibacterial peptides, including widely
"lytic/ionic" mechanism. A permeabilising effect on the
cecropins and ins. act by a
of these "lytic"
bacterial cytoplasm membrane is discussed as a common mechanism of action
ion n) channels in a
peptides. A cationic. amphipathic ure, which forms hydrophilic
the occurrence of ions. the membrane
lipid bilayer. is the basis for this activity. Through
' is destroyed and the cell is thus killed.
potential necessary for many fundamental life processes
membranes at
These lytic peptides often have a toxic effect on mammalian higher
medicaments. lf proline is inserted into
concentrations, which limits their suitability as possible
the ability of the peptides to permeabilise
the sequence of the a—helical antimicrobial peptides,
the cytoplasm membrane of E. coli falls, in dependence on the number of proline
residues.
of the most active. natural antibacterial
From this point of view, it is amazing that some
peptides. at least in relation to some gram-negative pathogens, belong to the family of the
proline-rich es (Otvos, L. et al. 2000. Protein Sci. 9: 742—749).
The above-described side-effects could be overcome by antimicrobial peptides (AMP) which
specifically recognise a bacterial n or other intra- or extra-cellular components, without
cross-reactivity with mammalian analogs. This appears to apply to proline-rich
. exhibiting
antimicrobial peptides, including apidaecins, drosocin and pyrrhocoricin, which were originally
biochemical properties, it is
isolated from insects. With the us variation in the size and
not surprising that the structure-action and conformation-action relationships are the focus of
antibacterial peptide ch. A complete study of the natural, antibacterial peptide repertoire
for biological strength is not only important for general biochemical questions but is also of
industry. Despite the ms of in vitro tests with
sustained interest for the pharmaceutical
peptide-based antibiotics, some natural, cationic antibacterial peptides have already reached
of these es exhibited activity
the clinical trial phase (Boman. H.G. 1995 ebd.). While some
as topical (local) agents in the early clinical trial phase, others were active
in systemic therapy.
For example. the cationic protein rBPI 21, which is used for the parenteral treatment of
H.G. 1995 ebd.).
meningococcemia, has completed the third phase of clinical testing (Boman,
(e.g. apidaecin, drosocin and coricin) kill bacteria The family of the proline-rich peptides
not by permeabilisation of their membrane but bind specifically to one or more target
DnaK has hitherto been
ns. These possible interaction partners, the heat shock protein
the e-rich peptides
thoroughly researched (inter alia Boman, H.G. 1995), are inhibited by
cell death. In
and presumably the correct protein folding is inhibited, which tely leads to
addition, proline-rich es, in stark st to AMPs with a defined secondary structure
or toxically on
such as melittin or gramicidin. do not appear in vitro to act either haemolytically
eukaryotic cells. In addition to the antimicrobial activity, the ity in mammalian serum (25%)
antibiotics. For
especially has a decisive influence on the development of new peptide-based
example. drosocin is degraded within 120
an hour, while pyrrhocoricin, with half-lives of
minutes, is considerably more stable to proteases. Presumably, not only are the N- and C-
termini cleaved by amino and carboxy peptidases, but the peptides are also digested by
stable to r decomposition but
endoproteases. Some of the lites formed thereby are
in most cases lose the crobial activity (MIC values 2 64 tug/ml).
J Invertebr Pathol. 78: 135-40),
In experiments conducted by der M. and Dorn A. (2001.
large milkweed bug Oncopeltus fasciatus from the family of the
nymphs and pupae of the
Lygaeidae were infected with two different egative Pseudomonas species, and their
of O. fasciatus with the human
immune response was analysed. While infection of the nymphs
pathogen Pseudomonas aeruginosa resulted in the death of all individuals after 48 hours, 71%
survived for at least 96
of the individuals infected with the less pathogenic Pseudomonas putida
hours. If the nymphs of the large milkweed bug were infected
first with P. putida and after 24
individuals within the first 24
hours with P. aeruginosa, the survival rate of the doubly infected
hours increased significantly to 73%. The probable induction of the sis of antibacterial
peptides, by means of which s defend themselves against incoming microorganisms
within the context of their innate immune system, was then investigated. Four peptides
(oncopeltus antibacterial peptide 1-4) were identified with molecular weights of 15, 8, 5 and
sible for the antibacterial activity. Sequence analysis according to
2 kDa and made
Edman ed. in addition to a partial sequence 34 amino acids long for peptide
1 (15 kDa).
also the incomplete sequence of the proline-rich 2 kDa peptide 4. It was not possible y to
19. The
identify the amino acids at positions 11 and the C-terminal sequence from position
exact molecular weight is unknown.
Table 1:
A ion of hitherto known sequences of antibiotic peptides is listed in
Table 1:
Peptide Species Sequence SEQ
Apidaecin 1a Apis mellifera GNNRPVYIPQPRPPHPRI
Apidaecin 1b Apis mellifera GNNRPVYIPQPRPPHPRL
Drosophila GKPRPYSPRPTSHPRPIRV
melanogaster
Myrmecia GRPNPVNNKPTPYPHL
Formaecm 1
gulosa
Pyrrhocoris VDKGSYLF’RPTPPRPIYNRN-NHZ
Pyrrhoconcrn apterus
Palomena VDKPDYRPRPRPPNM
Metalnikowm 1
Oncopeltus Oncopeltus EVSLKGEGGSNKGFIQGSGTKTL
antibacterial peptide fasciatus FQDDKTKLDGT
Oncopeltus Oncopeltus VDKPPYLPRP(XIP)PPRRIYN(NR)
antibacterial peptide fasciatus
Apidaecin tives are disclosed in W02009013262A1. Derivatives of oncopeltus
antibacterial peptide 4 are disclosed in W02010086401A1.
Different approaches at influencing the pharmacokinetic properties of pharmacological active
ingredients are described in the ture. Organic polymers (such as eg. polyethylene glycol)
are also used thereby.
The use of polyethylene glycol (PEG) in pharmaceutical dosage forms for the controlled release
of an active ient is known. A distinction must be made between two forms:
1. The introduction of the active ingredient into a crosslinked PEG hydrogel;
2. The direct bonding of a linear or branched PEG molecule to the active ingredient (known
as PEGyIation).
The two forms differ not only in the pharmacokinetics but also especially in the possible
stration routes. The hydrogel is administered locally. The PEGyIated active ingredient is
stered systemically ally enously).
els for the controlled release of an active ingredient are known inter alia from US
7,291,673, US 2008/0014149 A1 and Yang J et al. 2010 (Macromol. Biosci., 10, 445—454).
The tion of polypeptides is used in particular in order on the one hand to achieve
controlled release over a desired period of time (retard effect) and on the other hand to delay
the excretion of the active ingredient via the kidneys.
For the PEGyIation of pharmaceutical active ingredients, reference may be made inter alia to
US 4,179,337, and also to the overview articles , M et al. 1998 (Prog Polym Sci.
23: 1233—71) and Veronese FM, Harris JM 2002 (Adv Drug Deliv Rev 54: 453—456).
Because an irreversible bonding of PEG impairs the pharmacological action of the active
ingredient, a large number of known approaches for irreversible PEGyIation exist.
Veronese FM (2001. Biomaterials 22: 405-17) describes in a general overview article the
reversible PEGyIation inter alia enzyme—catalysed to ine side chains by the enzyme
transglutaminase. In an overview article by Roberts MJ et al. 2002 (Adv Drug Deliv- Rev 54:
459-476) too. several techniques for reversible PEGyIation of peptides and proteins are
mentioned, in particular hydrolysable ester bridges, reducible disulfide bridges.
EP1897561A, W09930727, US 6,180,095, U86,720,306, 663 be the use of a
linker, which is cleaved by a 1,4— or 1,5-benzyl elimination. In several publications, this linker is
combined in what is known as a double prodrug approach with an enzymatically cleavable
et al. 1999. J. Med. Chem. 42: 3657-3667;
group or a hydrolysable ester group (Greenwald RB
Lee S et al. 2001 Bioconjug Chem 12, 163-169; Greenwald RB et al. 2003. Bioconjug Chem.
14(2): 395-403).
Another approach to reversible PEGylation is trimethyl lock lactonization (TML), which is
sed inter alia in US 5,965,119 and US 6,303,569.
US 7,585,837 describes an approach to reversible PEGyIation by derivatisation of functional
groups with 9-fluorenylmethoxycarbonyl (Fmoc) or 2—sulfofluorenylmethoxycarbonyl (FMS),
which are readily cleaved by bases.
Guiotto et al. 2004 (J. Med. Chem. 47: 1280—9) describes the synthesis, characterisation and
first in vivo tests of PEG conjugates of the antitumour agent 10-aminoethylcamptothecin.
A protease-based prodrug strategy is based on peptide linkers which are bonded to
conventional chemical active ingredients ("small molecules") and contain cutting sites for
ses. The proteases that are used are mainly tissue-specific proteases or ses which
play a part in tissue lling (such as eg. Cathepsin B, PSA (prostate specific antigen) and
matrix metalloproteases (MMP), but also the serum proteases plasmin and urokinase. The field
of application of the active ients is substantially cancer therapy. The protease cutting site
is used for tumour targeting. In this connection, reference may be made to the overview article
of Law B, Tung CH. 2009 (Bioconjug Chem. 20(9):1683-95). US 2004192769 mentions the
approach of configuring the drug delivery system in such a manner that the peptide linker is
cleaved not by serum ses but only after being taken up into the target cell.
Li H ef al. A 2010 (Angew. Chem. Int. Ed. 49, 4930—4933) apply a protease-based prodrug
strategy to peptide active ingredients. Therapeutically active peptides are bonded via the
peptide linker to an albumin binding domain (ABD). In the blood, this fusion peptide first binds to
the serum protein albumin and is then d by the protease thrombin (or human factor Xa).
