# Patent

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

## 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.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a continuation of pending application of pending prior application Ser. No. 13/243,153 filed Sep. 23, 2011, entitled “METHODS FOR PURIFING PHYCOTOTXIONS, PHARMACEUTICAL COPMPOSITIONS CONTAINING PURIFIED PHYCOTOXINS, AND METHODS OF USE THEREOF”, by Marcel Santiago Lagos González, which is a continuation-in-part of PCT/IB2010/051187 filed Mar. 18, 2010, which claims priority to Chilean Patent Application No. 723-2009, U.S. Ser. No. 13/243,153 is also a continuation-in-part of PCT/IB2010/051188 filed Mar. 18, 2010, which claims priority to Chilean Patent Application No. 723-2009 filed Mar. 24, 2009, all of which are incorporated by reference.


FIELD OF THE INVENTION
This invention is in the field of methods of purification of phycotoxins from natural sources, particularly methods for the continuous production and purification of phycotoxins from cyanobacteria and use of the purified phycotoxins in pharmaceutical compositions.
BACKGROUND OF THE INVENTION
Phycotoxins ([phyco=seaweeds and algae] plus toxins) are a diverse group of substances produced by various aquatic plants in marine and fresh waters throughout the world. Not all aquatic plants produce toxins; and among those that do, not all, even from the same genera and species, produce toxins at all times and under all circumstances. Such toxins are associated with large fish kills and major kills of marine mammals. These toxins can also be found in shellfish.
Neosaxitoxin and saxitoxin are two specific phycotoxins produced by dinoflagellate species of the genera Alexandrium sp., Piridinium sp. and Gimnodinium sp. and cyanobacteria.
Neosaxitoxin and saxitoxin acts as specific blockers of the voltage-dependent sodium channels present in excitable cells. Due to the inhibition of sodium channels, the transmission of a nervous impulse is blocked preventing the release of neurotransmitters at the neuromotor junction, which prevents muscular contraction. Due to these physiological effects, these compounds are potentially useful in pharmacology when used as muscular activity inhibitors in pathologies associated with muscular hyperactivity, such as muscular spasms and focal dystonias, when administered locally. These compounds can also inhibit sensory pathways and generate an anesthetic effect when administered locally.
However, these compounds are not available commercially in the quantities necessary to manufacture pharmaceutical compositions. Therefore, there exists a need for improved methods to produce and purify phycotoxins having a definite compositional profile, particularly phycotoxin mixtures containing only neosaxitoxin and saxitoxin or GTX 2 and GTX 3.
Therefore, it is an object of the invention to improved methods for producing and purifying phycotoxins having a definite compositional profile, particularly phycotoxin mixtures containing only neosaxitoxin and saxitoxin or GTX 2 and GTX 3.
It is also an object of the invention to provide pharmaceutical compositions containing the purified phycotoxin(s) and methods of use thereof.
SUMMARY OF THE INVENTION
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, and the phycotoxin(s) (e.g., 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, and further purified by HPLC.
In a particular embodiment, the phycotoxins are produced in a continuous process in which one or more organic nutrients are added to the culture medium in order to form a mixture of phycotoxins having a definite compositional profile. In one embodiment, the process produces only neosaxitoxin and saxitoxin. In another embodiment, the process produces only GTX2/3.
Pharmaceutical compositions containing the purified phycotoxin(s) are also described herein. The compound(s) can be combined with one or more pharmaceutically acceptable excipients to prepare pharmaceutical compositions. The compounds can be formulated for parenteral administration, such as intravenous administration. In another embodiment, the compound(s) are formulated in an implant, such as an implant that provides extended or sustained release of the compound(s).



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a chromatogram of an extract of the phycotoxins produced by the cyanobacterium Aphanizomenon gracile as analyzed high-performance liquid chromatography (HPLC) equipment. The detection method used was fluorescence online detection. Peak 1, RT=2.640 minutes, cyanobacterial pigments. Peak 2, RT=7.980 minutes, neosaxitoxin. Peak 3, RT=11.970 minutes, saxitoxin.
FIG. 2 is a chromatogram of 10 microliters of a purified extract from Aphanizomenon gracile purified by preparative size exclusion high-performance liquid chromatography (separation by molecular size). The detection method used was fluorescence online detection. Peak 1, RT=3.067 minutes, cyanobacterial pigments. Peak 2, RT=9.373 minutes, neosaxitoxin. Peak 3, RT=12.953 minutes, saxitoxin.
FIG. 3 shows a chromatogram of an analytical standard including neosaxitoxin and saxitoxin as its main components, with a small amount of a mixture of gonyaulatoxins (NL2 standard), determined and quantified by analytical high-performance liquid chromatography with online fluorescence detection. Peak 1, RT=3.653 minutes, mixture of gonyaulatoxins; peak 2, RT=5.040 minutes, neosaxitoxin; peak 3, RT=7.440 minutes, saxitoxin.
FIG. 4 is a chromatogram of a neosaxitoxin sample purified from extracts of Aphanizomenon gracile cyanobacteria (RT=4.933 minutes). This corresponds to the fraction eluted from a preparative high-performance liquid chromatography using ion exchange columns with online fluorescence detection.
FIG. 5 shows a chromatogram of analytical standards of gonyaulatoxins. From left to right: Peak 1: GTX4 (gonyaulatoxin 4); Peak 2: GTX1 (gonyaulatoxin 1); Peak 3: GTX5 (gonyaulatoxin 5); Peak 4: GTX3 (gonyaulatoxin 3); Peak 5: GTX2 (gonyaulatoxin 2).
FIG. 6 shows a chromatogram of a sample of extract from the cyanobacterium C. raciborskii. Peak 1, RT=8.873 minutes, peak 2, RT=10.927 minutes, peak 3, RT=13.260 minutes; peak 4, RT=16.647 minutes.
FIG. 7 is a chromatogram of a fraction from C. raciborkii partially purified by size exclusion high-performance liquid chromatography, with a dominant presence of the epimers GTX 3 and GTX 2, respectively.
FIG. 8 shows a chromatogram of the final pure fraction of the epimers GTX 3 and GTX 2 obtained from a continuous culture of C. raciborkii cyanobacteria.
FIG. 9 shows an elution plot from a size exclusion preparative chromatography column showing the obtained fractions and identifying the range of fractions where the different compounds are present (organic compounds, phycotoxins and salts).
FIG. 10 shows an elution plot from an anion exchange preparative chromatography showing the obtained fractions and identifying the range of fractions where the different compounds are present (phycotoxins and salts).
FIG. 11 shows an elution plot from a cation exchange preparative chromatography showing the obtained fractions and identifying the range of fractions where the different compounds are present (phycotoxins and salts).
FIG. 12 shows an elution plot from a size exclusion preparative chromatography showing the obtained fractions and identifying the range of fractions where the different purified phycotoxins are present (saxitoxin and neosaxitoxin).



DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
“Definite compositional profile”, as used herein, refers to a mixture of particular phycotoxins. For example, a definite compositional profile can refer to a mixture containing only one, two, or three phycotoxins. In one embodiment, the mixture contains only two phycotoxins. In one embodiment, the mixture contains only neosaxitoxin (NEO) and saxitoxin (STX), wherein NEO is the major component (e.g., at least 60-80%). In another embodiment, the mixture contains only GTX2 and GTX3, wherein GTX 2 is the major component.
“Substantially pure”, as used herein, means greater than 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, or 99.5% purity. “Substantially pure” is typically used to describe the purity of one phycotoxin relative to one or more other phycotoxins, for example, the purity of neosaxitoxin relative to saxitoxin.
II. Methods of Purifying Phycotoxins
Methods of purifying phycotoxins, particularly phycotoxins produced in a continuous process, are described herein. The purified phycotoxins are suitable for use in pharmaceutical compositions and/or cosmetic applications.
A. Purification Steps
In one embodiment, phycotoxins are purified from a mixture of phycotoxins produced in a continuous process. In a particular embodiment, the phycotoxins are produced in a continuous process in which one or more organic nutrients are added to the culture medium to form a mixture of phycotoxins having a definite compositional profile. In one embodiment, the process produces only neosaxitoxin and saxitoxin. In another embodiment, the process produces only GTX2/3. The production process is described in PCT/IB2010/05186. The purification methods maintain the pharmacological activity of the phycotoxin(s) and provide a means for purifying phycotoxins on a commercial scale.
B. Production of Phycotoxin
Useful cyanobacteria are from genera Cylindrospermopsis sp, Anabaena sp, Gomophosphaeria sp, Oscillatoria sp, Aphanizomenon sp, Lyngbya sp, and combinations thereof. In a preferred embodiment, the species is Cylindrospermopsis raciborskii, Anphanizomenon flos-aquae, Aphanizomenon (APh) issatschenkoi (usaceb) Proskina-Lavrenco, Aphanizomenon gracile (Lemm) Lemm, Anabaena circinalis, Lyngbya wollei, and combinations thereof. In the most preferred embodiment, the cyanobacterium is Aphanizomenon gracile (Lemm) Lemm. 
Phycotoxins can be produced in a continuous culture of Aphanizomenon gracile. The organisms are harvested from the culture using centrifugation or filtration to produce a cell pellet. The phycotoxin can be extracted from the cell pellet as well as the supernatant.
C. Extraction of Phycotoxin from Cells
1. Lysis of the Cells
The cell pellet is treated to lyze the cell walls. The cell wall can be lyzed using a solvent, such as one or more organic solvents. In a preferred embodiment, this is achieved using a mixture of organic solvents such as 1:1 chloroform:methanol+50 mM acetic acid in a 50% proportion with chloroform:methanol, pH 5.0, added to the cell pellet. Other methods including mechanical lysis can be used to break the cell wall.
Several methods are commonly used to physically lyse cells, including mechanical disruption, liquid homogenization, high frequency sound waves, freeze/thaw cycles and manual grinding.
Liquid-based homogenization is the most widely used cell disruption technique for small volumes and cultured cells. Cells are lysed by forcing the cell or tissue suspension through a narrow space, thereby shearing the cell membranes. Three different types of homogenizers are in common use. A Dounce homogenizer consists of a round glass pestle that is manually driven into a glass tube. A Potter-Elvehjem homogenizer consists of a manually or mechanically driven PTFE pestle shaped to fit a rounded or conical vessel. The number of strokes and the speed at which the strokes are administered influences the effectiveness of Dounce and Potter-Elvehjem homogenization methods. Both homogenizers can be obtained in a variety of sizes to accommodate a range of volumes. A French press consists of a piston that is used to apply high pressure to a sample volume of 40 to 250 ml, forcing it through a tiny hole in the press. Only two passes are required for efficient lysis due to the high pressures used with this process. The equipment is expensive, but the French press is often the method of choice for breaking bacterial cells mechanically.
Sonication is the third class of physical disruption commonly used to break open cells. The method uses pulsed, high frequency sound waves to agitate and lyse cells, bacteria, spores and finely diced tissue. The sound waves are delivered using an apparatus with a vibrating probe that is immersed in the liquid cell suspension. Mechanical energy from the probe initiates the formation of microscopic vapor bubbles that form momentarily and implode, causing shock waves to radiate through a sample. To prevent excessive heating, ultrasonic treatment is applied in multiple short bursts to a sample immersed in an ice bath. Sonication is best suited for volumes <100 ml.
The freeze/thaw method is commonly used to lyse bacterial and mammalian cells. The technique involves freezing a cell suspension in a dry ice/ethanol bath or freezer and then thawing the material at room temperature or 37° C. This method of lysis causes cells to swell and ultimately break as ice crystals form during the freezing process and then contract during thawing. Multiple cycles are necessary for efficient lysis, and the process can be quite lengthy. However, freeze/thaw has been shown to effectively release recombinant proteins located in the cytoplasm of bacteria and is recommended for the lysis of mammalian cells in some protocols.
Manual grinding is the most common method used to disrupt plant cells. Tissue is frozen in liquid nitrogen and then crushed using a mortar and pestle. Because of the tensile strength of the cellulose and other polysaccharides comprising the cell wall, this method is the fastest and most efficient way to access plant proteins and DNA.
Cells can be treated with various agents to aid the disruption process. Lysis can be promoted by suspending cells in a hypotonic buffer, which cause them to swell and burst more readily under physical shearing. Lysozyme (200 μg/ml) can be used to digest the polysaccharide component of yeast and bacterial cell walls. Alternatively, processing can be expedited by treating cells with glass beads in order to facilitate the crushing of cell walls. This treatment is commonly used with yeast cells. Viscosity of a sample typically increases during lysis due to the release of nucleic acid material. DNase can be added to samples (25-50 μg/ml) along with RNase (50 μg/ml) to reduce this problem. Nuclease treatment is not required for sonicated material since sonication shears chromosomes. Finally, proteolysis can be a problem whenever cells are manipulated; therefore, protease inhibitors should be added to all samples undergoing lysis.
