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Preparation of Slightly Hydrophobic Heparin Derivatives which Can Be Used for Solvent Casting in Polymeric Formulation
Thrombosis Research 92 (1998) 149–156
REGULAR ARTICLE
Preparation of Slightly Hydrophobic Heparin Derivatives which Can Be Used for Solvent Casting in Polymeric Formulation Yong-kyu Lee, Hyun Tae Moon and Youngro Byun Department of Materials Science and Engineering, Kwangju Institute of Science and Technology, Kwangju, Korea. (Received 11 May 1998 by Editor N. Sakuragawa; revised/accepted 1 July 1998)
Abstract Heparin is clinically administered mainly by intravenous injection because of its highly hydrophilic property. A slightly hydrophobic heparin derivative which can be dissolved in organic solvent can be widely used in polymeric devices for clinical applications. In this study, hydrophobic heparin derivatives were prepared by coupling heparin with deoxycholic acid, cholesterol, lauric acid, and palmitic acid, respectively. The hydrophobicity of these heparin derivatives depended on the feed mole ratio of heparin to hydrophobic agents, and they showed good solubility in the co-solvent of acetone and water, as well as in water alone. Also, these heparin derivatives showed high anticoagulant activity. This approach for preparing hydrophobic heparin is expected to advance the drug delivery system by further extending the applications of heparin to medical devices such as cardiopulmonary bypass circuits, heart lung oxygenators, and kidney dialyzers. 1998 Elsevier Science Ltd. Key Words: Hydrophobic heparin; Deoxycholic acid; Cholesterol; Alkanoic acid; Solvent casting Abbreviations: ATIII, antithrombin III; DOCA, deoxycholic acid; DCC, N,N9-dicyclohexylcarbodiimide; DMF, N,N-dimethylformamide; FXa, factor Xa; APTT, activated partial thromboplastin time; PCL, polycaprolactone; PEG, polyethylene glycol. Corresponding author: Youngro Byun, Department of Materials Science and Engineering, Kwangju Institute of Science and Technology, 572 Sangam-dong, Kwangsan-ku, Kwangju 506-712, Korea. Tel: 182(62)970 2312; Fax: 182(62)970 2304; E-mail: ,yrbyun@ kjist.ac.kr..
H
eparin is a potent anticoagulant agent that interacts strongly with antithrombin III (ATIII) to prevent the formation of fibrin clot [1,2]. Clinically, a small amount of heparin is injected repeatedly into the body since the main adverse effect of heparin is bleeding and the halflife of heparin is approximately 3 hours [3,4]. However, the controlled release of heparin can maintain the heparin concentration in plasma in the desired minimum therapeutic range for a longer period and reduce the side effects of heparin [5]. Several methods have been developed for the formulation of heparin-releasing system. One method utilizes ionically bound heparin, where anionic heparin is bound to a cationic polymer matrix. The release of heparin in this case is controlled by the ion-exchange mechanism [6–10]. Another method utilizes the dispersed-heparin device, where heparin is physically blended within a polymer and the release of heparin is controlled by diffusion [11–13]. The most efficient and simplest method for preparing a polymeric device enclosing drug is the solvent casting method [14]. However, the solvent casting method cannot be used for preparing polymeric devices enclosing heparin since heparin cannot be dissolved in organic solvents, which dissolve the polymer being used as a device. Heparin is dissolved in water or formamide only, but formamide is not a good solvent for the solvent casting method since formamide is highly viscous and nonvolatile. If heparin is derivatized to have hydrophobic property, thereby dissolving in volatile organic solvents, the hydrophobic heparin derivative can
0049-3848/98 $–see front matter 1998 Elsevier Science Ltd. Printed in the USA. All rights reserved. PII S0049-3848(98)00124-8
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be simply loaded into the polymer matrix by solvent casting. However, highly hydrophobic heparin is not dissolved in water and cannot be released out of the polymer matrix. Such heparin derivative can be used for the surface immobilization but not for the controlled release system. The best way for preparing a heparin-releasing device is therefore to prepare a slightly hydrophobic heparin, which can be dissolved in organic solvents as well as in water. Barzu et al. [15] prepared LMW heparin by periodate depolymerization of heparin and modified it by O-acylation. Although these derivatives had low anticoagulant activity, they showed a high antiproliferative activity for smooth muscle cells. Linhardt et al. [16] enhanced hydrophobicity by coupling lauryl(C12) and stearyl(C18) to a single heparin chain; however, they found that a small linear aliphatic chains (C8, C10, C12) were ineffective in enhancing the hydrophobicity of heparin. The objective of this work was to synthesize slightly hydrophobic heparin derivatives while maintaining high anticoagulant activity. We also attempted to show that the heparin formulation was possible by the solvent casting method.
1. Materials and Methods 1.1. Materials Heparin sodium (140 U/mg), whose average molecular weight was about 12000 daltons, was obtained from Pharmacia Hepar Co. (Franklin, OH). Phenylsepharose CL-4B was obtained from Pharmacia Biotech (Uppsala, Sweden). Deoxycholic acid (DOCA), cholesterol, and N,N9-dicyclohexylcarbodiimide (DCC) were obtained from Sigma Chemical Co. (St. Louis, MO). N,N9-dimethylformamide (DMF) was purchased from Merck (Darmstadt, Germany) and used without further purification. All other chemicals included palmitic acid and lauric acid, were purchased from Aldrich (Milwaukee, WI). Filtration equipments and membrane filters were purchased from Nihon Millipore LTD. (Yonezawa, Japan). The dialysis membrane (MWCO 3,500) was obtained from Spectrum Co. (Houston. TX). Factor Xa (FXa), ATIII, aPTT reagent, and substrate, S-2238, were purchased from Sigma Chemical Co. Human plasma was ob-
tained from the blood bank of Red Cross Center (Kwangju, Korea).
