Biodegradable, semi-crystalline, phase separated, thermoplastic multi block copolymers for controlled release of biologically active compounds

Abstract

This invention is directed to a biodegradable, semi-crystalline, phase separated thermoplastic multi-block copolymer, a process for preparing said multi-block copolymer, a composition for the delivery of at least one biological active compound, and to a method for delivering a biologically active compound to a subject in need thereof. A multi-block copolymer of the invention is characterized in that: a) it comprises at least one hydrolysable pre-polymer (A) segment and at least one hydrolysable pre-polymer (B) segment, b) said multi-block copolymer having a T.sub.g of 37? C. or less and a T.sub.m of 110-250? C. under physiological conditions; c) the segments are linked by a multifunctional chain-extender; d) the segments are randomly distributed over the polymer chain; e) at least part of the pre-polymer (A) segment is derived from a water-soluble polymer.

Claims

1. Biodegradable, semi-crystalline, phase separated, thermoplastic multi-block copolymer, the copolymer being characterised in that: a) it comprises at least one hydrolysable pre-polymer (A) segment and at least one hydrolysable pre-polymer (B) segment, b) said multi-block copolymer having a Tg of 37? C. or less and a Tm of 110-250? C. under physiological conditions; c) the segments are linked by a multifunctional chain-extender, wherein said chain extender is a diisocyanate; d) the segments are randomly distributed over the polymer chain; e) at least part of the pre-polymer (A) segment is derived from a water-soluble polymer, and wherein said pre-polymer (B) segment has a Tm of 110-250? C. and is based on poly(l-lactic acid), poly(d-lactic acid), polyglycolic acid, or combinations thereof, and wherein said water-soluble polymer is derived from poly(ethylene glycol) (PEG).

2. Biodegradable, semi-crystalline, phase separated, thermoplastic multi-block copolymer according to claim 1, wherein said chain-extender is a difunctional aliphatic chain-extender.

3. Biodegradable, semi-crystalline, phase separated, thermoplastic multi-block copolymer according to claim 1, wherein pre-polymer (A) comprises reaction products of cyclic monomers and/or non cyclic monomers.

4. Biodegradable, semi-crystalline, phase separated, thermoplastic multi-block copolymer according to claim 1, wherein a water-soluble polymer is present as an additional pre-polymer.

5. Biodegradable, semi-crystalline, phase separated, thermoplastic multi-block copolymer according to claim 1, wherein said pre-polymer (B) segment comprises a crystallisable polymer derived from hydroxyalkanoate, glycolide, 1-lactide or d-lactide.

6. Biodegradable, semi-crystalline, phase separated, thermoplastic multi-block copolymer according to claim 1, having a swelling ratio under physiological conditions varies from 1 to 4.

7. Biodegradable, semi-crystalline, phase separated, thermoplastic multi-block copolymer according to claim 1, wherein said copolymer has an intrinsic viscosity of at least 0.1 dl/g.

8. A composition for the delivery of at least one biologically active compound to a host, comprising at least one biologically active compound encapsulated in a matrix, wherein said matrix comprises at least one biodegradable, semi-crystalline, phase separated, thermoplastic multi-block copolymer according to claim 1.

9. A composition according to claim 8, wherein said at least one biologically active compound is a non-peptide non-protein small sized drug, or a biologically active polypeptide.

10. A composition according to claim 9, wherein said non-peptide, non-protein small sized drug comprises one or more selected from the group consisting of an anti-tumour agent, an anti-microbial agent, a sephalosporin, an aminoglycoside, a macrolide, a tetracycline, a chemotherapeutic agent, a urinary tract antiseptic, a drug for anaerobic infections, a drug for tuberculosis, a drug for leprosy, an antifungal agent, an antiviral agent, an anti-helminthiasis agent, an anti-inflammatory agent, an anti-gout agent, a centrally acting (opoid) analgesic, a local anaesthetic, a drug for Parkinson's disease, a centrally acting muscle relaxant, a hormone or hormone anti-agonist, a corticosteroid, a glucocorticosteroid, an androgen, an androgenic steroid, an anabolic steroid, an anti-androgen, an estrogen, an estrogenic steroid, an anti-estrogen, a progestin, a thyroid drug and an anti-thyroid drug.

11. A composition according to claim 9, wherein said biologically active polypeptide comprises one or more selected from the group consisting of a protein/peptide drug, an enzyme, a receptor ligand, a neurotransmitter, an inhibitory peptide, a regulatory peptide, an activator peptide, a cytokine, a growth factor, a monoclonal antibody, a monoclonal antibody fragment, an anti-tumour peptide, an antibiotic, an antigen, a vaccine, and a hormone.

12. Composition according to claim 8, wherein said biologically active compound is a non-peptide, non-protein small molecule having an Mn which is 1000 Da or less, preferably said multi-block copolymer contains poly(ethylene glycol), as a segment of pre-polymer (A) and/or as an additional pre-polymer, wherein said poly(ethylene glycol) i) has a molecular weight of from 200 to 1500 g/mol, preferably from 600 to 1000 g/mol; and/or ii) is present in an amount of from 5 wt. % to 20 wt. %, preferably of from 5 wt. % to 10 wt. %.

13. Composition according to claim 8, wherein said biologically active compound is a biologically active polypeptide having a molecular weight which is 10000 Da or less, preferably said multi-block copolymer contains poly(ethylene glycol), as a segment of pre-polymer (A) and/or as an additional pre-polymer, and wherein said poly(ethylene glycol) i) has a molecular weight of from 400 to 3000 g/mol, preferably from 600 to 1500 g/mol; and/or ii) is present in an amount of from 5 wt. % to 60 wt. %, preferably of from 5 wt. % to 40 wt. %.

14. Composition according claim 8, wherein said biologically active compound is a biologically active polypeptide having a molecular weight of 10 000 Da or more, preferably said multi-block copolymer contains poly(ethylene glycol), as a segment of pre-polymer (A) and/or as an additional pre-polymer, and wherein said poly(ethylene glycol) i) has a molecular weight of from 600 to 5000 g/mol, preferably of from 1000 to 3000 g/mol; and/or ii) is present in an amount of from 5 wt. % to 70 wt. %, more preferably of from 10 wt. % to 50 wt. %.

15. Composition according to claim 8, in the form of microspheres, microparticles, nanoparticles, nanospheres, rods, implants, gels, coatings, films, sheets, sprays, tubes, membranes, meshes, fibres, or plugs.

16. Composition according to claim 8, wherein the average diameter of the microspheres and/or microparticles is preferably in the range of 0.1-1000 ?m, more preferably in the range of 1-100 even more preferably in the range of 10-50 ?m.

17. Composition according to claim 16, wherein the biologically active compound is dissolved or dispersed throughout the polymer matrix.

18. Composition according to claim 16, wherein the microsphere comprises a reservoir wherein biologically active compound is contained, surrounded by a polymer in mononuclear or polynuclear state.

19. Composition according to claim 8 for treating rheumatoid arthritis, hepatitis, diabetes, metabolic syndromes, osteoarthritis, renal disease, inflammation, local pain processes, local infections, local skin diseases, tumours (or their sites after surgical removal as a postoperative treatment to destroy any tumour cells possibly remaining), prostate or breast cancer, agromegaly, ocular diseases, local brain diseases, and cardiovascular diseases.

20. A method for delivering a biologically active compound to a subject in need thereof, comprising administering an effective dose of a composition according to claim 8.

21. A method of manufacturing a composition according to claim 16, comprising the successive steps of a) emulsifying an aqueous solution of a water-soluble biologically active compound in a solution of a biodegradable, semi-crystalline, phase separated, thermoplastic multi-block copolymer comprising at least one hydrolysable pre-polymer (A) segment and at least one hydrolysable pre-polymer (B) segment; said multi-block copolymer having a Tg of 37? C. or less and a Tm of 110-250? C. under physiological conditions; the segments are linked by a multifunctional chain-extender; the segments are randomly distributed over the polymer chain; and at least part of the pre-polymer (A) segment is derived from a water-soluble polymer, in an organic solvent; b) subsequently emulsifying the resultant emulsion of a) in an aqueous solution comprising a surfactant, thereby yielding a water-in-oil-in-water (W/O/W) emulsion; and c) extracting the organic solvent to solidify microspheres.

22. A method of manufacturing a composition according to claim 16, comprising the successive steps of a) dispersing the biologically active compound as a solid powder in a solution of a biodegradable, semi-crystalline, phase separated, thermoplastic multi-block copolymer comprising at least one hydrolysable pre-polymer (A) segment and at least one hydrolysable prepolymer (B) segment: said multi-block copolymer having a Tg of 37? C. or less and a Tm of 110-250? C. under physiological conditions; the segments are linked by a multifunctional chain-extender; the segments are randomly distributed over the polymer chain; and at least part of the pre-polymer (A) segment is derived from a water-soluble polymer, in an organic solvent; b) emulsifying the resultant dispersion of a) in an aqueous solution comprising a surfactant, thereby yielding a solid-in-oil-in-water (S/O/W) emulsion; and c) extracting the organic solvent to solidify the microspheres.

