Patent Document:

the cartilage used in this invention may be obtained from a mixture of shark species , principally blue shark ( prionace glauca ), but may include other species such as school shark ( galeorhinus galeus ), rig or smooth dogfish ( mustelus lenticulatus ) and spiky dog fish ( squalus acanthias , squalus mitsukurii ). the meat adhering to the cartilage may first be removed by any suitable means known to a person skilled in the art such as manual scraping , mild protease treatment or high pressure water treatment . the cartilage may then be dried by freeze drying , air drying or any other suitable means . the cartilage is preferably milled , ground or pulverised to provide a powder having a particle size of less than approximately 500 microns , preferably less than 300 microns . the cartilage powder is then suspended in water or in water containing salts or electrolytes such as a phosphate buffered saline solution . the water may contain one or more water miscible organic solvents . however , it is known that the use of a mixture having greater than approximately 50 % v / v of an organic solvent results in low yields of products having low activity . chaotropic salts ( those salts which extract proteins ) are not desired as their use causes reduced angiogenesis activation of the aqueous extracts . the amount of cartilage suspended in the water is preferably in the range of between 10 and 50 mg / ml . the suspension is preferably stirred or shaken for a time suitable to enable formation of the desired aqueous extract , preferably up to 3 or 4 days , more preferably approximately 20 hours . during the extraction , the temperature of the suspension is preferably maintained between approximately 4 ° c . and 50 ° c . the extraction is typically carried out at a temperature of approximately 18 - 20 ° c . the aqueous extract and the insoluble residue may be separated by any suitable method , for example , centrifugation , decantation or filtration including ultrafiltration or dialysis . the solid residue and the aqueous extract are preferably dried , for example , by freeze drying or air drying , or they may be used directly . the aqueous extract produced by using shark cartilage that has been cleaned of adhering flesh and minced but not dried does not exhibit angiogenesis activation . the extract is preferably stored at − 18 ° c . optionally following lyophilisation . after 8 months the angiogenesis activating activity of redissolved extract was approximately 85 - 90 % of the activity of freshly prepared extract . each product of the process of this invention ( the angiogenesis activator and the angiogenesis inhibitor ), may be combined with suitable carriers to provide formulations , such as tablets , capsules , liquids , emulsions , suspensions , creams , ointments , or other formulations , suitable for use as angiogenesis activators or angiogenesis inhibitors . an angiogenesis inhibitor or an angiogenesis activator of this invention may be administered in any form suitable for the control of angiogenesis such as orally , topically , rectally , intravenously , intraperitoneally , intramuscularly , or by surgical implantation . the following examples illustrate the invention but are not to be construed as limiting : powdered shark cartilage ( 100 g ) was suspended in water ( 900 ml ). the suspension was shaken for 20 hours at room temperature ( 18 - 20 ° c .). the insoluble residue was then separated from the aqueous extract by filtration . both the insoluble residue and the aqueous extract was dried . powdered shark cartilage ( 50 g ) was suspended in water ( 1000 ml ). the suspension was stirred at 20 ° c . for 20 hours . the insoluble residue was then separated from the aqueous extract by centrifugation . both the insoluble residue and the aqueous extract was dried . powdered shark cartilage was suspended in water ( 50 mg / ml ). the suspension was stirred at 4 ° c . for 20 hours . the insoluble residue was then separated from the aqueous extract by centrifugation . both the insoluble residue and the aqueous extract were dried . powdered shark cartilage was suspended in phosphate buffered saline solution ( 50 mg / ml ). the suspension was stirred at 4 ° c . for 20 hours . the insoluble residue was then separated from the aqueous extract by centrifugation . both the insoluble residue and the aqueous extract were dried . the determination of angiogenesis modulatory activity of the fractions involved the development of an assay using rings of rat aorta . thoracic aortas were removed from male rats ( 6 - 10 weeks of age ) the excised aortas were transferred immediately to a culture dish containing culture media mcdb131 . for all experimental procedures involving mcdb131 the media was supplemented with penicillin ( 100 u / ml ), streptomycin ( 100 mg / ml ) and aminocaproic acid 300 mg / ml . the fibroadipose tissue surrounding the aorta was removed carefully with particular attention paid to minimising damage to the aortic wall . rings ( 1 mm thickness ) were sectioned from the aorta and rinsed three times with mcdb131 . the aortic rings were cultured at 37 ° c . in 3 % co 2 , 97 % air for 40 min . the assays were performed using 24 well culture plates ( nune ). each assay was run in triplicate ( at least ). in a separate tube 30 ml of thrombin ( 10 nih units / ml ) was added to mcdb131 ( 1 . 5 ml ) containing fibrinogen . following rapid mixing 0 . 4 ml of the solution was added to each well . after the gel formation , an aortic ring was placed in the centre of each well . the rings were then covered with a further 0 . 4 ml of the fibrinogenthrombin mix . the aortic rings were cultured at 37 ° c . in 3 % co 2 / 97 % air in a humidified atmosphere . for analysis of the solid residue , up to approximately 2 mg of shark cartilage or derived material was added to the assay following mixing of the thrombin with fibrinogen . for analysis of the extract , a sample of the extract was added to the assay following mixing of the thrombin with fibrinogen and / or at the time of adding the mcdb131 ( 1 . 5 ml ) to each well . the extent of angiogenesis was determined by measuring the area occupied by the new vessels using a microscope with an ocular grid or by taking a photographic record of each well and measuring the area occupied by new vessels using computer image analysis and quantification . the residue obtained after extraction of the shark cartilage was compared for angiogenesis activity with crude shark cartilage . the experiment was conducted using cartilage from four different commercial sources a to d . with the exception of source a , it was found that the residue exhibited a marked increase in angiogenesis inhibition relative to the crude cartilage . a soluble extract of shark cartilage from a single source was prepared by extraction with water and another by extraction with aqueous mcdb131 medium . by incorporating aliquots of these extracts into the mcdb131 media above the aortic rings , the angiogenesis activity of these extracts was determined at varying concentrations of the extract &# 39 ; s dry weight . analysis of the assay results below showed activation of angiogenesis relative to a control . the results show an apparent increasing correlation between concentration of shark cartilage extract and angiogenesis activation . experimental rats were fed shark cartilage at 6 g / kg of food for 11 - 12 weeks . serum from these and from control animals fed a normal diet were assessed biochemically . the serum alanine - lactate aminotransferase ( alt ) activity was found to be increased by a mean of 45 % in rats which had been fed shark cartilage not subjected to the process of this invention . for the rats fed the solid residue from the aqueous extraction process of the invention , the increase was only 27 %. alt is a marker of liver function . elevated activity is suggestive of liver dysfunction . long - term consumption of shark cartilage may result in greater elevation of alt activity indicating increased liver damage . the alt activity for animals fed the solid residue from the extraction process is reduced . thus , the extraction process appears to remove one or more hepatotoxic substances from the shark cartilage . although the invention has been described by way of example and with reference to embodiments thereof , it should be appreciated that variations and modifications may be made thereto , without the departing from the scope of the claims . the angiogenesis inhibitor of this invention is useful as a therapeutic agent for the treatment or prevention of diseases or disorders such as cancer where reduced formation of new blood vessels is desired . the angiogenesis activator of the invention is useful as a therapeutic agent where the promotion of new blood vessels is desired , for example , for wound healing .

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