1.2. Preparation of the Heparin Derivatives DOCA (196 mg) was mixed with DCC (165 mg) and N-hydroxylsuccinimide (HOSu, 92 mg) in 15 ml of DMF. The feed mole ratio of DOCA, DCC, and HOSu was 1:1.6:1.6. The concentrations of DCC and HOSu were slightly higher than that of DOCA to activate DOCA completely. The mixture was reacted for 5 hours at room temperature under vacuum and then the precipitated dicyclohexylurea was removed. The unreacted DCC was removed by dropping of distilled water and then by filtering. The remaining HOSu was also removed by adding 15 ml of distilled water. The activated DOCA was precipitated and then lyophilized. The activated DOCA was then reacted with heparin in the co-solvent of DMF and water (1:1) for 4 hours at room temperature. The excess activated DOCA was removed by precipitating in water. After lyophilizing the heparin-DOCA solution, the heparin-DOCA conjugate was obtained as white powder. In the case of the heparin-cholesterol conjugate, most of experimental procedures were the same with those of heparin-DOCA conjugate except the carboxylation step. Cholesterol has only a hydroxyl group, and this hydroxyl group should be substituted by a carboxyl group to couple with the amine group of heparin. The hydroxyl group of cholesterol was substituted by the carboxyl group of chloroacetic acid. Lauric acid and palmitic acid were also coupled with heparin, respectively. The method of coupling was the same with that of the heparin-DOCA conjugates, that is, the carboxyl group of alkanoic acids was coupled with the amine groups of heparin, where the coupling agents were also HOSu and DCC.
1.3. Fractionation of the Heparin Derivatives A phenyl-sepharose CL-4B column was used for fractionations of heparin-DOCA, heparin-cholesterol, and heparin-alkanoic acid conjugates, respectively. The column (HR 16/30 I.D.) was washed with 100 ml of distilled water, and then equilibrated by washing with 40 ml of 50 mM phosphate buffer (pH 7.0) for 20 minutes. The column was washed by 40 ml of ammonium sulfate solution
Y.-K. Lee et al./Thrombosis Research 92 (1998) 149–156
(50 mM phosphate buffer (pH 7.0)11.7M ammonium sulfate), and then by 40 ml of 50 mM phosphate buffer. The solutions (1 mg/ml) of heparin or heparin derivatives in phosphate buffer were loaded on the column and eluted with the concentration gradient of ammonium sulfate, respectively. The flow rate was 1 ml/min, and each fraction (2 ml) was collected by a fraction collector. Finally, the column was washed with 100 ml of distilled water and 1.7 M ammonium sulphate to remove the heparin-DOCA conjugates remaining in the column. The fractionated samples were mixed with 1 ml of azure A solution (0.01 mg/ ml) for 1 minute and the absorbances of each solution were measured at 500 nm spectrophotometrically. The concentrations of heparin or hydrophobic heparin derivatives in the fractions were calculated from the absorbances.
1.4. Characterizations of the Heparin Derivatives The amounts of coupled hydrophobic agents in the heparin derivatives were determined by measuring their molecular weights by light scattering (series 4700; Malvern Instruments Ltd, Worcestershire, England), using the blue line (488 nm) of an argon ion laser. Four different concentrations of a heparin derivative such as 1.25, 2.5, 5, and 10 mg/ml in water or the co-solvent (methanol and water) were prepared. The scattered intensities were obtained at different scattering angles (40–1408) through a 500-mm aperture. Anticoagulant activities of the heparin derivatives were determined by FXa chromogenic assay and activated partial thromboplastin time (aPTT), respectively. In FXa chromogenic assay, all reactions were proceeded at 378C. Twenty five microliters of a heparin derivative (2.5 mg/ml) was diluted with 0.2 ml of ATIII (0.1 IU/ ml). This mixture was incubated for 2 minutes, after which 0.2 ml of FXa (4 nKat/ml) was added and incubated for an additional 1 minute. The substrate, S-2238 (0.2 ml, 0.8 mmol/ml), was then added and incubated for 5 minutes. The reaction was terminated by adding 0.2 ml of acetic acid (50% solution). The absorbance of the mixture was monitored spectrophotometrically at 405 nm using a Perkin-Elmer (CT, USA) Lambda-Vis 7 spectrophotometer. The bioactivities of the heparin derivatives were evaluated by comparing the absorbances with heparin.
151
Fig. 1. FT-IR spectrum of heparin (a) and heparin-lauric acid conjugate (b). (amide bond: 1585 cm21 and C5O double bond of DOCA: 1720 cm21).
APTT was determined using a Fibrometer with the following procedure. Twenty five microliter of heparin derivative (2.5 mg/ml) was added to 0.1 ml of citrated plasma, followed by adding 0.1 ml of aPTT reagent. This mixture was incubated at 378C for 2 minutes. The pre-warmed CaCl2 (0.1 ml, 0.02 M) was added and the time for fibrin clotting was recorded. The bioactivities of the heparin derivatives were evaluated by comparing the clotting time with heparin.
