Particles comprising polyesteramide copolymers for drug delivery

Abstract

The present disclosure relates to microparticles or nanoparticles comprising a polyesteramide (PEA) having a chemical formula described by structural formula (IV), ##STR00001##
wherein m+p varies from 0.9-0.1 and q varies from 0.1 to 0.9; m+p+q=1 whereby m or p could be 0; n is about 5 to about 300; (pref. 50-200); R.sub.1 is independently selected from the group consisting of (C.sub.2-C.sub.20) alkylene, (C.sub.2-C.sub.20) alkenylene, (R.sub.9COOR.sub.10OCOR.sub.9), CHR.sub.11OCOR.sub.12COOCR.sub.11 and combinations thereof; R.sub.3 and R.sub.4 in a single backbone unit m or p, respectively, are independently selected from the group consisting of hydrogen, (C.sub.1-C.sub.6)alkyl, (C.sub.2-C.sub.6)alkenyl, (C.sub.2-C.sub.6)alkynyl, (C.sub.6-C.sub.10)aryl, (C.sub.1-C.sub.6)alkyl, (CH.sub.2)SH, (CH.sub.2).sub.2S(CH.sub.3), CH.sub.2OH, CH(OH)CH.sub.3, (CH.sub.2).sub.4NH.sub.3+, (CH.sub.2).sub.3NHC(NH.sub.2+)NH.sub.2, CH.sub.2COOH, (CH.sub.2)COOH, CH.sub.2CONH.sub.2, CH.sub.2CH.sub.2CONH.sub.2, CH.sub.2CH.sub.2COOH, CH.sub.3CH.sub.2CH(CH.sub.3), (CH.sub.3).sub.2CHCH.sub.2, H.sub.2N(CH.sub.2).sub.4, Ph-CH.sub.2, CHCCH.sub.2, HO-p-Ph-CH.sub.2, (CH.sub.3).sub.2CH, Ph-NH, NH(CH.sub.2).sub.3C, NHCHNCHCCH.sub.2; R.sub.5 is selected from the group consisting of (C.sub.2-C.sub.20)alkylene, (C.sub.2-C.sub.20)alkenylene, alkyloxy or oligoethyleneglycol; R.sub.6 is selected from bicyclic-fragments of 1,4:3,6-dianhydrohexitols of structural formula (III); R.sub.7 is (C.sub.6-C.sub.10)aryl (C.sub.1-C.sub.6)alkyl; R.sub.8 is (CH.sub.2).sub.4; R.sub.9 or R.sub.10 are independently selected from C.sub.2-C.sub.12 alkylene or C.sub.2-C.sub.12 alkenylene; and R.sub.11 or R.sub.12 are independently selected from H, methyl, C.sub.2-C.sub.12 alkylene or C.sub.2-C.sub.12 alkenylene; whereby a is at least 0.05 and b is at least 0.05 and a+b=1.

