Antimycotic polyene-alginate oligomer conjugates

Abstract

The invention provides an antimycotic polyene-alginate oligomer conjugate comprising an antimycotic polyene connected covalently to at least one alginate oligomer, wherein one or other terminal uronic acid residues of each alginate oligomer in the conjugate is connected to the antimycotic polyene via a direct covalent bond or a covalent molecular linker, or a pharmaceutically acceptable salt, solvate, hydrate, diastereoisomer, tautomer, enantiomer, or active metabolite thereof. Also provided are methods for the preparation of said conjugate, pharmaceutical compositions comprising said conjugate and the use thereof in a method for the treatment or prevention of a fungal infection in a subject with, suspected to have, or at risk of, a fungal infection.

Description

[0001] Antimycotic polyene-alginate oligomer conjugates

[0002] The present invention provides a novel modified form of polyene antimycotics having advantageous properties. More specially, it has been recognised that covalently conjugating antimycotic polyenes and alginate oligomers via one of the terminal uronic acid residues of the alginate increases the aqueous solubility and reduces the host toxicity of the polyene antimycotic without significantly reducing antimycotic efficacy or breadth of target susceptibility thereof. In other words, antimycotic polyene-alginate oligomer conjugates are a class of novel chemical entities having antimycotic efficacy (which may be at least essentially the same as or similar to the antimycotic efficacy of the non-conjugated polyene antimycotic) but significantly less host toxicity and significantly greater aqueous solubility. The antimycotic polyene-alginate oligomer conjugates of the invention therefore have a wider therapeutic window and so represent improved treatments for fungal infections in human and non-human animal subjects. The invention therefore provides medical uses and methods of treatment reflecting these properties of the antimycotic polyene-alginate oligomer of the invention, namely the use of the antimycotic polyene-alginate oligomer conjugates of the invention in the treatment or prevention of fungal infections. The invention further provides methods for preparing the conjugates of the invention.

[0003] Polyene antimycotics (which are also referred to interchangeably herein as antimycotic polyenes, polyene antifungals, and antifungal polyenes) are a well-known and well- characterised class of antifungal / anti mycotic agents comprising an amphipathic macrolactone (cyclic carboxylic ester) ring containing a series of at least 3 conjugated double bonds assembled via repetitive condensation of small carboxylic acids by the polyketide synthase (PKS) enzymes. The region with conjugated double bonds on the polyene macrolide structures is often referred to as chromophore. The number of double bonds in the chromophore region determines the UV absorption spectra of the compound. The chromophore region creates a hydrophobic part to the molecule, and a number of polar groups such as carbonyls, hydroxyls and epoxides are located directly opposite the chromophore on the macrolactone ring to render the ring amphipathic.

[0004] A common structural feature for most of the antifungal polyenes used today in human therapy is also a region comprising eight carbon atoms within the macrolactone ring that are linked to the chromophore and form a C8 hemiketal region. The macrolactone ring is contracted via formation of this hemiketal. In this region a sugar, e.g. an aminosugar, is linked via an O-glycosidic bond to the first carbon after the chromophore region (counterclockwise direction when the chromophore is displayed at the bottom of the lactone ring and lactone group is displayed to the left, as shown in Figure 1). In many of the glycosylated polyenes, this sugar is represented by mycosamine or perosamine. Another functionally important chemical group in the C8 region is a carboxyl group, typically linked to the 4th carbon atom, counting from the chromophore counter-clockwise as defined above. Both the latter carboxyl and an amino group of mycosamine are ionizable, making the part of the molecule they are attached to polar. The part of the polyene macrolide molecules opposite to the C-8 region and adjacent to the lactone group is usually quite hydrophobic, as it often contains either methyl groups, or aromatic moieties (e.g. aminoacetophenone, as found in candicidin).

[0005] In summary, the overall chemistry of a typical antifungal glycosylated polyene molecule can be described as substituted macrolactone ring having a polar hydrophilic head, opposed chromophore and polyol chains, and a hydrophobic tail. Specific examples include amphotericin B, nystatin, natamycin (pimaricin), candidicin, rimocidin, candidin, hamycin, perimycin, tricomycin (hachimycin) and tetramycin.

[0006] Polyene antibiotics display a unique mode of action by making the fungal cell membranes permeable to ions and other small molecules via polyene-ergosterol interactions. This gives them highly efficacious, broad spectrum antifungal activity and restricts the chances of resistance developing. However, it is likely because of this property that polyene antifungal agents are rather toxic to the animal subject (or “host”) to which they are administered, and may cause such serious side-effects as infusion-related reactions, haemolytic toxicity, renal tubular damage and thrombophlebitis, especially upon intravenous administration. Much of the toxicity of the polyene antimycotics arises from their weak affinity for cholesterol, the main component of the mammalian membrane. They interact with cellular cholesterol component and then penetrate the mammalian membrane to create transmembrane channels that leads to the leakage of metabolites and monovalent ions triggering host cell death. As the polyene antimycotic molecules accumulate extensively in vital organs of the human body, particularly the kidney, their weaker affinity for cholesterol becomes deleterious to host cells via the disruption of membrane integrity, causing severe cellular damage.

[0007] Acute infusion-related toxicity is due to the fact that polyene antimycotics are molecules of microbial origin and so may be recognized by TLR2 (Toll-like receptor 2) and CD14 on mononuclear immune cells, leading to the initiation of an inflammatory response. Nephrotoxicity is thought to be caused by increased exposure of the polyene to renal cells via low-density lipoprotein (LDL) receptor mediated endocytosis. Moreover, polyene antimycotics cause vasoconstriction in afferent renal arterioles, which decreases renal blood flow and glomerular filtration.

[0008] Further complicating their use as antifungal medicaments is the fact that polyene antimycotics, especially those which are widely used today, are very poorly soluble in water, and can easily form aggregates of micelles in aqueous media. This property complicates formulation strategies (oral administration is extremely difficult) and leads to reduced bioavailability during parental administration due to the poor uptake and distribution of the compounds in organs and tissues.

[0009] Together these issues narrow the therapeutic window of the polyene antimycotics significantly, sometimes making them unusable in certain subjects, despite the advantageous repertoire of antifungal effects. Attempts have been made to expand the therapeutic window by formulating with lipids (liposomes, micellar suspensions, colloidal suspensions, emulsions) or as nanostructured formulations (e.g. nanocrystals, nanotubes, polymer nanocarriers, cubosomal and cochleate nanoparticles). In other approaches polyene antimycotics such as amphotericin B have been modified structurally to address toxicity and solubility disadvantages (Tevyashova, A.N., et al.; Discovery of Amphamide, a Drug Candidate for the Second Generation of Polyene Antibiotics. ACS Infect. Dis. 2020, 6, 2029- 2044).

[0010] There is however a continued need for novel forms of polyene antimycotics having advantageous properties, e.g. increased aqueous solubility and reduced host toxicity, without significant reduction in antimycotic efficacy.

[0011] Alginate oligomers have been described in the literature at length. Briefly, alginates are linear polymers of (1-4) linked p-D-mannuronic acid (M) and / or its C-5 epimer a-L-guluronic acid (G). The primary structure of alginates can vary greatly. The M and G residues can be organised as homopolymeric blocks of contiguous M or G residues, as blocks of alternating M and G residues and single M or G residues can be found interspacing these block structures. An alginate molecule can comprise some or all of these structures and such structures might not be uniformly distributed throughout the polymer. In the extreme, there exists a homopolymer of guluronic acid (polyguluronate) or a homopolymer of mannuronic acid (polymannuronate). Alginate oligomers may be obtained from alginate polymers which are typically isolated from natural sources as large high molecular weight polymers (e.g. an average molecular weight in the range 300,000 to 500,000 Daltons). Such large alginate polymers may be degraded, or broken down, e.g. by chemical or enzymatic hydrolysis to produce alginate structures of lower molecular weight (i.e. alginate oligomers).

[0012] As shown in the Examples, it has now been found that covalently conjugating antimycotic polyenes and alginate oligomers via one of the terminal uronic acid residues of the alginate creates a novel chemical entity with antimycotic efficacy (which may be at least essentially the same as, or similar to, or at least not significantly reduced as compared to, the antimycotic efficacy and / or the breadth of target susceptibility thereof of the non-conjugated form of the polyene) but with reduced host toxicity and / or increased aqueous solubility as compared to the non-conjugated form of the polyene antibiotic.

[0013] Accordingly, in a first aspect the invention provides an antimycotic polyene-alginate oligomer conjugate comprising an antimycotic polyene connected covalently to at least one alginate oligomer, wherein one or other terminal uronic acid residues of each alginate oligomer in the conjugate is connected to the antimycotic polyene via a direct covalent bond or a covalent molecular linker.

[0014] The antimycotic polyene-alginate oligomer conjugates of the invention may therefore also be described by Formula I:

[0015] P-(L-[U-A])n(Formula I) wherein P- is a polyene antimycotic, L is a direct covalent bond or a covalent molecular linker, -[U-A] is an alginate oligomer with terminal uronic acid residue II, and n is an integer of 1 to 10, e.g. 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 2 or 1.

[0016] In accordance with the invention the polyene antimycotic may be any member of the class of antifungal / antimycotic agents comprising an amphipathic macrolactone (cyclic carboxylic ester) aliphatic ring containing a series of conjugated double bonds and a plurality of polar groups (e.g. carbonyl, hydroxyl and epoxide) located directly opposite the series of double bonds on the macrolactone ring.

[0017] An antifungal / antimycotic agent is considered to be a molecule that inhibits the viability and / or growth of a fungus of colony thereof when applied in sufficient amounts at least in in vitro conditions, and preferably in in vivo conditions. In other words, a molecule which has antimycotic efficacy. The means to assess this are described below. Such agents may therefore be capable of treating or preventing infection / colonisation of a human or nonhuman animal. In certain embodiments the polyene antimycotic of use in the invention is an established treatment for fungal infection of a human or non-human animal, or has the potential to be if not for the agent having a therapeutic window which prevents practical use in therapy.

[0018] In certain embodiments there may be at least 3, e.g. at least 4, 5, 6, 7, 8, 9 or 10, or no more than 10, 9, 8, 7, 6, 5, or 4) conjugated double bonds in the amphipathic macrolactone (cyclic carboxylic ester) aliphatic ring. In certain embodiments there may be at least 3, e.g. at least 4, 5, 6, 7, 8, 9 or 10, or no more than 10, 9, 8, 7, 6, 5, or 4) polar groups (e.g. carbonyls, hydroxyls and epoxides) located directly opposite the series of double bonds on the macrolactone ring. The number of polar groups and the number of conjugated double bonds will be sufficient to provide an amphipathic macrolactone ring.

[0019] In certain embodiments the antifungal polyene of the conjugates of the invention also have a region, e.g. of 6, 7, 8, 9, 10 or 12 carbon atoms, linked to the series of conjugated double bonds which comprises a cyclic hemiketal group within the macrolactone ring. In certain embodiments the cyclic hemiketal group may be a pyranose ring.

[0020] In further embodiments, e.g. in the hemiketal region, a monosaccharide, e.g. an amino monosaccharide (by which it is meant that a hydroxyl group has been replaced with an amine group), is linked via an O-glycosidic bond, which may be in an a or a configuration, to the first carbon after the series of conjugated double bonds (counter-clockwise direction when the linked to the series of conjugated double bonds is displayed at the bottom of the lactone ring and lactone group is displayed to the left, as shown in Figure 1). In certain embodiments this monosaccharide may be a. a triose, a tetrose, a pentose, a hexose, a heptose, an octose, a nonose or a decose in pyranose or furanose form and / or L- or D- form where appropriate, and amine derivatives thereof. Pentose or hexose saccharides / residues are preferred, e.g. mannose (e.g. D-mannose), galactose (e.g. D- galactose), glucose (e.g. D-glucose), fructose, fucose (e.g. L-fucose), N-acetyl-glucosamine, N-acetylgalactosamine, rhamnose, galactosamine, glucosamine (e.g. D-glucosamine), galacturonic acid, glucuronic acid, acid, N-acetylneuraminic acid, methyl D-mannopyranoside (mannoside), a-methyl- glucoside, galactoside, ribose, xylose, arabinose, and amine derivatives thereof. In certain embodiments the monosaccharide is not mannuronate or guluronate. In particular embodiments the monosaccharide is mycosamine or perosamine.

[0021] In further embodiments the hemiketal region comprises at least one carboxyl group, e.g. linked to the 2nd, 3rd, 4th, or 5thcarbon atom, counting away from the series of conjugated double bonds (e.g. counter-clockwise as defined above). In certain embodiments at least one carboxyl group is present on the cyclic hemiketal, e.g. pyranose, group

[0022] In certain embodiments, the part of the polyene molecule opposite to the hemiketal region and adjacent to the lactone group (more specifically the carboxylic ester group of the lactone ring) is hydrophobic and may comprise alkyl, alkenyl, alkynyl, alkoxy, aryl, aryloxy or aminoacetophenone side groups. For instance, linear or branched, unsubstituted, aminoacetophenone substituted, and / or N-methyl-aminoacetophenone substituted, C1-8 alkyl, alkenyl, alkynyl or alkoxy groups, or unsubstituted Cs-s aryl or arlyoxy groups. Such groups may be found adjacent, e.g. directly adjacent, to the -O- group of the lactone group.

[0023] In certain embodiments the antifungal polyene of the conjugates of the invention does not have a monosaccharide bound covalently to the part of the polyene molecule opposite to the hemiketal region and adjacent to the lactone group, e.g. within 1 , 2, 3 or 4 carbon atoms of the -O- group of the lactone group counting in a clockwise direction from the lactone group in a structure as defined above. In certain embodiments, such monosaccharides may be glucose, galactose or mannose. In other embodiments the antifungal polyene of the conjugates of the invention is not amphotericin B with a glucose, galactose or mannose bound covalently to the part of the polyene molecule opposite to the hemiketal region and adjacent to the lactone group, e.g. within 1 , 2, 3 or 4 carbon atoms of the -O- group of the lactone group counting in a clockwise direction from the lactone group in a structure as defined above. The monosaccharide may be covalently bound via a glycosidic bond, e.g. an O-glycosidic bond.

[0024] Specific examples the antifungal polyene of the conjugates of the invention include amphotericin B, nystatin, natamycin (pimaricin), candidicin, rimocidin, candidin, hamycin, perimycin, tricomycin (hachimycin), partricin and tetramycin (Figure 1).

[0025] In certain embodiments the invention provides a conjugate of a polyene antimycotic and an alginate oligomer, said conjugate comprising a polyene antimycotic having a structure Wn-X- O-Y-Zm, wherein W is a carboxylic acid group

[0026] X is an amphipathic polyene cyclic carboxylic ester aliphatic ring comprising one or more regions of conjugated double bonds and one or more regions of saturated bonds, optionally wherein two or more carbon atoms of the ring are linked by an O, N or S atom to form a heterocyclic group, and wherein one or more hydrogen atoms in a saturated region is substituted with a hydroxyl group, a C1-8 carbonyl containing group, a linear or branched, unsubstituted, hydroxyl substituted, halo substituted, aminoacetophenone substituted, and / or N-methyl-aminoacetophenone substituted, C1-8 alkyl, C1-8 alkenyl, C1-8 alkynyl or Ci-8 alkoxy group, or an unsubstituted, hydroxyl substituted, or halo substituted, C3-8 aryl or C3-8 arlyoxy group, and

[0027] O is an O-glycosidic bond

[0028] Y is a monosaccharide

[0029] Z is an amine group n is an integer of 1-10 m is an integer of 1-10 and at least one alginate oligomer connected covalently thereto, wherein one or other terminal uronic acid residues of each alginate oligomer in the conjugate is connected to at least one W and / or at least one Z of the antimycotic polyene via a direct covalent bond or a covalent molecular linker.

[0030] C1-8 may be any of Ci , C2, C3, C4, C5, Ce, C7, or Cs, or any range having such values as endpoints.

[0031] C3-8 may be any of C3, C4, C5, Ce, C7, or Cs, or any range having such values as endpoints.

[0032] In certain embodiments the amphipathic polyene cyclic carboxylic ester aliphatic ring has at least 20 carbon atoms in its aliphatic backbone (i.e. not counting substituent groups). In other embodiments the amphipathic polyene cyclic carboxylic ester aliphatic ring has at least 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44 or 45 carbon atoms in its backbone. In other embodiments the amphipathic polyene cyclic carboxylic ester aliphatic ring has no more than 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44 or 45 carbon atoms in its backbone. Any ranges which may be constructed from these values are specifically contemplated. As used herein aliphatic refers to a covalently linked chain of carbon atoms which may consist of single, double and / or triple carbon to carbon bonds. The term is interchangeable with optionally substituted saturated or unsaturated hydrocarbon chain.

[0033] The chain of carbon atoms in the aliphatic ring may include one or more carbonyl (C=O) groups, that is the one or more of the carbons in that chain is part of a carbonyl group.

[0034] In certain embodiments the amphipathic polyene cyclic carboxylic ester aliphatic ring contains at least one series of conjugated double bonds having at least 3, e.g. at least 4, 5, 6, 7, 8, 9 or 10, or no more than 10, 9, 8, 7, 6, 5, or 4 conjugated double bonds. Any ranges which may be constructed from these values are specifically contemplated. The polyene of the conjugate can therefore be classified, in certain embodiments, as a triene, tetraene, pentaene, hexaene, heptaene, octaene, nonaene and decaene.

[0035] In certain embodiments the amphipathic polyene cyclic carboxylic ester aliphatic ring has a plurality of substituting groups in said one or more regions of saturated bonds. This may be sufficient to render the region hydrophilic. In certain embodiments there may be at least 2, e.g. at least 3, 4, 5, 6, 7, 8, 9 or 10, or no more than 10, 9, 8, 7, 6, 5, 4 or 3 substitutions. Any ranges which may be constructed from these values are specifically contemplated.

[0036] In certain embodiments the amphipathic polyene cyclic carboxylic ester aliphatic ring has a plurality of polar substituting groups (e.g. C1-8 carbonyl containing groups and hydroxyl) and / or carbonyl groups in said one or more regions of saturated bonds. In certain embodiments said substitutions are located directly opposite the one or more regions of conjugated double bonds on the macrolactone ring. In certain embodiments there may be at least 3, e.g. at least 4, 5, 6, 7, 8, 9 or 10, or no more than 10, 9, 8, 7, 6, 5, or 4 polar groups located directly opposite the series of double bonds on the macrolactone ring. The number of polar groups and the number of conjugated double bonds will be sufficient to provide an amphipathic macrolactone ring.

[0037] In certain embodiments the two or more carbon atom pairs linked by an O, N or S atom to form a heterocyclic group form an epoxide or a cyclic hemiketal group. The cyclic hemiketal may have 5, 6, 7 or 8 carbons. In certain embodiments the cyclic hemiketal group may be a pyranose ring. In certain embodiments the hemiketal group is situated in a region, e.g. of 6, 7, 8, 9, 10 or 12 carbon atoms, linked to one of the regions of conjugated double bonds.

[0038] In certain embodiments the hemiketal group is directly adjacent to or separated by 1 or 2 carbons from the glycosidic bond.

[0039] In certain embodiments the amphipathic polyene cyclic carboxylic ester aliphatic ring has a plurality of non-polar substituting groups (e.g. a linear or branched, unsubstituted, aminoacetophenone substituted, and / or N-methyl-aminoacetophenone substituted C1-8 aliphatic alkyl, alkenyl, alkynyl or alkoxy group, or an unsubstituted, C3-8 aryl or arlyoxy group) in said one or more regions of saturated bonds. In certain embodiments said substitutions are located directly opposite to the hemiketal region and adjacent to the lactone group.

[0040] In accordance with the invention the amphipathic polyene cyclic carboxylic ester aliphatic ring (X) further carries 1 to 10 carboxylic acid groups (namely, the W groups in the above formula). In certain embodiments the ring may carries 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 2 or 1 carboxylic acid groups. In certain embodiments at least one carboxylic acid group is linked to the 2nd, 3rd, 4th, or 5thcarbon atom from the least one of the series of conjugated double bonds. In certain embodiments at least one carboxyl group is present on the cyclic hemiketal, e.g. pyranose, group. In certain embodiments the at least one carboxyl group is separated by 2, 3 or 4 carbons from the glycosidic bond.

[0041] In further embodiments the amphipathic polyene cyclic carboxylic ester aliphatic ring carries a single (i.e. no more than one) carboxylic acid group, preferably on the cyclic hemiketal, e.g. pyranose, group. In certain embodiments the at least one carboxyl group on the hemiketal group is separated by 2, 3 or 4 carbons from the glycosidic bond. In certain embodiments one or other terminal uronic acid residues of an alginate oligomer is connected to this carboxyl group via a direct covalent bond or a covalent molecular linker. In more specific arrangements of this embodiment, no other alginate is conjugated to the polyene antimycotic.

[0042] In certain embodiments this carboxyl group is part of amide group which links to one or other terminal uronic acid residues of an alginate oligomer either directly or via a covalent molecular linker. In accordance with the invention monosaccharide Y is an amino monosaccharide (by which it is meant that at least one hydroxyl group has been replaced with an amine group). In certain embodiments this monosaccharide may be an amino form of a triose, a tetrose, a pentose, a hexose, a heptose, an octose, a nonose or a decose in pyranose or furanose form and / or L- or D- form where appropriate, and amine derivatives thereof. Pentose or hexose saccharides / residues are preferred, e.g. mannose (e.g. D-mannose), galactose (e.g. D- galactose), glucose (e.g. D-glucose), fructose, fucose (e.g. L-fucose), N-acetyl-glucosamine, N-acetylgalactosamine, rhamnose, galactosamine, glucosamine (e.g. D-glucosamine), galacturonic acid, glucuronic acid, acid, N-acetylneuraminic acid, methyl D-mannopyranoside (mannoside), a-methyl-glucoside, galactoside, ribose, xylose, arabinose, and amine derivatives thereof. In certain embodiments the monosaccharide is not mannuronate or guluronate. In particular embodiments the monosaccharide is mycosamine or perosamine.

[0043] In certain embodiments, the number of amine groups on the monosaccharide may by 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 2 or 1 , dependent on the size of the monosaccharide. In certain embodiments, the monosaccharide has a single amine group. In certain embodiments one or other terminal uronic acid residues of an alginate oligomer is connected to this amine group via a direct covalent bond or a covalent molecular linker. In more specific arrangements of this embodiment, no other alginate is conjugated to the polyene antimycotic.

[0044] In certain embodiments this amine group is part of amide group which links to one or other terminal uronic acid residues of an alginate oligomer either directly or via a covalent molecular linker.

[0045] In accordance with the invention monosaccharide (Y) is linked to the amphipathic polyene cyclic carboxylic ester aliphatic ring (X) via an O-glycosidic bond, which may be in an a or a configuration. In certain embodiments the carbon of the polyene ring contributing to the O- glycosidic bond is directly adjacent to or separated by 1 or 2 carbons from one of the series of conjugated double bonds.

[0046] In certain embodiments the carbon of the polyene ring contributing to the O-glycosidic bond is situated between one of the series of conjugated double bonds and the cyclic hemiketal and subject to the features described immediately above in relation to numbers of intervening carbon atoms. In certain embodiments the carbon of the polyene ring contributing to the O-glycosidic bond is situated between one of the series of conjugated double bonds and a (or the) carbon carrying a carboxylic acid and subject to the features described immediately above in relation to numbers of intervening carbon atoms.

[0047] In certain embodiments the amphipathic polyene cyclic carboxylic ester aliphatic ring (X) may have the ring structure, and optionally the non-carboxyl group substitution pattern of amphotericin B, nystatin, natamycin (pimaricin), candidicin, rimocidin, candidin, hamycin, perimycin, tricomycin (hachimycin), partricin or tetramycin, preferably as shown in Figure 1.

[0048] In certain embodiments the amphipathic polyene cyclic carboxylic ester aliphatic ring (X) and W may have the ring structure and substitution pattern of amphotericin B, nystatin, natamycin (pimaricin), candidicin, rimocidin, candidin, hamycin, or tricomycin (hachimycin), partricin or tetramycin,, preferably as shown in Figure 1.

[0049] In certain embodiments the polyene antimycotic of the conjugate may be selected from amphotericin B, nystatin, natamycin (pimaricin), candidicin, rimocidin, candidin, hamycin, perimycin, tricomycin (hachimycin), partricin and tetramycin, preferably as shown in Figure 1.

[0050] In certain embodiments the polyene antimycotic of the conjugate may be amphotericin B.

[0051] In certain embodiments the polyene antimycotic of the conjugate is amphotericin B and one or other terminal uronic acid residues of an alginate oligomer is connected to the carboxyl group on carbon 16 (C16, counting clockwise starting from the carbonyl group in the lactone group) of the antimycotic polyene via a direct covalent bond or a covalent molecular linker. In certain embodiments this carboxyl group is part of amide group which links to one or other terminal uronic acid residues of an alginate oligomer either directly or via a covalent molecular linker.

[0052] In certain embodiments the polyene antimycotic of the conjugate is nystatin and one or other terminal uronic acid residues of an alginate oligomer is connected to the carboxyl group on carbon 16 (C16, counting clockwise starting from the carbonyl group in the lactone group) of the antimycotic polyene via a direct covalent bond or a covalent molecular linker. In certain embodiments this carboxyl group is part of amide group which links to one or other terminal uronic acid residues of an alginate oligomer either directly or via a covalent molecular linker. In certain embodiments the polyene antimycotic of the conjugate is natamycin and one or other terminal uronic acid residues of an alginate oligomer is connected to the carboxyl group on carbon 12 (C12, counting clockwise starting from the carbonyl group in the lactone group) of the antimycotic polyene via a direct covalent bond or a covalent molecular linker. In certain embodiments this carboxyl group is part of amide group which links to one or other terminal uronic acid residues of an alginate oligomer either directly or via a covalent molecular linker.

[0053] In certain embodiments the polyene antimycotic of the conjugate is candidicin and one or other terminal uronic acid residues of an alginate oligomer is connected to the carboxyl group on carbon 18 (C18, counting clockwise starting from the carbonyl group in the lactone group) of the antimycotic polyene via a direct covalent bond or a covalent molecular linker. In certain embodiments this carboxyl group is part of amide group which links to one or other terminal uronic acid residues of an alginate oligomer either directly or via a covalent molecular linker.

[0054] In certain embodiments the polyene antimycotic of the conjugate is rimocidin and one or other terminal uronic acid residues of an alginate oligomer is connected to the carboxyl group on carbon 14 (C14, counting clockwise starting from the carbonyl group in the lactone group) of the antimycotic polyene via a direct covalent bond or a covalent molecular linker. In certain embodiments this carboxyl group is part of amide group which links to one or other terminal uronic acid residues of an alginate oligomer either directly or via a covalent molecular linker.

[0055] In certain embodiments the polyene antimycotic of the conjugate is candidin and one or other terminal uronic acid residues of an alginate oligomer is connected to the carboxyl group on carbon 16 (C16, counting clockwise starting from the carbonyl group in the lactone group) of the antimycotic polyene via a direct covalent bond or a covalent molecular linker. In certain embodiments this carboxyl group is part of amide group which links to one or other terminal uronic acid residues of an alginate oligomer either directly or via a covalent molecular linker.

[0056] In certain embodiments the polyene antimycotic of the conjugate is hamycin and one or other terminal uronic acid residues of an alginate oligomer is connected to the carboxyl group on carbon 18 (C18, counting clockwise starting from the carbonyl group in the lactone group) of the antimycotic polyene via a direct covalent bond or a covalent molecular linker. In certain embodiments this carboxyl group is part of amide group which links to one or other terminal uronic acid residues of an alginate oligomer either directly or via a covalent molecular linker. In certain embodiments the polyene antimycotic of the conjugate is tricomycin (hachimycin) and one or other terminal uronic acid residues of an alginate oligomer is connected to the carboxyl group on carbon 18 (C18, counting clockwise starting from the carbonyl group in the lactone group) of the antimycotic polyene via a direct covalent bond or a covalent molecular linker. In certain embodiments this carboxyl group is part of amide group which links to one or other terminal uronic acid residues of an alginate oligomer either directly or via a covalent molecular linker.

[0057] In certain embodiments the polyene antimycotic of the conjugate is partricin and one or other terminal uronic acid residues of an alginate oligomer is connected to the carboxyl group on carbon 18 (C18, counting clockwise starting from the carbonyl group in the lactone group) of the antimycotic polyene via a direct covalent bond or a covalent molecular linker. In certain embodiments this carboxyl group is part of amide group which links to one or other terminal uronic acid residues of an alginate oligomer either directly or via a covalent molecular linker.

[0058] In certain embodiments the polyene antimycotic of the conjugate is tetramycin and one or other terminal uronic acid residues of an alginate oligomer is connected to the carboxyl group on carbon 12 (C12, counting clockwise starting from the carbonyl group in the lactone group) of the antimycotic polyene via a direct covalent bond or a covalent molecular linker. In certain embodiments this carboxyl group is part of amide group which links to one or other terminal uronic acid residues of an alginate oligomer either directly or via a covalent molecular linker.

[0059] The above references to counting clockwise apply to structures in which the chromophore is displayed at the bottom of the lactone ring and lactone group is displayed to the left, as shown in Figure 1) and the carbonyl group in the lactone group (more specifically the carboxylic ester group of the lactone ring) is counted as carbon 1.

[0060] In certain embodiments the polyene antimycotic of the conjugate may be selected from amphotericin B, nystatin, natamycin (pimaricin), candidicin, rimocidin, candidin, hamycin, perimycin, tricomycin (hachimycin), partricin and tetramycin, preferably as shown in Figure 1, and one or other terminal uronic acid residues of an alginate oligomer is connected to the amine group on the amino monsaccharide of these polyene antimycotics via a direct covalent bond or a covalent molecular linker. In certain embodiments this amine group is part of amide group which links to one or other terminal uronic acid residues of an alginate oligomer either directly or via a covalent molecular linker. In these embodiments describing the position to which the alginate oligomer is liked covalently, the conjugate comprises a single alginate oligomer, i.e. no other alginate is conjugated to the polyene antimycotic.

