# US 7,407,779 B2 · Modification of solid 3-sn-phosphoglycerides

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- Number: US 7,407,779 B2
- Document ID: US7407779B2
- Status: Granted
- Kind: B2
- Application number: 10/492,252
- Filed: 2002-10-11
- Published: 2004-12-23
- Granted: 2008-08-05
- Earliest claimed: 2001-10-11
- Assignees: BioMolecular Products, Inc.
- Inventors: David W. Yesair; Walter A. Shaw; Stephen W. Burgess; Robert Travis McKee
- Web page: https://patentgrep.com/patent/US7407779B2

## CPC

- A61K 47/24: containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
- A61K 9/0014: {Skin, i.e. galenical aspects of topical compositions (non-active ingredients are additionally classified in A61K47/00; A61K9/0009, A61K9/0021, A61K9/7015, A61K9/7023 take precedence; cosmetic preparations A61K8/00, A61Q; preparations for wound dressings or bandages A61L26/00)}
- A61K 9/1274: Non-vesicle bilayer structures, e.g. liquid crystals, tubules, cubic phases or cochleates; Sponge phases
- A61P 11/00: Drugs for disorders of the respiratory system
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C12P 7/6481: Phosphoglycerides (phosphoglycerides having carboxylic acids with less than seven carbon atoms C12P7/62)
- C12P 9/00: Preparation of organic compounds containing a metal or atom other than H, N, C, O, S or halogen {(phosphoglycerides, C12P7/6481)}
- C12P 13/001: {Amines; Imines}
- C12P 13/06: Alanine; Leucine; Isoleucine; Serine; Homoserine
- 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
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}

## Abstract

Methods for hydrolyzing solid ungranulated lysophosphatidylcholine with phospholipase A2 are provided. Also disclosed are methods for making a lipid matrix of lysophosphatidylcholine, monoglyceride and fatty acid, and lipid matrices of particular structure.

## Claims

### Claim 1

A method for modifying ungranulated/solid matrix 3-sn-phosphoglyceride molecules, comprising
forming a reaction mixture by contacting ungranulated/solid matrix 3-sn-phosphoglyceride molecules with an amount of phospholipase A2 sufficient to modify an ester bond of the 3-sn-phosphoglyceride molecules,
adding to the reaction mixture one or more fatty acids and one or more agents selected from the group consisting of monoglyceride; diglyceride; polyglycerol fatty acid ester; sucrose fatty acid ester; sorbitan fatty acid ester; glycerol and other alcohol functional groups including serine and ethanolamine; and solvents; and
incubating the reaction mixture to hydrolyze the 2-acyl bond,
wherein the ungranulated/solid matrix 3-sn-phosphoglyceride molecules are hydrolyzed with >99% efficiency and wherein the hydrolysis of the ungranulated/solid matrix 3-sn-phosphogglyceride molecules is complete within 7 hours.

### Claim 2 (depends on 1)

The method of claim 1, wherein the 3-sn-phosphoglyceride is phosphatidylcholine.

### Claim 3 (depends on 1)

The method of claim 1, wherein the fatty acids comprise 8-24 carbon atoms and 0-6 cis or trans double bonds with or without methyl branches and/or hydroxyl groups at any carbon atom.

### Claim 4 (depends on 1)

The method of claim 1, wherein the agent is monoglyceride.

### Claim 5 (depends on 4)

The method of claim 4, wherein the monoglyceride has an acyl group and the acyl group comprises 8-24 carbon atoms and 0-6 cis or trans double bonds with or without methyl branches and/or hydroxyl groups at any carbon atom.

### Claim 6 (depends on 1)

The method of claim 1, further comprising adding calcium ions or other multivalent ions.

