ANTI-MICROBIAL METHODS AND MATERIALS

Abstract

The present invention provides methods for making and using antibacterial polymeric materials loaded with additives, as well as antibacterial materials comprising additives. Certain additives or combinations of additives show unexpected combinatorial or synergistic antibacterial activity. The invention also provides medical devices comprised of antibacterial polymeric materials, and methods of making and using such devices, which can have unexpected combinatorial or synergistic antibacterial activity.

Claims

1. A method of making an antibacterial polymeric material wherein the method comprises: a. Providing a first polymeric material, b. Providing two or more therapeutic agents with synergistic antibacterial activity; c. Blending the polymeric material with the therapeutic agents, thereby making a therapeutic agent-blended polymeric material, d. Processing the therapeutic agent-blended polymeric material, thereby making a processed antibacterial, polymeric material.

2. A method of making an antibacterial medical implant wherein the method comprises: a. Providing a first polymeric material, b. Providing two or more therapeutic agents with synergistic antibacterial activity; c. Blending the polymeric material with the therapeutic agents, thereby making a therapeutic agent-blended polymeric material, d. Processing the therapeutic agent-blended polymeric material, thereby making a processed antibacterial, polymeric material e. Fashioning a medical implant from the processed antibacterial, polymeric material, thereby making an antibacterial medical implant.

3. A method of making an antibacterial polymeric material wherein the method comprises: a. Providing a first polymeric material, b. Providing an antibiotic and a sodium-channel blocker with synergistic antibacterial activity; c. Blending the polymeric material with at least the antibiotic and the sodium-channel blocker, thereby making a therapeutic agent-blended polymeric material, d. Processing the therapeutic agent-blended polymeric material, thereby making a processed antibacterial, polymeric material.

4. A method of making an antibacterial polymeric material of claim 3, wherein the provided polymeric material is an ultra-high molecular weight polyethylene.

5. A method of making an antibacterial polymeric material of claim 3, wherein the antibiotic is gentamicin.

6. A method of making an antibacterial polymeric material of claim 3, wherein the sodium-channel blocker is bupivacaine.

7. A method of making an antibacterial polymeric material of claim 3, wherein the processing of the therapeutic agent-blended polymeric material is done by compression molding.

8. A method of making an antibacterial polymeric material of claim 3, wherein the processing of the therapeutic agent-blended polymeric material is photopolymerization.

9. A method of making an antibacterial polymeric material wherein the method comprises: a. Providing a first polymeric material, b. Providing an antibiotic and a NSAID with synergistic antibacterial activity; c. Blending the polymeric material with at least the antibiotic and the NSAID, thereby making a therapeutic agent-blended polymeric material, d. Processing the therapeutic agent-blended polymeric material, thereby making a processed antibacterial, polymeric material.

10. A method of making an antibacterial polymeric material of claim 9, wherein the provided polymeric material is an ultra-high molecular weight polyethylene.

11. A method of making an antibacterial polymeric material of claim 9, wherein the antibiotic is gentamicin.

12. A method of making an antibacterial polymeric material of claim 9, wherein the NSAID is ketorolac.

13. A method of making an antibacterial polymeric material of claim 9, wherein the NSAID is tolfenamic acid.

14. A method of making an antibacterial polymeric material of claim 9, wherein the processing of the therapeutic agent-blended polymeric material is done by compression molding.

15. A method of making an antibacterial polymeric material of claim 9, wherein the processing of the therapeutic agent-blended polymeric material is photopolymerization.

16. A method of making an antibacterial polymeric material wherein the method comprises: a. Providing a first polymeric material, b. Providing an NSAID and a sodium-channel blocker with synergistic antibacterial activity; c. Blending the polymeric material with at least the sodium-channel blocker and the NSAID, thereby making a therapeutic agent-blended polymeric material, d. Processing the therapeutic agent-blended polymeric material, thereby making a processed antibacterial, polymeric material.

17. A method of making an antibacterial polymeric material of claim 16, wherein the provided polymeric material is an ultra-high molecular weight polyethylene.

