WO2022236088A1

WO2022236088A1 - Lysin polypeptide compositions and methods of use

Info

Publication number: WO2022236088A1
Application number: PCT/US2022/028121
Authority: WO
Inventors: Chandrabali GHOSE-PAUL
Original assignee: Bioharmony Therapeutics Inc.
Priority date: 2021-05-06
Filing date: 2022-05-06
Publication date: 2022-11-10

Abstract

The present disclosure provides compositions and methods of use of combinations of lysin polypeptides and monobactam antibiotics effective against drug-resistant Gram-negative pathogens.

Description

TITLE

Lysin polypeptide compositions and methods of use

INVENTOR

Chandrabali Ghose-Paul

CROSS-REFERENCE TO RELATED APPLICATION This application claims priority to U.S. Provisional Application Serial No. 63/185,127, which was filed on May 6, 2021, the entire content of which is incorporated herein by reference.

STATEMENT REGARDING SEQUENCE LISTING The Sequence Listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference in the specification. The name of the text file containing the Sequence Listing is 101457-4_sequences_ST25.txt. The text file is 735 bytes, created on May 5, 2022, and is being submitted electronically via EFSWeb.

FIELD

The present disclosure is generally directed towards methods and compositions for the prevention, inhibition and treatment of infections caused by Gram-negative bacteria.

BACKGROUND

Intensive care units (ICU) are a primary source for the rise of drug resistant bacteria due to increased selection pressures caused by sterile environments in combination with increased use of alternative classes of antibiotics to treat infections in subjects. ICU subjects also exhibit an increased risk of infection due to their reduced or delayed immune response and use of invasive devices including mechanical ventilators and a variety of catheterizations. Pseudomonas aeruginosa (P. aeruginosa ) represents one of the most concerning pathogens involved in antibiotic resistance, with nosocomial infection rates of these drug resistance bacteria being significant, and treatment for such infections often being hampered by a bacteria’s resistance to more than one antibiotic. Currently, multiple drug resistant /¹ aeruginosa accounts for 13-19% of nosocomial infections each year in the US, with the increasing levels of resistance being attributed to patient- to-patient transmission of resistant strains as well as newly acquired resistance owing to previous antibiotic exposure.

P. aeruginosa is a Gram-negative rod-shaped bacterium and an infrequent component of human microflora in healthy individuals. P. aeruginosa is widespread in natural environments and is an opportunistic pathogen in humans, leading to a broad spectrum of conditions including respiratory infections and septicemia. It is the primary cause of ventilator-associated pneumonia in hospital intensive care units.

Bacterial infections in the lungs caused by P. aeruginosa are a serious problem in patients with cystic fibrosis, chronic obstructive pulmonary disease (COPD), or ventilator-associated pneumonia, with cystic fibrosis patients being particularly susceptible as a result of abnormal mucus production in the lungs and airways. P. aeruginosa is found in approximately half of all individuals with cystic fibrosis, with approximately 60% of adults being infected and a substantial portion of those adults carrying drug resistant/¹ aeruginosa.

Most commonly, the mucoid form of P. aeruginosa is highly resistant to conventional antibiotics as well as the patient’s immune-mediated killing, causing a rapid decline in lung function and a poor overall clinical prognosis. As of 2007, the median life expectancy of a patient with cystic fibrosis is 36.9 years. However, patients with a P. aeruginosa infection have shown a life expectancy of only 30 years, compared with 40 years in patients not infected with P. aeruginosa , while experiencing a more rapid decline in pulmonary function and more frequent hospitalizations

Currently, antibiotic use in the treatment of cystic fibrosis and COPD patients with chronic bacterial respiratory infections is increasing, and correlates to a higher prevalence of antibiotic- resistant strains. Further exacerbation of the condition of an individual with a P. aeruginosa infection may involve endocarditis, septicemia, urinary tract infections, pneumonia, and surgical wound infections.

The recent increase in nosocomial infections caused by P. aeruginosa have been recognized as a steadily growing acute problem in hospitals due to its innate resistance to many antibiotics and antiseptics, the ability to acquire further resistance mechanism to multiple classes of antibiotics, and its ability to propagate in moist environments. Various mechanism of innate drug resistance in P. aeruginosa include the presence of over-expressed efflux pumps and low permeability of its outer membrane. Acquired resistance results from the acquisition of a resistance gene or mutation in genes encoding porins, efflux pumps, penicillin-binding proteins, and chromosomal //-lacta ase, which contribute to //-lactam, carbapenem, aminoglycoside, and fluoroquinolone resistance. Additionally, P. aeruginosa demonstrates the ability to cause infection by manipulating host pathogen interactions. These mechanisms may exist simultaneously to confer combined resistance to a wide array of antibiotics including, for example, cefepime, ciprofloxacin, gentamicin, imipenem, levofloxacin, and meropenem, thereby limiting treatment indications for drug resistant/¹ aeruginosa. P. aeruginosa therefore has become a cause of a major public health problem because of severely limited and effective therapeutic options combined with the shortage of new and effective antibiotics. Thus, novel treatments for/¹ aeruginosa infections are needed in the face of prevalence multi-drug-resistant strains.

Therapeutic treatment of multi-drug resistant infectious pathogens has more recently included the application of bacterial peptidoglycan hydrolases and phage lysins to degrade the peptidoglycan of the bacterial cell wall. Peptidoglycan hydrolases include lysozymes, such as glucosaminidases and muramidases, that cleave the sugar backbone of peptidoglycan, endopeptidases, that cleave the stem-peptide or cross-bridge, and L-alanine amidases, that cleave the amide bond between the sugar and peptide moieties, that, with recombinant technologies can be expressed, purified, and added exogenously to cause lysis to bacteria. Also referred to as “lysis from without,” this strategy has been applied as an antibacterial treatment for several Gram positive bacterial pathogens.

Phage lysins are generally species or subspecies specific, and are effective only against bacteria from which they were produced. While some lysins act upon the cell walls of several bacterial genus or species, some “broad-spectrum” lysins also have been found. Since Gram negative bacteria possess an outer membrane that prevents extracellular lysin molecules from digesting peptidoglycan, lysins have been used as a treatment mainly against Gram positive bacteria. However, even though lysins showing activity against Gram-negative bacteria are known, there remains an unmet need for effective therapeutic agents for the treatment of Gram negative bacterial infections including those caused by multi-drug resistance P. aeruginosa.

However, the use of lysins for treatment of Gram-negative bacterial infections has been limited or partially effective because of the additional outer membrane layer within the bacterial cell wall which limits access of lysins to the peptidoglycan substrates in the cell wall. SUMMARY

The present disclosure may comprise one or more of the following features and combinations thereof.

The present disclosure provides compositions of lysin peptides used in combination with certain antibiotics, provides one or more of features and combinations thereof as described herein, and methods of use thereof. As provided herein, application of the lysin peptides in combination with certain antibiotics to a bacterial infection may increase the susceptibility of the infectious bacteria to treatment. In one embodiment of the present disclosure is provided a pharmaceutical composition having an isolated polypeptide with the amino acid sequence NAKDYKGAAAEFPKWNKAGGRVLAGLVKRRKSQSRESQC (SEQ ID NO: 1) and a monobactam antibiotic. In certain aspects, the monobactam antibiotic is effective against Gram negative bacterial infections when used in combination with SEQ ID NO: 1. In certain aspects, the pharmaceutical composition is effective against antibiotic resistant Gram-negative bacterial infections. The Gram-negative bacterial infection causative agent can be any one or more of Enterobacteriaceae including Escherichia coli and Klebsiella pneumonia , Neisseria gonorrhoeae, Acinetobacter baumanii , Pseudomonas aeruginosa , Salmonella spp., and Shigella spp. In other certain aspects, the monobactam antibiotic of the pharmaceutical composition is one or more of aztreonam, nocardicin A, tabtoxin, and tigemonam. In other embodiments of the present disclosure, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

In certain embodiments of the present disclosure is provided a method of inhibiting the growth, or reducing the population, or the killing of at least one species of an aerobic Gram negative bacteria with a composition comprising the amino acid sequence SEQ ID NO: 1 and one or more monobactam antibiotics, wherein the isolated polypeptide and the one or more monobactam antibiotics has the property of inhibiting the growth, or reducing the population, or the killing of at least one species of aerobic Gram-negative bacteria. In other embodiments of the present disclosure is provided a method of treating a bacterial infection caused by P. aeruginosa and optionally one or more additional species of aerobic Gram-negative bacteria, comprising administering to a subject diagnosed with, at risk for, or exhibiting symptoms of a bacterial infection, the pharmaceutical composition of SEQ ID NO: 1 and one or more monobactam antibiotics. In other aspects of the present disclosure is provided a method of treating topical or systemic pathogenic bacterial infection caused by P. aeruginosa and optionally one or more species of an aerobic Gram-negative bacteria in a subject, comprising administering to a subject the pharmaceutical composition comprising SEQ ID NO: 1 and one or more monobactam antibiotics. In still other embodiments is provided a method for augmenting the efficacy of an antibiotic suitable for treating an aerobic Gram-negative bacterial infection, comprising co administering an isolated polypeptide comprising the amino acid sequence SEQ ID NO: 1 in combination with a monobactam antibiotic, wherein administration of the combination is more effective in inhibiting the growth of, or reducing an initial population of, or killing the aerobic Gram-negative bacteria than administration of either the monobactam antibiotic or the isolated polypeptide comprising the amino acid sequence SEQ ID NO: 1 individually. In certain embodiments is a method for producing a polypeptide with the amino acid sequence SEQ ID NO: 1 by either expressing the amino acid sequence in a cell or chemically synthesizing the amino acid sequence SEQ ID NO: 1, each with methods known in the art. These and other objects, advantages, and features of the present disclosure will become apparent to those skilled in the art upon reading the details of compounds according to the present disclosure and uses thereof, as more fully described below.

