CA2204366A1 - Polysaccharides having a high iduronic acid content - Google Patents
Polysaccharides having a high iduronic acid contentInfo
- Publication number
- CA2204366A1 CA2204366A1 CA002204366A CA2204366A CA2204366A1 CA 2204366 A1 CA2204366 A1 CA 2204366A1 CA 002204366 A CA002204366 A CA 002204366A CA 2204366 A CA2204366 A CA 2204366A CA 2204366 A1 CA2204366 A1 CA 2204366A1
- Authority
- CA
- Canada
- Prior art keywords
- epimerization
- heparan sulfate
- buffer solution
- solution
- product
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
- C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof
Abstract
Process for the preparation of polysaccharides having a high iduronic acid content comprising: a) N-deacetylation of the polysaccharide K5 from E. coli or of the heparan sulfate or O-desulfation of heparin or heparan sulfate; b) N-sulfation of the product obtained from the stage a); c) epimerization in presence of the C5 epimerase enzyme; d) sulfation of at least some free hydroxy groups, wherein the stage c) is carried out in a reaction medium constituted by a classical buffer solution formed by HEPES, potassium chloride, EDTA and TRITON X-100 to which a suitable additive is added.
Description
CA 02204366 1997-0~-02 POLYSACCHARIDES HAVING A HIGH IDURONIC ACID CONTENT
PRIOR ART
The glycosaminoglycans which are substances obtained by extraction from animal tissues having various origin, for instance intestinal mucosa, lung, etc. belong particularly to the class of the polysaccharides containing iduronic acid. Heparin, heparan sulfate, condroitin sulfates and hyaluronic acid belong to the family of glycosaminoglycans.
The various glycosaminoglycans have different chemical structures and they are formed by polysaccharide chains constituted by the repetition of an uronic acid and a hexosamine. In particular in heparin and heparan sulfate the uronic acid is constituted by glycuronic or iduronic acid and the hexosamine by glycosA~i n~ .
The glycosamine may be preferentially N-acetylated (heparan sulfate) or preferentially N-sulfated (heparin) and 6-O sulfated. Moreover a sulfate group may be found also in the position 3 of the glycosamine.
The uronic acid may be 2-O sulfated.
The heparin has great importance in the clinical practice as anticoagulant and antithrombotic.
20 Besides this therapeutic use an useful utilization for the heparin and the heparan sulfate in several other pathologies, for instance with antilipemic, antiproliferative, antiviral, antitumoral and antiangiogenic function is expected.
The utilization of the heparin and the heparan sulfate in these new therapeutic applications involves the necessity to obtain these CA 02204366 l997-0~-02 W 096/14425 PCT~P95/04241 products, or similar products, by processes different from the extractive one, in particular by more flexible processes allowing the preparation of different structures.
Moreover the extractive process from animal tissues does not guarantee to obtain a virus free product.
A process for the preparation of anticoa~llant glycosaminoglycans by biosynthesis is described in the Patent Application No. WO 92/17507.
In this process the polysaccharide K5 from E. coli which is submitted to the following sequence of reactions:
- N-deacetylation;
- N-sulfation;
- epimerization in order to transform at least some residuals of D-glycuronic acid into residuals of L-iduronic acid; and - sulfation of at least some free hydroxy groups, is used as a starting compound.
In this process the critical stage is constituted by the epimerization which is limited to a 20 % AXi ~llm . The epimerization is carried out at room temperature with two days duration in presence of the D-glycuronyl-L-iduronyl-C5-epimerase enzyme in a classical reaction medium to pH 7.4 constituted by HEPES, potassium chloride, EDTA and TRITON X-100. An epimerization limited to one third of the uronic acid had been formerly described (M. Hook et al., The J. of Biol. Chem.
249, 12 3908-3915, June 25, 1974). And this epimerization degree seemed till now insuperable. Moreover it must be considered that in this document the epimerization in cellular environment is described CA 02204366 l997-0~-02 in the murine mastocytoma in conditions in which every factor concerning the biosynthetic process is present.
However an epimerization degree as obtained from the known art does not allow to obtain a product with the requested characteristics for the various therapeutic treatments.
In fact the iduronic acid gives a superior flexibility to the product with respect to the glycuronic acid (Casu B., Petitou M., Provasoli M., and Sinay P. (1988) Conformational flexibility: a new concept for explaining binding and biological properties of iduronic acid contAining glycosaminoglycans. Trends Biochem. Sci. 13, 221-225). It follows that the products contAining high percentages of iduronic acid are more active with respect to those containing glycuronic acid as it is pointed out by the greater anticoagulant and antithrombotic activity of the heparin towards heparan sulfate and by other activities such as that one on the basic fibroblast growth factor (bFGF) wherein the iduronic acid is recognized to be essential part of the active site (Maccarana ,~., Casu B., and ~in~Ah1 U. (1993). ~ini Al sequence in Heparin/Heparan Sulfate Required for Binding of Basic Fibroblast Growth Factor. J. Biol. Chem. 268, 23898-23905) .
Chem. ;bstr., vol. 115, no. 17, 1991. abstr. no. 180062w, describes experiments on the incubation of cultured fibroblasts with p-nitrophenyl-~-D-xyloside, resulting in a concentration dependent increase in galactosaminoglycan synthesis. In particular at low concentration of added xyloside, the galactosaminoglycan formed on the xyloside is mainly composed of L-iduronic acid.
Clearlv, said experiments do not teach an industrial process.
p~E~O~ S~
Therefore there is the problem to find a process allowing to obtain a product having a high degree of epimerization with yield and times acceptable accordlng to the industrial point of view.
SU~ARY
We have found that polysaccharides having a high iduronic acid content may be obtained starting from the poiysaccharide K5 from E. coli or from heparin or from the heparan sulfate by a process comprising:
p~E~J~E~ SH~
CA 02204366 1997-0~-02 PCT~P95/04241 a) N-deacetylation of said polysaccharide K5 or of the heparan sulfate or O-desulfation of the heparin or of the heparan sulfate;
b) N-sulfation of the product obtained from the stage a);
c) one or more treatments of epimerization in presence of the D-glycuronyl-L-iduronyl-C5-epimerase enzyme;
d) sulfation of at least some free hydroxy groups, characterized in that the stage of epimerization is carried out in a reaction medium constituted by a classical buffer solution at pH 7.4 formed by HEPES, potassium chloride and EDTA to which TRITON X-100 and an additive, or more additives, selected from the group formed by ethylene glycol, glycerol, polyvinylpyrrolidone, polyethylene glycol and phosphatidylcholine are added.
Polysaccharides having iduronic acid content greater than 50% with respect to uronic acids total content are obtained by the process according to the present invention.
DETAILED DESCRIPTION OF THE INVENTION
The characteristics and the advantages of the process according to the present invention and of the obtained polysaccharides ~ill be mostly pointed out during the following detailed description.
The polysaccharide K5 from E. coli described by Manzoni M., Bergomi S., Cavazzoni V. (Journal of Bioactive and Compatible Polymers. Vol.
VIII, July 1993, 251-257) is a substance particularly suitable to the use as a starting material for the process according to the present invention.
The heparin and the heparan sulfate may be used too as starting CA 02204366 1997-0~-02 W O96/14425 PCT~P95/04241 material with some variation of the operative conditions of the first stages of the process.
The starting substances may have a molecular weight ranging from 2,000 to more than 50,000 D.
