NOVEL HYDROGEL CONJUGATES

Abstract

The present invention relates to conjugates comprising backbone moieties that are crosslinked via particular crosslinker moieties to which a plurality of drug moieties are covalently and reversibly conjugated. It also relates to their use as medicaments and their use in the diagnosis, prevention and treatment of diseases.

Claims

1. A conjugate comprising a water-insoluble hydrogel Z, wherein said conjugate comprises a plurality of moieties -L.sup.2-L.sup.1-D covalently conjugated to Z, wherein each -D is drug moiety; each -L.sup.1- is independently a linker moiety to which -D is covalently and reversibly conjugated; each -L.sup.2- is independently either a chemical bond or a spacer moiety; Z is a PEG-based hydrogel comprising a plurality of backbone moieties that are crosslinked via crosslinker moieties —CL-, either directly or via a spacer moiety —SP— between a crosslinker moiety and —CL-, and wherein —CL- is of formula (A) ##STR00117## wherein dashed lines indicate attachment to a backbone moiety or to a spacer moiety —SP—; —Y.sup.1— is of formula ##STR00118## wherein the dashed line marked with the asterisk indicates attachment to -D.sup.1- and the unmarked dashed line indicates attachment to -D.sup.2-; —Y.sup.2— is of formula ##STR00119## wherein the dashed line marked with the asterisk indicates attachment to -D.sup.4- and the unmarked dashed line indicates attachment to -D.sup.3-; -E.sup.1- is of formula ##STR00120## wherein the dashed line marked with the asterisk indicates attachment to —(C═O)— and the unmarked dashed line indicates attachment to —O—; -E.sup.2- is of formula ##STR00121## wherein the dashed line marked with the asterisk indicates attachment to -G.sup.1- and the unmarked dashed line indicates attachment to —(C═O)—; -G.sup.1- is of formula ##STR00122## wherein the dashed line marked with the asterisk indicates attachment to —O— and the unmarked dashed line indicates attachment to -E.sup.2-; -G.sup.2- is of formula ##STR00123## wherein the dashed line marked with the asterisk indicates attachment to —O— and the unmarked dashed line indicates attachment to —(C═O)—; -G.sup.3- is of formula ##STR00124## wherein the dashed line marked with the asterisk indicates attachment to —O— and the unmarked dashed line indicates attachment to —(C═O)—; -D.sup.1-, -D.sup.2-, -D.sup.3-, -D.sup.4-, -D.sup.5- and -D.sup.6- are identical or different and each is independently of the others selected from the group comprising —O—, —NR.sup.11—, —N.sup.+R.sup.12R.sup.12a—, —S—, —(S═O)—, —(S(O).sub.2)—, —C(O)—, —P(Q)R.sup.13—, —P(O)(OR.sup.13) and —CR.sup.14R.sup.14a—; —R.sup.1, —R.sup.1a, —R.sup.2, —R.sup.2a, —R.sup.3, —R.sup.3a, —R.sup.4, —R.sup.4a, —R.sup.5, —R.sup.5a, —R.sup.6, —R.sup.6a, —R.sup.7, —R.sup.7a, —R.sup.8, —R.sup.8a, —R.sup.9, —R.sup.9a, —R.sup.10, —R.sup.10a, —R.sup.11, —R.sup.12, —R.sup.12a, —R.sup.13, —R.sup.14 and —R.sup.14a are identical or different and each is independently of the others selected from the group consisting of —H and C.sub.1-6 alkyl; optionally, one or more of the pairs —R.sup.1/—R.sup.1a, —R.sup.2/—R.sup.2a, —R.sup.3/—R.sup.3a, —R.sup.4/—R.sup.4a, —R.sup.1/—R.sup.2, —R.sup.3/—R.sup.4, —R.sup.1a/—R.sup.2a, —R.sup.3a/—R.sup.4a, —R.sup.12/—R.sup.12a, and —R.sup.14/—R.sup.14a form a chemical bond or are joined together with the atom to which they are attached to form a C.sub.3-8 cycloalkyl or to form a ring A or are joined together with the atom to which they are attached to form a 4- to 7-membered heterocyclyl or 8- to 11-membered heterobicyclyl or adamantyl; A is selected from the group consisting of phenyl, naphthyl, indenyl, indanyl and tetralinyl; r1, r2, r5, r6, r13, r14, r15 and r16 are independently 0 or 1; r3, r4 are independently 0, 1, 2, 3, or 4, with the provision that r3+r4≥1; r7, r8, r9, r10, r11, r12 are independently 0, 1, 2, 3, or 4; r17, r18, r19, r20, r21 and r22 are independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; s1, s2, s4, s5 are independently 1, 2, 3, 4, 5 or 6; and s3 ranges from 1 to 900.

2. The conjugate of claim 1, wherein r3 and r4 are both 1.

3. The conjugate of claim 1 or 2, wherein r1, r2, r5 and r6 are 0.

4. The conjugate of any one of claims 1 to 3, wherein s3 ranges from 15 to 100.

5. The conjugate of any one of claims 1 to 4, wherein s3 ranges from 20 to 50.

6. The conjugate of any one of claims 1 to 5, wherein a moiety —CL- has a molecular weight ranging from 0.2 kDa to 25 kDa

7. The conjugate of any one of claims 1 to 6, wherein a moiety —CL- is selected from the group consisting of ##STR00125## ##STR00126## ##STR00127## ##STR00128## ##STR00129## ##STR00130## ##STR00131## ##STR00132## wherein dashed lines indicate attachment to a backbone moiety or to a spacer moiety —SP—.

