Methods for preparing purified lipopeptides

专利摘要:
The present invention relates to daptomycin, a lipopeptide antibiotic having effective bactericidal activity against gram positive bacteria, including crystalline and crystalline like forms of lipopeptides, such as strains resistant to conventional antibiotics. The present invention also relates to a method for purifying daptomycin, which is a lipopeptide antibiotic having effective bactericidal activity against gram positive bacteria, including lipopeptides such as strains resistant to conventional antibiotics. The present invention relates to pharmaceutical compositions comprising tablet forms of lipopeptides and methods of using these compositions.
公开号:KR20030081353A
申请号:KR10-2003-7008117
申请日:2001-12-17
公开日:2003-10-17
发明作者:키이스데니스；라이장-지
申请人:큐비스트 파마슈티컬즈 인코포레이티드；
IPC主号:

专利说明:

Method for producing purified lipopeptides {METHODS FOR PREPARING PURIFIED LIPOPEPTIDES}
[5] The rapid increase in the incidence of Gram-positive infections, including infections caused by antibiotic-resistant bacteria, has led to renewed interest in the development of new classes of antibiotics. One such class is lipopeptide antibiotics, which include daptomycin. Daptomycin has in vitro effective bactericidal activity against clinically relevant Gram-positive bacteria causing serious and life-threatening diseases. Non-limiting examples of these bacteria include resistant pathogens such as vancomycin resistant enterococci (VRE), methicillin resistant Staphylococcus aureus (MRSA), glycopeptide mediated susceptible Staphylococcus aureus (GISA), coagulase-negative staphylococcus (CNS), and penicillin-resistant Streptococcus pneumoniae (PRSP), and the like, with few alternative therapies. See, eg, Tally et al., 1999, Exp. Opin. Invest. Drugs 8: 1223-1238. The inhibitory effect of daptomycin is a rapid concentration dependent bactericidal effect in vitro and in vivo, showing a concentration dependent effect after antibiotic use for a relatively long time in vivo.
[6] Daptomycin is described by Baltz in Biotechnology of Antibiotics, 2nd Ed., Ed. WR Strohl (New York: Marcel Dekker, Inc.), 1997, pp. 415-435. Daptomycin, also known as LY 146032, is a cyclic lipopeptide antibiotic that can be induced by Streptomyces roseosporus fermentation. Daptomycin is a member of the Factor A-21978C type ₀ antibiotic of S. roseosporus and consists of a decanoyl side chain bound to the N-terminal tryptophan of the cyclic 13-amino acid peptide (FIG. 1). Daptomycin is quite effective against most Gram-positive bacteria; It is a bactericide that works very quickly; The activity profile is good because of low resistance and effective against antibiotic resistant organisms. As a non-limiting example, compounds are currently being developed for use in a variety of formulations for the treatment of serious bacterial-induced infections, including methicillin resistant Staphylococcus aureus (MRSA) and vancomycin resistant Enterococcus (VRE).
[7] Many US patents disclose A-21978C ₀ antibiotics and daptomycin associated lipopeptides, including daptomycin (LY 146032). These patents also disclose methods for making and isolating A-21978C ₀ antibiotics and daptomycin related lipopeptides.
[8] U.S. Patents RE32,333, RE32,455, 4,800,157, 4,874,843, and 4,885,243 describe methods for synthesizing and separating daptomycin from fermentation cultures of Streptomyces roseosporus. U.S. Patents RE32,310, RE32,311, 4,537,717, 4,482,487 and 4,524,135 disclose methods of deacylating A-21978C ₀ antibiotics and A-21978C ₀ antibiotics and reacylating peptide nuclei, and prepared by such methods. Antibiotic derivatives are disclosed. US Pat. No. 5,912,226 (hereinafter referred to as' 226 patent) discloses the identification and separation of the β-isomers of two kinds of impurities, anhydro-daptomycin and daptomycin, produced during the preparation of daptomycin. None of these US patents disclose a method for precipitation or crystallization of lipopeptides in a manner that increases the purity of the lipopeptides.
[9] U.S. Patent 4,439,425 (the '425 patent) discloses a method for crystallizing crystalline lipopeptides and lipopeptides. Lipopeptides disclosed in the '425 patent are structurally different from daptomycin and daptomycin related lipopeptides. U.S. Patent 5,336,756 (hereinafter '756 patent) also discloses crystalline cyclic lipopeptides, including hexapeptides. The crystalline cyclic lipopeptides disclosed in the '756 patent are also structurally different from daptomycin and daptomycin related lipopeptides. The '756 patent describes that lipopeptides, which are ethinocandine type compounds, can be obtained when aqueous n-propanol is used as the crystallization solvent. See, eg, 1-2 columns of the '756 patent. Neither the '425 patent nor the' 756 patent discloses crystallization or precipitation of daptomycin or daptomycin related lipopeptides, nor does it disclose a method of crystallizing or precipitation of lipopeptides produced by Streptomyces.
[10] It is advantageous to develop methods for crystallizing or precipitating daptomycin and daptomycin related lipopeptides to provide improved purification methods for these lipopeptides. In addition, crystalline or highly purified precipitated forms of daptomycin or other daptomycin related lipopeptides are useful for formulating pharmaceutical compositions for treating bacterial infections. In addition, crystalline or highly purified precipitated forms of daptomycin or daptomycin related lipopeptides are useful in the manufacture of sterile products, particularly bulky sterile products. Thus, there is a need for a method for preparing crystalline or precipitated daptomycin and daptomycin related lipopeptides and crystalline or precipitated forms of the lipopeptides thus prepared. However, there is no simple and robust method effective to crystallize or precipitate daptomycin or daptomycin related lipopeptides to make the lipopeptides more pure than before crystallization or precipitation.
[1] Cross Reference of Related Application
[2] This application discloses US Provisional Application No. 60 / 256,268, filed December 18, 2000; Provisional Application 60 / 274,741, filed March 9, 2001; Provisional Application No. ____, filed December 13, 2001; And claiming application No. ____, filed on December 13, 2001.
[3] Technical Field of the Invention
[4] The present invention relates to daptomycin, a lipopeptide antibiotic having effective bactericidal activity against gram positive bacteria, including crystalline and crystalline like forms of lipopeptides, such as strains resistant to conventional antibiotics. The present invention also relates to methods for preparing crystalline or crystalline type forms of lipopeptides and to methods for purifying lipopeptides, including daptomycin. The present invention relates to pharmaceutical compositions comprising tablet forms of lipopeptides and methods of using these compositions.
[15] 1 shows the structure of daptomycin.
[16] FIG. 2 shows photomicrographs of sea urchin-shaped crystals or crystal-like particles of daptomycin prepared by the method disclosed in Example 12.
[17] 3 shows photomicrographs of acicular crystals of daptomycin.
[18] 4 shows rod-shaped light micrographs of daptomycin.
[19] 5 shows a photomicrograph of a 100 × magnified daptomycin sample. Photomicrographs of amorphous daptomycin using planar transmitted light (A) and amorphous daptomycin using cross polarized light (B) are shown. Photomicrographs of daptomycin crystals using planar transmitted light (C and E) and daptomycin crystals using cross polarized light (D and F) are shown. Daptomycin crystals were prepared by the protocol described in Example 7.
[20] 6 shows the x-ray powder diffraction pattern of amorphous daptomycin.
[21] 7 shows the x-ray powder diffraction pattern of daptomycin crystals prepared by the protocol disclosed in Example 7. FIG.
[22] FIG. 8 shows the x-ray powder diffraction pattern of a second daptomycin crystal sample prepared by the protocol disclosed in Example 7.
[23] 9 shows the birefringence of crystal-like particles of daptomycin upon exposure to polarized light. Crystal-like particles were prepared by the method disclosed in Example 12.
[24] 10 shows a process diagram of an exemplary crystallization method.
[25] 11 shows a process diagram of an exemplary manufacturing method without using crystallization or precipitation. In this method, bacterial fermentation produces a fermentation culture containing daptomycin, followed by microfiltration, anion exchange chromatography, size exclusion ultrafiltration, hydrophobic interaction chromatography, solvent anion exchange chromatography, and pyrogen removal ultrafiltration. Filtration and reverse osmosis are used to purify daptomycin and place daptomycin in the bottle. See, eg, International PCT Publication No. WO 01/44274, published June 21, 2001, which is incorporated by reference for details on this kind of method.
[26] FIG. 12 shows a process diagram of an exemplary method for preparing a lipopeptide compound comprising fermentation, microfiltration, anion exchange chromatography, size exclusion ultrafiltration, crystallization or precipitation, drying crystals or precipitates, and dry dosing of the compound into the bottle. do. See eg Example 13.
[27] FIG. 13 shows a process diagram of an exemplary method for preparing a lipopeptide compound comprising fermentation, microfiltration, anion exchange chromatography, crystallization or precipitation, crystallization or precipitation drying and dry introduction of the compound into the bottle. See, eg, Example 14.
[28] FIG. 14 shows a process diagram of an exemplary method for preparing a lipopeptide compound comprising fermentation, microfiltration, size exclusion ultrafiltration, crystallization or precipitation, crystal or precipitate drying and dry introduction of the compound into the bottle. See eg Example 15.
[29] FIG. 15 shows a process diagram of an exemplary method for preparing a lipopeptide compound comprising fermentation, microfiltration, crystallization or precipitation, crystallization or precipitation drying and dry introduction of the compound into the bottle. See eg Example 16.
[30] Figure 16 depicts the structure of CB-131547, a cyclic lipopeptide analog of daptomycin.
[31] Detailed description of the invention
[32] Purpose of the Invention
[33] It is an object of the present invention to provide a method for crystallization or precipitation of lipopeptides. In one embodiment, this method is used to crystallize or precipitate daptomycin or daptomycin related lipopeptides. In another embodiment, the method increases the purity of the lipopeptide compared to the purity of the lipopeptide prior to crystallization or precipitation. The method includes providing an amorphous preparation of the lipopeptides, crystallizing or precipitation of the lipopeptides under conditions in which the crystalline or precipitated crystal-like lipopeptides are purer than the amorphous lipopeptides. In one embodiment, the purity of the amorphous formulation is 92% or less, but the purity of the crystalline or crystalline-like lipopeptides purified therefrom is at least 95% and may be at least 96%, 97% or 98%. In another embodiment, the amorphous formulation has a purity of 80% or less, but the crystalline or crystalline like lipopeptides purified therefrom are at least 95% pure and may be at least 96%, 97% or 98%. In another embodiment, the amorphous formulation has a purity of 60% or less, but the crystalline or crystalline like lipopeptides purified therefrom are at least 95% pure and may be at least 96%, 97% or 98%. In another embodiment, the amorphous formulation has a purity of 40% or less, but the crystalline or crystalline like lipopeptides purified therefrom are at least 95% pure and may be at least 96%, 97% or 98%. In another embodiment, the amorphous formulation has a purity of 20% or less, but the crystalline or crystalline like lipopeptides purified therefrom are at least 95% pure and may be at least 96%, 97% or 98%. In a further preferred embodiment, the amorphous formulation has a purity of 10% or less, but the crystalline or crystal-like lipopeptides purified therefrom are at least 95% pure and may be at least 96%, 97% or 98%.
[34] It is a further object of the present invention to provide a process for the preparation and purification of lipopeptides, in particular comprising crystallizing or precipitation of the lipopeptides. In one embodiment, the crystallization or precipitation step is used to purify the lipopeptides. In a preferred embodiment, the crystallization or precipitation is carried out by batch crystallization or precipitation. In another embodiment, the process is a large scale process for industrially preparing lipopeptides, preferably daptomycin or daptomycin related lipopeptides. In one embodiment, the lipopeptides are produced by fermentation. The fermentation product is then purified by various purification techniques, including crystallization or precipitation. In one embodiment, a crystallization or precipitation step can be used in parallel with other purification techniques including microfiltration, size exclusion ultrafiltration and / or anion exchange chromatography. In one embodiment, a crystallization or precipitation step is used instead of one or more purification techniques used in purification processes that do not utilize crystallization or precipitation. In another embodiment, a crystallization or precipitation step is used to increase the degree of purification compared to other steps that do not include a crystallization or precipitation step. In a preferred embodiment, the method comprises recovering crystalline or crystalline like lipopeptides after crystallization or precipitation.
[35] It is another object of the present invention to provide lipopeptides in highly purified, such as sterile, crystalline or crystalline like forms. In one embodiment, the lipopeptides are daptomycin or daptomycin related lipopeptides. The crystalline or crystal-like form of the lipopeptides may be in the form of sea urchins (visually similar to sea urchins as clusters of connected needles) (see FIG. 2), needles (FIG. 3), rods (FIG. 4), flat or flaky It can have any crystalline or crystalline like shape, including a form. In one embodiment, the crystalline or crystalline pseudolipopeptide is at least 80% pure and at least 85% pure, at least 90% pure. In another embodiment, the crystalline or crystalline like form of lipopeptides are at least 95% pure and may be at least 96%, 97%, 98%.
[36] It is another object of the present invention to provide a pharmaceutical composition comprising a crystalline or crystalline like form of a lipopeptide. In one embodiment, the lipopeptides are daptomycin or daptomycin related lipopeptides. In one embodiment, the pharmaceutical composition may be in the form of enteric coatings for oral administration and formulated in the form of micronized particles or microspheres. In another embodiment, the present invention provides a method of administering a pharmaceutical composition to a subject in need thereof.
[37] Justice
[38] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. The practice of the invention, unless otherwise specified, uses conventional chemistry, biochemistry, biophysics and microbiology techniques and basic terminology used herein.
[39] The term “lipopeptide” means a molecule comprising a lipid-like moiety covalently bound to a peptide moiety and salts, esters, amides and ethers thereof. The term “lipopeptide” also includes protective forms of lipopeptides in which one or more amino, carboxylate or hydroxyl groups are protected. For example, for examples of protecting groups, see "Protective Groups in Organic Synthesis" by Theodora W. Greene, John Wiley and Sons, New York, 1981. In one embodiment, the lipopeptides are antibiotics. In another embodiment, the lipopeptides are LY 303366, echinocandines, pneumocandines, aculeacins, biscosine, surfactin, flippastatin B1, ampomycin or lipopeptide derivatives disclosed in US Pat. No. 5,629,288. These lipopeptides are known in the art. See, for example, US Pat. No. 5,202,309 and International PCT Publication WO 00/08197. In another embodiment, the lipopeptides are daptomycin related molecules. In another embodiment, the lipopeptides are daptomycin.
[40] A “daptomycin related molecule” refers to daptomycin, A54145 or other lipopeptides structurally related to daptomycin (eg, daptomycin related lipopeptides) such as all stereoisomers that can be made at any chiral center present in these molecules. It includes.
[41] “Daptomycin-related lipopeptides” include, but are not limited to, the lipopeptides disclosed in US Pat. no. In addition, daptomycin-associated lipopeptides are disclosed in International PCT Publication No. WO 01/44272, published June 21, 2001, International PCT Publication No. WO 01/44274, published June 21, 2001 and June 2001. And those disclosed in International PCT Publication No. WO 01/44271, published on 21 December, which are incorporated herein by reference. The daptomycin-associated lipopeptides disclosed in the foregoing applications relate to synthetic and semisynthetic lipopeptides with modified ornithine and / or kynurin residues, and / or fatty acid side chains of daptomycin. The daptomycin-associated lipopeptides also show that the n-decanoyl fatty acid side chain of daptomycin is n-octanoyl, n-nonanoyl, n-undecanoyl, n-dodecanoyl, n-tridecanoyl or n-tetra A-21978C ₀ antibiotics substituted with decanoyl fatty acid side chains.
