Patent Application: US-42072503-A

Abstract:
therapeutic polymers containing hydroxamate group that binds and thus inhibits zinc containing enzymes such as matrix metalloproteinases . the implantation of such material inhibits remodeling in its vicinity .

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 fig1 r 1 represents the polymer main chain and r 2 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 shapcs , 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 fig1 r 1 represents ch 2 ═ c — ch 3 and r 2 represents hydrogen . hx homopolymer synthesized from the hx monomer can also be grafted onto any derivatizable polymer to produce additional mmp - inhibiting polymers . in the example , with reference to fig1 r 1 represents any chemical group of a derivatizable polymer and r 2 represents hydrogen . small beads of hx polymer were injected in the vicinity of diseased or damaged tissue . alternatively hx polymer can be incorporated into devices in contact with tissue . the incorporation of hx beads into the implant site of biomaterial tubes made from gelatin inhibited the remodeling and degradation of the gelatin tubes in a murine model . fig2 illustrates the difference in degradation ( at day 11 ) of unimplanted ( control ) tubes versus tubes from untreated sites ( no beads ) and sites incorporating hx beads . the results showed that hx beads are capable of inhibiting tissue remodeling and destruction , controlling inflammation and restricting cell migration . the hydrokamate 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 , polyhydroxyethyimethacrylate , 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 . [ 0062 ] fig2 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 . fig3 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 fig4 . 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 ( fig5 ) 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 % feci 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 . although the invention describes and illustrates a preferred embodiment of the invention , it is to be understood that the invention is not so restricted and includes all alternative embodiments thereof . 1 . ashley r . and the sdd clinical research team ( 1999 ) clinical trials of a matrix metalloproteinase inhibitor in human periodontal disease , ann . new york acad . sci ., 878 , 159 - 79 . 2 . chen , j . j ., y . p . zhang , s . hammond , n . dewdney , t . ho et al . 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