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WO2019232059A1 - Antisense oligonucleotides for the treatment of p. aeruginsoa, a. baumannii and k. pneumoniae infections - Google Patents

Antisense oligonucleotides for the treatment of p. aeruginsoa, a. baumannii and k. pneumoniae infections Download PDF

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Publication number
WO2019232059A1
WO2019232059A1 PCT/US2019/034408 US2019034408W WO2019232059A1 WO 2019232059 A1 WO2019232059 A1 WO 2019232059A1 US 2019034408 W US2019034408 W US 2019034408W WO 2019232059 A1 WO2019232059 A1 WO 2019232059A1
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Prior art keywords
compound
seq
aea
acid
aeea
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French (fr)
Inventor
Nrusingh P. MOHAPATRA
Denis Guenette
Bruhaspathy Miriyala
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TECHULON Inc
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TECHULON Inc
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    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
    • A61K47/6455—Polycationic oligopeptides, polypeptides or polyamino acids, e.g. for complexing nucleic acids
    • 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
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides

Definitions

  • N is an antisense molecule that inhibits the growth of the bacteria P. aeruginosa
  • A. baumannii, or K. pneumoniae comprising a polynucleotide sequence that is antisense to the coding region of a P. aeruginosa, A. baumannii, or K. pneumoniae protein and hybridizes to the coding region under physiological conditions;
  • L is a linker or a bond
  • Z is a cell penetrating molecule.
  • the bacteria is P. aeruginosa and N is at least 85% identical to gcc ate age cgt get (SEQ ID NO: 12).
  • the compound is gcc ate age cgt get (SEQ ID NO: 12)-AEEA-AEA-B-RIR FKK AKK LFK RIR (SEQ ID NO: 13), wherein
  • AEEA is 8-Amino-3,6-dioxaoctanoic acid
  • AEA is 5-amino-3-oxapentanoic acid, and in the cell penetrating molecule:
  • B is beta-alanine
  • R is L- Arginine
  • I is L-Isoleucine
  • F is L-phenylaalanine
  • K is L-Lysine
  • A is L-Alanine
  • L is L-Leucine
  • the bacteria is A. baumannii and N is at least 85% identical to ett tac gca tga gag (SEQ ID NO: 14).
  • the compound is ett tac gca tga gag (SEQ ID NO: 14)- AEE A- AEA-BX-RXR-RXR-RXR-RXR (SEQ ID NO: 15), wherein
  • AEEA is 8-Amino-3,6-dioxaoctanoic acid
  • AEA is 5-amino-3-oxapentanoic acid
  • B is beta-alanine
  • R is L- Arginine
  • X is 6-amino-hexanoic acid.
  • the bacteria is K. pneumoniae and N is at least 85% identical to tcc att gat tct gtt (SEQ ID NO: 16).
  • the compound is tcc att gat tct gtt (SEQ ID NO: 16)- AEEA-AEA-RIR FKK AKK LFK RIR (SEQ ID NO: 17), wherein
  • AEEA is 8-Amino-3,6-dioxaoctanoic acid
  • AEA is 5-amino-3-oxapentanoic acid, and in the cell penetrating molecule:
  • R is L- Arginine
  • I is L-Isoleucine
  • F is L-phenylaalanine
  • K is L-Lysine
  • A is L-Alanine
  • L is L-Leucine
  • the bacteria is K. pneumoniae and N is at least 85% identical to tcc ata gtg tta cct (SEQ ID NO: 18).
  • the compound is tcc ata gtg tta cct (SEQ ID NO: 18)-AEEA-AEA- RRWYRWWRR (SEQ ID NO: 19), wherein AEEA is 8-Amino-3,6-dioxaoctanoic acid,
  • AEA is 5-amino-3-oxapentanoic acid
  • R is L- Arginine
  • X is 6-amino-hexanoic acid
  • W is L-Tryptophan
  • Y is L-Tyrosine
  • the compound is a PNA. In another embodiment, the
  • the compound has the structure as set forth in Fig. 11. In another embodiment, the compound has the structure as set forth in Fig. 12. In another embodiment, the compound has the structure as set forth in Fig. 13. In another embodiment, the compound has the structure as set forth in Fig. 14.
  • the bacteria is P. aeruginosa and the compound is gcc ate age cgt get (SEQ ID NO: 12)-AEEA-AEA-B- RIR FKK AKK LFK RIR (SEQ ID NO: 13).
  • the bacteria is A. baumannii and the compound is ett tac gca tga gag (SEQ ID NO: 14)-AEEA-AEA-BC- RXR-RXR-RXR-RXR (SEQ ID NO: 15).
  • the bacteria is K.
  • the bacteria is K. pneumoniae and the compound is tcc ata gtg tta cct (SEQ ID NO: 18)-AEEA- AEA- RRWYRWWRR (SEQ ID NO: 19).
  • Fig. 1 is a graph showing the minimum inhibitory concentration (MIC) of PPNA
  • Fig. 2 is a graph showing the MIC of PPNA 23X and colistin against two strains of P. aeruginaosa (ATCC-47085 and CDC-242) over 30 passages, showing little development of drug resistance for the PPNA.
  • Fig. 3 is a graph showing the MIC of PPNA 40 and colistin against two strains of
  • A. baumannii (ATCC-1605 and CDC-274) over 50 passages.
  • Fig. 4 is a graph showing the MIC of PPNA 40 and colistin against two strains of
  • A. baumannii (ATCC-1605 and CDC-274) over 50 passages.
  • Fig. 5 is a graph showing the MIC of PPNA 23X and colistin against two strains of P. aeruginaosa (ATCC-47085 and CDC-239) over 50 passages.
  • Fig. 6 is a graph showing the MIC of PPNA 23X and colistin against two strains of P. aeruginaosa (ATCC-47085 and CDC-239) over 50 passages.
  • Figs. 7A-7B are graphs showing the results of a murine neutropenic thigh infection model that shows that PPNA 40 is effective in vivo against A. baumannii strains UNT191.1 and UNT237.1 when compared to tigecycline and infection controls.
  • Fig. 8 is a graph showing the results of a murine neutropenic lung infection model that shows that PPNA 40 is effective in vivo against A. baumannii strains UNT191.1 and UNT237.1 when compared to tigecycline and infection controls.
  • Fig. 9 is a graph showing the results of a murine neutropenic lung infection model that shows that PNA 40 is effective in vivo against A. baumannii strains UNT191.1 and UNT237.1 when compared to tigecycline and infection controls.
  • Fig. 10 is a graph showing the results of a murine neutropenic lung infection
  • Fig. 11 depicts the structure of PPNA 23X.
  • Fig. 12 depicts the structure of PPNA 40.
  • Fig. 13 depicts the structure of PPNA Kl.
  • Fig. 14 depicts the structure of PPNA K2.
  • Specific aspects of the invention include a conjugate having the formula N-L-Z that is useful for the treatment of Gram negative bacterial infection and/or inhibiting the growth of P. aeruginosa, A. baumannii or K. pneumoniae.
  • the N-L-Z conjugates of the invention comprise N, an antisense molecule that inhibits the growth of P. aeruginosa, A. baumannii or K. pneumonia , a linker L, and Z, a cell penetration peptide (CPP).
  • the cell penetration peptide may have one or more functions to facilitate cell targeting and/or membrane permeation of Gram negative bacteria in a host.
  • the cell penetration peptide provides for membrane disruption of bacteria provides specificity and reduces toxicity.
  • Bulk synthesis can be carried out by contract manufacturers, such as Neo Group,
  • the N-L-Z conjugate is part of a composition comprising a buffer.
  • Suitable buffers in the composition of the invention provide a basic pH when dissolved or dispersed in water.
  • the buffer has a pKa of greater than about 7. See, for example, "Handbook of Pharmaceutical Excipients," 5 th ed., Rowe et al. (eds.) (2006); and SIGMA Life Sciences, "Products for Life Science
  • the composition may comprise one or more buffers.
  • buffers include— but are not limited to— phosphate buffers, bicarbonate buffers, ethanolamine buffers, borate buffers, imidazole buffers, tris buffers, and zwitterionic buffers (e.g., HEPES, BES, PIPES, Tricine, and other so-called “Good's Buffers”). See, for example, Good et al ., "Hydrogen Ion Buffers for Biological Research," Biochemistry , 5(2):467-477 (1966).
  • the buffer is a bicarbonate, such as sodium bicarbonate or carbonate.
  • the buffer has a pKa between about 6 and about 14, between about 7 and about 13, and between about 7 and about 12. In another embodiment, the buffer has a pKa between about 7 and about 8.
  • the N-L-Z conjugate is combined with a delivery polymer.
  • the polymer-based nanoparticle drug delivery platform is adaptable to a diverse set of polynucleotide therapeutic modalities.
  • the delivery polymer is cationic.
  • the delivery polymer comprises phosphonium ions and/or ammonium ions.
  • the N-L-Z conjugate is combined with a delivery polymer, and the composition forms nanoparticles in solution.
  • nanoparticle polyplexes are stable in serum and have a size in the range of about 30 nm - 5000 nm in diameter.
  • the particles are less than about 300 nm in diameter.
  • the nanoparticles are less than about 150 nm in diameter.
  • the delivery vehicle comprises a cationic block copolymer comprising phosphonium or ammonium ionic groups as described in PCT/US 12/42974.
  • the polymer is diblock-/W j' [(ethylene glycol)9 methyl ethyl methacralate][stirylphosphonium].
  • the delivery polymer comprises glycoamidoamines as described in Tranter et al. Amer Soc Gene Cell Ther , Dec 2011; polyhydroxylamidoamines, dendritic macromolecules, carbohydrate- containing polyesters, as described in US20090105115; and US20090124534.
