BIODEGRADABLE POLYESTERAMIDE USED FOR THE TREATMENT OF ARTHRITIC DISORDERS

Abstract

The present invention relates to a formulation sized for injection comprising a biodegradable polyesteramide co-polymer comprising at least a diol of bicyclic-1,4:3,6-dianhydrohexitol and analgesics for use in the treatment of arthritic disorders. More specific the invention relates to a formulation comprising injectable microparticle according to claim 1 wherein the polyesteramide co-polymer further comprises a diacid, a diol different from bicyclic-1,4:3,6-dianhydrohexitol and at least two different amino-acids. The formulation is used to treat pain or inflammation in a patient comprising administering to said patient a therapeutically effective amount of the formulation once or twice a year.

Claims

1. A formulation comprising articles sized for injection comprising a biodegradable polyesteramide co-polymer comprising at least a diol of bicyclic-1,4:3,6-dianhydrohexitol for use in the treatment of arthritic disorders.

2. A formulation according to claim 1 wherein the polyesteramide co-polymer further comprises a diacid, a diol different from bicyclic-1,4:3,6-dianhydrohexitol and at least two different amino-acids.

3. A formulation according to any one of the claims 1-2 wherein the polyesteramide co-polymer comprises structural formula (I) ##STR00006## wherein m varies from 0.01- 0.99; p varies from 0.99-0.01; and q varies from 0.99-0.01; n varies from 5-100 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 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.2COOH, —(CH.sub.2)COOH, —CH.sub.2—CO—NH.sub.2, —CH.sub.2CH.sub.2—CO—NH.sub.2, —CH.sub.2CH.sub.2COOH, CH.sub.3—CH.sub.2—CH(CH.sub.3)—, —(CH.sub.3).sub.2—CH—CH.sub.2—, H.sub.2N—(CH.sub.2).sub.4—, phenyl—CH.sub.2—, —CH═CH—CH.sub.3, HO—p—phenyl—CH.sub.2—, (CH.sub.3).sub.2—CH—, phenyl—NH—, NH.sub.2—(CH.sub.2).sub.3—CH.sub.2— or NH.sub.2—CH═N—CH═C—CH.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. R.sub.6 is selected from bicyclic-fragments of 1,4:3,6-dianhydrohexitols of structural formula (II); ##STR00007## R.sub.7 is hydrogen, (C.sub.6-C.sub.10) aryl, (C.sub.1-C.sub.6) alkyl or a protecting group such as benzyl; R.sub.8 is independently (C.sub.1-C.sub.20) alkylene or (C.sub.2-C.sub.20)alkenyl;

4. A formulation according to claim 3 wherein the polyesteramide co-polymer of Formula (I) comprises m+p+q=1, q=0.25, p=0.45 whereby R.sub.1 is —(CH.sub.2).sub.8—; R.sub.3 and R.sub.4 in the backbone units m and p is leucine, —R.sub.5 is —(CH.sub.2).sub.6—, R.sub.6 is a bicyclic-fragments of 1,4:3,6-dianhydrohexitols of structural formula (II); R.sub.7 is a benzyl group and R.sub.8 is —(CH.sub.2).sub.4—.

5. A formulation according to any one of the claims 1-4 further comprising an analgesic selected from the group of anti-inflammatory drugs, local anesthetic drugs and opioids or comprising a disease-modifying antirheumatic drug.

6. A formulation according to claim 5 wherein the analgesic is selected from an NSAID COX-2 inhibitor. A formulation according to claim 5 wherein the analgesic is a corticosteroid.

8. A formulation according to claim 7 wherein the corticosteroid is selected from triamcinolone acetonide.

9. A formulation according to claim 1 wherein the articles are selected from the group of microparticles, fibers, tubes or rods.

10. A formulation according to any one of the claims 1-9 wherein the article is an microparticle.

11. A formulation according to any one of the claims 9-10 wherein the size of the microparticle varies from 0.1-1000 micrometer.

