WO2012154109A1 - Self -aggregating antimicrobial cyclic peptides comprising beta - tri substituted amino acid residues - Google Patents

Abstract

The present, invention pertains to cyclic peptides, combinations of cyclic peptide having potent antibacterial effects, as well as pharmaceutical compositions for use as medicaments, in particular for treating bacterial infections. The backbone - cyclic peptides are made of an even number of 6-20 amino acid residues, wherein every second residue has a fully substituted beta-carbon atom (i.e. beta - trisubstituted amino acid, such as tert-Leucine and penicillamine). The peptides contain a cluster of at least 3 residues of the same stereo configuration, whereas the remaining residues are present as a L/D alternating structure. The combination of cyclic peptides having complementary stereo configurations provide antibacterial properties in view of their ability to form nanotubes, i.e. the complementary configurations act synergistically.

Description

SELF -AGGREGATING ANTIMICROBIAL CYCLIC PEPTIDES COMPRISING BETA - TRISUBSTITUTED AMINO ACID RESIDUES

Technical field

The present invention pertains to cyclic peptides, combinations of cyclic peptide having potent antibacterial effects, as well as pharmaceutical compositions comprising the combinations of cyclic peptides, or combinations of pharmaceutical compositions comprising individual cyclic peptides. Further, present invention relates to the use of the combinations of cyclic peptides, pharmaceutical compositions comprising the cyclic peptides or combinations of pharmaceutical compositions, for use as medicaments, in particular for treating bacterial infections.

Background art

Resistance against bacterial infections has developed into a major healthcare concern and numerous researchers and medical doctors continuously emphasize that we may, within a near future, lack the possibility to combat infectious diseases that were previously easily treated with currently available, mainstay antibiotics. Consequently, it is imperative to restrict the use of antibiotics, both as an in-patient and as an out- patient treatment alternative, as well as handling existing antibiotic agents in such a way so as to minimize resistance development. New hospital routines may indeed reduce the development of bacterial resistance, but novel antibiotic agents are crucial for successfully battling what may be one of the biggest threats to the healthcare system of today.

A number of factors have contributed to the fact that only a diminishing number of antibiotic agents are being developed. The pharmaceutical industry has long

l considered antibiotics as generating lower revenues than other classes of pharmacological agents (for instance therapeutic agents aimed at treating lifestyle- associated diseases, such as cardiovascular diseases and obesity), partially as an implication of the fact that multidrug-resistant infections still are relatively uncommon, at least in certain parts of the world. Another issue for the pharmaceutical companies pertains to the rapid evolution of drug resistance towards conventional antibiotics.

Antimicrobial peptides (AMPs) constitute a class of antibiotic substances that have been known for a relatively long time, but, in spite of their in many respects desirable properties, only a very limited number of these antibiotics have been developed into clinically useful drugs. AMPs are produced naturally in animals, including humans, and constitute part of the innate defense against infections. A substantial number of AMPs, both natural and synthetic, have been reported in the scientific literature over the years. The majority of these exert their antibacterial effects through binding to the cell wall of bacteria, causing disturbances in cell membrane and thereby killing the bacteria. Despite exhibiting rapid bactericidal activity and low development of resistance, the clinical use of AMPs has yet to materialize. Peptide-based antibiotics normally exhibit a number of undesirable pharmacokinetic properties, for instance metabolic instability and protease susceptibility. Further, AMPs are not orally active, meaning that other routes of administration, for instance intravenous injection, must be utilized. Alternatively, treatment of superficial infections via topical application could also be contemplated. Further, the costs associated with production of AMPs are normally higher than the cost for preparing conventional antibiotics, particularly if AMPs must be prepared through chemical synthesis. Additionally, like any pharmaceutical agent, AMPs may give rise to side-effects. Side-effects associated with AMP use have not been studied to any greater extent, but the mechanism behind the side-effects is likely analogous to the antibacterial effects exerted by AMPs. Both mammalian cells and bacteria have phospholipid membranes, which are disturbed by AMPs. However, AMPs exhibit a certain selectivity for bacterial cell membranes, as a function of a more hyperpolarized membrane potential and the presence of an anionic cell wall, as well as differences in lipid composition.

Ghadiri and colleagues (WO2002090503-A2, Ghadiri et al., Nature, 1993, 366, 324- 327, Dartois et al., 2005, Antimicrob. Agents Chemother. 49 3302) have previously shown that cationic cyclic peptides having an even number of amino acid residues, and wherein every other amino acid is present in L-configuration and every other amino acid is present in D-configuration, possess antibacterial properties. The antibacterial effects originate from the ability of these cyclic peptides to assemble into extended tube-like structures called peptide nanotubes, which leads to pore formation and subsequent bacterial death. The assembly into peptide nanotubes was shown to be strictly dependent on the exact structure of the cyclic peptide (i.e. a cyclic peptide having an even number of amino acid residues, and wherein every other amino acid is present in L-configuration and every other amino acid is present in D-configuration), with the nanotube formation being reliant on antiparallel hydrogen bonding between homochiral amino acid residues on adjacent cyclic peptides. In vitro and in vivo studies confirmed the antibacterial effects of these cyclic nanotube-forming peptides, and further studies showing that resistance was only slowly developing was of equal importance. In fact, the development of resistance was in parity with the resistance seen with the glycopeptide antibiotic vancomycin, a drug of last resort used in the treatment of infections caused by Gram-positive bacteria (notably methicillin-resistant Staphylococcus aureus) and known for its slow resistance development.

Figure 1 shows a segment of a peptide nanotube of the prior art assembled by space- filling CPK molecular models of the peptide cyc/orD-Val-L-Ala-D-Val-L-Ala-D-Val- L-Ala-D-Val-L-Ala] (where amino acid residues in the D-configuration are underlined). This model is in accordance with the model of peptide nanotube structure suggested by Ghadiri and co-workers. A prerequisite for the planar structure of the peptide is that the peptide has alternating D and L amino acid residues. If an amino acid residue in the structure would not follow the strictly alternating pattern, the side- chain of that residue would interfere with the formation of the peptide bonds causing a distortion in the packing of the peptide bonds within the peptide nanotube. It can also be seen that the orientation of the peptide bonds are so that the cyclic peptide in the middle of the structure is orientated NH-CO while the cyclic peptides at the top and at the bottom has the opposite orientation; CO-NH. This mode of packing of the peptide bonds gives rise to a structure closely resembling anti-parallel beta-sheets.

Summary of the invention

The previously described peptide nanotubes assemble (normally at relatively high concentrations) from a single cyclic peptide having amino acids with strictly alternating L and D configuration (e.g. D-Xaa-L-Xaa-D-Xaa-L-Xaa-D-Xaa-L-Xaa-D- Xaa-L-Xaa). For the sake of clarity, amino acid residues present in the D-configuration are henceforth underlined.

The strictly alternating sequence of amino acids having opposite chirality has previously been seen as an absolute necessity for an ordered nanotube formation and hence for the antibacterial properties, as previously suggested for instance in WO2002090503-A2, Ghadiri et al., Nature, 1993, 366, 324-327. The present inventor studied the formation of peptide nanotubes comprising a single cyclic peptide (in analogy with the studies carried out by Ghadiri and colleagues) having tert-leucine (Tie), with a /er/-butyl group as side-chain a representative example of a beta- trisubstituted amino acid, in every other position (Rosenthal-Aizman et al., J. Am. Chem. Soc, 126, 3372-3373). The conclusion from these studies was that the presence of ri-leucine (either all in D- or all in L-configuration) strongly disfavoured the formation of anti-parallel beta-sheets and subsequent aggregation into peptide nanotubes, as a result of steric hindrance between the bulky side chains of amino acids present in the same configuration.

This is illustrated in figure 2B for the peptide cyclo[L-Ala-D-Tle-L-Ala-D-Tle-L-Ala- D-Tle-L-Ala-D-Tle-L-Ala] . If this peptide should form peptide nanotubes according to the model suggested by Ghadiri and co-workers the bulky tert-butyl of the side-chain of the L-Tle residues must be stacked on top of each other. This will lead to a sterical hindrance for the formation of the peptide bonds and restrict the rotation of tert-butyl group and thereby interfere with the efficient formation of peptide nanotubes (Rosenthal-Aizman et al., J. Am. Chem. Soc, 126, 3372-3373).

