CA1050460A - Production of antibiotic mm 13902 by streptomyces - Google Patents

Abstract

ABSTRACT
A method for producing a novel antibiotic designated MM 13902 of the general formula (I)

Description

~o50460 BACKGROUND TO THE INVENTION `
British Patent No. 1,363,075 disclosed that a useful B- lactamase inhibitor could be obtained by the fermentation of certain strains of Streptomyces olivaceus. Until our present invention, it was believed that the material disclosed in British Patent No. 1,363,075 was substantially , pure. However, we have disclosed that this is not so and - that a minor component of that material can be isolated and ' has potent antibacterial activity. This new material is . ~ .,. . - .
designated MM 13902 and it is now believed to be responsible for a part of any antibacterial activity present in the material of British Patent No. 1,363,075 although it i~
responsible for only a very minor part of B-lactamase inhibitory activity exhibited by that material. Naturally, ~ nothing in this specification should be construed as claiming c~ mat~rial as dsiclosed in British Patent No. 1,363,075. Other ~-lactamase inhib;tors are known to be produced by strains of Streptomyces, for example those disclosed in German Published . ,. ~ ~ . .
~ - 2 -cj ~ .-,,:: : ~
. ~ ~
j .. . ... .
:

i .. . . , - - . , . . . ;. . , ~ 50460 Patent Application No. 23400Q5, but such known materials have not been demonstrated as having the potent antibacterial activity of the kind possessed by MM 13902.

DESCRIPTION OF THE INVENTION
The present invention provides the antibacterial agent designated herein MM 13902 and its salts.

The structural formula for MM 13902 is believed to be as follows: CH3 HO350 ~ ; - NH - CO - CH3 ~, MM 13902 is a solid carboxylic acid which in the form of a substantially pure sodium salt has the following characteris-tics:

i. It is highly soluble in wat:er, soluble in methanol and substantially insoluble in chloro-` form, diethylether and hydrocarbons.
., `j ~i. In a~ueous solution, it has a characteristic ultraviolet spectrum with absorption maxima one of which is at about 305 nm.

iii. When present at 0.4% w/w in a freshly prepared KBr disc, it has a characteristic infra-red spectrum which has absorption maxima at inter '~ 3 ,~ .

, 1~50460 alia about 3450, 2950, 1750, 1620~ 1510, 1400 and 1260 cm 1, iv. It has a characteristic n.m.r. spectrum when taken in D20 ~7hich spectrum possesses inter alia (a) a pair ,~ ~

,, ~ .
. Z O : ' ~''", '~i ,. . . .
:~
., 3a ~OS04~0 of low field doublets centred at approximately

2.85Z and 4.00Z with coupling constants of approximately 14 Hz; (b) a doublet centred at approximately 8.55 Zand (c) a sharp singlet at approximately 8.00~.
v. It possesses antibacterial activity against various species including inter alia, strains of StaPh lococcus aureus, Bacillus subtilis, Escherichia coli, Klebsiella aerogenes, Proteus mirabilis, Salmonella typhi and Pseudomonas aeruginosa.
vi. When mixed with ampicillin it synergyses its activity agains organisms including strains of Escherichia coli, Klebsiella aerogenes, Proteus mirabilis, Proteus morganii and Staphy~ococcu~ aureus Russell.
The pure di-sodium salt of MM 13902 is a4 -lactamase inhibitor and has an I50 (as hereinafter described) of between 0.001 ~g/ml and 0.0001 ~g~ml against the ~-lactamase of Escherichia coli B 11.
When run on cellulose in a thin layer chromatography ,` . system the pure di-sodium salt of MM 13902 has the following approximate Rf values:-ta~ n-butanol/isopropanol/water - 7:7:6 v/v;
Rf - 0.72 ! (b) isopropanol/water - 7:3 v/v; Rf = 0.85 tc) n-butanol/ethanol/water - 7:7:6; Rf = 0.81 td) n-propanol/water - 4:1; Rf _ 0.75 I From another viewpoint, MM 13902 may be characterised :.1 as a sulphur containing antibacterial agent, salts of which are !
: - 4 -~' .,' . .

produced during the cultivation of Streptomyces olivaceus ATCC 31126 and in the form of an aqueous solution of its di-sodium salt has UV absorption maxima at about 305 nm and at about 225 nm substantially as shown in Fig. 1 herein. ;~

.

Alternatively, MM 13902 may be characterised as an anti-bacterial agent which in the form of its substantially pure di-sodium salt has an infra-red absorption spectrum sub-stantially as shown in Fig. 2 when taken in a freshly prepared 0.4% W/W KBr disc and which in the form of its substantially pure di-sodium salt has n.m.r. spectrum substantially as shown in Fig. 3 when taken in a freshly prepared solutîon in D20.

. J~ .
The material MM 13902 tends to be unstable when in the unsalted acid form. Aec~rdingly a preferred aspect of this invention provides salts of MM 13902. Suitably the salts of MM 13902 are di-basic salts. Most suitably these salts are those formed with pharmace~ically acceptable ions such as 8~dium, potassium, calcium, magnesium, aluminium, conventional substituted ammonium ions and the like.
Particularly suitable salts are alkali metal salts such as The sodium or potassium salts, for example 9 the di-sodium or di-potassium salts.

Suitably the MM 13902 and its salts as provided by this invention are at least 50~ pure, more suitably at least 70 pure and preferably 80% pure, for example 90-100~ pure.

. ~ .
!' f .. . .
.'" ~ '.
:~ , . . .

~OS04ti0 In a further aspect this provides a pharmaceutical composition which comprises MM 13902 as hereinbefore described or a salt -~
thereof together with a pharmaceutically acceptable carrier.

Most suitably the compositions of this invention contain a sodium or potassium salt of MM 13902, for example, the di-sodium or di-potassium salt of MM 13902.

Such compositions may be in a form suitable for oral, topical or parenteral use. For example, tablets, capsules, creams, syrups, reconstituable powders and sterile forms suitable for injection or infusion may be used. Such compositions may contain conventional pharmaceu~i:cally acceptable materials such as diluents, binders, colours, flavours, preservatives, disintegrants and the like in accordance with conventional pharmaceutical practice in a manner well known ¦ to those skilled in the formulation of antibiotics such as penicillins and cephalosporins.
.. ,.j .
1 The MM 13902 or its salts may be present in the composition ¦ of the invention as sole therapeu~i-c agent or it may be present together with a ~-lactam antibiotic. Suitable , 20 ~-lactam antibiotics include those known to be susceptible lactamases and also having some intrinsic resistance to ~ lactamases. Such ~-lactam antibiotics include ampicillin, ;~ amoxycillin~ benzylpenicillin, phenoxymethylpenicillin, l~ propicillin, cephaloridine, cefoxitin, cephalothin, cephalexin, j carbenicillin, ticarcillin and in vivo hydrolysable esters of '1,` `: ~.

1 . .

lOS0~60 such compounds such as the phenyl, tolyl or indanyl esters of carbenicillin or ticarcillin or the acetoxymethyl, pivaloyl-oxymethyl or phthalidyl esters of ampicillin, benzylpenicillin, amoxycillin, cephaloridine, cephalogylcin and the like.

The ratio of MM 13902 or salt thereof to ~ lactam antibiotic is normally between 10:1 and 1:10, for example, between 3:1 and 1:3.

The total quantity of antibacterial agents present in any unit dosage form will normally be between 50 and 1500 mgs 10 and will usually be between 100 and 1000 mgs.

Preferred unit dosage compositions according to this invention may be administered one or more times a day, for example, 2 to 4 times a day, in the treatment of diseases of the urinary tract, respiratory tract, soft tissues and the like.
Thus the compositions may be used in the treatment of such diseases as bronchitis, gonorrhea, otitus media, mastitis , and the like.

. ' .
In one of its aspects, this invention provides a process for the preparation of MM 13902 or a salt thereof which comprises 20 chromatographically separating a solution of MM 4550 (Complex) as hereinafter described into fractions consisting essentially of a solution of MM 13902 or a salt thereof and other fractions and isolating the MM 13902 or a salt thereof from solution.

~ 7 .. . . . . . . ..

~050~60 By those fractions "consisting essentially of a solution of MM 13902 or a slat thereof" is meant that either the only antibiotic material present in that solution is MM 13gO2 or a salt thereof or that if any other antibiotic material i5 present then it is there to a lesser extent than the MM 13902 or salt thereof. More sultably MM 13902 or a salt thereof represents 70%, most suitably 80% and preferably 90-100%, of the antibiotic material present in the fraction. We have ~ound that an acceptable method of determining which fractions consist essentially of solution of MM 13902 or a 8alt thereof is to moniter the uv absorption spectrum of each sample. Thoseefractions showing a uv spectrum similar to that of Fig. 1 normally contain MM 13902 or a salt thereof substantially free from other antibiotic material such as MM 4550 as herein~fter described. In general those fractions showing a distinct uv absorption maximum at about 305 nm are of the desired purity.

.
Normally the preceeding process is used for the isolation of , MM 13902 as a dibasic salt. If a monobasic salt of MM 13902 ~ 20 or the free acid MM 13902 per se is required, they may be -~ prepared by the acidification of a dibasic salt of MM 13902 ¦ because in general the monobasic salts of MM 13902 and the free acid M~ 13902 are not sufficiently stable to isolate from mixtures such as MM 4550 (Complex) as hereinafter , described. These monobasic salts and free acid are not i readily obtainable owing to their low stability.

,` -:
: 1 ' ' . ..

lOS046V
A~ previously stated, we believe that the chromato-graphic isolation of MM 13902 is best carried out using a ,alt of MM 13902 such as the di-sodium salt.
Salts of'MM 13902 are more soluble in aqueous solvent systems than in highly lipophilic solvents and con-sequently it is preferred to use aqueous solvent systems in the chromatographic purifications used in this invention.

In our hands aqueous solutions o~ electrolytes buffered to approximately neutrality have proved suitable for use in conjunction with polar support ~aterials such as basic ion-exchange resins. Thus an aqueous solu-tion of sodium chloride buffered to about pH 7 with a conventional buffer such as a phosphate buffer may be used in conjunction with support materials which may contain tertiary amino gro~lps or ~uaternary ammonium groups. We have found that basic ion-exchange celluloses and basic ion-exchange cross linked dextrans are suitable support materials and that ' 20 QAE Sephadex A25 in particular is a highly suitable support material especially when the solvent system comprises C.7m sodium chloride in pH7 phosphate buffer.
~ ,Sephadex' is a Registered Trade Mark).