12. A formulation according to any one of the claims 1-11 for use in the treatment of arthritis.

13. A formulation according to claim 12 for use in the treatment of osteoarthritis.

14. A formulation according to claim 13 for use in the treatment of osteoarthritis of the knee, hip, spine or shoulder.

15. A formulation according to any one of the claims 1-11 for use in the treatment of arthritic disorders wherein the formulation is administered in 1 or 2 injections per year.

16. A method of treating pain or inflammation in a human or veterinary patient comprising administering to said patient a therapeutically effective amount of the formulation according to any one of the claims 1-11.

17. A method of slowing, arresting or reversing progressive structural tissue damage associated with chronic inflammatory disease in a human or veterinary patient comprising administering to said patient a therapeutically effective amount of the formulation according to any one of the claims 1-11.

18. The method of any of the claims 16-17 wherein the formulation according to claims 1-11 is administered in 1 or 2 injections per year.

19. The method of any one of claims 16-17 wherein the human or veterinary patient has osteoarthritis, rheumatoid arthritis, psoriatic arthritis, autoimmune arthritis, septic arthritis or synovitis.

Description

FIGURES

[0059] FIG. 1: SEM photograph of microparticles

[0060] FIG. 2: Size distribution of a plurality of microparticles

[0061] FIG. 3: Shows the biological effect in inflammation reduction (cell-assay based in vitro experiment. PGE-2 levels from human chondrocytes in presence of PEA-III-Bz particles.

[0062] FIG. 4: Cumulative fluorescein release from PEA-III-Bz films. Effect of the inflammatory cells lysate: stage 1 diffusion driven release, stage 2 in presence of (solution of broken inflammatory cells provides lysate)lysate, stage 3 diffusion driven release after partial degradation by lysates.

[0063] FIG. 5: Fluorescein release rate. Effect of the inflammatory cells lysate.

[0064] FIG. 6: Size distribution by light scattering of PEA-I-Bz microparticles after being immersed in water for 10 minutes. The second pic of the size distribution is a sign of agglomeration of particles

[0065] FIG. 7: Size distribution by light scattering of PEA-I-Bz microparticles after being immersed in water for 400 minutes. We can observed and increased of large particles as well as a widening of the size distribution compared to the measurement performed at 10 minutes post immersion.

[0066] FIG. 8: Size distribution by light scattering of PEA-III-Bz microparticles after being immersed in water for 10 minutes

[0067] FIG. 9: Size distribution by light scattering of PEA-III-Bz microparticles after being immersed in water for 400 minutes

[0068] FIG. 10: (light) microscopic representation of microparticles of PEA-I-Bz after being immersed in water for 10 minutes. Agglomerates are circled.

[0069] FIG. 11: (light) microscopic representation of microparticles of PEA-I-Bz after being immersed in water for 400 minutes. Agglomerates are circled.

[0070] FIG. 12: (light) microscopic representation of microparticles of PEA-III-Bz after being immersed in water for 10 minutes. No agglomerates of particles are observed.

[0071] FIG. 13: (light) microscopic representation of microparticles of PEA-III-Bz after being immersed in water for 400 minutes. No agglomerates of particles are observed.

MATERIALS

[0072] PGE-2 concentration is compared to levels produced in normal condition (Control—experiment). [0073] Presence of TnF-α increases the production of PGE-2 from chondrocytes (experiment Control+) to 300% [0074] Presence of empty particles does not influence on the production of PGE-2 from chondrocyte compare to Control+experiment. [0075] 0.1μM of TAA stops the production of PGE-2 by chondrocytes. This illustrates that Triamcinolone acetonide reduces inflammation. [0076] Chondrocytes incubated with TAA-loaded Microparticles do not produce PGE-2. This conditions shows that microparticles release an active agent that limits the inflammation of human chondrocyte, this, even when stimulated by TnF-α. [0077] PEA-III-Bz polymers are used in the following examples. A more extended description of PEA-III-Bz is poly-8-[(L-Leu-DAS).sub.0.45(L-Leu-6).sub.0.3[L-Lys(Bz)].sub.0.25. Structure is given in Formula III. The fractions indicate overall fractions of the monomers in the synthesis given in scheme 1. [0078] PEA-I-Bz polymers are used in microparticles for comparison with PEA-III-Bz microparticles. Structure of PEA-I-Bz polyesteramides is given in below Formula (IV).