[0061] In the above discussion of the polyene part of the conjugates of the invention all references to chemical structure extend to any stereoisomeric (e.g. enantiomeric or diastereoisomeric) or tautomeric forms thereof which have antifungal / antimycotic effect as defined herein.

[0062] In the above discussion of the polyene part of the conjugates of the invention all references extend pharmaceutically acceptable salts (e.g. those disclosed below), solvates or hydrates thereof.

[0063] As noted above, alginates typically occur as polymers of an average molecular mass of at least 35,000 Daltons, i.e. approximately 175 to approximately 190 monomer residues, although typically much higher. An alginate oligomer according to the present invention will, on the other hand, contain 2 to 100 monomer residues, more typically 3, 4, 5 or 6 to 100, and may contain 2, 3, 4, 5 or 6 to 75, 2, 3, 4, 5 or 6 to 50, 2, 3, 4, 5 or 6 to 40, 2, 3, 4, 5 or 6 to 35 or 2, 3, 4, 5 or 6 to 30 residues. Thus, an alginate oligomer for use according to the invention will typically have an average molecular weight of 350, 550, 700, 900 or 1000 to 20,000 Daltons, 350, 550, 700, 900 or 1000 to 15,000 Daltons, 350, 550, 700, 900 or 1000 to 10,000 Daltons, 350, 550, 700, 900 or 1000 to 8000 Daltons, 350, 550, 700, 900 or 1000 to 7000 Daltons, or 350, 550, 700, 900 or 1000 to 6,000 Daltons.

[0064] Alternatively put, the alginate oligomer may have a degree of polymerisation (DP), or a number average degree of polymerisation (DPn) of 2 to 100, preferably 2 to 75, preferably 2 to 50, more preferably 2 to 40, 2 to 35, 2 to 30, 2 to 28, 2 to 25, 2 to 22, 2 to 20, 2 to 18, 2 to 17, 2 to 15 or 2 to 12.

[0065] Other representative ranges (whether for the number of residues, DP or DPn) include any one of 3, 4, 5, 6, 7, 8, 9, 10 or 11 to any one of 50, 45, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31 ,

[0066] 30, 29, 28, 27, 26, 25, 24, 23, 22, 21 , 20, 19, 18, 17, 16, 15, 14, 13 or 12.

[0067] Other representative ranges (whether for the number of residues, DP or DPn) include any one of 8, 9, 10, 11, 12, 13, 14 or 15 to any one of 50, 45, 40, 39, 38, 37, 36, 35, 34, 33, 32,

[0068] 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21 , 20, 19, 18, 17 or 16. Other representative ranges (whether for the number of residues, DP or DPn) include any one of 11, 12, 13, 14, 15, 16, 17 or 18 to any one of 50, 45, 40, 39, 38, 37, 36, 35, 34, 33, 32,

[0069] 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20 or 19.

[0070] It may in some embodiments be advantageous to select a larger alginate oligomer so as to create a conjugate of greater size. Larger conjugates may help deliver the polyene antimycotic selectively to sites and locations of infection because the vascular permeability of such areas within a subject is typically greater than in the vasculature of non-infected areas. Consequently, larger conjugates are less likely to enter non-infected areas from the blood stream, but would able to enter the more permeable infected areas. A representative size range for such a larger oligomer may for example be 20 to 100 residues (or DP or DPn of 20 to 100) or any one of 20, 21 , 22, 23, 24 or 25, to any one of 100, 90, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35 or 30 residues (or DP or DPn of any one of these ranges) or any one of 30, 31,

[0071] 32, 33, 34 or 35, to any one of 100, 90, 80, 75, 70, 65, 60, 55, 50, 45 or 40 residues (or DP or DPn of any one of these ranges). Alternatively, these results might also be achieved by increasing the numbers of alginate oligomers, even those of smaller size, in the conjugate.

[0072] An alginate oligomer will, as noted above, contain (or comprise) guluronate or guluronic acid (G) and / or mannuronate or mannuronic acid (M) residues or units. An alginate oligomer according to the invention will preferably be composed solely, or substantially solely of (i.e. consist essentially of) uronate / uronic acid residues, more particularly alduronic acid residues, e.g. hexalduronic acid residues. An alginate oligomer according to the invention will most preferably be composed solely, or substantially solely of (i.e. consist essentially of) G and / or M residues. Alternatively expressed, in the alginate oligomer of use in the present invention, at least 80%, more particularly at least 85, 90, 95 or 99% of the monomer residues may be uronate / uronic acid residues, or more particularly alduronic acid residues, e.g. hexalduronic acid residues, or most particularly G and / or M residues. In other words, preferably the alginate oligomer will not comprise other residues or units (e.g. other saccharide residues, or more particularly other uronic acid / uronate residues). The terminal residues of the alginate oligomer are uronate residues, more particularly alduronic acid residues, e.g. hexalduronic acid residues, or most particularly, G and / or M residues.

[0073] The alginate oligomer is preferably a linear oligomer.

[0074] In certain embodiments at least 30% of the monomer residues of the alginate oligomer are G residues (i.e. guluronate or guluronic acid). Specific embodiments thus include alginate oligomers with (e.g. containing) 30 to 70% G (guluronate) residues or 70 to 100% G (guluronate) residues. Thus, a representative alginate oligomer for use according to the present invention may contain at least 70% G residues (i.e. at least 70% of the monomer residues of the alginate oligomer will be G residues). Specific embodiments thus include alginate oligomers with (e.g. containing) 70 to 100% G (guluronate) residues.

[0075] In certain embodiments, at least 40%, 45%, 50%, 55% or 60%, more particularly at least 75% or 80%, more particularly at least 85% or 90%, even more particularly at least 91 , 92, 93, 94, 95, 96, 97, 98 or 99% of the monomer residues are guluronate. In one embodiment the alginate oligomer may be an oligoguluronate (i.e. a homooligomer of G, or 100% G).

[0076] In a further preferred embodiment, the above described alginates of the invention have a primary structure wherein the majority of the G residues are in so called G-blocks. Preferably at least 50%, more preferably at least 70 or 75%, and most preferably at least 80, 85, 90, 92 or 95% of the G residues are in G-blocks. A G block is a contiguous sequence of at least two G residues, preferably at least 3 contiguous G residues, more preferably at least 4 or 5 contiguous G residues, most preferably at least 7 contiguous G residues.

[0077] In particular, at least 90% of the G residues are linked 1-4 to another G residue. More particularly at least 95%, more preferably at least 98%, and most preferably at least 99% of the G residues of the alginate are linked 1-4 to another G residue.

[0078] More specifically at least 70% of the monomer residues in the oligomer are G residues linked 1-4 to another G-residue, or more preferably at least 75%, and most preferably at least 80, 85, 90, 92, 93, 94, 95, 96, 97, 98, 99% of the monomers residues of the oligomer are G residues linked 1-4 to another G residue. This 1-4 linkage of two G residues can be alternatively expressed as a guluronic unit bound to an adjacent guluronic unit.

[0079] Such alginate oligomers of use in the invention are commonly referred to by the skilled person as "high G" or "G-block" oligomers i.e. having a high content of G residues or G- blocks (e.g. wherein at least 70% of the monomer residues are G, preferably arranged in G- blocks).

[0080] The alginate oligomer of use in the invention is preferably a 3- to 35-mer, more preferably a 3- to 28-mer, in particular a 4- to 25-mer, e.g. a 5- to 20-mer, especially a 6- to 22-mer, in particular an 8- to 20-mer, especially a 10- to 15-mer, e.g. having a molecular weight in the range 350 to 6400 Daltons or 350 to 6000 Daltons, preferably 550 to 5500 Daltons, preferably 750 to 5000 Daltons, and especially 750 to 4500 Daltons or 2000 to 3000 Daltons or 900 to 3500 Daltons. Other representative alginate oligomers include, as mentioned above, oligomers with 5, 6, 7, 8, 9, 10, 11 , 12 or 13 to 50, 45, 40, 35, 28, 25, 22 or 20 residues.

[0081] The alginate oligomer of the invention may have a number of residues, a degree of polymerisation (DP), or a number average degree of polymerisation (DPn), of 3-28, 4-25, 6- 22, 8-20 or 10-15, or 5-18 or 7-15 or 8-12, especially 10.

[0082] The alginate oligomer of the invention may have a number of residues, a degree of polymerisation (DP), or a number average degree of polymerisation (DPn), of 3-24, 4-23, 5-

[0083] 22, 6-21 , 7-20, 8-19, 9-18, 10-17, 11-16, 12-15 or 13-14 (e.g. 13 or 14).

[0084] The alginate oligomer of the invention may have a number of residues, a degree of polymerisation (DP), or a number average degree of polymerisation (DPn), of 4-25, 5-24, 6-

[0085] 23, 7-22, 8-21 , 9-20, 10-19, 11-18, 12-17, 13-16, 14-15 (e.g. 14 or 15).

[0086] The alginate oligomer of the invention may have a number of residues, a degree of polymerisation (DP), or a number average degree of polymerisation (DPn), of 5-26, 6-25, 7-

[0087] 24, 8-23, 9-22, 10-21 , 11-20, 12-19, 13-18, 14-17 or 15-16 (e.g. 15 or 16).

[0088] The alginate oligomer of the invention may have a number of residues, a degree of polymerisation (DP), or a number average degree of polymerisation (DPn), of 4-50, 4-40, 4- 35, 4-30, 4-28, 4-26, 4-22, 4-20, 4-18, 4-16 or 4-14.

[0089] The alginate oligomer of the invention may have a number of residues, a degree of polymerisation (DP), or a number average degree of polymerisation (DPn), of 5-50, 5-40, 5-

[0090] 25, 5-22, 5-20, 5-18, 5-23, 5-20, 5-18, 5-16 or 5-14.

[0091] The alginate oligomer of the invention may have a number of residues, a degree of polymerisation (DP), or a number average degree of polymerisation (DPn), of 6-50, 6-40, 6- 35, 6-30, 6-28, 6-26, 6-24, 6-20, 6-19, 6-18, 6-16 or 6-14. The alginate oligomer of the invention may have a number of residues, a degree of polymerisation (DP), or a number average degree of polymerisation (DPn), of 8-50, 8-40, 8- 35, 8-30, 8-28, 8-25, 8-22, 8-20, 8-18, 8-16 or 8-14.

[0092] The alginate oligomer of the invention may have a number of residues, a degree of polymerisation (DP), or a number average degree of polymerisation (DPn), of 9-50, 9-40, 9- 35, 9-30, 9-28, 9-25, 9-22, 9-20, 9-18, 9-16 or 9-14.

[0093] The alginate oligomer of the invention may have a number of residues, a degree of polymerisation (DP), or a number average degree of polymerisation (DPn), of 10-50, 10-40,

[0094] 10-35, 10-30, 10-28, 10-25, 10-22, 10-20, 10-18, 10-16 or 10-14.

[0095] The alginate oligomer of the invention may have a number of residues, a degree of polymerisation (DP), or a number average degree of polymerisation (DPn), of 11-50, 11-40,

[0096] 11-35, 11-30, 11-28, 11-25, 11-22, 11-20, 11-18, 11-16 or 11-14.

[0097] The alginate oligomer of the invention may have a number of residues, a degree of polymerisation (DP), or a number average degree of polymerisation (DPn), of 12-50, 12-40,

[0098] 12-35, 12-30, 12-28, 12-25, 12-22, 12-20, 12-18, 12-16 or 12-14.

[0099] The alginate oligomer of the invention may have a number of residues, a degree of polymerisation (DP), or a number average degree of polymerisation (DPn), of 13-50, 13-40,

[0100] 13-35, 13-30, 13-28, 13-25, 13-22, 13-20, 13-18, 13-16 or 13-14 (e.g. 13 or 14).

[0101] The alginate oligomer of the invention may have a number of residues, a degree of polymerisation (DP), or a number average degree of polymerisation (DPn), of 14-50, 14-40,

[0102] 14-35, 14-30, 14-28, 14-25, 14-22, 14-20, 14-18, or 14-16.

[0103] The alginate oligomer of the invention may have a number of residues, a degree of polymerisation (DP), or a number average degree of polymerisation (DPn), of 15-50, 15-40,

[0104] 15-35, 15-30, 15-28, 15-25, 15-22, 15-20, or 15-18.

[0105] The alginate oligomer of the invention may have a number of residues, a degree of polymerisation (DP), or a number average degree of polymerisation (DPn), of 18-50, 18-40, 18-35, 18-30, 18-28, 18-25, 18-22 or 18-20. The alginate oligomer of the invention may have a number of residues, a degree of polymerisation (DP), or a number average degree of polymerisation (DPn), of 20-100, 20-90, 20-80, 20-75, 20-70, 20-65, 20-60, 20-55, 20-50, 20-45, 20-40, 20-35, 20-30 or 20-25.

[0106] The alginate oligomer of the invention may have a number of residues, a degree of polymerisation (DP), or a number average degree of polymerisation (DPn), of 30-100, 30-90, 30-80, 30-75, 30-70, 30-65, 30-60, 30-55, 30-50, 30-45, 30-40 or 30-35.

[0107] Preferably the alginate oligomer of use in creating the conjugates of the invention is substantially free, preferably essentially free, of alginate oligomers having a degree of polymerisation outside of the ranges disclosed herein. This may be expressed in terms of the molecular weight distribution of the alginate oligomer of use in creating the conjugates of the invention, e.g. the percentage of each mole of the alginate oligomer being used to create the conjugates of the invention which has a DP outside the relevant range. The molecular weight distribution is preferably such that no more than 10%, preferably no more than 9, 8, 7, 6, 5, 4, 3, 2, or 1 % mole has a DP of three, two or one higher than the relevant upper limit for DPn. Likewise, it is preferred that no more than 10%, preferably no more than 9, 8, 7, 6, 5, 4, 3, 2, or 1 % mole has a DP below a number three, two or one smaller than the relevant lower limit for DPn.

[0108] Suitable alginate oligomers are described in W02007 / 039754, W02007 / 039760, WO 2008 / 125828, and W02009 / 068841 , the disclosures of which are explicitly incorporated by reference herein in their entirety.

[0109] Representative suitable alginate oligomers have a DPnin the range 5 to 30, a guluronate fraction (FG) of at least 0.80, a mannuronate fraction (FM) of no more than 0.20, and at least 95 mole% of DP no more than 25.

[0110] Further suitable alginate oligomers have a number average degree of polymerization in the range 7 to 15 (preferably 8 to 12), a guluronate fraction (FG) of at least 0.85 (preferably at least 0.90), a mannuronate fraction (FM) of no more than 0.15 (preferably no more than 0.10), and having at least 95% mole with a degree of polymerization less than 17 (preferably less than 14).

[0111] Further suitable alginate oligomers have a number average degree of polymerization in the range 5 to 18 (especially 7 to 15), a guluronate fraction (FG) of at least 0.80 (preferably at least 0.85, especially at least 0.92), a mannuronate fraction (FM) of no more than 0.20 (preferably no more than 0.15, especially no more than 0.08), and having at least 95% mole with a degree of polymerization less than 20 (preferably less than 17).

[0112] Further suitable alginate oligomers have a number average degree of polymerization in the range 5 to 18, a guluronate fraction (FG) of at least 0.92, a mannuronate fraction (FM) of no more than 0.08, and having at least 95% mole with a degree of polymerization less than 20.

[0113] Further suitable alginate oligomers have a number average degree of polymerization in the range 5 to 18 (preferably 7 to 15, more preferably 8 to 12, especially about 10), a guluronate fraction (FG) of at least 0.80 (preferably at least 0.85, more preferably at least 0.90, especially at least 0.92, most especially at least 0.95), a mannuronate fraction (FM) of no more than 0.20 (preferably no more than 0.15, more preferably no more than 0.10, especially no more than 0.08, most especially no more than 0.05), and having at least 95% mole with a degree of polymerization less than 20 (preferably less than 17, more preferably less than 14).

[0114] Further suitable alginate oligomers have a number average degree of polymerization in the range 7 to 15 (preferably 8 to 12), a guluronate fraction (FG) of at least 0.92 (preferably at least 0.95), a mannuronate fraction (FM) of no more than 0.08 (preferably no more than 0.05), and having at least 95% mole with a degree of polymerization less than 17 (preferably less than 14).

[0115] Further suitable alginate oligomers have a number average degree of polymerization in the range 5 to 18, a guluronate fraction (FG) of at least 0.80, a mannuronate fraction (FM) of no more than 0.20, and having at least 95% mole with a degree of polymerization less than 20.

[0116] Further suitable alginate oligomers have a number average degree of polymerization in the range 7 to 15, a guluronate fraction (FG) of at least 0.85, a mannuronate fraction (FM) of no more than 0.15, and having at least 95% mole with a degree of polymerization less than 17.

[0117] Further suitable alginate oligomers have a number average degree of polymerization in the range 7 to 15, a guluronate fraction (FG) of at least 0.92, a mannuronate fraction (FM) of no more than 0.08, and having at least 95% mole with a degree of polymerization less than 17. Further suitable alginate oligomers have a number average degree of polymerization in the range 5 to 20, a guluronate fraction (FG) of at least 0.85 and a mannuronate fraction (FM) of no more than 0.15.

[0118] Further suitable alginate oligomers have a number average degree of polymerization in the range 5 to 20, a guluronate fraction (FG) of 0.9-0.95 and a mannuronate fraction (FM) of 0.05- 0.1 , which may be expressed as an alginate oligomer having 90-95% G residues and an average molecular weight of 2600 Da.

[0119] Further suitable alginate oligomers have a number average degree of polymerization about 13 (e.g. 12, 13 or 14), a guluronate fraction (FG) of at least about 0.80, 0.85, 0.87, 0.88, 0.90 or 0.93 (e.g. 0.92, 0.93 or 0.94) and a corresponding mannuronate fraction (FM) of no more than about 0.20, 0.15, 0.13, 0.12, 0.10, or 0.07 (e.g. 0.08, 0.07 or 0.06).

[0120] Further suitable alginate oligomers have a number average degree of polymerization about 21 (e.g. 20, 21 or 22), a guluronate fraction (FG) of at least about 0.80 (e.g. 0.85, 0.87, 0.88, 0.90, 0.92, 0.94 or 0.95) and a corresponding mannuronate fraction (FM) of no more than about 0.20 (e.g. 0.15, 0.13, 0.12, 0.10, 0.08, 0.06, 0.05).

[0121] Further suitable alginate oligomers have a number average degree of polymerization about 6 (e.g. 5, 6 or 7), a guluronate fraction (FG) of at least about 0.80 (e.g. 0.85, 0.87, 0.88, 0.90, 0.92, 0.94 or 0.95) and a corresponding mannuronate fraction (FM) of no more than about 0.20 (e.g. 0.15, 0.13, 0.12, 0.10, 0.08, 0.06, 0.05).

[0122] It will thus be seen that a particular class of alginate oligomers favoured according to the present invention is alginate oligomers defined as so-called "high G" or "G-block" oligomers i.e. having a high content of G residues or G-blocks (e.g. wherein at least 70% of the monomer residues are G, preferably arranged in G-blocks). However, other types of alginate oligomer may also be used, including in particular "high M" or "M-block" oligomers or MG- block oligomers, as described further below. Alginate oligomers with high proportions of a single monomer type, and with said monomers of this type being present predominantly in contiguous sequences of that monomer type, that represent oligomers that are particularly preferred.

[0123] In a further embodiment at least, or more particularly more than, 50% of the monomer residues of the alginate oligomer may be M residues (i.e. mannuronate or mannuronic acid). In other words the alginate oligomer will contain at least or alternatively more than 50% mannuronate (or mannuronic acid) residues. Specific embodiments thus include alginate oligomers with (e.g. containing) 50 to 70% M (mannuronate) residues or e.g. 70 to 100% M (mannuronate) residues. Further specific embodiments also include oligomers containing 71 to 85% M residues or 85 to 100% M residues. Thus, a representative alginate oligomer for use according to this embodiment of the present invention will contain more than 70% M residues (i.e. more than 70% of the monomer residues of the alginate oligomer will be M residues).

[0124] In other embodiments at least 50% or 60%, more particularly at least 70% or 75%, even more particularly at least 80, 85, 90, 95 or 99% of the monomer residues are mannuronate. In one embodiment the alginate oligomer may be an oligomannuronate (i.e. a homooligomer of M, or 100% M).

[0125] In a further embodiment, the above described alginates of use in the invention have a primary structure wherein the majority of the M residues are in so called M-blocks. In this embodiment preferably at least 50%, more preferably at least 70 or 75%, and most preferably at least 80, 85, 90 or 95% of the M residues are in M-blocks. An M block is a contiguous sequence of at least two M residues, preferably at least 3 contiguous M residues, more preferably at least 4 or 5 contiguous M residues, most preferably at least 7 contiguous M residues.

[0126] In particular, at least 90% of the M residues are linked 1-4 to another M residue. More particularly at least 95%, more preferably at least 98%, and most preferably at least 99% of the M residues of the alginate are linked 1-4 to another M residue.

[0127] Other preferred oligomers are alginate oligomers wherein at least 70% of the monomer residues in the oligomer are M residues linked 1-4 to another M-residue, or more preferably at least 75%, and most preferably at least 80, 85, 90, 92, 93, 94, 95, 96, 97, 98, 99% of the monomers residues of the oligomer are M residues linked 1-4 to another M residue. This 1-4 linkage of two M residues can be alternatively expressed as a mannuronic unit bound to an adjacent mannuronic unit.

[0128] In a still further embodiment, the alginate oligomers of use in the invention comprise a sequence of alternating M and G residues. A sequence of at least three, preferably at least four, alternating M and G residues represents an MG block. Preferably the alginate oligomers of the invention comprise an MG block. Expressed more specifically, an MG block is a sequence of at least three contiguous residues consisting of G and M residues and wherein each non-terminal (internal) G residue in the contiguous sequence is linked 1-4 and 4-1 to an M residue and each non-terminal (internal) M residue in the contiguous sequence is linked 1-4 and 4-1 to a G residue. Preferably the MG block is at least 5 or 6 contiguous residues, more preferably at least 7 or 8 contiguous residues.

[0129] In a further embodiment the minority uronate in the alginate oligomer (i.e. mannuronate or guluronate) is found predominantly in MG blocks. In this embodiment preferably at least 50%, more preferably at least 70 or 75% and most preferably at least 80, 85, 90 or 95% of the minority uronate monomers in the MG block alginate oligomer are present in MG blocks. In another embodiment the alginate oligomer is arranged such that at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, e.g. 100% of the G and M residues in the oligomer are arranged in MG blocks.

[0130] Although at its broadest, the invention extends to embodiments wherein at least 1% but less than 100% of the monomer residues of the oligomer are G residues (i.e. guluronate or guluronic acid), more particularly, and as defined further below, at least 30% of the monomer residues are G residues. Thus, at its broadest the MG block containing alginate oligomer may contain at least 1%, but less than 100%, guluronate (or guluronic acid) residues, but generally the MG block containing alginate oligomer will contain at least 30% (or at least 35, 40 or 45% or 50% G) but less than 100% G. Specific embodiments thus include MG block containing alginate oligomers with (e.g. containing) 1 to 30% G (guluronate) residues, 30 to 70% G (guluronate) residues or 70 to 99% G (guluronate) residues. Thus, a representative MG block containing alginate oligomer for use according to the present invention may contain more than 30%, but less than 70%, G residues (i.e. more than 30%, but less than 70%, of the monomer residues of the MG block alginate oligomer will be G residues).

[0131] Preferably more than 30%, more particularly more than 35% or 40%, even more particularly more than 45, 50, 55, 60 or 65%, but in each case less than 70%, of the monomer residues of the MG block containing alginate oligomer are guluronate. Alternatively, less than 70%, more preferably less than 65% or 60%, even more preferably less than 55, 50, 45, 40 or 35%, but in each case more than 30% of the monomer residues of the MG block containing alginate oligomer are guluronate. Any range formed by any combination of these values may be chosen. Therefore for instance the MG block containing alginate oligomer can have e.g. between 35% and 65%, 40% and 60% or 45% and 55% G residues. In another embodiment the MG block containing alginate oligomer may have approximately equal amounts of G and M residues (e.g. ratios between 65% G / 35% M and 35% G / 65% M, for instance 60% G / 40% M and 40% G / 60% M; 55% G / 45% M and 45% G / 55% M; 53% G / 47% M and 47% G / 53% M; 51% G / 49% M and 49% G / 51% M; e.g. about 50% G and about 50% M) and these residues are arranged predominantly, preferably entirely or as completely as possible, in an alternating MG pattern (e.g. at least 50% or at least 60, 70, 80, 85, 90 or 95% or 100% of the M and G residues are in an alternating MG sequence).

[0132] In certain embodiments the terminal uronic acid residues of the oligomers of use in the invention do not have a double bond, especially a double bond situated between the C4 and C5 atom. Such oligomers may be described as having saturated terminal uronic acid residues. The skilled man would be able to prepare oligomers with saturated terminal uronic acid residues without undue burden. This may be through the use of production techniques which yield such oligomers, or by converting (saturating) oligomers produced by processes that yield oligomers with unsaturated terminal uronic acid residues.

[0133] The alginate oligomer will typically carry a charge and so counter ions for the alginate oligomer may be any physiologically tolerable ion, especially those commonly used for charged drug substances, e.g. sodium, potassium, ammonium, chloride, mesylate, meglumine, etc. Ions which promote alginate gelation e.g. group 2 metal ions may also be used. Others may include the salt-forming ions mentioned below.

[0134] In certain embodiments the structure of the alginate oligomer component of the conjugates of the invention, e.g. its number of residues and / or G and M composition and / or arrangement thereof, is selected to achieve predetermined functional criteria in the conjugate, for instance solubility, toxicity and / or efficacy.

[0135] While the alginate oligomer may be a synthetic material generated from the polymerisation of appropriate numbers of guluronate and mannuronate residues, the alginate oligomers of use in the invention may conveniently be obtained, produced or derived from natural sources such as those mentioned above, namely natural alginate source materials.

[0136] Polysaccharide to oligosaccharide cleavage to produce the alginate oligomer useable according to the present invention may be performed using conventional polysaccharide lysis techniques such as enzymatic digestion and acid hydrolysis. In one favoured embodiment acid hydrolysis is used to prepare the alginate oligomers on the invention. In other embodiments enzymatic digestion is used with an additional processing step(s) to saturate the terminal uronic acids in the oligomers.

[0137] Oligomers may then be separated from the polysaccharide breakdown products chromatographically using an ion exchange resin or by fractionated precipitation or solubilisation or filtration. US 6,121 ,441 and WO 2008 / 125828, which are explicitly incorporated by reference herein in their entirety, describe a process suitable for preparing the alginate oligomers of use in the invention. Further information and discussion can be found in for example in “Handbooks of Hydrocolloids”, Ed. Phillips and Williams, ORC, Boca Raton, Florida, USA, 2000, which textbook is explicitly incorporated by reference herein in its entirety.

[0138] The alginate oligomers may also be chemically modified, including but not limited to modification to add charged groups (such as carboxylated or carboxymethylated glycans) and alginate oligomers modified to alter flexibility (e.g. by periodate oxidation). However, in certain embodiments the alginate oligomer is not in an oxidised form, e.g. has not undergone periodate oxidation.

[0139] Alginate oligomers (for example oligoguluronic acids) suitable for use according to the invention may conveniently be produced by acid hydrolysis of alginic acid from, but not limited to, Laminaria hyperbora and Lessonia nigrescens, dissolution at neutral pH, addition of mineral acid reduce the pH to 3.4 to precipitate the alginate oligomer (oligoguluronic acid), washing with weak acid, resuspension at neutral pH and freeze drying.

[0140] The alginates for production of alginate oligomers of the invention can also be obtained directly from suitable bacterial sources e.g. Pseudomonas aeruginosa or Azotobacter vinelandii.

[0141] In embodiments where alginate oligomers which have primary structures in which the majority of the G residues are arranged in G-blocks rather than as single residues are required, algal sources are expected to be most suitable on account of the fact that the alginates produced in these organisms tend to have these structures. The bacterial sources may be more suitable for obtaining alginate oligomers of different structures. The molecular apparatus involved in alginate biosynthesis in Pseudomonas fluorescens and Azotobacter vinelandii has been cloned and characterised (WO 94 / 09124; Ertesvag, H., et al, Metabolic Engineering, 1999, Vol 1 , 262-269; WO 2004 / 011628; Gimmestad, M., et al (supra)-, Remminghorst and Rehm, Biotechnology Letters, 2006, Vol 28, 1701-1712; Gimmestad, M. et al, Journal of Bacteriology, 2006, Vol 188(15), 5551-5560) and alginates of tailored primary structures can be readily obtained by manipulating these systems.

[0142] The G content of alginates (for example an algal source material) can be increased by epimerisation, for example with mannuronan C-5 epimerases from A. vinelandii or other epimerase enzymes. Thus, for example in vitro epimerisation may be carried out with isolated epimerases from Pseudomonas or Azotobacter, e.g. AlgG from Pseudomonas fluorescens or Azotobacter vinelandii or the AlgE enzymes (AlgE1 to AlgE7) from Azotobacter vinelandii. The use of epimerases from other organisms that have the capability of producing alginate, particularly algae, is also specifically contemplated. The in vitro epimerisation of low G alginates with Azotobacter vinelandii AlgE epimerases is described in detail in Ertesvag et al (supra) and Strugala et al (Gums and Stabilisers for the Food Industry, 2004, 12, The Royal Society of Chemistry, 84 - 94).

