U.S. patent application number 11/146065 was filed with the patent office on 2005-12-22 for method and means for detecting inflammatory processes.
Invention is credited to Lundqvist, Thomas, Pettersson, Anders.
Application Number | 20050281738 11/146065 |
Document ID | / |
Family ID | 20417512 |
Filed Date | 2005-12-22 |
United States Patent
Application |
20050281738 |
Kind Code |
A1 |
Pettersson, Anders ; et
al. |
December 22, 2005 |
Method and means for detecting inflammatory processes
Abstract
A method of detecting inflammatory processes comprises (a)
administering per-orally or per-rectally a composition comprising a
diagnostically effective amount of
L-[guanido-.sup.15N.sub.2]-arginine, L-[guanido-.sup.15N]-arginine,
their mixtures and pharmaceutically acceptable salts thereof, and a
pharmaceutically acceptable carrier; (b) collecting a sample of
exhaled air, saliva or urine; (c) determining .sup.15NO in the air
sample or its transformation products .sup.15N-nitrite and/or
.sup.15N-nitrate in the saliva sample or urine. Also disclosed is a
corresponding diagnostic composition for use in diagnosing
inflammatory processes and a method for its manufacture.
Inventors: |
Pettersson, Anders; (Kode,
SE) ; Lundqvist, Thomas; (Uppsala, SE) |
Correspondence
Address: |
DICKSTEIN SHAPIRO MORIN & OSHINSKY, LLP
1177 Avenue of the Americas
New York
NY
10036
US
|
Family ID: |
20417512 |
Appl. No.: |
11/146065 |
Filed: |
June 7, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11146065 |
Jun 7, 2005 |
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10110040 |
Aug 26, 2002 |
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6962687 |
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10110040 |
Aug 26, 2002 |
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PCT/SE00/02054 |
Oct 24, 2000 |
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Current U.S.
Class: |
424/1.11 |
Current CPC
Class: |
A61K 51/1206 20130101;
A61B 5/0813 20130101; A61K 51/02 20130101; A61B 5/412 20130101 |
Class at
Publication: |
424/001.11 |
International
Class: |
A61K 051/00 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 26, 1999 |
SE |
9903885-3 |
Claims
1. A method of detecting the existence of an inflammatory process
comprising: administering per-orally or per-rectally to a person a
composition comprising a diagnostically effective amount of
L-[guanido-.sup.15N.sub.2]-arginine, L-[guanido.sup.15N]-arginine,
their mixtures and pharmaceutically acceptable salts thereof, and a
pharmaceutically acceptable carrier, collecting a sample of exhaled
air, saliva or urine, determining .sup.15NO in the air sample or
its transformation products .sup.15N-nitrite and/or
.sup.15N-nitrate in the saliva sample or urine.
2. The method of claim 1, wherein administration is per-oral is for
release in the small intestine or in the upper part of the large
intestine or both.
3. The method of claim 1, wherein the administration is per-rectal
for release in the lower part of the large intestine.
4. (canceled)
5. The method of claim 2, wherein the composition is in form of a
tablet, a capsule, or granules.
6. The method of claim 2, wherein the inflammatory process is
selected from the group consisting of ulcerative colitis, Crohn's
disease and celiac disease.
7. The method of claim 3, wherein the composition is in form of an
enema, a suppository or a foam.
8. The method of claim 3, wherein the inflammatory process is
ulcerative colitis.
9-10. (canceled)
11. The method of claim 1, wherein determination is by
IR-spectrometry, laser spectrometry, mass spectrometry, gas
chromatography, and their combinations.
12. (canceled)
13. A diagnostic composition for per-oral administration, including
pulmonary administration, and per-rectal administration, for use in
diagnosing inflammatory processes, comprising a diagnostically
effective amount of L-[guanido-.sup.15N.sub.2]-arginine,
L-[guanido-.sup.15N]-argin- ine, their mixtures, and
pharmaceutically acceptable salts thereof, and a pharmaceutically
acceptable per-oral or per-rectal carrier, in a form selected from
the group consisting of tablet, capsule, granules, enema,
suppository and foam.
14. The composition of claim 13, wherein said amount is from 1 mg
to 2,000 mg.
15. A method of manufacture of a diagnostic composition for
per-oral administration, including pulmonary administration, and
per-rectal administration, for use in diagnosing the existence of
an inflammatory process, comprising combining a diagnostically
effective amount of L-[guanido-.sup.15N.sub.2]-arginine,
L-[guanido-.sup.15N]-arginine, their mixtures, and pharmaceutically
acceptable salts thereof, and a suitable pharmaceutically
acceptable per-oral or per-rectal carrier into a form selected from
the group consisting of tablet, capsule, granules, enema,
suppository and foam.
