Classifications

• • 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
  • A61K9/10—Dispersions; Emulsions
  • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
  • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
• • 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
  • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
  • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
  • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
• • 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
  • A61K47/02—Inorganic compounds
• • 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
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
  • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
• • 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
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
  • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
  • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
• • 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
  • A61K9/10—Dispersions; Emulsions
  • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
  • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
  • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers comprising non-phosphatidyl surfactants as bilayer-forming substances, e.g. cationic lipids or non-phosphatidyl liposomes coated or grafted with polymers
• • 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
  • A61K9/10—Dispersions; Emulsions
  • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
  • A61K9/1277—Preparation processes; Proliposomes
• • 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
  • A61K9/10—Dispersions; Emulsions
  • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
  • A61K9/1277—Preparation processes; Proliposomes
  • A61K9/1278—Post-loading, e.g. by ion or pH gradient
• • 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
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
  • A61P31/06—Antibacterial agents for tuberculosis

Definitions

• the present disclosure relates to liposome compositions comprising oxazolidinone compounds, methods of their making and use of the aminoalkyl oxazolidinone compounds in the treatment of Mycobacterium tuberculosis and other gram-positive bacterial infections.
• Liposome compositions are useful for the delivery of therapeutic compounds.
• Liposome compositions can comprise liposomes encapsulating a therapeutic compound within a vesicle formed by a membrane formed by lipids. Liposomes are usually characterized by having an interior space sequestered from an outer medium by a membrane of one or more bilayers forming a microscopic sack, or vesicle.
• liposomes encapsulating therapeutic compounds can degrade during storage and prior to therapeutic administration.
• oxidative degradation of liposome components and changes in liposome particle size or polydispersity index (PDI) can occur during storage of liposome compositions comprising therapeutic compounds.
• PDI polydispersity index
• Liposome compositions and methods of treating a methicillin resistant Staphylococcus aureus (MRSA) bacterial infection are provided herein.
• aspects of the disclosure relate to a liposome composition of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, Formula (I) wherein R 1 is a tetrazole ring substituted at position 2’ with an aminoalkyl; and R 2 is an amine or an acetamide; wherein the compound of Formula (I) or pharmaceutically acceptable salt thereof is encapsulated in liposomes in an aqueous medium having a pH greater than 6.7; and wherein the liposomes comprise a phosphatidylcholine, cholesterol and a PEG polymer-conjugated lipid with 50-65 mol% cholesterol relative to the sum of cholesterol and non-pegylated phospholipid in the liposomes.
• R 2 is an acetamide (NHCOCH 3 ).
• R 1 is selected from the group consisting of:
• the PEG polymer-conjugated lipid is in an amount of 5 mol% relative to phosphatidylcholine.
• a sulfate salt of the compound of Formula (I) is encapsulated in the liposomes comprising the phosphatidylcholine, cholesterol and PEG polymer-conjugated lipid in a 45:55:2.25 molar ratio.
• the phosphatidylcholine is distearoylphosphatidylcholine (DSPC) or hydrogenated soy phosphatidylcholine (HSPC).
• the PEG polymer-conjugated lipid is PEG(Mol.
• the liposome composition further comprises a chelator selected from the group consisting of deferoxamine (DFO) and EDTA, wherein the chelator is at a concentration of 0.1-1 mM.
• DFO deferoxamine
• EDTA EDTA
• the compound of Formula (I) is a compound selected from AKG-38, AKG-39 and AKG-40 or a pharmaceutically acceptable salt thereof:
• the compound of Formula (I) is a sulfate salt of AKG-38.
• the pH of the liposome composition is over 7 and no more than 8. In some embodiments, the pH of the liposome composition is 7.3 - 7.7. In some embodiments, the pH of the liposome composition is 7.5.
• the compound of Formula (I) is a sulfate salt of AKG-38
• the liposome composition further comprises a chelator, wherein the chelator is deferoxamine (DFO) and wherein the chelator is at a concentration of 0.1-1 mM.
• the drug/lipid ratio of the AKG-38 to a total phospholipid (PhL) in the composition is 430-680 g/mol.
• the drug/lipid ratio of the AKG-38 to a total phospholipid (PhL) in the composition is 600 g/mol.
• the liposome composition comprises mono- or oligolamellar vesicles having z- average diameter of 90-130 nm; and the liposome composition has a polydispersity index of less than 0.15.
• liposome composition has a proportion of encapsulated AKG- 38 to overall AKG-38 of at least 90%.
• the aqueous medium further comprises sodium chloride.
• the aqueous medium has an osmolality of 270- 330 mOsmol/kg; the sodium chloride is at a concentration of 130-150 mM; and the chelator is at a concentration of 0.5 mM.
• the aqueous medium comprises an ammonium ion at a concentration of 20-60 mM, and the sodium chloride is at a concentration of 50-80 mM.
• the liposome composition further comprises a HEPES or phosphate buffer.
• aspects of the disclosure relate to an AKG-38 liposome composition having a pH of at least 7.0 and not more than 8.0, the liposome composition comprising lipids HSPC, cholesterol, and PEG(2000)-DSPE in a molar ratio of 45:55:2.25 or in a mass ratio of 5:3:1 and a pharmaceutically acceptable salt of AKG-38
• the liposome composition is further characterized by any one or more of the following characteristics: (a) the liposome composition comprises mono- or oligolamellar vesicles having z- average diameter of 90-130 nm or the liposome composition comprises mono- or oligolamellar vesicles have a z-average diameter of 100-130 nm; (b) the liposome composition has a poly dispersity index of less than 0.15 or the liposome composition has a poly dispersity index of less than 0.10; (c) the drug/lipid ratio of the AKG-38 to the total phospholipid (PhL) in the liposome composition is 430-480 g/mol, or the drug/lipid ratio of the AKG-38 to the total phospholipid (PhL) in the liposome composition is 500-650 g/mol; or the drug/lipid ratio of the AKG-38 to the total phospholipid (PhL) in the liposome composition is 430-650
• aspects of the disclosure relate toisotonic AKG-38 liposomal dispersion formulated with (5R)-3- ⁇ 3-Fluoro-4-[6-(2-(2-dimethylaminoethyl)-2H-tetrazol-5-yl)-3- pyridinyl]phenyl ⁇ -5-(methylacetamido)-l,3-oxazolidin-2-one, or a pharmaceutically acceptable salt thereof, encapsulated in liposomes comprising hydrogenated soy phosphatidylcholine (HSPC), cholesterol, and (PEG(Mol.
• HSPC hydrogenated soy phosphatidylcholine
• cholesterol cholesterol
• PEG(Mol PEG(Mol
• PEG- DSPE PEG(2000)-DSPE
• a chelator selected from the group consisting of: deferoxamine (desferrioxamine, Desferal), ethylenediamine tetraacetic acid (EDTA), diethylenetriamine pentaacetic acid (DTP A), nitrilotriacetic acid (NTA), ethyleneglycol- 0, O'-bis(2-aminoethyl)-N, N, N', N' -tetraacetic acid (EGTA), N-(2- hydroxyethyl)ethylenediamine-N, N', N' -triacetic acid (HEDTA), and 1,4,7,10- tetraazacyclododecane- 1,4,7,10-tetraacetic acid (DOTA).
• DETA deferoxamine
• EDTA ethylenediamine tetraacetic acid
• DTP A diethylenetriamine pentaacetic acid
• NTA nitrilotri
• the isotonic AKG-38 liposomal dispersion has a pH of greater than 6.7 and not more than 8.0. In some embodiments, the isotonic AKG-38 liposomal dispersion has a pH of 7.5. In some embodiments, the liposomes are formed from hydrogenated soy phosphatidylcholine (HSPC), cholesterol and PEG(2000)-DSPE in a molar ratio of 45:55:2.2. In some embodiments, the chelator is deferoxamine.
• the liposomal dispersion comprises lipid vesicles formed from a composition comprising a phosphatidylcholine, 55 mol% cholesterol and 5 mol% PEG-DSG or 5 mol% or PEG-DSPE.
• Aspect of the disclosure relate to a method of treating a methicillin resistant Staphylococcus aureus (MRSA) bacterial infection, the method comprising administering to a subject in need thereof a therapeutically effective amount of the liposomal composition.
• MRSA methicillin resistant Staphylococcus aureus
• Aspect of the disclosure relate to method of making liposome composition
• method of making liposome composition comprising the steps of (a) dissolving one or more phospholipid, cholesterol and a PEG-lipid derivative in ethanol to obtain a lipid solution; (b) combining the lipid solution of step (a) with a trapping agent solution to obtain a uniform lipid suspension having a desired phospholipid concentration; (c) extruding the lipid suspension of step (b) through membranes having defined pore sizes, such as polycarbonate track-etched (PCTE) membranes with the nominal pore size of 50-200 nm; (d) purifying liposomes from extraliposomal trapping agent in the extruded lipid suspension to obtain a purified extruded liposome preparation; (e) contacting the liposomes with the compound of Formula (I) in an aqueous medium to effect encapsulation of the compound in the liposomes; (f) optionally removing unencapsulated compound; and (g) providing the
• the trapping agent solution of step (b) comprises ammonium sulfate at the concentration of 0.5M.
• the chelator is deferoxamine.
• the extruded liposomes in step (c) are mono-or oligolamellar vesicles having a z- average diameter of 90-130 nm.
• Liposome compositions and methods of treating a mycobacterial infection are provided herein.
• the liposome composition comprises the compound of Formula (I) or a pharmaceutically acceptable salt thereof, wherein R 2 is an amine (NH 2 ).
• R 1 is selected from the group consisting of:
• the compound of Formula (I) or pharmaceutically acceptable salt thereof is encapsulated in liposomes in an aqueous medium having a pH greater than 6.7; and the liposomes comprise a phosphatidylcholine, cholesterol and a PEG polymer- conjugated lipid with 50-65 mol% cholesterol relative to the sum of cholesterol and non-pegylated phospholipid in the liposomes.
• the PEG polymer-conjugated lipid is in an amount of 5 mol% relative to phosphatidylcholine.
• a sulfate salt of the compound of Formula (I) is encapsulated in the liposomes comprising the phosphatidylcholine, cholesterol and PEG polymer-conjugated lipid in a 45:55:2.25 molar ratio.
• the phosphatidylcholine is di stearoylphosphatidylcholine (DSPC) or hydrogenated soy phosphatidylcholine (HSPC).
• the PEG polymer-conjugated lipid is PEG(Mol. weight 2,000)-distearoylglycerol (PEG-DSG) or PEG(Mol.
• the liposome composition further comprises a chelator selected from the group consisting of deferoxamine (DFO) and EDTA, wherein the chelator is at a concentration of 0.1-1 mM.
• DFO deferoxamine
• EDTA EDTA
• the compound of Formula (I) is a compound selected from AKG-28, AKG-29,
• the compound of Formula (I) is a sulfate salt of AKG-28 the compound is encapsulated in liposomes formed from hydrogenated soy phosphatidylcholine (HSPC), cholesterol and PEG(2000)-DSPE in a 45:55:2.25 molar ratio, in an aqueous medium at a pH of 7.3-7.7.
• HSPC hydrogenated soy phosphatidylcholine
• cholesterol cholesterol
• PEG(2000)-DSPE in a 45:55:2.25 molar ratio
• aspects of the disclosure relate to an AKG-28 liposome composition
• the liposomes comprising lipids HSPC, cholesterol, and PEG(2000)-DSPE in a molar ratio of 45:55:2.25 or in a mass ratio of 5:3:1, and a pharmaceutically acceptable salt of AKG-28 encapsulated into said liposomes
• the liposome composition is further characterized by any one or more of the following characteristics: (a) the liposome composition comprises mono- or oligolamellar vesicles having z-average diameter of 90-130 nm, or the liposome composition comprises mono- or oligolamellar vesicles having a z-average diameter of 100-130 nm; (b) the liposome composition has a poly dispersity index of less than 0.15, or the liposome composition has a poly dispersity index of less than 0.10; (c) the drug/lipid ratio of the AKG-28 to the total phospholipid (PhL) in the composition is 99-530 g/mol PhL, or 85-456 g/mol as AKG-28 free base (FB); 99-470 g/mol PhL, or 85-400 g/mol as AKG-28 free base (FB); 230-280 g/mol, or 190-240 g/mol as AKG-28 free base (FB
• aspects of the disclosure relate to method of making liposome composition, the method comprising the steps of: (a) dissolving one or more phospholipid, cholesterol and a PEG-lipid derivative in ethanol to obtain a lipid solution; (b) combining the lipid solution of step (a) with a trapping agent solution to obtain a uniform lipid suspension having a desired phospholipid concentration; (c) extruding the lipid suspension of step (b) through membranes having defined pore sizes, such as polycarbonate track-etched (PCTE) membranes with the nominal pore size of 50-200 nm; (d) purifying liposomes from extraliposomal trapping agent in the extruded lipid suspension to obtain a purified extruded liposome preparation; (e) contacting the liposomes with the compound of Formula (I) in an aqueous medium to effect encapsulation of the compound in the liposomes; (f) optionally removing unencapsulated compound; and (g) providing the lip
• the trapping agent solution of step (b) comprises ammonium sulfate at the concentration of 0.5M.
• the chelator is deferoxamine.
• the extruded liposomes in step (c) are mono-or oligolamellar vesicles having a z-average diameter of 90-130 nm.
• the chelator is deferoxamine and the extruded liposomes in step (c) are mono-or oligolamellar vesicles having a z-average diameter of 90-130 nm.
• aspects of the disclosure relate to method of treating a mycobacterial infection, the method comprising administering to a subject in need thereof a therapeutically effective amount of the liposomal composition.
• the mycobacterial infection is an infection with Mycobacterium tuberculosis, or an infection with a multi-drug resistant (MDR) strain of Mycobacterium tuberculosis, or an infection with an extremely drug resistant (XDR) strain of Mycobacterium tuberculosis.
• MDR multi-drug resistant
• XDR extremely drug resistant
• a liposomal composition for treating a mycobacterial infection.
• a liposomal composition for treating a methicillin resistant Staphylococcus aureus (MRSA) bacterial infection.
• MRSA methicillin resistant Staphylococcus aureus
• liposome preparations of oxazolidinone compounds with improved storage stability are provided.
• oxazolidinone liposome compositions comprising greater than 50 mol% cholesterol relative to sum of cholesterol and non- pegylated phospholipid in the liposome composition and having a pH of 7 or greater have surprisingly improved storage stability properties.
• adding a chelator such as deferoxamine or EDTA reduced the oxidative degradation of cholesterol during storage of oxazolidinone liposome compositions.
• oxazolidinone liposome compositions comprising ammonium displaced from the liposomes comprising an ammonium sulfate trapping agent during oxazolidinone drug loading (e.g., by omitting a post-loading buffer exchange) exhibited improved phosphatidylcholine storage stability.
• oxazolidinone liposome compositions provided herein consist of lipids consisting ofHSPC, cholesterol andPEG-DSPE in a mass ratio of about 5:3:1. In some embodiments, oxazolidinone liposome compositions provided herein consist of lipids consisting of HSPC, cholesterol and PEG-DSPE in a molar ratio of 45:55:2.25. In some embodiments, oxazolidinone liposome compositions comprise an oxazolidinone consisting of AKG-28 or a pharmaceutically acceptable salt thereof. In some embodiments, oxazolidinone liposome compositions comprise an oxazolidinone consisting of AKG-38 or a pharmaceutically acceptable salt thereof.
• liposome compositions comprising liposome vesicles and an oxazolidinone are provided.
• oxazolidinone liposome compositions comprise liposome vesicles encapsulating an oxazolidinone sulfate are provided.
• the liposome vesicles are in an aqueous medium.
• an oxazolidinone liposome composition can be obtained by a process comprising the step of combining oxazolidine compounds with a purified, extruded lipid suspension under conditions effective to form the oxazolidinone liposomes.
• the purified, extruded lipid suspension can comprise lipid components consisting of a phospholipid, cholesterol and optionally aPEG-lipid derivative combined in an aqueous medium at a desired concentration and a trapping agent such as ammonium sulfate (AS).
• the lipid components of the extruded lipid suspension consist of HSPC, cholesterol and PEG(2000)-DSPE. In some embodiments, the lipid components of the extruded lipid suspension comprises HSPC and cholesterol in a molar ratio of 45:55. In some embodiments, the lipid components of the extruded lipid suspension comprises HSPC and cholesterol in a weight ratio of 5:3. In some embodiments, the lipid components of the extruded lipid suspension consist of HSPC, cholesterol and PEG(2000)-DSPE in a molar ratio of 45:55:2.25. In some embodiments, the lipid components of the extruded lipid suspension consist of HSPC, cholesterol and PEG(2000)-DSPE in a weight ratio of 5:3 : 1.
• the purified, extruded lipid suspension is obtained by a process comprising the steps of: (a) dissolving one or more phospholipid, cholesterol and a PEG- lipid derivative in ethanol; (b) combining the lipid solution of step (a) with a trapping agent solution (e.g., 0.5 M ammonium sulfate) to obtain a uniform lipid suspension having a desired phospholipid concentration (e.g., 60 mM phospholipid); (c) extruding the lipid suspension of step (b) through membranes having defined pore sizes, such as polycarbonate track-etched (PCTE) membranes with the nominal pore size of 50-200 nm; and (d) purifying liposomes from extraliposomal trapping agent in the extruded lipid suspension (e.g., by tangential flow filtration on a hollow fiber cartridge) to obtain a purified extruded liposome preparation.
• a trapping agent solution e.g., 0.5 M ammoni
• the liposomes are mono-or oligolamellar vesicles having a z-average diameter of 90-130 nm or 100-130 nm. In some embodiments, the liposomes are mono-or oligolamellar vesicles having a poly dispersity index of less than 0.15 or less than 0.10.
• the purified extruded liposomes can be loaded with an oxazolidinone drug in a subsequent drug loading step.
• a drug stock solution of an oxazolidinone drug compound or salt thereof can be combined at a desired drug to phospholipid concentration with the purified, extruded lipid suspension of step (d) to form a drug-liposome mixture under conditions effective to load the drug into the liposomes within the purified extruded liposome preparation.
• the drug loading step comprises an exchange, across the liposome bilayer membrane, of the trapping agent ammonium cation with the oxazolidinone compound, resulting in generation of extraliposomal ammonium in the drug-liposome mixture that is displaced from within the liposomes during the drug loading process.
• unencapsulated drug compound can be purified from the drug-liposome mixture (e.g., by size exclusion chromatography, SEC, dialysis, or diafiltration, such as, tangential flow filtration), and the composition comprising oxazolidinone drug liposomes can be isolated and stored.
• the compound is entrapped in the liposome vesicle with a trapping agent, wherein the trapping agent comprises a polyanion.
• the trapping agent is triethylammonium sucrose octasulfate or ammonium sulfate.
• the trapping agent is tri ethyl ammonium sucrose octasulfate.
• the trapping agent is ammonium sulfate.
• the liposomal composition comprises a salt of the compound, wherein the salt is sulfate, citrate, sucrosofate, a salt with a phosphorylated or sulfated polyol, or a salt with a phosphorylated or sulfated polyanionic polymer.
• the liposomal composition comprises a sulfate salt of the compound.
• the liposomal composition comprises a sulfate or hydrosulfate salt of an oxazolidinone compound of Formula (I).
• the liposomal composition comprises a sulfate or hydrosulfate salt of (AKG-28).
• the liposomal composition comprises a sulfate or hydrosulfate salt of (AKG-38).
• the compound in the liposome vesicle has an aqueous solubility less than 1 mg/mL. In some embodiments, the compound in the liposome vesicle has an aqueous solubility less than 0.1 mg/mL.
• the liposome vesicle comprises a membrane comprising phosphatidylcholine and cholesterol. In some embodiments, the liposome vesicle comprises a membrane comprising phosphatidylcholine and cholesterol, wherein the membrane separates the inside of the liposome vesicles from the aqueous medium.
• the phosphatidylcholine is distearoylphosphatidylcholine (DSPC) or hydrogenated soy phosphatidylcholine (HSPC). In some embodiments, the phosphatidylcholine to cholesterol molar ratios is from about 60:40 to 35:65.
• the phosphatidylcholine to cholesterol molar ratio is from about 55:45 to about 35:65. In some embodiments, the phosphatidylcholine to cholesterol molar ratio is from about 50:50 to about 40:60. In some embodiments, the phosphatidylcholine to cholesterol molar ratio is from about 50:50 to about 45:55.
• the membrane further comprises a polymer-conjugated lipid. In some embodiments, the liposome vesicle comprises HSPC, cholesterol and polymer-conjugated lipid in about 45:55:2.75 molar ratio.
• the liposome vesicle comprises HSPC, cholesterol and polymer-conjugated lipid in a 45:55:2.25 molar ratio.
• the polymer-conjugated lipid is PEG(Mol. weight 2,000)-distearoylglycerol (PEG-DSG) orPEG(Mol. weight 2,000)-distearoylphosphatidylethanolamine (PEG-DSPE).
• the liposomes in the liposome composition have Z-average particle size from about 80 to about 130 nm.
• the drug liposomes are provided in an aqueous medium comprising sodium chloride and optionally further comprising ammonium displaced from the liposome during the drug loading process.
• the concentration of sodium chloride in the liposome composition is 50-80 mM.
• the concentration of sodium chloride in the liposome aqueous composition is 130-150 mM.
• the drug liposomes are provided in an aqueous medium comprise 20-60 mM ammonium displaced from the liposome during the drug loading process.
• the concentration of the ammonium in the liposome aqueous medium is less than 0.5 mM.
• the osmolality of the aqueous medium of the liposome composition is 270-330 mOsmol/kg. In some embodiments, the osmolality of the aqueous medium of the liposome composition is 270-310 mOsmol/kg.
• the oxazolidinone liposome composition has a pH greater than about 6.7. In some embodiments, the oxazolidinone liposome composition has a pH of 7-8. In some embodiments, the oxazolidinone liposome composition further comprises a buffer to bring the pH of the liposome aqueous medium to about 7.3-7.7. In some embodiments, the oxazolidinone liposome composition further comprises a buffer to bring the pH of the liposome aqueous medium to about 7.5. In some embodiments, oxazolidinone liposome composition comprises a buffer substance selected from the group consisting of HEPES and phosphate.
• oxazolidinone liposome composition comprises HEPES buffer. In some embodiments, oxazolidinone liposome composition comprises phosphate buffer. In some embodiments, oxazolidinone liposome composition comprises a buffer substance selected from the group consisting of HEPES and phosphate at a concentration of 5-50 mM. In some embodiments, oxazolidinone liposome composition comprises a buffer substance selected from the group consisting of HEPES and phosphate at a concentration of 20 mM.
• the oxazolidinone liposome composition further comprises a chelator. In some embodiments, the oxazolidinone liposome composition further comprises a chelator selected from the group consisting of: deferoxamine (DFO) and EDTA. In some embodiments, the oxazolidinone liposome composition further comprises a chelator selected from the group consisting of: deferoxamine (DFO) and EDTA at a concentration of 0.1-1 mM. In some embodiments, the oxazolidinone liposome composition further comprises a chelator selected from the group consisting of: deferoxamine (DFO) and EDTA at a concentration of 0.5mM.
• DFO deferoxamine
• EDTA EDTA
• oxazolidinone drug compounds were efficiently (>95%) loaded into extruded liposomes at increased drug to lipid ratios (Example 51) with blood PK characteristics close to that of liposomes with lower drug to lipid ratios (Example 53).
• oxazolidinone liposome preparations can be stabilized by retaining ammonium displaced from the trapping agent within the liposomes during the drug loading process (e.g., by omitting the buffer exchange step).
• the oxazolidinone drug compound is AKG-28
• the liposome composition comprises a sulphate salt of AKG-28 formed within the liposomes during the drug loading process.
• the AKG-28 liposome is prepared using a drug stock solution obtained by dissolving a salt form of (5R)-3- ⁇ 3-Fluoro-4-[6-(2-(2- dimethylaminoethyl)-2H-tetrazol-5-yl)-3-pyridinyl]phenyl ⁇ -5-(methylamino)-l,3-oxazolidin-2- one (AKG-28).
• salts of AKG-28 are provided, including hydrochloride salts of AKG-28.
• the salts of AKG-28 are useful in preparing the drug stock solution for loading the AKG-28 liposomes.
• the AKG-28 ion exchanges with the ammonium displaced from an ammonium sulfate trapping agent within the liposome, forming an AKG-28 salt within the AKG-28 liposome.
• the AKG-28 liposome composition has AKG-28 at a drug/lipid ratio of 230-380 g/mol total phospholipid (PhL). In some embodiments, the AKG-28 liposome composition has AKG-28 at a drug/lipid ratio of 230-290 g/mol total phospholipid (PhL). In some embodiments, the AKG-28 liposome composition has AKG-28 at a drug/lipid ratio of 290-360 g/mol total phospholipid (PhL). In some embodiments, the AKG-28 liposome composition has AKG-28 at a drug/lipid ratio of 300-340 g/mol total phospholipid (PhL).
• the AKG-28 liposome composition has AKG-28 at a drug/lipid ratio of about 250 g/mol total phospholipid (PhL). In some embodiments, the AKG-28 liposome composition has AKG-28 at a drug/lipid ratio of about 330 g/mol total phospholipid (PhL). In some embodiments, the overall (or total) concentration of AKG-28 in a liposome composition is 8-15 mg/ml. In some embodiments, the overall concentration of AKG-28 in a liposome composition is 9-11 mg/ml. In some embodiments, the proportion of encapsulated AKG-28 to overall AKG-28 in the AKG-28 liposome composition is at least 90%, at least 95%, at least 97% or at least 98%.
• the oxazolidinone drug compound is AKG-38
• the liposome composition comprises a sulphate salt of AKG-38 formed within the liposomes during the drug loading process.
• the AKG-38 liposome is prepared using a drug stock solution obtained by dissolving a salt form of (5R)-3- ⁇ 3-Fluoro-4-[6-(2-(2- dimethylaminoethyl)-2H-tetrazol-5-yl)-3-pyridinyl]phenyl ⁇ -5-(methylacetamido)-I,3- oxazolidin-2-one (AKG-38).
• salts of AKG-38 are provided, including hydrochloride salts of AKG-38.
• the salts of AKG-38 are useful in preparing the drug stock solution for loading the AKG-38 liposomes.
• the AKG-38 ion exchanges with the ammonium displaced from an ammonium sulfate trapping agent within the liposome, forming an AKG-38 salt within the AKG-38 liposome.
• the AKG-38 liposome composition has AKG-38 at a drug/lipid ratio of 430-680 g/mol total phospholipid (PhL). In some embodiments, the AKG-38 liposome composition has AKG-38 at a drug/lipid ratio of 500-650 g/mol total phospholipid (PhL). In some embodiments, the AKG-28 liposome composition has AKG-38 at a drug/lipid ratio of 550-650 g/mol total phospholipid (PhL). In some embodiments, the AKG-38 liposome composition has AKG-38 at a drug/lipid ratio of about 450 g/mol total phospholipid (PhL).