  • the nucleic acid delivery vehicle comprises a cationic polypeptide or cationic lipid.
  • An example of a cationic polypeptide is polylysine. See U.S. Pat. 5,521,291.
  • the N-L-Z conjugate is part of a composition comprising delivery or carrier polymers.
  • the N-L-Z conjugate is part of nanoparticle polyplexes capable of transporting molecules with stability in serum.
  • the polyplex compositions comprise a synthetic delivery polymer (carrier polymer) and biologically active compound associated with one another in the form of particles having an average diameter of less than about 500 nm, such as about 300 nm, or about 200 nm, preferably less than about 150 nm, such as less than about 100 nm.
  • the invention encompasses particles in the range of about 40 nm - 500 nm in diameter.
  • the delivery or carrier polymer comprises a cationic block copolymer containing phosphonium or ammonium ionic groups as described in
  • the delivery or carrier polymer comprises glycoamidoamines as described in Tranter et al. Amer Soc Gene Cell Ther , Dec 2011; polyhydroxylamidoamines, dendritic macromolecules, carbohydrate- containing polyesters, as described in US20090105115; and US20090124534.
  • the polyglycoamidoamine (PGAA) polymer system which is a proprietary, localized and biodegradable nanoparticle system, represents another delivery or carrier polymer.
  • Poly(galactaramidoamine) is an efficient cationic polymeric vehicle with low cytotoxicity (Wongrakpanich et al. Pharmaceutical Development and Technology , January 12, 2012).
  • the nanoparticle delivery system disclosed in Hemp et al. Biomacromolecules , 2012 13:2439-45 represents another delivery or carrier polymer useful in the present invention.
  • the delivery or carrier polymer comprises a cationic polypeptide or cationic lipid.
  • Polymers such as /w/j'-L-lysine (PLL), polye thyleneimine (PEI), chitosan, and their derivatives are also encompassed by the invention. Nucleic acid delivery using these compounds relies on complexation driven by electrostatic interactions between the gene and the polycationic delivery agent. Polymer- DNA complexes condense into particles on the order of 60 nm - 120 nm in diameter. Polymers such as linear PEI and PLL have high transfection rates in a variety of cells.
  • nucleic acid delivery has size constraints requiring a sufficiently small polyplex to enable long circulation times and cellular uptake.
  • polyplexes must resist salt- and serum-induced aggregation.
  • Serum stability is generally associated with a particle size of about sub-l50 nm hydrodynamic radius or below maintainable for 24 h.
  • the nanoparticles of the invention which comprise nucleic acid therapeutic and delivery polymer, have the hydrodynamic radius and material properties for serum stability.
  • the delivery polymer when combined with the nucleic acid, protects the therapeutic cargo under physiological conditions.
  • the delivery polymers are designed to have characteristics of spontaneous self-assembly into nanoparticles when combined with polynucleotides in solution.
  • the invention also contemplates other delivery polymers that form serum-stable nanoparticles.
  • the invention is not limited to the type of delivery polymer and may be adaptable to nucleic acid characteristics, such as length, composition, charge, and presence of coupled peptide.
  • the delivery polymer may also be adaptable for material properties of the resultant nanoparticle, such as hydrodynamic radius, stability in the host bloodstream, toxicity to the host, and ability to release cargo inside a host cell.
  • the N-L-Z conjugate is administered in the form of a salt.
  • the salt may be any pharmaceutically acceptable salt comprising an acid or base addition salt.
  • pharmaceutically acceptable salts with acids include those formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, hydroiodic acid, hydrofluoric acid, phosphorous acid, and the like.
  • salts that are formed with organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, alkanedioic acids, aromatic acids, aliphatic and. aromatic sulfonic acids, etc.
  • acetic acid trifluoroacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like.
  • Exemplary salts thus include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates,
  • Acid addition salts of basic molecules may be prepared by contacting the free base forms with a sufficient amount of the desired acid to produce the salt according to methods and techniques with which a skilled artisan is familiar.
  • inorganic base or an organic base to the free acid.
  • Pharmaceutically acceptable base addition salts may be formed with metals or amines, such as alkali and alkaline earth metals or organic amines.
  • Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like.
  • Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, for example, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, 2-dimethylaminoethanol, 2- diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, N,N-dibenzylethylenediamine, chloroprocaine, hydrabamine, choline, betaine, ethylenediamine, ethylenedianiline, N-methylglucamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like.
  • the N-L-Z conjugate is administered as part of a
  • compositions comprising a pharmaceutically acceptable diluent, excipient or carrier.
  • Suitable diluents, excipients and carriers are well known in the art and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gernnaro Ed., 1985).
  • the pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form must be sterile, fluid and stable under the conditions of manufacture and storage.
  • the liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, saline, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, buffers or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the N-L-Z conjugate in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization.
  • the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.
  • the composition comprising the N-L-Z conjugate is in contact with a fabric.
  • the fabric may comprise natural fibers, synthetic fibers, or both.
  • textile fabrics include, but are not limited to, nylon, cotton, nylon-cotton blends, wool, silk, linen, polyester, rayon, and worsted.
  • the fabric is cotton.
  • the fabric is nylon.
  • the fabric is a nylon-cotton blend.
  • the ratio of nylon to cotton in the nylon-cotton blend fabric can be between about 1 :99 and about 99: 1, between about 10:90 and about 90:10, between about 20:80 and about 80:20, between about 30:70 and about 70:30, between about 40:60 and about 60:40, and between about 45:55 and about 55:45.
  • the fabric is a 50:50 nylon-cotton blend. - lO -
  • the fabric has a high tensile strength-to- weight ratio.
  • the fabric with a high tensile-to-weight ratio is a fabric comprising aramid fibers.
  • the aramid fiber is a para-aramid fiber (e.g., the para-aramid fiber commercially known as KEVLAR).
  • the aramid fiber is a meta-aramid fiber (e.g., the meta-aramid fiber commercially known as NOMEX).
  • the antimicrobial fabric is capable of treating a P.
  • the antimicrobial fabric is capable of treating a P. aeruginosa, A.
  • the duration of a wash cycle is between about 10 minutes and about 90 minutes, between about 10 minutes and about75 minutes, between about 10 minutes and about 60 minutes, between about 10 minutes and about 45 minutes, between about 10 minutes and about 30 minutes, and between about 10 minutes and about 15 minutes.
  • the water temperature in the wash cycles is between about l6°C and about 60°C, between about 27°C and about 49°C, or between about 37° and about 44°C.
  • the antimicrobial fabric is capable of treating a P.
  • AATCC 147 from American Association of Textile Chemists and Colorists (AATCC).
  • composition comprising the N-L-Z
  • the composition may be in the form of solution that can be applied to a fabric, e.g., by rinsing, dipping, or spraying.
  • the fabric can be an antimicrobial fabric or a non-antimicrobial fabric.
  • application of the solution to the fabric provides a fabric that is capable of treating a P. aeruginosa, A. baumannii or K.
  • application of the solution to the fabric increases the fabric's capability of treating a P. aeruginosa, A. baumannii or K. pneumoniae infection or inhibiting growth of P. aeruginosa, A. baumannii or K. pneumoniae.
  • application of the solution to an antimicrobial fabric with low antimicrobial activity increases the antimicrobial activity of the fabric.
  • the wound healing dressing is an adhesive dressing. In another embodiment, the wound healing dressing is a non-adhesive dressing. In one embodiment, the dressing comprises a foam, gel, or cream. In another embodiment, the dressing comprises a fiber based material (e.g. , gauzes or waddings). In one embodiment, the fiber-based material is cotton. In another embodiment, the fiber- based material is rayon. In another embodiment, the fiber-based material is a gel-forming fiber, such as a carboxymethylated cellulosic material. In another embodiment, the fiber- based material is a synthetic polymer. In another embodiment, the wound healing dressing is THERAGAUZE (Soluble Systems, LLC, Newport News, VA).
  • the invention also provides a method of treating P. aeruginosa, A. baumannii or
  • K. pneumoniae infection and a method of inhibiting the growth of P. aeruginosa, A.
  • the animal undergoing treatment for P. aeruginosa, A. baumannii or K. pneumoniae infection exhibits one or more symptoms of P. aeruginosa, A. baumannii or K. pneumoniae infection including fever and chills, body aches, light-headedness, rapid pulse and breathing, nausea and vomiting, diarrhea, (P. aeruginosa), infections of the lungs, blood, brain, urinary track, and wound infections (A. baumannii), fever, confusion, neck stiffness, sensitivity to light, bloodstream infection, fever, chills, rash, light-headedness, altered mental state ⁇ K.
  • pneumonia as well as puss production in the infected area, acne, boils, abscesses, carbuncles, stys, cellulitis, diarrhea, botulism, and gas gangrene.
  • the animal may also exhibit signs of sepsis or pneumonia.
  • the N-L-Z conjugate is administered by intravenous injection.
  • the N-L-Z conjugate is administered by intramuscular injection. In another embodiment, the N-L-Z conjugate is administered by peritoneal injection. In another embodiment, the N-L-Z conjugate is administered topically, e.g. to a tissue suspected to be infected by P. aeruginosa, A. baumannii or K. pneumoniae. In another embodiment, the N-L-Z conjugate is administered orally.
  • the N-L-Z conjugate may be formulated as part of a pharmaceutical composition coated with an enteric coating that will protect the N-L-Z conjugate from the acid environment of the stomach and release the N-L-Z conjugate in the upper gastrointestinal tract. In another embodiment, the N-L-Z conjugate may be formulated as part of a sustained release formulation that will release the N-L-Z conjugate on a substantially continuous basis over a period of time.