When the polysaccharide K5 is used its structure is modified first by N-deacetylation which is carried out by treatment with a mixture of hydrazine and hydrazine sulfate or in a basic environment with sodium hydroxide or potassium hydroxide.
Then one proceeds with the N-sulfation by treatment with triethylamine-sulfur trioxide or with trimethylamine-sulfur trioxide.
A variously N-sulfated product, for instance from 25% to 100%, may be obtained with these operations.
The reactions of N-deacetylation and N-sulfation are carried out according to the known techniques, for example according to the Patent Application No. W0 92/17507.
When the heparin is used as starting substance first the 0-desulfation and then the N-sulfation are carried out in order to resulfate the amino-positions which lost the sulfate groups during the 0-desulfation.
When the heparan sulfate is used as starting substance both the N-deacetylation and the 0-desulfation and the resulting N-resulfation are carried out.
The N-sulfated product obtained from the polysaccharide K5 or from the heparan sulfate as described above, is submitted to the epimerlzation orocess in order to convert the glycuronic acid into iduronic acid while the product obtained from heparin is treated with the enzyme in - order to obtAin the glycuron1c acid from the iduronic acid.
The epimerization ~s carried out ln presence of the D-gly~u~uu~-L-iduronyl-C5-eplmerase enzyme (later on simply indicated wi.th C5 epimerase) extractea from cattle liver and purified with the mèthod described by A. ~Almctron in J. B. C. 255, 3878-3883 (19oO).
The Applicant ha_ surpris~ng1y found that modifying the cl~
reaction medium with suitable additives a very high degree of epimerization is obtaiced.
The reaction medium according to the present i~vention is a p~_7.4 buffer solution c~nstituted by HEPES, pot~cs~ chloride. EDrA and ~ 0N X-100 and zaded with one or more additives selected from-.the group formed by ethylene g~ycol. glycerol. polyvinylpyrrol~Qne.
particularly polyv~nylpyrroli~n~ having lec~ A~ weight from 15~000 to 90.000, polyethylene glycol. and phosphatidyl~holine i3 amounts 1~ suitable to increase the buffer solution viscosity to values r ~ging from 1.1 to 3 mm /s (1.1 to 3 centistokes).
In particular the reaction medium is prepared starting from the following buffer solution having pH 7.4: HEPES 0.04 M. KCl 0.4 M and ~ul-A 0.06 M, and to 25 ml of th~s buffer soiution from l00 to 1000 ~1 of l~l-l0~ X-l00, ..-o~ 0.5 ml to 60 ml of additive and distilled water to lOO ml are added The polysaccharide to submit to epimerization is added to said reaction medium i~ an amount from 5 to 1000 m~ per l00 ml obt~ining the solution A.
z5 The C5 epimerase :s separately dissolved in the same above-mentiOned ~ S~
CA 02204366 l997-0~-02 W O 96/14425 PCT~P95/04241 reaction medium in amounts from 21 to 2000 ,ug per 100 ml obtaining the solution B.
~ The solution B is added to the solution A in such a proportion to obtain a content from 1.5 to 15.000 ~g of C5 epimerase per lOO ml of mixture to submit to epimerization. The mixture is homogenized by agitation and warmed at a temperature ranging from 3O to 4O ~C in a constant-temperature chamber for a time ranging from 9O minutes to 15 hours.
The reaction is stopped warming the mixture at lOO ~C for 5 minutes.
The product is purified through a DEAE-Sephacel column using (NH4)HCO3 O.O5 M as buffer and eluting the product with (NH4)HCO3 2M buffer.
The gathered fractions are desalted by Sephadex G-15 column, the fraction contAining the product is lyo~hili~ed and the product is analyzed by lH-NMR.
The content of D-glycuronic acid and of the L-iduronic acid is computed by lH-N~R spectrum. The product obtained can be redissolved in solution A and treated again with solution B obtaining, with further treatments of epimerization, an increase of the L-iduronic acid content.
In order to estimate the anticoagulant and antithrombotic activity, the epimerizated product is O-sulfated using pyridine-sulfur trioxide as sulfating agent for instance with the method described by Ogamo et al. in the abstracts of the XIV International Carbohydrate Symposium (August 14-19, 1988), Stockholm. The products of the Examples 1, 3, 4, 5, 6, 7 and 9, obtained with the process according to the present invention, as later on reported, submitted to O-sulfation, have shown an exceilent an~icoaguian~ and antithrombotiC activlty wAile the producls of the ~xamples ~ and 8, cbtained according :o the ~cwn tecnnique, ;.as snown mucn lower acti~ity.
The obta~ned results snow that the products accordin~ tO the pre~ent invention nave cnaracteris.ics su1table to the clinical use with anticoagulant and an~ithromootic function and therefore they may be used for the prepara~ion of the pert~-ning p~rmnceutical compositiors mixed with aajuvant and P~ ri ~nt substances.
In order ~o expi~i n ~me process accorosng to the present nvention the following exampies ~re re~or~ed.
A buffer solution ccnt~ining 0.04 M h~ , 0.06 M EDTA. 0.4 M RCl p~
7.4 was prepared and ~7 ,ul af TRITON X-100, 9 ml of 10~
polyvinylp~rroiidone ~15 :n water. .62 ~i of ethylene g}ycol a~d water ;o a total :~lume ~f 18 ml were added to ~., ml of this solution.
The solution showed a viscosity of 1.41 mm /s (1.41 centistokes).
In this solution 1 mg of lOOX N-deacetylated N-sulfated K5 was dissolved obt~i ni n~ solution A.
1.6 ml o- '0~ polyvlnylpyr~olidone ~.'5 in water, 288 ~i of ethylene glycol and ~ater to a tota~ volume of 3.2 ml were added ~o 0.8 mi of the same startlng cuffer solution p~ 7.'' cont~ininF ~.9 ~g of C5 epimera~se, -bt~ininv solut on B.
Solution ~ was mixed with '.6 ml of solution B and t~.e msxture was kept at 37'~ .or u nours :n a warm room. .~fter 4 hours, ~.6 ml of ~E~ S~
CA 02204366 1997-0~-02 solution B were added and the reaction was kept at 37~C overnight.
The reaction was stopped by heating at 100~C for 5 minutes.
The product was purified by a DEAE-Sephacel column (2 x 1.5 cm) using 0.05 M (NH4)HC03 as buffer and the product was eluted with 2 M
(NH4)HC03.
The fractions cont~i ni ng the product were desalted on a Sephadex G-15 column (0.3 x 5 cm) and freeze-dried.
The product was analysed by lH-NMR and the spectrum is shown in Fig.
1.
The percentage of L-iduronic acid over the total uronic acids was 55.
EXAMPLE 2 (Comparison) The Example 1 has been repeated with the difference that the polyvinylpyrrolidone and the ethylene glycol have not been added.
The used buffer solution had a viscosity about equal to the water, namely about zero.
The lH-NMR spectrum relative to the obtained product is reported in the fig. 2. The L-iduronic acid content with reference to the sum of the iduronic acid and the glycuronic acid turned out to be equal to 18% .
The Example 1 has been repeated with the difference that the N-deacetylated and 50% N-sulfated polysaccharide K5 has been used as starting substance.
The obtained product has been analyzed by lH-NMR and the relative spectrum is reported in the Fig. 3.
