Polymer conjugation is a strategy able to enhance the tumour selectivity of anticancer drugs and to prolong their half life. Seventeen polymer-drug conjugates containing traditional chemotherapy (e.g. paclitaxel) have entered clinical trials and novel conjugates containing experimental drugs (e.g. the anti-angiogenic agent TNP-470) are emerging. Dopamine (DA) is an endogenous regulator of angiogenesis (the process of new blood vessels formation) and is able to retard the development of angiogenesis-dependent tumours when administered at a low non-toxic dose. However, the clinical application of DA in cancer treatment is hindered by its short half life (2 min) and severe side effects. DA is also a natural substrate of tyrosinase, a physiological enzyme present in the skin and over-expressed in malignant melanoma. Derivatives of DA have been designed as a new class of melanoma-specific anticancer prodrugs but their development is still limited to the preclinical stage. This study focuses on the development of novel polymer conjugates of DA and DA derivatives with the aim of enhancing their therapeutic potential as anti- angiogenic drugs and melanoma-specific agents, respectively. Polyglutamic acid (PGA) was selected as the polymeric carrier and the conjugation of DA was optimised using a carbodiimide and N-hydroxysuccinimide mediated coupling. The synthesis of the PGA-DA conjugate was confirmed by IH-NMR and 13C_NMR spectroscopic analysis. The total DA content was quantified by IH-NMR spectroscopic analysis and found to be in the range 10.8-14.9% w/w. The free DA content was determined by HPLC analysis and always found to be below 1 % of the total DA. PGA-DA as an anti-angiogenic drug. DA exerts its anti-angiogenic effect by binding to its D2 receptor and inhibiting the pro-angiogenic action of the vascular endothelial growth factor (VEGF). Thus, the anti-angiogenic activity of PGA-DA was assessed in two VEGF- dependent systems: an in vitro scratch assay, concerning the migration of human umbilical vein endothelial cells (HUVEC) and an in vivo Miles assay, evaluating vascular permeability. After a short incubation (1 h), both DA and PGA-DA reduced HUVEC migration to a similar extent (-50% VEGF inhibition). In contrast, after a longer incubation time (24 h), DA proved inactive while PGA-DA was even more effective (complete inhibition of VEGF). Similarly, a single injection of PGA-DA reduced the VEGF-induced vascular permeability for longer timeframes (24 h, 60% reduced dye extravasation) than DA (inactive at 24 h). Binding and degradation studies were performed to investigate the mechanism of action of PGA-DA. The inability of PGA-DA to bind to the D2 receptor and its degradation in the presence of cathepsin B suggested that the anti- angiogenic activity of the conjugate was mediated by the release of DA. PGA-(DA derivative) as melanoma-specific conjugates. The ability of DA to act as a substrate of tyrosinase was exploited in the development of an enzymatically activated conjugate. DA was chemically modified to act as a linker between the polymer and the drug. A model drug (p-nitroaniline (pNA)) or a cytotoxic drug (nitrogen-mustard) were coupled to DA. The ability of these DA derivatives to undergo enzymatic activation was assessed by a UV -Vis assay and oximetry. There were two main findings: (i) the linker- mustard was a substrate of tyrosinase; (ii) the linker-pNA was not recognised by tyrosinase as a substrate. Computational studies were used to probe this difference and showed that pNA prevented the activation of the DA derivative by altering its conformation in the active site of tyrosinase. An attempt to conjugate PGA to a DA derivative was also carried out using the method optimised for DA. Further characterisation is required to confirm the identity of the product. To conclude, the relevance of this work is two-fold: (i) polymer conjugation extended the anti-angiogenic activity of DA, making its translation to the clinic a plausible target; (ii) the development of the first melanoma-specific polymer-drug conjugate was undertaken.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:553045 |
| Date | January 2011 |
| Creators | Fante, Cristina |
| Publisher | University of Reading |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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