BACITRACIN-ALGINATE OLIGOMER CONJUGATES

Abstract

A bacitracin-alginate oligomer conjugate including a bacitracin-class antibiotic connected covalently to at least one alginate oligomer 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 the conjugate, pharmaceutical compositions comprising the conjugate and the use thereof in a method for the treatment or prevention of a bacterial infection in a subject with, suspected to have, or at risk of, a bacterial infection.

Claims

1. A bacitracin-alginate oligomer conjugate comprising a bacitracin-class antibiotic connected covalently to at least one alginate oligomer 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 bacitracin-alginate oligomer conjugate of claim 1, wherein said bacitracin-class antibiotic is selected from the group consisting of bacitracin A1, A2, B1, B2, B3, C, D1, D2, D3 and E and functionally equivalent derivatives thereof.

3. The bacitracin-alginate oligomer conjugate of claim 1, wherein said bacitracin-class antibiotic is represented by Formula II ##STR00005## wherein Leu is leucine; Glu is glutamic acid; Lys is lysine; Orn is ornithine; Phe is phenylalanine; His is histidine; Asp is aspartic acid; Asn is asparagine; Y is valine, isoleucine, leucine or 5-methylene-isoleucine; Z is valine, isoleucine, leucine or 5-methylene-isoleucine; and X is W.sup.[1]-Cys.sup.[2] or V.sup.[1]-Thz.sup.[2]; wherein W is valine, isoleucine, leucine or 5-methylene-isoleucine and Cys is cysteine; and V is
H.sub.2N—C(R)H— wherein R is the α side chain of valine, isoleucine, leucine or 5-methylene-isoleucine; and Thz is a thiazoline ring ##STR00006##  which is 2′ coupled to V and 4′ coupled to the α-carbon of Leu.sup.[3], wherein none or one or more, of amino acids Leu.sup.[3], Glu.sup.[4], Orn.sup.[7], Phe.sup.[9], His.sup.[10] or Asp.sup.[11] is replaced by another amino acid residue which may be selected from natural or non-genetically encoded amino acids.

4. The bacitracin-alginate oligomer conjugate of claim 3, wherein said natural or non-genetically encoded amino acid is selected from the group consisting of leucine, threonine, acid, phenylalanine, arginine, histidine, lysine, asparagine, serine, cysteine, homolysine, ornithine, diaminobutyric acid, diaminopimelic acid, diaminopropionic acid, homoarginine, trimethylysine, trimethylornithine, 4-aminopiperidine-4-carboxylic acid, 4-amino-1-carbamimidoylpiperidine-4-carboxylic acid and 4-guanidinophenylalanine.

5. The bacitracin-alginate oligomer conjugate of claim 1, wherein said bacitracin-class antibiotic is selected from the group consisting of bacitracin A (A1 and/or A2) and bacitracin B (B1 and/or B2).

6. The bacitracin-alginate oligomer conjugate of claim 1, wherein said bacitracin-class antibiotic is complexed with divalent metal cations.

7. The bacitracin-alginate oligomer conjugate of claim 1, wherein said alginate oligomer has an average molecular weight of less than 35,000 Daltons.

8. The bacitracin-alginate oligomer conjugate of claim 1, wherein the alginate oligomer has a degree of polymerisation (DP), or a number average degree of polymerisation (DPn) of 4 to 100.

9. The bacitracin-alginate oligomer conjugate of claim 1, wherein the alginate oligomer has at least 70% G residues.

10. The bacitracin-alginate oligomer conjugate of claim 9, wherein at least 80% of the G residues are arranged in G-blocks.

11. The bacitracin-alginate oligomer conjugate of claim 1, wherein the alginate oligomer has at least 70% M residues.

12. The bacitracin-alginate oligomer conjugate of claim 11, wherein at least 80% of the M residues are arranged in M-blocks.

13. The bacitracin-alginate oligomer conjugate of claim 1, wherein said direct covalent bond is part of an ester, carbonate ester, orthoester, ketal, hemiketal, ether, acetal, hemiacteal, peroxy, methylenedioxy, amide, amine, imine, imide, azide, azo, oxime, sulfide, disulfide, sulfinyl, sulfonyl, carbonothioyl, thioester, phosphine or phosphodiester functional group

14. The bacitracin-alginate oligomer conjugate of claim 13, wherein said direct covalent bond is part of an ester or an amide.

