U.S. patent application number 14/596926 was filed with the patent office on 2015-05-07 for compositions and kits for diagnosing infections.
The applicant listed for this patent is STC.UNM. Invention is credited to Seong Won Choi, Graham Timmins, Terry Wu.
Application Number | 20150125898 14/596926 |
Document ID | / |
Family ID | 47009961 |
Filed Date | 2015-05-07 |
United States Patent
Application |
20150125898 |
Kind Code |
A1 |
Timmins; Graham ; et
al. |
May 7, 2015 |
COMPOSITIONS AND KITS FOR DIAGNOSING INFECTIONS
Abstract
The present invention is directed to the use of isotopically
labeled citrulline for diagnosing the presence or absence of a
bacterial infection in the lungs of a patient.
Inventors: |
Timmins; Graham;
(Albuquerque, NM) ; Wu; Terry; (Albuquerque,
NM) ; Choi; Seong Won; (Albuquerque, NM) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
STC.UNM |
Albuquerque |
NM |
US |
|
|
Family ID: |
47009961 |
Appl. No.: |
14/596926 |
Filed: |
January 14, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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14111066 |
Oct 10, 2013 |
|
|
|
PCT/US12/33260 |
Apr 12, 2012 |
|
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14596926 |
|
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61517072 |
Apr 13, 2011 |
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Current U.S.
Class: |
435/34 ;
435/283.1; 562/560 |
Current CPC
Class: |
C12Q 1/04 20130101; C07B
2200/07 20130101; C12Q 2304/48 20130101; C07B 59/001 20130101; C07C
275/16 20130101; C07B 2200/05 20130101; C12Q 2304/46 20130101 |
Class at
Publication: |
435/34 ; 562/560;
435/283.1 |
International
Class: |
C12Q 1/04 20060101
C12Q001/04; C07B 59/00 20060101 C07B059/00; C07C 275/16 20060101
C07C275/16 |
Claims
1-17. (canceled)
18. A composition adapted for pulmonary administration to a patient
to be diagnosed for the existence or absence of a bacterial lung
infection, said composition consisting essentially of a diagnostic
effective amount of isotopically labeled citrulline or a
pharmaceutically acceptable salt thereof, in combination with a
pharmaceutically acceptable excipient, a propellant and optionally,
a solvent and a dispersant, said composition being further adapted
for single use.
19. The composition according to claim 18 wherein said isotopically
labeled citrulline, 1 or pharmaceutically acceptable salt thereof
is present in an amount ranging from about 0.25 mg to about 10
mg.
20. The composition according to claim 18 wherein said composition
is an aerosol.
21. The composition according to claim 18 adapted for delivery
through an inhaler.
22. The composition according to claim 18 wherein said solvent is a
mixture of water and ethanol and said propellant is a CFC
substitute fluorinated hydrocarbon.
23. A composition adapted for oral administration to a patient to
be diagnosed for the existence or absence of a bacterial lung
infection, said composition consisting essentially of a diagnostic
effective amount of isotopically labeled citrulline or a
pharmaceutically acceptable salt thereof, in combination with a
pharmaceutically acceptable carrier, additive, or excipient.
24. The composition according to claim 23 having an enteric
coating.
25. The composition according to claim 23 wherein said isotopically
labeled citrulline or pharmaceutically acceptable salt thereof is
present in an amount ranging from about 0.5 mg to about 100 mg.
26. A unit dosage form kit comprising a single use container and a
diagnostically effective amount of isotopically labeled citrulline,
or a pharmaceutically acceptable salt thereof, in combination with
a pharmaceutically acceptable carrier, additive, or excipient.
27. A kit comprising at least one unit dosage of isotopically
labeled citrulline or a pharmaceutically acceptable salt thereof,
and at least one device for the delivery of the isotopically
labeled citrulline to a patient or at least one breath collection
device, or a combination of such devices.
28. The kit of claim 26 comprising a plurality of unit dosages of
isotopically labeled citrulline, and further comprising
instructions for assaying the presence or absence of a bacterial
lung infection in a patient.
29. The kit according to claim 27 wherein the device for the
delivery of the isotopically labeled citrulline is an inhaler.
30. The kit according to claim 26, wherein the breath collection
device is a bag or a vial.
31. Isotopically labeled citrulline, or a pharmaceutically
acceptable salt thereof, comprising .sup.13C, .sup.14C, .sup.17O,
.sup.18O, .sup.15N, or mixture thereof, wherein the .sup.13C,
.sup.14C, .sup.17O, .sup.18O, or .sup.15N is present in an amount
greater than natural abundance.
32. The isotopically labeled citrulline of claim 31, wherein the
citrulline is ##STR00009##
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/517,072, filed Apr. 13, 2011, the entirety of
which is incorporated herein.
TECHNICAL FIELD
[0002] This invention relates to a rapid and effective test for
detecting and diagnosing bacterial lung infections in patients
using isotopically labeled citrulline, or a pharmaceutically
acceptable salt thereof.
BACKGROUND
[0003] Bacterial infections of the lungs can be life threatening if
left untreated. For example, pneumonic tularemia can be fatal if
not properly treated. Francisella tularensis is a bacteria that can
result in a pneumonic tularemia infection if inhaled. F. tularensis
is so infectious that is has been identified by the Centers for
Disease Control as a potential biological warfare agent.
[0004] Many bacterium have more that one biovar, i.e., more than
one strain. F. tularensis has biovars, for example, Type A (Schu
S4) and Type B (holarctica). Type A results in a more severe
infection than Type B. Early detection of lung infections results
in earlier treatment intervention and better prognosis for
recovery. In addition, distinguishing between bacterial biovars
allows for health care practitioners to prescribe the most
appropriate treatment. As such, methods of detecting lung
infections, and methods of distinguishing biovars of an infection,
are needed.
SUMMARY
[0005] Methods for diagnosing the presence or absence of a
bacterial infection in the lungs of a patient are described. These
methods comprise administering an effective amount of an
isotopically-labeled citrulline, or a pharmaceutically acceptable
salt thereof, to the lungs of the patient; collecting one or more
breath, urine, whole blood, plasma, or serum samples from the
patient; and analyzing the one or more samples to determine the
amount of isotopically labeled carbon dioxide, isotopically labeled
ammonia, or a mixture thereof in said samples; said amount
indicating the presence or absence of the bacterial infection.
[0006] Compositions and kits for use with the methods of the
invention are also described.
BRIEF DESCRIPTION OF DRAWINGS
[0007] FIG. 1 depicts the amount of .sup.13C in headspace after
treatment of strains of Franscisella tularensis with ureido
.sup.13C-citrulline.
DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0008] The present invention is a breath test which can be used to
detect the presence of a bacterial infection in the lungs of a
patient. Certain bacteria have enzymes that can convert certain
compounds into cleavage or fermentation products. Certain bacteria
can convert citrolline into cleavage or fermentation products. It
is theorized that an enzyme referred to as "citrulline ureidase" is
responsible for the conversion of citrolline into cleavage or
fermentation products. Certain strains of Franscisella tularensis,
for example Type A (Schu S4), have this enzyme.
[0009] For example, citrulline is converted by citrulline ureidase
to 2,5-diamino-pentanoic acid, carbon dioxide, and ammonia
according to Scheme 1:
##STR00001##
[0010] It has now been discovered by the applicants that if
isotopically labeled citrulline is administered to a patient having
a bacterial infection, for example, a Franscisella tularensis
infection, wherein the bacteria can convert citrulline to cleavage
or fermentation products, isotopically labeled carbon dioxide
and/or isotopically labeled ammonia is generated by the conversion
of citrulline by citrulline ureidase. See. Schemes 2 and 3.
##STR00002##
##STR00003##
[0011] The applicants have also discovered that certain bacteria
can convert glycerol into cleavage or fermentation products, for
example, water, carbon dioxide, propane diol, and ethanol. See
Scheme 4. It has heretofore been discovered that if isotopically
labeled glycerol is administered to a patient having a bacterial
infection wherein the bacteria can convert glycerol to cleavage or
fermentation products, isotopically labeled water, isotopically
labeled carbon dioxide, isotopically labeled propanediol, and/or
isotopically labeled ethanol, depending on the type and location of
the isotopic label, will be produced.
