ANTIMICROBIAL PEPTIDES MODIFIED FOR MAMMALIAN CELL

RECOGNITION AND/OR ADHESION

Technical Field

The present disclosure relates generally to cationic peptides having antimicrobial activity and modified to include a cell recognition and/or adhesion moiety, and to uses of such peptides.

Background

The use of biomaterial implants and medical devices is becoming increasingly common. Recent estimates suggest that up to 150 million intravascular devices and 1.25 million hip and knee replacements are performed in the United States each year. One of the most common medical devices in use today is the indwelling catheter which can be used in the treatment of patients needing dialysis, delivery of drugs or food for cancer patients, use with low-birth-weight newborns, and sampling of blood. However all medical devices may be predisposed to infection. Infection is the most common reason for failure of implanted devices and infections associated with implanted devices account for approximately 45% of nosocomial infections. Catheters can lead to infection of the skin at the site of insertion, which may in turn lead to more serious bloodstream infections. Indeed catheters are now the commonest cause of bloodstream infections in Australia.

Failure of percutaneous catheters can occur through combinations of marsupialisation, permigration, infection and trauma. The reason for the high rates of catheter-related infection is the adhesion and colonisation of microbes (mainly bacteria, particularly S. aureus, S. epidermidis but also Pseudomonas aeruginosa, and Candida albicans) to catheters at the site of skin implantation and lack of appropriate skin cell attachment to the catheter surface which predisposes to infection and marsupialisation. Once the catheter is colonised, the microbes can invade into the bloodstream. Whilst aseptic technique during surgery can help reduce infection, the ability of the skin to heal around the catheter quickly and appropriately is also of importance. Further compounding the problem of bio material-associated infection are alterations in host tissue responses around the implant, including an inability to adhere to the implant surface, and loosening or extrusion of the implant, which result in increased susceptibility to infection.

Current strategies for preventing catheter-associated infections apart from sterile technique typically involve coating the implant with antibiotics or silver. However a common and increasing problem is the development of microbial resistance which makes these strategies short-lived. Other common problems with antimicrobial coating strategies are loss of activity after covalent attachment, and rapid loss of the antimicrobials due to out- diffusion. It has also been reported that antibiotic releasing biomaterials and silver impregnated biomaterials can also be cytotoxic to keratinocytes.

There is a clear need for the development of alternative approaches to providing antimicrobial activity.

Summary of the Disclosure

A first aspect of the disclosure provides a cationic peptide having antimicrobial activity and further comprising a cell recognition and/or adhesion moiety.

By way of example, the peptide having antimicrobial activity may be melimine, protamine, lactoferricin, protattin, or a variant or functional fragment thereof. Combination may also be used. In an exemplary embodiment, the peptide is melimine or a variant or functional fragment thereof. In an exemplary embodiment the peptide having antimicrobial activity may comprise an amino acid sequence set forth in SEQ ID NO:l or SEQ ID NO:2 or a variant or functional fragment thereof.

Typically the cell recognition and/or adhesion moiety is a mammalian cell recognition and/or adhesion moiety. In exemplary embodiments the mammalian cell may be selected from an epithelial cell (such as a keratinocyte), a fibroblast, an osteoblast, an osteoclast or an endothelial cell. Typically the cell recognition and/or adhesion moiety is a peptide moiety. By way of example, the peptide moiety may comprise a cell recognition and/or adhesion motif selected from RGD, VGVAPG, GEFYFDLRLKGDK, YIGSR, and WQPPRARI. In an exemplary embodiment, the motif is RGD.

The cell recognition and/or adhesion moiety may be incorporated at one or more positions within the peptide sequence or at the C-tenninus or the N-terrninus. In certain embodiments the peptide moiety is located at the C-teiminus of the peptide having antimicrobial activity.

The peptide may be further modified to facilitate attachment of the peptide to a surface, for example, by the introduction of a thiol functional group, such as a thiol-containing residue, into the peptide. The thiol-containing residue may be cysteine. The thiol functional group may be introduced into the peptide at a position of the peptide where it is desired to attach the peptide to the surface. For example, the thiol functional group, such as a cysteine residue, may be introduced into the peptide at the C-terminus or at the N-tenriinus. In certain embodiments a cysteine residue is introduced at the N-terminus of the peptide having antimicrobial activity.

In an exemplary embodiment, the peptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:3 or SEQ ID NO:4.

In an exemplary embodiment, the peptide of the first aspect comprises the amino acid sequence set forth in SEQ ID NO:5 or SEQ ID NO:6. In alternative embodiments, the peptide of the first aspect comprises an amino acid sequence set forth in any one of SEQ ID NOs:7 to 16.

Typically, the surface is a surface to which adhesion of mammalian cells is desired.

The surface may be a solid surface. The solid surface may be any solid surface capable of being colonised by microorganisms. The solid surface may comprise a polymer, for example a hydrogel, a silicon hydrogel, a polymer or copolymer of 2- hydroxyethylmethacrylate, silicon rubber, polyurethane, polypropylene, polyethylene, polyacrylamide, polytetrafluoroethylene (Teflon), or a biodegradable polymer, such as poly-lactide or combinations thereof.

The solid surface may be the surface of a medical device. The medical device may be, for example, a contact lens, a corneal onlay or inlay device, fluid collection bag, sensor, hydrogel bandage, tubing, stent, heart valve, an implant, such as a hearing implant, a knee implant, a hip implant, an implantable electrode, an implantable neuro prosthetic electrode array, a catheter, such as an indwelling catheter, or carrier for antibiotic, diagnostic or therapeutic agents.

The solid surface may be a glass or metal surface or a metal-containing surface, for example a transition metal surface or a transition metal-containing surface. In certain embodiments, the transition metal is titanium.

A second aspect of the present disclosure provides a nucleic acid molecule comprising a nucleotide sequence encoding a peptide of the first aspect.

A third aspect of the present disclosure provides an antimicrobial composition comprising a peptide according to the first aspect or a nucleic acid molecule according to the second aspect, optionally together with one or more pharmaceutically acceptable carriers, excipients or diluents.

In a fourth aspect the present disclosure provides a device, wherein a peptide of the first aspect is directly or indirectly attached or bound to a surface of the device.

The device may be a medical device, for example, a contact lens, a corneal only or inlay device, fluid collection bag, sensor, hydrogel bandage, tubing, stent, heart valve, an implant, such as a hearing implant, a knee implant, a hip implant, an implantable electrode, an implantable neuro prosthetic electrode array, a catheter, such as an indwelling catheter, or carrier for antibiotic, diagnostic or therapeutic agents. A surface of the device may comprise a polymer, for example a hydrogel, a silicon hydrogel, a polymer or copolymer of 2-hydroxyethylmethacrylate, silicon rubber, polyurethane, polypropylene, polyethylene, polyacrylamide, polytetrafluoroethylene (Teflon), a biodegradable polymer, such as poly-lactide, or combinations thereof

The peptide may be attached to a surface of the device via a thiol functional group, such as a thiol-containing residue.

