Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
  • A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
  • A61K31/407—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with other heterocyclic ring systems, e.g. ketorolac, physostigmine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
  • A61K31/42—Oxazoles
  • A61K31/422—Oxazoles not condensed and containing further heterocyclic rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
  • A61K31/425—Thiazoles
  • A61K31/427—Thiazoles not condensed and containing further heterocyclic rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/54—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame
  • A61K31/542—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame ortho- or peri-condensed with heterocyclic ring systems
  • A61K31/545—Compounds containing 5-thia-1-azabicyclo [4.2.0] octane ring systems, i.e. compounds containing a ring system of the formula:, e.g. cephalosporins, cefaclor, or cephalexine
  • A61K31/546—Compounds containing 5-thia-1-azabicyclo [4.2.0] octane ring systems, i.e. compounds containing a ring system of the formula:, e.g. cephalosporins, cefaclor, or cephalexine containing further heterocyclic rings, e.g. cephalothin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
  • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
  • A61K31/7036—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin having at least one amino group directly attached to the carbocyclic ring, e.g. streptomycin, gentamycin, amikacin, validamycin, fortimicins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
  • A61K31/716—Glucans
  • A61K31/722—Chitin, chitosan
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
  • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents

Definitions

• the present disclosure relates to antimicrobial compositions and formulations directed against a broad range of bacteria and Pseudomonas aeruginosa biofilms, without engendering resistance.
• the present disclosure further relates to antimicrobial compositions and formulations which, in combination with antibiotics, slow the development of antibiotic-resistance and greatly improve bacterial susceptibility to multiple classes of antibiotics.
• the ESKAPE pathogens Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.
• MDR multidrug- resistant
• Nitric oxide (NO) an endogenously produced free radical species involved in the immune response, has previously been shown to reduce airway mucus viscosity and also exert broad-spectrum antibacterial and antibiofilm action against several ESKAPE pathogens [Reighard, K.
• Antibiotic resistance in bacteria is a major global threat and the leading cause for healthcare-related morbidity and mortality [Friedman, N. D., Temkin, E. & Carmeli, Y. The negative impact of antibiotic resistance. Clin. Microbiol. Infect. 22, 416-422 (2016); Ragheb, M. N. et al. Inhibiting the Evolution of Antibiotic Resistance. Mol. Cell 73, 157-165.e5 (2019)].
• resistant infections are expected to kill 10 million people by 2050 [Centers for Disease Control. Antibiotic Resistance Threats in the United States, 2013. Antibiotic Resistant Threats in the United States (2013)].
• Resistant respiratory infections are particularly difficult to treat because they form protective biofilms inside airway mucus and can survive for decades [Kovach, K. et al. Evolutionary adaptations of biofilms infecting cystic fibrosis lungs promote mechanical toughness by adjusting polysaccharide production npj Biofilms Microbiomes 3, (2017)].
• Novel antimicrobial agents that are capable of fully eradicating resistant infections, even those deep inside the lower airway, are desperately needed along with strategies for combatting the rise of antibiotic resistant bacteria.
• the applicants show that nitric oxide behaves as a broad-spectrum antimicrobial and improves antibiotic efficacy.
• Embodiment 1 A method of increasing susceptibility of a microorganism to at least one antibiotic, comprising: contacting the microorganism with a nitric oxide releasing chitosan oligosaccharide (COS/NO) for a period, and contacting the microorganism with the at least one antibiotic. Said contacting may be concurrent, sequential, or a combination thereof.
• COS/NO nitric oxide releasing chitosan oligosaccharide
• Embodiment 2 The method of embodiment 1 , wherein the microorganism is selected from the genera consisting of: Enterococcus, Staphylococcus, Klebsiella, Acinetobacter, Pseudomonas, and Enterobacter.
• Embodiment 3 The method of embodiment 1 , wherein the microorganism is selected from the group consisting of: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.
