Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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18-MEMBERED MACROCYCLES AND ANALOGS THEREOF
FIELD OF INVENTION
The present invention relates generally to the 18-membered macrocyclic
antimicrobial
agents called Tiacumicins, specifically, the R-Tiacumicin B or Tiacumicin B
and its related
compounds. In particular, substantially pure R-Tiacumicin B, as a potent
antibiotic agent for
the treatment of bacterial infections, specifically GI infections caused by
toxin producing
strains of Clostridium dicile (C. docile), Staphylococcus aureus (S. aureus)
including
methicillin-resistant Staphylococcus aureus (MRSA) and Clostridium perfringens
(C.
perfringens).
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BACKGROUND OF THE INVENTION
Macrocycles are an important therapeutic class of antibiotics. These compounds
are
frequently produced as a family of closely related biogenetic congeners. The
Tiacumicins are
a series of 18-membered macrocyclic antibiotics in which the macrocyclic ring
is
glycosidically attached to one or two sugars. A seven-carbon sugar is
esterfied at various
positions with small fatty acids. The other sugar, when present, is esterified
with an isomer
of the fully substituted benzoic acid, everninic acid. (Journal of Liquid
Chromatography,
1988, 11: 191-201).
Tiacumicins are a family of related compounds that contain the 18-membered
ring
shown in Formula I below.
H3C H3C OH
8
CH3
H3C O
,'O R2
H3C 19 R3
Formula I
At present, several distinct Tiacumicins have been identified and six of these
(Tiacumicin A-F) are defined by their particular pattern of substituents RI,
R2, and R3 (US
Patent No. 4,918,174; J. Antibiotics, 1987, 40: 575-588), as shown in Table 1.
Table 1. Substituents Present In Tiacumcins A-F
R R
A I H H
HO OH
B gypõ OH
Hiinio
HO OH
H]CO OH O OH
HO
CI
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C OH
HO,111 p p CI
HO O H3CO OHO H
HO I
D OH OH
Ho-111 p O O CI
HO O OCH3 _ OH
HO I
E X o OH
H011", p o p CI
HO 0)1110 H3CO OH O / \ OH
HO
F \ OH
HO d b CI 10
O OH H3 Co OHO H
HO-
CI
Tiacumicins A-F have been characterized spectroscopically and by other
physical
methods. The chemical structures of Tiacumicins are based on spectroscopy: UV-
vis, IR and
1H and 13C NMR, see for example J. Antibiotics, 1987, 40: 575-588. Inspection
of Table 1
reveals that certain members of the family are structurally related isomers
and/or differ by the
presence or absence of certain moieties. Others differ in the nature of their
ester groups.
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, IL 61604, accession number NRRL 18085. The
characteristics of
strain AB 718C-41 are given in J. Antibiotics, 1987, 40: 567-574 and US Patent
No.
4,918,174.
C. difficile-associated diarrhea (CDAD) is a disease characterized by severe
and
painful diarrhea. C. difficile is responsible for approximately 20% of the
cases of antibiotic-
associated diarrhea (AAD) and the majority of the cases of antibiotic-
associated colitis
(AAC). These diseases are typically caused by toxin producing strains of C.
difficile, S.
aureus including methicillin-resistant S. aureus (MRSA) and Clostridium
perfringens (C.
perfringens). AAD represents a major economic burden to the healthcare system
that is
conservatively estimated at $3-6 billion per year in excess hospital costs in
the U.S. alone.
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Vancomycin-resistant enterococci, for which intestinal colonization provides a
constant reservoir for infection, has also emerged as a major nosocomial
pathogen associated
with increased health care cost and mortality. VRE can appear as coinfection
in patients
infected with C. d ff cile, or more commonly cause infection in certain high
risk patients such
as haematology and oncology patients, patients in intensive care units and
patients receiving
solid organ transplants.
Methicillin-resistant Staphylococci, such as MRSA, are increasing in
prevalence in
both the hospital and community settings. Staphylococci are found on the skin
and within
the digestive and respiratory tracts but can infect open wounds and bums and
can progress to
serious systemic infection. The emergence of multi-drug resistant
Staphylococci, especially,
in the hospital where antibiotic use is frequent and selective pressure for
drug-resistant
organisms is high, has proven a challenge for treating these patients. The
presence of
MRSA on the skin of patients and health care workers promotes transmission of
the multi-
drug resistant organisms.
Similar diseases, including but not limited to clostridial enterocolitis,
neonatal
diarrhea, antibiotic-associated enterocolitis, sporadic enterocolitis, and
nosocomial
enterocolitis are also significant problems in some animal species.
AAD is a significant problem in hospitals and long-term care facilities and in
the
community. C. difficile is the leading cause of AAD in the hospital setting,
accounting for
approximately 20% of cases of AAD and the majority of cases of antibiotic-
associated colitis
(AAC). The rising incidence of Clostridiuin difficile-associated diarrhea
(CDAD) has been
attributed to the frequent prescription of broad-spectrum antibiotics to
hospitalized patients.
The most serious form of the disease is pseudomembranous colitis (PMC), which
is
manifested histologically by colitis with mucosal plaques, and clinically by
severe diarrhea,
abdominal cramps, and systemic toxicity. The overall mortality rate from CDAD
is low, but
is much greater in patients who develop severe colitis or systemic toxicity. A
recent study has
shown that even when death is not directly attributable to C. difficile, the
rate of mortality in
CDAD patients as compared to case-matched controls is much greater.
Diarrhea and colitis are caused by the elaboration of one or more C. difficile
toxins.
The organism proliferates in the colon in patients who have been given broad-
spectrum
antibiotics or, less commonly, cancer chemotherapy. CDAD is diagnosed in
approximately
20% of hospitalized patients who develop diarrhea after treatment with such
agents.
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There are currently two dominant therapies for CDAD: vancomycin and
metronidazole. Vancomycin is not recommended for first-line treatment of CDAD
mainly
because it is the only antibiotic active against some serious life-threatening
multi-drug
resistant bacteria. Therefore, in an effort to minimize the emergence of
vancomycin-resistant
Enterococcus (VRE) or vancomycin-resistant S. aureus (VRSA), the medical
community
discourages the use of this drug except when absolutely necessary.
Metronidazole is recommended as initial therapy out of concern for the
promotion
and selection of vancomycin resistant gut flora, especially enterococci.
Despite reports that
the frequency of C. dill cile resistance may be >6% in some countries,
metronidazole remains
nearly as effective as vancomycin, is considerably less expensive, and can be
used either
orally or intravenously. Metronidazole is associated with significant adverse
effects including
nausea, neuropathy, leukopenia, seizures, and a toxic reaction to alcohol.
Furthermore, it is
not safe for use in children or pregnant women. Clinical recurrence occurs in
up to 20% of
cases after treatment with either vancomycin or metronidazole. Therapy with
metronidazole
has been reported to be an important risk factor for VRE colonization and
infection. The
current treatment regime against Gastrointestinal infections, e.g.,
Clostridium difficile-
associated diarrhea (CDAD) is rather cumbersome, requiring up to 500 mg four-
times daily
for 10 to 14 days. Thus, there is a need for better treatment for cases of
CDAD as well as for
cases of other Antibiotic-associated diarrhea (AAD) and Antibiotic-associated
colitis (AAC).
Tiacumicins, specifically Tiacumicin B, show activity against a variety of
bacterial
pathogens and in particular against C. dill cile, a Gram-positive bacterium
(Antimicrob.