8. The conjugate of any any one of claims 1 to 7, wherein a backbone moiety has a molecular weight ranging from 1 kDa to 20 kDa.

9. The conjugate of any one of claims 1 to 8, wherein -L.sup.1- is of formula (I): ##STR00133## wherein the dashed line indicates the attachment to a nitrogen, hydroxyl or thiol of -D; —X— is selected from the group consisting of —C(R.sup.4R.sup.4a)—, —N(R.sup.4)—, —O—, —C(R.sup.4R.sup.4a)—C(R.sup.5R.sup.5a)—, —C(R.sup.5R.sup.5a)—C(R.sup.4R.sup.4a)—, —C(R.sup.4R.sup.4a)—N(R.sup.6)—, —N(R.sup.6)—C(R.sup.4R.sup.4a)—, —C(R.sup.4R.sup.4a)—O—, —O—C(R.sup.4R.sup.4a)—, and —C(R.sup.7R.sup.7a)—, X.sup.1 is selected from the group consisting of C and S(O); —X.sup.2— is selected from the group consisting of —C(R.sup.8R.sup.8a)— and —C(R.sup.8R.sup.8a)—C(R.sup.9R.sup.9a)—; ═X is selected from the group consisting of ═O, ═S, and ═N—CN; —R.sup.1, —R.sup.1a, —R.sup.2, —R.sup.2a, —R.sup.4, —R.sup.4a, —R.sup.5, —R.sup.5a, —R.sup.6, —R.sup.8, —R.sup.8a, —R.sup.9 and —R.sup.9a are independently selected from the group consisting of —H and C.sub.1-6 alkyl; —R.sup.3 and —R.sup.3a are independently selected from the group consisting of —H and C.sub.1-6 alkyl, provided that in case one of —R.sup.3 and —R.sup.3a or both are other than —H they are connected to N to which they are attached through an sp.sup.3-hybridized carbon atom; —R.sup.7 is selected from the group consisting of —N(R.sup.10R.sup.10a) and —NR.sup.10—(C═O)—R.sup.11; —R.sup.7a, —R.sup.10, —R.sup.10a and —R.sup.11 are independently selected from the group consisting of —H and C.sub.1-6 alkyl; alternatively, one or more of the pairs —R.sup.1a/—R.sup.4a, —R.sup.1a/—R.sup.5a, —R.sup.1a/—R.sup.7a, —R.sup.4a/—R.sup.5a and —R.sup.8a/—R.sup.9a form a chemical bond; alternatively, one or more of the pairs —R.sup.1/—R.sup.1a, —R.sup.2/—R.sup.2a, —R.sup.4/—R.sup.4a, —R.sup.5/—R.sup.5a, —R.sup.8/—R.sup.8a and —R.sup.9/—R.sup.9a are joined together with the atom to which they are attached to form a C.sub.3-10 cycloalkyl or 3- to 10-membered heterocyclyl; alternatively, one or more of the pairs —R.sup.1/—R.sup.4, —R.sup.1/—R.sup.5, —R.sup.1/—R.sup.6, —R.sup.1/—R.sup.7a, —R.sup.4/—R.sup.5, —R.sup.4/—R.sup.6, —R.sup.8/—R.sup.9 and —R.sup.2/—R.sup.3 are joined together with the atoms to which they are attached to form a ring A; alternatively, R.sup.3/R.sup.3a are joined together with the nitrogen atom to which they are attached to form a 3- to 10-membered heterocycle; A is selected from the group consisting of phenyl; naphthyl; indenyl; indanyl; tetralinyl; C.sub.3-10 cycloalkyl; 3- to 10-membered heterocyclyl; and 8- to 11-membered heterobicyclyl; and wherein -L.sup.1- is substituted with -L.sup.2- and wherein -L.sup.1- is optionally further substituted, provided that the hydrogen marked with the asterisk in formula (II) is not replaced by -L.sup.2- or a substituent.

10. The conjugate of any one of claims 1 to 9, wherein -L.sup.2- is a spacer moiety.

11. The conjugate of any one of claims 1 to 10, wherein -D is an antibiotic moiety.

12. The conjugate of any one of claims 1 to 11, wherein -D is selected from the group consisting of aminoglycosides, tetracycline antibiotics, amphenicols, pleuromutilins, macrolid antibiotics, lincosamides, steroid antibiotics, antifolate antibiotics, sulfonamides, topoisomerase inhibitors, quinolones, fluoroquinolones, nitroimidazole antibiotics, nitrofuran antibiotics, rifamycins, glycopeptides, penicillins, cephalosporins, monobactams, beta-lactamase inhibitors, polymyxin antibiotics, lipopeptide antibiotics, oxazolidinon, antimicrobial peptides, antimicrobial proteins, porphyrins, azole antifungals, polyenes, antiprotozoal drugs, fosfomycin, cycloserine, and bacitracin.

13. The conjugate of any one of claims 1 to 12, wherein -D is daptomycin.

14. A pharmaceutical composition comprising the conjugate of any one of claims 1 to 13 and at least one excipient.

15. The conjugate of any one of claims 1 to 13 or the pharmaceutical composition of claim 14 for use as a medicament.

16. The conjugate of any one of claims 11 to 13 or the pharmaceutical composition of claim 14 for use in the in the diagnosis, prophylaxis or treatment of a disease that can be treated with the conjugates of the present invention.

