# US 2017/0280761 A1 · Method to concentrate apoa-1 mimetic peptides transgenically expressed in plants

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- Number: US 2017/0280761 A1
- Document ID: US20170280761A1
- Status: Published application
- Kind: A1
- Application number: 15/309,428
- Filed: 2015-05-15
- Published: 2017-10-05
- Earliest claimed: 2014-05-15
- Assignees: The Regents of the University of California
- Applicants: The Regents of the University of California
- Inventors: Alan M. Fogelman; Srinivasa T. Reddy; Mohamad Navab
- Web page: https://patentgrep.com/patent/US20170280761A1

## CPC

- A23L 33/18: Peptides; Protein hydrolysates
- A61K 38/10: Peptides having 12 to 20 amino acids {(A61K38/043 - A61K38/046 take precedence)}
- A61P 35/00: Antineoplastic agents
- C07K 7/08: having 12 to 20 amino acids (gastrins C07K14/595; somatostatins C07K14/655; melanotropins C07K14/68)
- C07K 14/775: Apolipopeptides
- C12N 15/8257: {for the production of primary gene products, e.g. pharmaceutical products, interferon}
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- C07K 1/14: Extraction; Separation; Purification
- A23L: FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF (preservation thereof A23B)
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

In various embodiments, methods of concentrating apolipoprotein A-I mimetic peptide activity in transgenic plant material expressing such peptides are provided. In certain embodiments the methods involve providing a tissue of a transgenic plant where the tissue contains a heterologous ApoA-I mimetic peptide expressed by the plant, and where the tissue is provided as a substantially dry powder; mixing the powder with a solution comprising ethyl acetate and acetic acid or with a solution comprising ethyl lactate and lactic acid, to form an extraction mixture; and collecting the liquid phase of said mixture and drying said liquid phase to provide a concentrated dry powder extract that displays the biological activity of the apoA-I mimetic peptide.

## Claims

### Claim 1

A method of concentrating transgenic apoA-I mimetic peptides expressed in a transgenic tomato plant, said method comprising:
providing a tissue of a transgenic plant wherein said tissue contains a heterologous ApoA-I mimetic peptide expressed by said plant, and wherein said tissue is provided as a substantially dry powder;
mixing said power with a solution comprising ethyl acetate and acetic acid or with a solution comprising ethyl lactate and lactic acid, to form an extraction mixture;
collecting the liquid phase of said mixture and drying said liquid phase to provide a concentrated dry powder extract that displays the biological activity of said apoA-I mimetic.

### Claim 2 (depends on 1)

The method of claim 1, wherein said mixing comprises mixing said power with a solution comprising ethyl acetate and acetic acid.

### Claim 3 (depends on 2)

The method of claim 2, wherein said solution comprises about 1% to about 25% acetic acid, or about 2% to about 20% acetic acid, or about 3% to about 15% acetic acid, or about 4% to about 10% acetic acid, or about 4% to about 8% acetic acid, or from about 4% to about 6% acetic acid.

### Claim 4 (depends on 2)

The method of claim 2, wherein said solution comprises about 5% acetic acid.

### Claim 5 (depends on 1)

The method of claim 1, wherein said mixing comprises mixing said power with a solution comprising ethyl lactate and lactic acid.

### Claim 6 (depends on 5)

The method of claim 5, wherein said solution comprises about 1% to about 25% lactic acid, or about 2% to about 20% lactic acid, or about 3% to about 15% lactic acid, or about 4% to about 10% lactic acid, or about 4% to about 8% lactic acid, or from about 4% to about 6% lactic acid.

### Claim 7 (depends on 5)

The method of claim 5, wherein said solution comprises about 5% lactic acid.

### Claim 8 (depends on 1)

The method of claim 1, wherein, wherein said providing tissue comprises providing said tissue as a freeze-dried powder.

### Claim 9 (depends on 1)

The method of claim 1, wherein said mixing said power with a solution of ethyl acetate and acetic acid or ethyl lactate and lactic acid comprises incubating said extraction mixture for at least 15 minutes, or at least ½ hour, or at least 1 hour, or at least 2 hours, or at least 3 hours, or at least 4 hours, or at least 5 hours, or at least 6 hours, or at least 7 hours, or at least 12 hours, or at least 18 hours, or at least 24 hours, or at least 48 hours, or at least 72 hours, or at least 96 hours.

### Claim 10 (depends on 1)

The method of claim 1, wherein said collecting the liquid phase and drying said liquid phase comprises drying the liquid phase to produce said dry powder extract.

### Claim 11 (depends on 1)

The method of claim 1, wherein said collecting the liquid phase and drying said liquid phase comprises:
drying the liquid phase to produce a dry residue;
resuspending said residue in water to provide a resuspended mixture; and
drying the resuspended mixture to provide said dry powder extract.

### Claim 12 (depends on 11)

The method of claim 11, wherein said resuspending comprises re-suspending said residue in distilled water, de-ionized water, or food grade water.

### Claim at position 13

13-14. (canceled)

### Claim 15 (depends on 11)

The method of claim 11, wherein said drying the resuspended mixture comprises lyophilizing said mixture to provide said dry powder extract.

### Claim 16 (depends on 1)

The method of claim 1, wherein said tissue of a transgenic plant comprises tissue of a tomato plant.

### Claim at position 16

17-20. (canceled)

### Claim 21 (depends on 1)

The method of claim 1, wherein said heterologous ApoA-I mimetic peptide expressed by said plant comprises the amino acid sequence DWLKAFYDKFFEKFKEFF (6F) (SEQ ID NO:1), FFEKFKEFFKDYFAKLWD (rev6F) (SEQ ID NO:20), DWFKAFYDKVAEKFKEAF (4F) (SEQ ID NO:11), FAEKFKEAVKDYFAKFWD (rev4F) (SEQ ID NO:18), LLEQLNEQFNWVSRLANL, or LVGRQLEEFL.

### Claim at position 18

22-27. (canceled)

### Claim 28 (depends on 1)

The method according of claim 1, wherein said dry powder extract is effective to decrease plasma levels of lyophosphatidic acid (LPA) in a mammal, and/or to decrease SAA levels in said mammal, and/or to increase plasma paraoxonase activity in said mammal when fed to said mammal alone or as a component of a food or diet.

### Claim at position 20

29-32. (canceled)

### Claim 33 (depends on 1)

A concentrated extract that contains an ApoA-I mimetic peptide where said extract is produced by a method according to claim 1.

### Claim at position 22

34-40. (canceled)

### Claim 41 (depends on 33)

A protein powder or a nutritional supplement, wherein at least a portion of said protein powder or nutritional supplement comprises a concentrated extract of claim 33.

### Claim at position 24

42-49. (canceled)

### Claim 50 (depends on 1)

A method for the treatment or prophylaxis of a pathology characterized by an inflammatory response, said method comprising administering to a mammal in need thereof an effective amount of:
an extract, a protein powder, or a nutritional supplement that contains an ApoA-I mimetic peptide extracted by a method according to claim 1.

### Claim at position 26

51-71. (canceled)

### Claim 72 (depends on 1)

A method of preventing or reducing the uptake of one or more dietary pro-inflammatory micro-lipid components in a mammal, said method comprising administering to the mammal an effective amount of:
an extract, a protein powder, or a nutritional supplement that contains an ApoA-I mimetic peptide extracted by a method according to claim 1.

### Claim at position 28

73-85. (canceled)