EP4021918A2

EP4021918A2 - Antimicrobial peptides from medicinal leeches

Info

Publication number: EP4021918A2
Application number: EP19957042.5A
Authority: EP
Inventors: Jürgen Hemberger; Daniela MÜLLER; Zecher GANNA; Hendrik Nolte HANNS
Original assignee: Bioresources Tech & Engineering Bite GmbH; Bioresources Technology & Engineering Bite GmbH
Current assignee: Bioresources Tech & Engineering Bite GmbH; Bioresources Technology & Engineering Bite GmbH
Priority date: 2019-08-26
Filing date: 2019-08-26
Publication date: 2022-07-06
Also published as: WO2021160234A2; WO2021160234A4; WO2021160234A3

Abstract

The present invention relates to a peptide comprising or consisting of a sequence of 5 to 25 amino acid residues, wherein at least 35% of said amino acid residues are Pro.

Description

Antimicrobial Peptides from Medicinal Leeches

The present invention relates to peptides comprising or consisting of a sequence of 5 to 25 amino acid residues. Said peptides are further characterized in that at least 35% of said amino acid residues are Pro.

In this specification, a number of documents including patent applications and manufacturer’s manuals are cited. The disclosure of these documents, while not considered relevant for the patentability of this invention, is herewith incorporated by reference in its entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

The rapid development of antimicrobial resistance is one of the major challenges facing the global healthcare system. Today, many bacterial species already develop pronounced resistance to a variety of antibiotics. The number of deaths caused by antibiotic resistant bacteria is estimated to be 48.000 annually for Europe and USA and 700.000 per year worldwide. Being aware of the size of this problem, the World Health Organization (WHO) started a global action plan in 2013. One major point of this action plan is to trigger research for new antibiotics, which would be able to combat multi-resistant bacteria.

Accordingly, there is a very high need for novel antibiotic agents, which novel antibiotic agents are either alternative or improved as compared to the prior art antibiotic agents. In this regard, it has to be noted that even a novel antibiotic agent which exhibits reduced efficacy as compared to prior art antibiotic agents might constitute an improved agent - this may be the case when a novel antibiotic agent can be used against a broader spectrum of bacteria owing to absence of resistance against the novel agent in said bacteria.

Silver and Graf (ISJ 8, 173 (2011)) describe innate and procured immunity inside the digestive tract of the medicinal leech. Nelson and Graf (Gut Microbes 3, 322 (2012)) describe bacterial symbioses of the medicinal leech.

In a first aspect, the present invention relates to a peptide comprising or consisting of a sequence of 5 to 25 amino acid residues, more preferred is a sequence of 5-15 amino acid residues, wherein at least 35% of said amino acid residues are Pro, more preferred are at least 40% of said amino acid residues are Pro. The term “peptide” has its art-established meaning. It refers to a polycondensate of amino acids. Typically, the length of a peptide does not exceed 50 amino acids. In accordance with the invention, the peptide in accordance with the first aspect has a length of 5 to 25 amino acids. Accordingly, peptides in accordance with the present invention may have a length of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids. A particularly preferred length range is from 5 to 15 amino acids. Preferred specific lengths are 9, 10 and 11 amino acids.

Preferred amino acids are L-a-amino acids. Especially preferred are the 20 proteinogenic amino acids. Having said that, a peptide in accordance with the present invention may also comprise one or more, preferably 1 , 2, 3, 4 or 5 amino acids which are selected from D-amino acids such as D-a-amino acids, b-amino acids as well as further non-proteinogenic amino acids, which non-proteinogenic amino acids may also include L-a-amino acids.

The peptides according to the present invention surprisingly exhibit antibiotic activity, wherein said antibiotic activity is even found in those instances where art-established antibiotic agents fail in view of resistances raised against them. Reference is made to the examples enclosed herewith. Preferred bacteria, especially resistant or multiple resistant bacteria against which the peptides in accordance with the present invention exhibit activity are subject of preferred embodiments disclosed further below.

Peptides in accordance with the present invention are characterized by further surprising and advantageous properties. These properties include one or more of the following:

(i) Activity against Gram positive as well as Gram negative bacteria, preferably against Gram negative bacteria;

(ii) Extraordinary stability. In a preferred embodiment, said extraordinary stability is determined by incubation for 24, 48 or 72 hours in simulated gastric fluid at 37°C wherein preferably after each of these time spans at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% activity are retained. For preferred peptides of the invention applies that after incubation for 24 hours under the mentioned conditions, structure and activity were retained at a level of approximately 100%.

In another preferred embodiment said extraordinary stability is determined by incubation of the peptides according to this invention with typical intestine proteases, namely chymotrypsin and trypsin at 37°C for 1 , 2, 5, 24 and 48 hours, wherein preferably after these timespans at least 50%, at least 60 %, at least 70% or at least 80% activity are retained. In yet another preferred embodiment said extraordinary stability is detemened by incubation of the peptides according to this invention in 100% human serum, at 37°C for 0.5, 1, 1.5, 2, 3, 4, 5, 6 and 24 hours, wherein preferably after these timespans at least 30%, at least 40%, at least 60 %, at least 70%, at least 80%, at least 90%, at least 95% or at least 100% activity are retained. Activity assays are disclosed further below.