17. The conjugate of any one of claims 11 to 13 or the pharmaceutical composition of claim 14 for use as an antibiotic.

18. The conjugate of any one of claims 11 to 13 or the pharmaceutical composition of claim 14 for use in a method of preventing or treating a joint infection.

Description

EXAMPLES

Materials and Methods

[0905] All materials were commercially available except where stated otherwise.

[0906] RP-HPLC purification:

[0907] For preparative RP-HPLC a Waters 600 controller and a 2487 Dual Absorbance Detector was used, equipped with the following column: Waters XBridge™ BEH300 Prep C.sub.1-8 10 μm, 150×30 mm, flow rate 40 mL/min. Gradients of solvent system A (water containing 0.1% TFA v/v) and solvent system B (acetonitrile containing 0.1% TFA v/v) were used. Products were detected at 215 nm. HPLC fractions containing product were pooled and lyophilized if not stated otherwise.

[0908] Flash Chromatography:

[0909] Flash chromatography purifications were performed on an Isolera One system or an Isolera Four system from Biotage AB, Sweden, using Biotage KP-Sil silica cartridges and CH.sub.2C.sub.1-2/MeOH, CH.sub.2Cl.sub.2/ACN, CH.sub.2Cl.sub.2/THF, n-heptane/ethyl acetate or n-heptane/methyl acetate as eluents. Products were detected at 215 nm, 254 nm or 280 nm.

[0910] RP-LPLC purification:

[0911] Low pressure RP chromatography purifications were performed on an Isolera One system or an Isolera Four system from Biotage AB, Sweden, using Biotage SNAP C18 cartridges. Gradients of solvent system A (water containing 0.1% TFA v/v) and solvent system B (acetonitrile containing 0.1% TFA v/v) were used. Products were detected at 215 nm. LPLC fractions containing product were pooled and lyophilized if not stated otherwise.

Analytical Methods

[0912] UPLC-MS analysis:

[0913] Analytical ultra-performance LC (UPLC)-MS was performed on a Waters Acquity system or an Agilent 1290 Infinity II equipped with a Waters BEH300 C18 column (2.1×50 mm, 1.7 μm particle size or 2.1×100 mm, 1.7 μm particle size; solvent A: water containing 0.04% TFA (v/v), solvent B: acetonitrile containing 0.05% TFA (v/v) or solvent A: water containing 0.1% FA (v/v), solvent B: acetonitrile containing 0.1% FA (v/v)) coupled to an LTQ Orbitrap Discovery mass spectrometer from Thermo Scientific or coupled to a Waters Micromass ZQ or coupled to Single Quad MS System from Agilent or coupled to an Agilent Triple Quad 6460 system.

[0914] SEC analysis:

[0915] Size-exclusion chromatography (SEC) was performed on an Agilent 1260 system, equipped with a Sepax Zenix SEC-150 column (150 Å, 7.8×300 mm; isocratic: 60:40 v/v mixture of water containing 0.05% TFA and acetonitrile containing 0.04% TFA) with detection at 215 nm and 280 nm.

[0916] Amine content determination on the PEG-hydrogel beads:

[0917] Amino group content of the PEG-hydrogel was determined by conjugation of an Fmoc-amino acid to the free amino groups on the hydrogel and subsequent Fmoc-determination as described by Gude, M., J. Ryf, et al. (2002) Letters in Peptide Science 9(4): 203-206.

[0918] Maleimide content determination on the PEG-hydrogel beads:

[0919] Maleimide group content of the PEG-hydrogel was determined by conjugation of Fmoc-cysteine to the maleimide residues on the hydrogel and subsequent Fmoc-determination following a procedure, which is based on Gude, M., Ryf, J. et al. (2002) Letters in Peptide Science 9(4): 203-206 and Smyth, D. G., Blumenfeld, O. O., Konigsberg, W. (1964) Biochemical Journal 91: 589.

Quantitative Amino Acid Analysis (QAAA):

[0920] Quantitative amino acid analysis was performed to determine the amount of daptomycin in a sample matrix with unknown content. For the content determination, a material sample containing daptomycin was hydrolysed using a TFA/HCl mixture and microwave irradiation. The resulting single amino acids was dye labelled and analysed chromatographically. The contents of aspartic acid, alanine and ornithine were calculated using calibration curves of the respective amino acid standards. The amount of daptomycin was calculated using the averaged content values of aspartic acid, alanine and ornithine.

Hydrogel Degradation Kinetics:

[0921] A hydrogel sample was incubated with degradation buffer of the desired pH in a water bath at the desired temperature. For each sampling time-point, the reaction mixture was homogenized, centrifuged, supernatant was withdrawn, filtered through a syringe filter and transferred into a sterile Eppendorf tube. Samples were further incubated at the same temperature. At the end of the incubation time, all samples were quenched with acetic acid, and analysed chromatographically. The obtained peak areas of the individual samples were used to calculate degradation kinetics.

Example 1

Synthesis of Linker Reagent 1f

[0922] Tinker reagent 1f was synthesized according to the following scheme:

##STR00112##

[0923] To a solution of N,N′-di methylethylenediamine (2.00 g, 22.69 mmol) and NaCNBH.sub.3 (1.35 g, 21.55 mmol) in MeOH (40 mL) was added 2,4,6-trimethoxybenzaldehyde (4.23 g, 21.55 mmol) over two hours. After complete addition, the mixture was stirred at r.t. for 1 hour, acidified with 1 M HCl (60 mL) and stirred for further 30 min. To the reaction mixture saturated NaHCO.sub.3 solution (70 mL) was added and the solution was extracted with CH.sub.2Cl.sub.2 (5×150 mL). The combined organic phases were dried over Na.sub.2SO.sub.4, filtered and the solvents were evaporated in vacuo. The resulting N,N-dimethyl-N′-Tmob-ethylenediamine 1a was dried in high vacuum and used in the next reaction step without further purification.

