Monofunctional hydroxy-terminated poly(N-vinylpyrrolidone) was obtained by radical polymerization in the presence of isopropoxyethanol for protein surface modification. Superoxide dismutase was modified to various degrees by reaction with PVP-OH activated by 4-nitrophenyl chloroformate with high retention of enzymatic activity. All the polymer-enzyme conjugates showed increased body residence time after intravenous injection in rats with respect to the native enzyme. The residence time depended upon the number of bound polymer chains. PVP was found to affect the pharmacokinetic properties of the enzyme after subcutaneous, intramuscular and intraperitoneal administration. The antigenicity of superoxide dismutase was reduced to one third following modification, while its immunogenicity after intravenous, subcutaneous and intraperitoneal injection totally disappeared. The conjugates exhibited increased solubility in organic solvents and increased stability towards thermal denaturation, whereas changes in the structural properties were observed by circular dichroism measurements.
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References
1.
1. Davis, F. F., G. M. Kazo, M. L. Nucci and A. Abuchowski. 1990. Peptide and Protein Drug Delivery, H. H. L. Vincent, ed., New York: Mercel Dekker Inc., pp. 831-864.
2.
2. Nucci, M. L., R. Shorr and A. Abuchowski. 1991. "The Therapeutic Value of Poly(ethylene glycol) Modified Proteins,"Advanced Drug Delivery Reviews, 6:133-151.
3.
3. Veronese, F. M., P. Caliceti, O. Schiavon and L. Sartore. 1991. Poly(ethylene glycol), Chemistry, Biochemical and Biomedical Applications, J. M. Harris, ed., New York: Plenum Publication Corp.
4.
4. Cao, S. G., Q. Zhao, Z. T. Ding, L. Ma, T. Yu, J. H. Wang, Y. Feng and Y. H. Cheng. 1990. "Chemical Modification of Enzyme Molecule to Improve Their Characteristics,"Enzyme Eng, 10:460-467.
5.
5. Delgado, C., G. E. Francis and D. Fisher. 1992. "The Uses and Properties of PEG-Linked Proteins,"Critical Rev. Ther. Drug. Carrier Syst., 9:249-304.
6.
6. Nandini, V. K.1993. "The Conjugation of Proteins with Poly(ethylene glycol) and Other Polymers: Altering Properties of Proteins to Enhance Their Therapeutic Potential,"Advanced Drug Delivery Reviews, 10:91-114.
7.
7. Lee, W. Y., A. H. Sehon and B. W. Von Secht. 1981. "Suppression of IgE Antibodies to the (N-Hydroxy-3-Iodo-5-Nitrophenyl) Acetyl (NIP) Group and Induction of NIP Specific Suppressor Cells with HIP Pbly-N-Vinylpyrrolidone Conjugates,"Eur. J. Immunol., 11:13-17.
8.
8. Vyas, S. P., S. Rahchadraiah, C. P. Jain and S. K. Jain. 1991. "Polymeric Pseudolatices Bearing Pilocarpine for Controlled Ocular Delivery,"J. Microencapsul., 9(3):347-355.
9.
9. Von Secht, B. W., H. Seinfeld and W. Brendel. 1973. "Polyvinylpyrrolidone as a Soluble Carrier for Proteins,"Hope-Seyler's Z. Physiol. Chem., 354:1659-1660.
10.
10. Von Specht, B.-U. and W. Brendel. 1977. "Preparation and Properties of Trypsin and Chymotrypsin Coupled Covalently to Poly(N-Vinylpyrrolidone),"BBA, 484:109-114.
11.
11. Sartore, L., E. Ranucci, P. Ferruti, P. Caliceti, O. Schiavon and F. M. Veronese. 1994. "Low Molecular Weight End-Functionalized PVP (N-Vinylpyrrolidone) for the Modification of Polypeptide Amino Groups,"Journal of Bioactive and Compatible Polymers, 9:411-428.
12.
12. Sims, G. E. C. and T. J. Snape. 1980. "A Method for the Estimation of Poly(ethylene glycol) in Plasma Protein Fractions,"Anal. Biochem., 107:60-63.
13.
13. Paoletti, F., D. Aldinucci, A. Mocali, A. Capanini. 1986. "A Sensitive Spectrophotometric Method for the Determination of Superoxide Dismutase Activity in Tissue Extracts,"; Anal. Biochem., 154:536-541.
14.
