United States Patent [191
`Hamill et al.
`
`[54]
`
`[75]
`
`A-21978 ANTIBIOTICS AND PROCESS FOR
`THEIR PRODUCTION
`
`Inventors: Robert L. Hamill, Greenwood;
`Marvin M. Hoehn, Indianapolis, both
`of Ind.
`
`[73]
`
`Assignee: Eli Lilly and Company, Indianapolis,
`Ind.
`
`[21]
`
`[22]
`
`[60]
`
`[51]
`[52]
`[5 8]
`
`Appl. No.: 206,749
`
`Filed:
`
`Nov. 14, 1980
`
`Related US. Application Data
`Continuation-impart of Ser. No. 41,274, May 21, 1979,
`abandoned, which is a division of Ser. No. 951,695,
`Oct. 16, 1978, Pat. No. 4,208,403.
`
`1m. c1.3 .......................................... .. c07c 103/52
`US. 01. ..................... .. 260/1125 R; 424/115
`Field of Search ............................... .. 260/ 112.5 R
`
`_ [111
`[451
`
`4,331,594
`May 25, 1982
`
`[56]
`
`References Cited
`PUBLICATIONS
`Shoji et al., J. Antibiotics, 1976, vol. 29, pp. 380-389,
`1268-1274, 1275-1280.
`Primary Examiner—Dale R. Ore
`Attorney, Agent, or Firm—Nancy J. Harrison; Arthur R.
`Whale
`ABSTRACT
`[57]
`Antibiotic A-2l978 complexes, in particular the A
`21978C complex, comprising microbiologically active,
`related factors, C0, C1, C2, C3, C4, and C5. A-2l978
`complex and A-21978C complex are produced by sub
`merged aerobic fermentation of Streptomyces roseosporus
`NRRL 11379. The individual A-29178C factors are
`separated and isolated by chromatography. The A
`21978 and A-2l978C complexes, the A-21978C factors,
`and pharmaceutically acceptable salts thereof are anti
`bacterial agents and improve growth promotion in
`poultry.
`
`18 Claims, 7 Drawing Figures
`
`PETITIONERS
`
`EXHIBIT NO. 1009 Page 1 of 25
`
`

`

`US. Patent May 25, 1982
`
`Sheet 1 of 7
`
`4,331,594
`
`
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`PETITIONERS
`
`EXHIBIT NO. 1009 Page 2 of 25
`
`

`

`US. Patent May 25, 1982
`
`Sheet 2 of 7
`
`4,331,594
`
`400250
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`161820253040
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`PETITIONERS
`
`EXHIBIT NO. 1009 Page 3 0f 25
`
`PETITIONERS
`
`EXHIBIT NO. 1009 Page 3 of 25
`
`

`

`U.S. Patent May 25, 1982
`
`Sheet 3 of7
`
`4,331,594
`
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`PETITIONERS
`
`EXHIBIT NO. 1009 Page 4 of 25
`
`

`

`US Patent May 25, 1982
`
`Sheet 4 of 7
`
`4,331,594
`
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`PETITIONERS
`
`EXHIBIT NO. 1009 Page 5 of 25
`
`

`

`U.S. Patent May 25, 1982
`
`Sheet 5 of7
`
`4,331,594
`
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`
`PETITIONERS
`
`EXHIBIT NO. 1009 Page 6 of 25
`
`

`

`US. Patent May 25, 1982
`
`Sheet 6 of 7
`
`4,331,594
`
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`
`PETITIONERS
`
`EXHIBIT NO. 1009 Page 7 of 25
`
`

`

`US. Patent May 25, 1982
`
`Sheet 7 of 7
`
`4,331,594
`
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`PETITIONERS
`
`EXHIBIT NO. 1009 Page 8 0f 25
`
`PETITIONERS
`
`EXHIBIT NO. 1009 Page 8 of 25
`
`