Alternatively, in our hands solvent syste~s comprising mixtures of water and water miscible organic solvents ~- such as a lower alkanol have proved suitable for vse :i :
':
~ _ g _ .. , : . ~ . . .. . .

" , . .. . .. .

~. .
: ; , .

~ S0460 in conjunction with inert support materials such as silica gel or cellulose. A particularly suitable solvent system for use with cellulose is a mixture of water an~ isopropanol, especially a 7:3~isopropanol :
water mixture.

However, the product of the above procedure using ion-exchange resins frequently contai~s a very high proportion of sodium chloride - so that it is beneficial to de-salt the pooled solutions.
De-salting may b~ effected by passing the solution down a column containing a lipophilic material onto which the I~M 13902 is adsorbed bu-t w~lich does not adsorb the sodiwn chloride. Suitabie column materials include polystyrenes such as Amberlite XAD4. Alternatively gel ~iltration may be utilised using filtration agents such as cross linked : 15 dextrons such as Sephadex G 25 and polyacrylamide gels such as Biogel P2 ('Amberlite', 'Sephadex' and 'Biogell are Trade Marks). The an~ikiotic tnay be eluted from the column using ~ater, aqueous methanol or the l~ke.
:
When the desired solu~ions are ob-tained by the above process ~0 ~e di-basic salt of MM 13902 may ~e obtained in solid form by the removal of the solvent under m ld conditions. We have found that an acceptable method of obtaining such solid material iS to evaporate under reduced pressure and freeze-dry the pooled fractions containing MM 13902.
If desired the preceeding process may be carried out in t~ro or more steps. For example, a solution of MM 4550 (Complex) .. - . . . . . .
., , , . : , . . . . . : ., , . . . . - .. :. - , . ,, . -.

~0504~iO

may be separated into fractions comprising MM 13902 or salt .
thereof contaminated with up to its own weight of other anti-bacterial agents, the fractions may be freeze dried to yield a ma~erial containing, for example, about 50-60t of MM 13902 or a ~alt thereof and this material may bhen be re-chromatographed to yield a material which contains, for example, 90-100% of MM 13902 or a salt thereof.
r, For the purpose of this specification the term "MM 4550 (Complex)" is used to describe the material originally designated as MM 4550 in ~ritish Patent No. 1,363,075.
.l MM 4550 (Complex) as produced on repetition of the Examples of British Patent No. 1,363,075 is an impure material which , contains in various proportions salts of MM 4550 (as described hereinafter) and salts of MM 13902 and considerable . quantities of other materials. MM 4550 (Complex) does not : have the characteristic ultra-violet spectrum of MM 13902.
The I50(as described hereinafter) of MM 4550 (Complex) :
produced by repetition of the Examples of British Patent No.
1,3631075 is not normally below 0.0004 ug/ml. The ratio of salts of MM 4550 and salts of MM 13902 present in MM 4550 ~ (Complex) i8 believed to be highly variable and is thought 91 to d~p*n~ on such factors as the strain of organism ~sed :
I and/or the exact isolation techniques used but it has been i generally found that the complex contains more MM 4550 then - MM 13902. The preparation of MM 4550 (Complex) is described ~`
hereinafter in the section relating to "Descriptions".

''~ ' 11 t. --... , - . - -. . .~ ............................ . .
, . , . -.~; . . ; . . ~ . : , ~0~0~60 For the purpose of this specification the term "MM 4550" i5 used to describe a ~ubstantially pure compound which is a potent B-lactamase inhibitor and which also possesses a certain degree of antibacterial activity. The properties of MM 4550 are described hereinafteri~ the section relating to "Descrip$ions".

In an alternative view, this invention provides a process for the preparation of MM 13902 and its salts which process comprises the cultivation of a MM 13902 producing strain of Streptomyces and thereafter recovering the MM 13902 or salt thereof from the culture.

For this Process we have found that MM 13902 producing strains are strains of Streptomyces olivaceus and related organisms as described hereinafter.

Most suitably the organism used is a strain of Streptomyces olivaceus suoh as ATCC 21379, 21380, 21381, 21382~ 31126 or mutants thereof.

A particularly preferred organism for use in this process is Streptomyce~ olivaceus ATCC 31126.

When~r~ herein, the term "cultivation" means the deliberate aerobic growth of an organism in the presence of assimilable sources of carbon, nitrogen, sulphur and mineral sa lts.
Such aerobic growth may take place in a solid or semi-solid ( ..

~' ~

iO50460 nutritive medium, or in a liquid medium in which the nutrients are dissolved or suspended. The cultivation may take place on an aerobic surface or by submerged culture. The nutritive medium may be composed of complex nutrients or may be chemically defined. In our hands we have found media containing complex nutrients such as yeast extract, soya bean flour and the like to be particularly suitable. We have also ; found that the addition of cobalt ions, sulphate ions and calcium carbonate to be beneficial.

We have found that cultivation at a temperature of 28 ~2C
gives acceptable yields of antibiotic and that a good time to harvest the broth is 2-3 days after the initiation of fermentation.
.~ :

When used herein the term "mutant" includes any mutant strain whioh arises spontaneously or through the effect of an external agent whether that agent is applied deliberately or ~, otherwise. Suitable methods of producing mutant ~trains include those outlined by H.I. Adler in Techniques for the Development of Micro-organisms in "Radiation and Radioisotope~
for Industrial Micro-organisms", Proceedings of a Symposium, ~ Vienna, 1972, page 241, International Atomic Energy Authority i and these include:

i. Ionising radiation (such as X- and ~- rays), uv light, ~' uv light plus a photosensitizing agent (such as -! 8-methoxypsoralen~ nitrous acid, hydroxylamine, ~ pyrimidine base analogues (such as 5-bromouracil), .

., :. ~

~ os~o acridines, alkylating agents (such as mustard gas, ethyl-methane sulphonate), hydrogen peroxide, phenols, formaldehyde, heat, and ii. Genetic techniques such as recombination, transformation, transduction, lysogenisation, lysogenic conversion and selective techniques for spontaneous mu-tants.

We have found that use ofa mutation promoting agent can lead to the production of organisms which have the ability to produce enhanced qunatities of the desired antibiotics.
For example, irradiation of cultures of Streptomyces olivaceus ATCC 21379 followed by isolation of the resulting strain which appeared to produce the largest zone of activity on the KAG
assay as hereinafter described lead to the isolation of Streptom~ces olivaceus ATCC 31126 which is a preferred strain for use in this invention. ATCC 31126 has also been depo~ited in the Netherlands as CBS 155.75.
In general, all isolation and purification procedures used in obtaining the desired anti~iotic should take place at non-elevated temperatures, for example, below 20C and more ~uitably not above 12C.

~he desired product is normally obtained predominantely from the culture filtrate so that the preferred initial step in the isolation process is the removal of solid material from the fermentation, for example by filtration.

An impure preparation of MM 13902 or a slat thereof may be .

;

obtained from the clariied culture filtrate by absorbing the MM 13902 or a salt thereof onto an active material such as active carbon or the like and thereafter eluting the desired substance from the active material using a solvent such as aqueous acetone. Normally this process is carried out on a di-basic salt of MM 13902.

Alternatively an impure preparation of MM 1390~ or a salt thereof may be obtained from the culture filtrate by extraction using a lipophilic ammonium salt and a water immiscible solvent.
This is frequently more effective than the process described above. (The NM 13902 may be obtained as the substituted ammonium salt by evaporation of the organic solvent under reduced pressure.) Preferably the initial solution of the MM 13902 substituted ammonium salt is then back extracted into an aqueous phase by using '~ a solution of an alkali metal iodide such as sodium iodide.
T~is last process variant generally leads to preparation of an aqueous solution of an impure di-basic salt of MM 13902.
The impure forms of MM 13902 or its salts as described above are normally subjected to the chromatographic procedures hereinbefore described in order to produce material of , acceptable purity.
The following Descriptions elucidate general information useful in the isolation of antibacterial compounds. The ¦ following Examples are illustrative of aspects of the invention.
1~
. ~ .

~ b 1:

; '. , ~ ! . . . , ' .. .. .' . '' ' , ~

' , ' ' ' , " ' .'~ ' ' ' ', .. . . .
' ' ' ~ ' ' " ;' ' . ' ' ~050~6(~

Descr iption 1 I50 Determination The I50 value is the amount of material required to give 50% inhibition of hydrolysis of ampicillin by the ~-lactamase enzyme of Escherichia coli B11, an orqanism containing an R
factor controlled ~-lactamase. This ~-lactamase is classified as a type IIIa enzyme according to the classification of Richmond and Sykes ~Adv. in Microbiol. Physiol. 9 31 (1973)]
The rate of ~-lactamase hydrolysis of ampicillin to its penicilloic acid can be followed by a starch-iodometric assay in which one measures the rate of formation of penicilloic acid by following the decolorization of a starch-iodine complex.
This method and the preparation of the ~-lactamase are described in a paper by Cole M., Elson S., and Fullbrook P.D.
(Biochemical Journal 1972, 127, 295-308). A slight modification of the above method was used in that the sample of inhibitor was preincubated with the enzyme for 15 minutes at 37C prior to adding the substrate ampicillin. The procedure was as follows:-~ :, '~
~ -- 16 - ~
.

~050460 Reagents - `
suffer: 0.05M pH7 potassium phosphate buf~er Starch/iodine Prepared as described by Novick, solution: siochemical J. (1962) 83, 236 Substrate: Ampicillin 40 ~g/ml in buffer -lactamase Enzyme: Prepared from E.coli B11 as described by Cole et al, siochem. J (1972) 127,295 The dilution of the enzyme preparation in buffer was such as to give a fall of about 0.3 optical density units per 100 secs. in the uninhibited reaction. Other ~-lacta-mase producing strains of E.coli may be used, in particular those carrying an R
factor for example R TEM.
Conditions -The reactions were carried out in 1 cm cuvettes at 37C in an SP 800 Pye Unicam spectrophotometer. This instrument can carry four sample -, cuvettes and their corresponding blanks. The first cuvette was , 20 used for the control reaction and contained no inhibitor. The 2nd, , 3rd and 4th cuvettes were used for various dilutions of the inhibi~
, tor.
Thus:
Sample Blank Reagents Cuvette Cuvette Starch/iodine reagent 1.0 ml 1.0 ml E.coli ~-lactamase 0.1 ml Buffer 0.3 ml 0.4 ml Inhibitor of buffer 0.1 ml 0.1 ml 30 Substrate (added after incubation1.0 ml 1.0 ml of above mixtures for 15 mins.
at 37C) ~i~ The reactions were followed by recording optical density change at 1~ 590 nm. and measuring the velocity of the reaction as optical . . .
., ~ ~ - 17 -:;j ~ ,.

