BE624816A - - Google Patents

Description

       

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 EMI1.1
 loa oowaetfcanta just got the attention & ttir4o on a Hardware error occurred on page 62 of .4111011 '. description, in line 10 (point 11) where the percentage 40 B must and read 8152% and not 9.2 this error roof ttro Qon.i4'i. 00. "obvious since the total of all percentapel. Pa.rUiI \ 1.UH..t when the pcturothttgw of 11027ent was dQ.m1n '* by difference" ooame in the These present - oit 4 be ac) nl $ 100.00% whereas..1, we add up the percentages as mentioned above, we arrive at IOI.QO%, We would therefore like to ask you to include this letter in the file of the broiret to be worth oogme do right and for a quiz copy to be appended to any copy of the patent which o * r4
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 delivered to liters,
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 We pr1cn you.

   besides 4 * noua a4r .... r the duplicate of this letter of correction (Idatin4 to 8 be attached to the Title Otfio1el.
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  Would you find, attached,? 15 in a fiscal stamp, raised the official tax.
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 July approve) MeNaKMy <f) no Blttt & tiORB '. diatj / f - <'*' * <'3t'

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 lift presents învelltioxi concert * of processes, rl Y VpiwrY to prepare by sees micic-blologiquo do new antibiotics, jpy pa y (, i $ Miorobiologj.quo d <t nottveau otjhtibi & tiquea, and to concentrate, to isolate ot to purify or * produced.



  According to the invention, the new antibiotics are obtained by fermentation of certain species of the genus MiAïaQnos ZM (order don ltinotarotxes) prinolpaleaent y.g1.Sfflffft.oia..Fytt! ci! ur9. and y ha, Ho. |> hy.tlfta. (including their mutants and variants, it being understood that the tome "mutants" encompasses natural and artificial mutants, for example those obtained from the pecities by $

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 EMI3.1
 Tolu mutating agents as High Frequency Radiation
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 (r, uybhg-x # ultraviolet) of aetihophagee and% dlâzoto # mustard or mioforgünQmè. 4qu1val.nts until it closes anti1Qt1u.ment active products and by isolation of the substrate thus obtained antlbiotiquoment activated fractions.



  These fractions can 4tr..nuui separated and the ant1b1ot1- ques thus obtained can be transformed into their usual derivatives such as their salts with acids, their derivatives $ t- and / of the 0 <* aoyl <S6 and their reaction products of bisUltit1qU.1 dlaldebyde, and ketotest addition compounds In particular, the invention relates to the preparation of basic antibiotics from suitable onpeci of "1oro-, p, r, prai leo plus 1 load of dob antibiotics being degraded overnight. "dontamyoihi * t iiht1b1Gt1qu .. produced.



  . nved Gn1amYQ1bbH in addition to 00 which .and'called overnight * "antibiotics of the H * -45lM group and ktntibïotîqüea of the Sî'-30" group low culture of the ViVante have been deposited and are part of the Culture Collection of the US Department of AlOutur., "Northern Usage Bonotreh and Development 1YisiOrt", roortal 1111n011, USA, where they are available under their respective ttRftl identifications.



  Patml les which Ü, of which tèvdldo usable to prepare the GsntY1n. and water ed-prûduiltt antibiotic., liuh dbëhtto eu * to itê designated p & r tè.isitâmii {iÈP, QX. & noupe Ufpôlê dana 4a 8Ui âxpgtga) ta fOU 1 the bed NRRL 2953 and was ïdelè at librigine from a sample of oily earth from Syracuse in the State of otki USA * Ki is characterized by having an average diameter of 0.5 microns} it does not produce a true aerial al1; 11 produces separate apoeans with an average diameter of 1.0

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 micron, at the ends of #implesi sporophores is non-aoido * resistant; gram-positive; digests certain types of protein and starch; is aerobic; ben grows between 22 and 37 C but not at 46 C or above.

   The shape and orange color of the colony on the richest organic agar-agar media is
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 typical. On other low nitrogen agaragar media, the colony color varies from reddish purple to purple. While this reddish purple pigment is characteristic of the freshly isolated product, it may disappear temporarily or permanently in the streaks obtained by repeated isolation and transfer.
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  Another species, essentially equivalent to 1,, urp "reA! In the preparation of antibiotics, received the designation. Cromon08tA lobincerori .8p. (Hereinafter referred to as H. na," ^ ,, ra}. This species has was isolated from a sample of ptelevio mud at Jamei3T: 1.11C \, State of Heur Yor) ç, USA and received the NO NRRt 2985; pq ta, '. t grow in a medium consisting of 3.05 yeast extract (Itac) o 1 fe dextrose and 0.1 calcium carbonate in distilled water and that it was done incubate at a 4, turning at z0 for 30 days, is characterized by a long ttyoeiua, | branched, regular, without septao, about 5 microns in diameter. ' * - * ').' '"' 'The culture is purple The spores are pjo () 4Ùi: ±', '.: Q <ib4.aIiinî "nt separately under the tome of extrem1t4.rP11é ,, :) Lt.t cen- Irai.

   The spores and sticks are rustic. The opcre aur is roughly spherical with Ùk d: 1.- j being about 1.0 to 5 microns. By ioroBope contras t of 1 phase (x 1000 ) t \ 1E1 spores appear cooiae 6tan '"t \ 1U'u. The etwctronlques micrographs indicate that the roughage is due to thorns which protrude regularly from the surface of the
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 spores occasionally reaching a length '"1 | e 0.2 mi-' 'crono (In this culture broth in, 1eJ-Oontn 1nd1-

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 EMI5.1
 above, K. purpureti does not seem to produce sports) *.

   echinoapore, digests certain types of proteins and starch, is aerobic and also grows well in the temperature range.
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 res ranging from 22 to 37 * C but not above about 5000.



  When grown in a medium consisting of 0.3
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 beef extract, 0.5 tryptose, U, 1 K dextrose, z soluble starch, 0.5 yeast extract, 0.1 calcium carbonate and 1.5 agar-agar , all in tap water, shaking at 28 C for 30 days, a pig-
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 diffusible purple shade. This characteristic diffusible pigment may disappear momentarily in strains obtained by repeated isolation and transfer. (M. purpurea in the medium just described does not usually produce such
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 picment diffuoible).



   A microorganism which has been shown to be useful in the preparation of group E-451 antibiotics has received the designation
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 Microcionospora carbonabea n.sp. (hereinafter called M oarbonacea). received Zd NRRL 2972 and was originally isolated from a soil sample taken at Olean in New York State, USA. M. carbonacea is characterized by the development of charcoal-black areas of spore accumulation on a colony of vegetative mycelium which is, if not orange, when
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 grew in 0.5% yeast extract, dextrose, 0.1 CaCa-1.5% agar and incubated for 20 days at 2800.

   The spores of M. oarbonaoea vary in shape from oblong to ellipsoidal, and the average dimension of the mature spore is 1.0 by 1.5 microns, When the spores are examined completely under a microscope and direct light. , we see that they are black. The sporophorea appear in large concentrations in par- ticular areas of the vegetative mycelium rather than being distributed ha-
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 eard over the entire length of the mycelium. Normally the lI2, foel: l -

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 EMI6.1
 vegetative um does not seem to break into polymorphic elements 1 It phea (fragments but retains its integrity until there is eventual autosis. It has an average diameter of z 1 micron} it is non-aoidorealant and is gram-positive.

   Ltorgal "% 4 nisnre digests certain types of protein and starch, is
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 # aerobic and grows well between 16 and 37 "Ci but not at 50" 0 or above. Aïar, ue - li'cr¯aré and the orange color (with 9! Black-brownish mottling) of the colony are typical of ê-1: produced; ! freshly isolated from colts these characteristics may disappear momentarily or permanently in effects obtained by repeated isolation and transfer.
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 . A microorganism which has been shown to be usable in the preparation of antibiotics in the SP-30 group has been designated M1oromonoDPora halopt1o n.sp.

   (called in the cult. phyt1o), received N NRRL 2998 and was originally isolated; from a mud at the bottom of a salt pond 4 Syracuse in
 EMI6.4
 New York State, US. M. lohyti9a is characterized by a long branched mycelium approximately 0.5 microns in diameter and abundant sporulation. The spores vary in shape from that of an ellipsoid to that of a sphere and have a large diameter equal to an average of 1.2 microns. In ancient cultures, they appear with smooth walls and a black color.

   It appears that the spores are carried randomly along the mycelium on short or long sporophores; possibly, they are sessile. The vegetative mycelium does not normally fragment into polymorphic elements but appears
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 maintain its integrity. The mycelium is nonacidoresiotic and gram-positive.



  To isolate the microorganisms, a portion of the soil or the dried mud sample is shaken respectively in *
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 sterile distilled water and, after making a suitable dilution, the suspension is deposited on an agar-agar medium. j

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 The medium should contain 1; b of glycerol, 0.2? 6 of sodium nitrate, 0.1 of phosphate diPOtaBOIqUOP 0.05% of magnesium sulphate, 0005-e of potassium chloride, 0.001 f> of sulphate ferrous and 1.5% aear-agari it preferably contains 0.5 tryptose, z soluble starch, 0.5 i calcium propionate and 2.0% dfagnr-agar, all of these constituent being in distilled water.



   Cultures are tested for their antibiotic activity by first growing them in the following medium for up to 60 days at 26 * Ci 0.3 extract.
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 beef, 0.5 tryptoeia, 0.1 dextrose, 2.4 soluble starch, z, 5% yeast extract, 1.5% agar, all components being in ' tap water.

   Then all of the aqueous solution of acar-agar is extracted with butanol and the butanol-water extract is concentrated. Sufficient antibiotic is extracted with the butanol-water mixture to obtain a conc.
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 centered which, in the disk test, prevents the growth of sert-, phyloCQCC1! S aureue, and of Doillu8 subtilleo and in some cases also of Eschercha eoli, Peeu9mona $ aeruinosa. lebaie7.- the rnaumonix $ and:, yaobzoterium pmegmntis.

   Antibiotic activity against the same organisms is observed after growth in submerged fermentation for 96 hours in medium.
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 aqueous cor-holding nutrients oonvenablea, -telwnue # - = in the case of M. purpurea and M. eguinosp-orlo a medium containing 1 yeast extract, read cerelose and 0.1 carbonate calcium or a medium containing the constituents * described above in this paragraph; in the case of M. carbona-
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 esea, a medium containing 0.5 tf of yeast extract, 0.1 x of soluble fish products, 0.1 of calcium carbonate, 0.1 of spray-dried corn maceration liquor
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 and 3 lactose;

   and in the case of !. halopht1o ,. a medium containing 1 NZ type z amine (which is a peptone resulting

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 #i of 1st pancreatic digestion of casein, and that one villit and .procure? at Sheffield Chemicàl Cor Norwict ;, New York, OSA) # and 5 y soluble starch, Difco Lborator1e8 Ine. 1, Detroit, Michigan). - Tables 1 to 5 below compare the new lees
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 species mentioned above with some known species.
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 by Mcromon9sor. In the places where there is only a dash them.
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 values are missing or have not been reported in the tables * # - lioe observations on the colonies given 4B table 1 were made after 14 days of incubation e-24-2E "C ûaa - ¯ the indicated media.

   In lit, description of: Coma'UQna des colorationa O He used the references and the following system He the first designation is a color name t; Lr4 from the book "Descriptive Color Name ictipnary", by Teylor, 4och .., . GAnvl11e, published by the Container Corporation or 9rla, 1950 'with a color number corresponding to the name of çoului1 taken from the work "Color Harmony Kanual", 4th edition, 1958, 4th also published. by Container Corporation and Amer: L * 4; the 9 -? conde designation. is constituted by a r-om'de. color and one! number with the same meaning as the glue found, in .Circular 533 41, {National Bureau of Standard * {u.> 8 ?, ne- wrruàrq .955 .. - "- ;; ,,: 'c1.t' 'tt': "" 't. ",.

   With regard to j. b & 3, fiytlpla * .. "i! 1.", pi04 "4i table 1, ûeluioi is characterized, ps.y, the * 4bleai4 l, ceu1.Oto1 eat octér14 per 'P \. bI'. i .. in the Pji range from 6.8 to 7 Qau PftjEt. 4 J many midpoints of - 'r. ftitwjw. are in, ta, 3 fts r' '"'! .. ', - *' 6 'i "1t r - ù' ir but turn taking a brown tint With the - r, t. .Tl, .¯ i the light brown., (Tan), the bf 4rape and, the lywd '# <?! & Common color uiitri 'ations *,, The colony becomes. % n, tt sfftt6 -'.'.-; dark, it never becomes black t 'Se ml0rO-Or $); pite an abundant sporulation, .particularly d:' t laa 'f ôo-r v, old nies colored in br \ Ú \ - Qn obtains hab ; l.1u '\ ,! J. and \. .! ty3, s e ..

   T .. '. i S r s

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   Place diffuaible reddish brown on an agar-agar medium composed of 0.5 yeast extract and 1% of any of the following sugars: galactose, lactose, levulose, mannose, raffinose and trehalose.



   Table 2 provides information on colonies which were incubated for 14 days at 26 ° C. in various media. Information concerning the known species with which those of the invention are compared was taken from Bergey's work "Manual of Determinative Bacteriology", 7th edition, 1957, published by The Williams and Wilkine.
Company, Baltimore, Maryland, USA.



   In Table 3A and 3B, the growth and color of the colonies of the new species according to the invention in various media are compared with those presented by three species of Micromonospora currently available. The media employed are those which are currently used for the determinations of Streptomyces. The colony color is identified by the two systems used for the information in Table 1.

   In addition to what is shown in Table 3A, an observation was made on M. halophytica colonies in Waksman glucose-yeast extract -agar-agar medium, with the following result: good growth; colony:
Brick red-g5NG, Intense brown-55 colors,
The microorganisms according to the invention are capable of using various sources of carbon and nitrogen.



   In Table 4, their carbon utilization is compared with that of certain known species of Micromonospora by a visual estimation of the growth in agar agar veneers in media consisting of 0.5 "Difeo yeast extract. "(product of Difco Laboratories Inc.). 1.5 agar agar and 1% of the corresponding test carbohydrate, all of these components being in distilled water.

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 pine plus-of what is contained in table 4't i'utili- # '\;

   carbon cation of M. hnlp2) iXli2.% is determined in an identical manner, ayeo the Situants oona Living -oarbohydratea melibioae, trehalose, adonH-ol, duloitol and ribosotte good oroi-aeance being obaerv, be 'vea the first two this uan- etituanto while with the three will untie the. aro, health is
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 poor;
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 In Table 5 we have compared the use of d-O ots' don *.



  Micro-orgaitiaceajaelo.n the invention with that of other species of Kîcromort2U2rg by visual estimation of the crolvoance Our, slaps of agar in a medium consisting of 1%. glucroae, 1.5 agar-agar and, the particular source of nitrogen. ! used (according to the concentration indicated), all these co-agents being in diluted water.

   The color code used in Table 5 conforms to that given.
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 Table 1, It has been indicated above that the invention encompasses the use of mutants of the new species according to the invention to prepare antibiotics as well as the use of heavy variant * As examples of such variant $ which differ of the eapooes described on a. secondary point such as by a pro-
 EMI10.5
 Morphological or biochemical property, we can cite two variants of M. echitiospo-1 called M. echinosjpora r. ;

  , u ,,, i and M. echinoapora var. 2allida # and a variant of ï. o.arb-ona.c..a.t called M. carbonecea var. aur tiae ". the cultures of these va- *
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 laughing have also been deposited and are part of the collection - of the United States Department of Agriculture, Northern Use
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 Research and Development Division, where they are available under their respective identifications, namely? RN '299r NRRL 2996 and NRRL 2997,' respectively.

   These variant have euisi
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 was. originally isolated from mud samples near.
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 Jameaville, New York and from the ground at 0aa, New 'Or,' tJBA ",!

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 respectively and are taorxom3.which have the same as X, ecunogpora. or 1J ft% rMbnpn1aol.efo respectively but with the difference! following 9 * & il e, chino8, û.ra. var. ferrufrinfra does not reduce nitrates believes well on eollînbs2gè ribose and M. eohinoapora var.

   both have poor growth on this pantoae), these colonies tend to have a reddish or brown color
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 yellowish (eventually turning reddish to purple-brown with age) and is occasionally encountered in myoe-
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 lims of brown pigmenta * However, sporulation has generally
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 rise poor in certain environments (for example
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 # tomatu-f oat-agar-agar urine plus Otl% ¯ of sodium carbonate) in ancient cultures we find that the spore
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 and the sporophores have the characteristic rough wall *
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 In the latter medium and - on Bennett's agar-agar, the growth is good; 3 a colony is plicate;

   and the color is red- This mahogany-g6 1/2 el, deep reddish brown-41 *. eat; 3noc, zo, vax * rallicla does not produce dark purple pigmentation of mycelium on agar media. The neck-
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 their colony on most agar media varies
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 p51e orange with light tan ("café au lait *). On Bennett's agar-acar and with, tociate-oatmeal fart -: a.araar z + 0) 1% sodium carbonate , the growth is good and the colony plicate. your colony color in the first medium is apricot-s4GAf light orange '* 52; in the second medium the pr3phkt.a has the color shown below
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 while the center becomes darker.
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  S c, a, 7bonac.aā vair. agrantigoa does not reduce the nitrate produced oecauionnellenent tut, picaont diffused yellow ïnxaqu'.7. cro, ft in a cerbohydrate medium based on taannose or X3, 10e; and shows limited spore production * The colony color on most Agar4 - aear media is

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 , living orange to orange brown with time, La eurfaoe orant
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 , occasionally has small, black areas, formed)
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 almost oolor 'spores. in * '"o1rt and 1 is prlnoipa-, lemeniT formed of vegetative mycelium. On ligar,; - agar.de Be = e ^ tti the growth of the variant is good, the QQ10he' 1evéi the color of the latter being burnt orange <"g5NC !, intense reddish orange-35.

   On a medium 4'agar of toaate and oatmeal paste with 0 # 1 0 of 8odiltm carbonate the growth is quite good; the high colony! the color ### * of this last mhre-0TG, orange outen53. ' '* #.



  The variants listed are equivalent to the tspeoen described above to produce antbót1qùea in general and Gentamyoine or R-451t rea11ot1 v-eIIHmt in particular.
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 EMI13.1
 TABLE-1
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 Comparative taxonomy of 14icromonoopora species Part A Medium containing 0.1 amino NZ type 1, 1 dextrose, - .57 agar-acar Organism 1 Observations
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 aorù3cop1que Microscopic M ur42 r¯ea, No aerial mycelium;

   Mycelium long, ramifia, ¯ ".¯ïrZ 2953 colony stray high, receding * without septa diameter zaza '" "** 7? Chains, waxy! Good enough 0.5 / uni little eoropliorte ¯
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 <tb> growth; <SEP> not <SEP> of <SEP> pigment <SEP> and <SEP> of <SEP> scores; <SEP> spores <SEP> of <SEP> 1.0
 <tb>
 <tb> diff. <SEP> product. <SEP> Color: <SEP> on- <SEP> microns <SEP> of <SEP> diameter,
 <tb>
 
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 Ace: dark wine-C7PI, dark rtucoish brown-44; reverse wine-C7PG, dark red-16.



  M. cr'orlCeEJ, No Aerial Mycelium; Mycelium long, branched, lRf 2972 flat colony, moist; grow- r6ful., aa.ns septa, diameter wuu & (± aanaa quite good; pua of 0.6 u, dark color; spores pilent diff. produced. Cou- abundant oblong to ellipa their: surface: periphery old. 1, 0 x 1.5 Jx, litters light orange-c41iA, orange on anaez of aporophoroe. Vit-48, black-brownish center Inver3e: periphery light orange-4I: A, bright orange-48,
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 <tb> against <SEP> black <SEP> brownish.
 <tb>
 
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  M.chai ce a No aerial mycelium; Mycelium long, branched - AXCC 12452 colony slowly high or ul., Without septa, very thin, '' "" ''. , 252 chains, waxing machine; fairly good mo.Lna 0.5 in diameter;
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 <tb> growth, <SEP> not <SEP> do <SEP> pound <SEP> spores <SEP> abundant, Spherical <SEP>
 <tb>
 
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 Diff. Product. Color: aurfa- to ellipsoid, brown, ce: caf6-C3PU, olive brown 1.0 y in diameter, dark spores-96; reverse: caYé-3PN, mostly free but some dark olive brown-96. one lides isolation to aporo-
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 <tb> phorea <SEP> long.
 <tb>
 
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  I, i.fuaca No aerial mycelium; short, irregular mycelium, .. ", .., n qa slightly elevated colony, producing numerous elements NIUIL B'9'3 slightly elevated colony, producing numerous elements ''" "" û-yp moist, cranular-in chai- polymorphic , J 0, U-2, 0) l of nes; fairly good growth; diameter; spores 1.01 of dia- trea leiai, pigment diff. meter, hard to find
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 <tb> brown <SEP> yellowish <SEP> product, <SEP> among <SEP> many <SEP> shapes <SEP> poly-
 <tb>
 
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 Color: surface: red-orange orange (olive brown) with 83ish-g4PC wall, deep orange-deep.
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 <tb> 51 <SEP>; <SEP> reverse: <SEP> skin <SEP> tanned-
 <tb>
 
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 C4H :::, intense brown-55.



  '± 2. No aerial mycelium; Mycelium long, branched, "" fp-p ir \ MC colony Incrementally high, regular, no septa, 0 # 5¯u of v 1.OG126 che, Bl, ranulairetencha2nea; diameter; very abundant spores no diff pigment. product. dantea, in clusters and staves Color: aur: face: periphery separately; damn diameter
 EMI13.16
 
 <tb> cafe-g3PN, <SEP> brown <SEP> olive <SEP> dark- <SEP> be, <SEP> some <SEP> shapes <SEP> polymor-
 <tb>
 <tb> 96, <SEP> center Reddish <SEP> <SEP> orange- <SEP> phes, <SEP> lea <SEP> spores <SEP> and <SEP> the <SEP> for-
 <tb>
 
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 g4PC # sustained orange-51;

   my polymorphs are reverse walled: cafd-g32N periphery, dark.
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 <tb> brown <SEP> olive <SEP> dark-96, <SEP> center
 <tb>
 
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 mussaster orange-g4PU, soutar orange..u-51.

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 EMI14.1
 1 Mtt 4
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 8urt Uilim # lnt * nàfti fPJP ** * # 2 A # if * t y *
 EMI14.3
 Ob..n-a1 :: f..M8 OrJ8Sl1'.8, "(Il 1 '' 1 J", - 1, ..!,, .. '"' '' '' '' '' '' '' '' '\ -' '' '' 'Iii..lü. EBMBMaBJ * BaaBBBMBàa> aaàBaaaa * Ba Blikkeaie âaKNisttâi & ettiteiNB * aaafeaasttiJaBSaBa' '..l'. '! Iflltt'oo, 1cru- aerial;

   NealitM 1 <M 1 #mnr 2 "OOlonia high, curved, nD4" "mstm pê & tiikji $ .0 J * * m '?' '*' '' Waxy * growth aboidw Aiaxib <i'ee, ia5 & tef no join 4i ± f # arpurdlr icr ïeis 'ttjjfM' foM! < <C'Mly <euyfee <s t wr rr at IligtiiéiiiiàiM cuita-g5Ê *. t.ptm-55! lnr r i 1 # O a dls dlBïtt; t ** # 'µ5 * rou * âtî'e' * 5Kt, dark brown * ¯ ¯t,> t, 1 55.



  M. ohlno pora fac 4. Y.O "li.U8 .'rien, 1 ¯Ol 1¯ tfaafilfté-colony 1.",., OJ! 'Entl' .... riDJ1ier, saia #t, 0 # 5j; ! curved 4ir.u .., good 40 e ;, # $ & * êm * $ smt ro1.sance .. lje '! "pie; 8ent il 41tt., ambrth Ooul, 1U" ulU "taO" red brown4tr..outem .



  $$ l / 2Blt bruxt roug âtr fofwè ** 44} lu variai vougo4t .... 1'0li4l- ru .., 1u ;; ::; r.cet, M.carbûMaoet No aerial NyoelitMt; Myêlim lon # 5 krpt 0OT9 high colony, chain, regular. , n & r mmêi * 6j, KRRL 2972} 'le. of 810e11.118 a'ncm; p ¯6.11.- 1 ", \ ifIt diff. product. Color * isolated - ii * rç # fe i- eurfce terre cttite-g5tE, laolia, oUi'Ionott àwUAxM" intense brown-55; inverse 1 14 & 1'8. 1 * 0 * 10 Ai timmêmêâ î terra cui te-s5D, intenee brown-99 * ... mm ,, f 1 (, a, t "ir ,,, No aerial 810611ua; U1ceUu long,: ttllldt4.t J , lCO 12452 colonlo high, chain-linked, regulated; without a.irtJ .; 0 5 J mv / V a: * 34 waxy; enough growth of ci1aatre; | N * * n good; no pigment 41ft. , po: t1; '.. 8t gts Color t surface copper brown- en aima, ephii * lli | iéoî- g5.PI, intense brown-55; invar- daleet 1.0 m <te '4iâliitn.



