Compositions and uses of antimicrobial materials with tissue-compatible properties

Abstract

To provide an antimicrobial device having a surface coated with a composition having antimicrobial activity and barrier activity.SOLUTION: The present invention provides an antimicrobial device having a surface coated with a mixture containing one or more synthetic cationic polypeptide having antimicrobial activity, and a second pharmacologically acceptable polymer that is not the synthetic cationic polypeptide.SELECTED DRAWING: Figure 20B

Description

[Technical field]

[0001] <Incorporated by reference all priority applications> This application claims the benefit of U.S. Provisional Application No. 61 / 615,150, filed March 23, 2012, U.S. Provisional Application No. 61 / 625,757, filed April 18, 2012, U.S. Provisional Application No. 61 / 625,760, filed April 18, 2012, and U.S. Provisional Application No. 61 / 716,242, filed October 19, 2012, the disclosures of each of which are incorporated by reference in their entirety herein to the extent permitted by law. [Background technology]

[0002] When the barrier is breached, infection occurs. Surgery, trauma and burns, medical device use (e.g., catheter insertion, artificial respiration), chronic wounds (e.g., diabetic foot ulcers), and various diseases disrupt the human natural defense barrier. In almost all cases, these "wounds" become contaminated by microorganisms. The wound terrain then provides an excellent environment for microbial growth. The battle begins, and it is trench warfare. Damaged tissue ("fissures and crevices"), altered blood flow and exudate production, changes in local temperature, pH, and tissue oxygenation, and lack of commensal bacteria can all contribute. Bleeding and vascular leakage can also provide fluids and nutrients that ultimately support microbial growth. Bacterial or fungal colonization and / or overt infection can occur. Microbial biofilms can help the microorganism create its own local environment. A variety of surgical procedures, trauma, and medical conditions all deserve special attention because they involve the breakdown of the human natural defense barrier and the outcomes can range from rapid healing to fatal sepsis.

[0003] Natural barriers are usually referred to by their anatomical site, such as skin, pulmonary epithelium, gastrointestinal mucosa, etc. These names may encompass unrecognized levels of simplicity. These barriers are often either passive or active. They may involve a variety of cells, secreted glycoproteins, matrix components, and body fluids that act in concert to provide an effective defense against microbial invasion. At some sites, resident microorganisms contribute to the barrier action against other possible invaders. Under most circumstances, these physical and functional barriers are highly effective. However, they can be relatively easily disrupted by mechanical or chemical attack. In addition, certain systemic diseases can weaken the human natural barriers and increase the risk of disruption, as occurs with diabetic foot ulcers or cystic fibrosis. Finally, a first infection can weaken the host's defenses against a second infection, as occurs with influenza followed by bacterial pneumonia or vaginal trichomonas followed by certain sexually transmitted diseases (e.g., HIV).

[0004] When the defense barrier is breached, the host becomes susceptible to infection with a wide variety of microorganisms, ranging from normally benign commensals to aggressive pathogens. Generally, humans are their own source of microorganisms, contaminating their own wounds. The human body hosts a large number of bacteria, primarily on the skin, in the oral cavity, and in the lower gastrointestinal tract. Within the space of a single human body, there are approximately 100 mammalian cells ( 13 ) more bacterial cells (10 14 ) are estimated to be present in the human body. Despite this close association with microorganisms, most human tissues (including blood, subcutaneous tissue, muscle, and brain) remain sterile until natural barriers are disrupted. Other human and environmental sources of microorganisms are also important, especially in healthcare settings. Once barriers are disrupted, microbial contamination, critical levels of colonization, biofilm formation, and / or the onset of infection can occur. Polymicrobial colonization and / or infection is common in certain situations (e.g., diabetic foot ulcers, complicated intraperitoneal infections) and may involve aerobes, anaerobes, or both.

[0005] Prior art approaches to the prevention and treatment of such infections have demonstrated significant weaknesses. Both lack of efficacy and tissue toxicity have been problematic. Antimicrobial agents often fail to reach the correct tissue space and / or remain active for a sufficient period of time to prevent or treat infection. Complex surfaces such as the abdominal cavity, or large burns, are particularly difficult to treat effectively. Finally, safely administering sufficient antimicrobial agent to a particular tissue space (such as by laparoscopic or arthroscopic device) can be problematic. Antimicrobial agents that are easily administered by such methods tend to be solution-based substances with limited ability to bind to tissue and remain active for extended periods of time.

[0006] Biofilms present a particular problem. There is growing evidence demonstrating the resistance of bacterial biofilms to various antimicrobial approaches and their role in adverse patient outcomes. Such microbial populations resist conventional disinfectants and antibiotics through several mechanisms, including but not limited to their own production of extracellular polymeric substances, often negatively charged (anionic). Penetration of such substances by conventional antimicrobial agents is often limited. For example, in acute wounds (e.g., surgery and trauma), devitalized tissue and foreign objects (e.g., prosthetic implants) may support the formation of biofilms, thereby increasing the likelihood of infection developing. In chronic wounds (e.g., diabetic foot ulcers), biofilms may persist, leading to delayed wound healing. Medical devices such as ventilators and catheters may be sites of biofilm formation and may provide a source of infection.

[0007] Antibiotic treatment of initial infections may alter the course of infection, resulting in more resistant and dangerous infections. Common antibacterial strategies center on the use of selective antibiotics (e.g., penicillin for gram-positive organisms) to avoid the development of bacteria resistant to broad-spectrum antibiotics. Coincidentally, this important strategy may result in poor prognosis for individual patients, where targeted antibiotics result in the emergence of aggressive, different microorganisms (e.g., Pseudomonas). In this way, treated wounds may become the site of a "parade of pathogens," where the initial dominant microbial species (e.g., Staph aureus) is replaced by a second (e.g., MRSA, methicillin-resistant Staphylococcus aureus), and possibly even a third and fourth microbial species (e.g., multidrug-resistant gram-negative species).