Claims

1. Microparticles with an average diameter of from 1 to 100 m or nanoparticles with an average diameter of from 10 to less than 1000 nm comprising a bioactive agent and a biodegradable polyesteramide copolymer (PEA) according to the following formula: ##STR00008## wherein m+p is from 0.9-0.1 and q is from 0.1 to 0.9; m+p+q=1 whereby one of m or p could be 0; n is about 5 to about 300; a is at least 0.05, b is at least 0.05, a+b=1, qa=q*a, and qb=q*b; wherein units of m (if present), units of p (if present), units of qa, and units of qb are all randomly distributed throughout the copolymer; R.sub.1 is independently selected from the group consisting of (C.sub.2-C.sub.20) alkylene, (C.sub.2-C.sub.20) alkenylene, (R.sub.9COOR.sub.10OCOR.sub.9), CHR.sub.11OCOR.sub.12COOCR.sub.11, and combinations thereof; R.sub.3 and R.sub.4 in a single backbone unit m or p, respectively, are independently selected from the group consisting of hydrogen, (C.sub.1-C.sub.6)alkyl, (C.sub.2-C.sub.6)alkenyl, (C.sub.2-C.sub.6)alkynyl, (C.sub.6-C.sub.10)aryl, (C.sub.1-C.sub.6)alkyl, (CH.sub.2)SH, (CH.sub.2).sub.2S(CH.sub.3), CH.sub.2OH, CH(OH)CH.sub.3, (CH.sub.2).sub.4NH.sub.3+, (CH.sub.2).sub.3NHC(NH.sub.2+)NH.sub.2, CH.sub.2COOH, (CH.sub.2)COOH, CH.sub.2CONH.sub.2, CH.sub.2CH.sub.2CONH.sub.2, CH.sub.2CH.sub.2COOH, CH.sub.3CH.sub.2CH(CH.sub.3), (CH.sub.3).sub.2CHCH.sub.2, H.sub.2N(CH.sub.2).sub.4, Ph-CH.sub.2, CHCCH.sub.2, HO-p-Ph-CH.sub.2, (CH.sub.3).sub.2CH, Ph-NH, NH(CH.sub.2).sub.3C, NHCHNCHCCH.sub.2; R.sub.5 is selected from the group consisting of (C.sub.2-C.sub.20)alkylene, (C.sub.2-C.sub.20)alkenylene, alkyloxy or oligoethyleneglycol; R.sub.6 is selected from bicyclic-fragments of 1,4:3,6-dianhydrohexitols of structural formula (III); ##STR00009## R.sub.7 is selected from the group consisting of (C.sub.6-C.sub.10) aryl (C.sub.1-C.sub.6)alkyl; R.sub.8 is (CH.sub.2).sub.4; R.sub.9 or R.sub.10 are independently selected from C.sub.2-C.sub.12 alkylene or C.sub.2-C.sub.12 alkenylene; R.sub.11 or R.sub.12 are independently selected from H, methyl, C.sub.2-C.sub.12 alkylene or C.sub.2-C.sub.12 alkenylene.

2. The microparticles or nanoparticles according to claim 1, wherein a is at least 0.5.

3. The microparticles or nanoparticles according to claim 1, wherein a is at least 0.75.

4. The microparticles or nanoparticles according to claim 1, wherein p=0, m=0.75, and a=0.5; wherein the m, qa, and qb units are randomly distributed; R.sub.1 is (CH.sub.2).sub.8, R.sub.3 is (CH.sub.3).sub.2CHCH.sub.2, R.sub.5 is hexyl, and R.sub.7 is benzyl.

5. The microparticles or nanoparticles according to claim 1, wherein m=0.3, p=0.45, q=0.25, and a=0.5; wherein the m, p, qa, and qb units are randomly distributed; R.sub.1 is (CH.sub.2).sub.8; R.sub.3 and R.sub.4 respectively, are (CH.sub.3).sub.2CHCH.sub.2; R.sub.5 is (C.sub.2-C.sub.20)alkylene; and R.sub.7 is benzyl.

6. The microparticles or nanoparticles according to claim 1, wherein m=0.3, p=0.45, q=0.25, and a=0.75; wherein the m, p, qa, and qb units are randomly distributed; R.sub.1 is (CH.sub.2).sub.8; R.sub.4 is (CH.sub.3).sub.2CHCH.sub.2; and R.sub.7 is benzyl.

7. The microparticles or nanoparticles according to claim 1, wherein m=0.6, p=0.30, q=0.1, and a=0.5; wherein the m, p, qa, and qb units are randomly distributed; R.sub.1 (CH.sub.2).sub.4; R.sub.3 and R.sub.4 respectively, are (CH.sub.3).sub.2CHCH.sub.2; and R.sub.7 benzyl.

8. The microparticles or nanoparticles according to claim 1, wherein R.sub.1 is independently selected from (C.sub.2-C.sub.20)alkylene.