   # rouèâtro-g4PI brown,; brown in * e'lflll "55 *, M.fuaoa Pdi of myc.l1ua ser1.,. MYc.l1ua 1r 'gas B-9.' high colony, granulai! '. elements: p01: ;; Itw, - BSRï <B-945 roaeier, dull! good growth - 0.5-j !, 0 of 'Ui, "1'". i gazlC., t1 '&' light pilent diff. olive, por .. -fJJIat.jt 1.0.



  Ir yellowish brown. Colors eurtot- de 41 am..tr .; .. 411t .. ' <w this <terracotta -g5 ± ±, brown polynorphea forms of 0 * intenD -55j invars s brown '2,0ju da 4iaâaff * t tga-çggg, btUn 1I1t nl .- ".. J' j ....... .... u.tjt.u..ir '.: -..'--! - r -.-- rT ----.'.- .. rL .-.!' LL- .. ' 'r ---'-- i' -'-'- '".'-" -'- (..;, u ..' '' 'of 1rI1.c.lium .41'1' 'dyos. ; t of 2 types one long 4100 lo 2S High colony, granular and regp1., the other short and ATOO dense icoze, ..c .. grows very good * 1n '' "pi? ôdtti # uii ** # *.



  1 fi ne. Color! p4r1pbé surface. , br.wc: elements bo \ 1 * orph .... rie rouge rou0B & tre-g4M, 0,8 2,0 ju de a1t '"intense orange-50, o.ntr ... pore..j1e' ttbôMtea, akaaolat.g4Pt brown, gri- port brown '.. in ameié 1.O aA.



  ; sâtre foneé-62} inverse i diameter, iphéfia.ttejràr * # spice brown tont- ± ..iL1 .... run ellipsoidal "" -r- .... #) #. # .. #. # lPIr J l 't. ..... 11 ... i "'t" Aoo. "" R ,, ....................... & uL'M

  <Desc / Clms Page number 15>

 
 EMI15.1
 ut '&. e ..



  .Ehi ië! /!
 EMI15.2
 ! '1:: leu I:'; rrurell Cttl "bonE! A iftàloDhrlja .ChA'tOI & K.tJ, t.Ì 11.rXi" ltttl211 ,, (iflit1lâ;: r-: Hj ',? ¯L, " :;, JG, 7 ,. J, 'j Glucose? A of myce- Growths Croi .. & ncel ra6 of myc²- Cro1.ac'J Oro1; o & nc "CrQ1gSfio'J iijafiO aerial li id1ocr. E8ez good lium aii .: T1òuru., M'41ocr. Mtd1o, r8 '41ocr. Cr ..: i 3SaI \ ce: 1: <": 4ref ria8r.case: 71 have quite good .coulitrs vivacious. 8 (t.lub-1e, no pir-; eran :::; ec; 4L.1 Pio: rent Color * Eî soluble ornnc" vi! - SJoIu1> on: oran ,. pa.-
 EMI15.3
 
 <tb> Color: <SEP> Colors: <SEP> glove ' <SEP> brown
 <tb>
 
 EMI15.4
 f & 1e, .clair- pink colony plI.- 1. onc. aT. g51Aoran & 8 flat dark orange layer 4.reddish with black spore layer z: oder-J-1. of spores .cri. (be 1.



  No CG11- r..oir nn! - brown! Tr. ches de. spot 1. ur - &! res eosbrea ue ¯¯¯¯¯¯ ¯¯¯¯¯¯¯¯ "" el t1ne Liquefacton Uaaé! 'actien Lic14é: f1iction Liquefac'tionlL1.qué! ac'tiOD L1t "act : 1oD L1 ... u ': t & et1onIL2.qu-ae: 1o.



  \ .f a: ni01e JU .. 1 "aible 1:" ible- #imsr t-.:;-# <LCO & ± 1Ü .1.1 '.....



  Lai't e.ee l.n'te :: tent}) 'i. coaculate occa-teI :: ent pa ... uC sne .. p.pton1. ' coa S ## Utili.) Utiliaé Used 3SSSL # 1J1y.raé Not tnT.rai InTersed fo1EAstarch l! y4rolysed.) Hydrolysis Hydrolysis Hydrolysis S.:drolY15é Hydrolyzed Hydrolyzed 1! 7 tWofml ïl Decoposed .b.spid: aeI! '' ..Attacked Pa8 not decorating 4'com- Pa # ûâmamCellulose auell1ue 1'eU BéeeEpos d EÍ, c Ot: ln 0 s1! ouelcge eu "o8é 'Dose êS - ### Reduct1.on Variable J; i'tri'te .. l; itr: i: tea: atr1tea Variable .No d8 ..1tnt.a as of nitrate a - rrodui: ts rodui 'ta' Crodu1 'ta rductlon' Drodu1: te: redudtIiIID # "##### .3onne Good surprise - Growth. CrotOT8nce 2 growth to op't1muD growth., ...



  22-5T'C, pse I6 '' Cs not 18-40'S, not between '4e croinsan- de crciessn- de croisuan- JO-5-c ee A 47 * C ce à 50'C ce à 50'C ## ####### ######### ### '##### ####### -: Cro1scsance Aérobl. Aerobi. Aerobi. Aerobic Aerobic Aerobic [Aerobic [Aerobic * aerobic- or Aerobic A & ob: 1st Aerobic Aerobic Aerobic Aerobic Aerobic .1erobic anaerobic ¯¯¯¯¯¯¯ ¯¯¯¯¯¯¯¯ ##
 EMI15.5
 vJ have The properties of! - ech1nospora are identical with respect to these Bilieux.

  <Desc / Clms Page number 16>

 
 EMI16.1
 



  . <-, 4). ¯ .., ......, ,, "...., ............. ¯ - .. ... 1 1 -. - - - -, .1 - 1-. IJiiL 3A
 EMI16.2
 Ki1ieu "U, .MUE2urea .ecàinosnora 1 !. l !. ha10'Dbvtica M. cbe.1.cea M.fusea lt.8'O .., Hiu 11ffi -KRRL 2985 -lr.RRL 297 b-xCdL 298 ATCC 12452 -b1rRL -94J -èc 10026 Agar "" 'agar Growth.- Growth: Growth: Growth: Growth': Growth ';' Cxo1 $ sanee: b.onne, of good; good; good; fairly good; good; good; 'Peripheral colors: Èe Bennett Color: Color: elected colony- fold colony Color:

   Color: intense earth-55. brown baked terracotta and cate, curved terracotta- intense brown-55, -m5PE, nu-g71f2Py, chains. vee, Color: terracotta- g5PE, brown -centre: brown nail orange bru- brown roe- Color: copper-g5LC, SP brxzn intense-55 from glTo'f1! -3PL; tna-54 grayish orange orange bru- intnse-55MJ dark yellowish brown - 47 95rtA, nish orange-54 dark brown center-78 bright-48 (mar- chocp1at- - blackbré g4Pll, b ',;. brownish): dark-59 ',.:'.



  AagaT Croissa'ncet Oroissancte: Growth: Crci.ce: Growth .-- growth; Croisnance: bOI1.D; e è. ::: ersoD bOn! 1e-; bonnet 'quite good; 1 bo.nn.e; 'good; thick headed; C oul'êur: brown 'Color-. color: ele colony- plus colony Color: soft Color: cane4i, ûran orange red tuile- v6e. Neck: plicate. bitter-85pc "tangerine- intense-55 le-g51lCt s5Ï #, terracotta brown- Color; orange g53.A, -8 "" ôU-: Sne-55. g51! E. br.ln drunk-e5LC, sustained-51 bright orange 'gâtr.e -intense ... S, 5 dark orange, "i: n: tense-35 .¯¯¯¯¯ nitre-54.," .. .. ': 1 q 4bzz iï.Pl .v.8 $$' ''. Eti f4. # 331G? B sr0'i $ 3, ± 'CY .: riüipt.tci -. <# -n. 3 "1. $$ aTiC $: 8ro $ s $ nL'e: enough alît-e of .Srûtsance. Pr..sanq.:; Ascending, ..rc .. ¯ce <-onne. Color: tomato; ' pretty good; . 'ïar.e; good, good; .-... '"good.

   Color: flour Color: Color: ('::: 10n10 in' oolcnie pli- Color: color * peach arin.enta '. Oats- #:' àJir, e orange pous- rnaines. Cate.Color., Periphery ' che bxi1.- g5là, orange rou- -azar-agar brna.e-1J.t. 18t: r.ux-g4LC; Couleur-. pé ::: ipnér1e te1 "re cui! .e. lant-8511, gfâtre moderate-37 - angel, u- 'oe ¯ terracotta- g5P $., brown,: red orange- - zstàtre, c1! .fté-t': ...: wa.nge-g5U g5PE, intense brown -, 55.) 4th - e "'. "ite-¯ rlan-ge intmiae-55, moderate brown center-37," ---. h; 'te: lèe-5 () .. brown center .. ehQcalt-4TS "" "".' '; black center c1air-g4! HJ, dark brown-59. "t ', y'" r hzaua, be brown 55 intense-, w. .... <-¯¯¯ ##. - #

  <Desc / Clms Page number 17>

 
 EMI17.1
 TAMT-EA, -, '3A (continued)
 EMI17.2
 Kilieu K. purpurea M.echinosrora L: .carbonacea .haloytica .chalcea M.fusca BD.



  -1; RliL 2953 -! # ... 'iL 2b -? it-FaL 2972 KEEL 2;, D8 ATCC 12452 - p.K ± L 3-943 ATCC 10026
 EMI17.3
 
 <tb> Glucose- <SEP> Growth * <SEP> Growth: <SEP> Growth: <SEP> Growth: <SEP> Growth: <SEP> Growth: <SEP> Growth:
 <tb>
 
 EMI17.4
 aspara- quite good; poor poor fairly good to good; fairly good, mediocre gine- Color: mediocre; Color: Color: far- -nêcixe clair- flat colony, terracotta- sun orange Sar orange pocket Color: s5PE, brown -s5LA, orange aear reddish orange-g4LA intense-55. intense-50, noderate-37 - orange
 EMI17.5
 
 <tb> moderate-37 <SEP> intense-50.
 <tb>



  Glucose- <SEP> Growth: <SEP> Growth: <SEP> Growth <SEP>: <SEP> Growth <SEP>: <SEP> Growth: <SEP> Growth: <SEP> Growth: good;
 <tb> extract <SEP> good; <SEP> good; <SEP> enough <SEP> good; <SEP> good <SEP>; <SEP> good <SEP>; <SEP> good; <SEP> Color: <SEP> brown
 <tb>
 
 EMI17.6
 of Color: Cree colony - colony in fold - Color:. Color: oak-g4PI, brown
 EMI17.7
 
 <tb> yeast <SEP> brown <SEP> bunker <SEP> nelée-granu- <SEP> strings, <SEP> cate. <SEP> brown <SEP> intense <SEP> land <SEP> cooked- <SEP> intense-55
 <tb>
 
 EMI17.8
 agar- nu-g5PL, lar.

   Color: Coule;: r: cr., -G4PL, aspe brown, brown. dark brown agar-59 Color: russet orange- light spice- dark-59 intense-55 maple-e4lE, saster-4ï: C, g4LG, light brown brown-57 soda orange-58.
 EMI17.9
 
 <tb> held-51.
 <tb>
 

  <Desc / Clms Page number 18>

 
 EMI18.1
 mTi. ':' x.4..4.-x.E. : .xas. V -s.AtrfCI. A'r, -W '4WH S ^' x.rtW 'w.as ".is3'Y" S' 'Y., awlti ""' y ''. RurMfr 'w .r ^ v79wf4YIY ^ W ec ^ AAfM ! 'lRIiY.Y'X5'. '. -.-.... 1 -. * ..-.



  .il v 7 - fTtftTT 38 ¯. ¯, .. ¯
 EMI18.2
 middle L '..' ta r958 Â.zCv .. 4 26 Slice d. Growth, Believe .: Believe .: '0 83âKS S? apples of mediocre fairly good mediocre tabie-g6PG, b aere-g5s bitter-95n reddish earth dark orange-51 dark orange-51 er-43 Slice of ssasr- Growth: growth: G pissances: good 0growth: Growth: aese earotta mediocre mediocre mediocre uleur: ciocolate poor bo = e.

   Carrot color mediocre mediocre nëdiccre - ir = -g4PN, burnt orange brown-g5EC, ¯, .. f ce-59 reddish orange Sucro8e- Growth and Growth * Growth: Growth: none Growth mile Growth -.null
 EMI18.3
 
 <tb> nitrate <SEP> mediocre <SEP> null <SEP> enough <SEP> good
 <tb>
 
 EMI18.4
 agar-agar to mediocre (Czapek's agar (plate). Color: Czapek) oak brown- 94PI, Bright brown -55 p

  <Desc / Clms Page number 19>

 
 EMI19.1
 ARRAY} B (continued} ..
 EMI19.2
 



  Kilieu l: .l'mrpurea 31) J.cll1 "bonllcea K.halorhytica .chalcea ### E: fusca ... # 2. sr.



  -lairlL G55: a: ia <, 1.,) "{2 -l.I # L 2;) ') e -ATvG 12452 -l ;; tiL B - :: 4} AC lúÚ6
 EMI19.3
 
 <tb> Tyrosine <SEP> Growth: <SEP> Growth: <SEP> Growth: <SEP> Growth <SEP>: <SEP> Growth: <SEP> Growth.
 <tb>
 
 EMI19.4
 acrr-agar pretty good. quite bryrille. quite 1 gold: this bonus is quite stupid. pretty good.



  (observations Color: Color: tr .r .. Color: no Color: no to 2. 7 & 14 pa ae r3- no pil ,, - (1v6e and in diffusible black. ES "diffu- KS- difi ' u days; :: slow iffu- r: .ent diffyi- chains). siLle sible.



  Gordon & SEith.sible sible Color: J. Bact 14 no reaction.
 EMI19.5
 
 <tb>



  Peptone-iron <SEP> Growth: <SEP> Growth <SEP>: <SEP> Growth: <SEP> Growth. * Good <SEP> Growth <SEP>: <SEP> Growth:
 <tb>
 
 EMI19.6
 agar-aga--, - bonne-, quite bonrte. pretty good. Color: not good enough. pretty good.



  (ôobservationa; Color; "Color: 'Color: reaction.,. color. of Color.- no a 2, 1. no; ¯ no no reaction, reactive.
 EMI19.7
 
 <tb> days) <SEP> reaction. <SEP> reaction. <SEP> reaction.
 <tb>



  Purple <SEP> of <SEP> Growth: <SEP> Growth: <SEP> Growth: good <SEP> Growth: <SEP> Growth: <SEP> good
 <tb>
 
 EMI19.8
 bromocresol- good. good. Dieestion. pretty good. Digestion. milk 'D!. management. D4.gestion Color: no Dicestion. Color: no partial -Color. *. change in pH Color: no change in pRe no change in. interfere with pH PU.

  <Desc / Clms Page number 20>

 
 EMI20.1
 



  TJLEESAÏÏ 4
 EMI20.2
 Test with ... Durrurea Lv.echîros2cra K. carbonaeea M.halonh7tica M.chalcea ::. Uraa 1I.bp carbohydra- li, 7 I: 5 11LRL 2 72 -1; R3.L 298 iTcc 12452 mua. B-S45 1ÏCC 10026 te used ##### - #### Arabinosg good good good good good good good good '. l
 EMI20.3
 
 <tb> Cellulose <SEP> mediocre <SEP> mediocre <SEP> mediocre <SEP> mediocre <SEP> mediocre <SEP> mediocre
 <tb> Glucose <SEP> good <SEP> good <SEP> good <SEP> good <SEP> good <SEP> good <SEP> good
 <tb> Galactose <SEP> medium <SEP> medium <SEP> good <SEP> good <SEP> good <SEP> good <SEP> medium
 <tb>
 
 EMI20.4
 Lactose tle4.1. & Uédicuu '. good good average mediocre mediocre Levulose poor mediocre good good average '. mediocre tnediocre Kannosis medium medium good good good.

   good good "Raffinose mediocre mediocre mediocre seems to grow mediocre mediocre 1 \ leiiiocre -Rhamnos8 poor bonae mediocre mediocre mediocre mediocre 1.



  . Starch .. good '', good- good good .good - bucket onne '- sucrose good' ton good good good bornât good - bteane Xylose good bucket good good good- mediocre mediocre -Inosi't9. ] 1méì'oer *; -mdtocn mediocre mediocre mediocre mediocre mediocre -. ^ a1 ..: ;; .poor "" mediocre. mediocre mediocre mediocre good good Sorbitol mediocre mediocre mediocre mediocre 'mediocre mediocre mediocre o. ¯ #
 EMI20.5
 
 <tb> from <SEP> yeast <SEP> mediocre <SEP> mediocre <SEP> mediocre <SEP> mediocre <SEP> mediocre <SEP> mediocre <SEP> mediocre
 <tb>
 
 EMI20.6
 witness' "1 '. -. -'

  <Desc / Clms Page number 21>

 
 EMI21.1
 TABLE 5.
 EMI21.2
 



  Source of nitrogen i..7., R, J.rurea '*' 3.i.carbor.scea H. halprhytica: Z. chalce3, 1, .fusca: t! .81'1.



  , a.lra1 2953 j-fcRKI 572 -l;: - 'i..qt 2Y98 ATOC lr'¯4 l5: 13-943 -ATCO 10026
 EMI21.3
 
 <tb> 0.5 <SEP> extract <SEP> Growth: <SEP> Croissanse; <SEP> Growth * <SEP> Growth: <SEP> Growth: <SEP> Growth: <SEP> good
 <tb> from <SEP> yeast <SEP> good. <SEP> enough <SEP> good; <SEP> good <SEP>; <SEP> plica- <SEP> good <SEP>; <SEP> Color; <SEP> good; Color: <SEP> Color: <SEP> soft-
 <tb>
 
 EMI21.4
 Difco Color: te colony tile.

   Color :, 1 stable red- red orange- azre-C5PC, -rougēg5 ** S, high and spice brown, j '± 6PG' brown 4PC, orange dark orange-51 bright brown-55 in chains. clear-4ZG, bright reddish-50
 EMI21.5
 
 <tb> Color <SEP>: <SEP> brown <SEP> moderate- <SEP> moderate-43
 <tb> periphery <SEP> 58.
 <tb>
 
 EMI21.6
 orange -, -, 5THERE, bright-orange-50, i
 EMI21.7
 
 <tb> center: black
 <tb> brownish
 <tb>
 
 EMI21.8
 carbide ###. ¯¯¯¯¯¯ ###. ### ###### ¯- ¯ ¯¯¯¯¯¯¯¯¯¯¯. ^ 'i 1! # 1 # - t # ...
 EMI21.9
 
 <tb>



  1.0 <SEP>% <SEP> amine <SEP> Growth: <SEP> Growth: <SEP> Growth: <SEP> Growth: <SEP> Growth: good <SEP> Growth: <SEP> good
 <tb> NZ <SEP> type <SEP> A <SEP> good; <SEP> enough <SEP> good <SEP> enough <SEP> good, <SEP> good; <SEP> Color: <SEP> henna- <SEP> Color <SEP>: <SEP> land
 <tb> Color: <SEP> colony <SEP> el- <SEP> colony <SEP> el- <SEP> Color: <SEP> g5PG, <SEP> brown <SEP> vivid-55 <SEP> baked-g5PE, <SEP> brown
 <tb>
 
 EMI21.10
 burgundy-g7 vee and vee and her.né-f5PG, lively-55 1% 2P, brown chains.'or- chains.Cou- bright brown-55 dark red:

   their orange: grayish orange - g.FC, bralé-g5lTC,
 EMI21.11
 
 <tb> sâtre-47 <SEP> orange <SEP> dark- <SEP> orange <SEP> lively
 <tb> 51 (light <SEP> reddish marbling <SEP> 35.
 <tb> black-brownish
 <tb>
 

  <Desc / Clms Page number 22>

 
 EMI22.1
 * "-''- '"' Sf * '' - "'" - f-.t -! * -! - # ** - <. ### f. <## -. t - #### .---. 't <..-. '# .-.,. ,, -.-.,. - - -11, 1 ,, -, .r .. ,,., ¯., - ## <- .. - '1 ............,., ............, -: t., .....



    TABLE 5 (continued)
 EMI22.2
 Nitrogen source X42urnurea 3e) b? 1e & M.halophvtica M.Chalcea K.fuaea 3d.s.



  KERL 295T '' 'i''T2 - "TSEHL 29yµ A2CC Ï2452"' .4? Ç * "ÀïcÔ" "10'Ô2ê 1 aspa: rag.1ne Growth: .Growth: Growth'1ssance: 'Growth': '' Growth: 'Coissâne':
 EMI22.3
 
 <tb> enough <SEP> good; <SEP> mediocre <SEP> mediocre <SEP> enough <SEP> good. <SEP> mediocre <SEP> mediocre
 <tb> Color: <SEP> Color: brown
 <tb>
 
 EMI22.4
 burgundy-g? giro nail- 1 / 2PL, brown fle-g3PLlbrun
 EMI22.5
 
 <tb> dark <SEP> redâ- <SEP> dark <SEP> yellowish <SEP> grayish- <SEP> tre-78
 <tb> 47
 <tb> 1% <SEP> acid <SEP> Growth: <SEP> Growth: <SEP> Growth: <SEP> Growth: <SEP> Growth; <SEP> Growth <SEP>: <SEP>
 <tb> glutsaic <SEP> enough <SEP> good. <SEP> mediocre <SEP> mediocre <SEP> enough <SEP> good. <SEP> mediocre <SEP> mediocre
 <tb>
 
 EMI22.6
 Color: ôeur- .- = # -, Color:

   gogne brown-ST 1/2 giro nail <gogne-g'i 1/2 'giro- PZ nail, dark brown le-3FZ, brown
 EMI22.7
 
 <tb> reddish <SEP> yellowish
 <tb> grayish-47 <SEP> dark-78
 <tb> 1% <SEP> nitrate <SEP> Growth: <SEP> Growth <SEP>: <SEP> Growth: <SEP> Growth: <SEP> Growth: <SEP> Growth <SEP>: <SEP> ' <SEP>
 <tb> from <SEP> sodium <SEP> mediocre <SEP> to <SEP> null <SEP> mediocre <SEP> null <SEP> null <SEP> null
 <tb> null
 <tb> 1% <SEP> nitrate <SEP> Growth: <SEP> Growth: <SEP> Growth: <SEP> Growth: <SEP> Growth: <SEP> Growth
 ammonium <tb> <SEP> mediocre <SEP> to <SEP> null <SEP> mediocre <SEP> null <SEP> null <SEP> null
 <tb> null
 <tb>
 
 EMI22.8
 JE) the results for H. e, chinospora are identical to these.

  <Desc / Clms Page number 23>

 



    FORMATION AND ISOLATION OF ANTIBIOTICS.
 EMI23.1
 the new mioro-oreaniamea described oi-desaua and the other micro-orcanisms generally envisaged by the invention, produce antibiotic substances by fermentation processes essentially such as those which will be described. After fermentation (incubation), the products
 EMI23.2
 antibiotics are found 3. 1a fcia in the mycelium and in the broth; following the separation of the broth from the mycelium, it is preferable to use, as a source of antibiotic product
 EMI23.3
 ticks, the mycelium in the case of M.purpurea. of M'eohinoapora, and their variants, the broth in the case of Nocarbpnooea and its variants, and!. 'halophyti E! -. In all these cases, one obtains at the beginning of the mixture of antibiotics.



  In particular, according to ohromatographic studies on betting, it appears that the antibiotic substances produced by xi. purpurea and by M. ech1nosora and their equivalents have three constituents at the corners. The main constituent was called Gentamyoine and the other constituents which were isolated were called by the references BA-3 (fraction A) and 3A-3 (fraction B), respectively.
 EMI23.4
 



  Antibiotic substances produced by t., Rbon, Ecea and its equivalents include. At least five, constituents, identi-
 EMI23.5
 hereafter linked by R-45iko R-451B, R-451C, R-451D and R-4513 # respectively, tooth separation depends on the process employed, In a partition chromatography system, the constituent whose rate of migration is analogous to that of A, B, 0 and D are isolated in a croup which is antibiotic and which is identified under the name R-451. R-451D turns out to be one of the main constituents of this group.



   Finally, it appears that the antibiotic substances pro-
 EMI23.6
 taken by it, hdlorhztîca and; its equivalents include at least four constituents. The composite product of anti-

  <Desc / Clms Page number 24>

 
 EMI24.1
 biotiqùea produced by. h .a, h is identified by the code SP-30 and each of the above four constituents by SP-30ILt 8'-308, 'SP-300 and SP-30D.



   To form the antibiotic substances, the microorganisms will be grown at a temperature of about 25 to 40 C,
 EMI24.2
 under aerobic conditions submerged in a m.l, e, wutx .; . aqueous .containing a source of assimilable nitrogen and carbon.



  Arbohydrates such as starch, dextrin, certain sugars and the like are suitable carbon sources. Suitable nitrogen sources can be organic or inorganic, with preference being given to the former such as soybean flours, peptones, etc.
 EMI24.3
 



  The fermentation is carried out at a pH of between about 6 and. 8, for about 24-48 hours in the case of
 EMI24.4
 ! 1 * 2urpurea and M.echinoo2or ± for about 60 to 72 hours in the case of M. ca.r :, g, and for about 96 to 120 hours; in the case of M, halophytic. Towards the end of the respective periods, a maximum production of a, ut3, biotic substance is reached. The mycelium is then separated from the broth, and the antibiotics which appear to have been enriched are isolated *
 EMI24.5
 Thus, in the case of M4, Uurtiurea and da'.e "ahi, s * o, where most of the activity resides in the mycelium, the latter is separated by filtration and the filtrate is removed.

