Patent Abstract:
therapeutic polymers containing a hydroxamate group that preferentially binds to active forms of a matrix metalloproteinases , thus inhibiting their enzymatic activity . the implantation of such material modifies tissue turnover and remodelling in its vicinity .

Detailed Description:
hx polymer is synthesized by surface modification of cross - linked polymethacrylic acid ( pmaa )- co - methyl methacrylate ( maa ) beads ( resulting in a novel composition of pmaa - mma - hx ). in the example , with reference to fig2 , r1 represents the polymer main chain and r2 represents hydrogen . this method results in beads that are not soluble , but are useable as such ; the surface modification method can be applied to other shapes , but the materials will need to be in their final form prior to modification . polymerizable hx monomer was synthesized . this monomer can be used to synthesize an hx homopolymer or copolymerized with any other suitable comonomers to produce polymers with a variety of properties . these polymers are suitable for coating other materials ( e . g ., stainless steel ) or ones made into a solid material after conventional thermoplastic processing ( moulding , extrusion , etc .) or beads or nanoparticles made by spray drying , solvent evaporation or any other conventional polymer processing method . in the example , with reference to fig2 , r1 represents ch 2 ═ c — ch 3 and r2 represents hydrogen . hx homopolymer synthesized from the hx monomer can also be grafted onto any derivatizable polymer to produce additional mmp - inhibiting polymers . small beads of hx polymer may be injected in the vicinity of diseased or damaged tissue . alternatively hx polymer can be incorporated into devices in contact with tissue . the hydroxamate beads may be incorporated into a thermoreversible gel that can be applied to a wound as a liquid and then removed by washing with cool saline . an example of such thermoreversible gel is disclosed in pct published application serial number pct / ca01 / 00325 ( publication number wo 01 / 68768 ) filed on mar . 15 , 2001 in the name of cheng and lin , the specification of which is incorporated herein by reference . thermoreversible gels undergo structural changes in response to changes in the environment . within the composition , the copolymer undergoes a phase transition from liquid to gel in response to changes in an environmental parameter such as for example temperature , ph , ionic strength of the composition or combinations of these parameters . the thermoreversible gel can be used as a protective coating for a wound . in this embodiment , the hydroxamate beads are incorporated into the gel itself , which is then applied to the wound as a liquid . the gel is then removed by washing with a cool saline . one example of a thermoreversible gel comprises a copolymer and a solvent , the copolymer having the structure a ( b ) n , wherein a is soluble in the solvent , b is convertible between soluble and insoluble in the solvent depending on an environmental condition , and n is greater than 1 , the composition being convertible from liquid to gel under an environmental condition where b is insoluble . the environmental condition to conversion from liquid to gel may be temperature , ph , ionic strength and a combination thereof . in the preferred structure of the gel , a is polyethylene glycol ( peg ), polyvinyl pyrrolidone , polyvinyl alcohol , polyhydroxyethylmethacrylate , and hyaluronic acid , and b is poly - n - isopropyl acrylamide ( pnipaam ), hdroxypropylmethyl cellulose and other methyl cellulose derivatives , poly ( thylene glycol vinyl ether - co - butyl vinyl ether ), polymers of n - alky acrylamide derivatives , poly ( amino acid ) s , peptide sequences , poly ( methacryloy l - alanine methyl ester ), poly ( methacryloy l - alanine ethyl ester ) and nitrocellulose . the copolymer may be present in the solvent at a level from 5 to 50 % by weight , preferably , from 10 to 25 % by weight . also , the integer n may represent 2 , 4 or 8 with the preferred embodiment being greater or equal to 4 . in a specific preferred embodiment of the gel , the letter a represents polyethyleneglycol ( peg ) and b represents poly - n - isopropyl acrylamide ( pnippaam ) and the solvent is aqueous . this gel may be formed by a process comprising the steps of : ( i ) forming a copolymer having the structure a ( b ) n , wherein a is soluble in a solvent of interest , b is convertible between soluble and insoluble in the solvent depending on an environmental condition , and n is greater than 1 ; ( ii ) solubilizing said copolymer in the solvent in an amount adequate to convert the composition from liquid to gel under an environmental condition where b is insoluble . crosslinked poly ( methyl methacrylate - co - methacrylic acid ) ( pmma - maa ) beads were suspended in a suitable organic solvent ( e . g . dmf , thf , diethyl ether ) at approximately 10 % wt / vol and allowed to equilibrate in solvent for at least 30 min at 0 ° c . while stirring . a 100 % molar excess of n - methyl morpholine and chloroformate , relative to the maa content of the beads , was added to the bead suspension . the reaction proceeded at 0 ° c . for 30 min . the beads were filtered from suspension and washed with dmf . the beads were transferred to a vessel containing a 100 % molar excess of hydroxylamine solution in water and the reaction proceeded at ambient temperature for at least 1 hour . the beads were then filtered and washed with water , 0 . 