##STR00004##
[0012] This isotopically labeled ammonia, carbon dioxide, water,
propanediol, and ethanol generated by the cleavage and/or
fermentation of isotopically labeled citrulline and/or isotopically
labeled glycerol can be detected in the breath, urine, whole blood,
plasma or serum samples from the patient who has been administered
isotopically labeled citrulline and/or isotopically labeled
glycerol. By measuring the amount, for example, the concentration,
of isotopically labeled cleavage or fermentation product of
citrulline, for example, isotopically labeled carbon dioxide or
isotopically labeled ammonia, it is possible to determine whether
the patient has a bacterial infection. Moreover, using the methods
described herein, it is possible to identify the biovar of the
bacterial infection. By using the methods of the invention,
bacterial infections can readily be identified and appropriate
treatment can be administered.
[0013] The present invention is directed to methods for diagnosing
the presence or absence of a bacterial infection in the lungs of a
patient comprising administering an effective amount of an
isotopically-labeled citrulline, or a pharmaceutically acceptable
salt thereof, to the lungs of the patient; collecting one or more
breath, urine, whole blood, plasma, or serum samples from the
patient; and analyzing the one or more samples to determine the
amount of isotopically labeled carbon dioxide, isotopically labeled
ammonia, or a mixture thereof in said samples; said amount
indicating the presence or absence of the bacterial infection.
[0014] The isotopically labeled citrulline, or pharmaceutically
acceptable salt thereof, used within the scope of the invention
comprises .sup.13C, .sup.14C, .sup.17O, .sup.18O, .sup.15N,
.sup.2H, or mixture thereof, wherein the .sup.13C, .sup.14C,
.sup.17O, .sup.18O, .sup.15N, .sup.2H, is present in an amount
greater than natural abundance. In some embodiments, the
citrulline, or pharmaceutically acceptable salt thereof, includes a
plurality of isotopic labels. Preferably, the isotopic label is
.sup.13C, .sup.17O, .sup.18O, .sup.15N, or a mixture thereof. More
preferably, the isotopic label is .sup.13C or .sup.15N. Even more
preferably, the isotopic label is .sup.13C.
[0015] Isotopically labeled citrulline and isotopically labeled
glycerol can be prepared according to methods known in the art. For
example, one isotopically labeled citrulline of the invention can
be prepared according to the following Scheme 5.
##STR00005##
[0016] According to preferred embodiments of the invention, the
isotopically labeled citrulline is delivered to the lungs of the
patient by a pulmonary route of administration. For example, the
isotopically labeled citrulline can be administered via aerosol
inhalation or intratracheal instillation. Alternatively, the
isotopically labeled citrulline can be administered orally. Those
skilled in the art will readily understand that the amount of time
between citrulline administration and bodily fluid collection will
vary depending on the mode of administration of the citrulline.
[0017] In preferred embodiments of the invention, a breath sample
is collected from the patient. The breath sample can be taken for a
predetermined time to ensure that an adequate sample can be
collected for analysis.
[0018] Predetermined breath sample volumes can also be collected to
ensure that an adequate sample is collected for analysis.
[0019] In preferred embodiments of the invention, the breath
sample, or other bodily fluid sample, will be analyzed for the
presence of isotopically labeled carbon dioxide. Preferably, the
isotopically labeled carbon dioxide is .sup.13C-labeled carbon
dioxide.
[0020] In other embodiments, the breath sample, or other bodily
fluid sample, will be analyzed for the presence of isotopically
labeled ammonia. Preferably, the isotopically labeled ammonia is
.sup.15N-labeled ammonia.
[0021] In certain embodiments, the breath sample, or other bodily
fluid sample, will be analyzed for the presence of isotopically
labeled carbon dioxide and isotopically labeled ammonia.
Preferably, the isotopically labeled carbon dioxide is
.sup.13C-labeled carbon dioxide and the isotopically labeled
ammonia is .sup.15N-labeled ammonia.
[0022] In preferred analytic methods, the analysis step of the
methods of the invention comprises comparing at least one ratio of
isotopically labeled carbon dioxide, isotopically labeled ammonia,
or a mixture thereof to non-isotopically carbon dioxide,
non-isotopically labeled ammonia, or a mixture thereof in the
breath sample of the patient after the administration of the
isotopically-labeled citrulline to a control ratio of isotopically
labeled carbon dioxide, isotopically labeled ammonia, or a mixture
thereof to non-isotopically carbon dioxide, non-isotopically
labeled ammonia, or a mixture thereof in the breath sample of the
patient prior to the administration of the isotopically-labeled
citrulline.
[0023] More preferably, the analysis step of the methods of the
invention comprises comparing at least one ratio of isotopically
labeled carbon dioxide to non-isotopically carbon dioxide in the
breath sample of the patient after the administration of the
isotopically-labeled citrulline to a control ratio of isotopically
labeled carbon dioxide to non-isotopically carbon dioxide in the
breath sample of the patient prior to the administration of the
isotopically-labeled citrulline.
[0024] The methods of the invention are useful in identifying, for
example, Francisella tularensis, and in particular, biovar A of
Francisella tularensis, in the lungs of a patient or subject. F.
tularensis presence can result in the development of pneumonic
tularemia in a patient. Specifically, the present invention can be
used to diagnose the presence of Francisella tularensis by the
administration of a safe and effective amount of isotopically
labeled citrulline and/or isotopically labeled glycerol, or a
pharmaceutically acceptable salt of the citrulline and/or the
glycerol, to a subject and then detecting the amount, for example,
the concentration, of at least one of the cleavage or fermentation
products of the isotopically labeled citrulline and/or isotopically
labeled glycerol.
[0025] In the case of citrulline, detection of isotopically labeled
ammonia and/or isotopically labeled carbon dioxide in the exhaled
breath of the subject after a suitable period of time after
administration (depending upon the route of administration) can
provide diagnostic information about the existence of bacteria, for
example, F. tularensis, or biovar Type A of F. tularensis in the
subject's lungs.
[0026] In the case of glycerol, isotopically labeled carbon
dioxide, isotopically labeled water, isotopically labeled
propanediol, isotopically labeled ethanol or mixtures thereof,
measured in the exhaled breath of the patient or subject, after a
suitable time period has elapsed, can also provide diagnostic
information about the existence of bacteria, for example, F.
tularensis, or biovar Type A of F. tularensis in the subject's
lungs.
[0027] In an alternative embodiment, isotopically labeled
citrulline and/or isotopically labelled glycerol are administered
to the subject to be tested for bacterial infection and, depending
on the existence of isotopically labeled cleavage or fermentation
products (or their absence) measured in the exhaled breath of the
subject, diagnostic information may be used to determine whether or
not there is a bacterial infection. Methods of the invention can be
used to determine if F. tularensis, and in particular a biovar Type
A or biovar Type B of F. tularenesis is present in the lungs of the
subject.
[0028] In an aspect of the invention, in order to determine that an
infection is present in the lungs, measurements of isotopically
labeled nitrogen-15, carbon-13, hydrogen-2 and/or oxygen-17 or
oxygen-18 in the breath of the tested subject are made and compared
with concentrations of the naturally occurring nitrogen-14,
carbon-12, hydrogen-1 and/or oxygen-16 in the breath of the tested
subject for particular cleavage products. Preferably, at least a
majority of the exhaled breaths, and most preferably every exhaled
breath, is sampled for a given time period or until the
determination of the level of activity has reached a preset
accuracy.
[0029] A graph or curve may be generated showing the ratio of the
isotopically labeled cleavage or fermentation product ("the
isotopically labeled element") to the naturally occurring cleavage
or fermentation product in the breath of the tested subject, as a
function of time. A curve showing an increase in the ratio of the
isotopically labeled element to non-isotopically labeled element
over time is evidence of the existence of a bacterial infection.