[0924] To a solution of Fmoc-A-Me-Asp(OBn)-OH (4.63 g, 10.07 mmol) in CH.sub.2Cl.sub.2 (108 mL) EDC (2.51 g, 13.09 mmol), OxymaPure® (2.00 g, 14.09 mmol) and 2,4,6-collidine (2.53 mL, 2.32 g, 19.13 mmol) were added and the mixture was stirred for 5 min. A solution of crude 1a (3.00 g, max. 11.18 mmol) in CH.sub.2Cl.sub.2 (27 mL) was added and the solution was stirred at r.t. for 1 hour. The reaction was quenched by addition of 0.1 M HCl (300 mL) and the acidified mixture was extracted with CH.sub.2Cl.sub.2 (5×40 mL). The combined organic layers were washed with saturated NaHCQ.sub.3 solution (2×90 mL). The organic phase was dried over Na.sub.2SQ.sub.4, filtered and the solvent was evaporated in vacuo. Crude 1b was purified by flash chromatography.

[0925] Yield: 5.31 g (7.48 mmol, 74% over two steps)

[0926] MS: m/z 710.23=[M+H].sup.+, (calculated monoisotopic mass: [M]=709.34.)

[0927] To a solution of 1b (5.31 g, 7.48 mmol) in THF (53 mL) DBU (1.31 mL, 1.33 g, 8.75 mmol) was added and the solution was stirred at r.t. for 12 min. The reaction mixture was submitted to flash chromatography and 1c was isolated from the product fractions by evaporation of the solvents in vacuo.

[0928] Yield: 3.16 g (6.48 mmol, 87%)

[0929] MS: m/z 488.13=[M+H].sup.+, (calculated monoisotopic mass: [M]=487.27.)

[0930] To a solution of 1c (3.16 g, 6.48 mmol), PyBOP (4.05 g, 7.78 mmol) and DIPEA (3.39 mL, 2.51 g, 19.44 mmol) in CH.sub.2Cl.sub.2 (32 mL), a solution of 6-tritylmercaptohexanoic acid (3.04 g, 7.78 mmol) in CH.sub.2Cl.sub.2 (32 mL) was added and the mixture was stirred for 24 hours. Additional 6-tritylmercaptohexanoic acid (633 mg, 1.62 mmol) and PyBOP (843 mg, 1.62 mmol) were added and the mixture was stirred for additional 5 hours. After dilution with CH.sub.2Cl.sub.2 (600 mL), the organic layer was washed with 0.1 M HCl (3×300 mL) and brine (300 mL), dried over Na.sub.2SO.sub.4, filtered and the solvent was evaporated in vacuo. Crude Id was purified by flash chromatography.

[0931] Yield: 5.06 g (5.88 mmol, 91%)

[0932] MS: m/z 860.45=[M+H].sup.+, (calculated monoisotopic mass: [M]=859.42.)

[0933] To a solution of Id in a mixture of THF (61 mL) and water (61 mL) LiOH (423 mg, 17.66 mmol) was added and the solution was stirred at r.t. for six hours. After dilution with CH.sub.2Cl.sub.2 (500 mL), the organic layer was washed with a mixture of 0.1 M HCl/brine (1:1 v/v, 3×300 mL). The aqueous layers were re-extracted with CH.sub.2Cl.sub.2 (5×100 mL). The combined organic layers were washed with brine (200 mL), dried over Na.sub.2SO.sub.4, filtered and the solvents were evaporated in vacuo. Crude 1e was dried in high vacuum and used without further purification in the next step.

[0934] To a solution of crude 1e (5.05 g, max. 6.56 mmol) in CH.sub.2Cl.sub.2 (60 mL), NHS (1.13 g, 9.85 mmol) and EDC (1.89 g, 9.85 mmol) were added and the mixture was stirred at r.t. for 130 min. After evaporation of the solvent in vacuo, the residue was dissolved in a mixture of MeCN/water/TFA (8:2:0.002 v/v, 10 mL) and the resulting solution was purified by automated RP-LPLC to yield pure 1f after lyophilization.

[0935] Yield: 4.15 g (4.52 mmol, 76%, 96% purity by UV215)

[0936] MS: m/z 867.44=[M+H].sup.+, (calculated monoisotopic mass: [M]=866.39.)

Example 2

Synthesis of Daptomycin Linker Thiol 2b

[0937] Daptomycin linker thiol 2b was synthesized according to the following scheme:

##STR00113## ##STR00114##

[0938] To a mixture of daptomycin (1.08 g, approx. 0.63 mmol) and 1f (0.99 g, 1.01 mmol) in DMSO (38 mL) DIPEA (0.97 mL, 0.72 g, 5.69 mmol) was added and it was stirred for 380 min. After quenching with TFA (0.44 mL, 0.66 g, 5.69 mmol), the mixture was added to MTBE in 50 mL Falcon tubes (1 mL solution and 40 mL MTBE per tube) to precipitate the conjugate. The tubes were shaken and centrifuged. After decanting the supernatants, the residues were combined and dried in high vacuum overnight. Crude 2a was used for the next step without further purification.

