JP3541007B2 - Amphiphilic polysaccharide derivatives - Google Patents

Description

[0001]
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 large number of 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 suppression 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]
To improve the properties of such heparin, Rinhardt et al. (RJ Linhardt, et al., 83, J. Pharm. Sci. 1034-1039, 1994) reported that lauryl (C.₁₂) And stearyl (C₁₈) Groups were coupled to a single heparin chain to produce derivatized heparin with increased hydrophobicity but low anticoagulant activity. The results show that coupling a small linear aliphatic chain to heparin is not effective in enhancing the hydrophobicity of heparin while preserving activity. That is, 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.RTM. Heparin mixed with (2-hydroxybenzolyl) amino] caprylate was used and the possibility of oral delivery of heparin was disclosed. (Dryjski et al., Investigations on Plasma Activity of Low Molecular Weight Weighing Heparin after Intraventr. I described the possibility of
[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 producing such a heparin device is the solvent casting method. However, solvent casting cannot be used to make 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.
[0006]
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.
[0007]
SUMMARY OF THE INVENTION
It is an object of the present invention to synthesize amphiphilic heparin derivatives having high heparin bioactivity.
[0008]
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.
[0009]
It is another object of the present invention to provide heparin derivatives that can be used in controlled release systems to prevent blood clotting on surfaces.
[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.
[0011]
It is a further object of the present invention to provide heparin derivatives, including heparin coupled to bile acids such as deoxycholic acid and glycocholic acid, or hydrophobic agents such as cholesterol, or alkanoic acids. .
[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 polysaccharides are those selected from the group consisting of heparin, sodium heparin, sulfonated polysaccharides, cellulose, hydroxymethylcellulose and hydroxypropylcellulose. A particularly preferred polysaccharide is heparin. Such heparin preferably 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, glycocolic acid 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, valeric, caproic, caprylic, capric, lauric, myristic, palmitic, stearic 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 is a pharmaceutical composition comprising a pharmaceutically effective amount of (a) a complex comprising a polysaccharide to which a hydrophobic agent is covalently attached, and (b) a pharmaceutically acceptable carrier. including. Pharmaceutically acceptable carriers may be oral drug carriers, sustained release carriers, carriers for parenteral administration, and the like. Preferred sustained release carriers include 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. Examples include polymer matrices well known in the art.
[0014]
Yet another aspect of the invention is a medical device for contacting blood, comprising coating a medical device with a pharmaceutical composition comprising a polymer matrix intimately mixed with a complex comprising heparin to which a hydrophobic agent is covalently attached. And a method for inhibiting blood clotting in the device. Typically, medical devices are coated using film casting techniques as are well known in the art.
[0015]
DETAILED DESCRIPTION OF THE INVENTION
Prior to disclosing and describing the amphiphilic polysaccharide compositions of the present invention, and methods of making and using the same, the present invention is hereby described because its constitution, process steps and materials can be varied to some extent. It should be understood that the invention is not limited to the particular configurations, process steps, and materials disclosed. 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.
[0016]
As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should be noted that That is, 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" includes two or more. It includes references to mixtures of the above sterols.
[0017]
In the specification and claims of the present invention, the following terminology will be used in accordance with the definitions set out below.
[0018]
As used herein, "bile acid" refers to the natural and synthetic derivatives of steroids, cholanic acid, cholic acid, deoxycholic acid, chenodeoxycholic acid, lithocholic acid, ursocholic acid, ursodeoxycholic acid, isoulsoic acid. Deoxycholic acid, lagodeoxycholic acid, glycocholic acid, taurocholic acid, glycodeoxycholic acid, glycochenodeoxycholic acid, dehydrocholic acid, hyocholic acid, hyodeoxycholic acid and mixtures thereof, but are not limited thereto. Not something.
[0019]
As used herein, "sterol" means alcohols structurally related to steroids, including cholestanol, coprostanol, cholesterol, epicholesterol, ergosterol, ergocalciferol and mixtures thereof. However, the present invention is not limited to these.
[0020]
As used herein, "alkanoic acid" means a saturated fatty acid having about 4 to 20 carbon atoms. Examples of alkanoic acids include, but are not limited to, butyric, valeric, caproic, caprylic, capric, lauric, myristic, palmitic, stearic, and mixtures thereof.
[0021]
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 because heparin derivatives have increased hydrophobicity as compared to native heparin and have both hydrophilic and hydrophobic moieties, ie, amphipathic.
[0022]
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. Hydrophobic agents such as bile acids, eg, deoxycholic acid (DOCA); sterols, eg, cholesterol; and alkanoic acids, eg, 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, while 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^ThirteenIt was confirmed by detecting an amide bond by C-NMR analysis.
[0023]
The yield of the coupling reaction was about 70 to 80%, and there was no significant change even when the hydrophobic agent or the feed molar ratio was changed. For the heparin-DOCA conjugate, as the feed ratio increased, the amount of DOCA in the conjugate also increased. The weight percent of DOCA in heparin-DOCA was 24% when the feed molar ratio of heparin was 1: 200 with respect 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.
[0024]
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. By way of further example, hydrophobic heparin derivatives are used as coating materials for medical devices such as catheters, cardiopulmonary bypass circuits, cardiopulmonary oxygenators, renal dialysis machines, restenosis prevention stents or balloon coatings. can do. 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.
[0025]
After modification, heparin-hydrophobicity was demonstrated as indicated by chromatography on phenyl sepharose (trade name) with high performance protein liquid chromatography (FPLC) (eluted with ammonium sulfate buffer rather than phosphate buffer). It has also been found that the agents tend to exhibit hydrophobic interactions with the hydrophobic medium. 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 confirmed that the biological activity of the modified heparin derivatives was not significantly affected by the binding to the hydrophobic agent. The role of the hydrophobic agent bound to heparin was examined for two biological activities of heparin as measured by anticoagulation and factor Xa analysis. Hydrophobicity is accompanied by 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 in pharmaceutical applications.
