US008058238B2
`US008058238B2
`
`Illlllllll
`
`(12) United States Patent
`(12) United States Patent
`Kelleher et al.
`Kelleher et al.
`
`(io) Patent No.:
`(10) Patent N0.:
`(45) Date of Patent:
`(45) Date of Patent:
`
`US 8,058,238 B2
`US 8,058,238 B2
`Nov. 15, 2011
`Nov. 15, 2011
`
`(54) HIGH PURITY LIPOPEPTIDES
`(54)
`HIGH PURITY LIPOPEPTIDES
`
`(75)
`(75)
`
`Inventors: Thomas Kelleher, Weston, MA (US);
`Inventors: Thomas Kelleher, Weston, MA (US);
`Jan-Ji Lai, Westborough, MA (US);
`J an-Ji Lai, Westborough, MA (U S);
`Joseph P. DeCourcey, Charlestown, MA
`Joseph P. DeCourcey, CharlestoWn, MA
`(US); Paul Lynch, Arlington, MA (US);
`(US); Paul Lynch, Arlington, MA (US);
`Maurizio Zenoni, Milan (IT); Auro
`Maurizio Zenoni, Milan (IT); Auro
`Tagliani, Pavia (IT)
`Tagliani, Pavia (IT)
`
`(73) Assignee: Cubist Pharmaceuticals, Inc.,
`(73)
`Assignee: Cubist Pharmaceuticals, Inc.,
`Lexington, MA (US)
`Lexington, MA (US)
`
`( * ) Notice:
`Notice:
`
`Subject to any disclaimer, the term of this
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`U.S.C. 154(b) by 0 days.
`
`(21) Appl.No.: 11/739,180
`(21)
`Appl. No.: 11/739,180
`
`(22) Filed:
`Filed:
`(22)
`
`Apr. 24, 2007
`Apr. 24, 2007
`
`(65)
`(65)
`
`Prior Publication Data
`Prior Publication Data
`US 2007/0191280 Al
`Aug. 16, 2007
`US 2007/0191280 A1
`Aug. 16,2007
`
`Related U.S. Application Data
`Related U.S. Application Data
`(60) Continuation of application No. 10/747,485, filed on
`(60)
`Continuation of application No. 10/747,485, ?led on
`Dec. 29, 2003, now abandoned, which is a division of
`Dec. 29, 2003, noW abandoned, Which is a division of
`application No. 09/735,191, filed on Nov. 28, 2000,
`application No. 09/735,191, ?led on Nov. 28, 2000,
`now Pat. No. 6,696,412.
`noW Pat. No. 6,696,412.
`(60) Provisional application No. 60/177,170, filed on Jan.
`(60)
`Provisional application No. 60/177,170, ?led on Jan.
`20, 2000.
`20, 2000.
`
`(52)
`(52)
`
`(51) Int. CI.
`(51)
`Int. Cl.
`C07K 7/50
`(2006.01)
`(2006.01)
`C07K 7/50
`C07K 7/00
`(2006.01)
`(2006.01)
`C07K 7/00
`U.S. CI
`514/9; 514/11; 514/2; 514/14;
`U.S. Cl. .................. .. 514/9; 514/11; 514/2; 514/14;
`530/317; 530/322; 530/344; 435/886
`530/317; 530/322; 530/344; 435/886
`(58) Field of Classification Search
`514/9, 11,
`(58)
`Field of Classi?cation Search .............. .. 514/9, 11,
`514/2, 14; 530/317, 322, 344; 435/886
`514/2, 14; 530/317, 322, 344; 435/886
`See application file for complete search history.
`See application ?le for complete search history.
`
`(56)
`(56)
`
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`HoroWtZ, Sarah, et al; “Isolation and Characterization of a Surfactant
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`Kirsch, Lee E., et al.; “Kinetics of the Aspartyl Transporation of
`Daptomycin, a Novel Lipopeptide Antibiotic," Pharmaceutical
`Daptomycin, a Novel Lipopeptide Antibiotic,” Pharmaceutical
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`Lin, S.-C. et al., "General Approach for the Development of High-
`Lin, S.-C. et al., “General Approach for the Development of High
`Performance Liquid Chromatography Methods for Biosurfactant
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`Lin, S.-C. et al.; “Recovery and Puri?cation of the Lipopeptide
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`(Continued)
`(Continued)
`Primary Examiner — Chih-Min Kam
`Primary Examiner * Chih-Min Kam
`
`ABSTRACT
`(57)
`ABSTRACT
`(57)
`The invention discloses highly purified daptomycin and to
`The invention discloses highly puri?ed daptomycin and to
`pharmaceutical compositions comprising this compound.
`pharmaceutical compositions comprising this compound.
`The invention discloses a method of purifying daptomycin
`The invention discloses a method of purifying daptomycin
`comprising the sequential steps of anion exchange chroma­
`comprising the sequential steps of anion exchange chroma
`tography, hydrophobic interaction chromatography and
`tography, hydrophobic interaction chromatography and
`anion exchange chromatography. The invention also dis­
`anion exchange chromatography. The invention also dis
`closes a method of purifying daptomycin by modified buffer
`closes a method of purifying daptomycin by modi?ed buffer
`enhanced anion exchange chromatography. The invention
`enhanced anion exchange chromatography. The invention
`also discloses an improved method for producing daptomycin
`also discloses an improved method for producing daptomycin
`by fermentation of Streptomyces roseosporus. The invention
`by fermentation of Slreplomyces roseosporus. The invention
`also discloses high pressure liquid chromatography methods
`also discloses high pressure liquid chromatography methods
`for analysis of daptomycin purify. The invention also dis­
`for analysis of daptomycin purity. The invention also dis
`closes lipopeptide micelles and methods of making the
`closes lipopeptide micelles and methods of making the
`micelles. The invention also discloses methods of using
`micelles. The invention also discloses methods of using
`lipopeptide micelles for purifying lipopeptide antibiotics,
`lipopeptide micelles for purifying lipopeptide antibiotics,
`such as daptomycin. The invention also discloses using
`such as daptomycin. The invention also discloses using
`lipopeptide micelles therapeutically.
`lipopeptide micelles therapeutically.
`
`192 Claims, 11 Drawing Sheets
`192 Claims, 11 Drawing Sheets
`
`PETITIONERS
`
`EXHIBIT NO. 1001 Page 1 of 39
`
`