9. The microparticles or nanoparticles according to claim 1, wherein R.sub.5 is (C.sub.2-C.sub.20)alkylene.

10. The microparticles or nanoparticles according to claim 8, wherein R.sub.5 is (C.sub.2-C.sub.20)alkylene.

11. The microparticles or nanoparticles according to claim 1, wherein the bioactive agent is a small molecule drug, or prodrug or metabolite thereof.

12. The microparticles or nanoparticles according to claim 5, wherein the bioactive agent is a small molecule drug, or prodrug or metabolite thereof.

13. The microparticles or nanoparticles according to claim 10, wherein the bioactive agent is a small molecule drug, or prodrug or metabolite thereof.

14. The microparticles or nanoparticles according to claim 1, comprising an inner core and an outer shell structure.

15. A composition comprising the microparticles or nanoparticles according to claim 11.

16. A composition comprising the microparticles or nanoparticles according to claim 12.

17. A composition comprising the microparticles or nanoparticles according to claim 13.

18. An article or device comprising the microparticles or nanoparticles according to claim 11.

19. An article or device comprising the microparticles or nanoparticles according to claim 12.

20. An article or device comprising the microparticles or nanoparticles according to claim 13.

Description

(1) The present invention will now be described in detail with reference to the following non limiting Figures and examples which are by way of illustration only.

(2) FIG. 1: Hydrolytic degradation of PEA-I, PEA-III, PEA H/Bz and PLGA

(3) FIG. 2: Aggregation behavior of PEA-III-H/Bz compared to PEA-III-Bz.

(4) FIG. 3: Release of Dexamethasone from microparticles comprising either PEA-III-Bz; PEA-III-H/Bz 25% H; PEA-III-H/Bz 50% H or PLGA 50:50.

(5) FIG. 4: pH of the buffer during the degradation study of PEA-I-H/Bz 25% H, PEA-I-H/Bz 50% H and PLGA.

(6) FIG. 5: ARES2-rheometer with disposable geometries.

(7) FIG. 6: Experimental set up of degradation study

EXAMPLE I

(8) Protocol used PEA-1-Bz, copolymers of PEA-I-H/Bz 25% H, 50% H, PEA-IV-Bz, and PLGA 50/50, PLGA 75/25.

(9) 20 g oil phase comprised 5 wt % polymer, 0.5 wt % fluorescein, 9.45 wt % dimethyl sulfoxide (DMSO) and 85.05 wt % dichloromethane (DCM). Usually, 1 g of polymer was dissolved in 9 g DCM.

(10) The water phase comprised 2.5 wt % NaCl, 1 wt % PolyVinyl Acetate (PVA) (9-10 kDa, 80% hydrolyzed) and 96.5 wt % demi water. The PVA was dissolved in warm water (80 C.) and let under stirring overnight at 75 C. The concentration used was 5% PVA in water. 200 mL of cold water phase was used per 20 g of oil phase.

(11) For the particle formation, the water phase was poured into a 300 mL VWR beaker, 12 cm high, 6.7 cm diameter. The emulsification was done with an Ultraturrax IKA T25 coupled with a S25NK-19G stirrer. The stirring speed used was 4000 rpm. The polymer was injected via a 20 mL syringe with a bent 12 cm, 0.80 mm diameter needle. The stirring was let on 3 min after the injection end. Then the mixture was let under magnetic stirring overnight with an aluminum sheet with small holes on top of the beaker to let the solvent evaporate.

(12) The solution used contains 0.4 mg/mL Tween 20 in water solution. Around 400 mL of washing solution was used per 20 g oil phase.

(13) The mixture previously obtained was divided into four 50 mL falcon tubes and kept in ice. The beaker was rinsed with washing solution which was added to the tubes. They were centrifuged at 1000 rpm for 5 min. The supernatant was removed and replaced by 40 mL of washing solution. The particles were re-dispersed by gentle shaking. The tubes were centrifuged at 1000 rpm for 2 min. Once again, the supernatant was removed and replaced by 40 mL of washing solution. This washing was repeated twice and the supernatant was removed and replaced by 5 mL of washing solution. The fractions were blended into one tarred 50 mL falcon tube. The tubes were rinsed with washing solution which was added to the tarred tube.

