WO2006045156A1 - Therapy for multi-drug resistant microorganisms - Google Patents

Abstract

A pharmaceutical or veterinary composition comprising an antimicrobial peptide and a macrolide component together with a pharmaceutically or veterinarily acceptable carrier, wherein said antimicrobial peptide and macrolide component interact synergistically against a multi-drug resistant microorganism, a method of treatment and/or prophylaxis of an infection caused by a multi-drug resistant microorganism using said composition in the manufacture of a medicament for said treatment.

Description

THERAPY FOR MULTI-DRUG RESISTANT MICROORGANISMS

This invention relates to pharmaceutical preparations with antimicrobial and anti-pathogenic activity against multi-drug resistant microorganisms, in particular, multi¬ drug resistant gram-negative microorganisms. The invention particularly targets polymyxin-resistant bacteria such as Pseudomonas aeruginosa..

BACKGROUND OF THE INVENTION

All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.

The world is facing an enormous and growing threat from the emergence of microorganisms that are resistant to a wide range of currently available antibiotics. For example, infections caused by multi-drug resistant gram- negative bacteria, particularly Pseudomonas aeruginosa and Acinetobacter baumannii, are becoming a critical challenge in compromised hospital patients (e.g.. patients in intensive care units, patients with cystic fibrosis or diffuse panbronchiolitis^1-6) and with seemingly trivial infections in sites such as middle and central ear ^{7. 8} and eyes ⁹. The level of resistance to front line anti- pseudomonal agents is alarmingly high.² of particular concern are reports on antibiograms of P. aeruginosa. and A. baumannii in hospital outbreaks, where colistin, a member of the polymyxin class of antibiotics, is the only effective antibiotic.⁴' ⁶ Unfortunately, polymyxin-resistant P. aeruginosa has been isolated from patients¹⁰ with eye infections,⁹ ear infections,⁷ and particularly in the sputum of patients with cystic fibrosis¹¹' ¹²'¹³. The appearance of polymyxin-resistant gram-negative pathogens is of great concern.

The present invention addresses the need for an effective therapy against multi-drug resistant microorganisms. It also specifically addresses multi-drug resistant gram-negative bacteria. Examples of these are polymyxin-resistant gram-negative bacteria such as P. aeruginosa, and A. baumannii.

The present invention also addresses the problem of pathogenic factors released by living microorganisms in a host which lead to destructive effects on tissues at the site(s) of infection.

SUMMARY OF THE INVENTION

In a first aspect the present invention provides a pharmaceutical or veterinary composition comprising an antimicrobial peptide and a macrolide component together with a pharmaceutically or veterinarily acceptable carrier, wherein said antimicrobial peptide and macrolide component interact synergistically against a multi-drug resistant microorganism.

The present invention also provides a pharmaceutical or veterinary composition, comprising an antimicrobial peptide and a macrolide component together with, a pharmaceutically or veterinarily acceptable carrier, wherein said antimicrobial peptide and macrolide component interact with bi-directional antimicrobial synergy against a multi-drug resistant microorganism.

The pharmaceutical or veterinary composition may conveniently foe in the form of a kit in which the antimicrobial peptide and macrolide component axe held separately for separates, sequential or simultaneous use, It has been found that the antimicrobial peptide and macrolide component are effective in preventing or inhibiting growth of a multi-drug resistant microorganism.

Accordingly, the invention further provides the use of an antimicrobial peptide and a macrolide component, for preventing or inhibiting growth of a multi-drug resistant microorganism.

The invention still further provides a method of preventing or inhibiting growth of a multi-drug resistant microorganism comprising the step of administering to a subject in need thereof an effective amount of an antimicrobial peptide and a macrolide component.

The method above includes the administration of an effective amount of an antimicrobial peptide and a macrolide component separately, simultaneously or sequentially to a subject in need thereof.

It also provides a method of killing and/or preventing or inhibiting growth of a multi-drug resistant microorganism comprising the step of administering to a subject in need thereof an effective amount of a pharmaceutical or veterinary composition comprising an antimicrobial peptide and a macrolide component together with a pharmaceutically or veterinarily acceptable carrier, wherein said antimicrobial peptide and macrolide component interact synergistiσally against a multi-drug resistant microorganism.

The invention also provides the use of an antimicrobial peptide and a macrolide component, in the manufacture of a medicament, for preventing or inhibiting growth of a multi-drug resistant microorganism.

It also provides the use of an antimicrobial peptide, in the manufacture of a medicament for preventing or inhibiting growth of a multi-drug resistant microorganism in a subject being treated with a medicament comprising a macrolide component.

It also provides the use of a macrolide component in the manufacture of a medicament for preventing or inhibiting growth of a multi-drug resistant microorganism in a subject being treated with a medicament comprising an antimicrobial peptide.

It also provides the use of an antimicrobial peptide and a macrolide component in the manufacture of a medicament for the killing, prevention or inhibition of growth of a multi-drug resistant microorganism, wherein said antimicrobial peptide and macrolide component interact synergistically against said multi-drug resistant microorganism.

The term "being treated" as used above includes a subject who has been treated, is currently being treated and/or is going to be treated.

It has also been found that the antimicrobial peptide and macrolide component are effective in the treatment and/or prophylaxis of an infection caused by a multi-drug resistant microorganism.

Accordingly, the present invention also provides the use of an antimicrobial peptide and macrolide component in the treatment and/or prophylaxis of an infection caused by a multi-drug resistant microorganism.

The invention still further provides a method for treatment and/or prophylaxis of an infection caused by a multi-drug resistant microorganism comprising the step of administering to a subject in need thereof an effective amount of an antimicrobial peptide and a macrolide component.

The method above includes the administration of an effective amount of an antimicrobial peptide and macrolide component separately, simultaneously or sequentially to a subject in need thereof.

It also provides a method of treatment and/or prophylaxis of an infection caused by a multi-drug resistant microorganism comprising the step of administering to a subject in need thereof an effective amount of a pharmaceutical or veterinary composition comprising an antimicrobial peptide and a macrolide component together with a pharmaceutically or veterinarily acceptable carrier, wherein εaid antimicrobial peptide and macrolide component interact synergistically against a multi-drug resistant microorganism. The invention, also provides the use of an antimicrobial peptide and a macrolide component in the manufacture of a medicament for the treatment and/or prophylaxis of an infection caused by a multi-drug resistant microorganism. It also provides the use of an antimicrobial peptide and a macrolide component in the manufacture of a medicament for the treatment or prophylaxis of an infection caused by a multi-drug resistant microorganism, wherein said antimicrobial peptide and macrolide component interact synergistically against said multi-drug resistant microorganism.

It also provides the use of an antimicrobial peptide, in the manufacture of a medicament for the treatment and/or prophylaxis of an infection caused by a multi-drug resistant microorganism in a subject being treated with a medicament comprising a macrolide component.

It also provides the use of a macrolide component, in the manufacture of a medicament for the treatment and/or prophylaxis of an infection caused by a multi-drug resistant microorganism in a subject being treated with a medicament comprising an antimicrobial peptide.

Examples of infections referred to above include infections selected from the group consisting of bacterial wound infections, mucosal infections, enteric infections, septic conditions, infections in airways, cerebrospinal fluid, blood, eyes, ears and skin.

In one embodiment, the multi-drug resistant organisms are multi-drug resistant gram-negative microorganisms. In a further embodiment, the multi-drug resistant microorganism is resistant to at least one member of the polymyxin class of antibiotics and synthetic derivatives thereof. polymyxin class of antibiotics and synthetic derivatives thereof.

In an even further embodiment, the multi-drug resistant microorganism is polymyxin resistant Pseudomonas aeruginosa, Acinetobacter baumannii, Stenotrophomonas maltophilia, Salmonella spp, Klebsiella pneumonia, and/or Shigella spp.

It has also been found that the antimicrobial peptide and macrolide component are effective in increasing the established anti-pathogenic effects of macrolides alone.

Accordingly, in a second aspect, the present invention provides a pharmaceutical or veterinary composition comprising an antimicrobial peptide and a macrolide component, together with a pharmaceutically or veterinarily acceptable carrier, wherein said antimicrobial peptide enhances the anti-pathogenic activity of the macrolide component.

The invention further provides the use of an antimicrobial peptide and a macrolide component for inhibiting or preventing production of at least one pathogenic factor by a microorganism.

The invention further provides a method of inhibiting or preventing production of at least one pathogenic factor by a microorganism comprising the step of administering to a subject in need thereof an effective amount of an antimicrobial peptide and a macrolide component.

The invention further provides the use of an antimicrobial peptide and a macrolide component in the manufacture of a medicament for inhibiting or preventing production of at least one pathogenic factor by a microorganism.

It also provides the use of an antimicrobial peptide in the manufacture of a medicament for inhibiting or preventing production of a pathogenic factor by a microorganism in a subject being treated with a medicament comprising a macrolide component. It also provides the use of a macrolide component in the manufacture of a medicament for inhibiting or preventing production of a pathogenic factor by a microorganism in a subject being treated with a medicament comprising an antimicrobial peptide.

It has also been found that the antimicrobial peptide has effective antipathogenic effects against a polymyxin- susceptible and -resistant microorganism.

Accordingly, in a third aspect, the present invention provides a pharmaceutical or veterinary composition comprising an antimicrobial peptide, together with a pharmaceutically or veterinarily acceptable carrier, wherein, said antimicrobial peptide inhibits or prevents production of a pathogenic factor by a microorganism. The invention further provides the use of an antimicrobial peptide for inhibiting or preventing production of a. pathogenic factor by a microorganism.

The invention further provides a method of inhibiting or preventing production of a pathogenic factor by a microorganism comprising the step of administering to a subject in need thereof an effective amount of an antimicrobial peptide.

It also provides the use of an antimicrobial peptide in the manufacture of a medicament for inhibiting or preventing production of a pathogenic factor by a microorganism.

In one embodiment, the microorganism referred to in this aspect of the invention is resistant to said antimicrobial peptide. The pathogenic factors referred to above include production of biofilm, alginate production, expression of flagellin, cytokine production, alteration of polymorphonuclear cell function , and/or pyocyanin production. The terms "antimicrobial peptide" and "macrolide component" as used herein include pharmaceutically acceptable salts or derivatives, pro-drugs, tautomers and/or isomers thereof.

