HK1012009B - Lipopeptides from actinoplanes sp. endowed with pharmacological activity, process for their preparation and their use - Google Patents

Description

The invention relates to lipopeptides with very homologous amino acid sequences but different fatty acid residues (lipid content) synthesized by Actinoplanes sp. during fermentation and released into the culture medium, a method for isolating lipopeptides from the culture medium and purifying them, the use of lipopeptides as pharmacological agents, particularly against gram-positive bacteria, and Actinoplanes sp. DSM 7358 for the production of the o. g. lipopeptides.

Err1:Expecting ',' delimiter: line 1 column 245 (char 244)

The main use of this secondary metabolite is in the treatment of infectious diseases, but its widespread use often leads to resistance, which creates a constant need for new antibiotics and active substances with new mechanisms of action (Neu H.C., Science 257, 1992, pp. 1064-1073).

In addition, the indications for microbial active substances have been extended to include non-infectious diseases (e.g. tumour therapy, immunomodulation or fat metabolism regulation) and plant protection (herbicides and insecticides), although the active substances used are often still lacking in their efficacy, with unsatisfactory levels of action, excessive toxicity and/or undesirable side effects.

Lipopeptides are described in the literature whose amino acid fraction is identical in sequence or very similar in amino acid composition to the lipopeptides of the invention, but which differ in principle in their lipid fraction from the lipopeptides of the invention.

Examples of lipopeptides, as mentioned above, are: The use of the active substance in the active substance shall be limited to the maximum of the active substance, and the maximum residue concentration shall be at least 0,5%.

These lipopeptides, known as amphomycin-type lipopeptides, are synthesized by microorganisms of the genus Streptomyces. They exert their antibiotic activity against gram-positive bacteria, such as streptococci, staphylococci and enterococci. In particular, strains of the genus Staphylococci and enterococci have recently become increasingly problematic.

A group of strains of microorganisms that have developed resistance are, for example, methicillin-resistant Staphylococcus aureus strains, known as MRSA strains for short.

In addition to the above-mentioned compounds from streptomycetes, a compound from Actinoplanes nipponensis ATCC 31145 is known to have structural similarities to the lipopeptides of the invention due to its spectrum of action and the physicochemical properties described and is called Compound 41.012 (US Patent 4,000.397).

The purpose of the invention is to provide microbial natural substances with improved properties.

This task is solved by fermentation of Actinoplanes sp. in a nutrient solution with carbon and nitrogen source and the usual inorganic salts until lipopeptides, preferably the lipopeptides described below, accumulate in the culture medium, then isolation of the lipopeptides from the culture medium and separation of the mixture into its individual components.

The invention thus relates to: 1. lipopeptides of formula I, in which the R1 means a monounsaturated, monounsaturated, saturated or independently branched or unbranched fatty acid or hydroxy fatty acid with a chain length of 6 to 22 carbon atoms inclusive.2. (a) iC13 fatty acid, (b) iC14 fatty acid, (c) iC13 fatty acid or (d) iC15 fatty acid. R1 = or R1 = or R1 = or R1 = means a process for the production of one or more lipopeptides of formula I, as characterised in 1., one or more lipopeptides of formula II, as characterised in 2., or one or more lipopeptides of formula II, as characterised in 3., characterised by fermentation of Actinoplanes sp. in a culture medium until one or more lipopeptides accumulate in the culture medium and by clearing one or more lipopeptides of formula I from the culture medium.5.

The following describes the invention in detail, in particular in its preferred embodiments, and the content of the claims determines the invention.

Err1:Expecting ',' delimiter: line 1 column 349 (char 348)

Err1:Expecting ',' delimiter: line 1 column 57 (char 56)

All percentages refer to weight unless otherwise stated, and mixing ratios for liquids refer to volume unless otherwise stated.

The method of the invention can be used for fermentation on the laboratory scale (millilitre to litre range) and on the industrial scale (cubic metre scale).

Actinoplanes sp. is isolated from a soil sample. For purification from the soil, the soil is washed with a dilution series of physiological NaCl (0.9%) solution. The various dilutions (100-106) are then spread on actinomycetes fertile soil. After breeding the cultures at 30°C for 2 to 14 days, actinomycetes colonies are formed, which can be isolated and isolated by several successive purification steps.

The genus is determined by morphological and taxonomic criteria according to methods known to the expert.

Due to successive isolation and purification steps, a colony can be isolated from Actinoplanes sp. which releases one or more lipopeptide compounds, preferably lipopeptides A 1437 A, B, C, D, E, F, G, H, K, L and/or M, very efficiently into the culture medium and is referred to as the main producer.

A primary producer is an isolate that produces or releases into the culture medium one or more compounds of the lipopeptides of the invention in a 10 to 100-fold increase compared to isolates of the same actinoplane species.

A highly productive colony of Actinoplanes sp. is being propagated. An isolate was deposited with the German Collection of Microorganisms and Cell Cultures GmbH, Mascheroder Weg 1B, 3300 Braunschweig, Germany, on 18 June 1990 under the following number: Actinoplanes sp. DSM 7358.

Actinoplanes sp. DSM 7358 has an orange mycelium and is characterised by globose sporangions.

In a nutrient solution (also known as culture medium) containing a carbon source and a nitrogen source and the usual inorganic salts, Actinoplanes sp. produces one or more compounds of the lipopeptides of the invention, preferably DSM 7358.

The DSM 7358 strain may also be replaced by mutants and variants of the strain which synthesize one or more of the compounds of the lipopeptides of the invention, which can be produced in a known manner by physical means, such as irradiation, such as by ultraviolet or X-rays, or by chemical mutagenic agents, such as ethyl methan sulphonate (EMS), 2-hydroxy-4-methoxy-benzophenone (MOB) or N-methyl-N'-nitro-N-nitrosoguanidin (MNNG).