There is a ued need for new antibiotics.
bie properties for peptide antibiotics are:
(i) and
an increased half-life in mammalian serum through a higher protease ance
(ii) unchanged or preferably increased antimicrobial activity against one or more
bacterial strains, particularly human pathogens, or fungi or other microbial infections,
(iii) a d antigenic action and, as a , a reduced immune reaction, and
(iv) the peptides are not toxic to human cells, including erythrocytes.
is very complex, because they must penetrate
The action of proline-rich antimicrobial peptides
in order to inhibit a specific intracellular bacterial
the cell membrane and pass into the cytoplasm
Another
toxic effect on mammalian cells and blood cells.
target molecule, but without having a
ant point is the stability of the peptides or peptide derivatives to degradation by
a high
peptidases or proteases in blood and the bacteria. The ideal peptide therefore has
antibacterial activity (low MIC values), no cell toxicity, no ytic activity and a half—life of
several hours in blood.
increased
novel peptide antibiotics, preferably having
It is an object of the invention to provide
stability, reduced immune reaction and improved pharmacokinetics.
by the polymer polyethylene glycol
In the invention, peptide antibiotics are reversibly protected
in order to reduce the degradation of the
(PEG) via an enzymatically ble peptide chain,
of the active ingredient, as well as to
active ingredient in serum by proteases and the toxicity
organism (improved pharmacokinetics). The active
achieve a longer dwell time in the
defined compound by the proteases of the host
ingredients are ed from this chemically
to the
(e.g. patient, in ular serum proteases) or the ia. The choice ing
allows
invention of the polymer (structure, length) and of the e linker (length, sequence)
action of the
to be adapted to the mechanism of
the kinetics of the active ingredient release
ingredient and its lisation or excretion. Thus, by a clever choice of structure
active
active
linker), a constant concentration of the ingredient can be achieved over a
(polymer,
prolonged treatment period.
derivative, which has a ce
The specification discloses a modified peptide, an apidaecin
according to the l a 1, 2 or 3:
NT—X1X2NX3PVYIPX4X5RPPHP-CT (formula 1)
NT-X1NX2X3PVYIPX4X5RPPHP-CT (formula 2)
NT—XZNNXaPVYIPX4X5RPPHP-CT (formula 3)
under
wherein X1 is an amino acid residue whose side chain is positively charged
physiological conditions, preferably 0 (ornithine),
amino group in the side chain,
wherein X2 is an amino acid residue having an
n X3 is an amino acid e whose side chain is positively charged under
physiological conditions, ably‘R (arginine),
wherein X. is an amino acid residue whose side chain is positively d under
physiological conditions, preferably R,
wherein X5 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 (Arg-Leu),
wherein NT is the N-terminus, which is preferably guanidated.
The remaining amino acid residues each have the meaning ing to the IUPAC one-letter
code.
Modified peptides according to formulae 1 and 2 are particularly preferred.
The modified peptide according to the invention is characterised in that a linear or branched
hylene glycol polymer chain is bonded via a peptide linker to the amino group in the side
chain of X2, and the peptide linker is from 3 to 10 amino acid residues long and contains at least
one arginine or lysine.
X2 is preferably an L-ornithine.
The peptide linker contains a recognition sequence for trypsin-like serum proteases and is
preferably ed from linkers having from 4 to 10 amino acid residues which contain at least
one arginine or . The linker preferably has the ing general formula:
(L1)n'R"(L2)m 0r (L1)n'K'(l-2)m
wherein n and m are rs from 1 to 8 and n + rn is from 3 to 9,
wherein the individual L1 and L2 are selected independently of one another from amino acids
having from 2 to 6 carbon atoms, preferably 2 or 3 carbon atoms, in particular glycine. alanine
and . Particularly preferably, m = 2 or 3. Preferably, (L2)m = SG.
More preferably, the linker has the following formula:
G(L)kRSG
where L is selected from alanine, glycine and serine and k is selected from integers from 1 to 6,
preferably 1, 2, 3 or 4.
Particularly preferred linkers are selected from the peptides GRSG, GARSG, GAARSG,
GAAARSG and GAAAARSG (SEQ ID No. 61 to 65). The polyethylene glycol polymer chain is
preferably bonded to the alpha-amino group of the first amino acid e of the linker,
preferably a glycine. The carboxyl group of the last amino acid e of the linker is bonded to
the amino group in the side chain of X2 (preferably the delta-amino group of an L-ornithine
residue). Trypsin and related serum proteases each cut downstream of the R (or K) in the
peptide , so that the peptide is released from the polyethylene glycol polymer chain. The
released peptide still contains the amino acid residues of the linker which were originally located
to the right (C-terminal) of the R (or K). these are preferably the amino acid residues SG. These
residues of the linker remaining in the released peptide are still bonded to the amino group in
the side chain of X2 (preferably the amino group of an L—ornithine residue).
Surprisingly, these remaining residues of the linker do not impair or ly impair the activity
of the antimicrobial peptide (apidaecin derivative).
Particularly preferred modified es (apidaecin derivatives) have the following peptide
ces:
ONORPVYIPRPRPPHPRL (SEQ ID No. 9) or
OONRPVYIPRPRPPHPRL (SEQ ID No. 10) where O = thine
wherein the alpha-amino group of the first ornithine is guanidated and a linear or branched
polyethylene glycol polymer chain is bonded to the delta-amino grOUp of the second ornithine
via a peptide linker chosen as above. preferably selected from GRSG, GARSG and GAARSG
(SEQ ID No.61 to 63).
Preliminary tests showed that a modification of the C-terminus leads to a large activity loss. A
certain activity loss is observed on incorporation of the side chain at position 1 (formula 3. Orn-1
in SEQ ID No. 11). therefore the substitution of Asn-2 and Asn-3 for Orn (SEQ ID No. 9 and 10)
is particularly red.
Somewhat less red modified peptides (apidaecin derivatives) have the following peptide
sequence:
ONNRPWIPRPRPPHPRL (SEQ ID No. 11) where O = L-ornithine
wherein the alpha-amino group of the first ornithine is guanidated and a linear or branched
polyethylene glycol polymer chain is bonded to the delta-amino group of the same ine via
a peptide linker selected from GRSG, GARSG and GAARSG (SEQ ID No. 61 to 63).
Preferred modified peptides have the following structures:
PEG75°-linker
Guan-ONNRPWIPRPRPPHPRL—OH
PEG5°°°-Iinker
Guan-ONNRPWIPRPRPPHPRL-OH
PEG750'Iinker
Guan-OONRPVYIPRPRPPHPRL-OH
PEG5°°°-linker
Guan-OONRPVYlPRPRPPHPRL—OH
PEG750-linker
Guan-ONORPWIPRPRPPHPRL-OH
PEG5°°°—linker
Guan-ONORPVYlPRPRPPHPRL—OH,
wherein linker represents the peptide linker, which is chosen as above, particularly preferably
GRSG.
cterial 4.
The invention provides a modified peptide derived from oncopeltus peptide
4 to 6:
which has a ce according to one of the general formulae
NT'X1'Dz‘Ka—PrPs-Ys'L7'P8'R9'P10'X2'P12'P13'R14-X3-l16'Y17-N18'X4'CT
(formula 4)
NT'X1'D2'Kit—P4“P5'Y6‘L7‘P8'R9'P10'X2"P12‘P13'R14'X3‘l16'Y17'N18'N19'x4'CT
(formula 5)
NT'X1'DZ'Ka—P4‘P5'Y6'l—7'PB‘RS'P1O'X2‘P12'P13‘R14'X3'I1S'P17'N18'X4‘CT
(formula 6)
amino acid residue whose side
X1 is a residue having a non-polar, hydrophobic side chain or an
net charge or a side
chain is velycharged under logical conditions, having a positive
chain that is positively charged under physiological conditions;
D2 is an aspartic acid or glutamic acid residue,
K3 is a residue having a positive net charge or a side chain that is vely charged under
physiological conditions, preferably lysine or arginine,
net charge
X2 and X4 are ed independently of one r from residues having a positive
or a side chain that is positively charged under logical conditions;
X3 is a radical having a positive net charge or a side chain that is positively charged under
physiological ions or proline or a proline tive;
L7 and I15 are selected independently of one another from residues having a non-polar,
hydrophobic side chain, preferably leucine, cine, valine and fert-leucine,
each asparagine or
Y6 and Y17 are each tyrosine. R9 and R14 are each arginine. N13 and N19 are
glutamine, P4, P5, P3, P10, P12, P13 and P17 are selected independently of one another from
proline and proline derivatives or hydroxyproline and hydroxyproline derivatives,
wherein P13 and R14 are optionally interchanged. and/or
optionally one or two residues selected from D2, P4, P5, P3. P10, P12 P13, P17 and Y17 are replaced
by any d residue,
NT is the N-terminus of the amino acid X1.
modified C-
CT is the free C-terminal carboxyl group of the C—terminal amino acid (~COOH) or a
terminal carboxyl group.
This peptide (oncopeltus peptide derivative) is distinguished in that a linear or branched
polyethylene glycol polymer chain is bonded via a peptide linker to NT, wherein the peptide
linker is from 3 to 10 amino acid residues long and contains at least one arginine or .
The linker preferably has the following general formula:
(L1)n'R or ~ (L1)n'K
wherein n is an r from 1 to 9 and the individual L1 are selected independently of one
another from amino acids having from 2 to 6 carbon atoms, preferably 2 or 3 carbon atoms.
particular glycine, alanine and serine. Particularly preferably, n = 1 to 3.
More preferably, the linker has the following formula:
G(L)kR
where L is selected from alanine, glycine and serine and k is selected from 1. 2 and 3.
Particularly preferred linkers are selected from the peptides GR, GAR and GAAR (SEQ ID No.
66).
X5 and X5 are optionally additional residues. Where X5 and X6 are absent, the last arginine (Arg)
in the above-mentioned sequence has a free C-terminal carboxyl group or is bonded to CT.
Where at least one residue X5 and X6 is present, the peptide has, for example, a sequence
ing to one of the general formulae 7 to 9:
NT-X1-D-K——P~P-Y-L-P-R-P—Xz-P-P-R-Xa-l-Y-N-X4—X5-X6-COR3
la 7)
NT-X1-D-K—-P-P-Y—L—P-R—P-Xz-P-P-R-Xs-l-Y-N-X4-X5-COR3
(formula 8)
D-K——P-P-Y-L-P-R-P—Xz-P-P-R—X3-l-Y-N—X4-X6-COR3
(formula 9)
X5 is selected from proline, proline derivatives or a neutral residue having a polar side chain
(such as asparagine, glutamine). Preferred residues X5 are selected from the groups comprising
proline, cishydroxyproline, transhydroxyproline, cishydroxyproline, trans—3-
hydroxyproline, B-cyclohexylalanine, 3,4—cis-methanoproline, 3,4-dehydroproline, homoproline,
pseudoproline as well as asparagine. glutamine, citrulline, N~methylserine, N—methylglycine,
dihydroxyphenylalanine, N-ethylasparagine, N—ethylglycine, homoserine. penicillamine,
ydropyranylglycine, allo-threonine and 3,5-dinitrotyrosine.
X6 is selected from proline, proline derivatives, a polar residue (such as serine) or a hydrophobic
residue. red residues Xe are selected from the groups comprising proline. cishydroxy-
proline, transhydroxyproline, cishydroxyproline, transhydroxyproline, B-cyclohexyl-
alanine, 3,4-cis-methanoproline, 3,4-dehydroproline, homoproline or pseudoproline, serine,
threonine, 6-hydroxylysine, citrulline, homoserine or allo-threonine as well as phenylalanine, N-
methylleucine, leucine, cine, valine, methionine, ted-butylglycine, cyclohexylalanine,
e, B—alanine, 1-aminocyclohexylcarboxylic acid
, N-methylisoleucine, norleucine,
norvaline, N-methylvaline, or it is a short peptide sequence having ably from one to three
residues, which residues
are preferably selected from proline, isoleucine or one of the
mentioned above.
