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Bioconjugate Chem. 2008, 19, 1346–1351
ARTICLES Preparation of Sodium Deoxycholate (DOC) Conjugated Heparin Derivatives for Inhibition of Angiogenesis and Cancer Cell Growth Kwang Jae Cho,†,‡ Hyun Tae Moon,†,§ Go-eun Park,‡ Ok Chul Jeon,§ Youngro Byun,*,| and Yong-kyu Lee*,⊥ Department of Otolaryngology, Head and Neck Surgery, The Catholic University of Korea, College of Medicine Uijeongbu, St. Mary’s Hospital, Kyunggi-Do 480-717, Korea, Mediplex Corporation, Seoul 135-729, Korea, College of Pharmacy, Seoul National University, San 56-1, Sillim-dong, Gwanak-gu, Seoul 151-742, Korea, and Department of Chemical and Biological Engineering, Chungju National University, Chungbuk 380-702, Korea. Received April 28, 2008; Revised Manuscript Received May 19, 2008
We describe new DOC (sodium deoxycholate)-heparin nanoparticles for in ViVo tumor targeting and inhibition of angiogenesis based on chemical conjugation and the enhanced permeability and retention (EPR) effect. Heparin has been used as a potent anticoagulant agent for 70 years, and has recently been found to inhibit the activity of growth factors which stimulate the smooth muscle cells around tumor. From the results, DOC and heparin were conjugated by bonding carboxyl groups of heparin with amine groups of aminated sodium deoxycholate. Larger antitumor effects of the DOC-heparin VI (8.5 mol of DOC coupled with 1.0 mol heparin) were achieved in animal studies, compared to heparin alone. We confirmed that the conjugated heparin retained its ability to inhibit binding with angiogenic factor, showing a significant decrease in endothelial tubular formation. These results provide new insights into the nontoxic anticancer drug carrier as well as the design of multifunctional bioconjugates for targeted drug delivery.
INTRODUCTION Tumors produce a number of growth factors stimulating angiogenesis via affinity for receptors on endothelial cells. Low molecular weight heparin (LMWH) appears to have a greater inhibitory effect on angiogenesis than unfractioned heparin (1–4). It has been shown that LMWH, as a result of its smaller size and in contrast to unfractioned heparin, can reduce binding of growth factors to their receptors. Fragments of fewer than 18 saccharides reduce the activity of vascular endothelial growth factor (VEGF), and fragments of fewer than 10 saccharides inhibit the activity of basic fibroblast growth factor (bFGF) (5, 6). Small molecular heparins (smaller than 6000 Da) are more effective than unfractionated heparin (UFH) for inhibition of VEGF- and bFGF-mediated angiogenesis in ViVo (3, 4). Heparin has highly hydrophilic properties due to the negatively charged group such as sulfonyl, carboxyl, and hydroxyl within its structure (7–9). To treat tumor with heparin, we need new structure designs to allow the use of a reduced dose to achieve the same therapeutic response with a consequent decrease in systemic toxicity and side reactions. Recently, a heparin-based drug carrier has been developed to deliver an anticancer drug to tumor sites by the EPR effect (10, 11). In our previous study, a chemically modified unfractionated heparin * Correspondingauthors.E-mail:
[email protected]@cjnu.ac.kr. Tel.: +82-2-880-7866, Fax: +82-2-872-7864 (Y. Byun). Tel.: +8243-841-5224, Fax: +82-43-841-5220 (Y. Lee). † Dr. Cho and Dr. Moon are equal contributors to this article. ‡ The Catholic University of Korea. § Mediplex Corporation. | Seoul National University. ⊥ Chungju National University.