   The
 EMI24.6
 antibiotics produced (which, taking into account the quantitative conditions The extracts, can be simply called "crude entamyol" *), are separated from the mycelium by extraction with a mineral acid at a pH equal to approximately 2. The extracts by the acid are neutralized to a pH equal to 6 approximately and can be sent through an ion exchange resin, preferably of the type
 EMI24.7
 Jtjmberlito IRC-50 $ or be precipitated by formation of an insoluble Bel ', for example a helianthate, a reineckate, a dod-
 EMI24.8
 cylbenzenesulfonate (santomerse-S salt).

   Examples of 'resins'

  <Desc / Clms Page number 25>

 of the Amberlite type which can be used here, both for anion exchange and for cation exchange, is found
 EMI25.1
 wind in the open "handboek of Chemistry and Physics, 42 th edition, Cheaical Rubber Publiahing Co., Cleveland, Ohio, USA (I960).



   When using the "resin technique", the above solution of "crude gentamycin" is passed at pH 6 through the ion-exchanger resin and the crude gentamycin eluted from the resin using sulfuric acid. diluted. The eluate is made alkaline to a pH of about 10 with sodium hydroxide. When adding acetone, the mineral salts precipitate. The supernatant liquor is adjusted to pH 4.5 and concentrated in vacuo to give a concentrated solution of crude gentamycin sulfate.

   Methanol is added to this solution, ot the sulphate of
 EMI25.2
 Crude gentarnycin precipitates. , when the "insoluble salt method" is used, the neutralized mycelium extract (pH = 6) is treated for example with
 EMI25.3
 Santomeras-S ex is removed by filtration the precipitated salts, The precipitate is washed with water and dissolved in methanol, The methanolic solution is filtered, then it is passed through an anion exchange resin, for example of the Amberlite IRA-401 S type. The eluate is concentrated and its pH adjusted to 4.5 with sulfuric acid which gives a concentrated solution comprising essentially Gentamyoin sulfate.



  This sulfate is obtained by adding methanol to the solution and the precipitated salt is separated by filtration. It can be further purified by reprecipitation in the form of an insoluble salt followed by regeneration.
 EMI25.4
 



  Crude ce4twnycin as isolated by resin technology can be used as such or can be further divided into gentamycin proper and its antibiotic products. If you only want to prepare Genta-

  <Desc / Clms Page number 26>

 
 EMI26.1
 myoinop-one obtains this result preferably by fractional precipitation of its soil Bantomerse-S (sodium dodecylbenzenesulfonate).



   If the antibiotic constituents' BA-3 (Fractions A and B) produced at the same time are also to be isolated, they are separated.
 EMI26.2
 preferably a concentrated methanolic solution of otnta, crude mycin with the addition of acetone (fractional precipitation). (We take advantage of the greater solubility of Ganta-
 EMI26.3
 Myoin in the acetone-methanol mixture, relative to that of fraction A and b that of fraction B of BA "3) After washing with acetone, the precipitate is dried, which consists essentially of a mixture of BA-3 (fractions JL and 3).

   Following a variant for isolating this mixture from Gentaaycine, advantage is taken of the greater solubility of the dodeoyibenzene-sulfonate salts of these two constituents, compared with that of the salt; Gentamyoine correspondent. Gentamycin dad6or, teard-aulfonate is precipitated by adding a solution of Santomersoe S to a solution of crude gentamycin sulfate.

   After filtering and washing the cake remaining on the filter (cake which can be treated to give purified Gentamyoin or an al thereof), the filtrates which represent a solution are combined.
 EMI26.4
 contains sole doddoylbonzèteoalfonates of BA-3 (fraction A) and BA-3 (fraction B) and; these constituents can be isolated from the solution.



   From the mixture obtained by either of the preceding methods, fraction A and fraction B can be separated from each other using paper chromatography methods.
 EMI26.5
 



  In the case of .cart'inacea and; of. y, halop-hy ti oa, since most of the activity resides in the broth, the mycelium is filtered off and removed. The antibiotic substances are separated from the broth by extraction with

  <Desc / Clms Page number 27>

   using a Maniera solvent described below. The component separation is effected by partition chromatography, essentially as described below.



   The antibiotic substances are first isolated from the extract obtained from the broth by evaporating the solvent until a residue remains. The residue is examined by uhromatography on paper to guide the partition chromatography. The paper chromatograms are performed in various solvent systems and the RF values of the anointed constituents determined by standard bioautographic methods which consist of developing and drying a paper chromatogram which is then deposited on a plate. agar-agar seeded with Staphylococcus aureus.

   After a contact time of fifteen minutes, the paper is removed from the plate which is then incubated at 37 ° C. for sixteen to twenty hours ** By checking the location of the zones of inhibition, the values can be determined. of Rp of antibiotically active compounds *
Tables 6 and 7 below show the various solvent systems employed and the RF values determined for the components. In each of the systems in Table 6, a descending solvent was used; in the systems of Table 7, descending and ascending solvents were used as indicated.

  <Desc / Clms Page number 28>

 
 EMI28.1
 



  , LJ} At 6, ',,' (! Study cAro # atograph1u. 4es jeb4tAnco an; 1b1otiqued:.; Produced by fe1'll1.X), taton of 1S! ,. .... lim ,, .....! 'f) L'. '; Solvent system r-value
 EMI28.2
 Imm - çolo zoo1 Benzene- 10 pr v. V ''? , -ri. ther pètroit CI * 14 tr, i '.!' ;, ', i' ')' P.ï. 0. 0 '-' .; aetozi 5 p.v. ,, 4 ,,, y '$ | f "|' .b'j4 '" Tetapa $ a dev7, opPana' #,. ° ,,,, ,,;:. A'.4. 1.5 hours "i..L .. ..." '' '' cyon t, oiubat 20 PV * J # * fj n-fetttB 0l I | 5 ffV> Distilled water 7oO p.tr .. n, ' ; / # ;; j Bthey of petroleum n-to 042 'ûA * "0.71' ('tt, 6t. I) 195 peyo. 019 0.42 0 iti I. ..



  Time '*;> Af-. t1 -1,495 hours izth pd + tr5. OIP ** 'ig' "';, r ,.;; F4.' Methanol 8 POT * 'f., 9 i, # ..,.,;.'"; Aoet & ted'etbyle5p ..., # r .:. "'V'Vt / * \' 4% - 4 - '* lvcA.; - ir- * i> i, Distilled water 2 1.'r. ¯., # # '"? #' -% '. <* - 'T * Tempo of aéveioppezer4t f ;; ,,, .. ti, '/ -' "2 # ###"., # ju> ki '.
 EMI28.3
 w) p.v. partite 621 al ,, xra; - rr: t'r d I "> x ''. '; s 1 pv, 0, S68 .it,. ¯; l';. V bzz these tottpdrat% i-res ilidicate dis, ft.- ,, - t ...

  <Desc / Clms Page number 29>

 



    TABLE 7.
 EMI29.1
 



  Chromatographic study of antibiotic substances produced by fermentation of M. halophytica
 EMI29.2
 
 <tb> system <SEP> Value <SEP> 'from <SEP> of <SEP> spots Main <SEP>
 <tb>
 
 EMI29.3
 A-descending 0.0,: 0.35. 0.60, 0.72 - ascending 0.02 0.11, 0.95 O-ascending Oi95 'D-ascending 0.95' B-ascending 1.0
 EMI29.4
 
 <tb>? -ascendant <SEP> 1.0
 <tb>
 
 EMI29.5
 agoendant 1.0 11-from 0.01 / 0, '(4, 0.65, 0.83
 EMI29.6
 
 <tb> Systems
 <tb>
 A - Benzene chloroform (95.: 7 by volume) saturated with
 EMI29.7
 formamide. The chromatography paper, before use, is soaked in 25 mdtbanolque torm1, and air dried for 5 minutes to remove methanol.



  B '- Benzene methane ,! (9 s 1) o - Methanol. water 80 <20) containing 3% d..od1 chloride.



  The paper is buffered with a sulphate solution of
 EMI29.8
 sodium 0.95 m z sodium bisulfate 005 X and is adébé before development.



  D - Propanol'acetic acid! Water, (50 s 5 t 40) Acetic butanolgaçi4o (water (4 1 1 i 5) y <- Propnnol.pyr1dine.acid, aoetic.water (15 s 10 s 3 s 12) G - Phenol! water (80 g 20 QM3) H - Methylene chloride, carbon tetrachloride (80 t 20) saturated with formamide. Chromatographic paper, before
 EMI29.9
 use, is soaked in methanolic forcamide a
50% and air dried for 5 minutes to remove methanol.



   The observed RF value differentiates SF-30 from all *
 EMI29.10
 known antibiotics y OQmp "1B Ilolégn4pi4yoitet the novobloolnes penicillin Q and oyo1nt6ryt4.

  <Desc / Clms Page number 30>

 



   The following examples illustrate suitable methods for fermenting the preferred microorganisms used, for extracting antibiotic substances from mycelium or culture broth, respectively, and for separating them into their main constituents. In some of the examples,

     an indication of the activity of the antibiotic produced is given by a value expressed in units per milligram. Determination is carried out microbiologically using an agar-agar diffusion technique (and * Donald C. Grove et
William A. Bandell:

       "Assay Method of Antibiotics - A Laboratory
Manual ", New York - 1955) by a standard test of the disc type (in the case of Oentamyoin and its antibiotic co-products and antibiotics of the SP-30 group) or of the cup type (in the case of antibiotics of group R-451), using Staphylococcus aureus (ATCC 6538P) as test organism. A standard curve is prepared by plotting the dosage and response values of the antibiotic diluted in a buffer of phosphate at pH 8 in a medium comprising:

   
 EMI30.1
 
 <tb> peptone <SEP> 0.6 <SEP>%
 <tb>
 <tb>
 <tb> product <SEP> of <SEP> direction Pancreatic <SEP>
 <tb>
 <tb> from <SEP> casein <SEP> 0.4 <SEP>%
 <tb>
 <tb>
 <tb>
 <tb> extract <SEP> of <SEP> yeast <SEP> 0.3 <SEP>%
 <tb>
 <tb>
 <tb>
 <tb> extract <SEP> of <SEP> beef <SEP> 0.15%
 <tb>
 <tb>
 <tb>
 <tb> dextrose <SEP> 0.15%
 <tb>
 <tb>
 <tb>
 <tb> ' <SEP> agar-agar <SEP> 0.5 <SEP>%
 <tb>
 <tb>
 <tb>
 <tb> fold <SEP> 6.6
 <tb>
 
The used suspension of the test organism (Staphylococcus aureua)

     is standardized * to ensure 20% transmission lA 1,660 m in a colorimeter. The power of the whole sample determined from the reference curve ot is expressed in units per milligram} by setting the unit, in the teet of the disk type count being the amount of substance

  <Desc / Clms Page number 31>

 test necessary to produce an 18 mm inhibition zone with a 13 disc,

  5mm or in the oae of the cup-type test as the amount needed to produce a 15mm zone of inhibition with a 6.5mm OD steel cylinder.



   EXAMPLE 1 Preparation of Gentamycin by fermentation of
M. purpurea in the laboratory
A. Germination stage
A lyophilized culture of M. purpurea is added to a 300 cm3 flask stirred and containing 100 cm3 of the sterile medium according to t
 EMI31.1
 
 <tb> extract <SEP> of <SEP> bacto-beef <SEP> 3 <SEP> g
 <tb>
 <tb> tryptose <SEP> 5 <SEP> g
 <tb> dextrose <SEP> 1 <SEP> g
 <tb>
 <tb> starch <SEP> soluble <SEP> 24 <SEP> g
 <tb>
 <tb> extract <SEP> of <SEP> yeast <SEP> 5 <SEP> g
 <tb>
 <tb> water <SEP> of <SEP> tap <SEP> 1000 <SEP> cm3 <SEP> m)
 <tb>
 m) In this composition and in the water compositions given below, the solvent (usually water) is sometimes indicated in absolute amounts; there this by * - times followed by the expression "q.s.".

   While these two indications are in fact different, they can be substituted for each other at will, within the limits of precision necessary for these compositions. The flask and its contents are incubated for 5 minutes. days at 37 * 0 on a rotary stirrer (280 revolutions / minute, 5 cm stroke).



  B. Fermentation stage
A 25 cm3 inoculum obtained is passed to the following. of the geraination stage above in 4 flasks of 2 liters, * on .., each holding 500 cm3 of the following medium:
 EMI31.2
 
 <tb> extract <SEP> of <SEP> yeast <SEP> 10 <SEP> g
 <tb> dextrose <SEP> 10 <SEP> g
 <tb>
 <tb> carbonate <SEP> of <SEP> calcium <SEP> 1 <SEP> g
 <tb>
 <tb> water <SEP> of <SEP> tap <SEP> 1000 <SEP> we?
 <tb>
 The flasks and their contents are incubated for 5 days at 26 ° C. on a rotary shaker.

   We gather the contents of

  <Desc / Clms Page number 32>

   balloons. An aliquot of the broth is acidified to pH 2
 EMI32.1
 total, .using- & -c * t¯efet of 6N sulfuric acid. and centrifuging. The broth which floats on the surface is collected and neutralized with 6N sodium hydroxide. We try the activity of the game
 EMI32.2
 aliquot of the neutral broth with respect to µtad) iv ocoo ui SUER341 by the technique of diffusion in Ilagar-agate The detorations carried out on the broths obtained in * the rewritten manner give 15 - 20 un1t4e / c 3 antibiotic.



  C. Isolation of crude Gentamyoine (sulphate form from the fermentation substrate
The pH of all of the broth from step 8 of this example (approximately 2 liters) is adjusted to 2 with sulfuric acid.
 EMI32.3
 tU1 "'fhi 6N. Stir and filter. Adjust the fold of the filtrate t 7eO with 6N sodium hydroxide. The neutral broth is passed through a column of cation-exchanged resin (¯1I,):' 111t8 IRC -50.1 - 10 g of resin / liter of broth) The eluate is removed The column is eluted with sulfuric acid at pH
 EMI32.4
 2 # 0.

   Cn neutralizes the eluate with 6N soda at 7t VU The solution is concentrated in vacuum to approximately tenth of the * feather *
 EMI32.5
 The pH 1 is adjusted to 10.0 with a 6N sodium hydroxide solution and 4 volumes of acetone are added while stirring. The mixture is cooled and filtered. Adjust the pH of the filtrate to 5.0 with 6N sulfuric acid and concentrate in vacuo & temperature.
 EMI32.6
 room until the activity of the concentrate is close to 20,000 µg / om '. 10 volumes of methanol are added while stirring and the precipitated gentamyoin sulfate is filtered off. It is dried in a vacuum.

   Total yield: about 100 mg, assaying 343 units / mg by diffusion in agar agar against -
 EMI32.7
 tIPhllo.co90u ffus D. S!: E: 2âii25-S-SSÊ5 & ëX2BËES.Ë S33; â * Dissolve 15 g of the crude salt obtained as described c1.d'DeU8, (assaying approximately '40 ... 450 unitsa / # g) in 50 om3 of ea, we do

  <Desc / Clms Page number 33>

 passing the solution through an anion exchanged resin.



   (Amberlite IRA-400, OH form) in a column 38 mm in diameter and 61 cm high. The column was washed with 2 liters of water. The eluate is concentrated to dryness in vacuo and the residue is dissolved in 50 cc of methanol. While stirring, the solution is added to 1 liter of acetone. Filter and wash the precipitate with acetone. The filtrate is evaporated to dryness and the residue is taken up in 50 cm3 of methanol. We add the solution to
500 cm3 of ether while stirring. Or filter and wash the precipitate with ether. The filtrate is evaporated to give a residue consisting of purified Gentamyoin, approximately 3 g, assaying 961 units / mg. 'EXELTLE 2 - Preparation of BA-3 (nelanga from fractions A and B) in the laboratory.



   The procedure is as in Example 1 (parts A to C). Then following part D of this example 1, the addition of acetone to the methanolic solution of the concentrated eluate causes the precipitation of BA-3 (mixture of fractions A and B). The precipitate, after washing with acetone and drying, is sufficiently pure to be used.



  EXAMPLE 3 - Preparation of Gentamycin and BA-3 (fractions
A and B) by fermentation of M.echinospora or its variants in the laboratory.



   By replacing the lyophilized culture of M. purpurea used in Example 1 (part-1) with a corresponding culture of M. echinoapora (or one of its variants M. echinospor @ va? Ferruginea and M. echinospora var . pallida), and by essentially following the procedure of Examples 1 and / or 2, Gentamyoin and / or BA-3 (fractions A and B), respectively, can be obtained in the same manner.

  <Desc / Clms Page number 34>

 
 EMI34.1
 



  EXAMPLE 4 - Industrial preparation of Gentamyoine pu * fermentation of M.purpurea 1. !!! ¯6! E !!!!!!!]! 2 !!
Germination is carried out as indicated in Example 1 (part A).
 EMI34.2
 



  8. !!! ç !! ¯ !! ¯E2E! I!]! M¯211¯ !!! Lt1! -We pass 25 em3 of the first inoculum obtained following the preliminary stage of germination, in four flask * of 2 liters each containing 500 cm3 of the sterile medium used for germination.

   The flasks and their contents are incubated for five days at 28 ° C. on a rotary shaker (280 rpm, 5 cm stroke). We collect the contents of the ballona. 25 cm3 of the second inoculum thus obtained is added to each of the flasks of a series of twenty 2-liter flasks each containing 500 cm3 of the following medium soybean flour 30 g dextrose (oerelose) 40 g calcium carbonate 1 g tap water 1000 cm3
 EMI34.3
 The flasks and their contents are incubated for 3-5 days:

  at 2800 on a rotary agitator (280 revolutions / minute, 5'on stroke). The broth is pooled and aaeptically passed through a sterile inoculum flask with side tubing (total volume: approximately 10 liters).
 EMI34.4
 



  This stage of fermentation in tank 1 The 10 liters of inoculum were then transferred to a 240-liter fermenter containing about 185 liters *. the following sterile medium:
 EMI34.5
 bacto-beef extract 600 g bacto-tryptout 1000 g
 EMI34.6
 
 <tb> dextrose <SEP> (cerelosis) <SEP> 200 <SEP> g
 <tb>
 <tb> starch <SEP> soluble <SEP> 4800 <SEP> g
 <tb>
 <tb>
 <tb> extract <SEP> of <SEP> yeast <SEP> 1000 <SEP> g <SEP>
 <tb>
 <tb> agent <SEP> anti-mouase <SEP> to <SEP> base
 <tb>
 <tb> from <SEP> silicone <SEP> 100 <SEP> cm3 <SEP> /
 <tb>
 <tb>
 <tb> water <SEP> of <SEP> tap <SEP> that * <SEP> 200 <SEP> liters
 <tb>
 <tb>
 <tb>
 

  <Desc / Clms Page number 35>

 The pH is adjusted to 6.9 - 7.0 before sterilization.

   Aerobic fermentation is carried out for 20 to 30 hours (until the volume of packed cells is approximately 10 to 15%) under the following conditions: temperature 37 C sterile air intake approximately 150 liter% / minute pressure about 0.5 atm. above the
 EMI35.1
 pressure ata.norma7:

  stirring 180 revolutions, / Minute
 EMI35.2
 The contents of the fermenter are passed aeptically through a 2500 liter fermentation cellar containing about 1665 liters of a sterile medium having the following composition yeast extract 17 kg dextrose 17 kg calcium carbonate 1.7 kg anti-foam agent 400 cm3 water qs 1665 liters
 EMI35.3
 The commercial product ", Anti-foamer (HE 60" from General Electric Co.) was used, but other anti-foaming agents are also suitable.



  Ferment at 35 * 0 while stirring at 120 rpm and introducing air at about 0.5 atmosphere above normal atmospheric pressure at about 425 liters / minute, for 24 to 31 hours.



   At the end of this period, the potency of the antibiotics * produced reaches a maximum which remains practically constant.



  During fermentation, the pH practically remains between 6.6 and 7.4. The volume of packed cells reaches a constant value between 2 and 2.5 cm3. The potency of the antibiotic produced is approximately 25 units / cm3.
 EMI35.4
 



  D.! 22 !!! 2nw! - !!! - Z2! ¯1! ¯1!.! 2! ¯! ¯! Gt !!) delighting in the .termentatio auve
25 kg of filtering material (Colitis) are added to the approximately 1850 liters of the fermentation broth obtained according to

  <Desc / Clms Page number 36>

 part C of this example, and we filter. The filter material and the mycelium adhering to it are suspended in 370 liters.
 EMI36.1
 Very much water, just pH 42 with 6N etu7, iic acid and stir for 15 minutes. Filter and remove the mycelium cake. The pH of the filtrate is adjusted to 7.0 by adding 4N sodium hydroxide. The neutralized filtrate is loaded into a cation exchange adsorption column which contains 18
 EMI36.2
 to 20 liters of Amberllte Ire-50, in sodium form.

   The eluate is removed, the resin washed with water and eluted with acid.
 EMI36.3
 2N sulfuric acid, bringing together the acidic 3.uata. The elute is continued until the pH of the eluate is about 0.5.



  The pooled eluata are neutralized to a pH of approximately 4-7 with 2N sodium hydroxide. The vacuum is concentrated to about 3.7 liter * ten at a temperature below <t5 * C. We r ,, has the e of the concentrate to 10.5 with a 2N sodium hydroxide solution. Add 5 vials of acetone, cool, filter the precipitated and wash with acetone, then combine the filtrate and the washings. Adjust the pH of the combined filtrate to 4.5 with 2N sulfuric acid, concentrate in vacuo
 EMI36.4
 below 4500 to 100 cw3. 1 liter of metuax% olp is added and the filtrate is filtered off.

   The precipitate (crude Genteayoliae sulphate *) is dissolved in a sufficient quantity of water (100 cm3) to achieve a solution to 20%. The pH is adjusted to 10.5 with 2N sodium hydroxide and 5 volumes of sodium hydroxide are added. acetone.



  It is cooled, filtered and the precipitated salts are removed.
 EMI36.5
 adjust the pH of the filtrate to 4.5 with 2X # tallow acid and concentrate in vt-de at 50 em3 * Add 500 em3 of non-methmi-4, filter and wash the precipitate with cold metricol.



  The solid is dried in vacuum with phosphorus pentoxide, to obtain about 20 to 30 g of crude gentamyein sulfate (activity! T approximately 500 units / mg) constitutes; pr, t. cally by Gentaayeine sulphate mixed with the 'sul-

  <Desc / Clms Page number 37>

 
 EMI37.1
 fates of BA-3 (fraction A) 'and BA-3 (fraction B).



  E. Other (22Lzēform de-sulfate).



   The broth obtained according to part C of this example is filtered and the mycelium is suspended in 350 liters of water. While stirring, 6N sulfuric acid is added to adjust the pH to 2. The mycelium is filtered and washed with water. The filtrate and the washings are combined. The pH is adjusted to 6.5 with 4N sodium hydroxide. Then 2 kg of diatomaceous earth filter material and 750 cm3 of a solution are added.
 EMI37.2
 aqueous z Santomerae-S (sodium iodeoylbenxenesulcnate). Stir vigorously for 15 minutes and wash the filter cake with water. This cake is partially air dried and then suspended in 40 liters of methanol.

   Stir for 10 minutes and filter. The cake is extracted again with 40 liters of methanol and combined
 EMI37.3
 methane extracts. The methanol solution (about 100 liters) is passed through an anion exchange absorption column which contains 5.0 liters of Amberlite 4018 in methanol (resin in hydroxy form). The column is washed with 20 liters of methanol. The eluates are combined, concentrated in vacuo to about 10 liters and filtered. We adjust
 EMI37.4
 The filtrate is pH 7.0 with 2N sulfuric acid, centralized to 400 cm3 and filtered.

   The pH of the filtrate is adjusted to 4.5 with 2N sulfuric acid and 5 to 10 volumes of methanol are added. The precipitated crude gentamyony sulfate is filtered off, washed with methanol and dried at 40-60 ° C. in
 EMI37.5
 empty. Yield: approximately 56 g (titer 625 units / mg).



  F. !!! 2 !! 2¯¯ !!! 2 ... ± 2¯g! 2! L2 !! ¯È ± !! ¯ !! 2! T 55 g of crude Gentamyony sulfate is dissolved. tll that obtained by following part D or E of this example, in 10 liters of water, the pH is adjusted to 6.5 with 2N sodium hydroxide and the pH is adjusted.

  <Desc / Clms Page number 38>

 add 300 g of diatomaceous earth filter material.
 EMI38.1
 



  While stirring, 250 cc of an aqueous solution was added at t? 9 of Santomerae-S. It is filtered and the precipitate is washed with water. and air dry. On. And the filter cake suspended in 10 liter. of methanol, stirred and filtered. The methanol extraction of the cake was repeated and the filtrates were combined. The filtrates are passed through a column
 EMI38.2
 anion exchange containing 5 liter. 4'jmberl, t. tR110S (hydroxy form) in ethanol, at a flow rate of about 500 cm3; rai, aute. The column is washed with 10 liters of methanol and the dZua.te.

