Compositions and uses for topically applied antimicrobial synthetic cationic polypeptides with enhanced efficacy and safety - Patents.com

Abstract

Antibacterial pharmaceutical compositions are provided that have a low risk of local tissue toxicity and / or a low risk of systemic / remote organ toxicity. The antimicrobial pharmaceutical compositions are formulated to allow for local application in vivo at doses that provide antimicrobial efficacy with low risk of local tissue toxicity and / or low risk of systemic / remote organ toxicity. In various embodiments, the antimicrobial pharmaceutical compositions comprise an antimicrobial synthetic cationic polypeptide dispersed in an aqueous carrier and formulated to achieve a desired degree of polymer self-assembly and / or viscosity of the composition.

Description

[Technical field]

[0001] <Related application information> This application claims priority to U.S. Provisional Patent Application No. 62 / 482,630, filed April 6, 2017, which is incorporated by reference in its entirety.

[0002] This disclosure relates to antimicrobial pharmaceutical compositions comprising cationic antimicrobial agents and methods of using them to prevent and / or treat infection. [Background technology]

[0003] Topically applied antimicrobial agents, including antiseptics and antibiotics, often fail to achieve efficacy in preventing and treating infection, resulting in significant patient morbidity and mortality. Specific uses (including application to specific tissues) and doses (concentration, volume, number of applications) may be limited by one or more toxicity risks. Concerns regarding such toxicities may lead to clinical application of insufficient doses in certain pathophysiological situations or to avoid use altogether.

[0004] A wide variety of cationic antimicrobial agents are known for their ability to bind and disrupt bacterial membranes, including certain antibiotics, bisbiguanides, polymeric biguanides, quaternary ammonium compounds, natural antimicrobial peptides, and synthetic cationic polypeptides. However, mammalian toxicity has consistently remained a problem.

[0005] No. 9,017,730 describes synthetic cationic copolypeptides containing various ratios of cationic amino acid repeat units (such as lysine (K)) and hydrophobic amino acid units (such as leucine (L), isoleucine (I), valine (V), phenylalanine (F) or alanine (A)). No. 9,017,730 shows that segmented or block structures containing stretches of cationic amino acids and stretches of hydrophobic amino acids can improve compatibility with mammalian cells as measured in in vitro cytotoxicity assays. Furthermore, No. 9,017,730 shows that incorporation of selected cationic block copolypeptides into emulsions can improve compatibility with mammalian cells in vitro as measured in cytotoxicity assays. Although No. 9,017,730 describes topical application of a number of synthetic cationic copolypeptide formulations, it does not provide evidence of toxicity that may result from using these products in vivo.

[0006] US Patent No. 9,446,090 describes synthetic cationic polypeptides together with mutually water-miscible mixtures comprising such polypeptides and a second pharmacologically acceptable polymer. Specific examples describe antibacterial activity against certain bacteria using specific mixtures of synthetic cationic polypeptides and a second polymer, such as polyethylene glycol (PEG), hydroxyethyl cellulose (HEC) and poloxamer 407. Although the second polymer alone was not known to have significant antibacterial activity, data showed that the addition of the second polymer in the mixture maintained the antibacterial activity of the synthetic cationic polypeptide in vitro and in some cases enhanced the overall antibacterial potential in vivo. However, US Patent No. 9,446,090 does not provide evidence of toxicity that may result from the use of these mixtures of synthetic cationic polypeptides and other pharmacologically acceptable polymers in vivo. Moreover, it leaves open the possibility that the second polymer may increase the toxicity of the mixture compared to the synthetic cationic polypeptide.

[0007] Although U.S. Patent Nos. 9,017,730 and 9,446,090 describe significant advances in the prior art, many challenges remain, particularly with regard to the development of pharmacologically acceptable preparations of topically applied cationic antimicrobial agents that have both high efficacy and high safety in vivo. Summary of the Invention

[0008] We have developed cationic antimicrobial pharmaceutical compositions and methods of use that allow for local application in vivo at doses that provide antimicrobial efficacy with low risk of local tissue toxicity and / or low risk of systemic / remote organ toxicity.

[0009] In developing the present invention, we have found that toxicity risk following topical application of cationic antimicrobials in vivo is particularly relevant when applied in large quantities to tissue sites or in pathophysiological situations other than healthy, intact skin. Such sites and pathophysiological situations may include open wounds, body cavities, body orifices, diseased skin, and others. These tissue sites and pathophysiological situations may demonstrate greater local susceptibility and / or greater absorption and systemic distribution of the topically applied antimicrobial or excipients compared to healthy, intact skin. Greater local tissue toxicity and greater systemic / remote organ toxicity may ensue.

[0010] Furthermore, we have made a series of discoveries that have helped guide the development of the present invention. First, we have discovered that altering the material design of synthetic cationic polypeptides can produce different effects on in vivo antimicrobial efficacy and toxicity; second, we have discovered that altering the formulation of synthetic cationic polypeptides can produce different effects on in vivo antimicrobial efficacy and toxicity; third, we have discovered that sterilizing synthetic cationic polypeptide products can adversely affect molecular integrity and biophysical properties, thus affecting antimicrobial efficacy, toxicity, or both. Thus, we have discovered a combination of problems for which a solution has been found.

[0011] In various embodiments, pharmaceutical compositions and methods of use are provided that allow for local application of effective amounts of antimicrobial synthetic cationic polypeptides to various tissue sites and / or in various pathophysiological conditions with low risk of local and / or systemic toxicity. Without limiting the scope of the invention, in various embodiments, this is accomplished by one or more of (A), (B) and (C) as follows:

[0012] (A) Design of antibacterial synthetic cationic polypeptides, particularly the hydrophobic content and sequence arrangement, to promote increased viscosity in water. Unexpectedly, this design feature was found to enhance in vivo safety.

[0013] (B) Formulating antimicrobial synthetic cationic polypeptides into pharmaceutical compositions in such a way that they can exhibit viscosity enhancing properties. Without limiting the scope of the invention, this approach is believed to have the effect of improving tissue coverage and / or enhancing retention time, resulting in greater efficacy for the dose applied. Furthermore, such improved formulations have been found to unexpectedly enhance safety. Without limiting the scope of the invention, excipients include sodium chloride, potassium chloride, sugars and sugar alcohols such as glucose, mannitol, glycerol, xylitol and sorbitol, and surfactants and combinations thereof.

[0014] (C) sterilizing the pharmaceutical composition in such a manner that the sterilized pharmaceutical composition exhibits a viscosity equivalent to that of a non-sterile pharmaceutical composition. Without limiting the scope of the present invention, this technique is intended to provide a method for determining the molecular structure and function of the antimicrobial synthetic cationic polypeptides. It is believed to have the effect of protecting, as well as reducing the risk that the product will become contaminated by certain microbial organisms (eg, fungal dermatophytes) that exhibit resistance to the effects of the antimicrobial synthetic cationic polypeptides.

[0015] An embodiment is an antibacterial pharmaceutical composition comprising an aqueous carrier; and an antibacterial synthetic cationic polypeptide dispersed in the aqueous carrier at a concentration of about 0.01-5% by weight based on the total weight of the antibacterial pharmaceutical composition. In an embodiment, the antibacterial synthetic cationic polypeptide comprises a plurality of neutral pH positively charged amino acid units. In an embodiment, the antibacterial synthetic cationic polypeptide has a viscosity of 2 centistokes (cSt) or greater at 37° C. at a concentration of 2% by weight in deionized water. In an embodiment, the aqueous carrier comprising 2% by weight of the antibacterial synthetic cationic polypeptide has a viscosity at 37° C. that is greater than the viscosity of an aqueous carrier comprising 2% by weight of albumin in place of the antibacterial synthetic cationic polypeptide. In an embodiment, the aqueous carrier comprising 2% by weight of the antibacterial synthetic cationic polypeptide has a viscosity at 37° C. that is at least about 20% greater than the viscosity of an aqueous carrier comprising 2% by weight of albumin in place of the antibacterial synthetic cationic polypeptide. In an embodiment, an aqueous carrier comprising 2% by weight of the antimicrobial synthetic cationic polypeptide has a viscosity at 37° C. that is at least about 50% greater than the viscosity of an aqueous carrier comprising 2% by weight of albumin in place of the antimicrobial synthetic cationic polypeptide. In an embodiment, an aqueous carrier comprising 2% by weight of the antimicrobial synthetic cationic polypeptide has a viscosity at 37° C. that is at least about 50% greater than the viscosity of an aqueous carrier comprising 2% by weight of albumin in place of the antimicrobial synthetic cationic polypeptide. In an embodiment, the aqueous carrier comprising 2% by weight of the antimicrobial synthetic cationic polypeptide has a viscosity at 37° C. that is at least about 100% greater than the viscosity of the aqueous carrier comprising 2% by weight of albumin in place of the antimicrobial synthetic cationic polypeptide. In an embodiment, the antimicrobial pharmaceutical composition has low toxicity as measured by 50% or greater mouse survival at 72 hours after being injected intraperitoneally into healthy, young adult mice at a dose of 10 mL / kg.

[0016] Another embodiment provides a method of preventing microbial contamination of intact, healthy tissue other than skin, comprising: identifying a mammalian subject having an intact, healthy tissue site other than skin that is at risk for microbial contamination; and administering to the site an antimicrobial pharmaceutical composition described herein in an amount effective to at least partially protect the tissue site from microbial contamination.

[0017] Other embodiments provide a method of reducing microbial load in or on tissue other than intact, healthy skin, the method comprising: identifying a mammalian subject having an intact, healthy tissue site other than skin that has a microbial load; and administering to the tissue site an antimicrobial pharmaceutical composition described herein in an amount effective to at least partially reduce the microbial load.

[0018] These and other embodiments are described in more detail below. [Brief description of the drawings]

