A significant amount of work has been devoted to demonstrating biological activities of various seaweed components, including polysaccharide components such as fucoidan and ulvan. Fucoidan is a sulfated polysaccharide found in the cell walls of many species of brown seaweed. In vitro studies show that fucoidan has antitumor, antiangiogenic (Maruyama H, Tamauchi H, Hashimoto M, Nakano T. Antitumor activity and immune response of Mekabu fucoidan extracted from Sporophyll of Undaria pinnatifida. In Vivo 2003; 17 (3):245-249; Haneji K, Matsuda T, Tomita M et al. Fucoidan extracted from cladosiphon okamuranus tokida induces apoptosis of human T-cell leukemia virus type 1-infected T-cell lines and primary adult T-cell leukemia cells. Nutr. Cancer 2005; 52 (2):189-201; Liu J M, Bignon J, Haroun-Bouhedja F et al. Inhibitory effect of fucoidan on the adhesion of adenocarcinoma cells to fibronectin. Anticancer Res. 2005; 25 (3B):2129-2133; Koyanagi S, Tanigawa N, Nakagawa H, Soeda S, Shimeno H. Oversulfation of fucoidan enhances its anti-angiogenic and antitumor activities. Biochem. Pharmacol. 2003; 65 (2):173-179; Alekseyenko T V, Zhanayeva S Y, Venediktova A A, et al. Antitumor and antimetastatic activity of fucoidan, a sulfated polysaccharide isolated from the Okhotsk Sea Fucus evanescens brown alga. Bull. Exp. Biol. Med. 2007 June; 143 (6):730-2; Nagamine T, Hayakawa K, Kusakabe T, et al. Inhibitory effect of fucoidan on Huh7 hepatoma cells through downregulation of CXCL12. Nutr. Cancer 2009; 61 (3):340-7), antiviral (Lee J B, Hayashi K, Hashimoto M, Nakano T, Hayashi T. Novel antiviral fucoidan from sporophyll of Undaria pinnatifida (Mekabu). Chem. Pharm. Bull. (Tokyo) 2004 September; 52 (9):1091-4; Hayashi K, Nakano T, Hashimoto M, Kanekiyo K, Hayashi T. Defensive effects of a fucoidan from brown alga Undaria pinnatifida against herpes simplex virus infection. Int. Immunopharmacol. 2008 January; 8 (1):109-16.), and immunomodulatory effects (Raghavendran H R, Srinivasan P, Rekha S. Immunomodulatory activity of fucoidan against aspirin-induced gastric mucosal damage in rats. Int. Immunopharmacol. 2011 February; 11 (2):157-63). These effects are brought about by stimulating natural killer cells and by down regulating AP-I involved in cellular proliferation. Fucoidan also exhibited neuroprotective (Do H, Pyo S, Sohn E H. Suppression of iNOS expression by fucoidan is mediated by regulation of p38 MAPK, JAK/STAT, AP-1 and IRF-1, and depends on up-regulation of scavenger receptor B1 expression in TNF-alpha- and IFN-gamma-stimulated C6 glioma cells. J. Nutr. Biochem. 2009 Jul. 1; Luo D, Zhang Q, Wang H, et al. Fucoidan protects against dopaminergic neuron death in vivo and in vitro. Eur. J. Pharmacol. 2009 Sep. 1; 617 (1-3):33-40, radioprotective (Byon Y Y, Kim M H, Yoo E S, et al. Radioprotective effects of fucoidan on bone marrow cells: improvement of the cell survival and immunoreactivity. J. Vet. Sci. 2008 December; 9 (4):359-65), and antiulcer (Choi J I, Raghavendran H R, Sung N Y, et al. Effect of fucoidan on aspirin-induced stomach ulceration in rats. Chem Biol Interact. 2010 Jan. 5; 183 (1):249-54) properties.
