US008129342B2
`
`(12) United States Patent
`Kelleher et a].
`
`(16) Patent No.:
`(45) Date of Patent:
`
`US 8,129,342 B2
`*Mar. 6, 2012
`
`(54) HIGH PURITY LIPOPEPTIDES
`
`(75) Inventors: Thomas J. Kelleher, Thousand Oaks,
`CA (Us); JaII-Ji Lai, Westborough, MA
`(US); Joseph P. DeCourcey, Boston,
`
`5,912,226 A
`5,955,509 A
`
`6,696,412 B1
`6,852,689 B2
`
`6/1999 Baker
`9/1999 Webber
`3%? et 31
`2/2004 Kelleher
`2/2005 Oleson, Jr. et al.
`
`
`
`
`
`4/2006 Baker et 31' RE39’071 E : 8,058,238 B2 11/2011 Kelleher et al. ............. .. 435/886
`
`EP
`EP
`
`EP
`W0
`W0
`W0
`W0
`W0
`W0
`W0
`W0
`W0
`
`FOREIGN PATENT DOCUMENTS
`095295 A1
`11/1983
`178152 A2
`4/1986
`
`386951 A2
`WO 99/27954
`WO 99/27957
`W0 99/40ll3
`W0 99 43700
`WO 00/18419
`W0 00/81419
`WO 01/44271
`WO 01/44272
`WO 01/44274
`
`9/1990
`6/1999
`6/1999
`8/1999
`9 1999
`4/2000
`4/2000
`6/2001
`6/2001
`6/2001
`
`OTHER PUBLICATIONS
`
`g
`y
`A reement between Cubist Pharmaceuticals, Inc. and Eli Lill and
`Company dated NOV. 7, 1997 (redacted form from SEC Edgar).
`Agreement between Cubist Pharmaceuticals, Inc. and Eli Lilly and
`Company dated Oct. 6, 2000 (redacted form from SEC Edgar).
`Assignment of US RE 39,071 from Eli Lilly and Company to Cubist
`Pharmaceuticals, Inc. recorded on Apr. 23, 2007 Reel/Frame:
`019l81/0916‘
`Debono, M‘ et a1‘; “Enzymatic and Chemical Modi?cations of
`Lipopeptide Antibiotic A21978C: The Synthesis and Evaluation of
`Daptomycin (LY146032),” J. Antibiotics; 41; 1988; pp. 1093-1105.
`_
`(Continued)
`
`Primary Examiner * Chih-Min Kam
`(74) Attorney, Agent, or Firm *Cubist Pharmaceuticals,
`1110
`
`
`
`MA (US); Paul D. Lynch, Arlington, MA (Us); Maurizio Zenoni’ Ferentino
`
`Frosinone (IT); Auro R. Tagliani, Pavia
`(1T)
`
`(73) Assignee: Cubist Pharmaceuticals, Inc., Lexington MA (US)
`
`’
`
`( * ) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U'S'C' 154(1)) by 0 days‘
`This patent is subject to a terminal dis-
`Claimer
`'
`
`(22) F1led:
`
`Sep. 22, 2010
`
`(65)
`
`Prior Publication Data
`I
`I
`I
`Us 201 1/0207658 A1
`Aug' 25’ 2011
`
`Related U_s_ Application Data
`_
`_
`_
`_
`(63) cont1nuat1on of apphcanon NO‘ 11/739’180’ _?1ed_ On
`Apr.‘ 24, ‘2007, noW'Pat'. No. 8,058,238, Wh1ch 1s a
`cont1nuat1on of application No. 10/747,485, ?led on
`Dec: 293 2003, 110W‘ abandoned, Whlch 15 a
`cont1nuat1on 0f apphcatlon NO- 09/735,191, ?led 0n
`Nov. 28, 2000, noW Pat. No. 6,696,412.
`
`(60) Provisional application No. 60/177,170, ?led on Jan.
`20, 2000.
`
`(51) Int- Cl-
`(2006.01)
`C07K 7/50
`(2006.01)
`C07K 7/00
`(52) U.S. Cl. .................. .. 514/9; 514/11; 514/2; 514/14;
`530/317; 530/322; 530/344; 435/886
`(58) Field of Classi?cation Search .............. .. 514/9, 11,
`
`514/2’ 14; 530/317’ 322’ 344; 435/886
`See application ?le for complete search history.
`
`(56)
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`4,331,594 A
`5/1982 Hamill
`4,482,487 A 11/1984 Abbott
`4,524,135 A
`6/1985 Abbott
`4,537,717 A
`8/1985 Abbott
`RE32,310 E
`12/1986 Debono
`RE32,311 E
`12/1986 Debono
`RE32,333 E
`1/1987 Hamill
`RE32,455 E
`7/1987 Hamill
`4,800,157 A
`1/1989 Eaton
`4,874,843 A 10/1989 Baker
`4,882,164 A 11/1989 Ferro
`4,885,243 A 12/1989 Huber
`5,271,935 A 12/1993 Franco
`5,387,670 A
`2/1995 Roy
`5,573,936 A 11/1996 Kreuzman
`5,629,288 A
`5/1997 Lattrell
`
`(57)
`.
