U.S. patent application number 12/365230 was filed with the patent office on 2010-02-04 for medium for the production of tiacumicin b.
Invention is credited to Yuan-Ting Chen, Ming-Hsi Chiou, Chi-Jen Frank Du, Jonathan James Duffield, Franklin W. Okumu, Youe-Kong Shue, Mei-Chiao Wu.
Application Number | 20100028970 12/365230 |
Document ID | / |
Family ID | 31715689 |
Filed Date | 2010-02-04 |
United States Patent
Application |
20100028970 |
Kind Code |
A1 |
Shue; Youe-Kong ; et
al. |
February 4, 2010 |
Medium for the Production of Tiacumicin B
Abstract
Methods, processes and materials for the production and recovery
of Tiacumicins produced by culturing a microorganism belonging to
the species Dactylosporangium aurantiacum subspecies hamdenensis
having the ability to produce and accumulate one or more Tiacumicin
in a nutrient medium comprising a carbon source, a nitrogen source,
trace elements such as inorganic salts, and an adsorbent, wherein
said nitrogen source comprises fish powder, and wherein said
Tiacumicin is produced in a yield greater than about 50 mg/L
broth.
Inventors: |
Shue; Youe-Kong; (Carlsbad,
CA) ; Du; Chi-Jen Frank; (Taipei, TW) ; Chiou;
Ming-Hsi; (Taoyuan City, TW) ; Wu; Mei-Chiao;
(Jungli City, TW) ; Chen; Yuan-Ting; (Jian Shiang,
TW) ; Okumu; Franklin W.; ( San Diego, CA) ;
Duffield; Jonathan James; (San Diego, CA) |
Correspondence
Address: |
COHEN, PONTANI, LIEBERMAN & PAVANE LLP
551 FIFTH AVENUE, SUITE 1210
NEW YORK
NY
10176
US
|
Family ID: |
31715689 |
Appl. No.: |
12/365230 |
Filed: |
February 4, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10520863 |
Jul 13, 2005 |
7507564 |
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PCT/US03/21977 |
Jul 15, 2003 |
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12365230 |
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60399956 |
Jul 29, 2002 |
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Current U.S.
Class: |
435/180 |
Current CPC
Class: |
A61P 1/00 20180101; C12R
1/01 20130101; A61P 31/04 20180101; C12N 1/20 20130101; C12P 19/62
20130101 |
Class at
Publication: |
435/180 |
International
Class: |
C12N 11/08 20060101
C12N011/08 |
Claims
1-16. (canceled)
17. A medium for the production of tiacumicin B, said medium
comprising a microorganism capable of producing tiacumicin B, a
carbon source, a nitrogen source, trace elements, and an adsorbent
resin capable of adsorbing the tiacumicin B.
18-21. (canceled)
22. The nutrient medium according to claim 17 wherein said nitrogen
source is fish powder.
23. The medium according to claim 17 wherein said microorganism is
Dactylosporangium aurantiacum NRRL 18085.
24-27. (canceled)
28. The nutrient medium according to claim 17 wherein the nutrient
medium comprises 0.5%-15% by weight of the adsorbent resin.
29. The nutrient medium according to claim 17 wherein the adsorbent
resin is selected from the group consisting of Amberlite.RTM.
XAD16, XAD16HP, XAD2, XAD7HP, XAD1180, XAD1600, IRC50, Duolite.RTM.
XAD761 and a reverse phase silica gel.
30. The nutrient medium according to claim 17 wherein the trace
elements are inorganic salts.
31. The nutrient medium according to claim 17 wherein the nutrient
medium comprises 0.2-10% by weight of the carbon source.
32. The nutrient medium according to claim 17 wherein the nutrient
medium comprises 0.1-5.0% by weight of the nitrogen source.
33. The nutrient medium according to claim 17 wherein the nutrient
medium comprises 0.02-2.0% by weight of the trace elements.
Description
RELATED APPLICATIONS
[0001] Benefit of priority under 35 U.S.C. 119(e) is claimed herein
to U.S. Provisional application No. 60/399,956, filed Jul. 29, 2002
and International Application No. PCT/US2003/021977 filed 15 Jul.
2003. The disclosures of the above-reference applications are
incorporated by reference in their entirety herein.
FIELD OF INVENTION
[0002] This invention relates generally to fermentation production
of antibiotics and to media used in the same.
BACKGROUND
[0003] Tiacumicins are a family of structurally related compounds
that contain the 18-membered macrolide ring shown below.
