Organic compounds -- part of the class 532-570 series – Organic compounds – Heterocyclic carbon compounds containing a hetero ring...
Reexamination Certificate
2001-04-30
2004-08-03
Shippen, Michael L. (Department: 1621)
Organic compounds -- part of the class 532-570 series
Organic compounds
Heterocyclic carbon compounds containing a hetero ring...
C548S484000, C548S187000, C548S214000, C548S469000, C549S293000, C549S479000, C549S289000, C560S013000, C560S034000
Reexamination Certificate
active
06770764
ABSTRACT:
BACKGROUND OF THE INVENTION
Urinary trypsin inhibitor (“UTI”) is a glycoprotein that inhibits the enzyme reactivity of trypsin and &agr;-chymotrypsin, hyaluronidase, and creatine phosphokinase. UTI can be present in minute quantities in the urine of healthy individuals.
Trypsin inhibitor activity has been suggested for use in a screening test for diagnosing bacterial infection. When bacterial infections occur, white blood cells are mobilized, and the elastase activity of the white blood cells is activated. During the acute phase response, interleukin-1 induces the production of inter-&agr;-trypsin inhibitor, which is decomposed by the elastase activity into low molecular weight trypsin inhibitors. These trypsin inhibitors appear to act on the inflamed sites, showing anti-inflammatory and anti-shock activities before being excreted in the urine. Piette et al. (
European J. Med
. 1, 273 (1992)) reports that urinary trypsin inhibitor activity can be a useful marker, particularly in patients with fever of unknown origin or elevated erythrocyte sedimentation rate.
Quantitative changes in UTI are useful as an index of infection or inflammation. Kuwajima et al. (
Clin. Biochem
. 23, 167 (1990)) reports that the assay of UTI may be used for the clinical diagnosis of acute phase response. UTI levels are elevated under other circumstances such as malignant tumors, kidney disease, myocardial infarction and post surgery.
Serum C-reactive protein, sialic acid and erythrocyte sedimentation rate have been used as markers of infection and inflammation. However, all of these markers are serum-based, which requires a blood sample. Using blood samples requires time for coagulation, centrifugation, and separation of the blood sample before analysis.
Measuring UTI concentration has been accomplished several ways, including enzyme inhibition, antibody stains, latex agglutination methods and radioimmunoassay methods. Enzyme inhibition has been used to measure UTI concentration, and calorimetric enzyme substrates have been used to measure the extent of the inhibition. The method has been recently adapted to automated measurement on clinical analyzers (S. Kuwajima, et al., loc,. cit.). Such analytical techniques typically involve contacting the urine sample with a trypsin substrate attached to a chromophore at either arginine or lysine, because trypsin cleaves arginine and lysine. The concentration of UTI in the urine sample is inversely proportional to the intensity of the colored response of the chromophore since UTI inhibit trypsin activity according to their concentration in the fluid test sample.
Several calorimetric and fluorogenic trypsin substrates are commercially available, including N&agr;-benzoyl-L-arginine p-nitroanilide (BAPNA), N&agr;-benzoyl-D,L-arginine &bgr;-naphthylamide (BANA) and N&agr;-benzoyl-L-arginine-7-amido-4-methylcournarin.
Known indicating trypsin substrates are aromatic amides of N&agr;-protected arginine. When trypsin hydrolyzes these known substrates, the amide bond is cleaved and an aromatic amine is released. In the case of BAPNA, the amide bond is cleaved and yellow-colored p-nitroaniline is liberated and measured with a spectrophotometer. With BANA, 2-amino-naphthalene is produced, and it is detected by diazotization and coupling with N-(1-naphthyl)-ethylenediamine to form an azo dye (Goldberg, et al.,
Cancer
11, 283 (1958)). 7-Amino-4-methylcoumarin is released by hydrolysis of N&agr;-benzoyl-L-arginine-7-amido-4-methylcoumarin, and this fluorescent product is measured with a fluorometer. These substrates are used for measuring trypsin activity in liquid-phase assays but are not well suited for use in dry-phase formats, such as dip-sticks, which are typically read visually or with simple reflectance instruments.
