The synthesis and physico-chemical characterisation of a biodegradable multiblock polymer drug carrier based on poly(ethylene glycol) (PEG) is described. The blocks of PEG (Mw 2,000) are connected by an enzymatically degradable tripeptide derivative consisting of one lysine and two glutamic acid residues. Doxorubicin (Dox), was attached to the polymer carrier via a hydrazone bond susceptible to acid hydrolysis at pH 5.0. Human immunoglobulin (IgG) was covalently linked to the polymer-Dox conjugate by the reaction of 2-pyridyldisulfanyl groups of the polymer with thiol groups of the antibody modified with 2-iminothiolane. The resulting antibody-polymer-drug conjugates were characterised by size-exclusion chromatography, UV/VIS spectrophotometry, electrophoresis and amino acid analysis. All polymers studied (both with and without IgG) showed high anti-proliferative activity against concanavalin A-stimulated murine splenocytes and various cancer cell lines in vitro. The polymer-Dox conjugate (without IgG) exhibited a significant anti-tumor efficacy against murine EL4 T-cell lymphoma and human colorectal carcinoma SW620 in vivo.
Ř íhová, B. , Jelínková, M. , Strohalm, J. , Šubr, V. , Plocová, D. , Hovorka, O. , Novák, M. , Plundrová, D. , Germano, Y. and Ulbrich, K. (2000). Polymeric Drugs Based on Conjugates of Synthetic and Natural Macromolecules II. Anticancer Activity of Antibody or (Fab)2-Targeted Conjugates and Combined Therapy with Immunomodulators , J. Controlled Release, 64: 241-261 .
2.
Ř íhová, B. , Strohalm, J. , Kubáčková, K. , Jelínková, M. , Hovorka, O. , Kovář, M. , Plocová, D. , Šírová, M. , Štastný, M. , Rozprimová, L. and Ulbrich, K. (2002). Acquired and Specific Immunological Mechanisms Co-Responsible for Efficacy of Polymer-Bound Drugs , J. Controlled Release, 78: 97-114 .
3.
Ulbrich, K. , Šubr, V. , Strohalm, J. , Plocová, D. , Jelínková, M. and Říhová, B. (2000). Polymeric Drugs Based on Conjugates of Synthetic and Natural Macromolecules I. Synthesis and Physico-Chemical Characterisation , J. Controlled Release, 64: 63-79 .
4.
Dvořák, M. , Kopečková, P. and Kopeček, J. (1999). High-Molecular Weight HPMA Copolymer-Adriamycin Conjugates , J. Controlled Release, 60: 321-332 .
5.
Wang, D. , Pechar, M. , Li, W.J. , Kopečková, P. , Bromme, D. and Kopeček, J. (2002). Inhibition of Cathepsin K with Lysosomotropic Macromolecular Inhibitors , Biochemistry, 41: 8849-8859 .
6.
Nathan, A. , Zalipsky, S. , Ertel, S.I. , Agathos, S.N. , Yarmush, M.L. and Kohn, J. (1993). Copolymers of Lysine and Polyethylene Glycol: A New Family of Functionalized Drug Carriers , Bioconjugate Chem., 4: 54-62 .
7.
Nathan, A. , Zalipsky, S. and Kohn, J. (1994). Strategies for Covalent Attachment of Doxorubicin to Poly(PEG-Lys), a New Water-Soluble Poly(Ether Urethane) , J. Bioact. Compat. Polym., 9: 239-251 .
8.
Zalipsky, S. (1995). Chemistry of Polyethylene Glycol Conjugates with Biologically Active Molecules , Adv. Drug Delivery Rev., 16: 157-182 .
9.
Greenwald, R.B. , Conover, C.D. and Choe, Y.H. (2000). Poly(Ethylene Glycol) Conjugated Drugs and Prodrugs: A Comprehensive Review , Crit. Rev. Ther. Drug Carrier Syst., 17: 101-161 .
10.
Pechar, M. , Ulbrich, K. , Šubr, V. , Seymour, L.W. and Schacht, E.H. (2000). Poly(Ethylene Glycol) Multiblock Copolymer as a Carrier of Anti-Cancer Drug Doxorubicin , Bioconjugate Chem., 11: 131-139 .
11.
Jelínková, M. , Strohalm, J. , Etrych, T. , Ulbrich, K. and Ř íhová, B. (2003). Starlike Versus Classic Macromolecular Prodrugs: Two Different Antibody-Targeted HPMA Copolymers of Doxorubicin Studied In Vitro and In Vivo as Potential Anticancer Drugs , Pharm. Res., 20: 1558-1564 .
12.
Etrych, T. , Chytil, P. , Jelínková, M. , Ř íhová, B. and Ulbrich, K. (2002). Synthesis of HPMA Copolymers Containing Doxorubicin Bound Via a Hydrazone Linkage. Effect of Spacer on Drug Release and In Vitro Cytotoxicity , Macromol. Biosci., 2: 43-52 .