`

`1
`
`4,331,594
`
`A-21978 ANTIBIOTICS AND PROCESS FOR THEIR
`PRODUCTION
`
`20
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`This application is a continuation-in-part of applica
`tion Ser. No. 41,274, ?led May 21, 1979, now aban
`doned which in turn is a divisional of application Ser.
`No. 951,695, ?led Oct. '16, 1978, which issued as US.
`Pat. No. 4,208,403, on June 17, 1980.
`BACKGROUND OF THE INVENTION
`1. Field of the Invention
`Although there are many known antibacterial agents,
`the need for improved antibiotics continues. Antibiotics
`differ in their effectiveness against pathogenic organ
`isms, and organism strains which are resistant to cur
`rently used antibiotics continually develop. In addition,
`individual patients often suffer serious reactions to spe
`ci?c antibiotics, due to hypersensitivity and/ or to toxic
`effects. There is, therefore, a continuing need for new
`25
`and improved antibiotics. '
`i
`2. The Prior Art
`The A-21978C antibiotics are closely related, acidic
`peptide antibiotics. Members of this class of antibiotics
`which were previously known include crystallomycin,
`amphomycin, zaornycin, aspartocin, and glumamycin
`> [see T. Korzybski, Z. Kowszyk-Gindifer and W._Kury
`lowicz, “Antibiotics-Origin, Nature and Properties,” -
`Vol. I,- Perg'amon Press, New York, N.Y.,ii 1967, pp.
`397-401 and 404-408]; tsushimycin [J. Shoji, et al., J.
`Antibiotics 21, 439-443 (1968)]; laspart'omycin [H.
`Naganawa, et al., J. Antibiotics 21, 55-(1968)]; brevistin
`[J . Shoji and T. Kato, J. Antibiotics 29, 380-389 (1976)];
`cerexin A [J. Shoji, et al., J. Antibiotics 29, 1268-1274
`(1976)] and cerexin B [J . Shoji and T. Kato, J. Antibiot
`its 29, 1275-1280 (1976)]. Of these antibiotics, brevistin,
`cerexin A and cerexin B are believed to be‘ the prior art
`antibiotics which are most closely related to the new
`45
`A-21978C antibiotics.
`
`30
`
`35
`
`SUMMARY OF THE INVENTION
`This invention‘relates tov antibiotic substances. In
`particular, it relates to antibiotic complexes comprising
`several factors. The A-21978 complex contains major
`factor C and as yet uncharacterized factorzsvA, B, Dand
`E. A-2l978 factor C'is a complex of closely related
`antibiotic factors, including individual A_-21978C fac
`tors c0, c1, c2, c3, c4,‘ and c5. A-21978 factor 0 is,
`therefore, designated herein as A-21978C complex. The
`salts of the A-21978 and A-21978C complexes and of
`individual A-2l978C factors C0, C1, C2, C3, C4 and C5
`are also part of this invention.
`-
`The term “complex”. as used in the fermentation art
`and in this speci?cation refers to a ‘mixture of copro
`duced individual antibiotic factors. As will be recog
`nized by those familar with antibiotic production by
`65
`fermentation, the number and ratio of individual factors
`produced in an antibiotic complex will vary, depending
`upon the fermentation conditions used. In the A-21978C
`
`55
`
`2
`complex, factors C1, C2, ‘and C3 are major factors, and
`factors C0, C4, and C5 are minor factors.
`The antibiotic substances of this invention are arbi
`trarily designated herein as A-21978 antibiotics. In dis
`cussions of utility, the term “A-21978 antibiotic” will be
`used, for the sake of brevity, to denote a member se
`lected from the group consisting of A-21978 complex,
`A-21978C complex and A-21978C factors C0, C1, C2,
`C3, C4, and C5, and the pharmaceutically acceptable
`salts thereof.
`The A-2l978 complex is produced by culturing Strep
`tomyces roseosporus NRRL 11379 under submerged aer
`obic fermentation conditions until a substantial level of
`antibiotic activity is produced. The A-21978 complex is
`separated by ?ltering the fermentation broth, lowering
`the pH of the ?ltrate to about pH 3, allowing the com
`plex to precipitate, and separating the complex by ?ltra
`tion. The separated complex may be further puri?ed by
`extraction techniques. For isolation of the individual
`A~21978C complex and factors, chromatographic sepa
`rations are required. The A-21978 antibiotics of this
`invention inhibit the growth of pathogenic organisms,
`especially gram-positive bacteria.
`
`DESCRIPTION OF THE DRAWINGS
`Infrared absorption spectra (KBr pellet) of the fol
`lowing A-2l978C antibiotics (as sodium salts) are pres
`ented in the accompanying drawings as follows:
`FIG. 1—A-21978C Complex
`FIG. 2—A-2l978C Factor C1
`FIG. 3-~A-21978C Factor C2
`FIG. 4-A-2'1978C Factor 03
`FIG. 5—A-21978C Factor C0
`FIG. 6—-A-21978C Factor C4
`FIG. 7—A-21978C Factor C5
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`The A-2l978C factors of this invention are closely
`related peptide antibiotics.‘ As many as six antibiotic
`factors are recovered from the fermentation and are
`obtained as a mixtue, the A-21978C complex. Individual
`factors C0, C1, C2, C3, C4 and C5 are isolated as individ
`ual compounds as hereinafter described.
`The A-2l978C factors are closely related, acidic,
`cyclic polypeptide antibiotics bearing a fatty acid acyl
`group at the terminal amino group. Upon hydrolysis,
`each of the factors yielded the following amino acids:
`
`Amino Acid
`Aspartic acid"
`Glycine
`Alanine
`Serine
`Threonine
`Tryptophan
`Ornithine
`Kynurenine
`3-Methylglutamic
`acid“
`
`‘one of which could be asparaginc
`“could be from J-methylglutamine
`
`No. of moles
`4
`
`1
`
`PETITIONERS
`
`EXHIBIT NO. 1009 Page 9 of 25
`
`