Aromatic esters of arginine are not known to those of skill in the art as trypsin substrates. Esters are much more labile toward hydrolysis than amides, and are often incorporated into protease substrates in place of amides to give more sensitive, easily hydrolysed analogs. They are also more prone to non-enzymatic hydrolysis by nucleophiles. This is significant for arginine esters, which have the nucleophilic guanidino group as part of their structure. Gray, et al. (
Enzyme Microb. Technol
. 5, 137 (1983)) states that efforts to prepare the N&agr;-benzoyl-arginine esters of 2-hydroxynaphthol and 7-hydroxy-4-methylcoumarin were unsuccessful because of the lability of the ester group.
A trypsin substrate is needed that addresses the short-comings of prior art including, among other things, the requirement of a blood sample.
SUMMARY OF THE INVENTION
This invention provides aromatic esters of N&agr;-(&agr; amino group) and N
G
-(guanidino group) bis-protected arginine that are trypsin substrates. Surprisingly, trypsin hydrolyzes esters of arginine with protecting groups on the guanidino moiety. The esters of the present invention may be used to produce visible colors in dry-phase analytical elements to detect quantities of UTI in biological sample such as urine.
In one aspect of the invention, a compound of the formula (I) comprises:
wherein R
1
is a protecting group for N&agr;, R
2
is a protecting group for N
G
; and R
3
is aryl; and wherein the compound of formula (I) is a trypsin substrate such that trypsin cleaves the O—C single bond, which liberates R
3
—OH.
In another aspect of the invention, a diagnostic device comprises a carrier matrix and a compound of the formula (I).
In another aspect of the invention, a method of preparing a diagnostic device comprises (a) contacting a carrier matrix with a buffer solution, (b) drying the carrier matrix, and (c) contacting the carrier matrix with a solution comprising the trypsin substrate of formula (I).
In still another aspect of the invention, a method for detecting levels of urinary trypsin inhibitor in a biological sample comprises (a) contacting a biological sample with a predetermined amount of trypsin, a predetermined amount of a diazonium salt, and a diagnostic device comprising a trypsin substrate of the formula (I) wherein R
1
is a protecting group for N&agr;; R
2
is a protecting group for N
G
; and R
3
is aryl; and wherein the compound of formula (I) is a trypsin substrate such that trypsin cleaves the O—C single bond, which liberates R
3
—OH; and wherein the compound R
3
—OH reacts with a diazonium salt to form a visible color such that the greater the intensity of the color, the less urinary trypsin inhibitor is in the biological sample.
In still another aspect of the invention, a diagnostic kit for determining the presence of urinary trypsin inhibitor in a biological fluid comprises trypsin and a trypsin substrate of the formula (I).
The present invention provides the foregoing and other features, and the advantages of the invention will become further apparent from the following detailed description of the presently preferred embodiments. The detailed description is merely illustrative of the invention and does not limit the scope of the invention, which is defined by the appended claims and equivalents thereof.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Definition of Terms
“Alkyl” as used herein is the radical of saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups and cycloalkyl groups. Particularly preferred alkyl substituents include methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, iso-butyl, tert-butyl, sec-butyl, pentyl, hexyl, cyclohexyl, etc. Unless the number of carbons is otherwise specified, “lower alkyl” as used herein means an alkyl group, as defined above, but having from one to ten carbons, more preferably from one to six carbon atoms in its backbone structure. The aliphatic cyclic groups can be single or polycyclic containing between about 1 to 12 carbons per ring, but preferably between 1 and 9 carbons per ring.
“Aryl” as used herein includes 5-15 membered aromatic monocyclic or fused polycyclic moieties which may include from zero to four heteroatoms selected from the group consisting of oxygen, sulfur and nitrogen.
Corey Paul F.
Felman Steven W.
Pugia Michael J.
Rehm Gary E.
Bayer Corporation
Shippen Michael L.
Zucker Paul A.
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