Patent Application
20070104678
The present invention relates to synthetic antimicrobial peptides, known herein as ancient defense polymers.
Zasloff (Nature, Vol. 415, Jan. 24, 2002, p. 389) reviewed the role of antimicrobial peptides as an evolutionary ancient, non-specific defense mechanism that is conserved throughout the plant and animal kingdoms. These peptides can rapidly act to destroy a broad range of microbes, including bacteria, fungi, viruses, and protozoa. Although the hundreds of these peptides that have been isolated in recent years display diversity in size, composition, and structure, they share several common features that are believed to confer antimicrobial activity. Namely, the antimicrobial peptides have a net positive charge, are hydrophobic and are able to form amphipathic structures (i.e. they contain clusters of hydrophobic and cationic regions that are spatially organized in discrete sectors of the molecule).
The antimicrobial peptides work by targeting a fundamental difference in the design of microbes and multi-cellular animals, best understood for bacterial targets. Bacterial targets have a negatively charged cell surface, whereas the surface of plant and animal cells has no net charge. One model proposes that the net positive charge of the peptides allows them to interact with the negatively charged bacterial cell membrane surfaces, followed by displacement of lipids, alteration of membrane structure, and in certain cases entry of the peptide into the interior of the target cell, all of which lead to cell death. In contrast, normal mammalian cell membranes are spared because they exhibit no net charge.
Since the target of the antimicrobial peptides is the microbe membrane, cell death is virtually immediate making adaptation and development of resistance difficult. In contrast, traditional antibiotics affect specific internal cell targets that leave cell morphology intact, enabling the bacteria to adapt and develop resistance.
The general nature of the antimicrobial mechanism employed by the natural peptides allows a variety of molecular sizes, structures, and compositions to exhibit activity. Therefore, analogous synthetic or semi-synthetic polymers that fulfil the basic physicochemical criteria for antimicrobial activity (i.e. cationic, hydrophobic and membrane active) are anticipated to serve as a new type of antimicrobial compound. Synthetic polymers have several distinct advantages over their natural counterparts. These include lower cost, well-defined and high purity source materials, well-developed industrial synthetic techniques, tunable degradation or resistance to degradation, and the ability to be shaped and processed into devices and products.
It is an object of the present invention to provide a synthetic polymer analog for antimicrobial peptides.
Thus, in one aspect, the invention provides an ancient defense polymer having antimicrobial activity, the polymer comprising (i) one or more discrete hydrophobic segments, and (ii) one or more hydrophilic segments containing cationic functionality.
Further, the invention relates to a method of forming an ancient defense polymer comprising the step of forming a biologically active polymer containing a hydrophobic region or regions and a hydrophilic region or regions that carry a net cationic charge. The polymer is thus amphipathic, cationic and cell membrane-active.
Further, the invention includes an apparatus, in which the ancient defense polymer is bound in or attached to a surface of the apparatus to impart antimicrobial activity to said apparatus.
Other aspects and features of the present invention will become apparent to those ordinarily skilled in the art upon review of the following description of specific embodiments of the invention.
Embodiments of the present invention will now be described, by way of example only, with reference to the attached Figures, in which:
Generally, the present invention provides a new type of biologically active polymer, and materials and devices formed from or incorporating the biologically active polymer.
The invention relates to an ancient defense polymer, so named because it utilizes a similar “ancient” defense as peptides possessing antimicrobial activity. The polymer has discrete hydrophilic regions containing cationic charge(s), and discrete hydrophobic regions (making it amphipathic) to affect a selected mechanism of antimicrobial activity.
Examples of representations of such antimicrobial synthetic polymers are shown in
Exemplary blocks or regions for use as A, B and C are listed below. However, the invention is not only limited to these. Other blocks or regions which fall within the scope of A, B, and C may also be used, as would be clear to one of skill in the art.
A=a hydrophobic block (which may also be referred to herein as a segment or region) which may comprise:
B=a hydrophilic block (which may also be referred to herein as a segment or region) containing cationic charge at neutral or near-neutral pH, which may comprise:
C=an optional block (which may also be referred to herein as a segment or region) containing functional groups available for derivatization, which may comprise:
Blocks A and B are hydrophobic and hydrophilic blocks, respectively. These terms are used herein relatively. Thus, A must be hydrophobic relative to B, and B must be hydrophilic relative to A. Both A and B may contain various moieties some of which are hydrophilic and some of which are hydrophobic. However, on the whole A must be hydrophobic with respect to B, and vice versa. Furthermore, any one of blocks A, B, and C may contain degradable portions, to aid in degradation of the polymer in a patient.
The individual monomers listed above do not necessarily correspond to the monomers that are added to the reaction vessel for polymerization, but correspond to the monomers that make up the final polymerized product.
In another embodiment, the polymer of the invention may be entrapped in a degradable matrix to permit controlled release of the antimicrobial polymer.
