Physicochemical Conjugation with Deoxycholic Acid and

May 31, 2011 - Department of Otolaryngology, Asan Medical Center, College of ... and Development Center, Mediplex Corp., Seoul 151-742, Republic of ...
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Physicochemical Conjugation with Deoxycholic Acid and Dimethylsulfoxide for Heparin Oral Delivery Sang Kyoon Kim,†,‡ June Huh,†,§ Sang Yoon Kim,|| Youngro Byun,‡ Dong Yun Lee,*,^,# and Hyun Tae Moon*,0 ‡

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WCU Department of Molecular Medicine and Biopharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea § Department of Materials Science and Engineering, Yonsei University, Seoul 120-749, Republic of Korea Department of Otolaryngology, Asan Medical Center, College of Medicine, University of Ulsan, Seoul 138-736, Republic of Korea ^ Department of Bioengineering, College of Engineering, and Institute for Bioengineering and Biopharmaceutical Research, Hanyang University, Seoul 133-791, Republic of Korea # Hanyang University Institute of Aging Society, Seoul 133-791, Republic of Korea 0 Research and Development Center, Mediplex Corp., Seoul 151-742, Republic of Korea

bS Supporting Information ABSTRACT: Heparin, as therapeutic medications, cannot be administered orally because of its hydrophilic and high molecular weight. Here, we present a new technology to enhance the absorption of heparin in the intestine through its chemical conjugation with deoxycholic acid (DOCA) that can interact with bile acid transporter in the intestine. For the ampiphilic property and complete dissolution, the modified heparin was physically complexed with dimethylsulfoxide (DMSO). The DOCA-conjugated heparin could form nanoparticles in aqueous solution, whereas it was completely dissolved when treated with above 10% DMSO solution. Molecular dynamics computation study and two-dimensional homonulcear 1H nuclear overhauser effect spectroscopy (NOESY) NMR spectra demonstrated that one heparin molecule was chemically conjugated with two DOCA molecules that were physically interacted with six DMSO molecules within 4 Å via hydrophobic interactions and partly via hydrogen bonding. Its therapeutic efficacy was also pharmaceutically analyzed. When the DMSO-bound DOCA-conjugated heparin was orally administered into mice, its therapeutic efficacy was enhanced according to the amount of bound DMSO. Also, after oral administration of fluorescence-labeled DMSO-bound DOCA-conjugated heparin, it was circulated in the whole body for above 2 h. However, the DOCA-conjugated heparin without DMSO binding was fast eliminated after oral absorption. This study demonstrates that the interaction of structural constraints, DOCA and DMSO, with heparin can serve as a platform technology for potential macromolecule oral delivery.

’ INTRODUCTION Macromolecules such as proteins, polysaccharides, and DNA are rapidly being developed as specific and potent therapeutic materials. In drug delivery systems, by far the most convenient and preferred route is the oral route. For macromolecule oral delivery, formulation and delivery as much as the development of the medication itself are significant hurdles to clinical applications. Heparin, one of macromolecules, is the most potent anticoagulant for deep vein thrombosis (DVT) and pulmonary embolism (PE) prevention. Venous thromboembolisms, i.e., DVT and PE, are diagnosed in hospitalized patients annually. Currently, 5 days of parenteral heparin injections followed by 3 months of oral warfarin therapy prevents PE in 95% of patients with proximal DVT.1 However, although heparin and warfarin are effective anticoagulants, oral warfarin therapy varies by individual patients because warfarin has a slow onset due to predominant protein binding and drugdrug interactions.2 However, heparin does not r 2011 American Chemical Society

