Methods for preparing purified lipopeptides

Abstract

The present invention relates to crystalline and crystal-like forms of lipopeptides, including daptomycin, a lipopeptide antibiotic with potent bactericidal activity against gram-positive bacteria, including strains that are resistant to conventional antibiotics. The present invention relates to methods of purifying lipopeptides, including daptomycin, a lipopeptide antibiotic with potent bactericidal activity against gram-positive bacteria, including strains that are resistant to conventional antibiotics. The present invention also relates to pharmaceutical compositions comprising the purified form of the lipopeptide and methods of using these compositions.

1 - 56 . (canceled)  
   
   
       57 . A crystalline or crystal-like lipopeptide or a salt thereof wherein the lipopeptide is selected from the group consisting of a daptomycin, an A54145, an A-21978C analog, and a daptomycin-related lipopeptide.  
   
   
       58 . The crystalline or crystal-like lipopeptide or salt thereof, according to  claim 57 , wherein the salt is a divalent calcium salt.  
   
   
       59 . The crystalline or crystal-like lipopeptide antibiotic or salt thereof, according to  claim 57 , wherein the lipopeptide antibiotic is daptomycin.  
   
   
       60 . The crystalline or crystal-like lipopeptide or salt thereof, according to  claim 59 , wherein the salt is a divalent calcium salt.  
   
   
       61 . The crystalline daptomycin according to  claim 59 , wherein an x-ray diffraction pattern of the crystalline daptomycin, using a Cu (λ=1.54 Å) x-ray source, has a diffraction angle (2θ)=10.9, 19.2 and 23.3 (degree) or a diffraction angle (2θ)=19.2, 23.2, 23.4 and 23.6 (degree).  
   
   
       62 . The crystal-like lipopeptide according to  claim 57 , wherein crystal-like means the compound has crystalline characteristics by birefringence, but does not have crystalline characteristics by x-ray powder diffraction.  
   
   
       63 . The crystalline or crystal-like daptomycin according to  claim 59 , wherein the crystalline or crystal-like daptomycin comprises urchin-like or a cluster of needles, needle-like, flake-like, plate-like, or rod-like crystals.  
   
   
       64 . The crystalline or crystal-like lipopeptide according to  claim 59 , wherein the crystalline daptomycin has a purity of at least 95%.  
   
   
       65 . The crystalline or crystal-like lipopeptide according to  claim 59 , wherein the crystalline daptomycin or salt thereof has a purity of at least 97%.  
   
   
       66 . The crystalline or crystal-like daptomycin according to  claim 59 , wherein the crystalline daptomycin or salt thereof contains no single impurity greater than 1%.  
   
   
       67 . The crystalline or crystal-like daptomycin according to  claim 64 , wherein the purity is measured by HPLC.  
   
   
       68 . The crystalline or crystal-like lipopeptide according to  claim 57 , wherein a crystal of the lipopeptide is at least 5 μm.  
   
   
       69 . The crystalline or crystal-like lipopeptide according to  claim 68 , wherein the crystal is at least 50 μm.  
   
   
       70 . The crystalline or crystal-like lipopeptide according to  claim 68 , wherein the lipopeptide is daptomycin.  
   
   
       71 . The crystalline or crystal-like lipopeptide according to  claim 57 , wherein the crystalline lipopeptide exhibits a higher stability than an amorphous form of the lipopeptide.  
   
   
       72 . The crystalline or crystal-like lipopeptide according to  claim 71 , wherein the crystalline lipopeptide exhibits higher stability to heat, light, degradation or humidity than the amorphous form.  
   
   
       73 . The crystalline or crystal-like lipopeptide according to  claim 72 , wherein the stability is measured by antibiotic activity or degradation of the lipopeptide antibiotic.  
   
   
       74 . The crystalline or crystal-like lipopeptide according to  claim 71 , wherein the lipopeptide is daptomycin.  
   
   
       75 . The crystalline or crystal-like daptomycin according to  claim 74 , wherein the crystalline lipopeptide exhibits lower conversion to anhydro-daptomycin or the P-isomer of daptomycin than the amorphous form of daptomycin.  
   
   
       76 . The crystalline or crystal-like lipopeptide according to  claim 57 , which is a daptomycin-related lipopeptide.  
   
   
       77 . A pharmaceutical composition comprising a crystalline or crystal-like lipopeptide antibiotic and a pharmaceutically acceptable carrier, wherein the lipopeptide antibiotic is selected from the group consisting of a daptomycin, an A54145, an A-21978C analog, and a daptomycin-related lipopeptide.  
   
