U.S. patent application number 10/103880 was filed with the patent office on 2002-12-05 for vancoresmycin, a process for its production and its use as a pharmaceutical.
Invention is credited to Aszodi, Jozsef, Bhat, Ravi Gaianan, Hopmann, Cordula, Kurz, Michael, Le Beller, Dominique, Naker, Shantilal Dayaram, Oak, Uttara Vinayak, Ramakrishna, Nirogi Venkata Satya, Seibert, Gerhard, Sreekumar, Eyyammadichiyil Sankaranarayanan, Tanpure, Rajendra Prakash, Vijayakumar, Erra Koteswara Satya, Wink, Joachim.
Application Number | 20020183267 10/103880 |
Document ID | / |
Family ID | 8232941 |
Filed Date | 2002-12-05 |
United States Patent
Application |
20020183267 |
Kind Code |
A1 |
Ramakrishna, Nirogi Venkata Satya ;
et al. |
December 5, 2002 |
Vancoresmycin, a process for its production and its use as a
pharmaceutical
Abstract
The present invention relates to a compound named Vancoresmycin
which is obtainable by cultivation of the microorganism HIL-006734
(DSM 12216), and to its pharmaceutically acceptable salts. The
present invention further relates to a process for the production
of Vancoresmycin, to the microorganism HIL-006734 (DSM 12216), to
the use of Vancoresmycin and its pharmaceutically acceptable salts
as pharmaceuticals, and in particular to their use as antibiotics,
and to pharmaceutical compositions comprising Vancoresmycin or a
pharmaceutically acceptable salt thereof.
Inventors: |
Ramakrishna, Nirogi Venkata
Satya; (Mumbai, IN) ; Bhat, Ravi Gaianan;
(Mumbai, IN) ; Sreekumar, Eyyammadichiyil
Sankaranarayanan; (Ambarnath (East), IN) ;
Vijayakumar, Erra Koteswara Satya; (Mumbai, IN) ;
Naker, Shantilal Dayaram; (Mumbai, IN) ; Oak, Uttara
Vinayak; (Charai, IN) ; Tanpure, Rajendra
Prakash; (Manmad, IN) ; Hopmann, Cordula;
(Frankfurt am Main, DE) ; Kurz, Michael; (Hofheim,
DE) ; Wink, Joachim; (Rodermark, DE) ;
Seibert, Gerhard; (Darmstadt, DE) ; Le Beller,
Dominique; (Jaux, FR) ; Aszodi, Jozsef;
(Pontault Combault, FR) |
Correspondence
Address: |
FINNEGAN, HENDERSON, FARABOW, GARRETT &
DUNNER LLP
1300 I STREET, NW
WASHINGTON
DC
20005
US
|
Family ID: |
8232941 |
Appl. No.: |
10/103880 |
Filed: |
March 25, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10103880 |
Mar 25, 2002 |
|
|
|
09436718 |
Nov 9, 1999 |
|
|
|
6387943 |
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Current U.S.
Class: |
514/43 ; 435/74;
536/17.4 |
Current CPC
Class: |
A61P 31/04 20180101;
C12P 17/16 20130101; C07H 15/04 20130101; C12P 19/44 20130101 |
Class at
Publication: |
514/43 ;
536/17.4; 435/74 |
International
Class: |
A61K 031/7052; C07H
017/02; C12P 019/44 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 9, 1998 |
EP |
98121299.6 |
Claims
We claim:
1. Vancoresmycin, a compound of the formula: 3and its
pharmaceutically acceptable salts and derivatives, in all their
stereoisomeric and tautomeric forms.
2. Vancoresmycin, a compound of the molecular formula
C.sub.71H.sub.126N.sub.2O.sub.21, obtainable by cultivation of the
microorganism Amycolatopsis species HIL-006734 (DSM 12216) or one
of its variants or mutants under aerobic conditions in a nutrient
medium containing sources of carbon and nitrogen, followed by
isolation and purification, and its pharmaceutically acceptable
salts, and derivatives, in all their stereoisomeric and tautomeric
forms.
3. A process for the production of a compound as claimed in claim 1
comprising the steps of: cultivating the microorganism
Amycolatopsis species HIL-006734 (DSM 12216) or one of its variants
or mutants under aerobic conditions in a nutrient medium containing
sources of carbon and nitrogen to produce Vancoresmycin, isolating
the Vancoresmycin, and purifying the Vancoresmycin.