`

`3
`Each of the A_-21978C factors contains a fatty acid.
`Table I summarizes carbon content, and the 1dent1ty
`where known, of the fatty acid contained by each of the
`
`4,331,594
`
`4
`,continued
`
`.
`
`.,
`
`_
`D-Ala
`/I\
`
`_
`I Gly
`\1/
`
`1
`,
`‘D-Ser
`
`,
`
`5
`
`10
`
`v
`'
`
`-
`
`.
`
`.
`
`,
`
`L-Asp
`
`‘
`
`/1\
`
`L_O'"
`
`.
`
`3MG
`
`\
`
`'
`
`G1)’
`
`‘
`
`g
`
`L-Kyn
`01/
`
`/
`
`A~21978C factors.
`
`'
`
`TABLE I
`
`Fatty Acid
`
`,
`
`'
`
`>
`
`’“
`
`,
`
`Identity
`8-meth'yldecanoic
`acid
`IO-methylundecanoic
`acid '
`
`l0—methyldodecanoic
`acid
`
`A-21978C
`
`Factor
`C1
`
`C2
`
`C3
`
`C0
`
`Carbon
`
`Content
`C11
`
`C11
`
`C13
`
`C10
`
`—
`
`15
`
`L-Thr .
`
`.
`
`C4
`
`C5
`
`'
`
`C12
`
`C12
`
`-—
`
`—
`
`_.
`
`Subtractive Edman degradation reactions indicate
`that tryptophan is the N-terminal amino acid and that an 20
`aspart1c acid molety IS the next adjacent ammo. acid.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`
`
`Gas-chromatographic mass-spectral studies on ‘A- 21978C factor C2 indicate that one of the two following
`
`I
`
`i
`
`.
`
`.
`
`.
`
`.
`
`.
`
`sequences could be the structure of this factor (Asx
`1nd1cates aspart1c acid or asparagme and MeGlx indi-
`cates 3-methylglutamic acid or 3-methylglutamine):
`
`.
`
`.
`
`25
`
`Ir
`L-Asp
`
`L'-A.§P
`
`l~"TTP
`
`NH
`R
`
`|
`
`wherein 3MG represents L-threo-3-methy1glutamic
`
`‘Identity not yet determined
`
`Enzymatic hydrolysis of A-2l978C factor C3, using
`»
`carboxypeptides Y con?rmed that kynurenine is the 40
`The A_21973C complex and factors ‘(as Na Salts) are
`C-terminal amino acid and that the C-terminal COOH
`soluble in water and in acidic and alkaline solutions,
`group may esterify the hydroxyl group of the t1" eolline
`except at pH levels of below about pH ‘3.5; in lower
`moiety-
`_
`alcohols such as methanol, ethanol, propanol, and buta
`Based on the foregoing Studies,- the Structure of the‘
`A-2l978C antibiotics is believed to be as follows:
`' 45 n01; and in dimethylfol-mamide, dimethy] smfo'xide,
`dioxane, and tetrahydrofuran; but are onlyrslightly solu
`ble or are insoluble in acetone, chloroform, diethyl
`ether, benzene, ethyl acetate, and hydrocarbon sol
`50 vents. The salt forms of the A-21978C complex and
`factors are soluble‘ in water, methanol, dimethylform
`amide, and dimethyl sulfoxide; but are insoluble in sol
`vents such as ethanol, butanol, andldioxane.
`Table II summarizes the approximate percentage
`55 elemental composition of the sodium salt of each of the
`A-2l978C factors.
`'
`'
`
`L—~Asp
`/ \
`
`i
`
`‘
`
`"
`
`'
`
`TABLE II
`A—21978C Factor
`C5
`_
`C3
`C4
`C7
`C1
`C0
`Calcd Found Calcd Found ‘ Caled Found Found Calcd Found Found
`
`52.61
`6.07
`13.63
`26.28 '
`1.40
`
`52.07
`5.95
`12.73 v‘
`25.84
`3.41
`
`52.89
`6.14
`13.52
`26.06
`1.39
`
`52.47
`5.93
`13.38 -
`26.19
`2.03
`
`53.17
`6.21
`13.41
`25.84
`1.38
`
`51.37
`6.05
`13.66
`25.86
`2.56
`
`52.73
`5.99
`14.07
`25.81
`1.40
`
`53.44 _
`6.28
`13.29
`25.63
`1.36
`
`52.76
`54.18
`6.71
`6.35
`13.97
`13:34
`25.06 > 25.60
`1.07
`0.96
`
`Element
`
`Carbon
`Hydrogen
`Nitrogen
`Oxygen
`Sodium‘
`‘by difference
`
`PETITIONERS
`
`EXHIBIT NO. 1009 Page 10 of 25
`
`

