Combination Therapy with Polymeric Nanomedicines. Kopecek's laboratory was the first to use combination therapy with polymeric nanomedicines. Combination chemotherapy and photodynamic therapy with HPMA copolymer conjugates may result in a synergistic response, resulting in a better cure rate than monotherapy. On two cancer models, Neuro 2A neuroblastoma and human ovarian carcinoma heterotransplanted in the nude mice, combination therapy with HPMA copolymer - anticancer drug [DOX and meso chlorin e6 mono(N-2-aminoethylamide) {Mce6}] conjugates showed tumor cures that could not be obtained with either chemotherapy or photodynamic therapy alone. Cooperativity of the action of both drugs contributed to the observed effect. Based on biodistribution data, it was hypothesized that combination therapies of s.c. human ovarian carcinoma OVCAR-3 xenografts in nude mice using multiple doses/irradiation of P-GFLG-Mce6 (P is the HPMA copolymer backbone) and PGFLG-DOX may acquire low effective doses without sacrificing the therapeutic efficacy. Indeed, 10 out of 12 tumors exhibited complete responses in the group of mice receiving multiple PDT plus multiple chemotherapy. Finally, the advantages of targeted combination chemotherapy and photodynamic therapy using OV-TL16-targeted HPMA copolymer-DOX and HPMA copolymer- Mce6 conjugates were demonstrated. OV-TL16 antibodies are complementary to the OA-3 antigen (CD47) present on the majority of ovarian cancers. The immunoconjugates preferentially accumulated in human ovarian carcinoma OVCAR-3 xenografts in nude mice with a concomitant increase in therapeutic efficacy when compared with non-targeted conjugates. The targeted conjugates suppressed tumor growth for the entire length of the experiment (> 60 days).
Recently, we have quantitatively evaluated the synergism of HPMA copolymer bound SOS thiophene, DOX, and Mce6 in human renal carcinoma and, for antibody targeted conjugates, in human ovarian carcinoma models.
At present we are focusing on the use of HPMA copolymer-retinoid conjugates, which act as Biological Response Modifiers and induce differentiation in ovarian carcinoma cells resulting in enhanced susceptibility of pretreated cells to chemotherapy and/or photodynamic therapy.
See excellent results on combination therapy using two anti-angiogenic agents bound to one polymer chain.
New Targeting Strategies. A unique design of targetable conjugates from our laboratory was their synthesis using polymerizable Fab' antibody fragments and their successful use in the treatment of experimental ovarian cancer.
Research is ongoing with the aim to identify peptides as targeting moieties. Due to their low size, numerous targeting moieties can be attached to one macromolecule, resulting in targeting enhancement mediated by the multivalency effect. Two of several approaches used are highlighted below.
The first approach uses genetically engineering methods to design scaffolds for epitope display. Plasmids encoding specific protein structures containing epitopes recognizable by immunocompetent cells were designed and expressed in E. coli. The purified proteins, antiparallel coiled-coil stem loop (AP-CCSL) structures, contained recognizable epitopes in the loop. When self-assembled on a solid substrate the epitopes displayed at the solid-liquid interface in an organized manner. The unique design of the DNA sequence encoding the AP-CCSL incorporated restriction sites permitting an easy replacement of epitope encoding sequences. The interaction of exposed epitopes with immunocompetent cells, determination of the relationship between the sequence and/or secondary structure of epitopes and biorecognition, and binding studies in the presence of monoclonal antibodies specific for particular receptors was performed and resulted in identification of epitopes for binsing to the CD21 receptor. The identified sequence was incorporated into HPMA copolymer-DOX conjugates and the multivalency effect proven in vivo.
The second approach involves comb
inatorial approaches. Suitable peptides, complementary to the CD21 receptor on B-cells, were selected by phage display and chemically prepared peptide libraries using the one-bead one-compound (OBOC) method. The relationship between detailed structures of HPMA copolymer conjugates containing targeting peptides, their conformation, and efficacy was evaluated using numerous techniques including FRET (fluorescence resonance energy transfer).
Subcellular Targeting. In the past, macromolecular therapeutics were designed based on the hypothesis that they are internalized by clathrin-mediated endocytosis with an ultimate location in the lysosomes. However, recent research in our and other laboratories indicates that other mechanism of internalization take place. The relationship between the structure of conjugates and their mechanism of cell entry by clathrin-mediated endocytosis, caveolae-mediated endocytosis, macropinocytosis, and non-clathrin, non-caveolae mediated endocytosis is being evaluated.
Fig.: P-GFLG-DOX (HPMA copolymer bound DOX) and Alexa fluor 633 labeled transferrin were co-incubated in growth media for 2 h followed immediately by confocal microscopy. The yellow dots signify endosomes containing both transferrin and P-GFLG-DOX. Images were taken in live cells.
Fig.: Cells were exposed to Alexa fluor 647-Dextran 10 kd overnight and images were taken in the presence of P-GFLG-DOX after 2h. Staining was done with antibody against Lamp-1(lysosome associated membrane protein-1).
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Current ovarian anticancer regimens are constrained by decreased efficacy and the development of side effects in the patient after administration, resulting in low survival rates, especially in advanced stages of presentation. It is essential to develop anticancer agents that demonstrate good efficacy without causing systemic toxicity. The cytotoxicity of anticancer agents can be increased by targeting them specifically to the tumor cells. It can be further augmented by directing the anticancer agent to specific subcellular compartments where the drug exerts its action. The aim of our projects is to combine tumor targeting with subcellular targeting (to the nucleus or to mitochondria) and to develop efficacious chemotherapeutics with fewer side effects.
Fig.: Subcellular targeting of HPMA polymer bound drug to mitochondria. Combined field indicates colocalization of mitochondrial marker and the released drug.
Mechanism of Action. The hypothesis that free and polymer-bound drugs activate different signaling pathways is based on their internalization mechanisms. Free drug may initiate signaling pathways by interaction with membrane-bound proteins. In contrast, a polymer-bound drug may be hidden in the hydrophilic corona of the random polymer coil and prevented from interaction with membrane proteins during internalization. It may interact with proteins and DNA after being released from the carrier in the lysosomes and translocated into the cytoplasm. Differences in gene expression profiles of sensitive A2780 and doxorubicin-resistant A2780/AD human ovarian carcinoma cells were detected in our laboratory after exposure to free and HPMA copolymer-bound doxorubicin in vitro and on xenografts in mice in vivo. The differences in the expression of genes were related to multidrug resistance, DNA repair, and detoxification clearly indicating the advantages of macromolecular therapeutics. In addition, it was shown that the HPMA copolymer-doxorubicin conjugate (with a GFLG spacer) induces apoptosis in ovarian carcinoma cells through the mitochondrial pathway by simultaneous activation of both caspase-dependent and caspase-independent pathways of DNA damage. Experiments with free and HPMA copolymer-bound geldanamycin, a heat shock protein inhibitor, have revealed the possibility to modulate cellular stress responses by attaching drugs to polymeric carriers.
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