15. The bacitracin-alginate oligomer conjugate of claim 1, wherein said covalent linker is or comprises molecular groups selected from: (i) an amino acid or a peptide; (ii) monosaccharide or an oligosaccharide other than guluronate or mannuronate or polymers formed therefrom; (iii) a ribonucleotide or a deoxyribonucleotide; (iv) a straight chain, branched or cyclic, substituted or unsubstituted, alkyl, alkenyl or alkynl group; (v) an acetyl, succinyl, aconityl (cis or trans), glutaryl, methylsuccinyl, trimellityl cysteamine, penicillamine, N-(2-mercaptopropionyl)glycine, 2-mercaptopropionic acid, homocysteine, 3-mercaptopropionic acid or deamino-penicillamine group

16. The bacitracin-alginate oligomer conjugate of claim 1, wherein said direct covalent bond, a functional group containing said covalent bond or said covalent molecular linker is (i) acid labile; (ii) sensitive to reactive oxygen species; and/or (iii) degraded by an enzyme secreted by a bacterium or an immune cell.

17. The bacitracin-alginate oligomer conjugate of claim 1, wherein said conjugate consists of at least one alginate oligomer covalently bonded to a bacitracin-class antibiotic via: (a) an ester bond formed from a carboxyl group on the alginate and hydroxyl group on the bacitracin, or (b) an amide bond formed from a carboxyl group on the alginate and an amine group on the bacitracin.

18. (canceled)

19. The bacitracin-alginate oligomer conjugate of claim 1, wherein the alginate oligomer contains 2 to 100 monomer residues.

20. The bacitracin-alginate oligomer conjugate of claim 16, wherein the alginate oligomer has at least 70% G residues.

21. A pharmaceutical composition comprising a bacitracin-alginate oligomer conjugate as defined in claim 1 and a pharmaceutically acceptable excipient, carrier or diluent.

22. A method for the preparation of a bacitracin-alginate oligomer as defined in claim 1, said method comprising (ia) providing an alginate oligomer and a bacitracin-class antibiotic and forming a direct covalent bond between two molecular groups thereon; or (ib) providing an alginate oligomer, a bacitracin-class antibiotic and a covalent molecular linker and forming a direct covalent bond between two molecular groups on the alginate oligomer and the linker molecule and forming a direct covalent bond between two molecular groups on the bacitracin-class antibiotic and the linker molecule; or (ic) providing an alginate oligomer and a bacitracin-class antibiotic wherein one or both carry a covalent molecular linker molecule covalently bonded thereto and covalently linking the alginate oligomer to the bacitracin-class antibiotic via at least one of the linker molecules; and optionally (ii) separating at least a portion of the bacitracin-alginate oligomer conjugate from the reaction mixture.

23. The method of claim 22, said method comprising (i) providing an aqueous solution of an alginate oligomer having an available carboxyl group; (ii) contacting said alginate solution with 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) in an amount and under conditions sufficient to activate at least one carboxyl group in the alginate oligomer; (iii) optionally contacting said carboxyl activated alginate oligomer with sulfo N-hydroxysuccinimide (sulfo-NHS) in an amount and under conditions sufficient to form an amine-reactive sulfo-NHS ester; (iv) contacting said carboxyl activated alginate oligomer of step (ii) or the amine-reactive sulfo-NHS ester of step (iii) with an bacitracin-class antibiotic having an available primary amine group in an amount and under conditions sufficient to form an amide bond between the alginate oligomer and the bacitracin-class antibiotic; and (v) separating at least a portion of the bacitracin-alginate oligomer conjugate from the reaction mixture.

24. The method of claim 22, said method comprising (i) providing a solution of an alginate oligomer having an available carboxyl group, preferable an organic (e.g. DMF and/or DMSO) solution; (ii) contacting said alginate solution with dicyclohexylcarbodiimide (DCC) in an amount and under conditions sufficient to form an O-acylisourea intermediate; (iii) contacting said O-acylisourea intermediate with an bacitracin-class antibiotic having an available hydroxyl group and 4-N,N-dimethylaminopyridine (DMAP) in amounts and under conditions sufficient to form an ester bond between the alginate oligomer and the bacitracin-class antibiotic; and (iv) separating at least a portion of the bacitracin-alginate oligomer conjugate from the reaction mixture; wherein steps (ii) and (iii) may be performed simultaneously.

25. (canceled)

26. A method for the treatment or prevention of a bacterial infection in a subject with, suspected to have, or at risk of, a bacterial infection, said method comprising administering to said subject an effective amount of a bacitracin-alginate oligomer conjugate as defined in claim 1 or a pharmaceutical composition comprising a bacitracin-alginate oligomer conjugate as defined in claim 1 and a pharmaceutically acceptable excipient, carrier or diluent.

