Classifications

• • 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
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/438—The ring being spiro-condensed with carbocyclic or heterocyclic ring systems
• • 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
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/439—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom the ring forming part of a bridged ring system, e.g. quinuclidine
• • 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
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0043—Nose
• • 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
  • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
• • 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
  • A61K9/007—Pulmonary tract; Aromatherapy
  • A61K9/0073—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy
• • 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
  • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
• • 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
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/007—Pulmonary tract; Aromatherapy
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• the present invention discloses compositions and methods for the activation of the A. baumannii ferric-coprogen (FhuE) receptor and the use of high systemic exposures of rifabutin (Cmax and AUC) that are effective for treating A. baumannii infection.
• FhuE ferric-coprogen
• Cmax and AUC rifabutin
• MDR multi-drug resistant
• XDR extensively-drug resistant
• IV administration is the only method of delivery sufficient to provide antibiotic treatment.
• most antibiotics have not been formulated for IV administration because they are difficult or impossible to formulate into soluble forms.
• Rifabutin (also known as LM 427 and Mycobutin®) is a spiro-piperidyl-rifamycin derived from rifamycin-S. Mycobutin® was approved by FDA as an oral formulation in 1992. Mycobutin® (150 mg capsules) capsules are indicated for the prevention of disseminated Mycobacterium avium complex (MAC) disease in patients with advanced HIV infection. Rifabutin is widely known to have low outer membrane permeability against Gram-negative pathogens, thus limiting its cellular uptake into those life-threatening ESKAPE pathogens. As such, formulations or alternative routes of administration of rifabutin have not been fully explored, a situation that is compounded by its poor solubility.
• MAC Mycobacterium avium complex
• rifamycin molecules such as rifampin (also known as rifampicin)
• AUC/MIC concentration time curve-to-MIC ratio
• Cmax/MIC free peak concentration-to-MIC ratio
• the post-antibiotic effect duration was also most closely related to the Cmax/MIC ratio (see Gumbo, 2007, Concentration-dependent Mycobacterium tuberculosis killing and prevention of resistance by rifampin, Antimicrobial Agents and Chemotherapy, 51(ll):3781-3788, incorporated by reference).
• rifabutin product monograph In healthy adult volunteers a nominal therapeutic oral dose of 300 mg rifabutin produces a mean Cmax of 0.375 mg/L which is attained at approximately 3 hours after oral administration (rifabutin product monograph).
• the PK of rifabutin is linear after single administration of 300, 450 and 600 mg PO to healthy volunteers with a Cmax in the range of 0.4 to 0.7 mg/L (rifabutin product monograph).
• rifabutin Since rifabutin is approximately 90% protein bound the free drug concentrations after oral administration are very low. In healthy adult volunteers at least 53% of the oral dose is absorbed whereas the absolute bioavailability assessed in HIV positive patients in a multiple dose study was 20% on day 1 and 12% on day 28.
• the oral administration of rifabutin to patients with such infections will likely lead to rapid development of resistance.
• the invention provides systems and methods for increased clinical efficacy of rifabutin against A. baumannii.
• the discovery by the inventors of a novel mechanism of action has resulted in the ability of rifabutin to maintain significant activity against A. baumannii , while having only minimal activity against other Gram-negative pathogens.
• the invention takes advantage of the discovery of a ferric-coprogen (FhuE) receptor that is responsible for the uptake of rifabutin, but not of the closely related drug rifampin, into A. baumannii cells.
• FhuE ferric-coprogen
• the invention provides high AUC and Cmax of administered rifabutin, which allow for increased clinical efficacy via activation of the FhuE receptor and, at the same time, reducing the likelihood of resistance - a significant problem in standard oral formulations of rifabutin and related drugs.
• the inventors discovered that high exposures and in particular Cmax cannot be achieved by the currently available oral route of administration but can be achieved by intravenous injection or by methods known in the art for modified oral released or by inhalation if locally high Cmax and AUC are required.
• rifabutin liquid formulations are provided for intravenous (IV) administration, the formulations being effective against Acinetobacter baumannii.
• Rifabutin liquid formulations may exploit siderophores that modulate a ferric-coprogen (FhuE) receptor in bacterial cell membranes to transport rifabutin across bacteria cells membranes.
• FhuE ferric-coprogen
• Formulations of rifabutin disclosed herein are useful for treating bacterial infections where oral administration of an antimicrobial is impractical or impossible.
• the formulations and methods of the present invention provide the medical community with novel treatments for patients incapacitated by life-threatening bacterial infections.
• rifabutin uses the presence of siderophores that modulate TonB- dependent siderophore receptors in bacteria cells.
• the TonB-dependent siderophore receptors may be ferric-coprogen (FhuE) receptors.
• a preferred siderophore is one that modulates a bacterial TonB-dependent siderophore receptor.
• a preferred siderophore may be any iron chelator that mediates a TonB-dependent siderophore receptor's uptake of rifabutin.
• the siderophore may be an apotransferrin or may be a transferrin.
• the siderophore may be loaded with an iron complex.
• the siderophore that is iron-loaded may be a transferrin.
• Rifabutin may use the presence of siderophores to modulate a TonB-dependent siderophore receptor of Acinetobacter baumannii.
• methods of the invention are based on the recognition that FhuE receptor activation allow entry of rifabutin in A. baumannii and, in addition, that high Cmax and AUC of rifabutin can be achieved in patients for effective treatment of infections caused by this pathogen and for reducing likelihood of resistance development.
