U.S. patent application number 16/276635 was filed with the patent office on 2019-06-13 for methods for preventing or treating infectious diseases caused by extracellular microorganisms, including antimicrobial-resistant.
This patent application is currently assigned to CAHN SCHOOL OF MEDICINE AT MOUNT SINAI. The applicant listed for this patent is ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI. Invention is credited to Sharon Moalem, Daniel P. Perl.
Application Number | 20190175644 16/276635 |
Document ID | / |
Family ID | 39794789 |
Filed Date | 2019-06-13 |
United States Patent
Application |
20190175644 |
Kind Code |
A1 |
Perl; Daniel P. ; et
al. |
June 13, 2019 |
METHODS FOR PREVENTING OR TREATING INFECTIOUS DISEASES CAUSED BY
EXTRACELLULAR MICROORGANISMS, INCLUDING ANTIMICROBIAL-RESISTANT
STRAINS THEREOF, USING GALLIUM COMPOUNDS
Abstract
The present invention relates to methods for preventing or
treating infectious diseases caused by extracellular
microorganisms, such as bacteria and fungi, by systemically
administering to a patient a compound containing gallium. The
extracellular microorganisms targeted by the present methods
include methicillin-resistant Staphylococcus aureus (MRSA),
vancomycin-resistant Enterococcus faecalis (VRE), E. coli O157:H7,
fluoroquinolone-resistant Salmonella typhi, and the like.
Furthermore, in the present methods, gallium compounds can be
co-administered with one or more conventional antimicrobial agents
to treat infectious diseases with reduced risks of creating
multi-drug resistant pathogens. The methods of the present
invention is also applicable to those microorganisms, such as
ulcer-causing Helicobacter pylori, complete eradication of which so
far has been difficult to achieve.
Inventors: |
Perl; Daniel P.; (Bethesda,
MD) ; Moalem; Sharon; (New York, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI |
New York |
NY |
US |
|
|
Assignee: |
CAHN SCHOOL OF MEDICINE AT MOUNT
SINAI
New York
NY
|
Family ID: |
39794789 |
Appl. No.: |
16/276635 |
Filed: |
February 15, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15782860 |
Oct 13, 2017 |
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16276635 |
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15259100 |
Sep 8, 2016 |
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15782860 |
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14829675 |
Aug 19, 2015 |
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15259100 |
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14530766 |
Nov 2, 2014 |
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14829675 |
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12058484 |
Mar 28, 2008 |
8895077 |
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14530766 |
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60909658 |
Apr 2, 2007 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 1/04 20180101; A61K
9/0053 20130101; Y02A 50/47 20180101; A61K 38/08 20130101; Y02A
50/473 20180101; A61K 9/0019 20130101; Y10S 424/06 20130101; A61P
1/00 20180101; A61K 33/24 20130101; A61P 43/00 20180101; Y02A 50/30
20180101; A61K 31/5377 20130101; A61P 31/00 20180101; Y02A 50/483
20180101; Y02A 50/402 20180101; A61P 31/04 20180101; A61K 31/28
20130101; Y02A 50/478 20180101; A61P 31/10 20180101 |
International
Class: |
A61K 33/24 20060101
A61K033/24; A61K 38/08 20060101 A61K038/08; A61K 9/00 20060101
A61K009/00; A61K 31/5377 20060101 A61K031/5377; A61K 31/28 20060101
A61K031/28 |
Claims
1. A method for treating an infectious disease caused by
methicillin-resistant Staphylococcus aureus ("MRSA"),
vancomycin-resistant enterococci ("VRE"), E. coli O157:H7,
fluoroquinolone-resistant Salmonella typhi, ceftazidime-resistant
Klebsiella pneumoniae, or fluoroquinolone-resistant Neisseria
gonorrhoeae in the bloodstream of a subject, the method comprising:
administering to the subject a therapeutically effective amount of
a gallium compound selected from the group consisting of gallium
nitrate, gallium maltolate, gallium citrate, gallium phosphate,
gallium chloride, gallium fluoride, gallium carbonate, gallium
formate, gallium acetate, gallium sulfate, gallium tartrate,
gallium oxalate, and gallium oxide, wherein said therapeutically
effective amount is sufficient to reduce the number of, to suppress
the growth of, or to kill the methicillin-resistant Staphylococcus
aureus ("MRSA"), vancomycin-resistant enterococci ("VRE"), E. coli
O157:H7, fluoroquinolone-resistant Salmonella typhi,
ceftazidime-resistant Klebsiella pneumoniae, or
fluoroquinolone-resistant Neisseria gonorrhoeae in the bloodstream
of the subject.
2. The method of claim 1, further comprising co-administering a
therapeutically effective amount of at least one additional
antimicrobial agent.
3. The method of claim 2, wherein the additional antimicrobial
agent is vancomycin and/or linezolid.
4. The method of claim 1, wherein the gallium compound is
administered orally, intravenously, intramuscularly,
subcutaneously, intraperitoneally, or by suppositories.
5. A method for treating an infection in the bloodstream of a
subject, the infection caused by methicillin-resistant
Staphylococcus aureus ("MRSA"), vancomycin-resistant enterococci
("VRE"), E. coli O157:H7, fluoroquinolone-resistant Salmonella
typhi, ceftazidime-resistant Klebsiella pneumoniae, or
fluoroquinolone-resistant Neisseria gonorrhoeae, said method
comprising: administering to the subject a therapeutically
effective amount of a gallium compound selected from the group
consisting of gallium nitrate, gallium maltolate, gallium citrate,
gallium phosphate, gallium chloride, gallium fluoride, gallium
carbonate, gallium formate, gallium acetate, gallium sulfate,
gallium tartrate, gallium oxalate, and gallium oxide, wherein said
therapeutically effective amount is sufficient to reduce the number
of, to suppress the growth of, or to kill the methicillin-resistant
Staphylococcus aureus ("MRSA"), vancomycin-resistant enterococci
("VRE"), E. coli O157:H7, fluoroquinolone-resistant Salmonella
typhi, ceftazidime-resistant Klebsiella pneumoniae, or
fluoroquinolone-resistant Neisseria gonorrhoeae in the bloodstream
of the subject.
6. The method of claim 5, further comprising co-administering a
therapeutically effective amount of at least one additional
antimicrobial agent.
7. The method of claim 6, wherein the additional antimicrobial
agent is vancomycin and/or linezolid.
8. The method of claim 5, wherein the gallium compound is
administered orally, intravenously, intramuscularly,
subcutaneously, intraperitoneally, or by suppositories.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This is a Continuation of co-pending U.S. patent application
Ser. No. 15/782,860, filed Oct. 13, 2017, which is a Continuation
of U.S. patent application Ser. No. 15/259,100, filed Sep. 8, 2016,
which is a Continuation of U.S. patent application Ser. No.