[0008] The high number of adverse patient outcomes in today's advanced medical landscape highlights the shortcomings of the prior art in preventing and treating such infections. Some important weaknesses include:

[0009] 1. Low antibacterial activity in tissue environments and on biofilms;

[0010] 2. Inadequate distribution into the appropriate tissue space;

[0011] 3. Limited barrier activity, if any;

[0012] 4. Narrow spectrum of antimicrobial activity allows for a "parade of pathogens";

[0013] 5. Inadequate treatment may further promote antimicrobial resistance; and / or

[0014] 6. Tissue toxicity.

[0015] Wound infections and other barrier-disrupting conditions are common and costly. In the United States, approximately 12 million traumatic injuries are treated in emergency departments each year. In addition, over 50 million surgical procedures are performed (inpatient and outpatient). The U.S. Department of Health and Human Services notes that there are over 1.7 million healthcare-associated infections per year, resulting in approximately 100,000 deaths and $30 billion in healthcare costs each year. Many of these healthcare-associated infections are initiated by a breakdown in barriers. Examples include surgical site infections (SSIs), catheter-associated urinary tract infections, and ventilator-associated pneumonia. Chronic wounds associated with pressure sores (bed sores) and diabetic foot ulcers present their own unique problems.

[0016] In addition to infection, several other wound-related outcomes remain a major problem. These include blood loss, tissue adhesion / scarring, and poor wound healing. And in some cases, known antimicrobial treatments exacerbate these problems. Certain antimicrobial wound treatments (including antibiotic washes) can result in excessive tissue response (e.g., tissue adhesion or scarring). Certain antiseptics / antimicrobials can alter wound healing, resulting in inadequate tissue response (e.g., poor wound healing, poor wound strength).

[0017] Effective hemostasis in wounds also remains a major problem. Hemostatic agents have been reported and used in various situations, including trauma and surgery. Although effective in some situations, these agents do not provide an ideal solution to the problem. First, hemostasis may be insufficient and bleeding may be excessive, which may result in fatal outcomes. In some such cases, initial hemostasis is achieved, but rebleeding occurs later. This may be due to fibrinolytic activity. In addition to problems due to blood loss, extravasated blood components in tissues may contribute to additional adverse outcomes, including infection and fibrotic responses seen with tissue adhesions after surgery. Second, in some cases, hemostatic agents may cause problems by entering the bloodstream and inducing clotting (thrombosis) in blood vessels, which may result in fatal outcomes. Third, in some cases, wound treatment agents (including hemostatic agents) may act as a site for subsequent infection or may result in abnormal tissue responses, such as adhesion formation and / or tissue scar formation, resulting in adverse medical outcomes. Improved approaches to hemostasis are needed. Summary of the Invention [Problem to be solved by the invention]

[0018] According to an embodiment of the present invention, a mixture of an antimicrobial cationic polypeptide and a second pharmacologically acceptable polymer is used for the treatment and prevention of infections that occur when the human natural defense barrier is breached. Such novel compositions can provide two functions: direct antimicrobial activity and barrier activity. An embodiment of the present invention addresses one or more of the weaknesses of previous antimicrobial agents mentioned above. In particular, the effectiveness of most previous antiseptics and antibiotics is based on the determination of antimicrobial activity in solution with microorganisms in suspension (MIC assay), which gives results that are not necessarily indicative of, and therefore may be distant from, the determination of efficacy in tissue. In contrast, the inventors have focused on the design and selection of agents for broad antimicrobial activity, especially in the surface topography of tissue surfaces and wounds ("cracks and crevices"). This involves the formation of a barrier containing cationic (positively charged) elements that can inhibit the movement of certain substances or cells (e.g., microorganisms) that exhibit anionic elements. In one embodiment, the synthetic cationic polypeptides of these compositions contain at least one cationic segment and at least one hydrophobic segment, consist essentially of natural amino acids, and have broad antibacterial properties (i.e., against gram-positive and gram-negative bacteria). They can also be designed to self-assemble, based in part on the interaction of their hydrophobic segments. Furthermore, the synthetic polypeptides are blended with a second pharmacologically acceptable polymer to provide compositions that have direct antibacterial properties and effectively coat tissue. These mixtures can also exhibit hemostatic properties. In certain embodiments, the second pharmacologically acceptable polymer is not polyethylene glycol (PEG).

[0019] Embodiments of the present invention may be used alone or in combination with other agents that provide similar or complementary activities. [Means for solving the problem]

[0020] An embodiment provides an aqueous composition for the prevention, inhibition, or treatment of infection, comprising a mixture comprising: one or more synthetic cationic polypeptides having antibacterial activity; and a second pharmacologically acceptable polymer that is not a synthetic cationic polypeptide having antibacterial activity; wherein the amount of each of the one or more synthetic cationic polypeptides and the second pharmacologically acceptable polymer is at least about 100 μg / mL based on the total volume of the aqueous composition; wherein the amount of the second pharmacologically acceptable polymer is at least about 10% by weight based on the weight of the one or more synthetic cationic polypeptides; and wherein the synthetic cationic polypeptides and the second pharmacologically acceptable polymer are mutually water-miscible.

[0021] One or more synthetic cationic polypeptides having antimicrobial activity and a second pharmacologically acceptable polymer are considered to be miscible with each other if, after mixing in water at a concentration of 1 mg / mL of each polymer and maintaining at room temperature for 24 hours, at least about 90% of the polymer components remain soluble in each other upon visual inspection.

[0022] In another embodiment, one or more of the one or more synthetic cationic polypeptides in the aqueous composition comprises a segment having a chain length of at least 40 amino acid residues.

[0023] In another embodiment, the one or more synthetic cationic polypeptides in the aqueous composition comprise subunits that are substantially all naturally occurring amino acids.

[0024] In another embodiment, the one or more synthetic cationic polypeptides in the aqueous composition are characterized by at least one segment containing at least five consecutive cationic amino acid residues and at least one segment containing at least five consecutive hydrophobic amino acid residues.

[0025] In another embodiment, the second pharmacologically acceptable polymer in the aqueous composition is selected from the group consisting of cellulose, alginate, collagen, polymeric surfactants, polyethylene glycol, polyvinyl alcohol, polyurethane, polyvinylpyrrolidinone (PVP), fibrin (fibrinogen), blood proteins, and tissue proteins.