2. Results The prepared heparin derivatives were characterized by FT-IR and 13C-NMR to prove the coupling between heparin and hydrophobic agents. A clue of the coupling is the presence of an amide bond, which is produced by the coupling of the amine group of hepar in with the carboxyl group of a hydrophobic agent. In the FT-IR spectrum, the intensity of the band at 1585 cm21 showed the presence of amide bond between heparin and the hydrophobic agents. The peaks representing N-H groups in the heparin part of the heparin derivatives appeared around 3500 and 1620 cm21 (Figure 1). In the 13C-NMR spectra of the heparin-DOCA conjugate, there was a peak at 178 ppm by the carbon at the amide bond which was not detected in the 13C-NMR spectra of heparin (Figure 2). For heparin-DOCA, heparin-cholesterol, heparin-lauric acid, and heparin-palmitic acid conju-
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Fig. 2. The 300MHz 13C-NMR spectrum of (a) heparin and (b)heparin-DOCA conjugate. The D2O is used as a solvent. (amide bond: 178 ppm).
gates, Table 1 shows the production yields, molecular weights, and weight fractions of hydrophobic agents in the heparin derivatives according to the mole ratio of reactants. The production yield of heparin-DOCA conjugates was in the range of 71 to 77%. The molecular weight of heparin was determined as 12386 dalton 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. As the feed mole ratio of deoxycholic acid to heparin was increased from 1:6 to 1:200, the amount of DOCA in heparin-DOCA conjugate was also increased from 7 to 24%. In the heparin-cholesterol conjugates, the production yields were ranged from 73 to 78%. The amount of cholesterol in the hydrophobic heparin, however, was slightly lower than the amount of DOCA in the heparin-DOCA conjugates under the same experimental conditions. In heparin-lauric acid and heparin-palmitic acid conjugates, the production yield and amounts of alkanoic acids in the heparin derivatives were similar to those in the heparin-DOCA conjugate. Heparin can be dissolved in water or formamide only. But the heparin derivatives have a slightly hydrophobic property, thereby dissolving in the cosolvent of acetone and water (Table 2). In the case of the heparin-DOCA conjugates, as the amount of DOCA was increased, the solubility of the conjugate in the co-solvent of acetone and water was increased. In 14% DOCA, the heparin-DOCA conjugates were dissolved in the volume ratio of 50:50 acetone and water whereas the conjugates were not dissolved in the volume ratio of 70:30 acetone and water. On the other hand, when the amount of coupled DOCA was 24% in the heparin-DOCA conjugate, the solubility of the conjugate in the co-
Table 1. The change of molecular weights of the heparin derivatives, the amount of coupled hydrophobic agents, and the yield of the product according to the feed mole ratios of heparin to hydrophobic agents (molecular weight of heparin measured by light scattering is 12386 daltons)
Conjugate
Reaction mole ratio
Molecular weight
Amount of coupled hydrophobic agents
1:6 1:16 1:60 1:85 1:200 1:6 1:16 1:60 1:85 1:200 Heparin:Lauric acid (1:60)
13357 13706 14403 14759 16320 13151 13288 13791 14077 15500 13400
7.0% 9.6% 14.0% 16.0% 24.0% 5.8% 6.8% 10.0% 12.0% 20.0% 7.56%
77.0 74.8 71.8 72.9 73.0 77.0 78.0 74.0 72.9 73.0 76.0
Heparin:Palmitic acid (1:60)
13500
8.25%
77.0
Materials (feed mole ratio)
Heparin-DOCA
Heparin-cholesterol
Heparin-alkanoic acid [CH3(CH2)nCOOH, n510, 14]
Yield of product (%)
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Table 2. Solubilities of the heparin derivatives in the co-solvent of acetone and water 1 w/v% solution
Heparin
Heparin-DOCA (7%)
Heparin-DOCA (14%)
Heparin-DOCA (24%)
Water 50% water (50% acetone) 70% water (30% acetone) 100% acetone
S
S
S
S
I
S
S
S
I I
I I
I I
S I
S, soluble; I, insoluble.
solvent was maximized in the 50:50 volume ratio of acetone and water. Figure 3 shows the change of elution curve of the heparin-DOCA conjugates in the reversephase chromatography for different coupling ratios of heparin and DOCA. As the concentration of ammonium sulfate in the eluting solution was increased in FPLC, the hydrophobicity of the eluted heparin conjugate was also increased. Thus, the elution curves in Figure 3 shows the distributions of the heparin-DOCA conjugates according to their
hydrophobicity. Heparin was eluted with phosphate buffer only since heparin itself is very hydrophilic [17]. The heparin-DOCA conjugate was eluted not by phosphate buffer but by the ammonium sulfate solution. As the amount of DOCA was increased, a portion of the eluted conjugate was increased at a high concentration of ammonium sulfate as much as 1 M. Also, the heparinDOCA conjugate was completely eluted in the 1.3 M ammonium sulfate solution even if the content of DOCA was increased. The hydrophobic distribu-
Fig. 3. The FPLC of (a) unmodified heparin, (b) heparin-DOCA (7%), (c) heparin-DOCA(9.6%), (d) heparinDOCA(14%), and (e) heparin-DOCA (24%) DOCA.
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tions of different heparin derivatives were compared in Figure 4. Heparin-DOCA and heparin-cholesterol conjugates showed more broad hydrophobic distributions than the heparin-lauric acid and heparin-palmitic acid conjugates. All heparin conjugates, however, were completely eluted in the 1.3 M ammonium sulfate solution. The antithrombogenic activities of the heparin derivatives were measured by FXa chromogenic assay and aPTT, respectively (Figure 5). The bioactivities of all of the heparin derivatives prepared in this study were above 70% compared with the bioactivity of heparin by itself. There were no differences in the bioactivity attributable to different kinds of hydrophobic agent used, such as DOCA, cholesterol, lauric acid, or palmitic acid. But the bioactivity of the heparin derivatives was slightly decreased when the amount of the hydrophobic agent was increased. When 7wt% of DOCA was coupled with heparin, the relative bioactivity of the heparin-DOCA conjugate was 93% in aPTT and 80% in the FXa assay. On the other hand, when the coupled DOCA to heparin was 24wt%, the relative bioactivity of the heparin-DOCA con-
jugate was decreased as much as 71.5% in aPTT and 70.1% in the FXa assay.
