Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1997-12-11
2002-06-18
Prats, Francisco (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S100000, C435S101000, C536S004100, C536S020000, C536S021000, C536S017200, C536S018500, C536S018700, C536S055000, C536S055100, C536S055200, C536S055300, C536S102000, C536S123100, C536S124000, C536S126000, C525S032200
Reexamination Certificate
active
06406894
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention is directed to a process for using enzymatic glycosylation reactions to prepare polyvalent carbohydrate-containing polymers. These carbohydrate-containing polymers do not cause intolerance reactions in vivo, either in their intact state, or in the form of metabolites produced by physiological degradation processes. The process may also be used for preparing carbohydrate building blocks.
The role of carbohydrates as information carriers in physiologically relevant recognition processes has recently been a subject of intense study. The presence of carbohydrates as ligands on cell surfaces allows them to play, via binding to specific carbohydrate receptors, a crucial part in intercellular communication and in intercellular recognition processes. Carbohydrate ligands on cell surfaces act as recognition elements for, for example, viruses, bacteria, toxins and lectins. Carbohydrates therefore play a crucial role part in, inter alia, bacterial and viral infections, and in the initiation of inflammatory processes such as rheumatoid arthritis, allergies, post-infarct syndrome, shock, stroke and sepsis. Recent investigations have shown that during inflammatory processes the interaction between a carbohydrate ligand and a selectin expressed by endothelial cells mediates adhesion of leukocytes to inflammatory foci. Carbohydrate ligands that are particularly important for cell adhesion are sialylated and/or fucosylated carbohydrates such as sialyl-Lewis X and sialyl-Lewis A.
Therapeutic approaches to treating inflammatory disorders using free oligosaccharides to block the binding of natural ligands to receptors have been largely unsuccessful due to the low affinities observed between the receptor and the oligosaccharide (e.g. the dissociation constant, K
D
~10
−4
M for the interaction between a monovalent galactoside and the corresponding lectin, D. T. Connolly et al.,
J. Biol. Chem
. 257, 939, (1982)). These low affinities lead to a requirement for administering very high dosages of carbohydrate. Some divalent structures have, however, been show to have somewhat better binding affinities for a particular receptor. See. for example, Wong et al.,
J. Amer. Chem. Soc
. 115:7549 (1993) and U.S. Pat. No. 5,254,676.
It has been shown that increased interaction between. receptor and ligand can be achieved by coupling a plurality of ligands to a surface. For example, the ligand-receptor interaction between neuraminic acid and the viral protein hemagglutinin is significantly enhanced by coupling multiple carbohydrate moieties to a polymer. Thus the K
D
for the monovalent interaction is 2×10
−4
M, and the K
D
for the polyvalent interaction is 3×10
−7
M. Spaltenstein et al.,
J. Amer. Chem. Soc
. 113:686 (1991).
Surfaces used to date have been liposomes (Yamazaki,
Int. J. Biochem
. 24:99 (1991); WO 91/19501; WO 91/19502), polyacrylamides (Rathi et al.,
J. Polym. Sci
.: Part A:
Polym. Chem
. 29:1895 (1991), Nishimura. et al.,
Macromolecules
24:4236 (1991)), and polylysine or sulfated polysaccharides. These polyvalent structures have the disadvantage either of having only low stability in vivo or of not being tolerated in vivo due to degradation to toxic metabolites. In the case of polylysine or sulfated polysaccharides non-specific interactions with cell surface structures are also observed. European Published Patent Specifications EP 0 089 938, EP 0 089 939 and EP 0 089 940 describe carbohydrate compounds of varying chain length that are identical to ligands located on cell surfaces or receptors located on microorganisms. These carbohydrate compounds are intended to block receptors located on the microorganisms in vitro and in vivo in order to diagnose and treat diseases. The carbohydrate compounds in these cases may be coupled to a carrier, which may be used, inter alia, to produce antibodies. Similarly, WO 92/02527 discloses an oligosaccharide building block coupled to a solid carrier that may be used to diagnose inflammatory processes. The solid carrier is inert toward physiological systems and is thus not physiologically degraded.
In contrast, EP 0 601 417 A2, which is hereby incorporated by reference in its entirety, discloses a physiologically degradable polymer-based carbohydrate receptor blocker that carries oligosaccharide building blocks on the polymer surface. Improved pharmaceutical activity is obtained by enhanced interaction of the carbohydrate building blocks, which are present polyvalently on the polymer surface, with receptors, and by blocking specific structures. The carbohydrate receptor blocker is physiologically well tolerated, and has a preferred molecular weight of less than about 70 kD.