In other studies, fucoidan demonstrated anticoagulant (Colliec S, Fischer A M, Tapon-Bretaudiere J, et al. Anticoagulant properties of a fucoidan fraction. Thromb. Res. 1991 Oct. 15; 64 (2):143-54; Irhimeh M R, Fitton J H, Lowenthal R M. Pilot clinical study to evaluate the anticoagulant activity of fucoidan. Blood Coagul. Fibrinolysis. 2009 Aug. 18) and antithrombotic (Church F C, Meade J B, Treanor R E, Whinna H C. Antithrombin activity of fucoidan. The interaction of fucoidan with heparin cofactor II, antithrombin III, and thrombin. J. Biol. Chem. 1989 Feb. 25; 264 (6):3618-23) activities, and can have additive effects when taken with anticoagulants.
Fucoidan has also shown particular promise in the prevention of post-surgical adhesions. The study of Cashman et al (Cashman J D, Kennah E, Shuto A, Winternitz, Springate C M K, Fucoidan Film Safely Inhibits Surgical Adhesions in a Rat Model, J. Surg. Res. 2011 December 171 (2):495-503) trialled a number of compounds but identified fucoidan as the safest and most efficacious. Fucoidan loaded films reduced adhesion scores by approximately 90% compared with control films. A total of 50% to 100% of animals were adhesion free at fucoidan film loadings of 0.33% to 33% w/w compared with all control film animals having adhesions. No adverse effects were observed from 33% w/w fucoidan films equivalent to approximately 30 mg fucoidan/kg body weight.
Fucoidan is also proposed to be useful in wound management, and particularly the healing of burns. Reference is made to the work of Sezer et al (Sezer A D, Hatipoglu F, Cevher E, O{hacek over (g)}urtan Z, Bas A L, and Akbu{hacek over (g)}a J, Chitosan film containing fucoidan as a wound dressing for dermal burn healing: Preparation and in vitro/in vivo evaluation, AAPS Pharm. Sci. Tech. 2007 June; 8 (2): E94-E101). These authors demonstrated in a rabbit burn model that the best regenerated dermal papillary formation, best reepithelization, and the fastest closure of wounds were found in a fucoidan-chitosan film treatment group.
Ulvan (which is analogous to fucoidan) is extracted from green seaweed, and has potential in clinical applications. For example, ulvan holds significant promise as a scaffold in the manufacture of biomaterials, as a drug delivery vehicle, and also as an immune modulator.
It will be evident from the foregoing that polysaccharides extracted from marine organisms have the potential for use in diverse and clinically important applications. A significant problem in exploiting the benefits of these molecules is the presence of pyrogenic agents in the extracts. Where the disease indication requires parenteral administration, direct application to a wound or a surgical site, or implantation within the body, the use of these polysaccharide extracts is contraindicated due to the associated dangers of pyrogen-induced fever, toxic shock and even death.
“Microbial pyrogen” as opposed to “gram negative bacterial endotoxin” has become a general descriptive term for many different substances. However, pyrogenic substances can be produced by some gram positive bacteria, mycobacteria, fungi and also viruses, but the pyrogens produced by gram negative bacteria, i.e., the endotoxins, are of significance to the pharmaceutical and medical implant industry.
The prior art discloses a range of methods for depyrogenation of solutions for use in medicine. Many methods rely on the destruction of the pyrogenic agent. However, such methods often result in damage or destruction of a desired molecule in solution. This is particularly the case where the desired molecule is naturally derived.
Other methods of depyrogenation are directed to the physical removal of the pyrogenic agent by means such as chromatography and filtration. While such methods are clearly more suitable for the treatment of solutions containing labile molecules, significant problems arise in the scale up of these methods to the processing of commercial quantities. Often, industrial scale separative methods are not economically feasible due to the cost of media, and the need to constantly replace or regenerate the medium. Seaweed extracts in particular are known to contain residues and precipitates that cause blockage or fouling of separative media.
A further problem with seaweed extracts is the presence of an undesirable brown discolouration that often occurs during processing. This colouration is thought to be caused by the use of alkaline conditions, and typically carries through to the final product. Where the extract is formulated as a human injectable composition, an uncoloured solution is highly preferred to facilitate visual inspection of the injectate prior to administration.
It is an aspect of the present invention to overcome or alleviate a problem of the prior art by providing improved methods for treating seaweed extracts to decrease pyrogenicity and/or colouration. It is a further aspect to provide an alternative to existing methods of treatment.
The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.
USPTO Backgrounds
USPTO Backgrounds