U.S. patent application number 13/046375 was filed with the patent office on 2011-07-07 for 18-membered macrocycles and analogs thereof.
Invention is credited to Farah Babakhani, Yu-Hung Chiu, Chan-Kou Hwang, Franklin Okumu, Alex Romero, Pamela Sears, Youe-Kong SHUE.
Application Number | 20110166090 13/046375 |
Document ID | / |
Family ID | 39887707 |
Filed Date | 2011-07-07 |
United States Patent
Application |
20110166090 |
Kind Code |
A1 |
SHUE; Youe-Kong ; et
al. |
July 7, 2011 |
18-Membered Macrocycles and Analogs Thereof
Abstract
The present invention relates generally to the 18-membered
macrocyclic antimicrobial agents called Tiacumicins, specifically,
OPT-80 (which is composed almost entirely of the R-Tiacumicin B),
pharmaceutical compositions comprising OPT-80, and methods using
OPT-80. In particular, this compound is a potent drug for the
treatment of bacterial infections, specifically C. difficile
infections.
Inventors: |
SHUE; Youe-Kong; (US)
; Hwang; Chan-Kou; (US) ; Chiu; Yu-Hung;
(US) ; Romero; Alex; (US) ; Babakhani;
Farah; (US) ; Sears; Pamela; (US) ;
Okumu; Franklin; (US) |
Family ID: |
39887707 |
Appl. No.: |
13/046375 |
Filed: |
March 11, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11882219 |
Jul 31, 2007 |
7906489 |
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13046375 |
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PCT/US2005/002887 |
Jan 31, 2005 |
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11882219 |
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Current U.S.
Class: |
514/28 |
Current CPC
Class: |
A61P 31/04 20180101;
A61K 31/7048 20130101; Y10S 514/867 20130101; A61P 1/12 20180101;
C07H 17/08 20130101 |
Class at
Publication: |
514/28 |
International
Class: |
A61K 31/7048 20060101
A61K031/7048; A61P 1/12 20060101 A61P001/12 |
Claims
1-16. (canceled)
17. A method of treating reoccurring diarrhea caused by C.
difficile gastrointestinal infection in a human patient previously
treated for the diarrhea, comprising orally administering to said
patient a therapeutically effective amount of a compound having the
formula (IV): ##STR00023## or a pharmaceutically acceptable salt
combined with one or more pharmaceutically acceptable carriers,
wherein the compound having formula (IV) is greater than 90% by
weight stereomerically pure.
18. The method of claim 17, wherein the method consists of
administering to the human patient a therapeutically effective
amount of the compound having formula (IV) or a pharmaceutically
acceptable salt thereof combined with one or more pharmaceutically
acceptable carriers.
19. The method of claim 17, wherein the compound of formula (IV) is
formulated as a tablet.
20. The method of claim 17, wherein the compound of formula (IV) is
formulated as a capsule.
21. The method of claim 17, wherein the compound of formula (IV) is
greater than 93% by weight stereomerically pure.
22. The method of claim 17, wherein the compound of formula (IV) is
greater than 95% by weight stereomerically pure.
23. The method of claim 17, wherein the compound of formula (IV) is
greater than 97% by weight stereomerically pure.
24. The method of claim 17, wherein the compound of formula (IV) is
substantially free of other diastereomers of the compound.
25. The method of claim 17, wherein the patient suffers from
colitis.
26. A method of treating reoccurring diarrhea caused by C.
difficile gastrointestinal infection in a human patient previously
treated for the diarrhea, consisting of orally administering to
said patient a therapeutically effective amount of a compound
having the formula (IV): ##STR00024## or a pharmaceutically
acceptable salt thereof combined with one or more pharmaceutically
acceptable carriers, wherein the compound of the formula (IV) is
greater than 93% by weight stereomerically pure.
27. The method of claim 26, wherein the compound of formula (IV) is
formulated as a tablet.
28. The method of claim 26, wherein the compound is formulated as a
capsule.