The L-iduronic acid content turned out to be equal to 51% with reference tO the total of the uronlc acids loc~ to a N-sulfated gl l-cosz'~i nf' -EXA~LE 4 O . 06 M EDTA . O . 4 ~5 KCl rH 7 . 4 . 30 ui Of TEUTON X-100, 10 .6 ml of 10~
polyvinylpyrrolidone X15 in water. o.85 ml of ethylene glycol and 4.42 ml Of water were added to 5.3 ml of a buffer solution cont~ining 0.04 ~ HEPES.
The solution snowed a vlscosity of 1.2 mm /s (1.2 centistokes).
PRIOR ART
The glycosaminoglycans which are substances obtained by extraction from animal tissues having various origin, for instance intestinal mucosa, lung, etc. belong particularly to the class of the polysaccharides containing iduronic acid. Heparin, heparan sulfate, condroitin sulfates and hyaluronic acid belong to the family of glycosaminoglycans.
The various glycosaminoglycans have different chemical structures and they are formed by polysaccharide chains constituted by the repetition of an uronic acid and a hexosamine. In particular in heparin and heparan sulfate the uronic acid is constituted by glycuronic or iduronic acid and the hexosamine by glycosA~i n~ .
The glycosamine may be preferentially N-acetylated (heparan sulfate) or preferentially N-sulfated (heparin) and 6-O sulfated. Moreover a sulfate group may be found also in the position 3 of the glycosamine.
The uronic acid may be 2-O sulfated.
The heparin has great importance in the clinical practice as anticoagulant and antithrombotic.
20 Besides this therapeutic use an useful utilization for the heparin and the heparan sulfate in several other pathologies, for instance with antilipemic, antiproliferative, antiviral, antitumoral and antiangiogenic function is expected.
The utilization of the heparin and the heparan sulfate in these new therapeutic applications involves the necessity to obtain these CA 02204366 l997-0~-02 W 096/14425 PCT~P95/04241 products, or similar products, by processes different from the extractive one, in particular by more flexible processes allowing the preparation of different structures.
Moreover the extractive process from animal tissues does not guarantee to obtain a virus free product.
A process for the preparation of anticoa~llant glycosaminoglycans by biosynthesis is described in the Patent Application No. WO 92/17507.
In this process the polysaccharide K5 from E. coli which is submitted to the following sequence of reactions:
- N-deacetylation;
- N-sulfation;
- epimerization in order to transform at least some residuals of D-glycuronic acid into residuals of L-iduronic acid; and - sulfation of at least some free hydroxy groups, is used as a starting compound.
In this process the critical stage is constituted by the epimerization which is limited to a 20 % AXi ~llm . The epimerization is carried out at room temperature with two days duration in presence of the D-glycuronyl-L-iduronyl-C5-epimerase enzyme in a classical reaction medium to pH 7.4 constituted by HEPES, potassium chloride, EDTA and TRITON X-100. An epimerization limited to one third of the uronic acid had been formerly described (M. Hook et al., The J. of Biol. Chem.
249, 12 3908-3915, June 25, 1974). And this epimerization degree seemed till now insuperable. Moreover it must be considered that in this document the epimerization in cellular environment is described CA 02204366 l997-0~-02 in the murine mastocytoma in conditions in which every factor concerning the biosynthetic process is present.
However an epimerization degree as obtained from the known art does not allow to obtain a product with the requested characteristics for the various therapeutic treatments.
In fact the iduronic acid gives a superior flexibility to the product with respect to the glycuronic acid (Casu B., Petitou M., Provasoli M., and Sinay P. (1988) Conformational flexibility: a new concept for explaining binding and biological properties of iduronic acid contAining glycosaminoglycans. Trends Biochem. Sci. 13, 221-225). It follows that the products contAining high percentages of iduronic acid are more active with respect to those containing glycuronic acid as it is pointed out by the greater anticoagulant and antithrombotic activity of the heparin towards heparan sulfate and by other activities such as that one on the basic fibroblast growth factor (bFGF) wherein the iduronic acid is recognized to be essential part of the active site (Maccarana ,~., Casu B., and ~in~Ah1 U. (1993). ~ini Al sequence in Heparin/Heparan Sulfate Required for Binding of Basic Fibroblast Growth Factor. J. Biol. Chem. 268, 23898-23905) .
Chem. ;bstr., vol. 115, no. 17, 1991. abstr. no. 180062w, describes experiments on the incubation of cultured fibroblasts with p-nitrophenyl-~-D-xyloside, resulting in a concentration dependent increase in galactosaminoglycan synthesis. In particular at low concentration of added xyloside, the galactosaminoglycan formed on the xyloside is mainly composed of L-iduronic acid.
Clearlv, said experiments do not teach an industrial process.
p~E~O~ S~
Therefore there is the problem to find a process allowing to obtain a product having a high degree of epimerization with yield and times acceptable accordlng to the industrial point of view.
SU~ARY
We have found that polysaccharides having a high iduronic acid content may be obtained starting from the poiysaccharide K5 from E. coli or from heparin or from the heparan sulfate by a process comprising:
p~E~J~E~ SH~
CA 02204366 1997-0~-02 PCT~P95/04241 a) N-deacetylation of said polysaccharide K5 or of the heparan sulfate or O-desulfation of the heparin or of the heparan sulfate;
b) N-sulfation of the product obtained from the stage a);
c) one or more treatments of epimerization in presence of the D-glycuronyl-L-iduronyl-C5-epimerase enzyme;
d) sulfation of at least some free hydroxy groups, characterized in that the stage of epimerization is carried out in a reaction medium constituted by a classical buffer solution at pH 7.4 formed by HEPES, potassium chloride and EDTA to which TRITON X-100 and an additive, or more additives, selected from the group formed by ethylene glycol, glycerol, polyvinylpyrrolidone, polyethylene glycol and phosphatidylcholine are added.
Polysaccharides having iduronic acid content greater than 50% with respect to uronic acids total content are obtained by the process according to the present invention.
DETAILED DESCRIPTION OF THE INVENTION
The characteristics and the advantages of the process according to the present invention and of the obtained polysaccharides ~ill be mostly pointed out during the following detailed description.
The polysaccharide K5 from E. coli described by Manzoni M., Bergomi S., Cavazzoni V. (Journal of Bioactive and Compatible Polymers. Vol.
VIII, July 1993, 251-257) is a substance particularly suitable to the use as a starting material for the process according to the present invention.
The heparin and the heparan sulfate may be used too as starting CA 02204366 1997-0~-02 W O96/14425 PCT~P95/04241 material with some variation of the operative conditions of the first stages of the process.
The starting substances may have a molecular weight ranging from 2,000 to more than 50,000 D.
When the polysaccharide K5 is used its structure is modified first by N-deacetylation which is carried out by treatment with a mixture of hydrazine and hydrazine sulfate or in a basic environment with sodium hydroxide or potassium hydroxide.
Then one proceeds with the N-sulfation by treatment with triethylamine-sulfur trioxide or with trimethylamine-sulfur trioxide.
A variously N-sulfated product, for instance from 25% to 100%, may be obtained with these operations.
The reactions of N-deacetylation and N-sulfation are carried out according to the known techniques, for example according to the Patent Application No. W0 92/17507.
When the heparin is used as starting substance first the 0-desulfation and then the N-sulfation are carried out in order to resulfate the amino-positions which lost the sulfate groups during the 0-desulfation.