However, the interaction between a cyclic peptide having an amino acid sequence that is strictly alternating throughout the sequence between L- and D-configuration (e.g. cyc/o[D-Xaa-L-Tle-D-Xaa-L-Tle-D-Xaa-L-Tle-D-Xaa-L-Tle] with a another cyclic peptide having an amino acid sequence with strictly opposite chirality (i.e. cyclo[L- Xaa-D-Tle-L-Xaa-D-Tle-L-Xaa-D-Tle-L-Xaa-D-Tle], and with Tie residues in exactly the same positions (all L in the first peptide and all D in the second peptide), enabled the formation of peptide nanotubes, through close anti-parallel hydrogen bonding. This is illustrated in figure 2 C. The present inventor thus realized that an absolutely necessary condition for the efficient formation of peptide nanotubes was the strictly alternating chirality (i.e. that every other amino acid must be present in L- configuration and every other amino acid in D-configuration, e.g. c c/o [L-D-L-D-L-D- L-D] of the cyclic peptides and that beta-trisubstituted amino acid residues such as ter eucine (Tie) severely disfavoured the formation of peptide nanotubes if they had the same chirality in different peptides (Rosenthal-Aizman et al., J. Am. Chem. Soc, 126, 3372-3373). Any form of disturbances in the alternating chirality, not only in terms of Tie residues but throughout the cyclic peptides, caused substantial disturbances in the ability to form hydrogen bonds, meaning that the nanotube formation would be abrogated, or at least severely hampered. In contrast to the established scientific paradigm with regard to peptide nanotube formation, and through a highly surprising and serendipitous discovery resulting from an erroneous peptide synthesis process, the present inventor realized that two cyclic peptides with an even number of amino acids residues could in fact form cyclic peptide nanotubes, distorted peptide nanotube-like structures or some other previously not characterized structure despite not having strictly alternating chirality throughout the peptide sequences. Antibacterial trials with for instance a combination of two cyclic peptides having the sequences cvc/o[D-Tle-D-Xaa-D-Tle-L-Ala-D-Tle-L-Ala- D-Tle-L-Alal and cyc/orL-Tle-L-Xaa-L-Tle-D-Ala-L-Tle-D-Ala-L-Tle-D-Alal, where underlined amino acid residues are present in D-configuration and where Xaa represents lysine or arginine, unexpectedly exhibited potent antibacterial effects when combined. However, the individual cyclic peptides possess very low antibacterial effects on their own, whereas a combination of cyclic peptides, comprising at least two cyclic peptides, wherein the at least two cyclic peptides have beta-trisubstituted amino acids in the same amino acid positions, and wherein the beta-trisubstituted amino acids of at least one cyclic peptide of said combination are all present in D-configuration and wherein the beta-trisubstituted amino acids of at least one cyclic peptide of said combination are all present in L-configuration, displays potent antibacterial properties and additionally has a substantial specificity for bacteria over the mammalian cells of the infected individual.

The present invention aims to solve the above-identified problems pertaining to the need for novel antibiotics and the development of antibiotic resistance, as well as to improve the efficacy, side-effect profile, selectivity, and overall clinical utility of peptide antibiotics, through providing a combination of cyclic peptides that assemble into peptide nanotubes, distorted peptide nanotube-like structures, or some other previously not characterized structure having potent, specific, and selective antibacterial properties, combined with favourable side effect profiles, for instance in terms of low or no hemolytic activity. The combination of cyclic peptides, comprising at least two cyclic peptides, wherein the at least two cyclic peptides have beta- trisubstituted amino acids in the same amino acid positions, and wherein the beta- trisubstituted amino acids of at least one cyclic peptide of said combination are all present in D-configuration and wherein the beta-trisubstituted amino acids of at least one cyclic peptide of said combination are all present in L-configuration, leads to the in vivo and in vitro formation of peptide nanotubes, peptide nanotube-like structures, or some new previously not characterized peptide structure, which are surmised to cause disturbances in bacterial membranes, bestowing the combinations of cyclic peptides in accordance with the present invention with strong antibacterial properties. Further, since the mode of action of these peptides, requiring at least two complementary peptides to obtain antimicrobial effects, the invention represents a new strategy to deliver antimicrobial peptides. These peptides or analogues thereof might therefore provide an important tool in the fight against increasing antibiotic resistance and nosocomial infections.

In a first aspect, the present invention pertains to cyclic peptides, or pharmaceutically acceptable salts, solvates, or hydrates thereof, comprising an even number of amino acids from 6 to 20, the cyclic peptides having beta-trisubstituted amino acids in every second position, characterized in that at least three consecutive amino acids are all present in D-configuration or in L-configuration, and characterized in that the amino acids not forming part of the at least three consecutive amino acids are present in alternating D- and L-configuration, with every other amino acid being present in D- configuration and every other amino acid being present in L-configuration.

In an additional aspect, the present invention relates to combinations of cyclic peptides, comprising at least two cyclic peptides in accordance with the invention, wherein the at least two cyclic peptides have beta-trisubstituted amino acids in the same amino acid positions, and wherein the beta-trisubstituted amino acids of at least one cyclic peptide of said combination are all present in D-configuration and wherein the beta- trisubstituted amino acids of at least one cyclic peptide of said combination are all present in L-configuration. In a further aspect, the present invention relates to the combination of cyclic peptides for use as a medicament, more specifically for use in the prophylaxis and/or treatment of bacterial infection, in particular for use in the prophylaxis and/or treatment of bacterial infection caused by Gram-positive or by Gram-negative bacteria. The combination of cyclic peptides may for instance be administered simultaneously, sequentially, or separately, to a subject in need thereof.

In yet another aspect, the present invention relates to pharmaceutical composition comprising at least one cyclic peptide in line with the invention, or pharmaceutical compositions comprising a combination of cyclic peptides in accordance with the invention, together with at least one pharmaceutically acceptable excipient, diluent, and/or carrier. The pharmaceutical compositions may be for use in the prophylaxis and/or treatment of bacterial infection, in particular prophylaxis and/or treatment of bacterial infection caused by Gram-positive or by Gram-negative bacteria.

In a further aspect, the present invention pertains to a combination of at least two pharmaceutical compositions, wherein a first pharmaceutical composition comprises at least one cyclic peptide as per the invention, and a second pharmaceutical composition comprises at least one cyclic peptide as per the invention, wherein the at least one cyclic peptide present in said first pharmaceutical composition and the at least one cyclic peptide present in said second pharmaceutical composition have beta- trisubstituted amino acids in the same amino acid positions, and wherein the beta- trisubstituted amino acids of the at least one cyclic peptide present in said first pharmaceutical composition are all present in D-configuration and wherein the beta- trisubstituted amino acids of the at least one cyclic peptide present in said second pharmaceutical composition are all present in L-configuration, in combination with at least one pharmaceutically acceptable excipient, diluent, and/or carrier. The combination of pharmaceutical compositions may be for use in the prophylaxis and/or treatment of bacterial infection, in particular prophylaxis and/or treatment of bacterial infection caused by Gram-positive or by Gram-negative bacteria. In a further aspect, the combination of pharmaceutical compositions may be administered simultaneously, sequentially, or separately, to a subject in need thereof In an additional aspect, the present invention pertains to a method of treating a bacterial infection comprising administering to a subject in need thereof a therapeutically effective amount of a combination of the cyclic peptides, a pharmaceutical composition, or a combination of pharmaceutical compositions in accordance with the present invention.

