JP2002516355A - Amphiphilic polysaccharide derivatives - Google Patents

Abstract

  (57) [Summary] Polysaccharides, especially heparin, which are widely used as anticoagulants, can only be clinically administered by intravenous or subcutaneous injection due to their strong hydrophilicity and high negative charge. By binding to bile acids, sterols and alkanoic acids respectively, amphiphilic heparin derivatives are synthesized. The hydrophobicity of a heparin derivative depends on the molar ratio of heparin to hydrophobic agent fed. The heparin derivative is slightly hydrophobic and exhibits good solubility in water-acetone solvents as well as water. The heparin derivative has a high anticoagulant effect. These slightly hydrophobic heparin derivatives can be absorbed from the gastrointestinal tract and used as oral dosage forms. The heparin derivative can be used to modify the surface of medical devices such as extracorporeal devices and implant devices to prevent blood coagulation.

Description

DETAILED DESCRIPTION OF THE INVENTION

BACKGROUND OF THE INVENTION The present invention relates to polysaccharide derivatives having increased hydrophobicity as compared to unmodified polysaccharides. More particularly, the present invention relates to amphiphilic polysaccharide derivatives, such as amphipathic heparin derivatives, which preserve the biological activity of the polysaccharide. Further, the present invention relates to a method for producing and using such an amphiphilic polysaccharide derivative.

[0002] Heparin is a polysaccharide composed of sulfated D-glucosamine and D-glucuronic acid residues. Due to its numerous ionizable sulfate groups, heparin has a strong electronegative charge. Heparin is also a relatively strong acid that readily forms water soluble salts (eg, sodium heparin). Heparin is found in mast cells and can be extracted from many body organs, especially those with abundant mast cells. The liver and lungs are particularly rich in heparin. Circulating blood does not contain heparin except after severe destruction of mast cells. Heparin has many physiological functions such as blood anticoagulation and inhibition of smooth muscle cell proliferation. In particular, heparin is a powerful anticoagulant that interacts strongly with antithrombin III and prevents the formation of fibrin clots. However, the application of heparin in vivo is very limited. Heparin is not efficiently absorbed from the gastrointestinal tract, nasal or oral mucosal layers, etc. due to its hydrophilicity and high negative charge. Therefore, the only routes of administration used clinically are intravenous and subcutaneous injections. In addition, heparin is only soluble in a relatively small number of solvents, making it difficult to use for coating or delivery systems on medical devices.

[0003] In order to improve such properties of heparin, RJ Linhardt, et al.
et al., 83, J. Pharm. Sci. 1034-1039, 1994) couple lauryl (C ₁₂ ) and stearyl (C ₁₈ ) groups to a single heparin chain, increasing the hydrophobicity but increasing the anti- Derivatized heparin with low clotting activity was produced. The results indicate that coupling a small linear aliphatic chain to heparin is not effective in increasing the hydrophobicity of heparin while preserving activity. That is, the known heparin derivatives were not effective in preserving anticoagulant activity.

[0004] Rivera et al., Oral Delivery of Heparin in Combination with
Sodium N- [8- (2-Hydroxybenzolyl) amino] caprylate; Pharmacological Conside
rations, 14 Pharm. Res. p1830-1834, 1997) is N- [8- (2-hydroxybenzoyl)
) Heparin mixed with [amino] caprylate was used and the possibility of oral delivery of heparin was disclosed. Dryzski et al., Investigations on Plasma A
ctivity of Low Molecular Weight Heparin after Intravenous and Oral Admin
istrations, 28 Br. J. Clin. Pharma. 188-192, 1989) described the potential for oral absorption of low molecular weight heparin using enhancers.

[0005] Two basic methods have been developed for the formulation of heparin-release systems. One method is to attach heparin to the cationic polymer matrix by ionic bonding. Heparin release is controlled by an ion exchange mechanism. Another method involves the dispersion of heparin, where heparin is first physically mixed with the polymer, and then the release of heparin is controlled by diffusion. The simplest and most efficient method for the manufacture of such heparin devices is the solvent casting method. However, solvent casting cannot be used to fabricate heparin devices because heparin does not dissolve in the organic solvents used to dissolve the polymer. If a heparin derivative with increased hydrophobicity can be produced while maintaining biological activity,
This heparin derivative can be easily immobilized on a polymer matrix by a solvent casting method.