   The combined eluates are concentrated in vacuo to 1 liter, and dilute sulfuric acid is added until complete precipitation. (According to a variant before
 EMI38.3
 Afterwards, sulfuric acid is added until the pH is reduced to 4.5, then, while stirring, 18 g of pulverized activated charcoal is added, stirring is continued for 15 minutes. minutes, filter, wash the carbon cake with water, combine the filtrate and the washings and add this combination to 10 volumes of methanol).



    , Gentamyoine thus formed and dried. Yield: around 37g! your t about 780 units / mg.
 EMI38.4
 



  '' 3. BËl2525ÊB5B2iSR-Bl3Lt & âS <.ËSS3Sï2 '
10 g of purified gentamycin sulfate (from part? Of this example) was dissolved in 1500 cm3 of water and the solution was passed through an ani- exchange column.
 EMI38.5
 one containing 100 cm3 of Amberlite IRA-400 (OR form). The eluate is concentrated and lyophilized to give about 6.7 g of Gentamycin assaying about 1120 units / mg.
 EMI38.6
 



  ; ' ! nLR - 4iuotrielle of BA-'3 (mixture of fractions A and B)
One operates as in parts A to C then D or E dw Example 4, Then following part? from example 4, we

  <Desc / Clms Page number 39>

        The filtrate from the precipitate obtained by the addition of the Santomerse-S solution is combined with the washing water from the filter cake and the whole is passed through a column of a freshly regenerated anion exchange resin * ( preferably Amberlite IRA-401S, OH form). The eluate is concentrated to 150 cm 3 and added dropwise to 10 volumes of acetone while stirring so that the salts precipitate.



  These salts are filtered off, washed with acetone and removed. The combined filtrate is concentrated to remove acetone to give an aqueous solution of BA-3 (fraction A) and BA-3 (fraction B). The pH of the solution is adjusted to 4.0 with 2N sulfuric acid and filtered.



  10 volumes of methanol are added and the resulting precipitate is filtered off, washed with methanol and dried. The dried amorphous material consists essentially of the sulphates of BA-3 (fraction A) and BA-3 (fraction B).



  EXAMPLE 6 - Industrial preparation of Gentamycin and BA-3 (fractions A and E) by fermentation of M. echinospora or of a variant
By carrying out the germination described in part A of Example 4 with a lyophilized culture of M. echinospora (or one of the variants M. echinospora var. Ferrugines and M. echinospora, var. Pallida) instead of M. purpurea . and then essentially following the procedure of Examples 4 and / or 5, Gentamycin and / or BA-3 (moieties A and B), respectively, can be obtained in an analogous manner.



  EXAMPLE 7 - Preparation of Gentamycin hydrochloride
10 g of purified gentamyoin sulfate (from part F of example 4) are dissolved in 1500 cm3 of water, adsorption is carried out as described in part G of example 4 and set. the pH of the eluate to 4.5 with hydrochloric acid. The solution is concentrated to dryness in a vacuum.

  <Desc / Clms Page number 40>

 



    The residue is dissolved in 125 cm3 of methanol and added
 EMI40.1
 750 ca3 of acetone. The precipitated Geataaycine hydrochloride is collected on a filter, washed with acetone and obtained.
 EMI40.2
 vacuum-dried at 40 - 6000. Yield s 8 # 9 g approximately; titer about 820 units / mg.



  F =! '- PLE 8 - Preparation of Oentamycin helianthate 6.4 g of crude gentamycin sulphate are dissolved (obtained in accordance with one or other of parts D and 3 of example 4) in 65 cm3 of water. A hot solution (75 * 0) of 32 g of helianthine in about 300 to 350 cm3 of water is added. The mixture is stirred and heated to 75 ° C., and filtered while hot. The precipitated helianthate is washed with water and dried in
 EMI40.3
 vacuum over phosphorus pentoxide.

   By mixing in hot aqueous methanol, approximately 12.4 reddish-brown needles are obtained; Fusion point ! 26! Ci (a! At decomposition).



  Aneli0.2 - c 1 52.74, H s 6943%, X 8 13 6l, <. 0 1 19420 # bzz has 7.64%.



  The helienthate thus formed from crude Centamolyne sulfate may contain small amounts of the reactive salts of the antibiotics BA-3 (fraction A) t Bi-3 - (fraction B) co-produced. However # the preparation of the helianthate. in the manner described above effectively represents a purification, since during the conversion to hydrochloride ilitate (by dissolving in hydrochloric acid is filtered through a bed of charcoal, by concentrating the filtrate, adding 6 volumes of acetone, separating the hydrochloride thus precipitated by filtration, and drying), we
 EMI40.4
 can obtain a product grading approximately 760 .I / ..



  R "" w i 9 - Preparation of Roiriockate of Gentaayoine 2.2 g of Gentamyoine sulfate are dissolved. gross (taking note of one or the other of parts D and 2 of example 4) in

  <Desc / Clms Page number 41>

 22 cm3 of water. 82 cm3 of a saturated aqueous solution of Reinecke's salt are added while stirring. The mixture is stirred at room temperature for 1 hour, then allowed to stand at about 5 ° C. overnight. The precipitate is filtered, washed with ice water and dried in vacuo over phosphorus pentoxide. The Reineckate is recrystallized from hot aqueous methanol to give purple needles; melting point: 270 C (with decomposition).
 EMI41.1
 



  Analysis. 0 t 24.5; fi t 4t49; R: 22, .Q; 8 r 27.92, - Cr: 9.52.



   The Reineckate thus obtained may contain small amounts of the corresponding salts of antibiotics produced at the same time, but can be used without further purification, since by reconversion to the state of sulfate - by dissolving the Reineckate in methane! aqueous; by adsorbing it on a strongly basic anion exchange resin (Amberlite IRA-401S) by concentrating the eluate; adjusting the pH to 4.5 with dilute sulfuric acid; by adding 5 volumes of methanol and filtering the precipitated purified Gentamyoin sulfate, a product can be obtained assaying about 700 units / mg.



  EXAMPLE 10 - Preparation of Poly-N-Methylenesulfonic Acid Sodium Salt of Gentamyoine
16.4 g of Gentamyony sulfate (from part? Of Example 4) are dissolved in 108 cm3 4 * water * 30 g of sodium formaldehyde bisulfite are added per 100-200 mg portion.
 EMI41.2
 Stir for 30 minutes, add triethyl, m.ne Jua until the pH rises to 6.8 and continue stirring for 18 hours. The reaction mixture is added to 5 volumes of methanol and cooled to 5 ° C. The mixture is filtered and washed with methanol, then with ether. It is air dried.



  A colorless amorphous powder is obtained.



   A superior product can be obtained by operation.

  <Desc / Clms Page number 42>

 
 EMI42.1
 j. ra.n't as follows: 200 g of Gantamyo3.ua j sulfate (obtained according to Example 4?) are dissolved in 1.5 liters of water.



  366 g of sodium formaldehyde bisulfite are added in small portions over a 20 minute period. We adjust the pH
 EMI42.2
 ! 1A 6.7 with 135 es3 of triethylamine and stirred for 16 hours at room temperature. The solution is added to 7.5 liters of methsnol while stirring. The precipitate is filtered off and washed with methanol. Combine the filtrate and methane wash liquids, and add 9.8 pounds of acetone, everything. shaking. The precipitate is filtered off, washed
 EMI42.3
 , with acetone and dried.

   Yield 1178 g of wh. white amorphous powder; activity; s 580 units / mg.
 EMI42.4
 t Analysis - 0126.44o; S <6.18%; N: 5.54! S t 14e6t% î Na 1 7 # 49JÊJ
Total ash content: 22.2%.



    'EXAMPLE 11 - Preparation of R-451 by fermentation of
 EMI42.5
 LE, aarbonacea in laboratory #A. !!! Q! ¯S! I! .. i! 2 !!! 2 !! A lyophilized culture of 1i is added aaeptically. obonl} cel in a stirred 300 aza'3 flask containing 100 aa3 of the medium described in Example 1 A. The flask is incubated
 EMI42.6
 and Son sontenu-penda ... oar 3? 'C on a rotary stirrer (280 rpm, 5 cm stroke).



   B. Fermentation stage
 EMI42.7
 ! 25 cm3 of the inoculum obtained following the i germination stage above are passed through four 2-liter flasks i each containing 500--0 = 'of the following medium - - ------ yeast 5 g soluble fish products 1 g
 EMI42.8
 e - corn maceration liquor (dry) 1 g, calcium carbonate 1 g - lactose 30 g tap water 1000 cm3.

  <Desc / Clms Page number 43>

 



  The flasks are incubated. and their content for 2 to 3 days at 26 C on a rotary shaker. We collect the contents of the balloons.
 EMI43.1
 



  C. !! g !! 2ē !! ¯ !!! B2 !!.! E !!!! 2 !!!!
To the pooled fermenta from Part B above, add a filtration aid (diatomaceous earth) and filter. The filtrate is extracted with ethyl acetate or chloroform, maintaining the pH of the aqueous phase at
7 or above, but preferably as close as possible to
7. Concentrate the extract until it emits a residual oil by evaporating the solvent in vacuo. The residue is dissolved in excess chloroform and the solution clarified by filtration.

   The chloroform solution is concentrated in vacuo to a small volume and, while stirring, the concentrate solution is added to 10 volumes of petroleum ether (boiling range: 30 - 60 C). The precipitate is removed by filtration (repeated precipitation may be necessary in chloroform if an oil first forms rather than a filterable solid), this precipitate containing most of the antibiotic substances. . This subject
 EMI43.2
 is active against the StarhLioc3ceus in the mouth when solutions containing proportions as low as 10 g / cm3 are tried in the disk diffusion test.
 EMI43.3
 



  D. Pur3.içAtion of the antiriatfg eubetanee, abtained brute - !! ¯2¯! 2E ± !! ± -g¯ ±!:! G! This operation uses purification by column chromatography on alumina. The main eluate is defined as being R-451, and it appears that it did not contain
 EMI43.4
 constituent R-451E.

   Purification on alurrine makes B-451 soluble in water and therefore allows the preparation of aqueous or more soluble forms of the antibiotic.
 EMI43.5
 A chromatographic adsorption column is prepared. have

  <Desc / Clms Page number 44>

 alumina which is packed by pouring a suspension of ohromatographic-grade alumina (commercial product bearing No. 71 707 from Merck & Co.) in a mixture of chloroform and methanol (3: 1) in a column in glass having an inner diameter of about 51 mm, so that the height of alumina is about 61 cm.



   10 g of the crude antibiotic material obtained as indicated above was dissolved in 50 cc of chloroform and the solution was slowly introduced into the column. The latter is eluted with a chloroform-methanol mixture (3: 1), collecting 12 fractions of 500 om 3 each, then the elution is continued with a methanol-chloroform mixture (3:

   1) by collecting 37 additional fractions of 500 cm3 each. Fractions are tested by airing small samples and testing them for Staphylococcusaureus. The fractions are pooled according to their activity against Staphylococcus aureus. It appears that most of the eluted material * and the maximum activity is in the fractions * 13 to 30.

   These fractions are combined, concentrated in vacuo and precipitation is carried out by introducing the concentrate into petroleum ether. The R-451 antibiotic precipitates is filtered and air dried; yield: 1.7 g; activity shown by at least 1 g / cm3 against Staphylococcus aureus (disk diffusion test).



   Table 8 below gives the values which show the particular fractions pooled, indicates how the solid antibiotic material is isolated from the eluate, gives the results of the Staphylococcus aureus disk test and the values of RF - for the fractions collected in the particular system used. RF values are calculated in the usual way following the Staphylococcus aureus bioautography of the developed paper chromatogram.

  <Desc / Clms Page number 45>

 



    TABLE 8
 EMI45.1
 
 <tb> Antibiotics <SEP> E-451 <SEP>: <SEP> Chromatography <SEP> on <SEP> alumina
 <tb> Fractions <SEP> Treatment <SEP> and <SEP> yield <SEP> Zone <SEP> inhibition <SEP> System <SEP> bioautographiues <SEP> on
 <tb> gathered <SEP> Treatment <SEP> and <SEP> yield <SEP> (mm) <SEP>., <SEP> paper
 <tb> against <SEP> S. aureus <SEP> to <SEP> values <SEP> of <SEP> R
 <tb> various <SEP> concentrating <SEP> values <SEP> of <SEP> RF
 <tb> tions <SEP> (g / cm3)
 <tb>
 
 EMI45.2
 ] 1G00 100 10 .... a ere
 EMI45.3
 
 <tb> departure <SEP> 20 <SEP> 14 <SEP> 8 <SEP> 0 <SEP> 0.0 <SEP>, <SEP> 0.15 <SEP>, <SEP> 0.29 <SEP>, <SEP> 0.60, <SEP> 0.74
 <tb> inactive <SEP> ¯¯¯¯ <SEP> 0 <SEP> Y <SEP> Y <SEP> -
 <tb> 2 <SEP> residue <SEP> dissolved <SEP> in <SEP> chloroform, <SEP> precipitated <SEP> 23 <SEP> 17 <SEP> 10 <SEP> 0 <SEP> 0.0 <SEP>,

    <SEP> 0.12 <SEP> (trace), <SEP> 0.62, <SEP> 0.74
 <tb> with <SEP> 10 <SEP> vol. <SEP> ether <SEP> of <SEP> petroleum.
 <tb>
 
 EMI45.4
 



  Yield: 102 mg ¯¯¯¯¯¯¯¯¯¯ ¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯
 EMI45.5
 
 <tb> 3-4 <SEP> processed <SEP> like <SEP> in <SEP> 2. <SEP> Yield <SEP>: <SEP> 725 <SEP> mg <SEP> 15 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0.0
 <tb>
 
 EMI45.6
 5-8 treated as in 2. Yield: 541 mg 18 11 0 thread 0.0, 0.18 (trace), 0.62 (trace) ¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯ ¯¯ # # i '* ...... ## ,,! T ......



  9-12 inactive 0 0 16 o Î 8 -..-- u ...., ü-, 0.14 t .- 0.2F3, ¯ 0.64
 EMI45.7
 
 <tb> 13-30 <SEP> processed <SEP> like <SEP> in <SEP> 2. <SEP> Yield <SEP> 1.73 <SEP> g <SEP> 26 <SEP> 22 <SEP> 16 <SEP> 8 <SEP> 0.0 <SEP>, <SEP> 0.14 <SEP>, <SEP> 0.28 <SEP>, <SEP> 0.64
 <tb> 31-34 <SEP> 1 <SEP> processed <SEP> like <SEP> in <SEP> 2. <SEP> Yield <SEP>: <SEP> 32.5 <SEP> mg <SEP> 25 <SEP> 22 <SEP> 16 <SEP> 8 <SEP> 0.0 <SEP>, <SEP> 0.15 <SEP>, <SEP> 0.29 <SEP>, <SEP> 0.60
 <tb>
 
 EMI45.8
 35..2 dissolved in water, lyophilized. 22 17 11 0 0.0, fl, 13 (weakened,
 EMI45.9
 
 <tb> Yield <SEP>: <SEP> 111 <SEP> mg <SEP> 0.24 <SEP> (weak), <SEP> 0.59
 <tb> 43-49 <SEP> processed <SEP> like <SEP> in <SEP> 35-42. <SEP> 22 <SEP> 16 <SEP> 9 <SEP> 8 <SEP> 0.0 <SEP>, <SEP> 0.13 <SEP>, <SEP> 0.25 <SEP>, <SEP> 0.58
 <tb> Yield <SEP>:

    <SEP> 67.5 <SEP> mg
 <tb>
   *) 6.35 mm diameter filter paper disc.



  **) benzene-petroleum ether-acetone system (10: 2.5: 5), descending;
 EMI45.10
 Q1II.atographic paper. (llhatmen B-1); development temple, 1.5 hours.

  <Desc / Clms Page number 46>

 
 EMI46.1
 



  LEtiE 12 - Preparation of R-451 by fermentation of Ë *, c ", rbonacea var. 2urU.ti & c.% In the laboratory By replacing the lyophilized culture of M. carbonacea 4 in part. J, 4.- l i.piu 11 by corresponding cultivation of Me 2arbonaoe & var. fturantiaea, and essentially following the procedure of this example, R-451 can analogously be obtained.
 EMI46.2
 



  EXAMPLE there - Industrial Preparation of R-: 451 by fermentation of Xi, carbonate a - .¯ ......



  µ1µ1! -41-gormination - - µSâ2-S-.EEBi5S25 !!! ¯! ¯S! t! i! 2 A lyophilized culture 'Ce M. carbon2c2A is aseptically introduced into a stirred 300 cm3 flask containing 100 0 = 3 of the sterile culture medium following t
 EMI46.3
 baato extract .-- Drink 3 g tryptoae 5 g dextrose 1 g soluble starch 24 g extract of yeast carbonate from caiaiuas 1 g tap water 1000 cm3 The flask and its contents are incubated for four days at 35 C (or until so that good germination is obtained) on a rotary stirrer (280 revolutions / minute, 5 cm stroke).
 EMI46.4
 



  B. SSÊS-S-EEiS55S.BSBS252S
Into each of the flasks of a series of three stirred 300 cm3 flasks, each containing 100 cm3 of the above sterile culture medium, is introduced 5 cm3 of the first inoculum obtained following the germination stage described above. The flasks and their contents are incubated for 72 hours at 30 ° C. on the rotary shaker.
 EMI46.5
 



  3. !! ¯Ut! A:!:! 2U! ¯ !! 2g! A !!!!
25 cm3 of the second inoculum obtained following the pre-inoculation stage above are passed through each of the flasks of a series of ten 2-liter flasks, each containing

  <Desc / Clms Page number 47>

 500 cm3 of sterile culture medium used for germination. The flasks and their contents are incubated for 72 hours at 30 ° C. on a rotary stirrer (280 revolutions / minute, 5 oa stroke). The contents of the flasks are pooled and the broth is passed aseptically into a sterile inoculum flask with side tubing (total volume: about 5 liters).



  D. Fermentation stage in tank
The 5 liters of inooulum are aseptically passed through a 130 liter fermenter containing 90 liters of the sterile culture medium used in the germination stage. Ferment aerobically for 20 to 30 hours (until the packed cell volume is approximately 20 to 30%, as determined by centrifugation of a 10 µm3 sample at 2800 rpm for 5 minutes) under the following conditions temperature 35 C sterile air inlet approximately 153 liters / minute pressure approximately 0,

  5 atm above normal atmospheric pressure @ stirring 160 rpm When the volume of packed cells reaches at least 2 oz? the contents of the fermenter are aseptically passed through a 2500 liter fermentation vessel containing approximately 1665 liters of the following sterile medium:

   yeast extract 8.5 kg soluble fish products 1.7 kg male maceration liquor (dry) 1.7 kg calcium carbonate 1.7 kg lactose 51.0 kg anti-foam agent 500 cm3 fresh water approx. 1665 titrated Ferment at 35 ° C while stirring at 120 rpm and introducing air at approximately 0.5 atmosphere above normal atmospheric pressure at a rate of approximately 425 liters *

  <Desc / Clms Page number 48>

 / minute, for 50 to 70 hours. At the end of this period, the potency of the antibiotic produced reaches a maximum which is practically constant, as shown by a sample and test against Staphylococcus aureus.



   During fermentation, the pH practically remains between 7.0 and 7.3. The volume of the packed cells reaches a constant value equal to about 2.0 µm3.



  E. Isolation of antibiotic substances after in-tank fermentation 25 kg of filter material (colitis) are added to the approximately 1850 liters of fermentation broth from the. part D. of this example, stirred and filtered. The mycelium cake is removed. The filtrate is extracted twice with an equal volume of toluene.

   The extracts are combined with toluene and concentrated in vacuo to dryness, so that a dark brown oily residue is obtained. The residue is suspended in 1000 cm 3 of chloroform, filtered and, while stirring vigorously, this solution in chloroform is added to 10 volumes of petroleum ether. Stirring is continued for 5 minutes.

   The oily anti-solid precipitate is separated by decantation from the supernatant liquid. The precipitate is redissolved in 600 cm3 of chloroform and the solution is added to 5 volume of petroleum ether while stirring vigorously. Filtered and the chloroform solution added, with vigorous stirring, to 10 volumes of petroleum ether.

   Stirring is continued for 5 minutes. The semi-solid oily precipitate is separated by decantation from the supernatant liquid, the precise fraction is redissolved in 600 µm 3 of chloroform and the 5 volume solution of petroleum ether is added with vigorous stirring. The mixture is filtered, the amorphous precipitate washed with petroleum ether and dried in air. Yield! 12.6 g; Minimal inhibitory concentration against Staphylococcus aureus: 1 - 5 g / cm3.

  <Desc / Clms Page number 49>

 
 EMI49.1
 



  Y. lES-3É2â2-âiiS23; 5Ï2S - '& - EX5âRS.S.li2il3S * 5! 22¯ !!!!!! 2E¯!. !!
Instead of the method described in part E. above, the extraction method described in part C. of example 11 can also be used. In this way, about 125 g of solid material having a activity comparable to that of the product of Example 11 C.
 EMI49.2
 



  C. SerLtII2-2¯of antibiotic substances-in their conatitu an t C. êE !!!!! 2¯2¯ !! - 2 !!! ±! 2 !! g !! ¯-1r¯g!:! : - !!
This process uses column chromatography on diatomaceous earth, which effectively separates the antibiotic substances and isolates the constituent R-451A, R-451B, R-451D, R-451E, and then the constituent. R-451C1.



   The separation of the antibiotic substances obtained according to part E or, less preferably, according to part F of this example, is carried out by partition ohromatography using a two-phase solvent system and purified diatomaceous earth ( commercial product "Chromoaorb W", Johns Manville and Company, washed acid free, part size 0.25 - 0.15 mm) as inert support for the heavier phase as follows
A two-phase solvent system is prepared having the following composition:

   petroleum ether
 EMI49.3
 (boiling range 60-9000) 25 part ethyl acetate 75 part methanol 70 parts distilled water 30 parts 1.9 kg of the inert support is equilibrated with 950 cm3 of the heavier phase of the two-part solvent mixture. above phases and the mixture is slowly poured into a glass column 9 cm in diameter provided with a sintered disc at its lower part, this column having been previously filled to three quarters of its length with the light phase.

     We leave the earth of diato-

  <Desc / Clms Page number 50>

 They are then deposited in the column forming a compact bed, then compressed under a nitrogen pressure of about 0.07 to 0.15 atmospheres above normal atmospheric pressure.



     (the height of the column thus prepared is 137 cm).



     7.0 g of the amorphous mixture obtained following the process described in either part B 'are then dissolved. and,? of this example, in 38 cc of the heavy phase of the two phase solvent system. 19 g of the diatomaceous earth was added to the solution and the solid mixture was introduced to the top of the column bed and strongly compressed. Eluted with the light phase of the two-phase solvent system. .



   The eluates are collected at a rate of 50 cm3 / minute. The number of individual fractions is optional but is related to the "stop" volume (HOV) of the column. (HUV is defined as the volume which is occupied by the light phase of the two-phase system in the total column bed volume).

   In this column, the HUV is about 3500 m³. The first HUV collected is discarded. Subsequently, a paper chromatography of a sample of each fraction collected, using the solvent system I (Table 6) and a bioautography of the developed and dried chromatograms, was carried out, away from the.
Staphylococcus aureus The fractions are combined, according to their paper chromatographic system, into groups which are then concentrated to dryness in a vacuum, the residues being weighed and again chromatographed on paper and bioautographed to determine the relative composition of the group. particular of fractions.

   The results reported in Table 9 below are thus obtained,

  <Desc / Clms Page number 51>

   TABLE 9.
 EMI51.1
 
 <tb>



  Chromatography <SEP> on <SEP> land <SEP> of <SEP> diatoms
 <tb>
 <tb> of <SEP> antibiotics <SEP> of <SEP> group <SEP> R-451
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> HUV <SEP> Eluent <SEP> Weight <SEP> of <SEP> residue <SEP> System <SEP> bioautographic
 <tb>
 <tb>
 <tb> cm3 <SEP> mg <SEP> on <SEP> paper <SEP>; <SEP> RF
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 1 <SEP> 3500
 <tb>
 <tb>
 <tb>
 <tb> 2 <SEP> 3500 <SEP> 4300 <SEP> 0.77
 <tb>
 <tb>
 <tb>
 <tb> 2.1-2.9 <SEP> 2600 <SEP> 190 <SEP> 0.79
 <tb>
 <tb>
 <tb>
 <tb> 3-10 <SEP> 24500 <SEP> 2200 <SEP> 0.3 <SEP>; <SEP> 0.65; <SEP> 0.75
 <tb>
 <tb>
 <tb>
 <tb> 11-13 <SEP> 7000 <SEP> 100 <SEP> 0.14; <SEP> 0.28;

    <SEP> 0.64
 <tb>
 <tb>
 <tb>
 <tb> 14-16 <SEP> 7000 <SEP> 100 <SEP> 0
 <tb>
 
1 g of the material obtained from HUV 3-10 is chromatographed using the inert support (Chromosorb W) for the heavy phase of the following two-phase solvent system: petroleum ether (boiling range 30-60 C ) 10 parts toluene 200 parts n-butyl alcohol 15 parts ethyl alcohol 15 parts distilled water 70 parts 600 g are equilibrated at the start. of the diatomaceous earth support with 300 cc of the heavy phase of the above two-phase solvent mixture and slowly poured into a 5 cm diameter glass column partially filled with the light phase of the above solvent system.