In a fifth aspect the present disclosure provides a method for encouraging or stimulating tissue adhesion to a surface, the method comprising attaching to the surface, or coating the surface with, a peptide that is antimicrobial and comprises a cell recognition and/or adhesion moiety. In certain embodiments, the surface is the surface of a device, such as a medical device.

In a sixth aspect, the present disclosure provides a method for encouraging or stimulating tissue adhesion to a medical device, comprising attaching to a surface, or coating a surface, of the device with a peptide that is antimicrobial and comprises a cell recognition and/or adhesion moiety. In one embodiment, the medical device is an implantable medical device.

In one embodiment, the peptide that is antimicrobial and comprises a cell recognition and/or adhesion moiety comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15 and 16 or a variant or a functional fragment thereof.

In a seventh aspect, the present disclosure provides a method for encouraging or stimulating tissue adhesion and integration of an implantable medical device, the method comprising attaching (either directly and/or indirectly) to a surface of the device, or coating a surface of the device with, a peptide that is antimicrobial and comprises a cell recognition and/or adhesion moiety. In one embodiment, the peptide is a peptide according to the first aspect. In an exemplary embodiment, the tissue comprises mammalian cells.

In an eighth aspect the present disclosure provides a method for preventing the occurrence of microbial infection on or around the surface of a medical device inserted into a patient, or at or near the site of insertion of the medical device, the method comprising attaching to a surface of the device, or coating a surface of the device with, a peptide of the first aspect. In certain embodiments, the present disclosure provides a method for reducing, substantially preventing or preventing the occurrence of microbial infection on or around the surface of a medical device inserted in a patient, or at or near the site of insertion of the medical device, the method comprising attaching to a surface of the device, or coating a surface of the device with, a peptide of the first aspect.

In a ninth aspect the present disclosure provides a method for eliminating or inhibiting the growth of one or more microorganisms or the colonisation of an environment by the microorganisms, the method comprising contacting the one or more microorganisms, or an environment inhabited by the microorganisms, with an effective amount of a peptide of the first aspect, a composition of the third aspect or a device of the fourth aspect.

The one or more microorganisms may be selected from bacteria, fungi, yeast and protozoa.

The bacteria may be Gram-negative or Gram-positive bacteria. In certain embodiments, the bacteria are Staphylococcus spp., such as S. aureus, or Pseudomonas spp., such as P. aeruginosa.

Brief Description of the Drawings

Embodiments of the present disclosure will now be described, by way of example only, with reference to the accompanying drawings.

Figure 1. Representative images of adhesion of human epithelial cells and S. aureus to Cys-Mel-RGD-coated glass. A, confluent epithelial cells. B, demonstrates anti-S. aureus activity of the melimine-RGD coating, bright/green dots (marked with white arrows) = viable cells; dark/red dots = dead cells).

Figure 2. Representative images of adhesion of human epithelial cells to Cys-Mel-4 coated fluorinated ethylene propylene (FEP). A, unmodified FEP; B, poly(heptylamineamine) surface coating; C, process control; D, surface coated with mammalian cell recognition and/or adhesion motif (amino acid sequence GRGDSPC) (positive control); E, surface coated with Cys-Mel-4; F, surface coated with negative control peptide.

The subject specification contains amino acid and nucleotide sequence information prepared using the programme Patentln Version 3.4, presented herein in a Sequence Listing. Sequences are referred to by a sequence identifier number (SEQ ID NO:). The SEQ ID NOs: correspond numerically to the sequence identifiers <400>1 (SEQ ID NO: l), <400>2 (SEQ ID NO:2), etc. Specifically, the native melimine amino acid sequence is shown in SEQ ID NO: 1. The amino acid sequence of a truncated melimine ("Mel4") is shown in SEQ ID NO: 2. SEQ ID NOs: 3 and 4 show the sequences of melimine and Mel4 with cysteine residues at the N-terminus ("Cys-Mel" and "Cys-Mel4", respectively). SEQ ID NOs: 5and 6 show the amino acid sequences of Cys-Mel and Cys-Mel4, respectively, with an RGD tripeptide at the C-terminus ("Cys-Mel-RGD" and "Cys-Mel4- RGD"). SEQ ID NOs: 7 to 16 show the amino acid sequences of Cys-Mel Cys-Mel4 peptides with alternative cell recognition and/or adhesion moieties incorporated at the C- terminus (as described herein below). SEQ ID NOs: 17 to 19 show the amino acid sequences of the cationic peptides protamine, lactoferricin and protattin, respectively, while SEQ ID NOs: 20 to 23 show the amino acid sequences of other variant peptides having antimicrobial activity.

Detailed Description

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. In the context of this specification, the terms "a" and "an" are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

In the context of this specification, the term "about," is understood to refer to a range of numbers that a person of skill in the art would consider equivalent to the recited value in the context of achieving the same function or result.

In the context of this specification, the term "environment inhabited by" in the context of microorganisms encompasses one or more environments (solid, fluid or gaseous, including surfaces of cells, tissues, organs or inanimate objects) that are inhabited or colonised by, or is capable of being inhabited or colonised by, microorganisms.

In the context of this specification, the term "antimicrobial composition" is understood to mean a composition that is capable of eliminating, preventing, inhibiting or retarding the growth of at least one microorganism, the colonisation of an environment by the microorganism or the adherence to a surface by the microorganism.

In the context of this specification, the term "microorganism" is used in its broadest sense and is therefore not limited in scope to prokaryotic organisms. Rather, the term "microorganism" includes within its scope bacteria, archaea, viruses (such as enveloped viruses), yeast, fungi, protozoa and algae.

In the context of this specification, the term "effective amount" refers to an amount of the modified peptide which is sufficient to cause a Log reduction in the number of microorganisms of at least 1.0, which means that less than 1 microorganism in 10 remains. The modified peptides of the present disclosure may provide Log reductions in the number of microorganisms of at least about 2.0, or at least about 3.0, or at least about 4.0, or at least about 5.0, or at least about 6.0, or at least about 7.0.

In the context of this specification, the term "medical device" refers to one or more devices that are designed for use within, or in contact with cells, tissue or organs of a human or animal body.

The expression "promoting tissue adhesion to a surface" as used herein refers to "encouraging" and/or "stimulating" tissue to adhesion to a surface.

A "cell recognition moiety and/or adhesion moiety" is a moiety which promotes adhesion through interaction with one or more cell components and/or one or more tissue components.

As used herein, the term "coating the surface" refers to applying to at least a portion of the surface, and may include non-covalent attachment to the surface.

As used herein, "integration or an implantable medical device" means that the device is connected to the tissue to an extent that is similar to, substantially the same as, or the same as, that of the naturally occurring component which the device replaces.