• the AKG-38 liposome composition has AKG-38 at a drug/lipid ratio of about 600 g/mol total phospholipid (PhL).
• the overall concentration of AKG-38 in a liposome composition is 12-25 mg/ml.
• the overall concentration of AKG-38 in a liposome composition is 13.5-16.5 mg/ml.
• the overall concentration of AKG-38 in a liposome composition is about 15 mg/ml.
• the overall concentration of AKG-38 in a liposome composition is about 20 mg/ml.
• the proportion of encapsulated AKG-38 to overall AKG-38 in the AKG-38 liposome composition is at least 90%, at least 95%, at least 97% or at least 98%.
• Cholesterol and HSPC degradation was observed in certain AKG-28 and AKG-38 liposome compositions during accelerated stability testing of oxazolidinone liposome preparations (Examples 54, 65).
• FIG. 17 is a scheme showing the two major cholesterol oxidation degradation products, 7-hydroxy-cholesterol (alpha- and beta- isomers), and 7-ketochol esterol.
• FIG. 17 is a scheme showing the two major cholesterol oxidation degradation products, 7-hydroxy-cholesterol (alpha- and beta- isomers), and 7-ketochol esterol.
• HSPC Hydrogenated soy phosphatidylcholine
• HSPC is a 1,2-diacyl-sn- glycero-phosphocholine, where the 1 and 2 acyl chain positions are saturated fatty acids C16 to C22, being primarily stearic (C18) and palmitic (C16) acid.
• Distearoylphosphatidylcholine is the largest component of HSPC.
• AKG-28 liposomes can comprise displaced ammonium in an amount equal to or greater than the molar equivalent of AKG-28 drug loaded into the liposomes.
• the degradation of HSPC was minimized during accelerated stability testing in AKG-38 liposomes without post-drug loading buffer exchange (Example 55).
• the liposome composition is stable against degradation of the liposome lipid components and has pH > 7.0. It was discovered that the rate of lipid degradation, in particular, degradation of cholesterol depends on the liposome formulation pH and is lower at pH above 7.0 (Example 65). In some embodiments, the liposome composition has the pH of at least 7.1, at least 7.2, or at least 7.3, and no more than pH 8.0, no more than pH 7.7, or no more than pH 7.6. In some embodiments, the degree of cholesterol degradation after 3 months at 37°C is less than 10%, less than 5%, or less than 1% of the total cholesterol.
• the degree of phospholipid degradation after 6 weeks at 37°C is less than 10%, less than 5%, or less than 1% of the total phospholipid.
• the phospholipid is phosphatidylcholine.
• the phospholipid is HSPC, and the pH is between pH 7.3-7.6.
• the liposome composition comprises cholesterol and is stable against degradation of cholesterol, the degree of cholesterol degradation after 3 months at 37°C being less than 10%, less than 5%, or less than 1% of the total cholesterol. Avoiding degradation of cholesterol is important because the products of cholesterol degradation are toxic and may cause vascular endothelial injury (Rong et al., Arteriosclerosis, Thrombosis, and Vascular Biology, 1998, vol. 18, p.1885-1894; Sevanian et al., JLipidRes, 1995, vol. 36, p.1971-1986).
• the liposome composition comprises a chelator.
• the chelator is a chelator known to be tolerated in humans.
• the chelator is deferoxamine (Desferal, DFO), ethylenediamine tetraacetic acid (EDTA), diethylenetriamine pentaacetic acid (DTP A), nitrilotriacetic acid (NTA), ethyleneglycol-O, O'-bis(2-aminoethyl)-N, N, N', N' -tetraacetic acid (EGTA), N-(2-hydroxyethyl)ethylenediamine-N, N', N'-triacetic acid (HEDTA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), including their pharmaceutically acceptable salts.
• DFO deferoxamine
• EDTA ethylenediamine tetraacetic acid
• DTP A diethylenetriamine pentaacetic acid
• NTA nitrilotriacetic acid
• EGTA O'-bis(2-aminoethy
• the chelator is present in the composition at the concentration of at least 0.01 mM, at least 0.05 mM, at least 0.1 mM, at least 0.2 mM, or at least 0.5 mM, and not more than 1 mM, nor more than 2 mM, not more than 5 mM, or not more than 10 mM.
• the chelator is deferoxamine or deferoxamine mesylate, and the chelator concentration is about 0.5 mM.
• the external medium of the liposome composition has less than 0.5 mEq/L(milligram-equivalents per liter) of ammonium or substituted ammonium.
• the liposome composition contains in the liposome external medium an ammonium or substituted ammonium in the concentration of at least 10 mEq/L, at least 15 mEq/L, or at least 20 mEq/1, and no more than 200 mEq/L, no more than 150 mEq/L, no more than 100 mEq/L, no more than 80 mEq/L, or no more than 60 mEq/L.
• the liposome composition contains in the liposome external medium an ammonium or substituted ammonium in the concentration of at least 10 mEq/L, at least 15 mEq/L, or at least 20 mEq/1, and no more than 200 mEq/L, no more than 150 mEq/L, no more than 100 mEq/L, no more than 80 mEq/L, or no more than 60 mEq/L, and is stable against phospholipid degradation, the degree of phospholipid degradation after 6 weeks at 37°C being less than 10%, less than 5%, or less than 1% of the total phospholipid.
• the phospholipid is phosphatidylcholine.
• the phospholipid is HSPC
• the ammonium salt is ammonium chloride, ammonium sulphate, or a combination thereof, at the ammonium concentration of 10-80 mM, or 15-60 mM.
• the normality of ammonium in the external medium of the liposome composition is within 90-110% of the normality of encapsulated drug at the drug loading step, normality being the concentration expressed in gram-equivalents/L (eq/L).
• the liposome composition comprises encapsulated compound of Formula 1b atthe drug/lipid (DL) ratio of 300-350 g/mol PhL. In some embodiments, the liposome composition comprises encapsulated compound of Formula 1b at the DL ratio of 300-350 g/mol PhL and is characterized by the in vivo drug release half-life in the blood of a CD- 1 mouse of more than 80 hours, more than 200 hours, or more than 300 hours.
• the liposome composition comprises encapsulated compound of Formula 1c at the DL ratio of 500-650 g/mol PhL.
• the liposome composition comprises liposomes in an aqueous medium, the liposomes composed of HSPC, cholesterol, and PEG(2000)-DSPE in the molar ratio of 45:55:2.25 or in the mass ratio of 5:3:1, the liposomes being mono- or oligolamellar vesicles having z-average diameter of 90-130 nm or 100-130 nm, and poly dispersity index of less than 0.15 or less than 0.10, the liposomes containing encapsulated compound AKG-28 at the drug/lipid (DL) ratio of 230-280 g/mol phospholipid (PhL), 290-360 g/mol PhL, 300-340 g/mol PhL, about 250 g/mol PhL, or about 330 g/mol PhL, the overall concentration of AKG-28 in the composition being 8-15 mg/ml or 9-11 mg/ml, and the proportion of encapsulated AKG-28 to overall AKG-
• DL drug/lipid
• the aqueous medium comprises sodium chloride and optionally an ammonium ion.
• the osmolality of the aqueous medium is 270-330 mOsmol/kg or 270- 310 mOsmol/kg.
• the ammonium concentration in the aqueous medium is 20-60 mM, and the concentration of sodium chloride is 50-80 mM.
• the concentration of ammonium in the aqueous medium is less than 0.5 mM, and the concentration of sodium chloride is 130-150 mM.
• the composition also contains a buffer substance to bring the pH of the aqueous medium to about 7.3-7.7, or about pH 7.5.
• the buffer substance is HEPES or phosphate, at the concentration of 5-50 mM, or of about 20 mM.
• the composition can also contain a chelator, the chelator being deferoxamine (DFO) or EDTA, at the concentration of 0.1-1 mM, or about 0.5 mM.
• the liposome composition is storage-stable.
• the liposome composition comprises liposomes in an aqueous medium, the liposomes composed of HSPC, cholesterol, and PEG(2000)-DSPE in the molar ratio of 45:55:2.25 or in the mass ratio of 5:3:1, the liposomes being mono- or oligolamellar vesicles having z-average diameter of 90-130 nm or 100-130 nm and polydispersity index of less than 0.15, or less than 0.10, the liposomes containing encapsulated compound AKG-38 at the drug/lipid ratio of 430-480 g/mol phospholipid (Phi,), 500-650 g/mol PhL, 550-650 g/mol PhL, about 450 g/mol PhL, or about 600 g/mol PhL, the overall concentration of AKG-38 in the composition being 12-25 mg/ml, 13.5-16,5 mg/ml, about 15 mg/ml, or about 20 mg/ml, and
• the aqueous medium comprises sodium chloride and optionally an ammonium ion.
• the osmolality of the aqueous medium is 270-330 mOsmol/kg or 270-310 mOsmol/kg.
• the ammonium concentration in the aqueous medium is 20-60 mM, and the concentration of sodium chloride is 50-80 mM, In some embodiments, the concentration of ammonium in the aqueous medium is less than 0.5 mM, and the concentration of sodium chloride is 130-150 mM.
• the composition also contains a buffer substance to bring the pH of the medium to about 7.3-7.7, or about pH 7.5.
• the buffer substance is HEPES or phosphate, at the concentration of 5-50 mM, or of about 20 mM.
• the composition can also contain a chelator, the chelator being deferoxamine (DFO) or EDTA, at the concentration of 0.1-1 mM, or about 0.5 mM.
• the liposome composition is storage-stable.
• the liposome composition is stable against degradation of the encapsulated compound upon storage.
• the degradation of the encapsulated compound upon storage under the accelerated degradation conditions (37 °C), as measured by the decrease of the compound purity, expressed in percentage points, is less than 5%, less than 4%, less than 3%, less than 2%, or about 1% or less after three months of storage.
• the degradation of the encapsulated compound upon storage under the accelerated degradation conditions (37°C), as measured by the decrease of the overall concentration of the intact compound in the liposome composition is less than 20%, less than 10%, or less than 5% after three months of storage.
• the encapsulated compounds are AKG-28 or AKG-38.
• a liposomal composition of AKG-38 stored at 37 °C for three months, showed remarkably low decrease of AKG-38 purity from 98.99% to 98.07% (0.92 percentage points) and the low overall decrease in the intact AKG-38 concentration from 19.9 mg//ml to 19.06 mg/ml (4.2% decrease) (Example 68).
• FIG. 1 is a graph showing the effect of pH on the liposome loading of compounds AKG-3, AKG-5, and AKG-16.
• FIG. 2A and FIG. 2B are graphs showing the encapsulation of compounds AKG-3, AKG-5, and AKG-16 into liposomes with TEA-SOS trapping agent at different drug-to-lipid (DL) ratios
• FIG. 2A shows the effect of the added drug-to-lipid (DL0) ratio, in grams of the drug per mole of liposome phospholipid (PhL), on the liposome payload, expressed as post-load drug-to- lipid ratio (DL).
• FIG. 2B shows the effect the DL0 ratio (drug-to-lipid input ratio) on liposome loading efficiency, calculated as percent of post-load DL relative to DL0.
• FIG. 3A, FIG. 3B FG. 3C, and FIG. 3D are graphs showing the encapsulation of compounds AKG-3, AKG-5, and AKG-16 into liposomes with 0.5M ammonium sulfate as a trapping agent at different DL ratios.
• FIG. 3 A shows the effect the DL0 ratio on liposome payload for AKG-5, and AKG-16.
• FIG. 3B shows the effect the DL0 ratio on liposome loading efficiency for AKG-5, and AKG-16.
• FIG. 3C shows the effect the DL0 ratio on liposome payload for AKG- 3.
• FIG. 3D shows the effect the DL0 ratio on liposome loading efficiency for AKG-3.
• FIG. 4A and FIG. 4B are graphs showing the encapsulation of AKG-28 and AKG- 38 with TEA-SOS and ammonium sulfate as trapping agents at different DL0 ratio.
• FIG. 4A shows the effect the DL0 ratio on liposome payload.
• FIG. 4B shows the effect the DL0 ratio on loading efficiency.
• FIG. 5A, FIG. 5B, FIG. 5C, and FIG. 5D are graphs showing the dependence of fast drug leakage from the liposomes encapsulating compounds AKG-28 (FIG. 5A, FIG. 5C) and AKG-38 (FIG. 5B, FIG. 5D) upon in vitro contact with blood plasma of a mouse (denoted “mouse”) or a human (denoted “human”) as described in Example 19 below.
• Liposomes contained 5 mol% ofPEG(2000)-DSPE (denoted “DSPE”) or PEG-DSG (denoted “DSG”).
• Trapping agents 0.5M ammonium sulfate (AS) (FIG. 5 A, FIG. 5B), IN tri ethyl ammonium sucrose octasulfate (TEA-SOS) (FIG. 5C, FIG. 5D).
• FIG. 6 represents the numbered ring structure of a compound of Formula (I).
• FIG. 7 is a graph showing the plasma concentration versus time profiles for total drug in Sprague-Dawley rats after administration of a single intravenous dose (IV x 1) of Ls- AKG28 at 10 mg/kg (diamonds), 20 mg/kg (squares), and 40 mg/kg (circles).
• IV x 1 intravenous dose
• PO x 1 single oral dose
• 5 % methyl cellulose pH 3-4
• FIG. 8 is a graph showing the plasma concentration versus time profiles for total drug in Sprague-Dawley rats after administration of a single intravenous dose (IV x 1) of Ls- AK.G38 at 20 mg/kg (diamonds), 40 mg/kg (squares), and 80 mg/kg (diamonds).
• IV x 1 intravenous dose
• FIG. 9A, FIG. 9B, and FIG. 9C are graphs showing the plasma concentration versus time profiles for total drug in Sprague-Dawley rats after administration of Ls-AKG28 at 10 mg/kg (FIG. 9A), 20 mg/kg (FIG. 9B), and 40 mg/kg (FIG. 9C), IV x 1, on day 1 (circles), day 15 (squares), day 29 (diamonds), and day 43 (triangles). The mean and SD concentration are presented at each time point.
• FIG. 10A, FIG. 10B, and FIG. 10C are graphs showing the plasma concentration versus time profiles for total drug in Sprague-Dawley rats after administration of Ls-AKG38 at 20 mg/kg (FIG. 10 A), 40 mg/kg (FIG. 10B), and 80 mg/kg (FIG. 10C), IV x 1, on day 1 (circles), day 15 (squares), day 29 (diamonds), and day 43 (triangles). The mean and SD concentration are presented at each time point.
• FIG. 11 A, FIG. 1 IB, and FIG. 11C are graphs showing the plasma concentration versus time profiles of both lipid (using nonexchangeable DiIC18(3)-DS label), drug for liposomal AKG-28 (FIG. 11A) and liposomal AKG-38 (FIG. 1 IB), and the change in plasma drug-to-lipid ratio, a measure of drug release rate from the liposomes, for both Ls-AKG28 and Ls-AKG38 (FIG. 11C) in CD-I mice after single intravenous injection in CD-I mice. The mean and SD are presented at each time point.
• FIG. 12 is a graph showing the plasma drug concentration presented as % injected dose for Ls-AKG28 and Ls-AKG38 were compared were multiple formulations of liposomal AKG-28 and liposomal AKG-38 after the first and fourth weekly doses. Mice were injected with the indicated dose and formulation once per week for a total of 4 injections.
• FIG. 13A is a graph showing the effect of Ls-AKG28 dose escalation on female CD-I mice body weight over time.
• FIG. 13B is a graph showing the effect of Ls-AKG38 dose escalation on female CD-I body weight in mice over time.
• FIG. 13C are graphs showing the effects of Ls-AKG28 and Ls-AKG38 in combination with BP or BPM on hematology (RBC, HTC, PLT, WBC) and blood biochemistry (ALT, AST) parameters in female CD-I mice.
• FIG. 13D is a heat map showing the effect of monotherapy Ls-AKG28 or Ls- AKG38 on tissue pathological findings in female CD-I mice.
• FIG. 14A is a graph showing the effect of Ls-AKG28 in combination with bedaquiline and pretomanid (BP) or bedaquiline, pretomanid, and moxifloxacin (BPM) on female CD-I mice body weight over time.
• BP bedaquiline and pretomanid
• BPM moxifloxacin
• FIG. 14B is a graph showing the effect of Ls-AKG38 in combination with BP or BPM on female CD-I mice body weight over time.
• FIG. 14C are graphs showing the effect of Ls-AKG28 and Ls-AKG38 in combination with BP or BPM on hematology (RBC, HTC, PLT, WBC) and blood biochemistry (ALT, AST) parameters in female CD-I mice.
• FIG. 14D is a heat map showing the effect of Ls-AKG28 and Ls-AKG38 in combination with BP or BPM on tissue pathology findings in female CD-I mice.
• FIG. 15A is a graph showing the body weight change in female CD-I mice treated with Ls-AKG28 injected twice a week (2qw) at 50 mg/kg or once a week (Iqw) at 100 mg/kg alone or in in combination with BP over time.
• FIG. 15B is a graph showing the body weight change in female CD-I mice treated with Ls-AKG38 injected 2qw at 100 mg/kg or Iqw at 200 mg/kg alone or in combination with BP.
• FIG. 15C are graphs showing the hematology and blood biochemistry parameters in female CD-I mice treated with Ls-AKG28 (2qw at 50 mg/kg or Iqw at 100 mg/kg) or Ls- AKG28 (2qw at 100 mg/kg or Iqw at 200 mg/kg) alone or in combination with BP.
• FIG. 15D is a heat map showing the histopathology results of female CD-I mice treated with Ls-AKG28 (2qw at 50 mg/kg or Iqw at 100 mg/kg) or Ls-AKG28 (2qw at 100 mg/kg or Iqw at 200 mg/kg) alone, or in combination with BP.
• FIG. 16A is a graph showing the effect of Ls-AKG28 on body weight in male Sprague-Dawley rats treated chronically for a total of eight weeks over time.
• FIG. 16B is a graph showing the effect of Ls-AKG38 on body weight in male Sprague-Dawley rats treated chronically for a total of eight weeks over time.
• FIG. 17 is a scheme showing the two major cholesterol oxidation degradation products, 7 -hydroxy-cholesterol (alpha- and beta- isomers), and 7-ketochol esterol.
• FIG. 18 is a scheme showing breakdown of distearoylphosphatidylcholine (DSPC) to lysophosphatidylcholine and stearic acid.
• Hydrogenated soy phosphatidylcholine (HSPC) is a 1,2-diacyl-sn-glycero-phosphocholine, where the 1 and 2 acyl chain positions are saturated fatty acids C16 to C22, being primarily stearic (C18) and palmitic (Cl 6) acid.
• Distearoylphosphatidylcholine is the largest component of HSPC.
• FIG. 19A is a graph showing data for cholesterol degradation of AKG-38 liposome compositions for 12 weeks at room temperature.
• FIG. 19B is a graph showing data for cholesterol degradation of AKG-28 liposome compositions for 12 weeks at room temperature.
• FIG. 20A, FIG. 20B, and FIG. 20C are graphs showing the plasma concentration versus time profdes of AKG-28 drug, liposome lipid (using nonexchangeable DiIC18(3)-DS label), and plasma drug-to-lipid ratio for liposomal AKG-28 lots Ls-338 (sample 71), Ls-339 (sample 74), and Ls-340S (sample 76) after single intravenous injection in CD-I mice.
• the liposome characteristics are given in Example 59.
• the datapoints are the mean of three animals.
• FIG. 21 is a graph showing the data for cholesterol degradation of AKG-38 liposome composition lot Ls-371 (Example 67) upon storage at 37°C in the presence of various concentration of deferoxamine.
• FIG. 22 is a graph showing the data for HSPC degradation of AKG-38 liposome composition lot Ls-371 (Example 67) upon storage at 37°C in the presence of various concentration of deferoxamine.
• Fig. 23 is a graph showing the changes of pH in the AKG-38 liposome composition lot Ls-371 (Example 67) upon storage at 37°C in the presence of various concentration of deferoxamine.
• Fig. 24 shows synthesis Scheme-1 according to embodiments of the disclosure.
• Fig. 25 shows synthesis Scheme-2 according to embodiments of the disclosure.
• Fig. 26 shows synthesis Scheme-3 according to embodiments of the disclosure.
• Fig. 27 shows synthesis Scheme-4 according to embodiments of the disclosure.
• Fig. 28 shows synthesis Scheme-5 according to embodiments of the disclosure.
• the liposome compositions comprise compound of Formula (I) encapsulated in lipid vesicles.
• the liposome compositions comprise an oxazolidinone compound as a pharmaceutically acceptable salt thereof, and lipid vesicles comprising a phospholipid and cholesterol.
• the liposome compositions comprise an oxazolidinone compound as a pharmaceutically acceptable salt thereof, and lipid vesicles comprising a phospholipid and more than 50 mol% cholesterol relative to the sum of cholesterol and non- pegylated phospholipid in the liposome composition.
• the liposome compositions comprise an oxazolidinone compound as a pharmaceutically acceptable salt thereof, and lipid vesicles comprising a phospholipid and more than about 50 mol% cholesterol relative to the sum of cholesterol and non-pegylated phospholipid in the liposome composition.
• the liposome compositions comprise an oxazolidinone compound as a pharmaceutically acceptable salt thereof, and lipid vesicles comprising a phospholipid and between 50-65 mol% cholesterol relative to the sum of cholesterol and non-pegylated phospholipid in the liposome composition.
• the liposome compositions comprise an oxazolidinone compound as a pharmaceutically acceptable salt thereof, and lipid vesicles comprising a phospholipid and between 50-60 mol% cholesterol relative to the sum of cholesterol and non-pegylated phospholipid in the liposome composition.
• the liposome compositions comprise an oxazolidinone compound as a pharmaceutically acceptable salt thereof, and lipid vesicles comprising a phospholipid and between 50-55 mol% cholesterol relative to the sum of cholesterol and non-pegylated phospholipid in the liposome composition.
• the liposome compositions comprise an oxazolidinone compound as a pharmaceutically acceptable salt thereof, and lipid vesicles comprising a phospholipid and about 50 mol% cholesterol relative to the sum of cholesterol and non-pegylated phospholipid in the liposome composition.
• the liposome compositions comprise an oxazolidinone compound as a pharmaceutically acceptable salt thereof, and lipid vesicles comprising a phospholipid and about 55 mol% cholesterol relative to the sum of cholesterol and non-pegylated phospholipid in the liposome composition.
• oxazolidinone liposome compositions are provided that are characterized by reduced amounts of phospholipid or cholesterol degradation during storage.
• oxazolidinone liposome compositions having a pH of about 7 or greater (e.g., 7-8) and comprising a phospholipid and more than 50 mol% cholesterol (e.g. 50-65 mol%, 50-60 mol%, 50-55 mol%, about 50 mol%, or about 55 mol%) relative to the sum of cholesterol and non- pegylated phospholipid in the liposome composition.
• oxazolidinone liposome compositions further comprise a chelator such as DFO or EDTA in combination with a phospholipid and more than 50 mol% cholesterol relative to the sum of cholesterol and non- pegylated phospholipid in the liposome composition.
• oxazolidinone liposome compositions having a pH of 7-8 further comprise a chelator such as DFO or EDTA in combination with a phospholipid and more than 50 mol% cholesterol relative to the sum of cholesterol and non-pegylated phospholipid in the liposome composition.
• oxazolidinone liposome compositions further comprise extra-liposomal ammonium in combination with a vesicle comprising phospholipid and more than 50 mol% cholesterol relative to the sum of cholesterol and non-pegylated phospholipid in the liposome composition.
• oxazolidinone liposome compositions further comprise extra-liposomal ammonium generated during the drug loading of a oxazolidinone into liposome vesicles comprising phospholipid and more than 50 mol% cholesterol relative to the sum of cholesterol and non-pegylated phospholipid in the liposome composition.
• compositions, methods, and respective component(s) thereof are used in reference to compositions, methods, and respective component(s) thereof, that are present in a given embodiment, yet open to the inclusion of unspecified elements.
• the term "consisting essentially of' refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional character! stic(s) of that embodiment of the disclosure.
• compositions, methods, and respective components thereof refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.
• the term “about” means acceptable variations within 20%, within 10% and within 5% of the stated value. In certain embodiments, "about” can mean a variation of +/-!%, 2%, 3%, 4%, 5%, 10% or 20%.
• the term "effective amount” as used herein with respect to a compound or the composition means the amount of active compound (also referred herein as active agent or drug) sufficient to cause a bactericidal or bacteriostatic effect. In one embodiment, the effective amount is a "therapeutically effective amount” meaning the amount of active compound that is sufficient alleviate the symptoms of the bacterial infection being treated.
• subject refers to an animal, preferably a mammal, most preferably a human that receives either prophylactic or therapeutic treatment.
• administration includes all means of introducing the compounds or the pharmaceutical compositions to the subject in need thereof, including but not limited to, oral, intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, inhalation, buccal, ocular, sublingual, vaginal, rectal and the like. Administration of the compound or the composition is suitably parenteral.
• the compounds or the composition can be preferentially administered intravenously but can also be administered intraperitoneally or via inhalation like is currently used in the clinic for liposomal amikacin in the treatment of mycobacterium avium (see Shirley et al., Amikacin Liposome Inhalation Suspension: A Review in Mycobacterium avium Complex Lung Disease. Drugs. 2019 Apr; 79(5): 555-562)
• treat refers to therapeutic or preventative measures such as those described herein.
• pharmaceutically acceptable salt refers to a relatively non-toxic, inorganic or organic acid addition salt of a compound of the present disclosure which salt possesses the desired pharmacological activity.
• alkyl means saturated carbon chains which may be linear or branched or combinations thereof, unless the carbon chain is defined otherwise.
• alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec- and tert-butyl, pentyl, hexyl, heptyl, octyl, and the like.
• aminoalkyl means an alkyl wherein at least one carbon of an alkyl carbon chain forms the bond with an amino group, wherein said amino group is primary amino group, mono-alkyl-substituted (secondary) amino group, di-alkyl-substituted (tertiary) amino group, or an alkyl-substituted amino group where the amine nitrogen atom and the alkyl chain that substitutes for amine hydrogens form a heterocycle.
• liposomes means vesicles composed of a bilayer (unilamellar) and/or a concentric series of multiple bilayers (multi-lamellar) separated by aqueous compartments formed by amphipathic molecules such as phospholipids that enclose a central aqueous compartment.
• the drug substance is generally contained in liposomes.
• water soluble drugs are contained in the aqueous compartment(s) and hydrophobic drugs are contained in the lipid bilayer(s) of the liposomes. Release of drugs from liposome formulations, among other characteristics such as liposomal clearance and circulation half-life, can be modified by the presence of polyethylene glycol and/or cholesterol or other potential additives in the liposome.