.'~:, : " ,,' - ' ' . -. : '~ '' ,", ' ,; ' ; : ' ' " .

densîty change per 100 secs. during the 3 - 6 minute time interval.
The inhibitor sample was diluted until a dilution was reached which gave 50% of the rate of reaction seen in the no-inhibitor control.

. . :

j - 17a -~ ~ .
` ~ .,:
,~ ~

', " ' , . ' " ~ ': . " ' ' .' ' '' ': ' , ., ' ' ' , . ' . .. ' , . "' ': ', ' ' ' ' ' ' , ': , . ' '. ' .' ' : . - . . . ' ~OSQ4~;0 Description 2 The KAG As s ay me KAG assay is a method for determining the presence of a ~-lactamase inhibitor in fermentation broths or during stages in the isolation. Molten nutrient agar at 45C is seeded with a suitable ~-lactamase producing strain of Klebsiella aerogenes and then mixed with a sufficient quantity of a sterile solution of penicillin G to give a concentration of 6 ~g/ml of penicillin G in the agar. The agar is then poured into a petri dish and after solid-ification equally spaced cylindrical wells are made in the layer of agar by using a sterile metal cutter. The solutions to be tested are introduced into the wells. The dish is then incubated at a constant temperature between 27C and 37C. During the period of incubation, any inhibitor in the test solution diffuses out from the well into the agar and there inhibits the action of ~-lactamase produced by the Klebsiella cells. The penicillin G is thus protected from destruction by ~-lactamase and is present in sufficient concentration to prevent the growth of the Klebsiella. In those parts of the agar to which the inhibitor has not diffused in sufficient concentration, the penicillin G is destroyed by the ~-lactamase, allowing dense growth of the Klebsiella to develop. Clear circular zones of inhibition of Klebsiella growth are thus formed around the wells containing inhibitor the size of each zone depending on the concentration of inhibitor in the solution under test. The potency (arbitrary units/ml) of test solutions is obtained by reference to a standard line of log.conc. inhibitor against zone diameter. This assay system may also be used for bioautography.
`', ' ' .
~' .
., ., .

~050460 n6~scription 3 Preearation of MM 4550 (Complex) comparable to that disclosed in British Patent No. 1,363,075 Streptomyces olivaceus ATCC 2137~, was grown for 7 days at 28C
on a solid agar slant in a Roux bottle. The agar medium had the following composition:-Constituent Amount (g/l) Yeast Extract 10.0 Glucose Monohydrate 10.0 Agar 15.0 Tap Water to1 1 (The "Yeast extract" was "Yeatex"~; as supplied by Bovril FoodIngredients, P.O. Box 18, Wellington Road, Burton-on-Trent, U.K., and the "Agar" was supplied by Oxoid Limited, Southwark Bridge Road, London, S.E.l., ~.K.) The medium was adjusted to pH 6.8 before sterilisation. 50 ml.
of sterile deionised water containing 0.02% Tween 80 (Registered Trade Nark: Tween 80 is a polyoxyethylene sorbitan mono-oleate) was added to a Roux bottle culture and the spores suspended by shaking. This spore suspension was then added as inoculum to 75 1 of sterilised seed stage medium in a 100 1 stainless steel i fermenter. The composition of the seed stage medium was as follows:-*Trade Mark .
.~, , ~:

:i . ~ .. .. . . . :- . : , . . .. ..
, . .
~ , .: , 105~)460 Consti-tuent Am_unt (g/l) Soya-bean Flour lO.0 Glucose Monohydrate 20.0 Tap Water to 1 1 (The "Soya-bean Flour" was Arkasoy* 50 as supplied by the British Arkady Co.Ltd., Old Trafford, Manchester).

To control foaming 50 ml. of 10% v/v Pluronic L81 (Registered Trade Mark) in soya-bean oil was added to the fermentation medium before sterilisation. (Pluronic L81 was supplied by Jacobsen van den Berg U.K. Ltd., 231 The Vale, London, W.3.~ U.K.~ and is a block polymer of ethylene oxide and propylene oxide).
The medium was steam sterilised in the fermenter for 20 mins at 120C. The seed stage culture was stirred at 340 r.p.m. with a 8.5 inch diameter vaned disc agitator and supplied with 150 l/min sterile air through an open ended sparger. The culture vessel was fitted with baffles. The temperature was controlled at 28C and after incubation under these conditions for 45 hours, 7.5 l of this seed culture was added as inoculum to 150 l sterile fermentation medium in a 300 l stainless steel fermenter. The fermentation medium had the following compositions:-Constituent Amount (g/l) Soya-bean Flour (Arkasoy lO.0 S O ) Glucose Monohydrate20.0 Chalk ~Precipitated Calcium Carbonate) 0.2 ' Cobalt Chloride (CoCl2,6H20) 0.001 Tap Water to l l *Trade Mark ( ,.' .

" ,' , . " . ' ' 300 mi. of 10% Pluronic L81 in soya-bean oil was added to prevent foaming. The fermentation was harvested after 48 hours and clari-fied by centrifugation. The clari~ied brew gave 50% inhibition in the enzyme assay at a dilution of 1 in 100,000. 100 1 of the clari-fied brew was stirred with 12 kg. wet weight of Whatman DE32 (Registered Trade Mark) ion exchange cellulose in the acetate form, (as supplied by H. Reeve Angel & Co., 14 New Bridge Street, London E.~.4, U.K; the material is a microerystaline cellulose substituted by diethylaminoethyl groups).
The slurry was filtered and the MN 4550 (Complex) was eluted from the cellulose with 12 1 of 0.5M potassium sulphate. The extract was concentrated to 6 1 in a climbing film evaporator under vacuum and below 30C. Much of the potassium sulphate was precipitated by the addition of 12 1 of acetone. The solution was filtered and concentrated to 200 ml. by evaporation under vacuum below 30C.
The concentrate was loaded onto a 76 mm x 2 m column of Amberlite XAD-4 resin (Registered Trade Mark) (as manufactured by Rhom &
Hass Co., Philadelphia, U.S.A.; the material is a non-ionic polystyrene resin), eluted with deionised water and the elutate was collected in 140 ml. fractions. Active fractions, as detected by the KAG assay were bulked (2.2 1) and concentrated to 275 ml.
by ultrafiltration using an Amicon UM-05 membrane (150 mm diameter) (Registered Trade Mark) (as supplied by Amicon Ltd., 57 Queen's Road, High Wycombe) under nitrogen pressure of 60 p.s.i. The concentrate was freeze dried to yield 2.2g. of brown powder (I50 0.004~ug/ml.).

: ~ .
. i ,.
!, - 21 -,.~ ~ .. -.

,. .. - . . . . .

.. : ,. ~ ~ . :.~ . - . . , , -: , . : -. .. , ... . : . . . .: .

~o5Q460 1 g. of the powder was dissolved in 1 l of 0.2M sodium sulphate and mixed with l l of 2~ w/v tetra-n-butylammonium hydrogen sulphate (as supplied by AB Astra, 5Odertalje, Sweden) in dichloro~ethane.
The dichloromethane phase was separated by gravity, cooled to -70~C, filtered to remove ice and concentrated by evaporation to 20 ml.
400 ml. of 40-60C petroleum spirit was added to the concentrate and the precipitate collected by centrifugation. The precipitate was redissolved in dichloromethane (tO ml.) and extracted with water (10 ml.) containing barium iodide (80 mg.) and barium carbonate (70 mg.). The phases were separated, the aqueous phase filtered and adjusted to pH 6.5. The solution was freeze dried to yield a yellow powder. The solid was washed with acetone to dissolve out excess barium iodide and the pale yellow solid recovered by centrifugation and dried in vacuo. The yield was 13 mg.

15 (I50 0-0004 ~g/ml-)-, J

', , . .

~. ~ . , .

~.

.

~050460 Descrlption 4 Demonstration of Carbon Adsorption of Culture Filtrate useful in obtaining material as described in British Patent No. 1,361,075 Culture filtrate obtained as in Description 3 gave a zone diameter of 17 mm in a hole-in-plate agar diffusion antibacterial assay against Klebsiella ~ , a æone diameter of 38mm in the KAG assay and I50 at a dilution of 1 in 100,000. The clarified culture filtrate (170 1) at 5C was percolated by upward flow through a 9" diameter column packed to a height of 16" with active charcoal (Farnell*B0, as supplied by Dearborn Chemicals Ltd., Dilton, Widnes, Lancs., 60-80 mesh pre-washed with N HCl and buffered at pH 6 with phosphate) at a flow rate of 800-1000 ml/min.
Brew was washed from the carbon with deionised water (10 1) and the column eluted with 20% acetone at 20 C. The active fractions amounting to 10 1 were concentrated by evaporation in vacuo, below 30C to 6 1 and freeze dried to yield 282 g. of crude MM 4550 (Complex) preparation giving an I50 of 0.05/ug/ml. The recovery of enzyme inhibitory activity was 22~.

