# US 8,541,549 B2 · Annexin-based apoptosis markers

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 8,541,549 B2
- Document ID: US8541549B2
- Status: Granted
- Kind: B2
- Application number: 12/416,772
- Filed: 2009-04-01
- Published: 2009-10-01
- Granted: 2013-09-24
- Earliest claimed: 2008-04-01
- Assignees: University of Southern California
- Applicants: Ralf Langen
- Inventors: Ralf Langen
- Web page: https://patentgrep.com/patent/US8541549B2

## CPC

- G01N 33/92: involving lipids, e.g. cholesterol {, lipoproteins, or their receptors (steroid hormones G01N33/743)}
- A61K 49/0021: {the fluorescent group being a small organic molecule}
- A61K 49/0056: {Peptides, proteins, polyamino acids}
- C07K 14/4721: {Lipocortins}
- G01N 33/542: with steric inhibition or signal modification, e.g. fluorescent quenching
- G01N 33/575: for cancer
- G01N 2333/4718: Lipocortins
- G01N 2510/00: Detection of programmed cell death, i.e. apoptosis
- 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)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- 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

The present invention describes an annexin derivative and a method of using the annexin derivative as a biosensor for real-time visualization of phosphatidylserine exposure, apoptosis, live-cell imaging and monitoring of cell health.

## Claims

### Claim 1

An annexin derivative, comprising:
the amino acid sequence of SEQ ID NO: 1 wherein residues 101 and 260 of SEQ ID NO: 1 have been substituted with cysteine residues, wherein said cysteine residues are conjugated to polarity sensitive fluorophore N,N′-Dimethyl-N-(iodoacetyl)-N′(7-nitrobenz-2-oxa-1,2-diazol-4-yl)ethylenediamine (IANBD) or 6-Bromoacetyl-2-dimethylaminonaphthalene (BADAN), and wherein residues 113 and 302 of SEQ ID NO: 1 are substituted with an amino acid other than cysteine or lysine.

### Claim 2 (depends on 1)

The annexin derivative of claim 1, wherein the polarity sensitive fluorophore is N,N′-Dimethyl-N-(iodoacetyl)-N′(7-nitrobenz-2-oxa-1,2-diazol-4-yl)ethylenediamine (IANBD).

### Claim 3 (depends on 1)

The annexin derivative of claim 1, wherein the polarity sensitive fluorophore is 6-Bromoacetyl-2-dimethylaminonaphthalene (BADAN).

### Claim 4 (depends on 1)

A method of detecting apoptosis, comprising: contacting the annexin derivative of claim 1 to a sample comprising cells; and detecting a fluorescence resulting from the fluorophore in the sample.

### Claim 5 (depends on 4)

The method of claim 4, wherein detecting the fluorescence comprises detecting an increase of fluorescence intensity and/or a shift in the wavelength of the fluorescence.

### Claim 6 (depends on 5)

The method of claim 5, wherein the increase of fluorescence intensity is one or more orders of magnitude.

### Claim 7 (depends on 5)

The method of claim 5, wherein the polarity sensitive fluorophore is N,N′-Dimethyl-N-(iodoacetyl)-N′(7-nitrobenz-2-oxa-1,2-diazol-4-yl)ethylenediamine (IANBD) and the fluorescence is detected at a λmax of about 500 to about 600 nm.

### Claim 8 (depends on 7)

The method of claim 7, wherein detection is performed in a real time fluorescence detection and/or performed in a high-throughput screening system.

### Claim 9 (depends on 5)

The method of claim 5, wherein the polarity sensitive fluorophore is 6-Bromoacetyl-2-dimethylaminonaphthalene (BADAN) and the fluorescence is detected in the blue light range from 420 nm to 470 nm.

### Claim 10 (depends on 9)

The method of claim 9, wherein the detection of the fluorescence is performed in a real time fluorescence detection and/or in a high-throughput screening system.

### Claim 11 (depends on 1)

A method of monitoring cell health, comprising:
contacting the annexin derivative of claim 1 to a sample comprising cells; and
monitoring the fluorescence resulting from the fluorophore, wherein an increase in the fluorescence intensity and/or a shift in the wavelength of the fluorescence indicate that a cell is undergoing apoptosis and an absence of an increase in the fluorescence intensity and/or an absence of a shift in the wavelength of the fluorescence indicate that a cell is healthy.

### Claim 12 (depends on 11)

The method of claim 11, further comprising administering a therapeutic drug upon detection of one or more cells undergoing apoptosis.

### Claim 13 (depends on 11)

The method of claim 11, wherein the polarity sensitive fluorophore is N,N′-Dimethyl-N-(iodoacetyl)-N′(7-nitrobenz-2-oxa-1,2-diazol-4-yl)ethylenediamine (IANBD).

### Claim 14 (depends on 13)

The method of claim 13, wherein detection is performed in a real time fluorescence detection and/or in a high-throughput screening system.

### Claim 15 (depends on 11)

The method of claim 11, wherein the polarity sensitive fluorophore is 6-Bromoacetyl-2-dimethylaminonaphthalene (BADAN).

### Claim 16

A kit comprising: an annexin derivative comprising the amino acid sequence of SEQ ID NO:1 wherein residues 101 and 260 of SEQ ID NO:1 have been substituted with cysteine residues and said cysteine residues are conjugated to a polarity sensitive fluorophore N,N′-Dimethyl-N-(iodoacetyl)-N′(7-nitrobenz-2-oxa-1,2-diazol-4-yl)ethylenediamine (IANBD) or 6-Bromoacetyl-2-dimethylaminonaphthalene (BADAN), and wherein residues 113 and 302 of said SEQ ID NO:1 are substituted with an amino acid other than cysteine or lysine; and instructions for using the annexin derivative to detect phosphatidylserine exposure.

### Claim 17 (depends on 16)

The kit of claim 16, wherein the annexin derivative is in a cell culture medium.