DETAILED DESCRIPTION OF THE INVENTION For the purpose of this specification, it will be clearly understood that the word "comprising" means "including but not limited to", and that the word "comprises" have a corresponding meaning.

As used in the specification the singular forms "a", "an" and "the" include the plural references unless the context clearly dictates otherwise. Thus, for example, reference to "an antimicrobial peptide" includes mixtures of antimicrobial peptides, reference to "a macrolide component" includes mixtures of two or more such components, and the like.

The term "microorganism" includes any microscopic organism or taxonomically related macroscopic organism within the categories algae, bacteria, fungi, yeast and . protozoa or the like. It includes susceptible and resistant microorganisms. Examples of infections produced by such microorganisms are provided herein.

The microorganisms targeted in the first aspect of the present invention, the prevention or inhibition of growth of, and/or the treatment and/or prophylaxis of an infection caused by, multi-drug resistant microorganisms. Preferably, gram-negative microorganisms are targeted.

The anti-pathologenic aspects of the invention target the broader class of "microorganism" as defined herein. However, given that a multi-drug resistant microorganism is so difficult to treat, the antimicrobial peptide and macrolide component in the context of the anti-pathogenic aspect of the invention is most suited to treating a multi-drug resistant microorganism.

Unless stated otherwise, in the context of this. specification, the use of the term "microorganism" alone is not limited to "multi-drug resistant organism", and encompasses both drug-susceptible and drug-resistant microorganisms.

The term "multi-drug resistant microorganism" refers to those organisms that are, at the very least, resistant to more than two antibiotics in different antibiotic classes. This includes those microorganisms that have more resistance i.e. those that are resistant to three or more antibiotics in a single antibiotic class. This also includes microorganisms that are resistant to a wider range of antibiotics, i.e. microorganisms that are resistant to one or more classes of antibiotics.

The resistance or susceptibility of a microorganism to an antibiotic is classified by using defined minimum inhibitory concentration (MIC) breakpoints, MIC breakpoints for an antibiotic are determined by the MIC distributions of pathogens in a clinical indication and its pharmacokinetics and pharmacodynamics in humans. While a microorganism may literally be susceptible to a high concentration of an antibiotic in vitro, the microorganism may in fact be resistant to that antibiotic at physiologically realistic concentrations. If the concentration of drug required to inhibit growth of or kill the microorganism is greater than the concentration that can safely be achieved without toxicity to the subject, the microorganism is considered to be resistant to the antibiotic To facilitate the identification of antibiotic resistance or susceptibility using in vitro test results, the National Committee for Clinical Laboratory Standards (NCCLS, now known as clinical and Laboratory Standards Institute) has formulated standards for antibiotic susceptibility that correlate clinical outcome to in vitro determinations of the MIC of antibiotics. This will be discussed further below. Generally, MIC values indicate resistance or susceptibility of a microorganism. For example, MIC valves of a microorganism to colistin/polymyxin B are as follows: MIC ≤ 4mg/L =susceptible, MIC ≥ 8 mg/L =resistant and MIC ≥ 128mg/L =highly resistant. A suitable multi-drug resistant microorganism is an antimicrobial peptide resistant organism and/or a macrolide component resistant microorganism. An antimicrobial peptide resistant microorganism is a microorganism resistant to at least the antimicrobial peptide being used against it in the invention. A macrolide component resistant microorganism is a microorganism resistant to at least the macrolide component being used against it in the invention. Preferably, the multi-drug resistant microorganism is selected from the class of gram-negative microorganisms.

In one embodiment of the invention the antimicrobial peptide is a member of the polymyxin class of antibiotics. In this embodiment, the term "polymyxin resistant" refers to those microorganisms that are resistant to the member of the polymyxin class of antibiotics being used in the embodiment.

The term "prevent or inhibit growth" of a multi-drug resistant microorganism refers to the interference with growth or replication of the microorganism, which can include but does not necessarily extend to killing of the microorganism.

The term "treatment and/or prophylaxis" refers generally to affecting a subject, tissue or cell to obtain a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or sign or symptom thereof, and/or may be therapeutic in terms of. a partial or complete cure of a disease. The term "synergy" refers to the total increase in activity of both antimicrobial components over their additive antimicrobial activity. It includes the increase in activity of only one of the antimicrobial components. In the present invention, the antimicrobial peptide and macrolide component show at least synergistic anti- pathogenic activity.

The term "bidirectional synergy" refers to the increase in activity of each antimicrobial component when used in conjunction with the other antimicrobial component, and not merely an increase in activity of one of the antimicrobial components. In the present invention, the antimicrobial peptide and macrolide component show at least synergistic antimicrobial activity. Advantageously, the antimicrobial peptide and macrolide component show bidirectional synergistic antimicrobial activity.

The antimicrobial peptide and macrolide component have surprisingly been found to act synergistically to reverse the substantial resistance to each of these when used alone against a multi-drug resistant microorganism, in particular, a multi-drug resistant gram-negative microorganism, including polymyxin resistant bacteria, such as polymyxin resistant P. aeruginosa. in the present invention, the use of an antimicrobial peptide such as colistin and a macrolide component such as erythromycin, against a multi-drug resistant microorganism such as colistin resistant P. aeruginosa, leads to a bidirectional synergistic effect wherein each component increases the antimicrobial activity of the other. This is reflected in a significant decrease in the MIC of each component required to kill or inhibit the multi-drug resistant microorganism. The multi-drug resistant microorganism is therefore rendered susceptible.

The mechanism(s) for the bidirectional synergistic effect are not fully understood. It is known that antimicrobial peptides such as polymyxins have a permeabilising effect on the bacterial outer membrane of gram-negative microorganisms. Without wishing to be bound by theory, it is hypothesized that this permeabilising effect allows greater access to less water-soluble antibiotics such as macrolides which are not themselves active against certain gram-negative microorganisms. In the present invention, synergistic activity is obtained in a multi-drug resistant microorganism, particularly a multi-drug resistant gram-negative microorganism such as a polymyxin resistant "microorganism," (some highly resistant isolates with MIC of colistin (sulfate) ≥ 128 mg/L) e.g. P. aeruginosa. One would not expect that a macrolide or macrolide component would be useful against a microorganism such as polymyxin-resistant bacteria, particularly given that the macrolide or macrolide component is not generally active against a microorganism such as polymyxin-susceptible gram-negative bacteria. Accordingly, one would not expect a macrolide or macrolide component to interact with antimicrobial peptides to reverse the antimicrobial peptide resistance of a multi¬ drug resistant microorganism, thus making the microorganism susceptible to the antimicrobial peptide. The synergistic activity against a. multi-drug resistant microorganism is not fully explained by the permeabilising effect of an antimicrobial peptide such as polymyxins. We have found that the fractional inhibition concentration (ETC) of the antimicrobial peptide, when used with a macrolide component, as in the present invention, is reduced significantly. Therefore, bidirectional synergy is observed and it is not only the activity of the macrolide being increased, but also the activity of the antimicrobial peptide. This is even more surprising as it confirms that another mechanism, perhaps in addition to the permeabilising effect is occurring. A physico-chemical interaction between the two components is not expected as they are highly likely not to interact with each other bearing in mind the cationic nature of an antimicrobial peptide such as polymyxin, and the generally neutral nature of a macrolide component.. This is supported by circular dichroism data.

The fractional inhibition concentration (FIC) mentioned above is employed to indicate the effects of antibiotic combinations on the killing and/or inhibiting activity and is calculated by:

amp = antimicrobial peptide, mc = macrolide component.

The minimum inhibitory concentration (MIC) mentioned. above is the lowest concentration of antibiotic at which, with the inoculum of 10⁵ to 10⁶ CFU/ml, there is no visible growth after 18 to 24h incubation at 35°C.

The term "anti-pathogenic" refers to activity in inhibiting or preventing production of a pathogenic factor released by living microorganisms in a host which leads to destructive effects of tissues at the site(s) of infection. This includes the inhibition of the production of biofilm by disruption of a quorum sensing system¹⁴, and consequent inhibition of alginate production, inhibition of the expression of flagellin in P. aeruginosa., perturbing of cytokine production and altering action of polymorphonucteer cell functions in vivo and ex vivo, and/or the inhibition of production of pyocyanin, which is produced by microorganisms such as P. aeruginosa and is a blue pigment which disrupts human ciliary beating in vitro¹⁵, inhibits epidermal cell growth¹⁵ and also impedes lymphocyte proliferation.

Some bacteria, including P. aeruginosa, avidly form tightly arranged multi-cell structures in vivo known as biofilm. The production of biofilm is important for the persistence of infectious processes such as seen in pseudomonal lung infections in patients with cystic fibrosis and diffuse panbronchiolitis and many other diseases. Biofilm is resistant to phagocytosis by host immune cells and the effectiveness of antibiotics at killing bacteria in biofilm structures may be reduced by 10 to 1000 fold. Biofilm production and arrangement is governed by quorum sensing systems. The disruption of the quorum sensing system in bacteria such as P. aeruginosa is an important anti-pathogenic activity as it disrupts the biofilm formation and also inhibits alginate production. Alginate induces antigen-antibody reactions and increases the fluid viscosity in the airways of CF patients. The anti-pathogenic effect of alginate inhibition is beneficial by reducing the inflammation response and increasing phagocytosis as well as the ability for antibiotics to reach sensitive bacteria, resulting in enhanced clearance of bacteria.

Such anti-pathogenic factors are examples of conditions that are associated with the microbial peptide and macrolide component. The compositions are therefore useful in the treatment of conditions associated with microbial infections.

