WO2023055434A1 - Endolysin polypeptide compositions and methods of use - Google Patents
Endolysin polypeptide compositions and methods of use Download PDFInfo
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- WO2023055434A1 WO2023055434A1 PCT/US2022/029250 US2022029250W WO2023055434A1 WO 2023055434 A1 WO2023055434 A1 WO 2023055434A1 US 2022029250 W US2022029250 W US 2022029250W WO 2023055434 A1 WO2023055434 A1 WO 2023055434A1
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- endolysin
- pylori
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- pharmaceutical composition
- antibiotic
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01017—Lysozyme (3.2.1.17)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
Definitions
- the present disclosure is generally directed towards methods and compositions for the prevention, inhibition and treatment of infections caused by Gram-negative bacteria.
- Helicobacter pylori is a spiral-shaped, Gram-negative, pathogenic bacterium.
- H. pylori infection occurs through colonization in the human stomach. With over 4 billion people in the world infected with H. pylori, it is considered the most wide-spread chronic bacterial infection globally. All patients infected with H. pylori develop chronic gastritis, 20% develop peptic ulcer(s), and approximately 1% develop adenocarcinoma or mucosa-associated lymphoid tissue (MALT)-type gastric lymphoma.
- MALT mucosa-associated lymphoid tissue
- H. pylori infection is considered to be the leading risk factor for the development of gastric cancer and is categorized as a type-1 carcinogen, accounting for over one-third of all new infection- related cancer cases.
- H. pylori virulence factors also have been associated with cancer malignancy.
- the most frequently associated H. pylori virulence factors are the VacA vacuolating cytotoxin and the Cag pathogenicity island (cagPAT).
- cagPAT Cag pathogenicity island
- H. pylori In addition to the high degree of virulence of H. pylori, a growing clinical challenge has been to develop optimal treatment programs which take into account the extremely high levels of H. pylori antibiotic resistance. Antibiotic resistance is conferred in part through proliferation in an acidic pH environment. Since H. pylori transforms into its antibiotic-resistant coccoid form at an acidic pH, current treatments incorporate a proton pump inhibitor (PPI) in combination with antibiotics. However, the PPI decreases acidic pH and thereby increasing antimicrobial activity and the half-life of antibiotics, and antibiotic resistance is now becoming significant with regard to antibiotic standard of care treatments. Resistance levels have demonstrably increased globally, with some regions identifying near complete resistance to at least one of the standard-of-care antibiotics. As of 2018, current levels of antibiotic resistance in the United States are approximately 35% to clarithromycin, 30% to metronidazole, and 20% to levofloxacin, with multiple drug resistance strains progressing in other global regions, particularly the Asiatic countries.
- H. pylori cure rates using standard-of-care antibiotic treatment have been reported to be as low as 57%, whereas the minimum acceptable rate for a first treatment is approximately 90%.
- treatment failure rates are found to be substantially greater when applied to patients with antibiotic resistant H. pylori.
- a statistically significant association between eradication treatment failure and antibiotic resistance detected before treatment was observed for all the standard of care antibiotics.
- Patients with clarithromycin-resistant H. pylori infections were shown to have a risk of failing eradication approximately 7-fold higher than patients with susceptible strains when treated with a clarithromycin-containing regimen.
- 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.
- Phage endolysins are generally species or subspecies specific, and are effective only against bacteria from which they were produced. While some endolysins act upon the cell walls of several bacterial genus or species, some broad-spectrum endolysins have been found. However, the use of endolysins 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 endolysins to the peptidoglycan substrates in the cell wall, and therefore endolysins have been used as a treatment mainly against Gram-positive bacteria. Even though certain endolysins have demonstrated 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 antibiotic resistant//, pylori.
- the present disclosure may comprise one or more of the following features and combinations thereof.
- compositions of endolysin peptides used in combination with certain antibiotics provides one or more of features and combinations thereof as described herein, and methods of use thereof.
- application of the endolysin peptides in combination with certain antibiotics to a bacterial infection may increase the susceptibility of the infectious bacteria to treatment.
- a pharmaceutical composition for the treatment of an 7/ pylori infection comprising an isolated endolysin peptide having the activity of degrading the cell wall of Heliobacter pylori.
- the endolysin can be an isolated lysozyme.
- the endolysin can be an isolated bacteriophage endolysin.
- the pharmaceutical composition endolysin peptide can be an H. pylori bacteriophage endolysin.
- the pharmaceutical composition can be delivered to a patient in sufficient quantities to enhance effectiveness of standard of care antibiotic treatment.
- the pharmaceutical composition endolysin peptide can be one or more of SEQ ID NOS: 1 - 11.
- the pharmaceutical composition can further comprise at least one antibiotic, including H. pylori infection standard of care antibiotics, for example clarithromycin, amoxicillin, levofloxacin, metronidazole, rifabutin, tinidazole, tetracycline, etc.
- the pharmaceutical composition can further comprise at least one proton pump inhibitor, for example omeprazole, lansoprazole, dexlansoprazole, rabeprazole, pantoprozal, esomeprazole, etc.
- the pharmaceutical composition can further comprise bismuth subsalicylate.
- the pharmaceutical composition can further comprise at least one antihistamine, for example cimetidine, famotidine, nizatidine, ranitidine, etc.
- the pharmaceutical composition can be delivered to a patient in sufficient quantities to reduce an H. pylori infection by at least 50%, by at least 60%, by at least 70%, by at least 80%, by at least 90%, by at least 95%.
- a method of inhibiting the growth, or reducing the population, or the killing of H. pylori with a pharmaceutical composition comprising an isolated endolysin having the activity of degrading the cell wall of Heliobacter pylori, and one or more of an H. pylori infection standard of care antibiotic, antihistamine, PPI and bismuth subsalicylate, wherein the pharmaceutical composition has the property of inhibiting the growth, or reducing the population, or the killing of at least one species of H. pylori.
- pylori bacterial infection comprising coadministering an isolated endolysin having the activity of degrading the cell wall of Heliobacter pylori in combination with one or more of an H. pylori infection standard of care antibiotic, antihistamine, PPI and bismuth subsalicylate, wherein administration of the combination is more effective in inhibiting the growth of, or reducing an initial population of, or killing the H. pylori bacteria than administration of either the one or more of an H. pylori infection standard of care antibiotic, antihistamine, PPI and bismuth subsalicylate or the isolated endolysin having the activity of degrading the cell wall of Heliobacter pylori individually.
- 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.
- 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).
- 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).
- connecting elements are described to convey a connection, relationship, or association between or among two or more other 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 apply even in the absence of a discussion thereof.
- a connecting element may include, be embodied as, or otherwise represent, multiple connections, relationships, or associations between elements.
- a connecting element provides or 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.
- bacteriaicidal 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.
- 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.
- antibacterial in a context of an agent is used generically to include both bacteriostatic and bactericidal agents.
- 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.
- certain strains of Heliobacter pylori 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.
- 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.
- pharmaceutically acceptable carriers are suitable for use with subjects without undue adverse side effects (such as toxicity, irritation, and allergic response).
- 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.
- excipients such as urea can be useful to improve stability.