`
`.
`
`.
`
`ABSTRACT
`.
`.
`
`.
`
`The 1nvent1on d1scloses ~highly puri?ed daptomycin and to
`phanpaceullcal .composmons compnsmg lhls compounq'
`The invention d1scloses a method of purifying daptomyc1n
`comprising the sequential steps of anion exchange chroma
`tography, hydrophobic interaction chromatography and
`anion exchange chromatography. The invention also dis
`closes a method of purifying daptomycin by modi?ed buffer
`enhanced anion exchange chromatography. The invention
`also discloses an improved method for producing daptomycin
`by fermentation of Streptomyces roseosporus. The invention
`also discloses high pressure liquid chromatography methods
`for analysis of daptomycin purity. The invention also dis
`closes lipopeptide micelles and methods of making the
`micelles. The invention also discloses methods of using
`lipopeptide micelles for purifying lipopeptide antibiotics,
`such as daptomycin. The invention also discloses using
`lipopeptide micelles therapeutically.
`
`54 Claims, 11 Drawing Sheets
`
`
`1 of 34
`
`FRESENIUS-KABI, Exh. 1002
`
`

`
`US 8,129,342 B2
`Page 2
`
`OTHER PUBLICATIONS
`
`Desai, J. D., et al.; “Microbial Production of Surfactants and Their
`Commercial Potential,” Microbiology and Molecular Biology
`Review; vol. 61; No. 1; 1997; pp. 47-64; American Society for
`Microbiology.
`Fostel, Jennifer M., et al.; “Emerging Novel Antifungal Agents,”
`DDT; vol. 5; No. 1; Jan. 2000; pp. 25-32; Elsevier Science Ltd.
`Horowitz, Sarah, et al; “Isolation and Characterization of a Surfactant
`Produced by Bacillus licheniformis 86,” J. Industrial Microbiol.; 6;
`1990; pp. 243-248; Society for Industrial Microbiology.
`Kirsch, Lee E., et al.; “Kinetics of the Aspartyl Transporation of
`Daptomycin, a Novel Lipopeptide Antibiotic,” Pharmaceutical
`Research; vol. 6; No. 5; 1989; pp. 387-393; Plenum Publishing
`Corporation.
`Lasic, Dan D., et al.; “Novel Applications of Liposomes,” Trends
`Biotechnology; vol. 16; Jul. 1998; pp. 307-321; Elsevier Science Ltd.
`Lasic, Danilo D., et al.; “Mixed Micelles in Drug Delivery,” Nature;
`vol. 355; Issue No. 6357; Jan. 16, 1992; pp. 279-280.
`Lin, S. C., et al., “General Approach for the Development of High
`Performance Liquid Chromatography Methods for Biosurfactant
`Analysis and Puri?cation,” J. Chromatography; 825; 1998; pp. 149
`159.
`Lin, S. C. et al.; “Recovery and Puri?cation of the Lipopeptide
`Biosurfactant of Bacillus subtillis by Ultra?ltration,” Biotechology
`Techniques; vol. 11; No. 6; Jun. 1997; pp. 413-416; Chapman Hall.
`Miao, Vivian et al.; “Daptomycin Biosynthesis in Streptomyces
`roseosporus: Cloning and Analysis of the Gene Cluster and Revision
`of Peptide Stereochemistry,” Microbiology, (2005), 151, pp. 1507
`1523.
`Molloy, M. et al., Abstract,“Structure & Anhydro-Daptomycin and
`Iso-Daptomycin,” ACS 200th Meeting, 1990.
`Molloy, M. et al., Poster, “Structure & Anhydro-Daptomycin and
`Iso-Daptomycin,” ACS 200th Meeting, 1990.
`Mulligan, Catherine N., et al., “Recovery of Biosurfactants by
`Ultra?ltration,” J. Chem. Tech. Biotechnology; 47; 1990; pp. 23-29;
`Society of Chemical Industry; Printed in Great Britian.
`Schott, H.; “Colloidal Dispersions,” Remington: The Science and
`Practice of Pharmacy; vol. 1; 19th Edition; 1995; pp. 252-277; Mack
`Publishing Company; Easton, Pennsylvania USA.
`
`Shaw, Duncan J .; “Liquid-Gas and Liquid-Liquid Interfaces,” Intro
`duction to Colloid and Surface Chemistry; 1989; pp. 64-114; 4th
`Edition; Butterworth-Heinemann Ltd. Great Britian.
`Sterling, John; “Membrane-Based System Combines Selective Sepa
`ration with High-volume Throughput,” Genetic Engineering News;
`vol. 19; No. 20; Nov. 15, 1999; pp. 1, 34.
`Supersaxo, Andreas et al.; “Mixed Micelles as Proliposomal,
`Lymphotropic Drug Carrier,” Pharmaceutical Research; vol. 8; No.
`10; 1991; pp. 1286-1291; Plenum Publishing Corporation.