4. A process for the production of a compound as claimed in claim 2
comprising the steps of: cultivating the microorganism
Amycolatopsis species HIL-006734 (DSM 12216) or one of its variants
or mutants under aerobic conditions in a nutrient medium containing
sources of carbon and nitrogen to produce Vancoresmycin, isolating
the Vancoresmycin, and purifying the Vancoresmycin.
5. A process as claimed in claim 3, further comprising the step of
reacting the Vancoresmycin with a suitable agent to form a
pharmaceutically acceptable salt or derivative.
6. A process as claimed in claim 4, further comprising the step of
reacting the Vancoresmycin with a suitable agent to form a
pharmaceutically acceptable salt or derivative.
7. Amycolatopsis species HIL-006734 (DSM 12216) or one of its
variants or mutants.
8. A pharmaceutical composition, comprising an effective amount of
Vancoresmycin or a pharmaceutically acceptable salt or derivative
thereof as claimed in claim 1 and a pharmaceutically acceptable
carrier.
9. A pharmaceutical composition, comprising an effective amount of
Vancoresmycin or a pharmaceutically acceptable salt or derivative
thereof as claimed in claim 2 and a pharmaceutically acceptable
carrier.
10. A method of treating an infection comprising administering to a
patient in need thereof an effective amount of a compound as
claimed in claim 1.
11. A method of treating an infection comprising administering to a
patient in need thereof an effective amount of a compound as
claimed in claim 2.
12. A method of treating a Staphylococcus aureas infection
comprising administering to a patient in need thereof an effective
amount of a compound as claimed in claim 1.
13. A method of treating a Staphylococcus aureas infection
comprising administering to a patient in need thereof an effective
amount of a compound as claimed in claim 2.
14. The method of claim 12, wherein the Staphylococcus aureas is
vancomycin-resistant and/or teicoplanin-resistant.
15. The method of claim 13, wherein the Staphylococcus aureas is
vancomycin-resistant and/or teicoplanin-resistant.
16. The method of claim 10, wherein the infecting organism is a
Staphylococcus.
17. The method of claim 11, wherein the infecting organism is a
Staphylococcus.
18. The method of claim 10, wherein the infecting organism is an
Enterococcus.
19. The method of claim 11, wherein the infecting organism is an
Enterococcus.
Description
[0001] Under the provisions of Section 119 of 35 U.S.C., Applicants
hereby claim the benefit of the filing date of European Patent
Application Number 98121299.6, filed Nov. 9, 1998, which
Application is hereby incorporated by reference.
FIELD OF THE INVENTION
[0002] This invention relates to a compound named Vancoresmycin,
which is obtainable by cultivation of the microorganism HIL-006734
(DSM 12216), and to its pharmaceutically acceptable salts and
derivatives. The present invention further relates to a process for
the production of Vancoresmycin, to the microorganism HIL-006734
(DSM 12216), to the use of Vancoresmycin and its pharmaceutically
acceptable salts and derivatives as pharmaceuticals, including
their use as antibiotics, and to pharmaceutical compositions
comprising Vancoresmycin or a pharmaceutically acceptable salt or
derivative thereof.
BACKGROUND OF THE INVENTION
[0003] Methicillin resistant Staphylococcus aureus (MRSA)
infections are known to be predominant in several infectious
conditions, for example, wounds and burns. Vancomycin and
teicoplanin, belonging to the glycopeptide class, are the only two
antibiotics clinically used for the treatment of MRSA infections.
Due to the recent emergence of vancomycin- and
teicoplanin-resistant strains, however, these infections are
reported to have become menacing and fatal. In response, an
intensive search for a structurally different class of compounds
active against these vancomycin- and teicoplanin-resistant strains
has been initiated. For instance, methylsulfomycin I, a cyclic
thiopeptide, has been described earlier (European Patent
Publication No. 0818539 filed Jul. 11, 1996) as an antibiotic
active against vancomycin- and teicoplanin-resistant strains.
[0004] It has now been found that a novel compound named
Vancoresmycin has antibiotic activity.
DETAILED DESCRIPTION OF THE INVENTION
[0005] The present invention relates to Vancoresmycin, a compound
of the formula: 1
[0006] and to its pharmaceutically acceptable salts and
derivatives, such as esters, ethers, and obvious chemical
equivalents, including all stereoisomeric forms and all tautomeric
forms.
[0007] Vancoresmycin has the molecular formula
C.sub.71H.sub.126N.sub.2O.s- ub.21 (MW1343.80) and may be
characterized by any one or more of its physico-chemical and
spectral properties given below, such as its .sup.1H NMR
spectroscopic data and its .sup.13C NMR spectroscopic data, both
provided in Table 2.
