Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Patent
1987-02-09
1989-11-07
Griffin, Ronald W.
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
536124, C07H 504, C07G 1700
Patent
active
048793752
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a method for the preparation of hyaluronic acid and more particularly to a method for producing high purity hyaluronic acid derived from the synovial fluid of selected animal species.
Hyaluronic acid is known to occur in many animal species including man. In the majority of animals, it is widely distributed in tissues and extra cellular fluid. In particular, in man, it is found in synovial fluid, the vitreous humour of the eye, Wharton's jelly of the umbilical cord and articular-bone cartilage. It also occurs in small quantities in the connective tissue of animals and in some micro-organisms.
It is to be noted that hyaluronic acid is a mucopolysaccharide and as such, the molecular weight of the compound varies between animal species and also within a single animal species depending on its location in the tissues.
Hyaluronic acid is known to perform a number of functions in man and other animals including the lubrication of the joints, the maintenance of the gel-like character of the vitreous humour of the eye and the contribution to the ground substance around cells where it functions as an inter-cellular lubricant and flexible cement.
In man, hyaluronic acid has been used to maintain the hydration and condition of the eye during various surgical procedures such as corneal grafts. More recently, because of its joint lubricant function, investigations have been directed in an attempt to elucidate its potential to alleviate the inflammatory joint conditions such as arthritis. In animals such as the horse, it is currently used as a method of treatment of imflammatory joint conditions.
In addition, because it is known to be a constituent of the ground substance of cells, hyaluronic acid is being incorporated into various cosmetic preparations for the skin. In this role it is proposed that the addition of hyaluronic acid to the skin is able to raise the level of hyaluronic acid present in the cells coats in the dermal layers thereby improving the condition of the skin.
As used in this specification, "hyaluronic acid" refers to the acid and to its metallic salts.
In the prior art, the methods of preparation of hyaluronic acid have been directed towards extraction from the tissue sources in which hyaluronic acid occurs in high concentration in man and other animal species. In particular, the coxcomb of the chicken and the human umbilical cord have been identified as suitable sources. However, the combination of these sources and the many steps required to isolate hyaluronic acid in the prior art have resulted in the cost of hyaluronic acid being exceptionally high. Further the present inventor believes that some of the prior art sources and preparation methods may result in a hyaluronic acid compound which has an unsatisfactory biological activity in man. It is postulated that the unsatisfactory activity is due to methods of preparation which disrupt the composition and structure of the molecule unduly, including producing a high level of protein impurity and a hyaluronic acid of unsatisfactory molecular weight.
Representative of the prior art is U.S. Pat. No. 4,141,973 (Balazs). This discloses a long and complex procedure for the isolation of "ultrapure" hyaluronic acid using coxcombs and human umbilical cords as the hyaluronic acid containing starting material. The basic steps in the disclosed process for the production of hyaluronic acid are
(1) Cleaning and freezing starting material, slicing the frozen material and extracting the material with 95% ethanol;
(2) Extracting the material with water and chloroform;
(3) Addition of sodium chloride to the water/chloroform extracts, rejection of chloroform phase; acidification of aqueous phase to pH4-5 followed by extraction with chloroform; as an alternative to chloroform extraction, DNase or RNase enzymes may be used;
(4) Adjust pH to 6.0-7.0 and extract aqueous phase with chloroform; reject chloroform; centrifugation at 70,000 to 110, 000 g may also be used; and
(5) Filtration of aqueous phase through 0.2 micron filter, followed b
REFERENCES:
patent: 2599172 (1952-06-01), Hadidian
patent: 3211616 (1965-10-01), Yosizawa
patent: 3396081 (1968-08-01), Billek
patent: 4141973 (1979-02-01), Balazs
patent: 4517295 (1985-05-01), Bracke et al.
patent: 4713448 (1987-12-01), Balazs et al.
Griffin Ronald W.
White Everett
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