18. A method of making an antibacterial polymeric material of claim 16, wherein the sodium-channel blocker is procaine.

19. A method of making an antibacterial polymeric material of claim 16, wherein the NSAID is tolfenamic acid.

20. A method of making an antibacterial polymeric material of claim 16, wherein the processing of the therapeutic agent-blended polymeric material is done by compression molding.

21. A method of making an antibacterial polymeric material of claim 16, wherein the processing of the therapeutic agent-blended polymeric material is photopolymerization.

22. A method of making an antibacterial polymeric material wherein the method comprises: a. Providing a first polymeric material, b. Providing an antioxidant and a sodium-channel blocker with synergistic antibacterial activity; c. Blending the polymeric material with at least the sodium-channel blocker and antioxidant, thereby making a therapeutic agent-blended polymeric material, d. Processing the therapeutic agent-blended polymeric material, thereby making a processed antibacterial, polymeric material.

23. A method of making an antibacterial polymeric material of claim 22, wherein the provided polymeric material is an ultra-high molecular weight polyethylene.

24. A method of making an antibacterial polymeric material of claim 22, wherein the antioxidant is resveratrol.

25. A method of making an antibacterial polymeric material of claim 22, wherein the antioxidant is curcumin.

26. A method of making an antibacterial polymeric material of claim 22, wherein the sodium-channel blocker is lidocaine.

27. A method of making an antibacterial polymeric material of claim 22, wherein the sodium-channel blocker is bupivacaine.

28. A method of making an antibacterial polymeric material of claim 22, wherein the sodium-channel blocker is prilocaine.

29. A method of making an antibacterial polymeric material of claim 22, wherein the sodium-channel blocker is procaine.

30. A method of making an antibacterial polymeric material of claim 22, wherein the processing of the therapeutic agent-blended polymeric material is done by compression molding.

31. A method of making an antibacterial polymeric material of claim 22, wherein the processing of the therapeutic agent-blended polymeric material is photopolymerization.

32. A method of making an antibacterial polymeric material wherein the method comprises: a. Providing a first polymeric material, b. Providing an NSAID and an antioxidant with synergistic antibacterial activity; c. Blending the polymeric material with at least the antioxidant and a NSAID, thereby making a therapeutic agent-blended polymeric material, d. Processing the therapeutic agent-blended polymeric material, thereby making a processed antibacterial, polymeric material.

33. A method of making an antibacterial polymeric material of claim 32, wherein the provided polymeric material is an ultra-high molecular weight polyethylene.

34. A method of making an antibacterial polymeric material of claim 32, wherein the antioxidant is curcumin.

35. A method of making an antibacterial polymeric material of claim 32, wherein the antioxidant is resveratrol.

36. A method of making an antibacterial polymeric material of claim 32, wherein the NSAID is tolfenamic acid.

37. A method of making an antibacterial polymeric material of claim 32, wherein the NSAID is ketorolac.

38. A method of making an antibacterial polymeric material of claim 32, wherein the processing of the therapeutic agent-blended polymeric material is done by compression molding.

39. A method of making an antibacterial polymeric material of claim 32, wherein the processing of the therapeutic agent-blended polymeric material is photopolymerization.

40. A method of making an antibacterial polymeric material wherein the method comprises: a. Providing a first polymeric material, b. Providing an antibiotic and an antioxidant with synergistic antibacterial activity; c. Blending the polymeric material with at least the antioxidant and the antibiotic, thereby making a therapeutic agent-blended polymeric material, d. Processing the therapeutic agent-blended polymeric material, thereby making a processed antibacterial, polymeric material.

41. A method of making an antibacterial polymeric material of claim 40, wherein the provided polymeric material is an ultra-high molecular weight polyethylene.

42. A method of making an antibacterial polymeric material of claim 40, wherein the antioxidant is curcumin.

43. A method of making an antibacterial polymeric material of claim 40, wherein the antioxidant is resveratrol.

44. A method of making an antibacterial polymeric material of claim 40, wherein the antibiotic is gentamicin.

45. A method of making an antibacterial polymeric material of claim 40, wherein the antibiotic is vancomycin.

46. A method of making an antibacterial polymeric material of claim 40, wherein the processing of the therapeutic agent-blended polymeric material is done by compression molding.

47. A method of making an antibacterial polymeric material of claim 40, wherein the processing of the therapeutic agent-blended polymeric material is photopolymerization.