Agents Chemother. 1991, 1108-1111). C. docile 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. C. docile 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.
Tiacumicin antibiotics are described in U.S. Patent No. 4,918,174 (issued Apr.
17,
1990), J. Antibiotics 1987, 40: 575-588, J. Antibiotics 1987, 40: 567-574, J.
Liquid
Chromatography 1988, 11: 191-201, Antimicrobial Agents and Chemotherapy 1991,
35:
1108-1111, U.S. Patent No. 5,583,115 (issued Dec. 10, 1996), and U.S. Patent
No. 5,767,096
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(issued Jun. 16, 1998) . Related compounds
are the Lipiarmycin antibiotics (c.f., J. Chem. Soc. Perkin Trans. I, 1987,
1353-1359 and J.
Antibiotics 1988, 41: 308-315) and the Clostomicin antibiotics (J. Antibiotics
1986, 39:
1407-1412).
SUMMARY OF THE INVENTION
The present invention relates to new pharmaceutical compositions containing R-
Tiacumicins, specifically the optically pure R-Tiacumicin B, and to the use of
these new
compositions in combination with existing drugs to treat infections caused by
gram-positive
anerobes.
One embodiment of the present invention is directed towards the discovery that
the
chiral center at C-19 of Tiacumicin B has great effect on biological activity.
It has now been
discovered that a substantially pure preparation of higher activity R-
Tiacumicin B, which has
an R-hydroxy group at C-19 has surprisingly lower MIC values than the
optically pure S-
isomer of Tiacumicin B and other Tiacumicin B related compounds.
In another embodiment of the present invention the substantially pure R-
Tiacumicin B
has an unusually long post-antibiotic activity (PAE).
This invention encompasses the composition of novel antibiotic agents,
containing
substantially pure R-Tiacumicins, by submerged aerobic fermentation of the
microorganism
Dactylosporangium aurantiacum subspecies hamdenensis. The production method is
covered by WO 2004/014295 A2 .
BRIEF DESCRIPTION OF TILE FIGURES
Figure 1 shows the Oak Ridge Thermal Ellipsoid Plot Program (ORTEP) chemical
structure of R-Tiacumicin B.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
The term "antibiotic-associated condition" refers to a condition resulting
when
antibiotic therapy disturbs the balance of the microbial flora of the gut,
allowing pathogenic
organisms such as enterotoxin producing strains of C. diicile, S. aureus and
C. perfringens
to flourish. These organisms can cause diarrhea, pseudomembranous colitis, and
colitis and
are manifested by diarrhea, urgency, abdominal cramps, tenesmus, and fever
among other
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symptoms. Diarrhea, when severe, causes dehydration and the medical
complications
associated with dehydration.
The term "asymmetrically substituted" refers to a molecular structure in which
an
atom having four tetrahedral valences is attached to four different atoms or
groups. The
commonest cases involve the carbon atom. In such cases, two optical isomers (D-
and L-
enantiomers or R- and S- enantiomers) per carbon atom result which are
nonsuperposable
mirror images of each other. Many compounds have more than one asymmetric
carbon. This
results in the possibility of many optical isomers, the number being
determined by the
formula 2", where n is the number of asymmetric carbons.
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.
The term "C-19 Ketone" refers to a Tiacumicin B related compound shown below
in
Formula II:
OH
/ $S
O OH 11
HO/,,. R O OH
2 CI
O \ O m0
O OH
O `O O
O HO CI
S
19
O
Formula II
The term "diastereomers" refers to stereoisomers that are not mirror images of
each
other.
The term "enantiomer" refers to a non-superimposable mirror image of itself.
An
enantiomer of an optically active isomer rotates plane polarized light in an
equal but opposite
direction of the original isomer. A solution of equal parts of an optically
active isomer and its
enantiomer is known as a racemic solution and has a net rotation of plane
polarized light of
zero. Enantiomers will have the opposite prefixes of each other: D- becomes L-
or R-
becomes S-. Often only one enantiomer is active in a biological system,
because most
biological reactions are enzymatic and the enzymes can only attach to one of
the enantiomers.
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The term "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.
The term "halogen" includes F, Cl, Br and I.
The term "isomeric mixture" means a mixture of two or more configurationally
distinct chemical species having the same chemical formula. An isomeric
mixture is a genus
comprising individual isomeric species. Examples of isomeric mixtures include
stereoisomers (enantiomers and diastereomers), regioisomers, as might result
for example
from a pericyclic reaction. The compounds of the present invention comprise
asymmetrically
substituted carbon atoms. Such asymmetrically substituted carbon atoms can
result in
mixtures of stereoisomers at a particular asymmetrically substituted carbon
atom or a single
stereoisomer. As a result, racemic mixtures, mixtures of diastereomers, as
well as single
diastereomers of the compounds of the invention are included in the present
invention.
The term "Lipiarmycin A4" refers to a Tiacumicin B related compound shown
below
in Formula III:
OH
/ 8 S
_H
i O OH
HO~i.,,,,01111, R
1 CI
O 0 O -111110
O OH
0
0 HO CI
S,
19
OH
Formula III
The term "lower alkyl," alone or in combination, refers to an optionally
substituted
straight-chain or optionally substituted branched-chain having from 1 to about
8 carbons
(e.g., C1, C2, C3, C4, C5, C6, C7, C8,), more preferably 1 to 4 carbons (e.g.,
C1, C2, C3, C4,).
Examples of alkyl radicals include methyl, ethyl, n-propyl, isopropyl, n-
butyl, isobutyl, sec-
butyl, tert-butyl. A "lower alkyl" is generally a shorter alkyl, e.g., one
containing from 1 to
about 4 carbon atoms (e.g., C1, C2, C3, C4,).
The term "macrocycles" refers to organic molecules with large ling structures
usually
containing over 10 atoms.
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The term "18-membered macrocycles" refers to organic molecules with ring
structures containing 18 atoms.
The term "membered ring" can embrace any cyclic structure, including
carbocycles
and heterocycles as described above. The term "membered" is meant to denote
the number
of skeletal atoms that constitute the ring. Thus, for example, pyridine, pyran
and thiopyran
are 6 membered rings and pyrrole, furan, and thiophene are 5 membered rings.
The term "MIC" or "minimum inhibitory concentration" refers to the lowest
concentration of an antibiotic that is needed to inhibit growth of a bacteria]
isolate in vitro. A
common method for determining the MIC of an antibiotic is to prepare several
tubes
containing serial dilutions of the antibiotic, that are then inoculated with
the bacterial isolate
of interest. The MIC of an antibiotic can be determined from the tube with the
lowest
concentration that shows no turbidity (no growth).
The term "MIC50" refers to the lowest concentration of antibiotic required to
inhibit
the growth of 50% of the bacterial strains tested within a given bacterial
species.
The term "MIC9o" refers to the lowest concentration of antibiotic required to
inhibit
the growth of 90% of the bacterial strains tested within a given bacterial
species.
The term "OPT-80" refers to a preparation containing approximately 70-100%,
preferably, 90% (with respect to the whole antibiotic substance, by HPLC
assay) of the
optically pure R-Tiacumicin B (which has an R-hydroxy group at C-19, see
Formula IV). The
remaining portions consist essentially of small amounts of Tiacumicin B
related compounds
(including, but not limited to Lipiarmycin A4 and C-19 Ketone). Preparations
of this type are
described in detail in PCT application PCT/US03/21977, having an international
publication
number of WO 2004/014295 A2.