14. Gornall, A. G., C. J. Bardawill and M. M. David. 1949. "Determination of Serum Protein by Means of Biuret Reaction,"J. Biol. Chem., 177:751-766.
15.
15. Habeeb, A. F. S. A.1966. "Determination of Free Amino Groups in Proteins by Trinitrobezensulfonic Acid,"Anal. Biochem., 14:328-336.
16.
16. Veronese, F. M., P. Caliceti, A. Pastorino, O. Schiavon, L. Sartore, L. Banci and L. Monsu Scolaro. 1989. "Preparation, Physicochemical and Pharmacokinetic Characterization of Monomethoxypoly(ethylene glycol) Derivatized Superoxide Dismutase,"J. Controlled Release, 10:145-154.
17.
17. Kaksmojo, H., Y. Kojma, K. Ishii, K. Onove and H. Maeda. 1993. "The Suppressive Effect of Gelatin Conjugated Superoxide Dismutase on Disease Development and Severity of Collagen Induced,"Clin. Exp. Immunol., 94(2):241-246.
18.
18. Oda, T., T. Akaike, T. Hamamoto, F. Suzuki, T. Hirano and H. Maeda. 1989. "Oxygen Radicals in Influence Induced Pathogenesis and Treatment with Pyran Polymer Conjugated SOD,"Science, 244(4907):974-976.
19.
19. Hirota, M., M. Inove, Y. Ando and Y. Morito. 1990. "Inhibition of Stress Induced Gastric Mucose Injury by a Long Acting Superoxide Dismutase That Circulates Bond to Albumin,"Arch. Biochem. Biophys.,280:269-273.
20.
20. Maksimenko, A. V., E. L. Grigoryeva, A. D. Morozkin, E. G. Tischenko, E. B. Mikovsky and V. P. Torchilin. 1991. "The Estimation of Composition and Structure of Covalent Complexes between Superoxide Dismutase and Aldehyde Dextran by ANalytical Ultracentrifugation,"Biochimiye, 56:1330-1336.
21.
21. Hirano, T., T. Todoroki, S. Kazo, H. Yamamoto, P. Caliceti, F. M. Veronese, H. Maeda and S. Ohashi. 1994. "Synthesis of the Conjugate of Superoxide Dismutase (SOD) with the Copolymer of Divinyl Ether and Maleic Anhydride (DIVEMA) Retaining Enzymatic Activity,"J. Controlled Release, 28:203-209.
22.
22. Kojima, Y., A. Haruta, T. Imai, M. Otagiri and H. Maeda. 1993. "Conjugation of Cu,Zn-Superoxide Dismutase with Succinylated Gelatin: Pharmacological Activity and Cell-Lubrificating Function,"Bioconjugate Chem., 4:490-498.
23.
23. Fujita, T., Y. Yasuda, Y. Takakura, M. Hashida and H. Sezaki. 1990. "Alteration of Biopharmaceutical Properties of Drugs by Their Conjugation with Water-Soluble Macromolecules: Uricase-Dextran Conjugate,"J. Controlled Release, 11:149-156.
24.
24. Fujita, T., Y. Yasuda, Y. Takakura, M. Hashida and H. Sezaki. 1991. "Tissue Distribution of 111In-Labelled Uricase Conjugated with Charged Dextrans and Polyethylene Glycol,"J. Pharmacobio-Dyn., 14:623-629.
25.
25. Nucci, M. L., J. Olejarczyk and A. Abuchowski. 1986. "Immunogenicity of Polyethylene Glycol-Modified Superoxide Dismutase and Catalase,"J. Free Rad. Biol. Med., 2:321-325.
26.
26. Sartore, L., P. Caliceti, O. Schiavon and F. M. Veronese. 1991. "Enzyme Modification by MPEG with an Amino Acid or Peptide as Spacer Arm,"Appl. Biochem. Biotechnol., 27:45-54.
27.
27. Snider, J., C. Neville, L-C Yuan and J. Bullock. 1992. "Characterization of the Heterogeneity of Polyethylene Glycol-Modified Superoxide Dismutase by Chromatographic and Electrophoretic Techniques,"J. Chrom., 599:141-155.
28.
28. Caliceti, P., F. M. Veronese, T. Hirano and S. Ohashi. "Immunological, Pharmacokinetic and Physico-Chemical Characterization of Superoxide Dismutase Conjugated to the Copolymer of Divinyl Ether and Maleic Anhydride,"J. Controlled Release (submitted).