[42] The term “daptomycin” refers to an n-decanoyl derivative of a factor A-21978C type ₀ antibiotic comprising an α-aspartyl group. "Daptomycin" is synonymous with LY 146032.
[43] The term "anhydrous-daptomycin" refers to daptomycin-associated lipopeptides in which the α-aspartyl group of daptomycin is cyclized to a succinoimido group. See, for example, the '226 patent for the structure of anhydrous-daptomycin.
[44] The term "β-isomer" or "β-isomer of daptomycin" refers to daptomycin-associated lipopeptides that include β-aspartyl groups instead of α-aspartyl groups. See, for example, the '226 patent for the β-isomeric structure of daptomycin.
[45] The term “isolated” means a compound or product wherein the compound present in the mixture is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%. In addition, the term "isolated" means that at least 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70% of the compounds present in the mixture group , 70 to 80% or 80 to 90%. The percentage of compounds in the mixture can be measured by means known in the art, as described below for measuring the purity of the compounds.
[46] "Almost pure" means a sample comprising at least 95% of the desired compound. Preferably, daptomycin is “almost pure” when at least 95% to 97% of the sample is daptomycin. Similarly, daptomycin related lipopeptides are "almost pure" when at least 95% to 97% of the samples are daptomycin related lipopeptides.
[47] The daptomycin or daptomycin related lipopeptides are "substantially pure" if at least 98% to 99% of the samples in the sample are daptomycin or daptomycin related lipopeptides, respectively.
[48] The daptomycin or daptomycin related lipopeptides “nearly contain” another compound if other compounds are present in less than 1% of the amount of the daptomycin or daptomycin related lipopeptide preparation, respectively.
[49] The daptomycin or daptomycin related lipopeptides “substantially do not contain” another compound when the other compound is present at 0.5% or less of the amount of daptomycin or daptomycin related lipopeptide preparation, respectively.
[50] The daptomycin or daptomycin related lipopeptides “do not contain” another compound when other compounds are present at 0.1% or less of the amount of daptomycin or daptomycin related lipopeptide preparation, respectively. Alternatively, daptomycin or daptomycin-associated lipopeptides may be further reduced if the compound cannot be detected by HPLC under conditions of maximum sensitivity at which the detection limit is about 0.05% or less of the amount of daptomycin or daptomycin-related lipopeptide preparation, respectively. Does not include a compound.
[51] By “purified” daptomycin is meant nearly pure daptomycin, substantially pure daptomycin, or a salt thereof, or daptomycin or a salt thereof that contains little, substantially no, or no other compounds. Likewise, “purified” daptomycin related lipopeptides contain little, substantially no or substantially no pure daptomycin related lipopeptides, substantially pure daptomycin related lipopeptides, or salts thereof, or other compounds. Daptomycin related lipopeptides or salts thereof.
[52] "Crude" daptomycin means daptomycin or a salt thereof that is less than 90% pure. Likewise, "crude" daptomycin related lipopeptides refer to daptomycin related lipopeptides or salts thereof having a purity of less than 90%.
[53] "Semi-purified" daptomycin means daptomycin or a salt thereof having a purity of at least 90% and less than 95%. Similarly, "semi-purifying" daptomycin related lipopeptides refer to daptomycin related lipopeptides or salts thereof having a purity of at least 90% and less than 95%.
[54] The purity of daptomycin, daptomycin related lipopeptides or another lipopeptides means lipopeptides prior to formulation into pharmaceutical compositions. The purity of the lipopeptide is referred to as "purity (%)". The level of purity is not a measure of the crystallinity of the crystalline formulation. Purity can be measured by any means, such as nuclear magnetic resonance (NMR), gas chromatography / mass spectroscopy (GC / MS), liquid phase chromatography / mass spectroscopy (LC / MS), or microbiological analysis. One preferred means of measuring the purity of daptomycin is by analytical high pressure liquid chromatography (HPLC). Two methods of analytical HPLC are disclosed in WO 01/53330, published July 26, 2001, which is incorporated by reference.
[55] "Lipopeptide crystal" means one or more crystals of a lipopeptide or lipopeptide salt. The determination of lipopeptides as crystals can be measured by any means, including optical microscopy, electron microscopy, x-ray powder diffraction, solid state nuclear magnetic resonance (NMR) or polarization microscopy. Microscopes can be used to measure crystal length, diameter, width, size and shape, and to determine whether the crystal is present as single particles or polycrystalline.
[56] Lipopeptides, when measured by one means, such as the naked eye or by optical microscopy, have been determined to have crystalline properties when measured by polarized light, but do not appear to have crystalline properties when measured by another means, such as x-ray powder diffraction. Lipopeptide particles are in "crystal-like" form. "Crystal-like" lipopeptides may be crystalline under certain conditions, but may be amorphous under other conditions.
[57] "Crystalline lipopeptide" or "crystalline form of a lipopeptide" means a preparation of a lipopeptide or salt thereof comprising a lipopeptide crystal. In one embodiment, the crystalline lipopeptides may comprise a certain amount of amorphous lipopeptides. In one embodiment, the crystalline lipopeptides comprise at least 50% by weight lipopeptide crystals. In another embodiment, the crystalline lipopeptides comprise 60%, 70%, 80%, 90% or 95% of the lipopeptide crystals. Crystalline lipopeptides may comprise 50-60%, 60-70%, 70-80%, 80-90% or 90-95% lipopeptide crystals. In another embodiment, the crystalline lipopeptides comprise at least 95% lipopeptide crystals, such as at least 96%, 97%, 98% or 99% lipopeptide crystals, or 100% lipopeptide crystals. Crystalline lipopeptides may generally comprise between 95 and 100% lipopeptide crystals. Weight percent of lipopeptide crystals refers to a lipopeptide preparation prior to formulation into a pharmaceutical composition.
[58] "Amorphous" form of a lipopeptide means a lipopeptide preparation with little or no lipopeptide crystals or crystal-like lipopeptides (or crystal-like particles). In one embodiment, the amorphous lipopeptides comprise less than 20% by weight of lipopeptide crystals or crystalline like lipopeptides. In another embodiment, the amorphous lipopeptides comprise less than 10% by weight of lipopeptide crystals or crystal-like lipopeptides. In another embodiment, the amorphous lipopeptides comprise less than 5% by weight of lipopeptide crystals or crystal-like lipopeptides. In another preferred embodiment, the amorphous lipopeptides comprise less than 1% by weight of lipopeptide crystals or crystal-like lipopeptides.
[59] By "batch crystallization" is meant a method in which the lipopeptides are crystallized in solution by mixing the lipopeptides with a crystallization reagent in solution. By "batch precipitation" is meant a method in which the lipopeptides are mixed with the precipitation reagents in solution to precipitate the lipopeptides in solution. In one embodiment, the crystallized or precipitated formulation is collected from the solution. In another embodiment, the crystallization or precipitation formulation is collected by filtration or centrifugation.
[60] "Organic precipitant" means polyethylene glycol (PEG) or polyethylene glycol monomethyl ether (PEG MME) or chemically similar compounds.
[61] "Salt" means an ionic compound. These ionic compounds can act as precipitants.
[62] A "low molecular weight alcohol" is an organic compound comprising at least one alcohol functional group and up to eight carbon atoms. For example, non-limiting examples of low molecular weight alcohols include methanol, isopropanol and t-butanol.
[63] A "polyalcohol" is a compound that contains more than one alcohol group and less than eight carbon atoms. Examples of polyhydric alcohols include, but are not limited to, 1,6-hexanediol, ethylene glycol, propylene glycol, glycerol, 1,2-propanediol, 2-methyl-2,4-pentanediol and 1,4-butanediol It is not.
[64] "Container" means a receptacle that holds a product. For example, the container may be, but is not limited to, an ampoule, glass bottle, tube, bottle, or cylinder.
[65] Process for preparing purified lipopeptides
[66] One of the objects of the present invention is to provide a method for purifying a lipopeptide comprising providing an amorphous preparation of a lipopeptide and crystallizing or precipitating the lipopeptide. In one embodiment, the lipopeptides are of higher purity after the run than before the crystallization or precipitation. Lipopeptides can be crystallized by hydration, sitting drop or sandwich drop vapor diffusion, liquid-liquid or free interfacial diffusion, microdialysis or dialysis, delayed solvent evaporation, sublimation, or microbatch or batch crystallization. In general, lipopeptides can be precipitated in a similar manner, and it is preferred to precipitate the lipopeptides by batch precipitation. In a preferred embodiment, the crystallized or precipitated lipopeptides are daptomycin or daptomycin related lipopeptides. In a more preferred embodiment, the crystallized or precipitated lipopeptides are daptomycin.
[67] Lipopeptides may be crystallized or precipitated according to the teachings herein. In one embodiment, a solution comprising a lipopeptide as provided below is provided with a salt comprising a low molecular weight alcohol or a polyhydric alcohol, a pH buffer and a monovalent or divalent cation, causing precipitation or crystallization to cause the lipopeptides to It can be crystallized or precipitated. In another embodiment, the salt has buffering capacity such that no additional pH buffer is present in the solution. In another embodiment, the salt comprises a divalent cation. In a preferred embodiment, provided solutions do not comprise PEG or PEG-MME, or chemically similar compounds. In one embodiment, the method of precipitating or crystallizing the lipopeptide is generally
[68] a) mixing a lipopeptide with a salt comprising a monovalent or divalent cation, an optional pH buffer and a low molecular weight alcohol or a polyhydric alcohol; And
[69] b) precipitation or crystallization of the lipopeptides from solution under appropriate temperature conditions.
[70] In particular, microscopy can monitor the sample for crystal or precipitate formation, and the output can be analyzed spectrophotometrically. In a preferred embodiment, the crystallized or precipitated lipopeptides are daptomycin or daptomycin related lipopeptides.
[71] In another embodiment, lipopeptides can be crystallized by providing a solution comprising a low molecular weight or polyhydric alcohol (s), salts and organic precipitants as disclosed below. In a more preferred embodiment, the crystallized lipopeptide is daptomycin. In general, in a batch crystallization method, lipopeptides are dissolved in a solution, and low molecular weight alcohols, salts, buffers and / or organic precipitants are added to the solution. The sample is then crystallized under appropriate temperature conditions with or without stirring. In particular, microscopy can monitor the sample for crystal formation and analyze the output with a spectrophotometer.
[72] As mentioned above, the lipopeptides, preferably daptomycin or daptomycin related lipopeptides, crystallize or precipitate in the presence of one or more alcohols. In a preferred embodiment, the alcohol is a low molecular weight alcohol or a polyhydric alcohol. Non-limiting examples of low molecular weight alcohols or polyhydric alcohols include methanol, isopropanol, t-butanol, 1,6 hexanediol, ethylene glycol, propylene glycol, glycerol, 1,2-propanediol, 2-methyl-2,4- Pentanediol and 1,4-butanediol. In a preferred embodiment, the alcohol is isopropanol, t-butanol, glycerol, 1,6-hexanediol, 1,2-propanediol, 1,4-butanediol, propylene glycol and / or ethylene glycol. In a more preferred embodiment, the alcohol is isopropanol.
[73] Salts include, in particular, magnesium formate, sodium formate, ammonium sulfate, ammonium dihydrogen phosphate, calcium acetate, zinc acetate, trisodium citrate dihydrate, magnesium acetate, sodium acetate, magnesium chloride, cadmium chloride, ammonium acetate, sodium chloride and lithium sulfate It includes. In one embodiment, the salt comprises a monovalent cation such as sodium. In a preferred embodiment, the salt comprises a divalent cation. In a more preferred embodiment, the salt comprises a calcium cation, magnesium cation or manganese cation. In a further preferred embodiment, the salt comprises a calcium divalent cation. In one embodiment, the salt is calcium chloride, calcium acetate, zinc acetate, sodium citrate, trisodium citrate dihydrate, magnesium chloride, lithium sulfate, sodium chloride, magnesium acetate, sodium acetate or manganese salts such as manganese acetate or manganese chloride. In a preferred embodiment, the salt is calcium acetate. Examples of other salts comprising divalent cations such as calcium cations are known in the art and include those set forth in the 2000 Sigma Catalog, which are specifically incorporated by reference. Without being bound by any theory, it is believed that salt cations can neutralize negative charges on lipopeptides, such as four carboxylic acids on daptomycin. Organic precipitants include, in particular, polyethylene glycol (PEG) or polyethylene glycol monomethyl ether (PEG-MME), which may vary in average molecular weight from 300 to 10,000. In a preferred embodiment, the organic precipitant is PEG 300, PEG 600, PEG 2000, PEG 4000, PEG 8000 or PEG 10,000.
[74] Lipopeptides are precipitated or crystallized from a solution buffered at pH 5.0-9.5. In one embodiment, the pH of the solution prior to buffering is about 1.5, 2.0 or 3.0. In one embodiment, daptomycin or daptomycin related lipopeptides are precipitated or crystallized from a solution having a pH of about 5.5 to about 7.5. In yet another embodiment, the buffer has a pH of about 5.9 to about 6.3. In one embodiment, the buffered solution can be obtained using a pH buffer. Non-limiting examples of pH buffers include tris, phosphate, citrate, HEPES, CHES, sodium acetate or 2-morpholinoethanesulfonic acid (MES), sodium borate, sodium cacodylate, imidazole and trisodium citrate dihydrate. There is this. In a preferred embodiment, the salts are sodium cacodylate, sodium acetate, trisodium citrate dihydrate, HEPES, MES, CHES, imidazole, calcium acetate and tris-HCl. In a more preferred embodiment, the pH buffer is calcium acetate pH 6.1, sodium acetate pH 6.1, sodium cacodylate pH 6.5, trisodium citrate dihydrate pH 5.6, HEPES pH 7.5, imidazole pH 8, MES pH 6.0, calcium acetate pH 6 And Tris-HCl pH 8.5. In another embodiment, salts with buffering capacity can be used to buffer the solution. In a preferred embodiment, the pH buffer is calcium acetate pH 6.1.
[75] Lipopeptides are precipitated or crystallized from solutions containing 2-40% low molecular weight alcohols or polyhydric alcohols, 0.001-0.5 M salts and 0.005-0.2 M pH buffers using a conventional vapor diffusion method. In a preferred embodiment, the lipopeptides are precipitated or crystallized from a solution comprising 3-30% low molecular weight alcohol or polyhydric alcohol, 0.01-0.3 M salt and 0.01-0.1 M pH buffer. In a more preferred embodiment, the lipopeptides are precipitated or crystallized from a solution containing 5-20% low molecular weight alcohol or polyhydric alcohol, 0.02-0.1 M salt and 0.02-0.07 M pH buffer. The provided solution may or may not include polyethylene glycol (PEG) or polyethylene glycol monomethyl ether (PEG-MME).
[76] Batch crystallization is used to precipitate or crystallize the lipopeptides from a solution comprising 65-95% low molecular weight alcohol or polyhydric alcohol, 0.001-0.5 M salt and 0.001-0.2 M pH buffer. In a preferred embodiment, the lipopeptides are precipitated or crystallized from a solution containing 70-90% low molecular weight alcohol or polyhydric alcohol, 0.005-0.04 M salt and 0.005-0.04 M pH buffer. In some embodiments, the lipopeptides are crystallized from a solution comprising 3-8% organic precipitator. In a more preferred embodiment, the lipopeptides are precipitated or crystallized from a solution containing 80-85% low molecular weight alcohol or polyhydric alcohol, 0.01-0.03 M salt and 0.01-0.03 M pH buffer. In some embodiments, the solution further comprises about 4-5% organic precipitant, such as PEG or PEG-MME. In other embodiments, provided solutions may or may not include polyethylene glycol (PEG) or polyethylene glycol monomethyl ether (PEG-MME).
[77] Lipopeptides are precipitated or crystallized at a temperature of about 0 to about 30 ° C. to form precipitates or crystals, respectively. In a preferred embodiment, the lipopeptides are crystallized or precipitated at a temperature of about 20-30 ° C. In a more preferred embodiment, the mixture is crystallized or precipitated at about 23-28 ° C. In even more preferred embodiments, the mixture is crystallized or precipitated at about 27 ° C. The mixture may be crystallized or precipitated for any time that may cause crystallization or precipitation, preferably from about 1 hour to about 2 weeks. In a preferred embodiment, the mixture is stored for a period of time, preferably about 3 to about 24 hours, more preferably about 8 to 18 hours.
