Compositions with enhanced efficacy and safety and use of topically applied antibacterial synthetic cationic polypeptides

Abstract

To provide an antibacterial pharmaceutical composition with low risk of local tissue toxicity and / or low risk of systemic / remote organ toxicity.SOLUTION: An antibacterial pharmaceutical composition is formulated to allow topical application in vivo at a dose that provides antibacterial efficacy with low risk of local tissue toxicity and / or low risk of systemic / remote organ toxicity. In various embodiments, the antibacterial pharmaceutical composition comprises antibacterial synthetic cationic polypeptides dispersed in an aqueous carrier and formulated to achieve a desired degree of polymer self-assembly and / or composition viscosity.SELECTED DRAWING: Figure 32

Description

Technical Field

[0001] <Information on Related Applications> This application claims the priority of U.S. Provisional Patent Application No. 62 / 482,630, filed on April 6, 2017, the entire content of which is incorporated herein by reference.

[0002] This disclosure relates to antibacterial pharmaceutical compositions containing cationic antibacterial agents and methods of using them to prevent and / or treat infections.

Background Art

[0003] Topically applied antibacterial agents, including disinfectants and antibiotics, often fail to achieve their effectiveness in preventing and treating infections, resulting in significant patient morbidity and mortality. Specific uses (including application to specific tissues) and dosages (concentration, volume, number of applications) can be limited by one or more toxicity risks. Concerns about such toxicity can lead to clinical application at inadequate dosages or complete avoidance of use in certain pathophysiological situations.

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

[0005] U.S. Patent No. 9,017,730 describes synthetic cationic copolypeptides containing 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)) in various ratios. U.S. Patent No. 9,017,730 shows that a segmented or block structure 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. Further, U.S. Patent 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. U.S. Patent No. 9,017,730 describes the topical application of many synthetic cationic copolypeptide formulations but provides no evidence of toxicity that may result from using these products in vivo.

[0006] U.S. Patent No. 9,446,090 describes synthetic cationic polypeptides in a mutually water-miscible mixture containing such polypeptides and a second pharmacologically acceptable polymer. Specific examples describe the antibacterial activity against specific bacteria using specific mixtures of a synthetic cationic polypeptide 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, the data showed that addition of the second polymer to the mixture maintained the antibacterial activity of the synthetic cationic polypeptide in vitro and, in some cases, enhanced the overall antibacterial ability in vivo. However, U.S. Patent No. 9,446,090 provides no evidence of toxicity that may result from using these mixtures of synthetic cationic polypeptides and other pharmacologically acceptable polymers in vivo. Further, it leaves open the possibility that the second polymer may increase the toxicity of the mixture compared to the synthetic cationic polypeptide.

[0007] U.S. Patent Nos. 9,017,730 and 9,446,090 describe significant advancements in the prior art, but many challenges remain, particularly regarding the development of pharmacologically acceptable products of topically applied cationic antibacterial agents that have both high efficacy and high safety in vivo. SUMMARY OF THE INVENTION

[0008] We have developed a cationic antimicrobial pharmaceutical composition and method of use that permits topical application in vivo at a dosage that provides antibacterial efficacy with a low risk of local tissue toxicity and / or a low risk of systemic / remote organ toxicity.

[0009] In developing the present invention, we have found that the risk of toxicity after topical application of a cationic antibacterial agent in vivo is particularly relevant to the time of high-dose application at the tissue site or pathophysiological conditions other than healthy intact skin. Such sites and pathophysiological conditions can include open wounds, body cavities, body orifices, affected skin, and others. These tissue sites and pathophysiological conditions can demonstrate higher local sensitivity and / or higher absorption and systemic distribution of the topically applied antibacterial agent or excipient compared to healthy intact skin. Greater local tissue toxicity and greater systemic / remote organ toxicity can 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 changing the material design of synthetic cationic polypeptides can result in differences in the effects on antibacterial efficacy and toxicity in vivo; second, we have discovered that changing the formulation of synthetic cationic polypeptides can result in different effects on antibacterial efficacy and toxicity in vivo; third, we have discovered that sterilizing the products of synthetic cationic polypeptides can detrimentally affect the molecular integrity and biophysical properties and thus can affect antibacterial efficacy, toxicity, or both. Accordingly, we have discovered a combination of solutions to the problems found.

[0011] In various embodiments, pharmaceutical compositions and methods of use are provided, whereby an effective amount of antibacterial synthetic cationic polypeptides are provided to various tissue sites and / or under various pathophysiological conditions with reduced risk of local and / or systemic toxicity such that topical application is tolerated. Without limiting the scope of the invention, in various embodiments, this is achieved by one or more of the following (A), (B), and (C):

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

[0013] (B) Formulating antibacterial synthetic cationic polypeptides in a pharmaceutical composition in such a way that they can exhibit viscosity enhancing properties. Without limiting the scope of the invention, this approach is thought to have the effect of improving tissue coverage and / or enhancing retention time, resulting in greater efficacy at the applied dose. Furthermore, it has been unexpectedly found that improving the formulation in this way enhances safety. Without limiting the scope of the invention, excipients include saccharides and sugar alcohols such as sodium chloride, potassium chloride, glucose, mannitol, glycerol, xylitol, and sorbitol, and surfactants and combinations thereof.

[0014] (C) Sterilizing the pharmaceutical composition in such a way that the sterilized pharmaceutical composition can exhibit a viscosity equivalent to that of the non-sterilized pharmaceutical composition. Without limiting the scope of the invention, this approach is thought to protect the molecular structure and function of antibacterial synthetic cationic polypeptides, and to have the effect of reducing the risk of contamination by certain microbial life forms (e.g., fungal dermatophytes) to which the product may develop resistance to the effects of antibacterial synthetic cationic polypeptides. Protecting, as well as reducing the risk of contamination by certain microbial life forms (e.g., fungal dermatophytes) to which the product may develop resistance to the effects of antibacterial synthetic cationic polypeptides.

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

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

[0017] Another embodiment provides a method for reducing the microbial load in or on tissues other than intact, healthy skin, the method comprising: identifying a mammalian subject having a tissue site other than intact, healthy skin that has a microbial load; and administering to the tissue site an antibacterial 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]

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Mode for Carrying Out the Invention

[0020] Definitions As used in the context of describing an antibacterial synthetic cationic polypeptide herein, the term "antibacterial" 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 Staphylococcus aureus (S. aureus), Staphylococcus epidermidis (S. epidermidis), Pseudomonas aeruginosa (P. aeruginosa) and Escherichia coli (E. coli).

[0021] As used in the context of describing an antibacterial synthetic cationic polypeptide in this specification, the term "polypeptide" has its ordinary meaning as understood by one of ordinary skill in the art, and thus includes polymers containing two or more amino acid repeating units (also referred to as amino acid residues or more simply units or residues) linked to one another by peptide bonds. A copolypeptide is a type of polypeptide that contains two or more different amino acid repeating units. The molecular weight of the polymer is a weight average as determined by size exclusion chromatography (SEC) with a molecular weight standard or using light scattering detection.

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

[0023] As used in the context of describing an antibacterial synthetic cationic polypeptide herein, the term "hydrophobic" block has its ordinary meaning as understood by one of ordinary skill in the art, and thus a block or segment includes an array arrangement that contains a plurality of hydrophobic amino acid units therein. Examples of hydrophobic amino acid units are known to one of ordinary 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 one of ordinary skill in the art, and thus a block or segment includes an array arrangement that contains a plurality of hydrophilic amino acid units therein. Examples of hydrophilic amino acid units are known to one of ordinary skill in the art and include serine (S), threonine (T), aspartic acid (D), and glutamic acid (E), as well as the positively charged amino acids lysine (K), arginine (R), histidine (H), and ornithine (O).