However, for exclusive use in non-humans crude "OPT-80" that contains less
than
70% of the optically pure R Tiacumicin B (with respect to the whole antibiotic
substance, by
HPLC assay) may be used.
The term "ORTEP" refers to the Oak Ridge Thermal Ellipsoid Plot computer
program, written in Fortran, for drawing crystal structure illustrations. Ball-
and-stick type
illustrations of a quality suitable for publication are produced with either
spheres or thermal-
motion probability ellipsoids, derived from anisotropic temperature factor
parameters, on the
atomic sites. The program also produces stereoscopic pairs of illustrations
which aid in the
visualization of complex arrangements of atoms and their correlated thermal
motion patterns.
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The term "PAE" or "post-antibiotic effect" refers to a well-established
pharmacodynamic parameter that reflects the persistent suppression of
bacterial growth
following antibiotic exposure.
The term "patient" refers to a human or animal in need of medical treatment.
For the
purposes of this invention, human patients are typically institutionalized in
a primary medical
care facility such as a hospital or nursing home. However, treatment of a
disease associated
with the use of antibiotics or cancer chemotherapies or antiviral therapies
can occur on an
outpatient basis, upon discharge from a primary care facility, or can be
prescribed by a
physician for home-care, not in association with a primary medical care
facility. Animals in
need of medical treatment are typically in the care of a veterinarian.
The term "pharmaceutically acceptable c arrier" refers to a carrier o r d
iluent that i s
pharmaceutically acceptable.
The term "pharmaceutically acceptable salts" refers to those derived from
pharmaceutically acceptable inorganic and organic bases. Salts derived from
appropriate
bases include alkali metal (e.g., sodium or potasium), alkaline earth metal
(e.g., magnesium),
ammonium and N(C1-C4 alkyl)4+ salts, and the like. Illustrative examples of
some of these
include sodium hydroxide, potassium hydroxide, choline hydroxide, sodium
carbonate, and
the like.
The term "pharmaceutical 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
pharmaceutical composition is to facilitate administration of a compound to an
organism.
The term "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.
The term "pseudomembranous colitis" or "enteritis" refers to the formation of
pseudomembranous material (i.e., material composed of fibrin, mucous, necrotic
epithelial
cells and leukocytes) due to inflammation of the mucous membrane of both the
small and
large intestine.
The terms "R" and "S" configuration, as used herein, are as defined by the
11JPAC
1974 Recommendations for Section E, Fundamental Stereochemistry, Pure Appl.
Chern.
(1976)45,13-30. Chiral molecules can be named based on the atomic numbers of
the atoms
or groups of atoms, the ligands that are attached to the chiral center. The
ligands are given a
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priority (the higher the atomic number the higher the priority) and if the
priorities increase in
a clockwise direction, they are said to be R-. Otherwise, if they are
prioritized in a
counterclockwise direction they are said to be S-.
The term "R-Tiacumicin B" refers to the optically pure (R)-isomer of
Tiacumicin B
with an (R)-hydroxy group at C-19, as shown below in Formula IV:
OH
s
OH 1
I O OH
HOB/,,, ~dOl~ro R
1z CI
O O ,i10
O OH
O
O O O
O HO CI
S
R
19
OH
Formula IV
The term "S-Tiacumicin B" refers to the optically pure (S)-isomer of
Tiacumicin B
with an (S)-hydroxy group at C-19, as shown below in Formula V:
OH
/ 8 S
OH 11 /
HO/i,,, \\Olu-I, R O OH
tz CI
O \ O un0
O OH
O O -
HO CI
S
s,-
19
OH
Formula V
The term "stereoisomers" refers to compounds whose molecules have the same
number and kind of atoms and the same atomic arrangement, but differ in their
spatial
arrangement.
The term "sugar" generally refers to mono-, di- or oligosaccharides. A
saccharide may
be substituted, for example, glucosamine, galactosamine, acetylglucose,
acetylgalactose, N-
acetylglucosamine, N-acetyl-galactosamine, galactosyl-N-acetylglucosamine, N-
acetylneuraminic acid (sialic acid), etc., as well as sulfated and
phosphorylated sugars. For
the purposes of this definition, the saccharides are in their pyranose or
furanose form.
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The term "Tiacumicin" as used herein refers to a family of compounds all of
which
comprise the 18-membered macrocycle shown below in Formula I:
H3C H3C OH
R~ / s
CH3
H3C 0
1 10 R2
H3C 19 R3
Formula I
The term "Tiacumicin B" as used herein refers to the 18-membered macrocycle
shown below in Formula VI:
OH
/ 8 S
OH 11
HO/,,, ill. n 0 OH
2 CI
O 0 0 InO
0 OH
0 `O 0
0 HO CI
S
19
OH
Formula VI
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.
This invention encompasses the composition of novel antibiotic agents,
Tiacumicins,
by submerged aerobic fermentation of the microorganism DactylospOrangiufn
aurantiacum
subspecies hamdenensis. The production method is covered by WO 2004/014295 A2.
The present invention relates to new antibacterial compositions containing R-
Tiacumicins, specifically the R-Tiacumicin B (which has an R-hydroxy at C-19),
and to the
use of these new compositions in combination with existing drugs to treat
infections caused
by gram-positive anerobes.
The present invention further relates to the novel OPT-80 preparation which
contains
70-100% preferably, 90% (with respect to the whole antibiotic substance, by
HPLC assay) of
the R-Tiacumicin B. The remaining portions consist essentially of small
amounts of
Tiacumicin B related compounds (including, but not limited to Lipiarmycin A4
and C-19
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Ketone). Preparations of this type are described in detail in PCT application
PCT/US03/21977, having an international publication number of WO 2004/014295
A2.
However, for exclusive use in non-humans crude OPT-80 that contains less than
70% of the
R-Tiacumicin B (with respect to the whole antibiotic substance, by HPLC assay)
may be used.
In accordance with the present invention there are provided compounds with the
structure of Formula VII:
OH
/ 8 s
OH
HO/,,,. \~~Ol~i . R \ O OH
1i X CI
O -1111110111 O OH
Z O PO
S
HO CI
R
19
Y
Formula VII
wherein:
X is selected from lower alkyl, and wherein the term "lower alkyl" as used
herein refers to
branched or straight chain alkyl groups comprising one to two carbon atoms,
including
methyl, ethyl, n-propyl, isopropyl, and the like; and
Y is selected from OH or a ketone (=O); and
Z is selected from H or lower alkyl, and wherein the term "lower alkyl" as
used herein refers
to branched or straight chain alkyl groups comprising one to five carbon
atoms, including
methyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, and the like.
Preferred compounds of the invention are compounds of Formula VII wherein X is
methyl or ethyl, Y is ketone (=O) or OH and Z is isopropyl.
More preferred compounds of the invention are the compound of the Formula VII
wherein X is ethyl, Y is ketone (=O) or OH and Z is isopropyl.
The most preferred compounds of the invention are the compounds of Formula VII
wherein X is ethyl, Y is OH R and Z is isopropyl.
One embodiment of the present invention is directed towards the discovery that
the
chiral center at C-19 of Tiacumicin B has great effect on biological activity.