   
       78 . The pharmaceutical composition according to  claim 77 , wherein the crystalline or crystal-like lipopeptide is daptomycin.  
   
   
       79 . The pharmaceutical composition according to  claim 78 , wherein the crystalline or crystal-like daptomycin is enterically coated for oral administration.  
   
   
       80 . The pharmaceutical composition according to  claim 78 , wherein the crystalline or crystal-like daptomycin is formulated in a dose of 3 to 75 mg/kg.  
   
   
       81 . The pharmaceutical composition according to  claim 78 , wherein the carrier enhances the oral availability of daptomycin.  
   
   
       82 . The pharmaceutical composition according to  claim 77 , which is in the form of micronized particles or microspheres.  
   
   
       83 . A container comprising the pharmaceutical composition according to  claim 77 .  
   
   
       84 . The pharmaceutical composition according to  claim 82 , which is used as an aerosol.  
   
   
       85 . A formulation comprising a crystalline or crystal-like lipopeptide antibiotic and a pharmaceutically acceptable carrier, wherein the lipopeptide antibiotic is selected from the group consisting of daptomycin, A54145, an A-21978C analog, and a daptomycin-related lipopeptide.  
   
   
       86 . A container comprising the formulation of  claim 85  and a pharmaceutically acceptable buffer.  
   
   
       87 . The formulation according to  claim 85 , which is a pharmaceutical formulation, a food formulation, a feed formulation, a veterinary formulation, a cosmetic formulation or a personal care formulation.  
   
   
       88 . The formulation according to  claim 85 , that is a pharmaceutical formulation, wherein the formulation further comprises another antibiotic, a stabilizing agent, an agent to aid in absorption, a pH buffering agent or an inorganic salt.  
   
   
       89 . The formulation according to  claim 85  that is a feed formulation, wherein the formulation further comprises animal feed and may optionally comprise another antibiotic or vitamins.  
   
   
       90 . A method for administering a crystalline or crystal-like lipopeptide, a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof, wherein the lipopeptide is selected from the group consisting of a daptomycin, an A54145 and a daptomycin-related lipopeptide, comprising the step of administering to a patient in need thereof a therapeutically effective amount of the crystalline or crystal-like lipopeptide, the pharmaceutically acceptable salt thereof or the pharmaceutical composition thereof.  
   
   
       91 . The method according to  claim 90 , wherein the lipopeptide has a purity of greater than 95%.  
   
   
       92 . The method according to  claim 91 , wherein the lipopeptide is daptomycin.  
   
   
       93 . The method according to  claim 92 , wherein the daptomycin is a crystalline daptomycin and an x-ray diffraction pattern of the crystalline daptomycin has a diffraction angle (2θ) 10.9, 19.2 and 23.3 (degree) using a Cu (λ=1.54 Å) x-ray source.  
   
   
       94 . The method according to  claim 92 , wherein the daptomycin is a crystal-like daptomycin and the crystal-like daptomycin has crystalline characteristics by birefringence but does not have crystalline characteristics by x-ray powder diffraction.  
   
   
       95 . The method according to  claim 90 , wherein the crystalline or crystal-like lipopeptide is administered as a micronized particle.  
   
   
       96 . The method according to  claim 90 , wherein the crystalline or crystal-like lipopeptide is administered as a targeted release form.  
   
   
       97 . The method according to  claim 95 , wherein the lipopeptide is daptomycin.  
   
   
       98 . The method according to  claim 90 , wherein the administration is done subcutaneously, intravenously or intramuscularly.  
   
   
       99 . A method for storing a lipopeptide, wherein the lipopeptide is selected from the group consisting of a daptomycin, an A54145, an A-21978C analog, and a daptomycin-related lipopeptide, comprising the steps of 
 a) providing a dissolved solution of a lipopeptide;    b) crystallizing or precipitating the lipopeptide;    c) collecting and drying the lipopeptide; and    d) storing the lipopeptide;    wherein the crystalline or crystal-like lipopeptide is more stable than an amorphous form of the lipopeptide.    
   
   
       100 . A method for manufacturing a crystalline or crystal-like lipopeptide, wherein the lipopeptide is selected from the group consisting of a daptomycin, an A54145, an A-21978C analog, and a daptomycin-related lipopeptide, comprising the steps of 
 a) providing an amorphous form of a lipopeptide;    b) crystallizing or precipitating the lipopeptide; and    c) collecting the crystalline or crystal-like lipopeptide.    
   
