Patent Application: US-201515311779-A

Abstract:
there is described an ulvan containing composition . this composition is a viscosupplement composition and can be used in the treatment or prophylaxis of arthritis . also described is a method of treating a musculoskeletal disease , such as 5 arthritis , by administering the ulvan containing composition .

Description:
the following examples are merely illustrative and should not be construed to limit the scope of the disclosure . ulvan was solubilized in h 2 o at room temperature , and then hydrolysed with h 2 so 4 1 m at 100 ° c . for 2 . 5 hours . neutral sugars were determined as alditol acetates using gas chromatography analysis as described by coimbra et al . ( 1996 ). uronic acids were determined by an adapted 3 - phenylphenol colorimetric method described by coimbra et al . ( 1996 ). linkage analysis was executed by methylation as adapted by coimbra et al . ( 1996 ). results are shown in table 1 . ulvan ( 1 % w / v ) was solubilized in deuterium oxide at room temperature . sample was analysed by 1 h nmr acquired on a varian unity plus ( varian , usa ) spectrometer at 60 ° c . ( fig2 ). 1 h - nmr spectra were also obtained for the monomers mostly present within ulvan composition ( glucuronic acid , rhamnose and xylose , fig3 - 5 ). these monomers were prepared at 1 % w / v solution in deuterium oxide , as for the ulvan solution . the chemical shifts of the main repetition unit of the produced ulvan were identified based on the literature ( barros et al ., 2013 ; lahaye , inizan , & amp ; vigouroux , 1998 ; robic , sassi , & amp ; lahaye , 2008 ). the intensity of rharnose and glucuronic acid peaks was compared with the remaining peaks of the spectra , in order to identify the relative percentage of the repetition unit , such as the aldobiouroninc a3s (& gt ; 4 - beta - d - glucuronic acid -( 1 & gt ; 4 )- alpha - l - rhamnose 3 - sulphate - 1 & gt ;). by the relative composition of monomers and analysis of their linkage , the relative composition of aldobiouronic and ulvanobiose repetition unit can also be determined . results are shown in table 1 . sulphate content of ulvan sample was calculated based on 1 h nmr spectrum analyses assuming that : ( i ) all the rhamnose units are sulphated ; and ( ii ) there exists one unit of rhamnose per glucuronic acid . considering these assumptions , the calculation is made by the following developed equation 1 . results are in table 1 . equation 1 — equation developed to calculate ulvan sulphate percentage using 1 h nmr spectrum . where w s : sulfur weight ; w total : total weight ; mw : molecular weight ; a : anomeric c peak integration : rhms : sulphated rhamnose ; ga : glucuronic acid ; s : sulfur based on sulphate content and molecular weight of ulvan , the sulphation degree was estimated using equation 2 . results are in table 1 . equation 2 — equation developed to estimate ulvan sulphation degree ( sd ). where s t : total sulfur mass in the molecule ; mw : molecular weight of ulvan ; s %: sulphate content of ulvan , n s : estimated number of sulfur atoms ; mw s : sulfur molecular weight ; o t : total oxygen mass in the molecule ; mw o : oxygen molecular weight ; oh t : total number of hydroxyl groups in the molecule ; mw a : molecular weight of a repeating unit without sulfur ; note that molecular weight of a3s and b3s repeating units are equivalent , equation applies equally to both . ulvan was solubilized in nacl 0 . 3 m ( eluent solution ) at a final concentration of 0 . 1 % w / v . the solution was analysed by gel permeation chromatography in the equipment viscotek tda 305 , with the three detectors : light scattering , refractive index and viscometer . column set was composed by a guard pre - column aq . guard ( viscotek ) and a plaquagel - oh mixed 8 μm ( polymer laboratories ). elution was performed at 30 ° c . using a flow rate of 1 ml / min . triple detection calibration using polyethylene oxide as narrow standard was performed for molecular weight ( mw ) calculation . results are in table 1 . ulvan samples obtained from different processes were characterized for its chemical properties . notorious differences were registered between ulvan samples , especially on the average molecular weight ( mw ). additionally , the composition of the tested samples is also remarkably distinct , whereas aldobiouronic units were the most present in analysed samples . this fact indicates that ulvan 121314 , with 90 % aldobiouroninc , possibly a3s by 1 h nmr analysis , is expected to present less than 5 mol % of the monosaccharide xylose , whereas the ulvan prfd presents almost 30 mol %. osteoarthritic joints are characterized for expression of enzymes that degrade the cartilage matrix components , namely hyaluronidase . additionally , hyaluronidase promotes rapid degradation of hyaluronic acid based viscosupplements accelerating its clearance . ulvan was dissolved in phosphate buffer saline ( pbs ) at room temperature at the final concentrations of 0 . 