The present invention relates to novel antibacterially active materials obtainable from a microorganism, to processes for their production, and to their pharmaceutical use.

A large number of microorganisms have been isolated from nature and certain of those microorganisms have been found to produce various metabolites, which can be isolated and some of which have useful antibacterial activity. Two such metabolites are substances which have been designated MM

- ^~ , ~, compounds and have been found to have useful antibacterial activity.

The present invention accordingly provides the novel substances MM 56597 and 56598.

It is believed that the substances MM 56597 and 56598 have the structures outlined in FIG I.

They have the molecular weights and hplc retention times set out hereinbelow in the experimental section.

Each of MM 56597 and MM 56598 is believed to consist of a glycopeptide compound which may exist as a mixture of isomers.

The present invention also provides a process for the production of a substance or compound of the invention which comprises cultivating a producing microorganism and subsequently isolating the substance or compound or a derivative thereof from the culture.

The present invention furthermore provides a process for the preparation of a substance or compound of the invention

which comprises separating the substance or compound or a derivative thereof from a solution thereof in admixture with other antibacterially active substances and/or inactive substances by adsorption onto an affinity resin.

The term 'cultivation' (and derivatives of that term) as used herein means the deliberate aerobic growth of an organism in the presence of assimilable sources of carbon, nitrogen, sulphur and mineral salts. Such aerobic growth may take place in a solid or semi-solid 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.

It has been found that suitable microorganisms for use in the cultivation process according to the invention include bacterial strains belonging to the genus Amycolatopsis. It has further been found that an example of such a strain is sp. NCIB 40089 and also mutants thereof, which has been isolated from nature.

The term 'mutant' as used herein includes any mutant strain which arises spontaneously or through the effect of an external agent whether that agent is applied deliberately or otherwise. Suitable methods of producing mutant strains including those outlined by H.I. Adler in 'Techniques for the Development of Microorganisms' in 'Radiation and Radioisotopes for Industrial Microorganisms' , Proceedings of a Symposium, Vienna, 1973, page 241, International Atomic Energy Authority, and these include:

(i) Ionizing radiation (e.g. X-rays and λ-rays) , u.v. light, u.v. light plus a photosensitizing agent (e.g. 8-methoxypsoralen) ,

nitrous acid, hydroxylamine, pyrimidine base analogues (e.g. 5-bromouracil) , acridines, alkylating agents (e.g. mustard gas, ethyl methane sulphonate) , hydrogen peroxide, phenols, formaldehyde, heat, and

(ii) Genetic techniques, including, for example, recombination, transformation, transduction, lysogenisation, lysogenic conversion, protoplast fusion and selective techniques for spontaneous mutants.

Sp. NCIB 40089 has been deposited at the National Collections of Industrial and Marine Bacteria Ltd. (N.C.I.B), Aberdeen, Scotland under number 40089 on 6th December, 1988. ^• • -

The fermentation medium for cultivating sp. NCIB 40089 suitably contains sources of assimilable carbon and assimilable nitrogen together with inorganic salts. Suitable sources of nitrogen include yeast extract, soyabean flour, meat extract, cottonseed, flour, malt, distillers dried solubles, amino acids, protein hydrolysates and ammonium and nitrate nitrogen. Suitable carbon sources include glucose, lactose, maltose, starch and glycerol. Suitably the culture medium also includes alkali metal ions (for example, sodium), halogen ions (for example, chloride), and alkaline earth metal ions (for example calcium and magnesium) , as well as trace elements such as iron and cobalt.

The cultivation may suitably be effected at a temperature of about 20 to 35°C , advantageously 20 to 30°C, and the culture may suitably be harvested up to 7 days, advantageously about 3 to 5 days, after the initiation of fermentation in order to give an optimum yield of the product.

The desired product or a derivative thereof may then be isolated from the culture medium and worked up and purified using conventional techniques for glycopeptide compounds. All such isolation and purification procedures may conveniently be effected at cool to ambient temperature, for example at a temperature within the range of from 4 to 30°C, conveniently from 20 to 25°C.