It has now been
discovered that R-Tiacumicin B, which has an R-hydroxy group at C-19 has
significantly
higher activity than the S-Tiacumicin B and other Tiacumicin B related
compounds
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(Lipiarmycin A4 and C-19 Ketone). The higher activity is shown by much lowered
MIC
values, which can be seen below in Example 3, Tables 3 and 4 for several
strains of C.
docile, S. aureus, E. faecalis, and E. faecium. This effect of the C-19 chiral
center on
biological activity is an unexpected and novel discovery.
In another embodiment of the present invention OPT-80 (which is composed
almost
entirely of the R-Tiacumicin B) has an unusually long post-antibiotic effect
(PAE). This is
discussed below in Example 4, where it is shown that OPT-80 has a PAE of
greater than 24
hours. This PAE is unexpectedly longer than the usual antibiotic PAE of 1-5
hours.
The present invention also relates to the disclosure of pharmaceutical
compositions,
which comprise a compound of the present invention in combination with a
pharmaceutically
acceptable carrier.
Yet another aspect of the invention discloses a method of inhibiting or
treating
bacterial infections in humans, comprising administering to the patient a
therapeutically
effective amount of a compound of the invention alone or in combination with
another
antibacterial or antifungal agent.
Production
The 18-membered macrocycles and analogs thereof are produced by fermentation.
Cultivation of Dactylosporangium aurantiacum subsp. hamdenensis AB 718C-41
NRRL
18085 for the production of the Tiacumicins is carried out in a medium
containing carbon
sources, inorganic salts and other organic ingredients with one or more
absorbents under
proper aeration conditions and mixing in a sterile environment.
The microorganism to produce the active antibacterial agents was identified as
belonging to the family Actinoplanaceae, genus Dactylosporangium (Journal of
Antibiotics,
1987, 40: 567-574 and US patent 4,918,174). It has been designated
Dactylasporangium
aurantiacum subspecies hanadenensis 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, IL. 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, 40: 567-574 and US patent 4,918,174.
This invention encompasses the composition of novel antibiotic agents,
Tiacumicins,
by submerged aerobic fermentation of the microorganism Dactylosporangium
aurantiacum
subspecies hamdenensis. The production method is covered by WO 2004/014295 A2
14
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WO 2006/085838 PCT/US2005/002887
Pharmaceutical Formulation and Administration
Pharmaceutical compositions of the Tiacumicin compounds of the present
invention,
specifically OPT-80 (which is composed almost entirely of the R-Tiacumicin),
according to
the invention may be formulated to release an antibiotic substantially
immediately upon
administration or at any predetermined time or time period after
administration.
The latter types of compositions are generally known as modified release
formulations, which include formulations that create a substantially constant
concentration of
the drug within the intestinal tract over an extended period of time, and
formulations that have
modified release characteristics based on temporal or environmental criteria
as described in
Modified-Release Drug Delivery Technology, ed. M. J. Rathbone, J. Hodgraft and
M. S.
Roberts. Marcel Dekker, Inc. New York.
Any oral biologically-acceptable dosage form, or combinations thereof, can be
employed in the methods of the invention. Examples of such dosage forms
include, without
limitation, chewable tablets, quick dissolve tablets, effervescent tablets,
reconstitutable
powders, elixirs, liquids, suppositories, creams, solutions, suspensions,
emulsions, tablets,
multi-layer tablets, bi-layer tablets, capsules, soft gelatin capsules, hard
gelatin capsules,
osmotic tablets, osmotic capsules, caplets, lozenges, chewable lozenges,
beads, powders,
granules, particles, microparticles, dispersible granules, ingestibles,
infusions, health bars,
confections, animal feeds, cereals, cereal coatings, foods, nutritive foods,
functional foods
and combinations thereof. The preparation of any of the above dosage forms is
well known
to persons of ordinary skill in the art. Additionally, the pharmaceutical
formulations may be
designed to provide either immediate or controlled release of the antibiotic
upon reaching the
target site. The selection of immediate or controlled release compositions
depends upon a
variety of factors including the species and antibiotic susceptibility of Gram-
positive bacteria
being treated and the bacteriostatic/bactericidal characteristics of the
therapeutics. Methods
well known in the art for making formulations are found, for example, in
Remington: The
Science and Practice of Pharmacy (20th ed.), ed. A.R. Gennaro, 2000,
Lippincott Williams &
Wilkins, Philadelphia, or in Encyclopedia of Pharmaceutical Technology, eds.
J. Swarbrick
and J. C. Boylan, 1988-1999, Marcel Dekker, New York.
Immediate release formulations for oral use include tablets or capsules
containing the
active ingredient(s) in a mixture with non-toxic pharmaceutically acceptable
excipients.
These excipients may be, for example, inert diluents or fillers (e.g.,
sucrose, sorbitol, sugar,
mannitol, microcrystalline cellulose, starches including potato starch,
calcium carbonate,
CA 02596387 2012-04-12
sodium chloride, lactose, calcium phosphate, calcium sulfate, or sodium
phosphate);
granulating and disintegrating agents (e.g., cellulose derivatives including
microcrystalline
cellulose, starches including potato starch, croscarmellose sodium, alginates,
or alginic acid);
binding agents (e.g., sucrose,-glucose, mannitol, sorbitol, acacia, alginic
acid, sodium alginate,
gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium
aluminum
silicate, carboxymethylcellulose sodium, methylcellulose, hydroxypropyl
methylcellulose,
ethylcellulose, polyvinylpyrrolidone, or polyethylene glycol); and lubricating
agents, glidants,
and antiadhesives (e.g., magnesium stearate, zinc stearate, stearic acid,
silicas, hydrogenated
vegetable oils, or talc). Other pharmaceutically acceptable excipients can be
colorants,
flavoring agents, plasticizers, humectants, buffering agents, and the like as
are found, for
example, in The Handbook of Pharmaceutical Excipients, third edition, edited
by Arthur H.
Kibbe, American Pharmaceutical Association Washington DC.
Dissolution or diffusion controlled release can be achieved by appropriate
coating of
a tablet, capsule, pellet, or granulate formulation of compounds, or by
incorporating the
compound into an appropriate matrix. A controlled release coating may include
one or more
of the coating substances mentioned above and/or, e.g., shellac, beeswax,
glycowax, castor
wax, carnauba wax, stearyl alcohol, glyceryl monostearate, glyceryl
distearate, glycerol
palmitostearate, ethylcellulose, acrylic resins, dl-polylactic acid, cellulose
acetate butyrate,
polyvinyl chloride, polyvinyl acetate, vinyl pyrrolidone, polyethylene,
polymethacrylate,
methylmethacrylate, 2-hydroxymethacrylate, methacrylate hydrogels, 1,3
butylene glycol,
- ethylene glycol methacrylate, and/or polyethylene glycols. In a controlled
release matrix
formulation, the matrix material may also include, e.g., hydrated
methylcellulose, carnauba
wax and stearyl alcohol, Carbopol TM 934, silicone, glyceryl tristearate,
methyl acrylate-methyl
methacrylate, polyvinyl chloride, polyethylene, and/or halogenated
fluorocarbon.
A controlled release composition may also be in the form of a buoyant tablet
or
capsule (i.e., a tablet or capsule that, upon oral administration, floats on
top of the gastric
content for a certain period of time). A buoyant tablet formulation of the
compound(s) can
be prepared by granulating a mixture of the antibiotic with excipients and 20-
75% w/w of
hydrocolloids, such as hydroxyethylcellulose, hydroxypropylcellulose, or
hydroxypropyhnethylcellulose. The obtained granules can then be compressed
into tablets.