[0019] [Figure 1] Schematic representation of synthetic cationic polypeptides having various amino acid sequence arrangements. Particular arrangements may be referred to as blocks or segments. [Diagram 2] Synthetic cationic polypeptides are classified into three groups based on the length (number of amino acid units) of the cationic segment. [Diagram 3] Examples of synthetic cationic polypeptides are classified into groups (I) long cationic segment (200 or more units) polypeptides, (II) medium cationic segment (100-199 units) polypeptides, and (III) short cationic segment (10-99 units) polypeptides. A cationic segment comprises multiple cationic amino acid units, but does not have to be entirely composed of cationic amino acid units. A hydrophobic segment comprises multiple hydrophobic amino acid units, but does not have to be entirely composed of hydrophobic amino acid units. [Figure 4]Example synthetic cationic polypeptide having a cationic-hydrophobic block sequence arrangement based on lysine and enantiopure L-leucine amino acid units. The average overall chain length of the polypeptide preparation is approximately 160 amino acid units. The ratio of lysine to leucine, or K:L ratio, was found to be 3.2. The synthetic cationic polypeptide is designated as KL-160 / 3.2. Analysis using size exclusion chromatography shows two overlapping curves based on two injections. Dispersity (D) = weight average molecular weight (Mw) / number average molecular weight (Mn). [Diagram 5] Antibacterial assay of an example group 2 (middle cationic segment) synthetic cationic polypeptide having an enantiopure lysine-leucine block structure and an overall chain length of approximately 160 with a K:L ratio of 3.2. The synthetic cationic polypeptide is designated KL-160 / 3.2. A 60 minute in vitro time-kill assay was used to determine the bactericidal activity (log CFU reduction) at different sample concentrations from 1.6 μg / mL to 100 μg / mL (Lot BAC003) against Staphylococcus aureus (S. aureus) (ATCC6538), Pseudomonas aeruginosa (P. aeruginosa) (ATCC27853) and Candida albicans (C. albicans) (ATCC24433). [Figure 6] Summary of multiple antimicrobial time-kill assays with synthetic cationic polypeptide KL-160 / 3.2 against various microorganisms. 60-minute time-kill assays were performed with synthetic cationic polypeptide (Lot BAC003) at a concentration of 100 μg / mL. The term "100%" CFU reduction indicates that no microorganisms were detected. MDR = multidrug resistant; ESBL = extended-spectrum β-lactamase; KPC = K. pneumoniae carbapenemase. Bold = Centers for Disease Control and Prevention (CDC) "greatest threat; *Clinical isolates, RM Alden Research Laboratory. [Figure 7]An example synthetic cationic polypeptide having a cationic-hydrophobic block sequence arrangement based on lysine and racemic D,L-leucine amino acid units. The overall average chain length of the polypeptide preparation is about 100 amino acid units. The ratio of lysine to leucine, or K:L ratio, was found to be 5.7. The synthetic cationic polypeptide is designated KrL-100 / 5.7. Analysis using size exclusion chromatography shows two overlapping curves based on two injections. Dispersity (D) = weight average molecular weight (Mw) / number average molecular weight (Mn). [Figure 8] Antibacterial assay of an example Group 3 (short cationic segment) synthetic cationic polypeptide having a lysine-racemic leucine block structure and an overall chain length of approximately 100 with a K:(rac-L) ratio of 5.7. The molecule is designated KrL-100 / 5.7. Bactericidal activity (log CFU reduction) was determined at different sample concentrations from 1.6 μg / mL to 100 μg / mL (Lot BAC002) against important pathogens using a 60 minute in vitro time-kill assay. [Figure 9] Summary of multiple antimicrobial time-kill assays with synthetic cationic polypeptide KrL-100 / 5.7 against various microorganisms. 60-minute time-kill assays were performed with synthetic cationic polypeptide (Lot BAC002) at a concentration of 100 μg / mL. The term "100%" CFU reduction indicates that no microorganisms were detected. MDR = multidrug resistant; ESBL = extended-spectrum β-lactamase; KPC = K. pneumoniae carbapenemase. Bold = Centers for Disease Control and Prevention (CDC) "greatest threat; *Clinical isolates, RM Alden Research Laboratory. [Figure 10] Synthetic cationic polypeptides with block sequence arrangements can be designed and formulated to self-assemble into multimeric structures. Two examples are depicted here: KL-160 / 3.2 was designed to self-assemble into fibrous structures to form barrier hydrogels, and KrL-100 / 5.7 was designed to self-assemble into micellar structures with surfactant properties. [Figure 11]Critical aggregation concentrations (CAC) of various synthetic cationic polypeptides as determined by pyrene fluorescence. [Figure 12] Critical aggregation concentration (CAC) of the synthetic cationic polypeptide KrL-100 / 5.7 (Lot BAC002) as measured by the surface tension method. Grey depicts the surface tension across concentrations, black depicts the best-fit line for the two sets of values. The CAC was determined in this assay by the tolerance of the two best-fit lines, and is shown here as ~100 μg / mL. [Figure 13] (a) Dynamic viscosity of various synthetic cationic polypeptides at 1 wt. % in water measured using a glass capillary viscometer (Ubbelohde viscometer) at 37° C. (b) Dynamic viscosity of KrL-100 / 5.7 (lot BAC002) versus bovine serum albumin; water is provided as a reference. The data show that albumin at these concentrations has little effect on the viscosity of the aqueous formulation. [Figure 14] Kinematic viscosity of a synthetic cationic polypeptide having a lysine-L-leucine block sequence arrangement (KL) at 0.5 wt % in water measured using a glass capillary viscometer (Ubbelohde viscometer) at 37°C. [Figure 15] Kinematic viscosity of various synthetic cationic polypeptides with lysine-L-leucine (KL) and lysine-D,L-leucine (KrL) block sequence arrangements at 1% and 2% by weight in water measured using a glass capillary viscometer (Ubbelohde viscometer) at 40°C. [Figure 16] Viscosity of the synthetic cationic polypeptide KL-120 / 2.5 at 1.5 wt%, 2.0 wt%, and 3.0 wt% in water evaluated against increasing shear forces. Measured at room temperature for 2 minutes using a Brookfield RVDV (EQ-AL-2014-16) rotational viscometer with a size 14 spindle and a size 6R chamber. [Figure 17]An example synthetic cationic polypeptide, KL-120 / 2.5, forms viscous solutions and hydrogels in water. KL-120 / 2.5 was prepared at concentrations of 0.5 wt%, 1.0 wt%, 1.5 wt%, 2.0 wt%, and 3.0 wt% in DI water and evaluated for gel formation by tilt tube assay, hardness by texture analysis, and viscosity. (a) Visual gelation test by tilt tube assay. (b) Concentration-dependent hardness in water measured by texture analysis. Hardness values ​​were obtained at a probe depth of 8 mm. The inset image depicts KL-120 / 2.5 at 2 wt% in water applied to an artificial skin substrate (VITRO-SKIN; IMS Inc.). (c) Graphical representation showing the change in physical properties as the concentration of KL-120 / 2.5 increases (white = fluid; black = firm gel); based on data from the viscometer for 0.5 wt% and 1.0 wt% and data from texture analysis and tilt tube assay for higher concentrations. (d) KL-120 / 2.5 at 1.5 wt % in water resists penetration by two different stainless steel balls (BB). [Figure 18] A partial list of pharmacologically acceptable non-ionic excipients (additives) that may be used in the preparation of synthetic cationic polypeptides and antibacterial pharmaceutical compositions containing them. [Figure 19] Viscosity of the synthetic cationic polypeptide KL-120 / 2.5 (BAC004) at 2.0 wt % in water, 4.5% mannitol in water, 2.33% glycerol in water, or 2.8% histidine in water, evaluated against increasing shear forces, measured for 2 min at room temperature using a Brookfield RVDV (EQ-AL-2014-16) rotational viscometer with a size 14 spindle and a size 6R chamber. [Figure 20] Kinematic viscosity of two example synthetic cationic polypeptides with lysine-L-leucine (KL) and lysine-D,L-leucine (KrL) block sequence configurations prepared at 1 wt % in water, in water alone, in 0.9% saline, or in 4.4% aqueous xylitol solution, measured using a glass capillary viscometer (Ubbelohde viscometer) at 40 °C. [Figure 21]Viscosity of a formulation of synthetic cationic polypeptide KL-120 / 2.5 (BAC004) at 4.5% mannitol and 2.0 wt% in water evaluated against increasing shear forces. Measured at room temperature for 2 minutes using a Brookfield RVDV (EQ-AL-2014-16) rotational viscometer with a size 14 spindle and a size 6R chamber. [Figure 22] The example synthetic cationic polypeptide KrL-120 / 5.0 is shown to have reduced surface tension compared to two other cationic antibacterial agents, chlorhexidine gluconate (CHG) and polyhexamethylene biguanide (PHMB). Samples were prepared at 0.1 mM and evaluated at room temperature using a TA.XT2 Texture Analyzer with a 500 g load cell, a 5 cm diameter Du Nouy ring, and a probe speed of 0.2 mm / s. [Figure 23] Surface tension of various formulations of synthetic cationic polypeptides in water. Samples were prepared at 0.1 mM and evaluated at room temperature using a TA.XT2 Texture Analyzer with a 500 g load cell, a Du Nouy ring of 5 cm diameter and a probe speed of 0.2 mm / s. [Figure 24] Interfacial activity properties of KrL-100 / 5.7. (a) Surface tension of aqueous KrL-100 / 5.7 (lot BAC002) 10 mg / mL (1 wt %) compared to water using a tensiometer equipped with a Du Nouy ring. (b) Interfacial tension of KrL-100 / 5.7 at 1 wt % in water with n-hexane over a 10 min period. [Diagram 25] Surface tension: effect of acetic acid as an additive. The example synthetic cationic polypeptides KrL-130 / 3.3 (lot 77) and KL-130 / 3.3 (lot 72) reduce surface tension when formulated in water or in water with 0.25% or 1.0% acetic acid. The additive of acetic acid has a synergistic effect. [Figure 26]Interfacial tension: effect of acetic acid as an additive. The example synthetic cationic polypeptides KrL-130 / 3.3 (lot 77) and KL-130 / 3.3 (lot 72) have reduced interfacial tension when formulated in water or in water with 0.25% or 1.0% acetic acid. The additive of acetic acid has a synergistic effect. [Figure 27] Emulsifying properties. Example synthetic cationic polypeptides KrL-100 / 5.0 and KrL-160 / 3.2 in water form stable emulsions when mixed with soybean oil. (a) Photograph of the assay demonstrating the emulsion / liquid interface (white arrow); b) Emulsion index (E24; % emulsion at 24 hours). [Figure 28] Emulsifying Properties in Water, Saline, and Xylitol. Example synthetic cationic polypeptides KrL-110 / 4.0 (lot D-301-37-05) and KL-100 / 5.7 (lot D-301-67-03) formulated in water, saline (0.9%), or xylitol (4.4%) form stable emulsions when mixed with soybean oil. [Figure 29] Evaluation of Rabbit Skin Irritation Model Results. Negligible responses to intact and abraded skin were observed following application of various formulations and concentrations of synthetic cationic polypeptides KL-140 / 2.5 or KrL-120 / 5.0. Each rabbit (New Zealand White, N=3 per test article) had a total of eight sites: two intact controls, two intact test articles, two abraded controls, and two abraded test articles. Each site was approximately 2.5×2.5 cm and received 0.5 mL of test article for 24 hours. Sites were scored for erythema, edema, and eschar formation at 1, 24, 48, and 72 hours. [Diagram 30]Evaluation of Guinea Pig Skin Sensitization Results. No visual changes were observed after application of KL-140 / 2.5 (Lot 73) or KrL-120 / 5.0 (Lot 93) at 1% by weight in water. Hartley albino guinea pigs (n=11 for test group, n=6 for positive control, n=6 for negative control) were exposed to 0.3 mL of test article for 6 hours / day, 3 consecutive days per week for 3 weeks. The challenge exposure was performed 14±1 days after the last lead-in exposure. Application sites were scored 24 and 48 hours after removal of the challenge patch. [Diagram 31] Evaluation of oral toxicity results in rats. No abnormalities were observed after KL-140 / 2.5 in water or KrL-120 / 5.0 in water was administered to rats by oral gavage at doses of 0.625 mg / kg to 160 mg / kg. 2 mL was administered per male juvenile Sprague-Dawley rat (N=5 per group). Clinical observations were performed for 3 days. [Diagram 32] Evaluation of mouse systemic toxicity after intraperitoneal administration. CD-1 mice received intraperitoneal administration of 40 mL / kg of water or test articles K-100, KL-140 / 2.5-RAN, or KrL-120 / 5.0 diluted to 2% by weight in water. Final dose per animal was 800 mg / kg. N=5 CD-1 mice per group. Clinical observations were performed for 3 days. [Diagram 33] Evaluation of systemic toxicity in mice after intraperitoneal administration. CD-1 mice received intraperitoneal administration of 40 mL / kg of saline or KrL-130 / 3.3 diluted in water at various concentrations. Final doses per animal ranged from 12.5 mg / kg to 800 mg / kg. N=5 CD-1 mice per group. Clinical observations were performed for 3 days. [Diagram 34] Evaluation of systemic toxicity in mice after intraperitoneal administration. CD-1 mice received intraperitoneal administration of 40 mL / kg of water or KrL-120 / 5.0 Lot 93 or Lot 94 diluted in water at various concentrations. Final doses per animal ranged from 50 mg / kg to 800 mg / kg. N=5 CD-1 mice per group. Clinical observations were performed for 3 days. [Diagram 35]Evaluation of systemic toxicity in mice after intraperitoneal administration. CD-1 mice received intraperitoneal administration of 40 mL / kg of water or KL-170 / 3.3 or KL-140 / 2.5 diluted in water at various concentrations. The final dose per animal was 6.25 mg / kg to 400 mg / kg. N=5 CD-1 mice per group. Clinical observations were performed for 3 days. [Diagram 36] Evaluation of mouse systemic toxicity after intraperitoneal administration. CD-1 mice received intraperitoneal administration of 40 mL / kg of water or KL-140 / 2.5 in various formulations and concentrations. The final dose per animal ranged from 1.25 mg / kg to 400 mg / kg. N=5 CD-1 mice per group. Clinical observations were performed for 3 days. [Figure 37] Evaluation of mouse systemic toxicity after intraperitoneal administration. CD-1 mice received intraperitoneal administration of 40 mL / kg of water or KrL-120 / 5.0 in various formulations and at multiple concentrations. Final doses per animal ranged from 50 mg / kg to 800 mg / kg. N=5 CD-1 mice per group. Clinical observations were performed for 3 days. [Figure 38] SEC chromatograms of KL-140 / 2.5 (Lot 73) before and after gamma sterilization at doses of 25-40 kGy. The data indicate that the molecular structure degrades after gamma irradiation. [Figure 39] SEC chromatograms of KL-120 / 5.0 (Lot 94) before and after gamma sterilization at doses of 25-40 kGy. The data indicate that the molecular structure degrades after gamma irradiation. [Diagram 40] SEC chromatograms of KL-160 / 3.2 (Lot BAC003) before and after e-beam sterilization at a dose of 25 kGy. The data shows that the molecular structure is degraded after e-beam sterilization. [Diagram 41]Texture analysis of two KL-140 / 2.5 lots (73 and 95) before and after gamma sterilization at doses of 25-40 kGy. Measured using a TA.XT2 Texture Analyzer with a probe speed of 0.5 mm / s. The data shows that the hardness of many of these compositions is reduced after gamma irradiation. [Diagram 42] SEC chromatograms of KL-140 / 2.5 (Lot 73) prepared in water before and after autoclaving. The data shows that molecular structure is maintained after autoclaving. [Diagram 43] SEC chromatograms of KL-120 / 5.0 (Lot 94) prepared in water before and after autoclaving. The data shows that molecular structure is maintained after autoclaving. [Diagram 44] SEC chromatograms of KL-160 / 3.2 (Lot BAC003) prepared at 2 wt% in water before and after autoclaving. The data shows that molecular structure is maintained after autoclaving. [Diagram 45] SEC chromatograms of KL-160 / 3.2 (Lot BAC003) prepared at 2% by weight in water with 2% propylene glycol before sterilization, after autoclaving, and after sterile filtration. The data shows that molecular structure is maintained after autoclaving and sterile filtration. [Figure 46] SEC chromatograms of KL-160 / 3.2 (Lot BAC003) prepared at 2% by weight in water with 2.33% glycerol before sterilization, after autoclaving, and after sterile filtration. The data shows that molecular structure is maintained after autoclaving and sterile filtration. [Figure 47]Viscosity of synthetic cationic polypeptide KL-120 / 2.5 (Lot BAC004) at 2.0 wt % in water with 2.1% propylene glycol before and after filter sterilization, measured at room temperature for 2 minutes using a Brookfield RVDV (EQ-AL-2014-16) rotational viscometer with a size 21 spindle and a size 13R chamber. [Figure 48] Viscosity of synthetic cationic polypeptide KL-160 / 3.2 (Lot BAC003) at 2.0 wt% in water before and after autoclaving, measured using a Brookfield RVDV (EQ-AL-2014-16) rotational viscometer for 2 minutes. [Figure 49] Viscosity of synthetic cationic polypeptide KL-160 / 3.2 (Lot BAC003) at 2.0 wt% in water with 2.33% glycerol before and after autoclaving. Measured for 2 minutes using a Brookfield RVDV (EQ-AL-2014-16) rotational viscometer. [Figure 50] Texture analysis of two lots of synthetic cationic polypeptide KL-140 / 2.5 (98 and 99) at 1 and 2 wt% in water with and without 1% hydroxyethylcellulose (HEC) before and after autoclaving. Measured using a TA.XT2 Texture Analyzer with a probe speed of 2 mm / s. The data shows that the favorable characteristic firmness is maintained after autoclaving. Some overall decrease in firmness is observed in the high concentration formulations and in formulations formulated with HEC. [Figure 51] Texture analysis of synthetic cationic polypeptide KL-140 / 2.5 (Lot 99) at 1 wt% in water with 1 wt% hydroxypropyl cellulose (HEC), hydroxypropyl methylcellulose (HPMC) or methylcellulose (MC) before and after autoclaving. Measured using a TA.XT2 Texture Analyzer with a probe speed of 2 mm / s. [Figure 52]Reduction in surface tension by a preparation of synthetic cationic polypeptide KrL-120 / 5.0 (Lot 94) in water before and after autoclaving. Samples were prepared at 0.1 mM and evaluated at room temperature using a TA.XT2 Texture Analyzer with a 500 g load cell, a 5 cm diameter Du Nouy ring and a probe speed of 0.2 mm / s. [Figure 53] Diagrammatic depiction of wound classification. [Figure 54] Results of pre-inoculation of ex vivo porcine skin models. The antimicrobial barrier properties of KL-120 / 2.5 (Lot BAC003) and KL-160 / 3.2 (Lot BAC004) formulations were demonstrated in ex vivo porcine skin. Data show log CFU of viable P. aeruginosa 3 hours after inoculation of skin grafts pretreated for 30 minutes with water (control; N=8) or pretreated with (a) 0.5-2.0 wt% KL-120 / 2.5 (N=8) or (b) 0.5-2.0 wt% KL-160 / 3.2 (N=8). In the "not tilted" group, the grafts remained horizontal throughout pretreatment with the synthetic cationic polypeptide formulation; in the "tilted" group, the grafts were tilted 90° to a vertical position for 15 minutes to encourage runoff of the synthetic cationic polypeptide formulation coating prior to inoculation. *No microorganisms were detected. Error bars depict SEM. [Figure 55] Results of pretreatment of microbial inoculation in a porcine open wound model. Formulations of the synthetic cationic polypeptide KL-160 / 3.2 (Lot BAC003) prevented microbial contamination in a porcine model (N=5 wounds; 4 for "NI"). a) S. epidermidis b) P. aeruginosa. NI=no inoculation. Full thickness wounds were pretreated with the depicted concentrations of KL-160 / 3.2 or 1.0 mL water 15 min prior to inoculation with a mixed culture of S. epidermidis and P. aeruginosa. Total microbial counts as well as selective S. epidermidis and P. aeruginosa counts were assessed 4 h later. In all cases, differences between control and KL-160 / 3.2 were significant at p<0.01. *No microorganisms were detected. [Figure 56]Pretreatment results of microbial inoculation in a porcine open wound model. KL-160 / 3.2 (Lot BAC003) prevented microbial contamination in a porcine model (N=4 wounds per group). a) S. epidermidis b) P. aeruginosa. Full thickness wounds were pretreated with the depicted concentrations of KL-160 / 3.2 or 1.0 mL of water 24 h, 4 h or 1 h prior to inoculation with a mixed culture of S. epidermidis and P. aeruginosa. Total microbial counts as well as selective S. epidermidis and P. aeruginosa counts were assessed 4 h after inoculation. [Figure 57] Results of pre-treatment of microbial inoculation in a rodent closed wound model with surgical mesh. KL-160 / 3.2 (Lot BAC003) (1% by weight, in water) shows activity against (a) MRSA (ATCC33593) and (b) P. aeruginosa (ATCC27317) in a rodent closed wound model with different bodies (KL-160 / 3.2 N=6; control N=8; Sprague-Dawley rats). Indicated Log CFU per gram tissue of biopsy samples and per implanted polypropylene mesh. KL-160 / 3.2 formulations were applied 15 min prior to inoculation with microorganisms; microbial load was assessed 48 h later. Differences between control and KL-160 / 3.2 groups for both microorganisms were significant at p<0.0001. *No microorganisms were detected. [Figure 58] Anti-biofilm activity. Minimum biofilm disinfecting concentration (MBEC) assay of synthetic cationic polypeptide KrL-100 / 5.7 (Lot BAC002). Activity against P. aeruginosa (ATCCBAA-47) in biofilms. P. aeruginosa biofilms were exposed to different concentrations of synthetic cationic polypeptide (0.001%, 0.01%, 0.1% and 1.0% in water) for 20 minutes or 3 hours and samples were processed for evaluation of colony forming units (CFU). *No microorganisms were detected. [Figure 59]Anti-biofilm activity. Minimum biofilm disinfecting concentration (MBEC) assay of synthetic cationic polypeptide KrL-100 / 5.7 (Lot BAC002) and KL-100 / 5.7 (Lot D-301-67-03) formulations. Activity against P. aeruginosa (ATCC BAA-47) in biofilms. P. aeruginosa biofilms were exposed to different concentrations of synthetic cationic polypeptide formulations (0.01%, 0.1% and 1.0%) in water for 3 hours and samples were processed for evaluation of colony forming units (CFU). [Figure 60] Results of pre-treatment of microbial inoculation in a rodent closed wound model with surgical mesh. Formulations of the synthetic cationic polypeptide KrL-100 / 5.7 (Lot BAC002) show activity against MRSA (ATCC33593) in a rodent closed wound model with different bodies (KrL-100 / 5.7 N=6; control N=8; Sprague-Dawley rats). Indicated Log CFU per gram tissue of biopsy samples and per implanted polypropylene mesh. KrL-100 / 5.7 formulations (0.01%, 0.1% and 1.0%) were applied 15 min after inoculation with microorganisms; microbial load was assessed 48 h later. Data are expressed as mean + SEM. *No microorganisms were detected. [Figure 61] Pre-treatment results of microbial inoculation in a rodent closed wound model with surgical mesh. (a) Histopathology of tissue biopsies and mesh inoculated with MRSA. Samples were processed for histology and stained with H&E. (b) Inflammation scores were determined by microscopic analysis by an experienced veterinary pathologist. [Figure 62]Results of pre-treatment microbial inoculation of a rodent closed wound model with surgical mesh. Formulations of the synthetic cationic polypeptide KrL-100 / 5.7 (Lot BAC002) show activity against P. aeruginosa (ATCC 27317) in a rodent closed wound model with different bodies (KrL-100 / 5.7 N=6; control N=8; Sprague-Dawley rats). Indicated Log CFU survival per gram tissue of biopsy samples and per implanted polypropylene mesh. KrL-100 / 5.7 formulations (0.01%, 0.1% and 1.0%) were applied 15 min after inoculation with microorganisms; microbial load was assessed 48 h later. Data are expressed as mean + SEM. *No microorganisms were detected. [Figure 63] Pre-treatment results of microbial inoculation in a rodent closed wound model with surgical mesh. (a) Histopathology of tissue biopsies and mesh inoculated with P. aeruginosa. Samples were processed for histology and stained with H&E. (b) Inflammation scores were determined by microscopic analysis by an experienced veterinary pathologist. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] definition As used herein in the context of describing antimicrobial synthetic cationic polypeptides, the term "antimicrobial" has its ordinary meaning as understood by one of ordinary skill in the art, and thus includes polypeptides that exhibit bactericidal activity as measured by a 60 minute time-kill assay against at least one bacterium selected from the group consisting of S. aureus, S. epidermidis, P. aeruginosa, and E. coli.