##STR00003##

Wherein m varies from 0.1 to 0.9; p varies from 0.9 to 0.1; n varies from 50 to 150; [0079] each R1 is independently (C.sub.1-C.sub.20)alkylene; each R.sub.2 is independently hydrogen, or (C.sub.6-C.sub.10)aryl(C.sub.1-C.sub.6)alkyl such as benzyl. [0080] each R.sub.3 is independently hydrogen, (C1-C.sub.6) alkyl, (C.sub.2-C.sub.6)alkenyl, (C.sub.2-C.sub.6)alkynyl, or (C.sub.6-C.sub.10)aryl(C.sub.1-C.sub.6)alkyl; and each R4 is independently (C.sub.2-C.sub.20)alkylene.

[0081] PEA-I is a copolymer comprising alpha -amino acids, diols and an aliphatic dicarboxylic acids, which is copolymerized with aliphatic dicarboxylic acid and lysine.

##STR00004##

Synthesis of PEA-III-Bz

[0082] Trietylamine (30.9 mL, 0.222 mole, 2.2eq) and N,N-dimethylformamide (53.07 mL, 0.689 mole) were added to a mixture of Di-OSu-sebacinate (39.940 g, 0.1008 mole, 1.0eq), L-leucine(6)-2TosOH (20.823 g, 0.0302 mole, 0.30eq), L-leucine-(DAS)-2TosOH (32.503 g, 0.0453 mole, 0.45eq) and L-lysine(Bz)-2TosOH (14.628 g, 0.0252 mole, 0.25eq) in a nitrogen flushed 500mL round bottomed flask equipped with a overhead stirrer at room temperature. The subsequent mixture was heated to 60° C. to allow the reaction to proceed and monitored by GPC analysis in THF. After 36 hours a stable molecular weight was obtained, subsequently a portion of L-leucine(6)-2TosOH (4.338 g, 0.0063 mole) along with triethylamine (1.76 mL, 0.0126 mole) and N,N-dimethylformamide (4.54 mL, 0.0590 mole) was added to terminate the polymerization reaction. The mixture was heated additionally for 24 hours after which the viscous solution was further diluted with N,N-dimethylformamide (407.85g, 5.301 mole) and allowed to cool to room temperature. At room temperature acetic anhydride (1.89 mL, 0.0199 mole) was added to acylate the amino functional end groups of the polymer. The mixture was stirred at room temperature for 24 hours. In scheme 1 the general reaction is shown.

[0083] The obtained crude polymer mixture was precipitated in water in a 10:1 ratio (water: reaction mixture). The polymer was collected and dissolved in ethanol (500 mL, 8.57 mole) and the procedure was repeated a second time. The polymer was again dissolved in ethanol (500 mL, 8.57 mole) and precipitated in ethylacetate (5000 mL, 50.91 mole) by drop wise addition to a stirring solution. The precipitated polymer was washed with two portions ethylacetate (100mL, 1.00 mole), dried and dissolved in ethanol (500 mL, 8.57 mole) and filtered over a 0.2 μm PTFE membrane filter. The filtered polymer solution was dried under reduced pressure at 65° C.

[0084] Yield 75%, Mn=50 kDa (Gel Permeation Chromatography (GPC) in THF relative to polystyrene standards. Glass transition temperatures were determined by Differential Scanning calorimetry (DSC). Measurements were taken from second heating, with a heating rate of 10° C./min., Tg=48° C.

##STR00005##

Principle on Degradation Controlled Release.

[0085] Drug loaded microspheres with TAA as a drug are used in the study. As reference diffusion driven release has been performed in cell culture medium. The expected release curve is anticipated to closely resemble hypothetical FIG. 3 A.