`Sweadner, Kathleen J. et al., “Filter Removal of Endotoxin
`(Pyrogens) in Solution in Different States of Aggregation,” Applied
`and Environmental Microbiology; vol. 34; No. 4; 1977; pp. 382-385;
`American Society for Microbiology; Printed in the USA.
`Tally, F.P., et al.; “Daptomycin: A Novel Agent for Gram Positive
`Infections,” Exp. Opinion Invest. Drugs; 8; 1999; 1223-1238.
`Thimon, L. et al., “Surface-Active Properties of Antifungal
`Lipopeptides Produced by Bacillus substillis,” J. Am. Oil Chem.
`Soc.; 69; 1992; pp. 92-93.
`Yakimov, Michail M. et al.; “Characterization of a New Lipopeptide
`Surfactant Produced by Thermotolerant and Halotolerant Subsurface
`Bacillus licheniformis BAS50,” Applied and Environmental
`Microbiology; vol. 61; No. 5; 1995; pp. 1706-1713; American Soci
`ety for Microbiology;.
`Bayer, A. et al.; LY146032 Compared with Pencillin G in Experi
`mental Aortic Valve Endocarditis Caused by Group G Streptococci;
`Antimicrobial Agents and Chemotherapy, Jan. 1988, p. 141-143.
`Eliopoulos, G. M., et al.; “In Vitro and In Vivo Activity ofLY 146032,
`a New Cyclic Lipopeptide Antibiotic,” Antimicrobial Agents and
`Chemotherapy, Oct. 1986, p. 532-535.
`Ramos, M. C., “Comparison of Daptomycin, Vancomycin, and
`Ampicillin-Gentamicin for Treatment of Experimental Endocarditis
`Caused by Penicillin-Resistant Enterococci,” Antimicrobial Agents
`and Chemotherapy; Sep. 1992, p. 1864-1869.
`US. Appl. No. 07/060,148, ?led Jun. 10, 1987, Baker et al.
`Cubicin® (daptomycin for injection) Label 1004*Sep. 2003.
`Cubicin® (daptomycin for injection) Label 1004-1iRevised Aug.
`2004.
`Cubicin® (daptomycin for injection) Label 1004-2iRevised Jun.
`2005.
`Cubicin® (daptomycin for injection) Label 1004-10-1 Aug. 2010.
`
`* cited by examiner
`
`
`2 of 34
`
`

`
`U.S. Patent
`
`Mar. 6, 2012
`
`Sheet 1 of 11
`
`US 8,129,342 B2
`
`0
`
`NH2
`
`COBH2
`N
`
`H
`
`O
`fi__L|_(CH2,8CH3
`H
`
`/
`
`CONH
`
`N
`
`H
`
`o 2 H o
`NJ‘-(CH2)3CH3
`
`/ NH
`
`H020H/NK(NH
`HOJEOO
`
`NH
`
`0
`
`I 2
`
`O
`
`O
`
`H
`
`H020
`
`HO2C¢O
`HN\n)\NJj/N
`
`0
`
`H
`
`NH
`
`o
`CO2H
`
`O
`O
`0 EN 3
`$=o
`HN
`
`H
`
`NH
`
`2
`
`O
`
`Fig. 1
`
`0
`
`NH2
`
`H020 / NH
`H”
`HO\/270 O
`O
`
`O
`
`O
`
`NH
`
`0
`
`NH
`
`0
`
`N
`
`H
`
`H
`N
`
`O
`CO2H
`
`30f34
`
`
`3 of 34
`
`

`
`U.S. Patent
`
`Mar. 6, 2012
`
`Sheet 2 of 11
`
`US 8,129,342 B2
`
`0
`
`NH2
`
`CONH
`O 2
`
`N
`
`H
`
`o
`
`H
`
`N——”—(CH2)8CH3
`
`/ N
`
`H
`
`0
`
`R
`
`O
`CO2H
`
`NH2
`
`Fig. 3
`
`H020 / NH
`
`HN
`
`O
`
`Ho
`
`0 O
`
`O
`
`NH
`
`O
`
`0
`
`N
`
`NH
`
`3
`
`o
`
`oflzo
`
`0
`
`HN
`
`N
`H
`0 H020
`
`HN
`
`H
`N
`
`O
`
`COSHQ
`N
`
`H
`
`O
`fi_L|_(CH2)8CH3
`
`/
`
`NH
`
`0
`
`NH2
`
`NH
`
`OH
`O HO
`O
`
`O
`
`N
`H
`
`NH
`
` 0
`
`HN
`
`O
`
`N
`H
`
`H
`N
`
`O
`
`R
`
`O
`CO2H
`
`NH2
`
`"020
`
`Fig. 4
`
`40f34
`
`HO2C
`
`O
`
`HN
`
`O
`
`HOZC
`
`HN
`
`O O
`
`I
`
`$4
`o\)Lz
`£5
`
`
`4 of 34
`
`

`
`US. Patent
`
`Mar. 6, 2012
`
`Sheet 3 or 11
`
`US 8,129,342 B2
`
`NH2
`
`O H
`
`OH
`0
`O H
`O
`
`n
`
`NH
`
`3:0
`
`HN
`
`CONH2
`0 Hi
`N
`(CH2)8CH3
`
`N
`
`H % N
`
`H
`
`N
`O
`co2H
`
`NH2
`
`NH
`
`NH
`
`H020
`HN
`Hojo O
`NH
`
`O
`
`O
`H G
`
`{TEN/yr
`
`NH O H
`
`HN w Lk
`
`0 H2
`
`
`5 of 34
`
`

`
`U.S. Patent
`
`Mar. 6, 2012
`
`Sheet 4 of 11
`
`US 8,129,342 B2
`
`H020
`
`NH
`
`HN
`
`O
`
`O
`
`CONH
`O 2
`
`O
`
`o
`
`H
`
`H
`
`N
`
`o
`
`N
`
`H
`
`H 0
`N-U—(CH2)8CH3
`
`/ NH
`
`CO2H
`
`60f34
`
`
`6 of 34
`
`

`
`U.S. Patent
`
`Mar. 6,2012
`
`Sheet 5 of 11
`
`US 8,129,342 B2
`
`0
`
`H
`H020
`
`0
`
`_
`Fig. 9
`
`H o
`N—”-—(CH2)8CH3
`
`/ N
`
`CONH2
`o
`
`N
`H
`
`H
`
`O
`CO2H
`
`o
`
`N
`H
`
`o
`
`H0
`O
`
`NH
`
`HN
`
`HOWNH2
`
`0
`
`.