Although physical methods have traditionally been used to disrupt cells, there are some inherent disadvantages to their use. Localized heating within a sample can occur with many of the techniques described, leading to protein denaturation and aggregation. To avoid this problem it is essential to pre-chill equipment and keep samples on ice at all times. Reproducibility with homogenization and grinding methods can be challenging due to inexact terminology used to define sample handling. Furthermore, cells disrupt at different times so the viscosity of the medium constantly changes, and released subcellular components are subjected to disruptive forces. In addition to sample handling problems, some physical disruption methods require fairly expensive equipment, such as the French press and sonicator.
2. Extraction of the Lysed Cells
In the preferred embodiment, the lysed cells are subjected to a cold extraction and phase separation.
The lysed cells are filtered through a filtration system at room temperature, passing the volume harvested through a 5 micron filter. The supernatant is removed and the remaining pellet is acidified with glacial acetic acid (e.g., 500 ml).
To fracture the pellet, the pellet is resuspended with shaking for 10 minutes and then frozen at −80° C. for approximately 24 hours. If the fracture is not complete, disruption can be performed at room temperature with pure nitrogen (grade 5) at a pressure between 1000 to 2000 PSI for 30 minutes. The precipitate and supernatant are separated by centrifugation (e.g., 4,400 RPM for 30 minutes). A sample of the supernatant is analyzed for phycotoxin (e.g., neosaxitoxin) content by HPLC.
Following separation, the extract is purified by preparative chromatographic column. For example, 50 mL of toxin extract is introduced to a column of Bio Gel P-2 fine (available from Bio Rad), which has a size exclusion limit of 1,800 Daltons. The column is eluted with acetic acid (e.g., 50 mL, flow rate of 13 mL/minute). Materials having a molecular weight greater than 1800 Daltons, such as cell components and pigments, are retained on the column, while the phycotoxins are eluted from the column. The resulting phycotoxin fragment typically contains a relatively small amount of contaminants (e.g., less than 30%). Typically, 5 fractions, each having a volume of approximately 50 ml), are obtained from a single sample. Each fraction is then analyzed by HPLC to confirm conformity to the specifications.
Following HPLC analysis, the volume of the combined phycotoxin fraction is reduced to approximately 50 mL. The sample is introduced to a cation exchange chromatographic column, such as a Bio Rex-70 (available from Bio-Rad). The column is treated with acetic acid (e.g., 500 ml with a flow of 13 mL/min) to obtain 15 fractions of 50 mL each, which are analyzed by HPLC.
3. Purification of the Cell Extract
The aqueous portion of the extract is then concentrated, typically using centrifugation, then passed through a diatomaceous earth column. The column is washed, for example, with 10 times its volume of 50 mM acetic acid. After the washing step, phycotoxin (e.g., neosaxitoxin) retained on the column is extracted with an alcoholic extraction mixture such as 2:1:1 vol/vol/vol ethanol:water:5 mM acetic acid.
The eluate from the column is passed through activated charcoal columns, which are washed with distilled water to remove the retained pigments and impurities. Any retained phycotoxins are eluted with an alcoholic elution mixture such as 3:2:1 vol/vol/vol ethanol:water:1 mM acetic acid at pH 5.
The eluate from the charcoal column is again passed through a diatomaceous earth column and washed with 50 mM acetic acid. Subsequently, the toxin is eluted from the column with a 1:1:1 vol/vol/vol chloroform:methanol:water mixture.
The organic solvents from the eluate from the second diatomaceous earth column are removed by evaporation. The partially purified neosaxitoxin is retained in the aqueous phase. The partially purified extract is subjected to a size exclusion preparative HPLC (preparative high-performance liquid chromatography).
III. Purified Phycotoxin
The processes described herein provide one or more purified phycotoxins. For those embodiments where the phycotoxin is a mixture of two or more phycotoxins, the relative amounts of the phycotoxins can be controlled in the production process, for example by the addition of one or more organic nutrients. Moreover, purification conditions can be varied to separate one or more phycotoxins from one or more other phycotoxins. In one embodiment, the process yields a mixture of neosaxitoxin/saxitoxin with a ratio of 8.47. In percentages, an average of 11.8% saxitoxin and 88.2% neosaxitoxin was obtained. These are then separated for formulation.
In the preferred embodiment, the formulations are for injection at a particular site for immediate relief from pain. In other formulations, these are formulated for topical or sustained release.
A. Sustained Release Compositions
In one embodiment, the compound(s) are formulated for sustained or extended release. Sustained or extended release dosage forms provides release of an effective amount of the compound(s) for an extended period of time, such as at least one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, or six months. Sustained or extended release dosage forms can be administered parenterally, e.g., by injection, topically, or transdermally.
In one embodiment, the compound(s) are formulated as microparticles and/or nanoparticles that provide extended or sustained release of the compound. In some embodiments, the compounds can be incorporated into a matrix, wherein the matrix provides sustained or extended release. The matrix can contain one or more polymeric and/or non-polymeric materials. In other embodiments, microparticles and/or nanoparticles of drug can be coated with one or more materials that provide sustained or extended release.
Polymers which are slowly soluble in vivo and form a gel in an aqueous environment, such as hydroxypropyl methylcellulose or polyethylene oxide, may be suitable as materials for preparing sustained release drug containing microparticles. Other polymers include, but are not limited to, polyanhydrides, poly(ester anhydrides), polyhydroxy acids, such as polylactide (PLA), polyglycolide (PGA), poly(lactide-co-glycolide) (PLGA), poly-3-hydroxybutyrate (PHB) and copolymers thereof, poly-4-hydroxybutyrate (P4HB) and copolymers thereof, polycaprolactone and copolymers thereof, and combinations thereof.
Alternatively, the compound can be incorporated into microparticles prepared from materials which are insoluble in aqueous solution or slowly soluble in aqueous solution, but are capable of degrading by enzymatic degradation and/or mechanical erosion. As used herein, the term “slowly soluble in water” refers to materials that are not dissolved in water within a period of 30 minutes. Preferred examples include fats, fatty substances, waxes, wax-like substances and mixtures thereof. Suitable fats and fatty substances include fatty alcohols (such as lauryl, myristyl stearyl, cetyl or cetostearyl alcohol), fatty acids and derivatives, including but not limited to fatty acid esters, fatty acid glycerides (mono-, di- and tri-glycerides), and hydrogenated fats. Specific examples include, but are not limited to hydrogenated vegetable oil, hydrogenated cottonseed oil, hydrogenated castor oil, hydrogenated oils available under the trade name Sterotex®, stearic acid, cocoa butter, and stearyl alcohol. Suitable waxes and wax-like materials include natural or synthetic waxes, hydrocarbons, and normal waxes. Specific examples of waxes include beeswax, glycowax, castor wax, carnauba wax, paraffins and candelilla wax. As used herein, a wax-like material is defined as any material which is normally solid at room temperature and has a melting point of from about 30 to 300° C.