[78] Lipopeptide crystals or crystal-like particles may be needle-shaped, rod-shaped, sea urchin-shaped, flaky, plate-shaped or clustered forms thereof, but are not limited thereto. In one embodiment, the lipopeptide crystal or crystal like particle is sea urchin, rod or needle. The shape of a crystal or a crystal like particle can be determined especially by an optical microscope or an electron microscope. In yet another embodiment, the lipopeptide crystal or crystal like particle may be any size with any linear dimension of about 0.5 μm or greater. In a more preferred embodiment, the lipopeptide crystal or crystal like particle is at least 5 μm, more preferably at least 10 μm. In even more preferred embodiments, the lipopeptide crystal or crystal like particle is at least 50 μm, more preferably at least 100 μm. The size of the crystals can be determined by any method known in the art. See, eg, United States Pharmacopeia (USP), pp. 1965-67.
[79] The nature of the crystalline or crystalline like lipopeptides can be determined by any method known to those skilled in the art. Properties that can be measured include the size, shape, birefringence properties, powder x-ray diffraction properties, solid state NMR properties, melting point overheating, stability to light, humidity, and degradation of crystalline or crystal-like lipopeptides. In a preferred embodiment, one skilled in the art can determine whether the lipopeptides are crystalline by powder x-ray diffraction. Powder x-ray diffraction is extremely useful for determining whether a formulation is crystalline when the sample is a collection of randomly oriented small crystals. Diffraction by randomly oriented microcrystalline masses yields a series of lines or rings (depending on the detector) for the molecule being studied and its structure. In a preferred embodiment, powder diffraction is measured with an automated powder diffraction apparatus to determine whether the lipopeptides are crystalline. See, eg, Atkins et al., Physical Chemistrv, pp., For a discussion of the Debye-Scherrer method for powder diffraction. 710-716 (1978). The diffraction pattern can be measured using any powder diffractometer known in the art equipped with any detector for powder diffraction.
[80] In a preferred embodiment of the invention, the lipopeptides are crystallized or precipitated using buffers, salts and alcohols of about pH 5.0 to 9.5 for about 3 to 24 hours at a temperature of about 24 to 28 ° C. In a preferred embodiment, the salt comprises a divalent cation as a buffer, the alcohol is a low molecular weight alcohol, and the pH is about 5.5 to 7.5. In a more preferred embodiment, the salt is a calcium salt, the alcohol is isopropanol and the pH is about 5.9-6.3. In embodiments where the solution comprises an organic precipitant, it is preferred that the organic precipitant is PEG 4000 or PEG 8000. In another embodiment, the lipopeptides are precipitated or crystallized from a solution containing 12-18% glycerol, 0.3-0.8 M salt, 0.03-0.08 M pH buffer, and 12-18% PEG 600. In another preferred embodiment, the lipopeptides are daptomycin or daptomycin related lipopeptides. Examples 2-3 disclose methods for precipitating highly pure crystal-like daptomycin. One skilled in the art can modify the crystallization / precipitation conditions provided in the Examples from the teachings herein to crystallize or precipitate daptomycin, daptomycin related lipopeptides, or other lipopeptides as appropriate. In addition, although the teachings of the present specification disclose that a crystallization or precipitation step is performed once in a method of purifying a lipopeptide, a person skilled in the art performs a plurality of crystallization or precipitation steps in the process of purifying a lipopeptide from the teachings of this specification. You can do it. Performing multiple crystallizations or precipitations disclosed herein may further increase the purity of the lipopeptides and may be advantageous.
[81] After crystallization or precipitation, the crystalline material or crystalline like precipitate can be recovered by any method known in the art. In a preferred embodiment, the crystalline material or crystalline like precipitate is recovered by centrifugation or filtration. In an even more preferred embodiment, the crystalline material or crystalline like precipitate is recovered by filtration, since a filtration step can easily be introduced during the large scale process of preparing the lipopeptides. After the crystalline material or crystalline like precipitate is recovered, the wash may remove excess crystallization or precipitation reagent. Washing solvents known in the art can be chosen so long as the crystalline material or crystal-like precipitate is not soluble enough to be perceived. Examples of wash solvents are disclosed in Example 12. After washing the crystallized material or crystal-like precipitate, it can be dried by any method known in the art. Examples of drying methods include air drying, lyophilization (freeze-drying) or desiccation. In a preferred method, the crystalline material or crystalline like precipitate is dried by desiccation. See eg Example 12. In another embodiment, the stability of the crystalline lipopeptides can be measured by residual antibiotic activity or degradation products thereof. Antibiotic activity can be determined by standard agar diffusion assays for various bacterial strains. See, eg, Example 32 of US Pat. No. 4,537,717, which is incorporated herein by reference. The amount of degradation products can be measured, in particular, by HPLC analysis as disclosed in International PCT Publication No. WO 01/53330 published on July 26, 2001. In a preferred embodiment, the stability of the crystalline lipopeptides is greater than that of the lipopeptides in amorphous form. The stability of crystalline lipopeptides can be determined by exposing the crystalline lipopeptides and their amorphous forms to heat, light, humidity, and then measuring the degree of degradation of the crystalline forms relative to the resolution of the amorphous forms.
[82] The degree of degradation of the lipopeptide can be determined by measuring the biological activity or any applicable physical parameter of the lipopeptide. In one embodiment, the specific biological activity of the lipopeptides can be measured after application of heat, light, humidity, pH change, or the pH of the extremes, and the degree of degradation can be measured compared to the same biological activity of the lipopeptides prior to any stability test. . The amount of degradation can be measured, for example, by determining the percentage of biological activity remaining after the stability test. The remaining percentage of biological activity can be compared to that of the amorphous form of the lipopeptides tested. In one embodiment, if the lipopeptides are antibiotics, the crystalline lipopeptides can be tested for antibiotic activity before and after the stability test and compared to those in the amorphous form tested before and after the degradation test. In a preferred embodiment, the lipopeptides are daptomycin or daptomycin related lipopeptides and the biological activity test measures the amount of antibiotic activity of the lipopeptides against Gram-positive bacteria.
[83] Lipopeptide degradation can also be determined by physical analysis. In one embodiment, the degree of degradation can be determined by measuring the proportion of complete crystalline lipopeptides remaining after the stability test. The percentage of complete lipopeptide remaining can be compared to the lipopeptide in amorphous form, which has been tested the same for stability. In a preferred embodiment, degradation of the lipopeptides can be measured by HPLC, ultraviolet spectroscopy, infrared spectroscopy, NMR or mass spectroscopy. In an even more preferred embodiment, the percentage of complete lipopeptide remaining after the stability test is performed on the crystalline form of the lipopeptide using HPLC.
[84] Regardless of any theory, Applicants believe that daptomycin is crystallized by the method disclosed above. However, washing and / or drying of daptomycin crystals is believed to restore the daptomycin crystalline material to an amorphous but crystalline like form material. Nevertheless, if the disclosed method precipitates rather than crystallizes daptomycin or other lipopeptides, this method is also advantageous since the lipopeptides are purified by this method.
[85] The present invention also provides crystalline or crystalline like lipopeptides prepared by the methods disclosed above. In one embodiment, the crystalline or crystalline like lipopeptides comprise small amounts of one or more impurities compared to the lipopeptides prior to crystallization or precipitation. In one embodiment, the crystalline or crystalline like lipopeptides are daptomycins containing small amounts of the β-isomers of anhydrous-daptomycin and / or daptomycin compared to daptomycin prior to crystallization or precipitation. In another embodiment, the crystalline or crystalline like daptomycin comprises a small amount of all impurities as compared to amorphous daptomycin. Similarly, in another embodiment, the crystalline or crystalline like lipopeptides are daptomycin related lipopeptides as described above, comprising a small amount of one or more impurities as compared to the amorphous form of daptomycin related lipopeptides. In another embodiment, the crystalline or crystalline like daptomycin related lipopeptides comprise small amounts of all impurities as compared to the amorphous form of daptomycin related lipopeptides.
[86] Crystalline or crystal-like lipopeptides prepared by the methods disclosed above include monovalent or divalent cations and water. In a preferred embodiment, the crystalline or crystalline like lipopeptides are daptomycin or daptomycin related lipopeptides comprising a divalent cation. In a more preferred embodiment, the divalent cation is a calcium cation. In even more preferred embodiments, the crystalline or crystalline like daptomycin or daptomycin related lipopeptides comprise about 1-10% by weight of divalent calcium cations and about 0-15% by weight of water as determined by atomic absorption or thermogravimetric analysis. . In a more preferred embodiment, the crystalline or crystal-like lipopeptides are daptomycin comprising about 5% by weight divalent calcium cation and about 10% by weight of water and the purity of the crystalline or crystal-like daptomycin as determined by HPLC analysis is related to At least 95%, 96%, 97% or 98%, or any value between 95 and 98% for materials and organic contaminants. Alternatively, the crystalline or crystal-like daptomycin or daptomycin related lipopeptides include monovalent cations (eg sodium). Without being bound by any theory, it is believed that daptomycin or daptomycin related lipopeptides can form salts with monovalent or divalent cations upon crystallization or precipitation.
[87] The crystalline form of the lipopeptide may exhibit increased solubility and increased reconstitution rate in solution than the amorphous form of the lipopeptide. Whether crystalline lipopeptides exhibit increased solubility or increased reconstitution rate can be determined by methods known in the art. For example, a predetermined amount of crystalline lipopeptides may be dissolved in an aqueous solution and the concentration of dissolved lipopeptides measured, and then compared to the concentration of dissolved lipopeptides prepared by dissolving the same amount of amorphous lipopeptides in an aqueous solution. Likewise, crystalline lipopeptides can be added to an aqueous solution and then the concentration of dissolved lipopeptides measured over time, and the reconstitution rate of crystalline lipopeptides can be determined by comparison with the reconstitution rates of amorphous lipopeptides measured in the same manner. The concentration of lipopeptides is measured by HPLC.
[88] The methods disclosed above provide methods for the preparation of crystalline or crystal-like lipopeptides that are more pure than amorphous lipopeptides upon crystallization or precipitation. In one embodiment, the lipopeptides are daptomycin or daptomycin related lipopeptides. In another embodiment, the daptomycin or daptomycin related lipopeptides have a purity of 92% or less before crystallization, but after crystallization or precipitation as crystal-like lipopeptides, the purity is at least about 95%, 96%, 97% or 98%, or Or a value between 95 and 98%. In even more preferred embodiments, the daptomycin or daptomycin related lipopeptides are at most 90% pure before crystallization and at least about 97% or at least 98% after crystallization.
[89] In another embodiment, daptomycin has a purity of 80% or less, preferably 70% or less, more preferably 60% or less, and, after purification, purity of about 95%, 96%, 97% or 98 before crystallization or precipitation. % Or more, or any value between 95 and 98%. In another embodiment, daptomycin has a purity of 50% or less, preferably 40% or less, more preferably 30% or less before crystallization, and after purification by crystallization or precipitation, the purity is about 95%, 96%, 97 % Or at least 98%, or any value between 95 and 98%. In a further preferred embodiment, daptomycin has a purity of 20% or less, preferably 15% or less, more preferably 10% or less before crystallization and about 95%, 96%, 97% or 98% purity after purification. The value is above, or any value between 95 and 98%.
[90] In a more preferred embodiment, the lipopeptides are daptomycin. The daptomycin formulations are described, for example, in US Patents RE32,333, RE32,455, 4,800,157, RE32,310, RE32,311, 4,537,717, 4,482,487, 4,524,135, 4,874,843, which are incorporated herein by reference. Obtainable by any of the methods disclosed in either 4,885,243 or 5,912,226. The daptomycin preparation can also be obtained by one of the methods disclosed in International PCT Publication No. WO 01/53330 published on July 26, 2001. Once the lipopeptide formulation is prepared, the lipopeptide formulation is crystallized or precipitated according to the teachings disclosed herein, which is more pure than the lipopeptide formulation used to prepare it or contains a small amount of certain impurities such as an-daptomycin. Produces crystalline or crystal-like lipopeptides.
[91] Method for Preparing Purified Lipopeptides from Fermentation Cultures
[92] Another embodiment of the invention relates to a process combining crystallization or precipitation with process chromatography steps to yield purified lipopeptides. In a preferred embodiment, the method comprises the steps of preparing lipopeptides by any method known in the art, such as fermentation of naturally occurring or recombinant organisms, microfiltration, anion exchange chromatography, hydrophobic interactions for lipopeptide preparations. Subjecting any one or more purification methods, such as functional chromatography and / or size exclusion chromatography (typical size exclusion chromatography medium or ultrafiltration) to yield partially purified lipopeptide formulations, and crystallizing the lipopeptide formulations Or precipitation to obtain purified crystalline or crystal-like lipopeptides. In a preferred embodiment, the lipopeptides are daptomycin or daptomycin related lipopeptides. Steps related to fermentation, microfiltration, anion exchange chromatography, hydrophobic interaction chromatography and ultrafiltration are described, for example, in US Patents RE32,333, RE32,455, 4,800,157, RE32,310, RE32,311, 4,537,717 4,482,487, 4,524,135, 4,874,843, 4,885,243 or 5,912,226, International Publication No. WO 01/53330 published on July 26, 2001.
[93] The method may optionally include the step of collecting and / or washing the crystalline or crystalline like material after the crystallization or precipitation step. In a preferred embodiment, the crystalline lipopeptide formulation can be recovered by filtration. In another embodiment, the crystalline or crystalline like material is dried.
[94] In one embodiment, the purification method comprises expressing Streptomyces roseosporus to obtain a fermentation culture comprising daptomycin. In one embodiment, S. Roseosporus can be fermented as disclosed in US Pat. No. 4,885,243. In another embodiment, the fermentation conditions under which A-21978C ₀ containing crude product is produced by Streptomyces roseosporus, as disclosed in International PCT Publication No. WO 01/53330 published on July 26, 2001, are modified. Increases daptomycin production and s. It reduces impurities and related contaminants produced by Roseosporus fermentation cultures. WO 01/53330 applies a modification to maintain the decanoic acid feed at the lowest possible concentration without reducing the total yield of fermentation, as disclosed in the '243 patent. It describes the fermentation of roseosporus.