(iii) Peptides in accordance with the present invention were found to trigger no hemolysis, even at concentrations up to 100 times the effective IC₅₀ concentration.

(iv) No cytotoxicity has been observed for peptides in accordance with the present invention up to a concentration of 10 mM (which corresponds to about 140 times the the effective IC₅₀ concentration).

Peptides in accordance with the present invention, especially in view of their shortness, are easily accessible via chemical synthesis. In the alternative, they may be obtained from natural sources, especially from leeches. Preferred leeches include Hirudo medicinalis, Hirudo orientalis and Hirudo verbana.

As recited in accordance with the first aspect, peptides in accordance with the present invention contain a high proportion of proline residues. In structural terms, and consistent with such high proportion of proline residues, circular dichroism spectroscopy did not reveal a distinct secondary structure in aqueous solution for peptides of the invention.

In a preferred embodiment, said sequence comprises at least one positively charged amino acid, said positively charged amino acid preferably being selected from K, R, H and ornithine.

Preferably, said positively charged amino acid (in case there would be one positively charged amino acid), or at least one of the positively charged amino acids are located in a position of the sequence of the peptide which is after a cluster of proline residues. Between said cluster and the positively charged amino acid, one or more further amino acids may occur, but do not have to occur. In a particular preferred embodiment, there is one intervening amino acid between the proline cluster and a positively charged amino acid. Preferably, said one amino acid is glycine.

In a particularly preferred embodiment, said peptide comprising or consisting of a sequence of 9 or 10 or 11 amino acids, wherein the amino acids in positions 1 to 10, counted from the N- terminus, are as follows: (1) P, G, X or absent; (2) G; (3) P, G or X; (4) P; (5) P; (6) P; (7) G; (8) K, H, R or ornithine; (9) P or X; (10) Q, N or X, where X depicts any amino acid residue that carries no charge. Especially preferred is that the peptide has a sequence as set forth in SEQ ID NO: 1 or 2. As regards structural and functional properties of these two particularly preferred peptides, reference is made to the examples enclosed herewith.

In preferred embodiments, the peptides of the invention may be derivatized as compared to a mere polycondensate of amino acids. In particular, preferred are peptides wherein

(a) the N-terminus of said peptide is free or protected, preferably acylated, for example with Ci to C₈ acyl, preferred are acyls with C₂ to C₄; to C₈ may be branched or unbranched, unbranched being preferred.

(b) the N-Terminus of said peptide is modified by 1 to 10 additional amino acids selected from motifs of the so-called cell penetrating peptides, preferred are 3 to 5 additional amino acids. The preferred motifs are well known to a person skilled in the art and include but are not limited to sequences like MIIFR or RWRW;

(c) the C-terminus of said peptide is free or protected, preferably amidated, for example with ammonia or Ci to C₄alkylamine;

(d) the C-terminus of said peptide is prolonged by 1-10 amino acids, which contain 2-5 Pro and 1 to 3 positive charges, preferrably 3-4 Pro and 1 or 2 positive charges

(e) the N-terminus, the C-terminus and/or the side-chain N of Lys are linked to natural or synthetic polymers like PEG, HES and/or PAS or are linked to a carrier protein like serum albumins, antibodies or any fragments thereof.

Modifications according to (e) increase the half-life in the patients body. In fact, any art- established measure for half-life extension (HLE) may be used. In that regard, it is of note that a loss of activity or efficacy is tolerated, especially if the half-life can be significantly increased at the same time.

As regards the option recited in item (e) of this preferred embodiment, PEGylation is especially preferred (for reference see: Fee & Domadaran, Eur. Pharm. Rev., Issue 1 (2010))

HESylation is a covalent modification of a peptide by the attachment of hydroxyacyl starch (Liebner et. al„ Eur J Pharm Biopharm., 87(2):378-85 (2014), US7541328 B2).

PASylation refers to the attachment of a conformationally disordered polypeptide chain comprising or consisting of Pro, Ala and Ser residues to a peptide of the present invention. Typically, fusion constructs are obtained by PASylation (Ahmadpour & Hosseinimehr, Curr Drug Deliv. 15(3):331-341 (2018)). The mentioned derivatization can lead to a significantly increased hydrodynamic volume and/or molecular weight. An additional molecular weight of between 1 and 500 kDa is deliberately envisaged, a preferred range being between 30 and 250 kDa. In terms of pharmacokinetics, this provides for a dramatic reduction of peptide loss owing to filtration in the kidneys. Given that significantly more active agent is retained in the body, half-life is dramatically extended, for example between 10- and 100-fold, preferably between 30- and 50-fold, such as 40-fold.

In another embodiment according to (e) the linker between peptide and carrier is any cleavable linker, preferred is a disulfide linker, which may be cleaved under reducing conditions.

