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Functional Analysis of Recombinant Human Serum Albumin Domains for Pharmaceutical Applications

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Abstract

Purpose. Functional analysis of the three recombinant human serum albumin (rHSA) domains and their potential as stand-alone proteins for use as drug delivery protein carriers.

Methods. Protein structure was examined by fluorescence and CD spectroscopy. Ligand binding was estimated by ultrafiltration. Antioxidant activity was estimated by measuring the quenching of dihydrorhodamine 123. Esterase-like activity and enolase-like activity were estimated from the rate of hydrolysis of p-nitrophenyl acetate and conversion of dihydrotestosterone from the 3-keto to 3-enol form, respectively. The domains of human serum albumin (HSA) were radiolabeled with 111In to evaluate their pharmacokinetics.

Results. The ligand binding ability of subsites Ia and Ib could not be detected in domain II. However, the binding of ligands to subsite Ic and site II were preserved in domain II and domain III, respectively. Domain III retained about 45% of its esterase-like activity, and weaker esterase-like activity was also observed in domain I. All domains showed low enolase-like activity in a pH 7.4 phosphate buffer, but domain II had higher activity in a pH 9.2 carbonate buffer. Domain I exhibited antioxidant activity comparable to that of rHSA. All three of the 111In-radiolabeled domains were rapidly eliminated from HSA, with high accumulation in the kidneys.

Conclusion. Domain I of HSA has great potential for further development as a drug delivery protein carrier, due to its favorable properties and the presence of a free cysteine residue.

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REFERENCES

  1. T. Peters Jr. All about Albumin: Biochemistry, Genetics, and Medical Applications, Academic Press, San Diego, 1996.

    Google Scholar 

  2. F. Kratz, J. Drevs, G. Bing, C. Stockmar, K. Scheuermann, P. Lazar, and C. Unger. Development and in vitro efficacy of novel MMP2 and MMP9 specific doxorubicin albumin conjugates. Bioorg. Med. Chem. Lett. 11:2001–2006 (2001).

    Google Scholar 

  3. P. Kremer, G. Hartung, U. Bauder-Wust, H. H. Schrenk, A. Wunder, S. Heckl, U. Zillmann, and H. Sinn. Efficacy and toler-ability of an aminopterin-albumin conjugate in tumor-bearing rats. Anticancer Drugs 13:615–623 (2002).

    Google Scholar 

  4. A. Wunder, U. Muller-Ladner, E. H. Stelzer, J. Funk, E. Neu-mann, G. Stehle, T. Pap, H. Sinn, S. Gay, and C. Fiehn. Albumin-based drug delivery as novel therapeutic approach for rheuma-toid arthritis. J. Immunol. 170:4793–4801(2003).

    Google Scholar 

  5. B. L. Osborn, H. S. Olsen, B. Nardelli, J. H. Murray, J. X. Zhou, A. Garcia, G. Moody, L. S. Zaritskaya, and C. Sung. Pharmaco-kinetic and pharmacodynamic studies of a human serum albu-min-interferon-_ fusion protein in cynomolgus monkeys. J. Phar-macol. Exp. Ther. 303:540–548(2002).

    Google Scholar 

  6. B. L. Osborn, L. Sekut, M. Corcoran, C. Poortman, B. Sturm, G. Chen, D. Mather, H. L. Lin, and T. J. Parry. Albutropin: a growth hormone albumin fusion with improved pharmacokinetics and pharmacodynamics in rats and monkeys Eur. J. Pharmacol. 456:149–158(2002).

    Google Scholar 

  7. W. Halpern, T. A. Riccobene, H. Agostini, K. Baker, D. Stolow, M. L. Gu, J. Hirsch, A. Mahoney, J. Carrell, E. Boyd, and K. J. Grzegorzewski. Albugranin, a recombinant human granulocyte colony stimulating factor (G-CSF) genetically fused to recombi-nant human albumin induces prolonged myelopoietic effects in mice and monkeys. Pharm. Res. 19:1720–1729(2002).

    Google Scholar 

  8. P. Yeh, D. Landais, M. Lemaitre, I. Maury, J. Y. Crenne, J. Becquart, A. Murry-Brelier, F. Boucher, G. Montay, R. Fleer, P. H. Hirel, J. F. Mayaux, and D. Klatzmann. Design of yeast-secreted albumin derivatives for human therapy: Biological and antiviral properties of a serum albumin-CD4 genetic conjugate. Proc. Natl. Acad. Sci. USA 89:1904–1908(1992).