Using methods of the invention, a F. tularensis infection, in
particular biovar Type A of F. tularensis, for example, can be
detected.
[0030] An exemplary graph generated by comparing the ratio of
isotopic nitrogen, carbon, oxygen and/or hydrogen in cleaved
products in the exhaled breath of the patient can be made. By way
of example, a graph of nitrogen-15/nitrogen-14 in ammonia,
carbon-13/carbon-12 and/or oxygen-16/oxygen-18 or oxygen-17 in
carbon dioxide, hydrogen-2/hydrogen-1 and/or oxygen-16/oxygen-18 or
oxygen-17 in water, carbon-13/carbon-12 and/or oxygen-16/oxygen-18
or oxygen-17 in propanediol and/or ethanol in the exhaled breath of
the subject to be diagnosed is/are made. The concentrations or
amounts (ratio) of isotopically labeled elements in cleavage
products are compared to a standard concentration (ratio) of those
same cleavage products in a healthy patient and a curve is
generated. From the curve, the presence or absence of F. tularensis
biovar A may be determined or diagnosed directly. In the absence of
F. tularensis biovar A (determined from the analysis above) and the
presence of other symptoms or conditions of pneumonia tularemia,
the presence of F. tularensis biovar B may determined or diagnosed
indirectly.
[0031] A curve may be fitted to these measured concentrations and
is then analyzed, preferably by determining the rate of rise of the
curve. Such an analysis (rising rate) indicates the level of
activity of F. tularensis, in particular biovar A in the subject,
which can be used to diagnose the presence and extent of infection
of F. tularensis, biovar A in the lungs of the patient.
Alternatively, the absence of a rise in measured concentration in
comparison to standard, along with symptoms of pneumonic tularemia
will indicate the presence of F. tularensis, biovar B. This same
approach may be used, with modification, to determine the
effectiveness of therapy of a F. tularensis infection and the
prognosis for inhibition and/or a cure of infection.
[0032] Examples of appropriate labels for the substrate, and hence
for the cleavage product or products, are those which can be
detected by an appropriate measuring instrument, but which are
substantially not harmful or toxic to the subject including, but
not limited to, carbon-13 or carbon-14, oxygen-18 (oxygen-17) or
nitrogen-15, hydrogen-2 isotope-labelling.
[0033] An isotope is a form of an element, such as carbon, with a
specific mass. For example, carbon-12 has a mass of 12 atomic mass
units. In the present invention, the naturally more abundant
isotope of each of these elements is at least partially replaced by
a less abundant isotope. For example, the naturally more abundant
carbon-12 atoms could be at least partially replaced by the less
abundant carbon-13 atoms, permitting the cleavage product or
products which carry the label to be more easily detected, since
the less abundant isotope can be distinguished from the naturally
more abundant isotope. Furthermore, the advantage of certain
isotopes such as carbon-13 is that they are stable, so that they
are not radioactive, unlike isotopes such as carbon-14. Therefore,
preferably stable, non-radioactive isotopes such as carbon-13 are
used as labels.
[0034] Compositions for oral administration or pulmonary
administration are as otherwise described herein. Oral compositions
include powders or granules, suspensions or solutions in water or
non-aqueous media, sachets, capsules or tablets. Thickeners,
diluents, flavorings, dispersing aids, emulsifiers or binders may
be desirable. Such compositions adapted for oral administration can
also include an enteric coating. Compositions for pulmonary
administration include a pharmaceutically acceptable carrier,
additive or excipient, as well as a propellant and optionally, a
solvent and/or a dispersant to facilitate pulmonary delivery to the
subject.
[0035] Following the step of administering the isotopically labeled
citrulline and/or isotopically labeled glycerol to the subject, the
exhaled breath of the subject is analyzed to detect an amount of
isotopically labeled cleavage product or products as otherwise
described herein, which indicate the presence of bacteria, for
example, F. tularensis in the lungs of the subject. The amount of
isotopically labeled product or products are detected by analyzing
a gas sample of the exhaled breath of the subject with a measuring
instrument. Such a gas sample can be obtained in a number of ways
including, but not limited to, having the subject exhale or blow
into a tube connected to the measuring instrument. Preferably, a
nasal cannula is used. Such a cannula includes a section of tubing,
usually plastic, with two prongs. Each prong is inserted into a
nostril and the cannula is then connected to the measuring
instrument. As the patient exhales through the nose, the exhaled
air flows through the cannula to the measuring instrument.
[0036] The type of measuring instrument used to detect the
isotopically labeled product or products depends upon the type of
label. For example, if a carbon-13 isotopically-labelled substrate
is used, the carbon-13 isotopically-labelled cleavage product or
products can be detected by using a measuring instrument including,
but not limited to a mass spectrometer or a gas analyzer, which is
sensitive to the carbon-13 isotope. This is true for the other
labels used (Nitrogen-15, oxygen-18 or H-2). By way of nonlimiting
example (and as applied to the other elements by analogy), the
ratio of the concentration of carbon-13 isotopically-labelled
cleavage product or products to the concentration of carbon-12
cleavage product or products is then determined. Since carbon-12 is
the more abundant isotope in nature, carbon-12 atoms are more
abundant in unlabelled molecules. Thus, a higher
carbon-13/carbon-12 ratio indicates a higher concentration of the
carbon-13 isotopically-labelled cleavage product or products, which
positively indicates the presence of F. tularensis in the lungs of
the subject.
[0037] In certain preferred aspects of the invention, at least one
of the cleavage products is carbon-13 isotopically-labelled carbon
dioxide. Examples of measuring instruments which can be used with
carbon-13 isotopically-labelled carbon dioxide include, but are not
limited to, an infrared spectrometer. These infrared spectrometers
are well known in the art, and have the advantage of being both
rapid and accurate, as well as sensitive. Examples of such infrared
spectrometers are disclosed in U.S. Pat. No. 5,063,275, herein
incorporated by reference.
[0038] Alternatively, an analytical assay is described which is
based on the use of C-13 labeled expired CO.sub.2 as measured in
the present assay. In this method isotope ratio mass spectroscopy
(IRMS) is used as a detection method for C-13, a non-radioactive
isotope that occurs naturally in food and animal tissues.
Non-dispersive infrared spectroscopy (NDIRS) analysis and analysis
methods which are well known in the art may be employed. The test
protocol is as follows: isotopically labeled citrulline and/or
glycerol is administered a subject or patient. The administration
may be by oral or preferably by pulmonary administration, using an
inhaler or other device adapted to deliver an effective amount
(generally, an effective amount within the range of about 0.1 to
about 10 mg, about 0.25 to about 5 mg, about 0.5 to about 2.5 mg.
of isotopically labeled citrulline and/or glycerol, including both
isotopically labeled citrulline and glycerol to the lungs of the
patient or subject. After an appropriate period of time to allow
any bacteria, for example, F. tularensis, within the lungs of the
subject to convert the isotopically labeled citrulline and/or
isotopically labeled glycerol to a cleavage product. Breath samples
are collected before the dose and then a number of hours after
administration of the citrulline and/or glycerol reflecting the
conversion of substrate to cleavage product, and the isotopically
labeled cleavage product in expired breath from the subject will be
measured by an IRMS method.
[0039] Advantages of this test include, but are not, limited to the
following: it is practical, sensitive and specific; the validity of
the test is not influenced by stress, exercise, hormone imbalances,
or some drugs and medications it is a non-invasive method; it is
simple to perform and can be readily used in physicians' offices or
medical laboratories; it is safe since carbon-13 is a naturally
occurring isotope found in all carbon-containing substances; it
involves no radioactivity, and may be used in children and
women.
[0040] Alternatively and preferably, at least one of the cleavage
products is nitrogen-15 isotopically labeled ammonia. Of course,
both carbon-13 isotopically labeled carbon dioxide and nitrogen-15
isotopically-labelled ammonia could be present, providing that the
substrate has both labels. Both ammonia and carbon dioxide have the
advantage of being molecules which are present in the exhaled
breath of the subject.