[0008] Vancoresmycin is a new antibiotic active against vancomycin-
and teicoplanin-resistant strains. It has a hitherto unreported
structure with a tetramic acid moiety bearing an acyl substituent
at the 3-position. This acyl substituent has a highly oxygenated
long alkyl chain substituted with an amino sugar. A chemical
abstract literature search established Vancoresmycin to be a new
compound.
[0009] Vancoresmycin may be obtained by cultivating a microorganism
referred to as culture No. HIL-006734 (henceforth referred to as
HIL-006734). This microorganism was isolated from a soil sample
collected from National Park, Borivli, Mumbai, India. The
microorganism HIL-006734 belongs to the order of Actinomycetales,
genus Amycolatopsis. It was deposited on Jun. 4, 1998 with the
German Collection of Microorganisms and Cell Cultures
(DSMZ--Deutsche Sammlung von Mikroorganismen und Zellkulturen
GmbH), Braunschweig, Germany, and has been given the accession
number DSM No. 12216.
[0010] The present invention further provides a process for the
production of Vancoresmycin from amycolatopsis species HIL-006734,
its mutants and variants, comprising the steps of: growing the
amycolatopsis species HIL-006734 under aerobic conditions in a
nutrient medium containing one or more sources of carbon and one or
more sources of nitrogen and optionally nutrient inorganic salts
and/or trace elements; isolating the Vancoresmycin compound; and
purifying the Vancoresmycin compound in a customary manner.
[0011] Mutants and variants of the microorganism HIL-006734 may
also be able to synthesize the compound according to the present
invention. Such mutants may be produced in a known manner by
physical means, for example irradiation such as with ultraviolet-
or X-rays, or chemical mutagens, such as ethylmethylansulfonate
(EMS), 2-hydroxy-4-methoxy-benzophenone (MOB) or
N-methyl-N'-nitro-N-nitrosoguanidine (MNNG).
[0012] The screening for suitable mutants and variants which can
produce the compound according to the invention can be confirmed by
determination of the biological activity of the active compounds
accumulated in the culture broth, for example by testing the
antibacterial action.
[0013] The nutrient medium preferably contains sources of carbon,
nitrogen and nutrient inorganic salts. The carbon sources are, for
example, starch, glucose, sucrose, dextrin, fructose, molasses,
glycerol, lactose, or galactose. A typical carbon source is starch.
The sources of nitrogen are, for example, soybean meal, peanut
meal, yeast extract, beef extract, peptone, malt extract, corn
steep liquor, gelatin, casamion acids. Peptone and yeast extract
are typical. The nutrient inorganic salts are, for example, sodium
hydrogen phosphate, potassium hydrogen phosphate, sodium chloride,
calcium chloride, calcium carbonate, potassium nitrate, ammonium
sulphate, and magnesium sulphate. Calcium carbonate, sodium
chloride, and magnesium sulphate are typical.
[0014] The cultivation of HIL-006734 may be carried out at
temperatures between 25-35.degree. C. and pH between 6.0 and 8.0.
Typically, HIL-006734 is cultivated at 30.degree. C. (.+-.1.degree.
C.) and pH 7.0.
[0015] A good yield of the antibiotic of the invention may be
obtained by cultivating HIL-006734 for 60-96 hours. Typically,
cultivation is carried out by fermentation for 68-96 hours under
submerged conditions, for example in shake flasks, as well as in
laboratory fermenters. The progress of fermentation and formation
of the Vancoresmycin can be detected by High Pressure Liquid
Chromatography (HPLC) and by measuring the bioactivity of the
culture broth against Staphylococci and Enterococci species by the
known microbial agar plate diffusion assay method. The preferred
culture is Staphylococcus aureus 3066, which is a resistant strain
to methicillin, a .beta.-lactam antibiotic reported in the
literature, and Entrococcus faecium (E. faecium VR-1), which is
resistant to vancomycin. In the resulting culture broth,
Vancoresmycin is present in the culture filtrate as well as in
mycelium and can be isolated using known separation techniques.
Thus, it can be recovered from the culture filtrate by extraction
at pH 5-8 with a water immiscible solvent such as ethyl acetate,
dichloromethane, chloroform, or butanol, or by hydrophobic
interaction chromatography using polymeric resins such as "Diaion
HP-20.RTM." (Mitsubishi Chemical Industries Limited, Japan),
"Amberlite XAD.RTM." (Rohm and Hass Industries U.S.A.), activated
charcoal, or by ion exchange chromatography at pH 5-8. Typically,
the active material is extracted with ethyl acetate. The active
material can also be recovered from mycelium by extraction at pH
5-8 with a water miscible solvent such as methanol, acetone,
acetonitrile, n-propanol, or iso-propanol. Alternatively, it may be
extracted at pH 5-8 with a water immiscible solvent such as ethyl
acetate, dichloromethane, chloroform, or butanol. Typically, the
active material is extracted at pH 5-8 with ethyl acetate.