Alternatively, X6 is a branched linker which contains a plurality of peptide units. This is formed
by the residue of an amino acid which contains a plurality of amino groups, such as, for
example, lysine, hydroxylysine, ornithine, 2,4—diaminobutyric acid, 2,3—diaminopropionic acid,
2,2'—diaminopimelic acid, desmosine, isodesmosine.
The C-terminal amino acid is, for example, X4 in formula 1, X5 (in formula 3) or X6 (in ae 2
and 4).
Particularly red modified peptides (oncopeltus e derivatives) contain the following
peptide seqences:
VDKPPYLPRPRPPROIYNO-NHZ (SEQ ID No. 12) or
VDKPPYLPRPRPHprHypTIeYNO-NHZ(SEQ ID No. 13)
where O = L~ornithine,
Hyp = Lhydroxyproline and
Tie = L~tertiary-leucine (L-tertiary—butylglycine),
wherein a linear or branched hylene glycol polymer chain is bonded via the peptide linker,
preferably GAR, to the N-terminal amino group (alpha-amino group of the first amino residue V).
The C—terminus (CT) is here a carboxylic acid amide (i.e. the carboxyl group of the last ornithine
has been converted into the amide).
The comments and preferred variants mentioned below apply generally to all the modified
peptides according to the invention (apidaecin derivatives and oncopeltus peptide derivatives):
The modified peptides according to the invention preferably contain at least 18 amino acid
residues, preferably up to 50 amino acid es.
For all the ed peptides according to the invention (apidaecin derivatives and oncopeltus
peptide tives), the polyethylene glycol polymer chain ably has a molecular weight of
from 500 to 40,000 Da, particularly preferably at least 5000 Da. The polyethylene glycol r
chain is in each case bonded covalently to the peptide linker. preferably to the N—terminal amino
to the
group of the linker, particularly preferably the alpha-amino group of the glycine. Coupling
peptide linker is carried out either directly (e.g. with an NHS ester-activated polyethylene glycol
to the amino group) or by means of a short organic linker (preferably C1 to C10) and/or by
formation of a thioether bond (e.g. by derivatisation of the N-terminal amino group of the ,
particularly preferably the alpha-amino group of the glycine, with etic acid and reaction
with a thiol-modified polyethylene glycol). The hylene glycol polymer chain is preferably
Hnean
In all the peptides according to the invention, the tryptic cutting site is inserted immediately
n the polymer and the antimicrobial peptide. An additional cleavable group, which is
removed, for example, by imination, is not present.
es having a side chain that is positively charged under physiological conditions are
preferably selected from arginine, lysine, 6~hydroxylysine. homoarginine, 2.4-diaminobutyric
acid, B—homoarginine, D-arginine, arginal (—COOH in arginine is replaced by -CHO), 2-amino
guanidinopropionic acid, nitroarginine rably N(G)-nitroarginine), nitrosoarginine (preferably
N(G)-nitrosoarginine), methylarginine (preferably N-methyl-arginine), s-N-methyllysine, allo-
hydroxylysine, 2,3-diaminopropionic acid, iaminopime|ic acid. ornithine, sym-
dimethylarginine, imethylarginine, 2,6—diaminohexinic acid, p-aminobenzoic acid and 3-
aminotyrosine and, less ably, histidine, 1-methylhistidine and ylhistidine. X1, X2 and
X3 are preferably selected independently of one another from this list.
The expression 'proline derivative‘ denotes an amino acid residue derived from proline, which is
obtained from proline preferably by structural alteration of a functional group. Preferred proline
derivatives are selected from B-cyclohexylalanine, 3.4-cis-methanoproline, 3.4-dehydroproline,
homoproline or pseudoproline. The term hydroxyproline includes inter alia cishydroxyproline,
transhydroxyproline, cishydroxyproline and 3-hydroxyproline. The expression
hydroxyproline derivative correspondingly denotes an amino acid residue derived from
hydroxyproline, which is obtained from hydroxyproline preferably by structural alteration of a
onal group. Preferred hydroxyproline derivatives are ed from hydroxy-B-
cyclohexylalanine and the above-mentioned proline derivatives, which are tuted by a
hydroxyl group.
A neutral residue is a residue having a side chain that is ged under physiological
conditions.
A polar residue preferably has at least one polar group in the side chain. These are preferably
selected from hydroxyl, sulfhydryl, amine, amide and ester groups or other groups which permit
the formation of hydrogen bridges.
Preferred neutral polar residues are selected from gine, cysteine, glutamine, serine,
threonine, tyrosine, citrulline, N~methylserine, homoserine, allo-threonine and 3,5-dinitrotyrosine
and B-homoserine,
The residues having a non-polar, hydrophobic side chain are residues that are uncharged under
physiological conditions, preferably with a hydropathy index above 0, ularly preferably
above 3. Preferred non-polar, hydrophobic side chains are selected from alkyl, alkylene, ,
alkenoxy, alkylsulfanyl and alkenylsulfanyl residues having from 1 to 10, preferably from 2 to 6,
carbon atoms, or aryl residues having from 5 to 12 carbon atoms. Preferred residues having a
non-polar. hydrophobic side chain are selected from leucine, isoleucine, valine, methionine,
alanine, phenylalanine, N-methylieucine, fen-butylgiycine, cyclohexylalanine, ine, 1—
aminocyclohexylcarboxylic acid, ylisoleucine, norleucine, ine and N-methylvaline.
“Under physiological conditions' is to be tood as meaning a pH of from 6 to 8 and a
ature of from 30°C to 40°C, preferably a temperature of 37°C, a pH of 7.4 and an
osmotic pressure of 300 mosmol/kg.
NT is the free N-terminus of X1 or a modified N-terminal amino group. CT is the free C-terminal
carboxyl group of the C-terminal amino acid (-COOH) or a modified C—terminal carboxyl group.
"Modified N-terminal amino group" and "modified C-terminai carboxyl group" means that the
amino group or carboxyl group has been altered (e.g. reduced or substituted).
NT accordingly ents the free N-terminus of the amino acid X1 or a modification of the N-
terminal amino group (which replaces the N-terminal amino group of the amino acid X1 by NT)
having the general formula NR1R2. NT = NR1R2, wherein R1 and R2 are independent of one
r and are preferably selected from hydrogen or from the following groups:
(i) a straight-chain, branched, cyclic or heterocyclic alkyl group, such as, for example,
methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or exyl;
(ii) a straight-chain. branched, cyclic or heterocyclic alkanoyl group, such as, for
example, acetyl or methanoyl (formyl), propionyl, n-butyryl, isobutyryl, pentanoyl,
hexanoyl or cyclohexanoyl;
(iii) a reporter group, preferably a fluorescent dye (such as eg. fluorescein, Alexa488) or
(iv) together with CORa (see below) a linker between the N- and C-terminus in order to
obtain a cyclic peptide, for example based on guanidine, ethylene glycol oligomers,
2,4-diaminobutyric acid. 2,3-diaminopropionic acid, iaminopimelic acid.
desmosine or isodesmosine.
(v) a linker for the coupling of a further peptide or peptide derivative (Y1) via a ic
al or enzymatic reaction, for example based on iodo-, bromo- or chloro-
alkanoic acids (e.g. etic acid)~ or maleimide for coupling to a thiol-containing
peptide, or also a different reactive group (e.g. amino group. thiol group) for the
coupling of a second peptide or peptide derivative (e.g. as active ester, aldehyde or
thioester) as carrier protein.
(vi) a linker as mentioned in (v), to which a further peptide or peptide derivative Y1 is
coupled.
Examples of N-terminal modifications are ated, formylated and preferably guanidated N-
termini.
Preferably. a further peptide or peptide derivative Y1 is coupled via NT. Y1 is preferably a
biopolymer (e.g. peptide) which introduces the antimicrobial peptide according to formula 1 into
bacteria and thereby increases the activity of the antimicrobial peptide towards that bacterium
and/or introduces it into mammalian cells and thereby permits the treatment of bacteria which
are concealed in mammalian cells. Y1 is linked via NT to X1 of the peptide either permanently
(e.g. peptide or amide bond for NT=NH2 or thioether for NT=SH, etate or maleimide) or
by a bond which is cleavable under certain conditions (such as e.g. ide bridges or acid-
labile s). Preferred sequences for Y1 are enetrating peptides (CPP). for example
penetratin, Tat peptides, model athic peptides and transportans (Langel, U. in Handbook
of Cell—Penetrating Peptides 5—28. CRC - Taylor & Francis Group, 2006).
A linker is a name for les or molecule groups which are used to link two substances,
preferred linkers contain two reactive groups (such as e.g. iodoacetate, maleimide, imido- or
NHS-ester or hydrazide), which are bonded by a molecule bridge (e.g. polyethylene glycol)
having preferably from 10 to 20 carbon atoms.
CT is the free C-terminal yl group of the C-terminal amino acid (-COOH) or a modified C-
terminal carboxyl group, preferably having the l formula CORg (R3 replaces the hydroxyl
group of the last amino acid), Xs-COR3 or Xs—CORa or X5X6-COR3.
COR3 is preferably selected from the ing groups:
i. carboxyl (R3 is a free hydroxyl , an ester (R3 is an alkoxy group), an amide (R3 is
an amine) or an imide;
ii. a linker which, together with NT, bridges the N- and C-termini to a cyclic peptide;
iii. COR3_ wherein R3 is either an additional amino acid residue selected from the group
comprising Pro, lle, Leu, Arg and Gin, or wherein R3 is a peptide having preferably from
two to six amino acids, of which at least one 'amino acid is selected from the group
comprising Pro, lle, Leu, Arg and Gln, wherein this is substituted by a member from the
group with carboxyl (R3 is a free hydroxyl group), an ester (R3
is an alcohol, such as
methanol, ethanol, propanol, isopropanol or butanol), an amide (R3 is an amide) or an
imide (R3 is an mine or dialkylamine, such as methylamine, ethylamine,
dimethylamine or cyclohexylamine).
iv. COR; wherein R3 is an additional, branched amino acid, in order to form a dimeric or
oligomeric structure, such as, for example, lysine, hydroxylysine, ornithine, 2,4-
diaminobutyric acid, 2,3-diaminopropionic acid. 2,2‘-diaminopimelic acid, desmosine,
mosine or a combination of these branched amino acids.
v. a linker for the coupling of a further peptide or peptide derivative (Y1) via a specific
chemical or enzymatic on, for example based on iodo-, bromo- or chloro-alkanoic
acids (e.g. iodoacetic acid) or ide for coupling to a thiol-containing peptide or also
another reactive group (e.g. amino group, thiol group) for the coupling of a second
peptide or peptide derivative (e.g. as active ester, aldehyde or thioester) as carrier
protein.
vi. a linker as mentioned in (v), to which a further peptide or peptide derivative Y1 is
coupled.
