Patent Application: US-201414150297-A

Abstract:
sterile micron - sized gold particles contained in capped vials , a kit of part including the vial and a liquid capable of suspending the particles , as well as their preparation , medical devices or medicaments prepared by their preparation as well as uses thereof in treating inflammation . also , gold coated implants preferably for use in combination with the medical devices .

Description:
the micron sized gold particles according to the present invention have a purity of 99 . 99 % w / w pure gold . gold particles according to the invention are available from for example hildebrand & amp ; cie sa , thônex - genève , suisse . the size of the gold particles is selected so that the diameter or largest cross - section of a solid metal gold piece is at least 20 μm , separated by sieving through sieves having the appropriate mesh size . when the micron - sized gold is above this size it is not phagocytosed by macrophages . in the context of the present invention the term dissolucytosis , is an extracellular liberation of gold ions from the surface of gold particles bigger than 20 microns . such particles are particles that cannot be phagocytozed by macrophages . the dissolution membrane makes it possible for the macrophages to control the chemical milieu at the gold surface and the dissolucytosis of gold ions is most likely caused by the capacity of the macrophages to manipulate releasing cyanide ions and altering the oxygen tension and the ph in their vicinity ( larsen et al . 2006 ; ferre and claria 2004 ). the process of dissolucytosis is limited by the size of the gold surface , the amount of dissolucytotic macrophages , and their state of activity . the slow speed of the process results in a limited liberation of gold ions taken up solely by cells close to the implant , while inflammation will cause a dramatic increase in the amount of bio - released gold ions , that later decline to the ‘ at rest ’ stadium . dissolucytosis , the bio - release of gold ions by adhering macrophages , can only take place if the particles stay outside the cells , i . e . are not phagocytized as the dissolucytotic release takes place only extracellularly . therefore the lower range of 20 micron is an important aspect for an efficient treatment . in the context of the present invention the liquid capable of maintaining micron - sized gold particles in suspension is any liquid capable of carrying the gold particles or flakes in any embodiment described herein for at least 1 minute . in terms of parameters the liquid may be characterized by its viscosity as the liquid must be relatively viscous if gold particles have to be suspended while gold flakes can be suspended by less viscous solutions having a viscosity close to the viscosity of water . in the context of the present invention “ liquid ” is contemplated as being either a concentrated liquid or a solution of a compound such as an aqueous solution . when the micron sized gold are particles the viscosity of the liquid is preferably in the range 1 - 150 cp , more specifically 40 - 110 cp , such as approximately 50 cp at 25 ° c . an example is discovisc ® mentioned above . the liquid may be selected from e . g . collagen , silicones or cellulose that are compatible with the human body , such as medical grade silicones , hyaluronic acid as well as derivatives and combinations thereof . it is also contemplated that the liquid includes but is not limited to fluids extracted from the recipient him -/ herself and other constituents forming part of the human and animal body . it is contemplated that any of the above can be used in concentrated form or in solution such as in an aqueous solution . hyaluronic acid is a presently preferred liquid . it is a glycosaminoglycan ( gag ) composed of disaccharides chains , composed of d - glucuronic acid and d - n - acetylglucosamine , linked together via alternating 62 - 1 , 4 and β - 1 , 3 glycosidic bonds . hyaluronic acid is distributed widely throughout for example connective , epithelial , neural tissues and is the major component of the synovia ( korenek et al . 1992 ). approximately , one third of hyaluronic acid in a human is degraded and synthesized every day . thus , hyaluronic acid is ubiquitous in human and animal tissues , where it exhibits significant structural , rheological and physiological functions . according to the invention hyaluronic acid is contemplated in all forms such as free acid and any pharmaceutically acceptable salt , ester or hydrate thereof . an example of salt derivatives of the acid is selected from but not restricted to sodium and potassium as well as salts of heavy metals as described in e . g . u . s . pat . no . 4 , 746 , 504 , u . s . pat . no . 4 , 784 , 991 and u . s . pat . no . 