Table Of ContentEnhancing Cisplatin Delivery and Anti-tumor Efficacy Using Hyperthermia
by
Chelsea Dawn Landon
Department of Pathology
Duke University
Date:_______________________
Approved:
___________________________
Mark W. Dewhirst, Supervisor
___________________________
Soman N. Abraham
___________________________
Laura P. Hale
___________________________
Dennis J. Thiele
___________________________
Zeljko Vujaskovic
Dissertation submitted in partial fulfillment of
the requirements for the degree of Doctor of Philosophy
in the Department of Pathology
in the Graduate School
of Duke University
2013
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ABSTRACT
Enhancing Cisplatin Delivery and Anti-tumor Efficacy Using Hyperthermia
by
Chelsea Dawn Landon
Department of Pathology
Duke University
Date:_______________________
Approved:
___________________________
Mark W. Dewhirst, Supervisor
___________________________
Soman N. Abraham
___________________________
Laura P. Hale
___________________________
Dennis J. Thiele
___________________________
Zeljko Vujaskovic
An abstract of a dissertation submitted in partial fulfillment of
the requirements for the degree of Doctor of Philosophy
in the Department of Pathology
in the Graduate School
of Duke University
2013
Copyright by
Chelsea Dawn Landon
2013
Mild hyperthermia (39°C-43°C) has numerous therapeutic benefits as an adjuvant
therapy in the treatment of a variety of tumor types. Hyperthermia increases tumor blood flow
and vascular permeability, promoting drug delivery and tumor oxygenation. Hyperthermia
enhances the uptake and efficacy of numerous chemotherapeutic agents, including cisplatin,
resulting in increased cytotoxicity. In addition to these biological responses, hyperthermia can
be used as a drug-release trigger for temperature-sensitive nanoparticles, resulting in an
improved and more targeted drug delivery system. Cisplatin was chosen because 1) it shows
broad spectrum activity against a wide range of heatable cancers (i.e., those in sites such as the
pancreas, colon and rectum, cervix and bladder, and 2) the same hyperthermic temperatures
that enable temperature-sensitive lipsome-drug release also enhance cisplatin-induced
cytotoxicity.
The role of hyperthermia in enhancing cisplatin delivery and cytotoxicity was
investigated at both the cellular and tissue levels. While hyperthermia treatment is applicable to
a variety of tumor types, the focus of this work was on bladder cancer. The synergistic effects of
hyperthermia and cisplatin were investigated, along with the role of copper transport protein 1
(Ctr1) in this process. In addition, cisplatin was encapsulated within temperature-sensitive
liposomes, which were used in combination with hyperthermia for targeted drug delivery. These
studies demonstrated that the combination of cisplatin and hyperthermia improved drug
delivery, and potentially anti-tumor efficacy, and that targeted delivery was enhanced through
incorporation of temperature-sensitive liposomes. As many current methods for administering
bladder hyperthermia have drawbacks, such as invasiveness and regional heating, the final aim
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of this study was to develop and test a less-invasive and more focused preclinical bladder
heating device in a rat model.
Hyperthermia sensitizes cells to the cytotoxic effects of the commonly used
chemotherapeutic agent cisplatin by increasing drug accumulation and subsequent platinum-
DNA adduct formation. However, the molecular mechanisms underlying this enhancement
remain unclear. Understanding the fundamental mechanisms involved in the synergistic
interaction is necessary to increase the therapeutic benefits of this combination in the clinic. The
synergism between the anti-cancer benefits of cisplatin and the drug delivery benefits of
hyperthermia may offer a novel and more effective treatment for many cancer patients. We
hypothesized that hyperthermia increases cisplatin accumulation and efficacy in part by
modulating the function of Ctr1, a major regulator of cellular cisplatin uptake. To test this
hypothesis, we examined the significance of Ctr1 during combined hyperthermia and cisplatin
therapies and assessed the importance of cisplatin- and hyperthermia-induced Ctr1
multimerization in enhancing cisplatin cytotoxicity. We observed increased Ctr1 multimerization
following hyperthermia treatment (41°C) in vitro, compared to normothermic controls (37°C),
suggesting that this may be a mechanism for increased cisplatin uptake in heat-treated cells. The
impact of increased Ctr1 multimerization was evaluated by measuring platinum accumulation in
wild-type (WT) and Ctr1-/- cells. WT cells contained greater levels of platinum compared to Ctr1-/-
cells. A further increase in platinum was observed following hyperthermia treatment, but only in
the WT cells. Hyperthermia enhanced cisplatin-mediated cytotoxicity in WT cells with a dose-
modifying factor (DMF) of 1.8 compared to 1.4 in Ctr1-/- cells. Our data suggest that heat
increases Ctr1 activity by increasing multimerization, resulting in enhanced drug accumulation.
Although we recognize that the effect of heat on cells is multi-factorial, our results support the
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hypothesis that Ctr1 is, in part, involved in the synergistic interaction observed with cisplatin
and hyperthermia treatment.
In addition to assessing cisplatin delivery at the cellular level, we evaluated cisplatin
delivery at the tissue level, using novel cisplatin-loaded temperature-sensitive liposomes. We
hypothesized that delivering cisplatin encapsulated in liposomes under hyperthermic conditions
would improve the pharmacokinetic profiles of cisplatin, increase drug delivery to the tumor,
decrease normal tissue toxicity, and enhance the anti-tumor activity of cisplatin. We successfully
prepared temperature-sensitive liposomes loaded with cisplatin and demonstrated that heat
(42°C) sensitizes cisplatin-resistant cells to the cytotoxic effects of cisplatin in vitro.