In this manner, inal peptide derivatives can be obtained as ester (R3 = ), amide (R3
= amine, e.g. -NH2 or imine, e.g. —NH03H7) or imide or a peptide that has been lengthened by
r amino acids selected from the group containing Pro, lle, Arg and Val and have likewise
again been modified at the C-terminus as ester, amide or imide. Further peptide derivatives can
be formed by modifications of the N-terminal or C-terminal ends of the peptides. These changes
can be, for example, an additional alkyl or alkanoyl group (either with a straight chain or
branched, cyclic or heterocyclic) or an additional ino group or an additional
macromolecule or a reporter e, which is linked either permanently or by a compound that
is cleavable under certain conditions (such as disulfide bridges or acid-labile linkers).
A modification of the C-terminus preferably takes place by means of thioester synthesis and
subsequent substitution with y amines.
All the l amino acids, non-natural amino acids or amino acid derivatives (such as e.g.
imino acids) which form the peptides or peptide derivatives according to the invention can be
present in either the L- or D-conformation. Unless specified otherwise, the structural units in the .
ces are, r, preferably in the L—conformation.
such
The modifications of the N- and ini allow the peptides to be coupled to other groups,
as, for example, other amino acid sequences (multimeric peptides or proteins optionally being
created thereby) or other biomolecules which have the on of a carrier or label, for example
of Y1 via NT. In a specific embodiment, the molecule functions as a r for combating the
bacterial infection in mammalian cells or transporting the cterial peptide and peptide
derivative into bacteria into which the antibacterial peptide cannot penetrate on its own (e.g.
gram-positive bacteria). Examples of such cell-penetrating peptides (CPP) are, for example,
penetratins, Tat peptides, model amphipathic es and transportans. in addition, the site of
the infection can be recognised by the coupled ure t le) and the antibiotic
substance can thereby be brought into the vicinity of the (bacterial) cell in order to combat it.
Such target molecules are, for example, molecules that are known to bind to lipopolysaccharide
(LPS) molecules, which form the outside of the gram-negative bacteria. Known compounds for
this application are, for example, anchor peptides, such as the AcmA motif from Lactobacillus or
is preferred because it also
an antibody directed against iysaccharide. The latter variant
of the
has the
an intrinsic antibiotic effect and can therefore be used to increase activity
peptides according to the invention.
By the coupling of a cell-penetrating peptide sequence, such as penetratin, it is possible on the
one hand to increase the activity towards gram-negative and gram-positive bacteria, or
extend the spectrum of action to other gram-positive and gram-negative bacteria, and on the
other hand to introduce the antimicrobial peptides into mammalian cells, so that bacteria, fungi
in those cells can also be reached. The coupling of penetratin via a
or viruses concealed
thioether bridge forms part of the invention. The inus of the atin was thereby
at the N-terminus
lengthened by a cysteine and coupled to the antimicrobial peptide labelled
with etic acid.
The term "peptide" as used here denotes a ce of amino acids which are linked via a
peptide bond, wherein the amino acids are preferably selected from the twenty proteinogenic
amino acids and wherein the amino acids can be present in the L-configuration or D-
configuration, or in the case of isoleucine and threonine also in the D—allo-configuration (only
inversion of one of the two chiral centres). Also included are peptides which have been altered
by substitutions and/or modifications of one or more amino acid residues by chemical groups,
such
those chemical groups being other than the natural protein-forming amino acid residues,
acids or peptide having an d
as. for example. non-proteinogenic a-amino acids, B-amino
backbone. The sion "altered backbone" means that at least one peptide bond has been
chemically modified. that is to say replaced by a bond that is not cleavable under physiological
conditions and cannot be cut by endoproteases.
The non-cleavable bond is preferably a modified peptide bond such as, for example, a reduced
e bond, an alkylated amide bond or a thioamide bond. A reduced amide bond is a peptide
bond in which the carbonyl group (C=O) has been reduced to a hydroxyl group (HCOH) or a
methylene group (CH2). An alkylated amide bond is a peptide bond alkylated at either the
nitrogen (N-alpha) or carbon atom (C-alpha). The alkyl residue has preferably from 1 to 3
carbon atoms. An example is N-methylation.
The expression ‘altered backbone‘ additionally includes other groups which are suitable for
forming a covalent bond both with the COOH group of the ing amino acid residue and
with the NH2 group of the following amino acid residue, and which therefore do not necessarily
maintain the peptide backbone structure, such as, for example, sugar amino acid dipeptide
isosters, azapeptides, 6-homopolymers, gamma-peptides, depsipeptides (ester bridges in the
backbone), Y-lactam analogs, oligo(pheny|eneethylene)s, vinylogous sulfone peptides, poly-N-
substituted glycines or arbamates. Modifications of the backbone are preferred at
ons that are susceptible to enzymatic degradation, particularly at the six C-terminal
es of the peptides according to formulae 4 to 9 (oncocin or oncopeltus peptide
derivatives), particularly preferably positions 14 to 19, R-Xa—l16-Y17-N18-X4). Therefore, preferably
at least one of the bonds n X3416 (e.g. Arg-lle), N18-X4 (e.g. g), X4-NH2 (e.g. Arg-
NH2), Xe—X7 (e.g. Arg-Leu or Arg-lle) is a bond that is not ble for proteases. This non-
cleavable bond is preferably selected from the group of the reduced amide bonds, alkylated
amide bonds or ide bonds.
The es according to the invention are preferably linear. atively, the peptides, in
particular the apidaecin derivatives, are also cyclic, that is to say preferably the first (N-terminus)
and the last amino acid (C—terminus) are linked via a peptide bond or a linker. However,
cyclisations between a side chain (e.g. lysine) and the C-terminus, a side chain (e.g. glutamic
acid or aspartic acid) and the N-terminus or between two side chains (e.g. lysine and glutamic
acid or aspartic acid) are also included.
Methods for the preparation of the above-mentioned novel compounds having antibiotic activity
also form part of this invention.
‘The peptides or peptide derivatives of this invention can be prepared either synthetically or,
where applicable, recombinantly by conventional s. Preferably, the peptides or peptide
derivatives of this invention are prepared tionally using the known synthesis techniques,
as described, for example, by Merrifield. Alternatively, the peptides bed
in this invention
contains a nucleic
are prepared by recombinant techniques, by cloning a DNA fragment which
acid sequence that codes for one of the above-described peptides and expressing it, for
example, in a microorganism or a host cell. The coding nucleic acid sequences can be ed
synthetically or obtained by side-specific mutagenesis of an existing nucleic acid sequence (e.g.
sequence coding for the wild-type ltus 4). The coding sequence so prepared can be
ied by RNA (or DNA) with correspondingly ed primers in a polymerase chain
reaction (PCR) by known ques. After purification, for example by means of agarose gel
electrophoresis, the PCR product is ligated into a vector and the host cell is finally transformed
with the corresponding recombinant d. Recombinant techniques are known for various
host cells, for example E. coli, Bacillus, Lactobacillus, Streptomyces, mammalian cells (e.g.
CHO (Chinese hamster ovary) or COS-1 cells), yeast cells (e.g. Saccharomyces,
Schizophyllum), insect cells or viral expression systems (e.g. baculovirus system). After
conventional recombinant ation, the peptides of this invention can be isolated from the
host cells, either by tional cell disruption techniques or from the cell medium by
conventional methods, for example liquid chromatography, in particular affinity chromatography.
The antimicrobial peptide can be expressed as a single peptide or as an oligomer. The
oligomers can thereby contain a plurality of e sequences, which are linked via the N— or C-
terminus, or even contain an N- or C-terminal tag, which permits easier purification of the
recombinant peptides or protein ucts. Conventional techniques of molecular biology and
side-specific mutagenesis can be used to alter the sequence further and thus obtain the desired
non-native e sequences. These recombinant techniques have already been used for
many antimicrobial peptides including apidaecin (see eg. Maeno M et al. 1993. Biosci
Biotechnol Biochem 57: 1206—7).
It is also possible to introduce amino acids that do not occur naturally into the peptides by
genetic engineering (Noren C et at. 1989. Science 244: 182-8; Ellman J et al. 1991 Methods
Enzymol. 202: 301 -36).
The peptides can then be isolated from the host cell culture or the in vitro ation system.
This can be ed using the conventional techniques for protein purification and isolation,
which are known from the prior art. Such techniques can include, for example,
immunoadsorption or affinity chromatography. It is additionally possible to provide the peptides
with a tag (e.g. histidine tag) during the synthesis, which permits rapid binding and purification.
The tag can subsequently be cleaved enzymatically in order to obtain the active e
sequence.
If the peptide itself cannot be coded or expressed but is very similar to a codable or expressible
peptide, the method can first be applied to the similar peptide, in order subsequently to convert
it chemically or tically in one or more steps into the desired peptide or peptidomimetic.
The modified peptides according to the invention can be used individually, in combination, as
multimers or as branched multimers. Expedient combinations of the peptides according to the
invention e concatamers, in which the es ing to the invention are linked with
one another sequentially or via spacers, for example in the form of a peptide dimer or a peptide
trimer etc., by the individual peptides being strung together. This multimer can be composed of
peptides or peptide derivatives having identical ces or ent sequences according to
formula 1.
The modified peptides can additionally be coupled to a biocompatible protein, for example
human serum albumin, humanised antibodies, liposomes, micelles, tic polymers,
nanoparticles and phages. atively, multimers, in which the peptides or peptide tives
according to the invention are combined individually, can be prepared in the form of dendrimers
or rs, wherein three or more peptides are bonded to one centre.
In one ment, a plurality of peptides can be prepared as eric constructs or
arrangement. Thus, for example, optional amino acids (eg. Gly-Ser—) or other spacers based on
amino acids or other chemical compounds can be attached to the N- or C-terminus in order to
link two or more peptides with one another or couple them to a carrier. This arrangement can
take the form of one or more of the above-described synthetic peptides d to a carrier
protein. atively, an arrangement contains a plurality of peptides, each expressed as a
multiple antigenic peptide, optionally coupled to a r protein.- In a further variant. the
selected peptides are linked sequentially and are expressed as recombinant protein or
polypeptide. In one embodiment, a plurality of es are linked sequentially, with or without
amino acids as spacers. in order to obtain a larger recombinant n. Alternatively, the
recombinant protein can be fused to a carrier protein.
In another embodiment, the multimeric constructs contain at least two peptides, wherein one
peptide is coupled to the other peptide via any desired amino acid. Any desired 'number of
further peptides can be attached to any desired further amino acids of these peptides. In a
further embodiment of a multimeric arrangement which contains at least two peptides, the
second peptide or the further peptides is/are coupled to a branched structure of the other
es of the basic structure. Alternatively, each further peptide is linked covalently via the
group NT or CT to another peptide of the arrangement.
In another embodiment of a multimeric construct or an ement having at least two
peptides. at least one or more peptides are bonded to a carrier. In another embodiment, one or
more of the mentioned peptides is/are a synthetic peptide fused to a carrier protein.
Furthermore, there is the alternative of combining a plurality of the above-described peptides
tially, with or without flanking sequences, to form a linear polypeptide. The peptides or
the polypeptide are either d to the same carrier, or different peptides can be coupled
individually as peptides to one or different immunologically inert carrier proteins.