As described and exemplified herein the inventors have demonstrated that bifunctional peptides can be produced, which peptides have antimicrobial activity and mammalian cell recognition and/or adhesion properties. In certain embodiments, the peptide that is antimicrobial and comprises a cell recognition and/or adhesion moiety is chimeric.

The inventors have found (Examples 1 to 5) that the addition of a cell recognition and/or adhesion moiety to an antimicrobial peptide produces a peptide that is bifunctional, i.e. the addition of the cell recognition and/or adhesion moiety to the antimicrobial peptide retains the antimicrobial activity and facilitates cell recognition and/or adhesion.

As described and exemplified herein, the inventors have also found (Example 6) that a surface coated with a peptide with the amino acid sequence set forth in SEQ ID NO: 4 shows resistance to bacterial growth and promotes adhesion of mammalian cells to the surface. Without wishing to be bound to a particular theory, it is believed that in certain embodiments the amino acid sequence set forth in SEQ ID NO: 2 includes a cell recognition and/or adhesion moiety. This suggests that, when attached to a surface, a peptide which includes the amino acid sequence set forth in SEQ ID NO: 2, such as the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 4 or a variant or a functional fragment thereof, may be both antimicrobial and promote cell recognition and/or adhesion to the surface.

Accordingly, one aspect provides a method for promoting tissue adhesion to a surface, such as a surface of a device, the method comprising attaching to at least a portion of the surface, or coating at least a portion of the surface with, a peptide that is antimicrobial and comprises a cell recognition and/or adhesion moiety.

Promoting tissue adhesion and integration on the surface of an implantable device using a peptide that is both antimicrobial and possesses cell recognition and/or adhesion properties is advantageous in that is assists in the reduction of post-implantation infection, and assists in the integration of the device post-implantation. The dual acting surface coatmgs of the present disclosure, therefore, may assist in the reduction of the likelihood of post- implantation infection by providing a surface coating that is both antimicrobial and promotes cell recognition and/or adhesion to the coated surface. By promoting tissue adhesion, the dual acting surface coatings disclosed herein may also assist in the integration of the device post-implantation. In certain embodiments, the dual acting surface coatings of the present disclosure comprise a peptide that is both antimicrobial and a mammalian cell adhesion promoter. In certain embodiments, the dual acting surface coatings of the present disclosure comprise a peptide that is both antimicrobial and/or a mammalian cell adhesion promoter.

In one embodiment, there is provided a method for promoting tissue adhesion to a surface, such as a surface of a device, the method comprising attaching to at least a portion of the surface a peptide that is antimicrobial and comprises a cell recognition and/or adhesion moiety. In other embodiments, there is provided a method for promoting tissue adhesion to a surface, such as a surface of a device, the method comprising attaching to at least a portion of the surface a peptide that is antimicrobial, comprises a cell recognition and/or adhesion moiety. In one embodiment, there is provided a method for promoting tissue adhesion to a surface, such as a surface of a device, the method comprising coating at least a portion of the surface with a peptide that is antimicrobial and comprises a cell recognition and/or adhesion moiety. In other embodiments, there is provided a method for promoting tissue adhesion to a surface, such as a surface of a device, the method comprising coating at least a portion of the surface with a peptide that is antimicrobial, comprises a cell recognition and/or adhesion moiety.

In one embodiment, there is provided a method for encouraging or stimulating tissue adhesion to a surface, such as a surface of a device, the method comprising attaching to at least a portion of the surface, or coating at least a portion of the surface with, a peptide that is antimicrobial and comprises a cell recognition and/or adhesion moiety. In other embodiments, there is provided a method for encouraging or stimulating tissue adhesion to a surface, such as a surface of a device, the method comprising attaching to at least a portion of the surface, or coating at least a portion of the surface with, a peptide that is antimicrobial, comprises a cell recognition and/or adhesion moiety.

In one embodiment, there is provided a method for encouraging tissue adhesion to a surface, such as a surface of a device, the method comprising attaching to at least a portion of the surface, or coating at least a portion of the surface with, a peptide that is antimicrobial and comprises a cell recognition and/or adhesion moiety. In certain embodiments, there is provided a method for encouraging tissue adhesion to a surface, such as a surface of a device, the method comprising attaching to at least a portion of the surface, or coating at least a portion of the surface with, a peptide that is antimicrobial, comprises a cell recognition and/or adhesion moiety.

In one embodiment, there is provided a method for stimulating tissue adhesion to a surface, such as a surface of a device, the method comprising attaching to at least a portion of the surface, or coating at least a portion of the surface with, a peptide that is antimicrobial and comprises a cell recognition and/or adhesion moiety. In other embodiments, there is provided a method for stimulating tissue adhesion to a surface, such as a surface of a device, the method comprising attaching to at least a portion of the surface, or coating at least a portion of the surface with, a peptide that is antimicrobial, comprises a cell recognition and/or adhesion moiety.

In one embodiment, there is provided a method for encouraging or stimulating tissue adhesion to a surface, such as a surface of a device, the method comprising attaching to the surface, or coating the surface with, a peptide with the amino acid sequence set forth in one or more of SEQ ID NOs: 1 to 16 or a variant or functional fragment thereof.

The device may be devices that may come into contact with mammalian cells. Such a device may be coated with the peptides described herein to prevent microbial growth while promoting mammalian cell attachment.

In one embodiment, the device is an implantable device. The implantable device may be a device comprising a surface which may be coated with the peptide described herein and implanted in a subject. The implantable device may be any implantable device described herein.

The peptides may be attached to at least a portion of a surface, such as the surface of an implantable medical device. In certain embodiments, a portion, a substantial portion, or all surfaces of the implantable medical device that are in contact with tissue post-implantation are coated with the peptide. In some embodiments, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, 98% or 99% of the surface that will be in contact with tissue post-implantation is coated with the peptide.

In one embodiment, the peptide is attached to the surface to achieve a density in the range of from 0.01 nmole/cm ² to 20 nmole/cm ² , 0.1 to 20 nmole/cm ² , 0.3 to 18 nmole/cm ² , 0.5 to 15 nmole/cm ² or 0.5 to 13 nmole/cm ² , of peptide on the surface.

In one embodiment, there is provided a method for promoting integration of an implantable medical device, the method comprising attaching to at least a portion of the device, or coating at least a portion of the device with, a peptide that is antimicrobial and comprises a cell recognition and/or adhesion moiety. In one embodiment, there is provided a method for promoting integration of an implantable medical device, the method comprising attaching to at least a portion of the device, or coating at least a portion of the device with, a peptide that is antimicrobial, comprises a cell recognition and/or adhesion moiety.

Another aspect of the disclosure provides a cationic peptide having antimicrobial activity and further comprising a cell recognition and/or adhesion moiety.

The peptide may be arginine rich. In one embodiment the peptide may be arginine rich in the C-terminal portion. The peptide may comprise at least 10%, or at least 20%, or at least 30%, or at least 40% arginine. The peptide may be arginine and lysine rich. In one embodiment the peptide may be arginine and lysine rich in the C-terminal portion. The peptide may comprise at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50% arginine and lysine. The peptide may comprise between about 10 and about 60 amino acids.