##STR00001##
[0004] At present, several distinct Tiacumicins have been
identified and six of these (Tiacumicin A-F) are defined by their
particular pattern of substituents R.sup.1, R.sup.2, and R.sup.3
(U.S. Pat. No. 4,918,174; J. Antibiotics, 1987, 575-588).
[0005] The Lipiarmycins are a family of natural products closely
related to the Tiacumicins. Two members of the Lipiarmycin family
(A3 and B3) are identical to Tiacumicins B and C respectively (J.
Antibiotics, 1988, 308-315; J. Chem. Soc. Perkin Trans I, 1987,
1353-1359).
[0006] The Tiacumicins and the Lipiarmycins have been characterized
by numerous physical methods. The reported chemical structures of
these compounds are based on spectroscopy (UV-vis, IR and .sup.1H
and .sup.13C NMR), mass spectrometry and elemental analysis (See
for example: J. Antibiotics, 1987, 575-588; J. Antibiotics, 1983,
1312-1322).
[0007] Tiacumicins are produced by bacteria, including
Dactylosporangium aurantiacum subspecies hamdenensis, which may be
obtained from the ARS Patent Collection of the Northern Regional
Research Center, United States Department of Agriculture, 1815
North University Street, Peoria, Ill. 61604, accession number NRRL
18085. The characteristics of strain AB 718C-41 are given in J.
Antibiotics, 1987, 567-574 and U.S. Pat. No. 4,918,174.
[0008] Lipiarmycins are produced by bacteria including Actinoplanes
deccanensis (U.S. Pat. No. 3,978,211). Taxonomical studies of type
strain A/10655, which has been deposited in the ATCC under the
number 21983, are discussed in J. Antibiotics, 1975, 247-25.
[0009] Tiacumicins, specifically Tiacumicin B, show activity
against a variety of bacterial pathogens and in particular against
Clostridium difficile, a Gram-positive bacterium (Antimicrob.
Agents Chemother. 1991, 1108-1111). Clostridium difficile is an
anaerobic spore-forming bacterium that causes an infection of the
bowel. Diarrhea is the most common symptom but abdominal pain and
fever may also occur. Clostridium difficile is a major causative
agent of colitis (inflammation of the colon) and diarrhea that may
occur following antibiotic intake. This bacterium is primarily
acquired in hospitals and chronic care facilities. Because
Tiacumicin B shows promising activity against C. difficile, it is
expected to be useful in the treatment of bacterial infections,
especially those of the gastrointestinal tract, in mammals.
Examples of such treatments include but are not limited to
treatment of colitis and treatment of irritable bowel syndrome.
Tiacumicins may also find use for the treatment of gastrointestinal
cancers.
[0010] Fermentation processes are used to obtain antibiotics,
including Tiacumicins. Antibiotics may be produced by culturing a
microorganism in a medium containing readily assimilated sources of
carbon, nitrogen, and inorganic salts under submerged aerobic
fermentation conditions, until a substantial amount of antibiotic
activity is produced as deduced from in-process analyses. Because
of rising worldwide demand for antibiotics, there is an ongoing
need for improved methods to produce antibiotics.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 shows an HPLC chromatogram of crude fermentation
products produced according to Example 1; Tiacumicin B has a
retention time of approximately 12.6 min.
[0012] FIG. 2 shows an HPLC chromatogram of fermentation products
produced according to Example 2; Tiacumicin B has a retention time
of approximately 11.8 min.
[0013] FIG. 3 shows an HPLC chromatogram of purified (by HPLC)
Tiacumicin B produced by fermentation according to Example 2;
Tiacumicin B has a retention time of approximately 12.0 min.
[0014] FIG. 4 shows an HPLC chromatogram of Tiacumicin B produced
by fermentation and purified by reverse phase medium pressure
liquid chromatography followed by trituration according to Example
3; Tiacumicin B has a retention time of approximately 10.1 min.
SUMMARY OF THE INVENTION
[0015] The present invention presents methods, processes and
materials for the production of Tiacumicins. The present invention
also presents Tiacumicins produced using the fermentation methods,
processes, and materials described herein.
[0016] One embodiment of the present invention comprises an
improved process for producing Tiacumicins which comprises
culturing a microorganism belonging to the species
Dactylosporangium aurantiacum subspecies hamdenensis having the
ability to produce Tiacumicins in a nutrient medium and
accumulating at least one Tiacumicin in the nutrient medium,
wherein the yield of at least one Tiacumicin is greater than about
50 mg/L broth.