Concentration and lyophilization of the extracts gives the active
crude material.
[0016] The antibiotic Vancoresmycin of the present invention may,
for example, be recovered from the crude material as follows:
[0017] By fractionation using any of the following techniques:
normal phase chromatography (using alumina or silica gel as
stationary phase; eluents such as petroleum ether, ethyl acetate,
methylene chloride, acetone, chloroform, methanol, or combinations
thereof; and additions of amines such as NEt.sub.3); reverse phase
chromatography (using reverse phase silica gel such as
dimethyloctadecylsilylsilica gel, (RP-18) or dimethyloctylsilyl
silica gel (RP-8) as stationary phase; and eluents such as water,
buffers (for example, phosphate, acetate, citrate (pH 2-8)), and
organic solvents (for example, methanol, acetonitrile, acetone,
tetrahydrofuran, or combinations of these solvents)); gel
permeation chromatography (using resins such as .RTM.Sephadex LH-20
(Pharmacia Chemical Industries, Sweden), TSKgel .RTM.Toyopearl HW
(TosoHaas, Tosoh Corporation, Japan) in solvents such as methanol,
chloroform, acetone, ethyl acetate, or their combinations, or
.RTM.Sephadex G-10 and G-25 in water); or by counter-current
chromatography (using a biphasic eluent system made up of two or
more solvents such as water, methanol, ethanol, iso-propanol,
n-propanol, tetrahydrofuran, acetone, acetonitrile, methylene
chloride, chloroform, ethyl acetate, petroleum ether, benzene, and
toluene). These techniques may be used repeatedly, alone or in
combination. A typical method is chromatography over reverse phase
silica gel (RP-18).
[0018] The compound Vancoresmycin may be converted into
pharmaceutically acceptable salts and derivatives, like esters and
ethers, and other obvious chemical equivalents, which are all
covered by the present invention. The salts and derivatives can be
prepared by standard procedures known to one skilled in the art.
Salts like sodium and potassium salts, for example, may be prepared
by treating Vancoresmycin with suitable sodium or potassium
bases.
[0019] Esters and ethers may be prepared by the methods given in
the literature, for example, in Advanced Organic Synthesis,
4.sup.th Edition, J. March, John Wiley & Sons., 1992.
[0020] The amino group of the sugar moiety can be alkylated or
acetylated, e.g. with acid chlorides by standard procedures known
to one skilled in the art.
[0021] Chemical equivalents may be stable complexes with metal
ions, e.g. transition metals like La.sup.3+, Sm.sup.3+, Eu.sup.3+,
Gd .sup.3+, which are typical for tetramic acid derivatives and may
be prepared by the methods given in the literature (K. Tanaka et.
al., Chem. Pharm. Bull. 1979, 27, 1901; K. Matsuo, Chem. Pharm.
Bull. 1980, 28, 2494).
[0022] The double bonds of the alkyl side chain may be reduced by
the methods given in the literature, for example in P. N. Rylander,
"Hydrogenation Methods", Academic Press, New York (1985), Chpt. 2,
or may be hydrohalogenated by methods described by H. O. House in
"Modern Synthetic Reactions", W. A. Benjymin, Inc., New York
(1972), pp 446-452. Hydroxylated derivatives may be produced by
reaction of the double bonds with reagents such as OsO.sub.4 as
described in the literature, e.g. in Chem. Rev. 1980, 80, 187.
[0023] Derivatives may also be formed by conversion of the double
bonds into epoxides by oxidation, e.g. with MCPBA, as described in
Advanced Organic Synthesis, 4.sup.th Edition, J. March, John Wiley
& Sons., 1992.
[0024] Vancoresmycin has antibacterial activity. Minimum inhibitory
concentrations of Vancoresmycin against a wide range of bacterial
are given in Table 3 below. Vancoresmycin and its pharmaceutically
acceptable salts and derivatives can be administered to animals,
such as mammals, including humans, as pharmaceuticals on their own,
in mixtures with one another, and in the form of pharmaceutical
compositions that permit parenteral administration. Accordingly,
the present invention also relates to Vancoresmycin and its
pharmaceutically acceptable salts and derivatives for use as
pharmaceuticals and to the use of Vancoresmycin and its
pharmaceutically acceptable salts and derivatives for the
production of medicaments having antibacterial activity. The
present invention further relates to pharmaceutical compositions
which contain an effective amount of Vancoresmycin and/or one or
more pharmaceutically acceptable salts and/or derivatives thereof,
together with a pharmaceutically acceptable carrier.