14/829,675, filed Aug. 19, 2015, which is a Continuation of U.S.
patent application Ser. No. 14/530,766, filed Nov. 2, 2014, which
was a Continuation of U.S. patent application Ser. No. 12/058,484,
filed Mar. 28, 2008, now issued as U.S. Pat. No. 8,895,077, which
claimed the benefit of U.S. Provisional Application No. 60/909,658,
filed Apr. 2, 2007, the foregoing applications are incorporated
herein by reference in their entirely.
1. INTRODUCTION
Field
[0002] The present invention relates to methods of preventing or
treating infectious diseases caused by various extracellular
microorganisms, including bacteria and fungi, using gallium
compounds. In particular, the invention relates to the
pharmaceutical use of gallium compounds in preventing or treating
infectious diseases by systemic administration thereof, such as
oral administration, intravenous administration, intramuscular
administration, subcutaneous administration, and the like.
Infectious diseases that are preventable or treatable by the
present invention include those caused by microorganisms that are
known to be resistant to conventional antibiotics and/or drugs.
2. BACKGROUND
[0003] The emergence of an increasing number of deadly pathogenic
microorganisms that are resistant to conventional antibiotics and
other antimicrobial agents has become a great concern to public
health worldwide. The over-prescription/over-use of antibiotics in
humans and farm animals has contributed to a rapid development of
antibiotics-resistant strains of various microorganisms. For
example, Staphylococcus aureus, known to be a common cause of
hospital-acquired ("nosocomial") infections that can spread to the
heart, bones, lungs, and bloodstream with fatal consequences if not
treated, was well controlled by penicillin in the early 1940s.
However, by the late 1960s, more than 80 percent of Staphylococcus
aureus had developed resistance against penicillin and, by 1972, 2
percent of Staphylococcus aureus were found to be
methicillin-resistant. The percentage of methicillin-resistant
bacteria continued to rise to 57.1 percent by 2002 ("Bad Bugs, No
Drugs" by Infectious Diseases Society of America ("IDSA"), July
2004, based on Centers for Disease Control ("CDC") National
Nosocomial Infections Surveillance System, August 2003).
[0004] Similarly, the percentage of enterococci, an important cause
of endocarditis, as well as other nosocomial infections, including
urinary tract and wound infections and bacteremia, that are
resistant to vancomycin (VRE) has increased since the late 1980s
and, in 2002, more than 27 percent of the tested enterococci
samples from intensive care units were resistant to vancomycin (by
IDSA, 2004, supra). Other bacteria known to have developed
antibiotics resistance include methicillin resistant,
coagulase-negative staphylococci ("CNS"), ceftazidime resistant
Pseudomonas aeruginosa, amipicillin resistant Escherichia coli,
ceftazidime resistant Klebsiella pneumoniae, penicillin resistant
Streptococcus pneumoniae, and the like. In addition,
drug-resistance is no longer limited to hospital-acquired
infections, but has spread to community-acquired infections, as
evidenced by, for example, a total of 12,000 cases of
community-acquired methicillin-resistant Staphylococcus aureus
(MRSA) infections found in correctional facilities in Georgia,
California, and Texas between 2001 and 2003 (2004, IDSA,
supra).
[0005] Antibiotic-resistant microorganisms cause an enormous
economic burden to society. Infectious diseases caused by
drug-resistant microorganisms require longer hospitalizations,
higher costs for alternative medications, more lost work days and
so forth, and often result in death. According to the report by the
Institute of Medicine ("TOM") (1998, Antimicrobial Resistance:
Issues and Options), infections caused by MRSA cost an average of
$31,400 per case to treat. The total cost to U.S. society of
drug-resistant microorganisms is said to be at least $4 billion to
$5 billion annually.
[0006] Despite the urgent need for new drugs to control
antimicrobial resistance, development of new antibiotics has slowed
considerably in recent years as the focus of product development in
the pharmaceutical fields has increasingly shifted toward chronic
diseases, rather than to acute illness, such as acute bacterial
infections, mainly due to higher profitability associated with the
treatment of the former (March 2004, by U.S. Food and Drug
Administration ("FDA"), Innovation/Stagnation: Challenge and
Opportunity on the Critical Path to New Medical Products; and
December 2003, by Sellers, L. J., "Big Pharma bails on
anti-infectives research", Pharmaceutical Executive 22). According
to 10M and FDA, only two new classes of antibiotics have been
developed in the past 30 years: oxazolidinones in 2000 and
lipopeptides in 2003, and resistance to oxazolidinones have already
been reported.
[0007] Gallium is a group Ma semi-metallic element that has been
used for many years for diagnosing neoplasms and inflammation in
the field of nuclear medicine. Gallium has also shown some efficacy
in the treatment of cancers (Adamson et al., 1975, Cancer Chemothe.
Rept 59:599-610; Foster et al., 1986, Cancer Treat Rep
70:1311-1319; Chitambar et al., 1997, Am J Clin Oncol 20:173-178),
symptomatic cancer-related hypercalcemia (Warrell et al., 1989, in
"Gallium in the treatment of hypercalcemia and bone metastasis",
Important Advances in Oncology, pp. 205-220, J. B. Lippincott,
Philadelphia; Bockman et al., 1994, Semin Arthritis Rheum
23:268-269), bone resorption (Warrell et al., 1984, J Clin Invest
73:1487-1490; Warrell et al., 1989, supra), autoimmune diseases and
allograft rejection (Matkovic et al., 1991, Curr Ther Res
50:255-267; Whitacre et al., 1992, J Newuro immunol 39:175-182;
Orosz C. G. et al., 1996, Transplantation 61:783-791; Lobanoff M.
C. et al., 1997, Exp Eye Res 65:797-801), stimulating wound healing
and tissue repair (Bockman et al., U.S. Pat. No. 5,556,645; Bockman
et al., U.S. Pat. No. 6,287,606) and certain infections, such as
syphilis (Levaditi C. et al., 1931, C R Hebd Seances Acad Sci Ser D
Sci Nat 192:1142-1143), intracellular bacterial, fungal or
parasitic infections, such as tuberculosis, histoplasmosis, and
leishmaniasis, respectively (Olakanmi et al., 1997, J. Invest. Med.
45:234 A; Schlesinger et al., U.S. Pat. No. 6,203,822; Bernstein,
et al., International Patent Application Publication No. WO
03/053347), Pseudomonas aeruginosa infection (Schlesinger et al.,
U.S. Pat. No. 6,203,822), and trypanosomiasis (Levaditi C. et al.
supra).