[0143] To obtain G-block containing alginates or alginate oligomers, epimerisation with one or more Azotobacter vinelandii AlgE epimerases other than AlgE4 is preferred as these enzymes are capable of producing G block structures. On the other hand AlgE4 epimerase can be used to create alginates or alginate oligomers with alternating stretches of M / G sequence or primary structures containing single G residue as it has been found that this enzyme seems preferentially to epimerise individual M residues so as to produce single G residues linked to M residues rather than producing G blocks. Particular primary structures can be obtained by using different combinations of these enzymes.

[0144] Mutated versions of these enzymes or homologues from other organisms are also specifically contemplated as of use. WO 94 / 09124 describes recombinant or modified mannuronan C-5 epimerase enzymes (AlgE enzymes) for example encoded by epimerase sequences in which the DNA sequences encoding the different domains or modules of the epimerases have been shuffled or deleted and recombined. Alternatively, mutants of naturally occurring epimerase enzymes, (AlgG or AlgE) may be used, obtained for example by site directed or random mutagenesis of the AlgG or AlgE genes. A different approach is to create Pseudomonas and Azotobacter organisms that are mutated in some or all of their epimerase genes in such a way that those mutants produce alginates of the required structure for subsequent alginate oligomer production, or even alginate oligomers of the required structure and size (or molecular weight). The generation of a number of Pseudomonas fluorescens organisms with mutated AlgG genes is described in detail in WO 2004 / 011628 and Gimmestad, M., et al, 2003 (supra). The generation of a number of Azotobacter vinelandii organisms with mutated AlgE genes is disclosed in Gimmestad, M., et al, 2006 (supra).

[0145] A further approach is to delete or inactivate the endogenous epimerase genes from an Azotobacter or a Pseudomonas organism and then to introduce one or more exogenous epimerase genes, which may or may not be mutated (i.e. may be wild-type or modified) and the expression of which may be controlled, for example by the use of inducible or other "controllable promoters". By selecting appropriate combinations of genes, alginates of predetermined primary structure can be produced.

[0146] A still further approach would be to introduce some or all of the alginate biosynthesis machinery of Pseudomonas and / or Azotobacter into a non-alginate producing organism (e.g. E. coli) and to induce the production of alginate from these genetically modified organisms.

[0147] When these culture-based systems are used, the primary structure of the alginate or alginate oligomer products can be influenced by the culture conditions. It is well within the capabilities of the skilled man to adjust culture parameters such as temperature, osmolarity, nutrient levels / sources and atmospheric parameters in order to manipulate the primary structure of the alginates produced by a particular organism.

[0148] References to "G residues / G" and "M residues / M" or to guluronic acid or mannuronic acid, or guluronate or mannuronate are to be read interchangeably as references to guluronic acid / guluronate and mannuronic acid / mannuronate (specifically a-L-guluronic acid / guluronate and p-D-mannuronic acid / mannuronate), and further include derivatives thereof in which one or more available side chains or groups have been modified without resulting in a capacity to reduce the host toxicity and / or increase the aqueous solubility of the polyene antimycotic to which they are conjugated without significantly reducing their antifungal efficacy or spectrum of activity. Common saccharide modifying groups would include acetyl, sulphate, amino, deoxy, alcohol, aldehyde, ketone, ester and anhydro groups. The alginate oligomers may also be chemically modified to add charged groups (such as carboxylated or carboxymethylated glycans), and to alter flexibility (e.g. by periodate oxidation). However, in certain embodiments the alginate oligomer is not in an oxidised form, e.g. has not undergone periodate oxidation. The skilled person would be aware of still further chemical modifications that can be made to the monosaccharide subunits of oligosaccharides and these can be applied to the alginate oligomers of use in the invention. One or more of the side groups on the terminal uronic acid residues may be modified to permit the formation of a particular covalent bond between the alginate oligomer and the antimycotic polyene, or between the alginate oligomer and the covalent molecular linker.

[0149] In certain embodiments one or both of the terminal uronic acid residues in the alginate oligomer is an alduronic acid. In certain embodiments one or both of the terminal uronic acid residues in the alginate oligomer is a hexuronic acid, e.g. a hexalduronic acid. In certain embodiments one or both of the terminal uronic acid residues in the alginate oligomer is independently a guluronate residue or a mannuronate residue.

[0150] In certain embodiments the terminal uronic acid residue covalently conjugated to the antimycotic polyene is an alduronic acid, e.g. a hexalduronic acid, and is at the reducing end of the alginate molecule. The structure of this residue exists between a cyclic form with an ether bridge and an open chain form with a carbonyl group, as exemplified below:

[0151] ...MMG type In the above discussion of the alginate part of the conjugates of the invention all references to chemical structure extend to any stereoisomeric (e.g. enantiomeric or diastereoisomeric) or tautomeric forms thereof.

[0152] In the above discussion of the alginate part of the conjugates of the invention all references extend pharmaceutically acceptable salts (e.g. those disclosed below), solvates or hydrates thereof. Salts formed with monovalent cations, e.g. sodium and potassium may be of note.

[0153] In certain embodiments, the alginate oligomer will not be further covalently linked to the polyene antimycotic at a residue which is not a terminal residue of the molecule.

[0154] In certain embodiments, the alginate oligomer is covalently linked to the polyene antimycotic by one or other terminal residue, i.e. not both.

[0155] The direct covalent bond between the alginate oligomer and the antimycotic polyene is a covalent bond formed by an atom of one of the terminal uronic acid residues of the alginate oligomer and an atom of the antimycotic polyene. The atoms contributing to the bond may together or independently be carbon, oxygen, sulphur, nitrogen and / or phosphorous. Prior modification of the terminal uronic acid residue of the alginate oligomer and / or groups on the antimycotic polyene to include sulphur and / or phosphorus atoms / groups may be required and / or substitution of atoms in the terminal uronic acid residue of the alginate or the antimycotic polyene during bond formation may take place. The bond may be single, double or triple. In certain embodiments the bond is part of an organic functional group. The skilled person would be entirely familiar with the options available for suitable organic functional groups which could act as a link between a terminal uronic acid residue of the alginate oligomer and the antimycotic polyene. Non-limiting examples thereof may include ester, carbonate ester, orthoester, ketone, ketal, hemiketal, ketene, ether, acetal, hemiacteal, peroxy, methylenedioxy, carbamate, amide, amine, amine oxide, hydroxamic acid, hydroxylamine, imine, imide, imidate, nitrone, azine, azide, azo, oxime, carbodiimide, carbazone, hydrozone, dihydropyridazine, triazole, disulfide, sulfoxide, sulfonyl, thioamide, thioester, thioether, thioketone, thioketal, sulphonate ester, dithiocarbamate, semicarbazone, phosphine or phosphodiester functional groups. Amine, amide, ester, dihydropyridazine, pyridazine, isoxazolidine, and triazole (e.g. 1-4 triazole or 1-5 triazole) groups may be convenient and advantageous. In certain embodiments, the covalent bond may involve atoms from a carboxyl, amine or hydroxyl group on the antimycotic polyene and a hydroxyl, carboxyl, ketone or aldehyde group on the terminal uronic acid of the alginate oligomer. In certain embodiments the aldehyde group of the reducing terminal uronic acid residues may be covalently conjugated to an amine group on the polyene antifungal by reductive amination.

[0156] The covalent molecular linker may be any molecule or plurality of molecules, or part thereof, typically organic, which has a structure formed from covalently bonded atoms which is capable of bonding covalently with a terminal uronic acid residue of an alginate oligomer and a site on an antimycotic polyene. Within the conjugate there will be a continuous series of covalently bonded atoms from a terminal uronic acid residue of the alginate oligomer to the antimycotic polyene via the molecular linker. In preferred embodiments at least one of the covalent bonds in said series is as defined above. The molecular linker may however further comprise non-covalent, e.g. ionic bonds, in parts of the molecule which are not contributing to the covalent linkage between the antimycotic polyene and the alginate oligomer.

[0157] The covalent molecular linker may have a linear, circular and / or branched structure or combination thereof. In certain embodiments the molecular linker will have a molecular weight of equal to or less than 1500 Daltons, e.g. equal to or less than 1250, 1000, 900, 800, 700, 600, 500, 400, 300, 200 or 100 Daltons.

[0158] In certain embodiments at least one direct covalent bond between a terminal uronic acid residue of the alginate oligomer and the covalent molecular linker is as defined above. In certain embodiments at least one direct covalent bond between the antimycotic polyene and the covalent molecular linker is as defined above. Each bond may be the same or different. The covalent linker may also comprise at least one covalent bond, e.g. as defined above, preferably in the part of that molecule which contributes to the continuous series of covalently bonded atoms from a terminal uronic acid residue of the alginate oligomer to the antimycotic polyene via the molecular linker.

[0159] In certain embodiments one or more bonds selected from the bond between the antimycotic polyene and the linker, the bond between the terminal uronic acid of the alginate oligomer and the linker and a bond within the linker is within a functional group formed by a orthogonal bioconjugation technique, e.g. Cu-catalysed Azide-Alkyne Cycloaddition (CuAAC), the inverse electron-demand Diels-Alder reaction (iEDDA) (e.g. the Tetrazine Ligation reaction), Strain-Promoted Alkyne-Azide Cycloaddition (SPAAC), Strain Promoted Alkyne Nitrone cycloaddition (SPANC) and the Staudinger Ligation Reaction.

[0160] In Cu-catalyzed azide-alkyne cycloaddition reagents with terminal alkynes can be reacted with azide-bearing organic compounds or biomolecules (R-N3) to yield a stable triazole (e.g. 1-4 triazole or 1-5 triazole) linkage.

[0161] In the inverse electron-demand Diels-Alder reaction tetrazines (organic compounds composed of a six-membered aromatic ring with four nitrogen atoms) and suitable dienophiles react in an inverse electron demand hetero-Diels-Alder (ihDA)-retro-Diels-Alder (rDA) reaction cascade to form dihydropyridazines which can be further oxidised to the corresponding pyridazines. Suitable dienophiles include trans-bicyclo[6.1.0]nonene, transcyclooctene (TCO), methylcyclopropene, bicyclo[6.1.0]nonyne, cyclooctyne, norbornene, cyclopentene, and styrene.

[0162] The tetrazine ligation reaction is based on an inverse-demand Diels-Alder cycloaddition reaction between a trans-cyclooctene and tetrazine reaction pair, forming a dihydropyridazine / pyridazinne bond.

[0163] Strain-promoted alkyne-azide cycloaddition, also known as Cu-free click reaction, refers to the rapid cycloaddition between a strained cycloalkyne and an azide (R-N3) to yield a stable triazole linkage. Suitable cycloalkynes include cyclooctyne (OCT), difluorocyclo-octyne (DIFO), dibenzocyclooctyne (DIBO or DBCO) aza-dibenzocyclo-octyne (DIBAC), bicyclo[6.1.0]nonyne (BCN), aza-dimethoxycyclooctyne (DIMAC); biarylazacyclooctynone (BARAC), difluorobenzocyclooctyne (DIFBO), 3,3,6,6-tetramethyl-thiacycloheptyne (TMTH).

[0164] Strain promoted alkyne nitrone cycloaddition refers to the rapid cycloaddition between a strained cycloalkyne and an nitrone (RIR2C=NR3+O" where R3 is not H) to yield an isoxazolidine linkage. Suitable cycloalkynes include cyclooctyne (OCT), difluorocyclo-octyne (DIFO), dibenzocyclooctyne (DIBO or DBCO) aza-dibenzocyclo-octyne (DIBAC), bicyclo[6.1.0]nonyne (BCN), aza-dimethoxycyclooctyne (DIMAC); biarylazacyclooctynone (BARAC), difluorobenzocyclooctyne (DIFBO), 3,3,6,6-tetramethyl-thiacycloheptyne (TMTH).

[0165] The stepwise synthesis of amines starting with triphenylphosphine and azides is known as the Staudinger ligation reaction. In this process triphenylphosphine reacts with azides to form an intermediate iminophosphorane with the release of nitrogen gas. This intermediate quickly breaks down in aqueous environments to produce triphenylphosphine oxide and a primary amine.

[0166] Thus, in certain embodiments one or more bonds selected from the bond between the antimycotic polyene and the linker, the bond between the terminal uronic acid of the alginate oligomer and the linker and a bond within the linker is within a triazole group which is the reaction product of a terminal alkyne and an azide-bearing organic compound, or the reaction product of a strained cycloalkyne (OCT, DIFO, DIBO (DBCO), DIBAC, BCN, DIMAC, BARAC, DIFBO, TMTH) and an azide-bearing organic compound.

[0167] Thus, in certain embodiments one or more bonds selected from the bond between the antimycotic polyene and the linker, the bond between the terminal uronic acid of the alginate oligomer and the linker and a bond within the linker is within a dihydropyridazine or pyridazine group which is the reaction product of a tetrazine and a dienophile (TCO, trans- bicyclo[6.1.0]nonene, methylcyclopropene, BCN, OCT, norbornene, cyclopentene, and styrene).

[0168] Thus, in certain embodiments one or more bonds selected from the bond between the antimycotic polyene and the linker, the bond between the terminal uronic acid of the alginate oligomer and the linker and a bond within the linker is within an isoxazolidine group which is the reaction product of a nitrone and a strained cycloalkyne (OCT, DIFO, DIBO (DBCO), DIBAC, BCN, DIMAC, BARAC, DIFBO, TMTH).

[0169] Thus, in certain embodiments the linker may comprise a portion resulting from orthogonal bioconjugation, e.g. a triazole reaction product of a terminal alkyne and an azide-bearing organic compound, or a triazole reaction product of a strained cycloalkyne (OCT, DIFO, DIBO (DBCO), DIBAC, BCN, DIMAC, BARAC, DIFBO) and an azide-bearing organic compound, or a dihydropyridazine or pyridazine reaction product of a tetrazine and a dienophile (TCO, trans-bicyclo[6.1.0]nonene, methylcyclopropene, BCN, OCT, norbornene, cyclopentene, and styrene) or an isoxazolidine reaction product of a nitrone and a strained cycloalkyne (OCT, DIFO, DIBO (DBCO), DIBAC, BCN, DIMAC, BARAC, DIFBO, TMTH).

[0170] In preferred embodiments, the linker may comprise a triazole reaction product of a (OCT, DIFO, DIBO (DBCO), DIBAC, BCN, DIMAC, BARAC, DIFBO, TMTH) and an azide-bearing organic compound, more preferably a triazole reaction product of a DIBO (DBCO), and an azide-bearing organic compound. In these embodiments such portions may be directly covalently connected to a molecular group on one or other terminal uronic acid residues of an alginate oligomer (e.g. an aldehyde group), and / or a molecular group of an antimycotic polyene (e.g. a carboxyl or an amine group). In other embodiments such portions are directly covalently connected to a further linker portion (also termed “spacer” herein) or plurality thereof (e.g. those described below) and either a molecular group on one or other terminal uronic acid residues of an alginate oligomer (e.g. an aldehyde group), or a molecular group of an antimycotic polyene (e.g. a carboxyl or an amine group). In still further embodiments such portions are directly covalently connected to a further linker portion (also termed “spacer” herein) or plurality thereof (e.g. those described below) which in turn is covalently connected to either a molecular group on one or other terminal uronic acid residues of an alginate oligomer (e.g. an aldehyde group), or a molecular group of an antimycotic polyene (e.g. a carboxyl or an amine group). Those positions of such groups described above may be used.

[0171] In certain embodiments the covalent molecular linker may be or comprise as a portion thereof a straight chain, branched, cyclic or polycyclic, saturated or unsaturated, aliphatic (hydrocarbon) group or an aromatic group or combinations thereof, e.g. an alkyl, alkenyl, alkynyl, or aryl group. Typically, such linkers may be C2-30, e.g. C4-30, C6-30, Cs-3o, C10-30, C12-30, C14-30, C16-30, C18-30, C20-30, C22-30, C24-30, C26-30, C28-30, C3-29, C3-27, C3-25, C3-23, C3-21 , C3-19, C3-18, C3-17, C3-15, C3-13, C3-11 , C3-9, C3-7, C3-5, or any ranges which may be formed from the above recited integers. In certain embodiments the linker may be, or comprise as a portion thereof, side chain or heteroatom substituted forms of said hydrocarbon, alkyl, alkenyl, alkynyl, or aryl groups (e.g. phenyl and extended aromatic groups based thereon). Side chain substitutions include, hydroxyl, carbonyl, carboxyl, alkoxy, sulphate, phosphate, halo, amide and amine. The side chain substitutions are typically polar, e.g. hydroxyl, carbonyl, epoxide, to maintain aqueous solubility. Heteroatom substituted forms include oxygen, nitrogen, sulphur and phosphorous substitutions.

[0172] In certain embodiments the covalent molecular linker may be or comprise as a portion thereof a polyol group. Polyol groups include PEG (polyethylene glycol) or a polypropylene glycol. Typically such linkers may be C2-30, e.g. C4-30, C6-30, Cs-3o, C10-30, C12-30, C14-30, C16-30, C18-30, C20-30, C22-30, C24-30, C26-30, C28-30, C3-29, C3-27, C3-25, C3-23, C3-21 , C3-19, C3-18, C3-17, C3-15, C3-13, C3-11 , C3-9, C3-7, C3-5, or any ranges which may be formed from the above recited integers. In certain embodiments the covalent molecular linker may be or comprise as a portion thereof a PEG of Formula II, wherein n is an integer of 1 to 20, e.g. 1 to 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 to 20. Any ranges which may be formed from the above integers is expressly contemplated: (Formula II)

[0173] In certain embodiments the covalent molecular linker may be or comprise as a portion thereof an amino acid or a peptide, e.g. of equal to or fewer than 15 amino acid residues, e.g. of equal to or fewer than 12, 10, 8, 6, 5, 4, 3 or 2 amino acid residues. The amino acid may be and the peptide may comprise any natural or non-genetically encoded amino acids, e.g. alanine, glycine, proline, aspartate, glutamate, valine, leucine, isoleucine, threonine, phenylalanine, arginine, histidine, lysine, asparagine, glutamine, methionine; serine, cysteine, phenylalanine, tyrosine, tryptophan, homolysine, ornithine, diaminobutyric acid (e.g. a,y-diaminobutyric acid), diaminopimelic acid, diaminopropionic acid, homoarginine, trimethylysine, trimethylornithine, 4-aminopiperidine-4-carboxylic acid, 4-amino-1- carbamimidoylpiperidine-4-carboxylic acid and 4-guanidinophenylalanine. Specific examples of peptide linkers which may be used include but are not limited to peptides of Gly and / or Ser residues (e.g. (Gly)2-s, (Ser)2.8, (GGGGS)i-3); (EAAAK)i-3; A(EAAAK)I.3A; Leu-Glu; (Xaa- Pro)i-6(e.g. (Glu-Pro)i-6, (Lys-Pro)i-6, (Ala-Pro)i-6; VSQTSKLTRJ.AETVFPDV (Factor Xla / Factor Vila sensitive cleavage); PLGJ.LWA (matrix metalloprotease-1 sensitive cleavage); RVLJ.AEA (HIV-1 protease sensitive cleavage; EDVVCCJ.SMSY (NS3 protease sensitive cleavage; GGIEGRJ.GS (Factor Xa sensitive cleavage); TRHRQPRJ.GWE (furin sensitive cleavage); AGNRVRRJ.SVG (furin sensitive cleavage); GFLGJ. (Cathepsin B sensitive cleavage).

[0174] In certain embodiments, the covalent molecular linker may be or comprise as a portion thereof a monosaccharide or an oligosaccharide other than guluronate or mannuronate or a polymer formed therefrom, e.g. a saccharide of equal to or fewer than 12 monosaccharide residues, e.g. equal to or fewer than 10, 8, 6, 5, 4, 3 or 2 monosaccharide residues. Thus the covalent molecular linker may be a monosaccharide, disaccharide or trisaccharide or sugar derivatives thereof such as aldonic and uronic acids, deoxy or amino sugars, sulfated sugars, and sugar alcohols.

[0175] The monosaccharide or one or more of the monosaccharide residues of the disaccharide or trisaccharide may be a triose, a tetrose, a pentose, a hexose, a heptose, an octose, a nonose or a decose in pyranose or furanose form and / or L- or D- form where appropriate and / or sugar derivatives thereof. Pentose or hexose saccharides / residues are preferred, e.g. mannose (e.g. D-mannose), galactose (e.g. D- galactose), glucose (e.g. D-glucose), fructose, fucose (e.g. L-fucose), N-acetyl-glucosamine, N-acetylgalactosamine, rhamnose, galactosamine, glucosamine (e.g. D-glucosamine), galacturonic acid, glucuronic acid, N- acetylneuraminic acid, methyl D-mannopyranoside (mannoside), a-methyl-glucoside, galactoside, ribose, xylose, arabinose, saccharate, mannitol, sorbitol, inositol, glycerol and derivatives of these monomers. The disaccharide may be exemplified by acarviosin, allolactose, cellobiose, chitobiose, galactose-alpha-1 ,3-galactose, dentiobiose, isomalt, isomaltose, isomaltulose, kojibiose, lactitol, lactobionic acid, lactose, lactulose, laminaribiose, maltitol, maltose, mannobiose, melibiose, melibiulose, neohesperidose, nigerose, robinose, rutinose, sambubiose, sophorose, sucralfate, sucralose, sucrose, sucrose acetate isobutyrate, sucrose octaacetate, trehalose, truranose, xylobiose or derivatives of these disaccharides. In particular embodiments, the monosaccharide is not an amino monosaccharide, e.g. mycosamine or perosamine.

[0176] In certain embodiments, the covalent molecular linker may be or comprise as a portion thereof a nucleotide or an oligonucleotide, i.e. a nucleic acid, e.g. a ribonucleotide or a deoxyribonucleotide.

[0177] In certain embodiments, the covalent molecular linker may be or comprise as a portion thereof acetyl, succinyl, aconityl (cis or trans), glutaryl, adiptyl (hexanedioic), methylsuccinyl, trimellityl cysteamine, penicillamine, N-(2-mercaptopropionyl)glycine, 2-mercaptopropionic acid, homocysteine, 3-mercaptopropionic acid, aminohexanoic acid, and deaminopenicillamine groups.

[0178] In certain embodiments, the covalent molecular linker may be or comprise as a portion thereof an artificial polymeric molecule, e.g. a polyolefin, in particular polyethylene, polypropylene, polystyrene, polyvinyl chloride, poly lactic-co-glycolic acid (PLGA). In certain embodiments the covalent linker molecule may be a plurality of the molecules and / or groups described above.

[0179] In certain embodiments the direct covalent bond or the covalent linker molecule (more specifically a covalent bond within the linker molecule, a covalent bond between the linker molecule the alginate oligomer and / or a covalent bond between and the linker molecule and the antimycotic polyene) is selected for its ability to be lysed under conditions representative, or advantageously essentially unique to, a target site or location within a subject, e.g. conditions representative of a fungal infection, the respiratory tract (especially the lower respiratory tract including the lungs, more particularly the lungs of a patient with cystic fibrosis) or wounds (in particular chronic wounds). In this way delivery of the antimycotic polyene may be made more selective for the target site.

[0180] In specific embodiments a covalent bond, a functional group containing said covalent bond or linker molecule may be selected which is sensitive to (labile at, degrades at, lyses at) a pH which is lower than normal physiological pH (pH 7.2), i.e. acidic pH, e.g. a pH of from about 3 to about 7, 6.5, 6, 5.5, 5, 4.5, 4, or 3.5. Sites or locations of inflammation, especially inflammation caused by infection typically have a pH in these ranges. Functional groups including esters, cis-aconityl, disulphides and hydrozones may be sensitive to lower pHs, i.e. may be described as acid labile.

[0181] In specific embodiments a covalent bond, functional group or linker molecule may be selected which is sensitive to reactive oxygen species. Sites or locations of inflammation, especially inflammation caused by infection typically have high levels of reactive oxygen species. Functional groups including thioketals and thioethers may be sensitive to reactive oxygen species

[0182] In further specific embodiments a covalent bond, functional group or linker molecule may be selected which is lysed by enzymes produced or secreted only at the target site or overproduced or oversecreted at the target site. This may include enzymes such as glycosidases, nucleases and peptidases, in particular those secreted by infecting bacteria and those secreted by inflammatory cells of the host, e.g. lysozyme, alginate lyase, DNasel, restriction endonucleases, neutrophil elastase, cathepsins, phospholipases and - lactamases. It may however be advantageous to choose a covalent bond, functional group or linker molecule which is not lysed by enzymes capable of degrading the alginate oligomer or the antimycotic polyene and as such separation of the alginate oligomer from the antimycotic polyene will occur separately to the degradation of the alginate oligomer or antimycotic polyene.

[0183] In other embodiments the direct covalent bond or the covalent linker molecule may be selected for its stability under conditions representative, of advantageously essentially unique to, a target site or location within a subject, e.g. the location described above or locations or sites the conjugate may encounter en route to those locations and sites following administration and / or which the conjugate may encounter during its bodily distribution. An amide bond, a thioether bond or a Gly-Gly peptide linker may be, for example, suitable here.

[0184] In these embodiments the above described further covalent linker portions may be directly covalently connected to a molecular group on one or other terminal uronic acid residues of an alginate oligomer (e.g. an aldehyde group, in particular via a resultant amine group), and / or a molecular group of an antimycotic polyene (e.g. a carboxyl or an amine group, in particular via a resultant amide group). In other embodiments the above described further covalent linker portions may be directly covalently connected to a portion resulting from orthogonal bioconjugation, (e.g. those described above) and either a molecular group on one or other terminal uronic acid residues of an alginate oligomer (e.g. an aldehyde group, in particular via a resultant amine group), or a molecular group of an antimycotic polyene (e.g. a carboxyl or an amine group, in particular via a resultant amide group). The references above to an amine group include hydroxylamine, and so the linkage between the one or other terminal uronic acid residues of an alginate oligomer and the other component may be a hydroxylamine linkage.

[0185] Thus, in certain embodiments the linker may be considered to have the formula E-F or F-E wherein the E is a portion resulting from orthogonal bioconjugation, e.g. those described above, and F is one or more of the above described further covalent linker portions. In certain embodiments E is directly, or via a spacer, covalently connected to a molecular group on one or other terminal uronic acid residues of an alginate oligomer (e.g. an aldehyde group, in particular via a resultant amine group), and F is directly, or via a spacer, covalently conjugated to a molecular group of an antimycotic polyene (e.g. a carboxyl or an amine group, in particular via a resultant amide group). In certain embodiments F is directly, or via a spacer, covalently connected to a molecular group on one or other terminal uronic acid residues of an alginate oligomer (e.g. an aldehyde group, in particular via a resultant amine group), and E is directly, or via a spacer, covalently conjugated to a molecular group of an antimycotic polyene (e.g. a carboxyl or an amine group, in particular via a resultant amide group). The references above to an amine group include hydroxylamine, and so the linkage between the one or other terminal uronic acid residues of an alginate oligomer and F or E or the spacer may be a hydroxylamine linkage.

[0186] In certain embodiments F is a PEG group or saturated or unsaturated aliphatic (hydrocarbon) chain or side chain or heteroatom substituted form thereof (e.g. as defined above) and E is a triazole reaction product of a strained cycloalkyne (OCT, DIFO, DIBO (DBCO), DIBAC, BCN, DIMAC, BARAC, DIFBO) and an azide-bearing organic compound, or a dihydropyridazine or pyridazine reaction product of a tetrazine and a dienophile (TCO, trans-bicyclo[6.1.0]nonene, methylcyclopropene, BCN, OCT, norbornene, cyclopentene, and styrene) or an isoxazolidine reaction product of a nitrone and a strained cycloalkyne (OCT, DIFO, DIBO (DBCO), DIBAC, BCN, DIMAC, BARAC, DIFBO). In preferred embodiments, E is a triazole reaction product of a (OCT, DIFO, DIBO (DBCO), DIBAC, BCN, DIMAC, BARAC, DIFBO) and an azide- bearing organic compound, more preferably a triazole reaction product of a DIBO (DBCO), and an azide-bearing organic compound.

[0187] The dash in E-F and F-E formulas above may or may not represent a direct linkage of the recited portions. In certain embodiments the dash at these points may be a covalent spacer.

[0188] In the above discussion, a spacer may be a straight or branched, unsaturated or unsaturated, hydrocarbon chain of the below n numbers, or heteroatom substituted forms thereof (e.g. as defined above), each optionally comprising one or more amide, amine, aldehyde, ketone, ester, or sulpho groups, or a further PEG group with an n number repeating units as recited below, optionally comprising one or more amide, amine, aldehyde, ketone, ester, or sulpho groups.