In view of the above, it will be appreciated that the development of hydrophobic or amphiphilic heparin derivatives with high biological activity is a significant advance in the art. Such hydrophobic heparin derivatives can be used in controlled release systems for modifying the surface of medical devices for oral administration or for improving biocompatibility. Such heparin derivatives will further extend the medical applications of heparin.

SUMMARY OF THE INVENTION It is an object of the present invention to synthesize amphiphilic heparin derivatives having high heparin bioactivity.

It is a further object of the present invention to provide hydrophobic heparin derivatives that dissolve in solvents such as acetone / water as well as water.

Another object of the present invention is to provide a heparin derivative that can be used in a controlled release system to prevent blood clotting on a surface.

[0010] Yet another object of the present invention is to provide heparin derivatives that can be absorbed from the gastrointestinal tract, thereby facilitating oral delivery to prevent blood clotting.

Yet another object of the present invention is to provide bile acids such as deoxycholic acid and glycocholic acid,
Another object of the present invention is to provide a heparin derivative including a heparin coupled with a hydrophobic agent such as cholesterol or an alkanoic acid.

[0012] These and other objects can be addressed by providing a composition of matter comprising a polysaccharide to which a hydrophobic agent is covalently attached. Preferred as polysaccharides are heparin, heparin sodium,
It is selected from the group consisting of sulfonated polysaccharides, cellulose, hydroxymethylcellulose and hydroxypropylcellulose. A particularly preferred polysaccharide is heparin. Preferably, such heparin has a molecular weight of about 200 to 100,000. In a preferred embodiment of the invention, the hydrophobic agent is one selected from the group consisting of bile acids, sterols and alkanoic acids. Preferred bile acids include cholic acid, deoxycholic acid, chenodeoxycholic acid, lithocholic acid, ursocholic acid, ursodeoxycholic acid, isoulsodeoxycholic acid, lagodeoxycholic acid, glycocholic acid, taurocholic acid, and glycocide. Deoxycholic acid, glycochenodeoxycholic acid, dehydrocholic acid, hyocholic acid, hyodeoxycholic acid and mixtures thereof. Preferred sterols include cholestanol, coprostanol, cholesterol, epicholesterol, ergosterol, ergocalciferol and mixtures thereof. Preferred alkanoic acids include those having about 4 to 20 carbon atoms, such as butyric acid, valeric acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid and mixtures thereof. Preferably, the polysaccharide and the hydrophobic agent are present in a molar ratio of about 1: 1 to 1: 1000.

[0013] Yet another aspect of the present invention provides a pharmaceutically effective amount of (a) a composite material comprising a polysaccharide to which a hydrophobic agent is covalently bonded, and (b) a pharmaceutically acceptable carrier;
And a pharmaceutical composition comprising: Pharmaceutically acceptable carriers may be oral drug carriers, sustained release carriers, carriers for parenteral administration, and the like. Preferred sustained release carriers include
Well-known in the art, including those selected from the group consisting of poly (ethylene oxide) -poly (ε-caprolactone) copolymers, polyurethane polymers, silicone polymers, ethylene vinyl acetate polymers, hydrogels, collagen, gelatin and mixtures thereof. Polymer matrices.

[0014] Yet another aspect of the invention involves contacting blood with a pharmaceutical composition comprising a polymer composition that is intimately mixed with a complex comprising heparin to which a hydrophobic agent is covalently attached. A method for inhibiting blood clotting in a medical device. Typically, medical devices are coated using film casting techniques as are well known in the art.

DETAILED DESCRIPTION OF THE INVENTION Before disclosing and describing the amphiphilic polysaccharide compositions of the present invention, and methods of making and using the same, the present invention requires certain modifications in the structure, process steps, and materials of the same. As such, it is to be understood that the invention is not limited to the particular configurations, process steps, and materials disclosed herein. Furthermore, because the scope of the present invention is to be limited only by the appended claims and equivalents thereof, the terms used herein are used only to describe a particular embodiment. It should be understood that no limitation of the scope of the invention is intended.

