U.S. patent application number 13/607138 was filed with the patent office on 2013-03-14 for methods for treating intrapulmonary infections.
This patent application is currently assigned to Cubist Pharmaceuticals, Inc.. The applicant listed for this patent is Gurudatt A. Chandorkar, Jennifer A. Huntington, Tara Parsons, Obiamiwe C. Umeh. Invention is credited to Gurudatt A. Chandorkar, Jennifer A. Huntington, Tara Parsons, Obiamiwe C. Umeh.
Application Number | 20130065874 13/607138 |
Document ID | / |
Family ID | 47830390 |
Filed Date | 2013-03-14 |
United States Patent
Application |
20130065874 |
Kind Code |
A1 |
Chandorkar; Gurudatt A. ; et
al. |
March 14, 2013 |
METHODS FOR TREATING INTRAPULMONARY INFECTIONS
Abstract
This disclosure relates to the treatment of intrapulmonary
bacterial infections, including treatment of nosocomial pneumonia
lung infections with pharmaceutical compositions containing the
cephalosporin ceftolozane.
Inventors: |
Chandorkar; Gurudatt A.;
(Waltham, MA) ; Huntington; Jennifer A.; (Reading,
MA) ; Parsons; Tara; (Hanover, MA) ; Umeh;
Obiamiwe C.; (Acton, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Chandorkar; Gurudatt A.
Huntington; Jennifer A.
Parsons; Tara
Umeh; Obiamiwe C. |
Waltham
Reading
Hanover
Acton |
MA
MA
MA
MA |
US
US
US
US |
|
|
Assignee: |
Cubist Pharmaceuticals,
Inc.
Lexington
MA
|
Family ID: |
47830390 |
Appl. No.: |
13/607138 |
Filed: |
September 7, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61532914 |
Sep 9, 2011 |
|
|
|
61657386 |
Jun 8, 2012 |
|
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|
Current U.S.
Class: |
514/196 ;
514/192 |
Current CPC
Class: |
A61K 31/546 20130101;
Y02A 50/30 20180101; A61K 31/431 20130101; A61K 9/0019 20130101;
A61P 31/04 20180101; A61K 9/007 20130101; Y02A 50/473 20180101;
A61K 31/431 20130101; A61K 2300/00 20130101; A61K 31/546 20130101;
A61K 2300/00 20130101 |
Class at
Publication: |
514/196 ;
514/192 |
International
Class: |
A61K 31/431 20060101
A61K031/431; A61P 31/04 20060101 A61P031/04; A61K 31/43 20060101
A61K031/43; A61P 31/00 20060101 A61P031/00 |
Claims
1. A method of treating an intrapulmonary infection comprising the
step of intravenously administering about every 8 hours to a
subject in need thereof a pharmaceutical composition comprising 3.0
g of ceftolozane.
2. The method of claim 1, wherein the pharmaceutical composition
further comprises tazobactam.
3. The method of claim 2, wherein the pharmaceutical composition
comprises ceftolozane and tazobactam and the infection comprises
Gram-negative bacteria.
4. A method of treating an intrapulmonary infection comprising the
step of administering a therapeutically effective amount of a
pharmaceutical composition comprising ceftolozane.
5. The method of claim 1, wherein the intrapulmonary infection
includes an infection in the lung.
6. The method of claim 1, wherein the intrapulmonary infection is
pneumonia.
7. The method of claim 1, wherein the intrapulmonary infection is
nosocomial pneumonia.
8. The method of claim 1, wherein the pharmaceutical composition is
parenterally administered.
9. The method of claim 1, wherein the pharmaceutical composition is
intravenously administered.
10. The method of claim 1, wherein the pharmaceutical composition
is intravenously administered about once every 8 hours as an
infusion.
11. The method of claim 10, wherein the pharmaceutical composition
is intravenously administered as a 60-minute infusion.
12. The method of claim 1, wherein the infection comprises
Pseudomonas aeruginosa, Enterobacteriaceae, or a combination
thereof.
13. The method of claim 1, wherein the infection comprises
Pseudomonas aeruginosa.
14. The method of claim 1, wherein the infection comprises a
pathogen with minimum inhibitory concentration for ceftolozane and
tazobactam of .ltoreq.8 .mu.g/ml.
15. The method of claim 1, wherein the infection comprises a
pathogen with minimum inhibitory concentration for ceftolozane of
.ltoreq.8 .mu.g/ml.
16. A method of providing tazobactam or ceftolozane in the
epithelial lining fluid of a subject in an amount effective to
treat an intrapulmonary infection, comprising the step of
intravenously administering to the subject a pharmaceutical
composition comprising ceftolozane.
17. The method of claim 16, wherein the pharmaceutical composition
further comprises tazobactam and the pharmaceutical composition is
CXA-201.
18. The method of claim 1, wherein the method comprises
administering about 1.5 g of ceftolozane and tazobactam every 8
hours.
19. The method of claim 1, wherein the amount of the ceftolozane in
the epithelial lining fluid of the subject effective to treat an
intrapulmonary infection is at least about 8 .mu.g/ml.
20. The method of claim 1, wherein the ELF concentration of
ceftolozane in the ELF reaches at least about 8 .mu.g/ml after
administration of the pharmaceutical composition.
21. The method of claim 1, wherein the subject is a human having,
or believed to be at risk of having, nosocomial pneumonia.
22. The method of claim 21, wherein the patient has ventilator
acquired pneumonia or hospital acquired pneumonia.
23. The method of claim 1, wherein the treatment comprises
administering ceftolozane every 8 hours.
Description
RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application No. 61/532,914, filed Sep. 9, 2011, titled "Methods for
Treating Intrapulmonary Infections," and U.S. Provisional
Application No. 61/657,386, filed Jun. 8, 2012, titled "Methods for
Treating Intrapulmonary Infections." The contents of any patents,
patent applications, and references cited throughout this
specification are hereby incorporated by reference in their
entireties.
TECHNICAL FIELD
[0002] This disclosure relates to the treatment of intrapulmonary
bacterial infections, including the treatment of nosocomial
pneumonia infections, with a cephalosporin.