derivative for delivery of the anticancer doxorubicin was developed and proven a safe drug carrier without the risk of inducing hemorrhage and other side effects (11). Other studies have developed polysaccharides such as chitosan and curdlan as anticancer drug carriers with inhibition of tumor growth (12, 13). In this study, we propose a new anticancer drug conjugate system for the treatment of tumor and tumor vasculature by using DOC-heparin nanoparticles. To obtain the optimized anticancer effect of heparin, we designed DOC-heparin conjugates by modification of the C3-hydroxyl group of DOC with 4-nitrophenyl chloroformate (4-NPC) and ethylenediamine (EDA). Through the conjugation method, many DOC molecules were allowed to bind freely with one heparin structure. In addition, the conjugated heparin retained its ability to inhibit binding with angiogenic factors, which can induce proliferation of smooth muscle cells. The rationale for conjugating DOC to the polysaccharide heparin was to specifically target tumor and tumor vasculature and to circumvent adverse reactions from drug toxicity and bleeding by reducing the binding affinity with anticoagulant factors such as factor Xa.
MATERIALS AND METHODS Materials. Fraxiparin (101 IU/mg, heparin) of average molecular weight ca. 5000 Da was purchased from GlaxoSmithKline (Brentford, Middlesex, UK). Sodium deoxycholate (DOC), 4-nitrophenyl chloroformate, triethylamine, dicyclohexylcarbodiimide (DCC), hydroxysuccinimide (HOSu), 4-methylmorpholine, ethylene diamine, dimethyl sulfoxide (DMSO), and ethyl acetate were purchased from Sigma Chemical Co. (St. Louis, MO). Formamide was obtained from Merck (Darmstadt, Germany). Coatest Factor Xa assay kits were from Chromogenix
10.1021/bc800173m CCC: $40.75 2008 American Chemical Society Published on Web 06/28/2008
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Figure 1. Synthesis and schematic structure of DOC-heparin conjugate.
(Milano, Italy). All reagents were of analytical grade and were used without further purification. Preparation of DOC-Heparin Conjugates (Figure 1). For amination of sodium deoxycholate (DOC), DOC (0.93 mmol) in 5 mL DMSO was reacted with 4-nitrophenyl chloroformate (4-NPC, 4.65 mmol) and triethylamine (5.58 mmol) for 6 h at room temperature. After reaction, the precipitant was removed by 0.45 µm filter membrane. The filtrate was extracted with 25 mL ethyl acetate and 25 mL water. The crude product from aqueous solution was washed with ethyl acetate three times, and then DOC carbonate was obtained as a powder type after freeze-drying. To obtain aminated DOC, DOC carbonate was reacted with 4-methylmorpholine (1.42 mmol) and ethylenediamine (0.071 mmol) overnight at room temperature. The product was concentrated by rotary evaporation and then precipitated by adding acetonitrile. The precipitated product was dried under vacuum for 24 h. For preparation of the DOC-heparin conjugate, heparin (0.01 mmol) was dissolved in water and adjusted to pH 5.0 by adding 0.1 M HCl solution. The solution was mixed with EDAC (0.04 mmol), NHS (0.04 mmol), and aminated DOC (0.044 mmol). After 30 min, the mixture was dialyzed (MWCO: 2000) against water to remove unreacted NHS, EDAC, and aminated DOC. The final product, DOC-heparin, was obtained and stored at 4 °C after freeze-drying. The dried DOC-heparin conjugate was analyzed by 1H NMR and FT-IR (Bruker, Germany). Values for 1H NMR of heparin (D2O) were: δ 5.38 [H1 of glucosamine residue (A)], δ 5.04 [H1 of iduronic acid residue (I)], δ 4.84 [I-5], δ 4.36-4.23 [A-6], δ 4.12-4.40 [I-3], δ 4.08j [I-4], δ 4.02 [A-5], δ 3.78 [I-2], δ 3.71 [A-4], δ 3.65-3.69 [A-3], δ 3.24 [A-2]. Values for 1H NMR of aminated DOC (D2O) were: δ 1.2-1.9 [m, five and six rings of DOC, 1H], δ 2.1-2.3 [m, CH3 of DOC, 1H], δ 3.15[d, 12R-OH of DOC, 2H], δ 8.0 [H of CONH]. Values of 1H-NMR of DOC-heparin conjugates (D2O) were: δ 1.2-1.9 [m, five and six rings of DOC, 1H], δ