   The support is allowed to settle by gravity forming a compact bed and is understood by a nitrogen pressure of about 0.07 to 0.15 atmosphere above normal atmospheric pressure. The height of the thus-prepared column is 107 cm, and its HUY is 900 cm3.



   The tiatière to be chromatographed is then dissolved in 10 cm3 of the heavy phase of the aforementioned solvent system, 5 g of the inert support are added and mixed. The solid mixture is introduced at the top of the column bed and strongly compressed downwards. Elution is carried out with

  <Desc / Clms Page number 52>

 the light phase of the above two-phase solvent system, at a flow rate of 100 cm3 per minute. We continuously collect-
 EMI52.1
 addition of fractions from 50 to 900 em3 (corresponding to 1/8 # * 1 times the HUV of the column bed). The first HUV obtained was removed immediately after applying the test material and the fractions started to be collected with the second HUV.



     A sample of each fraction collected is immediately chromatographed on paper, using the
 EMI52.2
 solvent system 1 (Table 6) and a bloautography of the developed and dried ohro "matogram, with respect to 8, th oeat, aureus (preference is given to system I because it is the one which disappears most quickly). The fractions are combined according to their diagram or chromatographic system on paper, and the fractions are concentrated in vacuo to dryness. The residues are weighed and a new chromatography on paper followed by a bioautography is carried out to obtain μM oe. .., mation of the composition.

   The result of 1 a4: ps, rkt3, o4 chroma, tographic in the column, thus operated is given in the
 EMI52.3
 table 10 oi-dsnsauis.



  TBBEAtr 10 ..



  Chromatography of IIUV 3-10 of antibiotics of the R-451 group
 EMI52.4
 Huy Eiuant Residual weight System bloautoggaphi4uW
 EMI52.5
 
 <tb> mg <SEP> on <SEP> paper <SEP> RF
 <tb>
 <tb>
 <tb>
 <tb> 900-
 <tb>
 <tb>
 <tb> 1-1.5 <SEP> 450 <SEP> 320 <SEP> 0.78
 <tb>
 
 EMI52.6
 1.5-14 11000 (580 Q, 6 *, a 0974 EXEMPT 14 - Industrial preparation of R-451 By
 EMI52.7
 fermentation of M. earfrûflaeea Q..U <, ...



  We replaced it. culture 1YQph111'1ée of 11. aart) oa.t 'utili *. a ± * in part 1 of example 13 by a dulturo <!! 9ri'eapaaf danta de gs 2 ¯arb2neepa var tlitrantiaca and we follow elsewhere

  <Desc / Clms Page number 53>

 the process of Example 13 (parts A to D, E or? and G). R-451 is obtained in an analogous manner.



  EXAMPLE 15 - Separation of constituent R-451D from R-451
100 mg of the residue obtained from HUVs 1.5 to 14 according to Table 10 (Example 13 G), or the corresponding fraction of Example 14, are suspended in 40 cm3 of ether while stirring vigorously at Room temperature. The filtrate is filtered and cooled overnight. The precipitate which forms is filtered off and washed with ice-cold ether.

   It is air dried to give approximately 81 mg of an almost colorless powder assaying 1155 units per mg of highly purified R-451D.
When tested quantitatively against Staphyl-occccua aureus, the R-451D purified thus obtained appears to be biologically homogeneous. When it is chromatographed on a thin layer of silica gel following the Stahl technique using a mixture of acetone and benzene (1: 1) as the eluting agent, followed by treatment with sulfuric acid of the plaque developed, dried, no substance other than R-451D is detectable.



  EXAMPLE 16 - Preparation of R-451D Acetate
25 mg of R-451D are dissolved in 1 µm3 of pyridine. 0.4 cc of acetic anhydride is added and the mixture is allowed to stand overnight. The precipitate is filtered, washed with water to remove pyridine and acetic acid, and dried over phosphorus pentoxide under high vacuum overnight.



  Yield: 11 mg of a colorless amorphous powder; melting point 135 - 136 "C. A second crop of sea liquor can be isolated from the precipitate mentioned above by cooling overnight in a refrigerator (5 C), filtering the precipitate, washing it to get rid of it. pyridine and acetic anhydride and drying it as above.

  <Desc / Clms Page number 54>

 



    Yield: 6 mg of a colorless amorphous powder * When performing an ohromatography on a thin layer,
 EMI54.1
 using silica gel (brand "Silikagel 00 of 3. ÎIIfs1.11. Ai, Go darastadt, Germany) as adsorbent and a mixture of rra6.- ton # -" benzene (1 <1) as an elution system and that one puivéri- eo of 1 sulfuric acid on the developed plate and eeehee, We obtain a single constituent having a value of% equal to 0.87 * (In the mimes conditions E-451D has a value of flp 'pttlw at j 0949Y ,, upon alkaline b, rdrol; re in rdtrisrol, a-51i! is regenerated from t .451Tr acetate.



  SXEMPBE 17 - Separation of constituent R * 451E
This non-polar component of the antibiotic mixture is usually found in high concentrations in the liquor which floats during precipitation in chloroform and
 EMI54.2
 petroleum ether described in part 3 of example 13 # #% in the corresponding fraction obtained according to example 14.



   It is also present, to a lesser degree, in precipitated antibiotic substances * from where it can be recovered using]
 EMI54.3
 the column chromatographic partition process described in part G of Example 13.



  To obtain R-451B, the HuV 1 to 1 # 5 - * (Table 10) are concentrated to dryness in vacuo and air dried * Approximately 320 mg of an amber material is obtained which is still purified by precipitation from a mixture of ether and hexane, giving
 EMI54.4
 nant 280 mg of an almost colorless amorphous powder reptiognizing purified R-451E. on paper chromatography in System I (Table 6) and bioautography against
 EMI54.5
 ptaphylooocoua aureus and Bacillus subtilis. the R-451E antibiotic thus obtained appears to be biologically homogeneous.



    EXAMPLE 18 - Separation of the constituents R-451B, R-451C1 and
 EMI54.6
 R'451A¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯ First ---- tion of rw.w

  <Desc / Clms Page number 55>

 
Separation of this * constitute * crude antibiotic substances is initiated by partition chromatography analogous to that described in part G of Example 13 using a two-phase solvent system% and one. purified diatomaceous earth (product sold under the trademark "Chromosorb W" by Johna Manville and Company, washed acid-free, particle size:

     0.25 - 0.15 mm) as inert support for the heavy phase, as follows:
The two-phase system (petroleum ether, ethyl acetate, methanol water) described in Example 13 is first prepared. 2.5 kg of the inert support is equilibrated with 1250 cm3 of the heavy phase of the solvent mixture. phase and the column bed was prepared as described in Example 13G. The HUV of this column is about 8000 cc.



   7.4 g of the amorphous mixture obtained as described in Example 13 E, or the corresponding fraction * obtained according to Example 14, are dissolved in 40 cm3 of the heavy phase of the two-phase solvent system. 20 g of diatomaceous earth are added and the solid mixture is introduced at the top of the column bed, followed by strong compression. Elution is carried out with. light phase of the two-fold solvent system * Following the column chromatographic separation process described in Example 13G, the results given in Table 11 below were obtained. It should be noted that the constituents R-451B and R-451C1 have been enriched in the group of HUY fractions 1.9 to 2.8, while the constituent R-451A has been enriched in.

   the croup of HUY fractions 4.0 to 5.0.

  <Desc / Clms Page number 56>

    



  TABLE 11.



  TABLEAV First separation of antibiotic substances from the R-451 group by chromatography on diatomaceous earth
 EMI56.1
 HUY Eluent Residue weight Bioautographic system
 EMI56.2
 
 <tb> cm3 <SEP> mg <SEP> on <SEP> paper <SEP> RF
 <tb>
 <tb> 0.9 <SEP> 7000
 <tb>
 <tb> 1 <SEP> -1.1 <SEP> 800 <SEP> 186 <SEP> 0.8 <SEP>; <SEP> 0.55
 <tb>
 <tb> 1.2-1.8 <SEP> 5000 <SEP> 900 <SEP> 0.56 <SEP> 0.43
 <tb>
 
 EMI56.3
 199-218 7200 5700 z.6 0.23; 0.433 '. -5 2.9-3.9 8000 100 0 0.16 0923; 0.55 4.0-5.0 8000 120 0 (tràc) *) elution with acetone
 EMI56.4
 mu) 50% acetone in an elution of methwiol B, 8 aration çtu R 4 13 and xMjj'Çj1C l'vn of the other 1.8 g of the residue obtained is chromatographed from * HtI # '.



  1.9 to 2.8 of the preceding chromatographlqub separation, on 1800 g of cel.uloue powder (product sold under $ 1 "Ihatman" brand, washed acid-free $ fine quality) filling a column 7.5 cm in diameter aux- to a height of 150 cc, using the mixture of single-phase solvents follow acetone ¯¯ 1 part
 EMI56.5
 # ¯ & mother of oil (boiling range 3O-60eO) boiling 25 parts benzene i 50 parts The material is dissolved in 68 <! n3 of.

   mixture of solvents, 50 g of cellulose are mixed with the solution and
 EMI56.6
 the mixture is charged to the top of the bed at ia 0005 ne * A deposit is made at a flow rate of 50 oz. per minute . Tractions of 100 cc are continuously collected. The HUV of
 EMI56.7
 this column is about 6000 am3. The eluata are combined as indicated in Table 12 below. It should be noted that the constituent R-451B was enriched in the group of j! Frae.tio4e

  <Desc / Clms Page number 57>

 t HUV 17.7 to 27.7, while the constituent R-451C1 was enriched in HUV 1.7 to 17.6.



   TABI-WATER 12.



     Chromatography of HUV 1.9 to 2.8 from Table 11
 EMI57.1
 
 <tb> HUY <SEP> Eluent <SEP> Weight <SEP> of <SEP> residue <SEP> System <SEP> bioautographic
 <tb>
 <tb>
 <tb> cm3 <SEP> mg <SEP> on <SEP> paper <SEP> RF
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 0.7 <SEP> 4200
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 0.8 <SEP> - <SEP> 1.6 <SEP> 4800 <SEP> 1100 <SEP> 0.55
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 1.7-17.6 <SEP> 96000 <SEP> 300 <SEP> 0.43
 <tb>
 <tb>
 <tb>
 <tb> 17.7-27.7 <SEP> 60000 <SEP> 200 <SEP> 0.16 <SEP>; <SEP> 0.23
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> .27.7-28.1 <SEP> 2400 <SEP> 70 <SEP> 0 <SEP>; <SEP> 0.15
 <tb>
 C. Purification of R-451B
200 mg of the residue obtained from HUY 17.7 to 27.7 (Table 12) are dissolved in 5 cm3 of acetone.

   While stirring vigorously, the above solution is added to 50 cm3 of hexane. The precipitate which forms is filtered off and washed with hexane.



  Dry at room temperature in vacuum over phosphorus pentoxide to give about 120 mg of a slightly soiled white powder.



   The R-451B thus obtained can be soiled by traces of other constituents. However, when chromatographed according to the Stahl technique on a thin layer of silica gel using a mixture of acetone and benzene (1: 1) as the eluting agent, following this operation with a treatment with sulfuric acid from the developed and dried plate. Only one substance is detected which eliminates the need for further purification.



  D. Purification of R-451C1
100 mg of the residue obtained from HUV 1.7 to 17.6 (Table 12) are dissolved in 5 cm3 of acetone. While stirring vigorously, this solution is added to 50 cm3 of isopropyl ether. We thread-

  <Desc / Clms Page number 58>

 the precipitate which forms and washed with iso- ether.
 EMI58.1
 pro, l1qu. Dry at room temperature in vacuum over phosphorus pentoxide to give about 70 mg of a slightly dirty white powder of R-451C1.



  The purified R-4510 thus obtained is essentially biologically homogeneous as appears from a quantitative determination.
 EMI58.2
 t1v centers the 0aphdloc coue ua received. E. Purification of .R-451A The residue obtained from the HUY 490 to 5.0 fraction group from the diatomaceous earth column (Table II) was slurried with 10% of dichloromethane. Filter and wash the solid residue with dlohloromethane. Dry at room temperature
 EMI58.3
 ambient temperature in the vacuum on phosphorus pent5xyde. which gives approximately 70 mg of a whiteish powder consisting of R-451A.



    EXAMPLE 19 Preparation of crude SP-30 by fermentation of
 EMI58.4
 halo2hytica in the laboratory A. Germination stage
 EMI58.5
 A lyophilized culture of M. halophytica is introduced into a flask of 300 µm 3 shaken containing 100 cm 3 of the medium described in part A of Example 1. Incubated on a rotary shaker (280 heifer / minute; 5 cm 3). course) for five days at 37 C.
 EMI58.6
 



  Fementation stage "'-"' - "" "" "" '' '' 'IQ U4- n 25 cm3 of the innoculum obtained after the above germination stage are passed through each of the flasks $ of a series of four 2-liter balloons, each containing 500 cm3 of the following medium:
 EMI58.7
 am.dsa eo3uble starch soluble 50 g amine NZ type.! 10 g fresh water q.s. 1 litre
 EMI58.8
 '.' pH after etdrelization 7i45.



  * The flasks and their contents are incubated for 96 to 120

  <Desc / Clms Page number 59>

 hours at 28 C on a rotary stirrer.
 EMI59.1
 0. Isolation of SP-30 from the fermentation substrate
To 3.5 liters of the collected substrate from part B above is added a filter material (diatomaceous earth) and filtered. The filter cake is washed with several hundred cm3 of water and the washings are added to the filtrate (total volume approximately 3600 cm3). Then add an equal volume of ethyl acetate and stir for 30 minutes.



  The ethyl acetate layer is separated and the aqueous layer is reextracted with an equal volume of ethyl acetate. The extracts are pooled and concentrated on a film evaporator in vacuo to obtain a residue. The residue weighs about 4 g and, on agar plates, shows antibiotic activity against Staphylococcus aureus ATCC 209P down to 1 g / cm3.



    EXAMPLE 20 - Industrial preparation of SP-30 by fermentation of M. halophytica.
 EMI59.2
 



  A. !! ¯1¯! U! I2 Comne described in Example 19 A.



  B.! H! ¯; crlJi2 !! ¯! .1: 2culW! 25 cm3 of the first inoeulum obtained following the previous germination stage are passed through each of the flasks of a series of 4 2-liter flasks, each containing 500 cm3 of the sterile medium used for germination. The flasks and their contents are incubated for 5 days at 28 ° C., on a rotary shaker (280 rpm, 5 cm stroke). We collect the contents of the balloons. 25 cc of the second inoculum thus obtained is added to each of the flasks in a series of 10 2 liter flasks, each containing 500 cc of the sterile medium described above. The flasks and their contents are incubated for 3 to 5 days at 28 ° C. on a rotary shaker (280 rpm, 5 cm stroke).

   We collect the contents of the balloons

  <Desc / Clms Page number 60>

 
 EMI60.1
 and pouring the broth into a sterile linoleum flask having a side tubing (total volume about 5 liters).
 EMI60.2
 0 * 3rd stage of ermentation in a vat The 5 liters of inoculum are passed aeeptically
 EMI60.3
 obtained as a result of Part S above, in a 130 liter fevaente'r * "containing approximately 90 liters of the following sterile medium. Soluble starch 4500 g
Amino NZ type A 900 g
 EMI60.4
 anti-soft agent ce 100 om3 fresh water q.s.

   90 litrea Fermented under aerobic conditions for 55-65 hours (until packed cell volume roof.
 EMI60.5
 from about 4 # 5 to 5.0 as zero-rated: .need when counter fuge a sample of 10 om3 at 2800 revolutions / minute for 5 minutes) under the following conditions: temperature 26 C intake d 'sterile air about 76-80 liters / minute
 EMI60.6
 pressure about 0.5 .., Q, g natta. aut-deeeua pression ait, nortaale agitation 1 # O-J65 revolutions / Minute 'The information c.L below is ax "aa'x.etiqtea of such a fermentation operation:
 EMI60.7
 Crois- Kemp.

   Inlet Pressure t / m pH Volume 1> 'to1na.t1or1 aanoe 00 air atm. au- cellu- .U'dt8qU.
 EMI60.8
 
 <tb> hours <SEP> the minute <SEP> above <SEP> the <SEP> against
 <tb>
 
 EMI60.9
 normal packed Statih. us
 EMI60.10
 
 <tb> 0 <SEP> 26 <SEP> 76 <SEP> 0.49 <SEP> 160 <SEP> 6.80
 <tb> 8 <SEP> 26 <SEP> 76 <SEP> 0.55 <SEP> 160 <SEP> 6.95 <SEP> 1.0
 <tb>
 
 EMI60.11
 16 26 7Ci 0.53 160 6le5 ito 23 26 76 0.52 160 7 * 10 1.5 <14 ma ion
 EMI60.12
 
 <tb> 31 <SEP> 26 <SEP> 76 <SEP> 0.53 <SEP> 160 <SEP> 7.35 <SEP> 1.5
 <tb>
 <tb>
 <tb> 38 <SEP> 26 <SEP> 79 <SEP> 0.49 <SEP> 165 <SEP> 7.60 <SEP> 2.5 <SEP> -
 <tb>
 <tb>
 <tb>
 <tb> 46 <SEP> 26 <SEP> 7? <SEP> 0.54 <SEP> 165 <SEP> 7.75 <SEP> 2.5 <SEP> zone <SEP> of.

    <SEP> 21 <SEP> mm
 <tb>
 <tb>
 <tb>
 <tb> 54 <SEP> 26 <SEP> 79 <SEP> 0.52 <SEP> 165 <SEP> 7.90 <SEP> 4.5
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 65 <SEP> 26 <SEP> 79 <SEP> 0.51 <SEP> 165 <SEP> 7.80 <SEP> 5.0 <SEP> zone <SEP> of <SEP> 25 <SEP> mm
 <tb>
 

  <Desc / Clms Page number 61>

 D. Isolation of SP-30 from the fermentation tank
The mycelium is separated by filtration from the broth, using a material to aid filtration. 90 liters of the broth are extracted with 2 volumes of ethyl acetate after having first adjusted the pH to 7.0. The extract is concentrated to 3 liters in vacuo. The layer of water which separates after standing is removed and further concentrated to about 200 cm3. The concentrate is cooled overnight and filtered.

   The filtrate is added to 10 volumes of a mixture of petroleum ether and ethyl ether (1: 1) and filtered. The precipitate is washed with several portions of the solvent mixture and the liquids are added. of filtrate washings. The filtrate plus the washing liquids containing the antibiotics are concentrated in vacuo and a reaction consisting of a dark brown oil weighing 18 g is obtained. This crude SP-30 pockets growth of Staphylococcus aureus 209P on agar medium at dilutions as low as 1 g / cm3.



  EXAMPLE 21 - Separation of SP-30 into its constituents
The separation of the SP-30, as obtained according to either of Examples 19 and 20, is carried out by column chromatography using purified diatomaceous earth (brand "Chromosorb W ", from Johns Manville and Company, non-acidic lava, particle size 0.25-0.15 mm) as an inert support.



   A glass column of about 1.7 mm in diameter and about 305 mm in height is first prepared by packing it with a suspension of "Chromosorb W" in formamide. A small portion of "Chromosorb W" is mixed with 1 µm3 of a methanolic solution containing 408 mg of SP-30 (obtained by following the procedure described in Example 20 D). The mixture is dried in vacuo to remove methanol, then the upper part of the prepared column is carefully packed.

  <Desc / Clms Page number 62>

 



   The column is eluted in turn with 100 cm3 of the following solvent mixtures:
 EMI62.1
 -at. benzene-chloroform (95 t 5 saturated with foncamidej ^ 'br benzene-chloroform (90 t 10) saturated with' 011iatlde; Ja. benzene-chloroform (75 4 25) saturated with formaoidei. d. bontene-chloroform (50 t 50) saturated with formaide (e. benzene-chloroform bzz t @ 15) saturated with formaide.



  The column is then eluted with 600 cm3 of chloroform and = aemexu with 1000 cm3 of methanol. A total of 84 fractions are obtained in 25 cm 2 portions. We perform a bioautography - of each 25 om3 portion against 0 .aureua! Λ20C -6538P and OR pool the portions which exhibit similar antibiotic activity and the constituents. . each set in vacuum, water is added, and each set is extracted with chloroform. We focus each ex-
 EMI62.2
 tra.t.uparly to dryness. The residues obtained from each set are bioautographed as previously against
 EMI62.3
 , taphylococoua aureua and constituents are identified by position.

   The activity is determined by a normal process! -; is on a disc using a 6.3 mm diameter disc impregnated
 EMI62.4
 a, born from Staphylococcus aureus. The results of this research are given in table 13 below.
TABLE 13.



   Diatomaceous Earth Chromatography of Antibiotics of the SP-30 Group
 EMI62.5
 portion of 3.ut Set N * T eneur ..... eno0j9 a tenant
 EMI62.6
 
 <tb> 1 <SEP> none
 <tb>
 <tb> 2-5 <SEP> B <SEP> D <SEP> (trace)
 <tb>
 <tb>
 <tb> 6-16 <SEP> C <SEP> D, <SEP> C <SEP> (trace)
 <tb>
 
 EMI62.7
 17 * 23 C, D (trace)
 EMI62.8
 
 <tb> 24-43 <SEP> E <SEP> C
 <tb>
 
 EMI62.9
 bzz6 F, 6, B
 EMI62.10
 
 <tb> 47-57 <SEP> G <SEP> B, <SEP> C <SEP> (tï-aoe)
 <tb> 58-80 <SEP> H <SEP> none
 <tb>
 
 EMI62.11
 i, 81-84 A, B (trace), 0 (traee)

  <Desc / Clms Page number 63>

   PHYSICAL AND CHEMICAL PROPERTIES OF PREPARED ANTIBIOTICS
 EMI63.1
 FROM THE PREFERRED MIpO-ORGANIS # S INIQUE HIGHER.



  G?: IWAr'YCI :, 1, as prepared by the above method, is a white amorphous powder having the following physical and chemical ara, atritics: I.] Melting point (block Kofler): Softening at 102 * Ci complete fusion at 108 C.



  II. Analysis
 EMI63.2
 s)! E! g! - !!!!! a s 50.20 '; H 8 9 * 52% 1 N 1 13r'i 1 27.81 (by diffe- rence) no other element.
 EMI63.3
 b) Te8tāqualitatifBet¯uantitatfēdeB¯groupos
 EMI63.4
 
 <tb> 1) <SEP> groups <SEP> methoxy <SEP> none
 <tb>
 <tb> 2) <SEP> groups <SEP> N-methyl <SEP> 2.75 <SEP>% <SEP>
 <tb>
 
 EMI63.5
 3) a-methyl groups 2.97? T
 EMI63.6
 
 <tb> 4) <SEP> nitrogen <SEP> Van <SEP> Slyke <SEP> 10.18 <SEP>%
 <tb>
 <tb> 5) <SEP> test <SEP> of <SEP> the <SEP> ninhydrin <SEP> positive
 <tb>
 
 EMI63.7
 6) Elaon-Morgan test (amino sweet) positive 7) Maltol reaction test negative
 EMI63.8
 
 <tb> (not <SEP> of <SEP> training <SEP> of <SEP> maltol <SEP> after
 <tb> heating <SEP> with <SEP> of <SEP> alkali)
 <tb>
 <tb> 8)

    <SEP> test <SEP> of <SEP> furfural <SEP> negative
 <tb>
 <tb> (none <SEP> product <SEP> volatile <SEP> absorbent <SEP> the <SEP> ultraviolet
 <tb> formed <SEP> after <SEP> heating <SEP> with <SEP> of <SEP> acid <SEP> sulfuric
 <tb>
 
 EMI63.9
 aqueous at 40; at 10Q C)
 EMI63.10
 
 <tb> 9) <SEP> test <SEP> of <SEP> Sakaguchi <SEP> (for <SEP> the <SEP> guanidinea) <SEP> negative
 <tb>
 III. Molecular weight
Calculated (assuming the group
 EMI63.11
 u-ciethyl per molecule) 545 Found (because ebullioacopio in methanol) 543 IY. ovo, rotary ir, specific {o <) 5 = + 1468 (1% of water) V.

   Solubility Extremely soluble in water; soluble in polar media such as pyridine and dimethylformamide, as well as

  <Desc / Clms Page number 64>

 in acidic media with formation of salts} sparingly soluble in methanol, ethanol and acetone! insoluble in ether, benzene and halogenated hydrocarbons.



  VI. Spectrum in the ultra-violet Completely transparent in the range between 200 and 400 m VII. Infrared spectra Figure 1 of the drawing shows the spectrum in Nujol (Nujol being the trademark for a mineral oil based on hydrocarbons sufficiently pure for infrared spectroscopy, produced by Plow Inc. , Memphis, Tennessee, USA) where # represents wavelength in microns, represents frequency in cm-1 and A indicates absorption.