BACKGROUND
[0003] The cephalosporin
(6R,7R)-3-[5-Amino-4-[3-(2-aminoethyl)ureidol-1-methyl-1H-pyrazol-2-ium-2-
-ylmethyl]-7-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2-RZ)-1-carboxy-1-methylet-
hoxyimino]acetamido]-3-cephem-4-carboxylic acid (also referred to
as "CXA-101" and previously designated FR264205) is an
antibacterial agent. CXA-101 can be provided as the compound shown
in FIG. 1. The antibacterial activity of CXA-101 is believed to
result from its interaction with penicillin binding proteins (PBPs)
to inhibit the biosynthesis of the bacterial cell wall which acts
to stop bacterial replication. CXA-101 can be combined (e.g.,
mixed) with a .beta.-lactamase inhibitor ("BLI"), such as
tazobactam. Tazobactam is a BLI against Class A and some Class C
.beta.-lactamases, with well established in vitro and in vivo
efficacy in combination with active .beta.-lactam antibiotics. The
combination of CXA-101 and tazobactam in a 2:1 weight ratio is an
antibiotic pharmaceutical composition ("CXA-201") for parenteral
administration. CXA-201 displays potent antibacterial activity in
vitro against common Gram-negative and selected Gram-positive
organisms. CXA-201 is a broad-spectrum antibacterial with in vitro
activity against Enterobacteriaceae including strains expressing
extended spectrum .beta.-lactamases-resistant (MIC.sub.90 =1
.mu.g/mL), as well as Pseudomonas aeruginosa (P. aeruginosa)
including multi-drug resistant strains (MIC.sub.90=2 .mu.g/mL).
CXA-201 is a combination antibacterial with activity against many
Gram-negative pathogens known to cause intrapulmonary infections,
including nosocomial pneumonia caused by P. aeruginosa.
[0004] Intrapulmonary infections, such as nosocomial pneumonia,
remain a major cause of morbidity and mortality, especially
infections caused by drug resistant pathogens such as P.
aeruginosa. One challenge in treating intrapulmonary infections
with systemic administration of an antibiotic is determining the
antibiotic dose that will provide a therapeutically safe and
effective concentration of the antibiotic at the site of an
infection on the mucosal side of the bronchi in the lung (i.e., in
the bronchial secretions). Many antibiotics diffuse poorly from the
bloodstream across the bronchi [e.g., Pennington, J. E.,
"Penetration of antibiotics into respiratory secretions," Rev
Infect Dis 3(1):67-73 (1981)1, which can result in the
administration of higher doses of antibiotic than would be
prescribed for a truly systemic infection. Furthermore, the
purulent sputum that characterizes infected patients tends to
compromise the potency of many antibiotics (See e.g., Levy, J., et
al., "Bioactivity of gentamicin in purulent sputum from patients
with cystic fibrosis or bronchiectasis: comparison with activity in
serum," J Infect Dis 148(6):1069-76 (1983)). In some cases, the
result is the prescription of large amounts of a systemically
administered antibiotic to treat an intrapulmonary infection.
[0005] The efficacy of an antibiotic depends in part on the
concentration of the drug at the site of action. Efficacy of
antimicrobial therapy requires adequate antibiotic concentrations
at the site of bacterial infection, and some authorities believe
that epithelial lining fluid (ELF) concentrations are a reasonable
surrogate for predicting effective concentrations for treating
intrapulmonary infections such as pneumonia. For many antibiotics,
clinical data correlating ELF concentrations to clinical outcome is
unavailable and the clinical significance of differences in
pulmonary penetration of antibiotics is unknown or poorly
characterized. Few studies have quantified the penetration of
.beta.-lactam agents into the lung, as measured by the ratio of
area under the concentration-time curve (AUC) in ELF to AUC in
plasma (AUC(ELF)/AUC(plasma) ratio). For some published studies,
the concentration of antibiotics measured in the ELF of the lung
has varied widely. For example, the reported penetration ratio of
telavancin in healthy human volunteers ranges widely between 0.43
and 1.24 (Lodise, Gottfreid, Drusano, 2008 Antimicrobial Agents and
Chemotherapy). Thus, predicting the penetration of a drug into the
ELF a priori, based on the structure, molecular weight, size and
solubility is difficult due to the limited data available on the
effect of physicochemical properties on the lung penetration of
drugs.
[0006] Accordingly, the efficacy of a particular drug in treating
intrapulmonary infections, in particular nosocomial pneumonia,
cannot be predicted solely on the basis of data, such as in vitro
data relating to the activity of that drug against a particular
bacterium, which does not give any indication as whether the drug
will accumulate at a therapeutically safe and effective
concentration at the site of an infection on the mucosal side of
the bronchi in the lung (i.e., in the bronchial secretions). For
instance, tigicycline, a glycylcycline antimicrobial, has in vitro
activity against many species of Gram-positive and Gram-negative
bacteria, including P. aeruginosa, and it has been approved by the
FDA for the treatment of complicated skin and skin structure
infections, complicated intra-abdominal infections, and community
acquired pneumonia. However, tigicycline is not approved for the
treatment of nosocomial pneumonia, in view of an increased
mortality risk associated with the use of tigicycline compared to
other drugs in patients treated for nosocomial pneumonia.
SUMMARY
[0007] The present invention provides methods for treating
intrapulmonary infections, including nosocomial pneumonia, with
systemic administration of a pharmaceutical composition comprising
ceftolozane. The invention is based in part on results from a human
clinical study designed to assess the ELF penetration of CXA-201 in
comparison to piperacillin/tazobactam, indicated for the treatment
of nosocomial pneumonia. The study described herein quantified the
penetration of CXA-201 into the lung, as measured by the ratio of
area under the concentration-time curve (AUC) in epithelial lining
fluid (ELF) to AUC in plasma (AUC(ELF)/AUC(plasma) ratio). The
results of the study indicate that CXA-201 penetrated into the ELF
of human patients, with a ceftolozane ELF/plasma AUC ratio of 0.48.
The measured ELF concentrations of ceftolozane exceeded 8 .mu.g/mL
for 60% of the 8-hour dosing interval, a concentration that is
predicted to inhibit 99% of Pseudomonas aeruginosa based on current
surveillance data.
[0008] The study showed that CXA-201 penetrated well into the ELF
of healthy volunteers compared to piperacillin/tazobactam, an agent
widely used for treatment of lower respiratory infections. The
intrapulmonary pharmacokinetics measured in the study supports the
use of CXA-201 as a parenteral (e.g., intravenous) antibiotic for
treatment of intrapulmonary infections, such as nosocomial
pneumonia or other lower respiratory tract infections.
BRIEF DESCRIPTION OF DRAWINGS
[0009] FIG. 1 is the chemical structure of a salt of ceftolozane
hydrogen sulfate salt.
