(12) United States Patent
`Kelleher et al.
`
`(10) Patent N0.:
`(45) Date of Patent:
`
`US 8,058,238 B2
`Nov. 15, 2011
`
`USOO8058238B2
`
`(54)
`
`(75)
`
`HIGH PURITY LIPOPEPTIDES
`
`Inventors: Thomas Kelleher, Weston, MA (US);
`J an-Ji Lai, Westborough, MA (U S);
`Joseph P. DeCourcey, Charlestown, MA
`(US); Paul Lynch, Arlington, MA (US);
`Maurizio Zenoni, Milan (IT); Auro
`Tagliani, Pavia (IT)
`
`(73)
`
`Assignee: Cubist Pharmaceuticals, Inc.,
`Lexington, MA (US)
`
`EP
`EP
`EP
`EP
`EP
`W0
`W0
`W0
`W0
`W0
`W0
`W0
`
`FOREIGN PATENT DOCUMENTS
`0095295 A1 11/1983
`0178152 A2
`4/1986
`0294990 A2 12/1988
`0337731 61
`10/1989
`0386951 A2
`9/1990
`WO 99/27954
`6/1999
`WO 99/27957
`6/1999
`WO 99/43700
`9/1999
`WO 00/18419
`4/2000
`WO 01/44271
`6/2001
`WO 01/44272
`6/2001
`WO 01/44274
`6/2001
`
`OTHER PUBLICATIONS
`
`Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`
`(21)
`
`Appl. No.: 11/739,180
`
`(22)
`
`Filed:
`
`Apr. 24, 2007
`
`(65)
`
`(60)
`
`(60)
`
`(51)
`
`(52)
`
`(58)
`
`(56)
`
`Prior Publication Data
`
`US 2007/0191280 A1
`
`Aug. 16,2007
`
`Related U.S. Application Data
`
`Continuation of application No. 10/747,485, ?led on
`Dec. 29, 2003, now abandoned, which is a division of
`application No. 09/735,191, ?led on Nov. 28, 2000,
`now Pat. No. 6,696,412.
`
`Provisional application No. 60/177,170, ?led on Jan.
`20, 2000.
`
`Int. Cl.
`(2006.01)
`C07K 7/50
`(2006.01)
`C07K 7/00
`U.S. Cl. .................. .. 514/9; 514/11; 514/2; 514/14;
`530/317; 530/322; 530/344; 435/886
`Field of Classi?cation Search .............. .. 514/9, 11,
`514/2, 14; 530/317, 322, 344; 435/886
`See application ?le for complete search history.
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`4,331,594 A
`5/1982 Hamill etal.
`4,482,487 A 11/1984 Abbott etal.
`4,524,135 A
`6/1985 Abbott etal.
`4,537,717 A
`8/1985 Abbott etal.
`RE32,310 E
`12/1986 Debono
`RE32,311 E
`12/1986 Debono
`RE32,333 E
`1/1987 Hamill etal.
`RE32,455 E
`7/1987 Hamill etal.
`4,800,157 A
`1/1989 Eaton etal.
`4,874,843 A 10/1989 Baker
`4,882,164 A 11/1989 Ferro et al.
`4,885,243 A 12/1989 Huber et a1.
`5,271,935 A 12/1993 Franco et al.
`5,387,670 A
`2/1995 Royet a1.
`5,573,936 A 11/1996 Kreuzman et al.
`5,629,288 A
`5/1997 Lattrellet al.
`5,912,226 A
`6/1999 Baker et a1.
`5,955,509 A
`9/1999 Webberetal.
`6,194,383 B1
`2/2001 Hammann etal.
`RE39,071 E
`4/2006 Baker et a1.
`
`DeBono, M. et al.; “Enzymatic and Chemical Modi?cations of
`Lipopeptide Antibiotic A21978C: The Synthesis and Evaluation of
`Daptomycin (LY146032),” J. Antibiotics; 41; 1988; pp. 1093-1105.
`Desai, J. D., et al.; “Microbial Production of Surfactants and Their
`Commercial Potential,” Microbiology and Molecular Biology
`Review, v01. 61; N0. 1; 1997; pp. 47-64; American Society for
`Microbiology.
`Fostel, Jennifer M., et al.; “Emerging Novel Antifungal Agents,”
`DDT; v01. 5; N0. 1; Jan. 2000; pp. 25-32; Elsevier Science Ltd.
`Horowtz, Sarah, et a1; “Isolation and Characterization of a Surfactant
`Produced by Bacillus licheniformis 86,” J. Industrial Micr0b101.; 6;
`1990; pp. 243-248; Society for Industrial Microbiology.
`Kirsch, Lee E., et al.; “Kinetics of the Aspartyl Transporation 0f
`Daptomycin, a Novel Lipopeptide Antibiotic,” Pharmaceutical
`Research; v01. 6; N0. 5; 1989; pp. 387-393; Plenum Publishing
`Corporation.
`Lasic, Dan D., et al.; “Novel Applications of Liposomes,” Trends
`Biotechnology; v01. 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; v01. 11; N0. 6; Jun. 1997; pp. 413-416; Chapman Hall.
`(Continued)
`
`Primary Examiner * Chih-Min Kam
`
`ABSTRACT
`(57)
`The invention discloses highly puri?ed daptomycin and to
`pharmaceutical compositions comprising this compound.
`The invention discloses a method of purifying daptomycin
`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 Slreplomyces 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.
`
`192 Claims, 11 Drawing Sheets
`
`
`1 of 39
`
`FRESENIUS-KABI, Exh. 1001
`
`