  The absorption peaks (w = weak, m = moderate, m-s = moderate to "strong, s = strong, going very strong, sh = plateau) are located at the following wavelengths:
 EMI64.1
 
 <tb> @ <SEP> microns <SEP> Intensity <SEP> back <SEP> pics
 <tb>
 <tb> 3.00 <SEP> m-s
 <tb> 3.10 <SEP> six
 <tb> 3.25 <SEP> ah
 <tb> 3.42 <SEP> s
 <tb> 4.30 <SEP> w
 <tb> 6.35
 <tb> 6.85
 <tb> 7.25 <SEP> m-s
 <tb> 7.50 <SEP> sh <SEP> (large)
 <tb> 7.80 <SEP> sh
 <tb> 8.77 <SEP> w
 <tb> 9.06 <SEP> m <SEP> (large)
 <tb> 9.52 <SEP> s
 <tb> 9.78 <SEP> s
 <tb> 10.45 <SEP> m-s
 <tb> 11.58 <SEP> w
 <tb>
 VIII. Sala formation Gentamycin is a moderately strong base which readily forms salts with any strong organic or mineral acid.

  <Desc / Clms Page number 65>

 ral.

   In general, the mineral diacid salts are completely soluble in water. (The salts are obtained by concentrating their aqueous solutions and precipitating with a water-miscible organic solvent, preferably a lower aliphatic alcohol or a ketone).
 EMI65.1
 A. 2l2XâIS Melting point 194-209 * 0 (with decomposition) Specific optical rotation ([alpha]) D25 = + 1130 (1% in water)
 EMI65.2
 Elemental analysis; C s 35.47 / '; li s 7p27% i s 9.69? Sf 0 22.4h; Cl j t 2., 9Uy, Solubility very soluble in water and methanol; slightly soluble in ethanol; insoluble in other common organic solvents.



  Infra-red spectrum (Nujol) - (see figure 2 of
 EMI65.3
 ce a s in).
 EMI65.4
 
 <tb> (Microns) <SEP> Intensity <SEP> of <SEP> pics
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 3.00 <SEP> m-s
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 3.45 <SEP> a
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 4.27 <SEP> ah
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 4.95 <SEP> w
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 6.27 <SEP> m-a
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 6.64 <SEP> eh
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 6.85 <SEP> s
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 7.27 <SEP> a
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 7.58 <SEP> w
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 7.95 <SEP> ah
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 8.27 <SEP> w
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 8.95 <SEP> w <SEP> (large)
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 9.20 <SEP> m <SEP> (large)

  
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 10.25 <SEP> sh <SEP> (large)
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 10.98 <SEP> w
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 11.49 <SEP> w <SEP> (large)
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 13.90 <SEP> m
 <tb>
 

  <Desc / Clms Page number 66>

   Sulfate
Melting point 218-237 C (with decomposition)
 EMI66.1
 Elementary analysis s 0 i 31,22 #? H! 1 6f, 71 N t Sf465 * j 0 s $ 41 63% 1 80 4 <31.22%.



  Specific optical rotation 1 (o (} 5 "102. (1 in Iteau) Infra-red spectrum (in Nujol) - (see figure 3 of the drawing);
 EMI66.2
 
 <tb> / \, <SEP> (microns) <SEP> - <SEP> Intensity <SEP> of <SEP> pics
 <tb>
 
 EMI66.3
 z, 95 3.25 s (wide)
 EMI66.4
 
 <tb> 3.45 <SEP> go
 <tb>
 <tb> 4.27 <SEP> eh
 <tb>
 <tb> 4.80 <SEP> w <SEP> (large)
 <tb>
 <tb> 6.14 <SEP> m-s
 <tb>
 <tb> 6.55 <SEP> m-s
 <tb>
 <tb> 6.86 <SEP> s
 <tb>
 <tb> 7.26
 <tb>
 <tb> 7.45 <SEP> sh
 <tb>
 <tb> 7.77 <SEP> w
 <tb>
 
 EMI66.5
 8.70 ..r9 t "15 va (wide)
 EMI66.6
 
 <tb> 10.27 <SEP> m
 <tb>
 <tb> 11.38 <SEP> w <SEP> (large)
 <tb>
    C. Other salts Gentamyoine by the action of appropriate acids (or
 EMI66.7
 , soluble salts of these acids a) in aqueous media can be converted into insoluble or slightly -aolu-- # blea hearing salts.

   When the acid anion is pharmaceutically acceptable, the salt formed with Gentamycin becomes capable of being incorporated into an aqueous suspension composition or in oil1. Such preparations, when administered parenterally, exhibit depot effects with slow release of an1b10t1-; - than. In general, the acids. rig having 8 carbon atoms or
 EMI66.8
 . more give such Gentamyoin salts having reduced solubility. As examples of such acids, mention may be made of
 EMI66.9
 lauric, stearic, palmitic, oleic ,. etc.

   In was; ' .

  <Desc / Clms Page number 67>

 other acids behave in an analogous manner to give with Gentamyoin salts in which their hydrophilic properties are combated by a hydrophobic component supplied by the anion aoido. Comzae other types of acids which
 EMI67.1
 which may be used, there may be mentioned aloanoic acids, aryl-aubetituéo, such as phenylbutyric acid; aromatic arboxylic acids, such as naphthalene-1-arboxylic acid;

   substituted sulfuric acids and suppurative su tonic acids, such as laurylsultur1qu acid. and dodecylbenzene acid tallow on'.que, respectively! oholic acid, lemholz acid and derivative compounds, such as tannic acid * IX. Miscellaneous properties 1. No reaction with methanolic hydrochloric acid.
1.8 N at reflux after 2½ hours; quantitative recovery of the starting material.



  2. The antibiotic activity of Gentamycin was completely destroyed by hydrolysis with 6N hydrochloric acid at
 EMI67.2
 147 C in 15 minutes. With hydrochloric acid 2N z,
100 C, it takes 7 hours of reaction to completely destroy the activity.



  3. Stability: The activity of Gentamycin is not significantly altered when an aqueous solution of the anti. biotique is subjected to a temperature of 100 C for
30 minutes in the full range of folds from 2 to 12.



  4. Other derivatives Gentamycin, like other antibiotics having primary amino groups, is converted * to
 EMI67.3
 derivatives of K-methylene-sulph acid unique by the action of sodium sulphate for "maldehyde bisulphite (preferably by sulphate
 EMI67.4
 of Gentasycine). the degree of conversion of the primary amino groups of Gentumyo1no to the derivative state of the V-methylbneeulron1que balance is a function of the amount of the formaldehyde-sodium bieulfite addition compound useful in

  <Desc / Clms Page number 68>

      the reaction. the pH of the medium from which the product is isolated determines whether the product is a methanesulfonic acid or a salt thereof.

   Other analogous sulfonate derivatives are prepared by reacting on Gentamyoin adducts of ketones or aliphatic or aromatic aldehydes with bisulfite. These derivatives present therapeutic indices altered compared to those of the main antibiotic.

   The gentamyoin derivative in which all of the primary amino groups appear to have been converted to their respective N-methylenesulfonic acids is prepared as in Example 10. The properties of this derivative are as follows solubilized in water, insoluble in methanol; ([alpha]) D25: + 49.7 (1% in water); absorption bands in the infrared at 2.90 microns (s), 6.08 microns (m-s), 6.50 microns (sh), 8.72 microns (va) and
9.62 micron (s).



   X. Chromatographic properties on paper
Gentamycin, as determined by paper chromatograms, is different from other basic antibiotics. To obtain the chromatograms, we use an ascending system. Table. 14 gives the comparative values of RF in seven different systems (the values which are connected with a dash indicate a line while those which are not related indicate distinct and separate spots **).



   The antibiotics produced by the known Micromonospora species differ broadly from Gentamyoine in that they are not bases, do not have a broad spectrum of antibiotic activity, and cannot be extracted * with an Amberlite resin of the IRC-50 type as described in the present case.

  <Desc / Clms Page number 69>

 



   TABLE 14.



  Comparison of RF values of Gentamyoin in various systems with those of other basic antibiotics
 EMI69.1
 
 <tb> Antibiotic <SEP> System <SEP> ' <SEP> and <SEP> value <SEP> of <SEP> RF
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> A <SEP> B <SEP> C <SEP> D <SEP> E <SEP> F <SEP> G
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> Gentamycin <SEP> 0.59 <SEP> 0.26 <SEP> 0.1 <SEP> 0.3 <SEP> 0.45 <SEP> 0.0- <SEP> 0.0-
 <tb>
 <tb>
 <tb>
 <tb> 0.25 <SEP> 0.48
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> Neomycin <SEP> 0.22 <SEP> 0.12 <SEP> 0.29 <SEP> 0.0 <SEP> 0.05- <SEP> 0.0- <SEP> 0.0-
 <tb>
 <tb>
 <tb>
 <tb> 0.20 <SEP> 0.13 <SEP> 0.50
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> Kanamycin <SEP> 0.30 <SEP> 0.18 <SEP> 0.25 <SEP> 0.0- <SEP> 0.15- <SEP> 0.02 <SEP> 0.76
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 0.35 <SEP> 0,

  25
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> famine <SEP> 0.30 <SEP> - <SEP> 0.30 <SEP> 0.05 <SEP> 0.75
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> Streptomycin <SEP> 0.57 <SEP> 0.40 <SEP> 0.21 <SEP> 0.06 <SEP> 0.03- <SEP> 0.0 <SEP> 0.0-
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 0.19 <SEP> 0.33
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> Streptothricin <SEP> 0.36 <SEP> 0.30 <SEP> 0.27 <SEP> 0.27 <SEP> 0.09 <SEP> 0.0 <SEP> 0.18
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> Paromomycino <SEP> 0.33 <SEP> 0.11 <SEP> 0.38 <SEP> 0.0 <SEP> 0.03- <SEP> 0.03 <SEP> 0.25
 <tb>
 <tb>
 <tb>
 <tb> 0.29
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> Viomycin <SEP> 0.27 <SEP> 0.19 <SEP> 0.51 <SEP> - <SEP> 0- <SEP> 0.0 <SEP> 0.43-
 <tb>
 <tb>
 <tb>
 <tb> 0.25 <SEP> 0.65
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> Dihydrostrepto- <SEP> 0.53 <SEP> 0.36 <SEP> 0.18- <SEP> - <SEP> 0.03- <SEP> 0,

  0 <SEP> 0.22
 <tb>
 <tb>
 <tb>
 <tb> myoine <SEP> 0.45 <SEP> 0.3
 <tb>
 *) Systems A. 80% methanol (in water) + 3% NaCl, using, pH 2.3 buffered paper with sulfate - bisulfate, B. Propanol - pyridine - acetic acid - water.



     (15! 10: 3: 12).



  C. Propanol - water - acetic acid * (50: 40: 5).



  D. 80% aqueous phenol.



    E. n-butanol saturated with water containing 2% p-toluenesulfonic acid.



  F. Butanol - water (84:16) + 2% piperidine.



  G. Water -butano. (94: 6) + 0.25% p-toluenesulfonic acid.

  <Desc / Clms Page number 70>

 
 EMI70.1
 



  1 ES BA-3 (BUNDLE OF MOTIONS A BT 1!), Prepared as above, exhibits the buoyant physical and chemical properties:
I. Analysis a) Qualitative elementary analysis
0, H, N, 0: no other elements present,
 EMI70.2
 b)) t !!!! ¯9.!}! t !!!!!! ¯! ¯S!: 2!:; e !! f '1. lfinhydrin positive 2.: E1son ... Morgan positive
3. Maltol negative, 4. Furfural negative
Sakaguchi negative
 EMI70.3
 '6. Groupa methoxy negative
II. Solubility (((Similar to that of Gentamyoine
 EMI70.4
 II 1. Site where tradana the transparent ultraviolet in the range of 220 to 400 m.
 EMI70.5
 IY.

   Rormatipn de Bels Sulfate: white amorphous powder; soluble in water, insoluble in ethanol, methanol, acetone and other common organic solvents.
 EMI70.6
 



  V. Çhromator, geometry on paper Presents the same values of Bp as the Gentam: works in systems A, B, C, D,! and G (see Table 14). In the j E system, a separation is observed with Zal-3 (Fraction A) Sréaeri. j being a Br value equal to 0.15 and BA-3 (Fraction .3} having an RF value equal to 0.23 compared to the RF value of Gentamycin in this system, which value is equal at 0.45. i
 EMI70.7
 R-451 HRUT, as produced by the method of Example 11 D is a colorless amorphous substance having the "traote"! following physical and chemical characteristics:

  <Desc / Clms Page number 71>

 I. Melting point
200 to 204 "0.
 EMI71.1
 II, nal se teat u 1it tüe des grounèb) 1.

   Ninhydrin test negative 2. Ferric chloride negative
 EMI71.2
 III.Specific rotational power, - "-.- ¯¯ (OC) 25- ..16 (0.5% 'in chloroform), ¯l" r¯ - # :. "" IV. Solubility - "- - Soluble in water, insoluble in petroleum ether! Soluble in most organics .. co \! RtJlt. Dialyzable through an" OellophaRe "membrane in a methanol-methanol system .



  V. Ultraviolet Spectrum Figure 4 shows the absorption spectrum in methanol with a concentration of 5 g / 100 cm3. (X represents the wavelength in m, D represents the optical density):
 EMI71.3
 Àmax at 288 mu (E1 = 50).



  VI. Spectrum in the infra-rotî, -i (in the "Nujol") - (see figure 5). The absorption peaks are located at the following wavelengths:
 EMI71.4
 >% (microns) Pica intensity
 EMI71.5
 
 <tb> 2.92 <SEP> s
 <tb>
 <tb> 5.84 <SEP> m
 <tb>
 <tb> 6.00 <SEP> m <SEP> (large)
 <tb>
 <tb> 6.12
 <tb>
 <tb> 6.45 <SEP> m-s
 <tb>
 <tb> 8.00 <SEP>. <SEP> sh
 <tb>
 <tb> 8.07
 <tb>
 <tb> 8.33 <SEP> m-s
 <tb>
 <tb> 8.55 <SEP> eh
 <tb>
 <tb> 9.10 <SEP> a <SEP> (large)
 <tb>
 <tb> 9.60 <SEP>, <SEP> s <SEP> (large)
 <tb>
 <tb> 10.26 <SEP> - <SEP> m-s
 <tb>
 <tb> 10.55 <SEP> m-s
 <tb>
 <tb> 11.0? <SEP> w
 <tb>
 

  <Desc / Clms Page number 72>

 VII. Unstable stability below pH 7 at 20 C. Activity retained above pH 7 even after heating for 30 minutes at 100 C.
 EMI72.1
 



  VIII. When a caromatogram on paper is prepared in the manner indicated above and the spots are located autographically against the ta h loooca s ure oovaxt) described previously, the Ro values of R-451 from a cLiff, 6., renia systems are determined as follows
 EMI72.2
 
 <tb> System <SEP> of <SEP> solvents <SEP> used <SEP> RF
 <tb>
 
 EMI72.3
 A. 80 ?? of aqueous methanol + 3 99 of elllorurit
 EMI72.4
 
 <tb> from <SEP> sodium; <SEP> ascending; <SEP> paper <SEP> previously
 <tb>
 <tb>
 <tb> buffered <SEP> to <SEP> pH <SEP> 2.3 <SEP> with <SEP> of <SEP> sulfate-
 <tb>
 <tb>
 <tb> bisulfate <SEP> 0.60
 <tb>
 
 EMI72.5
 B. n-propano1-pyridine-water-acetic acid
 EMI72.6
 
 <tb> (15 <SEP>: <SEP> 10 <SEP>: <SEP> 3 <SEP>: <SEP> 12) <SEP>;

    <SEP> ascending <SEP> 1.0
 <tb>
 <tb> C. <SEP> n-propanol-water-aoid <SEP> acetic
 <tb>
 
 EMI72.7
 (50! Z 40 <5) 0.93 D. 80 aqueous phenol; ascending 0.95
 EMI72.8
 
 <tb> E. <SEP> benzene-methanol <SEP> (9 <SEP>: <SEP> 1); <SEP> ascending <SEP> 1.0
 <tb> F. <SEP> benzene-chloroform <SEP> (95 <SEP>: <SEP> 5); <SEP> descendant}
 <tb>
 
 EMI72.9
 formamido-methanol salt first paper
 EMI72.10
 
 <tb> (1 <SEP>: <SEP> 1) <SEP> and <SEP> dried <SEP> to <SEP> air <SEP> for <SEP> eliminate <SEP> on
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> methanol <SEP> before <SEP> use <SEP> 0.0
 <tb>
 
 EMI72.11
 G. bonsene-petroleum ether (range t.1bi1111 t1Qa 3P to 60 "c) -aetotia (10 8 5.5 t 5)! 0 oi 0.14 'descending. O, 2eIO64 r
 EMI72.12
 Parai 9 NT. ', 1'T, ISOUBS of R-451, constituted it * & ± ±! &# | as produced by the process of exempt 1.

   I, 'Mit uni amorphous substance ihc:> lore having the property,' \ l1Y "'t #? T. Ultraviolet spectrum * Tranaper the range of? 20 to 360 syu II. Spectrum dtffl 2xue; t (deJis le "Nu; Jo11i) .. 1 a #fgue --îg # peaks of '' absorptipn are located at'1pn. of waves sui "antel3; they are all broad and intenàvii 29d u, 6.25 rta;, 8.25 to 10.00 lU.

  <Desc / Clms Page number 73>

 R-451B, as produced by the process of Example 18C, is a colorless amorphous substance having the following properties: I. Ultraviolet spectrum
Transparent in the range from 220 to 360 m.
 EMI73.1
 



  H. Spectrum in llînfra-roure s (in "3tujsl") (see figure 7). The absorption peaks are located at the following wavelengths with the intensities indicated:
 EMI73.2
 
 <tb> # <SEP> (microns) <SEP> Intensities <SEP> of <SEP> pics
 <tb>
 <tb> 2.94 <SEP> m-s
 <tb> 5.72
 <tb> 5.80 <SEP> m-s
 <tb> 7.92 <SEP> m-s
 <tb> 8.60 <SEP> m-s
 <tb> 9.10 <SEP> s <SEP> (large)
 <tb> 9.64 <SEP> s <SEP> (large)
 <tb> 12.50 <SEP> m-s <SEP> (large)
 <tb> 13.90 <SEP> m-s <SEP> (large)
 <tb>
 R-451D, as produced according to the invention, is a white amorphous powder having the physical characteristics. and chemicals:
 EMI73.3
 I. Melting point (blooKof 1er)
138 and 140 C.



  II. Analysis a) Elementary
 EMI73.4
 VS <52.02, H i 6.81 #, N s 0.72, Cl 5104%, b)! N! L2. !!!!!! 2-! ± 2E! 2 OCH3 14., 15? 5.



  Tess¯s.uaj: a, ts¯dgsgrouggg 1.neinhydrin test negative 2. Elson-Morgan test positive 3.Alkaline KMnO4 test positive 4.greyish blue anthrone test
 EMI73.5
 5.tent of 2,4-dini1 ;: r, o- ph (! Nylhydra.z1ne positive

  <Desc / Clms Page number 74>

 
 EMI74.1
 6. Ehrlioh test. (diphenylamine) negative 7, triphenyl-tetrazolium test negative III, ouyo1r; specific ott01re (C () 5. + 29.2 '(1 in dioxane) IV .. Solubility very soluble in chloroforma, methylene chlorine, acetone and methanol, sparingly soluble in ether;
 EMI74.2
 insoluble dent petroleum ether, toluene, beiisene, water.



  -Peotri in 1-tultr violet. Figure 4 shows the spectrum in methanol (concentration - 'tiO1 2.97 mg / 100 om3) <.a 290 up to ± 951 = / 1.1 '(E1% = 37).



   In a solution of 6 cm3 of normal potash diluted with
 EMI74.3
 methanol at 100 om3, ............ 31% a 79.



  VI. Speotre in the infra-red t (in the * XuJolO) - (see figure 8). The absorption peaks are located at the following wavelengths
 EMI74.4
 
 <tb> (microns) <SEP> Intensity <SEP> of <SEP> pawn
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 2.88 <SEP> s
 <tb>
 <tb>
 <tb>
 <tb> 5.73 <SEP> s
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 6.13 <SEP> m-s
 <tb>
 <tb>
 <tb>
 <tb> 6.33 <SEP> m-s
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 6.45 <SEP> s
 <tb>
 <tb>
 <tb>
 <tb> 7.98 <SEP> s
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 8.32 <SEP> s
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 8.50
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 10.00 <SEP>)
 <tb>
 <tb>
 <tb>
 <tb> 10.22 <SEP> s
 <tb>
 <tb>
 <tb>
 <tb> 10.55
 <tb>
 <tb>
 <tb>
 <tb> 11.00 <SEP> m-s
 <tb>
 
 EMI74.5
 11 e5 m-a
 EMI74.6
 
 <tb> 12.30 <SEP> w
 <tb>
 <tb>
 <tb> 12.98 <SEP> w
 <tb>
 <tb>
 <tb> 13.55 <SEP> m-s
 <tb>
 <tb>
 <tb>
 <tb> 13,

  87 <SEP> m-s
 <tb>
 

  <Desc / Clms Page number 75>

 VII. Formation, derivative
 EMI75.1
 a) rem ±! * 16 melting point 135 to 136 C; ultraviolet spectrum, maximum absorption at
 EMI75.2
 285 M / U (Ele - 9.2). elementary analysis. s 0 9 53.00?:, H s 7.4r x a O # 77? fj absorption bands in the inlraira8a (in the "Dujo I as follows:
 EMI75.3
 
 <tb> \ <SEP> (microns) <SEP> Intensity
 <tb>
 <tb> 2.85 <SEP> w <SEP> (large)
 <tb> 5.57 <SEP> m-s
 <tb> 5.71 <SEP> a
 <tb> 6.14 <SEP> w
 <tb> 6.45 <SEP> m-s
 <tb> 8.02 <SEP> s
 <tb>
 
 EMI75.4
 8.38 M-9 8.88 # 9.17 9.55 VIII. Stability
R-451D is stable at 0 C and in the dark. At room temperature in direct sunlight, antibiotic activity decreases slowly after 24 hours and is completely destroyed after about 3 weeks.

   In methanol as a solvent, R-451D is stable for at least 2 weeks. Rapid deactivation occurs at pH below 5.5 while at pH above 7 and up to 14 activity is retained for a few days. Antibiotic activity is destroyed by treatment with decinormal methanolic hydrochloric acid at room temperature for 16 hours.

   Treatment of the acid mixture with a stoichiometric amount of sodium bicarbonate (aqueous solution
 EMI75.5
 to 2), followed by aethanol elimination by concentration

  <Desc / Clms Page number 76>

 in vacuum and extraction with chloroform, gives a
Balance of five components, as determined by chroma '*' tography in thin layer on silica gel using a mixture of benzene-acetone (75:25) as solvent and sulfuric acid as reactive spray. All the constituents are less polar than R-451D. By treatment with normal sodium hydroxide at room temperature for 16 hours, the activity of R-451D is completely retained.
 EMI76.1
 



  IE B-4ftlE, * isolated and purified as previously described (see Example 17) ent an almost colorless amorphous powder having the following physical and chemical properties t I. Melting point
 EMI76.2
 ## 59 96fT "T., na3 ee
 EMI76.3
 
 <tb> a) <SEP> Analysis Elementary <SEP>, <SEP> quantitative <SEP>
 <tb>
 
 EMI76.4
 c s s5, nr; H t 7.54%;

   N 1 1.31 and o 24.2 '.' b) Qualitative test of groups 1.tif ninhydrin killer 11 tif (2. positive E loori-14organ test
 EMI76.5
 
 <tb> 3. <SEP> test <SEP> with <SEP> of <SEP> KMnO4 <SEP> alkaline <SEP> positive
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 4. <SEP> test <SEP> of <SEP> anthrone <SEP> positive
 <tb>
 
 EMI76.6
 5.test dt la i, 4 '^ li, riitz'D.p7; 11jrx.ilydr.Z.tx .positive. 6.Uhtlich test (with d1phenyl.mine) negt1f - 7.teat of triphenyl-ttrazolium popfltif
 EMI76.7
 III. optical rotation 8péoitiQ (0O | 5 + '6.1 (1 in oxane).



  IV. Solubility 'Soluble in ohloroformet chloride and sthylMt' 'acetone, methanol and ether ;, moderately eluble: dtinl benzene, toluene and hexane; insoluble in t "lfS \ h V. Spftotro '? Q) ultrvglel Figure 4 shows the spectrum in methanol With a' *

  <Desc / Clms Page number 77>

 concentration of 3.0 mg / 100 cm3: #max = 240 m (E1% = 232).



  VI. Spectrum in the infrared (in the "Nujol") - (see figure 9). The absorption peaks are located at the following wavelengths with the intensity indicated below:
 EMI77.1
 
 <tb> # <SEP> (microns) <SEP> Intensity <SEP> of <SEP> pics
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 2.94 <SEP> m-a
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 5.68 <SEP> s
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 5.76 <SEP> s
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 5.83 <SEP> a
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 6 <SEP>, <SEP> 00 <SEP> ' <SEP> go
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 6.16.

    <SEP> m-s
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 6.22 <SEP> m-s
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 7.87 <SEP> go
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 8.31 <SEP> w
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 8.55 <SEP> w
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 8.96 <SEP> w
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 9.33 <SEP> w
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 9.55
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 9.77 <SEP> m-a
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 10.50 <SEP> w
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 10.90 <SEP> w
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 11.26 <SEP> m-a
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 11.42 <SEP> w
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 12.22 <SEP> w
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 12.62 <SEP> m-a
 <tb>
 SP-30 GROUP ANTIBIOTICS are soluble in most organic solvents such as ethyl acetate, chloroform, methylene chloride (and other halogenated hydrocarbons)

     and lower alkanols (eg, methanol and ethanol); they have minimal solubility in water. From the fomenting broth at a pH of 2 to 9.5, the SP-30 is easily extracted with butanol, ethyl acetate, methylene chloride, chloroform, carbon tetrachloride, benzene or ether.



