Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
2001-12-18
2003-01-07
Aulakh, Charanjit S. (Department: 1625)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C548S204000, C548S187000, C548S181000, C546S270400, C435S004000, C435S023000, C435S805000, C424S009100
Reexamination Certificate
active
06503725
ABSTRACT:
FIELD OF THE INVENTION
This invention relates generally to compounds, compositions, devices, and methods useful for detecting the presence of leukocytes through the activity of leukocyte esterases and proteinases in urine.
BACKGROUND OF THE INVENTION
The presence of leukocytes in human urine is associated with infection or malfunction in the kidney or urinary tract. Accurate detection has a significant meaning for the physiological treatment or diagnosis of the patient.
A basic method to measure the number of leukocytes in urine by microscope has been widely available for some time. However, the disadvantages of this method include the investment of time and money in obtaining and installing the appropriate instrumentation. Further, false negatives can be obtained when samples are allowed to sit too long before analysis.
Research has been directed to developing other methods, such as indicator assays, that are suitable for detecting leukocytes more easily, conveniently, and accurately. Typical indicator assays use a specific chemical substance (e.g., a substrate) that is degraded by one or more enzymes present in leukocytes to create a product suitable for effectuating a visible color change.
One such leukocyte assay is disclosed in U.S. Pat. No. 3,087,794 and involves the use of peroxidase that is contained in a granular leukocyte. This assay includes a filter paper stained with hydrogen peroxide and o-tolidine, which shows a colored oxidative product when contacted with leukocytes. Yet this assay is less than desirable because peroxidase can be dangerous and reductive materials in urine may make actual application impractical.
Other methods designed to confirm the presence of esterase and proteinase in leukocytes have also been developed. For example, one method uses a colorless or pale-colored ester compound as a substrate that is degraded by esterase into a colorless acid moiety and an alcoholic moiety. Then, under diazonium or oxidative reaction, the alcoholic moiety is converted to a second densely colored product. This method was derived from a process in which enzymatic degradation of the substrate naphtol-AS-D chloroacetate produced chloroacetate and naphtol-AS. Reaction of the naphtol-AS product with a diazonium salt resulted in the formation of a colored azo compound.
This assay allows for the determination of leukocyte concentration by the naked eye. Yet the use of this assay is less than desirable because the diazonium salt may react with urobilinogen or bilirubin contained in urine. As a result, concentrations of leukocytes greater than 500 cells/&mgr;l are often necessary to avoid a false-negative reading.
U.K. Patent No. 1,128,371 discloses other possible substrates, i.e., colorless indoxyl or thioindoxylesters. These substrates are degraded into indoxyl or thioindoxyl by esterase. Colored indigo or thioindigo may then be produced by reaction with oxygen in the air or by an oxidizer. Yet the use of an assay with these substrates is less than desirable because this is not sensitive and fails to detect leukocytes at a concentration less than 10,000 cells/&mgr;l.
Similarly, U.S. Pat. No. 4,278,763 suggests indoxyl-type substrates by disclosing a method of using indoxyl or thioindoxylamino acid ester as a substrate.
Other assays using pyrrole derivatives as substrates have also been disclosed. For example, U.S. Pat. No. 4,704,460 discloses use of an amino acid ester of a pyrrole derivative as a substrate. When this derivative is degraded in the presence of diazonium salt, a change in sample color to deep violet results. Moreover, the addition of a nucleophilic alcohol, such as decanol, greatly facilitates reaction rate, and in turn, allows detection of leukocyte concentrations as low as 10 cells/&mgr;l within 90 seconds. Yet this assay is less than desirable because the syntheses of these two substrates is very difficult.
The above-described assays are characterized by numerous disadvantages. Some disadvantages include the detection of false negatives, the interference of urobilinogen or bilirubin, the lack of sensitivity, and the requirement of less-than-desirable substrates. Thus, it would be desirable to identify new compounds and methods of using the compounds to detect the presence of leukocytes in urine.
SUMMARY OF THE INVENTION
The invention is directed to thiazole esters suitable for detecting leukocytes in urine, compositions containing thiazole esters, diagnostic devices suitable for detecting leukocytes in urine, and methods of using the thiazole esters or compositions thereof for detecting leukocytes in urine.
In one aspect, this invention is directed to novel thiazole esters.
One thiazole ester according to this invention is of the formula:
or a salt or solvated salt thereof, in which
A is an N-blocked amino acid residue or an N-blocked peptide chain, preferably N-blocked alanine or N-blocked polyalanine; and
R
1
is unsubstituted or substituted heteroaryl; alkenyl substituted with unsubstituted or substituted aryl; or alkenyl substituted with unsubstituted or substituted heteroaryl. For example, R
1
may be thienyl, pyridyl, furyl, styryl, pyrrolyl, or indolyl.
Another thiazole ester according to this invention is of the formula:
or a salt or solvated salt thereof, in which
A is an N-blocked amino acid residue or an N-blocked peptide chain, preferably N-blocked alanine or N-blocked polyalanine; and
R
2
is unsubstituted or substituted fused hydrocarbyl rings in which at least one ring is aromatic. For example, R
2
may be naphthyl or anthryl.
In another aspect, this invention is directed to compositions that include a thiazole ester of formula I or II. Compositions of the invention may also include a diazonium salt such as 2-methoxy-4-morpholinobenzene diazonium chloride, zinc chloride double salt.
In still another aspect, this invention is directed to diagnostic devices that include a compound or composition of the invention. Such diagnostic devices include a substrate having a thiazole ester of formula I or II deposited thereon.
The compounds, compositions, and diagnostic devices of the invention are also suitable for use in methods for detecting leukocytes in urine. Methods for detecting leukocytes in urine include contacting a thiazole ester of the invention and a diazonium salt with a urine sample.
The compounds and compositions suitable for use in devices and methods of the invention are typically pharmaceutically acceptable.
DETAILED DESCRIPTION OF THE INVENTION
The invention is directed to detecting leukocytes in urine. The presence of leukocytes is typically detected by detecting the presence of enzymes known in the art as leukocyte esterases and leukocyte proteinases that are present with leukocytes.
Compounds of the invention include thiazole esters that contain an ester functionality, which is hydrolyzed upon exposure to leukocytes in urine. The hydrolysis of the ester of a thiazole ester of the invention provides a useful diagnostic technique because thiazolyl can react with a diazonium salt to produce an azo dye.
The compounds according to this invention are thiazole esters that are suitable for use in compositions, diagnostic devices, and methods that can be used to detect leukocytes in urine.
One thiazole ester suitable for use in compositions and methods of the invention is of the formula:
or a salt or solvated salt thereof, in which A is an N-blocked amino acid residue or an N-blocked peptide chain, preferably N-blocked alanine or N-blocked polyalanine (e.g., alanine-alanine, alanine-alanine-alanine, and the like), being blocked at the amino terminus by protecting groups known in the art, such as, for example, benzyloxycarbonyl, t-butoxycarbonyl, and p-toluenesulfonyl; and R
1
is unsubstituted or substituted heteroaryl; alkenyl substituted with unsubstituted or substituted aryl; or alkenyl substituted with unsubstituted or substituted heteroaryl.
The term “heteroaryl” includes heterocyclic aromatic derivatives having at least one heteroatom, such as, for example, nitrogen, oxygen, phosphorus, or sulfur, and includes, for example
Aulakh Charanjit S.
Hypoguard America Limited
Merchant & Gould P.C.
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