23. A method of manufacturing a composition according to claim 1, comprising the successive steps of a) emulsifying an aqueous solution of a water-soluble biologically active compound in a solution of a biodegradable, semi-crystalline, phase separated, thermoplastic multi-block copolymer comprising at least one hydrolysable pre-polymer (A) segment and at least one hydrolysable pre-polymer (B) segment; said multi-block copolymer having a Tg of 37? C. or less and a Tm of 110-250? C. under physiological conditions; the segments are linked by a multifunctional chain-extender; the segments are randomly distributed over the polymer chain; and at least part of the pre-polymer (A) segment is derived from a water-soluble polymer, in an organic solvent; b) adding a polymer precipitant, to the resultant emulsion of a) to form embryonic microparticles; and c) extracting the polymer precipitant and the organic solvent to solidify the microspheres.

24. A method of manufacturing a composition according to claim 16, comprising the successive steps of a) dispersing the biologically active compound as a solid powder in a solution of a biodegradable, semi-crystalline, phase separated, thermoplastic multi-block copolymer comprising at least one hydrolysable pre-polymer (A) segment and at least one hydrolysable prepolymer (B) segment: said multi-block copolymer having a Tg of 37? C. or less and a Tm of 110-250? C. under physiological conditions; the segments are linked by a multifunctional chain-extender; the segments are randomly distributed over the polymer chain; and at least part of the pre-polymer (A) segment is derived from a water-soluble polymer, in an organic solvent; b) adding a polymer precipitant, to the resultant dispersion of a) to form embryonic microparticles; and c) extracting the polymer precipitant and the organic solvent to solidify the microspheres.

25. Biodegradable, semi-crystalline, phase separated, thermoplastic multi-block copolymer according to claim 1, wherein said aliphatic chain-extender is 1,4-butane diisocyanate.

26. Biodegradable, semi-crystalline, phase separated, thermoplastic multi-block copolymer according to claim 3, wherein said non cyclic monomers are selected from the group consisting of succinic acid, glutaric acid, adipic acid, sebacic acid, lactic acid, glycolic acid, hydroxybutyric acid, ethylene glycol, diethylene glycol, 1,4-butanediol and/or 1,6-hexanediol, and wherein said cyclic monomers are selected from the group consisting of glycolide, lactide, ?-caprolactone, ?-valerolactone, trim ethylene carbonate, tetramethylenecarbonate, 1,5-dioxepane-2-one, 1,4-dioxane-2-one (para-dioxanone) and/or cyclic anhydrides.

27. Biodegradable, semi-crystalline, phase separated, thermoplastic multi-block copolymer according to claim 1, wherein said water-soluble polymer is derived from poly(ethylene glycol) (PEG) having a Mn of 150-5000 g/mol.

28. Biodegradable, semi-crystalline, phase separated, thermoplastic multi-block copolymer according to claim 5, wherein said pre-polymer (B) segment comprises 1-lactide pre-polymers and d-lactide pre-polymers in such amounts and ratio that stereocomplexation between 1-lactide and d-lactide is achieved.

29. Biodegradable, semi-crystalline, phase separated, thermoplastic multi-block copolymer according to claim 28, wherein said pre-polymer (B) segment is poly(1-lactic acid) with an Mn of 1000 g/mol or more.

30. Biodegradable, semi-crystalline, phase separated, thermoplastic multi-block copolymer according to claim 29, wherein said pre-polymer (B) segment is poly(l-lactic acid) with an Mn of 2000 g/mol or more.

31. Biodegradable, semi-crystalline, phase separated, thermoplastic multi-block copolymer according to claim 29, wherein said pre-polymer (B) segment is poly(l-lactic acid) with an Mn of 3000 g/mol or more.

32. Biodegradable, semi-crystalline, phase separated, thermoplastic multi-block copolymer according to claim 6, wherein said swelling ratio under physiological conditions varies from 1 to 2.

33. Biodegradable, semi-crystalline, phase separated, thermoplastic multi-block copolymer according to claim 32, wherein said swelling ratio under physiological conditions varies from 1 to 1.5.

34. Biodegradable, semi-crystalline, phase separated, thermoplastic multi-block copolymer according to claim 7, wherein said copolymer has an intrinsic viscosity of between 0.2 and 2 dl/g.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1A: DSC thermograms of 50LP10L20-LL40

(2) FIG. 1B: DSC thermograms of 30LP30L40-LL40

(3) FIG. 1C: DSC thermograms of 50CP10C20-LL40

(4) FIG. 2: Cumulative release of lysozyme from films composed of 30LP10L20-LL40, 50LP10L20-LL40, 70LP10L20-LL40, 50CP10C20-LL40 and 30CP30C40-LL40. Films were loaded with 10 wt. % lysozyme. Release was measured at 37? C. in phosphate buffer pH 7.4 (n=3).

(5) FIG. 3: Cumulative release of bovine serum albumin (BSA) from films composed of 30LP10L20-LL40, 50LP10L20-LL40, 70LP10L20-LL40, 30LP30L40-LL40 and 30CP30C40-LL40. Films were loaded with 10 wt. % BSA. Release was measured at 37? C. in phosphate buffer pH 7.4 (n=3).

(6) FIG. 4: Effect of composition of the hydrophilic block of multi-block copolymers on cumulative release of lysozyme from films. Films were composed of 50LP10L20-LL40, 50GP10C20-LL40 or 50CP10C20-LL40 (25 wt. % PEG1000) and were loaded with 10 wt. % lysozyme. Release was measured at 37? C. in phosphate buffer pH 7.4 (n=3).

(7) FIG. 5: Activity of lysozyme released from films composed of 30LP10L20-LL40 or 50LP10L20-LL40 containing 10 wt. % lysozyme (37? C., phosphate buffer pH 7.4) and lysozyme activity of lysozyme solutions (0.01 wt. %, phosphate buffer pH 7.4) stored at 4 and 37? C. as a function of time (n=3).

(8) FIG. 6: In vitro release of BSA from microspheres composed of 30LP10L20-LL40 and 50CP10C20-LL40 loaded with 3-4 wt. % of BSA at 37? C. in phosphate buffer pH 7.4 (n=3).

(9) FIG. 7: In vitro release of IGF-1 from IGF-1 loaded 50CP30C40-LL40 and 30CP30C40-LL40 films made by solvent casting of W/O. Release was measured at 37? C. in phosphate buffer pH 7.2 (n=3). Solid lines represent IGF-1 release as measured by UPLC. Dotted lines represent IGF-1 release as measured by ELISA.

(10) FIG. 8: SEM photo of 0.2 wt. % IGF-1 loaded 50CP10C20-LL40 microspheres prepared via a W/O/W double emulsion route.

(11) FIG. 9: In vitro release of IGF-1 from 0.2 wt. % IGF-1 loaded microspheres prepared of 50CP10C20-LL40 with different IVs. Release was measured at 37? C. in phosphate buffer pH 7.2 (n=3).

(12) FIG. 10: SDS PAGE results of IGF-1 released from 50CP10C20-LL40 microspheres with 0.2 wt % IGF-1 target loading and prepared using various ultra-turrax speeds after 1 and 2 weeks.

(13) FIG. 11: In vitro release Protein A (MW 15 000 Da) from films composed of 20LP10L20-LL40, 30LP6L20-LL40 and 30CP10C20-LL40 (Protein A content 5 wt. %; film thickness 80-120 ?m). Release was measured at 37? C. in phosphate buffer pH 7.4 (n=3).

(14) FIG. 12: SEM photo of 3-4 wt. % Protein A loaded microspheres composed of 30CP10C20-LL40 (IV 0.71 dl/g) prepared using various amounts of inulin in the inner aqueous phase A: 0% inulin, B: 2% inulin. 1: Overview. 2: Zoom-in.

(15) FIG. 13: In vitro release of Protein A from microspheres composed of 30CP10C20-LL40 at 3-4 wt. % Protein A target loading with optionally 2 or 5 wt. % of inulin co-encapsulated, at 37? C. in phosphate buffer pH 7.4 (n=3).

(16) FIG. 14: In vitro release of Protein A from microspheres composed of 30CP10C20-LL40 at 3-4 wt. % Protein A target loading and different polymer IV, at 37? C. in phosphate buffer pH 7.4 (n=3).

(17) FIG. 15: SDS-PAGE results of Protein A released from 30CP10C20-LL40 microspheres with 4 wt. % Protein A and 2 wt. % inulin target loading after 1 (lane 4), 7 (lane 7), 14 (lane 8) and 21 (lane 9) days. Lane 5: Molecular weight markers. Lane 6: Protein A standard. Note that the dark smears are due to colouring of phosphate buffer salts.