In yet another aspect, the present invention relates to a kit comprising a first pharmaceutical composition comprising at least one cyclic peptide as per the invention, a second pharmaceutical composition comprising at least one cyclic peptide as per the invention, and instructions for using the kit

The present invention provides a completely novel, unexpected approach, associated with numerous advantages, to the need for new antibiotic agents. Without wishing to be bound by any theory, it is surmised that the at least two cyclic peptides of the present invention assemble into peptides nanotubes, peptide nanotube-like structures or some new previously not characterized peptide structure that create membrane disturbances in bacterial membranes, leading to bacterial growth inhibition and/or death. The combination of at least two cyclic peptides represents a completely novel, binary mechanism of action, meaning that the selectively for bacteria over the mammalian cells of the host are significantly increased, leading to lower toxicity. Additionally, since the cyclic peptides possess virtually no, or very little, antibacterial effect on their own, the development of antibiotic resistance may be substantially hampered. The cyclic peptides in line with the present invention may be tailored specifically depending on the pathogen to be treated, using suitable amino acid substitutions. Further, the cyclic peptides and the combination of cyclic peptides in accordance with the present invention display very low hemolytic properties, which is a crucial property for future clinical use associated with intravenous administration. It is well-known that the membrane-disturbing properties of antimicrobial peptides (AMPs) exerting effects also on the mammalian cells, predominantly the erythrocytes, of the host organism, caused primarily by hemolysis. It has previously been realized, by for instance Ghadiri and colleagues, that intravenous administration of peptides having the ability to self-aggregate (i.e. in contrast to the present invention where the nanotube formation only occurs when at least two cyclic peptides of opposite chirality are mixed) can result in an almost instantaneous drop in blood pressure, most likely stemming from peptide aggregation and subsequent disturbance of erythrocytes or disturbance of blood vessel cells (Dartois et al., 2005, Antimicrob. Agents Chemother. 49, 3302). In contrast, the cyclic peptides in accordance with the present invention possess high water solubility, as a result of the deviation from a perfectly alternating chirality throughout the amino acid sequence, meaning that they are unlikely to aggregate in an uncontrolled, toxic (hemolytic) fashion upon administration to a patient. For instance, the combination of cyclic peptide in accordance with the present invention could for instance be administered to a patient in need thereof through sequential intravenous injections, i.e. by first injecting a first cyclic peptide and subsequently, after said first cyclic peptide has been distributed approximately evenly in the circulation, by injecting a second cyclic peptide, for instance at another injection site. This would further reduce the risk of aggregation and associated toxicity at the site of injection, resulting in additional advantages originating from the combination of cyclic peptides in line with the present invention.

Brief Descriptions of the Drawings

Figure 1 shows CPK molecular models of a tripeptide segment of a peptide nanotube peptide assembeled from the peptide cyc/o[D- Val-L-Ala-D-Val-L-Ala-D-Val-L-Ala- D-Val-L-Ala] according to the model suggested by Ghadri and co-workers. Figure 1A shows the orientation of the peptide bonds in the three peptides, and Figure IB shows stacking of the peptides on top of each other by forming peptide bonds closley resembeling anti-parallel beta-sheets. IB. The pore of the peptide nanotube segment described in A. For the sake of clarity, amino acid residues in the D-configuration are underlined.

Figure 2A shows the orientation of the peptide bonds of the peptides in the peptide nanotube segment. Figure 2B shows a tripeptide segment of a peptide nanotube peptide assembeled from the peptide cyc/o[L-Tle-D-Ala-L-Tle-D-Ala-L-Tle-D-Ala-L- Tle-D-Ala] analogus to figure IB and in accordance to the model suggested by Ghadri and co-workers. The alanine residues are stacked on top of other alanine residues and tert-leucine are stacked upon fert-leucine residues. This packing of the ter/-leucine residues introduces sterical hindrance for the interaction between the peptide bonds. Figure 2C shows a tripeptide segment of a peptide nanotube peptide assembeled from the two peptides cvc/o[L-Tle-D-Ala-L-Tle-D-Ala-L-Tle-D-Ala-L-Tle-D-Ala] and cyc/o[D-Tle-L-Ala-D-Tle-L-Ala-D-Tle-L-Ala-D-Tle-L-Ala]. Enantiomeric pairs of peptides with a beta-trisubstituted such as tert-leucine allows the formation of peptide nanotubes as the bulky fert-butyl groups are stacked upon the methyl groups of alanine residues, thereby providing a more efficient packing into peptide nanotube. For the sake of clarity, amino acid residues in the D-configuration are underlined.

Figure 3 shows inhibition of the growth of Bacillus megaterium in an agar diffusion assay of the peptides:

A. cvc/orD-Tle-D-Lvs-D-Tle-L-Ala-D-Tle-L-Ala-D-Tle-L-Alal,

B. cvclo rL-Tle-L-Lvs-L-Tle-D-Ala-L-Tle-D- Ala-L-Tle-D- Ala] ,

C. cyc/orL-Tle-D-Lvs-L-Tle-D-Ala-L-Tle-D-Ala-L-Tle-D-Alal,

D. cvc/orD-Tle-L-Lvs-D-Tle-L-Ala-D-Tle-L-Ala-D-Tle-L-Alal For the sake of clarity, amino acid residues in the D-configuration are underlined. Figure 4 shows the concentration dependence of the inhibition of the growth of bacillus megaterium in a microdiution assay of the peptides:

A. cvc/orD-Tle-D-Lvs-D-Tle-L-Ala-D-Tle-L-Ala-D-Tle-L-Alal

B. cvc/orL-Tle-L-Lys-L-Tle-D-Ala-L-Tle-D-Ala-L-Tle-D-Alal C. cvc/o L-Tle-D-Lys-L-Tle-D-Ala-L-Tle-D-Ala-L-Tle-D-Alal

D. cvc/orP-Tle-L-Lys-D-Tle-L-Ala-D-Tle-L-Ala-D-Tle-L-Alal For the sake of clarity, amino acid residues in the D-configuration are underlined

Figure 5 shows the hemolytic activity of the peptides A-D shown in Figure 3 and Figure 4.

A. cvc/orD-Tle-D-Lvs-D-Tle-L-Ala-D-Tle-L-Ala-D-Tle-L-Alal

B. cvc/orL-Tle-L-Lvs-L-Tle-D-Ala-L-Tle-D-Ala-L-Tle-D-Alal

C. cvc/orL-Tle-D-Lys-L-Tle-D-Ala-L-Tle-D-Ala-L-Tle-D-Alal,

D. cvc/orP-Tle-L-Lys-D-Tle-L-Ala-D-Tle-L-Ala-D-Tle-L-Alal For the sake of clarity, amino acid residues in the D-configuration are underlined.

Detailed description of the invention

The present invention pertains to cyclic peptides, combinations of cyclic peptide having potent antibacterial effects, as well as pharmaceutical compositions comprising the combinations of cyclic peptides, or combinations of pharmaceutical compositions comprising individual cyclic peptides. Further, present invention relates to the use of the combinations of cyclic peptides, pharmaceutical compositions comprising the cyclic peptides or combinations of pharmaceutical compositions, for use as medicaments, in particular for treating bacterial infections, Gram-positive and/or Gram-negative.

Where features, embodiments, or aspects of the present invention are described in terms of Markush groups, a person skilled in the art will recognize that the invention may also thereby be described in terms of any individual member or subgroup of members of the Markush group. The person skilled in the art will further recognize that the invention may also thereby be described in terms of any combination of individual members or subgroups of members of Markush groups. Additionally, it should be noted that embodiments and features described in the context of one of the aspects and/or embodiments of the present invention may also apply mutatis mutandis to all the other aspects and/or embodiments of the invention. For instance, the simultaneous, sequential, or separate administration described in connection with the cyclic peptides may naturally also apply mutatis mutandis in the context of other aspects/embodiments of the invention, and, further, the cyclic peptides described in connection with one aspect/embodiment of the present invention also apply mutatis mutandis in the context of other aspects/embodiments of the invention, all in accordance with the present invention as such.

All words and abbreviations used in the present application shall be construed as having the meaning usually given to them in the relevant art, unless otherwise indicated. For clarity, some terms are however specifically defined below.

The term "amino acids" shall be understood to comprise any types of amino acids, e.g. natural and/or synthetic amino acids and/or proteinogenic amino acids and/or alpha, beta, and/or gamma amino acids and/or any amino acid derivates, etc. Any chemical compound comprising an amino group, or derivatives thereof, and a carboxylic acid, or derivatives thereof, is for the purpose of the present invention considered to be an amino acid. As will be apparent from the description and the examples, the term "at least three consecutive amino acids" shall be understood to relate to a sequence/stretch of at least three amino acids, for instance Tle-Arg-Tle, or Tle-Lys-Tle, or Tle-Glu-Tle. The term "strictly alternating chirality" shall be understood to relate to amino acid sequences having every other amino acid present in D-configuration and every other amino acid present in L-configuration, for instance c c/o [D-L-D-L-D-L-D-L-D-L-D- L-D-L-D-L], where D represents any amino acid present in D-configuration and L represents any amino acid present in L-configuration. The term "beta-trisubstituted amino acid" shall be understood to pertain to any amino acid, natural or synthetic, having three substituents on the beta carbon. Representative examples of beta- trisubstituted amino acids are tert-leucine (Tie) and penicillamine (Pen). The term "the amino acids not forming part of the at least three consecutive amino acids are present in alternating D- and L-configuration" shall be interpreted in such a way that all the remaining amino acids are present in alternating D- and L-configuration starting immediately adjacent to "the at least three consecutive amino acids", meaning that if "the at least three consecutive amino acids" are present in D-configuration then the amino acids in the positions next to the "the at least three consecutive amino acids" are present in L-configuration (for instance like in the following peptides, wherein positions 1-3 represent "the at least three consecutive amino acids": cyclo[D-Tle-D- Xaa-D-Tle-L-Xaa-D-Tle-L-Xaa-D-Tle-L-Xaal, cyclorL-Xaa-L-Tle-L-Xaa-D-Tle-L- Xaa-D-Tle-L-Xaa-D-Penl, or cvclorD-Tle-D-Xaa-D-Tle-L-Xaa-D-Pen-L-Xaa-D-Pen- L-Xaa-D-Tle-Xaal).