In some cases, it may be desirable to alter the rate of water penetration into the microparticles/nanoparticles. To this end, rate-controlling (wicking) agents may be formulated along with the fats or waxes listed above. Examples of rate-controlling materials include certain starch derivatives (e.g., waxy maltodextrin and drum dried corn starch), cellulose derivatives (e.g., hydroxypropylmethyl-cellulose, hydroxypropylcellulose, methylcellulose, and carboxymethyl-cellulose), alginic acid, lactose and talc. Additionally, a pharmaceutically acceptable surfactant (for example, lecithin) may be added to facilitate the degradation of such microparticles.
Proteins which are water insoluble, such as zein, can also be used as materials for the formation of drug containing microparticles. Additionally, proteins, polysaccharides and combinations thereof which are water soluble can be formulated with drug into microparticles and subsequently cross-linked to form an insoluble network. For example, cyclodextrins can be complexed with individual drug molecules and subsequently cross-linked.
Encapsulation or incorporation of drug into carrier materials to produce drug containing microparticles can be achieved through known pharmaceutical formulation techniques. In the case of formulation in fats, waxes or wax-like materials, the carrier material is typically heated above its melting temperature and the drug is added to form a mixture comprising drug particles suspended in the carrier material, drug dissolved in the carrier material, or a mixture thereof. Microparticles can be subsequently formulated through several methods including, but not limited to, the processes of congealing, extrusion, spray chilling or aqueous dispersion. In a preferred process, wax is heated above its melting temperature, drug is added, and the molten wax-drug mixture is congealed under constant stirring as the mixture cools. Alternatively, the molten wax-drug mixture can be extruded and spheronized to form pellets or beads. These processes are known in the art.
For some carrier materials it may be desirable to use a solvent evaporation technique to produce drug containing microparticles. In this case drug and carrier material are co-dissolved in a mutual solvent and microparticles can subsequently be produced by several techniques including, but not limited to, forming an emulsion in water or other appropriate media, spray drying or by evaporating off the solvent from the bulk solution and milling the resulting material.
In some embodiments, drug in a particulate form is homogeneously dispersed in a water-insoluble or slowly water soluble material. To minimize the size of the drug particles within the composition, the drug powder itself may be milled to generate fine particles prior to formulation. The process of jet milling, known in the pharmaceutical art, can be used for this purpose. In some embodiments drug in a particulate form is homogeneously dispersed in a wax or wax like substance by heating the wax or wax like substance above its melting point and adding the drug particles while stirring the mixture. In this case a pharmaceutically acceptable surfactant may be added to the mixture to facilitate the dispersion of the drug particles.
Polysaccharides can also be cross-linked to form a water-insoluble network. For many polysaccharides, this can be accomplished by reaction with calcium salts or multivalent cations which cross-link the main polymer chains. Pectin, alginate, dextran, amylose and guar gum are subject to cross-linking in the presence of multivalent cations. Complexes between oppositely charged polysaccharides can also be formed; pectin and chitosan, for example, can be complexed via electrostatic interactions.
Formulations may be prepared using a pharmaceutically acceptable carrier. As generally used herein “carrier” includes, but is not limited to, diluents, preservatives, binders, lubricants, disintegrators, swelling agents, fillers, stabilizers, and combinations thereof.
B. Parenteral Formulations
The microparticles and/or nanoparticles can be formulated for parenteral administration, typically for administration by injection or direct application. Parenteral formulations can be prepared as aqueous compositions using techniques is known in the art. Typically, such compositions can be prepared as injectable formulations, for example, solutions or suspensions; solid forms suitable for using to prepare solutions or suspensions upon the addition of a reconstitution medium prior to injection; emulsions, such as water-in-oil (w/o) emulsions, oil-in-water (o/w) emulsions, microemulsions, liposomes, or emulsomes.
The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, one or more polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), oils, such as vegetable oils (e.g., peanut oil, corn oil, sesame oil, etc.), and combinations thereof. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and/or by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride.
Solutions and dispersions of the active compounds as the free acid or base or pharmacologically acceptable salts thereof can be prepared in water or another solvent or dispersing medium suitably mixed with one or more pharmaceutically acceptable excipients including, but not limited to, surfactants, dispersants, emulsifiers, pH modifying agents, viscosity modifying agents, and combination thereof.
Suitable surfactants may be anionic, cationic, amphoteric or nonionic surface active agents. Suitable anionic surfactants include, but are not limited to, those containing carboxylate, sulfonate and sulfate ions. Examples of anionic surfactants include sodium, potassium, ammonium of long chain alkyl sulfonates and alkyl aryl sulfonates such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium bis-(2-ethylthioxyl)-sulfosuccinate; and alkyl sulfates such as sodium lauryl sulfate. Cationic surfactants include, but are not limited to, quaternary ammonium compounds such as benzalkonium chloride, benzethonium chloride, cetrimonium bromide, stearyl dimethylbenzyl ammonium chloride, polyoxyethylene and coconut amine. Examples of nonionic surfactants include ethylene glycol monostearate, propylene glycol myristate, glyceryl monostearate, glyceryl stearate, polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG-150 laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbates, polyoxyethylene octylphenylether, PEG-1000 cetyl ether, polyoxyethylene tridecyl ether, polypropylene glycol butyl ether, Poloxamer® 401, stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallow amide. Examples of amphoteric surfactants include sodium N-dodecyl-.beta.-alanine, sodium N-lauryl-.beta.-iminodipropionate, myristoamphoacetate, lauryl betaine and lauryl sulfobetaine.
The formulation can contain a preservative to prevent the growth of microorganisms. Suitable preservatives include, but are not limited to, parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. The formulation may also contain an antioxidant to prevent degradation of the active agent(s).