[0939] Crude 2a (2.50 g, max. 0.63 mmol) was dissolved in a mixture of HFIP/TES (39:1 v/v, 57 mL) and the solution was stirred at r.t. for 5 min. TFA (4.01 mL) was added and the reaction mixture was stirred at r.t. for two hours. All volatiles were removed in vacuo and the residue was dissolved in a mixture of DCM/TFA (98:2 v/v, 3.0 mL). The solution was added to MTBE in 50 mL Falcon tubes (1 mL solution and 40 mL MTBE per tube) to precipitate the material. The tubes were shaken and centrifuged. After decanting the supernatants, the combined residues were dried in high vacuum overnight. Crude 2b was purified by RP-LPLC to afford pure and mixed product fractions. Pure product fractions were lyophilized to afford a first crop of pure linker thiol. The mixed fractions were additionally purified by preparative RP-HPLC to afford a second crop of pure linker thiol. Both product batches were combined to afford pure 2b.

[0940] Yield: 1.00 g (0.46 mmol, 72%, 99% purity at 215 nm)

[0941] MS: m/z 975.92=[M+2H].sup.2+, (calculated monoisotopic mass: [M]=1948.89.)

Example 3

Synthesis of Cross-Linker Reagent 3d

[0942] Cross-linker reagent 3d was synthesized according to the following scheme. Theoretical calculations of the Mw of the polydisperse PEG conjugates were exemplarily performed for a PEG 1000 with 23 ethylene glycol units that has a Mw of 1031.22 g/mol (exact mass: 1030.61 g/mol):

##STR00115##

[0943] Glutaric acid monobenzyl ester (40.0 g, 180 mmol), ethylene glycol (101 mL, 1.80 mol) and DMAP (2.20 g; 18.0 mmol) were dissolved in CH.sub.2Cl.sub.2 (400 mL). DCC (44.6 g, 216 mmol) was added to the solution, and the mixture was stirred at room temperature for one hour. The reaction mixture was filtered and the filter cake was washed with additional CH.sub.2Cl.sub.2 (50 mL). The filtrate was washed with 0.1 N hydrochloric acid (2×250 mL) and brine (1×250 mL). The organic phase was dried over MgSO.sub.4, filtered and all volatiles were evaporated in vacuo.

[0944] The residue was purified by flash chromatography to afford intermediate 3a.

[0945] Yield: 41.9 g (157 mmol, 87%)

[0946] MS: m/z 267.00=[M+H].sup.+, (calculated monoisotopic mass: [M]=266.16.)

[0947] Intermediate 3a (41.0 g, 154 mmol), glutaric acid anhydride (31.6 g, 277 mmol) and DMAP (3.76 g, 30.8 mmol) were dissolved in CH.sub.2Cl.sub.2 (164 mL). DIPEA, (53.8 mL, 308 mmol) was added and the mixture was stirred at r.t. for two hours. The mixture was washed with 1 M hydrochloric acid (1×400 mL, 1×200 mL) and brine (200 mL). The organic phase was dried over MgSO.sub.4, filtered and all volatiles were evaporated in vacuo. The residue was purified by flash chromatography to afford intermediate 3b.

[0948] Yield: 34.9 g (91.7 mmol, 60%)

[0949] MS: m/z 381.05=[M+H].sup.+, (calculated monoisotopic mass: [M]=380.15.)

[0950] Poly(ethylene glycol) (PEG 1000, 19.0 g), intermediate 3b (25.3 g, 66.5 mmol) and DMAP (116 mg, 0.95 mmol) were dissolved in CH.sub.2Cl.sub.2 (95 mL). DCC (13.7 g, 66.50 mmol) was added at 0° C. and the mixture was afterwards stirred at r.t. for 16 hours. The mixture was diluted with MTBE (95 mL), filtered and all volatiles of the filtrate were evaporated in vacuo. The residue was dissolved in CH.sub.2Cl.sub.2 (120 mL) and the solution was diluted with MTBE (1800 mL) and n-heptane (100 mL) and split in two halves. The mixtures were cooled to −20° C. for 20 h. The supernatants were decanted and the precipitates suspended in a −20° C. cold mixture of MTBE/n-heptane (9:1 v/v, 2× approx. 900 mL). The mixtures were stored at −20° C. for one hour before supernatants were decanted. The precipitates were again suspended in a −20° C. cold mixture of MTBE/n-heptane (9:1 v/v, 2× approx. 900 mL) and the resulting suspensions were combined and filtered. The filter cake was washed with a −20° C. cold mixture of MTBE/n-heptane (9:1 v/v, 500 mL) and was afterwards dried in high vacuum to afford pure intermediate 3c.

[0951] Yield: 28.2 g

[0952] MS: m/z 878.33=[M+2H].sup.2+, (calculated monoisotopic mass: [M]=1754.89.) Compound 3c (28.1 g, 16.0 mmol) was dissolved in THE (281 mL) and palladium on charcoal (10% Pd, 0.68 g) was added. The reaction mixture was stirred at 50° C. under a hydrogen atmosphere for one hour. The mixture was filtered through a pad of Celite 503, which was flushed with additional THE (50 mL). To the combined filtrates, TSTU (19.3 g, 64.0 mmol) and DIPEA (11.2 mL, 64.0 mmol) were added and the reaction mixture was stirred at r.t. for three hours. The mixture was filtered and the filter cake was washed with THL (50 mL). All volatiles were removed from the combined filtrates in vacuo and the residue was dissolved in CH.sub.2Cl.sub.2 (1200 mL). The solution was washed with 0.5 M phosphate buffer pH 7.4 (2×600 mL) and brine (2×200 mL) and was afterwards dried over MgSO.sub.4.