(14) The particles were re-dispersed (sonication bath can be used) and frozen in liquid nitrogen. At this step, the tube can be stored in a freezer. Holes were pierced in a cap which fit to the falcon tube. Then the tarred falcon tube with the pierced cap was placed in a freeze dryer (0.04 mbar, 40 C.) for at least four days.

EXAMPLE II

(15) This study was carried out with microparticles prepared according to example I, the microparticles were loaded with fluorescein. Samples of about 25 mg of dry particles were introduced in a 15 mL flacon tube. Eighteen tubes were used per polymer studied (six data points in triple). 12 mL of 0.1 M PBS buffer, pH 7.4 with 0.05% NaN.sub.3 was added to each tube. Then the tubes were placed in a tube rack under shaking in a climate chamber as represented on FIG. 6.

(16) Then, for each data point, the pH of the buffer was measured and 2 mL of the buffer was filtered and stored in HPLC vial in a freezer.

(17) After that, the buffer was removed and particles washed with demineralized water. The particles were then monitored by microscope and dried under vacuum at 37 C. overnight. The next step was to dissolve 5 mg of particles in 2 mL of THF to measure their molecular weight distribution.

(18) The fluorescein loaded micro particles were challenged in the degradation study while monitoring the changes of the polymer molecular weight and particles capability to retain the loaded fluorescien. It was shown that PEA-H particles do swell quickly releasing the dye molecule. More hydrophobic PEA-Bz particles do not swell and retain the loaded fluorescien much better however the polymers do not show any sign of degradation during the experiment (13 weeks).

(19) Results of the degradation study are shown in FIG. 1.

EXAMPLE III

(20) W/O/W emulsion technique for preparation of PEA microparticles.

(21) The polymers used in this study were PEA-III-Bz, PEA-III-H/Bz and PLGA.

(22) Water 1 (W1) solution: 10 mg/ml Fitc-BSA containing 100 mg/mL trehalose .2H.sub.2O Oil composition: 5% Wt of the corresponding polymer was dissolved in chloroform. Water 2 (W2) solution: 80 gram 5% PVA and 320 gram Demiwater and 20 gram NaCl. For the fabrication of the microparticles Falcon tube (50 mL) and syringe (10 mL) were used. After adding the W1 solution to the oil, the mixture was vortexed for 30 seconds. After removing the plunger a needle was attached to the syringe the mixture was poored in the syringe (10 mL). Plunger was added when the needle of the syring was in the W2 layer. O/W mixture was added in circa 60 seconds at 4000 RPM. Mixture was stirred for additional 3 minutes at 4000 RPM. Particles were stirred overnight with magnetic stirrer and nitrogen flow.

(23) In a stock solution of Tween 20 in H2O at 0.4 mg/mL which was prepared and stored in fridge the microparticles were suspended. Next the particle suspension was added to 4 falcon tubes. The tubes were centrifuged at 1000 rpm for 5 min and placed directly in ice. The supernatant was replaced it with 5 mL of the cold Tween 20 solution and 4 fractions were collected in a falcon tube.

(24) The samples were re-dispersed immediately by gentle shaking and short sonication when need. Then the samples were centrifuged again and supernatant replaced. The washing procedure was repeated twice.

(25) After a re-dispersion step the particles were immediately frozen into liquid nitrogen. Next the caps of the tubes were pierced and samples were attached to the freeze-dryer.

(26) Approximately 20-40 mg of the freeze-dried micro particles were accurately weighted and transferred to 5 ml sample vials. Next was added two mL of stock solution to each vial containing 0.1 M PBS buffer containing 0.05 wt % NaN.sub.3 and 0.05 wt % Tween 20. The vials were placed in a climate chamber at 37 C. under gentle agitation. Samples were assessed and pictures were taken after 7 and 24 hours, 3, 4, 8, 21 days. Particles of PEA-III-H/Bz 50% H were floating freely in solution in contrast with PEA-III-Bz particles which formed agglomerates already in the first in 24 hours.