- Other excipients include bulking agents, buffering agents, tonicity modifiers, surfactants, preservatives and co-solvents.
- suitable pharmaceutically acceptable excipients include, but are not limited to, starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like.
- suitable pharmaceutically acceptable excipients include, but are not limited to, water, glycols, oils, alcohols, flavoring agents, preservatives, and the like.
- 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.
- an 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.
- co-administer is intended to embrace separate administration of an endolysin polypeptide, any one or more of a standard of care antibiotic, any one or more of an antihistamine, any one or more of a PPI, bismuth subsalicylate, any other standard of care composition as known in the art for treatment of H. pylori infection, 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 endolysin polypeptides with any one or more of a standard of care antibiotic, any one or more of an antihistamine, any one or more of a PPI, bismuth subsalicylate, any other standard of care composition as known in the art for treatment of H. pylori infection 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.
- the endolysin could be administered only initially within 24 hours of the first of any one or more of a standard of care antibiotic, any one or more of an antihistamine, any one or more of a PPI, bismuth subsalicylate, any other standard of care composition as known in the art for treatment of H. pylori infection, use and then the use of any one or more of a standard of care antibiotic, any one or more of an antihistamine, any one or more of a PPI, bismuth subsalicylate, any other standard of care composition as known in the art for treatment of H. pylori infection, may continue without further administration of endolysin.
- subject refers to a subject to be treated and generally includes a mammal.
- mammal subjects include, for example, humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals.
- 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.
- 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 20 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 an endolysin polypeptide as described below and maintaining the endolysin function. A polypeptide can be a naturally occurring polypeptide or an engineered or synthetically produced polypeptide.
- a particular endolysin 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 al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989)).
- conventional peptide synthesis techniques e.g., solid phase synthesis
- molecular biology techniques such as those disclosed in Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989)).
- variantants of endolysin polypeptides are also encompassed having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% sequence identity with the endolysin polypeptides provided herein.
- 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.
- 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.
- fusion polypeptide can be used interchangeably with the term “fusion protein.”
- the open-ended expression “a polypeptide comprising” a certain structure includes larger molecules than the recited structure such as fusion polypeptides.
- heterologous refers to nucleotide, peptide, or polypeptide sequences that are not naturally contiguous.
- 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 endolysin 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 endolysin activity. Included in this definition are two or more endolysin polypeptides or active fragments thereof. These can be used to make a fusion polypeptide with endolysin activity
- 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 an endolysin active to bore through the outer membrane and hydrolyze the coating of Gram-negative bacteria, whether by cleaving a sugar backbone or peptide bond.
- amphipathic peptide refers to a peptide having both hydrophilic and hydrophobic functional groups.
- secondary structure places hydrophobic and hydrophilic amino acid residues at different ends of the peptide. These peptides often adopt a helical secondary structure.
- cationic peptide refers to a peptide having positively charged amino acid residues.
- a cationic peptide has a pKa-value of 9.0 or greater.
- cationic peptide in the context of the present disclosure also encompasses polycationic peptides.
- polycationic peptide 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.
- 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.
- hydrophobic amino acids include glycine (Gly), alanine (Ala), valine (Vai), leucine (Leu), isoleucine (He), proline (Pro), phenylalanine (Phe), methionine (Met), and tryptophan (Trp).
- the term “augmenting” within the context of the present disclosure refers to a degree of any one or more of a standard of care antibiotic, any one or more of an antihistamine, any one or more of a PPI, bismuth subsalicylate, any other standard of care composition as known in the art for treatment of H. pylori infection, activity that is higher than it would be otherwise.
- “Augmenting” encompasses additive as well as synergistic (superadditive) effects.
- synergy or synergestic or synergism refers to a beneficial effect brought about by two or more active substances that exceeds that produced by each substance administered or applied alone.
- One or more 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.
- 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.
- 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 Gramnegative bacteria responsible for an infection or contamination.
- 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.
- 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 endolysin 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 endolysin polypeptide.
- a protective or capping group on a reactive moiety
- a detectable label such as antibody and/or fluorescent label
- glycosylation such as glycosylation
- a bulking group such as PEG
- 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)
- GFP green fluorescent protein
- RFP red fluorescent protein
- CFP cyan fluorescent protein
- YFP yellow fluorescent protein
- GFP green fluorescent protein
- RFP red fluorescent protein
- CFP cyan fluorescent protein
- YFP yellow fluorescent protein
- GFP green fluorescent protein
- RFP red fluorescent protein
- CFP cyan fluorescent protein
- YFP yellow fluorescent protein
- Polyethylene glycol (PEG) conjugation to proteins has been used as a method for extending the circulating half-life of many pharmaceutical proteins.
- derivative includes endolysin polypeptides chemically modified by covalent attachment of one or more PEG molecules. It is anticipated that pegylated endolysin polypeptides will exhibit prolonged circulation half-life compared to the unpegylated endolysin polypeptides, while retaining biological and therapeutic activity.
- percent amino acid sequence identity refers to the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific endolysin 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 endolysin peptides as well.
- substantially identical will encompass mutated, truncated, fused, or otherwise sequence-modified variants of isolated endolysin 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 endolysin 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 endolysin polypeptide are substituted with a similar or conservative amino acid substitution, and wherein the resulting endolysin have the profile of activities, antibacterial effects, and/or bacterial specificities of endolysin polypeptides disclosed herein.
- 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.
- antimicrobial peptide or “antimicrobial polypeptide” (AMP) refers to a member of a wide range of short (commonly 6 to 50 amino acid residues in length, but can be longer) 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.
- 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 of using the disclosed pharmaceutical compositions.
- 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.
- Endolysins are capable of cleaving the major bonds in the peptidoglycan layer of bacterial cell walls. Endolysins may be classified into five types, depending on the bonds these enzymes cleave in the peptidoglycan layer: (1) N-acetylmuramidases (e.g., lysozyme) and (2) lytic transglycolases cleave the N-acetylmuramoyl— 1,4-N-acetyl glucosamine bond between the alternating MurNAc and GlcNAc disaccharide backbone of the peptidoglycan; (3) N-acetyl— D- glucosaminidases cleaves the other glycosidic bond between the disaccharides (between the N- acetylglucosaminyl— 1,4-N-acetylmuramine bond), (4) N-acetylmuramoyl L-alanine amidases, which cleave between the
- H. pylori is a Gram-negative bacterium, it possesses a thin peptidoglycan layer in the bacterial cell wall formed from linear chains of two alternating amino sugars, namely N- acetyl glucosamine (GlcNAc or NAGA) and A-acetylmuramic acid (MurNAc or NAM A).
- the alternating sugars are connected by a p-(l,4)-glycosidic bond.
- This thin outer membrane is located outside the cell wall and prevents or inhibits some endolysins from interaction with the cell wall.
- N-acetylmuramidases including as a non-limiting example, lysozymes, and phage endolysins, for example H. pylori phage endolysins.