[0086] Second and third series of the same particle batch (also in triplicate) will be started at the same time. The first phase of the release will be performed similar to the control experiment (stage 1 in FIG. 3 B). At stage 2 the cell culture medium will be replaced with cell culture medium containing HL-60 neutrophil like cell lysates. To series two pure lysate will be added, to series 3 10× diluted lysate will be added. The enzymes present in the lysate will cause surface degradation of PEA microparticles and as a result an increase in the release of API is anticipated.

EXAMPLES

Example 1

Fabrication of TAA-Loaded PEA-III-Bz Microparticles.

[0087] 300 mg of PEA-III-Bz was dissolved in Dichloromethane. 75 mg of TAA was added to the solution and homogenized by ultrasound. The suspension was added to 20 ml of cold water containing 1 wt % of poly(vinyl alchohol) under high shear, using a ultra-Turrax®. After a stable suspension was obtained the particles were let hardened in 100 ml of water containing 1 wt % of poly(vinyl alchohol) for 12 hours. Excess of water and surfactant was removed by rinsing and centrifugation. Finally, particles were frozen and dried under vacuum. A picture of the microparticles is given in FIG. 1. The size distribution of the microparticles is given in FIG. 2.

Example 2

Culture of Chondrocyte in the Presence of TAA-Loaded PEA-III-Bz Microparticles.

[0088] OA chondrocytes from three human donors were harvested and cultivated from total knee replacement. The cells were incubated with 5 μg of microparticles produced in example 1 in a transwells. Every three days the cells and medium were collected and the transwells were transferred to wells with freshly plated OA chondrocytes from the same donor. Quantities of PGE-2 (prostaglandin-E 2) produced by the cells during the time of incubation were determined by ELISA. The total duration of the experiment was of 28 days. Cells were cultured in the following conditions: [0089] 1. Control experiment; without particles [0090] 2. Positive control, chondrocytes stimulated by TNF-alpha, without particles [0091] 3. Chondrocytes stimulated by TNF-alpha, incubated with 5 μg of empty PEA-III-Bz particles [0092] 4. Chondrocytes stimulated by TNF-alpha, incubated with 5 μg of PEA-III-Bz particles loaded with 20 wt % of Triamcinolone acetonide [0093] 5. Chondrocytes stimulated by TNF-alpha, incubated with a constant concentration of Triamcinolone acetonide of 0.1 μM
Results are represented in FIG. 3.

Example 3

[0094] Cumulative Release of Fluorescein from PEA-III-Bz Films

[0095] A proof of principle study has been executed with fluorescein as a dye that is slowly released from a film.

[0096] During acute inflammation, polymorphonuclear neutrophil cells (PMNs) are the first type of cells to migrate to an inflammatory site, where they will produce several pro-inflammatory mediators including chemokines that attract other PMNs and other cell types like monocytes-macrophages and lymphocytes, corresponding to chronic inflammation. Neutrophils and macrophages are two cells types known to interact with biomaterials, which could lead to possible degradation of a biomaterial. Modelling the interaction of neutrophil like cells with a biomaterial can be done with differentiated HL60 neutrophil like cells. Neutrophils are the first-responders of inflammatory cells to migrate toward the site of inflammation and are known to have a high level of interaction with foreign bodies such as biomaterials.

[0097] The effect of acute inflammation on the release of fluorescein was assessed on 10 wt % fluorescein loaded PEA-III-Bz films. A comparison release series was performed in PBS buffer with 0.05 wt % sodium azide, sodium azide was added as a biocide. The effect of inflammation on the release of the dye (fluorescein) was demonstrated by the addition of cell lysates from differentiated HL60 neutrophil like cells.

Samples Preparation

[0098] 101.3 mg fluorescein and 999.3 mg PEA-III-Bz were dissolved in 19 ml ethanol. The solution was left overnight to dissolve under gentle agitation on an orbital shaker.

[0099] 8 ml of the polymer fluorescein solution was pipetted in a Teflon mold with a diameter of 5 cm placed in a desiccator. Under a gentle nitrogen flow the solvent was allowed to evaporate in 18 hours. The nitrogen flow dried films were removed and dried further under vacuum at 70° C. for 48 hours. A sample of the film was analyzed for residual ethanol by 1H-NMR analysis in CDCl3, no ethanol peaks were detected. 6 mm round disks were punched out of the dried film and were used for the release experiment.