Suitable carriers can improve the stability, the administration or the production, or can alter the
activity spectrum of the peptides. Examples of carriers are human albumin, polyethylene glycol
or other biopolymers or other naturally or non-naturally occurring rs. In an embodiment.
the main component is preferably a protein or other molecule which can se the peptide
stability. An experienced person can easily select a suitable coupling unit.
in yet another embodiment, the peptides are arranged in the form of a multiple antigenic peptide
(MAP). This system uses a central unit of lysine residues onto which several copies of the same
peptide according to the invention are synthesised. Each MAP contains several copies of one or
more of the peptides according to the invention. One form of an MAP ns at least three but
preferably four or more peptides. A person skilled in the art can easily prepare any desired
number of multimeric compounds according to the peptides identified in the above formula. All
such multimeric ements and constructs are to form part of this invention.
Further combinations in the form of multimers can be prepared at the e of les, the
peptides or peptidomimetics being ted on their surface. The le can then function as
a carrier of a peptide or peptidomimetic and can at the same time act as a detectable marker.
ers can be obtained, for example, by N—terminal biotinylation of the N-terminal end of the
peptide or peptidomimetic chains and subsequent complex formation with streptavidin. Because
streptavidin is able to bind four biotin molecules or conjugates with high affinity, very stable
tetrameric peptide complexes are obtained by this method. ers can be prepared from
identical or ent peptides or peptidomimetics according to the invention. Preferably, the
multimers according to the invention contain two or more es or peptidomimetics, in which
each component makes a certain bution to the biocidal activity (target recognition,
antimicrobial activity, purification).
This invention also provides the use of the peptides or peptide derivatives according to the
ion in'medicine or pharmacy, for example for y with an antibiotic or in a composition
having antimicrobial (in particular bactericidal) activity.
The ion also provides the peptides modified according to the invention for use in
ne, as an antibiotic, in a disinfectant or cleaning agent, as a preservative or in a
packaging material. The modified peptides according to the invention are ularly suitable for
the treatment of microbial, bacterial or fungal infections.
The invention also provides the use of the peptides modified according to the invention for the
preparation of a medicament, in particular of an antibiotic, in particular for the treatment of
microbial infections, for example by bacteria, viruses and fungi.
.This invention r es pharmaceutical compositions which comprise one or more
peptides modified according to the invention or multimeric constructs, independently of the,
presence of other pharmaceutically active compounds.
The use of the peptides according to the invention as a pharmaceutical agent and/or for the
preparation of an active ingredient that can be used as an antibiotic also forms part of this
invention.
The ed peptides can also be used individually in pharmaceutical products. Alternatively,
one or more modified peptides, as described above, can be fused or conjugated to another
nd in order to increase the pharmacokinetics or bioavailability without triggering an
immune response. Any desired number of individual es or eric constructs can be
mixed with one another in order to prepare an individual composition.
A pharmaceutical composition according to the invention comprises a therapeutically effective
amount of one or more ed peptides of this invention. Once composed, the pharmaceutical
composition according to the invention can be stered directly to the subject in order to
treat microbial (in particular bacterial) ions. To that end, a therapeutically effective amount
of a composition ing to the invention is administered to the subject to be treated.
The compositions according to the invention are ed to treat infections of a mammal,
including a human being, infected with bacteria or fungi. At least one or alternatively also a
plurality of peptides or multimeric constructs according to the invention can be mixed with a
pharmacologically acceptable carrier or other components to form a composition having
antimicrobial (in particular antibacterial or fungicidal) activity. For the use of such a composition,
the chosen peptide is preferably prepared synthetically or also recombinantly, as described
above.
The direct stration of this composition is carried out locally or systemically, preferably
orally, parenterally, intraperitoneally, enously, intramuscularly, pulmonally or interstitially
into the tissue.
The pharmaceutical composition can comprise funher le and pharmaceutically acceptable
carriers, extenders or solvents and can be in the form of a capsule, tablet, pastille, dragée, pill,
drop, suppository, powder, spray, vaccine, ointment, paste, cream, inhalant, patch, aerosol or
the like. There can be used as ceutically acceptable carriers solvents, extenders or
other liquid s such as sing or suspension aids, surface-active agents, isotonic
active ingredients, thickeners or emulsifiers, preservatives, encapsulating agents, solid binders
or ts, according to what is most suitable for the particular dosage and at the same time
ible with the peptide, peptidomimetic (peptide derivative), peptide conjugate or
peptidomimetic conjugate.
The pharmaceutical composition therefore preferably comprises a pharmaceutically acceptable
carrier. The expression "pharmaceutically acceptable carrier" in this case also es a r
for administration of the therapeutic composition, such as, for example, antibodies or
polypeptides, genes or other therapeutic agents. The expression relates to any desired
pharmaceutical carrier which does not itself trigger the tion of antibodies, which could be
harmful for the individual to whom the formulation has been administered, and which does not
possess excessive toxicity. Suitable "pharmaceutically acceptable carriers" can be large, slowly
degradable macromolecules, such as, for example, proteins, polysaccharides, polylactonic
acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactivated virus
tuents. Such carriers are well known to the person skilled in the art.
Salts of the peptides or functionally equivalent compounds can be prepared by known methods,
which lly means that the peptides, peptidomimetics, peptide conjugates or peptidomimetic
conjugates are mixed with a pharmaceutically able acid to form an acid salt or with a
pharmaceutically acceptable base to form a basic salt. Whether an acid or a base is
pharmaceutically acceptable can easily be determined by a person skilled in the art with
knowledge of the application and the formulation. Thus, for example, not all acids and bases
that are acceptable for ex vivo applications can also be transferred to therapeutic formulations.
Depending on the particular application, pharmaceutically acceptable acids can be both organic
and inorganic in nature, for example formic acid, acetic acid, propionic acid, lactic acid, glycolic
acid, oxalic acid, c acid, succinic acid, maleic acid, malonic acid, cinnamic acid, sulfuric
acid, hydrochloric acid, hydrobromic acid, nitric acid, perchloric acid, phosphoric acid and
thiocyanic acid, which form ammonium salts with the free amino groups of peptides and
onally equivalent compounds. Pharmaceutically acceptable bases, which form
carboxylates with the free carboxylic acid groups of the peptides and functionally equivalent
compounds, include ethylamine, methylamine, dimethylamine, ylamine, isopropylamine,
diisopropylamine and other mono-, di- and tri-alkylamines, as well as arylamines.
Pharmaceutically acceptable solvents are also included.
Pharmaceutically acceptable salts can be used therein, such as, for example, salts of mineral
acids, such as hydrochlorides, hydrobromides, phosphates, sulfates and the like; but also salts
of organic acids, such as acetates, propionates, tes, benzoates and the like. A
comprehensive discussion of pharmaceutically acceptable ingredients will be found in
Remington's ceutical Sciences (Mack Pub. Co., N.J., 1991).
Pharmaceutically acceptable carriers in the therapeutic compositions can contain liquids, for
example water, salt water, glycerol and ethanol. In addition, auxiliary substances can be added,
such as humectants or emulsifiers, pH-buffering substances and similar compounds can be
present in such . Typically, the therapeutic compositions are prepared either in liquid
form or as a suspension for injection, solid forms for dissolution or suspension in r s
prior to injection are likewise possible. mes are also included in the definition of a
aceutically acceptable r“.
For therapeutic treatment, modified peptides or peptide conjugates, as described above, can
produced and administered to a subject requiring them. The peptide or peptide conjugate can
be administered to a subject in any d suitable form, preferably in the form of a
pharmaceutical composition which is adapted to the dosage form and is present in a dose
appropriate for the desired treatment.
The pharmaceutical compositions of this invention can comprise further active compounds, for
example tional antibiotics (e.g. vancomycin, streptomycin, tetracycline, penicillin) or
other antimicrobially active compounds, such as fungicides, for e intraconazole or
zole. Other nds which alleviate the symptoms associated with the infection,
such as fever (salicylic acid) or skin eruption, can also be added.
In addition to the therapeutic use for the treatment of infections, or also in biological warfare, it is
further possible to use the es or peptide derivatives according to the invention in
disinfectants or cleaning agents (e.g. a bactericidal composition) which can be used for the
disinfection or cleaning of surfaces or objects. Another field of application is packaging, where
peptides can be bound to packaging material or incorporated therein, or as preservatives
other als which can y be degraded by microorganisms. The peptides or peptide
derivatives according to the invention are suitable in particular for the packaging of foodstuffs,
because they are not toxic either on contact or on ingestion.
A method for treating mammals ed with microbes (in particular ia or fungi), including
active
the administration of an effective, therapeutically effective amount of the pharmaceutically
composition according to the invention, forms another part of this invention.
The expression "therapeutically effective amount" used here denotes the amount a
therapeutic agent, that is to say of a peptide, peptidomimetic, peptide conjugate or
peptidomimetic ate according to the invention, which is capable of reducing or completely
preventing the multiplication and colony formation of the ia or of achieving measurable
eutic or prophylactic success. The effect can be determined, for example, for biopsies
culture, by testing the antibacterial activity or by another suitable method for assessing the
extent and degree of a bacterial infection. The exact ive amount for a subject depends
its size and state of health, on the nature and extent of the disease and on the therapeutic
agents or the combination of several therapeutic agents chosen for the treatment.
compositions according to the invention can be used in particular for reducing or preventing
Methods of
bacterial ions and/or biological or associated physical symptoms (e.g. fever).
determining the initial dose by a doctor are known from the prior art. The doses determined
must be safe and sful.
that is necessary for
The amount of a protein, peptide or nucleic acid according to the invention
consideration of the pathogen that causes
an antibacterially effective dose can be determined in
the infection, the severity of the infection, as well as the age, weight, , l physical
condition etc. of the patient. The necessary amount of the active component, in order to be
antibacterially and antimycotically effective without e side-effects, depends on the
pharmaceutical formulation used and the possible presence of further constituents such as
effective dose
antibiotics, antimycotics, etc. For the fields of use according to the invention, an
between 0.2 nmollkg and 10 nmollkg
can be between 0.01 nmollkg and 50 nmollkg, preferabiy
of the peptide, peptidomimetic, peptide conjugate or peptidomimetic conjugate in the treated
individual.
Initial doses of the peptides, omimetics, ers, peptide ates or peptidomimetic
conjugates according to the ion can optionally be ed by repeated
administration.
is preferably between
The frequency of the doses depends on the factors identified above and
of approximately from three days to not more
one and six doses per day over a treatment period
than one week.
in a specific amount, for
In a further embodiment, the compounds are administered pulmonally
formulations
e by an inhaler, atomiser, aerosol spray or a dry powder inhaler. Suitable
Transdermal or rectal application, as well
can be prepared by known methods and ques.
as administration into the eye, may in some cases be appropriate.
invention more effectively
It can be advantageous to administer the substances according to the
Thus, if the
by advanced forms of drug delivery ced drug delivery or targeting methods).
digestive tract is to be avoided, the dosage form can comprise any desired substance or
that increases the ilability. This can be ed, for example, by reducing the
degradation, for example by an enzyme inhibitor or an antioxidant. It is better if the
bioavailability of the nd is achieved by increasing the permeability of the absorption
in this manner;
barrier, in most cases the mucosa. Substances that facilitate penetration can act
increase the fluidity of the mucosa while others widen the interstices between the
some
mucosal cells. Yet others reduce the viscosity of the mucous on the mucosa. The preferred
uptake rators include amphiphilic substances such as cholic acid tives,
phospholipids, ethanol, fatty acids, oleic acid, fatty acid derivatives, EDTA, carbomers,
rbophil and chitosan.