The desired product is- generally obtained predominantly from the culture filtrate, and it is therefore convenient for the first isolation step to involve removal of solid material from the fermentation broth by, for example, filtration or centrifugation, to give a clarified culture filtrate.

Further isolation of the desired product from the clarified culture filtrate may conveniently be effected by adsorption onto an affinity resin such as D-alanyl-D-alanine-sepharose affinity resin.

The desired product may readily be identified in a routine manner by testing for antibacterial activity and/or by monitoring the h.p.l.c. retention time.

Suitably, the separation procedure may include a high-performance liquid chromatography step, preferably as the last step. Elution may be effected using aqueous aH2P0 ₄ /acetonitrile.

The products according to the invention are suitably provided in substantially .pure form, for example at least 50% pure, suitably at least 60% pure, advantageously at least 75% pure, preferably at least 85% pure, more preferably at least 95% pure, especially at least 98% pure, all percentages being calculated as weight/weight. An impure or less pure form of a product according to the invention may, for example, be used in the preparation of a more pure form of the same product or of a related product

(for example a corresponding derivative) suitable for pharmaceutical use.

The products of the invention have antibacterial properties and are useful for the treatment of bacterial infections in animals, especially mammals, including humans, in particular humans and domesticated animals (including farm animals) . The products may be used for the treatment of infections caused by a wide range of organisms including, for example, those mentioned herein.

The present invention provides a pharmaceutical composition comprising a product of the invention or a pharmaceutically ^• acceptable derivative thereof together with a pharmaceutically acceptable carrier or excipient.

The present invention also provides a method of treating bacterial infections in animals, especially in humans and in domesticated mammals, which comprises administering a product of the invention or a pharmaceutically acceptable derivative thereof, or a composition according to the invention, to a patient in need thereof.

The products according to the invention may be formulated for administration in any convenient way for use in human or veterinary medicine, by analogy with other antibiotics.

The products according to the invention may be formulated for administration by any route, for example oral, topical or parenteral. The compositions may, for example, be made up in the form of tablets, capsules, powders, granules, lozenges, creams, syrups, or liquid preparations, for example solutions or suspensions, which may be formulated for oral use or in sterile form for parenteral administration by injection or infusion.

Tablets and capsules for oral administration may be in unit dosage form, and may contain conventional excipients including, for example, binding agents, for example, syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone; fillers, for example lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine; tabletting lubricants, for example magnesium stearate, talc, polyethylene glycol or silica; disintegrants, for example potato starch; and pharmaceutically acceptable wetting agents, for example sodium lauryl sulphate. The tablets may be coated according to methods well known in normal pharmaceutical practice.

Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for reconstitution with water or another suitable vehicle before use. Such liquid preparations may contain conventional additives, including, for example, suspending agents, for example sorbitol, methyl cellulose, glucose syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenated edible fats; emulsifying agents, for example lecithin, sorbitan monooleate or acacia; non-aqueous vehicles (which may include edible oils) , for example almond oil, oily esters (for example glycerine) , propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid; and, if desired, conventional flavouring and colour agents.

Compositions according to the invention intended for topical administration may, for example, be in the form of ointments, creams, lotions, eye ointments, eye drops, ear drops, impregnated dressings, and aerosols, and may contain

appropriate conventional additives, including, for example, preservatives, solvents to assist drug penetration, and emollients in ointments and creams. Such topical formulations may also contain compatible conventional carriers, for example cream or ointment _v bases, and ethanol or oleyl alcohol for lotions. Such carriers may constitute from about 1% to about 98% by weight of the formulation; more usually they will constitute up to about 80% by weight of the formulation.

Compositions according to the invention may be formulated as suppositories, which may contain conventional suppository bases, for example cocoa-butter or other glycerides.