[0010] FIG. 2A is a graph showing the ELF Concentration vs. Time
Profile for ceftolozane hydrogen sulfate salt (Median and Range)
for CXA-201.
[0011] FIG. 2B is a graph showing the ELF Concentration vs. Time
Profile for Tazobactam (Median and Range) for CXA-201.
[0012] FIG. 3A is a graph showing the (Comparative) ELF
Concentration vs. Time Profile for Piperacillin (Median and Range)
for a piperacillin/tazobactam comparator (ZOSYN.RTM.).
[0013] FIG. 3B is a graph showing the (Comparative) ELF
Concentration vs. Time Profile for Tazobactam (Median and Range)
for a piperacillin/tazobactam comparator (ZOSYN.RTM.).
[0014] FIGS. 4A and 4B are synthetic schemes for preparing
ceftolozane hydrogen sulfate salt.
DETAILED DESCRIPTION
[0015] The present disclosure relates to the treatment of
intrapulmonary infections, including nosocomial pneumonia, with
systemic administration of a pharmaceutical composition comprising
ceftolozane, including the parenteral administration of a
therapeutically effective amount of a pharmaceutical composition
comprising ceftolozane and tazobactam. As used herein, the term
"ceftolozane" means CXA-101 in a free-base or salt form, preferably
a hydrogen sulfate form (illustrated in FIG. 1). In one embodiment,
ceftolozane is CXA-101 in its free-base form. In another
embodiment, ceftolozane is CXA-101 in its salt form, preferably a
hydrogen sulfate form.
[0016] In a preferred embodiment, ceftolozane (in free base or salt
form, preferably hydrogen sulfate form) and tazobactam are in a 2:1
(ceftolozane:tazobactam) weight ratio. In a particular embodiment,
provided herein are methods of treating intrapulmonary infections,
including nosocomial pneumonia, with systemic administration of a
pharmaceutical composition comprising ceftolozane hydrogen sulfate
and tazobactam in a 2:1 weight ratio. The combination of
ceftolozane hydrogen sulfate and tazobactam in a 2:1 weight ratio
is referred to herein and in the examples as "CXA-201."
[0017] In one aspect, the invention provides a method of treating
an intrapulmonary infection comprising administering a
therapeutically effective amount of a pharmaceutical composition
comprising ceftolozane. The method may comprise administering a
pharmaceutical composition comprising ceftolozane in combination
with tazobactam.
[0018] In another aspect, the invention provides a method of
treating an intrapulmonary infection comprising the step of
intravenously administering about every 8 hours to a subject in
need thereof a pharmaceutical composition comprising 3.0 g of
ceftolozane. The method may comprise administering a pharmaceutical
composition comprising ceftolozane in combination with tazobactam.
In one embodiment, the method comprises administering CXA-201 and
the infection comprises Gram-negative bacteria. In another aspect,
the invention provides a method of treating an intrapulmonary
infection comprising the step of intravenously administering every
8 hours to a subject in need thereof a pharmaceutical composition
comprising 3.0 g of ceftolozane.
[0019] In another aspect, the invention provides a method of
providing tazobactam or ceftolozane in the epithelial lining fluid
of a subject in an amount effective to treat an intrapulmonary
infection, comprising the step of intravenously administering to
the subject a pharmaceutical composition comprising ceftolozane.
The method may comprise administering a pharmaceutical composition
further comprising tazobactam, optionally wherein the
pharmaceutical composition is CXA-201. The method may comprise
administering about 1.5 g of ceftolozane and tazobactam in total
every 8 hours. In one embodiment, the amount of the ceftolozane in
the ELF of the subject effective to treat an intrapulmonary
infection is at least about 8 .mu.g/ml. The ELF concentration of
ceftolozane in the ELF may reach at least about 8 .mu.g/ml after
administration of the pharmaceutical composition. The subject is
typically a human having, or believed to be at risk of having,
nosocomial pneumonia. The subject (or patient) may, in some
embodiments, have ventilator acquired pneumonia or hospital
acquired pneumonia.
[0020] In another aspect, the invention provides the use of
ceftolozane in the manufacture of a medicament for the treatment of
an intrapulmonary infection comprising administering a
therapeutically effective amount of a pharmaceutical composition
comprising the ceftolozane. The use may comprise administering the
pharmaceutical composition comprising ceftolozane, in combination
with tazobactam.
[0021] In another aspect, the invention provides the use of
ceftolozane in the manufacture of a medicament for the treatment of
an intrapulmonary infection comprising intravenously administering
a pharmaceutical composition comprising 3.0 g of the ceftolozane
every 8 hours to a subject in need thereof. The use may comprise
administering the pharmaceutical composition comprising ceftolozane
in combination with tazobactam. In one embodiment, the use
comprises administering ceftolozane and tazobactam and the
infection comprises Gram-negative bacteria.
[0022] In another aspect, the invention provides the use of
ceftolozane in the manufacture of a medicament for the treatment of
an intrapulmonary infection comprising intravenously administering
a pharmaceutical composition comprising the ceftolozane, wherein
tazobactam or ceftolozane is provided in the epithelial lining
fluid of a subject in an amount effective to treat the
intrapulmonary infection. The use may comprise administering a
pharmaceutical composition further comprising tazobactam,
optionally wherein the pharmaceutical composition is CXA-201. The
use may comprise administering about 1.5 g of ceftolozane and
tazobactam every 8 hours. In one embodiment, the amount of the
ceftolozane in the ELF of the subject effective to treat an
intrapulmonary infection is at least about 8 .mu.g/ml. The ELF
concentration of ceftolozane in the ELF may reach at least about 8
.mu.g/ml after administration of the pharmaceutical composition.
The subject is typically a human having, or believed to be at risk
of having, nosocomial pneumonia. The subject (or patient) may, in
some embodiments, have ventilator acquired pneumonia or hospital
acquired pneumonia. In the methods and uses of the invention, the
pharmaceutical composition may be administered parenterally. The
pharmaceutical composition may be administered intravenously. In
some embodiments, the pharmaceutical composition is intravenously
administered about once every 8 hours as an infusion. The
pharmaceutical composition may be intravenously administered as a
60-minute infusion.
