It is known that activities and functions of hyaluronic acid (henceforth also referred to as “HA”) change depending on the molecular size. For example, it has been reported that HA having a molecular weight of 1,200,000 shows NF-κB inactivation activity, neovascularization inhibition activity and so forth (Neumann A., Schinzel R., Palm D., Riederer P. and Munch G., “High molecular weight hyaluronic acid inhibits advanced glycation endproduct-induced NF-κB activation and cytokine expression”, FEBS Lett., 453(3):283-7, Jun. 25, 1999; Feinberg R. N., Beebe D. C., Hyaluronate in vasculogenesis, Science 220:1177-1179, 1983), whereas HA having a molecular weight of 500,000 or less has inverse activities (Noble P. W., McKee C. M., Cowman M. and Shin H. S., “Hyaluronan fragments activate an NF-κB/I-κB alpha autoregulatory loop in murine macrophages”, J. Exp. Med. 183:2373-2378, 1996.; West D. C. and Shaw D. M., “Tumour hyaluronan in relation to angiogenesis and metastasis”, In: Laurent T. C. ed., The chemistry, biology and medical applications of hyaluronan and its derivatives, London: Portland Press, 1998: 227).
It can be said that this well suggests possibilities of finding of various activities also for HA oligosaccharides and finding of specific activities depending on sizes of HA oligosaccharides. Therefore, it can be considered that, if HA oligosaccharides having different sizes are combined, they may exert an additive or synergistic effect, or conversely, against an activity of an HA oligosaccharide of a certain size, another HA oligosaccharide having a different size may act as an antagonist.
Assuming as described above, if a fraction containing HA oligosaccharides of various sizes is used for search of activities thereof for development of drugs utilizing HA oligosaccharides, not only it cannot be found which size of HA oligosaccharide constitutes an entity of a certain activity, but also an activity of HA oligosaccharide of a certain size may be compensated by an HA oligosaccharide of another size. Thus, important physiological activities or functions hidden in HA oligosaccharides may be overlooked.
Further, when an HA oligosaccharide of a certain specific size is used as a drug, it is necessary to eliminate oligosaccharides of other sizes that inhibit the function exerted by the HA oligosaccharide of specific size as much as possible, and there has been desired a fraction of high purity that does not substantially contain substances undesirable for drugs.
As described above, for creation and provision of novel drugs, there has been desired a HA oligosaccharide fraction of high purity that consists of HA oligosaccharides of substantially uniform sizes and does not substantially contain HA oligosaccharides of other sizes and other impurities.
Meanwhile, heat shock proteins (henceforth also referred to as “Hsp”) are also called stress proteins, which are proteins that inhibit affections caused by various stress reactions, and various families thereof have been known. The heat shock proteins of Hsp70 family, which is one of the families, are considered to prevent cell injury or cell death through actions of inhibiting structural changes of proteins, production of abnormal proteins and so forth caused by factors generating environmental stresses such as heat shock, hydrogen peroxide, heavy metals, amino acid analogues and glucose depletion and stress factors such as fervescence, inflammation, ischemia, viral infection, metabolic disorders, hypercardia, oxidative stresses, cellular tissue affections, oncogenes and carcinogenic substances or actions of regenerating functions of proteins.
In connection with the above, a stress protein expression promoter containing HA as an active ingredient is disclosed in Japanese Patent Unexamined Publication (Kokai) No. 9-227386. In this patent document, it is described that HA consisting of about 2 to 20 saccharides is preferred as the active ingredient (page 3), and it was suggested that HA consisting of ten or less saccharides was involved in enhancement or induction of expression of stress proteins (page 6). There is also described an experiment demonstrating that an unsaturated HA disaccharide enhanced expression of a stress protein and inhibited cell death (Example 2).
However, the above patent document does not specifically disclose various oligosaccharides as in the present invention, and it does not teach nor suggest at all the extremely notable activity for enhancement of expression of stress protein specifically observed for HA tetrasaccharide mentioned later.
Further, considering the functions of the aforementioned stress proteins, the stress proteins are considered to be involved also in protection of cells and tissues.
Concerning the above, Japanese Patent Unexamined Publication No. 11-246301 discloses an organ preservation solution for use in organ transplantation containing HA and/or a physiologically acceptable salt thereof. Although this patent document describes that the average molecular weight of HA is preferably 100,000 or more, it does not describe nor suggest HA oligosaccharides and superior effects exerted by them.