When the heparan sulfate is used as starting substance both the N-deacetylation and the 0-desulfation and the resulting N-resulfation are carried out.
The N-sulfated product obtained from the polysaccharide K5 or from the heparan sulfate as described above, is submitted to the epimerlzation orocess in order to convert the glycuronic acid into iduronic acid while the product obtained from heparin is treated with the enzyme in - order to obtAin the glycuron1c acid from the iduronic acid.
The epimerization ~s carried out ln presence of the D-gly~u~uu~-L-iduronyl-C5-eplmerase enzyme (later on simply indicated wi.th C5 epimerase) extractea from cattle liver and purified with the mèthod described by A. ~Almctron in J. B. C. 255, 3878-3883 (19oO).
The Applicant ha_ surpris~ng1y found that modifying the cl~
reaction medium with suitable additives a very high degree of epimerization is obtaiced.
The reaction medium according to the present i~vention is a p~_7.4 buffer solution c~nstituted by HEPES, pot~cs~ chloride. EDrA and ~ 0N X-100 and zaded with one or more additives selected from-.the group formed by ethylene g~ycol. glycerol. polyvinylpyrrol~Qne.
particularly polyv~nylpyrroli~n~ having lec~ A~ weight from 15~000 to 90.000, polyethylene glycol. and phosphatidyl~holine i3 amounts 1~ suitable to increase the buffer solution viscosity to values r ~ging from 1.1 to 3 mm /s (1.1 to 3 centistokes).
In particular the reaction medium is prepared starting from the following buffer solution having pH 7.4: HEPES 0.04 M. KCl 0.4 M and ~ul-A 0.06 M, and to 25 ml of th~s buffer soiution from l00 to 1000 ~1 of l~l-l0~ X-l00, ..-o~ 0.5 ml to 60 ml of additive and distilled water to lOO ml are added The polysaccharide to submit to epimerization is added to said reaction medium i~ an amount from 5 to 1000 m~ per l00 ml obt~ining the solution A.
z5 The C5 epimerase :s separately dissolved in the same above-mentiOned ~ S~
CA 02204366 l997-0~-02 W O 96/14425 PCT~P95/04241 reaction medium in amounts from 21 to 2000 ,ug per 100 ml obtaining the solution B.
~ The solution B is added to the solution A in such a proportion to obtain a content from 1.5 to 15.000 ~g of C5 epimerase per lOO ml of mixture to submit to epimerization. The mixture is homogenized by agitation and warmed at a temperature ranging from 3O to 4O ~C in a constant-temperature chamber for a time ranging from 9O minutes to 15 hours.
The reaction is stopped warming the mixture at lOO ~C for 5 minutes.
The product is purified through a DEAE-Sephacel column using (NH4)HCO3 O.O5 M as buffer and eluting the product with (NH4)HCO3 2M buffer.
The gathered fractions are desalted by Sephadex G-15 column, the fraction contAining the product is lyo~hili~ed and the product is analyzed by lH-NMR.
The content of D-glycuronic acid and of the L-iduronic acid is computed by lH-N~R spectrum. The product obtained can be redissolved in solution A and treated again with solution B obtaining, with further treatments of epimerization, an increase of the L-iduronic acid content.
In order to estimate the anticoagulant and antithrombotic activity, the epimerizated product is O-sulfated using pyridine-sulfur trioxide as sulfating agent for instance with the method described by Ogamo et al. in the abstracts of the XIV International Carbohydrate Symposium (August 14-19, 1988), Stockholm. The products of the Examples 1, 3, 4, 5, 6, 7 and 9, obtained with the process according to the present invention, as later on reported, submitted to O-sulfation, have shown an exceilent an~icoaguian~ and antithrombotiC activlty wAile the producls of the ~xamples ~ and 8, cbtained according :o the ~cwn tecnnique, ;.as snown mucn lower acti~ity.
The obta~ned results snow that the products accordin~ tO the pre~ent invention nave cnaracteris.ics su1table to the clinical use with anticoagulant and an~ithromootic function and therefore they may be used for the prepara~ion of the pert~-ning p~rmnceutical compositiors mixed with aajuvant and P~ ri ~nt substances.
In order ~o expi~i n ~me process accorosng to the present nvention the following exampies ~re re~or~ed.
A buffer solution ccnt~ining 0.04 M h~ , 0.06 M EDTA. 0.4 M RCl p~
7.4 was prepared and ~7 ,ul af TRITON X-100, 9 ml of 10~
polyvinylp~rroiidone ~15 :n water. .62 ~i of ethylene g}ycol a~d water ;o a total :~lume ~f 18 ml were added to ~., ml of this solution.
The solution showed a viscosity of 1.41 mm /s (1.41 centistokes).
In this solution 1 mg of lOOX N-deacetylated N-sulfated K5 was dissolved obt~i ni n~ solution A.
1.6 ml o- '0~ polyvlnylpyr~olidone ~.'5 in water, 288 ~i of ethylene glycol and ~ater to a tota~ volume of 3.2 ml were added ~o 0.8 mi of the same startlng cuffer solution p~ 7.'' cont~ininF ~.9 ~g of C5 epimera~se, -bt~ininv solut on B.
Solution ~ was mixed with '.6 ml of solution B and t~.e msxture was kept at 37'~ .or u nours :n a warm room. .~fter 4 hours, ~.6 ml of ~E~ S~
CA 02204366 1997-0~-02 solution B were added and the reaction was kept at 37~C overnight.
The reaction was stopped by heating at 100~C for 5 minutes.
The product was purified by a DEAE-Sephacel column (2 x 1.5 cm) using 0.05 M (NH4)HC03 as buffer and the product was eluted with 2 M
(NH4)HC03.
The fractions cont~i ni ng the product were desalted on a Sephadex G-15 column (0.3 x 5 cm) and freeze-dried.
The product was analysed by lH-NMR and the spectrum is shown in Fig.
1.
The percentage of L-iduronic acid over the total uronic acids was 55.
EXAMPLE 2 (Comparison) The Example 1 has been repeated with the difference that the polyvinylpyrrolidone and the ethylene glycol have not been added.
The used buffer solution had a viscosity about equal to the water, namely about zero.
The lH-NMR spectrum relative to the obtained product is reported in the fig. 2. The L-iduronic acid content with reference to the sum of the iduronic acid and the glycuronic acid turned out to be equal to 18% .
The Example 1 has been repeated with the difference that the N-deacetylated and 50% N-sulfated polysaccharide K5 has been used as starting substance.
The obtained product has been analyzed by lH-NMR and the relative spectrum is reported in the Fig. 3.
The L-iduronic acid content turned out to be equal to 51% with reference tO the total of the uronlc acids loc~ to a N-sulfated gl l-cosz'~i nf' -EXA~LE 4 O . 06 M EDTA . O . 4 ~5 KCl rH 7 . 4 . 30 ui Of TEUTON X-100, 10 .6 ml of 10~
polyvinylpyrrolidone X15 in water. o.85 ml of ethylene glycol and 4.42 ml Of water were added to 5.3 ml of a buffer solution cont~ining 0.04 ~ HEPES.
The solution snowed a vlscosity of 1.2 mm /s (1.2 centistokes).
2 m~ of 100% ~-deacetylated N-suifated K5 were dissoled in 9 ml of t-~is solutlon. obt~i n~ nF solution A.