As used in the specification of the present invention and the appended claims, the singular forms “a,” “an,” and “the” refer to a plural reference unless the context clearly dictates otherwise. It should be noted that it includes. Thus, for example, reference to "bile acid" includes a mixture of two or more bile acids, reference to "alkanoic acid" includes reference to one or more alkanoic acids, and reference to "sterol" may include a mixture of two or more bile acids. It includes references to mixtures of the above sterols.

In the description and claims of this invention, the following terms will be used in accordance with the definitions set forth below.

As used herein, “bile acid” refers to natural and synthetic derivatives of steroids, cholanic acid, cholic acid, deoxycholic acid, chenodeoxycholic acid, lithocholic acid, ursocholic acid, ursodeoxycholic acid. , Isoursodeoxycholic acid, lagodeoxycholic acid, glycocholic acid, taurocholic acid, glycodeoxycholic acid, glycochenodeoxycholic acid, dehydrocholic acid, hyocholic acid,
Examples include, but are not limited to, hyodeoxycholic acid and mixtures thereof.

As used herein, “sterol” refers to alcohols structurally related to steroids, such as cholestanol, coprostanol, cholesterol, epicholesterol, ergosterol, ergocalciferol and mixtures thereof. And the like, but are not limited thereto.

“Alkanoic acid” as used herein refers to a saturated fatty acid having about 4 to 20 carbon atoms. Examples of alkanoic acids include butyric, valeric, caproic,
Examples include, but are not limited to, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, and mixtures thereof.

As used herein, “hydrophobic heparin derivative” and “amphiphilic heparin derivative” are used interchangeably. Heparin is a very hydrophilic substance. Increasing the hydrophobicity of heparin by attaching a hydrophobic agent to heparin produces the amphipathic heparin derivative or hydrophobic heparin derivative referred to herein. Both terms are considered appropriate since heparin derivatives have increased hydrophobicity as compared to native heparin and have both hydrophilic and hydrophobic moieties, ie, amphipathic.

It is well known to use heparin as an antithrombotic agent to prevent blood clotting. Heparin is highly hydrophilic because a high density of negative charge is provided by the sulfone and carboxyl groups. Due to this hydrophilicity, heparin is usually administered by intravenous or subcutaneous injection. Here, a heparin derivative having a slightly hydrophobic property or an amphipathic property and a high biological activity will be described. Bile acids such as deoxycholic acid (DOCA); sterols such as cholesterol;
And hydrophobic agents such as alkanoic acids, for example lauric and palmitic acids, were coupled with heparin. Both deoxycholic acid and cholesterol are non-toxic because they are naturally occurring compounds found in the body. The amine group of heparin was coupled with the carboxyl group of the hydrophobic agent. The terminal carboxyl groups in DOCA, lauric acid and palmitic acid were used directly in the coupling reaction, whereas the cholesterol hydroxy groups were activated by reaction with chloroacetic acid prior to coupling. It has been found that attaching such a hydrophobic moiety to the amine group of heparin has little effect on the biological activity of heparin.
The coupling between heparin and the hydrophobic agent is FT-IR and ¹³ C-NM
Confirmed by detecting the amide bond by R analysis.

[0023] The yield of the coupling reaction was about 70-80%, and there was no significant change when the hydrophobic agent or the feed molar ratio was changed. In the case of the heparin-DOCA conjugate, as the feed ratio increased, the amount of DOCA in the conjugate also increased. The weight percentage of DOCA in heparin-DOCA was 24% when the feed molar ratio of heparin was 1: 200 to DOCA. This molar ratio was much higher than the ratio of amine groups in heparin to DOCA. Therefore, this supply ratio is estimated to be excessive DOCA.

The hydrophobic heparin derivatives of the present invention have many medical uses. For example, hydrophobic heparin can be administered orally. Oral administration of heparin can greatly expand the use of heparin as an oral anticoagulant. The heparin derivative is formulated with a pharmaceutically acceptable carrier well known in the art. To give further examples, hydrophobic heparin derivatives can be used for catheters, cardiopulmonary bypass circuits (
circuit), a cardiopulmonary oxygenator, a renal dialysis machine, a restenosis prevention stent, or a coating material for medical devices such as a balloon coating. The hydrophobic heparin derivative is typically mixed with a carrier and subsequently coated on the surface of the medical device by film casting techniques as are well known in the art.