[0026]
Example 1
Heparin- DOCA Synthesis of conjugate
After mixing 5 ml of N-hydroxylsuccinimide (HOSu, 92 mg / 5 ml) in dimethylformamide (DMF) with 5 ml of dicyclohexylcarbodiimide (DCC, 165 mg / 5 ml) in DMF, DOCA (196 (mg / 5 ml) 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 at room temperature under vacuum 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.
[0027]
Heparin derivatives prepared by this procedure were characterized by FT-IR and NMR according to methods well known in the art to confirm successful coupling between heparin and the hydrophobic agent. did. 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, 1740-1500 cm^-1Significant changes were observed between the spectra in the range. 1585cm^-1A strong band was observed and was 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 is 3500 and 1620 cm^-1Indicated around each. Heparin-DOCA conjugate^ThirteenThe C-NMR spectrum was as follows: δ 7.58 (carbon at the amine bond), 5.5 (H-1 of glucosamine 2,6-disulfate), δ 5.35 (H-1 of glucosamine 2-sulfate), δ 5.2 (Iduronic acid 2-sulfuric acid H-1) showed a characteristic absorption peak. Comparing heparin-DOCA and heparin,^ThirteenThe C-NMR spectrum showed a different peak at 178 ppm (carbon at the amine bond). 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.
[0028]
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 was reacted with HOSu and DCC in 10 ml of 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. Water was added to remove unreacted DCC and HOSu, 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.
[0029]
Example 3
Synthesis of heparin-alkanoic acid conjugate
Lauric and palmitic acids 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.
[0030]
For heparin-DOCA, heparin-cholesterol, heparin-alkanoic acid, the yield, molecular weight, and the molar ratio of binding 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. Increasing the feed molar ratio of deoxycholic acid to heparin from 1: 6 to 1: 200 increased the amount of DOCA in the heparin-DOCA conjugate 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 was slightly less than the amount of DOCA in the heparin-DOCA conjugate. In heparin-lauric and heparin-palmitic acid conjugates, similar amounts of alkanoic acid were coupled to heparin.
[0031]
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 example, this was investigated by evaluating the solubility in a mixture of acetone and water as a solvent. In the case of the heparin-DOCA conjugate, the solubility of the conjugate in the solvent increased as the weight percentage of DOCA increased. When DOCA was 14% by weight, the heparin-DOCA conjugate dissolved in 50:50 acetone-water, but this conjugate did not dissolve 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. The solubility of the heparin-DOCA (24%) conjugate in the solvent was maximized when the volume ratio of acetone to water was 50:50.
[0032]
Example 5
4 Phenyl-Sepharose at ℃ CL − 4B Analysis for separation of heparin on gel  Phenyl-Sepharose CL-4B was used to remove unreacted heparin from heparin-DOCA, heparin-cholesterol, and heparin-alkanoic acid. This was also useful for assessing the degree of hydrophobicity of the coupled heparin derivative. Commercially available sodium heparin (anticoagulant activity, 140 USP per mg) prepared from bovine lung was purchased from Pharmacia Hepar Co. (Franklin, Ohio). Phenyl Sepharose CL-4B gel column was obtained from Pharmacia Biotech (Sweden). The column (HR 16/30 ID) is washed with 10 volumes of water, followed by at least 40 ml of 50 mM phosphate buffer (pH 7.0) for 20 minutes, then 50 mM containing 1.7 M ammonium sulfate. Equilibration was done before use by washing with 40 ml of phosphate buffer (pH 7.0) and then with 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 eluted with a gradient solvent, respectively. The flow rate was 1 ml / min. Then, each 2-ml fraction was collected by a fraction collector. After elution on the column, the column was washed with 100 ml of water and 1.7 M ammonium sulfate to remove any remaining heparin or heparin-DOCA conjugate, and the collected fraction was subjected to Azure A (Azure A, concentration: 0. 0%). 01 mg / ml) for 1 minute. Each fraction containing heparin or hydrophobic heparin was quantified by spectrophotometric monitoring of the absorbance at 500 nm on a Varian CARY 1E UV / VIS spectrometer.
[0033]
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 with PBS but was eluted with 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 1.3M ammonium sulfate solution even when the amount of DOCA was increased.
[0034]
Example 6
Biological activity of heparin derivatives
The anticoagulant activity of the modified heparin derivatives was measured by activated partial thromboplastin time (APTT) and factor Xa chromogenic analysis, respectively. 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 had a capacity of 140 units per mg. The biological activity of all heparin derivatives produced in this study was greater 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 was increased. When conjugates containing 7% by weight of DOCA were tested, the relative biological activity of the heparin-DOCA conjugate was 93% by APTT analysis and 80% by FXa analysis. In contrast, when conjugates containing 24% by weight DOCA were tested, the relative biological activities of such heparin-DOCA conjugates were 71.5% (APTT) and 70.1% (FXa analysis). Decreased to.
[0035]
Example 7
Heparin oral delivery
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 administered a single oral dose of heparin, high molecular weight heparin-DOCA or low molecular weight heparin-DOCA. Blood samples (0.5 ml) were collected sequentially through heparin-coated capillaries mixed with 3.8% sodium citrate. Samples were collected 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 <0> C. Plasma heparin activity was measured by APTT analysis in each sample. The APTT bioassay was performed according to the procedure of Example 6. Plasma APTT units were determined from clotting times measured by fibrometer.
[0036]
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.
[0037]
Example 8
In vitro release rates of heparin derivatives
In vitro testing was performed by first casting the derivatized heparin on a polyethylene disc (2.22 cm diameter) into a PEO / PCL multi-block copolymer. PEO / PCL is a multi-block copolymer composed of alternating blocks of poly (ethylene oxide) (molecular weight of about 2,000) and poly (ε-caprolactone) (molecular weight of about 2,000). 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 immersed in 20 ml of PBS buffer (pH 7.4, I = 0.15) and randomly assigned to a shaking water bath (Han Back Scientific Co., Korea) at 37 ° C. and 80 rpm. Put in position. 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 in 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 (closed symbol) and chromogenic (open symbol) assays: Δ and □ deoxycholic acid (DOCA); ○ indicates cholesterol; ▼ and ▽ indicate palmitic acid; ▲ and △ indicate lauric acid.
FIG. 2 shows the clotting time as a function of time when orally administered low molecular weight heparin-DOCA.
FIG. 3 shows the clotting time as a function of time for oral administration of high molecular weight heparin DOCA-.
FIG. 4 shows the cumulative heparin-DOCA conjugate release from a poly (ethylene oxide) -poly (ε-caprolactone) (PEO-PCL) polymer matrix as a function of time; -% By weight of DOCA: (▽), 5% DOCA; (○), 10% DOCA; (△), 20% DOCA; (□), 30% DOCA