• Embodiment 4 The method of any one of embodiments 1-3, wherein the COS/NO: has a minimum inhibitory concentration (MIC) relative to the microorganism; and is provided in an amount selected from about 0.1 to about 4, about 0.2 to about 4, about 0.25 to about 4, about 0.3 to about 4, about 0.4 to about 4, about 0.5 to about 4, about 0.6 to about 4, about 0.7 to about 4, about 0.75 to about 4, about 0.8 to about 4, about 0.9 to about 4, about 1 to about 4, about 2 to about 4, about 3 to about 4, about 0.1 to about 3, about 0.1 to about 2, about 0.1 to about 1 , about 0.1 to about 0.9, about 0.1 to about 0.8, about 0.1 to about 0.75, about 0.1 to about 0.7, about 0.1 to about 0.6, about 0.1 to about 0.5, about 0.1 to about 0.4, about 0.1 to about 0.3, about 0.1 to about 0.25, about 0.1 to about 0.2, about 0.1 , about 0.2, about 0.25, about 0.3, about 0.4 to about 4,
• Embodiment 6 The method of any one of embodiments 1-5, wherein the period is selected from the group consisting of at least 0.25, at least 0.5, at least 0.75, at least 1 , at least 1 .25, at least 1 .5, at least 1 .75, at least 2, at least 2.25, at least 2.5, at least 2.75, at least 3, at least 3.25, at least 3.5, at least 3.75, at least 4, at least 4.25, at least 4.5, at least 4.75, at least 5, at least 5.25, at least 5.5, at least 5.75, at least 6, at least 6.25, at least 6.5, at least 6.75, at least 7, at least 7.25, at least 7.5, at least 7.75, and at least 8 hours.
• Embodiment 7 The method of any one of embodiments 1-6, wherein the increased susceptibility is lower viability of the at least one microorganism, measured as colony-forming units (CFU) per unit volume, after said contacting with the COS/NO for a period and subsequent contacting with the at least one antibiotic, as compared to contacting with either the COS/NO alone or the at least one antibiotic alone.
• CFU colony-forming units
• Embodiment 8 The method of any one of embodiments 1-7, wherein the at least one microorganism is in a biofilm.
• Embodiment 9 The method of any one of embodiments 1-8, wherein the COS/NO is in a composition formulated for: topical, oral, nasal, ophthalmic, intrathecal, parenteral, intraperitoneal, intravenous, subcutaneous, or intramuscular administration.
• Embodiment 10 The method of any one of embodiments 1-9, wherein the at least one antibiotic is in a composition formulated for: topical, oral, nasal, ophthalmic, intrathecal, parenteral, intraperitoneal, intravenous, subcutaneous, or intramuscular administration.
• Embodiment 11 The method of any one of embodiments 1-10, wherein the formulation is in the form of a paste, a liquid, a cream, a gel, a salve, a foam, an aerosol, a lotion, an ointment, a soap, a shampoo, a surgical drape, a suture, a bandage, a gauze, or a medical implant.
• Embodiment 12 The method of any one of embodiments 1-11, wherein the at least one antibiotic is selected from the group consisting of: aminoglycosides, ansamycins, beta-lactams, carbacephems, carbapenems, cephalosporins, fluoroquinolones, glycopeptides, lincosamides, macrolides, monobactams, oxazolidinones, penicillins, phenicols, polypeptides, quinolones, streptogramins, sulfonamides, and tetracyclines.
• the at least one antibiotic is selected from the group consisting of: aminoglycosides, ansamycins, beta-lactams, carbacephems, carbapenems, cephalosporins, fluoroquinolones, glycopeptides, lincosamides, macrolides, monobactams, oxazolidinones, penicillins,
• Embodiment 13 The method of any one of embodiments 1-12, wherein the at least one antibiotic is selected from the group consisting of: aztreonam, ceftazidime, ciprofloxacin, colistin, meropenem, and tobramycin.
• Embodiment 14 A method of reducing the development or progression, in a microorganism, of resistance to at least one antibiotic, comprising: contacting the microorganism with a nitric oxide-releasing chitosan oligosaccharide (COS/NO) for a period, and subsequently contacting the microorganism with the at least one antibiotic.
• COS/NO nitric oxide-releasing chitosan oligosaccharide
• Embodiment 15 The method of embodiment 14, wherein the microorganism is selected from the genera consisting of: Enterococcus, Staphylococcus, Klebsiella, Acinetobacter, Pseudomonas, and Enterobacter.