- a pharmaceutical composition comprising the isolated lysozyme of H. pylori R038b, comprising the amino acid sequence: MILVASFLIVDLEGFSPSIYTDKTGNPTIGYGYNLSVYSYEGKRITKAYGLLTDILKENYK AILS YGWYKNLDAMRRMVILDLSYNLGLNGLLKFKQFIKAIEDKNY ALA VERLQKSPYF NQVKKRASRNMEILKLGGCEKHCKKKYTIEKVIKEVGLELKSKSVMPYFFNEYDLTKN ANREEPERLRSQIISILMKTPKGREILKEYLNEGCILLGE (SEQ ID NO: 1).
- a pharmaceutical composition comprising the isolated lysozyme of H. pylori OK310 comprising the amino acid sequence: MDAMRRMVILDLSYNLGLSGLLKFKQFIKAIEDKNYALAVERLQKSPYFNQVRKRASR NMEVLKLGGCEKHCKKKYTIKKVIKEGGA (SEQ ID NO: 2).
- a pharmaceutical composition comprising the isolated lysozyme H. pylori UM066 comprising the amino acid sequence: MLCLSKLILAASFLIVDSEGFSPSVYTDKTGHPTIGYGYNLSVYSYESKRITKPQAYGLLT DILKENHKALLSYGWYKNLDAMRRMVILDLSYNLGLSGLLKFKQFIKGYRG (SEQ ID NO: 3).
- endolysins can also be used for the present disclosure.
- a pharmaceutical composition comprising the isolated endolysin Heliobacter phage endolysin YP 007237642 comprising the amino acid sequence:
- composition comprising the isolated endoendolysin YP 007005686 comprising the amino acid sequence: MDLTNLEDALNNGNFKEQVYSGLDGVYRISKVLNQLDLLKNFSEHDLEIVGGNGWVFH EHSQAIVYEILK (SEQ ID NO: 5).
- composition comprising the isolated endoendolysin of H. pylori P12 comprising the amino acid sequence: MILAASFLIVDSEGFSPSIYTDKTGHPTIGYGYNLSVYSYEGKRITKAYGLLTDILKENYK ALLSYGWYKNLDAMRRMVILDLSYNLGLSGLLKFKQFIKAIEDKNYALAVERLQKSPY
- FNQVKKERQGIWKF (SEQ ID NO: 6).
- a pharmaceutical composition comprising the isolated endolysin of H. pylori 0339, (ATCC strain 26695) comprising the amino acid sequence:
- composition comprising the isolated endolysin of H. pylori A-14 comprising the amino acid sequence:
- composition comprising the isolated endolysin of H. pylori H-l 1 comprising the amino acid sequence:
- composition comprising the isolated endolysin of H. pylori PeCan4 comprising the amino acid sequence:
- NEYDLIKKRQQRRA (SEQ ID NO: 10).
- a pharmaceutical composition comprising the isolated endolysin of H. pylori CPY1313 comprising the amino acid sequence: MDSEGFSPSIYTDKTGHPTIGYGYNLSVYSYESKRITKPQAYGLLTDILKENHKALLSYG WYKNLDAMRRMVILDLSYNLGLSGLLKFKQFIKAIEDKNYALAVERLQKSPYFNQVRK RASRNMEILKLGGCEKHCKKKYTIEKVIKEGGA (SEQ ID NO: 11).
- a fragment of any one or more of SEQ ID NOs. 1 - 11 are conjugated to an antimicrobial peptide to yield a conjugated polypeptide having antibacterial activity.
- any combination of 2 or more of SEQ ID NOS: 1 - 11 are used for the pharmaceutical composition of the present disclosure.
- any combination of 2 or more fragments of SEQ ID NOS: 1 - 11 are used for the pharmaceutical composition of the present disclosure.
- the peptides SQSRESQC (SEQ ID NO: 12) and CSQRQSES (SEQ ID NO: 13) are derived from hepatitis C virus and has been shown to have antimicrobial activity against gram-positive and gram-negative bacteria.
- SEQ ID NO: 12 a fusion polypeptide of SEQ ID NO: 12 or SEQ ID NO: 13 and any one or more of SEQ ID NOS: 1 - 11.
- a fusion polypeptide comprises the eight amino acids C, S, Q, R, S, E and S in any order and having antimicrobial activity is conjugated to any one or more of SEQ ID NOS: 1 - 11.
- SEQ ID NO: 12 or SEQ ID NO: 13 is conjugated to the C-terminus of any one or more of SEQ ID NOS: 1 - 11. In still other embodiments of the present disclosure, SEQ ID NO: 12 or SEQ ID NO: 13 is conjugated to the N- terminus of any one or more of SEQ ID NOS: 1 - 11.
- Modifications and changes can be made in the structure of the endolysin polypeptides of the disclosure and still obtain a molecule having similar characteristics as the endolysin polypeptide (e.g., a conservative amino acid substitution).
- 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 an endolysin polypeptide sequence and nevertheless obtain a endolysin 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, Vai), (Leu: Be, Vai), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Tip: Tyr), (Tyr: Trp, Phe), and (Vai: He, Leu).
- Embodiments of this disclosure thus contemplate functional or biological equivalents of a polypeptide as set forth above.
- 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.
- Polypeptides of the present invention can be produced by any known method.
- polypeptides can be produced in bacteria including, without limitation, E. coh. 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.
- the nucleic acid molecule encoding the peptide may also encode a leader sequence that permits the secretion of the mature peptide from the cell.
- 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.
- nucleic acid that encodes an endolysin polypeptide comprising an amino acid sequence that has at least 90% sequence identity to any of the amino acid sequences of SEQ ID NOS: 1 - 11, wherein the endolysin polypeptide has antibacterial activity.
- the nucleic acid encodes an endolysin polypeptide comprising an amino acid sequence of any one of SEQ ID NOS: 1 - 11, wherein the endolysin polypeptide has antibacterial activity.
- the nucleic acid encodes an endolysin polypeptide consisting of an amino acid sequence nucleic acid of any one of SEQ ID NOS: 1 - 11, wherein the endolysin polypeptide or fragment has antibacterial activity.
- Another embodiment is an expression vector that comprises a nucleic acid that encodes an endolysin polypeptide comprising an amino acid sequence that has at least 90% sequence identity to any one of the amino acid sequences of SEQ ID NOS: 1 - 11, wherein the endolysin polypeptide has antibacterial activity.
- the expression vector comprises a nucleic acid that encodes an endolysin polypeptide comprising any one of the amino acid sequences of SEQ ID NOS: 1 - 11, wherein the endolysin polypeptide has antibacterial activity.
- the expression vector comprises a nucleic acid that encodes an endolysin polypeptide consisting of an amino acid sequence nucleic acid of any one of SEQ ID NOS: 1 - 11, wherein the endolysin polypeptide has antibacterial activity.
- the nucleic acid that encodes an endolysin 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.
- GST glutathione S-transferase
- 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.
- DNA sequence encoding a protease recognition site will be fused between the reading frames for the affinity tag and the peptide of interest.
- Endolysin polypeptides and variants thereof can be synthesized by the solid-phase method using an automated peptide synthesizer.
- the peptide can be synthesized on Cyc(4- CH2Bxl)-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).
- a method of treatment comprising administering to a subject in need thereof an effective amount of an endolysin polypeptide, for example the polypeptide comprising any combination of one or more of SEQ ID NOS: 1 - 11.