In a preferred embodiment, said peptide has antibiotic activity, preferably against bacteria, more preferably against Gram negative bacteria such as E. coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterococci, including those that are resistant to state-of-the-art antibiotics.

Examples of peptides and peptide derivatives according to the invention have the sequences according to SEQ ID No. 3 to 62 (for reference see Table 2 in example 5).

For the purpose of determining antibiotic activity, art-established assays may be used. In a preferred embodiment, dilution series of a given peptide of the invention are prepared and antibiotic activity is determined as a function of concentration. This approach allows the determination of IC₅₀ values. Antibiotic activity comprises or consists of retardation of growth, inhibition of growth and/or killing of bacteria.

Peptides having antibiotic activity in accordance with the present invention preferably have an IC₅₀ value below 100 mM, below 10 mM, below 1 mM, below 100 nM, below 10 nM or below 1 nM.

Exemplary data can be found, for example, in Figure 9 as enclosed herewith.

In a second aspect, the present invention provides a pharmaceutical composition comprising one or more peptides as defined in the first aspect of the invention.

In a preferred embodiment, (a) said pharmaceutical composition comprises a pharmaceutically acceptable carrier, excipient or diluent; and/or (b) (i) said peptide(s) is/are the only pharmaceutically active agents comprised in said pharmaceutical composition; or (ii) said pharmaceutical composition comprises one or more antibiotics selected from penicillins, cephalosporins, polymyxins, rifamycins, lipiarmycins, quinolones, sulfonamides, macrolides, lincosamides, tetracyclines, lipopeptides, glycylcyclines, oxazolidinones and lipiarmycins.

As noted above, the pharmaceutical composition may further comprise pharmaceutically acceptable carriers, excipients and/or diluents. Examples of suitable pharmaceutical carriers, excipients and/or diluents are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc. Compositions comprising such carriers can be formulated by well known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose. Administration of the suitable compositions may be affected by different ways, e.g., by oral, intravenous, intraperitoneal, subcutaneous, intramuscular, topical, intradermal, intranasal or intrabronchial administration. In view of the peptides’ surprising high resistance to gastric juice and typical intestinal proteases like chymotrypsin and trypsin (see the enclosed examples), oral administration is deliberately envisaged and preferred.

The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. For example, peptides of the invention may be present in amounts between 1 pg and 50 mg/kg body weight per dose; however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors.

In a further preferred embodiment, the pharmaceutical composition is a broad-spectrum antibiotic.

The term “broad-spectrum antibiotic” has its art-established meaning. It refers to an antibiotic that acts against a wide range of bacteria. In the widest sense, said bacteria include both Gram positive and Gram negative bacteria. In accordance with the invention, preference is given to broad-spectrum antibiotics which act against a range of Gram negative bacteria.

In a further preferred embodiment, said pharmaceutical composition is active against multidrug resistant bacteria.

As noted in the introductory part herein above, multiple resistances present a manifest problem in the treatment of bacterial infections as well as nosocomial infections. Peptides in accord- ance with the present invention surprisingly exhibit efficacy also against those bacterial strains which are known to exhibit multiple resistances against art-established antibiotics. Such bacterial strains present a major threat and there is an urgent need for suitable agents for combating such bacterial infections.

The term “multi-drug-resistance” has its art-established meaning. Preferred embodiments disclosed further below illustrate this term.

In a third aspect, the present invention relates to a peptide in accordance with the first aspect or the pharmaceutical composition in accordance with the second aspect for use in a method of treating, ameliorating or preventing an infection by bacteria, preferably Gram negative bacteria including multi-drug resistant Gram negative bacteria.

Typically, bacterial infections are the medical indications amenable to treatment with the peptides or pharmaceutical compositions of the present invention. For the avoidance of doubt, and even though bacterial infections may affect different parts of the body, the notion of a bacterial infection defines a medical indication as it would be diagnosed by a practitioner or clinician. The medical indication which is a bacterial infection may, but does not have to be further characterized in terms of the causative bacteria. In fact, in many instances including cases of urgency, antibiotic agents are administered against bacterial infections which bacterial infections are not further defined in terms of their causative agent.

In a preferred embodiment said bacteria are selected from E. coli, Staphylococci, Klebsiella spp, Pseudomonas aeruginosa, Acinetobacter baumannii and Enterococci.

In a particularly preferred embodiment, said bacteria are multidrug resistant strains and selected from E. coli including ESBL and cephalosporin-resistant strains, Methicillin-resistant Staphylococcus aureus (MRSA), Carbapenem-resistant and ESBL Klebsiella pneumonia including strains KPC-2, SHV-11 , IHIT 19222, Vancomycin-resistant Enterococci, multidrug resistant Pseudomonas aeruginosa, Acinetobacter baumannii including CTX-M15 strains.

In a further preferred embodiment, said infection is an infection of the respiratory tract, of the urogenital tract, a wound infection or a systemic infection, which may lead to sepsis.

In a particularly preferred embodiment, said infection of the respiratory tract is pneumonia. In a further preferred embodiment, the said infection (a) is a nosocomial infection; and/or (b) affects a human or an animal such as a mammal.