[0953] After filtration, all volatiles were removed in vacuo to afford crude NHS ester. The crude material was dissolved in toluene (1000 mL) and the solution was split in two halves. To each portion MTBE (450 mL) was added and the resulting mixtures were stored at −20° C. overnight. The supernatants were decanted and the solids were collected by filtration and washed with −20° C. cold MTBE (500 mL). The filter residue was transferred into a 100 mL flask and dried for 4 h in high vacuum. The residue was dissolved in CH.sub.2Cl.sub.2 (600 mL) and the solution was split in three portions. To each portion MTBE (800 mL) was added and the resulting mixtures were were stored at −20° C. overnight. The supernatants were decanted from the precipitated oils and all volatiles were removed. The residues were combined with the precipitated oils and the combined crude material was dissolved in THL (1200 mL) and the solution was split in four portions. To each portion MTBE (700 mL) was added and the resulting mixtures were were stored at −25° C. overnight. The supernatants were decanted and the solids were collected by filtration and washed with −20° C. cold MTBE (1000 mL). Pure cross-linker reagent 3d was obtained after drying in high vacuum.

[0954] Yield: 17.5 g

[0955] MS: m/z 885.25=[M+2H].sup.2+, (calculated monoisotopic mass: [M]=1768.83.)

Example 4

Synthesis of Backbone Reagent 4

[0956] Backbone reagent 4 was synthesized as HCl salt using L-lysine building blocks, analogously to an earlier described procedure (WO2013/053856, example 1, compound 1g therein):

##STR00116##

Example 5

Synthesis of PEG-Hydrogel Beads 5a, 5b, and 5c Containing Free Amino Groups

[0957] The weights of the PEG-hydrogel beads 5a, 5b and 5c were estimated by the volume of the aqueous hydrogel bead suspensions, calculating with 1 g of the dry PEG-hydrogel beads 5a, 5b or 5c swelling to a volume of approx. 20 mL under aqueous conditions. All liquids, solvents and reagent solutions were filtered through 0.2 μm PES filters (for aqueous solutions) or 0.2 μm PTFE filters (all others) before use.

[0958] A cylindrical 250 mL reactor with bottom outlet, diameter 60 mm, equipped with baffles, was charged with an emulsion of Cithrol™ DPHS (0.25 g) in heptane (75 mL). The reactor content was stirred with a pitch-blade stirrer, diameter 45 mm, at 520 rpm, at r.t. A solution of cross-linker 3d (3129 mg) and backbone reagent 4 (2600 mg) in DMSO (22.92 g) was added to the reactor and stirred for 10 min to form an emulsion. TMEDA (11.6 mL) was added to effect polymerization and the mixture was stirred at r.t. for 16 h. Acetic acid (17.8 mL) was added while stirring. After 10 min, a sodium chloride solution (15 wt %, 90 mL) was added under stirring. After 10 min, the stirrer was stopped and phases were allowed to separate. After 30 min, the aqueous phase containing the PEG-hydrogel beads was drained.

[0959] For bead size fractionation, the water-hydrogel suspension was diluted with ethanol (40 mL) and wet-sieved on 125, 100, 75, 63, and 50 μm (mesh opening) stainless steel sieves, diameter 200 mm using a sieving machine for 15 min. Sieving amplitude was 1.5 mm, liquid flow was 300 mL/min. First, a sodium chloride solution (20 wt %, 3000 mL), then water (1000 mL) was used as the liquid for wet-sieving. The bead fractions on the different sieves were transferred into 50 mL Falcon tubes (max. 14 mL bead suspension per tube) and successively washed with AcOH (0.1% v/v, 3× ˜40 mL) and ethanol (5-7× ˜40 mL) by addition, shaking, centrifugation and decantation. The bead fractions were transferred into 20 mL syringes with PE frits (max. ≈600 mg hydrogel beads per syringe) and dried in high vacuum for 16 hours to yield amine hydrogels 5a, 5b and 5c. The amine content of the hydrogels was determined for bead fraction 5a, representatively for all batches, by conjugation of an Fmoc-amino acid to the free amino groups on the hydrogel and subsequent Fmoc determination.

[0960] Yields: 5a (63 μm sieve fraction): ≈125 mg [0961] 5b (75 μm sieve fraction): ≈600 mg [0962] 5c (100 μm sieve fraction): ≈1400 mg

[0963] Amine content: 0.877 mmol/g

Example 6

[0964] Synthesis of transient daptomycin-linker PEG-hydrogel conjugate 6b Amine hydrogel beads 5c (approx. 600 mg) were placed into a 20 mL syringe reactor with PE frit. The beads were washed with NMP (3×12 mL) and NMP/DIPEA (98:2 v/v, 2×12 mL) and all solvents were expelled afterwards. N-succinimidyl 3-maleimidopropionate (416 mg, 1.56 mmol) was dissolved in NMP (7.2 mL) and the resulting solution was drawn to the hydrogel in the syringe reactor. The suspension was allowed to incubate for two hours at r.t. under gentle agitation. The liquids were expelled and the hydrogel beads were washed with NMP (5×12 mL), AcOH (0.1% v/v, 5×12 mL) and ethanol (5×12 mL). Maleimide hydrogel 6a was obtained by drying in high vacuum for 5 days. The maleimide content of the functionalized PEG-hydrogel beads 6a was determined by conjugation of Fmoc-cysteine to the maleimide residues on the hydrogel and subsequent Fmoc determination.

