BE538487A - - Google Patents

Description

       

    <Desc / Clms Page number 1>
 
 EMI1.1
 



  Antibiotic product.
 EMI1.2
 



  The present invention relates to 1H '.) C "1' -: '::; n'.juv' .- t1: and useful in preparing an antibiotic which 1 = Â Dfiù :, n; 1 = 1 ,; r;;;, p,> <# 1 O'nog .. 'ilycin and that is <J ctu el18nstl, .iair known: J'Il lp l1) il: ttr3cycline. More particulisrC :: i'l "mt, it sir report" '31 df3 particular procedures to separate it and 1 "c ncs, at7: ra; J;> luti: J,> 5 brut'3s, and: 1ot:' ¯TI;: JE: nt broths .le f;:;. I. '"LC'.1t 1'> .i ':: t p.111r purify it.



  In c,>, ir 5 of these s ci emi 1 5 r * = 1; rin, Ô es, a deer in n'.J.! L1Jl "'{'., R.xw-u.itn du, l1é : -! r.Joll '> lld de 1 :: c2ois.¯ ncrm7.e, 1>: 1.c.t'r.i <is,,; li l'JLJl'J1: .ilGC'G. ^. Ci3luiS have lté i3ol'fs ,,; t; oa = C'c. "u; L,:; 1.é"!) '!!

  <Desc / Clms Page number 2>

 
 EMI2.1
 possessed interesting therapeutic properties. Among
 EMI2.2
 these products one can mention the pnieil-liiiee the streptomycin the graalicidin the tyrocidin the bwci racie, the subtilize the St'1 "ç? t0 t3.r7. ^ ii e,. lI rJ; 3 :: IC? ne C Ciî. O'¯'t trc C; ß C i12 e, the 1 'T'c'J1 j% C'? 3.



  (oiy + t4t1-a.cyrcline) and .'2'Z '?' S.'eI'c '-. C3.I2S of these products are useful by their action on the pathogenic f3r S ..' ". 5:?



  .DJ'eutres n-lort rillylun (- T2.ï. z15.



  Others have only limited utility for various examples because of their toxicity. Chlortetracycline, oxy-tetracycline and tetracycline are useful. by their broad spectrum of activity. P = ti: i1 ce, s der. ';. iers. tetra- eycline is an ar-tibiotic with spectrum = Tg # c, ci, 1r- = rne of better blood levels and --aoi-ns of reactions Z "3 ± î = Îlô.SÀl'eS that r = 1 = 1, <J x <"c é t i <z = r r: 'ton e and 3. -'; ārTCl jîC and 33t in particular pu; s:. \ ble que la = i = 'cr% 1 * é.l:>; a, ;; j; el, 1 = I in the ..i12i, e =: x ". Icplins.



  The object of the present invention is to provide methods of preparing tetrscyclina suitable for commercial applications.



  The present invention follows ;, in a process to pre- J ... s¯ '] ¯1 1 L2 :: = # gi: njrei = # e, a space of I3' vl ß VCJ.t: (f ; .. 7> 1 '> ± ..i 1 # "±'. <- from 1 '= ùv: to g to: nj, r cin% in an aqueous solution î.i.32T, -t :.'? ' of c .-. r'-ne <,.> t = oJc-: 1. # iu a nitrogenous nutrient in o3Oi2àl ulOiXs S .-- iD'C12S sub. '. iergées¯, j: sc¯iââ ce no é, C'lf, Iîi % E an appreciable bacterial be given this solution i # "tooi µ? 5n separates 1? # 1> 1; Éjj. #: -.; Iycin ..1.¯ produce fermentation broth J .. precipitate" - tion has the "lin e. i? ti ::: ': 2"' '?: e: obtained by the process of l3. present invention is a substance ¯: '3t' ..r - '.: 2: L! ¯ le i: 'sT #,?' 3 ': = 2:: Âc3 bacteria with Grar.i-positive and with Gr? .- n- = tif and .-: G2T.' 'Tlbl3 de: 3T S! Z -.



  '? ".. l? r .18s salts = -Tec 1,5s" 1 ..:.:. WJ si -L's'.' .. 'auj' j! .cm li b? 'c'? 7 " "ej? res? on 1: for ....! l3? ' 1 "to" CprIIJ '.'; "'-" "A -t tî q = .- e n <.i t 1, 1 7flJ -µL 7¯à. C - '. N? 3 # 1 S co -aà 5 * = = ir, u "L; 1't W7..ï.t z'l L f.a7j.¯ ¯ / -. - z7' 'J * - 2 /> 5 L "..i .. f¯ '. .i; m '.'-. 1 ..- t: ..f 1 1, -'? ?> .- '*' ";; - fl ...;:

   D 1

  <Desc / Clms Page number 3>

 shows absorption peaks in the ultra-violet at
267m (@ 17, 400) and 355m @ 13, 500) in 0.1 N hydrochloric acid and, when suspended in mineral oil, exhibits characteristic absorption in the infrared region of the spectrum at the following frequencies, expressed in cm-1:

    3490, 1672, 1607, 1524, 1259, 1222, 1130,
1065, 1040, 990, 963, 948, 932, 861, 838, 803, 786, 741,739 and 668, of which the hydrochloride practically melts at 217-219 C. on decomposing, and corresponds to the empirical formula
C22H24N2o3.HCI, has an optical rotation [Ó] D27 = -253 at a concentration of 0.5% in 0.1 N hydrochloric acid and exhibits, when suspended in mineral oil, a characteristic absorption in the infrared region of the spectrum at the following frequencies expressed in cm-1:

  3340, 1678, 1623, 1597,
1315, 1248, 1229, 1175, 1140, 1061, 1036, 1002, 964, 949, 864, 823, 796, 781, 743, 719, 692 and 667, and whose formate melts practically at 176 -177 C and corresponds to the empirical formula C22H24N2O8. HCOOH.



     Omegamycin exerts an inhibitory action on the development of numerous bacteria, as will be described in detail later.



   Omegamycin is prepared, among other methods, by culturing under specially controlled conditions a species of microorganism not previously described which has been tentatively called Streptomyces BL 567201 and which has been isolated from a soil sample. The description of this organism is given below.



   The organism BL 567. 201 sp. nov. which produces Omegamycin belongs to the genus called Streptomyces. The development of this organism is good on glycerol-asparagine-beef extract-agar at 30 C. On this medium mouse gray aerial hyphae are formed, and a yellow green pigment is secreted in the agar. The

  <Desc / Clms Page number 4>

 
 EMI4.1
 mycelium is composed of branched hyphae, the most
 EMI4.2
 young people are Gram-positive. Conidia are produced on aerial hyphae.
 EMI4.3
 



  Ol11eg: n.yclne can also be prepared, among other methods, by culturing teeth under conditions particularly 1 '(>. ::; lée3: of any of a number of species of icroornis - Bies not described so far which have been provisionally pollinated Streptoolyees BL 567714, Streptomyces BL 6 03 Str29-Go "lyces BL b 703., Strepto: llyces 67 $ 040, Streptomyces E1, 67106; Strepto-nyces 673079. Streptomyces 67 & l10, Streptomyces BL 456667 and streptomyces BL 456667A. All of these org: niS! 116S were isolated from soil samples. They all belong to the .E'4. '..w? î7; W: .¯t> ē:. of each of these orgeni 2es on gl'.1 cos ,, D ... ,, (.!' .- "" and ", ', '. de,:,' "..., ''" "" - d n -'- 1 to "'Z" "C"' 6 '' .spp.r "gine-extract become-agpr during ¯l .:. days at 32'C ran and the results were noted.

   The observations made for each Orb3 ..} 'liçs ": C.-' can be found in the table below.
 EMI4.4
 



  0rgznis: = ae Characteristic and Color in a pigment color of the mycelium secreted in the agaz 'BL 567714 Abon aerial mycelium- Soluble pigment dē2t and pineapple white pores
 EMI4.5
 
 <tb> to <SEP> pearl <SEP> smoke
 <tb>
 
 EMI4.6
 EL 673033 Moderate aerial mycelium pilent soluble light gray snanas EL 673035 Colorless colonies pilent soluble Svi1S lTCe.j.L3i.1 aerial pineapple BL 673040 Colorless colonies p # in.>; Jt soluble without aerial iycéliui'1, I1211è ', S EL 6710 / p6 Colorless colonies with PL; '; "rl" "t soluble: Jjr c 41ii.i A aerial white In? ms
 EMI4.7
 
 <tb> very <SEP> rare
 <tb>
 
 EMI4.8
 BL ô7î? 079 Colorless colonies # with soluble pillent mycliuf1- aé1'-iêÍl- s .sn: ': 1.?-3
 EMI4.9
 
 <tb> rare, <SEP> gray <SEP> cement
 <tb>
 
 EMI4.10
 EL 673110 Colorless colonies .t'i: 1f: 'lt soluble:

  1 T. ^. 41.W .t..L aerial "n- nas very laughing, gray cLn11 t

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Growth of Streptomyces BL 456667 on glucose-asparagine-agar gives gray hyphae and a buff reverse.



   Streptomyces BL 456667A is isolated from Streptomyces
BL 4.56667 which was itself isolated from a soil sample.



   Glucose-asparagine-agar plates are thus prepared containing
0.01% 2,3,5-triphenyl-tetrazolium chloride (TTZ), according to the method described by Huddleson & Baltzer (Science 112: 651 (195 @) A culture of Streptomyces is spread on the surface of the agar. BL456667 using sterile glass rods so as to obtain well isolated colonies The plates are incubated at 30-32 C and observed daily. After incubation for five days, two distinct colonies are observed. Streptomyces BL 456667A exhibit white aerial hyphae, lavender reverse.



   The color descriptions come from the Maerz & Paul Color Dictionary, 1st edition.



   For the preparation of omegamycin, the Applicant does not wish to be limited to this particular organism or to organisms fully corresponding to the description given above, purely by way of illustration. It particularly wishes to reserve the use of organisms which are mutation products obtained from the organism described by mutation agents such as x-rays, ultra-violet rays, nitrogen gases, etc.



     Omegamycin is a useful therapeutic agent, for example in human or veterinary medicine. Omegamycin has the particular advantage of a broad spectrum of activity, high blood levels and low toxicity. Omegamycin has an extremely useful resistance to degradation by heat or water in acidic or alkaline medium. Certain metals and acid addition salts of tetracycline are even more useful than base because of their lower hygroscopicity and greater solubility in water.

  <Desc / Clms Page number 6>

 
 EMI6.1
 



  The antibiotic, Ompga.nyc3.ne, is active in vitro against
 EMI6.2
 a number of Gram-positive and Gram-negative bacteria.
 EMI6.3
 



  The following table shows the antibiotic activity of 1-'Omegamycin (lot 25), Aureomycin i3Cl, lerranycine HCl and
 EMI6.4
 tetracycline placed in a trench made in a heart agar-infusion plate (pH 7.0):
 EMI6.5
 OMEGAi PLATE SPECTRUM.V1YCIN 5m g cm.3
 EMI6.6
 Zone d5îninhibiti-on en. rnm.



  Omegaamycin Tetracycline Aureomycin Terramycin lot 253 (: Brparée en (.3 mg! Em3) (1 mg! E: n3) (5mg / c3i) dechlorantia ciil.o r tetracy-
 EMI6.7
 
 <tb> cline
 <tb>
 
 EMI6.8
 (5 mg! Cm3) Organized (mg / cm3);
 EMI6.9
 
 <tb> Organized <SEP> of
 <tb>
 
 EMI6.10
 Bodenheimcr 6 8 '' 11 9 Prot2us XI3 7 8 8 10 Sh.

   SOIDlei 5 8 10 Il S. zte: ci tidi s 9 11 'il 13 S. par; tyii'i A 10 12 13 15 S. j3iAll-tJ ± Èl? l 8 11 11 13! 1 .. eo ra en e s 5 '6 9 11 Ps.fh1.Ol'BSCel1.S 1 7 9 13 A1 c. fcc: ¯l? s 6 11 th <5 - PT'.3rul; a-ris 0 6 il 0 lT. 9llJl Qtae 8 Il l3 lez Neisscria sp. 8 7 il C.xerosis 11 14 Il> 27 B> uy = co 5..i e 10 10 16> 27 B. ce s 10 11 13 1l S. m rcescens 0 2 2 0 1.1. tetr'Jgénu s Il 15 27> 27 s, ii em ei, 1 14 11 10 14 S. QvsetGrie 9 Il 10 15 C. 21 '; Di c ::, n s 6 .0' 0 0
 EMI6.11
 
 <tb> S <SEP> t <SEP> io <SEP> h. <SEP> to <SEP> r <SEP> had <SEP> 10 <SEP> 13 <SEP> 13 <SEP> 18
 <tb>
 
 EMI6.12
 E.tJPhS2 7 9 10 13 F. coli 4 7 sil 11 S. p: r typh3.

   B 9 ¯ ¯ 11 16 14 .l \ .. p11J; U: 1l :: mi? - e 5 (D 16 14 P s. 2eru ino se. Cl 4 9 S. llin.-n rui-1 -8 II 12 13 B.ntllrcis' 7 10 19,>, 27 S. 5chott'J.ul1eri 10 12 12 15 B.subtilis 10 9 il 13 B. mycoides 0 13 8 15 20

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 The spectrum test is performed as follows: About 30 cm3 of sterile heart infusion broth (Difco) with 2% agar added as a solidifying agent is placed in
 EMI7.1
 ru v, -. S ade sterile Petri dish (90 ksi d-iameter), and allowed to harden. A trench of 8 by 40 ins is then made in the agar using a sterile spatula. The bottom of the trench is closed with a drop or two of molten agar.

   We then make a trail using a small loop starting from the edge of the trench and going towards the wall of the Petri vessel, with a culture in nutrient broth of 24 hours of each.
 EMI7.2
 of the test t6ri'ds, previously incubated at 37 C. We re: r! l1:; rlj.t: xiii; 1. <this the trench of a solution at 5 µg / c-, z3 of l; '1a: ntibj.ot: .q1 ..:. e.



  On. =;]. #., This vase at 37 C for 18 to 2.4 hours. Linear measurements of the zone of inhibition are taken from the ba :: 4 of the ti.z <: i <# li, 4th until the point where 1 "test organism v .. se" - yc I'ïr. '.. 5 le = 3 where; bî'eb ",' \, ... e'l - la, ".- '1 Gt \ rt -:;. i {) r) Y) 81, êpres -The chosen number of hours and: .Îúr, c.cn period in \ 5 otlée.



  > One will find below a 1. "'É21.1;: é tests 8.' end vitro autibacterian activity of hydrochloride û '.' Tiïl'xE? Ga2: 'iß'L'ïnG' crystallized and de three other antibiotics The test is carried out by a dilution tube procedure and the minimum concentrations of the antibiotic which completely inhibits the growth of bacteria are determined Heart infusion broth is used as medium for all the organisms of test, unless otherwise indicated.