48. A method of making an antibacterial polymeric material wherein the method comprises: a. Providing a first polymeric material, b. Providing two different sodium-channel blockers with synergistic antibacterial activity; c. Blending the polymeric material with at least two different sodium-channel blockers, thereby making a therapeutic agent-blended polymeric material, d. Processing the therapeutic agent-blended polymeric material, thereby making a processed antibacterial, polymeric material.

49. A method of making an antibacterial polymeric material of claim 48, wherein the provided polymeric material is an ultra-high molecular weight polyethylene.

50. A method of making an antibacterial polymeric material of claim 48, wherein the sodium-channel blocker is lidocaine, or bupivacaine, or tetracaine, procaine, or prilocaine.

51. A method of making an antibacterial polymeric material of claim 48, wherein the processing of the therapeutic agent-blended polymeric material is done by compression molding.

52. A method of making an antibacterial polymeric material of claim 48, wherein the processing of the therapeutic agent-blended polymeric material is photopolymerization.

53. A method of making an implant comprising: a. Providing a polymeric material; b. Blending the polymeric material with two different drugs, including sodium-channel blocker, NSAID, antibiotic, antioxidant; c. Providing a second polymeric material; d. Blending the second polymeric material with a cross-linking agent; e. Layering the dual drug-blended polymeric material and the cross-linking agent-blended second polymeric material; f. Consolidating the layered polymeric materials; thereby obtaining a cross-linked implant with antibiotic-rich regions.

54. A method of making an implant comprising: a. Providing a polymeric material; b. Blending the polymeric material with two different drugs, including sodium-channel blocker, NSAID, antibiotic, antioxidant; c. Layering the dual drug-blended polymeric material polymeric and the polymeric material without drugs; d. Consolidating the layered polymeric materials; thereby obtaining an implant with drug-rich regions; e. Exposing at least parts of the implant to radiation.

55. A method of making a degradable, additive-blended polymeric material comprising: a. Providing liquid polymerizable macromer composed of a cross-linkable moiety ((B.sub.m-A.sub.n-B.sub.m-R) or (R-B.sub.m-A.sub.n-B.sub.m-R)), b. Blending the liquid, polymerizable mixture with at least two therapeutic agents (Drug X and Drug Y) with synergistic antibacterial activity c. Exposing the additive-blended, liquid polymerizable macromer(s) to as external stimulus which can create free radicals for a period of time, thereby forming a degradable, additive-blended gel.

56. A method of making an antibacterial polymeric material of claim 55, wherein the drug X can be sodium-channel blocker, or antibiotic, or anesthetic, or antioxidant, or NSAID. Drug Y can be sodium-channel blocker, or antibiotic, or anesthetic, or antioxidant, or NSAID.

57. A method of making a degradable, additive-blended medical implant comprising: a. Providing liquid polymerizable macromer composed of a cross-linkable moiety ((B.sub.m-A.sub.n-B.sub.m-R) or (R-B.sub.m-A.sub.n-B.sub.m-R)), b. Blending the liquid, polymerizable mixture with at least two therapeutic agents (Drug X and Drug Y) with synergistic antibacterial activity c. Exposing the additive-blended, liquid polymerizable macromer(s) to as external stimulus which can create free radicals for a period of time, thereby forming a medical implant.

58. A method of making an antibacterial polymeric implant of claim 57, wherein the drug X can be sodium-channel blocker, or antibiotic, or anesthetic, or antioxidant, or NSAID. Drug Y can be sodium-channel blocker, or antibiotic, or anesthetic, or antioxidant, or NSAID.