[0024] As used in the context of describing an antibacterial synthetic cationic polypeptide herein, the terms "positively charged" and "cationic" have their ordinary meaning as understood by one 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, if one or more of these positively charged amino acid units are present in a polypeptide (in an amount exceeding other anionic units), the polypeptide can be made cationic.

[0025] As used in the context of describing a sterilized antimicrobial pharmaceutical composition herein, the term "sterilized" has its ordinary meaning as understood by one of ordinary skill in the art, and thus, a composition that has undergone a sterilization process or in which known pathogens in the composition are surely absent or reduced to such an extent that it is clinically acceptable for topical administration to a body orifice (such as intranasal administration) or to open skin such as at a surgical site for administration to an open wound. Compositions that have undergone such a sterilization process include, by way of non-limiting example, heat sterilization (such as autoclaving), filtration sterilization, treatment with chemicals such as radiation and / or ethylene oxide.

[0026] As used in the context of describing a self - organizing polypeptide herein, the term "self - organizing" has its ordinary meaning as understood by one of ordinary skill 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 segments or blocks of the polypeptide are loosely bound to each other by intermolecular attractions between specific segments or blocks of the polypeptide in the medium. For example, as referred to in U.S. Patent No. 9,017,730, self - organization of block cationic copolypeptides is observed in aqueous solution, giving rise to various hierarchical structures that depend on the arrangement of the hydrophobic domains and the effect on intermolecular attractive interactions between the polymer chains. In contrast, it is shown in U.S. Patent No. 9,017,730 that random copolypeptides do not exhibit self - organization. One of ordinary skill in the art will be familiar with various techniques for determining whether a synthetic cationic polypeptide self - organizes (see, for example, U.S. Patent No. 9,017,730). Compared to other synthetic cationic polypeptides that present a random arrangement and do not exhibit self - organization in dilute solution, synthetic cationic polypeptides that self - organize generally exhibit a high viscosity.

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

[0028] As used herein in the context of describing synthetic cationic block copolypeptides that self - organize as compared to other equivalent randomly synthesized cationic copolypeptides As such, the term "other equivalent random synthetic cationic copolypeptides" has the ordinary meaning as understood by one of ordinary skill in the art, and thus, the approximate molecular weight and the relative numbers of hydrophobic and hydrophilic amino acid repeat units are the same as those of self-assembling synthetic cationic block copolypeptides, but the sequence arrangement of those amino acid repeat units of equivalent copolypeptides is more random than that of blocks, and includes copolypeptides. For example, with respect to a self-assembling block copolypeptide having a hydrophilic (positively charged) lysine block with an average length of about 120 units and a hydrophobic leucine block with an average length of about 30 units, other equivalent random synthetic cationic copolypeptides include, on average, about 120 lysine units and about 30 leucine units per copolypeptide chain, but the sequence arrangement of those units along the chain of the random copolypeptide is a statistical distribution that reflects the concentrations of lysine and leucine monomers in the polymerization mixture.

[0029] As used in the context of describing administering a sterilized antimicrobial pharmaceutical composition in a therapeutically effective amount to at least partially prevent and / or treat an infection at a body site of a mammal, the term "therapeutically effective amount" or "effective amount" has its ordinary meaning as understood by one of ordinary skill in the art and thus includes administering an amount of the copolypeptide that is at least 10-fold greater than the dosage required to achieve the desired preventive and / or therapeutic effect. Typically, administering a total therapeutic amount of an antimicrobial pharmaceutical composition that includes administering to a 70 kg human an amount of the synthetic cationic polypeptide of 1 g, representing 14.3 mg / kg, is considered a therapeutically effective amount. One of ordinary skill in the art understands that bioactive substances are generally administered within a "therapeutic window" that includes the dosage range within which the desired therapeutic response is achieved without causing significant adverse effects in the subject to which they are administered. This dosage range is generally between the minimum effective concentration (MEC) and the maximum toxic concentration (MTC) and is typically determined in advance for each bioactive substance and communicated to the subject and / or caregiver in the form of a recommended dosage. However, in some situations, such as topically administering an antimicrobial composition to a body orifice and / or an open wound of a mammalian subject, it may not be practical to determine the MEC, and thus the flexibility to administer the antimicrobial composition in a therapeutically effective amount is highly advantageous. For example, when treating an open wound in an emergency situation where time is of the essence, it is highly advantageous to have the flexibility to apply the antimicrobial composition to the open wound without concern that the caregiver will administer an amount that exceeds the MTC in a therapeutically effective amount (e.g., an amount that is at least 10-fold greater than the MEC). The MEC of a particular sterilized antimicrobial pharmaceutical composition can be determined by methods known to one of ordinary skill in the art, such as those described in the examples below (e.g., the amount effective to achieve 3-log CFU kill in an in vitro time-kill assay).

[0030] As used in the context of describing an antibacterial pharmaceutical composition comprising an aqueous carrier and an antibacterial synthetic cationic polypeptide dispersed therein or consisting thereof, the term "aqueous carrier" has its ordinary meaning as understood by one of ordinary skill in the art and thus includes various aqueous carrier systems that can optionally contain 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 antibacterial pharmaceutical composition comprising an aqueous carrier and an antibacterial synthetic cationic polypeptide dispersed therein or consisting thereof. The amount of the cationic polypeptide dispersed in the aqueous carrier can vary over a wide range mainly depending on the desired viscosity of the antibacterial pharmaceutical composition. For example, in various embodiments, the amount of the synthetic cationic polypeptide of the antibacterial pharmaceutical composition is about 0.001% to about 10% by weight based on the total weight of the antibacterial pharmaceutical composition. In some embodiments, the amount of the synthetic cationic polypeptide dispersed in the aqueous carrier is about 0.01% to about 5% by weight based on the total weight of the antibacterial pharmaceutical composition.

[0032] In various embodiments, the antibacterial synthetic cationic polypeptide dispersed in the aqueous carrier contains amino acid units (at neutral pH) with multiple positive charges. In one embodiment, the synthetic cationic polypeptide contains at least 40 amino acid units, at least some of which are positively charged. In one embodiment, 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 amino acid units with multiple positive charges at neutral pH. In one embodiment, the multiple positively charged amino acid units of the synthetic cationic polypeptide include positively charged lysine units.

[0033] In various embodiments, the antimicrobial synthetic cationic polypeptide has a viscosity of 2 centistokes (cSt) or greater as measured at 37 °C at a concentration of 2% by weight in deionized water. 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 degree to which the polypeptide self-organizes. In certain embodiments, the antimicrobial synthetic cationic polypeptide has a viscosity greater than that of bovine serum albumin as measured at 37 °C at a concentration of 2% by weight in deionized water.

[0034] The antimicrobial synthetic cationic polypeptide can include other monomer units in addition to the positively charged amino acid units. For example, in various embodiments, the antimicrobial synthetic cationic polypeptide may further include a plurality of hydrophobic amino acid units. In various embodiments, the number of hydrophobic amino acid units in 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 certain embodiments, the plurality of hydrophobic amino acid units of the synthetic cationic polypeptide includes leucine units.

[0035] The arrangement of the amino acid units of the synthetic cationic polypeptide can be random, block-like, or a combination thereof. For example, in certain embodiments, the arrangement of the hydrophobic and 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 certain embodiments, the synthetic cationic polypeptide is a block copolymer including hydrophobic leucine units and positively charged lysine units.

[0036] In various embodiments, the antimicrobial synthetic cationic polypeptide self-organizes in water and other aqueous carriers into a multimeric structure. Examples of multimeric structures include micelles, sheets, vesicles, and fibrils (see U.S. Patent No. 9,017,730). In certain embodiments, the antimicrobial synthetic cationic polypeptide forms a self-standing hydrogel at a 3 wt% concentration in deionized water at 37°C. In certain embodiments, the antimicrobial synthetic cationic polypeptide exhibits surface activity as measured by a decrease in surface tension of at least 10% or at least 20% compared to deionized water alone in deionized water at 37°C. In certain embodiments, the self-organization of the antimicrobial synthetic cationic polypeptide is evidenced by a critical aggregation concentration that is 1000 μg / mL or less at 37°C in deionized water for the polypeptide. In certain embodiments, the self-organization of the antimicrobial synthetic cationic polypeptide is evidenced by a critical aggregation concentration that is 100 μg / mL or less at 37°C in deionized water for the polypeptide.