3. Discussion Heparin derivatives with slightly hydrophobic property were developed in this study by coupling amine groups of heparin with carboxyl groups of the hydrophobic agents. Even if the anticoagulant activity of the heparin derivatives was slightly decreased when the amount of the coupled hydrophobic agents was increased, at least 70% of the anticoagulant activity was still maintained in the heparin derivatives. The reason for such a high anticoagulant activity of the heparin derivatives was that the amine groups of heparin involved in the coupling reaction had little effect on the anticoagulant activity of heparin [18,19]. However, the hydrophobic agents coupled with the amine group slightly reduced the bioactivity of the heparin derivatives by steric hindrance. If the hydrophobic agents were coupled near the active site of heparin, the steric effect on the bioactivity might have been more
Fig. 4. The FPLC of (a) heparinDOCA (9.6%), (b) heparin-cholesterol (10%), (c) heparin-lauric acid (7.26%), and (d) heparinpalmitic acid conjugate (8.25%), all of which include the similar amount of hydrophobic agents.
Y.-K. Lee et al./Thrombosis Research 92 (1998) 149–156
Fig. 5. Bioactivity of hydrophobic heparin by APTT and chromogenic assay. The closed symbols represent the values measured by APTT (j: DOCA, d: cholesterol, .: palmitic acid, m: lauric acid), and the open symbols represent the chromogenic assay values (h: DOCA, s: cholesterol, n: lauric acid, ,: palmitic acid).
significant, thereby reducing the bioactivity of the heparin derivative as much as 30%. For the heparin-DOCA conjugates, the amount of DOCA in the conjugate was 24% when the feed ratio of heparin to DOCA was 1:200. This molar ratio was very high compared to the ratio of amine groups in heparin to DOCA. Therefore, this feed ratio could be estimated as an excess condition, that is, the amine groups of heparin might have been saturated by DOCA. The heparin derivatives have a distribution of hydrophobicity since heparin has a broad molecular weight distribution. Also, the amount of coupled hydrophobic agents to heparin might be different in each heparin molecules. As the amount of hydrophobic agents coupled to heparin was increased, the distribution curve became more narrow and the peaks shifted to a more hydrophobic region. However, the peak did not shift above 1.3 M ammonium sulfate. This was the maximum hydrophobicity which the water-soluble heparin-DOCA conjugates could have. The maximum hydrophobicity of the heparin derivatives was not changed by the kind of hydrophobic agents used in this study. Under this maximum hydrophobic condition, the heparin derivatives were soluble in the co-solvent of acetone and water (70:30) as well as in water alone. Bae et al. synthesized the multi-block copolymers of polycaprolactone (PCL) and polyethylene
155
glycol (PEG), which have good solubility in the co-solvent of acetone and water (data are not published). Therefore, both the heparin derivatives and the PCL-PEG multi-block copolymer could be dissolved in the same solvent. If the heparin derivatives are used with the PCL-PEG multiblock copolymer, the heparin derivatives can be loaded into a polymer matrix by a simple casting method and the controlled release device for heparin could be developed. In conclusion, the heparin derivatives prepared in this study have high anticoagulant activity and good solubility for the co-solvent (acetone and water). And these results indicate that the heparin derivatives can be used in device formulations by a solvent casting method and to be widely used in medical applications. In future works, the controlled release device for heparin will be developed by using the heparin derivatives and PCL-PEG multi-block copolymer.