By way of example, the peptide having antimicrobial activity may be melimine, protamine, lactoferricin, protattin, or a variant or functional fragment thereof. In an exemplary embodiment, the peptide is melimine or a variant or functional fragment thereof. In a particular exemplary embodiment the peptide having antimicrobial activity may comprise an amino acid sequence set forth in SEQ ID NO: l or SEQ ID NO:2 or a variant or functional fragment thereof

The protamine peptide may have the amino acid sequence set forth in SEQ ID NO: 17, the lactoferricin peptide may have the amino acid sequence set forth in SEQ ID NO: 18, and the protattin peptide may have the amino acid sequence set forth in SEQ ID NO: 19. In alternative embodiments the peptide having antimicrobial activity may comprise one of the following amino acid sequences: TLISWIQRPRVS (SEQ ID NO:20); TLISWIKN R QRPRVS (SEQ ID NO:21); TLI S WIQRPRVSRRRRRRGGRRRR (SEQ ID NO:22); and KN RKRRRRRRGGRRRR (SEQ ID NO:23). In certain embodiments the cell recognition and/or adhesion moiety is a peptide moiety. In exemplary embodiments the peptide moiety may comprise a cell recognition and/or adhesion motif selected from GD, RGDSPASSKP, VGVAPG, GEFYFDLRLKGDK, YIGSR, and WQPPRARI. In another embodiment, the cell recognition and/or adhesion moiety may be comprised within a peptide with an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 4. However the skilled addressee will appreciate that the scope of the disclosure is not limited to these specific cell recognition and/or adhesion motifs. Other suitable motifs or peptide moieties as may be known to those skilled in the art may also be employed.

In some embodiments, the peptides of the first aspect may be further modified to facilitate attachment of the peptide to a surface, for example, by the introduction of a thiol functional group, such as a thiol-containing residue, into the peptide. The thiol-containing residue may be cysteine. The thiol functional group or residue that comprises a thiol functional group may be introduced into the peptide at any position, and in embodiments of the disclosure is introduced at the C-terminus or at the N-terminus of the peptide. In particular embodiment a cysteine residue is introduced at the N-terminus of the peptide.

In exemplary embodiments, the peptide having antimicrobial activity comprises one of the following amino acid sequences:

CTLISWIKNKRKQRPRVSRRRRRRGGRRRR (SEQ ID NO:3)

CKN RKRRRRRRGGRRRR (SEQ ID NO:4)

In exemplary embodiments, the peptide of the first aspect, having antimicrobial activity and possessing a cell recognition and/or adhesion moiety (underlined), comprises or consists of one of the following amino acid seqtiences:

CTLISWIKNKRKQRPRVSRRRRRRGGRRRRRGD (SEQ ID NO:5)

CKNKRKRRRRRRGGRRRRRGD (SEQ ID NO:6)

In alternative embodiments, the peptide of the first aspect having antimicrobial activity and possessing a cell recognition and/or adhesion moiety (underlined), comprises, or consists of one of the following amino acid sequences: CTLI S WIKNKR ORPRVSRRRRR GGR RRRGDSP AS S KP (SEQ ID NO:7);

CTLISWIK KRKQRPRVSRRRRRRGGRRRRVGVAPG (SEQ ID NO: 8);

CTLISWIKNKR ORPRVSRRRRRRGGRRRRYIGSR (SEQ ID NO:9);

CTLISWIKNKRKORPRVSRRRRRRGGRRRRWQPPRARI (SEQ ID NO: 10);

CTLISWIKNKRKORPRVSRRRRRRGGRRRRGEFYFDLRL GD (SEQ ID NO: l 1); CKNKRKRRRRRRGGRRRRRGDSPASSKP (SEQ ID NO: 12);

CKNKRKRRRRRRGGRRRRVGVAPG (SEQ ID NO: 13);

CKNKRKRRRRRRGGRRRRYIGSR (SEQ ID NO: 14);

CKNKRKRRRRRRGGRRRRWQPPRARI (SEQ ID NO: 15); or

CKNKRKRRRRRRGGRRRRGEFYFDLRLKGDK (SEQ ID NO: 16).

Those skilled in the art will appreciate that the cell recognition and/or adhesion moiety may be incorporated at one or more positions within the peptide sequence or at the C- tenninus or the N-tenriinus. In one embodiment, the peptide moiety is located at the C- terrninus of the peptide having antimicrobial activity. In another embodiment, the peptide moiety is located at the N-terminus of the peptide having antimicrobial activity. In another embodiment, the peptide moiety is located between the C-terminus and the N-terminus of the peptide having antimicrobial activity.

Also encompassed by the present disclosure are functional fragments of the peptides disclosed herein. The term "functional fragment" refers to a peptide that is a constituent of an antimicrobial peptide disclosed herein, which retains antimicrobial activity. The antimicrobial activity of the fragment need not be qualitatively or quantitatively the same as that of the peptide from which the fragment is derived. Similarly the fragment need not only include amino acid residues that are contiguous in the peptide from which the fragment is derived.

Also encompassed by the present disclosure is the modification of variants of the peptides disclosed herein. The term "variant' as used herein refers to substantially similar sequences. Generally, peptide sequence variants possess qualitative biological activity in common. Further, these peptide sequence variants may share at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with peptides disclosed herein. Variant peptides also encompass peptides that include both D- and L-amino acids, modified amino acids (for example methylated amino acids) and unconventional or non- naturally occurring amino acids (for example L- 3-phenyl-phenylalanine and L- biphenylalanine). Suitable modified, unconventional and non-naturally occurring amino acids are well known to those skilled in the art and the present disclosure is not limited by reference to those specifically recited by way of example above.

Embodiments of the present disclosure also provide methods for encouraging or stimulating tissue adhesion and integration of a surface, such as the surface of a device, comprising attaching to a surface, or coating a surface with, a peptide disclosed herein possessing antimicrobial activity and including a cell recognition and/or adhesion moiety. In some embodiments, the device is an implantable medical device. The implantable medical device may be, for example, a hearing implant, a knee implant, a hip implant, an implantable electrode, an implantable neuro prosthetic electrode array, a catheter, such as an indwelling catheter, or carrier for antibiotic, diagnostic or therapeutic agents.

Peptides of the present disclosure can, when attached to, or coating, a surface of an implantable medical device facilitate or enable cells from the recipient of the device to adhere to the surface, migrate along the surface and proliferate on the surface. Accordingly, such peptides provide a scaffold for cell adhesion allowing improved tissue integration around the device, whilst also preventing infection. In one embodiment, the peptide which can when attached to, or coating, a surface facilitate or enable cells from the recipient of the device to adhere to the surface, migrate along the surface and proliferate on the surface has an amino acid sequence set forth in one or more of SEQ ID Nos: 1 to 16 or a variant or a functional fragment thereof.