[0189] In certain embodiments, the invention provides a conjugate of a polyene antimycotic and an alginate oligomer having the following structures in which n is an integer of 1 to 25 , e.g. 1 to 25, 24, 23, 22, 21 , 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, or 24 to 25, and as applied to “PEG” represents the number of repeating units of Formula II in a PEG linker, the antimycotic polyene and the alginate oligomer are as defined above and the link to the alginate is via one or other of the terminal uronic acid residues of the alginate oligomer. Dashes in the following structures positioned between the references to “Amide” and “Amine” may or may not represent direct linkages of the groups recited and the term “between” should be construed accordingly. In certain embodiments the dash at these points may be a covalent spacer, e.g. a straight or branched, unsaturated or unsaturated, hydrocarbon chain of the above n numbers, or heteroatom substituted forms thereof (e.g. as defined above), each optionally comprising one or more amide, amine, aldehyde, ketone, ester, or sulpho groups, or a further PEG group with an n number repeating units as recited above, optionally comprising one or more amide, amine, aldehyde, ketone, ester, or sulpho groups. i. Antimycotic polyene-Amide-DBCO-triazole-PEGn-Amine-alginate oligomer. In certain embodiments the amide bond between the polyene and the DBCO is formed from a carboxyl group on the polyene ring, e.g. those described above, or from the amine group on the monosaccharide ii. Antimycotic polyene-Amide-PEGn-triazole-DBCO-Amine-alginate oligomer. In certain embodiments the amide bond between the polyene and the PEG is formed from a carboxyl group on the polyene ring, e.g. those described above, or from the amine group on the monosaccharide. iii. Antimycotic polyene-Amide-triazole-PEGn-Amine-alginate oligomer. In certain embodiments the amide bond between the polyene and the triazole is formed from a carboxyl group on the polyene ring, e.g. those described above, or from the amine group on the monosaccharide. In certain embodiments the triazole is a 1-4 or 1-5 triazole iv. Antimycotic polyene-Amide-PEGn-triazole-Amine-alginate oligomer. In certain embodiments the amide bond between the polyene and the PEGnis formed from a carboxyl group on the polyene ring, e.g. those described above, or from the amine group on the monosaccharide. In certain embodiments the triazole is a 1-4 or 1-5 triazole v. Antimycotic polyene-Amide-triazole-Cnalkyl-Amine-alginate oligomer. In certain embodiments the amide bond between the polyene and the triazole is formed from a carboxyl group on the polyene ring, e.g. those described above, or from the amine group on the monosaccharide. In certain embodiments the triazole is a 1-4 or 1-5 triazole vi. Antimycotic polyene-Amide-Cnalkyl-triazole -Amine-alginate oligomer. In certain embodiments the amide bond between the polyene and the Cnalkyl is formed from a carboxyl group on the polyene ring, e.g. those described above, or from the amine group on the monosaccharide. In certain embodiments the triazole is a 1-4 or 1-5 triazole vii. Antimycotic polyene-Amide-PEGn-Amine-alginate oligomer. In certain embodiments the amide bond between the polyene and the PEG is formed from a carboxyl group on the polyene ring, e.g. those described above, or from the amine group on the monosaccharide. viii. Antimycotic polyene-Amide-Cnalkyl-Amine-alginate oligomer. In certain embodiments the amide bond between the polyene and the Cnalkyl is formed from a carboxyl group on the polyene ring, e.g. those described above, or from the amine group on the monosaccharide. ix. Antimycotic polyene-Amide-phenyl-PEGn-Amine-alginate oligomer. In certain embodiments the amide bond between the polyene and the phenyl is formed from a carboxyl group on the polyene ring, e.g. those described above, or from the amine group on the monosaccharide. In certain embodiments the phenyl group is part of an extended aromatic ring system. x. Antimycotic polyene-Amide-PEGn-phenyl-Amine-alginate oligomer. In certain embodiments the amide bond between the polyene and the PEGnis formed from a carboxyl group on the polyene ring, e.g. those described above, or from the amine group on the monosaccharide. In certain embodiments the phenyl group is part of an extended aromatic ring system. xi. Antimycotic polyene-Amide-phenyl-Amine-alginate oligomer. In certain embodiments the amide bond between the polyene and the phenyl is formed from a carboxyl group on the polyene ring, e.g. those described above, or from the amine group on the monosaccharide. In certain embodiments the phenyl group is part of an extended aromatic ring system. xii. Antimycotic polyene-Amide-extended heteroaromatic system-PEGn-Amine-alginate oligomer. In certain embodiments the amide bond between the polyene and the extended heteroaromatic system is formed from a carboxyl group on the polyene ring, e.g. those described above, or from the amine group on the monosaccharide. xiii. Antimycotic polyene-Amide-PEGn-extended heteroaromatic system- Amine-alginate oligomer. In certain embodiments the amide bond between the polyene and the PEGnis formed from a carboxyl group on the polyene ring, e.g. those described above, or from the amine group on the monosaccharide. xiv. Antimycotic polyene-Amide-extended heteroaromatic system-Amine-alginate oligomer. In certain embodiments the amide bond between the polyene and the extended heteroaromatic system is formed from a carboxyl group on the polyene ring, e.g. those described above, or from the amine group on the monosaccharide. xv. Antimycotic polyene-Amide-TCO-dihydropyridazine(or pyridazine)-PEGn-Amine- alginate oligomer. In certain embodiments the amide bond between the polyene and the TCO is formed from a carboxyl group on the polyene ring, e.g. those described above, or from the amine group on the monosaccharide. xvi. Antimycotic polyene-Amide- PEGn-dihydropyridazine(or pyridazine)-TCO-Amine- alginate oligomer. In certain embodiments the amide bond between the polyene and the PEGn is formed from a carboxyl group on the polyene ring, e.g. those described above, or from the amine group on the monosaccharide. xvii. Antimycotic polyene-Amide-DBCO-isoxazolidine-PEGn-Amine-alginate oligomer. In certain embodiments the amide bond between the polyene and the DBCO is formed from a carboxyl group on the polyene ring, e.g. those described above, or from the amine group on the monosaccharide. xviii. Antimycotic polyene-Amide-PEGn-isoxazolidine-DBCO-PEGn-Amine-alginate oligomer. In certain embodiments the amide bond between the polyene and the PEGn is formed from a carboxyl group on the polyene ring, e.g. those described above, or from the amine group on the monosaccharide. xix. Antimycotic polyene-Amide-Spacer-DBCO-triazole-PEGn-Amine-alginate oligomer. In certain embodiments the amide bond between the polyene and the Spacer is formed from a carboxyl group on the polyene ring, e.g. those described above, or from the amine group on the monosaccharide. In these embodiments the amine group in the conjugate structures may be a group formed by the reductive amination of an aldehyde group and an aminoxy group, e.g. the group between the alginate oligomer and the PEG group in the specific structures below, i.e. -O- NH-, hydroxylamine.

[0190] In these embodiments the references to an alkyl group is interchangeable with a reference to a saturated hydrocarbon chain of length n, and references to an alkenyl group or an alkyny group are interchangeable with a reference to an unsaturated hydrocarbon chain comprising at least one 0=0 or 0=0 bond, respectively, of length n.

[0191] In certain embodiments, the invention provides a conjugate of a polyene antimycotic and an alginate oligomer having the following structures, wherein n is an integer of 1 to 40, e.g. 1 to 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16,

[0192] 15, 14, 13, 12, 11 , 10, 9, 8, 7, 6, 5, 4, 3 or 2, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15,

[0193] 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39 to 40, or 1 to 20, e.g. 1 to 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2, or 2, 3,

[0194] 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, or 19 to 20, and wherein Ri and R2may independently be any group other than H which does not interfere with formation of the molecule, e.g. methyl, ethyl, propyl, hydroxyl, methoxy, ethoxy or halo, and wherein A and Z are heteroatoms (e.g. N, P, S) and X-Y-B is a repeating linker unit of C and N, S, P or O, e.g. PEG, and Spacer may be a straight or branched, unsaturated or unsaturated, hydrocarbon chain of the above n numbers, or heteroatom substituted forms thereof (e.g. as defined above), each optionally comprising one or more amide, amine, aldehyde, ketone, ester, or sulpho groups, or a PEG group with an n number repeating units as recited above, optionally comprising one or more amide, amine, aldehyde, ketone, ester, or sulpho groups.

[0195]

[0196]

[0197] Further specifically contemplated are equivalents of these structures in which conjugation is instead or in addition via an amide bond formed from the amine group of the monosaccharide residue, for example.

[0198]

[0199] In certain embodiments, the invention provides a conjugate of a polyene antimycotic and an alginate oligomer having the following structures, wherein n is an integer of 1 to 20, e.g. 1 to 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, or 19 to 20, and Ri and R2 may independently be any group other than H which does not interfere with formation of the molecule, e.g. methyl, ethyl, propyl, hydroxyl, methoxy, ethoxy or halo.

[0200]

[0201] wherein A and Z are heteroatoms (e.g. N, O, P, S) and X-Y-B is a repeating linker unit of C and N, S, P or O, e.g. PEG

[0202]

[0203] Further specifically contemplated are equivalents of these structures in which conjugation is instead or in addition via an amide bond formed from the amine group of the monosaccharide residue, for example.

[0204] In these formulas the polyene antibiotic being represented is amphotericin B. Further specifically contemplated are equivalents of these structures in which the antimycotic polyene is any of those as defined above. In particular, the polyene part of the conjugates of the invention described above may be any stereoisomeric (e.g. enantiomeric or diastereoisomeric) or tautomeric form thereof which has antifungal / antimycotic effect as defined herein. In addition, in the above discussion of the polyene part of the conjugates of the invention all references extend pharmaceutically acceptable salts (e.g. those disclosed below), solvates or hydrates thereof.

[0205] In the above structures, the alginate oligomer portion is represented by a series of three G residues, but this is merely representative and, in practice, the alginate oligomer portion may be any described above, e.g. of any size and G and / or M content. Thus, the alginate oligomer portion could be represented by a series of M residues, or the following structures, adapted accordingly.

[0206] ...MMG type

[0207] The alginate oligomer portion of the above structures will preferably contain 2 to 100 monomer residues and / or the alginate oligomer may have at least 70% G residues. In particular, the alginate part of the conjugates of the invention described above may be any stereoisomeric (e.g. enantiomeric or diastereoisomeric) or tautomeric form thereof. In addition, in the above discussion of the alginate part of the conjugates of the invention all references extend pharmaceutically acceptable salts (e.g. those disclosed below), solvates or hydrates thereof.

[0208] Multivalent arrangements are contemplated in which more than one alginate oligomer is covalently linked to the antimycotic polyene. The alginate oligomers may be the same or different and a terminal uronic acid residue of each may be linked to the antimycotic polyene via the same type of covalent bond or covalent molecular linker, or different type of covalent bond or covalent molecular linker. In other embodiments the antimycotic polyene-alginate oligomer conjugate of the invention has no more than one alginate oligomer. References to the antimycotic polyene-alginate oligomer conjugates of the invention extends to pharmaceutically acceptable salts, solvates or hydrates thereof, diastereoisomers, tautomers, enantiomers, and active metabolites thereof. Suitable salts include acid addition salts from inorganic acids such as hydrochloric, sulphuric, phosphoric, nitric, carbonic, boric, sulfamic, and hydrobromic acids, or salts of pharmaceutically acceptable organic acids such as acetic, propionic, butyric, tartaric, maleic, hydroxymaleic, fumeric, citric, lactic, mucic, gluconic, benzoic, succinic, oxalic, phenylacetic, methanesulphonic, toluenesulphonic, benezenesulphonic, salicyclic, sulphanilic, aspartic, glutamic, edetic. stearic, palmitic, oleic, lauric, pantothenic, tannic, ascorbic, fendizoic, 4-4’-methylenebis-3-hydroxy-2-naphthoic acid, o-(p- hydroxybenzoyl)benzoic, 4-4’-dihydroxytriphenylmethane-2-carboxylic acid and valeric acids. Base salts include, but are not limited to, those formed with pharmaceutically acceptable cations, such as sodium, potassium, lithium, calcium, magnesium, ammonium and alkylammonium.

[0209] In a further aspect the invention provides an antimycotic polyene conjugates described by Formula III:

[0210] P-(L-[U])n(Formula III) wherein P- is a polyene antimycotic, L is a direct covalent bond or a covalent molecular linker, -[II] is a single guluronic acid residue or a single mannuronic acid residue, and n is an integer of 1 to 10, e.g. 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 2 or 1.

[0211] All specific embodiments described above and below apply mutatis mutandis insofar as the alginate oligomer is instead a single guluronic acid residue or a single mannuronic acid residue.

[0212] In a further aspect the invention provides a method for the preparation of an antimycotic polyene-alginate oligomer conjugate of the invention, said method comprising

[0213] (ia) providing an alginate oligomer and an antimycotic polyene and forming a direct covalent bond between a molecular group on the antimycotic polyene and a molecular group on one or other terminal uronic acid residue of the alginate oligomer; or

[0214] (ib) providing an alginate oligomer, an antimycotic polyene and a covalent molecular linker and forming a direct covalent bond between a molecular group on one or other terminal uronic acid residue of the alginate oligomer and a molecular group on the linker molecule and forming a direct covalent bond between a molecular group on the antimycotic polyene and a molecular group on the linker molecule; or

[0215] (ic) providing an alginate oligomer and an antimycotic polyene wherein one or both carry a covalent molecular linker molecule, or portion thereof, covalently bonded thereto, wherein if the alginate oligomer carries a linker molecule the linker molecule is covalently connected to one or other terminal uronic acid residue of the alginate oligomer, and covalently linking one or other terminal uronic acid residue of the alginate oligomer to the antimycotic polyene via at least one of the linker molecules or portions thereof; or

[0216] (id) providing an alginate oligomer and a first portion of a covalent molecular linker and forming a direct covalent bond between a molecular group on one or other terminal uronic acid residue of the alginate oligomer and a molecular group on the first portion of the linker molecule, and providing an antimycotic polyene and a second portion of the covalent molecular linker and forming a direct covalent bond between a molecular group on the antimycotic polyene and a molecular group on the second portion of the linker molecule, and forming a direct covalent bond between molecular groups on the first and second portions of the linker molecule; and optionally

[0217] (ii) separating at least a portion of the antimycotic polyene-alginate oligomer conjugate from the reaction mixture.

[0218] The bonds formed in the above method may be any of those described herein.

[0219] In a further embodiment the invention provides a method for the preparation of an antimycotic polyene-alginate oligomer conjugate of the invention, said method comprising

[0220] (i) providing an alginate oligomer and aminoxy-PEGn-Ns and forming an amine group (e.g. -O-NH- or hydroxylamine) from the aminooxy group and an aldehyde group on one or other terminal uronic acid residue of the alginate oligomer, e.g. by reductive amination;

[0221] (ii) providing an antimycotic polyene and an amine containing reagent selected from DCBO-amine, OCT-amine, DIFO-amine, DIBAC-amine, BCN-amine, DIMAC- amine, BARAC-amine, DlFBO-amine, or TMTH-amine, and forming an amide group from the amine group of said reagent and a carboxylic acid group on the amphipathic polyene cyclic carboxylic ester aliphatic ring, e.g. those carboxylic acid groups specified above; and (iii) forming a triazole group, e.g. by a strain-promoted alkyne-azide cycloaddition reaction, between the reaction products of (i) and (ii); and optionally

[0222] (iv) separating at least a portion of the antimycotic polyene-alginate oligomer conjugate from the reaction mixture, wherein steps (i) and (ii) may be performed in any order.

[0223] In a further embodiment the invention provides a method for the preparation of an antimycotic polyene-alginate oligomer conjugate of the invention, said method comprising

[0224] (i) providing an alginate oligomer and aminoxy-PEGn-Ns and forming an amine group (e.g. -O-NH- or hydroxylamine) from the aminooxy group and an aldehyde group on one or other terminal uronic acid residue of the alginate oligomer, e.g. by reductive amination;

[0225] (ii) providing an antimycotic polyene and a carboxylic acid containing reagent selected from DCBO-carboxylic acid, OCT-carboxylic acid, DIFO-carboxylic acid, DIBAC-carboxylic acid, BCN-carboxylic acid, DIMAC-carboxylic acid, BARAC- carboxylic acid, DlFBO-carboxylic acid, or TMTH-carboxylic acid, and forming an amide group from the carboxylic acid group of said reagent and an amine group on the monosaccharide of the antimycotic polyene, e.g. those amine groups specified above; and

[0226] (iii) forming a triazole linkage, e.g. by a strain-promoted alkyne-azide cycloaddition reaction, between the reaction products of (i) and (ii); and optionally

[0227] (iv) separating at least a portion of the antimycotic polyene-alginate oligomer conjugate from the reaction mixture, wherein steps (i) and (ii) may be performed in any order.

[0228] In a further embodiment the invention provides a method for the preparation of an antimycotic polyene-alginate oligomer conjugate of the invention, said method comprising

[0229] (i) providing an alginate oligomer and aminoxy-PEGn-tetrazine and forming an amine group (e.g. -O-NH- or hydroxylamine) from the aminooxy group and an aldehyde group on one or other terminal uronic acid residue of the alginate oligomer, e.g. by reductive amination;

[0230] (ii) providing an antimycotic polyene and an amine containing reagent selected from TCO-amine, trans-bicyclo[6.1.0]nonene-amine, methylcyclopropene-amine, bicyclo[6.1.0]nonyne-amine, cyclooctyne-amine, norbornene-amine, cyclopenteneamine, or styrene-amine, and forming an amide group from the amine group of said reagent and a carboxylic acid group on the amphipathic polyene cyclic carboxylic ester aliphatic ring, e.g. those carboxylic acid groups specified above; and

[0231] (iii) forming a dihydropyridazine or pyridazine group, e.g. by an inverse electrondemand Diels-Alder reaction, in particular a tetrazine ligation reaction, between the reaction products of (i) and (ii); and optionally

[0232] (iv) separating at least a portion of the antimycotic polyene-alginate oligomer conjugate from the reaction mixture, wherein steps (i) and (ii) may be performed in any order.

[0233] In a further embodiment the invention provides a method for the preparation of an antimycotic polyene-alginate oligomer conjugate of the invention, said method comprising

[0234] (i) providing an alginate oligomer and aminoxy-PEGn-tetrazine and forming an amine group (e.g. -O-NH- or hydroxylamine) from the aminooxy group and an aldehyde group on one or other terminal uronic acid residue of the alginate oligomer, e.g. by reductive amination;

[0235] (ii) providing an antimycotic polyene and a carboxylic acid containing reagent selected from TCO-carboxylic acid, trans-bicyclo[6.1.0]nonene-carboxylic acid, methylcyclopropene-carboxylic acid, bicyclo[6.1.0]nonyne-carboxylic acid, cyclooctyne-carboxylic acid, norbornene-carboxylic acid, cyclopentene-carboxylic acid, or styrene-carboxylic acid, and forming an amide group from the carboxylic acid group of said reagent and an amine group on the monosaccharide of the antimycotic polyene, e.g. those amine groups specified above; and

[0236] (iii) forming a dihydropyridazine or pyridazine group, e.g. by an inverse electrondemand Diels-Alder reaction, in partiuclar a tetrazine ligation reaction, between the reaction products of (i) and (ii); and optionally

[0237] (iv) separating at least a portion of the antimycotic polyene-alginate oligomer conjugate from the reaction mixture, wherein steps (i) and (ii) may be performed in any order.

[0238] In a further embodiment the invention provides a method for the preparation of an antimycotic polyene-alginate oligomer conjugate of the invention, said method comprising

[0239] (i) providing an alginate oligomer and aminoxy-PEGn-nitrone and forming an amine group (e.g. -O-NH- or hydroxylamine) from the aminooxy group and an aldehyde group on one or other terminal uronic acid residue of the alginate oligomer, e.g. by reductive amination; (ii) providing an antimycotic polyene and an amine containing reagent selected from DCBO-amine, OCT-amine, DIFO-amine, DIBAC-amine, BCN-amine, DIMAC- amine, BARAC-amine, DlFBO-amine, or TMTH-amine, and forming an amide group from the amine group of said reagent and a carboxylic acid group on the amphipathic polyene cyclic carboxylic ester aliphatic ring, e.g. those carboxylic acid groups specified above; and

[0240] (iii) forming a isoxazolidine linkage, e.g. by a strain promoted alkyne nitrone cycloaddition reaction, between the reaction products of (i) and (ii); and optionally

[0241] (iv) separating at least a portion of the antimycotic polyene-alginate oligomer conjugate from the reaction mixture, wherein steps (i) and (ii) may be performed in any order.

[0242] In a further embodiment the invention provides a method for the preparation of an antimycotic polyene-alginate oligomer conjugate of the invention, said method comprising

[0243] (i) providing an alginate oligomer and aminoxy-PEGn-nitrone and forming an amine group (e.g. -O-NH- or hydroxylamine) from the aminooxy group and an aldehyde group on one or other terminal uronic acid residue of the alginate oligomer, e.g. by reductive amination;

[0244] (ii) providing an antimycotic polyene and a carboxylic acid containing reagent selected from DCBO-carboxylic acid, OCT-carboxylic acid, DIFO-carboxylic acid, DIBAC-carboxylic acid, BCN-carboxylic acid, DIMAC-carboxylic acid, BARAC- carboxylic acid, DlFBO-carboxylic acid, or TMTH-carboxylic acid and forming an amide group from the carboxylic acid group of said reagent and an amine group on the monosaccharide of the antimycotic polyene, e.g. those amine groups specified above; and

[0245] (iii) forming a isoxazolidine linkage, e.g. by a strain promoted alkyne nitrone cycloaddition reaction, between the reaction products of (i) and (ii); and optionally

[0246] (iv) separating at least a portion of the antimycotic polyene-alginate oligomer conjugate from the reaction mixture, wherein steps (i) and (ii) may be performed in any order.

[0247] In a further embodiment the invention provides a method for the preparation of an antimycotic polyene-alginate oligomer conjugate of the invention, said method comprising

[0248] (i) providing an alginate oligomer and aminoxy-PEGn-Ns and forming an amine group (e.g. -O-NH- or hydroxylamine) from the aminooxy group and an aldehyde group on one or other terminal uronic acid residue of the alginate oligomer, e.g. by reductive amination;

[0249] (ii) providing an antimycotic polyene and a terminal alkyne-amine and forming an amide group from the amine group of said terminal alkyne-amine and a carboxylic acid group on the amphipathic polyene cyclic carboxylic ester aliphatic ring, e.g. those carboxylic acid groups specified above; and

[0250] (iii) forming a triazole linkage, e.g. by a Cu-catalysed azide-alkyne cycloaddition reaction, between the reaction products of (i) and (ii); and optionally

[0251] (iv) separating at least a portion of the antimycotic polyene-alginate oligomer conjugate from the reaction mixture, wherein steps (i) and (ii) may be performed in any order.

[0252] In a further embodiment the invention provides a method for the preparation of an antimycotic polyene-alginate oligomer conjugate of the invention, said method comprising

[0253] (i) providing an alginate oligomer and aminoxy-PEGn-Ns and forming an amine group (e.g. -O-NH- or hydroxyl amine) from the aminooxy group and an aldehyde group on one or other terminal uronic acid residue of the alginate oligomer, e.g. by reductive amination;

[0254] (ii) providing an antimycotic polyene and a terminal alkyne-carboxylic acid, and forming an amide group from the carboxylic acid group of said terminal alkynecarboxylic acid and an amine group on the monosaccharide of the antimycotic polyene, e.g. those amine groups specified above; and

[0255] (iii) forming a triazole linkage, e.g. by Cu-catalysed azide-alkyne cycloaddition reaction, between the reaction products of (i) and (ii); and optionally

[0256] (iv) separating at least a portion of the antimycotic polyene-alginate oligomer conjugate from the reaction mixture, wherein steps (i) and (ii) may be performed in any order.

[0257] In any of these methods the carboxylic acid containing reagents may be replaced by NHS (N-hydroxysuccinimide) ester containing reagents, or the terminal alkyne-carboxylic acid may be replaced by a terminal alkyne-NHS ester, and an amide is formed between said NHS groups and an amine group on the monosaccharide of the antimycotic polyene.

[0258] In any of these methods the amine containing reagent may be a sulpho amine containing reagent, and an amide is formed from the sulpho amine group and a carboxylic acid group on the amphipathic polyene cyclic carboxylic ester aliphatic ring. In any of these methods the carboxylic acid, amine, NHS ester and / or sulpho amine containing reagents may comprise a spacer, e.g. those described above (e.g. a straight or branched, unsaturated or unsaturated, hydrocarbon chain of the above n numbers, or heteroatom substituted forms thereof (e.g. as defined above), each optionally comprising one or more amide, amine, aldehyde, ketone, ester, or sulpho groups, or a PEG group with an n number repeating units as recited above, optionally comprising one or more amide, amine, aldehyde, ketone, ester, or sulpho groups), between the two reactive portions of the reagent.

[0259] In these methods, PEGnmay have Formula II as defined above.

[0260] In these methods, the antimycotic polyene-alginate oligomer conjugates so prepared may have no more than one alginate oligomer in the conjugate.

[0261] In further embodiments PEGn may be replaced by a straight chain C2-30 alkyl, C2-30 alkenyl or C2-30 alkynyl, e.g. C4-30, C6-30, Cs-3o, C10-30, C12-30, C14-30, C16-30, C18-30, C20-30, C22-30, C24-30, C26-30, C28-30, C3-29, C3-27, C3-25, C3-23, C3-21, C3-19, C3-18, C3-17, C3-15, C3-13, C3-11, C3-9, C3-7, C3-5, alkyl / alkenyl / alkynyl (i.e. a saturated or unsaturated hydrocarbon chain of said numbers of C atoms) or any ranges which may be formed from the above recited integers, or heteroatom substituted forms thereof (e.g. as defined above), each optionally comprising one or more amide, amine, aldehyde, ketone, ester, or sulpho groups.

[0262] As mentioned above, antimycotic polyene-alginate oligomer conjugates are a class of novel chemical entities having antimycotic efficacy (which may be at least essentially the same as, or similar to, or at least not significantly reduced as compared to, the antimycotic efficacy and / or the breadth of target susceptibility thereof of the non-conjugated form of the polyene), but with reduced host toxicity and / or increased aqueous solubility as compared to the nonconjugated form of the polyene antibiotic.

[0263] Antimycotic efficacy may for example be assessed on the basis of minimal inhibitory concentration (MIC) values (Jorgensen et al., Manual of Clinical Microbiology, 7th ed.

[0264] Washington, D.C: American Society for Microbiology, 1999; 1526-43), i.e. that concentration of anti-fungal agent that completely inhibits growth of that fungus. An antimycotic polyene- alginate oligomer conjugate may be judged as having essentially the same antimycotic efficacy as the unconjugated antimycotic polyene if the MIC value of the antimycotic polyene- alginate oligomer (e.g. as calculated based on the mass of the conjugate per se, or calculated based on the polyene content of the conjugate) is no greater than 2 times and no less than half the MIC of the unconjugated antimycotic polyene. An antimycotic polyenealginate oligomer conjugate may be judged as having a similar antimycotic efficacy to the unconjugated antimycotic polyene if the MIC value of the antimycotic polyene-alginate oligomer (e.g. as calculated based on the mass of the conjugate per se, or calculated based on the polyene content of the conjugate) is no greater than 4 times and no less than a quarter the MIC of the unconjugated antimycotic polyene. An antimycotic polyene-alginate oligomer with antimycotic efficacy which is not significantly reduced as compared to the unconjugated antimycotic polyene has a MIC which is no more than 10 times, e.g. no more than 9, 8, 7, 6, or 5 times that of the unconjugated antimycotic polyene. However, a significantly increased MIC, e.g. a MIC which is no more than 10 times, e.g. more than 12, 15, or 20 times that of the unconjugated antimycotic polyene may be tolerated by the skilled person (acceptable to the skilled person) if the conjugate has substantially reduced toxicity or substantially increased aqueous solubility as compared to the unconjugated antimycotic polyene. In some embodiments the antimycotic polyene-alginate oligomer conjugates of the invention may have a greater antimycotic efficacy as compared to the unconjugated antimycotic polyene. Such conjugates will have a MIC value (e.g. as calculated based on the mass of the conjugate per se, or calculated based on the polyene content of the conjugate) which is less than one quarter, e.g. one eighth or one sixteenth of that of the unconjugated antimycotic polyene.

[0265] An antimycotic polyene-alginate oligomer conjugate may be judged as having essentially the same breadth of target susceptibility, i.e. antifungal spectrum, as the unconjugated antimycotic polyene if the conjugate is effective (antifungal or fungistatic as appropriate) against the same strains of fungus. In certain embodiments the conjugate will be effective against at least 80%, e.g. at least 85%, 90%, 95% or 99% of the fungal strains against which the unconjugated antimycotic polyene is effective, preferably at equivalent concentration and duration of exposure.

[0266] The host cell toxicity of the antimycotic polyene-alginate oligomer conjugates of the invention and / or antimycotic polyenes may be assessed and compared by any convenient means for determining the viability of a host cell (i.e. a cell of the subject to be treated by the compound in question). Many routine assays are available to determine if is alive (viable) or dead. One option is to place the cell in conditions that would normally support the growth of that cell and monitor the growth of the cell by appropriate standard means, e.g. by monitoring the size of the cell, the morphology of the cell, the number of cells in the culture vessel over time, the consumption of nutrients in the culture media, etc.

[0267] Another option is to assess cells for morphologies characteristic of cell death, e.g. necrotic or apoptotic bodies, membrane blebs, nuclear condensation and cleavage of DNA into regularly sized fragments, ruptured cell walls or membranes and leakage of cell contents into the extracellular environment.

[0268] Other methods exploit the characteristic loss of cell membrane integrity in dead cells. Membrane impermeable dyes (e.g. trypan blue and propidium iodide) are routinely used to assess membrane integrity. These dyes are excluded from intact cells and so no staining occurs in such cells. If cell membrane integrity is compromised, these dyes can access the cells and stain intracellular components. Alternatively, or in addition, dyes that only stain cells with intact membranes are used to give an indication of the viability of the cell. The Live / Dead Assay of Invitrogen Ltd is an assay that uses two dyes, one to stain dead cells, the other to stain live cells. Another approach to assessing membrane integrity is to detect the release of cellular components into the culture media, e.g. lactate dehydrogenase.

[0269] A still further option is to measure the metabolism of cells. This can be done routinely in a number of ways. For instance, the levels of ATP can be measured. Only living cells with intact membranes can synthesis ATP and because ATP is not stored in cells, levels of ATP drop rapidly upon cell death. Monitoring ATP levels therefore gives an indication of the status of the cell. A yet further option is to measure the reducing potential of the cell. Viable cells metabolising nutrients use reducing reactions, by applying a marker that gives different outputs whether in reduced or oxidised form (e.g. a fluorescent dye) to the cell, the cell's reducing potential can be assessed. Cells that lack the ability to reduce the marker can be considered to be dead. The MTT and MTS assays are convenient examples of this type of assay. The Promega CellTiterGlo assay is a further option.