The formulation is typically buffered to a pH of 3-8 for parenteral administration upon reconstitution. Suitable buffers include, but are not limited to, phosphate buffers, acetate buffers, and citrate buffers.
Water soluble polymers are often used in formulations for parenteral administration. Suitable water-soluble polymers include, but are not limited to, polyvinylpyrrolidone, dextran, carboxymethylcellulose, and polyethylene glycol.
Sterile injectable solutions can be prepared by incorporating the active compounds in the required amount in the appropriate solvent or dispersion medium with one or more of the excipients listed above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The powders can be prepared in such a manner that the particles are porous in nature, which can increase dissolution of the particles. Methods for making porous particles are well known in the art.
C. Injectable/Implantable Solid Implants
The compounds can be incorporated into injectable/implantable solid or semi-solid implants, such as polymeric implants. In one embodiment, the compounds are incorporated into a polymer that is a liquid or paste at room temperature, but upon contact with aqueous medium, such as physiological fluids, exhibits an increase in viscosity to form a semi-solid or solid material. Exemplary polymers include, but are not limited to, hydroxyalkanoic acid polyesters derived from the copolymerization of at least one unsaturated hydroxy fatty acid copolymerized with hydroxyalkanoic acids. The polymer can be melted, mixed with the active substance and cast or injection molded into a device. Such melt fabrication typically requires polymers having a melting point that is below the temperature at which the substance to be delivered and polymer degrade or become reactive. The device can also be prepared by solvent casting where the polymer is dissolved in a solvent and the drug dissolved or dispersed in the polymer solution and the solvent is then evaporated. Solvent processes require that the polymer be soluble in organic solvents. Another method is compression molding of a mixed powder of the polymer and the drug or polymer particles loaded with the active agent. ATRIGEL® is another example of a formulation which forms a solid implant in situ upon contact with aqueous fluids.
Alternatively, the compound described herein can be incorporated into a polymer matrix and molded, compressed, or extruded into a device that is a solid at room temperature. For example, the compound can be incorporated into a biodegradable polymer, such as polyanhydrides, polyhydroalkanoic acids (PHAs), PLA, PGA, PLGA, polycaprolactone, polyesters, polyamides, polyorthoesters, polyphosphazenes, proteins and polysaccharides such as collagen, hyaluronic acid, albumin and gelatin, and combinations thereof and compressed into solid device, such as disks, or extruded into a device, such as rods. In other embodiments, the solid implant is in the form of a silastic implant.
The release of the compound from the implant can be varied by selection of the polymer, the molecular weight of the polymer, and/or modification of the polymer to increase degradation, such as the formation of pores and/or incorporation of hydrolyzable linkages. Methods for modifying the properties of biodegradable polymers to vary the release profile of the compound from the implant are well known in the art.
D. Topical Formulations
The compound can be administered topically. Suitable dosage forms for topical administration include creams, ointments, salves, sprays, gels, lotions, emulsions, and transdermal patches. The compositions may contain one or more excipients suitable for topical administration, such as chemical penetration enhancers, membrane permeability agents, membrane transport agents, emollients, surfactants, stabilizers, and combinations thereof.
“Emollients” are an externally applied agent that softens or soothes skin and are generally known in the art and listed in compendia, such as the “Handbook of Pharmaceutical Excipients”, 4th Ed., Pharmaceutical Press, 2003. These include, without limitation, almond oil, castor oil, ceratonia extract, cetostearoyl alcohol, cetyl alcohol, cetyl esters wax, cholesterol, cottonseed oil, cyclomethicone, ethylene glycol palmitostearate, glycerin, glycerin monostearate, glyceryl monooleate, isopropyl myristate, isopropyl palmitate, lanolin, lecithin, light mineral oil, medium-chain triglycerides, mineral oil and lanolin alcohols, petrolatum, petrolatum and lanolin alcohols, soybean oil, starch, stearyl alcohol, sunflower oil, xylitol and combinations thereof. In one embodiment, the emollients are ethylhexylstearate and ethylhexyl palmitate.
“Surfactants” are surface-active agents that lower surface tension and thereby increase the emulsifying, foaming, dispersing, spreading and wetting properties of a product. Suitable non-ionic surfactants include emulsifying wax, glyceryl monooleate, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polysorbate, sorbitan esters, benzyl alcohol, benzyl benzoate, cyclodextrins, glycerin monostearate, poloxamer, povidone and combinations thereof. In one embodiment, the non-ionic surfactant is stearyl alcohol.
“Emulsifiers” are surface active substances which promote the suspension of one liquid in another and promote the formation of a stable mixture, or emulsion, of oil and water. Common emulsifiers are: metallic soaps, certain animal and vegetable oils, and various polar compounds. Suitable emulsifiers include acacia, anionic emulsifying wax, calcium stearate, carbomers, cetostearyl alcohol, cetyl alcohol, cholesterol, diethanolamine, ethylene glycol palmitostearate, glycerin monostearate, glyceryl monooleate, hydroxpropyl cellulose, hypromellose, lanolin, hydrous, lanolin alcohols, lecithin, medium-chain triglycerides, methylcellulose, mineral oil and lanolin alcohols, monobasic sodium phosphate, monoethanolamine, nonionic emulsifying wax, oleic acid, poloxamer, poloxamers, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, propylene glycol alginate, self-emulsifying glyceryl monostearate, sodium citrate dehydrate, sodium lauryl sulfate, sorbitan esters, stearic acid, sunflower oil, tragacanth, triethanolamine, xanthan gum and combinations thereof. In one embodiment, the emulsifier is glycerol stearate.
Suitable classes of penetration enhancers are known in the art and include, but are not limited to, fatty alcohols, fatty acid esters, fatty acids, fatty alcohol ethers, amino acids, phospholipids, lecithins, cholate salts, enzymes, amines and amides, complexing agents (liposomes, cyclodextrins, modified celluloses, and diimides), macrocyclics, such as macrocylic lactones, ketones, and anhydrides and cyclic ureas, surfactants, N-methyl pyrrolidones and derivatives thereof, DMSO and related compounds, ionic compounds, azone and related compounds, and solvents, such as alcohols, ketones, amides, polyols (e.g., glycols). Examples of these classes are known in the art.
In some embodiments, the compounds can be applied topically in the form of a lotion, cream, gel, ointment, emulsion, or foam. These dosage forms typically contain hydrophilic and hydrophobic materials, for example, to form an emulsion.
