Drug delivery composition comprising proteins and biodegradable polyesteramides
09789189 · 2017-10-17
Assignee
Inventors
Cpc classification
C08L89/00
CHEMISTRY; METALLURGY
A61K47/34
HUMAN NECESSITIES
C08L89/00
CHEMISTRY; METALLURGY
C12Y111/01007
CHEMISTRY; METALLURGY
International classification
A61K47/34
HUMAN NECESSITIES
A61K47/42
HUMAN NECESSITIES
Abstract
Drug delivery compositions are provided having proteins and biodegradable polymers that may be used for controlled and long term release of proteins into a biological environment. According to some embodiments, the drug delivery composition may be provided with at least a protein and a biodegradable polymer possessing at least two different functional groups selected from the group of chosen among carboxyl, ester, amine, amide, thiol, thioester or hydroxyl groups pendant to the main polymer chain whereby the composition absorbs between 5-10% w/w water when exposed to physiological conditions for at least 20 days. A drug delivery system for controlled protein release which includes the drug delivery composition is also provided whereby the drug delivery system may be in the form of microparticles, films, coatings, fibers, pellets, cylinders, discs, implants, microcapsules, microspheres, nanospheres, wafers, micelles, liposomes or woven or non-woven fabrics.
Claims
1. A drug delivery composition which comprises, based on total weight of the composition: at least 0.1 wt. % to 99 wt. % of a protein, and from 99.9 wt. % to 0.1 wt. % of a biodegradable polymer, wherein the biodegradable polymer comprises at least two different functional groups being unprotected hydrophilic groups and protected hydrophobic groups pendant to the polymer backbone, which functional groups are selected from the group of carboxyl, amine, hydroxyl, ester, amide, thiol or thioester groups, and wherein the unprotected hydrophilic groups and the protected hydrophobic groups are present in a ratio of the unprotected hydrophilic groups to the protected hydrophobic groups of 0.17-3, and wherein the polymer absorbs between 2-20% w/w water if exposed to physiological conditions for at least 20 days.
2. The drug delivery composition according to claim 1, wherein the two different functional groups comprise at least an unprotected hydrophilic functional group selected from a carboxyl group, an amine group, a thiol group or a hydroxyl group, and at least a protected hydrophobic functional group selected from an ester group, an amide group or a thioester group.
3. The drug delivery composition according to claim 2, wherein the biodegradable polymer possesses pendant carboxyl and ester functional groups.
4. The drug delivery composition according to claim 1, wherein the biodegradable polymer is a polyesteramide.
5. The drug delivery composition according to claim 4, wherein the polyesteramide is a polyesteramide copolymer according to structural Formula (I): ##STR00007## wherein m+p varies from 0.9-0.1 and a+b varies from 0.1 to 0.9; m+p+a+b=1, wherein m or p could be 0; n varies from 5 to 300 and wherein a is at least 0.01, b is at least 0.015 and the ratio of a to b (a:b) is from 0.1:9 to 0.85:0.15, wherein the m unit and/or p unit, and the a and b units, are randomly distributed; and wherein: R.sub.1 is independently selected from the group consisting of (C.sub.2-C.sub.20) alkyl, 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.3).sub.2—CH—CH.sub.2—, —CH(CH.sub.3).sub.2, —CH(CH.sub.3)—CH.sub.2—CH.sub.3), —CH.sub.2—C.sub.6H.sub.5, —(CH.sub.2).sub.4—NH.sub.2, and mixtures thereof; R.sub.5 is independently selected from the group consisting of (C.sub.2-C.sub.20)alkyl, (C.sub.2-C.sub.20)alkylene, R.sub.6 is selected from bicyclic-fragments of 1,4:3,6-dianhydrohexitols of structural formula (II); ##STR00008## R.sub.7 is independently selected from the group consisting of (C.sub.6-C.sub.10) aryl (C.sub.1-C.sub.6) alkyl or a protecting group; and —R.sub.8 is —(CH.sub.2).sub.4—.