(18) FIG. 16: In vitro release of Peptide A (MW 2500) from films composed of 20LP10L20-LL40 (peptide load 5 and 10 wt. %; film thickness 80-100 ?m) loaded. Release was measured at 37? C. in phosphate buffer pH 7.4 (n=3).

(19) FIG. 17: In vitro release of Peptide A (MW 2500) from microspheres composed of 20LP10L20-LL40 (particle size 30 ?m; peptide load 10 wt. %). Release was measured at 37? C. in phosphate buffer pH 7.4 (n=3).

(20) FIG. 18 In vitro release of rapamycin from microspheres composed of various blends of 20LP1020-LL40- and 10LP10L20-LL40.

(21) FIG. 19 SEM pictures of goserelin-loaded 20LP1020-LL40 microspheres prepared via the W/O/O method

(22) FIG. 20 In vitro release of goserelin from 20LP1020-LL40 microspheres prepared via the W/O/O method

EXAMPLES

(23) In the following examples various biodegradable semi-crystalline, phase separated multi-block copolymers were synthesised and evaluated for their processing and controlled release characteristics. The polymers were composed of a crystalline L-lactide-based hard segment B with a melting point (T.sub.m) and a hydrophilic poly(ethylene glycol) (PEG)-based segment A having a glass transition temperature (T.sub.g) that was below body temperature under physiological conditions. In the following examples PEG is denoted with its molecular weight (MW). For example PEG.sub.1000 refers to PEG with MW 1000 g/mol.

Example 1

(24) In this example, general procedures for the preparation of poly(DL-lactide-co-PEG) prepolmer (A) are provided. Monomers were weighed into a three-necked bottle under nitrogen atmosphere and dried at 50? C. in case of glycolide and D,L-lactide for at least 16 h under reduced pressure. PEG was dried at 90? C. under reduced pressure for at least 16 h. PEG was added to the monomer(s) under nitrogen atmosphere. Subsequently, stannous octoate was added and the mixture was magnetically stirred and reacted at 140? C. for several days. .sup.1H-NMR was performed on a VXR Unity Plus NMR Machine (Varian) operating at 300 MHz. The d.sub.1 waiting time was set to 20 s, and the number of scans was 16. Spectra were recorded from 0 to 14 ppm. Conversion and pre-polymer M.sub.n was determined from .sup.1H-NMR. .sup.1H-NMR samples were prepared by dissolving 10 mg of polymer into 1 ml of deuterated chloroform.

Example 2

(25) This example describes the preparation of poly(DL-lactide-co-PEG.sub.1000) (pLP10L20) with M.sub.n 2000 g/mol. 149.84 grams (1.04 mol) of D,L-lactide (Purac) was weighed and 149.21 g (0.149 mol) of PEG MW1000 (Ineos, PU grade) was added. 71.6 mg of stannous octoate (Sigma Corp) was added (monomer/catalyst molar ratio=5900) and the mixture was magnetically stirred and reacted at 140? C. during 245 h. .sup.1H-NMR showed 94.8% monomer conversion. The calculated molecular weight (M.sub.n) from in-weights was 2000 g/mol. Molecular weight as determined by .sup.1H-NMR was 1950 g/mol.

Example 3

(26) This example describes the preparation of poly(DL-lactide-co-PEG.sub.3000) (pLP30L40) with M.sub.n 4000 g/mol. 50.35 g (0.349 mol) of D,L-lactide (Purac) was weighed and 151.08 g (50.4 mmol) of PEG MW3000 (Sigma Corp) was added. 37.5 mg of stannous octoate (Sigma Corp) was added (monomer/catalyst molar ratio=4300) and the mixture was magnetically stirred and reacted at 140? C. during 90 h. .sup.1H-NMR showed 93.4% monomer conversion. The calculated molecular weight (M.sub.n) from in-weights was 4000 g/mol. Molecular weight as determined by .sup.1H-NMR was 3940 g/mol.

Example 4

(27) This example describes the preparation of poly(?-caprolactone-co-PEG.sub.1000) pre-polymer (pCP10C20) with M.sub.n 2000 g/mol. 100.81 g (0.101 mol) of PEG MW1000 (Ineos, PU grade) was weighed into a three-necked bottle under nitrogen atmosphere and dried at 90? C. for at least 16 h under reduced pressure. 101.76 g (0.892 mol) of ?-caprolactone (Acros, previously dried and distilled over CaH.sub.2 under reduced pressure) was added to the PEG under nitrogen atmosphere and the mixture was heated to 135? C. 57.9 mg of stannous octoate (Sigma Corp) was added (monomer/catalyst molar ratio=6200) and the mixture was magnetically stirred and reacted at 135? C. during 76 h. .sup.1H-NMR showed 100% monomer conversion. The calculated molecular weight (M.sub.n) from in-weights was 2010 g/mol. Molecular weight as determined by .sup.1H-NMR was 1950 g/mol.

Example 5

(28) This example describes the preparation of poly(?-caprolactone-co-PEG.sub.3000) pre-polymer (pCP30C40) with M.sub.n 4000 g/mol. 176.60 g (58.9 mmol) of PEG MW3000 (Ineos, PU grade) was weighed into a three-necked bottle under nitrogen atmosphere and dried at 90? C. for at least 16 h under reduced pressure. 59.4 g (0.520 mol) of ?-caprolactone (Acros, previously dried and distilled over CaH.sub.2 under reduced pressure) was added to the PEG under nitrogen atmosphere and the mixture was heated to 135? C. 69.6 mg of stannous octoate (Sigma Corp) was added (monomer/catalyst molar ratio=3000) and the mixture was magnetically stirred and reacted at 135? C. during 243 h. .sup.1H-NMR showed 100% monomer conversion. The calculated molecular weight (M.sub.n) from in-weights was 2010 g/mol. Molecular weight as determined by .sup.1H-NMR was 1950 g/mol.

Example 6

(29) This example describes the preparation of poly(L-lactic acid) pre-polymer (LL4000) with M.sub.n=4000 g/mol initiated by 1,4-butanediol (BDO). 399.89 g (2.77 mol) of L-lactide (Purac) was weighed into a three-necked bottle under nitrogen atmosphere and dried at 50? C. for at least 16 h under reduced pressure. 9.36 g (0.104 mol) of BDO (Acros, previously distilled under reduced pressure) was added to the L-lactide under nitrogen atmosphere. 434 ml of dioxane (Acros, previously dried and distilled over sodium wire) was added to dissolve the L-lactide and BDO and the mixture was heated to 80? C. 87.8 mg of stannous octoate (Sigma Corp) was added (monomer/catalyst molar ratio=12 800). The mixture was magnetically stirred and reacted at 80? C. during 50.6 h. The polymer was retrieved from dioxane by freeze-drying for 72 h to a final temperature of 50? C. In case of polymer dissolved in dioxane, the dioxane was first removed under reduced pressure at 50? C. .sup.1H-NMR showed 96.5% monomer conversion. The calculated molecular weight (M.sub.n) from in-weights was 3940 g/mol. Molecular weight as determined by .sup.1H-NMR was 3900 g/mol. After freeze-drying dioxane content was determined by .sup.1H-NMR (300 MHZ, 50 mg of polymer dissolved into 1 ml of deuterated chloroform, d.sub.1=30 s, 32 scans). 5 mg of dibromobenzene (Acros) was dissolved in the sample for quantification of the dioxane. Dioxane content was found to be 1193 ppm.

Example 7

(30) This example describes the general procedures used for the preparation of multi-block copolymers. ?-Caprolactone-co-PEG-co-?-caprolactone (CPC) or D,L-lactide-co-PEG-co-D,L-lactide pre-polymers (LPL) (M.sub.n 2000 g/mol) were heated to 50-80? C. until they became more liquid. The appropriate amounts of LL4000 pre-polymer (M.sub.n 4000 g/mol) and CPC or LPL pre-polymer were weighted into a glass ampoule supplied with nitrogen inlet and dried at 50? C. for at least 48 h. Subsequently, the glass ampoule was supplied with a mechanical stirrer. 1,4-Dioxane (Acros, distilled over sodium) was added to a polymer concentration of 30 wt. % and the contents of the ampoule were heated to 80? C. to dissolve the pre-polymers. 0.900-0.990 equivalent (with respect to the pre-polymer hydroxyl groups) of 1,4-butanediisocyanate (Bayer, distilled at reduced pressure) was added and the reaction mixture was stirred mechanically for 16-22 h. Non-distilled dioxane was added to a polymer concentration of 20 wt. % to quench unreacted isocyanate groups. The reaction mixture was further diluted with non-distilled dioxane to a polymer concentration of 10 wt. %. The ampoule was cooled to room temperature, the reaction mixture was poured into a tray and frozen at ?18? C. Subsequently, dioxane was removed by placing the frozen reaction mixture under vacuum at 30? C. The polymer was stored in a sealed package at ?18? C. A small part of the batch was analysed for thermal properties (mDSC), dioxane content (gas chromatography), intrinsic viscosity and polymer composition (.sup.1H-NMR). Thermal analysis was performed by Modulated Differential Scanning calorimetry (mDSC). Samples of 5-10 mg were weighed in a DSC pan. The measurement was performed on a DSC Q1000 (TA Instruments) using a modulated temperature program. Amplitude was set to 1? C., the modulation period to 60 s and the heating rate to 5? C./min. Samples were heated from ?80? C. to 100-200? C. (depending on the type of polymer). Intrinsic viscosity was measured using an Ubbelohde Viscosimeter (DIN), type 0C, 0a or I, Schott Ger?te supplied with a Schott AVS-450 Viscosimeter including a water bath. The measurements were performed in chloroform at room temperature. The polymer concentration in chloroform was such that the relative viscosity was in the range of 1.2 to 2.0. Dioxane content was determined using a GC-FID headspace method. Measurements were performed on a GC-FID Combi Sampler supplied with an Agilent Column, DB-624/30 m/0.53 mm. Samples were prepared in DMSO. Dioxane content was determined using dioxane calibration standards.