`Fig. 10
`
`70f34
`
`
`7 of 34
`
`

`
`U.S. Patent
`
`Mar. 6,2012
`
`Sheet 6 of 11
`
`US 8,129,342 B2
`
`CONH
`0 2 H O
`NJ-'—(CH2)7CHCH3)CH3
`
`N
`
`H
`
`N
`H
`
`0
`
`NH2
`
`NH
`
`H020
`
`H”
`H0\‘£=o 0
`
`NH
`
`O
`
`O
`
`0
`
`0
`
`N
`
`H
`
`n
`
`0
`
`HN
`
`HO2C\_2:O
`HN
`
`0
`
`N
`H
`
`0
`
`H020
`
`0
`
`<::
`HN
`
`O
`
`H
`N
`
`NH2
`
`Fig. 11
`
`80f34
`
`
`8 of 34
`
`

`
`US. Patent
`
`Mar. 6, 2012
`
`Sheet 7 or 11
`
`US 8,129,342 B2
`
`010ml
`
`1.:
`
`% w 5 5 4 4 3 3 2 2 l }
`
`5 O 5 0 5 O 5 O 5 O 5 0
`
`1 :BF
`
`1 NF
`
`1 Q
`
`| S.
`
`6
`
`81012141618 20 22 24 26 28
`TlME(min)
`Fig. 12
`
`
`9 of 34
`
`

`
`US. Patent
`
`Mar. 6, 2012
`
`Sheet 8 or 11
`
`US 8,129,342 B2
`
`9 .mE
`
`mu
`
`0N
`
`vm
`
`mu
`
`ow
`
`2
`
`2 3
`
`
`
`E2: 52;
`
`QO'Sl-E
`
`00
`
`cm
`
`on
`
`on
`
`om
`
`on
`
`ON
`
`(AUJ) EISNOdSEIEI
`
`
`10 of 34
`
`

`
`US. Patent
`
`Mar. 6, 2012
`
`Sheet 9 or 11
`
`US 8,129,342 B2
`
`Fig. 14C
`
`
`11 of 34
`
`

`
`US. Patent
`
`Mar. 6, 2012
`
`Sheet 10 or 11
`
`US 8,129,342 B2
`
`
`
`EuG: .>.:>CUDOZOU
`
`MIN":
`
`I
`I
`I
`8000
`6000
`4000
`2000
`10000 12000
`0
`DAPTOMYCIN CONCENTRATION, ug/mL
`Fig. 15
`
`
`12 of 34
`
`

`
`US. Patent
`
`Mar. 6, 2012
`
`Sheet 11 0f 11
`
`US 8,129,342 B2
`
`INTENSITY
`SIZE DISTRIBUTIONS]
`
`fo
`
`5
`
`.==+
`5'0 100
`500
`DIAMETER (nm)
`
`VOLUME
`
`SIZE DISTRIBUTIONlS)
`
`I
`
`I
`
`560 '
`50 100
`DIAMETER (nm)
`
`Fig. 16
`
`
`13 of 34
`
`

`
`US 8,129,342 B2
`
`1
`HIGH PURITY LIPOPEPTIDES
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`
`The present application is a continuation of US. patent
`application No. 11/739,180, ?led Apr. 24, 2007, now US.
`Pat. No. 8,058,238, Which is a continuation of US. patent
`application Ser. No. 10/747,485, ?led Dec. 29, 2003, noW
`abandoned, Which is a continuation of US. patent application
`Ser. No. 09/735,191 ?led Nov. 28, 2000, now US. Pat. No.
`6,696,412, Which claims the bene?t of US. Provisional
`Application No. 60/177,170, ?led Jan. 20, 2000, all of Which
`are incorporated by reference herein in their entireties.