[0037] The self-organization of the antimicrobial synthetic cationic polypeptide can be controlled in various ways. For example, in certain embodiments, the antimicrobial synthetic cationic polypeptide includes an arrangement of hydrophobic amino acid units and positively charged amino acid units configured to promote the self-organization of the antimicrobial synthetic cationic polypeptide into a multimeric structure. For example, the self-organization of the polypeptide is promoted by a block-like arrangement of hydrophobic amino acid units and positively charged amino acid units. The self-organization in an aqueous carrier tends to be enhanced when the content of hydrophobic amino acid units in the polypeptide is high and / or the blocks of hydrophobic amino acid units are long.

[0038] The antibacterial synthetic cationic polypeptides described herein can be dispersed in an aqueous carrier to form an antibacterial 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 nonionic additive, or a combination thereof. Salts tend to inhibit the self-assembly of polypeptides and thus excessive salts should be avoided. Normal saline, half-strength saline, quarter-strength saline, and phosphate-buffered saline are examples of suitable aqueous carriers containing pharmacologically acceptable salts. In certain embodiments, the aqueous carrier contains sodium chloride. In various embodiments, the aqueous carrier is an aqueous solution containing a nonionic additive. Examples of suitable nonionic 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, nonionic additives, or combinations thereof. In various embodiments, the aqueous carrier contains a pharmacologically acceptable salt in an amount of 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 certain embodiments, the amount of additive in the aqueous carrier is selected to control the viscosity of the antibacterial pharmaceutical composition. In certain embodiments, the aqueous carrier contains an additive in an amount that increases the viscosity of the antibacterial pharmaceutical composition. In certain embodiments, the aqueous carrier contains an additive in an amount that decreases the viscosity of the antibacterial pharmaceutical composition. In certain embodiments, the nonionic additives are present in an amount effective to increase the osmotic concentration of the antibacterial pharmaceutical composition by at least 10% more than that of the antibacterial pharmaceutical composition without said additives. In various embodiments, the concentration of the additive in the antibacterial pharmaceutical composition is about 0.1 wt% to about 10 wt% based on the total weight. In various embodiments, the concentration of the nonionic additive in the antibacterial pharmaceutical composition is about 0.01 wt% to about 2 wt%, or about 0.05 wt% to about 5 wt% based on the total weight.

[0040] In various embodiments, the antimicrobial pharmaceutical compositions as described herein are sterilized by sterilization techniques configured to achieve a sterilized antimicrobial pharmaceutical composition. In one embodiment, the sterilization techniques are configured to have minimal impact on the chemical structure of the synthetic cationic polypeptide and / or the property of self-organization of the synthetic cationic polypeptide. Such sterilization techniques are illustrated in FIGS. 42-52. In one embodiment, the antimicrobial pharmaceutical composition as described herein is sterilized by a sterilization technique configured to achieve a sterilized 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 said sterilization technique. In one embodiment, the antimicrobial pharmaceutical composition is sterilized by said sterilization technique configured to achieve a sterilized antimicrobial pharmaceutical composition having a viscosity level at 37° C. equivalent to that of said antimicrobial pharmaceutical composition not sterilized by the sterilization technique. In one embodiment, the viscosity at 37° C. of the sterilized antimicrobial pharmaceutical composition is 20% to 200% of the viscosity of the unsterilized antimicrobial pharmaceutical composition, with other things being equal.

[0041] In certain embodiments, the antibacterial pharmaceutical composition has low toxicity as measured by a mouse survival rate of 50% or more at 72 hours after injection into the peritoneal cavity of a plurality of mice at a dosage of 10 mL / kg. In certain embodiments, the antibacterial pharmaceutical composition has low toxicity as measured by a mouse survival rate of 50% or more at 72 hours after injection into the peritoneal cavity of a plurality of mice at a dosage of 20 mL / kg. In certain embodiments, the antibacterial pharmaceutical composition has low toxicity as measured by a mouse survival rate of 50% or more at 72 hours after injection into the peritoneal cavity of a plurality of mice at a dosage of 40 mL / kg. In certain embodiments, the antibacterial pharmaceutical composition has bactericidal activity equivalent to that of an otherwise equivalent non-sterile antibacterial pharmaceutical composition, and the bactericidal activity is 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).

[0042] Incorporating other active pharmaceutical ingredients into the antibacterial pharmaceutical compositions described herein can enhance antibacterial ability and / or reduce the risk of toxicity, both locally and systemically. In particular, incorporating other antibacterial agents, including antibiotics, antiseptics, iodine compounds, and / or silver compounds, can work in concert with the synthetic cationic polypeptide to assist in preventing and / or treating infections. Additionally, incorporating one or more anti-inflammatory agents can enhance ability and / or reduce the risk of toxicity, both locally and systemically. Local inflammation can contribute to the cause of various disease situations, including microbial contamination or infection. Examples include otitis externa, chronic rhinosinusitis, pulmonary conditions, and symptoms of certain wounds. Such conditions could be treated with a combination of the synthetic cationic polypeptide and an anti-inflammatory agent, such as a corticosteroid, antihistamine, and / or anti-cytokine agent. Thus, including an anti-inflammatory agent in an antibacterial composition containing a synthetic cationic polypeptide can provide benefits.

[0043] In certain embodiments, the antibacterial pharmaceutical composition comprises an anti-inflammatory compound. For example, in certain embodiments, the anti-inflammatory agent is selected from the group consisting of corticosteroids, histamine inhibitors, and cytokine inhibitors. Examples of corticosteroids include betamethasone dipropionate, clobetasol propionate, diflorasone diacetate, 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 polypeptide is made in the manner taught in U.S. Patent Nos. 9,017,730 and / or 9,446,090, and all purposes including teaching such methods for making cationic polypeptides are hereby expressly incorporated by reference herein. The antibacterial pharmaceutical composition can be made by combining the antibacterial synthetic cationic polypeptide with an aqueous carrier and thereby dispersing (e.g., dissolving) the polypeptide in the aqueous carrier. For example, such a combination can be achieved by stirring and mixing the components (cationic polypeptide, aqueous carrier, and any optional components such as inflammatory compounds) at a temperature of about 20°C to 90°C for a time period effective to disperse (e.g., dissolve) the polypeptide. The components can be mixed together in any order, although those skilled in the art may prefer a particular order in individual cases.

[0045] Method for preventing microbial contamination Various embodiments provide a method for preventing microbial contamination of tissues that are particularly suitable for tissues other than intact, healthy skin. For example, in certain embodiments, such a method includes identifying a mammalian subject having a tissue site other than intact, healthy skin that is at risk of being microbially contaminated Including steps. Examples of such tissue sites include affected skin, surgical sites, traumatic wounds, dissected tissues, abdominal cavities, lung airways, sinuses, and urinary tracts. In certain embodiments, the method includes administering to the tissue site an antimicrobial pharmaceutical composition as described herein in an amount sufficient to at least partially protect the tissue site from being contaminated by microorganisms. For example, in some embodiments, the method at least partially protects the tissue site from infection by 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). The method further includes collecting the tissue site and evaluating its microbial load, for example, the load of 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). In certain embodiments, the antimicrobial pharmaceutical composition is administered during surgery to the surgical site. Administration of the antimicrobial pharmaceutical composition to the tissue site can be achieved by direct topical administration.

