Classifications

• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
  • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
  • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
  • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
  • C08B37/0072—Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates

Definitions

• the technical solution relates to the method of preparation of highly substituted amide derivatives of hyaluronic acid, which can be used in medicine, pharmacy and for preparation of hydrogels in tissue engineering.
• Hyaluronic acid is a linear heteropolysaccharide, composed of repetitive disaccharide units of D-glucuronic acid and N-acetyl-D-glueosamine.
• This npn r branched polysaccharide when isolated from natural source, may have molecular weight within the range from 50 000 to 5 000 000 Da, depending on the isolation method and teh source material.
• Hyaluronic acid is the main component of intercellular substance and due to its visco-elastic properties is essential part of rhedlogical,' physiological arid biological functions in organism. Due to its high lubrication capacity, ability of high sorptibh and water retention it is often applied in eye surgery. Hyaluronic acid eliminates free oxygen radicals and affects the proliferation and differentiation of cells. Furthermore, it prevents collagen deposition and in that way promotes healing of wounds and prevents formation of scars.
• th ' complications consist especially in a very quick degradation thereof in solutions and in the relatively poor mechanical properties thereof.
• Chemical modification of hyaluronan allows to improve these properties and at the same time to increase its resistance to degradation.
• hydrogels are hydrophilic polymer networks which can be used for controlled distribution of medicaments, bioactive and other substances in organisms, in the form of scaffolds or cutaneous filling substances.
• An important condition for the preparation of hydrogels is the stability of the derivatives which are used as input precursors for the cross-linking reaction.
• the low degree of hyaluronan ariiide derivatives can be increased by an application of specific reaction conditions.
• the main' subject matter 'of his invention is a method of preparation of hyaluronic acid amides,' where highly substituted products are obtained by selective amidation of hyaluronic 1 acid earboxylic group.
• the principle of the reaction consists in the activation of hyaluronan carbbxy!ic group by ethylchloroformiate in the presence of a base, wherein the resulting intermediate - activated hyalurprian - consequently reacts with a primary amine R-NH 2 and hyaluronic acid amide is formed ⁇ according to Scheme 1 :
• R is an alkyl linear or branched chain Ci - C30, optionally containing aromatic or heteroaromatic groups.
• the preparation is preferably carried out in a polar aprotic environment, particularly in dimethylsulphoxide.
• the hyaluronic acid is preferably in an acidic form and has a weight average molecular weight within the range 10 to 500 kDa, particularly within the range 350 to 500 kDa.
• the reaction is carried out at 25 °C for 8 to 24 hours, preferably for 12 hours.
• the degree of substitution of hyaluronic acid in the method of preparation according to the invention is preferably controlled 'by the molar quantity of Ihe added amine R-NH 2 , wherein the primary amine is preferably added to the reaction mixtiire at time 0.1 to 2 hours after the addition of ethylchloroformiate and in the molar quantity of 3' to 5 equivalents with respect to the molar amount of hyaluronan dimer.
• the primary amine R-NH 2 can be for example CH ⁇ C-CH 2 -NH 2> NH 2 -CH 2 -CH 2 i -CH 2 -N 3 or NH 2 (CH 2 ) 5 -CH 3 .
• the molar amount of the added ethylchloroformiate is in a molar ratio Of 3 to 5 equivalents with respect to the molar amount of hyaluronan dimer.
• the base used in the method of 'the invention can be trialkylamine, such as triethylamine, preferably in the molar amount in a molar ratio of 3 to 5 equivalents with respect to the molar amount of hyaluronan dimer.
• ⁇ ' 1 trialkylamine, such as triethylamine, preferably in the molar amount in a molar ratio of 3 to 5 equivalents with respect to the molar amount of hyaluronan dimer.
• Hyaluronan amide derivatives prepared by this method show a controllable degree of substitution within the range from ' 10 to 99%.
• the substitution degree is controlled by the molar ratio of the primary amine, ethylcfiloroformiate and the base.
• Amide derivatives are highly stable to degradation and hydrolysis and therefore, they can be used for the preparation of stable hydrogels. In case of a high substitution degree only a very small amount of catalyst is needed for the cross-linking reaction.
• hyaluronic acid (acidic ' fo' m) having the molecular weight of 100 kDa and polydispersion of 1.9 was dissolved in 50 mL of DMSO at 60°C. After the dissolution, the hyaluronic acid solution was allowed to cool to the roorrt temperature. After cooling, 0.922 mL of triethylamine (5 eq) was added' 'and the reaction mixture was stirred for 10 minutes. Then 0.378 mL of ethylchlorofohniate' (3 eq) was added arid the reaction mixture was stirred for 1 hour.
