POLY (AMIDO AMINE) DENDRIMERS

237 Pages·2010·7.17 MB·English

by  Deborah Sweet Goldberg

ABSTRACT

Title of Document: POLY (AMIDO AMINE) DENDRIMERS:
TRANSEPITHELIAL TRANSPORT
MECHANISMS AND APPLICATIONS IN
ORAL DRUG DELIVERY

Deborah Sweet Goldberg, Doctor of Philosophy,
2010

Directed By: Professor Hamidreza Ghandehari, Fischell
Department of Bioengineering

Small molecule chemotherapy drugs used in clinical practice are plagued by
dose-limiting side effects due to off-target toxicities. In addition, because of their
low water solubility and poor bioavailability, they must be administered
intravenously, leading to high treatment costs and recurring hospital visits. There is a
significant need for therapies that improve the bioavailability of chemotherapy agents
and enhance specific drug release in the tumor environment.
Dendrimers, a class of highly-branched, nanoscale polymers, share many
characteristics with traditional polymeric carriers, including water solubility, high
capacity of drug loading and improved biodistribution. Poly (amido amine)
(PAMAM) dendrimers have shown promise as oral drug carriers due to their compact
size, high surface charge density and permeation across the intestinal epithelial
barrier. Attachment of chemotherapy drugs to PAMAM dendrimers has the potential
to make them orally administrable and reduce off-target toxicities.
In this dissertation we investigate the transport mechanisms of PAMAM
dendrimers and their potential in oral drug delivery. We demonstrate that anionic
G3.5 dendrimers are endocytosed by dynamin-dependent mechanisms and their
transport is governed by clathrin-mediated pathways. We show that dendrimer
cellular internalization may be a requisite step for tight junction opening. We also
demonstrate that conjugation of small poly (ethylene glycol) chains to anionic
dendrimers decreases their transport and tight junction opening due to reduction in
surface charge, illustrating that small changes in surface chemistry can significantly
impact transepithelial transport. Knowledge of transport mechanisms and the impact
of surface chemistry will aid in rational design of dendrimer oral drug delivery
systems.
The potential of dendrimers as oral drug delivery carriers is demonstrated by
the evaluation of G3.5 PAMAM dendrimer-SN38 conjugates for oral therapy of
hepatic colorectal cancer metastases, a pathology present in over 50% of colorectal
cancer cases that is responsible for two-thirds of deaths. Conjugation of SN38, a
potent chemotherapy drug with poor solubility and low bioavailability, to PAMAM
dendrimers via a glycine linker increased intestinal permeability, decreased intestinal
toxicity and showed selective release in the presence of liver carboxylesterase,
illustrating that PAMAM dendrimers have the potential to improve the oral
bioavailability of potent anti-cancer therapeutics.
POLY (AMIDO AMINE) DENDRIMERS: TRANSEPITHELIAL TRANSPORT
MECHANISMS AND APPLICATIONS IN ORAL DRUG DELIVERY

By

Deborah Sweet Goldberg

Dissertation submitted to the Faculty of the Graduate School of the
University of Maryland, College Park, in partial fulfillment
of the requirements for the degree of
Doctor of Philosophy
2010