1.7 ~g of C5 epmmerase were dissoived in 8 ml of the same soluti ~
obt~ninF solutlcn B.
Solutlons A and B were mixed and the mixture was kept at 37-C for 4 hours in a warm room. The react~om was stopped by heating at lOO-C for , minutes.
~he product ~~as purified as descrmbed in Example ifter freeze-arylng ~e purif~ed product was d ssolved in 1 ml o~
solution A and mixed with 3.2 ml of solution B cont~inin~ 8.9 ug of C5 epimerase.
he mlxture was kept at 37-C overnight and the e.~zyme was inactlvated by heating at lOO'C for 5 minutes. The product was purified as described in Exampie 1 and anaiysed by lH-NMR as shown in Fig. 4.
rhe percentage o~ L-iduronic acid over the total uronic acids was 53.
'. buffer soiutlon cont~inin~ 0.04 M HEPES. 0.06 .~ EDTA. 0.4 M KCl p~
h~ ~~) S~
7.4 was F.epared ~.d ~2.8 ~l of TRITON X-100. 1 m~ of 100X N-deacetylated. '~-suifated :~5, 4 ml of a solution of 20S
polyvinyipyrroiidone ~15 cont~inin~ 40Z ethylene glycol and water to a tot~l volum of 8 mi bere added to 2 ml of this solution. 6 ml of a solution c~nt~ininv s'.O1 .~ HEPES, 0;015 M EDTA. 0.01 M KCl, 0.015%
TRITON X-100 and 134.4 ug of C5 epimerase were added to 6 ml of this solution.
The solution obtained showed a vi~co-eity of 1.36mm /s (1.3~ ~entisb~~s) The mixture was kept at 37-C for 1 hour. The reaction was stopped by ~oilin~ at lOO-C for 5 minutes~
The product was purl~~ied as described in ~mrle 1.
After freeze-arying the product wa~ dissolved in buffer solut1on cont~inin~ 6.25 ml of 0.04 ~ HEPES. o.o6 M EDTA. 0.4 ~ KCl p~ 7.4, 2.5 g of polyvinylpyrroiidone K15, 2.5 ml of ethylene glycol. 71.2 ~1 of rRITON X-iOO and water t~ a total volume of 25 nl.
1.9 ug o~ C~ eplmerase 5~ ssolved in 12 ml of ~.01 M HEPES. O.015 M
EDTA. 0.1 ~! KCl pH 1,7 c~nr~inin~ 0.015% of TPITON X-100 were added to 12 ml of the obtained mix~ure.
The mixture showed a ~iscosity of 1.4 mm /s tl.4 centistokes).
The rection was kept at ~7-C overnight. .~t the end of the reaction the enzyme was ;nactivated by heati~g the solution at lOO-C for 5 minutes.
The produc~ was purified as described in ~P~rle 1 and the lH-NMR
spectrum was performed (.-ig. ,).
The percentage of L-~duronic 2cid over the total uronic acids was 52.
zs ~XAMPLE 6 The Ex~mpie ~ was repe~ted except that in the first step the reaction ~Or~ S~
~as kept at 37~C for ~ ~.ours.
After purificat1on :- t-e same conditions of Example 1 and after freeze-drying the proauc~ ~as dissoived in 1 ml or water and added to a buffer solution con~-r--~ 50 mi or O.O4 M HEPES. 0.06 M EDTA, 0.4 M
KCl p~ /.4, 83., __ of ~RITON X-lOO. 100 ~1 of lOS
poly~inylpyrrolidone X15, 20 ml of ethylene glycol. 2 ~l of 9OS
glycerom. 1.9 ug or C~ epL~erase ard water to a total volume of 200 ml. The mixture sAowea a viscosity of 1.41 mm /s (1.41 centistokes).
The reaction was ~ept --t 37-C for 4 hours and the enzyme was lnact~Yated at 100 ~~ for -, ~inutes.
The pr~auct was pur~f_ed ~s described in Exampie 1 and the lH-NMR
spectrum wPC perfor~ea (~lg. 6).
The percentage of L-~durcnic acid over the total uronic acids was 55.
EXAMpr F
l.5 mg of 100Z ~.-deace~ ated, ~r-suifated K5 were dissoived in a buffer solution c-n~ini-~ 7 ml or 0.04 M HEP~C. ~.o6 M EDTA, 0.4 M
~Cl pH ,.~. 210 ~i cr ~RrTC~ X-i00, ;.4 g of poiyvlnyipyrroiidone K15, 2.8 mi of ethylene glyco~. .5 mg of C5 epimerase ana water to a total volume o f 28 ml.
The soiution showea a ~isccsity of 1.36 mm /s (1.36 centistokes).
The roaction was ~ept _- 37-C for 4 hours and the enzyme wa~
inac~ ated at 100-C for ~, ~inutes.
The product was pur1riea 2S described in Exampie l and freeze-dried.
~he product was .:en rissolved _~ 24 ml o~ .he same solution cont~i nin~ 1. 3 mg c~ C, eplmerase and the react~on was kept at 37'C
o~o S~
CA 02204366 l997-05-02 ~or 4 hours and the enzy~e ~as inactivated at lOO-C for 5 minutes.
~he product ~as purifled _ descrlbed in ~xamp}e 1 and fr2ez~ a~-ied.
he product :~as then --ssolYed in 8.4 ml of the same solution ~ont~inin~ 1 m~ of C5 epl~erase and the reaction was kept at 37'C for " hours.
The product was purified ,c described in Exu~nple 1 and the L~-NMR
spectrum was performed (r g. 7~.
The percenta~e of L-1duror~c acid over the total uronic acids was &0.
~YAMPLE 8 (com~arison) : _g of 100% '~-deacetyla-~ -sulfated K5 was ~issolved in a buffer soiution con~inin~ 3 ~1 .' 0.04 M HEPES. 3.06 M EDTA. 0.4 M KCl p~
l.4, 18 ,ul of TRITON X-100. 1.2 ml of acetone. 1.9 ~g of C5 ep~ sse ana water to a total voi~D of 12 ml.
The solution showed a viscosity of ~-~9 mm2/s (0.09 centistokes).
~he reaction was kept 2t ,-C for 4 hours.
~he product was purifi~ ~s described 3 ~x~mr1 e ~ and the lX-NMR
,pectr~m was per~ormed (~ -. 8).
~he percentage of L-idurc.- c acid over the total uronic acids was 0.
~XAMPLE 9 : ~g of 100~ N-deacetyla~ed, ~-sulfated K5 was dissoived in a buffer solution cont~inin~ 2 ml _. 0.04 M HEPES. 0.06 M EDTA. 0.4 M KC1 pH
,.4. 12 ~1 of TRITON X- 00. 1.336 ml of 2.4X polyvinylpyrrolidone K90, ~04 ug of C5 epimerase ~n~ ~ater to a total voiume of 8 ml.
~he solution showed a visccsity of 1.29 m~ /s (1.29 centistokes).
~-~ reaction was kept 2~ 3--C for 4 hours. ~he enzyme was lnactivated lOO-C for -, minutes ~-~ the solution filtered in a 0.22 ~m device.
F~,~r~ S~
CA 02204366 1997-0~-02 W 096/14425 PCT~P95/04241 8 ml of the same mixture cont~ining 304 ug of C5 epimerase were added to the filtered solution and the reaction was kept at 37~C for 4 hours, the enzyme was inactivated at 100~C for 5 minutes and the solution filtered in a 0.22 ~m device.