- the method further comprises standard of care treatment, for example administering to a subject in need thereof an effective amount of an antihistimine.
- the antihistamine can be one or more of cimetidine, famotidine, nizatidine, ranitidine, etc.
- the method further comprises administering to a subject in need thereof an effective amount of bismuth subsalicylate.
- the method further comprises administering to a subject in need thereof an effective amount of an antibiotic.
- the antibiotic can be one or more of clarithromycin, amoxicillin, levofloxacin, metronidazole, tinidazole, tetracycline, etc.
- the method further comprises administering to a subject in need thereof an effective amount of a proton pump inhibitor.
- the proton pump inhibitor can be one or more of omeprazole, lansoprazole, dexlansoprazole, rabeprazole, pantoprozal, esomeprazole, etc.
- the method of administrating the pharmaceutical composition disclosed herein can be a co-administration one or more of the antihistamine, the PPI, bismuth subsalicylate, and an antibiotic with one or more of the endolysins effective against H. pylori.
- Methods of administration of the disclosed pharmaceutical compositions can be oral using for example, liquid or gel dosages, oral tablets, granules, powders, and sprinkle capsules.
- the pharmaceutical carrier can be microorganisms, for example Spirulina. spp, etc., expressing the peptides of the present disclosure, dried and stored in dosage specific capsules for oral delivery.
- compositions of the present disclosure and a pharmaceutically acceptable carrier, buffering agent, or preservative.
- pharmaceutically acceptable carrier 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.
- 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, and the like.
- 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.
- 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.
- NCBI reference sequence WP 010875465 corresponding to SEQ ID NO. 7 was first codon optimized for expression in E. coli BL21 (DE3). After codon optimization, the DNA sequence was synthesized and cloned into an E. coli expression vector pET30 with a 6-His tag at the N-terminus. The inserted sequence encoding for codon optimized HP -Lysozyme was validated by DNA sequencing analysis. The final lysozyme DNA sequence is shown as follows, in bold: TAGAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGCACCACCACCACCACCACCA
- BH05 The final lysozyme protein sequence (herein referred to as BH05), corresponding to SEQ ID NO. 7, is as follows:
- the pET30-6H-HP-Lyso was then transformed into host cells wherein expression of the lysozyme was induced by IPTG at a 1 mM concentration. Soluble lysate was recovered by centrifugating host cells into a pellet and lysing the cell pellet with lysis buffer. Samples were then sonicated three times at two minutes each, and centrifugated at 35K rpm for forty minutes. SDS gel analysis and Western blots indicated that a majority of HP -Lysozyme (>98%) presented in the inclusion body, and the lysozyme was purified of HP-Lysozyme from the pelleted inclusion body. The cell pellet was extracted three times to remove most soluble proteins.
- the inclusion body was then dissolved in buffer. After refolding, soluble lysozyme was then purified by Ni-sepharose followed by other conventional FPLC chromatographic methods until the protein fraction reaches >90% homogeneity. Refolded and purified protein was dialyzed in the formulation buffer (20mm Tris-Cl, pH7.9, lOOmM NaCl, 20% Glycerol and ImM EDTA and stored at a final concentration of 0.6mg/ml.
- H. pylori strains (shown in Table 1) were used to determine the antimicrobial efficacy of BH05. Bacteria were cultured under microaerobic conditions on brain heart infusion (BHI) agar plates supplemented with 10% (v/v) sheep blood.
- BHI brain heart infusion
- H. pylori were suspended in sterile phosphate-buffered saline (PBS), and adjusted to 0.02 ODeoo using BHI ( ⁇ 6 x 10 6 CFUs/mL). The inoculated agar was allowed to dry for 15 min. Discs with BH05 were applied onto the inoculated agar. 500, 250ug and 125 pg/ml of purified BH05 was tested by adopting a qualitative modified Kirby-Bauer (disc-diffusion) method. The plates with H. pylori were incubated in microaerophilic atmosphere at 37 °C for 72 h. After incubation, the diameters of complete growth inhibition zones were measured. Antibacterial activity was expressed as the mean of complete inhibition diameters (mm) produced by the different concentrations of endolysin. Results are shown in Table 1.
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Abstract
The present disclosure provides compositions of endolysin peptides used in combination with certain antibiotics, provides one or more of features and combinations thereof as described herein, and methods of use thereof.
Description
TITLE
Endolysin 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. 63/250,140, which was filed on September 29, 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 101457-5_sequences_ST25.txt. The text file is 18,420 bytes, created on May 13, 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
Helicobacter pylori (H. pylori') is a spiral-shaped, Gram-negative, pathogenic bacterium.
H. pylori infection occurs through colonization in the human stomach. With over 4 billion people in the world infected with H. pylori, it is considered the most wide-spread chronic bacterial
infection globally. All patients infected with H. pylori develop chronic gastritis, 20% develop peptic ulcer(s), and approximately 1% develop adenocarcinoma or mucosa-associated lymphoid tissue (MALT)-type gastric lymphoma.
H. pylori infection is considered to be the leading risk factor for the development of gastric cancer and is categorized as a type-1 carcinogen, accounting for over one-third of all new infection- related cancer cases. Although some factors for disease progression are likely to be inherent to the patient, certain H. pylori virulence factors also have been associated with cancer malignancy. For example, the most frequently associated H. pylori virulence factors are the VacA vacuolating cytotoxin and the Cag pathogenicity island (cagPAT). However, studies have shown that patients with early gastric cancer who received H. pylori treatment had lower rates of metachronous gastric cancer and more improvement from baseline in the grade of gastric corpus atrophy than patients who received placebo.
In addition to the high degree of virulence of H. pylori, a growing clinical challenge has been to develop optimal treatment programs which take into account the extremely high levels of H. pylori antibiotic resistance. Antibiotic resistance is conferred in part through proliferation in an acidic pH environment. Since H. pylori transforms into its antibiotic-resistant coccoid form at an acidic pH, current treatments incorporate a proton pump inhibitor (PPI) in combination with antibiotics. However, the PPI decreases acidic pH and thereby increasing antimicrobial activity and the half-life of antibiotics, and antibiotic resistance is now becoming significant with regard to antibiotic standard of care treatments. Resistance levels have demonstrably increased globally, with some regions identifying near complete resistance to at least one of the standard-of-care antibiotics. As of 2018, current levels of antibiotic resistance in the United States are approximately 35% to clarithromycin, 30% to metronidazole, and 20% to levofloxacin, with
multiple drug resistance strains progressing in other global regions, particularly the Asiatic countries.
H. pylori cure rates using standard-of-care antibiotic treatment have been reported to be as low as 57%, whereas the minimum acceptable rate for a first treatment is approximately 90%. Not unexpectedly, treatment failure rates are found to be substantially greater when applied to patients with antibiotic resistant H. pylori. For example, a statistically significant association between eradication treatment failure and antibiotic resistance detected before treatment was observed for all the standard of care antibiotics. Patients with clarithromycin-resistant H. pylori infections were shown to have a risk of failing eradication approximately 7-fold higher than patients with susceptible strains when treated with a clarithromycin-containing regimen. Strong associations were also observed for levofloxacin at approximately 8-fold, metronidazole at approximately 2.5- fold, and combined clarithromycin and metronidazole resistance at over a 9-fold failure rate. Therefore, with the prevalence and increasing development of H. pylori antibiotic resistance strains, there is necessitated the development of new therapeutic strategies.