[0008] Although the exact mechanism of gallium's activity against
bone resorption and hypercalcemia is not well known, its
antiproliferative properties against cancer cells and antimicrobial
activities are said to be likely due to its competition with ferric
iron (i.e., Fe.sup.3+) for uptake by cancer cells or microorganisms
(Bernstein, 1998, Pharmacol Reviews 50(4):665-682). Iron is an
essential element for most living organisms, including many
pathogens, and is required for DNA synthesis and various
oxidation-reduction reactions (Byers et al., 1998, Metal Ions Bio
syst 35:37-66; Guerinot et al., 1994, Annu Rev Microbiol
48:743-772; Howard, 1999, Clin Micobiol Reviews 12(3):394-404).
Ga.sup.3+ is known to have solution- and coordination-chemistries
similar to those of Fe.sup.3+ (Shannon, 1976, Acta
Crystallographica A32:751-767; Huheey et al., 1993, In Inorganic
Chemistry: Principles of Structure and Reactivity I, ed. 4, Harper
Collins, NY; Hancock et al., 1980, In Org Chem 19:2709-2714) and
behaves very similarly to Fe.sup.3+ in vivo by binding to the
iron-transport protein transferrin (Clausen et al., 1974, Cancer
Res 34:1931-1937; Vallabhajosula et al., 1980, J Nucl Med
21:650-656). It is speculated that gallium enters microorganisms
via their iron transport mechanisms and interferes with their DNA
and protein synthesis.
[0009] U.S. Pat. No. 5,997,912 discloses a method for inhibiting
growth of Pseudomonas aeruginosa by administering gallium compounds
intravenously, orally or by aerosol and U.S. Patent Application
Publication No. 2006/0018945 discloses a method of preventing or
inhibiting biofilm growth formation using gallium compounds.
[0010] U.S. Pat. No. 6,203,822 and International Patent Application
No. WO 03/053347 disclose methods for treating patients infected
with intracellular bacteria, in particular, species of the genus
Mycobacterium, by intravenously or orally administering gallium
compounds to patients infected by this class of bacteria (also see
Olakanmi et al., 2000, Infection and Immunity 68(10):5619-5627).
These organisms primarily infect macrophages, which are known to
store large amounts of iron and overexpress transferrin receptors.
Parenterally or orally administered gallium compounds are readily
taken up by macrophages through transferrin receptors and then,
within these cells, are taken up by the infecting organisms,
thereby interfering with the organisms' metabolism.
[0011] The antimicrobial activities of gallium against
microorganisms other than intracellular organisms have thus far not
been explored to a great extent.
[0012] Furthermore, the use of gallium compounds against the ever
increasing number of multi-antibiotic resistant microorganisms has
not been explored.
3. SUMMARY
[0013] This invention is based upon the inventors' finding that
gallium compounds are effective in controlling the growth of a
variety of pathogenic, extracellular microorganisms, including
those which are known to be resistant to conventional antibiotics
and/or drugs.
[0014] Accordingly, the present invention provides methods for
preventing and/or treating infectious diseases caused by
extracellular microorganisms, said method comprising systemically
administering to a subject in need thereof a prophylactically or
therapeutically effective amount of a gallium compound. In a
preferred embodiment, such extracellular microorganisms exclude
Pseudomonas aeruginosa and Legionella spp., but include other
iron-dependent, extracellular, pathogenic microorganisms. Such
microorganisms may be bacteria or fungi, which infect host
organisms, including mammals and birds, and most notably humans.
Those microorganisms include, but are not limited to, bacteria
within the genera, Staphylococcus, Enterococcus, Escherichia,
Streptococcus, Campylobacter, Salmonella, Helicobacter, Bacillus,
Clostridium, Corynebacterium, Chlamydia, Coxilla, Ehrlichia,
Francisella, Legionella, Pasteurella, Brucella, Proteus,
Klebsiella, Enterobacter, Tropheryma, Acinetobacter, Aeromonas,
Alcaligenes, Capnocytophaga, Erysipelothrix, Listeria, Yersinia,
and the like; and fungi, such as Candida albicans, Microsporum
canis, Sporothrix schenckii, Trichophyton rubrum, Trichophyton
mentagrophytes, Malassezia furfur, Pityriasis versicolor, Exophiala
werneckii, Trichosporon beigelii, Coccidioides immitis, Blastomyces
dermatitidis, Aspergillus fumigatus, Epidermophyton spp., Fusarium
spp., Zygomyces spp., Rhizopus spp., Mucor spp., and so forth.
[0015] In another preferred embodiment, the microorganisms targeted
by the present invention are resistant to at least one antibiotic
or antimicrobial compound other than gallium compounds. In a
specific embodiment, such drug-resistant microorganisms include,
but are not limited to, methicillin-resistant Staphylococcus aureus
(MRSA), vancomycin-resistant enterococci (VRE),
ampicillin-resistant E. coli (e.g., E. coli O157:H7),
fluoroquinolone-resistant Salmonella typhi, ceftazidime-resistant
Klebsiella pneumoniae, and fluoroquinolone-resistant Neisseria
gonorrhoeae, and the like.
[0016] In another aspect, the present invention provides a method
for preventing and/or treating infectious diseases caused by
extracellular microorganisms, other than Pseudomonas aeruginosa and
Legionella spp., said method comprising systemically
co-administering to a subject in need thereof a prophylactically or
therapeutically effective amount of a gallium compound and at least
one additional antimicrobial agent. Such additional antimicrobial
agents include, but are not limited to, antibacterial agents, such
as conventional antibiotics, antifungal agents, and other naturally
or synthetically derived agents with antimicrobial activities.
3.1 Definitions
[0017] The term "subject" as used herein refers to an animal,
including a fowl (e.g., chickens, turkeys, and the like), and a
mammal such as a non-primate (e.g., cows, pigs, horses, cats, dogs,
rats, etc.) and a primate (e.g., monkey and human), most preferably
a human.
[0018] The term "systemic" or "systemically" as used herein refers
to an administration of gallium compounds to a subject in a manner
whereby the compound is distributed throughout the entire body of
the subject mainly through the circulatory system, such as the
cardiovascular system including the heart and blood vessels and the
lymphatic system including lymph nodes, lymph vessels and ducts.
Thus, systemic administrations of the gallium compound include oral
administration and parenteral administration, including
intravenous, intramuscular, subcutaneous, and intraperitoneal
administrations, as well as suppositories, and the like. In certain
situations, systemic administration can also provide an advantage
of the direct contact of the compound with causative organisms,
without going through the circulatory systems. For example, the
gallium compound that is orally administered would exert its effect
not only via systemic distribution but also via direct contact with
the microorganisms that infect the digestive tracts.
[0019] The term "prophylactically effective amount" as used herein
refers to that amount of the gallium compounds sufficient to
prevent a disease or disorder associated with pathogenic
microorganisms. A prophylactically effective amount can refer to
the amount of the gallium compounds sufficient to prevent or
suppress the growth of pathogenic microorganisms or kill pathogenic
microorganisms in a subject.