`
`US 8,058,238 B2
`Page 2
`
`OTHER PUBLICATIONS
`
`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, Michell M. et al.; “Characterization of a New Lipopeplide
`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.
`U.S. Appl. No. 07/060,148, ?led Jun. 10, 1987, Baker et al.
`Agreement between Cubist Pharmaceuticals, Inc. and Eli Lilly 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:
`019181/0916.
`Maio, et a1 ., “Daptomycin bio synthesis in Streptomyces roseosporus:
`cloning and analysis of the gene cluster and revision of peptide
`sterochemistry,” Microbiology, (vol. 151), (p. 1507-1523), (2005).
`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.
`
`
`2 of 39
`
`

`
`U.S. Patent
`
`Nov. 15, 2011
`
`Sheet 1 of 11
`
`US 8,058,238 B2
`
`0
`
`NH2
`
`NH
`
`0
`
`O
`
`O
`
`0
`
`NH
`
`N
`H
`
`0
`
`H020
`
`HN
`H0\__<EOO
`
`N
`
`I
`
`O
`
`HN
`
`H020
`
`O
`
`O
`
`HN
`
`0
`
`HN
`
`H
`N
`
`O
`
`N
`H
`
`H020
`
`Fig. 1
`
`0
`
`NH2
`
`COEHZ
`N
`
`H
`
`H
`
`Q
`NJJ—(0H2)80H3
`
`/ f
`
`l
`
`CONH
`o 2 H 0
`N—U—-(CH2)8CH3
`
`N
`
`H
`
`/N
`
`H
`
`R,‘
`
`0
`002H
`
`NH2
`
`K,‘
`
`0
`002H
`
`NH2
`
`H020
`
`NH
`
`HN
`
`N
`
`0 0
`
`O
`
`O
`
`0
`
`O
`
`N
`
`H
`
`O
`
`NH
`
`O
`
`2 O
`H H
`
`ofio
`
`HN
`
`0
`
`HN
`
`H
`N
`
`0
`
`O
`
`N
`H
`H020
`
`Fig. 2
`
`30f39
`
`
`3 of 39
`
`

`
`US. Patent
`
`Nov. 15, 2011
`
`Sheet 2 0f 11
`
`US 8,058,238 B2
`
`0 NH2
`
`H020
`
`NH
`
`HN
`
`O
`
`0
`
`O
`
`CONH
`W([ o 2
`N
`H
`
`o
`
`
`4 of 39
`
`

`
`US. Patent
`
`Nov. 15, 2011
`
`Sheet 3 0f 11
`
`US 8,058,238 B2
`
`HN
`
`N
`H
`0 H020
`
`H
`N
`
`0
`
`NH2
`
`Flg. 5
`
`HO
`
`O
`
`O H
`N
`
`?
`NH
`
`0
`
`O
`002H
`
`CONH2
`0 Hi H
`N
`N
`(CH2)sC 3
`H
`
`/
`
`N
`H
`
`HO
`
`Fig. 6
`
`
`5 of 39
`
`

`
`US. Patent
`
`Nov. 15, 2011
`
`Sheet 4 0f 11
`
`US 8,058,238 B2
`
`0 NH2
`
`NH
`
`H026
`HN
`HO\_2:OO
`NH
`
`O
`
`O
`
`0
`
`O
`N
`
`H
`
`O
`
`HN
`
`Hogc?o
`
`HN
`
`0
`
`o
`
`N
`H
`H020
`
`NH
`
`o
`
`HN
`
`H
`N
`
`o
`
`003%
`N
`
`H
`
`O
`nil—(011980113
`
`0
`H2N
`
`m
`
`o
`002H
`
`NH2
`
`Fig. 7
`
`O
`
`n
`
`0
`00211
`
`OONH2
`O H O
`N
`Nil—(014980113
`
`/
`N
`H
`
`Fig. 8
`
`
`6 of 39
`
`