29. The method of claim 26, wherein the compound of formula (IV) is
greater than 95% by weight stereomerically pure.
30. The method of claim 26, wherein the compound of formula (IV) is
greater than 97% by weight stereomerically pure.
31. The method of claim 26, wherein the compound of formula (IV) is
substantially free of other diastereomers of the compound.
32. The method of claim 26, wherein the patient suffers from
colitis.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation-in-part
application of International Application PCT/US2005/002887, filed
Jan. 31, 2005, which is incorporated by reference in its
entirety.
FIELD OF INVENTION
[0002] 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 difficile (C. difficile), Staphylococcus aureus (S.
aureus) including methicillin-resistant Staphylococcus aureus
(MRSA) and Clostridium perfringens (C. perfringens).
BACKGROUND OF THE INVENTION
[0003] 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).
[0004] Tiacumicins are a family of related compounds that contain
the 18-membered ring shown in Formula I below.
##STR00001##
[0005] 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, 40: 575-588), as
shown in Table 1.
TABLE-US-00001 TABLE 1 Substituents Present In Tiacumcins A-F
R.sup.1 R.sup.2 R.sup.3 A ##STR00002## H H B ##STR00003##
##STR00004## OH C ##STR00005## ##STR00006## OH D ##STR00007##
##STR00008## OH E ##STR00009## ##STR00010## OH F ##STR00011##
##STR00012## OH
[0006] 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 .sup.1H and
.sup.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.
[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, 40: 567-574 and U.S. Pat. No. 4,918,174.
[0008] 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.
[0009] 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. difficile, 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.
[0010] 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
burns 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.
[0011] 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.
[0012] 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 Clostridium
difficile-associated diarrhea (CDAD) has been attributed to the
frequent prescription of broad-spectrum antibiotics to hospitalized
patients.
[0013] 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.
[0014] 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.
[0015] 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.
[0016] 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. difficile 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).
[0017] Tiacumicins, specifically Tiacumicin B, show activity
against a variety of bacterial pathogens and in particular against
C. difficile, a Gram-positive bacterium (Antimicrob. Agents
Chemother. 1991, 1108-1111). C. 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. C. 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.
[0018] Tiacumicin antibiotics are described in U.S. Pat. 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. Pat. No. 5,583,115 (issued Dec. 10, 1996), and U.S.
Pat. No. 5,767,096 (issued Jun. 16, 1998), which are all
incorporated herein by reference. 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), which are all
incorporated herein by reference.
SUMMARY OF THE INVENTION
[0019] 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.
[0020] 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.
[0021] In another embodiment of the present invention the
substantially pure R-Tiacumicin B has an unusually long
post-antibiotic activity (PAE).
[0022] 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, which is hereby
incorporated by reference.
BRIEF DESCRIPTION OF THE FIGURES
[0023] FIG. 1 shows the Oak Ridge Thermal Ellipsoid Plot Program
(ORTEP) chemical structure of R-Tiacumicin B.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0024] 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. difficile, 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 symptoms. Diarrhea, when severe, causes dehydration and the
medical complications associated with dehydration.
[0025] 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.sup.n, where n is the number of asymmetric
carbons.
[0026] As used herein, and unless otherwise indicated, the terms
"biohydrolyzable carbamate," "biohydrolyzable carbonate,"
"biohydrolyzable ureide" and "biohydrolyzable phosphate" mean a
carbamate, carbonate, ureide and phosphate, respectively, of a
compound that either: 1) does not interfere with the biological
activity of the compound but can confer upon that compound
advantageous properties in vivo, such as uptake, duration of
action, or onset of action; or 2) is biologically inactive but is
converted in vivo to the biologically active compound. Examples of
biohydrolyzable carbamates include, but are not limited to, lower
alkylamines, substituted ethylenediamines, aminoacids,
hydroxyalkylamines, heterocyclic and heteroaromatic amines, and
polyether amines.