`

`4,331,594
`5
`The infrared- absorption spectra of the A-2l978C
`complex and» factors (as Na salts) in KBr pellet are
`shown in FIGS. 1-7 of the accompanying-drawings.
`Table III summarizes the most signi?cant absorption
`maxima for each of these.
`TABLE III
`IR Maxima (cm‘l) of the A'-2l978C Complex and Factors
`’ Complex
`C0
`C1
`C2
`C3
`C4
`C5
`
`3300
`3050
`2910
`2840
`1650
`1540
`1445
`1395
`
`1215
`1155
`1060
`745
`
`3300 I 3310
`3040
`3050
`2910
`2910
`2840
`2840
`1650 I
`1665
`1535
`1535
`1450
`1450
`1395
`1400
`
`3310 ‘3320
`3040
`3050
`2910
`2920
`2835
`2850
`1650
`1655
`1535
`1525
`1450
`1455
`1395
`1395
`
`1220
`1160
`1065
`745 .
`
`1225
`1160
`31065
`745
`
`1225
`1160
`1060
`745 .
`
`1220
`1160
`1065
`740i
`
`3300
`3045
`2910‘ _
`2840
`1650 ‘
`1525
`1445
`1390.
`
`i
`
`‘
`
`1215
`1155
`1055
`735
`
`3310
`3050
`2910
`2840
`1655
`1540
`1450
`1395
`
`1310
`
`1240
`1160
`1065
`745
`645
`555
`518
`
`The approximate molecular weights and molecular
`formulas of the three major A-_21978C factorsare sum
`marized in Table IV.
`TABLE IV
`Molecular Weight
`1622
`1636
`1650
`1664
`~1650
`1650
`
`A-2l978C Factor
`(:0
`v(:1
`c2
`c3
`. c4
`c5
`
`' Formula
`C1zH1ooN16027
`C73Hl0ZN16027
`C74-HlO4N16O27
`C75Hl06Nl6027
`C74Hl04Nl6O27
`C74H104N16027
`
`Table V summarizes the absorption maxima of the
`ultraviolet absorption spectra of the three major A
`21978C factors (Na salt forms) in neutralethanol.
`TABLE V
`UV Maxima [ethanol-neutral)
`
`' ' \
`
`En’mlqa .
`
`10
`
`20
`
`30
`
`35
`
`40
`
`6
`TABLE VII
`Optical Rotations
`
`A-2l978C
`Factor
`c0
`C1
`C2 '
`C3.
`C4
`c5
`
`Rotation
`+119° (c 0.7, H20)
`+169n (c 0.7, H20)
`+18.6° (c 0.9, H20)
`+209“ (c 0.4, H20)
`+ 14.8° (C 0.7, H20)
`+179“ (c 0.7, H20)
`
`The A-2l978C factors may be separated by high-per
`formance liquid chromatography (HPLC), using the
`following conditions:
`Column: glass, l><2l cm
`Packing: silica gel/C18 (Quantum LP-l)
`Solvent: water:methanolzacetonitrile (95:30:75) con
`taining 0.2% acetic acid and 0.2% pyridine
`Detector: UV at 285 nm
`Pressure: 100 psi
`The retention times for the A-21978C factors (Na salts)
`are summarized in Table VIII.
`TABLE VIII
`HPLC Retention Times
`Time
`Bioassay (Microcaccus luteus)
`(minutes)
`(units/ mg)
`6
`966
`8
`1663
`9
`1410
`13
`1390
`14
`1246
`19
`803
`
`A-2 l 978C
`Factor
`co
`C1
`C4
`C2
`C5
`C3
`
`'
`
`The 'A-2l97'8C complex can be separated and distin
`guished from A-2l978 factors A, B, D and E by using
`silica-gel thin-layer chromatography (TLC). Acetoni
`trile:water (3:1) is a preferred solvent system, and bi
`oautography with Micrococcus luteus is a preferred de
`tection method. The approximate Rf values of these
`A-2l978 factors (Na salt forms) are given in Table IX.
`TABLE IX
`A_2l978 Factor
`A
`B
`C complex
`D
`E
`
`RfValue
`0.66
`0.57
`0.31
`0.51
`0.48
`
`The factors of the A-2l978C complex can be sepa
`rated and distinguished from each other most conve
`niently using reversed-phase silica-gel TLC (Quantum,
`C18)- A preferred solvent system is waterzmethanol
`:acetonitrile (45:15:40) which contains 02 percent pyri
`dine and 0.2 percent acetic acid. Long-wave UV light
`(365 nm) may be used for detection. The approximate
`Rfvalues of the A-2l978C factors (Na salt forms) in this
`system are given in Table X.
`TABLE X
`A-2l978C Factor
`Co
`C1
`C2
`C 3
`C4
`
`RfVaIue
`0.71
`0.64
`0.56
`0.47
`0.63
`
`nm
`223
`260
`280
`I 290
`360
`
`C1
`307
`62
`39
`35
`33
`
`C2
`303
`62
`41
`36
`33
`
`c3
`300
`63
`42
`38
`32
`
`Table VI summarizes ,the the electrometric titration
`data, as determined in 66% aqueous dimethylformam
`ide, for the three major A-21978C factors and the A
`21978C complex (Na salt forms).
`'
`TABLE VI
`Titration 166% DMF)
`A-2l978C
`pKa Values‘
`Factor C1“ 5.7, 5.9; 7.2, 7.6
`Factor C2“ 5.8, 5.93; 7.6, 7.63
`Factor C3“ 5.73, 5.75; 7.54, 7.58
`Complex
`562; 7.16
`‘All have lesser groups at 11.5-12
`"Two determinations
`
`The optical rotations of the-A-21978C factors (Na
`salts), [a]D25, when determined \in'water are summa
`rized in Table VII.
`'
`
`45
`
`55
`
`60
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`65
`
`PETITIONERS
`
`EXHIBIT NO. 1009 Page 11 of 25
`
`