• a preferred rifabutin formulation comprises rifabutin powder in water, a solvent, and an acid to promote dissolution of the rifabutin.
• the solvent may be present in a concentration from about 25% to about 75%, from about 30% to about 70%, from about 35% to about 65%, from about 40% to about 60%, from about 45% to about 55%, from about 45% to about 65%, from about 50% to about 65%, from about 50% to about 60%, from about 50% to about 55%, or about 50%.
• the solvent and the distilled water are in a 1:1 ratio.
• Preferred solvents include polyoxyethylene sorbitan monooleate (Tween 80), sorbitan monooleate polyoxyethylene sorbitan monolaurate (Tween 20), polyethylene glycol (PEG), propylene glycol, N-methyl-2-pyrrolidone (NMP), glycerin, ethanol, dimethylacetamide (DMA), diethylene glycol monoethyl ether (transcutol HP), or dimethyl isosorbide (DMI).
• the solvent is DMI.
• a reconstituted solution of the invention preferably contains about 250 mg/ml (1:1 solvent/water) or about 166.7 mg/m.
• concentrations of the reconstituted solution may be as high as about 300 mg/ml.
• a more dilute solution is required and that is obtained by adding more water to the solvent.
• rifabutin in a 1 :4 solvent/water ratio will result in a solution of about 50 mg/ml.
• Such formulations are useful for treating a variety of conditions caused by A. baumannii , such as, but not limited to, bacteremia, Ventilator- Associated Bacterial Pneumonia (VABP), Hospital-Acquired Bacterial Pneumonia (HABP) and Urinary Tract Infections (UTI).
• rifabutin has potent activity towards A. baumannii due to exploitation of the A. baumannii ferric-coprogen (FhuE) receptor. Moreover, the invention provides methods for determining susceptibility of A. baumannii isolates to rifabutin treatment.
• compositions achieve critical Cmax and AUC that cannot be achieved using currently available oral formulations. This provides an unexpected clinical advantage previously unavailable for rifabutin.
• the intravenous administration of rifabutin provides a novel treatment for patients with life-threatening bacterial infections such as those caused by Acinetobacter baumannii.
• Formulations of the invention include an acid.
• the solvent solution may also include an acid.
• the acid may be added to the solvent solution to form a reconstitution solvent.
• the acid may be in a concentration sufficient to cause dissolution of rifabutin when adding rifabutin to the reconstitution solvent.
• the acid may be in a concentration from about 1.0% to about 5.0%, from about 1.1% to about 4.9%, from about 1.2% to about 4.8%, from about 1.3% to about 4.7%, from about 1.4% to about 4.6%, from about 1.5% to about 4.5%, from about 1.6% to about 4.4%, from about 1.7% to about 4.3%, from about 1.8% to about 4.2%, from about 1.9% to about 4.1%, from about 2.0% to about 4.0%, from about 2.1% to about 3.9%, from about 2.2% to about 3.8%, from about 2.3% to about 3.7%, from about 2.4% to about 3.6%, from about 2.5% to about 3.5
• the acid may be hydrochloric, methanesulfonic, phosphoric, 1-tartaric, d-glucuronic, 1-malic, d-gluconic, 1 -lactic, acetic or 1-aspartic.
• the acid is acetic acid.
• the formulation containing an acid has a pH from about 3.0 to about 10.0, from about 3.0 to about 9.0, from about 3.0 to about 8.0 from about 3.0 to about 7.0, from about 3.0 to about 6.0, from about 4.0 to about 10.0, from about 4.0 to about 9.0, from about 4.0 to about 8.0 from about 4.0 to about 7.0, from about 4.0 to about 6.0, from about 5.0 to about 6.0, from about 5.1 to about 5.9, from about 5.2 to about 5.8, from about 5.3 to about 5.7, from about 5.4 to about 5.6, from about 5.5 to about 5.6, from about 5.5 to about 5.7, from about 5.5 to about 5.8, from about 5.5 to about 5.9, or a pH of > 4.5.
• the pH is from about 5.0 to about 6.0.
• An intravenous rifabutin formulation may be manufactured by a process comprising preparing a solution comprising a solvent and distilled water in a 1:1 ratio in the presence of an acid suitable to promote dissolution of said rifabutin.
• Rifabutin may be present in a solid form or a powder form that is soluble in a liquid medium.
• Rifabutin may be dissolved in a solvent.
• Rifabutin may be soluble in an aqueous solution of 50% solvent (i.e., 1:1 solvent-distilled water) in the presence of an acid.
• rifabutin may be dissolved in a reconstitution solution containing an acid in an aqueous solution of a solvent in distilled water.
• Rifabutin may be added to the reconstitution solvent in an amount sufficient to produce a final solution having rifabutin in a concentration from about 150 mg/mL to about 350 mg/mL, from about 160 mg/mL to about 325 mg/mL, from about 170 mg/mL to about 300 mg/mL, from about 180 mg/mL to about 275 mg/mL, from about 190 mg/mL to about 265 mg/mL, from about 200 mg/mL to about 255 mg/mL, from about 210 mg/mL to about 250, mg/mL, from about 225 mg/mL to about 255 mg/mL, from about 235 mg/mL to about 255 mg/mL, from about 245 mg/mL to about 255, mg/mL, or from about 250 mg/mL to about 255 mg/mL.