[0017] In one embodiment of the invention, improved media and
conditions for the fermentative production of Tiacumicin B are
described. Thus, one embodiment of the present invention is a
nutrient medium for production of Tiacumicins comprising a carbon
source, a nitrogen source, trace elements such as inorganic salts,
and an adsorbent, wherein said nitrogen source comprises fish
powder, and wherein said nutrient medium is used to produce one or
more Tiacumicin in a yield greater than about 50 mg/L broth.
[0018] In another embodiment of the invention, an improved recovery
method, resin adsorption of Tiacumicin B is described.
[0019] Another embodiment of the invention involves using bacterial
strains related to Dactylosporangium aurantiacum subspecies
hamdenensis, as organisms for producing Tiacumicins. Thus, the
present invention includes a Tiacumicin produced by culturing a
microorganism belonging to the species Dactylosporangium
aurantiacum subspecies hamdenensis having the ability to produce
and accumulate one or more Tiacumicin in a nutrient medium
comprising a carbon source, a nitrogen source, trace elements such
as inorganic salts, and an adsorbent, wherein said nitrogen source
comprises fish powder, and wherein said Tiacumicin is produced in a
yield greater than about 50 mg/L broth.
[0020] Another embodiment of the invention involves the use of
reverse phase medium pressure liquid chromatography and/or
liquid/liquid partition and/or trituration for the purification of
Tiacumicins from the crude fermentation product.
[0021] These improvements, alone or together, allow the
fermentative production and recovery of Tiacumicin B in much
improved yield (>50 mg/L broth) over currently available
methods.
DETAILED DESCRIPTION OF THE INVENTION
[0022] All patents, publications and patent applications referred
to herein are hereby incorporated by reference in their entireties.
Unless otherwise defined, all technical and scientific terms used
herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs.
Exemplary methods and materials are described below. However,
methods and materials similar or equivalent to those described
herein can be also used to obtain variations of the present
invention. The materials, methods, and examples are illustrative
only and not intended to be limiting.
[0023] The compositions containing the Tiacumicins of the invention
can be administered for prophylactic and/or therapeutic treatments.
In therapeutic applications, the compositions are administered to a
patient already suffering from an infection, as described above, in
an amount sufficient to cure or at least partially arrest the
symptoms of the infection. An amount adequate to accomplish this is
defined as "therapeutically effective amount or dose." Amounts
effective for this use will depend on the severity and course of
the infection, previous therapy, the patient's health status and
response to the drugs, and the judgment of the treating physician.
In prophylactic applications, compositions containing the
Tiacumicins of the invention are administered to a patient
susceptible to or otherwise at risk of a particular infection. Such
an amount is defined to be a "prophylactically effective amount or
dose." In this use, the precise amounts again depend on the
patient's state of health, weight, and the like.
[0024] Once improvement of the patient's conditions has occurred, a
maintenance dose is administered if necessary. Subsequently, the
dosage or the frequency of administration, or both, can be reduced,
as a function of the symptoms, to a level at which the improved
condition is retained. When the symptoms have been alleviated to
the desired level, treatment can cease. Patients can, however,
require intermittent treatment on a long-term basis upon any
recurrence of the disease symptoms.
[0025] In general, a suitable effective dose of the Tiacumicins of
the present invention will be in the range of 0.1 to 1000
milligrams (mg) per recipient per day, preferably in the range of 1
to 500 mg per day. The desired dosage is preferably presented in
one, two, three, four or more subdoses administered at appropriate
intervals throughout the day. These subdoses can be administered as
unit dosage forms, for example, containing 5 to 1000 mg, preferably
10 to 200 mg of active ingredient per unit dosage form. Preferably,
the compounds of the invention will be administered in amounts of
between about 1.0 mg/kg to 250 mg/kg of patient body weight,
between about one to four times per day.
[0026] A "pharmacological composition" refers to a mixture of one
or more of the Tiacumicins described herein, or physiologically
acceptable salts thereof, with other chemical components, such as
physiologically acceptable carriers and/or excipients. The purpose
of a pharmacological composition is to facilitate administration of
a compound to an organism.
[0027] "Pharmaceutically acceptable salts" of the compounds of the
invention include those derived from pharmaceutically acceptable
inorganic and organic acids and bases. Examples of suitable acids
include hydrochloric, hydrobromic, sulfuric, nitric, perchloric,
fumaric, maleic, phosphoric, glycolic, gluconic, lactic, salicylic,
succinic, toluene-p-sulfonic, tartaric, acetic, citric,
methanesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic,
benzenesulfonic, 1,2 ethanesulfonic acid (edisylate),
galactosyl-D-gluconic acid, and the like. Other acids, such as
oxalic acid, while not themselves pharmaceutically acceptable, may
be employed in the preparation of salts useful as intermediates in
obtaining the compounds of this invention and their
pharmaceutically acceptable acid addition salts. Salts derived from
appropriate bases include alkali metal (e.g., sodium), alkaline
earth metal (e.g., magnesium), ammonium and N(C.sub.1-C.sub.4
alkyl).sub.4.sup.+ salts, and the like. Illustrative examples of
some of these include sodium hydroxide, potassium hydroxide,
choline hydroxide, sodium carbonate, and the like.