[0041] In any case, the measuring instrument used to detect the
cleavage product or products desirably have a number of
characteristics. The measuring instrument must be able to measure
the concentration of the product or products extremely rapidly.
Furthermore, either the measuring instrument itself, or an
associated device, must be able to perform the associated analysis,
including both the fitting of the curve and the analysis of the
curve. Such analyses must be performed rapidly. Preferably, the
measuring instrument, alone or in conjunction with the associated
device, should be able to measure the concentration and perform the
associated analysis within about 10 seconds, and most preferably
within about 3 seconds, particularly if substantially every exhaled
breath of the subject is to be analyzed.
[0042] The term "suitable time period" refers to the length of time
required for a cleavage product or products to form and to be
exhaled in the breath of the subject. Thus, a number of events must
occur. First, the administered isotopically labeled citrulline
and/or isotopically labeled glycerol must be accessible to the
bacterial, for example, F. tularensis. Then, the administered
isotopically labeled citrulline and/or isotopically labeled
glycerol must be cleaved or acted on by citrulline ureidase or
glycerol fermentation of F. tularensis to form a cleavage product
or products. The cleavage product or products must be produced in
the lungs or alternatively, in the blood such that the cleavage
product makes its way to the lungs in sufficient quantities. Next,
the cleavage product or products must be exhaled in the breath of
the subject. Finally, the presence of the cleavage product or
products must be detected in the exhaled breath.
[0043] Furthermore, the "suitable time period" should be such that
the curve of measured concentrations of cleavage product or
products in the exhaled breath of the subject is substantially
linear. Generally, the concentration will rise rapidly initially so
that the fitted curve is substantially linear, and will then
plateau after a period of time (preferably no more than about 40-70
minutes), as the process of formation and exhalation of cleavage
product or products reaches a steady state. Eventually, as the
administered urea is cleaved, the concentration will decrease. The
analysis is preferably performed before the curve of measured
values reaches a plateau.
[0044] This fitting and analysis of a curve of measured
concentrations is useful for rapidly establishing the existence of
a bacterial infection, for example F. tularensis, in particular a
biovar A F. tularensis infection, in the lungs of the subject. The
method of the present invention allows repeated breath samples to
be rapidly obtained and then maximizes both the speed and the
accuracy of analysis by fitting the measured values to a curve and
then calculating the rate of increase of the curve, which is the
derivative and which provides information as the existence and
extent of infection.
[0045] Any method for identifying the concentration of isotopically
labeled ammonia, isotopically labeled carbon dioxide (from cleavage
of citrulline) or isotopically labeled ammonia, isotopically
labeled carbon dioxide, isotopically labeled propanediol,
isotopically labeled ethanol (from fermentation of glycerol) can be
used to determine the existence (or absence) of a bacterial
infection, for example, a F. tularensis infection, in the lungs of
a patient or subject. The measurement of isotopically labeled gas
(or liquid propane diol and/or ethanol in blood) as a cleavage
product by action of the bacteria, for example, F. tularensis, on
isotopically labeled citrulline and/or isotopically labeled
glycerol directly evidences the existence (or absence) of a biovar
of the bacteria, for example, biovar A F. tularensis in the lungs
of the subject or the existence (or absence) of biovar B F.
tularensis coupled with other symptoms of or analysis of F.
tularensis.
[0046] In the present invention it is preferred to determine a
ratio of an isotopically labeled element (carbon, nitrogen, oxygen,
hydrogen) to a non-isotopically labeled element in a cleavage
product (preferably gas) being analyzed. For example, if
isotopically labeled ammonia is being measured as a cleavage
product pursuant to citrulline administration, a ratio of
nitrogen-15 to nitrogen-14 in ammonia obtained from the breath of a
subject is determined. This may be determined readily using mass
spectroscopy or infrared laser spectroscopy. For example, in
preferred aspects of the invention, a ratio of nitrogen-15 to
nitrogen-14 in ammonia exhaled by a subject to be diagnosed before
administration of isotopically labeled citrulline is determined as
a baseline ratio or standard. This ratio is generally below 1%.
[0047] Specifically, an exemplary method of analysis involves the
following steps. A plurality of samples of exhaled breath of the
subject is collected rapidly, on the order of one sample about
every few seconds or so, preferably such that at least a majority,
and most preferably substantially all of the exhaled breaths of the
subject are sampled. Next, the concentration of a cleavage product
is measured and the concentration of an isotopically labeled
element, such as nitrogen-15, carbon-13, oxygen-17 or oxygen-18 or
hydrogen-2 is compared with its naturally occurring counterpart
(e.g. respectively, nitrogen-14, carbon-12, oxygen-16 and
hydrogen-1) in the breath of the subject. Where the ratio of
isotopically-labeled element to naturally occurring element is 0 or
close to zero, then bacteria, for example, F. tularensis is not
present. In cases where the ratio of isotopically-labeled element
to naturally occurring element increases as a function of time,
then the existence of bacteria, for example, F. tularensis is made
out. A curve is fitted to the measured concentrations. If the ratio
remains flat at the x-axis (essentially 0 or close to 0) as a
function of time, the presence of bacteria, for example, F.
tularensis is ruled out. The rate of rise of the curve may
calculated by calculating the integral or by derivation
(calculation of the derivative), preferably after the measurement
of the concentration of cleavage product(s) in each sample. The
analysis of the curve indicates the level of bacteria, for example,
F. tularensis activity in the lungs of the subject. A rapid rise in
the measured concentrations (a steeper curve), would evidence a
high level of bacteria, for example, F. tularensis activity in the
subject, whereas a slower rise in the measured concentrations (a
shallower curve) would evidence a lower level of bacteria, for
example, F. tularensis activity. Analogous approaches for providing
the same information are also contemplated.
[0048] For example, in an analogous manner, if isotopically labeled
citrulline and isotopically labeled glycerol (preferably having
different elements labeled so that cleavage products of citrulline
will be distinguishable from cleavage products of glycerol) are
used, these can provide evidence of the existence (or absence) of
F. tularensis produce concentrations of cleavage products which
evidence that these substrates are being cleaved.
[0049] The calculation of a derivative has a number of advantages
over other methods of analysis, such as the calculation of an
integral. First, the calculation of the derivative does not require
a reference breath sample to be obtained before isotopically
labeled citrulline or isotopically labeled glycerol is administered
to the subject. Since the derivative represents the rate of
increase of the measured concentrations of a cleavage product or
products, the starting concentration of that cleavage product or
products is unimportant. However, the initial concentration of the
cleavage product or products in the reference breath sample is
important for the proper calculation of the integral, since such an
initial concentration represents a background value which must be
subtracted from the measured concentrations after administration of
the substrate.
[0050] After the resultant measurement has reached a predetermined
level of accuracy, or after a predetermined time period has
elapsed, no more samples are collected.
[0051] The present method utilizing a breath assay has a number of
advantages. For example, the exhaled breath of the subject can be
analyzed in real time; that is, there is relatively little delay
between the time the bacterial activity, for example, F. tularensis
activity takes place, and the time such activity is measured.
Second, the samples of exhaled breath are obtained rapidly and are
analyzed immediately in a manner which substantially increases the
accuracy of the results. Third, since multiple samples preferably
are obtained, the accuracy of the test is increased. Fourth, there
is less statistical error since many samples are collected. Fifth,
since samples are preferably collected until a preset level of
accuracy is reached, ambiguous results can be substantially
eliminated, preventing the need for repeating the test.
[0052] The readout of isotopic ratios can be performed by sensitive
gas mass spec analysis, but also laser spectroscopy techniques
which may allow for more compact and portable devices.
[0053] Isotopically labeled citrulline and/or isotopically labeled
glycerol may be administered by oral or preferably, by a pulmonary
route of administration. Most preferably, the isotopically labeled
citrulline and/or isotopically labeled glycerol is inhaled by the
patient.