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 EMI8.1
 



  INHIBITION CONCENTRATION INmc cm3
 EMI8.2
 Organism Omëgamycin Chlor- (kyté- Chlors1I1-
 EMI8.3
 
 <tb> HCl <SEP> tetra- <SEP> tracy- <SEP> phenicol
 <tb> cycline <SEP> cline
 <tb>
 
 EMI8.4
 ¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯HC1 HC1
 EMI8.5
 
 <tb> Micrococcus <SEP> pyogenes
 <tb>
 <tb>
 <tb> var.aureus <SEP> 0.15 <SEP> 0.25 <SEP> 0.25 <SEP> 5.0
 <tb>
 <tb>
 <tb> Gaffkya <SEP> tetragena <SEP> 0.03 <SEP> 0.06 <SEP> 0.13 <SEP> 5.0
 <tb>
 
 EMI8.6
 Streptococcus pyogenes G203 2.5 0.06 0.06 0.63 Streptococcus agalactiae 7077 0.3 0.13 0.13 1.25
 EMI8.7
 
 <tb> Streptococcus <SEP> dysgalactiae <SEP> 9926 <SEP> 1.25 <SEP> 0.25 <SEP> 0.13 <SEP> 1.25
 <tb>
 <tb> Streptococcus <SEP> uberis <SEP> - <SEP> 0.13 <SEP> 0.13 <SEP> 2.5
 <tb>
 <tb> Diplococcus <SEP> pneumoniae <SEP> 0.15 <SEP> 0.06 <SEP> 0.06 <SEP> 1.25
 <tb>
 
 EMI8.8
 Lactobacillus acidophilus 356''0.6 0.50 1.0 2,

   5 Lactobacillus casei # 4646 * -Y 1.25 0.50 1.0 5.0 Lactobacillus 1eichmannii * 2.5 0.50 1.0 2.5
 EMI8.9
 
 <tb> Bacillus <SEP> anthracis <SEP> 0.0375 <SEP> 0.02 <SEP> 0.03 <SEP> 2.5
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> Bacillus <SEP> cereus <SEP> var. <SEP> mycoides <SEP> 0.0375 <SEP> 0.02 <SEP> 0.03 <SEP> 2.5
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> Bacillus <SEP> subtilis <SEP> 5.0 <SEP> 1.0 <SEP> 1.0 <SEP> 2.5
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> Corynebacterium <SEP> xerosis <SEP> 0, <SEP> 6 <SEP> 0, <SEP> 25 <SEP> 0, <SEP> 25 <SEP> 0.63
 <tb>
 
 EMI8.10
 C10stridium welchii 601 * -.1f 7 (- 0.06 0.06 0.13> 6.25 Clostridiun welchii M * ² *.

   0.25 0.13 0.25> 6.25 C1ostric1iuL1 sporogenes 8J "0.125 0.25 0, .25 6, 25 Clostridium sporogenes 40 *** 0.06 0.06 0.13 6, 25
 EMI8.11
 
 <tb> Salmonella <SEP> typhosa <SEP> 2.5 <SEP> 0.39 <SEP> 0.78 <SEP> 1.56
 <tb>
 <tb> Escherichia <SEP> coli <SEP> 2.5 <SEP> 1.56 <SEP> 1.56 <SEP> 12.5
 <tb>
 
 EMI8.12
 Shigella sonnei 1.25 0.39 0, ig 1, 56 Klebsiella pneumoniae 2.5 0, 78 0.78 I, 56
 EMI8.13
 
 <tb> Proteus <SEP> vulgaris <SEP>. <SEP> 10.0 <SEP> 12.5 <SEP> 25.0 <SEP> 6.25
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> Pseudomonas <SEP> aeruginosa <SEP> 10.0 <SEP> 6.25 <SEP> 1.56 <SEP> 50.0
 <tb>
 
 EMI8.14
 Neisseria sp. 2.5. p, 19 1.56.

   50.0 Candida albicans 520> 100 100 J> JOO> 50
 EMI8.15
 
 <tb> * Test <SEP> in <SEP> of <SEP> broth <SEP> of <SEP> serum <SEP> to <SEP> 10%
 <tb>
 <tb> + * Test <SEP> in <SEP> of <SEP> broth <SEP> of <SEP> juice <SEP> of <SEP> tomatoes
 <tb>
 
 EMI8.16
 '* ** "Test in thioglycollate broth
The diffusion plate assay method for determining Omegamycin activity is shown below.



  Culture centre
Streptomycin Agar (with Yeast Extract) is available from Baltimore Biological Laboratories, Baltimore, Maryland, and used as directed by
 EMI8.17
 The label. A suitable preparation can be obtained by suspending in one liter of distilled water at a final pH of 6.2 or 8.0 a mixture of 1.5 g. of beef extract, 3 grams of yeast extract, 6.0 grams of peptone (eg Gelysate) and 15 grams of agar. The suspension is left to stand for
 EMI8.18
 Mix for 5 minutes, until a uniform suspension is obtained and heat moderately with stirring. We boil

  <Desc / Clms Page number 9>

 suspension for one or two minutes, or until solution has formed.

   The culture medium is then distributed and sterilized at 121 ° C. (1 kg / cm2 of vapor pressure on a manometer for 15 minutes).



   Incculum
The test organism is Bacillus subtilis American Type Culture Collection 6633. A spore suspension containing 50,000,000 viable spores per cm3 is added to the molten test agar described above (cooled to 53 ° C) to obtain a final 2% inoculum.



    Plate preparation
21 cm3 of sterile test agar prepared as described above is placed in a series of level sterile Petri plates and allowed to solidify. 4 cm3 of inoculated agar are then evenly distributed over the surface of the base layer in each plate. Stainless steel plates with a series of holes are placed on the medium when it has cooled to room temperature and the samples of the antibiotic material to be tested are placed in the holes.



  Buffer   '
Buffer at pH 6.2 or 8.0 as appropriate to perform the dilutions. At pH 6.2, citrate buffer is used to prepare the dilutions. It is obtained by mixing 192.12 g. anhydrous citric acid with 106.3 g. of sodium hydroxide in one liter of distilled water and the mixture is diluted to 1 / 10th of the concentration with distilled water. The pH of the buffer should be checked potentiometrically, and if necessary adjusted to pH 6.2 by addition of citric acid or sodium hydroxide. Changes in the pH or in the concentration of the buffer strongly modify the dimensions of the zones of inhibition.

   We have not. not found necessary to sterilize the tam-, on. The stock solution is stored in chloroform or

  <Desc / Clms Page number 10>

 toluene and new working solutions are prepared Daily * At pH 8.0 phosphate buffer is used to prepare the dilutions. It is obtained by mixing 95 cm3
 EMI10.1
 of molar KzHP04 with 5 cm3 of molar KH 2 P04 and by diluting the mixture to 1 / 10th with distilled water. The pH of the buffer should be checked potentiometrically and if necessary adjusted to pH 8.0 by the addition of either phosphate solution.

   Changes in pH or concentration of
 EMI10.2
 buffer significantly modify the dimensions of the inhibition zones. It was not found necessary to sterilize the tampon.
 EMI10.3
 The stock solution is stored in chloroform or toluene and new working solutions are prepared daily.



  Test
Unknown samples are diluted in buffer if necessary. Three holes in each plate are used to accommodate the same dilution of the sample. After incubation at 32 ° C., the diameters of the zones are measured and their average is established.



   Streptomyce s BL 567201 is differentiated from a
 EMI10.4
 strain of S. aureoaciens (NRBL 2209Y -obtained from the Northern Regional Eeseereh Laboratory, Peorie., Illinois, '. or it had been deposited as an authentic strain producing aureomycin, by observation of the developmental characteristics on a medium. <"c4rol- asparagine-beef extract-agar and Cz.8-pek-Dox agar containing dextrin.

   The agar mixtures - used and the results obtained are as follows: Glyceroî-asnaraine-beef extract-agar
 EMI10.5
 
 <tb> Glycerol <SEP> 1%
 <tb>
 
 EMI10.6
 AsDaregine 0.05 Beef extre.it 0, 2d KzHP04 '0, 0 5
 EMI10.7
 
 <tb> Aar <SEP> 1.5%
 <tb>
 <tb> Water <SEP> sterile <SEP> q.s.

    <SEP> 100%
 <tb>
 pH 7.2

  <Desc / Clms Page number 11>

 
 EMI11.1
 
 <tb> BL <SEP> 567201 <SEP> Streptomyces
 <tb>
 <tb>
 <tb>
 <tb> aureofaciens
 <tb>
 <tb>
 <tb>
 <tb> Development <SEP> Good <SEP> Good
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> Sporulation <SEP> Good <SEP> Good
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> Pigment <SEP> diffusible <SEP> Yellowish-green <SEP> Not
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> Training <SEP> of
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> spirals <SEP> Abundant, little <SEP> coiled <SEP> Not
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> Hyphae Aerial <SEP> <SEP> Mouse-gray <SEP> Gray-pink
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> Reverse <SEP> Brown <SEP> Olive
 <tb>
 Czapek-Dox dextrin
 EMI11.2
 NaN 3 0, 2 K2HP04 0.1%
 EMI11.3
 
 <tb> MgSO <SEP> 0.05%
 <tb>
 
 EMI11.4
 KC1, o, 05 #
 EMI11.5
 
 <tb> FeSO, <SEP> trace
 <tb> Hagar <SEP> 1,

  5%
 <tb> Water <SEP> sterile <SEP> q.s' <SEP> 100%
 <tb>
 pH 7.2
 EMI11.6
 
 <tb> BL <SEP> 567201 <SEP> Streptomyces
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> @ <SEP> aureofaciens
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> Development <SEP> Enough <SEP> good <SEP> to <SEP> good <SEP> Enough <SEP> good
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> Sporulation <SEP> Good <SEP> Low
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> Pigment <SEP> diffusible <SEP> Not <SEP> not
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> Training <SEP> of <SEP> Abundant, little <SEP> coiled <SEP> Rare, <SEP> very <SEP> little <SEP> en-
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> Spirals <SEP> rolled
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> Hyphae Aerial <SEP> <SEP> Mouse-gray <SEP> Chamois <SEP> to <SEP> gray
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> Reverse <SEP> Brown <SEP> clear, <SEP> Chamois <SEP> to <SEP> brown
 <tb>
 <tb>
 <tb>
 <tb>,

  he
 <tb>
 
Streptomyces BL 567201 is further characterized by the production of an intense bluish-green pigment when grown in submerged culture in a medium containing 1% sucrose, 1% soy flour, 1% soy peptone, 1 , 5% of
 EMI11.7
 KH2PO4 'and 0.5, f of (NH4J2HPO4. Streptomyces aureofaciens (NRRL 2209) does not produce this pigment.



   Streptomyces BL 567201 is further distinguished from Streptomyces aureofaciens by the following observations, shown in the table.

  <Desc / Clms Page number 12>

 
 EMI12.1
 
 <tb>



  S. <SEP> aureofaciens <SEP> Streptomyces
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> Middle <SEP> NRRL <SEP> 2209 <SEP> BL <SEP> 567201
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> Hagar <SEP> nutritious <SEP> Good <SEP> development. <SEP> pro <SEP> Good <SEP> development.
 <tb>
 <tb>
 <tb> duction <SEP> of <SEP> hyphae Aerial <SEP> <SEP> Not <SEP> of <SEP> mycelium
 <tb>
 <tb>
 <tb> and <SEP> of <SEP> spores <SEP> is <SEP> some Aerial <SEP>, colony
 <tb>
 <tb>
 <tb> little <SEP> limited <SEP> and <SEP> is <SEP> white <SEP> brown <SEP> to <SEP> brown <SEP> clear.
 <tb>
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A culture of 1 living organism BL 567201 which was isolated from soil and named streptomyces viridifaciens was deposited isolated from named soil. these viridifaciens has been deposited in the American Type Culture Collection, Washington D.C. and cataloged in its permanent collection of microorganisms under number ATCC 11989.



   The present invention covers a method of culturing species of microorganisms at 24-30 C under submerged conditions of agitation and aeration on media consisting of sterile water containing a carbon source, a carbon source. of nitrogen a source of developing substances, mineral salts such as sodium chloride, potassium phosphate, magnesium sulfate, sodium nitrate and if desired, a buffering agent such as calcium carbonate.



   As a source of carbon in the nutrient medium, carbohydrates are satisfactory. The following carbohydrates can be used:

  <Desc / Clms Page number 13>

 Regular starch Xylose Soluble starch Arabinose
 EMI13.1
 Sucrose 1- P -, - in o s e
Glucose Fructose
Maltose Lactose
Dextrose Inulin
Glycerol Dextrins
Galactose Xannitol These carbon sources are introduced into the medium
 EMI13.2
 in the form of rur-iTi4e ov, in the form of concentrated products. The amount of these carbon sources produces an autobiotic production of approximately 1: 4, to 5% by weight of the total weight of the fermentation medium.



   Appropriate sources of nitrogen, for the process of
 EMI13.3
 fermentation, including some sources of developing substances and o-rga, n5-cu = s and i? 210rw¯'C'.7. (lv. S containing nitrogen and salt shakers vr-ot4iroiJes en particular con; - take very diverse substances naked canvas:

   The a11j ..,. '. Oé'. (; Ià.es The gluten of J1laïs #: yBroJ., "}" L The cas4izne, # = yd, 1, olYs6e or not with!. 'Acid Fish meal Wheat glutane Soy flour hydrolyzed with acid Meat extracts Peptone Liver cake Offal Urea Brewer's yeast Nitrates Cotton flour
 EMI13.4
 The conposes of am.r, loniuTtl. Lectalbumin. Grain porridge from the. Tryptone distillery.
 EMI13.5
 The corn maceration liquor The flour at 'h1.1iléàcp:': :) J
The maceration liqueur of.

   Flax flour wheat Peanut flour
Whey or concentrates Whey sunflower flour
 EMI13.6
 These proteinaceous ingredients should not be at> oliuv4s 8. a dep: re of high purity; less Hard materials do contain traces of developing substances and considerable amounts of mineral nutrients are suitable.
 EMI13.7
 



  It is not possible, due to the crude nature of many of these nitrogenous substances, to specify infinite proportions to add to the content. A guxntit4 varying from 0.1 to 50 nodes on the basis of the solids pres corresnonr) has the useful j7iri7-e of nitrogenous substances. add to nilieuy in most cases. Returns' "'1ep.ts r, prtiClù.i0rn: lp.nt r10V'! 'S obtained pnr .er1nloi (10' '1ilieu)" containing nr.o'f: E'l):!' - w;

  <Desc / Clms Page number 14>

 
 EMI14.1
 soybean or soy peptones, it is = u-d're Degradation products of the bean = soybean, in particular hydrolyzed soy flour.



     The mineral salts used in the medium include certain heavy metal salts which are useful in traces and often found to this extent in the raw natural ingredients of the medium, for example in the maize macerator. When they are not found naturally in other elements of the environment, these salts are added and increase
 EMI14.2
 the yield of Omega.mycin.