  • Animals that may be treated with the N-L-Z conjugate according to the invention include any animal that may benefit from treatment with the N-L-Z conjugate. Such animals include mammals such as humans, dogs, cats, cattle, horses, pigs, sheep, goats and the like.
  • N-L-Z conjugate is administered in an amount that is effective for the
  • the amount may vary widely depending on the mode of administration, the species of P. aeruginosa, A. baumannii or K. pneumoniae , the age of the animal, the weight of the animal, and the surface area of the animal.
  • the amount of N-L-Z conjugate, salt and/or complex thereof may range anywhere from 1 pmol/kg to 1 mmol/kg. In another embodiment, the amount may range from 1 nmol/kg to 10 mmol/kg.
  • the amount of N-L-Z conjugate, salt and/or complex thereof may range anywhere from 1 to 99 weight percent. In another embodiment, the amount of N-L-Z conjugate, salt and/or complex thereof may range anywhere from 1 to 10 weight percent.
  • the invention also provides N-L-Z conjugates comprising a linker.
  • N-L-Z conjugates comprising a linker.
  • N is an antisense molecule that inhibits the growth of a bacterium comprising a polynucleotide sequence that is antisense to the coding region of a bacterial protein and hybridizes to the coding region under physiological conditions;
  • L is a linker having the formula (Y') radical, where each Y' is independently glycine, cysteine, 8-amino-3,6- dioxaoctanoic acid (AEEA), or 5-amino-3-oxapentanoic acid (AEA), and n is an integer from 1 to 10; and Z is a cell penetrating molecule.
  • n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • L is a bond.
  • the cell penetrating molecule Z has the formula: (ABC) P -D, wherein A is a cationic amino acid which is Lysine or Arginine; B and C are hydrophobic amino acids which may be the same or different and are selected from the group consisting of Valine, Leucine, Isoleucine, Tyrosine, Phenylalanine, and Tryptophan; p is an integer with a minimal value of 2; and D is a cationic amino acid or is absent.
  • A is Lysine
  • B is Phenylalanine
  • C Phenylalanine
  • D Lysine
  • p is 3.
  • / 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • the cell penetrating molecule Z has the formula: B-X I -(R-X 2 -R) 4 , where B is beta-alanine or is absent, Xi is 6-amino-hexanoic acid or is absent, X 2 is 6- amino-hexanoic acid, and R is arginine or homo-arginine.
  • R is arginine, selected from the group consisting of L-arginine and D-arginine.
  • the cell penetrating molecule is a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-11 (See Table 1).
  • N comprises a modified backbone.
  • the modified backbone is a PNA backbone.
  • Modified nucleic acids are non-natural polymers that hybridize to natural DNA and RNA with sequence specificity according to Watson-Crick base paring rules.
  • modified nucleic acids are phosphorothioate-oligodeoxynucleotides (PS- ODNs), locked nucleic acids (LNAs), 2'-0-methyloligoribonucleotides (2'O-Mes), phosphorodiamidate morpholino oligonucleotides (PMOs), and peptide nucleic acids (PNAs).
  • PS- ODNs locked nucleic acids
  • LNAs locked nucleic acids
  • 2'O-Mes 2'-0-methyloligoribonucleotides
  • PMOs phosphorodiamidate morpholino oligonucleotides
  • PNAs peptide nucleic acids
  • Modified nucleic acids have modified backbones and are generally more resistant to degradation than natural nucleic acids.
  • the invention includes any type of synthetically-modified DNA or RNA that hybridizes to natural DNA and RNA. See, e.g.. U.S. Pat. Nos.
  • Antisense molecules of the invention may also be composed of non-natural amino acids
  • Atypical nucleoside bases may also be employed, such as methylated bases, phosphorylated bases, inosine, thiouridine, pseudouridine, dihydrouridine, queuosine, and wyosine, among others.
  • antisense polymers comprising atypical bases are disclosed in U.S. Pat. Nos. 7,875,733, 7,919,612, 7,939,677, 8,314,229, 8,372,969, and 8,377,898.
  • antisense polynucleotide refers to a nucleic acid molecule that is
  • Antisense molecules specifically hybridize with one or more nucleic acids encoding a preselected target nucleic acid.
  • target nucleic acid and nucleic acid encoding the target encompass DNA encoding the target, RNA (including pre-mRNA and mRNA) transcribed from such DNA, and also cDNA derived from such RNA.
  • the hybridization of an antisense compound with its target nucleic acid interferes with the normal function of the nucleic acid. This modulation of function of a target nucleic acid by compounds which specifically hybridize to it is generally referred to as antisense.
  • the functions of DNA to be interfered with include replication and transcription.
  • the functions of RNA to be interfered with include all vital functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in or facilitated by the RNA.
  • the overall effect of such interference with target nucleic acid function is modulation of the expression of the target.
  • modulation means either an increase (stimulation) or a decrease (inhibition) in the expression of a gene.
  • inhibition is the form of modulation of gene expression.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity and similarity between two sequences can be accomplished using a mathematical algorithm (see e.g., Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H.
  • the percent identity between two sequences is determined based on alignments generated with the Clustal W algorithm (Thompson, J. D. et al, 1994, Nucleic acids Res. 22:4673-4680). This algorithm is incorporated into many commercial software packages, in this case the alignX software program in the Vector NTI suite (version 8.0).
  • Proteins are polymers containing one or more chains of amino acids bonded
  • Proteins typically fold into a three dimensional form, facilitating a biological function.
  • a polypeptide is a polymer of amino acids bonded together by peptide bonds.
  • the terms protein and polypeptide and peptide are generally used interchangeably, although polypeptides and peptides are generally shorter in length than proteins.
  • the invention provides the following specific N-L-Zs: PPNA for Pseudomonas aeruginosa
  • PPNA 40 - ett tac gca tga gag (SEQ ID NO: l4)-AEEA-AEA-BX-RXR-RXR-
  • PPNAs were synthesized by following Merrifield Solid Phase Peptide Synthesis using AAPPTEC automated peptide synthesizers. Each compound was synthesized at a O. lmicromolar concentration using Rink-Amide resin in a 50 ml reaction vessel. The Rink-amide resins were deprotected by 20% piperidine in N-Methyl-2-pyrrolidone (NMP). Resins were washed with NMP for 7 times with 2 mins mixing.
  • NMP N-Methyl-2-pyrrolidone
  • Coupling of Fmoc-amino acids/Fmoc-PNAs was performed for 60 mins with continuous shaking and intermittent argon gas bubbling. After coupling, resins were washed four times with NMP with 2 mins mixing. The growing amino acids/PNAs on resins were capped with 1.5 M Acetic anhydride for 30 mins followed by 5 times washing of resins with NMP with 2 mins of shaking. The process of deprotection, coupling, and capping steps repeated till the end of synthesis of compound. After final capping of amino acid/PNA onto growing resins, the final product was deprotected from resins.
  • the crude product was cleaved from resin by 95% trifluoroacetic acid, 2.5% TIS and 2.5 water for 4 hrs at 37°C.
  • the cleavage product was precipitated in 5 volumes of cold ether and the precipitated compound was collected by centrifugation.
  • the ether precipitated compound was air dried for purification.
  • the compounds were purified in Waters Prep-l50 system. Thirty milligrams of compound was loaded in X-Bridge C18 columns (lOmm X 250mm) with a flow rate of mobile phase 5.5 ml/min. Mobile phase conditions are as follows:
  • the purified fractions were lyophilized and converted to HC1 salt or acetate salts.
  • the HC1 salts of the compounds were prepared by addition of lOmM HC1 solution into lyophilized compound. The solution was flash frozen and further lyophilized to collect the final compound. Acetate salt of the compounds were converted by passing through the HPLC columns in a 1% acetic acid-water and acetonitrile mobile phase. The purified fractions were lyophilized to collect the acetate salts of the compounds. A small fraction of the compound was used to run in an analytical HPLC column to determine the purity of the compound. The purity of all compounds were >95% (Data not shown). The HCl/acetate salt of compounds were screened for the antimicrobial activities against bacterial strains.
  • MIC minimal inhibitory concentration
  • MMC minimal bactericidal concentration
  • Bioscreen-C instrument was used to detect the MIC and MBC of PPNAs against corresponding pathogens. Different concentration of PPNAs were prepared in lOmM of sodium bicarbonate buffer pH-7.4 and added to ⁇ 5.0 LoglO CFUs in a Honey comb Bioscreen-C plate. The plates were incubated in Bioscreen C instrument and growth of bacteria was observed in every 5 mins by measuring the optical density at 420-580 nm with intermittent shaking.
  • ATCC Culture Collections (ATCC) (ATCC-47085, ATCC-27853) and clinical isolates from Center for Disease Control (CDC).
  • PPNA 40 used against Acinetobacter baumannii strain from ATCC (ATCC-BAA-
  • aeruginosa ATCC-47085, CDC#242
  • A. baumannii ATCC-BAA-1605, CDC#274 strains in resistance study, respectively. Colistin was used as the positive control for this assay.
  • the MIC assay was performed by following the Clinical Laboratory Standard Institute (CLSI) protocols.
  • the diluted cultures were exposed to different concentrations of PPNAs (0 -100 pg/mL) and colistin (0-100 pg/mL) and incubated at 35°C overnight.
  • the MIC of PPNAs and colistin against P. aeruginosa and A. baumannii were recorded.
  • the culture of P. aeruginosa and A. baumannii grown at highest concentration of PPNAs or colistin during MIC assay were collected, added 9-fold more TSB and allowed to grow further overnight.
  • the MIC assay was repeated as above. This process was continued for 30 cycles and MIC value of PPNAs and colistin in each cycle were recorded.