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`US. Patent
`
`Nov. 15, 2011
`
`Sheet 5 0f 11
`
`US 8,058,238 B2
`
`0
`
`NH2
`
`H020
`HN
`HOJOO 0
`
`NH
`
`I
`N
`
`O
`
`HN
`
`1102ch
`
`o
`
`HN
`
`N
`H
`0 H020
`
`0
`
`O
`
`O
`
`N
`H
`
`NH
`
`o
`
`HN
`H
`N
`
`O
`
`n
`
`o
`00214
`
`NH2
`
`Fig. 9
`
`COSHZ
`N
`
`H
`
`H
`
`O
`Nil—(011970113
`
`/
`'A‘
`
`HO
`O
`
`NH
`
`CONH2
`O H O
`O
`XKQ N
`N ll" (CH2)86H3
`H
`O
`COZH
`
`/
`N
`H
`
`Fig. 10
`
`
`7 of 39
`
`

`
`US. Patent
`
`Nov. 15, 2011
`
`Sheet 6 0f 11
`
`US 8,058,238 B2
`
`Pick—2:0 o
`NH
`
`O
`
`0
`
`0
`
`HN
`
`O
`N
`H
`NH
`
`O
`
`n
`
`CONH2
`O H O
`N
`NJ-L(CH2)7CHCH3)CH3
`o H
`COZH
`
`/
`N
`H
`
`HOZCLXIO
`
`HN
`
`0
`
`0
`
`N
`H
`H020
`
`HN
`H
`N
`
`O
`
`NH2
`
`Fig. 11
`
`
`8 of 39
`
`

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`US. Patent
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`Nov. 15, 2011
`
`Sheet 7 0f 11
`
`US 8,058,238 B2
`
`
`
`RESPONSE (mV) 8 8
`
`O
`
`2
`
`4
`
`6 810121416182022242628
`TIME{min)
`Fig. 12
`
`
`9 of 39
`
`

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`US. Patent
`
`Nov. 15, 2011
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`Sheet 8 0f 11
`
`US 8,058,238 B2
`
`Fig. 13
`
`28
`
`26
`
`24
`
`I|1lIIIHIIIHHIIIH‘llll'llll'llll‘llll'llIIIIIIIIIIIllll 18
`
`22
`
`20
`
`
`
`16 TIME (min)
`
`14
`
`10 1
`
`8
`
`6
`
`4
`
`(AW) HSNOdSHEI
`
`8031-?
`
`L011—
`
`
`10 of 39
`
`

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`US. Patent
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`Nov. 15, 2011
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`Sheet 9 0f 11
`
`US 8,058,238 B2
`
`Fig. 14C
`
`
`11 of 39
`
`

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`US. Patent
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`Nov. 15, 2011
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`Sheet 10 0f 11
`
`US 8,058,238 B2
`
`o
`
`'
`
`a
`
`100—
`
`E 80-
`U
`R
`D 60
`E
`2
`E; 40—
`D
`%
`O 20—
`L.)
`
`O_
`
`o
`
`o
`
`CMC
`;
`I
`
`0
`
`8
`
`|
`
`|
`
`10000 12000
`80100
`6000
`4000
`2000
`0
`DAPTOMYCIN CONCENTRATION, ug/mL
`Fig. 15
`
`|
`
`1
`
`
`12 of 39
`
`

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`US. Patent
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`Nov. 15, 2011
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`Sheet 11 0f 11
`
`US 8,058,238 B2
`
`f
`
`INTENSITY
`
`SIZE DISTRIBUTIONISI
`
`5 ID
`
`.
`
`.
`
`1
`
`500
`50100
`DIAMETER Inm)
`
`VOLUME
`
`SIZE DISTRIBUTIONISI
`
`% IN CLASS
`
`5 0
`
`560
`50160
`DIAMETER Inm)
`
`V
`Fig. 16
`
`
`13 of 39
`
`