[0020] The term "therapeutically effective amount" as used herein
refers to that amount of the gallium compounds sufficient to treat,
manage or ameliorate a disease or disorder caused by pathogenic
organisms in a subject. A therapeutically effective amount can
refer to the amount of the gallium compounds sufficient to reduce
the number of pathogenic microorganisms, to suppress the growth of
pathogenic microorganisms (i.e., stasis), or to kill pathogenic
microorganisms at the affected sites or in the bloodstream of a
subject. Further, a therapeutically effective amount of the gallium
compounds means that the amount of the gallium compounds alone, or
in combination with other therapies and/or other drugs, that
provides a therapeutic benefit in the treatment, management, or
amelioration of a disease or disorder.
4. DETAILED DESCRIPTION
4.1 Iron Transport and Gallium
[0021] Most microorganisms, with a few exceptions (e.g.,
Lactobacillus spp.--see Archibald, 1983, FEMS Microbiol Lett
19:29-32; Weinberg, 1997, Perspectives in Biology and Medicine
40(4):578-583; and Borrelia burgdorferi--see Posey et al., 2000,
Science 288:1651-1653), require iron for their survival (Weinberg,
1978, Microbiol Rev 42:45-66; Neilands, 1972, Struct Bond
11:145-170). Despite the fact that iron is one of the most abundant
metals, its availability to microorganisms is limited due to its
existence as insoluble compounds (oxides-hydroxides) in aerobic
environments (Guerinot, 1994, supra; Spiro, et al., 1966, J Am Chem
Soc 88:2721-2725; Vander Helm et al., 1994, In Metal ions in fungi
vol. 11, pp. 39-98, Marcel Dekker, Inc. New York, N.Y.).
Accordingly, microorganisms, such as bacteria and fungi, have
developed various mechanisms for acquiring iron in the face of its
limited availability in the environment (Howard, 1999, supra).
[0022] One such mechanism is the synthesis of potent iron-chelating
compounds called siderophores. Microorganisms produce siderophores,
which bind Fe.sup.3+ in the environment and are transported into
the cells of the microorganisms via specific transport systems,
where Fe.sup.sup.3+ is released as Fe.sup.2+ and then stored. Known
siderophores include hydroxamates, such as rhodotorulic acid,
coprogens, ferrichromes, and fusarinines; polycarboxylates;
phenolates-catecholates and desferioxamine (Howard, 1999, supra).
Other mechanisms include direct internalization of iron complexed
with siderophores or host iron transporters (e.g., transferrin and
lactoferrin), membrane-associated reductase mechanisms, and
receptor-mediated mechanisms as well as membrane-mediated
direct-transfer mechanisms (Howard, 1999, supra; Crosa, 1997,
Microbiol Mol Biol Rev 61:319-336; Payne, 1994, Methods Enzymol
235:329-344). The availability of iron through these mechanisms is
closely linked to the virulence of microorganisms (Litwin et al.,
1993, Clin Microbiol Reviews 6(2):137-149), and each organism may
have multiple alternative mechanisms for obtaining iron from
iron-scarce environments to support its growth and survival (for
example, see Spatafora et al., 2001, Microbiology
147:1599-1610).
[0023] It has been reported that gallium ion (Ga.sup.3+) and ferric
ion (Fe.sup.3+) have strong biochemical similarities, in
particular, with regard to their binding to proteins and chelators.
These similarities are mainly attributed to their comparable ionic
radii and the degrees of ionic (electrostatic) versus covalent
contributions to bonding (for review, see Bernstein, 1998, supra).
Because of these similarities, Ga.sup.3+ can mimic Fe.sup.3+ in
various biological processes. For example, Ga.sup.3+ binds to
transferrin (see, for example, Clausen et al., 1974, Cancer Res
34:1931-1937; Vallabhajosula et al., 1980, J Nucl Med 21:650-656)
and is transported into the cell via transferrin-mediated
endocytosis (Chitambar, 1987, Cancer Res 47:3929-3934).
[0024] Without intending to be bound by theory, it is believed that
Ga.sup.3+ can competitively bind to siderophores and be easily
taken up by microorganisms, where it can disrupt DNA and protein
syntheses or bind to bacterial proteins and impair the growth of
the microorganisms, thereby eventually leading to the stasis or
death of the organisms. Alternatively, it is possible that
Ga.sup.3+ may occupy membrane-reductases of the microorganisms and
prevent Fe.sup.3+ from binding to the reductases to be reduced to
Fe.sup.2+, which would be more bioavailable than Fe.sup.sup.3+.
Since the uptake of gallium does not immediately kill the
microorganisms but rather leads to an initial stasis (i.e., a state
where the growth or multiplication of microorganisms is inhibited),
it has a reduced risk for generating resistant microorganisms.
Furthermore, because iron is an essential element for pathogenic
microorganisms for their survival and the biochemical similarities
between iron and gallium are so close, it is additionally less
likely for the microorganisms to be able to develop mechanisms that
can discriminate iron from gallium and become resistant to gallium.
Gallium may also prevent a microorganism from producing toxins by
interfering with its toxin enzyme production.
[0025] The present invention takes advantage of these
characteristics of gallium compounds and provides methods for
preventing or treating infectious diseases caused by such
pathogens, including those that are resistant to at least one
antimicrobial agent other than gallium.
4.2 Gallium Compounds
[0026] Gallium compounds suitable for use in the present invention
include any gallium-containing compounds that are pharmaceutically
acceptable and safe for animal use, such as avian and mammalian
use, in particular, for human use. Gallium compounds have been used
diagnostically and therapeutically in humans and are known to be
safe for human use (see Foster et al., 1986, supra; Todd et al.,
1991, Drugs 42:261-273; Johnkoff et al., 1993, Br J Cancer
67:693-700).
[0027] Pharmaceutically acceptable gallium compounds suitable for
use in the present invention include, but not by way of limitation,
gallium nitrate, gallium maltolate, gallium citrate, gallium
phosphate, gallium chloride, gallium fluoride, gallium carbonate,
gallium formate, gallium acetate, gallium sulfate, gallium
tartrate, gallium oxalate, gallium oxide, and any other gallium
compounds which can safely provide effective levels of element
gallium in various applications. Furthermore, gallium complexes,
such as gallium pyrones, gallium pyridones, and gallium oximes, as
well as gallium bound to proteins, such as transferrin and
lactoferrin, or gallium bound to siderophores, such as
hydroxamates, polycarboxylates, and phenolates-catecholates,
desferioxamine and other iron-chelators, such as cysteine,
.alpha.-keto acids, hydroxy acids and pyridoxal isonicotinyl
hydrazone class (Richardson et al., 1997, Antimicrobial Agents and
Chemotherapy 41(9):2061-2063) and the like are also suitable for
use in the present invention.
4.3 Pharmaceutical Use of Gallium Compounds
[0028] The present invention is directed to methods for preventing
or treating infectious diseases by systemically administering to a
subject in need thereof a prophylactically or therapeutically
effective amount of gallium compounds.