6. The drug delivery composition according to claim 5, wherein b is at least 0.025 and a:b is from 0.1:0.9 to 0.75:0.25.
7. The drug delivery composition according to claim 5, wherein b is at least 0.05 and a:b is from 0.1:0.9 to 0.5:0.5.
8. The drug delivery composition according to claim 5, wherein p=0 and m+a+b=1, m=0.75, b is 0.125 and a+b=0.25, wherein R.sub.1 is (CH.sub.2).sub.8, R.sub.3 is (CH.sub.3).sub.2—CH—CH.sub.2—, R.sub.5 is hexyl, R.sub.7 is benzyl, R.sub.8 is —(CH.sub.2).sub.4—.
9. The drug delivery composition according to claim 5, wherein m+p+a+b=1, a+b=0.25, p=0.45 and m=0.3, b is 0.0625, wherein R.sub.1, —(CH.sub.2).sub.8; R.sub.3 and R.sub.4 respectively, are (CH.sub.3).sub.2—CH—CH.sub.2—, R.sub.5 is selected from the group consisting of (C.sub.2-C.sub.20)alkylene, R.sub.7 is benzyl, R.sub.8 is —(CH.sub.2).sub.4—; R.sub.6 is selected from bicyclic-fragments of 1,4:3,6-dianhydrohexitols of structural formula (II).
10. The drug delivery composition according to claim 5, wherein m+p+a+b=1, a+b=0.25, p=0.45 and m=0.3, b is 0.125, wherein R.sub.1 is —(CH.sub.2).sub.8; R.sub.4 is (CH.sub.3).sub.2—CH—CH.sub.2—, R.sub.7 is benzyl, R.sub.8 is —(CH.sub.2).sub.4; R.sub.6 is selected from bicyclic-fragments of 1,4:3,6-dianhydrohexitols of structural formula (II).
11. The drug delivery composition according to claim 5, wherein m+p+a+b=1, a+b=0.1, p=0.30 and m=0.6, b is 0.05, wherein R.sub.1—(CH.sub.2).sub.4; R.sub.3 and R.sub.4 respectively, are (CH.sub.3).sub.2—CH—CH.sub.2—; R.sub.7 benzyl, R.sub.8 is —(CH.sub.2).sub.4—; R.sub.5 is selected from the group consisting of (C.sub.2-C.sub.20)alkylene, R.sub.6 is selected from bicyclic-fragments of 1,4:3,6-dianhydrohexitols of structural formula (II).
12. The drug delivery composition according to claim 1, wherein the protein is at least one selected from the group consisting of peptides, insulin, hormones, vaccines, enzymes, antibiotics, antibodies, neuroactive agents, growth factors, cytokines, antigens and glycoproteins.
13. A drug delivery system for controlled protein release comprising the composition according to claim 1.
14. The drug delivery system according to claim 13, wherein the drug delivery system is in a form comprising microparticles, films, coatings, fibers, pellets, cylinders, discs, implants, microcapsules, microspheres, nanospheres, wafers, micelles, liposomes, woven or non-woven fabrics.
15. The drug delivery composition according to claim 2, wherein the unprotected hydrophilic groups and the protected hydrophobic groups are present in a ratio of the unprotected hydrophilic groups to the protected hydrophobic groups of 0.3-1.
16. The drug delivery composition according to claim 3, wherein the unprotected hydrophilic groups and the protected hydrophobic groups are present in a ratio of the unprotected hydrophilic groups to the protected hydrophobic groups of 0.3-1.
17. The drug delivery composition according to claim 2, wherein the composition comprises from 0.1 wt% to 20 wt% of the protein and from 60 wt% to 99.9 wt% of the biodegradable polymer.
18. The drug delivery composition according to claim 3, wherein the composition comprises from 0.1 wt% to 20 wt% of the protein and from 6 wt% to 99.9 wt% of the biodegradable polymer.
19. The drug delivery composition according to claim 5, wherein the composition comprises from 0.1 wt% to 20 wt% of the protein and from 60 wt% to 99.9 wt% of the biodegradable polymer.