“Hydrophilic” as used herein refers to substances that have strongly polar groups that readily interact with water. “Lipophilic” refers to compounds having an affinity for lipids. “Amphiphilic” refers to a molecule combining hydrophilic and lipophilic (hydrophobic) properties. “Hydrophobic” as used herein refers to substances that lack an affinity for water; tending to repel and not absorb water as well as not dissolve in or mix with water.
A “gel” is a colloid in which the dispersed phase has combined with the continuous phase to produce a semisolid material, such as jelly. An “oil” is a composition containing at least 95% wt of a lipophilic substance. Examples of lipophilic substances include but are not limited to naturally occurring and synthetic oils, fats, fatty acids, lecithins, triglycerides and combinations thereof.
A “continuous phase” refers to the liquid in which solids are suspended or droplets of another liquid are dispersed, and is sometimes called the external phase. This also refers to the fluid phase of a colloid within which solid or fluid particles are distributed. If the continuous phase is water (or another hydrophilic solvent), water-soluble or hydrophilic drugs will dissolve in the continuous phase (as opposed to being dispersed). In a multiphase formulation (e.g., an emulsion), the discreet phase is suspended or dispersed in the continuous phase.
An “emulsion” is a composition containing a mixture of non-miscible components homogenously blended together. In particular embodiments, the non-miscible components include a lipophilic component and an aqueous component. An emulsion is a preparation of one liquid distributed in small globules throughout the body of a second liquid. The dispersed liquid is the discontinuous phase, and the dispersion medium is the continuous phase. When oil is the dispersed liquid and an aqueous solution is the continuous phase, it is known as an oil-in-water emulsion, whereas when water or aqueous solution is the dispersed phase and oil or oleaginous substance is the continuous phase, it is known as a water-in-oil emulsion. Either or both of the oil phase and the aqueous phase may contain one or more surfactants, emulsifiers, emulsion stabilizers, buffers, and other excipients. Preferred excipients include surfactants, especially non-ionic surfactants; emulsifying agents, especially emulsifying waxes; and liquid non-volatile non-aqueous materials, particularly glycols such as propylene glycol. The oil phase may contain other oily pharmaceutically approved excipients. For example, materials such as hydroxylated castor oil or sesame oil may be used in the oil phase as surfactants or emulsifiers.
An emulsion is a preparation of one liquid distributed in small globules throughout the body of a second liquid. The dispersed liquid is the discontinuous phase, and the dispersion medium is the continuous phase. When oil is the dispersed liquid and an aqueous solution is the continuous phase, it is known as an oil-in-water emulsion, whereas when water or aqueous solution is the dispersed phase and oil or oleaginous substance is the continuous phase, it is known as a water-in-oil emulsion. The oil phase may consist at least in part of a propellant, such as an HFA propellant. Either or both of the oil phase and the aqueous phase may contain one or more surfactants, emulsifiers, emulsion stabilizers, buffers, and other excipients. Preferred excipients include surfactants, especially non-ionic surfactants; emulsifying agents, especially emulsifying waxes; and liquid non-volatile non-aqueous materials, particularly glycols such as propylene glycol. The oil phase may contain other oily pharmaceutically approved excipients. For example, materials such as hydroxylated castor oil or sesame oil may be used in the oil phase as surfactants or emulsifiers.
A sub-set of emulsions are the self-emulsifying systems. These drug delivery systems are typically capsules (hard shell or soft shell) comprised of the drug dispersed or dissolved in a mixture of surfactant(s) and lipophilic liquids such as oils or other water immiscible liquids. When the capsule is exposed to an aqueous environment and the outer gelatin shell dissolves, contact between the aqueous medium and the capsule contents instantly generates very small emulsion droplets. These typically are in the size range of micelles or nanoparticles. No mixing force is required to generate the emulsion as is typically the case in emulsion formulation processes.
A “lotion” is a low- to medium-viscosity liquid formulation. A lotion can contain finely powdered substances that are in soluble in the dispersion medium through the use of suspending agents and dispersing agents. Alternatively, lotions can have as the dispersed phase liquid substances that are immiscible with the vehicle and are usually dispersed by means of emulsifying agents or other suitable stabilizers. In one embodiment, the lotion is in the form of an emulsion having a viscosity of between 100 and 1000 centistokes. The fluidity of lotions permits rapid and uniform application over a wide surface area. Lotions are typically intended to dry on the skin leaving a thin coat of their medicinal components on the skin's surface.
A “cream” is a viscous liquid or semi-solid emulsion of either the “oil-in-water” or “water-in-oil type”. Creams may contain emulsifying agents and/or other stabilizing agents. In one embodiment, the formulation is in the form of a cream having a viscosity of greater than 1000 centistokes, typically in the range of 20,000-50,000 centistokes. Creams are often time preferred over ointments as they are generally easier to spread and easier to remove.
The difference between a cream and a lotion is the viscosity, which is dependent on the amount/use of various oils and the percentage of water used to prepare the formulations. Creams are typically thicker than lotions, may have various uses and often one uses more varied oils/butters, depending upon the desired effect upon the skin. In a cream formulation, the water-base percentage is about 60-75% and the oil-base is about 20-30% of the total, with the other percentages being the emulsifier agent, preservatives and additives for a total of 100%.
An “ointment” is a semisolid preparation containing an ointment base and optionally one or more active agents. Examples of suitable ointment bases include hydrocarbon bases (e.g., petrolatum, white petrolatum, yellow ointment, and mineral oil); absorption bases (hydrophilic petrolatum, anhydrous lanolin, lanolin, and cold cream); water-removable bases (e.g., hydrophilic ointment), and water-soluble bases (e.g., polyethylene glycol ointments). Pastes typically differ from ointments in that they contain a larger percentage of solids. Pastes are typically more absorptive and less greasy that ointments prepared with the same components.