• the rifabutin solution or salt solution thereof contains about 250 mg/mL rifabutin. Adding the rifabutin to the reconstitution solution forms a reconstituted solution of rifabutin.
• the reconstituted rifabutin solution or salt solution thereof may be a concentrated solution not yet ready for parenteral administration.
• the concentrated solution may be a sterile solution.
• the rifabutin solutions of the present invention may be in the form of a formulation for parenteral administration.
• the reconstituted rifabutin solutions may be diluted with a pharmaceutically accepted diluent for intravenous administration of a therapeutically effective dose of rifabutin.
• the reconstituted rifabutin solution may be added to a pharmaceutically accepted diluent to prepare it for sterilized injection into a subject.
• the diluent may be a sodium chloride solution.
• Formulations may include a solvent.
• the w/v ratio of rifabutin to solvent may be from about 4: 1 to about 1 :4, from about 2: 1 to about 1 :3, or from about 1 : 1 to about 1 :2.
• the w/v ratio of rifabutin to solvent may be about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, or about 1:4.
• the formulation may be provided to achieve systemic concentrations of rifabutin from about 2mg/L to about 50.0 mg/L.
• Formulations of the invention may be for any parenteral administration.
• the composition may be formulated for injection or infusion or inhalation.
• the injection may be subcutaneous or intravenous.
• the composition is formulated for intravenous administration.
• formulations of the invention may also include a pharmaceutically acceptable diluent.
• the pharmaceutically acceptable diluent may be in a concentration sufficient to deliver a therapeutically effective amount of rifabutin in IV formulation to a patient suffering from an infection.
• the pharmaceutically acceptable diluent may be saline.
• the diluent is 0.9% saline.
• the solution may be administered with a therapeutically effective amount of rifabutin to treat a patient suffering from an infection.
• the disclosure provides a method of treating baumannii infection.
• the method includes administering to a patient a composition comprising rifabutin at a dose sufficient for activation of a ferric-coprogen (FhuE) receptor of A. baumannii cells, to thereby facilitate entry of said rifabutin into said A. baumannii cells.
• the composition is administered intravenously.
• the dose may provide an AUC and Cmax associated with activation of the FhuE receptor, e.g., preferably a Cmax greater than about 2 mg/L.
• the Cmax may be greater than about 2 mg/L and less than about 50 mg/L. In some embodiments, Cmax > 2 mg/L but ⁇ 50 mg/L and AUC > 10 mg*h/L and ⁇ 300 mg*h/L.
• the rifabutin may be administered
• the composition includes the rifabutin, water, a solvent, and an acid.
• the composition is delivered intravenously at a rifabutin dose that is at least about 2 mg/kg q24h, 1 mg/kg ql2h, or 0.5 mg/kg q6h.
• the solvent may be, for example, polyoxyethylene sorbitan monooleate (Tween 80), sorbitan monooleate polyoxyethylene sorbitan monolaurate (Tween 20), polyethylene glycol (PEG), propylene glycol, N-methyl-2-pyrrolidone (NMP), glycerin, ethanol, dimethylacetamide (DMA), diethylene glycol monoethyl ether (transcutol HP), or dimethyl isosorbide (DMI).
• the composition may have about 2:1 v/w solventxifabutin.
• the method preferably includes delivering the formulation to the patient by intravenous injection, for example, at a dose that results in a Cmax is that is at least about 2 mg/L.
• the formulation may be delivered at a dose that results in: 2 mg/L ⁇ Cmax ⁇ 50 mg/L; and 10 mg*h/L ⁇ AUC ⁇ 300 mg*h/L.
• the formulation may be delivered at a dose that is at least about 2 mg/kg q24h, 1 mg/kg ql2h, or 0.5 mg/kg q6h.
• compositions for use in treating A. baumannii infection comprising rifabutin, water, a solvent, and an acid.
• the composition is an aqueous solution with a high concentration of rifabutin (e.g., 2:1 v/w solvent: rifabutin) that activates a ferric-coprogen (FhuE) receptor of A. baumannii cells, to thereby facilitate entry of said rifabutin into said A. baumannii cells.
• rifabutin e.g., 2:1 v/w solvent: rifabutin
• the solvent may be, for example, polyoxyethylene sorbitan monooleate (Tween 80), sorbitan monooleate polyoxyethylene sorbitan monolaurate (Tween 20), polyethylene glycol (PEG), propylene glycol, N-methyl-2-pyrrolidone (NMP), glycerin, ethanol, dimethylacetamide (DMA), diethylene glycol monoethyl ether (transcutol HP), or dimethyl isosorbide (DMI).
• the solvent is DMI or trascutol HP.
• the solvent to water ratio v/v is 1 : 1 or 1 :2.
• the acid may be hydrochloric, methanesulfonic, phosphoric, L-tartaric, D-glucuronic, L-malic, D-gluconic, L- lactic, acetic, or L-aspartic acid.
• the acid is acetic acid or D- glucuronic acid.
• the rifabutin to acid molar ratio is 1:1.
• the composition is provided as an aqueous solution for intravenous delivery.
• the composition may be provided for a patient identified as infected with A. baumannii , and the composition may include rifabutin for IV delivery at a dosage that is at least about 2 mg/kg q24h, 1 mg/kg ql2h, or 0.5 mg/kg q6h.