[0027] As used herein, and unless otherwise indicated, the term
"biohydrolyzable ester" means an ester of a compound that either:
1) does not interfere with the biological activity of the compound
but can confer upon that compound advantageous properties in vivo,
such as uptake, duration of action, or onset of action; or 2) is
biologically inactive but is converted in vivo to the biologically
active compound. Examples of biohydrolyzable esters include, but
are not limited to, lower alkyl esters, alkoxyacyloxy esters, alkyl
acylamino alkyl esters, and choline esters.
[0028] As used herein, and unless otherwise indicated, the term
"biohydrolyzable amide" means an amide of a compound that either:
1) does not interfere with the biological activity of the compound
but can confer upon that compound advantageous properties in vivo,
such as uptake, duration of action, or onset of action; or 2) is
biologically inactive but is converted in vivo to the biologically
active compound. Examples of biohydrolyzable amides include, but
are not limited to, lower alkyl amides, .alpha.-amino acid amides,
alkoxyacyl amides, and alkylaminoalkylcarbonyl amides.
[0029] 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.
[0030] The term "C-19 Ketone" refers to a Tiacumicin B related
compound shown below in Formula II:
##STR00013##
[0031] The term "diastereomers" refers to stereoisomers that are
not mirror images of each other.
[0032] 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.
[0033] 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.
[0034] The term "halogen" includes F, Cl, Br and I.
[0035] As used herein, the term "hydrate" means a compound of the
present invention or a salt thereof that further includes a
stoichiometric or non-stoichiometric amount of water bound by
non-covalent intermolecular forces.
[0036] 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.
[0037] The term "Lipiarmycin A4" refers to a Tiacumicin B related
compound shown below in Formula III:
##STR00014##
[0038] 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., C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6, C.sub.7, C.sub.8,),
more preferably 1 to 4 carbons (e.g., C.sub.1, C.sub.2, C.sub.3,
C.sub.4,). 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., C.sub.1, C.sub.2, C.sub.3,
C.sub.4,).
[0039] The term "macrocycles" refers to organic molecules with
large ring structures usually containing over 10 atoms.
[0040] The term "18-membered macrocycles" refers to organic
molecules with ring structures containing 18 atoms.
[0041] 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.
[0042] The term "MIC" or "minimum inhibitory concentration" refers
to the lowest concentration of an antibiotic that is needed to
inhibit growth of a bacterial 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).
[0043] The term "MIC.sub.50" refers to the lowest concentration of
antibiotic required to inhibit the growth of 50% of the bacterial
strains tested within a given bacterial species.
[0044] The term "MIC.sub.90" refers to the lowest concentration of
antibiotic required to inhibit the growth of 90% of the bacterial
strains tested within a given bacterial species.
[0045] The term "OPT-80" refers to a preparation containing
R-Tiacumicin B and Tiacumicin B related compounds (including, but
not limited to, Tiacumicins, 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 and which preparations and are
incorporated here by reference.
[0046] 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.
[0047] 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.
[0048] 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.
[0049] The term "pharmaceutically acceptable carrier" refers to a
carrier or diluent that is pharmaceutically acceptable.
[0050] 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 potassium), 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. The term "pharmaceutically acceptable
salt" also refers to salts prepared from pharmaceutically
acceptable non-toxic acids, including inorganic acids and organic
acids. Suitable non-toxic acids include inorganic and organic acids
such as, but not limited to, acetic, alginic, anthranilic,
benzenesulfonic, benzoic, camphorsulfonic, citric, ethenesulfonic,
formic, fumaric, furoic, gluconic, glutamic, glucorenic,
galacturonic, glycidic, hydrobromic, hydrochloric, isethionic,
lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric,
pamoic, pantothenic, phenylacetic, propionic, phosphoric,
salicylic, stearic, succinic, sulfanilic, sulfuric, tartaric acid,
p-toluenesulfonic and the like. Particularly preferred are
hydrochloric, hydrobromic, phosphoric, and sulfuric acids, and most
particularly preferred is the hydrochloride salt.