3.24-5.38 [A or I of heparin], δ 8.0-8.2 [H of CONH of DOC-heparin]. Ninhydrin Colorimetric Method. For the coupling ratio of DOC in the DOC-heparin conjugate, the ninhydrin colorimetric method was used as described previously (14). In brief, 80 µL of the ninhydrin solution was added to 300 µL aminated DOC and the test tube covered with a piece of paraffin film to avoid the loss of solvent due to evaporation. With gentle stirring, the solution was heated for 5 min at 100 °C. After cooling to room temperature in a cold water bath, the absorbance was recorded with a spectrophotometer at 570 nm in wavelength. With the standard curves of aminated DOC, the coupling ratios of DOC-heparin conjugates were determined by subtracting the OD values of remaining aminated DOC. Anticoagulant Activity of DOC-Heparin Conjugates. DOC-heparin conjugate (100 µL) was mixed with 100 µL of antithrombin III (ATIII) solution to make DOC-heparin conjugate-ATIII complexes, where ATIII concentration was in excess of the DOC-heparin conjugate concentration. The solution was incubated at 37 °C for 3 min, and 100 µL of FXa was added to the solution. The resulting solution was then incubated for an additional 30 s. The concentration of FXa was also in excess of the DOC-heparin conjugate concentration. The substrate (200 µL, 0.8 µmol/mL) was then added and incubated at 37 °C for 3 min. The reaction was terminated by adding 300 µL of 20% acetic acid. The bioactivity and the concentration of DOC-heparin conjugate were calculated from the absorbance at 405 nm. Endothelial Tubular Formation. Human endothelial cells were resuspended at 4 × 105 cells/mL phenol red-free RPMI containing glutamine (2 mM). Then, 100 µL growth factor-free Matrigel with or without SDF-1R (200 ng/mL) was plated into 96-well plates (Costar, Corning, NY) and incubated at 37 °C for 30 min for gelation. Thereafter, cells were seeded in gelated Matrigel in the presence of different stimuli. Plates were
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Table 1. Reaction Condition and Coupled Ratio of DOC in DOC-Heparin Conjugates
Fraxiparin (Ca2+,mol) EDAC (mol) HOSu (mol) Aminated DOC (mol) Coupling ratio
DOC-heparin I
DOC-heparin II
DOC-heparin III
DOC-heparin IV
DOC-heparin V
DOC-heparin VI
1 1.2 1.2 1.3 0.9
1 2.4 2.4 2.6 2.1
1 3.6 3.6 4 3.2
1 4.8 4.8 5.2 3.9
1 6 6 6.7 4.2
1 12 12 13.3 8.5
incubated for 24 h, and then tubular formation was analyzed after 6 h of incubation. For inhibition experiments, heparin or DOC-heparin conjugates were used at 10, 50, and 250 µg/mL and added together with the cells before seeding the cells on Matrigel. After 6 h of incubation, cell growth and threedimensional organization were observed through a reversephase-contrast photomicroscope (Olympus 1 × 71), and the results were expressed as the mean number of junctions/5 fields at × 100 original magnification. Tubular formation and inhibition of tubular formation experiments were performed in duplicate and repeated at least 3 times. Human Tumor Xenograft. Four- to six-week-old Athymic BALB/c-nu/nu female nude mice (14-18 g) were purchased from SLC Inc. (Japan) and maintained under specific pathogenfree conditions. All experiments were approved by the institutional guidelines of the Institutional Animal Care and Use Committee (IACUC) of the Catholic University of Korea College of medicine in accordance with the NIH Guidelines. Cultured KB cells (ATCC, Rockville, MD) were trypsinized, washed twice with serum-free EMEM medium (ATCC), and suspended at 1 × 107cells/mL PBS buffer. 