• the disclosure provides a use of rifabutin for the manufacture of a medicament for treating A. baumannii infection in a patient.
• the medicament may provide a dose that results in an AUC and Cmax associated with activation of the FhuE receptor.
• the medicament is prepared to be administered in a dosage regime that results in a Cmax in the patient that is that is at least about 2 mg/L.
• the medicament may include rifabutin, water, a solvent, and an acid.
• the solvent may be, for example, polyoxyethylene sorbitan monooleate (Tween 80), sorbitan monooleate polyoxyethylene sorbitan monolaurate (Tween 20), polyethylene glycol (PEG), propylene glycol, N-methyl-2-pyrrolidone (NMP), glycerin, ethanol, dimethylacetamide (DMA), diethylene glycol monoethyl ether (transcutol HP), or dimethyl isosorbide (DMI).
• the medicament may be provided within an IV bag.
• the medicament provides a dose that results in: 2 mg/L ⁇ Cmax ⁇ 50 mg/L; and 10 mg*h/L ⁇ AUC ⁇ 300 mg*h/L.
• the medicament provides a rifabutin dosage that is at least about 2 mg/kg q24h, 1 mg/kg ql2h, or 0.5 mg/kg q6h.
• the invention provides methods of preparing an intravenous formulation of rifabutin.
• the method may include preparing a solution containing a solvent and distilled water.
• the solution is in a 1:1 ratio.
• the solvent may be any solvent but is preferably DMI.
• An acid may be added to the solution.
• the acid may be suitable to promote dissolution of rifabutin.
• the acid may be any acid, but preferably acetic acid.
• Rifabutin may be introduced into the solution containing the acid. Thus, the acid causes the rifabutin to dissolve into the solution.
• the rifabutin solution may be added to a pharmaceutically acceptable diluent.
• the diluent may be 0.9% saline.
• the intravenous formulation of rifabutin may comprise rifabutin, DMI, and saline.
• the w/v ratio of rifabutin to solvent may be from about 4: 1 to about 1 :4, from about 2: 1 to about 1 :3, or from about 1 : 1 to about 1 :2.
• the w/v ratio of rifabutin to solvent may be about 4:1, about 3:1, about 2:1, about 1:1, about 1 :2, about 1 :3, or about 1 :4.
• rifabutin DMI and saline
• the amount and concentration of rifabutin, DMI and saline will depend on the size of the IV bag (in intravenous embodiments) and such variations will be apparent to those of ordinary skill in the art to achieve preferred doses in the range of 6-9 mg/kg with the DMI amount determined based on the above ratios.
• the intravenous formulation of rifabutin may be administered to a subject in need of treatment.
• the invention provides methods of increasing the efficacy of an antibiotic.
• the methods include modulating a TonB-dependent siderophore receptor in a bacteria cell to increase uptake of an antibiotic.
• Modulating a TonB-dependent siderophore receptor may include administering an IV formulation of an antibiotic that mediates transporting the antibiotic across a cell membrane of the bacterial cell.
• the TonB-dependent siderophore receptor may be an FhuE receptor.
• Administration of the IV formulation of the antibiotic increases bioavailability of the antibiotic, thereby increasing uptake of the antibiotic through the bacterial cell membrane.
• the antibiotic may be any form of rifabutin capable of being administered by IV.
• a siderophore may be bound to an iron complex.
• Rifabutin may bind to the iron-loaded siderophore or may use the mediation of the TonB-dependent siderophore receptor of the iron-loaded siderophore to cross the cell membrane.
• the IV formulation may be that of any IV formulation of rifabutin described herein or dose thereof.
• the invention provides methods of treating a bacterial infection.
• the methods may include administering a liquid formulation of rifabutin to a subject with a bacterial infection.
• the liquid formulation may include rifabutin or a salt thereof, a solvent and an acid.
• the liquid formulation may be a solution of rifabutin and a diluent to be administered intravenously to a subject with a bacterial infection.
• the rifabutin may then travel across an outer membrane of a bacteria cell causing the infection and eradicate the bacteria cell.
• the bacterial infection may be any infection caused by A.
• the formulation for IV administration may include a pharmaceutically acceptable solvent.
• the method may include administering an IV formulation of any formulation of rifabutin described herein to a subject suffering from a bacterial infection.
• methods of the invention may include identifying compounds having Acinetobacter baumannii antibacterial activity.
• the methods may include providing a medium comprising at least an iron complex and an iron chelator, introducing a plurality of A. baumannii bacteria cells into the medium, and exposing the medium containing the plurality of bacteria cells to a compound.
• the method may also include quantifying the plurality of bacteria cells using any acceptable methods of bacteria quantification. Identifying a decrease of a number of the plurality of bacteria cells is indicative of antibacterial activity of the compound.
• a decrease in the number of the plurality of bacteria cells may occur when the compound crosses an outer membrane of the bacteria cells, thereby destroying the bacteria cells.
• the compound may cross the outer membrane in the presence of the iron complex or the iron chelator, thus enabling Ton-B siderophore receptor mediated uptake of the compound.
• the receptor may be an FhuE receptor.
• the medium may be Roswell Park Memorial Institute (RPMI) medium, 10% fetal calf serum, or a combination thereof.
• the medium may be any medium or may be any media that either contains iron and an iron chelator or is capable of having iron and an iron chelator added to the media.