[0028] A "physiologically acceptable carrier" refers to a carrier
or diluent that does not cause significant irritation to an
organism and does not abrogate the biological activity and
properties of the administered compound.
[0029] An "excipient" refers to an inert substance added to a
pharmacological composition to further facilitate administration of
a compound. Examples of excipients include but are not limited to,
calcium carbonate, calcium phosphate, various sugars and types of
starch, cellulose derivatives, gelatin, vegetable oils and
polyethylene glycols.
[0030] The term "nutrient medium" as used herein describes a
mixture of synthetic or naturally occurring ingredients. In
general, a nutrient medium comprises a carbon source, a nitrogen
source, trace elements such as inorganic salts, and optionally
vitamins or other growth factors, and an adsorbent.
[0031] The term "broth" as used herein refers to the fluid culture
medium as obtained during or after fermentation. Broth comprises a
mixture of water, the desired antibiotic(s), unused nutrients,
living or dead organisms, metabolic products, and the adsorbent
with or without adsorbed product.
[0032] The term "Tiacumicin" as used herein refers to a family of
compounds all of which comprise the 18-membered macrolide ring
shown below:
##STR00002##
[0033] The term "Tiacumicin B" refers to molecule having the
structure shown below:
##STR00003##
[0034] The term "yield" as used herein refers to an amount of crude
Tiacumicin re-constituted in methanol to the same volume as the
original fermentation broth. Yield is determined using standard
HPLC techniques. Yield is reported in units of mg/L.
[0035] One embodiment of the invention comprises a process suitable
for producing antibiotic agents, for example Tiacumicins, by
submerged aerobic fermentation of the microorganism. One embodiment
of such an organism is Dactylosporangium aurantiacum subspecies
hamdenensi. According to one embodiment of the invention,
Tiacumicins, for example Tiacumicin B, are recovered in exceptional
yield (>100 mg/L broth) from the fermentation broth by resin
absorption and eluted from the resin and mycelium by washing with
solvents of various polarities. Purification may be furthered by
solvent extraction and/or chromatographic separation such as
Sephadex, silica gel, High-Performance Liquid Chromatography (HPLC)
or reverse phase medium pressure liquid chromatography and/or
recrystallization with one or more solvents and/or trituration with
one or more solvents.
[0036] One microorganism employed in this invention was identified
as belonging to the family Actinoplanaceae, genus Dactylosporangium
(Journal of Antibiotics, 1987, p. 567-574 and U.S. Pat. No.
4,918,174). It has been designated Dactylasporangium aurantiacum
subspecies hamdenensis 718C-41. The subculture was obtained from
the ARS Patent Collection of the Northern Regional Research Center,
United States Department of Agriculture, 1815 North University
Street, Peoria, Ill. 61604, U.S.A., where it was assigned accession
number NRRL 18085. The characteristics of strain AB 718C-41 are
given in the Journal of Antibiotics, 1987, p. 567-574 and U.S. Pat.
No. 4,918,174.
[0037] Additional microorganisms capable of producing Tiacumicins
include mutant species, which show advantageous properties compared
with species known in the art. Such bacterial strains can be
generated by mutagenesis of a parent strain. Strategies and methods
of mutagenesis, procedures for screening and isolation of mutated
bacterial strains, compositions of media used in producing the
mutant strains of the invention are known in the art.
Microorganisms designated as strains may embody advantages such
increased production of desired macrolide, more efficient usage of
nutrient media, or decreased requirement of oxygen for aerobic
growth.
[0038] In the preferred embodiment, cultivation of
Dactylosporangium aurantiacum subspecies hamdenensis AB 718C41 NRRL
18085 for the production of the Tiacumicins is carried out in a
medium containing readily assimilable carbon sources, nitrogen
sources, inorganic salts and other organic ingredients with one or
more absorbents under proper aeration conditions and mixing in a
sterile environment. Compositions of nutrient media used in
producing antibiotics of the invention will be described in detail
in the examples.