[0023] In the methods and uses of the invention, the intrapulmonary
infection may be an infection in the lung. The intrapulmonary
infection may be pneumonia. In a preferred embodiment, the
intrapulmonary infection is nosocomial pneumonia. The
intrapulmonary infection may comprise Pseudomonas aeruginosa,
Enterobacteriaceae, or a combination thereof. Typically, the
intrapulmonary infection comprises Pseudomonas aeruginosa. The
intrapulmonary infection may comprise a pathogen with minimum
inhibitory concentration for CXA-201 of .ltoreq.8 .mu.g/ml. The
intrapulmonary infection may comprise a pathogen with minimum
inhibitory concentration for ceftolozane of .ltoreq.8 .mu.g/ml.
[0024] In another aspect, the invention provides ceftolozane, for
use in a method of treating an intrapulmonary infection. In one
embodiment, the ceftolozane is parenterally administered.
Typically, the ceftolozane is intravenously administered. In some
embodiments, the ceftolozane is administered about once every 8
hours as an infusion. In some embodiments, the ceftolozane is
intravenously administered as a 60-minute infusion.
[0025] In one embodiment, the ceftolozane is for use in a method of
treating an intrapulmonary infection wherein the intrapulmonary
infection comprises an infection in the lung. The intrapulmonary
infection may be pneumonia. In a preferred embodiment, the
ceftolozane is for use in a method of treating nosocomial
pneumonia. The intrapulmonary infection may comprise Pseudomonas
aeruginosa, Enterobacteriaceae, or a combination thereof.
Typically, the intrapulmonary infection comprises Pseudomonas
aeruginosa. The intrapulmonary infection may comprise a pathogen
with minimum inhibitory concentration for ceftolozane and
tazobactam of .ltoreq.8 .mu.g/ml. The intrapulmonary infection may
comprise a pathogen with minimum inhibitory concentration for
ceftolozane of .ltoreq.8 .mu.g/ml.
[0026] The invention also provides ceftolozane, for use in a method
of treating an intrapulmonary infection, comprising administration
of ceftolozane in combination with tazobactam. In one embodiment,
the ceftolozane and/or tazobactam is parenterally administered.
Typically, the ceftolozane and/or tazobactam is intravenously
administered. In some embodiments, the ceftolozane and/or
tazobactam is administered about once every 8 hours as an infusion.
In some embodiments, the ceftolozane and/or tazobactam is
intravenously administered as a 60-minute infusion. In one
embodiment, both the ceftolozane and tazobactam are parenterally
administered. In another embodiment, both the ceftolozane and
tazobactam are intravenously administered. In some embodiments,
both the ceftolozane and tazobactam are administered about once
every 8 hours as an infusion. In some embodiments, both the
ceftolozane and tazobactam are intravenously administered as a
60-minute infusion. In one embodiment, the ceftolozane is for use
in a method of treating an intrapulmonary infection wherein the
intrapulmonary infection comprises an infection in the lung. The
intrapulmonary infection may be pneumonia. In a preferred
embodiment, the ceftolozane is for use in a method of treating
nosocomial pneumonia. The intrapulmonary infection may comprise
Pseudomonas aeruginosa, Enterobacteriaceae, or a combination
thereof. Typically, the intrapulmonary infection comprises
Pseudomonas aeruginosa. The intrapulmonary infection may comprise a
pathogen with minimum inhibitory concentration for ceftolozane and
tazobactam of .ltoreq.8 .mu.g/ml. The intrapulmonary infection may
comprise a pathogen with minimum inhibitory concentration for
ceftolozane of .ltoreq.8 .mu.g/ml.
[0027] In another aspect, the invention provides tazobactam, for
use in a method of treating an intrapulmonary infection, comprising
administration of tazobactam in combination with ceftolozane. In
one embodiment, the tazobactam and/or ceftolozane is parenterally
administered. Typically, the tazobactam and/or ceftolozane is
intravenously administered. In some embodiments, the tazobactam
and/or ceftolozane is administered about once every 8 hours as an
infusion. In some embodiments, the tazobactam and/or ceftolozane is
intravenously administered as a 60-minute infusion. In one
embodiment, both the tazobactam and ceftolozane are parenterally
administered. In another embodiment, both the tazobactam and
ceftolozane are intravenously administered. In another embodiment,
both the tazobactam and ceftolozane are administered about once
every 8 hours as an infusion. In another embodiments, both the
tazobactam and ceftolozane are intravenously administered as a
60-minute infusion.
[0028] In one embodiment, the tazobactam is for use in a method of
treating an intrapulmonary infection wherein the intrapulmonary
infection comprises an infection in the lung. The intrapulmonary
infection may be pneumonia. In a preferred embodiment, the
tazobactam is for use in a method of treating nosocomial pneumonia.
The intrapulmonary infection may comprise Pseudomonas aeruginosa,
Enterobacteriaceae, or a combination thereof. Typically, the
intrapulmonary infection comprises Pseudomonas aeruginosa. The
intrapulmonary infection may comprise a pathogen with minimum
inhibitory concentration for ceftolozane and tazobactam of
.ltoreq.8 .mu.g/ml. The intrapulmonary infection may comprise a
pathogen with minimum inhibitory concentration for ceftolozane of
.ltoreq.8 .mu.g/ml.
[0029] In another aspect, the invention provides ceftolozane and
tazobactam, as a combined preparation for simultaneous, separate or
sequential use in a method of treating an intrapulmonary infection.
In one embodiment, the ceftolozane and tazobactam are parenterally
administered. Typically, the ceftolozane and tazobactam are
intravenously administered. In some embodiments, the ceftolozane
and tazobactam are administered about once every 8 hours as an
infusion. In some embodiments, the ceftolozane and tazobactam, are
intravenously administered as a 60-minute infusion.
[0030] In one embodiment, the ceftolozane and tazobactam are for
use in a method of treating an intrapulmonary infection wherein the
intrapulmonary infection comprises an infection in the lung. The
intrapulmonary infection may be pneumonia. In a preferred
embodiment, the ceftolozane and tazobactam are for use in a method
of treating nosocomial pneumonia. The intrapulmonary infection may
comprise Pseudomonas aeruginosa, Enterobacteriaceae, or a
combination thereof. Typically, the intrapulmonary infection
comprises Pseudomonas aeruginosa. The intrapulmonary infection may
comprise a pathogen with minimum inhibitory concentration for
ceftolozane and tazobactam of .ltoreq.8 .mu.g/ml. The
intrapulmonary infection may comprise a pathogen with minimum
inhibitory concentration for ceftolozane of .ltoreq.8 .mu.g/ml.