On contact with the gastric juice, the tablet forms a substantially water-
impermeable gel
barrier around its surface. This gel barrier takes part in maintaining a
density of less than one,
thereby allowing the tablet to remain buoyant in the gastric juice. Other
useful controlled
16
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WO 2006/085838 PCT/US2005/002887
release compositions are known in the art (see, for example, U.S. Patent Nos.
4,946,685 and
6,261,601).
A modified release composition may be comprised of a compression-coated core
whose geometric configuration controls the release profile of the encapsulated
antibiotic. By
varying the geometry of the core, the profile of the antibiotic release can be
adjusted to
follow zero order, first order or a combination of these orders. The system
can also be
designed to deliver more beneficial agents at the same time, each having a
different release
profile (see, for example U.S. Patent Nos. 4,111,202 and 3,279,995).
Formulations that target the Tiacumicin compounds of the present invention,
specifically OPT-80 (which is composed almost entirely of the R-Tiacumicin),
that release to
particular regions of the intestinal tract can also be prepared. The
Tiacumicin compounds of
the present invention, specifically OPT-80, can be encapsulated in an enteric
coating that
prevents release degradation and release from occurring in the stomach, but
dissolves readily
in the mildly acidic or neutral pH environment of the small intestine. A
formulation targeted
for release of antibiotic to the colon, utilizing technologies such as time-
dependent, pH-
dependent, or enzymatic erosion of polymer matrix or coating can also be used.
The targeted delivery properties of the Tiacumicin compounds of the present
invention, specifically OPT-80 (which is composed almost entirely of the R-
Tiacumicin B),
containing formulation may be modified by other means. For example, the
antibiotic may be
complexed by inclusion, ionic association, hydrogen bonding, hydrophobic
bonding, or
covalent bonding. In addition polymers or complexes susceptible to enzymatic
or microbial
lysis may also be used as a means to deliver drug.
Microsphere encapsulation of the Tiacumicin compounds of the present
invention,
specifically OPT-80 (which is composed almost entirely of the R-Tiacumicin B),
is another
useful pharmaceutical formulation for targeted antibiotic release. The
antibiotic-containing
microspheres can be used alone for antibiotic delivery, or as one component of
a two-stage
release formulation. Suitable staged release formulations may consist of acid
stable
microspheres, encapsulating the compounds of the present invention,
specifically OPT-80
(which is composed almost entirely of the R-Tiacumicin B), to be released
later in the lower
intestinal tract admixed with an immediate release formulation to deliver
antibiotic to the
stomach and upper duodenum.
Microspheres can be made by any appropriate method, or from any
pharmaceutically
acceptable material. Particularly useful are proteinoid microspheres (see, for
example, U.S.
Patent Nos. 5,601,846, or 5,792,451) and PLGA-containing microspheres (see,
for example,
17
CA 02596387 2007-07-31
WO 2006/085838 PCT/US2005/002887
U.S. Patent Nos. 6,235,224 or 5,672,659). Other polymers commonly used in the
formation
of microspheres include, for example, poly-c-caprolactone, poly(e-caprolactone-
Co-DL-
lactic acid), poly(DL-lactic acid), poly(DL-lactic acid-Co-glycolic acid) and
poly(s-
caprolactone-Co-glycolic acid) (see, for example, Pitt et al., J. Pharm. Sci.,
68:1534,1979).
Microspheres can be made by procedures well known in the art including spray
drying,
coacervation, and emulsification (see for example Davis et al. Microsphere and
Drug
Therapy, 1984, Elsevier; Benoit et al. Biodegradable Microspheres: Advances in
Production
Technologies, Chapter 3, ed. Benita, S, 1996, Dekker, New York;
Microencapsulation and
Related Drug Processes, Ed. Deasy, 1984, Dekker, New York; U.S. Patent No.
6,365,187).
Powders, dispersible powders, or granules suitable for preparation of aqueous
solutions or suspensions of the Tiacumicin compounds of the present invention,
specifically
OPT-80 (which is composed almost entirely of the R-Tiacumicin B), by addition
of water are
convenient dosage forms for oral administration. Formulation as a suspension
provides the
active ingredient in a mixture with a dispersing or wetting agent, suspending
agent, and one or
more preservatives. Suitable dispersing or wetting agents are, for example,
naturally-
occurring phosphatides (e.g., lecithin or condensation products of ethylene
oxide with a fatty
acid, a long chain aliphatic alcohol, or a partial ester derived from fatty
acids) and a hexitol or
a hexitol anhydride (e.g., polyoxyethylene stearate, polyoxyethylene sorbitol
monooleate,
polyoxyethylene sorbitan monooleate, and the like). Suitable suspending agents
are, for
example, sodium carboxymethylcellulose, methylcellulose, sodium alginate, and
the like.
18
CA 02596387 2012-04-12
EXAMPLES
The following examples are provided by way of describing specific embodiments
of
the present invention
Example 1- Exact Structure of R Tiacumicin B
The exact structure of the R-Tiacumicin B (the major most active component of
OPT-
80) is shown below in Formula IV. The X-ray crystal structure of the R-
Tiacumicin B was
obtained from a colorless, parallelepiped-shaped crystal (0.08 x 0.14 x 0.22
mm) grown in
methanol and is shown as an ORTEP diagram in Figure 1. This x-ray structure
confirms the
structure shown below in Formula IV. The official chemical name is 3-[[(6-
Deoxy-4-O-(3,5-
dichloro-2-ethyl-4,6-dihydroxybenzoyl)-2-O-methyl-R-D-mannopyranosyl)oxy)-
methyl]-
12(R)-[[6-deoxy-5-C-methyl-4-O-(2-methyl-l -oxopropyl)-o-D-lyxo-
hexopyranosyl]oxy]-
11(S)-ethyl-8(S)-hydroxy-I8(S)-(I(R)-hydroxyethyl)-9,13,15-
trimethyloxacyclooctadeca-
3,5,9,13,15-pentaene-2-one.
OH
$S
OH
Ho" ,=~.OI R O OH
1s CI
0
O 0 ,m0
0 OH
O :O O
110 HO CI
S
19
OH
Formula N
Example 2 - Analytical Data of OPT-80 and Related Substances
The analytical data of OPT-80 (which is composed almost entirely of the R-
Tiacumicin B, which is the most active component of OPT-80) and three related
compounds
(S-Tiacumicin B, Lipiarmycin A4, and C-19 ketone) are summarized below. The
structures
of these compounds are shown in Formula VIII and Table 2 below.
19
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WO 2006/085838 PCT/US2005/002887
OH
8
OH 11
HOs,,,, ,,\OI n, i n p OH
X CI
O
O p n10
O OH
Z p O
HO CI
S
19
Y
Formula VIII
Table 2: Structure of R-Tiacumicin B (the major most active component of OPT-
80) and
related substances
Compound X Y Z
R-Tiacumicin B Ethyl (R)-OH Isopropyl
S-Tiacumicin B Ethyl (S)-OH Isopropyl
Li iarm cin A4 Methyl (S)-OH Isopropyl
C-19 Ketone Ethyl =0 Isopropyl
Analytical data of R-Tiacumicin B
mp 166 - 169 C (white needle from isopropanol);
[od D20 -6.9 (c 2.0, MeOH);
MS m/z (ESI) 1079.7(M + Na)+;
1H 1H NMR NMR (400 MHz, CD3OD) S 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, 111),
4.71 (m, 111), 4.71 (br
s, 1H), 4.64 (br s, 1H), 4.61 (d, 111), 4.42 (d, 111), 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, 3 H),
1.17 (d, 3H), 1.16
(d, 3 H), 1.14 (s, 3H), 1.12 (s, 3H), 0.87 (t, 3H);
13C NMR (100 MHz, CD3OD) 6 178.4, 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.