601 , 790 . it is contemplated that throughout the description and claims when hyaluronic acid or hyaluronate is mentioned it is contemplated that any form of the above is covered . it is also contemplated in any suitable concentration such as 10 mg / ml in 0 . 9 % saline water . hyaluronic acid , also called hyaluronan , is manufactured in large scale in physiological acceptable molarities and is commercial available as a medical device for injection in joints , eyes , skin ( fillers ), ventricular system in the brain , the urinary bladder and more . hyaluronic acid suitable in the present invention is available for example under the trade name suplasyn ® available from bioniche pharma , ireland . typically hyaluronic acid / hyaluronane is available as an 8 - 10 mg / ml physiological aqueous solution , but in the context of the present invention solutions in the range 5 - 15 mg / ml is suitable , such as 10 mg / ml . the glass beads according to the invention have a size compatible with a commercial available capped vial . in preferred embodiments the glass beads have a diameter of 3 to 8 mm . the size of the glass beads should be chosen so that proper agitation or mixing and distribution of the particles in the liquid can be obtained . the capped vial according to the invention is any suitable vial , but most traditional are commercially available eppendorf ® 4 ml vials . other vials are equally applicable . the size depends on the location to be treated and the corresponding amount of liquid and gold . the specific choice of the vial is a trivial choice which is within the skill of the art . the syringe comprising the liquid contemplates any syringe suitable of holding a liquid and penetrating a capped vial . numerous syringes are available in the art such as the pre - filled syringes used in commercial hyaluronic products sold under the trade name the suplasyn ® and provisc ® or any other suitable syringe . for the purpose of the medical device or medicament and the methods of the invention it is contemplated that the syringe is prefilled or filled on the site , from e . g . a 10 ml or 25 ml capped vial of hyaluronic acid , before injecting into the capped vial . the amount of gold in the vial can be any amount . from an economic perspective the amount should however be minimized as much as possible without loosing clinical efficiency . it is preferred that the amount is sufficient to provide a lifelong supply of gold ions , at a specific location in the human or animal body once the medicament / medical device is administered . thus , in most application the amount of gold in the vial is in the range 2 to 16 mg , more preferred 6 - 14 mg , even more preferred 8 - 12 mg , such as 10 mg . for suspending an amount effective and economical in a lifelong local treatment the amount of liquid could be in the range 1 - 5 ml , depending of the circumstances . a suitable amount depends on the amount of gold suspended and is within the skill of the art . when the micron - sized gold is particles the ratio between gold and liquid is in the range 2 - 100 mg / 1 ml . when the micron - sized gold is flakes the ratio between gold and liquid is in the range 1 - 100 mg / 1 ml . the invention also relates to an implant such as prostheses , stents , hearing aids and the like which may be made from titanium alloys , zirconium alloys , or ceramics , or any other implant with a gold gildable surface . various applications of gold are possible in the method of the present invention . for example , gold may be applied to the surface of an implant by gilding , amalgamating , patching , or dotting . the method of applying the gold to an implant is within the skill of the art . in the context of the present invention the terms medical device and medicament are used interchangeably . the terms cover the gold particles or flakes suspended in the liquid according to the invention . neither of the terms should be interpreted narrowly . the compositions of the invention may be seen as both medical devices and medicaments as they constitute compositions capable of treating the human body . whether denoted medical device or medicament / medicinal products depend on the legislation under which they are approved . it is also contemplated that the medical device or medicament is denoted cosmetic composition when applied for this purpose . the disorders that can be treated according to the invention are selected from but not limited to inflammatory disease selected from the group consisting of e . g . arthrose , multiple sclerosis , alzheimers disease , psoriasis , eczema , autoimmune diseases and inflammation related conditions , such as wounds , as well as cosmetic conditions such as wrinkles . in a study by pedersen et al . ( 2012 ), 8 - week - old rats were used to evaluate the release of gold ions from gold particles suspended in hyaluronic acid . the animals were housed under standard conditions , i . e . a 12 - hour light / dark cycle at 22 ° c ., and with free access to food and water . all experimental procedures were performed in accordance with danish law . the experimental animals were injected with 2 × 2 . 