Example 8

(31) This example describes the preparation of 20(D,L-Lactide-co-PEG.sub.1000-co-D,L-lactide).sub.2000-80(L-lactide).sub.4000 (20LP10L20-LL40). 42.02 g of LL40 pre-polymer (M.sub.n 4040 g/mol, 10.40 mmol) and 10.16 g of D,L-lactide-co-PEG.sub.1000-D,L-lactide pre-polymer (M.sub.n 2000 g/mol, 5.08 mmol) were weighed and dissolved in 100 ml of 1,4-dioxane at 80? C. 1.8466 g (13.2 mmol) of 1,4-butanediisocyanate was added (0.851 equivalent with respect to the pre-polymer hydroxyl groups) with 20 ml of 1,4-dioxane After 17 h the reaction was quenched with 88 ml of non-distilled dioxane and further diluted with 255 ml of non-distilled dioxane. The dioxane was removed by placing the frozen reaction mixture under vacuum at 30? C.

Example 9

(32) This example describes the preparation of 30(D,L-lactide-co-PEG.sub.1000-co-D,L-lactide).sub.2000-70(L-lactide).sub.4000 (30LP10L20-LL40). 34.44 g of LL40 pre-polymer (M.sub.n 4020 g/mol, 8.57 mmol) and 14.95 g of D,L-lactide-co-PEG.sub.1000-D,L-lactide pre-polymer (M.sub.n 2040 g/mol, 7.33 mmol) were weighed and dissolved in 100 ml of 1,4-dioxane at 80? C. 2.7386 g (19.5 mmol) of 1,4-butanediisocyanate was added (1.231 equivalent with respect to the pre-polymer hydroxyl groups) with 20 ml of 1,4-dioxane After 20 h the reaction was quenched with 85 ml of non-distilled dioxane and further diluted with 240 ml of non-distilled dioxane. The dioxane was removed by placing the frozen reaction mixture under vacuum at 30? C.

Example 10

(33) This example describes the preparation of 50(D,L-lactide-co-PEG.sub.1000-co-D,L-lactide).sub.2000-50(L-lactide).sub.4000 (50LP10L20-LL40). 19.59 g of LL40 pre-polymer (M.sub.n 4060 g/mol, 4.83 mmol) and 19.57 g of D,L-lactide-co-PEG.sub.1000-D,L-lactide pre-polymer (M.sub.n 2040 g/mol, 9.59 mmol) were weighed and dissolved in 78 ml of 1,4-dioxane at 80? C. 2.0018 g (14.3 mmol) of 1,4-butanediisocyanate was added (0.991 equivalent with respect to the pre-polymer hydroxyl groups) in 20 ml of 1,4-dioxane. After 20 h the reaction was quenched with 67 ml of non-distilled dioxane and further diluted with 189 ml of non-distilled dioxane. Dioxane was removed by placing the frozen reaction mixture under vacuum at 30? C.

Example 11

(34) This example describes the preparation of 70(D,L-lactide-co-PEG.sub.1000-co-D,L-lactide).sub.2000-30(L-lactide).sub.4000 (70LP10L20-LL40). 8.59 g of LL40 pre-polymer (M.sub.n 4020 g/mol, 2.14 mmol) and 19.96 g of D,L-lactide-co-PEG.sub.1000-D,L-lactide pre-polymer (M.sub.n 2040 g/mol, 9.78 mmol) were weighed and dissolved in 48 ml of 1,4-dioxane at 80? C. 1.648 g (11.8 mmol) of 1,4-butanediisocyanate was added (0.986 equivalent with respect to the pre-polymer hydroxyl groups) with 20 ml of 1,4-dioxane After 21 h the reaction was quenched with 49 ml of non-distilled dioxane, and further diluted with 147 ml of non-distilled dioxane. The dioxane was removed by placing the frozen reaction mixture under vacuum at 30? C.

Example 12

(35) This example describes the preparation of 30(D,L-lactide-co-PEG.sub.3000-co-D,L-lactide).sub.4000-70(L-lactide).sub.4000 (30LP30L40-LL40). 29.96 g of LL40 pre-polymer (M.sub.n 4030 g/mol, 7.43 mmol) and 14.01 g of D,L-lactide-co-PEG.sub.1000-D,L-lactide pre-polymer (M.sub.n 4000 g/mol, 3.50 mmol) were weighed and dissolved in 83 ml of 1,4-dioxane at 80? C. 1.52 g (10.8 mmol) of 1,4-butanediisocyanate was added (0.992 equivalent with respect to the pre-polymer hydroxyl groups) with 20 ml of 1,4-dioxane. After 21 h the reaction was quenched with 74 ml of non-distilled dioxane and further diluted with 222 ml of non-distilled dioxane. The dioxane was removed by placing the frozen reaction mixture under vacuum at 30? C.

Example 13

(36) This example describes preparation of 50(?-caprolactone-co-PEG.sub.1000-co-?-caprolactone).sub.2000-50(L-lactide).sub.4000 (50CP10C20-LL40). 24.34 g of LL40 pre-polymer (M.sub.n 4030 g/mol, 6.04 mmol) and 23.87 g of ?-caprolactone-co-PEG.sub.1000-?-caprolactone pre-polymer (M.sub.n 2010 g/mol, 11.9 mmol) were weighed and dissolved in 95 ml of 1,4-dioxane at 80? C. 2.4098 g (17.2 mmol) of 1,4-butanediisocyanate was added (0.960 equivalent with respect to the pre-polymer hydroxyl groups) with 20 ml of 1,4-dioxane. After 18 h the reaction was quenched with 82 ml of non-distilled dioxane and further diluted with 246 ml of non-distilled dioxane. The dioxane was removed by placing the frozen reaction mixture under vacuum at 30? C.

Example 14

(37) This example describes preparation of 30(?-caprolactone-co-PEG.sub.3000-co-?-caprolactone).sub.4000-70(L-lactide).sub.4000 (30CP30C40-LL40). 35.84 g of LL40 pre-polymer (M.sub.n 4030 g/mol, 8.89 mmol) and 14.79 g of ?-caprolactone-co-PEG.sub.3000-?-caprolactone pre-polymer (M.sub.n 4010 g/mol, 3.69 mmol) were weighed and dissolved in 100 ml of 1,4-dioxane at 80? C. 1.7428 g (12.4 mmol) of 1,4-butanediisocyanate was added (0.988 equivalent with respect to the pre-polymer hydroxyl groups) with 20 ml of 1,4-dioxane. After 18 h the reaction was quenched with 83 ml of non-distilled dioxane and further diluted with 240 ml of non-distilled dioxane. The dioxane was removed by placing the frozen reaction mixture under vacuum at 30? C.

Example 15

(38) The synthesised multi-block copolymers were analysed for chemical composition, molecular weight and residual dioxane content. Table 1 shows the collected analysis results for 20LP10L20-LL40, 30LP10L20-LL40, 50LP10L20-LL40, 70LP10L20-LL40, 30LP30L40-LL40, 50CP10C20-LL40, 30CP30C40-LL40. The actual composition of the copolymers, as determined by .sup.1H-NMR from L/P and C/P molar ratios resembled the target composition well. All polymers had an intrinsic viscosity between 0.7 and 1.1 dl/g. Dioxane contents were well below 1000 ppm indicating effective removal of dioxane by vacuum-drying.