[0029] Examples of infectious diseases treatable by the present
invention are those as to which the subject to be treated can
benefit from a systemic administration of gallium compounds and
include, but are not limited to, those caused by extracellular
bacteria of the species of Staphylococcus, such as Staphylococcus
aureus, Staphylococcus epidermidis, and the like; of Enterococcus,
such as Enterococcus faecalis, Enterococcus faecium, and the like;
of Salmonella, such as Salmonella typhi, Salmonella typhimurium,
Salmonella enterica, and the like; of Escherichia, such as
Escherichia coli, and the like; of Streptococcus, such as
Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus
agalactiae, and the like; of Helicobacter, such as Helicobacter
pylori, and the like; of Campylobacter, such as Campylobacter
jejuni, and the like; as well as the species of genera, Yersinia,
Chlamydia, Coxilla, Ehrlichia, Francisella, Legionella,
Pasteurella, Brucella, Proteus, Klebsiella, Enterobacter,
Tropheryma, Acinetobacter, Aeromonas, Alcaligenes, Capnocytophaga,
Bacillus, Clostridium, Corynebacterium, Erysipelothrix, Listeria
and the like. Examples of infectious diseases treatable by the
present invention also include infections caused by fungi, such as
Candida albicans, Microsporum canis, Sporothrix schenckii,
Trichophyton rubrum, Trichophyton mentagrophytes, Malassezia
furfur, Pityriasis versicolor, Exophiala werneckii, Trichosporon
beigelii, Coccidioides immitis, Blastomyces dermatitidis,
Aspergillus fumigatus, Epidermophyton spp., Fusarium spp.,
Zygomyces spp., Rhizopus spp. Mucor spp., and so forth.
[0030] Gallium compounds can be administered by any methods that
result in systemic distribution or delivery of the gallium
compounds and include oral administration and parenteral
administration, such as intravenous administration, intramuscular
administration, subcutaneous administration, intraperitoneal
administration, and the like. In certain infections, oral
administration of gallium compounds provides not only systemic
distribution/delivery of the gallium compounds to the affected area
but also a direct contact of the compounds with the causative
microorganisms in the affected area, such as within the digestive
tracts. Thus, oral administration of the gallium compounds is
especially useful in preventing or treating digestive tract
infections caused by various microorganisms, including, but not
limited to, Staphylococcus aureus, Enterococcus faecalis,
Enterococcus faecium, Salmonella typhi, Salmonella typhimurium,
Salmonella enterica, Escherichia coli, Campylobacter jejuni,
Clostridium difficile, Clostridium perfringens, and the like.
Helicobacter pylori that causes gastric and duodenal ulcers,
gastritis, duodenitis, and gastric cancer, is also a good target
for the methods of the present invention.
[0031] Furthermore, the methods of the present invention can be
applied to preventing or treating infectious diseases caused by
microorganisms that are resistant to at least one antimicrobial
agent other than gallium compounds. The term "antimicrobial agent"
used herein refers to any naturally or synthetically derived agent
that kills microorganisms or inhibits the growth thereof, directly
or indirectly, and includes conventional antibiotics as well as
synthetic chemotherapeutic agents, such as sulfonamides, isoniazid,
ethambutol, AZT, synthetic peptide antibiotics, and the like. Thus,
in a specific embodiment, the infectious diseases preventable or
treatable by the present invention are caused by
antimicrobial-resistant strains of microorganisms mentioned above,
in particular, of Staphylococcus aureus, Enterococcus faecium,
Enterococcus faecalis, E. coli, Salmonella typhi, Campylobacter
jejuni, Klebsiella pneumoniae, Neisseria gonorrhoeae, Candida
albicans, and the like. More specifically, such
antimicrobial-resistant organisms include methicillin-resistant
Staphylococcus aureus (MRSA), vancomycin-resistant enterococci
(VRE), ampicillin-resistant E. coli (e.g., E. coli O157:H7),
fluoroquinolone-resistant Salmonella thyphi, ceftazidime-resistant
Klebsiella pneumoniae, fluoroquinolone-resistant Neisseria
gonorrhoeae, and the like. The methods of the present invention can
be applied to any other pathogenic microorganisms which have become
resistant to antimicrobial agents other than gallium, as far as
they are dependent on iron for their growth and survival.
[0032] Gallium compounds to be used in the present invention can be
formulated in conventional manner using one or more
pharmaceutically acceptable carriers or excipients. As used herein
the phrase "pharmaceutically acceptable carriers or excipients" is
intended to include any and all solvents, dispersion media,
coatings, isotonic and absorption delaying agents, and the like,
which are compatible with pharmaceutical administration. The use of
various pharmaceutically acceptable carriers or excipients for
pharmaceutically active substances is well known in the art. With
regard to gallium compounds, an injectable formula of gallium
nitrate (Ganite.TM.) is commercially available from Genta Inc
(Berkeley Heights, N.J.). Ganite.TM. is an aqueous solution of
Ga(NO.sub.3).9H.sub.2O and sodium citrate dehydrate. An oral
formula of gallium maltolate developed by Titan Pharmaceuticals,
Inc. (San Francisco, Calif.) is currently in Phase II clinical
testing in patients with metastatic prostate cancer and refractory
multiple myeloma.
[0033] The therapeutically effective amount (i.e., dosage) of a
gallium compound can vary based on the nature and severity of the
infection to be treated, the types of etiologic microorganism, the
location of the affected area, the method of administration, the
age and immunological background of a subject, the types of gallium
compound used, as well as other factors apparent to those skilled
in the art. Typically, a therapeutically effective amount of a
gallium compound can be that amount which gives a gallium
concentration at the affected area of the body or in blood plasma,
of at least about 1 .mu.M, at least about 50 .mu.M, at least about
100 .mu.M, at least about 500 .mu.M, at least about 1 mM, at least
about 10 mM, at least about 50 mM, at least about 100 mM, at least
about 200 mM, up to about 500 mM. Due to gallium's low toxicity,
the amount may be liberally increased to more than 500 mM but less
than that amount which causes any toxicity. For reference, it has
been reported that healthy adults can tolerate at least about 200
mg/m.sup.2/day gallium nitrate intravenous infusion for at least 7
days (see U.S. Pat. No. 6,203,822, supra). Also, an oral
administration of 100 mg to 1400 mg per 24 hours as a single agent
did not cause major toxicity in ovarian cancer patients and lung
cancer patients (see Collery et al., 2002, "Gallium in cancer
treatment", Oncology/Hematology 42:283-296). Thus, for the methods
of the present invention, what is contemplated is administration of
the gallium compounds at dosages of, at least about 10
mg/m.sup.2/day, at least about 50 mg/m.sup.2/day, at least about
100 mg/m.sup.2/day, at least about 200 mg/m.sup.2/day, at least
about 300 mg/m.sup.2/day, at least about 500 mg/m.sup.2/day, at
least about 600 mg/m.sup.2/day, at least about 700 mg/m.sup.2/day,
or at least about 800 mg/m.sup.2/day, but less than that dosage
which causes any toxicity.
