# US 2014/0154803 A1 · Anti-inflammatory proteins and methods of preparation and use thereof

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- Number: US 2014/0154803 A1
- Document ID: US20140154803A1
- Status: Published application
- Kind: A1
- Application number: 13/997,031
- Filed: 2011-12-22
- Published: 2014-06-05
- Earliest claimed: 2010-12-22
- Assignees: ManukaMed Limited
- Inventors: Amanda Bean; Peter Molan; Ray Cursons; Richard Wilkins
- Web page: https://patentgrep.com/patent/US20140154803A1

## CPC

- A61K 38/1767: {from invertebrates}
- A61K 35/644: Beeswax; Propolis; Royal jelly; Honey
- A61K 47/54: the modifying agent being an organic compound
- A61P 29/00: Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
- C07K 14/43572: {from bees}
- G01N 21/64: Fluorescence; Phosphorescence
- G01N 33/15: Medicinal preparations {; Physical properties thereof, e.g. dissolubility}
- G01N 33/68: involving proteins, peptides or amino acids {(involving lipoproteins G01N33/92)}
- 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)
- G01N 2333/43565: from bees
- 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)
- 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)

## Abstract

The present disclosure relates to anti-inflammatory proteins, their uses, methods of preparation and methods of their detection. In particular, the invention relates to major royal jelly proteins modified by methyglyoxal and fragments thereof from manuka honey.

## Claims

### Claim 1

An isolated apalbumin protein or fragment thereof, which has been chemically modified by methylglyoxal (MGO).

### Claim 2 (depends on 1)

The protein of claim 1 being a modified apalbumin 1 (Apa1) protein or a fragment thereof.

### Claim 3 (depends on 1)

The protein of claim 1 being isolated from manuka honey.

### Claim 4 (depends on 2)

The protein of claim 2 having at least 17 amino acid residues modified by MGO.

### Claim 5 (depends on 2)

The protein of claim 2 having between 17 and 32 amino acid residues modified by MGO.

### Claim 6 (depends on 2)

The protein of claim 2 having about 32 amino acid residues modified by MGO.

### Claim 7 (depends on 2)

The protein of claim 2 wherein the amino acid residues that are modified are either lysine or arginine.

### Claim 8

(canceled)

### Claim 9 (depends on 1)

A composition comprising an isolated protein of claim 1.

### Claim 10

(canceled)

### Claim 11

An isolated MGO-modified apalbumin protein or fragment thereof that has anti-inflammatory capacity and at least 85% sequence identity to an amino acid sequence to the amino acid sequence set forth in SEQ ID NO 1.

### Claim 12

(canceled)

### Claim 13 (depends on 11)

An isolated MGO-modified apalbumin protein or fragment thereof of claim 11, wherein the protein or fragment thereof is isolated from manuka honey.

### Claim 14 (depends on 11)

A composition comprising the protein or fragment thereof of claim 11.

### Claim 15

A method of producing an anti-inflammatory molecule by modifying royal jelly, the method including the step of reacting royal jelly with MGO at a concentration of at least 0.1%, 0.5% or 1.0% MGO.

### Claim at position 16

16-17. (canceled)

### Claim 18 (depends on 15)

The method of claim 15 which further includes the step of isolating the MGO modified apalbumin (MRJP 1) protein from the royal jelly product.

### Claim 19 (depends on 8)

A method of reducing inflammation in a cellular tissue, comprising the step of contacting the composition of claim 8 to the cellular tissue.

### Claim 20 (depends on 8)

A method of reducing the rate of phagocytosis by immune system cells, comprising the step of administering the composition of claim 8 to immune system cells.

### Claim 21 (depends on 8)

A method of inhibiting the receptors for phagocytosis on immune system cells, comprising the step of administering the composition of claim 8 to immune system cells.

### Claim 22 (depends on 8)

A method of reducing the respiratory burst and release of reactive oxygen species in inflammatory cells, comprising the step of administering the composition of claim 8 to the inflammatory cells.

### Claim 23

A method of identifying (i) the anti-inflammatory capacity or (ii) MGO-modified apalbumin concentration of a sample of honey, comprising the steps of:
assaying the fluorescence of the honey sample, and
b) correlating the measure of fluorescence of the honey sample with the anti-inflammatory
capacity of the honey sample, by comparing the measure of fluorescence of a sample of honey and the anti-inflammatory capacity of one or more samples of honey with previously measured capacity to inhibit phagocytosis.

### Claim 24 (depends on 23)

The method of claim 23 wherein the MGO-modified apalbumin is a modified apalbumin 1 protein (modified MRJP1).

### Claim 25 (depends on 23)

The method of claim 23, wherein the method is used to enable a bee keeper to determine the right time to harvest honey from a hive in order to obtain a honey sample containing a desired anti-inflammatory capacity or MGO-modified apalbumin content.

### Claim 26 (depends on 23)

The method of claim 23, wherein the method is used to enable a honey producer to determine a desired length of time to store honey, in order to obtain a honey sample with a desired anti-inflammatory capacity and MGO-modified apalbumin content.

### Claim 27

A method of increasing the anti-inflammatory capacity of one or more apalbumin proteins, by chemically treating the one or more proteins with MGO, formaldehyde, glyoxal and/or glutaraldehyde.

### Claim 28

A method of increasing the anti-inflammatory capacity and MGO-modified apalbumin protein content of a sample of honey, comprising the step of adding MGO or a MGO-precursor molecule to a honey sample.

### Claim 29 (depends on 27)

The method of claim 27 wherein the apalbumin protein is a modified apalbumin 1 protein.

### Claim 30 (depends on 28)

The method of claim 28 wherein the apalbumin protein is a modified apalbumin 1 protein.

### Claim 31 (depends on 14)

A method of reducing inflammation in a cellular tissue, comprising the step of contacting the composition of claim 14 to the cellular tissue.

### Claim 32 (depends on 14)

A method of reducing the rate of phagocytosis by immune system cells, comprising the step of administering the composition of claim 14 to immune system cells.

### Claim 33 (depends on 14)

A method of inhibiting the receptors for phagocytosis on immune system cells, comprising the step of administering the composition of claim 14 to immune system cells.

### Claim 34 (depends on 14)

A method of reducing the respiratory burst and release of reactive oxygen species in inflammatory cells, comprising the step of administering the composition of claim 14 to the inflammatory cells.