Methods for purifying phycotoxins, pharmaceutical compositions containing purified phycotoxins, and methods of use thereof

• US 8,952,152 B2
• Filed: 09/23/2011
• Issued: 02/10/2015
• Est. Priority Date: 03/24/2009
• Status: Active Grant

• Current Assignee: Proteus Corporation
• Original Assignee: Proteus Corporation
• Inventors: Lagos Gonzlez, Marcelo Santiago
• Primary Examiner(s): Kosar, Andrew D
• Assistant Examiner(s): Kenyon, John S
• Application Number:
  US13/243,153
• Publication Number:
  US 20120065217A1
• Time in Patent Office: 1,236 Days
• Field of Search: 514/267, 544/251
• US Class Current: 544/251
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: C07D 473/00 Heterocyclic compounds containing purine ring systems
• CPC Class Codes: C07D 487/14 Ortho-condensed systems
• CPC Class Codes: C12N 1/12 Unicellular algae; Culture media therefor as new plants A01H13/00
• CPC Class Codes: C12P 17/182 Heterocyclic compounds containing nitrogen atoms as the only ring heteroatoms in the condensed system alloxazine or isoalloxazine, e.g. riboflavine C12P25/00

Abstract

Phycotoxins are purified from a mixture of phycotoxins produced in a continuous process. Cyanobacteria are produced in a continuous culture, then lyzed, the cells pelleted and extracted, and the extract purified using an organic solvent-aqueous mixture and repeated passage through a diatomaceous earth column. The column is washed with acetic acid, then the neosaxitoxin extracted with an alcohol-water mixture. The eluate is passed through activated charcoal columns, which are washed with distilled water to remove the retained pigments and impurities, the further purified by HPLC. In one embodiment, the process produces only neosaxitoxin and saxitoxin. In another embodiment, the process produces only GTX2/3.

Claims

1. A method for the isolation and purification of neosaxitoxin produced by cyanobacteria which produce neosaxitoxin and saxitoxin, the method comprising:

(a) Lysing the cyanobacteria,

(b) cold extracting the phycotoxins from the lysed cyanobacteria at pH 5 to obtain a concentrate,

(c) passing the concentrate through a molecular weight separation column to obtain a phycotoxin eluate containing predominately neosaxitoxin, and

(d) passing the eluate through a cation-exchange column, washing the column with acid and eluting the neosaxitoxin with an acidic alcoholic extraction solution to produce a highly pure neosaxitoxin eluant.

2. The method according to claim 1, in which the extraction in step (b) is repeated.

3. The method according to claim 1, further comprising passing the eluate of the molecular weight column or cation exchange column through a diatomaceous earth column washed with acetic acid and eluting the neosaxitoxin with an alcohol solution.

4. The method according to claim 1, further comprising passing the eluate of the molecular weight column or cation exchange column through activated charcoal and eluting the neosaxitoxin using an alcohol solution.

5. The method according to claim 3, in which the alcohol solution is a 1:

1;

1 vol/vol/vol chloroform;

methanol;

water.

6. The method according to claim 1, wherein the size exclusion chromatography has an 1,800 Dalton exclusion.

7. The method according to claim 1, wherein the cyanobacteria are selected from the group consisting of Cylindrospermopsis sp, Microcystis sp, Anabaena sp, Gomphosphaeria sp, Oscillatoria sp, Aphanizomenon sp and Lyngbya wollei.

8. The method of claim 1, wherein the concentrate obtained from step (b) contains less than 13% saxitoxin.