Therapeutic treatment of mono- and multi-drug resistant infectious pathogens has more recently included the application of bacterial peptidoglycan hydrolases and phage endolysins 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 Grampositive bacterial pathogens.
Phage endolysins are generally species or subspecies specific, and are effective only against bacteria from which they were produced. While some endolysins act upon the cell walls of several bacterial genus or species, some broad-spectrum endolysins have been found. However, the use of endolysins 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 endolysins to the peptidoglycan substrates in the cell wall, and therefore endolysins have been used as a treatment mainly against Gram-positive bacteria. Even though certain endolysins have demonstrated 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 antibiotic resistant//, pylori.
SUMMARY
The present disclosure may comprise one or more of the following features and combinations thereof.
The present disclosure provides compositions of endolysin 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 endolysin peptides in combination with certain antibiotics to a bacterial infection may increase the susceptibility of the infectious bacteria to treatment. Provided herein is a pharmaceutical composition for the treatment of an 7/ pylori infection comprising an isolated endolysin peptide having the activity of degrading the cell wall of Heliobacter pylori. The endolysin can be an isolated lysozyme. The endolysin can be an isolated bacteriophage endolysin. The pharmaceutical composition endolysin peptide can be an H. pylori bacteriophage endolysin. The pharmaceutical composition can be delivered to a
patient in sufficient quantities to enhance effectiveness of standard of care antibiotic treatment. The pharmaceutical composition endolysin peptide can be one or more of SEQ ID NOS: 1 - 11. The pharmaceutical composition can further comprise at least one antibiotic, including H. pylori infection standard of care antibiotics, for example clarithromycin, amoxicillin, levofloxacin, metronidazole, rifabutin, tinidazole, tetracycline, etc. The pharmaceutical composition can further comprise at least one proton pump inhibitor, for example omeprazole, lansoprazole, dexlansoprazole, rabeprazole, pantoprozal, esomeprazole, etc. The pharmaceutical composition can further comprise bismuth subsalicylate. The pharmaceutical composition can further comprise at least one antihistamine, for example cimetidine, famotidine, nizatidine, ranitidine, etc. The pharmaceutical composition can be delivered to a patient in sufficient quantities to reduce an H. pylori infection by at least 50%, by at least 60%, by at least 70%, by at least 80%, by at least 90%, by at least 95%. Provided herein is a method of inhibiting the growth, or reducing the population, or the killing of H. pylori with a pharmaceutical composition comprising an isolated endolysin having the activity of degrading the cell wall of Heliobacter pylori, and one or more of an H. pylori infection standard of care antibiotic, antihistamine, PPI and bismuth subsalicylate, wherein the pharmaceutical composition has the property of inhibiting the growth, or reducing the population, or the killing of at least one species of H. pylori. Provided herein is a method for augmenting the efficacy of an antibiotic suitable for treating an H. pylori bacterial infection, comprising coadministering an isolated endolysin having the activity of degrading the cell wall of Heliobacter pylori in combination with one or more of an H. pylori infection standard of care antibiotic, antihistamine, PPI and bismuth subsalicylate, wherein administration of the combination is more effective in inhibiting the growth of, or reducing an initial population of, or killing the H. pylori bacteria than administration of either the one or more of an H. pylori infection standard of care
antibiotic, antihistamine, PPI and bismuth subsalicylate or the isolated endolysin having the activity of degrading the cell wall of Heliobacter pylori individually.
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.
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).
Some structural or method features, such as those representing devices, modules, instructions blocks and data elements, may be described as have 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. Additionally, the inclusion of a structural or method feature 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, where connecting elements are described to convey a connection, relationship, or association between or among two or more other 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 apply even in the absence of a discussion thereof. In some instances, a connecting element may include, be embodied as, or otherwise represent, multiple connections, relationships, or associations between elements. For example, where a connecting element provides or 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.
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 Heliobacter pylori 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 endolysin 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 endolysin 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 an endolysin polypeptide, any one or more of a standard of care antibiotic, any one or more of an antihistamine, any one or more of a PPI, bismuth subsalicylate, any other standard of care composition as known in the art for treatment of H. pylori infection, 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 endolysin polypeptides with any one or more of a standard of care antibiotic, any one or more of an antihistamine, any one or more of a PPI, bismuth subsalicylate, any other standard of care composition as known in the art for treatment of H. pylori infection, 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 an antibacterial agent to treat, e.g., a bacterial ulcer or an infected diabetic ulcer, the endolysin could be administered only initially within 24 hours of the first of any one or more of a standard of care antibiotic, any one or more of an antihistamine, any one or more of a PPI, bismuth subsalicylate, any other standard of care composition as known in the art for treatment of H. pylori infection, use and then the use of any one or more of a standard of care antibiotic, any one or more of an
antihistamine, any one or more of a PPI, bismuth subsalicylate, any other standard of care composition as known in the art for treatment of H. pylori infection, may continue without further administration of endolysin.
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 20 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 an endolysin polypeptide as described below and maintaining the endolysin function. A polypeptide can be a naturally occurring polypeptide or an engineered or synthetically produced polypeptide. A particular endolysin 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 al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989)). Variants of endolysin polypeptides are also encompassed having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% sequence identity with the endolysin 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 endolysin 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 endolysin activity. Included in this definition are two or more endolysin polypeptides or active fragments thereof. These can be used to make a fusion polypeptide with endolysin 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 an endolysin 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 (Vai), leucine (Leu), isoleucine (He), proline (Pro), phenylalanine (Phe), methionine (Met), and tryptophan (Trp).
The term “augmenting” within the context of the present disclosure refers to a degree of any one or more of a standard of care antibiotic, any one or more of an antihistamine, any one or more of a PPI, bismuth subsalicylate, any other standard of care composition as known in the art
for treatment of H. pylori infection, activity that 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 or more active substances that exceeds that produced by each substance administered or applied alone. One or more 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 Gramnegative bacteria responsible for an infection or contamination.
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 endolysin 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 endolysin polypeptide. Commonly used protective groups that may be added to endolysin
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 endolysin polypeptide or fused to a endolysin polypeptide without interfering with normal functions of cellular proteins. Typically, a polynucleotide encoding a fluorescent protein is inserted upstream or downstream of the endolysin polynucleotide sequence. This will produce a fusion protein (e.g., Endolysin Polypeptide GFP) that does not interfere with cellular function or function of a endolysin 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 endolysin polypeptide derivatives, the term “derivative” includes endolysin polypeptides chemically modified by covalent attachment of one or more PEG molecules. It is anticipated that pegylated endolysin polypeptides will exhibit prolonged circulation half-life compared to the unpegylated endolysin polypeptides, while retaining biological and therapeutic activity.