[0021] As used herein in the context of describing antimicrobial synthetic cationic polypeptides, the term "polypeptide" has its ordinary meaning as understood by those skilled in the art, and thus includes polymers that contain two or more amino acid repeat units (also called amino acid residues or more simply units or residues) linked together by peptide bonds. Copolypeptides are a type of polypeptide that contain two or more different amino acid repeat units. The molecular weight of the polymer is weight average as determined by size exclusion chromatography (SEC) with molecular weight standards or using light scattering detection.

[0022] The term "block" or "blocky" copolypeptide has its ordinary meaning as understood by those of skill in the art, and thus, a copolypeptide includes a sequence arrangement of amino acid units that includes a segment ("block") or multiple segments that are at least 10 amino acid units in length, in which one or more amino acid units are relatively abundant compared to the overall composition of the copolypeptide. Generally, synthetic block copolypeptides have a sequence arrangement that reflects the intended control over the copolymerization process. Similarly, the term "random" copolypeptide has its ordinary meaning as understood by those of skill in the art, and thus, includes a sequence arrangement of amino acid units that is a statistical distribution that reflects the concentration of the corresponding amino acid monomers in the polymerization mixture.

[0023] As used herein in the context of describing antibacterial synthetic cationic copolypeptides, the term "hydrophobic" block has its ordinary meaning as understood by those of skill in the art, and thus, the block or segment includes sequence arrangements that include multiple hydrophobic amino acid units therein. Examples of hydrophobic amino acid units are known to those of skill in the art and include glycine (G), leucine (L), isoleucine (I), valine (V), proline (P), tryptophan (W), cysteine ​​(C), methionine (M), phenylalanine (F) and alanine (A). Similarly, the term "hydrophilic" block has its ordinary meaning as understood by those of skill in the art, and thus, the block or segment includes sequence arrangements that include multiple hydrophilic amino acid units therein. Examples of hydrophilic amino acid units are known to those of skill in the art and include serine (S), threonine (T), aspartic acid (D) and glutamic acid (E), as well as positively charged amino acids such as cyclic amines (C, D, E, E, F ... It contains the amino acids lysine (K), arginine (R), histidine (H) and ornithine (O).

[0024] As used herein in the context of describing antimicrobial synthetic cationic polypeptides, the terms "positively charged" and "cationic" have their ordinary meaning as understood by those of ordinary skill in the art, and thus include amino acid units or polypeptides that are positively charged at neutral pH. Examples of amino acid units that are positively charged at neutral pH include lysine, arginine, histidine and ornithine, and thus the presence of one or more of these positively charged amino acid units in a polypeptide (in an amount greater than other anionic units) can render the polypeptide cationic.

[0025] As used herein in the context of describing sterile antimicrobial pharmaceutical compositions, the term "sterile" has its ordinary meaning as understood by those skilled in the art, and thus includes compositions that have undergone a sterilization process or process that has the effect of ensuring that known pathogens in the composition are absent or reduced to an extent that renders the sterilized composition clinically acceptable for topical administration to a bodily orifice (such as intranasal administration) or to open skin, such as administration to an open wound, such as a surgical site. Non-limiting examples of such sterilization processes include heat sterilization (such as autoclaving), filtration sterilization, radiation, and / or treatment with chemicals such as ethylene oxide.

[0026] As used herein in the context of describing self-assembling polypeptides, the term "self-assemble" has its ordinary meaning as understood by those skilled in the art, and thus includes the arrangement of the polypeptide when dispersed in a medium (such as other components of a pharmaceutical composition) in which the intermolecular attractive forces between certain segments or blocks of the polypeptide loosely bind them to each other. For example, as mentioned in U.S. Pat. No. 9,017,730, self-assembly of block cationic copolypeptides has been observed in aqueous solutions, resulting in various hierarchical structures that depend on the arrangement of hydrophobic domains and their effect on the intermolecular attractive interactions between polymer chains. In contrast, U.S. Pat. No. 9,017,730 indicates that random copolypeptides do not exhibit self-assembly. Those skilled in the art will be familiar with various techniques for determining whether synthetic cationic polypeptides self-assemble (see, for example, U.S. Pat. No. 9,017,730). Synthetic cationic polypeptides that self-assemble generally exhibit high viscosity when compared to otherwise equivalent synthetic cationic polypeptides that exhibit random arrangements and do not exhibit self-assembly in dilute solutions.

[0027] As used herein in the context of describing molecular characteristics or parameters that promote the self-assembly of polypeptides, terms such as "promote" and "promoting" have their ordinary meaning as understood by those skilled in the art, and thus include allowing or promoting such self-assembly. For example, U.S. Patent Nos. 9,017,730 and 9,446,090 describe various sequence arrangements of hydrophobic and hydrophilic amino acid units configured to promote the self-assembly of copolypeptides in water. Similarly, a sterilization technique configured to create a sterile condition that promotes the self-assembly of a polypeptide is one that allows or promotes self-assembly when applied to such polypeptides or compositions of polypeptides dispersed in an aqueous carrier. Similarly, a composition of an aqueous carrier selected to promote the self-assembly of a polypeptide is one that allows or promotes self-assembly when such polypeptides are dispersed in an aqueous carrier.

[0028] Others are used herein in the context of describing synthetic cationic block copolypeptides that self-assemble in comparison to equivalent randomly synthesized cationic copolypeptides. As used herein, the term "otherwise equivalent randomly synthesized cationic copolypeptide" has its ordinary meaning as understood by those of skill in the art, and thus includes copolypeptides that have the same approximate molecular weight and relative number of hydrophobic and hydrophilic amino acid repeat units as a self-assembling synthetic cationic block copolypeptide, but the sequence arrangement of those amino acid repeat units of the equivalent copolypeptide is more random than block. For example, for a self-assembling block copolypeptide having a hydrophilic (positively charged) lysine block having an average length of about 120 units and a hydrophobic leucine block having an average length of about 30 units, an otherwise equivalent randomly synthesized cationic copolypeptide will contain, on average, about 120 lysine units and about 30 leucine units per copolypeptide chain, but the sequence arrangement of those units along the random copolypeptide chain will be a statistical distribution that reflects the concentration of lysine and leucine monomers in the polymerization mixture.