8 ml of the same mixture containing 304 ,ug of C5 epimerase were added to this solution and the reaction was kept at 37~C for 4 hours, the enzyme was inactivated at 100~C for 5 minutes and the solution filtered in a 0.22 um device.
The product was purified as described in Example 1 and the lH-NMR
spectrum was performed (Fig. 9).
The percentage of L-iduronic acid over the total uronic acids was 59.
1.7 ~g of C5 epmmerase were dissoived in 8 ml of the same soluti ~
obt~ninF solutlcn B.
Solutlons A and B were mixed and the mixture was kept at 37-C for 4 hours in a warm room. The react~om was stopped by heating at lOO-C for , minutes.
~he product ~~as purified as descrmbed in Example ifter freeze-arylng ~e purif~ed product was d ssolved in 1 ml o~
solution A and mixed with 3.2 ml of solution B cont~inin~ 8.9 ug of C5 epimerase.
he mlxture was kept at 37-C overnight and the e.~zyme was inactlvated by heating at lOO'C for 5 minutes. The product was purified as described in Exampie 1 and anaiysed by lH-NMR as shown in Fig. 4.
rhe percentage o~ L-iduronic acid over the total uronic acids was 53.
'. buffer soiutlon cont~inin~ 0.04 M HEPES. 0.06 .~ EDTA. 0.4 M KCl p~
h~ ~~) S~
7.4 was F.epared ~.d ~2.8 ~l of TRITON X-100. 1 m~ of 100X N-deacetylated. '~-suifated :~5, 4 ml of a solution of 20S
polyvinyipyrroiidone ~15 cont~inin~ 40Z ethylene glycol and water to a tot~l volum of 8 mi bere added to 2 ml of this solution. 6 ml of a solution c~nt~ininv s'.O1 .~ HEPES, 0;015 M EDTA. 0.01 M KCl, 0.015%
TRITON X-100 and 134.4 ug of C5 epimerase were added to 6 ml of this solution.
The solution obtained showed a vi~co-eity of 1.36mm /s (1.3~ ~entisb~~s) The mixture was kept at 37-C for 1 hour. The reaction was stopped by ~oilin~ at lOO-C for 5 minutes~
The product was purl~~ied as described in ~mrle 1.
After freeze-arying the product wa~ dissolved in buffer solut1on cont~inin~ 6.25 ml of 0.04 ~ HEPES. o.o6 M EDTA. 0.4 ~ KCl p~ 7.4, 2.5 g of polyvinylpyrroiidone K15, 2.5 ml of ethylene glycol. 71.2 ~1 of rRITON X-iOO and water t~ a total volume of 25 nl.
1.9 ug o~ C~ eplmerase 5~ ssolved in 12 ml of ~.01 M HEPES. O.015 M
EDTA. 0.1 ~! KCl pH 1,7 c~nr~inin~ 0.015% of TPITON X-100 were added to 12 ml of the obtained mix~ure.
The mixture showed a ~iscosity of 1.4 mm /s tl.4 centistokes).
The rection was kept at ~7-C overnight. .~t the end of the reaction the enzyme was ;nactivated by heati~g the solution at lOO-C for 5 minutes.
The produc~ was purified as described in ~P~rle 1 and the lH-NMR
spectrum was performed (.-ig. ,).
The percentage of L-~duronic 2cid over the total uronic acids was 52.
zs ~XAMPLE 6 The Ex~mpie ~ was repe~ted except that in the first step the reaction ~Or~ S~
~as kept at 37~C for ~ ~.ours.
After purificat1on :- t-e same conditions of Example 1 and after freeze-drying the proauc~ ~as dissoived in 1 ml or water and added to a buffer solution con~-r--~ 50 mi or O.O4 M HEPES. 0.06 M EDTA, 0.4 M
KCl p~ /.4, 83., __ of ~RITON X-lOO. 100 ~1 of lOS
poly~inylpyrrolidone X15, 20 ml of ethylene glycol. 2 ~l of 9OS
glycerom. 1.9 ug or C~ epL~erase ard water to a total volume of 200 ml. The mixture sAowea a viscosity of 1.41 mm /s (1.41 centistokes).
The reaction was ~ept --t 37-C for 4 hours and the enzyme was lnact~Yated at 100 ~~ for -, ~inutes.
The pr~auct was pur~f_ed ~s described in Exampie 1 and the lH-NMR
spectrum wPC perfor~ea (~lg. 6).
The percentage of L-~durcnic acid over the total uronic acids was 55.
EXAMpr F
l.5 mg of 100Z ~.-deace~ ated, ~r-suifated K5 were dissoived in a buffer solution c-n~ini-~ 7 ml or 0.04 M HEP~C. ~.o6 M EDTA, 0.4 M
~Cl pH ,.~. 210 ~i cr ~RrTC~ X-i00, ;.4 g of poiyvlnyipyrroiidone K15, 2.8 mi of ethylene glyco~. .5 mg of C5 epimerase ana water to a total volume o f 28 ml.
The soiution showea a ~isccsity of 1.36 mm /s (1.36 centistokes).
The roaction was ~ept _- 37-C for 4 hours and the enzyme wa~
inac~ ated at 100-C for ~, ~inutes.
The product was pur1riea 2S described in Exampie l and freeze-dried.
~he product was .:en rissolved _~ 24 ml o~ .he same solution cont~i nin~ 1. 3 mg c~ C, eplmerase and the react~on was kept at 37'C
o~o S~
CA 02204366 l997-05-02 ~or 4 hours and the enzy~e ~as inactivated at lOO-C for 5 minutes.
~he product ~as purifled _ descrlbed in ~xamp}e 1 and fr2ez~ a~-ied.
he product :~as then --ssolYed in 8.4 ml of the same solution ~ont~inin~ 1 m~ of C5 epl~erase and the reaction was kept at 37'C for " hours.
The product was purified ,c described in Exu~nple 1 and the L~-NMR
spectrum was performed (r g. 7~.
The percenta~e of L-1duror~c acid over the total uronic acids was &0.
~YAMPLE 8 (com~arison) : _g of 100% '~-deacetyla-~ -sulfated K5 was ~issolved in a buffer soiution con~inin~ 3 ~1 .' 0.04 M HEPES. 3.06 M EDTA. 0.4 M KCl p~
l.4, 18 ,ul of TRITON X-100. 1.2 ml of acetone. 1.9 ~g of C5 ep~ sse ana water to a total voi~D of 12 ml.
The solution showed a viscosity of ~-~9 mm2/s (0.09 centistokes).
~he reaction was kept 2t ,-C for 4 hours.
~he product was purifi~ ~s described 3 ~x~mr1 e ~ and the lX-NMR
,pectr~m was per~ormed (~ -. 8).
~he percentage of L-idurc.- c acid over the total uronic acids was 0.
~XAMPLE 9 : ~g of 100~ N-deacetyla~ed, ~-sulfated K5 was dissoived in a buffer solution cont~inin~ 2 ml _. 0.04 M HEPES. 0.06 M EDTA. 0.4 M KC1 pH
,.4. 12 ~1 of TRITON X- 00. 1.336 ml of 2.4X polyvinylpyrrolidone K90, ~04 ug of C5 epimerase ~n~ ~ater to a total voiume of 8 ml.