Description

EXAMPLES

Example 1. Assessment of Antibacterial Activity of Single Drugs

[0099] Antibacterial activity of sodium channel blockers (lidocaine, bupivacaine, tetracaine, procaine, prilocaine), NSAIDs (ketorolac and tolfenamic acid), antioxidants (resveratrol and curcumin), and antibiotics (gentamicin and vancomycin) was performed against methicillin-sensitive Staphylococcus aureus (MSSA, ATCC 12600). The bacterial susceptibility tests were conducted as outlined in the Clinical and Laboratory Standard Institute (CLSI) protocol M07-A10. Bacterial suspensions with a concentration of approximately 5*10.sup.5 CFU/ml were prepared in Lysogeny broth and mixed with the tested drugs. Following 24-hour incubation at 37° C., turbidity of the resulting suspensions was visually evaluated, and the minimum inhibitory concentrations (MIC) was determined as the lowest concentration of a single drug that inhibits bacteria proliferation (clear solution). Minimum bactericidal concentration (MBC) was determined as the minimum concentration of the tested drug needed to kill 99.9% of bacteria (or 3−log 10 reduction). The obtained results suggested that tested analgesics possessed moderate antimicrobial properties against MSSA. The MIC values are presented in Table 1. The MBC was measured for lidocaine, bupivacaine, ketorolac, and gentamicin. The MBC of lidocaine and gentamicin were found to be 128 mg/ml and 2 μg/ml, respectively. The MBC of bupivacaine and ketorolac were not determined, as these concentrations were higher than the drug solubility limits in growth medium, which are approximately 15 mg/ml and 18 mg/ml, respectively.

Example 2. Assessment of the Combinational Antibacterial Effects

[0100] Combined antibacterial effects of lidocaine, bupivacaine, prilocaine, procaine, tetracaine, tolfenamic acid ketorolac, curcumin, resveratrol, gentamicin, and vancomycin was evaluated using the checkerboard test against two MSSA strains (ATCC® 12600 and 14775), and S. epidermidis strain (ATCC 12228). Drug combinations with various ratios were prepared and mixed with approximately 5*10.sup.5 CFU/ml of MSSA in Lysogeny broth. Following 24-hour incubation at 37° C. in a 96 well-plate, the turbidity of the resulting suspension was visually observed, and synergistic ratios were determined. The fractional inhibitory concentration indices (ΣFIC) were calculated. Drug combinations were considered to be synergistic if ΣFIC≤0.5; an additive/indifference effect was observed if 0.5<ΣFIC<4; and ΣFIC>4 represented antagonism. The results are presented in tables 2, 3, and 4.

Example 3. Time-Kill Experiments

[0101] Time-kill curves employing 3 time-points (0, 4, 24 hours) over the period of 24 hours were obtained for characterization of the antibacterial activity of selected combinations against MSSA (ATCC® 14775). Tested combinations of drugs were incubated with 5*10.sup.5 CFU/ml of bacteria in Lysogeny broth at 37° C. At a given time-point, aliquots were collected, and bacterial concentrations were determined using the spread-plate method. The minimum accurately countable concentration was 10.sup.2 CFU/ml. A combination was defined to be synergistic if 2−log 10 kill between the combination, and its most active constituent was observed after 24 hours. 1− to 2−log 10 reduction in bacteria when comparing a combination with the most active single agent was an evidence of improved activity. The increase of bacterial concentration represented antagonism. FIG. 1 shows that when used concurrently, 4 mg/ml of bupivacaine and 20 mg/ml of lidocaine showed pronounced synergistic effect at 4 h. At 24 h synergistic kill was achieved if bupivacaine at 1 mg/ml and lidocaine at 40 mg/ml were used. Other combinations of bupivacaine and lidocaine showed 1− to 2−log 10 kill, indicating improved antibacterial activity. Time-kill curves obtained for the ketorolac/gentamicin combinations (FIG. 2) were in good agreement with the checkboard test showing distinct synergism at 4 h when 2 mg/ml of ketorolac and 1 μg/ml of gentamicin were used concurrently. At lower concentrations, this drug combination also yielded synergistic effect at 24 h. Improved antibacterial activity was evident for all combinations at the 4 h time point. The obtained time-kill curves showed that gentamicin monotherapy regimens yielded a regrowth phase between 4 and 24 h, while there was no regrowth with ketorolac/gentamicin combinations. The combination regimens were significantly more active over the first 4 h showing substantially higher initial kill when compared with monotherapy. Similar effects were observed for bupivacaine/lidocaine combinations.

Example 4. Preparation of Dual Drug-Loaded UHMWPE

[0102] Drugs were mixed at a desired ratio, passed through a 75 μm sieve, and then blended with UHMWPE resin (GUR 1020). Dual drug-loaded UHMWPE blocks were prepared using compression molding in a custom mold and molded for 54 minutes at 170° C. under a pressure of 25 MPa.