[0025] Vancoresmycin can be administered orally, intramuscularly,
intravenously, or by other modes of administration. Pharmaceutical
compositions which contain Vancoresmycin or a pharmaceutically
acceptable salt or derivative thereof with other pharmaceutically
active substances can be prepared by mixing the active compounds
with one or more pharmacologically tolerated auxiliaries and/or
excipients such as, for example, fillers, emulsifiers, lubricants,
masking flavors, colorants, or buffer substances, and converting
the mixture into a suitable pharmaceutical form such as, for
example, tablets, coated tablets, capsules, granules, powders,
emulsions, suspensions, or solutions suitable for parenteral
administration.
[0026] Examples of auxiliaries and/or excipients which may be
mentioned are tragacanth, lactose, talc, agar-agar, polyglycols,
ethanol, and water. Suitable and preferred for parenteral
administration are suspensions or solutions in water. It is also
possible to administer the active substances as such, without
vehicles or diluents, in a suitable form, for example, in
capsules.
[0027] As is customary, the galenic formulation and the method of
administration as well as the dosage range which are suitable in a
specific case depend on the species to be treated and on the state
of the respective condition or disease, and can be optimized using
methods known in the art. On average, the daily dose of active
compound in a patient of about 75 kg weight is at least 0.001 mg to
at most 10 mg, typically at most 1.0 mg.
[0028] The following are provided as illustrative examples of the
present invention and do not limit the scope thereof:
EXAMPLE 1
Isolation of the Culture No. HIL-006734 From Soil
[0029] a) Composition of Nutrient Isolation Medium
1 Corn starch 10.0 g Casein 1.0 g Peptone 1.0 g Beef extract 1.0 g
K.sub.2HPO.sub.4 0.5 g Agar powder 13.0 g Demineralized water 1.0
liter pH 7.5
[0030] b) Soil Plating and Isolation
[0031] 10 g of soil collected from National Park, Borivli, Mumbai,
India were added to 90 ml of sterilized water in a 250 ml
Erlenmeyer flask which was shaken for 2 hours on a rotary shaker
(220 rpm). The above soil suspension was serially diluted in steps
of 10 up to 10.sup.-5. From the last dilution, 1 ml of suspension
was placed at the center of a sterile glass petri plate (15 cm
diameter) to which was poured approximately 50 ml of the above
isolation medium supplemented with 25 .mu.g/ml of amphotericin B as
antifungal agent and cooled to 45.degree. C. and the plate swirled
thoroughly. The mixture of soil suspension and medium was allowed
to settle and incubated at 28.degree. C. (.+-.1.degree. C.) for 7
days. The petri plate was periodically observed and HIL-006734
(culture No. Y-9439786) was isolated from amongst the growing
microorganisms.
EXAMPLE 2
Maintenance of the Culture No. HIL-006734
[0032] Composition of Maintenance Medium
[0033] HIL-006734 was maintained on the following medium:
2 Malt extract 10.0 g Glucose 4.0 g Yeast extract 4.0 g Actidiol
0.05 g Agar powder 13.0 g Demineralized water 1 liter pH
7.0-7.5
[0034] After dissolving the ingredients thoroughly by heating, the
resultant solution was distributed in test tubes and sterilized at
121.degree. C. for 20 min. The test tubes were cooled and allowed
to solidify in a slanting position. The agar slants were streaked
with the growth of HIL-006734 by a wire loop and incubated at
28.degree. C. (.+-.1.degree. C.) until a good growth was observed.
The well-grown cultures were stored in the refrigerator at
+8.degree. C.
[0035] Preparation of Glycerol Working Seed
[0036] Composition of Medium
3 Yeast extract 4.0 g Soluble starch 15.0 g K.sub.2HPO.sub.4 1.0 g
MgSO.sub.4 .times. 7 H.sub.2O 0.5 g Demineralized water 1 liter PH
7.0
[0037] The above medium was distributed in 100 ml amounts in 300 ml
Erlenmeyer flasks and autoclaved at 121.degree. C. for 20 minutes.