The physiologically tolerated and physiologically degradable polymer-based carbohydrate receptor blocker disclosed in EP 0 601 417 A2 has the following structure:
carbohydrate side chains—spacer—hydrophilic biodegradable polymer—potentiator (optional),
where the carbohydrate side chains consist of 1 to naturally occurring, identical or different monosaccharide units that are coupled via one or more bifunctional spacers of natural or synthetic origin to a hydrophilic, biodegradable polymer. The hydrophilic, biodegradable polymer optionally is linked to a potentiator consisting of one or more groups with hydrophobic, hydrophilic or ionic properties, or the potentiator is a crosslinker or enhances solubility.
The carbohydrate portion of the receptor blocker disclosed in EP 0 601 417 A2 may comprise, for example, the following sugar residues:
Gal&bgr;1-4GlcNAc-;
Gal&bgr;1-3GlcNAc-;
SA&agr;2-6Gal&bgr;1-4GlcNAc-;
SA&agr;2-3Gal&bgr;1-4GlcNAc-;
SA&agr;2-3Gal&bgr;1-3GlcNAc-;
Gal&bgr;1-4(Fuc&agr;1-3)GlcNAc-;
Gal&bgr;1-3(Fuc&agr;1-3)GlcNAc-;
SA&agr;2-3Gal&bgr;1-3(Fuc&agr;1-4)GlcNAc-;
SA&agr;2-3Gal&bgr;1-4(Fuc&agr;1-3)GlcNAc-.
Other examples of preferred embodiments of the carbohydrate portion are: sialyl-Lewis X, sialyl-Lewis A, VIM-2 and the following blood-group determinants: Lewis A, B, X, Y and A type
1
, A type
2
, B type
1
, B type
2
and H type
1
and H type
2
(see Lemieux,
Chem. Soc. Rev
., (1978) p. 423 and
Chem. Soc. Rev
., (1989) p. 347). Particularly preferred embodiments of the carbohydrate portion are sialyl-Lewis X, sialyl-Lewis A or VIM-2.
The formula of sialyl-Lewis X is: NeuNA&agr;2-3Gal1-4-(Fuc&agr;1-3)GlcNAc. The formula of sialyl-Lewis A is: NeuNA&agr;2-3Gal&bgr;1-3-(Fuc&agr;1-4)GlcNAc. The formula of VIM-2 is: NeuNA&agr;2-3Gal&bgr;1-4GlcNAc&bgr;1-3Gal&bgr;1-4(Fuc&agr;1-3)GlcNAc.
EP 0 601 417 A2 discloses a process for the preparation of the carbohydrate receptor blocker that is suitable for use on a laboratory scale. The carbohydrate receptor blocker accordingly can be synthesized only in milligram to gram quantities. The non-carbohydrate intermediates necessary for the synthesis, i.e., the hydrophilic biodegradable polymer, the bifunctional spacer and the potentiator can, however, be prepared in gram to kilogram amounts.
The limitation in the scale of the overall synthesis of the blocker is due to the synthetic schemes necessary to prepare the carbohydrate portion of the blocker. These schemes can only readily yield amounts of carbohydrate up to one gram. Known synthetic schemes for the preparation of oligosaccharides involve steps that do not proceed with a quantitative yield, and the product mixture obtained after each reaction must be purified by silica gel chromatography. This purification process is generally too costly and elaborate for preparing industrial quantities of material, and is used at the most for purifying final products or valuable intermediates. Additionally, oligosaccharide synthesis frequently uses heavy metal compounds as reagents, which is problematic for subsequent regulatory approval of the blockers as pharmaceutical products, due to possible heavy metal contamination.
In the process described in EP 0 601 417 A2, the required oligosaccharide is linked to the biodegradable polymer by means of a spacer only after the oligosaccharide has been assembled via a large number of c
Ahlers Michael
Bartnik Eckart
Hoersch Brigitte
Kretzschmar Gerhard
Seiffge Dick
Glycorex AB
Prats Francisco
LandOfFree
Process for preparing polyvalent and physiologically... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Process for preparing polyvalent and physiologically..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Process for preparing polyvalent and physiologically... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2916344