`

`US 8,058,238 B2
`US 8,058,238 B2
`Page 2
`Page 2
`
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`(Pyrogens) In Solution in Different States of Aggregation," Applied
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`and Environmental Microbiology; vol. 34; No. 4; 1977; pp. 382-385;
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`Tally, F.P, et al.; "Daptomycin: A Novel Agent for Gram Positive
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`Infections,” EXp. Opinion Invest Drugs; 8; 1999; 1223-1238.
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`Thimon, L et al., "Surface-Active Properties of Antifungal
`Thimon, L et al., “Surface-Active Properties of Antifungal
`Lipopeptides Produced by Bacillus substillisJ. Am. Oil Chem.
`Lipopeptides Produced by Bacillus substillis,” J. Am. Oil Chem.
`Soc.; 69; 1992; pp. 92-93.
`Soc.; 69; 1992; pp. 92-93.
`Yakimov, Michell M. et al.; "Characterization of a New Lipopeplide
`Yakimov, Michell M. et al.; “Characterization of a New Lipopeplide
`Surfactant Produced by Thermotolerant and Halotolerant Subsurface
`Surfactant Produced by Thermotolerant and Halotolerant Subsurface
`Bacillus
`licheniformis BAS50," Applied and Environmental
`Bacillus licheniformis BAS50,” Applied and Environmental
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`U.S. Appl. No. 07/060,148, filed Jun. 10, 1987, Baker et al.
`U.S. Appl. No. 07/060,148, ?led Jun. 10, 1987, Baker et al.
`Agreement between Cubist Pharmaceuticals, Inc. and Eli Lilly and
`Agreement between Cubist Pharmaceuticals, Inc. and Eli Lilly and
`Company dated Nov. 7, 1997. (Redacted form from SEC Edgar).
`Company dated Nov. 7, 1997. (Redacted form from SEC Edgar).
`Agreement between Cubist Pharmaceuticals, Inc. and Eli Lilly and
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`Company dated Oct. 6, 2000. (Redacted form from SEC Edgar).
`Company dated Oct. 6, 2000. (Redacted form from SEC Edgar).
`Assignment of US Re 39,071 from Eli Lilly and Company to Cubist
`Assignment of US Re 39,071 from Eli Lilly and Company to Cubist
`Pharmaceuticals, Inc. recorded on Apr. 23, 2007. Reel/Frame:
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`Iso-Daptomycin," ACS 200th Meeting, 1990.
`Iso-Daptomycin,” ACS 200th Meeting, 1990.
`
`PETITIONERS
`
`EXHIBIT NO. 1001 Page 2 of 39
`
`