Release Experiment

[0100] Two release series were started both in triplicate. At each time point the solutions were refreshed. Results are shown in FIGS. 4 and 5.

Principle of Inflammation Induced Release

[0101] Series 1 released in PBS buffer for the entire period (diffusion driven release). The release of fluorescein in series 2 is divided in four distinct phases.

[0102] Phase 1 release was performed in PBS buffer similar to series 1. In this phase both release curves closely resemble each other.

[0103] Phase 2, after 26 days of release HL60 neutrophil like cell lysate was added to the samples of series 2. The addition of the lysate results in an increased release rate of fluorescein.

[0104] Phase 3, after 33 days the action of the lysate was eliminated by a pre-treatment with 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride. The release rate in this phase closely resembled the release in PBS buffer. In phase 4 again fresh lysate was added and the release rate increased before the entirely fluorescein load to be released. See FIG. 4 for the cumulative release of fluorescein and FIG. 5 for the release rate.

[0105] The experiment clearly shows the surprising effect of the inflammation cell lysate on the release rate of from the polymer matrix. The results of the model system suggest that the release of an API from PEA III Ac Bz can be interactively tuned by the level of inflammation for a patient benefit.

[0106] Strong inflammation.fwdarw.Increased release rate.fwdarw.slowing down the inflammation.fwdarw.lower release rate.

Example 4

Fabrication of PEA-III-Bz Microparticles as Described in Example 1 Fabrication PEA-I Microparticles.

[0107] 300 mg of PEA-I-Bz was dissolved in Dichloromethane. The suspension was added to 20 ml of cold water containing 1 wt % of poly(vinyl alchohol) under high shear, using a ultra-Turrax®. After a stable suspension was obtained the particles were let hardened in 100 ml of water containing 1 wt % of poly(vinyl alchohol) for 12 hours. Excess of water and surfactant was removed by rinsing and centrifugation. Finally, particles were frozen and dried under vacuum.

[0108] Stability of PEA Microparticles in Water at Room Temperature; Agglomeration of PEA-I-Bz vs PEA-III-Bz

[0109] 30 mg of PEA-III-Bz and PEA-I-Bz microspheres were suspended with 0.5 ml water and put on a shaker at 20° C. at 120 rpm. Size and agglomeration of microparticles were monitored by size distribution measurement (using light scattering technic) and visual evaluation (light microscopic technic) for both type of particles after 10 and 400 minutes of immersion in water.

[0110] It could be seen that while PEA-III-Bz did not agglomerated during the course of the experiment, PEA-I-Bz particles were showing agglomeration after only 10 minutes of immersion in water. Agglomerated particles reduces the syringe-ability of those particles through a narrow needle.

[0111] Particle Size Measurement by Light Scattering After 10 and 400 Minutes;

[0112] The size distribution is defined by D10, D50, D90 and SPAN; where D10 corresponds to the value of the particle diameter at 10% in cumulative distribution, where D50 corresponds to the value of the particle diameter at 50% in cumulative distribution and where D90 corresponds to the value of the particle diameter at 90% in cumulative distribution as can be seen in the FIGS. 6-9.

[0113] The SPAN is calculated as SPAN=(D90-D10)/D50.

TABLE-US-00001 PEA-I-Bz Duration of immersion D(10) D(50) D(90) SPAN  10 Min 16.177 34.513  85.802 2.017 400 Min 18.653 39.393 127.754 2.77 
Agglomeration is clearly visible by an increase of all values in time.

TABLE-US-00002 PEA-III-Bz Duration of immersion D(10) D(50) D(90) SPAN  10 Min 12.001 33.667 59.064 1.398 400 Min 12.158 36.432 62.236 1.375

[0114] With respect to experimental accuracy, PEA-III-Bz microparticles do not show evidence of agglomeration after 400 minutes of immersion in water.