Embodiments of the present disclosure also provide methods for preventing the occurrence of microbial infection on or around the surface of a medical device inserted into a patient, or at or near the site of insertion of the medical device, comprising attaching to a surface of the device, or coating a surface of the device with, a peptide disclosed herein possessing antimicrobial activity and including a cell recognition and/or adhesion moiety. Thus, a surface to which a peptide disclosed herein has been attached or coated has the dual advantage of inhibiting colonisation of the surface of the device by microorganisms, and at the same time promoting tissue adhesion and/or integration of the device with the tissue.

In one embodiment, the peptide that is antimicrobial and comprises a cell recognition and/or adhesion moiety is selected from the group consisting of:

(a) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 1 ;

(b) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 2;

(c) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 3;

(d) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 4;

(e) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 5;

(f) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 6;

(g) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 7;

(h) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 8;

(i) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 9;

(j) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 10;

(k) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 11 ;

(1) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 12;

(m) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 13;

(n) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 14;

(o) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 15;

(P) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 16;

(q) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 17;

(r) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 18;

(s) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 19;

(t) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 20;

(u) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 21 ;

(v) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 22;

(w) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 23;

00 a peptide consisting essentially of the amino acid sequence set forth in SEQ ID

NO: 1 ;

(y) a peptide consisting essentially of the amino acid sequence set forth in SEQ ID

NO: 2; (Z) a peptide consisting essentially of the amino acid sequence set forth in SEQ ID

NO: 3;

(aa) a peptide consisting essentially of the amino acid sequence set forth in SEQ ID NO: 4;

(bb) a peptide consisting essentially of the amino acid sequence set forth in SEQ ID NO: 5;

(cc) a peptide consisting essentially of the amino acid sequence set forth in SEQ ID NO: 6;

(dd) a peptide consisting essentially of the amino acid sequence set forth in SEQ ID NO: 7;

(ee) a peptide consisting essentially of the amino acid sequence set forth in SEQ ID NO: 8;

(ff) a peptide consisting essentially of the amino acid sequence set forth in SEQ ID

NO: 9;

(gg) a peptide consisting essentially of the amino acid sequence set forth in SEQ ID NO: 10

(hh) a peptide consisting essentially of the amino acid sequence set forth in SEQ ID

NO: 11

(ϋ) a peptide consisting essentially of the amino acid sequence set forth in SEQ ID

NO: 12

(jj) a peptide consisting essentially of the amino acid sequence set forth in SEQ ID

NO: 13

(kk) a peptide consisting essentially of the amino acid sequence set forth in SEQ ID NO: 14

(11) a peptide consisting essentially of the amino acid sequence set forth in SEQ ID

NO: 15

(mm) a peptide consisting essentially of the amino acid sequence set forth in SEQ ID NO: 16

(nn) a peptide consisting essentially of the amino acid sequence set forth in SEQ ID NO: 17

(oo) a peptide consisting essentially of the amino acid sequence set forth in SEQ ID ( p) a peptide consisting essentially of the amino acid sequence set forth in SEQ ID NO: 19;

(qq) a peptide consisting essentially of the amino acid sequence set forth in SEQ ID NO: 20;

(rr) a peptide consisting essentially of the amino acid sequence set forth in SEQ ID NO: 21 ;

(ss) a peptide consisting essentially of the amino acid seqvience set forth in SEQ ID NO: 22;

(tt) a peptide consisting essentially of the amino acid sequence set forth in SEQ ID NO: 23;

(uu) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 ;

(vv) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 2;

(ww) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 3;

(xx) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 4;

(yy) ^a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 5;

(zz) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 6;

(aaa) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 7;

(bbb) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 8;

(ccc) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 9;

(ddd) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 10;

(eee) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 1 ;

(fff) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 12;

(ggg) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 13;

(hhh) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 14;

(iii) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 15; jjj) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 16;

(kkk) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 17;

(111) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 18;

(mmm) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 19;

(nnn) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 20;

(ooo) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 21 ;

(ppp) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 22; or (qqq) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 23.

The peptides according to the present disclosure may be prepared using standard peptide synthesis methods that are well known to those skilled in the art. For example, the peptides may be synthesized by standard solution phase methodology, as described in Hruby, Victor J., Meyer, Jean-Philippe, Chemical synthesis of peptides. University of Arizona, USA. Editor(s): Hecht, Sidney, M. Bioorganic Chemistry: Peptides and Proteins (1998), pp 27-64, Oxford University Press, New York, N.Y.

The peptides may also be synthesized by solid phase methodology using Boc chemistry, as described by Schnolzer et al, 1992, Int. J. Pept Protein Res. 40,180-193. Following deprotection and cleavage from the solid support the reduced peptides may be purified vising preparative chromatography.

The peptides may also be synthesized by solid phase methodology using Fmoc chemistry, for example as described below:

1) Peptide is synthesized by Fmoc solid-phase peptide synthesis using an automatic synthesizer.

2) Peptide is synthesized from its C-terminus by stepwise addition of amino acids.

3) the first Fmoc-amino acid is attached to an insoluble support resin via an acid labile linker.

4) After deprotection of Fmoc by treatment with piperidine, the second Fmoc-amino acid is coupled utilising a pre-activated species or in situ activation.

5) After the desired peptide is synthesized, the resin bound peptide is deprotected and detached from the resin via TFA cleavage.

6) Following deprotection and cleavage from the solid support the reduced peptides are purified using preparative chromatography.

In accordance with the present disclosure peptides may also be produced using standard techniques of recombinant DNA and molecular biology that are well known to those skilled in the art. Guidance may be obtained, for example, from standard texts such as Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989 and Ausubel et al., Current Protocols in Molecular Biology, Greene Publ. Assoc. and Wiley-Intersciences, 1992. Methods described in Morton et al, 2000 (Immunol Cell ft ^' o/78:603-607), Ryan et al, 1995 (J BiolChem 270:22037-22043) and Johnson et al., 2005 (J BiolChem 280:4037-4047) are examples of suitable purification methods for relaxin polypeptides, although the skilled addressee will appreciate that the present disclosure is not limited by the method of purification or production used and any other method may be used to produce relaxin for use in accordance with the methods and compositions of the present disclosure.

By way of example, a nucleotide sequence encoding the desired peptide sequence may be inserted into a suitable vector, operably linked to a suitable promoter, and the peptide expressed in an appropriate expression system. The host cell may be any suitable prokaryotic, yeast or higher eukaryotic cell. Appropriate host cells can be selected without undue experimentation by a person skilled in the art. The host cell may then be cultured in conventional nutrient media modified for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. Culture conditions, such as media, temperature, pH, and the like, can be selected without undue experimentation by the person skilled in the art. The cells may then be selected and assayed for the expression of the peptide using standard procedures.

Encompassed by the present disclosure are nucleic acid molecules comprising nucleotide sequences encoding modified peptides as disclosed herein.