   
       101 . The method according to  claim 99 , wherein said collecting is performed by filtration.  
   
   
       102 . The method according to  claim 101 , further comprising the step of washing the lipopeptide after step b).  
   
   
       103 . The method according to  claim 100 , further comprising the step of drying the lipopeptide after step c).  
   
   
       104 . The method according to  claim 103 , further comprising the step of sterilizing the lipopeptide after drying.  
   
   
       105 . The method according to  claim 100 , wherein step c) is performed under sterile conditions.  
   
   
       106 . The method according to  claim 105 , wherein step b) is performed under sterile conditions.  
   
   
       107 . The method according to  claim 106 , further comprising the step of drying the lipopeptide after step c) under sterile conditions.  
   
   
       108 . The method according to  claim 99 , wherein the purity of the crystalline or crystal-like lipopeptide is higher than the amorphous form of the lipopeptide.  
   
   
       109 . The method according to  claim 108 , wherein the purity of the amorphous form is approximately 90% and the purity of the crystalline or crystal-like form is greater than 95%.  
   
   
       110 . The method according to  claim 108 , wherein the purity of the amorphous form is approximately 93%, and the purity of the crystalline or crystal-like form is greater than 95%.  
   
   
       111 . The method according to  claim 108 , wherein the purity of the amorphous form is approximately 93%, and the purity of the crystalline or crystal-like form is approximately 98%.  
   
   
       112 . A method for preparing daptomycin, comprising the steps of providing an amorphous form of daptomycin and crystallizing the daptomycin from a crystallization solution comprising a cation from a salt, a buffer, an organic precipitant, and a low molecular weight alcohol.  
   
   
       113 . The method according to  claim 112 , wherein the buffer is selected from the group consisting of HEPES, Tris HCl, imidazole, MES, CHES, a citrate salt and a cacodylate salt.  
   
   
       114 . The method according to  claim 112 , wherein the alcohol is selected from the group consisting of ethylene glycol, propylene glycol, t-butanol, glycerol, isopropanol, 1,4-butanediol, 1,2-propanediol and methanol.  
   
   
       115 . The method according to  claim 112 , wherein the organic precipitant is polyethylene glycol or polyethylene glycol monomethyl ether.  
   
   
       116 . The method according to  claim 112 , wherein the crystallizing solution further comprises a divalent cation.  
   
   
       117 . The method according to  claim 116 , wherein the divalent cation is calcium, zinc or magnesium.  
   
   
       118 . The method according to  claim 112 , wherein the pH of the crystallization solution is in the range of 5 to 8.5.  
   
   
       119 . The method according to  claim 118 , wherein the pH of the crystallization solution is in the range of 5.5 to 7.5.  
   
   
       120 . The method according to  claim 119 , wherein the pH of the crystallization solution is in the range of 5.9 to 6.6.  
   
   
       121 . The method according to  claim 112 , wherein the crystallization is done by the hanging drop method or by batch crystallization.  
   
   
       122 . The method according to  claim 112 , wherein a crystal of the daptomycin is an urchin-like or a cluster of needles form.  
   
   
       123 . The method according to  claim 112 , wherein a crystal of the daptomycin is a rod-like form.  
   
   
       124 . The method according to  claim 112 , further comprising the step of collecting the daptomycin crystals.  
   
   
       125 . The method according to  claim 124 , wherein said collecting is done by centrifugation, precipitation or filtration.  
   
   
       126 . The method according to  claim 112 , further comprising washing the crystalline daptomycin.  
   
   
       127 . The method according to  claim 112 , wherein the daptomycin is at a starting purity of at least 90%.  
   
   
       128 . The method according to  claim 112 , wherein the daptomycin is at a starting purity of at least 93%.  
   
   
       129 . The method according to  claim 112 , wherein said crystallizing is performed at a temperature below 20° C.  
   
   
       130 . The method according to  claim 129 , wherein said crystallizing is performed at about 4° C.  
   
   
       131 . The method according to  claim 112 , wherein said crystallizing is performed at above 20° C.  
   
   
       132 . The method according to  claim 112 , wherein said crystallizing is performed with stirring.  
   
   
       133 . A method for preparing a crystalline or crystal-like daptomycin, comprising the steps of 
 a) providing a solution comprising daptomycin, a salt comprising a monovalent or divalent cation, a pH buffering agent and a low molecular weight or polyhydric alcohol; and    b) allowing the daptomycin to crystallize or precipitate from the solution to obtain a crystalline or crystal-like daptomycin preparation, respectively.    
   