Indications for which the modified es according to the invention, their conjugates or
multimers can be used are bacterial infections with both gram-positive and gram-negative
bacteria, for example Escherichia coli, Enterobacter cloacae, Erwlnla amylovora, Klebsiella
Salmonella
pneumoniae, Morganella morganll, Pseudomonas aeruglnosa, Salmonella lyphimun'um,
typhi, Shlgella dysenten‘ae, Yerslnia enterocolitica, obacter calcoaceticus, Agrobacten'um
tumefaciens, Franolsella tularensis, Legionella pneumophila, Pseudomonas syn'ngae, Rhizobium
melilotl, Haemophllus influenzae.
The invention is explained below by the following exemplary embodiments and figures,
limiting the invention thereto:
72 and Oncl10 (right)
Fig. 1 shows the serum stability of Api 300 ($6), Api 301 (86) , Onc
and the degradation
in mouse serum (100 %). The respective starting products (solid line)
products (broken line), where detected, are shown.
Fig. 2 shows the serum stability of Api 137 (sPEG75°-GRSG) (left, solid line) and Api 137
(PEGm-GARSG) (right, solid line) in 25% aqueous mouse serum (100 %). The active
ingredient ed Api 137 (SG) (broken line) und and the resulting degradation product,
ned at the C-terminus, Api 137 (SG) 1-16 (dotted line).
and Api 300 (tPEGSm-GRSG)
Fig. 3 shows the serum stability of Api 300 (tPEGm-GRSG) (left)
the active ingredient
(right) in mouse serum (100 %). The respective starting products (triangle),
released Api 300 (SG) (lozenge) and its degradation product shortened at the C-terminus
Api300 (SG) 1-16 (asterisk) are shown.
and Api 301 (tPEG5°°°-GRSG)
Fig. 4 shows the serum stability of Api 301 (tPEGm-GRSG) (left)
the active ingredient
(right) in mouse serum (100 %). The respective starting products (triangle),
released Api 301 (SG) (lozenge) and its degradation product shortened at the C-terminus
Api301 (SG) 1—16 (asterisk) are shown.
G 000_5 Fig. 5 shows the serum stability of Api 300 (tPEGm-GARSG) (left) and Api 300 (tPE
the active
GARSG) (right) in mouse serum (100 %). The respective startingproducts (triangle),
ingredient released Api 300 (SG) (lozenge) and its degradation product shortened
at the C-
terminus Api300 (SG) 1-16 (asterisk) are shown.
(tPEG5°°°- Fig. 6 shows the serum stability of Api 301 5°-GARSG) (left) and Api 301
the active
GARSG) (right) in mouse serum (100 %). The tive starting products (triangle),
ingredient released Api 301 (SG) (lozenge) and its degradation product shortened at the C—
us Api301 (SG) 1-16 (asterisk) are shown.
Fig. 7 shows the serum stability of Api 300 (tPEG75°-GAARSG) (left) and Api 300 (tPEG5°°°-
the active
GAARSG) (right) in mouse serum (100 %). The respective starting ts gle),
its degradation t ned at the C- ingredient released Api 300 ($6) (lozenge) and
terminus Api300 (SG) 1-16 (asterisk) are shown.
Fig. 8 shows the serum stability of Api 301 (tPEG75°—GAARSG) (left) and Api 301
(tPEG5°°°~
the active
GAARSG) (right) in mouse serum (100 %). The respective starting products (triangle),
at the C—
ient released Api 301 (SG) (lozenge) and its degradation t shortened
terminus Api301 (SG) 1-16 (asterisk) are shown.
and tPEG5°°°—GAR-Onc 72 (right)
Fig. 9 shows the serum stability of tPEG75°-GAR-Onc 72 (left)
and the active ingredient
in mouse serum (100 %). The tive starting products (triangle)
of Onc72 were not detected.
released Onc72 (lozenge) are shown. Degradation products
Fig. 10 shows the serum stability of tPEGm-GAR-Onc 110 (left) and tPEGmo-GAR-Onc 110
the active
(right) in mouse serum (100 %). The respective starting ts (triangle) and
of One 110 were not
ingredient released Onc 110 (lozenge) are shown. Degradation products
detected.
Example 1 Peptide synthesis and modification
1. e synthesis:
for the peptide synthesis were obtained, unless othenlvise ted, from
All the chemicals
Fluka Chemie GmbH (Buchs, rland) in the greatest possible purity.
using
The peptides were synthesised by means of conventional solid-phase peptide synthesis
obtained from MultiSynTech GmbH
the Fmoc/'Bu strategy. All standard Fmoc amino acids were
Transhydroxyproline
(Witten, Germany) or Orpegen Pharma GmbH (Heidelberg, Germany).
Biosciences GmbH,
(t-4—Hyp) and tert—Ieucine were obtained from Novabiochem (Merck
Darmstadt, Germany).
The peptides were synthesised on a 25 umol scale with the
aid of a SYRO 2000 peptide
42 mg of Leu-Wang resin
synthesis robot SynTech GmbH, Witten, Germany). To that end.
swollen for 30 minutes in DMF.
(load: 0.6 mmol/g) were weighed out per sis reactor and
with the
After cleavage of the N-terminal Fmoc protecting group, the peptides were synthesised
following synthesis cycle (Table 2).
2000)
Table 2: Synthesis cycle of the automatic multiple solid-phase peptide synthesis (SYRO
for coupling of an amino acid.
Synthesis step ts Reaction time
Amino acid coupling 8 eq. amino acid in HOBt/DMF (0.5 mol/l) and 60 min
DIC/DMF (2.0 moi/l)
1 min
Washing 3 x with 600 pl DMF each time
3 min
Fmoc cleavage 400 14'400/0 piperidine/DMF
min
500 pl 20% piperidine/DMF
1 min
Washing 6 x with 600 pl DMF each time
microwave peptide synthesis robot
For synthesis on a 100 pmol scale with the aid of a Liberty
(CEM GmbH, Kamp-Lintfort, Germany). 156.3 mg of commercially available Leu—Wang
resin
r and swollen for
from NovaBiochem (load: 0.64 mmol/g) were d out per synthesis
the peptides were
s in DMF. After cleavage of the N-terminal Fmoc protecting group,
inter alia sised with the following synthesis cycle (Table 3).
for coupling of
Table 3: sis cycle of the automatic multiple phase synthesis (Liberty)
the individual amino acids.
Cycle Synthesis step ts Reaction
time
Amino acid Washing 7.0 ml DMF
(0.10-single) Fmoc cleavage 7.0 ml 20% piperidine 0.5 min
watts at 75°C
Washing 5.0 ml DMF
Fmoc cleavage 7.0 ml 20% piperidine
watts at 75°C
Washing 4 x with 7.0 ml DMF each time
Coupling 2.5 ml 0.2 molll amino acid in DMF
1.0 ml 0.5 mot/l HBTU
watts at 75°C
Washing 3 x with 7.0 ml DMF each time
The peptides were synthesised either as acid on Wang resin or Leu-Wang
resin or as acid
amide Rink amide 4-methylbenzylhydrylamine (MBHA) resin (0.67 mmol/g) from
MultiSynTech GmbH (Witten. Germany).
There were used as side chain protecting groups nylmethyl (trityl) for Cys. Asn,
His and
tert—butyl ether (‘Bu) for Tyr, Ser and Thr, ted-butyl ester (O'Bu) for Asp and Glu, w-N- Gln,
6,7-pentamethyldihydrobenzofuran—S-sulfonyl (be) for Arg, tert—butyloxy-carbonyl (Boc)
cleaved for Lys and Om. The temporary Fmoc protecting group was with 40% piperidine
and again with fresh 20%
(Biosolve BV, Valkenswaard, Netherlands) in DMF (v/v) for 5 minutes
piperidine in DMF (v/v) for 10 minutes.
2. Modification of the peptides:
a.) Guanidation of the N-terminus:
The resin (1 eq.) was first swollen for 20 minutes in DMF. it was then ted for 3 hours at
in DMF (0.5 moi/l. 10 eq.) and NMM (10 eq.).
room temperature and with shaking with HBTU
The reaction solution was then renewed and the suspension was incubated
for a further 4
off and the resin
hours. When the reaction was complete, the reaction solution was separated
was washed six times with DMF or DCM.
b.) lodoacetylation
acid (8 eq.) and HOBt in
The resin (1 eq.) was first swollen for 20 minutes in DMF. lodoacetic
activated with BIG (8 eq.). The
DMF (0.5 mol/l, 8 eq.) were then added, and the reaction was
reaction mixture was then incubated for 16 hours at room temperature
in the dark and with
off and the resin
g. When the reaction was complete. the reaction solution was separated
then dried.
was washed six times with DMF or DCM and
of the peptide or of the side chain was
The completeness of the modification at the N-terminus
ted at 95°C with 0.28
checked by means of the Kaiser test. To that end. some resin was
molll ninhydrin (Riedel de Haen, Seelze, Germany) in ethanol (Carl Roth GmbH + Co. KG,
Karisruhe, Germany), 0.2 mmolll potassium e in pyridine and 76% phenol
in ethanol in
amino groups, the
the ratio . If a bIUe colouration occurred, which indicates free primary
ng was repeated.
3. Coupling of the polyethylene glycol:
e polyethylene glycol has hygroscopic properties, it was dried under a high vacuum
before each coupling reaction.
The following polyethylene glycol derivatives (all Iris Biotech GmbH. Marktredwitz, Germany, >
described below:
95 % purity) were used as ng materials for the ngs
MeO-PEGZ°°°°-
a-Methoxy-w-carboxylic acid polyethylene glycol (20000 Da)
COOH
MeO-PEG75°-
a-Methoxy-w-carboxylic acid polyethylene glycol (750 Da)
COOH
MeO—PEGs°-°°~
d-Methoxy-w-carboxylic acid polyethylene glycol (5000 Da)
COOH
MeO-PEGma-Methoxy
—w-mercapto polyethylene glycol (750 Da)
MeO-PEG5°°°-
d-Methoxy-w-mercapto polyethylene glycol (5000 Da)
for MeO-PEG-COOH) via
a.) Coupling of polyethylene glycol with a free acid group (sPEGl e.g.
DIC and HOBt: .
The polyethylene glycol derivative (5
The resin (1 eq.) was first swollen for 20 minutes in DMF.
reaction was ted
eq.) dissolved in HOBt/DMF (0.5 moi/I, 5 eq.) was then added. and the
hour at RT and with shaking.
with BIG (5 eq.). The reaction mixture was then incubated for one
carried out for a further 30 minutes.