• Embodiment 16 The method of embodiment 14, wherein the microorganism is selected from the group consisting of: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.
• Embodiment 18 The method of any one of embodiments 14-17, wherein the at least one antibiotic: has a minimum inhibitory concentration (MIC) relative to the microorganism; and is provided in an amount selected from about 0.1 to about 4, about 0.2 to about 4, about 0.25 to about 4, about 0.3 to about 4, about 0.4 to about 4, about 0.5 to about 4, about 0.6 to about 4, about 0.7 to about 4, about 0.75 to about 4, about 0.8 to about 4, about 0.9 to about 4, about 1 to about 4, about 2 to about 4, about 3 to about 4, about 0.1 to about 3, about 0.1 to about 2, about 0.1 to about 1 , about 0.1 to about 0.9, about 0.1 to about 0.8, about 0.1 to about 0.75, about 0.1 to about 0.7, about 0.1 to about 0.6, about 0.1 to about 0.5, about 0.1 to about 0.4, about 0.1 to about 0.3, about 0.1 to about 0.25, about 0.1 to about 0.2, about 0.1 , about 0.2, about 0.25, about 0.3, about 0.4 to about 4,
• Embodiment 19 The method of any one of embodiments 14-18, wherein the period is selected from the group consisting of at least 0.25, at least 0.5, at least 0.75, at least 1 , at least 1.25, at least 1.5, at least 1.75, at least 2, at least 2.25, at least 2.5, at least 2.75, at least 3, at least 3.25, at least 3.5, at least 3.75, at least 4, at least 4.25, at least 4.5, at least 4.75, at least 5, at least 5.25, at least 5.5, at least 5.75, at least 6, at least 6.25, at least 6.5, at least 6.75, at least 7, at least 7.25, at least 7.5, at least 7.75, and at least 8 hours.
• Embodiment 20 The method of any one of embodiments 14-19, wherein the increased susceptibility is lower viability of the at least one microorganism, measured as colony-forming units (CFU) per unit volume, after said contacting with the COS/NO for a period and subsequent contacting with the at least one antibiotic, as compared to contacting with either the COS/NO alone or the at least one antibiotic alone.
• CFU colony-forming units
• Embodiment 21 The method of any one of embodiments 14-20, wherein the at least one microorganism is in a biofilm.
• Embodiment 22 The method of any one of embodiments 14-21 , wherein the COS/NO is in a composition formulated for: topical, oral, nasal, ophthalmic, intrathecal, parenteral, intraperitoneal, intravenous, subcutaneous, or intramuscular administration.
• Embodiment 23 The method of any one of embodiments 14-22, wherein the at least one antibiotic is in a composition formulated for: topical, oral, nasal, ophthalmic, intrathecal, parenteral, intraperitoneal, intravenous, subcutaneous, or intramuscular administration.
• Embodiment 24 The method of any one of embodiments 14-23, wherein the formulation is in the form of a paste, a liquid, a cream, a gel, a salve, a foam, an aerosol, a lotion, an ointment, a soap, a shampoo, a surgical drape, a suture, a bandage, a gauze, or a medical implant.
• Embodiment 25 The method of any one of embodiments 14-23, wherein the formulation is in the form of a paste, a liquid, a cream, a gel, a salve, a foam, an aerosol, a lotion, an ointment, a soap, a shampoo, a surgical drape, a suture, a bandage, a gauze, or a medical implant.
• the at least one antibiotic is selected from the group consisting of: aminoglycosides, ansamycins, beta-lactams, carbacephems, carbapenems, cephalosporins, fluoroquinolones, glycopeptides, lincosamides, macrolides, monobactams, oxazolidinones, penicillins, phenicols, polypeptides, quinolones, streptogramins, sulfonamides, and tetracyclines.
• Embodiment 26 The method of any one of embodiments 14-25, wherein the at least one antibiotic is selected from the group consisting of: aztreonam, ceftazidime, ciprofloxacin, colistin, meropenem, and tobramycin.