[0054] The pharmaceutical compositions of the invention for
pulmonary administration are usually used as an inhalant. The
compositions can be formed into dry powder inhalants, inhalant
suspensions, inhalant solutions, encapsulated inhalants and like
known forms of inhalants. Such forms of inhalants can be prepared
by filling the pharmaceutical compositions of the invention into an
appropriate inhaler such as a metered-dose inhaler, dry powder
inhaler, atomizer bottle, nebulizer etc. before use. Of the above
forms of inhalants, powder inhalants may be preferable.
[0055] When the pharmaceutical compositions of the invention are
used in the form of a powder, the mean particle diameter of the
powder is not especially limited but, in view of the residence of
the particles in the lungs, is preferably in the range of about 0.1
to 20 .mu.m, and particularly about 1 to 5 .mu.m, because that size
results in deep penetration of the particles within the lungs of
the subject. Although the particle size distribution of the powder
pharmaceutical compositions of the invention are not particularly
limited, it is preferable that particles having a size of about 25
.mu.m or more account for not more than about 5% of the particles,
and preferably, 1% or less to maximize delivery into the lungs of
the subject.
[0056] The pharmaceutical compositions in the form of a powder of
the invention can be produced by, for example, the
drying-micronization method, the spray drying method and the
like.
[0057] According to the drying-pulverization method, the
pharmaceutical compositions in the form of a powder can be prepared
by drying an aqueous solution (or aqueous dispersion) containing a
pharmacologically active substance and carrageenan and
microparticulating the dried product. Stated more specifically,
after dissolving (or dispersing) carrageenan in an aqueous medium,
a pharmacologically active substance is added and dissolved (or
dispersed) by stirring using a homogenizer, etc. to give an aqueous
solution (or aqueous dispersion). The aqueous medium may be water
alone or a mixture of water and a lower alcohol. Examples of usable
lower alcohols include methanol, ethanol, 1-propanol, 2-propanol
and like water-miscible alcohols. If glycerol is to be used as a
substrate, ethanol may be used, but must be eliminated from the
final formulation so as not to jeopardize measurements of ethanol
from glycerol fermentation. After the obtained aqueous solution (or
aqueous dispersion) is dried by blower, lyophilization, etc., the
resulting product is pulverized or microparticulated into fine
particles using jet mills, ball mills or like devices to give a
powder having the above mean particle diameter. If necessary,
additives as mentioned above may be added in any of the above
steps.
[0058] According to the spray-drying method, the pharmaceutical
compositions in the form of a powder of the invention can be
prepared, for example, by spray-drying an aqueous solution (or
aqueous dispersion) containing a pharmacologically active
substances and excipients, additives or carriers for
microparticulation. The aqueous solutions (or aqueous dispersion)
can be prepared following the procedure of the above
drying-micronization method. The spray-drying process can be
performed using a known method, thereby giving a powdery
pharmaceutical composition in the form of globular particles with
the above-mentioned mean particle diameter.
[0059] The inhalant suspensions, inhalant solutions, encapsulated
inhalants, etc. can also be prepared using the pharmaceutical
composition in the form of a powder produced by the
drying-micronization method, the spray-drying method and the like,
or by using carrageenan and a pharmacologically active substance
that can be administered via the lungs, according to known
preparation methods.
[0060] Furthermore, the inhalant comprising the pharmaceutical
compositions of the invention are preferably used as an aerosol.
The aerosol can be prepared, for example, by filling the
pharmaceutical composition of the invention and a propellant into
an aerosol container. If necessary, dispersants, solvents and the
like may be added. The aerosols may be prepared as 2-phase systems,
3-phase systems and diaphragm systems (double containers). The
aerosol can be used in any form of a powder, suspension, solution
or the like.
[0061] Examples of usable propellants include liquefied gas
propellants, compressed gases and the like. Usable liquefied gas
propellants include, for example, fluorinated hydrocarbons (e.g.,
CFC substitutes such as HCFC-22, HCFC-123, HFC-134a, HFC-227 and
the like), liquefied petroleum, dimethyl ether and the like. Usable
compressed gases include, for example, soluble gases (e.g., carbon
dioxide, nitric oxide), insoluble gases (e.g., nitrogen) and the
like.
[0062] The dispersant and solvent may be suitably selected from the
additives mentioned above. The aerosol can be prepared, for
example, by a known 2-step method comprising the step of preparing
the composition of the invention and the step of filling and
sealing the composition and propellant into the aerosol
container.
[0063] As a preferred embodiment of the aerosol according to the
invention, the following aerosol can be mentioned: Examples of the
compounds to be used include isotopically labeled citrulline,
isotopically labeled glycerol or mixtures thereof. Excipients and
additives include carageenan, and those mainly comprising
iota-carrageenan are preferable. As propellants, fluorinated
hydrocarbons such as HFC-134a, HFC-227 and like CFC substitutes are
preferable. Examples of usable solvents include water, ethanol,
2-propanol and the like. Water and ethanol are particularly
preferable for citrulline and should be minimized or avoided when
glycerol is included. In particular, a weight ratio of water to
ethanol in the range of about 0:1 to 10:1 may be used for
citrulline formulation. Formulations which comprise glycerol should
substitute isopropanol for ethanol to avoid any impact on analysis
(glycerol is fermented to ethanol in certain aspects of the
invention).
[0064] The aerosol of the invention may contain carrageenan in an
amount of about 0.01 to 10.sup.4 wt. % (preferably about 0.1 to
about 10.sup.3 wt. %), propellant in an amount of about 10.sup.2 to
10.sup.7 wt. % (preferably about 10.sup.3 to 10.sup.6 wt. %),
solvent in an amount of about 0 to 10.sup.6 wt. % (preferably about
10 to 10.sup.5 wt. %), and dispersant in an amount of 0 to 10.sup.3
wt. % (preferably about 0.01 to 10.sup.2 wt. %), relative to the
weight of pharmacologically active substance.
[0065] The pharmaceutical compositions of the invention are safe
and effective for use in the diagnostic methods according to the
present invention. Although the dosage of the composition of the
invention may vary depending on the type of active substance
administered (isotopically labeled citrulline, isotopically labeled
glycerol or mixtures thereof) as well as the nature (size, weight,
etc.) of the subject to be diagnosed, the composition is
administered in an amount effective for allowing the
pharmacologically active substance (substrate) to be
cleaved/fermented to cleavage products to be measured. For example,
the composition is preferably administered such that the active
ingredient can be given to a human adult in a dose of about 0.001
to about 100 mg, about 0.01 mg to about 25 mg, about 0.05 mg to
about 15 mg, about 0.1 mg to about 10 mg, about 0.5 mg to about 5
mg, about 1 to about 3 mg, administered in a single dose.
[0066] The form of the pharmaceutical compositions of the invention
such as a powder, solution, suspension etc. may be suitably
selected according to the type of substance to be administered and
the action of a target enzyme on the isotopically labeled active
compound(s).
[0067] As an administration route, direct inhalation via the mouth
using an inhaler is usually preferable. Since the pharmaceutical
compositions of the invention allow direct local administration
into the airways and in particular, directly to pulmonary tissue,
the active substance contained therein produces immediate effects.
Furthermore, the composition is formulated as an immediate release
product so that cleavage and analysis can begin soon after
administration.
[0068] The present invention allows rapid diagnosis of a bacterial
infection, for example, a F. tularensis infection, in a patient or
subject. The diagnosis is rapid and effective and displays a number
of ancillary advantages as well, include direct diagnosis of
perhaps a more virulent form of the bacteria, for example, biovar A
F. tularensis, The present invention also allows the indirect
diagnosis of, for example, F. tularensis biovar B, when coupled
with other diagnostic and/or analytical methods.
[0069] The present invention also allows for the monitoring of a
bacterial infection, for example, an F. tularensis infection, in
the lungs of a patient, during therapy to determine the impact that
an antibiotic regimen is having on resolving the bacterial
infection.