(27) Results are shown in FIG. 2.

(28) It can be observed that micro particles consisting of PLGA 50:50 formed aggregates which could be re-dispersed after vigorous stirring. Micro particles consisting of PEA-III-Bz formed a minor amount of aggregates however the aggregates were not easy re-dispersable. Surprisingly micro particles of PEA-III-H/Bz 25% H and PEA-III-H/Bz 50% did not show agglomeration at all.

EXAMPLE IV

(29) Micro particles were prepared via solid in oil in water (S/O/W) emulsion technique. Briefly, 100 mg dexamethasone was dispersed in 20 g CHCl.sub.3 polymer solution that contained 5% polymer (oil phase). The polymers used were respectively PEA III Ac Bz, PEA III H/Bz 25% H, PEA III H/Bz 50% H and PLGA 50:50. The obtained oil phase dispersions were injected into the water phase that contained 1% PVA 9-10 kDa 88% hydrolyzed and 2.5% NaCl under ultra turrax mixing. The obtained microparticle suspension was stirred for 18 hours under ambient conditions prior to centrifugation at 1000 RPM for 5 minutes. After which the supernatant was decanted off. The microparticle residue was resuspended in 10 ml distilled water that contained 0.4 mg/ml Tween 20. The suspension was again centrifuged at 1000 RPM for 5 minutes and the supernatant was decanted off. The microparticle residue was resuspended in 10 ml distilled water that contained 0.4 mg/ml Tween 20. The obtained microparticle suspension was freeze-dried and stored at 20 C.

(30) Dexamethasone loading was determined with 1H-NMR.

(31) Drug loading 5-7%, particle size range 10-35 m.

(32) Particle size was determined using SLS (Static Light Scattering).

(33) Results are given in FIG. 3.

(34) In duplicate approximately 20 mg freeze-dried micro particles were accurately weighted and transferred to 10 ml sample vials. To the vials 4 ml PBS buffer containing 0.05% NaN.sub.3 and 0.05% Tween 20 was added. The vials were placed at 37 C. under gentle agitation. Sampling took place on a bi-weekly basis followed by a weekly sampling. During the sampling the microparticles were allowed to sediment for at least 1 hour after which 2 ml of the buffer was replaced with fresh buffer. The dexamethasone concentration was determined in the release buffer using a RP-HPLC method with DAD detection at 238 nm. The graph illustrates sustained release of dexamethasone up to 62 days from the polyesteramide matrices.

(35) The release from PLGA followed a bimodal release curve associated with the bulk degradation property of the material. Dexamethasone release from PEA micro particles did not illustrate this behavior and showed a sustained release over the test period of 62 days. Polymers with an increasing H % exhibited increased polarity and swelling properties associated with water uptake. However surprisingly the release kinetics did not correlate with the increased H % it was anticipated that PEA-III-H/Bz 50% H would release fastest and PEA-III-Bz would release slowest.

EXAMPLE V

(36) This study was carried out with microparticles made with the oil in water method as described in example I. 20-25 mg of microparticles was introduced in a 15 mL flacon tube. Each data point was in triple. 12 mL of PBS buffer with 0.05% NaN.sub.3 was introduced to each tube. The pH was measured with a Metrohm 848 Titrino plus. The calibration was checked before each use with pH buffers of pH=7 and pH=2 or 4 and was performed with pH=2 and pH=9.

(37) For each data point, the pH of the buffer was measured and 2 mL of the buffer was filtered and stored in HPLC vial in a freezer.

(38) After that, the buffer was removed and particles washed with demineralized water. The particles were then monitored by microscope and dried under vacuum at 37 C. overnight. The next step was to dissolve 5 mg of particles in 2 mL of THF to measure their molecular weight distribution. Some of the polymers studied didn't dissolve in THF after being in PBS buffer lists the issues and the solutions found. Results are shown in FIG. 4.