• Unilamellar liposomes also referred to as “unilamellar vesicles,” are liposomes that include one lipid bilayer membrane which defines a single closed aqueous compartment.
• the bilayer membrane includes two layers of lipids; an inner layer and an outer layer (leaflet).
• Lipid molecules in the outer layer are oriented with their hydrophilic (“head”) portions toward the external aqueous environment and their hydrophobic (“tail”) portions pointed downward toward the interior of the liposome.
• the inner layer of the lipid lays directly beneath the outer layer, the lipids are oriented with their heads facing the aqueous interior of the liposome and their tails toward the tails of the outer layer of lipid.
• Multilamellar liposomes also referred to as “multilamellar vesicles” or “multiple lamellar vesicles,” include more than one lipid bilayer membrane, which membranes define more than one closed aqueous compartment. The membranes are concentrically arranged so that the different membranes are separated by aqueous compartments.
• encapsulation and “entrapped,” as used herein, refer to the incorporation or association of the oxazolidinone pharmaceutical agent in or with a liposome.
• DL DL ratio
• D/L D/L ratio
• mol% with regard to cholesterol refers to the molar amount of cholesterol relative to the sum of the molar amounts of cholesterol and non-PEGylated phospholipid expressed in percentage points.
• 55 mol.% cholesterol in a liposome containing cholesterol and HSPC refers to the composition of 55 mol. parts of cholesterol per 45 mol. parts of HSPC.
• mol% refers to the ratio of the molar amount of PEG-lipid and non-PEGylated phospholipid expressed in percentage points.
• “5 mol.% PEG-DSPE” in a liposome containing HSPC and PEG-DSPE refers to the composition having 5 mol. parts of PEG-DSPE per 100 mol. parts of HSPC.
• sucrose octasulfate refers the same compound, sucrose octasulfuric acid or an anion thereof, and are used herein interchangeably.
• FB concentration is used to express the mass concentration of a salt-forming compound in its free base form.
• the mass-based concentration or ratio e.g., mg/ml or g/mol phospholipid
• concentration of the compounds isolated in the form of a salt is also expressed as the equivalent concentration of the compound as an anhydrous free base (a FB concentration).
• the calculated molecular weight of the compound in the free base form is divided by the calculated molecular weight of the salt form, and the concentration is multiplied by this factor.
• the molecular weight of AKG-28 as free base is 426.46, and the dihydrochloride form (in which this compound is isolated) has molecular weight of 499.37.
• the correction is also made for a known water content.
• the mass concentration of compounds isolated as free bases e.g., AKG-38
• FB concentration is always expressed as a FB concentration.
• the mass concentration or amount of, e.g., AKG-28, as quoted herein on the basis of its isolated synthetic product form of a dihydrochloride salt, into a molar concentration the mass concentration or amount is divided by the AKG-28 dihydrochloride molecular weight of 499.4 g/mol.
• the concentration is quoted on the compound free base basis, the mass amounts and concentration are divided by the molecular weight of the compound free base.
• the quoted 20 mg/mL concentration of AKG-38 is expressed in molar terms as 20/468.5 - 42.7 mM.
• liposome compositions comprising an oxazolidinone compound are provided.
• Oxazolidinones are synthetic antibiotics that exert their function by inhibiting protein synthesis.
• Linezolid is an oxazolidinone compound that exhibits bacteriostatic activity against M. tuberculosis.
• administration of LZD may cause severe side effects such as anemia, thrombocytopenia, and peripheral neuropathy.
• Tedizolid is an oxazolidinone compound which has been shown to inhibit gram positive bacteria.
• the side effects for tedizolid phosphate are similar, but generally less severe than observed for linezolid, although the experience with prolonged dosing such as that required for the treatment of tuberculosis has been limited for tedizolid phosphate compared to the extensive experience with linezolid.
• aspects of the disclosure relate to compounds that are aminoalkyl derivatives of oxazolidinone (see FIG. 6).
• the aminoalkyl is a dimethylaminoalkyl.
• the aminoalkyl derivatives of oxazolidinone compounds include either an amine or acetamide group at the R 2 positions of the oxazolidinone ring and a dimethylaminoethyl group on the tetrazole ring.
• the compounds having the following chemical Formula (I) and pharmaceutically acceptable salts thereof Formula (I) wherein R 2 is an amine (NH2) or an acetamide (NHCOCH 3 ), and wherein R 1 is a tetrazole ring substituted 1’ with an aminoalkyl.
• the compounds of the present disclosure can exist in free form, e.g. as a free base, or as a free acid, or as a zwitterion, or can exist in the form of a salt.
• Said salt may be any salt, either an organic or inorganic addition salt or a cocrystal, particularly any pharmaceutically acceptable organic or inorganic addition salt or a cocrystal, customarily used in pharmacy. It is understood that the chemical formula showing a compound in a particular salt form or ionic form also discloses this compound in its non-dissociated, free base (or free acid) form.
• the present disclosure encompasses all stereoisomeric forms of the compounds.
• the compounds of Table 1 below are substantially pure (i.e. at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, e.g. 100%) TABLE 1
• the compound has the following chemical formula: Formula 1a
• the compound has the following chemical formula:
• the compound of the Formula 1b is crystallized from aqueous ethanol. In some embodiments the compound of the Formula lb is the form of a dihydrochloride or dihydrochloride monohydrate
• the compound has the following chemical formula: Formula 1c
• the compound has the following chemical formula: Formula Id
• the compound has the following chemical formula: Formula 1e
• the compounds have the chemical formula la, lb, 1c, Id or le. In some embodiments, the compounds have the chemical formula lb.
• the compounds of Formula (I) have a minimum inhibitory concentration (MIC), for example against Mycobacterium tuberculosis, ranging from 0.1 ⁇ g/ml to 1 ⁇ g/ml, from 0.25 ⁇ g/ml to 1 ⁇ g/ml, from 0.5 ⁇ g/ml to 1 ⁇ g/ml, from 0.1 ⁇ g/ml to 0.25 ⁇ g/ml, from 0.1 ⁇ g/ml to 0.5 ⁇ g/ml, from 0.25 ⁇ g/ml to 0.
• MIC minimum inhibitory concentration
• the compounds of Formula (I) have a minimum inhibitory concentration (MIC), for example against Mycobacterium tuberculosis of less than 1 ⁇ g/ml, less than 0.25 ⁇ g/ml, or less than 0.1 ⁇ g/ml.
• MIC minimum inhibitory concentration
• the compounds of Formula (I) have a MIC ranging from 0.01 ⁇ g/ml to 0.25 ⁇ g/ml.
• the compound of Formula (I) have a MIC ranging from 0.01 ⁇ g/ml to 0.1 ⁇ g/ml. It should be appreciated that the MIC values can be lower or than the ranges provided herein depending on the bacteria.
• the compound for the treatment of mycobacterium, for example M. tuberculosis, has a MIC below 0.1 ⁇ g/mL. In some embodiments for the treatment of mycobacterium, for example M. tuberculosis, the compound has a selectivity index (SI) for killing M. tuberculosis vs human kidney cells (VERO) of at least 1,000. In some embodiments for the treatment of mycobacterium, for example M. tuberculosis, the compound has a MIC below 0.1 ⁇ g/mL and a selectivity index (SI) for killing M. tuberculosis vs human kidney cells (VERO) of at least 1,000.
• SI selectivity index
• the compound has the structure of AKG-28 (Formula 1b) or AKG-38 (Formula 1c).
• the MIC is less than 0.05 ⁇ g/mL and the selectivity index for MIC in M. tuberculosis relative to mitochondrial protein synthesis inhibition (SI-MPS) is greater than 20, such as for AKG-28.
• the compounds described herein have a 2-to-20 fold increase (about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20) in potency adjusted dose compared to linezolid for M. tuberculosis.
• the compound has a MIC against MRSA strains of less than 2 ⁇ g/mL. In some embodiments for the treatment of methicillin-resistant Staphylococcus aureus (MRSA), the compound has an IC50 of greater than 100 ⁇ g/ml against human VERO kidney cells. In some embodiments for the treatment of methicillin-resistant Staphylococcus aureus (MRSA), the compound has a MIC against MRSA strains of less than 2 ⁇ g/mL and an IC50 of greater than 100 ⁇ g/mL against human VERO kidney cells. In some embodiments, the compound has the structure of AKG-38 (Formula 1c), AKG-39 (Formula 1e) , and AKG-40 (Formula Id). Aqueous solubility
• the compounds are in the form of salts, e.g., a hydrochloride or mesylate salt and are soluble in water at greater than 1 mg/ml, and preferably greater than 10 mg/ml (and up to 1 g/ml) prior to encapsulation in liposomes.
• Additional salts prior to encapsulation can include, but are not limited to, besylate, bitartrate, carbonate, citrate, esylate, gluconate, glutamate, glycolate, lactate, malate, maleate, mandelate, methyl sulfate, napsylate, phosphate, propionate, salicylate, succinate, tartrate, and tosylate.
• the compounds are in the form of hydrate or solvate or a cocrystal prior to encapsulation in the liposomes.
• the drug is entrapped in the interior of the liposomes in a different salt form with a reduced aqueous solubility, for example less than 1 mg/mL and preferably less than 0.1 mg/mL (0.1 - 0.001 mg/mL).
• the salt of the compound once entrapped in the liposomes includes, but not limited to sulfate, citrate, phosphate, sucrosofate, or various phosphorylated or sulfated polyols or polyanionic polymers.
• Exemplary polyols include, but not limited to, sucrose, erythritol, mannitol, xylitol, sorbitol, inositol, and combinations thereof.
• Exemplary polyanionic polymers include but not limited to, polyvinyl sulfonate, polyvinyl sulfate, polyphosphate, copolymers of acrylic acid and vinylalcohol sulfate, and combinations thereof.
• Working stocks of the compounds were prepared as follows: to an aliquot of a compound (free base) in a powder form 1-1.5 equivalents of HC1 in the form of 1 N aqueous solution was added, and the mixture was vortex ed until homogeneity. To the resulting cake or syrup, water was added typically to the final 10 mg/ml, and complete dissolution was observed. In some instances, 0.95 equivalents of HC1 were added to the free base form of the drug, and 20 mg/ml stock solution was prepared.
• Aqueous solubility of the compounds of the present disclosure is illustrated by the following observations of obtaining visually clear solutions: [00154] These results show that the compounds provided herein have an aqueous solubility that is higher than the known aqueous solubilities of:
• the aqueous solubility of the compounds described herein, prior to encapsulation into the liposomes is at least 5 times, at least 10 times, at least 20 times, at least 30 times, or at least 40 times of the above oxazolidinones.
• an amphiphilic weak base has a pKa of between 7 and 12 and a logP between 1 and 6.
• a weak base property of the compounds of the present disclosure is characterized by an electrolytic dissociation constant in the pKa range of 7.0-12.0, 7.5-11.0, 7.8-10.5, or 8.0 -10.0.
• the amphiphilic property of the compounds described herein is characterized by a logP parameter in the range of 0.5-5.0, 1.0-4.0, 1.0-3.5, or 1.0-3.0.
• compositions and use of the compositions for the treatment of tuberculosis, as well as other mycobacterial and gram positive bacterial infections are disclosed.
• These compositions provided herein contain a highly potent and selective oxazolidinone encapsulated with high efficiency to maximize dosing potential of low toxicity drugs, and are stable in the presence of plasma.
• the compositions are long circulating and retain their encapsulated drug while in the circulation following intravenous dosing to allow for efficient accumulation at the site of the bacterial or mycobacterial infection.
• high doses that can be achieved when combined with the long circulating properties and highly stable retention of the drug allow for a reduced frequency of administration when compared to daily or twice daily administrations of other drugs typically utilized to treat these infecti ons.
• compositions for treating bacterial infections in particular a Mycobacterium tuberculosis infection.
• the pharmaceutical composition is a liposomal composition comprising a polyanion or a sulfate containing polyanion and an aminoalkyl oxazolidinone compound.
• compositions relate to a method of treating bacterial infection, the method comprising administering to a subject in need thereof a therapeutically effective amount of the liposomal composition provided herein.
• the bacterial infection is Mycobacterium tuberculosis infection.
• the compound in the liposome vesicle has a minimum inhibitory concentration (MIC) ranging from about 0.01 ⁇ g/ml to about 0.25 ⁇ g/ml. In some embodiments, the compound in the liposome vesicle has a minimum inhibitory concentration (MIC) ranging from about 0.01 ⁇ g/ml to about 0.1 ⁇ g/ml.
• the composition comprises liposomes in a medium, wherein the intraliposomal space comprises an aqueous phase with a polyanion and the compound of Formula (I).
• the composition comprises liposomes in a medium, wherein the intraliposomal space comprises a polyanion or a sulfate containing polyanion and the compound AKG-16, AKG-28, or AKG-38.
• the medium is an aqueous medium, where the primary composition in that media is the compound of Formula (I) and a corresponding trapping agent.
• the compound of Formula (I) can be entrapped within the liposome with a suitable polyanion, such as sucrose octasulfate (e.g. derived from tri ethyl ammonium sucrose octasulfate, (TEA-SO S) gradients) or sulfate (e.g. derived from ammonium sulfate gradients).
• a suitable polyanion such as sucrose octasulfate (e.g. derived from tri ethyl ammonium sucrose octasulfate, (TEA-SO S) gradients) or sulfate (e.g. derived from ammonium sulfate gradients).
• Additional polyanion trapping agents include but are not limited to inositol hexaphosphate, inositol hexasulfate, polyvinyl sulfonate, dextran sulfate, citrate,
• the exterior aqueous medium is typically composed of a suitable buffer and an isotonicity agent.
• Suitable buffers may include histidine, citrate, HEPES, MOPS, MES, TRIS, phosphate, glycine, and imidazole, borate, carbonate, and succinate.
• Isotonicity agents may include salts such as sodium chloride, potassium chloride, sucrose, glycerin, dextrose, or mannitol.
• the composition comprises a compound of Formula (I) or the Formula 1 a, lb, 1c, or Id or pharmaceutical acceptable salt thereof, encapsulated with a polyanion in a primarily unilamellar vesicle formed from one or more phospholipid, a sterol and optionally a lipid conjugated to a hydrophilic polymer (a polymer-conjugated lipid).
• the composition can comprise a compound of Formula (I) or the Formula 1a, lb 1c, or Id, or pharmaceutical acceptable salt thereof, encapsulated with a poly anion in unilamellar and multilamellar vesicles (e.g. having two or three lamella).
• the phospholipid is hydrogenated soy phosphatidyl choline (HSPC), distearoylphosphatidylcholine (DSPC), or egg sphingomyelin (ESM).
• HSPC hydrogenated soy phosphatidyl choline
• DSPC distearoylphosphatidylcholine
• ESM egg sphingomyelin
• phospholipid as used herein refers to any one phospholipid or combination of phospholipids capable of forming liposomes.
• Neutral phospholipids can include diacylphosphatidylcholines, dialkylphosphatidylcholines, sphingomyelins, and diacylphosphatidylethanolamines.
• Phosphatidylcholines including those obtained from egg, soybeans or other plant sources or those that are partially or wholly synthetic, or of variable lipid chain length and unsaturation are suitable for use in the present compositions.
• Synthetic, semisynthetic and natural product phosphatidylcholines including, but not limited to, distearoylphosphatidylcholine (DSPC), hydrogenated soy phosphatidylcholine (HSPC), soy phosphatidylcholine (soy PC), egg phosphatidylcholine (egg PC), hydrogenated egg phosphatidylcholine (HEPC), dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) are suitable phosphatidylcholines for use in this disclosure.
• DSPC distearoylphosphatidylcholine
• HSPC hydrogenated soy phosphatidylcholine
• soy PC soy phosphatidy
• Charged phospholipids can include phosphatidylserines, phosphatidic acids, phosphatidylinositols, phosphatidylglycerols, cardiolipins, or headgroup modified lipids such as N-succinyl-phosphatidylethanolamines, N- glutaryl-phosphatidylethanolamines, and PEG-derivatized phosphatidylethanolamines.
• Polymer-conjugated lipids may include polyethylene glycol)-conjugated (pegylated)phospholipids (PEG-lipids) such as PEG(Mol. weight 2,000) methoxy-poly(ethylene glycol)- 1,2-di stearoyl -sn-glycerol (PEG(2000)-distearoylglycerol, PEG-DSG), PEG(Mol. weight 2,000) l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)- 2000] (PEG(Mol.
• PEG-lipids such as PEG(Mol. weight 2,000) methoxy-poly(ethylene glycol)- 1,2-di stearoyl -sn-glycerol (PEG(2000)-distearoylglycerol, PEG-DSG), PEG(Mol. weight 2,000) l,2-distea
• the molecular weight of the PEG portion in the PEG-lipid component can also vary from 500-10,000 g/mol, from 1,500-6000 g/mol, but is preferably about 2,000 MW.
• polymers used for conjugation to lipid anchors may include poly(2-methyl-2-oxazoline) (PMOZ), poly(2-ethyl-2-oxazoline) (PEOZ), poly-N-vinylpyrrolidone (PVP), polyglycerol, poly(hydroxy ethyl L-asparagine) (PHEA), and poly(hydroxy ethyl L-glutamine) (PHEG).
• PMOZ poly(2-methyl-2-oxazoline)
• PEOZ poly(2-ethyl-2-oxazoline)
• PVP poly-N-vinylpyrrolidone
• PHEA poly(hydroxy ethyl L-asparagine)
• PHEG poly(hydroxy ethyl L-glutamine)
• the sterol is cholesterol.
• Other exemplary sterols include, but are not limited to, ergosterol, phytosterols such as P-sitosterol, and hopanoids.
• the ratio of the phospholipid(s) and the cholesterol is selected to provide a desired amount of liposome membrane rigidity while maintaining a sufficiently reduced amount of leakage of the compound of Formula (I) from the liposome.
• the optional polymer- conjugated lipid can be added to reduce the tendency of the liposomes to aggregate.
• the type and amount of polymer-conjugated lipid can be selected to provide desirable levels of protein binding, liposome stability and circulation time in the blood stream.
• the liposome vesicle comprises phosphatidylcholine (e.g. DSPC or HSPC) and cholesterol in an about 45:55 molar ratio.
• Phosphatidylcholine to cholesterol molar ratios can vary from about 60:40 to 35:65, about 50:50 to 35:65, about 50:50 to about 45:55.
• the liposome can comprise a vesicle consisting of HSPC, cholesterol and polymer-conjugated lipid (PEG-DSG or PEG-DSPE) in a about 45:55:2.75 molar ratio, corresponding to a PEG-lipid concentration of 5 mol % relative to the concentration of phospholipid.
• the concentration of PEG-lipid can vary from 0.5-to-10 mol % relative to (non-PEGylated) phospholipid, with a preferred ratio of 3-10 mol %, and an even more preferred ratio of 4-8 mol %.
• liposomes compositions provide desirable pharmacokinetic properties such as extended plasma half-life, measured as the percentage of the injected dose (ID) (or injected amount) remaining in blood after 6 or 24 hours following injection intravenously in immunocompetent mice, and stable encapsulation of drug over 24 hours in plasma as determined by changes in the drug-to-lipid ratio (DL ratio) following iv administration in mice.
• ID the percentage of the injected dose
• DL ratio drug-to-lipid ratio
• the percentage of drug remaining in blood is greater than 20 %, preferably greater than 30 %, and most preferably greater than 40 % of the injected dose at 6 hours.
• the percent retained in blood after 24 h is preferably greater than 10 %, and more preferably greater than 20 % of the injected dose.
• the DL ratio is greater than 20 % at 24 hours, preferably greater than 50 %, and most preferably greater than 80 % of the originally injected liposomal drug. Desirable liposome compositions also display stable encapsulation in the presence of human plasma in vitro using a burst release method, with liposomes retaining greater than 50 % of the drug over 20 min, greater than 60%, greater than 70%, preferably greater than 80 %, and most preferably greater than 90 % of encapsulated drug over 20 min.
• Liposomes of the present disclosure can be made by any method known in the art. See, for example, G. Gregoriadis (editor), Liposome Technology, vol. 1-3, 1st edition, 1983; 2nd edition, 1993; 3 rd edition, 2006; CRC Press, Boca Raton, Fla.
• methods suitable for making liposome composition of the present disclosure include membrane extrusion, reverse phase evaporation, sonication, solvent (e.g., ethanol) injection (including microfluidic, Y-junction and T-junction mixing), microfluidization, detergent dialysis, ether injection, and dehydration/rehydration.
• the size of liposomes can be controlled by controlling the pore size of membranes used for extrusions or the pressure and number of passes utilized in microfluidization or any other suitable methods.
• the desired lipids are first hydrated by thin- film hydration or by ethanol injection and subsequently sized by extrusion through membranes of a defined pore size, such as, 50 nm, 80 nm, 100 nm, or 200 nm, or the combinations thereof, producing the liposomes with the average size in the range of 70-150 nm, or 80-130 nm, and poly dispersity index of 0.1 or less.
• the drug compound to be encapsulated can be added to the liposome lipids prior to the liposome formation, dissolved in the aqueous medium in which the liposomes are formed by the above methods, whereby the drug is sequestered within the liposomes.
• the drug compound is encapsulated in the liposomes using a trapping agent incorporated into the interior space of the liposomes (see Drummond, D.C., et al. (2006) in: Liposome Technology, Third Edition (Ed. Gregoriadis, G.) Volume 2, p.149-168).
• the method of making liposome composition of the present disclosure comprises the steps of: (i) preparing the liposomes comprising phospholipid, cholesterol, and PEG-lipid, and having an interior space containing a trapping agent, in a medium substantially free from said trapping agent; (ii) contacting said liposomes with the compound of the present disclosure in an aqueous medium to effect encapsulation of the compound in the liposomes; (iii) removing unencapsulated compound; and (iv) providing the liposomes in a physiologically acceptable medium suitable for parenteral use.
• the step (iii) is high enough typically >95%, >97%, or >99%
• the step (iii), removing of unencapsulated compound is omitted.
• the process to generate the liposomes with the compound therein includes the steps of (a) preparing a liposome containing a trapping agent composed of an ammonium or substituted ammonium salt of a polyanion, (b) subsequently removing extra- liposomal trapping agent to form an electrochemical gradients across the membrane, and (c) contacting the liposome with the compound under conditions effective for the compound to enter the liposome and to permit a corresponding amount of the ammonia or substituted ammonia to leave the liposome (thereby exhausting or reducing the pH gradient across the resulting liposome).
• Liposome compositions containing a trapping agent in the interior of the liposome can be made by formation of the liposomes in a solution of the trapping agent.
• the transmembrane concentration gradient of the trapping agent can be formed across the liposome by the removal of the trapping agent outside of the or dilution of the liposomes either following liposome formation or before loading (entrapping) of the drug.
• the contacting step includes incubation of the liposomes with the drug in an aqueous medium at the temperature above ambient and below the boiling point of water, preferably between 30°C and 90°C, between 40°C and 80°C, between 50°C and 80°C, or between 60°C and 75°C.
• the incubation is carried at ionic strength of less than that equivalent to 50 mM NaCl, or more preferably, less than that equivalent to 30 mM NaCl.
• a concentrated salt, e.g., NaCl, solution may be added to raise the ionic strength to higher than that of 50 mM NaCl, or of about 100 mM NaCl.
• the increase of ionic strength after the drug loading incubation step aided in reducing post-loading aggregation of the liposomes.
• the incubation times may range from few minutes to several hours. In some embodiments, the incubation times are from 5 to 40 min, from 10 to 30 min, or from 15-25 min.
• the liposomes are cooled down and then allowed to reach the ambient temperature. In some embodiments, the liposomes are cooled down to 2-15 °C. In some embodiments, the liposomes are cooled down to 4-10°C.
• a concentrated salt, e g., NaCl, solution may be added to raise the ionic strength to higher than that of 50 mM NaCl, or of about 100 mM NaCl.
• the increase of ionic strength after the drug loading incubation step aided in reducing post-loading aggregation of the liposomes.
• the loading is performed in the presence of ionic agent, such as agent NaCl, KC1, NH 4 C1, Na 2 SO 4 , K 2 SO 4 , or (NH 4 ) 2 SO 4 . at 20-350 mEq/L, 20-100 mEq/L, or 50-80 mEq/L. Contrary to the convention in the field that low ionic strength (low salt concentration), it was found that loading of the compounds of present disclosure, in particular AKG-28, into the liposomes was more efficient in the presence of relatively high ionic strength agents such as NaCl, in particular when the loading was performed at higher concentrations of the drug.
• ionic agent such as agent NaCl, KC1, NH 4 C1, Na 2 SO 4 , K 2 SO 4 , or (NH 4 ) 2 SO 4 .
• the loading of the compounds described herein is performed at 20-350 mEq/L, 20-100 mEq/L, or 50-80 mEq/L of an ionic strength agent.
• the ionic agent is NaCl.
• the concentration of the added ionic strength agent is selected so that the post-loading liposomes are isotonic (have osmolality of 280-310 mOsmol/L, or osmolarity 270- 310 mOsmol/kg).
• the drug is AKG-28
• the ionic strength agent is NaCl
• the loading is preformed at about 12-13 mg/ml of the drug and the NaCl concentration 50-80 mM.
• the encapsulation efficiency of 95% or more, 97% or more, or 98% or more can be achieved.
• the contacting step also includes incubation of the liposomes with the drug in aqueous medium in the presence of an osmotic (tonicity) balancing agent.
• the osmotic balancing agent also referred herein as osmotic agent
• exemplary non-ionic osmotic agents include, but are not limited to, dextrose (glucose), sucrose, trehalose, lactose, mannitol, sorbitol, and polyvinylpyrrolidone.
• the concentration of osmotic agent has osmotic concentration (expressed as osmolarity or osmolality) equal to the osmotic concentration of the trapping agent solution in the interior space of the liposomes prior to drug loading.
• the osmotic concentration of the trapping agent solution can be measured by method known in the art before the solution is combined with the lipids to form liposomes.
• the concentration of osmotic agent provides osmotic concentration that is lower than the osmotic concentration of the trapping agent solution, and is less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, or less than about 10% of the osmotic concentration of the trapping agent solution.
• the concentration of osmotic agent during the drug loading process is in the range of 200-400 mmol/kg, preferably 250-350 mmol/kg.
• the osmotic agent is dextrose, and the concentration is 45 g/L.
• no osmotic agent is used during the incubation of the liposomes with the drug.
• the incubation is performed in the presence of a ionic strength adjusting agent.
• a ionic strength adjusting agent is sodium chloride, added to the liposome-drug solution for example at the concentration between 5 and 50 mM, between 10 and 20 mM, or about 10 mM.
• the compounds of the present disclosure are loaded into the liposomes of the present disclosure in a stable and highly efficient manner even if, during the drug-liposome contacting step, the amount of osmotic agent provides osmotic concentration that is lower than the osmotic concentration of the trapping agent solution (osmotically imbalanced liposomes), up to complete absence of the added osmotic agent.