Compositions according to the invention intended for parenteral administration may conveniently -be in fluid unit dosage forms, which may be prepared utilizing the active and a sterile vehicle, water being preferred. The active, depending on the vehicle and concentration used, may be either suspended or dissolved in the vehicle. In preparing solutions, the active may be dissolved in water for injection and filter-sterilised before being filled into a suitable vial or ampoule, which is then sealed. Advantageously, conventional additives including, for example, local anaesthetics, preservatives, and buffering agents can be dissolved in the vehicle. In order to enhance the stability of the solution, the composition may be frozen after being filled into the vial, and the water removed under vacuum; the resulting dry lyophilized powder may then be sealed in the vial and an accompanying vial of water for injection may be supplied to reconstitute the liquid prior to use. Parenteral suspensions may be prepared in substantially the same manner except that the active is suspended in the vehicle instead of being dissolved and sterilisation cannot be accomplished by filtration. The

active may instead be sterilised by exposure to ethylene oxide before being suspended in the sterile vehicle. Advantageously, a surfactant or wetting agent is included in such suspensions in order to facilitate uniform distribution of the active.

A product according to the invention may suitable be administered to the patient in an antibacterially effective amount.

A composition according to the invention may suitably contain from 0.1% by weight, preferably from 10 to 60% by weight, of a product according to the invention (based on the total weight of the composition) , depending on the method of administration.

The products according to the invention may suitably be administered to the patient at a daily dosage of from 1.0 to 50 mg/kg of body weight. For an adult human (of approximately 70 kg body weight) , from 50 to 3000 mg, for example about 1500 mg, of a product according to the invention may be administered daily. Suitably, the dosage for adult humans is from 5 to 20 mg/kg per day. Higher or lower dosages may, however, be used in accordance with normal clinical practice.

When the compositions according to the invention are presented in unit dosage form, each unit dose may suitably comprise from 25 to 1000 mg, preferable from 50 to 500 mg, of a product according to the invention.

The following Examples illustrate the preparation of products according to the present invention.

Example 1

a) Fermentation

A 1ml vegetative cell suspension of culture NCIB 40089 stored in 20% glycerol and 10% lactose under nitrogen, was used to inoculate 100ml of fermentation medium contained in a 500ml conical flask, stoppered with a foam plastic bung. The fermentation medium had the following composition:-

Constituent Amount /1

CaCl ₂ .2H ₂ 0 10.0 MgCl ₂ .6H ₂ 0 10.0 NaCl 10.0 FeCl ₃ 3.0 ZnCl ₂ 0.5

CuCl ₂ .2H ₂ 0 0.5 MnS0 ₄ .4H ₂ 0 0.5

The medium was adjusted to pH 7.3 before sterilisation at 117°C for 15 minutes. (The soya bean flour was Arkasoy '50' supplied by Arkady - ADM, Manchester, UK) .

5 After incubation for 72 hours at a temperature of 28°C and 240rpm on a gyratory shaker, 4ml aliquots were transferred to 500ml conical flasks containing 100ml fresh medium. These were incubated at 28°C and 240rpm for a further 48 hours. 0

15 litres of fermentation medium together with 0.1% antifoaming agent, polypropylene glycol P2000, was sterilised in situ for 60 minutes at 121°C in a 2-0 litre, fully baffled fermenter. The fermenter was stirred by a 5 bottom driven agitator fitted with three vaned-disc* impellers at 200rpm during both sterilisation and cultivation. 400ml vegetative inoculum from the second stage seed flasks was used to inoculate the fermenter and incubation was carried out at 28°C for 45 hours before Q transfer to the production stage. During the fermentation the fermenter was supplied with sterile filtered air at 0.23 volume per volume per minute and an overpressure of air of 0.5bar was maintained throughout.