BRIEF DESCRIPTION OF FIGURES

The invention described herein is illustrated by way of example and not by way of limitation in the accompanying figures. For simplicity and clarity of illustration, elements illustrated in the figures are not necessarily drawn to scale. For example, the dimensions of some elements may be exaggerated relative to other elements for clarity. Further, where considered appropriate, reference labels have been repeated among the figures to indicate corresponding or analogous elements.

FIG. 1 shows a plot of a time-kill study against/¹ aeruginosa ATCC 27853;

FIG. 2 shows a plot of a time-kill study against/¹ aeruginosa CDC 0241; and

FIG. 3 shows a plot of a time-kill study against CDC 051.

DETAILED DESCRIPTION

While the concepts of the present disclosure are susceptible to various modifications and alternative forms, specific embodiments thereof have been shown by way of example in the drawings and will be described herein in detail. It should be understood, however, that there is no intent to limit the concepts of the present disclosure to the particular forms disclosed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives consistent with the present disclosure and the appended claims. References in the specification to “one embodiment,” “an embodiment,” “an illustrative embodiment,” etc., indicate that the embodiment described may include a particular feature, structure, or characteristic, but every embodiment may or may not necessarily include that particular feature, structure, or characteristic. Moreover, such phrases are not necessarily referring to the same embodiment. Further, when a particular feature, structure, or characteristic is described in connection with an embodiment, it is submitted that it is within the knowledge of one skilled in the art to effect such feature, structure, or characteristic in connection with other embodiments whether or not explicitly described. Additionally, it should be appreciated that items included in a list in the form of “at least one A, B, and C” can mean (A); (B); (C); (A and B); (A and C); (B and C); or (A, B, and C). Similarly, items listed in the form of “at least one of A, B, or C” can mean (A); (B); (C); (A and B); (A and C); (B and C); or (A, B, and C).

In the drawings, some structural or method features, such as those representing devices, modules, instructions blocks and data elements, may be shown in specific arrangements and/or orderings for ease of description. However, it should be appreciated that such specific arrangements and/or orderings may not be required. Rather, in some embodiments, such features may be arranged in a different manner and/or order than shown in the illustrative figures. Additionally, the inclusion of a structural or method feature in a particular figure is not meant to imply that such feature is required in all embodiments and, in some embodiments, may not be included or may be combined with other features.

Further, in the drawings, where connecting elements, such as solid or dashed lines or arrows, are used to illustrate a connection, relationship, or association between or among two or more other schematic elements, the absence of any such connection elements is not meant to imply that no connection, relationship, or association can exist. In other words, some connections, relationships, or associations between elements may not be shown in the drawings so as not to obscure the disclosure. In addition, for ease of illustration, a single connecting element may be used to represent multiple connections, relationships, or associations between elements. For example, where a connecting element represents a communication of signals, data or instructions, it should be understood by those skilled in the art that such element may represent one or multiple signal paths (e.g., a bus), as may be needed, to effect the communication.

As used herein, the following terms refer to the description provided unless the context clearly indicates otherwise.

“Gram-negative bacteria” generally refers to bacteria which produce a crystal violet stain that is decolorized in Gram staining, i. e. they do not retain crystal violet dye in the Gram staining protocol. As used herein, the term “Gram-negative bacteria” may describe, without limitation, one or more (i.e., either alone or in combination) of the following bacterial species: Acinetobacter baumannii, Acinetobacter haemolyticus, Actinobacillus actinomycetemcomitans, Aeromonas hydrophila, Bacteroides fragilis, Bacteroides theataioatamicron, Bacteroides distasonis, Bacteroides ovatus, Bacteroides vulgatus, Bordetella pertussis, Brucella melitensis, Burkholderia cepacia, Burkholderia pseudomallei, Burkholderia mallei, Prevotella corporis, Prevotella intermedia, Prevotella endodontalis, Porphyromonas asacchitrolytica, Campylobacter jejuni, Campylobacter coli, Campylobacter fetus, Citrobacter freundii, Citrobacter koseri, Edwarsiella tarda, Eikenella corrodens, Enterobacter cloacae, Enterobacter aerogeries, Enterobacter agglomerans, Escherichia coli, Francisella tularensis, Haemophilus influenzae, Haemophilus ducreyi, Helicobacter pylori, Kingella kingae, Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella rhinoscleromatis, Klebsiella ozaenae, Legionella pemimophila, Moraxella catarrhalis, Morganella morganii, Neisseria gonorrhoeae, Neisseria meningitidis, Pasteurella multocida, Plesiomonas shigelloides, Proteus mirabilis, Proteus vulgaris, Proteus penneri, Proteus myxofaciens, Providencia stuartii, Providencia rettgeri, Providencia alcalifaciens, Pseudomonas aeruginosa, Pseudomonas fluorescens, Salmonella typhi, Salmonella paratyphi, Serratia marcescens, Shigella flexneri, Shigella boydii, Shigella sonnei, Shigella dysenteriae, Stenotrophomonas maltophilia, Streptobacillus moniliformis, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio alginolyticus, Yersinia enter ocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Chlamydophila pneumoniae, Chlamydophila trachomatis, Ricketsia prowazekii, Coxiella burnetii, Ehrlichia chaffeensis, or Bartonella hensenae . The compositions of the present disclosure will be useful in preventing or inhibiting pathogenic bacterial growth, or for the treatment of one or more bacterial infections, particularly but not necessarily exclusively involving Gram-negative bacteria and notably P. aeruginosa.

The term “bactericidal” in the context of an agent conventionally means having the property of causing the death of bacteria or capable of killing bacteria to an extent of at least a 3- log (99.9%) or better reduction among an initial population of bacteria.

The term “bacteriostatic” conventionally means having the property of inhibiting bacterial growth, including inhibiting growing bacterial cells, thus causing a 2-log (99%) or better and up to just under a 3-log reduction among an initial population of bacteria.

The term “antibacterial” in a context of an agent is used generically to include both bacteriostatic and bactericidal agents.

The term “drug resistant” in a context of a pathogen and more specifically a bacterium, generally refers to a bacterium that is resistant to the antimicrobial activity of a drug. When used in a more particular way, drug resistance specifically refers to antibiotic resistance. In some cases, a bacterium that is generally susceptible to a particular antibiotic can develop resistance to the antibiotic, thereby becoming a drug resistant microbe or strain. A “multi-drug resistant” pathogen is one that has developed resistance to at least two classes of antimicrobial drugs, each used as monotherapy. For example, certain strains of Pseudomonas aeruginosa have been found to be resistant to nearly all or all antibiotics including aminoglycosides, cephalosporins, fluoroquinolones, and carbapenems. One skilled in the art is able to determine if a bacterium is drug resistant using routine laboratory techniques that determine the susceptibility or resistance of a bacterium to a drug or antibiotic.

The term “pharmaceutically acceptable carrier” refers to, for example, solvents, additives, excipients, dispersion media, solubilizing agents, coatings, preservatives, isotonic and absorption delaying agents, surfactants, propellants, etc., that are physiologically compatible with a subject. The carrier(s) are “acceptable” in the sense of not being overly harmful to the subject to be treated in amounts typically used in medicaments. Pharmaceutically acceptable carriers are compatible with the other ingredients of the pharmaceutical composition without rendering the pharmaceutical composition unsuitable for its intended purpose. Furthermore, pharmaceutically acceptable carriers are suitable for use with subjects without undue adverse side effects (such as toxicity, irritation, and allergic response). Side effects are “undue” when their risk outweighs the benefit provided by the pharmaceutical composition. Non-limiting examples of pharmaceutically acceptable carriers or excipients include any of the standard pharmaceutical carriers such as phosphate buffered saline solutions, water, and emulsions such as oil/water emulsions and microemulsions. For solid compositions comprising a lyophilized lysin polypeptide, excipients such as urea can be useful to improve stability. Other excipients include bulking agents, buffering agents, tonicity modifiers, surfactants, preservatives and co-solvents. For solid oral compositions comprising lysin polypeptide, suitable pharmaceutically acceptable excipients include, but are not limited to, starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like. For liquid oral compositions, suitable pharmaceutically acceptable excipients include, but are not limited to, water, glycols, oils, alcohols, flavoring agents, preservatives, and the like. For topical solid compositions such as creams, gels, foams, ointments, or sprays, suitable excipients include, but are not limited to, a cream, a cellulosic or oily base, emulsifying agents, stiffening agents, rheology modifiers or thickeners, surfactants, emollients, preservatives, humectants, alkalizing or buffering agents, and solvents. Suitable excipients for the formulation of the foam base include, but are not limited to, propylene glycol, emulsifying wax, cetyl alcohol, and glyceryl stearate. Potential preservatives include methylparaben and propylparaben.

The term “effective amount” refers to an amount which, when applied or administered in an appropriate frequency or dosing regimen, is sufficient to prevent or inhibit bacterial growth or prevent, reduce or ameliorate the onset, severity, duration or progression of the disorder being treated (here bacterial pathogen growth or infection), prevent the advancement of the disorder being treated, cause the regression of the disorder being treated, or enhance or improve the prophylactic or therapeutic effect(s) of another therapy, such as antibiotic or bacteriostatic therapy.

The term “co-administer” is intended to embrace separate administration of a lysin polypeptide and an antibiotic or any other antibacterial agent in a sequential manner as well as administration of these agents in a substantially simultaneous manner, such as in a single mixture/composition or in doses given separately, but nonetheless administered substantially simultaneously to the subject, for example at different times in the same day or 24-hour period. Such co-administration of lysin polypeptides with one or more additional antibacterial agents can be provided as a continuous treatment lasting up to days, weeks, or months. Additionally, depending on the use, the co-administration need not be continuous or co-extensive. For example, if the use were as a topical antibacterial agent to treat, e.g., a bacterial ulcer or an infected diabetic ulcer, the lysin could be administered only initially within 24 hours of the first antibiotic use and then the antibiotic use may continue without further administration of lysin.

The term “subject” refers to a subject to be treated and generally includes a mammal. Examples of mammal subjects include, for example, humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. In certain embodiments, the subject is a human, for example, a human subject suffering from, at risk of suffering from, or susceptible to a Gram-negative bacterial infection, whether such infection be systemic or confined to a particular organ or tissue.