• the iron complex may be any iron complex capable of binding to a siderophore.
• the iron chelator may be any iron chelator capable of crossing a bacterial cell membrane.
• the iron chelator is pyridoxal isonicotinoyl hydrazone (PIH).
• the chelator may be present in concentrations from about 0.05 mM to 0.25 mM, from about 0.075 mM to 0.225 mM, from about 0.1 mM to 0.2 mM from about 0.125 mM to 0.15 mM.
• the iron chelator is present at about 0.1 mM.
• methods include treating a bacterial infection in a subject.
• the method may include administering a therapeutically effective amount of an intravenous formulation of rifabutin or a salt thereof.
• the formulation has a pharmaceutically acceptable solvent present at a ratio of 4: 1 to about 1 :4, from about 2: 1 to about 1 :3, or from about 1 : 1 to about 1 :2 w/v of rifabutin to solvent.
• the bacterial infection may be any infection caused by A. baumannii , such as, but not limited to, bacteremia, Ventilator-Associated Bacterial Pneumonia (VABP), Hospital-Acquired Bacterial Pneumonia (HABP) and Urinary Tract Infections (UTI).
• the bacteria species is A. baumannii.
• Aspects of the invention may also include an in vitro method of determining susceptibility of a species of bacteria to rifabutin.
• the method may include exposing a plurality bacteria cells to an iron complex, an iron chelator, and rifabutin.
• the method may also include quantifying a number of the plurality of bacteria cells and/or identifying a decrease in the number of bacteria cells. A decrease in the number of bacteria cells is indicative of the susceptibility of a species of bacteria to rifabutin.
• the method may also include administering rifabutin to a subject suffering from a bacterial infection of the species of bacteria
• FIG. 1 is a graph illustrating the quantification of fhuE expression levels in A. baumannii HUMC1 in different media.
• FIG. 2 is a graph of the activity of IV administration of rifabutin in a murine neutropenic sepsis model.
• FIG. 3 shows effects of rifabutin in neutropenic lung infection mouse models with CD-I mice infected with A. baumannii UNT091.
• FIG. 4 shows effects of rifabutin in neutropenic lung infection mouse models with CD-I mice infected with A. baumannii UNT093.
• FIG. 5 provides results of dose fractionation experiments indicating a clear dose response relationship for which both Cmax and AUC are critical for activity.
• FIG. 6 is a graph showing CFU per lung pair in mice inoculated with A. baumannii.
• FIG. 7 shows a schematic of TonB -dependent transport.
• FIG. 8 shows activity of rifabutin and rifampicin antibiotics upon plasmid mediated expression of FhuE- variants in CA-MHB.
• Embodiments of the disclosure provide methods, uses, and compositions for treating, or making a medicament for treating, A. baumannii infection.
• A. baumannii infection For background, see Howard, 2012, Acinetobacter baumannii. An emerging opportunistic pathogen, Virulence 3(3):243-250 and Peleg, 2008, Acinetobacter baumannii. Emergence of a successful pathogen, Clin Microbiol Rev 21(3):538-582, both incorporated by reference.
• Methods and compositions of the disclosure operate through the activation of a ferric-coprogen (FhuE) receptor to facilitate entry of said rifabutin into said A. baumannii cells.
• FeE ferric-coprogen
• Methods preferably include obtaining a sample from a patient suspected of having an infection; performing a test on the sample to identify an infection of A. baumannii in the patient; and providing a formulation of rifabutin for treating the patient that, when administered to the patient, maximizes a resultant AUC and Cmax.
• the method may include administering the formulation of rifabutin to the patient.
• the formulation includes rifabutin, water, a solvent, and an acid.
• Preferred solvents include polyoxyethylene sorbitan monooleate (Tween 80), sorbitan monooleate polyoxyethylene sorbitan monolaurate (Tween 20), polyethylene glycol (PEG), propylene glycol, N-methyl-2-pyrrolidone (NMP), glycerin, ethanol, dimethylacetamide (DMA), diethylene glycol monoethyl ether (transcutol HP), dimethyl isosorbide (DMI), or another polar solvent.
• the formulation may about 250 mg/ml (1 : 1 v/v solvent/water) or about 166.7 mg/ml (2:1 solvent/water), however concentrations of the reconstituted solution may be as high as about 300 mg/ml.
• the formulation is delivered to the patient, e.g., by intravenous injection.
• the IV injection results in a Cmax is that greater than about 2 mg/L and optionally less than about 50 mg/L.
• the formulation comprises a dose of rifabutin having a Cmax > 2 mg/L but ⁇ 50 mg/L and AUC > 10 mg*h/L and ⁇ 300 mg*h/L.
• the formulation may be delivered at a dose that is at least about 2 mg/kg q24h, 1 mg/kg ql2h, or 0.5 mg/kg q6h.
• the formulation is preferably delivered via IV at a dose that is at least about 2 mg/kg q24h, 1 mg/kg ql2h, or 0.5 mg/kg q6h.
• FIG. 1 shows that fhuE is at least 10-fold overexpressed when A. baumannii is grown in nutrient depleted medium (Roswell Park Memorial Institute (RPMI) medium plus 10% fetal calf serum (FCS)) compared to standard testing conditions (cation-adjusted Mueller Hinton broth; CA-MHB).