have teratogenic effects and has rapid onset with a half-life of 7 Å, both curves collapsed similarly, since there was no preferred DMSO orientation when the DMSODOCA distance was large (Figure 3C). However, in the region of r < 7 Å, the two curves start to separate from each other: the peak in site D correlation curves (red line) emerged at r = 56 Å slightly shorter than at the peak position (r = 7 Å) in the site C correlation curve (blue line). Obviously, this result suggests that the methyl groups of DMSO preferably interacted with the nonpolar 4-ring of DOCA via hydrophobic interaction as the sulfoxide groups of DMSO interacted favorably with water molecules. The hydrophobic correlation curves at sites C and D were nearly similar to the DMSODOCA interaction in Figure 3A, meaning that the broad peak appearing at r = 48 Å in the DOCADMSO is largely attributed to the hydrophobic interaction between methyl group of DMSO and the hydrophobic 4-ring of DOCA. Therefore, the solubilization mechanism of DMSO-bound DOCA-conjugated heparin was due to the amphiphilicity of DMSO molecule that was bound to DOCA largely via hydrophobic interaction at the site D while the hydrophilic sulfoxide group shielded the DOCA from aggregation. With increased time, the number of the DMSO molecules bound to DOCA (red line) increased, but the amount to heparin (blue line) was negligible (Figure 3D). On the other hand, the number of water molecules bound to heparin (blue line) was highly saturated, whereas that to DOCA (red line) did not change

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Figure 4. Molecular dynamics simulation of dispersing DOCA-conjugated heparin by DMSO binding. In aqueous solution, the bound DMSO could dissociate the aggregation of two chains of DOCAconjugated heparin according to the different time scale. The simulation time is indicated in each snapshot.

(Figure 3E). These tendencies of DMSO and water molecules interacting with DOCA-conjugated heparin were visualized in Figure 3F, where DMSO and water molecules positioned within 4 Å from the heparin backbone were displayed. The heparin backbone was surrounded by water molecules, whereas DMSO molecules were bound predominantly to DOCA. In addition, the hydrogens of water molecules tend to face toward the heparin backbone, which supports the aforementioned notion that the heparinwater interaction was driven mainly by sulfate or carboxylate groups rather than hydrogen bonding between water oxygen and hydroxyl group. Figure 4 shows the DMSO effect on the dissociation of the aggregated DOCA-conjugated heparin according to a different time scale. At zero time, two heparin backbones were aggregated via hydrophobic interaction of DOCA molecules. At 2 ns after the presence of DMSO molecules in aqueous solution, 56 DMSO molecules were strongly interacting with DOCA molecules, thereby inducing dissociation of the aggregated heparins. Finally, we simulated the mobility of one or two chains of DMSO-bound DOCA-conjugated heparin in solution (Supporting Information Movie S1). NMR Spectrometry Study of DMSO-Bound DOCA-Conjugated Heparin. In our previous study, we indirectly showed that the physical binding of DMSO to DOCA-conjugated heparin could produce the shifted or stretched peaks in the FT-IR spectrum and thermal analysis such as differential scanning calorimetry and thermal gravimetry analysis, which was caused by the binding of DMSO to DOCA-conjugated heparin via secondary interactions.20 For the precise evaluation of DMSO binding to DOCA-conjugated heparin, we used 1H NMR and 2D-NOESY NMR spectrometry. In this experiment, to clearly detect the peak shift of 1H of heparin and DOCA in DMSO-d6 and D2O solution, original peaks of heparin and DOCA were observed in the regions 3.05.0 ppm and 0.52.5 ppm, respectively (Figure 5A and B, purple color). After binding with DMSO-d6, the peaks of heparin and DOCA were shifted (Figure 5A and B, green color). In the 2D-MOSEY NMR spectrum of heparin and DOCA, heparin had a lot of cross-peaks by inter- or intramolecular interactions, while the cross-peaks of DOCA were simple (Figure 5C and D, purple color). Chemical shifts of backbone and residues of heparin were altered by the binding of DMSO to heparin (Figure 5C, green). We also found that NMR peaks for DOCA were also shifted by DMSO binding (Figure 5D, green). These results indicated that both heparin and DOCA in DOCA-conjugated heparin were affected by the interaction with DMSO molecules. 1455