`

`U.S. Patent
`
`Nov. 15,2011
`
`Sheet 1 of 11
`
`US 8,058,238 B2
`
`0
`it
`
`NHo
`
`i ^
`
`HO2C
`
`HN
`
`NH
`
`o o
`
`d
`
`HO
`
`NH
`
`O
`
`HN
`
`O
`
`H02C
`
`O
`
`HN
`
`N
`H
`0 H02C
`
`0.
`
`0
`
`O
`
`N
`H
`NH
`
`H
`N
`
`O
`'CO2H
`
`CONH2 O
`N
`H
`
`O
`
`HN
`H
`N
`
`0
`
`NH2
`
`Fig. 1
`
`O
`
`I I
`
`NH9
`
`I ^
`
`HO2C
`
`HN
`
`HQ
`
`NH
`
`O O
`
`0
`
`NH
`
`O
`
`NH OH
`O
`
`HN
`
`O
`
`O.
`
`O
`
`O
`
`N
`H
`NH
`
`H
`N
`
`O
`'C02H
`
`,CONH2 O
`N
`H
`
`0
`
`N
`H
`HO-C
`
`O
`
`HN
`H
`N
`
`O
`
`NH2
`
`Fig. 2
`
`0
`NJL(CH2)8CH3
`
`V
`N
`H
`
`(CH2)8CH3
`
`O
`
`H
`N
`
`'/
`N
`H
`
`PETITIONERS
`
`EXHIBIT NO. 1001 Page 3 of 39
`
`

`

`U.S. Patent
`
`Nov. 15,2011
`
`Sheet 2 of 11
`
`US 8,058,238 B2
`
`O
`
`I I
`
`NHp
`
`i
`
`H02C'
`
`HN
`
`NH
`
`HO
`
`0 O
`
`O
`
`O
`
`NH
`
`O
`
`N
`
`O
`
`HN
`
`O
`
`N
`H
`0 H02C
`
`O.
`
`O
`
`N
`H
`NH
`
`O
`
`H
`N
`
`0
`'C02H
`
`CONHp
`0
`
`N
`H
`
`0
`N—ILI—(CH2)gCH3
`
`N
`H
`
`O
`
`HN
`H N
`
`O
`
`NH2
`
`Fig. 3
`
`O
`
`I I
`
`NH?
`
`I ^
`
`NH
`
`OH
`O HO
`
`0
`
`O
`
`N
`H
`NH
`
`O
`
`HN
`
`H
`N
`
`O
`
`H02C
`HN
`
`HO
`
`O O
`
`NH
`
`O
`
`HN
`
`H02C
`
`0
`
`HN
`
`O
`
`N
`H
`0 H02C
`
`CONH2
`O
`
`N
`H
`
`0
`N —^— (C^JgCHg
`
`'/
`N
`H
`
`H
`N
`
`O
`'CO2H
`
`NH2
`
`Fig. 4
`
`PETITIONERS
`
`EXHIBIT NO. 1001 Page 4 of 39
`
`

`

`U.S. Patent
`
`Nov. 15,2011
`
`Sheet 3 of 11
`
`US 8,058,238 B2
`
`O
`ii
`
`NH2
`l
`
`HO2C
`
`HN
`
`NH
`
`OH
`
`HO
`
`O O
`
`O HO
`
`NH
`
`O
`
`O
`
`CONHo
`O
`
`o
`
`H
`N
`
`0
`'C02H
`
`H
`
`NHg
`
`N
`H
`
`O
`
`N
`H
`
`NH
`
`O
`
`HN
`
`H
`N
`
`O
`
`HNOH
`O
`
`O
`
`O
`
`HN
`
`N
`H
`0 H02C'
`
`HO
`
`}—N
`o=< H
`NH
`
`0
`
`HO
`
`Fig. 5
`
`O
`
`H
`N
`
`CONHo
`O
`
`O
`^—(CHg^CHg
`
`H
`
`O
`'C02H
`
`N
`H
`
`Fig. 6
`
`PETITIONERS
`
`EXHIBIT NO. 1001 Page 5 of 39
`
`