The flasks were cooled to room temperature and inoculated with the
abovementioned agar slant. The incubation was carried out for five
days on a rotary shaker at 180 rpm and 28.degree. C. 1.5 ml of this
culture was mixed with 1.5 ml glycerol (99%) and stored at
-20.degree. C.
EXAMPLE 3
Fermentation of the Culture No. HIL-006734 in Shake Flasks
[0038] Composition of Seed Medium
4 Glucose 15.0 g Soybean meal 15.0 g Corn steep liquor 5.0 g
CaCO.sub.3 2.0 g NaCl 5.0 g Demineralized water 1 liter pH
6.8-7.0
[0039] The medium could be used with or without corn steep
liquor.
[0040] The above medium was distributed in 100 ml amounts in 500 ml
Erlenmeyer flasks and autoclaved for 20 mins. The flasks were
cooled to room temperature and each flask was inoculated with a
loopful of the above mentioned well-grown culture of Example 2 and
shaken on a rotary shaker for 72 hours at 240 rpm at 27.degree. C.
(.+-.1.degree. C.) to give seed culture.
[0041] Composition of Production Medium
5 Glucose 20.0 g Soybean meal 10.0 g CaCO.sub.3 0.2 g
CoCl.sub.2.6H.sub.2O 0.001 g Demineralized water 1 liter pH 6.8 or
Starch 10.0 g Glucose 10.0 g Glycerol 99% 10.0 g Corn steep liquor
2.5 g Peptone 5.0 g Yeast extract 2.0 g NaCl 1.0 g CaCO.sub.3 3.0 g
Demineralized water 1 liter pH 7.2 (before sterilization)
[0042] The production medium was distributed in 100 ml amounts in
500 ml Erlenmeyer flasks and autoclaved at 121.degree. C. for 20
min. The flasks were cooled to room temperature and inoculated with
the abovementioned seed culture (2% v/v). The fermentation was
carried out on a rotary shaker at 240 rpm and 27.degree. C.
(.+-.1.degree. C.) for 48 hours. The production of the antibiotic
was determined by testing the bioactivity against S. aureus 3066
and Ent. faecium VR-1 using the well diffusion method in a known
manner.
EXAMPLE 4
Cultivation of the Culture No. HIL-006734 in Fermenters
[0043] Preparation of Seed Culture in Shake Flasks
[0044] The seed medium of Example 3 was distributed in 150 ml
amounts in 1000 ml Erlenmeyer flasks and autoclaved at 121.degree.
C. for 20 mins. The seed culture was grown in these flasks as
described in Example 3.
[0045] Large Scale Fermentation
[0046] Composition of production medium:
6 Glucose 20.0 g Soybean meal 10.0 g CaCO.sub.3 0.2 g
CoCl.sub.2.6H.sub.2O 0.001 g Demineralized water 1 liter pH 7.0 or
Starch 10.0 g Glucose 10.0 g Glycerol 99% 10.0 g Corn steep liquor
2.5 g Peptone 5.0 g Yeast extract 2.0 g NaCl 1.0 g CaCO.sub.3 3.0 g
Demineralized water 1 liter pH 7.2 (before sterilization)
[0047] 20 liters of the production medium in 22 liter fermenter (in
two fermenters) and 9 liters of the production medium in 12 liter
fermenter (in two fermenters) along with 1 ml(/10 liter fermenter)
of .RTM.Desmophen as antifoaming agent was sterilized in situ for
40 mins. at 121.degree. C., cooled to 27.degree. C. (.+-.1.degree.
C.) and seeded with 1.5 liter(/22 liter fermenter) or 0.75 liter
(/12 liter fermenter) of the seed culture mentioned above.
[0048] The fermentation was run with the following parameters:
7 Temperature 27.degree. C. (.+-.1.degree. C.) Agitation 200 rpm
Aeration 15 lpm/22 liter fermenter 10 lpm/12 liter fermenter
Harvest time 64 hours
[0049] The production of the antibiotic was determined by testing
the bioactivity against S. aureus 3066 and Ent. faecium VR-1 and
HPLC analysis. The final pH of the culture broth was 7.0-7.5. The
culture broth was harvested and centrifuged and the antibiotic was
isolated and purified from the culture filtrate and the mycelium by
the method described in the Example 5 or 6.