The term “polypeptide” is used interchangeably with the term “protein” and “peptide” and refers to a polymer made from amino acid residues and having at least about 30 amino acid residues. The term includes not only polypeptides in isolated form, but also active fragments and derivatives thereof (defined below). The term “polypeptide” also encompasses fusion proteins or fusion polypeptides comprising a lysin polypeptide as described below and maintaining the lysin function. A polypeptide can be a naturally occurring polypeptide or an engineered or synthetically produced polypeptide. A particular lysin polypeptide can be, for example, derived or removed from a native protein by enzymatic or chemical cleavage, or can be prepared using conventional peptide synthesis techniques (e.g., solid phase synthesis) or molecular biology techniques (such as those disclosed in Sambrook, J. et ah, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989)). Variants of lysin polypeptides are also encompassed having at least 80% or at least 85% or at least 90% or at least 95% or at least 98% sequence identity with the lysin polypeptides provided herein. The term “fusion polypeptide” refers to an expression product resulting from the fusion of two or more nucleic acid segments, resulting in a fused expression product typically having two domains or segments with different properties or functionality. In a more particular sense, the term “fusion polypeptide” also refers to a polypeptide or peptide comprising two or more heterologous polypeptides or peptides covalently linked, either directly or via an amino acid or peptide linker. The polypeptides forming the fusion polypeptide are typically linked C-terminus to N-terminus, although they can also be linked C-terminus to C-terminus, N-terminus to N-terminus, of N- terminus to C-terminus. The term “fusion polypeptide” can be used interchangeably with the term “fusion protein.” Thus the open-ended expression “a polypeptide comprising” a certain structure includes larger molecules than the recited structure such as fusion polypeptides.

The term “heterologous” refers to nucleotide, peptide, or polypeptide sequences that are not naturally contiguous. For example, in the context of the present disclosure, the term “heterologous” can be used to describe a combination or fusion of two or more peptides and/or polypeptides wherein the fusion peptide or polypeptide is not normally found in nature, such as for example a lysin polypeptide or active fragment thereof and a cationic and/or a polycationic peptide, an amphipathic peptide, or a hydrophobic peptide and/or an antimicrobial peptide which may have enhanced lysin activity. Included in this definition are two or more lysin polypeptides or active fragments thereof. These can be used to make a fusion polypeptide with lysin activity

The term “active fragment” refers to a portion of a full-length polypeptide disclosed herein which retains one or more functions or biological activities of the isolated original polypeptide. A biological activity of particular interest herein is that of a lysin active to bore through the outer membrane and hydrolyze the coating of Gram-negative bacteria, whether by cleaving a sugar backbone or peptide bond. The term “amphipathic peptide” refers to a peptide having both hydrophilic and hydrophobic functional groups. Preferably, secondary structure places hydrophobic and hydrophilic amino acid residues at different ends of the peptide. These peptides often adopt a helical secondary structure.

The term “cationic peptide” refers to a peptide having positively charged amino acid residues. Preferably, a cationic peptide has a pKa-value of 9.0 or greater. The term “cationic peptide” in the context of the present disclosure also encompasses polycationic peptides.

The term “polycationic peptide” as used herein refers to a synthetically produced peptide composed of mostly positively charged amino acid residues, in particular lysine and/or arginine residues. The amino acid residues that are not positively charged can be neutrally charged amino acid residues and/or negatively charged amino acid residues and/or hydrophobic amino acid residues.

The term “hydrophobic group” refers to a chemical group such as ah amino acid side chain which has low or no affinity for water molecules but higher affinity for oil molecules. Hydrophobic substances tend to have low or no solubility in water or aqueous phases and are typically apolar but tend to have higher solubility in oil phases. Examples of hydrophobic amino acids include glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (lie), proline (Pro), phenylalanine (Phe), methionine (Met), and tryptophan (Trp).

The term “augmenting” within the context of the present disclosure refer to a degree of antimicrobial activity is higher than it would be otherwise. “Augmenting” encompasses additive as well as synergistic (superadditive) effects.

The term “synergistic” or “superadditive” in relation to an effect refers to a beneficial effect brought about by two active substances that exceeds that produced by each substance administered or applied alone. One or both active ingredients may be employed at a subthreshold level, i.e., a level at which if the active substance is employed individually produces no or a very limited effect. Exemplary references for the quantitative evaluation of synergy or synergestic or synergism, as used herein, include Clinical and Laboratory Standards Institute (CLSI). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically, 11th ed. CLSI standard M07. (CLSI, 950 West Valley Road, Suite 2500, Wayne, Pennsylvania 19087 USA) 2018, CLSI; Performance Standards for Antimicrobial Susceptibility Testing; 30th ed. CLSI supplement M100. (CLSI, 950 West Valley Road, Suite 2500, Wayne, Pennsylvania 19087 USA) 2020; Odds FC. 2003; “Synergy, antagonism, and what the chequerboard puts between them” J Antimicrob Chemother 52(1): 1 ; Eliopoulos G and Moellering R. 1991. “Antimicrobial combinations” in Antibiotics in Laboratory Medicine , Third Edition, edited by V. Lorian. (Williams and Wilkins, Baltimore, MD) pp. 432-492.

The term “treatment” refers to any process, action, application, therapy, or the like, wherein a subject, including a human, is subjected to medical aid with the object of curing a disorder, or eradicating a pathogen, or improving the subject's condition, directly or indirectly. Treatment also refers to reducing incidence, or alleviating symptoms, eliminating recurrence, preventing recurrence, preventing incidence, improving symptoms, improving prognosis or combinations thereof. “Treatment” further encompasses reducing the population, growth rate or virulence of the bacteria in the subject and thereby controlling or reducing a bacterial infection in a subject or bacterial contamination of an organ or tissue or environment. Thus “treatment” that reduces incidence is effective to inhibit growth of at least one Gram-negative bacterium in a particular application, whether it be a subject or an environment. “Treatment” of an already established infection also can refer to reducing the population or killing, including eradicating the Gram- negative bacteria responsible for an infection or contamination. Treatment can occur via delivery of a pharmaceutical composition with methods as known in the art, and may include but not be limited to topical delivery, oral delivery, parenteral delivery, pulmonary delivery (intra- tracheobronchial, pulmonary, and/or nasal administration), etc. As used herein, “topical” refers to local delivery of a pharmaceutical composition to a specific organ or anatomically defined region, for example to lung, skin, dermis, bladder, eye, etc.

The term “preventing” refers to the prevention of the incidence, recurrence, spread, onset or establishment of a disorder such as a bacterial infection. It is not intended that the present disclosure be limited to complete prevention or to prevention of establishment of an infection. In some embodiments, the onset is delayed, or the severity of a subsequently contracted disease is reduced, and such constitute examples of prevention. Contracted diseases in the context of the present disclosure encompass both those manifesting with clinical or subclinical symptoms, such as the detection of as well as the detection of growth of a bacterial pathogen when symptoms associated with such pathology are not yet manifest.

The term “derivative” in the context of a peptide or polypeptide (which as stated herein includes an active fragment) refers to, for example, a polypeptide modified to contain one or more- chemical moieties other than an amino acid that do not substantially adversely impact or destroy the lysin activity exhibited by the polypeptide. The chemical moiety can be linked covalently to the peptide, e.g., via an amino terminal amino acid residue, a carboxy terminal amino acid residue, of at an internal amino acid residue. Such modifications include the addition of a protective or capping group on a reactive moiety, addition of a detectable label, such as antibody and/or fluorescent label, addition or modification of glycosylation, or addition of a bulking group such as PEG, and other changes that do not substantially adversely impact or destroy the activity of the lysin polypeptide. Commonly used protective groups that may be added to lysin polypeptides include, but are not limited to t-Boc and Fmoc. Commonly used fluorescent label proteins such as, but not limited to, green fluorescent protein (GFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), and yellow fluorescent protein (YFP), are compact proteins that can be bound covalently or noncovalently to a lysin polypeptide or fused to a lysin polypeptide without interfering with normal functions of cellular proteins. Typically, a polynucleotide encoding a fluorescent protein is inserted upstream or downstream of the lysin polynucleotide sequence. This will produce a fusion protein (e.g., Lysin Polypeptide GFP) that does not interfere with cellular function or function of a lysin polypeptide to which it is attached. Polyethylene glycol (PEG) conjugation to proteins has been used as a method for extending the circulating half-life of many pharmaceutical proteins. Thus, in the context of lysin polypeptide derivatives, the term “derivative” includes lysin polypeptides chemically modified by covalent attachment of one or more PEG molecules. It is anticipated that pegylated lysin polypeptides will exhibit prolonged circulation half-life compared to the unpegylated lysin polypeptides, while retaining biological and therapeutic activity.

The term “percent amino acid sequence identity” with respect to the lysin polypeptide sequences refers to the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific lysin polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for example, using publicly available software such as BLAST or Megalign (DNASTAR) software. Two or more polypeptide sequences can be anywhere from 0-100% identical, or any integer value there between. In the context of the present disclosure, two polypeptides are “substantially identical” when at least 80% of the amino acid residues (preferably at least about 85%, at least about 90%, and preferably at least about 95%) are identical. The term “percent (%) amino acid sequence identity” as described herein applies to lysin peptides as well. Thus, the term “substantially identical” will encompass mutated, truncated, fused, or otherwise sequence-modified variants of isolated lysin polypeptides and peptides described herein, and active fragments thereof, as well as polypeptides with substantial sequence identity (e.g., at least 80%, at least 85%, at least 90%, or at least 95% identity as measured for example by one or more methods referenced above) as compared to the reference polypeptide.

Two amino acid sequences are “substantially homologous” when at least about 80% of the amino acid residues (preferably at least about 85%, at least about 90%, and preferably at least about 95%) are identical, or represent conservative substitutions. The sequences of lysin polypeptides of the present disclosure, are substantially homologous when one or more, or several, or up to 10%, or up to 15%, or up to 20% of the amino acids of the lysin polypeptide are substituted with a similar or conservative amino acid substitution, and wherein the resulting lysin have the profile of activities, antibacterial effects, and/or bacterial specificities of lysin polypeptides disclosed herein.

