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

• US 9,249,150 B2
• Filed: 12/15/2014
• Issued: 02/02/2016
• Est. Priority Date: 03/24/2009
• Status: Active Grant

• Current Assignee: Proteus Corporation
• Original Assignee: Proteus Corporation
• Inventors: Lagos Gonzalez, Marcelo Santiago
• Primary Examiner(s): Jarrell, Noble
• Assistant Examiner(s): Kenyon, John S
• Application Number:
  US14/570,360
• Publication Number:
  US 20150099878A1
• Time in Patent Office: 414 Days
• US Class Current: 1/1
• 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 phycotoxins produced by cyanobacteria, the method comprising

a. Lysing the cyanobacteria cells of sonication to obtain a lysate,

b. isolating the liquid component of the lysate by centrifugation,

c. passing the liquid lysate through size exclusion gel filtration and collecting the eluted fractions,

d. identifying and isolating eluted fractions containing a phycotoxins,

e. passing the isolated fractions through cation-exchange chromatography, and collecting the eluted fractions,

f. identifying and isolating eluted fractions containing the purified phycotoxins extracted from the lysed cyanobacteria,

wherein the phycotoxin in the extract is selected from the group consisting of neosaxitoxin, saxitoxin, gonyaulatoxin 2 and gonyaulatoxin 3.

2. The method of claim 1, wherein the cyanobacteria is selected from the genera Cylindrospermopsis sp, Anabaena sp, Gomophosphaeria sp, Oscillatoria sp, Aphanizomenon sp, Lyngbya sp, and combinations thereof.

3. The method of claim 2, wherein the species is selected from the group consisting of Cylindrospermopsis raciborskii, Anphanizomenon flos-aquae, Aphanizomenon (APh) issatschenkoi (usaceb) Proskina-Lavrenco, Aphanizomenon gracile (Lemm) Lemm, Anabaena circinalis, Lyngbya wollei, and combinations thereof.

4. The method of claim 1, wherein the cyanobacterium is Aphanizomenon gracile (Lemm) Lemm.

5. The method of claim 1, wherein the extract contains less than 13% by weight of saxitoxin.

6. The method of claim 1, wherein the extract contains at least 98% pure neosaxitoxin.

7. The method of claim 6, wherein the structure of the neosaxitoxin is

8. The method of claim 1, wherein the extract contains at least 98% pure saxitoxin.

9. The method of claim 8, wherein the structure of the saxitoxin is