The term "antimicrobial peptide" refers to peptides which have antimicrobial activity. The polymyxin class is one class of antimicrobial peptides. The term "polymyxin" is used in its broadest sense to encompass all members of the well known polymyxin class of antibiotics and synthetic derivatives thereof. Derivatives within this class are the non-cyclic derivatives of cyclic polymyxins, derivatives containing amino acid variations, derivatives containing substitutes of the fatty acid components with other fatty acids or substituents, derivatives with D- and L- amino acid conversions, and derivatives substituted with any one or more optional substituents identified below. Classic polymyxins include polymyxin A, B1, B2, C, D1, D2, E1 and/or E2, F, G, M, P, S and T. The polymyxins are cationic detergents and are relatively simple basic peptides with molecular masses of about 1000-1200 daltons. Examples of members of the polymyxin class are polymyxin B (B₁ and B₂) and polymyxin E (colistin A and B) . Both these members include a cyclic heptapeptide ring with a tripeptide side chain. It is envisaged that declyclisation of the ring may result in a peptide with effective antimicrobial activity. Accordingly, non-cyclic derivatives of the polymyxins and similar peptides are encompassed within the term "antimicrobial peptide" . Also included within the scope of "antimicrobial peptide" are all the components of polymyxin B and polymyxin E, as well as synthetic derivatives thereof.

It is envisaged that one or more amino acid sequence(s) of the peptides above can be varied without significant effect on the structure or function of the peptide. Thus, the invention further includes variations of the peptide which show antimicrobial activity. Such variations or mutants include amino acid deletions, insertions, inversions, repeats and type substitutions. The structural formula of polymyxin B (B₁ and B₂) is as follows:

Polymyxin B₁: R = (+) - 6-methyloctanyl Polymyxin B₂: R = = (+) - 6-methylheptanyl

Phe: Phenylalanine

Thr: Threonine

Leu: Leucine

Dab = α, γ-diaminobutyric acid, wherein α and γ indicate the respective -NH₂ involved in the peptide linkage.

Polymyxin E (colistin) has many different components.

Its basic structure is a cyclic heptapeptide ring and a tripeptide side chain covalently bound to a fatty acid at the N-terminus via an acyl group. At least 30 components have been isolated and thirteen identified. They differ from each other by the composition of amino acids and fatty acids. The two major components are colistin A and colistin B. The structural for mula is as follows:

Colistin A: R = 6-methyloctanoic acid Colistin B: R = 6-methylheptanoic acid Thr; Threonine

Lgu: Leucine

Dab: α, γ-diaminobutyric acid wherein α and γ indicate the respective -NH₂ involved in the peptide linkage. It is envisaged that variation of these components, for example, by substituting a D-amino acid residue for the same or different L-amino acid residue or vice versa, varying the R substituents and/or conservative amino acid substitutions, while maintaining the synergistic antimicrobial activity with the macrolide component of the invention, is encompassed within the scope of the invention.

Minor components of colistin include the polymyxin E₃ and E₄, norvaline-polymyxin E₁, valine-polymyxin E₁, and valine-polymyxin E₂, isoleucine-polymyxin E₁, isoleucine- polymyxin E₁, polymyxin E₇ and isoleucine-polymyxin E₀.

In a preferred embodiment, the antimicrobial peptide comprises any one or more components of colistin, further preferably colistin A and/or colistin B. The proportion of colistin A and colistin B in commercial material varies between pharmaceutical suppliers and batches, but it is generally between 4.5:1 to 0.9:1. Colistin is available commercially in two forms, colistin sulphate and sodium colistin methanesulphonate. Sodium colistin methanesulphonate hydrolyses in aqueous media and forms a complex mixture of partially sulphomethylated derivatives plus colistin. One or more of the above forms of colistin or its derivatives are encompassed within the scope of the invention.

In a preferred embodiment, antimicrobial peptide comprises salts of colistin, preferably salts of pharmaceutically acceptable cations such as sodium, potassium, lithium and the like, acid addition salts of pharmaceutically acceptable inorganic acids such as hydrochloric, orthophosphoric, sulphuric and the like, and/or salts of pharmaceutically acceptable organic acids such as acetic, propionic, methanesulphonic, and the like.

In a preferred embodiment, antimicrobial peptide comprises colistin methanesulphonate and/or colistin sulphate. In a further preferred embodiment, antimicrobial peptide comprises colistin sulphate.

The term "amino acid" within the scope of the present invention is used in its broadest sense and is meant to include naturally occurring L α-amino acids or residues. The commonly used one and three letter abbreviations for naturally occurring amino acids are used herein.¹⁷ (The term includes D-amino acids as well as chemically modified amino acids such as amino acid analogs, naturally occurring amino acids that are not usually incorporated into proteins such as norleucine, and chemically synthesized compounds having properties known in the art to be characteristic of an amino acid. For example, analogs or mimetics of phenylalanine or proline, which allow the same conformational restriction of the peptide compounds as natural Phe or Pro are included, within the definition of amino acid. Such analogs and mimetics are referred to herein as "functional equivalents" of an amino acid. Other examples of amino acids are listed by Roberts and Vellaccio.¹⁸ which is incorporated herein by reference. Peptides synthesized by, for example, standard solid phase synthesis techniques, are not limited to amino acids encoded by genes. Commonly encountered amino acids which are not encoded by the genetic codes, include, for example, those described in International Publication No. WO 90/01940 such as, for example, 2-amino adipic acid (Aad) for GIu and Asp; 2-aminopimelic acid (Apm) for GIu and Asp; 2-aminobutyric (Abu) acid for Met, Leu, and other aliphatic amino acids; 2-aminoheptanoic acid (Ahe) for Met, Leu and other aliphatic amino acids; 2- aminoisobutyric acid (Aib) for GIy; cyclohexylalanine (Cha) for VaI, and Leu and Ile; homoarginine (Har) for Arg and Lys; 2,3-diaminopropionic acid (Dpr) for Lys, Arg and His; N-ethylglycine (EtGIy) for GIy, Pro, and Ala; N- ethylglycine (EtGIy) for GIy, Pro, and Ala; N- ethylasparigine (EtAsn) for Asn, and GIn; Hydroxyllysine (HyI) for Lys; allohydroxyllysine (AHyI) for Lys; 3- (and 4) hydroxyproline (3Hyp, 4Hyp) for Pro, Ser, and Thr,- allo-isoleucine (AIIe) for Ile, Leu, and val; p- amidinophenylalanine for Ala; ET-methylglycine (MeGIy, sarcosine) for GIy, Pro, and Ala; N-methylisoleucine (MeIIe) for Ile; Norvaline (Nva) for Met and other aliphatic amino acids; Norleucine (NIe) for Met and other aliphatic amino acids; Ornithine (Orn) for Lys, Arg and His; Citrulline (Cit) and methionine sulfoxide (MSO) for Thr, Asn and Gin; N-methylphenylalanine (MePhe) , trimethylphenylalanine, halo (F, Cl, Br and I) phenylalanine, triflouryphenylalanine, for Phe.

Conservative amino acid substitutions are shown in Table 1 under the heading of "exemplary substitutions" and "preferred substitutions" . If preferred substitutions do not result in a decrease or change in antimicrobial activity, then more substantial changes, denominated "exemplary substitutions" in Table 1, or as further described herein, may be introduced and the products tested for antimicrobial activity.

Table 1

The term "macrolide component" refers to a component having a lactone ring and which has antimicrobial activity. The term "macrolide" includes the well known class of macrolide antibiotics, including naturally occurring and synthetic derivatives thereof. Classic macrolides are the 14-15 lactone ring-based compounds to the 16-19 membered lactone ring-based compounds to which is attached one or more deoxy sugars, and which has antimicrobial activity. Examples of these are erythromycin, clarithromycin and azithromycin, included in the term "macrolide" are lincosamides (clindamycin), azalides (azithromycin) and ketolides (telithromycin) .

They differ from each other in that erythromycin and clarithromycin contain a 14-membered lactone ring and azithromycin contains a 15-membered lactone ring. Clarithromycin differs from erythromycin only by methylation of the hydroxyl group at the 6-position, and azithromycin differs by the addition of a methyl substituted nitrogen atom into the lactone ring. The macrolide lactone ring may be optionally substituted with various organic substituents.

In this specification "optionally substituted" means that one or more members of the lactone ring or a substituent on the lactone ring may or may not be further substituted with, one or more groups selected from alkyl, alkenyl, alkynyl, aryl, halo, haloalkyl, haloalkenyl, haloalkynyl, haloaryl, hydroxy, alkoxy, alkenyloxy, . aryloxy, benzyloxy, haloalkoxy, haloalkenyloxy, haloaryloxy, nitro, nitroalkyl, nitroalkenyl, nitroalkynyl, nitroaryl, nitroheterocyclyl, amino, alkylamino, dialkylamino, alkenylamino, alkynylamino, arylamino, diarylamino, benzylamino, dibenzylamino, acyl, alkenylacyl, alkynylacyl, arylacyl, acylamino, diacylamino, acyloxy, alkylsulphonyloxy, arylsulphenyloxy, heterocyclyl, heterocycloxy, heterocyclamino, haloheterocyclyl, alkylsulphenyl, arylsulphenyl, carboalkoxy, carboaryloxy, mercapto, alkylthio, benzylthio, acylthio, phosphorus-containing groups and the like. These may be protected by suitable protecting groups. Examples of macrolides containing substituents are disclosed in US 6,796,633 B2 dated 21 Sept 2001, US 6,358,942 filed 6 May 1998 and US 6,403,775 filed 28 Oct 1999 and are incorporated herein in their entirety by reference.

According to FIC values calculated as above, for the specific examples of clarithromycin/colistin sulphate, erythromycin/colistin sulphate, clarithromycin/colistin methanesulphonate, the following are preferred:

Clarithromycin and colistin sulphate are preferred over erythromycin and colistin sulphate.

Clarithromycin and colistin methanesulphonate are preferred over erythromycin and colistin methanesulphonate.

Colistin sulphate and a macrolide component are preferred over colistin methanesulphonate and a macrolide component as they showed 80% lower FlC values, thus expressing greater synergy. Therefore colistin sulphate and clarithromycin are preferred over colistin methanesulphonate and erythromycin.

The antimicrobial peptide and/or the macrolide may be used in the form of their salts, derivatives, pro-drugs, tautomers and/or isomers thereof.