[0039] Carbon sources capable of supporting microorganism growth
include but are not limited to glucose, sucrose, galactose,
fructose, starch, molasses, malt extracts, dextrins, whey,
glycerol, lipids, corn meal and the like and combinations thereof.
According to one embodiment of the invention, the carbon source is
present in the range between 0.2-10% by weight. Amounts of carbon
sources according to one embodiment of the invention are given in
Table 2.
[0040] Nitrogen sources capable of supporting microorganism growth
include but are not limited to beef extract, soybean meal,
cottonseed meal, whole yeast, yeast extract, soybean flour,
peptone, casamino acid, fish powder, corn steep liquor, ammonium
salts, casein, amino acids and the like and combinations thereof.
According to one embodiment of the present invention, the nutrient
medium contains fish powder (999 Prime quality fishmeal, TripelNine
Fish Protein, a.m.b.a. Fiskerihavnsgade 35, 6700 Esbjerg, Demark)
as the nitrogen source. According to one embodiment of the
invention, the nitrogen source is present in the range between
0.1-5.0% by weight. Amounts of nitrogen sources according to one
embodiment of the invention are given in Table 2.
[0041] Essential trace elements necessary for the growth and
development of the organism may occur as impurities in other
constituents of the media in amounts sufficient to meet the growth
and biosynthetic requirements of the organism. However, it may be
beneficial to incorporate in the culture media additional soluble
nutrient inorganic salts capable of assisting microorganism growth.
Inorganic salts capable of supporting microorganism growth include
but are not limited to K.sub.2HPO.sub.4, MgSO.sub.4.7H.sub.2O, KCl,
CaCO.sub.3 and the like. Essential trace elements are preferably
present in the range between 0.02-2.0% by weight. Amounts of
individual essential elements according to one embodiment of the
invention are given in Table 2.
[0042] Commercially available adsorbent resins were found to
enhance the yield and recovery efficiency of Tiacumicins during the
fermentation. Such adsorbents include but are not limited to
Amberlite.RTM. XAD16, XAD16HP, XAD2, XAD7HP, XAD1180, XAD1600, and
IRC50 (all Rohm & Haas Co., U.S.A.), Duolite.RTM. XAD761 (Rohm
& Haas Co., U.S.A.) and the like. Adsorbents are preferably
present in the range between 0.5-15% by weight. Amounts of
adsorbents according to one embodiment of the invention are given
in Table 2.
[0043] As is customary in aerobic submerged culture processes,
sterile air is dispersed through the culture medium. The oxygen
concentration was kept at higher than 3% (InPro 6000 series O.sub.2
sensors, Mettler Toledo). Under these conditions the growth of
cells is maintained at a level that prevents the growth conditions
becoming anaerobic. In some embodiments, the limiting component is
chosen from a carbon source, nitrogen source, or any other
component required by the cells (e.g., in the feed medium).
[0044] Bacteria are grown under suitable growth conditions. Such
suitable growth conditions are characterized by limiting the
availability of a component of the growth medium and/or feed medium
in such a way that aerobic conditions for the growth of said
bacterium are maintained. Such conditions can be also characterized
e.g. by maintaining a level of dissolved oxygen at a concentration
between about 2% to 30%. Such levels of dissolved oxygen can vary
depending on the specific technical equipment used for growing
bacteria and for measuring the dissolved oxygen concentration.
[0045] Tiacumicin-producing bacteria can be grown in vessels
ranging from shake flasks to large "batch" fermenters, by methods
known in the art. For producing substantial quantities of
Tiacumicins, submerged aerobic fermentation in tanks is utilized.
However, small amounts may be obtained by shake-flask culture. For
tank fermentation, it is preferable to use a vegetative inoculum.
The vegetative inoculum is prepared by inoculating a small volume
of culture medium with the spore form, mycelial fragments, or a
lyophilized pellet of the organism to obtain a fresh, actively
growing culture of the organism. The vegetative inoculum is then
transferred to a larger tank where, after a suitable incubation
time, the Tiacumicin antibiotic is produced in much improved
yield.
[0046] It may be necessary to add small amounts of an antifoam
agent to large-scale fermentation media if foaming becomes a
problem.