• the loading was found to be very effective (>95% loading, >97% loading and >98% loading) even at the higher end of the achievable DL ratio (AKG-28, 300-350 g/mol PhL; AKG-38, 500-600 g/mol PhL) and at high concentrations of the drug in the liposome-drug loading mixture (over 16 mg/ml for AKG-38, over 12 mg/ml for AKG-28).
• the liposome loading of AKG-28 is performed at 300-350 g/mol PhL and the drug concentration over 6 mg/ml, at least 10 mg/ml, or at least 12 mg/ml; while the liposome loading of AKG-38 is performed at 500-650 mg/ml, or 500- 600 mg/ml of the drug, and the drug concentration over 8 mg/ml, at least 12 mg/ml, or at least 16 mg/ml, and the efficiency of at least 95% loading, at least 97% loading, or at least 98% loading is achieved.
• the compounds of the present disclosure are loaded in the liposomes in the broad range of pH, such as pH 4.5-7.
• pH For AKG-28, the optimum loading efficiency of 95% or more, or 97% or more, was achieved in the range of pH 5.5-7.0 (Example 62).
• the loading pH is defined by the pH of the drug aqueous stock solution (40 mg/ml) which is selected in the range pH 5.3-7.0.
• pH of the 40 mg/ml AKG-28 stock solution is in the range pH 5.7-6.9, adjusted with NaOH.
• Liposomal and other lipid nanoparticle compositions are susceptible to degradation of the lipid components during storage which unfavorably effects their pharmaceutical qualities.
• Degradation of the lipids can be studied in accelerated stability study format where the liposome samples are stored at temperatures higher than the suggested storage temperature, so that the degradation takes place faster; generally being assumed to follow the Arrhenius law.
• the liposomes of present disclosure for example, containing the compounds AKG-28 and AKG-38 in the lipid compositions of PC and cholesterol, were found to accumulate both cholesterol oxidative degradation products (FIG. 17) and the products of phosphatidylcholine hydrolytic degradation (FIG. 18).
• Chelators are molecules that bind metal ions by forming one or more stable heterocyclic groups that include a metal and a coordination bond.
• Exemplary chelators are deferoxamine (desferri oxamine, Desferal) (abbreviated herein as DFO), ethylenediamine tetraacetic acid (EDTA), diethylenetriamine pentaacetic acid (DTP A), nitrilotriacetic acid (NTA), ethyleneglycol-O, O'-bis(2-aminoethyl)-N, N, N', N' -tetraacetic acid (EGTA), N-(2- hydroxyethyl)ethylenediamine-N, N', N'-triacetic acid (HEDTA), 1,4,7,10- tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA.
• DFO deferoxamine
• EDTA ethylenediamine tetraacetic acid
• DTP A diethylenetriamine pentaacetic acid
• NTA nitrilotriacetic acid
• EGTA N-
• the liposome composition comprises cholesterol and is stable against degradation of cholesterol, the degree of cholesterol degradation after 3 months at 37°C being less than 10%, less than 5%, or less than 1% of the total cholesterol.
• the liposome composition comprises a chelator.
• the chelator is deferoxamine (Desferal, DFO), ethylenediamine tetraacetic acid (EDTA), diethylenetriamine pentaacetic acid (DTPA), nitrilotriacetic acid (NTA), ethyleneglycol-O, O'-bis(2-aminoethyl)-N, N, N', N' -tetraacetic acid (EGTA), N-(2- hydroxyethyl)ethylenediamine-N, N', N'-triacetic acid (HEDTA), 1,4,7,10- tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), including their pharmaceutically acceptable salts.
• DFO deferoxamine
• EDTA ethylenediamine tetraacetic acid
• DTPA diethylenetriamine pentaacetic acid
• NTA nitrilotriacetic acid
• EGTA N-(2- hydroxyethyl)
• the chelator can be present in the composition at the concentration of at least 0.01 mM, at least 0.05 mM, at least 0.1 mM, at least 0.2 mM, or at least 0.5 mM, and not more than 1 mM, nor more than 2 mM, not more than 5 mM, or not more than 10 mM.
• the chelator is deferoxamine or deferoxamine mesylate, and the chelator concentration is about 0.5 mM. Deferoxamine was found to be particularly effective in preventing degradation of cholesterol in the liposomes of present disclosure.
• the lipids with encapsulated compounds of present disclosure was influenced by the pH of the liposome external medium. While general teaching in the field is that the optimum stability of the lipids in liposomes is achieved at pH around 6.5, it was discovered that for the liposomes of the present disclosure the optimum lipid stability for both cholesterol and PC components is achieved at pH over 7.0.
• the liposome composition has the pH of at least 7.1, at least 7.2, or at least 7.3, and no more than pH 8.0, no more than pH 7.7, or no more than pH 7.6.
• the degree of cholesterol degradation after 3 months at 37°C is less than 10%, less than 5%, or less than 1% of the total cholesterol. In some embodiments, the degree of phospholipid degradation after 6 weeks at 37°C is less than 10%, less than 5%, or less than 1% of the total phospholipid content.
• the phospholipid is phosphatidylcholine. In some embodiments, the phospholipid is HSPC, and the pH is between pH 7.3-7.6.
• ammonium salt is used as a trapping agent to effect the loading of the compounds described herein, such as AKG-28 or AKG-38, into the liposomes. Accordingly, for each molecule of the drug entering the liposome interior, one or two molecules of ammonia leave the interior of the liposome and accumulate in the liposome external medium, which is subsequently purged from the accumulated ammonium at the post-loading buffer exchange/unencapsulated drug removal step, such as by tangential flow fdtration, dialysis, or size exclusion chromatography.
• the external medium of the liposome composition has less than 0.5 mEq/L of ammonium or substituted ammonium.
• the liposome composition contains in the liposome external medium an ammonium or substituted ammonium in the concentration of at least 1 mEq/L., at least 2 mEq,/L, at least 5 mEq/L, at least 10 mEq/L, at least 15 mEq/L, or at least 20 mEq/1, and no more than 200 mEq/L, no more than 150 mEq/L, no more than 100 mEq/L, no more than 80 mEq/L, or no more than 60 mEq/L.
• the liposome composition contains in the liposome external medium an ammonium or substituted ammonium in the concentration of at least 1 mEq/L., at least 2 mEq,/L, at least 5 mEq/L, at least 10 mEq/L, at least 15 mEq/L, or at least 20 mEq/1, and no more than 200 mEq/L, no more than 150 mEq/L, no more than 100 mEq/L, no more than 80 mEq/L, or no more than 60 mEq/L, and is stable against phospholipid degradation, the degree of phospholipid degradation after 6 weeks at 37°C being less than 10%, less than 5%, or less than 1% of the total phospholipid.
• the liposome composition contains in the liposome external medium an ammonium or substituted ammonium in the concentration of at least 1 mEq/L., at least 2 mEq,/L, at least 5 mEq/L, at least 10 mEq/L, at least 15 mEq/L, or at least 20 mEq/1, and no more than 200 mEq/L, no more than 150 mEq/L, no more than 100 mEq/L, no more than 80 mEq/L, or no more than 60 mEq/L, and is stable against phospholipid degradation, the degree of phospholipid degradation after 3 months at 37°C being less than 10%, less than 7%, less than 5%, or less than 4% of the total phospholipid.
• the phospholipid is phosphatidylcholine. In some embodiments, the phospholipid is HSPC, and the ammonium salt is ammonium chloride, ammonium sulphate, or a combination thereof, at the ammonium concentration of 10-80 mM, or 15-60 mM. In some embodiments, the phospholipid is HSPC, and the ammonium salt is ammonium chloride, ammonium sulphate, or a combination thereof, at the ammonium concentration of 1-10 mM, or 2-5 mM.
• the normality of ammonium in the external medium of the liposome composition is within 90-110% of the normality of encapsulated drug at the drug loading step, normality being the concentration expressed in gram-equivalents/L (eq/L).
• the desired concentration of ammonium in the liposome external medium can be achieved by accumulation of the extraliposomal ammonium during the drug loading step at the expense of ammonium (used as part of a trapping agent) escape from the liposome interior as explained above.
• the desired levels of extraliposomal ammonium are contributed by the extraliposomal ammonium that remains in the liposomes after the removal of extraliposomal ammonium prior to the drug loading, or are achieved by addition of ammonium salt, such as ammonium chloride or ammonium sulfate, to the external medium of the liposome formulation.
• ammonium salt can be added to the liposomal preparation after the post-load buffer- exchange/unencapsulated drug removal step, or added to the exchange buffer,
• liposome compositions provided herein can further include in the liposome formulation, a lipophilic free-radical scavenger, such as .alpha. -tocopherol.
• a lipophilic free-radical scavenger such as .alpha. -tocopherol.
• oxazolidinone liposome compositions provided herein comprise HSPC, cholesterol and PEG-DSPE in a mass ratio of about 5 : 3 : 1. In some embodiments, oxazolidinone liposome compositions provided herein comprise HSPC, cholesterol and PEG- DSPE in a molar ratio of about 45:55:2.25. In some embodiments, oxazolidinone liposome compositions comprise an oxazolidinone consisting of AKG-28 or a pharmaceutically acceptable salt thereof. In some embodiments, oxazolidinone liposome compositions comprise an oxazolidinone consisting of AKG-38 or a pharmaceutically acceptable salt thereof.
• mycobacteria such as Mycobacterium tuberculosis
• gram positive bacteria such as methicillin-resistant Staphylococcus aureus (MRSA).
• MRSA methicillin-resistant Staphylococcus aureus
• Additional mycobacteria and gram positive bacteria include, but are not limited to, Mycobacterium avium complex, Mycobacterium leprae, Mycobacterium gordonae, Mycobacterium abscessus, Mycobacterium abscessus, Mycobacterium mucogenicum, streptococci, vancomycin-resistant enterococci (VRE), Staphylococcus pneumoniae, Enterococcus faecium, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, the viridans group streptococci, Listeria monocytogenes, Nocardia, and Corynebacterium.
• the compounds and compositions provided herein inhibit the growth of drug resistant strains of Mycobacterium tuberculosis. In some embodiments, methods of treating mycobacterial infections are provided. In some embodiments, the compounds and compositions provided herein can be used to treat nontuberculosis mycobacteria infections. In some embodiments, the method comprises administering a therapeutically effective amount of an aminoalkyl oxazolidinone of the disclosure and/or a pharmaceutical acceptable salt thereof to a subject in need thereof. In some embodiments, the method comprises administering a therapeutically effective amount of a liposomal composition comprising an aminoalkyl oxazolidinone compound of the disclosure and/or a pharmaceutical acceptable salt thereof to a subject in need thereof.
• Mycobacteria is a genus of bacteria responsible for tuberculosis (TB). According to the World Health Organization, worldwide, TB is one of the top 10 causes of death and the leading cause of death from a single infectious agent. Rifampicin is the most effective first-line drug to treat TB. However, there is a growing number of cases infected with mycobacterium tuberculosis that is resistant to rifampicin. Multidrug-resistant tuberculosis (MDR-TB) is a form of TB caused by bacteria that do not respond to isoniazid and rifampicin.
• MDR-TB Multidrug-resistant tuberculosis
• the composition is a liquid pharmaceutical formulation for parenteral administration.
• the liquid pharmaceutical formulation is a liposomal formulation containing a suitable amount of the oxazolidinone compound described herein, wherein the oxazolidinone compound is encapsulated in the interior of the liposomes.
• that compound is in a salt form in the interior of the liposome with a polyanion such as sulfate, citrate, sucrose octasulfate, inositol hexaphosphate.
• the compound is a precipitated or gelated salt with sulfate inside a liposome composed of multiple lipid excipients, including but not limited to, phosphatidylcholine, cholesterol, and pegylated phosphatidylethanolamine.
• the liposomes of the present disclosure show entrapment efficiencies of more than 85%, more than 90%, and more than 95%.
• the residual amount of the unentrapped drug is removed from the liposome composition. This can be achieved by various means, such as size exclusion chromatography, ion exchange, dialysis, ultrafiltration, tangential flow filtration, adsorption, or precipitation.
• the liposomes may be brought into a desired pharmaceutically acceptable carrier, for example, normal saline, isotonic dextrose, isotonic sucrose, Ringer's solution, or Hanks' solution.
• a buffer substance can be added to provide desired physiologically acceptable pH.
• the liposomal composition may be adjusted for desired drug concentration, and sterilized, e.g., by aseptic filtration through 0.2-0.22 pm filters.
• the compound concentration in the liposomal composition is in the range of 1-50 mg/ml, 3-30 mg/ml, or 5-25 mg/ml.
• compositions comprising the liposome composition provided herein may be sterilized by conventional, well known sterilization techniques.
• the aqueous solutions can then be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration.
• the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc.
• the lipidic suspension may include lipid-protective agents which protect lipids against free-radical and lipid-peroxidative damages on storage. Lipophilic free- radical quenchers, such as .alpha. -tocopherol are suitable.
• the liposomes are mixed with one or more additional excipients for isotonicity or pH control.
• the excipients include but are not limited to sodium chloride, Hepes buffer, phosphate buffer, and histidine buffer.
• the liposome compositions can also contain other pharmaceutically acceptable substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, and the like.
• the liposome suspension may include lipid-protective agents which protect lipids against free-radical and lipid-peroxidative damages on storage. Lipophilic free-radical quenchers, such as alpha-tocopherol , are suitable.
• the composition is an oral formulation.
• the composition is a liquid formulation.
• the composition is a solid formulation (e.g. tablet, capsule, pill, dragees, caplets etc.).
• tablets, troches, lozenges, aqueous or oil suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups or elixirs may be prepared (Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, Pa.).
• Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions.
• compositions may contain one or more agents including antioxidants, sweetening agents, flavoring agents, coloring agents and preserving agents, in order to provide a palatable preparation.
• Tablets containing the active ingredient in admixture with non-toxic pharmaceutically acceptable excipient or auxiliary agents which are suitable for manufacture of tablets are acceptable.
• Suitable excipients or auxiliary agents include but are not limited to, for example, inert diluents, solubilizers, suspending agents, adjuvants, wetting agents, sweeteners, perfuming or flavoring substances, isotonic substances, colloidal dispersants and surfactants.
• Tablets, dragees, capsules, pills, granules, suppositories, solutions, suspensions and emulsions, pastes, ointments, gels, creams, lotions, powders and sprays can be suitable pharmaceutical compositions.
• the compound or the composition can be administered local ly, orally, parenterally , intraperitoneally and/or rectally.
• Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, one or more doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.
• the dosage of the compounds and/or of their pharmaceutically acceptable salts or the liposomes comprising the compounds and/or of their pharmaceutically acceptable salts may vary within wide limits and should naturally be adjusted, in each particular case, to the individual conditions and to the pathogenic agent to be controlled.
• the compound or the pharmaceutical liposomal composition is administered once every 7 days (i.e., once every week), once every 14 days (i.e., once every' two weeks), once every 21 days (i.e., once every three weeks), once every' 28 days (i.e., once every' four weeks) and once every 42 days (i.e., once every six weeks) to the subject in need thereof.
• the average weekly dosage is from about 1 mg to about 1500 mg, about 10 to about 700 mg, about 25 to about 500 mg, or about 70 to about 250 mg.
• the average weekly dosage is from about 1 mg to about 10 mg, from about 10 mg to about 25 mg, from about 25 mg to about 50 mg, from about 50 mg to about 100 mg, from about 100 mg to about 200 mg, from about 200 mg to about 300 mg, from about 300 mg to about 400 mg, from about 400 mg to about 500 mg, from about 500 mg to about 600 mg, from about 600 mg to about 700 mg, from about 700 mg to about 800 mg, from about 800 mg to about 900 mg, from about 900 mg to about 1000 mg, from about 1000 mg to about 1100 mg, from about 1100 mg to about 1200 mg, from about 1200 mg to about 1300 mg, from about 1300 mg to about 1400 mg, from about 1400 mg to about 1500 mg.
• the compound or composition is administered for up to one month, up to two months, up to three months, up to four months or more.
• the specific therapeutically effective amount will depend on a variety of factors, including the bacterial infection being treated, the activity of the specific compound being administered, the pharmaceutical composition employed, the age, body eight, gender etc. of the subject, the route of administration, the severity of the bacterial infection, the optional drugs/active agents used in combination (sequentially or simultaneously) with the specific compound, and the like factors known to the medical doctor of ordinary skill.
• the compounds or the composition can be used for the treatment of tuberculosis or other mycobacterium infections.
• the compound can be used as a monotherapy.
• the treatment can include administering simultaneously and/or sequentially an effective amount of the compound described herein and an effective amount of one or more additional active agents to treat mycobacterium tuberculosis and other gram-positive bacterial infections.
• the treatment can include administering simultaneously and/or sequentially an effective amount of the compound described herein and an effective amount of two or more additional active agents (two, three, four, etc.) to treat mycobacterium tuberculosis and other gram-positive bacterial infections.
• a synergistic antibacterial effect denotes an antibacterial effect which is greater than the predicted purely additive effects of the individual compounds of the combination.
• the compound and the active agent can be contained in the same composition or in separate compositions.
• the composition comprising the compound and the composition comprising the additional active agent can be administered with a time separation (e.g. 20 minutes, 40 minutes, 60 minutes or more).
• the additional active agents can be administered using a different administration route or by different injections.
• the compounds of the disclosure can be administered intravenously and one or more additional agents can be administered orally.
• the administration of the compounds with one or more (e.g. one, two, three or four) additional active agents can result in a reduction of the length of the treatment duration.
• administration of the compounds with one or more e.g.
• one, two, three or four additional active agent can result in a treatment duration at least three times, at least twice, at least 1.5 times shorter than compared to the treatment with only one active agent.
• the additional agent(s) is an antibacterial agent.
• the additional active agent can include, but are not limited to, fluoroquinolines, such as moxifloxacin, gatifloxacin, or levofloxacin, bedaquiline and other diaryl quinoline analogs (e.g.
• the additional active agent can include, but are not limited to, vancomycin, gentamycin, daptomycin, teicoplanin, ceftaroline, ceftrobiprole, telavancin, dalbavancin, oritavancin, fluoroquinolines (e.g. delafloxacin), tetracyclines (e.g. eravacycline and omadacycline), sulfonamides (e.g. sulfamethoxazole), trimetrhoprim, lefamulin, and any combinations thereof.
• vancomycin e.g. delafloxacin
• tetracyclines e.g. eravacycline and omadacycline
• sulfonamides e.g. sulfamethoxazole
• trimetrhoprim etrhoprim
• the treatment can include administering simultaneously and/or sequentially an effective amount of the compound described herein and an effective amount of bedaquiline, pretomanid, pyrazinamide, moxifloxacin or a pharmaceutically acceptable salt of each thereof or a combination of the foregoing.
• Parental as used herein in the context of administration means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
• parenteral administration and “administered parenterally” as used herein refer to modes of administration other than enteral (i.e., via the digestive tract) and topical administration, usually by injection or infusion, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracap sul ar, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, inhalation, subcapsular, subarachnoid, intraspinal, epidural and intrastemal injection and infusion. Intravenous injection and infusion are often (but not exclusively) used for liposomal drug administration.
• the liquid composition is injected intravenously.
• the compound or the pharmaceutical composition is administered once every 7 days (i.e., once every week), once ever ⁇ ' 14 days (i.e., once every two weeks), once every 21 days (i.e., once every three weeks), once every 28 days (i.e., once every four weeks) and once every' 42 days (i.e., once every six weeks) to the subject in need thereof.
• the average weekly dosage is from about 1 mg to about 1500 mg, about 10 to about 700 mg, about 25 to about 500 mg, or about 70 to about 250 mg.
• the average weekly dosage is from about 1 mg to about 10 mg, from about 10 mg to about 25 mg, from about 25 mg to about 50 mg, from about 50 mg to about 100 mg, from about 100 mg to about 200 mg, from about 200 mg to about 300 mg, from about 300 mg to about 400 mg, from about 400 mg to about 500 mg, from about 500 mg to about 600 mg, from about 600 mg to about 700 mg, from about 700 mg to about 800 mg, from about 800 mg to about 900 mg, from about 900 mg to about 1000 mg, from about 1000 mg to about 1100 mg, from about 1100 mg to about 1200 mg, from about 1200 mg to about 1300 mg, from about 1300 mg to about 1400 mg, from about 1400 mg to about 1500 mg.
• the specific therapeutically effective amount will depend on a variety of factors, including the bacterial infection being treated, the activity of the specific compound being administered, the pharmaceutical composition employed, the age, body weight, gender etc., of the subject, the route of administration, the severity of the bacterial infection, the optional drugs/active agents used in combination (sequentially or simultaneously) with the specific compound, and the like factors known to the medical doctor of ordinary skill in the art.
• the liposomal composition is administered parenterally.
• the method comprises administering simultaneously or sequentially one or more additional active agent.
• the one or more active agents comprise bedaquiline, pretomanid, pyrazinamide, moxifloxacin, a pharmaceutically acceptable salt thereof or a combination thereof.
• the liposomal composition is administered once a week to once every six weeks.
• the percentage of compound remaining in blood is greater than 20 % of the administered amount at 6 hours following administration to the subject in need thereof. In some embodiments, the percentage of compound remaining in blood is greater than 10 % of the administered amount.
• aspects of the disclosure relate to method of making liposome composition
• method of making liposome composition comprising the steps of: (i) preparing the liposomes comprising phospholipid, cholesterol, and PEG-lipid, and having an interior space containing a trapping agent, in a medium substantially free from the trapping agent; (ii) contacting the liposomes with a compound disclosed herein in an aqueous medium to effect encapsulation of the compound in the liposomes; (iii) removing unencapsulated compound; and (iv) providing the liposomes in a physiologically acceptable medium suitable for parenteral use.
• the compound or the pharmaceutical oral composition is administered once or twice daily.
• the specific therapeutically effective amount will depend on a variety of factors, including the bacterial infection being treated, the activity of the specific compound being administered, the pharmaceutical composition employed, the age, body eight, gender etc., of the subject, the route of administration, the severity of the bacterial infection, the optional drugs/active agents used in combination (sequentially or simultaneously) with the specific compound, and the like factors known to the medical doctor of ordinary skill.
• An AKG-28 liposome composition comprising lipids HSPC, cholesterol, and PEG(2000)- DSPE in a molar ratio of 45:55:2.25 or in amass ratio of 5:3:1 and a pharmaceutically acceptable salt of AKG-28 (AKG-28).
• composition of embodiment 1, wherein the liposome composition comprises mono- or oligolamellar vesicles having z-average diameter of 90-130 nm.
• composition of embodiment 2, wherein the mono- or oligolamellar vesicles have a z- average diameter of 100-130 nm.
• composition of embodiment 1, wherein the liposome composition has a poly dispersity index of less than 0.15.
• composition of embodiment 4, wherein the liposome composition has a poly dispersity index of less than 0.10.
• composition of embodiment 1, wherein the drug/lipid ratio of the AKG-28 to the total phospholipid (PhL) in the composition is 230-290 g/mol.
• composition of embodiment 1, wherein the drug/lipid ratio of the AKG-28 to the total phospholipid (PhL) in the composition is 290-360 g/mol.
• composition of embodiment 1, wherein the drug/lipid ratio of the AKG-28 to the total phospholipid (PhL) in the composition is 300-340 g/mol.
• composition of embodiment 1, wherein the drug/lipid ratio of the AKG-28 to the total phospholipid (PhL) in the composition is about 250 g/mol.
• composition of embodiment 1, wherein the drug/lipid ratio of the AKG-28 to the total phospholipid (PhL) in the composition is about 330 g/mol.
• composition of embodiment 1, wherein the overall concentration of AKG-28 in the composition is 8-15 mg/mL.
• composition of embodiment 1, wherein the overall concentration of AKG-28 in the composition is 9-11 mg/mL.
• composition of embodiment 1, wherein the proportion of encapsulated AKG-28 to overall AKG-28 in the composition is at least 90%.
• composition of embodiment 1, wherein the proportion of encapsulated AKG-28 to overall AKG-28 in the composition is at least 95%.
• composition of embodiment 1, wherein the proportion of encapsulated AKG-28 to overall AKG-28 in the composition is at least 97%.
• composition of embodiment 1 wherein the proportion of encapsulated AKG-28 to overall AKG-28 in the composition is at least 98%.
• composition comprises liposome vesicles in an aqueous medium, the aqueous medium comprising sodium chloride and optionally comprising an ammonium ion.
• composition of embodiment 17, wherein the osmolality of the aqueous medium is 270- 330 mOsmol/kg.
• composition of embodiment 17, wherein the osmolality of the aqueous medium is 270- 310 mOsmol/kg.
• composition of embodiment 17, wherein the ammonium concentration in the aqueous medium is 20-60 mM.
• composition of embodiment 17, wherein the ammonium concentration in the aqueous medium is 50-80 mM.
• composition of embodiment 17, wherein the concentration of ammonium in the aqueous medium is less than 0.5 mM.
• composition of embodiment 17, wherein the concentration of ammonium in the aqueous medium is less than 130-150 mM.
• composition of embodiment 27, wherein the composition comprises HEPES or phosphate buffer at a concentration of 5-50 mM.
• composition of embodiment 27, wherein the composition comprises HEPES or phosphate buffer at a concentration of about 20 mM.
• composition of embodiment 27, wherein the composition comprises HEPES or phosphate buffer at a concentration of 20 mM.
• composition of embodiment 1 further comprising a chelator at a concentration of 0.1- 1 mM.
• DFO deferoxamine
• DFO deferoxamine
• DFO deferoxamine
• DFO deferoxamine
• composition of embodiment 1, wherein the composition is storage stable.
• An AKG-38 liposome composition comprising lipids HSPC, cholesterol, and PEG(2000)- DSPE in a molar ratio of 45:55:2.25 or in a mass ratio of 5:3:1 and a pharmaceutically acceptable salt of AKG-38
• composition of embodiment 41, wherein the liposome composition comprises mono- or oligolamellar vesicles having z-average diameter of 90-130 nm.
• composition of embodiment 41, wherein the liposome composition has a poly dispersity index of less than 0.15.
• composition of embodiment 44, wherein the liposome composition has a poly dispersity index of less than 0.10.
• composition of embodiment 41 wherein the drug/lipid ratio of the AKG-38 to the total phospholipid (PhL) in the composition is 430-480 g/mol. 47. The composition of embodiment 41, wherein the drug/lipid ratio of the AKG-38 to the total phospholipid (PhL) in the composition is 500-650 g/mol.
• composition of embodiment 41, wherein the drug/lipid ratio of the AKG-38 to the total phospholipid (PhL) in the composition is 430-650 g/mol.
• composition of embodiment 41, wherein the drug/lipid ratio of the AKG-38 to the total phospholipid (PhL) in the composition is about 450 g/mol.
