Arzneim.-Forsch./Drug Res. 45 (II), Nr. 8 (1995)
This excerpt reprinted with permission of publisher
© 1995 Editio Cantor Verlag

Biochemical and Pharmacokinetic Aspects of Oral Treatment
with Chondroitin Sulfate
A. Conte, N. Volpi, L. Palmieri, I. Bahous, and G. Ronca

Chondroitin sulfate (Condrosulf® ) was characterized for structure, physicochemical properties and purity. This glycosaminoglycan has a relative molecular mass of about 14,000, a sulfate-to-carboxyl ratio of 0,95 due to the high percentage of monosulfated disaccharides (38% 6-monosulfate and 5 % 4-monosulfate) and a low amount of disulfated disaccharides (1.1 %) inside the polysaccharide chains. No other glycosaminoglycans were detected in the preparation.

Chondroitin sulfate was labelled by reduction with sodium ³H-borohydride and administered by oral route in the rat and dog. More than 70% of radioactivity was absorbed and found in urine and tissues. The plasma radioactivity was fractionated by size-exclusion chromatography in three fractions: radioactivity associated with high, intermediate and low molecular mass compounds. The peak value of the concentration of high molecular mass radioactivity compounds in plasma was reached after 1.6 and 2.1 h for the rat and dog, respectively. After 36 h the high molecular mass radioactivity compounds were still present in plasma of dog and rat. After 24 h radioactivity was higher in the intestine, liver, kidneys, synovial fluid and cartilage than in other tissues. Condroitin sulfate was orally administered to man (healthy volunteer) in a single daily dose of 0.8 g and in two daily doses of 0.4 g The results showed that both forms of administration determined a significant increase of plasma concentration of chondroitin sulfate as compared with predose value over a full 24 h period. Elimination constant values and t_max (of the first administration in the case of fractionated dose) were almost the same for the two administrations.

Some biochemical parameters (number of leukocytes, proteins, sulfated glycosaminoglycans and hyaluronic acid amounts, and N-acetylglucosaminidase activity) of synovial fluid were evaluated in controls and treated osteoarthritic subjects. No variations were observed in the patient who did not receive chondroitin sulfate. Five days of chondroitin sulfate administration led to a significant increase of concentration and molecular mass of hyaluronan and a decrease of a lysosomal enzyme, N-acetylglucosaminidase. No significant differences in leukocyte count and protein content were detected.

The therapeutic uses of native glycosaminoglycans (hyaluronic acid, chondroitin sulfates, dermatan sulfate, heparan sulfate and heparin) as well of their low molecular mass derivatives (low molecular mass heparins, dermatan sulfate and chondroitin sulfate) and mixtures of different percentage of heteropolysaccharides have markedly increased with the knowledge of their pharmacological properties and biological functions [1,2].

An in-depth knowledge of the metabolism of exogenous glycosaminoglycans, administered both orally and paren-terally, would be particularly useful in view of their therapeutic applications. The low degree of sulfation of glycosaminoglycans allows their administration by both parenteral and oral routes [3,4,5]. In fact, they maintain their pharmacological properties when administered orally, unlike highly sulfated glycosaminoglycans and like heparin that is inactive as anticoagulant when administered orally [6] even if several fragments appear in the plasma [7]. Some evidence of heparin absorption by the gtomach mucosa was reported in a recent paper [8],describing that the administration of 60 mg/kg of sodium (or dextran sulfate) induced an anti-thrombotic rats. Jaques ct al. [8] report that heparin enters the body immediately on oral administration (2.4 and 6 min) and that this drug (or dextran sulfate) is recovered from the endothelium in large amounts and it is identified unchanged by electrophoretic technique. This gastric absorption might have been due to such a high dosage that a fraction of the drug or several of its active fractions which causes the antithrombotic effect had permeated.

Chondroitin sulfates are glycosaminoglycans composed of alternate sequences of differently sulfated residues of uronic acid (b-D-glucuronic) and a-D-N-acetyl-galactosamine linked by b(1-> 3) bonds [9]. The regular disaccharide sequence of chondroitin sulfate A, chondroitin-4-sulfate, is constituted by [(1->4)-O-(b-D-glucopyranosyluronic acid)-(l->3)--O-(2-N- acetamido-2-deoxyb-D-salactopyranosyl-4- sulfate)]. Chondrotin sulfate C or chondroitin-6-sulfate, is mainly composed of a disaccharide unit [(1->4)-0-(b-D- glucopyranosyluronic acid)-(l->3)-O-(2-N-acet-amido-2-deoxy-b-D-galacto-pyranosyl-6-sulfate)]. Disaccharides with different number and position of sulfate groups can be located, in different percentage, inside the polysaccharide chain, such as the non-sulfated or disulfated disaccharide in which two sulfate groups can be O-linked in position 2 of b-D-glucuronic acid and 6 of a-D-N-acetyl--galactosamme (disaccharide D) or in position 4 and 6 of a-D-N-acetylgalactosamine (disaccharide E) [9]. The hetero-geneity of the primary structure, besides the physicochemical properties such as the relative molecular mass and charge density, is responsible for different and more specialized biological and pharmacological functions of these glycosaminoglycans [10].