The salts of the antimicrobial peptide and macrolide component are preferably pharmaceutically acceptable, but it will be appreciated that non-pharmaceutically acceptable salts also fall within the scope of the present invention since they are useful as intermediates in the preparation of pharmaceutically acceptable salts. Examples of pharmaceutically acceptable salts include salts of pharmaceutically acceptable cations such as sodium, potassium, lithium, calcium, magnesium, ammonium and alkylammonium; acid addition salts of pharmaceutically acceptable inorganic acids such as hydrochloric, orthophosphoric, sulphuric, phosphoric, nitric, carbonic, boric, sulfamic and hydrobromic acids; or salts of pharmaceutically acceptable organic acids euch as acetic, propionic, butyric, tartaric, maleic, hydroxymaleic, fumaric, citric, lactic, mucic, gluconic, benzoic, succinic, oxalic, phenylacetic, methanesulphonic, trihalomethanesulphonic, toluenesulphonic, benzenesulphonic, salicylic, sulphanilic, aspartic, glutamic, edetic, stearic, palmitic, oleic, lauric, pantothenic, tannic, ascorbic and valeric acids. In addition, some antimicrobial peptides and/or macrolide components of the present invention may form solvates with water or common organic solvents. Such solvates are encompassed within the scope of the invention. By "pharmaceutically acceptable derivative" is meant any pharmaceutically acceptable salt, hydrate or any other compound which, upon administration to the subject, is capable of providing (directly or indirectly) an antimicrobial peptide and/or macrolide component or residue thereof.

The term "pro-drug" is used herein in its broadest sense to include those compounds which are converted in vivo to antimicrobial peptides and/or macrolide components of the present invention. The term "tautomer" is used herein in its broadest sense to include antimicrobial peptides and/or macrolide components which are capable of existing in a state of equilibrium between two isomeric forms. Such compounds may differ in the bond connecting two atoms or groups and the position of these atoms or groups in the compound.

The term "isomer" is used herein in its broadest sense and includes structural, geometric and stereo isomers. As the antimicrobial peptides and/or macrolide components may have one or more chiral centres, they are capable of existing in enantiomeric forms.

The term "microbial infection" is used herein in its broadest sense and refers to any infection caused by a microorganism and includes bacterial infections, fungal infections, yeast infections and protozoal infections. The term "microorganism" includes any microscopic organism or taxonomically related macroscopic organism within the categories algae, bacteria, fungi, yeast and protozoa, or the like. The microorganisms targeted in the first aspect of the present invention are multi-drug resistant microorganisms. Preferably, gram-negative microorganisms are targeted. Bacterial infections include, but are not limited to, infections caused by Bacillus cereus, Bacillus anthracis, Clostridium botulinum, Clostridium difficile, Clostridium tetani, Clostridium perfringens, Corynebacteria diphtheriae, Enterococcus (Streptococcus D) , Listeria monocytogenes, Pneumococcal infections (Streptococcus pneumoniae) , Staphylococcal infections and Streptococcal infections ; Gram-negative bacteria including Bacteroides, Bordetella pertussis, Brucella, Campylobacter infections, enterohaemorrhagic Escherichia coli (EHEC/E. coli 0157 : H7) enteroinvasive Escherichia, coli (EIEC) , enterotoxigenic Escherichia coli (ETEC) , Haemophilus influenzae, Helicobacter pylori, Klebsiella, pneumoniae, Legionella, spp., Moraxella catarrhalis, Neisseria gonnorrhoeae, Neisseria meningitidis, Proteus spp., Pseudomonas aeruginosa, Salmonella spp., Shigella spp.,

Vibrio cholera and Yersinia; acid fast bacteria including Mycobacterium tuberculosis, Mycobacterium avium- intracellulars, Myobacterium johnei, Mycobacterium leprae, atypical bacteria, Chlamydia, Mycoplasma, Rickettsia, Spirochetes, Treponema pallidum, Borrelia recurrentis,

Borrelia burgdorfii and Leptospira icterohemorrhagiae and other miscellaneous bacteria, including Actinomyces and Nocardia.

Further preferably the microbial infection is caused by gram-negative bacterium, for example, P. aeruginosa., A. baumannii, Salmonella spp, Klebsiella pneumonia, Shigella spp. and/or Stenotrophomonas maltophilia.

Examples of microbial infections include bacterial wound infections, mucosal infections, enteric infections, septic conditions, pneumonia, trachoma, ornithosis, trichomoniasis and salmonellosis, especially in veterinary practice. Examples of infections caused by P. aeruginosa include:

Nosocomial infections:

1. Respiratory tract infections in cystic fibrosis patients and mechanically ventilated patients;

2. Bacteraemia and sepsis;

3. Wound infections, particularly in burn wound patients;

4. Urinary tract infections; 5. Post-surgery infections on invasive devices;

5. Endocarditis by intravenous administration of contaminated drug solutions;

7. Infections in patients with acquired immunodeficiency syndrome, cancer chemotherapy, steroid therapy, haematological malignancies, organ transplantation, renal replacement therapy, and other situations with severe neutropenia..

Community-acquired infections;

1. Community-acquired respiratory tract infections; 2. Meningitis;

3. Folliculitis and infections of the ear canal caused by contaminated waters;

4. Malignant otitis externa in the elderly and diabetics; 5. Osteomyelitis of the calcaneus in children; Eye infections commonly associated with contaminated contact lens;

6. Skin infections such as nail infections in people whose hands are frequently exposed to water; 7. Gastrointestinal tract infections;

8. Musculoskeletal system infections.

Examples of infections caused by A. baumannii include: Nosocomial infections; 1. Bacteraemia and sepsis; 2. Respiratory tract infections in mechanically ventilated patients; 3. Post-surgery infections on invasive devices;

4. Wound infections, particularly in burn wound patients;

5. Infections in patients with acquired immunodeficiency syndrome, cancer chemotherapy, steroid therapy, haematological malignancies, organ transplantation, renal replacement therapy, and other situations with severe neutropenia;

6. Urinary tract infections; 7. Endocarditis by intravenous administration of contaminated drug solutions;

8. Cellulitis.

Community-acquired infections:

1. Community-acquired pulmonary infections; 2. Meningitis;

3. Cheratitis associated with contaminated contact lens;

4. War-zone community-acquired infections. Atypical infections: 1. Chronic gastritis.

Examples of infections caused by Stenotrophomonas maltophilia. include bacteremia, pneumonia, meningitis, wound infections and urinary tract infections. Some hospital breaks are caused by contaminated disinfectant solutions, respiratory devices, monitoring instruments and ice machines. Infections usually occur in debilitated patients with impaired host defense mechanisms.

Examples of infections caused by Klebsiella pneumoniae include community-acquired primary lobar pneumonia, particularly in people with compromised pulmonary function and alcoholics. It also caused wound infections, soft tissue infections and urinary tract infections.

Examples of infections caused by Salmonella, spp. are acquired by eating contaminated food products. Infections include enteric fever, enteritis and. bacteremia. Examples of infections caused, by Shigella, spp. include gastroenteritis (shigellosis) .

The antimicrobial peptides and/or macrolide components of the invention may also be used in various fields where antiseptic treatment or disinfection of materials is required, for example, surface disinfection.

The term "subject" as used herein refers to any animal having a disease or condition which requires treatment with a pharmaceutically-active agent. The subject may be a mammal, preferably a human, or may be a domestic or companion animal. While it is particularly contemplated that the antimicrobial peptides and/or macrolide components of the invention are suitable for use in medical treatment of humans, it ie also applicable to veterinary treatment, including treatment of companion animals such as dogs and cats, and domestic animals such as horses, ponies, donkeys, mules, llama, alpaca, pigs, cattle and sheep, or zoo animals such as primates, felids, canids, bovids, and ungulates. Suitable mammals include members of the orders Primates, Rodentia, Lagomorpha, cetacea, Carnivora, Perissodactyla and Artiodactyla. Members of the orders Perissodactyla and Artiodactyla are particularly preferred because of their similar biology and economic importance. For example, Artiodactyla comprises approximately 150 living species distributed through nine families: pigs (Suidae) , peccaries (Tayassuidae) , hippopotamuses (Hippopotamidae) , camels (Camelidae) , chevrotains (Tragulidae) , giraffes and okapi (Giraffidae) , deer (cervidae) , pronghorn (Antilocapridae) , and cattle, sheep, goats and antelope (Bovidae) . Many of these animals are used as feed animals in various countries. More importantly, many of the economically important animals such as goats, sheep, cattle and pigs have very similar biology and share high degrees of genomic homology. The order Perissodactyla comprises horses and donkeys, which are both economically important and closely related. Indeed, it is well known that horses and donkeys interbreed.

As used herein, the term "effective amount" is meant an amount of antimicrobial peptides and/or macrolide components of the present invention effective to yield a desired antibiotic activity.

The specific "effective amount" will, obviously, vary with such factors as the particular condition being treated, the physical condition of the subject, the type of subject being treated, the duration of the treatment, the nature of concurrent therapy (if any) , and the specific formulations employed, the ratio of the antimicrobial peptides and/or macrolide components to each other, the structure of each of these components or their derivatives.

The antimicrobial peptides and/or macrolide components of the present invention may additionally be combined with other medicaments to provide an operative combination. It is intended to include any chemically compatible combination of pharmacologically-active agents, as long as the combination does not eliminate the activity of the antimicrobial peptides and/or macrolide components. It will be appreciated that the antimicrobial peptides and/or macrolide components of the invention and the other medicament may be administered separately, sequentially or simultaneously.

Other medicaments which may be used when treating bacterial infections include salbutamol, ipratropium, dornase alpha, for example, for use in inhalation for respiratory infections such as cystic fibrosis.

As used herein, a "pharmaceutical carrier" is a pharmaceutically acceptable solvent, suspending agent or vehicle for delivering the combination of antimicrobial peptides and/or macrolide components to the subject. The carrier may be liquid or solid and is selected with the planned manner of administration in mind. Each carrier must be pharmaceutically "acceptable" in the sense of being compatible with other ingredients of the composition and non injurious to the subject.