   It is thus that we must introduce enough manganese, copper and particularly zinc, so that the quantities present, both as impurities in other materials and by addition are at least approximate.
 EMI14.3
 matively 0.00.033o 'of manganese in the form? of MnC12 4, H20, 0.00033 of copper in the form of Culs0 ,. 5 1-120, and ZnSO4 7 H2O '.



   The pH of the fermentation medium should be about 5.0-6.5 and preferably 5.8-6.2 at the start of fermentation.



  The preferred temperature of the fermentation process is about 24-30 C. Maximum product yield is generally obtained within 1 to 5 days, depending on the Streptomyces cultivation process.
 EMI14.4
 Omega-cine is active in vivo as well as in vitro and exerts marked chemotherapeutic activity on experimental infections in mice. The results of the tests and of the rooftop determinations are shown in the following table.
 EMI14.5
 



  CiMgamycin Weight test Algae toxicity CD50g / kg Lot n Reaction in mm. LDõmg / jcg / -sov.ris mouse, lntra, kidney -tntr8nri tonf :: Üe 1 25 to 1 mg / cm3 98 - 171 5 25 23 to 1/16 mg! cm --- 4.6
 EMI14.6
 The CD50 (Curative dose - 50) is the minimum dose of Om0Faycin yes ru4rit 50 of the subjects of a group of mice that had received intraperitoneal injections of 100 to 1000 LD50 doses of Dinlococcus One z.oniae, each dose LD50 ± both sufficient, ad: n "Í.-

  <Desc / Clms Page number 15>

 alone, to kill 50% of a group of mice. The infection
 EMI15.1
 is conuvuniüupe all at once after the second dose of the test drug. The drug under test will be administered in two equal doses, approximately 18 hours apart.

   The animals are observed for four days and the group deaths are expressed as% of the total animals per group. The proportion of deaths is converted into probit values and these are plotted and compared to the log dose in mg per kg of mouse weight. The point of intersection of the probit line 5 and the best line passing through the experimental points corresponds to the concentration of the drug which protects half of the animals under the conditions of the experiment. The antilo-
 EMI15.2
 The garitbme of this term is the CD50 value.



  EXAMPLE 1 To prepare the mega: myclne on a laboratory scale, the fermentation is carried out in shaken bottles open to air, but protected from contamination by lids.
 EMI15.3
 cotton wool or gauze. Streptoinyces BL 567? 01 is cultivated in a suitable nutrient medium, by the sul3.1 cultivation procedure (% e, the agitation and aeration of the culture being carried out by placing the bottles in a type of shaker. -and-comes which ensures the spraying, the projection or the agitation of the slurry in an atmosphere containing oxygen. In a typical case, 500 cm3 of culture medium are introduced into 4 liter bottles.
1% sucrose
1% soy flour
1% soy peptone
 EMI15.4
 1., 5 ?, KHI′0.



  0.5'l '(NËHPO 0.5 <(NHI. 8HPO ,,:;.



  :? au - '! - l 0' - -Tb we sterilize. After autoclaving is inoculated the, i} j.811 with 1.1 envlron, in vol'Ju =, a suspension I1f1UCmSi 1, r; <il> 1, e ae fxytJT '? 5 of Streptomyces from a layer C.i'iFu.îi' rirt e "t ±), 0 \ 6 ,? an d ± 1> ut of the fermentation. of

  <Desc / Clms Page number 16>

 The bottle is then incubated at 26-28 C for 48 hours while shaking at 130 strokes / minute with an amplitude of 11/4 inch (29 mm). At the end of the incubation period the fermentation liquid is bluish green, and when tested by the procedure described above gives zones of inhibition of about 27 mm when the broth is diluted. times. This broth contains
 EMI16.1
 omegaizycin which can be isolated as described below.



    EXAMPLE II
For a larger scale omegamycin preparation, an inoculum is prepared in a fermentation medium containing, by weight,
1% corn maceration liquor
1% sucrose
 EMI16.2
  , 55l] (NH4) 2HPO 4 1.5dlof KH2PO 4. o, 2. MgS04 "7 H20 Water q.s. 100%
 EMI16.3
 PHé 6? ¯-bs- expand it to a Volume of 2,500 cm3 and fill it into a 2 1/2 gallon (10 L) bottle. The medium is sterilized by steam at 118 to 120C for one hour. After cooling the Dent., About 0.5% by volume of a cloudy aqueous suspension of Streptomyces spores from an agar strain is inoculated.

   The contents of the bottle are then incubated at 26-28 C for 48 hours in a reciprocating type shaker and sterile air is blown onto the surface of the liquid. From the bou-
 EMI16.4
 bottle of the inoculun broth containing Streptoillyces s is poured into the fermentation tank under perfectly aseptic conditions (see Example III). At the desired time, the liquid can be processed as described below, and the Omega-mycin can be isolated.
 EMI16.5
 



  The antibiotic produced by Strento'lyces BL 567201., Omegamycin, can be prepared on a large scale by culturing

  <Desc / Clms Page number 17>

 
 EMI17.1
 Deep or S'U: 7: î1''v-rbe Fixed fermentation cures and appropriate agitation and aeration nuclei are useful for this purpose. A nutrient medium formed from 56.8 liters of
 EMI17.2
 maceration of corn, 56.8 k of sucrose, 28.4 k of (NTL, 4) 2.11po4y 5, 2 kg of KH 2 PO 4.p 11, 3 ka of KgS04. 7 H2O and water to make 1500 gallons (560 1) is suitable. The medium was put in a golden hearth - '. Na.r' in a glass-lined fermentation vessel with a capacity of 2,000 gallons (750 liters) fitted with a circulation jacket.
 EMI17.3
 water for temperature adjustment, a.

   D'2: itat '? Ur a1) P1: stainless steel oori and a sprinkler device for aeration.
 EMI17.4
 



  The middle is st <$ rilis6 by heating with steam under pressure and then cooled. After sterilization, the concentration of hydrogen ions in the medium must correspond to a, pp1 oxiina.iv <e <noent at pH 6. The nutrient medium is then inoculated with 15% by volume of a vegetative culture cul ti ve s :: rl.tdaas a i '!. T) 1) 8.y'cil of similar fenrmntation, previously inoculated with a described inoculini above, or an inoculun prepared in the laboratory. The culture in the 2000 gallon fermentation vessel is incubated at 83 F (28 C) for two or three days.

   During the incubation, the propeller is rotated at a speed of 90 rpm, and sterile air is introduced.
 EMI17.5
 c3 in the middle by the sprinkler at a rate of 100 cubic feet (2,, a m3) minute. At the end of the incubation period, the culture fluid normally contains enough antibiotic activity to give in the test against organisms a zone of inhibition of en-
 EMI17.6
 about 23 mm in diameter with a broth diluted 16 times. The OYn0,! ';: 3- mycin is isolated as described later.
 EMI17.7
 



  EX: PL 8 IV A fermentation liquid is prepared containing om4gai <iycin following the procedure of Example I, with the culture medium below.

  <Desc / Clms Page number 18>

 



  1% Wheat Gluten
 EMI18.1
 1 <Gl-rrcÂrol 0, 5 Na.Cl 0.05% Soluble products of distillation
 EMI18.2
 0, lfl C2C03
Water q.s. 100% EXAMPLE V
A fermentation liquid is prepared containing
 EMI18.3
 1 -'O, igac, α-ycin-su .- :. will proceed from Example I, with the medium. culture below.



   1% cotton flour
1% Glucose
 EMI18.4
 0.05 a.soluble matter from distillation 0.1% CaCO3 Water a. s. 100%
 EMI18.5
 EJCt'1PL VI A fermentation liquid containing 0m4ganycin is prepared according to the -process of 1-'ExemDle'-i., With the culture medium below- 1 Lioùeur de maize 1% Cerelose
 EMI18.6
 O ', 5? 4 NaCl 0.1% CaCO3 -
 EMI18.7
 Water C !. s. 100% EXI ;, PLS VII A fermentation liquid containing 1-IO.ciega-riiyein is prepared according to the procedure of Example I, with the culture medium below.



     1% soy flour
1% Cerelosis
0.5% NaCl
0.05% Yeast extract 0 1 le, 'CaCO3
Water q.s. 100% EXAMPLE VIII
A fermentation liquid is prepared containing
 EMI18.8
 1-'Oml? 'Gai-nycin according to the procedure of Example I, with the culture medium below * 3 Soy flour
0.5% Corn starch
 EMI18.9
 0.1f N-Z-k-1in B (En7ymatic digest of casein) 0.3% NaNO3
 EMI18.10
 0, 5 ,, f CaC0 Water q. s. 100%

  <Desc / Clms Page number 19>

 
EXAMPLE IX
A fermentation liquid containing Omegamycin was prepared according to the method of Example I, with the culture medium below.



   1% N-Z-Amine B '
1% Cerelosis
0.5% Yeast extract
0.5% NaCl
0.1% CaCO3
Water q.s. 100%
Once the fermentation is complete, and as shown in Examples X to XVI, XVIII to XXII, XXV, XXVII and XXIX, the omegamycin has so far been separated from the broth, by filtering to remove the mycelium, by stirring the broth (preferably at pH 8.5) with butanol or methyl isobutyl ketone, separating the solvent layer containing omegamycin, concentrating it to a small volume by distillation, and mixing it with a liquid lower hydrocarbon, / eg Skellysolve C, to precipitate solid Omegamycin in the form of the base, if the broth was an alkaline pH, eg pH 8,

   And in the form of the hydrochloride, if for example the broth had been acidified before extraction with hydrochloric acid. The solid omegamycin thus obtained was further purified by forming a suspension in ammonium hydroxide and also by adsorption from a solution of the free base onto a chromatographic adsorbent (for example a slice such as Florisil ), followed by washing (eg with acetone or methanol) to remove impurities, and elution with an acin.

   The acid salt of the purified Omegamycin in the eluate is then separated by crystallization, precipitation, lyophilization or by a similar process. The liquid lower hydrocarbons used as precipitating agents are commercial petroleum ethers, for example Skellysolve A, bp 28-38 C; Skellysolve B, bp 60-71 C; Skellysolve C, boiling point 85-100 C.

  <Desc / Clms Page number 20>

 
 EMI20.1
 



  According to the nerve methods of the present invention, and when the fermentation is complete, the omgmmycin, also called tetracycline, is separated from the fermentation broth by alkaline precipitation. The fermentation broth is adjusted to an alkaline pH in the range 8 to 11, and the mixture precipitates from mycelium and tetracycline is collected by filtration. As a variant of this process, the fermentation broth is acidified to a pH below 3,
 EMI20.2
 filtered to remove myceli1Fl, and the filtrate is treated as above, i.e., adjusted to an alkaline pH in the range of 8 to 11 inclusive, and the crude tetracycline precipitate is collected by filtration.

   Any water soluble alkali of sufficient concentration can be used
 EMI20.3
 preference being given, however, to 1 -'aiLmo.-i: iaciue or sodium hydroxide. The pH is set in the range of 8 to 11; an alkaline pH in the range of 9 to 10 is preferred. The tetracycline precipitate, with or without mycelium, is dissolved for handling by either of the methods described in aqueous acid below pH 3 or in aqueous alkali above pH 11. examples of such aqueous acids are acids soluble in water, providing the desired pH,
 EMI20.4
 for example hydrochloric acid, sulfuric acid, phosphoric acid and nitric acid.

   These same acids have the effect of lowering the pH of the fermentation broth below
 EMI20.5
 of pH 3, and preferably at about pH 2.0-2.5, to allow the separation of the tetracycline, which is in solution as an acid addition salt, from the solid mycelium by filtration before to use the alkaline precipitation described above.
 EMI20.6
 



  Various acid addition salts of Omegamycin can be prepared, very simply by adding the desired acid
 EMI20.7
 inorganic, (including mineral acids) or organic, 15a-nt - iliotic in water until a clear solution is

  <Desc / Clms Page number 21>

 obtained. Solid salts can be prepared by adjusting the
 EMI21.1
 pH of such solution of an om4gavqycin salt to a point lower than that which would cause separation of the antibiotic. The solution can then be dried, for example by subjecting the frozen solution to vacuum. Salts
 EMI21.2
 Acid of 1 OméaYllycine are obtained by RV8portation of a solution of the salt in water at a low pH.

   Mineral acids which can be used are hydrochloric acid, sulfuric acid and phosphoric acid. Organic acids which can be used are citric acid, tartaric acid, gluconic acid, benzoic acid, acetic acid, ascorbic acid, succinic acid, nicotinic acid, l formic acid, maleic acid etc. Since Omegamycin is amphoteric, salts of various metal elements with the antibiotic can be prepared, for example the sodium, potassium, calcium and magnesium salts.
 EMI21.3
 



  The alkali metal salts of Ompgamycin are formed by treating an aqueous suspension of the antibiotic with two equivalents of an alkali hydroxide. Solid metal salts
 EMI21.4
 and omeganiycin are obtained by evaporation in vacuo of the aqueous solution of the antibiotic at the appropriate pH. The preparation of omegamycin from natural solutions, for example, by lyophilization, gives the free base generally in the form of a trihydrate, if water is present and if one.
 EMI21.5
 does not apply heat. T0.tracycl; 'n. "" - trihydrate is also prepared by dissolving the base in an orra- solvent.
 EMI21.6
 nic soluble in water, for example methanol, ethanol., and by pouring the resulting solution into water.

   Tetracycline
 EMI21.7
 Solid calcium is converted to trihydrate of trihydrate by dissolution in aqueous acid, for example pH 3 or less, using sulfuric acid or chloryl acid ,. then by adding an agent to block calcium, for example sodium 'r #snate (acid' 'thylenediamine-ttr: 1p, ctil1ue),

  <Desc / Clms Page number 22>

 and finally adding an alkali to raise the pH to at least 6 but not significantly more than 8, which causes the
 EMI22.1
 precipitation of tetracycline trihydrate. This trit7A, drate is recrystallized from aqueous alcohols, for example from methanol, and converted to the anhydrous one by applying moderate heat in vacuum over a desiccant.
 EMI22.2
 



  The acid addition salts of on; egamwcine are prepared in different ways. For example, stoichiometric amounts of the acid are added to a solution of om4ga-mycin in llee-u or in an organic solvent, eg, butanol, acetone, methanol, to form the salt of. addition of acid;

   if desired, the solid salt is then isolated by freeze-drying the mixture obtained (for example in water), or by collecting the precipitated salt by filtration (for example
 EMI22.3
 ethyl acetate or by precipitating the salt by addition of another organic solvent (for example by adding a hydrocarbon, such as Skellysolve, to butanol or cyclo-
 EMI22.4
 hexane to "ethyl acetate" or by reducing the volu-ne of the solvent by vacuum distillation until the salt precipitates on cooling (eg butanol).

   Many other methods are used to prepare these salts, for example Onieganycine can be converted to uieganycin hydrochloride by adsorption of Omegalycin on a chromatographic adsorbent (eg a silica such as larisil followed by dilution. of OmgaTI1ycin with an acid, for example hydrochloric acid in aqueous, methanol or butanolic solution.