Under some circumstances it may be desirable to undertake oxidative bond formation of the expressed peptide as a chemical step following peptide expression. This may be preceded by a reductive step to provide the unfolded peptide. Those skilled in the art will be capable of determining appropriate conditions for the reduction and oxidation of the peptide.

The peptides of the present disclosure find use against a range of microorganisms, for example, bacteria, fungi (including mould and yeast), and protozoa. The bacteria may be Gram-negative or Gram-positive bacteria, such as for example Staphylococcus spp., Streptococcus spp., Enterococcus spp., Acinetobacter spp., Pseudomonas spp., Haemophilus spp., Proteus spp., Serratia spp., Escherichia spp., Salmonella spp., Klebsiella spp., Bacillus spp. and Listeria spp. Exemplary species include but are not limited to Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis, coagulase negative staphylococci, Enterococcus faecalis, Enterococcus faecium including vancomycin-resistant Enterococcus faecium, Streptococcus pneumoniae, Klebsiella pneumoniae, Haemophilus influenzae, Pseudomonas aeruginosa, Escherichia coli, Salmonella enterica, Salmonella typhi, Bacillus cereus, and Listeria monocytogenes.

Those skilled in the art will appreciate that the peptides may be useful in the control of other bacterial species in addition to those specifically recited above.

Yeast against which the peptides may find use include, but are not limited to, Candida albicans.

Mould against which the peptides may find use include, but are not limited to, Fusarium spp.

Protozoa against which the peptides may find use include, but are not limited to, Acanthamoeba spp.

The present disclosure also relates to antimicrobial compositions comprising one or more peptides as defined herein, optionally together with one or more pharmaceutically acceptable carriers, excipients or diluents. Compositions may be used to eliminate, reduce or inhibit microbial growth by allowing the composition to contact the microorganisms to be inhibited and/or the environment of the microorganisms. The compositions may be used in a range of different environments that are inhabited or colonised by, or susceptible to being inhabited or colonised by, unwanted microorganisms. The compositions may be adapted for application to a surface of an article or an area which may come into contact with, or is believed to harbour microorganisms. Examples of such articles and areas include, but are not limited to, surgical instruments, medical devices, medical establishments, such as doctor's surgeries and hospitals, and food preparation areas.

The compositions may take one or more suitable forms depending on the intended use thereof For example, the compositions may take the form of a solution which can be applied to a surface or area by spraying. Alternatively, the compositions may take the form of a solution into which articles may be immersed. In another embodiment the compositions may take the form of a wipe comprising the peptides which can be used to apply the modified peptides to a surface or article.

In exemplary embodiments the peptides disclosed herein are attached to a surface, for example the surface of a device, such as a medical device. The medical device may be, for example, a contact lens, a corneal only or inlay device, fluid collection bag, sensor, hydrogel bandage, tubing, stent, heart valve, an implant, such as a hearing implant, a knee implant, a hip implant, an implantable electrode, an implantable neuroprosthetic electrode array (such as those manufactured by Cochlear), a catheter (such as an indwelling catheter) or carrier for antibiotic, diagnostic or therapeutic agents.

The surface of the device may comprise a polymer, for example a hydrogel, a silicon hydrogel, a polymer or copolymer of 2-hydroxyethylmethacrylate, silicon rubber, polyurethane, polypropylene, polyethylene, polyacrylamide, polytetrafluoro ethylene (Teflon), a biodegradable polymer, such as poly-lactide or combinations thereof.

Those skilled in the art will appreciate that the peptides may be attached to the surface of other medical devices which are susceptible to contamination by microorganisms. The peptides may also be attached to the surface of devices intended for use in fields other than the medical field. In other embodiments the modified peptides may be attached to surfaces located in food preparation areas, for example kitchens. The peptides may be attached to a solid surface that is susceptible to contamination by microorganisms.

Attachment of the peptides to a solid surface may be either direct or indirect. Indirect attachment may involve attachment via linker and/or spacer molecules. Suitable linkers and spacer molecules will be well known to those skilled in the art.

The solid surface may be suitably functionalised so as to facilitate direct covalent attachment of the peptide thereto. Functionalisation of the solid surface may be achieved by use of a reagent that activates a functional group of the surface such that this functional group is capable of reacting with a functional group of the peptide. For example, an amino or thiol group of the modified peptide may be reacted with an isothiocyanate, an acyl azide, an N-hydroxysuccinimide ester, an aldehyde, a tosylate, an imido ester or an aryl halide group of the surface. In such embodiments, the surface preferably comprises a polymer, such as a polymer described herein.

Indirect attachment of the modified peptides to the surface may be achieved by use of cross-linking reagents, for example 4-azidobenzoic acid, 4-fluoro-3-nitro-azidobenzene, N, N'-carbonyldiimidazole and carbodiimides, such as EDC. Other suitable cross-linking reagents will be readily apparent to those skilled in the art.

In some embodiments, a linker is attached to the surface, the linker comprising or being, a functional group that is capable of reacting with a thiol functional group of the peptide. The linker may be a maleimide, which is able to undergo a Michael reaction with the thiol functional group thereby linking the modified peptide and the surface via a succinimidylene linker. The linker may be of the formula -NH(C=0)CH2-leaving group, which is able to undergo nucleophilic attack by the thiol functional group thereby linking the peptide and the surface by an -NH(C=0)CH ₂ - linker. In alternative embodiments, the modified peptide may be attached to the solid surface utilising native chemical ligation.

Indirect attachment of the modified peptide to the surface may also include the presence of a spacer located between the surface and the linker. The use of a spacer allows modulation of the distance between the surface and the peptide. Examples of spacers include, but are not limited to: alkylene, perfluoroalkylene, polyethylene glycol, phenylene and cycloalkylene.

In one embodiment, the spacer may be C1-C20 perfluoroalkylene C3-C6 cycloalkylene, Q- C2 ₀ alkylene, polyethylene glycol, or combinations thereof. These groups may be hydrophobic or hydrophilic. Alternatively, the spacer may be C ₃ -C ₆ cyclo alkylene, C]-C ₆ alkylene, between 2 and 20 ethylene glycol units, or combinations thereof.

In another embodiment the spacer has the following structure: wherein T is -(CH ₂ CH ₂ 0) _m - Ci-C ₆ alkylene or C ₃ -C ₆ cyclo alkylene, n is a number between 0 and 5 and m is a number between 1 and 10. In an alternative embodiment, T is Ci-C ₆ alkylene or C ₃ -C ₆ cyclo alkylene, and n is 0, 1, 2 or 3. In another embodiment T is C3-C6 cyclo alkylene, and n is 1 or 2. In still a further embodiment T is cyclohexylene, and n is 0 or 1.

The spacer molecule may be indirectly attached to the surface via a bivalent functional group comprising an amide.

The spacer molecule may be attached to the surface via a C-C bond, a C-N bond, Si-0 bond, Si-C bond, Ti-0 bond or an amide.