• the nitric oxide-releasing chitosan oligosaccharide (COS/NO) of Embodiment 1 is represented by the following structure disclosed in US Patent No. US 9850322 (incorporated by reference herein): comprising at least one structural unit of formula I: and optionally, at least one structural unit of formula II:
• Ri, R2, R3or R4 attached to the double bond-0 is absent; when Ri is absent, Rs is hydrogen, hydroxyl, C1-5 alkyl or Ci-salkoxy; when R3 1S absent, Re is hydrogen, hydroxyl, C1-5 alkyl or Ci-salkoxy; wherein in each instance where it is a single bond, Ri, R2, R3or R4 attached to the double bond-0 is present; when Ri is present, Rs is hydrogen; when R3 is present, R6 is hydrogen;
• Q is — (CRcRd)v — ; wherein R c and Rdare independently hydrogen or C1-5 alkyl, such as methyl, ethyl, n- propyl, isopropyl, t-butyl, n-butyl, isobutyl and pentyl.
• A is wherein, L is S, 0 or N; and G, in each instance, is independently, hydrogen, or is taken together with L to form a nitric oxide donor;
• X is hydrogen, C1-5 alkyl or is taken together with N to form a nitric oxide donor
• B is hydrogen or — Y — Z, wherein Y is a spacer and Z is a polymer or a terminus group; or B is absent;
• R-i, R2, Rsand R4, if present are each independently selected from the group consisting of hydrogen and C1-5 alkyl.
• R-i, R2, Rsand R4 1S C1-5 alkyl it is selected from methyl, ethyl, n-propyl, isopropyl, t- butyl, n-butyl, isobutyl and pentyl.
• Ri, R2, Rsand R4, if present is hydrogen or methyl.
• Ri, R2, Rsand R4, if present is hydrogen.
• each instance is a single or double bond. Preferably, it is a single bond in all instances.
• Q is — (CRcRd)v — ; wherein R c and Rd are independently hydrogen or Ci- 5 alkyl, such as methyl, ethyl, n-propyl, isopropyl, t-butyl, n-butyl, isobutyl and pentyl.
• R c and Rd are independently hydrogen, methyl or ethyl.
• Useful values of v are integers from 2 to 6.
• v is 2.
• Useful values of p include any integer from 1 to 100.
• p is an integer from 1 to 50. More preferably, p is an integer from 1 to 25. Most preferably, p is an integer from 1 to 10, such as, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10.
• L is N, S and O.
• L is N or S.
• G is independently hydrogen or a nitric oxide donor. Since the nitric oxide donor contributes to the amount of available NO on the COS/NO, it is preferable that G is a nitric oxide donor.
• at least 30% of G present on a COS/NO is a nitric oxide donor. More preferably, at least 50% of G present on a COS/NO is a nitric oxide donor. Even more preferably, at least 90% of G present on a COS/NO is a nitric oxide donor.
• At least 95% of G present on a COS/NO is a nitric oxide donor.
• Useful values of X are hydrogen, C1-5 alkyl or a nitric oxide donor. Since the nitric oxide donor contributes to the amount of available NO on the COS/NO, it is preferable that X is a nitric oxide donor. In preferred embodiments, at least 30% of X present on a COS/NO is a nitric oxide donor. More preferably, at least 50% of X present on a COS/NO is a nitric oxide donor. Even more preferably, at least 90% of X present on a COS/NO is a nitric oxide donor. Most preferably, at least 95% of X present on a COS/NO is a nitric oxide donor.
• B Useful values of B are hydrogen, — Y — Z, wherein Y is a spacer and Z is a monomer or polymer, or B is a terminus group. B may also be absent when L is O or S.
• a terminus group is any end-capping group at the terminus of a polymer or monomer. These groups are known in the art.
• B is a terminus group, it is hydrogen, hydroxyl or C1-5 alkyl.
• Useful values of Z include monomers and polymers known in the art, especially those used in active pharmaceutical ingredients. Particularly useful polymers or monomers include: wherein j, in each instance, is an integer from 1 to 100.
• Useful spacers, Y, in the formulae disclosed herein include spacers or linkers known in the art, especially those used in active pharmaceutical ingredients. Particularly useful spacers include the following: wherein, R P , R q , and Rt, in each instance, are independently, hydrogen or hydroxyl; and k is an integer from 1 to 20.
• any secondary amino group present on the oligosaccharide can be modified as described herein to form a NO-releasing oligosaccharide.