`
`US 8,058,238 B2
`
`1
`HIGH PURITY LIPOPEPTIDES
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`
`The present application is a continuation of Us. patent
`application Ser. No. 10/747,485, ?led Dec. 29, 2003 and now
`abandoned, which is a divisional of Us. patent application
`Ser. No. 09/735,191, ?led Nov. 28, 2000 (now U.S. 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.
`The present invention was the subject of a joint research
`agreement within the meaning of 35 U.S.C. §103(c)(3),
`between Cubist Pharmaceuticals, Inc. and Eli Lilly and Com
`pany, and said agreement was in effect on or before the date
`the claimed invention was made.
`
`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.
`
`20
`
`25
`
`30
`
`35
`
`BACKGROUND OF THE INVENTION
`
`40
`
`45
`
`The rapid increase in the incidence of gram-positive infec
`tions-including 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
`VlVO.
`Daptomycin is described by BaltZ in Biotechnology of
`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
`
`50
`
`55
`
`60
`
`65
`
`2
`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
`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 Us. 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.
`U.S. Pat. Re. 32,333, Re. 32,455 and Us. Pat. No. 4,800,
`157 describe a method of synthesiZing daptomycin by culti
`vating Streptomyces roseosporus NRL15998 under sub
`merged aerobic fermentation conditions. U.S. 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.
`U.S. Pat. Re. 32,310, Re. 32,31 1, Us. Pat. Nos. 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.
`U.S. Pat. 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 com
`plex was further puri?ed by ion exchange chromatography on
`IRA-68 and two rounds of silica gel chromatography. Indi
`vidual A-21978C factors were separated by reverse-phase
`silica gel or silica gel/C18. U.S. Pat. Re. 32,333 and Re.
`32,455 also disclose that A-2 1 978C may be puri?ed by batch
`chromatography using Diaion HP-20 resin followed by
`silica-gel column chromatography.
`U.S. 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.
`U.S. Pat. No. 5,912,226 describes the identi?cation and
`isolation of two impurities produced during the manufacture
`of daptomycin. Daptomycin, an (x-aspartyl peptide, becomes
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`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
`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-18 column, precipitated, and
`repuri?ed by reverse phase silica gel/C-18 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-18
`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 B-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
`
`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
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`ing the purity of daptomycin and detecting and characterizing
`other impurities in daptomycin, some of which were previ
`ously unknown.
`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-131010 (pre
`viously identi?ed as the [3-isomer, LY213846).
`FIG. 3 shows the structure of impurity 13, CB-130952
`(previously identi?ed as anhydro-daptomycin, LY178480).
`FIG. 4 shows the proposed structure of impurity 1,
`CB-131012 (previously identi?ed as LY212218).
`FIG. 5 shows the proposed structure of impurity 2,
`CB-13 101 1 .
`FIG. 6 shows the proposed structure of impurity 3,
`CB-131008 (previously identi?ed as LY213928).
`FIG. 7 shows the proposed structure of impurity 4,
`CB-13 1006.
`FIG. 8 shows the proposed structure of impurity 6,
`CB-130989 (previously identi?ed as LY213827).
`FIG. 9 shows the proposed structure of impurity 7,
`CB-13 1005.
`FIG. 10 shows the proposed structure of impurity 12,
`CB-13 1009.
`FIG. 11 shows the proposed structure of impurity 14,
`CB-131078 (previously identi?ed as LY109208).
`FIG. 12 shows an HPLC chromatogram for a bulk prepa
`ration of daptomycin, including impurities 1 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.
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`FIG. 16 shows the size distribution of daptomycin micelles
`by light scatter. The daptomycin micelles have an average size
`of 5.4 nm (54 A).
`
`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 ef?cient 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
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`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
`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 forrn 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.
`
`DEFINITIONS
`
`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
`preferred embodiment, the lipopeptide is an antibiotic. In
`another preferred embodiment, the lipopeptide is LY 303366,
`echinocandins, pneumocandins, aculeacins, surfactin, plip
`astatin Bl, amphomycin or the lipopeptide derivative dis
`closed in US. Pat. No. 5,629,288. These lipopeptides are
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`known in the art. See, e.g., U.S. Pat. No. 5,202,309 and
`International PCT Application WO 00/08197. In another pre
`ferred embodiment, the lipopeptide is a daptomycin-related
`molecule, including, inter alia, daptomycin, A54145, a dap
`tomycin-related lipopeptide disclosed in Us. Pat. Nos.
`4,537,717, 4,482,487, Re. 32,311, Re. 32,310, U.S. Pat. No.
`5,912,226, currently in reissue as U.S. Ser. No. 09/547,357,
`U.S. Provisional Applications Nos. 60/170,943, 60/170,946
`or 60/170,945, ?led Dec. 1