In a fourth aspect, the present invention relates to an animal feed comprising a peptide of the first aspect or a pharmaceutical composition of the second aspect of the present invention.

As noted further above, the peptides in accordance with the present invention surprisingly exhibit a high degree of resistance against gastric juice, intestinal proteases like chymotrypsin and trypsin and human serum. For that reason, they can advantageously be administered orally. For that reason, the present invention also provides an animal feed.

Deviant from antibiotic agents which are xenobiotic substances, the peptides of the invention are biodegradable. This confers a distinct advantage to their use in an animal feed as well as to their use in a clinical environment.

In a preferred embodiment, said peptide is present in an amount of 0.1 to 5% (w/w).

In a further aspect, the present invention provides a process of biotechnological production using a medium and/or culture, said process comprising the addition of a peptide in accordance with the first aspect to said medium or culture.

As a matter of fact, there is also an urgent need for antibiotic agents for the purpose of biotechnological production. To explain further, certain art-established antibiotics are not allowed for use in biotechnological production. This applies, for example, to streptomycin and gen- tamycin. Therefore, new biodegradable antibiotics like the peptides according to the invention would allow a safer and more efficient bioproduction and would avoid the well-known problem of the presence of antibiotics in waste water and in the environment, which lead to an increased emergence of antibiotic resistance.

In a further aspect, the present invention provides a cosmetic composition comprising a peptide in accordance with the first aspect.

In a further aspect, the present invention provides an in vitro method of controlling the growth of bacteria, preferably of Gram negative bacteria including multi-drug resistant Gram negative bacteria, said method comprising bringing into contact said bacteria with one or more peptides as defined in accordance with the first aspect. Related thereto, the present invention provides, in a further aspect, the use of one or more peptides as defined in accordance with the first aspect for controlling in vitro the growth of bacteria, preferably of Gram negative bacteria including multi-drug resistant Gram negative bacteria.

The latter two aspects generally relate to in vitro applications of the antibiotic peptides in accordance with the present invention. Said method and said use may overlap with the previously disclosed aspect relating to biotechnological production. Generally speaking, it is well-known that the culture media, including culture media which comprise cells in culture or bacteria, may suffer from bacterial superinfections. The peptides of the present invention are suitable means for controlling such superinfections.

In a preferred embodiment of the latter two aspects of the present invention, said controlling the growth comprises or consists of reducing, slowing down, inhibiting or abolishing said growth.

Said reducing and said slowing down are preferably by at least 10%, at least 20%, at least

30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100%.

The terms “inhibiting said growth” and “abolishing said growth” refer to situations where the administration of peptides in accordance with the present invention leads to non-detectable growth. It also extends to those cases where partial or complete killing of the bacteria occurs. Partial killing preferably refers to killing of at least 10%, at least 20%, at least 30%, at least

40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% of the bacteria.

In a further aspect, the present invention provides a method of preventing or reducing formation of a biofilm on a device for intracorporeal use and/or of removing of a biofilm from a device for intracorporeal use, wherein said device is not present in a human or animal body, said method comprising bringing said device into contact with a peptide in accordance with the first aspect.

Related thereto, the present invention provides a use of a peptide in accordance with the first aspect for preparing an intracorporeal device, wherein said device is not present in a human or animal body. Related to the latter two aspects, the present invention furthermore provides an intracorporeal device which is coated and/or loaded with in accordance with the first aspect.

Preferably, said device is a catheter, an implant, an endoscope, a drainage, a chirurgical instrument, a contact lens or a hearing aid.

As regards the embodiments characterized in this specification, in particular in the claims, it is intended that each embodiment mentioned in a dependent claim is combined with each embodiment of each claim (independent or dependent) said dependent claim depends from. For example, in case of an independent claim 1 reciting 3 alternatives A, B and C, a dependent claim 2 reciting 3 alternatives D, E and F and a claim 3 depending from claims 1 and 2 and reciting 3 alternatives G, H and I, it is to be understood that the specification unambiguously discloses embodiments corresponding to combinations A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D, G; B, D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H; C, D, I; C, E, G; C, E, H; C, E, I; C, F, G; C, F, H; C, F, I, unless specifically mentioned otherwise.

Similarly, and also in those cases where independent and/or dependent claims do not recite alternatives, it is understood that if dependent claims refer back to a plurality of preceding claims, any combination of subject-matter covered thereby is considered to be explicitly disclosed. For example, in case of an independent claim 1 , a dependent claim 2 referring back to claim 1 , and a dependent claim 3 referring back to both claims 2 and 1 , it follows that the combination of the subject-matter of claims 3 and 1 is clearly and unambiguously disclosed as is the combination of the subject-matter of claims 3, 2 and 1. In case a further dependent claim 4 is present which refers to any one of claims 1 to 3, it follows that the combination of the subject-matter of claims 4 and 1 , of claims 4, 2 and 1 , of claims 4, 3 and 1 , as well as of claims 4, 3, 2 and 1 is clearly and unambiguously disclosed.