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Analytical data of the S-Tiacumicin B:
UR\ OH
9H ' OH HO,,, .api,= O OH
HOo. a0
O OH O CI
n p m0
OO O " O NaBHq in McOH O p OH
/\ OH O HPLC separation HO CI
II SO HO CI s
p OH
Formula II (C- 19 Ketone) Formula V (S-Tiacumicin B)
NaBH4 (9 eq, 48mg) was added in three portions to a solution of C-19 Ketone
(150mg) in 3 mL MeOH. After 1 h, saturated NH4C1 solution was added. The
mixture was
extracted with CHC13, and then concentrated. S-Tiacumicin B was purified by
YMC-pack
ODS-A 75x30mm I.D. column (H20:MeOH:AcOH 28:72:1) yielding pure 35 mg of pure
S-
Tiacumicin B.
MS a/z 1074.5 (M + NH4)+;
1H NMR (400 MHz, CDC13) 6 7.15 (d, J = 11.4 Hz, 1 H), 6.58 (dd, J = 14.1, 11.4
Hz,
1 H), 5.82 (ddd, J = 14.1, 10.6, 3.5 Hz, 1 H), 5.78 (s, 1 H), 5.40 (dd, J =
7.8, 7.8 Hz, 1 H), 5.15
(dd, J = 9.5, 9.5 Hz, 1 H), 5.01 (d, J = 9.9 Hz, 1 H), 5.01 (d, J = 9.9 Hz, 1
H), 4.77 (ddd, J =
5.8, 5.3, 5.3 Hz, I H), 4.68 (d, J = 11.6 Hz, 1H), 4.65 (br s, 1H), 4.62 (br
s, 1H), 4.42 (d, J =
11.6 Hz, 1H), 4.28 (br s, 1H), 4.07-3.97 (m, 2H), 3.74-3.58 (m, 4H), 3.61 (s,
3H), 3.52 (dq, J
= 9.5, 5.8 Hz, 1H), 3.08 (dq, J= 12.6, 6.1 Hz, 1H), 3.01 (dq, J= 12.6, 6.1 Hz,
1H), 2.77-2.65
(m, 2H), 2.60 (heptet, J= 6.9 Hz, 1H), 2.55-2.44 (m, 3H), 1.95-1.84 (m, 1H),
1.80 (s, 3H),
1.76 (s, 3H), 1.66 (s, 3H), 1.34 (d, J = 5.8 Hz, 3H), 1.29-1.24 (m, 1H), 1.27
(d, J = 6.6 Hz,
3H), 1.21 (t, J= 6.1 Hz, 3H), 1.19 (d, J= 6.9 Hz, 3H), 1.18 (d, J= 6.9 Hz,
3H), 1.15 (s, 3H),
1.10 (s, 3H), 0.84 (t, J= 7.2 Hz, 3H);
13C NMR (100 MHz, CDC13) 8 177.4, 170.1, 168.8, 157.6, 152.8, 144.4, 143.1,
141.1,
136.7, 136.2, 134.9, 133.8, 128.7, 125.7, 125.2, 123.0, 113.9, 107.5, 107.2,
101.7, 94.9, 92.6,
80.8, 79.2, 76.6, 74.8, 73.5, 72.7, 71.9, 71.7, 70.2, 70.1, 69.5, 63.5, 62.3,
41.5, 36.6, 34.3,
29.5, 28.2, 26.2, 26.0, 19.4, 19.3, 18.9, 18.5, 17.8, 17.3, 15.3, 14.1, 13.7,
11.1;
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Analytical data of Lipiarmycin A4
MS m/ 1060.5 (M + NH4);
1H NMR (400 MHz, CDC13) 6 7.12 (d, J= 11.6 Hz, 1H), 6.59 (dd, J= 14.1, 11.6
Hz,
1H), 5.85 (br s, 1H), 5.83 (ddd, J = 14.1, 10.6, 4.8 Hz, 1H), 5.47 (dd, J =
8.3, 8.3 Hz, 1H),
5.12 (dd, J = 9.6, 9.6 Hz, 1 H), 5.00 (d, J = 10.1 Hz, 1 H), 4.98 (br d, J =
10.6 Hz, 1 H), 4.75-
4.69 (m, 1H), 4.68 (d, J= 11.4 Hz, 1H), 4.66 (br s, 1H), 4.62 (br s, 1H), 4.40
(d, J= 11.4 Hz,
1H), 4.26 (br s, 1H), 4.07-4.00 (m, I H), 4.02 (br d, J= 3.3 Hz, I H), 3.75-
3.61 (m, 4H), 3.62
(s, 3H), 3.55 (dq, J = 9.6, 6.1 Hz, 1H), 2.82-2.45 (m, 6H), 2.60 (s, 3H), 2.07-
1.97 (m, 1H),
1.92 (s, 3H), 1.81 (s, 3H), 1.67 (s, 3H), 1.32 (d, J= 6.1 Hz, 3H), 1.30-1.22
(m, 1H), 1.21 (d, J
= 6.6 Hz, 3H), 1.19 (d, J= 7.1 Hz, 3H), 1.18 (d, J= 7.1 Hz, 3H), 1.15 (s, 3H),
1.10 (s, 3H),
0.83 (t, J= 7.2 Hz, 3H);
13C NMR (100 MHz, CDC13) S 177.4, 170.5, 168.9, 157.8, 153.0, 144.3, 140.9,
137.7, 137.0,
136.3, 134.6, 134.4, 129.1, 127.9, 125.3, 123.2, 114.5, 107.4, 107.0, 101.8,
94.7, 92.5, 80.3,
79.6, 76.7, 74.9, 73.5, 72.7, 71.9, 71.6, 70.2, 70.1, 69.1, 63.6, 62.3, 41.9,
36.9, 34.4, 28.8,
28.2,25.9,20.0,19.3,19.0,18.6,18.5,17.8,17.2,15.5,13.8.11.2;
Analytical data of C-19 Ketone
MS m/z 1072.5 (M + NH4)+;
1H NMR (400 MHz, CDC13) S 7.27 (d, J = 11.4 Hz, 1 H), 6.61 (dd, J = 14.7, 11.4
Hz,
1H), 5.91 (ddd, J = 14.7, 9.1, 5.8 Hz, I H), 5.83 (s, 1H), 5.31 (dd, J = 7.9,
7.9 Hz, I H), 5.14
(dd, J = 9.7, 9.7 Hz, 1H), 5.06 (d, J = 10.6 Hz, 1H), 5.00 (d, J = 10.1 Hz,
1H), 4.98 (dd, J =
7.1, 4.8 Hz, 1H), 4.67 (d, J= 11.9 Hz, 1H), 4.66 (br s, 1H), 4.61 (br s, 1H),
4.42 (d, J= 11.9
Hz, 1H), 4.30 (br s, 1H), 4.02 (br d, J = 3.3 Hz, 1H), 3.63-3.60 (m, 4H), 3.62
(s, 3H), 3.51
(dq, J = 9.7, 6.1 Hz, 1H), 3.09 (dq, J = 14.4, 7.3 Hz, 1H), 3.03 (dq, J =
14.4, 7.3 Hz, 1H),
2.76-2.50 (m, 6H), 2.21 (s, 3H), 1.93-1.87 (m, 1H), 1.87 (s, 3H), 1.75 (s,
3H), 1.63 (s, 3H),
1.32 (d, J= 6.1 Hz, 3H), 1.27-1.22 (m, 1H), 1.21 (t, J= 7.3 Hz, 3H), 1.19 (d,
J= 7.1 Hz, 3H),
1.18 (d, J= 7.1 Hz, 3H), 1.14 (s, 3H), 1.10 (s, 3H), 0.84 (t, J= 7.3 Hz, 3H);
13C NMR (100 MHz, CDC13) S 205.5, 177.4, 170.1, 166.9, 157.6, 152.8, 145.7,
143.1,
142.0, 137.1, 136.8, 135.5, 133.7, 128.3, 124.8, 124.0, 122.8, 113.9, 107.3,
107.2, 101.3,
94.8, 92.4, 80.4, 77.7, 76.6, 74.7, 73.5, 72.6, 71.8, 71.7, 70.2, 70.0, 63.0,
62.3, 41.5, 36.5,
34.3, 29.6, 28.1, 26.2, 26.1, 26.0, 19.2, 18.9, 18.5, 17.8, 17.3, 15.2, 14.0,
13.3, 11.0
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Example 3: Biological activity
MIC values determined for several C. difficile strains
OPT-80 (which is composed almost entirely of the R-Tiacumicin B) and its
related
compounds were tested against C. diffzcile. The MIC values are reported below
in Table 3.