[0029] As used herein in the context of describing administering a sterile antimicrobial pharmaceutical composition to a mammalian body site in a large amount effective to at least partially prevent and / or treat an infection, the term "large amount" or "large amount" has its ordinary meaning as understood by those skilled in the art, and thus includes administering an amount of the copolypeptide that is at least 10 times greater than the dose required to achieve the desired preventative and / or therapeutic effect. Typically, a total therapeutic amount of an antimicrobial pharmaceutical composition that includes administering 1 g of synthetic cationic polypeptide or more to a 70 kg human, representing 14.3 mg / kg, would be considered a large dose. Those skilled in the art will appreciate that bioactive agents are generally administered in a "therapeutic window" that includes the range of doses in which the desired therapeutic response is achieved without causing significant adverse effects to the subject to whom they are administered. This dose range, typically between the minimum effective concentration (MEC) and maximum toxic concentration (MTC), is typically determined in advance for each bioactive agent and communicated to the subject and / or caregiver in the form of a recommended dosage. However, in some situations, such as administering the antimicrobial composition locally to a body orifice and / or open wound of a mammalian subject, it may be impractical to determine the MEC, and therefore the flexibility to administer the antimicrobial composition in large quantities is highly advantageous. For example, when treating an open wound in an emergency situation where time may be of the essence, it is highly advantageous for a caregiver to have the flexibility to apply the antimicrobial composition in large quantities (e.g., at least 10 times greater than the MEC) to the open wound without worrying about administering an amount that exceeds the MTC. The MEC of a particular sterile antimicrobial pharmaceutical composition can be determined by methods known to those skilled in the art, such as those described in the following examples (e.g., the amount effective to achieve 3-log CFU kill in an in vitro time-kill assay).

[0030] As used herein in the context of describing antimicrobial pharmaceutical compositions comprising or consisting of an aqueous carrier and an antimicrobial synthetic cationic polypeptide dispersed therein, the term "aqueous carrier" has its ordinary meaning as understood by those of skill in the art and thus includes a variety of aqueous carrier systems which may optionally include dispersed substances such as ionic additives (such as salts) or non-ionic additives (e.g., polymers, alcohols, sugars and / or surfactants). The substances dispersed in the aqueous carrier may be dissolved therein and / or dispersed in the form of small particles.

[0031] Antibacterial pharmaceutical composition Various embodiments provide an antimicrobial pharmaceutical composition comprising or consisting of an aqueous carrier and an antimicrobial synthetic cationic polypeptide dispersed in the aqueous carrier. The amount of cationic polypeptide dispersed in the aqueous carrier can vary over a wide range depending primarily on the desired viscosity of the antimicrobial pharmaceutical composition. For example, in various embodiments, the amount of synthetic cationic polypeptide in the antimicrobial pharmaceutical composition is about 0.001% to about 10% by weight based on the total weight of the antimicrobial pharmaceutical composition. In some embodiments, the amount of synthetic cationic polypeptide dispersed in the aqueous carrier is about 0.01% to about 5% by weight based on the total weight of the antimicrobial pharmaceutical composition.

[0032] In various embodiments, the antimicrobial synthetic cationic polypeptide dispersed in the aqueous carrier comprises a plurality of positively charged amino acid units (at neutral pH). In some embodiments, the synthetic cationic polypeptide comprises at least 40 amino acid units, at least some of which are positively charged. In some embodiments, the number of positively charged amino acid units of the synthetic cationic polypeptide is at least 10, at least 15, or at least 20. Lysine, arginine, histidine, and combinations thereof are examples of suitable multiple positively charged amino acid units at neutral pH. In some embodiments, the multiple positively charged amino acid units of the synthetic cationic polypeptide comprise a positively charged lysine unit.

[0033] In various embodiments, the antimicrobial synthetic cationic polypeptide has a viscosity of 2 centistokes (cSt) or greater when measured at a 2% by weight concentration in deionized water at 37° C. Suitable synthetic cationic polypeptides having higher and lower viscosity ranges (e.g., from about 1.5 cSt to about 16,000 cSt or from about 2.0 cSt to about 16,000 cSt) can be made by adjusting the molecular weight of the polypeptide, the level of positively charged amino acid units, and / or the extent to which the polypeptide self-assembles. In certain embodiments, the antimicrobial synthetic cationic polypeptide has a viscosity greater than that of bovine serum albumin when measured at a 2% by weight concentration in deionized water at 37° C.

[0034] The antimicrobial synthetic cationic polypeptides may include other monomer units in addition to the positively charged amino acid unit. For example, in various embodiments, the antimicrobial synthetic cationic polypeptides may further include a plurality of hydrophobic amino acid units. In various embodiments, the number of hydrophobic amino acid units of the cationic polypeptide is at least 5, at least 10, or at least 15. Examples of suitable hydrophobic amino acid units include leucine (L), isoleucine (I), valine (V), phenylalanine (F), alanine (A), and combinations thereof. In some embodiments, the plurality of hydrophobic amino acid units of the synthetic cationic polypeptide includes a leucine unit.

[0035] The sequence arrangement of the amino acid units of the synthetic cationic polypeptide can be random, block-like, or a combination thereof. For example, in some embodiments, the sequence arrangement of the hydrophobic amino acid units and the positively charged amino acid units of the synthetic cationic polypeptide is block-like. In many embodiments, such block copolypeptides can include various hydrophobic and hydrophilic amino acid units. For example, in some embodiments, the synthetic cationic polypeptide is a block copolypeptide that includes a hydrophobic leucine unit and a positively charged lysine unit.

[0036] In various embodiments, the antimicrobial synthetic cationic polypeptides self-assemble in water and other aqueous carriers into multimeric structures. Examples of multimeric structures include micelles, sheets, vesicles, and fibrils (see U.S. Pat. No. 9,017,730). In some embodiments, the antimicrobial synthetic cationic polypeptides form free-standing hydrogels at 3% by weight concentration in deionized water at 37° C. In some embodiments, the antimicrobial synthetic cationic polypeptides exhibit surface activity as measured by a reduction in surface tension of at least 10% or at least 20% in deionized water at 37° C. compared to deionized water alone. In some embodiments, the antimicrobial synthetic cationic polypeptides self-assemble as evidenced by a critical aggregation concentration for the polypeptide at 37° C. in deionized water that is 1000 μg / mL or less. In some embodiments, the antimicrobial synthetic cationic polypeptides self-assemble as evidenced by a critical aggregation concentration for the polypeptide at 37° C. in deionized water that is 100 μg / mL or less.

[0037] The self-assembly of the antimicrobial synthetic cationic polypeptide can be controlled in various ways. For example, in some embodiments, the antimicrobial synthetic cationic polypeptide comprises a sequence arrangement of hydrophobic amino acid units and positively charged amino acid units that is configured to promote the self-assembly of the antimicrobial synthetic cationic polypeptide into a multimeric structure. For example, the self-assembly of the polypeptide is promoted by a block-like sequence arrangement of hydrophobic amino acid units and positively charged amino acid units. A high content of hydrophobic amino acid units and / or long blocks of hydrophobic amino acid units in the polypeptide tend to enhance self-assembly in an aqueous carrier.

[0038] The antimicrobial synthetic cationic polypeptides described herein can be dispersed in an aqueous carrier to form an antimicrobial pharmaceutical composition. In various embodiments, the aqueous carrier is water. In other embodiments, the aqueous carrier is an aqueous solution containing a pharmacologically acceptable salt, a non-ionic additive, or a combination thereof. Salts tend to inhibit the self-assembly of the polypeptides, and therefore, excess salt should be avoided. Normal saline, half saline, quarter saline, and phosphate buffered saline are examples of suitable aqueous carriers containing a pharmacologically acceptable salt. In some embodiments, the aqueous carrier comprises sodium chloride. In various embodiments, the aqueous carrier is an aqueous solution containing a non-ionic additive. Examples of suitable non-ionic additives are aqueous solutions containing glucose, mannitol, glycerol, xylitol, sorbitol, surfactants, and combinations thereof.

[0039] The aqueous carrier can contain various amounts of additives, such as pharmacologically acceptable salts, non-ionic additives, or combinations thereof. In various embodiments, the aqueous carrier contains pharmacologically acceptable salts in an amount that is 9.0 g / L or less; or 8.0 g / L or less; or 7.0 g / L or less; or 6.0 g / L or less; or 5.0 g / L or less; or 4.5 g / L or less; or 4.0 g / L or less; or 3.0 g / L or less. In some embodiments, the amount of additive in the aqueous carrier is selected to control the viscosity of the antibacterial pharmaceutical composition. In some embodiments, the aqueous carrier contains the additive in an amount that increases the viscosity of the antibacterial pharmaceutical composition. In some embodiments, the aqueous carrier contains the additive in an amount that decreases the viscosity of the antibacterial pharmaceutical composition. In some embodiments, the non-ionic additives are present in an amount effective to increase the osmolality of the antibacterial pharmaceutical composition to at least 10% greater than that of the antibacterial pharmaceutical composition without the additives. In various embodiments, the concentration of the additive in the antibacterial pharmaceutical composition is about 0.1% to about 10% by weight based on the total weight. In various embodiments, the concentration of the non-ionic additive in the antibacterial pharmaceutical composition is about 0.01% to about 2% by weight, or about 0.05% to about 5% by weight based on the total weight.

[0040] In various embodiments, the antimicrobial pharmaceutical composition as described herein is sterilized by sterilization techniques configured to achieve a sterile antimicrobial pharmaceutical composition. In some embodiments, the sterilization techniques are configured to have minimal effect on the chemical structure of the synthetic cationic polypeptide and / or the self-assembling properties of the synthetic cationic polypeptide. Such sterilization techniques are illustrated in Figures 42-52. In some embodiments, the antimicrobial pharmaceutical composition as described herein is sterilized by sterilization techniques configured to achieve a sterile antimicrobial pharmaceutical composition having an antimicrobial synthetic cationic polypeptide with a weight average molecular weight and / or dispersity equivalent (e.g., within about 10%) to that of the antimicrobial synthetic cationic polypeptide of the antimicrobial pharmaceutical composition not sterilized by the sterilization technique. In some embodiments, the antimicrobial pharmaceutical composition is sterilized by the sterilization technique configured to achieve a sterile antimicrobial pharmaceutical composition having a viscosity level at 37°C equivalent to that of the antimicrobial pharmaceutical composition not sterilized by the sterilization technique. In some embodiments, the viscosity of the sterilized antimicrobial pharmaceutical composition at 37°C is 20%-200% of the viscosity of the otherwise equivalent non-sterile antimicrobial pharmaceutical composition.

[0041] In one embodiment, the antibacterial pharmaceutical composition has low toxicity when injected into the peritoneal cavity of a plurality of mice at a dose of 10 mL / kg, as measured by 50% or more mouse survival at 72 hours after injection. In one embodiment, the antibacterial pharmaceutical composition has low toxicity when injected into the peritoneal cavity of a plurality of mice at a dose of 20 mL / kg, as measured by 50% or more mouse survival at 72 hours after injection. In one embodiment, the antibacterial pharmaceutical composition has low toxicity when injected into the peritoneal cavity of a plurality of mice at a dose of 40 mL / kg, as measured by 50% or more mouse survival at 72 hours after injection. In one embodiment, the antibacterial pharmaceutical composition has bactericidal activity equivalent to that of an otherwise equivalent non-sterile antibacterial pharmaceutical composition, as measured by a 60 minute time-kill assay against at least one bacterium selected from the group consisting of S. aureus, S. epidermidis, P. aeruginosa, and E. coli.

[0042] Incorporation of other active pharmaceutical ingredients into the antimicrobial pharmaceutical compositions described herein may enhance antimicrobial capacity and / or reduce toxicity risk, both locally and systemically. In particular, incorporation of other antimicrobial agents, including antibiotics, antiseptics, iodine compounds and / or silver compounds, may work in synergy with the synthetic cationic polypeptide to help prevent and / or treat infection. In addition, incorporation of one or more anti-inflammatory agents may enhance capacity and / or reduce toxicity risk, both locally and systemically. Local inflammation may contribute to the pathogenesis of various disease conditions, including microbial contamination or infection. Examples include otitis externa, chronic sinusitis, pulmonary conditions and certain wound conditions. Such conditions could be treated by a combination of synthetic cationic polypeptides and anti-inflammatory agents, such as corticosteroids, antihistamines and / or anticytokine agents. As such, inclusion of anti-inflammatory agents in antimicrobial compositions comprising synthetic cationic polypeptides may provide benefits.

[0043] In some embodiments, the antibacterial pharmaceutical composition comprises an anti-inflammatory compound. For example, in some embodiments, the anti-inflammatory drug is selected from the group consisting of corticosteroids, histamine inhibitors and cytokine inhibitors. Examples of corticosteroids include betamethasone dipropionate, clobetasol propionate, diflorasone acetate, fluocinonide and halobetasol propionate. Examples of histamine inhibitors include those that inhibit histamine H1, H2, H3 and H4 receptors. Examples of cytokine inhibitors include glucocorticoids and pentoxifylline.

[0044] The antibacterial pharmaceutical compositions described herein can be made in a variety of ways. In certain embodiments, the antibacterial synthetic cationic polypeptides are made in the manner taught in U.S. Pat. Nos. 9,017,730 and / or 9,446,090, which are expressly incorporated herein by reference for all purposes, including teaching such methods for making the cationic polypeptides. The antibacterial pharmaceutical compositions can be made by combining the antibacterial synthetic cationic polypeptides with an aqueous carrier, thereby dispersing (e.g., dissolving) the polypeptide in the aqueous carrier. For example, such combination can be achieved by mixing the ingredients (cationic polypeptide, aqueous carrier, and any ingredients such as an anti-inflammatory compound) at a temperature of about 20° C. to 90° C. with stirring for a length of time effective to disperse (e.g., dissolve) the polypeptide. The ingredients can be mixed together in any order, although one of skill in the art may prefer a particular order in individual cases.