27. The method of claim 26 wherein the bacterial infection is (i) in a wound, preferably a chronic wound; (ii) a respiratory infection in a subject suffering from an underlying respiratory disorder or condition, preferably selected from CF, COPD/COAD, or asthma; (iii) a device related infection associated with implantable or prosthetic medical devices; or (iv) a systemic infection or an infection of multiple loci within or on the subject.

28. The method of claim 26, wherein the infection is a Gram negative bacterial infection.

29. The bacitracin-alginate oligomer conjugate of claim 4, wherein said diaminobutyric acid is α,γ-diaminobutyric acid.

30. The bacitracin-alginate oligomer conjugate of claim 6, wherein said divalent metal cations are selected from the group consisting of Zn.sup.2+, Mg.sup.2+, Mn.sup.2+, and Co.sup.2+.

Description

EXAMPLES

Example 1—Preparation of Bacitracin-Alginate Oligomer Conjugates

[0224] Preparation of Aliginate Oligomers by Acid Hydrolysis

[0225] Sodium alginate, containing >60% guluronate monomers (PRONOVA UP MVG), was dissolved in dH.sub.2O (10 mg/mL), the solution adjusted to 0.01 M HCl and then placed in a water bath at 80° C. for up to 24 h. To terminate the hydrolysis, the acid hydrolysate was cooled and pH was increased to pH 7 by addition of 10 M NaOH. Solutions were lyophilised and re-suspended in minimal dH.sub.2O.

[0226] To obtain acid-hydrolysed G-fragments (AHGs) with acceptable number average degree of polymerisation as well as molecular weight distribution, the solutions were filtered once by dialysis to remove LMW oligomers and salts. Typically, the following reaction times and dialysis membrane cut-offs were used to produce AHGs with a desired molecular weight:

TABLE-US-00002 Desired MW Approx, reaction time Dialysis membrane cut-off (g/mol) (h:min) (g/mol) 2,500 21:00  1,000 7,500 9:45 2,000 15,000 5:30 8,000

[0227] The final products were lyophilised and characterised by FT-IR and gel permeation chromatography (GPC). The GPC system comprised of two TSK G5000PW.sub.XL and G3000PW.sub.XL columns (Polymer Laboratories, UK) in series, mobile phase PBS (pH 7.4) and flow rate of 1 mL/min. Molecular weight and polydispersity were calculated relative to pullulan standards. Alginate standards are not commercially available, which necessitated the use of pullulan. These calibration standards do not give correct molecular weight for alginates since GPC separates according to hydrodynamic volume, which differs with varying macromolecular architecture. Alginates are more expanded than pullulans, resulting in a six-fold overestimate of alginate's molecular weight by GPC with pullulan. Consequently, a scaling factor (divide by 6) was applied to the apparent molecular weights calculated from pullulan calibration, to adjust for different macromolecular architecture of pullulan MW standards (Andersen T., et al., 2012, Alginates as biomaterials in tissue engineering. Carbohydrate Chemistry: Chemical and Biological Approaches, Vol. 37, 232-233).

[0228] Samples for GPC were prepared in PBS (3 mg/mL) and the eluate was monitored using a differential refractometer (Optilab t-Rex (Wyatt, UK)). PL Caliber Instrument software, version 7.0.4, from Polymer Laboratories (UK) was used for data analysis.

[0229] Acid-hydrolysed G-fragments (AHGs) were generated with molecular weights between 2,500-26,000 g/mol (polydispersity 2.5-3.5). Typical characteristics of AHGs used in these studies are summarised in the table below.

TABLE-US-00003 Apparent molecular Name weight (g/mol)* PDI DP.sub.n OligoG 15,600 [2,600] 1.8 13 ‘2.5 k’ 15,000 [2,500] 1.4 13 ‘6 k’ 36,207 [6,035] 4.0 30 ‘7.5 k’ 42,000 [7,000] 2.6 35 ‘9 k’ 52,000 [8,670] 3.0 45 ‘13.5 k’  81,543 [13,590] 2.9 68 ‘15 k’  72,000 [12,000] 3.5 60 ‘16 k’  93,500 [15,580] 3.6 81 ‘20 k’ 119,362 [19,894] 3.1 100 ‘26 k’ 152,500 [25,420] 3.5 132 PRONOVA UP MVG.sup.+  651,000 [108,500] 2.5 565 *relative to pullulan MW standards; value in [ ] indicates estimated actual MW after applying scaling factor to adjust for different macromolecular architecture of pullulan MW standards. .sup.+Elutes in void volume, therefore MW is likely underestimated. OligoG CF-5/20 for conjugation was produced as described previously (Khan et al. 2012, Antimicrobial Agents and Chemotherapy 56(10), 5134-5141). OligoG is a 5-20 mer alginate oligomer with at least 85% G residues.