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Bioconjugate Chemistry

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Figure 5. Correction between DMSO and DOCA-conjugated heparin molecules by NMR spectrometry. (A) 1H NMR spectrum of heparin with (green color) or without (purple color) 10% DMSO-d6 binding. (B) 1H NMR spectrum of DOCA with (green color) or without (purple color) DMSO-d6 binding. (C) 2D-NOESY spectrum of heparin with (green) or without (purple) 10% DMSO-d6 binding. (D) 2D-NOESY spectrum of DOCA with (green) or without (purple) 10% DMSO-d6 binding.

In Vivo Oral Administration of DMSO-Bound DOCAConjugated Heparin. After the physical binding of DOCA-

conjugated heparin was completed in 2%, 5%, 10%, and 20% DMSO solution, 10 mg/kg DMSO-bound DOCA-conjugated heparin in water solution (200 μL/mouse) was orally administered to mice. Heparin activity in plasma increased according to the increased amount of bound DMSO contents, as indicated by the maximum concentration (Cmax) values of heparin in plasma as well as the AUC (area under curve). For 0%, 3.3%, 11.9%, 20.5%, and 25.4% bound DMSO contents, the Cmax values were 0.34 ( 0.04, 0.33 ( 0.04, 0.34 ( 0.03, 0.48 ( 0.03, and 0.54 ( 0.10 IU/mL, respectively (Figure 6A). The AUC were 12.5 ( 0.9, 21.8 ( 2.3, 21.2 ( 1.8, 30.9 ( 1.6, and 31.5 ( 1.9 IU/mL/min, respectively (Figure 6B). To visualize the distribution of DMSO-bound DOCA-conjugated heparin in the body, optical imaging was carried out after the oral administration of 10 mg/kg of DMSO-bound Alexa-488 and DOCA-conjugated heparin (physical binding in 20% DMSO solution) or Alexa-488 and DOCA-conjugated heparin itself (control group) in mice (Figure 6C). At 10 min after oral administration, strong fluorescence intensity was observed in both cases. However, at 40 and 120 min after oral administration, the fluorescence intensity of DMSO-bound DOCA-conjugated heparin in the whole body was still strongly observed for 2 h. The reason was that the physically

bound DMSO could completely solubilize the nanoparticles of DOCA-conjugated heparin, and then, the conjugated DOCA molecules could be highly exposed to increase the binding opportunity onto bile acid transporters in the small intestine.6 However, the fluorescence intensity of DOCAconjugated heparin was gradually diminished in the body due to its low opportunity for binding bile acid transporter in the intestine. Therefore, physical binding of DMSO could improve the pharmacokinetics, i.e., bioavailability, of the orally administered heparin. On the other hand, in this study we did not show the toxicity of the orally administered DMSO-bound DOCA-conjugated heparin. Also, we previously showed no cytotoxicity to the Caco-2 cell monolayer in vitro.21 The reason was that the amount of bound DMSO was not too much to show cytotoxicity (DMSO content in 10 mg/kg of the modified heparin was about 20 wt %). It is known that the oral NOEL (no observable effects limit) of DMSO in rhesus monkey is 3 g/kg/day during an 18 month study. If extrapolated to humans, the oral monkey NOEL of 3 g/kg/day is comparable to an average human (70 kg) consuming approximately 210 g DMSO per day. In reality, the U.S. Food and Drug Administration (FDA) announced that DMSO was pharmaceutically placed in the safest category (class 3 solvents) with low toxicity potential. Therefore, we can conclude that the amount of bound DMSO in the heparin has no cytotoxicity. 1456

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Figure 6. Oral absorption of DMSO-bound DOCA-conjugated heparin in mice. (A) Absorption profiles of DMSO-bound DOCA-conjugated heparin according to different amounts of bound DMSO contents after oral administration into mice: 0% (b; control), 2% (O), 5% (9), 10% (0), and 20% (2). (B) Area under the curve (AUC; IU/mL/min) after its oral administration. (C) Optical imaging of the distribution of DMSO-bound Alexa-labeled DOCA-conjugated heparin in mice after oral administration according to different times. The data were expressed as the means ( SD (n = 5). *P < 0.05.