The synthesis of proteoglycans and hyaluronate has been clarified whilst their breakdown and the regulation of their turnover, in normal and pathological conditions are less known [11,12]. Chondroitin sulfates may be employed as chondroprotective [13] drugs with application in the therapy of tibiofibular osteoarthritis of the knee [14] and in the articular cartilage osteoarthritis by intramuscular and oral route. The oral route for chondroitin sulfates as chondro-protective drugs is very interesting, because it allows simplified use, which is more compatible with long administration periods.

The metabolic fate of orally administered exogenous chondroitin sulfate with defined structure and physico-chemical properties was studied in the experimental animal and in man using non-labelled and radioactive isotope labeled polysaccharide. Furthermore, we studied the plasma levels of chondroitin sulfate after repeated oral administration and the modification of cell number and biochemical parameters in synovial fluid during treatment.

The therapeutic effects of a high molecular mass natural compound, like chondroitin sulfate, administered orally is not surprising since many other natural macromolecules have pharmacological activity also when administered orally, e.g. heparan sulfate [39], dermatan sulfate [5] and bromeline. Recently, it has been reported that also collagen type II orally administered is effective in the treatment of rheumatoid arthritis [40].

The presence of exogenous chondroitin sulfate and its depolymerized derivatives in synovial fluid is very important to explain anti-inflammatory and chondroprotective effects of this polysaccharide. In fact, it has been shown that sulfated and desulfated poly- and oligosaccharides derived from chondroitin sulfate and hyalorunan degradation have regulatory effects [41,42,43]. Exogeneous chondroitin sulfate as well poly- and oligosaccharides may exert anti- inflammatory action on synovial cells (leukocytes) as well a regulatory activity on cartilage metabolism.

A number of clinical studies showed that chondroitin sulfate decreases pain and increases functional parameters in osteoarthritic patient [13,14,44-46]. Our observation give a first biochemical basis to the clinical outcomes. In fact, a few days after administration of exogenous chondroitin sulfate, some biochemical parameters of synovial fluid changes indicating that modifications take place in enzyme release and hyaluronan/glycosaminoglycan synthesis and/or degradation. Some of these effects may be related to the chondroprotective activity of exogenous chondroitin sulfate whereas others, such as the decrease of activity of lytic enzymes, may be also due to the anti-inflammmatory properties of this polysaccharide, which has been shown to modify some leulcocyte functions [47]. In synovial fluid, like in plasma, fractions with molecular mass higher than that of administered chondroitin sulfate were found, probably due to the binding of polysaccharide and its partially depolymerized derivatives with proteins. In fact, it has been observed that in plasma chondroitin sulfate is associated with proteins [25]. To date, we do not know if the observed quantitative variations of hyaluronan are due to its increased synthesis or decreased breakdown. In fact, chondroitin sulfate (and derivatives) stimulates hyaluronan formation [48] (besides the synthesis of proteoglycans and type II collagen [45]), and it protects hyaluronan from enzymatic degradation by inhibiting hyaluronidase activity and breakdown from free radicals.