The term “inhalable composition” refers to pharmaceutical compositions of the present disclosure that are formulated for direct delivery to the respiratory tract during or in conjunction with routine or assisted respiration (e.g., by intra-tracheobronchial, pulmonary, and/or nasal administration), including, but not limited to, atomized, nebulized, dry powder and/or aerosolized formulations deliverable by methods and devices as known in the art. The term “biofilm” refers to bacteria that attach to surfaces and aggregate in a hydrated polymeric matrix of their own synthesis. A biofilm is an aggregate of microorganisms in which cells adhere to each other on a surface. These adherent cells are frequently embedded within a self- produced matrix of extracellular polymeric substance (EPS). Biofilm EPS, which is also referred to as slime (although not everything described as slime is a biofilm) or plaque, is a polymeric conglomeration generally composed of extracellular DNA, proteins, and polysaccharides.

The term “suitable” in the context of an antibiotic being suitable for use against certain bacteria refers to an antibiotic that was found to be effective against those bacteria even if resistance subsequently developed.

The term “antimicrobial peptide” (AMP) refers to a member of a wide range of short (generally 6 to 50 amino acid residues in length) cationic, gene encoded peptide antibiotics that can be found in virtually every organism. Different AMPs display different properties, and many peptides in this class are being intensively researched hot only as antibiotics, but also as templates for cell penetrating peptides. Despite sharing a few common features (e.g., cationicity, amphipathicity and short size), AMP sequences vary greatly, and at least four structural groups (alpha-helical, beta-sheet, extended and looped) have been proposed to accommodate the diversity of the observed AMP conformations. Likewise, several modes of action of antibiotics have been proposed, and it was shown in certain instances that the primary target of many of these peptides is the cell membrane, whereas for other peptides the primary target is cytoplasmic invasion and disruption of core metabolic functions. AMPs may become concentrated enough to exhibit cooperative activity despite the absence of specific target binding, for example, by forming a pore in the membrane. The present invention provides pharmaceutical compositions having antibacterial activity and for methods for using the disclosed pharmaceutical compositions. As used herein, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. Terms such as “comprises”, “comprised”, “comprising”, “contains”, “containing” and the like have the meaning attributed in United States patent law; they are inclusive or open-ended and do not exclude additional, un-recited elements or method steps. Terms such as “consisting essentially of’ and “consists essentially of’ have the meaning attributed in United States patent law; they allow for the inclusion of additional ingredients or steps that do not materially affect the basic and novel characteristics of the claimed invention. The terms “consists of’ and “consisting of’ have the meaning ascribed to them in United States patent law; namely that these terms are close ended.

Lysin polypeptides in general can be an effective therapeutic against a bacterial infection. See, for example, U.S. Patent #10,744,189 (the ‘189 patent) and US Patent #10,590,403 (the ‘403 patent). In particular, the phage-encoded lytic antimicrobial peptide P307SQ-8C (also referred to interchangeably herein as “SEQ ID NO: 1” and “BH01”) of the ‘403 patent, a 39 amino acid phage- encoded, engineered lysin polypeptide, is being developed as a pharmaceutical composition for the treatment of A. baumannii infections, including multiple drug resistant (“MDR”) and carbapenem-resistant A. baumannii (“CRAB”) infections in patients with hospital acquired bacterial pneumonia (HABP) and ventilator-associated bacterial pneumonia (VABP) patients, for example cystic fibrosis patients. Lysin polypeptides are highly differentiated from small molecule antibiotics in that they generally display a narrow spectrum of activity, being genus or species specific. P307SQ-8C displays antimicrobial activity towards A. baumannii , and, in certain conditions such as high pH, activity against other Gram-negative bacteria such as E.coli. Fischetti, et. al, in the ‘403 patent, examined the in vitro bactericidal effect of P307SQ-8C in 50 mM Tris-HCL, pH 7.5 for 2 hours at room temperature. Among the bacterial species tested, A. baumannii strains were consistently the most sensitive to the peptides, showing an average of 6.2-log decrease with P307SQ-8C. Bacillus anthracis , P. aeruginosa , and Staphylococcus aureus were moderately sensitive to P307SQ-8C with an average 2.9-log decrease. Both Escherichia coli and Klebsiella pneumoniae were resistant to the peptides under these experimental conditions. However, P307SQ-8C, by itself, has no significant bactericidal activity against Pseudomonas spp., S. aureus or other bacterial pathogens that are commonly isolated from cystic fibrosis patients.

The present disclosure relates to novel antimicrobial compositions against Gram-negative bacteria. In particular, the present disclosure relates to a pharmaceutical composition comprising a combination of a lysin polypeptide and an antibiotic effective against Gram-negative bacteria such as P. aeruginosa. Examples of such a lysin polypeptide useful for the present disclosure are those having an amino acid sequence if SEQ ID NO: 1 and related amino acid sequences substantially similar to SEQ ID NO: 1. Examples of such an antibiotic useful for the present disclosure include one or more of the monobactam antibiotics aztreonam, nocardicin A, tabtoxin, and tigemonam. Such a pharmaceutical composition of the present disclosure demonstrates synergistic antibiotic activity against pathogens, particularly P. aeruginosa , a pathogen commonly found in cystic fibrosis patients.

In embodiments of the present disclosure is provided an antimicrobial pharmaceutical composition comprising a lysin polypeptide that further comprises an amino acid sequence having at least 90% identity, 95% identity, 100 % identity, to SEQ ID NO: 1, in combination with a monobactam antibiotic. In certain embodiments of the present disclosure, the monobactam antibiotic includes, but is not limited to, one or more of aztreonam, nocardicin A, tabtoxin, and tigemonam. In still other embodiments the pharmaceutical composition of the present disclosure is for the treatment of bacterial infections in a subject. The present disclosure provides for significant improvements in the efficacy of treatment of a bacterial infection, and in particular a P. aeruginosa bacterial infection, by a lysin polypeptide when used in combination with a monobactam antibiotic. In certain embodiments, the combination of the lysin polypeptide provided herein and a monobactam antibiotic for the treatment of a drug resistant bacterial infection, in particular a drug resistant P. aeruginosa infection, results in a synergistic increase in the efficacy of treatment than the efficacy of treatment with a monobactam antibiotic alone or a lysin polypeptide, as provided herein, alone. In other certain embodiments, the combination of the lysin polypeptide provided herein and a monobactam antibiotic for the treatment of a drug resistant bacterial infection, in particular a drug resistant P. aeruginosa infection, results in an increase in the efficacy of treatment that is greater than the efficacy of treatment with a monobactam antibiotic alone or a lysin polypeptide, as provided herein, alone. In still other certain embodiments, the combination of the lysin polypeptide provided herein and a monobactam antibiotic for the treatment of a drug resistant bacterial infection, in particular a drug resistant P. aeruginosa infection, and more particularly a P. aeruginosa resistant to a monobactam antibiotic, results in a reversal of bacterial resistance to the monobactam antibiotic. In certain embodiments, the combination of the lysin polypeptide provided herein and a monobactam antibiotic for the treatment of a multi-drug resistant bacterial infection, in particular a multi-drug resistant P. aeruginosa infection, results in a synergistic increase in the efficacy of treatment than the efficacy of treatment with a monobactam antibiotic alone or a lysin polypeptide, as provided herein, alone. In other certain embodiments, the combination of the lysin polypeptide provided herein and a monobactam antibiotic for the treatment of a multi-drug resistant bacterial infection, in particular a multi-drug resistant P. aeruginosa infection, results in an increase in the efficacy of treatment that is greater than the efficacy of treatment with a monobactam antibiotic alone or a lysin polypeptide, as provided herein, alone. In still other certain embodiments, the combination of the lysin polypeptide provided herein and a monobactam antibiotic for the treatment of a multi-drug resistant bacterial infection, in particular a multi-drug resistant/¹ aeruginosa infection, and more particularly a P. aeruginosa resistant to a monobactam antibiotic, results in a reversal of bacterial resistance to the monobactam antibiotic.

In specific embodiments, the present disclosure provides for significant improvements in the efficacy of treatment of a bacterial infection, and in particular a P. aeruginosa bacterial infection, by a polypeptide comprising SEQ ID NO: 1 when used in combination with the monobactam antibiotic aztreonam. In certain embodiments, the combination of the polypeptide comprising SEQ ID NO: 1 and aztreonam for the treatment of a drug resistant bacterial infection, in particular a drug resistant/¹ aeruginosa infection, results in a synergistic increase in the efficacy of treatment than the efficacy of treatment with aztreonam alone or the polypeptide comprising SEQ ID NO: 1 alone. In other specific embodiments, the combination of the polypeptide comprising SEQ ID NO: 1 and aztreonam for the treatment of a drug resistant bacterial infection, in particular a drug resistant P. aeruginosa infection, results in an increase in the efficacy of treatment that is greater than the efficacy of treatment with aztreonam alone or the polypeptide comprising SEQ ID NO: 1 alone. In still other specific embodiments, the combination of the polypeptide comprising SEQ ID NO: 1 and aztreonam for the treatment of a drug resistant bacterial infection, in particular a drug resistant P. aeruginosa infection, and more particularly a P. aeruginosa resistant to aztreonam, results in a reversal of bacterial resistance to aztreonam. In still other specific embodiments, the combination of the polypeptide comprising SEQ ID NO: 1 and aztreonam for the treatment of a multi-drug resistant bacterial infection, in particular a multi-drug resistant/¹, aeruginosa infection, results in a synergistic increase in the efficacy of treatment than the efficacy of treatment with aztreonam alone or the polypeptide comprising SEQ ID NO: 1 alone. In other specific embodiments, the combination of the polypeptide comprising SEQ ID NO: 1 and aztreonam for the treatment of a multi-drug resistant bacterial infection, in particular a multi-drug resistant P. aeruginosa infection, results in an increase in the efficacy of treatment that is greater than the efficacy of treatment with aztreonam alone or the polypeptide comprising SEQ ID NO: 1 alone. In still other specific embodiments, the combination of the polypeptide comprising SEQ ID NO: 1 and aztreonam for the treatment of a multi-drug resistant bacterial infection, in particular a multi-drug resistant P. aeruginosa infection, and more particularly a P. aeruginosa resistant to aztreonam, results in a reversal of bacterial resistance to the aztreonam.