References 1. Damus PS, Hicks M, Rosenberg RB. Anticoagulant action of heparin. Nature 1977;246:355. 2. Casu B. Structure and biological activity of heparin. Adv Carbohydr Chem Biochem 1985; 43:51–134. 3. Mcavoy TJ. The biologic half-life of heparin. Clin Pharmacol Ther 1979;25:372–9. 4. Perry PJ, Herron GR, King JC. Heparin halflife in normal and impaired renal function. Clin Pharmacol Ther 1974;16:514–9. 5. Ranade VV. Drug delivery systems 5A. Oral drug delivery. J Clin Pharmacol 1991;31:2–16. 6. Gott VL, Shiffen JD, Dutton RS. Heparin bonding on colloidal graphite surfaces. Science 1985;142:1297–8. 7. Leininger L, Cooper CW, Falb RD, Grode GA. Nonthrombogenic plastic surfaces. Science 1966;152:1625–6. 8. Grode GA, Falb RD, Crowly C. Biocompatible materials for use in the vascular system. J Biomed Mater Res Symp 1972;3:77–84. 9. Eloy R, Belleville J, Paul J, Pusine´ri C, Baguet J, Rissoan M-C, Cathignol D, Ffrench P, Ville D, Tartullier M. Thromboresistance of bulk heparinized catheters in human. Thromb Res 1987;45:223–33. 10. Tanzawa H, Mori Y, Harumiya N, Miyama H,
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Hori M, Oshima N, Idezuki Y. Preparation and evaluation of a new antithrombo genic heparinized hydrophilic polymer for use in cardiovascular system. ASAIO J 1973;14:188–94. Hufnagel CA, Conrad PW, Gillespie JF, Pifarre R, Ilano A, Yokoyama T. Characteristics of materials for intravascular applications. Ann NY Acad Sci 1968;146:262–70. Holland EF, Gidden HE, Mason RG, Klein E. Thrombogenicity of heparin-bound DEAE cellulose hemodialysis membranes. ASAIO J 1978;1:24–36. Jacobs H, Okano T, Lin J, Kim SW. PEG1heparin conjugate releasing polymers. J Control Rel 1985;2:313–9. Okor RS, Anderson W. Casting solvent effects on the permeability of polymer films of differing quaternary ammonium(cation) content. J Pharmacol 1987;39:547–8. Barzu T, Desmouliere A, Herbert JM, Level M, Herault JP, Petitou M, Lormeau J.-C, Gab-
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biani G, Pascal M. O-acylated heparin derivatives with low anticoagulant activity decrease proliferation and increase alpha-smooth muscle actin expression in cultured arterial smooth muscle cells. Eur J Pharmacol 1992;219:225–33. Liu I, Pervin A, Gallo CM, Desai UR, van Gorp CL, Linhardt RJ. New approaches for the preparation of hydrophobic heparin derivatives. J Pham Sci 1994;83:1034–9. Nagasawa K, Uchiyama H, Yajima D. Anticoagulant effect of low molecular weight fractions derived from a chemically modified heparin. Thromb Res 1991;64:521–5. Linhardt RJ. Carbohydrates. In: Ogura H, Hasegawa A, Suami T, editors. Carbohydrates: Synthetic Methods and Applications in Medicinal Chemistry. Tokyo: Kodansha/VCH Publishers; 1992. p. 387–403. Ebert CD, Kim SW. Immobilized heparin: spacer arm effects on biological interactions. Thromb Res 1982;26:43–57.
REGULAR ARTICLE
Preparation of Slightly Hydrophobic Heparin Derivatives which Can Be Used for Solvent Casting in Polymeric Formulation Yong-kyu Lee, Hyun Tae Moon and Youngro Byun Department of Materials Science and Engineering, Kwangju Institute of Science and Technology, Kwangju, Korea. (Received 11 May 1998 by Editor N. Sakuragawa; revised/accepted 1 July 1998)
Abstract Heparin is clinically administered mainly by intravenous injection because of its highly hydrophilic property. A slightly hydrophobic heparin derivative which can be dissolved in organic solvent can be widely used in polymeric devices for clinical applications. In this study, hydrophobic heparin derivatives were prepared by coupling heparin with deoxycholic acid, cholesterol, lauric acid, and palmitic acid, respectively. The hydrophobicity of these heparin derivatives depended on the feed mole ratio of heparin to hydrophobic agents, and they showed good solubility in the co-solvent of acetone and water, as well as in water alone. Also, these heparin derivatives showed high anticoagulant activity. This approach for preparing hydrophobic heparin is expected to advance the drug delivery system by further extending the applications of heparin to medical devices such as cardiopulmonary bypass circuits, heart lung oxygenators, and kidney dialyzers. 1998 Elsevier Science Ltd. Key Words: Hydrophobic heparin; Deoxycholic acid; Cholesterol; Alkanoic acid; Solvent casting Abbreviations: ATIII, antithrombin III; DOCA, deoxycholic acid; DCC, N,N9-dicyclohexylcarbodiimide; DMF, N,N-dimethylformamide; FXa, factor Xa; APTT, activated partial thromboplastin time; PCL, polycaprolactone; PEG, polyethylene glycol. Corresponding author: Youngro Byun, Department of Materials Science and Engineering, Kwangju Institute of Science and Technology, 572 Sangam-dong, Kwangsan-ku, Kwangju 506-712, Korea. Tel: 182(62)970 2312; Fax: 182(62)970 2304; E-mail: ,yrbyun@ kjist.ac.kr..
H
eparin is a potent anticoagulant agent that interacts strongly with antithrombin III (ATIII) to prevent the formation of fibrin clot [1,2]. Clinically, a small amount of heparin is injected repeatedly into the body since the main adverse effect of heparin is bleeding and the halflife of heparin is approximately 3 hours [3,4]. However, the controlled release of heparin can maintain the heparin concentration in plasma in the desired minimum therapeutic range for a longer period and reduce the side effects of heparin [5]. Several methods have been developed for the formulation of heparin-releasing system. One method utilizes ionically bound heparin, where anionic heparin is bound to a cationic polymer matrix. The release of heparin in this case is controlled by the ion-exchange mechanism [6–10]. Another method utilizes the dispersed-heparin device, where heparin is physically blended within a polymer and the release of heparin is controlled by diffusion [11–13]. The most efficient and simplest method for preparing a polymeric device enclosing drug is the solvent casting method [14]. However, the solvent casting method cannot be used for preparing polymeric devices enclosing heparin since heparin cannot be dissolved in organic solvents, which dissolve the polymer being used as a device. Heparin is dissolved in water or formamide only, but formamide is not a good solvent for the solvent casting method since formamide is highly viscous and nonvolatile. If heparin is derivatized to have hydrophobic property, thereby dissolving in volatile organic solvents, the hydrophobic heparin derivative can
0049-3848/98 $–see front matter 1998 Elsevier Science Ltd. Printed in the USA. All rights reserved. PII S0049-3848(98)00124-8
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be simply loaded into the polymer matrix by solvent casting. However, highly hydrophobic heparin is not dissolved in water and cannot be released out of the polymer matrix. Such heparin derivative can be used for the surface immobilization but not for the controlled release system. The best way for preparing a heparin-releasing device is therefore to prepare a slightly hydrophobic heparin, which can be dissolved in organic solvents as well as in water. Barzu et al. [15] prepared LMW heparin by periodate depolymerization of heparin and modified it by O-acylation. Although these derivatives had low anticoagulant activity, they showed a high antiproliferative activity for smooth muscle cells. Linhardt et al. [16] enhanced hydrophobicity by coupling lauryl(C12) and stearyl(C18) to a single heparin chain; however, they found that a small linear aliphatic chains (C8, C10, C12) were ineffective in enhancing the hydrophobicity of heparin. The objective of this work was to synthesize slightly hydrophobic heparin derivatives while maintaining high anticoagulant activity. We also attempted to show that the heparin formulation was possible by the solvent casting method.