16. The method of claim 15, wherein said amount is from 1 mg to
2,000 mg.
17. The composition of claim 13, wherein the carrier is a peroral
carrier and the composition is in form of a tablet, a capsule, or
granules.
18. The composition of claim 13, wherein the carrier is a
per-rectal carrier and composition is in form of an enema, a
suppository or a foam.
19. The method of claim 1, wherein the sample collected is exhaled
air or saliva.
20. The method of claim 2, wherein said amount is from 1 mg to
2,000 mg and the sample collected is exhaled air or saliva.
21. The method of claim 20, wherein the composition is in form of a
tablet, a capsule, or granules.
22. The method of claim 3, wherein said amount is from 1 mg to
2,000 mg and the sample collected is exhaled air or saliva.
23. The method of claim 22, wherein the composition is in form of
an enema, a suppository or a foam.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a method for determining
nitrogen oxide formed in inflammatory processes, to a means for
carrying out the method and to a method of manufacture of the
means.
BACKGROUND OF THE INVENTION
[0002] Nitrogen oxide (NO) has important biological functions. It
is the structurally simplest mediator in the human body as well as
one of the most important weapons of its immune defense. In its
later function it is excreted by activated macrophages to kill
foreign microorganisms and cells recognized as foreign. In this
second capability nitrogen oxide may be considered an inflammation
marker and has been recognized as such in, for instance,
inflammatory processes in the gastro-intestinal tract, such as
ulcerative colitis and Crohn's disease and celiac disease. In the
lung it may assume both roles. It is recognized that levels of
nitrogen oxide excretion are raised in asthmatics. Thus it may be
considered a marker for asthma which also comprises an important
inflammatory component.
[0003] In the body nitrogen oxide is formed from L-arginine by
hydroxylation of one of the guanidino nitrogens in a reaction
catalyzed by one of the isoforms of the enzyme nitrogen oxide
synthase (NOS). In a complex reaction the thus formed hydroxyimino
intermediate is oxidatively split into nitrogen oxide and
L-citrulline.
[0004] Nitrogen oxide may be sampled in situ and measured by, for
instance, chemoluminescense (WO 96/17244; WO 97/37587). In situ
sampling, for instance in the gastro-intestinal tract and in the
urinary tract, often is difficult or at least time-consuming.
Samples of exhaled air may contain nitrogen oxide formed in the
pulmonary system but also formed elsewhere because of the
considerable solubility of NO in water and lipids which makes it
freely diffusible in the body. Increased levels of NO in exhaled
air thus may be due to inflammation in the pulmonary system as well
as elsewhere. This detracts from the potential usefulness of
nitrogen oxide as an inflammation marker.
OBJECTS OF THE INVENTION
[0005] It is an object of the invention to provide an improved
method for determining nitrogen oxide formed in the body.
[0006] It is another object of the invention to provide a means for
carrying out said method.
[0007] Further objects of the invention will be apparent from the
following description of the invention and preferred embodiments
thereof, and from the appended claims.
SUMMARY OF THE INVENTION
[0008] According to the present invention is provided a method of
the aforementioned kind comprising the per-oral or per-rectal
administration of a composition comprising a diagnostically
effective amount of arginine terminally labeled with the stable
nitrogen isotope .sup.15N for determination, directly or
indirectly, of .sup.15NO in exhaled air or saliva. It is more
preferred for both terminal nitrogen atoms to be labeled. In this
specification is understood by `terminally labeled` the labeling of
one or both of the terminal guanido nitrogen atoms of L-arginine.
Direct determination of .sup.15NO implies that the compound is
measured as such, whereas indirect determination implies that a
product into which it has been transformed is measured such as, for
instance, .sup.15N-nitrite or .sup.15N-nitrate.
[0009] According to a first preferred aspect of the invention is
provided a method for per-oral administration of a diagnostic
composition comprising a diagnostically effective amount of
L-arginine terminally labeled with the stable nitrogen isotope
.sup.15N for release in the small intestine and/or in the upper
part of the large intestine.
[0010] According to a second preferred aspect of the invention is
provided a method of the aforementioned kind comprising the
per-rectal administration of a composition comprising a
diagnostically effective amount of arginine terminally labeled with
the stable nitrogen isotope .sup.15N for release in the lower part
of the large intestine.