  • PPNA 40 was administered with a 10 mg/kg intravenous bolus dose using a 10 mL/kg dose volume ( ⁇ .2 mL) and observed the effects before proceeding to the next higher dose. Mice were well tolerated at 10 mg/kg intravenous administration of both the drugs. However, higher concentration (>l5mg/kg) had shown adverse effect and death in mice within 30 mins of administration of the drug.
  • the MIC of PPNA 40 was determined in an additional fifteen (15) A. baumannii clinical isolates, in order to down-select the A. baumannii strains for use in an efficacy study in mice. Based on MIC assay, UNT191.1 and UNT237.1 strains were selected for efficacy studies in mice. UNT191.1 and UNT237.1 were susceptible to tigecycline and colistin, and the MIC of PPNA 40 against UNT191.1 and UNT237.1 was 2pg/mL. [0079] Female 5-6 week old CD-l (18-22 gm) were used in this study. Mice were quarantined for 48 hours before use and housed in groups of 5 with free access to food and water during the study.
  • Days -4 and -1 150 mg/kg of cyclohsphamide administered by
  • mice were inoculated intramuscularly into right thigh with 10 6
  • mice CFU/mouse of A. baumannii isolates.
  • Three groups of mice were administered with PPNA 40 (at +1, +9, or +l7hr) and one group with tigecy cline (at +1 & +9hr) via IV route.
  • Mice were euthanized by C02 inhalation at +1 and +24hrs post-infection and thighs were removed, and placed in 2 ml of sterile PBS, homogenized, serially diluted and plated to determine the CFU counts. Plates were incubated 18-24 hours and CFUs were counted.
  • Colony were counted and the number of colonies is converted to CFU/thigh by multiplying the number of colonies by the volume of the thigh homogenate spotted and the dilution at which the colonies were counted (5-50 colonies/spot). All count data were transformed into log 10 CFU/thigh for calculation of means and standard deviations.
  • Days -4 and -1 150 mg/kg of cyclohsphamide administered by
  • mice were inoculated via intranasal route with 10 8 CFU/mouse of A. baumannii strains (UNT191.1 and UNT237.1) into each nares.
  • Three groups of mice were administered with PPNA 40 (at +1, +9, or +l7hr) and one group with tigecy cline (at +1 & +9hr).
  • Mice were euthanized by C02 inhalation at +2 and +24hrs post-infection and, lungs were collected, and placed in 2 ml of sterile PBS, homogenized, serially diluted and plated to determine the CFU counts. Plates were incubated 18-24 hours and CFUs were counted.
  • Colony were counted and the number of colonies is converted to CFU/pair of lungs by multiplying the number of colonies by the volume of the lungs homogenate spotted and the dilution at which the colonies were counted (5-50 colonies/spot). All count data were transformed into log 10 CFU/pair of lungs for calculation of means and standard deviations.

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Abstract

Disclosed are antisense compounds useful for the treatment of P. aeruginosa, A. baumannii, and K. pneumoniae infections. The antisense compounds comprise a nucleotide sequence, a linker and a cell penetrating peptide. Also disclosed are methods for the treatment of P. aeruginosa, A. baumannii, and K. pneumoniae infections. Unexpectedly, the compounds of the invention are highly effective in inhibiting the growth of P. aeruginosa, A. baumannii, and K. pneumonia without developing significant resistance to the compounds over time.

Description

ANTISENSE OLIGONUCLEOTIDES FOR THE TREATMENT OF P.
AERUGINSOA, A. BAUMANNII AND K. PNEUMONIAE INFECTIONS
PRIOR RELATED APPLICATIONS
[0001] There are no prior applications related to the present application.
BRIEF SUMMARY OF THE INVENTION
[0002] Disclosed is a compound having the formula:
N— L— Z,
or pharmaceutically acceptable salt thereof, wherein
N is an antisense molecule that inhibits the growth of the bacteria P. aeruginosa,
A. baumannii, or K. pneumoniae comprising a polynucleotide sequence that is antisense to the coding region of a P. aeruginosa, A. baumannii, or K. pneumoniae protein and hybridizes to the coding region under physiological conditions;
L is a linker or a bond; and
Z is a cell penetrating molecule.
[0003] In one embodiment, the bacteria is P. aeruginosa and N is at least 85% identical to gcc ate age cgt get (SEQ ID NO: 12). In another embodiment, the compound is gcc ate age cgt get (SEQ ID NO: 12)-AEEA-AEA-B-RIR FKK AKK LFK RIR (SEQ ID NO: 13), wherein
AEEA is 8-Amino-3,6-dioxaoctanoic acid, and
AEA is 5-amino-3-oxapentanoic acid, and in the cell penetrating molecule:
B is beta-alanine,
R is L- Arginine,
I is L-Isoleucine,
F is L-phenylaalanine,
K is L-Lysine,
A is L-Alanine, and
L is L-Leucine.
[0004] In one embodiment, the bacteria is A. baumannii and N is at least 85% identical to ett tac gca tga gag (SEQ ID NO: 14). In another embodiment, the compound is ett tac gca tga gag (SEQ ID NO: 14)- AEE A- AEA-BX-RXR-RXR-RXR-RXR (SEQ ID NO: 15), wherein
AEEA is 8-Amino-3,6-dioxaoctanoic acid,
AEA is 5-amino-3-oxapentanoic acid,
B is beta-alanine,
R is L- Arginine, and
X is 6-amino-hexanoic acid.
[0005] In one embodiment, the bacteria is K. pneumoniae and N is at least 85% identical to tcc att gat tct gtt (SEQ ID NO: 16). In another embodiment, the compound is tcc att gat tct gtt (SEQ ID NO: 16)- AEEA-AEA-RIR FKK AKK LFK RIR (SEQ ID NO: 17), wherein
AEEA is 8-Amino-3,6-dioxaoctanoic acid, and
AEA is 5-amino-3-oxapentanoic acid, and in the cell penetrating molecule:
R is L- Arginine,
I is L-Isoleucine,
F is L-phenylaalanine,
K is L-Lysine,
A is L-Alanine, and
L is L-Leucine.
[0006] In one embodiment, the bacteria is K. pneumoniae and N is at least 85% identical to tcc ata gtg tta cct (SEQ ID NO: 18). In another embodiment, the compound is tcc ata gtg tta cct (SEQ ID NO: 18)-AEEA-AEA- RRWYRWWRR (SEQ ID NO: 19), wherein AEEA is 8-Amino-3,6-dioxaoctanoic acid,
AEA is 5-amino-3-oxapentanoic acid,
R is L- Arginine,
X is 6-amino-hexanoic acid,
W is L-Tryptophan, and
Y is L-Tyrosine.
[0007] In one embodiment, the compound is a PNA. In another embodiment, the
compound has the structure as set forth in Fig. 11. In another embodiment, the compound has the structure as set forth in Fig. 12. In another embodiment, the compound has the structure as set forth in Fig. 13. In another embodiment, the compound has the structure as set forth in Fig. 14.
[0008] Also disclosed is a pharmaceutical composition comprising the compound
described herein and a pharmaceutically acceptable carrier.
[0009] Also disclosed is a method of treating a bacterial infection, comprising
administering to an animal in need thereof an effective amount of the compound or the pharmaceutical composition described herein. In one embodiment, the bacteria is P. aeruginosa and the compound is gcc ate age cgt get (SEQ ID NO: 12)-AEEA-AEA-B- RIR FKK AKK LFK RIR (SEQ ID NO: 13). In another embodiment, the bacteria is A. baumannii and the compound is ett tac gca tga gag (SEQ ID NO: 14)-AEEA-AEA-BC- RXR-RXR-RXR-RXR (SEQ ID NO: 15). In another embodiment, the bacteria is K. pneumoniae and the compound is is tcc att gat tet gtt (SEQ ID NO: 16)- AEEA-AEA- RIR FKK AKK LFK RIR (SEQ ID NO: 17). In another embodiment, the bacteria is K. pneumoniae and the compound is tcc ata gtg tta cct (SEQ ID NO: 18)-AEEA- AEA- RRWYRWWRR (SEQ ID NO: 19).
BRIEF DESCRIPTION OF THE FIGURES
[0010] Fig. 1 is a graph showing the minimum inhibitory concentration (MIC) of PPNA
40 and colistin against two strains of A. baumannii (ATCC-1605 and CDC-274) over 30 passages, showing little development of drug resistance for the PPNA.
[0011] Fig. 2 is a graph showing the MIC of PPNA 23X and colistin against two strains of P. aeruginaosa (ATCC-47085 and CDC-242) over 30 passages, showing little development of drug resistance for the PPNA.
[0012] Fig. 3 is a graph showing the MIC of PPNA 40 and colistin against two strains of
A. baumannii (ATCC-1605 and CDC-274) over 50 passages.
[0013] Fig. 4 is a graph showing the MIC of PPNA 40 and colistin against two strains of
A. baumannii (ATCC-1605 and CDC-274) over 50 passages.
[0014] Fig. 5 is a graph showing the MIC of PPNA 23X and colistin against two strains of P. aeruginaosa (ATCC-47085 and CDC-239) over 50 passages.
[0015] Fig. 6 is a graph showing the MIC of PPNA 23X and colistin against two strains of P. aeruginaosa (ATCC-47085 and CDC-239) over 50 passages. [0016] Figs. 7A-7B are graphs showing the results of a murine neutropenic thigh infection model that shows that PPNA 40 is effective in vivo against A. baumannii strains UNT191.1 and UNT237.1 when compared to tigecycline and infection controls.
[0017] Fig. 8 is a graph showing the results of a murine neutropenic lung infection model that shows that PPNA 40 is effective in vivo against A. baumannii strains UNT191.1 and UNT237.1 when compared to tigecycline and infection controls.