[0045] How to prevent microbial contamination Various embodiments provide methods for preventing microbial contamination of tissue that are particularly suited to tissue that is other than intact, healthy skin. For example, in certain embodiments, such methods identify mammalian subjects having tissue sites other than intact, healthy skin that are at risk for microbial contamination. Examples of such tissue sites include affected skin, surgical sites, traumatic wounds, dissected tissue, abdominal cavities, pulmonary airways, sinuses, and urinary tracts. In certain embodiments, the method includes administering an antimicrobial pharmaceutical composition as described herein to the tissue site in an amount sufficient to at least partially protect the tissue site from microbial contamination. For example, in some embodiments, the method at least partially protects the tissue site from infection with at least one bacterium selected from the group consisting of S. aureus, S. epidermidis, P. aeruginosa, and E. coli. The method further includes harvesting the tissue site to assess its microbial load, e.g., the load of at least one bacterium selected from the group consisting of S. aureus, S. epidermidis, P. aeruginosa, and E. coli. In certain embodiments, the antimicrobial pharmaceutical composition is administered intraoperatively to the surgical site. Administration of the antimicrobial pharmaceutical composition to a tissue site can be accomplished by direct topical administration.

[0046] The amount of the antimicrobial pharmaceutical composition that is effective to at least partially protect a tissue site from contamination with microorganisms can be determined by one of skill in the art using routine experimentation informed by the guidance provided herein. In various embodiments, the antimicrobial pharmaceutical composition has a wide therapeutic window, thus providing a relatively wide range of doses over which the desired protection from infection is achieved. In some embodiments, this wide therapeutic window facilitates administering large amounts of the antimicrobial pharmaceutical composition to the tissue site.

[0047] Methods for reducing microbial load Various embodiments provide a method of reducing microbial load in or on tissue, which is particularly suitable for tissue other than intact, healthy skin. For example, in some embodiments, such a method includes identifying a mammalian subject having a tissue site other than intact, healthy skin that has a microbial load. Examples of such tissue sites include diseased skin, surgical sites, traumatic wounds, dissected tissue, abdominal cavity, pulmonary airways, sinuses, and urinary tract. In some embodiments, the tissue site is contaminated, infected, or both by microorganisms. In some embodiments, the method includes administering an antimicrobial pharmaceutical composition as provided herein to the tissue site in an amount effective to at least partially reduce the microbial load. For example, in some embodiments, the method at least partially reduces the microbial load at the tissue site of at least one bacterium selected from the group consisting of S. aureus, S. epidermidis, P. aeruginosa, and E. coli. The method further includes harvesting the tissue site to assess the microbial load, for example, the load of at least one bacterium selected from the group consisting of S. aureus, S. epidermidis, P. aeruginosa, and E. coli. In one embodiment, the antimicrobial pharmaceutical composition is administered intraoperatively to a tissue site that is contaminated, infected, or both, by a microorganism.

[0048] The amount of the antimicrobial pharmaceutical composition that is effective to at least partially reduce the microbial load can be determined by one of skill in the art using routine experimentation informed by the guidance provided herein. In various embodiments, the antimicrobial pharmaceutical composition has a wide therapeutic window, thus providing a relatively wide range of doses over which the desired reduction in microbial load is achieved. In some embodiments, this wide therapeutic window facilitates administering large amounts of the antimicrobial pharmaceutical composition to the tissue site. Administration of the antimicrobial pharmaceutical composition to the tissue site can be achieved by direct topical administration. EXAMPLES

[0049] Synthetic cationic polypeptides can be prepared with a variety of amino acid sequence arrangements. , some of which are illustrated diagrammatically in FIG. 1. Particular arrangements may be referred to as blocks or segments. Typically, these will be stretches of amino acid units that include a plurality of some type of amino acid (e.g., cationic, anionic, hydrophobic). The synthetic cationic polypeptides described herein have been made according to the synthetic methods described in U.S. Pat. Nos. 9,017,730 and 9,446,090, which are incorporated by reference in their entirety for all purposes, including for purposes of describing the synthetic cationic polypeptides and methods of making them.

[0050] The cationic block has multiple positive charges at neutral pH and can vary greatly in length from about 10 amino acid units to well over 300 amino acid units. The positively charged amino acid units can be selected from lysine (K), arginine (R), histidine (H) and ornithine (O). The cationic block need not be entirely composed of cationic amino acids. In addition to multiple cationic amino acids, the block segments may contain other amino acids, including polar amino acids such as serine (S) and threonine (T), which will help maintain hydrophilicity. A small proportion of negatively charged and / or hydrophobic amino acids may also be included in the cationic block, so long as there are more cationic units than anionic units in the block.

[0051] A hydrophobic block has multiple hydrophobic amino acids and can vary greatly in length, typically from about 5 to about 60 amino acid units. A hydrophobic block can also exhibit secondary structure (such as an alpha helix versus disorder). Hydrophobic amino acids are uncharged at pH 7.0. In addition, they have mostly carbon and hydrogen side chains, have very small dipole moments, and tend to be repelled by water. The hydrophobic amino acid units can be selected from a list including glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), and methionine (M). A hydrophobic block need not be entirely composed of hydrophobic amino acid units. In addition to multiple hydrophobic amino acids, a block segment may contain other amino acids, including polar amino acids such as serine (S) and threonine (T). A small percentage of charged amino acids may be included in the hydrophobic block.

[0052] Synthetic cationic polypeptides with cationic-hydrophobic block structures can be prepared with a wide range of total chain lengths, typically from about 20 amino acid units or less on the low side to about 400 amino acid units or more on the high side. When describing cationic polypeptides with their block structures, as depicted in FIG. 2, they can be classified into three groups: (I) long cationic segments (200 amino acid units or more); (II) medium cationic segments (100-199 amino acid units); and (II) short cationic segments (10-99 amino acid units). The ratio of cationic block length to hydrophobic block length can vary in a wide range, typically from about 1.5 on the low side to about 15 or more on the high side. The average molecular weight of these synthetic cationic polypeptides can vary in a wide range, typically from about 3,000 Da on the low side to about 70,000 Da on the high side. FIG. 3 shows the detailed composition of such synthetic cationic polypeptides with segmented structures.

[0053] As described in more detail below, we have synthesized and tested a large number of different synthetic cationic polypeptides with block sequence arrangements. For comparison, we have also synthesized cationic polypeptides with comparable amino acid composition but lacking blocked or segmented sequence arrangements. These synthetic cationic polypeptides were found to vary in multiple functional properties, including antimicrobial activity, hemostatic properties, barrier properties and surfactant properties. Furthermore, it has been demonstrated that both molecular design and formulation can affect these functional properties. With regard to molecular design properties, we have investigated the overall chain length and the cationic to hydrophobic block ratio. We have observed that the ratio of hydrophilic blocks to hydrophilic blocks is the property that has the greatest impact on function. Specific properties within each block also contribute, including amino acid choice and enantiopurity.

[0054] In this application, we use a nomenclature based on two main features of synthetic cationic polypeptides: overall chain length and the ratio of cationic block length to hydrophobic block length. As an example, KL-140 / 2.5 has an average chain length of about 140 amino acid units and a ratio of cationic segments to hydrophobic segments of about 2.5. In this polymer, the cationic amino acid unit is lysine (K) and the hydrophobic amino acid unit is enantiopure L-leucine (L). As another example, KrL-100 / 5.7 has an average chain length of about 100 amino acid units and a ratio of cationic segments to hydrophobic segments of about 5.7. In this polymer, the cationic amino acid unit is lysine (K) and the hydrophobic amino acid unit is racemic D,L-leucine (rL). As another example, RrL-75 / 2.8 has an average chain length of about 75 amino acid units. In this polymer, the cationic amino acid unit is arginine (R) and the hydrophobic amino acid unit is racemic D,L-leucine (rL).

[0055] The synthetic cationic polypeptides described in U.S. Pat. Nos. 9,017,730 and 9,446,090 (which use different nomenclature systems) are K55 、K 55 L5、K 55 L 10 、K 55 L 15 、K 55 L 20 、K 55 L 20 -RAN、K 55 L 25 、K 55 L 30 、K 55 (rac-L)5、K 55 (rac-L)5-RAN、K 55 (rac-L) 10 、K 55 (rac-L) 10 -RAN、K 55 (rac-L) 20 、R H 55 (rac-L) 20 、K 55 (rac-L) 20 -RAN、K 55 (rac-L) 30 、K 55 (rac-I) 20 、K 55 (rac-L / F) 20 、K 55 (rac-V) 20 、K 55 (rac-A) 20 、[K 65 (rac-L) 15 -RAN](rac-L) 20 、K 80 、K 80 (rac-L) 20 、K 90 (rac-L) 30 、K 90 (rac-L) 30 -RAN、K 99 L 36 、K 99 (rac-L) 36 、K 99 (rac-L) 36 -RAN、K 100 、K 100 L 20 、K 100 L 30 ,K100 L 40 、K 100 L 40 -RAN、K 100 L 50 、K 100 L 60 、K 100 (rac-L) 20 、K 100 (rac-L) 20 -RAN、K 100 (rac-L) 30 、K 100 (rac-L) 40 、K 100 (rac-L) 60 、K 120 (rac-L) 10 、K 120 (rac-L) 40 ,K 120 (rac-L) 40 -RAN、K 120 (rac-L) 50 、K 120 (rac-L) 50 -RAN、K 130 L 20 、K 130 L 30 、K 130 L 40 、K 130 L 40 -RAN、K 130 L 60 、K 130 (rac-L) 20 、K 130 (rac-L) 30 、K 130 (rac-L) 40 、K 130 (rac-L) 60 、K 150 L 30 、K 160 (rac-L) 20 、K 180 、K 180 L 18 、K 180 L 20 、K 180 L 36 、K 180 L 54 、K 180 (rac-L) 18 、K180 (rac-L) 20 , K 180 (rac-L) 36 , K 180 (rac-L) 54 , K 190 L 10 , K 200 L 50 , PEG 205 (rac-L) 20 , K 256 , K 324 L 36 , K 360 K 360 L 36 , K 360 L 36 -R.A.N., K. 360 L 54 , K 360 L 72 , K 360 (rac-L) 36 , K 360 (rac-L) 54 and K. 360 (rac-L) 72 Includes.

[0056] Example 1 Synthetic cationic polypeptides can be designed as shown in Figures 4-6 to enable formulations with robust bactericidal activity and barrier effect. Figure 4 describes an example synthetic cationic polypeptide having a cationic-hydrophobic block sequence arrangement based on lysine and enantiopure L-leucine amino acid units. The average overall chain length of the polypeptide formulation was found to be approximately 160 amino acid units, with a lysine to leucine or K:L ratio of 3.2. This synthetic cationic polypeptide was designated as KL-160 / 3.2, and the SEC chromatogram of the polymer had a relatively low polydispersity (D) of 1.1. A single peak is shown.

[0057] FIG. 5 depicts the concentration-dependent antibacterial activity of KL-160 / 3.2 in water. There is a clear concentration-dependent effect with activity against S. aureus, P. aeruginosa, and C. albicans at concentrations as low as 1.6 μg / mL. A summary of the antibacterial activity of KL-160 / 3.2 observed across many experiments is provided in FIG. 6. As noted below, when prepared at high concentrations in water (i.e., less than 2 wt%), KL-160 / 3.2 forms a free-standing hydrogel. These properties of the synthetic cationic polypeptides result in a desirable combination of antibacterial activity and barrier function. Importantly, it is recognized that the functional properties of these antibacterial formulations depend on the formulation (e.g., the presence and concentration of additives), as well as the presence, absence, or extent of certain process and / or handling procedures, including sterilization methods.

[0058] Example 2 Synthetic cationic polypeptides can be designed as shown in Figures 7-9 to enable formulations with robust bactericidal and surfactant activity. Figure 7 describes an example synthetic cationic polypeptide having a cationic-hydrophobic block sequence arrangement based on lysine and racemic D,L-leucine amino acid units. The average overall chain length of the polypeptide product was found to be approximately 100 amino acid units, with a lysine to racemic leucine or K:rL of 5.7. This synthetic cationic polypeptide was designated as KrL-100 / 5.7, and the SEC chromatogram of the polymer shows a single peak with a relatively low polydispersity (D) of 1.1.

[0059] FIG. 8 depicts the concentration-dependent antibacterial activity of KrL-100 / 5.7 in water. There is a clear concentration-dependent effect with activity against S. aureus, S. epidermidis, E. coli, P. aeruginosa and C. albicans at concentrations as low as 1.6 μg / mL. A summary of the antibacterial activity of KrL-100 / 5.7 observed across many experiments is provided in FIG. 9. As noted below, when prepared in water, KrL-100 / 5.7 demonstrates surfactant power. These properties of the synthetic cationic polypeptides provide a desirable combination of antibacterial activity and surfactant function, thereby enhancing the ability for biofilm and / or tissue debridement. Importantly, it is recognized that the functional properties of these antimicrobial formulations depend on the formulation (e.g., the presence and concentration of additives) and the presence, absence, or extent of certain process and / or handling procedures, including sterilization methods.

[0060] Example 3 It may be beneficial to self-assemble cationic antimicrobial peptides into multimeric complexes. Synthetic cationic polypeptides with block sequence arrangements can be designed and formulated to self-assemble into multimeric structures. Two examples are depicted in FIG. 10: KL-160 / 3.2 was designed to self-assemble into fibrous structures to form barrier hydrogels, and KrL-100 / 5.7 was designed to self-assemble into micellar structures with surfactant properties. Note that, as mentioned above, these two synthetic cationic polypeptides are also depicted in FIGS. 4-6 and 7-9.

[0061] Various embodiments of the present invention include antimicrobial compositions comprising at least one antimicrobial agent that forms a multimolecular complex with itself and / or with one or more other components of the composition. Explaining, the occurrence of such a multimolecular complex enhances intermolecular binding. Such binding is reversible. Such binding may cause, in whole or in part, a hydrophobic attraction effect. Such binding may be covalent or non-covalent. Such binding may be: Systemic absorption may be reduced or delayed. Such binding may reduce the risk of local and / or systemic toxicity.

