# US 2014/0193846 A1 · Novel apoc-i isoforms and their use as biomarkers and risk factors of atherosclerotic disease

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- Number: US 2014/0193846 A1
- Document ID: US20140193846A1
- Status: Published application
- Kind: A1
- Application number: 14/112,670
- Filed: 2011-12-23
- Published: 2014-07-10
- Earliest claimed: 2011-04-19
- Assignees: Scott & White Healthcare; The Johns Hopkins University; The Texas A&M University System
- Inventors: Ronald D. Macfarlane; Catherine J. McNeal; Subroto Chatterjee
- Web page: https://patentgrep.com/patent/US20140193846A1

## CPC

- G01N 33/92: involving lipids, e.g. cholesterol {, lipoproteins, or their receptors (steroid hormones G01N33/743)}
- C07K 14/775: Apolipopeptides
- G01N 2800/324: Coronary artery diseases, e.g. angina pectoris, myocardial infarction
- G01N 2800/50: Determining the risk of developing a disease
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

Novel isoforms of apolipoprotein C-I (apoC-I), namely apolipoprotein C-I1 (apoC-I1) and apolipoprotein C-I1′ (apoC-I1′), both of which have a molecular weight of approximately 90 daltons greater than native apolipoprotein C-I (SEQ ID NO:6) and native apolipoprotein C-I′ (SEQ ID NO:7), are shown to be both biomarkers for diagnosing atherosclerotic disease as well as risk factors for subjects having increased risk of developing an atherosclerotic disease.

## Claims

### Claim at position 1

-95. (canceled)

### Claim 96

A method of diagnosing or determining risk of developing coronary artery disease in a subject, comprising any one of:
(i) assessing a serum sample from the subject for presence of a first isoform of apolipoprotein C-I comprising 20 or more consecutive amino acids of SEQ ID NO:6, wherein the first isoform has a molecular weight of between 6.7 kD and 6.8 kD and presence of the first isoform in the sample indicates that the subject has coronary artery disease or is at increased risk of developing coronary artery disease compared to a subject without presence of the isoform;
(ii) assessing a serum sample from the subject for presence of a second isoform of apolipoprotein C-I comprising 20 or more consecutive amino acids of SEQ ID NO:7, wherein the second isoform has a molecular weight of between 6.5 kD and 6.55 kD and presence of the second isoform in the sample indicates that the subject has coronary artery disease or is at increased risk of developing coronary artery disease compared to a subject without presence of the isoform;
(iii) determining the molecular weight of apolipoprotein C-I isoforms of apolipoprotein C-I associated with high density lipoprotein2 (HDL2) or high density lipoprotein3 (HDL3) in a biological sample from the subject, and identifying the subject as being at risk for developing coronary artery disease if the apolipoprotein C-I isoforms associated with HDL2 or HDL3 in the sample have a molecular weight that is 50 daltons to 150 daltons greater than native apolipoprotein C-I (SEQ ID NO:6) or native apolipoprotein C-I′ (SEQ ID NO:7), respectively;
(iv) assessing whether a mass spectrum for HDL apolipoprotein C-I proteins recovered from the HDL2 or HDL3 fractions from a serum sample from the subject comprises a peak in the mass spectrum at between 6.7 kD and 6.8 kD, wherein presence of the peak indicates that the subject has coronary artery disease or is at increased risk of developing coronary artery disease compared to a subject without the peak in the mass spectrum at between 6.7 kD and 6.8 kD;
(v) assessing whether a mass spectrum for HDL apolipoprotein C-I or C-I′ proteins recovered from the HDL2 or HDL3 fractions from a biological sample obtained from the subject comprises a peak in the mass spectrum at between 6.5 kD and 6.55 kD, wherein presence of the peak indicates that the subject has coronary artery disease or is at increased risk of developing coronary artery disease compared to a subject without the peak in the mass spectrum at between 6.5 kD and 6.55 kD;
(vi) contacting HDL2 or HDL3 sub-fractions from a biological sample from the subject with an aortic smooth muscle cell, wherein the ability of the HDL2 or HDL3 sub-fractions to induce apoptosis of the aortic smooth muscle cell is indicative of the subject having or being at risk for developing coronary artery disease; or
(vii) contacting HDL2 or HDL3 sub-fractions from a biological sample from the subject with an endothelial cell, wherein the ability of the HDL2 or HDL3 sub-fractions to induce expression of nitric oxide and/or ICAM-1 at a greater level than HDL2 or HDL3 sub-fractions from a biological sample from a subject without coronary artery disease is indicative of the subject having or being at risk for developing coronary artery disease.