The polymer(s) may be shaped or cast to generate parts of or complete devices that exhibit antibacterial activity in a number of applications including surgical devices, sterile draping and dressings, clothing, food packaging, agricultural processing and bioreactor modification. Further, such polymers may be covalently or non-covalently bound to surfaces, to lend a permanent or semi-permanent biological activity to that surface. For example, an ancient defense polymer having antimicrobial activity may be bound to a biological implant, such as a catheter, a slow-release implant, a replacement valve, a stent, or another such apparatus as may be in contact with a subject. In this way, the apparatus itself would have an antimicrobial surface, and incidence of infection during use would be reduced.
In one aspect, the invention provides an ancient defense polymer made from 1-15 mol % BMA, 549 mol % AMA, and 5090 mol % PPO-Me. 100331 In one aspect, the invention provides an ancient defense polymer of claim made from 5-50 mol % AMA, and 50-95 mol % PPO-Me.
Antimicrobial Copolymers
The following antimicrobial polymers according to the invention were formulated as described herein, and possess antimicrobial properties. Copolymerization of 3-aminopropyl methacrylamide (AMA) and poly(propylene oxide) monomethacrylate (PPO-Me), both shown below, was carried out to generate a cationic, amphipathic polymer. The relative amounts of each monomer fed in the synthetic reaction was varied to generate a range of physicochemical properties.
The copolymers of Example 1 can be represented by
Synthesis and Purification
The desired amount of each monomer was dissolved in ethanol with stirring to make up a 20% (w/v) solution. Initiator (benzoyl peroxide) was then added at 1 wt % of the total mass of monomers fed, and the solution was heated to 70° C. The reaction proceeded at 70° C. for 6 h, then the reaction solution was poured into a beaker and the solvent was dried off at room temperature. The raw polymer solid was then further dried at room temperature under vacuum for at least 3 h to remove residual solvent. The dried polymer was then extracted with a low polarity organic solvent (e.g. ethyl ether, hexane) for at least 6 h using ˜50 mL solvent per gram of polymer. The remaining polymer solid was dried and dissolved in distilled, deionized water (˜50 mL per gram of polymer). The polymer was then precipitated in 0.2 M NaOH, rinsed with distilled, deionized water and dried under vacuum at room temperature for at least 16 h. Finally, the dried polymer was dissolved in methanol, filtered to remove insoluble impurities and cast into a film.
Material Characterization
The purified polymer films were qualitatively assessed by appearance, assayed for elemental composition at Galbraith Laboratories Inc. (Knoxville, Tenn.), solution properties (lower critical solution temperature) by differential scanning calorimetry (DSC, and molecular weight by gel permeation chromatography (GPC).
Bacterial Inhibition Characterization
Evaluation of bacterial inhibition by the polymers was assayed using a modified Kirby-Bauer zone-of-inhibition test. The polymers were incubated for 24 hr in contact with a model Gram-positive bacterium (Staph. aureus) and a model Gram-negative bacterium (P. Aeruginosa). The control material was a silicone polymer. Bacteria were grown in and on sterile culture media (typically Mueller Hinton™ (MH) broth and agar) that was prepared and autoclaved prior to use. The same medium was used to prepare serial dilutions of bacteria and to plate out serial dilutions to perform viable counts.
For contact inhibition experiments, bacterial lawns were prepared by swabbing MH plates in three directions with the standardized culture. The control and test materials (in duplicate) were placed on freshly prepared bacterial lawns and the plates incubated overnight at 37° C. in a humidified incubator. The following day, the plates were examined for zones of inhibition (the dimensions of which are measured if present) and the control and test materials were carefully removed with sterile forceps. Punches of the agar containing bacteria from the contact area under each film sample were taken. The punches were vortexed in sterile saline containing sterile glass beads to dislodge the bacteria, serially diluted and viable plate counts performed. In addition, the films were removed and vortexed in sterile saline to dislodge bacteria adherent to the films and counts were performed on these as well.
Red Cell Hemolysis Assay
Blood was obtained from healthy human donors. Red cells were isolated from whole, heparinized blood by centrifugation and removal of platelet-rich plasma. The red cells were washed three times with phosphate buffered saline (PBS, 145 mM NaCl, 10 mM Na2PO4) and made up as a 10% (v/v) solution in PBS. Copolymer samples were placed in 1.5 mL Eppendorf centrifuge tubes (50 mg per tube) and equilibrated in 400 microlitres PBS at 37° C. for 1 h. Then 100 microlitres of the 10% red cell suspension was added to make a final red cell concentration of 2%. The copolymer samples were incubated in the red cell suspension for 1 h at 37° C. After the incubation time was complete, the red cells were centrifuged out of solution and 100 microlitres of supernatant from each sample was transferred to a 96 well plate. The absorbance of each solution was measured in a plate reader at 540 nm and compared to the positive control (0.1% Triton X-100 incubated with red cell suspension) and the negative control (PBS incubated with red cell suspension). Less than 5% hemolysis was regarded as non-toxic.