[0230] Ester Conjugation (‘E’)

[0231] Briefly, for 2,500 g/mol AHG conjugation to bacitracin, alginate oligomer (200 mg, 0.08 mmol) was dissolved under stirring in anhydrous DMF (4 mL) (or anhydrous DMSO for Batches 3 and later) in a 10 mL round-bottomed flask. To this, DCC (16.5 mg, 0.08 mmol), DMAP (1.6 mg, 0.01 mmol) and bacitracin (37.5 mg, 0.03 mmol; Sigma) were added, and the reaction allowed to proceed at room temperature overnight, under stirring. To stop the reaction, the mixture was poured into excess chloroform (up to 20 mL), then the resulting precipitate was collected by filtration, re-dissolved in distilled water (dH.sub.2O, 2 mL) and purified by FPLC (see below).

[0232] Amide conjugation (‘A’)

[0233] Briefly, for 2,500 g/mol AHG conjugation to bacitracin, alginate oligomer (200 mg, 0.08 mmol) was dissolved under stirring in dH.sub.2O (2 mL) in a 10 mL round-bottomed flask. To this, EDC (19.9 mg, 0.1 mmol) and sulfo-NHS (22.6 mg, 0.1 mmol) were added, and the mixture was left stirring for 15 min. Subsequently, bacitracin (37.5 mg, 0.03 mmol) was added, and the reaction mixture was left stirring for 2 h at room temperature, the stored at −20° C. until purification by FPLC (see below).

[0234] Purification of Conjugates by FPLC

[0235] Conjugates were purified from the reaction mixture by fast protein liquid chromatography (FPLC) (AKTA FPLC; Amersham Pharmacia Biotech, UK) using a pre-packed HiLoad Superdex 75 16/600 PG column with a UV detector and data analysis using Unicorn 4.0 software (Amersham Pharmacia Biotech, UK). Samples of the reaction mixture (2 mL) were injected into a 2 μL loop using PBS (pH 7.4) at 0.5 mL/min as a mobile phase. Fractions (5 mL) were collected, dialysed against de-ionised water (8 water changes) and assayed for protein content (BCA assay) before pooling fractions containing conjugate. The final conjugate was lyophilised and stored at −20° C.

Example 2—Characterisation of Alginate Oligomer-Bacitracin Conjugates

[0236] Purity, Molecular Weight and Drug Content

[0237] Alginate oligomer-bacitracin conjugates were characterised by FPLC and GPC to assess purity and estimate molecular weight, and the total drug content of the conjugate was determined by the BCA assay using free drug as calibrant.

[0238] The FPLC system described above for purification was used again for final conjugate characterisation, with a pre-packed Superdex 75 10/300 GL column. Samples (200 μL) were dissolved in PBS (pH 7.4) and injected into a 100 mL loop at 0.5 mL/min. The GPC system described above as used again for final conjugate characterisation, and the same scaling factor was applied.

[0239] Results

[0240] Using these alginate oligomers, a library of bacitracin-alginate oligomer conjugates has been prepared, with a typical reaction yield of ˜40%, with molecular weights of 19,500-83,250 g/mol (relative to pullulan molecular weight standards); containing 1-7% w/w drug loading (equivalent to 3-5 oligomers: 1 bacitracin molecule).

[0241] Batch 1

TABLE-US-00004 Initial Drug drug loading Molecular Free ratio (% w/w) Molar ratio weight drug Conjugate (% w/w) (% yield) (1 antibiotic:x AHG) (g/mol) PDI (%) OligoG-ESTER- 18.2 6.8 (37%) 7.80 19,500 2.0 62.1 bacitracin 6k AHG-ESTER- 7.9 3.7 (47%) 6.17 42,250 2.9 41.3 bacitracin 13.5k AHG- 3.5 1.0 (29%) 10.44 83,250 3.6 3.5 ESTER-bacitracin *Purified by ion exchange instead of size exclusion chromatography

[0242] Batch 2

TABLE-US-00005 Drug loading Conjugate (% w/w) 7.5 k AHG-A-bacitracin 1.5 7.5 k AHG-E-bacitracin 0.7  15 k AHG-A-bacitracin 1.7

[0243] Batch 4

TABLE-US-00006 Mn* Mw* Free drug Drug loading Conjugate (g/mol) (g/mol) PDI (%) (% w/w) Bacitracin 9,250 10,750 1.1 OligoG-A-bacitracin 10,750 27,250 2.5 0.7 4.3 OligoG 7,000 16,750 2.4

Example 3—Antimicrobial Activity

[0244] Bacterial Isolates and Strains:

[0245] The strains used for susceptibility testing include both culture collection strains and clinical isolates are recited below. Their known relevant genotypes and origin have been described by Khan et al. supra.

