FORM 2
THE PATENT ACT 1970
(39 OF 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION (See section 10 and rule 13)
TITLE OF THE INVENTION
A PROCESS FOR THE PRODUCTION OF ANTIVIRAL POLYSACCHARIDES
FROM LABYRINTHULOMYCETES
APPLICANT(S)
(a) Name: Myko Tech Private Limited
(b) Nationality: Indian
(c) Address: #313 Vainguinnim valley,
Dona Paula,
Goa-403004,
India.

Priority Date
This complete specification claims priority from the provisional application number 3257/MUM/2010 filed in Mumbai branch of patent office on 29-November-2010.
The following specification particularly describes the invention and the manner in which it is to be performed.
Field of the Invention
The present invention relates to a process for the production of antiviral polysaccharides from Labyrinthulomycetes. The said antiviral polysaccharides are effective in treating herpes viruses. The invention provides a process for the production of antiherpes polysaccharides from strains of Labyrinthulomycetes, by growing them in culture media, harvesting the culture filtrate and extracting and purifying the extracellular polysaccharides.
Background of the Invention
Polysaccharides are polymers of different kinds of sugars and are produced by a wide variety of plants, animals and microorganisms. Many polysaccharides are of industrial use, for example, polysaccharides of plant and seaweed origin, such as starch, agar, alginate, carrageenan, arabic gum and guar gum are widely used in food, cosmetic and pharmaceutical industries. Microorganisms such as cyanobacteria, heterotrophic bacteria and fungi secret polysaccharides and other substances extracellularly into the growth medium and such polysaccharides are called as "extracellular polysaccharides" or, Exo Polymeric Substances (EPS) (Sutherland, I.W. 1996. Extracellular polysaccharides. In: Rhein, H.J. and Reed, G. (Eds.). Biotechnology. Vol. 6, VCH, Weinheim, pp. 615-627). Many microbial EPS, such as curdlan, gellan and xanthan are used in food and oil industry. There are several examples of the potential pharmaceutical uses of EPS, such as antimicrobial agents and anticancer agents (Selbmann, L., Stingele, F. and Petruccioli M. 2003. Exopolysaccharide production by filamentous fungi: the example of Botryosphaeria rhodina. Antonie van Leeuwenhoek 84: 135-145; M. Zhang, S.W. Cui, P.C.K. Cheung- and Q. Wang. 2006. Antitumor polysaccharides from mushrooms: a review on their isolation process, structural characteristics and antitumor activity. Trends in Food Science and Technology 18: 4-19). The prime advantage of microbial EPS is that due to short life cycle of the microorganism, it can be produced in large amounts in short duration of time resulting in a highly economical and cost-effective production process. Moreover, the EPS can be extracted in a pure form more easily.
Many microbial EPS and algal polysaccharides contain sulphate groups and are termed as "sulphated polysaccharides". These have many pharmaceutical uses (K. Sogawa et al., 1998, Marine microalgalpolysaccharides induces apoptosis in the human lymphoid cells. Journal of Marine Biotechnology 6; 35-38), including antiviral properties and capability of inhibiting the absorption and penetration of viruses by host cells. Several papers have

described the chemistry and application of extracellular sulfated polysaccharides from organisms such as a cyanobacterium (Aphanocapsa halophyiia) and a red alga {Porphyridium sp.) (S. Geresh and S. Arad, 1991, Bioresource Technology 38: 195-201; T. Matsunaga et al., 1998, Applied Microbiology and Biotechnology 45: 24-27). Dextran sulphate and red algal polysaccharides have been shown to inhibit HIV 1 and 2 retroviruses in vitro (Talyshinsky MM, YY Souprun and MM Huleihel. 2002. Antiviral activity of red micro-algal polysaccharides against retroviruses. Cancer Cell International 2: 1-7). Antiviral activities against influenza virus Type A has been shown in extracellular polysaccharides and their over sulfated derivatives of marine Pseudomonas (Matsuda M, Shigeta S, Okutani K. Antiviral Activities of Marine Pseudomonas Polysaccharides and Their Oversulfated Derivatives Mar Biotechnol 1999; 1:68-73).