[0270] The type of cells in which toxicity may be assessed is not limited and the skilled person would be able to select appropriate cell types which reflect the physiological targets underlying the host toxicity and other side effects elicited by antimycotic polyenes. In certain embodiments, the cells may be liver cells or kidney cells. The cells may be cell lines derived from primary host cells, e.g. HepG2 cells (liver) and LLC-PK1 cells (kidney). Toxicity of the antimycotic polyene-alginate oligomer conjugates of the invention and / or antimycotic polyenes may be assessed and compared by measuring the haemolytic activity of the test compound. Whole blood or fractions thereof containing red blood cells (erythrocytes) may be used.

[0271] In accordance with certain embodiments of the invention, the antimycotic polyene-alginate oligomer conjugate will have less toxicity than the unconjugated antimycotic polyene, e.g. as measured in one or more of the above assays. This might, for instance, be determined by measuring and comparing the amount of test compound which causes a certain level of experimental endpoint (e.g. proportion of cell viability, cellular component release, cell lysis etc.). The level chosen will vary depending on the context in which the experiment is run and the results obtained, but the levels set may be 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70% or maximum experimental endpoint (cell viability, cellular component release, cell lysis etc.). In certain embodiments, the antimycotic polyene-alginate oligomer conjugate will have no greater than 90%, e.g. no greater than 80, 70, 60, 50, 40, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 ,1% of the toxicity than the unconjugated antimycotic polyene based on the comparative measurements taken (e.g. as calculated based on the mass of the conjugate per se, or calculated based on the polyene content of the conjugate).

[0272] The antimycotic polyene-alginate oligomer conjugates of the invention are water soluble. Water soluble conjugates of the invention can be considered to be conjugates for which less than about 1000 ml pure water are required to fully solubilise 1 g of the conjugate at 25 °C and atmospheric pressure (101325 Pa). In other embodiments less than about 500 ml, e.g. less than about 250, 100, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 ml pure water are required to solubilise 1 g of peptide.

[0273] Expressed differently, a solution consisting of the antimycotic polyene-alginate oligomer conjugates of the invention and pure water may be prepared at 25 °C and atmospheric pressure (101325 Pa) to a concentration of at least 0.1%, e.g. at least 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% or at least 60% w / v.

[0274] The antimycotic polyene-alginate oligomer conjugates of the invention may have greater aqueous solubility than the unconjugated antimycotic polyene. This may be confirmed by assessing how much of each may be fully solubilised in pure water at 25 °C and atmospheric pressure (101325 Pa). Antimycotic polyene-alginate oligomer conjugates of the invention may be at least 2x, 3x, 4x, 5x, 10x, 15x, 20x 25x or 30x as soluble in pure water at 25 °C and atmospheric pressure as the unconjugated antimycotic polyene.

[0275] “% w / v” (or “percentage weight by volume”) is a commonly used expression of the amount of a certain compound or material in a liquid mixture, e.g. solution. 1% w / v equates to 1 gram of the compound or material per 100ml of liquid, 2% w / v equates to 2g of the compound or material per 100ml of liquid, and so on. Accordingly, % w / v may be expressed as g / 100ml, grams per 100 ml and g 100ml'1. 1% w / v also equates to 10 gram of the compound or material per litre of liquid. The skilled man would understand that through appropriate scaling calculations, % w / v can be expressed in terms of any SI unit of mass.

[0276] “% w / w” (or “percentage weight by weight”) is a commonly used expression of the amount of a particular compound or material in a composition, typically a solid composition. 1% w / w equates to 1 gram of the compound or material per 100g of composition, 2% w / w equates to 2g of the compound or material per 100g of compound, and so on. Accordingly, % w / w may be expressed as g / 100g, grams per 100g and g 100g'1. 1% w / w also equates to 10 gram of the compound or material per kilogram of solid. The skilled man would understand that through appropriate scaling calculations, % w / w can be expressed in terms of any SI unit of mass.

[0277] This combination of properties makes the conjugates of the invention advantageous in the treatment (particularly in the systemic treatment) of fungal infections compared both to polyene antimycotics per se, in particular via oral or intravenous routes.

[0278] Thus, in a further aspect the invention provides a pharmaceutical composition comprising an antimycotic polyene-alginate oligomer conjugate as defined herein and a pharmaceutically acceptable excipient, carrier or diluent. Suitable excipients, carriers or diluents are described and specific pharmaceutical compositions are detailed below.

[0279] In certain embodiments, the pharmaceutical composition does not comprise a significant amount of a lipid (e.g. a fatty acid, fatty alcohol, phospholipid, sterol, monoglyceride, diglyceride, triglyceride, prenol, sphingolipid, in particular cholate, phosphatidylcholine, cholesterol, cholesterol sulfate, distearoylphosphatidylglycerol, alpha tocopherol, dimyristoylphosphatidylcholine and dimyristoylphosphatidylglycerol) and / or a non-polar solvent. A significant amount in this context is an amount of lipid and / or non-polar solvent together or individually in the composition which is greater than 10 % w / w, e.g. greater than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% w / w.

[0280] In certain embodiments, the pharmaceutical composition does not comprise a liposome, micellar suspension, colloidal dispersion or emulsion, or is not suitable or capable of forming such formats.

[0281] In certain embodiments, the pharmaceutical composition does not comprise the antimycotic polyene-alginate oligomer conjugate in a nanostructured form, e.g. as nanocrystals, nanotubes, polymer nanocarriers, cubosomal and cochleate nanoparticles.

[0282] In certain embodiments, the pharmaceutical composition is a substantially aqueous formulation, e.g. greater than 80 % w / w, e.g. greater than 85%, 90%, 95%, 96%, 97%, 98%, or 99% w / w of the composition is water and / or water soluble components.

[0283] The invention further relates to the use of the antimycotic polyene-alginate oligomer conjugates as described herein and the pharmaceutical compositions comprising the same in the combat of fungal infection (also referred to interchangeably herein as mycotic infection or mycosis). The term “combat” as used herein includes both therapy and prophylaxis (i.e. the treatment or prevention of a fungal infection).

[0284] Thus in this aspect the invention provides the antimycotic polyene-alginate oligomer conjugates of the invention as described herein and pharmaceutical compositions comprising them for use in therapy, particularly for use in the treatment or prevention of a fungal infection.

[0285] Specifically, in a further aspect the invention provides a method for the treatment or prevention of a fungal infection in a subject with, suspected to have, or at risk of, a fungal infection, said method comprising administering to said subject an effective amount of a antimycotic polyene-alginate oligomer conjugate of the invention as defined herein.

[0286] The invention further provides an antimycotic polyene-alginate oligomer conjugate of the invention as defined herein, for use in the treatment or prevention of a fungal infection in a subject with, suspected to have, or at risk of, a fungal infection. An “effective”, more particularly a "pharmaceutically effective", amount of the antimycotic polyene-alginate oligomer conjugate is that amount of conjugate that provides a measurable treatment or prevention of the target fungal infection.

[0287] In particular, in such methods and uses the effective amount of antimycotic polyene-alginate oligomer conjugate causes at most manageable, preferably minor or negligible, or no appreciable or clinically relevant host toxicity in the subject. In any event the host toxicity is reduced as compared to the corresponding unconjugated antimycotic polyene.

[0288] In the above described embodiments, the primary physiological result to be achieved is the contact of the site of infection (in particular the fungi which are present in the infected site or location, and which may include multiple sites or locations of infection in the body, including also a systemic infection) and / or a site (e.g. a surface) at which the infection may occur (or is at risk of occurring) with the antimycotic polyene-alginate oligomer conjugate. The secondary physiological outcome from the administration of the conjugate of the invention and its subsequent contact of the site of infection is a reduced or limited degree of host toxicity in the subject undergoing treatment, as compared to the corresponding unconjugated antimycotic polyene, e.g. manageable, preferably minor or negligible, or no appreciable or clinically relevant host toxicity is encountered. Host toxicity may include one or more of infusion- related inflammatory reactions, haemolytic toxicity, nephrotoxicity (e.g. caused by increased exposure of the polyene to renal cells via low-density lipoprotein (LDL) receptor mediated endocytosis, decreased renal blood flow, or decreased glomerular filtration), renal tubular damage, thrombophlebitis, and hepatotoxicity. Host toxicity may manifest as kidney failure, liver failure or anaphylaxis.

[0289] Expressed alternatively, the invention further provides the use of an antimycotic polyene- alginate oligomer conjugate of the invention as defined herein for the manufacture of a medicament for use in the treatment or prevention of a fungal infection in a subject with, suspected to have, or at risk of, a fungal infection.

[0290] The term “fungal infection” (or "infected by" or "infected with" and the like) is used broadly herein to indicate that the subject may comprise, or contain, or carry, the fungi in question, i.e. that the fungi may simply be present in or on the subject, and this may include any site or location in or on the body of the subject. It is not necessary that the infection of the subject be manifest as a clinical disease (i.e. that the infection result in clinical symptoms in the subject), although this is of course encompassed. A subject who is suspected to be infected or who is at risk of infection may be a subject who has been exposed to the fungi or to an infected subject, or a subject presenting with clinical signs or symptoms of infection (in the case of a suspected infection), or a subject who is susceptible to infection, whether generally (e.g. due to the clinical status of the subject) or particularly to the fungi in question.

[0291] Also in accordance with certain aspects of the invention there may be a preceding step of identifying a subject as being a subject with, suspected to have, or at risk of, a fungal infection, or a step of diagnosing a subject as a subject with, suspected to have, or at risk of, a fungal infection. In particular, the fungal infection may be of a type which is known to be treated, or treatable, in usual clinical practice with an antimycotic polyene. In one embodiment the fungi are identified as or suspected to be fungi which are responsive to (i.e. sensitive to) an antimycotic polyene. In certain embodiments the sensitivity of that infection (or more particularly the fungi within the infection) to an antimycotic polyene may be determined.

[0292] Alternatively, or in addition to the above described preceding step, in accordance with the invention there may be a following step in which the subject’s clinical indicators of the fungal infection are assessed and preferably compared to a corresponding assessment made prior to, or earlier in, said treatment in order to determine any changes therein.

[0293] The diagnosis and monitoring of fungal infections based on readily observable physiological indicators is entirely routine for clinicians. Molecular biological and microbiological methods may also be used to more confirm diagnoses and to provide more information on the causative agents, e.g. taxonomic information, possible indications of virulence and their sensitivity to antibiotics (antimycotics).

[0294] Alternatively, or in addition to the above described preceding and / or following steps, in accordance with the invention there may be a following step in which the subject’s clinical indicators of antimycotic polyene toxicity are assessed and preferably compared to a corresponding assessment made prior to, or earlier in, said treatment in order to determine any changes therein. The monitoring of antimycotic polyene toxicity in a subject based on readily observable physiological indicators is entirely routine for clinicians. This may include assessment of the general condition of the subject or more specific molecular markers, e.g. inflammatory markers (e.g. circulating or localised cytokine levels), markers of kidney function (serum and / or urine levels of creatinine, albumin, uric acid) and / or markers of liver function (prothrombin time (PT / INR), activated Partial Thromboplastin Time (aPTT), and serum levels of albumin, bilirubin (total, direct and / or indirect), aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), and / or gamma-glutamyltransferase (GGT).

[0295] The invention encompasses the use of a single antimycotic polyene-alginate oligomer conjugate or a mixture (multiplicity / pl urality; two or more) of different antimycotic polyenealginate oligomer conjugates. Such mixtures may comprise conjugates carrying different antimycotic polyenes and the same alginate oligomer. Such mixtures may comprise conjugates carrying the same antimycotic polyene and different alginate oligomers. Such mixtures may comprise conjugates carrying different antimycotic polyenes and different alginate oligomers. In certain embodiments, when mixtures of conjugates of the invention having different alginate oligomer components are used the alginate oligomers may have different numbers of residues and / or different G and M compositions and / or structures. In certain embodiments these may be selected to achieve predetermined functional criteria, for instance solubility, toxicity and / or efficacy.

[0296] The fungal infection targeted according to the invention may be caused by any fungus, i.e. any eukaryotic organism with a cell wall containing chitin, or any organism classified as belonging to the taxonomic kingdom Fungi. The conjugates of the invention are believed to retain the broad spectrum of efficacy seen in unmodified antimycotic polyenes. More specifically the fungus may be a member of the taxonomic phyla Ascomycota (i.e. from the taxonomic class Neolectomycetes, Pneumocystidomycetes, Schizosaccharomycetes, Taphrinomycetes, Arthoniomycetes, Dothideomycetes, Geoglossomycetes, Eurotiomycetes, Laboulbeniomycetes, Lecanoromycetes, Leotiomycetes, Lichinomycetes, Orbiliomycetes, Pezizomycetes, Sordariomycetes, Saccharomycetes); Basidiomycota (i.e. from the taxonomic class Agaricomycetes, Dacrymycetes, Tremellomycetes, Agaricostilbomycetes, Attractiellomycetes, Classiculomycetes, Cryptomycocolacomycetes, Cystobasidiomycetes, Microbotryomycetes, Mixiomycetes, Pucciniomycetes, Ustilaginomycetes, or Exobasidiomycetes); Chytridiomycota (i.e. from the taxonomic class Chytridiomycetes or Monoblepharidomycetes); Glomeromycota (i.e. from the taxonomic class Glomeromycetes); Zygomycota (i.e. from the taxonomic class Trichomycetes or Zygomycetes); Microsporidia (i.e. from the taxonomic class Aquasporidia, Marinosporidia or Terresporidia);

[0297] Blastocladiomycota (i.e. from the taxonomic class Blastocladiomycetes); and Neocallimastigomycota (i.e. from the taxonomic class Neocallimastigomycetes). The term "fungus" extends to the spores that may be produced by certain species of fungus, e.g. the "fungus" may be a sporangiospore, a zygospore, an acospore, a basidiospore, an aeciospore, a urediospore, a teliospore, a conidiospore, or a mitospore.

[0298] The fungus may be a unicellular species or a species that may exist in a unicellular form at some point in its lifecycle. The fungus may therefore be a yeast. The fungus may be a species that exists as a part of a multicelled hyphae or mycelium or a species that may exist as a part of a multicelled hyphae or mycelium at some point in its lifecycle. The multicelled hyphae or mycelium may be microscopic or macroscopic. The fungus may therefore be a mould. Of note are fungus that are dimorphic, i.e. they may exist in a unicellular (yeast) form under certain conditions (e.g. at certain levels of nutrients, carbon dioxide, oxygen, pH, temperature, etc.) and may exist as part of a multicelled hyphae or mycelium under certain other conditions or different levels of the abovementioned conditions. It is common for a fungus that is an animal pathogen to exist in the environment as a part of a multicelled hyphae or mycelium, but in a unicellular form in the animal.

[0299] In other embodiments the fungus is an animal pathogen or parasite. The fungus may be an opportunistic pathogen in that usually it is benign to healthy subjects with an uncompromised immune system, but such a fungus can establish an infection in subjects whose immune system is compromised in some way. The fungus may also be a fungus that produces a mycotoxin that affects animals, typically by poisoning them or inducing allergic reactions.

[0300] By way of example the fungus may be the causative agent of an aspergillosis (i.e. fungi from the taxonomic genus Aspergillus, e.g. Aspergillus fumigatus, Aspergillus flavus, Aspergillus clavatus, Aspergillus terrus, Aspergillus nigery, a candidiasis (i.e. fungi from the taxonomic genus Candida, e.g. Candida albicans, Candida glabrata, Candida tropicalis, Candida lusitaniae, Candida dubliniensis, Candida parapsilossis, Candida krusei, Candida rugosay a coccidioidomycosis (e.g. Coccidioides immitis, Coccidioides posadasii a cryptococcosis (e.g. Cryptococcus neoformans, Cryptococcus gattii, Cryptococcus laurentii, Cryptococcus albidus a histoplasmosis (e.g. Histoplasma capsulatum, Histoplasma duboisii blastomycosis (e.g. Blastomyces dermatitidis a mycetoma (e.g. Actinomadura pelletieri, Acremonium strictum, Actinomadura madurae, Aspergillus nidulans, Noetestudina rosatii, Phaeoacremonium krajdenii, Pseudallescheria boydii, Curvularia lunata, Exophiala jeanselmei, Leptosphaeria senegalensis, Leptosphaeria tompkinsii, Madurella grisea, Madurella mycetomatis, Pyrenochaeta romeroiy paracoccidioidomycosis (e.g.

[0301] Paracoccidioides brasiliensisy pneumocystosis (e.g. Pneumocystis jirovecii fusariosis (e.g. the Fusarium solani complex: Fusarium oxysporum, Fusarium verticillioides, Fusarium proliferatum, Fusarium monilifromey a phaeohyphomycosis (e.g. fungi from the genus Alternaria, Exophiala jeanselmeiy an alternariosis (i.e. fungi from the genus Alternaria, e.g. Alternaria alternatay rhinosporidiosis (e.g. Rhinosporidium seeberi a microsporidiosis (e.g. Enterocytozoon bieneusi, Encephalitozoon intestinalisy basidiobolomycosis (e.g. Basidiobolus ranarurriy a conidiobolomycosis (e.g. Conidiobolus coronatus, Conidiobolus incongruusy a mucormycosis (e.g. Rhizopus oryzae, Mucor indicus Absidia corymbifera, Syncephalastrum racemosumy a trichosporonosis (e.g. Trichosporon spp, Trichosporon asahii, Trichosporon inkin, Trichosporon asteroides, Trichosporon cutaneum, Trichosporon mucoides, Trichosporon ovoides, Trichosporon pullulans, Trichosporon loubieri, Trichosporon japonicumy a chromoblastomycosis {Fonsecaea pedrosoi, Fonsecaea compacta, Phialophora verrucosay geotrichosis (e.g. Geotrichum candidurriy allescheriasis (e.g. Pseudallescheria boydii sporotrichosis (e.g. Sporothrix schenckiiy penicilliosis (e.g. Penicillium marneffeiy lobomycosis (e.g. Lacazia loboi a dermatophytosis (i.e. fungi from the genera Epidermophyton, Microsporum and Trichophyton, e.g. Epidermophyton floccosum, Microsporum canis, Microsporum audouinii, Microsporum gypseum, Trichophyton interdigitale / mentagrophytes, Trichophyton verrucosum, Trichophyton violaceum Trichophyton canis, Trichophyton tonsurans, Trichophyton schoenleini, Trichophyton rubrum, Trichophyton concentricum)', a piedra (e.g. Hortaea werneckii, Piedraia hortae, Malassezia furfur, Trichosporon spp, Trichosporon beigelii), a pityrosporum folliculitis I malassezia folliculitis (i.e. fungi from the genera Malassezia, e.g. Malassezia globosa, Malassezia restricta). Other fungal species capable of acting as an animal pathogen include, Malassezia pachydermatis, Scedosporium prolificans, Acremonium kiliense, and Paecilomyces lilacinus

[0302] In certain embodiments the fungus is a species from the taxonomic genus Candida, e.g. Candida albicans, Candida glabrata, Candida tropicalis, Candida lusitaniae, Candida dubliniensis, Candida parapsilossis, Candida krusei and Candida rugosa, in particular Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis, Candida krusei and Candida lusitaniae, and most particularly Candida albicans.

[0303] In certain embodiments the fungus is a species from the taxonomic genus Aspergillus, e.g. Aspergillus niger, Aspergillus fumigatus, Aspergillus flavus, Aspergillus clavatus and Aspergillus terrus, in particular Aspergillus flavus.

[0304] In certain embodiments the fungus is a species from the taxonomic genus Cryptococcus, e.g. Cryptococcus neoformans, Cryptococcus gattii, Cryptococcus laurentii, Cryptococcus albidus, in particular Cryptococcus neoformans. In certain embodiments the fungus is a species from the taxonomic genera Malassezia (e.g. Malassezia pachydermatis or Malassezia furfur"), Trichosporon (e.g. Trichosporon cutaneum), Fusarium (e.g. the Fusarium solani complex: Fusarium oxysporum, Fusarium verticillioides, Fusarium proliferatum, Fusarium monilifrome), Acremonium (e.g. Acremonium kiliense, Acremonium strictum), Paecilomyces (e.g. Paecilomyces lilacinus), Rhizopus (e.g. Rhizopus oryzae), Mucor (e.g. Mucor indicus), Scedosporium (e.g. Scedosporium prolificans) and Absidia (e.g. Absidia corymbifera).

[0305] Myctoxin producing fungi include those that produce an aflatoxin (e.g. Aspergillus species, in particular, Aspergillus flavus and Aspergillus parasiticus), an ochratoxin (e.g. Aspergillus and Penicillium species, in particular, Aspergillus ochraceus, Aspergillus carbonarius and Penicillium viridicatum), a citrinin (e.g. Aspergillus, Monascus and Penicillium species, in particular, Aspergillus niveus, Aspergillus ochraceus, Aspergillus oryzae, Aspergillus terreus, Monascus ruber, Monascus purpureus, Penicillium citrinum, Penicillium camemberti), an ergot alkaloid (e.g. Claviceps species, in particular, Claviceps africana, Claviceps fusiformis, Claviceps paspali, Claviceps purpurea), a patulin (e.g. Aspergillus and Penicillium species, in particular, Penicillium expansum), a trichothecene (e.g. Fusarium, Myrothecium, Trichoderma, Trichothecium, Cephalosporium, Verticimonosporium, and Stachybotrys species), or a fusarium toxin, which includes fumonisins, trichothecenes, zearalenone, beauvercin and enniatins, butenolide, equisetin, and fusarinspatulin (e.g. Fusarium species).

[0306] In certain aspects, the infection is a nosocomial infection, an infection in the respiratory tract of patients, e.g. in patients suffering from cystic fibrosis, chronic obstructive pulmonary disease, chronic obstructive airway disease (COAD) congestive obstructive airway pneumonia (COAP), pneumonia, emphysema, bronchitis or sinusitis; an infection in a wound, particularly a chronic wound (including burns), a device related infection associated with implantable or prosthetic medical devices e.g. prosthetic valve endocarditis or an infection of a line or a catheter or an artificial joints or a tissue replacements or an endotracheal or tracheotomy tube. Fungal infections, especially of the subjects mentioned above, are commonly encountered in healthcare institutions due in part to the close proximity of subjects with fungal infections and those that have compromised defences against microorganisms, but also because of the widespread use of antibiotics. Fungi, e.g. from the genera Candida, Aspergillus, Malassezia, Trichosporon, Fusarium, Acremonium, Paecilomyces, Rhizopus, Mucor, Scedosporium and Absidia, are often involved in nosocomial infections and accordingly the invention can be seen as providing treatments for fungal nosocomial infections, e.g. nosocomial infections involving Candida albicans, Candida glabrata, Candida tropicalis, Candida lusitaniae, Candida dubliniensis, Candida parapsilossis, Candida krusei, Candida rugosa, Aspergillus fumigatus, Aspergillus flavus, Aspergillus clavatus, Aspergillus terrus, Malassezia pachydermatis, Malassezia furfur, Trichosporon cutaneum, the Fusarium solani complex: Fusarium oxysporum, Fusarium verticillioides, Fusarium proliferatum, Fusarium monilifrome, Acremonium kiliense, Acremonium strictum, Paecilomyces lilacinus, Rhizopus oryzae, Mucor indicus, Scedosporium prolifican or Absidia corymbifera. Very often, but not always, these infections are invasive (systemic or disseminated) infections of the subjects.

[0307] The fungus targeted by the method of the invention may be resistant to more than one antifungal agent, or more particularly it may be resistant to more than one class of antifungal agent, e.g. the fungus may be resistant to at least 2 or 3, or at least 4, 5, 6, 7, 8, 9 or 10 antifungal agents or classes thereof. Those fungi that are resistant to more than 3 classes of antifungal agent are "multidrug resistant (MDR)" or "have an MDR phenotype" . By way of example, antifungal agents and classes thereof include, but are not limited to, polyene antifungals (e.g. amphotericin B, nystatin, natamycin (pimaricin), candidicin, rimocidin, candidin, hamycin, perimycin, tricomycin (hachimycin), partricin and tetramycin); azole antifungals (e.g. imidazole antifungals, in particular, miconazole, ketoconazole, clotrimazole, econazole, omoconazole, bifonazole, butoconazole, fenticonazole, isoconazole, oxiconazole, sertaconazole, sulconazole, tioconazole; triazole antifungals, in particular, fluconazole, fosfluconazole, itraconazole, isavuconazole, ravuconazole, posaconazole, voriconazole, terconazole, albaconazole; thiazole antifungals, in particular, abafungin); allylamine antifungals (e.g. terbinafine, naftifin, butenafine, amorolfine); echinocandin antifungals (e.g. anidulafungin, caspofungin, micafungin); ciclopirox; tolnaftate; and flucytosine. In certain embodiments the target fungus is not resistant to the antimycotic polyene of the antimycotic polyene-alginate oligomer conjugate to be used

[0308] Susceptibility, that is sensitivity, (and conversely resistance and tolerance) to an antifungal agent can be measured in any convenient way, e.g. with dilution susceptibility tests and / or disk diffusion tests. Preferably the susceptibility of a fungal strain to an antifungal agent is expressed in terms of the Minimum Inhibitory Concentration (MIC) of that antibiotic for that microorganism (Jorgensen et al., Manual of Clinical Microbiology, 7th ed. Washington, D.C: American Society for Microbiology, 1999; 1526-43), i.e. that concentration of antifungal agent that completely inhibits growth of that fungal strain. The skilled person would appreciate that the extent of the difference in tolerance / susceptibility sufficient to constitute resistance will vary depending on the antifungal agent and fungal strain under test and the test used. Many regulatory bodies, e.g. the European Committee on Antimicrobial Susceptibility Testing (ELICAST) set so called “breakpoints” for specific antimicrobials and microorganisms which are discriminatory antimicrobial concentrations used in the interpretation of results of susceptibility testing to define isolates as susceptible, intermediate or resistant to the antimicrobial agent under test. The skilled person can utilise such information to ascertain whether or not the fungal infection being treated by the invention is resistant to the antifungal agent in question under these definitions.

[0309] In certain embodiments the target infection will be fungi in a biofilm. However, in other embodiments the fungi will not be in a biofilm (e.g. will be growing planktonically or in a mycelium or a hypha). Put differently, the fungi will be, or will not be, in a biofilm mode of growth; or will be, or will not be, in a non-biofilm mode of growth.

[0310] By "biofilm" it is meant a community of microorganisms characterized by a predominance of sessile cells that are attached to a substratum or interface or to each other (some motile cells may also be present) and that are embedded in a matrix of extracellular polymers (more specifically extracellular polymers that they have produced) characterised in that the microorganisms of this colony exhibit an altered phenotype with respect to growth rate and gene transcription (for example as compared to their "non-biofilm" or free-floating or planktonic, mycelial, or hyphal counterparts). By "in a biofilm" it is meant that the fungi targeted by the method of the invention are within (completely or in part), on or associated with the polymer matrix of a biofilm. Viewed differently, fungi that are "not in a biofilm" are organisms that are either in isolation, e.g. planktonic, or if in an aggregation of a plurality of organisms, that aggregation is unorganised and / or is devoid of the matrix characteristic of a biofilm, e.g. is a mycelium or a hypha. In each case, the individual fungi do not exhibit an altered phenotype that is observed in their biofilm dwelling counterparts.

[0311] In particular embodiments the invention may provide for the treatment or prevention of respiratory infections or conditions associated therewith (e.g. cystic fibrosis, pneumonia, COPD, COAD, bronchitis, sinusitis, an infection in a chronic wound (including burns), a device related infection associated with implantable or prosthetic medical devices, bacteraemia, septicaemia, septic shock, or sepsis. In one preferred embodiment the fungal infection is a respiratory infection in a subject suffering from an underlying respiratory disorder or condition, including notably CF, COPD / COAD, or asthma.

[0312] "Treatment" when used in relation to the treatment of a fungal infection / medical condition in a subject in accordance with the invention is used broadly herein to include any therapeutic effect, i.e. any beneficial effect in relation to the infection or on the condition. Thus, not only included is eradication or elimination of the infection, or cure of the subject or infection, but also an improvement in the infection or condition of the subject. Thus included for example, is an improvement in any symptom or sign of the infection or condition, or in any clinically accepted indicator of the infection / condition (for example a decrease in wound size or an acceleration of healing time). Treatment thus includes both curative and palliative therapy, e.g. of a pre-existing or diagnosed infection / condition, i.e. a reactionary treatment.

[0313] "Prevention" as used herein refers to any prophylactic or preventative effect. It thus includes delaying, limiting, reducing or preventing the infection / condition or one or more symptoms or indications thereof, or the onset of the infection / condition or one or more symptoms or indications thereof, or the risk of contracting / developing the infection / condition or one or more symptoms or indications thereof, for example relative to the infection / condition, risk, symptom or indication prior to the prophylactic treatment. Prophylaxis thus explicitly includes both absolute prevention of occurrence or development of the infection / condition, or symptom or indication thereof, and any delay in the onset or development of the infection / condition or symptom or indication thereof, or reduction or limitation of the development or progression of the infection / condition or symptom or indication thereof, and any reduction or elimination of the risk of contracting / developing the infection / condition or one or more symptoms or indications thereof.

[0314] The subject may be any human or non-human animal subject, but more particularly may be a human or a non-human vertebrate, e.g. a non-human mammal, bird, amphibian, fish or reptile. In a preferred embodiment the subject is a mammalian subject. The animal may be a livestock or a domestic animal or an animal of commercial value, including laboratory animals or an animal in a zoo or game park. Representative animals therefore include dogs, cats, rabbits, mice, guinea pigs, hamsters, horses, pigs, sheep, goats and cows. Veterinary uses of the invention are thus covered. The subject may be viewed as a patient. Preferably the subject is a human. In some embodiments the subject is not a ruminant mammal. The term "in a subject" is used broadly herein to include sites or locations inside a subject or on a subject, e.g. an external body surface, and may include in particular infection of a medical device e.g. an implanted or "in-dwelling" medical device. The term "in a patient" should be interpreted consistently with this.