In certain embodiments of the present disclosure is provided a method for treatment of patients, for example patients with cystic fibrosis and a Gram-negative bacterial infection, with the polypeptide comprising SEQ ID NO: 1 and a monobactam antibiotic used together or in parallel with a mucolytic agent and/or mucolytic enzyme and/or bronehodilator, or as a separate indication to the mucolytic agent and/or mucolytic enzyme, and/or a bronehodilator. Mucolytic agents and mucolytic enzymes are a type of medications designed to help thin mucus in the lungs so patients can cough and expel it more easily, and are generally taken by inhalation using a nebulizer. Bronchodilators are a type of medication that make breathing easier by relaxing the muscles in the lungs and widening the airways and are used to treat long-term conditions including chronic obstructive pulmonary' disease (COPD) and pulmonary' complications arising from cystic fibrosis. Such bronchodilators may be either short-acting and used as short-term relief from for example, acute asthma attacks, or long-acting and used regularly to help control breathlessness in asthma, COPD, and cystic fibrosis patients and increase the effectiveness of corticosteroids in asthma.

In specific embodiments, the treatment comprises the method of providing an inbalable composition comprising by a polypeptide comprising SEQ II) NO: 1 and a monohactam antibiotic, and one or more mucolytic agents and/or mucolytic enzymes and/or bronehodilators. Such mucolytics agents and/or enzymes can also be administered by any one or more of the delivery methods as described herein. Non-limiting examples of such mucolytic agents include sodium or potassium citrate, potassium iodide, guaifenesin, tolu balsam, ammonium chloride, acetylcysteine, ambroxol, bromhexlne, carboeisteine, fudosteine, erdosteine, meeysteine, hypertonic saline, N- acetyl cysteine, etc. Non-limiting examples of such mucolytic enzymes include domase alia, gel sol in, thymosin beta4, etc. In certain specific embodiments of the present disclosure, the mucolytic agent and/or mucolytic enzyme is administered as a nebulized composition at the same time as the polypeptide comprising SEQ ID NO: 1 and a monohactam antibiotic, or as a pre- or post-treatment to the polypeptide comprising SEQ ID NO: 1 and a monohactam antibiotic. Non- limiting examples of such bronehodilators include beta-2 agonists including, but not limited to, as sa!butamol, sahneterol, formoterol, vilanterol, etc., anticholinergics including but. not. limited to ipratropium, tiotropium, aclidinium glyeopyrronium, etc., and theophylline.

In certain specific embodiments of the present disclosure, the bronchodilator is administered as a nebulized composition at the same time as the polypeptide comprising SEQ ID NO: 1 and a monohactam antibiotic, or as a pre- or post-treatment to the polypeptide comprising SEQ ID NO: 1 and a monohactam antibiotic, and may optionally include a mucolytic agent and/or mucolytic enzyme. In other embodiments of the present disclosure, a treatment for cystic fibrosis patients comprises the method of providing an inhalable composition comprising by a polypeptide comprising SEQ ID NO: 1 and a monobactam antibiotic, optionally one or more mucolytic agents and/or mucolytic enzymes and/or bronchodilators, and optionally one or more of cystic fibrosis transmembrane conductance (“CFTR”) modulators (or potentiator), CFTR correctors, and/or CFTR amplifiers. CFTR modulators contribute to the CFTR protein transporting salts across cell membranes by binding to the CFTR channel in an open conformation to increase salt transport. Such CFTR modulators include, but are not limited to, Kalydeco^® (ivacaftor), Orkambi^® (lumacaftor/ivacaftor), Symdeko^® (tezacaftor / ivacaftor), Trikafta^® (elexacaftor/ tezacaftor/ ivacaftor), etc. CFTR correctors are medications that bind to the CFTR protein and help it fold into the right shape so that more of the folded protein moves to the cell membrane. CFTR correctors can either bind directly to F508del-CFTR protein (chaperones) or work by creating conditions in the cell so that higher concentrations of CFTR can be made without being degraded (proteostasis regulators). Such CFTR modulators include, but are not limited to, Corr-4a (bisamionomethylbithiazole C4), VRT-325 (quinazolinone C3), etc. Other improved “next- generation” CFTR correctors include, but are not limited to, Lumacaftor (VX-809), Tezacaftor (VX-661), Cavosonstat (N91115), FDL169, etc. However, these treatments usually may not be sufficient to restore CFTR function and often are used in combination with a CFTR modulator, for example VX-455 and VX-659. CFTR amplifiers are treatments that increase the amount of CFTR protein that cells make so that amplifiers and modulators will be more effective. Amplifiers increase the amount of mutant CFTR messenger RNA, and consequently the amount of CFTR protein, thereby increasing the substrates for other CFTR modulators. Messenger RNA is an intermediate molecule containing a copy of the genetic code specifying the amino acid sequence of a protein. Amplifiers by themselves do not correct or improve the function of the CFTR protein. Two amplifiers, PTI-428 and PTI-CH, have shown promise in pre-clinical and clinical studies.

In one embodiment, the term “bacterial infection” may refer to a respiratory tract infection, especially but not exclusively to lower respiratory tract infections. In another embodiment, the term “bacterial infection” may refer to a sexually transmitted disease. In yet another embodiment, the term “bacterial infection” may refer to a urinary tract infection. In a further embodiment, the term “bacterial infection” may refer to acute exacerbation of chronic bronchitis. In still another embodiment, the term “bacterial infection” may refer to respiratory tract infections of patients having cystic fibrosis. In still a further embodiment, the term “bacterial infection” may refer to acute otitis media or neonatal septicemia. In yet a further embodiment, the term “bacterial infection” may refer to acute sinusitis. In one embodiment, the term “bacterial infection” may refer to an infection caused by drug resistant bacteria even multidrug-resistant bacteria. In another embodiment, the term “bacterial infection” may refer to catheter-related sepsis. In a further embodiment, the term “bacterial infection” may refer to community-acquired pneumonia or to nosocomial respiratory tract infections. In still a further embodiment, the term “bacterial infection” may refer to a complicated skin and skin structure infection. In yet a further embodiment, the term “bacterial infection” may refer to uncomplicated skin and skin structure infections. In one embodiment, the term “bacterial infection” may refer to endocarditis. In a further embodiment, the term “bacterial infection” may refer to hospital-acquired pneumonia. In still a further embodiment, the term “bacterial infection” may refer to osteomyelitis. In yet a further embodiment, the term “bacterial infection” may refer to sepsis.

In one embodiment, the present disclosure provides methods for treatment of the Gram negative bacterial infection in a subject caused by P. aeruginosa and optionally by at least one additional species of Gram-negative bacteria such as those selected from the group consisting of, Klebsiella spp., laHerobacter spp., P. coli, Citrobacter freundii, Salmonella typhimurium, Yersinia pestis, and Franciscella tulerensis , which are the Gram-negative bacteria most significant in human disease.

In one embodiment, the present disclosure provides methods for treatment of the Gram negative bacterial infection in a subject caused by P. aeruginosa. P. aeruginosa is an oxidase positive, Gram-negative, rod-shaped organism that is found ubiquitously in the environment. P. aeruginosa can grow in numerous habitats, including but not limited to soil, water, and on plant and animal tissue. It is an opportunistic organism and one of the most problematic nosocomial pathogens capable of causing localized or systemic disease in susceptible individuals such as people who have cystic fibrosis, cancer, bums, diabetic ulcers or an immune system deficiency. In a hospital setting in particular, it has become resistant to many commonly used antibiotics.

According to data from the US Centers for Disease Control and Prevention and the National Nosocomial Infection Surveillance System, P. aeruginosa is the second most common cause of nosocomial pneumonia, the third most common cause of urinary tract infection, the fourth most common cause of surgical site infection, the seventh most frequently isolated pathogen from the bloodstream, and the fifth most common isolate overall from all sites. Furthermore, P. aeruginosa is the most common multidrug-resistant Gram-negative pathogen causing pneumonia in hospitalized patients.

Nonlimiting examples of infections caused by P. aeruginosa include nosocomial infections such as respiratory tract infections especially in cystic fibrosis patients and mechanically- ventilated patients; bacteraemia and sepsis; wound infections, particularly those of burn victims; urinary tract infections; post-surgery infections on invasive devises; endocarditis by intravenous administration of contaminated drug solutions; infections in patients with acquired immunodeficiency syndrome, cancer chemotherapy, steroid therapy, hematological malignancies, organ transplantation, renal replacement therapy, and other conditions with severe neutropenia. Other non-limiting examples of infections caused by P. aeruginosa include those community- acquired infections such as: community-acquired respiratory tract infections; meningitis; folliculitis and infections of the ear canal caused by contaminated water; malignant otitis externa in the elderly and diabetics; osteomyelitis of the caleaneus in children; eye infections commonly associated with contaminated contact lens; skin infections such as nail infections in people whose hands are frequently exposed to water; gastrointestinal tract infections; and muscoskeletal system infections.

In some embodiments, the pharmaceutical compositions of the present disclosure are used to treat a subject at risk for acquiring an infection due to P. aeruginosa and/or another Gram negative bacterium. Subjects at risk for acquiring a P. aeruginosa or other Gram-negative bacterial infection include for example, but are not limited to, cystic fibrosis patients, neutropenic patients, patients with necrotising enterocolitis, burn victims, patients with wound infections, and more generally patients in a hospital setting, in particular surgical patients and patients being treated using an implantable medical device such as a catheter, for example a central venous catheter, or electrophysiologic cardiac devices, for example pacemakers and implantable defibrillators. Other patient groups at risk for infection with Gram-negative bacteria including P. aeruginosa include without limitation patients with implanted prostheses such a total joint replacement (for example total knee or hip replacement).