`
`TECHNICAL FIELD OF THE INVENTION
`
`The present invention relates to a highly puri?ed form of
`lipopeptides, including daptomycin, a lipopeptide antibiotic
`With potent bactericidal activity against gram-positive bacte
`ria, including strains that are resistant to conventional antibi
`otics. The present invention also relates to a process for pre
`paring the highly puri?ed form of the lipopeptide. The present
`invention further relates to micelles of lipopeptides. The
`present invention also relates to pharmaceutical compositions
`of the lipopeptide micelles and methods of using these com
`positions. The present invention also relates to methods of
`making lipopeptide micelles from non-associated monomers
`of the lipopeptides, and for converting lipopeptide micelles to
`non-associated monomers. The present invention also relates
`to a process for preparing lipopeptides using micelles that is
`easily scaled for commercial production.
`
`BACKGROUND OF THE INVENTION
`
`20
`
`25
`
`30
`
`35
`
`40
`
`The rapid increase in the incidence of gram-positive infec
`tionsiincluding those caused by antibiotic resistant bacte
`riaihas sparked reneWed interest in the development of
`novel classes of antibiotics. One such class is the lipopeptide
`antibiotics, Which includes daptomycin. Daptomycin has
`potent bactericidal activity in vitro against clinically relevant
`gram-positive bacteria that cause serious and life-threatening
`diseases. These bacteria include resistant pathogens, such as
`vancomycin-resistant enterococci (V RE), methicillin-resis
`tant Staphylococcus aureus (MRSA), glycopeptide interme
`diary susceptible Staphylococcus aureus (GISA), coagulase
`negative staphylococci (CNS), and penicillin-resistant
`Streptococcus pneumoniae (PRSP), for Which there are very
`feW therapeutic alternatives. See, e.g., Tally et al., 1999, Exp.
`Opin. Invest. Drugs 811223-1238, hereafter “Tally”. Dapto
`mycin’s inhibitory effect is a rapid, concentration-dependent
`bactericidal effect in vitro and in vivo, and a relatively pro
`longed concentration-dependent post-antibiotic effect in
`vivo.
`Daptomycin is described by BaltZ in Biotechnology of
`55
`Antibiotics, 2nd Ed., ed. W. R. Strohl (NeW York: Marcel
`Dekker, Inc.), 1997, pp. 415-435, hereafter “BaltZ.” Dapto
`mycin, also knoWn as LY 146032, is a cyclic lipopeptide
`antibiotic that can be derived from the fermentation of Strep
`tomyces roseosporus. Daptomycin is a member of the factor
`A-21978CO type antibiotics of S. roseosporus and is com
`prised of a decanoyl side chain linked to the N-terminal
`tryptophan of a cyclic 13-amino acid peptide (FIG. 1). Dap
`tomycin has an excellent pro?le of activity because it is
`highly effective against most gram-positive bacteria; it is
`highly bactericidal and fast-acting; it has a loW resistance rate
`and is effective against antibiotic-resistant organisms. The
`
`45
`
`50
`
`60
`
`65
`
`2
`compound is currently being developed in a variety of formu
`lations to treat serious infections caused by bacteria, includ
`ing, but not limited to, methicillin resistant Staphylococcus
`aureus (MRSA) and vancomycin resistant enterococci
`(V RE).
`A number of United States patents describe A-21978C
`antibiotics and derivatives thereof including daptomycin (LY
`146032) as Well as methods of producing and isolating the
`A-21978C antibiotics and derivatives thereof.
`US. Pat. Nos. Re. 32,333, Re. 32,455 and 4,800,157
`describe a method of synthesizing daptomycin by cultivating
`Streptomyces roseosporus NRL15998 under submerged
`aerobic fermentation conditions. US. Pat. No. 4,885,243
`describes an improved method of synthesiZing daptomycin
`by feeding a fermentation culture a decanoic fatty acid or
`ester or salt thereof.
`US. Pat. Nos. Re. 32,310, Re. 32,311, 4,537,717, 4,482,
`487 and 4,524,135 describe methods of deacylating the
`A-21978C antibiotic and reacylating the peptide nucleus and
`antibiotic derivatives made by this process. All of these pat
`ents describe a puri?ed deacylated A-21978C antibiotic
`nucleus or a derivative thereof Which Was isolated from the
`fermentation broth by ?ltration and then puri?ed by Diaion
`HP-20 chromatography and silica gel/C18 chromatography.
`US. Pat. Nos. Re. 32,333 and Re. 32,455 disclose a puri
`?cation method in Which a ?ltrate of Whole fermentation
`broth Was puri?ed through a number of precipitation and
`extraction steps to obtain a crude A-21978C complex. The
`crude complex Was further puri?ed by ion exchange chroma
`tography on IRA-68 and tWo rounds of silica gel chromatog
`raphy. Individual A-21978C factors Were separated by
`reverse-phase silica gel or silica gel/C18. US. Pat. Nos. Re.
`32,333 and Re. 32,455 also disclose that A-21978C may be
`puri?ed by batch chromatography using Diaion HP-20 resin
`folloWed by silica-gel column chromatography.