• RPMI Roswell Park Memorial Institute
• FCS fetal calf serum
• FIG. 2 is a graph illustrating the results of the effect of rifabutin and rifampin in a neutropenic sepsis mouse model. The results indicate that the IV administration of rifabutin protects against sepsis with a dose dependent response at 1 mg/kg whereas rifampin does not at 10 mg/kg supporting the potent activity of rifabutin observed in vitro.
• FIG. 3 shows effects of rifabutin in neutropenic lung infection mouse models with CD-I mice infected with A. baumannii UNT091.
• FIG. 4 shows effects of rifabutin in neutropenic lung infection mouse models with CD-I mice infected with A. baumannii UNT093.
• FIG. 5 provides results of dose fractionation experiments indicating a clear dose response relationship for which both Cmax and AUC are critical for activity.
• the disclosure provides methods of treating A. baumannii infection by administering rifabutin in an amount that maximize the AUC and Cmax.
• the administration may be achieved by intravenous injection or by methods known in the art for modified oral released or by inhalation if locally high Cmax and AUC are required.
• rifabutin is administered as an intravenous formulation in an amount that maximize the AUC and Cmax.
• rifabutin will reach therapeutic concentrations and reduce the frequency of resistance development.
• Cmax is greater than about 2 mg/L and less than about 50 mg/L.
• Embodiments of the disclosure provide a composition that includes rifabutin.
• the disclosure provides a composition for use in treating A. baumannii infection, the composition comprising rifabutin, water, a solvent, and an acid.
• the solvent may be polyoxyethylene sorbitan monooleate (Tween 80), sorbitan monooleate polyoxyethylene sorbitan monolaurate (Tween 20), polyethylene glycol (PEG), propylene glycol, N-methyl-2-pyrrolidone (NMP), glycerin, ethanol, dimethylacetamide (DMA), diethylene glycol monoethyl ether (transcutol HP), or dimethyl isosorbide (DMI).
• Tween 80 polyoxyethylene sorbitan monooleate
• Tween 20 sorbitan monooleate polyoxyethylene sorbitan monolaurate
• PEG polyethylene glycol
• NMP N-methyl-2-pyrrolidone
• the composition may include between about 1:1 and 2: 1 v/v solvent/water. Most preferably, the composition is provided within an IV bag. In preferred embodiments, the composition is provided for a patient identified as infected with the A. baumannii , and the composition includes rifabutin for IV delivery at a dosage that is at least about 2 mg/kg q24h, 1 mg/kg ql2h, or 0.5 mg/kg q6h.
• the composition may have a formulation that provides for a dose of rifabutin having a Cmax > 2 mg/L but ⁇ 50 mg/L and AUC > 10 mg*h/L and ⁇ 300 mg*h/L.
• the formulation is intended for intravenous delivery.
• the disclosure provides a use of rifabutin for the manufacture of a medicament for treating A. baumannii infection in a patient, in which the medicament is prepared to be administered in a dosage regime that results in a Cmax in the patient that is that is at least about 2 mg/L.
• the medicament comprises rifabutin, water, an acid, and a solvent (e.g., polyoxyethylene sorbitan monooleate (Tween 80), sorbitan monooleate polyoxyethylene sorbitan monolaurate (Tween 20), polyethylene glycol (PEG), propylene glycol, N-methyl-2- pyrrolidone (NMP), glycerin, ethanol, dimethylacetamide (DMA), diethylene glycol monoethyl ether (transcutol HP), or dimethyl isosorbide).
• the solvent is DMI or trascutol HP.
• the solvent to water ratio v/v is 1 : 1 or 1 :2.
• the acid may be hydrochloric, methanesulfonic, phosphoric, L-tartaric, D-glucuronic, L-malic, D-gluconic, L- lactic, acetic, or L-aspartic acid.
• the acid is acetic acid or D- glucuronic acid.
• the rifabutin to acid molar ratio is 1:1.
• the medicament is provided within an IV bag.
• the dosage regime results in: 2 mg/L ⁇ Cmax ⁇ 50 mg/L; and 10 mg*h/L ⁇ AUC ⁇ 300 mg*h/L.
• the dosage regime may preferably result in a dose that is at least about 2 mg/kg q24h, 1 mg/kg ql2h, or 0.5 mg/kg q6h.
• the rifabutin may be administered by inhalation in an amount that maximize the local AUC and Cmax.
• Such methods and compositions allow the rifabutin to reach therapeutic concentrations and reduce the frequency of resistance development.
• rifabutin is administered by a modified release oral drug delivery system to enhance Cmax and AUC and minimize tmax so to maximize the AUC and Cmax.
• a modified release oral drug delivery system to enhance Cmax and AUC and minimize tmax so to maximize the AUC and Cmax.
• Example 1 A number of approved drugs were tested against A. baumannii under standard testing conditions (cation-adjusted Mueller Hinton broth; CA-MHB) as well as nutrient depleted medium (Roswell Park Memorial Institute (RPMI) medium plus 10% fetal calf serum (FCS). The antibacterial activity of rifabutin against A. baumannii was greatly enhanced under non- standard testing conditions.
• Table 1 summarizes the results of the antimicrobial susceptibilities of carbapenem resistant A. baumannii strains HUMC1 and UNT091 under standard (CA-MHB, Mueller Hinton broth 2) and non-standard testing conditions. The results indicate that the two carbapenem resistant A.