• the secondary amino groups attached directly to the sugar backbone moieties or secondary amino groups pendant on the backbone sugar moieties can be functionalized with a NO releasing moiety.
• Useful NO releasing moieties include any NO releasing group known in the art. Particularly useful are residues of NO releasing groups, i.e. NO donors, are covalently bound to N, S or O on the COS/NO. The NO donor is taken together with the atom on the COS/NO to which it is bound to form a moiety selected from the group consisting of a diazeniumdiolate, — NO as part of a nitrosothiol group for example, a nitrosamine, a hydroxyl nitrosamine, a hydroxyl amine, a hydroxyurea, and combination thereof.
• the NO releasing moiety is a diazeniumdiolate.
• These groups may be present in the form of a salt.
• the NO donor is a N-diazeniumdiolate (i.e., a 1- amino-substituted deazen-1-lum-1 ,2-diolate) represented by the following:
• NXQB is represented by one of the following:
• NXQAB is represented by the following: Attorney Docket No. 035052/554484
• the COS/NO is represented by the following: [0049] In several embodiments, the COS/NO has a total releasable nitric oxide storage of at least 0.5 pmol of NO per milligram of COS/NO. In several embodiments, the COS/NO has a total releasable nitric oxide storage in a range of about 0.5 pmol to 2.5 pmol of NO per milligram of COS/NO.
• greater per milligram NO release is achieved, for example, at least about 2.5 pmol, about 3.0 pmol, about 3.5 pmol, about 4.0 pmol, about 4.5 mol, about 5 pmol or greater amounts of NO per milligram of COS/NO.
• the COS/NO has a half-life for nitric oxide release in a range of between about 0.7-4.2 hours.
• longer half-lives are achieved, such as for example, about 5 hours, about 6 hours, about 8 hours, about 10 hours, or any time between the listed times.
• the COS/NO has a total NO release after 4 hours in a range of between about 0.1- 4.0 pmol of NO per milligram of the COS/NO, including about 0.3-2.0 pmol of NO per milligram of the COS/NO, about 0.1-3.0 pmol of NO per milligram of the COS/NO, about 1.5-4 pmol of NO per milligram of the COS/NO, or, about 0.7- 3.0 pmol of NO per milligram of the COS/NO (or any range there between, including endpoints).
• FIG. 1 shows that nitric oxide increases cell permeability
• COS/NO exerts nitrosative and oxidative stress on the cell membrane through the production of multiple reactive byproducts. A more permeable cell membrane will allow for improved diffusion of antibiotics and other hydrophobic molecules
• c Fluorescent dye NPN fluorescence in P. aeruginosa strain K after exposure to tobramycin or colistin.
• FIG. 2 shows that combinations of COS/NO and antibiotics result in synergy for susceptible strains of P. aeruginosa
• FIG. 3 shows PAK viability after exposure to antibiotics without (solid line) or with NO-pretreatment at 25% (dashed line) or 100% (dotted line) COS/NO MIC for 4 h. Error is representative of the standard deviation of the mean for >3 biological replicates.
• FIG. 4 shows that nitric oxide pretreatment of P. aeruginosa biofilms results in improved tobramycin susceptibility
• PAK biofilm viability after exposure to tobramycin without pretreatment solid bars
• b-e MDR P. aeruginosa biofilm viability after exposure to tobramycin without pretreatment (solid bars) or with NO pretreatment of 4 h (striped bars).
• Error is representative of the standard deviation of the mean for >3 biological replicates.
• FIG. 5 shows that serial exposure to sub-inhibitory doses of NO does not result in any change in MIC for PAK or ATCC MRSA.
• NO doses from COS/NO were determined by chemiluminescence.
• FIG. 6 shows NO-release kinetics and totals of COS/NO in PBS pH 7.4.
• FIG. 7 shows representative 1 FI NMR of COS/NO in deuterium oxide.
• FIG. 8 shows representative FT-IR of 5 kDa chitosan (blue) and COS/NO
• FIG. 9 shows that nitric oxide pretreatment of MDR species of P. aeruginosa results in improved tobramycin susceptibility (a-d) P. aeruginosa viability after exposure to tobramycin with (dashed line) and without (solid line) NO pretreatment. Error is representative of the standard deviation of the mean for >3 biological replicates.