  In another process the base of omegamycin "in water at pH 8-9 approximately, is extracted with butanol, the butanol is separated and the base of omegamycin is trsfoY: T1e in hydrochloride d 'Om gai11ycin and it is passed to the used phase by extraction with aqueous hydrochloric acid;
 EMI22.5
 Om6xa> nycin drate thus prepared can be separated if desired, for example by lyophilization.

   In a nr6ç process, <rfi,

  <Desc / Clms Page number 23>

 
 EMI23.1
 the base :: '1'Oms :: a.:' 1Y0in8 is converted to salts of Omf.fl ;; 1ycina by dissolving the base in anhydrous acetone or anhydrous n-propanol, and adding the acid anhydrous, i.e. gaseous hydrochloric acid, sulfuric acid, sulfuric acid in anhydrous acetone, phosphoric acid, tartriaue acid,
 EMI23.2
 citric acid, nitric acid in anhydrous ¯rFtizyl - iso'utyl- ctone, and collecting by filtration the salt which -prci'oite, for example the hydrochloride of UmorTIycin or sulfate, phosphate, tartrate, citrate etc.
 EMI23.3
 



  EXfP L 8 X Omgat11ycin is easily extracted from alkaline fermentation liquids by non-polar organic solvents.
 EMI23.4
 



  The procedure below has 4 = been used.



  One liter of a fermentation broth of Entreotomvces II 5672Ql at pH 0.5 is extracted with 0.5 liter of rn: thzTl-i soutyl ketone. The organic solvent is separated washed with water, reduced by azeotropic distillation to a volume of about 20 cm3.
 EMI23.5
 and addition to 250 cm3 of Skellysolve C. 20 mg of 0.1 ± gai'ùjr = ine (batch 26) precipitate and are collected by filtration; they have an activity at 1: 1 µg / cm3, 1: 61+ dilution of 19.5 Tin (diameter of the zone in the B. subtilis plate assay on agar at pH 6.2); 24.2 1.mm at 1:16 and 27.7 nm at 1: 4. The initial broth gives 27.3 mm at a dilution of 1: 4.



  13XS :, 1Pt 15 XI In another process, pH 5.5, 4.5 liters are adjusted
 EMI23.6
 of a fermentation broth of Streptomyces BL 567201 at pH 6.7 and extracted with 3 liters of butanol. The butanol extract is separated, washed with water, reduced to a volume of about 35 cm3 by azeotropic distillation, and addition to 350 cm3 of Skellysolve C, (technical heptanes). Omgamycin (batch 27) precipitates in the solid form and is collected by filtration.
 EMI23.7
 (1.2 grams, having an activity at 1 mg / cm3 1:16 dilution of 23.2 mm; diameter of the area in the B. suhtilis a plaque assay, with agar at pH 6, 2).

  <Desc / Clms Page number 24>

 
 EMI24.1
 



  The residual broth at pH 5.5 after extraction with butanol is adjusted to p5.5 and extracted at 3 liters of butanol.



  The butanol is s6pat4, washed with water, reduced to a volume of about 30 cm3 and added to 350 cm3 of Skellysolve C. The crude omega-lycin (lot 28) precipitates under the yellow solid ATN roroma. -orange having an activity at 1 m ° / c3 1:16 'dilution of 25.2 mm (diameter of the test zone due to plaque on 3. subtilis on acts at nl 6 ,?). The initial broth gives 27.3% at a dilution of 1: I-.



  S {1 ': rJL t7-rT In a third procedure is adjusted to 1J3 674, 2.5 liters of a fermentation broth of Streptomyces 3L 56720l giving 25.7 yrc ,, n at a dilution of 1: .l- ',. and we entered it nar? liters of butanol. Butanol is separated from water, reduced to a vo L u :. e -'- nVl '..J! l 50 cri3 by allotropic distillation and addition 1 <one liter of Skellysolve C. r '; v?' 2 :: v'C: r2e dark yellow solid (500 11g) (batch 24) precipitates and is collected by 'f'1 ¯ tion; it has an activity at 1 = a; g / ci == 3 1:16 dilution of ls m'n (diameter of the area of the plate assay with B subtilis on -ity-2r at pl-17, z and 23 #: ceo1 = at a dilution of l: l ..



  The broth at pH 6.4, after extraction with bubanol, is kept in a cold room for three OS.rs9 nUls rg18 at pH 8.5 with sodium hydroxide and extracted with those liters of butanol. The buta.nol is senaré.7 washed with water, rduJ.:G at a voltne of about 30 c3 tzar az40tropiqu distillation = ¯ = added to 600 cIIl3 of Skellysolve C. The brown solid Om3a'vciLie, .wne4 (lsr0) (lot 25) precipitates and is collected by iltif¯tion; it has an activity at 1 '' La / C ?? 3 1:16 dilution of 2g m (diameter of the zone in plaoue tressai on B. subtilis S rsT? C of 8ar Pu pH E, 2.



  -.; ,, T 1; mi In another Eroc: d- we extract nar. cw3 C -, - n- o 1,760 rTl3 fermentation broth .'1? 3t.r2ptor'1'.cr:

  <Desc / Clms Page number 25>

 
 EMI25.1
 131 567 "'01. R; l to pH by 1 hydrochloride: 1 <e so, 3jirii. The n-butanol is separated, added to hydrochloric acid aaU811X "To obtain the pH 7.35, and the butanol is ± eliminated by vacuum distillation; 1 P produces concentra a.queuy remaining is extended to about 38 cm 3 with water and lyophilized to obtain 339 rlg solid., which has an activity at 1 zn; / crn3 of ta90n of 25 lncn (diameter of the zone in the plate test with B . sztilis on aear at pH 6.2 and 18.7 iiii at a dilution of 1: 3 The initial broth gives? 6.3 rilln (and 20.0 rtliii at a dilution of 1: 3) and the exhausted broth gives 19.0 rem (and 12, iffil1 at a dilution of 1: 3).



  EX'.IPL3 XIV In another nrocéd9 is extracted with 3 liters of n-butanol 5.5 liters of a fermentation broth of 3 brotpmyces L '67OI r9g1é at p3 5.5. The solvent is s6pay-4, ca.u washed, reduced to. a volume of about 35 cm 3 per distilln- .l; 1 <Jn xzòt.ropique, and added to 350 cm3 of S "kelJysolv \ '3 C. 1pra-, ll.'lcln'3 golden yellow solid (1.2 g) precipitates and is collected.
 EMI25.2
 
 EMI25.3
 1-, Ion (solid A). It has an activity at 1 Tnr / c; rl3 dilation 1:16 d.e 23.2 mrn. {outside the zone in the test. plate on 3.subtili.s on agar at pH 6.72) and 26.9 imn at a dilution of 1 :.



  The residual broth after extraction with but'anol at pH 5 is r <'g14 at p38, and extract nar 3 liters of butanol.



  The hutanol is sf98r ,. lav at 1 "e8U, reduced in evolution to about 30 r¯m3 by aze-otropic distillation and added to 350 cm3 of 8 ': r: lly-solvc C (technical heptanes). A new c1uantitp. ci'0; ar :, rc.īe yellow orange solid (05 F) (lot 30) nrôcii, 1.te and e.3t collected by filtration (solid B). It has an activity at 1 mÇcri3, dilution 1:16, of ? 5 ,? nr'i (diameter of the zom in the test on r-lanue 2vec .3 ',' i; llS? 2'pr pu nil 6e2): 'i r13 1-8,, 5 5 io, m lJY1'3 dilution of 1: 4. r, 'Ülr, f'. '1 Cjtl9 is identified vc c0rti.l.; l] rl "!, ri

  <Desc / Clms Page number 26>

 
 EMI26.1
 when it is colltasinated with similar orgaiiiauL2b chemical products because its' spactre 'of activity (i.e. the last of migration of fj values using a s -, ^' ry of solvents in vroc4d4¯ known as nolil on paper tape.

   This technique is a relatively new, but already well-known iJrocFd4 for the identification of organic coeàxwosôs. Co7niïe .les ..Jaxiiia in 1-ip-fra-rouze, the Rf values in a series of solvent systems are a unique and reproducible characteristic of a given chzrüauP product, and in a way constitute its fingerprints.
 EMI26.2
 



  The process used is as follows. Bands of filtered va1Jier, e; em13t of c ,, rilres, dense, and with high absorption capacity (for example 5S9 Blue Ribbon Specini of Carī Scl-icher Schell Co., K'eene, N, 15 .), .1 2 nbuee (13 pine) in width and 58 cm in length, are suspended from teric) .- er ,, constant ambient temperature in a prot6ijj4e rngion (nsr eyemole in a jar stand) at the edge of a cuvette The suoriess part of each strip is in contact with a certain amount of a particular solvent system (also called the development phase) in the cuvette;

   each of the strips is suspended from a different cuvette, containing a different solvent from a series of solvent systems used in the test. The bottom part . of each strip hangs freely and does not reach the amount of solvent system (or its volatile ingredients) placed below the suspended strip to facilitate saturation of the air with the chosen solvent.
 EMI26.3
 



  The exa7-! Inp 'product (in a fermentation broth or solid retort isolated) is placed at a location marked in the part. upper portion of the tape yes nend freely in the air.



  In the case of a solid matter. 9 it is dissolved in a solvent
 EMI26.4
 useful auelconue The ouantit4s used ce! -1-es. yes '-'on a 7one of garlic? en7i, JnJ 3DrTJMTiQS during fµ2 1'? SZôoi. rir.l¯, pt ce t "111i'nt2 t muvent 4fre dtpr''iTLeF 'opr un? ç; i; l'1 ç' 1 '. f¯. rnr r. ,, oe: i,;. 1 ,

  <Desc / Clms Page number 27>

 a useful amount is 5 microliters (0.005 cm3) of a solution containing 1 mg / cm3 of Omegamycin in a solvent for the B. subtilis assay. The strip is dried and then placed with its upper end in position in the cuvette which contains the chosen solvent system. The solvent is allowed to migrate downwards, i.e., develop the band, until the solvent front reaches the bottom of the band.

   This takes about 15 hours; the surrounding atmosphere is maintained at a constant temperature without drafts, and saturated with solvent vapors from a pool at the bottom of the jar.



   Each strip is then removed, air dried, and placed on an agar tray at a determined pH (, in this case 6.2) inoculated with a test organism, in this case B.subtilis.



  After staying in the refrigerator overnight, the bands developed in the different solvent systems are removed and the trays are marked to identify and locate the bands, incubate overnight and directly or photographed to obtain a permanent document *
The outline of the entire band is usually visible on the surface of the agar and often in the photograph.



  The entwining of each antiliotiae on the strip is indicated by a clear region, in contrast to the cloudy region where the test fungus developed. The band shown in the photograph is divided into zones representing 5,10, 10, 10,10, 10, 10, 10, 10, and 15% of the distance from the point of application of the sample to the part lower respectively, and these areas are described as before 'R @ values from 1 to 10 inclusive. A small spot in the center eyact, would therefore have a value Rf 6, whereas a large spot which would extend dens the adjacent zones would have a value Rf 5, 6, 7 since the entire zone is counted.



   Following this process, the Rf spectrum of

  <Desc / Clms Page number 28>

 
 EMI28.1
 O: t; aiCi2le (lot 30) with :; microlittes of a 1 mo: / c: rt3 solution of the antibiotic COTilll1 the sample for each band, and using B.subtilis on agar at pH 6.2., the bands being developed in the twelve \ solvent systems below:

   
 EMI28.2
 ------------------------------------------- ------- -------------- -1
 EMI28.3
 
 <tb> System <SEP> A <SEP> B <SEP> C <SEP> D <SEP> E <SEP> F <SEP> G <SEP> H <SEP> I <SEP> J <SEP> K <SEP> L
 <tb> solvent
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> Value <SEP> 6.7 <SEP> 5.6 <SEP> 4.5 <SEP> 4,5,6 <SEP> 1 <SEP> 8,9,10 <SEP> 6.7 <SEP> 8 <SEP> 5 <SEP> 9.10 <SEP> 6.7.8 <SEP> 3,4,5
 <tb>
 <tb> Rf
 <tb>
 
 EMI28.4
 -----------... --------------------- ...------------ ...------------ -'---- "'' '--- \ The composition of these solvent systems is as follows:

   A-Water B - Sodium citrate in 10% aqueous solution C - Sodium citrate in 40% aqueous solution D - Butanol saturated with water
 EMI28.5
 E - Anhydrous Mtthyl-isobutyl-ketone
F - Mixture of 100 parts of 80% methanol, and 5 parts of piperidine, adjusted to pH 9.5 with acetic acid.



   G - Mlane of 80 parts of methanol, 5 parts of glacial acetal acid and 15 parts of water by volume.



   H - Butanol saturated with water and containing 2% p-toluenesulfonic diacid.



     I - Mixture of 100 cm3 of water-saturated butanol and 5 cm3 of glacial acetic acid.



   J - Butanol saturated with water and containing 2% p- acid
 EMI28.6
 toluene Sl.Ù forL'1up '. and 2rtf. de-piperidine.



  K - 100 parts of water-saturated butanol and 2 grams of p - toluene sulf¯onia plus 2 cm3 of piperidine plus 2 seeds of laurium acid.



  L - Mixture of two parts of isoamyl alcohol and a nprtte of chloroform saturated with sodium citrate in; olutlon aaUC; US8 to 10 '. In this case, before aTI1Jli (nvr l'chn'ntillon, the 1 ') APr1'3S Gont saturated with podium citrate "; 11. Solutior anlzzl7; e 1,"' '. adjusted RU pH 5.7 nar ¯'ac¯raF; C1lrl.'118 and '-éhé "s.

  <Desc / Clms Page number 29>

 
 EMI29.1
 



  E.lPLE ZV Solid A of Example XIV is extracted because 50 cm3
 EMI29.2
 of ami'tioniuai hydroxide in aqueous solution at pH 6.4, which leaves 220 mg of a brown solid. Solid B (0.5 g) of Example XIV is extracted with 30 cm3 of aqueous ammonium hydroxide at pH 6.4, leaving 120 mg of solids. The
 EMI29.3
 Basic aqueous solutions of Ornég21yc3.ne are combined and passed through a chromium-containing column (0.5 by 4 inches) (1.3 x 10 cm) containing 7.5 g. of Florisil (chromatographic silica). About 600 mg of low activity brown solid are separated from the brown effluent.



   The column is then eluted with 125 cm3 of acetone and then
 EMI29.4
 125 cis3 of ethanol and the eluats are discarded. The column is then read with 1 'hydrochloric acid (100 cm 3).



  The acidic ethanol content is displaced by water (i.e. vacuum distilled with the addition of water until all the organic solvent has been removed) and water. is neutralism by
 EMI29.5
 ammonium hydroxide then lyophilized to obtain the oinEvJ; = solid ijrcine (batch 32) weighing 0.5 g and having an activity at 1 mg / cm3, dilution 1:16, of 24 mm (diameter of the zone in the test on
 EMI29.6
 planue with B.svutilis on agar at pH 6, 2).