• HUMC1 (BV374) (Spellberg/Luna Laboratory, University of Southern California, Los Angeles, CA) and UNT091-1 (BV378) (UNT Health Science Center, Fort Worth, TX).
• the HUMC1 isolate is a hyper-virulent drug-resistant clinical strain isolated from a blood-stream infection. Both strains are carbapenem resistant and colistin sensitive.
• the isolates were stored at -80 °C as 20 % (v/v) glycerol cultures.
• MICs were determined by the broth microdilution method following the guidelines of the Clinical Laboratory Standards Institute (CLSI) using RPMI supplemented with 10% (v/v) or cationic-adjusted Muller Hinton broth (CA-MHB) as assay medium.
• CLSI Clinical Laboratory Standards Institute
• CA-MHB cationic-adjusted Muller Hinton broth
• Serial 2-fold dilutions of the antibiotics were prepared in a separate 96-well plate polypropylene U-bottom plate (Ratiolab Cat. No. 6018111) in RPMI supplemented with 10 % (v/v) FCS at 10-fold of the final test concentrations and 10 m ⁇ of the dilutions were transferred to new 96-well polystyrene U-bottom microtiter plates with a parafilm plate cover.
• the plates were then inoculated with 90 pL per well of the prepared bacterial suspensions in using a multichannel pipette (Eppendorf), with the first column containing 4 wells each for growth control (no antibiotic).
• the plates were covered with parafilm and incubated at 35 °C for 20 - 24 h, after which the MIC was determined by visual inspection and the plates were scanned to record the data.
• the MIC was recorded as the lowest concentration of the compound that inhibited bacterial growth by visual inspection. MICs were determined at least in duplicates and in the case of variations, the higher values are provided.
• Example 2 FhuE overexpression: The level of ftiuE expression was evaluated in different media by qRT-PCR on the A. baumannii HUMC1 strain.
• FIG. 1 depicts a graph illustrating the quantification of fhuE expression levels in different media.
• fhuE is at approximately 10-fold overexpressed when A. baumannii is grown in RPMI + 10% FCS or CA-MHB supplemented with 0.1 mM pyridoxal isonicotinoyl hydrazone (PIH) compared to CA-MHB.
• PHI pyridoxal isonicotinoyl hydrazone
• fhuE was evaluated by quantitative reverse transcription-PCR (qRT- PCR). Isolates were grown in specified broth at 37 °C to mid-log phase (optical density at 600 nm [OD600] of 0.5), and total RNA was extracted using a PureLink RNA minikit (Ambion) according to the manufacturer’s recommendations. Residual DNA contaminations were removed using a Turbo DNA-free kit (Ambion). qRT-PCR was performed using a GoTaq 1-Step RT- qPCR System kit (Promega) on a CFX96 TouchTM Real-Time PCR Detection System (BioRad). As a housekeeping gene, the RNA polymerase sigma factor D (rpoD) was quantified and fhuE expression was normalized to that of rpoD using the comparative DDOT (where CT is threshold cycle) method.
• rpoD RNA polymerase sigma factor D
• Table 2 summarizes MICs of rifabutin in RPMI medium supplemented with 10 % (v/v) FCS for the fhuE deleted mutants and their parental strains.
• Deletion of fhuE resulted in elevated MICs towards rifabutin in RPMI + 10 % FCS however, surprisingly had no effect on the closely related compound rifampicin.
• FhuE is required for potent rifabutin activity in RPMI + 10% FCS and indicated that rifabutin activity in this medium is likely due to active uptake of the compound mediated by the A. baumannii siderophore receptor FhuE.
• the data from this experiment show that rifabutin is very active against A. baumannii because of a novel mechanism of entry in A. baumannii.
• the gene AWC45 RS10145 (HUMC1 genome) coding for the FhuE protein was deleted in the A. baumannii strains HUMC1 and UNT091 using a two-step recombination method. DNA fragments corresponding to 700-bp up and downstream genomic regions of fhuE were amplified by PCR and introduced into the pVT77 knockout plasmid using Gibson assembly. The resulting fhuE knockout plasmid was transferred in A. baumannii isolates by conjugation and trans- conjugants were selected on LB agar plates containing sodium tellurite.
• clones were screened for genomic plasmid integration by PCR and clones containing up- and downstream plasmid integrations were used for counter-selection on LB agar plates containing AZT for plasmid removal from the genome. Clones were screened for fhuE deletion and plasmid removal by PCR, and the genomic gene deletions were confirmed by DNA sequencing (Microsynth AG, Balgach, Switzerland).
• Plasmid-based expression of fhuE Plasmid-based expression of fhuE:
• Table 3 summarizes MICs of rifabutin in fhuE expressing A. baumannii strains in CA- MHB +/- 1 mM IPTG.
• rifabutin MIC was 1000-fold lower in the strain carrying the fhuE expressing plasmid compared to the strain carrying an empty plasmid as control. This data shows that activation of fhuE in A. baumannii results in a potent activity of rifabutin towards this organism.
• the fhuE gene (AWC45 RS10145) from the A. baumannii HUMC 1 strain was cloned into the E. coli / A. baumannii shuttle plasmid pVTl 11 under the control of the isopropyl -beta- D-l-thiogalactopyranoside (IPTG) inducible promoter Ptrc-lacO.
• IPTG isopropyl -beta- D-l-thiogalactopyranoside
• the resulting plasmid and the original pVTl 11 plasmid (control) were transferred to the A. baumannii strain ATCC- 17978 by conjugation and transconjugants were selected on LB agar plates containing kanamycin.