[0046] The amount of the antimicrobial pharmaceutical composition effective to at least partially protect the tissue site from being contaminated by microorganisms can be determined using routine experimentation informed by the guidance provided herein by those of ordinary skill in the art. In various embodiments, the antimicrobial pharmaceutical composition has a wide therapeutic window, and thus a relatively wide range of doses is provided where the desired protection from infection is achieved. In some embodiments, this wide therapeutic window facilitates the administration of large amounts of the antimicrobial pharmaceutical composition to the tissue site.

[0047] Method for reducing microbial load Various embodiments provide methods for reducing the microbial burden within or on tissues that are other than intact, healthy skin. For example, in certain embodiments, such methods include identifying a mammalian subject having a tissue site other than intact, healthy skin that has a microbial burden. Examples of such tissue sites include affected skin, surgical sites, traumatic wounds, debrided tissue, the abdominal cavity, the pulmonary airway, sinuses, and the urinary tract. In certain embodiments, the tissue site is contaminated, infected, or both, by microorganisms. In certain embodiments, the method includes administering to the tissue site an antibacterial pharmaceutical composition in an amount effective to at least partially reduce the microbial burden as provided herein. For example, in some embodiments, the method reduces at least partially the microbial burden at the tissue site of 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). The method further includes obtaining a tissue site and assessing the microbial burden, for example, the burden of 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). In certain embodiments, the antibacterial pharmaceutical composition is administered during surgery to a tissue site that is contaminated, infected, or both, by microorganisms.

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

Example

[0049] Synthetic cationic polypeptides can be prepared using various amino acid sequence arrangements , some of which are illustrated schematically in FIG. 1. Specific arrangements may be referred to as blocks or segments. Typically, these will be stretches of amino acid units that include multiple amino acids of a certain type (e.g., cationic, anionic, hydrophobic). The synthetic cationic polypeptides described herein are made according to the synthetic methods described in U.S. Patent Nos. 9,017,730 and 9,446,090, which are hereby incorporated by reference in their entirety for all purposes, including for the purpose of describing the synthetic cationic polypeptides and their manufacturing methods.

[0050] Cationic blocks have multiple positive charges at neutral pH and can vary significantly 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). Cationic blocks do not have to consist entirely of cationic amino acids. In addition to multiple cationic amino acids, the block segment may include other amino acids, such as serine (S) and threonine (T), which are polar amino acids and will help maintain hydrophilicity. A small proportion of negatively charged amino acids and / or hydrophobic amino acids may also be included in the cationic block as long as there are more cationic units than anionic units within the block.

[0051] The hydrophobic block has a plurality of hydrophobic amino acids and can have a length that can be significantly varied, typically from about 5 to about 60 amino acid units. The hydrophobic block can also exhibit a secondary structure (such as an α helix relative to disorder). Hydrophobic amino acids are not charged at pH 7.0. In addition, they mostly have side chains consisting of carbon and hydrogen, have a very small dipole moment, 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). The hydrophobic block does not have to consist entirely of hydrophobic amino acid units. In addition to the plurality of hydrophobic amino acids, the block segment may contain other amino acids, including polar amino acids such as serine (S) and threonine (T). A small proportion of charged amino acids may be included in the hydrophobic block.

[0052] Synthetic cationic polypeptides having a cationic-hydrophobic block structure can be prepared using a wide range of total chain lengths, typically with a lower side of about 20 amino acid units or less and an upper side of about 400 amino acid units or more. As depicted in Figure 2, when describing the cationic polypeptides with the block structure, they can be classified into three groups: (I) long cationic segments (200 amino acid units or more); (II) intermediate cationic segments (100 - 199 amino acid units); and (III) short cationic segments (10 - 99 amino acid units). The ratio of the length of the cationic block to the length of the hydrophobic block can vary over a wide range, typically with a lower side of about 1.5 and an upper side of about 15 or more. The average molecular weight of these synthetic cationic polypeptides can vary over a wide range, typically with a lower side of about 3,000 Da and an upper side of about 70,000 Da or more. Figure 3 shows the detailed composition of such synthetic cationic polypeptides having a segmented structure.

[0053] As described in more detail below, we synthesized and tested a very large number of different synthetic cationic polypeptides having a block array arrangement. For comparison, we also synthesized cationic polypeptides having an equivalent amino acid composition but lacking a block or segmented array arrangement. These synthetic cationic polypeptides were found to vary in multiple functional properties, including antibacterial activity, hemostatic properties, barrier properties, and surface activity. Furthermore, it has been demonstrated that both molecular design and formulation can affect these functional properties. With respect to molecular design properties, we observed that the overall chain length and the ratio of cationic / hydrophobic blocks to hydrophilic blocks are properties that have a high impact on function. Specific properties within each block, including amino acid selection and enantiomeric purity, also contribute.

[0054] In this application, we use a nomenclature based on two main features of synthetic cationic polypeptides: the overall chain length and the ratio of the cationic block length to the 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. Patent Nos. 9,017,730 and 9,446,090 (using 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 -RAN、 K 360 L 54 、 K 360 L 72 、 K 360 (rac-L) 36 、 K 360 (rac-L) 54 and K 360 (rac-L) 72 include.

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

[0057] Figure 5 depicts the concentration-dependent antibacterial activity of KL-160 / 3.2 in water. There is a clear concentration-dependent effect with activity against Staphylococcus aureus (S. aureus), Pseudomonas aeruginosa (P. aeruginosa), and Candida albicans (C. albicans) at concentrations as low as about 1.6 μg / mL. A summary of the antibacterial activity of KL-160 / 3.2 observed through numerous experiments is provided in Figure 6. As mentioned below, when prepared at high concentrations in water (i.e., less than 2 wt %), KL-160 / 3.2 forms self-standing hydrogels. These properties of the synthetic cationic polypeptide result in a desired combination of antibacterial activity and barrier function. Importantly, it is recognized that the functional properties of these antibacterial formulations depend on the presence, absence, or degree of specific processes and / or handling procedures, including formulation (e.g., the presence and concentration of additives), as well as the sterilization method.

[0058] Example 2 Synthetic cationic polypeptides can be designed as shown in Figures 7 - 9 to enable formulations with robust bactericidal activity and surfactant power. Figure 7 describes an example of a 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 is about 100 amino acid units, and the lysine to racemic leucine or K:rL was found to be 5.7. This synthetic cationic polypeptide is designated as KrL-100 / 5.7, and the SEC chromatogram of the polymer shows a single peak with a relatively low dispersity (D) of 1.1.

[0059] Figure 8 depicts the concentration-dependent antibacterial activity of KrL-100 / 5.7 in water. There is a clear concentration-dependent effect with activities against Staphylococcus aureus (S. aureus), Staphylococcus epidermidis (S. epidermidis), Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa) and Candida albicans (C. albicans) at concentrations as low as about 1.6 μg / mL. A summary of the antibacterial activity of KrL-100 / 5.7 observed through many experiments is provided in Figure 9. As mentioned below, when prepared in water, KrL-100 / 5.7 demonstrates surface activity. These properties of the synthetic cationic polypeptide result in a desired combination of antibacterial activity and surface-active function, thereby enhancing the ability against biofilms and / or debridement of tissue wounds. Importantly, it is recognized that the functional properties of these antibacterial formulations depend on the presence, absence or degree of specific processes and / or handling procedures, including formulation (e.g., the presence and concentration of additives), as well as the sterilization method.

[0060] Example 3 Self-organization of cationic antimicrobial peptides into multimeric complexes may be beneficial. Synthetic cationic polypeptides with a block sequence arrangement can be designed and formulated to self-organize into multimeric structures. Two examples are depicted in Figure 10: KL-160 / 3.2 was designed to self-organize into a fibrous structure to form a barrier hydrogel, and KrL-100 / 5.7 was designed to self-organize into a micellar structure to have surface-active properties. Note that these two synthetic cationic polypeptides are also described in Figures 4-6 and Figures 7-9 as mentioned above.