  F,: L \: E1,; PLE zei As in Example XV above, the broth is extracted
 EMI29.7
 adsorbed by Florisil by butanol at pH 8.5, and the product is dried for azeotropic distillation. nn pouring it into Skellysolve C, Ùr>; 6g> solid môrcine (500; -ntr; lot 36) D1-, ± <; j-jt == and is s' <pàr6e: it has an activity at 1 mg / C3 dilution 1.:16, of more than 30 mm (dif '..:': to be of the zone in the test on -0181113.9 with B.pubtilis on apar at the fold 6.2.



  Eµ1 "fl" PLE XVII We transform OIflgHY1wci! Î (of a ':: purity "" 1lCI)' r ') (nJP or, salt of SI) (H1J!' 1 by treating this material in the 'e [11) Nl1' (1 'hyr1roY; Tr] 8 d (80: 111.1'1 jusnu'1. What ln pH is f'11r /' f "" 'n A') 5. 0; on '": 1-G> n! î; .11it. (12 s01ntion 8t on 1" f.' tel .'r. ,, r, 11;

  <Desc / Clms Page number 30>

 vacuum to obtain the dry sodium salt as a stable water soluble powder.
 EMI30.1
 



  EX-FPLE VIII A Strepton fermentation broth is adjusted to pK $ 9.0 with sodium hydroxide: 1 yces BL L56667 A -500 cm3 giving 16.9 mm to the test on S. aureus not diluted (X) and 13.8
 EMI30.2
 mm diluted three times (3Xyprepared according to the methods described above, and extracted with 250 cm3 of n-butanol.

   The butanol is separated and adjusted to pH 6.90 by adding water and hydrochloric acid. Butanol is removed from this mixture by vacuum distillation leaving an aqueous concentrate.
 EMI30.3
 d.10-, ir'leaniyeir-9., currently called tetracycline which is extended to 25 cm3 by addition of water, and which gives an area of 19.5 mm in the test on S. aureus dilution three times, then we do it
 EMI30.4
 lyophilize to obtain 115 mg of solid OmRga8ycin (batch 6667A-3) giving a diameter of 19. mm on S. aureus at 1 mg / cm3 *
Another 500 cm3 of this bouillon.au pH 2.37 is adjusted with phosphoric acid and extracted with 250 cm3 of n-butanol.



  The butanol is separated, adjusted to pH 6.40, with water and sodium hydroxide. Butanol is removed from this mixture by vacuum distillation, leaving an aqueous concentrate.
 EMI30.5
 Ompga111rcine which is extended to 25 cm3 nar addition '. d-water., and yes gives a diameter of 19; 1! nm in the test on S. aureus at a dilution of three times,): and or'one then lyophilized to obtain 356 put of solid Omaycine (batch 6667A -4} giving 15.1 ma on S. Purel - ts at i; jcm3, The Ff spectra of the Qgn4camycin samples are determined as described above, with S.Pureu and are as follows:.
 EMI30.6
 



  Schan-System. * TiJ¯loT "l solvent¯¯¯¯" - BCEF "7 6667A-3 Ef value 4-7 5,63,4,5 1 8, g, 10 S, b ,, 9,10 7 , 8.9 6667A-4 R-value 5.6 5.67 4.5.6 1 8.10 - 6-9
 EMI30.7
 The solvent 7 systn'5 is bare mtt.h8TIol 80.f conenpnt

  <Desc / Clms Page number 31>

 
 EMI31.1
 2e of pipridine, 2 f of p-toluenesulfonoid acid and 2 " <lauric acid. The sol.vent 8 system is pyridine containing 1 water, 1% 1J-toluenesulfonia acid and 1% lauria diacid.



  TXPL = â ZIJ (A fermentation broth of Streptomyces BL 456667 L .10 was adjusted to pH 9.25 nar sodium hydroxide to give 20.1 mu on undiluped S. aureus (X and 15.0 1Jun diluted three times (3x) J prepared according to the above procedures, and extracted with 205 cm3 of n-butanol. The butanol is separated and adjusted to pH 7.30 by adding water and hydrochloric acid.



  Butanol is removed from this mixture by vacuum distillation "" this leaves an aqueous concentrated product of O) 11egamycin, currently called tetracycline, which is expanded to 21 cm3 by addition of water and which gives 18.0 in-ra in 15 runs on z. awreus dilution 3 times, harmfulonlelyonhilise to obtain 93 ng of solid 0m ± gamycin (batch 6667-1) 'giving 19.3: rrn at 1 rip., / cry, 3.



  Another 410 cc of this broth is adjusted to pH 2.59 with phosphoric acid and extracted with 205 cm3 of butan.ol.



  The hutanol is separated and adjusted to pH 6.9 in relation to water and SO2 hydroxide. Butanol is 01i1Y'.in this m41rnge by
 EMI31.2
 vacuum distillation, this yes leaves an aqueuy concentrate
 EMI31.3
 (POrn'gal11ycin which is increased to 21 cm3 by addition of water, which gives 20.0 rim on S. Rureus when diluted three times, and lyophilized to obtain 331 r at'0 m4 garoeycine solid, currently d / notum6e ttracycline (batch 6667-2) giving 17.3 mm on S. Fureus at 1 mg / cm3.



  The Rf spectra of Om ^ gamycin are determined as
 EMI31.4
 described above, on S. aureus, and are as follows:
 EMI31.5
 "Schan-System"
 EMI31.6
 
 <tb> tillon <SEP> Solvent <SEP> A <SEP> B <SEP> C <SEP> D <SEP> E <SEP> F <SEP> 7 <SEP> 8 <SEP> G
 <tb>
 
 EMI31.7
 6667-1 Rf value 6.,7 g7 1.5 1,2.3 1 10 7 9.10 8 9667-2 Rf value 6 6.7 4.5 2.3.4 1 10 7 9.10 7
 EMI31.8
 ey'lf1Pr ... 'ft: ex The distribution coefficient of t6tracycIine in ± T J

  <Desc / Clms Page number 32>

 butanol and water (concentration in butanol divided by concentration in water) is less than unity at any pH but is significantly changed by addition of calcium ions.

   Thus, the coefficient of distribution of tetracycline between butanol and calcium chloride in 2.0% aqueous solution is less than 2 (is often less than 1) from pH 1 to pH 6.6 approximately, but rises rapidly as the pH increases and is greater than 5 from pH 7.4 to 11, reaching a maximum of 9-10 between pH 9 and pH 10. A very similar shape of the coefficient curve. Distribution of tetracycline and pH is achieved using butanol and calcium chloride in 0.5% aqueous solution with a maximum of approx. /0. At approximately pH 10.

   A partition coefficient greater than 1.5 is not obtained for tetracycline between butanol and aqueous solutions containing 5 or 10% sodium sulphate or 2.5 or 10% sodium chloride from pH 4 to pH the partition coefficients between 2 and 4 can be obtained with 2 to 10% NaCl only at very acidic pH 2, which makes. the. operations. industrial 'difficult if not impossible
The extraction of an aqueous solution (broth) containing at least 250 meg / cm3 of tetracycline with a quarter of a volume of butanol at pH 8.5 in the presence of 0.5% of calcium chloride, gives a high separation, ( 40 to 50%) of tetracycline under the.

   forms a solid material of good potency after separation and concentration of butanol, back extraction with aqueous acid, such as hydrochloric acid, and isolation. The tetracycline is then extracted as described above.



   In this process, the calcium ion normally present in the broth (eg in corn maceration liquor) is used, together with added calcium ions. Calcium ion can be added in the form of any salt, for example calcium chloride or calcium carbonate, and preferably a total concentration of calcium ions is used.

  <Desc / Clms Page number 33>

 in the broth varying between 0.01 and 2.0 percent.

   The addition of about 0.1 percent calcium chloride is a useful measure; the amount to be added may also be the amount necessary to create in the broth the stoichiometric equivalent of the tetracycline present determined by testing to establish the amount of tetracycline and the amount of calcium ion.



  Excessive amounts of calcium ion are undesirable at times because they precipitate calcium phosphates which tend to take up tetracycline. This can only be recovered at the cost of additional operations.



   Further information on the increased efficiency of the extraction of tetracycline by butanol from fermentation broths containing calcium ions is provided by the observation that the addition of an ion sequestering or chelating agent calcium in broth, reduces the extraction of tetracyclic in butanol below the level observed when calcium chloride is not added to the broth.



   A fermentation broth containing teracycline and using corn maceration liquor in the medium is adjusted to pH 8.5 and divided into three equal portions to which are added the indicated amounts of calcium chloride.



  Butanol extraction is enhanced by calcium ions
 EMI33.1
 aJout / s, as indicated by the results shown in the table below., and obtained by biological tests.
 EMI33.2
 



  Ttracyc1ine prpsel1t n Cg / C! Jl3 Portion n fi Cad in butanol in broth frrJ 111 â .-. '
 EMI33.3
 
 <tb> 1 <SEP> ' <SEP> 0 <SEP> 136 <SEP> 21
 <tb>
 <tb> 2 <SEP> 0.1 <SEP> 196 <SEP> 20
 <tb>
 <tb> 3 <SEP> 0.5 <SEP> 196 <SEP> 20
 <tb>
 
 EMI33.4
 Y '' 'L' 2vvI Is added to a fermentation broth containing at least 100 mc !! / cm3 of t ftracy <31in approximately J1 'of C-, C12' ot one re (18 leu broth to the pil 8.5 - 9.0 approximately by 801 100 cqusi: 1-

  <Desc / Clms Page number 34>

 
 EMI34.1
 eue or ammonia, then the broth is extracted by about a fifth volume of n-butanol in an extraction in
 EMI34.2
 Several phases. The Bi-Mix is filtered and the tetracycline containing butanol is concentrated by vacuum distillation with the optional addition of enough water at the end to allow the formation of hydrates.

   Solids containing.
 EMI34.3
 tetracycline (at least 6tJ0 ¯L.cIQ .rcipitating as the base or the calqiun salt and are collected by filtration and dissolved in minimum amounts of n-propsnol.



  Hydrochloric acid gas is then passed through the
 EMI34.4
 n-propanol to precipitate crystallized hydrochloride of tetracyin, of theoretical strength.



  0Ti, ojv [F u f.-U. L .. J ¯W1 To a fenfl'entation broth containing 0.1% Cad- tetraeyclinc is added and the broth is adjusted to pH S, 5-90 approximately with sodium hydroxide or Dotasse. caustic or ammonia., it is extracted with n-butanol and using half
 EMI34.5
 volume for batch extraction or 1 / 4'a 1.5 volume for multi-phase extraction. ' The mixture is filtered. And the phase in the butanol is s4pa.r'.5e .Bi the - ,. Oroc'p * is used by
 EMI34.6
 lots. The butanol containing the tetracycline is concentrated to 1/200 of. broth by vacuum distillation.

   The butanol concentrate and the insoluble matters which it contains 4ventuell; f / i = nt, are extracted with 3 successive equal volumes of water (r41é) 'at pH 2.5 with hydrochloric acid * The extracts. aqueous are combined,
 EMI34.7
 filtered and concentrated by vacuum distillation to 1/400 or 1/500 of the volume of the broth. To this aqueous concentrate is added 1% calcium chloride (w / v) and the pH is adjusted to
 EMI34.8
 About 7.8-3.5 by addition of 3 'ar.soniu; hydroxide.

   The precipitate obtained from solid tetracycline calcium is collected by filtration and dry-ile 1- * in air or under vacuum, after having washed it with acetone.

  <Desc / Clms Page number 35>

 
 EMI35.1
 xr: PT .; S Y36II 1 1 gram of free tetracycline base is dissolved in a ú1Ínil ': u: .J. of anhydrous acetone and the stocchi-o-, 2ietriou quantity of hydrochloric acid T? -CeSSa: .I'¯ "2: 0'ü is passed under gas through the salt. tftracyc1ine
 EMI35.2
 A crystalline yellow solid precipitates and is collected by filtration.
 EMI35.3
 



  ] <'\ 1' f. "ROI [j 'V'7I The tttracycliD.9 is purified from the contô111inantes amounts of chlortS'tracycline and to'oxyt4.ti? Acycline, when these C01TJpOSS are present, by distribution in contrariety, for example dms 7¯'ar: Craig's Grill, using an n-h'a Jétn solvent system <Μl / 2.5 µl of acetic acid, as described, for example, in TTcisi7 "arer, Technique of Organic Gher: Istry, Vol. Xiii, Inte1'sci (nce Pu7l. Co. NY 1950 pages 171-311 et.". Nal Chem Vol 21 + e pages 66-70 (1952) and Vol 23 pages 41-44 (1951).



  :: T! :::; [11: X) [V
 EMI35.4
 \ Cationic, anio-
 EMI35.5
 niaues and nonionioues in fermentation broths of tt [, Dc "vcllne to increase the efficiency of the extraction of 1,> 'tracycline from broths by buta, nol or 8myl alcohol or thyl-isobutyl- The pH is carefully r = '. 1 "' in any case to obtain the azimurJ1 distribution in the o1'g solvent: mic, yes can be determined by a simple test. Examples of agents and agents Useful concentrations are a / .1'0801 OT (1.70 '), Ttreen 21 (05) and Teri o1 (0.1 =' j. The concentration used can reach 5.

   Other useful agents are the ionic agents, co1 ")?! 1-laurine acid, 1-sta1'in1 acid, esters of sulpurine acid, 7 napthalene-su1 napthalenic acids, napthalene-su1 acids. siilroni ues 8lkvl- é 'r01Î1 ::, higher tiues cationine wetting agents as the salts of;: rrl1' "" 'oni 1111 nua t 0rYJ.ai l'es (par e: re: irle, (CH )) 3NCH2 CU11F¯ CH!) OCR Cl "or OCR represent the acids j7, listed in 1.11 [1nr ()

  <Desc / Clms Page number 36>

 coconut oil), and - non-ionic wetting agents
 EMI36.1
 retort thers of û1'¯syl-ph nal and rolyalycol.



  In a simple process according to this principle, we use
 EMI36.2
 a vehicle for removing tetracycline from fermentation broth containing .100 mcg, / cm3 of tetracycline, into methyrlisobutyl-c4t <Jne. This solvent is separated; the addition of gaseous hydrochloric acid precipitates the solid crystalline tetracycline hydrochloride-
In another process, following this principle, we do
 EMI36.3
 pass the tetracycline from the broth into m4thyl-isobutyl-ketone by addition to the broth at pH 2-4 of about 0 ,? '. an anionic scouring agent is Tergitol 4 (sodium t3tradecyl sulfate, Tergitol 7 (sodium heptadecylsulfate) or Aerosol 0 (sodium dioctylsulf.osucpinate).