Advisory Committee:
Professor Hamidreza Ghandehari, Co-Chair
Professor William Bentley, Co-Chair
Professor Robert Briber
Professor Silvia Muro
Professor Peter Swaan
© Copyright by
Deborah Sweet Goldberg
2010
Dedication
To my wonderful husband Hirsh, for his constant, unwavering love and support.
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Acknowledgements
I have had the honor of working with many talented individuals during my
PhD studies who have helped me reach this milestone in my life. I would first like to
thank my advisor Dr. Hamid Ghandehari for his enduring commitment to my
training as a scientist. Although Dr. Ghandehari moved to the University of Utah
shortly after I began my research, he has always strived to maintain constant
communication and provide advice on my project. I appreciate his attention to detail
in his careful review of my abstracts, posters, presentations, publications and
dissertation. Dr. Ghandehari has always inspired me to do my best work, and I know
that the impact of his guidance will follow me into my future scientific endeavors.
I would also like to thank Dr. Peter Swaan for his contributions to my PhD
studies as both my co-advisor and local contact. Dr. Swaan has helped me feel at
home at the University of Maryland, Baltimore. I sincerely appreciate the many
research discussions that we had during group meetings and individual meetings. I
am also thankful for the fresh perspective he offered on my publications and
presentations. Dr. Swaan has taught me to think critically and continually question
everything, a mindset that will serve me well in the future.
I also wish to thank my committee members, Dr. Silvia Muro, Dr. Robert
Briber and Dr. William Bentley for their helpful suggestions during my research
proposal and committee meeting. Their insights have helped shape the trajectory of
my project, and I am grateful for their guidance.
In addition to my advisors and my committee members, two other professors
have had a significant impact on my PhD studies. I will be forever indebted to Dr.
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Rohit Kolhatkar for his guidance during the first few years of my PhD studies.
Rohit taught me experimental techniques and advised me on planning experiments,
interpreting results and troubleshooting chemical synthesis strategies. After Rohit left
to become an assistant professor at the University of Illinois, Chicago I realized that
his guidance had taught me everything I needed to think like a scientist and
successfully complete the remainder of my studies. I would also like to thank Dr.
Anjan Nan for graciously allowing me to conduct my research in his laboratory
space and use his equipment. Dr. Nan’s support of my long distance advisement by
Dr. Ghandehari made it possible for me to continue my research on dendrimers.
I wish to acknowledge all of my fellow lab members past and present in the
Ghandehari, Swaan and Nan labs who have had an influence on my day-to-day life in
the lab. Specifically, I would like to acknowledge Dr. Mark Borgman for his
endless technical advice on experiments and his wonderful friendship. Even after he
graduated, Mark was only a phone call or an email away, and has continued to be a
great friend and knowledgeable research consultant. I would also like to thank
Brittany Avaritt, Tatiana Claro da Silva and Paul Dowell for their friendship and
support over the years. Finally, I would like to thank Carl the janitor who always
managed to make me smile on his early morning rounds.
During my PhD experience I was fortunate to complete a summer internship
in the Department of Formulation Sciences at MedImmune LLC. Although this
internship experience was distinct from my graduate research, it gave me an
appreciation of the pharmaceutical industry and taught me new ways of approaching
problems, which positively impacted my graduate work after the internship. I would
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like to thank my supervisor Dr. Hasige Sathish and my formulation sub-group leader
Dr. Ambarish Shah for their positive contributions to my PhD experience and for
providing me with this unique opportunity.
Several fellowships and grants during my graduate studies have helped to
fund my research. I would like to thank the National Science Foundation for the
Graduate Research Fellowship, which sponsored my first three years of graduate
school. It was an honor to receive this fellowship and it had a positive impact on my
graduate school career, providing me with freedom in project selection. I would also
like to thank Dr. Robert Fischell for generously sponsoring the Fischell Fellowship
in Bioengineering, which funded my final years in graduate school. Dr. Fischell’s
commitment to fostering new Bioengineering research is admirable and his
enthusiasm is contagious. I also acknowledge NIH R01 EB007470 for funding
supplies for this research.
I would like to recognize my friends and family, whose support and
encouragement have been critical for the successful completion of my PhD studies.
My parents, Shari and Rick Sweet, have always been my biggest cheerleaders,
supporting all of my academic and personal pursuits. I truly appreciate their
excitement about all of my accomplishments from elementary school until today and
their constant encouragement to strive for my dreams. I would also like to thank my
mother and father in-law, Karen and Larry Goldberg for their interest and support
throughout my PhD studies. I would like to acknowledge the wonderful support of
my friends Marie Jeng, Chloe Marin, Rifat Jafreen and Kevin Nelson among
many others who have shown interest in my PhD progress over the years and offered
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countless words of encouragement. I wish to express my gratitude to the instructors
at Columbia Jazzercise for providing a fun and exhilarating stress release as well as
all of my friends at Jazzercise for their constant interest and encouragement. Finally,
I would like to thank my husband Hirsh who has been my greatest supporter through
all of the ups and the downs of my PhD research. Hirsh has always offered emotional
support and encouragement when I was frustrated with unsuccessful experiments and
has unselfishly taken over household responsibilities when I had to work late at night
or on the weekend to meet a deadline. I know that his love and unwavering support
helped me push through the most difficult challenges of my PhD and I could not have
done it without him. He truly deserves this degree as much as I do.
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Table of Contents

Dedication................................................................................................................ii

Acknowledgements.................................................................................................iii

List of Tables..........................................................................................................xi

List of Figures........................................................................................................xii

Abbreviations........................................................................................................xv

Chapter 1 : Introduction.........................................................................................1

1.1 Introduction....................................................................................................1
1.1.1 Polymer Therapeutics................................................................................1
1.1.2 Oral Drug Delivery...................................................................................2
1.1.3 Poly (amido amine) Dendrimers................................................................3
1.1.4 SN38.........................................................................................................4
1.2 Specific Aims..................................................................................................5
1.3 Scope and Organization...................................................................................6

Chapter 2 : Background..........................................................................................8

2.1 Introduction.....................................................................................................8
2.2 Polymeric Drug Delivery.................................................................................8
2.2.1 Therapeutic Advantages of Polymer-Drug Conjugates in Chemotherapy...9
2.2.2 Polymer-Drug Conjugates Currently in Clinical Trials............................12
2.3 Administration of Drugs via the Oral Route...................................................13
2.3.1 Physiology of the Gastrointestinal Tract..................................................14
2.3.1.1 Compartments and Functions...........................................................14
2.3.1.2 Intestinal Epithelial Barrier...............................................................17
2.3.1.3 Tight Junction Biology: Structure and Function................................18
2.3.2 Mechanisms of Transport Across the Intestinal Barrier...........................21
2.3.2.1 Paracellular Transport......................................................................21
2.3.2.2 Passive Diffusion.............................................................................23
2.3.2.3 Carrier-Mediated Transport..............................................................24
2.3.2.4 Endocytosis......................................................................................25
2.3.3 Physiochemical Properties that Govern Intestinal Absorption..................28
2.3.3.1 The Lipinski Rule of 5......................................................................28
2.3.3.2 The Biopharmaceutics Classification System (BCS).........................28
2.4 Models to Predict Oral Absorption and Oral Bioavailability...........................30
2.4.1 In Silico Models......................................................................................30
2.4.2 Parallel Artificial Membrane Permeability Assay....................................31
2.4.3 Caco-2 Monolayers.................................................................................33
2.4.4 Fast-Caco-2 Assay..................................................................................36
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