Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
1999-11-23
2002-01-15
Geist, Gary (Department: 1623)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C514S056000, C514S059000, C424S442000, C424S493000, C536S018700, C536S021000, C536S053000, C536S123100, C536S124000
Reexamination Certificate
active
06339074
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the homogeneous sulfation of polysaccharides and semisynthetic derivatives thereof, in particular glycosaminoglycans such as hyaluronic acid and its esters and tetraalkylammonium salts, for the preparation of new biomaterials useful in biomedical, health care, and pharmaceutical applications, and to such biomateriale per se. Such sulfated derivatives exhibit anti-thrombotic activity as evidenced by the lengthening of both the thrombin time and the whole blood clotting time. Moreover, the absence of hemolysis and the growth and shape of endothelial cells placed in contact with such sulfated derivatives indicate that these materials are promising heparin-like compounds.
DESCRIPTION OF THE RELATED ART
Many molecules of biological origin are polyelectrolytes, and their interactions are very important in a wide variety of biochemical reactions. Consequently, synthetic and/or semisynthetic polyelectrolytes have been in use for some time now. These polyelectrolytes mimic the biological characteristics of natural polyelectrolytes, and can have somewhat different characteristics compared to the starting material.
Polyelectrolytes of biological origin include sulfated polysaccharides, and in particular, heparin and its derivatives (D. A. Lane and U. Lindahl, Eds.,
Heparin
-
Chemical and Biological Properties, Clinical Applications
, Edward Arnold, London), which play an important role in cell-substrate interactions, particularly in the process of viral activity inhibition, in the process of blood coagulation, in lipid removal, etc.
Heparin is the most biologically reactive member of the family of sulfated glycosaminoglycans. It is well known for its antithrombotic and anticoagulant properties. In fact, it is extensively used in the management of cardiovascular diseases anti contributes enormously to the success of open heart surgery. Nevertheless, the structure of heparin is not simple and, due to the number of variations, is not entirely known. Commercial heparins consist of a spectrum of 21 heparins (Nader et al. (1974)
Biochem. Biophys. Res. Commun.
57:488) ranging in molecular weights from 3,000 to 37,500 in varying anticoagulant activities.
The blood anticoagulant activity of heparin is attributed to structural features, e.g., degree of sulfation, degree of dissociation, particular sequences of COO
−
and SO
−
3
groups, as well as to molecular shape and size. These factors appear to be related to biological activity by virtue of their importance in the ion binding capacity of heparin (Stivala et al. (1967)
Arch. Biochem. Biophys.
122:40). By virtue of its highly negatively charged nature, heparin has a strong affinity for cations, and its activity is pH-dependent.
Most of the readily available natural polysaccharides have been sulfated in an attempt to obtain heparin analogues (Hoffman et al. (982)
Carbohydrate Res.
2:115; Kindness et al. (1980)
Brit. J. Pharmac.
63:675; Horton et al. (1973)
Carbohydrate Res.
30:349; Okada et al. (1979)
Makromol. Chem.
180:813; Kikuchi et al. (1979)
Nippon Kagaku Kaishi
1:127; Manzac et al. (1981)
Proc. Third M.I.S.A.O.
5:504), and recently, sulfate, carboxylic, and sulfonate groups were attached to synthetic polymers such as polystyrene (Kanmaugue et al. (1985)
Biomaterials
6:297) and polyurethane (Ito et al. (1992)
Biomaterials
13:131). The anticoagulant activities of these materials were much lower than that heparin, and were dependent on the type and binding of the substituents, the degree of substitution, and sequences.
Some chemical reactions are known which make it possible to sulfate polysaccharides (WO 88/00211; EP 0 340 628; Nagasawa et al. (1986)
Carbohydrate Research
158:183-190), but it has not yet been possible to obtain sulfated polysaccharides which, besides the chemical and chemical-physical characteristics peculiar to such polysaccharides, also possess new characteristics, such as anticoagulant activity.
SUMMARY OF THE INVENTION
The present approach to studying the structural properties associated with the anticoagulant properties of polysaccharides was first to choose polymers possessing well-defined chemical groups consisting of regular repeating units, and secondly to modify their chemical structure.
Such molecules must therefore:
(1) Contain regular sequences of monomeric units, and
(2) Be chemically modifiable without destroying their structure.
Hyaluronic acid, the major component of the mammalian extracellular matrix, consists of alternating units of N-acetylglucosamine and glucuronic acid residues, and therefore seems a suitable macromolecule.
The sulfation of alcoholic hydroxyls present in the polymeric chain of a polysaccharide or of one of its semisynthetic derivatives by the use of a suitable sulfating agent can lead to the formation of new derivatives with chemical-physical characteristics, but most of all biological characteristics, which are different from those of the starting material.
The polyelectrolyte polyssaccharides which can be used as substrates in the present invention include glycosaminoglycans. First and foremost among these is hyaluronic acid and the semisynthetic derivatives thereof. Some particularly important semisynthetic derivatives of hyaluronic acid are esters thereof with alcohols of the aliphatic, araliphatic, heterocyclic and cycloaliphatic series, designated “HYAFF,” that are described in U.S. Pat. Nos. 4,851,521, 4,965,353, and 5,202,431, and EP 0 216 453. Sulfation of such pre-processed biomaterials is a novel feature of the present invention. In this case, the sulfation reaction no longer occurs in the homogeneous phase, but rather on the surface of the biomaterial in the heterogeneous phase, activating the exposed hydroxyl groups toward the reaction solvent.
The degree of sulfation that can be obtained directly on the biomaterial is an important characteristic, and requires careful kinetic control. To avoid the solubilization of the biomaterial, induced by the increased hydrophilic nature of the polymer which constitutes the matrix, the number of —SO
3
groups per dimeric unit must not exceed a certain level, generally less than 1.5-2, depending upon the degree of hydrophilicity of the starting biomaterial. For example, in the case of HYAFF 11 films, wherein all the carboxyls are involved in ester bonding with benzyl groups, the maximum degree of sulfation should not exceed 1.5.
The reagents commonly used for sulfation include the complex between sulfur trioxide and pyridine (SO
3
-pyridine).
The reaction is conducted by adding the sulfating reagent to a tetrabutylammonium salt of a polysaccharide in solution, or to a solution of a polysaccharide ester, which, in the case of partial esters, contains the remaining carboxy functions in the form of tetrabutylammonium salts, in aprotic solvents such as dimethylsulfoxide, N,N′-dimethylformamide, and N-methylpyrrolidone in the temperature range of from about 0° C. to about 60° C.
Different degrees of Bulfation, measured by the number of sulfate groups per disaccharide unit, are obtained by varying the quantity of SO
3
-pyridine. The ratio between moles of hydroxyls and moles of sulfating reagent can vary between 1:1 and 1:12.
Surprisingly, the present inventors succeeded in sulfating the polysaccharide chain of hyiluronic acid and its semisynthetic derivatives in a specific and homogeneous manner without causing loss of the polymer's characteristics, in particular its molecular weight, thus obtaining new polymers with biological and physico-chemical characteristics which hyaluronic acid and its semisynthetic derivatives did not previously possess.
By this method, it is possible to obtain new polymers with different levels of sulfation, but with the same molecular weight. Polymers with new biological characteristics can be obtained by using as starting materials biopolymers wherein the carboxy groups are salified with tetrabutylammonium salt. Such biopolymrs are not hemolytic.
A notable characteristic of these sulfated polysaccharides is thei
Barbucci Rolando
Cialdi Gloria
Magnani Agnese
Barbucci Rolando
Crane L. Eric
Fidia Advanced Biopolymers, Srl
Geist Gary
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