1. Materials and Methods 1.1. Materials Heparin sodium (140 U/mg), whose average molecular weight was about 12000 daltons, was obtained from Pharmacia Hepar Co. (Franklin, OH). Phenylsepharose CL-4B was obtained from Pharmacia Biotech (Uppsala, Sweden). Deoxycholic acid (DOCA), cholesterol, and N,N9-dicyclohexylcarbodiimide (DCC) were obtained from Sigma Chemical Co. (St. Louis, MO). N,N9-dimethylformamide (DMF) was purchased from Merck (Darmstadt, Germany) and used without further purification. All other chemicals included palmitic acid and lauric acid, were purchased from Aldrich (Milwaukee, WI). Filtration equipments and membrane filters were purchased from Nihon Millipore LTD. (Yonezawa, Japan). The dialysis membrane (MWCO 3,500) was obtained from Spectrum Co. (Houston. TX). Factor Xa (FXa), ATIII, aPTT reagent, and substrate, S-2238, were purchased from Sigma Chemical Co. Human plasma was ob-
tained from the blood bank of Red Cross Center (Kwangju, Korea).
1.2. Preparation of the Heparin Derivatives DOCA (196 mg) was mixed with DCC (165 mg) and N-hydroxylsuccinimide (HOSu, 92 mg) in 15 ml of DMF. The feed mole ratio of DOCA, DCC, and HOSu was 1:1.6:1.6. The concentrations of DCC and HOSu were slightly higher than that of DOCA to activate DOCA completely. The mixture was reacted for 5 hours at room temperature under vacuum and then the precipitated dicyclohexylurea was removed. The unreacted DCC was removed by dropping of distilled water and then by filtering. The remaining HOSu was also removed by adding 15 ml of distilled water. The activated DOCA was precipitated and then lyophilized. The activated DOCA was then reacted with heparin in the co-solvent of DMF and water (1:1) for 4 hours at room temperature. The excess activated DOCA was removed by precipitating in water. After lyophilizing the heparin-DOCA solution, the heparin-DOCA conjugate was obtained as white powder. In the case of the heparin-cholesterol conjugate, most of experimental procedures were the same with those of heparin-DOCA conjugate except the carboxylation step. Cholesterol has only a hydroxyl group, and this hydroxyl group should be substituted by a carboxyl group to couple with the amine group of heparin. The hydroxyl group of cholesterol was substituted by the carboxyl group of chloroacetic acid. Lauric acid and palmitic acid were also coupled with heparin, respectively. The method of coupling was the same with that of the heparin-DOCA conjugates, that is, the carboxyl group of alkanoic acids was coupled with the amine groups of heparin, where the coupling agents were also HOSu and DCC.
1.3. Fractionation of the Heparin Derivatives A phenyl-sepharose CL-4B column was used for fractionations of heparin-DOCA, heparin-cholesterol, and heparin-alkanoic acid conjugates, respectively. The column (HR 16/30 I.D.) was washed with 100 ml of distilled water, and then equilibrated by washing with 40 ml of 50 mM phosphate buffer (pH 7.0) for 20 minutes. The column was washed by 40 ml of ammonium sulfate solution
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(50 mM phosphate buffer (pH 7.0)11.7M ammonium sulfate), and then by 40 ml of 50 mM phosphate buffer. The solutions (1 mg/ml) of heparin or heparin derivatives in phosphate buffer were loaded on the column and eluted with the concentration gradient of ammonium sulfate, respectively. The flow rate was 1 ml/min, and each fraction (2 ml) was collected by a fraction collector. Finally, the column was washed with 100 ml of distilled water and 1.7 M ammonium sulphate to remove the heparin-DOCA conjugates remaining in the column. The fractionated samples were mixed with 1 ml of azure A solution (0.01 mg/ ml) for 1 minute and the absorbances of each solution were measured at 500 nm spectrophotometrically. The concentrations of heparin or hydrophobic heparin derivatives in the fractions were calculated from the absorbances.
1.4. Characterizations of the Heparin Derivatives The amounts of coupled hydrophobic agents in the heparin derivatives were determined by measuring their molecular weights by light scattering (series 4700; Malvern Instruments Ltd, Worcestershire, England), using the blue line (488 nm) of an argon ion laser. Four different concentrations of a heparin derivative such as 1.25, 2.5, 5, and 10 mg/ml in water or the co-solvent (methanol and water) were prepared. The scattered intensities were obtained at different scattering angles (40–1408) through a 500-mm aperture. Anticoagulant activities of the heparin derivatives were determined by FXa chromogenic assay and activated partial thromboplastin time (aPTT), respectively. In FXa chromogenic assay, all reactions were proceeded at 378C. Twenty five microliters of a heparin derivative (2.5 mg/ml) was diluted with 0.2 ml of ATIII (0.1 IU/ ml). This mixture was incubated for 2 minutes, after which 0.2 ml of FXa (4 nKat/ml) was added and incubated for an additional 1 minute. The substrate, S-2238 (0.2 ml, 0.8 mmol/ml), was then added and incubated for 5 minutes. The reaction was terminated by adding 0.2 ml of acetic acid (50% solution). The absorbance of the mixture was monitored spectrophotometrically at 405 nm using a Perkin-Elmer (CT, USA) Lambda-Vis 7 spectrophotometer. The bioactivities of the heparin derivatives were evaluated by comparing the absorbances with heparin.