20. The drug delivery composition according to claim 8, wherein the composition comprises from 0.1 wt% to 20 wt% of the protein and from 60 wt% to 99.9 wt% of the biodegradable polymer.
Description
FIGURES
(1)
(2)
(3)
(4)
(5)
(6)
(7) PEA III 100% H (polymer with only —COOH side groups)—the polymer fibers release the entire polymer load in the first several hours—typical burst release)
(8) PEA III Ac Bz (polymer with only benzyl ester side groups)—The polymer does not release significant amount of protein and the release properties of the fiber does not change over time.
(9) PEA III 75% H—Polymer fibers do release the entire protein load over three weeks and the activity of the released enzyme does not change along the release.
(10) PEA III 50% H—Polymer fibers do release the protein load between week 3 and week 11 with majority of the enzyme released between week 6 and week 10. The activity of the released enzyme does not change along the release.
(11) PEA III 25% H—Polymer fibers do start substantially to release in week 7. The release is not completed in the 11 weeks period of the experiment.
(12) The present invention will now be described in detail with reference to the following non limiting examples which are by way of illustration only.
EXAMPLES
Example 1
Swelling Behavior of PEA-III-H/Bz Copolymers
(13) The swelling behavior of PEA-III-Ac Bz (possessing pendant ester groups along the polymer chain), PEA-III-H/Bz 25% H (possessing both pendant ester and carboxyl groups), PEA-III-H/Bz 50% H (possessing both pendant ester and carboxyl groups) and PEA-III-H possessing pendant carboxyl groups were assessed on round polymeric films, prepared via solvent casting. The films were immersed in PBS buffer which contained 0.05% NaN.sub.3 as a biocide and were placed at 37° C. under gentle agitation. The swelling behavior was assessed based on the mass gain of the polymer films. The buffers were refreshed on each time point.
(14) Mass gain can be calculated according to the following Formula;
(15)
M.sub.t=mass at time point (t)
M.sub.0=initial mass
Films of PEA-III-Ac-Bz showed marginal swelling, up to 5% over the test period. In contrast PEA-III-H films gained mass up to 1000% compared to the original mass within a few days. Surprisingly films made from PEA-III-H/Bz 25% H and PEA-III-H/Bz 50% H showed a slow and sustained water absorption profile. This is shown in
Example 2
ABTS Activity Assay for HRP
(16) ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) is generally used as an activity assay for peroxidase activity (e.g. for HRP=horseradish peroxide). The assay is based on the formation of highly colored oxidized/radical form of the substrate that can be quantified photometric. The formation of the highly colored product is a direct measure for the activity of the measured enzyme. In scheme 1 the reaction of ABTS is shown.
(17) ##STR00006##
(18) The protocol for the assay was adapted from Putter, J. and Becker, R. (1983) Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.) 3rd ed., Vol III, pp. 286-293, Verlug Chemie, Deerfield Beach, Fla.
(19) A stock solution of ABTS with a concentration of 5 mg/mL was prepared in PBS buffer. A stock solution of 0.3 wt % H.sub.2O.sub.2 in water was prepared. In the reference cuvet 1 mL of the ABTS stock and 50 μL of the H.sub.2O.sub.2 stock were mixed. In the sample cuvet 1 mL of the ABTS stock and 50 μL of the H.sub.2O.sub.2 stock were mixed with 50 μL of the enzyme solution. The cuvet was directly placed in the photometer and the absorbance increase at 405 nm was measured over one minute with a time interval of 0.5s. The steepness of the absorbance increase is a measure for the activity of the enzyme and was determined based on the digital data of the photometer using Microsoft Excel. Eventually the activity of the enzyme was correlated to a concentration based on the activity of freshly prepared HRP solution and used for quantification.