The patent applications WO 2009027057 and WO 20090305 relate to sulfated polysaccharides selected from the group of carrageenans and fucoidans and pharmaceutical compositions made thereof, wherein said sulfated polysaccharides are present as antiviral active ingredients, for medical or veterinary use in the prevention or treatment of diseases caused by or associated with a virus entering an individual's body via the respiratory tract, the virus being selected from the group of ortho- myxoviridae, paramyxoviridae, adenoviridae and coronaviridae.
Among various viral diseases, the herpes, caused by Herpes Simplex Virus (HSV) is one of the common diseases affecting human beings. HSV establishes life long, latent infections in the peripheral nervous systems, escapes immune responses of the host and persists for a long time. It may also lie latent and often recur as chronic infections (Simmons AJ. 2002. Clinical manifestations and treatment considerations of herpes simplex virus infection. Infect Dis 186: 71-77).
The anti-viral agents used to treat HSV infections are acyclovir, valacyclovir, Famciclovir, Pencicloir, Cidofovir, Photoformatic Acid (PFA), also called foscarnet and Ara A or Vidarabine and Trifluorothymidine (TF). All these compounds are nucleoside analogues that target the viral DNA polymerase and block viral DNA replication. Of all these compounds, acyclovir is licensed for the treatment of primary HSV infections. Acyclovir selectively targets DNA replication primarily by getting phospharylated by the thymidine kinase (TK) enzyme of the virus, mistakenly followed by termination of DNA chain elongation due to its wrong incorporation in the DNA strand getting synthesized. ACV is available in topical, intravenous, oral formulations. Resistance of the virus to acyclovir may result owing to various reasons and cause serious problems, particularly in immunocompetent and immunocompromised individuals. Post treatment prophylaxsis has been suggested to reduce frequency of skin or CNS relapse.
Resistant acyclovir strains cause problematic mucocutaneous infections. Valacyclovir, the oral prodrug comparatively has been increasingly used, but it does not have significant bioavailability. Famciclovir is the oral prodrug of Penciclovir, the active

antiviral compound with a mechanism of action, similar to that of ACV. Valacyclovir is available in parenteral and ophthalmic preparations for treatment of HSV encephalitis, neonatal herpes and herpetic keratitis. It has been used as an alterative therapy in ACV-resistant infections. However, it is no longer a drug of choice for many, mainly because of its relatively low potency, rapid degradation and poor aqueous solubility. The drug is used for treatment of ACV-resistant HSV but is relatively toxic. Significant resistance to Foscarnet may develop in immunocompromised patients.
Due to the various problems associated with presently available drugs as above, there is a need to develop a more effective alternative to acyclovir for treatment of herpes.
Numerous antiherpes polysaccharides have been reported in literature for treatment of herpes. Early, preliminary results of Baba et al. (Baba M, Snoeck R, Pauwels R, de Clercq E. 1988. Sulfated polysaccharides are potent and selective inhibitors of various enveloped viruses, including herpes simplex virus, cytomegalovirus, vesicular stomatitis virus, and human immunodeficiency virus. Antimicrob Agents Chemother 32:, 1742-2745) have shown that sulfated polysaccharides are potent and selective inhibitors of various enveloped viruses, including herpes simplex virus.
Synthetic or semi-synthetic compounds for treatment of viruses have also been reported in prior art. An example is the treatment of orthopox and herpes viruses published by Prichard et al., 2009, Inhibition of Herpesvirus Replication by 5-Substituted 4_-Thiopyrimidine Nucleosides. Antimicrobial Agents and Chemotherapy 53: 5251-5258. US Patent 7,297,687 provides a method to inhibit the replication of herpes virus by administering an alpha-glycosidically linked starch polysaccharide derivative containing a degree of quaternary ammonium group substitution. Further US Patent 6,977,248 discloses polysaccharide preparations enriched in 3-OST-3 modified heparan sulfate and methods to treat herpes simples viral type-1 using pharmaceutical preparations containing the material.