.
An alternative activated charcoal which gives similar results is Darcogranular car~on (as supplied by Honeywill-Atlas Ltd., Mill Lane, Carshalton, Surrey).
,,.
*Trade Mark ., .

i . .. : . .. ~, , . : ., ,,: : :.. ... . . ..
,, . , . ' ' ~ ! ' ' ' ' . ' ' ' ' ' ' ' ; ' ~ ' lOS046() Description 5 Demonstration of Ion Exchange Resin Chromatography of Culture Filtrate useful in obtaining material as described in British Patent No.1~363,075 A 1 cm. internal diameter column was packed to a height of 6 cm.
(bed volume 4.7 ml.) with Dowex 21K ion-exchange resin (20-50 U.S.) mesh, chloride form)(Dowex~:21K was supplied by B.D.H. Chemicals Ltd., Poole, Dorset, ~.K. and is a polystyrene-divinylbenzene resin containing basic groups). The bed of resin was then washed with approximately 4 x 4.7 ml. of 5% aqueous methanol, followed by 1 x 4.7 ml. of distilled water. Two litres of culture filtrate was obtained essentially as described in Description 3 and applied to the column. The resin was then washed with 50% aqueous methanol to remove impurities.
- The MM 4550 (Complex) was eluted from the resin with 5% NaCl in 50%
aqueous methanol. Table 1 below shows the results obtained in terms of units of activity. The MM 4550 (Complex) content of culture filtrate was arbitrarily set at 8 units/ml. The eluted fractions from the column were assayed using an antibacterial diffusion method against Klebsiella aerogenes and the activity units were calculated by reference to a standard line prepared by plotting ~one diameter ~ ,~
against units/ml. for various dilutions of culture filtrate (i.e.
the culture filtrate was used as a standard).
It can be seen that the column was an effective way of concentrating - the MM 4550 (Complex). Fractions 2 - 5 inclusive contained 60% of the activity in a total volume of only 37 ml. The total dry weight ,. . . .

;.. . - ~ . , : . :

,. : . .

of these fractions was about 210 mg. whereas 35 g. of solids had been added to the column.

j .
.

^! ~

ii~
~ *Trade Mark ~ 24a -.~'.~ : : : ' ' .
' ,~'t,~
!~.. ~ .... ~; ... .. ... -... .- . . . ... . . . .. - .

T A B L E

.: :

Sample pHVolume Units/mlTotal Units .~ _ Culture filtrate 6.5 1970 15760 Percolate 16.9550 C1 ~100 Percolate 27.0525 C1 ~100 Percolate 37.0595 C1 ~100 Percolate 47.0300 L~ C100 Eluate 1 7.8 7.0 R4 588 Eluate 2 7.8 7.0 328 2296 Eluate 3 7.7 8.5 440 3740 Eluate 4 7.7 10.0 320 2200 Eluate 5 7.6 11.5 160 1840 Eluaté 6 7.5 11.0 80 880 Eluate 7 7.6 14.2 66 937 Eluate 8 7.6 20.0 33 660 Total:13141 __ _ _ . ~

:1 0: ~- ` `

.i: - 25 -~`

~os~o Description 6 Demonstration of Ion-Pair Extraction of Culture Filtrate useful in obtaining material as described in British Patent No. 1,363,075 Sodium sulphate (248 g.) was added to clarified culture filtrate (10 l) prepared as described in Description 3 and the solution extracted with 2~ tetra n-butyl-ammonium hydrogen sulphate in dichloromethane (10 l) by stirring for 30 minutes. The phases were separated and the dichloromethane phase cooled to 2C. A
small quantity of suspended water was removed by filtration through Whatman No. 1 PS paper. The dichloromethane solution was con-centrated to 20 ml. by evaporation ln vacuo below 30C. Theaddition of 400 ml. of petroleum ether (40 - 60) precipated a gum which contained the MM 4550 (Complex).

The gum was collected by centrifugation, redissolved in 50 ml. of dichloromethane and extracted with 50 ml. of water containing 1 9.
barium iodide and 1 g. barium carbonate by shaking for 2 minutes.
The solids present were filtered off and the two phases separated.
The aqueous phase containing the MM 4550 (Complex) as the barium ~` salt was adjusted to pH 6.5 and freeze dried to yield 317 mg. of a crude preparation of MM 4550 (Complex) with an I50 f 0.001 ~g/ml.

... .
.~ , .

~ . : - -.

., ~.
.' ., . ' ; .

, . . . . :

105~0 Description 7 Preparation of Partially Purified Antibiotic Complex containing MM 4550 and MM 13902 using Tetra n-butyl Ammonium Hydrogen Sulphate A crude preparation of MM 4550 (Complex) prepared as in Description 4 was redissolved in water (12.5 g. in 125 ml.) and extracted with 125 ml. of 10~ w/v tetra n-butyl ammonium hydrogen sulphate in dichloromethane. The two phases were separated and the dichloro-methane back extracted with 100 ml. water at 2C containing 190 mg.
sodium iodide by stirring slowly and adjusting the aqueous phase to p8 6.4 with 2% NaHCO3. The solution was freeze dried and the dry solid washed with acetone to yield 88 mg. of a preparation mixed sodium salts of MM 4550 and MM 13902 having an I50 f 0.0002 ~9 against Escherichia coli ~-lactamase. The recover of antibiotics was 70~.

The antibacterial activity of mixtures of ampicillin and material prepared as described in Description 7 was determined by the serial dilution method in nutrient agar. The minimum inhibitory concentrations which weee obtained are ~iven in T~ble ~

' ..

1~504~0 T A_B L E 2 .... ____ . :- .' Ampicillin + MM 4550 (Complex) at Ampicillin _ _ O r g a n i s mAlone _ _ 0.1 ~g/ml 1 ~g/ml10 ~g/ml . . . _ . _ E. coli B11 >500 ~ 500 500 50 E. coli JT417 250 250 250 50 E. coli JT39 > 500 >500 500 25 *
Shigelia sonnei S239 125 125 125 25 *
Klebsiella aerogenes A 125 25 5 * ~ 0.1 *
_ Klebsiella aerogenes -IP282 50 50 12.5 0.5 *
Proteus mirabilis 889 ~ 500 >500 50 0.5 Proteus mirabilis 247 >500 125 5.0 0.5 _ ` Proteus morganii F50 50 25 0.5 Proteus rettgeri R110 250 125 25 * 0.25 *
, ,. ~ , Staph.aur _s tRussell~ 250 125 C0.1 0.25 Staph.aureus ~Russell H) >500 ~0.1 ~0.1 ~ 0.1 ., . . , _. .. _ ' '' * Partial inhibition by MM 4550 (Complex) alone at these concentrations.
Similar synergistic effects were observed for mixtures of A~oxycillin and MM 4550 (Co;plex).

' :~
'~
.

:: ' , ' ' ~:'; ' '' `~' '' ', ., lOS()~tiV
Description 8 Preparation of Partially Purified Antibiotic Complex containing MM 4550 and MM 13902 using cetyldimethylbenzylammonium chloride Freeze dried product (I50 = 0.02 ~g/ml) from a Farnell carbon column eluted with acetone/water as in Description 4 was dissolved in distilled water at a concentration of 13 mg/ml. The solution was adjusted to pH 6.5.

;

100 ml. of the solution was extracted with an equal volume of 0.1 cetyldimethylbenzylammonium chloride in dichloromethane. The phases were separated by centrifugation and the organic phase back extracted with 100 ml. of 0.05~ sodium iodide solution. The phases were separated and any dichloromethane in the aqueous phase removed by maintaining the solution under reduced pressure for 10 minutes.

The aqueous solution was freeze dried and the product of freeze drying washed three times with 50 ml. portions of acetone. The acetone washed product was dried in a vacuum desiccator to yield a light brown powder (4.3 mg I50 = 0.00~ ~g/ml~.
;

'-~, ', .. .

:, :

.. . . .. ..

l(~S~?460 Deseription 9 Preparation of Partially Purified Antibiotic Complex contain~ng MM 4550 and MM 13902 using Gel Filtration .

1 g. of crude MM 4550 (Complex), I50 0.2 ~g/ml, prepared by the method of Description 4 was chromatographed on Sephadex G25 (fine grade), using acetone/water 4:6 v/v as eluant. The eolumn dimensions were 2.5em x 32 em and elution was carried out at 1.5 ml/min. The aetive fraetions were combined, eoneentrated ln vaeuo below 30C and freeze dried to yield 22 mg. amorphous buff eoloured solid having an I50 of 0.005 ~g/ml. The reeovery was 88% and purification 40-fold.

' ., :

1()50460 Description 10 Preparation of Purified Antibiotic Complex containing MM 4550 and MM 13902 using Cellulose Chromatography MM 4550 (Complex) (610 mg.) prepared as in Procedure 7 was chromatogr~phed on a 2.5cm. x 32cm. column of cellulose (Whatman~
CC 31) and eluted with a mixture of isopropanol/water, 7/3 (v/v) at 1.5 ml/min. The antibacterially active fractions were combined, concentrated in vacuo, below 30C and freeze dried to yield 40 mg. of brown amorphous solid preparation of antibiotic complex I50 of 0.00007~g/ml. The very small value of the I50 indicates that the active material is of improved purity.

On a different occasion the above procedure yielded a materlal having an I50 of 0.001 ~-g/ml. This material had the antibacterial activity shown in Table 3 when determined by a standard microtitre method in Oxoid sensitivity broth using light inocula tl% of overnight broth).

~tTrade Mark ', " , , ,: -. , . : .
, . , , , . ; .
,: : , . : :
.
~ ' : : -:

~)S0~60 ANTIBACTERIAL ACTIVITY OF MIXTURE OF MM 4550 and MM 13902 HAVING AN I5~ OF 0.001 ~g/ml _ . Organism MIC
. . .___ . . -., Staphylococcus aureus (Oxford) 7.5 Staphylococcus aureus (Russell) 7.5 Streptococcus faecalis 125 Bacillus subtilis 0.9 Escherichia coli (10418 3.7 ~ :
Escherichia coli (B11) 15 Klebsiella aerogenes 1.8 Enterobacter cloacae 62 .
.
Proteus mirabilis 7.5 . .
Providentia stuartii 7 5 ,, . ' ~:
Aclnetobacter anitratus 0.9 ~:
Pseudomonas aeruginosa 250 Serratia marcescens 7.5 Salmonella typhimurium 15 Shigella sonnei 7.5 .. j _ . .
: ~,', '' - ' . .

. ~ :

: . ::' ' ' .. : . . ' - ; . ,.... : ,.