[0034] The prophylactically effective amount of a gallium compound
may be that amount sufficient to prevent a disease or disorder
associated with pathogenic microorganisms and may vary based on the
location of the affected area, the types and the number of the
pathogenic organisms in the area, the types of gallium compound to
be used, as well as on the methods of application and other factors
apparent to those skilled in the art. Typically, the
prophylactically effective amount of a gallium compound may be that
amount which gives a gallium concentration at the affected area of
the body or in blood plasma, of at least about 0.1 .mu.M, at least
about 50 .mu.M, at least about 100 .mu.M, at least about 500 .mu.M,
at least about 1 mM, at least about 10 mM, at least about 50 mM, at
least about 100 mM, up to about 200 mM. Again, the amount of a
gallium compound for prophylactic purposes may be liberally
increased to more than 200 mM but less than the amount that causes
any toxicity.
[0035] In another aspect, the present invention provides a method
for preventing and/or treating infectious diseases caused by
extracellular microorganisms, said method comprising
co-administering to a subject in need thereof prophylactically or
therapeutically effective amounts, individually or collectively, of
a gallium compound and at least one additional antimicrobial agent.
The term "co-administration" or "co-administering" used herein
refers to the administration of gallium compound and at least one
additional antimicrobial agent either sequentially in any order or
simultaneously, by the same administration method or a combination
of different administration methods, for example, by an intravenous
administration of the gallium compound and an oral administration
of the additional antimicrobial agent, or vice versa. Such
co-administration of one or more additional antimicrobial agents
together with the gallium compound is especially beneficial because
the drugs attack the causative organisms by non-overlapping,
completely different mechanisms, and/or because the development of
antimicrobial resistance in the organisms may involve different
mechanisms for the different antimicrobial agents, thereby causing
nearly complete eradication of the organisms, by the drugs
themselves or in combination with the actions by the host's own
immune system and reducing or eliminating the chance for the
causative organisms to develop resistance to the drugs.
Furthermore, thanks to the low toxicity of gallium, by increasing
the dosage of gallium, a combination therapy can reduce the dosage
of an additional antimicrobial agent to an amount less than that
required when the latter is used alone, thereby reducing adverse
effects of the latter. Moreover, co-administration of a gallium
compound and an additional antimicrobial agent may result in a
synergistic effect and, thus, require less dosages than those
required when each is used alone.
[0036] Additional antimicrobial agents that can be co-administered
with gallium compounds can be antibacterial agents or antifungal
agents, depending on the type of the causative organisms. Examples
of antibacterial agents include, but not by way of limitation,
those in the classes of penicillins, including amipicillin,
flucloxacillin, dicloxacillin, methicillin, ticarcillin,
piperacillin, carbapenems, mecillinams, and the like; cephems,
including cephalosporin and cephamycins; sulfonamides;
aminoglycosides, including amikacin, gentamicin, kanamycin,
neomycin, netilmicin, paromomycin, streptomycin, tobramycin,
apramycin, and the like; chloramphenicol; tetracyclines, including
chlortetracycline, oxytetracycline, demeclocycline, doxycycline,
lymecycline, meclocycline, methacycline, minocycline,
rolitetracycline, and the like; macrolides, including erythromycin,
azithromycin, clarithromycin, dirithromycin, roxithromycin,
carbomycin A, josamycin, iktasamycin, oleandomycin, spiramycin,
troleandomycin, tylosin/tylocine, telithromycin, cethromycin,
ansamycin, and the like; lincosamides, including lincomycin,
clindamycin, and the like; streptogramins, including mikamycins,
pristinamycins, oestreomycins, virginiamycins, and the like;
glycopeptides, including acanthomycin, actaplanin, avoparcin,
balhimycin, bleomycin B (copper bleomycin), chloroorienticin,
chloropolysporin, demethylvancomycin, enduracidin, galacardin,
guanidylfungin, hachimycin, demethylvancomycin,
N-nonanoyl-teicoplanin, phleomycin, platomycin, ristocetin,
staphylocidin, talisomycin, teicoplanin, vancomycin, victomycin,
xylocandin, zorbamycin, and the like; rifamycins, including
rifampicin, rifabutin, rifapentine, and the like; nitroimidazoles,
including metronidazole, nitrothiazoles, and the like; quinolones,
including nalidixic acid, cinoxacin, flumequine, oxolinic acid,
piromidic acid, pipemidic acid, ciprofloxacin, enoxacin,
fleroxacin, lomefloxacin, nadifloxacin, norfloxacin, ofloxacin,
pefloxacin rufloxacin, balofloxacin, grepafloxacin, levofloxacin,
pazufloxacin mesilate, sparfloxacin, temafloxacin, tosufloxacin,
clinafloxacin, gemifloxacin, moxifloxacin, gatifloxacin,
sitafloxacin, trovafloxacin, and the like; dihydrofolate reductase
inhibitors, including trimethoprim; oxazolidinones, including
linezolid, eperezolid, and the like; lipopeptides, including
gramicidins, polymyxins, surfactin, and the like; and analogs,
salts and derivatives thereof. Examples of antifungal agents
include, but are not limited to, polyenes, such as amphotericin,
nystatin, pimaricin, and the like; azole drugs, such as
fluconazole, itraconazole, ketoco, and the like; allylamine and
morpholine drugs, such as naftifine, terbinafine, amorolfine, and
the like; antimetabolite antifungal drugs, such as
5-fluorocytosine, and the like; and analogs, salts and derivatives
thereof.
[0037] Which antimicrobial agent should be used in combination with
the gallium compounds in any given infection can be determined by
various simple and routine methods known to one skilled in the art.
