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Title: Synthesis and biological evaluation of a novel HPMA copolymer conjugate-based combination therapy designed for the treatment of breast cancer
Author: Greco, Francesca
ISNI:       0000 0004 2749 2009
Awarding Body: Cardiff University
Current Institution: Cardiff University
Date of Award: 2006
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Polymer-anticancer drug conjugates display passive tumour targeting by the enhanced permeability and retention effect. Several conjugates containing traditional chemotherapy (doxorubicin (Dox), paclitaxel and platinates) have entered clinical trials. N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-Dox (PK1) has shown activity in anthracycline-resistant breast cancer patients and was 5 times less toxic than Dox. The use of polymer conjugates to deliver endocrine agents is largely unexplored, therefore, the aim of this study was to design a novel HPMA copolymer conjugate carrying a combination of endocrine therapy (the aromatase inhibitor aminoglutethimide (AGM)) and chemotherapy (Dox) on the same polymer backbone. As patients with advanced metastatic breast cancer have a relatively poor prognosis (< 20 % survival at 5 years), it was hoped that this conjugate would elicit increased antitumour activity. A library of conjugates was synthesised to contain Dox, AGM, or both drugs attached to the same polymer chain. First, the conjugates were prepared by aminolysis of polymeric intermediates (Mw 20,000-25,000 g/mol Mw/Mn = 1.3- 1.5) where the C-terminus of the peptide side-chains were esterified with p-nitrophenol. The lower reactivity of the aromatic amine of AGM, however, favoured use of 1,3-dicyclohexylcarbodiimide (DCC)-mediated coupling, leading to improved yield of conjugation. NMR confirmed product identity and nuclear Overhauser effect (NOE) measurements verified the covalent binding of AGM. Total and free Dox were measured by HPLC and AGM content using an indirect method. Overall, the drug content of the conjugates was in the range 2.8 - 8.6 % w/w (free drug content < 1.4 % w/w total). Cytotoxicity of the conjugates was determined using the human oestrogen-dependent breast cancer cell line (MCF-7) and the aromatase-transfected derivative MCF-7ca combined with the MTT assay (72 h). Although HPMA copolymer-Dox was much less active than free Dox, and neither the HPMA copolymer-AGM nor mixtures of drug conjugates bearing AGM or Dox caused an elevation in cytotoxicity in either cell line, when AGM and DOX were covalently linked to the same polymeric backbone, cytotoxicity was significantly enhanced (more evident for MCF-7ca cells). To better understand the precise mechanism of action, the ability to inhibit aromatase was evaluated in 3 different in vitro systems. Free AGM and HPMA copolymer-AGM inhibited aromatase, but drug release was probably essential for the activity of the HPMA copolymer-AGM. To establish whether the rate or mechanism of uptake of HPMA copolymer-Dox ( AGM) were responsible for the different cytotoxicity, the uptake of the conjugates was evaluated by FACS. Internalisation and marked membrane binding were seen for both conjugates suggesting that the different cytotoxicity could not be attributed to differences in the uptake. Finally, immunocytochemistry studies showed that the HPMA copolymer-AGM-Dox reduced the level of the anti-apoptotic protein Bcl-2 in MCF-7, while the HPMA copolymer-Dox had no effect on this cellular marker. To conclude, the HPMA copolymer-AGM-Dox conjugate is the first polymer therapeutic to combine chemotherapy and endocrine therapy. The fact that AGM and Dox can act synergistically to produce markedly enhanced cytotoxicity in vitro compared to the HPMA copolymer-Dox conjugate, that has already shown activity in breast cancer patients clinically, underlines the potential importance of this polymer- drug combination.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available