Antiviral polysaccharides have been reported from many bacteria (Okutani K. 1992. Antiviral activities of sulfated derivates of a fucosamine containing polysaccharide of marine bacterial origin. Nippon Suisan Gakkaishi 58:927-30). EPS-1, a novel polysaccharide from the a strain of thermotolerant Bacillus licheniformis. isolated from a shallow marine hot spring of Vulcano Island (Italy) impaired HSV-2 replication in human peripheral blood mononuclear cells (PBMC) but not in WISH cells (Arena A, Maugeri TL, Pavone B, Iannello D, Gugliandolo C and Bisignano G. 2006. Antiviral and immunoregulatory effect of a novel exopolysaccharide from a marine thermotolerant Bacillus licheniformis. International Immunopharmacology 6: 8- 13). The compound of interest is a glycosaminoglycan and is already FDA approved and proven non-toxic to humans. Two fractions of the compound inhibited the growth of the HSV-1 in Vero cell line.
Freshwater and marine algae have been found to produce polysaccharides that have anti-

herpes activity. An inhibitor of enveloped virus replication, from a blue-green alga Spirulina platensis has been reported (Hayashi T, Hayashi M, Kojima I. A natural sulfated polysaccharide, calcium spirulan, isolated from Spirulina platensis: in vitro and ex vivo evaluation of anti-herpes simplex virus and anti-human immunodeficiency virus activities. J Nat Prod 1996;39:83- 7). US Patent 5089481 provides a process for using a protein-bound polysaccharide, having antiviral activity, obtained from seaweeds or marine algae belonging to the genera Nemacystus, Kjellmaniella, Laminaria, Undaria, Hizikia, Porphyra, Gelidium, Gloiopellis, Gracilaria, Hemineura, Viva, Spirogyra, Codium and Acetabularia. Harden et al. (Harden EA, Falshaw R, Carnachan SM, Kern ER, Prichard MN, 2009. Harden EA, Falshaw R, Carnachan SM, Kern ER, Prichard MR Virucidal activity of polysaccharide extracts from four algal species against herpes simplex virus. Antiviral Research 83: 282-289) have shown that the extracts from the marine algae Undaria pinnatifida, Splachnidium rugosum, Gigartina atropurpurea, and Plocamium cartilagineum were effective against the herpes viruses HSV-1 and HSV-2. The polysaccharide in the above examples has to be obtained from natural populations of large macroalgae or seaweeds.
Huleihel et al. (Huleihel M, Ishanu V, Tal J, Arad S: Antiviral effect of microalgal polysaccharides on Herpes simplex and Varicella zoster viruses. J. Appl. Phycol. 2001, 13:127-134) have shown that a microalgal, sulphated polysaccharide inhibited Herpes simplex virus. In yet another example, cell wall sulfated polysaccharide of the red microalga Porphyridium sp. has been shown to exhibit impressive antiviral activity against herpes simplex virus types 1 and 2 (HSV-1 and -2) both in vitro (cell culture) and in vivo (rats and rabbits) (Huheihel M, Ishanu V, Tal J. Arad SM. 2002. Activity of Porphyridium sp. polysaccharide against herpes simplex viruses in vitro and in vivo. J Biochem Biophys Methods. 2002 50:189-200). Depending on the concentration, this polysaccharide completely inhibited or slowed down the development of the cytopathic effect in HSV-infected cells, but did not show any cytotoxic effects on vera cells even when a concentration as high as 250 ug/ml was used. The glycosaminoglycan fraction obtained from the cyanobacteria inhibited the growth of HSV-1 and the anti-viral activity lasted for more than 72 hours in cell cultures at a concentration of 44 ug/ml. In both the above cases, the polysaccharides are extracted from the cell wall of the microalgae.
The production of antiviral EPS using microorganisms grown in large volumes in fermentors and which secrete EPS has several advantages as the process is cost-effective, economical and production can be carried out at large scale, while being environmentally sustainable as well. The process would be further economical if the organism also produces yet another compound of industrial application.