As we can see, OPT-80 was especially active when compared to S-Tiacumicin B
and
Lipiarmycin A4.
Table 3: MIC ml versus C. di zcile strains
C. difficile OPT-80 (composed S-Tiacumicin B Lipiarmycin A4 C-19
strains almost entirely of Ketone
R-Tiacumicin B
ATCC 9689 0.03 0.125 0.06 0.06
ATCC 43255 0.125 1 0.5 0.5
ATCC 17857 0.03 0.25 0.06 nd
LC # 1 (Clinical 0.125 1 0.5 0.5
isolate)
MIC values determined for various microorganisms
OPT-80 (which is composed almost entirely of the R-Tiacumicin B) and its
related
compounds were tested against several other pathogens. The MIC values are
reported below
in Table 4. As we can see, OPT-80 was especially active when compared to S-
Tiacumicin B
and Lipiarmycin A4.
Table 4: MIC ml against other microorganisms
Strain Organism OPT-80 S-Tiacumicin B Lipiarmycin
ID # (Composed almost A4
entirely of R-Tiacumicin
B)
1 S. aureus 4 64 8
(ATCC 29213)
2 S. aureus, (MRSA) 4 64 16
3 S. aureus, (MRSA) 4 64 8
4 E. faecalis 2 8 2
(ATCC 29212)
5 E. faecalis 4 32 16
Vanc. resistant
6 E. faecalis 1 16 4
Vanc. resistant
7 E. faeciuzn 1 8 4
Vanc. resistant
8 E. faeciunz 1 32 32
Vanc. resistant
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Example 4: Post-Antibiotic Effect of OPT-80 in C. difficile
The post-antibiotic effect (PAE) of OPT-80 (which is composed almost entirely
of the
R-Tiacumicin B) was measured versus two strains of C. difficile, ATCC 43255
and a clinical
isolate, LC3. Vancomycin and rifampin were tested additionally versus LC3.
The PAE at 4x the MIC was observed to be extremely long: greater than 24
hours, for
both strains. Because of the long duration of this effect, an exact PAE was
not calculated.
Vancomycin, on the other hand, had a more normal PAE of less than an hour when
used at 4x
the MIC versus strain LC3.
Example 5: In Vitro Activity of OPT-80
The in vitro efficacy of OPT-80 (which is composed almost entirely of the R-
Tiacumicin B),
metronidazole, and vancomycin were assessed versus 110 genetically distinct
clinical isolates
of C. docile via agar dilution. The MIC data are presented in Tables 5 and 6.
Table 5. Geometric mean, MIC ranges, MIC50, and MIC90 values for OPT-80against
110 C.
difficile clinical isolates, vancomycin, and metronidazole, in g/mL.
Range Geometric Mean MIC50 MIC90
OPT-80 0.015-0.25 0.08 0.125 0.125
Metronidazole 0.025-0.5 0.15 0.125 0.25
Vancomycin 0.06 - 4 0.8 1 1
Table 6. Raw MIC data for OPT-80, vancomycin (VAN), and metronidazole (MTZ)
versus
110 clinical isolates of C. difficile, in g/mL.
ORG ID OPT-80 MTZ VAN ORG ID OPT-80 MTZ VAN
Al 1535 0.125 0.25 1 COI 4652 0.25 0.125 1
B1832 0.06 0.125 1 CP1 5491 0.125 0.25 1
D11360 0.03 0.25 1 61 5930 0.03 0.25 1
El 816 0.06 0.125 1 63 6029 0.25 0.25 0.06
F11015 0.125 0.125 1 64 5940 0.125 0.25 1
G11077 0.125 0.125 1 65 5967 0.06 0.25 0.5
111389 0.125 0.125 1 66 6366 0.015 0.125 0.5
J15971 0.06 0.25 1 67 6367 0.125 0.25 1
J7 4224 0.03 0.125 1 68 6368 0.03 0.125 0.06
J9 4478 0.06 0.125 1 69 6370 0.25 0.25 0.5
K14305 0.125 0.25 0.5 70 6376 0.125 0.25 2
K14 5780 0.125 0.125 1 71 6379 0.125 0.25 1
L11423 0.125 0.125 0.5 72 6380 0.125 0.25 2
N1471 0.125 0.125 0.5 73 6382 0.25 0.25 1
011861 0.06 0.125 1 75 6388 0.125 0.125 0.5
24
CA 02596387 2007-07-31
WO 2006/085838 PCT/US2005/002887
ORG ID OPT-80 MTZ VAN ORG ID OPT-80 MTZ VAN
R1397 0.125 0.125 1 76 6389 0.125 0.25 0.5
R6 6015 0.015 0.25 2 77 6390 0.06 0.125 1
V11521 0.125 0.125 0.5 78 6392 0.015 0.03 0.5
W13931 0.125 0.5 1 80 6327 0.125 0.125 0.5
X11890 0.125 0.125 1 81 6328 0.125 0.125 0.5
Y15639 0.06 0.125 0.5 82 6329 0.06 0.03 0.5
Y21459 0.06 0.125 1 83 6330 0.06 0.125 0.5
Z13036 0.03 0.125 1 84 6331 0.125 0.25 0.5
AA2 4380 0.015 0.125 1 85 6332 0.06 0.125 1
AB2 1725 0.06 0.125 1 86 6333 0.03 0.125 0.5
AC1 1546 0.06 0.125 1 87 6334 0.125 0.125 0.5
AF11808 0.125 0.125 0.5 88 6335 0.125 0.25 0.5
AG1 3044 0.125 0.125 1 89 6336 0.25 0.5 1
AM 3430 0.125 0.25 0.5 90 6338 0.125 0.125 1
AJ1 1557 0.06 0.125 1 91 6339 0.125 0.125 1
AL11753 0.06 0.125 0.5 93 6341 0.125 0.125 1
AN1 464 0.125 0.125 0.5 94 6343 0.015 0.06 0.5
AO1287 0.125 0.125 1 95 6347 0.125 0.125 1
AS1 4099 0.125 0.125 1 96 6348 0.06 0.125 0.5
AT1 1216 0.125 0.125 1 97 6349 0.25 0.125 1
AV1 941 0.25 0.125 0.5 98 6350 0.125 0.5 1
CJ1893 0.125 0.025 1 1016354 0.015 0.06 1
AW14501 0.125 0.125 1 102 6355 0.016 0.125 1
BE1 4307 0.125 0.25 1 103 6068 0.06 0.125 1
Bill 4506 0.06 0.06 0.5 104 6060 0.03 0.25 1
BIl 1675 0.125 0.125 1 105 6071 0.03 0.125 0.5
BKl 4291 0.125 0.125 0.5 106 6078 0.03 0.25 0.5
BL1 716 0.125 0.125 1 107 6079 0.06 0.125 0.5
BMl 1453 0.06 0.125 1 109 6274 0.015 0.125 1
BN11322 0.125 0.25 1 1116279 0.03 0.125 1
BR1 1321 0.06 0.125 1 112 6280 0.06 0.125 0.5
BTl 706 0.06 0.125 1 113 6304 0.06 0.125 1