• composition of embodiment 41, wherein the drug/lipid ratio of the AKG-38 to the total phospholipid (PhL) in the composition is about 600 g/mol.
• composition of embodiment 41, wherein the overall concentration of AKG-38 in the composition is 12-25 mg/mL.
• composition of embodiment 41, wherein the overall concentration of AKG-38 in the composition is 13.5-16.5 mg/mL.
• composition of embodiment 41, wherein the overall concentration of AKG-38 in the composition is about 15 mg/mL.
• composition of embodiment 41, wherein the overall concentration of AKG-38 in the composition is about 20 mg/mL.
• composition of embodiment 41, wherein the proportion of encapsulated AKG-38 to overall AKG-38 in the composition is at least 90%.
• composition of embodiment 41, wherein the proportion of encapsulated AKG-38 to overall AKG-38 in the composition is at least 95%.
• composition of embodiment 41, wherein the proportion of encapsulated AKG-38 to overall AKG-38 in the composition is at least 97%.
• composition of embodiment 41, wherein the proportion of encapsulated AKG-38 to overall AKG-38 in the composition is at least 98%.
• composition of embodiment 41 wherein the composition comprises liposome vesicles in an aqueous medium, the aqueous medium comprising sodium chloride and optionally comprising an ammonium ion.
• composition of embodiment 59, wherein the osmolality of the aqueous medium is 270- 330 mOsmol/kg.
• composition of embodiment 59 wherein the osmolality of the aqueous medium is 270- 310 mOsmol/kg. 62. The composition of embodiment 59, wherein the ammonium concentration in the aqueous medium is 20-60 mM.
• composition of embodiment 59, wherein the ammonium concentration in the aqueous medium is 50-80 mM.
• composition of embodiment 59, wherein the concentration of ammonium in the aqueous medium is less than 0.5 mM.
• composition of embodiment 59, wherein the concentration of ammonium in the aqueous medium is less than about 0.5 mM
• composition of embodiment 59, wherein the concentration of sodium chloride is 130-
• composition of embodiment 41 further comprising a buffer, wherein the buffer buffers the composition at a pH of 7.3-7.7.
• composition of embodiment 41 further comprising a buffer, wherein the buffer buffers the composition at a pH of about 7.5.
• composition of embodiment 41 further comprising a buffer, wherein the buffer buffers the composition at a pH of 7.5.
• composition of embodiment 41 further comprising a HEPES or phosphate buffer.
• composition of embodiment 70 wherein the composition comprises HEPES or phosphate buffer at a concentration of 5-50 mM.
• composition of embodiment 70 wherein the composition comprises HEPES or phosphate buffer at a concentration of about 20 mM.
• composition of embodiment 70 wherein the composition comprises HEPES or phosphate buffer at a concentration of 20 mM.
• composition of embodiment 41 further comprising a chelator.
• composition of embodiment 41 further comprising a chelator at a concentration of 0.1-1 mM.
• composition of embodiment 41 further comprising a chelator at a concentration of about 0.5 mM.
• composition of embodiment 41 further comprising a chelator at a concentration of 0.5 mM.
• composition of embodiment 41 further comprising deferoxamine (DFO) or EDTA.
• composition of embodiment 41 further comprising deferoxamine (DFO) or EDTA at a concentration of 0.1-1 mM.
• composition of embodiment 41 further comprising deferoxamine (DFO) or EDTA at a concentration of about 0.5 mM.
• DFO deferoxamine
• composition of embodiment 41 further comprising deferoxamine (DFO) or EDTA at a concentration of 0.5 mM.
• DFO deferoxamine
• composition of embodiment 41, wherein the composition is storage stable.
• An AKG-28 liposome composition comprising lipids HSPC, cholesterol, and PEG(2000)- DSPE in a molar ratio of 45:55:2.25 or in a mass ratio of 5:3:1 and a pharmaceutically acceptable salt of AKG-28
• the liposome composition is further characterized by any one or more of the following characteristics: a. the liposome composition comprises mono- or oligolamellar vesicles having z-average diameter of 90-130 nm; or the liposome composition comprises mono- or oligolamellar vesicles having z-average diameter of 90-130 nm; or the liposome composition comprises mono- or oligolamellar vesicles having a z-average diameter of 100-130 nm; b. the liposome composition has a poly dispersity index of less than 0.15; or the liposome composition has a polydispersity index of less than 0.10; c.
• the drug/lipid ratio of the AKG-28 to the total phospholipid (PhL) in the composition is 230-280 g/mol; or the drug/lipid ratio of the AKG-28 to the total phospholipid (PhL) in the composition is 290-360 g/mol; or the drug/lipid ratio of the AKG-28 to the total phospholipid (PhL) in the composition is 300-340 g/mol; or the drug/lipid ratio of the AKG-28 to the total phospholipid (PhL) in the composition is about 250 g/mol; or the drug/lipid ratio of the AKG-28 to the total phospholipid (PhL) in the composition is about 330 g/mol; d.
• the overall concentration of AKG-28 in the composition is 8-15 mg/mL; or the overall concentration of AKG-28 in the composition is 9-11 mg/mL; e. the proportion of encapsulated AKG-28 to overall AKG-28 in the liposome composition is at least 90%; or the proportion of encapsulated AKG-28 to overall AKG-28 in the liposome composition is at least 95%; or the proportion of encapsulated AKG-28 to overall AKG-28 in the liposome composition is at least 97%; or the proportion of encapsulated AKG-28 to overall AKG- 28 in the liposome composition is at least 98%; f.
• the liposome composition comprises an aqueous medium comprising sodium chloride and optionally comprising an ammonium ion; g. the osmolality of the aqueous medium is 270-330 mOsmol/kg; or the osmolality of the aqueous medium is 270-310 mOsmol/kg; h. the ammonium concentration in the aqueous medium is 20-60 mM; or the ammonium concentration in the aqueous medium is 50-80 mM; or the concentration of ammonium in the aqueous medium is less than 0.5 mM; or the concentration of ammonium in said aqueous medium is less than 0130-150mM; i.
• the aqueous medium further comprises a buffer, wherein the buffer buffersthe liposome composition at a pH of 7.3-7.7; at a pH of about 7.5; or at a pH of 7.5; j .
• the aqueous medium further comprising a HEPES or phosphate buffer; or the aqueous medium comprises HEPES or phosphate buffer at a concentration of 5-50 mM; or the aqueous medium comprises HEPES or phosphate buffer at a concentration of about 20 mM; or the aqueous medium comprises HEPES or phosphate buffer at a concentration of 20 mM; k.
• the composition further comprises a chelator; or the composition further comprises a chelator at a concentration of 0.1-1 mM; or the composition further comprising a chelator at a concentration of about 0.5 mM; or the composition further comprises a chelator at a concentration of 0.5 mM; orthe composition further comprises deferoxamine (DFO) or EDTA; orthe composition further comprises deferoxamine (DFO) or EDTA at a concentration of 0.1-1 mM; or the composition further comprising deferoxamine (DFO) or EDTA at a concentration of about 0.5 mM; orthe composition further comprises deferoxamine (DFO) or EDTA at a concentration of 0.5 mM; or l.
• a chelator or the composition further comprises a chelator at a concentration of 0.1-1 mM; or the composition further comprising a a chelator at a concentration of about 0.5 mM; or the composition further comprises deferoxamine (
• AKG-28 is encapsulated within the liposome as a sulfate salt of AKG-28.
• An AKG-38 liposome composition comprising lipids HSPC, cholesterol, and PEG(2000)- DSPE in a molar ratio of 45:55:2.25 or in amass ratio of 5:3:1 and a pharmaceutically acceptable salt of AKG-38
• the liposome composition is further characterized by any one or more of the following characteristics: a. the liposome composition comprises mono- or oligolamellar vesicles having z-average diameter of 90-130 nm; b. the liposome composition comprises mono- or oligolamellar vesicles have a z-average diameter of 100-130 nm; c. the liposome composition has a poly dispersity index of less than 0.15 or the liposome composition has a poly dispersity index of less than 0.10; d.
• the drug/lipid ratio of the AKG-38 to the total phospholipid (PhL) in the liposome composition is 430-480 g/mol, or the drug/lipid ratio of the AKG-38 to the total phospholipid (PhL) in the liposome composition is 500-650 g/mol; or the drug/lipid ratio of the AKG-38 to the total phospholipid (PhL) in the liposome composition is 430-650 g/mol; or the drug/lipid ratio of the AKG-38 to the total phospholipid (PhL) in the liposome composition is about 450 g/mol; or the drug/lipid ratio of the AKG-38 to the total phospholipid (PhL) in the liposome composition is about 600 g/mol; e.
• the overall concentration of AKG-38 in the liposome composition is 12-25 mg/mL; or the overall concentration of AKG-38 in the liposome composition is 13.5-16.5 mg/mL; or the overall concentration of AKG-38 in the composition is about 15 mg/mL; or the overall concentration of AKG-38 in the liposome composition is about 20 mg/mL.
• the proportion of encapsulated AKG-38 to overall AKG-38 in the liposome composition is at least 90%; or the proportion of encapsulated AKG-38 to overall AKG-38 in the liposome composition is at least 95%; or the proportion of encapsulated AKG-38 to overall AKG-38 in the liposome composition is at least 97%; or the proportion of encapsulated AKG-38 to overall AKG- 38 in the liposome composition is at least 98%;
• the liposome composition comprises an aqueous medium comprising sodium chloride and optionally comprising an ammonium ion; h.
• the osmolality of the aqueous medium is 270-330 mOsmol/kg; or the osmolality of the aqueous medium is 270-310 mOsmol/kg; i. the ammonium concentration in the aqueous medium is 20-60 mM; or the ammonium concentration in the aqueous medium is 50-80 mM; or the concentration of ammonium in the aqueous medium is less than 0.5 mM; or the concentration of ammonium in the aqueous medium is less than 0.5 mM; j. the concentration of sodium chloride is 130-150 mM; k.
• the aqueous medium further comprises a buffer, wherein the buffer buffers the liposome composition at a pH of 7.3-7.7, at a pH of about 7.5; or at a pH of 7.5; l.
• the aqueous medium further comprises a HEPES or phosphate buffer, or the aqueous medium comprises HEPES or phosphate buffer at a concentration of 5-50 mM, or the aqueous medium comprises HEPES or phosphate buffer at a concentration of about 20 mM, or the aqueous medium comprises HEPES or phosphate buffer at a concentration of 20 mM; m.
• the aqueous medium further comprises a chelator; or further comprising a chelator at a concentration of 0.1-1 mM, or further comprises a chelator at a concentration of about 0.5 mM, or further comprises a chelator at a concentration of 0.5 mM, or further comprises deferoxamine (DFO) or EDTA, or further comprises deferoxamine (DFO) or EDTA at a concentration of 0.1-1 mM, or further comprises deferoxamine (DFO) or EDTA at a concentration of about 0.5 mM, or further comprises deferoxamine (DFO) or EDTA at a concentration of 0.5 mM; n. the liposome composition is storage stable; or o. the AKG-38 is encapsulated in the liposomes as a sulfate salt of AKG-38.
• HSPC hydrogenated soy phosphatidyl choline
• cholesterol cholesterol
• PEG-DSPE PEG(Mol. weight 2,000)- distearoylphosphatidylethanolamine
• the liposomal dispersion of embodiment 90 comprising 45-65 mol% cholesterol relative to the sum of cholesterol and non-pegylated phospholipid in the liposome vesicles.
• the liposomal dispersion of embodiment 90 comprising 50-60 mol% cholesterol relative to the sum of cholesterol and non-pegylated phospholipid in the liposome vesicles.
• the liposomal dispersion of embodiment 90 comprising about 50 mol% cholesterol relative to the sum of cholesterol and non-pegylated phospholipid in the liposome vesicles.
• the liposomal dispersion of embodiment 90 comprising about 55 mol% cholesterol relative to the sum of cholesterol and non-pegylated phospholipid in the liposome vesicles.
• liposomal dispersion of embodiment 90 wherein the liposomal dispersion lipid content consists of the HSPC, and PEG(2000)-DSPE.
• the liposomal dispersion of embodiment 95 comprising HSPC and cholesterol in a weight ratio of about 5:3.
• the liposomal dispersion of embodiment 95 comprising HSPC and cholesterol in a molar ratio of about 45:55.
• the liposomal dispersion of embodiment 95 comprising HSPC, cholesterol and PEG(2000)- DSPE in a weight ratio of about 5:3:1.
• HEPES 2-[4-(2- hydroxyethyl) piperazin-l-yl]ethanesulfonic acid
• 101 The liposomal dispersion of embodiment 100, further comprising sodium chloride at a concentration of 50-80 mM.
• the liposomal dispersion of any one of embodiments 104-106 comprising unilamellar lipid bilayer vesicles which encapsulate an aqueous space containing (5R)-3- ⁇ 3-Fluoro-4-[6-(2-(2- dimethylaminoethyl)-2H-tetrazol-5-yl)-3-pyridinyl]phenyl ⁇ -5-(methylacetamido)-l,3- oxazolidin-2-one as a sulfate salt.
• HSPC hydrogenated soy phosphatidyl choline
• cholesterol cholesterol
• PEG-DSPE PEG(Mol. weight 2,000)- distearoylphosphatidylethanolamine
• the liposomal dispersion of embodiment 108 comprising 50-65 mol% cholesterol relative to the sum of cholesterol and non-pegylated phospholipid in the liposome vesicles.
• the liposomal dispersion of embodiment 108 comprising 50-60 mol% cholesterol relative to the sum of cholesterol and non-pegylated phospholipid in the liposome vesicles.
• the liposomal dispersion of embodiment 108 comprising about 50 mol% cholesterol relative to the sum of cholesterol and non-pegylated phospholipid in the liposome vesicles.
• the liposomal dispersion of embodiment 108 comprising about 55 mol% cholesterol relative to the sum of cholesterol and non-pegylated phospholipid in the liposome vesicles.
• the liposomal dispersion of embodiment 108 wherein the liposomal dispersion lipid content consists of the HSPC, and PEG(2000)-DSPE.
• the liposomal dispersion of embodiment 113 comprising HSPC and cholesterol in a weight ratio of about 5:3.
• PEG(2000)-DSPE in a weight ratio of about 5:3:1.
• HEPES 2-[4-(2- hydroxyethyl) piperazin-l-yl]ethanesulfonic acid
• DFO deferoxamine
• the liposomal dispersion of embodiment 132 comprising 45-65 mol% cholesterol relative to the sum of cholesterol and non-pegylated phospholipid in the liposome vesicles.
• the liposomal dispersion of embodiment 132 comprising 50-55 mol% cholesterol relative to the sum of cholesterol and non-pegylated phospholipid in the liposome vesicles.
• the liposomal dispersion of embodiment 132 comprising about 50 mol% cholesterol relative to the sum of cholesterol and non-pegylated phospholipid in the liposome vesicles.
• the liposomal dispersion of embodiment 132 comprising about 55 mol% cholesterol relative to the sum of cholesterol and non-pegylated phospholipid in the liposome vesicles.
• the liposomal dispersion of embodiment 132 wherein the liposomal dispersion is obtained by a process comprising the step of dissolving (5R)-3- ⁇ 3-Fluoro-4-[6-(2-(2-dimethylaminoethyl)- 2H-tetrazol-5-yl)-3-pyridinyl]phenyl ⁇ -5-(methylamino)-l,3-oxazolidin-2-one hydrochloride in a drug loading solution and contacting the drug loading solution with extracted purified liposome vesicles comprising ammonium sulfate trapping agent to load the AKG-28 into the liposome vesicles.
• DFO deferoxamine
• lipid vesicles consist of HSPC, cholesterol and PEG(2000)-DSPE in a molar ratio of 45:55:2.25 or a mass ratio of 5:3:1.
• DFO deferoxamine
• lipid unilamellar lipid bilayer vesicles comprise HSPC and cholesterol in a molar ratio of 45:55 or in a mass ratio of 5:3.
• R 2 is an amine (NH 2 ) or an acetamide (NHCOCH 3 ); and c. a chelator selected from the group consisting of deferoxamine (DFO) or EDTA at the concentration of 0.1-1 mM. 162.
• DFO deferoxamine
• EDTA EDTA at the concentration of 0.1-1 mM.
• the liposomal dispersion of embodiment 161 further comprising a PEG-DSPE.
• oxazolidinone is (5R)-3- ⁇ 3-Fluoro-4-[6-(2-(2-dimethylaminoethyl)-2H-tetrazol-5-yl)-3- pyridinyl]phenyl ⁇ -5-(methylacetamido)-l,3-oxazolidin-2-one or a pharmaceutically acceptable salt thereof.
• oxazolidinone is a sulfate salt formed within liposome vesicles comprising an ammonium sulfate (AS) trapping agent within the liposomal dispersion.
• AS ammonium sulfate
• a liposomal dispersion comprising (5R)-3- ⁇ 3-Fluoro-4-[6-(2-(2-dimethylaminoethyl)-2H- tetrazol-5-yl)-3-pyridinyl]phenyl ⁇ -5-(methylamino)-l,3-oxazolidin-2-one or a pharmaceutically acceptable salt thereof and lipid vesicles formed from a phospholipid, 55 mol% cholesterol and 5 mol% PEG-DSG.
• a liposomal dispersion at a pH of 7-8 comprising (5R)-3- ⁇ 3-Fluoro-4-[6-(2-(2- dimethylaminoethyl)-2H-tetrazol-5-yl)-3-pyridinyl]phenyl ⁇ -5-(methylamino)-l,3-oxazolidin-2- one or a pharmaceutically acceptable salt thereof and lipid vesicles formed from a phospholipid and 55 mol% cholesterol.
• a liposomal dispersion comprising (5R)-3- ⁇ 3-Fluoro-4-[6-(2-(2-dimethylaminoethyl)-2H- tetrazol-5-yl)-3-pyridinyl]phenyl ⁇ -5-(methylamino)-l,3-oxazolidin-2-one or a pharmaceutically acceptable salt thereof; lipid vesicles formed from a phospholipid and 55 mol% cholesterol; and a chelator.
• chelator is selected from the group consisting of: deferoxamine (desferrioxamine, Desferal), ethylenediamine tetraacetic acid (EDTA), diethylenetriamine pentaacetic acid (DTP A), nitrilotriacetic acid (NTA), ethyleneglycol- 0, O'-bis(2-aminoethyl)-N, N, N', N' -tetraacetic acid (EGTA), N-(2- hydroxyethyl)ethylenediamine-N, N', N' -triacetic acid (HEDTA), and 1,4,7,10- tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA).
• deferoxamine deferoxamine
• EDTA ethylenediamine tetraacetic acid
• DTP A diethylenetriamine pentaacetic acid
• NTA nitrilotriacetic acid
• EGTA ethylenegly
• a liposomal dispersion comprising (5R)-3- ⁇ 3-Fluoro-4-[6-(2-(2-dimethylaminoethyl)-2H- tetrazol-5-yl)-3-pyridinyl]phenyl ⁇ -5-(methylacetamido)-l,3-oxazolidin-2-one or a pharmaceutically acceptable salt thereof and lipid vesicles formed from a phospholipid, 55 mol% cholesterol and 5 mol% PEG-DSG. 178.
• a liposomal dispersion at a pH of 7-8 comprising (5R)-3- ⁇ 3-Fluoro-4-[6-(2-(2- dimethylaminoethyl)-2H-tetrazol-5-yl)-3-pyridinyl]phenyl ⁇ -5-(methylacetamido)-l,3- oxazolidin-2-one or a pharmaceutically acceptable salt thereof and lipid vesicles formed from a phospholipid and 55 mol% cholesterol.
• a liposomal dispersion comprising (5R)-3- ⁇ 3-Fluoro-4-[6-(2-(2-dimethylaminoethyl)-2H- tetrazol-5-yl)-3-pyridinyl]phenyl ⁇ -5-(methylacetamido)-l,3-oxazolidin-2-one or a pharmaceutically acceptable salt thereof; lipid vesicles formed from a phospholipid and 55 mol% cholesterol; and a chelator.
• chelator is selected from the group consisting of: deferoxamine (desferrioxamine, Desferal), ethylenediamine tetraacetic acid (EDTA), diethylenetriamine pentaacetic acid (DTP A), nitrilotriacetic acid (NTA), ethyleneglycol- 0, O'-bis(2-aminoethyl)-N, N, N', N' -tetraacetic acid (EGTA), N-(2- hydroxyethyl)ethylenediamine-N, N', N' -triacetic acid (HEDTA), and 1,4,7,10- tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA).
• deferoxamine deferoxamine
• EDTA ethylenediamine tetraacetic acid
• DTP A diethylenetriamine pentaacetic acid
• NTA nitrilotriacetic acid
• EGTA ethylenegly
• a liposomal dispersion comprising (5R)-3- ⁇ 3-Fluoro-4-[6-(2-(2-dimethylaminoethyl)-2H- tetrazol-5-yl)-3-pyridinyl]phenyl ⁇ -5-(methylacetamido)-l,3-oxazolidin-2-one or a pharmaceutically acceptable salt thereof and lipid vesicles formed from a phospholipid, 55 mol% cholesterol and 5 mol% PEG-DSG.
• Additional embodiments include the oxazolidinone liposomal compositions described in the following additional embodiments below, and other combinations of features recited thereon:
• a liposomal pharmaceutical composition having a pH of 7.3-7.7 and comprising a. a chelator selected from the group consisting of DFO, EDTA and DTPA; b. liposomes vesicles comprising a phospholipid and greater than 40 mol% cholesterol relative to the total phospholipid in the liposomal composition, and c. a sulfate salt of a compound of Formula (I) encapsulated in the liposome vesicles
• R 2 is an amine (NH2) or an acetamide (NHCOCH 3 ), and wherein R 1 is a tetrazole ring substituted at position 2’ with an aminoalkyl.
• R 1 is a tetrazole ring substituted at position 2’ with an aminoalkyl.
• the liposomal composition of embodiment 1, the liposome vesicles comprising a compound of Formula 1c Formula 1c The liposomal composition of embodiment 1, the liposome vesicles comprising a compound of Formula Id or Formula 1e
• the liposomal composition of embodiment 7, wherein the trapping agent is triethylammonium sucrose octasulfate or ammonium sulfate.
• the liposomal composition of embodiment 7, wherein the trapping agent is triethylammonium sucrose octasulfate.
• the liposomal composition of embodiment 7, wherein the trapping agent is ammonium sulfate.
• the liposomal composition of any one of embodiments 1 to 5, comprising a salt of the compound, wherein the salt is sulfate.
• the liposomal composition of any one of embodiments 1 to 5, wherein the liposome vesicles comprise a membrane comprising phosphatidylcholine and cholesterol.
• the liposomal composition of embodiment 15, wherein the phosphatidylcholine to cholesterol molar ratios is from about 60:40 to about 35:65.
• the liposomal composition of embodiment 15, wherein the phosphatidylcholine to cholesterol molar ratio is from about 55:45 to about 35:65.
• the liposomal composition of embodiment 15, wherein the phosphatidylcholine to cholesterol molar ratio is from about 50:50 to about 45:55.
• the liposomal composition of embodiment 15, wherein the phosphatidylcholine to cholesterol molar ratio is from about 50:50 to about 40:60.
• the liposomal composition of any one of embodiments 1 to 5, wherein the liposome vesicles comprise HSPC, cholesterol and polymer-conjugated lipid in a about 45:55:2.75 molar ratio.
• PEG-DSG PEG(Mol. weight 2,000)-distearoylglycerol
• PEG-DSPE PEG(Mol. weight 2,000)-distearoylphosphatidylethanolamine
• a method of treating bacterial infection comprising administering to a subject in need thereof a therapeutically effective amount of the liposomal composition of any one of embodiments 1 to 5.
• the method of embodiment 27, wherein the bacterial infection is mycobacterium tuberculosis infection.
• the method of embodiment 27 or embodiment 28, wherein the compound in the liposome vesicles has a minimum inhibitory concentration (MIC) ranging from about 0.01 ⁇ g/ml to about 0.25 ⁇ g/ml.
• MIC minimum inhibitory concentration
• the method of embodiment 27 or embodiment 28, wherein the compound in the liposome vesicles has a MIC ranging from about 0.01 ⁇ g/ml to about 0.1 ⁇ g/ml.
• the method of embodiment 32, wherein the one or more active agents comprise bedaquiline, pretomanid, pyrazinamide, moxifloxacin, a pharmaceutically acceptable salt thereof or a combination thereof.
• the method of embodiment 31, wherein the liposomal composition is administered once a week to once every six weeks. 35.
• the method of embodiment 31, wherein the percentage of compound remaining in blood following administration to the subject in need thereof is greater than 20% of the administered amount at 6 hours.
• a method of making liposome composition comprising the steps of:
• Compounds AKG-1, AKG-2, AKG-6, AKG-8, AKG-9 and AKG-19 were synthesized by reacting Tedizolid mesylate (Tedizolid-MS) with respective amines at 60 °C in N- methyl-2-pyrrolidone (NMP) as a solvent (Scheme- 1).
• Tedizolid-MS was obtained by mesylation of the 1° hydroxyl group of Tedizolid with methanesulfonyl chloride in the presence of a base at room temperature (RT).
• Intermediate-2 was synthesized by boronation of commercially available aryl bromide using bis(pinocolato)diboron (Scheme-2). Suzuki coupling of Intermediate-2 with readily available 5-bromo-2-fluoropyridine resulted in Intermediate-3, which was heated in NMP in a sealed tube with the corresponding amine to give compounds AKG-11 to AKG-15.
• Intermediate- 13 was synthesized by mesylation of readily available aryl bromide.
• Intermediate- 15 was obtained by reducing Intermediate-14 with hydrazine (Scheme-5). Boc protection or acetylation of the primary amine in Intermediate- 15 followed by boronation resulted in Intermediates- 18 and 19, respectively. Suzuki (U.S. Pat. Appl. Publ. No. 20100022772, PCT Int. Appl. Publ. No.
• Fig. 24 shows synthesis Scheme- 1.
• Fig. 25 shows synthesis Scheme-2.
• Fig. 26 shows synthesis Scheme-3.
• Fig. 27 shows synthesis Scheme-4.
• Fig. 28 shows synthesis Scheme-5.
• Analytical HPLC was performed on an Agilent analytical HPLC system using a Sunfire column, 3.5pm (150 cm x 4.6 mm) and a gradient system (water (0.01%TFA)/ACN (0.01%TFA)) and a flow rate of 1 mL/min with detection at 254 and 214 nm. Flash Chromatographic (FC) purifications were performed with Silica Gel 60 from Santai Technologies (0.04-0.063 nm; 230-400 mesh).
• Procedure A The reaction mixture of Tedizolid-Ms (1.0 eq), R1R 2 NH (4.0 eq) in NMP (10 mL) was heated to 60 °C for 15 h in a sealed tube. Upon completion (LCMS), the reaction was diluted with H 2 O (40 mL) and extracted with EtOAc (2X50 mL). The combined extracts were washed with saturated brine dried over Na 2 SO 4 and filtered. The solvent was removed in vacuo and the residue was purified using FC to give the product with >95% purity.