• hyaluronic acid (acidic form) having the molecular weight of 248 kDa and polydispersion of 1.6 was dissolved in 50 mL of DMSO at- 60°C. After the dissolution, the hyaluronic acid solution was allowed to cool to the room temperature. After cooling, 0.922 mL of triethylamine (5 eq) was added and the reaction mixture was stirred for 10 minutes. Then 0.378 mL of ethylchloroformiate (3 eq) was added and the reaction mixture was stirred for 1 hour.
• hyaluronic acid (acidic form) having the molecular weight of 366 kDa and polydispersion of 1.6 was dissolved in 50 mL of DMSO ; at 60°C. After the dissolution, the hyaluronic acid solution was allowed to cool to the room temperature. After cooling, 0.922 mL of triethylamine (3 eq) was added and the reaction mixture was stirred for 10 minutes. Then 0.378 mL of ethylchlorofprmiate, (3, eq) was added 3 ⁇ 4trid the reaction mixture was stirred for 1 hour.
• hyaluronic acid (acidic form) having the molecular weight of 393 kDa and polydispersion of 1.6 was dissolved in 50 mL of DMSO at 60°C. After the dissolution, the hyaluronic acid solution was allowed tp cool to the room temperature. After cooling, 0.922 mL of triethylamine (5 eq) was added and the reaction mixture was stirred for 10 minutes. Then 0.378 mL of ethylchlorofoimiate, (3 eq) was added and the reaction mixture was stirred for 1 hour.
• hyaluronic acid (acidic ⁇ form) having the molecular weight of 485 kDa and polydispersion of 1.6 was dissolved ih 50 mL of DMSO at 60°C. After the dissolution, the hyaluronic acid solution was allowed to cool to the room temperature. After cooling, 0.922 mL of triethylamine (5 eq) was added and the reaction mixture was stirred for 10 minutes. Then 0.378 mL of ethylchloroformiate (3 eq) was added and the reaction mixture was stirred for 1 hour.
• hyaluronic acid (acidic form) having the molecular weight of 42 kDa and polydispersion of 1.6 was dissolved in 50 mL of DMSO at 60°C. After the dissolution, the hyaluronic acid solution was allowed to cool to the room temperature. After cooling, 0.922 mL of triethylamine (3 eq) was added and the reaction mixture was stirred for 10 minutes. Then 0.378 mL of ethylchloroformiate (4 eq) was added and the reaction mixture was stirred for 1 hour.
• hyaluronic acid (acidic form) having the molecular weight of 42 kDa and polydispersion of 1.6 was dissolved in 50 mL of DMSO at 60°C. After the dissolution, the hyaluronic acid solution was allowed to cool to the room temperature. After cooling, 0.922 mL of triethylamine (3 eq) was added and the reaction mixture was stirred for 10 minutes. Then 0.378 mL of ethylchloroformiate (5 eq) was added and the reaction mixture was stirred for 1 hour.
• hyaluronic acid (acidic form) having the molecular weight of 72 kDa and polydispersion of 1.7 was dissolved in 50 mL of DMSO at 60°C. After the dissolution, the hyaluronic acid solution was allowed to cool to the room temperature. After cooling, 0.922 mL of triethylamine (3 eq) was added and the reaction mixture was stirred for 10 minutes. Then ⁇ .378 mL of ethylchloroformiate ; (5,eq) was added , and the reaction mixture was stirred for 1 hour.
• hyaluronic acid (acidic form) having the molecular weight of 70 kDa and polydispersion of 1.7 was dissolved in 50 mL of DMSO at 60°C. After the dissolution, the hyaluronic acid solution was allowed to cool to the room temperature. After cooling, 0.922 mL of triethylamine (3 eq) was added and the reaction mixture was stirred for 10 minutes. Then 0.378 mL of ethylc oroformiate (5 eq) was added and the reaction mixture was stirred for 1 hour.
• hyaluronic acid (acidic form) having the molecular weight of 393 kDa and polydispersion of 1.7 was dissolved in 50 mL of DMSO at 60°C. After the dissolution, the hyaluronic acid solution was allowed to cool to the room temperature. After cooling, 0.922 mL of triethylamine (3 eq) was added and the reaction mixture was stirred for 10 minutes. Then 0.378 mL of ethylchloroformiate (3 eq) was added and the reaction mixture was stirred for 1 hour.
• hyaluronic acid (acidic form) having the molecular weight of 485 kDa and polydispersion of 1.7 was dissolved in 50 mL of DMSO at 60°C. After the dissolution, the hyaluronic acid solution was allowed to cool to the room temperature. After cooling, 0.922 mL of triethylamine (3 eq) was added and the reaction mixture was stirred for 10 minutes. Then 0.378 mL of ethylchloroformiaie (3 eq) was added arid the reaction mixture was stirred for 1 hour.
• hyaluronic acid (acidic ' form) having the molecular weight of 485 kDa and polydispersion of 1.7 was dissolved in 50 mL of DMSO at 60°C. After the dissolution, the hyaluronic acid solution was allowed to. cool to the room temperature. After cooling, 0.922 mL of triethylamine (5 eq) was added and the reaction mixture was stirred for 10 minutes. Then 0.378 mL of ethylchloroformiate (3 eq) was added and the reaction mixture was stirred for 1 hour.
• DOSY NMR log D (2.03 ppm CH5-CO-NH-Polymer)—10.5 m 2 /s

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• 2010
  • 2010-09-14 CZ CZ2010-687A patent/CZ305040B6/cs not_active IP Right Cessation
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