BVl 1183 0.125 0.25 1 114 386 0.06 0.125 4
BWl 3130 0.125 0.125 1 115 5985 0.015 0.25 2
BXl 4271 0.125 0.25 1 116 5702 0.06 0.125 1
CN1 667 0.25 0.25 117 6026 0.06 0.125 2
CB1 1584 0.25 0.125 120 6057 0.03 0.25 1
CFI 5922 0.125 0.125 1216072 0.06 0.25 0.5
CG1 1566 0.125 0.125 122 6111 0.25 0.25 0.5
CLl 3851 0.25 0.125 100 6353 0.125 0.25 1
CA 02596387 2012-04-12
Example 6: Activity of OPT-80 Compared Against Selected Anaerobic Species
The in vitro activity of OPT-80 was determined against 350 anaerobes. The
experimental procedure for which is outlined in Antimicrobial Agents and
Chemotherapy,
2004,48: 4430-4434.
All organisms, including the 21 C. docile strains, were separate isolates and
not
clonally related. All quality-control gram-negative and -positive strains
recommended by
NCCLS were included with each run: in every case, results (where available)
were in range.
Results of MIC testing are presented in Table 7.
Table 7 MICs ( g/ml) of OPT-80
Organism MIC range MIC50 MIC90
Bacteroides a 7is (19) 64 ->128 >128 >128
Non- a 'lis B. a 'lis group species 38) 64 - >128 >128 >128
Prevotella/Po h omonas species (42) 16 - >128 >128 >128
Fusobacteriunz nucleatum (14) 64 - >128 >128 >128
Fusobacterium morti eruzn (10) 64 - >128 >128 >128
Fusobacteriuzn species, miscellaneous (14) 16 - >128 >128 >128
Pe tostr tococcus tetradius (16) 0.25-2.0 1.0 1.0
Pe tostre tococcus asaccharol ticus (15) 0.25 -1.0 0.5 1.0
P tostre tococcus anaerobius (15) <0.016 - 0.03 <0.016 <0.016
Fine oldia magna 15 0.25-2.0 1.0 1.0
Micromonas micros (14) <0.016-0.06 0.03 0.06
P tostr tococcus prevotii (3) 0.25-1.0 NA NA
Pro ionibacteriuzn acnes (20) 0.5 -1.0 4.0 4.0
E erthella lento (10) <0.016-0.06 <0.016 <0.03
Miscellaneous gram-positive non-spore- <0.016-16 <0.125 16
forming rods (20)
Clostridiwn ped~ingens 35 <0.016-0.06 <0.016 0.03
Clostridium di zcile (21) <0.016 - 0.25 <0.016 0.125
Clostridium tertium 10 <0.016 - 0.06 <0.016 0.03
Clostridium species (19) <0.016-0.06 <0.016 0.03
Clostridium spp. (all) (85) <0.016 - 0.06 <0.016 0.06
26
CA 02596387 2012-04-12
Example 7: In Vitro Activities of OPT-80 Against Intestinal Bacteria
The in vitro activity of OPT-80 against intestinal bacteria was evaluated. The
experimental procedure for which is outlined in Antimicrobial Agents and
Chemotherapy,
2004, 48: 4898-4902.
Antimicrobial concentration ranges were selected to encompass or surpass the
levels
that would be achieved in the gut (to the extent that this information is
available), subject to
the limitations of solubility of the drugs in the testing medium. The range of
concentration of
OPT-80used during testing was 0.03 g/ml to 1024 g/ml.
For analysis, the bacteria tested were generally placed into genus, species,
or other
groups with at least 10 isolates. The ranges and the MICs at which 50 and 90%
of isolates
were inhibited were determined except for organisms with fewer than 10 strains
tested, for
which only the ranges are reported (Table 8).
OPT-80 had good activity against most anaerobic gram positive non-spore-
forming
rods and anaerobic gram-positive cocci. OPT-80 also showed good activity
against
enterococci and staphylococci.
Table 8 In vitro activity of OPT-80 against 453 bacterial isolates
Organism MIC range M1C59 M1C90
Bacteroides fragilis group spp. (50) 256 - >1024 256 >1024
Veillonella spp. (10) 16 -128 32 128
Other anaerobic gram negative rods (51) 0.06-1024 1024 >1024
All anaerobic gram-negative species (111) 0.06 - >1024 256 >1024
Clostridium biferlnentans (9) 0.06 NA NA
Clostridium bolteae (7) 1- 64 NA NA
Clostridium clostridioforme (4) 4-128 NA NA
Clostridium difcile (23) 0.06 - 2 0.12 0.25
Clostridium glycolicum (9) 0.06-1 NA NA
Clostridium innocuum (9) 32-128 NA NA
Clostridium paraputrificum (8) 0.06 - 8 NA NA
Clostridium perfringens (14) 0.06 0.062 0.062
Clostridium ramosum (10) 256 - 512 512 512
Clostridium sordellii (5) 0.06 NA NA
Other clostridial species (9) 0.06 - >1024 NA NA
27
CA 02596387 2012-04-12
=
All Clostridium species (107) 0.06->1024 0.062 128
Anaerobic non-spore-forming gram-positive 0.06 - >1024 1 32
rods (63)
Anaerobic gram-positive cocci (49) 0.06 - >1024 0.5 2
All anaerobic gram-positive species (219) 0.06->1024 0.12 64
Streptococcus, formerly S. milleri group (14) 16 - 64 32 32
Other Streptococcus species (9) 16-128 NA NA
Enterococcus species (21) 2.0-16 8 8
Staphylococcus aureus and Staphylococcus 0.25 - 2 0.5 2
epidern:idis (19)
Total for all strains (453) 0.06 - >1024 8 1024
OTHER EMBODIlVIENTS
The scope of the claims should not be limited by the preferred embodiments
set forth in the examples, but should be given the broadest interpretation
consistent
with the description as a whole.
28