After modification, heparin, as shown by high-performance protein liquid chromatography (FPLC: trade name) on phenyl sepharose (trade name), eluting with ammonium sulfate buffer rather than phosphate buffer -It has also been found that hydrophobic agents tend to exhibit hydrophobic interactions with hydrophobic media. These heparin derivatives had enhanced binding affinity compared to unmodified heparin. The increased interaction between the modified heparin derivative and phenyl sepharose (trade name) is due to its enhanced hydrophobicity and is the result of the presence of hydrophobic functional groups. These results suggest that hydrophobic heparin can be obtained by binding bile acids, sterols or alkanoic acids to heparin. In solubility tests, polar or organic solvents were suitable for dissolving the heparin-hydrophobic agent conjugate. For example, the heparin-deoxycholic acid conjugate dissolved well in a 65% acetone solution (35% water). Finally, it was determined that the biological activity of the modified heparin derivatives was not significantly affected by the binding to the hydrophobic agent.
The role of hydrophobic agents bound to heparin was examined for two biological activities of heparin as measured by anticoagulation and factor Xa analysis. Hydrophobicity has been associated with some reduction in anticoagulant and anti-factor Xa activity, but such a reduction in biological activity was not severe. These results indicate that blocking the heparin amine group has little effect on its biological activity. However, in the heparin-hydrophobic agent conjugate, when the amount of hydrophobic agent exceeded 20% by weight, the biological activity of heparin showed a gradual decrease. When the amount of hydrophobic agent in the conjugate was less than 20% by weight, the conjugate had more than 80% of the biological activity of unmodified heparin. A biological activity of 80% on hydrophobic heparin is suggested to be sufficient to support biological activity for pharmaceutical use.

Example 1 Synthesis of Heparin-DOCA Conjugate N-hydroxylsuccinimide (HOSu, 92) in dimethylformamide (DMF)
mg / 5 ml) 5 ml of dicyclohexylcarbodiimide (DCC, 165 mg / 5 ml) in DMF
After mixing with 5 ml, 5 ml of DOCA in DMF (196 mg / 5 ml) was added. The molar ratio of DOCA, HOSu and DCC was 1: 1.6: 1.6. The concentrations of HOSu and DCC were slightly higher than those of DOCA and activated DOCA completely. The resulting solution was reacted under vacuum at room temperature for 5 hours, followed by removal of dicyclohexylurea (DCU), a by-product precipitated during the reaction. Unreacted DCC was removed by adding a drop of distilled water and filtering. Further, residual HOSu was removed by adding 15 ml of distilled water. Activated DOCA was precipitated and subsequently lyophilized. Subsequently, the activated DOCA was dissolved in DMF and reacted with heparin at room temperature for 4 hours. The amount of heparin used in this reaction ranged from 40 to 400 mg. After the reaction, there were two forms of the product, a water-soluble product and a water-insoluble product. These products were separated by filtration through a 0.45 μm membrane filter, and the water-insoluble products were dried in a vacuum oven. The water-soluble product was dialyzed against water using a membrane (MWCO 3,500) for 1 hour, followed by freeze-drying heparin-DOCA.

The heparin derivative prepared by this procedure has been characterized by FT-IR and NMR according to methods well known in the art, and the coupling between heparin and the hydrophobic agent has been successful. It was confirmed. Evidence for heparin derivatives is an amide bond resulting from the coupling of the amine group of heparin with the carboxyl group of a hydrophobic agent. In the FT-IR spectrum,
Significant changes were observed between spectra in the range 1740-1500 cm ^-1 . 1585c
A strong band at m ^-1 was observed and assigned to amide vibration. This is related to the presence of an amide bond between heparin and the hydrophobic agent. The peak of the NH group in the heparin portion of the heparin derivative was shown around 3500 and 1620 cm ^-1 respectively. The ¹³ C-NMR spectrum of the heparin-DOCA conjugate was δ7.58 (carbon at the amine bond), 5.5 (H-1 of glucosamine 2,6-disulfate), δ5.35 (H-1 of glucosamine 2-sulfate). ) And δ5.2 (H-1 of iduronic acid 2-sulfuric acid) showed characteristic absorption peaks.
Comparing heparin-DOCA and heparin, the ¹³ C-NMR spectrum was 178 pp
m (carbon at the amine bond) showed a different peak. These results confirm the presence of an amide bond in the heparin-DOCA conjugate and demonstrate that the amine group of heparin is coupled to the carboxyl group of DOCA.