The term “percent amino acid sequence identity” with respect to the endolysin 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 endolysin 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 endolysin peptides as well. Thus, the term “substantially identical” will encompass mutated, truncated, fused, or otherwise sequence-modified variants of isolated endolysin 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 endolysin 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 endolysin polypeptide are substituted with a similar or conservative amino acid substitution, and wherein the resulting endolysin have the profile of activities, antibacterial effects, and/or bacterial specificities of endolysin polypeptides disclosed herein.
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” or “antimicrobial polypeptide” (AMP) refers to a member of a wide range of short (commonly 6 to 50 amino acid residues in length, but can be longer) 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 of 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.
Endolysins are capable of cleaving the major bonds in the peptidoglycan layer of bacterial cell walls. Endolysins may be classified into five types, depending on the bonds these enzymes cleave in the peptidoglycan layer: (1) N-acetylmuramidases (e.g., lysozyme) and (2) lytic transglycolases cleave the N-acetylmuramoyl— 1,4-N-acetyl glucosamine bond between the
alternating MurNAc and GlcNAc disaccharide backbone of the peptidoglycan; (3) N-acetyl— D- glucosaminidases cleaves the other glycosidic bond between the disaccharides (between the N- acetylglucosaminyl— 1,4-N-acetylmuramine bond), (4) N-acetylmuramoyl L-alanine amidases, which cleave between the sugar and the stem peptide bonds, (e.g., N-acetylmuramoyl L-alanine bonds); 5) Endopeptidases, which cleave between amino acids (aa) of the stem peptides or the interpeptide bridges.
Because H. pylori is a Gram-negative bacterium, it possesses a thin peptidoglycan layer in the bacterial cell wall formed from linear chains of two alternating amino sugars, namely N- acetyl glucosamine (GlcNAc or NAGA) and A-acetylmuramic acid (MurNAc or NAM A). The alternating sugars are connected by a p-(l,4)-glycosidic bond. This thin outer membrane is located outside the cell wall and prevents or inhibits some endolysins from interaction with the cell wall. Therefore, in certain embodiments of the present disclosure, are N-acetylmuramidases including as a non-limiting example, lysozymes, and phage endolysins, for example H. pylori phage endolysins.
In one embodiment of the present disclosure, is a pharmaceutical composition comprising the isolated lysozyme of H. pylori R038b, comprising the amino acid sequence: MILVASFLIVDLEGFSPSIYTDKTGNPTIGYGYNLSVYSYEGKRITKAYGLLTDILKENYK AILS YGWYKNLDAMRRMVILDLSYNLGLNGLLKFKQFIKAIEDKNY ALA VERLQKSPYF NQVKKRASRNMEILKLGGCEKHCKKKYTIEKVIKEVGLELKSKSVMPYFFNEYDLTKN ANREEPERLRSQIISILMKTPKGREILKEYLNEGCILLGE (SEQ ID NO: 1).
In one embodiment of the present disclosure, is a pharmaceutical composition comprising the isolated lysozyme of H. pylori OK310 comprising the amino acid sequence:
MDAMRRMVILDLSYNLGLSGLLKFKQFIKAIEDKNYALAVERLQKSPYFNQVRKRASR NMEVLKLGGCEKHCKKKYTIKKVIKEGGA (SEQ ID NO: 2).
In one embodiment of the present disclosure, is a pharmaceutical composition comprising the isolated lysozyme H. pylori UM066 comprising the amino acid sequence: MLCLSKLILAASFLIVDSEGFSPSVYTDKTGHPTIGYGYNLSVYSYESKRITKPQAYGLLT DILKENHKALLSYGWYKNLDAMRRMVILDLSYNLGLSGLLKFKQFIKGYRG (SEQ ID NO: 3).
Other endolysins can also be used for the present disclosure. In one embodiment is a pharmaceutical composition comprising the isolated endolysin Heliobacter phage endolysin YP 007237642 comprising the amino acid sequence:
MTNLENALNNGNFKEQ VYS SLEGVYQISKVLNQLDLLKNF SDHDLEINSF YQNKT ANLR DGNKIVSTYRTTCIFETPSEQADYKIAVFARKHKDLWVNMNYTANTSGFETSFLNNANF RGLTTQSIPTTYTTTGYFTSIPKRSFMKL (SEQ ID NO: 4).
In one embodiment is a pharmaceutical composition comprising the isolated endoendolysin YP 007005686 comprising the amino acid sequence: MDLTNLEDALNNGNFKEQVYSGLDGVYRISKVLNQLDLLKNFSEHDLEIVGGNGWVFH EHSQAIVYEILK (SEQ ID NO: 5).
In one embodiment is a pharmaceutical composition comprising the isolated endoendolysin of H. pylori P12 comprising the amino acid sequence: MILAASFLIVDSEGFSPSIYTDKTGHPTIGYGYNLSVYSYEGKRITKAYGLLTDILKENYK ALLSYGWYKNLDAMRRMVILDLSYNLGLSGLLKFKQFIKAIEDKNYALAVERLQKSPY
FNQVKKERQGIWKF (SEQ ID NO: 6).
In one embodiment is a pharmaceutical composition comprising the isolated endolysin of H. pylori 0339, (ATCC strain 26695) comprising the amino acid sequence:
MDSEGFSPSIYTDKTGHPTIGYGYNLSVYSYEGKRITKTYGLLTDILSYGWYKNLDAMR
RMVILDLSYNLGLNGLLKFKQFIKAIEDKNYALAVERLQKSPYFNQVKKERQGIWKF (SEQ ID NO: 7).
In one embodiment is a pharmaceutical composition comprising the isolated endolysin of H. pylori A-14 comprising the amino acid sequence:
MILAASFLIVDSEGFSPSIYTDKTEHPTIGYGYNLSVYSYEGKRITKAYGLLTDILKENYK
ALLSYGWYKNLDAMRRMVILDLSYNLGLNGLLKFKQFIKAIEDKNYALAVERLQKSPY FNQVKKRASRNMEILKLGGCEKHCKKKYTIEKVIKEVGLELKSKSVMPYFFNEYDLTKN
TNREELERLRSQIISILMKTPKGREILKEYLNEGCILLGE (SEQ ID NO: 8).
In one embodiment is a pharmaceutical composition comprising the isolated endolysin of H. pylori H-l 1 comprising the amino acid sequence:
MILAASFLIVDSEGFSPSIYTDKTEHPTIGYGYNLSVYSYESKRITKAYGLLTDILKENYK
ALLSYGWYKNLDAMRRMVILDLSYNLGLNGLLKFKQFIKAIEDKNYALAVERLQKSPY FNQVKKRASRNMEILKLGGCEKHCKKKYTIEKVIKEVGLELKSKSVMPYFFNEYDLTKN
ANREEPERLRSQIISILMKTPKGREILKEYLNEGCILLGE (SEQ ID NO: 9).
In one embodiment is a pharmaceutical composition comprising the isolated endolysin of H. pylori PeCan4 comprising the amino acid sequence:
MLCLSKLILAVSFLIVDSEGFSPSIYTDKTGHPTIGYGYNLSVYSYESKRITKPQAYGLLT DILKENHKALLSYGWYKNLDAMRRMVILDLSYNLGLNGLLKFKQFIKAIEDKNYALAV
ERLQKSPYFNQVRKRASRNMEILKLGGCEKHCKKKYTIEKVIKEVGLLEKSKSVMPYFF
NEYDLIKKRQQRRA (SEQ ID NO: 10).
