Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
1999-02-19
2001-10-23
Nolan, Patrick J. (Department: 1644)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S007100, C562S445000
Reexamination Certificate
active
06306576
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to novel methods for diagnosing and screening for diseases which involve accumulation of activated eosinophils at the disease site. More particularly, this invention is directed to methods for diagnosing asthma.
Asthma is clinically defined as a reversible obstructive airway disease. Symptoms of asthma range from chronic cough and wheezing to severe difficulty in breathing and respiratory failure. Acute severe asthma (status asthmaticus) refers to an attack of increased severity that is unresponsive to routine therapy and that, if severe enough, can lead to death.
Chronic inflammation of the respiratory mucosa plays a fundamental role in the pathogenesis of asthma. Chronic inflammation causes increased mucus production which, along with asthmatic induced bronchospasms, leads to narrowing of the airways of the lung. The net result is a decrease in airway exchange in the lungs, decreased oxygenation of blood and tissues, and increased work of breathing.
The inflammation associated with asthma involves a number of different inflammatory cell types including eosinophils. Indeed, the earliest cellular hallmark of asthma is the eosinophil. Eosinophils and their granule constituents are present in blood, sputum and bronchial tissue of asthmatics early during asthma exacerbation.
At present there are no specific biochemical methods for diagnosing asthma or for monitoring the extent of ongoing inflammatory tissue injury in a subject diagnosed as having asthma. Furthermore, although there are numerous therapeutic agents which can be used for combating asthma, their use is limited by the lack of objective diagnostic biochemical assays with which to monitor their efficacy. Instead, therapy is initiated and adjusted based upon clinical exam, a patient's subjective response to therapy, and pulmonary function testing which does not directly assess the level of airway inflammation present.
Accordingly, it is desirable to have new methods for diagnosing asthma and other diseases which are characterized by the presence of activated eosinophils. New methods for monitoring the effect of treatment with anti-asthmatic drugs are also desirable. Methods which are simple and based upon a diagnostic marker are especially desirable.
SUMMARY OF THE INVENTION
In accordance with the present invention, screening methods for asthma and analogous diseases in which activated eosinophils are found at the disease site are provided. The methods comprise: assaying for the presence of brominated tyrosine species in a bodily sample which has been obtained from a test subject. As used herein, the term “test subject” refers to a mammal, preferably a human, suspected of having or known to have the disease. The brominated tyrosine species are either free in the sample or protein bound. Preferably, the assay is designed to detect the presence of 3-bromo-tyrosine, 3,5-dibromotyrsosine, or combinations thereof in the bodily sample. The methods are especially useful for diagnosing, determining the prognosis of, and monitoring asthma in a test subject. The methods of the present invention are also useful for detecting the presence of oxidative stress in a subject, for screening for analogous diseases involving activated eosinophils, and for monitoring the effectiveness of therapeutic interventions for asthma and analogous diseases involving activated eosinophils.
In one embodiment, the assay comprises measuring the amount of brominated tyrosine species, particularly 3-bromotyrosine, 3,5-dibromotyrosine, or combinations thereof (referred to hereinafter collectively as the “diagnostic marker”) in a bodily sample from the test subject. Preferably, the concentration of the diagnostic marker in the bodily sample is determined and compared to the concentration of the diagnostic marker in corresponding bodily samples from normal subjects. Alternatively, the content of the diagnostic marker in the bodily sample is determined and compared to the content of the diagnostic marker in corresponding bodily samples from normal subjects.
In another embodiment for determining the prognosis of asthma in a test subject, the concentration or content of the diagnostic marker is determined in bodily samples taken from the test subject over successive time intervals. The concentrations are compared to determine the prognosis of the asthma. Alterations in the concentration or content correlate with the level of inflammatory tissue injury. An increase in concentration or content is indicative of increased inflammation.
In another embodiment of the invention for monitoring the response of the test subject to treatment with an anti-asthmatic drug, the concentration or content of the diagnostic marker is measured in bodily samples obtained from the test subject before and after such treatment. Preferably, the concentration or content of the diagnostic marker is measured in samples taken over successive time intervals following treatment. A decrease in the concentration or content of the markers following administration of the anti-asthmatic drug is indicative of decreased ongoing inflammatory tissue injury.
The present invention also relates to a diagnostic kit and to a diagnostic reagent for diagnosing asthma and analogous diseases which are associated with activated eosinophils. The diagnostic kit comprises an antibody reactive with a protein bound or peptide bound brominated tyrosine species. Preferably, the antibody is a monoclonal antibody reactive with proteins or peptides containing 3-bromotyrosine, 3,5-dibromotyrosine, or combinations thereof. The diagnostic reagent comprises a brominated tyrosine species selected from the group consisting of 3-bromotyrosine, 3,5-dibromotyrosine, a peptide or protein containing 3-bromotyrosine, and a peptide or protein containing 3,5-dibromotyrosine.
REFERENCES:
patent: 5710248 (1998-01-01), Grose
patent: 98/10294 (1998-03-01), None
Smith-Gill, Research in Immunology, vol. 145: 67-70, 1994.*
The Merck Manual of Diagnosis and Therapy 16thEdition, 1992.*
“3-Bromotyrosine and 3,5-Dibromotyrosine Are Major Products of Protein Oxidation by Eosinophil Peroxidase: Potential Markers for Eosinophil-Dependent Tissue Injury in Vivo” by Wu, et al.,Biochemistry, vol. 38, No. 12, 1999, pp. 3538-3548, Published on Web Mar. 5, 1999.
“3-Bormo-and 3,5-Dibromo-Tyrosine: Potential Markers for Eosinophil-Dependent Injury In Vivo” by Hazen, et al., Oxygen Society Annual Meeting, Washington, D.C., Nov. 1998,Free Rad. Biol. Med., Supplement I, 25:S74 (#209).
Hazen Stan
Schmitt David
Wu Weijia
Calfee Halter & Griswold LLP
Cleveland Clinic Foundation
Nolan Patrick J.
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