`

`7
`TABLE X-continued
`A-2l978C Factor
`C5
`
`RfValue
`0.53
`
`4,331,594
`8
`palmitic, cholic, pamoic, mucic, D-glutamic, d-cam
`phoric, glutaric, glycolic, phthalic, tartaric, lauric, stea
`ric, salicylic, methanesulfonic, benzenesulfonic, sorbic,
`picric, benzoic, cinnamic and like acids.
`'
`7
`It is well known in the veterinary pharmaceutical art
`that the form of an antibiotic is not ordinarily of great
`signi?cance when treating an animal with the antibiotic.
`In most cases, conditions within the animal change the
`drug to a form other than that in which it was adminis
`tered. The salt form in which it may be administered is,
`therefore, not of great signi?cance. The salt form may,
`however, be chosen for reasons of economy, conve
`nience, and toxicity.
`The novel antibiotics of this invention are produced
`by culturing an A-21978-producing strain of Streptomy
`ces roseosporus under submerged aerobic conditions in a
`suitable culture medium until substantial antibiotic ac~
`tivity is produced. The antibiotics are recovered by the
`use of various isolation and puri?cation procedures .
`recognized in the fermentation art.
`
`20
`
`The A-2l978C factors and the A-2l978C complex are
`stable in solutions having a pH of 2—9 at 5° C. and 25° C.
`for at least seven days. They are unstable at pH 11 after
`four hours (total inactivation) at both 5° C. and 25° C.
`The A-2l978 and A-2l978C complexes and individ
`ual A-21978C factors C0, C1, C2, C3, C4 and C5 are
`capable of forming salts. These salts are also part of this
`invention. Such salts are useful, for example, for sepa
`rating and purifying the complexes and the individual
`factors. In addition, pharmaceutically acceptable salts
`are especially useful. “Pharmaceutically-acceptable”
`salts are those in which the toxicity of the compound as
`a whole toward warm-blooded animals is‘ not increased
`relative to the non-salt form.
`It will be appreciated that the A-21978 antibiotics
`have as many as ?ve free carboxyl groups which can
`form salts. Partial, mixed and complete salts are, there
`fore, contemplated as part of this invention. In prepar
`ing these salts, pH levels greater than 10 should be
`avoided due to the instability of the antibiotics at such
`levels.
`The A-21978 antibiotics also have two free amino
`groups and can, therefore, form mono- or di-acid-addi
`tion salts.
`Pharmaceutically-acceptable alkali-metal, alkaline
`earth-metal and amine salts and acid-addition salts are
`particularly useful. Representative and suitable alkali
`metal and alkaline-earth metal salts of the A-2l978 anti
`biotics include the sodium, potassium, lithium, cesium,
`rubidium, barium, calcium and magnesium salts. Suit
`able amine salts of the A-21978 antibiotics include the
`ammonium and the primary, secondary, and tertiary
`C1—C4-alkylammonium and
`hydroxy-C3—C4-alkyl
`ammonium salts. Illustrative amine salts include those
`formed by reaction of an A-21978 antibiotic with am
`monium hydroxide, methylamine, sec-butylamine, iso
`propylamine, diethylamine, di-isopropylamine, ethanol
`amine, triethylamine, 3-amino-l-propanol and the like.
`The alkali-metal and alkaline-earth-metal cationic
`salts of the A-21978 antibiotics are prepared according
`to procedures commonly used for the preparation of
`cationic salts. For example, the free acid form of A
`21978C factor C1, is dissolved in a suitable solvent such
`as warm methanol or ethanol; 2. solution containing the
`stoichiometric quantity of the desired inorganic base in
`aqueous methanol is added to this solution. The salt thus
`formed can be isolated by routine methods, such as
`?ltration or evaporation of the solvent.
`The salts formed with organic amines can be pre
`pared in a similar manner. For example, the gaseous or
`liquid amine can be added to a solution of A-21978C
`factor C1 in a suitable solvent such as acetone; the sol
`vent and excess amine can be removed by evaporation.
`Representative and suitable acid-addition salts of the
`A-21978 antibiotics include those salts formed by stan
`dard reaction with both organic and inorganic acids
`such as, for example, hydrochloric, sulfuric, phos
`phoric, acetic, succinic, citric, lactic, maleic, fumaric,
`
`THE MICROORGANISM
`The microorganism of this invention was studied and
`characterized by Frederick P. Mertz and Ralph E.
`Kastner of the Lilly Research Laboratories.
`The new organism useful for the preparation of the
`A-2l978C antibiotics was isolated from a soil sample
`collected on Mount Ararat, Turkey. This organism is
`classi?ed as a novel strain of Streptomyces roseosporus,
`Falcao de Morias and Dalia Maia 1961. This classi?ca
`tion is based on a comparison with published descrip
`tions [R. E. Buchanan and N. E. Gibbons, “Bergey’s
`Manual of Determinative Bacteriology,” The Williams
`and Wilkins Company, 8th Ed, 1974; and E. B. Shirling
`and D. Gottlieb, “Cooperative Description of Type
`Strains of Streptomyces,” Intern. Journal of Systematic
`Bacterial, 808-809 (1972)].
`This classi?cation is based on methods recommended
`for the International Streptomyces Project [E. B. Shirl
`ing and D. Gottlieb, “Methods of Characterization of
`Streptomyces Species,” Intern. Journal of Systematic
`Bacterial 16, 313-340 (1966)] along with certain supple
`mentary tests. Carbon utilization was determined on
`ISP #9 basal medium to which carbon sources were
`added to equal a ?nal concentration of 1.0%. The car
`bon sources were sterilized by ?ltration; the basal me
`dium was sterilized by autoclaving. Plates were read
`after 14 days incubation at 30° C. The cell-wall sugars
`were determined using a modi?cation of the procedure
`of Lechevalier, (M. P. Lechevalier, “Chemical Meth
`ods as Criteria for the Separation of Actinomycetes into
`Genera,” Workshop sponsored by the Subcommittee on
`Actinomycetes of the American Society of Microbiol
`ogy, Dr. Thomas G. Pridham, Convenor; held at the
`Institute of Microbiology, Rutgers University, The
`State University of New Jersey, New Brunswick, N.J.,
`1971.) The isomer of diaminopimelic acid was deter
`mined using the method of Becker et al. [B. Becker, et
`al., “Rapid Differentiation Between Norcardia and
`Streptomyces by Paper Chromatography of Whole Cell
`rHydrolysates,” Appl. Microbiol. 11, 421-423 (1964)].
`
`40
`
`45
`
`55
`
`65
`
`PETITIONERS
`
`EXHIBIT NO. 1009 Page 12 of 25
`
`