25300 litres of fermentation medium, together with 0.1% antifoaming agent, polypropylene glycol P2000, was sterilised for 60 minutes at 121°C in a 450 litre, fully baffled fermenter. During sterilization the fermenter was stirred at lOOrpm by a bottom driven agitator, fitted with

30 three, vaned-disc impellers. 12 litres of vegetative inoculum from the 20 litre fermenter was used to inoculate the 450 litre fermenter and incubation was carried out at 28°C for 94 hours before harvest. During the fermentation, the fermenter was supplied with sterile filtered air at 0.5

35 volumes per volume per minute, with an agitator speed of 50rpm. An overpressure of air of 0.5bar was maintained throughout. The fermenter was harvested in 50L portions

which were adjusted to pH 10.9 by addition of 5M NaOH, prior to centrifugation. The resulting supernatant was adjusted to pH 6-8 by addition of 5M HC1.

b) Isolation of Glycopeptide Complex

The neutralised clarified broth (270L) was applied to a 22L column of Diaion HP20 at a flow rate of 1L min- ¹ . (HP 20 was supplied by Mitsubishi Chemical Industries, Tokyo, Japan) . The column was washed with 30L of deionised water and the percolate and water wash discarded. The active material was eluted from the column with 0.1M ammonia containing 50% propan-2-ol. 1 litre fractions were collected. Fractions with antibiotic activity,. (16-35), were bulked and evaporated in vacuo to 8.8 L.

8.8L of aqueous concentrate was mixed with 4.5L of butan-1-ol and the pH adjusted to 3.0 by the addition of

0.1M HC1. Phases were separated under gravity and the lower aqueous phase removed. The upper solvent phase and mixed phases were separated by centrifugation and the solvent phase recovered by aspiration. 4.5L of solvent phase was mixed with an equal volume of deionised water and the pH was adjusted to 10.0 by addition of 0.1M NaOH. Phases were separated under gravity and 4.7L of aqueous phase adjusted to pH7.0 with 0.1M HC1.

The aqueous phase was concentrated in vacuo until a gelatinous precipitate was formed, which was removed by filtration. The precipitate was resuspended in deionised water and refiltered via 541 filter papers [Filters supplied by Whatman, Springfield Mill, Maidstone, Kent, England] . The combined filtrates (6.3L) were stirred for 1 hour with

D-alanyl-D-alanine-sepharose affinity resin, (350ml wet volume) . [Prepared as described below] .

The mixture was filtered onto a glass scinter funnel and the affinity resin washed with deionised water (2 x 500 ml) . 7.45L of combined percolate and washings were retained. The affinity resin was then eluted with 0.1M ammonia containing 50% acetonitrile (5 x 800 ml)and washed with deionised water (3 x 500ml) to give combined eluates and washings which were evaporated jLn vacuo to give 2.3L of aqueous concentrate.

The 7.45L of affinity percolate was retreated with 350ml of affinity adsorbent as above to give a further 2.551* of- aqueous concentrate.

4.85L of combined concentrates were freeze dried to yield 15.2g of glycopeptide complex, MM 49728 which contained a mixture of MM 55266, MM 55267, MM 55268, MM 56597 and MM 56598 (see Published European Patent Application No. 0386940, USSN 490143) .

Preparation of Affinity Adsorbent.

The affinity adsorbent was prepared from D-alanyl-D-alanine immobilised on activated CM Sepharose 4B (6-aminohexanoic acid-activated-sepharose 4B was obtained from Sigma Chemical Co., Poole, Dorset, England).

The N-hydroxysuccinimide ester of 6-aminohexanoic acid Sepharose 4B (60g) was placed on a glass scinter and washed with 1 mM hydrochloric acid solution (2L) under suction. The wet cake was then added to a solution of D-alanyl-D-alanine (1.5g) in 0.1M sodium bicarbonate solution (60ml) and occasionally shaken over the next hour. The suspension was filtered under suction and the residue

suspended in 0.1M tris (hydroxymethyl)amino methane (TRIS) (100ml) for 1 hour and then refiltered through a glass scinter. The cake was washed successively with 0.1M sodium bicarbonate solution, 0.05M TRIS (containing 0.5M sodium chloride), 0.05M formate buffer at pH 4.0 (containing 0.5M sodium chloride) and finally distilled water. The affinity resin was then stored at 4°C in aqueous suspension.

Example 2

Isolation of MM 55268, MM 56597 and MM 56598.