The screening for mutants and variants synthesizing one or more compounds of the lipopeptides of the invention shall be carried out as follows: Separation of the mycelium after fermentation;Dropping of lipopeptides at pH 1 to 2 (4°C);Reception of precipitation in H2O/MeOH (1:1);Analysis by HPLC, DC or inhibition test.

The fermentation conditions described below apply to Actinoplanes sp., the deposited isolate DSM 7358, and mutants and variants thereof.

In a nutrient solution containing a carbon source and a nitrogen source and the usual inorganic salts, Actinoplanes sp., preferably DSM 7358, produces the lipopeptides A 1437 A-H and K, L, M.

The most suitable sources of carbon for aerobic fermentation are assimilatable carbohydrates and sugar alcohols, such as glucose, lactose or D-mannitic acid, and natural products containing carbohydrates, such as malt extract. The following nutrients, which contain nitrogen, are considered to be nitrogen: amino acids, peptides and proteins and their degradation products, such as peptones or tryptoins, meat extracts, ground seeds, such as corn, wheat, beans, soybeans or cotton, residues of distillation from alcohol production, meat flour or yeast extract, but also ammonium salts and nitrates.

The lipopeptides of the invention are particularly well formed in a nutritional solution containing approximately 0.1 to 5%, preferably 0.3 to 2% meat extract and 0.2 to 5%, preferably 0.5-2% sucrose and 0.05 to 5 g/l, preferably 0.1 to 0.5 g/l yeast extract and 0.05 to 2 g/l, preferably 0.1 to 1 g/l magnesium sulphate and 0.05 to 10 g/l, preferably 0.1 to 1 g/l potassium or sodium dihydrogen phosphate and 0 to 100 μM, preferably 5 to 20 μM iron-III chloride.

In this nutritional solution, Actinoplanes sp., preferably Actinoplanes DSM 7358, forms a mixture of lipopeptides, preferably consisting of 11 different detectable lipopeptides, of which the lipopeptides of the present invention are the following lipopeptides of formula I, designated A 1437 C, D, F and H.

Lipopeptide A 1437 C has an iso-C13 fatty acid not previously known in amphomycin-type lipopeptides.

A 1437 D has an iso-C14 fatty acid and thus the fatty acid known from Tsushimycin (Shoji et al., The Journal of Antibiotics 21, 439 (1968)). However, A 1437 D differs in its amino acid composition and sequence from the lipopeptides known so far from the state of the art. The amino acid composition of Amphomycin, Glumamycin, Zaomycin and Tsushimycin is identical according to the literature (Strong et al., Antimicrobial Agents and Chemotherapy 1970, 42; Bodanszley et al., J. Am. Chem. Soc. 95, 2352 (1973), Inoue, Bull. Chem. Soc. Jap. 35, 1556 (1962)).

A 1437 F is an ante-iso-C13 fatty acid, so it has the same fatty acid type as amphomycin.

The lipopeptide A 1437 H, like aspartocin, has an ante-iso-C15 fatty acid.

Err1:Expecting ',' delimiter: line 1 column 245 (char 244)

In addition to the lipopeptides of the present invention, Actinoplanes sp. prefers Actinoplanes DSM 7358 to form the following lipopeptides A, B, E, G, K, L and M of formula II:

Lipopeptide A 1437 A has an ante-iso-C13 fatty acid, which is not yet known in lipopeptides of the amphomycin type.

A 1437 B has a fatty acid of the type iso-C14 and thus the fatty acid known from Tsushimycin (Shoji et al., J. of Antibiotics 21, 439 (1968)) and possesses the amino acid sequence of amphomycin (Bodanszky et al., J. Am. Chem. Soc. 95, 2352 (1973)).

A 1437 E is identical to amphomycin known from streptomycetes.

The lipopeptide A 1437 G has an ante-iso-C15 fatty acid, as does aspartocin (Hausmann et al., Antimicrobial Agents and Chemotherapy 1963, 352). The amino acid sequence of A 1437 G is similar to that of amphomycin (Bodansky et al., J. Am. Chem. Soc. 95, 2352 (1973)).

A 1437 K, L and M are unbranched fatty acids with a chain length of C12-C14.

Err1:Expecting ',' delimiter: line 1 column 245 (char 244)

Depending on the composition of the nutritional solution, the quantitative content of one or more of the lipopeptides of the invention may vary, and the composition of the media may control the synthesis of individual lipopeptides, so that no lipopeptide is produced by the micro-organism or in an amount below the detection limit.

The culture medium of Actinoplanes sp., preferably DSM 7358, contains lipopeptides with a single or multiple unsaturated, saturated or independent of a branched or unbranched fatty acid or hydroxy fatty acid with a chain length of 6 to including 22 carbon atoms, preferably 10 to including 20 carbon atoms, particularly preferably 13,14 or 15 carbon atoms.

Such fatty acids are known to the professional, e.g. from the Römpp Chemie Lexikon, Prof. Falbe and Prof. Regitz, 9th ed. Georg Thieme Verlag Stuttgart, New York or from The Encyclopedia of Chemistry C.A. Hampel and G.G. Hawley, 3rd ed. Van Nostrand Reinhold Company, New York.

The following list of fatty acids is illustrative and does not claim to be exhaustive or restrictive.

Saturated, unbranched fatty acids on the lipopeptides of the invention are, for example, capron, Önanth, capryl, pelargon, caprin, undecan, laurin, tridecan, myristin, pentadecan, palmitin, margarine, stearin, nonadecan, arachin, benzoic acid.

Err1:Expecting ',' delimiter: line 1 column 228 (char 227)

Simple unsaturated, unbranched fatty acids on the lipopeptides of the invention are, for example, acrylic or crotonic acid.