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Fig. 1. FT-IR spectrum of heparin (a) and heparin-lauric acid conjugate (b). (amide bond: 1585 cm21 and C5O double bond of DOCA: 1720 cm21).
APTT was determined using a Fibrometer with the following procedure. Twenty five microliter of heparin derivative (2.5 mg/ml) was added to 0.1 ml of citrated plasma, followed by adding 0.1 ml of aPTT reagent. This mixture was incubated at 378C for 2 minutes. The pre-warmed CaCl2 (0.1 ml, 0.02 M) was added and the time for fibrin clotting was recorded. The bioactivities of the heparin derivatives were evaluated by comparing the clotting time with heparin.
2. Results The prepared heparin derivatives were characterized by FT-IR and 13C-NMR to prove the coupling between heparin and hydrophobic agents. A clue of the coupling is the presence of an amide bond, which is produced by the coupling of the amine group of hepar in with the carboxyl group of a hydrophobic agent. In the FT-IR spectrum, the intensity of the band at 1585 cm21 showed the presence of amide bond between heparin and the hydrophobic agents. The peaks representing N-H groups in the heparin part of the heparin derivatives appeared around 3500 and 1620 cm21 (Figure 1). In the 13C-NMR spectra of the heparin-DOCA conjugate, there was a peak at 178 ppm by the carbon at the amide bond which was not detected in the 13C-NMR spectra of heparin (Figure 2). For heparin-DOCA, heparin-cholesterol, heparin-lauric acid, and heparin-palmitic acid conju-
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Fig. 2. The 300MHz 13C-NMR spectrum of (a) heparin and (b)heparin-DOCA conjugate. The D2O is used as a solvent. (amide bond: 178 ppm).
gates, Table 1 shows the production yields, molecular weights, and weight fractions of hydrophobic agents in the heparin derivatives according to the mole ratio of reactants. The production yield of heparin-DOCA conjugates was in the range of 71 to 77%. The molecular weight of heparin was determined as 12386 dalton 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. As the feed mole ratio of deoxycholic acid to heparin was increased from 1:6 to 1:200, the amount of DOCA in heparin-DOCA conjugate was also increased from 7 to 24%. In the heparin-cholesterol conjugates, the production yields were ranged from 73 to 78%. The amount of cholesterol in the hydrophobic heparin, however, was slightly lower than the amount of DOCA in the heparin-DOCA conjugates under the same experimental conditions. In heparin-lauric acid and heparin-palmitic acid conjugates, the production yield and amounts of alkanoic acids in the heparin derivatives were similar to those in the heparin-DOCA conjugate. Heparin can be dissolved in water or formamide only. But the heparin derivatives have a slightly hydrophobic property, thereby dissolving in the cosolvent of acetone and water (Table 2). In the case of the heparin-DOCA conjugates, as the amount of DOCA was increased, the solubility of the conjugate in the co-solvent of acetone and water was increased. In 14% DOCA, the heparin-DOCA conjugates were dissolved in the volume ratio of 50:50 acetone and water whereas the conjugates were not dissolved in the volume ratio of 70:30 acetone and water. On the other hand, when the amount of coupled DOCA was 24% in the heparin-DOCA conjugate, the solubility of the conjugate in the co-
Table 1. The change of molecular weights of the heparin derivatives, the amount of coupled hydrophobic agents, and the yield of the product according to the feed mole ratios of heparin to hydrophobic agents (molecular weight of heparin measured by light scattering is 12386 daltons)
Conjugate
Reaction mole ratio
Molecular weight
Amount of coupled hydrophobic agents
1:6 1:16 1:60 1:85 1:200 1:6 1:16 1:60 1:85 1:200 Heparin:Lauric acid (1:60)
13357 13706 14403 14759 16320 13151 13288 13791 14077 15500 13400
7.0% 9.6% 14.0% 16.0% 24.0% 5.8% 6.8% 10.0% 12.0% 20.0% 7.56%
77.0 74.8 71.8 72.9 73.0 77.0 78.0 74.0 72.9 73.0 76.0
Heparin:Palmitic acid (1:60)
13500
8.25%
77.0
Materials (feed mole ratio)
Heparin-DOCA
Heparin-cholesterol
Heparin-alkanoic acid [CH3(CH2)nCOOH, n510, 14]
Yield of product (%)
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Table 2. Solubilities of the heparin derivatives in the co-solvent of acetone and water 1 w/v% solution
Heparin
Heparin-DOCA (7%)
Heparin-DOCA (14%)
Heparin-DOCA (24%)
Water 50% water (50% acetone) 70% water (30% acetone) 100% acetone
S
S
S
S
I
S
S
S
I I
I I
I I
S I
S, soluble; I, insoluble.