[1] Conrad, H.E., Ann. NY Acad. Sci. S56, 18 (1989)- [2] Casu, B., Semin. Thromb. Hemostasis, 17 (Suppl. 1-2), 9 (1991)- [3] Pescador, R.,Diamantini, G., Mantovani, M, Malandrino, S., Riva, A., Casu, B., Oreste, P., Arzneim.-Forsch./ Drug Res. 30 (II), 1893 (1980) [4]Segre, G., Min. Med. 77, 1859 (1986)- [5] Dawes, J., Hodson, B. A, Pepper, D. S, Thromb. Haem. 62, 945 (1989)- [6] Dal Pozzo, A., Acquasaliente, M., Geron, M. R., Thromb. Res. 56, 119 (1989)- [7] Larsen, A. K., Lund, D. P., Langer, R., Folkman, J., Proc. Natl. Acad. Sci. USA 83, 2964 (1986)- [8] Jaques, L.B., Hiebert, L.M., Wice, S. M., J. Lab. Clin. 117, 122 (1991)- [9] Murata, K., Yokoyama, Y., Anal. Biochem. 149, 261(1985)- [10] Volpi, N., Bolognani, L., Conte, A., Petrini, M., Leukemia Res. 17, 789 (1993)- [11] Prehm, P, Biochem. J. 267, 185 (1990)-[12] Redini, E, Daireaux, M., Mauviel, A., Galera, P, Loyau, Ci., Pujol, J. P., Biochim. Biophys. Acta 1093, 196 (1991)- [13] Pipitone,V. R., Drugs Exp. Clin. Res. 17, 3 (1991)- [14] Rovetta, G., Drugs Exp. Clin. Res 17, 53 (1991)- [15] Poblacion, C. A., Michelacci, Y. M., Carb. Res 147, 87 (1986)- [16] Volpi, N., Bolognani, L., J Chromatogr. 630, 390 (1993)- [17] Cassaro, C. M. E, Dietrich, C. P., J. Biol. Chem. 252, 2254 (1977)- [18] Cappelletti, R., Del Rosso, M., Chiarugi, V P, Anal. Biochem. 99, 311(1979)- [19] Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J., J. Biol. Chem. 193, 317 (1951)- [20] Volpi, N., Mascellani, G., Bianchini, P., Liverani, L., Il Farmaco 47 (Suppl. 5), 841 (1992)- [21] Friman, C., Eronen, I., Videman, T., Int. J. Sports Med. 7, 330 (1986)- [22] Lindqvist, U., Engstrom-Laurent, A, Laurent, U., Nyberg, A., Bjorklund, U., Eriksson, H., Pettersson, R., Tengblab,A., Scand. J. Clin. Lab. Invest. 48, 765 (1988)- [23] Calatroni, A., Vinci, R., Ital. J. Biochem. 36, 333A(1987)- [24] Blumenkrantz, N, Asboe-Hansen, G., Meth. Biochem. Anal. 24, 39 (1977)- [25] Staparans, I., Felts, J. M., J.Clin. Invest. 76, 1984 (1985)-[26] Boas, N. E, J. Biol. Chem. 204, 553 (1953)- [27] Denduchis, B., Kefalides, N. A., Berzkorovainy, A., Arch. Blochem. Biophys 138, 582 (1970)- [28] Bard, Y., Nonlinear Parameter Estimation, pp. 341, Academic Press, New York (1974)- [29] Carroll, G., Ann. Rheum. Dis 48, 17 (1989)- [30] Little, C. B., Hilbert, B. J., Wicksystrom, S., Hedlund, B. E., Am. J. Vet. Res. 51, 1534 (1990)- [31] Palmieri, L., Conte, A., Giovannini, L., Lualdi, P., Ronca, G., Aruleim.-Forsch./Drug Res. 40 (I), 319 (1990)- [32] Conte, A., de Bernardi, M., Palmieri, L., Lualdi, P, Mautone, G., Ronca, G., Arzneim.-Forsch./Drug Res. 41(11), 768 (1991)-[33] Conte, A., Palmieri, L., Segnini, D., Ronca, G., Drugs Exp.Clin. Res. 17, 27 (1991)- [34] Gervasi, G. B., Farina, C., Bartoll, C., Catalani, R., Carpita, G., Pistelli, N., Arzneim. Forsch./Drug Res. 43 0, 445 (1993)- [35] Dol, E, Houin, G., Dupouy, D., Cadroy, Y., Carnobe, C., Gabaig, A. M., Mardoguian, J., Sie, P., Boneu, B., Thromb. Haem. 59, 255 (1988)- [36] Dol, E, Houin, G., Rostin, M., Montastruc, J. L, Dupouy, U., Gianese, E, Sie, P, Boneu, B., Blood 74, 1577 (1989)- [37] Hopwood, J. J., in: Heparin. Chemical and Biological properties. Clinical applications, D. A. Lane, U. Lindahl (eds.), pp. 191-227, Edward Amold, London-Melbourne etc. (1989)- [38] Wood, K. M., Curtis, C. G., Powell, G. M., Wusteman, E S., Biochem. J. 158, 39 (1976) - [39] Agrati, A. M., De Bartolo, G., and Palmieri, G., Minerva Med. 83, 533 (1992) -[40]Trentham, D. E., Dynesius-Trentham, R. A, Orav, E. J., Combltem, D., Lorenzo, C., Lea Sewell, K., Hafler, D. A., Weiner, H. L., Science 261, 1727 (1993)- [41] Nethery, A., Giles, I., Jenkins, K., Jackson, C., Brooks, P., Burkhardt, D, Ghosh, P., Whitelock, J., O'Grady, R. L., Welgus, H. G., Schrieber, L., Biochem. Pharm. 44, 1549 (1 992)- [42] Nevo, Z., Dorfman, A., Proc. Natl. Acad. Sci. USA 69, 2069 (1972)- [43] Meier, S., Hay, E. D., Proc. Natl. Acad. Sci. USA 71, 2310 (1974)- [44] Pipitone, V., Ambanelli, U., Cervini, C., Cherier Lignlere, G., Tamborini, U. G., Ferraris, M. Marcolongo, R, P.V. Tegale, E, Semi, U., Trotta, E, Martino, E, Curr. Ther. Res. 52, 608 (1992)- [45] Bassleer, C., Henrotin, Y. and Franchimont, P, Int. J. Tiss. React. 14, 231 (1992)- [46] Setnikar, I., Int. J. Tiss. React. 14, 253 (1992)- [47] Conte, A., Petrini, M., Vaglini, E, Polidori, R., Litera Rheum. 14, 35 (1992)- [48] Hutadilok, N., Smith, M. M., Cullis-Hill, D., Brooks, P M., Cihosh, P, Curr. Ther. Res. 44, 845 (1988)