~050460 Description 11 MM 4550 may be recognised by its properties which are as follows:
MM 4550 is an acidic solid which in the form of a sodium salt has the following characteristics:-; 5 i. It is highly soluble in water, soluble in methanol and substantially insoluble in chloroform, diethylether and hydrocarbons.
ii. In aqueous solution, it has a characteristic ultra-violet spectrum with absorption maxima at about 238 nm. and at about 287 nm.
iii. When present at 0.4% w/w in a freshly prepared KBr disc, it has a characteristic infra-red spectrum which has absorption maxima at inter alia about 3450, 2950, 1765, 1695, 1510, 1390 and 1260 cm 1. If a further spectrum is taken about one week after the preparation of the KBr. disc, the spectrum shows considerable changes, for example, the large peak previously at about 1765 cm-1 is considerably reduced in size or is absent.
iv. It has a characteristic n.m.r. spectrum when taken in D20 which spectrum posses inter alia (a) a pair of low field doublets centered approximately at 2.45~ and 3.65 with coupling constants of approximately 15 Hz;
(b) a doublet centered at approximately 8.55t and .:
(c~ a sharp singlet at approximately 7.95~, :
. :
, _ 33 -~504~iO

v. When run over cellulose in a thin layer chromatography system, it has the following very approximate RE values:
(a) butanol/isopropanol/water - 7:7:6 v/v; Rf = 0~6 (b) isopropanol/water - 7:3 v/v; Rf = 0.7 (c) n-butanol/ethanol/water - 7:7:6 v/v; Rf = 0.7 (d~ n-propanol/water 4:1 v/v; Rf = 0.6 vi. It possesses antibacterial activity against various species including inter alia, strains of Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Klebsiella aerogenes, Proteus mirabills, Acinetobacter anitratus, Serratia marcescens and Shigella sonnei.
.:
vii. It possesses enzyme inhibitory activity against the 0-lactamases produced by various species including, inter alia, Escherichia coli, Klebsiella aerogenes and Staphylococcus aureus. It has an I50 (as herein-before defined) of less than 0.0001 ~g/ml. against the ~-lactamase of Escherichia coli B11.
viii. When mixed with ampicillin, it synergyses its activity against organisms including strains of Escherichia coli, Rlebsiella aerogenes, Proteus mirabilis, Proteus morganii and Staphylocoac=s aureas Rasse11.

:

~ - 34 -1(~50460 :

ix. Amino acid analysis indicates that the material is not a poly peptide or protein. No ~-aminoadipic acid is found after acid hydrolysis.

x. It reacts with Ehrlich's reagent (300 mg. of 4-dimethyl-aminobenzaldehyde dissolved in a mixture of 54 ml.
n-butanol, 9 ml. ethanol and 9 ml. concentrated hydrochloric acid~ to produce a blue colour on paper chromatograms and tlc sheets.

ix. It is not a general enzyme poison and does not inhibit the following enzymes at concentrations in excess of those required to inhibit the ~-lactamase of Escherichi coli; monoamine oxidase, carbonic anhydrase, dopa decarboxyla~e, trypsin, chytotryp~lin or urease.

': ~

( ~':

`: :

:'', :

.~ ..

. , .
',`'' 1~50460 Example 1.

Preparation of MM 4550 (Complex) and Separation into MM 4550 and The isolation procedures detailed in the Description section may be utilized in a variety of sequences. A particularly suitable sequence is carbon adsorption, ion pair extraction and cellulose chromatography as described below:

Culture filtrate (340 l) (obtained as in Description 3) was percolated by upward flow at 800 - 1200 ml/min. through a column (9" diameter x 21" high) packed with Farnell B0 carbon. The carbon had been used before for adsorption of MM 4550 (Complex) and was regenerated by ~ washing with the following reagents:-,, 0.5N NaOH; 0.2N NaOH/acetone (3:2); water; N HCl;
water; phosphate buffer pH 6; water.

The column was washed with water (20 l) to displace the culture filtrate and eluted by downward flow with acetone/water 2:8 v/v ' ; at 200 - 250 ml/min. One litre fractions were collected.
Fractions containing the MM 4550 (Complex) as determined by an antibacterial diffusion assay using Klebsiella aerogenes were combined l11 1) and concentrated by evaporation in vacuo below 30C to 5.5 1. The recovery of MM 4550 (Complex) at this staye was 20~.
, ~" ~

:' :

.

The concentrate was cooled to 2C, 5.5 l of 2% w/v tetra n-butyl-ammonium hydrogen sulphate in dichloromethane at -5C was added and the two phases mixed together for 2 minutes. The dichloromethane phase was separated and added to one litre of sodium iodide solution (0.6%) at 0C. The two phases were stirred gently and the aqueous phase gradually adjusted to between pH 6.5 - 7.0 with 2% aqueous solution of NaHCO3. The aqueous phase was separated and freeze dried. The dried solid was extracted with dry acetone to dissolve ; out excess sodium iodide and the acetone removed from the insoluble residue in vacuo, to yield a buff coloured powder (1.1 g.). Overall recovery of MM 4550 ~Complex) at this stage was 10%. The purification calculated on total dissolved solids in the culture filtrate was i 125-fold.
A 2.5cm. diameter column was packed with cellulose powder (Whatman CC 31) in isopropanol/water, ~:3 v/v, to a height of 32cm. 500 mg-` of the buff powder was dissolved in 2ml. isopropanol/water 7:3 v/v and chromatographed using the same solvent at a flow rate o 1.5 ml/min.
Fractions (3 ml.) were collected from the column and those showing W
.
absorption maxima at about 285 nm. were combined, concentrated and freeze dried to yield MM 4550 (Complex). Previous experiments have ~ -;:j . : , established that fractions absorbing at about 285 nm. contained enzyme inhibiting, antibacterially active material.
~,~ ~ ,,,;;.,.: :' ~ The yield of freeze dried MM 4550 (Complex) was 35 mg. It had a brown ,~ amorphous appearance and an I50 of0.0001~g/ml. The recovery of active material at this stage was 3% overall and the purification ~,~ 600-fold overall.

i ~ ' ~ ~ 37 ~
'; .

The antibacterial activity of this preparation was determined by the standard Microtitre method of Oxoid sensitivity test broth using light inocula. The resulting minimum inhibitory concentra-tions were similar to those shown in Table 3 hereinbefore.

Analysis of the preparation by thin layer chromatography on cellu-lose (Eastman Kodak "Chromagram"* sheets) developed with n-butanol;
isopropanol:water t7:7:6) separated two active substances, one with an approximate Rfof 0.58 which was designated MM 4550 and one with an approximate Rf of 0.72 which was de~ignated MM 13902. Both these materials may be detected by bioautography on a variety of organisms including B. subtilis, Staphylococcus aureus (Oxford), Staphylococcus aureus (penicillin resistant), Escherichia coli (penicillin sensitive and resistant), Salmonella typhimurium~
Proteus mirabilis, Proteus mor~anii and Klebsiella aerogenes. Some .. ..
of the results obtained are summarised in Table 4.

In a further experiment, the two substances were separated on a cellulose thin layer developed with isopropanol:water (8:2) and extracted from the cellulose with phosphate buffer. Each extract was assayed for ~-lactamase inhibition and both substances were shown to be inhibitors of Escherichia coli ~-lactamase. Both substances were also shown to be synergistic with benzylpenicillin against Kiebsiella aerogenes.

*Trade Mark .

: , , , . : :
..

. : . . . - , . .

~:)50460 BIOAUTOGRAPHY (TLC WITH CELLULOSE AND BUTANOL/ISOPROPYL
ALCOHOL/WATER 7:7:6 v/v) . ., Zone Diameter on Bioautograph O r g a n i s m . .
MM 4550 zone in MM 13902 zone in mm at Rf 0.58 mm at Rf 0.70 ~:
. -Klebsiella aerogenes20.0 mm 20.0 mm Proteus mirabilis 17.5 mm 13.5 mm Proteus morganii 16.0 mm 9.S mm Salmonella typhi 19.5 mm 21.0 mm Escherichia coli 10418 20.0 mm 13.5 mm Escherichia coli B1117.5 mm 10.5 mm . ~ ,:
.; Enterobacter aerogenes 6.5 mm No Zone .. ~ .
. .
Staphylococcus aureus(Oxford)13.0 mm 4.0 mm l' Staphylococcus aureus(Russell)12.0 mm 4.0 mm :
: ac;llus subtilis 24.0 mm . _ ~:

~.i, ~' .
.. ~` ~ ....

1, .
1 ~
~ I ~

1~ : ,, , .`~ .- .
~ 39 _ 1:
. ~ ~.. .. - .

10504~;0 Example 2.
Preparation of MM 4550 (Complex) and Separation into MM 4550 and MM 13902 Culture filtrate (1100 l) from the fermentation of streptomyces olivaceus ATCC 31126 at pH 6.5 and 5C were percolated at 3.2 l/min.
through a column (0.3m x 1m) packed with Darco granular carbon.
[The carbon had already been used for adsorption of MM 4550 (Complex) and was regenerated before use by washing with the following reagents:-0.5N NaOH; 0.2N NaOH/acetone ~3:2); water; ~ HCl;
water; phosphate buffer, pH 6; water].
The column was washed with water (60 1) to displace the culture filtrate and eluted with acetone/water (2:8 v/v) at 30C at 1.1 l/min.
60 1 was collected followed by 5 x 10 l fractions. Those fractions containing the MM 4550 (Complex), as determined by the antibacterial diffusion assay using Klebsiella aerogenes were pooled (40 1) and concentrated in vacuo below 30C to 32 1. The recovery of MM 4550 (Complex) at this stage was 15%.

Ths concentrate was cooled to 5C, 16 1 of 0.2% cetyldimethyl-benzylammonium chloride in dichloromethane at 5C was added and the two phases mixed for 5 mins. The dichloromethane phase was separated added to 3.75 1 sodium iodide solution (0.4%) at 5C. The two phases were mixed for 5 mins., the aqueous phase was separated and freeze dried. The dry solid was extracted with dry acetone to dissolve out excess sodium iodide and the residual solid was dried in vacuo to yield a buff coloured powder ~2.87 g). Overall recovery , oP MM 4550 (Complex) at this stage was 6%. The purification calculated on total dissolved solids in the culture filtrate was 160-fold.
. , .

~0~04~) A 63 mm diameter column was packed with cellulose powder tWhatman CC 31) in isopropanol-water (7:3 v/v) to a height of 300 nm.
2.0 g. of the buff coloured powder was dissolved in 5 ml. isopropanol/
water (7:3 v/v) and chromatographed in the same solvent at 3 ml/min.
S Fractions containing MM 4550 (Complex), as determined by assay against Klebsiella aerogenes were pooled, concentrated by evaporation in vacuo below 30C and freeze dried to yield a brown solid (207 mg.).
The recovery of MM 4550 (Complex) for this stage was 51% and the purification was 5-fold.