• the product was re-dissolved into H 2 O and 1 eq of aq. HC1 (0.02 M) was added. Freeze drying the product resulted in AKG-5 as a HC1 salt (600 mg, 42.7% yield).
• the salt was then re-dissolved into H 2 O and 1 eq of HC1 (0.02 M) was added, after freeze drying the product AKG-20 as a HC1 salt was obtained (0.61 g, 42% yield).
• Procedure B A mixture of Intermediate-3 (1.0 eq), R1R 2 NH (4.0 eq) and cat. amount of DMAP in NMP (10 mL) was heated to 100 °C for 16 h in a sealed tube. On completion of reaction (LCMS), it was diluted with H 2 O (50 mL) and extracted with EtOAc (2X50 mL). The combined organic extracts were washed with saturated brine followed by drying over Na 2 SO 4 and filtering. The solvent was removed in vacuo and the residue was purified using RPFC (Eluant with MeCN in 0.1% NH 4 HCO 3 /H 2 O, 0-40%, C18) to give the product.
• RPFC Eluant with MeCN in 0.1% NH 4 HCO 3 /H 2 O, 0-40%, C18
• a solution AKG-27-1 (0.9 g, 1.75 mmol) in dry DCM (16 mL) was added HC1 in dioxane (4.0 mL) under N2 atmosphere at RT. The reaction mixture was stirred at the same temperature for 6 hours and cooled down RT. The mixture reaction was evaporating the solvent under reduced pressure, gave AKG-27 (0.65 g, 82.5%) as a pale yellow solid.
• Procedure C A mixture of one of Intermediates-5/8/9/10/11 (l.Oeq), one of Intermediates-18/19 (L5eq), Pd(dppf)C12 DCM (O.leq), and K 3 PO 4 (2.0eq) in dioxane/H 2 O(10:l, 0.06M) was purged with N 2 and stirred at 90°C overnight. The mixture was diluted with EtOAc, washed with water and brine, dried over anhydrous magnesium sulfate, filtered and concentrated. The residue was purified by FC to give one of the compounds AKG-28-1/ AKG-29-1/ AKG-30- 1/ AKG-31-1/AKG-38/AKG-39/AKG-40.
• MICs were determined by broth microdilution assay with an Alamar blue endpoint (MABA), as described by Collins et al., 1997 (Collins L, Franzblau SG (1997). Microplate alamar blue assay versus BACTEC 460 system for high-throughput screening of compounds against Mycobacterium tuberculosis and Mycobacterium avium. AAC. 41(5): 1004- 1009) and Gruppo et al., 2006 (Gruppo V, Johnson CM, Marietta KS, Scherman H, Zink EE, Crick DC, Adams LB, Orme IM, Lenaerts AJ.
• MABA Alamar blue endpoint
• MABA is a 96-well colorimetric assay in which the redox indicator Alamar blue turns from blue to pink in the presence of mycobacterial growth activity in the broth medium.
• 7H9 complete media was prepared by adding Middlebrook 4.7 g of 7H9 broth powder (Millipore Sigma Cat #M0178), 2 mL glycerol, and 898 purified water in 1 L flask with mixing until dissolved, and subsequently adding 100 mL of ADC solution (6 g bovine serum albumin, 2 g dextrose, and 3 mg catalase dissolved in 100 mL water) to the same 1 L flask.
• ADC solution 6 g bovine serum albumin, 2 g dextrose, and 3 mg catalase dissolved in 100 mL water
• a series of nine 1:2 dilutions was prepared by adding 50 pl of drug solution in the first well to 50 pl of DMSO in the subsequent well and the carrying forward this process to the next eight wells in a drug preparation plate.
• Stocks of M.tuberculosis (M.tb) H34Rv and M.tb Erdman strains were diluted from their initial concentration of 3-4x10 7 CFU/mL with media to a final concentration of 5x10 5 CFU/mL, mixed thoroughly by pipetting up and down with a multi-channel pipettor.
• Assay plates were prepared by transferring 100 pl of the 5x10 5 CFU/mL inoculated media into all wells. Subsequently, 2.5 ⁇ L of each drug dilution from the drug preparation plate was transferred to the corresponding well in the assay plate. Assay plates were subsequently placed in ziplock bags and placed inside an incubator where they were incubated at 37 °C. The plates were subsequently read at OD 600 nm on a plate reader on days 3 and 10. After the day ten OD600 reading, 10 pl of Alamar Blue dye was added to each analytical well. On day 12, all assay plates were scanned on a flatbed color scanner.
• the lowest consecutive antimicrobial concentration (typically two-fold serial dilutions) that does not produce visible color change from blue to pink with Alamar Blue, and/or shows a > 80% reduction in OD600 relative to drug-free control wells, was regarded as the MIC for these compounds.
• Assays were conducted using two unique drug sensitive strains (M.tb Erdman and M.tb H37Rv). MIC assays can also be performed in presence of 4% (w/v) human serum albumin (huSA) (Sigma # Al 653) in order to assess potential protein binding (serum shift assay). Generally, a shift in MIC of two wells (4-fold shift in MIC) is considered to be significant. For PA-824 (positive control), a 4-fold shift in MIC is to be expected.
• huSA human serum albumin
• MICs were measured by the Alamar Blue (MABA) readout or by optical density readout (OD600) agreed or differed only by one 2-fold dilution, which is within the limits of the assay. All compounds tested showed consistency in MIC values against both Mtb Erdman and H37Rv, or were within one 2-fold dilution, with the exception of one compound AKG-40; which showed a higher MIC value of 1-2 ug/mL vs Erdman, and an MIC of 0.5 vs H37Rv. This discrepancy could be due to slower growth (lower OD readings) on the Erdman plate.
• MABA Alamar Blue
• OD600 optical density readout
• Linezolid showed an expected MIC value of 2 ⁇ g/mL, Tedizolid at 0.25 ⁇ g/mL and Bedaquiline at 0.125 ⁇ g/mL. These values are consistent with past MIC data and published values (Ruiz et al. Antimicrob. Agents Chemother. 2019, Mar 27; 63 (4), pii: eO 1939- 18 , Reddy et al. Antimicrob Agents Chemother. 2010 Jul;54(7):2840-6 , Torrea et al. J Antimicrob Chemother. 2015 Aug; 70(8):2300-5). AKG-28 showed an MIC of 0.03-0.015 ⁇ g/mL, significantly more active than Tedizolid.
• AKG-39 showed an MIC of 0.5 ⁇ g/mL, and AKG-40 an MIC of 1-0.5 ⁇ g/mL.
• AKG-38 with an MIC of 0.06 ⁇ g/mL also showed several folds greater activity than Tedizolid.
• Example 3 Assay for in vitro cytotoxicity to human kidney and human hepatocyte cells
• Adherent cells were grown to -80% confluency.
• the cells were trypsinized by adding 0.25 % trypsin-EDTA (Gibco # 25200-072) and the cells subsequently spun down, and 5 ml of growth medium (MEM media; Corning # 10010 CM) added to disperse the cells.
• MEM media growth medium
• the cell density was determined using a hemocytometer.
• Growth medium (MEM media containing 10% FBS; Corning # 35015 CV) was added to the cells to adjust to an appropriate concentration of cells.
• the increased toxicity to hepatocytes results in a comparatively low Selectivity Index for theoxazolidinones with a hydroxyl on the C5 side chain (AKG-23, AKG-25, AKG-26, and AKG-27) when compared to those with an amino or acetamide group at the same position on the C5 side chain (AKG28-31, AKG38-40, and AKG-3).
• a Selectivity Index was calculated to determine the relative inhibitory activities of the compounds on the two Mycobacterium tuberculosis strains, Erdman and H37Rv, compared to that on mammalian cells, namely, African green monkey kidney (VERO) or human hepatocyte-derived (HepG2) cells, as described in Experimental Examples 2 and 3, respectively.
• a high SI is preferable as it indicates preferred killing of the bacteria of tuberculosis strains at concentrations of the drug that are less harmful to normal cells in the body.
• the selectivity index was calculated using the formula below:
• the SI is shown as greater than (>) the ratio calculated using that highest concentration.
• the MIC for Erdman or H37Rv strains is greater than the highest concentration of drug tested (8 ⁇ g/ml)
• the SI is shown as less than ( ⁇ ) the ratio calculated using that highest concentration. Calculations where both numbers are above the highest concentrations tested are shown as not determined (nd). The results are shown in TABLE 4.
• the SI did not correlate directly to the activity of the molecules in either mycobacterial strains or mammalian cell lines, and increased potency in mycobacterial strains did not correlate directly to increased toxicity against the mammalian cell lines.
• AKG-38 demonstrated nanomolar MIC against both strains of mycobacterium tuberculosis, whereas it was relatively inactive against both VERO and HepG2 cell lines compared to other molecules in the panel, giving it a high SI. This was similarly seen for AKG-28. It is notable that both molecules, AKG-28 and AKG-38, had a dimethylaminoethyl substituent at the 2’ position of the tetrazole ring.
• the compounds of interest have a SI index for Erd/HepG2 and H37Rv/HepG2 higher than 100, higher than 200, higher 300, higher than 400, higher than 500, higher than 1000, higher than 1500, higher than 2000, higher than 2500, higher than 3000, higher than 3500, higher than 4000, higher than 4500, higher than 5000, higher than 5500, higher than 6000, higher than 6500, between 100 and 7000, between 100 and 6000, between 100 and 5000, between 100 and 4000, between 100 and 3000, between 100 and 2000, between 100 and 1000, between 100 and 900, between 100 and 800, between 100 and 700, between 100 and 600, between 100 and 500, between 100 and 400, between 100 and 300, between 100 and 200, between 200 and 7000, between 200 and 6000, between 200 and 5000, between 200 and 4000, between 200 and 3000, between 200 and 2000, between 200 and 1000, between 200 and 900, between 200 and 800, between 200 and 700, between 200 and 600, between 100 and 500, between 100 and 400,
• Example 5 Assay for in vitro activity against methicillin resistant Staphylococcus aureus (MRSA).
• the activity of the lead oxazolidinone inhibitors was measured to demonstrate sufficient potency against the gram positive bacterium methicillin resistant Staphylococcus aureus (MRSA) to justify their subsequent delivery in the form of liposomes for the treatment of the same.
• MRSA gram positive bacterium methicillin resistant Staphylococcus aureus
• the MIC in two of the three evaluated strains of less than 6 ⁇ g/mL. In some embodiments, the MIC in two of the three evaluated strains of less than 2 ⁇ g/mL less than 2 ⁇ g/mL is more preferred.
• Tedizolid showed an MIC of 0.206-0.617 ⁇ g/ml, similar to the 0.5 ⁇ g/ml described in US Patent No. 7,816,379.
• all of the molecules (AKG-3, AKG-28, AKG-29, and AKG-30) with a primary amine modification at R 2 of the oxazolidinone ring showed negligible activity against all three MRSA strains (>50 ⁇ g/ml).
• the molecules with an acetamide group at the same position (AKG-38, AKG-39, and AKG-40) were between 3 and 9-fold less active than tedizolid itself against the three MRSA strains.
• Example 6 Liposome compositions.
• lipid components phospholipid (PhL), cholesterol, and optionally - a PEG- lipid derivative and/or a lipid fluorescent label were combined in an amount of 100% ethanol equal to one-tenth of a volume (V) calculated to obtain lipid suspension with about 60 mM phospholipid and stirred at the temperature of 65-68 °C until complete dissolution of the lipids.
• Neutral phospholipids can include diacylphosphatidylcholines, dialkylphosphatidylcholines, sphingomyelins, and diacylphosphatidylethanolamines. Hydrogenated soyphosphatidylcholine, distearoylphosphatidylcholine, and egg sphingomyelin are some of the preferred phospholipids.
• PEG-lipid components may include PEG(Mol. weight 2, 000)-di stearoylglycerol (PEG-DSG), l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG-DSPE) or N-palmitoyl-sphingosine-l- ⁇ succinyl[methoxy(poly ethylene glycol)2000] ⁇ (PEG-ceramide).
• the molecular weight of the PEG-lipid component can also vary from 1,500-6,000 g/mol, but is preferably around 2,000 MW.
• Lipid fluorescent labels can include 1, l'-Dioctadecyl-3,3,3',3'- Tetramethylindocarbocyanine-5,5'-Disulfonic Acid (DiIC18(3)-DS), l,l'-Dioctadecyl- 3,3,3',3'-Tetramethylindodicarbocyanine-5,5'-Disulfonic Acid (DiIC8(5)-DS), 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-N-(Cyanine 7) (18:0 Cy7 PE), 1,2-distearoyl-sn- glycero-3-phosphoethanolamine-N-[amino(poly ethylene glycol)-2000]-N-(Cyanine 7) (DSPE PEG(2000)-N-Cy7), l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(Cyanine 7)
• Potential trapping agents may include but are not limited to diethylammonium or triethylammonium salts of sucrose octasulfate, ammonium sulfate, ammonium citrate, citric acid, dextran sulfate, polyvinyl sulfonate, or ammonium salts of inositol hexaphosphate, in the concentrations of 0.1-2 g-equivalents/L (0.1-2 N), preferably 0.2-1.5 N.
• Ammonium salts are typically employed and may include ammonium itself, monoalkyl-, dialkyl-, or trialkylammonium salts.
• the lipid suspension was extruded at least three times through a stack of track- etched polycarbonate membranes, typically, two or four membranes with the nominal pore size of 100 nm and one with the nominal pore size 200 nm (Whatman Nucl epore, USA), using a thermobarrel extruder (Lipex, Canada) at 65-68°C, at the pressure of 400-450 psi.
• the extrusion pressure was typically 260- 300 psi.
• the resulting liposomes have Z-average particle size (diameter) Xz between about 80 and about 130 nm, and PDI less than 0.1.
• the extruded lipid suspension (known to contain unilamellar and/or oligolamellar liposomes) was chilled in refrigerator (2-8 °C) and fdtered through a 0.2-micron Poly ethersulfone (PES) membrane fdter under positive pressure.
• PES Poly ethersulfone
• this step was performed using a tangential flow filtration (TFF) on a hollow fiber cartridge (Repligen Spectrum MicroKros PS or mPES membrane with MWCO of 500 KDa) effecting 8-10 volume exchanges (or until the conductivity of the liposome suspension dropped below 200 pS/cm) with Type 1 or USP “Water for injection” endotoxin-free water.
• TMF tangential flow filtration
• the lipid concentration in a purified extruded liposome preparation was determined using HPLC with UV detection, by measuring the concentration of cholesterol and correcting for the known phospholipid/cholesterol molar ratio Alternatively, a spectrophotometric blue phosphomolybdate method was used to directly quantify the phospholipid content.
• the drug was dissolved in Type 1 or endotoxin-free pure water in the form of a hydrochloric acid salt (e.g., AKG-3 and AKG-5 were used as monohydrochloride, AKG-28 and AKG-29 were used as dihydrochloride) at the concentration of 5-20 mg/ml of the drug.
• a hydrochloric acid salt e.g., AKG-3 and AKG-5 were used as monohydrochloride, AKG-28 and AKG-29 were used as dihydrochloride
• free base form e.g., AKG-16, AKG-38
• Tris tri s(hydroxymethyl)aminom ethane
• the amount of added osmotic agent e.g., dextrose at about 45 g/L
• the amount of added osmotic agent provided osmolality less that the measured osmolality of the trapping agent solution, and the loading was effected at 6-8 mg/ml of the drug.
• the drug-loaded liposomes were purified from the unencapsulated drug by size exclusion chromatography (SEC) on a gravity-feed Sepharose CL-4B column, eluent - 10 mM HEPES-buffer pH 7.0 in 140-144 mM NaCl (HBS-7). The liposome fractions were collected near the column void volume.
• SEC size exclusion chromatography
• the purification and buffer exchange were performed using TFF as described under item 5 above, using 10 volume exchanges with the HBS- 7 buffer. In a scaled-up process, about 8 volume exchanges were typically used.
• the purified liposomes were concentrated by continuing the TFF process without buffer feed.
• the purified, drug-loaded liposomes were aseptically filtered using 0.2-micron sterile PES filter under positive pressure and stored in refrigerator (2-8 °C).
• DL0 drug-to-phospholipid ratio in the liposome loading mixture before SEC or TFF purification
• DL is the drug-to-phospholipid ratio in the drug-loaded liposomes after purification (step 10).
• PDI Zetasizer Pro
• Example 7 In vivo stability and blood clearance of the liposomes.
• mice The stability of drug encapsulation and the blood clearance rates of the liposomes that encapsulate the compounds of the present disclosure was studied in mice according to the following general protocol.
• Mice of a given laboratory strain C3H female or CD-I male
• mice were injected with the drug-loaded liposomes via tail vein at the dose of 9 mg of the drug per kg of the body weight.
• timepoints 1 and 2 the blood was sampled from the retroorbital sinus, and the animals were sacrificed.
• the blood sampling timepoints included 5 min, 1 hour, 6 hours, and 24 hours post injection.
• the plasma was separated by centrifugation, extracted with acidified isopropanol, optionally containing a solubilizing agent (sodium octanesulfonate), and analyzed for the drug and the lipid (when a liposome the incorporated a lipid label, DilC18(3)-DS) by HPLC. Blood clearance of the liposomal drug was expressed at percent of injected dose remaining at a given timepoint. In vivo stability of the drug encapsulation was assessed by the percent change (decrease) of DL ratio in the plasma at a given timepoint compared to the pre-inj ection DL value.
• a solubilizing agent sodium octanesulfonate
• Trimethylammonium sucrose octasulfate trapping agent solution was prepared by passing a solution of commercial potassium sucrose octasulfate heptahydrate (40.2 g in 145 ml of water) through a 500-ml ion exchange column of Dowex 50Wx8 100-200 mesh in a hydrogen form and titration of the resulting free acid form of sucrose octasulfate with neat triethylamine to pH 6.2.
• the concentration of tri ethyl ammonium sucrose octasulfate (TEA-SOS) (1 N, corresponding to 0.125 M sucrose octasulfate) was estimated from the amount of triethylamine consumed in titration. Residual potassium was estimated using Horiba LAQUATwin K-ll potassium analyzer by the method of additions and was less than 0.1% of the initial potassium amount.
• Liposomes composed of hydrogenated soy phosphatidylcholine (HSPC) (Lipoid, Germany), cholesterol (3:2 molar ratio), and methoxypoly(ethyleneglycol) ether of 1, 2- di stearoylglycerol (PEG-DSG, PEG mol.weight 2000, NOF, Japan) (0.5 mol.% of HSPC) with 1 N trimethylammonium sucrose octasulfate (TEA-SOS) as a trapping agent were prepared essentially as described in the General protocol above.
• HSPC hydrogenated soy phosphatidylcholine
• PEG-DSG methoxypoly(ethyleneglycol) ether of 1, 2- di stearoylglycerol
• TAA-SOS trimethylammonium sucrose octasulfate
• the drug loading step was performed at the DL ratio (DL0) of 500 g/mol PhL in the presence of 16 mM morpholinoethanesulfonic acid (MES) -4 mM sodium citrate buffer having pH in the range of 4.3 -7.1, as well as without addition of any buffer substance (pH 5.2-5.9). All drugs were encapsulated into the liposomes with high efficiency (over 98%, except for AKG-16 at pH 4.38, that was loaded with the efficiency of 93.3%) in the whole studied range of pH (FIG. 1). Addition of a buffer substance was not required for efficient encapsulation.
• Example 9 Encapsulation of AKG-3, AKG-5, and AKG-16 into liposomes with TEA-SOS trapping agent at different DL ratios.
• Liposomes composed of HSPC, cholesterol (3:2 molar ratio), and PEG-DSG (0.5 mol.% of HSPC) with 1 N TEA-SOS as a trapping agent were prepared essentially as described in the General protocol (Example 6).
• the drug loading step was performed at the DLO ratios in the range of 750-1500 g/mol PhL without addition of a buffer substance (pH 4.98-6.22).
• Example 10 Encapsulation of AKG-3, AKG-5, and AKG-16 into liposomes with higher degree of PEGylation or with 0.25 M ammonium sulfate (AS) as a trapping agent
• Liposomes composed of HSPC and cholesterol (3:2 molar ratio) having various PEG-DSG content and trapping agents were prepared according to the General protocol and loaded with compounds AKG-3, AKG-5, and AKG-16, as in Example 9, at DLO ratios of 250 or 500 g/mol PhL. All three compounds were loaded into the liposomes with high efficiency as shown in the Table 6 below:
• Liposomes composed of HSPC and cholesterol (3:2 molar ratio) having 0.5 mol% or 5 mol% PEG-DSG (relative to PhL) and 0.5 M ammonium sulfate (AS) as a trapping agent were prepared according to the General protocol and loaded with compounds AKG-3, AKG-5, and AKG-16, as in Example 8, at DL0 ratios in the range of 500-1500 g/mol PhL. The results are shown on FIG. 3A, FIG. 3B, FIG. 3C and FIG. 3D. All three compounds were loaded in both liposomes to the DL ratio of 420-450 g/mol PhL with encapsulation efficiency of 93-100%; maximum drug payloads were as follows:
• Example 10 The loading was significantly improved over Example 10, where the loading efficiency was lower using 0.25 M ammonium sulfate, demonstrating that the higher ammonium sulfate concentration of 0.5 M, despite the higher osmolarity and potential for osmotic burst, is improved with respect to the amount of drug that can be loaded per mol of phospholipid, and preferable for anti-infectives where low toxicity and high dosing can lead to improved outcomes.
• Example 12 Loading of compounds AKG-3, AKG-5, AKG-16, and AKG-28 into liposomes of various compositions including a fluorescent lipid label
• Liposomes composed of HSPC and cholesterol (60:40 molar ratio) having 0.5 mol% PEG-DSG (relative to PhL), 0.15 mol.% lipid fluorescent label DiIC18(3)-DS (ThermoFisher, USA), and 0.5 M ammonium sulfate (AS) or 1 N TEA-SOS as trapping agents were prepared according to the General protocol and loaded with compounds AKG-3, AKG-5, and AKG-16, as in Example 11, at pH 4.7-5.8 (no added buffer substance).
• the liposomes had the following characteristics:
• Liposomes composed of various phospholipids (HSPC, distearoylphosphatidylcholine (DSPC, Avanti Polar Lipids, USA), or egg sphingomyelin (ESM, Lipoid, Germany) and cholesterol (60:40 molar ratio), containing various amounts of PEG-DSG or N-methoxypoly(ethyleneglycol)oxycarbonyl-l,2-distearoylphosphatidylethanolamine (PEG- DSPE, PEG mol.
• HSPC distearoylphosphatidylcholine
• ESM egg sphingomyelin
• PEG- DSPE N-methoxypoly(ethyleneglycol)oxycarbonyl-l,2-distearoylphosphatidylethanolamine
• Liposomes composed of HSPC and cholesterol (3:2 molar ratio) having 9.2 mol% PEG-DSPE (relative to PhL), 0.15 mol.% lipid label DiIC18(3)-DS, and 0.25 M ammonium sulfate (AS) as a trapping agent were prepared according to the General protocol and Example 12 with additional 50-nm extrusion, and loaded with AKG-28 at the drug-lipid ratio ( DL0) 150 g/mol PhL.
• the liposomes (Batch ID 98) has DL ratio of 73.8 g/mol PhL, Z-average liposome size 77.8 nm, and size polydispersity index (PDI) 0.090.
• Example 13 Blood persistence and in vivo encapsulation stability of the liposomes of Example 12 in mice.
• Example 14 Encapsulation of Compounds AKG-28 and AKG-38 into liposomes with various trapping agents, at different DL ratios
• Liposomes composed of HSPC and cholesterol (3:2 molar ratio) having 0.5 mol%
• PEG-DSG relative to PhL
• 0.15 mol.% lipid label DiIC18(3)-DS 0.15 mol.% lipid label DiIC18(3)-DS
• 0.5 M ammonium sulfate (AS) or 1 N TEA-SOS as trapping agents were prepared according to the General protocol and loaded with compounds AKG-28 and AKG-38, as in Example 8, at pH 4.95-5.17 (no added buffer substance) and DLO ratios in the range of 300-1050 g/mol PhL (AKG-28) or 400-1400 g/mol PhL (AKG-38).
• maximum drug loads for compounds AKG-28 and AKG-38 were in the range 404-424 g/mol PhL, and 818-842 g/mol PhL, respectively, and the loading efficiencies of more than 95% were at drug loads of 302 g/mol PhL (quantitative loading) and 387-764 g/mol PhL (95.5-96.7% loading), respectively.
• AKG-38 showed nearly quantitative loading between 400 and 800 g AKG-38/mol PhL, while the resulting drug-to-lipid ratio remained flat for AKG-28 over the range of 250-1000 g AKG-28/mol PhL suggesting a lower maximum drug load for AKG-28 than for AKG-38. It should be appreciated that the higher potency previously demonstrated for AKG-28 would allow liposome formulations of AKG-28 to be effective for treating infectious diseases like tuberculosis.
• Example 15 Encapsulation of Compounds AKG-28 and AKG-38 into liposomes with various phospholipid composition, degree of PEGylation, and trapping agents.
• Liposomes composed of a phospholipid (PhL) and cholesterol (3:2 molar ratio), PEG-DSG, and DiIC18(3)-DS (0.15 mol.% of PhL) with 0.5 M AS or 1 N TEA-SOS as trapping agents were prepared according to the General protocol and loaded with compounds AKG-28 and AKG-38 (in the absence of added buffer substance) at DLO ratios chosen to optimize the drug load and the encapsulation efficiency (EE). The results are in the Tables 10 and 11 below.
• Compound AKG-38 was loaded to significantly higher D/L ratios, between 525- 600 g/mol using 0.5 M AS or 1 N TEA-SOS when drug was added at 600 g AKG-38/mol PhL, or more than 735 g/mol when added at 800 g AKG-38/mol PhL. Loading for compound AKG-38 was less sensitive to the presence of sphingomyelin than was AKG-28.
• Example 16 Encapsulation of Compounds AKG-16, AKG-28, AKG-29, and AKG-38 into liposomes with increased PEGylation and 0.5 M ammonium sulfate as trapping agent .
• Liposomes composed of HSPC and cholesterol (3:2 molar ratio), PEG-DSG (5 mol.%), and DiIC18(3)-DS (0.15 mol.%) with 0.5 M ammonium sulfate as trapping agent were prepared according to the General protocol and loaded with compounds AKG-16, AKG-28, AKG- 29, or AKG-38 (in the absence of added buffer substance) at DLO ratios chosen to optimize the drug load and the encapsulation efficiency (EE). The results are in the Table 13 below.