50 µL of the suspended cells was subcutaneously injected into the back of the mice. On day 12-15 after tumor injection, the resulting tumors reached a volume of 45-55 mm3. According to body weight and tumor size, the animals were divided into four experimental groups of five mice each: groups A, B, C, and D, respectively, received through the tail vein injections of 100 µL of saline as control (Group A, n ) 5), heparin (10 mg/kg, Group B, n ) 5), DOC-heparin VI (5 mg/kg, Group C, n ) 5), and DOC-heparin VI (10 mg/kg, Group D, n ) 5). Each drug was administered twice a week for four weeks after tumor inoculation. Data are expressed as means ( SE. One-way
ANOVA was used to compare groups, where P values of C12-OH (15). We also found a sharp peak at δ 2.0 ppm, indicating the presence of a hydroxyl group at the C12-OH position. The amount of DOC conjugated to polysaccharide heparin estimated by the ninhydrin colorimetric method was maximized to 8.5 mol based on 1 mol of heparin (DOC-heparin VI) as shown in Table 1. By changing feed mole ratio of heparin, EDAC, HOSu, and aminated DOC, we controlled the coupling ratio between DOC and heparin. When dissolved in water, the DOC-heparin conjugates produced a clear solution at a concentration of 50 mg/mL. AFM and size measurement showed that the particles formed uniform spheres with a narrow size distribution as shown in Figure 2. The mean diameter of the nanoparticles (DOC-heparin IV) was 185 nm with a standard deviation of 2.8 nm, as determined by dynamic light scattering. A nanoparticle size of about 100-300 nm is considered to be suitable for passive targeted delivery utilizing the EPR effect. Several research groups using liposomes and other macromolecules have shown that particles with small size (less than 400 nm and ideally less than 200 nm) are more efficient in cell targeting than larger particles (16).
Figure 2. Size distribution and AMF image of DOC-heparin conjugate. (a) Size distribution of DOC-heparin conjugates by dynamic light scattering. (b) AFM image of DOC-heparin nanoparticles.
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Table 2. Bioactivity of DOC-Heparin Conjugates
sample
absolute bioactivity (IU/mg)
SD
Fraxiparine (Ca2+) DOC-heparin I DOC-heparin II DOC-heparin III DOC-heparin IV DOC-heparin V DOC-heparin VI
101 IU/mg 106.7 IU/mg 109.8 IU/mg 92.6 IU/mg 86.5 IU/mg 86.0 IU/mg 75.1 IU/mg
1.5 2.1 2.0 5.7 2.7 1.0
Biological Activity. Antifactor Xa activity of DOC-heparin conjugates measured by chromogenic assay decreased with the increase of coupled DOC in DOC-heparin as shown in Table 2. When the carboxyl group of heparin is modified by conjugation, its affinity to factor Xa and/or antithrombin and thus its anticoagulant activity diminishes (17–19). The conjugated
heparin presents several advantages as an anticancer drug carrier: (i) More DOC-heparin conjugates can access cancer cells, bypassing the coagulation cascade. (ii) Conjugation to DOC in DOC-heparin reduces the amount of negative charges of heparin, decreasing side effects such as heparin-induced thrombocytopenia (HIT) or bleeding that arise from the charge and size of heparin (20, 21). (iii) Through the intact sulfate group, the conjugated heparin retains its ability to inhibit binding with angiogenic factors, which can induce proliferation of smooth muscle cells. It is widely known that a high degree of sulfation and optimum saccharide chain length are essential for recognition of angiogenic growth factors such as bFGF and VEGF (22–27). All of the advantages suit our purpose to develop a biocompatible novel drug carrier that acts as an efficient antitumor agent, and is free of undesired interactions with blood and vessel components. DOC-Heparin Conjugates Inhibit Tumor Vasculature
Figure 3. Inhibition of tubular formation by heparin and DOC-heparin conjugates at different concentrations.
