# Patent

## Title
Core 2 β(1,6)-acetylglycosaminyltransferase as diagnostic marker for atherosclerosis

## Abstract

A method of indicating the presence of the atherosclerosis (particularly coronary artery atherosclerosis) in a subject is provided, comprising comparing the level of Core 2 GlcNAc-T in a tissue sample from a subject with a reference level determined for the same tissue. A level of Core 2 GlcNAc-T in the tissue sample from a subject that is higher than that of the reference level being indicative that the subject is afflicted with atherosclerosis (particularly coronary artery atherosclerosis—coronary artery disease—CAD). In preferred embodiments, the sample consists of leukocytes and the protein level is determined as the enzymatic activity using radiolabeled UDP-GlcNAc and a Galβ(1,3)-GalNAc derivative.

## Background
This application is the U.S. National Phase of International Application PCT/GB2006/002502, filed 6 Jul. 2006, which designated the U.S. PCT/GB2006/002502 claims priority to British Application No. 0513883.9 filed 6 Jul. 2005. The entire content of these applications are incorporated herein by reference.
The present invention is made in the field of methods for the diagnosis of vascular disease and in particular atherosclerosis.
BACKGROUND OF THE INVENTION
Atherosclerosis (AS) is known to have an inflammatory component and Core 2 GlcNAc-T (also known as UDP-GlcNAc:Galβ1,3GalNAc-R (GlcNAc to GalNAc) β-1,6 N-acetylaminotransferase or Core 2 β-1,6 N-acetylaminotransferase—EC 2.4.1.102) has been implicated in inflammation (WO 0031109). It has been speculated that Core 2 GlcNAc-T inhibitors may be useful in AS (e.g. WO0185748), however, no published studies have examined the levels of Core 2 GlcNAc-T in patients suffering atherosclerosis nor has the level of this enzyme been suggested as a marker for atherosclerosis in a subject.
SUMMARY OF THE INVENTION
The present inventors have surprisingly found that, the level of Core 2 GlcNAc-T activity in blood samples from patients with atherosclerosis is markedly raised compared to non afflicted healthy individuals consequently levels of Core 2 GlcNAc-T may be used to indicate the presence of atherosclerosis in a subject.
Blood samples may be particularly isolated preparations containing Polymorphonuclear Cells—PMNs and other leukocyte sub-populations and more particularly isolated preparations containing PMNs and Peripheral Blood Mononuclear Cells—PBMCs.
Atherosclerosis includes (coronary artery atherosclerosis/coronary artery disease—CAD/Ischemic Heart Disease—IHD/Arteriosclerotic Cardiovascular Disease —ASCVD/Coronary Heart Disease—CHD)
Accordingly a first aspect of the present invention provides a method of indicating the presence of atherosclerosis in a subject comprising comparing the level of Core 2 GlcNAc-T in a tissue sample from a subject with a reference level determined for the same tissue. A level of Core 2 GlcNAc-T in the tissue sample from a subject that is higher than the reference level being indicative that the subject is afflicted with atherosclerosis.
Typically the reference level is established in the tissue by determining the mean level of Core 2 GlcNAc-T in samples of the tissue from a population of one or more individuals associated with an absence of AS; preferably the reference level is established in the tissue by determining the mean level of Core 2 GlcNAc-T in samples of the tissue from a population of 5 or more individuals associated with an absence of AS; more preferably the reference level is established in the tissue by determining the mean level of Core 2 GlcNAc-T in samples of the tissue from a population of 10 or more individuals associated with an absence of AS
Conveniently the tissue sample is a blood sample.
Conveniently the level of Core 2 GlcNAc-T may be determined in Leukocytes which may be isolated from blood by methods well known in the art.
Particularly level of Core 2 GlcNAc-T may be determined in isolated preparations containing PMNs and other leukocyte sub-populations and more particularly in isolated preparations containing PMNs and PBMCs
The level of Core 2 GlcNAc-T may be the level of Core 2 GlcNAc-T RNA transcript, the level of Core 2 GlcNAc-T protein or the level of Core 2 GlcNAc-T enzyme activity; preferably it is the level of Core 2 GlcNAc-T enzyme activity.
Suitable assays for Core 2 GlcNAc-T enzyme activity include those using radio-labelled substrates or acceptor compounds, using fluorescently labelled substrates or acceptor compounds, or by derivatising a formed product prior to analysis (eg by HPLC) for example those described herein or in Chibber et al Diabetes 49, 1724-1730 (2000), Palmerini C. A. et al Glycoconj J. August; 13(4):631-6 (1996) or Kuhns W. et al Glycoconjugate Journal 10 381-394 (1993) (all of which are incorporated herein by reference). Leukocytes may be isolated from blood samples and the Core 2 GlcNAc-T activity determined as described in Chibber et al (2000) or by the protocol described herein under example 1.
The inventors have determined that the level of Core 2 GlcNAc-T enzyme activity in leukocyte preparations obtained from healthy individuals and assayed by the method of Chibber et al (2000) or as detailed in Example 1 is between 40 and 1000 pmoles/hr/mg protein and typically between 50 and 500 pmoles/hr/mg of protein.
The mean value may be between 50 and 1000, typically between 100 and 500 and more typically between 200 and 400 pmoles/hr/mg.
Levels of Core 2 GlcNAc-T in individuals afflicted with atherosclerosis will be in the region of at least 2 times, preferably at least 4 times, more preferably at least 6 times and most preferably at least 8 times the reference level of healthy non afflicted individuals when blood samples are treated and leukocytes assayed according to Chibber et al (2000) or as detailed in example 1.
The method of the invention can conveniently be carried out using a kit comprising components necessary for carrying out the method of the invention. Thus in a second embodiment of the invention is provided a kit for indicating the presence of atherosclerosis in a subject. The kit will preferably comprise an acceptor compound. An acceptor compound is a compound to which Core 2 GlcNAc-T is capable of transferring a monosaccharide residue. Preferably the acceptor compound is a derivative of Galβ(1,3)GalNAcα. Suitable acceptor compounds are for example Galβ(1,3)GalNAc-Bn, Galβ(1,3)GalNAc-p-nitrophenol or Galβ(1,3)GalNAcα-p-NHdansylphenyl.
Optionally the kit will also comprise UDP-GlcNAc, which may be radiolabelled. Conveniently the UDP-GlcNAc is labelled by 14C or 3H. Optionally the kit may further contain N-Acetylglucosamine (GlcNAc) and reagents for lysing leukocytes, such as a detergent (for example Triton-X100). The kit may comprise instructions for its use.
The present invention will now be described further by reference to the following non-limiting Examples, Schemes and Figures. Further embodiments falling within the scope of the claim will occur to those skilled in the art in the light of these