[0026] In another embodiment, the antimicrobial activity of the aqueous composition is greater than 3 log kill of Staphylococcus epidermidis and Escherichia coli in a standard 60 minute time-kill assay at a concentration of the synthetic cationic polypeptide of 100 μg / mL or less.

[0027] In another embodiment, the aqueous composition is further characterized by the ability to disrupt or inhibit biofilms in vitro at total polymer concentrations of 40 mg / mL or less.

[0028] In another embodiment, the aqueous composition is further characterized by a barrier activity as measured by a reduction in the diffusion rate of an anionic dye of greater than 2 log at a total polymer concentration of 40 mg / mL or less.

[0029] In another embodiment, the aqueous composition is further characterized by a storage modulus of at least 50 Pa at a total polymer concentration of less than 40 mg / mL.

[0030] In another embodiment, the aqueous composition is further characterized by a storage modulus of at least 50 Pa at a total polymer concentration of less than 40 mg / mL, and the ability to pass through a 20 g needle using a pressure of less than 60 N.

[0031] In another embodiment, the aqueous composition is further characterized by the ability to pass through a 20 g needle and to recover at least 70% of its strength, as measured by storage modulus, within 10 minutes.

[0032] In another embodiment, the aqueous composition is in the form of a solution, gel, cream, foam, or dressing.

[0033] In another embodiment, the aqueous composition is further characterized as being combined or associated with a dressing, including, but not limited to, gauze or a sponge.

[0034] In another embodiment, the aqueous composition has procoagulant activity, prohemostatic activity, or both.

[0035] In another embodiment, the aqueous composition further comprises a pharmacologically active ingredient (API) selected from the group consisting of steroids, pro-inflammatory agents, anti-inflammatory agents, anti-acne agents, preservatives, hemostatic agents, angiogenic agents, wound healing agents, anti-cancer agents, and other anti-bacterial agents.

[0036] Another embodiment provides a use of any one of the aqueous compositions described herein for any one or more selected from the group consisting of prevention of infection, treatment of infection, topical anti-infective treatment, treatment for microbial decontamination, wound treatment, surgical site treatment, trauma treatment, burn treatment, treatment of diabetic foot ulcers, eye treatment, treatment of vaginal infection, treatment of urinary tract infection, hand disinfection, for coating prosthetic devices and / or implants, food preservation, and solution preservation.

[0037] Another embodiment provides a method for the prevention and / or treatment of infection, comprising: contacting tissue of a subject patient with any of the aqueous compositions described herein.

[0038] In another embodiment, the method further comprises applying negative pressure to the wound.

[0039] In another embodiment, the method further includes treating the patient systemically with another antibiotic, and / or locally with another antibacterial agent, and / or at least one selected from the group consisting of an antibiotic, an anti-biofilm agent, a surfactant, and combinations thereof. [Brief description of the drawings]