For example, a monounsaturated, unsaturated fatty acid at the lipopeptides of the invention is sorbic acid.

Triple unsaturated, unbranched fatty acids on the lipopeptides of the invention are, for example, linoleic or elaeostearic acids.

For example, a quaternary unsaturated, unbranched fatty acid at the lipopeptides of the invention is arachidonic acid.

For example, a five-fold unsaturated, unbranched fatty acid at the lipopeptides of the invention is clupanodic acid.

For example, a hexaunsaturated, unbranched fatty acid at the lipopeptides of the invention is docosahexaenoic acid.

In addition, lipopeptides with multiple branched fatty acids, such as 2',4',6',8'-tetramethyldecanoic acid, may be present in the culture medium.

Err1:Expecting ',' delimiter: line 1 column 231 (char 230)

When 0,01 to 5%, preferably 0,02 to 0,1% L-valine is added to the nutrient solution described above, the Actinoplanes sp. strain gives preference to lipopeptides A 1437 B and D. When 0,01 to 5%, preferably 0,1% to 0,5% L-leucine is added to the nutrient solution described above, the Actinoplanes sp. strain gives preference to lipopeptides A 1437 A and C. When 0,01 to 5%, preferably 0,05 to 0,5% L-isoleucine is added to the above nutrient solution, the Actinoplanes sp. strain forms mainly the lipopeptides A 1437 E, F, G and H. When 0,01 to 5%, preferably 0,05 to 0,5% L-α-aminobutyric acid is added to the above nutrient solution, the Actinoplanes sp. strain forms the lipopeptide K. When 0,01 to 5%, preferably 0,05 to 0,5% L-norvaline is added to the above nutrient solution, the Actinoplanes sp. strain forms the lipopeptides L/SM and M. The same applies to the preferred strain D7358.

In addition to these amino acids, the corresponding α-keto acids of the aforementioned amino acids (α-ketoisovalerate, α-ketoisocaproate, α-keto-β-methylvalerate, α-ketovalerate) or their corresponding acids (isobutyrate, isovalerate, α-methylbutyrate, n-butyrate, propionate, valerate) may be used in the appropriate concentrations or other substances that may interfere with fatty acid biosynthesis. The culture of the micro-organism shall be performed aerobically, i.e. submerged for 6,5 hours or stirred in a shaker or fermenters, if necessary, by air or oxygen. The introduction of this micro-organism should be carried out in a temperature range of approximately 18 to 35 °C, preferably between 25 and 35 °C. The prevention of this should be carried out in general at a pH of between 32 and 300 °C. The micro-organism should be kept in a container for 24 to 35 hours, preferably between 14 and 28 °C. The pH of the container should be between 32 and 28 °C.

It is advantageous to cultivate in several stages, i.e. one or more pre-cultures are first produced in a liquid nutrient medium and then injected into the actual production medium, the main culture, for example in a 1:10 volume ratio. The pre-culture is obtained, for example, by injecting a mycelium into a nutrient solution and growing for about 36 to 120 hours, preferably 48 to 72 hours. The mycelium can be obtained, for example, by growing the stem for about 3 to 40 days, preferably 4 to 10 days, on a solid or liquid soil, for example yeast agar or nutrient broth agar (standard medium for microorganisms with the main component, eg, pectin, pectin and dicarbonate of agar).

The fermentation process can be monitored by the pH of the cultures or the mycelium volume, and by chromatographic methods such as thin film chromatography or high pressure liquid chromatography or biological activity testing.

The following isolation method is used to purify the lipopeptides of the invention, but preferably for the lipopeptides A 1437 C, A 1437 D, A 1437 F and A 1437 H.

The isolation or purification of a lipopeptide of the invention from the culture medium is carried out by known methods, taking into account the chemical, physical and biological properties of the natural substances. To test the concentration of the antibiotic in the culture medium or in the individual isolation steps, thin-film chromatography, e.g. on silica gel with isopropanol/25% NH3 as a solvent or HPLC, may be used. Detection by thin-film chromatographic separation may be carried out, for example, by dye agents such as anisaldehyde, whereby the amount of the formed substance is compared with an acrylic solution.

To isolate a lipopeptide according to the invention, the mycelium is first separated from the culture broth by the usual methods and then the culture filter is adjusted, preferably at 4°C, to a pH of 0,5 to including pH 4, preferably from pH 1,5 to including pH 2.5. The pH adjustment and thus the precipitation of lipopeptides A 1437 may be done with all commercial acids. The solution is incubated for up to 16 hours, preferably up to 4 hours, and the resulting precipitation is then centrifuged.

The precipitate containing all the lipopeptides is resuspended in 1/20 of the original volume of Aqua bidest and adjusted with NaOH to pH 6 to 7. The precipitate is completely dissolved, the solution is cooled to -20°C and lyophilized. The lyophilized solution, hereinafter referred to as the raw product, contains 5 to 30% lipopeptides and is used for further isolation.

The further purification of one or more lipopeptides of the invention is carried out by chromatography on suitable materials, preferably e.g. silica gel, aluminium oxide, ion exchangers or adsorbent resins and particularly preferably on strong or weak base anion exchangers.

The chromatography of lipopeptides is carried out with buffered aqueous solutions or mixtures of aqueous and alcoholic solutions.

For example, water, phosphate buffer, ammonium acetate, citrate buffer, borate buffer in a concentration of 0 to 1 M, preferably 1 to 100 mM, especially phosphate buffered solutions in a concentration of 1 to 100 mM are preferred.

Mixtures of aqueous or alcoholic solutions are all organic solvents which are miscible with water, preferably methanol, acetonitrile, in a concentration of 10 to 80% solvent, preferably 40 to 60% solvent or any buffered aqueous solutions which are miscible with organic solvents.

The separation of lipopeptides due to their different fatty acids is carried out by reverse phase chromatography, for example on MCI® (Mitsubishi Adsorbent Resin, Japan).