In one embodiment, the subject is suffering from a Gram-negative bacterial respiratory infection. In another embodiment, the subject is suffering from cystic fibrosis and each active ingredient is independently administered in an inhalable composition, an oral composition or a buccal composition. In a more specific embodiment, the subject is suffering from a Gram-negative bacterial respiratory infection associated with cystic fibrosis and each of the active ingredients is co-administered in an inhalable composition. In one embodiment, the subject is suffering from a wound that has been infected with/¹ aeruginosa or another Gram-negative bacterium. An example of a wound that is treatable by the methods of the present disclosure is an infected burn or a bum at risk of becoming infected. Such bums include thermal burns, cold temperature burns, chemical burns, electrical burns, or radiation burns.

Additionally, P. aeruginosa and other Gram-negative bacteria frequently colonize hospital food, sinks, taps, mops, and respiratory equipment. The infection is spread from patient to patient via contact with fomites or by ingestion of contaminated food and water.

In some embodiments, the pharmaceutical compositions further comprise a pharmaceutically acceptable carrier, buffering agent, or preservative. In some embodiments, the pharmaceutical composition is formulated for topical administration. In other embodiments, the pharmaceutical composition is formulated for subcutaneous delivery. In still other embodiments, the pharmaceutical composition is formulated for intravenous delivery. In yet other embodiments, the pharmaceutical composition is formulated for oral delivery. In yet other embodiments, the pharmaceutical composition is formulated for inhalable delivery. In still other embodiments, the pharmaceutical composition is engineered for inhalable delivery particulate size (e.g., micron, submicron, etc.) sufficient for proper anatomical placement in the pulmonary system. In some embodiments, the pharmaceutical composition is aerosolized sufficiently for effective and specific therapeutic pulmonary delivery methods, for example via oral-inhalable and/or intranasal pulmonary delivery methods. In other embodiments, the pharmaceutical composition is nano- designed sufficiently for effective therapeutic pulmonary delivery.

In some embodiments, the pharmaceutical composition further comprises a clotting agent. In some embodiments, the pharmaceutical composition is lyophilized.

The present invention also provides for methods for treating a subject in need thereof, comprising administering to the subject the pharmaceutical composition of the invention, comprising a polypeptide as disclosed herein, a monobactam antibiotic, and further comprising a pharmaceutically acceptable carrier, buffering agent, preservative and/or other agents including but not limited to as mucolytic agents or mucolytic enzymes, and/or a bronchodilator. The present invention particularly provides for methods for treating a subject in need thereof, comprising administering to the subject the pharmaceutical composition of the invention, comprising a polypeptide comprising SEQ ID NO: 1, aztreonam, and further comprising a pharmaceutically acceptable carrier, buffering agent, or preservative.

In one embodiment the method is a method for treating a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising a polypeptide of the invention, at least one monobactam antibiotic, and a pharmaceutically acceptable carrier, buffering agent, or preservative. In another related embodiment the method is a method for treating a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising a polypeptide comprising SEQ ID NO: 1, aztreonam, and a pharmaceutically acceptable carrier, buffering agent, or preservative.

In one embodiment is a method for treating a subject having a bacterial infection comprising administering to the subject a pharmaceutical composition comprising a polypeptide of the invention, a monobactam antibiotic, and a pharmaceutically acceptable carrier, buffering agent, or preservative. In a specific related embodiment is a method for treating a subject having a bacterial infection comprising administering to the subject a pharmaceutical composition comprising a polypeptide comprising SEQ ID NO: 1, aztreonam, and a pharmaceutically acceptable carrier, buffering agent, or preservative

In some embodiments, the subject has a bacterial infection that is non-responsive to other treatments. For example, the bacterial infection may be resistant to one or more antibiotics. In other embodiments, the bacterial infection is a wound infection. In still other embodiments is the method for prophylactically treating a subject comprising administering a pharmaceutical composition comprising a polypeptide of the invention, a monobactam antibiotic, and a pharmaceutically acceptable carrier, buffering agent, or preservative. In specifically related other embodiments is the method for prophylactically treating a subject comprising administering a pharmaceutical composition comprising a polypeptide comprising SEQ ID NO: 1, aztreonam, and a pharmaceutically acceptable carrier, buffering agent, or preservative. In some embodiments the subject has undergone, or is undergoing surgery and the surgical wound is contacted with a pharmaceutical composition of the invention. In certain embodiments, the surgical wound is irrigated with the pharmaceutical composition prior to closure of the wound. In other embodiments the pharmaceutical composition is applied to the wound after closure, for example the pharmaceutical composition is applied to the sutured or stapled area of the wound.

In other embodiments, the bacterial infection is a lung infection. In still other embodiments is the method for prophylactically treating a subject comprising administering a pharmaceutical composition comprising a polypeptide of the invention, a monobactam antibiotic, and a pharmaceutically acceptable carrier, buffering agent, or preservative in addition to other chemical such as mucolytic enzymes/agents, etc. In specifically related other embodiments is the method for prophylactically treating a subject comprising administering a pharmaceutical composition comprising a polypeptide comprising SEQ ID NO: 1, aztreonam, and a pharmaceutically acceptable carrier, buffering agent, or preservative. In some embodiments the subject needs a nebulizer or a ventilator in which the pharmaceutical composition of the invention is delivered. In certain embodiments, the lungs are irrigated with the pharmaceutical composition. In other embodiments the pharmaceutical composition is applied directly to the lungs.

In some embodiments, the method comprises administering a pharmaceutical composition of the invention in combination with an antibiotic. In some embodiments, the method comprises topically administering a pharmaceutical composition of the invention, for example through the skin, bladder, lungs, etc. In other embodiments, the method comprises administering a pharmaceutical composition of the invention subcutaneously. In still other embodiments, the method comprises administering a pharmaceutical composition of the invention by intravenous injection. In yet other embodiments, the method comprises administering a pharmaceutical composition of the invention orally. In still yet other embodiments, the method comprises administering a pharmaceutical composition via inhalation.

In some embodiments, the pharmaceutical composition is in a unit dosage form. In other embodiments, the pharmaceutical composition is in the form of a cream, ointment, salve, gel, lozenge, spray, or aerosol.

In some embodiments, the pharmaceutical composition is in a unit dosage form delivered to the lungs and/or pulmonary system, for example as an aerosolized composition or a nanoparticulate, each deliverable with a nebulizer, dry powder inhaler, etc. For aerosols used for inhalation delivery of a composition for use in the treatment of respiratory diseases, the formulation of the drug (excipient, active pharmaceutical ingredient, propellant, co-solvent) and delivery device (pressurized metered dose inhaler, dry powder inhaler, vibrating mesh nebulizer) must be considered in conjunction when considering the aerosol route for drug delivery. Aerosol particles are generated as either solid crystalline, amorphous or liquid droplet form, the latter as suspensions or solutions, undergoing rapid evolution in their size and composition in the aerosol phase. The formulations according to the invention may be inhaled orally or nasally.

The selection of device for pulmonary delivery can be an important factor in the formulation design. If the pharmaceutical composition is planned to a specific anatomical placement in the lungs, then the selected device should ideally be capable to generate and deliver the particles/droplets of specific aerodynamic diameter. The devices most commonly used for respiratory delivery includes nebulizers, metered-dose inhalers, and dry powder inhalers. Dry powder inhalers are of the most popular devices used to deliver drugs, especially proteins to the lungs. Some of the exemplary commercially available dry powder inhalers include Spinhaler and Rotahaler. Several types of nebulizers also are available and include jet nebulizers, ultrasonic nebulizers, and vibrating mesh nebulizers. Selection of a suitable device may depend on, for example, the specific characteristics of the pharmaceutical composition and its formulation, the site of action, and pathophysiology of the lung. Aqueous suspensions and solutions are nebulized effectively. Aerosols based on mechanically generated vibration mesh technologies also have been used successfully to deliver proteins to lungs and are currently being used in the clinical trials of protein and peptide-based pharmaceuticals. Recent applications of computational fluid dynamics have been helpful in design and development of DPI devices and understand the effect of airflow changes and deagglomeration in the inhaler device.

Also provided are methods for treating a bacterial infection comprising inhibiting the formation of or disrupting a bacterial biofilm comprising administering to a subject in need thereof, a composition comprising a polypeptide and a monobactam antibiotic of the invention in an amount effective to kill bacteria in the biofilm. Specific related methods for treating a bacterial infection comprise inhibiting the formation of or disrupting a bacterial biofilm comprising administering to a subject in need thereof, a composition comprising a polypeptide comprising SEQ ID NO: 1 and aztreonam of the invention in an amount effective to kill bacteria in the biofilm.

Additionally, provided herein are methods of disinfecting an article comprising contacting the article with a composition comprising a polypeptide and a monobactam antibiotic of the invention for a time sufficient to disinfect the article. Specific, related methods of disinfecting an article comprise contacting the article with a composition comprising a polypeptide comprising SEQ ID NO: 1, and aztreonam for a time sufficient to disinfect the article. In some embodiments, the article is a hard surface. In some embodiments, the article is a countertop, keyboard, surgical instrument, medical device, suture, implants, bandages, etc.

Additionally, provided herein are methods for inhibiting the formation of or disrupting a bacterial biofilm on an article comprising contacting the article with a lysin polypeptide, for example a polypeptide comprising SEQ ID NO: 1 and a monobactam antibiotic, for example aztreonam, in an amount effective to kill bacteria in the biofilm.

Also provided herein are articles of manufacture that contain a composition comprising a lysin polypeptide, for example a polypeptide comprising SEQ ID NO: 1, and a monobactam antibiotic, for example aztreonam. In some embodiments, the article of manufacture is a spray bottle that contains a lysin polypeptide, for example a polypeptide comprising SEQ ID NO: 1, and a monobactam antibiotic, for example aztreonam.