### Claim 7

A method for making lysophosphoglyceride comprising:
contacting a mixture of ungranulated/solid matrix 3-sn-phosphoglyceride, one or more fatty acids and an agent selected from the group consisting of monoglyceride; diglyceride; polyglycerol fatty acid ester; sucrose fatty acid ester; sorbitan fatty acid ester; glycerol and other alcohol functional groups including serine and ethanolamine; and solvents, with phospholipase A2 to form a reaction mixture wherein the ungradulated/solid matrix 3-sn-phosphoglyceride molecules are hydrolyzed with >99% efficiency, and wherein the hydrolysis of the ungradulated/solid matrix 3-sn-phosphoglyceride molecules is complete within 7 hours, and
recovering lysophosphoglyceride formed in the reaction mixture.

### Claim 8 (depends on 7)

The method of claim 7, wherein the fatty acids comprise 8-24 carbon atoms and 0-6 cis or trans double bonds with or without methyl branches and/or hydroxyl groups at any carbon atom.

### Claim 9 (depends on 7)

The method of claim 7, wherein the agent is monoglyceride.

### Claim 10 (depends on 9)

The method of claim 9, wherein the monoglyceride has an acyl group and the acyl group comprises 8-24 carbon atoms and 0-6 cis or trans double bonds with or without methyl branches and/or hydroxyl groups at any carbon atom.

### Claim 11 (depends on 7)

The method of claim 7, wherein the step of recovering comprises separating lysophosphoglyceride from one or more reaction mixture constituents selected from the group consisting of 3-sn-phosphoglyceride, fatty acid, and the agent.

### Claim 12 (depends on 11)

The method of claim 11, wherein separation of lysophosphoglyceride from the one or more reaction mixture constituents comprises extraction with acetone.

### Claim 13 (depends on 7)

The method of claim 7, wherein the 3-sn-phosphoglyceride in the reaction mixture is greater than about 40% by weight of the mixture.

### Claim 14 (depends on 13)

The method of claim 13, wherein the phosphatidyicholine in the reaction mixture is greater than about 50% by weight of the mixture.

### Claim 15 (depends on 14)

The method of claim 14, wherein the phosphatidyicholine in the reaction mixture is greater than about 60% by weight of the mixture.

### Claim 16 (depends on 7)

The method of claim 7, wherein the lysophosphoglyceride is lysophosphatidylcholine, and/or wherein the 3-sn-phosphoglyceride is phosphatidylcholine.

### Claim 17

A method for making a composition containing lysophosphoglyceride, monoglyceride and fatty acid, comprising:
contacting a reaction mixture of ungranulated/solid matrix 3-sn-phosphoglyceride, one or more fatty acids and monoglyceride with phospholipase A2 wherein the ungradulated/solid matrix 3-sn-phosphoglyceride molecules are hydrolized with >99% efficiency, and wherein the hydrolysis of the ungradulated/solid matrix 3-sn-pholphoglyceride molecules is complete within 7 hours, and
recovering a lipid complex containing lysophosphoglyceride, monoglyceride and fatty acid, wherein the molar ratio of lysophosphoglyceride:the sum of monoglyceride and fatty acid in the recovered lipid complex composition is between 1:3 and 1:12.

### Claim 18 (depends on 17)

The method of claim 17 wherein the molar ratio of lysophosphoglyceride:the sum of monoglyceride and fatty acid in the recovered lipid complex composition is between 1:5 and 1:6.

### Claim 19 (depends on 18)

The method of claim 18 wherein the recovered lipid complex composition has a lysophosphoglyceride:monoglyceride:fatty acid molar ratio between 1:4:2 and 1:2:4.

### Claim 20 (depends on 19)

The method of claim 19 wherein the recovered lipid complex composition has a lysophosphoglyceride:monoglyceride:fatty acid molar ratio selected from the group consisting of 1:4:2, 1:3:3 and 1:3:2.

### Claim 21 (depends on 17)

The method of claim 17 wherein the monoglyceride is derived from natural triglyceride.

### Claim 22 (depends on 17)

The method of claim 17, wherein the step of recovering the lipid complex comprises removal of water.

### Claim 23 (depends on 17)

The method of claim 17, wherein the lysophosphoglyceride is lysophosphatidylcholine.