(39) The multi-block copolymers were analysed for their thermal properties to confirm their phase separated morphology. Results are shown in Table 2. FIG. 1 shows typical DSC thermograms of 50LP10L20-LL40 (FIG. 1A), 30LP30L40-LL40 (FIG. 1B) and 50CP10C20-LL40 (FIG. 1C) multi-block copolymers. All multi-block copolymers exhibited a melting temperature (T.sub.m) at approximately 120-133? C., due to melting of the LL40 segment. As expected, the melting enthalpy (?H.sub.m) of the crystalline LLA40 segment increased with increasing amount of the segment. 70LP10L40-LL40, 50CP10C20-LL40 also exhibited a T.sub.m at approximately 85? C., which is attributed to melting of less perfect crystals of LL40. Copolymers containing PEG.sub.3000 showed a T.sub.m at approximately 40? C., due to melting of the PEG. The glass transition temperature (T.sub.g) of the multi-block copolymers is in general in between that of pre-polymer (A) and pre-polymer (B), indicating phase mixing of the amorphous pre-polymer (A) with the amorphous content of pre-polymer (B). The T.sub.g of LP10L20-LL40 type multi-block copolymers increased from ?18 to 50? C. when increasing the LLA40 segment from 30 to 80 wt. %. The T.sub.g of these multi-block copolymers is in between that of pre-polymer (A) (pLP10L20, T.sub.g?37? C.) and pre-polymer (B) (LL40, T.sub.g?50? C.) and is thus attributed to mixing of amorphous polylactide of the semi-crystalline LL40 block and PEG. 50CP10C20-LL40 had a T.sub.g of ?48? C., which is similarly attributed to mixing of amorphous PEG, polycaprolactone and polylactide. Table 3 shows the swelling degree of the multi-block copolymers. To measure the swelling characteristics of polymers, polymer films were made by pouring a 13 wt. % polymer solution in dichloromethane (approximately 300 mg of polymer with 1.5 ml of dichloromethane), on a glass plate and spreading the polymer solution with a casting knife or poured into a Teflon? mould. The dichloromethane was left to evaporate slowly overnight and the residual dichloromethane was removed by vacuum drying at 20? C. Resulting films had a thickness of 100-200 ?m. For the swelling tests, 15-40 mg of circular films with a diameter of approximately 25 mm were weighed and immersed in a flask containing 10 ml of phosphate buffer pH 7.4 (ISO-15814). The samples were stored in an oven at 37? C. At each sampling time point, samples were collected and excess buffer solution was removed from the surface where after the samples were weighed on a 4-decimal balance. All tests were performed in duplicate. The swelling degree was found to increase gradually with the content of PEG of the copolymers and with PEG MW at approximately constant PEG content.

(40) TABLE-US-00001 TABLE 1 Collected results regarding the chemical composition, intrinsic viscosity and residual dioxane content of multi-block copolymers 20LP10L20-LL40, 30LP10L20-LL40, 50LP10L20-LL40, 70LP10L20-LL40, 30LP30L40-LL40, 50CP10C20-LL40, 30CP30C40-LL40. 20LP10L20- 30LP10L20- 50LP10L20- 70LP10L20- 30LP30L40- 50CP10C20- 30CP30C40- LL40 LL40 LL40 LL40 LL40 LL40 LL40 Molar L/P ratio 126.1 78.2 42.1 26.3 137.4 27.8 130.1 in-weights Molar L/P ratio 128.5 75.9 42.6 25.7 129.9 26.8 131.8 .sup.1H-NMR Molar C/P ratio 8.8 7.8 in-weights Molar C/P ratio 8.2 8.8 .sup.1H-NMR Intrinsic viscosity 0.73 0.85 0.89 0.70 0.79 1.05 0.69 (dl/g) Dioxane content <200 256 <200 <200 <200 <200 <200 (ppm)

(41) TABLE-US-00002 TABLE 2 Thermal characteristics of multi-block copolymers (MBCP) 20LP10L20-LL40, 30LP10L20-LL40, 50LP10L20-LL40, 70LP10L20-LL40, 30LP30L40-LL40, 50CP10C20-LL40, 30CP30C40-LL40 and their pre-polymers (PP) A and B. 20LP10L20- 30LP10L20- 50LP10L20- 70LP10L20- 30LP30L40- 50CP10C20- 30CP30C40- LL40 LL40 LL40 LL40 LL40 LL40 LL40 T.sub.g (? C.) MBCP 50 5 ?15 ?18 ?48 T.sub.m (? C.) MBCP 134 126 123 85/120 37/132 87/126 43/133 ?H.sub.m (J/g) MBCP 50 39 31 2/4 1/40 4/13 35/25 T.sub.g (? C.) PP A ?37 ?37 ?37 ?37 ?39 ?67 ?67 T.sub.m (? C.) PP A 35/42 43 43 ?H.sub.m (J/g) PP A 37 (both peaks) 91 85 T.sub.g (? C.) PP B 43 46 48 46 57 57 57 T.sub.m (? C.) PP B 85/131 117/134 136 117/134 137 137 137 ?H.sub.m (J/g) PP B 24 (both peaks) 28 (both peaks) 32 28 (both peaks) 57 57 57

(42) TABLE-US-00003 TABLE 3 Composition and swelling of multi-block copolymers 20LP10L20-LL40, 30LP10L20-LL40, 50LP10L20-LL40, 70LP10L20-LL40, 30LP30L40-LL40, 50CP10C20-LL40, 30CP30C40-LL40. wt. % wt. % Swelling Segment Segment MW wt. % degree A B PEG PEG () 20LP10L20-LL40 20 80 1000 10 xx 30LP10L20-LL40 30 70 1000 15 1.03 50LP10L20-LL40 50 50 1000 25 1.13 70LP10L20-LL40 70 30 1000 35 1.26 30LP30L40-LL40 30 70 3000 22.5 1.16 50CP10C20-LL40 50 50 1000 25 1.18 30CP30C40-LL40 30 70 3000 22.5 1.67

Example 16

(43) In this example various hydrophilic phase separated multi-block copolymers described in the examples above were evaluated for their protein release characteristics using bovine serum albumin (BSA, 69 kDa) and lysozyme (14 kDa) as model proteins.

(44) Protein-loaded films containing 10 wt. % protein were prepared by mixing of approximately 150 ?l of 20 wt. % protein solution with 1.5 ml of dichloromethane containing 300 mg of polymer for 30 s with an Ultra turrax at 18 000 rpm. The emulsion was spread on a glass plate with a casting knife or poured into a Teflon? mould. The dichloromethane was left to evaporate slowly overnight and the residual dichloromethane was removed by vacuum drying at 20? C. Resulting films had a thickness of 80-120 ?m.

(45) For the release tests, 20 mg of protein loaded film were weighed and immersed in vials containing 5 ml of phosphate buffer pH 7.4 and stored in an oven at 37? C. At each sampling point, 1 ml of release medium was sampled and replaced with 1 ml of fresh buffer. The protein content of the release samples was determined with a Bicinchoninic Acid (BCA) assay (Pierce) using an Easys Expert 96 well plate reader.

(46) The biological activity of released lysozyme was measured by means of a bacteria lysis test. Lysozyme loaded films were prepared as described above. A 0.01 wt. % lysozyme solutions was prepared to serve as a control by weighing 2.1 mg of lysozyme and adding 20 ml of phosphate buffer. Lysozyme-loaded films were weighed and immersed in vials containing 5 ml of phosphate buffer pH 7.4. Vials containing lysozyme-loaded films as well as freshly prepared lysozyme solutions were stored in an oven at 37? C. At each sampling point, 1 ml of release medium was sampled and replaced with 1 ml of fresh buffer. The protein content of the release samples was determined by BCA as described above. The activity of (released) lysozyme was determined by following the change in turbidity at 450 nm for 3 min of a bacteria dispersion (Micrococcus lysodeikticus, Sigma, 0.21 mg/ml) to which 10 ?l of sample was added. A UV-VIS spectrometer (Varian) was used for this purpose. Samples were diluted if necessary to obtain a lysozyme concentration of 5-100 ?g/ml. The lysozyme activity of the samples was calculated by comparing the slope of the obtained curves (the slope relates to the lysozyme activity) with the slope of a curve obtained with a fresh lysozyme solution.

(47) FIGS. 2 and 3 show the release of respectively lysozyme and bovine serum albumin from the films. The results show that by changing the PEG content and PEG MW the release rate and profile can be varied. Lysozyme was released over periods varying from a few days up to approximately 3 months. Due to its larger size the release rate of BSA was lower resulting in release over periods ranging from a few days up to approximately 4 months. Furthermore, the release of lysozyme could be tuned by introducing different (combinations of) monomers adjacent to the PEG group in the hydrophilic block of the multi-block copolymers. The resulting multi-block copolymers (50LP10L20-LL40, 50GP10C20-LL40 and 50CP10C20-LL40) contained 25 wt. % PEG1000 and exhibited similar swelling degrees, but different degradation rates leading to various release profiles for the encapsulated lysozyme. (FIG. 4).

(48) FIG. 5 shows the activity of lysozyme released from 10 wt. % lysozyme-loaded films of 30LP10L20-LL40 or 50LP10L20-LL40 (phosphate buffer pH 7.4, 37? C.). As a control the activity of lysozyme of the 0.01 wt. % lysozyme solutions 4 stored at 4 or 37? C. was measured (phosphate buffer pH 7.4). The results show that lysozyme released from the films over a period of approximately one month retained its biological activity, indicating that the structural integrity and biological activity of lysozyme was not only preserved during the encapsulation process but also during long-term presence of lysozyme in the hydrated and swollen polymer matrix at 37? C. prior to release.