[0031] In another aspect, the invention provides ceftolozane for
use in a method of providing tazobactam or ceftolozane in the
epithelial lining fluid of a subject in an amount effective to
treat an intrapulmonary infection, comprising the step of
intravenously administering ceftolozane. In some embodiments,
ceftolozane is administered in combination with tazobactam.
Preferably, CXA-201 is administered. In preferred embodiments,
about 1.5 g of ceftolozane and tazobactam is administered every 8
hours. In one embodiment, the amount of the ceftolozane in the ELF
of the subject effective to treat an intrapulmonary infection is at
least about 8 .mu.g/ml. The ELF concentration of ceftolozane in the
ELF may reach at least about 8 .mu.g/ml after administration of the
ceftolozane. The subject is typically a human having, or believed
to be at risk of having, nosocomial pneumonia. The subject (or
patient) may, in some embodiments, have ventilator acquired
pneumonia or hospital acquired pneumonia.
[0032] The safe and effective treatment of intrapulmonary infection
with CXA-201 includes administration of an amount of the CXA-201
selected to provide a therapeutically effective dose of the CXA-201
antibiotic in the epithelial lining fluid (ELF). The penetration of
CXA-201 into the ELF compared to a piperacillin/tazobactam
comparator was assessed in a Phase 1 clinical study in healthy
adult volunteers. The piperacillin/tazobactam comparator contained
piperacillin/tazobactam in an 8:1 weight ratio with a total of 2.79
mEq of sodium per gram of piperacillin, FDA approved under the
tradename ZOSYN.RTM. ("Zosyn"). The study results evaluate the
penetration of intravenously administered CXA-201 into healthy
human lungs, as measured by the ratio of area under the
concentration-time curve (AUC) in epithelial lining fluid (ELF) to
AUC in plasma (AUC(ELF)/AUC(plasma)ratio).
[0033] In the study, a 4.5 g amount of piperacillin/tazobactam
incorporates the same dose of tazobactam (0.5 g) as 1.5 g of
CXA-201. A multiple-dose regimen was used in this study to ensure
that the concentrations of the analytes reached steady-state in
both plasma and ELF prior to assessment. Healthy volunteers were
chosen to standardize the subject population and minimize the
variability associated with using actively ill patients. The
objectives of the study included: (1) determination and comparison
of the ELF to plasma concentration ratios of multiple-doses of
intravenous CXA-201 compared to piperacillin/tazobactam in healthy
adult volunteers, and (2) assessment of the safety and tolerability
of multiple-doses of intravenous CXA-201 in healthy adult
volunteers.
[0034] The study was a Phase 1 prospective, randomized (1:1),
comparator controlled, open-label study of 50 healthy adult
volunteers. Each healthy volunteer received 3 doses of either
CXA-201(1.5 grams every 8 hours as a 60-minute infusion) or
piperacillin/tazobactam (4.5 grams every 6 hours as a 30-minute
infusion). Subjects received 3 doses of a study drug, underwent
serial blood draws at planned plasma sampling timepoints, and
underwent a single bronchoalveolar lavage (BAL) procedure at one of
the scheduled timepoints (Table 1).
TABLE-US-00001 TABLE 1 Plasma Sampling and BAL Timepoints Plasma
Sampling Timepoints BAL Timepoints Intensive plasma sampling 5
subjects per timepoint per from all 25 subjects for treatment
group; in hours from one dosing interval start of the third
infusion CXA-201 0 (pre-PK dose trough), 1, 2, 1, 2, 4, 6, 8 hours
post start 4, 6, 8 hours post start of of infusion of the third
dose of infusion of the third dose of CXA 201 CXA 201
Piperacillin/tazobactam 0 (pre-PK dose trough), 0.5, 1, 0.5, 1, 2,
4, 6 hours post start 2, 4, 6 hours post start of of infusion of
the third dose of infusion of the third dose of
piperacillin/tazobactam piperacillin/tazobactam
[0035] A total of 51 subjects were enrolled; 25 in the CXA-201
group and 26 in the piperacillin/tazobactam group. Key Inclusion
Criteria for the study were: (1) healthy adult male or non-pregnant
females between 18 and 50 years, inclusive; (2) body mass index
between 18.5 and 30; and (3) forced Expiratory Volume in 1 second
(FEV1) .gtoreq.80%. Key Exclusion Criteria for the study were: (1)
pregnancy or lactation; (2) clinically significant systemic disease
or the existence of any surgical or medical condition that may have
interfered with the distribution, metabolism, or excretion of
CXA-201; (3) history of asthma or any restrictive or obstructive
lung disease; (4) history of smoking or abuse of narcotics or
alcohol; (5) positive test for human immunodeficiency virus,
Hepatitis B surface antigen, or Hepatitis C antibodies; (6) any
condition or situation where bronchoscopy was not advisable; and
(7) impairment of renal function (CrCl<90 mL/min)
Determination of the ELF to Plasma Concentration Ratios of
Multiple-Doses of Intravenous CXA-201 Compared to
Piperacillin/Tazobactam in Healthy Adult Volunteers.
[0036] Plasma and BAL datapoints were used to construct one
concentration-time profile in the ELF using the mean concentrations
at each time point. After dosing, a single ELF sample was obtained
by bronchoalveolar lavage (BAL) from each healthy volunteer at one
of 5 scheduled time points (5 subjects/time point/treatment group).
The ELF to plasma concentrations of multiple-doses was determined.
Serial plasma samples were collected pre- and post-treatment over a
6-hour (piperacillin/tazobactam) or 8-hour (CXA-201) time period.
Urea levels in the plasma and BAL were used to calculate the ELF
drug concentrations (see Table 1). Pharmacokinetic parameters for
ELF were calculated by non-compartmental analysis using the mean
concentrations at each time point. The intrapulmonary penetration
of CXA-201 into the ELF was determined by dividing the ELF
AUC.sub.0-t by mean plasma AUC.sub.0-t.