[0018] Fig. 9 is a graph showing the results of a murine neutropenic lung infection model that shows that PNA 40 is effective in vivo against A. baumannii strains UNT191.1 and UNT237.1 when compared to tigecycline and infection controls.
[0019] Fig. 10 is a graph showing the results of a murine neutropenic lung infection
model that shows that PPNA 40 is effective in vivo against A. baumannii strains
UNT191.1 and UNT237.1 when compared to tigecycline and infection controls.
[0020] Fig. 11 depicts the structure of PPNA 23X.
[0021] Fig. 12 depicts the structure of PPNA 40.
[0022] Fig. 13 depicts the structure of PPNA Kl.
[0023] Fig. 14 depicts the structure of PPNA K2.
DETAILED DESCRIPTION OF THE INVENTION
[0024] The present invention may be understood by reference to the following detailed description of the embodiments of the invention and examples included herein. The terminology used herein is for the purpose of describing embodiments of the invention and is not intended to be limiting.
[0025] Specific aspects of the invention include a conjugate having the formula N-L-Z that is useful for the treatment of Gram negative bacterial infection and/or inhibiting the growth of P. aeruginosa, A. baumannii or K. pneumoniae.
[0026] The N-L-Z conjugates of the invention comprise N, an antisense molecule that inhibits the growth of P. aeruginosa, A. baumannii or K. pneumonia , a linker L, and Z, a cell penetration peptide (CPP). The cell penetration peptide may have one or more functions to facilitate cell targeting and/or membrane permeation of Gram negative bacteria in a host. The cell penetration peptide provides for membrane disruption of bacteria provides specificity and reduces toxicity. [0027] Bulk synthesis can be carried out by contract manufacturers, such as Neo Group,
Inc. (Cambridge, MA) or AmbioPharm, Inc. (North Augusta, SC) using standard methodologies including solid-scaffold protection/deprotection synthesis via high fidelity synthesizers.
[0028] In one embodiment of the invention, the N-L-Z conjugate is part of a composition comprising a buffer. Suitable buffers in the composition of the invention provide a basic pH when dissolved or dispersed in water. In some embodiments, the buffer has a pKa of greater than about 7. See, for example, "Handbook of Pharmaceutical Excipients," 5th ed., Rowe et al. (eds.) (2006); and SIGMA Life Sciences, "Products for Life Science
Research," Product Catalog (2008-2009). The composition may comprise one or more buffers. Such buffers include— but are not limited to— phosphate buffers, bicarbonate buffers, ethanolamine buffers, borate buffers, imidazole buffers, tris buffers, and zwitterionic buffers (e.g., HEPES, BES, PIPES, Tricine, and other so-called "Good's Buffers"). See, for example, Good et al ., "Hydrogen Ion Buffers for Biological Research," Biochemistry , 5(2):467-477 (1966). In one particular embodiment, the buffer is a bicarbonate, such as sodium bicarbonate or carbonate.
[0029] In one embodiment of the invention, the buffer has a pKa between about 6 and about 14, between about 7 and about 13, and between about 7 and about 12. In another embodiment, the buffer has a pKa between about 7 and about 8.
[0030] In another embodiment of the invention, the N-L-Z conjugate is combined with a delivery polymer. The polymer-based nanoparticle drug delivery platform is adaptable to a diverse set of polynucleotide therapeutic modalities. In one aspect of the invention, the delivery polymer is cationic. In another aspect of the invention, the delivery polymer comprises phosphonium ions and/or ammonium ions. In another example of the invention, the N-L-Z conjugate is combined with a delivery polymer, and the composition forms nanoparticles in solution. In a further embodiment, nanoparticle polyplexes are stable in serum and have a size in the range of about 30 nm - 5000 nm in diameter. In one embodiment, the particles are less than about 300 nm in diameter. For example, the nanoparticles are less than about 150 nm in diameter.
[0031] In one embodiment, the delivery vehicle comprises a cationic block copolymer comprising phosphonium or ammonium ionic groups as described in PCT/US 12/42974.
In one embodiment, the polymer is diblock-/Wj'[(ethylene glycol)9 methyl ethyl methacralate][stirylphosphonium]. In another embodiment of the invention, the delivery polymer comprises glycoamidoamines as described in Tranter et al. Amer Soc Gene Cell Ther , Dec 2011; polyhydroxylamidoamines, dendritic macromolecules, carbohydrate- containing polyesters, as described in US20090105115; and US20090124534. In other embodiments of the invention, the nucleic acid delivery vehicle comprises a cationic polypeptide or cationic lipid. An example of a cationic polypeptide is polylysine. See U.S. Pat. 5,521,291.
[0032] In one embodiment, the N-L-Z conjugate is part of a composition comprising delivery or carrier polymers. In another embodiment, the N-L-Z conjugate is part of nanoparticle polyplexes capable of transporting molecules with stability in serum. The polyplex compositions comprise a synthetic delivery polymer (carrier polymer) and biologically active compound associated with one another in the form of particles having an average diameter of less than about 500 nm, such as about 300 nm, or about 200 nm, preferably less than about 150 nm, such as less than about 100 nm. The invention encompasses particles in the range of about 40 nm - 500 nm in diameter.
[0033] In one embodiment, the delivery or carrier polymer comprises a cationic block copolymer containing phosphonium or ammonium ionic groups as described in
PCT/US 12/42974. In another embodiment of the invention, the delivery or carrier polymer comprises glycoamidoamines as described in Tranter et al. Amer Soc Gene Cell Ther , Dec 2011; polyhydroxylamidoamines, dendritic macromolecules, carbohydrate- containing polyesters, as described in US20090105115; and US20090124534. The polyglycoamidoamine (PGAA) polymer system, which is a proprietary, localized and biodegradable nanoparticle system, represents another delivery or carrier polymer.
Poly(galactaramidoamine) is an efficient cationic polymeric vehicle with low cytotoxicity (Wongrakpanich et al. Pharmaceutical Development and Technology , January 12, 2012). The nanoparticle delivery system disclosed in Hemp et al. Biomacromolecules , 2012 13:2439-45 represents another delivery or carrier polymer useful in the present invention.
[0034] In other embodiments of the invention, the delivery or carrier polymer comprises a cationic polypeptide or cationic lipid. Polymers, such as /w/j'-L-lysine (PLL), polye thyleneimine (PEI), chitosan, and their derivatives are also encompassed by the invention. Nucleic acid delivery using these compounds relies on complexation driven by electrostatic interactions between the gene and the polycationic delivery agent. Polymer- DNA complexes condense into particles on the order of 60 nm - 120 nm in diameter. Polymers such as linear PEI and PLL have high transfection rates in a variety of cells.
[0035] In vivo nucleic acid delivery has size constraints requiring a sufficiently small polyplex to enable long circulation times and cellular uptake. In addition, polyplexes must resist salt- and serum-induced aggregation. Serum stability is generally associated with a particle size of about sub-l50 nm hydrodynamic radius or below maintainable for 24 h. The nanoparticles of the invention, which comprise nucleic acid therapeutic and delivery polymer, have the hydrodynamic radius and material properties for serum stability. In particular, the delivery polymer, when combined with the nucleic acid, protects the therapeutic cargo under physiological conditions. The delivery polymers are designed to have characteristics of spontaneous self-assembly into nanoparticles when combined with polynucleotides in solution.
[0036] The invention also contemplates other delivery polymers that form serum-stable nanoparticles. The invention is not limited to the type of delivery polymer and may be adaptable to nucleic acid characteristics, such as length, composition, charge, and presence of coupled peptide. The delivery polymer may also be adaptable for material properties of the resultant nanoparticle, such as hydrodynamic radius, stability in the host bloodstream, toxicity to the host, and ability to release cargo inside a host cell.
[0037] In one embodiment, the N-L-Z conjugate is administered in the form of a salt.
The salt may be any pharmaceutically acceptable salt comprising an acid or base addition salt. Examples of pharmaceutically acceptable salts with acids include those formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, hydroiodic acid, hydrofluoric acid, phosphorous acid, and the like. Also included are salts that are formed with organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, alkanedioic acids, aromatic acids, aliphatic and. aromatic sulfonic acids, etc. and include, for example, acetic acid, trifluoroacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like. Exemplary salts thus include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates,
monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, trifluoroacetates, propionates, caprylates, isobutyrates, oxalates, malonates, succinate suberates, sebacates, fumarates, maleates, mandelates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, phthalates, benzenesulfonates, toluenesulfonates, phenylacetates, citrates, lactates, malates, tartrates, methanesulfonates, and the like. Also contemplated are salts of amino acids, such as arginates, gluconates, and galacturonates (see, for example, Berge S. M. et al.,
"Pharmaceutical Salts," Journal of Pharmaceutical Science, 66: 1-19 (1997). Acid addition salts of basic molecules may be prepared by contacting the free base forms with a sufficient amount of the desired acid to produce the salt according to methods and techniques with which a skilled artisan is familiar.
[0038] Pharmaceutically acceptable base addition salts are formed by addition of an
inorganic base or an organic base to the free acid. Pharmaceutically acceptable base addition salts may be formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, for example, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, 2-dimethylaminoethanol, 2- diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, N,N-dibenzylethylenediamine, chloroprocaine, hydrabamine, choline, betaine, ethylenediamine, ethylenedianiline, N-methylglucamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like.
[0039] In one embodiment, the N-L-Z conjugate is administered as part of a
pharmaceutical composition comprising a pharmaceutically acceptable diluent, excipient or carrier. Suitable diluents, excipients and carriers are well known in the art and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gernnaro Ed., 1985). The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form must be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, saline, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
[0040] Sterile injectable solutions are prepared by incorporating the N-L-Z conjugate in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.