`

`U.S. Patent
`US. Patent
`
`Nov. 15,2011
`Nov. 15, 2011
`
`Sheet 4 of 11
`Sheet 4 0f 11
`
`US 8,058,238 B2
`US 8,058,238 B2
`
`O
`
`I I
`
`NHo
`
`I ^
`
`HO2C
`
`HN
`
`HO
`
`NH
`
`O O
`
`O
`
`NH
`
`o
`
`HN
`HOoC
`2 V
`
`O
`
`o
`
`HN
`
`N
`H
`O H02C'
`
`o
`
`O
`
`— N
`H
`=<
`O
`NH
`o
`
`HN
`
`H
`N
`
`O
`
`H
`N
`
`O
`•CO2H
`
`NH2
`
`Fig. 7
`
`CONHp
`0
`
`N
`H
`
`O
`
`H
`N
`
`0
`H2N
`
`(CH2)8CH3
`(CH2)gCH3
`
`CONHn
`O
`
`NH
`N
`H
`
`O
`N-ii-(CH2)8CH3
`(CH2)8CH3
`
`0
`
`HO
`
`H
`N
`
`O
`'C02H
`
`N
`H
`
`Fig. 8
`
`PETITIONERS
`
`EXHIBIT NO. 1001 Page 6 of 39
`
`

`

`U.S. Patent
`US. Patent
`
`Nov. 15,2011
`Nov. 15, 2011
`
`Sheet 5 of 11
`Sheet 5 0f 11
`
`US 8,058,238 B2
`US 8,058,238 B2
`
`O
`0
`
`I I
`
`NH?
`NH2
`l ^
`
`NH
`NH
`
`H02C'
`H020
`HN
`HN
`0
`O O
`HOJOO O
`HQ
`NH
`I
`N
`
`O
`O
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`HN
`HN
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`O
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`HozcLfO
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`O
`o
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`HN
`HN
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`N
`N
`H
`H
`0 H020
`0 HO2C
`
`CONHo
`COSHZ
`O
`N
`N
`H
`H
`
`O
`O
`H
`N-^—(CH2)7CH3
`Nil-(011970113
`
`/
`'A‘
`N
`H
`
`O.
`0
`
`O
`O
`
`O
`O
`
`N
`N
`H
`H
`
`1MH
`NH
`
`0
`o
`
`HN
`
`HN
`H
`H
`N
`N
`0
`O
`
`H
`n
`N
`0
`o
`•CO2H
`00214
`
`m2
`NH2
`
`Fig. 9
`Fig. 9
`
`CONHp
`CONH2
`0
`O
`O
`O H O
`O
`H
`H
`(CH2)8CH3
`N
`KKK N
`N J" (CH2)sCH3
`N
`N
`N
`H
`H
`H
`O
`O
`'C02H
`NH
`CO2H
`NH
`
`HO
`HO
`O
`O
`
`O
`
`HN
`
`HO
`
`0
`
`/
`N
`N
`H
`H
`
`NH2
`
`Fig. 10
`Fig. 10
`
`PETITIONERS
`
`EXHIBIT NO. 1001 Page 7 of 39
`
`

`

`U.S. Patent
`
`Nov. 15,2011
`
`Sheet 6 of 11
`
`US 8,058,238 B2
`
`O
`
`M
`
`NHo
`
`I ^
`
`H02C'
`
`HN
`
`NH
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`O
`
`HO
`
`O O
`
`O
`
`O
`
`H
`
`,CONH2
`
`"
`
`(CH2)7CHCH3)CH3
`
`NH
`
`O
`
`HN
`
`HO2C
`
`O
`
`HN
`
`O
`
`N
`H
`O H02C'
`
`O
`'C02H
`
`NH2
`
`N H
`
`/-N K H
`
`NH
`
`O
`
`O
`
`HN
`H
`N
`
`O
`
`Fig. 11
`
`PETITIONERS
`
`EXHIBIT NO. 1001 Page 8 of 39
`
`

`

`K>
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`US 8,058,238 B2
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`Nov. 15, 2011
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`12
`
`PETITIONERS
`
`EXHIBIT NO. 1001 Page 9 of 39
`
`

`

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`Nov. 15, 2011
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`PETITIONERS
`
`EXHIBIT NO. 1001 Page 10 of 39
`
`