`US. Pat. No. 4,874,843 describes a daptomycin puri?ca
`tion method in Which the fermentation broth Was ?ltered and
`passed through a column containing HP-20 resin. After elu
`tion, the semipuri?ed daptomycin Was passed through a col
`umn containing HP-20ss, and then separated again on HP-20
`resin. The ’843 patent states that ?nal resolution and separa
`tion of daptomycin from structurally similar compounds by
`this method is impeded by the presence of impurities that are
`not identi?able by ultraviolet analysis of the fermentation
`broth. The ’843 patent further states that attempts to remove
`these impurities by reverse phase chromatography over silica
`gel, normal phase chromatography over silica gel or ion
`exchange chromatography also failed to signi?cantly
`improve the purity of daptomycin. The ’843 patent also dis
`closes a “reverse method” for puri?cation comprising the
`steps of contacting an aqueous solution of the fermentation
`product With a non-functional resin in aqueous phase, physi
`cally removing the Water from the charged resin, reWetting
`the charged resin With a polar organic solvent, Washing the
`resin With the organic solvent, eluting the fermentation prod
`uct from the resin by increasing the polarity of the solvent and
`recovering the fermentation product. The ’ 843 patent teaches
`that this method improves the ?nal purity from about 80% to
`about 93% and increases the yield from about 5% to about
`35%; hoWever, the ’843 patent does not disclose the type of
`impurities present in the daptomycin preparation.
`US. Pat. No. 5,912,226 describes the identi?cation and
`isolation of tWo impurities produced during the manufacture
`of daptomycin. Daptomycin, an ot-aspartyl peptide, becomes
`transpeptidated to form a stable intermediate in Which the
`aspartyl group becomes an anhydro-succinimido group (FIG.
`3). The ’226 patent teaches that the presence of this interme
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`US 8,129,342 B2
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`3
`diate, designated anhydro-daptomycin, is more pronounced
`at pH 4-6. Rehydration of the anhydro-succinimido form
`produces a second degradation product that contains an [3-as
`partyl group and is designated the [3-isomer form of dapto
`mycin (FIG. 2).
`The ’226 patent discloses that the t-BOC derivative of
`anhydro-daptomycin may be isolated by chromatography
`over reverse phase silica gel/C-l8 column, precipitated, and
`repuri?ed by reverse phase silica gel/C-l8 chromatography.
`The ’226 patent also teaches that the [3-isomer form of dap
`tomycin may be puri?ed by chromatography over a Diaion
`HP-20ss resin, desalted by chromatography over a Diaion
`HP-20 resin, and further puri?ed using a reverse-phase C-l8
`column folloWed by a HP-20 resin column in reverse mode.
`Kirsch et. al. (Pharmaceutical Research, 6:387-393, 1989,
`hereafter “Kirsch”) stated that anhydro-daptomycin and the
`[3-isomer Were produced in the puri?cation of daptomycin.
`Kirsch described methods to minimize the levels of anhydro
`daptomycin and the [3-isomer through manipulation of pH
`conditions and temperature conditions. HoWever, Kirsch Was
`unable to stabiliZe daptomycin and prevent the conversion of
`daptomycin to anhydro-daptomycin and its subsequent
`isomeriZation to [3-isomer. Kirsch Was also unable to prevent
`the degradation of daptomycin into other degradation prod
`ucts unrelated to anhydro-daptomycin and [3-isomer.
`The ’226 patent states that daptomycin may be prepared
`using these procedures so that the daptomycin contains no
`more than 2.5% by Weight of a combined total of anhydro
`daptomycin and [3-isomer, but gives no indication of the lev
`els of other impurities. In the method taught in US. Pat. No.
`4,874,843 and in large-scale preparations of daptomycin for
`clinical trials, the highest daptomycin purity levels observed
`has been about 90%-93%. There is a need for a commercially
`feasible method to produce more highly puri?ed daptomycin
`and, if possible, to increase its yield after puri?cation. Fur
`thermore, it Would be desirable to obtain puri?ed daptomycin
`that contains little or none of anhydro-daptomycin and the
`[3-isomer form of daptomycin. It Would also be desirable to
`reduce the levels of a number of other impurities in daptomy
`cin. HoWever, there has been no method available in the art
`that has been shoWn to be able to further reduce the levels of
`anhydro-daptomycin, [3-isomer form and other impurities in
`the daptomycin product.
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`SUMMARY OF THE INVENTION
`
`45
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`The instant invention addresses these problems by provid
`ing commercially feasible methods to produce high levels of
`puri?ed lipopeptides. In a preferred embodiment, the
`lipopeptide is daptomycin or a daptomycin-related lipopep
`tide. In one embodiment of the instant invention, commer
`cially feasible methods are disclosed that results in daptomy
`cin at a purity level of 95-97%. In another embodiment of the
`instant invention, a commercially feasible method is dis
`closed that almost completely eliminates the major impurities
`anhydro-daptomycin and [3-isomer as Well as other impurities
`in preparations of daptomycin. In another embodiment of the
`invention, commercially feasible methods are disclosed for
`purifying lipopeptides, including daptomycin or a daptomy
`cin-related lipopeptide, comprising separating lipopeptide
`micelles from loW molecular Weight contaminants and sepa
`rating non-associated lipopeptides from high molecular
`Weight contaminants. The invention also provides high per
`formance liquid chromatography (HPLC) methods of analyZ
`ing the purity of daptomycin and detecting and characterizing
`other impurities in daptomycin, some of Which Were previ
`ously unknown.