[0051] The term "pharmaceutical composition" refers to a
composition of the R-Tiacumicin 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 a mammal, including
humans.
[0052] 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.
[0053] As used herein, and unless otherwise indicated, the term
"prodrug" means a derivative of a compound that can hydrolyze,
oxidize, or otherwise react under biological conditions (in vitro
or in vivo) to provide the compound. Examples of prodrugs include,
but are not limited to, compounds that comprise biohydrolyzable
moieties such as biohydrolyzable amides, biohydrolyzable esters,
biohydrolyzable carbamates, biohydrolyzable carbonates,
biohydrolyzable ureides, and biohydrolyzable phosphate analogues.
Other examples of prodrugs include compounds that comprise --NO,
--NO.sub.2, --ONO, or --ONO.sub.2 moieties. When used to describe a
compound of the invention, the term "prodrug" may also to be
interpreted to exclude other compounds of the invention for example
racemates.
[0054] 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.
[0055] The terms "R" and "S" configuration, as used herein, are as
defined by the IUPAC 1974 Recommendations for Section E,
Fundamental Stereochemistry, Pure Appl. Chem. (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 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--.
[0056] 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:
##STR00015##
[0057] 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:
##STR00016##
[0058] 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.
[0059] As used herein, and unless otherwise indicated, the terms
"optically pure," "stereomerically pure," and "substantially
stereomerically pure" are used interchangeably and mean one
stereoisomer of a compound or a composition that comprises one
stereoisomer of a compound and is substantially free of other
stereoisomer(s) of that compound. For example, a stereomerically
pure compound or composition of a compound having one chiral center
will be substantially free of the opposite enantiomer of the
compound. A stereomerically pure compound or composition of a
compound having two chiral centers will be substantially free of
other diastereomers of the compound. A typical stereomerically pure
compound comprises greater than about 80% by weight of one
stereoisomer of the compound and less than about 20% by weight of
other stereoisomers of the compound, more preferably greater than
about 90% by weight of one stereoisomer of the compound and less
than about 10% by weight of the other stereoisomers of the
compound, even more preferably greater than about 95% by weight of
one stereoisomer of the compound and less than about 5% by weight
of the other stereoisomers of the compound, and most preferably
greater than about 97% by weight of one stereoisomer of the
compound and less than about 3% by weight of the other
stereoisomers of the compound.
[0060] 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.
[0061] 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:
##STR00017##
[0062] The term "Tiacumicin B" as used herein refers to the
18-membered macrocycle shown below in Formula VI:
##STR00018##
[0063] 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.
[0064] 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.
[0065] The present invention relates to new antibacterial
compositions containing R-Tiacumicins, specifically the
R-Tiacumicin B (which has an R-hydroxyl at C-19), and to the use of
these new compositions in combination with existing drugs to treat
infections caused by gram-positive anerobes.
[0066] The present invention further relates to stereoisomerically
pure Tiacumicin B, which contains 90-100% of the R-stereoisomer,
preferably at least 93% of the R-stereoisomer, more preferably 95%
of the R-stereoisomer, even more preferably 99% of the
R-stereoisomer.
[0067] In accordance with the present invention there are provided
compounds with the structure of Formula VII:
##STR00019##
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 (.dbd.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.
[0068] Preferred compounds of the invention are compounds of
Formula VII wherein X is methyl or ethyl, Y is ketone (.dbd.O) or
OH and Z is isopropyl.
[0069] More preferred compounds of the invention are the compound
of the Formula VII wherein X is ethyl, Y is ketone (.dbd.O) or OH
and Z is isopropyl.
[0070] 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.
[0071] 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 (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. difficile, S. aureus, E. faecalis, and E. faecium. This effect
of the C-19 chiral center on biological activity is an unexpected
and novel discovery.
[0072] 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.
[0073] 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.
[0074] 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
[0075] 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.