[0047] The production proceeds in a control medium with other
additives/ingredients to improve the production. A
liquid-submerged, stirred-culture process is used for the
production of Tiacumicins. Fermentation is carried out at a
temperature range of 25.degree. C. to 37.degree. C. The consumption
of the carbon source is carefully monitored and an additional
amount of carbon source is added as needed. The pH of the
fermentation is preferably maintained between about 6.0 to about
8.0. Tiacumicin B is produced and accumulated between 3 to 15 days
after inoculation of the fermentation. The standard control medium
consists of the following ingredients in the following
quantities:
TABLE-US-00001 Fish powder 0.1% to 5% Glucose 0.2% to 10%
K.sub.2HPO.sub.4 0.02% to 0.5% MgSO.sub.4.cndot.7H.sub.2O 0.02% to
0.5% KCl 0.01% to 0.3% CaCO.sub.3 0.1% to 2%
Other additives/ingredients consist of:
TABLE-US-00002 Casamino acid 0.05% to 2%, Yeast extract 0.05% to 2%
XAD-16 resin 0.5% to 15%
[0048] Upon completion of fermentation, the solid mass (including
the adsorbent resin) is separated from the broth by sieving.
Tiacumicins are eluted from the resin with organic solvents such as
ethyl acetate, methanol, acetonitrile or a mixture of two or more
organic solvents. The extract is then concentrated under reduced
pressure. This residue is further purified by trituration with low
polarity solvents such as hexanes, heptanes, methylcyclohexane, or
by partitioning between two phase solvent systems such as: ethyl
acetate/water; ethyl acetate/aqueous sodium chloride solution;
methanol/hexane, acetonitrile/hexane or other mixtures of two or
more solvents in various ratios and combinations or by Sephadex
column chromatography eluting with an appropriate organic solvent
system. If needed, Tiacumicins can be further purified either by
crystallization, and/or chromatographic separation and/or
High-Performance Liquid Chromatography (HPLC) and/or liquid/liquid
partitioning and/or trituration.
EXAMPLES
[0049] As can be appreciated from the disclosure above, the present
invention has a wide variety of applications. Accordingly, the
following examples are offered by way of illustration, not by way
of limitation.
Example 1
[0050] Dactylosporangium aurantiacum subsp. hamdenensis AB 718C-41
NRRL 18085 (-20.degree. C. stock), was maintained on 1 mL of Medium
No. 104 (Table 1). After standard sterilization conditions (30
min., 121.degree. C., 1.05 kg/cm.sup.2) the seed flask (250 mL)
containing Medium No. 104 (50 mL) was inoculated with AB 718C-41
NRRL 18085 on a shaker (set @ 250 rpm) at 30.degree. C. for 72 hr.
Five percent vegetative inoculum from the first passage seed flask
was then transferred aseptically to a fermentation flask containing
the same ingredients as in Table 1.
TABLE-US-00003 TABLE 1 Ingredients of Medium No. 104 Fish Casamino
Yeast powder Glucose K.sub.2HPO.sub.4 MgSO.sub.4.cndot.7H.sub.2O
KCl CaCO.sub.3 acid extract XAD-16 10 g/L 20 g/L 0.5 g/L 0.5 g/L
0.3 g/L 3 g/L 2.5 g/L 2.5 g/L 20 g/L
[0051] Fermentation flasks were incubated on a rotary shaker at
30.degree. C. for 3 to 12 days. Samples of the whole culture
fermentation broth were filtered. The filter cake was washed with
MeOH and solvents were removed under reduced pressure. The residue
was re-constituted in methanol to the same volume of the original
fermentation broth. Analysis was performed using a Waters BREEZE
HPLC system coupling with Waters 2487 2-channel UV/Vis detector.
Tiacumincins were assayed on a 50.times.4.6 .mu.m I.D., 5 .mu.m YMC
ODS-A column (YMC catalog # CCA AS05-0546WT) with a mobile phase
consisting of 45% acetonitrile in water containing 0.1% phosphoric
acid at a flow rate of 1.5 mL/minute. Tiacumicins were detected at
266 nm. An HPLC chromatogram of a crude product (Tiacumicin B
retention time @ 12.6 minutes) is shown in FIG. 1. In this example
the crude yield of Tiacumicin B was about 250 mg/L after 7 days.
After purification by HPLC, the yield of Tiacumicin B was about 100
mg/L.
Example 2
[0052] After standard sterilization conditions (30 min, 121.degree.
C., 1.05 kg/cm.sup.2) the seed flask (250 mL) containing Medium No.
104 (50 mL) was inoculated with AB 718C-41 NRRL 18085 and incubated
on a shaker (set @ 250 rpm) at 30.degree. C. for 72 hr. Five
percent vegetative inoculum from the first passage seed flask was
transferred aseptically to a seed flask containing the same
ingredients as in Table 1 and was incubated on a rotary shaker at
30.degree. C. for 72 hr. Five percent inoculum from the second
passage seed flasks was then used to inoculate with AB 718C-41 NRRL
18085 in a 5-liter fermenter containing Medium No. 104 (2.5 L).