   When subjected to certain qualitative tests of

  <Desc / Clms Page number 78>

      groups, SP-30 and its constituents isolated give the following test responses. ninhydrin and negative Sakaguchi (raw SP-30 as well as all its isolated constituents)
Reducing sugars (with ammoniacal silver nitrate) positive (Among the isolated constituents, @ is strongly positive, B, C and D are negative);
Reduction of positive ferrioyanide (Among the isolated constituents. Reacts negatively, B, 0 and D positively).



   . Se-30 and its constituents fractionated, in a methanolic solution, are absorbent in the ultraviolet in the range from 220 to 400 m with the following characteristics: the crude SP-30 has an absorption curve. not very described with inflection points at 260 m and 290 m, the pdint at
260 m / u having an E1% of about 28; the SP-30A has a similar type curve with one inflection at 258 m (E1% of about 70) and a second inflection at about 295 m / u; SP-30B shows a maximum at 268 m (E1% of about 111); SP-30C does not present maxima but inflections at 265 m (E1% of about 181) and at about 290 m;

   the SP-30D shows a maximum at 263 m (E1% of about 115). the infrared spectra of SP-30 and non-component isolated suspended in "Nujol"), are indicated by Figures 10-14, respectively.

   The absorption peaks and band characteristics are given in the following tables:

  <Desc / Clms Page number 79>

 
 EMI79.1
 
 <tb> SP-30 <SEP> (see <SEP> the <SEP> figure <SEP> 10) <SEP> Intensity <SEP> of <SEP> Pawn
 <tb>
 <tb>
 <tb> # <SEP> (microns)
 <tb>
 <tb>
 <tb>
 <tb> 2.98 <SEP> s
 <tb>
 <tb>
 <tb> 5.78 <SEP> a
 <tb>
 <tb> 6.00 <SEP> m-a <SEP> (large)
 <tb>
 <tb>
 <tb> 6.58 <SEP> w
 <tb>
 
 EMI79.2
 6, 81 maza
 EMI79.3
 
 <tb> 7.02 <SEP> w <SEP> (sh)
 <tb>
 <tb> 7.22 <SEP> m
 <tb>
 <tb> 7.71 <SEP> m-a <SEP> (sh)
 <tb>
 <tb>
 <tb> 7.83 <SEP> a
 <tb>
 <tb> 8.32 <SEP> w <SEP> (eh)
 <tb>
 <tb>
 <tb> 8 <SEP>, <SEP> 86 <SEP> m-s
 <tb>
 <tb> 9.28 <SEP> m-s
 <tb>
 <tb>
 <tb> 9.54 <SEP> wide
 <tb>
 <tb> 9.75 <SEP> wide
 <tb>
 <tb>
 <tb> 13.42 <SEP> m
 <tb>
 <tb> 14.20 <SEP> w <SEP> (large)

  
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> SP-30A <SEP> (see <SEP> the. <SEP> figure <SEP> 11) <SEP> Intensity <SEP> of <SEP> pics
 <tb>
 
 EMI79.4
 To (! Deny)
 EMI79.5
 
 <tb> 2.95 <SEP> 'a <SEP> (large)
 <tb>
 <tb>
 <tb>
 <tb> 3.42 <SEP> m
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 5.82 <SEP> s <SEP> (sh)
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 5.89 <SEP> a
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 6.15 <SEP> m <SEP> (ah)
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 6.60 <SEP> w
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 6.80 <SEP> w
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 7.16 <SEP> m-a
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 7.56 <SEP> m <SEP> (large)
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 8.50 <SEP> w
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 9.22 <SEP> w
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 9.46 <SEP> w
 <tb>
 

  <Desc / Clms Page number 80>

 
 EMI80.1
 SP-30B (see figure 12) Pioa intensity
 EMI80.2
 
 <tb> 2.98 <SEP> w <SEP> (large)

  
 <tb>
 
 EMI80.3
 5.72 m-s; 5, 80 mes
 EMI80.4
 
 <tb> 6.79 <SEP> m-s
 <tb>
 <tb> 7.15 <SEP> m-s
 <tb>
 
 EMI80.5
 7.2l - Vs \ # -
 EMI80.6
 
 <tb> 7.88
 <tb>
 <tb>
 <tb> 8.70 <SEP> s <SEP> (large)
 <tb>
 <tb>
 <tb> 10.00 <SEP> s <SEP> (large)
 <tb>
 <tb> 11.51 <SEP> m-s
 <tb>
 <tb>
 <tb> 12.00 <SEP> s <SEP> (large)
 <tb>
 <tb> 13.00 <SEP> (large)
 <tb>
 
 EMI80.7
 SP-300 (see.

   figure 13) Inteaaite 4th PiOt
 EMI80.8
 
 <tb> (microns)
 <tb> 2.95 <SEP> w <SEP> (large)
 <tb> 5.74 <SEP> s
 <tb>
 
 EMI80.9
 6.20, (eh)
 EMI80.10
 
 <tb> 6.79 <SEP> m-s
 <tb>
 <tb> 7.20
 <tb> 7.70 <SEP> (sh)
 <tb>
 <tb>
 <tb> 7.82
 <tb>
 <tb> 8.85
 <tb>
 <tb> 9.28 <SEP> s
 <tb> 9.56 <SEP> m
 <tb>
 <tb> 9.60 <SEP> (large)
 <tb>
 
 EMI80.11
 9.90 bzz rua (large)
 EMI80.12
 
 <tb> 11.50 <SEP> w <SEP> (large)
 <tb> 12.00 <SEP> (large)
 <tb>
 
 EMI80.13
 12.80 '', '' * m-a (wide)
 EMI80.14
 
 <tb> 13.35 <SEP> w <SEP> (large)
 <tb> 14.15 <SEP> (large)
 <tb>
 

  <Desc / Clms Page number 81>

 
 EMI81.1
 
 <tb> SP-30D <SEP> (see <SEP> the-figure <SEP> 14) <SEP> Intensity <SEP> of <SEP> pawn
 <tb>
 <tb> \ <SEP> (microns) <SEP>
 <tb>
 <tb> 2.95 <SEP> m
 <tb>
 <tb> 5.75 <SEP> s
 <tb>
 <tb> 6 <SEP>, <SEP> or.

    <SEP> m <SEP> (sh)
 <tb>
 <tb> 6.16 <SEP> w <SEP> (sh)
 <tb>
 <tb> 6.30 <SEP> w <SEP> (sh)
 <tb>
 <tb> 6.56 <SEP> w <SEP> (sh)
 <tb>
 <tb>
 <tb> 6.78 <SEP> m-s
 <tb>
 <tb> 7.20 <SEP> m
 <tb> 7.28 <SEP> w <SEP> (sh)
 <tb>
 <tb>
 <tb> 7.70 <SEP> a <SEP> (ah)
 <tb>
 <tb> 7.80 <SEP> a <SEP> (ah)
 <tb>
 <tb> 7.88
 <tb>
 <tb> 8.32 <SEP> w
 <tb>
 <tb> 8.52 <SEP> w <SEP> (ah)
 <tb>
 <tb> 8.88
 <tb>
 <tb> 9.28
 <tb>
 <tb> 9.42 <SEP> m-a <SEP> (large)
 <tb>
 <tb> 9.56 <SEP> - <SEP> m-s <SEP> (large)
 <tb>
 <tb> 9.72 <SEP> m-s <SEP> (large)
 <tb>
 <tb> 10.44 <SEP> w
 <tb>
 <tb> 11.30 <SEP> w <SEP> (large)
 <tb>
 <tb> 11.60 <SEP> w <SEP> (large)
 <tb>
 <tb> 11.82 <SEP> w <SEP> (sh)
 <tb>
 <tb> 12.42 <SEP> a
 <tb> 13.32 <SEP> m <SEP> (large)
 <tb>
 <tb>
 <tb> 13.48 <SEP> m <SEP> (large)
 <tb>
 <tb> 14.20 <SEP> m <SEP> (large)

  
 <tb>
 The stability of SP-30 varies from component to component. In particular, fraction A is stable at room temperature when mixed with an aqueous buffer at a pH between 2 and 4.



  At 100 ° C, its antibiotic activity decreases when the pH drops below 6. Its activity is destroyed at pH 2 by heating at 100 ° 0 for 30 minutes while no loss of activity is observed in the cell. conditions similar to pH 7 or higher. Fraction B is stable at a pH of between 2 and 10 at room temperature. At elevated temperature (100) it is stable for a pH down to 2 for 30 minutes, its activity decreasing from this point. Fraction 0 is stable over the pH range 2 to 10, even at 100 ° C. for 30 minutes. Fraction D is stable from pH 2 to pH 10 at ambient temperature and from pH 4 to fold 10 at 100 C for 30 min.

  <Desc / Clms Page number 82>

 
 EMI82.1
 



  BIOLOGICAL PROPERTIES D $ 8 T. ' BIOTT UE OBTAINED FROM THE FOREGOING EXAMPLES, IENT & N.INE has a broad anti-bacterial and anti-rickettoial spectrum. It is a useful anti-infectious agent capable of effectively inhibiting certain manifestations of diseases duos in Stphloooocua ureua, in I.r a.a.3. pneumoniae, and other UULRIIILIlJllh. | l irillirinLllJ I "MUIT - * #" - I "i" "J Vir 1ril'IL pathogenic organisms, It is also useful for treating
 EMI82.2
 z> mastitis of beetia.ux.



  It is effective in combating the disorders of the reticum caused by fatty ** negative organisms such as the eapeoea of Proteua and Patudomonaa. The number of infections due to gram-negative organisms which require hospitalization is increasing. Gentamycin appears particularly useful for the treatment.
 EMI82.3
 such chronic fatty-negative infections of the kidneys and bladder even in refractory patients. Gentamycin's high antibiotic activity against infections due to
 EMI82.4
 Protein and Pseudomonaa readily allows clinical use.

   As a particular example of such an effect, it can be stated that Gentamyoine has been used with success.
 EMI82.5
 to treat chronic queen infection due to Paeudomo-? nas, at a dose of 1 mg / kg given three times a day.



  . This infection, which did not respond well to other therapies, was - completely eliminated from the urine after 1 day of milking -; ment and has not reappeared after 10 days. Similar results have been obtained against several infections due to
 EMI82.6
 i. to Proteus. Gentamycin therefore appears to have an advantageous combination of unique types of activity together with low toxicity allowing its use as a pre-requisite to other antibiotics.
 EMI82.7
 The antibiotic activity of Gentamyoine is 1120 Uni- teamg, that of its hydrochloride 82t, and that of its aul

  <Desc / Clms Page number 83>

 800 units / mg fate.



   The comparative in vitro activity of Gentamyoin against various grau-positive, gram-negative and acid resistant organisms is reported in Table 15 below.
 EMI83.1
 

  <Desc / Clms Page number 84>

 
 EMI84.1
 TABLE 15.
 EMI84.2
 Antibiotic spectrum (In vitro activity) of Gentamyoine
 EMI84.3
 by comparison with:

  an81IlYQine and the 11 eomyoine
 EMI84.4
 Micro-organism tested Concentration nhib1tr1c Minimum designation (/ ug / cs3) Ho ayatématiue de Genta- W, ## strain x) myoin myoin myoino
 EMI84.5
 
 <tb> Gram-positive
 <tb>
 
 EMI84.6
 Bacillus careus var. mycoidea DA 30 0.5 # 0 0.5 l3acillua cereua
 EMI84.7
 
 <tb> var. <SEP> mycoidea <SEP> DA <SEP> 34 <SEP> 0.5 <SEP> 1.5 <SEP> 0.5
 <tb> Bacillus <SEP> cereua
 <tb>
 
 EMI84.8
 var. mycoldea ATCC 7064 0.75 3.0 2.0 'Bacillus r-egatherium DA 31 0.075 0.25 0.075 Baeïllus subtilie ATCC 6633 0.015 0.035 0.015 Bacillus sphaericua DA 32 0.1 K) 0.75 0125 Bacillua sphaerieun DA 33 0, 05 0.75 0.075 Brucella abortua DA 70 0.25 0.75 ot5 Staphylococous ATCC 6538P 0.075 0.75 0.1
 EMI84.9
 
 <tb> aureus
 <tb>
 
 EMI84.10
 aureus A2G0 12715 Q 055 005 0.035 Staphylococcus 68 0 17 0.75 Û> 375 aureus .;

   ; .1 "Otl75 Ot75 ob375 Staphylococcus ATC0 99Ó6 0 25 1 5 ob75 aureus Staphylooooeue ATCO 116 175 aurous ATC0 1163 0.175 Ot75 O 3? 5 SaSSu8 000CUB Grae Q 375 l S 25 Staphy1ooooQI.1S Smith 0 f:) 5 0.75 0.375 'Bï Sfeî q00Ua DA40 Oi25 l 5 /.0 <2. aureUI3 0025 1 # 5 StaphyloQoCQU8 375 0 2 "o # 5 * epiderznid1a DA 41 0.375 0.25 0 * 25 Sarcina lutea COC 9341À Ot25 0 75 '. 0025 Saroinalutea ATGC 9343 0.25 3.0 0., 2'5' Spyal> en9rCU8 DA 21 6,0. **) # 5 .O *. /Ô.O.



  Streptooodous Da 20 12 0) 100 0 'f 3.G O' fecalie ATCO 10541 12tO> 100.0 '46 * io Micrococcua flavue DA 60 0.075 6.0 - - '# 075.



  Corynebao; er1um DA 80 2015 z5. aimplex

  <Desc / Clms Page number 85>

 
 EMI85.1
 TABLE 15 (aulto) Antibiotic spectrum (In vitro activity) due to Gentamyoine compared to Kànamyoine and Neomycin
 EMI85.2
 Microorganism tried. - - Concentration inhibitrioe, t..f l) minimum dation (/ Ug / QM3) ..om eya o :: ca qU & of the Genta- Kana- Neo- strain x) mycine mycine - mycine ,.



  Gray-negative Aerobacter aerogenea DA 9D 0.75 94.0 24.0 Aeromonac salmonicida DA 1CO 0.25 3.0 0.25 Alca11eenea op. ATCc '10153 0.025 0.5 0.75 Alcalicenes fecalia ATOC b750 1.5 3.0 1.5 Escherichia coli DA 110 1.25 12.0 3.0 Eseherichia coli ATCC 10586 1.5 12.0 6.0 Escherichia lil 315 intermedia DA 111 '5 1.5 1.5 Klebaiella pneumoniae DA 1 O ,;

  S5 0.75 0.375 Proteus hydrophlla DA 120 C, 5 12.0 12.0 Proteus vulearis DA 121 6.0 16.0 12.0 aaruginosa ATGC iQ197 0'2 '25 '25 Paeudomonas X200 9027 0 25 12tO l 5 Pseudomonaa ATCC 9721 0.25 6.0 1- 0- --- Pseudomonas ATOC 10145 25 12tO 310 Psaudomonas ATCC 8709 0025 12to lo5 Salmonella 10 12 '' le5 schottmuelleri Salmonella DA11 6.0 12.0 12 0
 EMI85.3
 
 <tb> typhimurium
 <tb>
 
 EMI85.4
 Salmonella ATCC.9187. 3 # 0 1200 12 n
 EMI85.5
 
 <tb> Resistant <SEP> to <SEP> acid
 <tb>
 
 EMI85.6
 Myoobacterium amegmatia DA 150 0.15 # Uycobaoterium '7 EY 2O ++ * tuberculouis ¯¯¯ 37 "'" '"" ¯¯¯
 EMI85.7
 z) DA:

   reference to the 2dp of Collection of the Schering Corpo-
 EMI85.8
 ration.
 EMI85.9
 HH) 1 yeast extract, 1 yS dextrose medium ***) 5 human serum gas added
 EMI85.10
 +) Yeast-beef broth (Difco)
 EMI85.11
 * +) Dubos medium t "tween 80" (= ono-o: '\ 5ah of polyoxyethr. Ûne-sorbitaïl)

  <Desc / Clms Page number 86>

 
 EMI86.1
 Susceptibility of fluoroorganisms to antibiotic X # was determined by the standard tube dilution method as follows. In each example,
 EMI86.2
 105 dilutions of 4 hour culture broths as in particular the end points being taken after 24 hour incubation at 3 ° C.

   Unless otherwise indicated, the nutrient medium was constituted by a brain infusion broth (the ar. Of the table, the sulfates of the particular antibiotics, were used in the tests against the microorganisms listed. . However), the inhibited concentrations
 EMI86.3
 1 minimal bitripea are expressed relative to the pure basic antibiotic.



   In vivo activity against certain bacterial infections was also tested in mice. The mice were
 EMI86.4
 ' <- infected with an inoculum of the particular adtainial bacteria by intraperitoneal injection and They were then treated by subcutaneous injections of Gentamyoine sulfate (724 units per mg) dissolved in water, these injections being administered according to two equal daily doses * the protective dose for varying percentages of injected animals was determined as indicated in Table 16 below: ', TABLE 16.



   In vivo activity of Gentamycin
 EMI86.5
 tico.-organieme of infection Dose of Gentamy- Percentage
 EMI86.6
 
 <tb> groin <SEP> / ug / mouse / <SEP> of <SEP> survival
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> ' <SEP> day
 <tb>
 
 EMI86.7
 Klebaiells pneumoniae 0 (solution 0
 EMI86.8
 
 <tb> saline <SEP> witness)
 <tb>
 <tb>
 <tb>
 <tb> 20 <SEP> 20
 <tb>
 <tb>
 <tb>
 <tb> 30 <SEP> 50
 <tb>
 <tb>
 <tb>
 <tb> - <SEP> 40 <SEP> 80
 <tb>
 <tb>
 <tb> 50 <SEP> 100
 <tb>
 
 EMI86.9
 9t & phylocooousur6 & -ray 40 40.

   - ### - 60 .50
 EMI86.10
 
 <tb> 80 <SEP> 100
 <tb>
 
 EMI86.11
 Salmonella achottmuellort 80 50

  <Desc / Clms Page number 87>

 
The acute toxicity of Gentamyoine in mice was determined in the standardized manner by various routes.
 EMI87.1
 reading the Gentamyoine sulfate indicated above, on donated mice weighing 18 to 20 g, the following results were obtained:

   
 EMI87.2
 
 <tb> Mode <SEP> administration <SEP> 50 <SEP> (mg / kg <SEP> of <SEP> mouse)
 <tb>
 <tb> subcutaneous <SEP> 675
 <tb>
 
 EMI87.3
 intraperitoneal 550
 EMI87.4
 
 intravenous <tb> <SEP> 75
 <tb>
 buccal <tb> <SEP>> <SEP> 10000 <SEP>
 <tb>
 Gentamycin also exhibits antiviral activity
 EMI87.5
 in vitro against Rickett, eia ¯2¯¯k¯arie Groups of six day old embryonic eggs * were each infected with fifty liver LD 50 of Rickettaia akari. A single dose of 3.0 mg of Gentamycin, administered 3 hours before infection, provides protection at 50 i (P7j50). Proteotion is obtained when treatment is started with: a Gentamyoin two hours after infection.

   In mice infused by the intracere route
 EMI87.6
 brale with arproxiaativemetit 450 times the LD 50 of Rickettaia, akari, protection at 100 is provided by 5 mg of gentamycin, administered subcutaneously 4 hours before infection and the treatment being continued for 4 days at a rate of5 mg per day in 2 doses. When treatment with
 EMI87.7
 Gentamycino is delayed 48 hours after intrao'r4- infection. brale, 100% protection is still obtained by subcutaneous administration of 10 mg per day (two doses of 5 mg each) for 5 days.
 EMI87.8
 



  Gentamyoin and its salts have a toxic effect when tested in mice against mycob.act.eriun tuberculosis strain H 37 RY.



   BA-3 s using a solution of the mixture, the antibiotic activity of this titrating solution can be demonstrated.
 EMI87.9
 10 units / cn3 (based on the determination method used for "ventarayaine), against S taphylococcua ua reua,

  <Desc / Clms Page number 88>

 
 EMI88.1
 Streptococcus fecalia. 3aoiXlua subtilis, # Bscheriohl ftp'lf Belmenella achottmuelleri, Paeudotaonas ir, os, and Elobaiel g pneumoniae. The mixture appears to be ineffective in relation to Rickettaia akari in mice at 10 mg / day. Its aoti - quickly antibiotic (tried as for Gentamycin) is about 200 units / mg.



   R-451 GROUP ANTIBIOTICS, in particular
 EMI88.2
 R-451 and R-451D, and THOSE OF THE SP * 30 GROUP possess a wide range of activities against graM'-positive organisms Ha have particular value in the treatment of diseases produced by mioroorganniea resistant to pn , o3, .li. (see their tables 17, 21 and 24). They are also useful for treating diseases caused by the 3o au ,, a areuj9 ... Diplococcus pneumoniae and other iaiqro -Qr6'W * isro3B pa: .r5ia genes.



   Antibiotics of the R-451 group are also active against
 EMI88.3
 some micro-organtemau attacked by penicillin. They were, however, determined to be different from ponioillinea, as shown by the absence of deaaotivation of R-451 or R-451D by ponioillinase. In addition, these antibiotics can also be used on patients presenting or known -
 EMI88.4
 as exhibiting sensitivity resulting from b la therapy. penicillin and / or therapy using aybtbe derivatives:

   penicillin ticks, which is an xs, small / frequent phenomenon occurring in about 8-10% of the population -
 EMI88.5
 of the United States of America, that, 1 $ ELg¯4J> .l .BlttW ?, obtained as described in Example 11 and ,! inter alia a useful anti "infectious agent, capable of inhibiting certain manifestations of diseases due to a" "., d to r eu s. to Strclyxocoe es, to 1 ylooçç numoi.ae,. 'and to other pathogenic organisms Its activity in vitro compared against various organisms is reported in the ta-

  <Desc / Clms Page number 89>

 bleau 17 (this activity being determined as for Gentamycin, however using a nutrient medium formed from yeast extract, beef extract and glucose).

   Its activity in vivo against certain bacterial infections in
 EMI89.1
 mouse (18 in g) is determined cor.:n.e for Gontamyoine.



  Table 18 gives the results. 100% oral protection against Streptococcua pyoenes is also obtained by administering 400 µg (20 mg / kg mouse) in 2 equal doses administered one hour before infection for the first dose.

   The acute toxicity of R-451 has been determined
 EMI89.2
 as for Gentamycin and gave the following results s
 EMI89.3
 Method of administration LD 50 (mdkg mouse)
 EMI89.4
 
 <tb> subcutaneous <SEP> 1000
 <tb>
 <tb> intrapritonl <SEP> 1000
 <tb>
 intravenous <tb> <SEP> 850
 <tb>
 buccal <tb> <SEP>> <SEP> 2500
 <tb>
 
 EMI89.5
 R # 451A, obtained as described in Example 18 B, and R-451B, obtained as described in Example 18 C, by a determination cathode on a disc (disc of 6.3 mm diameter ),

   such that the zone of inhibition is measured after placing a disc impregnated with the corresponding antibiotic on an agar plate seeded with the test organism and incubated for 24 hours at 37 C , present
 EMI89.6
 They exhibit in vitro activity against certain microorganisms as indicated in Tables 19 and 20, respectively.

   The in vivo activity of R-451B was determined as for R-451 with the following results:
 EMI89.7
 S t c'hy 1 o c.pc eus sureun PD 50 * = 500 / ug / mouse (25 mg / ke of mouse), streptococcua yo; enee FI) 50 = F30 ug / fJQ'l1rie. (4 mg / kg of LER-451D sulfur, obtained as described in Example 15, pres-en-
 EMI89.8
 te an in vitro activity against various organisms c; ram-poElit1: f'a

  <Desc / Clms Page number 90>

 as shown in Table 21, the determination being the same as for R-451. Its in vivo activity was also determined as for R-451.

   The results of these tests are '
 EMI90.1
 given in Table 220 the acute toxicity of R-45IDs as determined in mice, is as follows
 EMI90.2
 
 <tb> Mode <SEP> administration <SEP> LD50 <SEP> (mg / kg <SEP> of <SEP> drunk) <SEP> - <SEP>
 <tb>
 
 EMI90.3
 mu ...... util> they lil | i # -. ##### $ oua- (1), ta.né '1000 intrapéri tonéal 500
 EMI90.4
 
 intravenous <tb> <SEP> 1.0
 <tb>
 For R-451E, obtained in accordance with Example 17, from
 EMI90.5
 the manner indicated for R-451A and R-451B, oa.a determined in vitro activity, the results of which are reported in Table 23.
 EMI90.6
 



  The in vitro activity of SP-3Q. against various gram-positive microorganisms, determined as for Gentamyoine main # except dat.ion - COntT.-e :: rre;: - in a nutrient medium consisting of a tryptoae phosphate broth, is indicated by the table 24. / * 'The substance used for the estais corresponds
 EMI90.7
 danta was obtained according to example 19 <"and had a potency of 4000 dilution units per tng, that is to say that | 1 mg of this material dissolved in 1 c3 of aoht tt a ** 1 # 5 born at a dilution 4000 times greater, also inhibits the growth of Sta h locoo ua aureus (ATCC 6'S38. 'In addition, crude P-30 as well as its isolated constituents (fractions)

     
 EMI90.8
 were tested by the eÙr, d18qU, determination technique. described with regard to R-451A and R-451B, against certaiwia ôrgô #% gram-positive niâmes. Table 25 gives the results of these tests. The in vivo activity against certain bacterial infections in mice was also tested as for
Gentamyoine.