A 16 mm. diameter column was packed with cellulose powder (~hatman CC 31) in n-propanol/water (4:1 v/v) to a height of 300 mm.
198 mg. of the brown solid was dissolved in water/n-propanol (1:1) and chromatographed in n-propanol/water (4:1 v/v) at a flow rate of 0.5 ml/min. Franctions (6 ml.) were collected, bioassayed against Klebsiella aerogenes and the uv spectrum of each fraction measured.
; Fractions 23 - 28, having a uv maximum at about 305 nm, contained the di-sodium salt of MM 13902. These fractions we~e pooled, con-centrated under vacuum and freeze dried to give 30 mg. of solid.
This preparation showed only one zone of antibiotic activity at Rf 0.77 on thin layer chromatography using Eastman Kodak cellulose plates with n-propanol/water (4:1 v/v). The zone was detected by bioautography on Bacillus subtilis. (Fractions 29 - 40, having a uv absorption maximum at about 285 nm, contained MM 4550. They were combined, concentrated under vacuum and freeze dried to give 53 mg. of solid which contained a salt of MM 4550 contaminated with a small quantity of a salt of MM 13902).

' ~"""'' ' .: . . .. .

~0504~0 Description 12 Organisms The property of producing MM ~550 ~Complex) was first discovered in the cultures ATCC 21379, ATCC 21380, ATCC 21381 and ATCC 21382 which had been isolated from soil samples obtained from Spain, New Zealand, South Africa and Israel respectively. In the laboratory, these cultures appeared identical and all were identified as Streptomyces olivaceus, by Dr. Bousfield, the actinomycete expert at the National Collection of Industrial Bacteria (NCIB), Torry Research Station, Aberdeen, Scotland, using ; the widely accepted 1967 classification of Hutter [Systematic der Streptomyceten, S. Karger, Basel, 382 pp]. MM 4550 production has since been found in other streptomyces species as may be seen from Table 4.
S. olivaceus was first described by Waksman in 1919 [Cultural Studies of Species of Actinomyces, Soil. Sci., 8,71 - 215].
Subsequently, in 1960, a group of Russian workers described a species with very similar characteristics but which they named Actinomycés (Streptomyces) fulvoviridis. [Kuchaeva et al., Trud. Inst. Mikrobiol., Akad Nauk. SSSR., 8, 226 - 253 (1960)].
Micro-organisms of the genus Streptomyces are extremely variable in their morphological and physiological characteristics depending on the conditions under which they are grown.
Descriptions of many species had been published before the existence of this extreme variability had been recognised, with consequent duplication and synonymy. H~tter (1967) lists 25 species as being synonymous with S. olivaceus including fulvov1ridis. To resolve the confusion is nomenclature and ., . , .

lOSq~460 classification, the Lnternational Streptomyces Pro~ect was begun in 1962, Shirling et al., Int.J.Syst.Bacteriol, 18, 69 - 189 (1968). Collaborators in this Project have carried out a series of studies aimed at producing accurate descriptions of species of Streptomyces under standard conditions. In this scheme, standard descriptions have been produced of type strains of some 400 named species including Streptomyces olivaceus and Streptomyces fulvoviridis.
The I.S.P. description of S. olivaceus and S. fulvoviridis differ in two characters only: (1) the form of the sporophore and (ii) ability to utilise inositol as sole carbon source.
S. olivaceus is described as follows:
Spore chain morphology: Section Spirales, with open spirals intergrading through flexuous spore chains suggestive of Section Rectiflexibiles. Mature spore chains generally long, often ~ith more than 50 spores per chain.
Carbon utilisation: D-Glucose, L-arabinose, D-xylose~
I i-inositol, D-mannitol, D-fructose and rhamnose are utilised for - growth. No growth or only trace of growth on sucrose and raffinose.
S. fulvoviridis is described as follows:-Spore chain morphology: Section Rectiflexibiles. Mature spore chains moderately short with 10-50 spores per chain. Carbon utilisation: D-Glucose, L-arabinose, D-Xylose, D-mannitol, D-fructose and rhamnose are utilised for growth; utilisation of sucrose is doubtful. No growth or only trace of growth on i-inositol or raffinose.

.

" , ,~ '.

. ~':, Characterisation of a number of cultures named, as, or synony-mous with S. olivaceus or S. fulvovirldis and other related species was determined using the standard methods and media (Shirling et al., Int. J. Syst. Bacteriol. 16, 313 - 340, t1966) reco~mended in the International Streptomyces Project (ISP).
The cultures were derived from different sources. ATCC 21379, 21380, 21381, 21382 were isolated from soil on the basis of pro-ducing MM 4550 (Complex). The other strains were obtained from various culture collections for comparison purposes.
Results of the tests for production of MM 4550 (Complex), colour of aerial mycelium, sporophore shape, colour of substrate myselium, soluble pigment production, melanin production and carbon source utilisation are given in Tables 4 - 8. For all strains the spore surface, as seen by electron microscopy, is smooth.
From the ISP description it is clear that the spiral growth of the sporophore in S. olivaceus is a variable character. True spirals were observed with a variable frequency in the t~rpe species of S. olivaceus, i.e. ATCC 3335. The majority of the , sporophores were long. No true spirals were observed from the cultures ATCC 21379, 21380, 21381 or 21382 although the sporophores were long and showed a tendency to spiral among a background of Rectiflexibiles types. However, in two S. olivaceus strains, NCIB 8238 and NCIB 8509, the majority of sporophores were of the Rectiflexibiles type. In NCIB 8238 they were of medium length while tho~e of NCIB 8509 were long.
The sporophores observed from ATCC 15863, the type species of S. fulvoviridis, were shorter than those seen from isolates :, :

' .:',,.' -': :::' : ., . ' . :.- . . , ~ .

~0S0460 ATCC 21379, 21380, 21381, 21382 and 31126 and were only of the Rectiflexibiles types. In respect of this character therefore, the isolates ATCC 21379, 21380, 21381, 21382 and 31126 correspond fairly well but not exactly with the S. olivaceus description.
Isolates ATCC 21379, 21380, 21381, 21382 and 31126 show some variability with regard to inositol utilisation which is the other differentîating character according to the I.S.P. type species descriptions between the species S. olivaceus and S. fulvoviridis (Table 8). ATCC 31126 and ATCC 21380 utilise _ _ inositol, which is characteristic of S. olivaceus, while the other strains show doubtful or negative utilisation. However, it is not generally considered satisfactory to separate off a species on the basis of the ability to utilise a single carbohydrate. Such a difference might arise by a single gene mutation and is often considered to be of strain significance only. The difference in sugar utilisatioD by the S. olivaceus strains NCIB 8138, NCIB 8509, ATCC 21549, ATCC 12019 and ATCC 3335 suggest that many authorities do not consider them of species signifioance. Thus ATCC 21379, 21380, 21381, 21382 and 31126 are properly designated S. oliva_eus.
Unless further studies uncover more important differences between those cultures presently named S. olivaceus and S. fulvoviridis, it is possible that they will eventually be inter-nationally recognised as a single species. In this case the correct name, by the priority rule, will be S. olivaceus, and this will also include those cultures listed by Hutter as synonymous with S. olivaceus.
. . .

' '.

;l~ - 45 -1 , i, .:

., ';.'. ~ ' ' " . ' ' ' ' ':; '' ', .. ' ,'''' ' .' .' ~" ' ' " ',. , ' ' ', ' , ' ; ', ' ,- , ' ' ` ' ' ' ~ '. ' ' ', ' " ', " ' ~ '' . . ' '; ; ,. ' " ~. : . ' ' :

~0504~0 Examination of culture filtrates of the strains of S. olivaceus and related species for the production of MM 4550 (Complex) may be carried out as follows:-Culture filtrates of the listed strains were spotted onWhatman No.l paper strips at 20~u 1 per origin and chromatographed in n-butanol/isopropanol/water (7:7:6) overnight in the cold.
Another set of strips were run in n-butanol/glacial acetic acid/
water (1~,3,5) also in the cold overnight. A partially purified sample of MM 4550 (Complex) was also run in the two systems at the same time as a market.
Alternatively, it is possible to extract 25 ml of clarified brew with 12.5 ml of 0.2% cetylbenzylammonium chloride in dichloromethane, separate the phases, retain the organic phase, add 2.5 ml of sodium iodide solution (0.5%), shake, separate the phases, retain the aqueous phase and use this chromatographically, for example, by spotting 5~ onto thin layer chromotography strips and run in n-butanol/isopropanol/water, 7:7:6.
Streptomyces olivaceus and related organisms have the follow-ing prime characteristics:
(a) They possesssporulating aerial mycelia in either the grey or yellow series.
(b) They possess a sporophone morphology of either the rextiflexibiles or spirales type.
(c) They possess smooth surfaced spores.
(d) They do not produce melanin.
Species possessing the preceding characteristics include those referred to in Tables S to 9.

.. . . .. .

:, . . :: ., : , - . : . :
. : : : , .:: . . ., : , . . , - : . . ,. :
... . : . . ~ - , ~ : . ., :
.' ' ~ . ~' , - . .:
' .-: -, , , . ' ' : ' ABILITY OF CULTURES STREPTOMYCES OLIVACEUS STREPTOMYCES FULVOVIRIDIS, .. ~ . .
AND RELATED SPECIES TO PRODUCE MM 4550 (COMPLEX~
. ~

Culture MM 4550 (complex~
Production ..... . .
Streptomyces olivaceus ATCC 21379 f " ATCC 31126 f " ATCC 21380 f ' ATCC 21381 f " ATCC 21382 f .--.
" NCIB 8238 f " NCIB 8509 f Streptomyces flavovirens ATCC 3320 f Streptomyces flavus ATCC 3369 f Streptomyces fulvoviridis ATCC 15863 f Streptomyces argenteolus ATCC 11009 f Streptomyces sioyaensis ATCC 13989 f Streptomyces lipmanii NRRL 3584 (Streptomyces olivaceus ATCC 21549, ATCC 12019 and ATCC 3335 di'd not produce MM 4550 (comple~), ATCC 15863 has also been deposited as RIA 660) ' "' '.