[0061] Various embodiments of the present invention include an antibacterial composition comprising at least one antibacterial agent that forms a multimeric complex, either by itself and / or with one or more other components of the composition. By way of explanation, when such a multimeric complex is generated, intermolecular bonds are enhanced. Such bonds are reversible. Such bonds can cause a hydrophobic attraction effect, either in whole or in part. Such bonds can be either covalent or non-covalent. Such bonds can reduce or delay systemic absorption. Such bonds can reduce the risk of local and / or systemic toxicity.

[0062] The critical aggregation concentration (CAC) is one criterion for the self-organization of molecules in an aqueous environment. The 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 measured by pyrene fluorescence. It is noteworthy that the polylysine chain (K-100) and the lysine-leucine synthetic cationic polypeptide lacking a 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 a block sequence arrangement demonstrate significantly 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 measured values of CAC by the surface tension method using the synthetic cationic polypeptide KrL-100 / 5.7.

[0063] Various embodiments of the present invention may include an antibacterial composition comprising at least one antibacterial agent and having a critical aggregation concentration (CAC) of 500 μg / mL or less when dissolved in water. This CAC can be measured by methods known in the art, such as those using pyrene fluorescence. Other antibacterial agents can have a lower CAC. For example, the antibacterial agent can 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 measure related to molecular properties as well as the properties of pharmaceutical compositions. Unexpectedly, we have discovered that increased viscosity can be a marker not only of increased safety but also of increased wound antibacterial ability. Many molecular design properties affect the viscosity of synthetic cationic polypeptides. These include the overall chain length, amino acid composition, ratio of cationic units to hydrophobic units, and the sequence arrangement of amino acid residues (e.g., random versus blocky). It should also be noted that other components that can be present within synthetic cationic polypeptides (e.g., counterions, other salts, and residual solvents) can affect the viscosity of aqueous formulations. Additionally, various additives (e.g., salts, nonionic tonicity-adjusting excipients, surfactants) that can be used in the preparation of pharmaceutical compositions in effective amounts to affect viscosity. As described below, the sterilization method can also significantly affect the viscosity.

[0065] Figure 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 evidenced by this data, even the presence of polylysine chains of approximately 100 amino acid units (K-100) or approximately 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, with a random / statistical array arrangement (i.e., not block) also has only a modest effect on viscosity (a value of 1.1 cSt). In comparison, a number of synthetic cationic polypeptides with a segmented or block array arrangement demonstrate enhanced viscosities in the high range of 290 cSt to 1.4 cSt with this set of data. The overall chain length was shown to increase viscosity; an increase in the length of the hydrophobic block was shown to increase viscosity; and the enantiomeric purity of the hydrophobic leucine amino acid was shown to increase viscosity. For comparison, Figure 13(b) shows that bovine serum albumin has only a minor effect, if any, on the viscosity of aqueous formulations at concentrations of 1 and 2 wt% under these conditions. Albumin is an abundant blood protein with a molecular weight of approximately 66.5 kDa and an overall chain length of approximately 583 amino acid units. These data indicate that molecular size alone is not sufficient to fully explain the viscosity effect.

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

[0067] The viscosity of synthetic cationic polypeptides can also be measured under the influence of shear force (viscosity). By this method, the shear thinning or shear thickening properties can be evaluated. These shear thinning or shear thickening properties can be important for both the overall ability in vivo as well as the ease of application to tissues. In particular, we have found that the shear thinning property facilitates manual spreading onto tissues, which can occur in various medical and surgical situations. As depicted in Figure 16, the viscosity of synthetic cationic polypeptide KL-120 / 2.5 at 1.5 wt%, 2.0 wt% and 3.0 wt% in water was evaluated using a rotational viscometer against shear forces that increase viscosity. A shear-dependent (increasing spindle speed) decrease in viscosity was observed (measured in centipoises (cP)). This "shear thinning" effect was seen at the three tested concentrations. A concentration-dependent increase in viscosity was also observed as shown in Figure 16.

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

[0069] As an example, a formulation of at least one antibacterial synthetic cationic polypeptide at a concentration of 10 mg / mL or 1 wt% in water has a viscosity of 1.25 centistokes to 500 centistokes (cSt; mm 2 / s) at 37 °C 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 antibacterial agent is greater than 500 cSt.

[0070] The synthetic cationic polypeptide can be designed and manufactured to form a self-standing hydrogel when dispersed in water. As depicted in FIGS. 17(a)-(d), for example, the exemplary synthetic copolymer polypeptide (KL-120 / 2.5) forms a viscous solution and a hydrogel 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 gel formation was evaluated by an inclined tube assay, hardness by texture analysis, and viscosity. The concentration-dependent effect on physical properties is evident. For example, when increasing to a concentration of 2 wt% in water, this synthetic cationic polypeptide formulation formed a self-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 hardness using texture analysis also clearly exhibited a concentration-dependent increase. This latter method also demonstrated the 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 the evaluation, we found that certain additives have an unexpectedly large effect on viscosity. We further discovered that these effects are dependent on the molecular design of the synthetic cationic polypeptide. For example, it has been demonstrated that adding NaCl (~0.9%) to an aqueous formulation of a Group II intermediate lysine-L-leucine (KL) polypeptide having a block amino acid sequence arrangement in a multi-faceted study results in a significant decrease in viscosity. Similarly, but to a lesser extent, an effect was observed with the lysine-D,L-leucine (KrL) polypeptide. This data indicates that both the molecular design and the nature of the additive should be considered together when preparing a pharmaceutical composition having the desired viscosity parameters. These observations also provide a reminder that certain substances found in manufactured formulations of synthetic cationic polypeptides, including salts, can have a significant impact on functionality and performance.

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

[0073] Figure 20 depicts the kinematic viscosities of two examples of synthetic cationic polypeptides having lysine-L-leucine (KL) and lysine-D,L-leucine (KrL) block array arrangements dissolved in only water, 0.9% saline or 4.4% xylitol aqueous solution at 1% by weight in water. In particular, when preparing KL-100 / 5.7 in 0.9% saline, a viscosity reduction of about 70% was demonstrated compared to the formulation in water. As a comparison, when preparing KrL-110 / 4.0 in 0.9% saline, a viscosity reduction of about 40% was demonstrated compared to the formulation in water. Further, from these studies, it was demonstrated that both synthetic cationic polypeptides in aqueous solutions of xylitol, a non-ionic additive, unexpectedly had enhanced viscosities compared to only water. Thus, xylitol offers potential benefits as an additive to pharmaceutical compositions when it is desirable to maintain high viscosities while increasing the tonicity and / or osmotic pressure. Additionally, in some embodiments, xylitol is an unexpectedly favorable pharmacologically acceptable additive for antibacterial compositions because, in addition to its effect on viscosity, it does not support the metabolism and growth of many microorganisms.

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

[0075] Various embodiments of the present invention include at least one antibacterial synthetic cationic polypeptide and at least one additive or excipient in an aqueous carrier in a combination and amount sufficient to result in a viscosity of a pharmaceutical composition of 1.25 centistokes to 500 centistokes (cSt; mm 2 / s) in an assay where the viscosity value of only water is below 0.9 cSt. They are 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 a pharmaceutical composition comprising at least one antibacterial 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 when measured using a rotational viscometer.

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

[0078] In accordance with the present invention, to reach an effective level of antibacterial activity, a significant amount of the pharmaceutical composition may be required for a particular patient (e.g., having large wounds and / or high microbial levels). We have found that an antibacterial agent can enhance both the effectiveness and safety of a topically applied antibacterial composition if designed in accordance with the teachings provided herein. An antibacterial agent that forms a multi-molecular complex by itself or with one or more other components of the composition increases the local concentration and tissue coating of the antibacterial agent, thereby achieving great effectiveness at a low dose. Further, 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 not only the antibacterial composition but also the antibacterial agent itself will affect the intermolecular interactions that allow for the formation of the multi-molecular complex.