The figures show:

Figure 1: Antimicrobial activity of the 3 kD-Ultrafiltration fraction from leech saliva compared to the total salivary gland excretion against E. coli K12 and Micrococcus luteus (n=5).

Figure 2: (A) Purification of several batches of the 3 kD fraction from leech saliva by RP-HPLC (B) Antimicrobial activity of the RP-HPLC fractionated 3 kD fraction against E. coli K12 and Micrococcus luteus (n=5). Figure 3: MS spectrum the peptide eluted at 12.1 min from the nano-LC column, mass is 486.27 doubly charged.

Figure 4: MS/MS spectrum with annotation of the b- und y- ion series, resolution 7,500 at 200 m/z.

Figure 5: (A) RP-HPLC Chromatogram after incubation of peptide SEQ ID 1 in simulated gastric fluid at 37°C, showing a recovery of the intact protein of >98% after incubation. A = incubation time 0 min, B = incubation time 24 hours. (B) Growth inhibiting activity of peptide (SEQ ID 1) after incubation in gastric juice against Micrococcus luteus

Figure 6: Stability testing (A), Recovered peptide SEQ ID 1 after incubation with chymotrypsin at 37°C . (B) Recovered peptide SEQ ID 1 after incubation with trypsin at 37°C. (C) Stability of peptide SEQ ID 1 in human serum at 37°C.

Figure 7 : Effect of peptide SEQ ID 1 on hemolysis in whole blood. Various concentrations of peptide were added to freshly drawn human blood and hemoglobin concentration was measured after 1 hour incubation at 37°C.

Figure 8: Cytotoxicity assay. Effect of peptide SEQ ID 1 at various concentrations on HeLa cells as determined by the MTT assay. Blank Z = control HeLa cells in medium; NC PBS = negative control (20% PBS in medium); PC DOX = positive control Doxorubicin (10mM in medium); PC SDS = positive control SDS (2.5 mg/ml in medium)

Figure 9: (A) Growth inhibition of a E. coli K12 strain by peptide according to this invention (SEQ ID 1). (B) Growth inhibition of a multi-resistant Klebsiella pneumoniae strain by peptide according to this invention. (C) Growth inhibition of a multi-resistant Acinetobacter baumanii strain by peptide according to this invention; dark = human clinical isolate, grey = clinical isolate from a dog.

The examples illustrate the invention.

Example 1

Estimation of antimicrobial activity Test bacteria were grown in an overnight culture in MHB-, LB medium or equivalent to an optical density between OD 2.0 and 4.0. 100 mI of the bacterial suspension containing about 5 x 10⁵ CFU/ml was pipetted into 96-well microtiter plates and 20 mI of the test solution in water/or 0.1% acetic acid was added. The plate was incubated for 18 h at 37°C and the optical density was read at 620 nm. E. coli K12 and Bacillus subtilis were used as model organisms for Gram negative and Gram positive bacteria respectively. The results were plotted as % growth reduction compared to the control with water instead of sample.

Example 2

Fractionation of crude leech saliva

Saliva from the leech Hirudo verbana was collected by standard methods as described for example in Rigbi et al. (1996).

The leech saliva was then fractionated by ultrafiltration (UF). Centrifuge UF units equipped with a membrane having a cut-off of 3 kD were loaded with crude saliva and centrifuged for 40 min at 4000 rpm at 4 °C. The filtrate was tested for antimicrobial activity as described in example 1.

Activity was found against Gram negative bacteria ( E . coli (ATCC 25404)) as well as Gram positive bacteria ( Micrococcus luteus (ATCC 4698)) as depicted in Fig. 1.

Example 3

Purification of peptides from leech saliva

The filtrate was further concentrated and subsequently purified by RP-HPLC on a C18-column. For elution, the gradient according to Table 1 was used:

Table 1: RP-HPLC gradient for further purification of antibiotic activity Detection of the peptides was done photometrically at 214 nm. Major peaks were collected manually and the antimicrobial activity measured as described above.

Fig. 2A shows an overlay of RP-HPLC runs from several batches of the 3 kD fraction performed under the same conditions.

Several fractions showed activity in the antimicrobial test, with fraction 10 being the most promising (fig. 2B).

Example 4

Identification of a leech-derived antimicrobial peptide bv LC/MS

C18-stage tips were activated with MeOH, washed with 80% ACN, 0.1% acetic acid and equilibrated with 0.1% acetic acid. RP-HPLC fraction 10 was mixed 1 :1 with 0.2% acetic acid and loaded onto the C18-stage tips and washed twice with 0.1% acetic acid. Elution was performed by addition of 80% ACN, 0.1% acetic acid and incubation for 30 min at room temperature. The purified fractions were further concentrated in a Speed-Vac and dissolved in 0.1% acetic acid. 4 pi of the purified concentrated sample were used for LC/MS studies.

LC was done on an Easy-LC chromatography station (Thermo Fisher). Elution conditions were 5% ACN to 35% ACN in 20 min at 250 nl/min and 40 °C. The column was washed for 10 min with 95% ACN and re-equilibrated for 5 min with 5% ACN. The total measurement time was 30 min per sample.