[0076] The microorganism to produce the active antibacterial agents
was identified as belonging to the family Actinoplanaceae, genus
Dactylosporangium (J. of Antibiotics, 1987, 40: 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, 40:
567-574 and U.S. Pat. No. 4,918,174.
[0077] Methods of isolating stereomerically pure isomers are known
in the art. Methods of isolating stereomerically pure R-Tiacumicin
include, but are not limited to, recrystallization of the crude
mixture in solvents including, aqueous methanol or isopropanol and
chiral HPLC.
[0078] 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,
which is hereby incorporated by reference.
Pharmaceutical Formulation and Administration
[0079] 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.
[0080] 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.
[0081] 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.
[0082] 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, 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 D.C.
[0083] 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 934,
silicone, glyceryl tristearate, methyl acrylate-methyl
methacrylate, polyvinyl chloride, polyethylene, and/or halogenated
fluorocarbon.
[0084] 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
hydroxypropyl-methylcellulose. 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 release compositions are
known in the art (see, for example, U.S. Pat. Nos. 4,946,685 and
6,261,601).
[0085] 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. Pat. Nos. 4,111,202 and
3,279,995).
[0086] 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.
[0087] 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.
[0088] 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.
[0089] Microspheres can be made by any appropriate method, or from
any pharmaceutically acceptable material. Particularly useful are
proteinoid microspheres (see, for example, U.S. Pat. No. 5,601,846,
or 5,792,451) and PLGA-containing microspheres (see, for example,
U.S. Pat. No. 6,235,224 or 5,672,659). Other polymers commonly used
in the formation of microspheres include, for example,
poly-.epsilon.-caprolactone, poly(e.about.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. Pat. No. 6,365,187).
[0090] 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.
EXAMPLES
[0091] The following examples are provided by way of describing
specific embodiments of the present invention without intending to
limit the scope of the invention in any way.
Example 1
Exact Structure of R-Tiacumicin B
[0092] 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.times.0.14.times.0.22 mm) grown in methanol and is shown as
an ORTEP diagram in FIG. 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-.beta.-D-mannopyranosyl]oxy]-methyl]-12(R)-[[6-deoxy-5-C-methyl-4-O-
-(2-methyl-1-oxopropyl)-.beta.-D-lyxo-hexopyranosyl]oxy]-11(S)-ethyl-8(S)--
hydroxy-18(S)-(1(R)-hydroxyethyl)-9,13,15-trimethyloxacyclooctadeca-3,5,9,-
13,15-pentaene-2-one.
##STR00020##
Example 2
Analytical Data of OPT-80 and Related Substances
[0093] 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.
##STR00021##
TABLE-US-00002 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 Lipiarmycin A4 Methyl (S)--OH Isopropyl C-19 Ketone Ethyl
.dbd.O Isopropyl
Analytical Data of R-Tiacumicin B
[0094] mp 166-169.degree. C. (white needle from isopropanol);
[0095] [.alpha.].sub.D.sup.20-6.9 (c 2.0, MeOH);
[0096] MS m/z (ESI) 1079.7 (M+Na).sup.+;
[0097] .sup.1H .sup.1H NMR 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);
[0098] .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.
Analytical Data of the S-Tiacumicin B
##STR00022##
[0100] NaBH.sub.4 (9 eq, 48 mg) was added in three portions to a
solution of C-19 Ketone (150 mg) in 3 mL MeOH. After 1 h, saturated
NH.sub.4Cl solution was added. The mixture was extracted with
CHCl.sub.3, and then concentrated. S-Tiacumicin B was purified by
YMC-pack ODS-A 75.times.30 mm I.D. column (H.sub.2O:MeOH:AcOH
28:72:1) yielding pure 35 mg of pure S-Tiacumicin B.