Any numbers recited in the present application shall be interpreted as comprising the actual number in question, i.e. "an even number from 6 to 20" shall be understood to comprise all even numbers from and including 6 to and including 20, that is 6, 8, 10, 12, 14, 16, 18, and 20. In a first aspect, the present invention pertains to a cyclic peptide, or a pharmaceutically acceptable salt, solvate, or hydrate thereof, comprising an even number of amino acids from 6 to 20, the cyclic peptide having beta-trisubstituted amino acids in every second position, characterized in that at least three consecutive amino acids are all present in D-configuration or in L-configuration, and in that the amino acids not forming part of the at least three consecutive amino acids are present in alternating D- and L-configuration, with every other amino acid being present in D- configuration and every other amino acid being present in L-configuration.

The beta-trisubstituted amino acids of the present invention may be the same or different. The beta-trisubstituted amino acids may be selected from the group comprising tert-leucine (Tie; also known as tert-butylglycine), penicillamine, beta- aminovaline, beta hydroxyvaline, and derivatives of valine wherein the beta-hydrogen has been substituted, or some other alpha amino acid where the beta carbon has been substitituted so that it forms three covalent bonds to any other atom except hydrogen. Beta-trisubstituted amino acids are especially preferred in the contexts of the present invention, since they disfavour the self-assembly of the individual peptide but facilitate self-assembly of two peptides where the chirality of the beta-trisubstituted amino acid residues are of different chirality, which is assumed, without wishing to be bound by any particular theory, to be important for the antibiotic mechanism of action.

Further in accordance with the present invention, the cyclic peptides may comprise at least one basic and/or at least one acidic amino acid, in order to modulate the specificity of the cyclic peptides, or the combination comprising at least two cyclic peptides, for bacteria having certain characteristics. The at least one basic amino acid may be selected from the group comprising lysine, diamino propionic acid, diamino butyric acid, arginine, arginine derivatives, histidine, ornithine or any functional group or combinations of atoms attached to the alpha carbon that will give rise to an cationic charge. The at least one acidic amino acid may be selected from the group comprising aspartic acid or glutamic acid. In a further aspect, the present invention pertains to a combination of cyclic peptides, comprising at least two cyclic peptides according to any one of the preceding claims, wherein the at least two cyclic peptides have beta-trisubstituted amino acids in the same amino acid positions, and wherein the beta-trisubstituted amino acids of at least one cyclic peptide of said combination are all present in D-configuration and wherein the beta-trisubstituted amino acids of at least one cyclic peptide of said combination are all present in L-configuration. The beta-trisubstituted amino acids may for instance be selected from the group comprising tert-leucine, penicillamine, beta-aminovaline, beta-hydroxyvaline or some other alpha-amino acid where the beta-carbon has been substitituted so that it forms three covalent bonds to any other atom then hydrogen.

The at least two cyclic peptides comprised in the combination of cyclic peptides may comprise at least one basic amino acid and/or at least one acidic amino acid. For instance, a first cyclic peptide may comprise at least one basic amino acid, and a second cyclic peptide may also comprise at least one basic amino acid. Conversely, a first cyclic peptide may comprise at least one acidic amino acid, and a second cyclic peptide may also comprise at least one acidic amino acid. Alternatively, a first cyclic peptide may comprise at least one acidic amino acid, and a second cyclic peptide may comprise at least one basic amino acid, or a first cyclic peptide may comprise at least one basic amino acid, and second cyclic peptide may comprise at least one acidic amino acid, and a third cyclic peptide may comprise at least one basic amino acid.

The antibacterial properties of the cyclic peptides as per the present invention may be tailored through introducing various types of derivatives, for instance through reacting a suitable derivative, or an intermediate, with a suitable functionality present on at least one of the cyclic peptides. Suitable functionalities for derivatizing the cyclic peptides may for instance be amine groups, which may be modified through the introduction of for instance a polycationic moiety connected to a lipophilic moiety. In one embodiment, polyarginine and/or polylysine and/or poly(Arg-Lys) is attached to an amino acid side chain of the cyclic peptide(s), followed by attachment of a fatty acid moiety to the free terminal end of the polycationic polyarginine and/or polylysine and/or poly(Arg-Lys). The fatty acid may be selected from the group comprising saturated, mono-unsaturated, di-unsaturated, tri-unsaturated, or poly-unsaturated fatty acids, either branched or linear, for instance octanoic acid or stearic acid or any other type of suitable fatty acid. Thus, the cyclic peptide(s) in accordance with the present invention may be derivatized with a polycationic moiety connected to a fatty acid moiety (for instance stearyl-modified polyarginine). In a further embodiment, the cyclic peptide(s) may be derivatized with 1 or 2 aminoethyl groups and/or 1 or 2 ethylguanidino groups, preferably on at least one of the basic (cationic) amino acid side chains.

The potent, synergistic antibacterial effect can be illustrated by the two peptides peptide A cyc/o[D-Tle-D-Lys-D-Tle-Ala-D-Tle-Ala-D-Tle-Ala] and peptide B cyc/o[Tle-Lys-Tle-D-Ala-Tle-D-Ala-Tle-D-Ala] in an agar diffusion assay against Bacillus megaterium. The peptide at a concentration of 1 mM is applied to the wells A and B (Figure 3). No inhibition of bacterial growth could be detected at the site of application of the either peptide. However, at the area where the two are mixed as a result of diffusion in the agar medium a significant inhibition of bacterial growth can be observed. These results should be compared with the effects of peptide C; cyclo[ - Tle-D-Lvs-L-Tle-D-Ala-L-Tle-D-Ala-L-Tle-D-Alal and peptide D; cvc/o D-Tle-L- Lys-D-Tle-L-Ala-D-Tle-L-Ala-D-Tle-L-Ala], peptides that have a strictly alternating pattern of D and L amino acids residues. These peptides when applied at 1 mM concentration at the wells C and D had antimicrobial activity both as individual peptides but an increased activity when they were mixed in the medium as a result of diffusion. It was also shown that when peptides with strict alternating sequence were mixed with peptides not having strictly alternating sequence in the agar medium as a result of diffusion a synergistic inhibition of bacterial growth could be observed.

This effect could also be observed in a microdilution assay where the growth of Bacillus megaterium was investigated as a function of increasing concentration of either peptide A or peptide B or peptide A and peptide B combined. These results clearly show that neither peptide A nor peptide B had any effect on the growth of Bacillus megaterium in concentrations up to 100 μΜ but when combined a potent inhibition can be observed.

A conclusion from these studies is that pairs of peptides not following the strictly alternation pattern of L and D amino acid residues displays a stronger synergistic mode of action as compared to analogous peptides with a strictly alternating sequence.

It is realized that potent antimicrobial activity alone is not a sufficient property of an antimicrobial peptide if the peptide should administrated intravenously to patients. Most antimicrobial peptides have at least some effects on the membrane of mammalian cells and these effects are usually investigated by measuring hemolysis; the ability of the peptide disrupt the membrane of mammalian red blood cells. Figure 5 shows the efficiency of peptide A and peptide B to cause hemolysis of red blood cells. In the experiment 100% hemolysis is induced by the addition of the detergent 0,1 %Triton X-100. Neither peptide A nor peptide B had any hemolytic activity in concentrations up to 1 mM (data not shown in figure). However, when combined a weak hemolytic activity could be recorded with half-maximal hemolysis at a concentration of 400 μΜ. These results should be compared to peptides cyclo[L-T\e- D-Lys-L-Tle-D-Ala-L-Tle-D-Ala-L-Tle-D-Alal and cyc/orD-Tle-L-Lys-D-Tle-L-Ala- D-Tle-L-Ala-D-Tle-L-Ala], peptides with a strict alternating pattern of D and L amino acids residues. Neither of these peptides shows any hemolytic activity in concentrations up to 100 μΜ. However, when combined these peptides have a hemolytic activity and half-maximal hemolysis occurring at a concentration of 25 μΜ.