[0315] The location of the infection may therefore be a surface in the oral cavity (e.g. teeth, gingiva, gingival crevice, periodontal pocket), the reproductive tract (e.g. cervix, uterus, fallopian tubes), the peritoneum, middle ear, prostate, the urinary tract, vascular intima, the eye, i.e. ocular tissue (e.g. the conjunctiva, corneal tissue, lachrymal duct, lachrymal gland, eyelid) the respiratory tract, lung tissue (e.g. bronchial and alveolial), heart valves, the gastrointestinal tract, skin, scalp, nails and the interior of wounds, particularly chronic wounds and surgical wounds, which may be topical or internal wounds. Other surfaces include the exterior of organs, particularly those undergoing transplantation, for example, heart, lungs, kidney, liver, heart valve, pancreas, intestine, corneal tissue, arterial and venous grafts and skin.

[0316] The infection may therefore also be present in body fluids (e.g. blood, plasma, serum, cerebrospinal fluid, Gl tract contents, semen, sputum and other pulmonary secretions) and tissues (e.g. adrenal, hepatic, renal, pancreatic, pituitary, thyroid, immune, ovarian, testicular, prostate, endometrial, ocular, mammary, adipose, epithelial, endothelial, neural, muscle, pulmonary, epidermis, osseous).

[0317] The infection may further be found on any “in-dwelling" medical or surgical equipment or devices. This may include any kind of line, including catheters (e.g. central venous and urinary catheters), prosthetic devices e.g., heart valves, artificial joints, false teeth, dental crowns, dental caps and soft tissue implants (e.g. breast, buttock and lip implants). Any kind of implantable medical device is included (e.g. stents, intrauterine devices, pacemakers, intubation tubes (e.g. endotracheal or tracheostomy tubes), prostheses or prosthetic devices, lines or catheters). An "in-dwelling" medical device may include a device in which any part of it is contained within the body, i.e. the device may be wholly or partly in-dwelling.

[0318] The infection may be acute, or alternatively chronic, e.g. an infection that has persisted for at least 5 or at least 10 days, particularly at least 20 days, more particularly at least 30 days, most particularly at least 40 days. A fungal infection can occur in any subject but some subjects will be more susceptible to infection that others. Subjects who are susceptible to fungal infection include, but are not limited to, subjects whose epithelial and / or endothelial barrier is weakened or compromised, subjects whose secretion-based defences to microbial infection have been abrogated, disrupted, weakened or undermined, and subjects who are immunocompromised, immunodeficient or immunosuppressed (i.e. a subject in whom any part of the immune system is net working normally, or is working sub-normally, in other words in whom any part of the immune response, or an immune activity is reduced or impaired, whether due to disease or clinical intervention or other treatment, or in any way).

[0319] Representative examples of subjects who are susceptible to fungal infection include, but are not limited to, subjects with a pre-established infection (e.g. with bacteria, viruses, fungi or parasites such as protozoa), especially subjects with HIV, subjects with bacteraemia, sepsis and subjects with septic shock; subjects with immunodeficiency, e.g. subjects preparing for, undergoing or recovering from chemotherapy and / or radiotherapy, organ (e.g. bone marrow, liver, lung, heart, heart valve, kidney, etc.) transplant subjects (including autograft, allograft and xenograft patients); subjects with AIDS; subjects resident in a healthcare institution, e.g. hospital, especially subjects in intensive care or critical care (i.e. those units concerned with the provision of life support or organ support systems to patients); subjects on respiratory ventilators; subjects suffering from trauma; subjects with burns, subjects with acute and / or chronic wounds; neonatal subjects; elderly subjects; subjects with cancer (defined broadly herein to include any neoplastic condition; malignant or non-malignant), especially those with cancers of the immune system (e.g. leukaemias, lymphomas and other haematological cancers); subjects suffering from auto-immune conditions such as rheumatoid arthritis, diabetes mellitus type I, Crohn's disease, especially those undergoing immunosuppression treatment for those diseases; subjects with reduced or abrogated epithelial or endothelial secretion (e.g. mucous, tears, saliva) and / or secretion clearance (e.g. subjects with poorly functioning cilia on mucosal tissue and / or patients with hyperviscous mucous (e.g. smokers and subjects with COPD, COAD, COAP, bronchitis, cystic fibrosis, emphysema, lung cancer, asthma, pneumonia or sinusitis)) and subjects fitted with a medical device.

[0320] As compared to the unmodified form of the antimycotic polyene in the antimycotic polyenealginate oligomer conjugates of the invention, the antimycotic polyene-alginate oligomer of the invention may be considered water soluble, or at least more water soluble than the nonconjugated form of the polyene antimycotic. In practical terms this means that the antimycotic polyene-alginate oligomer conjugates of the invention may be administered to the subject in any, almost any, or a greater number of convenient forms or by any, almost any, or a greater number of convenient means in order to deliver effective amounts to the fungi of the target infection and / or to the site carrying the invention or the site at risk of infection, e.g. by parenteral (e.g. intravenous, intraspinal, intramuscular, subcutaneous), topical, enteral (e.g. oral, buccal, sublingual, rectal), or by inhalation (including nasal inhalation). Administration may achieve systemic distribution or localised distribution, by which it is meant that delivery is effected to the bacteria of the target infection and / or to the site carrying the infection or to the site at risk of infection, but essentially no other location in the patient. The skilled person would be able to select an appropriate administration means to suit any particular target infection and / or site carrying the infection or site at risk of infection.

[0321] The comparatively low host toxicity of the antimycotic polyene-alginate oligomer conjugates of the invention make these entities more suitable than the unconjugated form of the antimycotic polyene for systemic use in subjects, that is systemic administration to treat a systemic infection and systemic administration to treat a localised infection, e.g. an infection in the lungs or a wound. In contrast the systemic use of antimycotic polyenes is very difficult, if not sometimes impossible because the associated host toxicity of these antibiotics reduces the therapeutic window to such an extent that effective antifungal amounts cannot be reached without resulting in unacceptable levels of toxicity. Systemic use of antimycotic polyenes is restricted to the most severe and grave cases of life threatening infection by fungi because in these scenarios the problems caused by host toxicity are outweighed by the immediate risk to the subject’s life posed by the infection.

[0322] The comparatively high aqueous solubility of the antimycotic polyene-alginate oligomer conjugates of the invention make these entities more suitable and convenient for systemic use, e.g. via intravenous or oral routes, than the unconjugated form of the antimycotic polyene. In contrast the use of antimycotic polyenes is very difficult and / or expensive because of the need to employ complex lipid-based or nano-structured formulations to increase systemic bioavailability via these routes of administration.

[0323] Thus in certain embodiments of the invention there is provided a method for the treatment or prevention of a fungal infection in a subject with, suspected to have, or at risk of, a fungal infection, said method comprising systemically administering to said subject, e.g. by intravenous or oral routes, an effective amount of an antimycotic polyene-alginate oligomer conjugate of the invention as defined herein. In certain embodiments the fungal infection is a systemic fungal infection e.g. sepsis (septicaemia) or an infection involving multiple loci in a subject.

[0324] The skilled person will be able to formulate the antimycotic polyene-alginate oligomer conjugates of the invention into pharmaceutical compositions that are adapted for the desired routes of administration and body distribution according to any of the conventional methods known in the art and widely described in the literature. In particular, the increased aqueous solubility of the antimycotic polyene-alginate oligomer conjugates of the invention allow the more convenient use of water-based (potentially less complex) formulations.

[0325] More specifically, the antimycotic polyene-alginate oligomer conjugates of the invention may be incorporated, optionally together with other active agents, with one or more conventional carriers, diluents and / or excipients, to produce conventional galenic preparations such as tablets, pills, granules (e.g. in free form or enclosed in capsules), powders (e.g. inhalable powders, including dry inhalable powders), lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), sprays (e.g. nasal sprays), compositions for use in nebulisers, ointments, creams, salves, soft and hard gelatine capsules, suppositories, pessaries, sterile injectable solutions, sterile packaged powders, and the like. Enteric coated solid or liquid compositions, e.g. enteric coated tablets and enteric coated granules (which may be provided in an enteric-coated capsule or in a non- enteric-coated capsule i.e. in which the coating may or may not be an enteric coating); sterile inhalable and sterile injectable compositions are of particular note.

[0326] Examples of suitable carriers, excipients, and diluents are lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, inert alginate polymers, tragacanth, gelatine, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water syrup, water, water / ethanol, water / glycol, water / polyethylene, hypertonic salt water, glycol, propylene glycol, methyl cellulose, methylhydroxybenzoates, propyl hydroxybenzoates, talc, magnesium stearate, mineral oil or fatty substances such as hard fat or suitable mixtures thereof. Excipients and diluents of note are mannitol and hypertonic salt water (saline).

[0327] The compositions may additionally include lubricating agents, wetting agents, emulsifying agents, suspending agents, preserving agents, sweetening agents, flavouring agents, and the like. Parenterally administrable forms, e.g. solutions suitable for delivery intravenously, should be sterile and free from physiologically unacceptable agents, and should have low osmolarity to minimize irritation or other adverse effects upon administration and thus solutions should preferably be isotonic or slightly hypertonic, e.g. hypertonic salt water (saline). Suitable vehicles include aqueous vehicles customarily used for administering parenteral solutions such as sterile water for injection, Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection and other solutions such as are described in Remington's Pharmaceutical Sciences, 15th ed., Easton: Mack Publishing Co., pp. 1405-1412 and 1461-1487 (1975) and The National Formulary XIV, 14th ed. Washington: American Pharmaceutical Association (1975) ), which is explicitly incorporated by reference herein in its entirety. The solutions can contain preservatives, antimicrobial agents, buffers and antioxidants conventionally used for parenteral solutions, excipients and other additives which are compatible with the antimycotic polyene-alginate oligomer conjugates and which will not interfere with the manufacture, storage or use of products.

[0328] Simple sterile water-based solutions of the antimycotic polyene-alginate oligomer conjugates or simple sterile liquid compositions comprising the antimycotic polyene-alginate oligomer conjugates may be especially convenient for use during surgical procedures and for delivery to the lungs, e.g. by nebuliser, or to the paranasal sinuses, e.g. by a nasal spray device.

[0329] Solid or liquid formulations of the antimycotic polyene-alginate oligomer conjugates may be provided with an enteric coating that prevents degradation in the stomach and / or other parts of the upper Gl tract but permits degradation in the lower Gl tract, e.g. the small intestine. Such coatings are routinely prepared from polymers including fatty acids, waxes, shellac, plastics, and plant fibres. Specific examples thereof include but are not limited to methyl acrylate-methacrylic acid copolymers, methyl methacrylate-methacrylic acid copolymers, cellulose acetate succinate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate (hypromellose acetate succinate), polyvinyl acetate phthalate (PVAP), cellulose acetate trimellitate, and sodium alginate polymer. Enteric coated tablets and enteric coated granules (which may be provided in an enteric-coated capsule or in a non-enteric coated capsule) are of particular note. Enteric coated granules may be prepared in accordance with the teachings of WO 1989008448 and Al-Khedairy, E.B.H, 2006, Iraqi J.Pharm.Sci., Vol.15 (1) 49, the contents of which are incorporated herein by reference, although the skilled person would be aware of further alternative techniques which may be used.

[0330] For topical administration the antimycotic polyene-alginate oligomer conjugates can be incorporated into creams, ointments, gels, salves, transdermal patches and the like. Further topical systems that are envisaged to be suitable are in situ drug delivery systems, for example gels where solid, semi-solid, amorphous or liquid crystalline gel matrices are formed in situ and which may comprise antimycotic polyene-alginate oligomer conjugate. Such matrices can conveniently be designed to control the release of the antimycotic polyene- alginate oligomer conjugates from the matrix, e.g. release can be delayed and / or sustained over a chosen period of time. Such systems may form gels only upon contact with biological tissues or fluids, e.g. mucosal surfaces. Typically, the gels are bioadhesive and / or mucoadhesive. Delivery to any site in a human or non-human animal body that can retain or be adapted to retain the pre-gel composition can be targeted by such a delivery technique. Such systems are described in WO 2005 / 023176.

[0331] In certain embodiments however, such compositions do not comprise a significant amount of a lipid (e.g. a fatty acid, fatty alcohol, phospholipid, sterol, monoglyceride, diglyceride, triglyceride, prenol, sphingolipid, in particular cholate, phosphatidylcholine, cholesterol, cholesterol sulfate, distearoylphosphatidylglycerol, alpha tocopherol, dimyristoylphosphatidylcholine and dimyristoylphosphatidylglycerol) and / or a non-polar solvent.

[0332] In certain embodiments, such pharmaceutical compositions do not comprise a liposome, micellar suspension, colloidal dispersion or emulsion, or are not suitable or capable of forming such formats.

[0333] In certain embodiments, such pharmaceutical compositions do not comprise the antimycotic polyene-alginate oligomer conjugate in a nanostructured form, e.g. as nanocrystals, nanotubes, polymer nanocarriers, cubosomal and cochletae nanoparticles.

[0334] The relative content of the antimycotic polyene-alginate oligomer conjugates in the compositions of the invention can vary depending on the dosage required and the dosage regime being followed but will be sufficient to achieve an effective amount at the target treatment site, i.e. the fungi of the target infection and / or the site carrying the infection, or the site at risk of infection, taking account of variables such as the physical size of the subject to be treated, the nature of the subject’s particular ailments, and the location and identity of the target treatment area. The skilled person would know that the amounts of the antimycotic polyene-alginate oligomer conjugates can be reduced if a multiple dosing regime is followed or increased to minimise the number of administrations or applications.

[0335] A representative aqueous solution for delivery of the antimycotic polyene-alginate oligomer conjugate of the invention by injection (e.g. by intravenous, intraspinal, intramuscular or subcutaneous injection) will be sterile and may contain 6 to 25%, e.g. 6 to 20%, 6 to 15%, 6 to 10%, 8 to 25%, 8 to 20%, 8 to 15 %, 9 to 25%, 9 to 20%, 9 to 15 %, 10 to 15%, 10 to 20%, 10 to 25%, 15 to 20%, or 15 to 25% w / v of the antimycotic polyene-alginate oligomer conjugate, the remainder being comprised of water and pharmaceutically acceptable excipients and / or other active agents if being used.

[0336] For administration to the nose or paranasal sinuses a sterile aqueous may be used; administered for instance by a nasal spray device, e.g. propellant-free or propellant-assisted. A representative formulation may contain 1 to 25%, 1 to 20%, e.g. 1 to 15%, 1 to 10%, 1 to 9%, 1 to 8%, 1 to 7% or 1 to 6%, 5 to 25%, 5 to 20%, 5 to 15%, 5 to 10%, 5 to 9%, 5 to 8%, 5 to 7%, 5 to 6%, 8 to 25%, 8 to 20%, 8 to 15%, 8 to 10%, 9 to 25%, 9 to 20%, or 9 to 15% w / v or w / w of the antimycotic polyene-alginate oligomer conjugate, the remainder being comprised of pharmaceutically acceptable excipients, e.g. water, and / or other active agents if being used.

[0337] A representative inhalable solution to be used to administer an antimycotic polyene-alginate oligomer conjugate of the invention to the upper respiratory tract typically will be sterile and may contain 6 to 25%, e.g. 6 to 20%, 6 to 15%, 6 to 10%, 8 to 25%, 8 to 20%, 8 to 15 %, 9 to 25%, 9 to 20%, 9 to 15 %, 10 to 15%, 10 to 20%, 10 to 25%, 15 to 20% or 15 to 25% w / v of the antimycotic polyene-alginate oligomer conjugate, the remainder being comprised of pharmaceutically acceptable excipients, e.g. water, and / or other active agents if being used.

[0338] A representative inhalable powder to be used to administer an antimycotic polyene-alginate oligomer conjugates of the invention to the lower respiratory tract may contain up to 90%, e.g. up to 85%, 80%, 75% or 70%, e.g. 50 to 90%, 55 to 90%, 60 to 90%, 65 to 90%, 70 to

[0339] 90%, 75 to 90%, 80 to 90%, 85 to 90%, 50 to 85%, 55 to 85%, 60 to 85%, 65 to 85%, 70 to

[0340] 85%, 75 to 85%, 80 to 85%, 50 to 80%, 55 to 80%, 60 to 80%, 65 to 80%, 70 to 80%, 75 to

[0341] 80%, 50 to 70%, 55 to 70%, 60 to 70%, or 65 to 70% w / v or w / w of the antimycotic polyene- alginate oligomer conjugate, the remainder being comprised of pharmaceutically acceptable excipients and / or other active agents if being used in the same composition.

[0342] In other embodiments a slow, delayed or sustained release formulation may be used for delivery, e.g. to the nose or paranasal sinuses. A representative formulation may be a powder containing the antimycotic polyene-alginate oligomer conjugate or a suspension of said powder, said powder containing up to 90%, e.g. up to 85%, 80%, 75% or 70%, e.g. 50 to 90%, 55 to 90%, 60 to 90%, 65 to 90%, 70 to 90%, 75 to 90%, 80 to 90%, 85 to 90%, 50 to 85%, 55 to 85%, 60 to 85%, 65 to 85%, 70 to 85%, 75 to 85%, 80 to 85%, 50 to 80%, 55 to 80%, 60 to 80%, 65 to 80%, 70 to 80%, 75 to 80%, 50 to 70%, 55 to 70%, 60 to 70%, or

[0343] 65 to 70% w / v or w / w of the antimycotic polyene-alginate oligomer conjugate, the remainder being comprised of pharmaceutically acceptable excipients and / or other active agents if being used. The powder may comprise a coating that controls release of the antimycotic polyene-alginate oligomer conjugate.

[0344] A representative topical formulation, e.g. a cream, ointment or salve, which may be used to administer a antimycotic polyene-alginate oligomer conjugate of the invention to the skin or cervix or other parts of the lower female reproductive system might contain 1 to 25%, 1 to 20%, 1 to 15%, 1 to 10%, 1 to 9%, 1 to 8%, 1 to 7%, 1 to 6%, 5 to 25%, 5 to 20%, 5 to 15%, 5 to 10%, 5 to 9%, 5 to 8%, 5 to 7%, 5 to 6%, 8 to 25%, 8 to 20%, 8 to 15%, 8 to 10%, 9 to 25%, 9 to 20%, or 9 to 15% w / v of the antimycotic polyene-alginate oligomer conjugate, the remainder being comprised of pharmaceutically acceptable excipients, and / or other active agents if being used. Delivery devices designed for the application of topical formulations to the female reproductive system are known and may be employed to deliver the above mentioned formulations if convenient.

[0345] A representative tablet to be used to administer an antimycotic polyene-alginate oligomer conjugate of the invention to the lower Gl tract may contain up to 99%, up to 95%, 90%, 85% or 80%, e.g. 50 to 95%, 55 to 95%, 60 to 95%, 65 to 95%, 70 to 95%, 75 to 95%, 80 to 95%, 85 to 95%, 90 to 95%, 50 to 90%, 50 to 90%, 55 to 90%, 60 to 90%, 65 to 90%, 70 to 90%, 75 to 90%, 80 to 90%, 85 to 90%, 50 to 90%, 55 to 85%, 60 to 80% or, 65 to 75% w / v or w / w of the antimycotic polyene-alginate oligomer conjugate, the remainder being comprised of pharmaceutically acceptable excipients and / or other active agents if being used. The tablet may be a multi-layered tablet. An enteric coated tablet may also be effective in administering an antimycotic polyenealginate oligomer conjugate of the invention to the lower Gl tract. A representative enteric coated tablet may contain up to 95%, e.g. up to 90%, 85% or 80%, e.g. 55 to 90%, 60 to 90%, 65 to 90%, 70 to 90%, 75 to 90%, 80 to 90%, 85 to 90%, 55 to 85%, 60 to 85%, 65 to 85%, 70 to 85%, 75 to 85%, 80 to 85%, 50 to 80%, 55 to 80%, 60 to 80%, 65 to 80%, 70 to 80%, or 75 to 80% w / v or w / w of the antimycotic polyene-alginate oligomer conjugate, the remainder being comprised of pharmaceutically acceptable excipients, including the enteric coating (e.g. polymers including fatty acids, waxes, shellac, plastics, and plant fibres) and / or other active agents if being used. The tablet may be a multi-layered tablet, e.g. as described above.

[0346] Enteric coated granules may also be effective in administering an antimycotic polyenealginate oligomer conjugate of the invention to the lower Gl tract. Such granules may be provided in a capsule which itself may or may not be provided with an enteric coating. A representative enteric coated granule may contain up to 95%, e.g. up to 90%, 85% or 80%, e.g. 55 to 90%, 60 to 90%, 65 to 90%, 70 to 90%, 75 to 90%, 80 to 90%, 85 to 90%, 55 to 85%, 60 to 85%, 65 to 85%, 70 to 85%, 75 to 85%, 80 to 85%, 50 to 80%, 55 to 80%, 60 to 80%, 65 to 80%, 70 to 80%, or 75 to 80% w / v or w / w of the antimycotic polyene-alginate oligomer conjugate, the remainder being comprised of pharmaceutically acceptable excipients, including the enteric coating (e.g. polymers including fatty acids, waxes, shellac, plastics, and plant fibres) and / or other active agents if being used.

[0347] A pessary may be used to administer an antimycotic polyene-alginate oligomer conjugate of the invention to the lower parts of the female reproductive tract. A representative formulation may contain 1 to 25%, 1 to 20%, e.g. 1 to 15%, 1 to 10%, 1 to 9%, 1 to 8%, 1 to 7%, 1 to 6%, 5 to 25%, 5 to 20%, 5 to 15%, 5 to 10%, 5 to 9%, 5 to 8%, 5 to 7%, 5 to 6%, 8 to 25%, 8 to 20%, 8 to 15%, 8 to 10%, 9 to 25%, 9 to 20%, or 9 to 15% w / v or w / w of the antimycotic polyene-alginate oligomer conjugate, the remainder being comprised of pharmaceutically acceptable excipients, including solid excipients, and / or other active agents if being used. Rectal suppositories may be formulated similarly.

[0348] The antimycotic polyene-alginate oligomer conjugates may be used at a daily dose of 0.1g to 10g, e.g. 0.5g to 5g, 0.8g to 3g, 1g to 2g, e.g. about 2g, which may be administered at one or more times per day (e.g. bis daily) and in one or more dosage forms or administration events (e.g. two tablets bis daily). The antimycotic polyene-alginate oligomer conjugates as defined herein may be used in conjunction or combination with one or more further therapeutically active agents, which may include other anti-microbial (e.g. antibacterial, antibiotic, antifungal and antiviral) agents, immunostimulatory agents, corticosteroids, non-steroidal anti-inflammatory drugs (NSAIDs), bronchodilators, mucus viscosity-reducing agents (i.e. an agent which reduces the viscosity of mucus and which terms are used interchangeably with the term “mucolytic agent”), CFTR modulators (also known as “CFTR modifiers”) or one or more non-conjugated alginate oligomers.

[0349] The combined use of an antimycotic polyene-alginate oligomer conjugate as herein defined and an antibiotic, a further antifungal, and / or a mucus viscosity reducing agent are especially preferred. A non-conjugated alginate oligomer, e.g. any of those described above, may be used as such agents or for other reasons.

[0350] In these embodiments wherein a non-conjugated alginate oligomer is used together with the antimycotic polyene-alginate oligomer conjugate, the alginate oligomer may be the same as that present in the conjugate, or may be different. A plurality of alginate oligomers of different structures, e.g. different sizes, G and M composition and sequence may be used.

[0351] In certain embodiments the further agent, e.g. antibiotic, is not conjugated to an alginate oligomer or to any other conjugating moiety.

[0352] The antimycotic polyene-alginate oligomer conjugate and the further therapeutically active agent may, for example, be administered together, in a single pharmaceutical formulation or composition, or separately (i.e. separate, sequential or simultaneous administration). Thus, the antimycotic polyene-alginate oligomer conjugate and the further therapeutically active agent may be combined, e.g. in a pharmaceutical kit or as a combined ("combination") product.

[0353] Thus a further aspect of the invention provides a product (e.g. a pharmaceutical combination or a kit) comprising an antimycotic polyene-alginate oligomer conjugate as defined herein together with a further therapeutically active agent (e.g. those described below) as combined preparation for separate, sequential or simultaneous use in treating or preventing a fungal infection in a subject. More generally this aspect of the invention also provides a kit comprising an antimycotic polyene-alginate oligomer conjugate as defined herein together with a further therapeutically active agent (e.g. those described below).

[0354] Combinations comprising an antimycotic polyene-alginate oligomer conjugate as herein defined and an antibiotic, an further antifungal, a CFTR modulator and / or a mucus viscosity reducing agent are especially preferred. A non-conjugated alginate oligomer, e.g. any of those described above, may be used as such agents. Such pharmaceutical products and pharmaceutical compositions are preferably adapted for use in the medical methods of the invention.

[0355] The further therapeutically active agent may conveniently be applied before, simultaneously with or following the antimycotic polyene-alginate oligomer conjugate. Conveniently the further therapeutically active agent is applied at substantially the same time as the antimycotic polyene-alginate oligomer conjugate or afterwards. In other embodiments the further therapeutically active agent may conveniently be applied or administered before the antimycotic polyene-alginate oligomer conjugate. The further therapeutically active agent can also be given (e.g. administered or delivered) repeatedly at time points appropriate for the agent used. The skilled person is able to devise a suitable dosage regimen. In long term treatments the antimycotic polyene-alginate oligomer conjugate can also be used repeatedly. The antimycotic polyene-alginate oligomer conjugate can be applied as frequently as the further therapeutically active agent, or more or less frequently. The frequency required may depend on the site or location in or on the patient to which the antimycotic polyene-alginate oligomer conjugate is administered and also the overall nature of the clinical condition displayed by the particular patient undergoing treatment.

[0356] The antimycotic polyene-alginate oligomer conjugate and the further therapeutically active agent may therefore be formulated together or separately, that is in the same or in different formulations or pharmaceutical compositions, and may be provided for administration by the same or different routes. The use of an antimycotic polyene-alginate oligomer conjugate as herein defined to manufacture such pharmaceutical products and pharmaceutical compositions for use in the medical methods of the invention is also contemplated.

[0357] Representative antibiotics which may be used in conjunction or combination with the antimycotic polyene-alginate oligomer conjugates as defined herein include, but are not limited to, the aminoglycosides (e.g. amikacin, gentamicin, kanamycin, neomycin, netilmicin, streptomycin, tobramycin); the p-lactams (e.g. the carbecephems (e.g. loracarbef); the 1st generation cephalosporins (e.g. cefadroxil, cefazolin, cephalexin); 2nd generation cephalosporins (e.g. cefaclor, cefamandole, cephalexin, cefoxitin, cefprozil, cefuroxime); 3rd generation cephalosporins (e.g. cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone); 4th generation cephalosporins (e.g. cefepime); the monobactams (e.g. aztreonam); the macrolides (e.g. azithromycin, clarithromycin, dirithromycin, erythromycin, troleandomycin); the monobactams (e.g. aztreonam); the penicillins (e.g. amoxicillin, ampicillin, carbenicillin, cioxacillin, dicloxacillin, nafcillin, oxacillin, penicillin G, penicillin V, piperacillin, ticarcillin); the polypeptide antibiotics (e.g. bacitracin, colistin, polymyxin B, but in certain embodiments not the polymyxin of the polymyxin-alginate oligomer conjugate to be used); the quinolones (e.g. ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin, ofloxacin, trovafloxacin); the sulfonamides (e.g. mafenide, sulfacetamide, sulfamethizole, sulfasalazine, sulfisoxazole, trimethoprim- sulfamethoxazole); the tetracyclines (e.g. demeclocycline, doxycycline, minocycline, oxytetracycline, tetracycline); the glycylcyclines (e.g. tigecycline); the carbapenems (e.g. imipenem, meropenem, ertapenem, doripenem, panipenem / betamipron, biapenem, PZ-601); other antibiotics include chloramphenicol; clindamycin, ethambutol; fosfomycin; isoniazid; linezolid; metronidazole; nitrofurantoin; pyrazinamide; quinupristin / dalfopristin; rifampin; spectinomycin; and vancomycin.

[0358] Representative antifungals include, but are not limited to the polyenes (e.g. natamycin, rimocidin, filipin, nystatin, amphotericin B, candicin); the imidazoles (e.g. miconazole, ketoconazole, clotrimazole, econazole, bifonazole, butoconazole, fenticonazole, isoconazole, oxiconazole, sertaconazole, sulconazole, tioconazole); the triazoles (e.g. fluconazole, itraconazole, isavuconazole, ravuconazole, posaconazole, voriconazole, terconazole); the allylamines (e.g. terbinafine, amorolfine, naftifine, butenafine); and the echinocandins (e.g. anidulafungin, caspofungin, micafungin).

[0359] Representative antivirals include, but are not limited to abacavir, acyclovir, adefovir, amantadine, amprenavir, arbidol, atazanavir, atripla, boceprevir, cidofovir, combivir, darunavir, delavirdine, didanosine, docosanol, edoxudine, efavirenz, emtricitabine, enfuvirtide, entecavir, famciclovir, fomivirsen, fosamprenavir, foscarnet, fosfonet, ganciclovir, ibacitabine , imunovir, idoxuridine, imiquimod, indinavir, inosine, interferon type III, interferon type II, interferon type I, lamivudine, lopinavir, loviride, maraviroc, moroxydine, nelfinavir, nevirapine, nexavir, oseltamivir, penciclovir, peramivir, pleconaril, podophyllotoxin, raltegravir, ribavirin, rimantadine, ritonavir, saquinavir , stavudine, tenofovir, tenofovir disoproxil, tipranavir, trifluridine, trizivir, tromantadine, truvada, valaciclovir, valganciclovir, vicriviroc, vidarabine, viramidine, zalcitabine, zanamivir, and zidovudine.

