Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
2002-10-21
2004-08-17
Wilson, James O. (Department: 1623)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C514S056000, C536S018700, C536S021000, C536S123100, C536S124000
Reexamination Certificate
active
06777398
ABSTRACT:
PRIOR ART
The chondroitin sulfates are natural products present in animal tissues with structural and physiological functions.
The chondroitin sulfates have antiinflammatory activity, they have been classified as SYSADOA (Symptomatic Slow Acting Drug Osteoarthritis) and used in the medium-long term therapy in the treatment of arthritic patients (Morreale P., Manopulo R., Galati M., Boccanera L., Saponati G. and Bocchi L. “Comparison of the antiinflammatory efficacy of chondroitin sulfate and diclofenac sodium in patients with knee osteoarthritis”. J. Rheumatol., 23,1385-1391, 1996).
The commercial chondroitin sulfates are obtained by extraction methods from animal tissues and consist of regular disaccharides formed by glucuronic acid and N-acetylgalactosamine sulfated in position 4 and/or 6 (YOSHIDA, K. et al.—Analytical Biochemistry, 177, pp. 327-332 (1989)).
The average molecular weight of the commercial products is about 18,000-20,000 D. The chondroitin sulfate with sulfate group in position 4 (Ch4S) is never present alone in the natural products, bur normally it is joined in the same chain to chondroitin sulfate with sulfate group in position 6 (Ch6S) in percentages ranging from about 10 to 73% and then these sequences are separable with difficulty. Generally a commercial chondroitin sulfate contains about 40% Ch4S and 60% Ch6S.
The K4 polysaccharide may be obtained by fermentation methodologies as disclosed in the patent WO 01/02597.
SUMMARY
Now we have found that by a series of reactions on the K4 polysaccharide from
Escherichia coli
it is possible to obtain chondroitin sulfates having the structure represented by at least 70% by weight by formula (I), the remainder to 100% consisting of non sulfated product.
In formula (I), R and R
1
equal or different, represent H or SO
3
Na provided that R and R
1
cannot be both H and n is an integer number ranging from 5 to 50.
In particular when R=R
1
=SO
3
Na the product is the 4,6-disulfate, when R=H and R
1
=SO
3
Na the product is the 6-sulfate and when R=SO
3
Na and R
1
=H the product is the 4-sulfate. The carboxyles/sulfates ratio ranges from 0.7 to 2. (Ia) corresponds to the glucuronic acid structure and (Ib) corresponds to N-acetyl galactosamine.
The above described three products are prepared from a common intermediate obtained from the K4 polysaccharide by a process including the following steps:
a) defructosilation of the K4 polysaccharide by treatment with HCl;
b) passage on ionic exchange column to obtain the acid polysaccharide or b1) selective protection of the carboxyl by formation of the methyl ester;
c) selective protection of the positions 4 and 6 of the galactosamine by dibenzylidenation;
d) protection of the positions 2 and 3 of the glucuronic acid by O-acetilation;
e) deprotection in position 4 and 6 of the galactosamine by the separation of the dibenzylidene by treatment with acetic acid obtaining the common intermediate having structure represented by at least 80% by formula (II)
wherein R
2
=acetyl, R
3
=Na or CH
3
and n=5-100.
In order to obtain the chondroitin sulfates having formula (I) according to the present invention, the product having formula (II) is selectively sulfated and deprotected, acting in the following conditions:
f) To obtain the chondroitin 4,6-disulfate (R=R
1
=SO
3
Na)
i.) The intermediate (II) is treated with sulfating agent consisting of pyridine-sulfotrioxide in conditions of temperature higher than 20° C. and with a sulfating agent/OH molar ratio between 1:1 and 3:1;
ii.) The product obtained is liberated from the acetyls of the glucuronic acids by basic treatment;
iii.) The product is recovered by diafiltration on membrane and drying.
g) To obtain the chondroitin 4-sulfate (R=SO
3
Na, R
1
=H)
i.) The intermediate (II) is treated with triphenylmethyl chloride to selectively protect the position 6 of galactosamine;
ii.) It is treated with sulfating agent consisting of pyridine-sulfotrioxide in conditions of temperature higher than 20° C. and with a sulfating agent/OH molar ratio between 1:1 and 3:1;
iii.) The product obtained from triphenylmethyl is liberated by acid treatment;
iv.) The product obtained is liberated from the acetyls of the glucuronic acid by basic treatment;
v.) The product is recovered by diafiltration on membrane and drying.
h) To obtain the chondroitin 6-sulfate (R=H, R
1
=SO
3
Na)
i.) The intermediate (II) is treated with sulfating agent consisting of pyridine-sulfotrioxide in conditions of temperature not higher than 5° C. and with a sulfating agent/OH molar ratio between 1:1 and 3:1;
ii.) The product obtained is liberated from the acetyls of the glucuronic acid by basic treatment;
iii.) The product is recovered by diafiltration on membrane and drying.
REFERENCES:
patent: 3895107 (1975-07-01), Morrison
patent: 6288044 (2001-09-01), Zoppetti et al.
patent: 2 055 872 (1981-03-01), None
patent: WO 98/34958 (1998-08-01), None
patent: WO 01/02597 (2001-01-01), None
H. W. Stuhlsatz and H. Greiling, “The Preparation of Chondroitin 4- and Chondroitin 6-Sulphates,”The Methodology Of Connective Tissue Research,pp. 129-136, 1976.
Casu, B. and Gennaro U., “A conductimetric method for the determination of sulphate and carboxyl groups in heparin and other mucopolysaccharides,”Carb. Research,39:168-176, 1975.
Harenberg, J. and De Vries, J. X., “Characterization Of Heparins By High-Performance Size Exclusion Liquid Chromatography,”J. Chromatography,261:287-292, 1983.
Holme, K. R. and Perlin, A. S., “Nuclear Magnetic Resonance Spectra Of Heparin In Admixture With Dermatan Sulfate And Other Glycosaminoglycans 2-D Spectra Of The Chrondroitin Sulfates,”Carb. Research,186:301-312, 1989.
Morreale, P. et al., “Comparison of the Antiinflammatory Efficacy of Chondroitin Sulfate and Diclofenac Sodium in Patients with Knee Osteoarthritis,”J. Rheumatology,23(8):1385-1391, 1996.
Suzuki, S. “Chondroitinase fromProteus vulgarisandFlavobacterium heparinum,” Chondroitinases From Bacteria,124:911-917.
Yoshida, K. et al., “Analysis of Unsaturated Disaccharides from Glycosaminoglycuronan by High-Performance Liquid Chromatography,”Analytical Biochemistry,177:327-332, 1989.
Oreste Pasqua
Zoppetti Giorgio
IBSA Institut Biochimique S.A.
Krishnan Ganapathy
Morgan & Finnegan L.L.P.
Wilson James O.
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