Example 2 Preparation of Heparin-Cholesterol Conjugate To generate free carboxyl groups, the hydroxyl groups of cholesterol were activated by reacting with chloroacetic acid. This modified cholesterol
Reacted with HOSu and DCC in 10 ml DMF. The molar ratio of cholesterol, HOSu and DCC was 1: 1.6: 1.6. The reaction was performed at room temperature for 5 hours. Unreacted DCC and HOSu
To remove, water was added and the solution was filtered through a 0.45 μm membrane. Next, the activated cholesterol was reacted with the heparin solution for 4 hours. Two products, a water-soluble product and a water-insoluble product, were obtained from the reaction. These products were processed according to the procedure described in Example 1 above.

Example 3 Synthesis of Heparin-Alkanoic Acid Conjugate Lauric acid and palmitic acid were coupled to heparin according to the procedure of Example 1. The carboxyl group of the alkanoic acid was coupled to the amine group of heparin to form an amide bond. The coupling agents were HOSu and DCC as well.

For heparin-DOCA, heparin-cholesterol, and heparin-alkanoic acids, the production yield, molecular weight and binding molar ratio between heparin and the hydrophobic agent varied with the molar ratio of the reactants. Heparin-DOCA conjugate yields ranged from 71 to 77%. The molar weight of heparin was measured at 12,386 daltons by light scattering. The amount of hydrophobic agent in the modified heparin derivative was calculated by subtracting the molecular weight of heparin from the measured molecular weight of each heparin derivative. By increasing the feed molar ratio of deoxycholic acid to heparin from 1: 6 to 1: 200,
The amount of DOCA in the heparin-DOCA conjugate increased from 7 to 24%. Heparin-cholesterol conjugate yields also ranged from 73 to 78%. However, the amount of cholesterol in such a hydrophobic heparin conjugate may vary from heparin-DOCA.
Slightly less than the amount of DOCA in the conjugate. Similar amounts of alkanoic acids were coupled to heparin in the heparin-lauric acid and heparin-palmitic acid conjugates.

Example 4 Heparin-DOCA Conjugate Solubility Test Heparin can be dissolved only in a relatively small number of solvents such as water and formamide. Because the heparin derivatives of the present invention are slightly hydrophobic, such derivatives were expected to be soluble in additional solvents. In the present embodiment, this is
It was investigated by evaluating the solubility in a mixture of acetone and water as solvents. In the case of the heparin-DOCA conjugate, the solubility of the conjugate in the solvent increased as the weight percentage of DOCA increased. At 14 wt% DOCA, the heparin-DOCA conjugate was dissolved in 50:50 acetone-water, but the conjugate was not soluble in 70:30 acetone-water. For 24 wt% DOCA, the solubility of the heparin-DOCA conjugate in the solvent increased as the acetone content of the solvent increased. Heparin-DOCA in solvent (24%)
The solubility of the conjugate was greatest when the volume ratio of acetone to water was 50:50.

Example 5 Analysis for Separation of Heparin on Phenyl-Sepharose CL-4B Gel at 4 ° C. Phenyl-Sepharose® CL-4B was prepared from heparin-DOCA, heparin-cholesterol, and heparin-alkanoic acid. Used to remove unreacted heparin. This was also useful for assessing the degree of hydrophobicity of the coupled heparin derivative. Commercially available heparin sodium (anticoagulant activity, 140 USP per mg) prepared from bovine lung was added to Pharmacia Hepar Co. (Franklin, Ohi).
o) obtained from. Phenyl Sepharose CL-4B gel column was used with Pharmacia Biotech
(Sweden). Wash the column (HR 16/30 ID) with 10 volumes of water,
This is followed by at least 40 ml of 50 mM phosphate buffer (pH 7.0) for 20 minutes, then 1.7 M
Equilibration was performed before use by washing with 40 ml of 50 mM phosphate buffer (pH 7.0) containing ammonium sulfate, followed by 40 ml of 50 mM phosphate buffer (pH 7.0). A solution of heparin (5 mg) and hydrophobic heparin (5 mg) in the same phosphate buffer (5 ml) was applied to the column and each was eluted with a gradient solvent. The flow rate was 1 ml / min., And each 2-ml fraction was collected by a fraction collector. After elution on the column, wash the column with 100 ml of water and 1.
Washing with 7M ammonium sulfate removes any remaining heparin or heparin-DOCA conjugate, and collects the collected fractions with Azure A (Azure A, concentration: 0.01 mg / ml).
Mix for minutes. Fractions containing heparin or hydrophobic heparin were collected using Varian CARY 1E
Quantification was performed by spectrophotometrically monitoring the absorbance at 500 nm on a UV / VIS spectrometer.