Example 3
SDS-PAGE
(20) All sodiumdodecyl sulfate (SDS) reagents and gels were obtained from Life Technologies (NuPAGE system). Release samples were mixed with 4×SDS loading buffer and 10× reducing agent. Protein denaturation was achieved by 5 minutes heating to 70° C. Typically 20 μl sample was loaded on the gel. 12% Bis-Tris or 4-12% Bis-Tris gels were used with MOPS or MES buffer, respectively. For detect of HRP (>1 ug), Coomassie staining (SimplyBlue safestain) was applied. Release media of PEA-III-50% H were analyzed using SDS-PAGE. In the illustration several release samples of HRP are shown. It can be observed that the released enzyme appears on the same spot as the HRP standard, confirming the release of the enzyme. This is shown in
Example 4
Aqueous SEC
(21) HRP release buffers were analyzed using an Agilent 1200 LC system equipped with a TSK gel G2000SWXL 7.8*300 mm (TOSOH Bioscience) column using DAD detection. The system was operated with a flow rate of 0.5 mL/min using 1.059 mM KH.sub.2PO.sub.4, 2.966 mM Na.sub.2HPO.sub.4, 300 mM NaCl, pH=7.4 and 10% EtOH as the mobile phase. A typical sample injection volume of 10 μL was used.
(22) DAD data obtained from several different release buffers of PEA III 50% H are illustrated and were used for quantification of the enzyme as complementary method to the ABTS activity assay. Both methods showed good correlation (90-110%) indicating that the released enzyme retained full activity. The peak at 10 min is also present in the blank and is not associated with the sample.
(23)
Example 5
Release of Horseradish Peroxidase (HRP) from Polymeric Matrices Processing and Loading
(24) 400 μL of a 10 wt % polymer solution in CHCl.sub.3 of PLGA 50:50, PEA-III-Ac Bz (possessing pendant ester groups along the polymer chain), PEA-III-H/Bz 50% H (possessing both pendant ester- and carboxyl groups) and PEA-III-100% H (possessing pendant carboxyl groups) was added to 2 mg lyophilized HRP. The HRP was mechanically dispersed in the solutions. The obtained suspensions of polymer and HRP were dried under reduced pressure at ambient temperature.
(25) Release of HRP
(26) Dry HRP loaded films were immersed in 3 mL PBS buffer and placed at 37° C. under gentle agitation. Over time the released HRP was quantified both with aqueous SEC and with a photometric activity assay using ABTS as the reagent. After one week the concentration of HRP was determined in the buffers with both methods. The data from both methods correlated well, indicating that the released HRP was active. PEA-III-100% H released already the majority of all HRP present in the film in the first week after 3 weeks the film was completely empty. From the PEA-III-Ac Bz practically no release was observed over the test period apart from the release in the first week. PLGA 50:50 showed release of HRP over a period of 5 weeks after that no active protein was released anymore. After 8 weeks the PLGA 50:50 matrix was completely degraded without any further release. Surprisingly from the PEA-III-H/Bz 50% H film active HRP was released over a time frame of 22 weeks with a relatively constant rate.
(27) It was observed that the release of HRP from the PEA films was in line with the water absorption properties of the subsequent polymers. The experiment shows the role of both types of pendant functional groups along the polymer chain. Only the composition comprising the polyesteramide comprising simultaneously both pendant ester and carboxyl groups (PEA-III-H/Bz 50% H) allows a complete and sustained release of the protein over a long period. Analysis of the polymer matrix after 22 weeks showed that no significant amount of enzyme is present confirming the hypothesis for complete release of the protein.
(28) PEA-III-100% H swells fast and released the HRP very fast, PEA-III-Ac-Bz shows a marginal swelling and almost no release of HRP, PEA-III-H/Bz 50% illustrates a gradual swelling in combination with a sustained release of HRP with an increased release rate beyond week 17. This is shown in
Example 6
Preparation of HRP Loaded PEA Polymer Fibres
(29) PEA-III-25% H polymer was dissolved in ethanol, methanol, chloroform, or dichloromethane for the experiments that tested the influence of solvents. For the release study PEA-III-AcBz and PEA-H/Bz polymers were dissolved in ethanol. Polymers solutions were prepared at a concentration of 10% (w/v). Subsequently, HRP was added under vigorous stirring in a Teflon beaker. After obtaining a homogeneous solution the solvent was allowed to evaporate. When the polymers became solid enough to manipulate they were transferred onto a Teflon sheet. The polymers were then shaped manually into a fibre by rolling it in between Teflon sheets and blocks. The fibers had a diameter between 0.8 and 1.2 mm and were 3 cm in length.