A “gel” is a semisolid system containing dispersions of small or large molecules in a liquid vehicle that is rendered semisolid by the action of a thickening agent or polymeric material dissolved or suspended in the liquid vehicle. The liquid may include a lipophilic component, an aqueous component or both. Some emulsions may be gels or otherwise include a gel component. Some gels, however, are not emulsions because they do not contain a homogenized blend of immiscible components. Suitable gelling agents include, but are not limited to, modified celluloses, such as hydroxypropyl cellulose and hydroxyethyl cellulose; Carbopol homopolymers and copolymers; and combinations thereof. Suitable solvents in the liquid vehicle include, but are not limited to, diglycol monoethyl ether; alklene glycols, such as propylene glycol; dimethyl isosorbide; alcohols, such as isopropyl alcohol and ethanol. The solvents are typically selected for their ability to dissolve the drug. Other additives, which improve the skin feel and/or emolliency of the formulation, may also be incorporated. Examples of such additives include, but are not limited, isopropyl myristate, ethyl acetate, C12-C15 alkyl benzoates, mineral oil, squalane, cyclomethicone, capric/caprylic triglycerides, and combinations thereof.
Foams consist of an emulsion in combination with a gaseous propellant. The gaseous propellant consists primarily of hydrofluoroalkanes (HFAs). Suitable propellants include HFAs such as 1,1,1,2-tetrafluoroethane (HFA 134a) and 1,1,1,2,3,3,3-heptafluoropropane (HFA 227), but mixtures and admixtures of these and other HFAs that are currently approved or may become approved for medical use are suitable. The propellants preferably are not hydrocarbon propellant gases which can produce flammable or explosive vapors during spraying. Furthermore, the compositions preferably contain no volatile alcohols, which can produce flammable or explosive vapors during use.
Buffers are used to control pH of a composition. Preferably, the buffers buffer the composition from a pH of about 4 to a pH of about 7.5, more preferably from a pH of about 4 to a pH of about 7, and most preferably from a pH of about 5 to a pH of about 7. In a preferred embodiment, the buffer is triethanolamine.
Preservatives can be used to prevent the growth of fungi and microorganisms. Suitable antifungal and antimicrobial agents include, but are not limited to, benzoic acid, butylparaben, ethyl paraben, methyl paraben, propylparaben, sodium benzoate, sodium propionate, benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, and thimerosal.
For topical applications, repeated application can be done or a patch can be used to provide continuous administration of the compounds over an extended period of time.
Implants can be used to provide sustained delivery. In one embodiment, the implant is the Alza minipump; in another it is an insulin type pump; in still another embodiment, it is a silastic tube of the type used to deliver birth control hormones, such as IMPLANON®.
IV. Methods of Use
Phycotoxins, such as neosaxitoxin and saxitoxin, act as specific blockers of the voltage-dependent sodium channels present in excitable cells. Due to the inhibition of sodium channels, the transmission of a nervous impulse is blocked preventing the release of neurotransmitters at the neuromotor junction, which prevents muscular contraction. Due to these physiological effects, these compounds are potentially useful in pharmacology when used as muscular activity inhibitors in pathologies associated with muscular hyperactivity, such as muscular spasms and focal dystonias, when administered locally. These compounds can also inhibit sensory pathways and generate an anesthetic effect when administered locally.
EXAMPLES
Example 1
Phycotoxins Obtained and Purified from Aphanizomenon gracile Maintained in Continuous Cultures Under Controlled Conditions
Methods and Materials
In Aphanizomenon gracile cultures produced from a selected single clone, neosaxitoxin is the main phycotoxin produced, with smaller amounts of saxitoxin.
The production of neosaxitoxin from Aphanizomenon gracile cyanobacteria was carried out from an initial amount of 10 milligrams of wet Aphanizomenon gracile cells. The purification as conducted on a cell pellet.
For 10 mg of cell pellet, 10 mL of the same volume in weight of the mixture of organic solvents (1:1 chloroform:methanol+50 mM acetic acid in a 50% proportion with chloroform:methanol, resulting pH 5.0) were added to the cell pellet to destroy the cell wall and the cytoplasmic membrane. The lysed cells were subjected to a cold extraction and phase separation.
The extracted aqueous phase was concentrated 10 times by volume using a rotary evaporator at room temperature. The resulting concentrate was centrifuged at 20,000×g for 30 minutes.
2 mL of the supernatant from the centrifugation was passed through a diatomaceous earth column. The column was washed with 10 times its volume of 50 mM acetic acid. After the washing step, neosaxitoxin retained in the column was extracted with 5 mL of an alcoholic extraction mixture (2:1:1 vol/vol/vol ethanol:water:5 mM acetic acid).
The eluate from the column obtained was passed through activated charcoal columns, which were washed with distilled water to remove the retained pigments and impurities. Phycotoxins remain retained in the columns, and were eluted with 10 mL of an alcoholic elution mixture (3:2:1 vol/vol/vol ethanol:water:1 mM acetic acid) at pH 5.
The eluate from the charcoal column was again passed through a diatomaceous earth column and washed with 50 mM acetic acid. Subsequently, the toxin was eluted from the column with 5 mL of a 1:1:1 vol/vol/vol chloroform:methanol:water mixture.
The organic solvents from the eluate from the second diatomaceous earth column were removed by evaporation using a “speed vac” (Savant, N.Y., USA). The partially purified neosaxitoxin was retained in the aqueous phase. The partially purified extract was subjected to a size exclusion preparative HPLC (preparative high-performance liquid chromatography).
Results
After 2 days of culturing, each reactor produced an average of 652.4 micrograms of total toxin per each collected liter. The average neosaxitoxin/saxitoxin ratio was 8.47. In percentages, an average of 11.8% saxitoxin and 88.2% neosaxitoxin was obtained.
FIGS. 1, 2 and 4 show chromatograms of HPLC runs detected by online fluorescence detection that describe a phycotoxin profile produced by the species Aphanizomenon gracile. FIGS. 1 and 2 describe HPLC profiles of fractions presenting several degrees of purification from Aphanizomenon gracile extracts.
FIG. 1 shows a chromatogram of an unpurified Aphanizomenon gracile extract carried out to know and quantify the profile of phycotoxins present in this original extract. The chromatogram shows a simple profile with a major proportion of neosaxitoxin and a lower amount of saxitoxin (less than 13%).
FIG. 2 shows the purification of 10 microliters of an extract from Aphanizomenon gracile grown in continuous culture under controlled conditions, partially purified by preparative size exclusion high-performance liquid chromatography (separation by molecular size). In this partially purified extract, a typical peak at RT=9.373 minutes that saturates the chromatogram, which corresponds to neosaxitoxin, is already present, indicating the presence of a large amount of neosaxitoxin in this fraction.
FIG. 4 shows a neosaxitoxin sample purified from extracts of Aphanizomenon gracile cyanobacteria (RT=4.933 minutes) that is obtained in the fraction eluted from HPLC with ion exchange columns, corresponding the last purification step (final step of the method described above). A single peak is obtained, corresponding to a single component, which is pure neosaxitoxin in this case.