[0041] In one embodiment, the composition comprising the N-L-Z conjugate is in contact with a fabric. The fabric may comprise natural fibers, synthetic fibers, or both. Examples of textile fabrics include, but are not limited to, nylon, cotton, nylon-cotton blends, wool, silk, linen, polyester, rayon, and worsted. In one particular embodiment of the invention, the fabric is cotton. In another embodiment, the fabric is nylon. In another embodiment, the fabric is a nylon-cotton blend. The ratio of nylon to cotton in the nylon-cotton blend fabric can be between about 1 :99 and about 99: 1, between about 10:90 and about 90:10, between about 20:80 and about 80:20, between about 30:70 and about 70:30, between about 40:60 and about 60:40, and between about 45:55 and about 55:45. In a preferred embodiment, the fabric is a 50:50 nylon-cotton blend. - lO -
[0042] In another embodiment of the invention, the fabric has a high tensile strength-to- weight ratio. In one embodiment, the fabric with a high tensile-to-weight ratio is a fabric comprising aramid fibers. In a particular embodiment, the aramid fiber is a para-aramid fiber (e.g., the para-aramid fiber commercially known as KEVLAR). In another particular embodiment, the aramid fiber is a meta-aramid fiber (e.g., the meta-aramid fiber commercially known as NOMEX).
[0043] In certain embodiments, the antimicrobial fabric is capable of treating a P.
aeruginosa, A. baumannii or K. pneumoniae infection or inhibiting growth of P.
aeruginosa, A. baumannii or K. pneumoniae after the fabric has been washed. In some embodiments, the antimicrobial fabric is capable of treating a P. aeruginosa, A.
baumannii or K. pneumoniae infection or inhibiting growth of a P. aeruginosa, A.
baumannii or K. pneumoniae after between about 10 and about 60 wash cycles, between about 20 and about 50 wash cycles, between about 20 and about 40 wash cycles, between about 20 and about 30 wash cycles, and between about 20 and about 25 wash cycles. In another embodiment, the duration of a wash cycle is between about 10 minutes and about 90 minutes, between about 10 minutes and about75 minutes, between about 10 minutes and about 60 minutes, between about 10 minutes and about 45 minutes, between about 10 minutes and about 30 minutes, and between about 10 minutes and about 15 minutes. In another embodiment, the water temperature in the wash cycles is between about l6°C and about 60°C, between about 27°C and about 49°C, or between about 37° and about 44°C. In one particular embodiment, the antimicrobial fabric is capable of treating a P.
aeruginosa, A. baumannii or K. pneumoniae infection or inhibiting growth of P.
aeruginosa, A. baumannii or K. pneumoniae following Laundry Test Method AATCC 147 from American Association of Textile Chemists and Colorists (AATCC).
[0044] In another embodiment, provided is a composition comprising the N-L-Z
conjugate. The composition may be in the form of solution that can be applied to a fabric, e.g., by rinsing, dipping, or spraying. The fabric can be an antimicrobial fabric or a non-antimicrobial fabric. In one embodiment, application of the solution to the fabric provides a fabric that is capable of treating a P. aeruginosa, A. baumannii or K.
pneumoniae infection or inhibiting growth of P. aeruginosa, A. baumannii or K.
pneumoniae. In other embodiments, application of the solution to the fabric increases the fabric's capability of treating a P. aeruginosa, A. baumannii or K. pneumoniae infection or inhibiting growth of P. aeruginosa, A. baumannii or K. pneumoniae. In a particular embodiment, application of the solution to an antimicrobial fabric with low antimicrobial activity increases the antimicrobial activity of the fabric.
[0045] In other embodiments of the invention, provide is a wound healing dressing
comprising the N-L-Z conjugate. In one embodiment, the wound healing dressing is an adhesive dressing. In another embodiment, the wound healing dressing is a non-adhesive dressing. In one embodiment, the dressing comprises a foam, gel, or cream. In another embodiment, the dressing comprises a fiber based material ( e.g. , gauzes or waddings). In one embodiment, the fiber-based material is cotton. In another embodiment, the fiber- based material is rayon. In another embodiment, the fiber-based material is a gel-forming fiber, such as a carboxymethylated cellulosic material. In another embodiment, the fiber- based material is a synthetic polymer. In another embodiment, the wound healing dressing is THERAGAUZE (Soluble Systems, LLC, Newport News, VA).
[0046] The invention also provides a method of treating P. aeruginosa, A. baumannii or
K. pneumoniae infection and a method of inhibiting the growth of P. aeruginosa, A.
baumannii or K. pneumoniae. In one embodiment, the animal undergoing treatment for P. aeruginosa, A. baumannii or K. pneumoniae infection exhibits one or more symptoms of P. aeruginosa, A. baumannii or K. pneumoniae infection including fever and chills, body aches, light-headedness, rapid pulse and breathing, nausea and vomiting, diarrhea, (P. aeruginosa), infections of the lungs, blood, brain, urinary track, and wound infections (A. baumannii), fever, confusion, neck stiffness, sensitivity to light, bloodstream infection, fever, chills, rash, light-headedness, altered mental state {K. pneumonia) as well as puss production in the infected area, acne, boils, abscesses, carbuncles, stys, cellulitis, diarrhea, botulism, and gas gangrene. The animal may also exhibit signs of sepsis or pneumonia.
[0047] In one embodiment, the N-L-Z conjugate is administered by intravenous injection.
In another embodiment, the N-L-Z conjugate is administered by intramuscular injection. In another embodiment, the N-L-Z conjugate is administered by peritoneal injection. In another embodiment, the N-L-Z conjugate is administered topically, e.g. to a tissue suspected to be infected by P. aeruginosa, A. baumannii or K. pneumoniae. In another embodiment, the N-L-Z conjugate is administered orally. When administered orally, the N-L-Z conjugate may be formulated as part of a pharmaceutical composition coated with an enteric coating that will protect the N-L-Z conjugate from the acid environment of the stomach and release the N-L-Z conjugate in the upper gastrointestinal tract. In another embodiment, the N-L-Z conjugate may be formulated as part of a sustained release formulation that will release the N-L-Z conjugate on a substantially continuous basis over a period of time.
[0048] Animals that may be treated with the N-L-Z conjugate according to the invention include any animal that may benefit from treatment with the N-L-Z conjugate. Such animals include mammals such as humans, dogs, cats, cattle, horses, pigs, sheep, goats and the like.
[0049] The N-L-Z conjugate is administered in an amount that is effective for the
treatment of P. aeruginosa, A. baumannii or K. pneumoniae infection or inhibition of the growth of P. aeruginosa, A. baumannii or K. pneumoniae. The amount may vary widely depending on the mode of administration, the species of P. aeruginosa, A. baumannii or K. pneumoniae , the age of the animal, the weight of the animal, and the surface area of the animal. The amount of N-L-Z conjugate, salt and/or complex thereof may range anywhere from 1 pmol/kg to 1 mmol/kg. In another embodiment, the amount may range from 1 nmol/kg to 10 mmol/kg. When administered topically, the amount of N-L-Z conjugate, salt and/or complex thereof may range anywhere from 1 to 99 weight percent. In another embodiment, the amount of N-L-Z conjugate, salt and/or complex thereof may range anywhere from 1 to 10 weight percent.
[0050] The invention also provides N-L-Z conjugates comprising a linker. In one
embodiment, N is an antisense molecule that inhibits the growth of a bacterium comprising a polynucleotide sequence that is antisense to the coding region of a bacterial protein and hybridizes to the coding region under physiological conditions; L is a linker having the formula (Y')„, where each Y' is independently glycine, cysteine, 8-amino-3,6- dioxaoctanoic acid (AEEA), or 5-amino-3-oxapentanoic acid (AEA), and n is an integer from 1 to 10; and Z is a cell penetrating molecule. In some aspects, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, L is a bond.
[0051] In some aspects, the cell penetrating molecule Z has the formula: (ABC)P-D, wherein A is a cationic amino acid which is Lysine or Arginine; B and C are hydrophobic amino acids which may be the same or different and are selected from the group consisting of Valine, Leucine, Isoleucine, Tyrosine, Phenylalanine, and Tryptophan; p is an integer with a minimal value of 2; and D is a cationic amino acid or is absent. In one embodiment, A is Lysine, B is Phenylalanine, C is Phenylalanine, D is Lysine, and p is 3. In another embodiment, is 2-10. In another embodiment, / is 2, 3, 4, 5, 6, 7, 8, 9 or 10.
[0052] In other aspects, the cell penetrating molecule Z has the formula: B-XI-(R-X2-R)4, where B is beta-alanine or is absent, Xi is 6-amino-hexanoic acid or is absent, X2 is 6- amino-hexanoic acid, and R is arginine or homo-arginine. In some embodiments, R is arginine, selected from the group consisting of L-arginine and D-arginine.
[0053] In some aspects, the cell penetrating molecule is a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-11 (See Table 1).
Table 1 : Cell Penetrating Peptides
Figure imgf000015_0001
[0054] In some embodiments, N comprises a modified backbone. In a particular
embodiment, the modified backbone is a PNA backbone.
[0055] Modified nucleic acids are non-natural polymers that hybridize to natural DNA and RNA with sequence specificity according to Watson-Crick base paring rules.