Results
The AMA monomer feed for the copolymer synthesis was varied from 10 to 50 mol % and the resulting copolymers' physical characteristics were qualitatively evaluated. Increasing AMA content resulted in increasingly stiff polymers, ranging from soft and tacky at 10 mol % AMA to semi-rigid and bendable at 37.5 mol %. The 50 mol % AMA feed copolymer was found to be water-soluble even at high pH and was therefore not characterized further. Since the 10 and 25 mol % AMA feed copolymers (90% and 75% PPO-Me, respectively) exhibited acceptable physical properties and low aqueous solubility, they were further characterized for physicochemical properties, bacterial inhibition and hemolytic potential.
Table 1 illustrates the elemental composition for the 10 and 25 mol % AMA copolymers. Elemental compositions were found to compare closely to expected, based on monomer feed ratios. In both cases, the measured nitrogen content was lower than expected indicating reduced cationic monomer incorporation in comparison to amount fed (the AMA monomer is the only nitrogen-containing species). However, the low absolute value of the nitrogen weight percent value amplifies any small differences between measured and feed values. This fact, in concert with the stated accuracy of the measurement technique (±10%) limits the precision of the calculation of polymer composition using this technique.
Mn, Mw, and P.D. values were determined for the 10% AMA copolymer. Mn was 217,970; M, was 509,650, and P.D. was 2.3. This demonstrates that the product is a relatively high molecular weight copolymer (i.e. not a combination of two homopolymers).
The copolymers showed sparing solubility (≦2%) in neutral or low pH aqueous solutions at low temperature. However, in testing the solubility properties, it was discovered that the copolymers display an inverse temperature solubility profile that was reversible (i.e. they are thermoreversible gels that precipitate from solution to form a gel at differing temperatures and redissolve if cooled below the gelation temperature, Tgel). Table 2 provides the gelation temperatures for the 10 and 25 mol % AMA feed copolymers as analyzed by DSC.
It appears that the gelation temperature increases with AMA content, probably due to a resultant increase in overall copolymer hydrophilicity. The reversibility of the gelation process was also demonstrated by cooling the samples in the DSC after heating. A dissolution exotherm was observed indicating reversibility.
Bacterial contact inhibition results for the 25 mol % AMA and 10 mol % AMA copolymers are shown in
Since any effective antimicrobial must not be toxic to mammalian cells, the Applicant assayed the cytocompatibility of the antimicrobial polymers using a common human red cell hemolysis test.
Antimicrobial Terpolymers
In addition to the copolymers described in Example 1, terpolymers were synthesized by adding a third monomer (e.g. n-butyl methacrylate, methyl methacrylate) during polymerization to further modify resulting polymer material and bacterial inhibition properties.
The terpolymers were made as described in Example 1 using three monomers, instead of two. The purification, material characterization, bacterial inhibition characterization, and red cell hemolysis assay were performed as for Example 1.
The terpolymers of Example 2 can be represented by
Results
The third monomer (e.g. n-butyl methacrylate, BMA and methylmethacrylate, MMA) were added at molar ratios ranging from 5 to 10% resulting in a wide variety of physical characteristics. The 10 mol % MMA terpolymer also contained 25 mol % AMA and 65 mol % PPO-Me and was found to be a clear, brittle material that showed relatively high solubility in aqueous solutions (˜2%). Therefore, since the Applicant were primarily interested in low-solubility, flexible materials, this polymer was not further characterized. Terpolymers containing BMA were found to be flexible, elastic, less water-soluble materials than corresponding MMA containing terpolymers. Therefore a 10 mol % BMA, 25 mol % AMA, 65% PPO-Me terpolymer (10% BMA) was selected for furlther characterization.
Table 3 shows the elemental composition for the 10% BMA terpolymer in comparison to the feed composition. Again, the measured composition compares closely with expected, with slightly lower than expected nitrogen which may indicate slightly lower AMA incorporation than fed.
Similar to the copolymers described in Example 1, the 10% BMA terpolymer displays a themoreversible gelation. However, the Tgel for the 10% BMA terpolymer is 17.2° C., intermediate to the 10 and 25% AMA copolymers suggesting introduction of BMA as a third monomer leads to increased hydrophobicity in comparison to the copolymer of equal AMA content (25% AMA).
Mn, Mw, and P.D. values were determined for the terpolymer. Mn was 143,970; Mw was 211,930, and P.D. was 1.5. This demonstrates that the product is a relatively high molecular weight terpolymer (i.e. not a combination of homopolymers and/or copolymers).
The bacterial contact inhibition results for 10% BMA are shown in
The red cell hemolysis assay was performed using the 10% BMA terpolymer to determine cytocompatibility.
The above-described embodiments of the present invention are intended to be examples only. Alterations, modifications and variations may be affected to the particular embodiments by those of skill in the art without departing from the scope of the invention, which is defined solely by the claims appended hereto.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/CA04/00529 | 4/7/2004 | WO | 10/7/2005 |
| Number | Date | Country | |
|---|---|---|---|
| 60460409 | Apr 2003 | US | |
| 60530260 | Dec 2003 | US |