These results have important implications for the invention. Successful general clinical application of membrane active antimicrobial peptides will in most cases require that the peptides should be injected into the blood. However, for these agents hemolytic activity is considered to be a major concern. Furthermore, the onset of the hemolytic effect (as well as the antimicrobial effects) is very fast. When injected into the organism the antimicrobial peptide will have to be administrated in a volume far smaller than the volume of the blood, thus the concentration of the drug given intravenously must be far higher than the desired plasma concentration levels. For example, if the desired concentrations of the peptide in a blood volume of 5 litre should be ΙΟμΜ and the volume of the injected peptide is set to 10 ml, the concentration of the peptide should be 5 mM or even higher if clearance and distribution into the extracellular fluid is taken into account. A conclusion from this line of reasoning is that it is very important that the hemolytic activity of antimicrobial peptides is very low. For peptide A and peptide B, when added as single peptides, we could not measure any hemolytic activity at concentrations up to 1 mM and only a weak hemolytic activity when added together. This implies that when given in a hypothetical clinical context the patient is unlikely to be exposed to hemolytic concentrations of the peptide not even at the site of injection. Hemolytic concentrations of the peptides will only be evident after two injections (peptide A followed by peptide B) and even in this case hemolytic effects will require very high concentrations of the peptides combined.

It is also of importance to stress that this mode action represents a new class of pharmacological synergy, that to the best of the inventors knowledge, has not previously been described for synthetic comounds. There are no evidences suggesting that synergy arise from the action of the two different peptides on separate pharmacological targets, such as enzymes. Considering the chemical structures of the peptides it can be assumed that in the bacterial membrane the two peptides aggregate and give rise to peptide nanotubes, peptide nanotube like structures or some new previously not characterized peptide aggregate that when accumulated leads to growth inhibition, killing of the bacteria or both these effects combined. A further aspect in accordance with the present invention pertains to the combination of cyclic peptides for use as a medicament. Further, the present invention relates to the combination of cyclic peptides as per the invention for use in the prophylaxis and/or the treatment of bacterial infection. The combination of cyclic peptides may, more specifically, be for use in the prophylaxis and/or treatment of bacterial infection caused by Gram-positive and/or by Gram-negative bacteria. The combination of cyclic peptides may be used for essentially any type of bacterial infection, for instance infection caused by Staphylococcus aureus, Methicilline-resistant Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Clostridium difficile, Enterococcus faecium, Vancomycine-resistant enterococcus, Neisseria gonorrhoeae, Pseudomonas aeruginosa, Eschericia coli, Salmonella, Acinetobacter baumannii, Klebsiella pneumoniae.

Further in accordance with the present invention, the combination of cyclic peptides may be administered simultaneously, sequentially, or separately, for instance through first administering a first cyclic peptide in accordance with the invention, and subsequently administering a second cyclic peptide in accordance with the invention. The administration of the second cyclic peptide may be carried out after the first cyclic peptide has been essentially completely distributed throughout the individual to be treated, but it may also be administered approximately simultaneously. Additionally, the present invention also pertains to administration of more than two cyclic peptides, for instance administration of 3-10 cyclic peptides, in order to modulate the antibacterial properties and/or the therapeutic effects to be achieved. Further in line with the present invention, the combination of cyclic peptides may also be administered to a patient in need thereof at different sites of the body, or via different routes of administration. For instance, a first cyclic peptide may be administered in the left arm of a human being in need of thereof, whereas a second cyclic peptide may be administered in the left arm of the same human being. This type of administration regimen would minimize the risk of local aggregation, which can be seen with conventional self-aggregating AMPs. The cyclic peptides in accordance with the present invention may be varied substantially as long as the major, necessary features of the present invention are maintained. Suitable cyclic peptides may for instance be selected from the group comprising the following peptides (and, naturally, their enantiomeric counterparts, as displayed below, having strictly opposite chirality throughout the amino acids sequences): For the sake of clarity, amino acid residues in the D-configuration are underlined.

cycloP-Tle-D-Xaa- D-Tle-L-Xaa-D-Tle-L-Xaa],

cyclo D-Tle-D-Xaa- D-Tle-L-Xaa-D-Tle-L-Xaa-D-Tle-L-Xaa\|

cvclorD-Tle-D-Xaa- D-Tle-L-Xaa-D-Tle-L-Xaa-D-Tle-L-Xaa-D-Tle-L-Xaal, cycloiD-Tle-D-Xaa- D-Tle-L-Xaa-D-Tle-L-Xaa-D-Tle-L-Xaa-D-Tle-L-Xaa-D-

Tle-L-Xaa],

cvclorD-Tle-D-Xaa-D-Tle-L-Xaa-D-Tle-L-Xaa-D-Tle-L-Xaa-D-Tle-L-Xaa-D- Tle-L-Xaa-D-Tle-L-Xaal ,

cvclo D-Tle-D-Xaa-D-Tle-L-Xaa-D-Tle-L-Xaa-D-Tle-L-Xaa-D-Tle-L-Xaa-D- Tle-L-Xaa-D-Tle-L- -Xaa-D-Tle-L-Xaal,

cyclofD-Tle-D-Xaa D-Tle-L-Xaa-D-Tle-L-Xaa-D-Tle-L-Xaa-D-Tle-L-Xaa-D- Tle-L-Xaa-D-Tle-L^•Xaa-D-Tle-L-Xaa-D-Tle-L-Xaal , and,

cvclorD-Tle-D-Xaa•D-Tle-L-Xaa-D-Tle-L-Xaa-D-Tle-L-Xaa-D-Tle-L-Xaa-D- Tle-L-Xaa-D-Tle-L-Xaa-D-Tle-L-Xaa-D-Tle-L-Xaa-D-Tle-L-Xaal, where underlined amino acid residues represent amino acids present in D- configuration, and wherein Xaa may represent any amino acid selected from the group comprising proteinogenic amino acids, natural amino acids, synthetic amino acids, alpha amino acids, and any amino acid derivatives thereof. More specifically, Xaa may represent any amino acid selected from the group comprising alanine, asparagine, aspartic acid, arginine, cysteine, glutamine, glycine, glutamic acid, histidine, isoleucine, lysine, leucine, phenylalanine, methionine, serine, proline, tryptophan, threonine, tyrosine, valine, diamino propionic acid, diamino butyric acid, ornithine, and any derivatives thereof.

The counterparts of the above cyclic peptides are having the following formulas:

cyclo L-Tle-L-Xaa-L-Tle-D-Xaa-L-Tle-D-Xaal.

cyclofL-Tle-L-Xaa-L-Tle-D-Xaa-L-Tle-D-Xaa-L-Tle-D-Xaal,

cyclorL-Tle-L-Xaa-L-Tle-D-Xaa-L-Tle-D-Xaa-L-Tle-D-Xaa-L-Tle-D-Xaal, cvclorL-Tle-L-Xaa-D-Tle-D-Xaa-L-Tle-D-Xaa-L-Tle-D-Xaa-L-Tle-D-Xaa-L

Tle-D-Xaal,

cyclo[L-Tle-L-Xaa-L-Tle-D-Xaa-L-Tle-D-Xaa-L-Tle-D-Xaa-L-Tle-D-Xaa-L- Tle-D-Xaa-L-Tle-D-Xaal,

cyclorL-Tle-L-Xaa-L-Tle-D-Xaa-L-Tle-D-Xaa-L-Tle-D-Xaa-L-Tle-D-Xaa-L- Tle-D-Xaa-L-Tle-D-Xaa-L-Tle-D-Xaal ,

cyclorL-Tle-L-Xaa-L-Tle-D-Xaa-L-Tle-D-Xaa-L-Tle-D-Xaa-L-Tle-D-Xaa-L- Tle-D-Xaa-L-Tle-D-Xaa-L-Tle-D-Xaa-L-Tle-D-Xaal , and,