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`The invention also provides puri?ed daptomycin that pos
`sesses a purity of at least 98% or that is substantially or
`essentially free of anhydro-daptomycin and [3-isomer. The
`invention provides puri?ed daptomycin that is free or essen
`tially free of anhydro-daptomycin and contains a much loWer
`level of the [3-isomer and of other contaminants than Was
`previously possible to obtain in the prior art. The invention
`also provides lipopeptide micelles. In a preferred embodi
`ment, the micelle comprises daptomycin or a daptomycin
`related lipopeptide. The invention also provides pharmaceu
`tical compositions comprising highly puri?ed daptomycin or
`a daptomycin-related lipopeptide micelles and methods of
`using these compositions.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`FIG. 1 shoWs the structure of daptomycin.
`FIG. 2 shoWs the structure of impurity 8, CB-l3l0l0 (pre
`viously identi?ed as the [3-isomer, LY213846).
`FIG. 3 shoWs the structure of impurity l3, CB-l30952
`(previously identi?ed as anhydro-daptomycin, LY178480).
`FIG. 4 shoWs the proposed structure of impurity l,
`CB-l3 1012 (previously identi?ed as LY212218).
`FIG. 5 shoWs the proposed structure of impurity 2,
`CB- 1 3 101 l .
`FIG. 6 shoWs the proposed structure of impurity 3,
`CB-l3 1008 (previously identi?ed as LY213928).
`FIG. 7 shoWs the proposed structure of impurity 4,
`CB- 1 3 1006.
`FIG. 8 shoWs the proposed structure of impurity 6,
`CB-l30989 (previously identi?ed as LY213827).
`FIG. 9 shoWs the proposed structure of impurity 7,
`CB- 1 3 1005.
`FIG. 10 shoWs the proposed structure of impurity l2,
`CB- 1 3 1009.
`FIG. 11 shoWs the proposed structure of impurity l4,
`CB-l3 1078 (previously identi?ed as LY109208).
`FIG. 12 shoWs an HPLC chromatogram for a bulk prepa
`ration of daptomycin, including impurities l to 14.
`FIG. 13 shoWs an HPLC chromatogram for a preparation
`of daptomycin after puri?cation on a Poros P150 resin.
`FIGS. 14A-14C shoW micellar structures. FIG. 14A shoWs
`a spherical micelle, in Which the hydrophobic tails of amphi
`pathic molecules are oriented toWard the center of the sphere
`While the hydrophilic heads of the amphipathic molecules are
`oriented toWards the outside of the sphere, in contact With the
`aqueous environment. FIG. 14A shoWs an example in Which
`the hydrophilic heads are negatively charged. FIG. 14B
`shoWs a lipid bilayer structure in Which tWo layers of amphi
`pathic molecules assemble such that the hydrophobic tails of
`each layer are oriented toWards each other While the hydro
`philic heads on either side of the bilayer are in contact With the
`aqueous environment. Lipid bilayers may be either spherical
`or planar. FIG. 14C shoWs a liposome, in Which a lipid
`bilayer, such as that shoWn in FIG. 14B, forms a spherical
`structure enclosing an aqueous interior. The hydrophilic
`heads of the liposome face the aqueous interior and the exter
`nal aqueous environment.
`FIG. 15 shoWs the results of an experiment to determine the
`critical micellar concentration (cmc) of daptomycin at pH
`4.0.
`FIG. 16 shoWs the siZe distribution of daptomycin micelles
`by light scatter. The daptomycin micelles have an average siZe
`of5.4 nm (54 A).
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`5
`DETAILED DESCRIPTION OF THE INVENTION
`
`Objects of the Invention
`One object of the present invention is to provide a method
`for purifying lipopeptides that is easily scaled for commercial
`production comprising a unique combination of anion
`exchange chromatography and hydrophobic interaction chro
`matography. In a preferred embodiment, the method is used to
`manufacture puri?ed daptomycin that is greater than 95%
`pure and exhibits reduced levels of impurities compared to
`daptomycin prepared by prior art methods. In another pre
`ferred embodiment, the method is used to manufacture dap
`tomycin using reduced levels of solvents compared to those
`used in prior art methods. In another preferred embodiment,
`the method is used to manufacture puri?ed daptomycin-re
`lated lipopeptides that are greater than 95% pure.
`Another object of the present invention is to provide a
`method for increasing the levels of a lipopeptide produced by
`a microorganism by feeding the fermentation culture a
`reduced level of a fatty acid. Using loWer levels of decanoic
`acid than those proposed for daptomycin fermentation in Us.