In one embodiment is a pharmaceutical composition comprising the isolated endolysin of H. pylori CPY1313 comprising the amino acid sequence: MDSEGFSPSIYTDKTGHPTIGYGYNLSVYSYESKRITKPQAYGLLTDILKENHKALLSYG WYKNLDAMRRMVILDLSYNLGLSGLLKFKQFIKAIEDKNYALAVERLQKSPYFNQVRK RASRNMEILKLGGCEKHCKKKYTIEKVIKEGGA (SEQ ID NO: 11).
In other embodiments of the present disclosure, a fragment of any one or more of SEQ ID NOs. 1 - 11 are conjugated to an antimicrobial peptide to yield a conjugated polypeptide having antibacterial activity. In other certain embodiments, any combination of 2 or more of SEQ ID NOS: 1 - 11 are used for the pharmaceutical composition of the present disclosure. In other certain embodiments, any combination of 2 or more fragments of SEQ ID NOS: 1 - 11 are used for the pharmaceutical composition of the present disclosure.
The peptides SQSRESQC (SEQ ID NO: 12) and CSQRQSES (SEQ ID NO: 13) are derived from hepatitis C virus and has been shown to have antimicrobial activity against gram-positive and gram-negative bacteria. In other embodiments of the present disclosure is a fusion polypeptide of SEQ ID NO: 12 or SEQ ID NO: 13 and any one or more of SEQ ID NOS: 1 - 11. In still other embodiments, a fusion polypeptide comprises the eight amino acids C, S, Q, R, S, E and S in any order and having antimicrobial activity is conjugated to any one or more of SEQ ID NOS: 1 - 11. In still other embodiments of the present disclosure, SEQ ID NO: 12 or SEQ ID NO: 13 is conjugated to the C-terminus of any one or more of SEQ ID NOS: 1 - 11. In still other embodiments of the present disclosure, SEQ ID NO: 12 or SEQ ID NO: 13 is conjugated to the N- terminus of any one or more of SEQ ID NOS: 1 - 11.
Modifications and changes can be made in the structure of the endolysin polypeptides of the disclosure and still obtain a molecule having similar characteristics as the endolysin
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 an endolysin polypeptide sequence and nevertheless obtain a endolysin 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, Vai), (Leu: Be, Vai), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Tip: Tyr), (Tyr: Trp, Phe), and (Vai: He, 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. coh. 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. coh. 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 an endolysin polypeptide comprising an amino acid sequence that has at least 90% sequence identity to any of the amino acid sequences of SEQ ID NOS: 1 - 11, wherein the endolysin polypeptide has antibacterial activity.
In another embodiment, the nucleic acid encodes an endolysin polypeptide comprising an amino acid sequence of any one of SEQ ID NOS: 1 - 11, wherein the endolysin polypeptide has antibacterial activity.
In yet another embodiment, the nucleic acid encodes an endolysin polypeptide consisting of an amino acid sequence nucleic acid of any one of SEQ ID NOS: 1 - 11, wherein the endolysin polypeptide or fragment has antibacterial activity.
Another embodiment is an expression vector that comprises a nucleic acid that encodes an endolysin polypeptide comprising an amino acid sequence that has at least 90% sequence identity
to any one of the amino acid sequences of SEQ ID NOS: 1 - 11, wherein the endolysin polypeptide has antibacterial activity.
In another embodiment, the expression vector comprises a nucleic acid that encodes an endolysin polypeptide comprising any one of the amino acid sequences of SEQ ID NOS: 1 - 11, wherein the endolysin polypeptide has antibacterial activity.
In yet another embodiment, the expression vector comprises a nucleic acid that encodes an endolysin polypeptide consisting of an amino acid sequence nucleic acid of any one of SEQ ID NOS: 1 - 11, wherein the endolysin polypeptide has antibacterial activity.
The nucleic acid that encodes an endolysin 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 endolysin 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 endolysin polypeptides in a biological system.
Endolysin 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-
CH2Bxl)-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).
Therapeutic and Prophylactic Compositions and their Use
In embodiments of the present disclosure is provided a method of treatment comprising administering to a subject in need thereof an effective amount of an endolysin polypeptide, for example the polypeptide comprising any combination of one or more of SEQ ID NOS: 1 - 11. In certain embodiments the method further comprises standard of care treatment, for example administering to a subject in need thereof an effective amount of an antihistimine. The antihistamine can be one or more of cimetidine, famotidine, nizatidine, ranitidine, etc. In other certain embodiments the method further comprises administering to a subject in need thereof an effective amount of bismuth subsalicylate. In certain embodiments the method further comprises administering to a subject in need thereof an effective amount of an antibiotic. The antibiotic can be one or more of clarithromycin, amoxicillin, levofloxacin, metronidazole, tinidazole, tetracycline, etc. In certain embodiments the method further comprises administering to a subject in need thereof an effective amount of a proton pump inhibitor. The proton pump inhibitor can be one or more of omeprazole, lansoprazole, dexlansoprazole, rabeprazole, pantoprozal, esomeprazole, etc.
In certain embodiments, the method of administrating the pharmaceutical composition disclosed herein can be a co-administration one or more of the antihistamine, the PPI, bismuth subsalicylate, and an antibiotic with one or more of the endolysins effective against H. pylori.
Methods of administration of the disclosed pharmaceutical compositions can be oral using for example, liquid or gel dosages, oral tablets, granules, powders, and sprinkle capsules. In particular embodiments, the pharmaceutical carrier can be microorganisms, for example Spirulina. spp, etc., expressing the peptides of the present disclosure, dried and stored in dosage specific capsules for oral delivery.
Such pharmaceutical compositions of the present disclosure 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. 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, and the like. 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.