[0079] We have found that the effectiveness and safety of topically administered antibacterial agents are affected by physical properties, including viscosity. For example, increasing viscosity can increase the tissue retention time of topically applied antibacterial agents, thereby enhancing effectiveness. This effect can also reduce the total amount of antibacterial composition required, thereby reducing the list of toxicities that limit dosage. Intermolecular interactions of the antibacterial agent with itself or other components of the composition contribute to the increased viscosity of the antibacterial composition.

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

[0081] The ability of various formulations of synthetic cationic polypeptides to reduce surface tension was evaluated (Figure 23). Several observations were made. Unexpectedly, lysine-leucine diblock copolypeptides prepared from 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., about 20 leucine units or less). In addition, lysine-leucine diblock copolypeptides prepared from racemic D,L-leucine demonstrated relatively high surfactant activity when compared to those prepared from enantiopure L-leucine with leucine block lengths of about 20 units or longer. Overall, it is shown that from the data, the surface activity is greatly affected by both the amino acid composition (including enantiopure vs. racemic) and the length of the hydrophobic block. Thus, the design factors can be used to obtain the desired surface-active properties of the synthetic cationic polypeptides.

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

[0083] Certain additives can also affect the surface and interfacial tensions. For example, from the data in Figure 25, 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 for the interfacial tension. (Figure 26)

[0084] The synthetic cationic polypeptides can be effective emulsifiers. As shown in Figure 27, we demonstrate that formulations of two examples of 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 (Figure 28). From the data in Figure 28, it is demonstrated that the emulsifying activity can also be demonstrated by synthetic cationic polypeptides formulated not only in water but also in physiological saline or water-soluble xylitol.

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

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

[0087] Unexpectedly, we have found that there is significant variability among different formulations of synthetic cationic polypeptides with respect to systemic safety. We have found that both molecular design and formulation greatly influence the risk of systemic toxicity when synthetic cationic polypeptide formulations are applied intraperitoneally. Intraperitoneal administration, while a type of topical application, facilitates significant systemic uptake and distribution of the therapeutic agent and excipients. Thus, intraperitoneal administration presents a higher risk of systemic toxicity than most topical applications of antimicrobial pharmaceutical compositions. It is important to note several things 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 safer antimicrobial agents that can be applied intraperitoneally. Third, there is a possibility that pharmaceutical compositions may be inadvertently administered intraperitoneally, intravenously, or to other sites that can dramatically increase systemic uptake. We have found that the formulations are highly effective and have low risks of both local and systemic toxicity even when applied to sites other than intact, healthy skin. We have now developed a topically applied antibacterial pharmaceutical composition with low toxicity.

[0088] Figure 29 depicts the results of a rabbit skin irritation model in which a composition of synthetic cationic polypeptides was applied to both intact and abraded skin. After applying synthetic cationic polypeptides KL-140 / 2.5 or KrL-120 / 5.0 at various concentrations and in various formulations, a negligible response was observed. Figure 30 depicts the results of a guinea pig skin sensitization test. After applying KL-140 / 2.5 (lot 73) or KrL-120 / 5.0 (lot 93) at 1 wt% in water, no visual changes were observed, indicating to those skilled in the art that synthetic cationic polypeptides are unlikely to cause sensitization. Figure 31 depicts the results of a rat oral toxicity test. After administering KL-140 / 2.5 in water or KrL-120 / 5.0 in water to rats at doses of 0.625 mg / kg to 160 mg / kg by oral gavage for 3 days, no abnormalities were observed.

[0089] The test for systemic toxicity after intraperitoneal administration yielded unexpected results. As depicted in Figure 32, when polylysine (K-100) of approximately 100 amino acid units was administered intraperitoneally at a concentration of 20 mg / mL and an overall dose of 800 mg / kg, 0 out of 5 animals survived, indicating significant toxicity. Similarly, the synthetic cationic polypeptide KL-140 / 2.5-RAN lacks a block sequence arrangement of amino acid units, and its formulation was found to be toxic, with 0 out of 5 animals surviving. In comparison, after administering KrL-120 / 5.0 at an equivalent concentration and dose in water, all animals (5 / 5) survived. As shown in Figure 33, an aqueous formulation of another cationic polypeptide, KrL-130 / 3.3, having segmented or block sequence arrangements of lysine and D,L-leucine, also showed a high level of safety with complete survival (5 / 5) at concentrations up to 20 mg / mL (800 mg / kg dose).

[0090] When testing two different lots (93 and 94) of a polymer with the same target structure, KrL-120 / 5.0, from additional tests, unexpected variations were revealed. As depicted in Figure 34, Lot 93 was found to cause significant toxicity and systemic mortality after intraperitoneal administration compared to Lot 94. This is particularly surprising since Lot 93 was shown to be safe for topical application in the guinea pig sensitization test described above in Figure 30. Lot 93 was also shown to be an antibacterial agent effective in vitro against both Gram-positive and Gram-negative bacteria. Furthermore, it was found to be safe and effective when used in vivo in both a porcine open wound model contaminated with microorganisms and a rodent non-open wound model.

[0091] In further analysis of Lots 93 and 94, notable differences were observed in the self-organization into multimeric structures evaluated by the critical aggregation concentration (CAC). Lot 93, which is the more toxic lot, was found to have a higher critical aggregation concentration (CAC) (130 μg / mL), indicating a weaker driving force for self-organization into multimeric structures compared to Lot 94 (CAC: 51 μg / mL).

[0092] We also found that synthetic cationic polypeptides that demonstrate low CAC and high viscosity when prepared in water are relatively safe when administered intraperitoneally. As depicted by the data in Figure 35, two formulations of lysine-L-leucine (KL) synthetic cationic polypeptides, KL-170 / 3.3 and KL-140 / 2.5, with a block sequence arrangement of amino acid units, were shown to be very safe for intraperitoneal administration. These synthetic cationic polypeptides and those with closely related structures demonstrated low CAC (typically in the single-digit μg / mL, see also Figure 11 above) and a high level of self-organization. We found that synthetic cationic polypeptides that demonstrate a high safety profile after intraperitoneal administration also demonstrate relatively high viscosity and / or hardness in the texture analysis of aqueous formulations. (see FIGS. 13 to 17). Accordingly, we have developed general principles as described herein to guide those skilled in the art in designing synthetic cationic polypeptides with 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 FIG. 36, formulations of KL-140 / 2.5 in water or in water and hydroxyethyl cellulose (HEC) demonstrated some differences in safety. In particular, formulations with a high HEC content were found to be more toxic, despite the fact that this additive is widely recognized as being safe. FIG. 37 shows additional examples of formulation changes that may enhance toxicity upon intraperitoneal administration.

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

[0095] The ratio of systemic toxicity that limits the dose to a local effective amount can depend on the tissue site and / or pathophysiological situation. Both the effective amount and the toxic amount can be determined after application to a specific site or pathophysiological situation. Alternatively, a "higher hurdle" type can be established by determining the systemic toxicity after application to a site with expected high systemic absorption (such as the peritoneal cavity). The local effective amount can further be determined after application to a specific site or pathophysiological situation (e.g., limb injury, soft tissue infection, sinus infection).

[0096] In embodiments, the antibacterial agent or antibacterial composition can be selected based in part on criteria of local or systemic toxicity in vivo.

[0097] In embodiments, the antibacterial agent or antibacterial composition can be selected based in part on criteria of toxicity in vivo after intraperitoneal administration.

[0098] Certain embodiments of the present invention can include an antibacterial agent or antibacterial composition that can be safely injected into the peritoneal cavity of a mouse at a dose higher than the dose expected to be effective in reducing the microbial load after local application to tissues other than the peritoneal cavity.

[0099] Example 8 The sterilization method is 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), self-organization into the multimeric structure of the polymer, and viscosity. In embodiments, the sterilization of the antibacterial pharmaceutical composition is carried out by a method that maintains the molecular integrity, multimeric structure, and viscosity.