Figure 4. Antitumor effects of DOC-heparin conjugates. (a) Shrinkage in tumor volume and (b) changes in body weight after treatment with saline (0), 10 mg/kg heparin (•), 5 mg/kg of DOC-heparin VI (1), and 10 mg/kg of DOC-heparin VI (∆), respectively. All data represent mean ( s.e.
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Growth. We sought to determine whether DOC-heparin conjugates would block angiogenesis that accompanies tumor growth. After heparin or DOC-heparin conjugates were added on Matrigel that included human endothelial cells, we observed the endothelial tubular formation and counted the number of tubes formed by a reverse-phase contrast photomicroscope as shown in Figure 3. When treated with heparin at 10, 50, and 250 µg/mL, no inhibitory effect of tubular formation was observed. On the other hand, DOC-heparin VI showed significant decrease in tubular formation at 50 µg/mL. For endothelial cells treated with 250 µg/mL of DOC-heparin VI, tubular formation was completely removed. From the results, we believe that blood vessels surrounding the tumor treated with DOC-heparin VI have almost disappeared whereas a number of capillaries near the tumor excised from nude mice treated with saline remained intact (data not in shown). A significant decrease of angiogenesis was seen in in Vitro treatment with DOC-heparin VI, indicating that DOCheparin inhibits tumor vasculature growth in mice. DOC-Heparin Conjugate Showed Remarkable Tumor Growth Inhibition. To determine whether heparin or DOCheparin conjugates can reduce tumor growth rate, athymic BALB/c-nu/nu female nude mice bearing KB tumors (human epidermoid carcinoma cells) were used. Tumor growth and body weight were monitored after treatment. The antitumor effect of DOC-heparin conjugate against KB tumor cells was remarkably better than that of heparin. The tumor volume of mice treated with injection of DOC-heparin VI was about 550 mm3, indicating shrinkage in initial tumor volume and inhibition of tumor growth by 37% (p < 0.01) compared to the control group (saline injection) as shown in Figure 4. The DOC-heparin conjugates did not induce death due to toxicity or body weight loss during treatment. By itself, however, polysaccharide heparin is ineffective in inhibiting solid tumor growth. When intravenously injected at 10 mg/kg, most of the polysaccharide heparin will be systemically distributed into blood vessels, bind preferably with Factor Xa, and act as a highly potent anticoagulant, leaving only a few polysaccharide heparin molecules available to reach cancer cells. A high concentration of heparin or anticancer drug may circumvent this problem, but not without the accompanying side effects such as bleeding, thrombocytopenia, and general complications similar to those of chemotherapy. Therefore, the need arises for a strategy to facilitate specific interaction between polysaccharide heparin and tumor cells. The binding affinity to Factor Xa is markedly diminished when the carboxyl groups of heparin are modified to anchor DOC (Figure 1). The extent of antiangiogenesis in Vitro would be ranked as follows: DOC-heparin VI > DOC-heparin IV > heparin > saline. The reduction of tumor vasculature when treated with the DOC-heparin VI implies that heparin, in addition to its targeted carrier role, may strongly interact with angiogenic stimulators to inhibit tumor vasculature growth. From the slowly halted tumor growth at 21 days, we concluded that it was likely that most of the residual solid tumors perished after treating with DOC-heparin VI for 21 days. The treated tumor cells had irregular structure with nuclei exposed to the outside of the cell membrane, indicating necrosis and cell malfunction. In addition, this study introduces new prospects for the delivery of macromolecular drugs. As an antitumor agent, DOC-heparin conjugate can efficiently be delivered to solid tumors via intravenous injection. The conjugates were shown to produce certain therapeutic effects on human tumor implanted in BALB/c mice. It may become a new compound, but further studies still need to be conducted to explore in ViVo metabolic
Cho et al.
profiles. Also, this approach can be used as a method to prove certain interaction between angiogenesis and tumor growth.