~he solution showed a visccsity of 1.29 m~ /s (1.29 centistokes).
~-~ reaction was kept 2~ 3--C for 4 hours. ~he enzyme was lnactivated lOO-C for -, minutes ~-~ the solution filtered in a 0.22 ~m device.
F~,~r~ S~
CA 02204366 1997-0~-02 W 096/14425 PCT~P95/04241 8 ml of the same mixture cont~ining 304 ug of C5 epimerase were added to the filtered solution and the reaction was kept at 37~C for 4 hours, the enzyme was inactivated at 100~C for 5 minutes and the solution filtered in a 0.22 ~m device.
8 ml of the same mixture containing 304 ,ug of C5 epimerase were added to this solution and the reaction was kept at 37~C for 4 hours, the enzyme was inactivated at 100~C for 5 minutes and the solution filtered in a 0.22 um device.
The product was purified as described in Example 1 and the lH-NMR
spectrum was performed (Fig. 9).
The percentage of L-iduronic acid over the total uronic acids was 59.
Claims (8)
1. Process for the preparation of polysaccharides having a L-iduronic acid content greater than 50% with reference to the uronic acids total content starting from the polysaccharide K5 of the E. coli or from the heparin or the heparan sulfate comprising:
a) N-deacetylation of said polysaccharide K5 or of the heparan sulfate or O-desulfation of said heparin or heparan sulfate;
b) N-sulfation of the product obtained from the stage a);
c) one or more treatments of epimerization in presence of the C5 epimerase enzyme;
d) sulfation of at least some free hydroxy groups, characterized in that the epimerization stage is carried out in a reaction medium consisting of a classical buffer solution at pH 7.4 constituted by HEPES, potassium chloride and EDTA to which TRITON X-100 and an additive or more additives in an amount suitable to increase the viscosity of said buffer solution to values ranging from 1.1 to 3 mm2/s (1.1 to 3 centistokes) are added, said additive or additives being selected from the group consisting of ethylene glycol, glycerol, polyvinylpyrrolidone, polyethylene glycol and phosphatidylcholine.
a) N-deacetylation of said polysaccharide K5 or of the heparan sulfate or O-desulfation of said heparin or heparan sulfate;
b) N-sulfation of the product obtained from the stage a);
c) one or more treatments of epimerization in presence of the C5 epimerase enzyme;
d) sulfation of at least some free hydroxy groups, characterized in that the epimerization stage is carried out in a reaction medium consisting of a classical buffer solution at pH 7.4 constituted by HEPES, potassium chloride and EDTA to which TRITON X-100 and an additive or more additives in an amount suitable to increase the viscosity of said buffer solution to values ranging from 1.1 to 3 mm2/s (1.1 to 3 centistokes) are added, said additive or additives being selected from the group consisting of ethylene glycol, glycerol, polyvinylpyrrolidone, polyethylene glycol and phosphatidylcholine.
2. Process as claimed in claim 1, characterized in that said additive is polyvinylpyrrolidone having molecular weight from 15,000 to 90,000.
3. Process as claimed in claim 1, characterized in that said additive, or additives, are added to said buffer solution in an amount from 0.5 to 60 ml with reference to 25 ml of said buffer solution.
4. Process as claimed in claim 1, characterized in that said starting compound is dissolved in said reaction medium in an amount from 5 to 1000 mg per 100 ml.
5. Process as claimed in claim 1, characterized in that said C5 epimerase is dissolved in said reaction medium in an amount from 21 to 2000 µg per 100 ml.
6. Process as claimed in claim 1, characterized in that the mixture for the epimerization contains from 1.5 to 15,000 µg of C5 epimerase per 100 ml of the mixture.
7. Process as claimed in claim 1, characterized in that said epimerization reaction is carried out in a constant-temperature chamber at a temperature ranging from 30 to 40 °C.
8. Process as claimed in claim 1, characterized in that said starting product is N-sulfated from 25% to 100%.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| ITMI942240A IT1271057B (en) | 1994-11-04 | 1994-11-04 | POLYSACCHARIDES HAVING A HIGH CONTENT OF HYDURONIC ACID |
| ITMI94A002240 | 1994-11-04 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2204366A1 true CA2204366A1 (en) | 1996-05-17 |
Family
ID=11369805
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002204366A Abandoned CA2204366A1 (en) | 1994-11-04 | 1995-10-30 | Polysaccharides having a high iduronic acid content |
Country Status (13)
| Country | Link |
|---|---|
| US (1) | US5958899A (en) |
| EP (1) | EP0789777B1 (en) |
| JP (1) | JP3640259B2 (en) |
| CN (1) | CN1094977C (en) |
| AT (1) | ATE195348T1 (en) |
| AU (1) | AU3926195A (en) |
| CA (1) | CA2204366A1 (en) |
| DE (1) | DE69518333T2 (en) |
| DK (1) | DK0789777T3 (en) |
| ES (1) | ES2150589T3 (en) |
| IT (1) | IT1271057B (en) |
| PT (1) | PT789777E (en) |
| WO (1) | WO1996014425A1 (en) |
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|---|---|---|---|---|
| IT1282994B1 (en) * | 1996-05-10 | 1998-04-03 | Inalco Spa | DERIVATIVES OF THE POLYSACCHARIDE K5 HAVING HIGH ANTI-COAGULANT ACTIVITY |
| SE9701454D0 (en) * | 1997-04-18 | 1997-04-18 | Ulf Lindahl | new DNA sequences and a process for enzyme production |
| US7223571B2 (en) * | 1998-04-02 | 2007-05-29 | The Board Of Regents Of The Universtiy Of Oklahoma | Targeted glycosaminoglycan polymers by polymer grafting and methods of making and using same |
| US8088604B2 (en) * | 1998-04-02 | 2012-01-03 | The Board Of Regents Of The University Of Oklahoma | Production of defined monodisperse heparosan polymers and unnatural polymers with polysaccharide synthases |
| US20080108110A1 (en) * | 1998-04-02 | 2008-05-08 | Deangelis Paul L | Targeted glycosaminoglycan polymers by polymer grafting and methods of making and using same |
| US20060188966A1 (en) * | 1998-04-02 | 2006-08-24 | Deangelis Paul L | Natural, chimeric and hybrid glycosaminoglycan polymers and methods of making and using same |
| US20040197868A1 (en) * | 2001-05-08 | 2004-10-07 | Deangelis Paul L. | Heparin/heparosan synthase from P. multocida, soluble and single action catalysts thereof and methods of making and using same |