TABLE-US-00007 Code Isolate Description V2 Pseudomonas MDR-PSA isolate, multi drug resistant aeruginosa (301) isolate defined as being resistant to piperacillin, ceftazidime, imipenem, and gentamicin. Origin; Poland. Khan et al., supra. V3 Klebsiella Khan et al., supra. pneumoniae V5 Escherichia coli Khan et al., supra. V19 Acinetobacter Khan et al., supra. baumannii E68 Staphylococcus Staphylococcus aureus control Khan etal., aureus NCTC supra. 6571 E75 Staphylococcus Methicillin-resistant Staphylococcus aureus aureus NCTC (MRSA) control NCTC strain 12493

[0246] Determination of Antimicrobial Activity

[0247] The MICs of alginate oligomers, bacitracin and alginate oligomer conjugates thereof were determined for isolates V2, V3, VS, V19, E68 and E75 using the broth microdilution method in accordance with standard guidelines (CLSI 2012). Test organisms were suspended in Mueller Hinton cation adjusted (MH) broth (100 μL, 1-5×10.sup.4 CFU/mL) and incubated in 96-well microtitre plates in serial two-fold dilutions of the test compounds. The MIC was defined as the lowest concentration of test compound that produced no visible growth after 16-20 hours. Unless otherwise stated, results show median values of 3 experiments.

[0248] None of the alginate fractions, or OligoG showed any antimicrobial activity, whereas unconjugated bacitracin showed high antimicrobial activity against Staphylococcus aureus (Gram positive) but not the Gram negative species tested (Pseudomonas aeruginosa, Klebsiella pneumonia, Escherichia coli and Acinetobacter baumannii.

[0249] On the hand alginate oligomer conjugated bacitracin shows good antimicrobial activity against (lower MIC values) against both the Gram negative strains (in particular the E. coli strain) and the Gram positive strains.

[0250] Antimicrobial activity was greatest for conjugates containing low molecular weight alginate oligomers (including OligoG).

[0251] Batch 1

[0252] MIC Values (μg/mL) for Each Compound Tested Against a Variety of Bacteria

TABLE-US-00008 Drug loading (% w/w) V2 V3 V5 V19 V33 E68 E75 OligoG — >1024 >1024 >1024 >1024 >1024 >1024 >1024 9k AHG — >1024 >1024 >1024 >1024 >1024 >1024 >1024 13.5k AHG — >1024 >1024 >1024 >1024 >1024 >1024 >1024 16k AHG — >1024 >1024 >1024 >1024 >1024 >1024 >1024 20k AHG — >1024 >1024 >1024 >1024 >1024 >1024 >1024 26k AHG — >1024 >1024 >1024 >1024 >1024 >1024 >1024 Bacitracin — >128 >128 >128  >128 >128 64 32 OligoG-E- 18.2  >128 128 16   64 >128 128 64 bacitracin 6k AHG-E- 7.9 >128 >128 32   64 >128 128 64 bacitracin 13.5k AHG- 3.5 >128 >128 32  >128* >128 >128 >128 E-bacitracin

[0253] Batch 2

[0254] MIC Values (μg/mL) for Each Compound Tested Against a Variety of Bacteria (n=3, Median)

TABLE-US-00009 Drug loading Conjugate (% w/w) V2 V3 V5 V19 E68 E75 Bacitracin — >8192 >8192 640 640 40 20 OligoG-A-bacitracin 3:1 3.2 >256 >256 32 128 >256 >256 OligoG-E-bacitracin 3:1 0.9 >256 >256 16 128 >256 128 OligoG-A-bacitracin 2:1 1.1 >256 >256 32 256 >256 128 OligoG-E-bacitracin 2:1 1.9 >256 >256 16 128 >256 256 OligoG-A-bacitracin 1:1 5 >256 >256 64 256 >256 256 OligoG-E-bacitracin 1:1 0.6 >256 >256 64 256 >256 128 7.5k AHG-A-bacitracin 1.5 >256 >256 32 256 >256 >256 7.5k AHG-E-bacitracin 0.7 >128 >128 128 >128 — >256 15k AHG-A-bacitracin 1.7 >256 >256 128 256 256 >256 OligoG-A-bacitracin 3:1 3.2 >256 >256 32 128 >256 >256 OligoG-E-bacitracin 3:1 0.9 >256 >256 16 128 >256 128