[95] Alternatively, daptomycin can be obtained by fermenting bacterial strains or other producing organisms that produce daptomycin by recombinant methods. In one embodiment, the recombinant bacterial strain or other recombinant organism comprises a daptomycin biosynthetic gene cluster. In another embodiment, the daptomycin biosynthetic gene cluster or portion thereof is introduced into an organism or bacterial strain via a bacterial artificial chromosome (BAC). In another embodiment, the recombinant bacterial strain used comprises S. a BAC containing a daptomycin biosynthetic gene cluster. Rosedale porous agarose or S. Libby is Tansu (S. lividans). U.S. Provisional Application 60 / 272,207, filed February 28, 2001, discloses S. A daptomycin biosynthetic gene Closter from Roseosporus and its use is disclosed, which application is incorporated by reference in its entirety.
[96] After fermentation, the fermentation broth is clarified by centrifugation, microfiltration or extraction as known in the art or as disclosed in WO 01/53330. In a preferred embodiment, the cleansing is by microfiltration. See, for example, Examples 13-16 and Figures 11-15. 11 shows an exemplary manufacturing process without using crystallization or precipitation.
[97] After clarifying the fermentation broth, the daptomycin concentration in the broth is about 5-10%. In one embodiment of the present invention, the daptomycin formulation is subjected to the crystallization / precipitation method disclosed above immediately after microfiltration. In one embodiment, the crystallization or precipitation is carried out under sterile conditions. After crystallization or precipitation is complete, crystalline or crystal-like daptomycin is recovered, washed and dried as necessary as described in further detail below. The bulk, anhydrous active drug can then be used to dry fill the sterile bottle. See, eg, Example 16 and FIG. 12.
[98] After clarification of the fermentation broth, the lipopeptides can be concentrated in the formulation as known in the art or by anion exchange chromatography as disclosed in WO 01/53330 or disclosed herein. See, eg, Examples 13-14 and Figures 12-13. After anion exchange chromatography, the purity of daptomycin in the broth is about 35-40%. In one embodiment of the invention, the daptomycin preparation is subjected to the crystallization or precipitation method described above immediately after anion exchange chromatography. In one embodiment, the crystallization or precipitation is carried out under sterile conditions. After crystallization or precipitation is complete, crystalline or crystal-like daptomycin is recovered, washed and dried as necessary as described in further detail below. The bulk, anhydrous active drug can then be used to dry fill the sterile bottle. See, eg, Example 14 and FIG. 13.
[99] In another embodiment of the present invention, size exclusion ultrafiltration is performed on the daptomycin preparation after anion exchange chromatography. Size exclusion ultrafiltration is disclosed in WO 01/53330. This application, published July 26, 2001, discloses a pyrogen removal, filtration and concentration method of daptomycin using an ultrafiltration membrane of 10,000 to 30,000 nominal molecular weight (NMW). This application discloses a method in which high molecular weight impurities such as endotoxins are filtered through a filter while lipopeptides pass through the ultrafiltration membrane. After passing the lipopeptide through the membrane, the pH, temperature and / or salt concentration of the lipopeptide solution is changed so that the lipopeptide forms micelles. The lipopeptide solution is then filtered through the ultrafiltration membrane under conditions in which lipopeptide micelles remain in the membrane and smaller impurities are passed through the filter. In this way, the lipopeptides are further purified. This application discloses conditions under which lipopeptide micelles can be formed and dissociated, and a method of filtering the lipopeptide solution to obtain more purified lipopeptide applications. In even more preferred embodiments, the lipopeptides are daptomycin or daptomycin related lipopeptides. Lipopeptides can then be crystallized as disclosed herein. After both anion exchange chromatography and size exclusion ultrafiltration, the daptomycin purity is about 80-90%. As discussed above, the aforementioned crystallization / precipitation method for daptomycin formulations is preferably performed under sterile conditions. Crystalline or crystalline like daptomycin may then be recovered, washed, dried and optionally used to dry fill the bottle as described above. See, eg, Example 13 and FIG.
[100] In another embodiment of the present invention, the crude daptomycin formulation is subjected to size exclusion ultrafiltration without anion exchange chromatography. After size exclusion ultrafiltration, the daptomycin purity is 35-40%. As described above, the lipopeptides can then be crystallized or precipitated, preferably by sterilization. As noted above, crystalline or crystalline like daptomycin may optionally be recovered, washed, dried and then used to dry fill the sterile bottle. See, eg, Example 15 and FIG. 14.
[101] In an alternative embodiment, after anion exchange chromatography or size exclusion filtration, the lipopeptide preparation is treated with hydrophobic interaction chromatography (HIC) as disclosed in WO 01/53330. Lipopeptides may be crystallized or precipitated as disclosed herein.
[102] After crystallization or precipitation, crystalline or crystal-like lipopeptides can be recovered by methods disclosed herein, such as by filtration or centrifugation. Crystalline or crystalline like lipopeptides are optionally washed to remove residual crystallization or precipitation solvent. Methods of washing crystals or crystal like materials are described below. See eg Example 3. The washed or unwashed crystal or crystal like material may be dried. Drying can be carried out by any method known in the art, including vacuum drying, spray drying, tray drying or lyophilization. In one embodiment, the drying is performed under sterile conditions. In another embodiment, the drying is carried out by vacuum drying. In an even more preferred embodiment, drying is carried out using a 0.65 m ³ Klein Hastelloy-B bubble cone vacuum dryer or equivalent apparatus. Dried crystalline or crystal-like lipopeptides are stable and easy to store.
[103] In one embodiment, the bottle is filled with any conventional amount of dried crystalline or crystalline like lipopeptides. In one embodiment, the bottle is filled and then capped under sterile conditions. In another embodiment, 50-5000 mg each of dried crystalline or crystalline-like lipopeptides is filled into a bottle. In yet another embodiment, the bottles are filled with 100-1000 mg each. In another embodiment, the bottles are filled with 200-500 mg each. In another embodiment, the dried crystalline or crystalline like lipopeptides are used for bulk packaging of the lipopeptides. Large packages generally contain at least 5000 mg of dried crystalline or crystalline like lipopeptides, respectively. In one embodiment, the large package is carried out under sterile conditions.
[104] In one embodiment, the crystallization or precipitation step is performed under sterile conditions. In this embodiment, a controlled working environment of sterile crystallization or precipitation reagents and sterile is used. In one embodiment, lipopeptide is filtered through the ultrafiltration membrane as described above and then mixed with sterile crystallization / precipitation reagent. After crystallization or precipitation, the crystalline or crystal-like lipopeptide formulation is collected by centrifugation or filtration under sterile conditions. In one embodiment, the lipopeptide formulation is collected by sterile filtration. In another embodiment, the crystalline or crystalline like lipopeptides are sterilized after harvesting. Sterile crystallization, precipitation and filtration methods and sterilization methods of the final pharmaceutical product are known in the art. See, eg, Remington: The Science and Practice of Pharmacy, Easton, Pennsylvania: Mack Publishing Company (1995), pp. 1474-1487.
[105] In another embodiment, the crystalline or crystalline like lipopeptides are not dried. In this embodiment, the crystalline or crystal-like lipopeptides are preferably stored in a solution that preserves the crystalline or crystal-like properties of the lipopeptides. Bottles can be filled with lipopeptides and solutions under sterile or non-sterile conditions. In one embodiment, the conditions are sterile conditions. Alternatively, large packages may be filled using crystalline or crystalline like lipopeptides and solutions.
[106] 10 and 11 are process diagrams illustrating an exemplary daptomycin preparation protocol using crystallization. Incorporating a sterile crystallization step into the preparation protocol significantly shortens the protocol and omits steps 3-4 of the process.
[107] Crystalline or crystalline like lipopeptides, pharmaceutical compositions and methods of using the same
[108] It is another object of the present invention to provide a pharmaceutical formulation comprising crystalline or crystalline like lipopeptides or salts thereof and crystalline or crystalline like lipopeptides or salts thereof. In one embodiment, the crystalline or crystalline like lipopeptides are daptomycin. However, all references herein to crystalline or crystalline like lipopeptides specifically include daptomycin, daptomycin related molecules, such as especially daptomycin, A54145 and daptomycin related lipopeptides, as described above.
[109] The daptomycin crystal or crystal-like particle and other lipopeptide crystal or crystal-like particle may, in particular, have a shape such as needle-like, plate-shaped, wedge-shaped, eccentric, sea urchin, or rod-shaped. In one embodiment, the daptomycin crystal or crystal like particle is sea urchin, needle-like, or rod-shaped. The size of the crystal or crystal like particle is about 0.5 μm to 100 μm or more. In one embodiment, the particle size is at least 5 μm. In a more preferred embodiment, the particle size is at least 10 μm, more preferably at least 50 μm. In even more preferred embodiments, the particle size is at least 100 μm.
[110] Also, in one embodiment, the daptomycin crystal has the x-ray diffraction pattern shown in FIGS. 6, 7 and 8. In another embodiment, the melting point of the lipopeptide crystal is different from the lipopeptide amorphous form.
[111] In one embodiment of the invention, the stability of the lipopeptide crystalline form is similar to or greater than the stability of the lipopeptide amorphous form. In a preferred embodiment, the crystalline form is daptomycin or daptomycin related lipopeptides. In another preferred embodiment, the crystalline lipopeptides are sterile. In another preferred embodiment, the stability of the crystalline lipopeptides is greater than the amorphous form of the lipopeptides. Crystalline lipopeptides can be more stable against heat, light, degradation or humidity than amorphous forms. The stability of the lipopeptide can be measured by any means, including, for example, antibiotic activity, lipopeptide degradation or conversion of daptomycin to anhydrous-daptomycin or daptomycin β-isomer. In another embodiment of the invention, the crystalline form of the lipopeptide can be reconstituted more rapidly in aqueous solution than the amorphous form of the lipopeptide.
[112] Crystalline or crystalline like lipopeptides such as daptomycin or daptomycin related lipopeptides, pharmaceutically acceptable salts, esters, amides, ethers and their protective forms, oral, intravenous for the treatment, experimental or prophylactic treatment of diseases, in particular bacterial infections. It may be formulated for internal, intramuscular, subcutaneous, aerosol, topical or parenteral administration. As used herein, "crystalline or crystalline like lipopeptides" or "crystalline or crystalline like daptomycin" includes pharmaceutically acceptable salts thereof. Crystalline or crystalline like lipopeptides, such as daptomycin, are micron-sized microsphere particles that allow convenient enteric-derived lipopeptide formulations for oral delivery, such as pharmaceutical compositions for aerosol delivery to the lung and sustained release lipopeptide combination formulations. It can be particularly advantageous for use in pharmaceutical compositions because it can be easily formulated. Crystalline or crystalline like lipopeptides and crystalline or crystalline like daptomycin can be more readily dissolved in aqueous solutions.
[113] Crystalline or crystalline like lipopeptides such as daptomycin or daptomycin related lipopeptides can be formulated using any pharmaceutically acceptable carrier or excipient compatible with the lipopeptides or daptomycins. For a general description of how to administer various antimicrobials for human therapy, see, for example, Handbook of Pharmaceutical Additives: An International Guide to More than 6000 Products by Trade Name, Chemical, Function, and Manufacturer, Ashgate Publishing Co., eds ., M. Ash and I. Ash, 1996; The Merck Index: An Encyclopedia of Chemicals, Drugs and Biologicals, ed. S. Budavari, annual; Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA; Martindale: The Complete Drug Reference, ed. K Parfitt, 1999; and Goodman & Gilman's The Pharmaceutical Basis of Therapeutics, Pergamon Press, New York, NY, ed. L. S. Goodman et al .: The contents of these documents are incorporated herein by reference. The compounds of the present invention can be mixed with conventional pharmaceutical carriers and excipients and used in the form of tablets, capsules, elixirs, suspensions, serums, oblates, creams and the like. The compounds of the present invention can be mixed with other therapeutic and antibiotic agents as disclosed herein. Compositions comprising a compound of the present invention comprise from about 0.1% to about 90%, more generally from about 10% to about 30% by weight of the active compound.
[114] Controlled (eg, capsule) or sustained release delivery systems (eg, biodegradable matrices) can be used to deliver the compositions of the present invention. Examples of delayed release delivery systems for drug delivery suitable for administration of the compositions of the present invention are disclosed in US Pat. Nos. 4,452,775 (Kent), 5,239,660 (Leonard), 3,854,480 (Zaffaroni).
[115] The composition may include corn starch or common carriers and excipients such as gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride and alginic acid. The composition may comprise croscarmellose sodium, microcrystalline cellulose, corn starch, sodium starch glycolate and alginic acid.
[116] Tablet binders that may be included include acacia, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone (Povidone), hydroxypropyl methylcellulose, sucrose, starch, ethylcellulose and the like.
[117] Lubricants that can be used include magnesium stearate or other metal stearates, stearic acid, silicone fluids, talc, waxes, oils, and colloidal silica.
[118] Flavoring agents, such as peppermint, presser oil, and cherry flavor, can be used. It may be desirable to add colorants to make the appearance of the formulation more beautiful or to aid in the identification of the product.
[119] As oral use, solid preparations such as tablets and capsules are particularly useful. Slow or enteric formulations may be devised. In another embodiment, crystalline or crystalline like lipopeptides can be supplied with a carrier composition that increases the oral availability of the lipopeptide. In a preferred embodiment, the crystalline or crystalline like lipopeptides are daptomycin. For pediatric and geriatrics, suspensions, syrups and chewable tablets are particularly suitable. Pharmaceutical compositions for oral administration are, for example, in tablet, capsule, suspension or liquid form. The pharmaceutical composition is preferably prepared in the form of a dosage unit containing a therapeutically effective amount of the active ingredient. Examples of such dosage units are tablets and capsules. For treatment, tablets and capsules may be used in addition to the active ingredient in conventional carriers, for example binders such as acacia gum, gelatin, polyvinylpyrrolidone, sorbitol or tragacanth; Fillers such as calcium phosphate, glycine, lactose, corn starch, sorbitol or sucrose; Lubricants such as magnesium stearate, polyethylene glycol, silica or talc; Disintegrating agents such as potato starch, flavoring or coloring agents, or acceptable wetting agents. Oral liquid preparations are generally in the form of aqueous or oily solutions, suspensions, emulsions, syrups or elixirs and may include conventional additives such as suspending, emulsifying, non-aqueous, preservative, coloring and flavoring agents. Oral liquid preparations may include lipopeptide micelles or monomeric forms of lipopeptides. Examples of additives for liquid preparations include acacia, almond oil, ethyl alcohol, distilled coconut oil, gelatin, glucose syrup, glycerin, edible hardened fat, lecithin, methyl cellulose, methyl or propyl para-hydroxybenzoate, propylene glycol, sorbitol Or sorbic acid.
[120] For use for intravenous (IV) administration, the water soluble forms of the compounds of the invention may be dissolved in any of the commonly used intravenous fluids and administered by infusion. Intravenous preparations may include carriers, excipients or stabilizers, including but not limited to calcium, human serum albumin, citrate, acetate, calcium chloride, carbonate and other salts. Non-limiting examples of intravenous fluids include physiological saline or Ringer's solution. Daptomycin or other lipopeptides can be placed in syringes, cannulas, catheters and lines.