13.
Ř íhová, B. , Strohalm, J. , Kubáčková, K. , Jelínková, M. , Rozprimová, L. , Šírová, M. , Plocová, D. , Mrkvan, T. , Kovář, M. , Pokorná, J. , Etrych, T. and Ulbrich, K. (2003). Drug-HPMA-HuIg Conjugates Effective Against Human Solid Cancer , Adv. Exp. Med. Biol., 519: 125-143 .
14.
Kovář, M. , Mrkvan, T. , Strohalm, J. , Etrych, T. , Ulbrich, K. , Š tastný, M. and Říhová, B. (2003). HPMA Copolymer-Bound Doxorubicin Targeted to Tumor Specific Antigen of BCL1 Mouse B Cell Leukemia , J. Controlled Release, 92: 315-330 .
15.
Pechar, M. , Strohalm, J. , Ulbrich, K. and Schacht, E. (1997). Biodegradable Drug Carriers Based on Poly(Ethylene Glycol) Block Copolymers , Macromol. Chem. Phys., 198: 1009-1020 .
16.
Pechar, M. , Ulbrich, K. , Jelínková, M. and Ř íhová, B. (2001). Enzymatically Degradable PEG Multiblock Copolymers with Hydrazone-Attached Doxorubicin in Cancer Therapy , J. Controlled Release, 72: 253-254 .
17.
Pechar, M. , Ulbrich, K. , Jelínková, M. and Ř íhová, B. (2003). Conjugates of Antibody-Targeted PEG Multiblock Polymers with Doxorubicin in Cancer Therapy , Macromol. Biosci., 3: 364-372 .
18.
Ř íhová, B. , Etrych, T. , Pechar, M. , Jelínková, M. , Štastný, M. , Hovorka, O. , Kovář, M. and Ulbrich, K. (2001). Doxorubicin Bound to a HPMA Copolymer Carrier Through Hydrazone Bond is Effective also in a Cancer Cell Line with a Limited Content of Lysosomes , J. Controlled Release, 74: 225-232 .
19.
Yoda, H. , Naito, S. , Takabe, K. , Tanaka, N. and Hosoya, K. (1990). Conjugate Addition to Chiral [Gamma]-Heterosubstituted [Delta]-Lactones as Pivotal Synthons from L-Glutamic Acid. Synthesis of an Optically Active Lignan Lactone; (-)-hinokinin , Tetrahedron Lett., 31: 7623-7626 .
20.
Pechar, M. , Strohalm, J. and Ulbrich, K. (1995). Synthesis of Poly(Ethylene Glycol) Block Copolymers as Potential Water-Soluble Drug Carriers , Collect. Czech. Chem. Commun., 60: 1765-1780 .
Pohl, J. , Baudyš, M. , Tomášek, V. and Kostka, V. (1982). Identification of the Active Site Cysteine and of the Disulfide Bonds in the N-Terminal Part of the Molecule of Bovine Spleen Cathepsin B , FEBS Lett., 142: 23-26 .
23.
Jelínková, M. , Strohalm, J. , Plocová, D. , Š ubr, V. , Štastný, M. , Ulbrich, K. and Říhová, B. (1998). Targeting of Human and Mouse T-lymphocytes by Monoclonal Antibody-HPMA Copolymer-Doxorubicin Conjugates Directed Against Different T-Cell Surface Antigens , J. Controlled Release, 52: 253-270 .
24.
Nathan, A. , Bolikal, D. , Vyavahare, N. , Zalipsky, S. and Kohn, J. (1992). Hydrogels Based on Water-Soluble Poly(Ether Urethanes) Derived from l-Lysine and Poly(Ethylene Glycol) , Macromolecules, 25: 4476-4484 .
25.
Jue, R. , Lambert, J.M. , Pierce, L.R. and Traut, R.R. (1978). Addition of Sulfhydryl Groups of Escherichia coli Ribosomes by Protein Modification with 2-Iminothiolane (Methyl 4-Mercaptobutyrimidate) , Biochemistry, 17: 5399 .
26.
Maeda, H. and Matsumura, Y. (1989). Tumoritropic and Lymphotropic Principles of Macromolecular Drugs , Crit. Rev. Ther. Drug Carrier Syst., 6: 193-210 .
27.
Diwu, Z. , Chen, C.S. , Stass, S.A. and Mixon, A.G. (1999). A Novel Acidotrophic pH Indicator and Its Potential Application in Labelling Acidic Organelles of Live Cells , Chem. Biol., 6: 411-418 .
28.
Ghose, T. , Guclu, A. , Tai, J. , Mammen, M. and Norvell, S.T. (1977). Immunoprophylaxis and Immunotherapy of EL4 Lymphoma , Eur. J. Cancer, 13: 925-935 .