[0965] Yield: not determined

[0966] Maleimide content: 0.7166 mmol/g

[0967] A suspension of the maleimide functionalized hydrogel beads 6a (346 mg, 0.248 mmol maleimides) in buffer (100 mM succinate, 0.05% Tween 20, pH 5.5, 15.0 mL) in a 50 mL Falcon tube was agitated for 5 min and then centrifuged. A part of the supernatant (approx. 11 mL) was discarded and a solution of daptomycin linker thiol 2b (820 mg, 0.376 mmol) in buffer (100 mM succinate, 0.05% Tween 20, pH 5.5, 32.8 mL) was added to the hydrogel suspension. The tube was agitated at r.t. and protected from light for 22 hours. The tube was centrifuged and the supernatant was partially removed to leave approx. 2 mL supernatant above the dense bead suspension. The beads were transferred into a 20 ml syringe reactor with a PE frit. The hydrogel beads were successively washed with buffer (100 mM succinate, 0.05% Tween 20, pH 5.5, 10×10 mF), AcOH (0.1% v/v, 10×10 mF), NMP/AcOH (97:3 v/v, 10×10 mL) and ethanol (10×10 mL). The transient daptomycin-linker PEG-hydrogel conjugate 6b was obtained after drying in high vacuum overnight. The daptomycin content of 6b was determined by QAAA.

[0968] Yield: 821 mg (99%, daptomycin content: 470.1 mg/g)

Example 7

Linker Release Kinetics for a Transient Daptomycin-Linker Hydrogel Conjugate

[0969] The linker kinetics with respect to the daptomycin species release from a transient daptomycin-linker hydrogel conjugate was investigated by incubation of transient daptomycin-linker PEG-conjugate 6b at pH 7.4 and 37° C. Daptomycin is prone to hydrolytic degradation and some minor different degradation pathways upon aqueous incubation. For determination of the linker kinetics on the carrier, the supernatant of the incubated suspension was analyzed by UPLC at 215 nm and all daptomycin-related peaks were taken into account for the calculation of the linker kinetics. The half-life of the linker with respect to daptomycin species release has been determined to be two weeks for the transient daptomycin-linker PEG-hydrogel conjugate 6b.

Example 8

Stability of Daptomycin in a Transient Daptomycin-Linker Hydrogel Conjugate

[0970] The relative stability of the covalently bound daptomycin in a transient daptomycin-linker hydrogel conjugate towards hydrolytic and other degradation pathways in comparison to free daptomycin was investigated. For that purpose, free daptomycin and transient daptomycin-linker PEG-conjugate 6b were incubated at pH 7.4 and 37° C. The supernatant of the carrier sample was exchanged five times within a week and the daptomycin purity in these samples was analyzed by UPLC. In parallel, analytical samples of the free daptomycin control solution were also analyzed by UPLC at the same incubation times. The purity of daptomycin in the samples was calculated as the ratio of the peak area of the intact daptomycin peak at 215 nm relative to the area sum of all daptomycin-related peaks identified at 215 nm. It was found that within the first 7 days of incubation under physiological conditions, the purity of the daptomycin, which was continuously released from transient daptomycin-linker hydrogel conjugate was constantly at around 85%, whereas the purity of the free daptomycin in the solution control sample dropped to 72% at day seven.

Example 9

Degradation Study of a Transient Daptomycin-Linker Hydrogel Conjugate

[0971] The transient daptomycin-linker hydrogel conjugate was analyzed regarding carrier degradation. For that purpose, the transient daptomycin-linker PEG-conjugate 6b was incubated at pH 7.4 and 37° C. The sample was visually checked for the presence of the solid carrier particles on a daily basis. As soon as no particles could be detected in the sample anymore, the material was deemed to be fully degraded to soluble products. It was found that the transient daptomycin-linker PEG-hydrogel conjugate 6b was fully degraded after about 40 days.

Example 10

Quantification of Daptomycin Concentrations in Rabbit Plasma

[0972] Daptomycin concentrations in rabbit plasma were determined after plasma protein precipitation via liquid chromatography separation and detection by LC-MS. As internal standard deuterated daptomycin-D5 peptide was used. LC-MS analysis was carried out by using a UHPLC system coupled to a triple quadrupole mass spectrometer via an ESI probe. Chromatography was performed on a C18 analytical UHPLC column. UPLC grade water containing 0.1% formic acid (v/v) was used as mobile phase A and UPLC grade acetonitrile with 0.1% formic acid as mobile phase B. The gradient system comprised a linear increase from 20% B to 45% B in 10 min. Mass analysis was performed in MRM mode with the selected transitions for daptomycin and the internal standard daptomycin-D5.

[0973] Calibration standards of daptomycin in blank plasma were prepared as follows: thawed K.sub.2-EDTA rabbit plasma was homogenized. The daptomycin formulation was spiked into blank plasma at concentrations between 1000 ng/mL and 2 ng/mL. These solutions were used for the generation of a calibration curve. Calibration curves were weighted 1/×.sup.2.