Example 17

(49) In this example 30LP10L20-LL40 (IV 0.85 dl/g) and 50CP10C20-LL40 (IV 1.06 dl/g) type phase separated copolymers were used to formulate BSA into microspheres.

(50) BSA loaded microspheres were prepared of 50CP10C20-LL40 (IV 1.06 dl/g) and 30LP10L20-LL40 (IV 0.85 dl/g) hydrophilic phase separated multi-block copolymers by a solvent evaporation method using procedures as disclosed by by Kissel et al., J. Contr. Rel. 1996, 39(2), 315-326 and Meinel et al., J. Contr. Rel. 2001, 70(1-2), 193-202. BSA (25-50 mg of) was dissolved in about 150 mg of ultra-pure water and emulsified with 2-3 ml of a solution of 50CP10C20-LL40 (15% w/v) or 30LP10L20-LL40 (23% w/v) in dichloromethane for 60 s using an Ultra turrax IKA T18 operated at 20 000 rpm yielding a water-in-oil (W/O) emulsion). The so-obtained primary emulsion was then emulsified in about 80-130 ml of UP-water containing 4.0 wt. % PVA for 30 s using an Ultra turrax IKA T18 operated at 14 000 rpm yielding a water-in-oil-in water (W/O/W) emulsion. The so-obtained secondary emulsion was gently stirred for 2 h at 600 rpm at room temperature. Due to the evaporation of the dichloromethane, the polymer precipitated from the solution to yield microspheres. After 3 h (the time necessary to achieve almost complete evaporation of the dichloromethane) the formed microspheres were collected by centrifugation, and the microspheres were washed three times with 100-200 ml of an aqueous solution of 0.05 wt. % Tween 20 in ultra-pure water. Finally, the microspheres were lyophilised.

(51) For IVR tests, 2 ml of 100 mM phosphate buffer (pH 7.4, 0.02 wt. % NaN.sub.3) in case of 30LP10L20-LL40 microspheres and 25 mM NaPi buffer (pH 7.2, 105 mM NaCl, 0.01 wt. % Tween 80, 0.02 wt. % NaN.sub.3) in case of 50CP10C20-LL40 microspheres was added to 20 mg of microspheres. The sample was incubated at 37? C. and at each sampling point 1.8 ml of sample was taken and refreshed with release buffer. BSA content was measured with BCA protein assay in case of 30LP10L20-LL40 microspheres and with UPLC (eluent A: 1 wt. % TFA in UP-water, eluent B: 0.085 wt. % TFA in acetonitrile, 95/5 v/v A/B to 5/95 A/B in 25 min) in case of 50CP10C20-LL40 microspheres.

(52) The particle size distribution of the microspheres was measured by Coulter counter. Approximately 1 mg of microspheres were dispersed in 50-100 ml of Isotron II solution by gently stirring and the particle size was measured with a Coulter counter equipped with a 100 ?m measurement cell.

(53) The BSA content of the microspheres was determined by dissolving 5-10 mg of microspheres, accurately weighted, in 5.0 ml of acetonitrile. After centrifugation, 4 ml of supernatant was removed and 5 ml of PBS was added. BSA content was measured with UPLC (eluent A: 0.1 wt. % TFA in UP-water, eluent B: 0.1 wt. % TFA in acetonitrile, 90/10 v/v A/B to 10/90 v/v A/B in 4 min).

(54) Table 4 lists the particle size, encapsulation efficiency (EE) of the BSA loaded microspheres prepared. FIG. 6 shows the in vitro release of BSA from 30LP10L20-LL40 microspheres with 5 wt. % BSA target loading and 50CP10C20-LL40 microspheres with 10 wt. % BSA target loading. BSA was released from 30LP10L20-LL40 microspheres for almost 3 months in a linear fashion without significant burst. 50CP10C20-LL40 microspheres release BSA for almost ?3 months in a linear fashion without significant burst, where after slower release followed for another ?1.5 months.

(55) TABLE-US-00004 TABLE 4 Average particle size, BSA content and encapsulation efficiency of BSA loaded 50CP10C20-LL40 and 30LP10L20-LL40 microspheres. Average size Content Polymer grade (?m) (wt. %) EE (%) 50CP10C20-LL40 14 2.8 33 30LP10L20-LL40 18 4.3 85

Example 18

(56) In this example various hydrophilic phase separated multi-block copolymers prepared as described in the examples above were used to prepare Insulin-like Growth Factor I (IGF-1) loaded film and microsphere formulations.

(57) IGF-1 loaded films were prepared by dissolution of 0.18 g of polymer into 1.46 g of dichloromethane and subsequent emulsification by Ultra turraxing with IGF-1 dissolved in ultra pure water at 18 000 rpm for 30 s or by using ultrasound at 100 W for 5 s. The emulsion was poured into a Teflon? mould. Dichloromethane was left to evaporate overnight and residual dichloromethane was removed by vacuum drying overnight. 20 mg films were cut and put on release at 37? C. with 1 ml of phosphate buffered saline (PBS, 25 M pH 7.2, 105 mM NaCl, 0.01 wt. % Tween 80 and 0.02 wt. % NaN.sub.3). At predetermined time points, samples were taken and the sampled amount was refreshed by fresh buffer.

(58) IGF-1 loaded microspheres were prepared by a solvent extraction/evaporation based W/O/W emulsification process. 2.78 mg of IGF-1 and 51.8 mg of BSA were dissolved in 143 ?l of UP-water in an Eppendorf cup and emulsified in a solution of 0.47 g of 50CP10C20-LL40 (IV 1.05 dl/g) in 2.62 g of dichloromethane using an Ultra turrax (20 000 rpm, 60 s). The so-obtained primary emulsion was then emulsified in 81 ml of UP-water containing 4.0 wt. % PVA using an Ultra turrax (14 000 rpm for 60 s), and stirred for 2 h at 600 rpm at room temperature. The resulting microspheres were collected on a 5 ?m membrane filter and washed with 1 l of UP-water containing 0.05 wt. % Tween 80. Finally, the microspheres were lyophilised.

(59) Approximately 1 mg of microspheres were dispersed in 50-100 ml of Isotron II solution by gently stirring and the particle size was measured with a Coulter counter equipped with a 100 ?m measurement cell.

(60) The IGF-1 and BSA content were determined by dissolving 5 mg of microspheres, accurately weighted, in 0.3 ml of acetonitrile. Subsequently, 1.2 ml of PBS was added and gently shaken. After centrifugation, the IGF-1 and BSA content in the supernatant were determined by UPLC. Procedure was performed in triplicate.

(61) Using a commercial sandwich ELISA (R&D Systems), the concentration of human insulin-like Growth Factor I (IGF-1) in a sample was measured to confirm that microencapsulated and released IGF-1 was still capable to bind with the capture and detection antibody after release and thus no protein degradation at that level has occurred. The capture and detection antibody of the kit were specific for natural and recombinant IGF-1 and as a standard recombinant IGF-1.

(62) To investigate the structural integrity of released IGF-1, 100-300 ng of IGF-1 collected from release samples was denaturated using Laemli/?-mercapto-ethanol buffer and loaded on an any KD TGX pre-cast mini gel and separated under denaturating conditions at 100-200 V using 1? Tris/Glycine/SDS as separating buffer, and stained overnight in colloidal CBB staining agent. A Dual Xtra Protein marker (Bio-Rad) was used to determine the protein size of the separated proteins.

(63) FIG. 7 shows the in vitro release of IGF-1 from 50CP30C40-LL40 and 30CP30C40-LL40 polymer films loaded with 0.6 wt. % of IGF-1 as measured by UPLC and ELISA. IGF-1 was released from the 50CP30C40-LL40 films in about 7 days whereas IGF-1 was slowly released from the 30CP30C40-LL40 polymer films with a cumulative release of about 40% after 28 days. Since the cumulative release of IGF-1 as measured by UPLC was nearly identical to the cumulative release of IGF-1 as measured by ELISA it was concluded that the released IGF-1 was structurally intact and biologically active.

(64) Microspheres with 0.5 wt. % of IGF-1 target loadings were prepared of 50CP10C20-LL40 with IV 1.05 and 0.68 dl/g by a double emulsification process. The microspheres had a smooth surface (FIG. 8) and encapsulation efficiencies varying between 40 and 60%. The volume average particle size (d.sub.50) as measured with a Coulter counter equipped with a 100 ?m measurement cell was 54.4 ?m with a CV (coefficient of variation) of 61%. FIG. 9 shows IGF-1 release from these microspheres in vitro. Complete release of IGF-1 within 2 days was obtained for microspheres composed of 50CP10C20-LL40 with IV 0.68 dl/g. IGF-1 release from microspheres composed of 50CP10C20-LL40 with IV 1.05 dl/g was slower with complete release achieved after approximately 6 days. Released IGF-1 was structurally intact as could be concluded from the SDS-PAGE results (FIG. 10), which did not show any degradation nor aggregation of the protein.