5 , 1 . 0 and 3 . 0 % w / v . hyaluronic acid ( ha ) was dissolved in phosphate buffer saline at room temperature for the final concentrations of 0 . 1 , 0 . 5 and 1 . 0 % w / v . solutions were incubated in optimal conditions ( ph 7 , 37 ° c ., 48 hours , 75 rpm ) with hyaluronidase ( ec 3 . 2 . 1 . 35 ). solutions were then evaluated for polysaccharide molecular weight alteration by gel permeation chromatography as described above . results are shown in table 2 . ha molecular weight was reduced by 97 . 3 ± 1 % by incubation with hyaluronidase , although ulvan had no molecular weight alteration . these results demonstrate that ulvan is resistant to hyaluronidase degradation in optimal conditions and hyaluronidase action over ha is dramatic . given that ha viscosity is described in the literature as being associated with its high molecular weight , the degradation power of hyaluronidase over ha is likely to reduce ha efficiency as a viscosupplement . the greater stability of ulvan towards hyaluronidase means that ulvan will remain intact over a longer timeframe than hyaluronic acid and as a consequence , lower dosages and fewer injections of ulvan should be required for effective pain relief . osteoarthritic joints present an oxidative environment , which is associated with inflammation and pain . additionally , parenteral administration of a viscosupplement , for example by intra - articular injection , presents high risk of bloodstream contact , for which it is important to avoid potential induction of blood clot formation . this example demonstrates the reducing power capacity of ulvan and proves that ulvan is non - thrombogenic , which is relevant for its potential application as an injectable formulation . ulvan samples and hyaluronic acid were dissolved at 0 . 5 % w / v in the mandatory solvent for each analysis performed . hyaluronic acid is the gold standard for osteoarthritis therapy , therefore it was used in this example as a basis for comparison . heparin is a well - known standard for anti - coagulant activity tests . the reducing power of samples was quantified by the following protocol . samples were prepared in pbs and mixed with potassium ferricyanide , then heated at 50 ° c . for 20 min . the reaction was stopped by the addition of trichloroacetic acid solution ( 10 % w / v ). the solution was centrifuged and supernatant mixed with distilled water and ferric chloride ( 0 . 1 % w / v ). absorbance was measured at 700 nm . ascorbic acid was used as standard for reducing power . results are in table 3 . anti - coagulant activity was quantified using heparin as a reference substance . measurement of prothrombin time ( pt ) and activated partial thrombiplastin time ( atpp ) was performed as previously described by subhapradha et al . results are shown in table 4 . ulvan presented 35 % reducing power compared to ascorbic acid . hyaluronic acid tested under the same conditions did not provide significant reducing power . this result indicates that ulvan can act as a potential anti - oxidant agent in osteoarticular environments unlike hyaluronic acid . ulvan presents anti - coagulant activity , by slightly increasing the coagulation time in pt test and very significantly in aptt test . when compared to heparin , it is possible to verify that ulvan &# 39 ; s anti - coagulant activity is not as high as heparin . this result indicates that ulvan will not promote an ischemic response , which is highly relevant taking into account its possible administration by parenteral routes . these results indicate that ulvan can act as anti - oxidant agent in osteoarticular environments without eliciting a thrombogenic response . every ulvan repetition unit presents at least one reactive group : hydroxyl , carboxyl and sulphated group . these available groups allow further chemical modification of ulvan polysaccharide . methacrylated ulvan was manufactured by application of the method described in wo 2011 / 119059 a1 . methacrylated ulvan , the reaction product , was solubilized in deuterium oxide at room temperature . the sample was analysed by 1 h nmr acquired on a varian unity plus ( varian , usa ) spectrometer at 70 ° c . substitution degree was calculated based on the method from m . hamcerencu m . ( 2008 ). methacrylated ulvan was dissolved in tris - hcl 2 m and h 3 bo 4 40 mm for final concentration of 4 % w / v . photo - initiator methyl benzoylformate ( mbf ) was added . the solution was exposed to uv - light and methacrylated ulvan hydrogels were produced . fig6 represents a 1 h nmr spectrum at 70 ° c . of reaction product dissolved in deuterium oxide . the signals in the spectral region of 5 . 