72 mg metallic gold suspended in sodium hyaluronate into 1 ) 20 neocortex 2 ) brain ventricles . the controls were injected hyaluronate only . the study showed a slowing of disease progression in terms of reduced weight loss in gold - treated animals as compared to vehicle - treated animals demonstrating the effect of therapy . ninety - nine percent pure metallic gold ( alfa - aesar , ge ) was sieved using 2 aluminum sieves ( retsch , ge ) to obtain solid metal gold particles in the size of 20 to 45 μm . 463 mg solid metal gold particles was mixed with 0 . 85 ml sodium hyaluronate ( 10 mg / ml in physiological water , i . e . 0 . 9 % saline ), provisc ®, alcon , in a water bath set at 40 ° c . prior to the injections , the animals were anesthetized with an injection of a mixture of narcoxyl - vet ( xylazin ) and ketominal - vet . the deeply anesthetized animals were placed in a benchmark stereotaxic instrument ( myneurolab . com , usa ) and a longitudinal incision was made in the skin covering the skull . two drill holes were made under the microscope according to the paxinos - franklin coordinates , and the solid metal gold sodium hyaluronate / sodium hyaluronate injections were given in the center of each drill hole . the amg developer consists of a 60 ml gum arabicum solution ( bidinger , aarhus , dk ) and 10 ml sodium citrate buffer ( 23 . 5 g sodium citrate ( merck 6448 , vwr , dk ) to 100 ml distilled water ), 15 ml reducing agent ( 0 . 85 g of hydroquinone ( merck 4610 , vwr , dk ) dissolved in 15 ml distilled water at 40 ° c . ), and 15 ml solution containing silver ions ( 0 . 12 g silver lactate ( fluka 85210 supplied by sigma - aldrich , vallensbk , dk ) in 15 ml distilled water at 40 ° c . ); the latter is added immediately before use while thoroughly stirring the amg solution . the amg development takes place in a water bath at 26 ° c . for 60 to 15 min under a dark hood . development is stopped by replacing the developer with a 5 % thio - sulphate solution for 10 min . finally , the tissue sections / slices are rinsed several times in distilled water ( for details see danscher 1981 and danscher & amp ; stoltenberg 2006 ). tissue slices to be analyzed in the light or electron microscope were treated in accordance with the following two procedures , respectively . after transcardial perfusion with 3 % glutaraldehyde ( merck 4239 , vwr , albertslund , dk ) the brains was immediately removed from the body and allowed to post - fixate in the same fixative . for light microscopical analyses the slices were placed in a 30 % solution of sucrose until they sank to the bottom of the glass . the slices were then frozen with co 2 , placed in a cryostat , and allowed a temperature fall to − 17 ° c . after amg development ( see below ), the sections were counterstained with a 0 . 1 % solution of aqueous toluidine blue ( ph 4 . 0 ), dehydrated in ascending concentrations of alcohol and xylene , embedded in depex and covered with a cover glass . for electron microscopy the slices were cut on a vibratome and the resulting 100 - μm - thick sections were developed in amg . the areas to be analyzed were cut out , placed in osmium tetroxide ( 1 % in phosphate buffer for 30 minutes ) and embedded in epon . from these epon blocks , 3 μm - thick sections were cut and amg developed . one of the three sections on each glass slide was counterstained with toluidine blue . after lm analysis , the sections to be analyzed further in the electron microscope were re - embedded on top of an epon block . ultrathin sections were cut , placed on a grid and counterstained with uranyl citrate and lead acetate ( danscher 1981 ; danscher and stoltenberg 2006 ). light microanalysis of amg developed sections showed fine traces of liberated gold ions in tissue near the metal gold deposit within the region of the lesion and its border zone . in this area , cytoplasmatic accumulation of silver enhanced nanogold particles , resulting from the bio - released gold ions taken up by the cells , was seen in both glia cells and neurons . the amg technique for demonstration gold ions bound to macromolecules in the tissue relay on reduction of the gold ions ( by radiating the sections with uv light ) to metallic gold atoms that accumulate in nanoparticles . it is these particles , originating from the bio - released gold ions that are made visible by amg silver enhancement . at ultrastructural levels the amg enhanced nanogold particles were found to be located in lysosome - like organelles . the number of gold labeled lysosomes was sparse as was the amount of amg grains in each lysosome . all control sections were void of amg staining . gold particles placed in the ventricular system of the brain showed dissolucytotic