The antimicrobial peptides and/or macrolide components may be administered topically, including local delivery to the gastrointestinal tract and other membrane surfaces including aerosol delivery for administration to lungs or nasal cavity, parenterally or orally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, and vehicles . The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrathecal, intraventricula, intracranial, injection or infusion techniques.

The present invention also provides suitable topical, parenteral and oral pharmaceutical formulations for use in the novel methods of treatment of the present invention. The combination of antimicrobial peptides and/or macrolide components may be administered orally as tablets, suspensions, lozenges, troches, powders, granules, emulsions, capsules, syrups or elixirs. The composition for oral use may contain one or more agents selected from the group of sweetening agents, flavouring agents, colouring agents and preserving agents in order to produce pharmaceutically elegant and palatable preparations. Suitable sweeteners include sucrose, lactose, glucose, aspartame or saccharin. Suitable disintegrating agents include corn starch, methylcellulose, polyvinylpyrrolidone, xanthan gum, bentonite, alginic acid or agar. Suitable flavouring agents include peppermint oil, oil of wintergreen, cherry, orange or raspberry flavouring. Suitable preservatives include sodium benzoate, vitamin E, alphatocopherol, ascorbic acid, methyl paraben, propyl paraben or sodium bisulphite. Suitable lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride or talc. Suitable time delay agents include glyceryl monostearate or glyceryl distearate. The tablets contain the active combination in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets.

These excipients may be, for example, (1) inert diluents, such as calcium carbonate, lactose, calcium phosphate or sodium phosphate; (2) granulating and disintegrating agents, such as corn starch or alginic acid; (3) binding agents, such as starch, gelatin or acacia; and (4) lubricating agents, such as magnesium stearate, stearic acid or talc. These tablets may be uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. Coating may also be performed using techniques described in the U.S. Pat. Nos. 4,256,108; 4,160,452; and 4,265,874 to form osmotic therapeutic tablets for control release. The antimicrobial peptides and/or macrolide components can be administered, for in vivo application, parenterally by injection or by gradual infusion over time independently or together. Administration may be intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intracavity, transdermal, inhalation, intracisternal, intraventricular, interathecal (intra-CSF) or infusion by, for example, infusion pump. Also included are ear drops, eye drops, gels for skin infections etc. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose) , and the like. Preservatives and other additives may also be present such as, for example, anti-oxidants, chelating agents, growth factors and inert gases and the like.

"Treating" as used herein covers any treatment of, or prevention of disease in a vertebrate, a mammal, particularly a human, and includes: (a) preventing the disease from occurring in a subject that may be predisposed to the disease, but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; or (c) relieving or ameliorating the effects of the disease, i.e., cause regression of the effects of the disease.

The invention includes various pharmaceutical compositions useful for ameliorating disease. The pharmaceutical compositions according to one embodiment of the invention are prepared by bringing antimicrobial peptides and/or macrolide components, analogues, derivatives or salts thereof, or combinations of these compounds and one or more other medicaments into a form suitable for administration to a subject using carriers, excipients and additives or auxiliaries. Frequently used carriers or auxiliaries include magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talc, milk protein, gelatin, starch, vitamins, cellulose and its derivatives, animal and vegetable oils, polyethylene glycols and solvents, such as sterile water, alcohols, glycerol and polyhydric alcohols . Intravenous vehicles include fluid and nutrient replenishers. Preservatives include antimicrobial, anti-oxidants, chelating agents and inert gases. Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like, as described, for instance, in Remington's Pharmaceutical Sciences, 20th ed. Williams &. Williams (2000) , the British National Formulary, 43^rd edition (British Medical Association and Royal Pharmaceutical Society of Great Britain, 2000) , the contents of which are hereby incorporated by reference. The pH and exact concentration of the various components of the pharmaceutical composition are adjusted according to routine skills in the art.¹⁹

The pharmaceutical compositions are preferably prepared and administered in dose units. Solid dose units may be tablets, capsules and suppositories. For treatment of a subject, depending on activity of the polymyxin and macrolide, manner of administration, nature and severity of the disorder, age and body weight of the subject, different daily doses can be used. Under certain circumstances, however, higher or lower daily doses may be appropriate. The administration of the daily dose can be carried out both by single administration in the form of an individual dose unit or else several smaller dose units and also by multiple administration of subdivided doses at specific intervals.

Typically, dosages used in vitro may provide useful guidance in the amounts useful for in situ administration of the pharmaceutical composition, and animal models may be used to determine effective dosages for treatment of the bacterial infections.²⁰ Formulations for oral use may be in the form of hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin. They may also be in the form of soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, such as peanut oil, liquid paraffin or olive oil.

Aqueous suspensions normally contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspension. Such excipients may be (1) suspending agent such as sodium carboxymethyl cellulose, methyl cellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; (2) dispersing or wetting agents which may be (a) naturally occurring phosphatide such as lecithin; (b) a condensation product of an alkylene oxide with a fatty acid, for example, polyoxyethylene stearate; (c) a condensation product of ethylene oxide with a long chain aliphatic alcohol, for example, heptadecaethylenoxycetanol; (d) a condensation product of ethylene oxide with a partial ester derived from a fatty acid and hexitol such as polyoxyethylene sorbitol monooleate, or (e) a condensation product of ethylene oxide with a partial ester derived from fatty acids and hexitol anhydrides, for example polyoxyethylene sorbitan monooleate.

The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleagenous suspension. This suspension may be formulated according to known methods using those suitable dispersing or wetting agents and suspending agents which have been mentioned above.

The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1, 3-butanediol. Among the acceptable vehicles and solvents that may be employed are water,

Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

The antimicrobial peptides and/or macrolide components of the present invention may also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine, or phosphatidylcholines.

The antimicrobial peptides and/or macrolide components of the present invention may also be presented for use in the form of veterinary compositions, which may be prepared, for example, by methods that are conventional in the art. Examples of such veterinary compositions include those adapted for:

(a) oral administration, external application, for example drenches (e.g. aqueous or non-aqueous solutions or suspensions) ; tablets or boluses; powders, granules or pellets for admixture with feed stuffs; pastes for application to the tongue;

(b) parenteral administration for example by subcutaneous, intramuscular or intravenous injection, e.g. as a sterile solution or suspension; or (when appropriate) by intramammary injection where a suspension or solution is introduced in the udder via the teat;

(c) topical applications, e.g. as a cream, gel, ointment or spray applied to the skin, delivery for local activity in the gut and lung, vagina, rectum, intrarectally as a suppository, cream or foam.

Dosage levels of the polymyxin and macrolide of the present invention may vary. The amount of the active ingredients that may be combined with the carrier materials to produce a single dosage will vary depending upon the host treated and the particular mode of administration.

It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the site of infection, the infecting pathogen, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease undergoing therapy. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a box and whisker plot for colistin sulphate in combination with macrolides against colistin- resistant and colistin-susceptible P. aeruginosa isolates demonstrating that total FICs were dramatically decreased (clarithromycin>erythromycin and resistant>sensitive) .

Figure 2 : FIC_{colistin sulphate} and FIC_erythromycin in combination against colistin-resistant and colistin- susceptible P. aeruginosa isolates, demonstrating the substantial bidirectional synergy, and the greater synergy with colistin-resistant isolates.

Figure 3 : FIC_colistin sulphate &nd FIC_{clarithromycin} in combination against colistin-resistant and colistin- susceptible P. aeruginosa isolates, demonstrating the substantial bidirectional synergy (greater synergy with clarithromycin compared to erythromycin in Figure 2) , and the greater synergy with colistin-resistant isolates.

Figure 4: Box and whisker plots for colistin methanesulphonate in combination with macrolides against colistin-resistant and colistin-susceptible P. aeruginosa isolates demonstrating that total FICs were dramatically decreased (clarithromycin>erythromycin and resistant>sensitive) .

Figure 5 : FIC_{colistin methanesulphonate} and FIC_erythromycin in combination against colistin-resistant and colistin- susceptible P. aeruginosa isolates, demonstrating the substantial bidirectional synergy, and the greater synergy with colistin-resistant isolates.

Figure 6 : FIC_{colistin methanesulphonate} and FIC_{clarithromycin} in combination against colistin-resistant and colistin- susceptible P. aeruginosa isolates, demonstrating the substantial bidirectional synergy, and the greater synergy with colistin-resistant isolates.

Figure 7 : Inhibition of pyocyanin by the combination for P. aeruginosa ATCC27853 demonstrating substantial enhancement of erythromycin-induced anti-pathogenic effect at a colistin sulphate concentration of 0.5mg/L (or 1/2XMIC) .

Figure 8 : Inhibition of pyocyanin by the combination for P. aeruginosa 19453 muc, demonstrating substantial enhancement of erythromycin-induced anti-pathogenic effects at increasing colistin sulphate concentrations at low fractions of the colistin sulphate MIC (MIC ≥ 128 mg/L) .

Figure 9: Inhibition of pyocyanin by the combination for P. aeruginosa. 19626 muc demonstrating substantial enhancement of erythromycin-induced anti-pathogenic effects at increasing colistin sulphate concentrations at low fractions of the colistin sulphate MIC (MIC ≥ 128 mg/L) , and substantial concentration-dependent anti- pathogenic effects of colistin sulphate alone (where erythromycin concentration was 0 mg/L.

Figure 10: FIC_{colistin sulphate} and FIC_erythromycin in combination against colistin-resistant and colistin- susceptible A. baumannii isolates, demonstrating the substantial bidirectional synergy, and the greater synergy with colistin-resistant isolates.

Figure 11 : FIC_{colistin sulphate} and FIC_erythromycin in combination against colistin-resistant and colistin- susceptible Stenotrophomonas maltophilia isolates, demonstrating the bidirectional synergy.

Figure 12 : FIC_{colistin methanesulphonate} and FIC_erythromycin in combination against colistin-resistant and colistin- susceptible A. baumannii isolates, demonstrating the modest bidirectional synergy, and the greater synergy with colistin-resistant isolates.