  EX% .4IÎJÉ YVI
A very simple, inexpensive and efficient method for isolating crude solid tetracycline from large volumes of fermentation broths is as follows. A broth of fermen-
 EMI36.4
 The treatment containing at least 250 mcg / cm3 of tetracycline, and preferably 1000 mcg / cm3 of tetracycline or more is adjusted to acidic pH and the mycelium is removed by filtration. The filtered broth. is then adjusted to pH 8 or more by adding caustic soda or ammonia for example. Solid, crude, active tetracycline precipitates as the free base or calcium salt, contaminated with calcium sulfate or other impurities and is collected by filtration. This crude product is further purified, if desired, by the procedures described above.

   For example, this product is dissolved in hydrochloric acid at pH 2 containing 10 sodium chloride, and extracted
 EMI36.5
 by butanol and butanol est-s0p8r and concentrated nat distillation in vacuum to precipitate tetracycline hydrochloride.



  EXE;,; PL 4 .: XXVII Several batches of 8mgam.ycin are available from

  <Desc / Clms Page number 37>

 
 EMI37.1
 Streptomyces BL-567201 fermentation broths as follows. To the whole broth (150-285 gallons), 285 to 532 g of calcimi chloride, and 50 to 120 gallons of wet n-butanol are added. The pH of the broth which is 4.9 to 5.5 is adjusted
 EMI37.2
 at e, 35 to 8.8 with 50% sodium hydroxide (z - 10.5 liters).



  After stirring, which gives an emulsion separated by filtration using Dicalite (diatomaceous earth), the phase of
 EMI37.3
 butanol containing 0m <4gaiycii) e is separated (100-122 gallons) and concentrated to a volume of about 2.5-7.0 liters by vacuum distillation at temperatures of Less than 100 F (38 C) and below 35 mm of.pressure.



   Concentrates in butanol, having pHs of about 7.0 to 7.5 are extracted three times in equal volumes. water
 EMI37.4
 and adjusted to pH 2.0 with gulfuric or hydrochloric acid.



  These aqueous extracts are combined, adjusted to pH 1.5 with hydrochloric acid, suspended with 1 or 2% activated charcoal (for example Darco KB), filters and the filtrate is concentrated to about 1/5 of its strength. volume by vacuum distillation.



  The pH of these aqueous concentrates is then adjusted to approximately 6.0
 EMI37.5
 with ammonium hydroxide, which precipitates the om-qamycin which is collected by filtration. A second quantity of Omegamycin is often obtained by concentrating the filtrate again to about a fifth of its volume.



   The different batches of Omegamycin obtained above (63 g) are combined and dissolved in 630 cm3 of water adjusted to
 EMI37.6
 pH 1.5 with hydrochloric acid. After filtration, 63 g is added to this solution. of Vrsine (ntl acid; rl = aetic ner3iarinetntr). The fold is then set to 6.0 by de 15bvrîro7v - ', r ,, dl, -im, tio- nian and i'0mFgaoiycin which -precipitates (30 g) and collects'] je onar filtration, sÀcii6e in the viie on P205 and two foj? nise in suspension with 120 cm3 dp: 1 ^ t'1n7] ¯, in rj and''nt 1.4, j with insoluble intlere. The FQl'iHor) ". Tith? 'Tioli"' u3 -ont. King''; and, r, r-r! p00 c-.yi3: 1 ', a .u.

   L3tJ (rl'ç; ",, rri-;, r, r ,, '. ,, ¯j' H" .., r-ip

  <Desc / Clms Page number 38>

 
 EMI38.1
 knotty solution by rest) llSCii7.'Ll 1-3nde-'lêil1 in chamber f> king1:;,. and. is collected by filtration (8.9% in two batches) and air dried.
 EMI38.2
 



  This 0: nEgamjveine is treated with carbon while au'elle is in solution in water at p3 1.5; the solution is filtered and precipitated by bringing the pH to 3.5 with al1HJ.onil1's hydroxide solution. The 6.9% of O: mOglliJwcin6 'are dissolved in 3.4 cm3 of water adjusted to pH 1.5 with 1-1 hydrochloric acid *; the solution is adjusted to pH 3.5 with ll-hvdro7rde arIl! lloniutl1 and seeded; the crystallized hydrated Om4gamycin base slowly precipitates and is collected by filtration (6.45 g), and air dried;

   it melts at. approx. 171.5-173.5 C, contains on ultraviolet ray analysis 890 mcg / mg (based on
 EMI38.3
 of 1000 mcg / m? OmÓ8111ycin or 96l. omegamycin trihydrate rncam}, and bioassay 892 mcg / mg on B. subtilis on pH z plates and is free of chlortetracycline and oxytetracycline or other impurities as shown in result of chromatography on a paper strip.



  This hydrated crystallized Omegamycin base contains 19 ?; of water and is an analyst after drying '
 EMI38.4
 Analysis: Calculated for C 22 H 24002
 EMI38.5
 
 <tb> Calculated <SEP> Found
 <tb>
 <tb> C <SEP> 59.45 <SEP> 59, <SEP> 6 <SEP>
 <tb> H <SEP> 5.44 <SEP> 5.45
 <tb> N <SEP> 6.30 <SEP> 6.29
 <tb>
 A suspension obtained by grinding this free base
 EMI38.6
 of ona8gany-cine hydrated and crystallized in Tinrale oil (Flujol) has many catact4ristL- absorption bands. aues in the infrared. Among these we noted the following ir4auences (in cm-l) 3J.0, 1672, 1607, 1524, 1259, 1222, 1130, 1065, lOl0,9, 963, 91 + 8, 932, 861, 838, 803 , 736, 741, 739 and 6C ".

   The snectre; ahSy'T? 'L.Oi1 in the Ínfré1-ro1) ee r'e this 1:> t "> 11 \ l1.ig,; x-1x the oil:' 1in6rle in lx 1- / zion csractrjstinu 4;> s' 'rs d'0YJnes 1350 5 650 on-1 is 1, nài ,, iJà on the fi "'. l zÏS 'SSaYt,"., .-'. S

  <Desc / Clms Page number 39>

 
 EMI39.1
 This crystallized hydrated 4'0môgaJycin base (2 p: raIi1111es) is dissolved in 20 cm3 of n-Dropanol containing about 0.5 cm3 of concentrated hydrochloric acid.

   After standing for 30 minutes at room temperature and 90 minutes at
 EMI39.2
 When cold, the OnÉ-.P ,, a7,, iycin crystallized, nr4ciDite hydrochloride and was collected by filtration (1.5 g); it melts at 217-219 C when decomposing *
 EMI39.3
 Analysis: Calculated for C22H 2408N 2 HCl:
 EMI39.4
 
 <tb> Calculated <SEP> Found
 <tb>
 <tb> C. <SEP> 54.9 <SEP> 55.0 <SEP>; <SEP>
 <tb>
 <tb> H. <SEP> 5.03 <SEP>, <SEP> 5.2;
 <tb>
 <tb> N. <SEP> 5.83; <SEP> 5.99 <SEP>; <SEP>
 <tb>
 <tb> Cl. <SEP> 7, <SEP> 37 <SEP>; <SEP> 7.5 <SEP>;
 <tb>
 A suspension obtained by grinding the hydrochloride
 EMI39.5
 of om4gam, ycin crystallized in mineral oil (Nujol) exhibits many characteristic absorption bands in the infrared.

   We note the following frequencies (in centimeters -1) 3340, 1678, 1623, 1597, 1315, 1248, 1229, 1175,
 EMI39.6
 1140, 1061, 1036, 1002, 964, 949, 864, 823, 796, 781, 7.3, 719, 692 and 667. The 1-infrared absorption spectrum of this mineral oil slurry in the characteristic region wave numbers between 1350 and 650 cm-1 is shown in fig. 2 of the accompanying drawings.
 EMI39.7
 



  Ompgamycin has a rotatory power. <-7 27 - 245
D at a concentration of 1% in methanol and exhibits
 EMI39.8
 absorption peaks in ultraviolet at 2671U (,, 17, 400) and 355rnt ((-e 13, 500) in 0.1N hydrochloric acid. 27 Omgamycin hydrochloride has a rotatory power -O <-7
D - 253 at a concentration of 0.5% in 0.1N hydrochloric acid.



  EXAMPLE XXVIII
Tetracycline is isolated from fermentation broth acidifying the broth to approximately pH 2.0-2.5, for example

  <Desc / Clms Page number 40>

 with sulfuric acid and filtering. The clear filtrate containing the tetracycline is then adjusted to pH 9-10 with alkali, for example sodium hydroxide or ammonia. A little sodium carbonate can be added towards the end to react with the calcium ion present and precipitate the calcium carbonate which adsorbs the tetracycline; The tetracycline which precipitates at pH 9-10 has a potency of at least 200 mcg / mg and is collected by filtration and purified according to the methods described.



   As an example of this purification, the product is dissolved in aqueous nitric acid at pH 2 - sodium nitrate is added thereto and the tetracycline is extracted with n-butanol. Butanol is separated and neutralized, this. aui precipitates tetracycline base * '
In another example, the product is suspended directly with dun-butanol and the pure tetracyclin is recovered, for example as the hydrochloride, by acidification and concentration.



   In a third process, the product is slurried with dilute sulfuric acid, filtered, and the filtrate is adjusted to about pH 6 by adding alkali thereto, which precipitates the solid purified tetracycline.



   The filtered acidic broth or the alkaline filtrate from the precipitation of tetracycline from the broth, are suitable in cases where they are collected or after drying as feed for animals or as additives to such feeds: These filtrates contain large amounts of factors important nutrients (eg proteins, carbohydrates, minerals, vitamins and especially vitamin B12 and vitamins of the same group and unidentified agents stimulating the development of the young, often called animal protein factors ) and can be added to animal feed especially those of origin

  <Desc / Clms Page number 41>

 vegetable, before or after concentration or drying.

   The value of such animal feeds and additives from these filtrates is increased by adding to the medium, prior to fermentation, a soluble cobalt salt, for example about 0.1 to 20 parts per million of the nitrate nutrient medium. cobalt, and adding to the medium before fermentation a substance (for example an alkali metal cyanide or an alkali metal ferrocyanide or ferricyanide) constituting a source of cyanide ion in an amount of 0.1 to 100 parts per million approximately.



    EXAMPLE XXIX
An effective method for obtaining crystallized tetracycline from fermentation broths is as follows. The broth is acidified to pH 2.0-2.5, approximately.
 EMI41.1
 for example with sulphurous acid it is filtered and one.,: 5 discard the solids - The filtrate is 1. ± g14 at a pH'a.ICalin, for example about 9.0 - 9.5, and extracted by a third of the volume of n-butanol. The butanol is separated, the aqueous phase is discarded (exhausted broth), it is acidified with sulfuric acid, for example to pH 5-6, and the mixture is concentrated by vacuum distillation to approximately one fifth of the initial volume of the broth. The tetracycline which precipitates is collected by filtration and has a potency of at least 200 mcg / mg.

   Butanol is recycled. 100 g of crude tetracycline are dissolved in 1 liter of water acidified to pH 1.5 with sulfuric acid; the subjects
 EMI41.2
 insoluble are 8 liRiinôes by filtration and discarded. The anuous solution is treated with activated charcoal (10-50 g) and filtered. At this point, 10 of Sequesterone (the acid: t "f1ylènc1i é '; Ün -t'tr "'rrt i oue) or another agent for s ooestr'f'1' the cn18ill" '. The pi of 1 *; () l11ti') n -lisp- is then 1 '/ 1 "' 1 â 3,5, by I? YO'1Yt !. '"1 l'r>' r" j {e 3 'by resting the pure base of tf, trecrr.ljy: 8 cri +. <, 11 i ^^. "T r.t, rwc zl'1¯ie by filtration *

  <Desc / Clms Page number 42>

 
Less pure tetracycline suitable for recycling can be further precipitated by bringing the pH of the final filtrate to about 6.



   Sequestering agents useful in removing calcium and thereby facilitating the precipitation of. the pure base of t4tracycline are in particular citric acid, tartriau.e acid, sodium phytate, gluconic acid, phosphates such as m4taphosphate. sodium and in general the members of this class referred to in part, see for example J. Chem. Educ. 25 482-488 (1948) and references therein.



  Many of these agents are compounds of weakly ionized polybasic acids, or hydroxylated organic carboxylic acids.



  EXAMPLE XXX
An efficient and simple procedure for isolating tetracyclin from fermentation broths without employing large volumes of solvent for the extraction is as follows. The broth is regulated to pH 9-10 by adding aclcali to it and filtered.



  Less than 100 mcg / cm3 of tetracycline in solution remains in the filtrate and the latter is discarded. This mixture with the mycelium of the absorbed and precipitated t4tracycline and other ingredients of the broth can be used as it is collected or after drying as feed or additives to such feed.

   The filtrate, aui contains little tetracycline, but large quantities of interesting dietary factors, (for example proteins, carbohydrates, minerals, vitamins, and particularly vitamin B12 and substances of the same group, and unidentified stimulants of animal development, often referred to as animal protein factors) may also be added slowly to foods, particularly those of plant origin, either after or before concentrating or drying.

   The value of these feeds and additives from the precipitate and filtrate is increased.

  <Desc / Clms Page number 43>

 by adding to the Medium before fermentation a soluble salt of cobalt, for example about 0.1 to 20 parts per million of the nutrient medium of cobalt nitrate and a substance, (for example an alkali metal cyanide or a ferrocyanide or ferric cyanide alkali metal) providing a source of cyanide ion at about 0.1 to 100 parts per million.



   The solid mass and the adsorbed tetracycline are extracted at a low pH (for example 1.5) with small volumes of aqueous diacid, for example sulfuric acid or oxalic diacid, to obtain a solution containing at least 3000 meg / cm3 of tetracycline *
In one method, tetracycline is isolated by neutralizing this acidic solution to pH 4-10 'and preferably at least pH 6, so as to precipitate the solid tetracycline in the base or in the calcium form, with a potency of. at least 100 mcg / mg.



   In another procedure the tetracycline is isolated by neutralizing this acidic solution to pH 9-10 and extracting the tetracycline with about a third of the volume of n-butanol. The butanol is separated and concentrated to about one-twentieth of its volume, which precipitates the tetracycline, either as a base or as a calcium, with a potency of at least 200 meg / mg.



  EXAMPLE XXXI
Wet and crude tetracycline base (eg 1 g containing 20% water) is converted to crystalline hydrochloride. by dissolving the base in a minimum quantity of anhydrous n-butanol (for example 5 cm3) by adding to it car¯centric hydrochloric lucide (for example 0.3 cm3), filtering and decolorizing with carbon if desired, then adding an additional amount of anhydrous butanol, for example 5 cm3.



  Pure tetracycline hydrochloride crystallizes on standing and is collected by filtration. The first filtration can be omitted.

  <Desc / Clms Page number 44>

 
 EMI44.1
 n.fe; in this, all of the butanol is added at once and the t4tracycline dissolves, then crystallizes readily as "ttracycline hydrochloride."