[121] Formulations for parenteral administration may be in the form of aqueous or non-aqueous isotonic sterile injectable solutions or suspensions. These solutions or suspensions may be prepared from the form of sterile powders or granules comprising one or more of the carriers mentioned for use in the formulation for oral administration. Crystalline or crystalline like lipopeptides can be dissolved in polyethylene glycol, propylene glycol, ethanol, corn oil, benzyl alcohol, sodium chloride and / or various buffers. For intramuscular, parenteral or intravenous preparations, a sterile blend of crystalline or crystalline like lipopeptide compounds or appropriate soluble salts of such compounds, such as hydrochloride, is prepared by pharmaceutical preparation such as water for injection (WFI), physiological saline or 5% glucose. It can be administered by dissolving in a diluent. Suitable insoluble forms of crystalline or crystalline like lipopeptides can be prepared and administered as suspensions in an aqueous base or a pharmaceutically acceptable oil base (eg, esters of long chain fatty acids such as ethyl oleate).
[122] Injectable depot forms can be prepared by forming microencapsulated matrices of crystalline or crystal-like lipopeptides in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer and the nature of the particular polymer used, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly (orthoesters) and poly (anhydrides). Depot injectable preparations are prepared by entrapping the drug in a microemulsion that is compatible with body tissues.
[123] For topical use, the compounds of the present invention may be prepared in a form suitable for application to the skin or mucous membranes of the nose and throat and may be in the form of creams, ointments, liquid sprays or inhalants, lozenges, or throat coatings. Can be taken. Such topical formulations may further comprise a chemical compound, such as dimethylsulfoxide (DMSO), which promotes surface penetration of the active ingredient. For topical formulations, sterile formulations comprising crystalline or crystalline like lipopeptides, such as crystalline or crystalline like daptomycin, suitable salts thereof, may be administered as creams, ointments, sprays or other topical surgical treatment ointments. The topical formulation can also be in the form of a bandage impregnated with the lipopeptide composition.
[124] For use in the eye or ear, the compounds of the present invention may be provided in liquid or semi-liquid form formulated in hydrophobic or hydrophilic bases such as ointments, creams, lotions, applicators or powders.
[125] For rectal administration, the compounds of the present invention may be administered in the form of suppositories mixed with conventional carriers such as cocoa butter, wax or other glycerides.
[126] For aerosol formulations, sterile formulations in the form of crystalline or crystalline like lipopeptides or salts of these compounds can be used in inhalers such as metered dose inhalers and nebulizers. Spray forms may be particularly useful for treating respiratory infections such as lung and sinus-family infections.
[127] Alternatively, the compounds of the present invention may be in powder crystalline or crystalline like form for reconstitution in a suitable pharmaceutically acceptable carrier upon delivery. In another embodiment, the unit dosage form of the compound may be a solution of the compound or salt thereof dissolved in a suitable diluent and placed in a sterile hemispherical sealing ampoule. The concentration of the compound in the unit dosage form may vary, for example from about 1 to about 50%, depending on the compound used and its solubility and the dosage desired by the physician. If the composition comprises dosage units, each dosage unit comprises about 10-5000 mg, more preferably 50-1000 mg, even more preferably 100-500 mg of the active substance. For adult treatment, the dosage used is preferably 100 mg to 3 g per day, depending on the route and frequency of administration.
[128] In a further aspect, the invention provides a method of treating an infection caused by Gram-positive bacteria in a subject. In a preferred embodiment, this method can be used to treat infection caused by Gram-positive bacteria. The term "treatment" means administering to a subject a therapeutically effective amount of a compound of the invention to prevent the occurrence of an infection and to control or eliminate an infection (eg, an established infection). The term "subject" as used herein is defined as a mammalian, plant or cell culture. As used herein, the term “therapeutically effective amount” refers to the amount of daptomycin, daptomycin-associated lipopeptides or other antimicrobial lipopeptides of the present invention that prevent the onset of bacterial infections, alleviate symptoms or stop progression. In a preferred embodiment, the subject is a human or other animal patient in need of lipopeptide treatment. The established infection can be acute or chronic. An effective amount is generally from about 0.1 to about 75 mg per kg of crystalline or crystalline like lipopeptides, such as crystalline or crystalline like daptomycin or daptomycin related lipopeptides, or pharmaceutically acceptable salts thereof. Preferred doses are from about 1 to about 25 mg / kg of crystalline or crystalline like daptomycin or daptomycin related lipopeptides, or pharmaceutically acceptable salts thereof. More preferred doses are about 1-12 mg per kg of crystalline or crystalline like daptomycin, crystalline or crystalline like daptomycin related lipopeptides, or pharmaceutically acceptable salts thereof. Even more preferred doses are about 3-8 mg / kg of crystalline or crystalline like daptomycin or daptomycin related lipopeptides, or pharmaceutically acceptable salts thereof. Exemplary procedures for delivering antimicrobials are disclosed in US Pat. No. 5,041,567 (Rogers) and International PCT Publication No. WO 95/05384, which are incorporated by reference in their entirety.
[129] Crystalline or crystalline like lipopeptides, such as daptomycin, may be administered in a single daily dose or in multiple daily doses. Therapies may require administration for extended periods of time, such as days or for 2-4 weeks. Dosage or total dose will vary depending on factors such as the nature and severity of the infection, the age and general health of the patient, the patient's tolerance to lipopeptides and the microorganism (s) associated with the infection. Methods of administration are disclosed in WO 00/18419, published April 6, 2000, incorporated herein by reference.
[130] The methods of the present invention comprise administering a compound of the present invention or a pharmaceutical composition thereof to a patient in need thereof in an amount effective to reduce or eliminate Gram-positive bacteria infection. Lipopeptides may be administered orally, parenterally, inhaled, topically, rectally, nasal, buccal, vaginal, or in implantable reservoirs, external pubs or catheters. Lipopeptides can be prepared for ophthalmic use or for spraying.
[131] The compounds of the present invention or pharmaceutical compositions thereof may be injected or administered directly into the abscess, ventricle or joint. Parenteral administration includes subcutaneous, intravenous, intramuscular, intraarticular, synovial, bath, intradural, intrahepatic, intralesional and intracranial injections or infusions. In a preferred embodiment, crystalline or crystal-like daptomycin, daptomycin related lipopeptides or other lipopeptides are administered intravenously, subcutaneously or orally.
[132] The method of the present invention can be used to treat bacterial infection patients caused or exacerbated by any type of Gram-positive bacteria. In a preferred embodiment, crystalline or crystalline like daptomycin, daptomycin related lipopeptides or other lipopeptides, or pharmaceutical compositions thereof are administered to a patient in accordance with the methods of the present invention. In another embodiment, nonlimiting examples include methicillin sensitive and methicillin resistant Staphylococcus (eg, Staphylococcus aureus, Staphylococcus epidermis)Staphylococcus epidermidis), Staphylococcus Hamilticus (Staphylococcus haemolyticus), Staphylococcus hominis (Staphylococcus hominis), Staphylococcus sapioticus (Staphylococcus saprophyticus), And coagulase-negative staphylococcus], glycopeptide mediated susceptible Staphylococcus aureus (GISA), penicillin sensitive and penicillin resistant Streptococcus [eg, Streptococcus pneumoniae, Streptococcus piogens (Streptococcus pyogenes), Streptococcus agalactia (Streptococcus agalactiae), Streptococcus avium (Streptococcus avium), Streptococcus Vorbis (Streptococcus bovis), Streptococcus lactis (Streptococcus lactis), Streptococcus Sangius (Streptococcus sangius) And Streptococcus group C, Streptococcus group G and Viridans streptococcus], Enterococcus [eg, vanacomycin sensitive and vanacomycin resistant strains such as Enterococcus faecalis (Enterococcus faecalis) And enterococcus fascium (Enterococcus faecium)], Clostridium difficile (Clostridium difficile), Clostridium ClostridiformeClostridium clostridiiforme), Clostridium InokumClostridium innocuum), Clostridium Perfringens(Clostridium perfringens), Clostridium Ramo Island (Clostridium ramosum), Haemophilus influenza (Haemophilus influenzae), Listeria monocytogenes (Listeria monocytogenes), Corynebacterium J-Caeum (Corynebacterium jeikeium), Bifidobacteria species (Bifidobacteriumspp.), and Eubacterium aerociens (Eubacterium aerofaciens), Yubacterium Lentum (Eubacterium lentum), Lactobacillus ashdophilus (Lactobacillus acidophilus), Lactobacillus casei (Lactobacillus casei), Lactobacillus plantarumLactobacilllus plantarum), Lactococcus species (Lactococcusspp.), leukonostock species (Leuconostocspp.), pediococcus (Pediococcus), Peptostreptococcus Annabius (Peptostreptococcus anaerobius), Peptostreptococcus asacaroticus (Peptostreptococcus asaccarolyticus), Peptostreptococcus magnus (Peptostreptococcus magnus), Peptostreptococcus micros (Peptostreptococcus micros), Peptostreptococcus prevoti (Peptostreptococcus prevotii), Peptostreptococcus products (Peptostreptococcus productus), Propionibacterium acins (Propionibacterium acnes), And actinomysis species (ActinomycesBacteria, such as spp.) can cause or worsen bacterial infections.
[133] In vitro experiments compare daptomycin antimicrobial activity against the classical "resistant" strain to that for the classic "sensitive" strain. In addition, the minimum inhibitory concentration (MIC) value of daptomycin for susceptible strains is typically four times lower than vancomycin. Thus, in a preferred embodiment, the pharmaceutical compositions of any of the compounds of the invention or crystalline or crystalline like lipopeptides are administered to a patient exhibiting a bacterial infection resistant to other antibiotics such as vancomycin according to the methods of the invention. In addition, daptomycin, unlike glycopeptide antibiotics, exhibits rapid concentration dependent bacterial activity against gram positive organisms. Thus, in a preferred embodiment, the pharmaceutical compositions of the compounds of the invention or any of the crystalline or crystalline like lipopeptides are administered to a patient in need of antibiotic treatment which is rapidly acting in accordance with the methods of the invention.
[134] The method of the present invention can be used for Gram-positive bacteria infection of any organ or tissue of the body. Non-limiting examples of these organs or tissues include skeletal muscle, skin, blood flow, kidneys, heart, lungs, bones, and the like. The methods of the present invention can be used to treat skin and soft tissue infections, bacteremia and urinary tract infections, but are not limited to such. The methods of the present invention can be used to treat locally acquired respiratory infections, including but not limited to otitis media, sinusitis, chronic bronchitis and pneumonia (eg, pneumonia caused by drug resistant Streptococcus pneumoniae or Haemophilus influenzae). . The methods of the invention can also be used to treat mixed infections including other types of Gram-positive bacteria such as aerobic, caprophilic or anaerobic bacteria. These types of infections include abdominal infections, pneumonia, bone and joint infections, and obstetric / gynecological infections. The methods of the present invention can be used to treat infections, as non-limiting examples, pericarditis, nephritis, septic arthritis and osteomyelitis. In a preferred embodiment any of the diseases disclosed above can be treated using a pharmaceutical composition of crystalline or crystalline like daptomycin, daptomycin related lipopeptides, antibacterial lipopeptides, or any of these crystalline or crystalline like lipopeptides. .
[135] Crystalline or crystalline like daptomycin, daptomycin related lipopeptides or other lipopeptides can be administered in the diet or feed of a patient or animal. When administered as part of the total meal intake, the amount of daptomycin or other lipopeptides may be less than 1% by weight, preferably 0.5% or less by weight of the meal. Animal feed may be a general feed with daptomycin or other lipopeptides added, or daptomycin may be added to the premix.
[136] The method of the present invention may be administered in another form of daptomycin or other lipopeptide antibiotic, such as not in a crystalline or crystalline like form, or in one or more antifungal agents other than crystalline or crystalline like daptomycin or other crystalline or crystalline like lipopeptide antibiotics. And / or in combination with one or more antibiotics. Concurrent administration of antifungal agents and antibiotics other than crystalline or crystalline like daptomycin or other lipopeptide antibiotics may be useful for mixed infections, such as those caused by other types of Gram-positive bacteria, or caused by bacteria and fungi. In addition, crystalline or crystalline like daptomycin or other lipopeptide antibiotics may improve the toxicity profile of one or more coadministered antibiotics. Administration of daptomycin and aminoglycosides has been shown to improve renal toxicity caused by aminoglycosides. In a preferred embodiment, the antibiotic and / or antifungal agent may be co-administered with the compound of the invention or administered in a pharmaceutical composition comprising the compound of the invention.
[137] Non-limiting examples of antimicrobial agents and classes thereof that can be coadministered with the compounds of the present invention include penicillin and related drugs, carbapenem, cephalosporins and related drugs, aminoglycosides, baccitracin, gramicidine, Mupirosine, chloramphenicol, thiamphenicol, pushdate sodium, lincomycin, clindamycin, macrolide, novobiocin, polymyxins, rifamycins, spectinomycin, tetracyclines, vancomycin, teicoplanin, Streptograms, antifolates such as sulfonamides, trimetaprim and combinations thereof and pyrimethamines, synthetic antimicrobial agents such as nitrofurans, metheneamine mandelate and metheneamine hypofurlate, nitroimidazoles, quinolones, Fluoroquinolones, isoniazid, ethambutol, pyrazineamide, paraaminosalicylic acid (PAS), cycloserine, capreomycin, ethionamide, pro On amide, thiazol theta zone, non-Oh, my God, Nino Avenue azithromycin, glycopeptides, glycyl cyclin, ketol lead flow, oxazolyl cooking dinon; Imifenen, amikacin, netylmycin, phosphomycin, gentamicin, ceftriaxone, ziracin, LY 333328, CL 331002, HMR 3647, linezolid, cinersid, attreonam and metronidazole, epiloprim , OCA-983, GV-143253, San Petrinem Sodium, CS-834, Viapenem, A-99058.1, A-165600, A-179796, KA 159, Dynemycin A, DX8739, DU 6681; Cefluprenam, ER 35786, cephalis, sanpetrinem selecetyl, HGP-31, cepypyrom, HMR-3647, RU-59863, mersacidine, KP 736, ripalazil; Kosan, AM 1732, MEN 10700, Renafenem, BO 2502A, NE-1530, PR 39, K130, OPC 20000, OPC 2045, Beneprim, PD 138312, PD 140248, CP 111905, Sulofenem, Liffeenam Acornsil, RO -65-5788, cyclothialidine, Sch-40832, SEP-132613, mycacosidin A, SB-275833, SR-15402, SUN A0026, TOC 39, carumone, zozofran, cepetetamet cladding and T 3811 and the like.
[138] In a preferred embodiment, non-limiting examples of antimicrobials that can be coadministered with the compounds of the invention include imifenen, amikacin, netylmycin, phosphomycin, gentamicin, ceftriaxone, teicoplanin, zira Shin, LY 333328, CL 331002, HMR 3647, linezolid, cinerside, aztreonam and metronidazole.
[139] Non-limiting examples of antifungal agents that can be coadministered with the compounds of the present invention include caspofunsen, boriconazole, sertaconazole, IB-367, FK-463, LY-303366, Sch-56592, citafloxacin, DB-289 polyenes such as amphotericin, nistatin, primaricin; Azoles such as fluconazole, itraconazole and ketoconazole; Allylamines such as naphthypine and terbinafine; And anti-metabolic agents such as flucitocin and the like. Non-limiting examples of other antifungal agents are disclosed in Fastel et al., Drug Discovery Today 5: 25-32 (2000), incorporated herein by reference. Fostel et al. Disclose compounds such as corynecandine, Mer-WF3010, fusacandines, artritintine / LL 15G256, sorbdarins, cispentasin, azoxyvacillin, aureobacidine and caprefungin.