[0101] MS m/z 1074.5 (M+NH.sub.4).sup.+;
[0102] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.15 (d, J=11.4
Hz, 1H), 6.58 (dd, J=14.1, 11.4 Hz, 1H), 5.82 (ddd, J=14.1, 10.6,
3.5 Hz, 1H), 5.78 (s, 1H), 5.40 (dd, J=7.8, 7.8 Hz, 1H), 5.15 (dd,
J=9.5, 9.5 Hz, 1H), 5.01 (d, J=9.9 Hz, 1H), 5.01 (d, J=9.9 Hz, 1H),
4.77 (ddd, J=5.8, 5.3, 5.3 Hz, 1H), 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);
[0103] .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 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;
Analytical data of Lipiarmycin A.sub.4
[0104] MS m/z 1060.5 (M+NH.sub.4).sup.+;
[0105] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 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, 1H), 5.00 (d, J=10.1 Hz, 1H), 4.98 (br d,
J=10.6 Hz, 1H), 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, 1H), 4.02 (br d, J=3.3 Hz, 1H), 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);
[0106] .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 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
[0107] MS m/z 1072.5 (M+NH.sub.4).sup.+;
[0108] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.27 (d, J=11.4
Hz, 1H), 6.61 (dd, J=14.7, 11.4 Hz, 1H), 5.91 (ddd, J=14.7, 9.1,
5.8 Hz, 1H), 5.83 (s, 1H), 5.31 (dd, J=7.9, 7.9 Hz, 1H), 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);
[0109] .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 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
Example 3
Biological Activity
MIC Values Determined for Several C. difficile Strains
[0110] OPT-80 (which is composed almost entirely of the
R-Tiacumicin B) and its related compounds were tested against C.
difficile. The MIC values are reported below in Table 3. OPT-80 was
surprisingly active when compared to its enantiomer S-Tiacumicin B
and Lipiarmycin A4.
TABLE-US-00003 TABLE 3 MIC (.mu.g/ml) versus C. difficile strains
R-Tiacumicin B (>90% C. difficile Stereomerically S-Tiacumicin
Lipiarmycin C-19 strains Pure) B A4 Ketone 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 0.125 1 0.5 0.5 (Clinical isolate)
MIC Values Determined for Various Microorganisms
[0111] 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. OPT-80 was suprisingly active when compared to S-Tiacumicin B
and Lipiarmycin A4.
TABLE-US-00004 TABLE 4 MIC (.mu.g/ml) against other microorganisms
R-Tiacumicin B (>90% Strain Stereomerically Lipiarmycin ID #
Organism Pure) S-Tiacumicin B A4 1 S. aureus 4 64 8 (ATCC 29213) 2
S. aureus, 4 64 16 (MRSA) 3 S. aureus, 4 64 8 (MRSA) 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. faecium 1 8 4 Vanc. resistant
8 E. faecium 1 32 32 Vanc. resistant
Example 4
Post-Antibiotic Effect of OPT-80 in C. difficile
[0112] 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.
[0113] The PAE at 4.times. 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 4.times. the MIC versus strain LC3.
Example 5
In Vitro Activity of OPT-80
[0114] 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.
difficile via agar dilution. The MIC data are presented in Tables 5
and 6.
TABLE-US-00005 TABLE 5 Geometric mean, MIC ranges, MIC.sub.50, and
MIC.sub.90 values for OPT-80against 110 C. difficile clinical
isolates, vancomycin, and metronidazole, in .mu.g/mL. Range
Geometric Mean MIC.sub.50 MIC.sub.90 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-US-00006 TABLE 6 Raw MIC data for OPT-80, vancomycin (VAN),
and metronidazole (MTZ) versus 110 clinical isolates of C.