5.5g of MM 49728, prepared as described in Example lb was dissolved in 800 ml of 5% methanol in water at pH -8.0. ^' This solution was applied to a 90ml column of Matrex C**_g reverse phase silica, (30μm particles, 60A pore diameter) , previously equilibrated in 0.05M NaH P0 ₄ pH 6.0 (Matrex supplied by Amicon, Upper Mill, Stonehouse, Gloucestershire, England) .

The column was washed with 200 ml of the equilibrating buffer before being eluted at 10ml.min- ¹ with the above buffer containing (i) 15% acetonitrile (200 ml) and finally (ii) 20% acetonitrile (200 ml) . These percolates., washings and eluates were discarded. The elution continued with the buffer containing (i) 20% acetonitrile (200 ml) and (ii) 30% acetonitrile (350 ml) . These eluates were pooled and evaporated in vacuo to an aqueous concentrate which was diluted with deionised water to 650 ml.

This solution was applied to a 275ml column of Matrex C ₁₈ reverse phase silica previously equilibrated in 0.05M NaH ₂ P0 ₄ pH 6.0. the column was washed with 500ml of the equilibrating buffer before being washed at 10ml.min- ¹ with the above buffer, containing (i) 15% acetonitrile (500 ml)

and (ii) 20% acetonitrile (500ml) . These percolates and washings were discarded.

Elution was started with the buffer containing a stepped gradient of acetonitrile, starting at 20%. After 10 minutes, the eluate was collected in 15 ml fractions. The acetonitrile concentration was changed as shown in Table 1.

Fractions were monitored by HPLC using a 4.6 X 150 mm Zorbax C-^g reverse phase silica column, 5μm particles, eluting with 0.1M NaH ₂ P0 ₄ pH 6.0, containing 28.4% acetonitrile at a flow rate of 1ml min- . [Column supplied by Du Pont, Hitchin, Herts, England] . The eluate was monitored for uv absorbance at 220nm. Under these conditions MM 55268 had a retention time of 8.8 minutes. Fractions (166-180), containing mainly MM 55268, but with some MM 56597 and MM 56598, were combined and evaporated in vacuo to 190 ml.

Table 1

Concentration of Acetonitrile in Eluant Related to Elution Time.

This aqueous concentrate was applied to a 100ml column of Diaion HP20. The column was washed with 300 ml of deionised water and eluted with 300 ml of methanol. The eluate was diluted with approximately 50 ml of deionised water and evaporated in vacuo to 91 ml and freeze dried to yield 680mg of material containing MM 55268, MM 56597 and MM 56598.

Example 3

Isolation of MM 56597

The preparative HPLC separation described in Example 3 also yielded fractions containing MM 56597, (70-78 from Dynamax 1) . The fractions were monitored by HPLC on a Zorbax column as described in Example 2. Under these conditions, MM 56597 had a retention time of 9.2 minutes.

Fractions 70-78 were pooled, diluted with deionised water, evaporated in vacuo to 25ml and applied to a 10ml column of Diaion HP20 equilibrated in water. The column waa washed and eluted as described in Example 3. The combined eluates were evaporated in vacuo and freeze dried to yeld 7.5mg of MM 56597.

FAB mass spectroscopy indicated a molecular ion corresponding to a molecular weight of 1982±1

Example 4

Isolation of MM 56598

The preparative HPLC separation described in Example 3 also yielded fractions containing MM 56598, (85-94 from Dynamax 1) . The fractions were monitored by HPLC on a Zorbax column as described in Example 2. Under these conditions, MM 56598 had a retention time of 10.8 minutes.

Fractions 85-94 were pooled, diluted with deionised water, evaporated in vacuo to 37ml and applied to a 12ml column of Diaion HP20 equilibrated in water. The column was washed and eluted as described in Example 3. The combined eluates were evaporated in vacuo and freeze dried to yield 7mg of MM

_

56598 .

FAB mass spectroscopy indicated a molecular ion corresponding to a molecular weight of 1820±1.

Detection Methods

a) Fermentation samples and column fractions were monitored for antibiotic activity by bioassay on Staphylococcus aureus V573, using the conventional hole in plate method.

b) MM 56597 and MM 56598 have a characteristic UV absorption maximum at 280nm. Purified samples can be assayed using direct measurement of this absorbance.