`

`Amino acid analysis was determined with washed cell
`wall fragments. Melanoid pigments were determined
`using ISP #1 (tryptone-yeast extract broth), ISP #6
`(peptone-yeast extract iron agar), ISP #7 (tyrosine
`agar), ISP #7 modi?ed (ISP #7 without tyrosine), and
`a tyrosine assay [Yuzuru Mikami, et al., “Modi?ed Arai
`and Mikani Melanin Formation Test of Streptomyces,”
`Intern. Journal of Systematic Bacterial. 27(3), 290 (1977)].
`Starch hydrolysis was determined by testing for the
`presence of starch with iodine.
`.
`.
`Temperature range, NaCl tolerance, pH range, and
`antibiotic sensitivity were done using ISP #2 agar me
`dium. The range of temperatures were: 25°, 28°, 30°,
`34°, 37°, 40°, 45°, 50° and 55° C. NaCl tolerance was
`measured by adding NaCl to the agar to equal: 0, 1, 2, 3,
`4, 5, 6, 8, 10 and 12%. These were incubated at 30° C.
`The pH range was measured by adjusting the agar from
`pH 3.0 to 11.0 at increments of 1.0 pH units, just prior to
`pouring. Antibiotic sensitivity was determined using
`sensitivity discs padded onto seeded agar plates.
`Color names were assigned according to the ISCC
`NBS method (K. L. Kelly and D. B. Judd, ‘_‘The ISCC
`NBS Methods of Designating Colors and a Dictionary
`of Color Names,” US. Department of Commerce Circ.
`553, Washington, DC, 1955).
`Figures in parentheses refer to the Tresner and
`Backus color series [H. D. Tresner, and E. J. Backus,
`“System of Color Wheels for Streptomycete Taxon
`. omy,” Appl. Microbiol. 11, 335-338 (1956)]. Color tab.
`designations are underlined. The Maerz and. Paul color .
`blocks. are enclosed in brackets (A. Maerz and M. R.
`Paul, “Dictionary of Color,” McGraw-Hill Book Com
`pany, Inc., New York, N.Y., 1950).
`CHARACTERIZATION OF
`A-21978-PRODUCING STRAIN
`Morphology
`The morphology of culture A-21978.6, the culture
`which produces the A21978 antibiotics, consists of spo
`rophores which are of the Rectus-Flexibilis (RF) classi
`?cation. Spore chains have > 10 spores per chain. Spore
`surface is smooth.
`'
`Culture A-2l978.6 is characterized by the production
`of a predominantly red aerial spore mass color, with a
`reddish-borwn reverse color. A light-brown water-sol
`uble pigment is also present. These characteristics are
`exhibited on three of 14 agar plating media (ISP #2, ISP
`#7, TPO). These three media are the only ones which
`supported abundant aerial and vegetative growth.
`'
`
`4,331,594
`10
`Two agar plating media, ISP #4 and glucose
`asparagine- agar, produced a white-to-gray aerial spore
`mass color, with a yellow reverse color. No water-‘solu
`ble pigment was observed. These two media supported
`good, but not abundant, aerial and vegetative growth.
`Nine other agar plating media were used, but these
`gave poor-to-no growth and sporulation. Aerial color
`when present, although poor, was in the white-to-gray
`color series.
`.Melanoid pigments are absent. Major constituents of
`the cell wall are: LL-DAP, glycine, glucose, and ribose.
`This indicates a Type I cell wall, and type C sugar
`pattern (R. E. Buchanan, and N. E. Gibbons, Eds.,
`“Bergey’s Manual of Determinative Bacteriology,” The
`Williams & Wilkins Company, 8th Edition, 1974, p.
`658).
`The following ?ve cultures were compared in labora
`tory tests to A-21978.6:
`Streptomyces albovinaceous ISP 5136; ATCC 15833
`Streptomyces candidus ISP 5141; ATCC 19891
`Streptomyces moderatus ISP 5529; ATCC 23443
`Streptomyces roseosporus ISP 5122; ATCC 23958
`Streptomyces setonii ISP 5395; ATCC 25497
`These cultures belong to the white and red color
`series, have RF type sporophore morphology, smooth
`spore surface ornamentation, and, according to the ISP
`descriptions, are melanin negative and do not have a
`distinctive reverse color or water-soluble pigments.
`These characteristics, together with carbon-utilization
`pattern and other secondary features, match those of
`culture A-2l978.6.
`When these cultures were compared with A-2l978.6
`under laboratory conditions, four were rejected. S.
`candidus and S. setonii exhibited a yellow aerial spore
`mass on many media, thereby differing from culture
`A-21978.6. S. albovinaceous and S. moderatus exhibited
`dark distinctive reverse color, water-soluble pigments,
`and produced melanoid pigments, all of which were
`different from culture A-21978.6. The ISP description
`of S. maderatus refers to. reddish brown or strong
`brown reverse color, but does not refer to such a char
`acteristic for S. albovinaceous. Neither culture is listed as
`melanin positive.
`Culture A-2l978.6 was classi?ed, therefore, as a
`strain of Streptomyces roseosporus, Falcao de Morias and
`Dalia Maia 1961. This classi?cation was based on com
`parison with published descriptions and direct labora
`tory comparisons. The following cultural characteris
`tics summarize the direct comparison studies.
`
`20
`
`25
`
`35
`
`40
`
`45
`
`CULTURAL CHARACTERISTICS
`Morphology
`
`Spores: Oblong to oval
`Average: 0.85 X 1.78 p.M
`Range: 0.65-0.97 X O.97—2.6 PM
`Growth
`Color
`
`MEL
`Sporophores straight to ?exuous (RF), with no hooks, loops or spirals observed.
`Chains of spores >10. The spore surface smooth as determined
`by scanning electron microscopy.
`Oblong to cylindrical
`1.01 X 2.47 ;1.M
`O.97—l.3 >< 1.63-3.25 p.M
`Growth
`Color
`was
`none
`good
`
`Aerial: g‘o‘od
`Vegetative: abundant
`
`gray 6 pink
`brown
`
`none
`yellow-brown
`
`PETITIONERS
`
`EXHIBIT NO. 1009 Page 13 of 25
`
`