Changes in the elution curve of the heparin-DOCA conjugate in FPLC were observed for different coupling ratios between heparin and DOCA. Heparin was eluted with PBS as an eluent, but was not eluted with ammonium sulfate because it was extremely hydrophilic. Heparin-DOCA conjugate was not eluted in PBS but eluted in ammonium sulfate solution. As the concentration of ammonium sulfate in the eluent increased, the hydrophobicity of the eluted heparin conjugate also increased. The heparin-DOCA conjugate was completely dissolved in the 1.3 M ammonium sulfate solution even when the amount of DOCA was increased.

Example 6 Biological Activity of Heparin Derivatives The anticoagulant activity of the modified heparin derivatives was determined by the activated partial thromboplastin time (AP
TT) and Factor Xa chromogenic analysis.
The antithrombotic activity of the heparin derivative was measured by FXa chromogen assay and APTT, respectively (FIG. 1). The biological activity of heparin used in these experiments was 140 units / mg
had s ability. The biological activity of all heparin derivatives produced in this study was more than 70% compared to the biological activity of unmodified heparin. There was no difference in the biological activity of the conjugate with respect to the hydrophobic agent used to make the conjugate. However, the biological activity of the heparin derivative decreased slightly as the amount of hydrophobic agent in the conjugate increased. When a conjugate containing 7% by weight of DOCA was tested, the relative biological activity of the heparin-DOCA conjugate was 93% by APTT analysis and 80% by FXa analysis. In contrast, when a conjugate containing 24% by weight DOCA was tested, the relative biological activity of such a heparin-DOCA conjugate was 71.5% (APTT)
And 70.1% (FXa analysis).

Example 7 Oral Delivery of Heparin Six rats bred at an animal breeding facility at the Korea Animal Center were fasted for 12 hours before dosing. Groups of rats weighing 250-300 g were given a single oral dose of heparin, high molecular weight heparin-DOCA or low molecular weight heparin-DOCA. Blood sample (0.5ml
) Were sequentially collected by a heparin-coated capillary mixed with 3.8% sodium citrate. Samples were taken before heparin or heparin derivative administration and at hourly intervals up to 10 hours after administration. Plasma was collected by centrifugation and frozen at below -20 ° C. Plasma heparin activity in each sample was measured by APTT analysis. The APTT bioassay was performed according to the procedure of Example 6. Plasma APTT units were determined from clotting times measured by a fibrometer.

In the case of low molecular weight heparin-DOCA (FIG. 2), the maximum clotting time was seen 4 hours after oral administration. The clotting time returned to baseline after 10 hours. In the case of high molecular weight heparin-DOCA (FIG. 3), the maximum clotting time was seen 8 hours after oral administration. The clotting time was maintained for more than 20 minutes even after 10 hours. High molecular weight heparin-DOCA had higher anticoagulant activity than low molecular weight heparin-DOCA.

