Doctor of Philosophy
School of Biological Sciences
Indira Chandran, Vineesh, Development of targeted anticancer agents using novel N-alkylisatin derivatives, Doctor of Philosophy thesis, School of Biological Sciences, University of Wollongong, 2012. http://ro.uow.edu.au/theses/3654
Targeted therapy involves selective targeting of tumour and/or tumour associated cells using agents that recognise specific biomarkers which are characteristic of the tumour cell population. The use of ligand-drug conjugates (LDCs) for targeted therapy includes agents that combine the selectivity and specificity of biomarker ligands with the high potency of chemotherapeutic drugs. This not only causes effective reduction of tumour growth, but also minimises off target cell killing and delays the development of resistance which otherwise arises from prolonged exposure to non-specific drugs.
This study focussed on the development of LDCs incorporating novel N-alkylisatin derivatives. We have previously reported the cytotoxic activity of a series of N-alkylisatin derivatives that destabilise microtubules and induce apoptotic cell death in a range of cancer cell lines. In this study, two of the most potent N-alkylisatin derivatives, 5,7-dibromo-N-(p-hydroxymethylbenzyl)isatin (NAI-1) and 5,7-dibromo-N-(pmethoxybenzyl)isatin (NAI-2), functionalised with an esterase-labile succinate (NAI-1-succ) and an imine (NAI-2-imine) functional linker respectively, were conjugated to well described tumour ligands, namely, transferrin (Tf), plasminogen activator inhibitor type 2 (PAI-2), and trastuzumab (TRZ). These were tested against various human breast carcinoma cell lines that differed in their cell surface expression of relevant targets for these ligands. Both NAI-1-succ-Tf and NAI-2-imine-Tf conjugates were selectively toxic against the cell lines tested. NAI-1-succ-Tf conjugate (IC50 0.75 μM) was greater than 10 × more active compared to the parent compound (IC50 7.67 μM) against the high Tf receptor (TfR) expressing SK-BR-3 cells after 48 h incubation. The NAI-2- imine-Tf conjugate was equipotent to the parent NAI-2 compound against cells expressing moderate to high TfR levels. As the conjugate incorporating the imine-based linker system was less selective for their target, evident by their equipotency to the parent NAI-2 and NAI-2-imine compounds, the imine-based linker system was not used in the development of other LDCs.
Previous studies performed in our laboratory found the NAI-1-succ-PAI-2 conjugate to be equivalent in efficacy to the free NAI-1 compound at 1/20th of the dose, in a metastatic, orthotopic human breast tumour xenograft mouse model. However, our laboratory has also shown that PAI-2 is rapidly excreted from the body. Therefore, to improve the pharmacokinetic and potential tumour uptake properties, a modified form of PAI-2 containing only three potential sulfhydryl sites, C161S-PAI-2, was covalently modified using polyethylene glycol molecules (PEGs) functionalised with a maleimide functional linker (mPEG) to form mPEG-C161S-PAI-2. Purification of mPEG-C161S-PAI- 2 by ion exchange was achieved after a series of optimisations and NAI-1-succ was then conjugated to the purified mPEG-C161S-PAI-2 to form mPEG-C161S-PAI-2- NAI-1-succ conjugate. In vitro studies showed mPEG-C161S-PAI-2-NAI-1-succ conjugate to be ~3.5 × more potent than the free NAI-1 and showed selectivity against high uPAR expressing breast cancer cells.
Recent studies have found over amplification of HER-2 and uPA receptors in 70 % of HER-2 amplified advanced invasive breast carcinoma cells. Further studies targeting HER-2 and uPA showed that depleting both HER-2 and uPA receptors suppressed cell growth and induced cell apoptosis in SK-BR-3 and ZR-75-1 mammary epithelial cells that over-express HER-2 and uPA receptors. In this direction, NAI-1-succ-TRZ conjugate was prepared by reacting the commercially available recombinant humanised monoclonal antibody (mAb) TRZ directed against HER-2 receptors, with a 40-fold molar excess of NAI-1-succ in sodium phosphate buffer (pH 6.0). The resultant NAI-1- succ-TRZ conjugate (IC50 0.61 μM) was ~8 × more toxic than free NAI-1 (IC50 5.19 μM) and was highly selective against high HER-2 expressing SK-BR-3 cells. This highly potent NAI-1-succ-TRZ along with NAI-1-succ-C161S-PAI-2 was utilised in a novel combination targeting approach (also called "double targeting") to specifically target metastatic breast cancer cells that over-express uPA/uPAR and HER-2 receptors. In vitro double targeting assays using these agents against high uPAR and HER-2 expressing ZR-75-1 breast cancer cells showed increased cell death. This warrants further in vivo study of NAI-1-succ-TRZ and NAI-1-succ-C161S-PAI-2 in combination in appropriate mouse models to ascertain their potential synergistic effect in reducing tumour growth compared to treatment with each drug alone. This approach is expected to result in superior efficacy over single agent therapy and due to minimal drug exposure, delay the development of drug resistance.
On the whole, this study successfully developed and proved the selective potency of 5,7-dibromo-N-(p-hydroxymethylbenzyl)isatin functionalised with an ester linker in ligand-drug conjugates, namely mPEG-C161S-PAI-2-NAI-1-succ and NAI-1-succ-TRZ in vitro. The utility of these conjugates in a novel double targeting treatment strategy to simultaneously target highly characteristic tumour-specific biomarkers in breast cancer warrants further development using appropriate animal tumour models.