• FIG. 10 shows that that nitric oxide pretreatment of MDR ESKAPE pathogens results in improved tobramycin susceptibility (a-d) ESKAPE pathogen viability after exposure to tobramycin with (dashed line) and without (solid line) NO pretreatment. Error is representative of the standard deviation of the mean for >3 biological replicates.
• FIG. 11 shows that nitric oxide pretreatment of P. aeruginosa biofilms results in improved tobramycin susceptibility
• PAK biofilm viability after exposure to tobramycin without pretreatment solid bars
• b-e MDR P. aeruginosa biofilm viability after exposure to tobramycin without pretreatment (solid bars) or with NO pretreatment of 4 h (striped bars).
• Error is representative of the standard deviation of the mean for >3 biological replicates.
• FIG. 12 shows that NO-pretreatment of P. aeruginosa biofilms does not significantly reduce viability. Error is representative of the standard deviation of the mean of n>3 separate measurements.
• Pseudomonas aeruginosa strain K was donated by Professor Matthew Wolfgang from the Department of Microbiology and Immunology at the University of North Carolina at Chapel Hill (Chapel Hill, NC).
• Staphylococcus aureus ATCC #29213
• Methicillin-resistant S. aureus MRSA, ATCC #33591
• Burkholderia cepacia complex BCC, ATCC #25416
• aeruginosa (AR 229, AR 230, AR 237, and AR 239)
• Klebsiella pneumoniae (AR 542) were obtained from the CDC & FDA Antibiotic Resistant Isolate Bank (Atlanta, GA).
• Chitosan oligosaccharides were subsequently modified with an aminoethyl Schiff base functional group through a tosylated nucleophilic substitution reaction to produce secondary amine-modified chitosan oligosaccharides (COS).
• Amine-modified chitosan 45 mg was dissolved in a mixture of water (450 pL), methanol (2.55 mL), and sodium methoxide (5.4 mM in methanol, 75 pL) and subsequently placed into a Parr hydrogenation vessel with continuous stirring. Oxygen was removed with three short argon purges (10 s, 7 bar) followed by three long argon purges (10 min, 7 bar). The reactor was pressurized with NO gas (10 bar) for 3 days.
• Nitric oxide-releasing chitosan oligosaccharides were precipitated in ethanol, collected via centrifugation, dried in vacuo, and stored at - 20 °C in vacuum sealed bags.
• Successful synthesis was confirmed with NMR and FTIR, and compared to previously published literature [Ahonen, M. J. R., Hill, D. B. & Schoenfisch, M. H. Nitric oxide-releasing alginates as mucolytic agents. ACS Biomater. Sci. Eng. 5, 3409-3418 (2019)]. Representative data are provided in Supporting Information (FIG. 7 and FIG. 8). [0072] Nitric oxide-release kinetics and totals
• Nitric oxide-release was measured in real time with a Zysense chemiluminescent nitric oxide analyzer (NOA, Boulder, CO). Approximately 1 mg of COS/NO was added into 30 ml_ PBS (10mM, pH 7.4) and carried to the instrument via nitrogen bubbled through solution at 200 mL/min. Analysis was stopped when measurements fell below 10 ppb NO per mg of COS.
• NOA Zysense chemiluminescent nitric oxide analyzer
• Frozen stocks of bacteria were reconstituted in TSB (3 ml_) and cultured overnight. The overnight cultures of bacteria (3 ml_) were inoculated into fresh TSB (30 ml_) and grown to 10 8 CFU/mL. Bacteria were diluted in TSB to a final concentration of 10 6 CFU/mL and exposed to serial dilutions of COS/NO or antibiotic (aztreonam, ceftazidime, ciprofloxacin, colistin, meropenem, and tobramycin) for 24 h. Inhibition was assessed with the resazurin assay and the minimum inhibitory concentration (MIC) was defined as the lowest concentration of antibacterial agent required to prevent the reduction of resazurin (/. e. , color change from blue to pink).
• MIC minimum inhibitory concentration
• the highest concentration for each antimicrobial tested was 2 c MIC.