1 m hcl , again with water , and then dried at 55 - 60 ° c . fig4 shows that the hydroxamate content ( as indicated by nitrogen content ) of the copolymer beads may be varied in this process by altering the acid content of the base copolymer from 15 to 80 mol % maa . ferric chloride stains hydroxamate groups with a purple colour . fig5 shows the gradient in the staining of beads composed of a base polymer containing between 10 and 80 % maa that has been derivatized with hydroxamate groups , as well as the lack of staining for the underivatized 80 % maa beads ( extreme right sample of beads in fig3 ). the capacity of the hydroxamate - derivatized beads ( from a 63 % maa base polymer ) to inhibit the activity mmp - 2 compared to underivatized beads is shown in fig6 . before incubation with mmp - 2 for 90 minutes at room temperature , hx and control beads were swollen in tris - hcl / ca buffer for 2 hours to eliminate any effects due to absorption . after ph adjustment with naoh ( to 7 . 6 ), the supernatant was incubated with fitc - gelatin for 60 minutes in the dark . mmp - 2 activity was proportional to the intensity of solution fluorescence produced by the by - products of fitc - gelatin degradation . polyacrylates may be derivatized via a nucleophilic displacement reaction by hydroxylamine in solution , yielding bulk modified , hydroxamate - containing copolymers . poly ( methylacrylate ) was dissolved in dmf at approximately 10 % wt / vol and the solution was placed in a sealed reactor and purged with dry , n 2 gas . the solution was heated to 45 ° c . and a 100 % molar excess ( relative to polymeric ester content ) of hydroxylamine and 300 % molar excess of n - methyl morpholine were added . the solution was stirred and the reaction was continued for 24 hr . the solution was cooled and the polymer was precipitated in a cacl 2 solution . the polymer precipitate was then washed with 1 n hcl and deionized water before drying at 55 ° c . methacrylic acid monomer was dissolved in a suitable solvent ( e . g . chloroform , diethyl ether ) at 7 % wt / vol and 0 ° c . a 25 % molar excess of 4 - methyl morpholine and 25 % molar excess of chloroformate ( relative to monomer carboxylic acid content ) were added to the monomer solution with stirring . the reaction proceeded for 15 min . at 0 ° c ., then the solution was filtered . the filtrate was added to a 25 % molar excess of hydroxylamine in water solution and the combined solution was stirred at room temperature for 1 hr . after completion of the reaction , a solution of 0 . 05m naoh was added to the reaction mixture . the aqueous layer was then separated from the organic phase and extracted three times with fresh organic solvent . the organic layer was extracted twice with 0 . 05 m naoh and all of the aqueous volumes were combined . the aqueous raw monomer solution was dried in a freeze - dryer , leaving a white tacky solid . the raw product was then purified using silica gel chromatography ( thin layer or column ) with ethyl acetate / methanol or diethyl ether / methanol as the eluting solvent system . the column - purified monomer was then further purified by recrystallization from a 75 / 25 ( vol / vol ) toluene / chloroform solution to yield a colourless crystalline solid . monomer purity was evaluated by nmr spectroscopy in d 6 - dmso ( fig7 ) and found to be approximately 95 mol %. the ferric hydroxamate test was performed on the raw , derivatized monomer . the monomer was dissolved in 0 . 1 m hcl , several drops of 5 wt % fecl 3 were added and the solution immediately turned dark burgundy confirming the presence of hydroxamate functionality . performing the test on underivatized maa resulted in no detectable colour change . in addition , the mmp inhibiting capacity of the purified monomer was demonstrated . modulation of mmp - 8 concentration and activity in buffer solution by hx beads hx bead dose response curves were generated with both pro -( inactive ) mmp - 8 and catalytic domain ( active ) mmp - 8 enzyme solutions . pro - mmp - 8 ( 15 ng / ml , r & amp ; d systems ) and catalytic domain mmp - 8 ( 100 u / ml , biomol international ) solutions were incubated with four different doses of hx beads ( 32 , 48 , 64 and 100 mg / ml ) for 1 . 