In some embodiments, the article of manufacture contains a pharmaceutical composition comprising a lysin polypeptide, for example a polypeptide comprising SEQ ID NO: 1, and a monobactam antibiotic, for example aztreonam, and a carrier, buffering agent or preservative. In some embodiments, the article of manufacture is a vial. In some embodiments, the article of manufacture is a delivery device. In some embodiments, the composition contained by the article of manufacture is lyophilized.

Modifications and changes can be made in the structure of the lysin polypeptides of the disclosure and still obtain a molecule having similar characteristics as the lysin polypeptide (e.g., a conservative amino acid substitution). For example, certain amino acids can be substituted for other amino acids in a sequence without appreciable loss of activity. Because it is the interactive capacity and nature of a polypeptide that defines that polypeptide's biological functional activity, certain amino acid sequence substitutions can be made in a lysin polypeptide sequence and nevertheless obtain a lysin polypeptide with like properties. Such amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include (original residue: exemplary substitution): (Ala: Gly, Ser), (Arg: Lys), (Asn: Gin, His), (Asp: Glu, Cys, Ser), (Gin: Asn), (Glu: Asp), (Gly: Ala), (His: Asn, Gin), (Be: Leu, Val), (Leu: Be, Val), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Tip: Tyr), (Tyr: Trp, Phe), and (Val: lie, Leu). Embodiments of this disclosure thus contemplate functional or biological equivalents of a polypeptide as set forth above. In particular, embodiments of the polypeptides can include variants having about 50%, 60%, 70%, 80%, 90%, and 95% sequence identity to the polypeptide of the invention.

“Identity” as known in the art, is a relationship between two or more polypeptide sequences, as determined by comparing the sequences. “Identity” can be readily calculated by known algorithms well known in the art. Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity are codified in publicly available computer programs. The percent identity between two sequences can be determined using analysis software (i.e., Sequence Analysis Software Package of the Genetics Computer Group, Madison Wis.) that incorporates the Needelman and Wunsch, (J. Mol. Biol., 48: 443-453, 1970) algorithm (e.g., NBLAST, and XBLAST).

Identity can be measured as “local identity” or “global identity”. Local identity refers the degree of sequence relatedness between polypeptides as determined by the match between strings of such sequences. Global identity refers to the degree of sequence relatedness of a polypeptide compared to the full-length of a reference polypeptide. Unless specified otherwise, as used herein identity means global identity. The percentages for global identity herein are calculated using the ClustalW algorithm used through the software MacVector, using the default settings; both for local and global identity.

Production of Polypeptides

Polypeptides of the present invention can be produced by any known method. For example, polypeptides can be produced in bacteria including, without limitation, E. coli , or in other existing system for polypeptide (e.g., Bacillus subtilis , baculovirus expression systems using Drosophila Sf9 cells, yeast or filamentous fungal expression systems, mammalian cell expression systems), or they can be chemically synthesized.

If a polypeptide is to be produced in bacteria, e.g., E. coli , the nucleic acid molecule encoding the peptide may also encode a leader sequence that permits the secretion of the mature peptide from the cell. Thus, the sequence encoding the peptide can include the pre sequence and the pro sequence of, for example, a naturally occurring bacterial ST peptide. The secreted, mature peptide can be purified from the culture medium.

The sequence encoding a peptide described herein is can be inserted into a vector capable of delivering and maintaining the nucleic acid molecule in a bacterial cell. The DNA molecule may be inserted into an autonomously replicating vector (suitable vectors include, for example, pGEM3Z and pcDNA3, and derivatives thereof). The vector may be a bacterial or bacteriophage DNA vector such as bacteriophage lambda or Ml 3 and derivatives thereof. Construction of a vector containing a nucleic acid described herein can be followed by transformation of a host cell such as a bacterium. Suitable bacterial hosts include but are not limited to, E. coli, B subtilis, Pseudomonas, Salmonella. The genetic construct also includes, in addition to the encoding nucleic acid molecule, elements that allow expression, such as a promoter and regulatory sequences. The expression vectors may contain transcriptional control sequences that control transcriptional initiation, such as promoter, enhancer, operator, and repressor sequences. A variety of transcriptional control sequences are well known to those in the art. The expression vector can also include a translation regulatory sequence (e.g., an untranslated 5' sequence, an untranslated 3' sequence, or an internal ribosome entry site). The vector can be capable of autonomous replication or it can integrate into host DNA to ensure stability during peptide production.

One embodiment of a nucleic acid according to the present invention is a nucleic acid that encodes a lysin polypeptide comprising an amino acid sequence that has at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1, wherein the lysin polypeptide has antibacterial activity.

In another embodiment, the nucleic acid encodes a lysin polypeptide comprising an amino acid sequence of SEQ ID NO: 1, wherein the lysin polypeptide has antibacterial activity. In yet another embodiment, the nucleic acid encodes a lysin polypeptide consisting of an amino acid sequence nucleic acid of SEQ ID NO: 1, wherein the lysin polypeptide or fragment has antibacterial activity.

Another embodiment is an expression vector that comprises a nucleic acid that encodes a lysin polypeptide comprising an amino acid sequence that has at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1, wherein the lysin polypeptide has antibacterial activity.

In another embodiment, the expression vector comprises a nucleic acid that encodes a lysin polypeptide comprising an amino acid sequence of SEQ ID NO: 1, wherein the lysin polypeptide has antibacterial activity.

In yet another embodiment, the expression vector comprises a nucleic acid that encodes a lysin polypeptide consisting of an amino acid sequence nucleic acid of SEQ ID NO: 1, wherein the lysin polypeptide has antibacterial activity.

The nucleic acid that encodes a lysin polypeptide described herein can also be fused to a nucleic acid encoding a peptide affinity tag, e.g., glutathione S-transferase (GST), maltose E binding protein, protein A, FLAG tag, hexa-histidine, myc tag or the influenza HA tag, in order to facilitate purification. The affinity tag or reporter fusion joins the reading frame of the peptide of interest to the reading frame of the gene encoding the affinity tag such that a translational fusion is generated. Expression of the fusion gene results in translation of a single peptide that includes both the peptide of interest and the affinity tag. In some instances where affinity tags are utilized, DNA sequence encoding a protease recognition site will be fused between the reading frames for the affinity tag and the peptide of interest.

Genetic constructs and methods suitable for production of immature and mature forms of the lysin polypeptides and variants described herein in protein expression systems other than bacteria, and well known to those skilled in the art, can also be used to produce lysin polypeptides in a biological system.

Lysin polypeptides and variants thereof can be synthesized by the solid-phase method using an automated peptide synthesizer. For example, the peptide can be synthesized on Cyc(4- CFb Bxl)-OCH2-4-(oxymethyl)-phenylacetamidomethyl resin using a double coupling program. Peptides can also be synthesized by many other methods including solid phase synthesis using traditional FMOC protection (i.e., coupling with DCC-HOBt and deprotection with piperdine in DMF). Lysin polypeptides and variants thereof of the present disclosure can be, as non-limiting examples, synthesized as a dimer, trimer or multiple peptides. Such multimer lysin polypeptides can be further stabilized using, for example, disulfide bonds between or among amino acids present in the natural polypeptide sequence or at amino acid position(s) engineered in to the amino acid sequence.

Therapeutic and Prophylactic Compositions and their Use

This invention provides methods of treatment comprising administering to a subject in need thereof an effective amount of a lysin polypeptide, for example the polypeptide comprising SEQ ID NO: 1, and a monobactam antibiotic, for example aztreonam. The subject is human or another animal, including but not limited to primates such as monkeys and chimpanzees; livestock animals such as cows, pigs, horse or chickens; and companion animals such as dogs, cats, and rodents. In a specific embodiment the subject is a human. In another specific embodiment the subject is a non human mammal. In another embodiment the lysin polypeptide, for example the polypeptide comprising SEQ ID NO: 1, and a monobactam antibiotic, for example aztreonam, are administered in combination with one or more other antibacterial agents. Methods of administration of the disclosed pharmaceutical compositions can be an inhalable aerosol, oral or parenteral and include but are not limited to intranasal, intratracheal, intraurethral, intradermal, intramuscular, intraperitoneal, intravenous, intra-articular, intra- synovial, subcutaneous, intranasal, epidural, topical and oral routes. The pharmaceutical composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. In addition, it may be desirable to introduce the pharmaceutical compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter. Pulmonary administration can also be employed, e.g., by use of an inhaler, ventilator, or nebulizer, and formulation with an aerosolizing agent or in addition to a mucolytic agent and/or mucolytic enzyme, etc., using such nebulizer or a ventilator. In a specific embodiment, it may be desirable to administer the pharmaceutical compositions of the invention locally to the area in need of treatment, such as topical use on the skin; any suitable method known to the art may be used.

In one aspect of the invention provides for pharmaceutical compositions comprising the polypeptides of the present disclosure, for example SEQ ID NO: 1, and a monobactam antibiotic, for example aztreonam, for therapeutic or prophylactic treatment of bacterial infections. An embodiment of the invention is a pharmaceutical composition formulated for topical treatment. Another embodiment of the invention is a pharmaceutical composition formulated for systemic infections. Such pharmaceutical compositions comprise a therapeutically effective amount of a polypeptide of the invention, a monobactam antibiotic, and a pharmaceutically acceptable carrier, buffering agent, or preservative. The term “pharmaceutically acceptable carrier” as used herein, includes, but is not limited to, solvents, diluents, or other liquid vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, solid binders, lubricants and the like, as suited to the particular dosage form desired. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition can also contain of wetting or emulsifying agents, preservatives, or pH buffering agents. These compositions can take the form of a solution, suspension, emulsion, tablet, pill, lozenge, capsule, powder, patches for topical administration and the like. For topical applications, the pharmaceutically acceptable compositions may be formulated in a suitable ointment, lotion or cream containing the active component suspended or dissolved in one or more carriers. Carriers for topical administration include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene-polyoxypropylene compounds, emulsifying wax, polysorbate 60, cetyl esters wax, ceteary alcohol, 2-octyldodecanol, benzyl alcohol and water. The composition can be formulated as a suppository with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. One of skill in the art is well versed in formulation of therapeutic agents.