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graph illustrating the levels of Core 2 GlcNAc-T activity in leukocytes from healthy control individuals and subjects with ischaemic heart disease (IHD). For controls n=19 for IHD patients n=13.



EXAMPLES
1. Determination of Core 2 GlcNAc-T Activity in Leukocvtes Isolated from the Blood of Patients Diagnosed with Ischemic Heart Disease
13 Middle aged patients of both genders with IHD and 19 age matched healthy controls were used in this study. Patients with diabetes were excluded. IHD patients included in the study suffered stable angina, unstable angina, or had recently suffered a myocardial infarction.
Blood was taken into heparinised tubes. The blood sample was layered onto an equal volume of HISTO-PAQUE 1077 (Pharmacia and available from Sigma, Poole, Dorset, UK) and centrifuged at 400 g for 30 mins. The Buffy coat (containing peripheral blood mononuclear cells (PBMNC) and polymorphonuclear (PMN) leukocytes) was washed in phosphate buffered saline. Isolated leukocytes were frozen and lysed in 0.9% NaCl 0.4% Triton-X100 1 mM PMSF and the Core 2 GlcNAc-T assayed. The reaction was performed in 50 mmol/l 2(N-morpholino) 2(N-morpholino) ethanesulfonic acid pH 7.0; 1 mmol/l UDP GlcNAc, 0.5 μCi UDP-6 [3H]-N-acetylglucosamine (16,000 dpm/nmol, NEN Life Science Products, Hounslow, U.K.); 0.1 mol/l GlcNAc; 1 mmol/l βgal (1-3)Da-GalNAc-p-nitrophenol and 15 μl cell lysate (100-200 μg protein) for a final volume of 30 μl. After incubating the mixture for 1 h at 37° C., the reaction was terminated by adding 1 ml of ice cold water and processed on a C18 SEP-PAK column (Waters-Millipore, Watford, U.K.). After washing the column with 20 ml water, the product was eluted with 5 ml methanol and radioactivity counted. Endogenous activity of Core 2 GlcNAc-T was measured in the absence of the added acceptor. The results are shown in FIG. 1.
Core 2 GlcNAc-T activity in healthy individuals was 287±147.2 pmoles/hr/mg or protein, whist in patients with IHD the value was 2376±461. These values are in agreement with those for three groups of healthy individuals in Chibber et al (2000) in which values were 249±35.9 (n=25), 334±86 (n=11) and 283±37 (n=31) pmols/hr/mg.

## Claims

1. A method of indicating possible atherosclerosis in a subject comprising comparing the level of Core 2 GlcNAc-T enzyme activity in a blood sample from a subject with a reference enzyme activity level determined for a blood sample, wherein a level of Core 2 GlcNAc-T enzyme activity in the blood sample from a subject that is higher than the reference enzyme activity level being indicative that the subject may be afflicted with atherosclerosis.

2. A method according to claim 1 wherein the level of core 2 GlcNac-T enzyme activity is measured in leukocytes.

3. A method according to claim 2 in which the leukocytes are isolated from the blood.

4. A method according to claim 2 wherein a level of Core 2 GlcNAc-T enzyme activity of the leukocytes of a subject is at least 2 times higher than that of the reference level is indicative of atherosclerosis in the subject.

5. A method according to claim 2 wherein a level of Core 2 GlcNAc-T enzyme activity of the leukocytes of a subject is at least 4 times higher than that of the reference enzyme activity level is indicative of atherosclerosis in the subject.

6. A method according to claim 2 wherein a level of Core 2 GlcNac-T enzyme activity of the leukocytes of a subject is at least 2 times higher than that of the reference enzyme activity is indicative of atheroscleosis in the subject.

7. A method according to claim 2 wherein a level of Core 2 GlcNAc-T enzyme activity of the leukocytes of a subject is at least 8 times higher than that of the reference level is indicative of atherosclerosis in the subject.