Lipopeptides are chromatographed using buffered or acidified aqueous solutions or mixtures of aqueous solutions with alcohols or other organic solvents which are miscible with water.

For example, buffered or acidified aqueous solutions are water, phosphate buffer, ammonium acetate, citrate buffer, borate buffer in a concentration of 0 to 0,5 M, and formic acid, acetic acid, trifluoroacetic acid or any commercial acid known to the professional, preferably in a concentration of 0 to 1%, with a particular preference being given to 0,1%.

Chromatography is carried out with a gradient starting with 100% water and ending with 100% solvent, preferably a linear gradient of 40 to 60% acetonitrile.

The order of the two chromatographies mentioned above (chromatography for separating lipopeptides by amino acids Asp or Asn and by fatty acid type) is reversible, preferring to separate lipopeptides by different amino acids first and then separating them by fatty acid type.

If the above raw material contains lipopeptides with a uniform fatty acid, the prescribed chromatography (separation of lipopeptides due to different fatty acids) is used to desalinate and further purify the lipopeptides.

Alternatively, gel chromatography or hydrophobic phase chromatography may be used.

The gel chromatography is performed on polyacrylamide or mixed polymer gels, such as Biogel-P2® (Fa. Biorad) or Fractogel TSK HW 40® (Fa. Merck, Germany or Toso Haas, USA).

The lipopeptides of the invention are stable in the solid state and in solutions in the pH range 4 to 8, in particular 5 to 7, and can therefore be incorporated into common galenic preparations.

One or more of the compounds of the lipopeptides of the invention are suitable for use as medicinal products due to their valuable pharmacological properties.

The substances of the invention have pharmacological efficacy in particular as antibiotics against gram-positive bacteria, particularly preferably against glycopeptide-resistant strains.

In the case of penicillin- and methicillin-resistant strains (MRSA) which have developed further resistance to antibiotics, only glycopeptides such as vancomycin or teicoplanin often have a therapeutic effect. However, strains resistant to these antibiotics are also increasingly appearing (FEMS Microbiol. Lett. 98 (1992) 109 to 116). One or more compounds of the lipopeptides of the invention also have an excellent effect against these problem germs.

The invention also relates to pharmaceutical preparations of one or more compounds of the lipopeptides of the invention.

One or more compounds of the lipopeptides of the invention, preferably one or more compounds of the lipopeptides A 1437 C, D, F and H, may be administered as such in substance, preferably in combination with suitable excipients or carrier materials. The usual feed mixtures or any pharmacologically compatible carrier materials and/or excipients may be used as carrier materials in veterinary medicinal products or in humans.

The medicinal products of the invention are generally administered orally or parenterally, but rectal administration is also possible in principle. Suitable solid or liquid galenic preparations are, for example, granules, powders, tablets, dragees, (micro) capsules, suppositories, syrups, emulsions, suspensions, aerosols, drops or solutions for injection in the form of ampoules, and preparations with prolonged release of active substance, the manufacture of which is usually carried out with carriers and additives and/or auxiliary explosives such as binders, transdermal agents, sprays, lubricants, lubricants, flavourings, sweeteners or solvents.

Where appropriate, oral dosing units may be microencapsulated to delay or extend administration over a longer period, such as by coating or embedding the active substance in particle form in suitable polymers, waxes or similar.

Preferably, the pharmaceutical preparations are manufactured and administered in dosage units, each unit containing as an active ingredient a specific dose of one or more of the compounds of the lipopeptides of the invention, in fixed dosage units such as tablets, capsules and suppositories, this dose may be up to about 200 mg, but preferably about 0.1 to 100 mg, and in solutions for injection in the form of ampoules up to about 200 mg, but preferably about 0.5 to 100 mg, per day.

The daily dose to be administered depends on the body weight, age, sex and condition of the breast-fed infant, but higher or lower daily doses may be appropriate.

The medicinal products of the invention are manufactured by introducing one or more compounds of the lipopeptides of the invention into the appropriate dosage form with conventional carriers and, where appropriate, additives and/or excipients.

The following examples explain the invention further. Percentages refer to weight. Mixing ratios for liquids refer to volume, unless otherwise stated.

Examples 1a) Production of a glycerol culture from Actinoplanes sp. DSM 7358

100 ml of food solution (4 g/l yeast extract, 15 g/l soluble starch, 1 g/l K2HPO4, 0.5 g/l MgSO4 x 7 H2O filled with water per 1000 ml, pH before sterilisation 7,0) in a sterile 300 ml Erlenmeyer flask are inoculated with the Actinoplanes sp. DSM 7358 strain and incubated for 7 days at 30 °C and 150 UpM on a rotary shaker. 1,5 ml of this culture are then diluted with 2,5 ml of 80% glycerol and stored at -20 °C.

(b) Production of a culture or a preculture in the Erlenmeyer flask of Actinoplanes sp. DSM 7358:

A sterile 300 ml flask of Erlenmeyer containing 100 ml of the following nutrient solution 30 g/l sucrose, 2 g/l KNO3, 1 g/l K2HPO4, 0.5 g/l MgSO4 x 7 H2O, 0.5 g/l KCl, 0.01 g/l FeSO4 x 7 H2O, 2 g/l yeast extract, 5 g/l peptone is inoculated with a culture grown on a strawberry (same nutrient solution but with 2% agar) or with 1 ml of a glycerin culture (see example 1a) and incubated on a shaker at 180 UpM and 30 °C. The maximum production of one or more of the compounds of the lipopeptides of the invention is reached in about 120 hours. For inoculation from 10 to 48 ml of 200 l is sufficient and 96 to 10% of the same nutrient subculture (animal) is used.

