Hyaluronic acid oligosaccharide fractions and drugs containing the same

Abstract

Hyaluronic acid oligosaccharides having a size selected from sizes of 4 to 60 saccharides, fractions containing the hyaluronic acid oligosaccharides and having particular physicochemical properties, and drugs containing the same. The hyaluronic acid oligosaccharides of the present invention are extremely useful, because they exert superior pharmaceutical effects as active ingredients of a heat shock protein expression promoter, cell death inhibitor, cell injury inhibitor and cell and tissue protecting agent (e.g., organ preservation agent, antiulcer agent, antihepatopathic agent, IL-10 production promoter or IL-8 production inhibitor) and exhibit high safety.

1 - 13 . (canceled) 
     
     
         14 : A process for preparing a fraction comprising a hyaluronic acid oligosaccharide of a substantially uniform size, the process comprising:
 degrading hyaluronic acid with an enzyme that degrades hyaluronic acid or dimethyl sulfoxide to a form a mixture of hyaluronic acid oligosaccharides of various sizes;   applying the mixture to a strongly basic anion exchanger so that the hyaluronic acid oligosaccharides are bound by the anion exchanger; and   eluting the bound hyaluronic acid oligosaccharides with a salt concentration gradient.   
     
     
         15 : The process of  claim 14 , wherein the hyaluronic acid oligosaccharide of a substantially uniform size is hyaluronic acid tetrasaccharide. 
     
     
         16 : The process of  claim 14 , wherein the strongly basic anion exchanger is an anion exchanger having at least one kind of anion exchange group selected from the group consisting of a trimethylammonium group, a trimethylammoniomethyl group, a β-hydroxyethyldimethylammonium group, a 2-hydroxypropylamino group, a diethyl-(2-hydroxypropyl)aminoethyl group, and a dimethylethanolammonium group. 
     
     
         17 : The process of  claim 16 , wherein the strongly basic anion exchanger comprises a trimethylammoniomethyl group. 
     
     
         18 : The process of  claim 14 , wherein the salt gradient concentration is a salt gradient concentration of NaCl, KCl, or LiCl. 
     
     
         19 : The process of  claim 14 , wherein the salt gradient concentration is increased from 0.1 M to 0.5 M during elution. 
     
     
         20 : The process of  claim 14 , wherein the salt concentration is increased with a rate of 0.1 M/40 to 450 hours. 
     
     
         21 : The process of  claim 14 , wherein the applying is conducted employing a column wherein the strongly basic anion exchanger is packed, and wherein the eluting is conducted by continuously eluting the bound hyaluronic acid oligosaccharides at a flow rate of 80 to 100 ml/hour. 
     
     
         22 : A fraction comprising a hyaluronic acid tetrasaccharide having the physicochemical properties defined in the following (1) to (6):
 (1) when the fraction is analyzed by the detection methods in the following (a) and (b) using gel filtration chromatography, the fraction shows a substantially single peak in both of the methods, and the peaks have peak areas defined in the following (a) and (b):   (a) when the detection is performed based on absorbance at 210 nm, a relative ratio of peak area of the tetrasaccharide to the sum of peak areas of total hyaluronic acid oligosaccharides in the fraction is 85% or more;   (b) when the detection is performed with a differential refractometer, a relative ratio of peak area of the tetrasaccharide to the sum of peak areas of total hyaluronic acid oligosaccharides in the fraction is 98% or more;   (2) when the fraction is analyzed based on absorbance at 210 nm employing anion exchange chromatography, the fraction shows a substantially single peak, and a relative ratio of peak area of the tetrasaccharide to the sum of peak areas of total hyaluronic acid oligosaccharides in the fraction is 90% or more;   (3) when the fraction is labeled with fluorescence and then analyzed by electrophoresis, a single band is detected, and bands of hyaluronic acid oligosaccharides of other sizes are not detected;   (4) when a theoretical value of monoisotopic molecular weight or average molecular weight of the tetrasaccharide constituting the fraction is taken as 1, an actual value of the same measured for the fraction by mass spectrometry is 0.997 to 1.003 (relative value);   (5) respective differences between theoretical values (weight %) of carbon (C), hydrogen (H) and nitrogen (N) contents in the tetrasaccharide constituting the fraction and values of the same actually measured for the elements by elemental analysis of the fraction (weight %) are all in the range of ±1 (weight %); and   (6) The fraction does not substantially contain proteins, DNA and endotoxins.   
     
     
         23 : The fraction according to  claim 22 , which further has the physicochemical property defined in the following (7):
 (7) results of  1 H-NMR and  13 C-NMR of the fraction do not contradict the structure of the tetrasaccharide represented by the following formula (1);   
       
         
           
           
               
               
           
         
       
       wherein in the formula, n is 1, M represents a proton or a monovalent cation, and Ac represents acetyl group. 
     
     
         24 : The fraction according to  claim 22 , wherein the hyaluronic acid tetrasaccharide is a saturated hyaluronic acid tetrasaccharide.