[140] The compounds of the invention, or any one or more pharmaceutical compositions of crystalline or crystalline like lipopeptides, may be administered according to the methods of the invention until bacterial infection is eradicated or reduced. In one embodiment, the crystalline or crystalline like lipopeptides are administered for about 3 days to about 6 months. In a preferred embodiment, the crystalline or crystalline like lipopeptides are administered for 7 to 56 days. In a more preferred embodiment, the crystalline or crystalline like lipopeptides are administered for 7 to 28 days. In even more preferred embodiments, the crystalline or crystalline like lipopeptides are administered for 7-14 days. Crystalline or crystalline like lipopeptides can be administered longer or shorter than this as necessary. In a preferred embodiment, the lipopeptides are daptomycin or daptomycin related lipopeptides.
[141] In order to more fully understand the present invention, the following examples are disclosed. These examples are disclosed for illustrative purposes only and should not be construed as limiting the scope of the invention in any way.
[11] Summary of the Invention
[12] The present invention solves the above problems by providing crystalline and crystalline like forms of lipopeptides, specifically daptomycin and daptomycin related lipopeptides and methods for their preparation. In one embodiment, the present invention provides a method for lipopeptide crystallization. In yet another embodiment, the method provides a more pure lipopeptides thereafter than before crystallization or precipitation.
[13] The present invention also provides a robust method for preparing and purifying peptides, particularly comprising crystallization or precipitation of lipopeptides. In one embodiment, the lipopeptides are purified using an in-process crystallization or precipitation step. In another embodiment, the process of the present invention is used for large scale production and / or industrial production of lipopeptides, preferably daptomycin.
[14] In addition, the present invention provides highly purified crystalline or crystalline like forms of daptomycin and daptomycin related lipopeptides. In one embodiment, crystalline or crystalline like forms of lipopeptides can be used in pharmaceutical compositions. In another embodiment, the present invention includes a method of using the pharmaceutical composition.
[142] Example 1
[143] Daptomycin was prepared by conventional techniques. The daptomycin formulation was a pale yellow amorphous powder with a solubility in water of at least 1 g / ml and a solubility in ethanol at 2.8 mg / ml at 25 ° C. The amorphous daptomycin formulation is hygroscopic and degraded at 215 ° C.
[144] The remaining examples disclose crystallization or precipitation of lipopeptides with or without organic precipitants (eg PEG).
[145] Example 2
[146] In microbatch crystallization, 25 μl of daptomycin stock (20 mg / ml in methanol) was added to 15 μl of reagent stock (200 mM calcium acetate, 0.1 M cacodylate (pH 6.5), 18% [w / v] PEG). 8000 and 15 μl ethylene glycol) to obtain a solution of 27.5% aqueous component, 45% methanol and 27.5% ethylene glycol. As measured by HPLC, sea urchin crystals of 98% purity were formed in 50% yield.
[147] Example 3
[148] The daptomycin stock solution was prepared by dissolving 440 mg of daptomycin in 1 ml of a buffer containing 25 mM sodium acetate (pH 5.0) and 5 mM CaCl ₂ . Crystallization was carried out by the vapor diffusion method, in which 5 µl of daptomycin stock solution was added to 0.5 µl of 0.1 M trisodium citrate dihydrate (pH 5.6) and 35 µl [v / v] t-butanol in water. To form. The drop was suspended over the reservoir solution (0.1 M sodium trisodium citrate dihydrate (pH 5.6) in water and 35% [v / v] t-butanol) under gas tight conditions until crystallization occurred. In this way, sea urchin type daptomycin crystals were obtained. See FIG. 2.
[149] Example 4
[150] 5 μl of daptomycin stock solution prepared as in Example 3 was added to 5 μl of a solution containing 0.1 M sodium cacodylate (pH 6.5), 0.2 M calcium acetate and 9% [w / v] PEG 8000. Crystallized by the vapor diffusion method as disclosed in Example 3. In this way, needle-like daptomycin crystals were produced. See eg FIG. 3.
[151] Example 5
[152] 5 μl of daptomycin stock solution prepared as in Example 3 was added to 5 μl of 0.1 μl benzamidine solution containing 0.1 M sodium cacodylate (pH 6.5), 0.2 M zinc acetate and 9% [w / v] PEG 8000. Was added to obtain daptomycin at a final concentration of 220 mg / ml. Crystallized by the vapor diffusion method described in Example 3. In this way, rod-like daptomycin crystals were produced. See eg FIG. 4.
[153] Example 6
[154] 1 ml of daptomycin (purity 97.1% as determined by HPLC) in water at a concentration of 20-25 mg / ml in water, 231 μl of water, 77 μl of calcium acetate (pH 6.0), 960 μl of propylene glycol and 50% [w / v] PEG 4000 231 μl were mixed sequentially. The solution was left at 4 ° C. for 4-5 hours. Sea urchin crystals were formed in 75% yield. Crystalline daptomycin was washed with isopropanol. Daptomycin had a purity of 98.4% as determined by HPLC.
[155] Example 7
[156] Daptomycin (200 mg, 97.1% purity) was dissolved in 2.54 mL of water. The daptomycin solution was mixed sequentially with 10.0 mL methanol, 0.78 mL 1M calcium acetate (pH 6.0), 9.50 mL propylene glycol and 2.20 mL 50% [w / v] PEG4000 to give a final volume of 25.02 mL. The mixture was spun in a hematology mixer (Fisher) for 10-14 hours at room temperature. Within minutes the crystals started to appear. The final yield was about 70-80% after 14 hours. The crystals were recovered by centrifugation at 1000 rpm for 15 minutes. The supernatant was removed and the crystals resuspended in 12.5 ml of isopropanol. The daptomycin suspension was transferred to a column (Biorad) and the isopropanol was removed in a drop by gravity. The crystals were dried under nitrogen. If there was a lump, it was broken during the drying process to obtain a uniform anhydrous sample. Crystals prepared in this manner were sea urchin shaped, with a purity of 98.37%.
[157] Example 8
[158] Daptomycin was crystallized according to Example 7 except that PEG 8000 was used instead of PEG 4000. The amount of reagent used is the same as in Example 7. Crystals prepared in this manner were sea urchin shaped and had a purity of 98.84%.
[159] Example 9
[160] Two daptomycin samples were prepared according to Example 7, and one amorphous sample was analyzed for crystallinity using USP <695> crystalline test. The daptomycin particles were placed in mineral oil on a glass slide and examined by polarization microscopy (PLM). The particles were determined to be crystalline if the particles were birefringent (having interference color) and had an absorbing position when the stage was rotating.
[161] Amorphous daptomycin samples consist of flaky particles of lace that are not birefringent. There were some silver-like regions in some flakes with weak birefringence, but the particles were mostly amorphous. In contrast, the daptomycin sample prepared according to Example 7 consisted of polycrystalline particles exhibiting weak birefringence and some absorption, indicating that the sample is predominantly crystalline. See FIG. 5.
[162] Example 10
[163] Two daptomycin samples were prepared according to Example 7, and one amorphous sample was analyzed for crystallinity by x-ray powder diffraction. Samples were analyzed on a Siemens D500 Automated Powder Diffractometer (ORS 1D No. LD-301-4), which used the ORS standard operating procedure EQ-27 Rev. Operated according to 9 The diffractometer was equipped with a graft monochromator and a Cu (λ = 1.54 mW) x-ray source operating at 50 kV, 40 mA. 2θ test is performed using NBS mica standard (SRM675). Samples were analyzed using the following device parameters:
[164] Measurement range for 2θ (°) 4.0-40.0
[165] Step Width (°) 0.05
[166] Measurement time per step (s) 1.2
[167] Beam Slits 1 (1 °), 2 (1 °), 3 (1 °), 4 (0.15 °), 5 (0.15 °).
[168] ORS Standard Operating Procedures Using a Zero Background Sample Plate MIC-7 Rev. Sample preparation was carried out according to 1.
[169] All samples were conducted using a Cu (λ = 1.54 kV) x-ray source. The amorphous daptomycin sample did not show any peaks by x-ray powder diffraction. See FIG. 6. In contrast, both daptomycin samples showed peaks by x-ray powder diffraction. The diffraction angles 2θ (FIG. 7) of the first daptomycin sample were 19.225, 23.242, 23.427, and 23.603 (°). The diffraction angles 2θ (FIG. 8) for the second daptomycin sample were 10.966, 19.205, and 23.344 (°). The first crystalline daptomycin sample also showed a small peak between 10 and 11 degrees. See FIG. 7.
[170] Example 11
[171] Daptomycin was dissolved in water. Sodium acetate was added to bring the final concentration to 187 mM. Calcium chloride was added to bring the final concentration to 28 mM. The daptomycin solution was mixed and isopropanol was added at a final concentration of 78.4%. The solution was mixed and incubated. Precipitated material formed after incubation. The precipitated material appeared to be sea urchin crystals with a diameter of about 60 μm as measured by an optical microscope. This material was then dried. The dry material contained about 30-40% salt. After drying, powder x-ray diffraction was performed. Powder x-ray diffraction did not confirm the presence of crystals in the dried daptomycin precipitate.
[172] Example 12
[173] 1 g of daptomycin (purity of about 91.5% as determined by HPLC) was added to 16.8 mL of distilled water and dissolved. 2.5 ml of 1 M calcium acetate (pH 6.1) and 60 ml of isopropanol were added. The solution was placed in a 27 ° C. water bath and equilibrated to bath temperature. A 5 ml aliquot of isopropanol was added slowly until the solution became cloudy (the total amount of isopropanol was about 30 ml). The solution was incubated overnight at 27 ° C. to form a precipitate. The precipitate was found to contain sea urchin crystals of about 60 μm when observed under an optical microscope. See FIG. 2.
[174] The daptomycin precipitate was poured into a pressure filter / drying funnel and filtered by gravity. The precipitate was washed twice with 25 ml of washing solution each time (80% isopropanol and 20% solution A, where solution A consisted of 18 ml of water and 2 ml of glacial acetic acid) and the liquid was dropped by gravity overnight. The precipitate was then transferred to a desiccator and dried under vacuum. After drying, powder x-ray diffraction was performed. Powder x-ray diffraction did not confirm the presence of crystals in the dried daptomycin precipitate. However, purity analysis of the precipitate material by HPLC revealed that the material was daptomycin with a purity of 98.2%. It is important that the daptomycin preparations after precipitation are significantly less anhydrous-daptomycin than the daptomycin preparations before precipitation.
[175] Without being bound by any theory, Applicants note that the conditions used to precipitate daptomycin in Examples 11 and 12 actually yield the crystalline form of daptomycin, but the subsequent washing and / or drying steps yield crystalline daptomycin. It is thought to restore to amorphous form. Nevertheless, amorphous daptomycin is still in crystal-like form as shown in FIG. 3 by birefringence of the crystal sample during polarization.
[176] Example 13
[177] s. Fermentation cultures of Roseosporus NRRL strain 15998 were subjected to controlled decanoic acid feed fermentation to optimize antibiotic production while minimizing the generation of contaminants. Residual decanoic acid feed is measured by gas chromatography and the target residual concentration is 10 ppm decanoic acid from induction induction (about time 30) to recovery. Centrifuge the cultures and analyze the broth clarified by HPLC to determine the production of daptomycin. The recovery titer is usually 1.0 to 3.0 g per liter of fermentation broth.
[178] Fermentation cultures are recovered by microfiltration using Pall-Sep or equivalent microfiltration, or by fully industrial scale centrifugation and turbidity filtration. The clarified broth is added to the anion exchange resin Mitsubishi FP-DA 13, washed with 30 mM NaCl at pH 6.5 and eluted with 300 mM NaCl at pH 6.0-6.5. Alternatively, the FP-DA 13 column is washed with 30 mM NaCl at pH 6.5 and eluted with 300 mM NaCl at pH 6.0-6.5. pH is adjusted to 3.0-4.8, and temperature is adjusted to 2-15 degreeC. Under these conditions, daptomycin forms micelles. The micelle daptomycin solution is filtered washed using a 10,000 NMW ultrafiltration (AG Technology Corp. UF hollow fiber or equivalent) of any shape. Daptomycin micelles remain in the filter, but most impurities are removed because they pass through a 10,000 NMW filter. Ultrafiltration of daptomycin micelles increases the purity of daptomycin to about 80-90%.
[179] The daptomycin formulation is then crystallized or precipitated under sterile conditions using one of the methods described above. In a preferred embodiment, daptomycin is crystallized or precipitated according to the protocols disclosed in Examples 7, 8 or 12, except for increasing the scale for large scale production of daptomycin. Filtration, preferably vacuum filtration, separates the crystalline or crystal-like daptomycin from the crystallization / precipitation solution. Crystalline or crystal-like daptomycin is washed with the wash solution (see Example 3). The crystalline or crystalline like daptomycin is vacuum dried using a 0.65 m ³ Klein Hastelloy-B bubble cone vacuum dryer or equivalent apparatus. The bottle is then filled with 250 mg or 500 mg of dried crystalline daptomycin per bottle. 9 shows a process diagram of the manufacturing method.
[180] Example 14
[181] s. Fermentation of Roseosporus, microfiltration of fermentation cultures and anion exchange chromatography are performed as described in Example 13. At this point the purity of the daptomycin formulation is about 35-40%. After anion exchange chromatography, daptomycin is crystallized or precipitated according to the protocol described in Example 13. Daptomycin is then washed and dried according to the protocol described in Example 13. Dried crystalline or crystal-like daptomycin is used to fill a sterile bottle as disclosed in Example 13. 6 shows a process diagram of the manufacturing method.
[182] Example 15
[183] s. Fermentation of Roseosporus and microfiltration of fermentation cultures are carried out as described in Example 13. After microfiltration, the fermentation cultures are treated with size exclusion ultrafiltration as described in Example 13. At this point the purity of the daptomycin formulation is about 35-40%. After ultrafiltration, daptomycin is crystallized or precipitated according to the protocol described in Example 13. Daptomycin is then washed and dried according to the protocol described in Example 13. Dried crystalline or crystalline like daptomycin is used to fill a sterile bottle as described in Example 13. 7 shows a process diagram of the manufacturing method.
[184] Example 16
[185] s. Fermentation of Roseosporus and microfiltration of fermentation cultures are carried out as described in Example 13. At this point the purity of the daptomycin formulation is 5-10%. After microfiltration, the fermentation cultures are crystallized or precipitated according to the protocol described in Example 13. Daptomycin was washed and dried as described in Example 13 and then used to fill a sterile bottle. 8 shows a process diagram of the manufacturing method.
[186] Example 17
[187] CB-131547 (see FIG. 16), a cyclic lipopeptide analog of daptomycin, was prepared from daptomycin via a semisynthetic route. CB-131547 is a pale yellow amorphous powder, having a solubility in general brine at -80 mg / ml at 25 ° C.
[188] CB-131547 (60 mg, ˜90% purity) was dissolved in 2.5 mL of water. The CB-131547 solution was mixed sequentially with 5.0 ml methanol, 0.2 ml 1 M calcium acetate (pH 6.0), 2.5 ml propylene glycol, and 1.0 ml 50% (w / v) PEG4000 to give a final volume of 11.2 ml. This solution was left at 4 ° C. for 4 to 24 hours. CB131547 crystals having a purity of -98.0% as measured by HPLC were obtained in -70% yield.
[189] Example 18
[190] CB-131547 (see FIG. 16), a cyclic lipopeptide analog of daptomycin, was prepared from daptomycin via a semisynthetic route. CB-131547 is a pale yellow amorphous powder, having a solubility in general brine at -80 mg / ml at 25 ° C.
[191] CB-131547 (60 mg, ˜90% purity) was dissolved in 2.5 mL of water. 0.2 ml 1 M calcium acetate (pH 6.0) and 8 ml isopropanol are added. Equilibrate the solution for 5 minutes at room temperature (25 ° C). A 1 ml aliquot of isopropanol is slowly added until the solution becomes cloudy. The solution is allowed to stand overnight at room temperature to form crystals.
[192] All publications and patent applications cited herein are hereby incorporated into the scope in which the publication or patent application is specifically and individually indicated by reference. While the invention has been described in some detail by way of example and illustration for purposes of clarity of understanding, some modifications and variations will be apparent to those skilled in the art without departing from the spirit or scope of the appended claims from the teachings of the invention.