Figure 13: Killing kinetics of colistin sulphate/erythromycin against P. aeruginosa (Table 1) . Figure 14: Killing kinetics of colistin sulphate /erythromycin and colistin methanesulphonate/erythromycin against P. aeruginosa (Tables 2 and 3) EXAMPLES

Numerous additional aspects and advantages of the invention will become apparent to those skilled in the art upon consideration of the following detailed description of the invention which describes presently preferred embodiments thereof.

1. MATERIALS AND METHODS

1.1 Antibiotics

All antibiotics were purchased from Sigma-Aldrich (Sydney, Australia) except clarithromycin from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) . 1.2 Killing effects 1.2.1 Fractional inhibitory concentration (FIC) test Before an experiment, isolates stored at -80°C were cultured on horse blood agar and incubated at 35°C for 18 to 36 h. Stock solutions of each drug were prepared freshly at a concentration of 5120 mg/L (colistin sulphate and methanesulphonate in Milli-Q water, erythromycin in ethanol and clarithromycin in dimethyl sulfoxide) . The stock solutions of colistin sulphate and colistin methanesulphonate were passed through a 0.22 μm filter. The stock solution was diluted with cation-adjusted Mueller-Hinton broth (CAMHB) using the National Committee for Clinical Laboratory Standards (NCCLS) protocol. FICs were determined at concentrations of 0.25 - 16 mg/L for colistin sulphate or 1.0 - 64 mg/L for colistin methanesulphonate with 0.125 - 128 mg/L for the macrolides using 96-well micro-plates. The initial inocula of P. aeruginosa, were 5x10⁵ to 10⁶ CFU/mL. Results were read at 24 h after incubation at 35°C.

• FICs of combinations of colistin sulphate or colistin methanesulphonate with erythromycin were tested against 22 P. aeruginosa isolates including 12 colistin-resistant isolates. • FICs of combinations of colistin sulphate or colistin methanesulphonate with clarithromycin were tested against 10 P. aeruginosa isolates including 8 colistin-resistant isolates, a subset of those tested with erythromycin combinations described above.

• FICs of combinations of colistin sulphate with erythromycin were tested against 5 Acinetobacter baumannii isolates including 3 colistin-resistant isolates. FICs of combinations of colistin methanesulphonate with erythromycin were tested against 2 Acinetobacter baumannii isolates including 1 colistin-resistant isolates.

• FICs of combinations of colistin sulphate with erythromycin were tested against 3 Stenotrophomonas maltophilia isolates including 2 colistin-resistant isolates . The FIC was calculated by

FIC: <=0.5 synergy (total effect of combination more than additive); >0.5 & <1.0 additive; >1 & <4 indifference; >=4 antagonism.

MIC is the minimum inhibitory concentration defined as the lowest concentration without visible growth after overnight incubation at 35°C with an inoculum of 5x10⁵ to 10⁶ CFU/mL.

1.2.2 Killing curves Killing curves were determined using a range of concentrations of colistin sulphate (or colistin methanesulphonate) together with a macrolide in a checkerboard design. Briefly, two days before an experiment, isolates stored at -80°C were cultured on horse blood agar and incubated at 35°C for 18 h for P. aeruginosa ATCC27853 (colistin susceptible and macrolide resistant) or 24 h for 20844 n/tnS (clinical isolate, resistant to both colistin and macrolide) . On the day before an experiment, 10 mL CAMHB was inoculated and incubated in a shaking water bath (80 cycles per minute) at 35°C. On the day of an experiment, 10 mL fresh CAMHB was inoculated with 1 mL of overnight culture and incubated in the shaking water bath (80 cycles per minute) at 35°C for 1 h to obtain bacteria in early log-phase growth. Then 19.8 mL fresh CAMHB containing appropriate concentrations of the combination (a macrolide with colistin sulphate or colistin methanesulphonate) were inoculated with 0.2 mL of the early log-phase broth. Samples (0.1 mL) were taken at 0, 1, 4, 8, 24 h for P. aeruginosa ATCC27853 and 0, 1, 4, 8, 24, 48, 72 h for 20844 n/mS Serial dilutions were conducted and 20 μL plated on nutrition agar plates in duplicate. Colonies were counted after 24 h incubation at 35°C. Synergy was defined as >=2 log₁₀ enhanced killing with the combination in comparison with the most active single drug alone. The killing effect of the combinations at different concentrations was quantified by calculation of the mean survival time (MST) at different time using

Where MST = mean survival time (in hours) , AUMC = area- under-the-curve of CFU/mL versus time multiplied by time of sampling in hours, AUC = area-under-the-curve of CFU/mL versus time. Areas were determined using the trapezoidal rule.

2.0 In vivo assay:

The results below demonstrate the effectiveness of the antimicrobial peptide/macrolide component combination as described. In vivo testing can be conducted according to the following assay. The assay is described with reference to the example of azithromycin, erythromycin and/or clarithromycin as the representative macrolide. Colistin methane sulphonate and/or colistin will be used as the representative antimicrobial peptide. Using the mouse thigh infection model²¹ combinations of the representative macrolide and antimicrobial peptide will be examined against polymyxin-resistant P. aeruginosa.

The subcutaneous doses of colistin methanesulphonate and colistin will be designed to achieve clinically relevant plasma concentrations (approximately 0.4 - 25 mg/L for CMS and 0.2 - 3.5 mg/L for colistin) ²²The subcutaneous dose of macrolide will be based not only upon the results from in vitro studies but also on knowledge of clinically achievable plasma concentrations (for example, in the case of azithromycin, approximately 0.2 - 4.5 mg/L; Product Information, Pfizer) . Both the antimicrobial peptide and the macrolide referred to throughout the specification are approved for administration to humans. Accordingly their toxicity profiles, bioavailability, half-life following administration and other relevant properties following administration in humans are well known to those in the art. The appropriate optimal dosage of each of the antimicrobial peptide and macrolide component to treat a particular condition in a subject can be readily determined by those in the art on the basis of information available in the art and described herein.

Following infection of the thigh, mice will be treated for 24 h with preferably the colistin methanesulphonate - macrolide or colistin - macrolide combination (n = 6) , colistin alone or colistin methanesulphonate alone at the same dose used in combination (n = 6) or vehicle (n = 6) . A macrolide-group alone will not be included because it is intrinsically inactive against P. aeruginosa. At 24 h, the mice will be killed and the thigh removed. Following homogenisation, counting for viable organisms will be conducted to conclude the antibacterial effect and the concentrations of the bacterial pathogenic factor, pyocyanin, will be determined by HPLC.

3. RESULTS AND DISCUSSION

3.1 Killing effects 3.1.1 FIC Tests

3.1.1.1 Synergy of the combination of colistin sulphate and macrolides The combinations of colistin sulphate with either erythromycin or clarithromycin led to substantial decreases in MICs of each component of the combination. The synergy was particularly marked for colistin and macrolide combinations against highly colistin-resistant (MIC ≥ 128 mg/L) versus colistin-sensitive (MIC < 4 mg/L) P. aeruginosa isolates (Figure 1-3) This was reflected by very substantial decreases in the total FICs and both the individual component FICs. Against the colistin-resistant isolates, all of FIC_{colistin sulphate} were < 0.0625, and the lowest FIC_{clarithromycin} and FIC_erythromycin reached 0.0039 and

0.0156, respectively (Figures 2-3) . According to standard definitions substantial bidirectional synergy was observed. Interestingly, clarithromycin and colistin sulphate combinations showed even greater synergy than colistin sulphate and erythromycin combinations.

Unexpectedly, the synergy was particularly marked for colistin and macrolide combinations against highly colistin-resistant (MIC≥128mg/L) versus colistin-sensitive (MIC ≤ 4 mg/L) P. aeruginosa isolates (Figure 1-3) . Similar synergy was also observed for the combination of colistin sulphate - erythromycin against colistin- susceptible (MIC ≤ 4 mg/L) and -resistant (MIC > 8 mg/L) A. baumannii (Figure 10) . The combination of colistin sulphate-erythromycin showed synergy against colistin-susceptible (MIC ≤ 4 mg/L) and - resistant (MIC > 8 mg/L) S. maltophilia (Figure 11) . 3.1.1.2 Synergy of the combination of colistin methanesulphonate and macrolides

The combinations of colistin methanesulphonate with either erythromycin or clarithromycin led to substantial decreases in MICs of each component of the combination. Unexpectedly, the synergy was particularly marked for colistin methanesulphonate and macrolide combinations against highly colistin-resistant (MIC > 128 mg/L) versus colistin-sensitive (MIC ≤ 4 mg/L) P. aeruginosa isolates (Figure 4-6) . Against the colistin-resistant isolates, all of FIC_{colistin methanesulphonate} were <= 0 . 5 , and the lowest

FIC_{clarithromycin} and FIC_erythromycin reached 0.0039 and 0.0078, respectively (Figures 4-6) . According to standard definitions substantial bidirectional synergy was observed. Interestingly, clarithromycin and colistin methanesulphonate combinations showed even greater synergy than colistin methanesulphonate and erythromycin combinations. The average FIC values for the macrolide and colistin methanesulphonate combinations (average FIC = 0.25) were approximately 80% of that observed with colistin sulphate and macrolide combinations (average FIC =0.19) in 2.1.1.1.

Modest synergy was observed for the combination of colistin methanesulphonate-erythromycin against colistin- susceptible (MIC ≤ 4 mg/L) and -resistant (MIC ≥ 8 mg/L) A. baumannii (Figure 12)

3.1.2 Killing Curves

3.1.2.1 Synergy of the combination of colistin sulphate and macrolides

The combination of colistin sulphate with erythromycin lead to substantial decreases in colony counts obtained from kill curves compared to control, or either antibiotic used alone. According to standard definitions of synergy used in kill curve analyses (ie.>= 2 Log₁₀ enhanced killing compared with by colistin sulphate alone) , substantial antibacterial synergy was observed with the colistin sulphate/macrolide combinations. Consistent with the FIC observations, synergy was particularly marked with highly colistin-resistant (MIC ≥ 128 mg/L) compared with colistin-sensitive P. aeruginosa isolates. Remarkably, the concentrations of both antibiotics required for significant antibacterial effects in resistant isolates was only a very small fraction of their respective MICs determined alone.