, - 47 -.
', .
- . . .- . .
- .. . . ~ , , ', . ' ' . , ,. " ~ ' ~, 10S0460 STREPTOMYCES OLIVACE~S, STPEPTOMYCES FULVOVIRIDIS AND RELATED SPECI~S
- COLOUR AND MORPHOLOGY OF MATURE SPORULATING AERIAL MYCELIUM
ON ISP MEDIA AFTER 14 DAYS GROWTH .
- .
_ _ . Sporophore .~Culture SS YM GA OM Morphology . mid size ATCC 21379 Grey Grey Grey/White Grey Long RF .
ATCC 31126Grey/~rown Grey/Brown Grey Grey Long RF , ATCC 21380 ; Grey Grey Grey Grey Long RF
ATCC 21381 Grey Grey Pale Grey Grey/White Long RF .
ATCC 21382 ~ Grey/~hite Grey Grey Grey Long RF .
NCI~ 8238 Grey/White Grey Grey Grey Medium RF
NCI9 8509 Grey Grey Grey Grey Long RF
ATCC 21549 Grey Grey Grey Grey Long S
ATCC 12019 Grey Grey Grey Grey Long RR/S.
ATCC 3335 Gr0y Grey Grey Grey Long RF/S
ATCC 15863 Grey/Nhite Grey Grey/White Grey Medium RF i ATCC 3320 Grey/Blue Grey Grey/Green Grey Long RF ;
ATCC 3369 Grey Grey Grey Pale grey~ . i . . PrownMedium RF/RA
.. ATCC 11009 Pale Grey Grey~brown Grey/White Grey/Green Medium RF/RA,. . ATCC 13898White/Grey White Whi.te/ Grey/White Short S ;
. _ _ yellow .

SS ~ Inorganic salts - starch agar (ISP Medium 4) YN = Yeast extract - malt extract agar (ISP Medium 2) ' GA ~ Glycerol - asparagine agar (ISP Medium 5) ~
OM ~ Oatmeal agar (ISP Medium 3) !
RF ~ Rectiflexibiles j RA ~ R~tinaculiaperti ~
S ~ Spirales :: :
.' ':
. . _ 48 -. ~ .
.
' -; ~ .

'; : ~ ' - ` i ~
.: ' ' s ~

-: - :. , . , .
-: :, .
: : ~ , . . : . .,: . .: '.

'` 1050460 T A 8 L E 7 :

STREPTOMYCES OLIVACEUS, STREPTOMYCES FULVOVIRIDIS AND RELATED SPECI~S ..
- COLOUR O~ SUBSTRATE ~YCELIU~I AS VIE~IED FROM THE REVERSE SIDE . .
_ _ _ Culture SS YM GA OM

ATCC 21379 olive olive brown grey/brown olive green ATCC 31126 olive brown olive brown brown olive brown ATCC 21380 olive olive green olive brown olive yellow ATCC 21381 dark brown dark brown olive green olive green .
ATCC 21382 olive olive brown olive green NCIB 8238 brown olive/brown brown olive yellow NCIB 8509 brown olive olive brown olive yellow .
ATCC 21549 buff/black brown/black brown/black buff/grey .
ATCC 12019 buff buff buff buff -ATCC 3335 brown brown brown grey .
ATCC 15863 brown/grey dark brown olive brown olive yellow ATCC 3320 yellow/brown olive/brown olive/brown orange/brown .
ATCC 33eg buff!grey yellow hrown buff/green yellow ATCC 11009 black black/yellow black/yellow grey/green .
ATCC 13989 orange yellow yellow colourless _ , _ ''. :'' ' .
. ' ' ' ' ,:
.' . , .',"..
~ ' ' ' ' " ' I ~ -;
; . ' ' ~ "

' ' , ' , ~ :' .
, .
: - 49 _ :~'', . ''~' :
- ,, ~ '' :

': ~ : ,. ' ' " :
:
'; ~, ' , ' : ' ;, , " .
~;~" :

, , ~ . . .

.

`
()50460 . .
STREPTOMYCES OLIVhCEUS, STREPTOMYC~S_FULVOVIRIDIS AND RELATED SP~CIES
- PRODUCTION OF PIGMENTS IN CULTURE MEDIA
.

:
r - ~ - _ _ ~ ruble Non-Melanoid Pigments ~Melanin Culture ' _ _. _ Production .
.~ SS YM GA OM
, _ __ _ _ _ ~
ATCC 21379 _ _ _ slight _ .
. yellow ATCC 31126 _ _ . _ _ i ATCC 21380 _ _ _ _ .. , ~TCC 21381 _ _ _ _ _ , ATCC 21382 _ _ _ _ _ .
NCI~ 8238 f _ f f _ i slight slight slight , 'brown brown 'brown NCIB 8509 _ _ _ Syefgllhtow _ ATCC 21549 _ . f f f _ . slight slight red/ slight brown brown brown ATCC 12019 _ _ _ _ _ ATCC 3335 _ . _. _ _ ~
: ., ATCC 15863 _ _ . _ _ .
ATCC 3320 f _ _ _ _ sliinhkt ATCC 3369 _ _ _ _ _ . . ATCC 11009 _ f _ _ _ slight .
. . 'brown : ATCC 13989 _ f _ _ _ : : slight orange _ _ _ _ _ , f = Pigment produced .
~ = Pigment not produced .
. * ' Tested on peptone-yeast-iron agar (ISP6), tyrosine .~ agar (ISPl7? and tryptone-yeast broth (ISP 1) :~
- 50 - ' : ' ' ' ` ' -' .
,~ . .
. ' '" ' ' , .
,, '~` --'' ; - : , - :
., . .:. ' . , ~ ::

~ . 1 460 1 ~
~r A B L E 9 ., . ,,..
STREPTOMYCES OLIVACEUS, STREPTOMYCES FULVOVIRIDIS AND RELATED SPECIES
.. ..
- CARBON UTILISATION TESTS
.

. ' ' ~ ., .; . ATCC 21379 f _ _ f f f f f f :
.i ATCC 31126 f _ _ f f f f f f .
ATCC 21380 f _ _ f f f f f f ATCC 21381 f _ _ _ f f f f f ATCC 21382 f f _ _ f f f f f . ,.
~ NCIB 8238 f _ _ _ f f f f f ;
; NCIB 3509 , f f _ _ f f f f f .' ATCC 21549 _ _ ._ f f f f f f ., . ATCC 12019 f f f f f f f f f , . ATCC 3335 f _ f f f f f f f .. ::
.~ ATCC 15863 .f _ _ _ f f f f f i .
. ATCC 3320 . f _ ~ _ f .f f f f . : . .
ATCC 3369 f f f f f f f f . .
. ~ ATCC 11009 f _ .. f f f f f f .
ATCC 13989 _ i f f f _ f f _ . f denotes the compouhd is utilised ' :
denotes the compound is not utilised , ~-f d-notes.utilis-tion is doubtful or very poor ~.. : ~
51- ' 'i.
`~i, '~ '~ . ' . .

J~ ;

`: 1050460 Exam.~le 3 A stock of spores of S.olivaceus ATCC 31126 was maintained by storage in tubes of dry soil in a closed container with a dessicant at a temperature of 20 C. A small quantity of soil stock (approximately 20 mg) was transferred aseptically to a 500 ml Erlenmeyer flask containing 100 ml of the following medium:
Constituent Amount (g/litre) Glucose monohydrate 20.0 Soya-bean flour 10.0 Deionised water to 1 litre The pH was adjusted to 6.5 before sterilisation. The soya bean flour was 'Arkasoy 50' as supplied by British Arkady Co. Ltd., Old Strafford, Manchester, England.
The flask was incubated on a rotary shaker (240 r.p.m.) for about 30 hours at 28C. 2 ml of the resulting vegetative growth was then used to inoculate a solid agar slant in a Roux bottle.
The agar medium had the following composition:-Constituent Amount V-8* vegetable juice 20.0 ml Agar 20.0 g Deionised water to 1 litre The pH was adjusted to 6.0 before sterilisation. (V-8 juice is obtainable from Campbell's Soups Ltd., Kings Lynn, Norfolk, England).
I The inoculation was spread on the agar surface by roc~ing the bottle which was then incubated upright at 30C. After two l~ *Trade Mark ,~ - 52 -;l ~
ij , . - - ,: ~ : : : .: : ~

.. . . , : . . .. , : ::: :.
. . . : . . . .

~5()460 days incubation, surplus liquid in the bottle was removed by pipette and incubation continued for a further 4 days.
It had been previously found that the development of actinophage in the slant culture is suppressed if this method of preparation of the Roux bottle culture is adopted.
50 ml of sterilised deionised water containing 0.02% Tween 80 was added to a Roux bottle culture and the spores suspended by shaking. This spore suspension was then added as inoculum to 75 litres of sterilised seed stage medium in a 100 litre 10 staînless steel fermenter. The composition of the seed stage medium was as follows:-Constituent Amount (g/l) Soya-bean flour ('Arkasoy 50') 10.0 Glucose monohydrate 20.0 Tap water to 1 litre To control foaming, 50 ml of 10 % v/v 'Pluronic L81' in soyal-bean oil was added to the fermentation medium before sterilisation.
The medium was steam sterilised in the fermenter for 20 minutes at 120C. The seed stage culture was stirred at 140 rpm with a 7.5 inch diameter vaned disc agitator and supplied with 75 litres/min sterile air through an open ended sparger.
The temperature was controlled at 28C and after incubation under these conditions for 48 hours the contents of the vessel were added as inoculum to 1500 litres of sterile fermentation medium in a 2000 litre stainless steel fully baffled fermenter.
The fermentation medium had the following composition:

~..

'.
.
,. .
.