[0037] The concentration of CXA-201 and piperacillin/tazobactam in
ELF were estimated from the concentration of drug in BAL fluid, the
volume of BAL fluid collected, and the ratio of urea concentration
in BAL fluid to that in plasma. Calculation of ELF volume was
determined by the urea dilution method, using urea as an endogenous
marker of ELF recovered by BAL. Concentration of CXA-201 and
piperacillin/tazobactam in ELF was estimated from the concentration
of drug in BAL fluid, the volume of BAL fluid collected, and the
ratio of urea concentration in BAL fluid to that in plasma. The
following formulas represent these calculations:
CXA-201 (CXA/T)=[CXA/T].sub.BAL.times.V.sub.BAL/V.sub.ELF
[0038] [CXAM.sub.BAL is the concentration of CXA-201 in BAL fluid;
V.sub.BAL is the volume of aspirated BAL fluid (total); V.sub.ELF
is V.sub.BAL.times.[urea].sub.BAL/[urea].sub.plasma, where
[urea].sub.BAL is the concentration of urea in the BAL fluid
(supernatant) and [urea].sub.plasma is the concentration of urea in
the plasma specimens.
Piperacillin/tazobactam=[PIP/T].sub.BAL.times.V.sub.BAL/V.sub.ELF
[0039] [PIP/T].sub.BAL is the concentration of
piperacillin/tazobactam in BAL fluid; V.sub.BAL is the volume of
aspirated BAL fluid (total); V.sub.ELF is
V.sub.BAL.times.[urea].sub.BAL/[urea].sub.plasma, where
[urea].sub.BAL is the concentration of urea in the BAL fluid
(supernatant) and [urea].sub.plasma is the concentration of urea in
the plasma specimens.
[0040] No oral antibiotic therapy was permitted. Safety was
monitored through the review of vital signs, laboratory and
physical examinations and the occurrence of adverse events (AEs).
Subjects who received three doses of study medication and had both
BAL and plasma samples collected were included in the
pharmacokinetic (PK) analysis population. All randomized subjects
who received any dose (including partial doses) of study medication
were included in the safety analysis population.
[0041] The results of the study (Table 2) indicate that CXA-201
penetrated well into ELF. The ceftolozane component of CXA-201
ELF/plasma AUC ratio was 0.48, compared to 0.26 for the
piperacillin component of piperacillin/tazobactam. The ELF
concentrations of ceftolozane exceeded 8 .mu.g/mL for 60% of the
8-hour dosing interval. The plasma concentrations for ceftolozane
were consistent with those seen previously at this dose.
[0042] The ELF concentration vs. time profiles for ceftolozane and
tazobactam components of CXA-201 are shown in FIGS. 2A and 2B,
respectively. Comparative data showing the ELF concentration vs.
time profiles for piperacillin and tazobactam components of the
comparator drug are shown in FIGS. 3A and 3B, respectively. The ELF
to plasma penetration ratios are shown in Table 2.
[0043] The PK parameters were determined by non-compartmental PK
analysis. PHOENIX.RTM. WinNonlin v 6.1 (PHARSIGHT.RTM., Mountain
View, Calif.) was used for the derivation of all PK individual
measures for each subject. The PK parameters for ELF were
calculated by taking the mean concentrations of the 5 subjects at
each time point and constructing a single profile over the duration
of sampling. In the event that the urea concentrations determined
in plasma or ELF were below quantifiable limits, thereby providing
only an estimate of concentration, those values were not used in
the calculation of mean concentration at that time point. The
ceftolozane, piperacillin, and tazobactam PK parameters that were
computed in plasma and ELF were: [0044] C.sub.max (.mu.g/mL):
Maximum plasma and ELF concentration over the entire sampling phase
directly obtained from the experimental plasma concentration time
data, without interpolation. [0045] T.sub.max (hr): Sampling time
at which C.sub.max occurred, obtained directly from the
experimental plasma and ELF concentration time data, without
interpolation. [0046] C.sub.last (.mu.g/mL): Plasma or ELF
concentration when last quantifiable concentration was observed,
relative to the end of infusion. [0047] T.sub.last (hr): Time when
the last quantifiable concentration was observed. [0048]
AUC.sub.0-t (.mu.g*hr/mL): An area under the concentration time
curve from the time of the dose to the end of the dosing interval.
[0049] Percent penetration into ELF: Calculated as the ratio of
AUC.sub.0-tELF and mean AUC.sub.0-tPlasma.
TABLE-US-00002 [0049] TABLE 2 Summary of ELF to Plasma Penetration
Ratios Mean Plasma ELF ELF AUC.sub.0-.tau. AUC.sub.0-.tau.
Penetration Analyte (.mu.g*hr/mL) (.mu.g*hr/mL) Ratio ceftolozane
158.5 75.1 0.48 (in CXA-201) Tazobactam 19.3 8.5 0.44 (in CXA-201)
Piperacillin 357.3 94.5 0.26 (in piperacillin/tazobactam)
Tazobactam 46.1 24.7 0.54 (in piperacillin/tazobactam)
[0050] The ELF/plasma AUC ratio for the ceftolozane component of
CXA-201 was 0.48, compared to 0.26 for the piperacillin component
of the comparator drug (piperacillin/tazobactam). The ELF/plasma
AUC ratio for tazobactam was 0.44 and 0.54 when given as part of
CXA-201 and piperacillin/tazobactam, respectively. The ELF
concentrations of ceftolozane exceeded 8 .mu.g/mL for 60% of the
8-hour dosing interval. The plasma and ELF concentrations of
tazobactam when given as piperacillin/tazobactam was approximately
2-fold higher than when an equivalent dose was given as
CXA-201.
[0051] The results show that ceftolozane and tazobactam (i.e.,
administered as CXA-201) penetrated well into the ELF of healthy
volunteers compared to piperacillin/tazobactam, an agent widely
used for treatment of lower respiratory infections. CXA-201's
intrapulmonary pharmacokinetics support use of CXA-201 as a
parenteral (e.g., intravenous) antibiotic for treatment of lower
respiratory tract infections, including infections caused by
pathogens with minimum inhibitory concentrations of .ltoreq.8
.mu.g/ml. The concentrations of ceftolozane in ELF exceeded 8
.mu.g/mL, a concentration that inhibits 99% of P. aeruginosa, for
approximately 60% of the 8-hour dosing interval for the CXA-201
regimen of 1.5 grams every eight hours as a 60 minute infusion.
[0052] Assessment of the safety and tolerability of multiple-doses
of intravenous CXA-201 in healthy adult volunteers.
[0053] Among the subjects, 50 of the 51 (98%) subjects received all
3 doses of study medication and completed the BAL procedure. One
subject prematurely discontinued piperacillin/tazobactam and
terminated their participation in the study due to an AE of
hypersensitivity that occurred during administration of the first
dose. Demographics and baseline characteristics are summarized in
Table 3, the two treatment arms were well balanced.