2) Comparative characterisation of Actinoplanes sp. DSM 7358

For the characterisation of the Actinoplanes sp. DSM 7358 strain, a comparison with closely related strains is made using the ISP method of Shirling and Gottlieb (Int. J. of Sys. Bacteriol. 16, 3 (1966) 313 to 340). Tabelle 1:

	Actinoplanes sp. DSM 7358	A.utahensis NRRL 12052	A.brasiliensis ATCC 25844	A.nipponensis ATCC 311455
Luftmycel	-	-	+ (ISP 3)	-
Sporangien	+	+	+	+
Medium
ISP 2	orange	orange	gelborange	orange
ISP 3	orange	orange	gelborange	orange
ISP 4	orange	orange	gelborange	orange
ISP 5	orange	orange	gelborange	orange
ISP 6	orange	orange	gelborange	orange
	rotes	braunrotes	rotes	rotes
	Exopigment	Exopigment	Exopigment	Exopigment
Melanin	-	-	(+)	(+)
Glucose	+	+	+	+
Arabinose	+	+	+	+
Saccharose	+	+	+	+
Xylose	+	+	(+)	-
Inosit	+	+	(+)	(+)
Mannit	+	+	+	-
Fructose	+	+	+	-
Rhamnose	+	+	+	+
Raffinose	+	+	(+)	-
Cellulose	+	+	(+)	-
Melibiose	-	-	-	-
Amygdalin	-	+	+	+
Gelatine (Hydrolyse)	+	+	+	+
Citrat (Hydrolyse)	-	-	+	-
Harnstoff (hydrolyse)	+	-	-	-
Arginin hydrolase	+	-	-	-
ß-Galactosidase	-	-	-	-
Tryptophanase	-	-	-	-
Lysindecarboxylase	+	-	-	-
Acetoin (Bildung)	+	+	+	+
Indol (Bildung)	-	-	-	-
	-	-	-	-
NaCl Toleranz	0 - 2,5 %	0 - 2,5 %	0 - 2,5 %	0 - 2,5 %

3a) Manufacture of lipopeptides A 1437 B and D

A 500 1 fermenter shall operate under the following conditions: Nutritional medium: 11 g/l of sucrose 6 g/l of meat extract 0,3 g/l of yeast extract 0,6 g/l MgSO4 0,01 g/l of KH2PO4 The following shall be added to the list of substances: 0,6 g/l L-valine The test chemical is used to determine the concentration of the test chemical in the test medium.

Repeated addition of polyethylene solution can suppress foaming, and the maximum production is reached after about 96 to 120 hours.

After the fermentation of Actinoplanes sp. DSM 7358 has been completed, the culture broth is filtered with the addition of about 2% filter agents (e.g. Celite®) and the culture filter cooled to 4°C and a pH of 1.5. After 4 hours, the culture broth is centrifuged at 10000 g and the precipitate is then resuspended in Aqua. By neutralizing the suspension, the substance is dissolved. It is frozen and lyophilized. The yield is about 1.5 g/l of raw product (= 750 g).

3b) Lone chromatographic separation of the raw product (contains B + D)

A 3.2 l chromatography column (10 cm ID x 40 cm H) is filled with DEAE®sepharose fast flow and balanced with 10 mM potassium phosphate buffer, pH 7.0 in 40% methanol (buffer A). Then 25 g of A 1437 B raw product (obtained as in example 3a), dissolved in 3.5 l of water, applied to the column and washed with 1 l of water, then with 6 l of buffer A. Impurities are deposited through and in the washing water of the raw product. Then a 10 to 100 mM potassium phosphate, pH 7.0 gradient in 40 % methanol is applied. With 25 to 35 mM potassium phosphate A 1437 D-Peptidate is obtained with 55 to 40 mM antibiotic and 1437 B antibiotic.The fractions are removed from the methanol in a vacuum. The desalination is carried out using a 1 l ®Dianion HP-20 column (Mitsubishi, Japan). The 9 l fractions containing pure B-peptide are then applied to the column and washed with 3 l of desalinated water. The water isopropanol is elavated in the gradient process (0 to 50% alcohol content). With 15 to 25% isopropanol, the pure A 1437 B is washed from the carrier. This column effluent is collected separately, compressed in a humid vacuum and freeze-dried.1 g of the antibiotic, 98% purity.

4a) Manufacture of lipopeptides A 1437 A and C

The production is as described in 3a, only the L-valine is replaced in the production medium by 4 g/l L-leucine and the fermentation is carried out in a 50 l bioreactor.

4b) Isolation of lipopeptides A 1437 A and C

10 g of the raw product were dissolved in 100 ml aqua dest. and processed according to the following scheme.

The following is the procedure:

5a) Manufacture of lipopeptides A 1437 E, F, G and H

The production is as described in example 3a, only the L-valine is replaced in the production medium by 1,5 g/l L-isoleucine and the fermentation is carried out in a 50 l bioreactor.

5b) Ion chromatographic separation of the raw peptide

On the column, as described in example 3a, 25 g of lipopeptide raw product obtained in example 5a are separated according to example 3a. The lipopeptide A 1437 F (yield: 1.8 g),18 to 25 mM buffer the A 1437 E (yield: 1.3 g),35 to 50 mM buffer the A 1437 H (yield: 2.7 g) and64 to 82 mM buffer the A 1437 G (yield: 1.9 g) are eluted.

The corresponding fractions are aggregated and released from the methanol in a vacuum.

5c) Cleaning of the components from example 5b to reverse phase RP-18

Fill a 500 ml HPLC preparation column (5.1 cm (ID) x 25 cm H) with ®LiChrosorbG RP-18, 10 μm, and apply the solution containing 1.9 g of the antibiotic to the saline A 1437 G solution. Eluvate in the gradient method with 5% acetonitrile in 10 mM potassium phosphate buffer, pH 7.0 to 36% acetonitrile in 10 mM potassium phosphate buffer, pH 7.0.