| CN1098354C (en) * | 1998-11-12 | 2003-01-08 | 武汉大学 | Process for synthesizing triazazole nucleoside by dienzyme catalysis method |
| US6608042B2 (en) | 2000-03-28 | 2003-08-19 | Aventis Pharma, S.A. | Pharmaceutical compositions containing oligosaccharides, the novel oligosaccharides and preparation thereof |
| IT1318432B1 (en) * | 2000-03-30 | 2003-08-25 | Inalco Spa | GLYCOSAMINOGLICANS DERIVED FROM THE K5 POLYSACCHARIDE HAVING HIGH ANTI-AGULATING AND ANTI-THROMBOTIC ACTIVITY AND PROCESS FOR THEM |
| US8227449B2 (en) | 2000-03-30 | 2012-07-24 | Glycores 2000 S.R.L. | Glycosaminoglycans derived from K5 polysaccharide having high anticoagulant and antithrombotic activities and process for their preparation |
| US20020062019A1 (en) | 2000-03-30 | 2002-05-23 | Pasqua Oreste | Glycosaminoglycans derived from K5 polysaccharide having high anticoagulant and antithrombotic activities and process for their preparation |
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| ITMI20032498A1 (en) | 2003-12-17 | 2005-06-18 | Pasqua Anna Oreste | ANTITROMBOTIC POLYSACCHARIDES AT LOW MOLECULAR WEIGHT E |
| ITMI20010397A1 (en) * | 2001-02-27 | 2002-08-27 | Giorgio Zoppetti | HIGHLY N, O-SULPHATED DERIVATIVES OF POLYSACCHARIDE K5 AND THEIR PREPARATION |
| US8580290B2 (en) | 2001-05-08 | 2013-11-12 | The Board Of Regents Of The University Of Oklahoma | Heparosan-based biomaterials and coatings and methods of production and use thereof |
| AU2002256501B2 (en) * | 2001-05-08 | 2008-05-29 | The Board Of Regents Of The University Of Oklahoma | Heparin/heparosan synthase and methods of making and using same |
| EP1518120A4 (en) | 2002-03-11 | 2008-08-13 | Momenta Pharmaceuticals Inc | Analysis of sulfated polysaccharides |
| US8513407B2 (en) * | 2002-06-18 | 2013-08-20 | Glycores 2000 S.R.L. | Process for the preparation of N-acyl-(epi)K5-amine-O-sulfate-derivatives and products thus obtained |
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| US20120253029A1 (en) | 2002-06-18 | 2012-10-04 | Pasqua Anna Oreste | Process for the preparation of highly o-sulfated epimerized derivatives of k5 polysaccharide and intermediates therein |
| GB0216861D0 (en) * | 2002-07-19 | 2002-08-28 | Univ Birmingham | Saccharide libraries |
| US20040171819A1 (en) | 2002-10-10 | 2004-09-02 | Aventis Pharma S.A. | Mixtures of polysaccharides derived from heparin, their preparation and pharmaceutical compositions containing them |
| ITMI20031618A1 (en) * | 2003-08-06 | 2005-02-07 | Inalco Spa | POLYSACCHARIDE DERIVATIVES EQUIPPED WITH HIGH ACTIVITY |
| FI20065035A (en) * | 2006-01-19 | 2007-07-20 | Polysackaridforskning I Uppsal | Essentially pure enzyme |
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| US9068957B2 (en) | 2011-02-21 | 2015-06-30 | Momenta Pharmaceuticals, Inc. | Evaluating heparin preparations |
| BR112014024291B1 (en) | 2012-03-30 | 2022-07-05 | The Board Of Regents Of The University Of Oklahoma | METHOD FOR THE RECOMBINANT PRODUCTION OF A HEPAROSAN POLYMER WITH HIGH MOLECULAR WEIGHT, USE OF SUCH TISSUE ANCREASEMENT POLYMER, ISOLATED POLYNUCLEOTIDE AND COMPOSITION OF BIOLOGICAL MATERIAL COMPRISING SUCH POLYMER |
| JP6670235B2 (en) | 2013-06-17 | 2020-03-18 | ザ ユニバーシティ オブ ノース カロライナ アット チャペル ヒルＴｈｅ Ｕｎｉｖｅｒｓｉｔｙ Ｏｆ Ｎｏｒｔｈ Ｃａｒｏｌｉｎａ Ａｔ Ｃｈａｐｅｌ Ｈｉｌｌ | Reversible heparin molecules, methods of making and using the same |
| US10259889B2 (en) * | 2015-03-31 | 2019-04-16 | Seikagaku Corporation | Method for sulfating glycosaminoglycan |
| CN116622006A (en) | 2017-03-10 | 2023-08-22 | 北卡罗来纳大学查珀尔希尔分校 | Short acting heparin-based anticoagulant compounds and methods |
| JP2021502470A (en) | 2017-11-03 | 2021-01-28 | ザ ユニバーシティ オブ ノース カロライナ アット チャペル ヒルＴｈｅ Ｕｎｉｖｅｒｓｉｔｙ Ｏｆ Ｎｏｒｔｈ Ｃａｒｏｌｉｎａ Ａｔ Ｃｈａｐｅｌ Ｈｉｌｌ | Sulfated oligosaccharide with anti-inflammatory effect |
| CN108486187A (en) * | 2018-02-02 | 2018-09-04 | 南京微构医药科技有限公司 | A kind of preparation process of iduronic acid |
| EP3810152A4 (en) | 2018-06-20 | 2022-04-27 | The University of North Carolina at Chapel Hill | Cell protective methods and compositions |
| CN110577559B (en) * | 2019-08-19 | 2023-02-17 | 江西科技师范大学 | Synthesis method of fluorosidase substrate for alpha-L-iduronidase determination |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2286193A (en) * | 1991-03-28 | 1995-08-09 | Italfarmaco Spa | Anticoagulants and processes for preparing such |
-
1994
- 1994-11-04 IT ITMI942240A patent/IT1271057B/en active IP Right Grant
-
1995
- 1995-10-30 AT AT95937026T patent/ATE195348T1/en not_active IP Right Cessation
- 1995-10-30 AU AU39261/95A patent/AU3926195A/en not_active Abandoned
- 1995-10-30 DE DE69518333T patent/DE69518333T2/en not_active Expired - Fee Related
- 1995-10-30 US US08/628,690 patent/US5958899A/en not_active Expired - Fee Related
- 1995-10-30 EP EP95937026A patent/EP0789777B1/en not_active Expired - Lifetime
- 1995-10-30 DK DK95937026T patent/DK0789777T3/en active
- 1995-10-30 PT PT95937026T patent/PT789777E/en unknown
- 1995-10-30 JP JP51501996A patent/JP3640259B2/en not_active Expired - Fee Related
- 1995-10-30 WO PCT/EP1995/004241 patent/WO1996014425A1/en active IP Right Grant
- 1995-10-30 ES ES95937026T patent/ES2150589T3/en not_active Expired - Lifetime
- 1995-10-30 CN CN95196040A patent/CN1094977C/en not_active Expired - Fee Related
- 1995-10-30 CA CA002204366A patent/CA2204366A1/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| ITMI942240A0 (en) | 1994-11-04 |
| DE69518333D1 (en) | 2000-09-14 |
| IT1271057B (en) | 1997-05-26 |
| DE69518333T2 (en) | 2001-02-22 |
| PT789777E (en) | 2001-01-31 |
| AU3926195A (en) | 1996-05-31 |
| JPH10508204A (en) | 1998-08-18 |
| ES2150589T3 (en) | 2000-12-01 |
| CN1162339A (en) | 1997-10-15 |
| WO1996014425A1 (en) | 1996-05-17 |
| EP0789777B1 (en) | 2000-08-09 |
| ATE195348T1 (en) | 2000-08-15 |
| JP3640259B2 (en) | 2005-04-20 |
| US5958899A (en) | 1999-09-28 |
| DK0789777T3 (en) | 2000-11-06 |
| ITMI942240A1 (en) | 1996-05-04 |
| EP0789777A1 (en) | 1997-08-20 |
| CN1094977C (en) | 2002-11-27 |
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| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| FZDE | Discontinued |