For example, an infectious microorganism isolated from a patient
can be tested for its sensitivity to various antimicrobial agents
using a standardized disk-diffusion method (e.g., Kirby-Bauer
disk-diffusion method). Briefly, in this method, an appropriate
agar plate is uniformly inoculated with the test organism and paper
disks impregnated with predetermined concentrations of different
antibiotics are placed on the agar surface. After incubation, the
diameter of a circular zone, around the disks, in which the growth
of the organism is inhibited is measured. The diameter of the
inhibition zone is a function of the amount of the antibiotic in
the disk as well as the susceptibility of the organism to the
antibiotic. The antibiotics to which the organism shows
susceptibility can be used for a combination treatment with the
gallium compounds. Other examples of antibiotic susceptibility
tests include, but are not limited to, a broth tube dilution method
for determining Minimum Inhibitory Concentration (MIC) and Minimum
Bactericidal Concentration (MBC) of a given antimicrobial agent
against a given organism. These methods are described in Section
6.1, infra. Thus, in a specific embodiment, an infection caused by
MRSA can be treated by co-administration of gallium compound and
vancomycin or linezolid (e.g., ZyVox.TM. by Pfizer, NY) to a
subject in need thereof. Vancomycin and Zyvox.TM., respectively,
are currently used as the antibiotics of choice to treat MRSA
infections. Likewise, in another specific embodiment, an infection
caused by VRE can be treated by co-administration of a gallium
compound and linezolid. In yet another specific embodiment, an
infection or a disease/disorder (e.g., peptic ulcers, gastritis,
duodenitis, gastric cancer, and the like) caused by Helicobacter
pylori can be treated by co-administration of a gallium compound
and clarithromycin, amoxicillin and/or metronidazole. Other agents
that directly or indirectly inhibit or suppress the growth of
Helicobacter pylori can be also co-administered with the gallium
compound. Such agents include, but are not limited to, proton pump
inhibitors, such as omeprazole that is currently used together with
clarithromycin and amoxicillin in triple therapy for peptic ulcers;
and urease inhibitors, such as fluorofamide, acetohydroxamic acid,
certain divalent metal ions, including Zn, Cu, Co, and Mn, and the
like; as well as other agents, such as bismuth compounds (e.g.,
bismuth subsalicylate) that not only protect the stomach lining by
coating the latter, but also suppress H. pylori growth (S. Wagner
et al., 1992, "Bismuth subsalicylate in the treatment of H2 blocker
resistant duodenal ulcers: role of Helicobacter pylori", Gut
33:179-183).
[0038] In another aspect, the present invention provides a kit
comprising one or more vials containing a gallium compound and one
or more additional antimicrobial agents.
5. EXAMPLES
[0039] The following examples are provided to further illustrate
the current invention but are not intended to in any way limit the
scope of the current invention.
5.1. In Vitro Study: Susceptibility of Microorganisms to
Gallium
Example 1
[0040] Susceptibility of various microorganisms to gallium was
tested by determining the minimum inhibitory concentration (MIC)
and minimum bactericidal concentration (MBC) for each microorganism
using gallium nitrate. In general, MIC is determined by (i) mixing
a series of broths, each containing a standard number of
microorganisms, with serially diluted solutions of the gallium
compound; and (ii) determining the MIC, after incubation, that is
the lowest concentration of the gallium compound that inhibits the
growth of the microorganism. The lower the MIC, the more
susceptible the organism is. The MBC is determined by subculturing
an aliquot of each sample from the MIC test on an appropriate agar
plate containing no gallium compound. After incubation, the MBC is
determined to be the lowest concentration of the gallium compound
at which no growth is observed.
[0041] Specifically, in the present experiment, two grams of
gallium nitrate powder were dissolved in 10 ml of filter-sterilized
deionized water and the resulting 20% (w/v) (i.e., 200 mg/ml)
solution was once again filter-sterilized. Two-fold serial
dilutions were prepared in sterile deionized water down to 0.156%
(i.e., 1.56 mg/ml) for the tests for most of the organisms, except
for the test for Candida albicans, in which 10%, 1%, 0.5%, 0.1%,
0.05%, 0.01%, 0.005% and 0.001% of gallium nitrate solutions were
prepared.
[0042] Table 1 shows the list of microorganisms tested for MIC and
MBC. All organisms were obtained from the American Type Culture
Collection (ATCC), Manassas, Va. Each microorganism was picked from
the seed culture (see Table 1) and inoculated in an appropriate
type of broth to obtain a 0.5 McFarland turbidity standard. The
standard suspension of the microorganism was then diluted to 1:100
with the broth and used for the tests.
TABLE-US-00001 TABLE 1 TEST ORGANISM ATCC # SEED CULTURE Candida
albicans 10231 On Sabouraud dextrose agar, at 25-30.degree. C. for
24-48 hours Methicillin-resistant 33592 On tryptic soy agar with
Staphylococcus aureus Staphylococcus aureus 5% (MRSA) sheep blood
(BAP), at 35- Vancomycin-resistant 51575 37.degree. C. for 24-48
hours Enterococcus faecalis (VRE) Escherichia coli O157:H7 35150
Salmonella typhi 6539 Campylobacter jejuni 29428 On Brucella agar
with 5% sheep blood, at 35-37.degree. C. for 48 hours under
microaerophilic conditions .sup.aAntibiotics resistance of the
organism was confirmed by CLSI (Clinical Laboratory Standards
Institute) Oxacillin disk-diffusion test. The zone of inhibition
was 6 mm (CLSI Oxacillin resistant range: .ltoreq.10 mm).
.sup.bAntibiotics resistance of the organism was confirmed by CLSI
Vancomycin disk-diffusion test. The zone of inhibition was 10 mm
(CLSI Vancomycin resistant range: .ltoreq.14 mm).
[0043] Each microorganism was tested in duplicate by either a
microdilution broth method in 96-well plates (i.e., 0.1 ml of the
gallium nitrate solution mixed with 0.1 ml of the microorganism
suspension) or a macrodilution broth method in test tubes (i.e., 1
ml of the gallium nitrate solution mixed with 1 ml of the
microorganism suspension) as follows:
[0044] Microdilution broth method: Candida albicans; Escherichia
coli O157:H7; and Campylobacter jejuni.
[0045] Macrodilution broth method: Methicillin-resistant
Staphylococcus aureus (MRSA); Vancomycin-resistant Enterococcus
faecalis (VRE); and Salmonella typhi.
[0046] The growth of the microorganisms were determined by visual
observation of turbidity in the samples.
[0047] The following controls were incubated together with the test
samples:
[0048] Viability control: A mixture of equal volumes of deionized
water and an appropriate broth inoculated with a test microorganism
but without gallium nitrate; and
[0049] Sterility control: A mixture of equal volumes of deionized
water and an appropriate broth without either microorganisms or
gallium nitrate.
[0050] Purity of each microorganism was confirmed by streaking an
appropriately diluted suspension of the microorganism onto an
appropriate agar plate to obtain isolated colonies and observing
colony morphology.
[0051] The concentrations of microorganisms in the suspension used
in MIC test were determined by inoculating serial dilutions of the
suspensions onto appropriate agar plates and counting the number of
colonies.
[0052] To determine MBC, 10 .mu.l of each sample used in MIC were
inoculated onto an appropriate agar plate and incubated. The lowest
concentration of the gallium nitrate that showed no growth was
determined to be the MBC.
[0053] The results are shown in Table 2 below.
TABLE-US-00002 TABLE 2 Final Conc Broth Agar Plate Incubation of
Organism MIC MBC (MIC) (MBC) Condition (CFU/ml) (mg/ml) (mg/ml)
Candida Sabouraud Sabouraud dextrose At 27.degree. for 9.75 .times.