For the MS/MS analyses a LTQ Orbitrap Velos (Thermo) with the higher-energy collisional induced dissociation (HCD) fragmentation mode was used. Applying a maximal aquisition time of 150 ms (target value: 1e6), the MS spectra were measured at a resolution of 30000 (200 m/z). For the HCD MS/MS spectra a target value of 5e4 and a resolution of 7500 at a normalized collision energy of 35 eV was chosen.

Fig. 3 displays the MS spectrum of the LC peak at 12.1 min with a major MS peak at 486.27 m/z corresponding to the doubly labelled mol peak.

In Fig. 4 the MS/MS spectrum of the peak at 486.27 m/z together with the annotation of the b- and y-ions is shown. The individual MS and MS/MS spectra were analyzed manually and the sequence implemented into a EST database from Hirudo verbana translated into all 6 frames using MaxQuant (Cox & Mann, 2008). The sequence could be validated as [P,G]PPPPGKPQ with the Andromeda Search Engine (Cox et al., 2011).

SEQ ID 1 PGPPPPGKPQ

SEQ ID 2 GPPPPPGKPQ

Example 5

Synthesis of antimicrobial peptides bv the solid phase method

The peptide synthesis was performed with the solid phase automated synthesizer "Liberty blue" (CEM). The amino acid sequence and conditions for the synthesis reaction were defined in the "Liberty" software. Amino acids were dissolved at a concentration of 0.2 mol/L in DMF and connected to the synthesizer. The reaction vessel was filled with the respective Wang res- in, activator and activator base were filled in at concentrations of 0.5 mol/L and 1 mol/L respectively.

When the synthesis programme was finished, the reaction vessel was washed twice with DMF followed by two washings with DCM, transferred into a 50 ml centrifuge tube and dried over night. Cleavage of the peptide from the resin is performed with 2.5 ml cleavage cocktail (95% TFA, 2.5% TIS and 2.5% purified water). The resin is removed by filtration and washed with cleavage cocktail. For precipitation of peptide ice-cold diethyl ether is added (4 times the volume of the peptide solution). The precipitate is isolated by centrifugation and washed again with ice-cold diethyl ether. The pellet is then dissolved in water and lyophilized. Purity of the synthetic peptides was checked by RP-HPLC, the sequence identity was validated by MALDI MS.

Table 2 depicts the peptides and peptide derivatives which were synthesized by this method. Table 2: List of peptides synthesized (small characters refer to D-aminoacids)

Example 6

Stability of the antimicrobial peptide 1. Stability in simulated gastric fluid

Stability of peptides according to SEQ ID 1 and 2 were tested by incubation in simulated gastric fluid at 37 °C. Simulated gastric fluid juice was prepared according to "Hagers Handbuch der pharmazeutischen Praxis" Volume 2 with the following composition: 0.20 % (m/v) NaCI

0.32% (m/v) Pepsin in 0.1 M HCI; pH 1.0-1.2

2.08 mg peptide were dissolved in 100 pi water, 50 mI of the peptide solution were pipetted to 450 mI simulated gastric fluid. After 2 h and 24 h respectively, the solution was neutralized by

22 mI of 2 N NaOH. 20 mI of the neutralized solution was analyzed by RP-HPLC and compared to untreated peptide.

Conditions for RP-HPLC column are as follows: Phenomenex Kinetex C18, 150x2.1 mm, 3 pm flow: 0.1 ml/min detection: 214 nm

The gradient is given in Table 3.

Table 3: RP-HPLC gradient fortesting the peptide stability

Chromatograms of the peptide in water and in simulated gastric fluid (after 0 and 24 h) are shown in Figure 5A. The pronounced chemical stability correlated with the retention of full antimicrobial activity (see. Fig. 5B)

2. Stability against the intestinal proteases chymotrypsin and trypsin

A 2 mM stock solution of the peptide SEQ ID 01 was prepared in DMSO. 10 mI peptide solution (20 mM) was added to 30 mI water, 50 mI of buffer and 10 mI of protease solution. This mix was incubated at 37°C for up to 48 hours. After 1, 2, 5, 24 and 48 hours 10 mI of the reaction mix was removed and added to 90 mI of a quenching solution. 25 mI of the quenched solution was applied to RP-HPLC as described above.

Conditions for chymotrypsin:

Enzym: 0.1 mg/ml with 1000 USP units/mg Buffer: 10 mM Phosphate buffer pH 7.4 Quencher: 0.08% TFA in water Conditions for trypsin:

Enzym: 0.5 mg/ml with 2500 USP units/mg Buffer: 100 mM NH₄HC0₃ + 0.1 mM CaCI₂ Quencher: 0.08% TFA in water

The results of the stabilty assays are shown in Fig. 6A (Chymotrypsin) and Fig. 6B (trypsin).