TABLE 2







Production of neosaxitoxin and saxitoxin as a


function of the number of filaments and the wet weight


of the Aphanizomenon gracile pellet













neoSTX
STX
neoSTX
STX
neoSTX
STX
Cyano.


mM
mM
μg/ml
μg/ml
pg/fil.
pg/fil.
fil./ml*
















8.98
3.50
2.83
1.05
2.42
0.90
1,170,000.00


13.66
4.91
4.30
1.47
3.44
1.18
1,250,000.00


3.42
1.03
1.08
0.31
1.71
0.49
630,000.00


17.82
4.78
5.61
1.43
9.42
2.41
596,000.00


17.81
4.02
5.61
1.21
13.17
2.83
426,000.00


7.66
1.47
2.41
0.44
4.94
0.91
488,000.00


10.87
2.19
3.42
0.66
10.25
1.97
334,000.00


10.52
2.07
3.31
0.62
4.38
0.82
756,000.00


10.77
1.96
3.39
0.59
7.47
1.30
454,000.00


17.24
2.99
5.43
0.90
13.12
2.16
414,000.00


5.31
0.41
1.67
0.12
5.19
0.38
322,000.00


12.26
2.17
3.86
0.65
9.47
1.59
408,000.00


9.84
1.94
3.10
0.58
17.42
3.27
178,000.00





STX: saxitoxin


neoSTX: neosaxitoxin


Cyano. fil.: cyanobacterial filaments


*Cyanobacterial filaments are associations of 20 to 100 cyanobacterial cells.






These cyanobacteria form filaments in solution and those filaments are counted using the magnification of an inverted microscope.

The yields obtained represent a surprising unexpected production of pure phycotoxins (neosaxitoxin and saxitoxin) per milligram of wet cyanobacteria, when compared to the production of filaments and cyanobacterial pellets in usual culture conditions described so far in small culture flasks without continuous micro-aeration and with light:day and no light:night cycles. In the example described herein, cyanobacteria are always in the logarithmic growth phase, with permanent illumination 24 hours a day and with permanent collection of cyanobacteria, inducing permanent growth by adding new nutrients in a volume that is equivalent to the volume collected in each harvest.
Example 2
Phycotoxins Obtained and Purified from Cylindrospermopsis raciborskii Maintained in Continuous Cultures Under Controlled Conditions
Methods and Materials
The cyanobacterium Cylindrospermopsis raciborskii was cultured according to the continuous massive semi-automatic culture method under controlled conditions in permanent logarithmic growth, described in PCT/IB2010/05186, then purified as described in Example 1.
Results
The unpurified extract of the strain Cylindrospermopsis raciborskii has a chromatographic profile according to FIG. 6. Cylindrospermopsis raciborskii has a toxin profile where GTX 3 and GTX 2 are predominant, with the longest retention times in the column (the last two peaks at right, respectively). The Cylindrospermopsis raciborskii extract from the continuous culture is subjected to the purification process corresponding to the steps described for Example 1, and already at the size exclusion preparative HPLC step, the GTX3 and GTX2 epimers are almost exclusively obtained, as shown in FIG. 7.
The final fraction of the phycotoxin purification method from Cylindrospermopsis raciborskii has only GTX3 and GTX2, as shown in FIG. 8 and fractions containing each gonyaulatoxin can be collected to obtain large amounts of pure GTX3 and GTX2.
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

## 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