Claims (12)

A complex substance comprising heparin covalently linked to bile acids, sterols or mixtures thereof. The heparin, 2 00 to the composite material of claim 1 having a molecular weight of 100,000. The bile acid is cholic acid, deoxycholic acid, chenodeoxycholic acid, lithocholic acid, ursocholic acid, ursodeoxycholic acid, isoulsodeoxycholic acid, lagodeoxycholic acid, glycocholic acid, taurocholic acid, glycodeoxycholic acid, glycoside The composite substance according to claim 1, which is a bile acid selected from the group consisting of chenodeoxycholic acid, dehydrocholic acid, hyocholic acid, hyodeoxycholic acid and a mixture thereof. The composite substance according to claim 1, wherein the sterol is a sterol selected from the group consisting of cholestanol, coprostanol, cholesterol, epicholesterol, ergosterol, ergocalciferol, and a mixture thereof. The composite substance according to claim 1, wherein the heparin and the bile acid or sterol are present in a molar ratio of 1 : 1 to 1: 1,000. (A) a bile acid, a composite material of pharmaceutically effective amount of those selected from the group consisting of sterols comprising a covalently bound heparin, pharmaceutical compositions containing responsible body may be acceptable (b) a pharmaceutically. The pharmaceutical composition according to claim 6, wherein the pharmaceutically acceptable carrier is an oral drug carrier. 7. The pharmaceutical composition according to claim 6, wherein the pharmaceutically acceptable carrier is a sustained release carrier . 9. The pharmaceutical composition according to claim 8, wherein said sustained release carrier is a polymer matrix or a polymer coated film. The sustained release carrier is poly (ethylene oxide) - poly (.epsilon.-caprolactone) copolymer, polyurethane polymers, silicone polymers, ethylene vinyl acetate polymers, arsenate Dorogeru, collagen, gelatin and the polymer matrix or polymer selected from the group consisting of a mixture thereof The pharmaceutical composition according to claim 8, which is a coating film. The pharmaceutical composition according to any one of claims 6 to 10, as a coating material for a medical device. 12. The pharmaceutical composition according to claim 11, wherein the medical device is a catheter, a cardiopulmonary bypass circuit, a cardiopulmonary oxygenator, a renal dialysis machine, or a stent or balloon for restenosis prevention.

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