    Google Scholar 

  9. E. G. DeMaster, B. J. Quast, B. Redfern, and H. T. Nagasawa. Reaction of nitric oxide with the free sulfhydryl group of human serum albumin yields a sulfenic acid and nitrous oxide. Biochem-istry 34:11494–11499(1995).

    Google Scholar 

  10. M. Anraku, K. Yamasaki, T. Maruyama, U. Kragh-Hansen, and M. Otagiri. Effect of oxidative stress on the structure and func-tion of human serum albumin. Pharm. Res. 18:632–639(2001).

    Google Scholar 

  11. K. Ikeda, Y. Kurono, Y. Ozeki, and T. Yotsuyanagi. Effects of drug bindings on esterase activity of human serum albumin. Dis-sociation constants of the complexes between the protein and drugs such as N-arylanthranilic acids, coumarin derivatives and prostaglandins. Chem. Pharm. Bull. 27:80–87(1979).

    Google Scholar 

  12. Y. Kurono, I. Kushida, H. Tanaka, and K. Ikeda. Esterase-like activity of human serum albumin. VIII. Reaction with amino acid p-nitrophenyl esters. Chem. Pharm. Bull. 40:2169–2172(1992).

    Google Scholar 

  13. Z. Drmanovic, S. Voyatzi, D. Kouretas, D. Sahpazidou, A. Pa-pageorgiou, and O. Antonoglou. Albumin possesses intrinsic enolase activity towards dihydrotestosterone which can differen-tiate benign from malignant breast tumors. Anticancer Res. 19:4113–4124(1999).

    Google Scholar 

  14. M. Dockal, D. C. Carter, and F. Ruker. The three recombinant domains of human serum albumin. Structural characterization and ligand binding properties. J. Biol. Chem. 274:29303–29310(1999).

    Google Scholar 

  15. M. Dockal, M. Chang, D. C. Carter, and F. Ruker. Five recom-binant fragments of human serum albumin-Tools for the char-acterization of the warfarin binding site. Protein Sci. 9:1455–1465 (2000).

    Google Scholar 

  16. S. M. Twine, M. G. Gore, P. Morton, B. C. Fish, A. G. Lee, and J. M. East. Mechanism of binding of warfarin enantiomers to recombinant domains of human albumin. Arch. Biochem. Bio-phys. 414:83–90(2003).

    Google Scholar 

  17. J. S. Stamler, O. Jaraki, J. Osborne, D. I. Simon, J. Keaney, J. Vita, D. Singel, C. R. Valeri, and J. Loscalzo. Nitric oxide circulates in mammalian plasma primarily as an S-nitroso adduct of serum albumin. Proc. Natl. Acad. Sci. USA 89:7674–7677(1992).

    Google Scholar 

  18. H. Watanabe, S. Tanase, K. Nakajou, T. Maruyama, U. Kragh-Hansen, and M. Otagiri. Role of arg-410 and tyr-411 in human serum albumin for ligand binding and esterase-like activity. Bio-chem. J. 349:813–819(2000).

    Google Scholar 

  19. R. F. Chen. Removal of fatty acids from serum albumin by char-coal treatment. J. Biol. Chem. 242:173–181(1967).

    Google Scholar 

  20. Y. Takakura, T. Fujita, M. Hashida, and H. Sezaki. Disposition characteristics of macromolecules in tumor-bearing mice. Pharm. Res. 7:339–346(1990).

    Google Scholar 

  21. J. R. Duncan and M. J. Welch. Intracellular metabolism of in-dium-111-DTPA-labeled receptor targeted proteins. J. Nucl. Med. 34:1728–1738(1993).

    Google Scholar 

  22. K. Yamaoka, Y. Tanigawara, T. Nakagawa, and T. Uno. A phar-macokinetic analysis program (multi) for microcomputer. J. Pharmacobiodyn. 4:879–885(1981).

    Google Scholar 

  23. T. Mukai, Y. Arano, K. Nishida, H. Sasaki, H. Saji, and J. Nakamura. In-vivo evaluation of indium-111-diethylene-triaminepentaacetic acid-labelling for determining the sites and rates of protein catabolism in mice. J. Pharm. Pharmacol. 51:15–20(1999).