• Six additional dosages at stepwise, 2-fold dilutions in concentration were evaluated, resulting in 49 total combinations of COS/NO and each antibiotic tested against each strain of bacteria.
• the lowest drug concentration in the array that did not support bacterial growth nor change color after incubation with resazurin was determined the most effective inhibitory concentration.
• the fractional bactericidal concentration index ( ⁇ FIC) was calculated using Equation 1 , reported by Elion et al. [Elion, G. B., Singer, S. & Hitchings, G. H. Antagonists of Nucleic Acid Derivatives: VIII. Synergism in combinations of biochemically related antimetabolites. J. Biol. Chem.
• MICA and MICB are the values determined for agents A and B in the single-agent assays, respectively
• MICAB and MICBA are the concentrations of agent A and B that constituted the most effective inhibitory combination as determined by the checkerboard assay.
• Checkerboard assays were conducted in at least duplicate for each bacterial strain and for each drug combination.
• Characterization of the combinations was performed using the following criteria based on ⁇ FIC values: ⁇ 0.25 is highly synergistic; ⁇ 0.5 is synergistic; ⁇ 1 is additive; ⁇ 4 is indifferent; > 4 is antagonistic.
• Time-kill assays were performed over 24 h in order to quantitatively probe the effect of each antibiotic-NO combination as a function of time [Belley, A. et al. Assessment by time-kill methodology of the synergistic effects of oritavancin in combination with other antimicrobial agents against Staphylococcus aureus. Antimicrob. Agents Chemother. 52, 3820-3822 (2008)]. Planktonic bacteria at a final concentration of 10 6 CFU/mL in TSB were incubated with combinations of COS/NO and antibiotic at 1 c MIC.
• Bacteria were cultured and diluted to a final concentration of 10 6 CFU/mL in TSB as described previously and incubated with subinhibitory concentrations (1 ⁇ 4 x MIC) of COS/NO for 1 , 2, or 4 h in a 1 -dram vial, added to serial dilutions of antibiotic (/. e. , aztreonam, colistin, meropenem, or tobramycin), and incubated for 20 h. After exposure, wells were serially diluted in sterile MilliQ water and spiral plated on TSA. Viability was assessed by colony counting.
• bacteria at a final concentration of 10 6 CFU/mL in TSB were incubated with subinhibitory concentrations (1 ⁇ 4 x MIC) of COS/NO for 4 h in a 15 mL centrifuge tube and subsequently incubated with an array of antibiotic combinations in TSB for 24 h at 37 °C.
• the highest antibiotic concentration tested was 2 x MIC.
• Six additional dosages at stepwise, 2-fold dilutions in concentration were evaluated, resulting in 49 total combinations of tobramycin and either colistin or aztreonam tested against each strain of P. aeruginosa.
• the lowest drug concentration in the array that did not support bacterial growth nor change color after incubation with resazurin was determined the most effective inhibitory concentration.
• the ⁇ FIC was calculated using Equation 1. Checkerboard assays were conducted in at least duplicate for each strain and characterized using the previously described criteria.
• Bacteria were cultured to a concentration of 10 8 CFU/mL in TSB and diluted to 10 6 CFU/mL in 200 pL TSB in a 96 well plate. Plates were incubated with shaking for 3 days until a non-surface attached viscous aggregate formed that was easily separated from growth medium. Biofilms (100 pl_) were removed, gently injected into PBS (200 mI_) to remove loosely adhered planktonic cells, and added to a sterile 96 well plate. To the wells, PBS or test agent dissolved in PBS (100 mI_) was added and incubated with shaking for 24 h.
• the biofilm (100 mI_) was removed from the well plate, added to 900 mI_ sterile MilliQ water, and disrupted with pipetting and vortexing. Disrupted biofilms were serially diluted and spiral plated on TSA. Viability was assessed with colony counting.
• the minimum biofilm eradication concentration (MBEC) was defined as the lowest concentration of test agent required to reduce viability by 5-log (/. e. , 10 8 to 10 3 CFU/mL).