[0040] [Figure 1] Examples of synthetic cationic polypeptides, Kx(rac-L)y and KxLy, are shown. These polypeptides contain cationic segments of lysine amino acids and hydrophobic segments of leucine amino acids. The cationic segments provide multimeric interactions with anionic substances, including bacterial surfaces. The hydrophobic segments associate in aqueous media, leading to the formation of various structures, such as dissolved multimers, micelles, sheets, and fibrils. These hydrophobic interactions are important in barrier formation. [Figure 2A] 1 is a table showing examples of synthetic cationic polypeptides synthesized using amine initiators (Table 1). [Figure 2B] 1 is a table (Table 1) showing examples of synthetic cationic polypeptides synthesized using the organometallic Co(PMe3)4 initiator. [Diagram 3] 1 is a gel permeation chromatogram of a chain extension experiment. [Figure 4] 1 is a gel permeation chromatogram of a representative deprotected diblock copolypeptide. [Diagram 5] Attenuated total reflectance Fourier transform infrared (ATR-IR) interferograms of the monomer. [Figure 6] Attenuated total reflectance Fourier transform infrared (ATR-IR) interferograms of the crude reaction mixture and the complete polymerization. [Figure 7A] Matrix-assisted laser desorption / ionization (MALDI) mass spectra of representative peptides are shown. [Figure 7B] Matrix-assisted laser desorption / ionization (MALDI) mass spectra of representative peptides are shown. [Figure 8] The liquid proton nuclear magnetic resonance (H1-NMR) spectrum of a representative deprotected copolypeptide in d-TFA is shown. [Figure 9]In vitro antimicrobial time-kill assay (60 min) against Staphylococcus epidermidis (RMA 18291) and Escherichia coli (ATCC 25922) using K100(rac-L)20. [Figure 10] In vitro antimicrobial time-kill assay (60 min) against Staphylococcus epidermidis (RMA 18291) and Escherichia coli (ATCC 25922) using K100L40. [Figure 11] Table 2 shows the in vitro antimicrobial time-kill of K100(rac-L)20 and 1:1 K100(rac-L)20:Poloxamer 407 solutions at concentrations of 10 and 100 μg / mL against Staphylococcus aureus (29213) and P. aeruginosa (27853) after 5 and 60 minutes. [Figure 12] 1 is a table (Table 3) showing the in vitro antimicrobial time-kill of K100(rac-L)20 solutions at concentrations of 10 and 100 μg / mL against various gram-positive and gram-negative bacteria and fungi after a contact time of 60 minutes. [Figure 13] We show that K100(rac‐L)20 and K100L40 at concentrations between 0.1 and 20 mg / mL are effective against P. aeruginosa biofilms in vitro (contact time 24 h). [Figure 14] In vitro antibacterial time-kill assays after 5 and 60 minutes against Staphylococcus aureus (29213) and Pseudomonas aeruginosa (27853) are shown for K100(rac-L)20 at concentrations of 10 and 100 μg / mL along with other ratios of excipients: 5:1 K100(rac-L)20:Poloxamer 407, 2:1 K100(rac-L)20:HEC, and 1:3 K100(rac-L)20:Peg 400. [Figure 15]Figure 2 shows in vitro antibacterial time-kill assays after 30 minutes against Staphylococcus aureus (29213) using K100L40 (10 μg / mL) alone and in combination with cellulose ethers (methylcellulose (MC), hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose (HPC), and hydroxyethyl cellulose (HEC)) in a 1:1 ratio. [Figure 16] Table 4 shows the in vitro antimicrobial time-kill assay against Staphylococcus aureus (29213), Staphylococcus epidermidis (RMA 18291), Pseudomonas aeruginosa (27853), and Escherichia coli (25922) after a contact time of 60 minutes. K100L40 at concentrations of 10 and 100 μg / mL was tested along with other ratios of hydroxyethylcellulose: 1:2 K100(rac-L)20:HEC and 1:20 K100(rac-L)20. [Figure 17] A barrier assay was presented in which a 2% synthetic cationic polypeptide formulation (K100L40) was shown to be highly effective at blocking the diffusion of a colored anionic dye over a 48 hour period. In contrast, the dye diffused readily (within 5 minutes) through a 2% polyethylene glycol (10,000) formulation. [Figure 18] Figure 1 shows a synthetic cationic polypeptide binding to the synthetic tissue analog Vitroskin®. As demonstrated with 1% solutions of FITC-K100(rac-L)20 and FITC-K100L40, the majority of the applied FITC-labeled copolypeptide was shown to remain associated with Vitroskin®. In comparison, the majority of the labeled BSA was removed by washing. The % remaining was determined using FITC fluorescence (λexc=495 nm, λem=521 nm) after 1-10 washes and calculated by subtracting the percent removed from 100%. [Figure 19]In vitro antibacterial activity of K100(rac-L)20 and K100L40 (100-3000 μg / mL) against Staphylococcus aureus on Vitroskin® surfaces after 0 and 5 washes (5 x 1 mL). Each 3 cm x 3 cm piece of Vitroskin® was incubated with Staphylococcus aureus for 60 minutes. [Figure 20A] Firmness values ​​are shown for mixtures of 1% synthetic cationic polypeptide solutions and 1% hydroxyethylcellulose (HEC, Natrosol HHX) in water. [Figure 20B] 1 shows adhesion work values ​​for mixtures of 1% copolypeptide solutions and 1% hydroxyethyl cellulose (HEC, Natrosol HHX) in water. [Figure 21] 5 is a table (Table 5) showing synthetic cationic polypeptide texture analysis profile data for pure polypeptide and HEC blends. a. Values ​​for 1% (wt / wt) polypeptide in water. b. Values ​​for 1% polypeptide / 1% HEC (wt / wt) in water. c. Interaction parameter: ΔF=F blend(poly / HEC)-(Fpoly+FHEC), where F blend(poly / HEC)=stiffness of 1% polypeptide / 1% HEC, Fpoly=stiffness of 1% polypeptide solution, and FHEC=stiffness of 1% HEC in water (Natrosol HHX). Similar data treatment was performed for the work of adhesion interaction parameter. [Figure 22A] Shown are A) strain and B) frequency sweep of a synergistic mixture of 1% K100L40 / 1% HEC and individual components at 1% in water. ■=1% K100L40 / 1% HEC, ●=1% K100L40, and ▼=1% HEC. G' values ​​= filled symbols and G” = open symbols. [Figure 22B] Shown are A) strain and B) frequency sweep of a synergistic mixture of 1% K100L40 / 1% HEC and individual components at 1% in water. ■=1% K100L40 / 1% HEC, ●=1% K100L40, and ▼=1% HEC. G' values ​​= filled symbols and G” = open symbols. [Figure 23]A synthetic cationic polypeptide solution (anti-infective solution; left) and a synthetic cationic polypeptide hydrogel (barrier gel; right) are shown to be effective in covering open wounds in a porcine model. The synthetic cationic polypeptide was fluorescently labeled. [Figure 24] Antibacterial activity of K100(rac-L)20 in a rodent infection model. Polypropylene meshes were inserted subcutaneously into rats followed by 107 MRSA (33593). After 15 min, 10, 2, or 0.4 mg / mL K100(rac-L)20 or water was added. After 2 days, the implanted meshes and surrounding tissues were analyzed for MRSA bacterial counts. [Diagram 25] Antibacterial activity of K100(rac‐L)20 in an open wound porcine model. Each wound was contaminated with bacteria (Staphylococcus epidermidis, Pseudomonas aeruginosa (porcine clinical isolate)). After 2 hours, the wounds were rinsed with saline and treated with 5 mL of test article or water. Test articles: 10, 2, 0.4 mg / mL K100(rac‐L)20, 10 mg / mL K100(rac‐L)20 and 30 mg / mL PEG400 in water, and deionized water as a control. Gauze soaked in the test article or water was placed on the wound. Wound biopsies were taken 4 hours after treatment. [Figure 26] We show that K100L40 hydrogel prevents infection in an open wound porcine model. For each wound, hydrogel was applied to the wound surface and gauze. Gauze soaked in hydrogel was placed on the wound. Saline was used as a control. After 15 minutes, each wound was contaminated with bacteria (Staphylococcus epidermidis, Pseudomonas aeruginosa (porcine clinical isolate)). Hydrogel test articles: K100L40 10mg / mL, K100L40 5mg / mL and 10mg / mL HEC, K100L40 2mg / mL and 15mg / mL HEC, and saline as a control. Wound biopsies were taken 4 hours after hydrogel application. [Figure 27] 1 shows a synthetic cationic polypeptide conjugated to a medically acceptable sponge material. This product concept illustrates one way in which a copolypeptide can be contacted with a wound to facilitate delivery of hemostatic and / or antimicrobial activity. [Figure 28] Shown are the results of an in vitro whole blood clotting assay with K100L40 at 10 and 100 μg / mL and a 1:1 mixture of K100L40 and HEC. Controls were HEC alone at 10 and 100 μg / mL, negative control saline, and positive control thromboplastin, TF (50 μL in 500 μL whole blood). [Figure 29] 1 shows the results of a platelet aggregation assay with K100L40 at 10 and 100 μg / mL and a 1:1 mixture of K100L40 and HEC. Controls were HEC alone at 10 and 100 μg / mL, saline as a negative control, and collagen as a positive control. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] According to an embodiment of the present invention, an aqueous composition comprising a mixture of an antimicrobial cationic polypeptide and a second pharmacologically acceptable polymer is used for the treatment and / or prevention of infections that occur when the human natural defense barrier is breached. This novel composition can exhibit two functions: direct antimicrobial activity and barrier activity. An embodiment of the present invention addresses one or more of the weaknesses of previous antimicrobial agents mentioned above. In particular, the inventors started from the realization that most previous antiseptics and antibiotics are based on antimicrobial activity in solution with microorganisms in suspension (MIC assay), a method that may be directed away from efficacy in tissue. In contrast, the inventors focused on designing and selecting agents for broad antimicrobial activity, especially in the surface topography of tissue surfaces and wounds ("cracks and crevices"). This involves the formation of a barrier containing cationic (positively charged) elements that can inhibit the movement of certain substances or cells (e.g., microorganisms) that exhibit anionic elements. In one embodiment, the synthetic cationic polypeptides of these compositions contain at least one cationic segment and at least one hydrophobic segment, consist essentially of natural amino acids, and have broad antibacterial properties (i.e., against gram-positive and gram-negative bacteria). They can also be designed to self-assemble, based in part on the interaction of their hydrophobic segments. Furthermore, the synthetic polypeptides are blended with a second pharmacologically acceptable polymer to provide compositions that have direct antibacterial properties and effectively coat tissue. These mixtures can also exhibit hemostatic properties. In certain embodiments, the second pharmacologically acceptable polymer is not polyethylene glycol (PEG).