Excessive foam formation was controlled by the addition of an
antifoaming agent (Sigma A-6426). This product is a mixture of
non-silicone organic defoamers in a polyol dispersion.
[0053] Glucose consumption was monitored as a growth parameter and
its level was controlled by the addition of the feeding medium.
Feeding medium and conditions in Example 2 were as follows:
Feeding Medium:
TABLE-US-00004 [0054] Yeast Casamino extract acid Glucose
K.sub.2HPO.sub.4 MgSO.sub.4.cndot.7H.sub.2O KCl 1.5% 1.5% 30% 0.5%
0.5% 0.3%
Fermenter Medium: No. 104
[0055] Fermenter Volume: 5 liters Sterilization: 40 minutes,
121.degree. C., 1.05 kg/cm.sup.2
Incubation Temperature: 30.degree. C.
[0056] Aeration rate: 0.5-1.5 volumes of air per culture volume and
minute
Fermenter Agitation: 300-500 rpm
[0057] The fermentation was carried out for 8 days and the XAD-16
resin was separated from the culture broth by sieving. After
washing with water the XAD-16 resin was eluted with methanol
(5-10.times. volume of XAD-16). Methanol was evaporated and the
oily residue was extracted three times with ethyl acetate. The
extracts were combined and concentrated under reduced pressure to
an oily residue. The oily residue was dried and washed with hexane
to give the crude product as a pale brown powder and its HPLC
chromatogram (Tiacumincin B retention time @ 11.8 minutes) is shown
in FIG. 2. This was purified by silica gel column (mixture of ethyl
acetate and hexane as eluent) and the resultant material was
further purified by RP-HPLC (reverse phase HPLC) to give Tiacumicin
B as a white solid. The purity was determined to be >95% by HPLC
chromatography and the chromatogram (Tiacumincin B retention time @
12.0 minutes) is shown in FIG. 3. Analysis of the isolated
Tiacumincin B gave identical .sup.1H and .sup.13C NMR data to those
reported in J. Antibiotics, 1987, 575-588, and these are summarized
below.
Tiacumicin B:
[0058] mp 129-140.degree. C. (white powder from RP-HPLC);
[0059] mp 166-169.degree. C. (white needles from isopropanol);
[0060] [.alpha.].sub.D.sup.20 -6.9 (c 2.0, MeOH);
[0061] MS m/z (ESI) 1079.7 (M+Na).sup.+;
[0062] .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 7.21 (d, 1H), 6.59
(dd, 1H), 5.95 (ddd, 1H), 5.83 (br s, 1H), 5.57 (t, 1H), 5.13 (br
d, 1H), 5.09 (t, 1H), 5.02 (d, 1H), 4.71 (m, 1H), 4.71 (br s, 1H),
4.64 (br s, 1H), 4.61 (d, 1H), 4.42 (d, 1H), 4.23 (m, 1H), 4.02
(pentet, 1H), 3.92 (dd, 1H), 3.73 (m, 2H), 3.70 (d, 1H), 3.56 (s,
3H), 3.52-3.56 (m, 2H), 2.92 (m, 2H), 2.64-2.76 (m, 3H), 2.59
(heptet, 1H), 2.49 (ddd, 1H), 2.42 (ddd, 1H), 2.01 (dq, 1H), 1.81
(s, 3H), 1.76 (s, 3H), 1.65 (s, 3H), 1.35 (d, 3H), 1.29 (m, 1H),
1.20 (t, 3H), 1.19 (d, 3H), 1.17 (d, 3H), 1.16 (d, 3H), 1.14 (s,
3H), 1.12 (s, 3H), 0.87 (t, 3H);
[0063] .sup.13C NMR (100 MHz, CD.sub.3OD) .delta. 178.4, 169.7,
169.1, 154.6, 153.9, 146.2, 143.7, 141.9, 137.1, 137.0, 136.4,
134.6, 128.5, 126.9, 125.6, 124.6, 114.8, 112.8, 108.8, 102.3,
97.2, 94.3, 82.5, 78.6, 76.9, 75.9, 74.5, 73.5, 73.2, 72.8, 71.6,
70.5, 68.3, 63.9, 62.2, 42.5, 37.3, 35.4, 28.7, 28.3, 26.9, 26.4,
20.3, 19.6, 19.2, 18.7, 18.2, 17.6, 15.5, 14.6, 14.0, 11.4.