[0070] The present invention represents a rapid, one-stop, easily
administered test. The present approach of obtaining a sample from
the lungs of the patient and processing the sample to determine the
existence of a bacterial infection, for example, an F. tularensis
infection, is laborious, time consuming and not cost effective. The
prior art approach creates logistical issues, especially given that
the risk element and principal use of the technology is anticipated
to be to identify bioweaponized bacterial infections, for example,
an F. tularensis infection. The present diagnostic test would be
administered in a manner which would obviate the need for precise
skill (compared to PCR and other diagnostic approaches) and would
reduce/remove laboratory error/chain of custody concerns and the
increased likelihood of infection of laboratory personnel. The
present systems allow faster treatment of infected and potentially
infectious patients. Finally, administration of a tracer tablet and
collecting breath or urine afterwards needs lower level medical
skills, making widespread testing by paramedics possible. Samples
can easily be mailed to central facilities.
[0071] The present invention allows for the monitoring of therapy
for a bacterial infection, for example, an F. tularensis infection.
This is potentially quite useful for "in the field" applications.
Thus, the present method not only can identify a bacterial
infection, for example, an F. tularensis infection, with great
accuracy, but can monitor the progression of therapy so that steps
can be taken to modify a therapeutic regimen (for example, by
adding an additional antibiotic or placing the patient on
alternative therapy) for a more successful outcome.
[0072] In preferred embodiments, breath samples from a patient who
has been administered isotopically labeled citrulline can be
analyzed for isotopically labeled carbon dioxide and/or
isotopically labeled ammonia after less than 8 hours post
isotopically labeled citrulline administration. Preferably,
analysis can be performed after less than 6 or 4 hours post
isotopically labeled citrulline administration. Even more
preferably, analysis can be performed after less than 2 or 3 hours
post isotopically labeled citrulline administration. Most
preferably, analysis can be performed after less than 1 hour post
isotopically labeled citrulline administration. In most preferred
embodiments, analysis can be performed after less than 45 minutes,
30 minutes, 20 minutes, 15 minutes, 10 minutes, or 5 minutes post
isotopically labeled citrulline administration.
[0073] Also within the scope of the invention are kits useful for
practicing the methods of the invention. Kits according to the
invention comprise at least one unit dosage of isotopically labeled
citrulline or a pharmaceutically acceptable salt thereof, and at
least one device for the delivery of the isotopically labeled
citrulline to a patient or at least one breath collection device or
a combination of such devices. Kits of the invention can include a
unit dosage form comprising a single use container and a
diagnostically effective amount of isotopicaly labeled citrulline,
or a pharmaceutically acceptable salt thereof, in combination with
a pharmaceutically acceptable carrier, additive, or excipient. The
delivery devices used within the kits of the invention can be any
one or more of the delivery devices described herein or known to
those skilled in the art. Preferred delivery devices include
inhalers.
[0074] Breath collection devices for use with the kits of the
invention include any of the collection devices described herein or
known to those skilled in the art. Preferred collection devices
include bags and vials.
[0075] Kits of the invention can optionally include instructions
for using the kits of the invention. These instructions will
preferably describe how to use the kit to assay for the presence or
absence of a bacterial lung infection in a patient.
[0076] All patents and publications referenced or mentioned herein
are indicative of the levels of skill of those skilled in the art
to which the invention pertains, and each such referenced patent or
publication is hereby incorporated by reference to the same extent
as if it had been incorporated by reference in its entirety
individually or set forth herein in its entirety. Applicants
reserve the right to physically incorporate into this specification
any and all materials and information from any such cited patents
or publications.
[0077] The specific methods and compositions described herein are
representative of preferred embodiments and are exemplary and not
intended as limitations on the scope of the invention. Other
objects, aspects, and embodiments will occur to those skilled in
the art upon consideration of this specification, and are
encompassed within the spirit of the invention as defined by the
scope of the claims. It will be readily apparent to one skilled in
the art that varying substitutions and modifications may be made to
the invention disclosed herein without departing from the scope and
spirit of the invention. The invention illustratively described
herein suitably may be practiced in the absence of any element or
elements, or limitation or limitations, which is not specifically
disclosed herein as essential. The methods and processes
illustratively described herein suitably may be practiced in
differing orders of steps, and that they are not necessarily
restricted to the orders of steps indicated herein or in the
claims.
[0078] As used herein and in the appended claims, the singular
forms "a," "an," and "the" include plural reference unless the
context clearly dictates otherwise. Thus, for example, a reference
to "a host cell" includes a plurality (for example, a culture or
population) of such host cells, and so forth. Under no
circumstances may the patent be interpreted to be limited to the
specific examples or embodiments or methods specifically disclosed
herein. Under no circumstances may the patent be interpreted to be
limited by any statement made by any Examiner or any other official
or employee of the Patent and Trademark Office unless such
statement is specifically and without qualification or reservation
expressly adopted in a responsive writing by Applicants.
[0079] The terms and expressions that have been employed are used
as terms of description and not of limitation, and there is no
intent in the use of such terms and expressions to exclude any
equivalent of the features shown and described or portions thereof,
but it is recognized that various modifications are possible within
the scope of the present invention.
[0080] The following terms shall be used to describe the present
invention. In instances where a term is not defined specifically,
that term shall be accorded the ordinary meaning ascribed to the
term by those of ordinary skill in the art when used within
context.
[0081] The specific methods and compositions described herein are
representative of preferred embodiments and are exemplary and not
intended as limitations on the scope of the invention. Other
objects, aspects, and embodiments will occur to those skilled in
the art upon consideration of this specification, and are
encompassed within the spirit of the invention as defined by the
scope of the claims. It will be readily apparent to one skilled in
the art that varying substitutions and modifications may be made to
the invention disclosed herein without departing from the scope and
spirit of the invention. The invention illustratively described
herein suitably may be practiced in the absence of any element or
elements, or limitation or limitations, which is not specifically
disclosed herein as essential.
[0082] The term "patient or subject" refers to a human or
domesticated animal, on whom the method of diagnosing an infection
of the lungs is performed.
[0083] The term "natural abundance" refers to the amount of an
isotope, for example, .sup.13C, .sup.14C, .sup.17O, .sup.18O, and
.sup.15N, as naturally found on earth. Isotopes present in the
isotopically labeled compounds of the invention are understood to
have been incorporated using synthetic techniques known in the art.
For example, the natural abundance of .sup.13C is about 1.1%.
.sup.14C exists in nature in only trace amounts. The natural
abundance of .sup.18O is about 0.2%. The natural abundance of
.sup.17O is about 0.04%. The natural abundance of .sup.15N is about
0.36%.
[0084] As used herein, "unit dosage" refers to a pre-determined
amount of a pharmaceutical composition or compound of the
invention.
[0085] The term "pneumonic tularemia" is used to describe a
particular disease state or condition caused by the infectious
agent Francisella tularensis, which is a small, rod-shaped
nonmotile bacteria. F. tularensis has been found in over 100
species of mammals, birds, amphibians, arthropods, and even fish,
and the disease occurs throughout North American and Eurasia. It is
most prevalent in the western and south-central parts of the
country. Typically, a few hundred cases of tularemia are reported
in the U.S. every year. Although relatively rare in nature,
pneumonic tularemia is feared to be a disease caused by
bioweaponized F. tularensis.
[0086] The main vectors of F. tularensis are small mammals such as
mice, moles, squirrels, rabbits and hares. Small mammal vectors
usually acquire the organism through the bite of an infected tick
or other arthropod or by contact with contaminants in the
environment, noting that the microbe can survive for a number of
weeks outside of a living host. In humans, infection is usually
caused by bites from Dermacentor or Amblyomma ticks in summer and
from contact with rabbit carcasses in winter, but other modes of
transmission occur, especially contact with contaminated water, air
or soil.
[0087] There are several different types of tularemia, which vary
in presentation and severity depending on the method of acquisition
and the dose and virulence of the specific infecting organisms.