cyclorL-Tle-L-Xaa-L-Tle-D-Xaa-L-Tle-D-Xaa-L-Tle-D-Xaa-L-Tle-D-Xaa-L- Tle-D-Xaa-L-Tle-D-Xaa-L-Tle-D-Xaa-L-Tle-D-Xaa-L-Tle-D-Xaal, where underlined amino acid residues represent amino acids present in D- configuration, and wherein Xaa may represent any amino acid selected from the group comprising proteinogenic amino acids, natural amino acids, synthetic amino acids, alfa amino acids, and any amino acid derivatives thereof. More specifically, Xaa may represent amino acid selected from the group comprising alanine, asparagine, aspartic acid, arginine, cysteine, glutamine, glycine, glutamic acid, histidine, isoleucine, lysine, leucine, phenylalanine, methionine, serine, proline, tryptophan, threonine, tyrosine, valine, diamino propionic acid, diamino butyric acid, ornithine, and any derivatives thereof. Albeit that there may, in certain instances, potentially be advantageous to have at least two beta-trisubstituted amino acids among the at least three consecutive amino acids all present in either D or in L configuration, it may also potentially be advantageous to have only one beta-trisubstituted amino acid among, for instance, three consecutive amino acids all present in either D or in L configuration. This can be exemplified by the following peptides:

cyclo L-Xaa-L-Tle-L-Xaa-D-Tle-L-Xaa-D-Tle1,

cvclo[L-Xaa-L-Pen-L-Xaa-D-Pen-L-Xaa-D-Tlel,

cvclo L-Xaa-L-Tle-L-Xaa-D-Pen-L-Xaa-D-Pen-L-Xaa-D-Tlel,

cvclorD-Xaa-D-Tle-D-Xaa-L-Pen-D-Xaa-L-Pen-D-Xaa-L-Penl

The present invention also pertains to cyclic peptide having more than three consecutive amino acids all present in D-configuration or in L-configuration, for instance the following peptides (and, naturally, their enantiomeric counterparts, having strictly opposite chirality throughout the amino acids sequences):

cvclo[L-Tle-L-Xaa-L-Tle-L-Xaa-D-Tle-L-Xaa-D-Tle-L-Xaal

cvclorL-Tle-L-Xaa-L-Tle-L-Xaa-L-Tle-D-Xaa-L-Tle-D-Xaa-L-Tle-D-Xaal, and,

cyclofL-Tle-L-Xaa-L-Tle-L-Xaa-D-Tle-L-Xaa-D-Tle-L-Xaa-D-Tle-L-Xaa-D-

where underlined amino acid residues represent amino acids present in D- configuration, and wherein Xaa may represent any amino acid selected from the group comprising proteinogenic amino acids, natural amino acids, synthetic amino acids, alfa amino acids, and any amino acid derivatives thereof. More specifically, Xaa may represent amino acid selected from the group comprising alanine, asparagine, aspartic acid, arginine, cysteine, glutamine, glycine, glutamic acid, histidine, isoleucine, lysine, leucine, phenylalanine, methionine, serine, proline, tryptophan, threonine, tyrosine, valine, diamino propionic acid, diamino butyric acid, ornithine, and any derivatives thereof.

In another aspect, the present invention relates to a pharmaceutical composition comprising at least one cyclic peptide, in combination with at least one pharmaceutically acceptable excipient, diluent, and/or carrier. Further, the present invention pertains to a pharmaceutical composition for use as a medicament, specifically for use for use in the prophylaxis and/or treatment of bacterial infection, in particular infection caused by Gram-positive or by Gram-negative bacteria. The pharmaceutical compositions of the present invention may be in the form of a pill, tablet, coated tablet, granule, capsule, hard or soft gelatin capsule, solution, syrup, emulsion, suspension, pastille, suppository, solution for injection, ointment, tincture, cream, lotion, powder, spray, transdermal therapeutic systems, nasal spray, aerosol mixture, microcapsule, and/or an implant.

In a further aspect, the present invention pertains to pharmaceutical compositions comprising a combination of cyclic peptides, together with at least one pharmaceutically acceptable excipient, diluent, and/or carrier. Pharmaceutically acceptable excipients, diluents, and/or carriers are well-known to the skilled person. The pharmaceutical compositions of the present invention may be in the form of a pill, tablet, coated tablet, granule, capsule, hard or soft gelatin capsule, solution, syrup, emulsion, suspension, pastille, suppository, solution for injection, ointment, tincture, cream, lotion, powder, spray, transdermal therapeutic systems, nasal spray, aerosol mixture, microcapsule, and/or an implant.

A further aspect in accordance with the present invention pertains to a pharmaceutical composition in line with the invention for use as a medicament. Further, the present invention relates to the pharmaceutical composition as per the invention for use in the prophylaxis and/or the treatment of bacterial infection. The pharmaceutical composition may, more specifically, be for use in the prophylaxis and/or treatment of bacterial infection caused by Gram-positive and/or by Gram-negative bacteria. The pharmaceutical composition may be used for essentially any type of bacterial infection, for instance infection caused by Staphylococcus aureus, Methicilline-resistant Staphylococcus aureus (MRS A), Streptococcus pyogenes, Streptococcus pneumoniae, Clostridium difficile, Enterococcus faecium, Vancomycine-resistant enterococcus, Neisseria gonorrhoeae, Pseudomonas aeruginosa, Eschericia coli, Salmonella, Acinetobacter baumannii, Klebsiella pneumoniae. In yet another aspect, the present invention pertains to a combination of at least two pharmaceutical compositions, wherein a first pharmaceutical composition comprises at least one cyclic peptide in accordance with the invention, and a second pharmaceutical composition comprises at least one cyclic peptide in accordance with the invention, wherein the at least one cyclic peptide present in said first pharmaceutical composition and the at least one cyclic peptide present in said second pharmaceutical composition have beta-trisubstituted amino acids in the same amino acid positions, and wherein the beta-trisubstituted amino acids of the at least one cyclic peptide present in said first pharmaceutical composition are all present in D-configuration and wherein the beta- trisubstituted amino acids of the at least one cyclic peptide present in said second pharmaceutical composition are all present in L-configuration, in combination with at least one pharmaceutically acceptable excipient, diluent, and/or carrier.

A further aspect in accordance with the present invention pertains to the combination of pharmaceutical compositions in line with the invention for use as a medicament. Further, the present invention relates to the combination of pharmaceutical compositions as per the invention for use in the prophylaxis and/or the treatment of bacterial infection. The combination of pharmaceutical compositions may, more specifically, be for use in the prophylaxis and/or treatment of bacterial infection caused by Gram-positive and/or by Gram-negative bacteria. The combination of pharmaceutical compositions may be used for essentially any type of bacterial infection, for instance infection caused by Staphylococcus aureus, Methicillineresistant Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Clostridium difficile, Enterococcus faecium, Vancomycine resistant enterococcus, Neisseria gonorrhoeae, Pseudomonas aeruginosa, Eschericia coli, Salmonella, Acinetobacter baumannii, Klebsiella pneumoniae.

The combination of pharmaceutical compositions in accordance with the present invention may be in the form of pills, tablets, coated tablets, granules, capsules, hard or soft gelatin capsules, solutions, syrups, emulsions, suspensions, pastilles, suppositories, solution for injections, ointments, tinctures, creams, lotions, powders, sprays, transdermal therapeutic systems, nasal sprays, aerosol mixtures, microcapsules, and/or implants, and/or any combinations thereof.

Further in accordance with the present invention, the combination of pharmaceutical compositions may be administered simultaneously, sequentially, or separately, for instance through first administering a first pharmaceutical composition in accordance with the invention, and subsequently administering a second pharmaceutical composition in accordance with the invention. The administration of the second pharmaceutical composition may be carried out after the first pharmaceutical composition and its active ingredient(s) has been essentially completely distributed throughout the individual to be treated, but it may also be administered approximately simultaneously. Additionally, the present invention also pertains to administration of more than two pharmaceutical compositions, for instance administration of 3-10 pharmaceutical compositions, in order to modulate the antibacterial properties and/or the therapeutic effects to be achieved.

In yet another aspect, the present invention relates to a method of treating a bacterial infection comprising administering to a subject in need thereof a therapeutically effective amount of a combination of cyclic peptides in accordance with the invention, a pharmaceutical composition in accordance with the invention, or a combination of pharmaceutical compositions in accordance with the invention. The subject in need of treatment may for instance be a mammal, such as a human, or any other type of animal. Further, the bacterial infection to be treated may be caused by one or more Gram- positive and/or Gram-negative bacterium, for instance selected from the group comprising Staphylococcus aureus, Methicillineresistant Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Clostridium difficile, Enterococcus faecium, Vancomycine resistant enterococcus, Neisseria gonorrhoeae, Pseudomonas aeruginosa, Eschericia coli, Salmonella, Acinetobacter baumannii, Klebsiella pneumoniae.