CONCLUSION This study demonstrated that the DOC-heparin conjugate can specifically target tumor and tumor vasculature with minimized side effects. The main advantage of these novel biomolecular nanoparticles is the superior antitumor effect in ViVo. By design, the self-assembled nanoparticles accumulate in tumors by passive targeting mechanisms. The conjugate inhibited tumor growth in mice without decrease in body weight. Our findings in animal studies suggest that the DOC-heparin VI has high antitumor activity and selective toxicity, yielding 34% tumor inhibition compared with tumor volume treated heparin. The results are consistent with tumor angiogenesis inhibition, showing a less detectable tumor vasculature after DOC-heparin VI treatment.
ACKNOWLEDGMENT This work was supported by a grant of the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea. (A060699). This work was also in part supported by the BioImaging Research Center at GIST, and the Catholic Cancer Center, the Catholic University of Korea College of Medicine.
LITERATURE CITED (1) Hejna, M., Raderer, M., and Zielinski, C. C. (1999) Inhibition of metastases by anticoagulants. J. Natl. Cancer Inst. 91, 22– 26. (2) Smorenburg, S. N., and Van Noorden, C. J. F. (2001) The complex effects on heparins on cancer progression and metastasis in experimental studies. Pharmacol. ReV. 53, 93–105. (3) Norrby, K. (1993) Heparin and angiogenesis: a low-molecularweight fraction inhibits and a high-molecular-weight fraction stimulates angiogenesis systemically. Haemostasis 23, 141–149. (4) Norrby, K., and Ostergaard, P. (1996) Basic-fibroblast-growthfactor-mediated de novo angiogenesis is more effectively suppressed by low-molecular-weight than by high-molecular-weight heparin. Int. J. Microcirc. Clin. Exp. 16, 8–15. (5) Jayson, G., and Gallagher, J. T. (1997) Heparin oligosaccharides: inhibitors of the biological activity of bFGF on Caco-2 ells. Br. J. Cancer 75, 9–16. (6) Soker, S., Goldstaub, D., Svahn, C. M., Vlodavski, I., Levi, B. Z., and Neufeld, G. (1994) Variation in the size and sulfation of heparin modulate the effect of heparin on the binding of VEGF 165 to its receptors. Biochem. Biophys. Res. Commun. 203, 1339– 1347. (7) Linhardt, R. J. (2003) Heparin: structure and activity. J. Med. Chem. 46, 2551–2564. (8) Ishihara, M., and Ono, K. (1998) Structure and function of heparin and heparan sulfate: heparinoid library and modification of FGF-activities. Trends Glycosci. Glycotechnol. 10, 223–233. (9) Casu, B., and Lindahl, U. (2001) Structure and biological interactions of heparin and heparan sulfate. AdV. Carbohydr. Chem. Biochem. 57, 159–206. (10) Yu, M. K., Lee, D. Y., Kim, Y. S., Park, K., Park, S. A., Son, D. H., Lee, G. Y., Nam, J. H., Kim, S. Y., Kim, I. S., Park, R. W., and Byun, Y. (2007) Antiangiogenic and apoptotic properties of a novel amphiphilic folate-heparin-lithocholate derivative having cellular internality for cancer therapy. Pharm. Res. 24, 705–714. (11) Park, K., Lee, G. Y., Kim, Y. S., Yu, M., Park, R. W., Kim, I. S., Kim, S. Y., and Byun, Y. (2006) Heparin-deoxycholic acid chemical conjugate as an anticancer drug carrier and its antitumor activity. J. Controlled Release 114, 300–306. (12) Na, K., Park, K., Kim, S. W., and Bae, Y. H. (2000) Selfassembled hydrogel nanoparticles from Curdlan derivatives: characterization, anticancer drug release and interaction with a