5 h at room temperature . following the bead incubations , pro - mmp - 8 supernatants were assayed for total mmp - 8 concentration using an enzyme linked immunosorbent assay ( elisa ; r & amp ; d systems ). to measure mmp activity , catalytic domain mmp - 8 supernatants were added to a chromogenic , broad - spectrum mmp substrate ( 1 mm , biomol international ). substrate digestion was monitored by measuring optical density ( absorbance ) over time for 30 min and mmp activity estimated from the slope of the absorbance vs . time curve . reductions in mmp levels for the two enzyme solutions as a function of hx bead concentration are shown in fig8 . mmp - 8 catalytic domain demonstrated a bead - dose dependent reduction in mmp activity ranging from 24 - 100 %. in contrast , pro - mmp - 8 concentrations were not reduced . these differences in mmp reductions for the two mmp - 8 forms demonstrate that hx beads have an affinity for the active form of mmp - 8 compared to the inactive form . modulation of mmp - 8 concentration and activity in human chronic wound fluid by hxbeads a subsequent bead - dose experiment was performed with human chronic wound fluid exudate , which contains a mixture of pro and active forms of mmp - 8 . following bead incubations , wound fluid supernatants were analyzed for both mmp - 8 concentration and mmp activity as described above . the hx bead dose response curves for this experiment are shown in fig9 . human wound fluid exhibited a dose dependent reduction in mmp activity ranging from 36 - 100 %, while mmp - 8 concentration changed by only 17 - 32 %. these results are consistent with those of example 1 . high reductions in mmp activity on the substrate assay are observed , reflective of the active , bead - bound component of mmp - 8 in the wound fluid sample , while the inactive constituent of mmp - 8 remains unaffected as seen by the modest reduction in mmp - 8 concentration by elisa . solutions of mmp - 3 , - 8 and - 13 catalytic domains ( biomol international ) were prepared at 100 u / ml . separate aliquots were incubated with four different doses of hx beads ( 32 , 48 , 64 and 100 mg / ml ) for 1 . 5 h at room temperature . following the bead incubations , solution mmp activities were determined using the chromogenic substrate assay . the relative rates of substrate digestion as a function of hx bead concentration are illustrated in fig1 and demonstrate a similar range of mmp inhibition ( from 21 - 100 %) for the different mmp subclasses . modulation of mmp activity in human arthritic synovial fluid by hxbeads samples of human arthritic synovial fluid , which contain a mixture of active mmps , were incubated with 100 mg / ml of hx beads and the inhibition of mmp activity subsequently measured with the chromogenic substrate assay . as illustrated in fig1 , synovial fluid mmp activity is reduced by 74 % following hx bead incubation . full length human mmp - 3 ( 115 nm , biomol international ) was activated by incubating the enzyme solution at 55 ° c . for 1 h [ koklitis et al ., 1991 ]. mmp solution activity was determined prior to and following the activation procedure using the chromogenic substrate assay . as illustrated in fig1 , untreated mmp - 3 has minimal activity towards the substrate . following treatment there is a significant increase in the slope of the absorbance vs . time curve (& gt ; 400 %) indicative of successful activation . in order to determine the effect of mmp - 3 activation on hx bead efficacy , samples of heat activated and untreated mmp - 3 ( 500 ng / ml ) were incubated with 100 mg / ml of hx beads for 1 . 5 h at room temperature . following the incubations , supernatants were diluted 1 : 50 in assay diluent and analyzed by elisa for total mmp - 3 concentration . fig1 shows the resulting reductions in mmp - 3 concentration for the two treatment groups . a 63 % reduction in concentration was achieved for the activated enzyme compared to 25 % for the untreated group . this difference in mmp inhibition illustrates preferential hx bead binding towards the active form of mmp - 3 . full - length , recombinant human mmp - 8 ( 23 nm ) was activated by incubating the enzyme solution with a 2 : 1 molar ratio of heat activated mmp - 3 for 12 h at 37 ° c . [ knauper et al ., 1993 ]. mmp solution activity was determined prior to and following the activation procedure using the chromogenic substrate assay . as illustrated in fig1 , untreated mmp - 8 is partially active . treatment with mmp - 3 increases the slope of the absorbance vs . time curve by 72 %, indicating a significant increase in the proportion of active form mmp - 8 with mmp - 3 treatment . notably , this increase in solution mmp activity exceeds the contribution from the added mmp - 3 as illustrated by the relatively low slope of mmp - 3 absorbance vs . time curve . in order to determine the effect of mmp - 8 activation on hx bead efficacy , samples of mmp - 3 activated and untreated mmp - 8 ( 500 ng / ml ) were incubated with 100 mg / ml of beads for 1 . 5 h at room temperature . following the incubations , supernatants were diluted 1 : 50 in assay diluent and analyzed by elisa for total mmp - 8 concentration . fig1 shows the resulting reduction in mmp - 8 concentration for the two treatment groups . a 32 % reduction in concentration was achieved for the activated enzyme compared to 2 . 5 % for the untreated group reflecting a preferential hx bead binding towards the activated mmp - 8 . a second mmp - 8 activation protocol was devised which combined the mmp - 3 activation procedure described above with the addition of 1 nm apma , a well established mmp activating reagent , for 12 h , 37 ° c . 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