• Biofilms were grown as described previously and exposed to COS/NO at 1 ⁇ 4 x MBEC for 1 , 2, or 4 h. The biofilm was then removed from its 96 well plate, added to a new 96 well plate containing tobramycin dissolved in PBS (100 mI_), and incubated for 20 h. The biofilm was disrupted in 900 mI_ sterile MilliQ water with pipetting and vortexing, serially diluted, and spiral plated on TSA. Viability was assessed with colony counting.
• Bacteria at a final concentration of 10 6 CFU/mL in TSB were incubated with serial dilutions of COS/NO and/or tobramycin for 24 h. Growth was assessed by measuring absorbance at 600 nm. The wells that had the highest concentration of test agent and an O ⁇ eoo corresponding to more than 10 8 CFU/mL were diluted to 10 6 cfu/mL in TSB and incubated with fresh solutions of COS/NO or tobramycin for 24 h. A single passage was defined as one exposure, incubation, and subsequent dilution. Bacteria were passaged for up to 70 days.
• Nitric oxide represents a potential solution to the threat of antibiotic resistance.
• NO As a diatomic radical, NO rapidly generates several reactive oxygen and nitrogen species in physiological conditions that kill bacteria through multiple mechanisms (e.g., lipid peroxidation, protein deamination, FIG. 1 , panel A)
• lipid peroxidation, protein deamination FIG. 1 , panel A
• FIG. 1 panel A
• ESKAPE pathogen susceptibility to antibiotics may improve by harnessing the disruptive capabilities of NO, which simultaneously exerts bactericidal action.
• NO exists naturally as a highly reactive gas
• the applicants deliver NO into solution via a water-soluble chitosan donor that releases NO under physiological conditions in a burst release profile (FIG. 6 and Table 1).
• Nitric oxide releasing chitosan (COS/NO) has been used previously by our lab to eradicate both planktonic bacteria and biofilms [Lu, Y., Slomberg, D. L. & Schoenfisch, M. H. Nitric Oxide-Releasing Chitosan Oligosaccharides as Antibacterial Agents.
• /V-diazeniumdiolate NO donor ligands are employed to facilitate NO storage and release from chemically modified chitosan oligosaccharides (COS/NO). Additional ligands are disclosed in U.S. Patent Nos. 98503222 and 10759877, incorporated by reference herein.
• the chitosan biopolymer releases two molecules of NO per secondary amine upon breakdown of the N-diazeniumdiolate at physiological pH (i.e. , protonation, FIG. 7), with the NO exerting broad-spectrum bactericidal action.
• Checkerboard assays characterize antimicrobial combinations as synergistic, additive, indifferent, or antagonistic using fractional inhibitory concentration indices ( ⁇ FIC).
• ⁇ FIC values are calculated using an equation that evaluates the inhibitory action of each test agent in the combination and alone, and when the effects of the combination are greater than the sum of the individual agents, the result in synergy.
• the opposite effect, antagonism occurs when the effects of the combination are worse than the individual agents, which is dangerous and undesirable for clinical applications.
• Ciprofloxacin Fluoroquinolone Inhibits DNA replication 1.25 Indifferent Polycation ic
• aeruginosa were more susceptible to tobramycin after NO-pretreatment (Table 4 and FIG. 9).
• Biofilms are inherently resistant to antibiotic penetration and action due primarily to the protective matrix and altered metabolic state of enclosed bacteria.
• Nitric oxide has previously been shown to disrupt P. aeruginosa biofilms, and rheological analysis has suggested that the biofilm matrix is compromised with NO treatment [Reighard, K. P., Hill, D. B., Dixon, G. A., Worley, B. V. & Schoenfisch, M. H. Disruption and eradication of P. aeruginosa biofilms using nitric oxide-releasing chitosan oligosaccharides. Biofouling 31, 775-87 (2015); Howlin, R. P. etal.
• Tobramycin+NO c _ 2000 _ 4000 _ 10_ a single passage is defined as the exposure to serial dilutions of test agent for 24 h and subsequent dilution to 10 6 CFU/mL. ⁇ Dose of NO from COS/NO determined by chemiluminescence. c MIC of tobramycin when delivered with 25% COS/NO MIC simultaneously.
• Nitric oxide improves antibiotic action in both planktonic and biofilm bacteria and this phenomenon occurs irrespective of bacteria species, antibiotic mechanism of action, or molecular mechanisms of resistance.

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