[0360] Representative immunostimulatory agents include, but are not limited to, cytokines e.g. TNF, IL-1 , IL-6, IL-8 and immunostimulatory alginates, such as high M -content alginates as described for example in US 5,169,840, WO91 / 11205 and W003 / 045402 which are explicitly incorporated by reference herein in their entirety, but including any alginate with immunostimulatory properties.

[0361] Representative examples of suitable corticosteroids include but are not limited to prednisone, flunisolide, triamcinolone, fluticasone, budesonide, mometasone, beclomethasone, amcinonide, budesonide, desonide, fluocinonide, fluocinolone, halcinonide, hydrocortisone, cortisone, tixocortol, prednisolone, methylprednisolone, prednisone, betamethasone, dexamethasone, fluocortolone, aclometasone, prednicarbate, clobetasone, clobetasol, and fluprednidene.

[0362] Representative NSAIDs include, but are not limited to, the salicylates (e.g. aspirin (acetylsalicylic acid), choline magnesium trisalicylate, diflunisal, salsalate, the propionic acid derivatives (e.g. ibuprofen, dexibuprofen, dexketoprofen, fenoprofen, flurbiprofen, ketoprofen, loxoprofen, naproxen, oxaprozin), the acetic acid derivatives (e.g. aceclofenac, diclofenac, etodolac., indomethacin, ketorolac, nabumetone, tolmetin, sulindac), the enolic acid derivatives (e.g. droxicam, isoxicam, lornoxicam, meloxicam, piroxicam, tenoxicam), the anthranilic acid derivatives (e.g. flufenamic acid, meclofenamic acid, mefenamic acid, tolfenamic acid) and the selective COX-2 inhibitors (Coxibs; e.g. celecoxib, etoricoxib, lumiracoxib, parecoxib, rofecoxib, valdecoxib). The propionic acid derivatives (e.g. ibuprofen, dexibuprofen, dexketoprofen, fenoprofen, flurbiprofen, ketoprofen, loxoprofen, naproxen, oxaprozin) are preferred, ibuprofen being most preferred.

[0363] Representative examples of suitable bronchodilators include but are not limited to the p2 agonists (e.g. the short-acting p2 agonists (e.g. pirbuterol, epinephrine, salbutamol, levosalbutamol, clenbuterol, terbutaline, procaterol, metaproterenol, fenoterol, bitolterol mesylate, ritodrine, isoprenaline); the long-acting p2 agonists (e.g. salmeterol, formoterol, bambuterol, clenbuterol); and the ultra-long-acting p2 agonists (e.g. indacaterol)), the anticholinergics (e.g. ipratropium, oxitropium, tiotropium) and theophylline. As used herein, the terms “mucolytic agent” and “mucus viscosity reducing agent” are intended to encompass agents which reduce the intrinsic viscosity of mucus and agents which reduce the attachment of mucus to underlying epithelium, in particular agents which directly or indirectly disrupt the molecular interactions within or between the components of mucus, agents which affect the hydration of mucus and agents which modulate the ionic microenvironment of the mucosal epithelium (particularly the levels of divalent cations, e.g. calcium). Representative examples of suitable mucus viscosity reducing agents include, but are not limited to, a nucleic acid cleaving enzyme (e.g. a DNase such as DNase I or dornase alfa), hypertonic saline, gelsolin, a thiol reducing agent, an acetylcysteine, an uncharged low molecular weight polysaccharide (e.g. dextran, mannitol), arginine (or other nitric oxide precursors or synthesis stimulators), an agonist of the P2Y2 subtype of purinergic receptors (e.g. denufosol) or an anionic polyamino acid (e.g. poly ASP or poly GLU). Ambroxol, bromhexine, carbocisteine, domiodol, eprazinone, erdosteine, letosteine, mesna, neltenexine, sobrerol, stepronin, tiopronin are specific mucolytics of note. DNase I and hypertonic saline are preferred.

[0364] CFTR modulators are small molecules which can redress, at least partially, a CFTR dysfunction. Present CFTR modulators fall into three main groups: CFTR potentiators, CFTR correctors and read-through agents (Derichs, N., Eur. Respir. Rev., 2013, 22(127), 58- 65; Petit, R.S. and Fellner, C., Pharmacy and Therapeutics, 2014, 39(7), 500-511 ; the contents of which are incorporated herein by reference). CFTR potentiators are CFTR modulators which increase the activity of the CFTR ion channel present on the epithelial cell surface. CFTR correctors are CFTR modulators which increase the amount of CFTR protein delivered or retained at the epithelial cell surface. Read-through agents (also known as “premature stop codon suppressors” (PSC suppressors) or “premature termination codon suppressors” (PTC suppressors, which terms are used interchangeably herein) are CFTR modulators which cause the translation machinery of the cell to pass over any premature termination codon in the CFTR mRNA thereby increasing the amount of substantially full length and functional CFTR produced.

[0365] Representative examples of suitable CFTR potentiators include, but are not limited to, ivacaftor (VX-770; N-(2,4-di-tert-butyl-5-hydroxyphenyl)-1 ,4-dihydro-4-oxoquinoline-3- carboxamide) and VRT-532 (4-methyl-2-(5-phenyl-1 H-pyrazol-3-yl)-phenol) of Vertex Pharmaceuticals™). Representative examples of suitable CFTR correctors include, but are not limited to, Prototypical lumacaftor (VX-809) and VX-661 of Vertex Pharmaceuticals™ and N6022 (3-[1- (4-carbamoyl-2-methylphenyl)-5-(4-imidazol-1-ylphenyl)pyrrol-2-yl]propanoic acid).

[0366] Representative examples of suitable read-through agents include, but are not limited to, ataluren (PTC124) of PTC Therapeutics and gentamicin.

[0367] The invention will be further described with reference to the following non-limiting Examples in which:

[0368] Figure 1 shows the structures of various polyene antimycotics: A) Rimocidin; B) Candidin; C) Hamycin; D) Perimycin; E) Candicidin; F) Tricomycin (Hachimycin); G) Partricin A and B; H) Tetramycin A and B; I) Amphotericin B; J) Nystatin; and K) Natamycin.

[0369] Figure 2 shows Candida albicans CCLIG 39343 growth curves in the presence of increasing concentrations of A) Amphotericin B (pM); B) Amphotericin B (pM); C) OligoG (pM); D) OligoG-PEG5-N3(pM); E) AmpB-C-DCBO (pM); F) AmpB-C-DBCO-PEGs-OligoG (pM); G) Amphotericin B (pg / ml); and H) AmpB-C-DBCO-PEGs-OligoG (pg / ml).

[0370] Figure 3 shows LDH leakage (%) in LLC-PK1 cells (A) and HepG2 cells (B) after 48 hrs exposure to increasing concentrations of test agents - Amphotericin B; AmpB-C-DCBO; OligoG; OligoG-PEGs-Ns; AmpB-C-DBCO-PEGs-OligoG; PAPA; and digitoin.

[0371] Figure 4 shows cell viability (%) in LLC-PK1 cells (A) and HepG2 cells (B) after 48 hrs exposure to increasing concentrations of test agents - Amphotericin B; AmpB-C-DCBO; OligoG; OligoG-Ns; AmpB-C-DBCO-PEGs-OligoG; PAPA; and digitoin.

[0372] Figure 5 shows percentage haemolysis in the presence of increasing concentrations of amphotericin B (A) and AmpB-C-DBCO-PEGs-OligoG (B)

[0373] Figure 6 shows Candida albicans CCLIG 39343 growth curves in the presence of increasing concentrations (pM) of (A) Amphotericin B, AmpB-C-DCBO and AmpB-C-DBCO-PEGs- OligoG; and (B) Amphotericin B, AmpB-N-DCBO and AmpB-N-DBCO-PEGs-OligoG.

[0374] Figure 7 shows Candida albicans CCLIG 39343 growth curves in the presence of increasing concentrations (pM) of (A) Amphotericin B, AmpB-C-DCBO, AmpB-C-DBCO-PEGs-OligoG, AmpB-C-DBCO-PEGs-G4, AmpB-C-DBCO-PEGs-Ge, AmpB-C-DBCO-PEGs-Gs, and AmpB- C-DBCO-PEG5-G10; and (B) Amphotericin B, AmpB-N-DCBO, AmpB-N-DBCO-PEGs-OligoG, AmpB-N-DBCO-PEGs-G4, AmpB-N-DBCO-PEGs-Ge, AmpB-N-DBCO-PEGs-Gs, and AmpB- N-DBCO-PEG5-G10.

[0375] Figure 8 shows cell viability (% of digitonin control) in HepG2 cells after 48 hrs exposure to increasing concentrations of test agents (A) Amphotericin B, AmpB-C-DCBO, AmpB-C- DBCO-PEG5-OligoG; (B) Amphotericin B, AmpB-N-DCBO, AmpB-N-DBCO-PEGs-OligoG;

[0376] (C) Amphotericin B, AmpB-C-DCBO, AmpB-C-DBCO-PEGs-OligoG, AmpB-C-DBCO-PEGs- G4, AmpB-C-DBCO-PEGs-Ge, AmpB-C-DBCO-PEGs-Gs, and AmpB-C-DBCO-PEGs-Gwi and

[0377] (D) Amphotericin B, AmpB-N-DCBO, AmpB-N-DBCO-PEGs-OligoG, AmpB-N-DBCO-PEGs- G4, AmpB-N-DBCO-PEGs-Ge, AmpB-N-DBCO-PEGs-Gs, and AmpB-N-DBCO-PEGs-Gw-

[0378] Figure 9 shows cell viability (% of digitonin control) in LLC-PK1 cells after 48 hrs exposure to increasing concentrations of test agents (A) Amphotericin B, AmpB-C-DCBO, AmpB-C- DBCO-PEG5-OligoG; (B) Amphotericin B, AmpB-N-DCBO, AmpB-N-DBCO-PEGs-OligoG;

[0379] (C) Amphotericin B, AmpB-C-DCBO, AmpB-C-DBCO-PEGs-OligoG, AmpB-C-DBCO-PEGs- G4, AmpB-C-DBCO-PEGs-Ge, AmpB-C-DBCO-PEGs-Gs, and AmpB-C-DBCO-PEGs-Gwi and

[0380] (D) Amphotericin B, AmpB-N-DCBO, AmpB-N-DBCO-PEGs-OligoG, AmpB-N-DBCO-PEGs- G4, AmpB-N-DBCO-PEGs-Ge, AmpB-N-DBCO-PEGs-Gs, and AmpB-N-DBCO-PEGs-Gw-

[0381] EXAMPLES

[0382] EXAMPLE 1 - Preparation of AmpB-C-DBCO-PEGs-OligoG is of OligoG CF5 / 20 PEG5-N3

[0383] OligoG CF-5 / 20 shown in its two equilibrium forms - reduced form on right:

[0384] The reducing end of OligoG CF-5 / 20 was activated by one-pot reductive amination with aminooxy-PEGs-Ns using OligoG CF-5 / 20 (10.05 mM, DPn 25), 2 molar equivalences of aminooxy-PEGs-Ns, and 10 molar equivalence pic-BH3 added twice (in total 20x) to compensate its time-dependent degradation. Aminooxy-PEGs-Ns was left at room temperature for 20 min before its use to acquire density solution corresponding to around 20° C. The reaction mixture left for 6 days and additional 10 molar equivalence of pic-BHs was added to the mixture 72 h from the beginning of the reaction’s setup. The reaction was terminated by dialysis (MWCO=3.5 kDa) against NaCI (50 mM, 2x with approximately 1 h per shift) to convert it on sodium form and was conducted until the insoluble pic-BHswas removed. Followed by dialysis against MQ-water until conductivity was below 2 pS / cm, and finally freeze-dried. Synthesis of AmpB-C-DBCO amide Amphotericin B (125 mg, 135 pmol, Sigma Aldrich), was dissolved in DMSO (12.5 ml, Merck Millipore) and stirred under Ar, protected from light. A solution of triethylamine (40 pl, 287 pmol, 2.1 equiv., Sigma Aldrich), and DBCO-NH2 (80 mg, 289 pmol, 2.1 equiv., Sigma Aldrich) in DMSO (6.25 ml) was added, followed by a solution of PyBOP (150 mg, 288 pmol, 2.1 equiv., Sigma Aldrich). The reaction was stirred at room temperature for 6h and sampled for MS analysis. The reaction was then quenched by pouring into a solution of 0.2 M K2CO3(aq) (100 ml, Sigma Aldrich). The precipitate was isolated by centrifugation and washed 0.2M K2CO3(aq) (2x45 ml), where each wash constituted re-dispersing the precipitated pellet using vortex mixing followed by centrifugation. The pellet was then dissolved in acetone (50 ml, Merck), and centrifuged to remove any insoluble matter. The yellow solution was transferred to a round bottom flask, and the solvent removed under reduced pressure to give the desired product as a bright yellow solid (160 mg) in a quantitative yield. ESI MS observed m / z 1182,6 expected 1182.6 for [M+H]+characterised the product as a mixture of two diastereomers.

[0385] Synthesis of AmpB-C-DBCO-PEGs-OligoG

[0386] AmpB-C-DBCO (13 mg, 11.02 pmol) was dissolved in DMSO (2 ml, Merck) and added to a solution of OligoG CF5 / 20 PEG5-N3 (61 mg, 11.6 pmol) dissolved in water (6 ml). The cloudy solution was stirred, protected from light at room temperature for 17 h, then diluted with water 4 ml. The solution was dialysed against water (5 x1 h x 1 L) in Specta / Por type 1 (6 / 8 KDa cutoff). The now clear yellow solution was freeze dried yielding the desired product as a pale-yellow powder (72.6 mg) in a 97% yield. The material was characterised by DOSY NMR which confirmed click reaction had occurred.

[0387] EXAMPLE 2 - Growth inhibitory effects of AmpB-C-DBCO-PEGs-OligoG and precursors on Candida albicans

[0388] • The test strain used was Candida albicans CCLIG 39343.

[0389] • Preparation of preculture: 100 l from a freeze stock of C. albicans CCLIG 39343 was inoculated to 6 ml YPD medium (Sigma, Y1375) in a 50 ml tube and cultivated at 37°C for 18 hours. The culture was then diluted in 0.5x YPD medium to an GD600 of 0.25.

[0390] • 30 pl of the preculture was inoculated to 96-well plates containing 120 pl 0.5xYPD medium with serial dilutions (0-25 pM) of the test compounds. The plates were incubated for 22 hours at 37°C and GD600 measured at regular intervals within the incubation period. Test compounds as in Table 1, indicated molecular weights used for calculation of assay concentrations (25 pM as the highest concentration)

[0391] Results shown in Figure 2. Calculated MIC as shown in Table 2

[0392] Inhibitory effect on growth of Candida: AmpB-C-DBCO-PEGs-OligoG approx. 6x less active than free AmpB (calculated using the estimation of 20% AmpB in AmpB-C-DBCO-PEGs- OligoG conjugate on a weight basis).

[0393] EXAMPLE 3- Cytotoxic effects of AmpB-C-DBCO-PEGs-OligoG, precursors, and cytotoxic controls

[0394] • LDH (membrane leakage) and MTT assay (metabolic activity, viability)

[0395] • 48 hours exposure, Digitonin as positive control

[0396] • Concentration gradient up to 1 mg / ml

[0397] The following standard protocol was used with HepG2 cells (liver) and LLC-PK1 cells (kidney). Cells are kept in a sub confluent state by routinely passaging twice or three times a week to seeding densities between 6 x 104- 1 x 105cells / cm2according to supplier’s instructions. Dilute cells to a density of 5 x 105cells / mL in RPMI1640 (10% FBS) cell culture media. Plate 100 pL cells / well. Incubate plates for 24 hr at 5% CO2, 37°C and 95% humidity. Cells are grown to approximately 80% confluence.

[0398] Remove time zero plate from the incubator and add 100 pL fresh culture media to all wells. Then add 10 pL of 20X Triton-X-100 to the positive control wells for a final concentration of 0.1% Triton-X-100. Let the plate set for 10 minutes at room temperature. Remove 50 pL of media from each well and transfer it to another plate (Greiner, 655163), maintaining plate format. Transfer 25 pL if half area plates are used for LDH assay (Costar, 3695). Use this plate immediately for the LDH assay. Remove remaining media from original plate and discard. Add 200 pL fresh media to all wells. Add 50 pL MTT to all wells. Cover with aluminum foil and incubate at 37°C for 3-4 hrs. Aspirate and discard media. Add 200 pL DMSO to all wells to solubilize the MTT formazan crystals. Add 25 pL glycine buffer to all wells. Cover with aluminum foil and place on shaker to mix for 10 minutes in room temperature. Read absorbance at 570 nm on plate reader using a reference wavelength of 680 nm.

[0399] Test Samples and Positive Control Addition are run in triplicates with 9 dilutions each Dilute the test compound in media, making a total of nine 1:4 dilutions at 2X of the desired final concentration. Add 100 pL of each sample dilution and positive control to 4, 24 and 48 hr exposure plates and place in 37°C incubator with 5% CO2 and 95% humidity for indicated time. At the end of each exposure point remove plate from the incubator and add 100 pL fresh culture media to all wells. Then add 10 pL of 20X Triton-X-100 to the positive control wells for a final concentration of 0.1% Triton-X-100. Let the plate set for 10 minutes at room temperature. Remove 50 pL of media from each well and transfer it to another plate (Greiner, 655163), maintaining plate format. Transfer 25 pL if half area plates are used for LDH assay (Costar, 3695). Use this plate immediately for the LDH assay. Remove remaining media from original plate and discard. Add 200 pL fresh media to all wells. Add 50 pL MTT to all wells. Cover with aluminum foil and incubate at 37°C for 3-4 hrs. Aspirate and discard media. Add 200 pL DMSO to all wells to solubilize the MTT formazan crystals. Add 25 pL glycine buffer to all wells. Cover with aluminum foil and place on shaker to mix for 10 minutes in room temperature. Read absorbance at 570 nm on plate reader using a reference wavelength of 680 nm. LDH Assay is adapted from Biovision LDH Cytotoxicity Assay Kit, K311-400.

[0400] Add 50 pL of the LDH Reaction Mixture to each well of transfer plate. Alternatively add 25 pL of the LDH Reaction Mixture if half area plates are used. Shake plate on an orbital shaker briefly to mix samples. Incubate at room temperature for up to 20 minutes in the dark. Read the plate on a plate reader at 490 nm using a reference wavelength of 680 nm.

[0401] All samples, positive, negative, and media controls are run in triplicate. Each well will be subtracted from its respective cell-free blank in the following calculations. The average of these three values should be used in the equations below for the positive and negative controls

[0402] MTT Assay:

[0403] % Cell Viability = sample absorbance - cell free sample blank x100 mean media control absorbance

[0404] LDH Assay:

[0405] % Total LDH = (sample absorbance - cell free sample blank)-mean media control absorbance X100 Leakage mean TritonX positive control absorbance-mean media control absorbance

[0406] Mean, SD and %CV should also be calculated for each positive control, negative control and unknown sample.

[0407] Results of membrane leakage assay shown in Figure 3.

[0408] • Approximately 50% LDH leakage at 6.5 pg / ml AmpB versus 873 pg / ml AmpB-C- DBCO-PEGs-OligoG for kidney cells

[0409] • Approximately 40% LDH leakage at 9 pg / ml AmpB versus 873 pg / ml AmpB-DBCO- PEGs-OligoG, for liver cells

[0410] • OligoG and OligoG-PEGs-Ns are not toxic even at highest concentration

[0411] • Precursor AmpB-C-DBCO is toxic above 10 pg / ml

[0412] • PAPA is toxic at the highest concentrations used, this compound was growth inhibitory in P. aeruginosa assay

[0413] Results of cell viability assay shown in Figure 4. Similar responses as in LDH assay

[0414] Approximately 65% viability at 10 pg / ml AmpB versus 873 pg / ml AmpB-C-DBCO- PEGs-OligoG for liver cells

[0415] Toxicity / viability on liver cells: AmpB-C-DBCO-PEGs-OligoG approx. 20x less toxic than free AmpB (calculated using the estimation of 20% AmpB in AmpB-DBCO-PEGs-OligoG conjugate on a weight basis).

[0416] EXAMPLE 4- Heamolytic effects of AmpB-C-DBCO-PEGs-OligoG, precursors, and cytotoxic controls

[0417] • Human blood

[0418] • AmpB and AmpB-C-DBCO dissolved in DMSO, other components in PBS

[0419] • 2x dilution series

[0420] The following standard protocol was used.

[0421] Take a 2-3 mL aliquot of the pooled blood and centrifuge 15 min at 800 x g. Collect supernatant. Keep at RT while preparing standard curve, quality controls, and total hemoglobin sample. The collected sample is used to determine plasma free hemoglobin (PFH). Add 200 pL of each calibration standard, quality control, and blank (CMH Reagent) per well on a 96 well plate. Fill 2 wells for each calibrator and 4 wells for each quality control (QC) and blank. Position test samples so as they are bracketed by QC. Add 200 pL of total blood hemoglobin (TBH) sample, prepared by combining 20 pL of the pooled whole blood and 5.0 mL of CMH Reagent. Fill 6 wells. Add 100 pL of plasma (PFH) per well on 96 well plate. Fill 6 wells. Add 100 pL of CMH Reagent to each well containing sample. Cover plate with plate sealer and gently shake on a plate shaker for 1-2 min (shaker speed settings should be vigorous enough to allow mixing, but avoid spillage and crosswell contamination; e.g. LabLine shaker speed 2-3). Read the absorbance at 540 nm to determine hemoglobin concentration. Use the dilution factor 2 for PFH samples and dilution factor 251 for TBH. Dilute pooled whole blood with Ca2+ / Mg2+free PBS to adjust total blood haemoglobin concentration to 10 ± 2 mg / mL (TBHd). Set up two racks. Rack 1 contains tubes for the test sample incubation with blood. Rack 2 contains tubes for the test sample only (no blood) control. Prepare 6 tubes for each test-sample and place 3 tubes into Rack 1 and 3 tubes into Rack 2. Place 4 tubes for the positive control, 2 tubes for the negative control and 2 tubes of the vehicle control into Rack 1. Add 100 pL of the test-sample or control in corresponding tubes in Rack 1 and Rack. Add 700 pL of Ca2+ / Mg2+free PBS to each tube in Rack 1 . Add 800 pL of Ca2+ / Mg2+free PBS into each tube in Rack 2. Add 100 pL of the whole blood prepared above to all tubes in Rack 1. Cover tubes and gently rotate to mix. Place the tubes in a water bath set at 37°C and incubate for 3 h ±15 min, mixing the samples every 30 min. Alternatively, tubes may be incubated on a tube rotator in an incubator set at 37°C. Remove the tubes from water bath or incubator. If a water bath was used, dry excess water with absorbent paper. Centrifuge the tubes for 15 min at 800 x g. Prepare a fresh set of calibrators and quality controls.

[0422] To a fresh 96 well plate, add 200 pL of blank reagent, calibrators, quality controls or total blood hemoglobin sample (TBHd) prepared by combining 400 pL of blood from above with 5.0 mL of CMH reagent. Fill 2 wells for each calibrator, 4 wells for blank and each quality control, and 6 wells for TBHd sample. As before, position all test samples between quality controls on the plate. Add 100 pL per well of test samples and controls (positive, negative, and vehicle with and without blood) prepared above. Test each sample in duplicate. Add 100 pL of CMH Reagent to each well containing sample and controls. Cover plate with plate sealer and gently shake on a plate shaker (LabLine shaker speed settings 2-3 or as appropriate for a given shaker). Read the absorbance at 540 nm to determine concentration of hemoglobin. Use the dilution factor 18 for samples and controls and dilution factor 13.5 for TBHd.

[0423] Percent Hemolysis: (Hemoglobin in test sample / TBHd) x 100 %

[0424] Results shown in Figure 5.

[0425] • AmpB gave 50% haemolysis at approx. 3 pg / ml

[0426] • AmpB-C-DBCO-PEGs-OligoG gave 50% haemolysis at approx. 350 pg / ml

[0427] • No haemolytic activity detected for OligoG, OligoG-PEGs-Na and OligoG-DBCO for any concentration tested (max 1000 pg / ml)

[0428] • PAPA gave 86 % haemolysis at max cone, tested (1000 pg / ml)

[0429] Haemolytic activity: AmpB-C-DBCO-PEGs-OligoG approx. 20x less hemolytic activity than free AmpB (calculated using the estimation of 20% AmpB in AmpB-DBCO-PEG5-OligoG conjugate on a weight basis).

[0430] EXAMPLE 5 - Preparation of AmpB-N-DBCO-PEG5-OligoG Synthesis of AmpB-N-DBCO-amide by peptide coupling

[0431] Amphotericin B (125 mg, 135 pmol, Sigma Aldrich), is dissolved in DMSO (12,5 ml, Merck Millipore) and stirring under Ar, protected from light. A solution of triethylamine (40 ul, 287 pmol, 2,1 equiv., Sigma Aldrich), and dibenzocyclooctyne-acid (96 mg, 289 pmol, 2.1 equiv., Sigma Aldrich) in DMSO (6.25 ml) is added to the stirring reaction mixture. A solution of PyBOP (150 mg, 288 pmol, 2.1 equiv., Sigma Aldrich) is then added to the stirring reaction mixture. The reaction is stirred at room temperature for an appropriate time between 1-24h until complete or conversion is slow as monitored by TLC, NMR or another appropriate analytical tool. The reaction is then quenched by pouring into a solution of 0.2 M K2CO3<aq) (100 ml, Sigma Aldrich). The crude precipitate is isolated by centrifugation and washed 0,2M K2CO3(aq) (2x45 ml), where each wash constitutes re-dispersing the precipitated pellet by vortex mixing followed by centrifugation. The crude material pellet is then dissolved in acetone (50 ml, Merck), and isolated by centrifugation to remove any insoluble matter. The crude product solution is then dried and used without further purification in the next step.

[0432] Synthesis of AmpB-N-DBCO-PEGs-OligoG AmpB-N-DBCO-amide (11.02 pmol) is dissolved in DMSO (2 ml, Merck) and added to a solution of OligoG-PEGs azide (61 mg, 11.6 pmol) dissolved in water (6 ml). The reaction is protected from light and stirred at room temperature for an appropriate time between 1-24h until complete or conversion is slow as monitored by TLC, NMR or another appropriate analytical tool. The reaction mixture is then diluted with water 4 ml. The solution is purified by an appropriate purification method, i.e. dialysis against water using an appropriate mass cut-off. The solution containing product is then dried to give the desired product.

[0433] EXAMPLE 6 - Preparation of “G-Block” alginate oligomers (oligoguluronates) of varying length

[0434] OligoG (AlgiPharma AS, Sandvika, Norway, FG = 0.89, Mn= 3200 g / mol) was acid precipitated to increase FG. OligoG was dissolved (20 mg / ml) in water (1.5 % NaCI), the pH was decreased to 3.05 using 0.1 M HCI and stored at 4 °C overnight. The solution was further centrifugated and washed several times with water (pH 3.05) and the supernatant was removed from the sample. The precipitate was dissolved (20 mg / ml) in water (1.5 % NaCI) and 1 M NaOH was added to the solution until everything was dissolved (pH 5.5). The pH was thereafter decreased to pH 3.8 by adding HCI. An acid hydrolysis was performed at 95 °C for 15 h. Prior to the acid hydrolysis, the solution was degassed using nitrogen gas for 15 min. After the acid hydrolysis the pH was increased to pH 7.0 by adding NaOH and the sample was freeze dried on a Christ alpha 1-4 LD freeze dryer (Germany) with an Edwards RV 5 vacuum pump (UK). G-blocks of varying length were prepared by injecting the sample on three serially connected Superdex30 columns (2.6 x 60 cm). The OligoG sample was dissolved (30 mg / ml) in buffer (0.1 M NaCI, pH 7.0) and filtered (0.45 pm) prior injection. Various fractions were collected and desalted using dialysis (3.5 kDa MWCO) and freeze dried. Fractions were analyzed using HPAEC-PAD to determine the degree of polymerization (DP).

[0435] EXAMPLE 7 - Preparation of Gn-PEGs-Ns conjugates

[0436] G-blocks were conjugated with aminoxy-PEGs-Ns by one-pot reductive amination. G-blocks were dissolved (10 mM) in buffer (0.5 M NaAc, pH 4.0) and aminooxy-PEGs-Nswas added to the solution (1 :1.2 molar ratio) and left on stirring overnight. The reducing agent a-picoline borane (PB) was added to the solution (10 molar ratio) and the mixture was incubated at 40 °C for 24 h. Undissolved PB was removed from the sample by dialysis (3.5 kDa MWCO). Excess aminoxy-PEGs-Ns was removed from the sample by size-exclusion chromatography (SEC). The sample was further dialyzed (to remove salt introduced by the SEC buffer, 3.5 kDa MWCO) and freeze dried.