[0011] According to a third preferred aspect of the invention is
provided a method of the aforementioned kind comprising the
per-oral administration of a diagnostic composition comprising a
diagnostically effective amount of L-arginine terminally labeled
with the stable nitrogen isotope .sup.15N for inhalation.
[0012] Preferred assaying methods for .sup.15N comprise:
IR-spectrometry, laser spectrometry, gas chromatography and mass
spectrometry, and their combinations. The determination of .sup.15N
as NO by any of these methods must take into account the fact that
atmospheric nitrogen is a mixture of .sup.14N (99.63%; isotopic
abundance) and .sup.15N (0.37%). In a sample of exhaled air is thus
the amount of .sup.15N in excess of the natural isotopic abundance
of .sup.15N that is representative of labeled nitrogen in arginine.
The determination of .sup.15N as NO by any of these methods must
also take into account the purity of the label. The fact that
.sup.14N and .sup.15N differ in their mass by 6.6%, in
consideration of the natural abundance of .sup.16O oxygen being
99.76%, makes .sup.15N particularly attractive as a marker. It is
thus .sup.15N.sup.16O which is determined at m/e=31 in the presence
of .sup.14N.sub.2 (m/e=28), .sup.14N.sup.15N (m/e=29),
.sup.14N.sup.16O (m/e=30) formed from non-labeled L-arginine,
.sup.16O.sub.2 (m/e=32), .sup.14N.sup.18O (m/e=32),
.sup.16O.sup.17O (m/e=33), .sup.16O.sup.18O (m/e=34). Because of
the low natural abundance of .sup.17O (0.037%) .sup.14N.sup.17O
(m/e=31) will be present in minute amounts only, and can be
disregarded from.
[0013] It is advantageous to partially or fully separate the
components of a gaseous sample containing .sup.15NO in a gas
chromatograph before injecting a sample of the fraction containing
nitrogen oxide in the mass spectrometer. Methods for such
separation are well known in the art.
[0014] Since NO formed from .sup.15N.sub.2-arginine is isotopically
quantitatively different from NO formed from unlabeled arginine,
the amount of NO formed at or near the site where the labeled
arginine is administered can be determined.
[0015] Thus, according to a further preferred aspect of the
invention, the site of administration of terminally
.sup.15N-labeled arginine is different from the site of detection
of .sup.15N-labeled NO formed therefrom. For instance, terminally
labeled .sup.15N-arginine can be administered orally or rectally to
a person suspected to suffer from ulcerative colitis or Crohn's
disease, and the intestinally formed .sup.15N-labeled NO be
determined in the exhaled air or, in the form of nitrite or
nitrate, in saliva.
[0016] According to still another preferred aspect of the invention
labeled nitrogen oxide formed from correspondingly terminally
labeled .sup.15N-arginine can be assayed by measurement of one or
several of the products to which it is biologically transformed, in
particular nitrate. It is known that a substantial amount of
nitrogen oxide entering the bloodstream is oxidized to nitrate in
which form it is excreted by the kidneys. It is thus within the
scope of the invention to determine the formation of labeled
nitrogen oxide by measuring labeled .sup.15NO.sub.3.sup.- in urine.
Another important path for excretion is from the salivary glands.
It is thus also within the scope of the invention to determine the
formation of labeled nitrogen oxide from correspondingly
.sup.15N-labeled L-arginine by measuring .sup.15N-labeled
NO.sub.3.sup.- in saliva and/or by measuring .sup.15N-labeled
NO.sub.2.sup.- in saliva to which nitrate is rapidly transformed by
the action of certain bacteria colonizing the surface of the
tongue.
[0017] According to still another preferred aspect of the invention
.sup.15N-labeled arginine can be administered to the lungs in form
of a spray or mist for the detection of inflammatory processes in
the respiratory system, in particular of asthma, nasal
inflammation, and sinusitis.
[0018] According to still another preferred aspect of the invention
.sup.15N-labeled arginine can be administered to the circulating
blood in form of an injection or infusion for the detection of
inflammatory processes in the blood (sepsis) or in tissues in
contact with circulating blood.
[0019] According to the present invention is also disclosed a
diagnostic composition for use in diagnosing inflammatory
processes, comprising a diagnostically effective amount of
terminally labeled .sup.15N-arginine and a pharmaceutically
acceptable carrier.