The antibacterial activities of MM 56597 and MM 56598 were determined by the microtitre method. Oxoid No. 2 broth (supplied by Oxoid Ltd., Wade Road, Basingstoke, Hampshire, UK. (Oxoid is a trade mark)) was used for all organisms except for the Streptococcus spp. which was tested using Todd Hewitt broth (supplied by Oxoid Ltd.) . Inocula were overnight broth cultures diluted ten-fold. The microtitre plates were incubated for 24 hours at 37°C. The results are shown in Tables 2 and 3.

Table 2

Antibacterial activity of MM 56597 against a range of organisms determined by the microtitre method (MIC μg/ml)

Organism MM 56597

Bacillus subtilis ATCC 6633 2 Corynebacterium xerosis NCTC 9755 0.25 Staphylococcus aureus - Smith 4

- Oxford 1.

- V573 MR* 0/5- Staph. saprophyticus - FL1 4

- FL2 2 Staph. epidermidis - 60137 0.5

- 54815 4 Streptococcus pyogenes - CN10 0.06

- 1950 0.25 Strep. agalactiae 2798 0.25 Strep, viridans 'Harding' 0.25 Strep, pneumoniae Pu7 1 Strep, faecalis I 1

* Multi-resistant (Methicillin , Tetracycline, Erythromycin and Gentamicin resistant) .

Table 3

Antibacterial activity of MM 56598 against a range of organisms determined by the microtitre method (MIC μg/ml)

*Multi-resistant (Methicillin, Tetracycline, Erythromycin and Gentamicin resistant) .

FIG I

MM 56597: R-= -(CH ₂ ) ₆ CH(CH ₃ ) ₂ , R ² = Mannosyl MM 56598: R ^x = -Cc,H ₁₉ , R ² = H

International Application No: PCT/

MICROORGANISMS

Optional Shaat tn cannectlaa wtth Ih* mtcracrganltm lateπaa ¹ M an paie. -2 .«.__ 2JL •I th* ***crt βon >

A. icHiimncATtOM or MPOMT «

Furth-M *Hn» » f Mwtua-X an it a*--«t*nal itml Q'

Nam* at dapaaH-aty iaaNtwttan <

NATIONAL COLLECTION OF INDUSTRIAL AND MARINE BACTERIA LIMITED

«Mrw at «(HMιy tMHtuβan 0"du«nt iMMal cad* ttn4 taunt*-,) «

Torry Research Station, PO Box 31 , 135 Abbey Road, Aberdeen, United Kingdom, AB9 8DG

Data •< tfaaaaK •

6th December , 1988 NCIB 40089 a. AOOmOMAL INDICATIONS » (laa«a Man* M Mt appttcaMa). TMa I irma-m** ta caMKwaa an a aaparata attach** MM Q

In respect of those designations in which a European Patent is sought, a sample of the deposited micro¬ organism will be made available until the publication of the mention of the grant of the European patent or until the date on which the application has been refused or withdrawn, only by the issue of such a sample to an expert nominated by the person requesting the sample .

C. OtSMNATIO STATM FOft WHICH INDtCATIONS AM MAOI • (H th* tadtcaUena ara na« far a* daaHnatad Stataa)

O. SIPARATI FURNISHIN* OF INDICATIONS • (laaoa Mank M net appMcaati)

Th* indication* hat** katow «rW * autontttad ta tha Inta attanal Buraaw iatar • (Spiel ty th* ganaral natwa at th* Indication ι - Aee*a*lan Nwmkar *f Dapoalt **)

C. ~~~ Thi* «h**t «r» r*c*ι-**d with th* international application whan filed (to b* checked fcy tha racantnf Office)

I I AΌΓII ΛΦfϊ (Authonta Officer) .<_.* - Λη C - >___v _ _» v

Th* data ol receipt (Irom th* applicant) b- th* Intarnational Buraau >•

(Authorlxed Officer)

Form PCT θ'*34 (January 1Mt)