Example 3
[0064] A crude sample of Tiacumicin B (15 g) was obtained by
fermentation as an oily residue after release from the resin as
described in Example 2. This was dissolved in ethyl acetate (300
mL) at 35.degree. C. and the solution was shaken in a separatory
funnel with water (300 mL) and allowed to settle for 1 minute.
Saturated aqueous sodium chloride solution (100 mL) was added and
the mixture was allowed to stand for a further 1 minute. The lower
phase and any solids present at the interface were discarded and
the upper phase was concentrated to a brown solid under reduced
pressure at 35.degree. C. The resulting foam was subjected to
reverse phase medium pressure liquid chromatography using a Biotage
75 L apparatus coupled to an Isco UA-6 UV/vis detector with the
following parameters:
Column: 1.2 kg, Biotage KP-C18-HS silica.
Equilibration: 50:50:1, MeCN/H.sub.2O/AcOH (6 L).
[0065] Loading: In methanol (20 mL) via sample injection module
containing 25 g of Biotage KP-C18-HS silica.
Eluent: 50:50:1, MeCN/H.sub.2O/AcOH.
[0066] Flow: 230 mL/min
Pressure: Solvent--90 psi
[0067] Radial--100 psi
Detector: Wavelength--254 nm
[0067] [0068] Path length--0.1 cm [0069] Sensitivity--2 [0070]
Chart speed--60 cm/hr. [0071] Noise filter--5 sec. Fraction
Collection Manual--began collection just after inflection between
main peak and previous peak, ended collection at 20% of main peak
height.
Column Conditioning: 100% MeCN (4 L)
[0072] Saturated aqueous sodium chloride solution (25% of the
fraction volume) was added to the collected fraction. The mixture
was shaken and allowed to separate into two phases. The upper phase
was removed and concentrated to dryness under reduced pressure at
30.degree. C. The resulting solid was dissolved in ethyl acetate
(75 mL) and washed with water (2.times.75 mL) to remove sodium
chloride. The organic phase was concentrated under reduced pressure
at 30.degree. C. to a yellow foam (recovery: 4.56 g, 30%; purity
.about.93%).
[0073] The material was combined with several other batches (total:
156.0 g, 90.8% purity) and to this was added isopropanol (1000 mL).
The mixture was sonicated with stirring at room temperature for 20
min. to produce an off-white suspension. At this point the material
was filtered and the filter cake was washed with isopropanol (300
mL). The solid was dried under high vacuum to leave an off white
powder (recovery: 146.2 g, 94%; purity: 91.1%) (FIG. 4). Mp
156-160.degree. C.; [.alpha.].sub.D.sup.20 -8.4 (c 2.0, MeOH); MS
m/z (ESI) 1079.7 (M+Na).sup.+; Calcd for
C.sub.52H.sub.74Cl.sub.2O.sub.18: C, 59.03; H, 7.05; Cl, 6.70.
Found: C, 58.75; H, 7.04; Cl, 6.91.
[0074] The inventions illustratively described herein can suitably
be practiced in the absence of any element or elements, limitation
or limitations, not specifically disclosed herein. Thus, for
example, the terms "comprising," "including," "containing," etc.
shall be read expansively and without limitation. Additionally, the
terms and expressions employed herein have been used as terms of
description and not of limitation, and there is no intention in the
use of such terms and expressions of excluding any equivalents of
the future shown and described or any portion thereof, and it is
recognized that various modifications are possible within the scope
of the invention claimed. Thus, it should be understood that
although the present invention has been specifically disclosed by
preferred embodiments and optional features, modification and
variation of the inventions herein disclosed can be resorted by
those skilled in the art, and that such modifications and
variations are considered to be within the scope of the inventions
disclosed herein. The inventions have been described broadly and
generically herein. Each of the narrower species and subgeneric
groupings falling within the scope of the generic disclosure also
form part of these inventions. This includes the generic
description of each invention with a proviso or negative limitation
removing any subject matter from the genus, regardless of whether
or not the excised materials specifically resided therein. In
addition, where features or aspects of an invention are described
in terms of the Markush group, those schooled in the art will
recognize that the invention is also thereby described in terms of
any individual member or subgroup of members of the Markush
group.
[0075] It is to be understood that the above description is
intended to be illustrative and not restrictive. Many embodiments
will be apparent to those of in the art upon reviewing the above
description. The scope of the invention should therefore, be
determined not with reference to the above description, but should
instead be determined with reference to the appended claims, along
with the full scope of equivalents to which such claims are
entitled. The disclosures of all articles and references, including
patent publications, are incorporated herein by reference.
* * * * *