3. Stability in human serum

10 ml of human blood was allowed to coagulate for 50 min at room temperature and centrifuged thereafter for 10 min at 1.400xg. 1 ml of the serum obtained was then centrifuged for 10 min at 15.000xg. 800 mI of the supernatant was allowed to adapt to a temperature of 37°C for 10 min. 200 mI of a 10 mM solution of peptide SEQ ID 1 was added and the mix was incubated for 24 hours. After 0.5, 1, 1.5, 2, 3, 4, 5, 6, and 24 hours 100 mI were drawn from separate vials. 20 mI 15% TCA were added to precipitate the serum proteins. After centrifugation for 15 min at 4°C at 15.000xg, the supernatant was diluted 1 :8 with water, sterile filtrated and 25 mI of this solution was analyzed by RP-HPLC as described above.

Fig 6C shows the results of the stability in human serum.

Example 7

Effect of the antimicrobial peptide SEQ ID 1 on hemolysis

Freshly drawn human blood was rinsed three times with PBS and resuspended in PBS to 4% (v/v). One hundred microliters of the suspension was added to 96-well microtiter plate containing equal volume of peptides to give final concentrations of peptides encompassing the range of 0.0625 - 10 mg/ml in PBS. PBS was used as blank control. Plates were incubated at 37 °C for 1 hr with gentle shaking. Subsequently, the plate was centrifuged and the supernatant was transferred to a new plate. The release of hemoglobin in the supernatant was monitored at absorbance of 450 nm using a Tecan plate reader (Tecan Group, Maennedorf, Switzerland). Results were pooled from three independent experiments and expressed as mean. Hemoglobin concentrations were calculated from a standard curve using purified hemoglobin (Hb).

The results showed, that up to the maximum solubility of the peptide, which was found to be 10 mg/ml, no significant hemolysis activity over control was observed; see Fig. 7. The highest concentration tested was at about 100-fold the effective IC₅₀ concentration..

Example 8

Effect of the antimicrobial peptide SEQ ID 1 on cvtotoxicitv

Assays on cytotoxicity of the peptides SEQ-ID 1 and SEQ-ID 2 were performed according to ISO 10993-5 (2009) - Annex C "MTT cytotoxicity test" using HeLa cells in 48 well plates. Peptides was disolved in water. All experiments were done in triplicate with an incubation time of 72 hrs at 37°C. Fig. 8 show means values + standard deviation. No significant difference was observed between the two peptides tested.

Example 9

Growth inhibitory effect of the antimicrobial peptide against different pathogenic bacteria The assay for the determination of growth inhibition was done according to the following procedure:

The test compound was, depending on sequence and solubility, dissolved in water or 0.1% acetic acid respectively. As growth medium for the bacteria Mueller-Hinton-Broth or equivalent was chosen. The assay was performed in 96-well microtiter plates with flat bottom and assigned as non-binding. Adsorption of the peptides to the plates has to be avoided.

100 pi of a bacterial suspension with 1-2 x 10⁷ cfu/ml was pipetted into each well followed by 20 pi peptide solution. Initial concentration of the peptide was 1000 pg/ml with 2-fold serial dilution down to 7.81 pg/ml, which resulted in final peptide concentrations of 166 to 1.3 pg/ml. Incubation proceeded for 18-24 hrs at 37 °C with gentle shaking. After the incubation time the OD at 600 nm was read.

Each peptide dilution was assayed in triplicate.

Calculation: growth inhibition— [1-(1/OD600 cellsw/o compound) (OD600T_estcompondOD600$tart)]

Results are shown in Fig 9. Fig. 9A shows the effect of the peptide on an E. coli K12 strain. 100% growth inhibition was reached at a concentration of 68.4 nM and the IC₅₀ was found to be about 6 nM. A similar pronounced effect was accomplished with a multiresitant clinical isolate of Klebsiella pneumoniae with an IC₅₀ also in the range of 6 nM, as shown in Fig. 9B. The peptide was also effective against clinical isolates resistant ESBL strains of Acinetobacter baumannii from humans and dogs respectively. As can be seen from Fig 9C the IC₅₀ of the human isolate was about 15 nM.

Table 4 lists peptides according to this invention together with their antibiotic activity against the Gram negative model strain E. coli K12. Table 4: Antibiotic activity of peptides according to this invention against E. coli K12

Claims

1. A peptide comprising or consisting of a sequence of 5 to 25 amino acid residues, wherein at least 35 % of said amino acid residues are Pro.

2. A peptide according to claim 1 with a sequence of 5 to 15 amino acid residues, wherein at least 40% of said amino acid residues are Pro.

3. A peptide according to claim 1 and 2, wherein said sequence comprises at least one positively charged amino acid, said positively charged amino acid preferably being selected from K, R, H and ornithine.

4. A peptide according to claim 1 and 2, said peptide comprising or consisting of a sequence of 9 or 10 or 11 amino acids, wherein the amino acids in positions 1 to 10, counted from the N-terminus, are as follows: (1) P, G, X or absent; (2) G; (3) P, G or X; (4) P; (5) P; (6) P; (7) G; (8) K, H, R or ornithine; (9) P or X; (10) Q, N or X, where X depicts any amino acid residue that carries no charge.