Marine single-celled organisms belonging to the protistan group of Labyrinthulomycetes produce sulphated EPS, as well as the omega-3 poly unsaturated fatty acid (PUFA), docosahexaenoic acid (DHA). Jain et al. (2005) have reported that thraustochytrids, a

group of Labyrinthulomycetes, secrete polysaccharides externally into the culture medium. However, no use of these has been reported in this article. WO/2007/074479 describes the simultaneous production of high yields of extracellular polysaccharides, as well as intracellular DHA in a mutant strain of Schizochytrium limacinum MTCC 5249. However, this patent does not demonstrate anti-herpes properties of these extracellular polysaccharides.
Thus, the above mentioned known prior art processes relating to the production of useful extracellular polysaccharides from various microorganisms are not derived from microbial EPS and are not derived from the group of Labyrinthulomycetes that simultaneously produce polyunsaturated fatty acids.
In the view of the foregoing, there is an ongoing need for an improved production of antiviral polysaccharides from Labyrinthulomycetes for the treatment of herpes disease.
Summary of the Invention
It is an object of the present invention to provide a process for the production of an anti-herpes EPS (Exo polymeric substance) effective against herpes viruses of family Herpesviridae. It is a further object of, the present invention to provide a process for the production of an anti-herpes EPS fromprotists belonging to the class labyrinthulomycetes. It is still further object of the present invention to provide a process for the production of anti-herpes EPS from thraustochytrids subgroup of labyrinthulomycetes.,
According to one aspect of the invention the method of producing EPS from throustochytirids comprises:
• Culturing the cells in a culture medium comprising carbon source, nitrogen source, vitamin source and seawater of sodium salt for desired growth.
• Incubating the cells at a temperature in the range of 25 -28° C for a period of 3 to 8 days to obtain the culture
• Separation of the cell biomass from filtrate by means of centrifugation and filtration
• Extraction of Exo-polymeric substances (EPS) from the said culture filtrate using 70 % isopropyl alcohol or by freezing the culture filtrate or combination thereof.
Detailed description of the Invention
The foregoing object of the present invention is achieved by the below described method:

Protists belonging to Labyrinthulomycetes group are grown in a culture media containing seawater or sodium salts and suitable source of carbon and nitrogen for the desired growth. The carbon sources may include and not limited to glucose, starch, glycerol and corn syrup. . The nitrogen source may include and not limited to , ammonium chloride, sodium nitrate, glutamate and peptone. The vitamin source may include and not limited to yeast extract, corn steep liquor, beef extract, malt extract and soy extract. The organism is allowed to grow for a suitable duration of time in order for EPS to accumulate in the culture medium. This incubation period may last from 3 to 8 days and thereafter, cell biomass is separated from the culture filtrate by various means such as centrifugation and filtration. The culture filtrate containing the EPS is further extracted by various techniques known in prior art, such as the addition of 70 % isopropyl alcohol, freezing the culture filtrate to precipitate the EPS and other methods, or a combination of these different methods. The precipitated water soluble EPS may then be centrifuged and later freeze-dried to yield a powder. It may be further purified using a variety of techniques, such as column chromatography, if required. The EPS possesses anti-herpes properties. This EPS may be further used to make various anti viral formulations, as is well known in prior art.
In order that this invention be more fully understood, the following preparative and testing examples are set forth. These examples are for the purpose of illustration only and are not to be construed as limiting the scope of the invention in any way.
Example 1
Extraction of EPS
A culture of the thraustochytrid, belonging to Schizochytrium limacinum, was used to extract the Extracellular Polysaccharides (EPS). The organism was grown in a culture medium containing 4.0 g glucose, 1.0 g peptone, 0.1 g Yeast extract and 100 ml seawater. The cultures were grown in Erlenmeyer flasks on a rotary shaker at 120 rpm at a room temperature of approximately 28°C. After growth for I week, the culture was centrifuged and the pelleted biomass was removed. The culture supernatant containing the EPS was concentrated to a tenth of the volume, using an Amicon Stirred Cell with an ultrafilter of molecular weight cut off(MWCO) of 10,000 daltons. Isopropyl alcohol was added to the concentrated culture filtrate to yield a final concentration of 70 % of the alcohol. This mixture was then stored in the refrigerator for 48 hours, by which time the EPS had precipitated. The EPS was collected by centrifugation and was freeze-dried to yield a powder. The powdered EPS was used for all further experiments.