   The test animals were administered the material obtained following Example 190 suspended in
 EMI90.9
 0.25 $ aqueous carboxymethyl cellulose 1.prao dose

  <Desc / Clms Page number 91>

 cover for 100% of animals infected with Staphvlo-
 EMI91.1
 cor u -eup, is 10 msourie / day (II 40,000 limited dilution per mouse per day) * The protective dose for 100% of *
 EMI91.2
 animals infected with Piplococcus pneuraoniae is 15 m & / mouse / day (= 60,000 dilution units / mouse / day}, this dose being administered for 2 days. The acute toxicity (LD50) of the same sample of SP-30 was also measured in the usual way,

   We form a suspension in 0.25 aqueous carboxymthyl cellulose with the following results! :
 EMI91.3
 
 <tb> Mode <SEP> administration <SEP> LD50 <SEP> (mg / kg <SEP> of <SEP> smile)
 <tb>
 
 EMI91.4
 sub-oiitane> 7500
 EMI91.5
 
 <tb> intraperitoneal <SEP> 7500
 intravenous <tb> <SEP> 800
 <tb>
 <tb>
 <tb>
 <tb>
 
 EMI91.6
 

  <Desc / Clms Page number 92>

 
TABLE, 17.



  R-451 antibiotic spectrum in vitro
 EMI92.1
 
 <tb> Microorganism <SEP> tried
 <tb>
 
 EMI92.2
 Hvatémfltiaue Hibitrloe concentration Ayatematic name Minimum designation (/ ug / cm3)
 EMI92.3
 
 <tb> dela
 <tb> strain
 <tb>
 
 EMI92.4
 Bacillus coreus TOC 7064 0.75
 EMI92.5
 
 <tb> Bacillus <SEP> cereus
 <tb>
 
 EMI92.6
 var. mycoides DA.39 cl5 Bacillus metfathoritua DA 31 0.75 Bacillus subtille ÂTOC 6633 0.25
 EMI92.7
 
 <tb> Staphylococcus <SEP> albua <SEP> DA <SEP> 2100 <SEP> 0.10
 <tb>
 <tb> Staphylococcus <SEP> aureus <SEP> ATCC <SEP> 6538P <SEP> 0.075
 <tb>
 
 EMI92.8
 Staphyloooccus aureun 1l, TCC 1163 Ot25 Staphylococcus aureue ACCO 12715 0.75
 EMI92.9
 
 <tb> Staphylococcua <SEP> aureus <SEP> Gray <SEP> 0.50
 <tb>
 <tb> Staphylocoocua <SEP> aureue <SEP> DA <SEP> 201 <SEP> @) <SEP> 0.10
 <tb>
 
 EMI92.10
 Staphylococoua aureua DA 202.

   0.25 Staphylococcus aureua DA 203 *** '0.30 Staphylococoua aureùa DA 1-04' ") 0.15 Staphyloooooua aureua DA 205 * '0.40 Streptocoocua fecalia DA 20 0.75 Streptococous pyogenea AÏCO 12384 0.175' - Streptoioccun pyogenes DA 21 0,? 75 tt) Strain resistant to Novobipeineo) Strain resistant to O? .ea.ndomyoiMe and BrytQ ..



  Penicillin reattached strains.



  ¯¯¯¯¯¯¯¯¯¯¯¯¯. ,, -. R ....... 3.

  <Desc / Clms Page number 93>

 
TABLE 18.



  In vivodu activity R-451
 EMI93.1
 Eiero-orzanism Dosage of E-451 Percentage of infection / ug / aouria / days survival after
 EMI93.2
 
 <tb> / <SEP> of <SEP> 24 <SEP> hours
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> Streptococcus <SEP> 0 <SEP> (solution <SEP> saline <SEP> 0
 <tb>
 <tb>
 <tb> pyogenes <SEP> witness)
 <tb>
 
 EMI93.3
 40 (2; / k;) 50
 EMI93.4
 
 <tb> 40 <SEP> (2 <SEP> mg / kg), <SEP> 100
 <tb>
 <tb> a <SEP> alone <SEP> inject
 <tb>
 <tb> tion <SEP> 1 <SEP> time
 <tb>
 <tb> after <SEP> infection
 <tb>
 
 EMI93.5
 Staphylocoocua aureua 40 (2 mg / PC) 50 Diplococcus 50 (2.5 mdlcg) 100 (after
 EMI93.6
 
 <tb> pneumoniae <SEP> 2 <SEP> days;
 <tb>
 
TABLE 19.



  In vitro activity of R-451A
 EMI93.7
 Concentration of the Zone of Inhibition, we diameter in mm.) ¯, against K-451A (/ ue / ct <i3) Staphylococcua Streptococoua Bacillus' aureua fecalia subtilie
 EMI93.8
 
 <tb> 1000 <SEP> 18 <SEP> 23 <SEP> 13
 <tb>
 <tb> 100 <SEP> 9 <SEP> 15 <SEP> 10
 <tb>
 <tb>
 <tb> 10 <SEP> 0 <SEP> 10 <SEP>, <SEP> 0
 <tb>
 <tb>
 <tb> 1 <SEP> 070
 <tb>
 
TABLE 20.



  In vitro activity of R-451B
 EMI93.9
 Zone of inhibition (diarnot.re in Mme) against R-451B (/ ug / cm3) Staphylococcus streptococcua. Daoi11u.
 EMI93.10
 
 <tb>



  / <SEP> aureus <SEP> fecalie <SEP> subtilis
 <tb>
 <tb>
 <tb>
 <tb> 1000 <SEP> 23 <SEP> 30 <SEP> 17
 <tb>
 <tb>
 <tb> 100 <SEP> 20 <SEP> 26 <SEP> 8
 <tb>
 <tb>
 <tb> 10 <SEP> 15 <SEP> 22 <SEP> 0
 <tb>
 <tb>
 <tb> 1 <SEP> 15 <SEP> 0
 <tb>
 

  <Desc / Clms Page number 94>

 TABLE 21.



  ; Antibiotic spectrum of R-451D
 EMI94.1
 (Áo: tiN.i-t-: l: 1î vitro), '¯¯¯¯¯ (Âcjtiirt-H; vitro) ...: .-
 EMI94.2
 ¯j¯¯ "- 'na-- # -¯-> é Concentration Microorganism tested Inhibitory designation i otx t.L t1' of the minimum of.



  Systematic name strain R-45LD </ Ug / oa3) 1.1 ... J # IITTI- - '- "\ t -" [# - "' # - Ba, r: i7.lua oereus AT CC 7064 0.25 Baoillus C4) rGUS var.



  1: mycoidea # DA, 9 'J; 075--' Bacillus meeatl'eriUÎl1 DA. 3 1 0.25 \ # BacÍl1ue megatheriua BZZ 3773 0.025; ..



  Bacillua me; athsriwn DA 38 0.25.



  Bacilli sphaerioua DA 32 0.75 -7 Baoillua ephaericus DA 33 "'0.25' Baoillus subtilis ATCO 6633 0.25 'Diplocooous pneumoniae.) DA 150 0.25
 EMI94.3
 
 <tb> Saroina <SEP> lutea <SEP> ATGC <SEP> 9431 <SEP> ' <SEP> 0.0075
 <tb>
 
 EMI94.4
 Staphylococcua albue DA 2100 0.25
 EMI94.5
 
 <tb> Staphylococcus <SEP> aureus <SEP> DA <SEP> 2027-2036 <SEP> each <SEP> 0.25
 <tb>
 <tb>
 <tb> (10 <SEP> products Isolated <SEP>
 <tb>
 <tb>
 <tb> clinics <SEP> resistant <SEP> to
 <tb>
 <tb>
 <tb> la- <SEP> Penicillin, <SEP>
 <tb>
 
 EMI94.6
 Ja-Ttraoyoline,
 EMI94.7
 
 <tb> the <SEP> Streptomycin,
 <tb> and <SEP> Erythromycin)
 <tb>
 <tb> Staphylococcus <SEP> aureus' <SEP> Gray <SEP> 0.25
 <tb>
 <tb> Streptococcua <SEP> fecalis <SEP> DA <SEP> 20 <SEP> $ 0.02
 <tb>
 
 EMI94.8
 streptocoooua pyogenea * 'DA 21 0.25
 EMI94.9
 
 <tb> *)

  Broth <SEP> infusion <SEP> brain-heart <SEP> + <SEP> 0.5 <SEP>% <SEP> of <SEP> serum
 <tb> human
 <tb>
 

  <Desc / Clms Page number 95>

   TABLE 22.



  In vivo activity of R-451D
 EMI95.1
 4.taraor; uniame Dose of R-45lD Percent survival infection / uc / aouri8 / daya after 24 hours
 EMI95.2
 
 <tb> Streptooocoua <SEP> 0 <SEP> (solution <SEP> saline <SEP> 0
 <tb>
 <tb> pyogenes <SEP> witness)
 <tb>
 <tb>
 <tb> 75 <SEP> (3.75 <SEP> mg / kg) <SEP> 50
 <tb>
 <tb>
 <tb> 100 <SEP> (5.0 <SEP> me / kg) <SEP> 100
 <tb>
 
 EMI95.3
 Staphylococcus 250 (12.5 NË / kg) 50 auroua 400 (20 mkg) 100 Dirlococcus 75 (3t75 mlikg) 50 (after 2 days) 1 pneumor.iae 100 (5 o rat> ykG) 100 (after 2 days)
 EMI95.4
 .TA3EKAU 2 ?.



  In vitro activity of R-451S - "j ¯ - -.
 EMI95.5
 Concentration of P ± pPn3 - & - oïl, in the inxi.bitian zone mm aan: R-451.E / us / cnt3 Staphylococcus Streptococcua Bacillue
 EMI95.6
 
 <tb> ' <SEP> aureus <SEP> fecalis <SEP> aubtilia
 <tb>
 
 EMI95.7
 1030 20 19 '¯ 24 ¯¯ ¯
 EMI95.8
 
 <tb> 100 <SEP> 15 <SEP> 13 <SEP> 19
 <tb>
 <tb> 10 <SEP> 8 <SEP> 15
 <tb>
 <tb> 0 <SEP> 0
 <tb>
 

  <Desc / Clms Page number 96>

 
TABLE 24.



  In vitro activity of SP-30
 EMI96.1
 ) Micro-gold, ganïame tried Concentration -nao. + ##! inhibitory Systematic name of the minimal strain (/ OM3) Bao111us cereus var-MYCOÏ406 AICC. 7064 6.0 Baelllue garous var.mycoldes DA 39 0.75 Baoillue cerous var.mycoides DA 30 12.0 Baclilus megatheriun DA 31 0.25 '
 EMI96.2
 
 <tb> Bacillua <SEP> meatherium <SEP> DA <SEP> 36 <SEP> 3.0
 <tb>
 
 EMI96.3
 Bacillus mecatherium DA 35 6eo Bac 1 Hua aphaoricua 'DA 32 zoo Bacillua ophacricue DA 33 $ 1 P Diplococcus pneumoniab DA 999 0.075
 EMI96.4
 
 <tb> Staphylococcus <SEP> albua <SEP> DA <SEP> 40.

    <SEP> 0.75
 <tb> Staphylococcus <SEP> aureus <SEP> ATCC <SEP> 6538 <SEP> 0.25
 <tb>
 <tb> Staphylococcua <SEP> aureus <SEP> ATCC <SEP> 6538P <SEP> .0.25
 <tb>
 
 EMI96.5
 Staphylococous aureus ATOC $ 1,271 2.0
 EMI96.6
 
 <tb> Staphylococous <SEP> aureus <SEP> ATCC <SEP> 1163 <SEP> 1.0
 <tb> Staphylococcus <SEP> aureus <SEP> 25
 <tb>
 <tb> var. <SEP> Gray.
 <tb>



  Staphylococcus <SEP> aureus
 <tb> var. <SEP> Smith <SEP> DA <SEP> 141 <SEP> 0.25
 <tb>
 
 EMI96.7
 Staphylococcus aureus DA 2026 0.25
 EMI96.8
 
 <tb> (Products Isolated <SEP> <SEP> clinics <SEP> DA <SEP> 2027 <SEP> 0.75
 <tb>
 <tb>
 <tb> resistant <SEP> to <SEP> the <SEP> penicillin) <SEP> DA <SEP> 2028 <SEP> 1.5
 <tb>
 <tb>
 <tb> DA <SEP> 2029 <SEP> 0.75
 <tb>
 <tb>
 <tb> DA <SEP> 2030 <SEP> 2.0
 <tb>
 <tb>
 <tb>
 <tb> VA <SEP> 2031 <SEP> 1.0
 <tb>
 <tb>
 <tb> / <SEP> DA <SEP> 2032 <SEP> 0.75
 <tb>
 <tb>
 <tb>
 <tb> DA <SEP> 2033 <SEP> 0.75
 <tb>
 <tb>
 <tb> DA <SEP> 2034 <SEP> 0.75
 <tb>
 <tb>
 <tb>
 <tb> DA <SEP> 2035. <SEP> 1.0
 <tb>
 
 EMI96.9
 Staphylooooûua apidemidie DA 41 0.25 Saroina lutea ATCC 9341 0.7 $ '## mierqoooiue flavue DA 60 0.05 atreptococcue pyogwes Ci, DA 21 0.025 streptooccoue teoalid' # ATCC 1041 16.0 Eaoher1chia 0011 DA 110,. 16.0 Escherichia illtnned1s.

   DA 111 2.0 Mycobanterium sl1 '! - :: Igulaiois' DA 150 4.0 Staphylooooous auteuat ATCC 9996 0 "$ 7.

  <Desc / Clms Page number 97>

 
TABLE 25.



  In vitro activity of SP-30 antibiotics
 EMI97.1
 
 <tb> Antibio- <SEP> Concentra- <SEP> Zone <SEP> inhibition <SEP> (mm) <SEP> against
 <tb>
 
 EMI97.2
 tick tion (/ Ug) Staphylococcus Streptococcua Baoillue 'aureus pyogenea eubt111s 1 ............ j <.....,., ....... ,,.,! ....! ..,!,. , .., ...., .. <..
 EMI97.3
 
 <tb>



  SP-30 <SEP> 1000 <SEP> 28 <SEP> 29 <SEP> 12
 <tb>
 <tb>
 <tb>
 <tb> 100 <SEP> 19 <SEP> 19
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 10 <SEP> 13 <SEP> 9 <SEP> 0
 <tb>
 <tb>
 <tb>
 <tb> 1 <SEP> 9 <SEP> 0 <SEP> 0 <SEP>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> SP-30A <SEP> 1000 <SEP> 28 <SEP> 31 <SEP> 9
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 100 <SEP> 17 <SEP> 12 <SEP> 0
 <tb>
 <tb>
 <tb>
 <tb> 10 <SEP> ¯ <SEP> 0 <SEP> 0 <SEP>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 1 <SEP> 0 <SEP> 0 <SEP> 0
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> SP-30B <SEP> 1000 <SEP> 28 <SEP> 35 <SEP> 11
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 100 <SEP> 20 <SEP> 20
 <tb>
 <tb>
 <tb>
 <tb> 10 <SEP> 11 <SEP> + <SEP> 0
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 1 <SEP> 0 <SEP> 0 <SEP> 0 <SEP>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> SP-300 <SEP> 1000 <SEP> 28 <SEP> 33 <SEP> 14
 <tb>
 <tb>
 <tb>
 <tb> 100 <SEP> 22 <SEP> 24 <SEP> 8
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 10 <SEP> 17 <SEP> 14 <SEP> 0
 <tb>
 <tb>

  
 <tb>
 <tb>
 <tb> 1 <SEP> 9 <SEP> ¯ <SEP> 0
 <tb>
 <tb>
 <tb>
 <tb> SP-30D <SEP> 1000 <SEP> 19 <SEP> 19 <SEP> 0
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 100 <SEP> 13 <SEP> 10 <SEP> 0
 <tb>
 <tb>
 <tb>
 <tb> 10 <SEP> ¯ <SEP> 0 <SEP> 0
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 1 <SEP>. <SEP> 0 <SEP> 0 <SEP> 0
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> "¯" <SEP> indicates <SEP> existence <SEP> of a <SEP> light <SEP> inhibition,
 <tb>
 <tb>
 <tb> difficult <SEP> to <SEP> evaluate <SEP> digitally.
 <tb>
 

  <Desc / Clms Page number 98>

 
 EMI98.1
 



  PHARMACEiC PRESENTATIONS AtîTIBIQT ic PRECI '.



   For administration to patients, the antibiotics according to the invention are presented under current strengths. used for antibiotics. For the preparation of these dosage forms, the usual pharmaceutical carriers can be used.
 EMI98.2
 Examples will be given of such pharmaceutical forms containing Gentamyoine (in the form of its sulfate) as an active principle. Of course, sulphate of
 EMI98.3
 .

   Gentamyoin can be replaced by other derivatives of Ontamyoin, by free Gentamyoin, by other antibiotics according to the invention (including their usual derivatives) or by mixtures of such antibiotics and / or derivative.
 EMI98.4
 EXËKPEE A - Solution for injection
For the preparation of a suitable injection solution, 50 g of Gentamycin (in the form of its sulfate) is dissolved in a sufficient quantity of injection water, and the resulting solution is made up with injection water. to obtain 1.0 liter of solution.

   The solution is filtered through a bacteria retaining filter and sterile ampoules are aseptically filled with this solution. The ampoules are autoclaved at 121 ° C. for 30 minutes.



    EXAMPLE B - Topical Ointment An ointment can be prepared from the following constituents:
 EMI98.5
 Gentarayoine (as sulfate) 5.0 methyl tri-p-hydroxybenzoate, 2 g Butyl p-hydroxy-benzoate 108 g mineral oil 100.0 g petrolatum q.s. 1 kg
 EMI98.6
 To prepare the ointment L using the above constituents, - the petrolatua is melted and mixed at 70-9040 with the methyl p-hydroxy-benzoate and the p-hydxoxywbsnoate of

  <Desc / Clms Page number 99>

 
 EMI99.1
 butyl. Once everything is dissolved, let the SOl \ t1i! -: - ,, - 1 re1'ro1d1r-to around 400q.

   The Gentamyoine .8uat.- is dispersed separately in the oil would undermine the dis-
 EMI99.2
 persion us through a uou23.i è colloids. The dispersion coming out of this apparatus is added to the-baB; 3 oi4deBsû ointment and the mixture is allowed to settle. spontaneously cool to room temperature with continuous stirring. We fill tubea or suitable jars with this product. The ointment thus obtained can also be used as an ophthalmic ointment.
 EMI99.3
 



  EXw # LE C - Ophthalmic drops: 1iQue Ophthalmic drops containing Gentamyoinet are obtained from the following constituents: z Ger.tsme3.ne (in the form of sulfate) 5.0 g p! Jnyl-ethyl alcohol 5 ,? g purified water q.e. 1 liter to prepare the ecuttes. the phenyl-ethyl alcohol is dissolved in approxifflativèraent 90 ;. of water, then the Gentamycin alfate is dissolved out in the phenyl ethyl alcohol solution and enough water is added to bring the product to 1 liter. The solution is filtered h through a filter retaining the
 EMI99.4
 bacteria and sterile dropper bottles are asoptically filled with this solution.


    

Claims (1)

ABSTRACT . EMI99.5 ¯¯ -..., .... ¯¯¯. ¯¯ .... ¯ The invention relates in particular to the / Use of. microorganisms of one of the species!,: ioromonoEll 'ra purpureag !! .. oc1.inoBJ) ora, M. carbonecea and LI. halophytica or a mutant or variant thereof, or equivalent microorganisms, for the preparation of new antibiotics in the usual biological manner; isolating these antibiotics from the culture; and, optionally, their transformation into usual derivatives, in particular their salts with acids, their N- and / or O-acylated derivatives and their pro- <Desc / Clms Page number 100> EMI100.1 "Reaction duita with compost from addit3.ow bieu. '. ticks of aldehydes or ketones. 2 * / A process according to 1 "/, further exhibiting the following peculiarities taken separately or according to the various possible combinations t EMI100.2 a) an aerpb incubation is carried out; the Nioro-orsania- mes used in an aqueous nutrient medium which contains EMI100.3 assimilable carbon and nitrogen until at least one compound having significant antibiotic activity is produced; b) to prepare new basic antibiotics, incubate a strain capable of straining antibiotics EMI100.4 basic, of the ruioro-oreanismen of Goitre, gromo o, until at least one compound having antibiotic activity nota- EMI100.5 If the product has been produced, the antibiotically active basic fractions are isolated from the substrate thus obtained and, optionally, it is converted into the usual derivatives, in particular into sole with EMI100.6 acids or reaction products i: .tveo let; bisulfite addition compounds of aldehydes, preferably Se tormalddhyeot or ketones; -c) .an ttti-3d-c <r¯tg'8 * Tn ± crO '"0X'g ± tniaines of one of the specieso- i R * purpurea and LI. echingapora, or cvloi & -' Oïiani9 # it * slow as the variant i, eah o, o, var. t2 ± ruie, ea and M. echinoapora var. pallidat for the preparation i # î t alo-olo myoine and / or its co-products antibioTics BA -3 (fractions. A and B in the usual biological manner, we isolated and evolved, we purify at least one of these cultural antibiotics, and, if broken down, it is transformed into their derivativesi d) a strain capable of producing la.0enâ * mycin, preferably one of the strains HRRL 2953e 1, MU Z985,. ttRRL 2995 and HRRL 29961 e) after formation of a product having significant antibiotic activity, the mycelium is extracted from the culture with a <Desc / Clms Page number 101> acid and the anti-biotic co-produced Gentasycin and / or Bas is isolated from the acid extract by adsorption, fractional precipitation of salts or a combination of both; f) M. carbonacea, or known equivalent microorganisms, the variant M. carbonacea var. aurantiaca, in order to prepare the new antibiotic R-451 in the usual biological manner, and; if necessary, at least one purifies. one of seo constituents R-451A, R-451B, R-451C, R-451D and R-451E of the culture, and, if necessary, they were transformed into their usual derivatives, in particular into N- and / or O-acylea such as the acetate of component R-451D; g) using a layer capable of producing R-451, preferably one of the strains NRRL 2972 and NRRL 2997; h) M. halophytica, or equivalent microorganisms, are used to prepare the new antibiotic SP-30 in the usual biological manner, one iuolates and, optionally, at least one of the constituents SP-30A, Si '-308, SP-30C and SP-30D from the culture, and, if desired, converted into their usual derivatives; i) a strain capable of producing SP-30 is used, preferably the strain NRRL 2998; j) after formation of a product having notable antibiotic activity, in accordance with one of paragraphs f) to i) above, the mycelium is separated from the culture, the remaining broth is extracted with an immiscible solvent with water, and from the extract thus obtained the desired antibiotics or mixtures of these antibiotics are isolated by adsorption, by partitioning, by fractional precipitation or by a combination of these methods; 3 / A process for the preparation of antibiotically active pharmaceutical compositions, characterized in that at <Desc / Clms Page number 102> at least one of the antibiotically active and / or derivative products obtained by a process according to 1 / or 2 / is present in a form suitable for pharmaceutical administration. 4 / A method according to 3 / characterized in that the derivative or mixed products, respectively, are mixed with a suitable pharmaceutical vehicle. An antibiotically active pharmaceutical preparation obtained by a method as specified in 3 / or 4 /. 6 / Basic antibiotics that can be obtained EMI102.1 by incubation of microorganisms of the genus Microtnoïioapora. 7 / antibiotically active products that can be EMI102.2 obtained by a process as specified in / or 26/1 y boa * taken their usual derivatives such as their salts with acid, their N- and / or 0-acylated derivatives, and their reaction products' with compounds of biaulfitic addition of aldehydes or ketonos, as well as mixtures of these products and / or derivatives. EMI102.3 80 / Oentamyoin, as a new antibiotic, and its usual derivatives, in particular its salts with acids and se *. EMI102.4 reaction product with bisulfite addition compounds of aldehydes and ketones. 9 * / Gentamicin sulfate. 7.0 / Gentamyoine hydrochloride 11 * / Gentamycin N-methylene sulfonate. 12 "/ Fractions A and B of the new antibiotic. 1 r. Their usual derivatives, in particular their salts with acids, and mixtures of these fractions and / or derivatives. 13 / As a new antibiotic, R-451 and its constituents - EMI102.5 ante 4 to E, in particular the constituents R-451BO R - t51D -and B-4> IB # their usual derivatives, in particular their N- and / or 0- acylated derivatives, and mixtures of these constituents and / or derivatives. EMI102.6 14 * / R-t51D acetate. '159 / As a new antibiotic SP-30 and its a'ongt3.tt' <Desc / Clms Page number 103> ants SP-30A, SP-30B, SP-300 and SP-30D, their usual derivatives EMI103.1 and mixed them from these conatitant and / or derivative. 16 / The processes described in Examples 1 to 21 and the clearly equivalent processes. EMI103.2 177 / the antibiotically active compounds which can be obtained according to Examples 1 to 21 or by a procedure which is an obvious equivalent of any of the example cases.