In conclusion, the mechanism study through the simulation revealed that DMSO molecules are rarely bound to the heparin backbone whereas water molecules are very crowded around the heparin backbone that is mainly driven by the electrostatic interaction between negatively charged sulfate (or carboxylate groups) and water hydrogen rather than hydrogen bonding between hydroxyl group and water oxygen. Also, the main driving force responsible for DMSODOCA binding is not hydrogen bonding but hydrophobic interaction between the methyl group of DMSO and the 4-ring of DOCA. The sulfoxides of DMSO facing outward with respect to DOCA shield DOCAs to prevent self-assembled particulation. Therefore, the solubilization mechanism of DMSO-bound DOCA-conjugated heparin is due to the amphiphilic action of DMSO molecules that is bound to DOCA largely via hydrophobic interaction, while the hydrophilic sulfoxide group shields the DOCA from their aggregation, which is reminiscent in many ways of colloidal particle stabilization in an amphiphilic system. Therefore, the observed main dissolution effect would be given via “hydrophobic hydration” by DMSO/water mixture. These results open new perspectives for improved oral formulation of macromolecules with DOCA and DMSO as structural constraints in nanoscale. Finally, the structural constraints-based oral formulation can open new opportunities to use DMSO and DOCA in a newly designed platform technology for oral delivery of macromolecules as drug additives.

’ ASSOCIATED CONTENT

bS

Supporting Information. Additional graphics as described in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Dong Yun Lee, Ph.D., Department of Bioengineering, College of Engineering, Institute for Bioengineering and Biopharmaceutical Research, Hanyang University, and Hanyang University Institute of Aging Society, Seoul 133-791, Republic of Korea; Phone (82-2) 2220-2348; Fax (82-2) 2220-4741; E-mail [email protected]. Hyun Tae Moon, Ph.D., Research and Development Center, Mediplex Corp., #21-315, College of Pharmacy, Seoul National University, San 56-1, Shinlim-dong, Gwanak-gu, Seoul 151-742, Republic of Korea; Phone (82-2) 880-2537; Fax (82-2) 872-7864; E-mail [email protected]. Author Contributions †

These authors contributed equally to this work as first authors.

’ ACKNOWLEDGMENT This study was supported by a grant from the Next Generation New Technology Development Program (grant no. 10011353) of the Korean Ministry of Commerce, Industry, and Energy (MOCIE), and supported by the World Class University 1457

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Bioconjugate Chemistry (WCU) program (grant no. R31-2008-000-10103), the Converging Research Center Program (2009-0081879) and the Basic Science Research Program (grant no. 2010-0002994) through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology, Republic of Korea.

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thrombosis: non-human primate study. J. Controlled Release 123, 155–63. (21) Kim, S. K., Lee, D. Y., Lee, E., Lee, Y. K., Kim, C. Y., Moon, H. T., and Byun, Y. (2007) Absorption study of deoxycholic acid-heparin conjugate as a new form of oral anti-coagulant. J. Controlled Release 120, 4–10.