   T4-: PL: i'¯IT T9tracycline is separated from the c011t1 Ün2.ntes of chlortstracycline 4ventuellaTient nrhse == tes by forming a SUSension of the free base in fapthanol. The base of chlor- t <straejrcl in: 3 does not dissolve; the dissolved tetracycline base is separated under a purified flomaie as above, for example by removing the solvent or by pouring the illth2nol into water.



  Tetracycline is effectively removed from the remaining amounts of chlortetracyeline 10r so that these are 1Jr ± - felt by suspending them in an aqueous acid, pra- tj, c, 18nt at pH 2.5, which only dissolves the chlort4tracycline. .



  The rC.ïÊ3! ' y} r (,. f4r; is hydrochloric acid l.'other a, cid = s, e.g.'1 ::. sulfuric acid, acid -oho s110riQu, J '' - tt .rl1't. be used, provided that the desired pX is obtained. The base or the salt of acyclic acid is suspended in hydrochloric acid. .ntitr sufficient to obtain a pi-1 of 2.,) or fe TIT <fiftenee, the base or the i2rnur salt of t tracycl irm, for example cJÜorhydl ">; -; C9, is dissolved in aqueous acid in <JE 1, 5 or: aoins, and we add enough base, by eLe'-1} the 0 '}' e1 "hydroyYde: l ': onÍurl or h, roro). :: sodium yde to obtain the pi, of 2, 5 The q ln-tat s tetracycline used n '' 3St nas a binding factor in gpnér81, it is 2vantageuy to use 50 to 100 Y: 10 'of base or of tetracycline salt by c3 à'ez-1J, acidified P1J.1 = x 2.5 F, - hydrochloric acid-ue.

   The n5: is a cri factor tt01Y :; this is 2.5, although any pH in the EUL of 2.0 to 3.0 is useful. L2 <iipptit8 de ct = 1 ± -rtfitra.c..rclir> e nr ± se-1t = under orp-e G 'iI; purity in tetY-acyrcli == e 1 purify Der ce p-rocé = 1é IL - 1-eu, exceed 9 "lQJ c :: l3 of acidified water and, from 'Jr: -ér8nce, not exceed 5 Tr <; / CI: 1; this line is - :: actle'le'1t: rlrrSri-rr-created by pupmsntant if necessary there Quantity of acidified water used n? iJ, r the J112e in suspension.

   These j,: p "ler3S S: .Ci 7¯i '¯, ^ - ¯. N; -r 8yc;, v'nl'"

  <Desc / Clms Page number 45>

 the ttracycline hydrochloride and the chlor-tetraclin hydrochloride in this purification operation are then mixed, that is to say, suspended in water
 EMI45.1
 acidified with hydrochloric acid nrāticruerPnt 'to pH 2.5 for a considerable period, for example 16 hours, enough
 EMI45.2
 long to allow the chlort4tracycline to dissolve.



  It can be determined by a simple test. The purified solid tetracyclin base which remains undissolved is then collected by filtration.



   For example, was suspended for 16 hours in 200 cm3 of water adjusted to pH 2.5, approximately with hydrochloric acid, 10 g of tetracycline hydrochloride
 EMI45.3
 cot) t'Tfinnt in the test 830 key tetracycline and 171 mce / mg of cb.Iarttracycline (83 of ttracycline) by differential absorption in the ultraviolet and containing approximately 10 of chlort4t1 to cycline, determined by chroratoara.nhie on strip of paper. The solid t6tacycline nurifipe base is then collected by filtration and dried. lille weighed 3.0 grams = 1 ms, contains 901 µg! mg of ttracycline and 39 T1lcg !: mg of chlortntraccline (96 of treicycline) a, r la. TI1f; absorption method clings to ultraviolet and contains 95-97% tetracycline according to the band chromatography process of
 EMI45.4
 paper.

   Recovery of t.tracycline reached = 7.



  A similar experiment carried out under nitrogen gives a 929 recovery of the tetracycline base containing zl.ero to ';! O-1' of chlort; 'tracycline, Duisssnce 929 Y1lCg! R: lg.



  1: r ::: rT l yXXIV Lp tF'.tr2CZrCllne is effectively nurij0 and $ T5 (ITrP des ("l1 ± 'n ti tf, s conté'-' in2nte ::: de c'1.lort! -TrRc : rclin e when C? lllCF-ci are T) r / s "'11te, in 1." l11et "! - a / l1.t. in S7J',. 'W'? nSl.Cn f1'ns ne i 'aicxolJ¯ b allTl-1> 1 a pll' r? yT7.'C0: 1 7.5- /), it nE dissolved naked ia chlor-; 4trxc; rcline. ['\: -: 1 "1s a 5 .ii re (0Y", 2 ":' r ::. Xér; 11ion, the b! '1se or the acid iCd'ition salt ost li S. ^, OL1S at TJTT;, 5 pt '7r' "C7.n '.' I. '' '[' I") n "). lj for-m of the base of t '"trpc7cline nur5, j!: e rn r. <rl.> nt 1, e ro! T

  <Desc / Clms Page number 46>

 
 EMI46.1
 to 7.5-q, 0 ,, preferably about 8.5. rare addition of acid, for example hydrochloric acid. The preferred alkali is ammonia, but other water-soluble alkalis such as sodium hydroxide or triethylamine are useful if they provide the necessary pll.

   The base or the acid addition salt of tetracycline, for example the hydrochloride, is suspended in water adjusted to pH 8, 5-9., 0 per.
 EMI46.2
 addition of alkali. The amount of tetracycline is not a limiting factor; in general it is advantageous to use 50
 EMI46.3
 at. 100 Hig per cia3 of water. The pH is a critical factor and should be nearly 7.5 to 9.0. The amount of chortrtracycline present as iln-purity in the tetracycline to be aurified by this C'roc cannot pass 12 mg / cm3 of alkaline water and should preferably be less than 2 µg / cm3; this limit esi x% ciieioEenc
 EMI46.4
 observed by increasing the amount of alkaline water if necessary
 EMI46.5
 used for suspension.

   Solids, iicr. example tetràcyeline and chlortetracycline in wptte -ourification are angc'5es, i-riises in suspension r¯cms â; rendered alevine with alonloniUt hydroxide at 139-.0 for a considerable time, eg 16 hours, times sufficient to allow the chlortetraeycline to dissolve.



  The exact tzps can be determined by a sinpie test. The
 EMI46.6
 base; solid tetracycline purified yes remains undissolved is
 EMI46.7
 then collected by filtration. The whole operation is preferably carried out in an e eate atmosphere of ex, yaene, by eye-ile under nitrogen, to reduce or remove any d-'cō: .os3i io.



  As an example, we --- and in suspension in nitrogen 16 hours dcns 200 c = 13 exu r-cree at fold 9.0 T3.I 'the hyrode d'sm.ioniuri hydrochloride b- 'ircyclim d'amioniiri 10 g of hydrochloride, te tXttac; rclino cc: ¯it-r, ¯ant on test 945 = c / ¯t2 e t4tr *, c.rjiiT = e -7t lil 1 = cv¯ / her? <niort4tr * c> .qhine (35? tetracycline) T)? 'r the proc <? 5. * .y.î = C3ï''1t10? '? differential in Z'üZ '?'% '.- V) ¯Oiwt? Cr.TI '¯; 2: rr; 1. "N 10 'cblort --etracycline, t ^ - r i. .1.I1", i p'r .C1 "L" t: f:' c ': i "

  <Desc / Clms Page number 47>

 
 EMI47.1
 on paper tape.

   The solid ttracyclilla base -puri 10 = is then collected by filtration and secii; zee. ille weighs'35 e, contains on test 9 ° 6 -lc / -ng of ttracvcl ine and 20 iics / <ng of clnlort4tracjrcline (98.1 'of tetracycline) by the differential deabsorb4lio-i procFd6 in the ultraviolet and contains 97-93;' of tetracycline, sorted by cro;? rtograpnis on strip of paper. The recovery of tetracycline reached 39 "'.



  The results of the tests are expressed as ¯ micrograles equivalents of t4tracycline hydrochloride. The theoretical tlnyl.::lUll powers in mcg / mg for: ir-tières solids, anhydrous are 1000 for tetracycline hydrochloride and 1080 for the base of t "tracycline- E] I a -... J .. X77V Tetracycline c is purified <i.1-Ciqle 'n in ..' extracting ppr üu rr t; anal in which it is insoluble and by dissolving it in> iFthaxio1 containing calcium chloride.

   The undissolved 1> npui? E.t, 5s are liolin.es nar filtration and the esu and anifioni ac at pH 8 are added to the solution to precipitate the purified t, atra.ctrc7..ne clcicle. i, 'a, +, "W] 10 -'- tr-t \ rr Ovn4r a..lycine of aueicon-wheel purity can be transformed into the hydrochloride by treating the; = 1a.tiére dpns'l water with hydrochloric acid until we obtain a linpid and bare solution, ie less than 3-4. The solution is = o1-xe- .Le and sfc? 1 <e under vacuum to obtain an easily soluble powder.



  'lY "?' 'l' iJ i VIT On:;: and in suspension 10 0 #rafxres (00275: .. El) of 1 = "'t1-icjrcline anhydrous in' 100 cm3 'e ¯-rronan.l and on. a joust 11 Fr? * 1 "e (02.! h. drown) 'diacid 4or; ni (-, ue to 9t". When' Le is heated 50-5511 making oueloues -min re- t'ro3.di, An abundant crystalline nrc.pit ^ appears. Resting for 15 hours at 0-5 C, the:. 'lane is filtered and read.

  <Desc / Clms Page number 48>

 
 EMI48.1
 i'u: -, :: i :: te .3.> t; tl'ê1cyclirh3 solid â # .l ':; t cit; 111: s: telll' tOUl tl2r du. ! 1-) ropEffi.) L and from 1 "ther en! Lyr1'2. O! 1 le SC'1 'l'., -. I 1 '.



  P0iè, s - 9.3, 7 (391 '). i? anointed with 'L¯S3OI1 17Ó, 5 -177 C s? l 7 ").: ie ... calculate -our ?? -? l '? 0- ... vv'" '"-.
 EMI48.2
 vG.L ^ ''. a - f¯ r: r011 '(C. 56.3; 5È, 6 E. 5.35. 5, 59. r2o, no loss at 6ù C in vacuum *
 EMI48.3
 ', ¯r ^ ¯w': ':% c: i32C of the present intention is lp'.; # = E 211 '). T'1rL8 had that (JU'Ol1 d / nO!' Ee current. :: 3111 vtrfG'irCl.! Ih zips r '! 0n les 1JI'OOri! Të <s are described in the Journal of t? ie f? y, L f C! '1 ce.1 Society, Volume 75., 1; su. = s ../.,!;1^t? 1953. in.si nar c etl.?)le 1 "7r. ', 7 t: a Sample of the free base of tetracycline according to the littratn8 from C! I.0it.tT'Ç'cyclinF, we find 0u' i1 ond at 170-175 C and contains 0, 7.Il chlorine, this yes indicates urine contamination .by about 10 'of chlor2trocycline notYel4.1t 1) 88 r'ai.

   Cat sample and another Sample free of chlorine and t tr = - cyline ure (melting point l169-171 C with decomposition) are the iÎ: 3rtles that a Sample of 0 = l = éga.nycînè (lot 30 ; 1JrQpar8 following eerile 3J'J above) and all the z¯puret'Ç are
 EMI48.4
 distinguished by examination of these samples and samples of
 EMI48.5
 ch10rttracTcline and C ', ¯'GWttraCtrCz2le alone and in: - ^' '1%?' zf? ' ? by chromatography on paper tape (particularly with the D and L solvent sersts).



  The fact cue .'i'T? T '' 'P¯'Tij, F (' ZI2e (lot 30) is t4tracycline is' ai.e: =. Ent proved by identical speed,: the loss d- * activity at pH Q, 0 to 37 C at about 0.7 # nullz3 (24 de erte in. hours); chlort4tracvcline loses about 50 of its activity in 14 hours and ortetracyclin8 dies about 50 "of its activity in 26 hours under r: ê'11gs conditions.n addition, 10'npa''nycine is clainittent r1i.sU¯nn'e of 1

  <Desc / Clms Page number 49>

 
 EMI49.1
 ci-il-ù2t-'tr-acYcline and àe 1'oxvt'-trac; clâms nar 1L wiped on, 1.i éWéH 'wv pT, 0, 01 \ la chlortt-racycline 8t o: xyt0t1 " 8.cycU.n ,:

   turn out to be much less active oue .'0: -ayc3¯n,
Solids assays were performed on plates of B, subtilis with agar at pH 6.2 and pH 8.0 and using the buffers described above as a diluent,
 EMI49.2
 corresponding to the pH of the agar. The tYUiOU8S test results are as follows:

   
 EMI49.3
 
 <tb> Antibiotic <SEP> Concentration <SEP> Test <SEP> on <SEP> plaaues <SEP> B.subtilis
 <tb>
 <tb> in <SEP> on <SEP> cy- <SEP> 3 <SEP> Dimensions <SEP> of <SEP> the <SEP> zone <SEP> in <SEP> mm
 <tb>
 
 EMI49.4
 linder mc cm3 (pi 6.2) ¯¯¯¯¯¯¯¯ (on '3.01 -, Chlorttracycline 0.5 19 NR 2 a0.2 NR GyYptracyc1ine 4 15, 5 NR Ttracyc1ine 50 z, 5 19, 0 Ch1orttracycline 1 21, 5 lot, 2
 EMI49.5
 
 <tb> more <SEP> @ <SEP> 21.5 <SEP> 18.2
 <tb>
 
 EMI49.6
 'l'trac, yr.line 50 Ornt uiycin (1ot 25) 100 20, 2,:

  .16, 6 Omgwayc1ne (batch 3 Q) 25 23, 3 15.1 12, 5 20.8 .'1 12.9 Om0f, 8myc5.ne (batch 36) 16 25.9, z, 2 ¯¯¯¯ ¯¯¯¯¯ ######. # ...... #. # T # - ##### - ##### .... #, # <## '#######
Omegamycin can thus be prepared in contaminating amounts of chlorteltracycline or oxytetracyclin by holding an aqueous solution at pH 8.0 until
 EMI49.7
 that all chlortptracycline or o7tetrac # cline is decopOS0P. and then isolating 1-lüm (Ig, -: u-oycin Durifl14e, as above.



   This is how the solid base of tetracycline containing
 EMI49.8
 about 26 of chlortetracycline can be dissolved in aqueous sodium hydroxide and further amounts of sodium hydroxide can be added to bring the concentration to
 EMI49.9
 0.17 - 1.17 normal. After rest at ordinary teJJ1prature or at. h0 C for 6, 20 or 45 minutes, the solution is acidified to pH 1.5-2.0, fil) t6e, and the tptracycine is precipitated and reduced by bringing the pH of the filtrate to 3.5 or 6.0 . The separate tPtracycUne has satisfactory potency and contains less than 2%
 EMI49.10
 'from chl () rttracrc1ine.