[0020] Also disclosed is a process for the manufacture of such
composition comprising formulating a diagnostically effective
amount of terminally labeled .sup.15N-arginine and a
pharmaceutically acceptable carrier into a pharmaceutical
composition for per-oral or per-rectal administration.
[0021] It is preferred for the compositions according to the
invention for per-oral administration to be selected from the group
consisting of: enteric delayed release compositions for per-oral
administration releasing .sup.15N-arginine in the small intestine
and/or the upper part of the large intestine; nebulizable aqueous
solutions of and powders containing terminally labeled
.sup.15N-arginine for administration to the bronchi and the
lung.
[0022] It is preferred for the compositions according to the
invention for per-rectal administration to be selected from enemas,
foams, and suppositories.
DESCRIPTION OF PREFERRED EMBODIMENTS
EXAMPLE 1
[0023] Terminally N-labeled arginine.
L-[guanido-.sup.15N.sub.2]-arginine is commercially available from
ICN Pharmaceuticals, Inc. (Costa Mesa, Calif.) and Tracer
Technology, Inc. (Somerville, Mass.). Fully .sup.15N-labeled
arginine can also be used. It can be produced by Corynebacterium
herculis or Brevibacterium flavum strains carrying the recombinant
DNA pCarg11 or pCarg110 according to the method disclosed in U.S.
Pat. No. 5,017,482, which is hereby incorporated by reference, with
the proviso that .sup.15N.sub.3 ammonium sulphate is substituted
for ammonium sulphate as the nitrogen source.
L-[guanido-.sup.15N.sub.2]-argi- nine can be used in form of the
free base or a pharmaceutically acceptable salt, such as the
hydrochloride or aspartate.
EXAMPLE 2
[0024] Enteric tablet. An enteric tablet for release in the small
intestine and the upper large intestine can be prepared according
to EP 0 502 092 A1 by substituting a terminally N-labeled arginine
for any of the glucocorticoids disclosed therein. A suitable amount
of terminally N-labeled arginine, such as
L-[guanido-.sup.15N.sub.2]-arginine, is 50 mg, in the form of the
free base or a pharmaceutically acceptable salt thereof, such as
the hydrochloride.
EXAMPLE 3
[0025] Suppository. A suitable type of suppository can be made by
use of the Salazopyrin.RTM. EN, Pharmacia & Upjohn, enema
composition in which the active principle sulfasalazine (500 mg) is
exchanged for 50 mg L-[guanido-.sup.15N.sub.2]-arginine and 450 mg
of suitable neutral constitutents, such as calcium carbonate.
EXAMPLE 4
[0026] Solution for inhalation spray. 100 mg
L-[guanido-.sup.15N.sub.2]-ar- ginine are dissolved in 10 ml of
sterile water. The solution is administered by a nebulizer capable
of dispensing measured doses.
EXAMPLE 5
[0027] Determination of inflammatory conditions in the small
intestine and the upper part of the large intestine. An enteric
tablet of EXAMPLE 2 is administered to the person suspected of
inflammation in a fasting state. Breath samples (100 ccm) are taken
in 10 min intervals starting at time of administration.
EXAMPLE 6
[0028] Determination of inflamatory conditions in the lower part of
the large intestine. A suppository of EXAMPLE 3 is per-rectally
administered to the person suspected of inflammation being in a
fasting state. Breath samples (100 ccm) are taken in 10 min
intervals starting at time of administration.
EXAMPLE 7
[0029] Determination of inflamatory conditions in the lung. A
metered dose (1 ccm) of the solution for inhalation of EXAMPLE 4 is
nebulized for inhalation by the patient using of a state-of-the-art
nebulizer. Breath samples (100 ccm) are taken in 5 min intervals
starting at time of administration.
EXAMPLE 7
[0030] Determination of inflammatory conditions in the blood. An
intravenous infusion of L-[guanido-.sup.15N.sub.2]-arginine is
administered to a person suspected of sepsis. Breath samples (100
ccm) are taken in 10 min intervals starting at time of
administration.
EXAMPLE 8
[0031] Determination of .sup.15NO in gaseous samples. See: D C
Macallan et al., Am. J. Physiol. 272 (6, part 2), p. R1888-R1896
(1997).
EXAMPLE 9
[0032] Determination of urinary .sup.15NO.sub.3.sup.-. See: P Forte
et al., Measurement of Nitric Oxide Synthesis in Humans Using
L-[.sup.15N.sub.2]Arginine. Methods in Enzymology 301 (1999) 92-98.
The method is easily modified for measurement of nitrate in
saliva.
* * * * *