权利要求:
Claims (56)
[1" claim-type="Currently amended] A crystalline or crystalline like lipopeptide or salt thereof, wherein the lipopeptide is selected from the group consisting of daptomycin, A54145 and daptomycin related lipopeptides.
[2" claim-type="Currently amended] The crystalline or crystalline like lipopeptides or salts thereof according to claim 1, wherein the salt is a divalent calcium salt.
[3" claim-type="Currently amended] The crystalline or crystalline like lipopeptides or salts thereof according to claim 1, wherein the lipopeptide antibiotic is daptomycin.
[4" claim-type="Currently amended] 4. The crystalline or crystalline like lipopeptides or salts thereof according to claim 3, wherein the salt is a divalent calcium salt.
[5" claim-type="Currently amended] 4. The x-ray diffraction pattern of crystalline daptomycin using Cu (λ = 1.54 kPa) x-ray source is characterized by diffraction angles (2θ) = 10.9, 19.2 and 23.3 (°) or diffraction angle (2θ) = 19.2, 23.2, 23.4 and 23.6 (°), wherein the crystalline or crystalline like lipopeptides or salts thereof.
[6" claim-type="Currently amended] The method according to any one of claims 1 to 4, wherein crystal-like means that the compound retains crystalline properties by birefringence but does not exhibit crystalline properties by x-ray powder diffraction. Crystalline or crystalline like lipopeptides or salts thereof.
[7" claim-type="Currently amended] 4. The crystalline or crystalline like lipopeptide or salt thereof of claim 3, wherein the crystalline or crystalline like daptomycin comprises sea urchin- or needle-like clusters, acicular or rod-like crystals.
[8" claim-type="Currently amended] 4. The crystalline or crystalline like lipopeptide or salt thereof of claim 3, wherein the crystalline daptomycin is at least 95% pure.
[9" claim-type="Currently amended] 4. The crystalline or crystalline like lipopeptide or salt thereof of claim 3, wherein the crystalline daptomycin or salt thereof is at least 97% pure.
[10" claim-type="Currently amended] 4. The crystalline or crystalline like lipopeptide or salt thereof of claim 3, wherein the crystalline daptomycin or salt thereof does not comprise at least 1% of a single impurity.
[11" claim-type="Currently amended] The crystalline or crystalline like lipopeptides or salts thereof according to any one of claims 8 to 10, wherein the purity is measured by HPLC.
[12" claim-type="Currently amended] The crystalline or crystal-like lipopeptides or salts thereof according to claim 1, wherein the lipopeptide crystals are at least 5 μm.
[13" claim-type="Currently amended] The crystalline or crystal-like lipopeptides or salts thereof according to claim 12, wherein the crystals are at least 50 μm.
[14" claim-type="Currently amended] The crystalline or crystalline like lipopeptides or salts thereof according to claim 12 or 13, wherein the lipopeptides are daptomycin.
[15" claim-type="Currently amended] The crystalline or crystalline like lipopeptide or salt thereof of claim 1, wherein the stability of the crystalline lipopeptides is higher than that of the lipopeptide in amorphous form.
[16" claim-type="Currently amended] 16. The crystalline or crystalline like lipopeptides or salts thereof according to claim 15, wherein the stability to heat, light, degradation or humidity of the crystalline lipopeptides is higher than in the amorphous form.
[17" claim-type="Currently amended] The crystalline or crystalline like lipopeptides or salts thereof of claim 16, wherein the stability is measured by antibiotic activity or degradation of the lipopeptide antibiotic.
[18" claim-type="Currently amended] The crystalline or crystalline like lipopeptides or salts thereof according to claim 15, wherein the lipopeptides are daptomycin.
[19" claim-type="Currently amended] 19. The crystalline or crystalline like lipopeptide or salt thereof according to claim 18, wherein the crystalline lipopeptides have a lower conversion of anhydro-daptomycin or daptomycin to the β-isomer than the amorphous form of daptomycin.
[20" claim-type="Currently amended] The crystalline or crystalline like lipopeptides or salts thereof according to claim 1, which are daptomycin related lipopeptides.
[21" claim-type="Currently amended] A pharmaceutical composition comprising a crystalline or crystalline like lipopeptide antibiotic and a pharmaceutically acceptable carrier, wherein the lipopeptide antibiotic is selected from the group consisting of daptomycin, A54145 and daptomycin related lipopeptides.
[22" claim-type="Currently amended] The pharmaceutical composition of claim 21, wherein the crystalline or crystalline like lipopeptides are daptomycin.
[23" claim-type="Currently amended] The pharmaceutical composition of claim 22, wherein the crystalline or crystalline like daptomycin is enteric coated for oral administration.
[24" claim-type="Currently amended] The pharmaceutical composition of claim 22, wherein the crystalline or crystalline like daptomycin is formulated at a dose of 3 to 75 mg / kg.
[25" claim-type="Currently amended] The pharmaceutical composition of claim 22, wherein the carrier enhances oral availability of daptomycin.
[26" claim-type="Currently amended] The pharmaceutical composition of claim 22, which is in the form of micron sized particles or microspheres.
[27" claim-type="Currently amended] A container comprising the pharmaceutical composition of claim 21.
[28" claim-type="Currently amended] The pharmaceutical composition of claim 26, which is used as an aerosol.
[29" claim-type="Currently amended] A combination comprising a crystalline or crystalline like lipopeptide antibiotic and a pharmaceutically acceptable carrier, wherein the lipopeptide antibiotic is selected from the group consisting of daptomycin, A54145 and daptomycin related lipopeptides.
[30" claim-type="Currently amended] The formulation of claim 29, which is a pharmaceutical formulation, a food formulation, a feed formulation, a veterinary formulation, a cosmetic formulation or a personal care formulation.
[31" claim-type="Currently amended] 30. The combination according to claim 29, which is a pharmaceutical combination further comprising another antibiotic, stabilizer, absorption aid, pH buffer or inorganic salt.
[32" claim-type="Currently amended] 30. The combination according to claim 29, which is a feed formulation which further comprises an animal feed and may optionally comprise another antibiotic or vitamin.
[33" claim-type="Currently amended] The formulation of claim 29, which is a personal care formulation that is a cleaning formulation, soap, shampoo, or a limiting agent.
[34" claim-type="Currently amended] 30. The combination according to claim 29, which is a veterinary combination which is a soap, shampoo or pharmaceutical composition.
[35" claim-type="Currently amended] A method of administering a crystalline or crystalline like lipopeptide, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, comprising administering to a patient in need thereof a therapeutically effective amount of a crystalline or crystalline like lipopeptiide, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition. Wherein the lipopeptide is selected from the group consisting of daptomycin, A54145 and daptomycin related lipopeptides.
[36" claim-type="Currently amended] The method of claim 35, wherein the lipopeptide antibiotic is at least 95% pure.
[37" claim-type="Currently amended] The method of claim 36, wherein the lipopeptide antibiotic is daptomycin.
[38" claim-type="Currently amended] The x-ray diffraction pattern of crystalline daptomycin using Cu (λ = 1.54 μs) x-ray source wherein the daptomycin is crystalline daptomycin and the diffraction angle (2θ) = 10.9, 19.2 and 23.3 (°). How to be.
[39" claim-type="Currently amended] The method of claim 37, wherein the daptomycin is crystal-like daptomycin and the crystal-like daptomycin exhibits crystalline properties by birefringence but not crystalline properties by x-ray powder diffraction.
[40" claim-type="Currently amended] 36. The method of claim 35, wherein the crystalline or crystalline like lipopeptides are administered as micron sized particles.
[41" claim-type="Currently amended] 36. The method of claim 35, wherein the crystalline or crystalline like lipopeptides are administered as a targeted release form.
[42" claim-type="Currently amended] 42. The method of claim 40 or 41, wherein the lipopeptides are daptomycin.
[43" claim-type="Currently amended] 36. The method of claim 35, wherein oral administration is effected subcutaneously, intravenously or intramuscularly.
[44" claim-type="Currently amended] a) providing a solution in which the lipopeptides are dissolved;
b) crystallizing or precipitation of the lipopeptides;
c) recovering and drying the lipopeptides; And
d) A lipopeptide storage method comprising the step of storing lipopeptides, wherein the lipopeptides are selected from the group consisting of daptomycin, A54145 and daptomycin related lipopeptides, wherein the crystalline or crystalline like lipopeptides are present in an amorphous form of the lipopeptides. More stable.
[45" claim-type="Currently amended] a) providing an amorphous form of the lipopeptide;
b) crystallizing or precipitation of the lipopeptides; And
c) A method for preparing a crystalline or crystalline like lipopeptide comprising recovering a crystalline or crystalline like lipopeptides, wherein the lipopeptides are selected from the group consisting of daptomycin, A54145 and daptomycin related lipopeptides.
[46" claim-type="Currently amended] 45. The method of claim 44, wherein said recovering step is performed by filtration.
[47" claim-type="Currently amended] The method of claim 46, further comprising step b) followed by washing of the lipopeptides.
[48" claim-type="Currently amended] 47. The method of claim 45 or 46, further comprising step c) followed by drying of the lipopeptides.
[49" claim-type="Currently amended] 49. The method of claim 48, further comprising sterilizing the lipopeptid after drying.
[50" claim-type="Currently amended] 46. The method of claim 45, wherein step c) is performed under sterile conditions.
[51" claim-type="Currently amended] 51. The method of claim 50, wherein step b) is performed under sterile conditions.
[52" claim-type="Currently amended] The method of claim 51, further comprising step c) followed by drying of the lipopeptides under sterile conditions.
[53" claim-type="Currently amended] 45. The method of claim 44, wherein the purity of the crystalline lipopeptides is higher than the lipopeptides in amorphous form.
[54" claim-type="Currently amended] The method of claim 53, wherein the purity of the amorphous form is about 90% and the purity of the crystalline or crystalline like form is at least 95%.
[55" claim-type="Currently amended] The method of claim 53, wherein the purity of the amorphous form is about 93% and the purity of the crystalline or crystalline like form is at least 95%.
[56" claim-type="Currently amended] The method of claim 53, wherein the purity of the amorphous form is about 93% and the purity of the crystalline or crystalline like form is about 98%.