33 - 6 . 18 ppm were attributed to the vinyl carbon - linked hydrogen ( c ═ ch2 ). the peaks shown at 1 . 90 - 1 . 97 ppm , were ascribed to methyl groups adjacent to the double bond ( ch3 - c ═ ch2 ). these signals indicate the successful formation of methacrylated ulvan . methacrylated ulvan substitution degree was 17 %± 1 . fig7 illustrates methacrylated ulvan hydrogels produced by photo - crosslinking . hydrogels present the shape of the used mold and a yellowish coloration , which is typical of ulvan solutions . example 5 : evaluation of the in vitro effect of ulvan formulations on a primary culture of human osteoarthritic articular chondrocytes this procedure may be applied to the evaluation of any ulvan formulation according to the invention . human articular cartilage was obtained from patients diagnosed with osteoarthritis and undergoing total knee replacement . chondrocytes were isolated and cultured following methods described by masuda and sah ( 2006 ). osteoarthritic chondrocytes were exposed to ulvan formulation ( z014 , see table 5 ) ( treated group ), or culture media only ( untreated group ) for 24 h . cells were further collected for gene expression of collagen type ii , collagen type i and collagen type x , by quantitative real time polymerase chain reaction ( qrt - pcr ). gene expression of treated group was normalised to untreated control group , and presented as normalised expression ratio , according to livak and schmittgen ( 2011 ). data is presented as average ± sd . fig8 represents the normalised expression ratio of three genes quantified by real time pcr ( qrt - pcr )- genes coding for collagen type ii , collagen type i and collagen type x proteins . after treatment , ulvan formulation induced a 2 . 9 × increase in the expression of collagen type ii , relatively to untreated cells , marker of healthy cartilage matrix . regarding expression of proteins related to unhealthy cartilage matrix , while collagen type i was upregulated 1 . 7 ×, collagen type x was downregulated 0 . 06 × relatively to untreated cells . this trend indicates that exposure of oa chondrocytes to ulvan formulation most notably promotes gene expression related with collagen type ii expression , which is a sign of healthy extracellular expression . example 6 : evaluation of the in vivo effect of ulvan formulations on rabbit osteoarthritic articular cartilage model this procedure may be applied to the evaluation of any ulvan formulation according to this invention . a rabbit osteoarthritis model was used to test the effects of ulvan formulations ( a132 , see table 5 ) on the progression of oa . an osteoarthritic condition was induced in the animal &# 39 ; s knee by medial meniscectomy following methods described by smith and little ( 2007 ). an 8 week treatment was tested by periodic intra - articular injection of ulvan formulation every 2 weeks . after treatment , articular cartilage samples were harvested for histological analysis . safranin o / fast green staining was performed to identify status of cartilage matrix . fig9 represents microscopic images of rabbit articular cartilage sections stained with safranin o / fast green . the left image presents staining of normal articular cartilage ( stained red ) and subchondral bone ( stained blue - green ). the middle image demonstrates staining of articular cartilage immediately after osteoarthritis induction . signs of oa are revealed such as delamination and fibrillation of the cartilage surface , fissures within the matrix as well as glycosaminoglycan loss within the tissue . the right image presents staining of osteoarthritic articular cartilage after 8 weeks of treatment with ulvan formulation ( 3 % w / v ). visible improvement of the physical condition of the articular cartilage is observed : stronger staining of the cartilage matrix as well as lower fibrillation and fissures . ulvan formulations are prepared under aseptic environment . ulvan in powder form can be dissolved in an appropriate liquid . table 5 presents ulvan formulations and their composition . under aseptic environment , 30 mg of ulvan in solid form is dissolved in 1 ml of sterile phosphate buffer saline , ph 7 . dissolution occurs at room temperature in 30 minutes under rotational agitation . coimbra , m . a ., delgadillo , i ., waldron , k . w . & amp ; selvendran , r . r . 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