Process for formulating an anionic agent

Abstract

Formulations comprising anionic agents such as nucleic acids within a lipid-containing particle methods of formulating a lipid-containing particle comprising an anionic agent such as a nucleic acid, methods for preparing a lipid-containing particle comprising an anionic agent such as a nucleic acid, methods for therapeutic delivery of an anionic agent to a patient in need thereof, where the anionic agent is formulated in a lipid-containing particle as described herein.

We claim: 
     
         1 . A method of producing a particle comprising a first lipid or sterol, a second lipid or sterol and a small molecule comprising:
 (a) combining a second lipid or sterol with a solvent to form a second lipid or sterol solution in said solvent, wherein said solvent is free of said first lipid or sterol; and   (b) combining said second lipid or sterol solution in said solvent with said first lipid or sterol thereby causing a concentration of said solvent in said first lipid or sterol to gradually increase from 0% to up to no more than 40% (v/v) as more of said second lipid or sterol in said solvent is added to said first lipid or sterol to form a solution of the second lipid or sterol and the first lipid or sterol, wherein the solubility of said first lipid or sterol in said solvent in the presence of said second lipid or sterol is higher than the solubility of said first lipid or sterol in said solvent in the absence of said second lipid or sterol; and   (c) combining the solution of the second lipid or sterol and the first lipid or sterol of step (b) with a small molecule,   thereby producing a particle comprising a first lipid or sterol, a second lipid or sterol and a small molecule.   
     
     
         2 . The method of  claim 1 , wherein said first lipid or sterol is a sterol selected from the group consisting of cholesterol, cholestanone, cholestenone, coprostanol, 3.beta.-[-(N—(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol (DC-cholesterol) and bis-guanidium-tren-cholesterol (BGTC). 
     
     
         3 . The method of  claim 1 , wherein said first lipid or sterol is present at a concentration selected from the group consisting of 10 mg/ml or more, 11 mg/ml or more, 12 mg/ml or more, 15 mg/ml or more, 20 mg/ml or more, 25 mg/ml or more, 30 mg/ml or more, 35 mg/ml or more, 37 mg/ml or more, 40 mg/ml or more, 45 mg/ml or more, 50 mg/ml or more, 55 mg/ml or more, 60 mg/ml or more, 65 mg/ml or more, 70 mg/ml or more, 74 mg/ml or more, 75 mg/ml or more, 80 mg/ml or more, 85 mg/ml or more, 90 mg/ml or more, 95 mg/ml or more, 100 mg/ml or more, 150 mg/ml or more, 200 mg/ml or more, 250 mg/ml or more, 500 mg/ml or more and 1 g/ml or more within said solution of the second lipid or sterol and the first lipid or sterol. 
     
     
         4 . The method of  claim 1 , wherein the total lipid content of said solution of the second lipid or sterol and the first lipid or sterol is selected from the group consisting of 12 mg/ml or more, 15 mg/ml or more, 20 mg/ml or more, 25 mg/ml or more, 30 mg/ml or more, 35 mg/ml or more, 37 mg/ml or more, 40 mg/ml or more, 45 mg/ml or more, 50 mg/ml or more, 55 mg/ml or more, 60 mg/ml or more, 65 mg/ml or more, 70 mg/ml or more, 74 mg/ml or more, 75 mg/ml or more, 80 mg/ml or more, 85 mg/ml or more, 90 mg/ml or more, 95 mg/ml or more, 100 mg/ml or more, 150 mg/ml or more, 200 mg/ml or more, 250 mg/ml or more, 500 mg/ml or more and 1 g/ml or more. 
     
     
         5 . The method of  claim 1 , wherein said first lipid or sterol is selected from the group consisting of DODMA, DOTMA and a cationic lipid selected from the group consisting of L-1 to L-49: 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
     
     
         6 . The method of  claim 1 , wherein said second lipid or sterol is selected from the group consisting of DODMA, DOTMA and a cationic lipid selected from the group consisting of L-1 to L-49: 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
     
     
         7 . The method of  claim 1 , wherein said solvent is selected from the group consisting of ethanol, saline, methanol, n-propanol, isopropanol, n-butanol, isobutanol, tert-butanol, glycerol, ethylene glycol, propylene glycol, polyethylene glycol, chloroform, dichloromethane, hexane, cyclohexane, acetone, ether, diethyl ether, dioxan, isopropyl ether, tetrahydrofuran and combinations thereof. 
     