• Example 17 Blood persistence and in vivo encapsulation stability of the liposomes of Examples 15 and 16 in mice.
• Example 18 Preparation and loading of AKG-28 and AKG-38 into pegylated liposomes with varying ratios of phospholipid-to-cholesterol.
• Liposomes containing 5 mol% PEG-DSG or PEG-DSPE (relative to PhL), 0.15 mol.% lipid label DiIC18(3)-DS, and 0.5 M ammonium sulfate (AS) or 1 N TEA-SOS as trapping agents, were prepared according to the General protocol and loaded with compounds AKG-28 and AKG-38, as in Example 8, at pH 5.07-5.82 (no added buffer substance).
• Z-average size (x Z ) and poly dispersity index (PDI) of the liposomes were determined by dynamic light scattering (DLS) cumulants method using Malvern Zetasizer Pro (Malvern Panalytical) at 173° measurement angle.
• Plasma 80 pl was mixed with liposomal drug formulations (20 pl) in a 0.5 ml Eppendorf tube. The mixture was subsequently incubated for 20 min at 37 °C and then put into chilled water. The mixture (0.1 mL) was chromatographed without delay on a 2 mL Sepharose CL-4B column, eluted with Hepes-buffered saline (pH 7.0) and 0.25 mL of liposomal drug was collected in the void volume fraction. The drug and DiI(3)-DS lipid label were then analyzed by HPLC as described in Example 7, and the % drug remaining encapsulated determined using the following formula:
• Ad /Ai /(Ad,0 /Ai,0)*100 % drug remaining encapsulated
• Ad - are of the drug peak
• Ai,o- are of the lipid label peak pre-incubation.
• FIG. 5A, FIG. 5B, FIG. 5C and FIG. 5D The results are shown on FIG. 5A, FIG. 5B, FIG. 5C and FIG. 5D.
• burst release phenomenon a rapid drop of the DL ratio signifying the drug release from the liposomes
• burst release phenomenon was observed in human plasma for the formulations containing 40 mol.% cholesterol, but not for the formulations with 45 mol.% or more of cholesterol.
• FIG.5B For the liposomes with encapsulated AKG-38 (FIG.5B), burst release phenomenon was observed in both human and mouse plasma for the formulations with cholesterol content of 40 mol.% and 45 mol.%, but not at cholesterol content of 50 mol.% or more.
• Example 20 In vitro plasma release and in vivo pharmacokinetics of 5 mol % PEG-lipid liposomes containing AKG-38 and 40 or 55 mol % cholesterol
• Example 18 Three of the liposome formulations of Example 18 using the 0.5 M AS trapping agent were evaluated in a two time point pharmacokinetic study in female CD-I mice as described in Example 7, measuring percent of the injected dose (% ID) of the liposome lipid remaining in the blood at both 5 min and 6 h, and measuring drug release from the liposomes through determination of the drug-to-lipid ratio (DL).
• % ID percent of the injected dose
• DL drug-to-lipid ratio
• Example 21 Inhibition of mitochondrial protein synthesis (MPS) by AKG-3, AKG-16, AKG-22, AKG-28, AKG-29, AKG-30, AKG-38, AKG-39, and AKG-40 and selectivity for M. tuberculosis (H37Rv) inhibition over MPS inhibition.
• MPS mitochondrial protein synthesis
• the levels of two mitochondrial proteins were measured simultaneously, including the mitochondrial DNA-encoded subunit I of Complex IV (COX-1) and the nuclear DNA-encoded 70kDa subunit of Complex II (SDH-A).
• COX-1 mitochondrial DNA-encoded subunit I of Complex IV
• SDH-A nuclear DNA-encoded 70kDa subunit of Complex II
• the H9C2 rat BDIX heart myoblast cell line was used in these studies in a 384 well plate assay format. Cells were grown in DMEM media with 10 % FBS and IxGlutamine at 37 °C and 5 % CO 2 . Cells were plated at a density of 1,500 cells/well in 384 well plates in 47.5 pl/well.
• An MPS selectivity index (SI-MPS) was determined by dividing the MPS IC50 in ug/ml by the MIC in the drug sensitive H37Rv M. tuberculosis strain as determined in Example 2.
• Example 22 Scaled-up preparation of liposomal AKG-28 lot 275.
• the resulting extruded liposomes were kept overnight in a refrigerator (2-8 °C) and filtered through 0.2-pm polyethersulfone (PES) filter under positive pressure.
• Extraliposomal trapping agent ammonium sulfate
• TFF buffer exchange for endotoxin-free water on a KrosFlo TFF system using polysulfone hollow fiber cartridge with MW cut-off 500 KDa (Spectrum Laboratories) until residual conductivity dropped to less than 200 pS/cm (143 pS/cm after 5.2 volume exchanges).
• the phospholipid concentration in the post-TFF liposome suspension was determined by blue phosphomolybdate method to be 57.4 mM.
• AKG-28 as dihydrochloride salt
• aqueous stock solution adjusted to pH 5.03 with NaOH
• post-TFF liposome suspension was combined with post-TFF liposome suspension to form the loading mixture at drug-to-phospholipid (DL) ratio of 250 g/mol in the presence of 45 mg/ml dextrose and AKG-28 concentration of 6 mg/ml.
• the mixture was quickly heated to 60-63 °C by external heating under constant stirring, and the incubation continued with stirring on the 65°C bath. After 20 min. incubation, the mixture was quickly chilled in an ice-water to less than 10 °C, and kept at this temperature for about 10 min.
• the drug-loaded liposomes were purified by TFF using polysulfone hollow fiber cartridge with molecular weight cutoff 500 KD.
• the liposomes were pre-concentrated by diafiltration to about 12 mg/ml of AKG-28 and purified from any extraliposomal drug by TFF exchange into 10 mM HEPES-Na buffer pH 7.0, containing 0.144 M NaCl made with endotoxin- free water (HBS-7 buffer) for the total of about 8 volume exchanges.
• the proportion of unencapsulated drug prior to purification was estimated spectrophotometrically at 305 nm in the pre-concentration diafiltrate and found to be about 0.9% (corresponds to 99.1% loading efficiency).
• the concentrated, purified liposomes were aseptically passed through 0.2-pm sterile filter and analyzed for the particle size by DLS, and for the drug and phospholipid concentration by spectrophotometry. This procedure was repeated three more times (lots 269, 271, 273). Obtained liposomes had the characteristics shown in TABLE 19.
• Example 23 Scaled-up preparation of liposomal AKG-38 lot 276.
• Lot 268 Lot 268.
• the protocol of Example 22 was used with the following differences: the stock aqueous solution of AKG-38 (as free base) was prepared by dissolving the drug in the equivalent amount of 1 N HC1 and adjusting the volume to obtain 20 mg/ml of AG-38 (as free base), pH 5.08.
• the loading mixture contained 1300 mg of AKG-38 and was prepared at 8 mg/ml of AKG-38 and DL ratio of 450 g/mol phospholipid, and additionally contained 10 mM NaCl.
• the post-loading liposomes were pre-concentrated to about 22 mg/ml of the drug; the proportion of unencapsulated drug prior to purification was estimated spectrophotometrically at 305 nm in the pre-concentration diafiltrate and found to be about 3.2% (corresponds to 96.8% loading efficiency). The process was repeated three more times (lots 270, 272, 274). Obtained liposomes had the characteristics shown in TABLE 20.
• the purified liposomes had 42.9 mM phospholipid, the particle sizeXz 113.7 nm, and PDI 0.0612. They were aseptically passed through 0.2-pm sterile filter and adjusted to 20 mM phospholipid with sterile HBS-7.
• Example 25 Liposomal AKG-38 lot 279.
• Example 6 The general procedure of Example 6 was followed. HSPC (Lipoid AG) 13.102 g (16.67 mmol), cholesterol (Dishman, High purity) 7.877 g (20.37 mmol), and PEG-DSPE (Lipoid AG) 2.250 g (0.833 mmol) (HSPC: Choi :PEG-DSPE 45:55:2.25 molar ratio) were combined with 25 ml of absolute ethanol (Sigma, E-7023) and heated with stirring on a 68°C bath until all lipids dissolved.
• HSPC Lipoid AG
• cholesterol Dishman, High purity
• PEG-DSPE Lipoid AG
• HSPC Choi :PEG-DSPE 45:55:2.25 molar ratio
• Extraliposomal trapping agent (ammonium sulfate) was removed by TFF buffer exchange for endotoxin-free water on a KrosFlo TFF system using polysulfone hollow fiber cartridge with MW cut-off 500 KDa (Spectrum Laboratories) until residual conductivity dropped to 180 pS/cm after 5.1 volume exchanges).
• the phospholipid concentration in the post-TFF liposome suspension was determined by blue phosphomolybdate method to be 54.97 ⁇ 0.32 mM.
• AKG-38 (free base) was mixed with 0.95 equivalents of 1 N HC1 and made up with endotoxin-free water to obtain 20 mg/ml aqueous stock solution (pH 5.16).
• the solution was passed through 0.2-pm filter, and the amount of filtrate containing 3958 mg of the drug was combined with the post-TFF liposome suspension to form the loading mixture at drug-to- phospholipid (DL) ratio of 450 g/mol in the presence of 44.5 mg/ml dextrose, 10 mM NaCl, and AKG-38 concentration of 8 mg/ml, pH 5.54.
• DL drug-to- phospholipid
• the mixture was heated to 61 °C by external heating under constant stirring over the period of 5 min, and the incubation continued with stirring on the 65°C bath for another 22 min. Then the mixture was transferred into ice-water bath, stirred for 7 minutes to let the temperature drop to 10 °C, and kept in the ice-water bath for another 8 min. After being taken out of the ice bath, having reached the ambient temperature, and adjustment to 0.1 M NaCl by addition of 3 M NaCl stock, the drug-loaded liposomes (pH 6.53) were purified by TFF using polysulfone hollow fiber cartridge with molecular weight cutoff 500 KD.
• the liposomes were pre-concentrated by diafiltration to about 22 mg/ml of AKG-38 and purified from any extraliposomal drug by TFF exchange into HBS-7 buffer for the total of 8 volume exchanges.
• the concentrated, purified liposomes were aseptically passed through 0.2-pm PES high-flow sterile filter and analyzed for the particle size by DLS, and for the drug and phospholipid concentration by spectrophotometry.
• the liposomes had the following characteristics: AKG-38 21.1 ⁇ 0.19 mg/ml, DL ratio 454 ⁇ 4.7 g/mol phospholipid, Xz 116.4 nm, PDI 0.0231. Yield of the formulated drug 3834 mg (96.9%).
• Example 26 Liposomal AKG-28 lot 281.
• Example 6 The general procedure of Example 6 was followed. Extruded liposomes composed of HSPC, cholesterol, and PEG-DSPE in the molar ratio of 45:55:2.25 containing 0.5 M ammonium sulfate were prepared as described in Example 25. Extraliposomal trapping agent (ammonium sulfate) was removed by TFF exchange for endotoxin-free water on a KrosFlo TFF system using polyethersulfone hollow fiber cartridge with MW cut-off 500 KDa (Spectrum Laboratories) until residual conductivity dropped to 150 pS/cm (4.1 volume exchanges). The phospholipid concentration in the post-TFF liposome suspension was determined by blue phosphomolybdate method to be 55.4 mM.
• Extraliposomal trapping agent ammonium sulfate
• AKG-28 as dihydrochloride salt
• aqueous stock solution adjusted to pH 5.24 with NaOH
• post-TFF liposome suspension was combined with post-TFF liposome suspension to form the loading mixture at drug-to-phospholipid (DL) ratio of 250 g/mol in the presence of 44.5 mg/ml dextrose and AKG-28 concentration of 6 mg/ml.
• the mixture was heated to 65.4 °C in 2.5 min by external heating under constant stirring, and the incubation continued with stirring on the 65°C bath. After 20 min. incubation, the mixture was chilled in ice-water to 9.3°C in 2.75 min, and kept in the ice-water bath for about 10 min.
• the mixture was allowed to reach the ambient temperature and adjusted to 0.1 M NaCl; pH 6.43. 133.4 g of the loading mixture was subjected to purification by TFF using polysulfone hollow fiber cartridge with molecular weight cutoff 500 KD.
• the liposomes were pre-concentrated by diafiltration to about 12 mg/ml of AKG-28 and purified from any extraliposomal drug by TFF exchange into HBS-7 buffer for the total of 8.1 volume exchanges.
• the proportion of unencapsulated drug prior to purification was estimated spectrophotometrically at 302 nm in the pre-concentration diafiltrate and found to be about 0.7% (corresponds to 99.3% loading efficiency).
• the concentrated, purified liposomes were aseptically passed through 0.2-pm sterile filter and analyzed for the particle size by DLS, and for the drug and phospholipid concentration by spectrophotometry.
• the liposomes had the following characteristics: AKG-28 13.26 ⁇ 0.21 mg/ml, DL ratio 258.2 ⁇ 3.7 g/mol phospholipid, Xz 117.3 nm, PDI 0.0421.
• Example 6 The general procedure of Example 6 was followed. Extruded liposomes composed of HSPC, cholesterol, and PEG-DSPE in the molar ratio of 45:55:2.25 containing 0.5 M ammonium sulfate were prepared essentially as described in Example 25. Extraliposomal trapping agent (ammonium sulfate) was removed by TFF exchange for endotoxin-free water on a KrosFlo TFF system using polyethersulfone hollow fiber cartridge with MW cut-off 500 KDa (Spectrum Laboratories) until residual conductivity dropped to 138 pS/cm (5.6 volume exchanges). The phospholipid concentration in the post-TFF liposome suspension was determined by blue phosphomolybdate method to be 53.1 mM.
• Extraliposomal trapping agent ammonium sulfate
• AKG-38 (free base) was mixed with 0.95 equivalents of 1 N HC1 and made up with endotoxin-free water to obtain 19.9 mg/ml aqueous stock solution (pH 5.13).
• the solution was passed through 0.2-pm filter, and the amount of filtrate containing 1400 mg of the drug was combined with the post-TFF liposome suspension to form the loading mixture at drug-to- phospholipid (DL) ratio of 450 g/mol in the presence of 44.5 mg/ml dextrose, 10 mM NaCl, and AKG-38 concentration of 8 mg/ml, pH 5.58.
• DL drug-to- phospholipid
• the mixture was heated to 63 °C by external heating under constant stirring over the period of 2.25 min, and the incubation continued with stirring on the 65°C bath for the total of 21 min. Then the mixture was transferred into ice-water bath, stirred for 3 minutes to let the temperature drop to 10.3 °C, and kept in the ice-water bath for another 7 min. After being taken out of the ice bath, having reached the ambient temperature, and adjustment to 0.1 MNaCl by addition of 3 MNaCl stock, the drug-loaded liposomes (pH 6.70) were purified by TFF using polysulfone hollow fiber cartridge with molecular weight cutoff 500 KD.
• the liposomes were pre-concentrated by diafiltration to about 22 mg/ml of AKG-38 and purified from any extraliposomal drug by TFF exchange into HBS-7 buffer for the total of 7.7 volume exchanges.
• the concentrated, purified liposomes had AKG-38 concentration of 23.1 mg/ml.
• the drug concentration was adjusted to 20 mg/ml with HBS-7 buffer, the liposomes were aseptically passed through 0.2-pm PES high-flow sterile filter and analyzed for the particle size by DLS, and for the drug and phospholipid concentration by spectrophotometry.
• the liposomes had the following characteristics: AKG-3820.35 ⁇ 0.26 mg/ml, DL ratio 437.8 ⁇ 6.5 g/mol phospholipid, Xz 121.1 nm, PDI 0.0200. Yield of the formulated drug 1355 mg (96.8%).
• Example 28 Liposomal AKG-28 lot 286. [00358] Extruded liposomes (HSPC:Chol:PEG-DSPE 45:55:2.25 molar ratio) containing 0.5M ammonium sulfate, free from extraliposomal trapping agent, were obtained as in Example 27.
• AKG-28 as dihydrochloride salt
• 20 mg/ml aqueous stock solution adjusted to pH 5.18 with NaOH
• post-TFF liposome suspension to form the loading mixture at drug-to-phospholipid (DL) ratio of 250 g/mol in the presence of 44.5 mg/ml dextrose and AKG-28 concentration of 6 mg/ml.
• the mixture was placed on a 65°C water bath with stirring and reached 60 °C in 4.5 min. The incubation continued with stirring for the total of 20 min, the mixture was chilled in ice-water to 10.0°C in 2 min, and kept in the ice-water bath for about 10 min.
• the mixture was allowed to reach the ambient temperature and adjusted to 0.1 M NaCl; pH 6.23. 104.6 g of the loading mixture was subjected to purification by TFF using polysulfone hollow fiber cartridge with molecular weight cutoff 500 KD.
• the liposomes were preconcentrated by diafiltration to about 12 mg/ml of AKG-28 and purified from any extraliposomal drug by TFF exchange into HBS-7 buffer for the total of 8.3 volume exchanges.
• the concentrated, purified liposomes were aseptically passed through 0.2-pm sterile filter (chased with HBS-7 buffer) and analyzed for the particle size by DLS, and for the drug and phospholipid concentration by spectrophotometry.
• the liposomes had the following characteristics: AKG-28 12.05 ⁇ 0.13 mg/ml, DL ratio 239.4 g/mol phospholipid, Xz 120.1 nm, PDI 0.0294. Yield of the formulated drug 555.5 mg (92.6%).
• Example 29 Liposomal AKG-38 lot 292.
• Extraliposomal trapping agent (ammonium sulfate) was removed by TFF buffer exchange for endotoxin-free water on a KrosFlo TFF system using polysulfone hollow fiber cartridge with MW cut-off 500 KDa (Spectrum Laboratories) until residual conductivity dropped to 152 pS/cm after 5.4 volume exchanges).
• the phospholipid concentration in the post- TFF liposome suspension was determined by blue phosphomolybdate method to be 57.76 ⁇ 0.53 mM.
• AKG-38 (free base) was mixed with 0.95 equivalents of 1 N HC1 and made up with endotoxin-free water to obtain 19.7 mg/ml aqueous stock solution (pH 5.11).
• the solution was passed through 0.2-pm filter, and the amount of filtrate containing 3509 mg of the drug was combined with the post-TFF liposome suspension to form the loading mixture at drug-to- phospholipid (DL) ratio of 450 g/mol in the presence of 44.5 mg/ml dextrose, 10 mM NaCl, and AKG-38 concentration of 8 mg/ml, pH 5.50.
• DL drug-to- phospholipid
• the mixture was heated to 61.6 °C by external heating under constant stirring over the period of 5 min, and the incubation continued with stirring on the 65°C bath for another 20 min. Then the mixture was transferred into ice-water bath, stirred for 7 minutes to let the temperature drop to 10 °C, and kept in the ice-water bath for another 8 min. After being taken out of the ice bath, having reached the ambient temperature, and adjustment to 0.1 M NaCl by addition of 3 M NaCl stock, the drug-loaded liposomes were purified by TFF using polysulfone hollow fiber cartridge with molecular weight cutoff 500 KD.
• the liposomes were preconcentrated by diafiltration to about 22 mg/ml of AKG-38 and purified from any extraliposomal drug by TFF exchange into HBS-7 buffer for the total of 7.8 volume exchanges.
• the concentrated, purified liposomes were aseptically passed through 0.2-pm PES high-flow sterile filter and analyzed for the particle size by DLS, and for the drug and phospholipid concentration by spectrophotometry.
• the liposomes had the following characteristics: AKG-3822.47 ⁇ 0.38 mg/ml, DL ratio 441.6 g/mol phospholipid, Xz 121.3 nm, PDI 0.0465. Yield of the formulated drug 3375 mg (96.2%).
• the drug-loaded liposomes were purified by TFF using polysulfone hollow fiber cartridge with molecular weight cutoff 500 KD. The liposomes were pre-concentrated by diafiltration to about 22 mg/ml of AKG-38 and purified from any extraliposomal drug by TFF exchange into HBS-7 buffer for the total of 8.1 volume exchanges.
• the concentrated, purified liposomes were aseptically passed through 0.2-pm PES high-flow sterile filter and analyzed for the particle size by DLS, and for the drug and phospholipid concentration by spectrophotometry.
• the liposomes had the following characteristics: AKG-38 22.84 ⁇ 0.41 mg/ml, DL ratio 452.7 g/mol phospholipid, Xz 120.3 nm, PDI 0.0522. Yield of the formulated drug 1407 mg (93.4%).
• Extraliposomal trapping agent (ammonium sulfate) was removed by TFF buffer exchange for endotoxin-free water on a KrosFlo TFF system using polysulfone hollow fiber cartridge with MW cut-off 500 KDa (Spectrum Laboratories) until residual conductivity dropped to 146 uS/cm after 5.5 volume exchanges.
• the phospholipid concentration in the post-TFF liposome suspension was determined by blue phosphomolybdate method to be 56.94 ⁇ 0.41 mM.
• AKG-38 (free base) was mixed with 0.95 equivalents of 1 N HC1 and made up with endotoxin-free water to obtain 20 mg/ml aqueous stock solution (pH 5.15).
• the solution was passed through 0.2-pm fdter, and the amount of filtrate containing 2315 mg of the drug was combined with the post-TFF liposome suspension to form the loading mixture at drug-to- phospholipid (DL) ratio of 450 g/mol in the presence of 44.5 mg/ml dextrose, 10 mM NaCl, and AKG-38 concentration of 8.02 mg/ml, pH 5.52.
• DL drug-to- phospholipid
• the mixture was heated to 64.4 °C by external heating under constant stirring over the period of 3.25 min, and the incubation continued with stirring on the 65°C bath for another 17 min. Then the mixture was transferred into ice-water bath, stirred to let the temperature drop to below 10 °C, kept in the ice-water bath for the total of 10 min, allowed to reach the ambient temperature, and adjusted to 0.1 M NaCl with 3 M NaCl stock; pH 6.63.
• the drug-loaded liposomes were purified by TFF using polysulfone hollow fiber cartridge with molecular weight cutoff 500 KD.
• the liposomes were pre-concentrated by diafiltration to about 22 mg/ml of AKG-38 and purified from any extraliposomal drug by TFF exchange into HBS-7 buffer for the total of 8.0 volume exchanges.
• the concentrated, purified liposomes were aseptically passed through 0.2-pm PES high-flow sterile filter and analyzed for the particle size by DLS, and for the drug and phospholipid concentration by spectrophotometry.
• the liposomes had the following characteristics: AKG-38 22.07 ⁇ 0.23 mg/ml, DL ratio 441.6 g/mol phospholipid, Xz 120.4 nm, PDI 0.0395. Yield of the formulated drug 2141 mg (92.5%).
• Lot 292. Lots 288 (150.3 g), 289 (61.2 g), and 290 (19.5 g) were combined to give 278.4 g of the lot 292 at 22.5 mg/ml of liposomally formulated AKG-38. All liposomal formulations were stored at 2-8 °C.
• Example 6 The general procedure of Example 6 was followed. HSPC (Lipoid AG) 940 mg (1.20 mmol), cholesterol (Dishman, High purity) 568 mg (1.47 mmol), PEG-DSPE (Lipoid AG) 163 mg (0.06 mmol), and 0.0018 mmol of the lipophilic fluorescent label DiIC 18 (3)-DS (AAT Bioquest, USA) (HSPC:Chol:PEG-DSPE:DiICi 8 (3)-DS 45:55:2.25:0.0675 molar ratio, 0.15 mol% DH3-DS relative to HSPC) were combined in 2 ml of absolute ethanol (Sigma, E-7023) and heated with stirring on a 68°C bath until all lipids dissolved.
• the resulting extruded liposomes were kept overnight in a refrigerator (2-8 °C) and filtered through 0.2-pm polyethersulfone (PES) filter under positive pressure.
• Extraliposomal trapping agent ammonium sulfate
• TFF buffer exchange for endotoxin-free water on a KrosFlo TFF system using polysulfone hollow fiber cartridge with MW cut-off 500 KDa (Spectrum Laboratories) until the conductivity of the retentate drops to 60 pS/cm (10 volume exchanges).
• the phospholipid concentration in the post- TFF liposome suspension was determined by blue phosphomolybdate method to be 37.56 ⁇ 0.62 mM.
• AKG-28 as dihydrochloride salt
• aqueous stock solution adjusted to pH 4.99 with NaOH
• DL drug-to-phospholipid
• the mixture was incubated with stirring on a 65°C bath for 20 min, quickly chilled in ice-water and kept in the ice-water bath for about 10 min. After reaching the ambient temperature and adjustment to 0.1 M NaCl with 3 M NaCl stock solution, the pH was 5.80.
• Drug-loaded liposomes were purified by TFF using polysulfone hollow fiber cartridge with molecular weight cutoff 500 KD.
• the liposomes were preconcentrated by diafiltration to about 5 mg/ml of AKG-28 and purified from any extraliposomal drug by TFF exchange into HBS-7 buffer for the total of about 10 volume exchanges.
• the purified liposomes were further concentrated two-fold by TFF using syringe-operated small 500 KD hollow fiber cartridge (MicroKros, Spectrum).
• the concentrated, purified liposomes were aseptically passed through 0.2-pm sterile filter and analyzed for the particle size by DLS, and for the drug and phospholipid concentration by spectrophotometry.
• the liposomes had the following characteristics: AKG-28 8.22 ⁇ 0.16 mg/ml, DL ratio 257.3 ⁇ 10.3 g/mol phospholipid, liposome size Xz 118.2 nm, PDI 0.0188. Yield of the formulated drug 41.4 mg (82.8%).
• Post-TFF extruded liposomes containing 0.5 M ammonium sulfate of Example 30 were used.
• AKG-38 (free base) was mixed with 0.95 equivalents of 1 N HC1 and made up with endotoxin-free water to obtain 20 mg/ml aqueous stock solution (pH 5.11).
• the solution was passed through 0.2-nm filter, and the amount of filtrate containing 70 mg of the drug was combined with the post-TFF liposome suspension (Example 30) to form the loading mixture at drug-to- phospholipid (DL) ratio of 450 g/mol in the presence of 140 mg/ml dextrose and AKG-38 concentration of 3 mg/ml.
• DL drug-to- phospholipid
• the mixture was incubated with stirring on a 65°C bath for 20 min, quickly chilled in ice-water and kept in the ice-water bath for about 10 min. After reaching the ambient temperature and adjustment to 0.1 M NaCl with 3 M NaCl stock solution, the pH was 6.33.
• Drug-loaded liposomes were purified by TFF using polysulfone hollow fiber cartridge with molecular weight cutoff 500 KD. The liposomes were pre-concentrated by diafiltration to about 6 mg/ml of AKG-38 and purified from any extraliposomal drug by TFF exchange into HBS-7 buffer for the total of about 10 volume exchanges.
• the purified liposomes were further concentrated twofold by TFF using syringe-operated small 500 KD hollow fiber cartridge (MicroKros, Spectrum).