An additional aspect in accordance with the present invention pertains to a kit comprising a first pharmaceutical composition comprising at least one cyclic peptide as per the invention, a second pharmaceutical composition comprising at least one cyclic peptide as per the invention, and instructions for using the kit, preferably in a clinical setting. The pharmaceutical compositions may be supplied in dedicated compartments, for instance vials, ampoules, syringes, or any other type of compartment and/or device. The kit may, further in line with the invention, optionally comprise only one pharmaceutical composition, comprising at least one cyclic peptide in accordance with the invention, and instructions for use. Supplying the antibacterial cyclic peptides of the present invention in the form of a kit implies numerous advantages in terms of inter alia handling, storage, and/or distribution.

The cyclic peptides in accordance with the present invention may be administered to a patient in need thereof in therapeutically effective dosages, or in accordance with therapeutically effective dosage regimens. Solutions, suspensions, solutions for injection, or any other types of essentially liquid pharmaceutical compositions may have concentrations of the cyclic peptides in accordance with the invention of between approximately comprise between 1 μΜ and 0,1 M. Tablets, capsules, pills, or any other types of essentially solid pharmaceutical compositions may comprise amounts of the cyclic peptides in accordance with the invention of between approximately 10 microgram and 5 gram. Further, the dose of the cyclic peptides may vary from between approximately 0.1 mg/kg body weight and 500 mg/kg body weight. However, the above values may naturally vary substantially depending on, inter alia, the animal, mammal, and/or human to be treated, and, additionally, depending on the antibacterial cyclic peptides per se, the pharmaceutical compositions comprising the cyclic peptides, as well as on the nature, severity, and localization of the pathogen(s). Further, a skilled person may also need to adjust the above values in accordance with the therapeutic situation at hand.

When contemplating intravenous injection as a potential mode of administration, it is crucial to bear in mind that the local concentrations of peptides will transiently be significantly higher than the concentrations needed to achieve therapeutic, antibacterial effects. Thus, since the cyclic peptides of the present invention may be administered separately, the hemolytic effects of individual cyclic peptides were also evaluated, using the same assay. These trials clearly show that the individual cyclic peptides caused essentially no hemolysis up to concentrations as high as 1 mM. The conclusions from these experiments are that the cyclic peptides of the present invention, in complete contrast to previously described AMPs, have very low propensity for causing hemolysis even at very high peptide concentrations, a fact that is highly surprising. The peptides described previously by for instance Ghadiri and colleagues (i.e. peptides in line with the previously established paradigm for antibacterial cyclic peptides; that is, cyclic peptides having strictly alternating chirality throughout the amino acid sequences and having tert-leucine in every other position) were also evaluated using the same assay. The peptides cyclo[Tle-Lys-Tle-Ala-Tle- Ala-Tle-Ala] och cyclo[Tle-Lys-Tle-Ala-Tle- Ala-Tie- Ala] (where underlined amino acids are present in D-configuration), i.e. peptides not in accordance with the present invention, exhibited an EC₅₀ of approximately 20 μΜ (Figure 5), which is more than 10 times lower concentrations than the peptides of the present invention. Thus, it is clear that the peptides of the present invention have highly surprising and advantageous properties in terms of both low hemolysis, in particular compared to the cyclic antibacterial peptides previously described within the art, and in terms of synergistic antibacterial effects. The above-outlined structural formulas merely depict exemplary embodiments according to the present invention. A person skilled in the art would hence immediately realize that the above structural formula is primarily meant for illustrative purposes, and that other structures and moieties not specifically mentioned and/or illustrated above are encompassed by the present invention. Further, a person skilled in the art would immediately recognize the schematic nature of the above formulas, and that additional suitable moieties may be incorporated in the formulas without departing from the scope of the present invention.

Examples

Peptide synthesis

The peptides were synthesized by manual solid phase peptide synthesis on a 2- chlorotrityl resin in the following order: 2-Cl-trityl resin-L-Ala-D-Tle-L-Ala-D-Tle-L- Ala-D-Tle-D-Lys(Boc)-D-Tle and 2-Cl-trityl resin-D-Ala-L-Tle-D-Ala-L-Tle-D-Ala- L-Tle-L-Lys(Boc)-L-Tle. Fmoc-alanine (0,5 mmol) was dissolved in dry 10 ml DCM and added to 1 g of 2-chlorotrityl resin followed by 2 mmol DIEA. The mixture was stirred for 2 hours and washed three times with DCM/MeOH/DIEA (17:2: 1) followed by washes with DCM, DMF and DCM (3 three times each). The Fmoc group of the first amino acid was removed with 20 % piperidine in NMP for 15 minutes and the resin was washed three times with NMP followed by four times with DCM.

Coupling of the following amino acid to the resin was conducted by activating the Fmoc amino acid dissolving 2 equivalents (eq) of Fmoc-protected amino acid, and 2 eq of COMU in a minimal amount of N-methylpyrrolidone (NMP). The solution was put on ice, 4 eq diisopropylethylamine was added, the solution was stirred for 1 minute and added to the resin resuspended in NMP. Fmoc-Tle-OH was coupled for 2 hours and 5 minutes, while for Fmoc-Ala and Fmoc-Lys(Boc)-OH were coupled for 1 hour. After coupling of the amino acids, the resin was washed three times with DMF, followed by three times with DCM. After completing the peptide sequence, the N- terminal Fmoc group was removed by 20% piperidin in NMP washed three times with NMP followed by four times with DCM.

The peptide was cleaved from the resin by adding a mixture of 0.5 % TFA in DCM for one minute followed by filtration into a solution of 9 ml DCM containing 1 ml pyridin in a 100 ml round bottom flask. . This procedure was repeated 4 times. The solvents were removed by reduced pressure and the residual material dissolved in 20 ml DCM and the solvent removed by reduced pressure. This procedure was repeated 4 times.

In order to remove the pyridine-trifluoro acetate salt formed at the cleavage of the peptide from the resin, the dry material was suspended in water followed by centrifugation at 4770 rpm for 10 minutes, the supenatant was removed and the pellet was resuspended in water and the centrifugation was repeated twice. The resulting pellet was frozen and dried at at reduced pressure on a lyophilizer.

Cyclization of the linear peptide was performed by dissolving the peptide in DMF (lOmg/ml) followed by addition of DIEA:HOBt:BOP (3:1.5: 1.5). The solution was stirred for 22 hours with a teflon stirring bar and additional DIEA:HOBt:BOP (3: 1.5: 1.5) was added to the mixture and stirred for additional 3 hours. DMF was removed by a vaccum pump connected to a solvent trap cooled by dry ice. The residual material was resuspended in DCM and the solvent was removed first by a rotary evaporator followed by a vaccum pump. This procedure was repeated twice in order to remove residual DMF. Water was added to the residual material and the mixture stirred for two days in order to get the coupling reagents into solution and the peptide into a homogenous suspension. The suspension was twice centrifuged at 4770 rpm for 10 minutes and and the pellet resuspended in water. The final pellet was frozen and dried on a lyophilizer. In order to remove the Gpsilon-Boc on the lysine residue the dry peptide was treated using 25 mL of a mixture containing 1 % triisopropysilane and 1 % H₂O in TFA for 40 minutes. TFA was removed on a rotary evaporator and the residual oily material was put on high vacuum for 10 minutes. The peptide was precipitated with diethyl ether, the diethylether removed by aspiration and residual solvents were removed by a high vacuum pump.

Purification of peptides were performed using a Discovery BIO WidePore C₁₈ column, 10x250, particle size 5-10 μηι and a pore diameter of 300 A. The mobile phase consisted of a gradient from 15 to 60 % MeCN/H₂O, 0.1 % TFA, at 2.0 mL/min flow rate over 50 minutes, hold at 60 % MeCN in 10 minutes, from 60 % MeCN to 100 % MeCN in 1 minutes and back to 15 % MeCN in 1 minute. The analysis was performed on a Discovery BIO WidePore C₁₈ column (Supelco) 4.6x150 mm, particle size 5 μηι, pore diameter 300 A and flow rate 0.8 mL/ml with the same gradient profile as mentioned above.

MALDI-TOF mass spectra was recorded on a ABI MALDI-ToF Voyager-DE STR. The matrix used was 2,6-dihydroxyacetophenone, 10 mg/mL in 50 % MeCN/H₂O, 0.1 % TFA.