[0062] Critical aggregation concentration (CAC) is one measure of molecular self-assembly in an aqueous environment. CAC can be measured by several techniques, including pyrene fluorescence and surface tension techniques. Figure 11 shows the CAC values ​​of various synthetic cationic polypeptides as measured by pyrene fluorescence. It is noteworthy that polylysine chains (K-100) and a lysine-leucine synthetic cationic polypeptide lacking block sequence arrangement (KL-130 / 3.3-RAN) demonstrate very high CACs of 1,600 and 2,700 μg / mL, respectively. In comparison, many lysine-leucine synthetic cationic polypeptides with block sequence arrangements demonstrate much lower CACs of 1 μg / mL to 160 μg / mL by this method, as shown by the remaining entries in Figure 11. Figure 12 depicts the measurements of CAC by surface tension using the synthetic cationic polypeptide KrL-100 / 5.7.

[0063] Various embodiments of the present invention may include antimicrobial compositions that include at least one antimicrobial agent, which when dissolved in water has a critical aggregation concentration (CAC) of 500 μg / mL or less. The CAC can be measured by methods known in the art, such as those using pyrene fluorescence. Other antimicrobial agents may have a lower CAC. For example, the antimicrobial agent may have a CAC of 200 μg / mL or less, 100 μg / mL or less, 50 μg / mL or less, and / or 20 μg / mL or less.

[0064] Example 4 Viscosity is a relevant measure of molecular properties as well as properties of pharmaceutical compositions. Unexpectedly, we have discovered that increased viscosity can be a marker of increased safety as well as increased intrawound antimicrobial potential. Many molecular design properties affect the viscosity of synthetic cationic polypeptides. These include overall chain length, amino acid composition, ratio of cationic units to hydrophobic units and sequence arrangement of amino acid residues (e.g., blocky versus random). It should also be noted that other components that may be present in the synthetic cationic polypeptides (e.g., counterions, other salts and residual solvents) can affect the viscosity of aqueous formulations. In addition, various additives (e.g., salts, non-ionic tonicity adjusting excipients, surfactants) may be used in the preparation of pharmaceutical compositions in amounts effective to affect viscosity. As described below, sterilization methods can also have a significant effect on viscosity.

[0065] FIG. 13(a) depicts the kinematic viscosity values ​​of various synthetic cationic polypeptides at 1 wt. % in water measured using a glass capillary viscometer (Ubbelohde viscometer). As demonstrated by this data, the presence of polylysine chains of about 100 amino acid units (K-100) or about 200 amino acid units (K-200) has only a modest effect on viscosity at this concentration. Similarly, the presence of a lysine-leucine synthetic cationic polypeptide, KL-170 / 3.3-RAN, having a random / statistical sequence arrangement (i.e., non-block), also has only a modest effect on viscosity (value of 1.1 cSt). In comparison, several synthetic cationic polypeptides having segmented or block sequence arrangements demonstrate enhanced viscosities in this set of data, ranging from 290 cSt to as high as 1.4 cSt. Overall chain length has been shown to increase viscosity; increasing hydrophobic block length has been shown to increase viscosity; and enantiopurity of the hydrophobic leucine amino acid has been shown to increase viscosity. For comparison, Figure 13(b) shows that bovine serum albumin has little, if any, effect on the viscosity of the aqueous formulation at concentrations of 1 and 2 wt% under these conditions. Albumin is an abundant blood protein with a molecular weight of about 66.5 kDa and an overall chain length of about 583 amino acid units. These data indicate that molecular size alone does not fully explain the viscosity effect.

[0066] Figure 14 shows viscosity data of two synthetic cationic polypeptides at 0.5 wt% in water at 37°C. In this case, both synthetic cationic polypeptides, KL-160 / 3.3 and KL-120 / 2.5, have similar length and design of hydrophobic segments (~35-40 enantiopure L-leucine units). Higher viscosity is found in KL-160 / 3.3, which has a longer cationic block structure and therefore a longer overall structure. However, it is also important to note that 0.5 wt% KL-120 / 2.5 (Figure 14) has a higher viscosity than 1.0 wt% KrL-160 / 3.3 (Figure 13). The latter has a longer overall chain length, a similar size of hydrophobic block, but a different composition of hydrophobic amino acid units (racemic D,L-leucine vs. enantiopure L-leucine). Figure 15 further demonstrates that both molecular design and concentration affect viscosity. The data was obtained using a glass capillary viscometer at 40° C. and includes four synthetic cationic polypeptides from different production lots than those mentioned above.

[0067] The viscosity of synthetic cationic polypeptides can also be measured under the influence of shear forces (viscosity). This technique allows the evaluation of shear-thinning or shear-thickening properties. These shear-thinning or shear-thickening properties can be important for both ease of application to tissues as well as overall performance in vivo. In particular, we have found that shear-thinning properties facilitate spreading into tissues by manual manipulation, which may occur in a variety of medical and surgical situations. As depicted in FIG. 16, the viscosity of synthetic cationic polypeptide KL-120 / 2.5 at 1.5%, 2.0% and 3.0% by weight in water was evaluated using a rotational viscometer against increasing shear forces. A shear-dependent (with increasing spindle speed) decrease in viscosity was observed (measured in centipoise (cP)). This "shear-thinning" effect was seen at the three tested concentrations. A concentration-dependent increase in viscosity was also observed as shown in FIG. 16.

[0068] Various embodiments of the present invention include antimicrobial compositions comprising at least one antimicrobial synthetic cationic polypeptide, which when dissolved in water induces a significant increase in viscosity. For example, in examples where the synthetic cationic polypeptide is a block copolymer, the increase in viscosity observed is greater than that observed for an otherwise equivalent random synthetic cationic polypeptide. The increase in viscosity can be measured using one or more types of viscometers by methods known in the art.

[0069] By way of example, a formulation of at least one antimicrobial synthetic cationic polypeptide at a concentration of 10 mg / mL or 1% by weight in water has a viscosity of between 1.25 centistokes and 500 centistokes (cSt; mm 2 / s) and is measured using a glass capillary viscometer, such as an Ubbelohde viscometer, in an assay where the viscosity value of water alone is below 0.9 cSt. In various embodiments, the viscosity of these formulations of at least one antimicrobial agent is greater than 500 cSt.

[0070] Synthetic cationic polypeptides can be designed and manufactured to form free-standing hydrogels when dispersed in water. As depicted in Figure 17(a)-(d), an example synthetic copolypeptide (KL-120 / 2.5) forms viscous solutions and hydrogels in water depending on the concentration. KL-120 / 2.5 was prepared at concentrations of 0.5, 1.0, 1.5, 2.0 and 3.0 wt% in DI water and evaluated for gel formation by tilt tube assay, firmness by texture analysis, and viscosity. Concentration-dependent effects on physical properties are evident. For example, upon increasing concentration to 2 wt% in water, this synthetic cationic polypeptide formulation formed a free-standing hydrogel. At 1.5 wt%, the formulation acted as a barrier and resisted penetration by two different stainless steel balls, or BBs. Quantitative measurement of firmness using texture analysis also clearly exhibited a concentration-dependent increase. This latter method also demonstrates barrier properties and resistance to penetration.

[0071] Example 5 Various additives can modify the viscosity of aqueous formulations of synthetic cationic polypeptides. We have found that various additives can be used to alter certain properties of the pharmaceutical compositions described herein, including pH and tonicity. During evaluation, we have found that certain additives have unexpectedly large effects on viscosity. We have further found that these effects are dependent on the molecular design of the synthetic cationic polypeptide. For example, multiple studies have demonstrated that the addition of NaCl (~0.9%) to aqueous formulations of Group II intermediate Lysine-L-Leucine (KL) polypeptides with blocked amino acid sequence arrangements significantly reduces viscosity. A similar, but less significant effect was observed for Lysine-D,L-Leucine (KrL) polypeptides. This data indicates that both the molecular design and the nature of the additive should be considered together when preparing pharmaceutical compositions with desired viscosity parameters. These observations also provide a reminder that certain substances, including salts, that may be found in manufactured formulations of synthetic cationic polypeptides can significantly affect functionality and potency.

[0072] Figure 18 shows a partial list of pharmacologically acceptable non-ionic excipients (additives) that may be used to prepare pharmaceutical compositions containing synthetic cationic polypeptides. Figure 19 depicts the viscosity of synthetic cationic polypeptide KL-120 / 2.5 (BAC004) at 2.0 wt % in water, 4.5% mannitol in water, 2.33% glycerol in water, or 2.8% histidine in water, evaluated against increasing shear forces. The histidine formulations showed very low viscosities at all spindle speeds. Formulations in mannitol and glycerol showed relatively high, shear-dependent viscosity profiles, albeit at somewhat lower levels than observed for the water-only formulations.

[0073] FIG. 20 depicts the dynamic viscosity of two example synthetic cationic polypeptides having lysine-L-leucine (KL) and lysine-D,L-leucine (KrL) block sequence configurations dissolved in water alone, 0.9% saline, or 4.4% xylitol aqueous solution at 1% by weight in water. In particular, preparation of KL-100 / 5.7 in 0.9% saline demonstrated a viscosity reduction of about 70% compared to formulation in water. In comparison, preparation of KrL-110 / 4.0 in 0.9% saline demonstrated a viscosity reduction of about 40% compared to formulation in water. Furthermore, these studies demonstrated that both synthetic cationic polypeptides in aqueous solution of xylitol, a non-ionic additive, unexpectedly exhibited enhanced viscosity compared to water alone. Thus, xylitol offers the potential for benefit as an additive in pharmaceutical compositions when it is desirable to maintain high viscosity while increasing tonicity and / or osmolality. Moreover, in some embodiments, xylitol is an unexpectedly preferred pharmacologically acceptable additive to the antimicrobial composition because, in addition to its effect on viscosity, it does not support microbial metabolism and the growth of many microorganisms.

[0074] Figure 21 depicts the viscosity of synthetic cationic polypeptide KL-120 / 2.5 (BAC004) at 2.0 wt% in 4.5% aqueous mannitol evaluated against increasing shear forces. It further depicts that this formulation is stable to heat treatment, including 50°C and 94°C. Stability to heat treatment can be beneficial for certain manufacturing steps, such as dissolving and slowly cooling the synthetic cationic polypeptide in an aqueous carrier, and reducing or eliminating any contaminating microorganisms by sterilization.

[0075] Various embodiments of the present invention provide a method for preparing at least one antimicrobial synthetic cationic polypeptide and at least one additive or excipient in an aqueous carrier, the method comprising the steps of: 2 / s) in a combination and amount sufficient to produce a pharmaceutical composition viscosity of water alone below 0.9 cSt in an assay. The viscosity of the pharmaceutical compositions is measured using a glass capillary viscometer, such as an Ubbelohde viscometer. In various embodiments, the viscosity of these pharmaceutical compositions is greater than 500 cSt.

[0076] Various embodiments of the present invention include pharmaceutical compositions comprising at least one antimicrobial synthetic cationic polypeptide in an aqueous carrier and an amount of at least one additive or excipient effective to impart a shear-thinning effect to the composition at 37° C. as measured using a rotational viscometer.

[0077] Various embodiments of the present invention include pharmaceutical compositions comprising at least one antimicrobial synthetic cationic polypeptide in an aqueous carrier and an amount of at least one additive or excipient effective to impart a viscosity of the composition greater than that of a comparable formulation in the aqueous carrier without the additive or excipient. For example, in the examples, the addition of an effective amount of xylitol or glycerol to a pharmaceutical composition, including compositions that may also include various salts such as NaCl, enhances the viscosity of the formulation compared to a comparable pharmaceutical composition without the xylitol or glycerol.

[0078] In accordance with the present invention, a significant amount of the pharmaceutical composition may be required for certain patients (e.g., those with large wounds and / or high microbial levels) to reach an effective level of antimicrobial activity. We have found that the antimicrobial agent can enhance both the efficacy and safety of a topically applied antimicrobial composition if designed in accordance with the educational content provided herein. An antimicrobial agent that forms a multi-molecular complex with itself or with one or more other components of the composition may increase the local concentration and tissue coating of the antimicrobial agent, thereby obtaining greater efficacy at a lower dose. Furthermore, this property may reduce or delay potential systemic absorption and distribution, and may reduce systemic toxicity. It may also reduce local toxicity. The design of the antimicrobial agent itself, as well as the formulation of the antimicrobial composition, will affect the molecular interactions that allow for the formation of multi-molecular complexes.

[0079] We have found that the efficacy and safety of locally administered antimicrobials are influenced by their physical properties, including viscosity. For example, increasing viscosity may increase the tissue retention time of a locally applied antimicrobial, thereby enhancing efficacy. This effect may also reduce the total amount of antimicrobial composition required, thereby reducing the list of dose-limiting toxicities. Molecular interactions of the antimicrobial with itself or with other components of the composition contribute to the increased viscosity of the antimicrobial composition.

[0080] Example 6 Synthetic cationic polypeptides can be designed to demonstrate surfactant power. Certain design features add more surfactant power; others add less. Surfactant power can be demonstrated in multiple assays. For example, a surfactant can be shown to reduce surface tension at a liquid-gas interface. As depicted in FIG. 22, the synthetic cationic polypeptide KrL-120 / 5.0 was shown to reduce the surface tension of water significantly compared to synthetic antimicrobial agents that are not synthetic cationic polypeptides; chlorhexidine and PHMB appear to have little or no effect.