`Pat. No. 4,885,243 results in improved economics in addition
`to producing a highly pure form of daptomycin or a dapto
`mycin-related lipopeptide. In a preferred embodiment, the
`method is used to increase the concentration and amount of
`daptomycin produced by Slreplomyces roseosporus While
`minimizing the production of related contaminants. LoWer
`levels of contaminants in the fermentation broth results in a
`more e?icient recovery and puri?cation of daptomycin,
`Which provides for a manufacturing process With a higher
`yield.
`Another object of the present invention is to provide a
`method for purifying daptomycin or daptomycin related
`lipopeptides comprising the use of modi?ed buffer enhanced
`anion exchange chromatography. In a preferred embodiment,
`the method is used to produce daptomycin that is at least 98%
`pure or that is substantially or essentially free of anhydro
`daptomycin or [3-isomer. In another preferred embodiment,
`the method is used to purify daptomycin-related lipopeptides
`to at least 98% purity.
`Another object of the present invention is to provide a
`process chromatography method to purify a lipopeptide com
`prising a novel combination of anion exchange chromatogra
`phy, hydrophobic interaction chromatography and modi?ed
`buffer enhanced anion exchange chromatography. In a pre
`ferred embodiment, the process chromatography method is
`used to purify daptomycin or a daptomycin-related lipopep
`tide. The modi?ed buffer unexpectedly permits a separation
`ofanhydro -daptomycin from daptomycin not previously pos
`sible in prior chromatography methods.
`Another object of the invention is to provide a method for
`purifying lipopeptides that is easily scaled for commercial
`production using lipopeptide micelles. In one embodiment,
`the method comprises converting a lipopeptide solution from
`a monomeric, nonmicellar state to a micellar state and back
`again during puri?cation procedures. In a preferred embodi
`ment, the method comprises subjecting the lipopeptides to
`conditions in Which micelles are formed, separating the
`lipopeptide micelles from loW molecular Weight contami
`nants by, e.g., a siZe separation technique. In another pre
`ferred embodiment, the method comprises subjecting the
`lipopeptides to conditions in Which the lipopeptides are in
`monomeric form and separating the monomeric lipopeptide
`molecules from high molecular Weight molecules or aggre
`gates by, e. g., a siZe separation technique. In a more preferred
`embodiment, the method comprises both steps: subjecting the
`lipopeptides to conditions in Which micelles are formed and
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`separating the lipopeptide micelles from loW molecular
`Weight contaminants, and then subjecting the lipopeptide
`micelles to conditions in Which the lipopeptides are in mono
`meric form and separating the lipopeptide monomers from
`high molecular Weight molecules or aggregates. These tWo
`steps may be performed in either order. In an even more
`preferred embodiment, the siZe separation technique is ultra
`?ltration or siZe exclusion chromatography.
`A further object of the present invention is to provide
`improved methods for measuring the purity of lipopeptides,
`including daptomycin, by high pressure liquid chromatogra
`phy (HPLC).
`Another object of the present invention is to provide puri
`?ed lipopeptides, such as daptomycin or a daptomycin-re
`lated lipopeptide, and pharmaceutically acceptable salts or
`formulations thereof. In a preferred embodiment, the present
`invention provides daptomycin or a daptomycin-related
`lipopeptide puri?ed by one of the methods described in the
`speci?cation. The present invention also provides pharma
`ceutical compositions of a puri?ed lipopeptide or its salts and
`methods of administering these compositions. In a preferred
`embodiment, the pharmaceutical composition comprises
`puri?ed daptomycin.
`Another object of the present invention is to provide
`lipopeptide micelles and pharmaceutically acceptable formu
`lations thereof. In a preferred embodiment, the present inven
`tion provides daptomycin micelles or a daptomycin-related
`lipopeptide micelle and pharmaceutically acceptable formu
`lations thereof. In another embodiment, the invention also
`provides methods of administering the lipopeptide micelles
`or pharmaceutical formulations thereof to patients in need
`thereof. In a preferred embodiment, the lipopeptide micelles
`are administered intravenously, parenterally, intramuscularly
`or topically.
`De?nitions
`Unless otherWise de?ned, all technical and scienti?c terms
`used herein have the meaning as commonly understood by
`one of ordinary skill in the art to Which this invention belongs.
`The practice of the present invention employs, unless other
`Wise indicated, conventional techniques of chemistry, bio
`chemistry and microbiology and basic terminology used
`therein.
`The term “isolated” refers to a compound or product that is
`refers to a compound Which represents at least 10%, prefer
`ably at least 20% or 30%, more preferably at least 50%, 60%
`or 70%, and most preferably at least 80% or 90% of the
`compound present in the mixture.
`The term “lipopeptide” refers to a molecule that comprises
`a lipid-like moiety covalently linked to a peptide moiety, as
`Well as salts, esters, amides and ethers thereof. The term
`“lipopeptide” also encompasses protected forms of lipopep
`tides in Which one or more amino, carboxylate or hydroxyl
`groups are protected. See, e.g., “Protective Groups in Organic
`Synthesis” by Theodora W. Greene, John Wiley and Sons,
`NeW York, 1981 for examples of protecting groups. In a
`preferr