TABLE-US-00003 TABLE 3 Demographics and Baseline Characteristics
(Safety Population) Piperacillin/ CXA-201 tazobactam 1.5 grams 4.5
grams (N = 25) (N = 26) Sex, n (%) Female 11 (44.0) 11 (42.3) Male
14 (56.0) 15 (57.7) Age, years Mean (SD) 32.6 (7.8) 34.2 (8.5)
Minimum, Maximum 21, 47 22, 49 Race, n (%) White 20 (80.0) 21
(80.8) Black or African American 2 (8.0) 2 (7.7) Asian 1 (4.0) 0
(0.0) American Indian or Alaska Native 0 (0.0) 1 (3.8) Native
Hawaiian or Pacific Islander 1 (4.0) 0 (0.0) Other 1 (4.0) 2 (7.7)
BMI, kg/m.sup.2 Mean (SD) 26.21 (2.6) 23.23 (2.4) Minimum, Maximum
22.3, 30.0 20.6, 29.9
[0054] During the study, treatment-emergent AEs (TEAEs) occurred in
20.0% (5/25) of subjects receiving CXA-201 and 23.1% (6/26) of
subjects receiving piperacillin/tazobactam. No serious AEs were
reported in either treatment group. All AEs were mild in severity.
The incidence and pattern of AEs were generally similar in the 2
treatment groups, Table 4.
TABLE-US-00004 TABLE 4 TEAEs by Preferred Term (Safety Population)
Subjects with at least 1 TEAE 5 (20.0) 6 (23.1) Diarrhea 1 (4.0) 3
(11.5) Viral Upper Respiratory Infection 1 (4.0) 0 (0)
Musculoskeletal Chest Pain 1 (4.0) 0 (0) Somnolence 1 (4.0) 0 (0)
Hematuria 1 (4.0) 0 (0) Cough 1 (4.0) 0 (0) Type I Hypersensitivity
0 (0) 1 (3.8) Alanine Aminotransferase Increased 0 (0) 1 (3.8)
Aspartate Aminotransferase Increased 0 (0) 1 (3.8) Blood Creatine
Phosphokinase Increased 0 (0) 1 (3.8) Hyperkalemia 0 (0) 1
(3.8)
[0055] Eight subjects had TEAEs assessed as related to study drug;
two in the CXA-201 group (diarrhea and somnolence in 1 subject
each) and six in the piperacillin/tazobactam group (diarrhea in 3
subjects, type I hypersensitivity in 1 subject, blood creatine
phosphokinase increased in 1 subject, and alanine aminotransferase
increased, aspartate aminotransferase increased, and hyperkalaemia
all in the same 1 subject). One piperacillin/tazobactam-treated
subject discontinued study drug due to an adverse event, type I
hypersensitivity. There were no clinically significant changes in
safety laboratory assessments or vital signs.
[0056] CXA-201 appeared safe and well tolerated in this group of
healthy adult subjects.
Determining Appropriate Dose
[0057] A Monte Carlo simulation was performed based on clinical
trial data to predict an effective CXA-201 dose for treating
nosocomial pneumonia using PHOENIX.RTM. NLME (PHARSIGHT.RTM.,
Mountain View, Calif.) software, a tool for data processing and
modeling for population PK/PD analysis. A population
pharmacokinetic (PK) model was developed using the CXA-201 plasma
concentration versus time data from a previously conducted Phase 2
study in patients with complicated intra abdominal infections.
Estimates of clearance and volume of distribution along with the
associated inter-individual variability were obtained from these
analyses. The outputs from the PK population model served as inputs
for a clinical trial simulation performed using PHARSIGHT.RTM.
Trial Simulator (PHARSIGHT.RTM.) software, a tool for defining and
testing interactive drug models, exploring and communicating study
design attributes, and performing statistical and sensitivity
analysis through graphical and statistical summaries. Based on the
mean ELF penetration data, an ELF/Plasma AUC ratio of 0.48 for
ceftolozane (modeled as a numerical range of 0.25-0.65) calculated
from the ceftolozane ELF study mentioned above was used to generate
a random/Plasma AUC ratio from the range 0.25-0.65 for each
simulated patient. This range reflects a conservative estimate of
the potential distribution in a patient population. Using the
results from the PK population model and the ELF/Plasma AUC ratio,
the model simulated plasma and ELF concentration of CXA-201 versus
time profiles for 1,000 hypothetical clinical trial patients with
nosocomial pneumonia. The model evaluated the probability of
clinical success of the 3.0 g every 8 hour (q8h) dose of CXA-201
against three key pathogens in nosocomial pneumonia. The MIC
distribution for these pathogens was imputed from 2008 United
States surveillance data. Clinical success was defined as the
achievement of an ELF or plasma concentration of ceftolozane higher
than the MIC(s) of the lower respiratory pathogen(s) for a given
patient. In vivo models have demonstrated that, as for typical
cephlaosporins, the relavent PK/PD driver for CXA-201 is the
percentage of time above MIC during the dosing interval. The target
is to achieve concentrations that exceed the MIC of the pathogen
for 45-50% of the time between each q8H dose. Thus, a threshold of
50% time above the minimum inhibitory concentration [T>MIC] on
Day 7 of treatment was used. Plasma and ELF concentrations were
estimated at 15 time-points post-administration on Day 7 when dosed
every 8 hours. The results of these simulations are shown in Table
5.
TABLE-US-00005 TABLE 5 Probability of Target Attainment versus Key
Pathogens in Nosocomial Pneumonia Using the Simulated 3.0 g versus
the 1.5 g Dose of Ceftolozane/tazobactam 50% 50% T > MIC T >
MIC Pathogen Dosing Regimen in Plasma in ELF P. aeruginosa 1.5 g q8
h 98.2 94.6 3.0 g q8 h 99.4 98.5 E. coli 1.5 g q8 h 96.3 94.2 3.0 g
q8 h 98.8 95.5 K. pneumoniae 1.5 g q8 h 90.2 87.3 3.0 g q8 h 92.6
89.3 Abbreviation: T > MIC = Time above minimum inhibitory
concentration.
[0058] These simulations demonstrate that the 3.0 g dose of CXA-201
administered every 8 hours is expected to provide adequate
concentrations for treatment of the vast majority of lower
respiratory infections caused by these pathogens.