    Google Scholar 

  24. K. Yamasaki, T. Maruyama, U. Kragh-Hansen, and M. Otagiri. Characterization of site I on human serum albumin: concept about the structure of a drug binding site. Biochim. Biophys. Acta 1295:147–157 (1996).

    Google Scholar 

  25. R. J. Hondal, A. K. Motley, K. E. Hill, and R. F. Burk. Failure of selenomethionine residues in albumin and immunoglobulin G to protect against peroxynitrite. Arch. Biochem. Biophys. 371:29–34 (1999).

    Google Scholar 

  26. S. C. Makrides, P. A. Nygren, B. Andrews, P. J. Ford, K. S. Evans, E. G. Hayman, H. Adari, H. Adari, M. Uhlen, and C. A. Toth. Extended in vivohalf-life of human soluble complement receptor type 1 fused to a serum albumin-binding receptor. J. Pharmacol. Exp. Ther. 277:534–542(1996).

    Google Scholar 

  27. P. Kurtzhals, S. Havelund, I. Jonassen, B. Kiehr, U. D. Larsen, U. Ribel, and J. Markussen. Albumin binding of insulins acylated with fatty acids: characterization of the ligand-protein interaction and correlation between binding affinity and timing of the insulin effect in vivo. Biochem. J. 312:725–731(1995).

    Google Scholar 

  28. L. Beljaars, G. Molema, B. Weert, H. Bonnema, P. Olinga, G. M. Groothuis, D. K. Meijer, and K. Poelstra. Albumin modified with mannose 6-phosphate: A potential carrier for selective delivery of antifibrotic drugs to rat and human hepatic stellate cells. Hepa-tology 29:1486–1493(1999).

    Google Scholar 

  29. H. Sakai, S. Takeoka, S. I. Park, T. Kose, H. Nishide, Y. Izumi, A. Yoshizu, K. Kobayashi, and E. Tsuchida. Surface modification of hemoglobin vesicles with poly(ethylene glycol) and effects on aggregation, viscosity, and blood flow during 90% exchange transfusion in anesthetized rats. Bioconjug. Chem. 8:23–30 (1997).

    Google Scholar 

  30. T. Kjeldsen, A. F. Pettersson, L. Drube, P. Kurtzhals, I. Jonassen, S. Havelund, P. H. Hansen, and J. Markussen. Secretory expres-sion of human albumin domains in Saccharomyces cerevisiaeand their binding of myristic acid and an acylated insulin analogue. Protein Expr. Purif. 13:163–169(1998).

    Google Scholar 

  31. E. Suzuki, K. Yasuda, N. Takeda, S. Sakata, S. Era, K. Kuwata, M. Sogami, and K. Miura. Increased oxidized form of human serum albumin in patients with diabetes mellitus. Diabetes Res. Clin. Pract. 18:153–158(1992).

    Google Scholar 

  32. W. P. Sheffield, J. A. Marques, V. Bhakta, and I. J. Smith. Modu-lation of clearance of recombinant serum albumin by either gly-cosylation or truncation. Thromb. Res. 99:613–621(2000).

    Google Scholar 

  33. K. Nakajou, H. Watanabe, U. Kragh-Hansen, T. Maruyama, and M. Otagiri. The effect of glycation on the structure, function and biological fate of human serum albumin as revealed by recombinant mutants. Biochim. Biophys. Acta 1623:88–97(2003).

    Google Scholar 

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Authors and Affiliations

  1. Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Kumamoto, 862-0973, Japan

    Sadaharu Matsushita, Yu Isima, Victor Tuan Giam Chuang, Hiroshi Watanabe, Sumio Tanase, Toru Maruyama & Masaki Otagiri

  2. Department of Pharmacy, Faculty of Allied Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300, Kuala Lumpur, Malaysia

    Victor Tuan Giam Chuang

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  1. Sadaharu Matsushita
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Matsushita, S., Isima, Y., Chuang, V.T.G. et al. Functional Analysis of Recombinant Human Serum Albumin Domains for Pharmaceutical Applications. Pharm Res 21, 1924–1932 (2004). https://doi.org/10.1023/B:PHAM.0000045248.03337.0e

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