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EP1908770A1|2008-04-09|

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HK1108582A1|2012-10-19|

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NZ571597A|2010-05-28|

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KR20110127732A|2011-11-25|

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KR20080036661A|2008-04-28|

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AT535539T|2011-12-15|

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ES2377931T5|2015-11-04|

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AU2002246687B2|2008-05-29|

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US20030045678A1|2003-03-06|

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WO2002056829A3|2003-03-27|

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引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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法律状态:
2000-12-18|Priority to US25626800P

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2000-12-18|Priority to US60/256,268

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2001-03-09|Priority to US27474101P

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2001-03-09|Priority to US60/274,741

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2001-12-13|Priority to US34052501P

---

2001-12-13|Priority to US34131501P

---

2001-12-13|Priority to US60/340,525

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2001-12-13|Priority to US60/341,315

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2001-12-17|Application filed by 큐비스트 파마슈티컬즈 인코포레이티드

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2001-12-17|Priority to PCT/US2001/048886

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2003-10-17|Publication of KR20030081353A

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2009-09-01|First worldwide family litigation filed

优先权:

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申请号 | 申请日 | 专利标题

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US25626800P| true| 2000-12-18|2000-12-18|

---

US60/256,268|2000-12-18|

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US27474101P| true| 2001-03-09|2001-03-09|

---

US60/274,741|2001-03-09|

---

US34131501P| true| 2001-12-13|2001-12-13|

---

US34052501P| true| 2001-12-13|2001-12-13|

---

US60/340,525|2001-12-13|

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US60/341,315|2001-12-13|

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PCT/US2001/048886|WO2002059145A1|2000-12-18|2001-12-17|Methods for preparing purified lipopeptides|