Examples of modified nucleic acids are phosphorothioate-oligodeoxynucleotides (PS- ODNs), locked nucleic acids (LNAs), 2'-0-methyloligoribonucleotides (2'O-Mes), phosphorodiamidate morpholino oligonucleotides (PMOs), and peptide nucleic acids (PNAs). Modified nucleic acids have modified backbones and are generally more resistant to degradation than natural nucleic acids. The invention includes any type of synthetically-modified DNA or RNA that hybridizes to natural DNA and RNA. See, e.g.. U.S. Pat. Nos. 5,116,195, 5,539,082, 5,527,675, 5,623,049, 5,714,331, 5,736,336, 5,773,571, 5,786,461, 5,811,232, 5,837,459, 5,874,564, 5,891,625, 5,972,610, 5,986,053,
6,107,470, 6,174,870, 7,098,192, 7,696,345, 8,124,745, 8,354,093, 8,357,664, Wagner et ah, Nucl. Acid Res. 79:5965-71 (1991); and Koshkin el at., Tetrahedron 54 :3607 -30 (1998).
[0056] Antisense molecules of the invention may also be composed of non-natural
polymers that hybridize to natural nucleic acids. Atypical nucleoside bases may also be employed, such as methylated bases, phosphorylated bases, inosine, thiouridine, pseudouridine, dihydrouridine, queuosine, and wyosine, among others. Examples of such antisense polymers comprising atypical bases are disclosed in U.S. Pat. Nos. 7,875,733, 7,919,612, 7,939,677, 8,314,229, 8,372,969, and 8,377,898.
[0057] The term antisense polynucleotide refers to a nucleic acid molecule that is
complementary to at least a portion of a target nucleotide sequence of interest and hybridizes to the target nucleotide sequence under physiological conditions. Antisense molecules specifically hybridize with one or more nucleic acids encoding a preselected target nucleic acid. The terms target nucleic acid and nucleic acid encoding the target encompass DNA encoding the target, RNA (including pre-mRNA and mRNA) transcribed from such DNA, and also cDNA derived from such RNA. The hybridization of an antisense compound with its target nucleic acid interferes with the normal function of the nucleic acid. This modulation of function of a target nucleic acid by compounds which specifically hybridize to it is generally referred to as antisense. The functions of DNA to be interfered with include replication and transcription. The functions of RNA to be interfered with include all vital functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in or facilitated by the RNA. The overall effect of such interference with target nucleic acid function is modulation of the expression of the target. In the context of the present invention, modulation means either an increase (stimulation) or a decrease (inhibition) in the expression of a gene. In the context of the present invention, inhibition is the form of modulation of gene expression. [0058] Polynucleotides are described as complementary to one another when hybridization occurs in an antiparallel configuration between two single-stranded polynucleotides.
[0059] The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity and similarity between two sequences can be accomplished using a mathematical algorithm (see e.g., Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). In a preferred embodiment, the percent identity between two sequences is determined based on alignments generated with the Clustal W algorithm (Thompson, J. D. et al, 1994, Nucleic acids Res. 22:4673-4680). This algorithm is incorporated into many commercial software packages, in this case the alignX software program in the Vector NTI suite (version 8.0). Default Clustal W parameters were used to generate pairwise alignments from which percent identity values were calculated (gap opening penalty of 10; gap extension penalty of 0.1). The percent identity is defined as the number of identical bases divided by the total number of bases and multiplied by 100. If sequences in the alignment are of different lengths (due to gaps or extensions), the length of the longest sequence will be used in the calculation, representing the value for total length.
[0060] Proteins are polymers containing one or more chains of amino acids bonded
together by peptide bonds. Proteins typically fold into a three dimensional form, facilitating a biological function.
[0061] A polypeptide is a polymer of amino acids bonded together by peptide bonds. The terms protein and polypeptide and peptide are generally used interchangeably, although polypeptides and peptides are generally shorter in length than proteins.
[0062] The invention provides the following specific N-L-Zs: PPNA for Pseudomonas aeruginosa
[0063] PPNA 23 X- gcc ate age cgt get (SEQ ID NO: l2)-AEEA-AEA-B-RIR FKK AKK
LFK RIR (SEQ ID NO: 13)
PPNA for Acinetobacter baumannii
[0064] PPNA 40 - ett tac gca tga gag (SEQ ID NO: l4)-AEEA-AEA-BX-RXR-RXR-
RXR-RXR (SEQ ID NO: 15)
PPNAs for Klebsiella pneumoniae
[0065] PPNA Kl - tec att gat tet gtt (SEQ ID NO: 16)- AEEA-AEA-RIR FKK AKK LFK
RIR (SEQ ID NO: 17)
[0066] PPNA K2 - tee ata gtg tta cct (SEQ ID NO: 18)- AEEA- AEA- RRWYRWWRR
(SEQ ID NO: 19)
wherein:
a-Adenine
g-Guanine
t-Thymine
c-Cytosine
AEEA- 8 - Amino-3 ,6-di oxaoctanoi c aci d
AEA- 5-amino-3-oxapentanoic acid
B- beta-alanine
R- L- Arginine
I-L-Isoleucine
F- L-phenylaalanine
K- L-Lysine
A-L- Alanine
L- L-Leucine
X- 6-amino-hexanoic acid
W- L-Tryptophan
Y-L-Tyrosine. EXAMPLES
Synthesis of PPNAs
[0067] PPNAs were synthesized by following Merrifield Solid Phase Peptide Synthesis using AAPPTEC automated peptide synthesizers. Each compound was synthesized at a O. lmicromolar concentration using Rink-Amide resin in a 50 ml reaction vessel. The Rink-amide resins were deprotected by 20% piperidine in N-Methyl-2-pyrrolidone (NMP). Resins were washed with NMP for 7 times with 2 mins mixing. Three equimolar concentration of Fmoc-amino acids/Fmoc-PNAs were mixed with 2.85 equimolar concentrations of l-Cyano-2-ethoxy-2-(oxoethylidenaminooxy) dimethylamino- morpholino- carbenium hexafluorophosphate in presence of NMP for 1 min and added to the deprotected resins with further addition of 0.3M N,N-Diisopropylethylamine (DIEA) and 0.3M of 2,6 Lutidine for coupling of Fmoc-amino acids/Fmoc-PNAs. Coupling of Fmoc-amino acids/Fmoc-PNAs was performed for 60 mins with continuous shaking and intermittent argon gas bubbling. After coupling, resins were washed four times with NMP with 2 mins mixing. The growing amino acids/PNAs on resins were capped with 1.5 M Acetic anhydride for 30 mins followed by 5 times washing of resins with NMP with 2 mins of shaking. The process of deprotection, coupling, and capping steps repeated till the end of synthesis of compound. After final capping of amino acid/PNA onto growing resins, the final product was deprotected from resins. The crude product was cleaved from resin by 95% trifluoroacetic acid, 2.5% TIS and 2.5 water for 4 hrs at 37°C. The cleavage product was precipitated in 5 volumes of cold ether and the precipitated compound was collected by centrifugation. The ether precipitated compound was air dried for purification.
Purification of the PPNAs.
[0068] The air-dried crude compounds were solubilized in 0.1% TFA in HPLC water.
The compounds were purified in Waters Prep-l50 system. Thirty milligrams of compound was loaded in X-Bridge C18 columns (lOmm X 250mm) with a flow rate of mobile phase 5.5 ml/min. Mobile phase conditions are as follows:
Figure imgf000019_0001
Figure imgf000020_0001
[0069] The purified fractions were lyophilized and converted to HC1 salt or acetate salts.
The HC1 salts of the compounds were prepared by addition of lOmM HC1 solution into lyophilized compound. The solution was flash frozen and further lyophilized to collect the final compound. Acetate salt of the compounds were converted by passing through the HPLC columns in a 1% acetic acid-water and acetonitrile mobile phase. The purified fractions were lyophilized to collect the acetate salts of the compounds. A small fraction of the compound was used to run in an analytical HPLC column to determine the purity of the compound. The purity of all compounds were >95% (Data not shown). The HCl/acetate salt of compounds were screened for the antimicrobial activities against bacterial strains.
Assays to determine the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) PPNAs against corresponding pathogens.
[0070] Bioscreen-C instrument was used to detect the MIC and MBC of PPNAs against corresponding pathogens. Different concentration of PPNAs were prepared in lOmM of sodium bicarbonate buffer pH-7.4 and added to ~5.0 LoglO CFUs in a Honey comb Bioscreen-C plate. The plates were incubated in Bioscreen C instrument and growth of bacteria was observed in every 5 mins by measuring the optical density at 420-580 nm with intermittent shaking.
[0071] PPNA 23X used against Pseudomonas aeruginosa strains from American Type
Culture Collections (ATCC) (ATCC-47085, ATCC-27853) and clinical isolates from Center for Disease Control (CDC).
[0072] PPNA 40 used against Acinetobacter baumannii strain from ATCC (ATCC-BAA-
1605) and clinical isolates from CDC.
[0073] PPNA Kl and PPNA K2 used against Klebsiella pneumoniae strain from ATCC
(ATCC-700603) and clinical isolates from CDC. Table 2: MIC and MBC of PPNA 23X against P. aeruginosa strains/isolates.
Figure imgf000021_0001
*AR- Antimicrobial resistance
Table 3: MIC and MBC of PPNA 40 against A. baumannii strains/isolates
Figure imgf000022_0001
Figure imgf000023_0001
*AR- Antimicrobial resistance
Table 4: MIC and MBC of PPNA Kl and PPNA K2
against Klebsiella pneumoniae strains or isolates
Figure imgf000023_0002
Resistance study:
[0074] A long-term resistance study was performed to determine the emergence of
resistance colonies against PPNAs. PPNA 23X and PPNA 40 were used against P.
aeruginosa (ATCC-47085, CDC#242) and A. baumannii (ATCC-BAA-1605, CDC#274) strains in resistance study, respectively. Colistin was used as the positive control for this assay. P. aeruginosa and A. baumannii strains were grown in Tryptic soy broth (TSB) overnight and optical density was adjusted to OD600=l.0. The culture was further diluted in 2X Mueller Hinton broth (MHB) to obtain 5X106 CFU/mL. The MIC assay was performed by following the Clinical Laboratory Standard Institute (CLSI) protocols. The diluted cultures were exposed to different concentrations of PPNAs (0 -100 pg/mL) and colistin (0-100 pg/mL) and incubated at 35°C overnight. The MIC of PPNAs and colistin against P. aeruginosa and A. baumannii were recorded. The culture of P. aeruginosa and A. baumannii grown at highest concentration of PPNAs or colistin during MIC assay were collected, added 9-fold more TSB and allowed to grow further overnight. The MIC assay was repeated as above. This process was continued for 30 cycles and MIC value of PPNAs and colistin in each cycle were recorded.