[0042] Embodiments of the present invention may be used alone or in combination with other agents that provide similar or complementary activities.

[0043] Synthetic Cationic Polypeptides A variety of synthetic cationic polypeptides can be used in the aqueous compositions described herein. In embodiments, the synthetic cationic polypeptides include a segment of repeating units or residues of positively charged amino acids (e.g., lysine, arginine) and another segment of repeating units or residues of hydrophobic amino acids (e.g., leucine, isoleucine, valine, alanine). An example is a K-type cationic polypeptide having an overall structure consisting of a segment or block of one or more repeating lysine amino acids and a segment or block of one or more repeating leucine amino acids. x L y (FIG. 1). In various embodiments, one or more of the synthetic cationic polypeptides includes a segment having a length of at least 40 amino acid residues. In various embodiments, the synthetic cationic polypeptides include subunits that are substantially all naturally occurring amino acids. In some embodiments, the synthetic cationic polypeptides include at least one segment that contains at least five consecutive cationic amino acid residues and at least one segment that contains at least five consecutive hydrophobic amino acid residues. For example, in some cases, the polypeptides include one or more segments that contain at least five consecutive repeating lysine amino acid units and one or more segments that contain at least five consecutive repeating hydrophobic amino acids (e.g., leucine). In some cases, the cationic polypeptides can be relatively long molecules with lysine blocks that contain from 50 to 200 or more lysine amino acid residues. Examples of synthetic cationic polypeptides include K 50 (rac‐L) 10 , K 50 (rac‐L) 20 , K 50 (rac‐L) 30 , K 50 (rac‐L) 40 , K 50 (rac‐L) 50 , K 50 L 10 , K 50 L 20 , K 50L 30 , K 50 L 40 , K 50 L 50 , K 100 (rac‐L) 10 , K 100 (rac‐L) 20 , K 100 (rac‐L) 30 , K 100 (rac‐L) 40 , K 100 (rac‐L) 50 , K 100 L 10 , K 100 L 20 , K 100 L 30 , K 100 L 40 , K 100 L 50 , K 200 (rac‐L) 10 , K 200 (rac‐L) 20 , K 200 (rac‐L) 30 , K 200 (rac‐L) 40 , K 200 (rac‐L) 50 , K 200 L 10 , K 200 L20, K 200 L 30 , K 200 L 40 , K 200 L 50 (FIG. 2 (Table 1)). Other synthetic cationic polypeptides are contemplated, e.g., with longer or shorter lysine segments and longer or shorter leucine segments. The invention also includes other synthetic cationic polypeptides in which the cationic segment may include other amino acids, such as arginine, and in which the hydrophobic block may contain one or more hydrophobic amino acids, including, but not limited to, leucine, alanine, valine, and / or isoleucine. In some cases, one or more of the segments may have a random sequence of amino acids, including hydrophobic and hydrophilic amino acids.

[0044] Examples of synthetic cationic polypeptides include those described in U.S. Patent Nos. 6,680,365; 6,632,922; 6,686,446; 6,818,732; 7,329,727; U.S. Patent Application Publication No. 2008 / 0125581; and U.S. Patent Application Publication No. 2011 / 048869. The aforementioned patents and patent applications are incorporated herein by reference, particularly for the purposes of describing synthetic cationic polypeptides and methods of making same.

[0045] A method for producing synthetic cationic polypeptides with highly reproducible and extremely narrow polydispersity has been developed, and such polymers can be made to various specifications to meet the needs of antimicrobial activity and barrier formation. For example, high-quality block copolypeptides are synthesized by combining high-quality α-amino acid N-carboxyanhydrides (NCAs) in anhydrous solvents with benzylamine initiators. These polymers are then deprotected and purified to obtain the final products.

[0046] Several analytical techniques have been developed and refined both for monitoring the synthesis of peptides and for the analysis of the size, properties, and residual impurities of the resulting polymer products. Infrared spectroscopy may be used for monitoring the progress of the reaction, while size-exclusion gel permeation chromatography may be used for monitoring the growth and state of the polymer at various stages of the process. Other analytical techniques such as nuclear magnetic resonance spectroscopy (NMR), matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS), and inductively coupled plasma mass spectrometry (ICP-MS) may be used as additional quality control tests, for example to ensure a certain reproducibility and purity (Figures 3-8).