• the presence of the plasmids in the receiver A. baumannii strains was then confirmed by PCR.
• Table 4 summaries the FoR results of the A. baumannii spontaneous resistance frequencies to rifabutin on RPMI + 10 % FCS agar medium. Dose-dependent FoR ranging from 10 5 to 10 9 was observed for the HUMC1 strain. High FoR’s around 10 5 were observed at rifabutin concentrations of 0.02 and 0.2 mg/L, followed by a step wise decrease to 10 7 at 1 mg/L and 10 9 at 2 mg/L and 20 mg/L rifabutin. Similar results were obtained for strains UNT091-1, ACC00445, LAC-4 and UNT238-1.
• agar plates were prepared using RPMI powder (Sigma R7755) dissolved at 10.3 g/L with agar at 15 g/L and boiled until complete agar melting. After cooling the media to 45 °C, 0.3 g/L L-glutamine (Sigma G7513), 25mM HEPES (Gibco 15630-056) and 10% (v/v) FCS (Gibco 10500-064) were added. Concentrations of 0.02, 0.1, 1.0, 2.0 and 20.0 mg/L of rifabutin were supplemented and 25 mL of the media was poured directly into 9 cm petri dishes.
• the culture inocula were prepared from a bacterial NaCl suspension at 0.5 McFarland diluted 200-times in 100 mL of RPMI (Sigma R8758) + 10 % FCS in 500 mL flasks to reach ⁇ 5 x 10 5 CFU/mL.
• the flasks were incubated for 24 h at 37 °C under shaking at 220 rpm. After incubation, the cells were pelleted by centrifugation (10 min, 7000 rpm at RT) and re-suspended in 1 mL PBS.
• the CD-I mice were IP infected with A. baumannii ACC00445 strain in the presence of 5 % mucin.
• the mice were treated with rifabutin and rifampin via IV administration at 1 h and 5 h post-infection.
• the results indicate that the IV administration of rifabutin protects against sepsis with a dose dependent response at 1 mg/kg whereas rifampin does not at 10 mg/kg supporting the potent activity of rifabutin observed in vitro.
• FIG. 5 summarizes the results of dose fractionation experiments in a neutropenic mouse model of infection.
• Rifabutin was administered IV either once daily (q24h), bi-daily (ql2h) or four-times within 24 hours (q6h).
• the results indicate a clear dose response relationship for which both Cmax and AUC are critical for activity.
• Table 5 is the target exposures required to treat A. baumannii infections based upon MIC.
• the estimations are based on efficacy models and using a sigmoid Emax model with variable slope to fit the dose and PK/PD index (PDI) responses to determine the PDI values of rifabutin resulting in 1-log reduction in lung CFUs using GraphPad Prism version 5.03 (GraphPad, Inc., San Diego, CA). From this data it is evident that the oral administration of rifabutin will not achieve the exposures required to treat >90% of the A. baumannii isolates (MIC ⁇ 1 mg/L).
• FIG. 6 is a graph showing CFU per lung pair in mice inoculated with A. baumannii strains.
• the treatment single IV dose
• the effect of RBT was blunted in mice infected with the UNT091-1 ::AfhuE strain supporting the role of fhuE as mediating RBT sensitivity both in vitro and in vivo.
• Example 7 The in vitro activity of rifabutin was determined towards a panel of clinical A. baumannii in the presence of an iron chelator leading to increased siderophore receptor expression.
• Rifabutin displays a potent in vitro activity against a large panel of recently isolated and mainly XDR A. baumannii including isolates non-susceptible to colistin, and carbapenems. Complexation of free iron by PIH allows robust rifabutin susceptibility testing in nutrient rich standard MHA.
• baumannii with rifabutin was performed using agar dilution method with Muller Hinton Agar (MHA) supplemented with O.lmM pyridoxal isonicotinoyl hydrazine (PIH), a potent nontoxic iron chelator. Comparator antibiotics were tested at CLSI standard conditions.
• Example 8 Modeling FhuE TonB effects on rifabutin activity
• FIG. 8 shows activity of rifabutin and rifampicin antibiotics upon plasmid mediated expression of FhuE-variants in CA-MHB.
• A. baumannii ATCC- 17978 was used as host strain, gene expression from plasmids was induced with 1 mM IPTG and a plasmid that did not encode fhuE was used as control.
• FIG. 7 shows a schematic of TonB-dependent transport adapted from Hickman, S. J., Cooper, R. E. M., Bellucci, L., Paci, E. & Brockwell, D. J. Gating of TonB-dependent transporters by substrate-specific forced remodelling. Nat. Commun. 8, 1-12 (2017), incorporated herein by refrence.
• TBDTs are so- called gated porins with their lumen occluded by an N-terminal plug domain preventing the substrate to transit across the outer membrane.
• the binding of substrate induces an allosteric rearrangement of the plug domain, releasing the Ton box into the periplasmic space, where it recruits the C-terminal domain of TonB in complex with ExbB and ExbD to form the energy transducing machinery.
• Linkage of the OM and IM via this non-covalent complex is required to trigger the full or partial unfolding of the plug domain, allowing the passage of the substrate.
• TonB-dependent transporter OM: outer membrane
• IM inner membrane
• PG peptidoglycan.

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