The corresponding purification of the A 1437 H solution obtained in Example 5b is carried out with the solvent gradient of 10 to 50% acetonitrile in 10 mM potassium phosphate buffer, pH 7.0.

5d) Purification of lipopeptide antibiotics A 1437 E and F from example 5b on MCI gel

After loading the carrier with the product to be separated, wash with buffer A (5 mM potassium phosphate buffer pH 7.0 with 20% acetonitrile) and elude in the gradient process against buffer B (5 mM potassium phosphate buffer pH 7.0 with 20% acetonitrile). 34 to 35% solvent proportions result in the elution of the pure antibiotic. Concentrate in vacuum and remove ®dianepione to 1.4-20 HP and the result is lipoptid A in 1437 g.

6a) Manufacture of lipopeptide A 1437 K

The production is as described in 4a, only the L-valine is replaced in the production medium by 500 mg/l L-α-aminobutyric acid (or 1 g/l of the racemate) and the fermentation is carried out in a 10 l bioreactor.

6b) Isolation of lipopeptide A 1437 K

10 g of the raw product were dissolved in 100 ml of aqua dest. and processed according to the following scheme.

The Commission has also adopted a proposal for a Regulation on the

10 g raw peptide in 100 ml aqua dest.Absorption to Q-sepharose fast flow (3.5 x 17 cm column) Buffer A: NaH2PO4 1 mM in 50% methanol, pH 5.9Buffer B: NaH2PO4 100 mM in 50% methanol, pH 5.320 Min: Buffer A --→ in 45 min to 25% buffer B, another 45 min at 25% buffer lyophilization of the A 1437 K fractions Desalination to biogel P2 (100-200 mesh) ((7 x 20 cm column) 700 mg A 1437 K (60 %ig) RP chromatography to nucleosil C18 7 μm (20 x 250 mm column) Order: 50 mg of the pre-cleaned productElution with the following gradientBuffer A: aqua bidest., 0.1% TFAPuffer B: acetonitrile10 min.: Buffer A / 5 % Buffer B -- in 60 min. to 70% Buffer B at 10 ml/min. The maximum level of the active substance is 0,05 mg/kg.

7a) Manufacture of lipopeptides A 1437 L and M

The production is as described in 4a, only the L-valine is replaced in the production mixture by 500 mg/l L-norvaline (or 1 g/l of the racemate) and the fermentation is carried out in a 10 l bioreactor.

7b) Isolation of lipopeptides A 1437 L and M

Dissolve 10 g of the raw product in 100 ml of aqua and process according to the following scheme.

The following is the procedure:

10 g raw peptide in 100 ml aqua dest.Absorption to Q-sepharosis fast flow (3,5 x 17 cm column) Elution with the following gradient Buffer A: NaH2PO4 1 mM in 50% methanol, pH 5.9Buffer B: NaH2PO4 100 mM in 50% methanol, pH 5.320 Min: Buffer A --- in 45 min. to 25% buffer B, another 45 min. at 25% buffer Blyophilization of the A 1437 L and M fractions Salination to biogel P2 (100-200 mesh) The test chemical is used to determine the concentration of the active substance in the test chemical. Order: 50 mg of the pre-cleaned productElution with the following gradientBuffer A: aqua bidest., 0.1% TFAPuffer B: acetonitrile10 min.: Buffer A / 5 % Buffer B --→ in 60 min. to 70% Buffer B at 10 ml/min. The total volume of the test chemical is approximately 5 mg/kg.

8) HPLC system for the detection of A 1437 lipopeptides

The system described below allows the separation and quantification of lipopeptides in the raw mixture and culture filter respectively, with retention times ranging from 11.5 minutes (A 1437 E) to approximately 15.9 minutes (A 1437 H). Other

Laufmittel	A	Kalium-Phosphatpuffer pH 7,0, 10 mM
	B	Acetonitril

Gradient	t[Min]	Fluß[ml/min]	A[%]	B[%]
	0	1,5	80	20
	15	1,5	50	50
	15,5	2	00	100
	16,5	2	00	100
	17	1,5	80	20
	22	1,5	80	20

The following shall be used for the calculation of the maximum mass of the vehicle: The dose of the active substance is calculated by dividing the dose of the active substance by the dose of the active substance.

9) Comparison between Actinoplanes nipponensis ATCC 31145 and Actinoplanes spec. DSM 7358

From both strains, a pre-culture is first attracted as described in example 1b and used to inoculate the following production media. Medium 1:as described in example 3a Medium 2:as Medium 1 but without L-valine Medium 3:Glucose 30 g/l; soy flour 20 g/l; Fe2(SO4) 3 0,3 g/l; MnCl2x4H2O 0,3 g/l and CoCl2x6 H2O pH 7,3 each 100 ml of medium in a 300 ml Erlenmeyer flask.

The incubation is carried out at 30°C in a rotary shaker. After 48, 96 and 144 hours, the concentration of A 1437 lipopeptides in the culture filter is determined by HPLC (see Bsp. 8). In medium 2 and medium 3 no lipopeptide is detectable in the strain Actinoplanes nipponensis ATCC 31145. In medium 1 some peptides can be detected in very small quantities after 144 hours. If an identical specificity is used.