Example 5. Drug Release from Dual Drug-Loaded UHMWPE

[0103] Drugs were mixed at a desired ratio, passed through a 75 μm sieve, and then blended with UHMWPE resin (GUR 1020). Dual drug-loaded UHMWPE blocks were prepared using phase-separation compression molding in a custom mold and molded for 54 minutes at 170° C. under a pressure of 25 MPa. The following formulations were prepared: 10 wt % Bupivacaine-loaded UHMWPE, 6 wt % Bupivacaine/4 wt % Tolfenamic acid-loaded UHMWPE, 5 wt % Bupivacaine/5 wt % Tolfenamic acid-loaded UHMWPE, 4 wt % Bupivacaine/6 wt % Tolfenamic acid-loaded UHMWPE, 3 wt % Bupivacaine/7 wt % Tolfenamic acid-loaded UHMWPE, 7 wt % Bupivacaine/3 wt % Tolfenamic acid-loaded UHMWPE, 10 wt % Tolfenamic acid-loaded UHMWPE. The obtained materials were cut into 3×5×20 mm strips, transferred to 1.7 ml of PBS, and incubated in syringes under mild shaking (100 rpm) at room temperature. At a given time point, the release medium was collected, the syringes were rinsed, and the medium was replaced. High-performance liquid chromatography (HPLC) was used to measure drug concentration in the eluent. Drug concentration was measured using a Waters Alliance 2695 separations module (Milford, Mass.) and a Waters 2487 UV detector at a detection wavelength of 210 nm. A Waters Nova-Pak C18 column with 4 μm particle size, 3.9 mm diameter and 150 mm length was used. An isocratic mixture of 50% acetonitrile and 50% deionized water with 0.2% phosphoric acid was used as a mobile phase. The flow rate was 1.0 ml/min, and the sample injection volume was 5 μl. 6 replicates were performed to obtain drug release kinetics. The results are shown in FIG. 3.

Example 6. Synthesis of MA-PLA.SUB.4.-PEG.SUB.9.-PLA.SUB.4.-MA

[0104] 2.51 g of PEG with a molecular weight of 400 g/mol was mixed with 3.60 g of DL lactide and 55 mg of stannous octoate. Then the mixture was heated in a microwave for 2 minutes to obtain PLA.sub.4-PEG.sub.9-PLA.sub.4. Methacrylic anhydride (2.59 g) was added and the obtain mixture was heated in a microwave for 2 minutes to synthesize MA-PLA.sub.4-PEG.sub.9-PLA.sub.4-MA.

Example 7. Incorporation of Drugs into MA-PLA.SUB.4.-PEG.SUB.9.-PLA.SUB.4.-MA

[0105] A photoinitiator solution (10 wt % Phenylbis(2,4,6-trimethylbenzoyl)phosphine oxide in acetone) was added to the macromer (MA-PLA.sub.4-PEG.sub.9-PLA.sub.4-MA) at a ratio of 50 μl solution:1 g macromer. Two different drugs (ketorolac and bupivacaine) were incorporated into the macromer via manual stirring. The macromer/drug mixture was then injected into a mold and irradiated with ultraviolet light with a wavelength of 365 nm for 5 minutes to produce a solid gel form of dual drug-loaded MA-PLA.sub.4-PEG.sub.9-PLA.sub.4-MA. The following formulations were prepared 2.5 wt % bupivacaine/2.5 wt % ketorolac—loaded hydrogel, 5 wt % bupivacaine/5 wt % ketorolac—loaded hydrogel, 10 wt % bupivacaine/10 wt % ketorolac—loaded hydrogel

Example 8. Drug Release from Dual Drug-Loaded MA-PLA.SUB.4.-PEG.SUB.9.-PLA.SUB.4.-MA

[0106] Ketorolac and Bupivacaine-loaded MA-PLA.sub.4-PEG.sub.9-PLA4-MA gels, described in example 7, with dimensions 3 mm×5 mm×20 mm were transferred into 2 ml of PBS. At given time points, PBS was replaced and the concentration of the eluted drugs in the solution was measured using high-performance liquid chromatography. The results of this experiment are in FIG. 4.