Broth microdilution assay of antimicrobial activity

A small culture of bacillus megaterium was inoculated into 2 mL nutrient broth (3 g Beef Extract and 5 g enzymatic digest of Gelatin, Heraco, Sweden) and put in a shaker at either 30 or 31 °C over night. The bacteria were diluted 2 fold to 0.4-0.6 OD₆₀₀ and aliquoted into 96-well polypropylene microtiter plate with cells 0.02 OD₆₀o per well (100 μΐ). The bacteria were either treated with two peptides at the same time or with only one peptide. The peptides were dissolved in water or in 5 % DMSO in water. The individual concentrations were measured in triplicates. The controls used in the experiment were 100 μΐ bacteria and 100 μΐ of water, 100 μΐ of nutrient broth or 100 μΐ with the highest concentration of DMSO used in the experiment. The bacteria were incubated at either 30 to 31 °C on a shaker for 18 hours and bacterial growth inhibition was determined by measuring the absorbance at 595 nm in a microplate reader.

Agar diffusion assay

A small culture of bacillus megaterium was inoculated into 2 mL nutrient broth (3 g Beef Extract and 5 g enzymatic digest of Gelatin, Heraco, Sweden) and put in a shaker at 30 °C over night. The following day, the bacteria were diluted 2 fold to 0.48 OD₆oo- A cotton swab was dipped into this solution and applied onto the agar by moving the tip of the swab in different directions, covering the whole plate. Thereafter 6 mm wells were punched in the agar and each well was filled with 23 μg peptide (25 μΐ). The plates were dried for 10 minutes in room temperature and incubated at 30 °C for 24 hours whereas after 30 minutes of incubation the plates were inverted and allowed to be further incubated. The following day the zones of growth inhibition were measured.

Evaluating the hemolytic properties of cyclic peptides separately and in combinations

Sheep blood in Alsever^'s solution was centrifuged for 10 minutes at 1000 x g. The red blood cells were washed one time with PBS and centrifuged once more and resuspended in PBS into a sheep red blood cell solution [4%(v/v) in final]. Aliquots (190 μΐ) of this solution were added into tubes followed by 10 μΐ peptide or 5 μΐ of one peptide and after 10 minutes the next peptide was added (5 μΐ). The samples were incubated at 37 °C for 20 minutes, followed by centrifugation for 10 minutes at 1000 x g. Hemoglobin release was monitored by measuring the supernatant absorbance at 540 nm on a microplate reader.

Claims

Claims

1. A cyclic peptide, or a pharmaceutically acceptable salt, solvate, or hydrate thereof, comprising an even number of amino acids from 6 to 20, said cyclic peptide having beta-trisubstituted amino acids in every second position, characterized in that at least three consecutive amino acids are all present in D-configuration or in L-configuration, and in that the amino acids not forming part of the at least three consecutive amino acids are present in alternating D- and L-configuration, with every other amino acid being present in D- configuration and every other amino acid being present in L-configuration.

2. The cyclic peptide according to claim 1 , wherein the beta-trisubstituted amino acids are the same or different.

3. The cyclic peptide according to any one of the preceding claims, wherein the beta-trisubstituted amino acids are selected from the group comprising tert- leucine, penicillamine, beta-aminovaline and beta-hydroxyvaline, and any derivatives thereof, and derivatives of valine wherein the beta-hydrogen has been substituted.

4. The cyclic peptide according to the any one of the preceding claims, comprising at least one basic and/or at least one acidic amino acid.

5. The cyclic peptide according to the any one of the preceding claims, wherein the at least one basic amino acid is selected from the group comprising lysine, diamino propionic acid, diamino butyric acid, arginine, arginine derivatives, histidine, ornithine, and any derivates thereof.

6. The cyclic peptide according to the any one of the preceding claims, wherein the at least one acidic amino acid is selected from the group comprising aspartic acid and glutamic acid.

7. A combination of cyclic peptides, comprising at least two cyclic peptides according to any one of the preceding claims, wherein the at least two cyclic peptides have beta-trisubstituted amino acids in the same amino acid positions, and wherein the beta-trisubstituted amino acids of at least one cyclic peptide of said combination are all present in D-configuration and wherein the beta- trisubstituted amino acids of at least one cyclic peptide of said combination are all present in L-configuration.

8. The combination of cyclic peptides according to claim 7, wherein the at least two cyclic peptides comprise at least one basic amino acid and/or at least one acidic amino acid.

9. The combination of cyclic peptides according to any one of claims 7 to 8 for use as a medicament.

10. The combination of cyclic peptides according to any one of claims 7 to 8 for use in the prophylaxis and/or treatment of bacterial infection.

1 1. The combination of cyclic peptides according to any one of claims 7 to 8 for use in the prophylaxis and/or treatment of bacterial infection caused by Gram- positive or by Gram-negative bacteria.

12. The combination of cyclic peptides according to any one of claims 9 to 1 1, wherein the at least two cyclic peptides are administered simultaneously, sequentially, or separately.

13. A pharmaceutical composition comprising at least one cyclic peptide according to any one of claims 1 to 6, in combination with at least one pharmaceutically acceptable excipient, diluent, and/or carrier

14. The pharmaceutical composition according to claim 13 for use as a medicament.

15. The pharmaceutical composition according to claim 13 for use in the prophylaxis and/or treatment of bacterial infection.

16. The pharmaceutical composition according to claim 13 for use in the prophylaxis and/or treatment of bacterial infection caused by Gram-positive or by Gram-negative bacteria.

17. A pharmaceutical composition comprising a combination of cyclic peptides according to any one of claims 7-8, in combination with at least one pharmaceutically acceptable excipient, diluent, and/or carrier.

18. The pharmaceutical composition according to claim 17 for use as a medicament.

19. The pharmaceutical composition according to claim 17 for use in the prophylaxis and/or treatment of bacterial infection.

20. The pharmaceutical composition according to claim 17 for use in the prophylaxis and/or treatment of bacterial infection caused by Gram-positive or by Gram-negative bacteria.

21. A combination of at least two pharmaceutical compositions according to any one of claims 13 or 17, wherein a first pharmaceutical composition comprises at least one cyclic peptide according to any one of claims 1 to 6, and a second pharmaceutical composition comprises at least one cyclic peptide according to any one of claims 1 to 6, wherein the at least one cyclic peptide present in said first pharmaceutical composition and the at least one cyclic peptide present in said second pharmaceutical composition have beta-trisubstituted amino acids in the same amino acid positions, and wherein the beta-trisubstituted amino acids of the at least one cyclic peptide present in said first pharmaceutical composition are all present in D-configuration and wherein the beta- trisubstituted amino acids of the at least one cyclic peptide present in said second pharmaceutical composition are all present in L-configuration, in combination with at least one pharmaceutically acceptable excipient, diluent, and/or carrier.

22. The combination of pharmaceutical compositions according to claim 21 for use as a medicament.

23. The combination of pharmaceutical compositions according to claim 21 for use in the prophylaxis and/or treatment of bacterial infection.

24. The combination of pharmaceutical compositions according to claim 21 for use in the prophylaxis and/or treatment of bacterial infection caused by Gram- positive or by Gram-negative bacteria.

25. The combination of pharmaceutical compositions according to any one of claims 21 to 24, wherein the at least two pharmaceutical compositions are administered simultaneously, sequentially, or separately.

26. A method of treating a bacterial infection comprising administering to a subject in need thereof a therapeutically effective amount of a combination of cyclic peptides according to any one of claims 7 to 8, a pharmaceutical composition according claim 13, a pharmaceutical composition according to claim 17, or a combination of pharmaceutical compositions according to claim 21.

27. The method according to claim 26, wherein the subject is a mammal.

28. The method according to any one of claims 26 to 27, wherein the subject is a human.

29. The method according to any one of claims 26 to 28, wherein the bacterial infection is a caused by one or more Gram-positive or Gram-negative bacterium.

30. The method according to claim 26, wherein the bacterial infection is a caused by one or more Gram-positive bacterium selected from the group comprising Staphylococcus aureus, Methicillineresistant Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Clostridium difficile, Enterococcus faecium, Vancomycine resistant enter ococcus.

31. The method according to claim 28, wherein the bacterial infection is a caused by one or more Gram-negative bacterium selected from the group comprising Neisseria gonorrhoeae, Pseudomonas aeruginosa, Eschericia coli, Salmonella enterica, Salmonell enterica, Salmonella typhi, Acinetobacter baumannii, Klebsiella pneumoniae, Legionella pneumophilia, Hemophilus influenzae.

32. A kit comprising:

o a first pharmaceutical composition comprising at least one cyclic peptide according to any one of claims 1 to 6;

o a second pharmaceutical composition comprising at least one cyclic peptide according to any one of claims 1 to 6; and,

o instructions for using said kit.