Example 8 In Vitro Release Rate of Heparin Derivatives First , heparin derivatized on polyethylene discs (2.22 cm diameter) was PE
In vitro tests were performed by casting on O / PCL multi-block copolymers. PEO / PCL is a multi-block copolymer composed of alternating blocks of poly (ethylene oxide) (molecular weight about 2,000) and poly (ε-caprolactone) (molecular weight about 2,000), and the total molecular weight of the copolymer is about 30,000. The heparin derivative was mixed with the polymer and dissolved in acetone / water and cast on a polyethylene disc, followed by evaporation of the solvent. Subsequently, the lower surface of the disk was placed on the bottom of a 50-ml vial. Each disc was washed with PBS buffer (pH 7.4, I
= 0.15) immersed in 20 ml, shake water bath at 37 ° C, 80 rpm (Han Back Scientific Co.
., Korea) at a randomly assigned location. At the specified time (determined that the concentration of the heparin derivative in the released medium does not exceed 10% of its saturation solubility at 37 ° C.), a sample is removed and mixed with Azure A before UV spectroscopy (530 nm ) Was analyzed for drug content. At each sampling time, all release media was removed and replaced with fresh pre-warmed PBS buffer. Following the release study, the initial amount of the heparin derivative in the PEO / PCL multi-block copolymer film was calculated by summing the cumulative amount released over 40 days and the amount remaining on the disk for 40 days. . This was compared to the initial amount calculated from the drug load. The cumulative amount of heparin derivative released was plotted against time, and the percentage released was used for statistical comparisons made by repeated measures analysis of variance.

[0038] The heparin derivative was released from the polymer matrix at an almost controlled release rate with a small burst effect. This burst effect appeared within one hour, with the amount released in the burst being about 10% of the input (FIG. 4).

[Brief description of the drawings]

FIG. 1 shows the biological activity of hydrophobic heparin measured by APTT assay (closed symbol) and chromogenic assay (open symbol) analysis: Δ and □
Indicates deoxycholic acid (DOCA); ● and ○ indicate cholesterol; ▼ and Δ indicate palmitic acid; ▲ and Δ indicate lauric acid.

FIG. 2 shows the clotting time as a function of time when low molecular weight heparin-DOCA is administered orally.

FIG. 3 shows the clotting time as a function of time for oral administration of high molecular weight heparin DOCA-.

FIG. 4 shows poly (ethylene oxide) -poly (ε-caprolactone) (PEO-PCL).
2) shows the cumulative heparin-DOCA conjugate release from the polymer matrix as a function of time; weight percent heparin-DOCA in the polymer matrix: (▽)
, 5% DOCA; (○), 10% DOCA; (△), 20% DOCA; (□), 30% DOCA

[Procedure for Amendment] Submission of translation of Article 19 Amendment of the Patent Cooperation Treaty

[Submission date] October 26, 1999 (1999.10.26)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Claims]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 47/34 A61K 47/34 47/42 47/42 47/48 47/48 A61L 33/10 A61P 7 / 02 A61P 7/02 C08B 15/00 C08B 15/00 37/10 37/10 A61L 33/00 A (31) Priority claim number 09/300, 173 (32) Priority date April 27, 1999 (1999) 4.27) (33) Priority Claimed States United States (US) (81) Designated States AE, AT, AU, BR, CA, CH, CN, DE, DK, ES, FI, GB, HU, ID, IN, JP, KP, KZ, MN, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, TR, UA, ZA (72) Inventor E, Yong Qew Korea, Jung-ranamdo 500 −480, Qua Nju, Book, Orion-Dong 500-712, Gwangju Institute of Science and Technology, Dormitory No. 4301 F-term (reference) 4C076 AA30 BB01 CC14 DD41 DD70 EE08 EE22 EE26 EE27 EE42 EE43 FF68 4C08A02A05 A064 EA26 EA27 MA03 MA05 MA09 NA03 NA12 ZA54 4C090 AA09 BA01 BA08 BA28 BA68 BB02 BB12 BB33 BB36 DA24

Claims (20)