difficile, in .mu.g/mL. R-Tiacumicin B (>90% ORG ID
Stereomerically Pure) MTZ VAN A1 1535 0.125 0.25 1 B1 832 0.06
0.125 1 D1 1360 0.03 0.25 1 E1 816 0.06 0.125 1 F1 1015 0.125 0.125
1 G1 1077 0.125 0.125 1 I1 1389 0.125 0.125 1 J1 5971 0.06 0.25 1
J7 4224 0.03 0.125 1 J9 4478 0.06 0.125 1 K1 4305 0.125 0.25 0.5
K14 5780 0.125 0.125 1 L1 1423 0.125 0.125 0.5 N1 471 0.125 0.125
0.5 O1 1861 0.06 0.125 1 R1 397 0.125 0.125 1 R6 6015 0.015 0.25 2
V1 1521 0.125 0.125 0.5 W1 3931 0.125 0.5 1 X1 1890 0.125 0.125 1
Y1 5639 0.06 0.125 0.5 Y2 1459 0.06 0.125 1 Z1 3036 0.03 0.125 1
AA2 4380 0.015 0.125 1 AB2 1725 0.06 0.125 1 AC1 1546 0.06 0.125 1
AF1 1808 0.125 0.125 0.5 AG1 3044 0.125 0.125 1 AH1 3430 0.125 0.25
0.5 AJ1 1557 0.06 0.125 1 AL1 1753 0.06 0.125 0.5 AN1 464 0.125
0.125 0.5 AO1 287 0.125 0.125 1 AS1 4099 0.125 0.125 1 AT1 1216
0.125 0.125 1 AV1 941 0.25 0.125 0.5 CJ1 893 0.125 0.025 1 AW1 4501
0.125 0.125 1 BE1 4307 0.125 0.25 1 BH1 4506 0.06 0.06 0.5 BI1 1675
0.125 0.125 1 BK1 4291 0.125 0.125 0.5 BL1 716 0.125 0.125 1 BM1
1453 0.06 0.125 1 BN1 1322 0.125 0.25 1 BR1 1321 0.06 0.125 1 BT1
706 0.06 0.125 1 BV1 1183 0.125 0.25 1 BW1 3130 0.125 0.125 1 BX1
4271 0.125 0.25 1 CN1 667 0.25 0.25 1 CB1 1584 0.25 0.125 1 CF1
5922 0.125 0.125 1 CG1 1566 0.125 0.125 1 CL1 3851 0.25 0.125 1 CO1
4652 0.25 0.125 1 CP1 5491 0.125 0.25 1 61 5930 0.03 0.25 1 63 6029
0.25 0.25 0.06 64 5940 0.125 0.25 1 65 5967 0.06 0.25 0.5 66 6366
0.015 0.125 0.5 67 6367 0.125 0.25 1 68 6368 0.03 0.125 0.06 69
6370 0.25 0.25 0.5 70 6376 0.125 0.25 2 71 6379 0.125 0.25 1 72
6380 0.125 0.25 2 73 6382 0.25 0.25 1 75 6388 0.125 0.125 0.5 76
6389 0.125 0.25 0.5 77 6390 0.06 0.125 1 78 6392 0.015 0.03 0.5 80
6327 0.125 0.125 0.5 81 6328 0.125 0.125 0.5 82 6329 0.06 0.03 0.5
83 6330 0.06 0.125 0.5 84 6331 0.125 0.25 0.5 85 6332 0.06 0.125 1
86 6333 0.03 0.125 0.5 87 6334 0.125 0.125 0.5 88 6335 0.125 0.25
0.5 89 6336 0.25 0.5 1 90 6338 0.125 0.125 1 91 6339 0.125 0.125 1
93 6341 0.125 0.125 1 94 6343 0.015 0.06 0.5 95 6347 0.125 0.125 1
96 6348 0.06 0.125 0.5 97 6349 0.25 0.125 1 98 6350 0.125 0.5 1 101
6354 0.015 0.06 1 102 6355 0.016 0.125 1 103 6068 0.06 0.125 1 104
6060 0.03 0.25 1 105 6071 0.03 0.125 0.5 106 6078 0.03 0.25 0.5 107
6079 0.06 0.125 0.5 109 6274 0.015 0.125 1 111 6279 0.03 0.125 1
112 6280 0.06 0.125 0.5 113 6304 0.06 0.125 1 114 386 0.06 0.125 4
115 5985 0.015 0.25 2 116 5702 0.06 0.125 1 117 6026 0.06 0.125 2
120 6057 0.03 0.25 1 121 6072 0.06 0.25 0.5 122 6111 0.25 0.25 0.5
100 6353 0.125 0.25 1
Example 6
Activity of OPT-80 Compared Against Selected Anaerobic Species
[0115] 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, which
is hereby incorporated by reference in its entirety.