For colistin sulphate/erythromycin against P. aeruginosa ATCC27853 (susceptible to colistin sulphate, MIC = 1 mg/L but resistant to erythromycin, MIC = 256 mg/L) , synergy was observed at the concentrations of 0.5 mg/L colistin sulphate and 32 mg/L erythromycin at 8 h with ΔLog10 of -2.14, compared with the killing by 0.5 mg/L colistin sulphate alone (Table 1) . No obvious inhibition of growth was observed with 32 mg/L erythromycin. At 2 h with the combination, maximal suppression of bacterial growth was achieved at 2h with a MST_2h of 0.69 h. Overall, the combination showed modest synergy against this colistin- susceptible isolate.

Table 1: Killing kinetics of colistin sulphate/erythromycin against P. aeruginosa ATCC27853 (colistin-sensitive and macrolide-resistant)

For colistin sulphate/erythromycin against P. aeruginosa 20844 n/mucS (resistant to colistin sulphate, MIC >128 mg/L and erythromycin, MIC = 512 mg/L) , the bacterial counts declined throughout the experiment with all combinations tested. Significant synergy was observed at 24 h for all the combinations with a range of ΔLog10 (-2.01 to -2.54), compared with the killing by 8 mg/L colistin sulphate alone (Table 2) . With the combinations of 8 mg/L colistin sulphate (still a small fraction of the MIC; <1/16XMIC) and 16 mg/L erythromycin (1/32xMIC) , the growth was significantly inhibited. The values of MST_24h for the combination treated groups were significantly lower than the control and single antibiotic treated ones (P < 0.00001) . The inhibition was still present at 48 h.

Table 2 : Killing kinetics of colistin sulphate/erythromycin against P. aeruginosa 20844 n/mucS (colistin and macrolide-resistant)

3.1.2.2 Synergy of the combination of colistin methanesulphonate and macrolides against P. aeruginosa 20844 n/mucS (colistin and macrolide-resistant) the combination of colistin methanesulphonate with erythromycin led to substantial decreases in colony counts obtained from kill curves compared to control, or either antibiotic used alone. However, colistin methanesulphonate/erythromycin combinations had a weaker synergistic effect than colistin sulphate/erythromycin combinations (3.1.2.1) . This is also consistent with the relative weaker antibacterial activity of colistin methanesulphonate, compared with colistin sulphate.¹² Significant synergy was observed at 24 h for all the combinations, except the combination of 64 mg/L colistin methanesulphonate and 4 mg/L erythromycin (ΔLog10 of -1.31), with a range of ΔLog10 (-2.21 to -5.83), compared with the killing by 32 mg/L colistin methanesulphonate alone (Table 3) . With the combinations of 32 mg/L colistin sulphate (<1/4xMIC) and 16 mg/L erythromycin (1/32xMIC) , the growth was considerably inhibited (ΔLog10 of -2.21) . The values of MST_24h for the combination treated groups were considerably lower than the control and single antibiotic treated ones . The inhibition persisted until 48h.

Table 3 : Killing kinetics of colistin methanesulphonate/erythromycin demonstrating significant yet weaker synergy than colistin sulphonate/erythromycin (Table 2) against P. aeruginosa 20844 n/mucS (colistin and macrolide-resistant

3.2 Anti-pathogenic effects 3.2.1 Measurement of pyocyanin

3.2.1.1 Anti-pathogenic effect of the combination of colistin sulphate and macrolides

As expected, erythromycin alone decreased the production of pyocyanin in a concentration-dependent manner in both colistin-susceptible (Figure 7) and colistin-resistant (Figures 8 and 9) isolates. In the colistin-susceptible strain (Figure 7) , the addition of colistin sulphate in concentrations of 0.125 mg/L and 0.25 mg/L to erythromycin had little or no enhancing effect on inhibiting the production of pyocyanin at any given concentration of erythromycin. At a concentration of 0.5 mg/L of colistin sulphate (being a concentration of half of the MIC for colistin sulphate against this strain) , however, there was a very substantial enhancement of the anti-pathogenic effect of erythromycin (Figure 7) . P. aeruginosa 19453 muc and 19626 muc are both highly colistin-resistant (MIC > 128 mg/L) and erythromycin- resistant (MIC > 512 mg/L for 19453 muc and MIC = 256 mg/L for 19626 muc) isolates. With these isolates also, erythromycin alone decreased the production of pyocyanin in a concentration-dependent manner. As was observed for the susceptible isolates, the addition of colistin sulphate led to an enhanced reduction in the production of this anti-pathogenic factor (Figures 8 and 9) . For 19453 muc, the enhancement of the anti-pathogenic effect was most evident at a colistin sulphate concentration of 8 mg/L, a concentration of l/64xMIC of colistin sulphate against this isolate. For the other colistin-resistant isolate (19626 muc) , colistin sulphate enhanced the reduction in production of pyocyanin caused by erythromycin over the colistin sulphate concentration range from 0.125 mg/L to 8 mg/L, once again concentrations much lower than the MIC. Unexpectedly, with both colistin- susceptible (Figure 7) and colistin-resistant (Figures 8 and 9) isolates, colistin in the absence of erythromycin caused a reduction in the production of pyocyanin. This was most evident with isolate 19626 muc (Figure 9) where there was a concentration-dependent reduction in the production of pyocyanine as colistin sulphate concentration was increased to 8 mg/L. The mechanism of enhancement of the anti-pathogenic effect of erythromycin by colistin sulphate, at concentrations below its MIC for the respective isolates is unknown. The anti-pathogenic effect of colistin sulphate alone, at concentrations well below its MIC against colistin-resistant isolates, was most unexpected; the mechanism of this phenomenon is also presently unknown.

It will be apparent to the person skilled in the art that while the invention has been described in some detail for the purposes of clarity and understanding, various modifications and alterations to the embodiments and methods described herein may be made without departing from the scope of the inventive concept disclosed in this specification. References cited herein are listed on the following pages, and are incorporated herein by this reference.

References:

1. Livermore, D. M. (2003) . The threat from the pink corner. Ann Med 35, 226-234.

2. Pitt, T. L., Sparrow, M., Warner, M. & Stefanidou, M. (2003) . Survey of resistance of Pseudomonas aeruginosa from UK patients with cystic fibrosis to six commonly prescribed antimicrobial agents . Thorax 58, 794-796.

3. Ayan, M., Durmaz, R., Aktas, E. & Durmaz, B. (2003) . Bacteriological, clinical and epidemiological characteristics of hospital-acquired Acinetobacter baumannii infection in a teaching hospital. J Hosp Infect 54, 39-45.

4. Boutiba-Ben Boubaker, I., Boukadida, J., Triki, O. , Hannachi, N. & Ben Redjeb, S. (2003) . Outbreak of nosocomial urinary tract infections due to a multidrug resistant Pseudomonas aeruginosa. Pathol Biol 51, 147-150.

5. Lopez-Hernandez, S., Alarcon, T. & Lopez-Brea, M. (2000) . Evolution of antimicrobial susceptibility of Acinetobacter baumannii clinical isolates . Rev Esp Quimioter 13, 394-400.

6. Levin, A. S., Mendes, C. M., Sinto, S. I., Sader, H. S., Scarpitta, C. R., Rodrigues, E., et al. (1996) . An outbreak of multiresistant Acinetobacter baumanii in a university hospital in Sao Paulo, Brazil. Infect Control Hosp Epidemiol 17, 366-368.

7. Cantrell, H. F., Lombardy, E. E., Duncanson, F. P., Katz, E. & Barone, J. S. (2004) . Declining susceptibility to neomycin and polymyxin B of pathogens recovered in otitis externa clinical trials. South Med J 97, 465-471.

8. Dohar, J. E., Kenna, M. A. & Wadowsky, R. M. (1996) . In vitro susceptibility of aural isolates of Pseudomonas aeruginosa to commonly used ototopical antibiotics. Am J Otol 17, 207-209. 9. Rhee, M. K., Kowalski, R. P., Romanowski, E. G., Mah, F. S., Ritterband, D. C. & Gordon, Y. J. (2004) . A laboratory evaluation of antibiotic therapy for ciprofloxacin-resistant Pseudomonas aeruginosa. Am J Ophthalmol 138, 226-230.

10. Oberhofer, T. R. (1980) . Cultural and biochemical characteristics of clinical isolates of unusual colistin-resistant pseudomonads . J Clin Microbiol 12, 156-160. 11. Denton, M., Kerr, K., Mooney, L., Keer, V., Rajgopal, A., Brownlee, K., et al. (2002) . Transmission of colistin-resistant Pseudomonas aeruginosa between patients attending a pediatric cystic fibrosis center. Pediatr Pulmonol 34, 257-261. 12. Li, J., Turnidge, J., Milne, R., Nation, R. L. & Coulthard, K. (2001) . In vitro pharmacodynamic properties of colistin and colistin methanesulfonate against Pseudomonas aeruginosa isolates from patients with cystic fibrosis. Antimicrob Agents Chemother 45, 781-785.

13. Nguyen, T., Louie, S. G., Beringer, P. M. & Gill, M. A. (2002) . Potential role of macrolide antibiotics in the management of cystic fibrosis lung disease. Curr Opin PuIm Med 8, 521-528. 14. Kobayashi, H. (1995) . Biofilm disease: its clinical manifestation and therapeutic possibilities of macrolides. Am J Med 99, 26S-30S.

15. Wilson, R., Pitt, T., Taylor, G., Watson, D., MacDermot, J., Sykes, D., et al . (1987) . Pyocyanin and 1-hydroxyphenazine produced by Pseudomonas aeruginosa inhibit the beating of human respiratory cilia in vitro. J Clin Invest 79, 221-229.

16. Cruickshank, C. N. & Lowbury, E. J. (1953) . The effect of pyocyanin on human skin cells and leucocytes. Br J Exp Pathol 34, 583-587.

17. Lehninger, A.L., Biochemistry, 2d ed. , pp. 71-92, (1975), Worth Publishers, New York. 18. (The peptides: Analysis, Synthesis, Biology,) Eds. Gross and Meiehofer, Vol. 5 p 341, Academic Press, Inc, N.Y. 1983.