In one embodiment the bivalent functional group comprising an amide is a group of the following formula:

- -(CH ₂ ) _P N j^. _n o _r r ^r wherein p is a number between 1 and 6. In an alternative embodiment p is 2, 3 or 4. In still a further embodiment p is 3.

In yet another embodiment of the disclosure the peptide may be attached to the surface via the following bivalent groups:

wherein p is a number between 1 and 3 and q is a number between 0 and 2. In an alternative embodiment, p is 3 and q is 1.

In the event that a solid surface does not possess suitable reactive functional groups for attachment of the peptide, such functional groups may be incorporated by conventional synthetic methods known to those skilled in the art. Alternatively, where the surface is a polymeric surface, appropriate reactive groups may be introduced thereto by the addition of polymerisable monomers comprising reactive groups into a monomer mixture used to prepare the polymer. Where the surface is not capable of chemical modification, plasma activation of the surface may be employed. Plasma modification may be appropriate where the surface does comprise reactive functional groups that are modified, or contains metal.

Examples of polymer surfaces onto which the peptides may be attached include surfaces formed from, for example, polymers and copolymers of styrene and substituted styrenes, ethylene, tetrafluoro ethylene, propylene, acrylates and methacrylates, N-vinyl lactams, acrylamides and methacrylamides, acrylonitrile, acrylic and methacrylic acids as well as polyur ethanes, polyesters, polydimethylsiloxanes, biodegradable polymers, such as poly- lactide, and mixtures thereof. Such polymers may include hydrogels and silicone hydrogels.

In some embodiments, lightly crosslinked polymers and copolymers of 2- hydroxyethylmethacrylate ("HEMA") are used. By "lightly crosslinked" it is meant that the polymer has a sufficiently low crosslink density such that it is soft and elastic at room temperature. Typically, a lightly crosslinked polymer possesses about 0.1 to about 1 crosslinking molecules per about 100 repeating monomer units. Examples of suitable lightly crosslinked HEMA polymers and copolymers include, but are not limited to etafilcon A and copolymers of glycerol methacrylate and HEMA or methyl methacrylate and HEMA (HEM A- MM A). Preferred silicone hydrogels include those comprising hydrophilic monomers, such as N,N-dimethylacrylamide.

Embodiments of the present disclosure also provide methods for eliminating or inhibiting the growth of one or more microorganisms or the colonisation of an environment by the microorganisms, comprising contacting the one or more microorganisms, or an environment inhabited by the microorganisms, with an effective amount of a peptide disclosed herein possessing antimicrobial activity and/or including a cell recognition and/or adhesion moiety. In order to facilitate contact of the microorganisms, or the environment inhabited by the microorganisms with the peptides, the peptides may be present in the form of a composition as described herein. Alternatively, the peptides may be attached to a solid surface, for example the surface of a device, such as a medical device as described herein.

Those skilled in the art will appreciate that the embodiments described herein are susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes such variations and modifications which fall within the spirit and scope. The disclosure also includes the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and combinations of two or more of said steps or features.

Certain embodiments will now be described with reference to the following examples which are intended for the purpose of illustration only and are not intended to limit the scope of the generality hereinbefore described.

Examples

Example 1 -Peptides used in the present study

Melimine (TLISWIK K QRPRVSRRRRRRGGRRRR; SEQ ID NO:l ) was synthesized by conventional solid-phase peptide synthesis protocols and was obtained from American Peptide Company (CA, USA). A truncated peptide derived from melimine in which amino acid residues 1-6 and 12-17 of the full length melimine sequence were deleted, and termed herein "Mel4" (KNKRKRRRRRRGGRRRR; SEQ ID NO:2), was also synthesized. A cysteine residue was added to the N-terminus of each of the peptides of SEQ ID Nos: l and 2, to generate peptides termed herein "Cys-melimine" (SEQ ID NO:3) and "Cys-Mel4" (SEQ ID NO:4). The Cys-Mel4 peptide was further modified by the inclusion of the RGD tripeptide at the C-terminus to generate a peptide termed herein "Cys-Mel4-RGD" (CKNKRKRRRRRRGGRRRRRGD; SEQ ID NO:6).

Example 2-Antimicrobial activity of modified peptides

Minimal inhibitory concentrations ^g/ml) for synthesized peptides against Staphylococcus aureus strains 31 and 004, Staphylococcus epidermidis strain 013 and Pseudomonas aeruginosa strains 6294 and PAOl were detemrined in a two-fold dilution series. The growth medium was Mueller-Hinton broth with 0.2% BSA and 0.01 % acetic acid. Bacterial strains were added at a final density of 10 ⁵ CFU/ml. The results are shown in Table 1.

Table 1: Minimal inhibitory concentrations of synthesized peptides

The truncated peptide Cys-Mel4 containing a cysteine residue at the N-terminus (SEQ ID NO:4) showed a similar or lower MIC as the parent antimicrobial peptide, Melimine. Antibacterial activity of the Mel4 peptide was retained following addition of RGD.

Example 3-Bacterial adherence to peptides attached to a polymer surface Covalent Attachment

Synthesized peptides were covalently attached to the polymer hydroxyethyl methacrylate- methyl methacrylate (HEMA-MMA). The zero-length crosslinker l-ethyl-3-[3- dimethylaminopropyljcarbodiimide hydrochloride (EDC) was used to crosslink carboxylic acid (COOH) groups on the polymer and amine (NH2) groups of the peptides. The ability of bacterial strains (Staphylococcus spp. as for Example 2, above, and P. aeruginosa strain 6294) to adhere to the peptide-coated HEMA-MMA surface was determined.

Bacteria from frozen stock cultures (stored at -80°C) were streaked onto agar plates. Single colonies were inoculated into tryptic soy broth and incubated at 37°C overnight. These broth colonies were used for experiments without further passage. Broth colonies were pelleted by centrifugation and resuspended in phosphate buffered saline (PBS) twice to remove growth medium. Bacteria were diluted to a final density of 10 ⁶ CFU/ml. Staphylococcus spp. were diluted in 10% tryptic soy broth (90% PBS) and P. aeruginosa 6294 was diluted in PBS only.

Samples of the polymer (14 mm diameter) were placed in the wells of a 24-well polystyrene tissue culture plate. 1 ml of bacterial dilution was added to each well and the plate was incubated at 37°C overnight. Samples were rinsed thrice in PBS to remove loosely associated bacteria, transferred to a 5 ml vial containing PBS and small bead, and vortexed at high speed to dissociate adherent bacteria. Aliquots of PBS were transferred to tryptic soy agar plates containing 0.5% (w/v) polysorbate 80 and 0.1% (w/v) lecithin. The number of adherent bacteria (CFU/mm ) was calculated from the resulting colonies, and is expressed as a Logl O reduction in adhesion, in comparison to the number of bacteria adherent to unmodified HEMA-MMA (negative control) in Table 2.