     
         8 . The method of  claim 1 , wherein said solvent is selected from the group consisting of acetic acid, acetone, acetonitrile, anisole, benzene, 1-butanol, 2-butanol, butyl acetate, tert-butylmethyl ether, carbon tetrachloride, chlorobenzene, chloroform, cumene, cyclohexane, 1,2-dichloroethane, 1,1-dichloroethene, 1,2-dichloroethene, dichloromethane, 1,2-dimethoxyethane, n,n-dimethylacetamide, n,n-dimethylformamide, dimethyl sulfoxide, 1,4-dioxane, ethanol, 2-ethoxyethanol, ethyl acetate, ethyleneglycol, ethyl ether, ethyl formate, formamide, formic acid, heptane, hexane, isobutyl acetate, isopropyl acetate, methanol, 2-methoxyethanol, methyl acetate, 3-methyl-1-butanol, methylbutyl ketone, methylcyclohexane, methylethyl ketone, methylisobutyl ketone, 2-methyl-1-propanol, n-methylpyrrolidone, nitromethane, pentane, 1-pentanol, 1-propanol, 2-propanol, propyl acetate, pyridine, sulfolane, tetrahydrofuran, tetralin, toluene, 1,1,1-trichloroethane, 1,1,2-trichloroethene, xylene, and combinations thereof. 
     
     
         9 . The method of  claim 1 , wherein said solvent is ethanol. 
     
     
         10 . The method of  claim 1 , wherein said solution of the second lipid or sterol and the first lipid or sterol comprises at least one lipid selected from the group consisting of a neutral lipid, a sterol, a cationic lipid and a modified lipid which prevents particle aggregation during lipid-anionic agent particle formation. 
     
     
         11 . The method of  claim 1 , wherein said particle possesses a property selected from the group consisting of improved size and/or PDI, improved efficacy in a subject administered said particle and improved tolerability in a subject administered said particle, as compared to an appropriate control particle formed by an appropriate control process that comprises exposing said first lipid or sterol to said solvent before said second lipid or sterol is exposed to said solvent. 
     
     
         12 . The method of  claim 1 , wherein step (c) is performed by in-line mixing said solution of the second lipid or sterol and the first lipid or sterol of step (b) and said small molecule. 
     
     
         13 . The method of  claim 10 , wherein the neutral lipid is one or more of DSPC, DPPC and DOPC. 
     
     
         14 . The method of  claim 10 , wherein the cationic lipid has the formula: 
       
         
           
           
               
               
           
         
         wherein each R 1  and R 2  is, independently, optionally substituted C 11-24  alkyl, optionally substituted C 11-24  alkenyl, optionally substituted C 11-24  alkynyl, optionally substituted C 11-24  heteroalkyl, optionally substituted C 11-24  heteroalkenyl, or optionally substituted C 11-24  heteroalkynyl; each n1 and n2 is, independently, an integer from 0 to 2; and R 5  is selected from the group consisting of H, optionally substituted C 1-6  alkyl, and optionally substituted heterocyclyl. 
       
     
     
         15 . The method of  claim 10 , wherein the cationic lipid has the formula: 
       
         
           
           
               
               
           
         
         wherein each R 1  and R 2  is, independently, optionally substituted C 11-24  alkyl, optionally substituted C 11-24  alkenyl, optionally substituted C 11-24  alkynyl, optionally substituted C 11-24  heteroalkyl, optionally substituted C 11-24  heteroalkenyl, or optionally substituted C 11-24  heteroalkynyl; R 3  is H or optionally substituted C 1-6  alkyl; L 1  is optionally substituted C 1-6  alkylene; and each R 5  and R 6  is, independently, H or optionally substituted C 1-6  alkyl, or where R 5  and R 6  combine to form an optionally substituted C 3-7  heterocyclyl. 
       
     
     
         16 . The method of  claim 10 , wherein said modified lipid which prevents particle aggregation during lipid anionic agent particle formation is a polyethylene glycol (PEG)-, PEG-ceramide, or ganglioside-modified lipid.