solvent was maximized in the 50:50 volume ratio of acetone and water. Figure 3 shows the change of elution curve of the heparin-DOCA conjugates in the reversephase chromatography for different coupling ratios of heparin and DOCA. As the concentration of ammonium sulfate in the eluting solution was increased in FPLC, the hydrophobicity of the eluted heparin conjugate was also increased. Thus, the elution curves in Figure 3 shows the distributions of the heparin-DOCA conjugates according to their
hydrophobicity. Heparin was eluted with phosphate buffer only since heparin itself is very hydrophilic [17]. The heparin-DOCA conjugate was eluted not by phosphate buffer but by the ammonium sulfate solution. As the amount of DOCA was increased, a portion of the eluted conjugate was increased at a high concentration of ammonium sulfate as much as 1 M. Also, the heparinDOCA conjugate was completely eluted in the 1.3 M ammonium sulfate solution even if the content of DOCA was increased. The hydrophobic distribu-
Fig. 3. The FPLC of (a) unmodified heparin, (b) heparin-DOCA (7%), (c) heparin-DOCA(9.6%), (d) heparinDOCA(14%), and (e) heparin-DOCA (24%) DOCA.
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tions of different heparin derivatives were compared in Figure 4. Heparin-DOCA and heparin-cholesterol conjugates showed more broad hydrophobic distributions than the heparin-lauric acid and heparin-palmitic acid conjugates. All heparin conjugates, however, were completely eluted in the 1.3 M ammonium sulfate solution. The antithrombogenic activities of the heparin derivatives were measured by FXa chromogenic assay and aPTT, respectively (Figure 5). The bioactivities of all of the heparin derivatives prepared in this study were above 70% compared with the bioactivity of heparin by itself. There were no differences in the bioactivity attributable to different kinds of hydrophobic agent used, such as DOCA, cholesterol, lauric acid, or palmitic acid. But the bioactivity of the heparin derivatives was slightly decreased when the amount of the hydrophobic agent was increased. When 7wt% of DOCA was coupled with heparin, the relative bioactivity of the heparin-DOCA conjugate was 93% in aPTT and 80% in the FXa assay. On the other hand, when the coupled DOCA to heparin was 24wt%, the relative bioactivity of the heparin-DOCA con-
jugate was decreased as much as 71.5% in aPTT and 70.1% in the FXa assay.
3. Discussion Heparin derivatives with slightly hydrophobic property were developed in this study by coupling amine groups of heparin with carboxyl groups of the hydrophobic agents. Even if the anticoagulant activity of the heparin derivatives was slightly decreased when the amount of the coupled hydrophobic agents was increased, at least 70% of the anticoagulant activity was still maintained in the heparin derivatives. The reason for such a high anticoagulant activity of the heparin derivatives was that the amine groups of heparin involved in the coupling reaction had little effect on the anticoagulant activity of heparin [18,19]. However, the hydrophobic agents coupled with the amine group slightly reduced the bioactivity of the heparin derivatives by steric hindrance. If the hydrophobic agents were coupled near the active site of heparin, the steric effect on the bioactivity might have been more
Fig. 4. The FPLC of (a) heparinDOCA (9.6%), (b) heparin-cholesterol (10%), (c) heparin-lauric acid (7.26%), and (d) heparinpalmitic acid conjugate (8.25%), all of which include the similar amount of hydrophobic agents.
Y.-K. Lee et al./Thrombosis Research 92 (1998) 149–156
Fig. 5. Bioactivity of hydrophobic heparin by APTT and chromogenic assay. The closed symbols represent the values measured by APTT (j: DOCA, d: cholesterol, .: palmitic acid, m: lauric acid), and the open symbols represent the chromogenic assay values (h: DOCA, s: cholesterol, n: lauric acid, ,: palmitic acid).
significant, thereby reducing the bioactivity of the heparin derivative as much as 30%. For the heparin-DOCA conjugates, the amount of DOCA in the conjugate was 24% when the feed ratio of heparin to DOCA was 1:200. This molar ratio was very high compared to the ratio of amine groups in heparin to DOCA. Therefore, this feed ratio could be estimated as an excess condition, that is, the amine groups of heparin might have been saturated by DOCA. The heparin derivatives have a distribution of hydrophobicity since heparin has a broad molecular weight distribution. Also, the amount of coupled hydrophobic agents to heparin might be different in each heparin molecules. As the amount of hydrophobic agents coupled to heparin was increased, the distribution curve became more narrow and the peaks shifted to a more hydrophobic region. However, the peak did not shift above 1.3 M ammonium sulfate. This was the maximum hydrophobicity which the water-soluble heparin-DOCA conjugates could have. The maximum hydrophobicity of the heparin derivatives was not changed by the kind of hydrophobic agents used in this study. Under this maximum hydrophobic condition, the heparin derivatives were soluble in the co-solvent of acetone and water (70:30) as well as in water alone. Bae et al. synthesized the multi-block copolymers of polycaprolactone (PCL) and polyethylene
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glycol (PEG), which have good solubility in the co-solvent of acetone and water (data are not published). Therefore, both the heparin derivatives and the PCL-PEG multi-block copolymer could be dissolved in the same solvent. If the heparin derivatives are used with the PCL-PEG multiblock copolymer, the heparin derivatives can be loaded into a polymer matrix by a simple casting method and the controlled release device for heparin could be developed. In conclusion, the heparin derivatives prepared in this study have high anticoagulant activity and good solubility for the co-solvent (acetone and water). And these results indicate that the heparin derivatives can be used in device formulations by a solvent casting method and to be widely used in medical applications. In future works, the controlled release device for heparin will be developed by using the heparin derivatives and PCL-PEG multi-block copolymer.
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