`

`U.S. Patent
`US. Patent
`
`Nov. 15,2011
`Nov. 15, 2011
`
`Sheet 9 of 11
`Sheet 9 0f 11
`
`US 8,058,238 B2
`US 8,058,238 B2
`
`© ©n©, ^ ©
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`
`PETITIONERS
`
`EXHIBIT NO. 1001 Page 11 of 39
`
`

`

`U.S. Patent
`US. Patent
`
`Nov. 15, 2011
`Nov. 15, 2011
`
`Sheet 10 of 11
`Sheet 10 0f 11
`
`US 8,058,238 B2
`US 8,058,238 B2
`
`lOOn
`100"
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`DAPTOMYCIN CONCENTRATION, ug/mL
`DAPTOMYCIN CONCENTRATION, ug/mL
`Fig. 15
`Fig. 1 5
`
`PETITIONERS
`
`EXHIBIT NO. 1001 Page 12 of 39
`
`

`

`U.S. Patent
`US. Patent
`
`Nov. 15,2011
`Nov. 15, 2011
`
`Sheet 11 of 11
`Sheet 11 0f 11
`
`US 8,058,238 B2
`US 8,058,238 B2
`
`INTENSITY
`INTENSiTY
`
`SIZE DISTRIBUTION(S)
`SIZE DISTRIBUTIONS]
`
`fc
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`DIAMETER (nm)
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`VOLUME
`VOLUME
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`SIZE DISTRIBUTION(S)
`SIZE DISTRIBUTIONS)
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`DIAMETER (nm)
`DIAMETER (nm)
`
`Fig. 16
`Fig. 16
`
`PETITIONERS
`
`EXHIBIT NO. 1001 Page 13 of 39
`
`