DOC-Heparin Derivatives hepatoma cell line (HepG2). J. Controlled Release 69, 225–236. (13) Kwon, S., Park, J., Chung, H. H., Kwon, I. C., and Jeong, S. Y. (2003) Physicochemical characteristics of self-assembled nanoparticles based on glyco chitosan bearing 5¥aˆ-cholanic acid. Langmuir 19, 10188–10193. (14) Hwang, M., and Ederer, G. M. (1975) Rapid hippurate hydrolysis method for presumptive identification of group B streptococci. J. Clin. Microbiol. 1, 114–115. (15) von Geldern, T. W., Tu, N., Kym, P. R., Link, J. T., Jae, H. -S., Lai, C., Apelqvist, T., Rhonnstad, P., Hagberg, L., Koehler, K., Grynfarb, M., Goos-Nilsson, A., Sandberg, J., Osterlund, M., Barkhem, T., Hoglund, M., Wang, J., Fung, S., Wilcox, D., Nguyen, P., Jakob, C., Hutchins, C., Farnegardh, M., Kauppi, B., Ohman, L., and Jacobson, P. B. (2005) Liver-selective glucocorticoid antagonists: a novel treatment for type 2 diabetes. J. Med. Chem. 48, 2724–2724. (16) Kong, G., Braun, R. D., and Dewhirst, M. W. (2000) Hyperthermia enables tumor-specific nanoparticle delivery: Effect of particle size. Cancer Res. 60, 4440–4445. (17) Lee, Y., Nam, J. H., Shin, H. C., and Byun, Y. (2001) Conjugation of low molecular weight heparin and deoxycholic acid for development of new oral anticoagulant agent. Circulation 104, 3116–3120. (18) Lee, Y., Moon, H. T., and Byun, Y. (1998) Preparation of slightly hydrophobic heparin derivatives which can be used for solvent casting in polymeric formulation. Thromb. Res. 92, 149– 156. (19) Lee, Y., Kim, S. K., Lee, D. Y., Lee, S., Kim, C. Y., Shin, H. C., Moon, H. T., and Byun, Y. (2006) Efficacy of orally active chemical conjugate of low molecular weight heparin and
Bioconjugate Chem., Vol. 19, No. 7, 2008 1351 deoxycholic acid in rats, mice and monkeys. J. Controlled Release 111, 290–298. (20) Petitou, M., He´rault, J. P., Bernat, A., Driguez, P. A., Duchaussoy, P., Lormeau, J. C., and Herbert, J. M. (1999) Synthesis of thrombin-inhibiting heparin mimetics without side effects. Nature 398, 417–422. (21) Casu, B. (1985) Structure and biological activity of heparin. AdV. Carbohydr. Chem. Biochem. 43, 51–134. (22) Parish, C. R., Freeman, C., Brown, K. J., Francis, D. J., and Cowden, W. B. (1999) Identification of sulfated oligosaccharidebased inhibitors of tumor growth and metastasis using novel in vitro assays for angiogenesis and heparanase activity. Cancer Res. 59, 3433–3441. (23) Jayne, D. G., Perry, S. L., Morrison, E., Farmery, S. M., and Guillou, P. J. (2000) Activated mesothelial cells produce heparinbinding growth factors: implications for tumor metastases. Br. J. Cancer 82, 1233–1238. (24) Li, H. L., Ye, K. H., Zhang, H. W., Luo, Y. R., Ren, X. D., Xiong, A. H., and Situ, R. (2001) Effect of heparin on apoptosis in human nasopharyngeal carcinoma CNE2 cells. Cell. Res. 11, 311–315. (25) Meyer, T., and Hart, I. R. (1998) Mechanisms of tumour metastasis. Eur. Cancer 34, 214–221. (26) Zacharski, L. R., and Ornstein, D. R. (1998) Heparin and cancer. Thromb. Haemost. 80, 10–23. (27) Elias, E. G., Shukla, S. K., and Mink, I. B. (1975) Heparin and chemotherapy in the management of inoperable lung carcinoma. Cancer 36, 129–136. BC800173M
