Preclinical evaluation of a novel drug delivery system for cisplatin

Venugopal, Balaji (2012) Preclinical evaluation of a novel drug delivery system for cisplatin. MD thesis, University of Glasgow.

Abstract

The aim of this body of work was to characterise a novel cisplatin drug delivery system and to develop new tools based on biophotonic imaging that could be used to enhance studies of drug delivery in vivo.

Cucurbiturils (CB) are macrocycles which are formed by acid catalysed condensation of glycoluril and formaldehyde. The internal cavity of CB[7] encapsulates a single molecule of cisplatin and the hypothesis was that encapsulation would reduce thiol degradation of the drug. Drug sensitivity studies in vitro with the cisplatin-sensitive human ovarian cancer cell line, A2780, and a cisplatin-resistant derivative, A2780/cp70, showed that the CB[7] encapsulated cisplatin retained activity but that this encapsulation drug delivery system was not able to overcome resistance to platinum. However, when these cell lines were grown as subcutaneous xenografts in nu/nu mice, the encapsulated cisplatin was able to reduce the growth of A2780/cp70 tumours which are resistant to the maximum tolerated dose of cisplatin in vivo. One possible explanation of this observation is that encapsulation might alter the pharmacokinetics of cisplatin and a method for the detection of platinum in biological samples by ICP-MS was established and validated. This assay was sufficiently sensitive to detect the low levels of platinum present in mouse plasma 24 hours after administration of either free or encapsulated cisplatin. Plasma and tissue pharmacokinetics show that encapsulation had no effect on the peak plasma concentration of cisplatin but did reduce the rate at which cisplatin was cleared from the plasma. The increased plasma AUC of cisplatin resulted in a non-selective increase in the delivery of cisplatin to both tumour and normal tissues. However, there was no apparent increase in toxicity which could be explained by the fact that encapsulation, unlike an increase in the dose of free cisplatin, had no effect on the peak plasma concentration.

Subcutaneous xenografts lack critical features of human tumours. The development of more complex models for use in drug development has been limited due to lack of a method for monitoring tumour growth. Biophotonic imaging was, therefore, investigated to determine whether it is sufficiently sensitive and reproducible to be able to evaluate growth of disseminated tumours in mice. The bioluminescent signal is dependent on the metabolism of luciferin by luciferase. Subcutaneous injection of luciferin was shown to produce a consistent signal in all injected mice. The bioluminescent signal was transient but reached a maximum intensity 6 minutes after injection and remained stable for about 4 minutes which defined the window during which measurements were taken. Sensitivity was shown to be dependent on the level of expression of luciferase by the cells. Injection of commercially available HCT116Luc cells, where the luciferase gene was inserted by a lentiviral system, was shown to allow detection of 10,000 cells in the lungs of mice. This sensitivity was about 10 fold greater than was obtained by lipofectamine based gene transfection. When HCT116Luc cells were grown as subcutaneous xenografts in mice, an exponential growth pattern was easily detected by bioluminescence imaging and the reproducibility between mice was comparable to that routinely obtained by calliper measurements.

Activity of encapsulated cisplatin was determined in a model of disseminated ovarian cancer. Rab25, a member of the RAS oncoprotein superfamily, is up-regulated in around 80% of ovarian cancer samples compared to normal ovarian epithelium. Rab25 contributes to tumour progression by enabling the tumour cells to invade the extracellular matrix by altering the trafficking of integrin. Transfection of Rab25 into A2780 cells results in cells that can grow in the peritoneal cavity of mice. A2780-Rab25 cells were 4 fold resistant to cisplatin in vitro which confirms a previous observation that Rab25 expression in A2780 makes them less sensitive to the induction of apoptosis in response to stress. A2780-Rab25 cells that express the luciferase gene (A2780-Rab25Luc) were injected into the peritoneal cavity of mice and growth was measured by biophotonic imaging. Exponential growth was clearly apparent at a stage at which no obvious abdominal distension was apparent. The disseminated A2780-Rab25Luc tumour xenografts were less sensitive to cisplatin than are subcutaneous xenografts of A2780. This is the first study that suggests that Rab25 over-expression results in reduced drug sensitivity in vivo. In contrast, a very significant growth inhibition was observed when mice were treated with an equivalent dose of encapsulated cisplatin regardless of whether it was administered by the intraperitoneal or subcutaneous route. These results are very encouraging since they confirm the enhanced activity of encapsulated cisplatin and also demonstrate the value of biophotonic imaging for measurement of tumour growth in vivo.

Pharmacodynamic measures of drug activity in vivo in animal models are often based either on measures of surrogate tissue response or on single measures on tumour tissue removed at the end of the experiment. Biophotonic imaging in vivo allows the translation of reporter assays used in cell lines in vitro to studies of tumour response in vivo. A plasmid was prepared that links the p53 transcriptional response element to the luciferase gene and it was then transfected in to A2780 cells which express wild type p53. Stable transfectants of A2780p53Luc were treated with cisplatin, doxorubicin and paclitaxel and induction of p53 determined by bioluminescence and confirmed by Western blotting. A very low bioluminescent signal was present in untreated cells and a clear dose dependent increase in bioluminescence was seen in response to all three drugs. When A2780p53Luc cells were grown as subcutaneous xenografts the bioluminescent signal was significant in untreated tumours but was markedly increased 24 hours after treatment of the mice with cisplatin. Induction of p53 in the tumours was confirmed by immunohistochemistry and this also confirmed significant expression of p53 in untreated tumours.

The possible implications of these findings for the improved delivery of cisplatin are discussed.

• Item Type: Thesis (MD)
• Qualification Level: Doctoral
• Keywords: Cisplatin, cucurbituril, drug delivery, bioluminescence, tumour models, ICP-MS, pharmacokinetics
• Subjects: R Medicine > RC Internal medicine > RC0254 Neoplasms. Tumors. Oncology (including Cancer)
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Cancer Sciences
• Supervisor's Name: Evans, Prof. Jeff
• Date of Award: 2012
• Depositing User: Dr Balaji Venugopal
• Unique ID: glathesis:2012-4198
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 26 Apr 2013 10:42
• Last Modified: 26 Apr 2013 10:42
• URI: https://theses.gla.ac.uk/id/eprint/4198