`

`11
`
`‘4,331,594
`
`‘ -conti‘nued
`
`CULTURAL‘CHARACTERISTICS ,
`
`'
`
`’
`
`Morphology
`
`'
`
`12
`
`A21978.6
`'
`S. raseosgorus
`Sporophores straight to flexuous (RF), with ‘no books, loops or'spirals observed.
`Chains of spores > 10. The spore surface smooth as determined
`by scanning electron microscopy. >_
`Oblong to cylindrical
`1.01 X 2.47 uM
`0.97-1.3 X 1.63-3.25 uM
`Growth
`Color
`
`Spores: Oblong to oval
`Average: 0.85 X 1.78 MM
`Range: 0.65-0.97 >< O.97-2.6 pM
`Growth
`Color
`
`no soluble pigment
`
`gray 6 pink
`Aerial: good
`brown
`Vegetative: abundant
`dark brown soluble pigment
`
`Aerial: fair
`Vegetative: good
`no soluble pigment
`
`(W)a white
`[lOAl] pale yellow green
`
`no soluble pigment
`Potato Plugs
`,none
`fair
`-'
`no soluble pigment
`[SP #l (Tryptone-yeast ext. agar!
`poor
`poor
`_
`no soluble pigment
`IS? #2 (Yeast-malt extract agar)
`abundant
`
`abundant
`
`Aerial: abundant
`
`Vegetative: abundant
`
`(R) Seb gy.
`yellow pink
`[SD10] lt.
`red brown
`
`light brown so]. pigment
`
`Aerial: fair
`(W)a white
`Vegetative: fair
`[lOAZ] pale yellow pink
`light brown sol. pigment
`
`none
`orange-brown
`
`‘
`
`1 "
`
`(W)a white
`[1082] pale yellow green
`
`(R) 30a pale orange yellow
`
`’
`
`[lZL7] lt. olive brown
`7
`'
`
`(W)a white .
`pale greenish gray
`
`(R) 3C2 pale orange yellow
`[1 115] grayish yellow
`
`light brown sol‘ pigment
`IS? #3 (Oatmeal agar)
`poor
`fair -
`no soluble pigment
`ISP #4 (Inorganic salts starch agar) -
`good
`Aerial: good
`(W)b white
`Vegetative: good
`[1081] pale yellow-green
`abundant
`light brown sol. pigment
`no soluble pigment
`lSP #5 (Glycerol - asparagine agar)
`(W) l3ba purplish white
`‘
`(W) b white
`,_l_‘air_
`[lOCZ] grayish yellow
`‘good
`[387] gy. yellow pink
`light brown sol. pigment
`.
`ISP #7 (Tyrosine agar)
`Aerial: abundant
`(R) '5cb gy. yell. pink
`(R) Scb gy. yell. pink
`abundant
`Vegetative: abundant
`[l IE5] yellow-brown
`[7Ll2] mod. red brown
`abundant
`dark brown sol. pigment
`light brown sol. pigment
`.
`Bennett‘s modi?ed agar
`'1
`abundant
`(R) Seb gy. yell. pink
`abundant
`[l lD4] grayish yellow
`light brown sol. pigment
`'
`Calcium malate agar
`v(W) a white
`poor
`pale yellow~grleen
`poor
`pale yell-green sol. pigment
`Czapek's solution agar
`none
`none
`
`Aerial: fair
`Vegetative: good
`gy. pink sol. pigment
`
`'
`
`'
`
`7
`
`Aerial: none
`Vegetative: poor
`no soluble pigment
`
`pale yellow br.
`
`Aerial: none
`Vegetative: fair
`[7Ll2] mod. red brown
`light brown sol. pigment
`
`Aerial: poor