[0047] Synthetic cationic polypeptides can be designed to exhibit broad-spectrum antimicrobial activity (see U.S. Patent Application Publication No. 2011 / 048869). Synthetic cationic polypeptides are ... Antibacterial activity is considered to be achieved when greater than 3 log kill of S. epidermidis and E. coli is achieved in a time-kill assay. In embodiments, the synthetic cationic polypeptides have antibacterial activity that achieves greater than 3 log kill of S. epidermidis and E. coli in a standard 60 minute time-kill assay at a synthetic cationic polypeptide concentration of 100 μg / mL or less. As shown in Figures 9-12, embodiments of these synthetic cationic polypeptides exhibit in vitro antibacterial activity against both gram-positive and gram-negative bacteria. This activity is demonstrated in an in vitro time-kill assay using the synthetic cationic polypeptides in aqueous media. Additionally, embodiments of the synthetic cationic polypeptides exhibit in vitro antibiofilm activity, as shown in Figure 13. These effects can likely be explained in part by the binding of the cationic polypeptides to anionic charges present in the biofilm matrix. In addition, the detergent-like activity of the synthetic cationic polypeptides may also contribute to the antibiofilm effect. It is also recognized that the compositions of the present invention may alter the structure and / or charge of a biofilm, which may allow other agents (e.g., locally or systemically administered antibiotics) to penetrate the biofilm, thereby enhancing their activity.

[0048] The mixture of synthetic cationic polypeptide and other polymers can retain antimicrobial activity in vitro. The second pharmacologically acceptable polymer is different from the one or more synthetic cationic polypeptides having antimicrobial activity. In embodiments, the second pharmacologically acceptable polymer has little or no antimicrobial activity by itself. For example, in embodiments, the antimicrobial activity of the second pharmacologically acceptable polymer is less than 10% of the antimicrobial activity of the synthetic cationic polypeptide(s).

[0049] The amount of each of the synthetic cationic polypeptide(s) and the second pharmacologically acceptable polymer in the aqueous medium is at least about 100 μg / mL, and may be higher, for example, about 1 mg / mL, about 5 mg / mL, or about 10 mg / mL, or about 20 mg / mL, or about 40 mg / mL, or more. The amount of the second pharmacologically acceptable polymer in the aqueous composition is at least about 10% by weight, and may be higher, for example, at least about 20% by weight, at least about 30% by weight, or at least about 50% by weight, based on the weight of the one or more synthetic cationic polypeptides. The synthetic cationic polypeptide(s) and the second pharmacologically acceptable polymer in the aqueous composition are selected such that the polymers are miscible with each other. As indicated above, the cationic polypeptide(s) having antimicrobial activity and the second pharmacologically acceptable polymer are considered to be miscible with each other if, after mixing in water at a concentration of 1 mg / mL of each polymer and maintaining at room temperature for 24 hours, at least about 90% of the polymer components remain soluble in each other upon visual inspection.Surprisingly, such miscibility of water, synthetic cationic polypeptide(s), and the second pharmacologically acceptable polymer with each other can be achieved, even though phase separation is typically expected due to the immiscibility of the polymers in aqueous solutions at a concentration of 1 mg / mL and the molecular weights described herein.The aqueous compositions described herein can be made by mixing the individual polymer components with water using conventional mixing methods known to those skilled in the art, for example, by stirring at room temperature. EXAMPLES

[0050] Example 1 As shown in Figures 14-16, embodiments of the synthetic cationic polypeptides have been shown to retain substantially all of their antimicrobial activity in the presence of other pharmacologically acceptable polymers, as shown by in vitro time-kill assays against Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Escherichia coli. In these studies, various cellulosic second polymers were evaluated. As shown in Figures 11 and 13, other studies have shown that other cellulosic second polymers can be used in combination with other pharmacologically acceptable polymers. It has been shown that antimicrobial activity is maintained in vitro in the presence of pharmacologically acceptable (polymeric surfactants). In embodiments, the antimicrobial activity of the aqueous composition is greater than 3 log kill of Staphylococcus epidermidis and Escherichia coli in a standard 60 minute time-kill assay at synthetic cationic polypeptide(s) concentrations of 100 μg / mL or less. In another embodiment, the aqueous composition is further characterized by its ability to disrupt or inhibit biofilms in vitro at total polymer concentrations of 40 mg / mL or less.

[0051] Mixtures of synthetic cationic polypeptides and other polymers can exhibit enhanced physical and viscoelastic properties in vitro. In developing compositions for use in patients, the need to maintain antimicrobial activity while increasing volume, tissue coverage, and / or biocompatibility is recognized. Various synthetic cationic polypeptides have been mixed with other pharmacologically acceptable polymers in a manner that maintains at least about 80% (e.g., at least 90%) of the antimicrobial activity of the synthetic cationic polypeptides, and in preferred embodiments also improves tissue coverage. It is believed that the amphoteric nature of the synthetic cationic polypeptides and their self-assembly in aqueous solution induces a stable phase-separated (collapsed or solvated) dispersion of hydrophobic and hydrated hydrophilic substances. In certain embodiments, it is believed that the chain length of the copolypeptide (e.g., degree of polymerization or n>50) provides a network of partially collapsed pockets of hydrophobicity that retards the diffusion of solutes and solvents. The barrier properties of a material are the product of its ability to trap water and reduce its mobility, thereby dramatically retarding the passage of any solutes or particles present in water. The length of the polypeptide strongly influences the barrier properties (e.g., by dramatically reducing diffusion). Therefore, the selection of a second polymer to mix with the synthetic cationic peptide(s) should be done carefully to avoid disruption of the influencing biophysical parameters and achieve mutual solubility in the aqueous composition. Those skilled in the art can use routine experimentation guided by the teachings provided herein to select the polymer components and amounts to form the aqueous compositions described herein.