[0081] The ability of synthetic cationic polypeptides to reduce the surface tension of various formulations was evaluated (FIG. 23). Several observations were made. Unexpectedly, lysine-leucine diblock copolypeptides prepared with enantiopure L-leucine demonstrated little surfactant activity when the hydrophobic block was 30 amino acid units or longer. Surfactant activity was observed with shorter enantiopure L-leucine blocks (e.g., less than about 20 leucine units). In addition, lysine-leucine diblock copolypeptides prepared with racemic D,L-leucine demonstrated relatively high surfactant activity when compared to those prepared with enantiopure L-leucine with leucine block lengths of about 20 units or longer. Overall, the data indicate that surfactant power is greatly influenced by both amino acid composition (including enantiopure versus racemic) and the length of the hydrophobic block. Thus, design factors can be used to obtain the desired surfactant properties of synthetic cationic polypeptides.

[0082] Surfactant power can also be demonstrated by other assays. One example is the reduction of interfacial tension in a water / oil system. As depicted in Figures 24(a) and 24(b), the synthetic cationic polypeptide KrL-100 / 5.7 was shown to reduce interfacial tension.

[0083] Certain additives can also affect surface and interfacial tension. For example, the data in Figure 25 show that formulations of the synthetic cationic polypeptides KrL-130 / 3.3 (Lot 77) and KL-130 / 3.3 (Lot 72) lower the surface tension when formulated in water alone or in water with 0.25% or 1.0% acetic acid. The presence of acetic acid has an additive effect. A similar effect is observed in the interfacial tension. (Figure 26)

[0084] Synthetic cationic polypeptides can be effective emulsifiers. As shown in FIG. 27, we demonstrate that formulations of two example synthetic cationic polypeptides, KrL-100 / 5.0 and KrL-160 / 3.2, having a racemic D,L-leucine hydrophobic segment, are effective in emulsifying soybean oil. This emulsifying effect was also observed for synthetic cationic polypeptides having an enantiopure leucine hydrophobic block (FIG. 28). The data in FIG. 28 demonstrate that emulsifying activity can be demonstrated by synthetic cationic polypeptides formulated not only in water, but also in saline or aqueous xylitol.

[0085] Synthetic cationic polypeptides can be designed and formulated to demonstrate surfactant properties. We have found that formulations of synthetic cationic polypeptides that combine direct bactericidal activity with surfactant properties are highly effective antibiofilm agents. Furthermore, we have demonstrated that such formulations are highly effective in vivo on tissues that are heavily contaminated with both Gram-positive and Gram-negative bacteria.

[0086] Example 7 Understanding that the potential for both local and systemic toxicity is important. Local tissue compatibility and safety are both desirable for the effective use of topically applied antimicrobial compositions. Overall, we have found that formulations of synthetic cationic polypeptides with blocked or segmented sequence configurations demonstrate relatively high tissue compatibility and local safety. Furthermore, appropriate formulations, dosages and application methods as described herein can be used to minimize tissue damage and aid in healing.

[0087] Unexpectedly, we found that there is a great deal of variability among different formulations of synthetic cationic polypeptides when it comes to systemic safety. We found that both molecular design and formulation greatly impact the risk of systemic toxicity when synthetic cationic polypeptide formulations are applied intraperitoneally. Although intraperitoneal administration is a type of topical application, it promotes significant systemic uptake and distribution of therapeutic agents and excipients. Thus, intraperitoneal administration presents a higher risk of systemic toxicity than most topical applications of antimicrobial pharmaceutical compositions. It is important to keep several things in mind in this regard. First, topical application to various tissues other than intact, healthy skin can increase systemic uptake. Second, intraperitoneal infections are very serious and there is a need for better and safer antimicrobials that can be applied intraperitoneally. Third, inadvertent administration of pharmaceutical compositions intraperitoneally, intravenously, or to other sites that can dramatically increase systemic uptake can occur. We found that the risk of both local and systemic toxicity was low even when applied to sites other than healthy, intact skin, which is highly effective. We have now developed a topically applied antibacterial pharmaceutical composition that has low risk.

[0088] FIG. 29 depicts the results of a rabbit skin irritation model in which compositions of synthetic cationic polypeptides were applied to both intact and abraded skin. Negligible responses were observed after application of synthetic cationic polypeptides KL-140 / 2.5 or KrL-120 / 5.0 at various concentrations and formulations. FIG. 30 depicts the results of a guinea pig skin sensitization study. No visual changes were observed after application of KL-140 / 2.5 (Lot 73) or KrL-120 / 5.0 (Lot 93) at 1% by weight in water, indicating to one of ordinary skill in the art that synthetic cationic polypeptides have no sensitizing potential. FIG. 31 depicts the results of a rat oral toxicity study. No abnormalities were observed after administration of KL-140 / 2.5 in water or KrL-120 / 5.0 in water to rats at doses ranging from 0.625 mg / kg to 160 mg / kg for three days by oral gavage.

[0089] Testing of systemic toxicity after intraperitoneal administration yielded unexpected results. As depicted in Figure 32, polylysine of approximately 100 amino acid units (K-100) was administered intraperitoneally at a concentration of 20 mg / mL and a total dose of 800 / mg / kg, proving to be highly toxic with 0 of 5 animals surviving. Similarly, the synthetic cationic polypeptide KL-140 / 2.5-RAN lacks a block sequence arrangement of amino acid units, and this formulation proved to be toxic with 0 of 5 animals surviving. In comparison, after administration of KrL-120 / 5.0 at an equivalent concentration and dose in water, all animals (5 / 5) survived. As shown in FIG. 33, an aqueous formulation of another cationic polypeptide, KrL-130 / 3.3, having a segmented or blocked sequence arrangement of lysine and D,L-leucine, also demonstrated a high level of safety with complete viability (5 / 5) at concentrations up to 20 mg / mL (dose of 800 mg / kg).

[0090] Further testing revealed unexpected variations when testing two different lots (93 and 94) of the polymer with the same target structure, KrL-120 / 5.0. As depicted in FIG. 34, Lot 93 was found to cause significant toxicity and systemic mortality after intraperitoneal administration compared to Lot 94. This was particularly surprising because Lot 93 had been shown to be safe for topical application in the guinea pig sensitization study described above in FIG. 30. Lot 93 was also shown to be an effective antimicrobial agent in vitro against Gram-positive and Gram-negative bacteria. Additionally, it was found to be safe and effective when used in vivo in both a microbially contaminated porcine open wound model and a rodent closed wound model.

[0091] Upon further analysis of Lots 93 and 94, notable differences were observed in the self-assembly into multimeric structures as assessed by the critical aggregation concentration (CAC). Lot 93 was found to be the more toxic lot and had a higher critical aggregation concentration (CAC) (130 μg / mL), indicating a weaker driving force for self-assembly into multimeric structures than Lot 94 (CAC: 51 μg / mL).

[0092] We have also found that synthetic cationic polypeptides that demonstrate low CAC and high viscosity when formulated in water are relatively safe when administered intraperitoneally. As depicted in the data in Figure 35, two formulations of Lysine-L-Leucine (KL) synthetic cationic polypeptides with a block sequence arrangement of amino acid units, KL-170 / 3.3 and KL-140 / 2.5, have been shown to be very safe for intraperitoneal administration. These synthetic cationic polypeptides and those with closely related structures have low measured CAC (typically in the single digits μg / mL, see also Figure 11 above) and demonstrate high levels of self-assembly. We have found that synthetic cationic polypeptides that demonstrate a high safety profile following intraperitoneal administration also demonstrate relatively high viscosity and / or hardness in the texture analysis of the aqueous formulations. (See Figures 13 to 17.) We have therefore developed general principles, as described herein, that will guide those skilled in the art in designing synthetic cationic polypeptides that have a low risk of systemic toxicity.

[0093] Formulation additives were also found to affect the safety profile of synthetic cationic polypeptide compositions administered intraperitoneally. As shown in Figure 36, formulations of KL-140 / 2.5 in water or water and hydroxyethylcellulose (HEC) demonstrated some differences in safety. Notably, formulations with higher HEC content were found to be more toxic, despite the fact that this additive is widely accepted as safe. Figure 37 shows additional examples of formulation changes that may increase toxicity upon intraperitoneal administration.

[0094] Taken together, these findings indicate that local tissue compatibility and systemic safety are not the same, and it is important to address both. Notably, both polymer design features and formulation parameters can increase or decrease the risk of systemic toxicity. With regard to polymer design features, our data indicate that safer synthetic cationic polypeptides can be made with design features that promote self-assembly with lower critical aggregation concentrations and / or promote high viscosity. Furthermore, we found that formulation additives should be selected to maintain or enhance the self-assembly of synthetic cationic polypeptides into multimeric structures and / or maintain or enhance polymer viscosity.

[0095] The ratio of dose-limiting systemic toxicity to locally effective doses may depend on the tissue site and / or pathophysiological situation. Both effective and toxic doses may be determined after application to a particular site or pathophysiological situation. Alternatively, a "higher hurdle" type may be established by determining systemic toxicity after application to a site with expected high systemic absorption (such as the peritoneal cavity). Locally effective doses may also be determined after application to a particular site or pathophysiological situation (e.g., limb injury, soft tissue infection, sinus infection).

[0096] In embodiments, antimicrobial agents or compositions may be selected based in part on criteria of local or systemic toxicity in vivo.

[0097] In embodiments, the antimicrobial agent or composition may be selected based in part on the criteria of in vivo toxicity following intraperitoneal administration.

[0098] Certain embodiments of the invention may include antimicrobial agents or compositions that can be safely injected into the peritoneal cavity of mice at doses higher than would be expected to be effective in reducing microbial load following topical application to tissues other than the intraperitoneal cavity.

[0099] Example 8 Sterilization methods are important. We have found that the safety of antibacterial pharmaceutical compositions based on synthetic cationic polypeptides depends on the molecular design (especially the cationic-hydrophobic block sequence arrangement), the self-assembly of the polymer into a polymeric structure, and the viscosity. In an embodiment, sterilization of the antibacterial pharmaceutical composition is carried out by a method that maintains the molecular integrity, polymeric structure, and viscosity.

[0100] Figures 38-41 demonstrate that traditional radiation sterilization techniques can destroy the molecular structure of synthetic cationic polypeptides and cause a decrease in the hardness of the compositions, as assessed by texture analysis. Electron beam sterilization of aqueous formulations of synthetic cationic polypeptides is also shown to cause a significant decrease in viscosity. The samples were observed to be as fluid as water. As such, radiation sterilization techniques need to be carefully adjusted and / or modified for use in sterilizing antibacterial pharmaceutical compositions having synthetic cationic polypeptides.

[0101] In comparison to radiation techniques, both sterile filtration and heat sterilization (autoclaving) provide effective sterilization of antimicrobial pharmaceutical compositions while maintaining the molecular structure of the synthetic cationic polypeptide and the desired physical properties of aqueous compositions containing it. Figures 42-46 show SEC chromatograms demonstrating the maintenance of molecular integrity after sterilization by filtration and autoclaving.

[0102] Sterilization by filtration and autoclaving also preserves key beneficial physical properties of aqueous formulations and compositions containing synthetic cationic polypeptides. Figure 47 demonstrates that an aqueous formulation of synthetic cationic polypeptide KL-120 / 2.5 (Lot BAC004) at 1.25% by weight in water with 4.5% by weight mannitol exhibited nearly identical viscosities before and after sterilization by filtration. Figures 48 and 49 demonstrate that an aqueous formulation of synthetic cationic polypeptide KL-160 / 3.2 (Lot BAC003) retains significant levels of shear-thinning viscosity after autoclaving, although the overall levels were somewhat reduced compared to the samples before autoclaving. Furthermore, when various aqueous formulations and compositions are autoclaved, the beneficial properties of firmness, as assessed by texture analysis, are maintained (Figures 50 and 51), although some reduction in firmness is noted. The data further demonstrate that a reduction in firmness is observed at high concentrations of synthetic cationic polypeptides and in the presence of certain additives, such as HEC. For example, the observed decrease in viscosity and hardness may be due to further dissolution of the multimeric structure and subsequent annealing that may occur during autoclave sterilization. Thus, these formulations are expected to maintain their viscosity and hardness benefits. Figure 52 shows that the surfactant power of the synthetic cationic polypeptide KrL-120 / 5.0 is maintained after autoclave sterilization.

[0103] Example 9 To be both effective in inhibiting or killing microorganisms and also to reduce the risk of local and systemic toxicity, the exemplary antimicrobial pharmaceutical compositions as described herein provide both antimicrobial activity and beneficial physicochemical properties. The latter is related to the better self-assembly of the synthetic cationic polypeptides into multimeric structures, as exemplified by albumin, higher viscosities than are found in aqueous formulations of many proteins, or both. In various embodiments, a further reduction in the risk of both local and systemic toxicity is achieved by sterilization of the antimicrobial pharmaceutical composition, so long as the sterilization process is not done in a manner that does not significantly adversely affect the molecular integrity of the synthetic cationic polypeptide or the beneficial physicochemical properties of the antimicrobial pharmaceutical composition. Finally, to achieve efficacy and reduced risk of local and systemic toxicity in an in vivo environment, the method of application (including how much and how often) is important to consider, especially when applying the antimicrobial pharmaceutical composition to tissues other than healthy, intact skin.

[0104] Without limiting the scope of the present invention, in some clinical situations, such as those involving large surgical or traumatic wounds, applying "large quantities" of antimicrobial pharmaceutical compositions as described herein may be important to achieve antimicrobial efficacy. As stated above, "large quantities" refers to a total therapeutic amount of an antimicrobial pharmaceutical composition comprising a synthetic cationic polypeptide, which represents 1 g for a 70 kg human, or 14.3 mg / kg, or more. When applying an antimicrobial pharmaceutical composition in large quantities, antimicrobial efficacy, physicochemical properties, and method of use should all be considered in combination to minimize the risk of local and systemic toxicity to the patient (human or animal). The antimicrobial pharmaceutical compositions described herein can be used in multiple and diverse clinical situations. In some embodiments, direct application to exposed surgical and traumatic tissues is among the most valuable applications. Figure 53 is a schematic depiction of wound classifications conventional in surgical situations. Wounds are classified as Class I (clean), Class II (clean / contaminated), Class III (contaminated), and Class IV (dirty / infected). Considering the method of use In both Class I and Class II wound situations, the antimicrobial pharmaceutical compositions described herein may be applied to the tissue prior to significant microbial contamination. In some embodiments, a combination of direct bactericidal activity and antimicrobial barrier properties is beneficial in such situations. Additionally, in Class III and Class IV wound situations, the antimicrobial pharmaceutical compositions described herein may be applied to the tissue after significant microbial contamination, or biofilm formation, or overt infection. In some embodiments, a combination of direct bactericidal activity and surfactant properties is beneficial in such situations.