   
       134 . The method according to  claim 133 , wherein the buffering agent is selected from the group consisting of HEPES, Tris HCl, imidazole, MES, CHES, sodium acetate, calcium acetate, a citrate salt and a cacodylate salt.  
   
   
       135 . The method according to  claim 133 , wherein the alcohol is selected from the group consisting of ethylene glycol, propylene glycol, t-butanol, glycerol, isopropanol, 1,4-butanediol, 1,2-propanediol and methanol.  
   
   
       136 . The method according to  claim 135 , wherein the alcohol is isopropanol.  
   
   
       137 . The method according to  claim 133 , wherein the salt comprises a divalent cation.  
   
   
       138 . The method according to  claim 137 , wherein the divalent cation is a magnesium cation, a zinc cation or a calcium cation.  
   
   
       139 . The method according to  claim 138 , wherein the divalent cation is a calcium cation.  
   
   
       140 . A method for preparing a crystalline or crystal-like daptomycin, comprising the steps of 
 a) providing a solution comprising daptomycin, a pH buffering agent that is a salt comprising a monovalent or divalent cation, and a low molecular weight or polyhydric alcohol; and    b) allowing the daptomycin to crystallize or precipitate from the solution to obtain a crystalline or crystal-like daptomycin preparation, respectively.    
   
   
       141 . The method according to  claim 140 , wherein the buffering agent comprises a divalent cation selected from a calcium cation or a magnesium cation.  
   
   
       142 . The method according to  claim 133  or  140 , wherein the pH of the solution is in the range of 5.0 to 9.5.  
   
   
       143 . The method according to  claim 142 , wherein the pH of the solution is in the range of 5.5 to 7.5.  
   
   
       144 . The method according to  claim 143 , wherein the pH of the solution is in the range of 5.9 to 6.3.  
   
   
       145 . The method according to  claim 133  or  140 , wherein said crystallizing or precipitating step is done at a temperature of 0-30° C.  
   
   
       146 . The method according to  claim 145 , wherein the temperature is 23-28° C.  
   
   
       147 . The method according to  claim 140 , wherein the solution comprises calcium acetate pH 6.1 and isopropanol.  
   
   
       148 . The method according to  claim 147 , wherein said crystallizing or precipitating step comprises adding isopropanol until the mixture becomes cloudy.  
   
   
       149 . The method according to  claim 148 , wherein said crystallizing or precipitating step is done for a period of time of from one hour to three weeks.  
   
   
       150 . The method according to  claim 140 , wherein said crystallizing or precipitating is done by batch crystallization or batch precipitation, respectively.  
   
   
       151 . The method according to  claim 133  or  140 , further comprising the step of collecting the crystalline or crystal-like daptomycin.  
   
   
       152 . The method according to  claim 151 , wherein said collecting step is performed by filtration or centrifugation.  
   
   
       153 . The method according to  claim 152 , wherein said collecting is performed by filtration.  
   
   
       154 . The method according to  claim 151 , further comprising the step of washing the crystalline or crystal-like daptomycin.  
   
   
       155 . The method according to  claim 133  or  140 , wherein the crystalline or crystal-like daptomycin has an urchin-like form.  
   
   
       156 . The method according to  claim 133  or  140 , wherein the daptomycin has a purity before crystallizing or precipitating of no greater than 90% and has a purity after crystallization or precipitation of at least 95%.  
   
   
       157 . The method according to  claim 156 , wherein the daptomycin has a purity before crystallizing or precipitating of no greater than 80% and has a purity after crystallization or precipitation of at least 95%.  
   
   
       158 . The method according to  claim 156 , wherein the daptomycin has a purity before crystallizing or precipitating of no greater than 60% and has a purity after crystallization or precipitation of at least 95%.  
   
   
       159 . The method according to  claim 156 , wherein the daptomycin has a purity before crystallizing or precipitating of no greater than 40% and has a purity after crystallization or precipitation of at least 95%.  
   
   
       160 . The method according to  claim 156 , wherein the daptomycin is at a starting purity of no greater than 10% and has a purity after crystallization or precipitation of at least 95%.  
   
   
       161 . The method according to  claim 156 , wherein the daptomycin has a purity after crystallization or precipitation of at least 96%.  
   
   
       162 . The method according to  claim 156 , wherein the daptomycin has a purity after crystallization or precipitation of at least 97%.  
   