[0052] The properties of the aqueous compositions described herein can be adjusted by controlling the ratio of the amount of cationic polypeptide to the second pharmacologically acceptable polymer(s). Non-limiting examples of these second polymers include cellulose (e.g., hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), hydroxymethyl cellulose (HMC)), alginate, collagen, polymeric surfactants, polyethylene glycol, polyvinyl alcohol, polyurethane, polyvinylpyrrolidinone (PVP), fibrin (fibrinogen), or blood or tissue proteins. In certain embodiments, the second pharmacologically acceptable polymer is not polyethylene glycol (PEG). The barrier of the composition is believed to be in dynamic equilibrium with its individual components, at least one of which is also antimicrobial. Leaching of aggregates can be advantageous since the released material can increase the effective surface area of ​​the barrier, which can enhance the effectiveness of the interaction between microorganisms and the barrier material (Figures 17-19). The barrier properties of the material can be further enhanced by the synergistic effect of polar biopolymers that further crosslink the self-assembled copolypeptide matrix. By blending with different biopolymers, the flow and mechanical properties of the material, as well as the strength of the barrier, can be adjusted and controlled.

[0053] Example 2 Texture analysis profiles were used to determine the effect of block copolypeptide composition and hydrophobic enantiomeric purity on the mechanical properties of block copolypeptide / HEC blends (FIG. 20). Measurement of the firmness values ​​of the individual components revealed that K 100 L 40 A 1% (wt / wt) solution of HE in water has a stiffness of 2.94 + / - 0.25 mN. It was shown that the solution of C exhibited a stiffness of 5.38 + / - 0.32 mN (Figure 21 (Table 5)). 100 L 40Mixing together 1% (wt / wt) of diblock copolypeptide K and 1% HEC resulted in a large increase in stiffness to a value of 22.77 + / - 0.90 mN, corresponding to an interaction parameter value of 14.45 mN, an overall increase of over 170% compared to that expected from the additive contribution of the individual components. A similar trend was observed for adhesion, as shown in Figure 20b. For comparison, the diblock copolypeptide K at a concentration of 1% (wt / wt) 100 (rac‐L) 40 The combination of K (containing a racemic hydrophobic block) with HEC at a concentration of 1% (w / w) also showed improved firmness and adhesion, but the increase was greater with K (containing an enantiomerically pure hydrophobic block) 100 L 40 The effect was not as pronounced as in the case of 1% (wt / wt) K. The length of the hydrophobic block was also shown to have an effect. 100 L 20 The mixture of 1% (wt / wt) HEC and 1% (wt / wt) HEC showed increased stiffness compared to either biopolymer alone, but the effect was not significant. 100 L 40 Interestingly, K 100 L 20 had the opposite effect on the work of adhesion. 100 (rac‐L) 20 showed no effect at all. Lysine homopolypeptide, K 100 and K. 200 did not enhance the firmness or adhesiveness of HEC alone, and in fact appeared to exhibit the opposite activity.

[0054] Example 3 Rheological measurements revealed that K 100 L 40 Further support for the synergistic interaction between HEC and 1% (wt / wt) K is seen in both the oscillatory strain sweep and the frequency dependent sweep in Figure 22. 100 L 40exhibited the characteristics of relatively brittle and weak gels. In strain sweep analysis, the brittle nature of the gel was observed by the breakdown of the gel network at low strain rates near γ = 0.01; frequency sweep analysis showed the formation of a weak gel with elastic modulus (G' = 22 Pa at 1 rad / s). In comparison, 1% (wt / wt) HEC exhibited rheological properties more characteristic of a viscous fluid. Strain sweep analysis of the 1% HEC solution showed a higher loss modulus (G") than the elastic modulus (G') throughout all strain rates tested (Figure 21A), and frequency sweeps also showed higher G" values ​​than G' until higher frequencies were reached (approximately 100 rad / s) (Figure 21B). The 1% (wt / wt) K 100 L 40 Significant changes in the rheological properties were observed when blended with 1% (wt / wt) HEC. In particular, strain sweep analysis of the blends showed an order of magnitude extension of the linear viscoelastic region and a decrease in G' near γ = 0.1. Frequency sweeps showed a large synergistic increase in modulus, G' = 141 Pa at 1 rad / s, which was significantly larger than that of the 1% (wt / wt) K 100 L 40 This is a seven-fold increase over the elastic modulus alone (G' = 22 Pa).

[0055] The amount and type of polymeric components in the aqueous compositions described herein can be selected to achieve various properties. In an embodiment, the aqueous compositions are characterized by a barrier activity measured by a reduction in the diffusion rate of an anionic dye of more than 2 logs at a total polymer concentration of 40 mg / mL or less. In an embodiment, the aqueous compositions are characterized by a storage modulus of at least 50 Pa at a total polymer concentration of less than 40 mg / mL. In an embodiment, the aqueous compositions are characterized by a storage modulus of at least 50 Pa and the ability to pass through a 20 g needle with a pressure of less than 60 N at a total polymer concentration of less than 40 mg / mL. In an embodiment, the aqueous compositions are characterized by the ability to pass through a 20 g needle and the ability to recover at least 70% of its strength, as measured by storage modulus, within 10 minutes. A person skilled in the art can use routine experimentation guided by the teachings provided herein to select polymeric components and amounts to form an aqueous composition having the properties described herein.

[0056] Example 4 Mixtures of synthetic cationic polypeptides and other polymers can exhibit enhanced antibacterial activity in vivo. In particular, the synthetic cationic polypeptides can be used in micellar solutions and in As hydrogels, cationic peptides can be used to coat tissues in vivo (Figure 23). This tissue coating may involve the binding of cationic peptides to anionic charges presented on damaged tissues (as well as anionic charges in bacterial biofilms) through charge interactions. In addition, self-assembly and cross-linking may increase the amount of material that binds to and coats tissues, thus affecting various biological activities and responses.

[0057] As shown in FIG. 24, these synthetic cationic polypeptides are antimicrobial when administered locally in vivo. In the quest to develop improved products for in vivo administration, it was recognized that the tissue binding and tissue coating properties of these materials can greatly affect their activity, including their antimicrobial properties, their antibiofilm properties, and their effects on tissue adhesion formation and tissue remodeling. The molecular properties of the synthetic cationic polypeptides (e.g., length, charge, etc.), as well as the structures they form in aqueous environments (e.g., micelles, sheets, fibrils, hydrogels), can affect tissue binding and tissue coating. It was also recognized that by mixing these synthetic cationic polypeptides with other polymers to form the aqueous compositions described herein, their biophysical properties in aqueous media and on tissues can be altered in various ways. Thus, the inventors tested mixtures of synthetic cationic polypeptides and other polymers in vitro.