(30) Fibres were allowed to dry in ambient conditions for 5 days before being used. Fibres for the release of HRP #P6782 were then cut to be used as triplicates in the release experiments. HRP used was Sigma #P6782.
(31) Release of HRP
(32) Release experiments were carried out in Dulbecco's Phosphate Buffered Saline (PBS) under continuous shaking at ±100 rpm at 37° C. HRP loaded PEA-III-AcBz, 25% H, 50% H, 75% H or 100% H were weight and analysed under the microscope before start of the experiments. All fibres had mass of about 50 mg (±5 mg). The first day buffer exchanged after 2, 5 and 24 hours and after that once a week. The old buffer was then analysed for protein quantity and HRP activity.
(33)
(34)
(35) PEA-III-100% H (polymer with only —COOH side groups)—the polymer fibers release the entire polymer load in the first several hours—typical burst release)
(36) PEA-III-Ac Bz (polymer with only benzyl ester side groups)—The polymer does not release significant amount of protein and the release properties of the fiber does not change over time.
(37) PEA-III-75% H—Polymer fibers do release the entire protein load over three weeks and the activity of the released enzyme does not change along the release.
(38) PEA-III-50% H—Polymer fibers do release the protein load between week 3 and week 11 with majority of the enzyme released between week 6 and week 10. The activity of the released enzyme does not change along the release.
(39) PEA-III-25% H—Polymer fibers do start substantially to release in week 7. The release is not completed in the 11 weeks period of the experiment
(40) Protein Analysis
(41) Quantification:
(42) Release samples were analysed using the BCA Quantipro assay (Sigma Aldrich) according to manufacturer's instructions. As a control, release samples from empty fibres (without HRP) were also analysed because PEA can give small signal when degrading. When necessary this signal was subtracted from the actual release samples. The BCA assay was measured with a Multiscan GO plate reader (Thermo Scientific).
(43) Activity Assay:
(44) A stock solution of ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (Sigma # A1888) with a concentration of 5 mg/mL was prepared in PBS buffer. A stock solution of 0.3 wt % H.sub.2O.sub.2 in water was prepared. In the reference cuvette 1 mL of the ABTS stock and 50 μL of the H.sub.2O.sub.2 stock were mixed. In the sample cuvette 1 mL of the ABTS stock and 50 μL of the H.sub.2O.sub.2 stock were mixed with 50 μL of the sample solution. The cuvette was directly placed in the photometer (Shimadzu UV-1800) and the absorbance increase at 405 nm was measured over one minute. The slope of absorbance was used to measure the activity of the enzyme. Assay was carried out at room temperature.
(45) Swelling Behaviour:
(46) Films (thickness 0.5 mm) of each polymer were prepared and dried under reduced pressure to reduce the water content of the tested materials below 0.3% (w/w). Discs with a diameter of 4 mm and a thickness of 0.5 mm were punched from the films.
(47) Prior to the incubation in PBS buffer (2 mL), samples were weighted on Sartorius micro balance and both thickness and diameter were recorded. Samples were immersed in 5 mL 10 mM PBS buffer, pH 7.4 containing 0.05% (w/w) NaN.sub.3. All polymer samples were incubated at 37° C. the water uptake was evaluated by measuring the sample weight increase at different time points. The weight increase was measured as follows:
Samples were taken out of the buffer solution and dried on dust-free absorbing paper (without pressing) in order to remove the adhering water from the discs. Next the samples were analysed by Sartorius micro balance and readouts were recorded. Each polymer sample has been analysed in triple. The weight was then compared to the original weight. The original weight was set as a 100%. At the read out time points buffer solutions were refreshed.
(48) The figure shows that the activity of the enzyme released from the polymer matrix remains at the same level over the entire 11 weeks period of the experiment. This suggests that neither polymer matrix nor the polymer degradation products do impact on enzyme activity and stability.