’ REFERENCES (1) Hyers, T. M., Hull, R. D., and Weg, J. G. (1995) Antithrombotic therapy for venous thromboembolic disease. Chest 108, 335S–351S. (2) Hull, R., Delmore, T., Carter, C., Hirsh, J., Genton, E., Gent, M., Turpie, G., and McLaughlin, D. (1982) Adjusted subcutaneous heparin versus warfarin sodium in the long-term treatment of venous thrombosis. N. Engl. J. Med. 306, 189–94. (3) Hardman, J. G., Limbird, L. E., Molinoff, P. B., Ruddon, R. W., and Gilman, A. G. (1996) The pharmacological basis of therapeutics, Vol. 9, McGraw-Hill, New York. (4) Lee, Y., Nam, J. H., Shin, H. C., and Byun, Y. (2001) Conjugation of low-molecular-weight heparin and deoxycholic acid for the development of a new oral anticoagulant agent. Circulation 104, 3116–20. (5) Lee, S., Kim, K., Kumar, T. S., Lee, J., Kim, S. K., Lee, D. Y., Lee, Y. K., and Byun, Y. (2005) Synthesis and biological properties of insulindeoxycholic acid chemical conjugates. Bioconjugate Chem. 16, 615–20. (6) Kim, S. K., Kim, K., Lee, S., Park, K., Park, J. H., Kwon, I. C., Choi, K., Kim, C. Y., and Byun, Y. (2005) Evaluation of absorption of heparinDOCA conjugates on the intestinal wall using a surface plasmon resonance. J. Pharm. Biomed. Anal. 39, 861–70. (7) Kim, S. K., Vaishali, B., Lee, E., Lee, S., Lee, Y. K., Kumar, T. S., Moon, H. T., and Byun, Y. (2006) Oral delivery of chemical conjugates of heparin and deoxycholic acid in aqueous formulation. Thromb. Res. 117, 419–27. (8) Haynes, W. M. (1999) (Haynes, W. M., Ed.) CRC Press/Taylor and Francis, Boca Raton, FL. (9) Wang, J., Wolf, R. M., Caldwell, J. W., Kollman, P. A., and Case, D. A. (2004) Development and testing of a general amber force field. J. Comput. Chem. 25, 1157–74. (10) Ponder, J. W., and Case, D. A. (2003) Force fields for protein simulations. Adv. Protein Chem. 66, 27–85. (11) Jakalian, A., Bush, B. L., Jack, B. D., and Bayly, C. I. (2000) Fast, efficient generation of high-quality atomic charges. AM1-BCC model: I. J. Comput. Chem. 21, 132–46. (12) Jorgensen, W. L., Chandrasekhar, J., Madura, J. D., Impey, R. W., and Klein, M. L. (1983) Comparison of simple potential functions for simulating liquid water. J. Chem. Phys. 79, 926–935. (13) Kale, L., Skell, R., Bhandarkar, M., Brunner, R., Gursoy, A., Krawetz, N., Phillips, J., Shinozaki, A., Varadarajan, K., and Schulten, K. (1999) NAMD2: greater scalability for parallel molecular dynamics. J. Comput. Phys. 151, 283–312. (14) Darden, T., York, D., and Pdersen, L. (1993) Particle mesh Ewald: An Nxlog(N) method for Ewald sums in large systems. J. Chem. Phys. 98, 10089–10092. (15) Freiman, D. G., Suyemoto, J., and Wessler, S. (1965) Frequency of pulmonary thromboembolism in man. N. Engl. J. Med. 272, 1278–80. (16) Casu, B. (1989) Structure of heparin and heparin fragments. Ann. N.Y. Acad. Sci. 556, 1–17. (17) Breddin, H. K., Hach-Wunderle, V., Nakov, R., and Kakkar, V. V. (2001) Effects of a low-molecular-weight heparin on thrombus regression and recurrent thromboembolism in patients with deep-vein thrombosis. N. Engl. J. Med. 344, 626–31. (18) Jaques, L. B. (1979) Heparins--anionic polyelectrolyte drugs. Pharmacol. Rev. 31, 99–166. (19) Lee, Y., Kim, S. H., and Byun, Y. (2000) Oral delivery of new heparin derivatives in rats. Pharm. Res. 17, 1259–64. (20) Kim, S. K., Lee, D. Y., Kim, C. Y., Nam, J. H., Moon, H. T., and Byun, Y. (2007) A newly developed oral heparin derivative for deep vein 1458

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