[Claims] 1. A composite substance comprising a polysaccharide to which a hydrophobic agent is covalently bonded, wherein the hydrophobic agent is selected from the group consisting of bile acids, sterols and alkanoic acids. 2. The composite material according to claim 1, wherein the polysaccharide is selected from the group consisting of heparin, heparin sodium, sulfonated polysaccharide, cellulose, hydroxymethylcellulose, and hydroxypropylcellulose. 3. The composite substance according to claim 2, wherein the polysaccharide is heparin. 4. The composite of claim 3, wherein said heparin has a molecular weight of about 200 to 100,000. 5. The method of claim 1, wherein the hydrophobic agent is cholic acid, deoxycholic acid, chenodeoxycholic acid, lithocholic acid, ursocholic acid, ursodeoxycholic acid, isoulsodeoxycholic acid, lagodeoxycholic acid, glycocholic acid, taurocholic acid, The composite substance according to claim 3, which is a bile acid selected from the group consisting of glycodeoxycholic acid, glycochenodeoxycholic acid, dehydrocholic acid, hyocholic acid, hyodeoxycholic acid and a mixture thereof. 6. The composite substance according to claim 3, wherein the hydrophobic agent is a sterol selected from the group consisting of cholestanol, coprostanol, cholesterol, epicholesterol, ergosterol, ergocalciferol and mixtures thereof. 7. The composite material according to claim 3, wherein the hydrophobic agent is an alkanoic acid having 4 to 20 carbon atoms. 8. The method according to claim 8, wherein the alkanoic acid is butyric acid, valeric acid, caproic acid, caprylic acid,
The composite substance according to claim 7, which is selected from the group consisting of capric acid, lauric acid, myristic acid, palmitic acid, stearic acid and a mixture thereof. 9. The method according to claim 8, wherein the polysaccharide and the hydrophobic agent are present in an amount of about 1: 1 to 1: 1.
The composite of claim 1, wherein the composite is present in a molar ratio of: 1000. 10. A pharmaceutical composition comprising: (a) a pharmaceutically effective amount of a complex substance containing a polysaccharide to which a hydrophobic agent is covalently bonded; and (b) a pharmaceutically acceptable carrier, wherein the hydrophobic agent is a bile acid. And a sterol and an alkanoic acid. 11. The pharmaceutical composition according to claim 10, wherein the pharmaceutically acceptable carrier is an oral drug carrier. 12. The pharmaceutical composition according to claim 11, wherein the pharmaceutically acceptable carrier is a sustained release carrier. 13. The method of claim 1, wherein said sustained release carrier is a polymer matrix.
3. The pharmaceutical composition according to 2. 14. The method of claim 14, wherein the sustained release carrier is poly (ethylene oxide) -poly (ε
-Caprolactone) copolymers, polyurethane polymers, silicone polymers,
14. A polymer matrix selected from the group consisting of ethylene vinyl acetate polymer, hydrogel, collagen, gelatin and mixtures thereof.
A pharmaceutical composition according to claim 1. 15. The polymer matrix of claim 1, wherein the polymer matrix is poly (ethylene oxide)-
The pharmaceutical composition according to claim 14, which is a poly (ε-caprolactone) copolymer. 16. The pharmaceutical composition according to claim 10, wherein said polysaccharide is heparin. 17. On a medical device comprising coating a medical device in contact with blood with a pharmaceutical composition comprising a polymer matrix intimately mixed with a complex comprising heparin to which a hydrophobic agent is covalently attached. For inhibiting blood clotting in the blood. 18. The method according to claim 17, wherein said hydrophobic agent is selected from the group consisting of bile acids, sterols and alkanoic acids. 19. The method according to claim 19, wherein the bile acid is cholic acid, deoxycholic acid, chenodeoxycholic acid, lithocholic acid, ursocholic acid, ursodeoxycholic acid, isoulsodeoxycholic acid, lagodeoxycholic acid, glycocholic acid, taurocholic acid, glycodeoxy acid. Cholic acid, glycochenodeoxycholic acid, dehydrocholic acid,
Wherein the sterol is selected from the group consisting of cholestanol, coprostanol, cholesterol, epicholesterol, ergosterol, ergocalciferol and mixtures thereof; hyocholic acid, hyodeoxycholic acid and mixtures thereof. The alkanoic acid is butyric, valeric, caproic, caprylic, capric, lauric, myristic,
19. The method of claim 18, wherein the method is selected from the group consisting of palmitic acid, stearic acid, and mixtures thereof. 20. The method according to claim 1, wherein the polymer matrix is poly (ethylene oxide)-
The method according to claim 17, wherein the method is selected from the group consisting of poly (ε-caprolactone) copolymer, polyurethane polymer, silicone polymer, ethylene vinyl acetate polymer, hydrogel, collagen, gelatin and mixtures thereof.