Decreased toxicity was observed in animals treated with the cisplatin liposome (± heat)
compared to the free drug treatments. A pharmacokinetic study of cisplatin-loaded
temperature-sensitive liposomes and free drug was performed in tumor-bearing mice under
normothermic and hyperthermic conditions. Cisplatin half-life in plasma was increased following
liposome treatment compared to free cisplatin, and cisplatin delivery to the tumors was
greatest in mice that received liposomal cisplatin under hyperthermia. These initial in vivo data
demonstrate the potential effectiveness of this cisplatin-loaded liposome formulation in the
treatment of certain types of cancer. To assess the anti-cancer efficacy of the liposome
treatment, a tumor growth delay study was conducted and demonstrated equivalent efficacy for
the cisplatin-loaded temperature-sensitive liposome compared to free drug.
In addition to the liposome work, we developed and evaluated a novel heating device
for the bladder. Despite the evidence that hyperthermia is an effective adjuvant treatment
strategy, current clinical heating devices are inadequate, warranting the development of a new
and improved system. We induced hyperthermia using ferromagnetic nanoparticles and an
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alternating magnetic field device developed by Actium Biosystems. Initial preclinical studies in a
rat model demonstrated preferential bladder heating. However, our preliminary studies show
severe toxicity with the direct instillation of the nanoparticles in the bladder, and further studies
are needed to potentially modify the nanoparticle coating, the catheterization procedure, as
well as to develop a different animal model.
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Dedication
This body of work is dedicated to my parents and my sister, who have always supported
me in my dreams and goals and have encouraged me to never settle for anything less than what
makes me happy in life.
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Contents
Abstract ........................................................................................................................................... iv
List of Tables .................................................................................................................................. xv
List of Figures ................................................................................................................................. xvi
List of Abbreviations ...................................................................................................................... xix
Acknowledgements........................................................................................................................ xxi
1. Introduction ................................................................................................................................. 1
1.1 Cancer as a Disease ............................................................................................................. 1
1.1.1 The Hallmarks of Cancer................................................................................................. 1
1.1.2 A Complex Disease to Target.......................................................................................... 4
1.2 Bladder Cancer .................................................................................................................... 5
1.2.1 Project Goal .................................................................................................................... 6
1.2.2 Treatment Strategies ...................................................................................................... 6
1.2.2.1 Transurethral Resections ........................................................................................ 6
1.2.2.2 Intravesical Therapy ................................................................................................ 7
1.2.2.3 Radical Cystectomy (Bladder Removal) ................................................................ 10
1.3 Hyperthermia as an Adjuvant Anti-Cancer Treatment Modality ...................................... 11
1.3.1 Hyperthermia at the Biological Level ........................................................................... 14
1.3.2 Hyperthermia in a Clinical Setting ................................................................................ 18
1.4 Cisplatin ............................................................................................................................. 23
1.4.1 Cisplatin Resistance ...................................................................................................... 27
1.4.2 Cisplatin and Hyperthermia ......................................................................................... 29
1.4.2.1 Cisplatin and Hyperthermia in the Clinic .............................................................. 30
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1.5 Copper Transport Protein 1 (Ctr1)..................................................................................... 31
1.6 Thermosensitive Liposomes .............................................................................................. 35
1.7 Dissertation Objective and Outline ................................................................................... 41
2.0 Liposomes and Hyperthermia: Temperature-Sensitive Liposomes ......................................... 43
2.1 Drug Delivery Problem ...................................................................................................... 43
2.2 Rationale for Using Liposomes and Hyperthermia............................................................ 45
2.2.1 Non-Thermosensitive Liposomes ................................................................................. 45
2.2.2 Temperature-Sensitive Liposomes ............................................................................... 48
2.2.2.1 Hyperthermia and Drug Delivery .......................................................................... 52
2.2.2.2 Preclinical Studies with Hyperthermia and Temperature-Sensitive Liposomes ... 55
2.2.2.3 New Paradigm of Temperature-Sensitive Liposome Drug Release ...................... 61
2.2.3 Clinical Progress............................................................................................................ 62
2.2.3.1 Non-thermal Sensitive Liposomes ........................................................................ 62
2.2.3.2 Non-thermal Sensitive Liposomes and Hyperthermia .......................................... 66
2.2.3.3 Temperature-Sensitive Liposomes and Hyperthermia ......................................... 68
2.2.3.4 Clinical Perspective on Cancers that would Benefit from Hyperthermia ............. 71
2.2.3.5 Important Considerations for Temperature-Sensitive Liposome-Device
Combinations .................................................................................................................... 78
2.3 Conclusions ........................................................................................................................ 79
3.0 A Role for Ctr1 in the Synergistic Interaction between Hyperthermia and Cisplatin Treatment:
Increasing cisplatin delivery and efficacy at the cellular level ....................................................... 81
3.1 Introduction ....................................................................................................................... 81
3.2 Material and Methods ....................................................................................................... 84
3.2.1 Reagents and Chemicals ............................................................................................... 84
3.2.2 Cell Lines ....................................................................................................................... 84
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