`

`US 8,058,238 B2
`US 8,058,238 B2
`
`CROSS-REFERENCE TO RELATED
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`APPLICATIONS
`
`5
`
`TECHNICAL FIELD OF THE INVENTION
`TECHNICAL FIELD OF THE INVENTION
`
`BACKGROUND OF THE INVENTION
`BACKGROUND OF THE INVENTION
`
`1
`1
`HIGH PURITY LIPOPEPTIDES
`HIGH PURITY LIPOPEPTIDES
`
`2
`2
`highly effective against most gram-positive bacteria; it is
`highly effective against most gram-positive bacteria; it is
`highly bactericidal and fast-acting; it has a low resistance rate
`highly bactericidal and fast-acting; it has a loW resistance rate
`and is effective against antibiotic-resistant oiganisms. The
`and is effective against antibiotic-resistant organisms. The
`compound is currently being developed in a variety of formu-
`compound is currently being developed in a variety of formu
`lations to treat serious infections caused by bacteria, includ­
`lations to treat serious infections caused by bacteria, includ
`ing, but not limited to, methicillin resistant Staphylococcus
`ing, but not limited to, methicillin resistant Staphylococcus
`The present application is a continuation of U.S. patent
`The present application is a continuation of Us. patent
`aureus (MRSA) and vancomycin resistant enterococci
`aureus (MRSA) and vancomycin resistant enterococci
`application Ser. No. 10/747,485, filed Dec. 29,2003 and now
`application Ser. No. 10/747,485, ?led Dec. 29, 2003 and noW
`(VRE).
`(V RE).
`abandoned, which is a divisional of U.S. patent application
`abandoned, Which is a divisional of Us. patent application
`A number of U.S. patents describe A-21978C antibiotics
`Ser. No. 09/735,191, filed Nov. 28, 2000 (now U.S. Pat. No.
`A number of Us. patents describe A-21978C antibiotics
`Ser. No. 09/735,191, ?led Nov. 28, 2000 (noW U.S. Pat. No.
`6,696,412), which claims the benefit of U.S. Provisional 1° and derivatives thereof including daptomycin (LY 146032) as
`and derivatives thereof including daptomycin (LY 146032) as
`6,696,412), Which claims the bene?t of Us. Provisional
`well as methods of producing and isolating the A-21978C
`Well as methods of producing and isolating the A-21978C
`application No. 60/177,170, filed Jan. 20, 2000, all of which
`application No. 60/177,170, ?led Jan. 20, 2000, all of Which
`antibiotics and derivatives thereof.
`antibiotics and derivatives thereof.
`are incorporated by reference herein in their entireties.
`are incorporated by reference herein in their entireties.
`U.S. Pat. Re. 32,333, Re. 32,455 and U.S. Pat. No. 4,800,
`U.S. Pat. Re. 32,333, Re. 32,455 and Us. Pat. No. 4,800,
`The present invention was the subject of a joint research
`The present invention Was the subject of a joint research
`157 describe a method of synthesizing daptomycin by culti-
`157 describe a method of synthesiZing daptomycin by culti
`agreement within the meaning of 35 U.S.C. § 103(c)(3),
`agreement Within the meaning of 35 U.S.C. §103(c)(3),
`between Cubist Pharmaceuticals, Inc. and Eli Lilly and Com- 15 vating Streptomyces roseosporus NRL15998 under sub-
`vating Streptomyces roseosporus NRL15998 under sub
`betWeen Cubist Pharmaceuticals, Inc. and Eli Lilly and Com
`meiged aerobic fermentation conditions. U.S. Pat. No. 4,885,
`merged aerobic fermentation conditions. U.S. Pat. No. 4,885,
`pany, and said agreement was in effect on or before the date
`pany, and said agreement Was in effect on or before the date
`243 describes an
`improved method of synthesizing
`243 describes an improved method of synthesiZing
`the claimed invention was made.
`the claimed invention Was made.
`daptomycin by feeding a fermentation culture a decanoic
`daptomycin by feeding a fermentation culture a decanoic
`fatty acid or ester or salt thereof.
`fatty acid or ester or salt thereof.
`20 U.S. Pat. Re. 32,310, Re. 32,311, U.S. Pat. Nos. 4,537,717,
`U.S. Pat. Re. 32,310, Re. 32,31 1, Us. Pat. Nos. 4,537,717,
`20
`4,482,487 and 4,524,135 describe methods of deacylating the
`The present invention relates to a highly purified form of
`4,482,487 and 4,524,135 describe methods of deacylating the
`The present invention relates to a highly puri?ed form of
`A-21978C antibiotic and reacylating the peptide nucleus and
`lipopeptides, including daptomycin, a lipopeptide antibiotic
`A-21978C antibiotic and reacylating the peptide nucleus and
`lipopeptides, including daptomycin, a lipopeptide antibiotic
`antibiotic derivatives made by this process. All of these pat-
`with potent bactericidal activity against gram-positive bacte-
`antibiotic derivatives made by this process. All of these pat
`With potent bactericidal activity against gram-positive bacte
`ents describe a purified deacylated A-21978C antibiotic
`ria, including strains that are resistant to conventional antibi-
`ents describe a puri?ed deacylated A-21978C antibiotic
`ria, including strains that are resistant to conventional antibi
`otics. The present invention also relates to a process for pre- 25 nucleus or a derivative thereof which was isolated from the
`nucleus or a derivative thereof Which Was isolated from the
`otics. The present invention also relates to a process for pre
`25
`paring the highly purified form of the lipopeptide. The present
`fermentation broth by filtration and then purified by Diaion
`fermentation broth by ?ltration and then puri?ed by Diaion
`paring the highly puri?ed form of the lipopeptide. The present
`HP-20 chromatography and silica gel/C18 chromatography.
`invention further relates to micelles of lipopeptides. The
`HP-20 chromatography and silica gel/C 18 chromatography,
`invention further relates to micelles of lipopeptides. The