[0059] Following these simulations, the safety and tolerability of
a 10 day course of CXA-201 3.0 g IV q8h was evaluated in healthy
human volunteers. Subjects were randomized to receive either 3.0 g
(2.0/1.0 g) CXA-201 (n=8), 1.5 g (1.0/0.5 g) CXA-201 (n=4), or
placebo (n=4). The data showed that CXA-201 was generally safe and
well tolerated in this study. There were no serious adverse events
or deaths reported in this study.
[0060] In conclusion, given the pharmacokinetic simulations
conducted, the favorable data from the intrapulmonary PK study and
demonstrated safety and tolerability of the higher dose of CXA-201
in the Phase 1 study mentioned above, the data provide
justification for the use of 3.0 g CXA-201 IV q8h for the treatment
of patients with nosocomial pneumonia caused by Gram-negative
pathogens.
Preparing CXA-201
[0061] CXA-201 can be prepared by combining ceftolozane and
tazobactam in a 2:1 weight ratio. CXA-201 can be obtained using
methods described in U.S. Pat. No. 7,129,232 and Toda et al.,
"Synthesis and SAR of novel parenteral anti-pseudomonal
cephalosporins: Discovery of FR264205," Bioorganic & Medicinal
Chemistry Letters, 18, 4849-4852 (2008), incorporated herein by
reference in its entirety.
[0062] According to the method disclosed in Toda et al., "Synthesis
and SAR of novel parenteral anti-pseudomonal cephalosporins:
Discovery of FR264205," Bioorganic & Medicinal Chemistry
Letters, 18, 4849-4852 (2008), ceftolozane can be obtained by the
synthetic schemes of FIGS. 4A and 4B. Referring to FIGS. 4A and 4B,
synthesis of ceftolozane can be performed via activation of the
thiadiazolyl-oximinoacetic acid derivative (I) with methanesulfonyl
chloride and K.sub.2CO.sub.3 in DMA at 10.degree. C., followed by
coupling with the 7-aminocephem (II) by means of Et.sub.3N in cold
EtOAc/H.sub.2O, affords amide (III) (1). Substitution of the
allylic chloride of compound (III) with
4-[(N-Boc-aminoethyl)carbamoylamino]-1-methyl-5-tritylaminopyrazole
(IV) in the presence of 1,3-bis(trimethylsilyl)urea (BSU) and KI in
DMF then affords the protected pyrazolium adduct (V), which, after
full deprotection with trifluoroacetic acid in
anisole/CH.sub.2Cl.sub.2, can be isolated as the hydrogensulfate
salt by treatment with H2SO4 in i-PrOH/H.sub.2O (1, 2). Scheme 1.
The pyrazolyl urea intermediate (IV) can be prepared as follows.
Treatment of 5-amino-1-methylpyrazole (VI) with NaNO.sub.2/HCl in
water at 5.degree. C. gives the 4-nitrosopyrazole derivative (VII),
which can be reduced to the diaminopyrazole (VIII) by catalytic
hydrogenation over Pd/C in the presence of H.sub.2SO.sub.4.
Selective acylation of the 4-amino group of compound (VIII) with
phenyl chloroformate in the presence of NaOH in H.sub.2O/dioxane at
10.degree. C. then yields the phenyl carbamate (IX). After
protection of the free amine group of carbamate (IX) with
chlorotriphenylmethane in the presence of Et.sub.3N in THF, the
resulting N-trityl derivative (X)can be coupled with
N-Boc-ethylenediamine (XI) in the presence of Et.sub.3N in DMF to
afford pyrazolyl urea (IV).
Biological Activity Assay
[0063] The antibacterial activity of the CXA-201 or other compounds
can be measured by the minimum inhibitory concentrations (MIC) of
the compounds against various bacteria measured by using the broth
microdilution method performed according to Clinical and Laboratory
Standards Institute (CLSI) guidelines with modifications as
described below (CLSI guidelines can be derived from the CLSI
document M7-A8 published in January 2009: "Methods for Dilution
Antimicrobial Susceptibility Tests for Bacteria That Grow
Aerobically; Approved Standard-Eighth Edition").
[0064] To prepare for MIC testing, individual colonies can be
isolated by streaking frozen glycerol material containing
Staphylococcus or Pseudomonas spp. onto rich, non-selective,
tryptic soy agar containing 5% sheep's blood (TSAB), and incubated
at 37.degree. C. for 18-24 hrs.
[0065] On the day of testing, primary cultures can be started by
scraping off 5-10 colonies from the TSAB plates. The material can
be suspended in .about.5 mL of cation adjusted Mueller Hinton Broth
(CAMHB) in 14 mL culture tubes and can be incubated at 37.degree.
C. with aeration (200 rpm) for .about.2 hrs until the OD600 was
.gtoreq.0.1.
[0066] Inoculum cultures can be prepared by standardizing the
primary cultures to OD600=0.1 and then adding 20 .mu.L of the
adjusted primary culture per 1 mL CAMHB for Pseudomonas and CAMHB
plus 4% NaCl for MRSA so that the final inoculum density was
.about.10.sup.5 colony forming units per milliliter. Diluted
inoculum cultures can be used to inoculate 50 .mu.L per well in 96
well broth microdilution assay plates. 50 .mu.L of CAMHB that
contained compound concentrations ranging from 64-0.06 .mu.g/mL in
two-fold dilutions can also be added to the broth microdilution
assay plates for a final volume 100 .mu.L per well, therefore final
culture OD.sub.600 was approximately 0.001 and the final NaCl
concentration for the MRSA strain was 2%.
[0067] Plates can be incubated for 18-20 hours at 37.degree. C.
with aeration (200 rpm). Following incubation, growth can be
confirmed visually placing plates over a viewing apparatus (stand
with a minor underneath) and then OD.sub.600 can be measured using
a SpectraMax 340PC384 plate reader (Molecular Devices, Sunnyvale,
Calif.). Growth was defined as turbidity that could be detected
with the naked eye or achieving minimum OD.sub.600 of 0.1. MIC
values were defined as the lowest concentration producing no
visible turbidity.
[0068] The examples and illustrative embodiments described herein
are provided by way of illustration, and do not constitute
additional limitations on the scope of the claims. While some
embodiments have been shown and described in the instant
specification, the specification as ready by one of ordinary skill
in the relevant arts also discloses various modifications and
substitutions of embodiments explicitly disclosed herein. The
exemplary embodiments from the specification are not provided to
read additional limitations into the claims.
* * * * *