Typically, tularemia is divided into six forms: ulceroglandular,
oculoglandular, glandular, oropharyngeal, typhoidal and pneumonic.
Of these, typhoidal tularemia is the most serious form.
[0088] Pneumonic tularemia commonly develops by inhalation of F.
tularensis, but can occur as a consequence of ulceroglandular
tularemia from a bite by a tick or from abrasion, as a secondary
disease state. Pneumonic tularemia may also develop as a
consequence of typhoid tularemia (septicemia tularemia), also as a
secondary disease state.
[0089] Because F. tularensis can exist in aerosolized form and as
few as 10-50 organisms are required to establish infection and
cause disease, there is increasing concern that tularemia could be
used as a bioweapon.
[0090] Symptoms of tularemia usually develop within three or four
days of infection, although in some cases it can take up to 10 days
for the disease to manifest. The organism is intracellular and
spreads via the lymphatic system, multiplying within macrophages.
Pneumonic tularemia is characterized by fever, exhaustion and
weight loss accompanied by lung infection. Typically, the patient
experiences a dry cough, respiratory difficulty and chest pain.
Meningitis is an uncommon but potentially serious complication of
tularemia.
[0091] Diagnosis of tularemia is based on the signs and symptoms
described above, ideally combined with a history of recent
arthropod bite or plausible environmental exposure to F.
tularensis. Standard blood tests have historically been unhelpful
in diagnosing tularemia, although about half of all patients will
exhibit non-specific abnormalities in liver function. Some patients
develop elevated creatine kinase levels as a result of
rhabdomyolosis; this is frequently associated with a poor
prognosis.
[0092] A number of specific tests exist for tularemia, but they are
not widely available. Direct examination of biopsy specimens or
secretions by fluorescent antibody or Gram or histochemical stains
is often helpful in diagnosis. F. tularensis can also be
demonstrated microscopically with fluorescent-labeled antibodies.
Antibodies are not typically present, however in the first ten days
to so after exposure. Polymerase chain reaction (PCR) tests can
also be utilized. F. tularensis can be grown in culture, although
such practice is somewhat risk in that workers can themselves
become infected.
[0093] Traditional parenteral antibiotic therapy with streptomycin
is the treatment of choice for tularemia. Gentamicin is considered
an acceptable alternative. The recommended treatment period is
about 7-10 days. Quinolone and fluoroquinolone (e.g., naldixic
acid, ciprofloxacin, levofloxacin, trovofloxacin, norifloxacin,
gemifloxacin, moxifloxacin, among others) are also effective
against F. tularensis, thus providing an additional option for
physicians. Tetracyclines and chloramphenicol can also be used, but
a higher rate of relapse is associated with these agents, as they
are bacteriostatic rather than bactericidal. Recommended treatment
with these medications is usually extended to 2-3 weeks or
more.
[0094] Although the mortality rate for tularemia is generally low,
around 1-2%, the mortality rate for untreated pneumonic tularemia
may be as high as one-third.
[0095] The term "compound" shall mean any specific compound which
is disclosed within this specification and typically means an
isotopically labeled compound. Pharmaceutically acceptable salts
are also compounds for use in the present invention. The term
compound, as it relates to the present invention, also refers to
isotopically labeled citrulline and glycerol, as well as
compounds/derivatives of these compounds.
[0096] The term "effective" when used in context, shall mean any
amount of a compound or component which is used to produce an
intended result within the context of its use. In the case of
compounds according to the present invention, the term effective
generally refers to an effective amount of citrulline and/or
glycerol, which are isotopically labeled and administered to a
patient or subject. In addition, a therapeutically effective amount
of compound which will inhibit the growth and/or produce a die-off
(bacteriacidal) of F. tularensis. This is distinguishable from a
diagnostically effective amount of citrulline and/or glycerol which
may be administered/used short term (for example, a few hours) to
diagnose the presence of a F. tularensis infection in a patient at
risk. Agents useful in treating pneumonic tularemia infections are
treated with an amount and for a duration effective in treating
(i.e., in eliminating or at least stabilizing) the infection in the
patient.
[0097] The terms "agents," "active agents", "active compounds"
"isotopically labeled active agents or compounds" describes, within
context, isotopically labeled metabolic precursors in this case
citrulline and/or glycerol which are acted on by enzyme pathways of
F. tularensis to produce identifiable and distinguishable cleavage
products or metabolites which can be analyzed using conventionally
available analytic tools and methodologies. Active agents according
to the present invention include citrulline, glycerol and their
pharmaceutically acceptable salts. In the present invention, if a
patient has a F. tularensis infection in his or her lungs, the
isotopically labeled compound, which is delivered to the lungs is
metabolized to at least one isotopically labeled cleavage product
(which is preferably a gas) selected from the group ammonia, carbon
dioxide, water, propanediol and ethanol. While propanediol and
ethanol are liquids at ambient temperature, these compounds may be
vaporized and measured using techniques as otherwise disclosed
herein for the gaseous cleavage products, or alternatively, may be
monitored in the blood of the subject.
[0098] The term "cleavage product of citrulline" refers, within
context, to ammonia and carbon dioxide and the term "cleavage
product of glycerol" refers, within context, to carbon dioxide,
water, propanediol and/or ethanol.
[0099] The term "cleaves" refers to the fact that bacterial, for
example, F. tularensis, in particular, biovar A, through enzymatic
process (e.g., citrulline ureidase or fermentation) can break at
least one chemical bond of citrulline and/or glycerol, forming a
plurality of products, by a chemical process including, but not
limited to, hydrolysis. A product so formed is a "cleavage or
fermentation product". The concentration of the cleavage product or
products indicates the level of activity of the bacteria, for
example, F. tularensis, in the lungs of the patient or subject,
which can be used to determine a diagnosis of infection. A positive
diagnosis can also indicate the presence of a biovar in the lungs
of the patient, for example, the present of F. tularensis, biovar
A. A negative diagnosis in the presence other symptology of
pneumonic tularemia may indicate the presence a different biovar
that does not cleave citrulline or glycerol, for example, F.
tularensis, biovar B. The method for diagnosis according to the
present invention is referred to as a "breath test."
[0100] The term "safe and effective amount of substrate" refers to
an amount of a substrate (i.e., isotopically labeled citrulline,
isotopically labeled glycerol or mixtures of these two) which is
sufficient to produce a detectable level of a cleavage product or
products, without an untoward level of adverse side effects, such
as toxicity, irritation, allergy or hypersensitivity responses. The
level of any such side effects should be commensurate with
acceptable risk/benefit ratios.
[0101] The term "isotope-labelling" is used to describe the
naturally more abundant isotope of each of these elements is at
least partially replaced by a less abundant isotope. For example,
the naturally more abundant carbon-12 atoms could be at least
partially replaced by the less abundant carbon-13 atoms, permitting
the cleavage product or products which carry the label to be more
easily detected, since the less abundant isotope can be
distinguished from the naturally more abundant isotope.
Furthermore, the advantage of certain isotopes such as carbon-13 is
that they are stable, so that they are not radioactive, unlike
isotopes such as carbon-14. Therefore, preferably stable,
non-radioactive isotopes such as carbon-13, nitrogen-15, oxygen-17
or 18 and hydrogen-2 preferably are used as labels, depending upon
the cleave product to be analyzed.
[0102] The following examples provide insight into the use of the
present invention. The examples are simply that, exemplary, and are
not to be construed to limit the present invention in any way.
Example 1
[0103] A patient is suspected of (pneumonic) lung tularemia as a
result of a potential bioweapon release in a Gulf state. The
patient inhales 1 to 100 mg of citrulline, with .sup.13C-label on
the ureide:
##STR00006##
using a dry powder inhaler, and exhaled breath .sup.13CO.sub.2
levels monitored over then next 5 to 120 minutes. An increase in
.sup.13CO.sub.2 breath concentrations after inhalation, above a
threshold value to be determined, would support a diagnosis of
pneumonic tularemia.