[0100] Figures 38 to 41 demonstrate that traditional radiation sterilization techniques can cause destruction of the molecular structure of synthetic cationic polypeptides and a decrease in the hardness of the composition, as evaluated 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. Thus, radiation sterilization techniques need to be carefully adjusted and / or modified for use in the sterilization of antibacterial pharmaceutical compositions having synthetic cationic polypeptides.

[0101] Compared to radiation techniques, both the sterilizing filtration method and the heat sterilization method (autoclaving) provide effective sterilization of antibacterial pharmaceutical compositions while maintaining the molecular structure of synthetic cationic polypeptides and the desired physical properties of the water-soluble compositions containing them. Figures 42 to 46 show SEC chromatograms demonstrating the maintenance of molecular integrity after sterilization by filtration and autoclaving.

[0102] Filtration and sterilization by autoclaving also maintain the key and beneficial physical properties of aqueous formulations and compositions containing synthetic cationic polypeptides. Figure 47 demonstrates that an aqueous formulation of 1.25 wt% synthetic cationic polypeptide KL-120 / 2.5 (lot BAC004) in water with 4.5 wt% mannitol exhibited nearly the same viscosity before and after filter sterilization. Figures 48 and 49 demonstrate that an aqueous formulation of synthetic cationic polypeptide KL-160 / 3.2 (lot BAC003) retains a significant level of shear-thinning viscosity after autoclave sterilization, although the overall level decreased somewhat compared to the sample before autoclave sterilization. Furthermore, while some decrease in hardness is noticed when various aqueous formulations and compositions are autoclave sterilized, the beneficial properties of hardness, as evaluated by texture analysis, are maintained (Figures 50 and 51). The data further shows that the decrease in hardness is seen when the synthetic cationic polypeptide is at a high concentration and in the presence of certain additives such as HEC. As an example, the observed decreases in viscosity and hardness can be caused by further dissolution of the multimeric structure that can occur during autoclave sterilization treatment and by re-chilling treatment. Therefore, these formulations are thought to maintain the benefits of viscosity and hardness. Figure 52 shows that the surface activity of synthetic cationic polypeptide KrL-120 / 5.0 is maintained after autoclave sterilization.

[0103] Example 9 To be effective in inhibiting or killing microorganisms and also to reduce the risk of local and systemic toxicity, examples of antimicrobial pharmaceutical compositions as described herein provide both antimicrobial activity and beneficial physicochemical properties. The latter is related to the better self-organization of synthetic cationic polypeptides into multimeric structures, as exemplified by albumin, a higher viscosity than that seen 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, provided that the sterilization process is 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 a reduction in the risk of local and systemic toxicity in the in vivo environment, the method of application (including how much and how frequently) 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 invention, in some clinical situations, such as those involving large surgical or traumatic wounds, applying the antimicrobial pharmaceutical composition as described herein "in large amounts" may be important to achieve antimicrobial efficacy. As described above, "in large amounts" refers to the total therapeutic amount of an antimicrobial pharmaceutical composition containing more than 1 g, or 14.3 mg / kg, of synthetic cationic polypeptide for a 70 kg human. When applying the antimicrobial pharmaceutical composition in large amounts, the 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 a plurality of diverse clinical situations. In some embodiments, direct application to surgically and traumatically exposed tissues is among the most valuable uses. Figure 53 is a schematic depiction of conventional wound classifications in a surgical setting. Wounds are classified as class I (clean), class II (clean / contaminated), class III (contaminated), class IV (dirty / infected). Considering the method of use Among them, in both Class I and Class II wound situations, the antimicrobial pharmaceutical compositions described herein can be applied to tissues before significant microbial contamination. In some embodiments, the combination of direct bactericidal activity and antimicrobial barrier properties is beneficial in such situations. Further, in Class III and Class IV wound situations, the antimicrobial pharmaceutical compositions described herein can be applied to tissues after significant microbial contamination, or biofilm formation, or apparent infection. In some embodiments, the combination of direct bactericidal activity and surfactant properties is beneficial in such situations.

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

[0106] Figure 55 depicts the results of an in vivo porcine open wound model where the synthetic cationic polypeptide KL-160 / 3.2 formulation was applied to full-thickness wounds 15 minutes prior to microbial contamination. When evaluated 4 hours after inoculation with a high-density mixed culture of Staphylococcus epidermidis and Pseudomonas aeruginosa, the treated wounds showed little to no tissue microbial contamination compared to the control, if any. Figure 56 demonstrates that the effect of pre-treating tissue prior to microbial contamination depends on both concentration and time. Thus, in a clinical setting, a preferred embodiment includes the step of administering an effective amount of the synthetic cationic polypeptide and involves repeated application over multiple extended periods to exposed tissue (e.g., extended surgical procedures and wounds with delayed closure are included). Figure 57 shows the results of pre-treatment of a rodent wound model (acting as a different body) with implanted surgical mesh prior to microbial inoculation. In this model, the wound was closed immediately after inoculation. As the data show, a single administration of the antimicrobial pharmaceutical composition having the synthetic cationic polypeptide KL-160 / 3.2 resulted in a very significant (multi-log) reduction in CFU when evaluated at 48 hours.

[0107] In summary, these data demonstrate (1) the benefits of direct bactericidal activity and microbial barrier properties; (2) the value of early treatment prior to significant microbial contamination; and (3) the importance of treatment planning that takes into account both the period of tissue exposure and the likely timing of microbial contamination.

[0108] The benefits of the combination of direct bactericidal activity and surfactant properties can be demonstrated in various test situations. As shown in FIG. 53, the antimicrobial compositions described herein can be applied in a number of diverse clinical situations where there is already significant microbial contamination of tissue, existing biofilms or overt infections, or combinations of two or more. Such situations include surgical procedures targeting class III and class IV wounds. These include the treatment of surgical sites, including those involving different bodies (e.g., periprosthetic joint infections, hernia mesh infections, breast implant infections). These situations also include not only surgical or non-surgical treatment of frequently contaminated areas such as the sinuses of the head (especially in patients with chronic rhinosinusitis), but also many traumatic wounds. Repair of surgical sites with different bodies should also be considered to have a high risk of biofilm formation, even if there is no overt infection in them. In these situations, as mentioned above, the antimicrobial pharmaceutical compositions described herein are likely to be applied to sites where microbial contamination exists. The combination of direct bactericidal activity and surfactant properties can be of particular benefit.

[0109] FIGS. 58 and 59 demonstrate potent activity against Pseudomonas aeruginosa (P. aeruginosa) grown within a biofilm using formulations of synthetic antimicrobial polypeptides having both bactericidal activity and surfactant power. FIGS. 60 - 63 depict results demonstrating these same principles in vivo. The antimicrobial compositions described herein, which demonstrate direct bactericidal activity and surfactant properties, have been shown to be highly effective when applied to tissue sites highly infected with both Gram-positive (such as methicillin-resistant Staphylococcus aureus (S. aureus)) and Gram-negative (such as Pseudomonas aeruginosa (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 partially due to a reduction in tissue inflammation, a recognized cause of tissue damage. In various embodiments, the reduction in inflammation can be obtained or enhanced by combining the antimicrobial compositions described herein with an anti-inflammatory agent.