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container is a notice in the form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals or biologic products, which notice reflects approval by the agency of manufacture, use or sale for human administration, directions for use, or both.

The above description is presented to enable a person skilled in the art to make and use the invention, and is provided in the context of a particular application and its requirements. Various modifications to the embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments and applications without departing from the spirit and scope of the invention. Thus, this invention is not intended to be limited to the embodiments described, but is to be accorded the widest scope consistent with the principles and features disclosed herein. Having generally described this invention, a further understanding can be obtained by reference to certain specific examples, which are provided herein for purposes of illustration only, and are not intended to be limiting unless otherwise specified.

While the concepts of the present disclosure are susceptible to various modifications and alternative forms, specific aspects and embodiments thereof have been shown by way of example in the drawings and will be described herein in detail. It should be understood, however, that there is no intent to limit the concepts of the present disclosure to the particular forms disclosed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives consistent with the present disclosure and the appended claims. References in the specification to “one embodiment,” “an embodiment,” “an aspect,” “one aspect,” etc., indicate that the aspect or embodiment described may include a particular feature, structure, or characteristic, but every aspect or embodiment may or may not necessarily include that particular feature, structure, or characteristic. Moreover, such phrases are not necessarily referring to the same aspect or embodiment. Further, when a particular feature, structure, or characteristic is described in connection with an aspect or embodiment, it is submitted that it is within the knowledge of one skilled in the art to effect such feature, structure, or characteristic in connection with other aspects or embodiments whether or not explicitly described. Additionally, it should be appreciated that items included in a list in the form of “at least one A, B, and C” can mean (A); (B); (C); (A and B); (A and C); (B and C); or (A, B, and C). Similarly, items listed in the form of “at least one of A, B, or C” can mean (A); (B); (C); (A and B); (A and C); (B and C); or (A, B, and C).

It will be apparent to one skilled in the art that various changes or equivalents may be made without departing from the scope of the invention.

While the disclosure has been illustrated and described in detail in the foregoing drawings and description, the same is to be considered as exemplary and not restrictive in character, it being understood that only illustrative embodiments thereof have been shown and described and that all changes and modifications that come within the spirit of the disclosure are desired to be protected.

EXAMPLES

Example 1. Here is examined the combination of the BH01 lysin polypeptide (SEQ ID NO: 1) with aztreonam, meropenem, cefepime, and tobramycin against primary respiratory pathogens of patients with cystic fibrosis, including drug resistant/¹ aeruginosa , Burkholderia cepacia and S. aureus. Determinations of synergy, antagonism or indifference were made based on testing the BH01 polypeptide and antibiotics alone to evaluate the minimum inhibitory concentration (“MIC”) and in combination to evaluate the fractional inhibitory concentration indices (“FICI”). MIC values for the and the resulting mean FIC indices are summarized in Table 1. FICI and mean FICI values indicated neither synergy nor antagonism for the BH01 lysin polypeptide in combination with aztreonam, meropenem, cefepime, or tobramycin against either Staphylococcus aureus ATCC 29213 (QC) or S. aureus ATCC 43300 (methicillin-resistant). Mean FICI values indicative of synergy (values <0.5) were observed with aztreonam when combined with the BH01 lysin polypeptide against the two P. aeruginosa isolates, P. aeruginosa ATCC 27853 (quality control strain) and P. aeruginosa MMX 10106 [(MDR), VIM] (Tables 1(a) and 1(b)). The mean FICI values observed for the BH01 lysin polypeptide plus antibiotic combinations against B. cepacia MMX 546 and B. cepacia MMX 1793 indicated neither synergy nor antagonism, with the exception of the BH01 lysin polypeptide-tobramycin combination, where antagonism was observed against the B. cepacia MMX 546 isolate only (FICI values >4). Despite the BH01 lysin polypeptide being developed for treatment of A. baumannii as monotherapy, the BH01 lysin polypeptide in combination with aztreonam demonstrated synergy in vitro against P. aeruginosa based on fractional inhibitory concentration (Tables 1 (a) and (b) and Table 2).

TABLE 1(a). Minimum Inhibitory Concentration, Fractional Inhibitory Concentration of BH01 and select antibiotics against target organisms.

QC results against A. coli ATCC 25922 and P. aeruginosa ATCC 27853

AMP/SUL, ampicillin/sulbactam; MEM, meropenem; LVX, levofloxacin; TIG, tigecycline; COL, colistin; RIF, rifampicin; TOB, tobramycin

TABLE 1(b). Minimum Inhibitory Concentration, Fractional Inhibitory Concentration of BH01 and select antibiotics against target organisms.

QC results for all FIC runs

Table 2. Summary of mean FICI data for BH01 (SEQ ID NO: 1) lysin polypeptide and comparators against pathogens from cystic fibrosis

MRSA, methicillin-resistant S. aureus; MDR, multi drug-resistant; * The mean FICI value indicates synergy.

Example 2. Synergistic antibiotic effects between BH01 (SEQ ID NO: 1) lysin polypeptide and the monobactam antibiotic aztreonam (AZT) were further evaluated for select P. aeruginosa isolates through a time-kill kinetic analysis. The analysis was performed with BH01 lysin polypeptide and aztreonam alone and in combination.

BH01 lysin polypeptide was active against A. baumannii ATCC BAA- 1797 with an MIC value of 32 pg/mL. Aztreonam MIC values used to determine time-kill test concentrations were based on those observed in a prior study fori⁵ aeruginosa ATCC 27853 (4 pg/mL), P. aeruginosa CDC 0241 (16 pg/mL), and P. aeruginosa CDC 051 (64 pg/mL). Time-kill (TK) analysis was performed on three P. aeruginosa isolated types with sub-inhibitory concentrations of BH01 lysin polypeptide and aztreonam alone or in combination. Viable counts were plotted over time by test isolate and the corresponding log CFU/mL values and variation in log CFU/mL values for the combination relative to either agent alone was reported for strains that are sensitive or resistant to Aztreonam.

All combinations of the BH01 lysin polypeptide plus aztreonam displayed synergistic activity (2-log decrease in CFU/mL in comparison to treatment of either agent alone) at the 6- and 24-hour marks against all three evaluated P. aeruginosa isolates. FIG. 1 shows results of P. aeruginosa ATCC 27853 time kill study. FIG. 2 shows results of P. aeruginosa CDC 0241 time kill study. FIG. 3 shows results of P. aeruginosa CDC 051 time kill study. Aztreonam and BH01 lysin polypeptide combinations showed synergy against all three evaluated P. aeruginosa isolates by the four-hour mark when testing BH01 lysin polypeptide at 128 pg/mL and aztreonam at 0.25X the MIC value. The combination of BH01 lysin polypeptide at 256 pg/mL and aztreonam at 0.25X the MIC displayed synergistic activity at 4 hours against two out of the three isolates tested. In summary, the combination of sub-inhibitory MIC values of both BH01 lysin polypeptide and aztreonam displayed synergistic killing of three P. aeruginosa isolates by 4 hours in a time-kill kinetic analysis. These data provide evidence that the BH01 lysin polypeptide in combination with aztreonam have a greater than expected combinatorial effect on efficacy in P. aeruginosa killing and evidences both the reversal of aztreonam resistance in the aztreonam-resistant strain

Pseudomonas strains to sensitive to aztreonam.

Claims

WHAT IS CLAIMED:

1. A pharmaceutical composition comprising an isolated polypeptide comprising the amino acid sequence SEQ ID NO: 1 and a monobactam antibiotic.

2. The pharmaceutical composition of claim 1, wherein the monobactam antibiotic is one or more of the group consisting of aztreonam, nocardicin A, tabtoxin, and tigemonam.

3. The pharmaceutical composition of claim 1, wherein the monobactam antibiotic is effective against aerobic Gram-negative bacteria.

4. The pharmaceutical composition of claim 1, wherein the aerobic Gram-negative bacteria is P. aeruginosa.

5. The pharmaceutical composition of claim 1 further comprising a pharmaceutically acceptable carrier.

6. The pharmaceutical composition of claim 1, comprising an isolated polypeptide comprising the amino acid sequence SEQ ID NO: 1 and aztreonam.

7. . The pharmaceutical composition of claim 1, further comprising one or more of the group consisting of a mucolytic enzyme, a mucolytic agent, a bronchodilator, a CFTR modulator, a CFTR amplifier, and a CFTR corrector.

8. A method of inhibiting the growth, or reducing the population, or the killing of at least one species of an aerobic Gram-negative bacteria with a composition comprising the amino acid sequence SEQ ID NO: 1 and one or more monobactam antibiotics, wherein the isolated polypeptide and the one or more monobactam antibiotics has the property of inhibiting the growth, or reducing the population, or the killing of at least one species of aerobic Gram-negative bacteria.

9. A method of treating a bacterial infection caused by P. aeruginosa and optionally one or more additional species of aerobic Gram-negative bacteria, comprising administering to a subject diagnosed with, at risk for, or exhibiting symptoms of a bacterial infection, the pharmaceutical composition of claim 1.

10. A method of treating a topical or systemic pathogenic bacterial infection caused by P. aeruginosa and optionally one or more species of an aerobic Gram-negative bacteria in a subject, comprising administering to a subject the pharmaceutical composition of claim 1.

11. A method for augmenting the efficacy of an antibiotic suitable for treating an aerobic Gram-negative bacterial infection, comprising co-administering an isolated polypeptide comprising the amino acid sequence SEQ ID NO: 1 in combination with a monobactam antibiotic, wherein administration of the combination is more effective in inhibiting the growth of, or reducing an initial population of, or killing the aerobic Gram-negative bacteria than administration of either the monobactam antibiotic or the isolated polypeptide comprising the amino acid sequence SEQ ID NO: 1 individually.