### Claim 97 (depends on 96)

The method of claim 96, wherein the coronary artery disease is selected from the group consisting of atherosclerosis-associated plaque rupture, myocardial infarction, angina, and coronary ischemia.

### Claim 98 (depends on 96)

The method of claim 96, wherein the subject is a mammal.

### Claim 99 (depends on 98)

The method of claim 98, wherein the mammal is a human.

### Claim 100 (depends on 96)

The method of claim 96, wherein in (i) or (ii), the first isoform of apolipoprotein C-I comprises 30 or more consecutive amino acids of SEQ ID NO:6 and the second isoform of apolipoprotein C-I comprises 30 or more consecutive amino acids of SEQ ID NO:7.

### Claim 101 (depends on 96)

The method of claim 96, wherein in (i) or (ii), the first isoform of apolipoprotein C-I comprises 40 or more consecutive amino acids of SEQ ID NO:6 and the second isoform of apolipoprotein C-I comprises 40 or more consecutive amino acids of SEQ ID NO:7.

### Claim 102 (depends on 96)

The method of claim 96, wherein in (i) or (ii), the first isoform has a molecular weight of between 6.7 kD and 6.75 kD and the second isoform has a molecular weight of between 6.51 kD and 6.53 kD.

### Claim 103 (depends on 96)

The method of claim 96, wherein in (iii), (v), and (vii), the biological sample is a blood sample or a serum sample.

### Claim 104 (depends on 96)

The method of claim 96, wherein in (iii), (v), and (vii), the biological sample was previously obtained from the subject.

### Claim 105 (depends on 96)

The method of claim 96, wherein in (iii), the apolipoprotein C-I isoforms associated with HDL2 or HDL3 in the sample have a molecular weight that is 80 daltons to 100 daltons greater than native apolipoprotein C-I (SEQ ID NO:6) or apolipoprotein C-I′ (SEQ ID NO:7), respectively.

### Claim 106 (depends on 96)

The method of claim 96, wherein in (iv), the mass spectrum comprises a peak between 6.7 kD and 6.75 kD or wherein in (v) the mass spectrum comprises a peak between 6.51 kD and 6.53 kD.

### Claim 107 (depends on 96)

The method of claim 96, wherein in (iv) or (v), the apolipoprotein C-I protein measured by mass spectrometry is oxidized.

### Claim 108 (depends on 96)

The method of claim 96, wherein in (vi), the aortic smooth muscle cell is a human aortic smooth muscle cell.

### Claim 109 (depends on 96)

The method of claim 96, wherein in (vi) or (vii), the HDL2 sub-fraction of the sample corresponds to a density range of 1.063 g/mL to 1.125 g/mL in a lipoprotein density profile and wherein the HDL3 sub-fraction of the sample corresponds to a density range of 1.125 g/mL to 1.210 g/mL in a lipoprotein density profile.

### Claim 110 (depends on 96)

The method of claim 96, wherein in (vii), the endothelial cell is a human aortic endothelial cell.

### Claim 111

A method for identifying a compound that inhibits the development of coronary artery disease and/or is useful in the treatment of coronary artery disease, the method comprising:
(a) administering the compound to a subject exhibiting coronary artery disease;
(b) obtaining a high density lipoprotein (HDL2) or high density lipoprotein3 (HDL3) sub-fraction from a biological sample from the subject; and
(c) determining the molecular weight of the apolipoprotein C-I isoforms in the HDL2 or HDL3 sub-fractions of the sample,
wherein the compound is determined to inhibit the development of coronary artery disease and/or be useful in the treatment of coronary artery disease if the molecular weight of the apolipoprotein C-I isoforms in the HDL2 or HDL3 sub-fractions of the sample is about the same molecular weight as apolipoprotein C-I isoforms in the HDL2 or HDL3 sub-fractions of an angiographically-normal subject.

### Claim 112 (depends on 111)

The method of claim 111, wherein the coronary artery disease is selected from the group consisting of atherosclerosis-associated plaque rupture, myocardial infarction, angina, and coronary ischemia.

### Claim 113

An isolated apolipoprotein C-I protein comprising 20 or more consecutive amino acids of SEQ ID NO:6 and having a molecular weight of 50 daltons to 100 daltons greater than the protein encoded by SEQ ID NO:6.

### Claim 114 (depends on 113)

The protein of claim 113, wherein the protein has a molecular weight of (i) 80 daltons to 100 daltons greater than the protein encoded by SEQ ID NO:6.

### Claim 115 (depends on 113)

The protein of claim 113, wherein the protein has a molecular weight between 6.7 kD and 6.8 kD.