Example 2
Toxicity studies of EPS
The anti-herpes simplex virus (anti-HSV) effect of the EPS from the thraustochytrid MTCC 5259 was tested. Vero Cell lines obtained from African Green Monkey kidneys was used. ATCC strain VR-73, which is a strain of Herpes Simplex Virus-1 (HSV-1) was obtained from the American Type Culture Collection. All experiments were carried out using Vero cells cultivated in multiwell tissue culture plates. These were inoculated with HSV-1 ATCC strain. The following experimental sets were kept; (1) Vero Cell Control was made up of the cell line cultures which were uninoculated; (2) Inoculated Cell control was made of Vero Cell lines inoculated with the HSV-1 culture, at a concentration of 1 Multiplicity of Infection (Mol) dose, corresponding to 10091.93 viral particles per well; and (3) Acyclovir Drug Control was made of Vero Cell lines. One ml of acyclovir (purchased as Zovirex) in water containing Minimum Inhibitory Concentration (MIC) of 2 μg was added to each well immediately after inoculation with the virus as above. (3) EPS assays were performed using Vero Cell lines inoculated with the HSV-1 standard strain as above. One ml of a DMSO solution of EPS from the thraustochytrid containing 5, 10 and 20 μg was added to each well immediately after inoculation with the virus. The cytopathic effect of the virus was assayed after 24 h, based on the number of plaques present. The percent reduction in the number of viral plaques was calculated to obtain the percent inhibition by the various concentrations of the drugs. The EPS from the thraustochytrid culture inhibited cytopathic effect. A concentration of 5 μg per ml resulted in an inhibition of 75-90%, and that of 10μg produced an inhibition of 90 %. A 100 % inhibition against viral infection was achieved with a concentration of 20 μg per ml EPS. In comparison, addition of 1 ml of Acyclovir to each well at a concentration of 4μg per ml showed 100% inhibition of the plaque formation. The EPS was not toxic at highest concentration tested (22μg per ml).
Example 3
Cytotoxic effect of EPS
The cytotoxic effect of EPS obtained from the thraustochytrid MTCC 5249 was tested as follows. Normal cell morphology of the Vero Cell Lines was first ascertained by phase contrast light microscopy. Subsequently various concentrations of the EPS from the thraustochytrid, ranging from 1 to 22 μg per ml were tested on the Vero cell lines. No difference in cell morphology was observed even at the highest concentration, indicating that the EPS did not have any cytotoxic effect on the Vero Cell Line at this concentration.

Claims
What is claimed is:
1. An anti-viral polysaccharide obtained from a marine alga belonging to the class labyrinthulomycetes, group thraustochytrids for use an active ingredient in a pharmaceutical composition or medicament for the therapeutic treatment of symptom, condition or disease caused by or associated with an infection by a simplex virus.
2. The anti-viral polysaccharide according to claim l, wherein the simplex virus is herpes simplex virus 1 (HSV-1) and herpes simplex virus 2 (HSV-2).
3. The anti-viral polysaccharide according to claim 1, wherein the thraustochytrid is Schizochytrium limacinum,
4. A process for production of anti-viral polysaccharide or Exo-polymeric substance extracellularly from thraustochytrid as claimed in claim 1, wherein the process comprising:
a. Culturing the cells of the marine alga belonging to the group
thraustochytrid in a culture medium comprising carbon source, nitrogen
source, vitamin source and seawater of sodium salt for desired growth;
b. Incubating the cells at a temperature in the range of 25 -28° C for a period
of 3 to 8 days to obtain the culture;
c. Separation of the cell biomass from filtrate by means of centrifugation and
filtration;
d. Extraction of Exo-polymeric substances (EPS) from culture filtrate using
70 % isopropyl alcohol or by freezing the culture filtrate or combination
thereof.
5. The process according to claim 4, wherein the carbon source is selected from the group consisting of glucose, starch, glycerol and corn syrup
6. The process according to claim 4, wherein the nitrogen source is selected from the group consisting of ammonium chloride, sodium nitrate, glutamate and peptone
7. The process according to claim 4, wherein the vitamin source is selected from a group consisting of yeast extract, corn steep liquor, beef extract, malt extract and soy extract.