[0075] At the end of 30 cycles, the cultures were passaged twenty (20) more times
without PPNA or colistin in TSB to evaluate the emergence of persister cells. At the end of each passage, a fraction of culture was used for glycerol stock for future studies.
MIC of Single Intravenous Dose Administration to Determine the Maximum
Tolerability Dose (MTD) of PPNA 40 in Mice.
[0076] Single ascending intravenous (IV) dose study was performed to determine
tolerability of the drug in mice.
[0077] Initially, PPNA 40 was administered with a 10 mg/kg intravenous bolus dose using a 10 mL/kg dose volume (Ό.2 mL) and observed the effects before proceeding to the next higher dose. Mice were well tolerated at 10 mg/kg intravenous administration of both the drugs. However, higher concentration (>l5mg/kg) had shown adverse effect and death in mice within 30 mins of administration of the drug.
Murine Neutropenic Thigh Infection Model to Determine the Antimicrobial Efficacy of PPNA 40 Against A. baumannii strains (UNT191.1 and UNT237.1)
[0078] The MIC of PPNA 40 was determined in an additional fifteen (15) A. baumannii clinical isolates, in order to down-select the A. baumannii strains for use in an efficacy study in mice. Based on MIC assay, UNT191.1 and UNT237.1 strains were selected for efficacy studies in mice. UNT191.1 and UNT237.1 were susceptible to tigecycline and colistin, and the MIC of PPNA 40 against UNT191.1 and UNT237.1 was 2pg/mL. [0079] Female 5-6 week old CD-l (18-22 gm) were used in this study. Mice were quarantined for 48 hours before use and housed in groups of 5 with free access to food and water during the study.
[0080] The animals were made neutropenic by administration of cyclophosphamide on
Days -4 and -1. On Days -4 150 mg/kg of cyclohsphamide administered by
intraperitoneal route and 100 mg/kg was administered on Days -1. Days listed are referenced from the date of infection (study day -Day 0).
[0081] On day 0, mice were inoculated intramuscularly into right thigh with 106
CFU/mouse of A. baumannii isolates. Three groups of mice were administered with PPNA 40 (at +1, +9, or +l7hr) and one group with tigecy cline (at +1 & +9hr) via IV route. Mice were euthanized by C02 inhalation at +1 and +24hrs post-infection and thighs were removed, and placed in 2 ml of sterile PBS, homogenized, serially diluted and plated to determine the CFU counts. Plates were incubated 18-24 hours and CFUs were counted.
[0082] Colony were counted and the number of colonies is converted to CFU/thigh by multiplying the number of colonies by the volume of the thigh homogenate spotted and the dilution at which the colonies were counted (5-50 colonies/spot). All count data were transformed into log 10 CFU/thigh for calculation of means and standard deviations.
Murine Neutropenic lung Infection Model to Determine the Antimicrobial Efficacy of PPNA 40 Against A. baumannii strains (UNT191.1 and UNT237.1)
[0083] Female 5-6 week old CD-l (18-22 gm) were used in this study. Mice were
quarantined for 48 hours before use and housed in groups of 5 with free access to food and water during the study.
[0084] The animals were made neutropenic by administration of cyclophosphamide on
Days -4 and -1. On Days -4 150 mg/kg of cyclohsphamide administered by
intraperitoneal route and 100 mg/kg was administered on Days -1. Days listed are referenced from the date of infection (study day -Day 0).
[0085] On day 0, animals were inoculated via intranasal route with 108 CFU/mouse of A. baumannii strains (UNT191.1 and UNT237.1) into each nares. Three groups of mice were administered with PPNA 40 (at +1, +9, or +l7hr) and one group with tigecy cline (at +1 & +9hr). Mice were euthanized by C02 inhalation at +2 and +24hrs post-infection and, lungs were collected, and placed in 2 ml of sterile PBS, homogenized, serially diluted and plated to determine the CFU counts. Plates were incubated 18-24 hours and CFUs were counted.
[0086] Colony were counted and the number of colonies is converted to CFU/pair of lungs by multiplying the number of colonies by the volume of the lungs homogenate spotted and the dilution at which the colonies were counted (5-50 colonies/spot). All count data were transformed into log 10 CFU/pair of lungs for calculation of means and standard deviations.

Claims

WHAT IS CLAIMED IS:
1. A compound having the formula:
N— L— Z,
or pharmaceutically acceptable salt thereof, wherein
N is an antisense molecule that inhibits the growth of the bacteria P. aeruginosa,
A. baumannii, or K. pneumoniae comprising a polynucleotide sequence that is antisense to the coding region of a P. aeruginosa, A. baumannii, or K. pneumoniae protein and hybridizes to the coding region under physiological conditions;
L is a linker or a bond; and
Z is a cell penetrating molecule.
2. The compound of claim 1, wherein the bacteria is P. aeruginosa and N is at least 85% identical to gcc ate age cgt get (SEQ ID NO: 12).
3. The compound according to claim 2, wherein the compound is gcc ate age cgt get (SEQ ID NO: 12)- AEE A- AE A-B -RIR FKK AKK LFK RIR (SEQ ID NO: 13), wherein
AEEA is 8-Amino-3,6-dioxaoctanoic acid, and
AEA is 5-amino-3-oxapentanoic acid, and in the cell penetrating molecule:
B is beta-alanine,
R is L- Arginine,
I is L-Isoleucine,
F is L-phenylaalanine,
K is L-Lysine,
A is L-Alanine, and
L is L-Leucine.
4. The compound of claim 1, wherein the bacteria is A. baumannii and N is at least 85% identical to ett tac gca tga gag (SEQ ID NO: 14).
5. The compound of claim 4, wherein the compound is ett tac gca tga gag (SEQ ID NO: 14)- AEEA-AEA-BX-RXR-RXR-RXR-RXR (SEQ ID NO: 15), wherein AEEA is 8-Amino-3,6-dioxaoctanoic acid,
AEA is 5-amino-3-oxapentanoic acid,
B is beta-alanine,
R is L- Arginine, and
X is 6-amino-hexanoic acid.
6. The compound of claim 1, wherein the bacteria is K. pneumoniae and N is at least 85% identical to tcc att gat tct gtt (SEQ ID NO: 16).
7. The compound of claim 6, wherein the compound is tcc att gat tct gtt (SEQ ID NO: 16)- AEEA-AEA-RIR FKK AKK LFK RIR (SEQ ID NO: 17), wherein
AEEA is 8-Amino-3,6-dioxaoctanoic acid, and
AEA is 5-amino-3-oxapentanoic acid, and in the cell penetrating molecule:
R is L- Arginine,
I is L-Isoleucine,
F is L-phenylaalanine,
K is L-Lysine,
A is L-Alanine, and
L is L-Leucine.
8. The compound of claim 1, wherein the bacteria is K. pneumoniae and N is at least 85% identical to tcc ata gtg tta cct (SEQ ID NO: 18).
9. The compounds of claim 8, wherein the compound is tcc ata gtg tta cct (SEQ ID NO: 18)- AEEA-AEA- RRWYRWWRR (SEQ ID NO: 19), wherein
AEEA is 8-Amino-3,6-dioxaoctanoic acid,
AEA is 5-amino-3-oxapentanoic acid,
R is L- Arginine,
X is 6-amino-hexanoic acid,
W is L-Tryptophan, and
Y is L-Tyrosine.
10. The compound of any one of claims 1-9, which is a PNA.
11. The compound of claim 1, wherein the compound has the structure as set forth in Fig. 11.
12. The compound of claim 1, wherein the compound has the structure as set forth in Fig. 12.
13. The compound of claim 1, wherein the compound has the structure as set forth in Fig. 13.
14. The compound of claim 1, wherein the compound has the structure as set forth in Fig. 14.
15. A pharmaceutical composition comprising the compound of any one of claims 1 to 14 and a pharmaceutically acceptable carrier.
16. A method of treating a bacterial infection, comprising administering to an animal in need thereof an effective amount of the compound of any one of claims 1 to 14 or
pharmaceutical composition of claim 15.
17. The method of claim 16, wherein the bacteria is P. aeruginosa and the compound is gcc ate age cgt get (SEQ ID NO: 12)-AEEA-AEA-B-RIR FKK AKK LFK RIR (SEQ ID NO: 13).
18. The method of claim 16, wherein the bacteria is A. baumannii and the compound is ett tac gca tga gag-AEEA-AEA-BX-RXR-RXR-RXR-RXR.
19. The method of claim 16, wherein the bacteria is K. pneumoniae and the compound is is tcc att gat tet gtt- AEEA-AEA-RIR FKK AKK LFK RIR.
20. The method of claim 16, wherein the bacteria is K. pneumoniae and the compound is tcc ata gtg tta cct-AEEA-AEA- RRWYRWWRR.
PCT/US2019/034408 2018-05-29 2019-05-29 Antisense oligonucleotides for the treatment of p. aeruginsoa, a. baumannii and k. pneumoniae infections Ceased WO2019232059A1 (en)

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