Example 19

(65) In this example various hydrophilic phase separated multi-block copolymers (20LP10L20-LL40 (IV 0.58 dl/g), 30LP6L20-LL40 (IV 0.60 dl/g) and 30CP10C20-LL40 (IV 0.71 dl/g)) prepared as described in the examples above were used to prepare film formulations loaded with a highly water-soluble biologically active polypeptide with a molecular weight of 15 kDa (Protein A). Furthermore, 30CP10C20-LL40 multi-block copolymers with various IV (0.81, 0.71 and 0.65 dl/g) were used to formulate Protein A into microspheres formulations.

(66) Protein A loaded films were prepared by a solvent casting method. 10 mg of Protein A was dissolved in 123 mg of UP-water and emulsified in a solution of 0.18 g of polymer in 1.46 g of dichloromethane using an Ultra turrax (18 000 rpm, 60 s). The so-obtained primary emulsion was poured in a Teflon? mould and the dichloromethane was evaporated overnight. Residual dichloromethane was removed by vacuum drying.

(67) Protein A loaded microspheres were prepared by a solvent extraction/evaporation based W/O/W emulsification process. 21 mg of Protein A (5 wt. % target loading) was dissolved in 156 ?l of UP-water optionally containing inulin in an Eppendorf cup and emulsified in a solution of 0.4 g of polymer in 2.1 g of dichloromethane using an Ultra turrax (20 000 rpm, 60 s). The so-obtained primary emulsion was then emulsified in 70 ml of UP-water containing 4.0 wt. % PVA using an ultraturrax (14 000 rpm for 60 s), and stirred for 2 h at 600 rpm at room temperature. The resulting microspheres were collected on a 5 ?m membrane filter and washed with three times 100 ml of UP-water containing 0.05 wt. % Tween 80. Finally, the microspheres were lyophilised.

(68) Approximately 10 mg of microspheres were dispersed in 50-100 ml of Isotron II solution by gently stirring and the particle size was measured with a Coulter counter equipped with a 100 ?m measurement cell.

(69) The Protein A content was determined by dissolving 5 mg of microspheres, accurately weighted, in 0.3 ml of acetonitrile. After centrifugation, the supernatant was removed and the residual ACN was evaporated. 1.95 ml of PBS was added. Protein A content was measured with UPLC (eluent A: 0.1 wt. % TFA in UP-water, eluent B: 0.1 wt. % TFA in acetonitrile, 80/20 v/v A/B to 10/90 A/B in 3 min).

(70) For SEM imaging, a small amount of microspheres was adhered to carbon conductive tape and coated with gold for 3 min. The sample was imaged using a 10 kV electron beam.

(71) The in vitro release kinetics of Protein A-loaded films and microspheres were measured in 100 mM of phosphate buffer pH 7.4 (20 mg of film in 2 ml). The samples were incubated at 37? C. At each sampling point, 1.8 nil of sample was taken and refreshed with 1.8 ml of phosphate buffer. Protein A content was measured with UPLC (eluent A: 0.1 wt. % TFA in UP-water, eluent B: 0.1 wt. % TFA in acetonitrile, 80/20 v/v A/B to 10/90 AB in 3 min).

(72) SDS-PAGE was performed in reducing mode with 4-20% Tris-HCl gels. Per slot 20 ?l of protein solution was applied for samples and Protein A standard. For the marker, 2 ?l was applied to the slot. The amount of protein added per slot was either 75 or 150 ng. Samples were prepared by dilution with 12 mM PBS pH 7.4 or UP-water to a Protein A concentration of either 150 or 300 ng/20 ?l. Subsequently, Laemmli working solution (Laemmli buffer containing 1% of mercaptoethanol) was added in ratio 1:1 v/v. The samples were heated to ?90? C. for 5 min and applied to the gels. The gels were clamped in the electrophoresis cell and running buffer (Tris/Glycine/SDS pH 8.3) was added. The samples and standards were applied to the gels, and the gels were run for 15 min at 100 kV. The voltage was subsequently set to 200 kV and the gels were run until a good separation of the molecular weight standards was obtained. The gels were washed with UP-water and stained with silver reagent.

(73) FIG. 11 shows the in vitro release of Protein A from 20LP10L20-LL40 (10 wt. % of PEG MW 1000), 30LP6L20-LL40 (9 wt. % of PEG MW 600) and 30CP10C20-LL40 (15 wt. % of PEG MW 1000). 30CP10C20-LL40-based films released Protein A relatively fast with a cumulative release of Protein A of 100% after 3 months. By replacing PEG1000 by PEG600, which leads to reduction of the swelling degree, the release of Protein A could be slowed down and near first-order diffusion controlled release kinetics were obtained leading to a cumulative release of ?75% after 4 months. Reduction of the release rate of Protein A could also be achieved by lowering the weight fraction of the hydrophilic LP10L20 block in the polymer. By using 20LP10L20-LL40 (10 wt. % PEG1000) release could be further slowed down, and after an initial small burst of less than 15%, well-controlled release kinetics of Protein A were obtained with cumulative release of ?65% in 6 months. The data clearly show that Protein A release kinetics can be controlled by the choice of polymer.

(74) Protein-A-loaded microspheres were prepared of 30CP10C20-LL40 loaded with 3-4 wt. % of Protein A. Optionally, 2 or 5 wt. % of inulin was co-encapsulated to enhance the release rate of Protein A. The effect of polymer molecular weight on protein release kinetics was studied by studying the release kinetics of Protein A from microspheres composed of 30CP10C20-LL40 polymers with different intrinsic viscosity. For all Protein A loaded microspheres, spherical microspheres were obtained. For microspheres with co-encapsulated inulin, the surface porosity increased with increasing inulin content, as shown on the SEM pictures in FIG. 12. Table 5 lists the particle size and encapsulation efficiency (EE) of Protein A of the microspheres. FIG. 13 shows that after a small initial burst, Protein A was released at a constant rate. It was observed that the burst decreased and linearity increased with decreasing inulin content. Without inulin present ?70% was released in 3 months, while 90-100% was released when 2 or 5 wt. % inulin was co-encapsulated. Release of Protein A from 30CP10C20-LL40 films containing 2 or 5 wt. % of co-encapsulated inulin were similar. Release data is shown up to ?4 months. Expected duration of release for Protein A from 30CP10C20-LL40 microspheres is approximately 6 months.

(75) FIG. 14 shows the release kinetics of Protein A from 30CP10C20-LL40 films with different intrinsic viscosity (IV) of the polymer. The release rate of Protein A increased with increasing polymer IV. For 30CP10C20-LL40 polymers with an IV 0.71 or 0.81 dl/g, sustained release of Protein A was obtained with cumulative release of 60-70% after 2 months. The release kinetics of Protein A from microspheres composed of 30CP10C20-LL40 with an IV of 0.58 dl/g were significantly different. The initial release rate up to one month was significantly lower, but Protein A release accelerated between 1 and 3 months, where after it slowed down again, giving a total duration of release of approximately 5 months. The data clearly show that Protein A can be released from microspheres in linear fashion for at least 4 months and that release kinetics can be controlled by the co-encapsulation of sugars, such as inulin, as well as by the intrinsic viscosity of the polymer.

(76) The structural integrity of Protein A released from microspheres was studied by SDS-PAGE. SDS-PAGE confirmed that Protein A released for at least 21 days consisted mainly of native Protein (FIG. 15). These results show that 30CP10C20-LL40 microspheres provide a suitable matrix for the long-term release of structurally intact Protein A.

(77) TABLE-US-00005 TABLE 5 Overview of Protein A loaded microspheres characteristics with 3-4 wt. % Protein A target loading. Polymer Particle Protein A EE IV Co-encapsulated size content Protein A MSP # (dl/g) inulin (wt. %) (?m) (wt. %) (%) #1 0.71 0 52 3.7 100 #2 0.71 2 57 3.3 90 #3 0.71 5 55 1.8 54 #4 0.57 0 33 4.0 100 #5 0.81 0 43 0.7 24

Example 20

(78) In this example the hydrophilic phase separated multi-block copolymer 20LP10L20-LL40 (IV 0.73 dl/g) prepared as described in the examples above was used to prepare film and microsphere formulations loaded with a biologically active polypeptide with a molecular weight of 2.5 kDa (Peptide A).

(79) Peptide A loaded films were prepared by a solvent casting method. 10 (for 5 wt. % loading) or 20 mg (for 10 wt. % loading) of Peptide A was dissolved in 123 mg of UP-water and emulsified in a solution of 0.18 g of 20LP10L20-LL40 (IV 0.76 dl/g) in 1.46 g of dichloromethane using an Ultra turrax (18 000 rpm, 30 s). The so-obtained primary emulsion was poured in a Teflon mould and the dichloromethane was evaporated overnight. Residual dichloromethane was removed by vacuum drying.

