Compositions of partially deacetylated chitin derivatives

Abstract

The present invention relates to compositions comprising biologically active chitinous oligomers and their endotoxin purified and partially deacetylated chitin polymer precursors, and their use in pharmaceutical compositions, biomaterial compositions, medical devices, and processes to produce the said oligomers. More specifically the present invention relates to novel compositions and processes to produce such compositions. The compositions include therapeutic hetero polymer and hetero oligomer compositions compricing specific sequences of N-acetyl glucosamine and glucosamine, developed to optimize chemical and structural features which are important for the therapeutic activity of these compositions. In addition, the present invention provides methods to use degree of deacetylation of a partially deacetylated chitin polymer in order to modulate physical and biological parameters in a calcium phosphate composite for bone implant applications.

1 . A method for producing highly pure and fully soluble partially deacetylated chitin polymer, the method comprising the steps of:
 partially deacetylating chitin with an alkali treatment to a degree of deacetylation (DD) between 25% and 70%;   neutralizing the partially deacetylated chitin;   dissolving the partially deacetylated chitin in acidic solution;   removing undissolved particles through sequential filtering steps;   adjusting the solution to a pH above 8;   raising the temperature of the solution to above 40° C.; and   precipitating the fully dissolved and purified partially deacetylated chitin, through   (i) elevated temperature above 40, and   (ii) addition of salt or through neutralization of the dissolved acid in solution,   
       wherein the precipitate is recovered and washed after precipitation through sieving or by centrifugation and wherein the temperature of the washing water and the precipitate is above 50° C. 
     
     
         2 . The method according to  claim 1 , wherein the degree of deacetylation (DD) of the partially deacetylated chitin polymer is between 30-60%. 
     
     
         3 . The method according to  claim 1 , wherein the degree of deacetylation (DD) of the partially deacetylated chitin polymer is between 30 and 55%. 
     
     
         4 . The method according to  claim 1 , wherein the step of adjusting the partially deacetylated chitin solution comprises raising the pH to between pH 8-13. 
     
     
         5 . The method according to  claim 1 , wherein the precipitating comprises addition of salt selected from sodium chloride; a salt of tricarboxylic, dicarboxylic or monocarboxylic organic acid; sulfate; urea; or guanidinium chloride. 
     
     
         6 . The method according to  claim 5 , wherein said addition of salt comprises adding salt to reach a salt concentration for precipitation in the range between 2% and saturation. 
     
     
         7 . The method according to  claim 1 , further comprising treating said chitin with inorganic or organic acid prior to the step of partially deacetylating. 
     
     
         8 . The method according to  claim 1 , further comprising a step of extensive treatment of the partially deacetylated chitin with a family 18 endo-chitinase. 
     
     
         9 . The method according to  claim 1 , wherein the precipitating comprises addition of salt selected from a salt of citric acid, malic acid, or acetic acid. 
     
     
         10 . A method for producing therapeutically active chitooligomers comprising extensive enzymatic hydrolysis with a Family 18 chitinase enzyme of partially deacetylated chitin polymer having a degree of deacetylation (DD) of between 25 to 70%, wherein the partially deacetylated chitin polymer is prepared by the method according to  claim 1 . 
     
     
         11 . The method according to  claim 10 , further comprising filtering the hydrolyzed chitin polymer with a 1 kDa ultrafiltration membrane, and collecting the retentate comprising chitooligomers. 
     
     
         12 . The method according to  claim 10 , further comprising drying the chitooligomers thereby producing a dry form of the chitooligomers selected from a powder, flakes and a fibrous material. 
     
     
         13 . The method according to  claim 10 , wherein said partially deacetylated chitin polymer has a degree of deacetylation (DD) of between 30 to 60%. 
     
     
         14 . A composition comprising therapeutically active chitooligomers of N-acetylglucosamine and glucosamine, wherein the chitooligomers are prepared by a method comprising a first step of partial deacetylation, of chitin to obtain partially deacetylated chitin polymer having a degree of deacetylation (DD) between 30% and 60% and a subsequent step of extensive enzymatic hydrolysis by a Family 18 chitinase enzyme of the obtained partially deacetylated chitin polymer, wherein the deacetylation step comprises:
 partially deacetylating chitin with an alkali treatment to a degree of deacetylation (DD) between 30% and 600%, wherein said alkali is cooled prior to mixing with the chitin, the decatylation reaction maintained at a temperature in the range of 5-60° C.   
     
     
         15 . The composition according to  claim 14 , wherein the hydrolyzed chitin polymer is filtered using a 1 kDa cutoff ultrafiltration membrane and chitooligomers retained by the membrane are used in the composition. 
     
     
         16 . The composition according to  claim 14 , wherein the chitooligomers are in a dry form selected from the group consisting of a powder, flakes and a fibrous material. 
     
     
         17 . The composition according to  claim 14 , wherein said partially deacetylated chitin polymer is prepared by a method comprising: partially deacetylating chitin with an alkali treatment to a degree of deacetylation (DD) between 25% and 70%, wherein said alkali is cooled prior to mixing with the chitin, the decatylation reaction maintained at a temperature in the range of 5-50° C.;
 neutralizing the partially deacetylated chitin; 
 dissolving the partially deacetylated chitin in acidic solution; 
 removing undissolved particles through sequential filtering steps; 
 adjusting the solution to a pH above 8; 
 raising the temperature of the solution to above 40° C.; 
 optionally adding salt; and 
 
       precipitating the fully dissolved and purified partially deacetylated chitin, wherein the precipitate is recovered and washed after precipitation through sieving or by centrifugation and wherein the temperature of the washing water and the precipitate is above 50° C. 
     
     
         18 . A composition comprising therapeutically active chitooligomers of N-acetylglucosamine and glucosamine, wherein the chitooligomers are prepared by a method comprising a first step of partial deacetylation of chitin to obtain partially deacetylated chitin with a degree of deacetylation (DD) between 30% and 60%, and a second step of extensive enzymatic hydrolysis by a Family 18 chitinase, and further comprising a step of filtering with a 1 kDa ultrafiltration membrane, retaining the retentate.