[0974] For sample preparation, 70 μL of sample were spiked with 20 μL of internal standard solution. Subsequently, the mixture was spiked with 40 μL of 0.5 M citrate buffer pH 4.0 and incubated for 30 min at room temperature. Protein precipitation was carried out by addition of 270 μL of room temperature methanol. 200 μL of the supernatant were transferred into a new well-plate and evaporated to dryness (under a gentle nitrogen stream at 45° C.). 50 μL of reconstitution solvent (H.sub.2O/MeOH 1:1+1.0% FA) were used to dissolve the residue by intensive shaking. 10 μL were injected into the LC-MS system.

Example 11

[0975] Pharmacokinetic Profiles of Daptomycin in New Zealand White Rabbits after Intraarticular (IA) Injections with a Transient Daptomycin-Linker Hydrogel Conjugate

[0976] This study was performed in order to investigate the systemic pharmacokinetics of daptomycin in male New Zealand White (NZW) rabbits following intraarticular administration of transient daptomycin-linker PEG-hydrogel conjugate 6b. Animals (n=9 per group) received a single IA injection of 300 μL transient daptomycin-linker PEG-hydrogel conjugate 6b formulation (15 mg daptomycin nominal) in the right knee and 300 μL vehicle in the left knee. Three animals from each group were sacrificed three days, two weeks, and six weeks after dosing. Blood samples for PK analysis were collected and processed to plasma at predose and 0.5, 1, 2, 4, 8, 12, 24, 48, 72, 96, 120, 144, 168, 336 hours post dose (PK blood samples were only collected until 72 hours post dose from animals with three days inlife). Moreover, blood was collected for clinical chemistry and hematology at predose, day three, day seven*, week two*, and week six* (*in the appropriate groups). Visual inspection and palpation (such as reddening/swelling) were performed in the first seven days after injection. Hereafter, visual inspection and palpation was done once a week. Upon sacrifice all knees were sampled for histopathological examination.

[0977] Results: Dose administrations were well tolerated with no visible signs of discomfort during administration and following administration. No dose site reactions were observed any time throughout the study and all animals showed normal behavior and no knee swelling or warming. After intraarticular injection of the transient daptomycin-linker PEG-hydrogel conjugate 6b, sustained PK plasma concentrations above 100 ng/mL were detected over the time course of one week after injection.

Abbreviations

[0978] ACN Acetonitrile [0979] AcOH Acetic Acid [0980] Asp Aspartic Acid [0981] Bn Benzyl [0982] Crl Charles River Laboratories [0983] DBU 1,8-Diazabicyclo[5.4.0]undec-7-ene [0984] DCC Dicyclohexylcarbodiimide [0985] DCM Dichloromethane [0986] DIPEA N, A-Diisopropylethylamine [0987] DMAP 4-(Dimethylamino)pyridine [0988] DMSO Dimethyl Sulfoxide [0989] DPHS Dipolyhydroxystearate [0990] EDC N-(3-Dimcthylaminopropyl)-N′-ethylcarbodiimide Hydrochloride [0991] EDTA Ethylenediaminetetraacetic Acid [0992] eqv. Equivalents [0993] ESI Electrospray Ionization [0994] EtOH Ethanol [0995] FA Formic Acid [0996] Fmoc Fluorenylmethyloxycarbonyl [0997] HFIP 1,1,1,3,3,3-Hexafluoro-2-propanol [0998] HOBt 1-Flydroxybenzotriazole [0999] FIPLC High-Performance Liquid Chromatography [1000] IA Intraarticular [1001] LC-MS Mass Spectrometry Coupled Liquid Chromatography [1002] LPLC Low Pressure Liquid Chromatography [1003] MeCN Acetonitrile [1004] MeOH Methanol [1005] MES 2-(N-Morpholino)ethanesulfonic acid [1006] MRM Multiple Reaction Monitoring [1007] MTBE tert-Butyl Methyl Ether [1008] Mw Molecular Weight [1009] NHS A-Hydroxysuccinimide [1010] NMP M- Methyl-2-pyrrolidone [1011] NZW New Zealand White Rabbits [1012] OD600 Optical Density Measured at 600 nm Wavelength [1013] OPA o-Phthalaldehyde [1014] OxymaPure® Ethyl cyano(hydroxyimino)acetate [1015] PE Polyethylene [1016] PEG Poly(ethylene glycol) [1017] PK Pharmacokinetic/s [1018] PTFE Polytetrafluoroethylene [1019] PyBOP Benzotriazol-1-yl-oxytripyrrolidinophosphonium Hexafluorophosphate [1020] QAAA Quantitative Amino Acid Analysis [1021] RP-HPLC Reversed Phase High-Performance Liquid Chromatography [1022] RP-LPLC Reversed Phase Low Pressure Liquid Chromatography [1023] r.t. Room Temperature [1024] SEC Size-exclusion chromatography [1025] TES Triethylsilane [1026] TFA Trifluoroacetic Acid [1027] THF Tetrahydro furane [1028] TMEDA N, N, N′,N′-Tetramethylethylenediamine [1029] Tmob 2,4,6-Trimethoxybenzyl [1030] Trt Trityl [1031] TSTU N,N,N′,N′-Tetramethyl-O—(N-succinimidyl)uronium Tetrafluorborate [1032] Tween 20 Polyethylene Glycol Sorbitan Monolaurate [1033] UHPLC Ultra High Performance Liquid Chromatography [1034] UPLC Ultra Performance Liquid Chromatography [1035] UPLC-MS Mass Spectrometry Coupled Ultra Performance Liquid Chromatography