Example 2
[0104] A patient is suspected of (pneumonic) lung tularemia as a
result of an accidental release in a research lab, and is
critically ill on an incubator. The patient has a solution
nebulized containing 1 to 100 mg of citrulline, with .sup.13C-label
on the ureide:
##STR00007##
and exhaled breath .sup.13CO.sub.2 levels monitored over then next
5 to 120 minutes. An increase in .sup.13CO.sub.2 breath
concentrations after inhalation, above a threshold value to be
determined, would strongly support a diagnosis of pneumonic
tularemia.
Example 3
[0105] Francisella tularensis strains Schu4, LVS and Holoarctica
were grown to mid log phase, harvested, and sonicated at different
powers (P08 to P10) or with bacterial protein extraction reagent
(B-PER, Pierce) to release intracellular citrulline ureidase. Whole
bacteria were removed by filtration, and lysate sterility assessed.
The assay system consisted of phosphate buffered saline 700 ul,
lytsate 50 ul and 250 ul of 1 mM ureido-.sup.13C-citrulline:
##STR00008##
[0106] The assay was incubated overnight, and headspace gas
analysed for d.sup.13CO.sub.2 using isotope ratio mass spectrometry
as previously (Jassal et al 2010, PloS One 27; 5 e12451). The
following lysates, including the method used to release
intracellular citrulline ureidase, were tested:
TABLE-US-00001 Strain Method Schu S4 PO8 Schu S4 PO9 Schu S4 PO10
Schu S4 B-PER Schu S4 B-PER LVS PO8 LVS PO9 LVS PO10 LVS B-PER LVS
B-PER Holartica PO8 Holartica PO9 Holartica PO10 Holartica B-PER
Holartica B-PER
[0107] The results are shown in FIG. 1.
Example 4
Preparation of an Isotopically Labeled Citrulline
[0108] 2 grams of l-ornithine sulfate were heated at reflux with an
excess of copper oxide (1 gram) in 10 ml of distilled water for 10
minutes, producing a blue solution of copper-l-ornithine complex.
Excess copper oxide was removed by filtration. The
copper-l-ornithine complex was added to 1 gram .sup.13C-urea in a
15 ml Q-tube, and reacted at 102.degree. C. for 3 hours, forming a
precipitate of copper-l-.sup.13C-ureido-citrulline which was
collected and washed with water (3 ml), and without isolation
treated with an excess of Al.sub.2S.sub.3 to produce a clear
solution free l-.sup.13C-ureido-citrulline and a precipitate of
copper sulfide which was removed by filtration and concentrated to
2 ml, and 10 ml of ethanol added and stored at 4.degree. C.
overnight for crystallization. Mp 220.degree. C. uncorrected, TLC
on silica using n-butanol-acetic acid-water 40:10:17, Rf 0.4, as
was l-citrulline standard.
[0109] Some embodiments of the present invention include methods
for diagnosing the presence of Francisella tularensis in the lungs
of a patient or subject, including the steps of:
[0110] (a) administering an effective amount of at least one
isotopically labeled agent selected from the group consisting of
citrulline, glycerol, a pharmaceutically acceptable salt thereof or
mixtures thereof to the patient or subject, said active compound
being acted upon by F. tularensis if present to form an
isotopically labeled cleavage product or fermentation product;
[0111] (b) collecting the breath, urine, whole blood, plasma or
serum samples from the subject; and
[0112] (c) analyzing said samples to determine a concentration or
concentrations of said cleavage product(s) or fermentation
product(s), said concentration(s) indicating the presence or
absence of F. tularensis including the presence or absence of type
A biovar of F. tularensis in the lungs of said patient or
subject.
[0113] In some embodiments, said agent is citrulline or a
pharmaceutically acceptable salt thereof. In other embodiments,
said agent is a mixture of citrulline and/or a pharmaceutically
acceptable salt of citrulline and glycerol.
[0114] In certain embodiments, said sample is the breath of said
subject taken for a predetermined time.
[0115] In other embodiments, said agent is isotopically labeled
with carbon-13, nitrogen-15, oxygen-17, oxygen-18, hydrogen-2 or
mixtures thereof. In still other embodiments, said agent is
citrulline or its pharmaceutically acceptable salt isotopically
labeled with carbon-13, nitrogen-15, oxygen-17 or oxygen-18. In yet
other embodiments, said citrulline is isotopically labeled with
carbon-13 or nitrogen-15. In other embodiments, said citrulline is
isotopically labeled with carbon-13.
[0116] In preferred embodiments of the invention, said agent is
glycerol isotopically labeled with carbon-13, oxygen-17, oxygen-18,
hydrogen-2 or mixtures thereof. Preferably, said glycine is
isotopically labeled with nitrogen-15. In other embodiments, said
glycerol is isotopically labeled with carbon-13, oxygen-17,
oxygen-18, hydrogen-2 or mixtures thereof. In yet other
embodiments, said glycerol is isotopically labeled with carbon-13
or oxygen-18.
[0117] In some embodiments, said cleavage product or said
fermentation product is selected from the group consisting of
ammonia, carbon dioxide, water and propanediol. In other
embodiments, said cleavage product or fermentation product is a
gas. Preferably, said gas is ammonia, carbon dioxide or mixtures
thereof. Also preferred are embodiments wherein said gas is a
mixture of isotopically labeled carbon dioxide, from the action of
F. tularensis on citrulline and/or glycerol and isotopically
labeled ammonia from the action of F. tularnesis on citrulline.
[0118] In some embodiments of the invention, said isotopically
labeled cleavage product or metabolite is selected from the group
consisting of propanediol, ethanol or mixtures thereof, which is
measured in the urine, whole blood, serum or plasma of the
patient.
[0119] In preferred embodiments of the invention, said analyzing
step comprises comparing at least one ratio of isotopically labeled
element(s) to non-isotopically labeled element(s) in said exhaled
breath of said subject to a control ratio of isotopically labeled
element(s) to non-isotopically labeled elements in the exhaled
breath of said subject or a control group prior to administration
of said isotopically labeled active agent.
[0120] In certain preferred embodiments of the invention, said
agent is administered to the lungs by a pulmonary route. In other
embodiments, said agent is administered orally.
[0121] Some embodiments of the invention include the optional steps
of fitting the concentrations of said cleavage products obtained to
a curve; and analyzing the curve or a plateau of said curve to
determine the extent of infection.
[0122] Also within the scope of the invention are compositions
adapted for pulmonary administration to a subject to be diagnosed
for the existence or absence of bacterial infection in the lungs of
a patient, said composition consisting essentially of a diagnostic
effective amount of isotopically labeled citrulline and/or
isotopically labeled glycerol, pharmaceutically acceptable salts or
mixtures thereof, in combination with a pharmaceutically acceptable
excipient, a propellant and optionally, a solvent and a dispersant,
said composition being further adapted for single use only. In
preferred compositions of the invention, said citrulline, glycerol
or pharmaceutically acceptable salts or mixtures thereof are
present in amounts ranging from about 0.25 mg to about 10 mg each
in said composition. Preferred compositions are aerosols. Other
preferred compositions are adapted for delivery through an inhaler.
In some embodiments, said solvent is a mixture of water and ethanol
and said propellant is a CFC substitute fluorinated
hydrocarbon.
[0123] Also within the scope of the invention are single dose
compositions adapted for oral administration to a subject, said
composition consisting essentially of a diagnostic effective amount
of isotopically labeled citrulline and/or isotopically labeled
glycerol, or pharmaceutically acceptable salts and mixtures
thereof, in combination with a pharmaceutically acceptable carrier,
additive or excipient. Preferably, the composition has an enteric
coating and is in enteric dosage form. In some embodiments, said
citrulline and/or glycerol together comprise about 0.5 mg to about
100 mg by weight.
[0124] Also within the scope of the invention are kits comprising a
single dose composition as disclosed herein, a bag or vial to
collect the breath of a subject after administration of said
composition; and an instruction manual. Preferably, the composition
administered using the kits of the invention is administered using
an inhaler.
* * * * *