ABSTRACT
Various conjugates of anticancer drug doxorubicin (Dox) covalently bound by the hydrolytically degradable hydrazone bond to the drug carrier based on N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers were synthesised. Structure of the conjugates differed in the type and the content of hydrophobic substituent (dodecyl, oleic acid and cholesterol moieties) introduced into the polymer structure. In aqueous solutions the conjugates self-assembled into high-molecular-weight supramolecular structures, such as polymeric micelles or stable hydrophilic nanoparticles 13-37 nm in diameter, depending on the type and the content of hydrophobic substituents. Treatment of mice bearing EL-4 T cell lymphoma with the conjugates in the therapeutic regime of drug administration (i.v.) resulted in significant tumour regression with up to 100% of long-term survivors, depending on the dose and the detailed structure of the carrier. The nanoparticles formed by the conjugate bearing cholesterol moiety exhibited prolonged blood circulation and enhanced tumour accumulation indicating an important role of the EPR effect in excellent anticancer activity of the conjugate.
Subject(s)
Antibiotics, Antineoplastic/pharmacology , Doxorubicin/analogs & derivatives , Drug Carriers/pharmacology , Methacrylates/pharmacology , Polymethacrylic Acids/pharmacology , Animals , Antibiotics, Antineoplastic/chemistry , Antibiotics, Antineoplastic/pharmacokinetics , Cell Line, Tumor , Cell Proliferation/drug effects , Doxorubicin/chemistry , Doxorubicin/pharmacokinetics , Doxorubicin/pharmacology , Drug Carriers/chemistry , Drug Carriers/pharmacokinetics , Humans , Hydrophobic and Hydrophilic Interactions , Methacrylates/chemistry , Methacrylates/pharmacokinetics , Mice , Mice, Inbred C57BL , Neoplasm Transplantation , Neoplasms/drug therapy , Neoplasms/metabolism , Polymethacrylic Acids/chemistry , Polymethacrylic Acids/pharmacokineticsABSTRACT
Conjugates based on N-(2-hydroxypropyl)methacrylamide (HPMA) represent a new generation of antibody-targeted polymeric anticancer drugs with both cytotoxic and immunoprotecting/immunomobilizing activity. 20-90% of mice that are cured of EL4 mouse T-cell lymphoma, BCL1 mouse B-cell leukaemia and 38C13 mouse B-cell lymphoma by injection of doxorubicin-HPMA conjugate develop a long-lasting memory and systemic antitumour resistance. It is suggested that the main activity of the polymeric drug, directly after application is - due to the high level of the drug - of cytotoxic and cytostatic nature. Thereafter, long-term conjugates persist at low concentration in the circulation, which are capable of mobilizing the defence mechanisms of the host. Until now, seven patients with generalized carcinoma were treated with doxorubicin-HPMA-human-Ig conjugate. Disease stabilization, lasting from 6 to more than 18 months, was recorded.
Subject(s)
Antibiotics, Antineoplastic/pharmacology , Doxorubicin/pharmacology , Immunity, Innate , Methacrylates/pharmacology , Neoplasms/drug therapy , Animals , Antibiotics, Antineoplastic/pharmacokinetics , Doxorubicin/pharmacokinetics , Humans , Methacrylates/pharmacokinetics , Neoplasms/immunologyABSTRACT
N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymer carrier containing the anticancer drug doxorubicin bound either by a proteolytically degradable bond (non-targeted PK1 or targeted with alpha-CD71 mAb) or by a hydrolytically degradable bond were synthesised and tested in vivo for various biological properties. Mouse 38C13 B-cell lympoma was used as a well established and defined cell line for this study. 38C13 cells are sensitive to free doxorubicin and IC50 was very low, about 0.014 microM. PK1 showed a strongly decreased cytostatic effect, IC50 being 12.6 microM. alpha-CD71 targeted conjugate, which can be considered as an antibody-targeted form of PK1, had IC50 0.358 microM. HPMA copolymer with doxorubicin bound via a hydrolytically sensitive bond (HYD conjugate) showed a high cytostatic effect with IC50 about 0.052 microM. We demonstrated that HYD conjugate inhibited DNA synthesis and induced p21(Waf1/Cip1) protein expression (p21(Waf1/Cip1) is cyclin-dependent kinase inhibitor which blocks cell cycle progression) as quickly as free doxorubicin, whereas PK1 acted much more slowly. Similarly, apoptosis induction measured by Annexin V binding and Caspase 3 activity was detected later after incubation of cells with PK1 or alpha-CD71 targeted conjugate. Apoptosis was manifested by elevation of bax and bad mRNA levels, which was much more rapid and intense in the case of free doxorubicin and HYD conjugate. Expression of antiapoptotic genes as well as cyclin-dependent kinases was surprisingly not affected.
Subject(s)
Acrylamides/chemical synthesis , Acrylamides/pharmacology , Doxorubicin/chemical synthesis , Doxorubicin/pharmacology , Ligands , Acrylamides/metabolism , Animals , Apoptosis/drug effects , Carrier Proteins/genetics , Carrier Proteins/metabolism , Caspase 3 , Caspases/adverse effects , Caspases/drug effects , Caspases/metabolism , Cell Cycle/genetics , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Cell Membrane/drug effects , Cell Membrane/pathology , Cell Proliferation/drug effects , Cyclin-Dependent Kinase Inhibitor p21 , DNA/antagonists & inhibitors , DNA/genetics , DNA/metabolism , Doxorubicin/metabolism , Drug Evaluation, Preclinical/methods , Gene Expression/drug effects , Gene Expression/genetics , Genes, myc/drug effects , Genes, myc/genetics , Hydrazones/chemical synthesis , Hydrazones/metabolism , Hydrazones/pharmacology , Hydrolysis , Inhibitory Concentration 50 , Mice , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , RNA, Messenger/genetics , Receptors, Transferrin/drug effects , Receptors, Transferrin/genetics , Thymidine/metabolism , Tritium , bcl-2-Associated X Protein , bcl-Associated Death ProteinABSTRACT
N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymer carrier containing the anticancer drug doxorubicin and targeted with B1 monoclonal antibody (mAb) to BCL1 leukemia cells was synthesised and tested in vitro and in vivo. BCL1 leukemia growing in syngenic Balb/c mice was selected as a tumor model system. B1 mAb recognising the idiotype of surface IgM on BCL1 cells was used as a targeting moiety. Both B1 mAb and doxorubicin were conjugated to HPMA copolymer carrier by aminolysis through a tetrapeptidic Gly-Phe(D,L)-Leu-Gly spacer to ensure the intracellular delivery and controlled release of the drug. B1 mAb-targeted conjugate was shown to possess strictly tumor-specific binding capacity to target BCL1 cells in vitro. A similar conjugate, but containing human nonspecific Ig (HuIg) instead of B1 mAb, failed to bind to BCL1 cells. In vitro, B1 mAb-targeted conjugate demonstrated 40-fold higher cytotoxic effect than nontargeted or human nonspecific Ig-containing HPMA copolymer-bound doxorubicin. Conjugate targeted with B1 mAb was also shown to bind to target BCL1 cells in vivo. B1 mAb-targeted conjugate was shown to be more efficient in the treatment of established BCL1 leukemia than free doxorubicin, nontargeted and human nonspecific Ig-containing conjugate. Antibody-targeted polymeric drugs are thus promising conjugates for cancer treatment.