[0437] EXAMPLE 8 - Preparation of AmpB-DBCO amide conjugates with various spacers

[0438] Synthesis of AmpB-C-Sulfo-DBCO amide

[0439] Amphotericin B (50 mg, 54 pmol, Sigma Aldrich), was dissolved in DMSO (5,0 ml, Merck Millipore) and stirred under Ar, protected from light. A solution of triethylamine (16 pl, 113 pmol, 2.1 equiv., Sigma Aldrich), and sulfo-DBCO-NH2 (49 mg, 113 pmol, 2.1 equiv., Broadpharm) in DMSO (2,5 ml) was added, followed by a solution of PyBOP (42 mg, 81 pmol, 1 ,5 equiv., Sigma Aldrich) in DMSO (2,5 ml). The reaction was stirred at room temperature for 6h and sampled for MS analysis. The reaction was then quenched by pouring into a solution of 0.2 M K2CO3(aq) (40 ml, Sigma Aldrich). The precipitate was isolated by centrifugation and washed 0.2M K2CO3<aq) (2x20 ml), where each wash constituted re-dispersing the precipitated pellet using vortex mixing followed by centrifugation. The pellet was then dissolved in acetone (20 ml, Merck), and centrifuged to remove any insoluble matter. The yellow solution was transferred to a round bottom flask, and the solvent removed under reduced pressure to give the desired product as a bright yellow solid. ESI MS observed m / z 1314.50 [M-H2O]+vs expected 1333.60 for [M+H]+characterised the product as a mixture diastereomers. Synthesis of AmpB-C-Sulfo-PEG4-DBCO amide

[0440] Amphotericin B (50 mg, 54 pmol, Sigma Aldrich), was dissolved in DMSO (5,0 ml, Merck Millipore) and stirred under Ar, protected from light. A solution of triethylamine (16 pl, 113 pmol, 2.1 equiv., Sigma Aldrich), and sulfo-PEG4-DBCO-NH2 (77 mg, 113 pmol, 2.1 equiv., Broadpharm) in DMSO (2,5 ml) was added, followed by a solution of PyBOP (42 mg, 81 pmol, 1 ,5 equiv., Sigma Aldrich) in DMSO (2,5 ml). The reaction was stirred at room temperature for 6h and sampled for MS analysis. The reaction was then quenched by pouring into a solution of 0.2 M K2CO3(aq) (40 ml, Sigma Aldrich). The precipitate was isolated by centrifugation and washed 0.2M K2CO3<aq) (2x20 ml), where each wash constituted re-dispersing the precipitated pellet using vortex mixing followed by centrifugation. The pellet was then dissolved in acetone (20 ml, Merck), and centrifuged to remove any insoluble matter to give the desired product as a bright yellow solid (79 mg, 93% yield). ESI MS observed m / z 1561.66 [M-H2O]+vs expected 1580.75 for [M+H]+characterised the product as a mixture diastereomers.

[0441] Synthesis of AmpB-N-Cs-DBCO amide

[0442] Amphotericin B (125 mg, 135 pmol, Sigma Aldrich), was dissolved in NMP (12,5 ml, Merck Millipore) and stirred under Ar, protected from light. A solution of diisopropylethyamine (56 pl, 284 pmol, 2.1 equiv., Sigma Aldrich), and DBCO-Ce-NHS ester(122 mg, 285 pmol, 2.1 equiv., Broadpharm) in NMP (12,5 ml) was added. The reaction was stirred at room temperature for 22 h and sampled for MS analysis. The reaction was then quenched by pouring into a solution of 4,5 wt% NaCI(aq) (100 ml, Merck). The precipitate was isolated by centrifugation and washed 4,5 wt% NaCI(aq) (2x20 ml), where each wash constituted re-dispersing the precipitated pellet using vortex mixing followed by centrifugation. The pellet was then washed with acetone (2x 50 ml, Merck), dried under reduced pressure to give the desired product as a bright yellow solid (198 mg I quantitative yield). ESI MS observed m / z 1238.62 [M vs expected 1238,61 for [M]’ characterised the product as a mixture diastereomers.

[0443] Synthesis of AmpB-N-PEG2-DBC0 amide

[0444] Amphotericin B (50 mg, 54 pmol, Sigma Aldrich), was dissolved in NMP (5,0 ml, Merck Millipore) and stirred under Ar, protected from light. A solution of diisopropylethyamine (20 pl, 113 pmol, 2.1 equiv., Sigma Aldrich), and DBCO-PEG2-NHS ester (59 mg, 113 pmol, 2.1 equiv., Broadpharm) in NMP (5 ml) was added. The reaction was stirred at room temperature for 22 h and sampled for MS analysis. The reaction was then quenched by pouring into a solution of 4,5 wt% NaCI(aq) (40 ml, Merck). The precipitate was isolated by centrifugation and washed 4,5 wt% NaCI(aq) (2x5 ml), where each wash constituted re-dispersing the precipitated pellet using vortex mixing followed by centrifugation. The pellet was then washed with acetone (2x 20 ml, Merck), dried under reduced pressure to give the desired product as a bright yellow solid (105 mg I quantitative yield). ESI MS observed m / z 1352,64 [M-H2O]+vs expected 1370.67 for [M+H]+characterised the product as a mixture diastereomers.

[0445] Synthesis of AmpB-N-PEG4-DBC0 amide

[0446] Amphotericin B (50 mg, 54 pmol, Sigma Aldrich), was dissolved in NMP (5,0 ml, Merck Millipore) and stirred under Ar, protected from light. A solution of diisopropylethyamine (20 pl, 113 pmol, 2.1 equiv., Sigma Aldrich), and DBCO-PEG4-NHS ester (77 mg, 113 pmol, 2.1 equiv., Broadpharm) in NMP (5 ml) was added. The reaction was stirred at room temperature for 22 h and sampled for MS analysis. The reaction was then quenched by pouring into a solution of 4,5 wt% NaCI(aq) (40 ml, Merck). The precipitate was isolated by centrifugation and washed 4,5 wt% NaCI(aq) (2x5 ml), where each wash constituted re-dispersing the precipitated pellet using vortex mixing followed by centrifugation. The pellet was then washed with acetone (2x 20 ml, Merck), dried under reduced pressure to give the desired product as a bright yellow solid (105 mg / quantitative yield). ESI MS observed m / z 1457.725 [M]+vs expected 1457.7245 for [M]+characterised the product as a mixture diastereomers.

[0447] Amphotericin B (50 mg, 54 pmol, Sigma Aldrich), is dissolved in DMSO (5,0 ml, Merck Millipore) and stirred under Ar, protected from light. A solution of triethylamine (16 pl, 113 pmol, 2.1 equiv., Sigma Aldrich), and DBCO-PEG2-NH2 (49 mg, 113 pmol, 2.1 equiv., Broadpharm) in DMSO (2,5 ml) is added, followed by a solution of PyBOP (42 mg, 81 pmol, 1 ,5 equiv., Sigma Aldrich) in DMSO (2,5 ml). The reaction is stirred at room temperature for 6h and sampled for MS analysis. The reaction is then quenched by pouring into a solution of 0.2 M K2CO3(aq) (40 ml, Sigma Aldrich). The precipitate is isolated by centrifugation and washed 0.2M K2CO3<aq) (2x20 ml), where each wash constitutes re-dispersing the precipitated pellet using vortex mixing followed by centrifugation. The pellet is then dissolved in acetone (20 ml, Merck), and centrifuged to remove any insoluble matter. The yellow solution is transferred to a round bottom flask, and the solvent removed under reduced pressure to give the desired product as a bright yellow solid.

[0448] Synthesis of AmpB-C-PEG-DBCO amide

[0449] Amphotericin B (50 mg, 54 pmol, Sigma Aldrich), is dissolved in DMSO (5,0 ml, Merck Millipore) and stirred under Ar, protected from light. A solution of triethylamine (16 pl, 113 pmol, 2.1 equiv., Sigma Aldrich), and DBCO-PEG4-NH2 (59 mg, 113 pmol, 2.1 equiv., Broadpharm) in DMSO (2,5 ml) is added, followed by a solution of PyBOP (42 mg, 81 pmol, 1 ,5 equiv., Sigma Aldrich) in DMSO (2,5 ml). The reaction is stirred at room temperature for 6h and sampled for MS analysis. The reaction is then quenched by pouring into a solution of 0.2 M K2CO3(aq) (40 ml, Sigma Aldrich). The precipitate is isolated by centrifugation and washed 0.2M K2CO3<aq) (2x20 ml), where each wash constitutes re-dispersing the precipitated pellet using vortex mixing followed by centrifugation. The pellet is then dissolved in acetone (20 ml, Merck), and centrifuged to remove any insoluble matter. The yellow solution is transferred to a round bottom flask, and the solvent removed under reduced pressure to give the desired product as a bright yellow solid.

[0450] Other AmpB-C-PEG-DBCO amide conjugates with PEG spacers of other lengths may be prepared analogously.

[0451] EXAMPLE 9 - Preparation of AmpB-N-DBCO-PEGs-Gn and AmpB-C-DBCO-PEGs-Gn

[0452] AmpB-N-DBCO-PEGs-Gn

[0453] AO-AmpB conjugates containing alginate of different numbers of residues (DP) were prepared by copper free click chemistry. AmpB-DBCO conjugates were dissolved in DMSO and the pegylated G-blocks of length n were dissolved in H2O. The two solutions were then mixed so that the reaction mixture was 40 % DMSO and left while stirring in RT for 24 h. All reactions were performed with a molar ratio of 1 :1.2 (20 % excess of AmpB-DBCO). The reaction solution was further dialyzed using 3.5 kDa MWCO, freeze dried and dissolved in 100 % DMSO to separate the solute and the precipitate. The solute contains the excess unconjugated DMSO-AmpB, while the precipitate contains the AO-AmpB conjugate. The AO-AmpB conjugates (Table 3) were further dissolved in water, dialyzed (3.5 kDa MWCO), freeze dried and characterized using NMR. Gnspecifies the number (n) of residues in the oligomer. NMR samples were prepared by dissolving approximately 2 mg in 200 pl D2O and transferred to a 5 mm NMR tube. The sample was inserted into a Bruker Avance III HD 800 MHz spectrometer (Bruker BioSpin AG, Falladen, Switzerland) using a 5 mm Z-gradient CP-TCI (H / C / N) cryogenic probe. The spectra were recorded using TopSpin 4.0.8 software (Bruker BioSpin) and processed and analyzed with TopSpin 4.3.0 software (Bruker BioSpin). The 800 MHz NMR spectrometer is located at the NV-NMR-Center / Norwegian University of Science and Technology (NTNU).

[0454] Table 3. An overview of the different AmpB-L-Gnconjugates prepared

[0455] Gn-PEGs-Ns conjugates AmpB-DBCO conjugates AmpB-L-Gnconjugates

[0456] G4-PEG5-N3 AmpB-CONH-DBCO AmpB-C-DBCO-PEG5-G4

[0457] G6-PEG5-N3 AmpB-CONH-DBCO AmpB-C-DBCO-PEG5-G6

[0458] G8-PEG5-N3 AmpB-CONH-DBCO AmpB-C-DBCO-PEG5-G8

[0459] G10-PEG5-N3 AmpB-CONH-DBCO AmpB-C-DBCO-PEGs-G

[0460] OligoG-PEG5-N3AmpB-CONH-DBCO AmpB-C-DBCO-PEGs-

[0461] OligoG

[0462] G4-PEG5-N3 AmpB-NHCO-DBCO AmpB-N-DBCO-PEG5-G4

[0463] G6-PEG5-N3 AmpB-NHCO-DBCO AmpB-N-DBCO-PEG5-G6

[0464] G8-PEG5-N3 AmpB-NHCO-DBCO AmpB-N-DBCO-PEG5-G8

[0465] G10-PEG5-N3 AmpB-NHCO-DBCO AmpB-N-DBCO-PEGs-G

[0466] OligoG-PEG5-N3AmpB-NHCO-DBCO AmpB-N-DBCO-PEGs-

[0467] OligoG

[0468] The calculated yield for AmpB-L-Gw-using AmpB-CONH-DBCO and AmpB-NHCO-DBCO was found to be 74 % and 75 % respectively.

[0469] EXAMPLE 10 - Preparation of AmpB-L-AO conjugates

[0470] AmpB-N-Spacer-DBCO-PEG-Alginate Oligomer

[0471] AmpB-C-Spacer-DBCO-PEG-Alginate Oligomer Wherein the spacer is as defined herein. Wherein any number of PEG units are used. Wherein any alginate oligomer is used.

[0472] AmpB-L-AO conjugates are prepared by copper free click chemistry, where any AO-PEGn-Na and any AmpB-C / N-Spacer-DBCO can be combined. AmpB-C / N-Spacer-DBCO conjugates are dissolved in DMSO and the Ns-pegylated alginate oligomers are dissolved in H2O. The two solutions are then mixed so that the reaction mixture is 40 % DMSO and stirred at RT for 24 h. All reactions are performed with a molar ratio of 1 :1.2 (20 % excess of AmpB-C / N-Spacer- DBCO). The reaction solution is dialyzed using 3.5 kDa MWCO, freeze dried and dissolved in 100 % DMSO to separate the solute and the precipitate. The solute contains the excess unconjugated DMSO-AmpB, while the precipitate contains the AmpB-L-AO conjugates. The AmpB-L-AO conjugates are redissolved in water, dialyzed (3.5 kDa MWCO), freeze dried and characterized using NMR.

[0473] The invention extends to any of the intermediate AmpB-DBCO conjugates and AO-PEG-N3 conjugates explicitly disclosed herein, as well as any combination of any AmpB-DBCO conjugates and any AO-PEG-N3 conjugates to form an AmpB-L-AO conjugate.

[0474] EXAMPLE 11 - Growth inhibitory effects of AmpB-C-DBCO-PEGs-OligoG, AmpB-N- DBCO-PEGs-OligoG, AmpB-L-Gnconjugates, and precursors on Candida albicans

[0475] • The test strain used was Candida albicans CCUG 39343.

[0476] • Preparation of preculture: 100 pl from a freeze stock of C. albicans CCUG 39343 was inoculated to 6 ml YPD medium (Sigma, Y1375) in a 50 ml tube and cultivated at 37°C for 18 hours. The culture was then diluted in 0.5x YPD medium to an OD600 of 0.25.

[0477] • 30 pl of the preculture was inoculated to 96-well plates containing 120 pl 0.5xYPD medium with serial dilutions in the range of 0-25 pM for AmpB and 0-100 pM for the other test compounds. The plates were incubated for 22 hours at 37°C and GD600 measured.

[0478] Test compounds as in Table 4, indicated molecular weights used for calculation of assay concentrations (25 pM as the highest concentration for AmpB, 100 pM for the other test compounds). Structures of AmpB-L-Gnconjugates as described in Table 3.

[0479] Results are shown in Figures 6 and 7. EXAMPLE 12- Cytotoxic effects of AmpB-C-DBCO-PEGs-OligoG, AmpB-N-DBCO-PEGs- OligoG. AmpB-L-Gn conjugates, and precursors

[0480] • Promega CellTiterGlo assay (metabolic activity, viability)

[0481] • 48 hours exposure, Digitonin as positive control

[0482] • Concentration gradient up to 150 pM

[0483] The following protocol was used with HepG2 (liver) and LLC-PK1 cells (kidney)

[0484] Cells are maintained in a sub-confluent state by routine passaging two to three times per week to seeding densities of 6 * 104— 1 x 105cells / cm2according to the supplier's instructions. For cytotoxicity testing, HepG2 cells are diluted to a density of 2 x 105cells / mL in RPMI 1640 cell culture medium with 10% FBS, 2 mM L-glutamine, and 100 U / rnL PenStrep, while LLC-PK1 cells are diluted to a density of 5 x 104cells / mL in Medium 199 with 3% FBS, 2 mM L-glutamine, and 100 U / rnL PenStrep. For preparation of cell culture assay plates, 30 pL of each cell suspension is seeded per well in 384-well microplates (Revity). The cell culture plates are incubated for 24 hours at 37°C in 5% CO2and 95% humidity.

[0485] After 24 hours, water-soluble samples are diluted to appropriate concentrations in cell culture medium and transferred to V-bottom 96-well plates (Greiner). 5 pl per well of the diluted sample are then transferred to the cell culture plates using a Beckman Coulter i7 robotic liquid handling instrument. AmpB is dissolved in DMSO and added to the cell culture plates using a Tecan D300e digital dispensing instrument. The cell culture plates are transported back to the incubator after sample addition and incubated for 48 hours at 37°C in 5% CO2and 95% humidity.

[0486] Cell viability assessed is by reading luminescence after 10 min incubation with the CellTiterGlo 2.0 reagent (20 pL / well, mixed 1 :1 with PBS prior to addition) in a Beckman Coulter liquid handlig robotic system with an integrated Molecular Devices SpectraMax i3x microplate reader. Cell viability is calculated based on the luminescence signal of the samples relative to the average luminance signal of plate internal control groups only added cell culture medium as samples.

[0487] Structures of AmpB-L-Gnconjugates as described in Table 3. Results are shown in Figures 8 and 9. All AmpB-alginate oligomer conjugates tested show significantly less toxicity than AmpB. This should allow higher doses, as compared to AmpB, to be administered, e.g. doses at which effective growth inhibition is achieved. In practice, this would help counter the comparatively more modest growth inhibitory effects of the AmpB- alginate oligomer conjugates as compared to AmpB. As such, the AmpB-alginate oligomer conjugates may be used to provide the same antimycotic efficacy as AmpB, but their formulation may be greatly simplified on account of their markedly increased water solubility.

Claims

CLAIMS1 . An antimycotic polyene-alginate oligomer conjugate comprising an antimycotic polyene connected covalently to at least one alginate oligomer, wherein each alginate oligomer in the conjugate has 2 to 100 monomer residues, and wherein one or other terminal uronic acid residues of each alginate oligomer in the conjugate is connected to the antimycotic polyene via a direct covalent bond or a covalent molecular linker, or a pharmaceutically acceptable salt, solvate, hydrate, diastereoisomer, tautomer, enantiomer, or active metabolite thereof.

2. The antimycotic polyene-alginate oligomer conjugate of claim 1 , wherein said conjugate comprises a polyene antimycotic having a structure Wn-X-O-Y-Zm, whereinW is a carboxylic acid groupX is an amphipathic polyene cyclic carboxylic ester aliphatic ring comprising one or more regions of conjugated double bonds and one or more regions of saturated bonds, optionally wherein two or more carbon atoms of the ring are linked by an O, N or S atom to form a heterocyclic group, optionally wherein one or more of the carbon atoms of the ring is part of a carbonyl group, and wherein one or more hydrogen atoms in a saturated region is substituted with a hydroxyl group, a C1-8 carbonyl containing group, a linear or branched, unsubstituted, hydroxyl substituted, halo substituted, aminoacetophenone substituted, and / or N-methyl-aminoacetophenone substituted, C1-8 alkyl, C1-8 alkenyl, C1-8 alkynyl or C1-8 alkoxy group, or an unsubstituted, hydroxyl substituted, or halo substituted, C3-8 aryl or C3-8 arlyoxy group, andO is an O-glycosidic bondY is a monosaccharideZ is an amine group n is an integer of 1-10 m is an integer of 1-10 and at least one alginate oligomer connect covalently thereto, wherein one or other terminal uronic acid residues of each alginate oligomer in the conjugate is connected to at least one W and / or at least one Z of the antimycotic polyene via a direct covalent bond or a covalent molecular linker.

3. The antimycotic polyene-alginate oligomer conjugate of claim 2, wherein the amphipathic polyene cyclic carboxylic ester aliphatic ring has at least 20 carbon atoms in its backbone.

4. The antimycotic polyene-alginate oligomer conjugate of claim 2 or claim 3, wherein the amphipathic polyene cyclic carboxylic ester aliphatic ring contains at least one series of conjugated double bonds having at least 3 conjugated double bonds.

5. The antimycotic polyene-alginate oligomer conjugate of any one of claims 2 to 4, wherein the amphipathic polyene cyclic carboxylic ester aliphatic ring carries a single carboxylic acid group.

6. The antimycotic polyene-alginate oligomer conjugate of any one of claims 2 to 5, wherein Y is an amino monosaccharide carrying a single amine group7. The antimycotic polyene-alginate oligomer conjugate of any one of claims 1 to 6, wherein Y is mycosamine or perosamine.

8. The antimycotic polyene-alginate oligomer conjugate of any one of claims 1 to 6, wherein the polyene antimycotic is selected from the group consisting of amphotericin B, nystatin, natamycin (pimaricin), candidicin, rimocidin, candidin, hamycin, perimycin, tricomycin (hachimycin), partricin, and tetramycin.

9. The antimycotic polyene-alginate oligomer conjugate of any one of claims 5 to 8, wherein the one or other terminal uronic acid residues of an alginate oligomer is connected to the single carboxylic acid group and / or the single amine group of the amino monosaccharide via a direct covalent bond or a covalent molecular linker.

10. The antimycotic polyene-alginate oligomer conjugate of claim 9, wherein the conjugate comprises no greater than one alginate oligomer.

11. The antimycotic polyene-alginate oligomer conjugate of any one of claims 1 to 10, wherein the alginate oligomer has a number of monomer residues, a degree of polymerisation (DP), or a number average degree of polymerisation (DPn) of(i) 4 to 100, 4 to 75, 4 to 50, 4 to 35, 4 to 30, 4 to 25, 4 to 22, 4 to 20, 4 to 18, 4 to 16 or 4 to 14.(ii) 6 to 50, 6 to 35, 6 to 30, 6 to 25, 6 to 22, 6 to 20, 6 to 18, 6 to 16 or 6 to 14,(iii) 8 to 50, 8 to 35, 8 to 30, 8 to 25, 10 to 25, 10 to 22, 10 to 20, 10 to 18, or 10 to 15, or(iv) 20 to 100, 20 to 90, 20 to 80, 20 to 75, 20 to 70, 20 to 65, 20 to 60 , 20 to 55, 20 to 50, 20 to 45, 20 to 40, 20 to 35, 20 to 30 or 20 to 25, or(v) 30 to 100, 30 to 90, 30 to 80, 30 to 75, 30 to 70, 30 to 65, 30 to 60, 30 to 55, 30 to 50, 30 to 45, 30 to 40 or 30 to 35.

12. The antimycotic polyene-alginate oligomer conjugate of any one of claims 1 to 11, wherein the alginate oligomer has at least 70% G residues, or at least 80%, or at least 85%, or at least 90%, or at least 95% G residues.

13. The antimycotic polyene-alginate oligomer conjugate of any one of claims 1 to 12, wherein the alginate oligomer has at least 70% M residues, or at least 80%, or at least 85%, or at least 90%, or at least 95% M residues.

14. The antimycotic polyene-alginate oligomer conjugate of any one of claims 1 to 13, wherein one or both of the terminal uronic acid residues in the alginate oligomer is independently a guluronate residue or a mannuronate residue.

15. The antimycotic polyene-alginate oligomer conjugate of any one of claims 1 to 14, wherein the terminal uronic acid residue covalently conjugated to the antimycotic polyene is at the reducing end of the alginate molecule.

16. The antimycotic polyene-alginate oligomer conjugate of any one of claims 1 to 14, wherein the direct covalent bond is part of an organic functional group selected from the group consisting of ester, carbonate ester, orthoester, ketone, ketal, hemiketal, ketene, ether, acetal, hemiacteal, peroxy, methylenedioxy, carbamate, amide, amine, amine oxide, hydroxamic acid, hydroxylamine, imine, imide, imidate, nitrone, azine, azide, azo, oxime, carbodiimide, carbazone, hydrozone, dihydropyridazine, triazole, disulfide, sulfoxide, sulfonyl, thioamide, thioester, thioether, thioketone, thioketal, sulphonate ester, dithiocarbamate, semicarbazone, phosphine and phosphodiester functional groups.

17. The antimycotic polyene-alginate oligomer conjugate of any one of claims 1 to 16, wherein one or more bonds selected from a bond between the antimycotic polyene and the linker, a bond between the terminal uronic acid of the alginate oligomer and the linker and abond within the linker is within a functional group formed by a orthogonal bioconjugation technique18. The antimycotic polyene-alginate oligomer conjugate of claim 17, wherein the orthogonal bioconjugation technique is selected from the group consisting of Cu-catalysed Azide-Alkyne Cycloaddition (CuAAC), the inverse electron-demand Diels-Alder reaction (iEDDA) (e.g. the Tetrazine Ligation reaction), Strain-Promoted Alkyne-Azide Cycloaddition (SPAAC), Strain Promoted Alkyne Nitrone cycloaddition (SPANC) and the Staudinger Ligation Reaction.

19. The antimycotic polyene-alginate oligomer conjugate of any one of claims 1 to 18, wherein one or more bonds selected from a bond between the antimycotic polyene and the linker, a bond between the terminal uronic acid of the alginate oligomer and the linker and a bond within the linker is(i) within a triazole group which is the reaction product of a terminal alkyne and an azide-bearing organic compound, or the reaction product of a strained cycloalkyne and an azide-bearing organic compound,(ii) within a dihydropyridazine or pyridazine group which is the reaction product of a tetrazine and a dienophile,(iii) within an isoxazolidine group which is the reaction product of a nitrone and a strained cycloalkyne.

20. The antimycotic polyene-alginate oligomer conjugate of any one of claims 1 to 19, wherein the linker may be or comprise as a portion thereof(i) a C2-30 straight chain, branched, cyclic or polycyclic, saturated or unsaturated aliphatic group or a C2-30 aromatic group or combinations thereof,(ii) a side chain or heteroatom substituted form of a C2-30 straight chain, branched, cyclic or polycyclic, saturated or unsaturated aliphatic group, or of a C2-3oaryl group,(iii) a polyethylene glycol or a polypropylene glycol(iv) an amino acid or a peptide;(v) a monosaccharide other than guluronate or mannuronate or oligosaccharide formed therefrom;(vi) a ribonucleotide or a deoxyribonucleotide;(vii) an acetyl, succinyl, aconityl (c / s or trans), glutaryl, methylsuccinyl, trimellityl cysteamine, penicillamine, N-(2-mercaptopropionyl)glycine, 2-mercaptopropionic acid, homocysteine, 3-mercaptopropionic acid, aminohexanoic acid, or deamino-penicillamine group’(viii) an artificial polymeric molecule selected from polyethylene, polypropylene, polystyrene, polyvinyl chloride, poly lactic-co-glycolic acid (PLGA).

21. The antimycotic polyene-alginate oligomer conjugate of claim 20, wherein said polyethylene glycol has the structure of formula II,(Formula II) and wherein n is an integer of 2 to 25, preferably 2 to 20, 2 to 15, 2 to 10, 2 to 5, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22 or 24.

22. The antimycotic polyene-alginate oligomer conjugate of any one of claims 1 to 21 , wherein the linker may be considered to have the formula E-F or F-E, wherein the E is a portion resulting from orthogonal bioconjugation, preferably those defined in claim 18, and F is one or more of the covalent linker portions defined in claims 20 and 21.

23. The antimycotic polyene-alginate oligomer conjugate of claim 22, wherein F is a linker portion as defined in claim 20 (i)-(iii) and claim 21 and E is(i) a triazole reaction product of a strained cycloalkyne and an azide-bearing organic compound, or(ii) a dihydropyridazine or pyridazine reaction product of a tetrazine and a dienophile, or(iii) an isoxazolidine reaction product of a nitrone and a strained cycloalkyne.

24. The antimycotic polyene-alginate oligomer conjugate of any one of claims 1 to 23 having the structureor an equivalent structure in which conjugation is instead, or in addition, via an amide bond formed from the amine group of the monosaccharide residue, preferablywherein n is an integer of 1 to 40, Ri and R2 may independently be methyl, ethyl, propyl, hydroxyl, methoxy, ethoxy or halo, A and Z are heteroatoms, X-Y-B is a repeating unit of C and N, S, P or O, preferably PEG, Spacer is a straight or branched, unsaturated or unsaturated, hydrocarbon chain of Cn, or a heteroatom substituted form thereof, each optionally comprising one or more amide, amine, aldehyde, ketone, ester, or sulpho groups, or a PEG group with an n repeating units, optionally comprising one or more amide, amine, aldehyde, ketone, ester, or sulpho groups, and the alginate oligomer is as defined in any of claims 12 to 15.

25. A pharmaceutical composition comprising an antimycotic polyene-alginate oligomer conjugate as defined in any one of claims 1 to 24 and a pharmaceutically acceptable excipient, carrier or diluent.

26. A method for the preparation of an antimycotic polyene-alginate oligomer conjugate as defined in any one of claims 1 to 24, said method comprising(ia) providing an alginate oligomer and an antimycotic polyene and forming a direct covalent bond between a molecular group on the antimycotic polyene and a molecular group on one or other terminal uronic acid residue of the alginate oligomer; or(ib) providing an alginate oligomer, an antimycotic polyene and a covalent molecular linker and forming a direct covalent bond between a molecular group on one or other terminal uronic acid residue of the alginate oligomer and a molecular group on the linker molecule and forming a direct covalent bond between a molecular group on the antimycotic polyene and a molecular group on the linker molecule; or(ic) providing an alginate oligomer and an antimycotic polyene wherein one or both carry a covalent molecular linker molecule, or portion thereof, covalently bonded thereto, wherein if the alginate oligomer carries a linker molecule the linker molecule is covalently connected to one or other terminal uronic acid residue of the alginateoligomer, and covalently linking one or other terminal uronic acid residue of the alginate oligomer to the antimycotic polyene via at least one of the linker molecules or portions thereof; or(id) providing an alginate oligomer and a first portion of a covalent molecular linker and forming a direct covalent bond between a molecular group on one or other terminal uronic acid residue of the alginate oligomer and a molecular group on the first portion of the linker molecule, and providing an antimycotic polyene and a second portion of the covalent molecular linker and forming a direct covalent bond between a molecular group on the antimycotic polyene and a molecular group on the second portion of the linker molecule, and forming a direct covalent bond between molecular groups on the first and second portions of the linker molecule; and optionally(ii) separating at least a portion of the antimycotic polyene-alginate oligomer conjugate from the reaction mixture.

27. An antimycotic polyene-alginate oligomer conjugate as defined in any one of claims 1 to 24 or a pharmaceutical composition as defined in claim 25 for use in in the treatment or prevention of a fungal infection.

28. A method for the treatment or prevention of a fungal infection in a subject with, suspected to have, or at risk of, a fungal infection, said method comprising administering to said subject an effective amount of an antimycotic polyene-alginate oligomer conjugate as defined in any one of claims 1 to 24 or a pharmaceutical composition as defined in claim 25.

29. The antimycotic polyene-alginate oligomer conjugate or the pharmaceutical composition for use of claim 27 or the method of claim 28, wherein an effective amount of the antimycotic polyene-alginate oligomer conjugate or the composition is administered to the subject systemically.

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