Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
1998-09-02
2001-09-18
Warden, Jill (Department: 1743)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C204S452000, C204S603000
Reexamination Certificate
active
06291439
ABSTRACT:
FIELD OF THE INVENTION
The present invention is in the field of medicine and biochemistry, particularly heparin chemistry. In particular, the invention provides a novel method for diagnosing atherosclerosis and predicting the risk for developing atherosclerosis by measuring the amount of endogenous heparin present in a mammal. The invention also provides novel methods for measuring levels of endogenous heparin in a mammal and methods for treating atherosclerosis by administering heparin to individuals identified according to the methods of the invention.
BACKGROUND OF THE INVENTION
Carbohydrates play a number of important roles in the functioning of living organisms. In addition to their metabolic roles, carbohydrates are structural components of the human body covalently attached to numerous other entities such as proteins and lipids (called glycoconjugates). For example, the human endothelium cell surface makeup includes a glycoprotein matrix. The carbohydrate portion of this matrix imparts important properties to the endothelial cell surface and internal blood vessel structure as well as affecting the fluidity of the blood which interacts with the endothelium surface.
Atherosclerosis is a disease characterized by changes in the endothelium and the underlying stromal compartment with cellular proliferation and accumulation of lipid-filled macrophages. This disease kills over a million Americans every year (American Heart Association, 1998
, Heart and Stroke Statistical Update
). The numbers of people affected but asymptomatic is probably higher due to the lack of early diagnostic techniques. Early in the disease process, clinical symptoms are absent and the disease is usually not recognized by the patient or physician. By the time clinical symptoms manifest, a significant amount of morbidity and mortality have occurred.
Preventive techniques to control the onset of atherosclerosis include physical activity, a healthy diet, cessation of smoking and weight loss (US Preventative Services Task Force,
Guide to the Clinical Preventative Services
, 2
nd
ed., Baltimore, Williams & Wilkins, 1996). These intervention therapies require accurate and sensitive measures of atherosclerosis to be able to adequately assess immediate and long-term efficacy. One of the best measures of susceptibility to atherosclerosis is assessment of coronary artery calcium (Arad et al.,
Circulation
93:1951-1953 (1996); Simon et al.,
American Heart Association
92:1414-1421 (1995)). Although assessing levels of coronary artery calcium yields measurements that have been shown to be highly correlated with risk factors, these measurements do not provide information on the chance for a thrombotic event. It is clear that more accurate and sensitive methods for early detection of coronary atherosclerosis and risk for coronary thrombosis are needed.
A number of biochemical measurements in the blood are associated with an increased risk of developing atherosclerosis. These include glucose, lipids, lipoproteins, apolipoproteins and homocystine. In assessing these new tests, it is important to establish that they can, in practice, be used to predict development of the actual condition of atherosclerosis rather than a “risk-factor” for the tendency to develop atherosclerosis. The validation of predictive factors is urgent due to the need to apply emerging new treatments for atherosclerosis to those individuals at high risk.
Some have speculated that endogenous heparin levels are inversely related to cholesterol and lipoprotein levels (Engelberg,
Circulation
23:573-577 (1961)). However, an effective way to measure endogenous heparin and correlate the levels present with atherosclerotic risk have not been available previously.
Glycosaminoglycans are sugar chains consisting of repeating polymers of acidic polysaccharides. These materials are composed of building blocks of the following sugars in various combinations: galactose, glucose, N-acetylglucosamine, N-acetylgalactosamine, glucuronic acid, galacturonic acid and iduronic acid. In addition these sugar units may be variably linked &agr; or &bgr; at their anomeric carbons and (1-3) or (1-4) to their ring carbons through an O-glycosidic bond. Finally they may be variably substituted with sulfates at their 2,3,4 or 6 carbons. Depending on the precise repeating disaccharide structure and location of sulfates, human connective tissue glycosaminoglycans are commonly classified as chondroitin sulfates, dermatan sulfates, heparan sulfates, heparin sulfates and keratan sulfates (Collins,
Carbohydrates
, London, Chapman Hall, (1987)). Glycosaminoglycans are carbohydrates that are integrally associated with the endothelium and are thought to be the major source of naturally-occurring anticoagulants in human blood.
Glycosaminoglycans (GAGs) are present in mammalian blood, urine and other body fluids and are sensitive markers for the diagnosis of lysosomal storage diseases (Klock et al.,
Internat Pediatr
, 9:40-48 (1994); Starr et al.,
Glycosylation
&
Disease
15 1:165-176 (1994)). Degradation products of GAGs are found in urine. The concentration of individual carbohydrates in a sample can be measured by FACE®, an acronym standing for the technique of fluorophore—assisted carbohydrate electrophoresis. The FACE technique is described in detail in U.S. Pat. Nos. 4,975,165, 5,035,786, 5,087,337, 5,094,731, 5,094,740, 5,205,917, 5,316,638, 5,340,453, and 5,472,582, the disclosures of which are herein incorporated by reference. FACE® permits the electrophoretic separation of a complex mixture of carbohydrates into distinct bands on a gel. Prior to electrophoresis, a carbohydrate mixture for analysis is treated with a charged fluorescent tag that combines with the reducing end of the carbohydrates for analysis. The fluorescent label permits the quantitative measurement of the labeled carbohydrates. The charged tag not only fluorescently labels the carbohydrates, but imparts an ionic charge, thus permitting hitherto uncharged carbohydrates to migrate in an electric field. Suitable fluorescent labels include 8-aminonapthalene -1,3,6-trisulphonic acid (ANTS), 1-amino-4-napthalene sulfonic acid (ANSA), 1-amino-6,8-disulphonic acid (ANDA),2-aminoacridone and lucifer yellow. After the carbohydrates have been labeled, the sample is subjected to polyacrylamide gel electrophoresis in order to separate and concentrate the labeled carbohydrates into bands. The separated carbohydrates may be visualized directly by fluorescence under ultraviolet light. Alternatively the separated carbohydrates may be visualized by means of a laser-scanner photomultiplier tube system, a charge coupled device (CCD). CCD's are semiconductor imaging devices that permit the sensitive detection of emitted light.
CCD's and their uses are described in U.S. Pat. No. 4,874,492, the disclosure of which is herein incorporated by reference. The image produced by the CCD may be subsequently transferred to a computer wherein the bands may be analyzed with respect to intensity, mobility, standards, and the like.
The development of FACE® technology (Jackson,
Biochem J
270:705-713 (1990)) and its adaptation for measuring carbohydrate analytes in several disease processes (Klock et al., “The Different Faces of Disease, FACE Diagnosis of Disease”,
Glycoimmunology
, eds. Alavi and Axford, (1995) pages. 13-25) have provided a new method for high-resolution separation of nanomolar quantities of monosaccharides and oligosaccharides derived from human tissues. The separation of carbohydrates using CE has been described in recent publications, such as Honda et al.,
Biochem
. 191:228-234 (1990) and Liu et al.,
J. Chromatography,
559:223-235 (1991). The present invention is based in part on the discovery that the increased sensitivity of the FACE® heparin assay now makes it possible to measure endogenous production of heparin in the blood and urine. The present invention also takes advantage of the increased sensitivity of the FACE® heparin assay to provide novel methods for monitoring heparin levels in patients r
Bigornia Luisa
BioMarin Pharmaceuticals
Halluin Albert P.
Howrey Simon Arnold & White , LLP
Starsiak Jr. John S.
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