• the concentrated, purified liposomes were aseptically passed through 0.2-pm sterile filter and analyzed for the particle size by DLS, and for the drug and phospholipid concentration by spectrophotometry.
• the liposomes had the following characteristics: AKG-389.04 ⁇ 0.16 mg/ml, DL ratio 463.9 ⁇ 19.8 g/mol phospholipid, liposome size Xz 119.3 nm, PDI 0.0267. Yield of the formulated drug 56 mg (80%).
• Example 32 Retention of encapsulated drugs in the liposomes of the lots 235 and 236 in vitro in the presence of plasma.
• liposomes were stable against burst-release of the drug in contact with blood plasma.
• Example 33 Preparation of liposomal AKG-28 and AKG-38 lots 231, 232 (HSPC:cholesterol:PEG-DSPE 45:55:2.25 molar ratio, trapping agent 0.5 M ammonium sulfate).
• Example 6 The general procedure of Example 6 was followed. HSPC (Lipoid AG) 4.255 g (5.41 mmol), cholesterol (Dishman, High purity) 2.56 g (6.62 mmol), andPEG-DSPE (Lipoid AG) 729 mg (0.27 mmol) (HSPC:Cholsterol:PEG-DSPE 45:55:2.25 molar ratio) were combined in 9 ml of absolute ethanol (Sigma, E-7023) and heated with stirring on a 70°C bath until all lipids dissolved.
• the resulting extruded liposomes were kept overnight in a refrigerator (2-8 °C) and filtered through 0.2-pm polyethersulfone (PES) filter under positive pressure.
• Extraliposomal trapping agent ammonium sulfate
• TFF buffer exchange for endotoxin-free water on a KrosFlo TFF system using polysulfone hollow fiber cartridge with MW cut-off 500 KDa (Spectrum Laboratories) until the conductivity of the retentate drops to 60 iiS/cm (10 volume exchanges).
• the phospholipid concentration in the post-TFF liposome suspension was determined by blue phosphomolybdate spectrophotometric method to be 46.97 ⁇ 0.80 mM.
• Lot 231 350 mg of AKG-28 (as dihydrochloride salt) in the form of 20 mg/ml aqueous stock solution (adjusted to pH 5.02 with NaOH) were combined with post-TFF liposome suspension to form the loading mixture at drug-to-phospholipid (DL) ratio of 250 g/mol in the presence of 137.6 mg/ml dextrose and AKG-28 concentration of 2.53 mg/ml.
• the mixture pH 5.60 was incubated with stirring on a 65°C bath for 20 min, quickly chilled in ice-water and kept in the ice-water bath for about 10 min.
• Drug-loaded liposomes were purified by TFF using polysulfone hollow fiber cartridge with molecular weight cutoff 500 KD.
• the liposomes were pre-concentrated by diafiltration to about 9 mg/ml of AKG-28 and purified from any extraliposomal drug by TFF exchange into HBS-7 buffer for the total of 10.9 volume exchanges.
• the purified liposomes were further concentrated to about 12 mg/ml of the drug by continuing TFF diafiltration without buffer feed.
• the concentrated, purified liposomes were aseptically passed through 0.2-pm sterile filter and analyzed for the particle size by DLS, and for the drug and phospholipid concentration by spectrophotometry.
• the liposomes had the following characteristics: AKG-28 11.42 ⁇ 0.09 mg/ml, DL ratio 247.7 ⁇ 7.1 g/mol phospholipid, liposome size Xz 116.5 nm, PDI 0.0511. Yield of the formulated drug 322.7 mg (92.2%).
• Lot 232 AKG-38 (free base) was mixed with 0.95 equivalents of 1 N HC1 and made up with endotoxin-free water to obtain 20 mg/ml aqueous stock solution (pH 5.09). The solution was passed through 0.2-pm filter, and the amount of filtrate containing 580 mg of the drug was combined with the post- TFF liposome suspension of this Example to form the loading mixture at drug-to-phospholipid (DL) ratio of 500 g/mol in the presence of 137.6 mg/ml dextrose, AKG-38 concentration of 2.53 mg/ml, pH 5.72.
• DL drug-to-phospholipid
• Drug-loaded liposomes were purified by TFF using polysulfone hollow fiber cartridge with molecular weight cutoff 500 KD.
• the liposomes were pre-concentrated by diafiltration to about 12 mg/ml of AKG-38 and purified from any extraliposomal drug by TFF exchange into HBS-7 buffer for the total of 8.5 volume exchanges.
• the purified liposomes were further concentrated two-fold by continuing TFF diafiltration without buffer feed.
• the concentrated, purified liposomes were aseptically passed through 0.2-pm sterile filter and analyzed for the particle size by DLS, and for the drug and phospholipid concentration by spectrophotometry.
• the liposomes had the following characteristics: AKG-38 16.03 ⁇ 0.07 mg/ml, DL ratio 487.3 ⁇ 13.9 g/mol phospholipid, liposome size Xz 120.0 nm, PDI 0.0069. Yield of the formulated drug 538.9 mg (92.9%).
• Example 34 Preparation of liposomal AKG-28 lot 233 (HSPC:cholesterol:PEG-DSG 60:40:3 molar ratio, trapping agent 1 N triethylammonium sucrooctasulfate)
• Example 6 The general procedure of Example 6 was followed. HSPC (Lipoid AG) 1.88 g (2.4 mmol), cholesterol (Dishman, High purity) 619 mg (1.6 mmol), and PEG-DSG (Sunbright GS- 020, NOF, Japan) 312 mg (0.12 mmol) were combined in 3 ml of absolute ethanol and heated with stirring on a 67°C bath until all lipids dissolved.
• HSPC Lipoid AG
• cholesterol Dishman, High purity
• PEG-DSG Unbright GS- 020, NOF, Japan
• the obtained suspension was stirred on a 65 °C bath for 5 min, and extruded three times at 400 psi through the stack of four 47-mm 100-nm pore size and one 200-nm pore size polycarbonate track- etched membranes (Whatman Nucleopore) using Lipex 100-ml thermobarrel liposome extruder (Northern Lipids, Inc.) heated with circulating 65°C water.
• the resulting extruded liposomes were kept overnight in a refrigerator (2-8 °C) and filtered through 0.2-pm polyethersulfone (PES) filter under positive pressure.
• PES polyethersulfone
• AKG-28 as dihydrochloride salt
• aqueous stock solution adjusted to pH 5.02 with NaOH
• DL drug-to-phospholipid
• the mixture was incubated with stirring on a 65°C bath for 20 min, quickly chilled in ice-water and kept in the ice-water bath for about 10 min. After reaching the ambient temperature and adjustment to 0.1 M NaCl with 3 M NaCl stock solution, the pH was 5.80.
• Drug-loaded liposomes were purified by TFF using polysulfone hollow fiber cartridge with molecular weight cutoff 500 KD.
• the liposomes were preconcentrated by diafiltration to about 9 mg/ml of AKG-28 and purified from any extraliposomal drug by TFF exchange into HBS-7 buffer for the total of 10.9 volume exchanges.
• the purified liposomes were further concentrated to about 12 mg/ml of the drug by continuing TFF diafiltration without buffer feed.
• the concentrated, purified liposomes were aseptically passed through 0.2-pm sterile filter (chased with HBS-7 buffer) and analyzed for the particle size by DLS, and for the drug and phospholipid concentration by spectrophotometry.
• the liposomes had the following characteristics: AKG-28 10.64 ⁇ 0.20 mg/ml, DL ratio 246.8 ⁇ 11.7 g/mol phospholipid, liposome size Xz 116.3 nm, PDI 0.0022. Yield of the formulated drug 118.2 mg (84.4%).
• Example 35 Preparation of liposomal AKG-38 lot 234 (HSPC:cholesterol:PEG-DSPE 45:55:2.25 molar ratio, trapping agent 1 N triethylammonium sucrooctasulfate)
• Example 6 The general procedure of Example 6 was followed. HSPC (Lipoid AG) 3.30 g (4.20 mmol), cholesterol (Dishman, High purity) 1.985 g (5.13 mmol), and PEG-DSPE (Lipoid AG) 567 mg (0.21 mmol) (HSPC:Cholsterol:PEG-DSPE 45:55:2.25 molar ratio) were combined in 7 ml of absolute ethanol (Sigma, E-7023) and heated with stirring on a 70°C bath until all lipids dissolved.
• aqueous tri ethylammonium sucrooctasulfate (TEA-SOS) solution (0.2-micron filtered) were preheated on a 70°C bath and poured with stirring into the hot lipid ethanolic solution.
• the obtained suspension was stirred on a 70°C bath for 10 min. and extruded eight times at 260 psi through the stack of two 47-mm 100-nm pore size and one 200-nm pore size polycarbonate track-etched membranes (Whatman Nucleopore) using Lipex 100-ml thermobarrel liposome extruder (Northern Lipids, Inc.) heated with circulating 70°C water.
• the resulting extruded liposomes were kept overnight in a refrigerator (2-8 °C) and filtered through 0.2-pm polyethersulfone (PES) filter under positive pressure. Phospholipid concentration was 54.6 mM. 11.33 g of the extruded liposomes were purified from the extraliposomal trapping agent (TEA-SOS) by TFF buffer exchange for endotoxin-free water on a KrosFlo TFF system using polysulfone hollow fiber cartridge with MW cut-off 500 KDa (Spectrum Laboratories) until the conductivity of the retentate drops to 64 pS/cm (13.8 volume exchanges). The phospholipid concentration in the post-TFF liposome suspension was determined by blue phosphomolybdate spectrophotometric method to be 28.67 ⁇ 1.01 mM.
• AKG-38 (free base) was mixed with 0.95 equivalents of 1 N HC1 and made up with endotoxin-free water to obtain 20 mg/ml aqueous stock solution (pH 5.09).
• the solution was passed through 0.2-pm filter, and the amount of filtrate containing 250 mg of the drug was combined with the post-TFF liposome suspension of this Example to form the loading mixture at drug-to-phospholipid (DL) ratio of 500 g/mol in the presence of 116.4 mg/ml dextrose, AKG-38 concentration of 2.53 mg/ml, pH 5.24.
• the mixture was incubated with stirring on a 65°C bath for 20 min, quickly chilled in ice-water and kept in the ice-water bath for about 10 min.
• Drug-loaded liposomes were purified by TFF using polysulfone hollow fiber cartridge with molecular weight cutoff 500 KD.
• the liposomes were pre-concentrated by diafiltration to about 10 mg/ml of AKG-38 and purified from any extraliposomal drug by TFF exchange into HBS-7 buffer for the total of 8.0 volume exchanges.
• the purified liposomes were further concentrated approximately two-fold by continuing TFF diafiltration without buffer feed.
• the concentrated, purified liposomes were aseptically passed through 0.2-pm sterile filter and analyzed for the particle size by DLS, and for the drug and phospholipid concentration by spectrophotometry.
• the liposomes had the following characteristics: AKG-38 15.71 ⁇ 0.33 mg/ml, DL ratio 518.6 ⁇ 18.4 g/mol phospholipid, liposome size Xz 114.3 nm, PDI 0.0284. Yield of the formulated drug 235.7 mg (94.3%).
• Example 36 Effect of osmotic agent concentration on the loading efficiency of AKG-28 and AKG-38 into the liposomes and drug retention by the liposomes in plasma.
• Example 6 The general protocol of Example 6 was followed. Extruded liposomes containing 0.5 M ammonium sulfate and the lipid composition of HPSC, cholesterol, PEG-DSPE, and DiIC18(3)-DS (fluorescent lipid label) in the molar ratio of 45:55:2.25:0.0675 were prepared as described in Example 30.
• the liposomes were purified from the extraliposomal ammonium sulfate by TFF exchange for endotoxin-free “water for injection”(WFI)-quality water (Hyclone) using syringe-operated MicroKros polysulfone hollow fiber cartridge (MWCO 500 KDa, Spectrum Laboratories) (13.8 volume exchanges, residual conductivity 88 pS/cm, phospholipid concentration 55.4 mM).
• the liposomes were loaded with AKG-28 or AKG-38 by incubation of the drugs (prepared as aqueous 20 mg/ml stocks as described in Examples 30 and 31) with the purified extruded liposomes in aqueous solution in a 65°C water bath for 20 min in the presence of various concentrations of osmotic agent (dextrose), at the drug concentration 2.22 mg/ml and DL ratio of 250 g/mol phospholipid (AKG-28) or 450 g/mol phospholipid (AKG-38).
• osmotic agent osmotic agent
• Unencapsulated drug was removed by size-exclusion chromatography on Sepharose CL-4B, eluent HBS-7 buffer, and the loading (encapsulation) efficiency was determined from the results of drug and phospholipid analysis.
• the osmotic agent concentration was expressed both in absolute terms and as percent of the 168 mg/ml dextrose concentration determined to be isoosmotic to the0.5 M ammonium sulfate solution used to form the liposomes.
• the drugs were effectively loaded into the liposomes of the disclosure (encapsulation efficiency more than 85%, and mostly more than 90%) even under hypoosmotic conditions (i.e., at the osmolality of the extraliposomal solution lower than that of the intraliposomal trapping agent solution), down to complete absence of the added osmotic balance agent (dextrose) (Table 22).
• hypoosmotic conditions i.e., at the osmolality of the extraliposomal solution lower than that of the intraliposomal trapping agent solution
• osmotic balance agent osmotic balance agent
• Linezolid at 50 mg/kg of the body weight was administered orally as a gavage formulated with 0.5 % methyl cellulose and acidified to pH 3-4 (Sigma M0430) at a concentration of 20 mg/mL.
• 0.5 ml blood was collected in lithium heparin tubes at 5 min, 15 min, 1 h, 3 h, 6 h, 24 h, 48 h, and 72 h. The samples were centrifuged and the resultant plasma was separated and transferred to duplicate clear polypropylene tubes, frozen immediately over dry ice, and stored at -80 °C until analysis. The plasma concentration in rats was determined by HPLC. Non-compartment PK analyses were performed using Phoenix WinNonlin (Version 7.0).
• this PK software was used to estimate the plasma maximum concentration (Cmax), plasma maximum concentration divided by dose (Cmax/dose), time of Cmax (T max ), last measured concentration (Ciast), time of last measured concentration (T last ), area-under the plasma concentration versus time curve from Oh to last time point (AUCo-iast) and Oh to infinity (AUCo-inf), AUCo-iast divided by dose (AUCo-iast/ dose), clearance (CL), volume of distribution (Vd), and elimination half-life (T’A).
• this PK software was used to estimate the same PK parameters except for apparent clearance (CL/F) and apparent volume of distribution (Vd/F).
• the plasma concentration versus time profdes for Ls-AKG28 were detectable from 5 min to 72 h. Based on the results of Cmax/dose and AUC/dose, the plasma PK of Ls-AKG28 is linear (dose proportional) after administration of 10, 20, and 40 mg/kg. At all doses the plasma clearance (CL) of Ls-AKG38 (-2.59 mL/h/kg) was greater than Ls-AKG28 (-1.67 mL/h/kg). At the same dose (20 or 40 mg/kg), the Vd of Ls-AKG28 was greater than for
• the plasma concentration versus time profiles for Ls-AKG38 were detectable from 5 min to 72 h. Based on the results of Cmax/dose and AUC/dose, the plasma PK of Ls-AKG38 is linear (dose proportional) after administration of 20, 40, and 80 mg/kg. At all doses the plasma clearance (CL) of Ls-AKG38 (-2.59 mL/h/kg) was greater than Ls-AKG28 (-1.67 mL/h/kg). At the same dose (20 or 40 mg/kg), the Vd of Ls-AKG28 was greater than for Ls-AKG38. TABLE 25. Summary of plasma PK parameters for total drug after administration of Ls-AKG38 at 20, 40, and 80 mg/kg IV
• the plasma concentration versus time profiles for total drug after administration of Ls-AKG28 at 10, 20, and 40 mg/kg IV x 1 and Ls-AKG38 at 20, 40, and 80 mg/kg IV x 1 are presented in FIG.7 and FIG. 8, respectively.
• the plasma PK of Ls-AKG28 is linear (dose proportional) after administration of 10, 20, and 40 mg/kg.
• the plasma PK of Ls-AKG38 is linear (dose proportional) after administration of 20, 40, and 80 mg/kg.
• Ls-AKG38 At all doses the plasma clearance (CL) of Ls-AKG38 (-2.59 mL/h/kg) was greater than Ls-AKG28 (-1.67 mL/h/kg). At the same dose (20 or 40 mg/kg), the Vd of Ls-AKG28 was greater than for Ls-AKG38.
• the total plasma PK exposure of Ls-AKG28 at 40 mg/kg and Ls-AKG38 were -73 -fold and -110-fold higher than plasma PK of linezolid (using AUC from 0 to last).
• Example 38 Plasma pharmacokinetics (PK) of the total form of (encapsulated + released drug) Ls-AKG28 and Ls-AKG38 after multiple IV doses in Sprague-Dawley rats.
• PK pharmacokinetics
• Example 39 Pharmacokinetic Studies of drug and liposome lipid of Ls-AKG28 and Ls- AKG38 in CD-I mice.
• the liposomes at the dose of 50 mg/kg (Ls-AKG28) or 90 mg/kg (Ls- AKG38) were injected in the lateral tail vein at time 0 and the blood was sampled at 0.083, 1, 3, 6, 24, and 48 hours post injection.
• the plasma concentration of AKG-28, AKG-38, and a fluorescent liposome lipid label (DilC is(3)-DS) was determined by HPLC. Plasma concentration of the liposome phospholipid was calculated from the fluorescent label quantification using liposome lots 235 and 236 as standards.
• the tissue affinity of non-encapsulated oxazolidinone drugs is expected to be many times higher than that of the liposome-encapsulated ones (as supported, for example, by the Vd of 2,291.26 mL/kg of non-encapsulated oxazolidinone, linezolid, in comparison with the Vd of 33.27-43.74 mL/kg for liposome-encapsulated AKG-28 in rats, see Example 37), the plasma drug concentration could be attributed predominantly to liposome-associated drug, and the plasma drug-liposome lipid (DL) ratio normalized to the original (pre-inj ection) DL value was taken as the measure of drug retention by the liposomes.
• DL plasma drug-liposome lipid
• Non-compartment PK analyses were performed using Summit Research Services, PK Solutions 2.0.
• this PK software was used to estimate the plasma maximum concentration (Cmax), plasma maximum concentration divided by dose (Cmax/dose), time of Cmax (Tmax), last measured concentration (Ci as t), time of last measured concentration (Ti as t), area-under the plasma concentration versus time curve from 0 h to last time point (AUCo-iast) and Oh to infinity (AUCo-inf), AUCo-iast divided by dose (AUCo-iast / dose), clearance (CL), volume of distribution (Vd), and elimination half-life.
• FIG. 11 A The plasma concentration versus time profiles for drug after administration of Ls- AKG28 (FIG. 11 A) and Ls-AKG38 (FIG. 11B) are presented.
• the summary of plasma PK parameters for Ls-AKG28 and Ls-AKG38 drug in plasma are presented in TABLE 28 and of the liposomal phospholipid is presented in TABLE 29.
• Dynamics of the DL ratio indicative of the stability of the drug encapsulation in vivo is presented in FIG. 11C and TABLE 30.
• Ls-AKG28 has a near perfect in vivo stability with an undetectable loss of drug up to 48 hours after IV injection in mice.
• Ls-AKG28 has a faster drug release rate.
• mice Five mice were used per group. The plasma concentration of AKG-28 and AKG-38 in mice was determined by HPLC. Mice were injected with the indicated dose and formulation once per week for a total of 4 injections. The drug was measured in the plasma at the 6 h time point after the 1 st and 4 th doses (Fig. 12). None of the groups tested had a significant accelerated clearance of the 4 th injection (2 -tailed, unequal variance t-test all p values >0.05). This data confirms that these liposomal oxazolidinones can be dosed chronically for multiple weekly cycles with no significant negative impact on drug exposure.
• Plasma drug concentration for Ls-AKG28 and Ls-AKG38 for liposomal phospholipid after administration of Ls-AKG28 and Ls-AKG38 Abbreviations: SOS, 1 N TEA-SOS; AS, 0.5 M ammonium sulfate; Choi, cholesterol content as mol% of the sum of cholesterol and HSPC; DL, drug-to-lipid ratio, g/mol liposome phospholipid; %ID -percent of injected dose, average per group; SD- standard deviation.
• mice Female CD-I mice of 20-22 grams (5 per each group) were administered with Ls-AKG28 (50, 65, 90 or 100 mg/kg/dose) or Ls-AKG38 (50, 90, 120 or 200 mg/kg/dose) by intravenous injection (tail vein) once weekly for 4 weeks.
• the liposomal formulations (Ls-AKG28 lot231 and Ls-AKG38 lot 232) were prepared as described previously in Example 33.
• the control group was injected once weekly for 4 weeks with an equal volume of HEPES buffered saline (HBS, pH 7). The body weights were measured 3 times a week throughout the study and data were presented as a percentage of body weight change relative to the body weight measured at day zero.
• HBS HEPES buffered saline
• the animals were humanely euthanized at the end of the study (72 hours post last treatment) using CO 2 inhalation.
• Blood samples were collected by a cardiac puncture and transferred to EDTA prefilled microtainers for hematology analysis (Homological ADVIA 120/2120i Analyzer) and to microtainers prefilled with lithium heparin for plasma separation.
• Plasma was separated from the cell fraction by centrifugation at 10000 rpm for 5 min and used for the biochemistry analysis (Cobas 6000 Analyzer).
• Tissue samples liver, spleen, kidney, ling, heart, small intestine, and column
• the tissues were embedded in paraffin, sectioned, stained with hematoxylin and eosin (H&E) and evaluated for histopathology by a board-certified veterinary pathologist.
• Example 42 In vivo tolerability of Ls-AKG28 and Ls-AKG38 combined with BDQ/PMD or BDQ/PMD/MOX in mice.
• CD-I mice (5 per each group) were treated with either Ls-AKG28 (lot 231) or LsAKG38 (lot 232) alone or together with bedaquiline (BDQ) and pretomanid (PMD) combination.
• Ls-AKG28 (Lot 231) and Ls-AKG38 (Lot 232) were prepared as described in Example 33. Additionally, mice were co-treated with a triple combination of BDQ, PMD and moxifloxacin (MO XI) and liposomal oxazolidinones.
• Ls-AKG28 50 mg/kg/dose
• LsAKG38 90 mg/kg/dose
• mice were treated with only BDQ/PMD/MOX or BDQ/PMD (25/100 mg/kg/dose respectively) plus linezolid (LNZ) given orally at 100 mg/kg/dose daily, five times a week for 4 weeks.
• LNZ linezolid
• the histopathology data showed no treatment related changes in case of Ls-AKG28 combined with BDQ/PMD.
• Ls-AKG28 + BDQ/PMD/MOX combination has minimal events associated with mixed cell and mononuclear cell infiltration in lung and heart.
• Treatment with Ls-AKG38 as a monotherapy was associated with minimal test article-related findings in the liver (inflammatory infiltration and hepatocellular necrosis).
• Administration of Ls- AKG38 + BDQ/PMD did not show any treatment related findings and combination of Ls-AKG38 + BDQ/PMD/MOX was associated with minimal mixed cell infiltration in lung.
• Example 43 Effect of dose scheduling on tolerability of Ls-AKG28 and Ls-AKG38 combined with BDQ/PMD in mice.
• BDQ/PMD 25 and 100 mg/kg/dose respectively
• HBS HEPES buffered saline
• FIG. 15D Histopathology analysis of collected tissues (FIG. 15D) showed minimal interstitial mixed cell infiltrates composed of macrophages and neutrophils in 2 out of 5 mice that received Ls-AKG28 at 50 mg/kg (2qw) and mild interstitial mixed cell infiltrates in 1 out of 5 animals that received Ls-AKG28 at 100 mg/kg (Iqw).
• mice that received Ls-AKG28 + BP at 50 mg/kg (Iqw) there were minimal interstitial infiltrates composed of macrophages (1 out of 5 animals) or mixed (macrophages and neutrophils) inflammatory cells (3 out of 5 mice).
• mice that received Ls- AKG28 + BP at 100 mg/kg (Iqw) there were minimal interstitial mixed cell infiltrates in 4 out of 5 animals.
• both Ls-AKG28 and Ls-AKG38 (alone or in combination with BDQ/PMD) administrated twice a week at doses 50 mg/kg and 100 mg/kg respectively or at doubled doses of 100 mg/kg and 200 mg/kg once a week were well tolerated in mice and did not affect body weight, hematology, or histopathology of the treated animals.
• Example 44 In vivo tolerability of Ls-AKG28 and Ls-AKG38 in rats.
• Ls-AKG28 (lot 275) and Ls-AKG38 (lot 276) were prepared as described in Examples 22 and 23, respectively.
• Male Sprague-Dawley rats were administered with Ls-AKG28 (10, 20 or 40 mg/kg/dose) or LsAKG-38 (20, 40 or 80 mg/kg/dose) by intravenous injection (tail vein) once weekly for 8 weeks.
• the control group was injected once weekly for 8 weeks with an equal volume of HEPES buffered saline (HBS, pH 7).
• NCV Nerve Conduction Velocity
• MAP Muscle Action Potential
• the amplitude of the evoked response reflects the number and synchrony of the activated fibers.
• Data were recorded with the active recording electrode positioned approximately 10 mm below the hair line on the tail (determined visually) and the stimulating cathode 50 mm further distal.
• the amplitude and the onset latency of the signal were recorded, and velocity was calculated by dividing the distance between the stimulating cathode and the active electrode by the absolute onset latency of the initial depolarizing current.
• Digital nerve NCV measures the speed of conduction in the sensory digital nerve.
• the digital nerve is the distal extreme of the sciatic nerve innervating the dorsal surface of the hind paw.
• Nerve conduction velocity is sensitive to the nodal and transmembrane currents, structure and mean cross-sectional diameter of the responding axons and the integrity of the associated myelin sheaths.
• Data were recorded with the active recording electrode positioned at the ankle behind the lateral malleolus and the stimulating cathode at the base of the second digit of the hind paw. The amplitude and the onset latency of the signal were recorded, and velocity was calculated by dividing the distance between the stimulating cathode and the active electrode by the absolute onset latency of the initial depolarizing current.
• Tibial motor conduction measures the response properties of the intrinsic muscles of the rat hind paw following stimulation of the motor fibers at the distal portion of the tibial nerve. Data were recorded with the active electrode positioned in a lateral dorsal muscle of the hind paw (equivalent to the extensor digitorum brevis muscle in humans) and the stimulating cathode positioned proximal to the ankle, behind the lateral malleolus. The speed of nerve conduction in the motor axons was estimated from the onset latency of the induced compound muscle action potential (CMAP). The amplitude of the CMAP was determined at the peak of the response following supramaximal stimulation of the associated nerve.
• CMAP induced compound muscle action potential

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