EXAMPLE 5
Isolation and Purification of Vancoresmycin
[0050] The culture broth (60 liters) was harvested and centifuged
to separate the mycelium (2.5 kg) and culture filtrate (50 liters,
pH 7.3). The mycelium was extracted with methanol (2.times.20
liters) and the active extracts were pooled and concentrated under
reduced pressure to get 70 g of crude material. The culture
filtrate (50 liters, pH 7.3) was adjusted to pH 5.5 with 2N
hydrochloric acid and passed through a column of .RTM.Diaion HP-20
(2.5 liters). The column was washed with water (10 liters) followed
by 15 liters of methanol:water (1:1). The active compound was found
to be present in 15 liters of methanol:water (3:1) and 30 liters
methanol eluates. The monitoring of the purification was done by
bioassay against S. aureus 3066 and Ent. faecium VR-1. The active
eluates were pooled and concentrated to get 15 g of crude material.
The combined crude was chromatographed on .RTM.Sephadex LH-20
column (2.5 cm.times.90 cm) in methanol repeatedly and the active
fractions were pooled and concentrated. This was further
chromatographed on .RTM.Toyopearl TSK HW 40F column (6 cm.times.35
cm) in methanol. The active fractions were pooled and concentrated
under reduced pressure to get 3 g of semipure material. The final
purification was done by preparative HPLC using the following
conditions:
[0051] The semi-pure material was finally purified by preparative
HPLC on a 25 mm.times.250 mm Hibar -Lichrospher RP-18 (10 .mu.
using 67.5:32.5 Methanol:Phosphate buffer (0.01 M, pH 6.5) as the
eluant at a flow rate of 23 ml/min and detection at 220 nm).
[0052] The active eluates were pooled and concentrated under
reduced pressure to remove the solvent and then desalted on
.RTM.Diaion HP-20 (50 ml) column, eluted with acetonitrile:water
(80:20) and concentrated under reduced pressure and lyophilized to
get 70 mg of pure compound.
EXAMPLE 6
Isolation and Purification of Vancoresmycin
[0053] The culture broth (200 liters) was harvested and centrifuged
to separate the mycelium and the culture filtrate. The mycelium was
exhaustively extracted with methanol (30-40 L) and the extract
concentrated 1:10 to obtain a colorless precipitation which was
filtered off to obtain 30-50 g of crude material. This material was
further purified by HPLC:
[0054] 1.) Column: Fractogel TSK-HW 40 (4 L, 500.times.100 mm)
[0055] Eluent: MeOH
[0056] Flow: 20 ml/min
[0057] Detection: 204 and 236 nm
[0058] The active fractions were eluted after 125 min. The pooled
fractions were concentrated under reduced pressure and freeze
dried.
[0059] 2.) Column: Silica Gel 60 (Merck); Erimatech (300.times.20
mm, 100 ml)
[0060] Eluent: A) CH.sub.2Cl.sub.2:MeOH 9:1 B)
CH.sub.2Cl.sub.2:MeOH 3:1+1% NEt.sub.3 C) MeOH
8 Gradient: min % A % B % C 0 100 0 0 23 100 0 0 24 0 100 0 51 0
100 0 52 0 0 100 93 0 0 100 Flow Rate: 25 ml/min Detection: 236 and
288 nm
[0061] The vancoresmycin-containing fractions eluted after 32 min.
The pooled fractions were concentrated under reduced pressure and
freeze dried. The yield of the two purification columns was
50%.
[0062] The physico-chemical and spectral properties of
Vancoresmycin are given in Tables 1 and 2. The minimum inhibitory
concentrations (MIC) against various bacteria are listed in Table
3.
9TABLE 1 Appearance White solid Solubility Methanol, DMSO Melting
point 141-143.degree. C. [.alpha.].sub.D -13.degree.(c 0.2,
Methanol) TLC (Thin Layer Rf: 0.6 Chromatography) [Silica gel plate
(Article No. 5554, E. Merck); n-BuOH:MeOH:water (4:1:2)] HPLC (High
Pressure Retention time: 14.70 min Liquid Chromatography) [Column:
LiChrocart (250 mm .times. 4 mm) RP Select B (5 .mu.; Eluant:
Gradient of 0.1% aqueous orthophospharic acid (pH 2.5) to
CH.sub.3CN in 20 min; Flow rate: 1 ml/min; Detection: 220 nm] Fig.
1 of the accompanying drawings or: Column: Purospher Star RP.18e
(Merck), 55 .times. 4 mm, 3 .mu.m Eluent: CH.sub.3CN/0,01%
H.sub.3PO.sub.4 (85%) Gradient: time % CH.sub.3CN 0.00 5.0 3.00
95.0 5.00 95.0 6.00 5.0 10.00 5.0 Flow: 2 ml/min Temp.: 40.degree.