`Vegetative: poor
`no soluble pigment
`
`(W)a white
`off-white
`
`—
`—
`
`—-
`[13L6]
`
`(W)b white
`[1282] gy. yellow
`
`[8Ll2] dk. gy. brown
`
`pale yellow-gray
`
`Aerial: poor
`Vegetative: abundant
`no soluble pigment
`
`Aerial: good
`Vegetative: good
`no soluble pigment
`
`Aerial: poor
`Vegetative: abundant
`brown soluble pigment
`
`Aerial: none
`Vegetative: poor
`no soluble pigment
`
`Aerial: abundant
`Vegetative: abundant
`brown soluble pigment
`
`Emerson's agar
`(R)5cb gy. yell. pink
`abundant
`[i115] gy. yellow
`abundant
`light brown sol. pigment .
`Glucose - asparagine agar
`fair
`good
`no soluble pigment
`Glycerol - glycine'agar
`(W)b white
`abundant
`[lOG3] light yellow
`abundant
`light brown sol. pigment
`Nutrient agar
`;
`fair
`good
`no soluble pigment
`(Tomato-paste Oatmeal agar!
`(R)5cb gy. yell. pink
`abundant
`(R) Scb gy. yell. pink
`[8Ll2] dk. gy. brown
`abundant
`[lZL7] yell.'brown '
`brown soluble pigment
`
`(W)b white
`[1282] pale yell. green
`
`(W)b white
`pale yellow gray
`
`PETITIONERS
`
`EXHIBIT NO. 1009 Page 14 of 25
`
`

`

`13
`
`Carbon Utilization
`A2 1978.6
`
`S. roseosporus
`
`Substrate
`L-Arabinose
`D-Fructose
`D-Galactose
`D-Glucose
`i-Inositol
`D-Mannitol
`D-Raf?nose
`L-Rhamnose
`Salicin
`Sucrose
`D-Xylose
`
`Key:
`+ = Positive utilization
`— = Negative utilization
`
`4,331,594
`
`l0
`
`15
`
`20
`
`25
`
`14
`.
`ces roseosporus NRRL 11379 which produce the A
`21978 antibiotics may be used in this invention.
`The culture medium used to grow Streptomyces ro
`seosporus NRRL 11379 can be any one of a number of
`media. For economy in production, optimal yield, and
`ease of product isolation, however, certain culture
`media are preferred. Thus, for example, a preferred
`carbon source in large-scale fermentation is tapioca
`dextrin, although glucose, fructose, galactose, maltose,
`mannose, cottonseed oil, methyl oleate, glycerol, re
`?ned soybean oil, and the like can also be used. A pre
`ferrednitrogen source is enzyme-hydrolyzed casein,
`although soluble-meat peptone, soybean ?our, soybean
`hydrolysate, soybean grits, yeast, amino acids such as
`L-asparagine and DL-leucine, and the like are also use
`ful. Nutrient inorganic salts which can be incorporated
`in the culture media are the soluble salts capable of
`yielding potassium, ammonium, chloride, sulfate, nitrate
`and like ions. Among these, K2804 is especially useful
`for antibiotic production. Molasses ash, ash dialysate
`and synthetic mineral mix are also useful.
`For production of the A-21978 a