[0116] All organisms, including the 21 C. difficile 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.
[0117] Results of MIC testing are presented in Table 7.
TABLE-US-00007 TABLE 7 MICs (.mu.g/ml) of R-Tiacumicin B (>90%
Stereomerically Pure) Organism MIC range MIC.sub.50 MIC.sub.90
Bacteroides fragilis (19) 64->128 >128 >128 Non-fragilis
B. fragilis group 64->128 >128 >128 species (38)
Prevotella/Porphyromonas species 16->128 >128 >128 (42)
Fusobacterium nucleatum (14) 64->128 >128 >128
Fusobacterium mortiferum (10) 64->128 >128 >128
Fusobacterium species, 16->128 >128 >128 miscellaneous
(14) Peptostreptococcus tetradius (16) 0.25-2.0 1.0 1.0
Peptostreptococcus 0.25-1.0 0.5 1.0 asaccharolyticus (15)
Peptostreptococcus anaerobius <0.016-0.03 <0.016 <0.016
(15) Finegoldia magna (15) 0.25-2.0 1.0 1.0 Micromonas micros (14)
<0.016-0.06 0.03 0.06 Peptostreptococcus prevotii (3) 0.25-1.0
NA NA Propionibacterium acnes (20) 0.5-1.0 4.0 4.0 Eggerthella
lenta (10) <0.016-0.06 <0.016 <0.03 Miscellaneous
gram-positive <0.016-16 <0.125 16 non-spore-forming rods (20)
Clostridium perfringens (35) <0.016-0.06 <0.016 0.03
Clostridium difficile (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
Example 7
In Vitro Activities of OPT-80 Against Intestinal Bacteria
[0118] 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, which
is hereby incorporated by reference in its entirety.
[0119] 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-80 used during testing was 0.03
.mu.g/ml to 1024 .mu.g/ml.
[0120] 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).
[0121] 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-US-00008 TABLE 8 In vitro activity of R-Tiacumicin B (>90%
Stereomerically Pure) against 453 bacterial isolates Organism MIC
range MIC.sub.50 MIC.sub.90 Bacteroides fragilis group spp.
256->1024 256 >1024 (50) Veillonella spp. (10) 16-128 32 128
Other anaerobic gram-negative 0.06-1024 1024 >1024 rods (51) All
anaerobic gram-negative 0.06->1024 256 >1024 species (111)
Clostridium bifermentans (9) 0.06 NA NA Clostridium bolteae (7)
1-64 NA NA Clostridium clostridioforme (4) 4-128 NA NA Clostridium
difficile (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 All Clostridium species (107) 0.06->1024
0.062 128 Anaerobic non-spore-forming 0.06->1024 1 32
gram-positive rods (63) Anaerobic gram-positive cocci (49)
0.06->1024 0.5 2 All anaerobic gram-positive 0.06->1024 0.12
64 species (219) Streptococcus, formerly S. milleri 16-64 32 32
group (14) Other Streptococcus species (9) 16-128 NA NA
Enterococcus species (21) 2.0-16 8 8 Staphylococcus aureus and
0.25-2 0.5 2 Staphylococcus epidermidis (19) Total for all strains
(453) 0.06->1024 8 1024
Other Embodiments
[0122] All references discussed above are herein incorporated by
reference in their entirety for all purposes. While this invention
has been particularly shown and described with references to
preferred embodiments thereof, it will be understood by those
skilled in the art that various changes in form and details may be
made therein without departing from the spirit and scope of the
invention as defined by the appended claims.
* * * * *