[0105] The benefit of combining direct bactericidal activity with antimicrobial barrier properties can be illustrated in a variety of test situations. Figure 54 depicts the results of a microbially contaminated porcine ex vivo skin model. In this model, skin grafts can be pretreated with synthetic cationic polypeptide formulations in a horizontal position or tilted vertically for a period of time to encourage outflow of the antimicrobial formulation. This method is designed to better mimic in vivo tissue application. As the data shows, both KL-120 / 2.5 and KL-160 / 3.2 formulations are very effective at both 1% and 2% by weight on horizontal tissue samples. However, when the samples are tilted vertically for a period of time, the higher concentration formulations have a higher viscosity and are shown to be more effective. Both formulations have significantly stronger antimicrobial activity than is required to treat a microbial load of this magnitude.

[0106] FIG. 55 depicts the results of an in vivo porcine open wound model, where a synthetic cationic polypeptide KL-160 / 3.2 formulation was applied to a full thickness wound 15 minutes prior to microbial contamination. When evaluated 4 hours after inoculation with a high-density mixed culture of S. epidermidis and P. aeruginosa, the treated wounds showed little, if any, tissue microbial contamination compared to the control. FIG. 56 demonstrates that the effectiveness of pretreating tissue prior to microbial contamination is both concentration and time dependent. Thus, in a clinical setting, a preferred embodiment includes administering an effective amount of a synthetic cationic polypeptide, including multiple repeated applications over an extended period of time to exposed tissue (e.g., extended surgery and wounds with delayed closure). FIG. 57 shows the results of pretreatment of a rodent wound model (acting as a distinct body) with an implanted surgical mesh prior to microbial inoculation. In this model, the wound was closed immediately after inoculation. As the data show, a single dose of the antibacterial pharmaceutical composition having the synthetic cationic polypeptide KL-160 / 3.2 resulted in a highly significant (multi-log) reduction in CFU when assessed at 48 hours.

[0107] Collectively, these data demonstrate: (1) the benefits of direct bactericidal activity and microbial barrier properties; (2) the value of early treatment before significant microbial contamination; and (3) the importance of treatment planning that considers both the duration of tissue exposure and the timing of potential microbial contamination.

[0108] The benefit of the combination of direct bactericidal activity and surfactant properties can also be illustrated in various test situations. As shown in FIG. 53, the antimicrobial compositions described herein can be applied in many diverse clinical situations where there is already significant microbial contamination of tissues, existing biofilms or overt infections, or a combination of two or more. Such situations include surgical procedures targeting Class III and Class IV wounds. These include treatment of surgical sites, including those involving dissimilar bodies (e.g. periprosthetic joint infections, hernia mesh infections, breast implant infections). These situations also include many traumatic wounds, as well as surgical or non-surgical treatment of frequently contaminated areas, such as the sinuses of the head (especially in patients with chronic sinusitis). It should also be considered that repair of surgical sites with dissimilar bodies is at high risk for biofilm formation, even if they do not have overt infections. In these situations, as mentioned above, the antimicrobial pharmaceutical compositions described herein are likely to be applied to sites where microbial contamination is present. The combination of direct bactericidal activity and surfactant properties can be of particular benefit.

[0109] Figures 58 and 59 demonstrate potent activity against P. aeruginosa grown in biofilms using a formulation of synthetic antimicrobial polypeptides that have both bactericidal and surfactant activity. Figures 60-63 depict results that demonstrate these same principles in vivo. The antimicrobial compositions described herein that demonstrate direct bactericidal activity and surfactant properties prove highly effective when applied to tissue sites that are highly infected with both gram-positive (e.g., methicillin-resistant S. aureus) and gram-negative (e.g., P. aeruginosa) microorganisms.

[0110] In addition to significantly reducing the number of microorganisms, in various embodiments, these treatments appear to be tissue protective, which may be due in part to a reduction in tissue inflammation, a recognized cause of tissue damage, hi various embodiments, a reduction in inflammation can be obtained or enhanced by combining the antimicrobial compositions described herein with an anti-inflammatory drug.

[0111] This example illustrates the benefits of combining barrier properties with direct bactericidal activity and combining direct bactericidal activity with surfactant properties. In particular, one of skill in the art can use the guidance provided herein to design synthetic cationic polypeptides that have all three functionalities: direct bactericidal activity, barrier properties, and surfactant properties. In the manufacture of pharmaceutical compositions with these multifunctional synthetic cationic polypeptides, it is important that formulation and sterilization are performed in a manner that supports these functionalities.

[0112] In various embodiments, antimicrobial compositions comprising synthetic cationic polypeptides as described herein are preferred where the polypeptides and overall composition are designed to provide high levels of antimicrobial activity coupled with low risk of local and systemic toxicity, and physicochemical properties including self-organization into multimeric structures and / or viscosity are desirable.

Claims

1. 1. An antibacterial pharmaceutical composition, the antibacterial pharmaceutical composition comprising: an aqueous carrier; and an antimicrobial synthetic cationic polypeptide dispersed in said aqueous carrier at a concentration of about 0.01% to 5% by weight based on the total weight of said antimicrobial pharmaceutical composition; Including; The antimicrobial synthetic cationic polypeptide comprises: at least 40 amino acid units, said at least 40 amino acid units being a cationic segment comprising at least 10 amino acid units that are positively charged at neutral pH; and a hydrophobic segment comprising a plurality of hydrophobic amino acid units; a ratio of the cationic segment length to the hydrophobic segment length of 1.5 to 15; said antimicrobial synthetic cationic polypeptide having a viscosity of 2 centistokes (cSt) or greater at 37° C. at a concentration of 2% by weight in deionized water; said aqueous carrier when containing 2% by weight of said antimicrobial synthetic cationic polypeptide has a viscosity at 37° C. that is greater than the viscosity of said aqueous carrier when containing 2% by weight of albumin in place of said antimicrobial synthetic cationic polypeptide; The antibacterial pharmaceutical composition has low toxicity, with a mouse survival rate of 50% or more at 72 hours after intraperitoneal injection of 50 mg / kg of the antibacterial synthetic cationic polypeptide into healthy young adult mice; Sterilized by heat sterilization and / or filtration sterilization, Antibacterial pharmaceutical composition.

2. The antibacterial pharmaceutical composition of claim 1, wherein the cationic segment contains at least 15 amino acid units that are positively charged at neutral pH.

3. The antibacterial pharmaceutical composition described in claim 1 or 2, wherein the aqueous carrier is water or an aqueous solution of a pharmacologically acceptable salt.

4. An antibacterial pharmaceutical composition described in any one of claims 1 to 3, wherein the aqueous carrier further contains a non-ionic additive.

5. The antibacterial pharmaceutical composition described in claim 4, wherein the non-ionic additive is present in an amount effective to increase the osmotic concentration of the antibacterial pharmaceutical composition to a value at least 10% greater than the osmotic concentration of the antibacterial pharmaceutical composition without the additive.

6. The antibacterial pharmaceutical composition described in claim 4 or 5, wherein the nonionic additive is selected from the group consisting of glucose, mannitol, glycerol, xylitol, sorbitol, surfactants and combinations thereof.

7. An antibacterial pharmaceutical composition described in any one of claims 1 to 6, wherein the aqueous carrier further contains an additive in an amount that increases the viscosity of the antibacterial pharmaceutical composition.

8. An antibacterial pharmaceutical composition described in any one of claims 1 to 7, wherein the aqueous carrier further contains an additive in an amount that reduces the viscosity of the antibacterial pharmaceutical composition.

9. The antibacterial pharmaceutical composition described in any one of claims 1 to 8, wherein the antibacterial pharmaceutical composition has a weight average molecular weight and dispersity equivalent to those of the antibacterial synthetic cationic polypeptide of the non-sterilized antibacterial pharmaceutical composition.

10. An antibacterial pharmaceutical composition described in any one of claims 1 to 9, wherein the antibacterial pharmaceutical composition has a viscosity level at 37°C equivalent to that of the non-sterile antibacterial pharmaceutical composition.

11. An antibacterial pharmaceutical composition described in any one of claims 1 to 10, wherein the viscosity of the antibacterial pharmaceutical composition at 37°C is 20% to 200% of the viscosity of an otherwise equivalent non-sterile antibacterial pharmaceutical composition.

12. An antibacterial pharmaceutical composition described in any one of claims 1 to 11, wherein the antibacterial pharmaceutical composition has bactericidal activity equivalent to an otherwise equivalent non-sterile antibacterial pharmaceutical composition, the bactericidal activity being measured by a 60-minute time-kill assay against at least one bacterium selected from the group consisting of Staphylococcus aureus (S. aureus), Staphylococcus epidermidis (S. epidermidis), Pseudomonas aeruginosa (P. aeruginosa) and Escherichia coli (E. coli).

13. An antibacterial pharmaceutical composition described in any one of claims 1 to 12, wherein the hydrophobic segment contains at least five hydrophobic amino acid units.

14. The antibacterial pharmaceutical composition described in claim 13, wherein the at least five hydrophobic amino acid units are selected from leucine (L), isoleucine (l), valine (V), phenylalanine (F) or alanine (A).

15. An antibacterial pharmaceutical composition described in any one of claims 1 to 14, wherein the hydrophobic segment contains at least 10 hydrophobic amino acid units selected from leucine (L), isoleucine (L), valine (V), phenylalanine (F) or alanine (A).

16. An antibacterial pharmaceutical composition described in any one of claims 1 to 15, wherein the hydrophobic segment contains at least 15 hydrophobic amino acid units selected from leucine (L), isoleucine (L), valine (V), phenylalanine (F) or alanine (A).

17. An antibacterial pharmaceutical composition described in any one of claims 1 to 16, wherein the hydrophobic amino acid unit includes a leucine unit.

18. An antibacterial pharmaceutical composition described in any one of claims 1 to 17, wherein the positively charged amino acid unit includes a lysine unit.

19. The antibacterial pharmaceutical composition of any one of claims 1 to 18, wherein the antibacterial synthetic cationic polypeptide is a block copolypeptide comprising a hydrophobic leucine unit and a positively charged lysine unit.

20. An antibacterial pharmaceutical composition described in any one of claims 1 to 19, wherein the antibacterial synthetic cationic polypeptide has an average molecular weight of 3,000 Da or more and 70,000 Da or less.

21. The antibacterial pharmaceutical composition of any one of claims 1 to 20, wherein the antibacterial synthetic cationic polypeptide comprises an arrangement of hydrophobic amino acid units and positively charged amino acid units configured to promote self-organization of the antibacterial synthetic cationic polypeptide into a multimeric structure.

22. The antibacterial pharmaceutical composition of claim 21, wherein the antibacterial synthetic cationic polypeptide self-assembles into multimeric structures when measured at a critical aggregation concentration of less than 1000 μg / mL in deionized water at 37°C.

23. An antibacterial pharmaceutical composition according to any one of claims 1 to 22, wherein the antibacterial synthetic cationic polypeptide forms a self-supporting hydrogel at a concentration of 3% by weight in deionized water at 37°C.

24. An antibacterial pharmaceutical composition described in any one of claims 1 to 23, wherein the antibacterial synthetic cationic polypeptide exhibits surfactant activity at a concentration of 1 wt% in deionized water at 37°C, as measured by a reduction in surface tension of at least 10% compared to deionized water alone.

25. An antibacterial pharmaceutical composition described in any one of claims 1 to 24, further comprising an anti-inflammatory compound.

26. The antibacterial pharmaceutical composition described in claim 25, wherein the anti-inflammatory compound is selected from the group consisting of corticosteroids, histamine inhibitors and cytokine inhibitors.

27. ​​An antibacterial pharmaceutical composition described in any one of claims 1 to 26 for preventing microbial contamination of tissue other than intact, healthy skin.

28. An antibacterial pharmaceutical composition described in any one of claims 1 to 26 for reducing the microbial load in or on tissue other than intact, healthy skin.

29. The antibacterial pharmaceutical composition described in claim 27 or 28, wherein the tissue is selected from affected skin, a surgical site, a traumatic wound, dissected tissue, an abscess, the abdominal cavity, the pulmonary airways, the sinuses and the urinary tract.

30. The composition of claim 1, wherein the aqueous carrier comprises 2% by weight of an antimicrobial synthetic cationic polypeptide, and the aqueous carrier has a viscosity of greater than 6 cSt at 40°C. The antibacterial pharmaceutical composition described above.

31. Any one of claims 1 to 30, wherein the aqueous carrier comprises 2% by weight of an antimicrobial synthetic cationic polypeptide, and the aqueous carrier has a viscosity of greater than 15 cSt at 40°C. The antibacterial pharmaceutical composition described in 32. Any one of claims 1 to 31, wherein the aqueous carrier comprises 2% by weight of an antimicrobial synthetic cationic polypeptide, and the aqueous carrier has a viscosity of greater than 20 cSt at 40°C. The antibacterial pharmaceutical composition described in 33. Any one of claims 1 to 32, wherein the aqueous carrier comprises 2% by weight of an antimicrobial synthetic cationic polypeptide, and the aqueous carrier has a viscosity of greater than 24 cSt at 40°C. The antibacterial pharmaceutical composition described in

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