TABLES

[0107]

TABLE-US-00001 TABLE 1 MIC of the tested drugs against MSSA (ATCC 12600). Drug MIC, mg/ml Gentamicin 0.004 Lidocaine 15.625 Ketorolac 3.125 Tolfenamic Acid 0.25 Bupivacaine 1.875 Tetracaine 0.9375 Procaine 100 Prilocaine 25 Vancomycin 0.000625 Curcumin 0.1875 Resveratrol 0.125

TABLE-US-00002 TABLE 2 FIC indices measured against MSSA (ATCC 12600) for various drug combinations. Drug combination FIC index Result Bupivacaine/Lidocaine 0.375 Synergy Bupivacaine/Prilocaine 1 Additive effect Bupivacaine/Procaine 0.75 Additive effect Bupivacaine/Tetracaine 0.5 Synergy Bupivacaine/Vancomycin 0.7504 Additive effect Curcumin/Bupivacaine 1.125 Additive effect Curcumin/Gentamicin 0.28125 Synergy Curcumin/Ketorolac 0.625 Synergy Curcumin/Prilocaine 2.25 Additive effect Curcumin/Procaine 2 Additive effect Curcumin/Tetracaine 1.5 Additive effect Curcumin/Tolfenamic Acid 0.375 Synergy Curcumin/Vancomycin 0.3754 Synergy Gentamicin/Ketorolac 0.375 Synergy Gentamicin/Prilocaine 2 Additive effect Gentamicin/Procaine 1 Additive effect Ketorolac/Lidocaine 2 Additive effect Ketorolac/Prilocaine 1 Additive effect Ketorolac/Procaine 0.75 Additive effect Ketorolac/Tetracaine 2 Additive effect Lidocaine/Prilocaine 0.75 Additive effect Lidocaine/Procaine 0.5 Synergy Prilocaine/Tetracaine 1 Additive effect Prilocaine/Vancomycin 2 Additive effect Procaine/Prilocaine 1 Additive effect Procaine/Tetracaine 1.5 Additive effect Procaine/Vancomycin 1.5008 Additive effect Resveratrol/ Ketorolac 2 Additive effect Resveratrol/Bupivacaine 0.5625 Synergy Resveratrol/Gentamicin 0.28125 Synergy Resveratrol/Lidocaine 0.75 Additive effect Resveratrol/Prilocaine 0.5 Synergy Resveratrol/Procaine 0.5 Synergy Resveratrol/Tetracaine 0.25 Synergy Resveratrol/Tolfenamic Acid 0.5 Synergy Resveratrol/Vancomycin 0.3754 Synergy Tetracaine/Vancomycin 1 Additive effect Tolfenamic Acid/Bupivacaine 1 Additive effect Tolfenamic Acid/Gentamicin 0.375 Synergy Tolfenamic Acid/Prilocaine 1 Additive effect Tolfenamic Acid/Procaine 0.3125 Synergy

TABLE-US-00003 TABLE 3 FIC indices measured against MSSA (ATCC 14775) for various drug combinations Drug combination FIC index Result Bupivacaine/Ketorolac 2 Additive effect Bupivacaine/Lidocaine 0.31 Synergy Lidocaine/Ketorolac 2 Additive effect Bupivacaine/Gentamicin 0.75 Additive effect Lidocaine/Gentamicin 0.75 Additive effect Ketorolac/Gentamicin 0.37 Synergy

TABLE-US-00004 TABLE 4 FIC indices measured against S. epidermidis (ATCC 12228) for various drug combinations. Drug combination FIC index Result Bupivacaine/Vancomycin 1.2504 Additive effect Bupivacaine/Tetracaine 3 Additive effect Tolfenamic Acid/Bupivacaine 0.75 Additive effect Resveratrol/Lidocaine 0.375 Synergy Resveratrol/Bupivacaine 0.079 Synergy Resveratrol/Tetracaine 0.7492 Additive effect Resveratrol/Vancomycin 0.75 Additive effect Resveratrol/Gentamicin 1 Additive effect Resveratrol/Tolfenamic Acid 0.5 Synergy