(80) Peptide A loaded microspheres were prepared by a solvent evaporation based double emulsion process. 50 mg of Peptide A was dissolved in PBS and emulsified in a solution of 0.5 g of 20LP10L20-LL40 (IV 0.73 dl/g) in 2 g of dichloromethane using an Ultra turrax (24 000 rpm, 60 s). The so-obtained primary emulsion was then emulsified in 200 ml of UP-water containing 4.0 wt. % polyvinyl alcohol using an ultraturrax (14 000 rpm for 30 s), and stirred for 3 h at 600 rpm at room temperature. The resulting microspheres were centrifuged, the supernatant was removed and the microspheres were washed three times with 200 ml of UP-water containing 0.05 wt. % Tween 20. Finally, the microspheres were lyophilised. The particle size distribution was measured with a Coulter Counter. Approximately 10 mg of microspheres were dispersed in 50-100 ml of Isotron II solution by gently stirring and the particle size was measured with a 100 ?m measurement cell.

(81) Peptide A content of microspheres was determined by dissolving 5-10 mg of microspheres, accurately weighted, in 5.0 ml of acetonitrile. After centrifugation, 4 ml of supernatant was removed and 5 ml of PBS was added. Peptide A content was measured with HPLC (eluent A: 1 wt. % TFA in UP-water, eluent B: 0.085 wt. % TFA in acetonitrile, 95/5 v/v A/B to 5/95 A/B in 25 min).

(82) The in vitro release kinetics of Peptide A from films and microspheres were measured in PBS pH at 37? C. Peptide A containing films or microspheres (5-20 mg) were weighed into a vial and 2 ml of PBS was added. The vials were incubated at 37? C. and sampled at pre-determined time-points. At each sampling point 75-90% of release medium was collected and replaced by fresh PBS. Peptide A content of release samples was determined with HPLC (eluent A: 1 wt. % TFA in UP-water, eluent B: 0.085 wt. % TFA in acetonitrile, 95/5 v/v A/B to 5/95 A/B in 25 min).

(83) FIG. 16 shows the in vitro release of Peptide A from 20LP10L20-LL40 films. Peptide A was released from 5 wt. % loaded 20LP10L20-LL40 films in a linear fashion for at least 5 months without significant burst. For 20LP10L20-LL40 films with a higher Peptide A loading (10 wt. %), burst release increased to 15%. After approximately 2 months, release was similar to the 5 wt. % loaded films.

(84) Peptide A loaded 20LP10L20-LL40 microspheres had an average particle size of 30 ?m and a Peptide A content of 10.3 wt. %, representing an encapsulation efficiency of 100%. FIG. 17 shows that Peptide A MSP exhibited a low burst release of approximately 10 wt. % followed by zero-order release kinetics for at least 40 days.

Example 21

(85) In this example, hydrophilic phase separated multi-block copolymers 20LP10L20-LL40 (Example 8) and 10LP10L20-LL40 were used to prepare microspheres loaded with rapamycin (MW 914 Da). The polyethylene glycol component of the polymers had a molecular weight of 1000 g/mol.

(86) Rapamycin loaded microspheres with a target load of 20 wt. % rapamycin were prepared by a solvent evaporation method using a single oil-in-water (O/W) emulsion route. The polymers were dissolved in various blend ratios in dichloromethane to a concentration of about 20 wt. %, and the required amount of rapamycin was added. The polymer/rapamycin solution was then emulsified in 200 ml of UP-water containing 4.0 wt. % polyvinyl alcohol (PVA) using an Ultra turrax (14 000 rpm for 30 s), and then stirred with a magnetic stirrer for 3 h at 300 rpm at room temperature. The microsphere dispersion was concentrated by centrifugation and the microspheres were washed three times with 50 ml of aqueous 0.05 wt. % Tween 20 solution. Finally, the microspheres were lyophilised.

(87) The particle size distribution was measured with a Coulter Counter. Approximately 10 mg of microspheres were dispersed in 50-100 ml of Isotron II solution by gently stirring and the particle size was measured with a 100 ?m measurement cell.

(88) Rapamycin content of microspheres was determined by dissolving 5-10 mg of microspheres, accurately weighted, in 5.0 ml of acetonitrile. After centrifugation, 4 ml of supernatant was removed and 5 ml of PBS was added. Rapamycin content was measured with HPLC (eluens: acetonitrile/water 70/30 v/v; 278 nm).

(89) The in vitro release kinetics of rapamycin from microspheres were measured at 37? C. in 10 mM PBS pH 7.4 containing 0.5 wt. % SDS rapamycin containing microspheres (5-20 mg) were weighed into a vial and 2 ml of release medium was added. The vials were incubated at 37? C. and sampled at pre-determined time-points. At each sampling point 75-90% of release medium was collected and replaced by fresh PBS. Rapamycin content of release samples was determined with HPLC.

(90) The so-prepared rapamycin microspheres had an average size of 35 ?m and a rapamycin content varying from 17 to 20 wt. %, representing encapsulation efficiencies of 89% to 100%. FIG. 18 shows the release of rapamycin from microspheres composed of various blends of 20LP10L20-LL40 and 10LP10L20-LL40. Rapamycin release from 20LP10L20-LL40-based microspheres was relatively fast, whereas release of rapamycin from 10LP10L20-LL40-based microspheres was very slow. By blending the two polymers microspheres with intermediate release profiles were obtained.

Example 22

(91) In this example, goserelin acetate loaded microspheres were prepared of the hydrophilic phase separated multi-block copolymer 20LP10L20-LL40 by means of a water-in-oil-in-oil process. 62.6 mg of goserelin acetate was dissolved in 150 ?l of UP-water (29.4 wt. %) and emulsified with a solution of 0.5 g of 20LP10-LLA40 polymer in 7.4 g of dichloromethane in a scintillation vial (Ultra turrax, 20 000 rpm, 60 s). 13.5 g of the polymer precipitant (silicon oil, 350 cSt) was then slowly added (2-5 min) under constant stirring (12 000 rpm) to form embryonic microparticles. The embryonic microparticles were then poured into 550 ml of heptane at room temperature (13.5:1 ratio of dichloromethane to heptane solvent). The extraction vessel was closed to prevent excessive evaporation of the extraction medium. After approximately 3 h of extraction, the microparticles were collected by vacuum filtration, rinsed with additional heptane and dried under vacuum. The microspheres had an average size of 67 ?m and a goserelin content of 8.3%, representing an encapsulation efficiency of 88%.

(92) The particle size distribution was measured with a Coulter Counter. Approximately 10 mg of microspheres were dispersed in 50-100 ml of Isotron II solution by gently stirring and the particle size was measured with a 100 ?m measurement cell.

(93) Goserelin content of microspheres was determined by dissolving 5-10 mg of microspheres, accurately weighted, in 5.0 ml of acetonitrile. After centrifugation, 4 ml of supernatant was removed and 5 ml of PBS was added. Goserelin content was measured with HPLC (eluens: water/acetonitrile/trifluoracetic acid 72/28/0.1, 220 nm).

(94) The in vitro release kinetics of goserelin from microspheres were measured in PBS (192 mM pH 7.4 containing 0.01% tween 80 and 0.02% sodium azide) at 37? C. Goserelin containing microspheres (5-20 mg) were weighed into a vial and 2 ml of release medium was added. The vials were incubated at 37? C. and sampled at pre-determined time-points. At each sampling point 75-90% of release medium was collected and replaced by fresh PBS. Goserelin content of release samples was determined with HPLC.

(95) The so-prepared goserelin-loaded 20LP10-LLa40 microspheres had a spherical and smooth appearance (FIG. 19), an average size of 71 ?M (CV 47%) and a goserelin content of 8.3% representing an encapsulation efficiency of 88%. FIG. 20 shows the release of goserelin from the microspheres.

Example 23

(96) In this example, lysozyme-loaded microspheres were prepared of the hydrophilic phase separated multi-block copolymer 30CP10L20-LL40 by means of a solid-in-oil-in-oil process (S/O/O). 0.43 g of 30CP10L20-LL40 was dissolved in 7.4 g of dichloromethane in a scintillation vial (5.4 wt. %), and 0.074 g of spray-dried inulin-stabilized lysozyme microparticles (lysozyme/inulin ratio: 1:2 w/w) with a particle size of 1-2 ?m were added to the polymer solution, and the dispersion was homogenised by Ultra turrax (20 000 rpm, 60 s). 11.46 g of the polymer precipitant (silicon oil, 350 cSt) was then slowly added (2-5 min) under constant stirring (12 000 rpm) to form embryonic microparticles. The embryonic microparticles were then poured into 550 ml of heptane at room temperature (13.5:1 ratio of dichloromethane to heptane solvent). The extraction vessel was closed to prevent excessive evaporation of the extraction medium. After approximately 3 h of extraction, the microparticles were collected by vacuum filtration, rinsed with additional heptanes and dried by vacuum filtration. The microspheres had an average size of 59 ?m and a lysozyme content of 4.1-5.6%, representing an encapsulation efficiency of 80-100%.