10) Effect of the A 1437 lipopeptides

The sensitivity of relevant germs to the A 1437 lipopeptide is determined by an agar dilution test. The agar is Müller-Hinton agar, which is added to 10 per cent horse blood in the case of S. pyogenes and the Enterococcus. The antibiotic-containing plates are inoculated with a multi-channel vaccine (5 x 104 cfu/vaccination site, a stationary culture of the respective strain). The MHK values are read at 37°C. The MHK value is defined as the concentration of the antibiotic at which no visible growth of the germ is detectable after 24 hours of incubation. The results are summarised in Table 2.The control amphomycin was obtained from Boehringer Mannheim (Germany), where it is available as a fine chemical. Tabelle 2:

A 1437 Substanzen: In vitro Aktivität (AB-Spektrum); Konzentration in µg/ml
A 1437 A	0.391	0.781	0.391	1.56	0.391	3.13
A 1437 B	0.098	0.195	0.098	0.195	0.049	0.391
A 1437 C	0.195	0.781	0.391	3.13	0.781	3.13
A 1437 D	0.049	0.195	0.049	0.781	0.391	0.781
A 1437 E	0.098	0.195	0.094	0.098	0.025	0.195
A 1437 F	0.098	0.391	0.195	1.56	0.781	1.56
A 1437 G	0.098	0.195	0.049	0.088	0.025	0.195
A 1437 H	0.049	0.098	0.049	0.195	0.049	0.195
Amphomycin	0.195	0.781	0.391	1.56	0.391	1.56

A 1437 Substanzen: In vitro Aktivität (Vancomycin-resistente Stämme); Konzentration in µg/ml
	Enterococcus faecium VR1	Enterococcus faecium VR2	Streptococcus pyogenes
A 1437 A	nicht bestimmt
A 1437 B	1	1	0.5
A 1437 C	4	4	4
A 1437 D	2	2	1
A 1437 E	4	4	1
A 1437 F	4	4	1
A 1437 G	0.25	0.25	0.125
A 1437 H	1	1	0.5
Amphomycin	4	4	2

The K, L, M compounds have similar in vitro activity to the A-H compounds.

Example 11a: Characterisation of A 1437 D

The lipopeptide A 1437 D is isolated as an amorphous solid The test shall be carried out on the test vessel. HPLC: retention time: 15.1 minutes Amino acids: 2 aspartic acids 1 Asparagine 1 β-methylaspartate 2 Glycine 2,3-Diaminobutyric acid 1 Proline 1 Pipecolinic acid The value of the FAB-MS: m/e = 1303, 6952 [M+H+] The following shall be added to the list of active substances: The CID-MS: m/z = 356, 491, 517, 520, 741, 761, 938, 982 and the CID-MS is the same as the CID-MS. The test chemical is used to determine the concentration of the active substance in the test chemical.

Example 11b: Characterisation of A 1437 B

The lipopeptide A 1437 B is isolated as an amorphous solid The test chemical is used to determine the concentration of methanol in the test vessel. HPLC: retention time: 12.8 minutes Amino acids:3 Aspartic acids 1 β-methylaspartate 2 Glycine 2,3-Diaminobutyric acid 1 Proline 1 Pipecolinic acid The following information shall be provided for the purpose of the calculation of the value of the product: The following shall be added to the list of substances which are to be authorised for use in the manufacture of the product: The CID-MS: m/z = 356, 407, 518, 521, 741, 762, 938, 982 and the CID-MS is the same as the CID-MS. The test chemical is used to determine the concentration of the active substance in the test chemical.

Example 11c: Characterisation of A 1437 C

The lipopeptide A 1437 C is isolated as an amorphous solid The test shall be carried out on the test vessel. HPLC: retention time: 14.1 minutes Amino acids: 2 aspartic acids 1 Asparagine 1 β-methylaspartate 2 Glycine 2,3-Diaminobutyric acid 1 Proline 1 Pipecolinic acid The following shall be added to the list of substances which are to be classified in Annex I to Regulation (EC) No 1907/2006: The CID-MS: m/z = 356, 392, 503, 503, 741, 747, 938, 981 and the CID-MS is the same as the CID-MS. The test chemical is used to determine the concentration of the active substance in the test chemical.

Example 11d: Characterisation of A 1437 A

The lipopeptide A 1437 A is isolated as an amorphous solid The test shall be carried out on the test vessel. HPLC: retention time: 11.8 minutes Amino acids:3 Aspartic acids 1 β-methylaspartate 2 Glycine 2,3-Diaminobutyric acid 1 Proline 1 Pipecolinic acid The following shall be added to the list of substances which are to be classified in Annex I to Regulation (EC) No 1907/2006: The CID-MS: m/z = 356.478, 504, 507, 741, 748, 938, 981 and the CID-MS is the same as the CID-MS: m/z = 356.478, 504, 507, 741, 748, 938, 981 The test chemical is used to determine the concentration of the active substance in the test chemical.

Example 11e: Characterisation of A 1437 F

The lipopeptide A 1437 F is isolated as an amorphous solid The test chemical is used to determine the concentration of methanol in the test vessel. HPLC: retention time: 13.8 minutes Amino acids: 2 aspartic acids 1 Asparagine 1 β-methylaspartate 2 Glycine 2,3-Diaminobutyric acid 1 Proline 1 Pipecolinic acid The following shall be added to the list of substances which are to be classified in Annex I to Regulation (EC) No 1907/2006: The CID-MS: m/z = 356, 392, 503, 506, 741, 747, 938, 981 and the CID-MS is the same as the CID-MS. The test chemical is used to determine the concentration of the active substance in the test chemical.

Example 11f: Characterisation of A 1437 E

The lipopeptide A 1437 E is isolated as an amorphous solid HPLC: retention time: 11.5 minutes Amino acids:3 Aspartic acids 1 β-methylaspartate 2 Glycine 2,3-Diaminobutyric acid 1 Proline 1 Pipecolinic acid The following shall be added to the list of substances which are to be classified in Annex I to Regulation (EC) No 1907/2006: The CID-MS: m/z = 356, 393, 504, 507, 741, 748, 938, 981 and the CID-MS is the same as the CID-MS. The test chemical is used to determine the concentration of the active substance in the test chemical.