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

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

[0113] As other aspects of the present disclosure, for example, the following can be mentioned. [1] An antimicrobial pharmaceutical composition, wherein the antimicrobial pharmaceutical composition comprises: An aqueous carrier; and An antimicrobial synthetic cationic polypeptide dispersed in the aqueous carrier at a concentration of about 0.01 wt% to 5 wt% based on the total weight of the antimicrobial pharmaceutical composition comprising; The antimicrobial synthetic cationic polypeptide comprises at least 40 amino acid units, and the at least 40 amino acid units comprise a cationic segment comprising at least 10 amino acid units that are positively charged at neutral pH and comprising a hydrophobic segment containing a plurality of hydrophobic amino acid units; the ratio of the length of the cationic segment to the length of the hydrophobic segment is 1.5 to 15; the antibacterial synthetic cationic polypeptide has a viscosity of 2 centistokes (cSt) or more at 37 °C at a concentration of 2% by weight in deionized water; when the aqueous carrier contains 2% by weight of the antibacterial synthetic cationic polypeptide, it has a viscosity greater than the viscosity of the aqueous carrier when it contains 2% by weight of albumin instead of the antibacterial synthetic cationic polypeptide at 37 °C; after the antibacterial pharmaceutical composition is injected into the peritoneal cavity of a plurality of healthy, young adult mice at a dose of 10 mg / kg of the antibacterial synthetic cationic polypeptide, the survival rate of the mice at 72 hours is 50% or more and the toxicity is low, antibacterial pharmaceutical composition. [2] The cationic segment contains at least 15 amino acid units that are positively charged at neutral pH, the antibacterial pharmaceutical composition according to [1]. [3] The aqueous carrier is water or an aqueous solution of a pharmaceutically acceptable salt, the antibacterial pharmaceutical composition according to [1] or [2]. [4] The aqueous carrier further contains a nonionic additive, the antibacterial pharmaceutical composition according to any one of [1] to [3]. [5] The nonionic additive is present in an amount effective to increase the osmotic concentration of the antibacterial pharmaceutical composition by at least 10% greater than the concentration of the antibacterial pharmaceutical composition without the additive, the antibacterial pharmaceutical composition according to [4]. [6] The nonionic additive is selected from the group consisting of glucose, mannitol, glycerol, xylitol, sorbitol, surfactants and combinations thereof, the antibacterial pharmaceutical composition according to [4] or [5]. [7] The aqueous carrier further contains an additive in an amount that increases the viscosity of the antibacterial pharmaceutical composition, the antibacterial pharmaceutical composition according to any one of [1] to [6]. ​[8] The aqueous carrier further contains an additive in an amount that reduces the viscosity of the antibacterial pharmaceutical composition, the antibacterial pharmaceutical composition according to any one of [1] to [7]. [9] The antibacterial pharmaceutical composition according to any one of [1] to [8], which is sterilized by a heat sterilization method and / or a filtration sterilization method.

[10] The antibacterial pharmaceutical composition according to [9], wherein the antibacterial pharmaceutical composition has a weight average molecular weight and a dispersity equivalent to those of the antibacterial synthetic cationic polypeptide of the non-sterilized antibacterial pharmaceutical composition.

[11] The antibacterial pharmaceutical composition according to [9] or

[10] , wherein the antibacterial pharmaceutical composition has a viscosity level at 37 °C equivalent to that of the non-sterilized antibacterial pharmaceutical composition.

[12] The antibacterial pharmaceutical composition according to

[11] , wherein the viscosity of the antibacterial pharmaceutical composition at 37 °C is 20% to 200% of the viscosity of a non-sterilized antibacterial pharmaceutical composition that is equivalent in other respects.

[13] The antibacterial pharmaceutical composition has a bactericidal activity equivalent to that of a non-sterilized antibacterial pharmaceutical composition that is equivalent in other respects, and the bactericidal activity is 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). The antibacterial pharmaceutical composition according to any one of [9] to

[12] .

[14] The hydrophobic segment contains at least 5 hydrophobic amino acid units, the antibacterial pharmaceutical composition according to any one of [1] to

[13] .

[15] The at least 5 hydrophobic amino acid units are selected from leucine (L), isoleucine (I), valine (V), phenylalanine (F), or alanine (A), the antibacterial pharmaceutical composition according to

[14] .

[16] The hydrophobic segment contains at least 10 hydrophobic amino acid units selected from leucine (L), isoleucine (I), valine (V), phenylalanine (F), or alanine (A), the antibacterial pharmaceutical composition according to

[14] .

[17] The hydrophobic segment contains at least 15 hydrophobic amino acid units selected from leucine (L), isoleucine (I), valine (V), phenylalanine (F), or alanine (A), and the antibacterial pharmaceutical composition according to

[14] .

[18] The hydrophobic amino acid unit contains a leucine unit, and the antibacterial pharmaceutical composition according to any one of

[14] to

[17] .

[19] The positively charged amino acid unit contains a lysine unit, and the antibacterial pharmaceutical composition according to any one of [1] to

[18] .

[20] The antibacterial synthetic cationic polypeptide is a block copolymer polypeptide containing a hydrophobic leucine unit and a positively charged lysine unit, and the antibacterial pharmaceutical composition according to any one of [1] to

[19] .

[21] The antibacterial synthetic cationic polypeptide has an average molecular weight of 3000 Da or more and 70000 Da or less, and the antibacterial pharmaceutical composition according to any one of [1] to

[20] .

[22] The antibacterial synthetic cationic polypeptide contains an arrangement of hydrophobic amino acid units and positively charged amino acid units configured to promote the self-organization of the antibacterial synthetic cationic polypeptide into a multimeric structure, and the antibacterial pharmaceutical composition according to any one of [1] to

[21] .

[23] When measured at a critical aggregation concentration of less than 1000 μg / mL at 37°C in deionized water, the antibacterial synthetic cationic polypeptide self-organizes into a multimeric structure, and the antibacterial pharmaceutical composition according to

[22] .

[24] The antibacterial synthetic cationic polypeptide forms a self-standing hydrogel at a concentration of 3% by weight at 37°C in deionized water, and the antibacterial pharmaceutical composition according to any one of [1] to

[23] .

[25] When measured at a concentration of 1% by weight at 37°C in deionized water, the antibacterial synthetic cationic polypeptide exhibits surface activity with a decrease in surface tension of at least 10% compared to deionized water alone, and the antibacterial pharmaceutical composition according to any one of [1] to

[24] .

[26] The antibacterial pharmaceutical composition according to any one of [1] to

[25] , further comprising an anti-inflammatory compound.

[27] The anti-inflammatory compound is the antibacterial pharmaceutical composition according to

[26] , which is selected from the group consisting of corticosteroids, histamine inhibitors, and cytokine inhibitors.

[28] The antibacterial pharmaceutical composition according to any one of [1] to

[27] for preventing tissues other than intact, healthy skin from being contaminated by microorganisms.

[29] The antibacterial pharmaceutical composition according to any one of [1] to

[27] for reducing the microbial load in or on tissues other than intact, healthy skin.

[30] The antibacterial pharmaceutical composition according to

[28] or

[29] , wherein the tissue is selected from affected skin, surgical sites, traumatic wounds, debrided tissues, abscesses, abdominal cavities, pulmonary airways, sinuses, and urinary tracts.

[31] The antibacterial pharmaceutical composition according to any one of [1] to

[30] , wherein the aqueous carrier contains 2% by weight of an antibacterial synthetic cationic polypeptide and the aqueous carrier has a viscosity higher than 6 cSt at 40°C.

[32] The antibacterial pharmaceutical composition according to any one of [1] to

[30] , wherein the aqueous carrier contains 2% by weight of an antibacterial synthetic cationic polypeptide and the aqueous carrier has a viscosity higher than 15 cSt at 40°C.

[33] The antibacterial pharmaceutical composition according to any one of [1] to

[30] , wherein the aqueous carrier contains 2% by weight of an antibacterial synthetic cationic polypeptide and the aqueous carrier has a viscosity higher than 20 cSt at 40°C.

[34] The antibacterial pharmaceutical composition according to any one of [1] to

[30] , wherein the aqueous carrier contains 2% by weight of an antibacterial synthetic cationic polypeptide and the aqueous carrier has a viscosity higher than 24 cSt at 40°C.

Claims

[Claim 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, the 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 10 mg / kg of the antibacterial synthetic cationic polypeptide into healthy young adult mice. Antibacterial pharmaceutical composition.

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