DIC (2.5 eq.) was then added again, and incubation was
off and the resin was
When the reaction was te, the reaction on was separated
of the Kaiser test.
washed six times with DMF. Complete coupling was checked by means
for PEG5000 and greater:
b.) Coupling after activation of the polyethylene glycol, in ular
glycol derivative with a free acid group was first activated
Here, the polyethylene as N-
hydroxysuccinimide (NHS) ester and then coupled in on.
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
PEG active ester (1 eq.)
dissolved in DMF until, after three hours, a fine precipitate formed. The
to the peptide (0.9 eq.) and incubated at RT. with
ved in the supernatant was added
of RP-HPLC and
g, in PBS (pH = 7.4) or DMF. The reaction was monitored by means
MALDl-TOF-MS or by LC-MS.
c.) Thioether ligation with thiol—modified polyethylene glycol (tPEG):
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
hours. The progress of the
tion was carried out under an N: atmosphere at 4°C for 18
reaction was monitored by means of RP—HPLC and MALDl-TOF-MS.
The ted peptides were obtained in a yield of from 25 to 40% and characterised by
means of RP—HPLC and MALDl-MS.
Table 4: Overview of the synthesised peptide sequences
pi 137* Guan-ONNRPVYIPRPRPPHPRL—OH
pi 137(sPEém) Guan-O(sPE575°)NNRPWIPRPRPPHPRL—OH
pi 137 (SG)* Guan-O(SG)NNRPVY|PRPRPPHPRL—OH
pi 137 (GRSG)" Guan-O(GRSG)NNRPVYIPRPRPPHPRL—OH
pi 137 (GARSGY’ Guan-O(GARSG)NNRPVYIPRPRPPHPRL—OH
pi 137 (sPEGmToRSG) Guan-O(sPEG?5°—GRSG)NNRPWIPRPRPPHPRL-OH
22 . pi 137 (sPEGWGARSG) Guan-O(sPEG75°:GARSG)NNRPVYIPRPRPPHPRL-OH
23 pi 137 (sPEGmEARSG) Guan-O(sPEGsm-_GARSG)NNRPWIPRPRPPHPRL-OH
24 pi 137 (sPEGZWEARSG) Guan-O(sPEGZ°°T°—_GARSG)NNRPWIPRPRPPHPRL—OH
2 5 . pi 300 (ser Guan-OO(SG)NRPVYIPRPRPPHPRL-OH
‘ pi 300 (GRSG)* Guan-OO(GRSG)NRPW|PRPRPPHPRL-OH
‘ pi 300 (GARSG)* Guan-OO(GARSG)NRPVYIPRPRPPHPRL-OH
‘ pi 300 (GAARSGY Guan-OO(GAARSG)NRPVYIPRPRPPHPRL-OH
. pi 300 (sPEGm-GRSG) Guan-OO(5PEG75°-GRSG)NRPVYIPRPRPPHPRL-OH
' pi 300 EU-GARSG) Guan-OO(SPEG:so-GARSG)NRPVYIPRPRPPHPRL-OH
‘ pi 300 (sPEGIsu-GAARSG) Guan-OO(sPEG‘5°—GAARSG)NRPVYIPRPRPPHPRL-OH
‘ pi 300 (tPEGiso-GRSG) Guan-OO(tPEG‘SU-GRSG)NRPWIPRPRPPHPRL-OH
- pi 300 -GARSG) Guan-OO(tPEG755-GARSG)NRPVYIPRPRPPHPRL-OH
pi 300 (tPEG5°-GAARSG) Guan-OO(tPEGm-GAARSGWRPWIPRPRPPHPRL-OH
pi 300 (tPEG5°°°-GRSG) Guan—OOUPEGsom-GRSG)NRPVYIPRPRPPHPRL-OH
‘ pi 300 (tPEGmO-GARSG) Guan—OO(tPEG5°°fi-GARSG)NRPWIPRPRPPHPRL-OH
‘ pi 300 (tPEGsUDo-GAARSG) O(tPEG5°UU—GAARSG)NRPVYIPRPRPPHPRL-OH
‘ pi 301 (SG)+ Guan-ONO(SG)RPVYIPRPRPPHPRL—OH
‘ pi 301 * Guan—ONO(GRSG)RPVYIPRPRPPHPRL—OH
pi 301 (GARSG)*
‘ pi 301 (GAARSG)*
‘ pi 301 (sPEGm- RSG) Guan-ONO(sPEG:5°—GRSG)RPVYIPRPRPPHPRL-OH
‘ pi 301 (sPEGiso-GARSG) Guan-ONO(SPEG:5U-GARSG)RPVYIPRPRPPHPRL-OH
pi 301 (sPEG75°—GAARSG) Guan-ONO(SPEGi 56-GAARSG)RPVY|PRPRPPHPRL-OH
pi 301 (tPEG-GRSG) Guan-ONO(tPEG: G)RPVYIPRPRF’PHPRL-OH
‘ pi 301 (tPEGm-GARSG) Guan-ONO(tPEEEo-GARSG)RPWIPRPRPPHPRL-OH
‘ pi 301 (tPEGm-GAARSG) Guan-ONOGPEG;55-GAARSG)RPVY|PRPRPPHPRL-OH
pi 301 (tPEEm-GRSG) Guan—ONO(tPEGm-_GRSG)RPWIPRPRPPHPRL-OH
~ pi 301 (tPEém-GARSG) Guan-ONO(tPEG5W-GARSG)RPVYIPRPRPPHPRL-OH
pi 301 (tPEGW’TGAARSG) Guan-ONO(tPEG5°°°-GAARSG)RPVYIPRPRPPHPRL-OH
Onc 72+ DKPPYLPRPRPPROIYNO-NH;
52 One 110+ DKPPYLPRPRPHprHypTIeYNO-NH2
53 GAR-Ono 72* GAR-VDKPPYLPRPRPPROIYNO—NH2
54 GAR—Ono 110* GAR-VDKPPYLPRPRPHypRHypTleYNO-NH2
~ PEG756—GAR-Onc 72 PEEm-GAR-VDKPPYLPRPRPPROIYNO-NH2
PEG75°-GAR-Onc 110 PEGWGAR-VDKPPYLPRPRPHprHypTIeYNO-NH2
PEGmTGAR-Onc 72* PEG75°-GAR-VDKPPYLPRPRPPROIYNO-NHZ
PEGm-GAR-Onc 11o Pam-GAR-VDKPPYLPRPRPHprHypTleYNO-NH;
PEGswo-GAR-Onc 72 mo-GAR—VDKPPYLPRPRPPROIYNO-NHz
PEésm-GAR—Onc 11o -GAR-VDKPPYLPRPRPHprHypTleYNO-NHZ
Guan = guanidino group at the N-terminus, Hyp = trans-4—hydroxyproline, L(N3) = Nor-(9-
fluorenylmethyloxycarbonyl)~s—azido—L-lysine, O = ornithine, Pra = Na-(9-fluorenyl-
methyloxycarbonyl)-L-progargylglycine, Tle = 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 +.
c.) Cleavage of protecting groups and purification:
the resins were
When the sis of the peptides and optional modification were complete.
d for 4
washed carefully with DMF and DCM and dried. The resin-bonded es were
hours at room ature with a mixture of water, m-cresol, thioanisole and ethanedithiol
chains were
(5:525:25) in 87.5% trifluoroacetic acid (TFA). and at the same time the side
ether and
deprotected. The peptides and peptide tives were precipitated with cold diethyl
dried and dissolved in
centrifuged off at 3000 x g. The pellet was washed twice with cold ether,
0.1% aqueous TFA (UV spectroscopy). The samples were stored at 20°C.
The cleaved (optionally ed) peptides were purified by means of RP-HPLC on an Akta
pm 300
HPLC System (Amersham Bioscience GmbH, FreiburgLGermany) with a Jupiter Cw
A, 250 x 10 mm or Jupiter Cw 15 pm, 300 A, 250 x 21 mm column (Phenomenex lnc., Torrance.
USA).
As eluent there was used in each case 0.1% aqueous TFA (eluent A) and 60% aqueous
A typical linear
acetonitrile (Biosolve BV, Valkenswaard, lands) with 0.1% TFA (eluent B).
minute with a flow
gradient began at 5% B and the elution took place at a gradient of 1% B per
rate of 10 mllmin (250 x 21 mm column ) or 5 mllmin (250 x 10 mm column). Detection was at
with the same HPLC
220, 230 and 240 nm. is of the purified es was carried out
lnc., Torrance,
system with a Jupiter C13 5 pm, 300 A, 150 x 4.6 mm column (Phenomenex
of 5-95% B in 30
USA). Elution was carried out at a flow rate of 1 mllmin with a linear gradient
minutes, and detection was at 220 nm. in addition, the purity was determined by means
matrix—assisted laser desorption/ionisation with time-of—flight mass spectrometry (MALDl-TOF-
To that end,
MS; 4700 Proteomic Analyzer. Applied Biosystems GmbH, Darmstadt, Germany).
acid (Bruker
0.5 pl of peptide solution was co-crystallised with 0.5 pl of a-cyanohydroxycinnamic
Daltonik GmbH; Bremen, Germany) as matrix (4 mglml in 60% acetonitrile in 0.1% aqueous
TFA).
Example 2: ination of the minimum inhibitory concentrations and growth kinetics
1. m inhibitory concentrations:
in a double The minimum inhibitory concentrations (MIC) of the peptides were ined
ination of triplicates with a positive control (gentamycin) and a negative control (0.9%
NaCl solution).
series with
To that end, the peptides were dissolved in water and diluted in a two-fold dilution
from 128
1% aqueous soybean medium (TSB) in sterile 96-well plates (Greiner Bio-One GmbH)
strain BL21Al
ug/ml in twelve dilution steps to 62.5 ng/ml. Overnight cultures of Escherichia coli
units per ml. In each case 50 pl of
were adjusted with 1% TSB to about 1.5 x 107 colony forming
peptide solution per well were then mixed with in each case 50 pl of the bacteria solution,
order to achieve a starting concentration of 4 x 105 bacteria per well. After incubation for 20
hours at 37°C. the absorption at 595 nm was determined (microplate reader, TECAN Trading
concentration at
AG). The minimum inhibitory concentration was identified as the lowest peptide
which no bacterial growth was demonstrated.
% mouse serum.
In addition, the antibacterial activity was also determined in the presence of
To that end, the MIC values were determined as described above, but 25 pl of mouse serum
were also added to each well before the incubation (25% final concentration).