release of gold ions into the cerebrospinal fluid and were taken up by glia cells around the ventriculocisternelle system . based on these observations it is shown that a safe local gold cure can be obtained by using small but non - phagocytizable i . e . & gt ; 20 μm gold particles hereby limiting any damaging effects of the injection of the gold particles , and investigations in our laboratory indicates that placing micron - sized solid metal gold particles / implants within the ventricular system is a feasible way to circumvent placing the pure gold metal directly in the brain tissue . the amount of gold ions liberated through dissolucytosis has shown to be so limited and localized that even micron - sized gold particle could serve as a life - long cure with a minimal risk of toxic side effects . the increased number and activity of microglia / macrophages during an ongoing inflammation will automatically increase the amount of gold ions liberated when it is most needed . the hyaluronic acid injections have no observable influence on the brain ( gorm danscher and agnete larsen “ effects of dissolucvtotic gold ions on recovering brain lesions ” histochem cell biol ( 2010 ) 133 : 367 - 373 . bio - liberated gold ions i . e . gold ions dissolucytotically liberated by macrophages by gold particles influence the immunological response by affecting the cells that are involved in the inflammatory process ( larsen a et al ., 2008 ; pedersen mø et al ., 2009 “ metallic gold reduces tnfalpha expression , oxidative dna damage and pro - apoptotic signals after experimental brain injury ”; pedersen mø et al ., 2010 ; pedersen mø et al ., 2009 , “ metallic gold treatment reduces proliferation of inflammatory cells , increases expression of vegf and fgf , and stimulates cell proliferation in the subventricular zone following experimental traumatic brain injury ”; pedersen ds et al ., 2012 ). these findings indicates that treatment with metallic gold implants with a large surface ‘ in casu ’ gold particles , is an approach with clinical dimensions for ameliorating aseptic inflammation and pain everywhere in the body , including in the brain . furthermore , the treatment is believed to have protective and regenerative potentials as shown in the mouse brain . aktas o , ullrich o , infante - duarte c , nitsch r , zipp f ( 2007 ) neuronal damage in brain inflammation . arch . neurol . 64 , 185 - 189 . aschner m , sonnewald u , tan , k h ( 2002 ) astrocyte modulation of neurotoxicity . brain pathol . 12 , pp . 475 - 481 . danscher g localization of gold in biological tissue . 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( 2006 ) silver enhancement of quantum dots resulting from ( 1 ) metabolism of toxic metals in animals and humans , ( 2 ) in vivo , in vitro and immersion created zinc - sulphur / zinc - selenium nanocrystals , ( 3 ) metal ions liberated from metal implants and particles . prog . histochem . cytochem . 41 , 57 - 139 . felson d t , anderson j j , meenan r f ( 1990 ) the comparative efficacy and toxicity of second - line drugs in rheumatoid arthritis . results of two metaanalyses . arthritis rheum . 34 , 134 - 213 . ferre n , claria , j ( 2006 ) new insight into the regulation of 30 liver inflammation and oxidative stress . mini rev . med . chem . 6 , 1321 - 1330 . futami , t , ohnishi h , taguchi n , kusakari h , oshima h , maeda t ( 2000 ) tissue response to titanium implants in the rat maxilla : ultrastructural and histochemical observations of the bone - titanium interface . j . periodontol . 71 , 287 - 298 . larsen a , kolind k , pedersen d s , doering p , pedersen m o , danscher g , et al ( 2008 ) gold ions bio - released from metallic gold particles reduce inflammation and apoptosis and increase the regenerative responses in focal brain injury . histochem cell biol . 130 ( 4 ): 681 - 92 . larsen a , stoltenberg m . & amp ; danscher , g ( 2007 ) in vitro liberation of charged gold atoms . autometallographic tracing of gold ions released by macrophages grown on metallic gold surfaces 128 , 1 - 6 histochem cell biol . mhatre m , floyd r a , hensley , k . ( 2004 ) oxidative stress and neuroinflammation in alzheimer &# 39 ; s disease and amylotrophic lateral sclerosis : common links and potential therapeutic targets . j . alzheimer dis . 6 , 147 - 157 . pedersen m o , larsen a , pedersen d s , stoltenberg m , penkowa m . ( 2009 ) metallic gold reduces tnfalpha expression , oxidative dna damage and pro - apoptotic signals after experimental brain injury . 0 . brain res 19 ; 1271 : 103 - 13 . pedersen mø , larsen a , stoltenberg m , penkowa m . ( 2010 ) bio - released gold ions modulate expression of neuroprotective and hematopoietic factors after brain injury . brain res . 2010 jan . 11 ; 1307 : 1 - 13 . pedersen mø , larsen a , pedersen d s , stoltenberg m , penkova m . 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