Example 11g: Characterisation of A 1437 H

The lipopeptide A 1437 H is isolated as an amorphous solid The test chemical is used to determine the concentration of methanol in the test vessel. HPLC: retention time: 15.9 minutes Amino acids: 2 aspartic acids 1 Asparagine 1 β-methylaspartate 2 Glycine 2,3-Diaminobutyric acid 1 Proline 1 Pipecolinic acid The following shall be added to the list of substances which are to be classified in Annex I to Regulation (EC) No 1907/2006: The CID-MS: m/z = 356, 420, 531, 534, 741, 775, 938, 981 and the CID-MS is the same as the CID-MS. The test chemical is used to determine the concentration of the active substance in the test chemical.

Example 11h: Characterisation of A 1437 G

The lipopeptide A 1437 G is isolated as an amorphous solid The test chemical is used to determine the concentration of methanol in the test vessel. HPLC: Retention time: 13.6 minutes Amino acids: 3 aspartic acids 1 β-methylaspartate 2 Glycine 2,3-Diaminobutyric acid 1 Proline 1 Pipecolinic acid The following shall be added to the list of substances which are to be classified in Annex I to Regulation (EC) No 1907/2006: The CID-MS: m/z = 356, 421, 532, 535, 741, 776, 938, 981 and the CID-MS is the same as the CID-MS. The test chemical is used to determine the concentration of the active substance in the test chemical.

Example 11i: Characterisation of A 1437 K

The lipopeptide A 1437 K is isolated as an amorphous solid HPLC: retention time: 12.5 minutes Amino acids:3 Aspartic acids 1 β-methylaspartate 2 Glycine 2,3-Diaminobutyric acid 1 Proline 1 Pipecolinic acid The following information shall be provided for the purpose of the calculation of the value of the product: The following shall be added to the list of substances which are to be authorised for use in the manufacture of the product: The CID-MS: m/z = 393, 504, 507, 741, 748, 938, 981 and the CID-MS is the same as the CID-MS. The test chemical is used to determine the concentration of the active substance in the test chemical.

Example 11j: Characterisation of A 1437 L

The lipopeptide A 1437 L is isolated as an amorphous solid HPLC: retention time: 13.0 minutes Amino acids:3 Aspartic acids 1 β-methylaspartate 2 Glycine 2,3-Diaminobutyric acid 1 Proline 1 Pipecolinic acid The following information shall be provided for the purpose of the calculation of the value of the goods: The following shall be added to the list of active substances: The CID-MS: m/z = 407, 518, 741, 761, 938, 981 and the CID-MS is the same as the CID-MS. The test chemical is used to determine the concentration of the active substance in the test chemical.

Example 11k: Characterisation of A 1437 M

The lipopeptide A 1437 M is isolated as an amorphous solid HPLC: retention time: 9.8 minutes Amino acids:3 Aspartic acids 1 β-methylaspartate 2 Glycine 2,3-Diaminobutyric acid 1 Proline 1 Pipecolinic acid The following information shall be provided for the purpose of the calculation of the value of the product: The following shall be added to the list of substances which are to be authorised for use in the manufacture of the product: The CID-MS: m/z = 379, 490, 493, 724, 741, 938, 981 and the CID-MS is the same as the CID-MS: m/z = 379, 490, 493, 724, 741, 938, 981 The test chemical is used to determine the concentration of the active substance in the test chemical.

Example 12: C13-chem.

The table shows the C13-chemical shifts of the CH signals from A 1437B Other

For comparison with the IH data, the NH-chemical shifts of the NH signals or, for Pip and Pro, the CαH shifts of the corresponding spin systems were given.

Claims (15)

1. Lipopeptides of the formula I in which

   R¹   is a singly or multiply unsaturated, saturated or, independently thereof, a branched or unbranched fatty acid or hydroxy fatty acid with a chain length of from 6 to 22, inclusive, carbon atoms.
2. A lipopeptide as claimed in claim 1, wherein R¹ has a chain length of from 10 to 20, inclusive, carbon atoms.
3. A lipopeptide as claimed in claim 1, wherein R¹ has a chain length of 12, 13, 14 or 15 carbon atoms.
4. A lipopeptide as claimed in claim 1, wherein R¹ is an

   a) iC₁₃-fatty acid,

   b) iC₁₄-fatty acid,

   c) aiC₁₃-fatty acid or a

   d) aiC₁₅-fatty acid,
5. A lipopeptide as claimed in claim 4, wherein R¹ =
6. A lipopeptide as claimed in claim 4, wherein R¹ =
7. A lipopeptide as claimed in claim 4, wherein R¹ =
8. A lipopeptide as claimed in claim 4, wherein R¹ =
9. A process for the production of a lipopeptide of the formula I as claimed in one or more of claims 1 to 8, which comprises fermenting Actinoplanes sp. in a culture medium until one or more lipopeptides accumulate in the culture medium and purifying the lipopeptide of the formula I from the culture medium.
10. The process as claimed in claim 9, wherein the purification takes place by precipitating the lipopeptides from the culture medium using acids at pH 0.5 to pH; 4, inclusive, then where appropriate purifying the lipopeptides obtained from the precipitation by chromatography on anion exchangers or chromatographic on a hydrophobic matrix, it being possible for the to chromatographies to be carried out as alternatives or consecutively in any desired sequence.
11. The process as claimed in claim 9 or 10, wherein Actinoplanes sp. DSM 7358 is fermented.
12. A pharmaceutical containing one or more lipopeptides of the formula I as claimed in one or more of claims 1 to 8 and, where appropriate, pharmaceutical vehicles.
13. The use of a lipopeptide of the formula I as claimed in any one of claims 1 to 8 for the production of an antibiotic against Gram-positive bacteria.
14. The use as claimed in claim 13, wherein the bacteria are glycopeptide-resistant bacteria.
15. Actinoplanes sp. DSM 7358.