Table 5: m inhibitory concentrations
e derivative MIC [umol/l] in MIC [umol/I] in
TSB TSB/mouse serum
Apidaecin 1b* 0.48 1.75
Api 88* 0.44 1.19
Api 134* 1.78 7.12
Api 137* 0.46 0.23
Api 137 (scar
(GRSG)*
Api 137 (GARSGY
Api 137 (sPEEm-GARSG)
Api 137 (sPE§6°°-GARSG)
Api 137 -GARSG)
Api 300 (so)+
Api 300 (sPEG750-GRSG)
Api 300 (sPEémZGARSG)
Api 300 (sPEGmL—GAARSG)
Api 300 (tPEG —GRSG)
Api 300 5°—GARSG)
Api 300 (tPEGWGAARSG)
Api 300 (tPEGsmr-GRSG)
Api 300 (tPEGWGARSG)
Api 300 (tPEG °°-GAARSG)
Api 301 (SG)+
Api 301 (sPEG 5°-GRSG)
Api 301 (sPEG75°-GARSG)
Api 301 (sPEEm-GAARSG)
Api 301 (tPEEm-GRSG)
Api 301 (tPE—Gm-GARSG)
Api 301 (tPEG75°-GAARSG)
Api 301 (tPEEmm-GRSG)
Api 301 (1PE_G5°°°EARSG)
Api 301 (tPESWEAARSG)
Onc 72+
GAR-Onc 72*
sPEG750-GAR-Onc 72
tPEG750-GAR-Onc 72
Onc 110+
GAR-Ono 110*
sPEG °-GAR-Onc 11o
tPEGW-GAR-Onc 110
with +.
comparison examples are marked with *, cleavage products are marked
Owing to the large difference in mass n the unmodified and the PEG-coupled peptides.
the MIC is not given in the conventional unit pg/ml but in pmolll.
The results show that the presence of the peptide linker (GAR-Ono 72 and GAR-Ono 120) or
residues thereof remaining in the cleavage product (Api 137 (8G), Api 300 (8G) and Api 301
(SG)) does not increase the MIC or ses it only insignificantly. In the dified
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: e of the peptide active ingredients in serum
1. Analysis in mouse serum:
of 50 pl
pl of a 1 mg/ml peptide solution were diluted with 120 pl of water and, after addition
of serum, incubated at 37°C for 0 hours, 0.5 hour, 1 hour, 2 hours or 4 hours. Alternatively, the
final
peptide derivatives having a starting concentration of 3 mg/ml were diluted with serum to a
concentration of 15 pg/ml. According to the stability of the peptides, the samples were analysed
were
at the times mentioned above or after 0 hours, 4 hours and 6 hours. The serum proteins
pl of
precipitated by addition of 50 pl of 15% TCA, and the atant was neutralised with
1 molll NaOH. The samples were made up to 250 pl with 5% B, 230 pl of the solution were
transferred into a HPLC vial, and 220 pl were injected.
RP-HPLC analysis Was carried out with a linear itrile gradient (Biosolve BV,
Fluka
Valkenswaard, Netherlands) in the presence of 0.1% oroacetic acid (TFA, UV grade,
with
Chemie GmbH, Buchs, Switzerland) as ion-pair reagent. The fractions were co-crystallised
d-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-TOFfI'OF-MS, 4700 Proteomics er; Applied Biosystems GmbH, Weiterstadt,
Germany) in positive-ion tor mode. The amount of intact peptides and their degradation
products identified and quantified at the individual times. 25% or metabolites could thus be
in parallel for the same time als, was used as
aqueous mouse serum, which was analysed
Table 6: Overview of the half-lives in 25% (v/v) and pure mouse serum, the different side-chain
in combination with PEG750 were compared; primary (red) and
cations secondary
cleavage site (blue), the half-lives marked with were quantified by means of MALDl-TOF MS.
Half-life (t.,,)
Peptide derivative_
in 25% serum in serum
Api 137* >360 min
Api 137 (GRSG)* 60 min
Api 137 (GARSG)* 30 min
Api 137 (sPEm-GRSG) 120 min
Api 137 (sPEGW‘tGARSG) 60 min
Api 300 (SG)* 240 min
Api 300 (sPEGmTGRSG) 50 min
Api 300 (tPEEm-GRSG) 40 min
Api 300 (tPEGmGRSG) 75% (6 h)**
Api 300 (sPEG75°-_GARSG) 35 min
Api 300 (tPEG’sD-GARSG) 45 min
Api 300 (tPEGm-GARSG) 69% (6 h)“
Api 300 (sPEém-GAARSG) 45 min
Api 300 (tPEGm-GAARSG) 35 min
Api 300 (tPEGW—GAARSG) 75% (6 h)**
Api 301 (SG)+ 60 % (4 h)**
Api 301 (sPEEm-GRSG) 30 min
Api 301 (tPEGmERSG) 55 min
Api 301 (iPEG‘W’TGRse) 75% (6 h)**
Api 301 (sPEGmYsARSG) 45 min
Api 301 (tPEGm-GARSG) 45 min
Api 301 (tPE—GWEARSG) 62% (6 h)**
Api 301 (sPEG -GAARSG) 55 min
Api 301 (tPEG’Si’EAARSG) 50 min
Api 301 (tPEGmTeAARSG) 78% (5 h)**
GAR-Ono 72* 85% (4 h)**
GAR-Ono 110* 75% (4 h)**
sPEG 5"-GAR-Onc 72 6 h
sPEGm-GAR-Onc 11o 73% (6 h)**
75°-GAR-Onc 72 55% (6 h)**
tpfim-GAR-Onc 110 64% (6 h)**
AR-Onc 72 86% (6 h)**
tPEé5m-GAR-Onc 110 96% (6 h)**
comparison examples are marked with *. cleavage products are marked with +.
“uncleaved peptide remaining after the indicated time (instead of half-life)
the case of the
The half-lives (or uncleaved peptide remaining after the indicated time) relate in
free peptides (Api 137, Api300 (SG) and Api 301 (SG)) to the cleavage site in the e
(downstream of Arg-16). In the case of the remaining derivatives, the values relate to the
and Api 301
cleavage in the linker in the side chain with release of Api 137 (SG), Api300 (SG)
(SG) or of One 72 and Onc 110.
The following abbreviations are used in the description of the ion:
Agp 2-aminoguanidinopropionic acid
800 tert—butyloxy-carbonyl
‘Bu tert-butyl ether
Dap aminopropionic acid
DCC dicyclohexylcarbodiimide
DCM dichloromethane
DIC diisopropylcarbodiimide
DMF dimethylformamide
E. coii Escherichia coli
eq. equivalents per mol
Fmoc fluorenylmethoxycarbonyl
Guan guanidino group (at the N-terminus)
Hyp transhydroxyproline
HBTU 2-(1H-benzotriazolyI)-1,1.3,3—tetramethyluronium uorophosphate
HOBt 1-hydroxybenzotriazole
LC-MS liquid chromatography with mass spectrometry coupling
MALDI-TOF matrix assisted laser desorption/ionization with time of flight analysis
MIC m tory concentration
MS mass spectrometry
Mtt 4-methyltrityl
NHS N-hydroxysuccinimide
NMM N-methylmorpholine
O ornithine
O‘Bu ted-butyl ester
PBS phosphate-buffered saline
PEG polyethylene glycol
sPEG PEG linked via an acid group
tPEG PEG linked via a thiol group
Pra Na—(9-fluoreny|methyioxycarbonyI)-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 c soy broth
Claims (9)
1. Modified e containing one of the following sequences: NT—X1'Dera—PrPs-Ys'L7-Pa-R9'P1o-X2-P12-P13-R14'X3'l16'Y17-N1a-X4‘CT NT'X1‘DZ'KS—‘PA'PS'YG'LT'PB'RQ'P10'X2'P12'P13'R14‘X3'l16'Y17'N18‘N19‘X4'CT NT’X1'D2'Ka—P4'Psi-Ye-L7'P8‘R9'P10'X2-P12‘P13'R14-X3'l16'P17-N13'X4-CT amino acid residue whose X1 is a residue having a non-polar, hobic side chain or an net charge side chain is positively charged under physiological conditions, having a positive under physiological conditions; or a side chain that is positively charged D2 is an aspartic acid or glutamic acid residue, chain that is positively charged under K3 is a e having a ve net charge or a side physiological conditions, preferably lysine or arginine, another from residues having a positive net X2 and X4 are selected ndently of one conditions; charge or a side chain that is positively charged under physiological chain that is positively charged under X3 is a residue having a positive net charge or a side physiological conditions or proline or a proline derivative; L7 and I16 are selected independently of one another from residues having a non-polar, hydrophhobic side chain, preferably leucine, isoleucine and valine, and N19 are each asparagine Y6 and Y" are each tyrosine, R9 and R14 are each ne, N13 from are selected independently of one another or glutamine. P4. P5, P3, P10, P12. P13 and P17 derivatives, proline and proline derivatives or hydroxyproline and hydroxyproline wherein P13 and R14 are optionally interchanged, and/or optionally one or two of the residues ed from 02, P4, P5, P3, P10, P12 P13. P17 and Ya are replaced by any desired residue, NT is the free or ed N-terminus of the amino acid X1, acid X4, CT is the free or modified C-terminus of the amino is bonded to NT characterised in that a linear or branched polyethylene glycol polymer chain es long via a e linker, and wherein the peptide linker is from 3 to 8 amino acid that is to and contains at least one arginine or lysine at the C-terminus of the e linker, say upstream of X1.
2. Modified peptide according to claim 1 containing one of the following sequences: LPRPRPPROIYNO-NH2 (SEQ ID No. 12) or VDKPPYLPRPRPHprHypTieYNO-NHZ(SEQ ID No. 13) where O = L-ornithine, Hyp = L-4—hydroxyproline, Tle = L—t-Ieucine (L-t-butylglycine), via the peptide wherein a linear or branched polyethylene glycol polymer chain is bonded amino acid residue V). linker to the N-terminal amino group (alpha—amino group of the first
3. Modified peptide according to either claim 1 or claim 2, characterised in that the other amino and serine. acid residues in the peptide linker are selected from glycine, alanine that the
4. Modified peptide according to in any one of claims 1 to 3, characterised polyethylene glycol polymer chain has a molecular weight of from 500 to 40,000
5. Use of a modified peptide ing to any one of claims 1 to 4 in a disinfectant or cleaning agent, as a preservative, or in a packaging material.
6. Use according to claim 5 as a disinfectant or cleaning agent against contamination surfaces or objects with microbes, bacteria or fungi. is bound to the
7. Use according to claim 5 in packaging material n the modified peptide packaging al or incorporated therein.
8. Use according to claim 7 wherein the packaging material is a packaging of foodstuffs.
9. Use of a modified peptide according to any one of claims 1 to 4 in the manufacture of a medicament for ng infections by microorganisms including bacteria and fungi. fat.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE102011118029.3 | 2011-06-20 | ||
DE102011118029A DE102011118029A1 (en) | 2011-06-20 | 2011-06-20 | Modified antibiotic peptides with variable systemic release |
PCT/EP2012/061780 WO2012175532A1 (en) | 2011-06-20 | 2012-06-20 | Modified antibiotic peptides having variable systemic release |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ618829A NZ618829A (en) | 2015-03-27 |
NZ618829B2 true NZ618829B2 (en) | 2015-06-30 |
Family
ID=
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