10.sup.5 10 >100 albicans Dextrose agar 48 hours Methicillin-
Muller Tryptic soy agar At 36.degree. for 5.2 .times. 10.sup.5 ND*
12.5 resistant Hinton with 5% sheep blood 48 hours Staphylococcus
aureus (MRSA) Vancomycin- Muller Tryptic soy agar At 36.degree. for
3.9 .times. 10.sup.5 ND 25 resistant Hinton with 5% sheep blood 48
hours Enterococcus faecalis (VRE) Escherichia Muller Tryptic soy
agar At 36.degree. for 1.38 .times. 10.sup.6 ND 6.25 coli O157:H7
Hinton with 5% sheep blood 48 hours Salmonella Muller Tryptic soy
agar At 36.degree. for 8.3 .times. 10.sup.5 ND 6.25 typhi Hinton
with 5% sheep blood 48 hours Campylobacter Muller Tryptic soy agar
At 36.degree. for 4.9 .times. 10.sup.5 ND <0.78 jejuni Hinton
with 5% sheep blood 48 hours *ND: Not determined due to
non-specific turbidity caused by the precipitation of gallium
nitrate at some dilutions.
5.2. In Vivo Study: Effect of Gallium Nitrate in Animal Models
Example 2
[0054] Methicillin-Resistant Staphylococcus aureus (MRSA)
[0055] Adult BALBc mice are inoculated with 1.times.10.sup.6
CFU/mouse of Staphylococcus aureus-MRSA strain (e.g., ATCC 33592)
by intraperitoneal injection. Following bacterial injections
(approximately 8 hours post-inoculation), each mouse receives a
single intravenous injection of one of the following: 0.9% saline
(control), 30 mg/kg, 45 mg/kg, or 60 mg/kg of gallium nitrate, 200
mg/kg of vancomycin, or 45 mg/kg of gallium nitrate and 200 mg/kg
of vancomycin, all in 0.9% saline. Initially, there are 5 mice in
each of the six groups. Following inoculation, the mice are
monitored twice daily for morbidity. Body temperature is obtained
twice daily and a mouse whose body temperature decreases by
4.degree. C. or greater will be considered moribund and euthanized.
Body weights are taken once daily for the duration of the study. On
Day 5, all remaining animals are euthanized. Spleen, lymph nodes
and kidneys are collected, homogenized in sterile PBS and serially
diluted for bacterial quantitation.
Example 3
[0056] Vancomycin-Resistant Enterococcus faecalis (VRE)
[0057] Adult CF1 mice are caged individually and total counts of
native enterococci and possible VRE in colony forming unit (CFU)
per gram of feces are determined as a baseline for each mouse. On
Day 1, each mouse receives 0.5 ml (about 10.sup.9 CFU/ml) of VRE
(e.g., ATCC 51575) suspension in Muller-Hinton broth (MHB), or MHB
alone (control), via gavage with a stainless steel feeding tube. At
specified intervals thereafter (e.g., 1, 7, 14 days and so on), 2
fresh fecal pellets from each mouse are collected, weighed, and
emulsified in MHB and the numbers of CFU of VRE, enterococci, and
gram-negative enteric bacilli per gram of feces are determined by
standard serial dilution and plating techniques. For example, total
enterococcal counts can be measured with bile-esculin agar, counts
of enteric bacilli with MacConkey agar, and counts of VRE with
Muller-Hinton II agar containing vancomycin (50 .mu.g/ml),
streptomycin (100 .mu.g/ml), polymyxin (100 .mu.g/ml) and nystatin
(2 .mu.g/ml) (see M. S. Whitman et al., 1996, "Gastrointestinal
tract colonization with vancomycin-resistant Enterococcus faecium
in an animal model", Antimicrobial Agents and Chemotherapy
40(6):1526-1530). Groups of mice (at least 5 mice/group) are
assigned to receive daily either sterile drinking water (control),
or drinking water containing 100 .mu.g/ml, 200 .mu.g/ml or 300
.mu.g/ml of gallium citrate, 250 .mu.g/ml of vancomycin, 250
.mu.g/ml of linezolid, or 200 .mu.g/ml of gallium citrate and 250
.mu.g/ml of linezolid, starting 24 hours after the inoculation of
the mice up to 10 days. Counts of VRE and total enterococci in
feces are determined for each group at specified intervals up until
40 days after the inoculation and compared with the baseline
counts.
Example 4
[0058] Helicobacter pylori
[0059] C57BL/6 mice are inoculated with the mouse-adapted
Helicobacter pylori SS1 strain (Lee A, O'Rouke et al., 1997, "A
standardized mouse model of Helicobacter pylori infection:
introducing Sydney strain", Gastroenterology 112:1386-97) by
intragastric delivery of 0.1 ml of the bacterial suspension
(approximately 1-2.times.10.sup.9 bacteria/ml) in an appropriate
medium (e.g., brucella broth). Control mice are given 0.1 ml of the
medium without the bacteria. Mice are left for 1-3 weeks for
bacterial colonization to become established. Groups of mice (at
least 5 mice/group) are assigned to receive daily, via intragastric
gavage, either sterile saline (control), or 60 mg/kg, 80 mg/kg or
100 mg/kg of gallium maltolate with or without 15 mg/kg of
omeprazole in saline solution for 14 days. Mice are euthanized 24
hours after the completion of the treatment. A longitudinal section
of gastric tissue is removed, fixed in formalin solution, embedded
in paraffin and cut at 8.mu.. to produce histologic sections. The
sections are prepared with Giemsa stain and examined
microscopically for Helicobacter pylori colonization of the gastric
mucosa. A second longitudinal section of gastric tissue is removed,
weighed and homogenized in 1 ml brucella broth. The homogenate is
diluted in phosphate-buffered saline and an aliquot is plated, in
duplicate, on a selective medium (e.g., blood agar supplemented
with 5% defibrinated sheep blood, 100 .mu.g/ml vancomycin, 3.3
.mu.g/ml polymixin B, 200 g/ml bacitracin, 10.7 .mu.g/ml nalidixic
acid and 50 .mu.g/ml amphotericin B (see J. I. Keena et al., 2004,
"The effect of Helicobacter pylori infection and dietary iron
deficiency on host iron homeostasis: A study in mice", Helicobacter
9(6):643-650). Growth of Helicobacter pylori is confirmed based on
Gram staining, morphology and urease production. The numbers of
colony forming unit (CFU) per gram of tissue are determined and
compared among the groups.
6. EQUIVALENTS
[0060] Those skilled in the art to which the present invention is
related will recognize, or be able to ascertain, many equivalents
to the specific embodiments of the invention described herein using
no more than routine experimentation. Such equivalents are intended
to be encompassed by the following claims.
[0061] All publications, patents and published patent applications
mentioned in this specification are herein incorporated by
reference into the specification.
[0062] Citation or discussion of a reference herein shall not be
construed as an admission that such is prior art to the present
invention.
* * * * *