  <Desc / Clms Page number 50>

 



   In another case, approximately 45,000 meg / cm3 of crude tetracycline was freed from contaminating chlort4tracycline by dissolving it for 24 hours at pH 8.5 at
 EMI50.1
 ordinary tension and then recovering it. The ttr CVC 2. '! E (O: -: 1-? G2f? 1ycin) of the present invention is useful in the form of the free base, including the anhydrous nettle and the hydrated forms and particularly the
 EMI50.2
 trihydrate. to combat many diseases caused by bacterial infections in humans and animals. "besides this aD'.71'CatOnp la trāc, yclir.¯e is associated with an au = ntit4 8 '': ï2Cc.ti'TC c'u.'J. vehicle or -1-lur- 2i, Cïr3it'lv -;

  laT¯7aCCIItiC <Llar.?Snt acceptable cooked new to be a solid Oil un 1i0uio.e. The GO ':? T7rJS' c '' -. IC ??: can have the fone of C0 "11'J:> ÏI> PS" of c # c '\ J!' I ': lt? S eff' e: students, of 1JOïJG.1: 'e, of ;;; rJ.nul'9s ,. capsules (hard-necked capsules and shell 1 "1011e) or suspensions in edible oils or e" utr9s forms suitable Dp..Ltic1.lLière'lel1t for a: irninistl.: e.tiol1 1J2rvoie bucc81e . The 1-d-ç; = iideg dilupts are used at 1 ' <'ta, t sterile for 2.c1ninistration. Daren t'rR19, Cp: r7.-â-â7re rar injection. The medium can be a sterile solvent or a. Elise's agent in suspension such as water or an oil for injection.

   The compositions can be in the form of the active material, that is to say tetracycline (term used in this description for the free base and its hydrates) in admixture with solid diluents and / or auxiliaries.
 EMI50.3
 pastillage coniine starch, lactoses, talc, staric acid, ¯a.ansiun stoz-rate, gonzes etc. All encapsulant or -castillan materials used in = Jt> ± 1r = ue naracn, - tique can be used where there is no incompatibility with ttracycline. Natieres can be transformed
 EMI50.4
 in com ori -. n-'s with or without auxiliaries.

   Alternatively, the ttra-cyclin can be placed in a known capsule of absorbable material, for example a gelatin capsule and administered
 EMI50.5
 under this form. In another form of reconciliation, tetracy, q in:

  <Desc / Clms Page number 51>

 
 EMI51.1
 can be balled in packs under a ton of powder <and used of this ilanière. Tetracycline can be, 7rFDà.r < <e in a ton of a palatable suspension in the water tetracycline is not soluble, for example coconut oil.



  In this case, it is possible to use coconut oil modified to have a setting rate of less than 60 F (15.5 C) and / or it can be gelled with aluminum stearate. This suspension
 EMI51.2
 can be administered orally, as is or as a capsule. Ttracvcline ointments and lotions are useful topically. Nasal drops, trochiids and suppositories can also be used in topin therapy. The tetracycline of the present invention is parti-
 EMI51.3
 cUl1.ere'rlent useful when administered by buccal or intramuscular route; a qaimne of useful doses POUl: '! This is approximately 10 to 1000 mg per dose.

   Doses are given one to six times per day depending on the patient's condition, infection, route of administration, etc.



   The proportion of active ingredients in these compounds
 EMI51.4
 sitions may vary. It is necessary that the active ingredients constitute such a proportion that one obtains a dose. Of course, several unit doses are new.
 EMI51.5
 Î't1 ' <: administered shortly thereafter. Bfcn has been observed 11f1rtic1.l1ière111ent for intravenous injections, '1U> 1.1TI0 in a proportion of less than 0.10' of t6tracYcline is effective, 3l stnr '' 'fr?' Ble not to use less than 0 , 10; tetracycline.



  'nctivit iupineritp- with the concentration of. tAtrAc ".rcU Yle. L:" 1 rrC'17c, rt: j.n? 1 d3acen'L. small can reach 10 'or 5.,; f, or iai <do <1 :: 1v ': nt.;: LC1' ('! Of 1 "substance:.: R}' 11ini.strf.e. By eyerJ1ple, some co," - t ';'; t'e can, 41; re px'T) -3T '^ r F'v'3c a 111'r "),", (") Y'tirn nE? 71 iYn1') nrt'Y1t -, ('! Tir', 1il1Jynt and 17.213; ') rte proportion ,,:>' ;; īF: r9 active, f) t: '>' 1 ('nl- primed ;; (,,' lnt'W; 1> 't "' : l'J ±? 103a '1:' 1 '"t" tllÎ' jrf: 1, i "fl nrnrlron [-" jY1t r "'t = f; it j¯n,.' 1 1, t 1J1 ' i1 .. ". r,, - c '?' i3 ei '' (, 1,; 1 T": l1tjro "f; .nlidr> t'-i1 j, ':)' '' 11. ' '' Lrn 1; n: "> 1" "" I ", '1, t")' '' 'C "1, (" qY', prt, ie j 1 i, ét rar> ycl 1 pu, " , p2.) '0Àr /.' "'' ''" '1.1 l'; - '11', ", n" 1 î '; 111T'nl;') 'years ..:'

  <Desc / Clms Page number 52>

 gelatin capsule.



   The solubility of ttracycline in water between
 EMI52.1
 pH 5 and pH 7 is very low (α-Dnro7i! n, -tive-bind O, β. mg, / cm3 ': The addition of calciuri chloride, for example the. (' in Doids / volu e allows to obtain much more concentrated solutions before a solubility exceeding 5 mg / cm3, for example at pH 6 or in the range of pH 5 to 7. These solutions are very viscous, but the viscosity can be changed by adjusting the pH in the high solubility range * Stable solutions or suspensions containing more tetracycline are thus easily obtained and
 EMI52.2
 have great utility in 1Jh.arniaceutiaues compositions.



   Highly concentrated aqueous solutions of tetracycline containing more than 20 mg / cm3 at pH 3.5 to 9 and suitable for oral and parenteral administration are obtained by mixing tetracycline, a non-toxic heavy metal salt, for example aluminum sulfate and tartaric acid or similar chelating agents like citric acid,
 EMI52.3
 pthylenediamine-tptra-acetic acid, gluconic acid and phytic acid.

   By heavy metal is meant here any metal other than the alkali metals; this is how alkaline earth metals such as calcium are included in this definition of heavy metals- A similar composition, useful
 EMI52.4
 and comparable qolubility, can be achieved by -A41anpant a chlating agent and a heavy salt of ttracycl 111P-, Although different forms of the invention have ('tir "f. =: -: ritp- in detail, it will be understood that I1.o ifications can be made to the 1Jroc <? Des described and nroducts without falling within the scope of the invention.

   Certain ¯agents, compounds or -.ni (by -acids, media, solvents, etc.)
 EMI52.5
 , t '7¯t: e: s c1.'t; :: they -7onn's nJar either e;:? - nle nu' 1'Y '"" i r.ti :: m " : 1roc.'js ": 1 - '; UV?" T qtre lltj 1 iss 00111' d ': 1u.tr'1,?; = T .---. ¯ .. n1J' r] t: w ¯


    

Claims (1)

CLAIMS- EMI53.1 ¯, ¯¯¯¯¯¯¯¯..¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯ 1.- Process for the preparation of tetracycline characterized in that Streptomyces BL 567201 is cultured in an aqueous carbohydrate solution containing nitrogenous nutrients under submerged aerobic conditions until appreciable antibacterial activity is imparted. to this solution, then the tetracycline thus obtained is separated by alkaline precipitation or extraction of the fermentation broth. 2. - Process for preparing tetracycline characterized in that Streptomyces BL 567201 is cultivated in an aqueous solution of carbohydrates containing nitrogenous nutrients under submerged aerobic conditions until appreciable antibacterial activity is communicated. to this solution, then the tetracycline thus obtained is separated by alkaline precipitation from the fermentation broth. 3. - Method according to claim 2, characterized in that the separation of the tetracycline comprises adjusting the fermentation broth to an alkaline pH in the range of 8 to 11 and the separation by filtration of the precipitated mixture of mycelium and tetracycline. EMI53.2 4. A process according to claim '#, characterized in that the separation of the tetracycline comprises adjusting the fermentation broth to an alkaline pH in the range of 8 to 11 and separating by filtration the precipitated mixture of mycelium and tetracycline and dissolving the tetracycline from this mixture in aqueous acid below pH 3. 5. - Process according to claim 2, characterized in that the separation of the tetracycline comprises adjusting the fermentation broth to an alkaline pH in the range of 8 to 11, the separation by filtration of the precipitated mixture of mycelium and tetracycline and dissolving the tetracycine from this mixture in an aqueous acid below pH 3 and adjusting the pH of this tetracycline solution above pH 3 <Desc / Clms Page number 54> to precipitate the solid tetracycline. 6. A process according to claim 2, characterized in that the separation of the tetracycline comprises adjusting the fermentation broth to an alkaline pH in the range of 8 to 11 and the separation by filtration of the precipitated mixture of mycelium and tetracycline and dissolving the tetracycline from this mixture in aqueous acid below pH 3 and adding to this acidic tetracycline solution a chelating agent in an amount sufficient to block substantially all of the calcium present and adjusting the pH of the tetracycline solution to a pH greater than 3 to precipitate the solid tetracycline. 7. - Process according to claim 2, characterized in that the separation of the tetracycline comprises the acidification of a fermentation broth containing the tetracycline to a pH below 3, the filtration and the alkalinization of the filtrate at a pH 8-11 to precipitate solid tetracycline. 8. A process according to claim 2, characterized in that the separation of the tetracycline comprises dissolving the solid tetracycline in aqueous acid below pH 3, filtering and adjusting the pH above the pH. 3 to precipitate the crystallized tetracycline base. 9. A process according to claim 2, characterized in that the separation of the tetracycline comprises dissolving the solid tetracycline in an aqueous acid below pH 3 adding enough chelating agent to block virtually all of it. calcium, filtration and adjusting the pH above 3 to precipitate the crystallized tetracycline base. 10. A process according to claim 2, characterized in that the separation of the tetracycline comprises dissolving the solid tetracycline in aqueous acid at a pH of approximately 1.5 to 2.5, filtration and adjustment of the pH. from 3 to 6 for <Desc / Clms Page number 55> precipitate the crystalline tetracycline base. 11. A method according to claim 2, characterized in that the separation of the tetracycline comprises dissolving the solid tetracycline in aqueous acid at pH 1.5 to 2.5 approximately, the addition of sufficient agent. chelation to block virtually all calcium, filtration and pH adjustment to 3-6 to precipitate crystallized tetracycline base. 12. A process according to claim 2, characterized in that the separation of the tetracycline comprises dissolving the solid tetracycline in aqueous acid at a pH of approximately 1.5 to 2.5, filtering and adjusting the pH to 3. -4 to precipitate the crystalline tetracycline base. 13. A method according to claim 2, characterized in that the separation of the tetracycline comprises acidification of the fermentation broth to a pH below 3, filtration, alkalinization of the filtrate to a pH in the mixture. range 8 to 11, extracting less than half a volume of n-butanol and separating and concentrating the tetracycline-rich butanol to precipitate solid tetracycline having a potency greater than 200 mg / cm3. 14.- Process according to claim 2, characterized in that the separation of the tetracycline comprises the extraction of an aqueous acidic liquid containing the tetracycline having a pH of less than 3 with a solution of an inorganic salt in an immiscible solvent. volatile selected from the group formed by lower aliphatic alcohols, lower alkyl esters of lower fatty acids, methyl isobutyl ketone and chloroform, in the presence of a water soluble inorganic salt selected from the group formed by halides and sulphates of alkali metals, alkaline earth metals, ammonium and lower alkyl amines, this solvent being present 'at least in an amount such that an amount of pure tetracycline equal to the initial tetracycline content of the liquid containing the <Desc / Clms Page number 56> tetracycline is soluble in this solvent, separating the extract by the solvent from the residual aqueous liquid and recovering the tetracycline from this extract in the solvent. 15. - Process according to claim 14, characterized in that the salt is sodium chloride and the solvent is n-butanol. 16. A method according to claim 15, characterized in that the weight of the sodium chloride added is between 2 and 20% of the weight of the aqueous liquid. 17. - Process according to claim 2, characterized in that the separation of the tetracycline comprises the extraction of an aqueous solution of tetracycline having a pH in the range of 8 to 11 and containing calcium ions in an amount of 0.1 at 2.0%, calculated by weight% calcium chloride per less than half a volume of n-butanol and the separation of the butanol rich in tetracyclin. 18.- Aqueous composition for combating infections due to bacteria characterized in that it comprises tetracycline and calcium chloride. 19.- Aqueous composition for combating infections due to bacteria characterized in that it comprises a non-toxic heavy metal salt of tetracycline and a chelating agent 20.- Aoueuse composition for combating. infections caused by bacteria characterized by comprising tetracycline, a chelating agent and a non-toxic water soluble salt of a heavy metal * 21.- Aoueux composition for combating infections due to bacteria characterized in that it comprises an aluminum salt of tetracycline and a chelating agent. 22.- Water-based composition for coma @ ttre infections due to bacteria characterized in that it comprises <Desc / Clms Page number 57> tetracycline., a chelating agent and a non-toxic, water-soluble aluminum salt. 23. - The method of claim 2, characterized in that the separation of tetracycline comprises maintaining an aqueous solution of tetracycline at a pH in the range of 7 to 11 at a temperature above 20 C until qu Virtually complete destruction of the chlortetracyclin present as an impurity and separation of the purified tetracyclin is obtained. 24.- Process according to claim 2, characterized in that the separation of the tetracycline comprises the suspension with activated carbon of an aqueous solution of tetracycline below pH 7 and the removal of the carbon by filtration to obtain a purified solution of tetracycline with virtually no loss of potency. 25.- Process for the preparation of a composition for combating infections due to bacteria, characterized in that tetracycline and calcium chloride are added in the presence of water. 26. - Process for preparing a composition for combating infections due to bacteria, characterized in that a non-toxic heavy metal salt of tetracyclin is added to a chelating agent in the presence of water. 27. - Process for the preparation of a composition nour coribattre infections due to bacteria characterized in that EMI57.1 add ttracycline, a beneficial agent and a toxic ron salt, soluble in water from a heavy stall in preference to water. EMI57.2 28.- Preparation process of a ':! 0!' !!, od t: JI) n to fight infections due to aill 2r '! .Ri "': se * ri = t ± ris ± en ce ou ' we add an aluniniun salt of t!. 'tra0 "! 02.irl'? Fi it '^. :: '1 "- ::! 1t this chl? TiOn on loan from e2a. 9.- Pr0cl. (! T de T.ç'sn2r.i pT'a r'tfx..F, ni'ïÇx t ir! Nou '' 't. <1, ¯ <Desc / Clms Page number 58> beating bacterial infections characterized by adding tetracycline, a chelating agent and a non-toxic water soluble aluminum salt in the presence of water. 30.- The present invention with all its novel features *