Table 2: Mean LoglO reduction in bacteria adherent to peptide-coated HEMA-MMA in comparison to unmodified HEMA-MMA

Mean LoglO reduction in bacterial adhesion

Peptide coating S. aureus 31 S. aureus 004 S. epidermidis P. aeruginosa

013 6294

Above limit of detection

These data indicate that Cys-Mel4 (SEQ ID NO:4) can produce a surface coating on the polymer that retains a range of antibacterial activity against various bacterial strains.

Ionic attachment

Peptides were ionically bound to HEMA-MMA via the interaction between negatively charged COOH groups of the polymer and positively-charged amino acid residues of the peptides. Bacterial adherence experiments were carried out using S. aureus Saur31 and the results shown in Table 3.

Table 3

* below limit of detection These data indicate that the peptide containing RGD is as active, when ionically bound, as the parent peptide melimine.

Example 4- Mam median cell recognition by modified peptides

The Cys-Mel4-RGD peptide was covalently bound onto a glass surface and both the antimicrobial activity of the peptide determined and the ability of human corneal limbal epithelial cells (HCLE) to migrate and proliferate on the coated surface were determined. Briefly, cells were maintained on plastic at 2 x 10 ⁴ /cm ² in a keratinocyte serum- free medium (Invitrogen-Gibco, Grand Island, NY) supplemented with 25 pg/mL bovine pituitaiy extract, 0.2 ng/mL epidermal growth factor (Invitrogen, Mount Waverley, VIC, Australia), and 0.4 mM CaCl ₂ and were grown at 37°C in a 5% carbon dioxide atmosphere. All experiments involving the culture of HCLE were performed at 37°C in a 5% C0 ₂ atmosphere. Cells were allowed to adhere to glass coverslips coated with Cys- Mel-RGD for 18 hours and then viewed by microscopy for number and morphology of cells. For bacterial adhesion, S. aureus Saur 31 was grown overnight in TSB and then washed three times in phosphate buffered saline (PBS) and resuspended in PBS to OD600 = 0.1. Bacterial cells were allowed to adhere to the Cys-Mel-RGD coated glass for up to 48 hours, and then glass was washed in PBS and stained with LIVE/DEAD stain BacLight Bacterial Viability Kit (Molecular Probes, Inc, Eugene, OR, USA). The glass slides with adherent bacteria were then viewed with a fluorescent microscope.

As shown in Figure 1, human epithelial cells adhered to the coated surface, and can subsequently migrate and proliferate, whilst antimicrobial activity was maintained.

Example 5-Cytotoxicity

Cytotoxicity of the Cys-Mel4-RGD peptide against mammalian cells was determined using two different methods, cell growth inhibition in liquid and direct contact. L929 murine fibroblasts were used, maintained and tested in Dulbecco's Modified Eagle Medium, (DMEM) with 10% serum. Cell growth inhibition in liquid

L929 cells were established at low density in 35 mm polystyrene petri dishes. After 24 hours, medium was replaced with fresh medium containing the test solution (25% test solution, 75% fresh medium). Test peptides were dissolved in PBSas. After a further 48 hours, cells were stained with trypan blue, which is excluded from viable cells, and counted by microscopy. Cytotoxicity is indicated by a greater than 30% reduction in viable cell numbers, as compared to non-challenged cultures. Experiments were conducted in accordance with ISO 10993-5, Biological evaluation of medical devices - Part 5: Tests for in vitro cytotoxicity.

The results shown in Table 4 indicate that the Cys-Mel4-RGD peptide is non-cytotoxic in solution to mammalian cells at 600ug/ml.

Table 4: Percentage reduction in viable cell count of L929 fibroblasts exposed to test solutions

Direct contact

L929 murine fibroblasts were grown to near confluency in 6-well polystyrene cell culture plates (diameter 35 mm). Medium was replaced and the sterile test material (as described in Table 5) was placed directly on the cell monolayer for 24 hours, during which time any cytotoxic components of the test material will disrupt cell growth. Cells were then stained with tiypan blue and counted by microscopy. Samples were graded according to the presence of degraded cells, and how far the zone of degraded cells extended from beneath the sample, as follows: 0 (None) - no detectable zone of reactivity around or under sample; 1 (Slight) zone limited to area under sample; 2 (Mild) zone extends less than 0.5cm beyond sample; 3 (Moderate) zone extends 0.5 to 1.0 cm beyond sample; and 4 (Severe) zone extends more than 1.0 cm beyond sample. Results are shown in Table 5.

Table 5: Reactivity grade observed in L929 fibroblast cultures exposed to solid samples.

* latex cut from powder-free latex examination gloves (Livingstone International)

All polymer samples caused some damage to the cell monolayer directly underneath the samples due to physical disruption, and were thus graded "1 ". These data indicate that the Cys-Mel4-RGD peptide is non-cytotoxic when covalently attached to a polymer surface and in direct contact with mammalian cells.

Example 6 - Mammalian cell adhesion

The synthesised peptides were covalently attached to a fluorinated ethylene propylene (FEP) substrate. FEP samples (1 cm square) were coated with a thin film of poly(heptylamine) by plasma polymerisation to provide amine (NH2) surface chemistry. The amine-functionalized surface was incubated with the amine-to-thiol crosslinker, succinimidyl-4-(N-maleimidomethyl)cyclohexane-l-carboxylate (SMCC), and then with peptides containing a cysteine residue. Peptide-coated FEP samples were sterilized and placed in 24- well plates for mammalian cell adhesion experiments.

HaCaT cells (human keratinocyte cell line) were maintained in DMEM with 10% serum; DMEM without serum was used for adhesion experiments. HaCaT cells suspended in DMEM without serum were added to 24-well plates containing peptide-coated FEP samples. After incubation for 48 hours at 37°C in a 5% C0 ₂ atmosphere, samples were rinsed with PBS and stained with LIVE/DEAD® Viability/Cytotoxicity Kit, for mammalian cells (Life Technologies, CA, USA) and viewed with a fluorescence microscope (Figure 2). As can be seen from Figure 2, viable HaCaT cells adhered to the coated surface, and can subsequently migrate and proliferate on the surface of the peptide coated FEP samples.

Sequence Listing

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Brien Holden Vision Institute

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<223> Synthetic sequence

<400> 21

Leu He Ser Trp He uys Asn Lys Arg Lys Gin Arg Pro Arg

5 10 15

Ser <210> 22

<211> 24

<212> PRT

<213> Artificial

<220>

<223> Synthetic sequence

<400> 22

Leu lie Ser Trp lie Gin Arg Pre Arg Val Ser Arg Arg Arg Arg

5 10 15

Arg Gly Gly Arg Arg Arg Arg

<210> 23

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Synthetic sequence

<400> 23

Lys Asn Lys Arg Lys Arg Arg Arg Arg Arg Arg Gly Gly Arg Arg Arg 1 5 10 15

Arg