C. Detection: 210 nm, 254, 280, 320, 380 t.sub.R: 2.19 min ESI-MS
(Electrospray 1342 (M - H) lonisation Mass) HR-FAB-MS (High
1343.89142 (M + H).sup.+ resolution Fast Atom [Calculated for
C.sub.71H.sub.127N.sub.2O.sub.21: Bombardment Mass) 1343.889915 (M
+ H).sup.+] Mol. formula: C.sub.71H.sub.126N.sub.2O.sub.21 UV/VIS:
MeOH, .lambda..sub.max (log.epsilon.) = 234 nm (4.39), 280 (4.29)
(Figure 2) IR: KBr, .nu. = 3384 cm.sup.-1 (br), 2934 (m), 1674 (m),
1616 (s), 1456 (s), 1381 (m), 1064 (Figure 3) .sup.1H NMR: see
Table 2 .sup.13C NMR: see Table 2
[0063]
10TABLE 2 .sup.1H and .sup.13C NMR Spectroscopic Data of
Vancoresmycin in MeOD at 300 K. .sup.1H .sup.13C 1 0.85 14.76 2
0.97 20.83 3 1.86 31.20 4 3.40 82.19 5 1.69 39.54 6 0.77 13.64 7
3.53 80.19 8 1.65 36.40 9 0.86 12.75 10 1.43 31.20 11 1.43 36.52 12
3.74 72.03 13 1.60/1.54 44.90 14 3.75 72.03 15 1.57/1.38 36.61 16
1.49/1.16 30.18 17 1.94 38.60 18 0.92 15.04 19 3.86 81.67 20 4.59
103.48 21 3.83 72.03 22 2.48 57.61 23 -- -- 24 3.08 75.08 25 3.21
74.66 26 1.27 18.29 27 1.52/1.42 39.35 28 3.96 65.82 29 1.49 42.39
30 3.79 72.47 31 4.31 73.26 32 6.61 142.09 33 -- 138.52 34 1.82
12.75 35 -- 206.04 36 4.20 41.01 37 1.14 17.77 38 5.36 128.35 39 --
138.72 40 1.67 12.75 41 3.98 80.53 42 1.63 41.30 43 0.88 8.10 44
3.58 73.85 45 1.48 36.52 46 1.62/1.32 23.51 47 1.55/1.32 33.36 48
3.79 75.08 49 1.73 42.71 50 0.77 11.91 51 3.40 77.82 52 1.61 36.28
53 0.85 12.70 54 1.54/1.33 31.34 55 1.61/1.44 36.28 56 3.77 71.83
57 1.67/1.62 44.53 58 3.90 71.28 59 2.29 37.53 60 5.75 129.78 61 --
141.66 62 1.82 13.98 63 -- 195.36 64 -- 174.52 65 3.22 27.78 66 --
136.51 67 -- 183.80 68 -- 100.31 69 5.35 116.05 70 3.07 26.50 71
1.09 24.46 72 1.09 24.46
[0064] 2
11 TABLE 3 Test organism MIC (.mu.g/ml) Staphylococcus aureus 209P
0.05 S. aureus 20240 0.05 S. aureus E-710 0.10 S. aureus SG511 0.05
S. aureus 3066 0.10 S. warneri 6563 II (2) 0.10 S. aureus E-712
0.10 S. aureus 20424 0.10 S. aureus Chantot 31153 0.05 S. aureus
Wein 11 0.05 S. aureus Wein 12 0.05 S. aureus 503 (Mers) 0.025 S.
aureus Brussel 4115 0.05 S. aureus MLS-16 0.39 S. haemolyticus 809
0.05 S. epidermidis 825 0.10 S. epidermidis 823 0.20 S. epidermidis
607 0.10 S. epidermidis 5747 IW 0.10 Enterococcus faecalis ATCC
29212 0.39 Ent. faecalis D 21777 0.39 Ent. faecalis D 23241 0.39
Ent. faecalis D 756 0.39 Ent. faecalis D 26777 0.39 Ent. faecalis D
7F 0.39 Ent. faecium D-65 0.39 Ent. faecium 5601 (H) 0.39 Ent.
faecium P-1 0.39 Ent. faecium P-2 0.39 Ent. faecium VR-1 0.39 Ent.
hirae 55 0.39 Ent. durans 4939H 0.39 Escherichia coli 9632 >100
E. coli super sen. 2231 >100 Pseudomonas aeruginosa M35
>100
[0065] The present invention may be embodied in other specific
forms without departing from its spirit or essential
characteristics. The described embodiments are to be considered in
all respects as illustrative only and not restrictive.
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