[0058] Example 5 Aqueous compositions containing a mixture of synthetic cationic polypeptides and two different polymers, polyethylene glycol 400 and hydroxyethyl cellulose, were both found to be effective in vivo. 100 (rac‐L) 20 was effective in a porcine open wound healing model, both alone and in combination with PEG400. 100 L 40was effective in preventing microbial contamination, both alone and in combination with hydroxyethylcellulose. Furthermore, the data suggest that the enhanced biophysical properties of the mixture may improve antimicrobial activity in vivo compared to the synthetic cationic copolypeptide alone. The aqueous compositions described herein may be used for any one or more treatments and / or administrations, including, but not limited to, prevention of infection, treatment of infection, topical anti-infective treatment, treatment for microbial decontamination, wound treatment, surgical site treatment, trauma treatment, burn treatment, treatment of diabetic foot ulcers, eye treatment, treatment of vaginal infection, treatment of urinary tract infection, hand disinfection, for coating prosthetic devices and / or implants, food preservation, and solution preservation.

[0059] The aqueous compositions described herein may be formulated as solutions, emulsions, particles, or hydrogels with various viscoelastic properties to enhance their antimicrobial properties, their barrier properties, or both. In one embodiment, the aqueous compositions described herein include wound cleansing products in which a single lysine-leucine block copolypeptide in water, saline, or other aqueous medium is mixed with a second polymer and a surfactant, usually poloxamer 407. In one embodiment, the aqueous compositions described herein may include viscous fluids / flowable gels that can be administered using a spray to cover various tissues. This can be used in open or laparoscopic procedures. These materials, alone or in combination with other materials, may be formed into various dressings or bandages. These may include constituting or coating various materials such as gauze or sponges. Examples include the aqueous compositions described herein (e.g., synthetic block copolypeptide KxLy-containing) in the form of a coating on gauze or alginate bandages. Another example includes a bilayer material in which an aqueous composition described herein (e.g., containing a synthetic block copolypeptide KxLy alone or with another polymer such as collagen) is coated onto the surface of a relatively inert sponge material (e.g., polyacrylate, polyurethane, or polyhema) (Figure 27). In this case, the embodiment may have the appearance of a rectangular sponge coated on one side, or a spherical sponge (reminiscent of a tennis ball) coated on all sides. This may be particularly advantageous for treating bleeding wounds where hemostasis is required (Figures 28-29).

[0060] An embodiment provides a method for preventing and / or treating infection, for example, in a wound, comprising contacting a tissue of a subject with the aqueous composition described herein. Another embodiment further comprises applying negative pressure to the treated wound. The subject may be an animal, preferably a human. In an embodiment, the subject is further treated systemically with an antibiotic, and / or with another antimicrobial agent, and / or at least one selected from the group consisting of an antibiotic, an antibiofilm agent, a surfactant, and combinations thereof.

[0061] The aqueous compositions described herein may further comprise one or more pharmacologically active ingredients (APIs), examples of which include steroids, pro-inflammatory agents, anti-inflammatory agents, anti-acne agents, preservatives, hemostatic agents, angiogenic agents, wound healing agents, anti-cancer agents, and other antimicrobial agents.

Claims

1. 1. A dressing for treating a wound site capable of biofilm formation, comprising: The dressing comprises a composition, said composition comprising one or more synthetic polypeptides having a chain length of at least 40 amino acid residues and having repeating segments of positively charged amino acid residues and other repeating hydrophobic amino acid residues; the one or more synthetic polypeptides are cationic at neutral pH; the one or more synthetic polypeptides form multimers in solution; the one or more synthetic polypeptides being capable of inhibiting or killing microorganisms in a biofilm; Dressing material.

2. The dressing of claim 1 , further comprising gauze or a sponge.

3. 3. The dressing of claim 1 or 2, wherein the wound site is selected from the group consisting of acute wounds, chronic wounds, and devitalized tissue.

4. The dressing material according to any one of claims 1 to 3, wherein the composition has procoagulant or prohemostatic activity.

5. The dressing material according to any one of claims 1 to 4, wherein the composition further comprises a pharmacologically acceptable polymer which is not a synthetic cationic polypeptide.

6. 6. The dressing of claim 5, wherein the pharmacologically acceptable polymer is selected from the group consisting of cellulose, alginate, collagen, polymeric surfactants, polyethylene glycol, polyvinyl alcohol, polyurethane, polyvinylpyrrolidinone (PVP), fibrin (fibrinogen), blood proteins, and tissue proteins.

7. The dressing of claim 6, wherein the pharmacologically acceptable polymer is cellulose.

8. 8. The dressing material according to claim 1, wherein the synthetic polypeptide is characterized by at least one segment having at least five consecutive cationic amino acid residues and at least one segment having at least five consecutive hydrophobic amino acid residues.

9. A dressing according to any one of claims 1 to 8, wherein one or more of the synthetic polypeptides comprises a segment having a chain length of between 50 and 200 lysine amino acid residues.

10. 10. The dressing of any one of claims 1 to 9, wherein the antimicrobial activity is greater than 3 log killing of Staphylococcus epidermidis and Escherichia coli in a standard 60 minute time-kill assay at a concentration of synthetic cationic polypeptide of 1 mg / mL or less.

11. A dressing according to any one of claims 5 to 10, wherein the amount of said pharmacologically acceptable polymer is at least 30% by weight relative to the weight of said one or more synthetic polypeptides.

12. A dressing according to any one of claims 5 to 11, wherein the synthetic polypeptide and the pharmacologically acceptable polymer are miscible with each other in water.

13. 13. The dressing of any one of claims 1 to 12, wherein the antimicrobial activity of the one or more synthetic polypeptides at a concentration of up to 100 μg / mL in an aqueous composition is greater than 3 log killing of Staphylococcus epidermidis and Escherichia coli in a standard 60 minute time-kill assay.

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