EXAMPLE 1 : LYSOZYME EXPRESSION AND PURIFICATION
NCBI reference sequence WP 010875465 corresponding to SEQ ID NO. 7 was first codon optimized for expression in E. coli BL21 (DE3). After codon optimization, the DNA sequence was synthesized and cloned into an E. coli expression vector pET30 with a 6-His tag at the N-terminus. The inserted sequence encoding for codon optimized HP -Lysozyme was validated by DNA sequencing analysis. The final lysozyme DNA sequence is shown as follows, in bold: TAGAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGCACCACCACCACCA
CCACGGCGGCGGGGAATTCATGGATTCTGAGGGGTTTTCCCCTTCTATTTATAC CGACAAGACTGGGCATCCGACCATTGGCTATGGTTACAATCTGTCTGTTTATTC
TTATGAGGGTAAGCGTATCACCAAAACCTATGGGCTTCTGACTGACATCCTGTC
TTATGGGTGGTATAAAAATCTGGACGCGATGCGTCGTATGGTCATCCTGGATCT
GTCTTACAATCTGGGCCTGAACGGTCTGCTCAAATTCAAGCAATTCATCAAGGC GATCGAGGATAAAAATTATGCTCTGGCTGTTGAGCGTCTGCAAAAATCTCCGTA TTTCAATCAAGTGAAAAAAGAGCGTCAAGGTATCTGGAAATTTTGACTCGAGCA
CCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTG CCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGG GGTTTTTTGCTGAAAGGAGGAACTATATCCGGATTGGCGAATGGGACGCGCCCTGTA GCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTT GCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCG CCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGC TTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCC ATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGT GGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATT TATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAA
AATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTAG (SEQ ID NO: 14)
The final lysozyme protein sequence (herein referred to as BH05), corresponding to SEQ ID NO. 7, is as follows:
MHHHHHHGGGEFMDSEGFSPSIYTDKTGHPTIGYGYNLSVYSYEGKRITKTYGLLTDILS
YGWYKNLDAMRRMVILDLSYNLGLNGLLKFKQFIKAIEDKNYALAVERLQKSPYFNQV KKERQGIWKF (SEQ ID NO: 15)
The pET30-6H-HP-Lyso was then transformed into host cells wherein expression of the lysozyme was induced by IPTG at a 1 mM concentration. Soluble lysate was recovered by centrifugating host cells into a pellet and lysing the cell pellet with lysis buffer. Samples were then
sonicated three times at two minutes each, and centrifugated at 35K rpm for forty minutes. SDS gel analysis and Western blots indicated that a majority of HP -Lysozyme (>98%) presented in the inclusion body, and the lysozyme was purified of HP-Lysozyme from the pelleted inclusion body. The cell pellet was extracted three times to remove most soluble proteins. The inclusion body was then dissolved in buffer. After refolding, soluble lysozyme was then purified by Ni-sepharose followed by other conventional FPLC chromatographic methods until the protein fraction reaches >90% homogeneity. Refolded and purified protein was dialyzed in the formulation buffer (20mm Tris-Cl, pH7.9, lOOmM NaCl, 20% Glycerol and ImM EDTA and stored at a final concentration of 0.6mg/ml.
EXAMPLE 2: PURIFIED LYSOZYME PROTEIN (BH05) ACTIVITY AGAINST H. PYLORI
H. pylori strains (shown in Table 1) were used to determine the antimicrobial efficacy of BH05. Bacteria were cultured under microaerobic conditions on brain heart infusion (BHI) agar plates supplemented with 10% (v/v) sheep blood.
H. pylori were suspended in sterile phosphate-buffered saline (PBS), and adjusted to 0.02 ODeoo using BHI (~6 x 106 CFUs/mL). The inoculated agar was allowed to dry for 15 min. Discs with BH05 were applied onto the inoculated agar. 500, 250ug and 125 pg/ml of purified BH05 was tested by adopting a qualitative modified Kirby-Bauer (disc-diffusion) method. The plates with H. pylori were incubated in microaerophilic atmosphere at 37 °C for 72 h. After incubation, the diameters of complete growth inhibition zones were measured. Antibacterial activity was expressed as the mean of complete inhibition diameters (mm) produced by the different concentrations of endolysin. Results are shown in Table 1.
All H. pylori strains were inhibited by BH05 at the concentrations tested as having zone of growth inhibition more than 8mm.
Claims
WHAT IS CLAIMED: A pharmaceutical composition for the treatment of an H. pylori infection comprising: an isolated endolysin peptide having the activity of degrading the cell wall of Heliobacter pylori. The pharmaceutical composition of claim 1, wherein said composition is delivered to a patient in sufficient quantities to enhance effectiveness of standard of care antibiotic treatment. The pharmaceutical composition of claims 1 and 2, wherein the endolysin is an H. pylori bacteriophage endolysin. The pharmaceutical composition of claims 1 - 3, wherein the endolysin is a lysozyme endolysin. The pharmaceutical composition of claims 1 - 4, wherein the endolysin comprises one or more of the group consisting of SEQ ID NOS: 1 - 11. The composition of claims 1 - 5, further comprising at least one antibiotic. The composition of claims 1 - 6, wherein the at least one antibiotic is selected from the group consisting of clarithromycin, amoxicillin, levofloxacin, metronidazole, rifabutin tinidizole and tetracycline. The composition of claims 1 - 7, further comprising at least one proton pump inhibitor. The composition of claims 1 - 8, wherein the at least one proton pump inhibitor is selected from the group consisting of omeprazole, lansoprazole, dexlansoprazole, rabeprazole, pantoprozal and esomeprazole. The composition of claims 1 - 9, further comprising bismuth subsalicylate.
33
The composition of claims 1 - 10, further comprising at least one antihistamine. The composition of claims 1 - 11, wherein the at least one antihistamine is selected from the group consisting of cimetidine, famotidine, nizatidine, and ranitidine. The pharmaceutical composition of claims 1 - 12, wherein said composition is delivered to a patient in sufficient quantities to reduce an H. pylori infection by at least 50%, by at least 60%, by at least 70%, by at least 80%, by at least 90%, by at least 95%. A method of inhibiting the growth, or reducing the population, or the killing of H. pylori with a pharmaceutical composition comprising an isolated endolysin having the activity of degrading the cell wall of Heliobacter pylori, and one or more of an H. pylori infection standard of care antibiotic, antihistamine, PPI and bismuth subsalicylate, wherein the pharmaceutical composition has the property of inhibiting the growth, or reducing the population, or the killing of at least one species of H. pylori. A method for augmenting the efficacy of an antibiotic suitable for treating an H. pylori bacterial infection, comprising co-administering an isolated endolysin having the activity of degrading the cell wall of Heliobacter pylori in combination with one or more of an H. pylori infection standard of care antibiotic, antihistamine, PPI and bismuth subsalicylate, wherein administration of the combination is more effective in inhibiting the growth of, or reducing an initial population of, or killing the H. pylori bacteria than administration of either the one or more of an H. pylori infection standard of care antibiotic, antihistamine, PPI and bismuth subsalicylate or the isolated bacteriophage endolysin having the activity of degrading the cell wall of Heliobacter pylori individually.
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US20020187136A1 (en) * | 2000-04-28 | 2002-12-12 | Lawrence Loomis | Use of bacterial phage associated lysing enzymes for treating various illnesses |
CN111378638A (en) * | 2018-12-29 | 2020-07-07 | 江苏万邦医药科技有限公司 | Helicobacter pylori phage lyase and preparation method thereof |
US10988520B2 (en) * | 2018-03-29 | 2021-04-27 | Contrafect Corporation | Lysin-antimicrobial peptide (AMP) polypeptide constructs, lysins, isolated polynucleotides encoding same and uses thereof |
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US20020187136A1 (en) * | 2000-04-28 | 2002-12-12 | Lawrence Loomis | Use of bacterial phage associated lysing enzymes for treating various illnesses |
US10988520B2 (en) * | 2018-03-29 | 2021-04-27 | Contrafect Corporation | Lysin-antimicrobial peptide (AMP) polypeptide constructs, lysins, isolated polynucleotides encoding same and uses thereof |
CN111378638A (en) * | 2018-12-29 | 2020-07-07 | 江苏万邦医药科技有限公司 | Helicobacter pylori phage lyase and preparation method thereof |
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