   
       163 . The method according to  claim 156 , wherein the daptomycin has a purity after crystallization or precipitation of at least 98%.  
   
   
       164 . A method for preparing a purified daptomycin, comprising the steps of 
 a) providing a solution comprising daptomycin, a pH buffering agent that is a salt comprising a monovalent or divalent cation, and a low molecular weight or polyhydric alcohol; and    b) allowing the daptomycin to crystallize or precipitate from the solution to obtain a purified daptomycin preparation.    
   
   
       165 . The method according to  claim 164 , wherein the purified daptomycin preparation is at least 95% pure.  
   
   
       166 . The method according to  claim 165 , wherein said purified daptomycin preparation is at least 96% pure.  
   
   
       167 . The method according to  claim 166 , wherein said purified daptomycin preparation is at least 97% pure.  
   
   
       168 . The method according to  claim 167 , wherein said purified daptomycin preparation is at least 98% pure.  
   
   
       169 . A method of preparing a crystalline form of a lipopeptide which comprises combining the lipopeptide with a crystallization solution comprising at least one cation and at least one alcohol chosen from the group consisting of polyhydric alcohols and low molecular weight alcohols and combinations thereof, wherein the lipopeptide is chosen from the group consisting of daptomycin and daptomycin analogs.  
   
   
       170 . The method of  claim 169 , wherein the polyhydric alcohol is chosen from the group consisting of ethylene glycol, propylene glycol, glycerol, 1,2-propane diol, 2-methyl-2,4-pentanediol, 1,6-hexanediol, and 1,4-butanediol.  
   
   
       171 . The method of  claim 169 , wherein the low molecular weight alcohol is chosen from the group consisting of methanol, isopropanol, tert-butanol, and n-propanol.  
   
   
       172 . The method of  claim 169 , wherein the cation is a divalent cation.  
   
   
       173 . The method of  claim 172 , wherein the divalent cation is chosen from the group consisting of manganese, magnesium, and calcium.  
   
   
       174 . The method of  claim 173 , wherein the divalent cation is calcium.  
   
   
       175 . The method of  claim 169 , wherein the crystallization solution consists of at least one salt and at least one low molecular weight alcohol.  
   
   
       176 . The method of  claim 169 , wherein the crystallization solution further comprises one or more additional components chosen from the group consisting of organic precipitants, pH buffers, low molecular weight alcohols and detergents.  
   
   
       177 . The method of  claim 176 , wherein the crystallization solution comprises as an organic precipitant polyethylene glycol.  
   
   
       178 . A method for treating a disease caused by a gram-positive pathogen in a subject which comprises administering to the subject the pharmaceutical composition of  claim 77  which comprises the crystalline lipopeptide in a therapeutically effective amount.  
   
   
       179 . The method of  claim 178 , wherein the disease is chosen from the group consisting of complicated skin and soft tissue infections, community-acquired pneumonia, complicated urinary tract infections, enteroccocal infections, endocarditis and bacteremia.  
   
   
       180 . A method for administering to a subject in need thereof a crystalline lipopeptide or salt thereof, wherein the crystalline or crystal-like lipopeptide is chosen from the group consisting of crystalline daptomycin and crystalline A-21978C analogs, which comprises administering to the subject a pharmaceutical composition comprising the crystalline or crystal-like lipopeptide or salt thereof and a pharmaceutically acceptable carrier.  
   
   
       181 . The method of  claim 180 , wherein the crystalline or crystal-like lipopeptide or salt thereof is administered to the subject by pulmonary administration as a micronized particle.  
   
   
       182 . The method of  claim 180 , wherein the crystalline or crystal-like lipopeptide or salt thereof is administered to the subject as a sustained release form.  
   
   
       183 . The method of  claim 180 , wherein the crystalline or crystal-like lipopeptide is administered orally.  
   
   
       184 . The method of  claim 180 , wherein the crystalline or crystal-like lipopeptide is administered subcutaneously.  
   
   
       185 . The method of  claim 180 , wherein the crystalline or crystal-like lipopeptide is administered intravenously or intramuscularly.  
   
   
       186 . A method of storing a lipopeptide selected from the group consisting of daptomyin and A-21978C analogs which comprises preparing the lipopeptide in crystalline form and storing the crystalline or crystal-like lipopeptide.  
   
   
       187 . In a method for preparing a lipopeptide selected from the group consisting of daptomycin and A-21978C analogs, the improvement which comprises preparing the lipopeptide in crystalline form.