`U.S. Pat. Re. 32,333 and Re. 32,455 disclose a purification
`present invention also relates to pharmaceutical compositions
`U.S. Pat. Re. 32,333 and Re. 32,455 disclose a puri?cation
`present invention also relates to pharmaceutical compositions
`of the lipopeptide micelles and methods of using these com-
`method in which a filtrate of whole fermentation broth was
`method in Which a ?ltrate of Whole fermentation broth Was
`of the lipopeptide micelles and methods of using these com
`positions. The present invention also relates to methods of 30 purified through a number of precipitation and extraction
`puri?ed through a number of precipitation and extraction
`positions. The present invention also relates to methods of
`30
`making lipopeptide micelles from non-associated monomers
`steps to obtain a crude A-21978C complex. The crude com-
`steps to obtain a crude A-21978C complex. The crude com
`making lipopeptide micelles from non-associated monomers
`of the lipopeptides, and for converting lipopeptide micelles to
`plex was further purified by ion exchange chromatography on
`plex Was further puri?ed by ion exchange chromatography on
`of the lipopeptides, and for converting lipopeptide micelles to
`non-associated monomers. The present invention also relates
`IRA-68 and two rounds of silica gel chromatography. Indi-
`IRA-68 and tWo rounds of silica gel chromatography. Indi
`non-associated monomers. The present invention also relates
`to a process for preparing lipopeptides using micelles that is
`vidual A-21978C factors were separated by reverse-phase
`vidual A-21978C factors Were separated by reverse-phase
`to a process for preparing lipopeptides using micelles that is
`35 silica gel or silica gel/C18. U.S. Pat. Re. 32,333 and Re.
`easily scaled for commercial production.
`silica gel or silica gel/C18. U.S. Pat. Re. 32,333 and Re.
`easily scaled for commercial production.
`35
`32,455 also disclose thatA-21978C may be purified by batch
`32,455 also disclose that A-2 1 978C may be puri?ed by batch
`chromatography using Diaion HP-20 resin followed by
`chromatography using Diaion HP-20 resin folloWed by
`silica-gel column chromatography.
`silica-gel column chromatography.
`U.S. Pat. No. 4,874,843 describes a daptomycin purifica-
`The rapid increase in the incidence of gram-positive infec­
`The rapid increase in the incidence of gram-positive infec
`U.S. Pat. No. 4,874,843 describes a daptomycin puri?ca
`tions-including those caused by antibiotic resistant bacte- 40 tion method in which the fermentation broth was filtered and
`tions-including those caused by antibiotic resistant bacte
`tion method in Which the fermentation broth Was ?ltered and
`40
`passed through a column containing HP-20 resin. After elu-
`ria—has sparked renewed interest in the development of
`riaihas sparked reneWed interest in the development of
`passed through a column containing HP-20 resin. After elu
`novel classes of antibiotics. One such class is the lipopeptide
`tion, the semipurified daptomycin was passed through a col­
`tion, the semipuri?ed daptomycin Was passed through a col
`novel classes of antibiotics. One such class is the lipopeptide
`antibiotics, which includes daptomycin. Daptomycin has
`umn containing HP-20ss, and then separated again on HP-20
`antibiotics, Which includes daptomycin. Daptomycin has
`umn containing HP-20ss, and then separated again on HP-20
`potent bactericidal activity in vitro against clinically relevant
`resin. The '843 patent states that final resolution and separa-
`potent bactericidal activity in vitro against clinically relevant
`resin. The ’843 patent states that ?nal resolution and separa
`gram-positive bacteria that cause serious and life-threatening 45 tion of daptomycin from structurally similar compounds by
`tion of daptomycin from structurally similar compounds by
`gram-positive bacteria that cause serious and life-threatening
`45
`diseases. These bacteria include resistant pathogens, such as
`this method is impeded by the presence of impurities that are
`this method is impeded by the presence of impurities that are
`diseases. These bacteria include resistant pathogens, such as
`vancomycin-resistant enterococci (VRE), methicillin-resis-
`not identifiable by ultraviolet analysis of the fermentation
`vancomycin-resistant enterococci (V RE), methicillin-resis
`not identi?able by ultraviolet analysis of the fermentation
`tant Staphylococcus aureus (MRSA), glycopeptide interme­
`broth. The '843 patent further states that attempts to remove
`tant Staphylococcus aureus (MRSA), glycopeptide interme
`broth. The ’843 patent further states that attempts to remove
`diary susceptible Staphylococcus aureus (GISA), coagulase-
`these impurities by reverse phase chromatography over silica
`diary susceptible Staphylococcus aureus (GISA), coagulase
`these impurities by reverse phase chromatography over silica
`negative staphylococci (CNS), and penicillin-resistant 50 gel, normal phase chromatography over silica gel or ion
`negative staphylococci (CNS), and penicillin-resistant
`gel, normal phase chromatography over silica gel or ion
`50
`Streptococcus pneumoniae (PRSP), for which there are very
`exchange chromatography also failed to significantly
`exchange chromatography also failed to signi?cantly
`Streptococcus pneumoniae (PRSP), for Which there are very
`few therapeutic alternatives. See, e.g., Tally et al., 1999, Exp.
`improve the purity of daptomycin. The '843 patent also dis­
`improve the purity of daptomycin. The ’843 patent also dis
`feW therapeutic alternatives. See, e.g., Tally et al., 1999, Exp.
`closes a "reverse method" for purification comprising the
`Opin. Invest. Drugs