19. See Goodman and Gilman's The Pharmacological Basis for Therapeutics (7th ed. , 1985) .

20. Langer, Science 249:1527(1990)

21 . Vogelman B, Gudmundsson S, Turnidge J, Leggett J, Craig WA. In vivo postantibiotic effect in a thigh infection in neutropenic mice . J Infect Dis 1988;157 (2) : 287-98)

22. Li J, Coulthard K, Milne R, et al. Steady-state pharmacokinetics of intravenous colistin methanesulphonate in patients with cystic fibrosis. J. Antimicrob. Chemother. 2003;52 (6) :987-992.

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A pharmaceutical or veterinary composition comprising an antimicrobial peptide and a macrolide component together with a pharmaceutically or veterinarily acceptable carrier, wherein said antimicrobial peptide and macrolide component interact synergistically against a multi-drug resistant microorganism.

2. A composition according to claim 1, wherein said antimicrobial peptide and macrolide component interact with bidirectional antimicrobial synergy against a multi¬ drug resistant microorganism.

3. A composition according to claim 1 or 2, wherein said antimicrobial peptide is a member of the polymyxin class of antibiotics and synthetic derivatives thereof .

4. A composition according to claim 3, wherein said polymyxin is selected from the group consisting of polymyxin A, B1, B2, D1, D2, E1 and/or E2, F, G, M, P, S and/or T.

5. A composition according to claim 4, wherein said polymyxin is selected from polymyxin Bl and/or polymyxin B2.

6. A composition according to claim 4, wherein said polymyxin is selected from colistin A and/or colistin B.

7. A composition according to claim 6, wherein said polymyxin is in the form of a colistin salt.

8. A composition according to claim 7, wherein said salt is a methane sulphonate and/or a sulphate salt.

9. A composition according to any one of claims 1 to 8, wherein said macrolide is selected from ketolides and/or lactone ring-based compounds containing a 14 to 19 membered lactone ring.

10. A composition according to claim 9, wherein said lactone ring-based compound contains a 14-15 membered lactone ring.

11. A composition according to claim 9 or 10, wherein said macrolide is selected from the group consisting of erythromycin, clarithromycin, azithromycin and/or telithromycin.

12. A composition according to any one of claims 1 to 11, wherein said antimicrobial peptide is colistin sulphate and/or colistin methane sulphonate, and said macrolide is erythromycin, clarithromycin and/or azithromycin.

13. A composition according to claim 12, wherein said antimicrobial peptide is colistin methane sulphonate and said macrolide is clarithromycin.

14. A pharmaceutical or veterinary composition comprising colistin methane sulphonate and clarithromycin together with a pharmaceutically or veterinarily acceptable carrier, wherein said colistin methane sulphonate and clarithromycin interact with bidirectional synergy against a colistin resistant microorganism.

15. A product or kit comprising a pharmaceutical or veterinary composition as defined in any one of claims 1 to 14, in which the antimicrobial peptide and macrolide component are held separately for separate, sequential or simultaneous use.

16 . A composition according to any one of claims 1 to 13 , wherein said multi-drug resistant microorganism is selected from the class of gram-negative microorganisms

17. A composition according to claim 16, wherein the multi-drug resistant microorganism is resistant to at least one member of the polymyxin class of antibiotics and synthetic derivatives thereof.

18. A composition according to claim 10, wherein the multi-drug resistant microorganism is polymyxin resistant Pseudomonas aeruginosa, Acinetobacter baumannii,

Stenotrophomonas maltophilia, Salmonella spp and/or Klebsiella pneumonia and/or Shigella spp..

19 A composition according to claim 10 or 11, wherein the multi-drug resistant microorganism is resistant to colistin and/or a colistin salt.

20. A method of killing and/or preventing or inhibiting growth of a multi-drug resistant microorganism comprising the step of administering to a subject in need thereof an effective amount of a pharmaceutical or veterinary composition comprising an antimicrobial peptide and a macrolide component together with a pharmaceutically or veterinarily acceptable carrier, wherein said antimicrobial peptide and macrolide component interact synergistically against a multi-drug resistant microorganism.

21. A method of killing and/or preventing or inhibiting growth of a multi-drug resistant microorganism comprising the step of administering to a subject in need thereof an effective amount of an antimicrobial peptide and a macrolide component, wherein said antimicrobial peptide and/or macrolide component are as defined in any one of claims 3 to 14.

22. A method of treatment and/or prophylaxis of an infection caused by a multi-drug, resistant microorganism comprising the step of administering to a subject in need thereof an effective amount of a pharmaceutical or veterinary composition comprising an antimicrobial peptide and a macrolide component together with a pharmaceutically or veterinarily acceptable carrier, wherein said antimicrobial peptide and macrolide component interact synergistically against a multi-drug resistant microorganism.

23. A method of treatment and/or prophylaxis of an infection caused by a multi-drug resistant microorganism comprising the step of administering to a subject in need thereof an effective amount of an antimicrobial peptide and a macrolide component, wherein said peptide and/or macrolide component are as defined in any one of claims 3 to 14.

24. A method according to claim 22 or 23, wherein said infection is selected from the group consisting of bacterial wound infections, mucosal infections, enteric infections, septic conditions, infections in airways, cerebrospinal fluid, blood, eyes, ears and skin.

25. A method according to any one of claims 22 to 24, wherein said infection is caused by a gram negative multi¬ drug resistant microorganisms.

26. A method according to any one of claims 20 to 25, wherein the multi-drug resistant microorganism is resistant to at least one member of the polymyxin class of antibiotics and synthetic derivatives thereof.

27. A method according to claim 25 or 26, wherein the multi-drug resistant microorganism is polymyxin resistant Pseudomonas aeruginosa, Acinetobacter baυmannii, Stenotrophomonas maltophilia, Salmonella spp Klebsiella pneumonia, and/or Shigella spp.

28. A method according to any one of claims 25 to 27, wherein the multi-drug resistant microorganism is resistant to colistin and/or a colistin salt.

29. Use of an antimicrobial peptide and a macrolide component in the manufacture of a medicament for the killing and/or prevention or inhibition of growth of a multi-drug resistant microorganism, wherein said antimicrobial peptide and macrolide component interact synergistically against said multi-drug resistant microorganism.

30. Use of an antimicrobial peptide and macrolide component in the manufacture of a medicament for killing and/or preventing or inhibiting growth of a multi-drug resistant microorganism, wherein said antimicrobial peptide and/or macrolide component are as defined in anyone of claims 3 to 14.

31. Use of an antimicrobial peptide and a macrolide component in the manufacture of a medicament for the treatment and/or prophylaxis of an infection caused by a multi-drug resistant microorganism.

32. Use of an antimicrobial peptide in the manufacture of a medicament for the treatment and/or prophylaxis of an infection caused by a multi-drug resistant microorganism in a subject being treated with a medicament comprising a macrolide component.

33. Use of a macrolide component in the manufacture of a medicament for the treatment and/or prophylaxis of an infection caused by a multi-drug resistant microorganism in a subject being treated with a medicament comprising an antimicrobial peptide.

34. Use of an antimicrobial peptide and a macrolide component in the manufacture of a medicament for the treatment or prophylaxis of an infection caused by a multi-drug resistant microorganism, wherein said antimicrobial peptide and macrolide component interact synergistically against said multi-drug resistant microorganism.

35. Use according to any one of claims 31 to 34, wherein said antimicrobial peptide and macrolide component are as defined in any one of claims 3 to 14.

36. Use according to any one of claims 31 to 35, wherein said infection is selected from the group consisting of bacterial wound infections, mucosal infections, enteric infections, septic conditions, infections in airways, cerebrospinal fluid, blood, eyes, ears and skin.

37. A pharmaceutical or veterinary composition comprising an antimicrobial peptide and a macrolide component together with a pharmaceutically or veterinarily acceptable carrier, wherein said antimicrobial peptide enhances the anti-pathogenic activity of the macrolide component.

38. A composition according to claim 37, wherein said antimicrobial peptide and macrolide component are as defined in any one of claims 3 to 14.

39. Use of an antimicrobial peptide and a macrolide component in the manufacture of a medicament for inhibiting or preventing production of at least one pathogenic factor by a microorganism.

40. Use of an antimicrobial peptide in the manufacture of a medicament for inhibiting or preventing production of at least one pathogenic factor by a microorganism in a subject being treated with a medicament comprising a macrolide component.

41. Use of a macrolide component in the manufacture of a medicament for inhibiting or preventing production of at least one pathogenic factor by a microorganism in a subject being treated with a medicament comprising an antimicrobial peptide.

42. Use according to any one of claims 39 to 41, wherein asid microorganism is a multi-drug resistant microorganism.

43. Use according to claim 42, wherein said microorganism is selected from the class of gram-negative microorganisms.

44. Use according to claim 43, wherein the multi-drug resistant microorganism is resistant to at least one member of the polymyxin class of antibiotics and synthetic derivatives thereof .

45. Use according to claim 44 multi-drug resistant microorganism is polymyxin resistant Pseudomonas aeruginosa, Acinetobacter baumannii, Stenotrophomonas maltophilia, Salmonella spp and/or Klebsiella pneumonia and/or Shigella spp.

46. Use according to claim 45, wherein the multi-drug resistant microorganism is resistant to colistin and/or a colistin salt.

47. Use of an antimicrobial peptide for inhibiting or preventing production of at least one pathogenic factor by a multi-drug resistant microorganism wherein said microorganism is resistant to said antimicrobial peptide.

48. Use according to any one of claims 39 to 47 wherein said antimicrobial peptide and macrolide component are as defined in any one of claims 3 to 14.

49. Use according to any one of claims 39 to 48, wherein said pathogenic factor is selected from the group consisting of production of biofilm, alginate production, expression of flagellin, cytokine production, alteration of polymorphonuclear cell function , and/or pyocyanin production.

50. A pharmaceutical or veterinary composition comprising an antimicrobial peptide, together with a pharmaceutical or veterinarily acceptable carrier, wherein said antimicrobial peptide inhibits or prevents production of a pathogenic factor by a microorganism resistant to said antimicrobial peptide.