10~04~0 Constituent Amount (g/litre) soya-bean flo~r ('Arkasoy 50') 10.0 Glucose monohydrate 20.0 Chalk (precipitated calcium carbonate) 0.2 Cobalt chloride (COC12.6H2O) 0.001 Sodium sulphate (anhydrous) l.o Tap water to 1500 litres The pH was adjusted to 6.0 with sodium hydroxide before sterilisation. 3 litres of 10% 'Pluronic L81' in soya bean oil was added before sterilisation to prevent foaming. After sterilisation the pH was again adjusted, to 7.0, with sterile sodium hydroxide solution. The fermentation was stirred at 106 r.p.mO, the stirrer shaft being fitted with two 19 inch diameter vaned disc impellers. Temperature was controlled at 30C and airflow at 1200 litres/min. The fermentation was harvested after 60 hours and clarified by centrifugation.
The material produced above was arbitrarily assigned an activity of 340 units/ml. Assays were determined as described in Description 2.
Culture filtrate (1050 1; 340 units/ml.) at 10C and pH8 was extracted with dichloromethane (310 1.) at 10C
containing cetyldimethylbenzylammonium chloride (1200 g.) by p~lping the two liquids at predetermined flow rates through an in-line mixer. The phases were separated in a Sharples continuous centrifuge having been admixed - 54 - ,~

.

~ l~)S04~
for about two minutes. The dichloromethane phase (300 1) was back extracted with aqueous sodium iodide. The back extraction was performed in four batches using a total of 7 1 water containing 210 g. sodium iodide. The phases were separated by gravity. The aqueous phase was adjusted from pH 7.7 to pH 7.0 with hydrochloric acid and filtered.
The sodium iodide extract (7 1) contained 21,900 units/ml.
An ion exchange column was prepared by packing QAE
Sephadex A25 (obtained from Pharmacia Ltd.) in pE17 phosphate 10 buffer (0.05M) containing sodium chloride (0.3M) into a 10 cm diameter glass column to a height of 40 cm. The sodium iodide extract (7 1) at 5C was percolated through the QAE
Sephadex at 50 ml./min. The column was eluted with 0.7M NaCl in 0.05M phosphate bufer, Pb 7 a1so ."' ..
. . ~

1' ~

' , :;' .. . . .

~05(~4~0 at 5C at a flow rate of 25 ml./min. 2 l eluate was discarded and 90 fractions (100 ml.) were collected. The fractions were scanned in a u.v. spectrophotometer and those fractions showing an absorption maximum at about 305 nm were pooled and adjusted to pH 7 (fractions 50 - 62, pooled volume 1440 ml. 71,000 units/ml. It had previously been shown that fractions having a maximum in this region contained MM 13902.

Sodium chloride (5 g./100 ml.~ was added to the pooled fractions which were then percolated at 5C through a 6.3 cm diameter column packed with A~berlite XAD 4 resin (as supplied by Rohm & Haas Ltd.) to a height of 30 cm. at a flow rate of 20 ml./min. MM13902 was adsorbed to the resin under these conditions whereas the inorganic impurities were not. MM 13902 was eluted at room temperature with distilled water (200 ml.) followed by 50%
aqueous methanol. The eluate (1 l) was evaporated below 30C under reduced pressure to 70 ml., adjusted to pH 7 and freeze dried to a brown solid (1.62 g.) which was a partially purified salt of MM 13902 with an activity of 37,000 units/mg.
.'.
Partially purified MM 13902 di-sodium salt (1.0 g., 37,000 units/mg.) was chromatographed on a cellulose column (3.8 cm. x 30 cm; Whatman , :
CC31 cellose) eluted with n-propanol/water ~4:1 v/v) at a flow rate !
of 2.5 ml./min. 170 ml. eluate was discarded and 100 x 15 ml.
! fractions collected. The fractions containing MM 13902 di-sodiumsalt (Nos. 37 - 43) as determined by uv absorption were pooled ~89 ml.), evaporated below 30C under reduced pressure to remove n-propanol and freeze dried to yield a yellowish powder ~219 mg.) with an activity of ; ~ 73,000 units/mg.

~: .

'~

~ 04~ :

The above material showed a uv absorption maximum at about 308 nm with an E 1 of about 343. This material had the i.r. and n.m.r.
spectra shown in Figs.2 and 3 respectively. The antibacterial activity of the material is shown in Table 10. Elemental analysis of this material indicated that it contained inter alia nitrogen, sulphur and sodium probably in the ratio N:S:Na - 2:2:2. The positive and negative maxima of the circular dichroism curves for di sodium MM 13902 were determined on a Cary-61 recording spectropolarimeter at concentrations of 0.37 mg/ml at a path length of 1 cm; the results were as follows:

(nm) ~ /m 186 + 67.24 x 10 4 ' 221 - 67.24 x 10 286 - 3.3 x 10 323 - 3.2 x ~

:
,' ~ .
.

;' ... . . ,, , , , - .: :. :, . :
: , : : . . , : , . , . . . . :

~05~4~0 (MICROTITRE METHOD - HEAVY INOCULUM, 1/100 OVERNIGHT BROTH) .

Organism MIC (ug/ml) , ' 7-Bacillus subtilis A 0.15 Staph. aureus Oxford 0.3 Staph. aureus Russell 0.6 Strep. Faecalis 3.1 Enterbacter cloacae N1 25 E. coli 10418 0.15 E. coli JT410 5 Kleb. aerogenes A 0.03 Proteus mirabilis 13 0.6 Proteus vulgaris WO 90 0.6 Prov. stuartii 0.07 Ps. aerug nosa A 50 Salmonella typhimurium CT 10 0.3 Serratia marcescens US 39 2.5 Shigella sonnei 0.6-0.3 Haemophilus influenzae P6 0.08 Neisseria gonorrhoeae 0.08 . " . _ . _ ~
-, ~.

' .

:

,

Claims (18)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE PROPERTY OR PRIVILEGE
IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A process for the preparation of the compound MM 13902 of the formula (I) and its pharmaceutically acceptable salts which compound in the form of a substantially pure sodium salt has the following characteristics:
(i) It is highly soluble in water, soluble in methanol and substantially insoluble in chloroform, diethylether and hydrocarbons.
(ii) In aqueous solution, it has a characteristic ultra-violet spectrum with absorption maxima one of which is at about 305 nm.
(iii) When present at 0.4% w/w in a freshly prepared KBr disc, it has a characteristic infra-red spectrum which has absorption maxima at inter alia about 3450, 2950, 1750, 1620, 1510, 1400 and 1260 cm-1.
(iv) It has a characteristic n.m.r. spectrum when taken in D2O which spectrum possesses inter alia (a) a pair of low field doublets centred at approximately 2.851 and 4.00 with coupling constants of approximately 14 Hz; (b) a doublet centred at approximately 8.55% and (c) a sharp singlet at approximately 8.00%.
(v) It possesses antibacterial activity against various species including inter alia, strains of Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Klebsiella aerogenes, Proteus mirabilis, salmonella typhi and Pseudomonas aeruginosa.

(vi) When mixed with ampicillin it synergises its activity against organisms including strains of Escherichia coli, Klebsiella aerogenes, Proteus mirabilis, Proteus morganii and Staphylococcus aureus Russell;

and which compound is further characterised in that its pure di-sodium salt has the following approximate Rf values when run on a cellulose thin layer chromatography system:
(a) n-butanol/isopropanol/water - 7:7:6 v/v;
Rf = 0.72 (b) isopropanol/water - 7:3 v/v;
Rf = 0.85 (c) n-butanol/ethanol/water - 7:7:6;
Rf = 0.81 (d) n-propanol/water - 4:1;
Rf = 0.75 and that the pure di-sodium salt has an I50 of between 0.001 µg/ml and 0.0001 µg/ml against the .beta.-lactamase of Escherichia coli B 11; which process comprises the cultivation of a MM 13902 producing strain of Streptomyces olivaceus and thereafter recovering the substantially pure MM 13902 or a pharmaceutically acceptable salt thereof from the culture.

2. A process as in claim 1 wherein the Streptomycete is Streptomyces olivaceus ATCC 31226 or a mutant thereof.

3. A process for the preparation of MM 13902 or a pharmaceutically acceptable salt thereof as claimed in claim 1 which comprises chromato-graphically separating a solution of MM 4550 (Complex) into fractions consisting essentially of a solution of MM 13902 or a salt thereof and other fractions and isolating the substantially pure MM 13902 or a pharmaceutically acceptable salt thereof from solution.

4. A process according to claim 3 adapted for the preparation of a substantially pure di-basic salt of MM 13902.

5. A process according to claim 3 adapted for the preparation of a substantially pure di-sodium or di-potassium salt of MM 13902.

6. A process according to claim 3 adapted so that a pharmaceutically acceptable salt at least 70% pure is obtained.

7. A process according to claim 3 adapted so that a pharmaceutically acceptable salt at least 80% pure is obtained.

8. A process according to claim 3 adapted so that a pharmaceutically acceptable salt at least 90% pure is obtained.

9. A process for the preparation of substantially pure MM 13902 or a pharmaceutically acceptable salt thereof as claimed in claim 1 wherein the MM 13902 or pharmaceutically acceptable salt thereof is recovered by chromatographically separating a solution of MM 4550 (Complex) into fractions consisting essentially of a solution of MM 13902 or a salt thereof and other fractions and isolating the substantially pure MM 13902 or a pharmaceutically acceptable salt thereof from solution.

10. The substantially pure compound MM 13902 as defined in claim i and its pharmaceutically acceptable salts whenever prepared by the process of claim 1 or an obvious equivalent thereof.

11. The substantially pure compound MM 13902 as defined in claim 1 and its pharmaceutically acceptable salts whenever prepared by the process of claim 2 or an obvious equivalent thereof.

12. The substantially pure compound MM 13902 as defined in claim 1 and its pharmaceutically acceptable salts whenever prepared by the process of claim 3 or an obvious chemical equivalent thereof.

13. The substantially pure compound MM 13902 as defined in claim 1 in the form of a di-basic salt whenever prepared by the process of claim 4 or an obvious equivalent thereof.

14. The substantially pure di-sodium or di-potassium salt of the compound MM 13902 whenever prepared by the process of claim 5 or an obvious equivalent thereof.

15. An at least 70% pure pharmaceutically acceptable salt of MM 13902 as defined in claim 1 whenever prepared by the process of claim 6 or an obvious equivalent thereof.

16. An at least 80% pure pharmaceutically acceptable salt of MM 13902 as defined in claim 1 whenever prepared by the process of claim 7 or an obvious equivalent thereof.

17. An at least 90% pure pharmaceutically acceptable salt of 13902 as defined in claim 1 whenever prepared by the process of claim 8 or an obvious chemical equivalent thereof.

18. The substantially pure compound MM 13902 as defined in claim 1 or a pharmaceutically acceptable salt thereof whenever prepared by the process of claim 9 or an obvious equivalent thereof.