5. A peptide according to claim 1 and 2, wherein said sequence is the sequence set forth in SEQ ID NO: 1 or 2.

6. A peptide of any one of the preceding claims, wherein

(a) the N-terminus of said peptide is free or protected, preferably acylated, for example with Ci to C₈ acyl, preferred are acyls with C₂ to C₄; C_! to C₈ may be branched or unbranched, unbranched being preferred.

(b) the N-Terminus of said peptide is modified by 1 to 10 additional amino acids selected from motifs of the so-called cell penetrating peptides, preferred are 3 to 5 additional amino acids. The preferred motifs include but are not limited to sequences like MIIFR or RWRW;

(e) the N-terminus, the C-terminus and/or the side-chain N of Lys are PEGylated, HESylated and/or PASylated or are linked to a carrier protein like serum albumins, antibodies or any fragments thereof.

7. The peptide of any one of the preceding claims, wherein said peptide has antibiotic activity, preferably against bacteria, more preferably against Gram-negative bacteria such as E. coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterococci, including multi-drug resistant Gram negative bacteria.

8. A pharmaceutical composition comprising one or more peptides as defined in any of the preceding claims.

9. The pharmaceutical composition of claim 8, wherein

(a) said pharmaceutical composition comprises a pharmaceutically acceptable carrier, excipient or diluent; and/or

(b) (i) said peptide(s) is/are the only pharmaceutically active agents comprised in said pharmaceutical composition; or

(ii) said pharmaceutical composition comprises one or more antibiotics selected from penicillins, cephalosporins, polymyxins, rifamycins, lipiarmycins, quinolones, sulfonamides, macrolides, lincosamides, tetracyclines, lipopeptides, glycylcyclines, oxazolidinones and lipiarmycins.

10. The pharmaceutical composition of claim 8 or 9, wherein said pharmaceutical composition is a broad-spectrum antibiotic.

11. The pharmaceutical composition of any one of claims 8 to 10, wherein said pharmaceutical composition is active against multi-drug resistant bacteria.

12. A peptide of any one of claims 1 to 7 or a pharmaceutical composition of any one of claims 8 to 11 for use in a method of treating, ameliorating or preventing an infection by bacteria, preferably Gram negative bacteria including multi-drug resistant Gram negative bacteria.

13. The peptide for use or the pharmaceutical composition for use of claim 12, wherein said bacteria are selected from E. coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterococci.

14. The peptide for use or the pharmaceutical composition for use of claim 13, wherein said bacteria are multidrug resistant strains and selected from E. coli including ESBL and cephalosporin-resistant strains, Methicillin-resistant Staphylococcus aureus (MRSA), Carbapenem-resistant and ESBL Klebsiella pneumonia including strains KPC-2, SHV-11, IHIT 19222, Vancomycin-resistant Enterococci, multidrug resistant Pseudomonas aeruginosa, Acinetobacter baumannii including CTX-M15 strains.

15. The peptide for use or the pharmaceutical composition for use of any one of claims 12 to 14, wherein said infection is an infection of the respiratory tract, of the urogenital tract, a wound infection or a systemic infection, which may lead to sepsis.

16. The peptide for use or the pharmaceutical composition for use of claim 15, wherein said infection of the respiratory tract is pneumonia.

17. The peptide for use or the pharmaceutical composition for use of any one of claims 12 to 16, wherein said infection

(a) is a nosocomial infection; and/or

(b) affects a human or an animal such as a mammal.

18. Animal feed comprising a peptide of any one of claim 1 to 7 or a pharmaceutical composition of any one of claims 8 to 11.

19. Animal feed according to claim 18, wherein said peptide is present in an amount of 0.1 to 5% (w/w).

20. A process of biotechnological production using a medium and/or culture, said process comprising the addition of a peptide of any one of claims 1 to 7 to said medium or culture.

21. A cosmetic composition comprising a peptide of any one of claim 1 to 7.

22. An in vitro method of controlling the growth of bacteria, preferably of Gram negative bacteria including multi-drug resistant Gram negative bacteria, said method comprising bringing into contact said bacteria with one or more peptides as defined in any one of claims 1 to 7.

23. Use of one or more peptides as defined in any one of claims 1 to 7 for controlling in vitro the growth of bacteria, preferably of Gram negative bacteria including multi-drug resistant Gram negative bacteria.

24. The method of claim 22 or the use of claim 23, wherein said controlling the growth comprises or consists of reducing, slowing down, inhibiting or abolishing said growth.

25. A method of preventing or reducing formation of a biofilm on a device for intracorpo- real use and/or of removing of a biofilm from a device for intracorporeal use, wherein said device is not present in a human or animal body, said method comprising bringing said device into contact with a peptide of any one of claims 1 to 7.

26. Use of a peptide of any one of claims 1 to 7 for preparing an intracorporeal device, wherein said device is not present in a human or animal body.

27. Intracorporeal device which is coated and/or loaded with a peptide of any one of claims 1 to 7.

28. The method of claim 25, the use of claim 26, or the device of claim 27, wherein said device is a catheter, an implant, an endoscope, a drainage, a chirurgical instrument, a contact lens or a hearing aid.